THE MEDICINAL CHEMISTRY OF 2-AMINOTETRALIN-DERIVED

A Novel Class of Potential Atypical Agents RIJKSUNIVERSITEIT GRONINGEN

THE MEDICINAL CHEMISTRY OF 2-AMINOTETRALIN-DERIVED BENZAMIDES

A Novel Class of Potential Agents

PROEFSCHRIFT

ter verkrijging van het doctoraat in de Wiskunde en Natuurwetenschappen aan de Rijksuniversiteit Groningen op gezag van de Rector Magnificus, Dr. D.F.J. Bosscher, in het openbaar te verdedigen op vrijdag 16 oktober 1998 om 16.15 uur

door

Evert Jan Homan

geboren op 17 mei 1970 te Vries Promotor Prof. Dr. H.V. Wikström

Co-promotor Dr. C.J. Grol Voor mijn ouders Promotiecommissie Prof. Dr. U. Hacksell Prof. Dr. J.P. Snyder Prof. Dr. J. Zaagsma

Paranimfen Henk-Frans Kwint Ruud Timmerman

Colophon

Copyright © 1998 by E.J. Homan. All rights reserved. No part of this book may be reproduced in any manner or by any means without written permission from the publisher.

An electronic version of this thesis is available at http://www.ub.rug.nl/eldoc/dis/science/e.j.homan

ISBN 90-367-0953-9 NUGI 746

Printing: Ponsen & Looijen BV, Wageningen, The Netherlands Cover design: Cor J. Grol

The research project described in this thesis was performed within the framework of the research school GUIDE, and was financially supported by Astra Arcus AB, Södertälje, Sweden. CONTENTS

1 INTRODUCTION 1

2 2-AMINOTETRALIN-DERIVED SUBSTITUTED BENZAMIDES WITH MIXED 57

DOPAMINE D2, D3, AND 5-HT1A RECEPTOR BINDING PROPERTIES: A NOVEL CLASS OF POTENTIAL ATYPICAL ANTIPSYCHOTIC AGENTS

3 STRUCTURAL ANALOGUES OF 5-OME-BPAT: SYNTHESIS AND 81

INTERACTIONS WITH D2, D3, AND SEROTONIN 5-HT1A RECEPTORS

4 C5-SUBSTITUTED DERIVATIVES OF 5-OME-BPAT: SYNTHESIS AND 99

INTERACTIONS WITH DOPAMINE D2 AND SEROTONIN 5-HT1A RECEPTORS

5 SYNTHESIS AND IN VITRO PHARMACOLOGICAL EVALUATION OF THE 119 ENANTIOMERS OF 5-OME-BPAT AND 5-OME-(2,6-DI-OME)-BPAT

6 IN VIVO PHARMACOLOGICAL EVALUATION OF THE ENANTIOMERS OF 131 5-OME-BPAT

7 MOLECULAR MODELING OF THE DOPAMINE D2 AND SEROTONIN 5-HT1A 153 RECEPTOR BINDING MODES OF THE ENANTIOMERS OF 5-OME-BPAT

SUMMARY 179

SAMENVATTING 183

ABBREVIATIONS 187

AMINO ACID CODES AND STRUCTURES 188

PUBLICATIONS AND PRESENTATIONS 189

CURRICULUM VITAE 190

DANKWOORD 191 1 INTRODUCTION

1.1

Although the disease nowadays called schizophrenia has probably been present since early civilization,182 it was not until the beginning of the nineteenth century that the first detailed descriptions of the illness appeared in literature.152,299 In 1896, Emil Kraepelin named the disorder dementia praecox or early-onset dementia, distinguishing the patients from those with a late-onset dementia and from those suffering from manic-depressive illness.205 Recognizing that the common characteristic between the patients was a ‘thought disorder’ or the ‘splitting of the fabric of thought’, Eugen Bleuler in 1911 renamed the disease schizophrenia [schízein (Gk.) = to split, phrèn (Gk.) = mind].37 Bleuler distinguished between ‘accessory’ and ‘fundamental’ symptoms, nowadays referred to as positive and negative symptoms, respectively. Positive symptoms of schizophrenia include hallucinations (sensory experiences without adequate external stimuli), delusions (belief held despite evidence to the contrary), positive formal thought disorder and bizarre or disorganized behaviour. Negative symptoms include alogia (poverty of speech), affective flattening (reduced emotional responsiveness), anhedonia (inability to feel pleasure), asociality (inability to initiate or maintain social contacts), avolition–apathy (lack of motivation, underachievement at work or school) and attentional impairment.9 Based on this differentiation in symptoms, a concept of two different syndromes in schizophrenia, referred to as Type I and Type II, has been postulated. In the Type I syndrome (or ‘florid state’) positive symptoms are most prominent, whereas in the Type II syndrome (or ‘deficit state’) negative symptoms dominate. Although the two syndromes seem to be relatively

1 Chapter 1 independent, they are not mutually exclusive, since both positive and negative symptoms of schizophrenia can be prominent in a single patient.80 Schizophrenia appears to have a worldwide incidence of about 1 percent. Throughout the world, the prevalence of schizophrenia seems to be fairly homogeneous, although there is evidence of geographical pockets with a relatively high prevalence. Incidence estimates, however, are highly dependent on the diagnostic criteria being used.375 Schizophrenia is highly variable across individuals and across time in the same individual. Whereas some individuals experience one or more episodes and return to normal or near-normal functioning, others have a gradual or intermittent course with increasing disability. Both men and women seem to be equally affected by schizophrenia, although men usually have a younger age of onset.410 Despite its ancient history and the vast amount of research that has been devoted to the disease during the last century, little is understood about the etiology of schizophrenia. A genetic predisposition for schizophrenia has been generally accepted, based on family studies, twin studies and studies with adoptees.197 In addition to this genetic hypothesis, environmental factors such as (prenatal) exposure to viruses,198,249 perinatal complications,223 autoimmunity,200 season of birth,172 and social-cultural status323 have been put forward as possible causes underlying schizophrenia. In view of the complexity of the disease, it seems likely that a combination of both genetic and environmental factors is a prerequisite for developing schizophrenia. For a more extensive review on the history, etiology, pathophysiology and treatment of schizophrenia, the reader is referred to ref. 35.

1.2 NEUROCHEMICAL HYPOTHESES OF SCHIZOPHRENIA

Whereas the true causes underlying schizophrenia are still subject of debate, evidence has been accumulating over the last three decades that the symptoms of the disease may be the result of neurochemical and/or anatomical abnormalities in the central nervous system (CNS). The implication of disturbed neurotransmission in schizophrenia was initiated by the findings of Carlsson and Lindqvist, who reported in 1963 that the antipsychotic drugs and enhanced the accumulation of the main metabolites of dopamine and noradrenaline in the rat brain, presumably by blockade of catecholamine receptors.56 These findings have laid the foundation for the dopamine hypothesis of schizophrenia, which has governed the development of antipsychotic drugs during the last thirty years.

1.2.1 THE DOPAMINE HYPOTHESIS OF SCHIZOPHRENIA

Dopamine (3,4-dihydroxyphenylethylamine, 1) is utilized as a neurotransmitter in specific neuronal pathways within the CNS. The localization of these pathways has been unraveled in the 1960’s. Three major systems can be distinguished in the mammalian CNS (for reviews and references see refs. 34, 72, 121, and 266):

2 Introduction

(1) the nigrostriatal dopamine system (A9), with cell bodies situated predominantly in the substantia nigra pars compacta and nerve terminals in the caudate nucleus/putamen (striatum), the globus pallidus and the subthalamic nucleus; (2) the mesolimbocortical dopamine system (A10), with cell bodies predominantly in the ventral tegmental area and projections to limbic cortical areas, including the piriform cortex, the entorhinal cortex, the perirhinal cortex, the prefrontal cortex and the cingulate cortex (mesocortical projections), as well as to other limbic structures, including the nucleus accumbens, the septum, the hippocampus, the olfactory tubercle and the amygdala (mesolimbic projections); (3) the tuberoinfundibular/tuberohypophyseal dopamine system (A12), with cell bodies lying in the arcuate and periventricular nuclei and nerve terminals in the median eminence and the pituitary, respectively.

HO NH2

HO 1

Chart 1.1 Chemical structure of dopamine (1).

Basically, the dopamine hypothesis of schizophrenia posited that the symptoms of the disease are a manifestation of a hyperdopaminergic state of the CNS, in particular of the mesolimbic dopamine system, which traditionally has been implicated in the control of mood and emotional behaviours like aggression, anxiety and sexuality (for reviews and references, see refs. 54, 135, and 337). Both preclinical and clinical studies have provided consistent evidence for overactivity in central dopaminergic neurotransmission in the pathophysiology of schizophrenia. First, patients suffering from Parkinson’s disease who are treated with L-DOPA, a precursor in the biosynthesis of dopamine, sometimes experience psychotic episodes as side-effects.181 Furthermore, drugs that stimulate dopaminergic neurotransmission (e.g. d- and L-DOPA) can exacerbate psychotic symptoms in schizophrenic patients.10 Chronic administration of high doses of such drugs to healthy individuals can induce a syndrome mimicking paranoid schizophrenia,10,11,348 which can be reversed by antipsychotic drugs.347 Second, a highly significant positive correlation between the affinity of antipsychotic drugs for brain dopamine D2 receptors and their clinical has been demonstrated.78,342 Third, post-mortem analyses on the brains of schizophrenics have revealed significantly elevated numbers of dopamine D2 receptors, which cannot be totally accounted for by 79,239,339,376 the history of the patients. In vivo measurements of dopamine D2 receptor densities in humans by positron emission tomography (PET) gave conflicting results in this regard, but these might be caused by differences in the radioligands and patients groups that were used.225,333,334 Despite these consistencies in evidence pointing towards a hyperdopaminergic state of the CNS in schizophrenia, there is now general agreement that the dopamine hypothesis, as posited in its original form, is too simple for explaining the complete symptomatology of the disease. Several observations support its shortcomings. First, substantial evidence for elevated levels of dopamine in the schizophrenic brain is still lacking.58,137 In fact, many patients – particularly those in which negative

3 Chapter 1 symptoms dominate – exhibit normal or subnormal levels of the dopamine metabolite homovanillic acid (HVA) in their cerebrospinal fluid (CSF).154,303,385 In addition, low doses of the indirect 62,134 52,287 dopamine agonists d-amphetamine and L-DOPA have been reported to improve negative symptoms in a subgroup of patients. Taken together, these findings suggest that at least in a subgroup of patients, the CNS shows an overall hypo- rather than a hyperdopaminergic activity.395,409 Several investigators conceived the possibility of the coexistence of areas with a hyper- and a hypodopaminergic activity in the brains of schizophrenics.85,137 Hypofrontality, a dysfunction of the prefrontal cortex, has been proposed as the cause of the hypodopaminergic state.404 In fact, animals and humans with frontal lobe damage show behaviour with striking similarities to negative symptoms of schizophrenia.70 These findings have led to a reconceptualization of the dopamine hypothesis of schizophrenia, which explains the existence of both positive and negative symptoms: as a consequence of hypofrontality a hypodopaminergic state of the prefrontal cortex arises, causing the more persistent negative symptoms of schizophrenia.58,85,137,385 This hypodopaminergic state of the prefrontal cortex can under certain circumstances (e.g. stress)162 lead to a dysinhibition of subcortical dopaminergic systems, in particular the mesolimbic dopamine system, and trigger an abnormally high release of dopamine in these brain areas.82,137 This latter phenomenon should in turn account for the occurrence of positive symptoms in schizophrenia. A second shortcoming of the dopamine hypothesis is its failure to explain the delayed onset of action of antipsychotic drugs. Thus, while antipsychotic drugs produce complete occupancy of dopamine receptors within minutes after administration,335 it usually takes days or weeks before their clinical effects become apparent.297 These observations, however, may be explained in terms of initial compensatory mechanisms (e.g. receptor upregulation) in the dopaminergic neurotransmission developing after blockade of dopamine receptors, which upon long-term drug treatment slowly adapt towards a new homeostasis situation. Finally, a significant population of schizophrenic patients persistently shows moderate to severe positive symptoms despite trials with several antipsychotic drugs of different classes. This subgroup of patients, referred to as ‘treatment-resistant’ or ‘treatment-refractory’, has been estimated to comprise 15 to 30% of all schizophrenics, depending on the diagnostic criteria.258 Taken together, these shortcomings of the dopamine hypothesis have lead to the believe that, in addition to dopamine, other neurotransmitters may be involved in the pathophysiology of schizophrenia.55 In this respect, serotonin in particular has regained much attention during the last few years.

1.2.2 THE SEROTONIN HYPOTHESIS OF SCHIZOPHRENIA

Serotonin (5-hydroxytryptamine, 5-HT, 2), was recognized to function as a neurotransmitter in the CNS in the early 1950’s.382 Similar to dopamine, neurotransmission is restricted to specific neuronal pathways in the brain (for reviews and references, see refs. 18, 177, and 377). The cell bodies of serotonergic neurons, designated as the clusters B1–B9, are located in the pons and upper brain stem of the CNS and can be divided into a superior and an inferior group. The superior group (B5–B9) consists of four main nuclei: the median raphe nucleus (MRN, B5 and B8), the dorsal raphe nucleus (DRN, B6 and B7), the caudal linear nucleus (CLN, B8), and the nucleus prosupralemniscus (NP, B9). The inferior group (B1–B4) consists of five main nuclei: the nucleus

4 Introduction

NH2

HO

N H

2

Chart 1.2 Chemical structure of serotonin (2). raphe obscurus (NRO, B2), the nucleus raphe pallidus (NRPa, B1 and B4), the nucleus raphe magnus (NRM, B3), the lateral paragigantocellular nucleus (LPGN), and the intermediate reticular nuclei (IRN, B1 and B3). The cells in the inferior group project to the ventral horn and central gray area of the spinal cord and therefore are probably irrelevant in the pathophysiology of schizophrenia. Cells in the superior group innervate the forebrain, their ascending afferents predominantly running via the medial forebrain bundle (MFB). Serotonergic fibers have been detected in virtually every area of the forebrain, but certain areas are innervated with relatively high densities. These include the cortex, the hippocampus, the suprachiasmatic nuclei of the hypothalamus, the substantia nigra zona compacta, the medial mammilary nucleus, the lateral septum, the periventricular nucleus of the thalamus, the ventrolateral geniculate, and the medial nucleus of the amygdala. In comparison with the dopaminergic system, the serotonergic projections are more diffuse: most cells in the superior group appear to innervate overlapping terminal fields. For example, the DRN and the CLN project to structures of the basal ganglia including the corpus striatum and the substantia nigra, while the MRN, but also the DRN project to limbic structures, such as the hippocampus and septum. Four major lines of evidence exist which implicate serotonin in the pathophysiology of schizophrenia (for reviews see refs. 36, 47, and 288). First, the structural similarities between serotonin and lysergic acid diethylamide (d-LSD), a drug known to induce effects resembling certain aspects of schizophrenia, raised the idea in the early 1950’s that d-LSD might exert these effects by blocking serotonin receptors.122 A deficiency of serotonin in the brains of schizophrenic patients should thus account for the observed symptoms.407 Following this ‘serotonin deficiency hypothesis of schizophrenia’, other researchers have put forward the ‘transmethylation hypothesis of schizophrenia’, proposing that certain forms of schizophrenia may be caused by the formation of hallucinogenic N,N-dimethyltryptamines from naturally occurring biogenic amines.127,193 However, no increases in the concentrations of such compounds in urine,57,127 whole blood230 and plasma,17 or CSF76 of schizophrenics, as compared to normal controls, could be detected. Furthermore, hallucinogenic and d-LSD predominantly induce visual hallucinations, symptoms which are actually quite rare in schizophrenia, but fail to induce other prominent features of the disease, such as formal thought disorders.116 These shortcomings, together with the increasing amount of evidence for a prominent role of dopamine in the pathophysiology of schizophrenia, which has been accumulated during the same period, have led to a gradual disbelief in these hypotheses. More recent pharmacological developments, however, have revived the interest in the possible role of serotonin in schizophrenia (see Section 1.6.4). Second, post-mortem studies on the brains of schizophrenic patients have revealed some consistencies related to disturbances in serotonergic neurotransmission, although a number of studies

5 Chapter 1 with conflicting data have been reported. Measured post-mortem parameters include brain levels of , serotonin and 5-hydroxyindoleacetic acid (5-HIAA), the major metabolite of serotonin, and serotonin receptor densities. Inconsistencies in these parameters arise from differences in patient parameters such as age, cause of death, medication history, and brain region under investigation in the different studies. Thus, several researchers have reported increased levels of 5-HT and/or 5- HIAA in various brain areas, including the basal ganglia,81,112,203,376 while others found decreased405 183 3 or unaffected levels. Initial measurements of serotonin 5-HT2 receptor densities, employing [ H]- LSD as the radioligand, gave conflicting results as well: some researchers found decreased numbers of [3H]-LSD binding sites in the frontal cortex of schizophrenics,31 while others reported unchanged311 or even increased cortical [3H]-LSD binding.398 The lack of receptor selectivity of LSD should probably in part account for these controversial observations, since studies performed with more specific radioligands gave more consistent results. Thus, more recently a group of Japanese researchers, employing the selective serotonin 5-HT2 receptor antagonists as a radioligand, found decreased densities of serotonin 5-HT2 receptors in the prefrontal cortices of schizophrenic patients.150,263 Other scientists, also using [3H]-ketanserin216 or [3H]-,13 were able to reproduce these results. In contrast to cortical areas, increased serotonin 5-HT2 receptor densities in the hippocampus, the nucleus accumbens and the ventral putamen have been observed.184 376 The increase in serotonin 5-HT2 receptor density in the putamen was confirmed by Toru et al.

Three post-mortem studies on the density of serotonin 5-HT1A receptors in schizophrenics have been published. Joyce reported increased numbers of serotonin 5-HT1A receptors in the prefrontal cortex and the hippocampus of schizophrenic patients.184 Hashimoto and coworkers also found increased prefrontal cortical serotonin 5-HT1A receptor binding, but no differences in other brain areas, 150,151 including the hippocampus. Increased serotonin 5-HT1A receptor densities in the prefrontal cortex of schizophrenics were confirmed by Sumiyoshi et al.364 Interestingly, there seems to be general agreement in these reports that the observed changes in receptor densities of both serotonin

5-HT2 and 5-HT1A receptors are unrelated to the medication histories of the investigated patients. A third line of evidence comes from measurements of cerebrospinal fluid (CSF) levels of 5-HIAA, and measurement of peripheral markers of serotonin function, including blood platelet serotonin and whole blood serotonin. CSF 5-HIAA levels can be taken as a crude indicator of central 5-HT metabolism. Initial data from a number of CSF 5-HIAA measurements were again inconsistent, although no increases were reported.36,288 Three independent studies later showed that cortical atrophy and ventricular enlargements, malformations of the brain which are frequently observed in schizophrenics with predominantly negative symptoms, were significantly correlated with a decrease in CSF 5-HIAA levels.232,284,305 In addition, there seems to be evidence for a relationship between low CSF 5-HIAA levels and suicidal behaviour in a subgroup of schizophrenic patients.20,73,279,386 Blood platelet serotonin content has been put forward as a measure of serotonin turnover in the CNS. The human blood platelets are neuroectodermal derivatives and therefore have many biochemical and morphological characteristics in common with CNS serotonergic synaptosomes.357 A number of studies on platelet and whole blood serotonin levels in schizophrenics has been reported and there seems to be general agreement on elevated levels in chronic schizophrenics.36,288 The clinical significance of these findings, however, is unclear.

6 Introduction

Finally, attempts have been made to challenge the serotonergic neurotransmission in schizophrenic patients with selective serotonergic agents, in order to evoke alterations in the symptoms and hence shed more light on the role of serotonin in the disease. Thus, treatment studies with high doses of L- tryptophan and 5-hydroxytryptophan, two precursors in the biosynthesis of serotonin, but also tryptophan hydroxylase inhibitors such as para-chlorophenylalanine, non-selective serotonin receptor agonists like meta-chlorophenylpiperazine, and serotonin uptake inhibitors like have been undertaken, but the results were in general disappointing.36,47,288 In summary, the inconsistencies in the studies described above indicate that no solid conclusions can be drawn about the implications of serotonin in schizophrenia. These inconsistencies might be a reflection of the heterogeneity of the disease. Nevertheless, a few consistent observations, particularly the decreased and increased densities in prefrontal cortical serotonin 5-HT2 and 5-HT1A receptors, respectively, and the elevations in blood and platelet serotonin levels at least suggest that serotonergic mechanisms are involved in the pathophysiology of schizophrenia.

1.2.3 INVOLVEMENT OF OTHER NEUROTRANSMITTERS IN SCHIZOPHRENIA

Since the implication of disturbed neurotransmission in schizophrenia by Carlsson and Lindqvist,56 virtually every neurotransmitter of importance in the CNS has been suggested to play a role in the pathophysiology of the disease.226 Thus, a noradrenaline hypothesis,384 a glutamate hypothesis,174 an acetylcholine hypothesis,371 and a g-aminobutyric acid (GABA) hypothesis356 of schizophrenia have been put forward to account for the symptoms observed in the disease. In addition, several neuropeptides, including neurotensin,194 cholecystokinine,344 and endogenous opioids,326 but also hormones, such as oestrogens313 and melatonin322 have been implicated in the pathophysiology of schizophrenia. In view of the complexity of the disease, it cannot be excluded that, in addition to dopamine and serotonin, other neurotransmitters also play a role in the pathophysiology of the disease. A discussion on the possible role of these compounds, however, is beyond the scope of this thesis. For reviews and references, the reader is referred to the references quoted.

1.3 DOPAMINE RECEPTORS

1.3.1 GENERAL STRUCTURAL FEATURES OF G-PROTEIN-COUPLED RECEPTORS

Upon release from dopaminergic neurons, dopamine exerts its action by interacting with specific dopamine receptors. At present, five subtypes of dopamine receptors have been identified and anatomically, and to a certain extent biochemically and pharmacologically characterized. All currently identified subtypes belong to the superfamily of G-protein-coupled receptors (GPCRs). Members of this family, to which among others the opsins, olfactory receptors and many neurotransmitter receptors belong, comprise large glycoproteins embedded in the cell membranes of the cells targeted by a specific endogenous ligand. GPCRs are, by definition, coupled to guanine- nucleotide-binding regulatory proteins (G-proteins), which link the receptors to intracellular effector mechanisms. The exact molecular structures of GPCRs are unknown, since attempts to crystallize

7 Chapter 1 these proteins have failed thus far. Nevertheless, biophysical, biochemical and molecular biological studies on various GPCRs suggest that these receptors have many structural features in common. Thus, hydropathy analyses of the primary amino acid sequences and sequences alignments of the currently identified GPCRs revealed the presence of seven relatively hydrophobic regions of about 25 amino acids in length, which are interconnected by six relatively hydrophilic regions of variable lengths. The seven hydrophobic regions are believed to form seven transmembranal (TM) a-helices, orientated in a more or less parallel manner. All GPCRs cloned thus far have been shown to possess a substantial degree of homology in their amino acid sequences, especially in the TM regions. The amino-terminal region of the protein, which contains one or more glycosylation sites, is located extracellularly, whereas the carboxyl-terminal region protrudes into the cytosol. The interconnecting loops are alternatively located intra- and extracellularly. The binding site for the endogenous ligand (the ‘active site’) is believed to be situated within the core formed by the seven TM domains, while the third cytoplasmatic loop is thought to be involved in the coupling of the receptor to the G- protein. Binding of the endogenous ligand to the active site presumably induces conformational changes in the receptor molecule, which trigger via the G-protein an intracellular response, e.g. the production of a second messenger molecule. In this way, the ‘information’ carried by the ligand is transduced over the plasma membrane into the cell (for reviews and references on GPCRs, see refs. 96, 97, and 363).

1.3.2 DOPAMINE RECEPTOR CLASSIFICATION

The application of new molecular biology techniques in the field of receptor research has accounted for a revolution during the last decade. Until ten years ago, only two subtypes of dopamine receptors were discriminated, based on biochemical and pharmacological observations: dopamine D1 receptors, mediating the stimulation of intracellular cyclic adenosine monophosphate

(cAMP) production by activating the enzyme adenylate cyclase, and dopamine D2 receptors, which mediate the inhibition of this second messenger system.196,360 The identification of the DNA sequence 95 encoding the hamster b2- receptor in 1986 proved to be a milestone in receptor research, since it opened the possibility to locate the coding sequences of a number of other GPCRs, including those of the dopamine receptors. Thus, screening of genomic libraries has resulted in the identification, cloning and expression of at least five different subtypes of human dopamine receptors, termed D1, D2, D3, D4, and D5. Based on similarities and differences in gene organization, molecular structure, pharmacology and biochemistry these subtypes have been classified into two subfamilies: dopamine ‘D1-like’ receptors, comprising the dopamine D1 and D5 receptor subtypes, and dopamine ‘D2-like’ receptors, comprising the dopamine D2, D3, and D4 receptor subtypes, respectively. The properties of the two subfamilies closely resemble those of the dopamine D1 and D2 receptor subtypes as originally defined in the late 1970’s. The most important characteristics of the cloned human dopamine receptor subtypes are summarized in Table 1.1.

8 Introduction

In addition to elucidating the molecular features of GPCRs, molecular cloning techniques have also allowed for the expression of GPCRs in cells that normally do not express such receptors. Thus,

TABLE 1.1 Summary of the characteristics of cloned human dopamine receptor subtypes.

D1-like D2-like

D1 D5 D2 D3 D4 Gene Reference 89, 366, 413 365 84, 361 351 388 Chromosome localization 5q35.1 4p16.3 11q22–23 3q13.3 11p15.5 Introns no no yes yes yes

Expression – – D2A/D2B – D4.2–D4.10

Protein Amino acids 446 477 443/414 400 387 3rd Cytoplasmatic loop short short long long long C-terminus long long short short short a Sequence homology D1 100 82 47 45 42

D5 100 44 40 45

D2 100 77 51

D3 100 40

D4 100

Localizationb CPut, NAc, Hipp, Thal, CPut, NAc, NAc, ICj, Sept, FC, OT, ICj, OT Hyp ICj, OT, Pit, Thal, Hyp, Cer Amyg, Mes, SN, VTA MO

Pharmacology Dopamine affinity (nM)c 2000 250 2000 30 450 Specific agonistd SKF 38393 (3) SKF 38393 N-0923 (5) PD 128907 (7) PD 168077 (9) Specific antagonistd SCH 23390 (4) SCH 23390 (6) S 14297 (8) L-745,870 (10)

Biochemistrye G-protein-coupled yes yes yes ? yes cAMP • • ¯ –/¯ –/¯

IP3 • ? • – – Ca2+ • ? ¯ ¯ ¯ Arachidonic acid ? ? • –/¯ • Dopamine release ? ? ¯ ¯ – Mitogenesis ? ? • –/• ? Acidification ? ? • • • aSequence homology in TM domains, expressed as percentages. bBased on rat brain mRNA distribution data. Abbreviations: CPut, caudate putamen; NAc, nucleus accumbens; ICj, islands of Calleja; OT, olfactory tubercle; Hipp, hippocampus; Thal, thalamus; Pit, pituitary; SN, substantia nigra; VTA, ventral tegmental area; Sept, septum; Cer, cerebellum; FC, frontal cortex; Amyg, amygdala; Mes, mesencephalon; MO, medulla oblongata (taken from ref. 176). cValues in the presence of a guanyl nucleotide (taken from ref. 354). dFor chemical structures, see Chart 1.3. e Abbreviations: cAMP, cyclic adenosine monophosphate; IP3, inositol triphosphate; •, increase; ¯, decrease; –, no effect; ?, unknown (taken from ref. 355).

9 Chapter 1 mammalian cell lines can be transiently or permanently transfected with cDNAs encoding the different dopamine receptor subtypes. Because these cells usually express a single receptor subtype in high density, they are very suitable for determining receptor binding affinities of drug candidates. Since most newly synthesized target compounds presented in the subsequent chapters of this thesis have been evaluated for their ability to bind to cloned human dopamine D2 and D3 receptors, the characteristics of these two receptor subtypes will be described in more detail in the next sections. For reviews and references on the other dopamine receptor subtypes, see refs. 176, 180, 362, and 387.

1.3.3 DOPAMINE D2 RECEPTORS

In 1988, Bunzow and coworkers identified and cloned the gene encoding the rat dopamine D2 receptor, by applying a cloning strategy which was based on the presumed structural homology 53 between different GPCRs. Thus, initially using the DNA sequence encoding the hamster b2- as a hybridization probe, in order to screen a rat genomic library for the coding sequences of other GPCRs, they ultimately were able to isolate the cDNA encoding a protein of 415 amino acids with the characteristics of a GPCR: the protein contained seven hydrophobic domains, a number of amino acids which are highly conserved among a large number of GPCRs, potential asparagine-linked glycosylation and phosphorylation sites in the N-terminal region, and a significant sequence homology with other GPCRs. Analysis of its mRNA distribution in the rat brain revealed high abundancies in brain areas classically associated with dopaminergic neurotransmission, including the striatum, nucleus accumbens, olfactory tubercle, pituitary, substantia nigra and ventral tegmental area. Expression of the receptor in different mammalian cell lines allowed for the establishment of the ligand specificity and the functional coupling of the receptor to effector systems. The receptor was shown to bind classical dopamine D2 like spiperone, (+)-, haloperidol and (–)- with high affinity and selectivity, and appeared to mediate the inhibition of adenylate cyclase activity, as well as prolactin secretion. Taken together, these findings strongly suggest that the cloned protein indeed corresponds to the classical dopamine D2 (for review and references see ref. 138).

A human homologue of the rat dopamine D2 receptor gene was cloned by the same research group in 1989 from human pituitary tissue.139 The protein encoded by the gene was 443 amino acids in length and showed an overall sequence similarity with the rat dopamine D2 receptor of approximately 96%. The difference in length between the two species isoforms was shown to be the result of alternative splicing of pro-mRNA during the process of gene expression. Thus, several research groups have reported the existence of two splice variants of the dopamine D2 receptor in rats66,84,129,264,286,307 and humans.84,139,343,361 In both species, the two isoforms differ by a stretch of 29 amino acid in the putative third cytoplasmatic loop. Both human isoforms are one amino acid shorter than their rat homologues. The long isoforms have been termed D2A [alternative nomenclature: D2L,

D444 (rat), or D443 (human)], while the short isoforms have been termed D2B [alternative nomenclature: D2S, D415 (rat), or D414 (human)].

The biological significance of the existence of two isoforms of the dopamine D2 receptor is unclear. All brain regions expressing dopamine D2 receptors that have been investigated thus far

10 Introduction

H H HO HO N NH N CH 3 S HO Cl OH 3 4 5

OH O O H Cl HO N N N O H N OCH3 O Cl 6 7 8

O H C Cl 3 N N CN CH H 3 N N O N 9 10

CHART 1.3 Chemical structures of SKF 38393 (3), SCH 23390 (4), N-0923 (5), raclopride (6), PD 128907 (7), S 14297 (8), PD 168077 (9), and L-745,870 (10). seem to express both splice variants, although their relative abundance in different brain regions is variable. In general, the short isoform is the least abundant of the two. Receptor binding studies have shown that the two isoforms can not be pharmacologically distinguished, although some compounds 61,241 seem to have some preference for the dopamine D2B receptor. Since the two isoforms differ only in the length of the putative third cytoplasmatic loop, differences in coupling to second messenger systems may be expected (for review and references, see ref. 106)

Virtually all compounds previously designated to be selective for the dopamine D2 receptor also bind with high affinity to the dopamine D3 receptor (see below). Therefore, in order to be able to further investigate the functional role of the dopamine D2 receptor, there is currently a strong need for selective dopamine D2 receptor agonists and antagonists.

Expression of cloned dopamine D2 receptors in various cell lines has revealed that they utilize different signal transduction systems, presumably via coupling to different G-proteins (for reviews see refs. 176 and 362). In all cellular environments inhibition of adenylate cyclase has been detected,3,27,153,192,231,240,275,383 but cell-specific signaling pathways may be present as well. Thus, dependent on the type of cells which express the receptors, stimulation of dopamine D2 receptors, in addition to inhibition of intracellular cAMP production, may result in: (1) enhancement of phosphoinositide (PI) hydrolysis by activation of the enzyme phospholipase C;231,383 (2) an increase153,231,383 or decrease383 in the intracellular Ca2+ concentration; (3) opening of K+ channels,383 and (4) extracellular release of arachidonic acid.192,300

11 Chapter 1

1.3.4 DOPAMINE D3 RECEPTORS

In 1990, the gene encoding a novel dopamine receptor, termed D3, was cloned by French 128,351 researchers. The human dopamine D3 receptor comprises a glycoprotein of 400 amino acid residues and shows 50% sequence homology with the human dopamine D2 receptor, or 77% when only the presumed TM domains are considered. In addition, the two subtypes have more structural features in common, such as a long third intracellular loop, a short carboxylic acid terminal segment and several glycosylation sites (Table 1.1). Furthermore, in analogy with the dopamine D2 receptor gene organization, the coding sequence of the dopamine D3 receptor contains introns, which may give rise to the expression of splice variants encoded by the same gene. Various different splice variants indeed have been identified in mouse, rat, and human brain, but in most cases the structures of the encoded proteins made it unlikely that they comprise fully functional receptors. The identification of 7-hydroxy-2-(N,N-di-n-propylamino)tetralin (7-OH-DPAT) as a selective dopamine D3 receptor agonist allowed for the autoradiographic localization of the dopamine D3 219 receptor in the brain. The overall abundance of the dopamine D3 receptor turned out to be about two orders of magnitude lower, and more importantly, the regional distribution to be much more 43 351 restricted than that of the dopamine D2 receptor. Northern blot and in situ hybridization analyses confirmed high density expression of dopamine D3 receptors predominantly in the olfactory tubercle/islands of Calleja complex, the anterior and shell parts of the nucleus accumbens, the bed nucleus of the stria terminalis, the mammilary nuclei of the hypothalamus, and the geniculate nuclei of the thalamus. The expression of the dopamine D3 receptor being restricted to these phylogenetically old brain areas, referred to as the limbic system, suggests an important role of the dopamine D3 receptor in the control of cognitive, emotional and reward processes, and hence as a major target for antipsychotic drug action. High densities of dopamine D3 receptors were also identified in the cerebellum, but the physiological function of this cerebellar expression is unclear.

The identification of low levels of dopamine D3 receptor expression in the substantia nigra pars compacta and the ventral tegmental area suggest that this receptor subtype may function as an autoreceptor.

The pharmacological profile of the dopamine D3 receptor is similar, but not identical to that of the dopamine D2 receptor, and supports a possible role as an autoreceptor. Thus, all dopamine D2 receptor agonists and antagonists bind with good affinities to dopamine D3 receptors as well, but some compounds, previously designated as putative dopamine D2 autoreceptor agonists (e.g. 7-OH-

DPAT) or antagonists [e.g. (+)-AJ 76 and (+)-UH 232], show preference for the dopamine D3 receptor.349,351 Remarkably, in comparison with the other dopamine receptor subtypes, the dopamine

D3 receptor displays an exceptional high affinity for dopamine itself (Table 1.1), the significance of 349 91 which is unclear. In addition, various selective dopamine D3 receptor agonists (e.g. PD 128907) and antagonists (see Section 1.6.1) have become available during the last few years, allowing to further study the functional role of the dopamine D3 receptor.

Whereas the signal transduction pathways of the dopamine D2 receptor has been unraveled to a large extent, the biochemistry of the dopamine D3 receptor is much less clear. Initial studies failed to demonstrate any coupling to G-proteins. Binding of agonists to dopamine D3 receptors, expressed in various cell types, was not or only weakly affected by guanide nucleotides, and no second messenger

12 Introduction generation was observed.42,119,240,332,349,351,354 Later studies, however, revealed functional coupling of dopamine D3 receptors to different transduction mechanisms in various cell lines. Thus, dopamine D3 receptor-mediated inhibition of cAMP production,67 aggregation of melanophore pigment,304 acidification of the extracellular environment,315 and inhibition of Ca2+ currents331 have been reported. Nevertheless, the magnitudes of the effects seem to be consistently lower than the corresponding dopamine D2 receptor-mediated effects. The relationships between these in vitro observations and the functional role of the dopamine D3 receptor in vivo remain to be established

(for reviews on the dopamine D3 receptor, see refs. 330, 353, and 354).

1.4 SEROTONIN RECEPTORS

1.4.1 SEROTONIN RECEPTOR CLASSIFICATION

In analogy with dopamine and other neurotransmitter substances, serotonin interacts with its own specific receptors after its release from serotonergic neurons. Gaddum and Picarelli in 1957 were the first to distinguish between two types of serotonin receptors, which they termed D and M.123 Receptors of the D-type mediated the serotonin-induced contraction of smooth muscle, which could be blocked by dibenzyline. M-type receptors mediated the serotonin-induced release of acetylcholine from postganglionic nerve terminals, which could be blocked by morphine. A new classification, based on radioligand binding studies, was proposed by Peroutka and Snyder in the late 1970’s: 3 3 receptors labeled by [ H]-5-HT were classified as 5-HT1, while those labeled by [ H]-spiperone were 296 termed 5-HT2. It was soon realized that both classification schemes were incomplete, but also complemented each other. Therefore, in 1986 a group of scientists decided to combine the two classification schemes, taking into account rank order and affinities for agonists and antagonists, and to some extent the second messenger systems involved. Thus, the different serotonin receptor subtypes were grouped in three main classes, designated ‘5-HT1-like’ (corresponding to some D receptors and 5-HT1 binding sites), 5-HT2 (corresponding to most D receptors and 5-HT2 binding 46 sites), and 5-HT3 (equivalent to M receptors). This classification scheme has formed the basis for the classification scheme of serotonin receptors currently in use, and which is based on a combination of operational, transductional and structural considerations (for reviews, see refs. 165, 166, and 325). Application of modern molecular biology techniques have revealed the existence of at least seven classes of serotonin receptors (5-HT1–7) to date, and some of these consist of several subtypes, adding up to a total of fourteen serotonin receptor subtypes. Some of these have been identified as gene products only, and therefore are denoted in small characters. The serotonin 5-HT1 class comprises the subtypes 5-HT1A, 5-HT1B, 5-HT1D, 5-ht1E, and 5-ht1F. The non-rodent form of the serotonin 5-HT1B receptor has previously been referred to as 5-HT1Db, while the serotonin 5-HT1D receptor has been formerly termed 5-HT1Da. The serotonin 5-HT2 class consists of subtypes 5-HT2A,

5-HT2B (previously named 5-HT2F), and 5-HT2C. The serotonin 5-HT2C receptor has been formerly referred to as the serotonin 5-HT1C receptor, but based on clear operational and transductional similarities with the 5-HT2A receptor, it was later assigned to the 5-HT2 class. The serotonin 5-ht5 class comprises the subtypes 5-ht5A and 5-ht5B. The serotonin 5-HT3 receptor is a ligand-gated ion

13 Chapter 1 channel, all other serotonin receptor subtypes belong to the superfamily of G-protein-coupled receptors. The most important features of the serotonin receptors have been summarized in Table 1.2. Since all of the compounds disclosed in this thesis have been evaluated for their affinities at the serotonin 5-HT1A receptor, the characteristics of this subtype will be addressed in more detail.

TABLE 1.2 Summary of the characteristics of the currently identified serotonin receptor subtypes. Receptor Subtype Locationa Specific agonistb Specific antagonistb Responsec References

5-HT1 5-HT1A Hipp, RNu 8-OH-DPAT (11) WAY 100,635 (12) cAMP¯ 4, 110, 201

5-HT1B SN, GP CP 93129 (13) SB 224289 (14) cAMP¯ 2, 244, 391

5-HT1D Hipp, OT L-694,247 (15) GR 127935 (16) cAMP¯ 19, 145, 146, 396 d 5-ht1E CPut – – cAMP¯ 142, 220, 248, 412

5-ht1F Hipp, FC LY 334370 (17) – cAMP¯ 1, 6, 233

5-HT2 5-HT2A FC DOI (18) ketanserin (19) IP3/DAG• 65, 185, 306, 321

5-HT2B not in CNS BW 723C86 (20) SB 204741 (21) IP3/DAG• 117, 118, 211, 212

5-HT2C SN Ro 60-0175 (22) SB 242084 (23) IP3/DAG• 186, 237, 238, 321

+ + 5-HT3 – m-CPBG (24) MDL 72222 (25) Na i•, K i¯ 161, 243, 251

5-HT4 – Hipp (26) GR 113808 (27) cAMP• 124

5-ht5 5-ht5A Hipp, Hyp – – cAMP¯ 105, 302, 309

5-ht5B Hipp – – – 105, 247

5-ht6 – Str, OT 5-CT (28) – cAMP• 202, 265, 319

5-HT7 – Hyp, Thal 5-CT SB 258719 (29) cAMP• 22, 301, 320, 381 aAbbreviations: Hipp, hippocampus; RNu, raphe nuclei; SN, substantia nigra; GP, globus pallidus; OT, olfactory tubercle; Hyp, hypothalamus; Thal, thalamus. bFor chemical structures, see Chart 1.4. cAbbreviations: cAMP, cyclic adenosine monophosphate; IP3, inositol triphosphate; DAG, diacyl glycerol; ¯, decrease; •, increase; i, intracellular. dNo selective ligand available.

14 Introduction

H N N OH OCH3 H N N O N N N N O H 11 12 13

O O H C 3 CH S N 3 H C 3 N O H NH N O 2 CH3 N N N N O O N H 14 15 H3C N O CH N 3 CH3 F N O CH O N 3 HN N HN

OCH N 3 H 16 17

O

OCH3 O NH N 2 N F CH I 3 N O H OCH3 18 19

S NH H 2 N O H C CH 3 O 3 N S HN

N N H CH3 20 21

CHART 1.4 Chemical structures of 8-OH-DPAT (11), WAY 100,635 (12), CP 93129 (13), SB 224289 (14), L- 694,247 (15), GR 127935 (16), LY 334370 (17), DOI (18), ketanserin (19), BW 723C86 (20), and SB 204741 (21).

15 Chapter 1

CH 3 CH Cl 3 NH O Cl N 2 O N H C 3 N N N F H 22 23

O H H CH3 Cl N N NH Cl N 2 O NH NH

Cl 24 25

F

O O O O H N N Cl CH N S 3 H OCH O O 3 N H2N OCH3 CH3 26 27

NH2

O O O CH3

H3C S H2N N N CH N 3 CH H 3 28 29

CHART 1.4 (continued) Chemical structures of Ro 60-0175 (22), SB 242084 (23), m-CPBG (24), MDL 72222 (25), cisapride (26), GR 113808 (27), 5-CT (28), and SB 258719 (29).

1.4.2 SEROTONIN 5-HT1A RECEPTORS

The heterogeneousity of the 5-HT1 class of serotonin receptors, as defined by Bradley and co- workers, was demonstrated by Pedigo et al., who showed that a subclass of the serotonin 5-HT1 receptors had relatively high affinity for spiperone. These sites were termed 5-HT1A, while the sites 294 with lower affinity for spiperone were named 5-HT1B. The simultaneous identification of 8- hydroxy-2-(N,N-di-n-propylamino)tetralin (8-OH-DPAT, 3) as a highly potent and selective 15,159 serotonin 5-HT1A receptor agonist turned out to be a major breakthrough in the characterization 3 242,291,390 of the serotonin 5-HT1A receptor. Autoradiography studies with [ H]-8-OH-DPAT in the rat 167,292 and human brain revealed high densities of serotonin 5-HT1A receptors in the dentate gyrus of the hippocampus (CA1 region), the lateral septum, the entorhinal and frontal cortex, and the amygdala, brain areas which are associated with mood control. Stimulation of these postsynaptic

16 Introduction

receptors in rats by selective serotonin 5-HT1A receptor agonists induces several aspects of a behavioural pattern called the ‘5-HT syndrome’, characterized by hyperlocomotion, lower lip retraction, flat body posture, Straub tail, hindlimb abduction, reciprocal forepaw treading or ‘piano playing’, wet dog shakes, and head weaving.32,379,380 In addition to these postsynaptic receptors, serotonin 5-HT1A receptors have also been found on the cell bodies of the serotonergic neurons in the median and dorsal raphe nuclei. Stimulation of these somatodendritic autoreceptors inhibits neuronal cell firing and release of serotonin onto the postsynaptic receptors.98

Serotonin 5-HT1A receptors are generally considered to be important targets for the treatment of mood disorders like anxiety and depression. An overactivity of the serotonergic system is thought to be responsible for developing anxiety. Therefore, stimulation of somatodendritic serotonin 5-HT1A autoreceptors may prove to be a suitable therapy against this disease.148 Depressions on the other hand seem to be related to a reduced serotonergic activity,38 and may be treated by stimulating 276 postsynaptic serotonin 5-HT1A receptors with selective agonists. In 1987 Kobilka and co-workers cloned a gene encoding a protein belonging to the family of GPCRs.201 The identity of the receptor encoded by the gene was resolved a year later and shown to 110 correspond to the human 5-HT1A receptor. The protein consisted of 421 amino acids and seemed to have many structural features in common with other GPCRs. In 1993, Chanda et al. reported that the Kobilka sequence lacked one amino acid.64 Two different amino acid sequences of the rat serotonin 5-HT1A receptor have also been reported. Both are constituted of 422 amino acids, but the 4,120 sequences differ at one position. Thus, the human and rat serotonin 5-HT1A receptor are equal in length and have an overall sequence identity of 89%, or 99% when only the putative TMs are considered.

Similar to the dopamine D2 receptor, the serotonin 5-HT1A receptor contains several asparagine- linked glycosylation sites in the extracellular N-terminus region, as well as potential sites for phosphorylation and palmitoylation have been identified in the intracellular loops. There is general agreement that stimulation of serotonin 5-HT1A receptors is predominantly associated with a decrease in intracellular cAMP production both in vitro and in vivo, mediated through an inhibitory effect on adenylate cyclase.87,88,111,149

Due to the early recognition of 8-OH-DPAT as a selective serotonin 5-HT1A receptor agonist, this serotonin receptor subtype is the best studied to date. Since its discovery, a number of potent and selective agonists (e.g. ) and antagonists (e.g. WAY 100,635), belonging to various chemical classes, have become available.131,144,273,308 However, the recent notification that 8-OH-

DPAT also has considerable affinity for the serotonin 5-HT7 receptor suggests that some of its activities, which have previously been attributed to its effects on serotonin 5-HT1A receptors, may be 102 mediated via stimulation of serotonin 5-HT7 receptors. Since it is quite well possible that new serotonin receptor subtypes will be identified in the future,295 it is therefore essential that the development of new selective serotonin 5-HT receptor ligands is continued.

17 Chapter 1

1.5 CLASSICAL VERSUS ATYPICAL ANTIPSYCHOTIC AGENTS: BENEFICIAL VERSUS SIDE-EFFECTS

In 1950, Charpentier at Rhône-Poulenc synthesized the derivative encoded 4560RP, later to be known as chlorpromazine. Searching for adjuvants in surgical anesthesia, the surgeon Laborit tested chlorpromazine and noted that the compound induced ataraxia (indifference). Soon it was realized that chlorpromazine might be used for treatment of psychiatric disorders. The first clinical tests were performed on manic patients, and moderate responses were observed. In 1952, Deniker and co-workers reported the results of a trial with 38 schizophrenic patients who had been resistant to all existing therapies. All patients showed considerable improvement of schizophrenic symptoms.90 The serendipitous discovery of chlorpromazine as an antipsychotic agent proved to be a milestone in the pharmacotherapeutic treatment of schizophrenia, and has laid the foundation for the dopamine hypothesis of schizophrenia. Since then, numerous compounds with antipsychotic efficacy have been developed and have become available for the treatment of schizophrenia and related disorders.

1.5.1 CHEMICAL CLASSIFICATION OF ANTIPSYCHOTIC AGENTS

Several classification schemes for the currently clinically available antipsychotic agents have been proposed.262 Compounds may be classified based on chemical structure, pharmacological profiles, potency and nonneurologic side-effect profile, or clinical efficacy and neurologic side-effect liability. A chemical classification is chosen here. However, this section is not intended to provide a detailed review on the structure-activity relationships of the currently available antipsychotic agents, but rather to give an impression of the chemical diversity of the available compounds. Most antipsychotic agents which are currently in use in the Netherlands belong to one of five main chemical classes: (1) ; (2) ; (3) ; (4) , and (5) benzamides (for reviews and references, see refs. 163, 168, 218, 274, and 296). Phenothiazines were the first antipsychotic drugs to become available, and are exemplified by the prototypical compound of this class, chlorpromazine (30). Large series of analogues of 30 have been made since its discovery, and these fall into three subclasses of phenothiazine derivatives, based on

S S S S

N Cl N CF3 N SCH3 Cl

CH CH CH N 3 N N 3 N 3

CH3 N CH3

OH 30 31 32 33

CHART 1.5 Chemical structures of chlorpromazine (30), (31), (32) and chlorprotixene (33).

18 Introduction the different alkyl side chains: compounds with an aliphatic side chain, as in 30; compounds with a side chain, exemplified by fluphenazine (31); compounds with a piperidine side chain, as present in thioridazine (32). The antipsychotic activity of phenothiazine derivatives is dependent on the nature of the side chain, the basic amine, and the aromatic substituents. Derivatives with a piperazine side chain are generally the most potent. Regardless of the type of side chain, a three- carbon chain between the phenothiazine nitrogen atom and the side chain nitrogen atom is crucial for optimal activity. The basic nitrogen atom requires three substituents for optimal potency. The presence of an electronwithdrawing substituent at the 2-position of the phenothiazine nucleus is also essential for high potency. Phenothiazine derivatives are characterized by high affinities for dopamine

D1, D2, and D3, serotonin 5-HT2, a1-adrenergic, histamine H1, and muscarinic receptors, the ratio of the affinities being dependent on the type of side chain. Thioxanthenes (e.g. chlorprotixene, 33) are structurally related to phenothiazines, and their SARs with respect to requirements for side chains, basic nitrogen, and aromatic substituents are similar, but thioxanthenes are generally less potent than their phenothiazine analogues. The presence of the double bond in the side chain infers the existence of geometric isomers, referred to as cis and trans. In the cis isomers, which are more potent than the trans isomers, the side chain is directed towards the aromatic 2-substituent. In the early 1950’s a synthesis program was set up by Paul Janssen and co-workers at the laboratories of Janssen Pharmaceutica, aimed at the development of analgesics more potent than morphine. A number of compounds emerging from this program showed chlorpromazine-like effects in addition to analgetic activity. Structural optimization of the antipsychotic-like activity resulted in the synthesis of haloperidol (34, Chart 1.6) in 1958. Since its discovery, over 5,000 structurally related analogues of 34 have been synthesized and pharmacologically evaluated. Nevertheless, 34 proved to be one of the best compounds in terms of activity and toxicity, and still is the most prescribed antipsychotic agent to date. Virtually all structural modifications to the para- fluorobutyrophenone side chain lead to a decrease in antipsychotic activity. Thus, the para-fluor substituent, the carbonyl moiety, and an unbranched propylene chain connecting the aryl group and

Cl

O OH N

O H F N 34 O N N F O H N F N 35 N

F 36

CHART 1.6 Chemical structures of haloperidol (34), spiperone (35), and (36).

19 Chapter 1 the basic nitrogen are essential for high potency. The structural requirements for the basic amine seem to be less stringent, although maximum activity is usually obtained when the basic nitrogen atom is incorporated in a ring system. In case of a piperidine ring, like in 34, the presence of a substituent at the piperidine 4-position is beneficial for the antipsychotic activity. This substituent does not necessarily have to be aromatic in nature, as demonstrated by the spirocarbocyclic analogue spiperone (35), a derivative with an extremely high affinity for dopamine D2 receptors. Radiolabeled spiperone is frequently used as a radioligand in dopamine D2 receptor binding studies. In general, butyrophenones like haloperidol are relatively selective for dopamine D2 receptors, but most of them also display high affinities for serotonin 5-HT2 and a1-adrenergic receptors. Pimozide (36) is the best-known representative of the class of diphenylbutylpiperidines. These compounds may be considered as structural analogues of the butyrophenones, in which the carbonyl oxygen atom has been replaced by a phenyl ring, and as such, the SARs of the two classes of compounds are fairly similar. Diphenylbutylpiperidines are characterized by a relatively high affinity for dopamine D2 receptors, a strong antipsychotic potency and a long duration of action. The interesting pharmacological and clinical profile of the derivative sulpiride (37) prompted researchers at Astra Läkemedel (presently Astra Arcus) to search for analogues of 37 with higher oral bioavailabilty and brain penetration capabilities, two important characteristics of a CNS drug which 37 lacks due to its relatively low lipophilicity. In order to achieve this goal they introduced halogen atoms and a second flanking alkoxy group at the benzamide nucleus. (38) was one of the most promising compounds that emerged from this project. It has been clinically available during a short period, and showed a superior antipsychotic profile compared to standard therapies. Substituted benzamides possess a unique pharmacological profile: they bind to dopamine D2 and D3 receptors only. Radiolabeled raclopride (6, Chart 1.3) is frequently employed as a radioligand in dopamine D2 and D3 receptor binding and PET studies. The antidopaminergic properties of this class of benzamides have been shown to reside predominantly in the (S)- enantiomers. Benzamides with different types of side chain have been reported, and they have different structural requirements for high activity. Thus, benzamides with 4-piperidinyl side chains,

O O H CO O H N H 3 H 2 S Br N N O H H N N OCH 3 OCH3

37 38

O N O Cl Cl N N N H H H C CH H N OCH 3 N OCH 3 2 3 H 3 39 40

Chart 1.7 Chemical structures of sulpiride (37), remoxipride (38), (39), and (40).

20 Introduction such as clebopride (39), and with 3-pyrrolidinyl side chains, such as nemonapride (40), are also potent dopamine D2 receptor antagonists, provided that they bear a large lipophilic N-substituent. A few compounds are available which do not belong to any of the aforementioned chemical classes, the most important representative being the dibenzodiazepine derivative (41). In addition, several compounds of different chemical classes, including (42), (43), and (44), have recently been launched on the market. The pharmacological and clinical profiles of these compounds will be addressed in more detail in Sections 1.5.3. and 1.6.

H N Cl N O

N O F N N N

N N CH3

CH3 41 42

Cl H N N S NH CH 3 N O N N N

N F CH3 43 44

CHART 1.8 Chemical structures of clozapine (41), risperidone (42), olanzapine (43), and sertindole (44).

1.5.2 CLASSICAL ANTIPSYCHOTIC AGENTS: THE NEUROLEPTICS

The observation by Carlsson and Lindqvist in 1963 that chlorpromazine and haloperidol affected the dopaminergic and noradrenergic neurotransmission in the CNS not only laid the basis for the dopamine hypothesis of schizophrenia56 (see Section 1.2.1), but also initiated the search for a common site of action of antipsychotic agents. The development of the radioligand binding technique in the early 1970’s allowed for the measurement of the affinities of drugs for specific binding sites. In 1976, Creese et al.78 and Seeman et al.342 independently reported that a highly significant correlation existed between the affinity of antipsychotic agents for central dopamine receptors in vitro and their clinical potency. After the identification of two distinct subtypes of dopamine receptors,196 it was soon generally accepted that the dopamine D2 receptor was the principal target for antipsychotic activity.337 As a consequence, development of new antipsychotic drugs during the last 30 years has been primarily focused on compounds which potently block dopamine D2 receptors.

Blockade of dopamine D2 receptors in the mesolimbic and mesocortical dopaminergic (A10) system of the CNS is generally believed to be the mechanism of action of the classical antipsychotic

21 Chapter 1 agents. Chronic treatment with antipsychotic agents presumably leads to a so-called ‘depolarization block’ of dopaminergic neurons projecting to the limbic and cortical areas, which prevents firing of these cells (for review and references, see ref. 285). Classical antipsychotic agents are frequently referred to as ‘neuroleptics’, a term which reflects their capabilities to ‘take control of the neurons’.

However, whereas blockade of dopamine D2 receptors in the A10 system probably is responsible for the antipsychotic effects, simultaneous blockade of dopamine D2 receptors in other brain areas may give rise to the induction of serious side-effects (for reviews and references, see refs. 24 and 293).

Antagonism of postsynaptic dopamine D2 receptors in the nigrostriatal dopaminergic system (A9) leads to a functional deficiency of dopamine in the basal ganglia, which results in the occurrence of typical motor side-effects, referred to as extrapyramidal side-effects (EPS).399 Acute EPS include , dystonia, akathisia, and dyskinesia. Neuroleptic-induced parkinsonism (secondary parkinsonism) is characterized by hypo- or bradykinesia (reduction in amplitude and velocity of voluntary movements), muscular rigidity and tremor. These symptoms closely resemble those of Parkinson’s disease (idiopathic parkinsonism), a neurodegenerative disorder caused by a deficiency of dopamine in the striatum as a result of the degeneration of the nigrostriatal dopaminergic projections. Acute dystonia is characterized by sustained muscle contractions which may result in repetitive movements or abnormal postures. Acute akathisia consists of a feeling of restlessness and compulsion to move the limbs, and this side-effect is the least tolerable by the patients. Acute dyskinesia comprises the involuntary movements of the limbs and orofacial musculature, the latter being characterized by protrusion or twisting of the tongue, smacking, chewing, pursing and sucking movements of the lips, puffing of the cheeks, and lateral jaw movements. These acute EPS, which have been estimated to occur in 10–50% of all patients, are usually observed within days after initiation of the treatment, and disappear soon after termination of the treatment. Upon chronic antipsychotic drug treatment, however, more persistent, so-called ‘tardive’ forms of EPS may become apparent. The tardive EPS tend to develop in a later phase or even after treatment, and, as opposed to acute EPS, dose reduction or discontinuation of drug treatment does not provide immediate relief but usually tends to worsen the symptoms. Of the tardive EPS, tardive dystonia (~2% incidence) and tardive dyskinesia (TD, ~15% incidence) seem to be the most severe, since they are frequently irreversible. Whereas the acute occurrence of acute EPS seems to be accounted for by the acute blockade of dopamine D2 receptors in the nigrostriatal system, the mechanisms causing the tardive EPS are less well understood. The most plausible theory at the moment seems to be the 125 ‘dopamine D1/D2 imbalance hypothesis’ proposed by Gerlach et al. According to this hypothesis, blockade of postsynaptic dopamine D2 receptors in the nigrostriatal system at treatment onset leads to acute EPS. Simultaneous blockade of presynaptic dopamine D2 receptors causes an increase in synthesis and release of dopamine, which stimulates the unoccupied postsynaptic dopamine D1 receptors. A resulting supersensitivity of the dopamine D1 receptors, due to relative overstimulation, in combination with dysfunctional dopamine D2 receptors, should then account for the occurrence of tardive EPS. Secretion of the hormone prolactin from the pituitary is inhibited by dopamine. Treatment with antipsychotic agents therefore frequently results in hyperprolactinaemia, as blockade of dopamine D2 receptors by the antipsychotic agents prevents dopamine to properly regulate the prolactin secretion.

22 Introduction

Hyperprolactinaemia can lead to gynaecomastia (breast growth in man), galactorrhea (increased milk production), amenorrhea (no menstruation), and loss of sexual drive.

In addition to these dopamine D2 receptor-mediated side-effects, most antipsychotic agents produce a number of other side-effects, the extent of which is related to their receptor binding profiles. Thus, blockade of muscarinic cholinergic receptors leads to autonomic side-effects, including dry mouth, blurred vision, constipation, urinary retention, orthostatic hypotension, and tachycardia. Blockade of muscarinic receptors, however, also has antiparkinsonian effects. Therefore, antipsychotic agents with high affinity for muscarinic receptors, such as chlorpromazine and thioridazine, usually show a lower incidence of EPS. Antipsychotic agents with high affinity for histamine H1 receptors have strong sedative effects, experienced by the patients as feelings of slowness, lethargy and weakness. In the majority of patients these effects are undesirable, but in agitated, excited patients they may be beneficial. Blockade of a1-adrenergic receptors also contributes to sedation, and in addition, may cause orthostatic hypotension. Taken together, phenothiazine derivatives, by virtue of their strong muscarinic, a1-adrenergic and histamine H1 receptor blocking properties, are very sedative, often induce autonomic side-effects, but have a relatively low EPS liability. derivatives generally have somewhat lower affinities at histamine H1 and muscarinic Therefore, they are less sedative than phenothiazine derivatives, but have a higher risk of inducing EPS. Butyrophenones and diphenylbutylpiperidines lack significant affinity for muscarinic and histamine H1 receptors, and therefore hardly induce autonomic side- effects, but due to their high affinity for dopamine D2 receptors, they have a strong potential to induce EPS. Besides these receptor binding-related side-effects, classical antipsychotic agents can induce various nonspecific side-effects which seem not to be related to their receptor binding properties. Of these side-effects, the neuroleptic malignant syndrome (NMS) and agranulocytosis are the most serious, since they are potentially lethal. NMS occurs usually within a few days after initiation of neuroleptic treatment, and is characterized by muscular rigidity, hyperpyrexia (high fever), autonomic dysregulation, and delirium. Incidence estimates range from 0.02% to 2.4%. The mortality rate has been estimated to be 20–30%. Agranulocytosis is characterized by a suppression of the production of white blood cells, which makes a patient very susceptible for infections. Phenothiazines have a reputation for inducing agranulocytosis, with an estimated incidence risk of 0.05%. The mechanisms which cause NMS and agranulocytosis are poorly understood. Other aspecific side-effects, which are frequently observed, but less threatening, are weight gain, dermatological reactions, ophthalmological reactions, and sexual dysfunction. In addition to these side-effects, treatment of schizophrenia with classical antipsychotic agents has two other major drawbacks. First, virtually all neuroleptics only improve the positive symptoms of the disease in as much as 60–70% of the patients, while the negative symptoms are only marginally or not at all affected.133 Second, the large number of treatment-resistant patients not only undermines the dopamine hypothesis of schizophrenia (see Section 1.2.1), but also emphasizes the need for with an improved clinical efficacy.

1.5.3 ATYPICAL ANTIPSYCHOTIC AGENTS

23 Chapter 1

A few compounds seem to have an antipsychotic profile which is different from those of the classical antipsychotic agents. These so-called ‘atypical antipsychotics agents’ are characterized by a significantly lower incidence of EPS, sometimes combined with an improved antipsychotic efficacy. Clozapine (42) is generally considered to be the prototypical atypical antipsychotic agents, since its superior clinical efficacy in combination with its low EPS liability has not been matched by any other compound thus far. The definition of the term ‘atypical antipsychotic’ has been the subject of debate.77,262 From a preclinical perspective, an atypical antipsychotic agents should show a large dose separation in animal models with predictive value for antipsychotic activity (e.g. conditioned avoidance responding, inhibition of locomotor activity, prepulse inhibition) and models predicting EPS liability (e.g. catalepsy), whereas from a clinical perspective, an atypical antipsychotic agent should combine a superior antipsychotic activity and a low propensity to induce EPS. Ideally, an atypical antipsychotic agent should fulfill the following clinical requirements: (1) improvement of both positive and negative symptoms; (2) low acute and tardive EPS liability; (3) efficacy in therapy- resistant patients, and (4) no increase in plasma prolactin levels. The history of clozapine is peculiar (for reviews see refs. 158, 250, and 358). The compound was synthesized in 1958 at Sandoz-Wander Ltd., as part of a research program devoted to the discovery of new . In animal studies it was soon noted that clozapine behaved more like an antipsychotic agent, but failed to induce catalepsy, an effect which at that time was generally accepted to be a prerequisite for any compound to show antipsychotic activity in man. Clinical trials started in the early 1960’s, and the results were encouraging.140 The compound was launched in a number of countries, and evidence for its superior profile started to accumulate.103,126 In 1975, however, shortly after its launch in Finland, 16 patients developed agranulocytosis as a results of treatment with clozapine, 8 of which were fatal.7 This resulted in the withdrawal of clozapine in several countries, while its use was restricted in many others. Nevertheless, a certain myth started to develop around clozapine during this period of restricted use, as it was consistently shown to possess a superior antipsychotic efficacy in comparison with classical compounds, to be effective in therapy- resistant patients, and to have a low propensity to induce acute and tardive EPS. These findings have been confirmed by several clinical trials performed in the 1980’s.59,71,187,209,229 Predominantly as a result of the multi-centre study by Kane and co-workers in 1988,190 the use of clozapine for treatment of neuroleptic-responsive and treatment-resistant patients has been re-approved in the USA and a number of European countries. Whereas the superiority of clozapine in treatment-resistant patients is beyond doubt, its efficacy against negative symptoms is less evident. Results from several clinical trials suggest that clozapine indeed is superior to classical antipsychotic agents in the treatment of negative symptoms,48,190,255 but these effects may in fact be the result of the reduction in EPS and depression caused by the compound.191 Despite almost 40 years of research, the mechanism of action responsible for the unique profile of clozapine is poorly understood. The compound has high affinities for a number of receptor subtypes

(see Table 1.3), the most prominent being dopamine D4, serotonin 5-HT2, 5-HT6, and 5-HT7, cholinergic muscarinic, a1- and a2-adrenergic, and histamine H1 receptors. In contrast to most classical antipsychotic agents, clozapine displays a relatively low affinity for dopamine D2 receptors. Furthermore, PET studies have shown that clozapine occupies a significantly lower number of

24 Introduction

107-109 central dopamine D2 receptors at therapeutic doses than classical antipsychotic agents. Moreover, whereas it has been generally accepted that antipsychotic agents exert their antipsychotic effect by blocking dopamine D2 receptors, observations from in vitro and in vivo studies suggest that 175,241 clozapine may behave as a dopamine D2 agonist. In addition, clozapine has also been shown to 411,414 314 possess intrinsic efficacy at muscarinic m4 and serotonin 5-HT1A receptors. Although clozapine seems to be the ideal antipsychotic agent, therapy with this drug has one major drawback: in approximately 1–2% of all patients it causes agranulocytosis.207 Consequently, the blood of patients treated with clozapine has to be monitored for leukocytes on a regular basis. The formation of free radical metabolites has been proposed as the cause of this potential lethal blood discrasia.115 In addition, clozapine is strongly sedative, and may induce seizures, weight gain, orthostatic hypotension, hypersalivation and constipation.60

Of the older antipsychotic agents, thioridazine (32), sulpiride (37) and remoxipride (38) have been categorized as atypical, since therapy with these compounds seems to be associated with a reduced incidence of EPS. The reduced EPS liability of thioridazine may be the result of its high affinity for muscarinic receptors. The low propensity of sulpiride and remoxipride is remarkable in view of their high selectivity for dopamine D2 receptors (Table 1.3), and re-emphasizes the hypothesis that dopamine D2 receptor blockade is the primary mode of antipsychotic drug action. Remoxipride was

a Table 1.3 Receptor binding affinities (Ki, nM) of haloperidol and putative atypical antipsychotic agents. Receptor Hal Cloz Remox Risp Olanz Sert Zipr Quet

D1 15 53 > 1,000 21 10 12 9.5 390

D2L 2.2 190 125 5.9 31 7.0 4.6 700

D3 7.8 280 969 14 49 10 10 340

D4.2 11 40 > 1,000 16 28 21 39 > 1,000

5-HT1A > 1,000 140 > 1,000 420 > 1,000 280 12 320

5-HT2A 25 3.3 > 1,000 0.2 1.9 0.9 0.3 120

5-HT2C > 1,000 13 > 1,000 63 7.1 1.3 13 > 1,000 b 5-HT6 > 1,000 4.0 > 1,000 420 2.5 – – –

5-HT7 380 21 > 1,000 1.6 120 28 4.9 290

AchM > 1,000 34 > 1,000 > 1,000 26 > 1,000 > 1,000 > 1,000

a1 19 23 > 1,000 2.3 60 1.8 12 58 a2 > 1,000 160 > 1,000 7.5 – > 1,000 – – b1 > 1,000 > 1,000 > 1,000 > 1,000 > 1,000 > 1,000 > 1,000 > 1,000 b2 > 1,000 > 1,000 > 1,000 > 1,000 > 1,000 > 1,000 > 1,000 > 1,000

H1 790 0.2 > 1,000 27 0.7 130 15 19

sigma1 1.1 > 1,000 60 950 > 1,000 120 110 220 aData adapted from refs. 12, 204, 316, 317 and 329. Abbreviations: Hal, haloperidol; Cloz, clozapine; Remox, remoxipride; Risp, risperidone; Olanz, olanzapine; Sert, sertindole; Zipr, ; Quet, . bNo data available.

