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The medicinal chemistry of arylpiperazines with potential efficacy Mensonides, Marguerite

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RIJKSUNIVERSITEIT GRONINGEN

The Medicinal Chemistry of Arylpiperazines with Potential Antidepressant Efficacy

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 5 januari 2001 om 16.00 uur

door

MARGUÉRITE MASCHA MENSONIDES-HARSEMA

geboren op 24 april 1968 te Harlingen

Promotor Prof. Dr. H.V. Wikström

Promotiecommissie Prof. Dr. C. Kruse Prof. Dr. V. Pike Prof. Dr. J. Zaagsma

Paranimfen Yuki Kawahara Fiona Westerhof

Colophon

Copyright © by M.M. Mensonides-Harsema. 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/m.m.mensonides

ISBN 90-367-1282-3 NUGI 746

Cover design: Tom Hightower: ‘Depression’. Printing: KFS, Lund, Sweden.

The research project described in this thesis was performed within the framework of the research school GUIDE, and was financially supported by Merck KGaA, Darmstadt, Germany. The printing of this thesis was financially supported by MPT Consultancy, Bjärred, Sweden, Merck KGaA, Darmstadt, Germany and AstraZeneca R&D Lund, Sweden, Research school GUIDE, Groningen, the Netherlands Rijksuniversiteit Groningen, Groningen, the Netherlands.

voor Poeske en Sammie

TABLE OF CONTENTS

1 INTRODUCTION 7

2 STRUCTURAL ANALOGUES OF THE 5-HT1A ANTAGONIST WAY100635: 69 SYNTHESIS AND IN VITRO AND IN VIVO FUNCTIONAL STUDIES

3 IN VIVO PHARMACOLOGICAL EVALUATION OF P-SUBSTITUTED 95 ANALOGUES OF WAY199635 AT PRE- AND POST-

SYNAPTICE 5-HT1A RECEPTORS

4 STRUCTURAL ANALOGUES OF THE 5-HT1B/1D ANTAGONIST GR127935 107 AND THE 5-HT1B INVERSE SB224289: SYNTHESIS AND IN VITRO AND IN VIVO FUNCTIONAL STUDIES

5 6-METHOXYMIANSERIN AND STRUCTURAL ANALOGUES: SYNTHESIS 131 AND IN VITRO AND IN VIVO PHARMACOLOGICAL EVALUATION

6 RESOLUTION OF 6-METHOXYMIANSERIN AND IN VITRO AND IN VIVO 151 PHARMACOLOGICAL EVALUATION OF THE ENANTIOMERS

SUMMERY 165

SAMENVATTING 169

LIST OF PUBLICATIONS AND ABSTRACTS 175

DANKWOORD 177

1 INTRODUCTION

1.1 Depression 7 1.1.1 The Archeology of Mood Disorders 7 1.1.2 The Discovery of the First Generation of 9 1.1.3 Antidepressants and Mono-amine Oxidase Inhibitors 11 1.2 Neurochemical Hypothesis of Depression 12 1.2.1 The Catechol-amine Hypothesis 12 1.2.2 The Hypothesis 14 1.2.3 Involvement of Other Neurochemical Systems 15 1.3 The Second Generation of Antidepressants 16 1.3.1 The Selective Serotonin Reuptake Inhibitors 16 1.3.2 Atypical Antidepressants 18 1.4 Serotonin Receptors in the Brain 20 1.4.1 G-Protein Coupled Receptors 20 1.4.2 The System 23 1.4.3 Serotonin Receptor Subtype Classification 26 1.5 Animal Models in Antidepressant Research 33 1.5.1 Acute Models for Depression 33 1.5.2 Chronic Models for Depression 34 1.6 Development of a Third Generation of Antidepressants; 37 Future Pharmacological Approaches 1.6.1 Reuptake Inhibition Combined with 5-HT Autoreceptor Blockade 38 1.6.2 Other Strategies 40 1.7 Scope of the Thesis 43 1.8 References 45

1.1 DEPRESSION

1.1.1 THE ARCHEOLOGY OF MOOD DISORDERS

The terms mania and melancholia were originally introduced by the Greeks. The modern meaning of these terms, however, only goes back one hundred years.1 Until early 1900, ca. 50% of the diagnoses made were for mania. Since World War I, in contrast, the diagnosis of mania has become a part of the diagnosis of manic- depression. Today no more than 1-2% of psychiatric patients are diagnosed with mania. Many terms used in psychiatry, including the terms neurosis, psychosis, mania and melancholia, changed in meaning at the turn of the 20th century. The diagnosis of depression did not even exist before 1900. Before this term was introduced, any state

7 that would lead to under-activity, was diagnosed as melancholia. Conditions that are now recognized as negative , obsessive-compulsive disorder, social phobia, panic disorder and major depression would all attract the diagnosis of melancholia. In 1919, the German psychiatrist Kraepelin introduced the term ‘manic-depressive insanity’.2 In contrast to schizophrenia, manic-depressive disorders showed a remitting course and the symptoms he described were very close to what would now be diagnosed as a major depressive disorder or endogenous or vital depression. The disorder was characterized by apathy, loss of energy, retardation of thinking and activity, as well as profound feelings of gloominess, despair and suicidal ideation. In addition, Kraepelin pointed to vegetative symptoms, such as poor sleep and loss of appetite. The development, in the 1950s, of the and the antidepressants vindicated Kraepelins’ point of view. Early 1960s, operational definitions to distinguish between endogenous and reactive (or neurotic) depression were proposed.3 Endogenous depression was characterized by vegetative disturbances such as early morning wakening, loss of appetite, and diurnal variation of mood as well as retardation of thinking and feeling. Accordingly it was only appropriately treated pharmacologically or with electric convulsive-shock treatment (ECT). Neurotic, or reactive depression in contrast stemmed from adversity and could probably be managed psychotherapeutically in the main. This formulation fitted nicely with the amine theories of depression that emerged in the mid 1960s and with the fact that the first antidepressant was an amine reuptake inhibitor. According to the American Psychiatric Association, major depression is a syndrome consisting of affective, cognitive, motor and somatic signs and symptoms. Symptoms should be prominent and persistent and represent a change from previous function. For clinical depression to be diagnosed, a patient will exhibit either depressed mood or a loss of interest or pleasure in activities (adhedonia), or both. Several other signs and symptoms, including decreased appetite, , fatigue, feelings of worthlessness or guilt, diminished ability to think or concentrate, or recurrent thoughts of death or suicide, also need to be present.4, 5 Patients with mania, by contrast, exhibit a triad of euphoric mood, pressured speech, and psychomotor agitation. During the period of mania, signs and symptoms such as inflated self-esteem, decreased need for sleep, pressure to keep talking, psychomotor agitation, distractibility and flight-of-ideas are evident. To treat the manic phase in manic-depression, lithium is generally used. Lithium, an alkali metal, was first isolated in 1817 from stone. Suggestions of the beneficial effects of spring waters with a high lithium content, in the treatment of mania, can be cited as a forerunner of modern lithium use. In 1949, lithium was tried on a number of manic, depressed and schizophrenic patients. The manic patients responded to the treatment.6, 7 In 1952, the usefulness of lithium, using a placebo and double-blind techniques and a specially devised rating scale, was explored. This study was one of the very first double-blind randomized placebo-controlled trial in psychiatry.8

8 Besides lithium carbonate (Eskalith/Camcolith), (1.1, Tegretol) and sodium valproate/valproic acid (1.2, Depakene/Epilim) are currently marketed to treat bipolar disorders or prophylaxis of recurrent disorders.