25 Chapter 1 launched in 1990 but had to be withdrawn in 1994, due to the induction of aplastic anemia in 8 out of 45,000 patients. During the short period the remoxipride has been clinically available, the compound has been shown to improve both positive and negative symptoms, being at least as effective as haloperidol in both first-episode and severely psychotic patients. In addition, it can prevent relapse in chronic schizophrenics and was shown to be effective in one third of therapy- resistant patients. Moreover, remoxipride showed a minimal tendency to induce EPS and does not affect plasma prolactin levels.221,222,394 Remoxipride may have proven to be the most significant advance in antipsychotic therapy since the reappraisal of clozapine, as it meets virtually all the requirements of the ideal atypical antipsychotic agent, and it is therefore truly unfortunate that this compound is no longer clinically available. Possible mechanisms underlying the atypical properties of remoxipride, which may also hold true for sulpiride, have been suggested to be either a selective effect on a subpopulation of dopamine D2 receptors, a preferential effect on subsets of functionally 290 coupled dopamine D2 receptors, or differential effects on subsystems in the corpus striatum. Like haloperidol, remoxipride also has considerable affinity for sigma binding sites in addition to dopamine D2 and D3 receptors. However, since clinical trials with selective sigma site antagonists have failed to shown that such compounds possess antipsychotic activity,68,178 this action of remoxipride is unlikely to contribute to its clinical profile.

1.6 PHARMACOLOGICAL APPROACHES FOR THE DEVELOPMENT OF POTENTIAL ATYPICAL ANTIPSYCHOTIC AGENTS

The considerations above make it clear that there is still need for the development of antipsychotic agents with an improved clinical profile, i.e. antipsychotic agents which combine the superior antipsychotic activity and low neurological side-effect liability of clozapine, but also lack its potential to induce agranulocytosis. Furthermore, such compounds may help to better understand the exact mode of action of antipsychotic agents and to unravel the etiology of schizophrenia and related disorders. Since it has been generally accepted that a dopamine D2 antagonistic component is required for antipsychotic activity, most pharmacological approaches which are currently under investigation rely on the development of compounds which interfere to some extent with dopamine

D2-like receptors. An atypical antipsychotic profile may possibly be achieved with compounds which selectively block a subtype of dopamine D2-like receptors (i.e. D3 or D4), compounds which possess some intrinsic activity at dopamine D2 receptors (i.e. dopamine D2 autoreceptor agonists or partial dopamine D2 receptor agonists), or by compounds which, in addition to dopamine D2 receptors, also bind to specific serotonin receptor subtypes (i.e. serotonin 5-HT2 or 5-HT1A). These different pharmacological approaches will be addressed in more detail in the subsequent sections.

1.6.1 SELECTIVE DOPAMINE D3 RECEPTOR ANTAGONISTS

The rationale for the development of selective dopamine D3 receptor antagonists as potential atypical antipsychotic agents is predominantly based on two observations. First, the specific regional distribution of the dopamine D3 receptor in the CNS suggests that this receptor subtype may be an interesting target for antipsychotic drug action (for reviews, see refs. 130, 330, 350, 352, and 354).

After the identification of the dopamine D3 receptor as a new dopamine receptor subtype,

26 Introduction

localization studies have shown that dopamine D3 receptors are predominantly expressed in limbic structures, including the olfactory tubercle/Islands of Calleja complex, the mammilary nuclei of the hypothalamus, the amygdala, the bed nucleus of the stria terminalis, the nucleus accumbens, the 43,155,214,215,219,259 septum and the hippocampus. In contrast, the expression of the dopamine D2 receptor in the midbrain is more restricted to areas which are associated with motor functions. This raised the idea that selective dopamine D3 receptor antagonists should alleviate psychotic symptoms by blocking dopamine D3 receptors in the limbic areas, while at the same time leaving the dopamine

D2 receptors in the extrapyramidal areas unaffected, and hence be devoid of EPS. Second, molecular genetic studies have suggested that an association may exist between the occurrence of schizophrenia and the gene encoding the dopamine D3 receptor (for reviews, see refs. 199, 267, and 268).

Encouraged by the identification of the dopamine D3 receptor as a potential target for atypical antipsychotic agents, much effort has been devoted to the development of selective dopamine D3 receptor antagonists during the last years, and various selective compounds have emerged (for review see ref. 374). (+)-AJ 76 (45, Chart 1.9) and (+)-UH 232 (46), two compound previously 298 designated as selective dopamine D2 autoreceptor antagonists, were the first compounds shown to 351 have some preference (Ki ratio D2/D3 of 3.0 and 4.4, respectively) for the dopamine D3 receptor. More selective agents are exemplified by S 14297 (8, Chart 1.3, 23-fold selective),260 which is structurally closely related to the selective dopamine D3 receptor agonist (R)-(+)-7-OH-DPAT, the 6-substituted 2-aminotetralin derivative GR 218231 (47, 400-fold selective),271 and the benzamide- related compounds (48, 10-fold selective)324 and GR 103691 (49, 125-fold selective).270 It should be noted however, that the claimed selectivity ratios are dependent on the radioligand 389 employed in the dopamine D2 binding assays. Despite the availability of various compounds, no

CH3 N N R O O S

OCH3 H3CO 45 R = H 47 46 R = n-propyl

H3CO

N H3CO O O N N N H H N

CN O

CH3 48 49

CHART 1.9 Chemical structures of the selective dopamine D3 receptor antagonists (+)-AJ 76 (45), (+)-UH 232 (46), GR 218231 (47), nafadotride (48), and GR 103691 (49).

27 Chapter 1

selective dopamine D3 receptor antagonist has been evaluated against schizophrenia in clinical trials thus far, presumably due to the lack of knowledge concerning the functional role of the dopamine D3 receptor in vivo.

1.6.2 SELECTIVE DOPAMINE D4 RECEPTOR ANTAGONISTS

The hypothesis that selective dopamine D4 receptor antagonists might have potential as atypical antipsychotic agents has predominantly been founded on the observation that clozapine possesses a 388 relatively high affinity for the dopamine D4 receptor. Subsequently, based in this presumed selectivity, Seeman and co-workers have proposed a concept of dopamine D4/D2 receptor occupancy of atypical antipsychotic drug action. Their hypothesis was based on analyses of the so-called ‘radioligand-independent’ affinities of a number of classical and atypical antipsychotic agents for dopamine D2 receptors, and estimations of free plasma concentrations of these compounds at therapeutic doses. Clozapine, at therapeutic plasma concentrations of 10–20 nM, would primarily occupy dopamine D4 receptors, while other antipsychotic agents would primarily occupy dopamine 338,340 D2 receptors. The hypothesis was based on clozapine having an affinity of 10 nM for the dopamine D4 receptor. However, significantly lower affinities, and hence selectivity ratios for clozapine have been reported by others.63,101,213,217 In addition, whereas the affinity of clozapine for dopamine D2 receptors is generally considered to be relatively low (~150 nM), Malmberg et al. reported an affinity for the cloned dopamine D2B receptor of 35 nM, again suggesting that clozapine 241 may not be as selective for dopamine D4 receptors as originally claimed. Moreover, the affinities of clozapine for serotonin 5-HT2, 5-HT6, 5-HT7, muscarinic, a1- and a2-adrenergic, and histamine H1 receptors are all in the order of magnitude of the estimated therapeutic plasma levels (Table 1.3), making it equally likely that clozapine exerts its unique profile by interacting with one or more of these receptor subtypes.

A six-fold increase in the density of dopamine D4 receptors in post-mortem brain tissue of schizophrenic patients was also claimed by Seeman and co-workers. Dopamine D4 receptor densities 3 were measured by determining the differences in Bmax values for the binding of [ H]-nemonapride, 3 which has high affinities for dopamine D2, D3, and D4 receptors, and [ H]-raclopride, which has high affinities for dopamine D2 and D3 receptors only. Although similar results were reported by others using the same methodology,269 they should be taken with caution. For example, Assié et al. have 16 reported an affinity of 4.5 nM of nemonapride for the serotonin 5-HT1A receptor. Simultaneous labeling of this receptor subtype therefore may also have accounted for the observed differences in

Bmax values. Furthermore, the fact that substituted benzamides such as raclopride and nemonapride consistently label almost twice as much binding sites as the prototypical dopamine D2 receptor ligand spiperone may also have contributed to the reported observations.341 For more reliable assessments of dopamine D4 receptor densities, a highly selective dopamine D4 receptor antagonist (see below) should be employed as a radioligand in such studies.

28 Introduction

In view of the larger difference in sequence homology between dopamine D4 receptors on the one hand and dopamine D2 and D3 receptors on the other hand (Table 1.1), it is not surprising that a considerable number of highly selective dopamine D4 receptor antagonists have become available recently (for reviews see refs. 228 and 374). The piperazinylazaindole L-745,870 (50, Chart 1.10) was reported to have 2200-fold and >5,000-fold selectivity for dopamine D4 over dopamine D2 and 210 D3 receptors, respectively. The same researchers recently reported the structurally related morpholine derivative 51, with >1,000-fold and >2,000-fold selectivity over dopamine D2 and D3 receptors.345 The isoxazole 52, which was also prepared in the same lab, is an isomer of the highly selective L-741,742 (10, Chart 1.3), but is slightly less selective.318 The optical pure isochromane derivative U-101387 (53) was shown to possess high affinity for the dopamine D4 receptors, while lacking significant affinity for dopamine D1, D2 and D3, serotonin 5-HT1A and 5-HT2, and a1- and a2- adrenergic receptors.373 This compound is currently undergoing clinical trials. YM-43611 (54) is a derivative of nemonapride (40, Chart 1.7) with 110-fold D4 selectivity and 10-fold D3 preference over dopamine D2 receptors. In addition, this benzamide was shown to have negligible affinities for a number of representative neurotransmitter receptors. Compound 55 was the most dopamine D4- selective representative of a series of napthoate esters, having a 1,260-fold selectivity over dopamine

D2 receptors. Furthermore, 55 lacked significant affinities for dopamine D3 and serotonin 5-HT1A, 5-

HT2A, and 5-HT2C receptors.

Cl N O N N O

N N N H N H Cl 50 51

N CH Cl 3 N N O

SO NH N O 2 2 52 53

O OCH N 3 Cl O N H O N HN OCH3 O

54 55

CHART 1.10 Chemical structures of the selective dopamine D4 receptor antagonists L-741,870 (50), 51, 52, U-101387 (53), YM-43611 (54), and 55.

29 Chapter 1

L-745,870 (50) has been evaluated for antipsychotic efficacy in schizophrenic patients. At a dose 49 of 15 mg/day this compound was ineffective, suggesting that blockade of dopamine D4 receptors alone is not sufficient for exerting antipsychotic effects. Nevertheless, such selective dopamine D4 antagonists may be useful in unraveling the functional role of the dopamine D4 receptor and the relationship it bears to the pathophysiology of schizophrenia.

1.6.3 DOPAMINE D2 AUTORECEPTOR AGONISTS AND PARTIAL DOPAMINE D2 RECEPTOR AGONISTS

Several lines of evidence indicate that compounds which possess intrinsic efficacy at dopamine D2 receptors may have potential as atypical antipsychotic agents (for reviews see refs. 29 and 30). The indirect dopamine receptor agonists d-amphetamine62,134 and L-DOPA52,287 have been shown to improve negative symptoms in some schizophrenic patients when co-administered with traditional antipsychotic agents. Furthermore, the dopamine D1/D2 receptor agonist alone at low doses has been reported to improve certain aspects of schizophrenia.370 In preclinical neurochemical and behavioural models, at low doses simultaneously inhibits dopamine synthesis and locomotor activity in rats. These effects have been ascribed to activation of dopamine D2 autoreceptors, located presynaptically at the nerve terminals of the dopaminergic neurons. Stimulation of these receptors leads to a decrease in synthesis and release of dopamine, and hence provides a feedback mechanism for regulation of the amount of dopamine available for stimulation of postsynaptic receptors in the synaptic cleft. Therefore, selective stimulation of terminal dopamine D2 autoreceptors by selective agents has been proposed as a mechanism for treatment of schizophrenia.278 In brain areas with dopaminergic hyperactivity, stimulation of terminal dopamine D2 autoreceptors would normalize the dopaminergic neurotransmission and hence reduce psychotic symptoms. Beneficial effects against negative symptoms of schizophrenia may stem from the compound’s ability to stimulate postsynaptic dopamine D2 receptors in areas with dopaminergic hypoactivity. Furthermore, some degree of intrinsic efficacy would also reduce the propensity to cause EPS by mild stimulation of postsynaptic dopamine D2 receptors in the nigrostriatal system. This hypothesis of dopamine D2 autoreceptor selectivity also implies that terminal dopamine D2 autoreceptors and postsynaptic dopamine D2 receptors represent two distinct pharmacological entities. However, Drukarch and Stoof showed that the two types of receptors display similar pharmacological characteristics, suggesting that they are 99 identical. Nevertheless, several compounds with putative preferential action on dopamine D2 autoreceptors have been developed during the last decade. Examples of such compounds are (S)-(–)- 3-PPP (preclamol, 56),403 B-HT 920 (, 57),8 SND 919 (, 58),328 EMD 49980 (, 59),44 and OPC-4392 (60).21 Detailed analyses of the pharmacological profiles of these compounds, however, has revealed that they merely act as partial dopamine D2 receptor agonists with varying degrees of intrinsic efficacy at both presynaptic and postsynaptic dopamine D2 receptors. Moreover, most of the compounds previously designated as putative dopamine D2 autoreceptor agonists have been shown to bind preferentially to dopamine D3 receptors, and a correlation between their dopamine D3 receptor binding properties and their potency to regulate dopamine autoreceptor-mediated cell activity has 206 been reported. Furthermore, the inhibitory effects of presumed selective dopamine D2

30 Introduction autoreceptor agonists on locomotor activity in rodents are probably mediated by stimulation of 367 postsynaptic dopamine D3 receptors. Taken together, the effects of these compounds, which have been previously ascribed to their alleged preferential action on terminal dopamine D2 autoreceptors, are probably the result of simultaneous stimulation of both pre- and postsynaptic dopamine D2 and

D3 receptors. The combination of the degree of intrinsic efficacy and preference for the dopamine D3 receptor presumably determines the overall profile of these compounds. Preclamol, talipexole, pramipexole, and roxindole have been evaluated in small clinical trials for efficacy in schizophrenia. In a single-blind trial with four patients, preclamol showed antipsychotic activity in two subjects and was well tolerated.368 Talipexole was evaluated in twelve schizophrenics in an open trial: in only four subjects significant amelioration of positive symptoms was observed.400,401 Pramipexole has recently been shown to significantly improve both positive and negative symptoms when used in combination with haloperidol.195 In addition, the compound has proven to be of value in the treatment of Parkinson’s disease,136 and has recently become available for this indication. Roxindole has been reported to be ineffective in patients in which positive symptoms dominate, but in a subgroup of patients with predominantly negative symptoms, moderate to significant improvements were observed.397,401 These effects of roxindole on negative symptoms may be attributed to properties of the compound: roxindole binds with high affinity to serotonin 5-HT1A receptors and inhibits the reuptake of serotonin. This antidepressant action was confirmed by an open clinical trial with patients suffering from major depression, in which roxindole proved to be very effective.141 In general, from these preliminary clinical data it can be concluded that the usefulness of putative selective dopamine D2 autoreceptor agonists as atypical antipsychotic agents is limited.

1.6.4 MIXED DOPAMINE D2/SEROTONIN 5-HT2 RECEPTOR ANTAGONISTS

The implication of serotonin in the pathophysiology of schizophrenia has been addressed in Section 1.2.2. A renewed interest in serotonergic agents as potential antipsychotic agents started

H N S S NH

H2N N H2N N N OH 56 57 58

HO N CH H 3 O N O N CH N 3 N H 59 60

CHART 1.11 Chemical structures of the putative selective dopamine D2 autoreceptor agonists (S)-(–)-3-PPP (56), B- HT 920 (57), SND 919 (58), EMD 49980 (59), and OPC-4392 (60).

31 Chapter 1 when in 1984 clozapine was shown to act as a potent antagonists at serotonin receptors.114 Altar and co-workers in 1986 suggested that the serotonin 5-HT2/dopamine D2 receptor affinity ratio might be the key to the unique profile of clozapine.5 Based on cluster analyses, performed on the receptor binding profiles of a number of classical and supposedly atypical antipsychotic agents, Meltzer and coworkers in 1989 hypothesized that compounds which combine 5-HT2 receptor antagonism and dopamine D2 receptor antagonism in an appropriate ratio (5-HT2/D2 pKi ratio ³ 1.12) should possess atypical antipsychotic properties. This hypothesis should thus account for the superior clinical profile of clozapine.256,257 Measurement of receptor occupancy in rat brain revealed that a number of 246,359 putative atypical antipsychotic agents preferably occupy serotonin 5-HT2 receptors in vivo. Furthermore, PET studies in schizophrenics have shown that clozapine displays a very high serotonin

5-HT2 receptor occupancy, while occupation of dopamine D2 receptors by clozapine is consistently lower than by classical antipsychotic agents.107,280,281

Support for a role of serotonin 5-HT2 receptors in the pathophysiology of schizophrenia and in the mode of action of atypical antipsychotic agents also comes from clinical studies with the partial serotonin 5-HT2 agonist m-chlorophenylpiperazine (MCPP). In unmedicated schizophrenic patients MCPP has been shown to exacerbate psychotic symptoms, but in healthy volunteers it does not induce symptoms resembling psychosis.208 In addition, the increase in plasma levels of prolactin, growth hormone and cortisol induced by MCPP in schizophrenic patients can by blocked by clozapine.289 Remarkably, patients who benefited most from treatment with clozapine were also those in which MCPP induced the strongest endocrine response when drug-free.188 The observation that fluphenazine, which predominantly blocks dopamine D2 receptors, was unable to counteract the MCPP responses, supports the hypothesis that the effects of clozapine in these studies are mediated 289 via blockade of serotonin 5-HT2 receptors.

Substantial evidence also comes from clinical studies with the selective serotonin 5-HT2 receptor antagonist . When added to the regular antipsychotic medication of chronic schizophrenics with severe EPS, ritanserin markedly reduced EPS in these subjects.33 Reyntjens and co-workers also reported significant improvement of EPS and negative symptoms by ritanserin in an add-on trial in chronic schizophrenics.312 In another add-on study ritanserin proved to be particularly effective against negative symptoms.100 Finally, Wiesel et al. reported that, in an open trial, ritanserin alone proved to be effective against both positive and negative symptoms, without exacerbating EPS.402

The latter findings suggest that blockade of serotonin 5-HT2 receptors alone might be sufficient for exerting an atypical antipsychotic profile. However, in order to validate this hypothesis, double-blind clinical trials are required. Particularly the findings with ritanserin have stimulated the development of new potential atypical antipsychotic agents based on the concept of mixed dopamine D2/serotonin5-HT2 receptor antagonism during the last decade (for reviews see refs. 92, 171, 234, 254, and 327). Risperidone (42, Chart 1.8) was the first compound developed on the basis of this concept, and was launched on the market in 1994. Risperidone has higher affinity for serotonin 5-HT2 receptors than for dopamine

D2 receptors (Table 1.3). In addition, it also possesses high affinities for dopamine D3, serotonin 5-

HT7, and a1- and a2-adrenergic receptors. In contrast to clozapine, risperidone lacks high affinities for muscarinic and histamine receptors. In several open and double-blind clinical trials, risperidone has been shown to be effective against both positive and negative symptoms of schizophrenia, being

32 Introduction superior to haloperidol and at least as effective as clozapine. Risperidone produces maximal efficacy and less EPS than classical antipsychotic agents at doses between 4–8 mg/day. At larger doses, however, the compound may induce marked EPS. As risperidone treatment does not seem to be associated with agranulocytosis, it may prove to be a valuable alternative for clozapine. However, the efficacy of risperidone in treatment-refractory patients remains to be established (for reviews and references, see refs. 39, 41, 143, and 258). Side-effects associated with risperidone treatment are mild sedation, orthostatic hypotension, prolactin increase and weight gain.60 Following risperidone, several new putatively atypical antipsychotic agents based on the concept of mixed dopamine D2/serotonin 5-HT2 antagonism have been developed. Two of these, olanzapine (43) and sertindole (44), have recently become clinically available. Olanzapine has strong structural resemblance to clozapine, and their receptor binding profiles are also similar (Table 1.3), but olanzapine has generally somewhat higher receptor affinities than clozapine. Therefore, olanzapine should be effective at lower doses, which should reduce the risk of aspecific side-effects such as agranulocytosis. Clinical trials have revealed that olanzapine is equally effective as haloperidol against positive and significantly more effective against negative symptoms, has a reduced incidence risk of EPS, and does not elevate plasma prolactin levels.28,39 Olanzapine also seems to be effective in treatment-resistant patients.245 Results of a recent double-blind multi-center comparison study between risperidone and olanzapine suggested that the latter has a superior clinical profile.378 The superiority of olanzapine to clozapine remains to be established since these compounds have not been compared in clinical trials thus far. Olanzapine is mildly sedative, may cause orthostatic hypotension, and tends to induce weight gain.60 Sertindole is structurally unrelated to any of the currently available antipsychotic agents. It has 261 particularly high affinities for dopamine D2, serotonin 5-HT2 and a1-adrenergic receptors. In several clinical trials, sertindole was shown to be equally effective as haloperidol against positive and significantly more effective against negative symptoms.39,369 Sertindole’s efficacy in treatment- resistant patients, as well as its relative efficacy compared to clozapine remain to be established. Prolongation of the QT interval, decreased ejaculatory volume, nasal congestion and weight gain have been frequently reported as side-effects of sertindole treatment.60 Ziprasidone (CP-88,059, 61) and quetiapine (seroquel, ICI 204,636, 62) are putative atypical antipsychotic agents, which have been developed based on the concept of mixed dopamine

D2/serotonin 5-HT2 receptor antagonism, and are on the verge of becoming clinically available. Ziprasidone has some structural features in common with risperidone, and their receptor binding

S N S

N N N N O N Cl N H O OH

61 62

CHART 1.12 Chemical structures of the mixed dopamine D2/serotonin 5-HT2 receptor antagonists ziprasidone (61) and quetiapine (62).

33 Chapter 1 profiles are also similar (Table 1.3). Unlike risperidone, however, ziprasidone also has high affinity 336 for serotonin 5-HT1A receptors, which may be beneficial for its EPS liability (see Section 1.6.5). In clinical trials ziprasidone was shown to be effective against both positive and negative symptoms of schizophrenia, and to have antidepressant and anxiolytic properties. Furthermore, it seems to have a low tendency to induce EPS and other side-effects.86 Its efficacy in treatment-resistant patients remains to be established. Quetiapine has structural resemblance with clozapine and like clozapine it has a broad receptor binding profile, but the affinities are generally lower (Table 1.3). Quetiapine displays relatively high affinities for histamine H1, a1-adrenergic, and serotonin 5-HT2 receptors. The outcome of several clinical trials with quetiapine in schizophrenic patients has revealed that it has an antipsychotic efficacy comparable to haloperidol, but with a more favorable side-effect profile.14,40,147,346 In summary, the clinical results obtained thus far with risperidone, olanzapine, sertindole, ziprasidone and quetiapine strongly suggest that the concept of mixed dopamine D2/serotonin 5-HT2 receptor antagonism, with a predominant occupancy of serotonin 5-HT2 receptors, has proven to be successful for the development of antipsychotic agents with an improved efficacy and side-effect profile. Nevertheless, none of the compounds has proven to be superior to clozapine thus far.

1.6.5 MIXED DOPAMINE D2 RECEPTOR ANTAGONISTS/SEROTONIN 5-HT1A RECEPTOR AGONISTS

The outcome of both preclinical and clinical investigations suggest that the serotonin 5-HT1A receptor may prove to be an interesting target for improving antipsychotic therapy. In preclinical behavioural and neurochemical models selective serotonin 5-HT1A receptor agonists have been shown to interact with antipsychotic agents. For example, several selective serotonin 5-HT1A receptor agonists, including 8-OH-DPAT, flesinoxan, , and , have consistently been shown to reverse catalepsy induced by dopamine D2 receptor antagonists, such as haloperidol and raclopride, in rats51,104,156,173,235,253,272,393 and monkeys.69,227 Furthermore, 8-OH- DPAT has been shown to possess antipsychotic-like properties157,392 and to enhance the antipsychotic properties of raclopride in animals models with predictive value for antipsychotic activity.393 Catalepsy in animals has been generally accepted as a model for EPS in man. Thus, antipsychotic agents which potently induce catalepsy in animals show a high propensity to cause EPS in humans.160 Clozapine has only weak cataleptogenic properties at high doses and is even capable of reversing catalepsy induced by other antipsychotic agents, such as and olanzapine.189 In addition, whereas clozapine acts as an antagonist at virtually all receptor subtypes it binds to, it has been shown to act as a partial serotonin 5-HT1A receptor agonist. For example, Rollema et al. recently reported that the preferential increase in prefrontal cortical extracellular dopamine induced by clozapine could be partially blocked by the selective serotonin 5-HT1A receptor antagonist WAY 314 100,635, suggesting that clozapine exerts this action by stimulation of serotonin 5-HT1A receptors. Nevertheless, Bartoszyk et al. demonstrated that the anticataleptic properties of clozapine are not 26 mediated by its action on serotonin 5-HT1A receptors.

Several clinical observations suggest a role for the serotonin 5-HT1A receptor in schizophrenia. First, post-mortem studies on the brains of schizophrenic patients have revealed increased densities of serotonin 5-HT1A receptors in the frontal cortex, which were unrelated to the medication history

34 Introduction of the patients (see Section 1.2.2). Second, clinical studies with buspirone, a compound with mixed 372 dopamine D2 receptor antagonistic and partial serotonin 5-HT1A receptor agonistic properties, suggest that it may have an atypical antipsychotic profile. Buspirone (63, Chart 1.13) has originally been developed and marketed as an anxiolytic agent. Early observations suggested that buspirone may also possess antipsychotic properties.252,406 When added to haloperidol in an open trial with schizophrenic patients, buspirone improved negative symptoms and EPS.132 Buspirone was also reported to reduce TD and EPS when given alone in an open trial.236 Furthermore, the compound has repetitively been reported to suppress akathisia induced by several neuroleptics,83 although worsening of movement disorders due to treatment with buspirone in a few patients has also been reported.50,224 Nevertheless, these results with buspirone suggest that it would be worthwhile to evaluate compounds with similar pharmacological profiles in preclinical models for antipsychotic activity and side-effect liability. Finally, it has been suggested that a serotonin 5-HT1A receptor agonistic component may be beneficial in relieving the anxiety that can trigger psychotic episodes in schizophrenics.282

In summary, these findings suggest that compounds which combine dopamine D2 receptor antagonism with serotonin 5-HT1A receptor agonism may have enhanced antipsychotic activity and a reduced EPS liability. However, the dopamine D2/serotonin 5-HT1A receptor affinity ratio, as well as the degrees of intrinsic efficacy at these receptor subtypes required for an optimal clinical profile need to be established. Nevertheless, several compounds with the indicated pharmacological profile have recently been disclosed (Chart 1.13). (RWJ-37796, 64) has high affinity for dopamine D2, D3, and serotonin 5-HT1A receptors (Ki values of 2.2, 1.8 and 1.7 nM, respectively) 310 but also for a1-adrenergic receptors (Ki = 1.3 nM). At serotonin 5-HT1A receptors, it behaves as a . The chromane derivative EMD 128130 (65) has high affinities for dopamine D2-like

(D2: Ki = 20 nM) and serotonin 5-HT1A (Ki = 1 nM) receptors only, the activity residing in the (R)- 25,45 enantiomer. EMD 128130 behaves in vivo as a dopamine D2 receptor antagonist and a serotonin

5-HT1A receptor agonist. PD 158771 (66) is the lead compound of a series of aminopyrimidine derivatives with high affinities for both dopamine D2, D3, and serotonin 5-HT1A receptors (Ki values of 5.2, 13.7 and 3.5 nM respectively). At both dopamine D2 and serotonin 5-HT1A receptors, 66 behaved as a partial agonist.408 Recently several compounds have been disclosed, which in addition to dopamine D2 and serotonin 5-HT1A receptors, also possess high affinity for serotonin 5-HT2 receptors. The anthranilamide 1192U90 (67) is the lead compound of a series of substituted benzamides with such receptor binding profiles.282,283 Like ziprasidone it bears a 4-(1,2- benzisothiazol-3-yl)-1-piperazine moiety, which presumably is responsible for the high serotonin 5-

HT2 receptor affinity. A similar receptor binding profile was reported for the structurally closely related thiazolidinone derivative P-9236 (HP-236, 68).169,170 Compounds 64–68 were all active in animal models with predictive value for antipsychotic activity (e.g. conditioned avoidance responding, inhibition of apomorphine-induced mouse climbing). Furthermore, evaluation in animal models with predictive value for EPS liability in man (e.g. catalepsy, inhibition of apomorphine- induced stereotyped behaviour) showed that they all are likely to have a low propensity to cause

EPS. These results are promising and suggest that compounds with mixed dopamine D2 receptor antagonistic and serotonin 5-HT1A receptor agonistic properties may have potential as atypical antipsychotic agents. Mazapertine and 1192U90 are currently undergoing clinical trials.

35 Chapter 1

O O N N O N N N N O N

N 63 64

N F N

O N N H N N N H 65 66

N S S

O N H C N 3 F N N N N H S

NH2 H3C O CH3 67 68

CHART 1.13 Chemical structures of the mixed dopamine D2 receptor antagonists/serotonin 5-HT1A receptor agonists buspirone (63), mazapertine (64), EMD 128130 (65), PD 158771 (66), 1192U90 (67), and P-9236 (68).

1.7 SCOPE OF THE THESIS

The Department of Medicinal Chemistry at the University of Groningen has a history of more than 25 years in the design, synthesis, and pharmacological evaluation of drugs acting at the CNS. Research within the Department has been focused in particular on 2-aminotetralin-derived and structurally closely related compounds, such as octahydrobenzo[f]quinolines, hexahydro- naphthoxazines and tetrahydrobenzopyranoxazines, with activity at dopaminergic, serotonergic or melatonergic receptors during the last 15 years (e.g. see refs. 23, 74, 93, 179 and 277). Examples of compounds that have been developed within the Department are the selective dopamine D3 receptor agonists (R)-7-OH-DPAT113 and PD 128907,94 the melatonin receptor agonist 8-methoxy-2- 75 164 acetamidotetralin, and the dopamine D2 receptor agonist N-0923, which is currently undergoing clinical trials for the treatment of Parkinson’s disease.

Encouraged by the promising concept of mixed dopamine D2 receptor antagonism and serotonin

5-HT1A receptor agonism for the development of potential atypical antipsychotic agents, the idea was raised to design compounds with such a pharmacological profile using the 2-aminotetralin system as a structural base. This thesis is the result of that idea. In the subsequent chapters, the design,

36 Introduction synthesis, pharmacological evaluation, and molecular modeling aspects of a series of 2-aminotetralin- derived benzamides and structurally closely related compounds are described. It is the hope that the medicinal chemistry of the compounds disclosed in this thesis will contribute to a better understanding of the pathophysiology of schizophrenia and the mechanism of action of potential atypical antipsychotic agents.

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encoded by a subfamily of two distinct genes: 5-HT1Da and 5-HT1Db. Proc Natl Acad Sci USA 89, 3630–3634. 397 Wetzel H, Hillert A, Grunder G and Benkert O (1994) Roxindole, a dopamine autoreceptor agonist, in the treatment of positive and negative schizophrenic symptoms. Am J Psychiatry 151, 1499–1502. 398 Whitaker PM, Crow TJ and Ferrier IN (1981) Tritiated LSD binding in frontal cortex in schizophrenia. Arch Gen Psychiatry 38, 278–280. 399 White FJ and Wang RY (1983) Differential effects of classical and atypical antipsychotic drugs on A9 and A10 dopamine neurons. Science 221, 1054–1057. 400 Wiedemann K, Benkert O and Holsboer F (1990) B-HT 920 – a novel dopamine autoreceptor agonist in the treatment of patients with schizophrenia. Pharmacopsychiatry 23, 50–55. 401 Wiedemann K, Krieg J-C, Loycke A and Holsboer F (1992) Novel dopamine autoreceptor agonists B-HT 920 and EMD 49980 in the treatment of patients with schizophrenia. In: Meltzer HY (Ed) Novel Antipsychotic Drugs. Raven Press, New York, pp 91–98. 402 Wiesel FA, Nordström AL, Farde L and Eriksson B (1994) An open clinical and biochemical study of ritanserin in acute patients with schizophrenia. Psychopharmacology 114, 31–38. 403 Wikström H, Sanchez D, Lindberg P, Hacksell U, Arvidsson L-E, Johansson A, Thorberg S-O, Nilsson JLG, Svensson K, Hjorth S, Clark D and Carlsson A (1984) Resolved 3-(3-hydroxyphenyl)-N-n-propylpiperidine, 3- PPP and its analogues: central dopamine receptor activity. J Med Chem 27, 1030–1036. 404 Williamson P (1987) Hypofrontality in schizophrenia: a review of the evidence. Can J Psychiatry 32, 399–404. 405 Winblåd B, Bucht G, Gottfries CG and Roos BE (1979) Monoamines and monoamine metabolites in brains from demented schizophrenics. Acta Psychiatr Scand 60, 17–28. 406 Wood PL, Nair NP, Lai S and Etienne P (1983) Buspirone: a potential atypical neuroleptic. Life Sci 33, 269–273. 407 Woolley DW and Shaw E (1954) A biochemical and pharmacological suggestion about certain mental disorders. Proc Natl Acad Sci USA 40, 228–231. 408 Wustrow D, Belliotti T, Glase S, Ross Kesten S, Johnson D, Colbry N, Rubin R, Blackburn A, Akunne H, Corbin A, Davis MD, Georgic L, Whetzel S, Zoski K, Heffner T, Pugsley T, Wise L (1998) Aminopyrimidines with high affinity for both serotonin and dopamine receptors. J Med Chem 41, 760–771. 409 Wyatt RJ (1986) The dopamine hypothesis: variations on a theme (II). Psychopharmacol Bull 22, 923–927. 410 Wyatt RJ, Alexander RC, Egan MF and Kirch DG (1988) Schizophrenia, just the facts – What do we know, how well do we know it? Schizophr Res 1, 3–18. 411 Zeng XP, Le F and Richelson E (1997) Muscarinic m4 receptor activiation by some atypical antipsychotic drugs. Eur J Pharmacol 321, 349–354. 412 Zgombick JL, Schechter LE, Macchi M, Hartig PR, Branshek TA and Weinshank RL (1992) Human gene S31

encodes the pharmacologically defined serotonin 5-hydroxytryptamine1E receptor. Mol Pharmacol 42, 180–185. 413 Zhou QY, Grandy DK, Thambi L, Kushner JA, Van Tol HHM, Cone R, Pribnow D, Salon J, Bunzow JR and

Civelli O (1990) Cloning and expression of human and rat D1 dopamine receptors. Nature 347, 76–80.

55 Chapter 1

414 Zorn SH, Jones SB, Ward KM and Liston DR (1994) Clozapine is a potent and selective muscarine M4 receptor agonist. Eur J Pharmacol 269, 1–2.

56 2-AMINOTETRALIN-DERIVED SUBSTITUTED BENZAMIDES

WITH MIXED DOPAMINE D2, D3, AND SEROTONIN 5-HT1A RECEPTOR BINDING PROPERTIES: A NOVEL CLASS OF 2 POTENTIAL ATYPICAL ANTIPSYCHOTIC AGENTS

ABSTRACT

A new chemical class of potential atypical antipsychotic agents, based on the pharmacological concept of mixed dopamine D2 receptor antagonism and serotonin 5-HT1A receptor agonism, was designed by combining the structural features of the 2-(N,N-di-n-propylamino)tetralins (DPATs) and the 2-pyrrolidinylmethyl-derived substituted benzamides in a structural hybrid. Thus, a series of 35 differently substituted 2-aminotetralin-derived benzamides was synthesized and the compounds were evaluated for their ability to compete for [3H]-raclopride binding to cloned 3 human dopamine D2A and D3 receptors, and [ H]-8-OH-DPAT binding to rat serotonin 5-HT1A receptors in vitro. The lead compound of the series, 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 17a), displayed high affinities for the dopamine D2A receptor (Ki = 3.2 nM), the dopamine D3 receptor (Ki = 0.58 nM), as well as the serotonin 5-HT1A receptor (Ki = 0.82 nM). The structure-affinity relationships of the series suggest that the 2-aminotetralin moieties of the compounds occupy the same binding sites as the DPATs in all three receptor subtypes. The benzamidoethyl side chain enhances the affinities of the compounds for all three receptor subtypes, presumably by occupying an accessory binding site. For the dopamine D2 and D3 receptors, this accessory binding site may be identical to the binding site of the 2-pyrrolidinylmethyl-derived substituted benzamides.

57 Chapter 2

2.1 INTRODUCTION

Dopamine and serotonin possess considerable conformational freedom, and it is therefore likely that they bind to the different dopamine and serotonin receptor subtypes in different conformations. Compounds with receptor subtype selectivity can be obtained by applying the ‘rigid analogue approach’, i.e. by constraining the structural elements of the endogenous ligand, which are essential for its biological activity, into a (semi)rigid chemical skeleton.18 The 2-aminotetralin (2-amino- 1,2,3,4-tetrahydronaphthalene system (1, Chart 2.1) has proven to be a suitable structural base for the development of conformationally restricted analogues of both dopamine and serotonin. In the 2- aminotetralin system the flexible aminoethyl side chain, as present in dopamine and serotonin, is fixed in an extended trans conformation, which retains the amine functionality close to the plane through the aromatic ring. The C2 carbon atom of the 2-aminotetralin system is chiral, hence racemic 2- aminotetralin comprises a mixture of two optical antipodes. When appropriately substituted, the pharmacological activity of the 2-aminotetralin system can be accurately tuned. This is nicely demonstrated by the 2-(N,N-di-n-propylamino)tetralins (DPATs), a class of compounds with intriguing structure-activity relationships (SARs). For example, 5-hydroxy- 2-(N,N-di-n-propylamino)tetralin (5-OH-DPAT, 2), mimicking the a-rotamer of dopamine,7 is a 16,36 highly potent dopamine D2 receptor agonist. In contrast, the 6-OH analogue (6-OH-DPAT, 3) displays only weak dopaminergic activity. Movement of the hydroxy group to the 7-position gives rise to 7-OH-DPAT (4), in which the dopaminergic activity, although somewhat weaker than 2, is retained.3,5,14,45,48,50 Moreover, 7-OH-DPAT, which mimicks the b-rotamer of dopamine,7 displays 30,47 considerable selectivity for the dopamine D3 receptor. Surprisingly, the 8-OH analogue (8-OH-

DPAT, 5) turned out to be a potent and selective serotonin 5-HT1A receptor agonist devoid of dopaminergic activity,3 demonstrating that the presence of an indole nucleus, as in serotonin, is not a prerequisite for high affinity at the serotonin 5-HT1A receptor. Similarly, the observation that 2- (N,N-di-n-propylamino)tetralin (DPAT, 6), which lacks a hydroxy substituent, possesses mixed dopaminergic and serotonergic properties,33,54 demonstrates that it is not always necessary to incorporate all the functional groups of the endogenous ligand into a synthetic analogue in order to mimic some of its biological activities.

8 N 7 NH2 2 R 6 5 1 2 R=5-OH 7 R=5-OCH3 3 R=6-OH 8 R=6-OCH3 4 R=7-OH 9 R=7-OCH3 5 R=8-OH 10 R=8-OCH3 6 R=H

Chart 2.1 Chemical structure and numbering of the 2-aminotetralin system (1), and chemical structures of the 2- (N,N-di-n-propylamino)tetralins (DPATs, 2–10).

58 2-Aminotetralin-derived Substituted Benzamides

The DPATs interact with the dopamine D2, D3, and serotonin 5-HT1A receptors in a highly stereoselective manner. Thus, whereas the dopaminergic activity of 2 has been shown to reside 36,45,50,53 predominantly in the (S)-enantiomer, (R)-2 behaves as a weak dopamine D2 receptor 28 34,48,51 antagonist. Both enantiomers of 2 show some preference for the dopamine D3 receptor. On the other hand, the dopaminergic activity of 4 can be ascribed almost exclusively to the (R)- 4,12,34,51 enantiomer. Both enantiomers of 5 have high affinity for the serotonin 5-HT1A receptor, but (R)-5 is a full agonist, while (S)-5 behaves as a partial agonist.6,9 Remarkably, the enantiomers of 6 show a stereoselective separation between dopaminergic and serotonergic activity, (S)-6 being a moderately potent dopamine D2/D3 receptor agonist, and (R)-6 being a fairly potent serotonin 5- 33,54 HT1A receptor agonist, respectively. The methoxy-substituted DPATs 7–9 usually have lower, but sometimes higher affinities for the 16 dopamine D2 and D3 receptor than their hydroxy analogues 2–4, dependent on the 34,51 stereochemistry. For example, (S)-7 binds about ten times weaker at both dopamine D2 and D3 receptors than (S)-2, but (R)-7 has about threefold higher affinities for both receptor subtypes than (R)-2. Replacement of the hydroxy group of 4 by a methoxy group leads to a large drop in affinities for both receptor subtypes, an effect predominantly caused by the strong loss of affinity of (R)-9 compared to (R)-4. In contrast, the enantiomers of 10 bind with about equal affinities to the serotonin 5-HT1A receptor as their hydroxy analogues (S)- and (R)-5. Moreover, it has been shown that the C8-substituent of 5 can be replaced by a variety of groups, including bulky substituents like 29,31–33,39 phenyl, without much loss of affinity for the serotonin 5-HT1A receptor. SAR studies have revealed that the structural requirements of the substituents at the basic nitrogen atom of the 2-aminotetralin system for high affinity at the dopamine D2, D3, or the serotonin 2,6,16,39,46,51 5-HT1A receptor are comparable. Thus, derivatives of 1 with at least one N-n-propyl group have the highest affinities at either of the receptor subtypes, dependent on the substitution pattern of the aromatic nucleus. The corresponding N-ethyl analogues are usually slightly less active, but the absence of an N-substituent results in dramatic loss of affinity. The structural demands for the second N-substituent are less stringent, but only one N-substituent larger than n-propyl is tolerated by the receptors.

As outlined in Section 1.6.5, compounds which combine dopamine D2 receptor antagonism with serotonin 5-HT1A receptor agonism may have potential as atypical antipsychotic agents. Encouraged by this hypothesis, we conceived it possible to design a new class of potential atypical antipsychotic agents with the indicated pharmacological profile, by combining the structural, and hence pharmacological features of the DPATs and the 2-pyrrolidinylmethyl-derived substituted benzamides in a structural hybrid. The SAR of the latter class of compounds, as exemplified by sulpiride (11, Chart 2.2), remoxipride (12) and raclopride (13) has been briefly addressed in Section 1.5.1. Thus,

O O H CO O OH O H N H 3 H H 2 S Br Cl N N N O H H H N N N OCH3 OCH3 OCH3 Cl 11 12 13

Chart 2.2 Chemical structures of sulpiride (11),remoxipride (12), and raclopride (13).

59 Chapter 2

O N N N R H R

A B

O N N R H R

C

FIGURE 2.1 Schematic representation of the structural hybridization of 2-(N,N-di-n-propylamino)tetralins (DPATs, A) and 2-pyrrolidinylmethyl-derived substituted benzamides (B), resulting in the concept of 2- aminotetralin-derived benzamides (C). by linking the basic nitrogen of differently substituted 2-(N-n-propylamino)tetralins via a two-carbon chain to the amide nitrogen of differently substituted benzamide moieties (Figure 2.1), a series of 35 N-(2-benzamidoethyl)-substituted 2-(N-n-propylamino)tetralins with various substitution patterns was synthesized. The ability of these compounds to compete for [3H]-raclopride binding to cloned 3 human dopamine D2A and D3 receptors and [ H]-8-OH-DPAT binding to rat serotonin 5-HT1A receptors was determined.

2.2 CHEMISTRY

The synthetic pathway employed to obtain the target compounds is outlined in Scheme 1. The appropriately substituted 2-(N-n-propylamino)tetralins 14a–14e, known from the literature,1,11,27 served as starting points for the synthesis of the target compounds. N-alkylation with bromoacetonitrile in boiling acetone, employing potassium carbonate as a base and potassium iodide as a catalyst, gave the cyanomethyl intermediates 15a–15e in good yields, which subsequently were reduced almost quantitatively with LiAlH4 to the corresponding primary amines 16a–16e. Three different methods were employed to obtain the amides 17a–17p, 18a–18d, 19a–19d, 20a–20d, and 21a–21d. First, the appropriate acid chloride (either commercially available or readily obtained from the corresponding carboxylic acid using standard procedures) was employed in the presence of sodium hydroxide and the biphasic medium water/dichloromethane according to the Schotten- Baumann procedure (Method A).8 Second, the appropriate acid chloride was allowed to react with the appropriate primary amine in boiling chloroform, without addition of a base (Method B).13 Third, the appropriate carboxylic acid was converted into a mixed anhydride using ethyl chloroformate and was then allowed to react with the appropriate primary amine in the presence of triethylamine and acetone as the solvent (Method C).17

60 2-Aminotetralin-derived Substituted Benzamides

NH N CN a b R R

14a R=5-OCH3 15a–15e 14b R=6-OCH3 14c R=7-OCH3 14d R=8-OCH3 14e R=H

O N N NH c or d or e N Ar R 2 R H

16a–16e 17a–17o R=5-OCH3 18a–18d R=6-OCH3 19a–19d R=7-OCH3 20a–20d R=8-OCH3 21a–21d R=H

Scheme 2.1 Reagents and conditions: (a) BrCH2CN, K2CO3, KI, acetone, D; (b) LiAlH4, THF, D; (c) ArCOCl, 10%

NaOH, CH2Cl2, RT; (d) ArCOCl, CHCl3, D; (e) ArCOOH, EtOCOCl, Et3N, acetone, 0 ºC.

Since in the DPAT series the hydroxy-substituted congeners usually have the highest affinities, compounds 17a, 18a, 19a, and 20a were demethylated with boron tribromide in dichloromethane,37 resulting in the corresponding hydroxy analogues 17p, 18e, 19e, and 20e respectively (Scheme 2.2).

O O N N N a N H3CO H HO H

17a 5-OCH3 17p 5-OH 18a 6-OCH3 18e 6-OH 19a 7-OCH 19e 7-OH 3 20e 8-OH 20a 8-OCH3

Scheme 2.2 Reagents and conditions: (a) BBr3, CH2Cl2, –50 ºC.

61 Chapter 2

2.3 PHARMACOLOGY

Compounds 17a–17p, 18a–18e, 19a–19e, 20a–20e, and 21a–21d were evaluated for their ability 3 to compete for [ H]-raclopride binding to cloned human dopamine D2A and D3 receptors, expressed in Ltk– and CHO cells respectively, as well as their ability to compete for [3H]-8-OH-DPAT binding to rat hippocampal membranes in vitro.

2.4 RESULTS AND DISCUSSION

By linking differently substituted benzamide moieties, as present in the 2-pyrrolidinylmethyl- derived class of substituted benzamides, with their amide nitrogen atom via a 2-carbon chain to the basic nitrogen atoms of differently substituted 2-(N-n-propylamino)tetralins, we have attempted to combine the pharmacological properties of these two distinct classes of compounds into a new chemical class of compounds, i.e. the 2-aminotetralin-derived benzamides. The results of the binding assays in Table 2.1 show that most compounds display moderate to high affinities for dopamine D2,

D3, and serotonin 5-HT1A receptors. When considering the effects of differently substituted benzamide moieties, several consistent trends can be observed in the binding data. First, compounds with a 2,3-dimethoxy substitution pattern on the benzamide moiety (17c, 19b, and 20b) always have a higher affinity for the dopamine

D2A receptor than for the dopamine D3 receptor, whereas for compounds with a 2,6-dimethoxy substitution pattern (17d, 19c, and 20c) the opposite is the case, i.e. they prefer the dopamine D3 receptor to the dopamine D2A receptor. Compounds 18b and 18c have not been evaluated for their affinities at dopamine D3 receptors, but since 18b has high affinity for the dopamine D2A receptor while 18c is devoid of affinity, it may be anticipated that these compounds will also obey to the rule mentioned above. These remarkable consistencies in affinities towards the two dopamine receptor subtypes should probably be explained in part by differences in conformational behaviour of the two types of substituted benzamides. Compounds with one methoxy substituent positioned ortho to the benzamide carbonyl group presumably adopt a conformation in which the ortho-methoxy group is oriented in a coplanar fashion with respect to the plane of the aromatic ring and amide group, while forming a hydrogen bond between its oxygen atom and the amide hydrogen atom (Figure 2.2). Benzamides with a 2,6-dimethoxy substitution pattern cannot adopt such a coplanar system due to

O O H3C O R R N N H O H O CH3 CH3 A B

FIGURE 2.2 Conformational differences between 2-methoxy- (A) and 2,6-dimethoxy-substituted (B) benzamides: intramolecular hydrogen bond formation in A results in a coplanar benzene ring and amide, whereas in B the presence of a second ortho-methoxy group causes steric hindrance with the carbonyl oxygen atom and hence an out-of-plane twisted benzene ring, which prevents the formation of an intramolecular hydrogen bond.

62 2-Aminotetralin-derived Substituted Benzamides steric hindrance between the second ortho-methoxy group and the carbonyl oxygen atom, resulting in an out-of-plane conformation of the entire aromatic ring with respect to the amide functionality. These assumptions are supported by X-ray diffraction10,21–24,49,52 and molecular modeling studies21,22,24,43,44 performed on 2-pyrrolidinylmethyl-derived benzamides with comparable substitution patterns. It should be noticed, however, that the dopamine D2A/D3 receptor affinity ratio is also affected by substituents at other positions of the benzamide moiety. This becomes obvious when the affinities of compounds 17a–17c, 17e, 17f, 17i, and 17k for these receptor subtypes are compared. Introduction of an ortho-methoxy group in 17a, resulting in 17b, decreases the affinities for both receptor subtypes considerably. Introduction of an additional methoxy group at the 3- position, as in 17c, restores the high affinity for the dopamine D2A receptor (cf. 17a), but decreases the dopamine D3 receptor affinity even more (cf. 17b). Thus, the preference of compounds 17c, 18b,

19b, and 20b for the dopamine D2A receptor, as compared to their 2,6-dimethoxy-substituted analogues 17d, 18c, 19c, and 20c, seems not only to be accounted for by the ability of 17c, 18b, 19b, and 20b to form an intramolecular hydrogen bond, as opposed to 17d, 18c, 19c, and 20c, but also by the presence of the 3-methoxy group, which enhances the dopamine D2A receptor affinity and at the same time decreases the dopamine D3 receptor affinity. The affinities of compounds 17e, 17f, 17i, and 17k reveal that substitution of the benzamide 5-position, in combination with an ortho- methoxy group (cf. 17g and 17h), also restores some of the affinity for the dopamine D2A receptor (cf. 17b). These observations are in line with SAR and QSAR studies performed on 2- pyrrolidinylmethyl-derived benzamides, which have shown that a 2,3-dimethoxy substitution pattern,20,25,26 but also the presence of a lipophilic and/or bulky substituent at the 5-position21,24,40–42 are favourable for high dopamine D2 receptor affinity. Therefore, the benzamide moieties of the 2- aminotetralin-derived benzamides presented here may occupy the same binding site as the 2- pyrrolidinylmethyl-derived benzamides.

63 Chapter 2

TABLE 2.1 Receptor binding data of compounds 17a–17p, 18a–18e, 19a–19e, 20a–20e, 21a–21d, haloperidol, and clozapine.

O 8 N 7 N Ar R H 6 5

a Ki (nM)

Compound R Ar D2A D3 5-HT1A b 17a 5-OCH3 Ph 3.2 ± 0.2 0.58 ± 0.05 0.82 ± 0.11

17b 5-OCH3 2-OCH3-Ph 22.9 ± 8.4 19.7 ± 5.6 15.2 ± 6.9

17c 5-OCH3 2,3-di-OCH3-Ph 6.7 ± 2.7 24.3 ± 1.4 12.7 ± 4.5 c 17d 5-OCH3 2,6-di-OCH3-Ph 47.9 ± 16.0 2.6 ± 0.1 27.3 ± 1.6

17e 5-OCH3 5-Br-2-OCH3-Ph 9.4 ± 1.0 17.4 ± 0.5 38.5 ± 23.0

17f 5-OCH3 5-I-2-OCH3-Ph 6.9 ± 2.3 16.3 ± 0.7 75 ± 45 d 17g 5-OCH3 5-Br-2-OH-Ph 79.8 ± 2.0 ND 10.4 ± 1.6

17h 5-OCH3 2-OH-5-I-Ph 266 ± 110 ND 21.9 ± 0.4

17i 5-OCH3 2-OCH3-5-SO2NH2-Ph 14.7 ± 2.7 20.3 ± 1.1 34.4 ± 6.4

17j 5-OCH3 5-Br-2,6-di-OCH3-Ph 42.8 ± 3.2 ND 53.9 ± 20.6

17k 5-OCH3 4-NH2-5-Cl-2-OCH3-Ph 2.9 ± 0.5 2.5 ± 0.2 40.5 ± 1.5

17l 5-OCH3 2-Thienyl 3.6 ± 0.2 0.69 ± 0.05 1.1 ± 0.1

17m 5-OCH3 3-Thienyl 5.4 ± 1.8 ND 4.6 ± 0.5

17n 5-OCH3 1-Naphthyl 21.9 ± 0.3 ND 13.0 ± 2.8

17o 5-OCH3 2-Naphthyl 18.5 ± 2.9 ND 20.2 ± 3.8 17p 5-OH Ph 1.4 ± 0.2 0.28 ± 0.03 1.5 ± 0.4

18a 6-OCH3 Ph 70.8 ± 7.6 ND 16.8 ± 4.3

18b 6-OCH3 2,3-di-OCH3-Ph 10.1 ± 1.0 ND 72.8 ± 12.4

18c 6-OCH3 2,6-di-OCH3-Ph 1,070 ± 70 ND 93.0 ± 23.7

18d 6-OCH3 2-Thienyl 112 ± 11 ND 61.1 ± 8.3 18e 6-OH Ph 13.4 ± 2.4 ND 17.4 ± 4.7 c 19a 7-OCH3 Ph 60.7 ± 0.9 14.3 ± 0.7 4.2 ± 1.1

19b 7-OCH3 2,3-di-OCH3-Ph 3.7 ± 0.1 6.8 ± 0.4 12.7 ± 1.2

19c 7-OCH3 2,6-di-OCH3-Ph 366 ± 51 56.2 ± 0.2 32.8 ± 0.6

19d 7-OCH3 2-Thienyl 110 ± 4 32.7 ± 6.6 12.2 ± 0.4 19e 7-OH Ph 3.7 ± 0.1 0.50 ± 0.03 3.0 ± 1.2

20a 8-OCH3 Ph 54.9 ± 1.8 4.5 ± 0.7 <0.3

20b 8-OCH3 2,3-di-OCH3-Ph 1.0 ± 0.1 14.6 ± 0.4 0.76 ± 0.11

20c 8-OCH3 2,6-di-OCH3-Ph 89.5 ± 1.3 15.0 ± 2.0 1.0 ± 0.1

20d 8-OCH3 2-Thienyl 60.3 ± 4.1 9.8 ± 1.8 0.73 ± 0.19 20e 8-OH Ph 55.2 ± 2.2 6.8 ± 2.3c <0.3 21a H Ph 10.0 ± 0.8 0.46 ± 0.01 0.56 ± 0.05

21b H 2,3-di-OCH3-Ph 0.63 ± 0.1 7.4 ± 0.1 3.5 ± 0.3

21c H 2,6-di-OCH3-Ph 59.8 ± 9.3 2.6 ± 0.1 3.7 ± 0.4 21d H 2-Thienyl 106 ± 18 ND 1.8 ± 0.3 Haloperidol 0.67 ± 0.11 2.7 ± 0.6 2,213 ± 585 Clozapine 59.8 ± 7.8 83.3 ± 9.9 304 ± 184 aMean values ± s.e.m. of 2–4 independent experiments; bPh: phenyl; cTwo binding sites significant; dND: not determined.

64 2-Aminotetralin-derived Substituted Benzamides

Whereas 2-pyrrolidinylmethyl-derived substituted benzamides generally need several lipophilic substituents on the aromatic nucleus for high affinity, this is not necessary for the 2-aminotetralin- derived benzamides: comparison of the affinities of 17a with those of 17b–17k shows that attachment of substituents on the benzamide nucleus generally leads to somewhat lower affinities. Furthermore, the benzene ring of the benzamide moiety of 17a can be replaced by aromatic isosters of comparable size, such as 2-thiophene (17l) and 3-thiophene (17m) without seriously affecting the affinities for the receptors. A similar isosteric replacement in 20a does not affect the receptor binding (cf. 20d) either, but in 18a, 19a, and 21a it results in lower affinities (cf. 18d, 19d, and 21d, respectively). Replacement by larger aromatic systems, such as 1-naphthalene (17n) or 2-naphthalene (17o) results in somewhat lower affinities when compared to their benzene analogue (17a). Apparently, these groups are to bulky to be accommodated optimally by the receptors. When the effects of the differently substituted 2-aminotetralin moieties are examined, several conclusions can be drawn. First, when comparing the dopaminergic affinities of 5-, 6- and 7- methoxy-substituted congeners with identically substituted benzamide moieties (e.g. 17a, 18a, and 19a), the general order of potency (except for the 2,3-dimethoxy-substituted benzamides) is 5-OMe > 7-OMe > 6-OMe. Furthermore, the hydroxy-substituted congeners (17p, 18e and 19e) all have higher affinities than their methoxy-substituted analogues. In addition, all congeners with an 8- methoxy substituent have a very high affinity for the serotonin 5-HT1A receptor. Replacement of the 8-methoxy group by a hydroxy group (cf. 20a vs. 20e) does not affect the affinity for the serotonin

5-HT1A receptor. Nevertheless, the binding data of compounds 21a–21d show that the presence of substituents on the 2-aminotetralin nucleus is not a prerequisite for high affinity. Taken together, these structure-affinity relationships are consistent with those of the DPATs (see Section 2.1) and suggest that the 2-aminotetralin parts of the molecules occupy the same binding sites as the DPATs. Finally, the binding data suggest that attachment of a 2-benzamidoethyl side chain to the basic nitrogen atom of differently substituted 2-(N-n-propylamino)tetralins enhances the affinities for the dopamine D2 and D3, as well as for the serotonin 5-HT1A receptor, when compared to their DPAT analogues. For example, whereas 5-OH-DPAT (2) and 7-OH-DPAT (4) have virtually no serotonergic activity, their benzamide analogues 17p and 19e have high affinities for the serotonin 5-

HT1A receptor. On the other hand, compound 20e has moderate and high affinity for the dopamine

D2 and D3 receptor, respectively, whereas 8-OH-DPAT (5) is devoid of dopaminergic activity. The observation that the large 2-benzamidoethyl side chain is tolerated well by the three receptor subtypes is consistent with the requirements for the nitrogen substituents of the DPATs, where only one substituent larger than n-propyl is allowed. Taken together, the findings suggest that the 2- benzamidoethyl side chain may occupy an accessory binding site in all three receptor subtypes, thereby enhancing the affinities for the receptors. For the dopaminergic receptors, this accessory binding site may prove to be identical to the binding site of the 2-pyrrolidinylmethyl-derived substituted benzamides, as noted earlier. Conversely, when the concept of 2-aminotetralin-derived substituted benzamides is approached by taking the class of 2-pyrrolidinylmethyl-derived substituted benzamides as a starting point, it can be concluded that by merely replacing the pyrrolidinylmethyl moiety by 2-(N-n-propylamino)tetralin, serotonergic activity has been introduced in these compounds. For example, whereas 2-pyrrolidinylmethyl-derived substituted benzamides, such as sulpiride (11) and remoxipride (13), are completely devoid of serotonergic activity,15 their 2-(N-n-

65 Chapter 2

propylamino)tetralin-derived analogues 17i and 17j have good affinities for the serotonin 5-HT1A receptor. Thus, by combining the structural features of the DPATs and the 2-pyrrolidinylmethyl- derived substituted benzamides into one structural hybrid, the pharmacological properties of these classes of compounds have also been joined together. Since both the DPATs and the 2-pyrrolidinylmethyl-derived substituted benzamides display high stereoselectivity in their interactions with the receptors, it may be anticipated that this will also be true for the 2-aminotetralin-derived benzamides. Therefore, 5-methoxy-2-[N-(2-benzamidoethyl)-N- n-propylamino]tetralin (5-OMe-BPAT, 17a) and its 2,6-dimethoxybenzamide analogue [5-OMe-

(2,6-di-OMe)-BPAT, 17d], which showed some preference for the dopamine D3 receptor, have been selected for enantiopure synthesis and assessment of the in vitro receptor binding profiles and the intrinsic efficacies at dopamine D2, D3, and serotonin 5-HT1A receptors of their enantiomers (see Chapter 5).

2.5 CONCLUSIONS

In conclusion, a series of compounds with mixed dopamine D2, D3, and serotonin 5-HT1A receptor binding profiles was designed by combining the structural, and hence pharmacological features of two distinct classes of compounds, i.e. the 2-pyrrolidinylmethyl-derived substituted benzamides and the DPATs, into a new basic skeleton. Several compounds display high affinities for both dopamine D2 and D3, and serotonin 5-HT1A receptors. Provided that they possess the desired intrinsic efficacies at the indicated receptor subtypes, these compounds may be interesting candidates for further evaluation of the dopamine D2/serotonin 5-HT1A hypothesis of atypical antipsychotic drug action.

2.6 EXPERIMENTAL SECTION

2.6.1 CHEMISTRY

General Remarks. Unless otherwise indicated, all materials were purchased from commercial suppliers and used without further purification. All basic amine products were converted to their corresponding hydrochloride or oxalate salts by adding an equimolar amount of a 1 M ethereal HCl solution or an ethanolic solution of oxalic acid to a solution of the free base in ether. All chemical data, except for TLC analyses and electron impact mass spectra, were obtained on the salt forms, unless otherwise stated. TLC analyses were carried out on aluminium plates (Merck) precoated with silica gel 60 F254 (0.2 mm), and spots were visualised with UV light and I2. Gravity column chromatography was performed using silica gel (Merck 60). Melting points were determined in open glass capillaries on an Electrothermal digital melting-point apparatus and are uncorrected. IR spectra were recorded on an ATI- Mattson Genesis Series FT-IR spectrophotometer, and only the important absorptions are indicated. Broad peaks (b) have been indicated as such. 1H NMR spectra were recorded at 200 MHz on a Varian Gemini-200 spectrometer or at 300 MHz on a Varian VXR-300 spectrometer. 1H NMR chemical shifts are denoted in d units (ppm) relative to the solvent and converted to the TMS scale, using 7.26 for CDCl3 and 3.30 for CD3OD. The following abbreviations are used to indicate spin multiplicities: s (singlet), as (apparent singlet), bs (broad singlet), d (doublet), dd (doublet of doublets), t (triplet), m (multiplet). 13C NMR spectra were recorded at 50 MHz on a Varian Gemini-200 spectrometer or at 75 MHz on a Varian VXR-300 spectrometer. 13C NMR chemical shifts are denoted in d units (ppm) relative to the solvent and converted to the TMS scale, using 76.91 for CDCl3 and 49.50 for CD3OD. Chemical ionization (CI)

66 2-Aminotetralin-derived Substituted Benzamides mass spectra were recorded on a Finnegan 3300 quadrupole mass spectrometer. Ammonia was used as the reactant gas and samples were introduced into the ion source by means of the direct insertion probe. Alternatively, chemical ionization mass spectra were recorded on a NERMAG R 3010 triple quadrupole mass spectrometer equipped with a home-built atmospheric pressure ionization source and ionspray interface. Electron impact (EI) mass spectra were recorded on a Unicam Automass mass spectrometer in conjunction with a gas chromatograph. Elemental analyses (C, H, and N) for target compounds were performed at the Micro Analytical Department, University of Groningen.

General Procedure for the Preparation of Compounds 15a–15e. The method adopted for the synthesis of 5- methoxy-2-(N-cyanomethyl-N-n-propylamino)tetralin·HCl (15a) is described: bromoacetonitrile (1.17 g, 9.8 mmol) 1 was added to a suspension of 14a·HCl (1.00 g, 3.9 mmol), K2CO3 (1.35 g, 9.8 mmol) and KI (0.16 g, 0.1 mmol) in acetone (50 mL). The reaction mixture was refluxed for 18 h under a nitrogen atmosphere. After cooling, the solids were removed by filtration and the filtrate was concentrated under reduced pressure, which yielded the crude nitrile as a dark brown oil. Purification by column chromatography (eluent: CH2Cl2) gave 0.86 g (3.3 mmol, 85%) of the pure base of 15a as a colourless oil, which was converted to the hydrochloride salt: mp 183–186 °C (EtOH); IR: cm–1 2924, 1 2832, 2739, 2347 (b), 1590; H NMR (base, 200 MHz, CDCl3): d 0.97 (t, J = 7.3 Hz, 3H), 1.46–1.75 (m, 3H), 2.14– 2.22 (m, 1H), 2.52–3.07 (m, 7H), 3.68 (s, 2H), 3.84 (s, 3H), 6.72 (dd, J = 8.4 Hz, 8.4 Hz, 2H), 7.14 (t, J = 7.8 Hz, 13 1H); C NMR (base, 50 MHz, CDCl3): d 11.6, 20.9, 26.5, 33.2, 38.6, 52.1, 55.2, 57.7, 107.2, 116.9, 121.5, 124.8, 126.4, 136.4, 157.1; MS (EI, 70 eV) m/z (rel. intensity) 104 (47), 123 (60), 134 (64), 161 (100), 229 (33), 258 (58, M+).

6-Methoxy-2-(N-cyanomethyl-N-n-propylamino)tetralin Hydrochloride (15b): using the general procedure, this compound was prepared from 14b·HCl.27 Yield 30%; mp 138–140 °C; IR: cm–1 2936, 2839, 2344 (b), 1611; 1H

NMR (base, 200 MHz, CDCl3): d 0.93, (t, J = 7.3 Hz, 3H), 1.46–1.78 (m, 3H), 2.07–2.17 (m, 1H), 2.65–3.03 (m, 7H), 3.66 (s, 2H), 3.77 (s, 3H), 6.63 (d, J = 2.7 Hz, 1H), 6.70 (dd, J = 8.3 Hz, 2.7 Hz, 1H), 7.00 (d, J = 8.3 Hz, 1H); 13C

NMR (base, 50 MHz, CDCl3): d 11.3, 20.7, 26.5, 28.8, 32.1, 38.4, 51.9, 55.0, 58.1, 112.0, 113.0, 116.8, 126.8, 130.1, 136.8, 157.7; MS (EI, 70 eV): m/z (rel. intensity) 91 (41), 134 (98), 161 (100), 199 (15), 229 (52), 258 (61, M+).

7-Methoxy-2-(N-cyanomethyl-N-n-propylamino)tetralin Hydrochloride (15c): this compound was synthesized from 14c·HCl1 using the general procedure. Yield 73%; mp 144–146 °C; IR: cm–1 2922, 2832, 2737, 2365 (b), 1616; 1 H NMR (base, 200 MHz, CDCl3): d 0.96 (t, J = 7.3 Hz, 3H), 1.44–1.71 (m, 3H), 2.09–2.17 (m, 1H), 2.66–3.00 (m, 13 7H), 3.64 (s, 2H), 3.77 (s, 3H), 6.64–6.74 (m, 2H), 7.00 (dd, J = 8.6 Hz, 1H); C NMR (base, 50 MHz, CDCl3): d 11.4, 20.7, 26.8, 27.8, 33.1, 38.4, 51.8, 55.0, 58.0, 112.3, 113.7, 116.9, 127.8, 129.3, 136.1, 156.7; MS (EI, 70 eV): m/z (rel. intensity) 91 (24), 134 (100), 161 (85), 218 (20), 229 (20), 258 (54, M+).

8-Methoxy-2-(N-cyanomethyl-N-n-propylamino)tetralin Hydrochloride (15d): using the general procedure, this compound was prepared from 14d·HCl.1 Yield 62%; mp 163–164 °C; IR: cm–1 2943, 2839, 2305 (b), 1584; 1H

NMR (base, 200 MHz, CDCl3): d 0.99 (t, J = 7.3 Hz, 3H), 1.48–1.75 (m, 3H), 2.13–2.20 (m, 1H), 2.53 (dd, J = 16.0 Hz, 9.6 Hz, 1H), 2.74 (dd, J = 7.2 Hz, 7.2Hz, 2H), 2.84–3.13 (m, 4H), 3.68 (s, 2H), 3.85 (s, 3H), 6.72 (dd, J = 8.4 Hz, 13 8.4 Hz, 2H), 7.14 (t, J = 7.8 Hz, 1H); C NMR (base, 50 MHz, CDCl3): d 11.4, 20.8, 26.1, 26.8, 29.0, 38.4, 52.0, 55.0, 57.9, 106.8, 116.9, 120.6, 123.7, 126.2, 137.2, 157.3; MS (EI, 70 eV): m/z (rel. intensity) 91 (16), 104 (27) 123 (20), 134 (28), 161 (100), 229 (24), 258 (47, M+).

2-(N-Cyanomethyl-N-n-propylamino)tetralin Hydrochloride (15e): using the general procedure, this compound was prepared from 14e·HCl.11 Yield 69%; mp 158–160 °C; IR: cm–1 3138, 3022, 2933, 2364 (b) 1578; 1H NMR (base,

200 MHz, CDCl3): d 0.97 (t, J = 7.3 Hz, 3H), 1.44–1.83 (m, 3H), 2.12–2.20 (m, 1H), 2.73 (dd, J = 7.5 Hz, 7.5 Hz, 13 2H), 2.82–3.09 (m, 5H), 3.69 (s, 2H), 7.14 (s, 4H); C NMR (base, 50 MHz, CDCl3): d 11.6, 20.9, 26.8, 28.8, 33.1, 38.9, 52.1, 58.1, 116.9, 125.8, 126.0, 128.6, 129.4, 135.0, 135.9; MS (EI, 70 eV): m/z (rel. intensity) 104 (22), 131 (100), 199 (18), 228 (16, M+).

67 Chapter 2

General Procedure for the Preparation of Compounds 16a–16e. The method adopted for the synthesis of 5- methoxy-2-[N-(2-aminoethyl)-N-n-propylamino]tetralin·(HCl)2 (16a) is described: a solution of the free base of 15a

(10.00 g, 38.7 mmol) in dry THF (75 mL) was added dropwise to a stirred, ice-cooled suspension of LiAlH4 (3 g) in dry THF (75 mL). When addition was complete, the reaction mixture was refluxed overnight under a nitrogen atmosphere. After cooling, excess LiAlH4 was quenched by subsequent addition of H2O (3 mL), 4N aqueous NaOH solution (3 mL) and H2O (9 mL). After drying over Na2SO4, the suspension was filtered and the filtrate was concentrated under reduced pressure, yielding 10.09 g (38.5 mmol, 99%) of the pure base of 16a as a clear yellow oil, which was converted to the dihydrochloride salt: mp 112–114 °C [lit.46 mp 110–112°C]; IR: cm–1 3406 (b), 2965, 1 2836, 2631 (b), 2520 (b), 2063 (b), 1588; H NMR (base, 200 MHz, CDCl3): d 0.88 (t, J = 7.3 Hz, 3H), 1.39–1.55 (m, 5H), 1.97–2.04 (m, 1H), 2.41–3.02 (m, 11H), 3.75 (s, 3H), 6.62 (dd, J = 15.6 Hz, 7.8 Hz, 2H), 7.04 (t, J = 7.9 Hz, 13 1H); C NMR (base, 50 MHz, CDCl3): d 11.6, 22.1, 23.7, 25.2, 32.0, 40.5, 52.3, 52.9, 54.8, 55.9, 106.6, 121.5, 125.0, 126.0, 137.9, 157.1; MS (EI, 70 eV): m/z (rel. intensity) 72 (10), 161 (50), 232 (100), 262 (1, M+).

6-Methoxy-2-[N-(2-aminoethyl)-N-n-propylamino]tetralin Dihydrochloride (16b): using the general procedure, this compound was prepared from 15b. Yield 98%; mp 113–115 °C; IR: cm–1 3414 (b), 2965, 2837, 2639 (b), 2517, 1 2055 (b), 1610; H NMR (base, 200 MHz, CDCl3):d 0.88 (t, J = 7.3 Hz, 3H), 1.40–1.69 (m, 3H), 1.95–2.15 (m, 1H), 2.43–2.95 (m, 11H), 3.75 (s, 3H), 6.61 (d, J = 2.2 Hz, 1H), 6.66 (dd, J = 8.3 Hz, 2.7 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H); 13 C NMR (base, 50 MHz, CDCl3): d 11.6, 22.1, 25.5, 30.0, 31.0, 40.4, 52.4, 52.9, 55.0, 56.5, 111.8, 113.0, 128.5, 130.1, 137.4, 157.5; MS (CI with AcOH): m/z (rel. intensity) 220 (7), 234 (14), 263 (100, M+1).

7-Methoxy-2-[N-(2-aminoethyl)-N-n-propylamino]tetralin Oxalate (16c): using the general procedure, this compound was prepared from 15c. Yield 97%; mp 168–170 °C; IR: cm–1 2937, 2836, 2670 (b), 2527 (b), 1719, 1612; 1 H NMR (base, 200 MHz, CDCl3): d 0.89 (t, J = 7.3 Hz, 3H), 1.40–1.65 (m, 3H), 1.93–2.02 (m, 3H), 2.43–2.59 (m, 13 4H), 2.67–2.96 (m, 7H), 3.75 (s, 3H), 6.61–6.69 (m, 2H), 6.95–6.99 (m, 1H); C NMR (base, 50 MHz, CDCl3): d 11.6, 22.1, 25.7, 28.9, 32.2, 40.3, 52.3, 52.8, 55.0, 56.3, 111.9, 113.7, 128.4, 129.3, 137.6, 157.4; MS (CI with AcOH): m/z (rel. intensity) 220 (7), 234 (5), 263 (100, M+1).

8-Methoxy-2-[N-(2-aminoethyl)-N-n-propylamino]tetralin Dihydrochloride (16d): using the general procedure, this compound was prepared from 15d. Yield 97%; mp 155–157 °C; IR: cm–1 3396 (b), 2939, 2837, 2637 (b), 2520 (b) 1 1587; H NMR (base, 200 MHz, CDCl3): d 0.91 (t, J = 7.3 Hz, 3H), 1.39–1.70 (m, 3H), 1.80 (bs, 2H), 1.91–2.03 (m, 1H), 2.36–3.01 (m, 11H), 3.82 (s, 3H), 6.68 (dd, J = 8.8 Hz, 8.8 Hz, 2H), 7.08 (t, J = 7.9 Hz, 1H); 13C NMR (base, 50

MHz, CDCl3): d 11.8, 22.4, 25.6, 30.3, 40.6, 52.6, 53.1, 55.1, 56.6, 106.7, 120.8, 125.3, 125.9, 137.8, 157.5; MS (CI with AcOH): m/z (rel. intensity) 220 (23), 234 (32), 263 (100, M+1).

2-[N-(2-Aminoethyl)-N-n-propylamino]tetralin Dihydrochloride (16e): using the general procedure, this compound was prepared from 15e. Yield 96%; mp 138–141 °C; IR: cm–1 3402 (b), 2965, 2643 (b), 2522 (b), 2043 (b), 1 1607; H NMR (base, 200 MHz, CDCl3): d 0.91 (t, J = 7.3 Hz, 3H), 1.39–1.68 (m, 5H), 1.96–2.06 (m, 1H), 2.46–3.01 13 (m, 11H), 7.10 (s, 4H); C NMR (base, 50 MHz, CDCl3): d 11.8, 22.3, 25.8, 29.9, 32.1, 40.7, 52.6, 53.1, 56.5, 125.6, 128.6, 129.4, 135.0, 135.5; MS (EI, 70 eV): m/z (rel. intensity) 72 (23), 131 (100), 202 (79), 232 (2, M+).

General Procedures for the Preparation of Compounds 17a–17o, 18a–18d, 19a–19d, 20a–20d and 21a–21d. Method A. The method adopted for the synthesis of 5-methoxy-2-[N-(2-benzamidoethyl)-N-n- propylamino]tetralin·HCl (17a) is described: benzoyl chloride (4.70 g, 33.4 mmol), dissolved in CH2Cl2 (25 mL), was added dropwise to a firmly stirred mixture of CH2Cl2 (225 mL), 10% aqueous NaOH solution (40 mL) and 16a

(4.00 g, 11.9 mmol). After stirring overnight at room temperature, the reaction mixture was poured into H2O (50 mL) and the phases were separated. The aqueous layer was extracted with CH2Cl2 (2 ´ 100 mL), the organic layers were combined and subsequently washed with saturated aqueous NaHCO3 solution (3 ´ 100 mL), H2O (100 mL) and brine

(100 mL). The organic layer was dried (Na2SO4), filtered and concentrated under reduced pressure, which gave the crude amide as a brown oil. Purification by column chromatography [eluent: MeOH/CH2Cl2, 1/15 (v/v)] yielded 1.68 g (4.6 mmol, 39%) of the pure base of 17a as a colourless oil, which was converted to the hydrochloride salt: mp 91–93

68 2-Aminotetralin-derived Substituted Benzamides

–1 1 °C; IR: cm 3267, 2938, 2836, 2611 (b), 2517 (b), 1654, 1588, 1540; H NMR (base, 200 MHz, CDCl3): d 0.92 (t, J = 7.3 Hz, 3H), 1.42–1.71 (m, 3H), 1.99–2.08 (m, 1H), 2.45–2.63 (m, 3H), 2.75–2.86 (m, 4H), 2.93–3.07 (m, 2H), 3.45– 3.55 (m, 2H), 3.80 (s, 3H), 6.67 (dd, J = 7.4 Hz, 7.4 Hz, 2H), 7.06–7.13 (m, 2H), 7.40–7.52 (m, 3H), 7.78–7.83 (m, 13 2H); C NMR (base, 50 MHz, CDCl3): d 11.6, 21.8, 23.5, 25.3, 31.9, 37.6, 48.3, 51.9, 55.0, 55.6, 106.8, 121.4, 124.9,

126.1, 126.7, 128.4, 131.1, 134.6, 137.3, 157.1, 167.1; MS (CI with NH3): m/z (rel. intensity) 78 (6), 161 (2), 232 (8),

367 (100, M+1); Anal. calcd for C23H30N2O2·HCl·¼H2O: C 67.78, H 7.94, N 6.70; obsd C 67.76, H 8.02, N 6.77.

Method B. The method adopted for the synthesis of 5-methoxy-2-{N-[2-(2,3-dimethoxy)benzamidoethyl]-N-n- propylamino}tetralin·HCl (17c) is described: a solution of 2,3-dimethoxybenzoyl chloride (0.72 g, 3.6 mmol) in

CDCl3 (10 mL) was added dropwise to a boiling solution of the free base of 16a (0.38 g, 1.4 mmol) in CDCl3 (15 mL). When addition was complete, the reaction mixture was allowed to cool to room temperature and stirring was continued overnight. The reaction mixture was transferred to a separatory funnel and subsequently washed with saturated NaHCO3 solution (3 ´ 20 mL), H2O (20 mL) and brine (20 mL). After drying (Na2SO4) and filtration, the organic layer was concentrated under reduced pressure, which gave the crude amide as a brown oil. Purification by column chromatography [eluent: MeOH/CH2Cl2, 1/20 (v/v)] yielded 0.28 g (0.7 mmol, 46%) of the pure base of 17c as a colourless oil, which was converted to the hydrochloride salt: mp 113–116 °C; IR: cm–1 3420 (b), 2981, 2838, 2667 1 (b), 2519 (b), 1660, 1611, 1538; H NMR (base, 200 MHz, CDCl3): d 0.89 (t, J = 7.3 Hz, 3H), 1.41–1.69 (m, 3H), 2.02–2.12 (m, 1H), 2.43–2.61 (m, 3H), 2.74–3.06 (m, 6H), 3.51–3.59 (m, 2H), 3.80 (s, 3H), 3.91 (s, 6H), 6.67 (dd, J = 8.5 Hz, 8.5 Hz, 2H), 7.01–7.19 (m, 3H), 7.71 (dd, J = 7.8 Hz, 1.7 Hz, 1H), 8.40 (bs, 1H); 13C NMR (base, 50 MHz,

CDCl3): d 11.6, 22.1, 23.6, 25.3, 31.7, 38.2, 48.7, 52.1, 55.0, 55.8, 56.1, 61.1, 106.7, 114.9, 121.5, 122.7, 124.1,

125.0, 126.0, 126.9, 137.7, 147.5, 152.5, 157.1, 165.0; MS (CI with NH3): m/z (rel. intensity) 182 (6), 232 (6) 427

(100, M+1); Anal. calcd for C25H34N2O4·HCl·H2O: C 62.41, H 7.77, N 5.82; obsd C 62.72, H 7.81, N 5.95.

Method C. The method adopted for the synthesis of 5-methoxy-2-{N-[2-(4-amino-5-chloro-2- methoxy)benzamidoethyl]-N-n-propylamino}tetralin·HCl (17k) is described: ethyl chloroformate (0.08 g, 0.7 mmol) was added to a stirred solution of 4-amino-5-chloro-2-methoxybenzoic acid (0.15 g, 0.7 mmol) and triethylamine (0.08 g, 0.7 mmol) in acetone (5 mL) at 0 °C. Stirring was continued for 1 h at 0 °C, upon which a white solid precipitated. Then 16a (0.25 g, 0.7 mmol) was added, immediately followed by a second amount of triethylamine (0.10 g, 1.0 mmol). Stirring was again continued for 1 h at 0 °C, and then the precipitate was removed from the reaction mixture by filtration. The filtrate was concentrated under reduced pressure, the resulting residue was dissolved in CH2Cl2 (50 mL) and the organic solution was subsequently washed with saturated aqueous NaHCO3 solution (3 ´ 25 mL), H2O (25 mL) and brine (25 mL). After drying (Na2SO4) and filtration, the organic solution was concentrated in vacuo, which yielded the crude amide as a light brown oil. After purification by column chromatography [eluent: MeOH/CH2Cl2, 1/15 (v/v)], 0.15 g (0.3 mmol, 43%) of the pure base of 17k was obtained as a colourless oil, which was converted to the hydrochloride salt: mp 135–137 °C; IR: cm–1 3394, 3319, 3204, 2940, 1 2835, 2434 (b), 1637, 1589, 1534; H NMR (base, 200 MHz, CDCl3): d 0.88 (t, J = 7.3 Hz, 3H), 1.40–1.68 (m, 3H), 2.02–2.11 (m, 1H), 2.42–2.58 (m, 3H), 2.66–2.81 (m, 4H), 2.94–3.05 (m, 2H), 3.46–3.52 (m, 2H), 3.79 (s, 3H), 3.87 (s, 3H), 4.48 (bs, 2H), 6.32 (s, 1H), 6.66 (dd, J = 6.7 Hz, 6.7 Hz, 2H), 7.07 (t, J = 7.8 Hz, 1H), 8.11 (s, 1H), 8.24 (bs, 13 1H); C NMR (base, 50 MHz, CDCl3): d 11.5, 21.8, 23.6, 25.1, 31.7, 38.0, 48.6, 52.1, 55.0, 55.7, 55.9, 97.6, 106.8,

111.2, 112.4, 121.4, 124.9, 126.1, 132.9, 137.5, 146.5, 157.1, 157.5, 164.4; MS (CI with NH3): m/z (rel. intensity) 72

(17), 161 (18), 201 (14), 220 (21), 232 (15), 446 (100, M+1); Anal. calcd for C24H31N3O3·HCl: C 64.62, H 7.25, N 9.42; obsd C 64.38, H 7.32, N 9.41.

5-Methoxy-2-{N-[2-(2-methoxy)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (17b): method B. Yield 92%; mp 88–92 °C; IR: cm–1 3335 (b), 2939, 2837, 2593 (b), 2499 (b), 1640, 1597, 1523; 1H NMR (base, 200

MHz, CDCl3): d 0.92 (t, J = 7.3 Hz, 3H), 1.43–1.71 (m, 3H), 2.02–2.12 (m, 1H), 2.46–3.10 (m, 9H), 3.53–3.61 (m, 2H), 3.81 (s, 3H), 3.98 (s, 3H), 6.68 (dd, J = 7.3 Hz, 7.3 Hz, 2H), 6.97–7.14 (m, 3H), 7.41–7.50 (m, 1H), 8.26 (dd, J = 13 7.9 Hz, 1.9 Hz, 1H), 8.43 (bs, 1H); C NMR (base, 50 MHz, CDCl3): d 11.8, 22.3, 23.9, 25.5, 32.1, 38.4, 48.7, 52.3, 55.2, 55.7, 56.1, 106.9, 111.2, 121.1, 121.5, 121.8, 125.1, 126.2, 132.2, 132.5, 137.8, 157.2, 157.6, 165.1; MS (CI

69 Chapter 2

with NH3): m/z (rel. intensity) 72 (21), 135 (41), 161 (62), 178 (14), 232 (100), 397 (38, M+1); Anal. calcd for

C24H32N2O3·HCl·½H2O: C 65.21, H 7.77, N 6.34; obsd C 65.05, H 7.82, N 6.35.

5-Methoxy-2-{N-[2-(2,6-dimethoxy)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (17d): method A. Yield 59%; mp 113–116 °C; IR: cm–1 3400 (b), 2939, 2837, 2630 (b), 2519 (b), 1655, 1595, 1524; 1H NMR (base,

200 MHz, CDCl3): d 0.86 (t, J = 7.3 Hz, 3H), 1.41–1.61 (m, 3H), 1.99–2.05 (m, 1H), 2.43–2.55 (m, 3H), 2.73–2.88 (m, 4H), 2.94–3.05 (m, 2H), 3.49–3.56 (m, 2H), 3.80 (s, 9H), 6.43 (bs, 1H), 6.56 (d, J = 8.6 Hz, 2H), 6.66 (dd, J = 7.1 13 Hz, 7.1 Hz, 2H), 7.08 (t, J = 7.8 Hz, 1H), 7.28 (t, J = 7.4 Hz, 1H); C NMR (base, 50 MHz, CDCl3): d 11.6, 21.8, 23.6, 25.2, 31.8, 37.6, 48.7, 51.9, 55.0, 55.6, 55.8, 103.7, 106.8, 116.1, 121.4, 124.9, 126.1, 130.3, 137.7, 157.1,

157.3, 165.6; MS (CI with NH3): m/z (rel. intensity) 232 (6), 427 (100, M+1); Anal. calcd for C25H34N2O4·HCl·½H2O: C 63.60, H 7.70, N 5.94; obsd C 63.66, H 7.86, N 5.93.

5-Methoxy-2-{N-[2-(5-bromo-2-methoxy)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (17e): method A. Yield 49%; mp 108–110 °C; IR: cm–1 3328 (b), 2938, 2837, 2596 (b), 2478 (b), 1649, 1590, 1522; 1H

NMR (base, 300 MHz, CDCl3): d 0.91 (t, J = 7.3 Hz, 3H), 1.56–1.70 (m, 3H), 2.11–2.13 (m, 1H), 2.47–2.66 (m, 3H), 2.82–3.13 (m, 6H), 3.55–3.62 (m, 2H), 3.79 (s, 3H), 3.96 (s, 3H), 6.66 (dd, J = 7.9 Hz, 3.8 Hz, 2H), 6.87 (d, J = 8.8 Hz, 1H), 7.08 (t, J = 7.9 Hz, 1H), 7.52 (dd, J = 8.8 Hz, 2.9 Hz, 1H), 8.30 (d, J = 2.6 Hz, 1H), 8.45 (bs, 1H); 13C NMR

(base, 75 MHz, CDCl3): d 11.6, 23.5, 38.0, 49.0, 55.1, 56.0, 107.0, 113.1, 113.6, 121.3, 123.3, 124.7, 126.3, 129.2,

134.7, 135.0, 156.6, 157.1, 163.8; MS (CI with NH3): m/z (rel. intensity) 72 (19), 161 (27), 232 (58), 475 (100,

M[Br=79]+1), 477 (100, M[Br=81]+1); Anal. calcd for C24H31N2O3Br·HCl·H2O: C 54.39, H 6.48, N 5.29; obsd C 54.57, H 6.48, N 5.44.

5-Methoxy-2-{N-[2-(5-iodo-2-methoxy)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (17f): method A. Yield 46%; mp 110–112 °C; IR: cm–1 3337 (b), 2937, 2837, 2598 (b), 2502 (b), 1644, 1586, 1523; 1H

NMR (base, 200 MHz, CDCl3): d 0.90 (t, J = 7.3 Hz, 3H), 1.45–1.68 (m, 3H), 2.03–2.08 (m, 1H), 2.47–2.59 (m, 3H), 2.72–2.83 (m, 4H), 2.97–3.05 (m, 2H), 3.51–3.57 (m, 2H), 3.80 (s, 3H), 3.94 (s, 3H), 6.66 (dd, J = 7.3 Hz, 7.3 Hz, 2H), 6.75 (d, J = 8.8 Hz, 1H), 7.08 (t, J = 7.8 Hz, 1H), 7.70 (dd, J = 8.8 Hz, 2.6 Hz, 1H), 8.32 (bs, 1H), 8.49 (d, J = 13 2.6 Hz, 1H); C NMR (base, 50 MHz, CDCl3): d 11.6, 21.9, 23.7, 25.3, 31.9, 38.2, 48.7, 52.2, 55.1, 55.8, 56.2, 106.9,

107.7, 113.5, 121.4, 123.8, 124.9, 126.1, 128.5, 140.6, 140.8, 157.1, 157.2, 163.5; MS (CI with NH3): m/z (rel. intensity) 161 (4), 232 (10), 523 (100, M+1); Anal. calcd for C24H31N2O3I·HCl·H2O: C 49.96, H 5.95, N 4.86; obsd C 49.96, H 5.82, N 4.86.

5-Methoxy-2-{N-[2-(5-bromo-2-hydroxy)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (17g): method B. Yield 15%; mp 106–109 °C; IR: cm–1 3225 (b), 2937, 2837, 2608 (b), 2508 (b), 1638, 1589, 1542; 1H

NMR (200 MHz, CD3OD): d 1.02 (t, J = 7.3 Hz, 3H), 1.74–1.95 (m, 3H), 2.31 (bs, 1H), 2.55–2.68 (m, 1H), 3.01–3.42 (m, 9H), 3.49–3.62 (m, 2H), 3.75 (s, 3H), 6.71 (m, 2H), 6.85 (d, J = 8.8 Hz, 1H), 7.06 (t, J = 7.9 Hz, 1H), 7.47 (dd, J 13 = 8.8 Hz, 2.6 Hz, 1H), 7.94 (d, J = 2.2 Hz, 1H); C NMR (base, 50 MHz, CDCl3): d 11.3, 17.7, 22.6, 23.8, 28.6, 35.7, 51.5, 54.5, 55.3, 60.6, 108.0, 110.9, 115.3, 119.9, 121.1, 124.5, 127.3, 130.0, 132.4, 137.2, 157.1, 160.5, 169.8; MS

(CI with NH3): m/z (rel. intensity) 72 (24), 91 (10), 161 (76), 232 (100), 461 (17, M[Br=79]+1), 463 (17,

M[Br=81]+1); Anal. calcd for C23H29N2O3Br·HCl·½H2O: C 54.49, H 6.18, N 5.53; obsd C 54.74, H 6.04, N 5.57.

5-Methoxy-2-{N-[2-(2-hydroxy-5-iodo)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (17h): method B. Yield 68%; mp 102–105 °C; IR: cm–1 3414 (b), 3216 (b), 2939, 2836, 2610 (b), 2510 (b), 1637, 1588, 1 1542; H NMR (300 MHz, CD3OD): d 1.06 (t, J = 7.1 Hz, 3H), 1.83–1.95 (m, 3H), 2.33–2.38 (m, 1H), 2.62–2.70 (m, 1H), 3.07–3.43 (m, 7H), 3.61–3.64 (m, 1H), 3.80 (s, 3H), 3.81–3.83 (m, 2H), 6.69–6.77 (m, 3H), 7.11 (t, J = 8.1 Hz, 13 1H), 7.68 (dd, J = 8.5 Hz, 2.2 Hz, 1H), 8.15 (d, J = 2.0 Hz); C NMR (300 MHz, CD3OD): d 11.3, 19.5, 23.6, 24.6, 30.6, 37.3, 52.2, 54.4, 55.8, 61.9, 109.0, 119.5, 120.6, 122.3, 124.5, 128.2, 134.5, 138.8, 143.7, 158.5, 159.8, 170.4;

MS (CI with NH3): m/z (rel. intensity) 180 (16), 223 (12), 383 (100), 509 (7, M+1); Anal. calcd for C23H29N2O3I·HCl: C 50.69, H 5.56, N 5.14; obsd C 50.74, H 5.69, N 5.21.

70 2-Aminotetralin-derived Substituted Benzamides

5-Methoxy-2-{N-[2-(2-methoxy-5-sulfamoyl)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (17i): method C. Yield 32%; mp 177–178 °C; IR: cm–1 3317, 3188 (b), 3072 (b), 2941, 2841, 2485 (b), 2363, 1632, 1591, 1 1528; H NMR (300 MHz, CD3OD): d 1.07 (t, J = 7.6 Hz, 3H), 1.87–1.96 (m, 3H), 2.34–2.42 (m, 1H), 2.62–2.70 (m, 1H), 3.07–3.45 (m, 6H), 3.63–3.69 (m, 1H), 3.80 (s, 3H), 3.83–3.89 (m, 3H), 4.05 (s, 3H), 6.68–6.77 (m, 2H), 7.11 (t, J = 7.3 Hz, 1H), 7.31 (d, J = 8.8 Hz, 1H), 8.04 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 8.50 (d, J = 2.0 Hz, 1H); 13C NMR (75

MHz, CD3OD): d 10.6, 18.8, 22.9, 23.8, 30.0, 37.3, 52.0, 53.9, 55.1, 56.7, 61.1, 108.3, 112.9, 121.3, 121.6, 123.9, 127.5, 130.1, 132.1, 133.9, 136.9, 157.8, 161.0, 167.8; MS (EI, 70 eV): m/z (rel. intensity) 86 (68), 161 (100), 204 + (49), 232 (71), 355 (7), 377 (9), 432 (4), 461 (7), 475 (13, M ); Anal. calcd for C24H34N3O5S·HCl: C 56.28, H 6.71, N 8.21; obsd C 55.96, H 6.84, N 8.06.

5-Methoxy-2-{N-[2-(5-bromo-2,6-dimethoxy)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (17j): method B. Yield 36%; mp 193–195 °C; IR: cm–1 3183, 2940, 2835, 2575 (b), 1661, 1587, 1541; 1H NMR (base,

200 MHz, CDCl3): d 0.85 (t, J = 7.3 Hz, 3H), 1.40–1.64 (m, 3H), 1.96–2.05 (m, 1H), 2.24–3.04 (m, 9H), 3.47–3.57 (m, 2H), 3.79 (s, 3H), 3.80 (s, 3H), 3.88 (s, 3H), 6.45 (bs, 1H), 6.58–6.70 (m, 3H), 7.10 (t, J = 7.8 Hz, 1H), 7.49 (d, J 13 = 8.8 Hz, 1H); C NMR (base, 50 MHz, CDCl3): d 11.5, 21.6, 23.5, 25.1, 31.7, 37.8, 48.5, 52.0, 55.0, 55.8, 62.2, 106.8, 108.0, 108.2, 121.4, 123.0, 124.9, 126.1, 133.7, 137.5, 154.6, 156.5, 157.1, 164.4; MS (CI with AcOH): m/z

(rel. intensity) 506 (94, M[Br=79]+1), 508 (100, M[Br=81]+1); Anal. calcd for C25H33N2O4Br·HCl·¼H2O: C 54.94, H 6.38, N 5.13; obsd C 54.77, H 6.27, N 5.14.

5-Methoxy-2-[N-(2-thiophen-2-carboxamidoethyl)-N-n-propylamino]tetralin Hydrochloride (17l): method A. Yield 62%; mp 102–105 °C; IR: cm–1 3238 (b), 2938, 2836, 2608 (b), 2497 (b), 1640, 1588, 1543; 1H NMR (300

MHz, CD3OD): d 1.00 (m, 3H), 1.84–2.18 (m, 3H), 2.52–2.65 (m, 2H), 2.96–3.04 (m, 2H), 3.08–3.47 (m, 4H), 3.61– 3.70 (m, 2H), 3.79 (s, 3H), 3.89–3.99 (m, 2H), 6.62 (dd, J = 33.5 Hz, 7.5 Hz, 1H), 6.75 (dd, J = 8.1 Hz, 2.20 Hz, 1H), 13 7.04–7.18 (m, 2H), 7.66–7.70 (m, 1H), 8.18 (dd, J = 7.3 Hz, 3.7 Hz, 1H); C NMR (50 MHz, CDCl3): d 11.0, 17.4, 22.4, 28.4, 30.0, 35.8, 51.5, 54.4, 55.0, 60.6, 107.7, 121.0, 123.1, 127.0, 128.1, 129.4, 130.5, 132.6, 138.3, 156.9,

162.7; MS (CI with NH3): m/z (rel. intensity) 161 (3), 232 (6), 373 (100, M+1); Anal. calcd for

C21H28N2O2S·HCl·¼H2O: C 60.99, H 7.20, N 6.78; obsd C 60.69, H 7.24, N 6.76.

5-Methoxy-2-[N-(2-thiophen-3-carboxamidoethyl)-N-n-propylamino]tetralin Hydrochloride (17m): method B. Yield 20%; mp 93–95 °C; IR: cm–1 3232 (b), 3061, 2938, 2835, 2479 (b), 1653, 1588, 1543; 1H NMR (base, 200

MHz, CDCl3): d 0.92 (t, J = 7.3 Hz, 3H), 1.46–1.66 (m, 3H), 2.00–2.05 (m, 1H), 2.55 (dd, J = 7.4 Hz, 7.4 Hz, 3H), 2.74–2.83 (m, 4H), 2.94–3.06 (m, 2H), 3.44–3.52 (m, 2H), 3.81 (s, 3H), 6.68 (dd, J = 6.7 Hz, 6.7 Hz, 2H), 6.94 (bs, 13 1H), 7.10 (t, J = 7.9 Hz, 1H), 7.32–7.41 (m, 2H), 7.85 (s, 1H); C NMR (base, 50 MHz, CDCl3): d 11.6, 21.6, 23.4, 25.2, 31.9, 37.3, 48.3, 51.9, 55.0, 55.7, 106.9, 121.4, 124.8, 125.8, 126.2, 126.3, 127.8, 137.2, 157.1, 162.8; MS (CI with AcOH): m/z 373 (M+1); Anal. calcd for C21H28N2O2S·HCl·¼H2O: C 60.99, H 7.20, N 6.78; obsd C 60.67, H 7.33, N 6.68.

5-Methoxy-2-[N-(2-naphthalen-1-carboxamidoethyl)-N-n-propylamino]tetralin Hydrochloride (17n): method B. Yield 45%; mp 152–153 °C; IR: cm–1 3232 (b), 2939, 2835, 2512 (b), 1655, 1588, 1522; 1H NMR (base, 200 MHz,

CDCl3): d 0.88 (t, J = 7.3 Hz, 3H), 1.43–1.62 (m, 3H), 1.98–2.06 (m, 1H), 2.54 (dd, J = 7.3 Hz, 7.3 Hz, 3H), 2.75– 2.79 (m, 4H), 2.89–3.08 (m, 2H), 3.50–3.61 (m, 2H), 3.81 (s, 3H), 6.65 (dd, J = 7.8 Hz, 4.2 Hz, 2H), 6.91 (bs, 1H), 7.11 (t, J = 7.8 Hz, 1H), 7.42–7.64 (m, 4H), 7.89 (dd, J = 7.8 Hz, 7.8 Hz, 2H), 8.39–8.44 (m, 1H); 13C NMR (base, 50

MHz, CDCl3): d 11.7, 21.9, 23.6, 25.2, 31.9, 38.0, 48.5, 52.1, 55.0, 55.8, 106.9, 121.5, 124.7, 124.8, 124.9, 125.5, 126.2, 126.8, 128.2, 130.1, 130.3, 133.6, 134.7, 137.5, 157.1, 169.4; MS (CI with AcOH): m/z 417 (M+1); Anal. calcd for C27H32N2O2·HCl·½H2O: C 70.18, H 7.43, N 6.06; obsd C 70.15, H 7.17, N 6.15.

5-methoxy-2-[N-(2-naphthalen-2-carboxamidoethyl)-N-n-propylamino]tetralin Hydrochloride (17o): method B. Yield 35%; mp 117–119 °C; IR: cm–1 3246, 2938, 2835, 2479 (b), 1653, 1588, 1534; 1H NMR (base, 200 MHz,

CDCl3): 0.92 (t, J = 7.3 Hz, 3H), 1.44–1.69 (m, 3H), 2.04–2.09 (m, 1H), 2.58 (dd, J = 7.3 Hz, 7.3 Hz, 3H), 2.79–2.83 (m, 4H), 2.95–3.05 (m, 2H), 3.51–3.61 (m, 2H), 3.78 (s, 3H), 6.65 (t, J = 7.5 Hz, 2H), 7.07 (t, J = 7.8 Hz, 1H), 7.32

71 Chapter 2

13 (bs, 1H), 7.49–7.59 (m, 2H), 7.82–7.94 (m, 4H), 8.34 (s, 1H); C NMR (base, 50 MHz, CDCl3): d 11.6, 21.6, 23.5, 25.1, 31.8, 37.7, 48.4, 52.1, 55.0, 55.9, 106.9, 121.4, 123.4, 124.8, 126.2, 126.5, 127.3, 127.4, 127.6, 128.3, 128.9,

131.8, 132.6, 134.5, 137.1, 157.1, 167.2; MS (CI with AcOH): m/z 417 (M+1); Anal. calcd for C27H32N2O2·HCl·H2O: C 68.84, H 7.50, N 5.95; obsd C 68.66, H 7.56, N 6.08. 6-Methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin Hydrochloride (18a): method A. Yield 77%; –1 1 mp 76–78 °C; IR: cm 3245 (b), 2938, 2835, 2479 (b), 1654, 1610, 1578, 1534; H NMR (base, 200 MHz, CDCl3): d 0.91 (t, J = 7.3 Hz, 3H), 1.45–1.63 (m, 3H), 1.95–2.18 (m, 1H), 2.54 (dd, J = 7.3 Hz, 7.3 Hz, 2H), 2.70–3.09 (m, 7H), 3.47–3.58 (m, 2H), 3.75 (s, 3H), 6.61–6.71 (m, 2H), 6.95–7.15 (m, 2H), 7.38–7.49 (m, 3H), 7.79–7.83 (m, 2H); 13C

NMR (base, 50 MHz, CDCl3): d 11.6, 21.7, 25.5, 29.7, 31.0, 37.7, 48.4, 52.0, 55.0, 56.3, 111.9, 113.0, 126.7, 130.1, 131.2, 134.6, 137.1, 157.6, 167.1; MS (EI, 70 eV): m/z (rel. intensity) 77 (28), 105 (36), 161 (100), 232 (83), 366 (2, + M ); Anal. calcd for C23H30N2O2·HCl·¼H2O: C 67.78, H 7.81, N 6.88; obsd C 67.69, H 8.13, N 6.77.

6-Methoxy-2-{N-[2-(2,3-dimethoxy)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (18b): method A. Yield 37%; mp 83–85 °C; IR: cm–1 3353 (b), 2937, 2835, 2465 (b), 1647, 1611, 1578, 1505; 1H NMR (base, 200

MHz, CDCl3): d 0.88 (t, J = 7.3 Hz, 3H), 1.40–1.72 (m, 3H), 1.98–2.16 (m, 1H), 2.49–2.60 (m, 2H), 2.68–3.04 (m, 7H), 3.50 3.58 (m, 2H), 3.74 (s, 3H), 3.89 (s, 3H), 3.90 (s, 3H), 6.59–6.69 (m, 2H), 6.94–7.17 (m, 3H), 7.70 (dd, J = 13 7.0 Hz, 1.7 Hz, 1H), 8.38 (bs, 1H); C NMR (base, 50 MHz, CDCl3): d 11.6, 22.1, 25.6, 29.9, 30.8, 38.3, 48.8, 52.2, 55.0, 55.8, 56.7, 61.1, 111.8, 113.0, 114.9, 122.6, 124.1, 126.9, 128.2, 130.1, 137.3, 147.5, 152.5, 157.5, 165.0; MS (EI, 70 eV): m/z (rel. intensity) 91 (12), 122 (9), 161 (100), 202 (7), 232 (98), 383 (2), 426 (2, M+); Anal. calcd for

C25H34N2O4·HCl·¼H2O: C 64.21, H 7.76, N 5.99; obsd C 64.07, H 7.74, N 5.86.

6-Methoxy-2-{N-[2-(2,6-dimethoxy)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (18c): method A. Yield 76%; mp 83–85 °C; IR: cm–1 3240 (b), 2939, 2838, 2606 (b), 2499 (b), 1655, 1596, 1503; 1H NMR (300

MHz, CD3OD): d 1.10 (t, J = 7.3 Hz, 3H), 1.88–2.00 (m, 3H), 2.35–2.37 (m, 1H), 2.98–3.27 (m, 5H), 3.36–3.44 (m, 2H), 3.60–3.65 (m 1H), 3.75 (s, 3H), 3.77 (s, 3H), 3.80 (s, 3H), 3.88–3.91 (m, 1H), 6.69–6.76 (m, 4H), 7.08 (d, J = 13 8.3 Hz, 1H), 7.35–7.39 (m, 1H); C NMR (75 MHz, CD3OD): d 11.3, 19.7, 24.5, 29.5, 37.9, 53.7, 54.3, 55.7, 56.4, 61.9, 105.1, 114.1, 115.1, 125.3, 131.2, 132.8, 137.1, 158.6, 159.9, 172.1; MS (EI, 70 eV): m/z (rel. intensity) 91 (18), + 161 (100), 232 (98), 267 (10), 383 (5), 426 (3, M ); Anal. calcd for C25H34N2O4·HCl·½H2O: C 63.60, H 7.70, N 5.94; obsd C 63.43, H 7.77, N 5.84.

6-Methoxy-2-[N-(2-thiophen-2-carboxamidoethyl)-N-n-propylamino]tetralin Hydrochloride (18d): method B. Yield 36%; mp 88–90 °C; IR: cm–1 3244 (b), 3059, 2937, 2837, 2609 (b), 2486 (b), 1640, 1611, 1543; 1H NMR (base,

200 MHz, CDCl3): d 0.91 (t, J = 7.3 Hz, 3H), 1.42–1.74 (m, 3H), 1.96–2.03 (m, 1H), 2.54 (dd, J = 8.2 Hz, 6.5 Hz, 2H), 2.63–3.09 (m, 7H), 3.39–3.51 (m, 2H), 3.76 (s, 3H), 6.60–6.71 (m, 2H), 6.95–7.10 (m, 3H), 7.43–7.53 (m, 2H); 13 C NMR (base, 50 MHz, CDCl3): d 11.6, 21.6, 25.4, 29.7, 30.9, 37.4, 48.2, 52.0, 55.0, 56.2, 111.9, 113.0, 127.5, 127.7, 127.8, 129.4, 130.1, 137.1, 139.2, 157.6, 161.7; MS (EI, 70 eV): m/z (rel. intensity) 72 (24), 111 (27), 161 + (100), 202 (14), 232 (63), 372 (3, M ); Anal. calcd for C21H28N2O2S·HCl·¼H2O: C 60.99, H 7.20, N 6.78; obsd C 61.01, H 7.04, N 6.73.

7-Methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin Hydrochloride (19a): method A. Yield 45%; mp 70–72 °C; IR: cm–1 3252 (b), 2937, 2836, 2590 (b), 2486 (b), 1655, 1611, 1578, 1533; 1H NMR (base, 200 MHz,

CDCl3): d 0.93 (t, J = 7.5 Hz, 3H), 1.44–1.76 (m, 3H), 1.97–2.07 (m, 1H), 2.57 (dd, J = 8.1 Hz, 6.4 Hz, 2H), 2.77– 2.83 (m, 6H), 3.00–3.11 (m, 1H), 3.46–3.58 (m, 2H), 3.77 (s, 3H), 6.61 (d, J = 2.6 Hz, 1H), 6.70 (dd, J = 8.4 Hz, 2.6 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 7.04 (bs, 1H), 7.41–7.52 (m, 3H), 7.79–7.84 (m, 2H); 13C NMR (base, 50 MHz,

CDCl3): d 11.8, 22.0, 26.0, 28.9, 32.3, 37.8, 48.6, 52.2, 55.2, 56.3, 112.2, 113.9, 126.8, 128.3, 128.5, 129.5, 131.3,

134.8, 137.3, 157.6, 167.2; MS (CI with NH3): m/z 367 (M+1); Anal. calcd for C23H30N2O2·HCl·½H2O: C 67.04, H 7.84, N 6.80; obsd C 67.06, H 7.89, N 6.78.

7-Methoxy-2-{N-[2-(2,3-dimethoxy)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (19b): method A. Yield 37%; mp 88–90 °C; IR: cm–1 3345 (b), 2938, 2835, 2461 (b), 1649, 1587, 1516; 1H NMR (200 MHz,

72 2-Aminotetralin-derived Substituted Benzamides

CD3OD): d 1.07 (t, J = 7.3 Hz, 3H), 1.85–2.03 (m, 3H), 2.27–2.47 (m, 1H), 2.56–2.77 (m, 1H), 3.03–3.70 (m, 8H), 3.80 (s, 3H), 3.85–3.88 (m, 2H), 3.90 (s, 3H), 3.92 (s, 3H), 6.68–6.79 (m, 2H), 7.09–7.26 (m, 3H), 7.37–7.45 (m, 1H); 13 C NMR (50 MHz, CD3OD): d 11.0, 19.1, 23.2, 24.0, 30.5, 37.7, 52.8, 54.3, 55.3, 56.2, 61.3, 61.6, 108.7, 117.2,

122.0, 122.2, 124.4, 125.1, 127.9, 134.5, 158.3; MS (CI with NH3): m/z (rel. intensity) 232 (6), 392 (4), 427 (100,

M+1); Anal. calcd for C25H34N2O4·HCl·½H2O: C 63.60, H 7.70, N 5.94; obsd C 63.76, H 7.74, N 5.91.

7-Methoxy-2-{N-[2-(2,6-dimethoxy)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (19c): method A. Yield 41%; mp 114–116 °C; IR: cm–1 3252 (b), 2939, 2837, 2490 (b), 1657, 1595, 1505; 1H NMR (base, 200 MHz,

CDCl3): d 0.85 (t, J = 7.3 Hz, 3H), 1.41–1.65 (m, 3H), 1.93–2.00 (m, 1H), 2.51 (dd, J = 7.4 Hz, 7.4 Hz, 2H), 2.73– 3.01 (m, 7H), 3.44–3.58 (m, 2H), 3.75 (s, 3H), 3.78 (s, 6H), 6.43 (bs, 1H), 6.54–6.69 (m, 4H), 6.97 (d, J = 8.3 Hz, 13 1H), 7.26 (t, J = 8.4 Hz, 1H); C NMR (base, 50 MHz, CDCl3): d 11.5, 21.7, 25.6, 28.7, 31.9, 37.6, 48.9, 52.0, 55.0,

55.6, 56.3, 103.7, 111.9, 113.8, 116.0, 128.2, 129.3, 130.3, 137.2, 157.3, 157.4, 165.7; MS (CI with NH3): m/z (rel. intensity) 161 (14), 182 (27), 232 (26), 267 (14), 427 (100, M+1); Anal. calcd for C25H34N2O4·HCl·¾H2O: C 63.00, H 7.74, N 5.88; obsd C 62.80, H 7.73, N 5.86.

7-Methoxy-2-[N-(2-thiophen-2-carboxamidoethyl)-N-n-propylamino]tetralin Hydrochloride (19d): method A. Yield 27%; mp 80–83 °C; IR: cm–1 3238 (b), 2937, 2837, 2606 (b), 2499 (b), 1638, 1611, 1543; 1H NMR (base, 200

MHz, CDCl3): d 0.94 (t, J = 7.3 Hz, 3H), 1.43–1.75 (m, 3H), 1.96–2.08 (m, 1H), 2.54 (dd, J = 8.1 Hz, 6.4 Hz, 2H), 2.69–2.86 (m, 6H), 2.94–3.05 (m, 1H), 3.42–3.52 (m, 2H), 3.77 (s, 3H), 6.62 (d, J = 2.6 Hz, 1H), 6.70 (dd, J = 8.1 Hz, 2.6 Hz, 1H), 6.89 (bs, 1H), 7.00 (d, J = 8.1 Hz), 7.08–7.12 (m, 1H), 7.45–7.52 (m, 2H); 13C NMR (base, 50 MHz,

CDCl3): d 11.8, 22.1, 26.0, 28.9, 32.4, 37.7, 48.4, 52.0, 55.2, 56.1, 112.1, 113.9, 127.6, 127.9, 128.3, 129.5, 137.2,

157.6, 161.8; MS (CI with NH3): m/z 373 (M+1); Anal. calcd for C21H28N2O2S·HCl·½H2O: C 60.33, H 7.25, N 6.70; obsd C 60.19, H 7.17, N 6.68.

8-Methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin Hydrochloride (20a): method A. Yield 48%; –1 1 mp 107–109 °C; IR: cm 3276 (b), 2981, 2840, 2480 (b), 1659, 1596, 1542; H NMR (base, 200 MHz, CDCl3): d 0.92 (t, J = 7.3 Hz, 3H), 1.43–1.73 (m, 3H), 1.95–2.02 (m, 1H), 2.42–2.62 (m, 3H), 2.73–3.08 (m, 6H), 3.40–3.62 (m, 2H), 3.81 (s, 3H), 6.70 (dd, J = 10.1 Hz, 7.9 Hz, 2H), 7.07–7.14 (m, 2H), 7.41–7.56 (m, 3H), 7.82–7.88 (m, 2H); 13C

NMR (base, 50 MHz, CDCl3): d 11.6, 22.0, 25.2, 25.7, 29.9, 37.6, 48.2, 52.0, 55.0, 55.9, 106.7, 120.7, 124.7, 126.0, 126.8, 128.4, 131.1, 134.7, 137.5, 157.4, 167.1; MS (CI with AcOH): m/z 367 (M+1); Anal. calcd for

C23H30N2O2·HCl·1½H2O: C 64.92, H 7.95, N 6.58; obsd C 65.19, H 7.99, N 6.73.

8-Methoxy-2-{N-[2-(2,3-dimethoxy)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (20b): method A. Yield 66%; mp 88–92 °C; IR: cm–1 3345 (b), 2936, 2837, 2585 (b), 2461 (b), 1647, 1578, 1514; 1H NMR (base,

200 MHz, CDCl3): d 0.89 (t, J = 7.3 Hz, 3H), 1.44–1.67 (m, 3H), 1.97–2.06 (m, 1H), 2.52–2.60 (m, 3H), 2.77–3.01 (m, 6H), 3.41–3.58 (m, 2H), 3.80 (s, 3H), 3.91 (s, 3H), 3.93 (s, 3H), 6.68 (dd, J = 9.8 Hz, 8.1 Hz, 2H), 7.02–7.19 (m, 13 3H), 7.73 (dd, J = 7.9 Hz, 2.9 Hz, 1H), 8.18 (bs, 1H); C NMR (base, 50 MHz, CDCl3): d 11.8, 22.4, 25.5, 25.7, 30.2, 38.5, 48.9, 52.4, 55.1, 56.0, 56.6, 61.3, 106.7, 115.0, 120.7, 122.8, 124.1, 125.1, 126.0, 126.9, 129.2, 137.7, 152.6,

157.7, 165.0; MS (CI with NH3): m/z (rel. intensity) 161 (47), 182 (97), 220 (24), 232 (50), 267 (12), 385 (8), 427

(100, M+1); Anal. calcd for C25H34N2O4·HCl·¾H2O: C 63.00, H 7.74, N 5.88; obsd C 63.18, H 7.62, N 5.76.

8-Methoxy-2-{N-[2-(2,6-dimethoxy)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (20c): method A. Yield 41%; mp 121–123 °C; IR: cm–1 3391 (b), 2938, 2838, 2605 (b), 2512 (b), 1654, 1596, 1524; 1H NMR (base,

200 MHz, CDCl3): d 0.85 (t, J = 7.3 Hz, 3H), 1.40–1.69 (m, 3H), 1.89–1.96 (m, 1H), 2.33–2.60 (m, 3H), 2.73–2.98 (m, 6H), 3.47–3.56 (m, 2H), 3.78 (s, 9H); 6.46 (bs, 1H), 6.55 (d, J = 8.3 Hz, 2H), 6.66 (dd, J = 8.6 Hz, 8.6 Hz, 2H), 13 7.07 (t, J = 7.8 Hz, 1H), 7.26 (t, J = 8.6 Hz, 1H); C NMR (base, 50 MHz, CDCl3): d 11.6, 21.9, 25.3, 30.0, 37.7,

48.8, 51.8, 54.9, 55.6, 56.1, 103.7, 106.6, 116.0, 120.7, 124.9, 125.9, 130.2, 137.5, 157.3, 165.7; MS (CI with NH3): m/z (rel. intensity) 60 (31), 139 (17), 161 (30), 182 (100), 220 (23), 267 (15), 427 (19, M+1); Anal. calcd for

C25H34N2O4·HCl·½H2O: C 63.60, H 7.70, N 5.94; obsd C 63.36, H 7.71, N 5.85.

73 Chapter 2

8-Methoxy-2-[N-(2-thiophen-2-carboxamidoethyl)-N-n-propylamino]tetralin Oxalate (20d): method A. Yield 66%; mp 135–137 °C; IR: cm–1 3257 (b), 2968, 2833, 2787 (b), 2662 (b), 1718, 1642, 1588, 1541; 1H NMR (200

MHz, CD3OD): d 1.03 (t, J = 7.3 Hz, 3H), 1.79–1.90 (m, 3H), 2.14–2.26 (m, 1H), 2.67–2.94 (m, 3H), 3.19–3.34 (m, 4H), 3.45–3.56 (m, 2H), 3.71–3.93 (m, 6H), 6.68–6.75 (m, 2H), 7.07–7.17 (m, 2H), 7.69–7.78 (m, 2H); 13C NMR (50

MHz, CD3OD): d 9.6, 17.8, 22.7, 23.1, 27.7, 36.0, 50.8, 53.1, 54.1, 60.5, 106.9, 120.1, 120.5, 126.8, 127.6, 129.3,

131.2, 135.7, 137.3, 157.1, 164.7; MS (CI with AcOH): m/z 373 (M+1); Anal. calcd for C21H28N2O2S·C2H2O4: C 59.71, H 6.55, N 6.06; obsd C 59.55, H 6.56, N 6.11. zHz 2-[N-(2-Benzamidoethyl)-N-n-propylamino)tetralin Hydrochloride (21a): method A. Yield 62%; mp 83–86 °C; –1 1 IR: cm 3436 (b), 3250 (b), 3060, 2936, 2610 (b), 2512 (b), 1654, 1577, 1541; H NMR (300 MHz, CD3OD): d 1.06 (t, J = 7.3 Hz, 3H), 1.82–1.99 (m, 3H), 2.31–2.42 (m, 1H), 2.95–3.01 (m, 2H), 3.12–3.42 (m, 5H), 3.60–3.65 (m, 1H), 3.77–3.96 (m, 3H), 7.07–7.18 (m, 4H), 7.49 (dd, J = 6.9 Hz, 6.9 Hz, 2H), 7.58 (t, J = 7.3 Hz, 1H), 7.92 (d, J = 7.0 Hz, 13 2H); C NMR (75 MHz, CD3OD): d 10.6, 18.9, 24.2, 28.5, 29.7, 37.0, 52.2, 53.8, 61.1, 126.7, 127.1, 127.9, 128.9,

129.1, 129.6, 132.7, 133.4, 135.3, 171.1; MS (CI with NH3): m/z (rel. intensity) 202 (14), 337 (100, M+1); Anal. calcd for C22H28N2O·HCl·¼H2O: C 70.00, H 7.89, N 7.42; obsd C 69.73, H 8.09, N 7.36.

2-{N-[2-(2,3-Dimethoxy)benzamidoethyl]-N-n-propylamino}tetralin Oxalate (21b): method A. Yield 25%; mp 70–72 °C; IR: cm–1 3360 (b), 2936, 2835, 2619 (b), 2520 (b), 1719, 1649, 1577, 1524; 1H NMR (base, 200 MHz,

CDCl3): d 0.90 (t, J = 7.3 Hz, 3H), 1.45–1.71 (m, 3H), 1.98–2.10 (m, 1H), 2.55 (dd, J = 7.5 Hz, 7.5 Hz, 2H), 2.76– 3.04 (m, 7H), 3.52–3.60 (m, 2H), 3.92 (s, 3H), 3.93 (s, 3H), 7.02–7.09 (m, 5H), 7.16 (t, J = 7.9 Hz, 1H), 7.73 (dd, J = 13 7.2 Hz, 1.8 Hz, 1H), 8.39 (bs, 1H); C NMR (base, 50 MHz, CDCl3): d 11.9, 22.3, 25.9, 29.9, 31.9, 38.5, 49.0, 52.4, 56.1, 56.7, 61.3, 115.1, 122.8, 124.2, 125.6, 125.7, 127.1, 128.6, 129.4, 136.3, 147.7, 152.5, 165.1; MS (CI with

NH3): m/z (rel. intensity) 190 (16), 202 (6), 397 (100, M+1); Anal. calcd for C24H32N2O3·C2H2O4·¼H2O: C 63.58, H 7.09, N 5.71; obsd C 63.52, H 7.26, N 5.56.

2-{N-[2-(2,6-Dimethoxy)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (21c): method A. Yield –1 1 75%; mp 107–110 °C; IR: cm 3241 (b), 2938, 2837, 2473 (b), 1656, 1596, 1522; H NMR (300 MHz, CD3OD): d 1.13 (t, J = 7.3 Hz, 3H), 1.92–2.04 (m, 3H), 2.38–2.42 (m, 1H), 2.98–3.09 (m, 2H), 3.16–3.35 (m, H), 3.48–3.57 (m, 1H), 3.73–3.79 (m, 2H), 3.83 (s, 6H), 3.88–3.97 (m, 1H), 6.75 (d, J = 8.6 Hz, 2H), 7.16–7.24 (m, 4H), 7.41(t, J = 8.4 13 Hz, 1H); C NMR (75 MHz, CD3OD): d 11.4, 19.9, 24.8, 29.3, 30.6, 38.2, 53.8, 54.4, 56.4, 61.6, 105.1, 115.3, 127.4,

127.7, 129.6, 130.3, 132.7, 133.8, 136.1, 158.6, 171.8; MS (CI with NH3): m/z (rel. intensity) 202 (14), 397 (100,

M+1); Anal. calcd for C24H32N2O3·HCl·½H2O: C 65.21, H 7.77, N 6.34; obsd C 64.89, H 7.68, N 6.38.

2-[N-(2-Thiophen-2-carboxamidoethyl)-N-n-propylamino]tetralin Hydrochloride (21d): method A. Yield 62%; mp 93–94 °C; IR: cm–1 3232 (b), 3059, 2937, 2609 (b), 2480 (b), 1639, 1543; 1H NMR (base, 200 MHz,

CDCl3): d 0.94 (t, J = 7.3 Hz, 3H), 1.44–1.76 (m, 3H), 1.98–2.07 (m, 1H), 2.56 (dd, J = 8.1 Hz, 6.5 Hz, 2H), 2.71– 3.12 (m, 7H), 3.39–3.57 (m, 2H), 7.04–7.14 (m, 6H), 7.45 (dd, J = 5.0 Hz, 1.1 Hz, 1H), 7.55 (dd, J = 3.7 Hz, 1.0 Hz, 13 1H); C NMR (base, 50 MHz, CDCl3): d 11.7, 21.7, 25.5, 29.5, 31.8, 37.6, 48.3, 52.0, 56.1, 125.6, 125.7, 127.6, 127.8, 128.5, 129.3, 129.5, 135.8, 136.1, 139.2, 161.7; MS (CI with AcOH): m/z 343 (M+1); Anal. calcd for

C20H26N2OS·HCl·¼H2O: C 62.63, H 7.24, N 7.31; obsd C 62.31, H 7.29, N 7.19.

General Procedure for the Preparation of Compounds 17p, 18e, 19e and 19e. The method adopted for the synthesis of 5-hydroxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin·HCl (17p) is described: under a nitrogen atmosphere, a 1 M solution of BBr3 in CH2Cl2 (2 mL, 4 mmol) was added dropwise to solution of 17a (0.25 g, 0.7 mmol) in CH2Cl2 which was cooled at –50 ºC. Stirring was continued at –50 ºC for 1 h, and then the reaction mixture was allowed to gradually warm to room temperature. After stirring overnight at room temperature, the reaction mixture was concentrated under reduced pressure and the residue was partitioned between CH2Cl2 and saturated aqueous NaHCO3 solution. The organic solution was dried (Na2SO4), filtered and evaporated to dryness, which gave the crude phenol as a brown oil. Purification by column chromatography yielded 0.21 g (0.6 mmol, 87%) of the pure base of 17p as a colourless oil, which was converted to the hydrochloride salt: mp 116–118 °C; IR: cm–1

74 2-Aminotetralin-derived Substituted Benzamides

1 3231 (b), 2966, 2623 (b), 1654, 1588, 1534; H NMR (base, 200 MHz, CDCl3): d 0.91 (t, J = 7.3 Hz, 3H), 1.47–1.60 (m, 3H), 1.99–2.05 (m, 1H), 2.48–2.60 (m, 3H), 2.78–3.02 (m, 6H), 3.47–3.58 (m, 2H), 6.64 (dd, J = 19.2 Hz, 7.7 Hz, 13 2H), 6.94 (t, J = 7.6 Hz, 1H), 7.40–7.52 (m, 3H), 7.79–7.83 (m, 2H); C NMR (base, 50 MHz, CDCl3): d 11.6, 21.6, 23.4, 25.2, 31.8, 37.8, 48.2, 52.1, 55.9, 112.0, 120.8, 123.1, 126.2, 126.8, 128.5, 131.4, 134.2, 137.4, 154.1, 167.7;

MS (CI with AcOH): m/z 353 (M+1); Anal. calcd for C22H28N2O2·HCl·H2O: C 65.65, H 7.65, N 6.96; obsd C 65.47, H 7.46, N 6.96.

6-Hydroxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin Hydrochloride (18e): yield 89%; mp 108–110 –1 1 °C; IR: cm 3244 (b), 2939, 2629 (b), 1654, 1577, 1534; H NMR (base, 200 MHz, CDCl3): d 0.90 (t, J = 7.3 Hz, 3H), 1.37–1.61 (m, 3H), 1.90–1.95 (m, 1H), 2.52–2.98 (m, 9H), 3.45–3.55 (m, 2H), 6.59–6.66 (m, 2H), 6.82–6.86 (m, 13 1H), 7.31 (bs, 1H), 7.40–7.52 (m, 3H), 7.82 (m, 2H); C NMR (base, 50 MHz, CDCl3): d 11.6, 21.4, 25.4, 29.4, 30.7,

37.7, 48.4, 52.2, 56.5, 113.3, 114.8, 126.6, 126.8, 128.5, 130.1, 131.4, 134.1, 137.0, 154.5, 167.6; MS (CI with NH3): m/z 353 (M+1); Anal. calcd for C22H28N2O2·HCl·¾H2O: C 65.65, H 7.65, N 6.96; obsd C 65.40, H 7.49, N 6.63.

7-Hydroxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin Hydrochloride (19e): yield 94%; mp 111–113 –1 1 °C; IR: cm 3236 (b), 2938, 2627 (b), 2513 (b), 1647, 1577, 1536; H NMR (200 MHz, CD3OD): d 0.98 (t, J = 7.3 Hz, 3H), 1.87–2.13 (m, 3H), 2.52–2.60 (m, 1H), 2.76–2.85 (m, 3H), 3.03–3.16 (m, 1H), 3.24–3.42 (m, 4H), 3.62–4.00 (m, 4H), 6.50–6.65 (m, 2H), 6.88 (d, J = 8.1 Hz, 1H), 7.43–7.57 (m, 3H), 8.14–8.19 (m, 2H); 13C NMR (50 MHz,

CD3OD): d 11.3, 18.8, 24.0, 24.8, 28.1, 36.5, 52.5, 54.7, 61.4, 114.9, 116.0, 126.3, 128.4, 129.1, 130.2, 132.2, 134.5,

134.8, 156.4, 167.2; MS (CI with NH3): m/z 353 (M+1); Anal. calcd for C22H28N2O2·HCl·½H2O: C 66.39, H 7.61, N 7.04; obsd C 66.50, H 7.65, N 7.07.

8-Hydroxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin Hydrochloride (20e): yield 74%; mp 116–119 –1 1 °C; IR: cm 3215 (b), 2968, 2937, 2615 (b), 2515 (b), 1654, 1588, 1635; H NMR (300 MHz, CDCl3): d 0.92–0.96 (m, 3H), 1.71–1.93 (m, 2H), 2.20–2.30 (m, 3H), 2.47–2.77 (m, 3H), 3.12–3.54 (m, 7H), 3.78–3.94 (m, 2H), 6.43–6.45 13 (m, 1H), 6.78–6.89 (m, 2H), 7.38–7.53 (m, 3H), 8.01–8.09 (m, 2H); C NMR (75 MHz, CDCl3): d 11.1, 18.0, 24.1, 28.1, 36.4, 51.5, 53.9, 55.2, 61.6, 112.7, 118.7, 119.7, 127.2, 127.6, 128.6, 132.0, 132.6, 135.6, 154.5, 168.7; MS (CI with NH3): m/z 353 (M+1); Anal. calcd for C22H28N2O2·HCl·¾H2O: C 65.65, H 7.65, N 6.96; obsd C 65.90, H 7.82, N 6.42.