1.1.2 THE DISCOVERY OF THE FIRST GENERATION OF ANTIDEPRESSANTS

In 1952, the first drug, (1.3, Thorazine®/Largactil®), was discovered.9 It was the critical event in the foundation of psychopharmacology. The history of the synthesis of chlorpromazine goes back to the development of coal- tar chemistry. In 1883, the first compound had been synthesized. Sixty years later, the were linked to histamine (1.4) and chlorpromazine was developed from this parent compound. The development of (1.5, Tofranil®) also came from an interest in . A series of forty-two related compounds, using iminodibenzyl (1.6) as the lead, was put together.10, 11 In the first clinical study of imipramine, it was examined for its antipsychotic qualities and compared to chlorpromazine. In 1955, a second study was set up, to look specifically at the effects of imipramine in patients who suffered from depression. The responses were so dramatic that there was little doubt that the treatment was effective.12, 13 The patients selected for this study, were all diagnosed with endogenous or vital depression. Treatment produced an increase in vivacity and a restoration of interest in activities in general and in social interaction in particular. Sleep was restored and would feel normal and refreshing, unlike the sleep that followed the then available . Appetite was stimulated. Although improvement might be apparent after two to three days, it was claimed that it could take up to four weeks for the clinical effect to become established. All the side effects now associated with tricyclic use – dry mouth, a tendency to sweat more profusely, some constipation, possible drops in and possible confusional conditions in patients with other brain disorders - were described. As a result, a dose range, that remains the same today, was put forward. In 1937, the enzyme monoamine oxidase (MAO) was discovered, which is responsible for the metabolism of (1.7), noradrenaline (1.8, NA), (1.9, DA) and 5-hydroxytrypamine (1.10, 5-HT or serotonin). In 1912, isoniazid (1.11) was synthesized. This compound was resynthesized in 1951, when a large stock of hydrazine that was left over from World War II became available to the different chemical companies.14 Using isoniazid as the starter, iproniazid was synthesized. Both compounds were marketed as tuberculostatics.15 In 1952, it was discovered that iproniazid, but not isoniazid, inhibited MAO and was therefore named a monoamine oxidase inhibitor (MAOI).16 In 1957, the MAO inhibitor iproniazid (1.12), whose development helped to create the antidepressant market, was introduced. The synthesis and development of iproniazid was triggered by several events.17-21

9 S N O N N Cl N OH 1.1 1.2 1.3 1.4 NH2 O NH2 N

OH OH

HO NH HO NH2 N N 1.5 HO 1.7HO 1.8 CH2 1.6 N O NH O HO 2 NH NH HO NH2 N HN N NH2 1.9N 1.10 1.11 1.12 HO H

Chart 1.1 Chemical structure of carbamazepine (1.1), valproic acid (1.2), chlorpromazine (1.3), histamine (1.4), imipramine (1.5), iminodibenzyl (1.6), adrenaline (1.7), noradrenaline (1.8), dopamine (1.9) serotonin (1.10), isoniazid (1.11) and iproniazid (1.12).

In 1953, (1.13) was isolated from Rauwolfia serpentina, a plant root used in India for the treatment of , snakebite and insanity.22, 23 Although reserpine was not successful as a drug, it immediately led to several biochemical and psychological hypotheses. The effects of reserpine were correlated to the lowering of brain 5-HT. This was the first bridge between neurochemistry and behavior. Thus, the biochemical psychopharmacology was established.24 Observations of the effects of reserpine on the release of catecholamines further extended this bridgehead in two important ways.25 It stimulated the debate between the 5-HT and the catecholamine camp and provided the pharmaceutical industry with a principle to guide drug development. Drugs could either be designed to produce similar depletions of 5-HT or catecholamines or, alternatively, they could be designed to modify or block the reserpine effect. Hence, drug development became systematic. In 1958, Merck approached several psychiatrists to investigate (1.14, Elavil®) for possible antischizophrenic properties. In the field, it was suggested that amitriptyline should also be investigated for antidepressant properties, since the molecular structure was so close to that of imipramine. Clinical studies revealed that amitriptyline was effective in much the same dose range as imipramine. It had a very similar profile of side effects and, like imipramine, took several weeks before the therapeutic effects would appear.26, 27 Amitriptyline was launched in 1961. Its discovery led to the final acceptance of imipramine as an antidepressant drug.

10 1.1.3 TRICYCLIC ANTIDEPRESSANTS AND MONO-AMINE OXIDASE INHIBITORS

In the 1960s, the Hamilton rating scale, that has become the standard for the assessment of depression and antidepressant effects, was introduced.28 Several compounds structurally related to imipramine have been developed - using this scale to ensure a basic level of quality of their clinical actions - and are currently marketed for the treatment of depression. They each have a three (tri)-joined ring (cyclic) structure with a side chain containing a tertiary or secondary amine attached to the central ring. The acronym used for the tricyclic antidepressants is TCA. Tertiary amine-containing TCAs include imipramine, amitriptyline, (1.15), doxepine (1.16) and (1.17). Secondary amine-containing TCAs are (1.18) and (1.19). Desipramine and nortriptyline are actually the desmethyl metabolites of imipramine and amitriptyline.29, 30 The most prominent action of TCAs is the blockade of reuptake of either NA or 5-HT from the synapse back into the nerve terminal, without blocking the reuptake of DA. The secondary amine-containing TCAs are 25- to 500-fold more potent in inhibiting NA than 5-HT reuptake as compared to tertiary amine-containing TCAs, which are only 3- to 5-fold more potent in inhibiting NA than 5-HT reuptake.

OMe X N H O OMe N H O H H OMe MeO MeO OMe 1.13 1.14 R =CH ; X=CH O N 1 3 2 1.16 R1=CH3; X=O R1 1.19 R1=H; X=CH2

NH H 2 N N NH2 R1 NH2 R2 1.20 1.21 1.22

N O

R3 N 1.15 H R1=H; R2=R3=CH3 N 1.17 R1=Cl; R2=H; R3=CH3 Cl 1.23 1.18 R1=R2=R3=H O

Chart 1.2 Chemical structure of reserpine (13), the TCAs amitriptyline (1.14), trimipramine (1.15), doxepine (1.16), clomipramine (1.17), desipramine (1.18) and nortriptyline (1.19) and of phenelzine (1.20), (1.21), (1.22) and (1.23).

11 Although the MAOI iproniazid was effective in the treatment of depression, it is no longer used clinically because of its hepatic toxicity. Two of the MAOIs currently marketed for the treatment of depression are phenelzine (1.20), a derivative of hydrazine, and tranylcypromine (1.21), which is structurally related to amphetamine (1.22). The strongest disadvantage of the MAOIs is their interaction with containing foods, like old cheese and wine, through the inhibition of the iso-enzyme MAO-B. This lead to the development of reversible monoamine oxidase A inhibitors (RIMAs), e.g. moclobemide (1.23). The advantage of the RIMAs is the absence of this so called ‘cheese-effect’, since tyramine can still be metabolized. It should be noted that chlorpromazine, imipramine and iproniazid were discovered by sensitive observations of their effects on patients. This method of discovery is very different from the means by which the antidepressant credentials of later antidepressants were established. Today, candidate drugs have to go through extensive in vitro (affinity studies for different receptor subtypes and effects on second messenger systems) and in vivo pharmacology, toxicology and metabolism/kinetic studies before they are even admitted into a clinical trial.

1.2 NEUROCHEMICAL HYPOTHESES OF DEPRESSION

1.2.1 THE CATECHOL-AMINE HYPOTHESIS

Julius Axelrod was the first to demonstrate the presence of a catecholamine and 5-HT uptake system and that these systems were inhibited by imipramine. He was awarded the Nobel Prize for his findings in 1970.31-33 The demonstration that imipramine inhibited catecholamine reuptake, came several years before it was shown that the TCAs inhibited 5-HT reuptake as well. This lead to the catecholamine hypothesis of depression which was put forward in 1965 by Schildkraut in the American Journal of Psychiatry.34 The argument was that as reserpine was known to deplete catecholamines and as it had been reported that reserpine could lead to depression and in some cases make people suicidal, there were strong suggestions that the low levels of catecholamines might be associated with the generation of depressive states. Because TCAs inhibited catecholamine reuptake, it was suggested that they increase catecholamine levels in the synaptic cleft. This would subsequently lead to a functional increase in the levels of these neurotransmitters. MAOIs, by blocking the metabolism, also led to an increase in catecholamine levels. The conclusion was that both major groups of drugs acted as antidepressants through this increase catecholamine levels. A similar argument could be, and indeed was later, made for 5-HT.35, 36 By then, however, it had been discovered that both imipramine and amitriptyline were metabolized to desipramine and nortriptyline. Both metabolites are much more potent inhibitors of the catecholamine uptake than of the 5-HT reuptake, strongly suggesting that the catecholamines were the more important neurotransmitters.

12 There were, however, several weak points in the catecholamine hypothesis. The only prospective study where reserpine was given to patients and the level of depression and suicidal tendency were monitored, showed that reserpine acts as an antidepressant and anxiolytic.37 This was in full contrast with reports of people committing suicide after being treated with reserpine for hypertension. Also, on the basis of the catecholamine hypothesis, one would expect that antidepressants would be unhelpful in the treatment of mania. However, both ECT and lithium (and more controversially the ) were used to treat both depression and mania.39 Schildkraut characterized his hypothesis in his 1965 article as “at best a reductionist oversimplification” that might be “of heuristic value” that “may soon require revision due to rapid progress in the basic sciences.” Despite this view and all the drawbacks, the catecholamine hypothesis persisted for more than two decades. Why did the catecholamine hypothesis hold ground despite conflicting findings? For a start, it gave the pharmaceutical industry a clear goal to aim at. Second, during the 1970s attention had switched from levels of NA and its precursors and metabolites to a focus on receptors. The introduction of receptors into the argument about the mechanism of action of antidepressants offered an answer to the fact that some of the newer antidepressants neither blocked reuptake or inhibit MAO. The classical statements of receptor theory were formulated around 1932 by Alfred Clark, who worked out the principles whereby drugs could be expected to bind to a receptor and the conditions under which there could be competition at the receptor site.41

k-1 L + R LR k 1 Figure 1.1 Schematic representation of the reversible binding of a ligand to a receptor;

L = free ligand; R = unoccupied receptor; LR = ligand-receptor complex; k1 and k-1 are the association and dissociation rate constants.

The first steps towards proof of this theory came in the early 1970s with the use of radio-labeled hormones, such as , and radio-labeled drugs, in particular those which bound to catecholamine receptors.42 Using radio-labeled drugs, it was possible to demonstrate binding to particular receptors and to estimate both the amount of receptors in a particular piece of membrane and the affinity of a particular drug. The estimates derived were in keeping with the predictions of classical receptor theory. Radio-labeled binding studies also led to the discovery of further drugs and from there it has been possible to isolate the receptor proteins and clone them. The antidepressants were found to down-regulate β- receptors.43 Even the ones that didn’t block catecholamine reuptake acted this way. This down-regulation took up to two weeks to appear, which appeared consistent with the delay there is between taking an antidepressant and onset of clinical action.

13 In 1976, in the same year that the dopamine hypothesis of schizophrenia was put forward, it was proposed that down-regulation of β-adrenoceptors was precisely the mechanism of action of antidepressants.44 The appeal of receptors came from the fact that, from the start, they had been portrayed in terms of ‘keys fitting locks’, terminology which was in line with ‘the magic bullet of therapeutic specificity’. During the 1970s the major psychiatric disorders became defined as disorders of single neurotransmitter systems and their receptors, with depression being a catecholamine disorder, anxiety a serotonergic disorder, dementia a disorder and schizophrenia a dopamine disorder. Thus the search had begun for receptor(subtype)-specific and selective compounds. Later it was found that an intact 5-HT system is necessary for adrenergic down- regulation to occur.45, 46 Until today, it has not been possible to set up a decisive experiment that would either confirm or deny the catecholamine hypothesis. There is no conclusive evidence of the presence of supersensitive -adrenoceptors in depressed patients, which would have fitted with the proposed action for antidepressants. It also has never been disproved as an antidepressant principle. Generally, findings of studies of the monoamine systems of depressed patients are explained with an appeal to either the heterogeneity of depression and/or a statement that ‘the findings are empirically robust but from a theoretical standpoint difficult to explain’.40

1.2.2 THE SEROTONIN HYPOTHESIS

The catecholamine theory began to fade with the introduction of the first selective serotonin reuptake inhibitors in the early 1980s. Since that time, scientific discussion on the mechanisms and backgrounds of depression has been dominated by the serotonin hypothesis. In 1967, a 5-HT version of the Schildkrauts’ catecholamine hypothesis was proposed.47 This hypothesis was based on the findings that adding , a 5-HT precursor, to a MAOI, boosted its antidepressant effect. It was also found that the concentration of the 5-HT metabolite, 5-hydroxyindoline acetic acid (5-HIAA) was apparently low in the cerebrospinal fluid of depressed patients – the implication was that there was an abnormality of 5-HT turnover in depression.48, 49 Furthermore, it was argued that, if imipramine and amitriptyline were only pro-drugs of desipramine and nortriptyline, they should have been associated with a grater delay in onset of action and possibly more side effects.50 The only dissociation between the tertiary and the secondary amine-containing TCAs was the impression that desipramine restored drive more clearly and that imipramine had grater mood-elevating properties. In the early 1970s, it was discovered that if a preferentially noradrenaline reuptake inhibitor was halogenated, it became a 5-HT reuptake inhibitor. Halogenating brompheniramine led to the production of zimeldine (1.24), the first specific 5-HT

14 reuptake inhibitor, which was marketed in Europe in 1982 as Zelmid®.51 Before it was marketed in the USA, however, it was associated with cases of Guillain-Barré syndrome and withdrawn. In the 1970s, the antidepressant market was still very small and despite the specific development of psychotropic drugs, psychiatry was still not part of real medicine. In 1972, (Prozac®, 1.25) was first synthesized.52 Although it was clearly a 5-HT reuptake inhibitor, the suggestion that it might be considered for the treatment of depression was not immediately appreciated.53, 54 It was only in the early 1980s, after the antidepressant credentials of zimeldine had become apparent and the perception of the size of the antidepressant market began to change, that the development of fluoxetine as an antidepressant was continued.

1.2.3 INVOLVEMENT OF OTHER NEUROCHEMICAL SYSTEMS

Since the implication that elevation of adrenergic and later serotonergic levels is of vital importance in the treatment with antidepressants, several other neurochemical systems have been suggested to play a role in the pathogenesis and therapy of depression. Thus, calcium channel antagonists have shown promise in both animal experiments and clinical trials as potential therapeutics in the treatment of mood disorders.55-57 Intracellular calcium plays a crucial role in multiple neuronal processes involved in such essential brain functions as excitability, re- and degeneration, and the synthesis, release and reuptake of neurotransmitters/neuropeptides. In addition, several drugs that modify mood, as well as ECT, have been shown to modify cellular 58-66 calcium trafficking. Another potential target is the N-methyl-D-aspartate (NMDA) receptor. Recent studies have shown, that NMDA receptor antagonists modulate both catecholamine and serotonin neurotransmission and antagonize stress- induced increases in dopamine metabolism.67-75 Neuropeptide Y (NPY), an endogenous ligand for the σ-receptor, may also play a role in the mechanism of action of antidepressant drugs and the pathology of mood disorders. For example, when rats are chronically treated with antidepressants or subjected to repeated ECT, there is an increase in NPY immune reactivity in several brain regions.76-78 Conversely, NPY immuno reactivity is decreased in the brains of rats that have been subjected to bilateral olfactory bulbectomy, a procedure that has been used in the development of a rodent model of depression.79 It has also been shown that NPY can modulate the release of NA and 5-HT.80, 81