2.6.2 PHARMACOLOGY

3 – Pharmacology. [ H]-Raclopride Binding to Cloned Dopamine D2A and D3 Receptors. Mouse fibroblast (Ltk ) cells expressing human dopamine D2A receptors (obtained from Dr. O. Civelli, Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, OR) and Chinese hamster ovary (CHO) cells expressing human dopamine D3 receptors (obtained from INSERM Institute, Paris, France) were grown and the cell membranes were prepared essentially as described by Malmberg et al.34 Briefly, the Ltk– cells were cultured in DMEM (Dulbecco’s Modified Eagles Medium) supplemented with 10 mM HEPES, 10% fetal calf serum (FCS, heat- inactivated) and selected with G-418 (Geneticin 0.7 mg/ml). CHO cells were grown in DMEM supplemented as above, except that the FCS was dialyzed and MEM Amino Acids solution (50 ´) without L-glutamine was added. The cells were detached with 0.05% trypsin and 0.02% EDTA, centrifuged (300 ´ g, for 10 min), washed twice with

DMEM and homogenized in 10 mM Tris-HCl and 5 mM MgSO4 (pH 7.4). The homogenate was washed (43,500 ´ g, for 10 min) in binding buffer (50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 4 mM MgCl2, 1 mM EDTA, pH 7.4) and stored at –70 °C until further use. On the day of the experiment the frozen homogenate was thawed, homogenized with a Branson 450 sonifier and suspended in binding buffer. The binding assays were performed in a total volume of 0.5 mL with a receptor concentration of about 100 pM (~30 mg protein/0.5 mL). 1 nM 3 [ H]-Raclopride (Kd’s for dopamine D2A and D3 1.20 and 1.60 nM, respectively; specific activity 80 Ci/mmol, Du Pont New England Nuclear, Boston, MA, or 41 Ci/mmol, Astra Arcus AB, Södertälje, Sweden) was incubated with the test compound (10–12 concentrations) at 22 °C for 60 min. Binding in the presence of 1 mM (+)-butaclamol (Research

75 Chapter 2

Biochemicals Inc., Natick, MA) was defined as nonspecific. The incubation was terminated by rapid filtration through Whatman GF/B glassfiber filters and subsequent washing with ice-cold buffer, using a Brandel cell harvester. Scintillation cocktail (Packard Ultima Gold, 4 mL) was added and the radioactivity was determined with a Liquid Scintillation Counter (Packard 2200CA or 2500TR) at about 50% efficiency. Alternatively, the incubation was terminated by rapid filtration through Wallac Printed Filtermat B and washed with cold buffer using a Tomtec harvester, and the radioactivity was determined in 205 Beta Plate (Wallac), with about 30% efficiency. Protein concentration was measured by the method of Markwell et al.35 3 [ H]-8-OH-DPAT Binding to Serotonin 5-HT1A Receptors. This assay was performed essentially as previously described by Hedberg et al.19 Briefly, male Sprague-Dawley rats weighing 150–220 g (B & K Universal AB, Sollentuna, Sweden) were decapitated and the hippocampi were dissected out on ice. The tissue was homogenized at 0 °C using an Ultra-Turrax, in 50 mM Tris-HCl buffer containing 10 mM EDTA (pH 7.4). The homogenate was centrifuged at 4 °C for 10 min at 17,000 × g, the pellet was resuspended in 50 mM Tris-HCl with 10 mM EDTA and recentrifuged. The final pellet was frozen in 0.32 M sucrose and stored at –70 °C until further use. On the day of the experiment the frozen homogenate was thawed and suspended in binding buffer containing 50 mM Tris-HCl, 2 mM

CaCl2, 1 mM MgCl2 and 1 mM MnCl2 (pH 7.4), to a final concentration of 2.0 mg original wet weight per 0.5 mL. In order to remove endogenous serotonin the membranes were preincubated for 10 min at 37 °C and subsequently 10 mM 3 was added. Competition experiments with 1 nM [ H]-8-OH-DPAT (Kd = 1.00 nM; specific activity 136, 149 or 163 Ci/mmol, Du Pont New England Nuclear, Boston, MA) and test compounds (10–12 concentrations) were performed at 37 °C for 45 min. Nonspecific binding was defined with 100 mM 5-HT (Sigma Chemical Co., St. Louis, MO). The incubations were terminated and the radioactivity was determined as described for the dopamine receptor binding assay.

Data Analysis. The Ki values (inhibition constants) of the test compounds were determined from inhibition curves using the iterative non-linear curve-fitting program LIGAND.38 One- and two-site curve fitting was tested in all experiments. The one-site model gave a better fit (p > 0.05; F test) unless otherwise stated. The Kd values (dissociation constants) of the various radioligands used to calculate the Ki values were determined by saturation studies.

2.7 REFERENCES

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reveals the potencies of N,N-disubstituted 2-aminotetralins as D2 dopamine agonists. Naunyn Schmiedebergs Arch Pharmacol 336, 487–493. 6 Björk L, Höök BB, Nelson DL, Andén N-E and Hacksell U (1989) Resolved N,N-dialkylated 2-amino-8-

hydroxytetralins: stereoselective interactions with 5-HT1A receptors in the brain. J Med Chem 32, 779–783. 7 Cannon JG (1983) Structure-activity relationships of dopamine agonists. Annu Rev Pharmacol Toxicol 23, 103– 130. 8 Copinga S, Tepper PG, Grol CJ and Dubocovich ML (1993) 2-Amido-8-methoxytetralins: a series of nonindolic melatonin-like agents. J Med Chem 36, 2891–2898.

76 2-Aminotetralin-derived Substituted Benzamides

9 Cornfield LJ, Lambert G, Arvidsson LE, Mellin C, Vallgårda J, Hacksell U and Nelson DL (1991) Intrinsic

activity of enantiomers of 8-hydroxy-2-(di-n-propylamino)tetralin and its analogs at 5-hydroxytryptamine1A receptors that are negatively coupled to adenylate cyclase. Mol Pharmacol 39, 780–787. 10 Csöregh I and Högberg T (1992) Molecular and crystal structure of the hydrochloride monohydrate of (R)-N-[[1-

(4-fluorobenzyl)-2-pyrrolidinyl]methyl-5-bromo-2,3-dimethoxybenzamide, NCQ 115, a novel dopamine D2 receptor antagonist. Acta Pharm Nord 4, 1–-4. 11 Cymerman Craig J, Moore B and Ritchie E (1959) Simplified analogues of lysergic acid. I. Derivatives of 1,2,3,4- tetrahydronaphthylamine. Aust J Chem 12, 447–452. 12 Damsma G, Bottema T, Westerink BH, Tepper PG, Dijkstra D, Pugsley TA, MacKenzie RG, Heffner TG and

Wikström H (1993) Pharmacological aspects of R-(+)-7-OH-DPAT, a putative dopamine D3 receptor ligand. Eur J Pharmacol 249, 9–10. 13 de Paulis T, Hall H, Kumar Y, Rämsby S, Ögren S-O and Högberg T (1990) Potential antipsychotic agents. 6. Synthesis and antidopaminergic properties of substituted N-(1-benzyl-4-piperidinyl)salicylamides and related compounds. QSAR based design of more active members. Eur J Med Chem 25, 507–517. 14 Feenstra MG, Rollema H, Dijkstra D, Grol CJ, Horn AS and Westerink BH (1980) Effect of non-catecholic 2- aminotetralin derivatives on dopamine metabolism in the rat striatum. Naunyn Schmiedebergs Arch Pharmacol 313, 213–219. 15 Hacksell U, Jackson DM and Mohell N (1995) Does the dopamine receptor subtype selectivity of antipsychotic agents provide useful leads for the development of novel therapeutic agents? Pharmacol Toxicol 76, 320–324. 16 Hacksell U, Svensson U, Nilsson JL, Hjorth S, Carlsson A, Wikström H, Lindberg P and Sanchez D (1979) N- Alkylated 2-aminotetralins: central dopamine-receptor stimulating activity. J Med Chem 22, 1469–1475.

17 Harrold MW, Wallace RA, Farooqui T, Wallace LJ, Uretsky N and Miller DD (1989) Synthesis and D2 dopaminergic activity of pyrrolidinium, tetrahydrothiophenium, and tetrahydrothiophene analogues of sulpiride. J Med Chem 32, 874–880. 18 Hart PA and Rich DH (1996) Stereochemical aspects of drug action I: Conformational restriction, steric hindrance and hydrophobic collapse. In: Wermuth CG (Ed) The Practice of Medicinal Chemistry. Academic Press, London, pp 393–412. 19 Hedberg MH, Johansson AM, Nordvall G, Yliniemelä A, Li HB, Martin AR, Hjorth S, Unelius L, Sundell S and Hacksell U (1995) (R)-11-Hydroxy- and (R)-11-hydroxy-10-methylaporphine: synthesis, pharmacology, and

modeling of D2A and 5-HT1A receptor interactions. J Med Chem 38, 647–658. 20 Högberg T, Bengtsson S, de Paulis T, Johansson L, Ström P, Hall H and Ögren S-O (1990) Potential antipsychotic agents. 5. Synthesis and antidopaminergic properties of substituted 5,6-dimethoxysalicylamides and related compounds. J Med Chem 33, 1155–1163. 21 Högberg T and Norinder U (1991) Theoretical and experimental methods in drug design applied on antipsychotic dopamine antagonists. In: Krogsgaard-Larsen P and Bundgaard H (Eds) A Textbook in Drug Design and Development. Harwood Academic Publishers, Chur, pp 59–70. 22 Högberg T, Norinder U, Rämsby S and Stensland B (1987) Crystallographic, theoretical and molecular modelling

studies on the conformations of the salicylamide, raclopride, a selective dopamine-D2 antagonist. J Pharm Pharmacol 39, 787–796. 23 Högberg T, Rämsby S, de Paulis T, Stensland B, Csöregh I and Wägner A (1986) Solid state conformations and antidopaminergic effects of remoxipride hydrochloride and a closely related slicylamide, FLA 797, in relation to dopamine receptor models. Mol Pharmacol 30, 345–351. 24 Högberg T, Rämsby S, Ögren S-O and Norinder U (1987) New selective dopamine D-2 antagonists as antipsychotic agents. Pharmacological, chemical, structural and theoretical considerations. Acta Pharm Suec 24, 289–328. 25 Högberg T, Ström P, de Paulis T, Stensland B, Csöregh I, Lundin K, Hall H and Ögren S-O (1991) Potential antipsychotic agents. 9. Synthesis and stereoselective dopamine D-2 receptor blockade of a potent class of substituted (R)-N-[(1-benzyl-2-pyrrolidinyl)methyl]benzamides. Relations to other side chain congeners. J Med Chem 34, 948–955.

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26 Högberg T, Ström P, Hall H and Ögren S-O (1990) Potential antipsychotic agents. Part 8. Antidopaminergic properties of a potent series of 5-substituted (–)-(S)-N-[(1-ethylpyrrolidin-2-yl)methyl]-2,3-dimethoxybenzamides. Synthesis via common lithio intermediates. Helv Chim Acta 73, 417–425. 27 Kanao M, Hashizume T, Ichikawa Y, Irie K and Isoda S (1982) Spasmolytic agents. 2. 1,2,3,4-Tetrahydro-2- naphthylamine derivatives. J Med Chem 25, 1358–1363. 28 Karlsson A, Björk L, Pettersson C, Andén N-E and Hacksell U (1990) (R)- and (S)-5-hydroxy-2- (dipropylamino)tetralin (5-OH-DPAT): assessment of optical purities and dopaminergic activities. Chirality 2, 90– 95. 29 Kline TB, Nelson DL and Namboodiri K (1990) Novel [(diazomethyl)carbonyl]-1,2,3,4-tetrahydronaphthalene

derivatives as potential photoaffinity ligands for the 5-HT1A receptor. J Med Chem 33, 950–955. 30 Levesque D, Diaz J, Pilon C, Martres MP, Giros B, Souil E, Schott D, Morgat JL, Schwartz J-C and Sokoloff P

(1992) Identification, characterization, and localization of the dopamine D3 receptor in rat brain using 7- [3H]hydroxy-N,N-di-n-propyl-2-aminotetralin. Proc Natl Acad Sci USA 89, 8155–8159. 31 Liu Y, Cortizo L, Svensson BE, Lewander T and Hacksell U (1995) C8-Substituted derivatives of 2- (dipropylamino)tetralin: exploration of the effect of C8-aryl and heteroaryl substitutents on the interaction with 5-

HT1A-receptors. Eur J Med Chem 30, 277–286. 32 Liu Y, Svensson BE, Yu H, Cortizo L, Ross SB, Lewander T and Hacksell U (1991) C8-Substituted derivatives of

2-(dipropylamino)tetralin: Palladium-catalyzed synthesis and interactions with 5-HT1A-receptors. Bioorg Med Chem Lett 1, 257–262. 33 Liu Y, Yu H, Svensson BE, Cortizo L, Lewander T and Hacksell U (1993) Derivatives of 2-

(dipropylamino)tetralin: effect of the C8-substituent on the interaction with 5-HT1A receptors. J Med Chem 36, 4221–4229. 34 Malmberg Å, Nordvall G, Johansson AM, Mohell N and Hacksell U (1994) Molecular basis for the binding of 2-

aminotetralins to human dopamine D2A and D3 receptors. Mol Pharmacol 46, 299–312. 35 Markwell MAK, Haas SM, Bieber LL and Tolbert NE (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87, 206–210. 36 McDermed JD, McKenzie GM and Freeman HS (1976) Synthesis and dopaminergic activity of (±)-, (+)-, and (–)- 2-dipropylamino-5-hydroxy-1,2,3,4-tetrahydronaphthalene. J Med Chem 19, 547–549. 37 McOmie JWF, Watts ML and Wets DE (1968) Demethylation of aryl methyl ethers by boron tribromide. Tetrahedron 24, 2289–2292. 38 Munson PJ and Rodbard D (1980) LIGAND: A versatile computerized approach for characterization of ligand- binding systems. Anal Biochem 107, 220–239. 39 Naiman N, Lyon RA, Bullock AE, Rydelek LT, Titeler M and Glennon RA (1989) 2-(Alkylamino)tetralin

derivatives: interaction with 5-HT1A serotonin binding sites. J Med Chem 32, 253–256. 40 Norinder U and Högberg T (1989) QSAR on substituted salicylamides. In: Fauchère JL (Ed) QSAR: Quantitative Structure-Activity Relationships in Drug Design. Alan R. Liss, Inc. New York, pp 369–372. 41 Norinder U and Högberg T (1991) QSAR on substituted salicylamides using PLS with implementation of 3D MEP descriptors. Quant Struct-Act Relat 10, 1–5.

42 Norinder U and Högberg T (1992) A quantitative structure-activity relationship for some dopamine D2 antagonists of benzamide type. Acta Pharm Nord 4, 73–78. 43 Petterson I and Liljefors T (1992) Conformational analysis of dopamine D-2 receptor antagonists of the benzamide series in relation to a recently proposed D-2 receptor interaction model. J Med Chem 35, 2355–2363. 44 Rognan D, Sokoloff P, Mann A, Martres M-P, Schwartz J-C, Costenin J and Wermuth C-G (1990) Optically active

benzamides as predictive tools for mapping the dopamine D2 receptor. Eur J Pharmacol 189, 59–70. 45 Seiler MP and Markstein R (1984) Further characterization of structural requirements for agonists at the striatal

dopamine D2 receptor and a comparison with those at the striatal dopamine D1 receptor. Studies with a series of monohydroxyaminotetralins on acetylcholine release from rat striatum. Mol Pharmacol 26, 452–457. 46 Seiler MP, Stoll AP, Closse A, Frick W, Jaton A and Vigouret JM (1986) Structure-activity relationships of dopaminergic 5-hydroxy-2-aminotetralin derivatives with functionalized N-alkyl substituents. J Med Chem 29, 912–917.

78 2-Aminotetralin-derived Substituted Benzamides

47 Sokoloff P, Andrieux M, Besançon R, Pilon C, Martres MP, Giros B and Schwartz J-C (1992) Pharmacology of

human dopamine D3 receptor expressed in a mammalian cell line: comparison with D2 receptor. Eur J Pharmacol 225, 331–337. 48 Sonesson C, Boije M, Svensson K, Ekman A, Carlsson A, Romero AG, Martin IJ, Duncan JN, King LJ and Wikström H (1993) Orally active central dopamine and serotonin receptor ligands: 5-, 6-, 7-, and 8- [[(trifluoromethyl)sulfonyl]oxy]-2-(di-n-propylamino)tetralins and the formation of active metabolites in vivo. J Med Chem 36, 3409-3416. 49 Stensland B, Högberg T and Rämsby S (1987) Structure of remoxipride, a new antipsychotic agents. Comparison of base and hydrochloride forms. Acta Cryst 43, 2393–2398. 50 Tedesco JL, Seeman P and McDermed JD (1979) The conformation of dopamine at its receptor: binding of monohydroxy-2-aminotetralin enantiomers and positional isomers. Mol Pharmacol 16, 369–381. 51 Van Vliet LA, Tepper PG, Dijkstra D, Damsma G, Wikström H, Puglsey TA, Akunne HC, Heffner TG, Glase SA

and Wise LD (1996) Affinity for dopamine D2, D3, and D4 receptors of 2-aminotetralins. Relevance of D2 agonist binding for determination of receptor subtype selectivity. J Med Chem 39, 4233–4237. 52 Wägner A, Stensland B, Csöregh I and de Paulis T (1985) Molecular structure and absolute configuration of the hydrochloride of a novel dopamine receptor antagonist: 2S-(–)-5-chloro-3-ethyl-N-[(1-ethyl-2- pyrrolidinyl)methyl]-6-methoxysalicylamide. Acta Pharm Suec 22, 101–110. 53 Wikström H, Andersson B, Sanchez D, Lindberg P, Arvidsson LE, Johansson AM, Nilsson JL, Svensson K, Hjorth S and Carlsson A (1985) Resolved monophenolic 2-aminotetralins and 1,2,3,4,4a,5,6,10b-octahydro- benzo[f]quinolines: structural and stereochemical considerations for centrally acting pre- and postsynaptic dopamine-receptor agonists. J Med Chem 28, 215–225. 54 Yu H, Liu Y, Malmberg Å, Mohell N, Hacksell U and Lewander T (1996) Differential serotonergic and dopaminergic activities of the (R)- and the (S)-enantiomers of 2-(di-n-propylamino)tetralin. Eur J Pharmacol 303, 151–162.

79 STRUCTURAL ANALOGUES OF 5-OME-BPAT:

SYNTHESIS AND INTERACTIONS WITH DOPAMINE D2, D3, 3 AND SEROTONIN 5-HT1A RECEPTORS

ABSTRACT

2-Aminotetralin-derived substituted benzamides constitute a series of compounds with mixed dopamine D2, D3, and serotonin 5-HT1A receptor binding properties. In order to gain insight in the importance of the various functional groups as present in the 2-aminotetralin-derived benzamides, and hence in the mode of binding of these compounds to the receptors, several structural analogues of 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-

BPAT, 1) were synthesized and evaluated for their ability to bind to dopamine D2A, D3, and serotonin 5-HT1A receptors in vitro. Replacement of the benzamido moiety of 1 by an acetamido group resulted in a strong decrease in the affinities for the receptors, while the affinities of the cyclohexylcarboxamido analogue were even enhanced, suggesting that a relatively large, lipophilic substituent at the amide carbonyl group, but not necessarily aromatic in nature, is required for high affinities at the receptors. The presence of the carbonyl group in 1 seems to be beneficial for its high affinity at the dopaminergic receptors, since replacement by a sulfonyl or methylene group resulted in decreased affinities for the dopamine D2A and D3, but not for the serotonin 5-HT1A receptor. Therefore, the carbonyl oxygen atom of 1 may act as a hydrogen bond acceptor when this compound binds to the dopaminergic receptors. The phthalimidoethyl analogue of 1 was devoid of dopaminergic and serotonergic affinity. In addition, alkylation of the amide nitrogen atom of 1 resulted in a strong decrease predominantly in dopaminergic affinity, suggesting that the amide hydrogen atom of 1 may be involved in hydrogen bonding while binding to the dopaminergic receptors. However, dynamic NMR and molecular modeling studies revealed that two stable rotamers of the synthesized N’-alkyl analogues, which may possess different pharmacological properties, are likely to coexist under pharmacological conditions. Furthermore, an out-of-plane orientated benzamide moiety in these compounds may contribute to their low dopaminergic affinities. Therefore, no definitive conclusions can be drawn on the role of the amide hydrogen atom of 1 while interacting with the dopaminergic receptors. Since the effects of the indicated carbonyl replacements and amide N’-alkylations on the affinity for the serotonin 5-HT1A receptor were less pronounced, the benzamidoethyl side chain of 1 probably enhances the affinity for this receptor subtype predominantly through hydrophobic interactions with an accessory binding site. The substituent requirements for the basic nitrogen atom, as revealed by the N-H, N- methyl, N-ethyl, N-allyl and N-benzyl analogues of 1, were similar to those of the DPATs, supporting the hypothesis that the 2-aminotetralin moieties of the compounds may occupy the same binding sites as the DPATs. Finally, chain

81 Chapter 3

elongation of the benzamidoethyl side chain with one methylene group reduced the affinities for the dopamine D2A and serotonin 5-HT1A receptor, but a similar insertion in the phthalimidoethyl analogue of 1 restored the affinities to a large extent, suggesting that the benzamidopropyl and phthalimidopropyl side chains of these chain-elongated analogues may occupy a different accessory binding site than the analogues with a benzamidoethyl side chain.

3.1 INTRODUCTION

2-Aminotetralin-derived substituted benzamides comprise a series of compounds with mixed 7 dopamine D2, D3, and serotonin 5-HT1A receptor binding properties. Provided that they behave as antagonists at the dopaminergic receptors and as agonists at the serotonin 5-HT1A receptor, these compounds may have potential as atypical antipsychotic agents. The lead compound of this series, 5- methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 1) has nanomolar affinities for all three receptor subtypes. Previously reported SAFIR7 indicated that the benzene ring of the benzamide moiety can be replaced by small aromatic isosters, while attachment of substituents to this ring generally leads to somewhat lower affinities. Furthermore, the benzamidoethyl side chain appears to enhance the affinities for both the dopaminergic and serotonin 5-HT1A receptors, and thus may occupy an accessory binding site in all three receptor subtypes. In addition, the SAFIR suggested that the aminotetralin parts of the molecules probably occupy the same binding sites as the corresponding DPATs. In order to shed more light on the importance of the various functional groups as present in the 2-aminotetralin-derived benzamides, and hence on the mode of binding of these compounds to the receptors, various structural analogues of 1 were synthesized and evaluated for their ability to bind to dopamine D2A, D3, and serotonin 5-HT1A receptors in vitro.

3.2 CHEMISTRY

In order to investigate whether an aromatic ring attached to the amide carbonyl moiety is required for high affinity, the acetamido- and the cyclohexylcarboxamido- analogue of 1 were synthesized (Scheme 3.1). 5-Methoxy-2-[N-(2-aminoethyl)-N-n-propylamino]tetralin (2)7 served as starting material for both amides: reaction with acetic anhydride in the presence of sodium acetate and the biphasic medium water/ethyl acetate gave the acetamide 3, while reaction with cyclohexylcarboxylic acid chloride in the presence of sodium hydroxide and the biphasic medium water/dichloromethane yielded the cyclohexylcarboxamide 4.4

O N N H

OCH3 1

Chart 3.1 Chemical structure of 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 1).

82 Structural Analogues of 5-OMe-BPAT

O O N N N CH N H 3 H

OCH3 OCH3 3 a b 4

N NH2

OCH3 c 2 d

O O O N N S N N H O OCH OCH3 3 5 6

SCHEME 3.1 Reagents and conditions: (a) Ac2O, NaOAc, EtOAc, H2O, RT; (b) C6H11COCl, 10% NaOH, CH2Cl2,

RT; (c) phthalic anhydride, NaOAc, AcOH, D; (d) C6H5SO2Cl, K2CO3, CHCl3, H2O, RT.

Also starting from 2, two analogues of 1 with different benzamide isosters were prepared (Scheme 3.1). The phthalimido- analogue 5 was prepared by allowing 2 to react with phthalic anhydride in boiling glacial acetic acid, while reaction of 2 with benzenesulfonyl chloride in the presence of potassium carbonate and the biphasic medium water/chloroform gave the benzenesulfonamido- analogue 6.

Reduction of the carbonyl group of 1 with LiAlH4 in boiling THF gave the N’-benzyl analogue 7 (Scheme 3.2). Selective alkylation of the amide nitrogen of 1 with the appropriate alkyl iodide, employing dimethyl sulfoxide as a solvent and solid potassium hydroxide as a base,8 gave the N’- methyl-, N’-ethyl- and N’-n-propyl analogues 8, 9, and 10, respectively. Compounds 5–10 should reveal whether the presence of a secondary amide functionality, capable of forming hydrogen bonds with both its carbonyl oxygen and N-H hydrogen atom, is essential for high affinities at the receptors.

83 Chapter 3

O N N H

OCH3 a 1 b

O N N N N H R

OCH3 OCH3

7 8 R=CH3 9 R=C2H5 10 R=n-C3H7

SCHEME 3.2 Reagents and conditions: (a) LiAlH4, THF, D; (b) RI, KOH, DMSO, RT.

As outlined in Section 2.1, the DPATs have specific structural requirements with respect to the substituents on the basic nitrogen atom. In order to investigate whether these structural requirements are similar for the 2-aminotetralin-derived benzamides, which would lend further support that they may share common binding sites, analogues of 1 with different nitrogen substituents next to the benzamidoethyl side chain, i.e. benzyl, hydrogen, methyl, ethyl, and allyl, were prepared. The synthesis of these analogues is outlined in Scheme 3.3. N-Alkylation of 5-methoxy-2-(N- benzylamino)tetralin (11)10 with bromoacetonitrile in boiling acetone, using potassium carbonate as a base and potassium iodide as a catalyst, gave the N-cyanomethyl intermediate 12, which was reduced with LiAlH4 in boiling THF to the corresponding primary amine 13. Amide formation with benzoyl chloride as described for 4 yielded 14, the N-benzyl analogue of 1. Catalytic debenzylation of 14 by hydrogenation gave 15, the N-H analogue of 1, which served as a starting point for the preparation of the N-methyl, N-ethyl and N-allyl analogues, 16, 17, and 18, respectively. Thus, reductive amination of the appropriate aldehydes with 15 and hydrogen, employing ethanol as a solvent and Pd/C (10%) as a catalyst, gave 16 and 17, while N-alkylation of 15 with allyl bromide in boiling acetonitrile, employing cesium carbonate as a base and potassium iodide as a catalyst, yielded 18. Finally, a chain-elongated analogue of 1 was prepared, as outlined in Scheme 3.4. 5-Methoxy-2- (N-n-propylamino)tetralin1 (19) was N-alkylated with N-(3-bromopropyl)phthalimide in boiling acetonitrile, employing cesium carbonate as a base and potassium iodide as a catalyst. The resulting phthalimide 20 was hydrolyzed to the corresponding primary amine 21 with hydrazine in ethanol. Amide formation with benzoyl chloride, as described for 4, resulted in the N-(3-benzamidopropyl) analogue 22.

84 Structural Analogues of 5-OMe-BPAT

NH N CN N a b NH2

OCH3 OCH3 OCH3 11 12 13

O O H N N c N d N H H

OCH3 OCH3 14 15

R O N e or f or g N H

OCH3 16 R=methyl 17 R=ethyl 18 R=allyl

SCHEME 3.3 Reagents and conditions: (a) BrCH2CN, K2CO3, KI, acetone, D; (b) LiAlH4, THF, D; (c) C6H5COCl,

10% NaOH, CH2Cl2, RT; (d) Pd/C (10%), H2, EtOH, RT; (e) formaldehyde, Pd/C, H2, MeOH, D; (f)

acetaldehyde, Pd/C (10%), H2, EtOH, D; (g) CH2CHCH2Br, Cs2CO3, KI, MeCN, D.

O

NH N N a b O

OCH3 OCH3 19 20

H N NH N N 2 c O

OCH3 OCH3 21 22

SCHEME 3.4 Reagents and conditions: (a) N-(3-bromopropyl)phthalimide, K2CO3, KI, acetone, D; (b), hydrazine

hydrate, EtOH, RT; (c) C6H5COCl, 10% NaOH, CH2Cl2, RT.

85 Chapter 3

3.3 PHARMACOLOGY

Compounds 3–15 were evaluated for their ability to compete for [3H]-raclopride binding to – cloned human dopamine D2A receptors (expressed in Ltk cells) and cloned human dopamine D3 receptors (expressed in CHO cells), and their ability to compete for [3H]-8-OH-DPAT binding to rat hippocampal membranes containing serotonin 5-HT1A receptors in vitro. The results of these binding studies are shown in Table 3.1. For comparison purposes, the previously reported affinities of 17 have been included.

3.4 RESULTS AND DISCUSSION

5-Methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 1) is one of the most potent representatives of a series of compounds with mixed dopamine D2, D3, and serotonin 5-

HT1A receptor binding properties. Previous SAFIR studies on the series have shown that the aromatic nucleus of the benzamide moiety of 1 can be replaced by small aromatic isosters, including 2-thiophene and 3-thiophene, without serious loss of affinity for the indicated receptor subtypes. Replacement of this ring by 1-naphthalene or 2-naphthalene, however, resulted in about 6- and 20- fold lower affinities for dopamine D2A and serotonin 5-HT1A receptors, respectively. The results in Table 3.1 show that replacement of the benzamide moiety by an acetamide group (3) strongly

TABLE 3.1 Receptor binding data of compounds 1, 3–10, 14–18, 20, and 22.

R1

N (CH2)n X N R3

R2

OCH3 a Ki (nM)

Compound R1 R2 R3 X n D2A D3 5-HT1A 1 n-propyl H phenyl C=O 1 3.2 ± 0.2 0.58 ± 0.05 0.82 ± 0.11 3 n-propyl H methyl C=O 1 150 ± 60 NDb 123 ± 9 4 n-propyl H cyclohexyl C=O 1 2.2 ± 0.1 0.73 ± 0.26 <0.5 5 n-propyl phthalimidec 1 >1,000 >1,000 >1,000

6 n-propyl H phenyl SO2 1 46.3 ± 7.0 20.5 ± 1.5 1.7 ± 0.3

7 n-propyl H phenyl CH2 1 16.2 ± 10 ND 1.8 ± 0.3 8 n-propyl methyl phenyl C=O 1 208 ± 22 51.6 ± 3.2 20.2 ± 0.7 9 n-propyl ethyl phenyl C=O 1 316 ± 177 ND 17.9 ± 1.9 10 n-propyl n-propyl phenyl C=O 1 260 ± 61 ND 4.6 ± 0.6 14 benzyl H phenyl C=O 1 264 ± 3 ND 223 ± 11 15 H H phenyl C=O 1 8.7 ± 0.3 8.9 ± 2.5 4.1 ± 0.9 16 methyl H phenyl C=O 1 11.7d ND 45.7e 17 ethyl H phenyl C=O 1 2.9 ± 1.1 3.2 ± 0.3 0.65 ± 0.20 18 allyl H phenyl C=O 1 9.1 ± 1.9 2.3 ± 0.1 0.28 ± 0.05 20 n-propyl phthalimidec 2 20.5 ± 0.7 3.3 ± 0.2 14.9 ± 0.4 22 n-propyl H phenyl C=O 2 46.3 ± 6.3 ND 15.9 ± 4.0 a b c d 3 Mean ± s.e.m. of 2–4 independent experiments. ND: not determined. R2-N-X-R3= phthalimide. [ H]-spiperone binding, rat striatum, n = 1. eRat frontal cortex, n = 1.

86 Structural Analogues of 5-OMe-BPAT

decreases the affinities for both the dopamine D2A and serotonin 5-HT1A receptor. Replacement of the benzene ring by a cyclohexyl ring (4), however, even enhances the affinities for these two receptor subtypes. Apparently, a lipophilic substituent with about the size of a phenyl ring, but not necessarily aromatic in nature, is optimal for high affinities for both the dopamine D2A and serotonin

5-HT1A receptor. The phthalimidoethyl analogue 5 showed no affinity for the receptors under investigation. Apparently, the presence of the additional carbonyl group in the phthalimide moiety prohibits binding to the receptors. This could be caused by steric or electronic factors, or a combination of both.

Replacement of the carbonyl group of 1 by a SO2 group results in about 15-fold, 35-fold, and 2-fold loss of affinity for the dopamine D2A, D3, and serotonin 5-HT1A receptor, respectively (cf. 6). Reduction of the carbonyl group to a methylene group (cf. 7) reduces the affinity for the dopamine

D2A receptor about 5-fold and the affinity for the serotonin 5-HT1A receptor about 2-fold. These results suggest that the presence of a single carbonyl group, as in the benzamide moiety, is beneficial for high dopamine D2A and D3 receptor affinities, but not for binding to serotonin 5-HT1A receptors. Therefore, the carbonyl group of 1 may act as a hydrogen bond acceptor when this compound binds to dopamine D2A and D3 receptors. Although the SO2 group of 6 can also act as a hydrogen bond acceptor, the directionality of the SO groups apparently is not optimal for hydrogen bond formation. One of the SO groups may be orientated in a coplanar fashion with the benzene ring, mimicking the carbonyl group of 1, but the trigonal bipyramidal configuration of the SO2 moiety then dictates the other SO group to be directed in an orientation almost perpendicular to the plane of the benzene ring, and pointing in the same direction as the amide hydrogen. This phenomenon could account for the lower affinities of 6 at the dopaminergic receptor than 1. Alkylation of the benzamide nitrogen atom resulted in a strong decrease predominantly in dopaminergic affinity: compounds 8–10 all have low affinities for the dopamine D2A receptor. In addition, the affinity of 8 for the dopamine D3 receptor, compared to 1, is also reduced. However, the affinities of 8–10 for the serotonin 5-HT1A receptor are still considerable. Moreover, elongation of the N’-alkyl substituent, going from methyl (8) via ethyl (9) to n-propyl (10), even seems to restore most of the serotonergic activity, suggesting that the N’-n-propyl group may reach an accessory binding site in the serotonin 5-HT1A receptor. At first sight, these observations suggest that the diminished affinities of compounds 8–10 for the dopamine D2A receptors are caused either by steric hindrance of the N’-alkyl groups with one or more amino acid residues in the binding site, or by preventing the formation of a hydrogen bond with a hydrogen bond accepting amino acid residue in the binding site. This latter hypothesis would also explain the lack of affinity for the dopamine D2A receptor of the phthalimide analogue 5, where the presence of the additional carbonyl oxygen atom would cause strong repulsive interactions with the hydrogen bond acceptor atom of the receptor.

87 Chapter 3

However, differences in conformational behaviour of the benzamide moieties of 8–10 and 1 may also account for the observed results. 1H and 13C NMR spectra of 8–10, taken at room temperature and using deuterated chloroform as the solvent, indicated the presence of two stable conformers in 1 an 1 to 1 ratio. Figure 3.1 shows the H NMR spectra of 1 and 8, as observed under the indicated conditions. This phenomenon is most clearly illustrated by the triplet at 0.92 ppm in the spectrum of 1, which arises from the protons at the terminal carbon of the N-n-propyl group. In the spectrum of 8 these protons produce two identical triplets at 0.78 and 0.93 ppm. The two singlets at 3.04 and 3.13 ppm in the spectrum of 8, belonging to the N’-methyl protons of the two conformers, are also illustrative. Dynamic NMR experiments, performed on 8–10 in deuterated DMSO, deuterated , and deuterated tetrachloroethane, showed coalescense of the resonances upon heating, the coalescence temperature being dependent on the solvent (Figure 3.2). After cooling to room

1 FIGURE 3.1 200 MHz H NMR spectra of 1 (top) and 8 (bottom) recorded in CDCl3.

88 Structural Analogues of 5-OMe-BPAT

1 FIGURE 3.2 500 MHz H NMR spectra of 8 recorded in C2D2Cl4, at 20 ºC (top) and 90 ºC (bottom). temperature, the spectra again indicated the presence of two stable conformers in an 1 to 1 ratio. Since these observations suggested that the rotation behaviour of the amide bonds in 8–10 might be responsible for the coexistence of two stable conformers at room temperature, molecular modeling studies were undertaken to rationalize this phenomenon. Thus, in order to determine the rotation barrier and the locations of the energy minima for the amide bonds of 1 and 8–10, N- methylbenzamide and N,N-dimethylbenzamide were taken as model compounds for 1 and 8, respectively, and the energies of all rotamers, obtained by rotating the amide torsion angle with a resolution of 10°, were calculated semi-empirically using the AM1 method.5 The results of these calculations are shown in Figure 3.3. Rotation of the torsion angle t1-2-3-4 of N-methylbenzamide, as defined in Figure 3.3, revealed three minimum energy rotamers, at t = 30° (DE = 9.3 kcal/mol), t =

89

Structural Analogues of 5-OMe-BPAT the amides in an 1 to 1 ratio accounts for the double resonances observed in the NMR spectra of these compounds at room temperature. Upon sufficient heating, the rotation barriers can be overcome and the cis and trans forms may isomerize, which is reflected by the coalescence of their resonances in the NMR spectra. Importantly, these observations suggest that the two forms may also exist under biological conditions. It can therefore not be excluded that the cis amides of 8–10 have different pharmacological properties than the trans rotamers, which complicates the interpretation of the receptor binding data of these compounds. In addition to the conformational behaviour as described above, another conformational aspect of the N-alkylated benzamides may account for their reduced dopamine D2A receptor affinities. Full geometry optimization of the minimum energy rotamers of N,N-dimethylbenzamide resulted in an out-of-plane orientation of the aromatic nucleus with respect to the amide moiety of approximately 52°. Previously reported SAFIR studies on 2-aminotetralin-derived substituted benzamides (see Chapter 2) have shown that benzamides with a 2,6-dimethoxy substitution pattern have consistently lower affinities for the dopamine D2A receptor than their 2,3-dimethoxy- or unsubstituted analogues, presumably because they cannot adopt a coplanar orientation of the aromatic ring and the amide function. Therefore, this conformational effect of amide N-alkylation may also contribute to the reduced dopamine D2A receptor affinities of 8–10. The receptor binding data of compounds 1 and 14–18 reveal that the presence of a substituent at the basic nitrogen atom with about the size of a n-propyl group is optimal for high affinities at all three receptor subtypes. The hydrogen- and N-methyl-substituted analogues 15 and 16 have somewhat lower affinities, while introduction of a second bulky substituent next to the N- benzamidoethyl side chain, as in 14, results in strong loss of affinity for both the dopamine D2A and 2,3,6,11,13,15 serotonin 5-HT1A receptor. These structural requirements are similar to those of the DPATs and support the previously stated hypothesis (see Chapter 2) that the 2-aminotetralin moieties of both classes of compounds may occupy the same binding sites. For the DPATs and structurally closely related compounds, these specific requirements for the nitrogen substituents have been rationalized by proposing the presence of a pocket in the dopamine D2 receptor, capable of accommodating unbranched N-substituents not larger than n-propyl.6,12,16 Using molecular modeling studies, Malmberg et al. have shown the presence of such a pocket in both the dopamine D2 and D3 receptor.9 In view of the similarities in the SAFIR of the DPATs and the 2-aminotetralin-derived benzamides, as noted above and in Chapter 2, it is likely that the N-alkyl substituents of 1, 17 and 18 protrude into this ‘propyl cleft’ while binding to the dopamine D2A or D3 receptor. Chain elongation of the benzamidoethyl side chain of 1 with one methylene group (22) results in about 15-fold reduction of the affinities for the dopamine D2A and the serotonin 5-HT1A receptor. Surprisingly, the phthalimidopropyl analogue 21, a synthetic intermediate for the preparation of 22 which was also evaluated in the receptor binding assays, and as such the chain-elongated analogue of the inactive 5, had affinities comparable to those of 22. Therefore, the benzamidopropyl and phthalimidopropyl side chains of 22 and 21 probably occupy a different accessory binding site than the analogues with a benzamidoethyl side chain. These observations further support the previously postulated hypothesis (see Chapter 2) that the benzamidoethyl side chain of the 2-aminotetralin- derived benzamides may occupy a specific binding site in the dopamine D2 and D3 receptors, which may be identical to the binding site of 2-pyrrolidinylmethyl-derived substituted benzamides.

91 Chapter 3

3.5 CONCLUSIONS

The SAFIR of the compounds presented in this chapter revealed that the aromatic ring of the benzamide moiety of 5-OMe-BPAT (1) contributes to the high affinities of this compound for dopamine D2, D3, and serotonin 5-HT1A receptors, although it can be replaced by a cyclohexane ring without loss of affinity. Furthermore, 1 may interact with the dopamine D2 and D3 receptors through hydrogen bond formation with its carbonyl group. Investigation of the role of the amide hydrogen atom by the preparation and evaluation of amide N’-alkylated analogues of 1 was not conclusive, since dynamic NMR and molecular modeling studies on these compounds revealed that conformational effects of amide N’-alkylation may be responsible for the decreased dopaminergic affinities of these compounds. The effects of the modifications of the amide moiety on the serotonin

5-HT1A receptor affinity were less pronounced, suggesting that the benzamidoethyl side chain of 1 as a whole enhances the affinity for this receptor subtype probably not by hydrogen bond formation with its amide moiety, but through hydrophobic interactions with an accessory binding site. The structural requirements for the substituents at the basic nitrogen atom of 1 are comparable to those of the DPATs, supporting the hypothesis that the 2-aminotetralin moieties of the two classes of compounds may share the same binding sites. The benzamidoethyl side chain of 1 probably occupies a specific binding site in the dopamine D2 and D3 receptors.

3.6 EXPERIMENTAL SECTION

3.6.1 CHEMISTRY

General Remarks. See Section 2.6.1. Dynamic 1H and 13C NMR experiments were performed on a Varian Unity Plus 500 MHz NMR spectrometer. Chemical ionisation mass spectra were recorded on a NERMAG R 3010 triple quadrupole mass spectrometer equipped with a home-built atmospheric pressure ionisation source and ionspray interface. Alternatively, chemical ionisation mass spectra were recorded on a Unicam Automass mass spectrometer. Ammonia was used as the reactant gas and samples were introduced into the ion source by means of the direct insertion probe.

5-Methoxy-2-[N-(2-acetamidoethyl)-N-n-propylamino]tetralin Hydrochloride (3): acetic anhydride (0.90 mL, 7 9.5 mmol) was added dropwise at room temperature to a firmly stirred mixture of 2·(HCl)2 (0.50 g, 1.5 mmol),

NaOAc (0.70 g, 0.9 mmol), EtOAc (16 mL) and H2O (5 mL). After stirring overnight at room temperature, the reaction mixture was diluted with H2O (10 mL), the phases were separated and the H2O layer was extracted with EtOAc (2 ´ 15 mL). Subsequently the EtOAc layers were combined and washed with saturated aqueous solutions of

NaHCO3 (3 ´ 20 mL) and NaCl (20 mL) and finally dried over Na2SO4. Evaporation of the solvent under reduced pressure gave the crude acetamide as a brown oil. Purification by silica column chromatography [eluent:

MeOH/CH2Cl2, 1/15 (v/v)] yielded 0.29 g (1.0 mmol, 64%) of the pure base of 3 as a colourless oil: mp 56–58 °C; IR: –1 1 cm 3421 (b), 3246 (b), 3059, 2940, 2837, 2627 (b), 1670, 1588, 1543; H NMR (base, 200 MHz, CDCl3): d 0.89 (t, J = 7.3 Hz, 3H), 1.36–1.66 (m, 3H), 1.96 (s, 3H), 2.45–2.77 (m, 7H), 2.85–3.04 (m, 2H), 3.22–3.29 (m, 2H), 3.79 (s, 13 3H), 6.30 (bs, 1H), 6.66 (dd, J = 8.7 Hz, 8.7 Hz, 2H), 7.06 (t, J = 7.8 Hz, 1H); C NMR (base, 50 MHz, CDCl3): d 11.5, 21.6, 23.0, 23.5, 25.2, 31.8, 37.5, 48.4, 51.9, 55.0, 55.7, 106.8, 121.4, 124.9, 126.1, 137.4, 157.1, 169.9; MS (CI with NH3): m/z (rel. intensity) 161 (28), 202 (11), 232 (48), 275(13), 305 (100, M+1); Anal. calcd for

C18H28N2O2·HCl·H2O: C 60.23, H 8.72, N 7.81; obsd C 60.14, H 8.90, N 7.89.

92 Structural Analogues of 5-OMe-BPAT

5-Methoxy-2-[N-(2-cyclohexylcarboxamidoethyl)-N-n-propylamino]tetralin Hydrochloride (4): a solution of cyclohexylcarboxylic acid chloride (0.55 g, 3.8 mmol) in CH2Cl2 (10 mL) was added dropwise to a firmly stirred, ice- cooled mixture of 2·(HCl)2 (0.50 g, 1.5 mmol), 10% aqueous NaOH solution (12 mL) and CH2Cl2 (50 mL). When addition was complete, the reaction mixture was allowed to warm to room temperature and stirring was continued overnight. The reaction mixture was poured into H2O (50 mL), the phases were separated, and the aqueous phase was extracted with CH2Cl2 (2 × 50 mL) The organic layers were combine and subsequently washed with saturated aqueous

NaHCO3 solution (3 × 50 mL), H2O (50 mL) and brine (50 mL). After drying (Na2SO4) and filtration, the solvent was evaporated, which gave the crude amide as an orange oil. Purification by silica column chromatography [eluent:

MeOH/CH2Cl2, 1/15 (v/v)] yielded 0.26 g (0.7 mmol, 47%) of the pure base of 4 as a colourless oil: mp 60–62 ºC; IR: –1 1 cm ; H NMR (base, 200 MHz, CDCl3): d 0.91 (t, J = 7.3 Hz, 3H), 1.18–2.15 (m, 16H), 2.44–3.06 (m, 8H), 3.23– 3.33 (m, 2H), 3.80 (s, 3H), 6.32 (bs, 1H), 6.67 (dd, J = 8.0 Hz, 8.2 Hz, 2H), 7.09 (t, J = 8.1 Hz, 1H); 13C NMR (base,

50 MHz, CDCl3): d 11.8, 21.9, 23.7, 25.4, 25.8, 29.7, 32.1, 37.2, 45.5, 48.7, 52.2, 55.2, 55.9, 107.0, 121.5, 125.0,

126.2, 137.4, 157.2, 175.9; MS (CI with NH3): m/z (rel. intensity) 161 (7), 232 (48), 373 (100, M+1); Anal. calcd for

C23H36N2O2·HCl·½H2O: C 66.07, H 9.18 , N 6.70; obsd C 65.95, H 9.30, N 6.62.

5-Methoxy-2-[N-(2-phthalimidoethyl)-N-n-propylamino]tetralin Hydrochloride (5): a suspension of phthalic anhydride (0.25 g, 1.7 mmol), NaOAc (0.50 g, 6.1 mmol) and 2·(HCl)2 (0.25 g, 0.7 mmol) in glacial acetic acid (5 mL) was refluxed under a nitrogen atmosphere for 30 min. After cooling, saturated aqueous NaHCO3 solution (25 mL) and CHCl3 (25 mL) were added, stirring was continued for a few minutes and then the phases were separated. The organic layer was washed with saturated aqueous NaHCO3 solution (4 ´ 25 mL), the aqueous layers were collected and washed with CHCl3 (2 ´ 25 mL). The collected CHCl3 layers were subsequently washed with H2O (25 mL), brine (25 mL) and then dried over Na2SO4. After filtration and evaporation of the solvent, the crude phthalimide was afforded as a brown oil. Purification by silica column chromatography [eluent: MeOH/CH2Cl2, 1/50 (v/v)] yielded 0.15 g (0.4 mmol, 51%) of the pure base of 5 as a light yellow oil: mp 228–230 °C dec; IR: cm–1 3461 (b), 2943, 2881, 2842, 1 2362 (b), 1771, 1709, 1590; H NMR (base, 200 MHz, CDCl3): d 0.82 (t, J = 7.3 Hz, 3H), 1.33–1.62 (m, 3H), 1.92– 2.02 (m, 1H), 2.42–3.03 (m, 9H), 3.70–3.79 (m, 5H), 6.64 (dd, J = 7.6 Hz, 7.6 Hz, 2H), 7.06 (t, J = 7.8 Hz, 1H), 7.68– 13 7.74 (m, 2H), 7.79–7.86 (m, 2H); C NMR (base, 50 MHz, CDCl3): d 11.4, 21.8, 23.6, 25.6, 31.8, 37.4, 47.7, 52.6,

55.0, 56.0, 106.7, 121.5, 122.9, 125.0, 125.9, 132.1, 133.6, 137.8, 157.1, 168.3; MS (CI with NH3): m/z (rel. intensity)

161 (2), 232 (4), 393 (100, M+1); Anal. calcd for C24H28N2O3·HCl·¼H2O: C 66.49, H 6.87, N 6.46; obsd C 66.67, H 6.86, N 6.38.

5-Methoxy-2-[N-(2-benzenesulfonamidoethyl)-N-n-propylamino]tetralin (6): benzenesulfonyl chloride (0.29 g,

1.6 mmol) was added dropwise at 0 °C to a vigorously stirred mixture of 2·(HCl)2 (0.50 g, 1.5 mmol), K2CO3 (0.41 g,

3.0 mmol), CHCl3 (50 mL) and H2O (40 mL). The mixture was stirred at room temperature for 1 h and then the phases were separated. The organic layer was washed with H2O (2 ´ 50 mL), dried (Na2SO4) and filtered. The filtrate was concentrated under reduced pressure to give the crude sulfonamide as a brown oil. Purification using silica column chromatography [eluent: MeOH/CH2Cl2, 1/15 (v/v)] yielded 0.51 g (1.3 mmol, 85%) of the pure base of 6 as a colourless oil: mp 104–106 °C; IR: cm–1 3422, 3065, 2939, 2881, 2837, 2600 (b), 2487 (b), 1588, 1329, 1160; 1H

NMR (base, 200 MHz, CDCl3): d 0.76 (t, J = 7.3 Hz, 3H), 1.19–1.48 (m, 3H), 1.83–1.91 (m, 1H), 2.24–2.75 (m, 8H), 2.88–2.99 (m, 3H), 3.78 (s, 3H), 5.27 (bs, 1H), 6.65 (dd, J = 7.9 Hz, 3.6 Hz, 2H), 7.07 (t, J = 7.9 Hz, 1H), 7.42–7.57 13 (m, 3H), 7.85–7.90 (m, 2H); C NMR (base, 50 MHz, CDCl3): d 11.7, 22.0, 23.7, 25.2, 31.9, 40.9, 48.2, 51.9, 55.2,

55.7, 107.0, 121.5, 124.8, 126.3, 127.0, 129.0, 132.6, 137.4, 139.5, 157.2; MS (CI with NH3): m/z (rel. intensity) 148

(21), 186 (56), 220 (33), 263 (33), 305 (17), 353 (27), 403 (100, M+1); Anal. calcd for C22H30N2SO3·HCl·¼H2O: C 59.57, H 7.17, N 6.32; obsd C 59.48, H 7.32, N 6.26.

5-Methoxy-2-[N-(2-benzylaminoethyl)-N-n-propylamino]tetralin Dihydrochloride (7): a solution of 17 (0.50 g,

1.4 mmol) in dry THF (75 mL) was added dropwise to a stirred suspension of LiAlH4 (2.00 g) in dry THF (75 mL). After refluxing under a nitrogen atmosphere overnight, the reaction mixture was cooled to room temperature and excess LiAlH4 was decomposed by subsequent careful addition of H2O (2 mL), 4N aqueous NaOH solution (2 mL) and

H2O (6 mL). The precipitate was removed by filtration and the filtrate was concentrated in vacuo. The resulting oil

93 Chapter 3

was taken up in CH2Cl2 (100 mL) and the solution was dried over Na2SO4. After filtration and evaporation of the solvent, the crude product was purified using silica column chromatography (eluent: CH2Cl2), which gave 0.34 g (1.0 mmol, 71%) of the pure base of 7 as a colourless oil: mp 120–121 °C; IR: cm–1 3404 (b), 2938, 2837, 2619 (b), 1588; 1 H NMR (base, 200 MHz, CDCl3): d 0.89 (t, J = 7.3 Hz, 3H), 1.41–1.63 (m, 3H), 1.99–2.03 (m, 1H), 2.45–3.08 (m, 12H), 3.83 (s, 3H), 3.85 (s, 2H), 6.70 (dd, J = 11.8 Hz, 7.9 Hz, 2H), 7.11 (t, J = 7.9 Hz, 1H), 7.25–7.37 (m, 5H); 13C

NMR (base, 50 MHz, CDCl3): d 11.8, 22.2, 23.9, 25.5, 32.1, 47.5, 49.6, 52.5, 53.9, 55.2, 56.1, 106.9, 121.6, 125.2,

126.1, 126.9, 128.1, 128.4, 138.0, 140.1, 157.2; MS (CI with NH3): m/z (rel. intensity) 72 (41), 161 (90), 232 (100),

353 (2, M+1); Anal. calcd for C23H32N2O·(HCl)2·¼H2O: C 64.24, H 8.10, N 6.52; obsd C 64.23, H 8.62, N 6.48.

5-Methoxy-2-{N-[2-(N’-methyl)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (8): powdered KOH (0.36 g, 6.4 mmol) was added to DMSO (2 mL) and the suspension was stirred for 5 min. Then a solution of 17

(0.57 g, 1.6 mmol) in DMSO (1 mL) was added, immediately followed by CH3I (0.45 g, 3.2 mmol). Stirring was continued overnight at room temperature, after which the reaction mixture was poured into H2O (20 mL) and extracted with CH2Cl2 (3 ´ 20 mL). After subsequent washing with saturated aqueous NaHCO3 solution (3 ´ 10 mL),

H2O (10 mL), and brine (10 mL), the organic layer was dried (Na2SO4) and filtered. After evaporation of the solvent, the residue was purified by silica column chromatography [eluent: MeOH/CH2Cl2, 1/20 (v/v)], which yielded g (mmol, %) of the pure base of 8 as a colourless oil: mp 91–93 °C; IR: cm–1 3416 (b), 2937, 2836, 2452 (b), 1630, 1588; 1H

NMR (base, 500 MHz, C2D4Cl2, 95 ºC): d 0.90 (as, 3H), 1.44–1.67 (m, 3H), 2.02–2.09 (m, 1H), 2.48–3.03 (m, 7H), 3.07 (s, 3H), 3.46–3.58 (m, 2H), 3.83 (s, 3H), 6.69 (dd, J = 13.5 Hz, 7.9 Hz, 2H), 7.09 (t, J = 7.9 Hz, 1H), 7.37–7.45 13 (m, 5H); C NMR (base, 125 MHz, C2D4Cl2, 95 ºC): d 13.7, 23.8, 25.4, 27.4, 33.8, 50.3, 55.3, 57.5, 60.2, 110.0,

123.6, 126.8, 128.5, 128.9, 130.4, 131.5, 138.6, 159.4, 173.6; MS (CI with NH3): m/z (rel. intensity) 69 (28), 153 (80),

220 (100), 247 (22), 381 (7, M+1); Anal. calcd for C24H32N2O2·HCl·½H2O: C 67.65, H 8.06, N 6.58; obsd C 67.68, H 8.19, N 6.50.

5-Methoxy-2-{N-[2-(N’-ethyl)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (9): this compound –1 was essentially prepared as described for 8, using ethyl iodide instead of CH3I. Yield 59%; mp 96–98 ºC; IR: cm 1 3421 (b), 2966, 2880, 2836, 2457, 1628, 1588; H NMR (base, 500 MHz, C2D4Cl2, 95 ºC): d 0.88 (as, 3H), 1.18 (as, 3H), 1.30–1.55 (m, 3H), 2.01–2.07 (m, 1H), 2.53–3.01 (m, 9H), 3.44–3.50 (m, 4H), 3.82 (s, 3H), 6.68 (dd, J = 16.1

Hz, 7.6 Hz, 2H), 7.09 (t, J = 6.0 Hz, 1H), 7.37–7.42 (m, 5H); MS (CI with NH3): m/z (rel. intensity) 69 (31), 112 (20),

150 (44), 220 (51), 395 (100, M+1); Anal. calcd for C25H34N2O2·HCl·½H2O: C 68.23 , H 8.26 , N 6.37 ; obsd C 68.15, H 8.38, N 6.28.

5-Methoxy-2-{N-[2-(N’-n-propyl)benzamidoethyl]-N-n-propylamino}tetralin Hydrochloride (10): this compound was essentially prepared as described for 8, using n-propyl iodide instead of CH3I. Yield 42%; mp 118–120 –1 1 °C; IR: cm ; H NMR (base, 500 MHz, C2D4Cl2, 95 ºC): d 0.88 (as, 6H), 1.34–1.75 (m, 6H), 1.96–2.10 (m, 1H), 2.37–3.12 (m, 8H), 3.27–3.58 (m, 4H), 3.82 (s, 3H), 6.66–6.69 (m, 2H), 7.09 (t, J = 7.9 Hz, 1H), 7.37–7.44 (m, 5H);

MS (CI with NH3): m/z (rel. intensity) 164 (14), 367 (6), 409 (100, M+1); Anal. calcd for C26H36N2O2·HCl·½H2O: C 68.76, H 8.45, N 6.17; obsd C 68.71, H 8.51, N 6.08.

5-Methoxy-2-(N-cyanomethyl-N-benzylamino)tetralin Hydrochloride (12): bromoacetonitrile (2.44 g, 20.34 10 mmol) was added to a mixture of 11·HCl (2.47 g, 8.1 mmol), K2CO3 (2.81 g, 20.3 mmol) and KI (0.34 g, 2.1 mmol) in acetone (100 mL). After refluxing overnight, the reaction mixture was cooled and the solids were removed by filtration. The filtrate was concentrated and the resulting oil was purified by silica column chromatography (eluent:

CH2Cl2), yielding 2.00 g (6.5 mmol, 80%) of the pure base of 12 as a light yellow oil: mp 173–175 °C (EtOH); IR: –1 1 cm 2985, 2928, 2889, 2829, 2262 (b), 1601, 1587; H NMR (base, 200 MHz, CDCl3): d 1.72–1.91 (m, 1H), 2.30– 2.36 (m, 1H), 2.61–2.78 (m, 1H), 2.89–3.18 (m, 4H), 3.52 (s, 2H), 3.84–4.00 (m, 2H), 3.88 (s, 2H), 6.79 (dd, J = 12.0 13 Hz, 7.8 Hz, 2H), 7.20 (t, J = 7.8 Hz, 1H), 7.32–7.46 (m, 5H); C NMR (base, 50 MHz, CDCl3): d 22.7, 26.4, 33.3, 38.2, 54.1, 55.1, 57.5, 107.2, 116.5, 121.4, 126.4, 127.6, 128.6, 128.8, 136.2, 137.8, 157.1; MS (CI with AcOH): m/z 307 (M+1).

94 Structural Analogues of 5-OMe-BPAT

5-Methoxy-2-[N-(2-aminoethyl)-N-benzylamino]tetralin Oxalate (13): a suspension of 12 (1.50 g, 4.4 mmol) and LiAlH4 (2.00 g) in dry THF (100 mL) was refluxed overnight under a nitrogen atmosphere. After cooling to room temperature, excess LiAlH4 was decomposed as described for 7. The precipitate was removed by filtration and the filtrate was dried over Na2SO4. After filtration and evaporation of the solvent, 1.29 g (4.2 mmol, 95%) of the pure base of 13 was obtained as a colourless oil: mp 120–121 °C (EtOH); IR: cm–1 3413 (b), 2937 (b), 2837, 2534 (b), 1719, 1 1589; H NMR (base, 200 MHz, CDCl3): d 1.60–1.74 (m, 1H), 1.95 (bs, 2H), 2.13–2.20 (m, 1H), 2.46–2.77 (m, 5H), 2.86–3.14 (m, 4H), 3.75 (s, 2H), 3.82 (s, 3H), 6.72 (dd, J = 18.8 Hz, 7.8 Hz, 2H), 7.13 (t, J = 7.9 Hz, 1H), 7.28–7.44 13 (m, 5H); C NMR (base, 50 MHz, CDCl3): d 23.7, 24.8, 31.9, 40.0, 52.6, 54.6, 55.0, 55.6, 106.8, 121.5, 125.1, 126.1, 126.7, 128.2, 128.4, 137.8, 140.8, 157.2; MS (CI with AcOH): m/z 311 (M+1).

5-Methoxy-2-[N-(2-benzamidoethyl)-N-benzylamino]tetralin Hydrochloride (14): this compound was essentially prepared as described for 4, starting from 13. Yield 53%; mp 112–114 °C; IR: cm–1 3261 (b), 2941, 2835, 1 2489 (b), 1655, 1588, 1535; H NMR (base, 200 MHz, CDCl3): d 1.64–1.72 (m, 1H), 2.18–2.23 (m, 1H), 2.50–2.64 (m, 1H), 2.87–2.96 (m, 4H), 3.05–3.16 (m, 2H), 3.48–3.54 (m, 2H), 3.71–3.87 (m, 5H), 6.73 (dd, J = 13.7 Hz, 7.7 Hz, 2H), 7.04–7.09 (m, 1H), 7.15 (t, J = 7.7 Hz, 1H), 7.23–7.57 (m, 8H), 7.79 (dd, J = 8.1 Hz, 1.7 Hz, 2H); 13C NMR

(base, 50 MHz, CDCl3): d 23.8, 25.1, 32.1, 38.0, 48.1, 54.6, 55.2, 56.1, 107.1, 121.6, 125.0, 126.4, 127.0, 127.3,

128.5, 128.6, 128.8, 131.3, 134.7, 137.3, 140.0, 157.2, 167.3; MS (CI with NH3): m/z (rel. intensity) 106 (21), 139

(20), 174 (32), 206 (78), 280 (14), 325 (28), 415 (100, M+1); Anal. calcd for C27H30N2O2·HCl·¾H2O: C 69.80, H 7.07, N 6.03; obsd C 69.95, H 7.09, N 6.11.

5-Methoxy-2-[N-(2-benzamidoethyl)amino]tetralin Oxalate (15): a solution of the free base of 14 (0.56 g, 1.4 mmol) in absolute EtOH (50 mL) was transferred to a Parr flask, 10% Pd-on-C catalyst (0.30 g) was added and the solution was hydrogenated overnight under 4 atm H2 at room temperature. The catalyst was removed by filtration and the solvent was evaporated, yielding 0.36 g (1.1 mmol, 82%) of the pure base of 15 as a colourless oil: mp 208–210 ºC –1 1 dec (MeOH); IR: cm 3281 (b), 3045, 2936, 2836, 2505, 1735, 1632, 1562; H NMR (base, 200 MHz, CDCl3): d 1.55–1.70 (m, 2H), 2.00–2.09 (m, 1H), 2.51–2.67 (m, 2H), 2.81–3.06 (m, 5H), 3.50–3.58 (m, 2H), 3.81 (s, 3H), 6.68 (dd, J = 7.8 Hz, 4.2 Hz, 2H), 6.96 (bs, 1H), 7.10 (t, J = 7.9 Hz, 1H), 7.36–7.53 (m, 3H), 7.53–7.79 (m, 2H); 13C NMR

(base, 50 MHz, CDCl3): d 21.4, 28.8, 36.4, 39.8, 45.4, 52.4, 55.0, 106.9, 121.4, 124.8, 126.1, 126.8, 128.4, 131.2,

134.5, 136.2, 157.0, 167.4; MS (CI with AcOH): m/z 325 (M+1); Anal. calcd for C20H24N2O2·C2H2O4: C 63.74, H 6.34, N 6.76; obsd C 63.59, H 6.36, N 6.76.

5-Methoxy-2-[N-(2-benzamidoethyl)-N-methylamino]tetralin Oxalate (16): a solution of 15 (0.10 g, 0.3 mmol) in MeOH (50 mL) was transferred to a Parr flask, 37% aqueous formaldehyde solution (5 mL) and 10% Pd-on-C catalyst (0.10 g) were added, and the reaction mixture was hydrogenated overnight under 4 atm H2 at 55 ºC. The catalyst was removed by filtration and the filtrate was concentrated. The residue was dissolved in CH2Cl2 and subsequently washed with 10% aqueous NaHCO3 solution, H2O and brine. After drying (Na2SO4) and filtering, the solvent was evaporated, which gave the crude product as a yellow oil. Purification by silica column chromatography

[eluent: MeOH/CH2Cl2, 1/20 (v/v)] yielded 80 mg (0.24 mmol, 77%) of the pure base of 16 as a colourless oil: mp –1 1 141–143 ºC; IR: cm 3395 (b), 3065, 2949, 2837, 1719, 1654, 1588, 1542; H NMR (base, 200 MHz, CDCl3): d 1.62– 1.73 (m, 1H), 2.03–2.09 (m, 1H), 2.37 (s, 3H), 2.47–2.64 (m, 1H), 2.74–3.05 (m, 6H), 3.51–3.59 (m, 2H), 3.81 (s, 3H), 6.69 (dd, J = 7.3 Hz, 7.3 Hz, 2H), 7.00 (bs, 1H), 7.10 (t, J = 7.8 Hz, 1H), 7.38–7.54 (m, 3H), 7.76 (d, J = 8.1 Hz, 13 2H); C NMR (base, 50 MHz, CDCl3): d 23.0, 25.4, 31.5, 36.9, 51.4, 55.0, 59.2, 106.9, 121.4, 124.9, 126.2, 126.8, 131.1, 134.6, 136.9, 157.1, 167.3; MS (CI with AcOH): m/z 325 (M+1); Anal. calcd for: C, H, N; obsd C, H, N.

5-Methoxy-2-[N-(2-benzamidoethyl)-N-ethylamino]tetralin Hydrochloride (17): this compound was prepared essentially as described for 16, using acetaldehyde instead of formaldehyde. Yield 88%; mp 104–105 °C; IR: cm–1 1 3422 (b), 3271 (b), 2938, 2836, 2617 (b), 2472 (b), 2357, 1649, 1588, 1534; H NMR (base, 200 MHz, CDCl3): d 1.12 (t, J = 7.3 Hz, 3H), 1.24–1.73 (m, 1H), 2.02–2.17 (m, 1H), 2.47–3.07 (m, 9H), 3.40–3.60 (m, 2H), 3.81 (s, 3H), 6.68 (t, J = 7.3 Hz, 2H), 7.10 (t, J = 8.1 Hz, 1H), 7.18 (bs, 1H), 7.39–7.54 (m, 3H), 7.80–7.85 (m, 2H); 13C NMR (base, 50

MHz, CDCl3): d 14.1, 23.6, 25.7, 32.3, 38.1, 44.1, 480, 55.2, 55.8, 107.0, 121.5, 125.0, 126.3, 126.9, 128.5, 131.2,

95 Chapter 3

134.7, 137.4, 157.2, 167.2; MS (CI with NH3): m/z (rel. intensity) 161 (14), 193 (20), 218 (57), 305 (7), 353 (100,

M+1); Anal. calcd for C22H28N2O2·HCl·¼H2O: C 67.15, H 7.57, N 7.12; obsd C 67.43, H 7.92, N 7.17. 5-Methoxy-2-[N-(2-benzamidoethyl)-N-allylamino]tetralin Hydrochloride (18): allyl bromide (0.10 g, 0.8 mmol) was added to a stirred suspension of 15 (0.10 g, 0.2 mmol), Cs2CO3 (0.23 g, 0.7 mmol) and a catalytic amount of KI in MeCN (50 mL). The reaction mixture was refluxed overnight, cooled to room temperature and then the solids were removed by filtration. The filtrate was concentrated in vacuo, which gave the crude tertiary amine as a yellow oil.

Purification by silica column chromatography [eluent: MeOH/CH2Cl2, 1/20 (v/v)] gave the pure base of 18 as a colourless oil. Yield 32 mg (0.1 mmol, 36%); mp 100–102 °C; IR: cm–1 3331 (b), 3065, 2930, 2836, 1633, 1601, 1 1584; H NMR (base, 300 MHz, CDCl3): d 1.56–1.70 (m, 1H), 2.03–2.08 (m, 1H), 2.48–2.60 (m, 1H), 2.75–2.90 (m, 4H), 2.96–3.09 (m, 2H), 3.28 (d, J = 6.2 Hz, 2H), 3.44–3.57 (m, 2H), 3.80 (s, 3H), 5.12 (d, J = 9.9 Hz, 1H), 5.23 (d, J = 17.0 Hz, 1H), 5.80–5.93 (m, 1H), 6.67 (t, J = 8.4 Hz, 2H), 6.90 (bs, 1H), 7.09 (t, J = 7.9 Hz, 1H), 7.42–7.53 (m, 13 3H), 7.77 (d, J = 8.1 Hz, 2H); C NMR (base, 75 MHz, CDCl3): d 23.8, 25.8, 32.5, 37.9, 48.1, 53.6, 55.4, 56.2,

107.2, 117.4, 121.7, 125.1, 126.5, 127.0, 128.8, 131.5, 135.0, 136.9, 137.5, 157.4, 167.4; MS (CI with NH3): m/z (rel. intensity) 77 (33), 105 (53), 161 (79), 203 (32), 230 (100), 324 (2), 365 (3, M+1); Anal. calcd for

C23H28N2O2·HCl·½H2O: C 67.37, H 7.39, N 6.83; obsd C 67.16, H 7.77, N 6.75.

5-Methoxy-2-[N-(3-phthalimidopropyl)-N-n-propylamino]tetralin Oxalate (20): a stirred suspension of N-(3- 1 bromopropyl)phthalimide (3.14 g, 11.7 mmol), K2CO3 (1.62 g, 11.7 mmol), KI (0.13 g, 0.78 mmol) and 19·HCl (1.00 g, 3.9 mmol) in MeCN (150 mL) was refluxed for 24 h. After cooling, the reaction mixture was filtered and the MeCN was evaporated from the filtrate. The crude product was purified by silica column chromatography [eluent: EtOAc/petroleum ether (bp 40–60), 1/3 (v/v)], which yielded 1.16 g (2.9 mmol, 74%) of the pure base of 20 as a colourless oil: mp 157–159 °C; IR: cm–1 2980, 2943, 2836, 2669 (b), 1768, 1698, 1591; 1H NMR (base, 200 MHz,

CDCl3): d 0.90 (t, J = 7.3 Hz, 3H), 1.41–1.56 (m, 3H), 1.76–1.90 (m, 2H), 1.94–2.07 (m, 1H), 2.47 (dd, J = 8.2 Hz, 6.5 Hz, 2H), 2.61 (t, J = 7.1 Hz, 2H), 2.76–3.03 (m, 5H), 3.71–3.78 (m, 5H), 6.65 (dd, J = 10.1 Hz, 8.0 Hz, 2H), 7.05 13 (t, J = 7.9 Hz, 1H), 7.66–7.70 (m, 2H), 7.79–7.84 (m, 2H); C NMR (base, 50 MHz, CDCl3): d 11.9, 22.2, 23.9, 25.5, 27.9, 31.9, 36.5, 47.9, 52.3, 55.1, 56.2, 106.8, 121.6, 123.0, 125.2, 126.1, 132.2, 133.8, 138.0, 157.2, 168.3; MS (CI with NH3): m/z 407 (M+1); Anal. calcd for C25H30N2O3·C2H2O4: C 65.30, H 6.51, N 5.64; obsd C 65.29, H 6.43, N 5.34.

5-Methoxy-2-[N-(3-aminopropyl)-N-n-propylamino]tetralin Oxalate (21): hydrazine hydrate (10 mL) was added slowly to a stirred solution of 20 (0.90 g, 2.2 mmol) in absolute EtOH (50 mL). The reaction mixture was stirred for 1 h at room temperature and then the EtOH was removed in vacuo. The resulting oil was taken up in

EtOAc and the solution was washed with a saturated aqueous K2CO3 solution. The H2O layer was extracted with

EtOAc (2 ´ 25 mL), the organic layers were combined and subsequently washed with H2O (25 mL) and brine (25 mL).

After drying over Na2SO4, the organic layer was filtered and evaporated, yielding 0.48 g (1.7 mmol, 79%) of the pure base of 21 as a colourless oil: mp 136–138 °C (EtOH); IR: cm–1 3412 (b), 2965, 2837, 2762, 2640, 2538, 2120 (b), 1 1592, 1518; H NMR (base, 200 MHz, CDCl3): d 0.89 (t, J = 7.3 Hz, 3H), 1.38–1.67 (m, 8H), 1.98–2.08 (m, 1H), 2.43–3.06 (m, 10H), 3.79 (s, 3H), 6.67 (dd, J = 13.0 Hz, 7.9 Hz, 2H), 7.08 (t, J = 7.9 Hz, 1H); 13C NMR (base, 50

MHz, CDCl3): d 11.9, 22.1, 23.9, 25.4, 32.0, 32.5, 40.8, 48.2, 52.3, 55.2, 56.0, 106.8, 121.6, 125.3, 126.1, 138.1, 157.2; MS (CI with AcOH): m/z 277 (M+1).

5-Methoxy-2-[N-(3-benzamidopropyl)-N-n-propylamino]tetralin Hydrochloride (22): this compound was prepared as described for 4, starting from 21. Yield 63%; mp 62–63 ºC; IR: cm–1 3408 (b), 3264 (b), 3057, 2937, 1 2836, 2620, 2494, 1647, 1588, 1541; H NMR (base, 200 MHz, CDCl3): d 0.90 (t, J = 7.3 Hz, 3H), 1.49–1.63 (m, 3H), 1.75–1.87 (m, 2H), 2.03–2.11 (m, 1H), 2.50–2.59 (m, 3H), 2.75–2.81 (m, 4H), 2.93–3.08 (m, 2H), 3.53–3.65 (m, 2H), 3.79 (s, 3H), 6.63 (dd, J = 7.2 Hz, 6.5 Hz, 2H), 7.08 (t, J = 7.9 Hz, 1H), 7.37–7.49 (m, 3H), 7.80–7.84 (m, 2H), 8.34 13 (bs, 1H); C NMR (base, 50 MHz, CDCl3): d 12.0, 21.5, 23.7, 24.7, 25.99, 31.7, 40.6, 49.9, 52.3, 55.2, 56.1, 107.0,

121.5, 124.9, 126.3, 127.0, 128.4, 131.1, 134.9, 137.2, 157.2, 167.4; MS (CI with NH3): m/z (rel. intensity) 220 (11),

381 (100, M+1); Anal. calcd for C24H32N2O2·HCl·¾H2O: C 66.95, H 8.07, N 6.51; obsd C 67.28, H 8.43, N 6.56.

96 Structural Analogues of 5-OMe-BPAT

3.6.2 PHARMACOLOGY

3 3 [ H]-Raclopride Binding to Cloned Dopamine D2A and D3 Receptors, [ H]-8-OH-DPAT Binding to Serotonin

5-HT1A Receptors, and Data Analysis were performed essentially as described in Section 2.6.2.

3.6.3 MOLECULAR MODELING

Calculations were performed on a Silicon Graphics Indy Workstation running IRIX 5.3. N-methyl- and N,N- dimethylbenzamide were build in the molecular modeling package SYBYL 6.314 from standard fragments using the sketch mode. After minimising the starting structures within the Tripos force field with default options selected, rotamers were generated by stepwise rotating the amide bonds with a resolution of 10º. The energies of the resulting rotamers were then calculated semi-empirically with the AM1 method5 as implemented in MOPAC 5.0 and accessed through SYBYL, applying the additional keywords 1SCF and MMOK(0).

3.7 REFERENCES

1 Ames DE, Evans D, Grey TF, Islip PJ and Richards KE (1965) The synthesis of alkoxy-1,2,3,4- tetrahydronaphthalene derivatives. Part I. 2-Amino-, alkylamino-, and dialkylamino-derivatives. J Chem Soc 2636–2641. 2 Arvidsson LE, Hacksell U, Johansson AM, Nilsson JL, Lindberg P, Sanchez D, Wikström H, Svensson K, Hjorth S and Carlsson A (1984) 8-Hydroxy-2-(alkylamino)tetralins and related compounds as central 5-hydroxytryptamine receptor agonists. J Med Chem 27, 45–51. 3 Björk L, Höök BB, Nelson DL, Andén N-E and Hacksell U (1989) Resolved N,N-dialkylated 2-amino-8-

hydroxytetralins: stereoselective interactions with 5-HT1A receptors in the brain. J Med Chem 32, 779–783. 4 Copinga S, Tepper PG, Grol CJ and Dubocovich ML (1993) 2-Amido-8-methoxytetralins: a series of nonindolic melatonin-like agents. J Med Chem 36, 2891–2898. 5 Dewar MJS, Zoebisch EG, Healy EF and Stewart JJP (1985) AM1: A new general purpose quantum mechanical molecular model. J Am Chem Soc 107, 3902–3909. 6 Hacksell U, Svensson U, Nilsson JL, Hjorth S, Carlsson A, Wikström H, Lindberg P and Sanchez D (1979) N- Alkylated 2-aminotetralins: central dopamine-receptor stimulating activity. J Med Chem 22, 1469–1475. 7 Homan EJ, Copinga S, Elfström L, Van Der Veen T, Hallema J-P, Mohell N, Unelius L, Johansson R, Wikström H

and Grol CJ (1998) 2-Aminotetralin-derived substituted benzamides with mixed dopamine D2, D3, and serotonin

5-HT1A receptor binding properties: A novel class of potential atypical antipsychotic agents. Bioorg Med Chem (in press). 8 Johnstone RAW and Rose ME (1979) A rapid, simple, and mild procedure for alkylation of phenols, , amides and acids. Tetrahedron 35, 2169–2173. 9 Malmberg Å, Nordvall G, Johansson AM, Mohell N and Hacksell U (1994) Molecular basis for the binding of 2-

aminotetralins to human dopamine D2A and D3 receptors. Mol Pharmacol 46, 299–312. 10 McDermed JD, McKenzie GM and Freeman HS (1976) Synthesis and dopaminergic activity of (±)-, (+)-, and (–)- 2-dipropylamino-5-hydroxy-1,2,3,4-tetrahydronaphthalene. J Med Chem 19, 547–549. 11 Naiman N, Lyon RA, Bullock AE, Rydelek LT, Titeler M and Glennon RA (1989) 2-(Alkylamino)tetralin

derivatives: interaction with 5-HT1A serotonin binding sites. J Med Chem 32, 253–256. 12 Seiler MP, Markstein R, Walkinshaw MD and Boelsterli JJ (1989) Characterization of dopamine receptor subtypes by comparative structure-activity relationships: dopaminomimetic activities and solid state conformation of monohydroxy-1,2,3,4,4a,5,10,10a-octahydrobenz[g]quinolines and its implications for a rotamer-based dopamine receptor model. Mol Pharmacol 35, 643–651.

97 Chapter 3

13 Seiler MP, Stoll AP, Closse A, Frick W, Jaton A and Vigouret JM (1986) Structure-activity relationships of dopaminergic 5-hydroxy-2-aminotetralin derivatives with functionalized N-alkyl substituents. J Med Chem 29, 912-917. 14 SYBYL Molecular Modeling Software version 6.3. Tripos Inc, 1699 S Hanley Rd, St Louis, MI 63144-2913, USA. 15 Van Vliet LA, Tepper PG, Dijkstra D, Damsma G, Wikström H, Puglsey TA, Akunne HC, Heffner TG, Glase SA

and Wise LD (1996) Affinity for dopamine D2, D3, and D4 receptors of 2-aminotetralins. Relevance of D2 agonist binding for determination of receptor subtype selectivity. J Med Chem 39, 4233-4237. 16 Wikström H, Andersson B, Sanchez D, Lindberg P, Arvidsson LE, Johansson AM, Nilsson JL, Svensson K, Hjorth S and Carlsson A (1985) Resolved monophenolic 2-aminotetralins and 1,2,3,4,4a,5,6,10b-octahydro- benzo[f]quinolines: structural and stereochemical considerations for centrally acting pre- and postsynaptic dopamine-receptor agonists. J Med Chem 28, 215–225.

98 C5-SUBSTITUTED DERIVATIVES OF 5-OME-BPAT: SYNTHESIS AND INTERACTIONS WITH 4 DOPAMINE D2 AND SEROTONIN 5-HT1A RECEPTORS

ABSTRACT

Eight new C5-substituted derivatives of the potential atypical antipsychotic agent 5-methoxy-2-[N-(2- benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 19) have been prepared by chemical conversion of the 5- trifluoromethylsulfonyloxy (triflate) analogue 22 via various Stille-type cross-couplings, a Heck reaction, and an amidation in moderate to good yields. The 5-acetyl, 5-cyano, 5-methyl, 5-(2-furyl), 5-phenyl, methyl 5-carboxylate, and the 5-carboxamido analogues 23–29 thus obtained, the previously disclosed 5-methoxy, 5-hydroxy, and 5- unsubstituted analogues 19–21, and the 5-triflate analogue 22 were evaluated for their ability to compete for [3H]- 3 spiperone binding to rat striatal membranes containing dopamine D2 receptors, and their ability to compete for [ H]-8-

OH-DPAT binding to rat frontal cortex membranes containing serotonin 5-HT1A receptors in vitro. Compounds 19–29 displayed weak to high affinities for the dopamine D2 receptor, with Ki-values ranging from 550 nM for the 5- carboxamido analogue to 4.9 nM for the 5-hydroxy analogue. Therefore, the 5-methoxy substituent of 19 is likely to be involved in the binding to this receptor subtype. The affinity ranking of the 5-methoxy, 5-hydroxy, and 5-unsubstituted analogues suggested that these analogues may bind to the same site and in a similar way as the 5-oxygenated DPATs, the 5-methoxy substituent of 19 functioning as a hydrogen bond acceptor. The inconsistencies in the SAFIR of the other analogues suggest that they may bind to the dopamine D2 receptor in a different way. The serotonin 5-HT1A receptor tolerates more chemical diversity at the C5-position of 19, since all analogues showed moderate to high affinities for this receptor subtype, with Ki-values ranging from 60nM for the 5-carboxamido analogue to 4.9 nM for the 5-unsubstituted analogue. PLS analysis of a set of 24 molecular descriptors, generated for each analogue, revealed no significant correlation between the dopamine D2 receptor affinities of 19–29 and their molecular properties, supporting the view that they may have different binding modes at this receptor subtype. A PLS model with moderate 2 predictability (Q = 0.49) could be derived for the serotonin 5-HT1A receptor affinities of 19–29. According to the model, a relatively lipophilic, nonpolar C5-substituent, incapable of forming hydrogen bonds, should be optimal for a high affinity at this receptor subtype.

99 Chapter 4

4.1 INTRODUCTION

During recent years trifluoromethanesulfonate esters of phenols (aryl triflates) have found more and more application in the field of medicinal chemistry, particularly in research devoted to diseases of the CNS (for examples, see refs. 3, 21, 29, and 48). The triflate group has strong electron- withdrawing properties,53 and therefore the aliphatic triflate substituent has excellent leaving group capabilities.54 In contrast, aryl triflates are highly stable, as demonstrated by their chemical stability under solvolytic conditions.56 These properties of aryl triflates have led to the idea that the triflate group may serve as a suitable bioisostere for electron-donating aromatic substituents such as hydroxy and methoxy, functionalities which are frequently encountered in compounds with CNS activity. Substitution of these functionalities by a triflate group should make such compounds less prone to metabolic degradation in vivo, thereby enhancing the bioavailability. In addition, the increased lipophilicity* of the triflate group should also be beneficial for the bioavailabilty and the capability of the compounds to penetrate into the CNS. Several examples of successful application of this triflate concept, resulting in compounds with improved bioavailabilty and metabolic stability, have been reported recently.15,49,50,52 It should be noted, however, that introduction of an aryl triflate group usually also affects the pharmacodynamic, i.e. receptor binding properties, of the compounds under investigation (e.g. see ref. 28). Despite their chemical stability, aryl triflates can be synthetically transformed under specific reaction conditions. The Heck reaction, the Stille reaction, and the Suzuki reaction offer powerful tools to transform an aryl triflate group into a variety of functionalities (for review and references, see ref. 42). Since these reactions tolerate the presence of many other functional groups, they can be employed to introduce chemical diversity in a series of compounds under investigation at a late stage in the synthetic route. The Heck reaction offers one of the most important methods for the creation of carbon-carbon bonds at unsubstituted vinylic positions (for reviews see refs. 9 and 19). In its original form this palladium-catalyzed reaction described the vinylation and arylation of organic halides,18,37 but vinyl and aryl triflates also proved to be suitable substrates. A schematic representation of the reaction mechanism of the Heck reaction between an aryl triflate and an olefin is shown in Scheme 4.1. The Heck reaction requires the presence of a palladium(0) species 1, which is usually generated in situ by reduction of a Pd(II) salt, presumably by oxidizing some of the olefin20,37 or the added phosphine.1,38 The phosphine-coordinated Pd(0) species then undergoes oxidative addition of the aryl triflate, resulting in the aryl palladium(II) triflate complex 2. Subsequent coordination of the olefin forms the cationic p-palladium(II) complex 3.

* The aromatic triflate substituent has an estimated p value of 1.23. Nilsson JE, personal communication.

100 C5-Substituted Derivatives of 5-OMe-BPAT

2 Pd X2

[L] + – BH OTf ArOTf 0 Pd L2 Base B 1

L L 2 2 L Pd OTf L Pd OTf H Ar Ar Ar R and 10 2 b a R R 8 9

+ + + L Ar L L 2 – 2 – 2 – L Pd OTf and L Pd OTf L Pd OTf H R H Ar R Ar R

6 7 3

L L 2 2 Ar L Pd OTf Ar L Pd OTf and H R R H 4 5

SCHEME 4.1 Schematic representation of the mechanism of the Heck reaction between an aryl triflate (ArOTf) and an olefin.

Syn-insertion of the palladium and the aryl group to the double bond gives rise to two possible s- alkylpalladium species 4 and 5. Instantaneous syn hydrogen elimination of a hydridopalladium species HPd(II), yields two possible new cationic p-palladium(II) complexes, 6 and 7. Subsequent decomposition of these complexes results in the b- and a-coupled products 8 and 9. The hydridopalladium(II) triflate 10 decomposes to the Pd(0) species 1 and triflic acid, which is neutralized by the added base. Isomerization of 6 and 7 may, in addition to 9, give rise to different mixtures of syn-b- and anti-b-coupled products (syn-8 and anti-8). For unsymmetrically substituted olefins the regioselectivity of the reaction depends on both steric and electronic properties of the substituents, but also on the nature of the substituents on the aryl substrates, and the type of phosphine ligand employed (for review and references see ref. 42). Heck coupling of aryl triflates with butyl vinyl ether (11, Scheme 4.2) can be utilized to prepare aryl methyl ketones, offering an alternative to Friedel-Crafts acylations and related reactions. Cabri and co-workers have shown that in the presence of 1,3-bis(diphenylphophino)propane (dppp) as a ligand for the palladium(0) species exclusively the a-arylated products are formed, independent on the electronic properties of the aryl substrate substituents.4 Subsequent acid hydrolysis of the aryl ethenyl ether 12 results in the aryl methyl ketone 13.

101 Chapter 4

Ar Ar + dppp H O + ArOTf O O 11 12 13

SCHEME 4.2 a-Regioselective Heck coupling of vinyl butyl ether (11) with an aryl triflate (ArOTf) under the control of 1,3-bis(diphenylphophino)propane (dppp). Acid hydrolysis of the resulting aryl ethenyl ether 12 yields the aryl methyl ketone 13. An alternative method for the formation of carbon-carbon bonds is the cross-coupling reaction between an organic electrophile and an organometallic reagent. Cross-couplings between organostannanes and vinyl or aryl triflates, which require the presence of a palladium catalyst, are commonly referred to as Stille reactions.11,47 A schematic representation of the Stille reaction between an aryl triflate and an organostannane is shown in Scheme 4.3 (for reviews and references see refs. 42 and 55). Oxidative addition of the aryl triflate ArOTf to a palladium(0) species 1 yields

2 Pd X2

[L]

0 Pd L2 Ar R 1 ArOTf

+ L L 2 2 – L Pd R L Pd OTf Ar Ar +L 16 14 -L

n-Bu3SnCl LiCl

L L S Pd 2 R L Pd 2 Cl n-Bu SnR LiOTf Ar 3 Ar 18 15

n-Bu3SnCl +L -L L S Pd 2 Cl n-Bu3SnR Ar 17

SCHEME 4.3 Schematic representation of the mechanism of the Stille reaction between an aryl triflate (ArOTf) and an organostannane.

102 C5-Substituted Derivatives of 5-OMe-BPAT the (s-aryl)palladium(II) complex 14, which has been shown to exist as a fully dissociated cationic complex in coordinating solvents, such as dimethylformamide (DMF) and N-methylpyrrolidone (NMP), with TfO– as counteranion.26 Reaction of the relatively instable complex 14 with lithium chloride, the presence of which is essential for completion of the reaction, yields the stable arylpalladium(II) chloride complex 15 and lithium triflate (LiOTf). Subsequent transmetalation with the organostannane yields a bisorganopalladium(II) complex 16, which upon reductive elimination results in the regeneration of 1 and the crosscoupled product Ar–R. In this catalytic cycle, as originally proposed by Scott and Stille, the transmetalation is the rate-determining step.47 When the typical Stille ligand triphenylphosphine is employed, this step proceeds relatively slow and elevated temperatures are required in order to complete the reaction. Large rate enhancements and reductions in reaction temperatures may be obtained by employing less strong electron-donating ligands such as triphenylarsine and tri(2-furyl)phosphine.12,13 In these cases, 15 presumably may lose one of its ligands L, resulting in the ‘ligandless’ complex 17, which is stabilized by the coordinating solvent S. Rapid transmetalation of 17 then yields 18, which upon re-addition of ligand results in 16. Thus, the reaction performance is compromised by the nature of the ligand: strongly donating ligands (e.g. triphenylphosphine) favor the oxidative addition of the aryl triflate, but therefore also will less readily dissociate from the organopalladium(II) complexes, and hence slowing down the transmetalation with the organostannane. The latter is favored by ligands with weaker electron-donating properties (e.g. triphenylarsine and tri(2-furyl)phosphine), but poorly donating ligands may insufficiently stabilize the palladium(0) species, which may lead to catalyst decomposition and precipitation of metallic palladium. The scope of the Stille reaction is also dependent on the nature of the aryl triflate. Sterically hindered aryl triflates with electron-donating substituents have been shown only to react in the presence of increased amounts of palladium catalyst, and excess triphenylphosphine and lithium chloride. In addition, elevated temperatures are required in order to complete such crosscouplings.45 In the present investigation, the Heck reaction and the Stille reaction have been employed to prepare several C5-substituted derivatives of the potential atypical antipsychotic agent 5-methoxy-2- [N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 19). Thus, in order to further explore the structure-affinity relationships of this type of compounds, and to investigate whether the dopamine D2 and serotonin 5-HT1A receptors show different structural requirements with respect to the C5-substituent, the 5-triflate, 5-acetyl, 5-cyano, 5-methyl, 5-(2-furyl), 5-phenyl, methyl 5- carboxylate, and the 5-carboxamido analogues of 19 were prepared and their affinities for dopamine

D2 and serotonin 5-HT1A receptors were determined in vitro.

4.2 CHEMISTRY

The synthetic steps employed to access the different 5-substituted analogues of 19 are outlined in Scheme 4.4. The 5-triflate analogue 22, which served as the starting point for the preparation of the other derivatives, was prepared from the previously reported hydroxy analogue 2024 by reaction with N,N-bis(trifluoromethanesulfonyl)aniline (N-phenyltriflimide) in dichloromethane, employing triethylamine as a base.23

103 Chapter 4

N R

CN N 24 N R R

CH d 3 O CH3 23 25 b, c e

N N N R a R h R

OH OSO2CF3 O OCH3 20 f 22 28

g i N R

O N N R R 26

O NH2 29 27 O

R = N H

SCHEME 4.4 Reagents and conditions: (a) N-phenyltriflimide, Et3N, CH2Cl2, RT; (b) butyl vinyl ether, Pd(OAc)2,

dppp, Et3N, DMF, D; (c) 5% HCl, D; (d) KCN, (PPh3)2NiCl2, PPh3, Zn, DMF, D; (e)

tetramethylstannane, (PPh3)2PdCl2, PPh3, LiCl, 2,6-di-tert-butyl-4-methylphenol, DMF, D; (f) tributyl(2-

furyl)stannane, (PPh3)2PdCl2, PPh3, LiCl, 2,6-di-tert-butyl-4-methylphenol, DMF, D; (g)

tributylphenylstannane, Pd(PPh3)4, LiCl, 2,6-di-tert-butyl-4-methylphenol, DMF, D; (h) CO(g), MeOH,

Pd(OAc)2, dppp, Et3N, DMSO, D; (i) formamide, 30% NaOCH3, DMF, D. A Heck reaction between 22 and vinyl butyl ether in the presence of 1,3- bis(diphenylphosphino)propane and a catalytic amount of palladium(II) acetate, employing triethylamine as a base and DMF as a solvent, was used to prepare the 5-acetyl analogue 23.4 The resulting 5-(2-butoxyethenyl) intermediate was then converted to 23 by hydrolysis with 5% aqueous hydrochloric acid solution.

104 C5-Substituted Derivatives of 5-OMe-BPAT

The 5-cyano analogue 24 was prepared according to a procedure reported by Hedberg et al.,22 which is a modification of the procedure originally described by Chambers and Widdowson.6 Thus, reaction of 22 with potassium cyanide in the presence of zinc dust, triphenylphosphine and bistriphenylphosphine-nickel(II) chloride as a catalyst, employing DMF as a solvent, gave complete conversion to 24 after 48 h at 120 ºC. Stille reactions, employing the modifications reported by Saá et al. for hindered electron-rich aryl triflates,45 were performed for the preparation of the 5-methyl (25), 5-(2-furyl) (26), and 5-phenyl (27) analogues. Thus, reaction of 22 with the appropriate organostannane reagent (tetramethylstannane, tributyl(2-furyl)stannane and tributylphenylstannane, respectively) in the presence of excess lithium chloride, triphenylphosphine, and catalytic amounts of bistriphenylphosphine-palladium(II) chloride and 2,6-di-tert-butyl-4-methylphenol (radical scavenger), employing DMF as a solvent and elevated reaction temperatures, gave the desired products in acceptable yields. Since the carboxamide moiety has been shown to be an interesting bioisostere for aromatic hydroxy and methoxy substituents in structurally related serotonergic ligands,32 we wanted to prepare the 5-carboxamide analogue of 19. However, different attempts to convert the 5-cyano analogue (24) directly to the 5-carboxamido analogue (29), employing different mild hydrolytic 8 conditions, failed in our hands. Thus, reaction with manganese dioxide in CH2Cl2, mercury(II) acetate in glacial acetic acid,40 aqueous sodium perborate without41 or with methanol,36 or hydrogen peroxide5 gave only starting material. Application of stronger reaction conditions, such as refluxing in aqueous sulfuric acid,22 resulted in the hydrolysis of both the nitrile group and the benzamide moiety. As an alternative route for the preparation of 29, the methyl 5-carboxylate analogue 28 was prepared from 22 by a palladium-catalyzed crosscoupling with carbon monoxide in the presence of excess methanol.10 The resulting methyl ester could be converted efficiently to the carboxamide 29 by reaction with formamide and 30% methanolic sodium methoxide.25

4.3 PHARMACOLOGY

The newly prepared C5-substituted analogues 22–29, as well as the previously described 5- methoxy, 5-hydroxy, and 5-substituted analogues 19, 20 and 2124 were evaluated for their ability to 3 compete for [ H]-spiperone binding to rat striatal membranes containing dopamine D2 receptors, and their ability to compete for [3H]-8-OH-DPAT binding to rat frontal cortex membranes containing serotonin 5-HT1A receptors in vitro. The results of these binding studies are shown in Table 4.1. In order to characterize the binding assay, the affinities of spiperone and 8-OH-DPAT, as well as of several classical and atypical antipsychotic agents have been included as well.

4.4 RESULTS AND DISCUSSION

In the previous two chapters we have elaborated on the structure-affinity relationships (SAFIRs) of a series of 2-aminotetralin-derived benzamides and structurally closely related analogues with mixed dopamine D2, D3, and serotonin 5-HT1A receptor binding properties. The SAFIRs suggested that the 2-aminotetralin moieties of the compounds may share the same binding sites in these

105 Chapter 4 receptor subtypes as the class of N,N-di-n-propylaminotetralins (DPATs). In the present study the effects of introduction of different substituents at the C5-position of the lead compound of the series,

5-OMe-BPAT (19) on the affinities for the dopamine D2 and serotonin 5-HT1A receptor have been investigated. Chemical diversity at this position was introduced by chemical transformation of the 5- triflate analogue 22, using several Stille-type crosscouplings and a Heck reaction. All reactions proceeded in moderate to good yields, ranging from 52 to 82%. Aryl triflates have been shown to be efficiently hydrogenated to their unsubstituted analogues in the presence of a palladium catalyst, when tributylamine is employed as the hydride source.44 This observation may explain why hydrogenolysis is sometimes observed as a side-reaction of palladium-catalyzed crosscouplings of aryl triflates, in cases where triethylamine is added as a base.51 However, there was no indication of the formation of C5-unsubstituted by-product during the preparation of 28. Compounds 19, 20, and 21 have been characterized previously under different assay conditions (see Chapter 2). The affinities reported here (Table 4.1) are somewhat lower than those previously reported. Particularly, 21 shows a low affinity for the dopamine D2 receptor, whereas at cloned 3 human dopamine D2 receptors, using [ H]-raclopride as a radioligand, it had an affinity of 10 nM. These differences are likely to be the result of differences in the assay conditions ([3H]-spiperone

TABLE 4.1 Receptor binding data of compounds 19–29, spiperone, 8-OH-DPAT, and various classical and atypical antipsychotic agents.

O N N H

R a Ki (nM)

Compound R D2 5-HT1A D2/5-HT1A

19 OCH3 6.3 2.4 2.6 20 OH 4.9 6.5 0.8 21 H 160 0.98 163

22 OSO2CF3 16 6.3 2.5

23 COCH3 120 9.3 13 24 CN 25 36 0.7

25 CH3 35 1.3 27 26 2-furyl 30 12 2.5 27 phenyl 120 8.3 14

28 COOCH3 110 8.3 13

29 CONH2 550 60 9.2 Spiperone – 0.15 98 0.002 8-OH-DPAT – > 1,000 2.5 > 400 Chlorpromazine – 7.6 > 1,000 < 0.008 Haloperidol – 1.3 > 1,000 < 0.001 Clozapine – 70 269 0.3 Olanzapine – 15 > 1,000 < 0.01 Risperidone – 3.1 603 0.005 aFor compounds 19–29: n = 1, for the reference compounds: n = 3.

106 C5-Substituted Derivatives of 5-OMe-BPAT versus [3H]-raclopride as a radioligand, tissue versus cloned receptors). Nevertheless, the affinity ranking of 19–21 at both receptor subtypes was the same under both assay conditions: at the dopamine D2 receptor, the ranking is 20 > 19 > 21, while at the serotonin receptor, the ranking is 21

> 19 > 20. The 5-unsubstituted analogue 3 has the highest affinity for the serotonin 5-HT1A receptor, and due to its low affinity for the dopamine D2 receptor, it is also the most selective serotonin 5-

HT1A receptor ligand of this series under these assay conditions. The dopamine D2 receptor affinity ranking, but also the relative affinities of 19–21 are consistent with what may be expected for 5- substituted DPAT analogues: the hydroxy-substituted congeners usually have the highest affinities, followed by the methoxy analogues, while unsubstituted analogues have only weak to moderate affinities.16,33,50,58,60 Presumably, the hydroxy group at the C5-position functions both as hydrogen bond donor and acceptor, while a methoxy group can only act as a hydrogen bond acceptor while 33 interacting with the dopamine D2 receptor. Unsubstituted DPATs are incapable of forming hydrogen bonds, which would explain their lower affinity for the dopamine D2 receptor. The differences observed in the receptor binding affinities of compounds 19–29 must be explained by differences in physicochemical properties of the C5-substituents. Table 4.2 lists several important aromatic substituent descriptors, describing the lipophilic (Hansch p), electronic (Hammett sm and sp) and steric (Molar Refractivity, MR) contributions of the C5-substituents of 19–29. In general, introduction of substituents at the C5-position other than hydroxy leads to a lower dopamine D2 receptor affinity. Particularly, compounds with a carbonyl moiety attached to C5 (cf. 23, 28, and 29) have a low affinity for the dopamine D2 receptor. This cannot be solely accounted for by the electron-withdrawing properties of these substituents, since the 5-triflate and 5-cyano analogues 22 and 24 possess considerable affinity for the dopamine D2 receptor, despite their strong electron- withdrawing properties, as expressed by their positive Hammett constants. Similarly, the relatively hydrophilic nature of the 5-acetyl and 5-carboxamido substituents being responsible for the low affinities of compounds 23 and 29, respectively, is in contradiction with the much higher affinities of 20 and 24. Furthermore, whereas the acetyl and methyl carboxylate substituents only differ considerably in their lipophilic properties, the affinities of 23 and 28 for the dopamine D2 receptor are about equally low. Similar to 21, the C5-methyl and C5-phenyl analogues 25 and 27 are both incapable of forming hydrogen bonds with their C5-substituent. Nevertheless, both congeners have a

TABLE 4.2 Aromatic substituent descriptors of the C5-substituents of compounds 19–29, describing their lipophilic a (p), electronic (sm and sp) and steric (MR) properties.

Compound R p sm sp MR

19 OCH3 –0.02 0.12 –0.27 0.79 20 OH –0.67 0.12 –0.37 0.28 21 H 0.00 0.00 0.00 0.10 b 22 OSO2CF3 – 0.56 0.53 1.45

23 COCH3 –0.55 0.38 0.50 1.12 24 CN –0.57 0.56 0.66 0.63

25 CH3 0.56 –0.07 –0.17 0.56 26 2-furyl – 0.06 0.02 1.79 27 phenyl 1.96 0.06 –0.01 2.54

28 COOCH3 –0.01 0.36 0.45 1.29

29 CONH2 –1.49 0.28 0.36 0.98 aAll data taken from ref. 17. bNo data available.

107 Chapter 4 higher affinity than 21, despite their increased steric properties. Taken together, the observations suggest that, whereas the binding affinities of 19–21 are consistent with those of 5-oxygenated DPATs and suggest that these compounds may share similar binding modes, it is very well possible that the other congeners of this series bind to the dopamine D2 receptors in a different way.

The serotonin 5-HT1A receptor seems to tolerate more chemical diversity at the C5-position of 5-

OMe-BPAT than the dopamine D2 receptor: all congeners have moderate to high affinity for the former receptor subtype, with Ki-values ranging from 60.3 nM for the 5-carboxamido analogue 29 to 0.98 nM for the 5-unsubstituted analogue 21. Given the considerable differences in the nature of the various C5-substituents, they apparently do not contribute much to the affinity for the serotonin 5-

HT1A receptor. Similar observations were reported for C8-substituted analogues of DPAT by Liu et al., who showed that the presence of a wide variety of alkyl, heteroalkyl, aryl and heteroaryl moieties 30–32 at this position was tolerated by the serotonin 5-HT1A receptor. In these studies, some of the derivatives behaved as full agonists, while others were devoid of intrinsic efficacy in vivo. Thus, whereas the nature of the C8-substituent seemed to be of little significance for the affinity, it appeared to be important for the intrinsic efficacy in vivo. In the current investigation only the affinities of the compounds for the serotonin 5-HT1A receptor were determined. Nevertheless, it is quite well possible that they possess varying degrees of intrinsic efficacy. Thus, whereas the 5- methoxy substituent of 19 does not seem to be of importance for the affinity for the receptor, it may prove to be essential for receptor stimulation. In cases like these, where no obvious patterns can be derived from the structure-affinity data, multiple linear regression (MLR) techniques, such as classical Hansch analysis, may be employed in order to attempt to correlate molecular descriptors (e.g. substituent parameters) with one or more response variables (e.g. receptor affinity).27 Prerequisites for MLR to provide statistically reliable results are that the compound to descriptor ratio should be large (>5) and that the descriptors are not correlated. Obviously the number of compounds considered in this study is too small to obtain statistically reliable results from such analysis techniques. Alternatively, when the number of descriptors exceeds the number of compounds, multivariate data analysis techniques such as Partial Least-Squares Projections to Latent Structures (PLS)59 should be employed to derive quantitative structure-activity relationships (QSARs). PLS is capable of extracting latent variables (components) hidden in the descriptor matrix, which are orthogonal to each other and explain the maximum amount of variance in the response variables. PLS tolerates large numbers of descriptors, which need not to be independent (i.e. they may describe similar properties), and a certain amount of missing data in the descriptor matrix is allowed. Here we have employed PLS in an attempt to derive quantitative relationships between the molecular properties of 19–29 and their affinities for the dopamine D2 and serotonin 5-HT1A receptor. Thus, 24 different descriptors, describing either whole molecule or C5-substituent properties, were generated for each compound (see Section 4.6.3). PLS analysis of the descriptor matrix (11 × 24; 11 compounds and 24 descriptors) and the corresponding receptor affinities revealed no statistically significant correlation between the molecular properties of 2 19–29 and their affinities for the dopamine D2 receptor, as reflected by a negative value of Q . The lack of predictability supports the previously stated hypothesis that the compounds do not all bind to the receptor in a similar fashion. For the serotonin 5-HT1A receptor affinities a statistically significant model was obtained, with the first two latent variables of the PLS model accounting for 80% of the

108 C5-Substituted Derivatives of 5-OMe-BPAT variance in the affinities. Figure 4.1 shows the correlation between the fitted and the observed (i.e. experimental) pKi values of 19–29 at the serotonin 5-HT1A receptor. The predictability of the model was evaluated by leave-one-out crossvalidation,14 resulting in a Q2 value of 0.49 after two components (Figure 4.1). Inspection of the regression coefficients after two components (Figure 4.2) shows that the descriptors 9, 10, and 21 contribute most to the serotonin 5-HT1A receptor affinity. These descriptors correspond to the number of oxygen atoms in the whole molecule (9), the number of sulphur atoms in the whole molecule (10), and the log P value of the C5-substituent (21). Large values of descriptors 4, 6, 7, 8, 20, 23, and 24 are less favourable for the serotonin 5-HT1A receptor affinity, as can be seen from the negative values of their corresponding regression coefficients. These descriptors correspond to the total dipole moment of the whole molecule (4), number of H-bond donors in the whole molecule (6), number of H-bond acceptors in the whole molecule (7), number of nitrogen atoms in the whole molecule (8), total dipole moment of the C5-substituent (20), number of H-bond donors in the C5-substituent (23), and the number of H-bond acceptors in the C5-substituent (24). Size-related properties, such as molecular surface (2 and 12), molecular volume (3 and 13), and the Verloop parameters (14–19) have little effect on the serotonin 5-HT1A receptor affinity. Taken together, relatively lipophilic, nonpolar substituents incapable of forming hydrogen bonds, but not necessarily small in size, are optimal for a high affinity at the serotonin 5-HT1A receptor. These findings are consistent with the observed affinities of compounds 19–29 for the serotonin 5-HT1A receptor, and explain why analogues with C5-substituents as different as hydrogen (21), triflate (22), methyl (25) and phenyl (27) share a high affinity for this receptor subtype.

21 9 21 9

25

i 25 K i 27

K 27 19 26 26 22 24 19 20 8 23 8 23 24 28 20 28 Fitted p Predicted p

22 29 29 7 7 7 8 9 7 8 9 Observed pK Observed pK i i

FIGURE 4.1 Plots showing the observed versus fitted (left) and observed versus predicted (right) pKi values of

compounds 19–29 at the serotonin 5-HT1A receptor after two PLS components.

109 Chapter 4

0.1

0.0

-0.1

-0.2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Regression coefficient

FIGURE 4.2 Plot showing the regression coefficients 1–24 for the serotonin 5-HT1A receptor affinities of compounds 19–29 after two PLS components. Of the reference compounds that have been included in the receptor binding assays, only 8-OH-

DPAT is highly selective for the serotonin 5-HT1A receptor. Spiperone, the classical antipsychotic agents chlorpromazine and haloperidol, and the presumed atypical antipsychotic agents olanzapine and risperidone have a high preference for the dopamine D2 receptor over the serotonin 5-HT1A receptor, which is consistent with previous findings.2,46 Spiperone has the highest affinity for the serotonin 5-HT1A receptor after 8-OH-DPAT, but due to its very high dopamine D2 receptor affinity it is still one of the most selective dopamine D2 receptor ligands in this study. Clozapine, the prototypical atypical antipsychotic agent, is the least selective of all reference compounds: it has moderate affinity for the dopamine D2 receptor and low affinity for the serotonin 5-HT1A receptor. Although the latter finding has been confirmed by others,34 recent neurochemical studies have suggested that clozapine, despite this low affinity, acts as a partial agonist at serotonin 5-HT1A receptors in vivo, which may be of significance for its unique clinical profile.43

4.5 CONCLUSIONS

The 5-methoxy substituent of 5-OMe-BPAT plays a role in the binding of the compound to the dopamine D2 receptor, since replacement of this substituent by other moieties affects the affinity for this receptor subtype. However, no clear structure-affinity relationship could be derived from the limited set of C5-substituted analogues. The 5-methoxy, 5-hydroxy, and 5-unsubstituted analogues probably share their binding sites and binding modes with the 5-oxygenated DPATs, but the other analogues may bind to the receptor in a completely different way. For high serotonin 5-HT1A receptor affinity, the C5-substituent should be relatively lipophilic, nonpolar, and have no hydrogen bond-forming capacities.

110 C5-Substituted Derivatives of 5-OMe-BPAT

4.6 EXPERIMENTAL SECTION

4.6.1 CHEMISTRY

General Remarks. For general remarks, see Section 2.6.1. All chemical ionization mass spectra were recorded on a NERMAG R 3010 triple quadrupole mass spectrometer equipped with a home-built atmospheric pressure ionization source and ionspray interface.

5-{[(Trifluoromethyl)sulfonyl]oxy}-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin Oxalate (22): triethyl- amine (2.87 g, 28.4 mmol) was added to a stirred solution of N-phenyltriflimide (6.09 g, 17.0 mmol) and 2024 (5.00 g,

14.2 mmol) in CH2Cl2 (100 mL). The reaction mixture was stirred overnight at room temperature under a nitrogen atmosphere. H2O (50 mL) was added to the reaction mixture, the phases were separated and the organic layer was subsequently washed with 10% aqueous Na2CO3 solution (3 ´ 50 mL), H2O (50 mL) and brine (50 mL). After drying

(Na2SO4) and filtration of the organic layer, the solvent was evaporated, which gave the crude triflate as a brown oil. Chromatographic purification [eluent: EtOAc/petroleum ether (bp 40–60), 1/2 (v/v)] gave 5.68 g (11.7 mmol, 83%) of the pure base of 22 as a light yellow oil: mp 147–149 °C (acetone); IR: cm–1 3377, 2973, 2880, 2651 (b), 2504 (b), 1 1718, 1660, 1579, 1525, 1417, 1210; H NMR (base, 300 MHz, CDCl3): d 0.85 (t, J = 7.3 Hz, 3H), 1.40–1.55 (m, 3H), 1.97–2.01 (m, 1H), 2.45–2.50 (dd, J = 7.0 Hz, 7.7 Hz, 2H), 2.60–3.02 (m, 7H), 3.37–3.43 (m, 2H), 6.97–7.10 (m, 3H), 13 7.25–7.41 (m, 4H), 7.77 (dd, J = 8.3 Hz, 1.3 Hz, 2H); C NMR (base, 75 MHz, CDCl3): d 11.7, 22.0, 24.1, 24.8, 32.1, 38.5, 48.8, 52.4, 55.7, 118.3, 118.6 (q, J = 320 Hz), 126.9, 128.4, 129.4, 131.2, 134.6, 140.0, 148.1, 167.3; MS (CI with AcOH): m/z 486 (M+1); Anal. calcd for C25H27N2O4SF3·C2H2O4: C 52.25, H 5.10, N 4.88; obsd C 51.87, H 5.04, N 5.36.

5-Acetyl-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin Hydrochloride (23): Pd(OAc)2 (6 mg, 0.03 mmol), dppp (11 mg, 0.03 mmol), Et3N (0.20 g, 1.98 mmol), and butyl vinyl ether (0.50 g, 5.0 mmol) were added to a solution of the free base of 22 (0.24 g, 0.50 mmol) in dry DMF (2 mL). The reaction mixture was stirred under a nitrogen atmosphere at room temperature for 15 min. Then the reaction vessel was sealed and the reaction mixture was heated at 120 °C for 24 h. After cooling to ~90 °C, 5% aqueous HCl solution (3 mL) was added to the reaction mixture and stirring was continued for 15 min at ambient temperature. The reaction mixture was then cooled to room temperature, poured into H2O (25 mL) and solid NaHCO3 was added until basic. The mixture was extracted with

CH2Cl2 (3 ´ 25 mL), the organic layers were collected and subsequently washed with H2O (3 ´ 25 mL) and brine (25 mL). After drying (Na2SO4) and filtering, the organic layer was concentrated under reduced pressure to give the crude product as a brown oil. Purification by column chromatography (eluent: EtOAc) yielded 0.154 g (0.4 mmol, 82%) of the pure base of 23 as a colourless oil: mp 81–83 °C; IR: cm–1 3256 (b), 2963, 2494 (b), 1676, 1654, 1601, 1578, 1 1533; H NMR (base, 200 MHz, CDCl3): d 0.88 (t, J = 7.3 Hz, 3H), 1.46–1.64 (m, 3H), 1.95–2.10 (m, 1H), 2.51 (s, 3H), 2.58–2.66 (m, 2H), 2.81–3.13 (m, 7H), 3.51–3.55 (m, 2H), 7.15 (d, J = 5.4 Hz, 2H), 7.35–7.51 (m, 5H), 7.80 (dd, 13 J = 7.7 Hz, 1.8 Hz, 2H); C NMR (base, 50 MHz, CDCl3): d 11.5, 20.8, 25.1, 27.7, 29.7, 32.1, 37.4, 48.8, 52.4, 56.0, 125.4, 126.8, 127.0, 128.4, 131.3, 132.8, 134.2, 135.7, 137.0, 137.7, 167.2; MS (CI with AcOH): m/z 379 (M+1);

Anal. calcd for C24H30N2O2·HCl·¾H2O: C 67.26, H 7.66, N 6.54; obsd C 67.03, H 7.32, N 6.44.

5-Cyano-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin Hydrochloride (24): Zn (dust, 103 mg, 1.6 mmol), (PPh3)2NiCl2 (172 mg, 0.3 mmol), PPh3 (140 mg, 0.5 mmol) and KCN (83 mg, 1.27 mmol) were added to a solution of the free base of 22 (0.50 g, 1.03 mmol) in dry DMF (5 mL). The mixture was stirred under a nitrogen atmosphere at room temperature for 15 min. Then the reaction vessel was sealed and stirring was continued at 120 °C for 48 h. After cooling to room temperature, the reaction mixture was poured into H2O (25 mL) and the mixture was extracted with Et2O (3 ´ 25 mL). The organic layers were collected and washed with brine (25 mL). After drying

(Na2SO4), the organic solution was concentrated in vacuo which gave the crude nitrile as an orange oil. Purification by column chromatography (eluent: EtOAc) yielded 0.260 g (0.72 mmol, 70%) of the pure base of 24 as a colourless oil: mp 117–120 ºC; IR: cm–1 3421 (b), 3246 (b), 3058, 2937, 2880, 2475 (b), 2224, 1654, 1578, 1534; 1H NMR (base,

111 Chapter 4

200 MHz, CDCl3): d 0.86 (t, J = 7.3 Hz, 3H), 1.39–1.70 (m, 3H), 1.96–2.05 (m, 1H), 2.45–2.52 (m, 2H), 2.68–3.15 13 (m, 7H), 3.33–3.47 (m, 2H), 7.05–7.43 (m, 7H), 7.75 (d, J = 7.1 Hz, 2H); C NMR (base, 50 MHz, CDCl3): d 11.6, 21.8, 24.8, 28.2, 31.8, 38.0, 48.5, 52.1, 55.3, 111.9, 117.8, 126.0, 126.7, 128.4, 130.2, 131.2, 133.9, 134.5, 137.8,

139.6, 167.2; MS (CI with AcOH): m/z 362 (M+1); Anal. calcd for C23H27N3O·HCl·¼H2O: C 68.63, H 7.15, N 10.44; obsd C 68.55, H 7.14, N 9.84.

5-Methyl-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin Hydrochloride (25): a solution of the free base of

22 (250 mg, 0.5 mmol) in dry DMF (3 mL) was added to a mixture of (PPh3)2PdCl2 (42 mg, 0.06 mmol), LiCl (181 mg, 4.27 mmol) and PPh3 (82 mg, 0.31 mmol) in dry DMF (7mL). The reaction mixture was stirred at room temperature for 10 min, and then (CH3)4Sn (0.29 mL, 2.09 mmol) and a few crystals of 2,6-di-tert-butyl-4- methylphenol were added. The reaction vessel was sealed a the reaction mixture was stirred overnight at 120 °C. After cooling, the reaction mixture was partitioned between CHCl3 and 10% aqueous NaHCO3 solution. The combined organic layers were subsequently washed with 10% aqueous KF solution, H2O and brine. After drying (Na2SO4) and filtering the organic layer, the solvent was evaporated, which gave the crude product as a brown oil. Purification by column chromatography [eluent: MeOH/CH2Cl2, 1/20 (v/v)] yielded 150 mg (0.43 mmol, 82%) of the pure base of 25 as a colourless oil: mp 122–124 °C; IR: cm–1 3249 (b), 2965, 2467 (b), 1655, 1601, 1578, 1533; 1H NMR (base, 200

MHz, CDCl3): d 0.92 (t, J = 7.3 Hz, 3H), 1.43–1.79 (m, 3H), 2.04–2.10 (m, 1H), 2.21 (s, 3H), 2.57 (dd, J = 7.3 Hz, 7.3 Hz, 2H), 2.65–3.08 (m, 7H), 3.46–3.56 (m, 2H), 6.92–7.09 (m, 4H), 7.41–7.56 (m, 3H), 7.80 (dd, J = 7.7 Hz, 1.6 13 Hz, 2H); C NMR (base, 50 MHz, CDCl3): d 11.6, 19.4, 21.6, 25.8, 27.0, 32.3, 37.5, 48.2, 51.9, 55.5, 125.5, 126.7, 127.2, 128.4, 131.2, 134.5, 134.6, 135.8, 136.2, 167.1; MS (CI with AcOH): m/z 351 (M+1); Anal. calcd for

C23H30N2O·HCl·¾H2O: C 68.97, H 8.20, N 7.00; obsd C 69.16, H 8.03, N 7.07.

5-(2-Furyl)-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin Hydrochloride (26): a mixture of (PPh3)2PdCl2

(43 mg, 0.1 mmol), LiCl (175 mg, 4.1 mmol), PPh3 (80 mg, 0.3 mmol) and the free base of 22 (220 mg, 0.45 mmol) in dry DMF (4 mL) was stirred at room temperature under a nitrogen atmosphere. After 5 min a solution of tributyl(2- furyl)stannane (0.325 mL, 1.03 mmol) in dry DMF (6 mL) was added, followed by a few crystals of 2,6-di-tert-butyl- 4-methylphenol. After stirring for 45 min at 120 °C under a nitrogen atmosphere, the reaction mixture was cooled and partitioned between CH2Cl2 and 10% aqueous NaHCO3 solution. The organic solution was subsequently washed with

H2O and brine, dried (Na2SO4) and concentrated under reduced pressure. The resulting crude product was purified by column chromatography (eluent: EtOAc), which yielded 140 mg (0.35 mmol, 77%) of the pure base of 26 as a colourless oil: mp 127–130 ºC; IR: cm–1 3416 (b), 3246 (b), 3057, 2939, 2880, 2611, 2475, 1654, 1578, 1534; 1H

NMR (base, 200 MHz, CDCl3): d 0.92 (t, J = 7.3 Hz, 3H), 1.46–1.67 (m, 3H), 2.03–2.09 (m, 1H), 2.53–2.60 (m, 2H), 2.75–3.11 (m, 7H), 3.46–3.56 (m, 2H), 6.48 (s, 2H), 7.03–7.06 (m, 2H), 7.18 (t, J = 7.7 Hz, 1H), 7.39–7.54 (m, 5H), 13 7.80 (dd, J = 7.7 Hz, 1.8 Hz, 2H); C NMR (base, 50 MHz, CDCl3): d 11.6, 21.7, 25.9, 28.6, 32.6, 37.6, 48.2, 51.9, 55.3, 108.8, 111.0, 125.4, 125.6, 126.7, 128.4, 129.1, 130.1, 131.2, 133.3, 134.6, 136.8, 141.6, 153.3, 167.1; MS (CI with AcOH): m/z 403 (M+1); Anal. calcd for C26H30N2O2·HCl·½H2O: C 69.69, H 7.21, N 6.25; obsd C 69.64, H 7.18, N 6.25.

5-Phenyl-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin Hydrochloride (27): a solution of tributylphenyl- stannane (228 mg, 0.62 mmol) in 1,4-dioxane (9 mL) was added to a mixture of the free base of 22 (250 mg, 0.52 mmol), LiCl (68 mg, 1.60 mmol) and Pd(PPh3)4 (30 mg, 0.03 mmol) in dry DMF (3 mL). Then a few crystals of 2,6- di-tert-butyl-4-methylphenol were added and the reaction mixture was stirred overnight at 120 °C under a nitrogen atmosphere. After cooling, the solids were removed from the reaction mixture by filtration (Celite®) and the filtrate was poured into H2O (25 mL). The aqueous solution was extracted with CHCl3 (3 × 25 mL) and the extracts were combined. The organic solution was subsequently washed with aqueous NaHCO3 solution (3 × 25 mL), H2O (25 mL) and brine. After drying (Na2SO4) and filtering, the organic solution was concentrated under reduced pressure, which gave the crude product as a brown oil. Purification by silica column chromatography [eluent: MeOH/CH2Cl2, 1/20 (v/v)] yielded 110 mg (0.27 mmol, 52%) of the pure base of 27 as a colourless oil: mp 103–105 °C; IR: cm–1 3256 (b), 1 3057, 2965, 2479 (b), 1654, 1601, 1578, 1533; H NMR (300 MHz, CDCl3): d 1.00 (as, 3H), 1.77–1.80 (m, 1H), 1.99– 2.06 (m, 2H), 2.32–2.47 (m, 1H), 2.69–2.79 (m, 2H), 3.05–3.22 (m, 4H), 3.31–3.44 (m, 1H), 3.51–3.60 (m, 1H),

112 C5-Substituted Derivatives of 5-OMe-BPAT

3.64–3.74 (m, 1H), 3.79–3.87 (m, 1H), 4.02–4.11 (m, 1H), 6.93–7.54 (m, 12H), 8.06–8.10 (m, 2H); 13C NMR (75

MHz, CDCl3): d 11.6, 21.3, 25.6, 28.2, 32.0, 37.4, 48.6, 52.2, 56.2, 125.6, 126.8, 127.4, 127.9, 128.5, 128.6, 129.0, 131.2, 133.6, 134.4, 135.9, 141.6, 141.8, 167.1; MS (CI with AcOH): m/z 413 (M+1); Anal. calcd for

C28H32N2O·HCl·½H2O: C 73.41, H 7.50, N 6.12; obsd C 73.14, H 7.70, N 6.28.

Methyl 2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin-5-carboxylate Hydrochloride (28): methanol (3.32 mL, 131 mmol) was added to a mixture of the free base of 22 (1.00 g, 2.1 mmol), Pd(OAc)2 (14 mg, 0.1 mmol), dppp

(26 mg, 0.1 mmol), Et3N (0.42 g, 4.2 mmol) and DMSO (15 mL). The mixture was flushed with nitrogen until all reagents were dissolved (1 h) and then the reaction mixture was saturated with CO gas (caution: highly toxic!) by flushing it at room temperature during 1 h. Then the reaction mixture was heated at 70 ºC under an atmosphere of CO. After 24 and 48 h of heating, the reaction mixture was cooled to room temperature, resaturated with CO and extra MeOH (1.0 mL) was added in order to complete the reaction. After 72 h of heating at 70 ºC TLC analysis revealed absence of starting material. The reaction mixture was cooled to room temperature and poured into H2O (50 mL). The aqueous phase was extracted with CH2Cl2 (5 × 15 mL), and the collected organic layers were washed with H2O (5 × 15 mL) and brine (15 mL). After drying (Na2SO4) and filtering of the organic layer, the solvent was evaporated, yielding the crude ester as a brown oil. Purification by column chromatography [eluent: MeOH/CH2Cl2, 1/20 (v/v)] yielded 0.50 g (1.3 mmol, 61%) of the pure base of 28 as a colourless oil: mp 90–91 ºC; IR: cm–1 3400 (b), 3247 (b), 3057, 1 2965, 2880, 2626 (b), 2492 (b), 1718, 1654, 1578, 1534; H NMR (base, 200 MHz, CDCl3): d 0.89 (t, J = 7.3 Hz, 3H), 1.43–1.62 (m, 3H), 1.99–2.05 (m, 1H), 2.53 (dd, J = 7.3 Hz, 7.3 Hz, 2H), 2.72–3.10 (m, 6H), 3.28–3.49 (m, 3H), 3.84 13 (s, 3H), 7.04 (bs, 1H), 7.12–7.21 (m, 2H), 7.36–7.47 (m, 3H), 7.66–7.79 (m, 3H); C NMR (base, 50 MHz, CDCl3): d 11.6, 21.7, 25.6, 28.0, 32.6, 37.7, 48.3, 51.6, 51.9, 55.2, 125.2, 126.7, 128.1, 128.4, 129.6, 131.1, 133.3, 134.6, 137.4, 137.7, 161.1, 168.2; MS (CI with AcOH): m/z (rel. intensity) 337 (8), 395 (100, M+1); Anal. calcd for

C24H30N2O3·HCl·½H2O: C 65.51, H 7.35, N 6.37; obsd C 65.35, H 7.32, N 6.48.

5-Carboxamido-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin Hydrochloride (29): formamide (69 mg, 1.6 mmol) was added to a solution of the free base of 28 (150 mg, 0.4 mmol) in dry DMF (20 mL). The reaction mixture was heated at 100 ºC under a nitrogen atmosphere and then 30% NaOCH3 solution in MeOH (0.1 mL) was added dropwise via a syringe. Heating was continued at 100 ºC for 1 h and then the reaction mixture was allowed to cool to room temperature. After concentrating the reaction mixture under reduced pressure, the residue was dissolved in CH2Cl2 (25 mL), and the resulting solution was subsequently washed with 10% aqueous NaHCO3 solution (3 × 25 mL), H2O (25 mL) and brine (25 mL). After drying (Na2SO4) and filtering, the solvent was evaporated, which gave the crude carboxamide as a yellow oil. Purification by silica column chromatography [eluent: MeOH/CH2Cl2, 1/10 (v/v)] yielded 110 mg (0.3 mmol, 76%) of the pure base of 29 as a colourless oil: mp 190–192 °C; IR: cm–1 3297 (b), 1 3169 (b), 2965, 2611 (b), 2520 (b), 1655, 1578, 1533; H NMR (base, 200 MHz, CHCl3): d 0.88 (t, J = 7.3 Hz, 3H), 1.39–1.67 (m, 3H), 1.97–2.02 (m, 1H), 2.52 (dd, J = 7.4 Hz, 7.4 Hz, 2H), 2.71–3.16 (m, 7H), 3.42–3.47 (m, 2H), 6.05 (bs, 1H), 6.26 (bs, 1H), 7.07–7.12 (m, 3H), 7.19–7.24 (m, 1H), 7.36–7.52 (m, 3H), 7.75 (dd, J = 7.9 Hz, 1.6 Hz, 2H); 13 C NMR (base, 50 MHz, CDCl3): d 11.6, 21.6, 25.3, 27.0, 32.3, 37.6, 48.3, 52.0, 55.4, 124.4, 125.5, 126.7, 128.4, 131.2, 131.4, 134.2, 134.4, 135.1, 137.3, 167.2, 172.4; MS (CI with AcOH): m/z (rel. intensity) 288 (3), 380 (100,

M+1); Anal. calcd for C23H29N3O2·HCl·¼H2O: C 65.69, H 7.33, N 9.99; obsd C 65.37, H 7.33, N 9.93.

4.6.2 PHARMACOLOGY

3 [ H]-Spiperone Binding to Dopamine D2 Receptors. Male Wistar rats, weighing 200–300 g, (TNO, The Netherlands) were decapitated and the striata were dissected. After weighing, the striata were taken up in 20 volume amounts of 50 mM Tris-HCl buffer (pH 7.7) and homogenized using a Potter-Elvehjem homogenizer (10 ´ 600 rpm). The resulting homogenate was centrifuged at 4 ºC during 10 min at 50,000 × g in a Sorvall RC-5 centrifuge. The supernatant was decanted and the pellet was resuspended in 20 volume amounts of 50 mM Tris-HCl buffer, using a Vortex mixer. The suspension was again homogenized (5 ´ 600 rpm) and centrifuged at 4 ºC during 10 min at 50,000 × g. After having repeated this washing procedure two more times, the tissue was taken up in 20 volume amounts of

113 Chapter 4

incubation medium (47 mM Tris-HCl buffer, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 0.1% ascorbic acid, 5 mM pargyline, pH 7.7) and homogenized (5 ´ 600 rpm). The membrane fraction was then taken up in 100 volume amounts of incubation medium, to a final concentration of 10 mg original wet weight per mL. The homogenate was incubated batchwise at 37 ºC during 10 min (activation of pargyline), distributed in 96-well plates (0.5 mL per well) by a Tecan dilution robot, and stored at 4 ºC until further use. Half an hour prior to the incubation the tissue was allowed to warm to room temperature. About 15 min prior to the incubation 50 mL of test compound, dissolved in 0.1% ascorbic acid solution, or 0.1% ascorbic acid solution (determination of total binding) was added to the tissue. The incubation was then initiated by adding 50 mL of [3H]-spiperone (New England Nuclear, Boston, MA; specific activity 20–40 Ci/mmol, Kd = 0.12 nM), dissolved in 0.1% ascorbic acid solution, resulting in a concentration of 0.5 nM during incubation. The incubation mixture was incubated at 25 ºC on a water bath during 20 min. Binding in the presence of haloperidol (0.1 mM) was defined as non-specific. The incubation was terminated by filtration of the plates through Whatman GF/B glassfiber filters and subsequent washing with 50 mM Tris-HCl buffer (2 × 5 mL) of 4 ºC, using Tomtek cell harvesters. The filters, each containing 96 samples, were dried and transferred in polyurethane envelops, together with solid scintillation sheets. The envelops were heat-sealed and counted in a Wallac betaplate counter.

3 [ H]-8-OH-DPAT Binding to Serotonin 5-HT1A Receptors. Male Wistar rats, weighing 200–300 g, (TNO, The Netherlands) were decapitated and the frontal cortices were dissected. After weighing, the frontal cortices were taken up in 10 volume amounts of 0.32 M sucrose solution and homogenized using a Potter-Elvehjem homogenizer (10 ´ 600 rpm). The resulting homogenate was centrifuged at 4 ºC during 10 min at 700 × g in a Sorvall RC-5 centrifuge. The supernatant was decanted and again centrifuged at 4 ºC during 10 min at 50,000 × g. The pellet was resuspended in 10 volume amounts of 50 mM Tris-HCl buffer (pH 7.7), using a Vortex mixer and homogenized during 10 sec using a Polytron (level 100). The resulting homogenate was incubated during 10 min at 37 ºC in order to remove endogenous serotonin. Again the homogenate was centrifuged at 4 ºC during 10 min at 50,000 × g. The resulting pellet was resuspended in 10 volume amounts of incubation medium (50 mM Tris-HCl buffer, 4 mM CaCl2, 0.1% ascorbic acid, 10 mM pargyline, pH 7.7) and homogenized (5 ´ 600 rpm). The membrane fraction was then taken up in 50 volume amounts of incubation medium, to a final concentration of 20 mg original wet weight per mL. The homogenate was incubated batchwise at 37 ºC during 15 min (activation of pargyline), distributed in 96-well plates (0.5 mL per well) by a Tecan dilution robot, and stored at 4 ºC until further use. Half an hour prior to the incubation the tissue was allowed to warm to room temperature. About 15 min prior to the incubation 50 mL of test compound, dissolved in 0.1% ascorbic acid solution, or 0.1% ascorbic acid solution (determination of total binding) was added to the tissue. The incubation was then initiated by adding 50 mL of [3H]-8-OH-DPAT (New England Nuclear, Boston,

MA; specific activity 158 Ci/mmol, Kd = 2.0 nM), dissolved in 0.1% ascorbic acid solution, resulting in a concentration of 1.0 nM during incubation. The incubation mixture (0.6 mL) was incubated at 37 ºC on a water bath during 10 min. Binding in the presence of serotonin (10 mM) was defined as non-specific. The incubations were terminated and the radioactivity was determined as described for the dopamine D2 receptor binding assay.