15 1.3 THE SECOND GENERATION OF ANTIDEPRESSANTS

1.3.1 THE SELECTIVE SEROTONIN REUPTAKE INHIBITORS

Today, several 5-HT reuptake inhibitors are used in the treatment of depression and other, related diseases like obsessive compulsive behaviors (OCD), social phobia, panic disorders and anorexic/bulimic nervosa. Fluvoxamine (1.26, Luvox®/Faverin®) was first marketed in 1983, citalopram (1.27, Cipramil®) in 1986, fluoxetine (Prozac®) in 1987, sertraline (1.28, Zoloft®/Lustral®) in 1990 and paroxetine (1.29, Paxil®/Seroxat®), which had first been developed in the 1970s, in 1991. In an effort to distinguish paroxetine from the other marketed antidepressants, it was referred to as a selective serotonin reuptake inhibitor (SSRI). The acronym SSRI was quickly adopted as a term to refer to all 5-HT reuptake inhibitors, and has since then been used in the distinguishing of venlafaxine (1.30, Efexor), which was introduced in 1993, a SNRI (serotonin and noradrenaline reuptake inhibitor) and (1.31, Edronax), which was launched in 1997, a SNaRI (selective noradrenaline reuptake inhibitor).

Figure 1.2 Putative structure of the rat serotonin transporter, showing possible phosphorylation (P) sites and glycolisation sites (on the large second extracellular loop).5

Compared to the tricyclic antidepressants, which were for the most part very similar molecules, the 5-HT reuptake inhibitors are structurally quite different and their range of actions across a number of receptor systems is also somewhat dissimilar. Although they all inhibit 5-HT reuptake and are termed SSRI by virtue of the fact that the parent molecules have little effect on catecholamine reuptake, they are not specific to the serotonergic system or 5-HT reuptake blockade. They also differ in potencies with which they block 5-HT reuptake. Fluvoxamine is the most potent 5-HT reuptake inhibitor of the currently marketed compounds, while citalopram is the most specific (see Figure 1.3).82 The variation across compounds indicates that 5-HT reuptake

16 inhibition might not be sufficient for antidepressant efficacy. Hence, depression might not be a disorder of one neurotransmitter or a particular receptor subtype, but rather a number of physiological systems which are compromised or shut down or desynchronized in some way.

4000 NA/5-HT 10 IC50 5-HT ratio

3000

2000 5

1000

0 0 1.25 1.26 1.27 1.28 1.29

Figure 1.3 Bar diagram showing the affinity of 1.25 – 1.29 for the 5-HT reuptake site (black bars; right y-axis) and their selectivity, as measured by the ratio between the affinities of the 5-HT and NA transporter (gray bars, left y-axis). Redrawn from ref. 82.

Br CH3 CF3 HN O H CH3 CH N 3 O N N CF3 O 1.26 CH 1.24 3 1.25

NH2 Cl H C F N 3 N O Cl O

CH 3 O O N 1.28 CH F 1.27 3 1.29

N OH H CH3 N

CH3 O O 1.30 O N 1.31 O H CH3

Chart 1.3 Chemical structure of the SSRIs zimeldine (1.24), fluoxetine (1.25), fluvoxamine (1.26), citalopram (1.27), sertraline (1.28) and paroxetine (1.29) and of the SNRI venlafaxine (1.30) and the SNaRI reboxetine (1.31).

17

1.3.2 ATYPICAL ANTIDEPRESSANTS

The tetracyclic azepin (1.32, Tolvon®), was introduced as an antidepressant by Organon in Europe in the 1970s. Its structure and in vitro profile were significantly different from the existing TCAs and MAOIs. Mianserin is devoid of or cardiovascular side effects that are associated with the first generation of antidepressants. The most common side effect associated with the use of mianserin is sleepiness, due to its high affinity for the histaminergic receptors. It appears significantly safer in overdose than the older drugs, which has led to its widespread use. In the mid 1980s, it was one of the two most commonly prescribed antidepressants in Britain. Despite its low affinity for the NA uptake site, the antidepressant action of mianserin is explained by a combination of inhibition of NA 83 reuptake and α2 adrenoceptor blockade. The successor of mianserin, (1.33, Remeron®) was introduced in 1994. It trades heavily on the supposed interactions between the serotonergic and noradrenergic system (Figure 1.4).84-88

Figure 1.4 Mechanisms for noradrenergic control of 5-HT release. Activation of somatodendritic α2 adrenergic autoreceptors decreases noradrenergic neuronal activity, and activation of terminal α2 adrenergic autoreceptors decreases release of NA. Noradrenergic terminals innervate 5-HT cell bodies, where stimulation of postsynaptic α1 adrenergic receptors serotonergic neuronal activity, increasing 5-HT release. Activation of α2 adrenergic heteroreceptors on 5-HT terminals inhibits 5-HT release. Redrawn from ref. 5.

18

Its antidepressant actions are attributed to the fact that it is an α2 adrenoceptor selective antagonist with no apparent affinity for the α1 adrenoceptor. In vivo, mirtazapine is shown to elevate both NA levels - supposedly through blockade of the

α2 autoreceptors located on the NA cell bodies in the locus coereleus - and 5-HT levels - supposedly through blockade of the α2 heteroreceptors located on the 5-HT nerve terminals. In the course of investigating clomipramine, it was discovered that this antidepressant had an effect on sexual (dis)functioning.89-92 The SSRIs also have effects on sexual functioning, affecting well over 50% of users. However, rather than direct development for the indication of anorgasmia, impotence and loss of libido, companies have waited for off-license prescribing by clinicians of e.g. clomipramine to lead to market development. In vivo experiments with SSRIs monitoring sexual functioning in animals strongly suggests that drugs, such as (1.34) - active on the 5-HT1A receptor - and (1.35) - which blocks the 5-HT2A receptor - should be reliable as .93 Trazodone even developed a reputation in this respect. Following its pre-Prozac launch in the United States as an antidepressant, its sales were substantial, partly because of its effects on sexual functioning.

O N H N N X H H N N (CH2)4 N N N H O 1.34 CH3O N OH 1.32 1.36 O X = CH CH3 1.33 X = N O CH3 Cl N O O HN N N N (CH ) N N S N 2 3 N N N O 1.35 H3C O CH3 CH3 1.37 CH3

O N CH CH N N 3 3 CH3 (CH2)3 Cl N CH O N H 3 1.38 Cl O 1.39

Chart 1.5 Chemical structure of mianserin (1.32), mirtazapine (1.33), buspirone (1.34), trazodone (1.35), (1.36), viagra (1.37), (1.38) and buproprion (1.39).

19 Despite the clinical evidence that the very similar compound yohimbine (1.36) has reliably aphrodisiac properties this area has been downplayed by the pharmaceutical industry right up to the introduction of viagra (1.37). For the post-SSRI compounds like nefazodone (1.38), moclobemide and buproprion (1.39) the possible aphrodisiac properties are now highlighted and this marketing pitch is likely to increase awareness of the potential to prescribe for premature ejaculation and anorgasmia, even though none of the companies may ever seek a license for these indications. The antidepressant actions of trazodone are explained by the fact that it inhibits reuptake of both NA and 5-HT. The inhibition of 5-HT reuptake is stronger than of NA. Trazodone, induces less anticholinergic and antihistaminergic side effects than the classical TCAs, although it is known to induce sleepiness. Nefazodone, like trazodone, is a 5-HT2A with weak affinity for the NA and 5-HT reuptake sites and is also known to be sedative. However, nefazodone causes less orthostatic than trazodone. Buproprion is relatively unique in that it more potently blocks the reuptake of DA than NA or 5-HT. In spite of this, it inhibits the firing of noradrenergic cells in the locus coeruleus more potently than that of cells in the midbrain.

1.4 SEROTONIN RECEPTORS IN THE BRAIN

Of the many brain neuronal systems, the serotonin system is currently the most common neurobiological target for antidepressant treatments. TCAs act on both serotonergic and noradrenergic neurons by inhibiting transmitter uptake, MAOIs increase serotonergic and noradrenergic transmission by preventing their metabolism and SSRIs selectively inhibit the 5-HT transporter thereby increasing the synaptic concentration of 5-HT. SSRIs display little affinity for aminergic receptors and lack the severe side effects associated with the first generation antidepressants. The clinical actions of SSRIs can be attributed to an enhanced activation of one or several postsynaptic 5-HT receptors. It has been postulated that the slow onset of clinical action following treatment with the current available antidepressants may be partly ascribed to the inhibition of 5-HT release by forebrain serotonergic nerve terminals after the administration of antidepressants. This negative feedback, which involves the activation of 5-HT autoreceptors, is also believed to be responsible for the attenuation of cell firing of 5-HT neurons.

1.4.1 G-PROTEIN COUPLED RECEPTORS

With the exception of the serotonin 5-HT3 receptor, which is a ligand gated ion channel, all serotonin receptor subtypes belong to the super family of G-protein coupled receptors (GPCRs). Signal transduction through GPCRs requires three membrane-bound components. First, a cell-surface receptor that determines to which signal the cell can respond. Second, a G protein on the intracellular side of the

20 membrane that is stimulated by the activated receptor. And third, either an effector enzyme that changes the level of a second messenger or an effector channel that changes ionic fluxes in the cell in response to the activated G protein (see Figure 1.5).

Neurotransmitter messenger) (1st Receptor G-protein Effector 2nd messenger mediator

Adenylate 5-HT 5-HT G cAMP PKA 7 s cyclase ATP

PIP2

ACh muscarinic Gp PLC DAG PKC +

IP3

CaM kinase + 2+ Ca other targets

Figure 1.5 Overview of G-protein signaling to protein kinases. PLC, phospholipase C; PIP2 phosphatidylinositol biphosphate; DAG, diacylglycerol; CaM, Ca2+-calmodulin-dependent;

IP3, inositol 1,4,5-triphosphate, PKA and PKC, protein kinase A and C.

There are three main families of GPCRs in mammals: the rhodopsin-like family, the metabotropic glutamate and chemosensor family and the glucagon/VIP/calcitonin family. As many as 1000 receptors for monoamines, chemokines, odorants, neuropeptides and lipid messengers couple to one or more of the 20 different G proteins. In 1983, the first G-protein coupled molecule, rhodopsin - a light sensing molecule which binds the chromophore retinal – was cloned. The most striking structural features of this photo-receptor were the seven hydrophobic segments (see Figure 1.6). These hydrophobic regions are believed to form seven transmembral (TM) α-helices, like the seven TM helices of the proton pump bacteriorhodopsin. The GPCRs possess a substantial degree of homology in their amino acid sequences, especially in the TM regions. The seven hydrophobic stretches that span the membrane are connected by loops ranging in length from 5 to 420 amino acid residues. The amino(N)-terminal region of the protein is located extracellularly, while the carboxyl(C)-terminal is located intracellularly. The second and third intracellular loop and the cytoplasmic C- terminus are involved in the coupling to the G protein. G-proteins are turmeric structures composed of two functional units: an α subunit that catalyzes the GTPase activity and a βγ dimer that tightly interacts with the α subunit when bound to GDP. G proteins are held in an inactive state because of very high affinity binding of GDP to

21 their α subunits. Coupling of the endogenous ligand to the active site of the 7TM- receptor will induce conformational changes in the receptor molecule. These conformational changes will trigger the dissociation of GDP. This permits GTP to bind to and activate the α subunit, which then dissociates from the βγ dimer. The GTP-α complex can interact with e.g. adenylate cyclase, which then catalyzes the synthesis of the second messenger cyclic AMP (cAMP) from ATP. The signaling is terminated when the α subunit hydrolyzes its bound GTP to GDP. The hydrolysis permits the GDP-α complex to dissociate from its effector and associate again with the βγ dimer.5, 94-99

N extracellular II IV VI β I III V VII α γ intracellular

C

Figure 1.6 G-protein mediated signal transduction across the cellular membrane. Small signal molecules, like serotonin, are bound in the cavity formed by the seven trans membrane helices of the GPCR. The signal is transmitted via cytosolic loops of the GPCR to the G protein complex. Redrawn from ref. 5.

Table 1.1 Some chemical messengers that act through the Rhodopsin family GPCRs.

1st Messenger Light/Odorants Retinal/ Multiple substances Ions/Proteases Ca2+/thrombin Amino acids Glutamate, GABA Mono amines 5-HT, adrenaline, NA, DA, , ACh, histamine Lipid messengers Prostaglandins, thromboxane, platelet activating factor, leukotrienes, Purines Adenosine, ATP Neuropeptides Substance P, gastrin releasing polypeptide, NPY, enkephalins Hormones Angiotensin, ACTH (peptides); LH/FSH, TSH (glycoprotein) Chemokines IL-8, formyl-Met-Phe-Leu, Complement C5a

22 1.4.2 THE SEROTONERGIC SYSTEM

It was already known in the 19th century, that blood serum contained a substance with vasoconstritive properties. In 1948, Rapport, Green and Page isolated this compound and named it serotonin.100 A year later, they were able to identify the compound as 5- hydroxytryptamine (5-HT).101 In 1953, the presence of 5-HT in the brain was demonstrated and shortly afterwards it was recognized as a neurotransmitter.102 In 1957, the first two types of serotonin receptors were distinguished, which were termed D and M.103 Receptors of the D-type mediated the serotonin-induced contraction of smooth muscle, which could be blocked by dibenzylin (1.40). The M-type receptors mediated the serotonin-induced release of (1.41) from postganglionic nerve terminals, which could be blocked by (1.42). In 1979, a new classification, based on radioligand studies, was proposed.104 In 1986, the two classification schemes were combined and regrouped in three main classes, designated

‘5-HT1-like’ (corresponding to some D receptors and 5-HT1 binding sites), 5-HT2 105 (corresponding to most D receptors and 5-HT3 (equivalent to M receptors).

Figure 1.7 Schematic drawing of the 5-HT pathways in the human brain; serotonergic neurons are indicated in black; areas that are innervated by 5-HT axons are shown in light gray.113

This classification scheme has formed the basis for the current classification of serotonin receptors, which is based on a combination of operational, transductional and structural considerations.106,107 The cell bodies of 5-HT neurons are located in the brain stem and consist of two divisions. The axons of the caudal serotonergic system - which stems from cell bodies located in the caudal raphe nuclei in the medulla oblongata and the caudal pons - project to the spinal cord. For the rostral serotonergic system, the cell bodies are located in the rostral raphe nuclei in the midbrain and

23 rostral pons. These neurons have projections to several tissues in the forebrain, including basal ganglia (striatum, pallidum), limbic system (hippocampus, amygdala), hypothalamus, thalamus and several parts of the cerebral cortex. In addition to the nine 5-HT nuclei (B1-B9) originally described in the mid 1960s, immunochemical localization of 5-HT has also detected reactive cells in the area postrema and in the caudal locus coereleus.108-111 The basal ganglia were initially thought to be involved in movement processes only, but it seems that they are also involved in several neuropsychiatric symptoms. The limbic system was originally identified as the seat of emotions, but later it was suggested to have a function in memory and aggressive behavior as well. Neuroendocrine regulation is an important function of the hypothalamus and it is involved in appetite and sexual behavior. The thalamus is important in pain perception and the cerebral cortex in numerous processes that concern cognition, skills and observation.112

BIOSYNTHESIS AND METABOLISM OF SEROTONIN

About one to two percent of the total amount of 5-HT in the body is found in the brain. Because 5-HT cannot cross the blood-brain barrier, it is clear that the brain cells must synthesize it in situ.