Data Analysis. Concentrations of unlabeled test compound causing 50% displacement of the specific binding of a labelled compound (IC50 values) were obtained by computerized log-probit linear regression analysis of data obtained in experiments in which four to six different concentrations of the test compound were used. Inhibition constants (Ki) 7 were calculated using the Cheng-Prusoff equation. Mean Ki-values were calculated from at least three values obtained from independent experiments, i.e. in experiments performed on different days with different membrane preparations. All incubations were done in triplicate.

4.6.3 QUANTITATIVE STRUCTURE-ACTIVITY RELATIONSHIPS

All calculations were performed on an Octane R10,000 Silicon Graphics workstation running IRIX 6.4. The program Tsar57 was used to generate 24 descriptors for compounds 19–29. The following whole molecule descriptors were generated: molecular mass (1); molecular surface area (2); molecular volume (3); total dipole moment (4);

114 C5-Substituted Derivatives of 5-OMe-BPAT number of heteroatoms (5); number of H-bond donors (6); number of H-bond acceptors (7); number of N atoms (8); number of O atoms (9); number of S atoms (10). In addition, the following descriptors were generated for the C5- substituents only: molecular mass (11); molecular surface area (12); molecular volume (13); Verloop L (14); Verloop B1 (15); Verloop B2 (16); Verloop B3 (17); Verloop B4 (18); Verloop B5 (19); total dipole moment (20); log P (21); molecular refractivity (22); number of H-bond donors (23); number of H-bond acceptors (24). A matrix was formed by putting the descriptors in the columns and the compounds in the rows. The first two columns were formed by the dopamine D2 and serotonin 5-HT1A pKi values of the compounds. After mean-centering and autoscaling of the matrix, PLS analysis was performed using the PLS_Toolbox39 for MatLab.35

4.7 REFERENCES

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44 Saá JM, Dopico M, Martorell G and García-Raso A (1990) Deoxygenation of highly hindered phenols. J Org Chem 55, 991–995. 45 Saá JM, Martorell G and García-Raso A (1992) Palladium-catalyzed cross-coupling reactions of highly hindered electron-rich phenol triflates and organostannanes. J Org Chem 57, 678–685. 46 Schotte A, Janssen PF, Gommeren W, Luyten WH, Van Gompel P, Lesage A, De Loore K and Leysen JE (1996) Risperidone compared with new and reference antipsychotic drugs: in vitro and in vivo receptor binding. Psychopharmacology Berl 124, 57–73. 47 Scott WJ and Stille JK (1986) Palladium-catalyzed coupling of vinyl triflates with organostannanes. Synthetic and mechanistic studies. J Am Chem Soc 108, 3033–3040. 48 Sonesson C (1995) Arylpiperidine and Arylpyrrolidine Derivatives with Potential Antipsychotic Efficacy. PhD thesis, Uppsala University. 49 Sonesson C, Barf T, Nilsson J, Dijkstra D, Carlsson A, Svensson K, Smith MW, Martin IJ, Duncan JN, King LJ and Wikström H (1995) Synthesis and evaluation of pharmacological and pharmacokinetic properties of monopropyl analogs of 5-, 7-, and 8-[[(trifluoromethyl)sulfonyl]oxy]-2-aminotetralins: central dopamine and serotonin receptor activity. J Med Chem 38, 1319–1329. 50 Sonesson C, Boije M, Svensson K, Ekman A, Carlsson A, Romero AG, Martin IJ, Duncan JN, King LJ and Wikström H (1993) Orally active central dopamine and serotonin receptor ligands: 5-, 6-, 7-, and 8- [[(trifluoromethyl)sulfonyl]oxy]-2-(di-n-propylamino)tetralins and the formation of active metabolites in vivo. J Med Chem 36, 3409–3416. 51 Sonesson C, Lin C-H, Hansson L, Waters N, Svensson K, Carlsson A, Smith MW and Wikström H (1994) Substituted (S)-phenylpiperidines and rigid congeners as preferential dopamine autoreceptor antagonists: synthesis and structure-activity relationships. J Med Chem 37, 2735–2753. 52 Sonesson C, Waters N, Svensson K, Carlsson A, Smith MW, Piercey MF, Meier E and Wikström H (1993) Substituted 3-phenylpiperidines: new centrally acting dopamine autoreceptor antagonists. J Med Chem 36, 3188– 3196. 53 Stang PJ and Anderson AG (1976) Hammett and Taft substituent constants for the mesylate, tosylate, and triflate groups. J Org Chem 41, 786. 54 Stang PJ, Hanack M and Subramanian LR (1982) Perfluoroalkanesulfonic esters: methods of preparation and applications in organic chemistry. Synthesis 85–126. 55 Stille JK (1986) The palladium-catalyzed cross-coupling reactions of organotin reagents with organic electrophiles. Angew Chem Int Ed Engl 25, 508–524. 56 Streitwieser Jr A and Dafforn A (1976) Solvolysis of aryl trifluoromethanesulfonates. Tet Lett 18, 1435-1438. 57 Tsar version 3.0. Oxford Molecular Group, Oxford OX4 4GA, UK. 58 Van Vliet LA, Tepper PG, Dijkstra D, Damsma G, Wikström H, Puglsey TA, Akunne HC, Heffner TG, Glase SA

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117 SYNTHESIS AND IN VITRO PHARMACOLOGICAL EVALUATION OF THE ENANTIOMERS OF 5-OME-BPAT 5 AND 5-OME-(2,6-DI-OME)-BPAT

ABSTRACT

The optically pure enantiomers of 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 1) and 5-methoxy-2-{N-[2-(2,6-dimethoxy)benzamidoethyl]-N-n-propylamino}tetralin [5-OMe-(2,6-di-OMe)-BPAT,

2] were synthesized and evaluated for their in vitro binding affinities at a1-, a2-, and b-adrenergic, muscarinic, dopamine D1, D2A, and D3, and serotonin 5-HT1A and 5-HT2 receptors. In addition, their intrinsic efficacies at dopamine D2A, D3, and serotonin 5-HT1A receptors were established in vitro. (S)- and (R)-1 had high affinities for dopamine D2A, D3, and serotonin 5-HT1A receptors, moderate affinities for a1-adrenergic and serotonin 5-HT2 receptors, and no affinity (Ki > 1,000 nM) for the other receptor subtypes. (S)- and (R)-2 had lower affinities for the dopamine D2A and the serotonin 5-HT1A receptor, compared to (S)- and (R)-1, and hence showed some selectivity for the dopamine D3 receptor. The interactions with the receptors were stereoselective, since the serotonin 5-HT1A receptor preferred the (S)-enantiomers, while the dopamine D2A and D3 receptors preferred the (R)-enantiomers of 1 and 2. The intrinsic efficacies at the serotonin 5-HT1A receptor were established by measuring their ability to inhibit VIP-induced cAMP production in GH4ZD10 cells expressing serotonin 5-HT1A receptors. Both enantiomers of 1 behaved as full serotonin 5-HT1A receptor agonists in this assays, while both enantiomers of 2 behaved as weak partial agonists. The intrinsic efficacies at dopamine D2A and D3 receptors were established by measuring their ability to induce dopamine

D2 and D3 receptor-mediated mitogenesis in CHO cells expressing rat dopamine D2A or D3 receptors, either alone or in the presence of the dopamine D2/D3 receptor agonist . All four compounds behaved as partial dopamine D2 receptor agonists in this assay, the (S)-enantiomers of both 1 and 2 having the highest intrinsic efficacies. (S)-1 displayed low intrinsic efficacy at the dopamine D3, receptor, while the other compounds behaved as antagonists at this receptor subtype. The intrinsic efficacies at the indicated receptor subtypes, together with their relatively clean receptor binding profiles compared to those of several reference antipsychotic agents, makes these compounds interesting pharmacological tools for further exploration of the dopamine D2/serotonin 5-HT1A hypothesis of atypical antipsychotic drug action.

119 Chapter 5

5.1 INTRODUCTION

In a previous report, we have disclosed a new class of compounds with mixed dopamine D2, D3, 8 and serotonin 5-HT1A receptor binding profiles. The lead compound of this series, 5-methoxy-2-[N- (2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 1, Chart 1), had high affinities for all three receptor subtypes, while its 2,6-dimethoxy substituted analogue, 5-methoxy-2-{N-[2-(2,6- dimethoxy)benzamidoethyl]-N-n-propylamino}tetralin [5-OMe-(2,6-di-OMe)-BPAT, 2], showed some preference for the dopamine D3 receptor. The basic skeleton for the series was conceived by combining the structural, and hence pharmacological features of the N,N-di-n-propyl-substituted 2- aminotetralins (DPATs) and the 2-pyrrolidinylmethyl-derived class of substituted benzamides into one structural hybrid. Since both classes of compounds display highly stereoselective pharmacological properties, it may be expected that the enantiomers of the 2-aminotetralin-derived benzamides also possess different pharmacological properties. Herein we report the synthesis, the receptor binding profiles and the intrinsic efficacies at dopamine D2A, D3, and serotonin 5-HT1A receptors of the enantiomers of 1 and 2.

O R N N H R

OCH3 1 R=H 2 R=OCH3

CHART 5.1 Chemical structures of 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 1) and 5-methoxy-2-{N-[2-(2,6-dimethoxy)benzamidoethyl]-N-n-propylamino}tetralin [5-OMe-(2,6-di- OMe)-BPAT, 2].

5.2 CHEMISTRY

The synthetic route employed to obtain the enantiomers of 1 and 2 is outlined in Scheme 5.1. As starting points served optically pure (S)- and (R)-5-methoxy-2-aminotetralin,11 (S)- and (R)-3, which were reacted with propionyl chloride in the presence of sodium hydroxide and the biphasic medium water/dichloromethane, according to the Schotten-Baumann procedure. The resulting propionamides, (S)- and (R)-4, were reduced with LiAlH4 in boiling THF to give the N-n-propyl analogues (S)- and (R)-5, respectively. N-Alkylation with bromoacetonitrile in boiling acetone, employing K2CO3 as a base, gave the corresponding N-cyanomethyl analogues (S)- and (R)-6.

Reduction of the nitriles with LiAlH4 in boiling THF yielded the corresponding primary amine derivatives (S)- and (R)-7, respectively. Finally, acylation of (S)- and (R)-7 with the appropriately substituted benzoyl chloride in the presence of sodium hydroxide and the biphasic medium water/dichloromethane, according to the Schotten-Baumann procedure, gave the pure enantiomers of 1 and 2.

120 Enantiomers of 5-OMe-BPAT and 5-OMe-(2,6-di-OMe)-BPAT

O

NH NH NH 2 a b

OCH3 OCH3 OCH3 (S)-3 (S)-4 (S)-5 (R)-3 (R)-4 (R)-5

N CN N c b NH2 d

OCH3 OCH3 (S)-6 (S)-7 (R)-6 (R)-7

O R N N H R

OCH3 (S)-1 R=H (R)-1 R=H

(S)-2 R=OCH3

(R)-2 R=OCH3

Scheme 5.1 Reagents and conditions: (a) propionyl chloride, 10% NaOH, CH2Cl2, RT; (b) LiAlH4, THF; (c)

BrCH2CN, K2CO3, KI, acetone, D; (d) ArCOCl, 10% NaOH, CH2Cl2, RT.

5.3 PHARMACOLOGY

The enantiomers of 1 and 2 were evaluated in the following in vitro receptor binding assays: a1-, a2-, and b-adrenergic, muscarinic, dopamine D1, D2A, and D3, and serotonin 5-HT1A and 5-HT2. The results of these binding studies are shown in Table 5.1. For comparison purposes, the previously 8 reported affinities of racemic 1 and 2 for dopamine D2A, D3, and serotonin 5-HT1A receptors, as well as the receptor binding data of six reference antipsychotic agents, which were evaluated under the same assay conditions and have been previously reported by Hacksell et al.,7 have been included. In addition, the intrinsic efficacies of the compounds at cloned human dopamine D2A, D3, and serotonin

5-HT1A receptors were determined in vitro. The intrinsic efficacies at serotonin 5-HT1A receptors were evaluated by testing the ability of the compounds to inhibit VIP-induced cAMP production in 5 GH4ZD10 cells expressing serotonin 5-HT1A receptors. The intrinsic efficacies at dopamine D2A and

D3 receptors were established by measuring the ability of the compounds to inhibit the quinpirole-

121 Chapter 5

3 induced incorporation of [ H]-thymidine in CHO cells transfected with rat dopamine D2A or D3 receptors.2,12

5.4 RESULTS AND DISCUSSION

The optically pure enantiomers of 5-methoxy-2-aminotetralin, (–)-3 and (+)-3, served as starting materials for the synthesis of the desired end products. These compounds are known to possess the absolute (S)- and (R)-configuration at the C2 carbon atom, respectively.11 In view of the reaction conditions employed during the various synthetic steps, it is unlikely that inversion of stereochemistry has taken place. Therefore it is safe to assume (–)-1 and (–)-2 have the (S)- configuration while (+)-1 and (+)-2 have the (R)-configuration at the C2 carbon. The results of the binding studies (Table 5.1) show that both enantiomers of 1 bind with high affinities to dopamine D2A and D3, as well as to serotonin 5-HT1A receptors. In addition, both compounds have moderate affinities for a1-adrenergic and serotonin 5-HT2 receptors, and no affinity for a2- and b-adrenergic, dopamine D1, and muscarinic receptors. The enantiomers of 2 bind weakly to a1-adrenergic and serotonin 5-HT2 receptors, and like (S)- and (R)-1, have no affinity for a2- and b-adrenergic, dopamine D1 and muscarinic receptors. Furthermore, (S)- and (R)-2 have somewhat lower affinities for the dopamine D2A and serotonin 5-HT1A receptors than (S)- and (R)-1, and hence, in consistency with racemic 2, show a certain selectivity for the dopamine D3 receptor. As anticipated, the enantiomers of both 1 and 2 display stereoselectivity in their receptor binding properties, although the absolute differences between the affinities of the two pairs of enantiomers are not large. Nevertheless, the (S)-enantiomers, when compared to their optical antipodes, have the highest affinities for the serotonin 5-HT1A receptor, while the (R)-enantiomers are preferred by the dopamine D2A and D3 receptors. The latter finding is consistent with the SAFIR of the enantiomers of 5-OMe-DPAT: the affinities of (R)-5-OMe-DPAT for the dopamine D2A and D3 receptor are higher than those of its optical antipode.14 The 5-methoxy substituent plays a role in this stereoselectivity, since the stereoselectivity of the enantiomers of 5-OH-DPAT is reversed: at both

a TABLE 5.1 Receptor binding data (Ki values in nM) of the racemates and enantiomers of 1 and 2, and six reference antipsychotic agents.b

Compound D1 D2A D3 a1 a2 b M 5-HT1A 5-HT2 1 NDc 3.2 0.58 ND ND ND ND 0.82 ND (S)-1 > 1,000 3.4 0.42 156 > 1,000 > 1,000 > 1,000 0.20 121 (R)-1 > 1,000 0.77 0.14 124 > 1,000 > 1,000 > 1,000 3.8 136 2 ND 48 2.6 ND ND ND ND 27 ND (S)-2 > 1,000 58 1.6 560 > 1,000 > 1,000 > 1,000 32 453 (R)-2 > 1,000 15 0.89 594 > 1,000 > 1,000 > 1,000 56 > 1,000 Chlorpromazin 22 1.1 1.2 0.75 300 > 1,000 47 980 4.9 e Haloperidol 79 0.67 2.7 8.4 > 1,000 > 1,000 > 1,000 > 1,000 31 Thioridazine 22 2.3 2.3 1.1 180 > 1,000 10 140 11 Risperidone 270 1.7 6.7 0.50 1.6 > 1,000 > 1,000 180 0.34 Clozapine 170 60 83 2.5 450 > 1,000 15 300 9.5 Remoxipride > 1,000 130 970 > 1,000 > 1,000 > 1,000 > 1,000 > 1,000 > 1,000 aMeans of at least two independent experiments in duplicate. bAdapted from ref. 7. cND: not determined.

122 Enantiomers of 5-OMe-BPAT and 5-OMe-(2,6-di-OMe)-BPAT receptor subtypes (S)-5-OH-DPAT has the highest affinities of the two.6,14,19 Remoxipride has the cleanest receptor binding profile of the reference antipsychotic agents: it has moderate affinity for dopamine D2A receptors only, and is inactive at the other receptor subtypes. All other reference compounds have high affinities for a1-adrenergic and serotonin 5-HT2 receptors. In addition, chlorpromazine, haloperidol, thioridazine and risperidone have high affinities for dopamine

D2A and D3 receptors. The latter compound also has high affinity for the a2-adrenergic receptor, while the former three possess considerable affinity for the dopamine D1 receptor. Chlorpromazine, thioridazine and clozapine share a relatively high affinity for the muscarinic receptor. Only thioridazine and risperidone show moderate affinity for the serotonin 5-HT1A receptor. As previously noted by Hacksell et al.,7 it is virtually impossible to derive a common denominator responsible for the antipsychotic activity of these drugs from these receptor binding data.

Serotonin 5-HT1A receptors are known to be functionally coupled to Gi-proteins mediating the inhibition of adenylate cyclase, the enzyme which catalyzes the conversion of ATP into cAMP.4 3 Vasoactive intestinal peptide (VIP) stimulates the formation of cAMP in GH4ZD10 cells.

Stimulation of rat serotonin 5-HT1A receptors, brought to expression in these cells, counteracts the VIP-induced cAMP formation, and this system can be utilized to establish the intrinsic efficacy of 5 serotonin 5-HT1A receptor ligands. Both enantiomers of 1 caused complete inhibition of VIP- induced cAMP production (Table 5.2), indicating that they behave as full serotonin 5-HT1A receptor agonists in this in vitro assay. Although (S)- and (R)-1 were less potent than the reference serotonin

5-HT1A agonists 5-HT, 8-OH-DPAT and flesinoxan in this assay, as illustrated by their lower IC50 values, they were the only compounds whose efficacy matched that of 5-HT. The enantiomers of 2 behaved as weak partial agonists in this assay, both having an intrinsic efficacy of 0.3. In accordance with their affinities for the serotonin 5-HT1A receptor, the (S)-enantiomers were more potent in this assay than the (R)-enantiomers.

Activation of dopamine D2 and D3 receptors, brought to expression in CHO cells, stimulates mitogenesis, which can be quantified by measuring the amount of [3H]-thymidine incorporated into the cells.2,12 The intrinsic efficacies of antagonists can be determined in this assay by measuring their ability to inhibit agonist-induced mitogenesis. Quinpirole was employed as the agonist in these studies. The data of these studies are shown in Table 5.3. All compounds behaved as partial agonists at the dopamine D2A receptor, and they all were more potent than quinpirole, but particularly the

TABLE 5.2 Effects of 5-HT receptor agonists and the enantiomers of 1 and 2 on serotonin 5-HT1A receptor-mediated a inhibition of VIP-induced cAMP production in GH4ZD10 cells. b Compound Efficacy IC50 (nM) 5-HT 100 60 (R)-8-OH-DPAT 87 30 (S)-8-OH-DPAT 62 40 Flesinoxan 79 10 Buspirone 43 100 (S)-1 100 < 100 (R)-1 100 100 (S)-2 30 300 (R)-2 30 1,000 aData of reference compounds taken from ref. 5. bResults are presented as relative ‘efficacy’, indicating the ratio of the effect of the test compound to the maximum response of 5-HT in percentage.

123 Chapter 5

TABLE 5.3 Effects of quinpirole and the enantiomers of 1 and 2 on dopamine D2A and D3 receptor-mediated mitogenesis in CHO cells. Agonist effects Antagonist effectsa

D2A D3 D2A D3

Compound Intrinsic efficacy (%) EC50 (nM) Intrinsic efficacy (%) EC50 (nM) EC50 (nM) EC50 (nM) Quinpiroleb 96 2.2 111 1.7 IAc IA (S)-1 62 0.006 30 0.003 IA ³ 100 (R)-1 36 0.02 IA > 10,000 ³ 1,000 ³ 0.1 (S)-2 72 0.5 IA > 10,000 IA ³ 1 (R)-2 53 0.1 IA > 10,000 ³ 1,000 ³ 1 aBlockade of quinpirole-induced effect. bData taken from ref. 12. cIA: Inactive. extremely high potency of (S)-1 in this assay is noteworthy. The enantiomers of 1 were more potent than those of 2, which is consistent with their higher affinities at the dopamine D2A receptor. The (S)- enantiomers of both 1 and 2 had the highest intrinsic efficacies, a finding which is consistent with the SAR of the 5-oxygenated DPATs, where the (S)-enantiomers also have higher intrinsic efficacies than their optical antipodes.11,14,15,17,18 This observation, together with the finding that the (R)- enantiomers of both 1 and 2 have the highest affinities for the dopaminergic receptors, supports the previously stated hypothesis (see Chapters 2 and 3) that the 2-aminotetralin-derived benzamides and the DPATs may share common binding sites in these receptor subtypes. (R)-1 and (R)-2 were able to block the dopamine D2A receptor-mediated mitogenesis induced by quinpirole at an EC50 of about 1 mM. At the dopamine D3 receptor only (S)-1 showed some intrinsic efficacy (30%), the other compounds behaved as antagonists, as shown by their ability to block the dopamine D3 receptor- mediated mitogenesis induced by quinpirole.

In summary, (S)-1 behaved in these in vitro assays as a mixed partial dopamine D2/D3 receptor agonist and as a full serotonin 5-HT1A receptor agonist. Its optical antipode, (R)-1 turned out to be a partial dopamine D2 receptor agonists with low intrinsic efficacy, a dopamine D3 receptor antagonist and a full serotonin 5-HT1A receptor agonist. The enantiomers of 2 both behaved as partial dopamine

D2 receptor agonists, dopamine D3 receptor antagonists, and partial serotonin 5-HT1A receptor agonists. In view of the relatively clean receptor binding profiles of these compounds compared to the reference antipsychotics, they may prove to be interesting pharmacological tools for further exploration of the concept of mixed dopamine D2 receptor antagonism and serotonin 5-HT1A receptor agonism of atypical antipsychotic drug action. However, neurochemical and behavioural tests need to be performed in order to establish their in vivo efficacies and to predict their antipsychotic potential and liability to cause EPS in man. Therefore, the enantiomers of 1 were selected for evaluation in in vivo models with predictive value for antipsychotic activity and side- effect liability.

124 Enantiomers of 5-OMe-BPAT and 5-OMe-(2,6-di-OMe)-BPAT

5.5 CONCLUSIONS

The optical pure enantiomers of 1 and 2 have been shown to possess high affinities for dopamine

D2A, D3, and serotonin 5-HT1A receptors only, and to interact with these receptor subtypes in a stereoselective manner. Particularly, the differences in intrinsic efficacies at the dopaminergic receptors of the enantiomers of 1, in addition to their full serotonin 5-HT1A receptor agonism, makes these compounds interesting candidates for further exploration of the dopamine D2/serotonin 5-HT1A hypothesis of atypical antipsychotic drug action.

5.6 EXPERIMENTAL SECTION

5.6.1 CHEMISTRY

General Remarks. See Section 2.6.1. All chemical ionization mass spectra were recorded on a NERMAG R 3010 triple quadrupole mass spectrometer equipped with a home-built atmospheric pressure ionization source and ionspray interface. Specific optical rotations were measured in methanol (c = 1.0) at 22 ºC on a Perklin-Elmer 241 polarimeter.

(S)-5-Methoxy-2-propionamidotetralin [(S)-4]: a solution of propionyl chloride (3.25 g, 35.1 mmol) in CH2Cl2 (50 mL) was added dropwise to an ice-cooled, vigorously stirred mixture of (S)-3·HCl (3.00 g, 14.0 mmol), 10% aqueous NaOH solution (90 mL) and CH2Cl2 (100 mL). The mixture was allowed to warm to room temperature and stirring was continued for 18 h at room temperature. The reaction mixture was poured into H2O (100 mL) and the phases were separated. The H2O layer was extracted with CH2Cl2 (2 ´ 50 mL), the organic layers were combined and subsequently washed with a saturated aqueous NaHCO3 solution (3 ´ 50 mL) and H2O (50 mL). After drying over

MgSO4, the CH2Cl2 layer was filtered and evaporated under reduced pressure which gave the crude amide as a white solid. Recrystallisation from acetone/n-hexane yielded 3.04 g (13.0 mmol, 93%) of the title compound as white 22 –1 1 needles: mp 141–143 °C; [a] D –53°; IR: cm 3300 (b), 2937, 2848, 1637, 1587, 1535; H NMR (200 MHz, CDCl3): d 1.13 (t, J = 7.7 Hz, 3H), 1.72–1.83 (m, 1H), 1.97–2.05 (m, 1H), 2.17 (q, J = 7.7 Hz, 2H), 2.56–2.78 (m, 3H), 3.08 (dd, J = 16.2 Hz, 4.7 Hz, 1H), 3.80 (s, 3H), 4.21–4.30 (m, 1H), 5.71 (bs, 1H), 6.66 (d, J = 8.1 Hz, 2H), 7.09 (t, J = 8.1 13 Hz, 1H); C NMR (50 MHz, CDCl3): d 9.9, 21.0, 28.0, 29.8, 35.6, 44.5, 55.2, 107.2, 121.5, 124.4, 126.4, 135.4, 157.2, 173.3; MS (EI, 70 eV): m/z (rel. intensity) 74 (24), 91 (20), 104 (17), 115 (12), 129 (21), 145 (21), 160 (100), 233 (2, M+).

(R)-5-Methoxy-2-propionamidotetralin [(R)-4]: this compound was prepared as described for (S)-4, starting from 22 –1 1 (R)-3·HCl. Yield 78%: mp 142–143 °C; [a] D +53°; IR: cm 3298 (b), 2939, 2850, 1633, 1587, 1543; H NMR (200

MHz, CDCl3): d 1.11 (t, J = 7.7 Hz, 3H), 1.66–1.77 (m, 1H), 1.95–2.04 (m, 1H), 2.15 (q, J = 7.7 Hz, 2H), 2.54–2.77 (m, 3H), 3.05 (dd, J = 16.2 Hz, 4.7 Hz, 1H), 3.78 (s, 3H), 4.17–4.25 (m, 1H), 5.94 (bs, 1H), 6.63 (d, J = 8.6 Hz, 2H), 13 7.06 (t, J = 8.1 Hz, 1H); C NMR (50 MHz, CDCl3): d 10.0, 21.2, 28.1, 29.8, 35.7, 44.6, 55.2, 107.2, 121.5, 124.4, 126.4, 135.5, 157.2, 173.4; MS (EI, 70 eV): m/z (rel. intensity) 74 (25), 91 (20), 104 (18), 115 (13), 129 (22), 145 (20), 160 (100), 233 (2, M+).

(S)-5-Methoxy-2-(N-n-propylamino)tetralin Hydrochloride [(S)-5]: a solution of (S)-4 (2.98 g, 12.8 mmol) in dry THF (75 mL) was added to a stirred suspension of LiAlH4 (3.00 g) in dry THF (75 mL) and the reaction mixture was refluxed under a nitrogen atmosphere for 42 h. After cooling, excess LiAlH4 was quenched with H2O and 4N aqueous NaOH solution, the precipitate was filtered off and the filtrate was concentrated under reduced pressure. The resulting oil was taken up in CH2Cl2 and dried over MgSO4. After filtration, the solvent was evaporated, yielding 2.68 g (12.2 mmol, 95%) of the pure base of (S)-5 as a colourless oil: mp 276–278 °C dec (EtOH) [lit.20 mp 278–280 °C]; 22 20 –1 1 [a] D –73° [lit. –63°]; IR: cm 2967, 2835, 2793, 2735, 2525, 2443, 2588; H NMR (base, 200 MHz, CDCl3): d

125 Chapter 5

0.97 (t, J = 7.3 Hz, 3H), 1.51–1.67 (m, 3H), 1.80 (bs, 1H), 2.05–2.13 (m, 1H), 2.55–2.74 (m, 4H), 2.85–3.05 (m, 3H), 13 3.81 (s, 3H), 6.69 (dd, J = 11.1 Hz, 7.7 Hz, 2H), 7.11 (t, J = 8.1 Hz, 1H); C NMR (base, 50 MHz, CDCl3): d 11.9, 22.2, 23.6, 29.2, 36.8, 49.1, 53.2, 55.2, 107.0, 121.5, 125.1, 126.2, 136.7, 157.2; MS (CI with AcOH): m/z 220 (M+1).

(R)-5-Methoxy-2-(N-n-propylamino)tetralin Hydrochloride [(R)-5]: this compound was prepared as described 20 22 20 for (S)-5, starting from (R)-4. Yield 91%; mp 275–277 °C dec (EtOH) [lit. mp 278–280 °C]; [a] D +71° [lit. –1 1 +70°]; IR: cm 2967, 2835, 2791, 2734, 2525, 2443, 1588; H NMR (base, 200 MHz, CDCl3): d 0.97 (t, J = 7.3 Hz, 3H), 1.34 (bs, 1H), 1.50–1.61 (m, 3H), 2.06–2.13 (m, 1H), 2.54–2.74 (m, 4H), 2.85–3.07 (m, 3H), 3.82 (s, 3H), 6.67 13 (dd, J = 9.0 Hz, 9.0 Hz, 2H), 7.11 (t, J = 8.1 Hz, 1H); C NMR (base, 50 MHz, CDCl3): d 11.9, 22.2, 23.6, 29.3, 36.9, 49.1, 53.2, 55.2, 107.0, 121.5, 125.1, 126.1, 136.8, 157.2; MS (CI with AcOH): m/z 220 (M+1).

(S)-5-Methoxy-2-(N-cyanomethyl-N-n-propylamino)tetralin Hydrochloride [(S)-6]: bromoacetonitrile (3.55 g,

29.6 mmol) was added dropwise to a stirred suspension of K2CO3 (4.10 g, 29.6 mmol) and (S)-5 (2.60 g, 11.9 mmol) in acetone (150 mL), and the reaction mixture was refluxed under a nitrogen atmosphere for 24 h. After cooling, the precipitate was removed by filtration and the filtrate was concentrated under reduced pressure. The resulting crude oil was purified by column chromatography (eluent: CH2Cl2), which gave 2.62 g (10.1 mmol, 85%) of the pure base of 22 –1 1 (S)-6 as a colourless oil: mp 189–191 °C; [a] D –66°; IR: cm 2922, 2833, 2737, 2368 (b), 2322 (b), 1590; H NMR

(base, 200 MHz, CDCl3): d 0.96 (t, J = 7.3 Hz, 3H), 1.45–1.72 (m, 3H), 2.12–2.22 (m, 1H), 2.59–3.06 (m, 7H), 3.68 13 (s, 2H), 3.83 (s, 3H), 6.71 (dd, J = 8.3 Hz, 8.3 Hz, 2H), 7.17 (t, J = 7.9 Hz, 1H); C NMR (base, 50 MHz, CDCl3): d 11.6, 20.9, 22.9, 26.5, 33.2, 38.6, 52.1, 55.2, 57.7, 107.2, 116.9, 121.5, 124.8, 126.3, 136.3, 157.1; MS (CI with AcOH): m/z (rel. intensity) 220 (100), 259 (86, M+1).

(R)-5-Methoxy-2-(N-cyanomethyl-N-n-propylamino)tetralin Hydrochloride [(R)-6]: this compound was 22 –1 prepared as described for (S)-6, starting from (R)-5. Yield 85%; mp 186–188 °C; [a] D +66°; IR: cm 2922, 2834, 1 2367 (b), 2330 (b), 1590; H NMR (base, 200 MHz, CDCl3): d 0.96 (t, J = 7.3 Hz, 3H), 1.49–1.72 (m, 3H), 2.15 (m, 1H), 2.60–3.06 (m, 7H), 3.68 (s, 2H), 3.83 (s, 3H), 6.71 (dd, J = 8.3 Hz, 8.3 Hz, 2H), 7.13 (t, J = 7.7 Hz, 1H); 13C

NMR (base, 50 MHz, CDCl3): d 11.6, 20.9, 22.9, 26.5, 33.2, 38.6, 52.1, 55.2, 57.7, 107.2, 116.9, 121.5, 124.8, 126.3, 136.3, 157.1; MS (CI with AcOH): m/z (rel. intensity) 220 (31), 259 (100, M+1).

(S)-5-Methoxy-2-[N-(2-aminoethyl)-N-n-propylamino]tetralin Dihydrochloride [(S)-7]: a solution of the free base of (S)-6 (2.35 g, 9.1 mmol) in dry THF (75 mL) was added to a stirred suspension of LiAlH4 (3.00 g) in dry THF (75 mL). After refluxing for 24 h under a nitrogen atmosphere, the reaction mixture was cooled to room temperature and excess LiAlH4 was decomposed by adding H2O and 4N aqueous NaOH solution. The precipitate was removed by filtration and the filtrate was concentrated under reduced pressure. The resulting oil was dissolved in CH2Cl2, the solution was dried over Na2SO4 and subsequently filtered. Removal of the solvent under reduced pressure gave 2.16 g 22 –1 (8.2 mmol, 90%) of the pure base of (S)-7 as a colourless oil: mp 111–112 °C; [a] D –53°; IR: cm 3399 (b), 2939, 1 2881, 2836, 2633, 2519, 1588; H NMR (base, 200 MHz, CDCl3): d 0.90 (t, J = 7.3 Hz, 3H), 1.39–1.69 (m, 3H), 1.80 (bs, 2H), 1.98–2.08 (m, 1H), 2.28–3.07 (m, 11H), 3.82 (s, 3H), 6.69 (dd, J = 11.7 Hz, 7.9 Hz, 2H), 7.10 (t, J = 7.9 Hz, 13 1H); C NMR (base, 50 MHz, CDCl3): d 11.8, 22.3, 23.9, 25.5, 32.8, 40.6, 52.6, 53.0, 55.2, 56.2, 106.8, 121.6, 125.2, 126.1, 138.1, 157.2; MS (CI with AcOH): m/z (rel. intensity) 220 (2), 263 (100, M+1).

(R)-5-Methoxy-2-[N-(2-aminoethyl)-N-n-propylamino]tetralin [(R)-7]: this compound was prepared as 22 –1 described for (S)-7, starting from (R)-6. Yield 97%; mp 111–113 °C; [a] D +54°; IR: cm 3400 (b), 2939, 2882, 1 2837, 2639, 2525, 1588; H NMR (base, 200 MHz, CDCl3): d 0.90 (t, J = 7.3 Hz, 3H), 1.42–1.68 (m, 3H), 1.84 (bs, 2H), 1.99–2.08 (m, 1H), 2.46–2.67 (m, 5H), 2.70–3.07 (m, 5H), 3.81 (s, 3H), 6.69 (dd, J = 12.2 Hz, 7.9 Hz, 2H), 7.10 13 (t, J = 7.7 Hz, 1H); C NMR (base, 50 MHz, CDCl3): d 11.8, 22.3, 23.9, 25.5, 32.2, 40.6, 52.6, 53.0, 55.2, 56.2, 106.8, 121.6, 125.2, 126.1, 138.1, 157.2; MS (CI with AcOH): m/z (rel. intensity) 220 (5), 263 (100, M+1).

(S)-5-Methoxy-2-[N-(2-(benzamidoethyl)-N-n-propylamino]tetralin Hydrochloride [(S)-1]: benzoyl chloride

(0.52 g, 3.7 mmol), dissolved in CH2Cl2 (10 mL), was added dropwise to a vigorously stirred mixture of (S)-7 (0.50 g,

126 Enantiomers of 5-OMe-BPAT and 5-OMe-(2,6-di-OMe)-BPAT

1.5 mmol), 10% aqueous NaOH solution (12 mL) and CH2Cl2 (50 mL). After stirring at room temperature for 3 h, the reaction mixture was poured into H2O (50 mL) and the organic phase was separated. The H2O layer was extracted with CH2Cl2 (2 ´ 50 mL) and the combined organic layers were subsequently washed with saturated aqueous NaHCO3

(3 ´ 50 mL), H2O (50 mL) and brine (50 mL). After drying over Na2SO4 the organic layer was concentrated in vacuo to obtain the crude amide as an orange oil. Purification by column chromatography [eluent: MeOH/CH2Cl2, 1/15 (v/v)] 22 –1 yielded 0.47 g (1.3 mmol, 86%) of the pure base of (S)-1 as a colourless oil: mp 91–93 °C; [a] D –37°; IR: cm 3255 1 (b), 2939, 2835, 2598 (b), 2502 (b), 1655, 1588, 1533; H NMR (base, 200 MHz, CDCl3): d 0.93 (t, J = 7.3 Hz, 3H), 1.47–1.64 (m, 3H), 2.00–2.05 (m, 1H), 2.52–2.62 (m, 3H), 2.74–2.83 (m, 4H), 2.92–3.09 (m, 2H), 3.45–3.55 (m, 2H), 3.79 (s, 3H), 6.67 (t, J = 8.8 Hz, 2H), 7.10 (t, J = 7.9 Hz, 1H), 7.23 (bs, 1H), 7.38–7.48 (m, 3H), 7.84 (dd, J = 7.9 Hz, 13 8.8 Hz, 2H); C NMR (base, 50 MHz, CDCl3): d 11.9, 22.1, 23.8, 25.6, 32.3, 38.2, 48.6, 52.3, 55.2, 56.0, 107.0, 121.6, 125.0, 126.3, 126.9, 128.5, 131.2, 137.5, 157.2, 167.2; MS (CI with AcOH): m/z 367 (M+1); Anal. calcd for

C23H30N2O2·HCl·½H2O: C 67.04, H 7.84, N 6.80; obsd C 66.70, H 7.76, N 6.75.

(R)-5-Methoxy-2-[N-(2-(benzamidoethyl)-N-n-propylamino]tetralin Hydrochloride [(R)-1]: this compound 22 –1 was prepared as described for (S)-1, starting from (R)-7. Yield 91%; mp 91–93 °C; [a] D +38°; IR: cm 3261 (b), 1 2939, 2836, 2619 (b), 1655, 1589, 1535; H NMR (base, 200 MHz, CDCl3): d 0.91 (t, J = 7.3 Hz, 3H), 1.47–1.65 (m, 3H), 1.97–2.06 (m, 1H), 2.47–2.60 (m, 3H), 2.75–2.82 (m, 4H), 2.95–3.04 (m, 2H), 3.42–3.56 (m, 2H), 3.80 (s, 3H), 6.67 (t, J = 8.2 Hz, 2H), 6.96 (bs, 1H), 7.08 (t, J = 8.1 Hz, 1H), 7.42–7.53 (m, 3H), 7.78 (dd, J = 8.1 Hz, 1.5 Hz, 2H); 13 C NMR (base, 50 MHz, CDCl3): d 11.6, 21.8, 23.5, 25.3, 31.9, 37.7, 48.3, 51.9, 55.0, 55.6, 106.9, 121.4, 124.9, 126.2, 126.7, 128.4, 131.2, 134.6, 137.4, 157.1, 167.1; MS (CI with AcOH): m/z 367 (M+1); Anal. calcd for

C23H30N2O2·HCl·½H2O: C 67.04, H 7.84, N 6.80; obsd C 67.18, H 7.71, N 6.80.

(S)-5-Methoxy-2-{N-[2-(2,6-dimethoxy)benzamidoethyl]-N-n-propylamino}tetralin Oxalate [(S)-2]: this 22 –1 compound was prepared as described for (S)-1. Yield 74%; mp 78–80 °C; [a] D –32°; IR: cm 3259 (b), 2939, 2840, 1 2642 (b), 2530 (b), 1655, 1597, 1523; H NMR (base, 200 MHz, CDCl3): d 0.86 (t, J = 7.3 Hz, 3H), 1.41–1.69 (m, 3H), 1.97–2.06 (m, 1H), 2.42–2.59 (m, 3H), 2.77–2.83 (m, 4H), 2.93–3.05 (m, 2H), 3.42–3.59 (m, 2H), 3.78 (s, 9H), 6.56 (d, J = 8.5 Hz, 2H), 6.66 (dd, J = 6.6 Hz, 6.6 Hz, 2H), 7.08 (t, J = 7.8 Hz, 1H), 7.26 (dd, J = 8.4 Hz, 8.4 Hz, 1H); 13 C NMR (base, 50 MHz, CDCl3): d 11.5, 21.4, 23.5, 25.0, 31.5, 37.5, 49.0, 52.1, 55.0, 55.6, 56.2, 103.7, 106.8, 115.9, 121.4, 124.8, 126.2, 130.3, 137.3, 157.1, 157.3, 165.8; MS (CI with AcOH): 427 (M+1); Anal. calcd for

C25H34N2O4·C2H2O4: C 62.76, H 7.04, N 5.42; obsd C 63.09, H 7.37, N 5.45.

(R)-5-Methoxy-2-{N-[2-(2,6-dimethoxy)benzamidoethyl]-N-n-propylamino}tetralin Oxalate [(R)-2]: this 22 –1 compound was prepared as described for (S)-1, starting from (R)-7. Yield 72%; mp 79–81 °C; [a] D +29°; IR: cm 1 3406 (b), 2938, 2838, 2627 (b), 2504 (b), 1654, 1596, 1542; H NMR (base, 200 MHz, CDCl3): d 0.85 (t, J = 7.3 Hz, 3H), 1.37–1.66 (m, 3H), 1.95–2.04 (m 1H), 2.41–2.59 (m, 3H), 2.72–2.80 (m, 4H), 2.87–3.05 (m, 2H), 3.47–3.56 (m, 2H), 3.79 (s, 9H), 6.43 (bs, 1H), 6.56 (d, J = 8.3 Hz, 2H), 6.65 (dd, J = 6.8 Hz, 6.8 Hz, 2H), 7.07 (t, J = 7.9 Hz, 1H), 13 7.27 (dd, J = 9.0 Hz, 7.8 Hz, 1H); C NMR (base, 50 MHz, CDCl3): d 11.5, 21.7, 23.6, 25.1, 31.7, 37.6, 48.8, 52.0, 55.0, 55.6, 55.9, 103.7, 106.8, 116.0, 121.4, 124.9, 126.1, 130.3, 137.6, 157.1, 157.3, 165.6; MS (CI with AcOH): m/z

427 (M+1); Anal. calcd for C25H34N2O4·C2H2O4: C 62.76, H 7.04, N 5.42; obsd C 62.80, H 7.40, N 5.53.

5.6.1 PHARMACOLOGY

Radioligand Binding Studies. The receptor binding assays were essentially performed as described by Malmberg 13 9 3 et al. and Jackson et al. The following receptors and radioligands were used: D1, rat striatum and [ H]-SCH 23390; – 3 D2A and D3, cloned human receptors (expressed in Ltk and CHO cells, respectively) and [ H]-raclopride; a1, rat 3 3 3 3 cortex and [ H]-; a2, rat cortex and [ H]-RX 821002; b, rat cortex and [ H]-DHA; M, rat cortex and [ H]- 3 3 QNB; 5-HT1A, rat hippocampus and [ H]-8-OH-DPAT; 5-HT2, rat cortex and [ H]-ketanserin.

127 Chapter 5

Inhibition of VIP-stimulated cAMP Production in GH4ZD10 Cells Expressing Serotonin 5-HT1A Receptors. 10 This assay was performed essentially as described by Johansson et al. Briefly, the GH4ZD10 (rat pituitary tumor) cells (obtained from dr. O. Civelli, Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, OR) were cultured in 175-cm2 flasks in Ham’s medium with 1 mM L-glutamine supplemented with 10% FCS, 10 mM HEPES, penicillin and streptomycin at 37 ºC. Cells in passages 8 to 11 were used. Geneticin (G418 sulfate, 700 mg/L) was used for selection of cells transfected with receptors. The test compounds were dissolved to a 20 mM concentration in dimethyl sulfoxide and stored at –20 ºC until further use. The stock solutions were further diluted with water containing 0.01% ascorbic acid and 0.1 mM IBMX. The 5-HT was freshly prepared in the solution above. The cAMP assay was performed according to the method described by Dorflinger and Schonbrunn3 with some minor modifications.5 The cells were detached from the cultured flasks with Earle’s balanced salt solution supplemented with 1 mM EDTA without Ca2+ and Mg2+. The cells were suspended in FCS-free Ham’s medium and the suspension was centrifuged at 250 × g for 6 min at room temperature. The pellets were resuspended to a density of 107 cells/mL in medium containing 0.01% ascorbic acid and 0.1 mM IBMX. Cells were preincubated in this solution for 1 h at 37 ºC and then diluted to a final density of 106 cells/mL. Aliquots (0.4 mL) of the cell suspension were added to Eppendorf tubes containing 0.1 mL VIP at a final concentration of 30 nM along with the test compounds and incubated for 20 min at 37 ºC. Each sample was carried out in duplicate. Reactions were stopped by placing the assay tubes in boiling water for 4 min, after which the samples were transferred to ice water. The lysates were then centrifuged at 12,000 rpm for 4 to 5 min at 4 ºC, the supernatants were decanted, frozen and stored at –20 ºC until analyzed. Cyclic AMP levels were determined according to the method of Brown and Elkins1 as modified by Nordstedt and Fredholm16, in which free [3H]-cAMP/cAMP is separated from that bound to the bovine adrenocortical protein kinase A on glass fiber filters with a semiautomatic cell harvester (Skatron AS, Tranby, Norway).

3 [ H]-Thymidine Incorporation in CHO Cells Expressing Rat Dopamine D2A or D3 Receptors. These assays were performed essentially as described by Lajiness et al.12 and Chio et al.2 Briefly, CHO L6 and CHO D3-3 cells, expressing dopamine rat D2A and D3 receptors, respectively, were seeded into 96-well plates at a density of 5,000 cells/well and were grown at 37 ºC in aMEM, with 10% fetal calf serum, for 48 h. The wells were rinsed three times with serum-free aMEM. Ninety microliters of fresh aMEM were added along with 10 mL of drug (diluted in sterile water and filtered through 0.2-mm filters) or sterile water alone. Eight wells of every plate received 100 mL of aMEM with 10% fetal calf serum. After culturing for 16–17 h, [3H]-thymidine (1 mCi/well) was added and the cell were incubated for 2 h at 37 ºC. The cells were trypsinized and harvested onto filter mats with a Skatron cell harvester. The filters were counted in a Betaplate counter. Dose-response curves were analyzed with a nonlinear least squares fitting program provided by F. Kezdy (The Upjohn Co.), in which the curves were fitted to the equation A = B ´ [C/(D + C)] 3 + G, where A is the [ H]-thymidine cpm, B is the maximal effect, C is the EC50, D is the concentration of agonist, and G is the [3H]-thymidine cpm in the absence of agonist. Parameters B, C, and G were determined by Simplex optimization.

5.7 REFERENCES

1 Brown BL and Ekins P (1972) Saturation assay for cyclic AMP using endogenous binding protein. Adv Cyclic Nucleotide Res 2, 25–40.

2 Chio CL, Lajiness ME and Huff RM (1994) Activation of heterologously expressed D3 dopamine receptors:

comparison with D2 dopamine receptors. Mol Pharmacol 45, 51–60. 3 Dorflinger LJ and Schonbrunn A (1983) Somatostatin inhibits vasoactive intestinal peptide-stimulated cyclic adenosine monophosphate accumulation in GH pituitary cells. Endocrinology 113, 1541–1550. 4 Fargin A, Raymond JR, Regan JW, Coteccia S, Lefkowitz RJ and Caron MG (1989) Effector coupling mechanism

of the cloned 5-HT1A receptor. J Biol Chem 264, 14848–14852.

5 Fowler CJ, Ahlgren PC and Brännström G (1992) GH4ZD10 cells expressing rat 5-HT1A receptors coupled to adenylate cyclase are a model for the postsynaptic receptors in the rat hippocampus. Br J Pharmacol 107, 141–145.

128 Enantiomers of 5-OMe-BPAT and 5-OMe-(2,6-di-OMe)-BPAT

6 Grol CJ, Nordvall G, Johansson AM and Hacksell U (1991) 5-Oxygenated N-alkyl- and N,N-dialkyl-2-amino-1-

methyltetralins. Effects of structure and stereochemistry on dopamine-D2-receptor affinity. J Pharm Pharmacol 43, 481–485. 7 Hacksell U, Jackson DM and Mohell N (1995) Does the dopamine receptor subtype selectivity of antipsychotic agents provide useful leads for the development of novel therapeutic agents? Pharmacol Toxicol 76, 320–324. 8 Homan EJ, Copinga S, Elfström L, Van Der Veen T, Hallema J-P, Mohell N, Unelius L, Johansson R, Wikström H

and Grol CJ (1998) 2-Aminotetralin-derived substituted benzamides with mixed dopamine D2, D3, and serotonin

5-HT1A receptor binding properties: a novel class of potential atypical antipsychotic agents. Bioorg Med Chem (in press). 9 Jackson DM, Ryan C, Evenden J and Mohell N (1994) Preclinical findings with new antipsychotic agents: what makes them atypical? Acta Psychiatr Scand Suppl 380, 41–48. 10 Johansson L, Sohn D, Thorberg S-O, Jackson DM, Kelder D, Larsson L-G, Rényi L, Ross SB, Wallsten C, Eriksson H, Hu P-S, Jerning E, Mohell N and Westlind-Danielsson A (1997) The pharmacological

characterization of a novel selective 5-hydroxytryptamine1A receptor antagonist, NAD-299. J Pharmacol Exp Ther 283, 216–225. 11 Karlsson A, Björk L, Pettersson C, Andén N-E and Hacksell U (1990) (R)- and (S)-5-hydroxy-2- (dipropylamino)tetralin (5-OH-DPAT): assessment of optical purities and dopaminergic activities. Chirality 2, 90– 95.

12 Lajiness ME, Chio CL and Huff RM (1993) D2 Dopamine receptor stimulation of mitogenesis in transfected Chinese hamster ovary cells: relationships to dopamine stimulation of phosphorylations. J Pharmacol Exp Ther 267, 1573–1581. 13 Malmberg Å, Jackson DM, Eriksson A and Mohell N (1993) Unique binding characteristics of antipsychotic

agents interacting with human dopamine D2A, D2B, and D3 receptors. Mol Pharmacol 43, 749–754. 14 Malmberg Å, Nordvall G, Johansson AM, Mohell N and Hacksell U (1994) Molecular basis for the binding of 2-

aminotetralins to human dopamine D2A and D3 receptors. Mol Pharmacol 46, 299–312. 15 McDermed JD, McKenzie GM and Freeman HS (1976) Synthesis and dopaminergic activity of (±)-, (+)-, and (–)- 2-dipropylamino-5-hydroxy-1,2,3,4-tetrahydronaphthalene. J Med Chem 19, 547–549. 16 Nordstedt C and Fredholm BB (1990) A modification of a protein-binding method for rapid quantification of cAMP in cell-culture supernatants and body fluid. Anal Biochem 89, 231–234. 17 Seiler MP and Markstein R (1984) Further characterization of structural requirements for agonists at the striatal

dopamine D2 receptor and a comparison with those at the striatal dopamine D1 receptor. Studies with a series of monohydroxyaminotetralins on acetylcholine release from rat striatum. Mol Pharmacol 26, 452–457. 18 Tedesco JL, Seeman P and McDermed JD (1979) The conformation of dopamine at its receptor: binding of monohydroxy-2-aminotetralin enantiomers and positional isomers. Mol Pharmacol 16, 369–381. 19 Van Vliet LA, Tepper PG, Dijkstra D, Damsma G, Wikström H, Puglsey TA, Akunne HC, Heffner TG, Glase SA

and Wise LD (1996) Affinity for dopamine D2, D3, and D4 receptors of 2-aminotetralins. Relevance of D2 agonist binding for determination of receptor subtype selectivity. J Med Chem 39, 4233–4237. 20 Wikström H, Andersson B, Sanchez D, Lindberg P, Arvidsson LE, Johansson AM, Nilsson JL, Svensson K, Hjorth S and Carlsson A (1985) Resolved monophenolic 2-aminotetralins and 1,2,3,4,4a,5,6,10b-octahydro- benzo[f]quinolines: structural and stereochemical considerations for centrally acting pre- and postsynaptic dopamine-receptor agonists. J Med Chem 28, 215–225.

129 IN VIVO PHARMACOLOGICAL EVALUATION 6 OF THE ENANTIOMERS OF 5-OME-BPAT

ABSTRACT

The enantiomers of 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT) were evaluated for (1) their ability to affect various aspects of spontaneous and d-amphetamine-induced locomotor activity in rats; (2) their ability to induce catalepsy in rats; and (3) their effects on neurochemical processes in different brain areas, using intracerebral microdialysis in freely moving rats. Low doses of (S)-5-OMe-BPAT suppressed spontaneous horizontal activity, peripheral activity and forward locomotion, but these parameters were restored at higher doses. Rearing on the other hand was strongly and dose-dependently suppressed. In combination with d-amphetamine (5 mg/kg s.c.), (S)-5-OMe-BPAT dose-dependently enhanced horizontal activity, but particularly peripheral activity and forward locomotion were strongly potentiated. Rearing was suppressed at low doses, but not at the highest dose tested. In contrast, (R)-5-OMe-BPAT dose-dependently inhibited most aspects of spontaneous as well as d-amphetamine- induced locomotor activity. Only peripheral activity in combination with d-amphetamine was not significantly affected. Both enantiomers failed to induce catalepsy in rats at doses up to 60 mmol/kg s.c. (S)-5-OMe-BPAT (25 mmol/kg s.c.) decreased extracellular levels of dopamine and 5-HIAA in the striatum, while DOPAC levels were increased. These effects were associated with simultaneous increases of the extracellular levels of dopamine, DOPAC and noradrenaline, and a decrease of 5-HIAA in the prefrontal cortex, as assessed by dual-probe intracerebral microdialysis in freely moving rats. In contrast, (R)-5-OMe-BPAT (25 mmol/kg s.c.) increased extracellular levels of both dopamine and DOPAC in the striatum. Similar to its optical antipode, (R)-5-OMe-BPAT induced a simultaneous increase of extracellular levels of dopamine, DOPAC and noradrenaline in the prefrontal cortex, while 5-HIAA levels were decreased in both brain areas. The increases in striatal and prefrontal cortical dopamine levels induced by (R)-5- OMe-BPAT after 60 min were not significantly different at this dose, but possible regional differences in its effects on dopaminergic neurotransmission may become apparent at lower doses. Both enantiomers (25 mmol/kg s.c.) markedly suppressed extracellular levels of serotonin and 5-HIAA in the ventral hippocampus. Changes in these neurochemical parameters induced by (S)-5-OMe-BPAT were associated with the specific behaviour indicative of serotonin 5-HT1A receptor stimulation. (R)-5-OMe-BPAT on the other hand appeared to suppress locomotor behaviour and only weakly

131 Chapter 6 induced several aspects of the 5-HT syndrome. Taken together, the results of these in vivo studies suggest that (S)-5-

OMe-BPAT is capable of stimulating dopamine D2 receptors, while the (R)-enantiomer behaves as a dopamine D2 receptor antagonist. In addition, both enantiomers behave as full serotonin 5-HT1A receptor agonists in vivo. These findings are largely consistent with their in vitro intrinsic efficacy profiles at these receptor subtypes, and as such, the enantiomers of 5-OMe-BPAT should be interesting pharmacological tools for further investigating the concept of mixed dopamine D2 receptor antagonism and serotonin 5-HT1A receptor agonism for atypical antipsychotic drug action. The lack of cataleptogenic activity of (R)-5-OMe-BPAT, in combination with its dopamine D2 receptor antagonistic and serotonin 5-HT1A receptor-stimulating properties, suggest that this compound in particular may possess enhanced antipsychotic efficacy and a low propensity to cause EPS in man.

6.1 INTRODUCTION

Since no true animal models of schizophrenia exist, the prediction of potential (atypical) antipsychotic activity in man, using preclinical animal models, is without doubt one of the most difficult stages in the development process of a new antipsychotic agent (for reviews, see refs. 26 and 63). As a result of the general acceptance of the dopamine hypotheses of schizophrenia and antipsychotic drug action (see Chapter 1), most behavioural models currently employed for this purpose are based on the ability of antipsychotic agents to affect the behaviour induced by psychostimulants known to interfere with dopaminergic neurotransmission in the CNS, such as d- amphetamine, apomorphine, , and . Examples of behavioural paradigms, with various degrees of validity and predictability of antipsychotic activity, that have emerged during the last three decades are: inhibition of stimulant-induced locomotor activity;58 inhibition of apomorphine-induced mouse climbing;21 differential activity in the paw test;27 and reversal of stimulant-induced deficits in prepulse inhibition (PPI).81 The rationale for using stimulant-induced behavioural models stems in part from the observations that compounds such as d- amphetamine can exacerbate psychotic symptoms in schizophrenic patients,5 and induce syndromes in healthy volunteers mimicking paranoid schizophrenia,5,6,79 which can be blocked by antipsychotic agents.78 The ability of a compound to inhibit d-amphetamine-induced locomotor activity in rodents has been frequently used for the initial screening of potential antipsychotic agents in the past, in part because locomotor activity can be easily measured in fully automated systems,29 using large numbers of animals. d-Amphetamine is an indirect dopamine receptor agonist, which acts by enhancing the release of dopamine from dopaminergic nerve terminals (for review and references see ref. 72). Relatively low doses (0.5–1.5 mg/kg) of d-amphetamine stimulate most aspects of locomotor activity in rats, including forward locomotion and rearing. Additionally, higher doses (2.5–10 mg/kg) evoke stereotyped behaviour, such as head bobbing, licking and gnawing. Virtually all clinically effective antipsychotic agents are able to antagonize these behavioural effects induced by d-amphetamine in rats.58 Stimulation of locomotor activity by d-amphetamine is presumably mediated by the mesolimbic and mesocortical dopaminergic systems, while the nigrostriatal dopaminergic system appears to be the neuronal substrate for the stereotyped behaviour.46,76 Since it has been generally accepted that antipsychotic agents exert their antipsychotic effects by blocking dopamine D2 receptors in the mesolimbic and mesocortical dopaminergic systems, while simultaneous blockade of dopamine D2 receptors in the nigrostriatal dopaminergic system presumably causes EPS, a potential atypical antipsychotic agent should possess a wide separation between the dose that blocks d-

132 In Vivo Pharmacological Evaluation of the Enantiomers of 5-OMe-BPAT amphetamine-induced locomotor activity and the dose that blocks d-amphetamine-induced stereotyped behaviour in rats. Spontaneous locomotor activity in rats can also be blocked by antipsychotic agents,58 while higher doses may even induce catalepsy, a state of tonic immobility, characterized by akinesia and the inability of the animal to correct its position when placed in an unusual posture. Most classical antipsychotic agents dose-dependently induce catalepsy in rats.58 Blockade of dopamine receptors in the nigrostriatal dopaminergic system seems to be the mechanism of the cataleptogenic effect.64 Whereas the catalepsy test has been used as a screening assay for identifying new antipsychotic agents in the past,55 today catalepsy has been generally accepted as a model for EPS in man,58 and the ability of a potential antipsychotic agent to induce catalepsy in rats is considered to be a measure for its liability to induce EPS in man.38 In vivo measurement studies, using monitoring techniques such as intracerebral microdialysis and electrophysiological single cell recordings, have revealed that acute administration of antipsychotic agents affects neurochemical and electrophysiological processes in the central nervous system (for review and references see ref. 8). Dependent on the receptor binding properties of a compound under investigation, the release of different neurotransmitters and their metabolites may be differently affected in different brain areas. Classical antipsychotic agents consistently have been shown to increase extracellular levels of dopamine in both the striatum and limbic brain areas, while atypical antipsychotic agents appear to preferentially increase dopaminergic activity in limbic brain areas, i.e. the nucleus accumbens and prefrontal cortex. Since preferential blockade of mesolimbocortical dopamine D2 receptors may prove to be the key to an antipsychotic profile with improved efficacy against negative symptoms of schizophrenia and a low propensity to cause EPS and TD, research has been focused on compounds which preferentially affect the dopaminergic neurotransmission in these brain areas. Additionally, it has been shown that clozapine and other alleged atypical antipsychotic agents also increase extracellular levels of noradrenaline in the prefrontal cortex, a feature which also may contribute to their improved clinical profiles.62,93 Therefore, complementary to studying the effects of potential atypical antipsychotic agents in behavioural models, establishing the regional effects of such compounds on neurochemical processes in the CNS may provide additional clues about their mechanism of action and lend further support for their alleged atypical properties. Intracerebral microdialysis is a suitable technique for this purpose, since it allows to measure the extracellular levels of a wide variety of neurotransmitters and their metabolites in almost any brain area of interest in conscious, freely moving animals (for reviews and references see refs. 11, 25, 92, and 94). In the current investigation, the effects of the enantiomers of the potential atypical antipsychotic agent 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT) on spontaneous and d-amphetamine-induced locomotor activity in rats, and their ability to induce catalepsy in rats were determined. Furthermore, their ability to affect the release of various neurotransmitters and metabolites in different brain areas was evaluated, using intracerebral microdialysis in freely moving rats.

133 Chapter 6

6.2 PHARMACOLOGY

Dose-dependent effects of (S)- and (R)-5-OMe-BPAT on spontaneous and d-amphetamine- induced locomotor activity in rats were determined in automated activity cages, allowing for measurement of horizontal activity, peripheral activity, forward locomotion, and rearing. The following paradigm was used: the test compound or saline was administered subcutaneously (s.c.), 30 minutes later the animals were placed in the activity cages, and their locomotor activity was measured during 30 minutes. Thus, activity measurements during this so-called ‘habituation phase’ reflect the ability of a compound to affect spontaneous locomotor activity. Then d-amphetamine (5 mg/kg s.c.) was administered and the activity measurements were continued during the next 60 minutes (for detailed descriptions of the experimental procedures, see Section 6.5). The ability of (S)- and (R)-5-OMe-BPAT to dose-dependently induce catalepsy in rats was established by measuring the catalepsy time on a grid, at ½, 1, 2, 4, 8, and 24 hours after subcutaneous administration of the compounds. Intracerebral microdialysis in freely moving rats, applying the dual-probe approach, was used to simultaneously establish the effects of (S)- and (R)-5-OMe-BPAT (25 mmol/kg s.c.) on the dopaminergic neurotransmission in the striatum and the prefrontal cortex, by monitoring the release of dopamine and its main metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). In addition, 5- hydroxyindoleacetic acid (5-HIAA), the main metabolite of serotonin, was measured in both areas, while in the prefrontal cortex the release of noradrenaline was monitored as well. Furthermore, intracerebral microdialysis was used to evaluate the effects of (S)- and (R)-5-OMe-BPAT (25 mmol/kg s.c.) on the release of serotonin and 5-HIAA in the ventral hippocampus, as a measure of their serotonin 5-HT1A receptor-stimulating properties.

6.3 RESULTS AND DISCUSSION

Although locomotor activity studies are relatively easy to perform, the interpretation of the results can be complicated, since various aspects of locomotor activity can be differently affected by blockade or stimulation of different subtypes of dopaminergic, but also non-dopaminergic receptors

(for review and references, see refs. 42 and 63). For example, both selective dopamine D1 (e.g. SCH

23390) and non-selective dopamine D2/D3 (e.g. raclopride) receptor antagonists can inhibit spontaneous as well as d-amphetamine-induced locomotor activity. Selective dopamine D3 receptor antagonists, however, have been reported to stimulate locomotor activity, presumably by blockade of 20,71,89 postsynaptic dopamine D3 receptors. Selective dopamine D1 receptor agonists, such as SKF

38393, predominantly stimulate sniffing and grooming. Low doses of nonselective dopamine D2/D3 receptor agonists, such as quinpirole, suppress locomotor activity at low doses, presumably by stimulating presynaptic autoreceptors at the nerve terminals. At higher doses, these compounds stimulate locomotor activity and induce stereotyped behaviour, such as intense licking and biting, effects which are probably mediated by stimulation of postsynaptic dopamine D2 receptors. Selective dopamine D3 receptor agonists (e.g. PD 128907) also appear to have an inhibitory effect at low doses, but fail to induce stereotyped behaviour at high doses.4,23,24,80 The functional role of the dopamine D4 receptor in relation to locomotor activity is less clear, in part due to the lack of

134 In Vivo Pharmacological Evaluation of the Enantiomers of 5-OMe-BPAT

selective agonists available for this receptor subtype. The selective dopamine D4 antagonist L- 745,870 was reported not to affect d-amphetamine-induced locomotor activity.14 Similar to dopamine receptor ligands, serotonergic agents can induce specific types of behaviour in animals. For example, selective serotonin 5-HT1A receptor agonists induce several aspects of the ‘5-HT syndrome’ in rats, including a flat body posture, Straub tail, hindlimb abduction, reciprocal forepaw treading (‘piano playing’), and lower lip retraction12,36,77,84,85. In addition, they also affect certain aspects of locomotor activity in rats. The most frequently studied compound in this respect is 8-OH-DPAT. Reports on the effects of 8-OH-DPAT have been somewhat conflicting: some researchers found in increase in locomotor activity,44,49,85 while others reported a decrease.1,16,51 Close examination of its effects has revealed that it decreases the total amount of activity, but increases the relative proportion of forward locomotion and peripheral activity. In other words, the rats tend to move slowly along the walls of the activity cages.2,34,35 Similar effects were reported for 3 the selective serotonin 5-HT1A receptor agonist flesinoxan. Furthermore, selective serotonin 5-HT1A receptor agonists, including 8-OH-DPAT, flesinoxan, buspirone, ipsapirone, and gepirone have consistently been reported to suppress rearing behaviour in a dose-dependent manner in rats.1,2,13,34,35,51,68 Taking the observations above into consideration, it is clear that the interpretation of behavioural effects of compounds with mixed dopamine and serotonin receptor binding profiles in relation to their neurochemical effects may be complicated. In the previous chapter, the enantiomers of 5-OMe-

BPAT were shown to possess different degrees of intrinsic efficacies at dopamine D2 and D3 receptors in vitro. Thus, the (S)-enantiomer behaved as a partial agonist at both dopamine D2 and D3 receptors, with intrinsic efficacies of 62% and 30%, respectively. The (R)-enantiomer also behaved as a partial agonist at dopamine D2 receptors, but with a lower intrinsic efficacy (36%). At dopamine

D3 receptors this compound was devoid of intrinsic efficacy. Furthermore, both enantiomers were shown to behave as full serotonin 5-HT1A receptor agonists in vitro. Being devoid of affinity for the dopamine D1 receptor, effects mediated by this receptor subtype can be ruled out. Thus, the behavioural and neurochemical effects induced by (S)- and (R)-5-OMe-BPAT described below should be interpreted in terms of their mixed dopamine D2, D3, and serotonin 5-HT1A receptor binding profiles, while particularly taking the differences in intrinsic efficacies at the dopaminergic receptors into account. In the locomotor activity experiments, (S)-5-OMe-BPAT affected all four monitored aspects of locomotor activity in the animals during the habituation phase (Figure 6.1). Horizontal and peripheral activity were significantly inhibited at all doses tested, the effect on both parameters being the strongest at a dose of 0.6 mmol/kg. Forward locomotion was also significantly decreased at this dose, but at higher doses forward locomotion was restored (1.8 mmol/kg) and even stimulated (5.4 mmol/kg). The inhibitory effects on these parameters at low doses are probably caused by stimulation of presynaptic dopamine D2 receptors. Rearing on the other hand was strongly and dose-dependently suppressed by (S)-5-OMe-BPAT. At the highest dose (5.4 mmol/kg), hardly any rearing was detected. This effect on rearing clearly reflects the serotonin 5-HT1A receptor-stimulating properties of (S)-5-OMe-BPAT. Therefore, it is likely that the restoring effect on peripheral activity and the increase in forward locomotion at this dose are also the result of serotonin 5-HT1A receptor stimulation. On the other hand, it may also reflect the dopamine D2 receptor agonist properties, since

135 Chapter 6

dopamine D2/D3 receptor agonists stimulate locomotor activity in rats at relatively high doses, due to stimulation of postsynaptic dopamine D2 receptors. d-Amphetamine increased the horizontal activity counts without specifically affecting any of the locomotor activity aspects in particular, as can be seen by comparing the control values of the different aspects in Figures 6.1 and 6.2. In combination with d-amphetamine (5 mg/kg) the effects of (S)-5-OMe-BPAT were more pronounced (Figure 6.2). Horizontal activity, and particularly peripheral activity and forward locomotion were strongly enhanced at the highest dose (5.4 mmol/kg). Rearing showed a U-shaped dose-response graph after treatment with d-amphetamine: at doses of 0.6 and 1.8 mmol/kg rearing was significantly decreased, but was restored to control values at the highest dose tested. These results suggest that (S)-OMe-BPAT has no dopamine D2 receptor blocking properties under these conditions, and hence must behave as an agonist. Provided that (S)-

OMe-BPAT can still compete with dopamine for binding to postsynaptic dopamine D2 receptors after a dose of 5 mg/kg of d-amphetamine, it is possible that the stimulating effects of the compound at higher doses are caused by postsynaptic dopamine D2 receptor stimulation. Alternatively, the potentiating effects may also be ascribed to the serotonin 5-HT1A receptor-stimulating properties of (S)-OMe-BPAT, because, whereas 8-OH-DPAT inhibits total spontaneous locomotor activity (see above), it has been reported to potentiate d-amphetamine-induced locomotor activity.41 This may explain why the effects of (S)-5-OMe-BPAT were more pronounced under these conditions. In addition, particularly the pronounced stimulation of peripheral activity and forward locomotion, indicating that the animals were moving forward while remaining close to the walls of the cages, suggest that (S)-5-OMe-BPAT induces effects similar to selective serotonin 5-HT1A receptor

2000 1000

750 * * *** ** *** *** 1000 500 *** *** 250 Peripheral activity Horizontal activity

0 0 0.0 0.2 0.6 1.8 5.4 0.0 0.2 0.6 1.8 5.4 Dose (mmol/kg) Dose (mmol/kg)

750 400 *

300 500

** 200 ** 250 *** Rearing 100 *** Forward locomotion *** *** 0 0 0.0 0.2 0.6 1.8 5.4 0.0 0.2 0.6 1.8 5.4 Dose (mmol/kg) Dose (mmol/kg)

FIGURE 6.1 Dose-dependent effects (mean + s.e.m.; n = 7–12; *, p < 0.05; **, p < 0.01; ***, p < 0.001) of (S)-5-OMe- BPAT on spontaneous locomotor activity (horizontal activity, top left; peripheral activity, top right; forward locomotion, bottom left; rearing, bottom right) in rats.

136 In Vivo Pharmacological Evaluation of the Enantiomers of 5-OMe-BPAT

7500 4000 *** *** 3000 5000 * 2000 * * 2500 * 1000 Peripheral actitivy Horizontal activity

0 0 0.0 0.2 0.6 1.8 5.4 0.0 0.2 0.6 1.8 5.4 Dose (mmol/kg) Dose (mmol/kg)

4000 500 *** 400 3000

300 ** 2000 200 * Rearing * 1000 100 Forward locomotion

0 0 0.0 0.2 0.6 1.8 5.4 0.0 0.2 0.6 1.8 5.4 Dose (mmol/kg) Dose (mmol/kg)

FIGURE 6.2 Dose-dependent effects (mean + s.e.m.; n = 5–8; *, p < 0.05; **, p < 0.01; ***, p < 0.001) of (S)-5-OMe- BPAT on d-amphetamine-induced locomotor activity (horizontal activity, top left; peripheral activity, top right; forward locomotion, bottom left; rearing, bottom right) in rats. agonists like 8-OH-DPAT and flesinoxan.3 On the other hand, the restoring effect on rearing at a dose of 5.4 mmol/kg suggests that stimulation of postsynaptic dopamine D2 receptors is also involved in the effects. In contrast to its optical antipode, (R)-5-OMe-BPAT inhibited most parameters, both during the habituation phase and in combination with d-amphetamine. Thus, spontaneous horizontal activity, forward locomotion, and rearing were significantly decreased at doses of 0.6 to 28.8 mmol/kg (Figure 6.3). The effects on peripheral activity seemed somewhat weaker: significant inhibition occurred at doses of 0.6, 7.2, and 28.8 mmol/kg, but not at 1.8 mmol/kg. The suppression of rearing is indicative of serotonin 5-HT1A receptor stimulation, although the effect was less pronounced than that of (S)-5-OMe-BPAT. This may reflect lower affinity of the (R)-enantiomer for the serotonin 5-

HT1A receptor (see Chapter 5). The suppression of the other parameters, given the wide dose range, suggest that this compound acts as a dopamine D2 receptor antagonist, which is consistent with the low intrinsic efficacy determined in vitro. In this in vitro assay (R)-5-OMe-BPAT also behaved as a dopamine D3 receptor antagonist. Although selective dopamine D3 receptor antagonists have been reported to stimulate spontaneous locomotor activity (see above), possible stimulating effects of (R)-

5-OMe-BPAT mediated through blockade of dopamine D3 receptors seem to be completely overruled by blockade of dopamine D2 receptors in the behavioural model discussed here. When combined with d-amphetamine, (R)-5-OMe-BPAT inhibited horizontal activity, forward locomotion, and rearing. Horizontal activity was significantly suppressed at doses ranging from 1.8 to 28.8 mmol/kg, while forward locomotion and rearing were significantly inhibited only at doses of

7.2 and 28.8 mmol/kg. These findings strongly suggest that this enantiomer acts as dopamine D2

137 Chapter 6 receptor antagonist in vivo. Remarkably, the effects of (R)-5-OMe-BPAT seem to be somewhat less pronounced in the presence of d-amphetamine, despite its dopamine D2 receptor-blocking properties. Peripheral activity in particular was not significantly affected at all doses tested. Similar to (S)-5-

OMe-BPAT, this may reflect the serotonin 5-HT1A receptor-stimulating properties of the compound, which seem to be more pronounced in the presence of d-amphetamine. Thus, at higher doses peripheral activity contributes the strongest to the total horizontal activity, but since forward locomotion is suppressed at these doses, these findings indicate that the animals place themselves close to the walls of the activity cages, where they remain seated. This is in contrast with the effects of (S)-5-OMe-BPAT, where the animals move along the walls. It is noteworthy that these effects of (R)-5-OMe-BPAT remarkably resemble those induced by buspirone in the same paradigm, as 2 reported by Ahlenius et al. Buspirone is a potent partial serotonin 5-HT1A receptor agonist, but also 57 a dopamine D2 receptor antagonist. In the catalepsy test both enantiomers of 5-OMe-BPAT were devoid of cataleptogenic activity at doses up to 60 mmol/kg (data not shown), suggesting that they will have a low propensity to cause

EPS in man. Selective serotonin 5-HT1A receptor agonists, including 8-OH-DPAT, flesinoxan, buspirone, ipsapirone and gepirone have been shown to be able to reverse catalepsy induced by dopamine D2 receptor antagonists, such as haloperidol and raclopride, in various animal species.15,28,33,40,48,50,56,88 These compounds presumably exert their anticataleptic action by stimulation 40 of somatodendritic serotonin 5-HT1A autoreceptors located in the dorsal and medial raphe nuclei. It has been speculated that stimulation of these autoreceptors in the raphe nuclei leads to an inhibition of serotonergic neurons projecting directly to the striatum. This inhibition should in turn cause a

2000 1000

750 * * * ** 1000 *** 500 *** *** 250 Peripheral activity Horizontal activity

0 0 0.0 0.2 0.6 1.8 7.2 28.8 0.0 0.2 0.6 1.8 7.2 28.8 Dose (mmol/kg) Dose (mmol/kg)

500 300

400 * * 200 300 ** **

200 *** *** *** Rearing 100 100 *** Forward locomotion

0 0 0.0 0.2 0.6 1.8 7.2 28.8 0.0 0.2 0.6 1.8 7.2 28.8 Dose (mmol/kg) Dose (mmol/kg)

FIGURE 6.3 Dose-dependent effects (mean + s.e.m.; n = 9–11; *, p < 0.05; **, p < 0.01; ***, p < 0.001) of (R)-5- OMe-BPAT on spontaneous locomotor activity (horizontal activity, top left; peripheral activity, top right; forward locomotion, bottom left; rearing, bottom right) in rats.

138 In Vivo Pharmacological Evaluation of the Enantiomers of 5-OMe-BPAT

4000 1500

3000 * 1000 ** 2000 *** 500 1000 Peripheral activity Horizontal activity

0 0 0.0 0.2 0.6 1.8 7.2 28.8 0.0 0.2 0.6 1.8 7.2 28.8 Dose (mmol/kg) Dose (mmol/kg)

750 500

400 500 300 * 200 250 ** Rearing ** 100 *** Forward locomotion

0 0 0.0 0.2 0.6 1.8 7.2 28.8 0.0 0.2 0.6 1.8 7.2 28.8 Dose (mmol/kg) Dose (mmol/kg)

FIGURE 6.4 Dose-dependent effects (mean + s.e.m.; n = 6–7; *, p < 0.05; **, p < 0.01; ***, p < 0.001) of (R)-5-OMe- BPAT on d-amphetamine-induced locomotor activity (horizontal activity, top left; peripheral activity, top right; forward locomotion, bottom left; rearing, bottom right) in rats. disinhibition of dopaminergic neurons in the striatum, resulting in increased levels of dopamine (but see also refs. 48, 59 and 82), capable of competetively displacing the dopamine D2 antagonist from 45 postsynaptic striatal dopamine D2 receptors. However, it has recently been shown that the anticataleptic effect of 8-OH-DPAT is not associated with increased levels of dopamine in the striatum.48 Worth mentioning in this context is also the observation that clozapine, which has been 69 shown to act as a partial serotonin 5-HT1A receptor agonist in vivo, is capable of reversing catalepsy induced by other antipsychotic agents.43 However, it was shown that these anticataleptic 10 properties of clozapine are not mediated by its serotonin 5-HT1A receptor-stimulating properties.

Nevertheless, it is likely that the serotonin 5-HT1A receptor-stimulating properties of (R)-5-OMe- BPAT prevent the compound from inducing catalepsy caused by simultaneous blockade of dopamine

D2 receptors. Since the (S)-enantiomer seems to stimulate dopamine D2 receptors, simultaneous stimulation of serotonin 5-HT1A receptors is probably not relevant for its lack of cataleptogenic activity. The results of the microdialysis experiments are shown in Figures 6.5–6.7. In the striatum (S)-5- OMe-BPAT caused a significant decrease in extracellular levels of dopamine (p < 0.05 at t = 135 and t = 150 min) to about 75% of basal levels (Figure 6.5). Extracellular levels of 5-HIAA were also decreased (p < 0.05 at t = 60–150 min), the extent of the effect being comparable to that on dopamine. In contrast, DOPAC levels were increased to about 140% of basal levels after 75 min (p <

0.05 at t = 30–150 min). It has been shown that acute administration of dopamine D2 receptor antagonists causes an increase in extracellular levels of striatal dopamine and DOPAC, as a consequence of blockade of dopamine D2 autoreceptors located at the nerve terminals in the

139 Chapter 6

150 250 DA

200 DOPAC 5-HIAA 100 150

100 50 DA 50 % of basal levels DOPAC % of basal levels 5-HIAA 0 0 -60 0 60 120 180 -60 0 60 120 180 time (min) time (min)

FIGURE 6.5 Effects (mean + s.e.m., n = 4) of (S)-5-OMe-BPAT (25 mmol/kg s.c., left) and (R)-5-OMe-BPAT (25 mmol/kg s.c., right) on extracellular levels of striatal dopamine (DA), DOPAC and 5-HIAA.

95 striatum. Stimulation of these autoreceptors by dopamine D2 receptor agonists therefore may be expected to cause a decrease in the release of dopamine and DOPAC.91 The decrease in striatal dopamine levels caused by (S)-5-OMe-BPAT clearly indicates that this compound has dopamine D2 receptor-stimulating properties, although the effect is rather weak in relation to the high dose used.

For comparison, the selective dopamine D2/D3 agonist (–)-N-0437, which has a dopamine D2 receptor affinity comparable to those of (S)-5-OMe-BPAT,86 has been reported to decrease both striatal dopamine and DOPAC levels at a dose of 1 mmol/kg i.p. to 40% and 75% of control values, respectively.83 Furthermore, the concomitant increase in extracellular levels of DOPAC induced by (S)-5-OMe-BPAT is also very remarkable. These opposing effects on dopamine and DOPAC levels may results from the differences in intrinsic efficacies of (S)-5-OMe-BPAT at the dopamine D2 and

D3 receptors, the decrease in dopamine being caused by stimulation of dopamine D2 autoreceptors, and the increase in DOPAC being caused by blockade of dopamine D3 autoreceptors. The decrease in 5-HIAA levels support the earlier notion that (S)-5-OMe-BPAT is also a serotonin 5-HT1A receptor agonist. Presumably, this inhibiting effect on the metabolism of 5-HT is mediated by stimulation of somatodendritic autoreceptors located at the serotonergic cell bodies in the raphe nuclei (see below). However, stimulation of serotonin 5-HT1A receptors is unlikely to account for the observed increase in DOPAC levels, since the selective serotonin 5-HT1A receptor agonist 8-OH- DPAT has consistently been reported not to affect striatal levels of dopamine and DOPAC.48,59,82 The effects of (R)-5-OMe-BPAT on extracellular levels of striatal dopamine and DOPAC strongly suggest that this compound is a dopamine D2 receptor antagonist (Figure 6.5). A transient increase in dopamine levels was observed, with a maximum increase of about 150% of basal levels after one hour (p < 0.05 at t = 30–135 min). The increase in DOPAC lasted longer, and the maximum increase of was reached after 75 min (p < 0.05 at t = 45–150 min). This profile is similar to those induced by typical dopamine D2/D3 receptor antagonists, such as sulpiride and haloperidol.39,48,53,95,97 Similar to the (S)-enantiomer, (R)-5-OMe-BPAT caused a weak but significant decrease in 5-HIAA levels (p < 0.05 at t = 120–150 min), suggestive of stimulation of serotonin 5-

HT1A autoreceptors. In contrast to the striatum, (S)-5-OMe-BPAT caused a strong but transient increase in extracellular dopamine in the prefrontal cortex (p < 0.05 at t = 15–150 min, Figure 6.6). A maximum increase of about 250% was reached after 45 min. DOPAC levels were also increased (p < 0.05 at t

140 In Vivo Pharmacological Evaluation of the Enantiomers of 5-OMe-BPAT

= 15–150 min), but to a lesser extent than dopamine. The increases in dopamine and DOPAC levels are remarkable, since a compound capable of stimulating dopamine D2 receptor agonist should be expected to cause a decrease in dopamine release and metabolism.70 Arborelius et al. reported that systemic administation of (R)-8-OH-DPAT to rats at low doses caused a preferential increase of dopamine in the prefrontal cortex as compared to the striatum and nucleus accumbens.7 Presumably, this effect of 8-OH-DPAT is mediated by preferentially modulating the activity of dopaminergic neurons in the ventral tegmental area, from which the prefrontal cortex receives its dopaminergic input. Others have reproduced this preferential effect of 8-OH-DPAT, and similar observations have 82,90 been reported for the serotonin 5-HT1A receptor agonists buspirone and ipsapirone. These findings suggest that the serotonin 5-HT1A receptor-stimulating properties of (S)-5-OMe-BPAT may be responsible for the increases in prefrontal cortical dopamine and DOPAC levels. (S)-5-OMe- BPAT also caused a rapid increase in extracellular levels of noradrenaline in the prefrontal cortex. A maximum increase of about 200% of basal values was reached after 30 min, and noradrenaline levels only slightly decreases over time (p < 0.05 at t = 15–150 min). 5-HIAA was decreased to about 60% of basal values ( p < 0.05 at t = 135 and 150 min). The effects produced by (R)-5-OMe-BPAT on the extracellular levels of dopamine, DOPAC, 5- HIAA and noradrenaline in the prefrontal cortex were fairly similar to those of the (S)-enantiomer (Figure 6.6). Thus, a short-lasting increase in dopamine was observed, with a maximum increase to about 180% reached after 45 min. After 150 min, basal levels had almost been reached again (p < 0.05 at t = 15–120 min). The increase in dopamine was associated with a slower increase of DOPAC levels, which seemed to lag behind somewhat, as the maximum increase (~50%) was reached only after 90 min (p < 0.05 at t = 45–150 min). In contrast to the effects in the striatum, the increases in cortical dopamine and DOPAC should probably not be solely attributed to its dopamine D2 and D3 receptor-blocking properties, since selective dopamine D2/D3 receptor antagonists, such as sulpiride and raclopride, have been reported not to or only moderately affect cortical dopamine and DOPAC 32,53,54,61,70 levels. Consequently, the serotonin 5-HT1A receptor-stimulating properties of (R)-5-OMe- BPAT must also contribute to these effects, in analogy to the presumed mechanism of action of the (S)-enantiomer. (R)-5-OMe-BPAT increased the extracellular levels of noradrenaline in a parallel fashion with dopamine. Thus, noradrenaline levels were increased to a maximum of about 170%

300 250 DA DA

250 DOPAC 200 DOPAC 5-HIAA 5-HIAA 200 NA 150 NA 150 100 100 50 % of basal levels

50 % of basal values

0 0 -60 0 60 120 180 -60 0 60 120 180 time (min) time (min)

FIGURE 6.6 Effects (mean + s.e.m., n = 4) of (S)-5-OMe-BPAT (25 mmol/kg s.c., left) and (R)-5-OMe-BPAT (25 mmol/kg s.c., right) on extracellular levels of dopamine (DA), DOPAC, 5-HIAA and noradrenaline (NA) in the prefrontal cortex.

141 Chapter 6 after 45 min and then gradually decreased almost back to basal values (p < 0.05 at t = 15–135 min). 5-HIAA levels were gradually decreased to about 70% after 150 min (p < 0.05 at t = 120–150 min). The increase in extracellular levels of noradrenaline caused by both enantiomers is intriguing, since this effect is unlikely to be mediated through adrenergic receptors: both enantiomers displayed low affinities for a1-adrenergic receptors, while being devoid of affinity for a2- and b-adrenergic receptors (see Chapter 5). Behavioural activation of the animals may have been responsible for this effect (see below). Microdialysis of serotonin and 5-HIAA in the ventral hippocampus revealed similar effects induced by the enantiomers of 5-OMe-BPAT (Figure 6.7). Both compounds markedly suppressed the extracellular levels of both serotonin and 5-HIAA. The effect of (S)-OMe-BPAT on serotonin was more pronounced (maximum decrease of 53% after 120 min) and longer lasting (p < 0.05 at t = 45–120 min) than the effect of the (R)-enantiomer (maximum decrease of 42% after 45 min; p < 0.05 at t = 15–135 min), which may reflect the higher affinity for the serotonin 5-HT1A receptor of the former. In a similar fashion, both enantiomers gradually decreased extracellular levels of 5-HIAA to about 70% of basal values after 120 min (p < 0.05 at t = 15–120 min). Similar effects have been reported for various serotonin 5-HT1A receptor agonists, including 8-OH-DPAT, buspirone, gepirone, and ipsapirone (e.g. see refs. 17, 60, and 73), and it has been demonstrated that these effects are mediated by stimulation of somatodendritic serotonin 5-HT1A autoreceptors located at the serotonergic cell bodies in the raphe nuclei.37,74,75 Stimulation of these receptors causes an inhibition of the firing of the serotonergic neurons projecting to the hippocampus. Thus, the effects of the enantiomers of 5-OMe-BPAT in this assay unequivocally demonstrate that they both behave as full serotonin 5-HT1A receptor agonists in vivo and support the previous notion that these properties may contribute to the effects observed in the other experiments. It should be mentioned that particularly the (S)-enantiomer of 5-OMe-BPAT induced strong behavioural effects during the microdialysis experiments at the dose tested. Thus, various aspects of the 5-HT syndrome, including a flat body posture, hindlimb abduction, Straub tail and piano playing could be observed in all animals, again clearly demonstrating serotonin 5-HT1A receptor-stimulating properties of the compound. These effects had an onset within a few minutes after administration, and were present for the duration of the experiments. Moreover, all animals showed typical peripheral locomotor behaviour, i.e. they constantly moved from one corner of the cage to the next,

125 125

100 100

75 75

50 50

25 25 % of basal levels 5-HT % of basal levels 5-HT 5-HIAA 5-HIAA 0 0 -60 0 60 120 -60 0 60 120 time (min) time (min)

FIGURE 6.7 Effects (mean + s.e.m., n = 4) of (S)-5-OMe-BPAT (25 mmol/kg s.c., left) and (R)-5-OMe-BPAT (25 mmol/kg s.c., right) on extracellular levels of serotonin (5-HT) and 5-HIAA in the ventral hippocampus.

142 In Vivo Pharmacological Evaluation of the Enantiomers of 5-OMe-BPAT while staying close to the walls of the cage. As a consequence of this behaviour, the animals had to be handled frequently in order to prevent obstruction of the dialysis tubings, and hence loss of data. It is known that behavioural activation and mild stressors, such as handling, feeding and novelty, can evoke increases in dopaminergic and noradrenergic release in the prefrontal cortex.18,22,30 Therefore, it cannot be excluded that the behavioural activation by (S)-5-OMe-BPAT, but also the handling during these experiments, have contributed to the observed increase in extracellular levels of dopamine and noradrenaline in the prefrontal cortex. Rats treated with (R)-5-OMe-BPAT displayed little locomotor activity during the microdialysis experiments, consistent with the findings in the locomotor activity experiments. Therefore, no handling of these animals was required. Despite its serotonin 5-HT1A receptor-stimulating properties, (R)-5-OMe-BPAT only weakly induced aspects of the 5-HT syndrome, including a flat body posture and the Straub tail. Presumably, the behavioural activation due to serotonin 5-HT1A receptor stimulation, as observed for the (S)-enantiomer was overruled by the inhibitory effects of concomitant dopamine D2 receptor blockade. Simultaneous measurement of dopaminergic release in the striatum and prefrontal cortex was undertaken in order to establish possible regional differences in the effects of the enantiomers of 5- OMe-BPAT. Several studies have revealed that the prototypical atypical antipsychotic agent clozapine preferentially activates dopaminergic neurons in the mesolimbic and mesocortical dopaminergic systems, as opposed to the nigrostriatal dopaminergic system, suggesting that this may underlay the unique clinical profile of this compound.52 Thus, microdialysis experiments consistently have shown that clozapine, in contrast to classical antipsychotic agents such as haloperidol, preferentially increases extracellular levels of dopamine in the prefrontal cortex.19,31,32,47,53,54,61,66,67,87

Rollema et al. recently demonstrated that stimulation of serotonin 5-HT1A receptors by clozapine in part contributes to this effect.69 A single, relatively high dose of the enantiomers of 5-OMe-BPAT was tested in the current investigation. This dose was chosen based on a few pilot experiments with

(R)-5-OMe-BPAT, which at this dose showed the neurochemical profile of a typical dopamine D2 receptor antagonist in the striatum. Obviously (S)-5-OMe-BPAT displays large regional differences at this dose, due to the opposing effect on dopamine levels in the prefrontal cortex and the striatum. Comparison of the effects after 60 min reveals that they differ highly significantly (Figure 6.8). In contrast, the increases in dopamine levels in the striatum and prefrontal cortex caused by (R)-5- OMe-BPAT after 60 min were not significantly different. However, it is very well possible that this compound will show a larger regional selectivity at lower doses, since the horizontal activity in the locomotor experiments in combination with d-amphetamine was already blocked significantly at a dose of 1.8 mmol/kg, indicating blockade of dopamine D2 receptors in the limbic system. Thus, in order to be able to draw conclusions about possible regional selectivity of these compounds, additional experiments with lower doses (for example 1, 2.5 and 10 mmol/kg) are required. Evaluation of (S)-5-OMe-BPAT at lower doses may also eliminate the behavioural activation contributing to the increases in prefrontal cortical dopamine and noradrenaline levels, and hence elucidate the true mechanism mediating these increases. Furthermore, the contribution of serotonin

5-HT1A receptor stimulation to the observed effects may be investigated by concomitant administration of a selective serotonin 5-HT1A receptor antagonist, such as WAY 100,635.

Since it has been generally accepted that dopamine D2 receptor antagonism is required for a compound to exert antipsychotic effects, and partial dopamine D2 receptor agonists c.q. dopamine

143 Chapter 6

250 ***

200

150

100

50 DA ( %of basal values) 0 (S)-5-OMe-BPAT (R)-5-OMe-BPAT

FIGURE 6.8 Effects (mean + s.e.m., n = 4) of the enantiomers of 5-OMe-BPAT on extracellular levels of dopamine (DA) in the striatum (open bars) and prefrontal cortex (hatched bars) after 60 min (***, p < 0.001).

D2 autoreceptor agonists have been proven to be of limited value in the treatment of schizophrenia (see Section 1.6.3), at this point the (R)-enantiomer of 5-OMe-BPAT seems to be the most promising candidate for further development as a potential atypical antipsychotic agent of the two.

However, for compounds which combine dopamine D2 and serotonin 5-HT1A receptor binding properties, the optimal combination of intrinsic efficacies, as well as the optimal affinity ratio at the two receptor subtypes, required for an optimal clinical profile, remain to be established. In this respect, it is noteworthy that compounds which combine dopamine D2 receptor antagonism with full 9 serotonin 5-HT1A receptor agonism, but also mixed partial dopamine D2/serotonin 5-HT1A receptor agonists96 have been reported to show beneficial properties in preclinical models with predictability for antipsychotic and extrapyramidal side-effects (see also section 1.6.5).

6.4 CONCLUSIONS

Consistent with the differences in intrinsic efficacies at the dopamine D2 receptor observed in vitro, (S)-5-OMe-BPAT displays dopamine D2 receptor-stimulating properties in vivo, while its optical antipode behaves as a dopamine D2 receptor antagonist. In addition, both compounds behave as full serotonin 5-HT1A receptor agonists in vivo. Therefore, these compounds are interesting pharmacological tools for further exploring the dopamine D2/serotonin 5-HT1A hypothesis of atypical antipsychotic drug action. Moreover, the lack of cataleptogenic activity of (R)-5-OMe-BPAT, in combination with its dopamine D2 receptor antagonistic and serotonin 5-HT1A receptor-stimulating properties, suggest that this compound in particular may possess enhanced antipsychotic efficacy and a low propensity to cause EPS in man.

144 In Vivo Pharmacological Evaluation of the Enantiomers of 5-OMe-BPAT

6.5 EXPERIMENTAL SECTION

6.5.1 INHIBITION OF D-AMPHETAMINE-INDUCED LOCOMOTOR ACTIVITY

Animals. Male Sprague-Dawley rats (B&K strain, B&K Universal, Sollentuna, Sweden), weighing ~225–440 g were used. The animals arrived in the laboratory at least 5 days before being used in the experiments, and were housed 5 per cage under controlled conditions of temperature (21 °C), relative humidity (55–65 %) and light-dark cycle (12:12 h, lights on at 6 a.m.). Food (R36, Ewos, Södertälje, Sweden) and tap water were freely available in the home cage. The experiments were performed between 7 a.m. and 5 p.m. The experiments were approved by the Stockholm South ethical committee for experiments with laboratory animals.

Drug Treatment. d-Amphetamine hydrochloride was dissolved in saline. (S)- and (R)-5-OMe-BPAT hydrochloride were dissolved in distilled water with the aid of minute amounts of glacial acetic acid. Rats were injected subcutaneously in the neck (2.0 mL/kg body weight). The test compounds or saline were administered 60 min prior to amphetamine, and the animals were put back in their home cages. After 30 min, the rats were transferred to the activity cages and their activity was monitored for 30 min (habituation phase). Then d-amphetamine (5 mg/kg) was administered and activity measurements were continued during 60 min.

Locomotor activity studies in automated cages. Seven perspex activity cages (Kungsbacka Mät- och Reglerteknik AB, Fjärås, Sweden) with a floor area of 70 × 70 cm, were used. They were housed in soundproofed ventilated boxes with no lighting. Two rows of photocells (total 32 photocells, one row of 16 photocells at floor level to measure activity at the floor level, the other row placed higher to measure rearing) enabled a computer-based system to determine the location of the animal at any time. In the present study, we measured and report the following variables: horizontal activity, representing the total number of times light beams in the lower row were interrupted; peripheral activity, representing the breaking of beams that are located closest to one of the four walls; forward locomotion, representing the successive breaking of beams in the lower rows, representing movement of an animal in a single direction; rearing, representing the breaking of the upper row of beams. It should be noted that these variables are not independent and that horizontal activity includes, for example, the counts generated in the peripheral activity variable.

Data Analysis. The number of beam crossings representing the different aspects of locomotor activity were accumulated for each animal and the average numbers of beam crossings were calculated for each dose of the test compound. Dose-dependent effects were tested for significance compared to control treatment by one-way ANOVA followed by Dunnett’s test, using the statistical software package SigmaStat for Windows (Jandel Corporation). A significance level of 0.05 was applied.

6.5.2 CATALEPSY

Animals. Male Sprague-Dawley (B&K Universal AB, Sollentuna, Sweden) weighing ~250–330 g were used. They were housed in Macrolon type IV cages in groups of 5 with a controlled light-dark cycle (12:12 h, lights on at 6 a.m.). The rats were fed with pellets (R36, Ewos, Södertälje, Sweden) and tap water, and housed in the ‘animal room’ for a minimum of 5 days before use in experiment. The experiments were approved by the Stockholm South ethical committee for experiments with laboratory animals.

Drug Treatment. (S)- and (R)-5-OMe-BPAT hydrochloride were dissolved in distilled water with the aid of minute amounts of glacial acetic acid. Rats were injected subcutaneously in the neck (2.0 mL/kg body weight).

Catalepsy Test. The rats were weighed and marked, and then placed in the experiment room at least 1 hour before the beginning of the experiment. During the whole experiment the rats were housed in their ‘home cages’ and had free access to food and water. The drug solutions were made fresh before the start of the experiment, while the animals

145 Chapter 6 were habituating to the room. Rats were injected with a time interval of 1 or 2 minutes during 30 minutes. The catalepsy of each rat was tested 6 times, i.e. ½, 1, 2, 4, 8 and 24 hours after injection. During the test, the rat was placed on the lower part of a grid (consisting of a wire netting of 5 ´ 5 mm with an outside measure of 23 ´ 33 cm placed at an angle of 60 ± 2°) with the head pointing upwards, and a stop-watch was started. When the rat moved one of the legs the stop-watch was stopped, the catalepsy time was noted in 1/100 parts of a minute, and the rat was taken down from the grid. The maximal catalepsy time was set to 3.0 minutes.

Data Analysis. Mean, medians, Mann-Whitney U-test (two-tailed) and ANOVA were calculated for each test occasion with a computer program (STAT-PACK). A dose/time curve of median catalepsy time and two curves with confidence interval and the ED50 dose based on the catalepsy time of all the animals at the peak of the dose/time curve were calculated. The ED50 value was set to either 1.0 minutes or 0.5 minutes of catalepsy. Any dose that did not cause catalepsy was not used in the calculations.

6.5.3 INTRACEREBRAL MICRODIALYSIS

Animals. Male albino rats of a Wistar-derived strain (Harlan, Zeist, The Netherlands), weighing ~250–350 g, were housed prior to surgery in groups of 6 animals in plastic cages (70 × 50 × 20 cm) under conditions of constant temperature (20 °C) and humidity, with a controlled light-dark cycle (12:12 h, lights on at 7 a.m.). Food and water were freely available. After surgery the rats were housed individually in Plexiglas cages (25 × 25 × 30 cm) with free access to food and water. Animal procedures were conducted in accordance with guidelines published in the NIH Guide for the Care and Use of Laboratory Animals and all protocols were approved by the Groningen University Institutional Animal care and Use Committee.

Drug Treatment. hydrobromide (Lundbeck, Denmark) was dissolved in saline (0.9% NaCl in distilled water). (S)- and (R)-5-OMe-BPAT hydrochloride were dissolved in saline with the aid of a small aliquot of glacial acetic acid and stocked in a concentration of 25 mmol/ml. These solutions were administered subcutaneously in a volume of 1.0 ml/kg body weight.

Surgery. The microdialysis probes used were of a vertical, concentric design.94 The dialysis tube (inner diameter: 0.22 mm, outer diameter: 0.31 mm) was prepared from polyacrylonitrile/sodium methyl sulphonate copolymer dialysis fiber (AN 69, Hospal, Bologna, Italy). Probes with different lengths of the exposed areas were used to sample the different brain structures under investigation: striatum, exposed area 3.0 mm; prefrontal cortex, exposed area 4.0 mm; ventral hippocampus, exposed area 4.0 mm. Animals were anaesthetized with chloral hydrate (400 mg/kg i.p.) and placed in a stereotaxic apparatus (David Kopf Instruments). During surgery, lidocaine hydrochloride (6% solution in saline, adjusted to pH = 6.0 with 1 N aqueous NaOH solution) was used as an adjuvant local anaesthetic. The skull was exposed and holes were drilled in the skull. Depending on the brain area under investigation, the microdialysis probes were implanted at the following coordinates according to the atlas of Paxinos and Watson:65 striatum, AP +0.5, ML –3.0 relative to bregma, and V –6.0 below dura; prefrontal cortex, AP +3.5, ML +0.9 relative to bregma, and V – 5.0 below dura; ventral hippocampus, AP –5.3, ML +4.8 relative to bregma, and V –8.0 below dura. The probes were secured to the skull with a screw and dental cement. Rats were allowed to recover from the surgical procedure for at least 24 h.

Microdialysis. During dialysis, the inlet and outlet of the microdialysis probes were connected via pieces of PEEK tubing (inner diameter: 0.12 mm, length: 45 cm) to a perfusion pump (CMA/100, Carnegie Medicin, Stockholm, Sweden), and to the injection valve of the HPLC system respectively. The dialysis tubing was continuously perfused with an artificial cerebrospinal fluid (aCSF) solution (striatum and prefrontal cortex: 147 mM NaCl, 4 mM KCl, 2.3 mM CaCl2 and 1.0 mM MgCl2; hippocampus: 147 mM NaCl, 3 mM KCl, 1.2 mM CaCl2 and 1.2 mM MgCl2) at a flow rate of 1.5 mL/min. The selective serotonin reuptake inhibitor citalopram (1 mM) was added to the aCSF in order to obtain measurable levels of serotonin. The injection valve (Valco), which was controlled by an electronic timer, was

146 In Vivo Pharmacological Evaluation of the Enantiomers of 5-OMe-BPAT held in the load position for 15 min, during which the sample loop (50 mL) was filled with dialysate. The valve was then switched to the inject position for 20 s. This procedure was repeated every 15 min, which was the time needed to record one complete chromatogram. The connection of the dialysis probe with the HPLC system introduced a lag time of ~8 min, for which the presented data were corrected. Drugs were administered to the animals when baseline output of the analytes under investigation was stable (i.e., less than 20% variation between consecutive samples). When dialysis had been finished, the rat was killed with an overdose of pentothal, brain tissue was fixated with 4% paraformaldehyde via intracardiac perfusion, and the location of the dialysis probe was verified.

Dialysate Analysis. Dopamine, DOPAC, noradrenaline and 5-HIAA were quantified in the striatum and prefrontal cortex by HPLC with electrochemical detection. The system consisted of an HPLC pump (Shimadzu LC- 10AD) in conjunction with an electrochemical detector (ESA Coulochem II). A reverse-phase column (Supelco, inner diameter: 4.6 mm, length: 15 cm) packed with C18 material (5 mm particles) was used for chromatographical separation. The mobile phase consisted of a mixture of aqueous sodium acetate (4.1 g/L), adjusted to pH 4.2 with acetic acid, 1-octanesulphonic acid (0.14 g/L), Na2EDTA (0.05 g/L), and methanol (10% v/v), and was delivered at a flow of 1.0 mL/min. Serotonin and 5-HIAA were quantified in the hippocampus by HPLC with electrochemical detection. The system consisted of an HPLC pump (Shimadzu LC-10AD) in conjunction with an electrochemical detector (Intro, Antec Leyden). A reverse-phase column (Phenomenex Hypersil 3: inner diameter 2.0 mm; length 10 cm) packed with C18 material (3 mm particles) was used for chromatographical separation. The mobile phase consisted of a mixture of aqueous diammonium sulfate (5.0 g/L), Na2EDTA (0.5 g/L), 1-heptanesulphonic acid (0.05 g/L), and triethylamine (30 mL/L), adjusted to pH 4.65 with acetic acid, and was delivered at a flow of 0.4 mL/min.

Data Analysis. Four consecutive microdialysis samples with less then 20% difference were considered as control values and set at 100%. Data are presented as percentages of control level (mean ± S.E.M.). Statistical analyses were performed using SigmaStat for Windows (Jandel Corporation). Treatment values were compared using two-way ANOVA for repeated measures, followed by Dunnett’s test, applying a significance level of 0.05.

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151 MOLECULAR MODELING OF THE

DOPAMINE D2 AND SEROTONIN 5-HT1A RECEPTOR BINDING MODES OF 7 THE ENANTIOMERS OF 5-OME-BPAT

ABSTRACT

Molecular modeling studies were undertaken in order to elucidate the possible dopamine D2 and serotonin 5-HT1A receptor binding modes of the enantiomers of 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe- BPAT, 1). For this purpose, a combination of indirect molecular modeling and direct construction of the seven transmembrane (7TM) domains of the receptors was employed in a stepwise, objective manner. Pharmacophore models and corresponding receptor maps were identified by superimposing selected sets of receptor agonists in their presumed pharmacologically active conformations, while taking the conformational freedom of the ligands into account. The 7TM models were then constructed around the agonist pharmacophore models, by adding the TM domains one-by-one. Initially, the relative positions of TM3, TM4, and TM5 were determined using the three- dimensional structure of bacteriorhodopsin, but subsequently the orientations of all TM domains were adjusted in order to mimic the topology of the TM domains of rhodopsin. The presumed dopamine D2 receptor binding conformations of (S)- and (R)-1 were determined by using the semirigid dopamine D2 receptor antagonist N- benzylpiquindone as a template for superposition. Similarly, the selective serotonin 5-HT1A receptor agonist flesinoxan was employed for identifying the serotonin 5-HT1A receptor binding conformations of the enantiomers of 1. After docking of the presumed pharmacologically active conformations in the 7TM models and subsequent optimization of the binding sites, specific interactions between the ligands and the surrounding amino acid residues, consistent with the structure-activity relationships, were observed. Thus, both enantiomers of 1 bound to the dopamine D2 receptor model in a similar fashion: a reinforced electrostatic interaction was present between the protonated nitrogen atoms and Asp114 in TM3; their carbonyl groups accepted a H-bond from Ser121 in TM3; their amide NH groups acted as H-bond donor to Tyr416 in TM7; and their benzamide phenyl rings were involved in a hydrophobic edge-to-face interaction with Trp386 in TM6. Differences were observed in the orientations of the 2-aminotetralin moieties, which occupied the agonist binding site. Whereas the (S)-enantiomer could form a H-bond between its 5-methoxy substituent and Ser193 in TM5, the (R)-enantiomer could not, which may account for the differences in their intrinsic efficacies at the dopamine D2 receptor. In the serotonin 5-HT1A receptor model, the benzamide phenyl rings of both enantiomers

153 Chapter 7 were involved in hydrophobic face-to-face interactions with Phe112 in TM3, while their protonated nitrogens atoms formed a reinforced electrostatic interaction with Asp116 in TM3. Consistent with the structure-affinity relationships of 1, the amide moieties were not involved in specific interactions. Both enantiomers of 1 could form a hydrogen bond between their 5-methoxy substituent and Thr200 in TM5, which may account for their full serotonin 5-HT1A receptor agonist properties.

7.1 INTRODUCTION

Dopamine and serotonin exert their physiological effects by interacting with specific receptors.

With the exception of the serotonin 5-HT3 receptor, all dopaminergic and serotonergic receptor subtypes currently identified belong to the superfamily of G-protein-coupled receptors (GPCRs; see Section 1.3.1). Although several hundred different members of this family have been identified to date,52 little is known about their molecular properties. Since it has not been possible to unequivocally establish the exact molecular structure of any of these receptors by techniques such as X-ray crystallography thus far, structural information has only been obtained from indirect methods, such as multiple sequence alignments,47 identification of evolutionary conserved amino acid residues,56 and hydropathy analyses.38 A common picture emerging from these indirect methods is that GPCRs are embedded in the cell membranes in a specific manner. Presumably they traverse the lipid bilayers seven times, with the amino-terminal and carboxy-terminal regions located extra- and intracellularly, respectively. The seven transmembrane (TM) domains, corresponding to relatively hydrophobic regions in the amino acid sequence, are believed to comprise a-helices, and to be interconnected via intracellular and extracellular loops of relatively hydrophilic nature. A similar pattern has been observed for bacteriorhodopsin (bR), from which a three-dimensional (3D)

FIGURE 7.1 Inside-to-outside (left) and orthogonal view (right) of the 3D electron cryo-microscopy-derived structure of bR, showing the relative arrangements of the TM domains, visualized as line ribbons, and the position of the retinal chromophore.

154 Molecular Modeling of the Enantiomers of 5-OMe-BPAT structure at a resolution of 7 Å has been obtained by electron cryo-microscopy (Figure 7.1).5,23 This structure has recently been confirmed to a large extent by an X-ray structure, which was resolved at 2.5 Å.55 bR constitutes a light-driven proton pump embedded in the cell membranes of Halobacterium salinarium, which responds to light by isomerization of a retinal molecule, that is covalently bound in the core of the protein. The net result of the subsequent conformational changes is the translocation of a proton from the inside to the outside of the bacterium (for review and references, see ref. 43). The apparent structural similarities between bR and GPCRs have prompted a number of researchers to use the molecular structure of bR as a structural template for the construction of molecular models of GPCRs (e.g. see refs. 16, 37, 51, 67 and 68), a technique which is referred to as homology modeling (for reviews and references, see refs. 13, 25 and 31). However, unlike GPCRs, bR is not coupled to a G-protein. Mammalian opsins do belong to the family of GPCRs, but nevertheless they also share some structural and functional features with bR. Thus, similar to bR, they function by photoisomerization of a covalently bound retinal molecule, but instead of proton extrusion, this results in an intracellular signal transduction mediated by the associated G- protein termed transducin (for review and references, see ref. 20). Therefore, the mammalian opsins have been suggested to form the evolutionary link between bR and ligand-binding GPCRs, which provides a rationale for using the structure of bR as a structural template for the construction of molecular models of GPCRs.13,54,65 Supportive of this hypothesis is a two-dimensional (2D) density projection map derived from bovine rhodopsin by electron cryo-microscopy, which showed a pattern that could be readily interpreted when assuming that this receptor forms a bundle of seven TM domains.58 However, the suitability of the structure of bR for homology-based modeling of GPCRs may be questioned, since no significant (<20%) sequence homology exists between bR and any of the GPCRs, including the opsins.14 Furthermore, theoretical2,24 as well as experimental23,58,69 data suggest that, despite similarities in the overall membrane topology, the arrangement of the individual TM domains of bR and rhodopsin may be considerably different (Figure 7.2). Alternatively, when the structure of the receptor is not available, indirect molecular modeling techniques, such as pharmacophore identification or receptor mapping, may be undertaken in order to gain insight in the structural properties of the molecular target (for reviews see refs. 18 and 73). A pharmacophore may be defined as an ensemble of structural features which are required for a ligand to be recognized by the receptor. The spatial arrangement of these structural features is referred to

FIGURE 7.2 Schematic representation of the topology of the TM domains of bR (left) and rhodopsin (right) based on helix assignment calculations by Herzyck and Hubbard. The view is from the intracellular side. Adapted from ref. 24.

155 Chapter 7 as a pharmacophoric pattern, while the spatial arrangement of structural features of the target molecule, complementary to those of the pharmacophore, is referred to as the receptor map. In order to develop a pharmacophore or a receptor map, capable of accommodating a set of ligands, the active analogue approach may be employed. Two stages can be recognized in this approach. First, the relative importance of the different functional groups present or absent in a set of known active or inactive ligands is determined, taking the SAR of the compounds into consideration. This may reveal information about the nature of the complementary functional groups in the receptor, involved in the binding of the ligands. Secondly, a hypothesis is proposed about the spatial arrangement between either the functional groups of the ligands, resulting in a pharmacophoric pattern, or the recognition points postulated to belong to the receptor, resulting in a receptor map. The hypothesis is evaluated by searching for a spatial arrangement of the functional groups c.q. interaction points which is common to all of the ligands. Since the global minimum energy conformation of a ligand does not necessarily need to be the pharmacologically active one, the conformational freedom of the ligands under investigation has to be taken into consideration during the processes of pharmacophore identification and receptor mapping. For flexible molecules, this poses a potential problem, since their presumed pharmacologically active conformations have to be deduced from a large number of minimum energy conformations. However, this problem may be tackled by including an active ligand with restricted conformational freedom in the data set, which can then be used as a template for matching the optimal conformations of the flexible ligands. In order to improve the quality of a GPCR model, the techniques of homology modeling and indirect molecular modeling may be combined. The active site of the receptor can be determined by probing the homology model with a pharmacophore c.q. receptor map obtained from indirect modeling approaches. The combination of these two approaches seems attractive, since it includes more experimental data than the homology-based model alone. Furthermore, a more realistic binding site, capable of accommodating structurally diverse ligands and rationalizing their affinities and activities, may be obtained. Several successful applications of this approach have appeared in literature.16,21,41,51 In the previous two chapters, the enantiomers of the potential atypical antipsychotic agent 5- methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 1) were shown to possess different intrinsic efficacies at dopamine D2 and D3 receptors both in vitro and in vivo.

Particularly the in vivo results suggested that (S)-5-OMe-BPAT [(S)-1] acts as a dopamine D2 receptor agonist, while (R)-5-OMe-BPAT [(R)-1] behaves as a dopamine D2 receptor antagonist.

Furthermore, both enantiomers were shown to act as full serotonin 5-HT1A receptor agonists in vitro

O O N N N N H H

OCH3 OCH3 (S)-1 (R)-1

CHART 7.1 Chemical structures of the enantiomers of 5-methoxy-2-[N-(2-benzamidoethyl)-N-n- propylamino]tetralin (5-OMe-BPAT, 1).

156 Molecular Modeling of the Enantiomers of 5-OMe-BPAT and in vivo. The interactions with the receptors in vitro were stereoselective, since (S)-1 had the highest serotonergic affinity, while (R)-1 preferred the dopaminergic receptors. Earlier investigations on the SAFIR of structural analogues and derivatives of racemic 1 revealed several important clues about the possible receptor binding modes of this compound. First, the 2-aminotetralin moiety of 1 is likely to occupy the same binding sites as the 2-aminotetralin moiety of the DPATs in both the dopaminergic and serotonergic receptors. Furthermore, the benzamidoethyl side chain seemed to enhance the affinities of 1 for both the dopaminergic and serotonergic receptors, but not in an identical way. Thus, the SAFIR suggested that the benzamide moiety of the side chain may occupy the same binding sites in the dopaminergic receptors as the benzamide moieties of the 2- pyrrolidinylmethyl-derived substituted benzamides, presumably by forming specific interactions between the amide functionality and certain amino acid residues in the receptors. At the serotonin 5-

HT1A receptor, the benzamidoethyl side chain as a whole seemed to act merely as a ‘spacer’, which enhanced the affinity due to interactions not involving the amide functionality. The 5-methoxy substituent of 1 appeared to be important for the dopaminergic affinities, but not for the serotonergic affinity. In the present investigation, molecular modeling studies were undertaken in order to rationalize the modes of binding of (S)- and (R)-1 to the dopamine D2 and serotonin 5-HT1A receptor. A combination of indirect molecular modeling, i.e. pharmacophore identification, using the active analogue approach, and homology-based modeling of the receptors was used for this purpose, while taking the SAFIRs as described above into account.

7.2 MOLECULAR MODELING

7.2.1 LIGAND SELECTION

Sets of well-known dopamine D2 and serotonin 5-HT1A receptor agonists of various chemical classes were selected, in order to be able to identify the agonist binding sites in the two receptor subtypes. The following dopamine D2 receptor agonists were taken into consideration: dopamine (2, Chart 7.2), (S)-5-hydroxy-2-(N,N-di-n-propylamino)tetralin [(S)-5-OH-DPAT, 3], (R)-7-hydroxy-2- (N,N-di-n-propylamino)tetralin [(R)-7-OH-DPAT, 4], and (R)-apomorphine (5). In addition, the pyrroloisoquinoline derivative N-benzylpiquindone66 (6) was used as a semirigid template for identifying the dopamine D2 receptor binding conformations of (S)- and (R)-1. Similarly, for the identification of a serotonin 5-HT1A receptor agonist receptor map, the following serotonin 5-HT1A receptor agonists were selected: serotonin (7, Chart 7.3), (R)-8-hydroxy-2-(N,N-di-n- propylamino)tetralin [(R)-8-OH-DPAT, 8], the partial LY 197,20660 (9), and (R)-10- methyl-11-hydroxyaporphine4 (10). The enantiomer of compound 9, which is homochiral to the (R)- enantiomer of 8, was considered. In addition, the selective serotonin 5-HT1A receptor agonist 36 flesinoxan (11) was chosen as a template molecule for determining the serotonin 5-HT1A receptor agonist conformations of (S)-and (R)-1.

157 Chapter 7

HO NH2 N HO N

HO OH 2 3 4

OH O C H HO 11 10 N A B D N N H H H CH3 5 6

CHART 7.2 Chemical structures of the dopamine D2 receptor agonists dopamine (2), (S)-5-OH-DPAT (3), (R)-7-OH-

DPAT (4) and (R)-apomorphine (5), and the dopamine D2 receptor antagonist N-benzylpiquindone (6).

NH2 OH OCH3 HO N N

N H N H 7 8 9

OH 1 C 4 H C 11 3 3 O 2 N O 10 A B D HO O N N N H H CH 3 F 10 11

CHART 7.3 Chemical structures of the serotonin 5-HT1A receptor agonists serotonin (7), (R)-8-OH-DPAT (8), LY 197,206 (9), (R)-10-methyl-11-hydroxyaporphine (10), and flesinoxan (11).

7.2.2 CONFORMATIONAL ANALYSES

Prior to the pharmacophore identification, all ligands considered in this investigation were submitted to extensive conformational analyses in MacroModel46 (for detailed descriptions of the experimental procedures, see Section 7.6). Only conformations with conformational energies within 3.0 kcal/mol above the global minimum energy conformation were considered to be relevant. All ligands were considered in their protonated, positively charged forms. Water was simulated as the solvent during the analyses. N-n-propyl groups were truncated to N-methyl groups, and benzamide aromatic rings were maintained in a coplanar fashion with the amide moieties during all

158 Molecular Modeling of the Enantiomers of 5-OMe-BPAT minimizations. The arylpiperazine moiety of 11 was fixed in the conformation as described by Kuipers et al.36 during all minimizations. For ligands containing a protonated nitrogen atom with three different substituents (i.e. 1, 5, 6 and 10), the possibility of inversion of this nitrogen atom was taken into account by calculating two subpopulations of minimum energy conformations for these ligands, which only differed in the orientation of the proton at the basic nitrogen.57 These subpopulations were then combined, and conformations with energies more than 3.0 kcal/mol above the global energy minimum were discarded.

7.2.3 PHARMACOPHORE IDENTIFICATION

The program APOLLO (Automated PharmacOphore Location through Ligand Overlap)61,62 was used for establishing the dopamine D2 and serotonin 5-HT1A receptor binding conformations of the ligands under investigation, and for the identification of different pharmacophoric patterns for binding at these receptor subtypes. Although APOLLO also allows to explicitly use specific functional groups c.q. atoms as pharmacophoric elements, the main purpose of the program is to identify from a set of ligands their mutual interaction points belonging to the receptor site. In this way additional flexibility is introduced, since different ligands are allowed to interact with the same receptor point from different directions. Using APOLLO, vector points emanating from essential functional groups are defined in all minimum energy conformations of the ligands under investigation. Subsequently the appropriate vector points of the different ligands are superimposed, while considering all possible combinations of the minimum energy conformations of the ligands. Different weights may be assigned to different fitting points, and in cases where functional groups may form interactions from different sides, APOLLO can choose which vector results in the best superposition. Specific matches are scored using root mean square (RMS) deviations, and conformational energies may be included in the scoring procedure. Finally, APOLLO can reproduce the superimposed ligands of all matches, and places water molecules at the mutual interaction points, mimicking H-bond donating or accepting amino acid residues belonging to the receptor, and thus representing a simple receptor map.

7.2.4 RECEPTOR CONSTRUCTION

Construction of the 7TM models of the dopamine D2 and serotonin 5-HT1A receptors was 64 performed in SYBYL. The amino acid sequences of the human dopamine D2A and the human 6,11 serotonin 5-HT1A receptor were obtained from the literature and were manually aligned with the sequences of other dopaminergic and serotonergic receptor subtypes. The alignment was guided by identifying evolutionary conserved amino acids. The amino acids to be included in the TM domains were visually selected, based on their relatively hydrophobic nature. The TM domains thus selected are shown in Figure 7.3. The 7TM models were constructed in a stepwise manner, with the aid of the receptor maps as identified by APOLLO, and the 3D structure of bR,23 which was retrieved from the Brookhaven Protein Databank (PDB code: 1BRD). Construction of the models was started by positioning TM3 and TM5 at opposite sides of the superimposed agonists, while superimposing specific amino acid residues, presumably involved in the primary interactions with the ligands (see

159 Chapter 7

D2A 36 Y Y A T L L T L L I A V I V F G N V L V C M A V S 60

5-HT1A 37 V I T S L L L G T L I F C A V L G N A C V V A A I A 62

D2A 96 V E L Y V V W P M V L T A V L L D A V A L S V I L 72

5-HT1A 99 L V Q Y L A A M P L V L V S V M L D T V A L S G I L 74

D2A 107 C D I F V T L D V M M C T A S I L N L C A I S I 130

5-HT1A 109 C D L F I A L D V L C C T S S I L H L C A I A L 132

D2A 174 L G F L L P C S I T F S L V W V I S I M V T V 152

5-HT1A 175 W G L M P P I S I L F G I L W T L S I L A A A 153

D2A 187 P A F V V Y S S I V S F Y V P F I V T L L V Y I 210

5-HT1A 192 D H G Y T I Y S T F G A F Y I P L L L M L V L Y G R 217

D2A 397 I N L I H T I F F P L W C I I F V G L V I A L M 374

5-HT1A 370 F P L V L A V I F F P L W C L I F T G M I I G L T 346

D2A 405 P V L Y S A F T W L G Y V N S A V N P I I Y T T 428

5-HT1A 379 T L L G A I I N W L G Y S N S L L N P V I Y A Y F N 404

FIGURE 7.3 Alignment of the seven putative TM domains of the human dopamine D2A and serotonin 5-HT1A receptors. The domains are displayed back and forth, starting from left to right with TM1 at the top. Numbers correspond to the amino acids at the start and end of the rows. Evolutionary conserved amino acids are shaded, and amino acids presumed to be important for binding of the endogenous ligands are boxed. Section 7.3), with their side chains replacing the water molecules included in the receptor maps. The other TM domains were then added one by one, in the following order: TM4, TM6, TM2, TM7, and TM1. The 3D structure of bR was used for their initial crude positioning, but subsequently their relative arrangements were manually adjusted in order to obtain 7TM models which more closely resemble the topology of the TM domains of rhodopsin, according to the model calculated by Herzyk and Hubbard24 (see Figure 7.2).

7.2.5 LIGAND DOCKING

All ligands considered in this investigation were individually and manually docked into the active sites of the final 7TM models in their presumed pharmacologically active conformations, as determined with APOLLO. The ligands were initially positioned in such a way that the interactions between their functional groups and the appropriate amino acid side chains, presumably primarily involved in the binding process, could be visualized as H-bonds. The interaction energies between the receptors and the ligands were then minimized by manually adjusting the positions of the ligands in the binding sites, while attempting to maintain the primary interactions displayed as H-bonds, using the Docking procedure as implemented in SYBYL.

160 Molecular Modeling of the Enantiomers of 5-OMe-BPAT

7.2.6 ACTIVE SITE OPTIMIZATION

The interactions between the amino acids in the binding sites and the individually docked ligands were finally optimized using the pseudoreceptor-generating program PrGen. This program was originally designed for the construction of mini- and pseudoreceptors around a set of ligands, superimposed in their presumed pharmacologically active conformations.72,77 For this purpose, PrGen relies on the Yeti force field, in which the directionalities of H-bonds and electrostatic interactions have been strongly implemented.71 Translational, rotational, and torsional degrees of freedom for both ligands and amino acid residues can be specified to the accuracy of single bonds. This makes the program very suitable for optimizing binding sites derived from homology-base models, since it allows the backbone atoms to be fixed, and hence to maintain the overall shape of the binding site.

7.3 RESULTS AND DISCUSSION

The purpose of the present investigation was to gain insight in how the enantiomers of 5-OMe- BPAT exert their effects at the molecular level, using molecular modeling techniques. Although both enantiomers have high affinities for dopamine D2, D3, and serotonin 5-HT1A receptors, only their binding modes at the dopamine D2 and the serotonin receptor were investigated. The results from the in vivo studies suggested that their effects are predominantly mediated via dopamine D2 and serotonin 5-HT1A receptors. Furthermore, since all dopamine receptor agonists, previously designated to be selective for the dopamine D2 receptor, also have comparable or even higher affinities for the dopamine D3 receptor, and since the dopamine D2 and D3 receptors share a sequence homology of about 80% in the TM domains, the modeling approach employed here would in all probability result in virtually identical binding sites for the two receptor subtypes. Dopamine D2 versus D3 receptor selectivities would then have to be explained based on very subtle differences in receptor conformations (e.g. see ref. 41). Therefore, the dopamine D3 receptor binding modes of (S)- and (R)-1 were not considered in this study. The process of receptor construction was started by mapping the agonist binding sites in the two receptor subtypes, using the active analogue approach. It is very likely that receptor agonists share the same binding site, since they also share the ability to stimulate the receptor during the binding process. Furthermore, sequence alignments and site-directed mutagenesis studies (see below) have identified several amino acid residues which are presumed to be important for agonist binding. Definition of the agonist binding sites was therefore considered to be a good starting point for the construction of the 7TM models. For the identification of the dopamine D2 receptor agonist pharmacophore, several structural features of the selected agonists were considered to be important for their activity (for reviews and references, see refs. 3, 27, 35, and 74). First, all dopamine receptor ligands known to date contain a basic nitrogen atom, which will to a large extent be protonated under physiological conditions. It is generally believed that the protonated nitrogen atoms are involved in a reinforced electrostatic interaction with a highly conserved aspartic acid residue in TM3

(Asp114 in D2A). This interaction was simulated by placing a vector point emanating from the protonated nitrogen atoms of 2–5. Second, similar to dopamine, all dopamine receptor agonists

161 Chapter 7

FIGURE 7.4 Stereo representation of the superposition of the dopamine D2 receptor agonists 2–5 in their presumed

dopamine D2 receptor binding conformations. The water molecules mimic putative amino acid residues belonging to the receptor, capable of forming interactions (dotted lines) with the ligands. For clarity purposes alkyl and aryl hydrogen atoms have been omitted in all pictures. contain an aromatic ring (or a bioisostere) at a certain distance from the basic nitrogen atom, capable of forming hydrophobic interactions. In order to include them for superposition, centroids and normals were defined in these rings (i.e. the A-ring of 5). Third, the meta-hydroxy substituent of dopamine is important for binding to the dopamine D2 receptor, but the para-hydroxy substituent is not. SARs suggest that the meta-hydroxy substituent functions both as H-bond donor and acceptor. Thus, the meta-hydroxy substituent of dopamine, the hydroxy substituents of 3 and 4, and the 11- hydroxy substituent of 5 were defined as both H-bond donor and acceptor. The best matching superposition, resulting from fitting the appropriate vector points emanating from the hydroxy substituents, the centroids and normals defined through the aromatic rings, and the vector points emanating from the protonated nitrogen atoms, is shown in Figure 7.4. In the best match, dopamine adopts a folded conformation, while the 2-aminoteralin moieties of 3 and 4 are in a half-chair conformation with a pseudo-equatorial amino group. The N-methyl substituent of 5 is also oriented in a pseudo-equatorial fashion. The aromatic rings and the hydroxy substituents, corresponding to the meta-hydroxy substituent of dopamine, are oriented in a similar fashion and show good overlap. However, the fact that all ligands form an interaction with their protonated nitrogens and a mutual putative receptor point, although they approach this point from different directions, clearly shows that explicit overlap of functional groups belonging to the ligands (in this case the protonated nitrogen atoms) is not required for a good interaction with the receptor. In this respect it is noteworthy that the superposition of 3 and 4, which possess opposite absolute configurations in their active enantiomers, strongly resembles the pharmacophore models proposed by Grol et al.17 and Johansson et al.,34 and hence differs substantially from the well-known McDermed model, in which the hydroxy substituents, the aromatic nuclei and the protonated nitrogen atoms of 3 and 4 explicitly overlap.44 Similar to dopamine receptor agonists, formation of a reinforced electrostatic interaction via a protonated basic nitrogen atom presumably is essential for binding of serotonin receptor ligands to the receptors. Furthermore, the 5-hydroxy substituent of serotonin may act as a H-bond acceptor, since the methoxy analogues of serotonin and hydroxy-containing synthetic serotonin 5-HT1A receptor agonists are usually equipotent. The indole NH of serotonin is also important for binding, since N-alkylation of this group decrease its affinity for the serotonin 5-HT1A receptor (for review, see ref. 48). Taking these observations into account, the appropriate vector points, centroids and normals were defined in 7–10 and superimposed. The A-ring of 10 was used for superposition of the aromatic rings. Despite its importance, the indole NH group could not be included in the fitting

162 Molecular Modeling of the Enantiomers of 5-OMe-BPAT procedure, since not all ligands contain this functionality. However, the best matching superposition (Figure 7.5) shows that the indole NH groups of 7 and 9 coincide and point in the same direction, indicating that they may interact with a mutual H-bond accepting amino acid residue. The side chain of serotonin is oriented in an all-trans conformation, lying in the plane of the indole nucleus. The cyclohexyl rings of 8 and 9 have adopted a half-chair conformation, bearing pseudo-equatorial amino substituents. The hydroxy groups of the ligands coincide well and have a mutual H-bond-donating interaction point. However, because the 11-hydroxy group of 10 can only be approached from one side due to steric hindrance caused by the C-ring, and as a consequence of using the A-ring for aromatic superposition, 10 is oriented almost perpendicular to the plane through 7–9. Nevertheless, it is still capable of forming the essential interactions with the receptor map. It is also noteworthy that 10 has an axial N-methyl substituent in the best match. During the process of receptor construction (see below) it soon became clear that 10 would be difficult to accommodate in the orientation as revealed by APOLLO, using the A-ring for superposition. Difficulties resulted from the 10-methyl group causing steric hindrance with TM5. Furthermore, due to the perpendicular orientation compared to the other agonists, the C-ring of 10 protruded in the direction of TM4, thus preventing this helix to be tightly packed with TM3 and TM5. Therefore, an alternative fitting mode of 7–10 was considered, using the C-ring of 10 for aromatic superposition. The same conformations of all ligands were identified, and the superposition of 7–9 was identical to the first approach (Figure 7.5). Furthermore, the mutual interaction points were located at almost identical positions in the two fitting modes. However, in this alternative orientation, 10 could be well accommodated by the binding site, since it is lying in the plane of the other agonists, while the 10- methyl group points more away from TM5, in the direction of the binding site crevice. Particularly

FIGURE 7.5 Stereo representations of the two superposition modes of the serotonin 5-HT1A receptor agonists 7–10 in their presumed receptor binding conformations, using the A-ring (top) and C-ring (bottom) of 10 for superposition. The water molecules mimic putative amino acid residues belonging to the receptor, capable of forming interactions (dotted lines) with the ligands.