Scheme1.1a The metabolic pathways available for the synthesis and metabolism of serotonin

NH2

O HO N 1.43

a NH OH HO 2 HO

O O HO N N 1.51 1.53 c b NH H HO 2 N H HO O N O d O e N N N 1.10 N-acetyl serotonin 1.54 a Enzymes: a) tryptophan hydroxylase; b) L-aromatic amino acid decarboxylase; c) mono- amine oxidase, aldehyde dehydrogenase; d) 5-hydroxytryptamine N-acetylase; e) 5-hydroxy indole O-methyl transferase.

24 The first step in this synthesis is the uptake of the amino acid tryptophan (1.43). Plasma tryptophan arises primarily from the diet, and elimination of dietary tryptophan can profoundly lower the levels of brain 5-HT and can even induce depression. Substrates for the active carrier pump that transports tryptophan across the blood-brain barrier are large amino acids – including aromatic amino acids, like (1.44) and (1.45), and branch-chain amino acids, like leucine (1.46), isoleucine (1.47) and valine (1.48), and others, like methionine (1.49) and histidine (1.50). The competitive nature of the large neutral amino acid carrier means that brain levels of tryptophan will be determined not only by the plasma concentration of tryptophan, but also by that of competing neutral amino acids. The next step in the synthetic pathway is hydroxylation of tryptophan at the 5 position to form 5-hydroxytryptophan (1.51). The tryptophan hydroxylation step can be specifically blocked by p-chlorophenylalinine (PCPA, 1.52), which binds irreversibly to tryptophan hydroxylase. The last step in the synthesis is decarboxylation of 5-hydroxytryptophan by l-aromatic amino acid decarboxylase to yield 5-hydroxytryptamine (5-HT, 1.11).

Cl HO OH H3C O O N + H C N CH O 3 3 H C H3C O 3 1.41 H 1.40 1.42 N O O CH3 H C CH H3C 3 3 OH OH O O NH2 NH HO 2 H3C 1.44 1.45 H N OH H2N OH 2 1.46 1.47 CH 3 O HN H C 3 O O Cl S N H3C OH H N OH 1.49 H N OH 2 NH 2 1.48 2 1.50 1.52 H NH2 N

HN O N N CH 3 H3C S N CH3 N N H O 1.56 H C 1.55 3

Cl

Chart 1.5 Chemical structure of dibenzylin (1.40), acetylcholine (1.41), morphine (1.42), the amino acids tyrosine (1.44), phenylalanine (1.45), leucine (1.46), isoleucine (1.47), valine (1.48), methionine (1.49) and histidine (1.50), PCPA (1.52), (1.55) and (1.56).

25 The only effective route of continued metabolism for 5-HT is deamination by MAO-A to 5-hydroxyindoleacetaldehyde. This can be further oxidized to 5-hydroxyindoleacetic acid (5-HIAA, 1.53) or reduced to 5-hydroxytryptophol, depending on the NAD+/NADH ratio in the tissue. In the pineal gland, 5-HT is converted to 5-methoxy-N-acetyltryptamine (melatonin, 1.54), via N-acetylation and 5-hydroxy-indole-O-methyl-transferase. Melatonin plays an important role in that it can e.g. manipulate the biological clock, regulate sleep- awake patterns and improve jet-lag. Considering the multiplicity of tissues that are innervated by 5-HT neurons and their functions, it is not surprising that 5-HT has been found to be involved in a variety of central functions and disorders. These central functions include mood, sleep, appetite, pain perception, memory and sexual behavior. 5-HT has been suggested to be involved in disorders such as depression, migraine, schizophrenia and anxiety, including obsessive-compulsive disorders. A major application of drugs that interact with the serotonergic system are the antidepressants (TCAs, RIMAs and SSRIs). Other drugs that act (partly) via 5-HT receptors are antipsychotics, like clozapine (1.55) and antimigraine drugs, like sumatriptan (1.56).

1.4.3 SEROTONIN RECEPTOR SUBTYPE CLASSIFICATION

The three main criteria for classifying and naming 5-HT receptors are based upon operational (recognitory), transductional and structural characteristics. None of these characteristics have precedence.106, 114-116 An additional classification principle is ‘Human Species Primacy’, the same receptor subtype name should be used for all species homologues of a gene, specifying the species with brief letter prefixes.117 Each of the seven currently recognized 5-HT receptor classes exhibits a quite distinct pharmacology, even though some of these classes utilize the same transduction pathway.106 Historically, the operational characteristics of 5-HT receptors, comprising functional pharmacological and radioligand binding data, were considered sufficient for their differential classification.105 The tendency for operational profiles to become increasingly conserved within a receptor class means that sub-type differentiation within a class is increasingly difficult. The human 5-HT1B and 5-HT1D receptor subtypes are classic examples. Although these two subtypes are distinct gene products with approximately 70% sequence homology, they exhibit pharmacological profiles that are almost indistinguishable.118 On the other hand, a high degree of sequence homology between receptors does not always result in similar operational characteristics. Hence, the rat and human homologues of the 5-HT1B receptors share 97% overall sequence identity, yet their operational profiles are quite distinct.119, 120

Thus, they were previously seen as different receptor subtypes (h-5-HT1D and r-5-

HT1B). Application of modern molecular biology techniques and the above described considerations have presently revealed the existence of seven different classes of

26 serotonin receptors (5-HT1-7). Some of these classes, like the 5-HT1 and 5-HT2 receptor families consist of several subtypes. Some 5-HT receptor subtypes have only been identified as gene products and are therefore denoted in small characters.

SEROTONIN 5-HT1 RECEPTORS

The 5-HT1 receptor class now comprises of the 5-HT1A, 5-HT1B, 5-HT1D, 5-ht1E and 107 5-ht1F subtypes. The 5-HT1A receptor is found both presynaptically (autoreceptors) on the cell bodies of 5-HT neurons in the raphé nuclei - where they regulate the 5-HT synthesis, release and electrical activity via a negative feedback control - and postsynaptically in the different projection areas, with high concentrations in the hippocampus, septum and some of the amygdaloid. Many of these regions are components of the pathways involved in the modulation of emotion, the limbic system. 5-HT1A receptors are also present in the neocortex, the hypothalamus and the 121-126 substantia gelatinosa of the spinal cord. In rats, activation of central 5-HT1A receptors by induces a rectal temperature decrease, a behavioral (5-HT) syndrome and inhibition of ultrasonic vocalization.127-130

The 5-HT1B and 5-HT1D receptors may be involved in a large number of functions, e.g. depression, anxiety, migraine and movement disorders. In 1996, the nomenclature 131 for the 5-HT1B/1D receptor subtypes was revised. In previous classification schemes the well-characterized rodent 5-HT1B receptor was recognized as a distinct entity. The subsequent identification of a pharmacologically different receptor with a similar distribution in non-rodents led to the recognition of the 5-HT1D class. In 1992, if was shown that the human 5-HT1D receptor in fact comprised two subtypes, encoded by 132 distinct genes. These were named the h-5-HT1Dα and h-5-HT1Dβ subtypes. It was subsequently shown that, in spite their pharmacological differences, the rodent 5-

HT1B and non-rodent 5-HT1Dβ receptors were in fact homologues. Accordingly, they are now called 5-HT1B receptors. The 5-HT1Dα receptor has retained the 5-HT1D notion, since its pharmacology is the same as that described for the 5-HT1D receptor from the outset. The terminal 5-HT autoreceptors are of the 5-HT1B type, whereas 5- 132-135 HT1D autoreceptors are colocalized with 5-HT1A receptors in the raphé nuclei.