163 Chapter 7 the alternative superposition mode of 7–10 stresses the conclusion drawn for the dopaminergic pharmacophore that corresponding functional groups of the different ligands may approach mutual interaction points belonging to the receptor from different angles. All G-protein-coupled receptors contain a highly conserved aspartic acid residue at similar positions in TM3, corresponding to Asp114 and Asp116 in the dopamine D2A and serotonin 5-HT1A receptor, respectively. Site-directed mutagenesis studies have revealed that mutation of Asp114 to an uncharged residue completely abolishes the binding of both dopamine D2 receptor agonists and antagonists,42 strongly suggesting that the negatively charged carboxy terminus of Asp114 is involved in forming a reinforced electrostatic interaction with the protonated, positively charged nitrogen atom of dopamine and synthetic dopamine receptor ligands. In addition, a highly conserved aspartic acid residue in TM2, corresponding to Asp80 and Asp82 in the dopamine D2A and serotonin 5-HT1A receptor, respectively, is present in all monoaminergic GPCRs. Site-directed mutagenesis of this residue in the dopamine D2 and a2-adrenergic receptor revealed its importance for the pH and sodium regulations of agonist affinities, suggesting that it is involved in stabilizing the receptor in a certain conformation required for signal transduction, by complexation of cations.28,49 Furthermore, catecholamine receptors contain a cluster of serine residues in TM5, corresponding to

Ser193, Ser194 and Ser197 in the dopamine D2 receptor, capable of forming H-bonds with a catecholamine moiety. Since the intracellular loop connecting TM5 and TM6 presumably is involved in the coupling of the receptor to its associated G-protein, the ability of a ligand to interact with one or more of these serine residues seems to be crucial for agonistic properties. However, the specific contributions of these serine residues in the binding process of dopamine and synthetic receptor agonists are not completely clear, since site-directed mutagenesis experiments on these residues resulted in different, sometimes contradicting effects for different ligands.9,10,42,76 Nevertheless, Ser193 and Ser197 seem to be of importance for agonist binding, while Ser194 may be involved in mediating the ability of dopamine to inhibit cAMP production. Finally, mutation studies have shown that Phe389 and Phe390 in TM6 are crucial for ligand binding.8 Possible interactions with the amino acids discussed above were taken into consideration when the 7TM model of the dopamine D2 receptor was constructed around the superimposed ligands 2–5. Thus, TM3 was positioned in such a way that the terminal carboxy terminus of Asp114 replaced the water molecule in the receptor map, which interacted with the protonated nitrogen atoms of 2–5. Similarly, the terminal hydroxy groups of Ser193 and Ser197 replaced the water molecules interacting with the hydroxy groups of 2–5. Ser193 was defined as H-bond donor and Ser197 as H- bond acceptor (Figure 7.6). TM4 was the placed on the correct side of TM3 and TM5 with the aid of the 3D structure of bR, resulting in a counterclockwise orientation of the TM domains when viewed from the outside. The positioning of TM4 could also be guided by the possibility to form a disulfide bridge between Cys118 in TM3 and Cys168 in TM4 (Figure 7.6), as proposed by Moereels and Leysen.45 Cys118 is a conserved amino acid, present in the sequences of a number of GPCRs. 70 Cys168, however, is only found in the dopamine D2 and D3 receptors. The possibility to form this disulfide bridge may therefore be important for maintaining the relative orientation of TM3 to TM4, as well as the overall shape of the dopamine D2 and D3 receptors. Javitch et al., however, claimed that Cys118 is exposed in the binding site, based on studies with sulfhydryl-specific methanethiosulfonate reagents that were allowed to react with cysteine residues, suggesting that

164 Molecular Modeling of the Enantiomers of 5-OMe-BPAT

FIGURE 7.6 Pictures illustrating the stepwise construction process of the dopamine D2 receptor model. The left picture shows the positioning of TM3 and TM5 with the aid of the indirectly obtained receptor map. The right picture gives a top-to-bottom view of the relative positions of TM3, TM4 and TM5. The positioning of TM4 could be guided by the possibility to form a disulfide bridge between Cys118 in TM3 and Cys168 in TM4, which is clearly visible. The backbones of the TM domains are displayed as line ribbons. Cys118 is not involved in disulfide bridge formation.33 Therefore, as suggested by Moereels and Leysen,45 Cys118 should be an interesting candidate for site-directed mutagenesis studies.

Site-directed mutagenesis studies have confirmed that Asp116 in TM3 of the serotonin 5-HT1A receptor probably is involved in the formation of a reinforced electrostatic interaction with the protonated nitrogen atoms of ligands binding to this receptor subtype. TM5 of the serotonin 5-HT1A receptor contains a serine (Ser199) and a threonine (Thr200) residue, both capable of forming H- bonds with the 5-hydroxy group of serotonin. Thr200 is conserved in all 5-HT1 subclass receptors. Since these receptor subtypes also require the presence of the 5-hydroxy substituent for serotonin to bind, Thr200 appears to be important for the affinity of serotonin, and presumably acts as a H-bond donor to the 5-hydroxy substituent of serotonin. The importance of Thr200 for serotonin binding was supported by site-directed mutagenesis studies.26 However, Ser199 also seems to be involved in serotonin binding, since mutation of this residue was found to decrease the affinity of serotonin as well.26 Kuipers et al. suggested that Ser199 may be involved in binding the indole NH of serotonin.37

This assumption was based on the observation Ser199 is conserved in serotonin 5-HT1A, 5-HT1B and

5-HT1D receptors, while in serotonin 5-HT2A and 5-HT2C receptors, this residue is replaced by a glycine. This appears to be consistent with the observations that an unsubstituted indole nitrogen of serotonin is required for high affinity at the 5-HT1 but not for the 5-HT2 receptor subtypes. In analogy with the dopaminergic 7TM model, the construction process was started by bringing TM3 and TM5 in close contact with the superimposed agonists 7–10. Asp116 in TM3 was directed at the protonated nitrogen atoms, thus merely replacing the water molecule in the receptor map, while the

165 Chapter 7 terminal hydroxy group of Thr200 was defined as H-bond donor towards the 5-hydroxy substituent of serotonin. It soon became evident that a simultaneous interaction between Ser199 and the indole NH groups of serotonin and 9 was unlikely, since the orientation of the cluster of agonists required for this to occur caused serious steric hindrance between the ligands and the backbone of TM5. During the construction process however, an interaction between the indole NH groups of serotonin and 9 and Ser168 in TM4 became possible, which was also useful for determining the position of TM4 relative to TM3 and TM5.

The 7TM models of the dopamine D2 and serotonin 5-HT1A receptor finally obtained are shown in Figure 7.7. The overall topological arrangement of the 7TM domains in both models is clearly dissimilar from bR (Figures 7.1 and 7.2), and show much more resemblance with rhodopsin (Figure 7.2). Nevertheless, differences in the relative positions of the TM domains are also clear. Particularly the relative arrangement of TM3, TM4 and TM5 in the serotonin 5-HT1A receptor model differs considerably from that of rhodopsin and the dopamine D2 receptor model. Deviations in these arrangements probably arise from differences in packing of the helices, due to differences in amino acid sequences and in the receptor maps used for the construction of the agonist binding sites. Since most published 7TM models of the dopamine D2 and serotonin 5-HT1A receptor were constructed either by mutating the amino acids in the 3D structure of bR,36,37,67 or by using the 3D structure of bR as an explicit template for positioning all TM domains,22,25,32,41,68 it will be difficult to compare those models with the results obtained in the present investigation. The interactions of the agonists with the receptors, revealed after individual docking and optimization of their binding sites, are exemplified by dopamine (Figure 7.8) and serotonin (Figure 7.9). Dopamine interacts with the receptor in a folded conformation. The protonated nitrogen atom forms a reinforced electrostatic interaction with the carboxylate group of Asp114 on TM3. The meta-hydroxy substituent is involved in two H-bonds: it acts as a H-bond donor to the oxygen of Ser197, while at the same time accepting a H-bond from Ser193. Furthermore, Ser197 donates a H- bond to the para-hydroxy substituent. Both hydroxy substituents of dopamine are rotated slightly out of the plane of the aromatic ring. Ser194 is pointing towards TM6 and not involved in the binding of dopamine. In a model reported by Moereels and Leysen45 dopamine was also found to

FIGURE 7.7 Pictures showing the topological arrangements of the TM domains of the final 7TM models of the

dopamine D2 (left) and serotonin 5-HT1A receptor (right) and the locations of the endogenous ligands in the active sites. The backbones of the TM domains are displayed as line ribbons. The view is from the intracellular side.

166 Molecular Modeling of the Enantiomers of 5-OMe-BPAT

FIGURE 7.8 Stereo representation of dopamine in its optimized binding site model. interact with Ser193 and Ser197. However in their model, the meta-hydroxy substituent acts as H- bond donor to Ser193, while the para-hydroxy substituent acts as H-bond donor to Ser197. Furthermore, Phe390 in TM6 is in close contact with the aromatic ring of dopamine, presumably contributing to its affinity by hydrophobic interactions. Compounds 3–5 occupied the agonist binding site in a similar fashion as dopamine (not shown). Thus, the hydroxy substituents of 3 and 4, and the 11-hydroxy substituent of 5 acted as H-bond donor and acceptor to Ser197 and Ser193 respectively. In addition, similar to the para-hydroxy substituent of dopamine, the 10-hydroxy substituent of 5 accepted a H-bond form Ser197. All synthetic agonist formed a reinforced electrostatic interaction with their protonated nitrogens atoms and Asp114. It should be noted, however, that the relative positions of 2–5 after individual docking and optimization of the binding sites had changed considerably compared to the relative positions in the pharmacophore model (Figure 7.4). The N-n-propyl groups of 3 and 4 could easily be accommodated by the binding site models. The so-called ‘propyl group phenomenon’ of the DPATs and structurally closely related compounds, which involves the observation that one of the N- substituents should be an unbranched group not larger than N-n-propyl, while the structural requirements for the other N-substituent are less demanding for optimal affinity, has been rationalized by the presence of a small lipophilic pocket in the dopamine D2 receptor, capable of accommodating the N-n-propyl substituent.19,59,75 Malmberg et al. were able to identify and visualize this ‘propyl pocket’ in the dopamine D2A and D3 receptor, using homology-based molecular

FIGURE 7.9 Stereo representation of serotonin in its optimized binding site model.

167 Chapter 7 modeling studies.41 Our model did not clearly reveal the presence of such a pocket. Differences with the Malmberg model presumably result from differences in the arrangements of the TM domains, since their model was explicitly based on the 3D structure of bR. The extended conformation of serotonin, as revealed by APOLLO, was maintained after optimization of its position in the binding site (Figure 7.9). Thr200 acts as H-bond donor to the 5- hydroxy substituent of serotonin, while Asp116 in TM3 forms a salt bridge with the protonated nitrogen atom. The indole NH acts as H-bond donor to Ser168 in TM4. Other neighbouring amino acid residues are Met172 in TM4, and Thr196, Ala203 and Phe204 in TM5. Similarly, the hydroxy substituents of 8 and 10, and the methoxy substituent of 9 accepted a H-bond from Thr200, while their protonated nitrogens atoms formed a reinforced electrostatic interaction with Asp116 (not shown). In addition, like serotonin, the indole NH of 9 donated a H-bond to Ser168. Based on homology-based modeling studies, this serine residue was proposed by Hedberg et al. to function as H-bond donor to the 11-hydroxy substituent of 10,21 although in an earlier report they proposed a similar interaction with Ser199 in TM5.22 In our model, Ser168 is not involved in the binding of 10, in all probability due to a completely different orientation of the ligand in the binding site and to differences in the overall TM domain topology as argued above. In view of the agonist properties of 10, an interaction with either Thr200 or Ser199 seems the most reasonable. SAFIRs of the 2-aminotetralin-derived benzamides suggested that the 2-aminotetralin moieties of the compounds may occupy the same binding site as the 2-aminotetralin moieties of the DPATs (i.e. the agonist binding site), while the 2-benzamidoethyl side chains may share the same binding site as the 2-pyrrolidinylmethyl-derived substituted benzamides in the dopamine D2 receptor. Obviously, the dopamine D2 receptor binding conformations of (S)- and (R)-1 can not be identified by including their 2-aminotetralin moieties in the fitting procedure as described for 2–5, since this would not reveal the relative position of the benzamidoethyl side chains. Therefore, a suitable template molecule, containing pharmacophoric elements corresponding to both the benzamidoethyl side chains and the 2-aminotetralin moieties of (S)- and (R)-1 needed to be considered. N-Benzylpiquindone (6) was selected for this purpose. This semirigid compound is a member of a series of pyrrolo[2,3- 66 g]isoquinoline derivatives, which are highly stereoselective dopamine D2 receptor antagonists. (–)- , the N-methyl analogue of 6, and lead compound of the series,53 has been frequently used as a rigid template for identifying the dopamine D2 receptor binding conformations of 2- pyrrolidinylmethyl-derived substituted benzamides.29,30,50,57 Although belonging to different chemical classes, (–)-piquindone and the 2-pyrrolidinylmethyl-derived substituted benzamides have several structural and pharmacological features in common, which is essential when employing the active analogue approach. Thus, both classes of compounds contain an aromatic ring capable of forming hydrophobic interactions, a carbonyl group, and a basic nitrogen atom at a certain distance from the aromatic ring. In addition, the binding of both classes of compounds is highly stereoselective and sodium-dependent, suggesting that they may share the same binding site and possess similar binding modes. Using homology-based receptor modeling, the dopamine D2 receptor binding mode of several derivatives of (–)-piquindone has been elucidated by Teeter et al.66 An interesting observation from their study was that 6 had a 55-times higher affinity for the dopamine D2 receptor than the lead compound, suggesting that the binding of 6 was strongly enhanced by the presence of the N-benzyl group. In the receptor model, the N-benzyl group was located in the agonist binding

168 Molecular Modeling of the Enantiomers of 5-OMe-BPAT

FIGURE 7.10 Stereo representations of (S)-1 and 6 (top), and (R)-1 and 6 (bottom), superimposed in their presumed

dopamine D2 receptor binding conformations. The water molecules mimic putative amino acid residues belonging to the receptor, capable of forming interactions (dotted lines) with the ligands. site, forming favourable interactions with Phe389 and Phe390 in TM6. Therefore, 6 was considered to be a suitable template for identifying the dopamine D2 receptor binding conformations of (S)- and (R)-1: the pyrrolo[2,3-g]isoquinoline skeleton could be used for fitting the benzamidoethyl side chains, while the N-benzyl group should identify the orientation of the 2-aminotetralin moieties. The pyrrole ring of 6, corresponding to the benzamide nuclei of (S)- and (R)-1, the carbonyl groups, the protonated nitrogen atoms, and the N-benzyl group of 6, corresponding to the 2-aminotetralin aromatic rings, were defined as pharmacophoric elements. The superpositions of the best matching conformations of (S)- and (R)-1 and 6 are shown in Figure 7.10. Whereas the conformations of the benzamidoethyl side chains of (S)- and (R)-1 are identical, obviously as a result of the identical fitting procedure, differences in the overall conformations occur in the orientations of the 2-aminotetralin moieties, as a consequence of the opposite chiralities of the C2 carbon atom.

Flesinoxan (11) is a selective serotonin 5-HT1A receptor agonist, which shares the presence of a benzamidoethyl side chain with 1. Modeling studies performed by Kuipers et al. suggested that the arylpiperazine moiety of 11 occupies the same binding site in the serotonin 5-HT1A receptor as serotonin and other agonists, including 8 and 10,36,37 supporting the observation that the arylpiperazine moiety, when appropriately substituted, can function as a bioisostere of the 2- aminotetralin moiety.48 According to Kuipers et al., the plane angle between the benzene and piperazine rings of 11 in its the receptor binding conformation is approximately 30º, corresponding to a dihedral angle t1-2-3-4 (see Chart 7.3) of about –11º. In the active site, the protonated arylpiperazine N4-atom interacted with Asp116, while the most distant oxygen atom of the dioxane ring accepted a H-bond from Thr200. The amide functionality of 11 was not involved in the formation of specific interactions. The lack of dopamine D2 receptor affinity of 11 was explained by the inability of this receptor subtype to accommodate the hydroxymethyl substituent at the dioxane ring. In view of the structural and pharmacological similarities with 1, 11 was considered to be a suitable template for identifying the serotonin 5-HT1A receptor binding conformations of (R)-and (S)- 1. Thus, vectors points emanating from the protonated N4-atom and the most distant dioxane

169 Chapter 7 oxygen atom, and centroids and normals through both aromatic rings were defined in 11. These points were matched with the appropriate points defined in the enantiomers of 1, i.e. vector points emanating from the protonated nitrogens and the 5-methoxy oxygen atoms, and centroids and normals through both aromatic rings. The best-matching conformations thus identified are shown in

Figure 7.11. Similar to the presumed dopamine D2 receptor binding conformations (Figure 7.10), the benzamidoethyl side chains of (S)- and (R)-1 have adopted identical conformations, whereas the orientations of the 2-aminotetralin moieties are different. However, the overall conformations of both enantiomers found here are completely different from their presumed dopamine D2 receptor binding conformations.

The presumed dopamine D2 receptor binding conformations of (S)- and (R)-1 were docked into the binding site of the 7TM model with their 2-aminotetralin moieties positioned in the agonist binding site, and their protonated nitrogen atoms interacting with Asp114. Consequently, the benzamidoethyl side chains were directed downwards, i.e. towards the intracellular side, in a more or less parallel fashion with TM3. Inspection of the surrounding amino acid residues revealed possible interactions between the amido carbonyl oxygen atoms and Ser121 in TM3, which is situated almost two turns below Asp114. In addition, a H-bond could be formed between the amide hydrogen atoms of the ligands and Tyr416 in TM7, which was directed towards TM3. These observations support the SAFIRs which revealed the importance of the carbonyl moiety and the presence of an unsubstituted amide nitrogen necessary for high affinity of 1 at the dopamine D2 receptor (see Chapter 3). Furthermore, edge-to-face interactions between the indole NH of Trp386 in TM6 and the benzamide moieties were present. The N-n-propyl groups of both enantiomers adopted an all- trans conformation and were directed towards TM7. Moreover, the 5-methoxy substituents of both enantiomers were located close to Ser193 in TM5, but after optimization of the complexes, only the 5-methoxy substituent of (S)-1 formed a H-bond with this amino acid residue (Figure 7.12). The

FIGURE 7.11 Stereo representations of (S)-1 and 11 (top), and (R)-1 and 11 (bottom), superimposed in their presumed

serotonin 5-HT1A receptor binding conformations. The water molecules mimic putative amino acid residues belonging to the receptor, capable of forming interactions (dotted lines) with the ligands.

170 Molecular Modeling of the Enantiomers of 5-OMe-BPAT

FIGURE 7.12 Stereo representations of (S)-1 (top) and (R)-1 (bottom) located in the optimized dopamine D2 receptor binding site models. aminotetralin moiety of the (R)-enantiomer was directed more towards TM6, which prevented its 5- methoxy substituent to interact with Ser193. Since stimulation of the dopamine receptors by agonists probably requires binding to one of the conserved serine residues in TM5, this subtle but important difference may explain why (S)-1 behaves as a dopamine D2 receptor agonist, while (R)-1 behaves as an antagonist. It is noteworthy that the 5-methoxy substituent of (S)-1 was rotated 49º out of the plane of the aromatic ring of the 2-aminotetralin nucleus, while the 5-methoxy substituent of (R)-1 was oriented in a coplanar fashion with this ring. Semi-empirical AM1 calculations12 revealed that this unfavourable orientation of the 5-methoxy substituent of (S)-1 can be adopted at the cost of 2.5 kcal/mol. This may explain the lower affinity of (S)-1 for the dopamine D2 receptor compared to (R)- 1, despite its apparent ability to form a H-bond with its 5-methoxy substituent. In contrast to their dopaminergic receptor binding modes, the benzamidoethyl side chains of (S)- and (R)-1 were directed upwards along TM3, i.e. towards the extracellular space, in the serotonin 5-

HT1A receptor model. No amino acid residues capable of H-bond formation were surrounding the benzamide moieties, but a specific face-to-face interaction between the benzamide aromatic rings and the aromatic ring of Phe112 in TM3 was possible. The N-n-propyl groups of both compounds were pointing towards TM4, both having adopted an all-trans conformation. In addition, the 5-methoxy substituents of both enantiomers were located close to Thr200 in TM5. Although their overall orientations within the binding site were not identical, both enantiomers formed a reinforced electrostatic interaction with their protonated nitrogens and Asp116, and a H-bond between their 5- methoxy substituent and Thr200 after optimization of the individual complexes. In addition, the benzamide moieties of both enantiomers were in close contact with Phe112, forming face-to-face interactions with their aromatic rings (Figure 7.13). The lack of specific interactions with the

171 Chapter 7 benzamide moieties is consistent with the findings from SAFIRs that this functionality does not contribute to the affinity of 1 for the serotonin 5-HT1A receptor (see Chapter 3). Due to steric hindrance of their 5-methoxy terminal CH3 groups with Thr200, the 5-methoxy substituents of (S)- and (R)-1 were rotated 56º and 64º out of plane, requiring energy penalties of 2.1 and 3.2 kcal/mol, respectively. The energy difference in favour of the (S)-enantiomer may explain its higher affinity for the serotonin 5-HT1A receptor (see Chapter 5). Out-of-plane orientation of the 5-methoxy substituents abolishes the steric hindrance and simultaneously favours the formation of an H-bond with Thr200. Although previous SAFIR studies on the role of the 5-methoxy substituent of 1 revealed that H-bond formation of the C5-substituent does not contribute to the serotonin 5-HT1A receptor affinity (see Chapter 4), it may prove to be essential for stimulation of the receptor, i.e. the intrinsic efficacy. Therefore, the ability of both enantiomers to interact with Thr200 may explain why they both behave as full serotonin 5-HT1A receptor agonists in vitro and in vivo.

FIGURE 7.13 Stereo representations of (S)-1 (top) and (R)-1 (bottom) located in the optimized serotonin 5-HT1A receptor binding site models.

172 Molecular Modeling of the Enantiomers of 5-OMe-BPAT

The unbiased nature of the receptor models presented here, resulting from the combination of indirect and direct modeling during the objective, stepwise construction process should be emphasized. A distinct advantage of this procedure seems to be the ability of the resulting receptor models to rationalize the binding modes and support the SARs of ligands (i.e. the enantiomers of 1) which have not been used during the construction process.

7.4 CONCLUSIONS

Models of the dopamine D2 and serotonin 5-HT1A receptor, which resemble the projection structure of rhodopsin in their TM domain topology, were developed by using a combination of indirect molecular modeling (i.e. the active analogue approach) and direct construction of the TM domains of the receptors, in a stepwise, objective manner. The models could be used to rationalize the dopamine D2 and serotonin 5-HT1A receptor binding modes of the enantiomers of 5-OMe-BPAT.

(S)- and (R)-5-OMe-BPAT bound to the dopamine D2 receptor model in a similar but not identical fashion, the most important difference being the ability of (S)-5-OMe-BPAT to interact with Ser193 in TM5, which may explain its dopamine D2 receptor agonist properties. The ability of the amide moieties to form specific interactions were consistent with the SAFIRs earlier observed. Similarly, the observed binding modes of (S)- and (R)-5-OMe-BPAT in the serotonin 5-HT1A receptor model were consistent with the SAFIR of 5-OMe-BPAT, and could explain their full agonist properties at this receptor subtype.

7.5 EXPERIMENTAL SECTION

7.5.1 CONFORMATIONAL ANALYSES

All calculations were performed on a Silicon Graphics IRIS Indigo XS/4000 Workstation or a Silicon Graphics Indy Workstation, running IRIX 5.3. Conformational analyses were performed in MacroModel version 4.5,46 using the Monte Carlo Multiple Minimum (MCMM) search protocol.7 Ligands with the correct stereochemistry were build from standard fragments. All ligands were considered in their protonated, positively charged forms. N-n-propyl groups were truncated to N-methyl groups during the conformational analyses in order to reduce the number of torsion angles. All minimizations were performed within the MM3* force field,1,39,40, while simulating a distance-dependent GB/SA water continuum,63 as implemented in MacroModel. Benzamide moieties were fixed in a coplanar fashion during all minimizations. Prior to submitting them to the MCMM protocol, all ligands were minimized with default options. In order to mimic the possibility of inversion of the protonated nitrogen atom, a second starting conformation was generated for ligands containing a protonated nitrogen atom bearing four different substituents, by inverting its ‘chirality’. The two starting conformations thus obtained for these ligands were independently submitted to the MCMM protocol. To search conformational space 1000–5000 MC steps were performed on each starting conformation, dependent on the number of torsion angles. Starting conformations for each step were systematically generated using the SUMM option.15 The number of torsion angles to be varies in each MC step was set between 2 and n–1, n being the total number of variable torsion angles. Ring closure bonds were defined in 5- and 6-membered non- aromatic rings in order to allow torsion angles within these rings to be varied as well. Ring closure distances were limited to 0.5–2.0 Å. The randomly generated structures were minimized using the Truncated Newton Conjugate Gradient (TNCG)minimizer, allowing for 250 iterations per structure, until an initial gradient of 0.01 kcal/Å mol–1 was reached. Least squares superposition of all non-hydrogen atoms was used to eliminate duplicate conformations. For non-chiral ligands, rejection of mirror images was prevented by specifying the NANT option. The minimum

173 Chapter 7 energy conformations thus obtained were submitted to a final minimization, using the Full Matrix Newton Raphson (FMNR) minimizer, allowing for 1000 iterations per structure, until a final gradient of 0.002 kcal/Å mol–1 was reached. An energy cut-off of 3.0 kcal/mol was applied to the search results. For ligands containing a ‘chiral’ protonated nitrogen atom, the search results of the independent analyses performed on the starting conformations with inverted nitrogen atoms were combined and subsequently filtered on energy (DE £ 3.0 kcal/mol) using the Filter module.

7.5.2 PHARMACOPHORE IDENTIFICATION

The sets of minimum energy conformations of the ligands, as obtained from the conformational analyses, served as input for the VECADD module of the pharmacophore-identifying program APOLLO.61,62 This module was used to add extension vectors pointing from H-bond donating or accepting groups towards putative complementary receptor points, and to define centroids and normals through the planes of aromatic rings in each conformation of each ligand. In all cases a minimum density of vectors was specified, representing the ideal position for H-bond formation. The RMSFIT module of APOLLO was then used to identify the conformation of the different ligands that exhibited the best overall least squares fit with respect to the specified fitting points. All fitting points were weighed equally. Extension vectors emanating from oxygen atoms, centroids, and the extremes of normals were defined as choices. Conformational energies and root mean square deviations were used to rank the matches. The best match was then extracted with the MMDFIT module of APOLLO.

7.5.3 RECEPTOR CONSTRUCTION

The 7TM models of the dopamine D2 and serotonin 5-HT1A receptors were constructed using the Biopolymer module of the integrated molecular modeling package SYBYL version 6.4.64 Right-handed a-helices (2 turns, f = – 57°, y = –47°, W = 180°) were build from the appropriate L-amino acids. Proline residues were fixed, hydrogen atoms were added and side chain torsion angles were scanned. Gasteiger-Hückel charges were defined on all atoms, and the helices were independently minimized within the Tripos force field. A distance-dependent dielectric constant of 5.0 and nonbonded cut-off value of 8.0 Å were specified. Minimizations were performed using the Conjugate Gradient minimizer. An RMS energy gradient of 0.1 kcal/Å mol–1 was set as the final convergence criterion. The complete 7TM models were constructed in a stepwise manner, starting with positioning TM3 and TM5, guided by the receptor maps as obtained from the pharmacophore identification procedure. The structure of bR was then used to determine the relative position of TM4. Subsequently, TM3, TM4 and TM5 were packed as tightly as possible, by slightly adjusting (i.e. tilting, rotating or shifting) their relative positions, without disturbing the primary interactions with the superimposed agonists. After each adjustment the TM domains were inspected for overlap of the van der Waals volumes of their side chains, using the Z-clipping option of SYBYL. This feature allows to cut slices of any desired width at any desired position through a molecule, and hence is very suitable for visualazing overlap of van der Waals volumes between adjacent TM domains. In a similar fashion, the other TM domains were added one by one, in the following order: TM6, TM2, TM7, and TM1. The more hydrophilic sides of the helices were directed towards the binding site crevice, while the more hydrophobic sides faced the hypothetical lipid bilayer. The TM domains were positioned according to the topological arrangement of rhodopsin. Each time a new TM domain had been added and appropriately positioned, the energy of the entire 7TM model was minimized.

7.5.4 LIGAND DOCKING

The presumed pharmacologically active conformations of the ligands, as identified by APOLLO, were individually docked into the active sites of the receptors. Where appropriate, N-n-propyl groups were introduced and extended in an all-trans conformation. Atomic charges were calculated semi-empirically on all ligands, using the AM112 method as implemented in MOPAC 5.0 and accessed through SYBYL. In all cases the additional keyword 1SCF was supplied,

174 Molecular Modeling of the Enantiomers of 5-OMe-BPAT while for benzamides the keyword MMOK(0) was supplied as well. Attractive interactions between the ligands and the receptors were then optimized and repulsive interactions minimized by manually rotating and translating the ligands in the binding sites, using the Docking procedure as implemented in SYBYL. H-bonds between the ligands and the receptors were dynamically displayed during this process. When necessary, side chain dihedral angles were manually adjusted in order to minimize overlap or to optimize attractive interactions between amino acid residues in the binding sites and the ligands.

7.5.5 ACTIVE SITE OPTIMIZATION

The pseudoreceptor-generating program PrGen version 2.077 was used for optimizing the interactions between the individual ligands and the amino acid residues surrounding them. For this purpose, all amino acid residues having one or more atoms located within a sphere of 4 Å surrounding a ligand, were extracted from the receptors in SYBYL, and transferred to PrGen. Force field atom types were assigned to the ligands, and their solvation energies, entropy corrections and reference energies were calculated. Minimizations in PrGen were performed within the Yeti force field,71 using the Conjugate Gradient type 2 minimizer. During the minimizations, only torsion angles in the side chains, conjugated bonds, and terminal CH3 groups of the amino acid residues were allowed to be varied. The ligands were allowed to be translated and rotated, and all freely rotatable torsion angles, conjugated bonds, and terminal CH3 groups were allowed to be varied, except for the coplanar benzamide moieties. A sufficient number of iterations was specified in order to reach a convergence criterion of 0.001 kJ/Å mol–1. All other convergence criteria were set at default values.

7.6 REFERENCES

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178 SUMMARY

Schizophrenia is a severe disease of the central nervous system which affects approximately one percent of the world population. The disease can not be cured presently, but since the early 1950’s, compounds have become available which to a certain extent can alleviate the symptoms. Although numerous antipsychotic agents have been developed during the last forty years, none of them displays an ideal clinical profile. Thus, whereas most compounds to a certain extent can control the positive symptoms of schizophrenia, they usually fail to improve the more persistent negative symptoms. In addition, approximately one third of all patients does not respond at all to treatment with these classical antipsychotic drugs. Moreover, these compounds frequently induce severe side- effects, the most prominent being extrapyramidal movement disorders such as Parkinsonism and tardive dyskinesias. An unique exception is formed by clozapine, a compound which possesses a superior antipsychotic profile associated with a low incidence of extrapyramidal side-effects. As such, clozapine is considered to be the prototype of the so-called ‘atypical’ antipsychotic agents. However, treatment with clozapine is restricted, since it can induce agranulocytosis, a potentially lethal blood dyscrasia. Furthermore, the mechanism of action of clozapine, responsible for its unique profile, is poorly understood, since the compound has high affinities for a wide range of receptor subtypes. Therefore, the search for new atypical antipsychotic agents is still continued. Such compounds may help to gain further insight the pathophysiology of schizophrenia, the mechanism of action of antipsychotic agents in general, and of clozapine in particular. This thesis describes the attempts to develop a novel class of potential atypical antipsychotic agents. A general introduction into the field of schizophrenia and antipsychotic agents is provided in Chapter 1, with special attention for the medicinal chemistry aspects. Emphasis is laid on the dopamine hypothesis of schizophrenia and the role of dopamine D2-like receptors. This subclass of dopamine receptors comprises the classical dopamine D2 receptor and the recently identified D3 and

D4 subtypes. Blockade of dopamine D2 receptors in the mesolimbic and mesocortical dopaminergic systems of the central nervous system has been generally accepted as the mechanism by which the antipsychotic effects are mediated, while blockade of dopamine D2 receptors in the nigrostriatal dopaminergic system presumably causes the extrapyramidal side-effects. Due to its different regional distribution, which shows relatively high abundance in the mesolimbic system, the dopamine D3 receptor is considered to be an interesting target for atypical antipsychotic activity, while the relatively high affinity of clozapine for the dopamine D4 receptor suggested that its unique effects may be mediated by this receptor subtype. Evidence has been accumulated during the last two decades that, in addition to dopamine, other neurotransmitters may play a role in the pathophysiology of schizophrenia and the mechanism of action of atypical antipsychotic agents. Particularly serotonin has received much attention in this respect, in part by the notion that clozapine and other alleged atypical antipsychotic agents block serotonin 5-HT2 receptors more potently than dopamine D2 receptors. Consequently, a number of novel putative atypical antipsychotic agents with a balanced dopamine D2/serotonin 5-HT2 receptor-blocking ratio have been developed during the last fifteen years, some of which recently have become clinically available. Clinical results suggest that these compounds have an improved efficacy against the negative symptoms of schizophrenia combined with a reduced liability to induce extrapyramidal side-effects. Whether they are superior to

179 Summary clozapine remains to be established. Alternatively, preclinical as well as clinical observations suggest that compounds, which combine dopamine D2 receptor antagonism with serotonin 5-HT1A receptor agonism, also may have potential as atypical antipsychotic agents. Several compounds with such a pharmacological profile, showing beneficial efficacy in preclinical models predictive of antipsychotic efficacy and side-effect liability, have been disclosed recently. This promising concept formed the basis for the medicinal chemistry of the compounds described in the rest of the thesis. The 2-(N,N-di-n-propylamino)tetralins constitute a series of compounds with high affinities for dopamine D2, D3 and/or serotonin 5-HT1A receptors, dependent on their aromatic substitution pattern. A class of highly potent and selective dopamine D2/D3 receptor antagonists, some of which possess atypical antipsychotic properties, is formed by the 2-pyrrolidinylmethyl-derived substituted benzamides. Hence, it was conceived possible to develop a class of compounds with the desired receptor binding profile by combining the structural features of these two classes of compounds in a basic skeleton. Chapter 2 describes the design, synthesis and structure-affinity relationships

(SAFIRs) with respect to dopamine D2, D3 and serotonin 5-HT1A receptors of a series of 35 2-(N-n- propylamino)tetralin-derived benzamides with various aromatic substitution patterns. Most compounds possessed high affinities for the three receptor subtypes. The SAFIRs of the series suggest that the 2-aminotetralin moieties of the compounds occupy the same binding sites as the 2- (N,N-di-n-propylamino)tetralins in all three receptor subtypes. The benzamidoethyl side chain enhances the affinities of the compounds for all three receptor subtypes, presumably by occupying an accessory binding site. For the dopamine D2 and D3 receptors, this accessory binding site may be identical to the binding site of the 2-pyrrolidinylmethyl-derived substituted benzamides. In Chapter 3, the SAFIRs of the 2-aminotetralin-derived benzamides were further explored, by synthesizing various structural analogues of the lead compound of the series, 5-methoxy-2-[N-(2- benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT), and evaluating them for their affinities at dopamine D2, D3 and serotonin 5-HT1A receptors. Structural modifications of the benzamide moiety predominantly affected the affinities for the dopaminergic receptor subtypes, suggesting that the amide functionality of 5-OMe-BPAT forms specific interactions while binding to these receptor subtypes. Chain elongation of the benzamidoethyl side chain decreased both the dopaminergic and the serotonergic affinities. Replacement of the N-n-propyl substituent by smaller and larger groups revealed that the substituent requirements for the basic nitrogen atom were comparable to those of the 2-(N,N-di-n-propylamino)tetralins, further supporting the hypothesis that the 2-aminotetralin moieties of both classes of compounds may occupy the same binding sites. The contribution of the 5-methoxy substituent to the affinities of 5-OMe-BPAT for the dopamine

D2 and serotonin 5-HT1A receptors was investigated by preparing several structural analogues of 5- OMe-BPAT bearing different C5-substituents. The synthesis and SAFIRs of these compounds is described in Chapter 4. Chemical diversity at the C5-position was introduced by conversion of the 5-trifluoromethanesulfonyloxy analogue, using several Stille-type reactions, and a Heck reaction. No clear relationships between the nature of the C5-substituents and the dopamine D2 receptor affinities were observed. Nevertheless, the results suggested that the 5-methoxy substituent of 5-OMe-BPAT is involved in the binding to the dopamine D2 receptor. The serotonin 5-HT1A receptor tolerated more structural variation at the C5-position of 5-OMe-BPAT. A quantitative structure-activity relationship, explaining the serotonin 5-HT1A receptor binding, could be derived from a set of

180 Summary physicochemical descriptors, using Partial Least Squares Projection to Latent Structures. A relatively lipophilic, nonpolar C5-substituent, incapable of forming hydrogen bonds, appeared be optimal for high affinity at the serotonin 5-HT1A receptor. The synthesis and in vitro pharmacological evaluation of the optically pure enantiomers of 5- OMe-BPAT and of its 2,6-dimethoxybenzamido analogue, 5-OMe-(2,6-di-OMe)-BPAT, is described in Chapter 5. Screening of the enantiomers on several relevant receptors subtypes in vitro revealed that they possess high affinities for dopamine D2, D3 and serotonin 5-HT1A receptors only, and show stereoselectivity at these receptor subtypes. Determination of the intrinsic efficacies at these receptor subtypes in vitro showed that both enantiomers of 5-OMe-BPAT behaved as full serotonin 5-HT1A receptor agonists, while both enantiomers of 5-OMe-(2,6-di-OMe)-BPAT turned out to be weak partial agonist with low intrinsic efficacy at this receptor subtype. At the dopamine D2 receptor, all four compounds behaved as partial agonists, the (S)-enantiomers having the highest intrinsic efficacies. At the dopamine D3 receptor, only the (S)-enantiomer of 5-OMe-BPAT possessed some intrinsic efficacy, the other compounds behaving as antagonists. Chapter 6 describes the effects of the enantiomers of 5-OMe-BPAT in two behavioural and a neurochemical model with relevance for prediction of antipsychotic activity and side-effect liability. It was shown that (S)-and (R)-5-OMe-BPAT differently affected spontaneous and d-amphetamine- induced locomotor activity in rats, the (S)-enantiomer behaving as a mixed dopamine D2 and serotonin 5-HT1A receptor agonist, and the (R)-enantiomer behaving as a mixed dopamine D2 receptor antagonist and a serotonin 5-HT1A receptor agonist, respectively. These findings were supported by establishing their effects on neurochemical processes in different brain areas, using intracerebral microdialysis in freely moving rats. Both enantiomers failed to induce catalepsy in rats at high doses, predicting a low propensity to induce extrapyramidal side-effects in man. It was concluded that the enantiomers of 5-OMe-BPAT may prove to be interesting pharmacological tools for further exploring the concept of mixed dopamine D2 receptor antagonism and serotonin 5-HT1A receptor agonism for atypical antipsychotic drug action. The lack of cataleptogenic activity of (R)-5-

OMe-BPAT, in combination with its dopamine D2 receptor antagonistic and serotonin 5-HT1A receptor-stimulating properties, suggest that this compound in particular may possess atypical antipsychotic properties. The results of molecular modeling studies, which were undertaken to gain insight in the binding modes of the enantiomers of 5-OMe-BPAT to the dopamine D2 and serotonin 5-HT1A receptors at the molecular level, are presented in Chapter 7. A combination of pharmacophore identification, based on carefully selected sets of known receptor ligands of different chemical classes, and direct construction of the seven-transmembrane domains of the receptors was employed for this purpose. It was shown that both enantiomers could bind to the dopamine D2 receptor in a similar fashion, their protonated tertiary amine atoms and their benzamide moieties being involved in specific interactions with certain amino acid residues of the receptor. Differences were observed in the interactions involving the 5-methoxy substituent of the aminotetralin moiety and a specific amino acid residue in the fifth transmembrane domain, which may account for the observed differences in intrinsic efficacies of the enantiomers at this receptor subtype. Likewise, both enantiomers were shown to bind to the serotonin 5-HT1A receptor in a similar way, but the presumed pharmacologically active conformations and their interactions with the receptor were different from those observed for the

181 Summary

dopamine D2 receptor. The modes of binding to the serotonin 5-HT1A receptor could explain the full agonist properties of both enantiomers and supported the findings from the SAFIRs that their amide moiety is not important for high affinity at this receptor subtype.

182 SAMENVATTING

Schizofrenie is een ernstige aandoening van het centrale zenuwstelsel, waaraan ongeveer één procent van de wereldbevolking lijdt. Genezing van deze ziekte is nog niet mogelijk, maar sinds het begin van de vijftiger jaren zijn er verbindingen beschikbaar, die aangewend kunnen worden om de symptomen te bestrijden. Hoewel er gedurende de laatste veertig jaren vele antipsychotica zijn ontwikkeld, vertoont geen enkele van deze verbindingen een ideaal klinisch werkingsprofiel. Zo bestrijden de meeste antipsychotica de positieve symptomen van schizofrenie in zekere mate, maar zijn niet in staat de negatieve symptomen te verbeteren. Daarnaast heeft ongeveer een derde deel van de patïenten in het geheel geen baat bij therapie met deze klassiek antipsychotica. Bovendien vertonen dergelijke verbindingen vaak ernstige bijwerkingen, met name extrapyramidale bewegingsstoornissen zoals Parkinsonisme en tardieve dyskinesiëen. Een unieke uitzondering vormt clozapine, een verbinding die een superieur antipsychotisch werkingsprofiel combineert met een lage incidentie van extrapyramidale bijwerkingen. Clozapine wordt daarom beschouwd als het prototype van de zogenaamde ‘atypische’ antipsychotica. Clozapine moet echter met terughoudendheid worden toegepast omdat het agranulocytose kan veroorzaken, een bloedbeeld- afwijking die fataal kan zijn. Het werkingsmechanisme dat verantwoordelijk is voor het unieke klinische profiel van clozapine is onduidelijk, omdat de stof hoge affiniteiten vertoont voor een groot aantal receptorsubtypes. Het zoeken naar nieuwe atypische antipsychotica wordt dus voortgezet, omdat zulke stoffen mogelijk meer inzicht kunnen geven in de pathofysiologie van schizofrenie, het werkingsmechanisme van antipsychotica in het algemeen, en van clozapine in het bijzonder. Dit proefschrift beschrijft de pogingen om een nieuwe klasse van mogelijk atypische antipsychotica te onwikkelen. Een algemene inleiding op het gebied van schizofrenie en antipsychotica, met speciale aandacht voor de farmacochemische aspecten, wordt gegeven in Hoofdstuk 1. Hierbij wordt de nadruk gelegd op de dopaminehypothese van schizofrenie en de rol die dopamine D2-achtige receptoren daarin spelen. Deze subklasse van dopamine receptoren bestaat uit de klassieke dopamine D2 receptor en de recentelijk ontdekte D3 en D4 subtypes. Algemeen wordt aangenomen dat antipsychotica hun antipsychotische effecten uitoefenen door dopamine D2 receptoren te blokkeren in de mesolimbische en mesocorticale dopaminerge systemen van het centrale zenuwstelsel, terwijl blokkade van dopamine D2 receptoren in het nigrostriatale dopaminerge systeem waarschijnlijk verantwoordelijk is voor het optreden van extrapyramidale bijwerkingen. De dopamine D3 receptor wordt als een interessant doelwit beschouwd voor atypische antipsychotica, omdat diens verdeling over de diverse hersengebieden een relatief hoge dichtheid in de limbische gebieden vertoont. De relatief hoge affiniteit van clozapine voor de dopamine D4 receptor suggereert echter dat het unieke werkingsprofiel wellicht door dit receptorsubtype gemedieerd wordt. Tijdens de laatste twee decennia is het steeds duidelijker geworden dat naast dopamine ook andere neurotransmitters mogelijk een rol spelen in de pathofysiology van schizofrenie en het werkingsmechanisme van atypische antipsychotica. Vooral serotonine staat in dit verband erg in de belangstelling, ten dele vanwege het feit dat clozapine en andere vermeend atypisch antipsychotica serotonine 5-HT2 receptoren sterker blokkeren dan dopamine D2 receptoren. Dientengevolge zijn er in de afgelopen vijftien jaren verscheidene vermeend atypische antipsychotica ontwikkeld met een uitgebalanceerde

183 Samenvatting

verhouding in hun dopamine D2 en serotonine 5-HT2 receptor-blokkerende vermogen, waarvan enkele recentelijk voor klinische toepassing beschikbaar zijn gekomen. De klinische resultaten duiden erop dat dergelijke verbindingen een verbeterde effectiviteit tegen de negatieve symptomen van schizofrenie combineren met een verminderd risico op het optreden van extrapyramidale bijwerkingen. Uit zowel preklinische als klinische studies is gebleken dat verbindingen, die dopamine D2 receptor antagonisme combineren met serotonine 5-HT1A receptor agonisme, mogelijk ook atypische antipsychotische eigenschappen bezitten. Recentelijk zijn een aantal verbindingen met een dergelijk farmacologisch profiel gepubliceerd, en deze bleken gunstige eigenschappen te vertonen in preklinische modellen met voorspellende waarde voor antipsychotische activiteit en extrapyramidale bijwerkingen. Dit veelbelovende concept heeft de basis gevormd voor de farmacochemie van de verbindingen die in het resterende deel van dit proefschrift wordt beschreven. De 2-(N,N-di-n-propylamino)tetralines (DPATs) vormen een klasse van verbindingen die, afhankelijk van hun aromatische substitutiepatroon, hoge affiniteit bezitten voor dopamine D2, D3, en/of serotonine 5-HT1A receptoren. Een klasse van zeer potente en selective dopamine D2/D3 receptor antagonisten, waarvan sommige atypische antipsychotische eigenschappen bezitten, wordt gevormd door de 2-pyrrolidinylmethyl-afgeleide gesubstitueerde benzamides. Het werd mogelijk geacht om een nieuwe klasse van verbindingen met het gewenste receptorbindingsprofiel te ontwikkelen door de strukturele eigenschappen van deze twee verbindingsklassen te combineren in een nieuw basisskelet. In Hoofdstuk 2 worden het ontwerp, de synthese en structuur- affiniteitsrelaties (SAFIRs) met betrekking tot dopamine D2, D3, en serotonine 5-HT1A receptoren van een serie van 35 2-(N-n-propylamino)tetraline-afgeleide benzamides met verschillende aromatische substitutiepatronen beschreven. De meeste verbindingen vertoonden hoge affiniteiten voor de drie receptorsubtypes. De SAFIRs suggereerden dat de 2-aminotetralinegroepen van de verbindingen dezelfde bindingsplaatsen bezetten in de drie receptorsubtypes als die van de DPATs. De benzamidoethyl zijketen verhoogt de affiniteiten van de verbindingen voor de drie receptorsubtypes, vermoedelijk door het bezetten van een allostere bindingsplaats. Mogelijk is dit in het geval van de dopamine D2 en D3 receptoren dezelfde bindingsplaats die door de 2- pyrrolidinylmethyl-afgeleide gesubstitueerde benzamides wordt bezet. In Hoofdstuk 3 worden de SAFIRs van de 2-aminotetraline-afgeleide benzamides verder onderzocht, door verschillende structurele analoga van de leadverbinding van de serie, 5-methoxy-2- [N-(2-benzamidoethyl)-N-n-propylamino]tetraline (5-OMe-BPAT) te synthetiseren en hun affiniteiten voor dopamine D2, D3, en serotonine 5-HT1A receptoren vast te stellen. Strukturele modificaties van de benzamidegroep beïnvloedden voornamelijk de affiniteiten voor de dopaminerge receptoren, hetgeen suggereert dat de amide functionaliteit van 5-OMe-BPAT specifieke interacties vormt bij de binding aan deze receptorsubtypes. Ketenverlenging van de benzamidoethyl zijketen resulteerde in lagere affiniteiten voor zowel de dopaminerge als de serotonerge receptoren. Vervanging van de N-n-propylsubstituent door kleinere en grotere groepen toonde aan dat de strukturele vereisten voor de substituenten op het basische stikstofatoom vergelijkbaar waren met die van de DPATs. Deze waarnemingen ondersteunen de hypothese dat de 2-aminotetralingroepen van beide verbindingsklassen dezelfde bindingsplaatsen bezetten. De bijdrage van de 5-methoxysubstituent aan de affiniteiten van 5-OMe-BPAT werd onderzocht door een aantal structurele analoga met verschillende C5-substituenten te synthetiseren en hun

184 Samenvatting

affiniteiten voor dopamine D2 en serotonine 5-HT1A receptoren te bepalen. De syntheses en SAFIRs van deze verbindingen worden beschreven in Hoofdstuk 4. Chemische diversiteit op de C5-positie werd geïntroduceerd door de 5-triflouromethaansulfonyloxy-analoog te converteren door middel van een aantal Stille reacties en een Heck reactie. Er konden geen duidelijke relaties worden aangetoond tussen de aard van de C5-substituenten en de affiniteiten van de verbindingen voor de dopamine D2 receptor. Niettemin duidden de resultaten erop dat de 5-methoxysubstituent van 5-

OMe-BPAT betrokken is bij de binding aan dit receptorsubtype. De serotonine 5-HT1A receptor tolereerde meer strukturele variatie op de C5-positie van 5-OMe-BPAT. Met behulp van Partial Least Squares Projection to Latent Structures kon een kwantitieve structuur-aktiviteitsrelatie worden afgeleid uit een set van physicochemische parameters, waamee de affiniteiten van de verbindingen voor de serotonine 5-HT1A receptor konden worden verklaard. Een relatief lipofiele, apolaire C5- substituent, die geen waterstofbruggen kan vormen, was volgens het model optimaal voor een hoge affiniteit voor dit receptorsubtype. De syntheses en farmacologische evaluatie in vitro van de enantiomeren van 5-OMe-BPAT en diens 2,6-dimethoxy-analoog, 5-OMe-(2,6-di-OMe)-BPAT, worden beschreven in Hoofdstuk 5. Screening van de enantiomeren op een aantal relevante receptorsubtypes liet zien dat de verbindingen uitsluitend hoge affiniteiten vertonen voor dopamine D2, D3, en serotonine 5-HT1A receptoren. Uit bepalingen van de intrinsieke activiteiten op deze receptorsubtypes bleek dat de enantiomeren van 5-OMe-BPAT zich gedroegen als volle serotonine 5-HT1A agonisten, terwijl die van 5-OMe-(2,6-di-OMe)-BPAT een lage intrinsieke aktiviteit bleken te vertonen voor dit receptorsubtype. Alle vier verbindingen gedroegen zich als partiële dopamine D2 receptor agonisten, waarbij de (S)-enantiomeren de hoogste intrinsieke aktiviteiten vertoonden. Alleen de (S)- enantiomeer van 5-OMe-BPAT vertoonde enige intrinsieke activiteit voor de dopamine D3 receptor, de andere verbindingen gedroegen zich als antagonisten op dit receptorsubtype. Hoofdstuk 6 beschrijft de effecten van de enantiomeren van 5-OMe-BPAT in twee gedragsmodellen en een neurochemisch model met relevantie voor het voorspellen van antipsychotische activiteit en het risico op extrapyramidale bijwerkingen. (S)-en (R)-5-OMe-BPAT beïnvloedden in verschillende mate spontaan lokomotiegedrag and d-amfetamine-geïnduceerd lokomotiegedrag in ratten. Het (S)-enantiomeer gedroeg zich in deze tests als een gecombineerde dopamine D2 en serotonine 5-HT1A receptor agonist, terwijl het (R)-enantiomeer zich gedroeg als een gecombineerde dopamine D2 receptor antagonist en serotonine 5-HT1A receptor agonist. Deze bevindingen werden ondersteund door de effecten van de verbindingen op neurochemische processen in verschillende hersengebieden, zoals die werden bepaald door middel van intracerebrale microdialyse in vrijbeweeglijke ratten. Hoge doses van beide enantiomeren induceerden geen katalepsie in ratten, hetgeen voorspelt dat het risico op het optreden van extrapyramidale bijwerkingen in de mens gering zal zijn. Geconcludeerd werd dat de enantiomeren 5-OMe-BPAT interessante farmacologische hulpmiddelen kunnen vormen om het concept van gecombineerd dopamine D2 receptor antagonisme en serotonine 5-HT1A receptor agonisme voor het bewerkstelligen van atypische antipsychotishe activiteit verder te onderzoeken. Met name het (R)- enantiomeer lijkt interessant als mogelijk atypisch antipsychoticum, omdat deze stof dopamine D2 receptor antagonisme combineert met serotonine 5-HT1A receptor agonisme, zonder katalepsie te induceren.

185 Samenvatting

Om inzicht te krijgen in de manier waarop de enantiomeren van 5-OMe-BPAT op een molekulair niveau binden aan de dopamine D2 en serotonine 5-HT1A receptoren, werden molekulaire modeling studies ondernomen. De resultaten hiervan worden beschreven in Hoofdstuk 7. Hiervoor werd gebruik gemaakt van een combinatie van farmacofooridentificatie, gebaseerd op sets van zorgvuldig gekozen liganden van diverse chemische klassen, en directe constructie van de zeven transmembrane domeinen van de receptoren. Aangetoond werd dat beide enantiomeren op een vergelijkbare wijze aan de dopamine D2 receptor kunnen binden. De geprotoneerde tertiaire stikstofatomen en de benzamidegroepen van de liganden vormden hierbij specifieke interacties met bepaalde aminozuren van de receptor. Verschillen werden waargenomen in de interacties van de 5- methoxysubstituenten en een specifiek aminozuur in het vijfde transmembrane domein, hetgeen de verschillen in intrinsieke activiteit van de enantiomeren voor dit receptorsubtype zou kunnen verklaren. De wijze van binding aan de serotonine 5-HT1A receptor bood een verklaring voor de agonistische eigenschappen van beide enantiomeren, en ondersteunde de waarnemingen uit de SAFIRs dat de amide functionaliteit niet belangrijk is voor hoge affiniteit voor dit receptorsubtype.

186 ABBREVIATIONS

5-HIAA 5-hydroxyindoleacetic acid 5-HT 5-hydroxytryptamine, serotonin aCSF artificial cerebrospinal fluid cAMP cyclic adenosine monophosphate CNS central nervous system CSF cerebrospinal fluid DMF dimethylformamide DMSO dimethylsulfoxide DOPAC 3,4-dihydroxyphenylacetic acid DPAT 2-(N,N-di-n-propylamino)tetralin dppp 1,3-bis(diphenylphophino)propane EPS extrapyramidal side-effects GABA g-aminobutyric acid GPCR G-protein-coupled receptor HVA homovanillic acid MLR multiple linear regression NA noradrenaline PCA principal component analysis PET positron emission tomography PLS partial least-squares projections to latent structures PPI prepulse inhibition QSAR quantitative structure-activity relationship RT room temperature s.c. subcutaneously SAFIR structure-affinity relationship SAR structure-activity relationship TD tardive dyskinesias TM transmembrane domain triflate trifluoromethanesulfonate

187 AMINO ACID CODES AND STRUCTURES

NH

CH3 A Ala Alanine * R Arg Arginine N NH * H 2

NH N Asn Asparagine 2 OH * D Asp Aspartic acid * O O

O C Cys Cysteine * SH E Glu Glutamic acid * OH

O Q Gln Glutamine H * NH2 G Gly Glycine *

CH3 CH H His Histidine 3 * NH I Ile Isoleucine * N

CH L Leu Leucine 3 NH2 * K Lys * CH3

S M Met Methionine CH * 3 F Phe *

P Pro Proline * S Ser Serine * OH N *

CH3 T Thr Threonine * OH W Trp Tryptophan * N H CH3 Y Tyr Tyrosine V Val Valine * * CH3 OH

One-letter codes, three-letter codes, full names, and side chain structures of amino acids occurring in G-protein- coupled receptors. Side chains are shown in the neutral form. Atoms marked with * are part of the protein backbone.

188 PUBLICATIONS AND PRESENTATIONS

Drijfhout WJ, Homan EJ, Grol CJ and Westerink BHC. A new method in circadian studies: long- term in vivo microdialysis in the pineal gland. 2nd ULLA Summerschool, Uppsala, Sweden, July 1–8, 1995. Poster.

Drijfhout WJ, Homan EJ, Grol CJ and Westerink BHC. A new method in circadian studies: long- term in vivo microdialysis in the pineal gland. 10th Camerino-Noorwijkerhout Symposium ‘Perspectives in Receptor Research’, Camerino, Italy, September 10–14, 1995. Poster.

Drijfhout WJ, Homan EJ, Brons HF, Oakly NR, Skingle M, and Grol CJ (1996) Exogenous melatonin entrains rhythm and reduces amplitude of endogenous melatonin: An in vivo microdialysis study. J Pineal Res 20, 24–32.

Homan EJ, Copinga S, Mohell N, Johansson R, and Grol CJ. 2-Aminotetralin-derived Benzamides: Potential Atypical Antipsychotics. 26e Vrije Mededelingendag Sectie Farmacochemie, Amsterdam, The Netherlands, December 13, 1996. Oral presentation.

Homan EJ, Copinga S, Unelius L, Mohell N, Jackson D, Johansson R, and Grol CJ. 2-Aminotetralin- derived Benzamides: Potential Atypical Antipsychotics. 11th Noorwijkerhout-Camerino Symposium ‘Trends in Drug Research’, Noordwijkerhout, The Netherlands, May 11–15, 1997. Oral presentation.

Homan EJ, Copinga S, Unelius L, Mohell N, Jackson D, Johansson R, Wikström HV, and Grol CJ.

2-Aminotetralin-derived benzamides with mixed dopamine D2/D3 and serotonin 5-HT1A receptor binding profiles as potential atypical antipsychotics. 27th Annual Meeting of the Society for Neuroscience, New Orleans, LA, October 25–30, 1997. Poster.

Nilsson J, Homan EJ, Smilde A, Grol CJ and Wikström HV. A multiway 3D QSAR analysis of a series of (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-methoxybenzamides (1998) J Comput-Aided Mol Design 12, 81–93.

Homan EJ, Copinga S, Elfström L, Van der Veen T, Hallema JP, Mohell N, Unelius L, Johansson R, Wikström HV and Grol CJ. 2-Aminotetralin-derived substituted benzamides with mixed dopamine

D2, D3, and serotonin 5-HT1A receptor binding profiles: a novel class of potential atypical antipsychotics agents (1998) Bioorg Med Chem (in press).

Homan EJ, Selditz U, Grol CJ, Wikström H and de Zeeuw RA. Mechanistic studies of selected 2- amidotetralins on a Pirkle-type stationary phase. Submitted.

CURRICULUM VITAE

189

DANKWOORD

Na vijf jaar doctoraal en vijf jaar docotoraat zit het er op, met dit proefschrift komt er voor mij een eind aan tien jaar ‘rondlopen’ bij de afdeling Farmacie. In zo’n periode leer je veel mensen kennen. Met een aantal van hen werk je intensief samen in een soort wetenschappelijke symbiose, terwijl anderen indirect een bijdrage geleverd hebben aan het tot stand komen van dit proefschrift, soms wellicht zonder dat zij zich dat gerealiseerd hebben. Een aantal van deze mensen, die voor mij belangrijk zijn geweest, wil ik op deze plaats noemen. In de eerste plaats ben ik dank verschuldigd aan Håkan Wikström, mijn promotor. Håkan, ik besef dat het niet zo eenvoudig moet zijn geweest om de begeleiding van een promovendus op je te nemen, die ging werken aan een project dat al was opgestart voordat je naar Groningen kwam. Je hebt dat opgelost door mij een grote mate van vrijheid te geven tijdens het onderzoek. Niettemin stond je deur altijd open voor raad en daad. Ik ben je erkentelijk voor deze benadering, want zo leer je echt de kunst van het onderzoek doen. Je enthousiasme voor de farmacochemie, je brede belangstelling buiten het vakgebied, en je gevoel voor humor maken je tot een plezierig persoon om mee samen te werken. Håkan, bedankt voor het vertrouwen, en het allerbeste voor de toekomst. De dagelijkse begeleiding van mijn onderzoek was in handen van Cor Grol, mijn co-promotor. Cor, als geestelijk vader van dit onderzoeksproject heb je aangetoond dat het rationeel ontwerpen van nieuwe verbindingen nog steeds een goed alternatief kan zijn voor technieken als combinatorial chemistry en high-throughput screening. Door vast te houden aan het belang van de integratie van organische synthese, farmacocolgie en molecular modeling, ben je er wederom in geslaagd een echte all-round farmacochemicus af te leveren. Je manier van begeleiden heb ik als bijzonder prettig ervaren. De bijkans filosofische gesprekken over het werkingsmechanisme van verbindingen op moleculair niveau tijdens de vele model-sessies heb ik altijd weten te waarderen. Andere dingen die me bij zullen blijven zijn de voetbalanalyses, je slechte imitaties, voortreffelijke barbeques, zeevissen, tripjes naar Rome, Parijs en New Orleans. Cor, ik ben je dankbaar voor het vertrouwen dat je in me hebt gesteld, en voor de farmacochemische bagage die je me hebt meegegeven. Toen ik aan dit onderzoeksproject begon, was de weg reeds enigszins geplaveid door Swier Copinga, mijn voorganger. Swier was ook degene die mij enthousiast heeft gemaakt voor de farmacochemie tijdens de eerste gerichte keuzevakken. Swier, ik hoop dat je tevreden bent met de lotgevallen van de SC-stofjes, en wil je bedanken voor je inzet vanuit Zweden. I wish to express my gratitude to Nina Mohell, Lena Unelius, David Jackson, and Rolf Johansson from CNS Preclinical R & D, Astra Arcus AB, Södertälje, Sweden, for providing me with the accurate behavioural and receptor binding data implemented in chapters 2, 3, 5 and 6. Rita Huff, Mary Lajiness and Kjell Svensson from Pharmacia & Upjohn, Kalamazoo, MI, are thanked for producing the dopaminergic intrinsic efficacy data included in chapter 5. Martin Tulp van Solvay Pharmaceuticals verdient een bijzondere vermelding vanwege het belangeloos testen van de verbindingen beschreven in hoofdstuk 4. De QSAR-analyses in dat hoofdstuk zijn uitgevoerd door Jonas Nilsson. Jonas, tack sa mycket! Een belangrijke bijdrage aan hoofdstuk 6 is geleverd door Jan ‘Pomtiedom’ de Vries en Thomas Cremers. Door het verrichten van de benodigde stereotactische operaties en het optimaal tunen van de diverse microdialyse-opstellingen zorgden zij er voor dat ik

191 slechts de stofjes hoefde toe te dienen, de tubings te ontwarren, en de piekjes te meten. Heren, hartelijk dank voor jullie inzet, en Thomas, ik zet het bier alvast koud in de States! Een aantal studenten hebben in de vorm van een keuze- en/of bijvak een bijdrage geleverd aan dit onderzoeksproject. Lotta, Trees, Jan-Pieter, Esther, Monica, Marjolein, Tom en Martin, bedankt voor jullie inzet, enthousiasme en de gezellige tijd. Zonder de expertise van een aantal specialisten op het gebied van structuurkarakterisering begin je als synthetisch chemicus weinig. Margot Jeronimus-Stratingh wil ik in dit verband bedanken voor het meten van de vele massaspectra. Dank ook aan Wim Kruizinga en Jan Herrema van het CASS voor de strakke NMR-spectra. Jan verdient in dit verband een bijzondere vermelding vanwege het uitvoeren van de temperatuurexperimenten beschreven in hoofdstuk 3. De heren Ebels en Draaijer worden bedankt voor hun nauwkeurige element-analyses, al moest er van mijn kant wel eens wat water bij de wijn. The members of the assessment committee, prof.dr. Hacksell, prof.dr. Snyder, and prof.dr. Zaagsma are gratefully acknowledged for their willingness to fulfill their tasks and their constructive criticism with regard to the manuscript. Tijdens het werk op het lab was ik veroordeeld tot een aantal kamergenoten. Dit heb ik echter nooit als straf ervaren. Het was gezellig ‘hokken’ met Kees, Gert en Pieter aan het eind van de gang op de eerste verdieping van het UCF, en na de verhuizing met Pieter en Erik in de ‘skybox’ van het MWF-gebouw. De welbespraaktheid, het gezang, en de typische humor van Kees zorgden elke dag weer voor het nodige vermaak. Met Gert had ik een bijzondere band vanwege onze Drentse ‘roots’, hetgeen regelmatig tot uitdrukking kwam in de Drentse dialogen die we voerden. Van zijn encyclopedische kennis van de organische chemie en zijn synthetische vaardigheden heb ik als farmaceut enorm veel geleerd en geprofiteerd. Jongens, bedankt voor de fijne tijd, en succes met jullie onderneming! Pieter vormt een onmisbare schakel binnen de werkgroep, want hij zorgt er voor dat alle apparatuur in een puike conditie verkeert en dat er orde op de zalen heerst. Niet alleen van je praktische vaardigheden, maar ook van je passie voor vogels en motorrijden heb ik veel opgestoken, hoewel dit laaste geen onverdeeld succes was. Pieter, je was voor mij meer dan een collega. Ik dank je voor je vriendschap. Erik, ik denk dat Cor in jou een waardig opvolger voor mij heeft gevonden. De sfeer is in ieder geval goed! De werkgroep Farmacochemie is zonder twijfel één van de gezelligste binnen het lab. De gemoedelijke sfeer op de synthese- en microdialysezalen, de discussies, verhalen, roddels en grappen tijdens koffie- en lunchpauzes, de labdagjes, de oliebollenborrels, verjaardagsfeestjes, barbeques, kroppkakor en andere etentjes, biljarten, avondjes bios, en uiteraard het afzakkertje op vrijdagmiddag in de Toeter zal ik dan ook zeker gaan missen. Arjen, Astrid, Bas-Jan, Ben, Dennis, Durk, Eytan, Hiroshi, Janita, Janneke, Jonas, Marguerite, Mark, Nienke, Olga, Peter, Sander, Sandrine, Tjeerd, Ulrik, Ulrike, Wia, Yi, en Yuki: bedankt voor deze fantastische periode! Dank ook aan de ‘men in black’ Henk-Frans en Ruud, paranimfen van het eerste uur, voor hun bereidheid deze taak op zich te willen nemen. Jongens, we maken er een dagje van! Tot slot wil ik mijn ouders Rika en Willem en mijn broer Otte danken voor hun onvoorwaardelijke steun en vertrouwen in tijden van voor- en tegenspoed, en het scheppen van de ideale condities vanaf mijn vroegste jeugd om dit resultaat te bereiken. Ik zie jullie in de States!

192