Postsynaptically, high densities of 5-HT1B receptor mRNA has been found in the striatum, cerebellum (Purkinje cell layer), hippocampus (pyramidal cell layer of CA1), entorhinal and cingulate cortex (layer IV), subthalamic nucleus, and nucleus 136-138 accumbens but not substantia nigra. High densities of 5-HT1D receptor mRNA has been found in the substantia nigra, globus pallidus, dorsal subiculum and superior colliculi.139-141 In 1996, an endogenous tetrapeptide (Leu-Ser-Ala-Leu) was isolated from both rat and bovine brain. This peptide was designated 5-HT-moduline and it appears to function as an endogenous ligand specific for the 5-HT1B receptor. 5-HT-Moduline is released from synaptosomal preparation by a Ca2+-dependent K+ stimulation, suggesting that the peptide is stored in excitable cells.142, 143

27 The 5-ht1E receptor was first identified in 1989 in homogenates of human frontal 144 cortex. Until now, the 5-ht1E receptor has not been cloned from any other species. Homogenate-binding studies indicate that the receptor is generally present in brain 145-147 regions similar to those of the 5-HT1D receptor in varying relative proportions.

The function of the 5-ht1E receptor is not yet known. The pharmacology of the 5-ht1F receptor is solely based on the isolation and expression of the encoding cDNA. Based on the sequence homology, the 5-ht1F receptor is closely related to the 5-ht1E receptor. From in situ hybridization studies, the mRNA for the human receptor protein has be identified in the brain, mesentery and uterus, but not in kidney, liver, spleen, heart, pancreas or testes. In the brain, the mRNA has been shown to be concentrated in the dorsal raphé, hippocampus and cortex. This distribution indicates a role as another 5- HT autoreceptor type.148, 149

SEROTONIN 5-HT2 RECEPTORS

The 5-HT2 receptor subclass consists of three subtypes. The 5-HT2A receptor, the

‘classical’ 5-HT2 receptor, the 5-HT2B receptor, which was formerly called the 5-HT2F receptor - F for stomach fundus - and the 5-HT2C receptor, which was originally designated as the 5-HT1C receptor. The 5-HT2A receptor has been reported to be involved in many peripheral as well as central functions, e.g. smooth muscle contraction, platelet aggregation, control of hormone or transmitter release, control of sexual activity, regulation of sleep, motor behavior and psychiatric disorders like epilepsy, migraine, anxiety, depression, schizophrenia and hallucination. Some behavioral effects in rodents, like head twitch and wet-dog shake, and neuronal 105 depolarization have been attributed to activation of central 5-HT2A receptors.

The 5-HT2A receptors are enriched in many areas of the cortex. In the neocortex, these sites are mainly concentrated in laminae I and IV (rat) and III and V (human). 5-HT2A sites are also found in the claustrum (connected to the visual cortex), the limbic system (olfactory nuclei) and parts of the basal ganglia.150, 151

Due to the lack of specific 5-HT2B receptor ligands, there is not much known about the function of 5-HT2B receptors. The rat stomach fundic strip has been known for a long time to be extremely sensitive to 5-HT, where the main effect appears to be contraction.152 Although the fundus receptor shared some characteristics with the 153, 154 classical 5-HT2 receptor, it was clear that it was not a 5-HT2A receptor. Based on the rank order of potency of a variety of agonists, the fundus receptor was shown to 155 also bear resemblance to the 5-HT2C receptor. In 1994, the h-5-HT2B receptor was 156 cloned. Human 5-HT2B mRNA has been detected in a variety of peripheral tissues and also, although in relatively low amounts, in the brain. No transcripts of 5-HT2B mRNA have been found in the rat brain. This has lead to the notion that the 5-HT2B receptor in the human brain may be involved in higher cognitive brain functions.157

5-HT2C receptors are highly expressed in the epithelial cells of the choroid plexus, and function in the control of the exchanges between the central nervous system and

28 the cerebrospinal fluid.158, 159 They are also present, although at lower densities than in the choroid plexus, in the limbic system and regions associated with motor 160 behavior. 5-HT2C sites appear to be more abundant in the basal ganglia of humans, particularly the globus pallidus and in the substantia nigra.124 It has been suggested that 5-HT2C receptors play a role in a variety of processes such as locomotion, feeding, anorexia nervosa, cerebrospinal fluid production, adrenocorticotropic hormone release, migraine, obsessive, compulsive disorders and anxiety.161-166 The best characterized 5-HT2C receptor –mediated effects are hypolocomotion and hypophagia.165 Other effects include penile erection, decreased social interaction and suppression of hypertonic saline consumption.167, 168

SEROTONIN 5-HT3 - 5-HT7 RECEPTORS

The 5-HT3 receptor is a ligand gated ion channel, that consists of 5 subunits 169 (pentamer). In the brain, the highest densities of 5-HT3 receptors are found in discrete nuclei of the lower brain stem, e.g. dorsal vagal complex and trigeminal nucleus, the area postrema en the nucleus tractus solitarius and the substantia 170, 171 gelatinosa of the spinal cord. Lower, but significant densities of 5-HT3 binding sites are also found in the cortex and areas of the limbic region such as the hippocampal formation, amygdala and medial nucleus of the habenula.171-176 In the periphery, 5-HT3 receptors are located on pre- and postganglionic autonomic neurons 177-182 and neurons of the sensory and enteric nervous systems. Activation of 5-HT3 182-184 receptors triggers a rapid depolarization. In the CNS, 5-HT3 antagonists profoundly influence animal behavior, implicating a role for 5-HT3 receptors in psychosis, anxiety, cognition and the rewarding and withdrawal effects from drugs of abuse and eating disorders.185, 186 187 The 5-HT4 receptor was first described in 1988. Effects of 5-HT mediated by 5-

HT4 receptors have been described in heart, adrenal, bladder and alimentary canal, which implies a role of this receptor subtype in diseases of the gastrointestinal tract, as well as in cardiac, urinary and endocrine functions.188 In the brain, high densities of 5-

HT4 receptors are found in several limbic areas, such as olfactory tubercles and nucleus accumbens, corpus striatum, globus pallidus and substantia nigra. This suggests a possible role in affective disorders, psychoses, motor coordination, arousal and visual perception, in addition to learning and memory.189-191 At present, there are no functional correlates or transductional characteristics known of the recombinant 5-ht5A and 5-ht5B receptors. Because of their high affinity to LSD (D-lysergic acid diethylamine, 1.57) and ergot derivatives, they are postulated to mediate some of the effects of these drugs. The chromosomal localization of the

5-ht5A receptors corresponds to regions in which mutations lead to abnormal brain development, which suggests a possible role for this receptor subtype in brain development. Both receptor genes show a strong sequence similarity (77%), but similarities to other 5-HT receptor subtypes are low.192-195

29 There is increasing circumstantial evidence to suggest that the putative 5-ht6 receptor is expressed endogenously in neuronal tissue. 5-ht6 receptors exhibit a high affinity for tricyclic antipsychotic drugs, like clozapine, amitriptyline, mianserin and (1.58), and because 5-ht6 receptor transcripts have been localized in limbic and cortical regions of the brain (striatum, olfactory tubercles, cerebral cortex, hippocampus) it has been suggested that the 5-ht6 receptor may play a role in several neuropsychiatric disorders that involve serotonergic systems.196, 197 There is no evidence for the presence of the 5-ht6 receptor in peripheral tissues. In ‘knock-out’ rats, a specific behavioral syndrome of yawning, stretching and chewing is revealed.198 Because (1.59) dose-dependently antagonizes this behavioral syndrome, the 5-ht6 receptors may be involved in the control of cholinergic transmission.

CH 3 F O N CH 3 O CH N 3 CH3 HO O N N N CH 3 S 1.59 H N O 1.58 N 1.57 F H

Chart 1.6 Chemical structure of LSD (1.57), ritanserin (1.58) and atropine (1.59).

In 1997, the 5-ht7 receptor was upgraded from putative serotonin orphan receptor to 199 5-HT7 receptor. The 5-HT7 receptor has been cloned from rat, mouse, guinea pig and human cDNA.200-205 It exhibits a high degree of interspecies homology (95%), but a low sequence homology with other 5-HT receptors (<40%). In the brain, the 5-HT7 receptor is located in the thalamus, hypothalamus and several limbic and cortical 204, 206, 207 regions. Furthermore, there are high densities of 5-HT7 receptors in certain peripheral nervous structures, such as lumbar dorsal root ganglia, and in vascular and non-vascular smooth muscle, including the external carotid vascular bed, the coronary artery and several regions of the gastrointestinal tract (stomach, descending colon, 205, 208, 209 ileum). The localization of 5-HT7 receptors in these tissues implies a role for these subtypes in depression, regulation of circadian rhythm, sensory processing, sleep disorders, schizophrenia, migraine, modulation of sympathetic afferent pathways and hypertension.

30

Table 2 1996 NC-IUPHAR classification and nomenclature for 5-HT receptors and some of their characteristics

Receptor Previous Location Agonist Antagonist Effector/ Structural name Response information

5-HT1A  Hipp, RNu 1.60 1.69 Gi/o h 421aa cAMP↓ 7TM

5-HT1B 5-HT1Dβ* Str, Hipp, OT 1.61 1.70 Gi/o h 390aa cAMP↓ 7TM

5-HT1D 5-HT1Dα RNu, SN, GP 1.62 1.71 Gi/o h 377aa cAMP↓ 7TM

5-ht1E  SN, GP   Gi/o h 365aa cAMP↓ 7TM

5-ht1F 5-HT1Eβ; Hipp, FC, RNu   Gi/o h 366aa

5-HT6 cAMP↓ 7TM

5-HT2A D; 5-HT2 FC, OT 1.63 1.72 Gq/ll h 471aa

IP3/DAG↑ 7TM

5-HT2B 5-HT2F peripheral 1.64 1.73 Gq/ll h 479aa

IP3/DAG↑ 7TM

5-HT2C 5-HT1C CP, SN, GP 1.65 1.74 Gq/ll h 458aa

IP3/DAG↑ 7TM

+ + 5-HT3 M Hipp, TgN 1.66 1.75 Na i↑, K i↓ m 487aa 5TM

5-HT4  OT, Str, GP, SN 1.67 1.76 Gs r 387aa cAMP↑ 7TM

5-ht5A 5-HT5α Hipp, Hyp   Gs? h 357aa 7TM

‡ 5-ht5B 5-HT5β Hipp   unknown m 370aa 7TM

5-ht6  Str, OT, Hipp, FC 1.68  Gs h 440aa, 7TM

5-HT7 5-HTx Hyp, Thal, 1.68 1.77 Gs h 445aa, cAMP↑ 7TM ‡ * Only the non-rodent form of the receptor was previously called 5-HT1D ; No human equivalent has been identified; Abbreviations: Hipp, hippocampus; RNu, raphé nuclei; SN, substantia nigra; TgN, trigeminal nucleus; GP, globus pallidus; OT, olfactory tubercle; CP, choroid plexus; FC, frontal cortex;

Hyp, hypothalamus; Str, striatum; Thal, thalamus; cAMP, cyclic adenosine monophosphate; IP3, inositol triphosphate; DAG, diacyl glycerol.

31 H N H NH OH Pr N 2 O H O N N S N Pr O H3C N 1.60 1.61 1.62 O N H N F H OMe S NH 2 NH O N O 2

I HN OMe 1.63 1.64 1.65 N H NH Cl CH3 N 2 H O H H N Cl N N NH NH2 2 F H2N 1.66 NH NH 1.67 1.68 N H

Chart 1.7 Chemical structure of 5-HT agonists 8-OH-DPAT (1.60), CP931259589 (1.61), L-694,247 (1.62), (R)-DOI (1.63), BW 723C86 (1.64), Ro 60-0175 (1.65), m-CPBG (1.66), (1.67) and 5-CT (1.68).

O O N N N N O N N N O 1.69 1.70 O N O H O N N O N O N O N N N H O 1.71 1.72 H N N O F N S HN H N N N 1.73 N O N O 1.74 Cl O O S Cl N O O O O N H Cl N N 1.75 S 1.76 O N 1.77 N O

Chart 1.8 Chemical structure of 5-HT antagonists WAY100635 (1.69), SB224289 (1.70), GR127935 (1.71), (1.72), SB204741 (1.73), SB242084 (1.74), MDL72222 (1.75), GR113808 (1.76) and SB258719 (1.77).

32 1.5 ANIMAL MODELS IN ANTI- RESEARCH

There are several different strategies in the antidepressant research. The first strategy focuses on a known property of existing anti- that is held responsible for the therapeutic effect of these drugs. The goal of this strategy is to maximize this property, while others - that are believed to induce the unwanted side- effects – are minimized. An example of this approach are e.g. the SSRIs, in which the 5-HT-reuptake-inhibition property is maximized, while the noradrenergic and acetyl cholinergic properties are minimized. A second strategy focuses on targeting the neurochemical effects that are identified as common to a variety of antidepressants. Examples of this approach are the 5-HT autoreceptor down-regulation or the NMDA receptor modulation. A third possible strategy would be the screening of novel compounds in behavioral tests that are predictive of antidepressant activity. Of course, in practice, in vivo models are used primarily to follow-up on leads that have emerged from neurochemical strategies. In theory, however, this approach would require no preconceptions as to mechanisms of action and could generate novel neurochemical hypotheses for antidepressant drugs.

1.5.1 ACUTE MODELS FOR DEPRESSION

For the screening of anti-depressant action of novel compounds, several acute, non- invasive models for depression have been developed. In 1977, Porsolt’s forced-swim test or behavioral despair test was first described.210 Rats are forced to swim in a cylinder of water and, after an initial period of active escape attempts, they adopt an immobile posture. On a second exposure, the immobile posture is entered more rapidly. Antidepressants delay the onset of immobility in this second test. Important false negatives are the SSRI’s. 5-HT1A agonists and NMDA antagonists, on the other hand, possess antidepressant-like activity according to this procedure.211,212 The learned helplessness test, although already described in dogs in 1967, was not applied in psychopharmacology until 1979.213 Learned helplessness refers to the incapacity of animals (rats or dogs), pre-exposed to a series of inescapable shocks, to subsequently learn to escape from shock, for example by pressing a lever in a Skinner Box or crossing to the other side of a shuttle box. The test derives its name from the assumption that the exposure to situations over which the animal has no control, induces feelings of helplessness and signs of cognitive incapacity. The procedure is time-consuming and difficult to reproduce in rats. However, learned helplessness is sensitive to a wide range of antidepressants, including SSRIs, 5-HT1A agonists and NMDA antagonists.214 The tail suspension test or restraint-induced depression was originally conceived as a dry version of the forced-swim test, in which simple suspension by the tail of the animal (rats or mice) induced immobility. The test shows similar sensitivity to the behavioral despair test, but differs in that it is sensitive to 212, 215 SSRIs, but not to 5-HT1A agonists (see table 1.3).

33 Table 1.3 Hits and misses of the acute models for antidepressant detection

Forced-swim test Learned helplessness Tail suspension Hits TCAs; MAOIs; RIMAs; TCAs; MAOIs; RIMAs; TCAs; MAOIs; Sertraline; Zimeldine; SSRIs; Atypicals; RIMAs;

Fluoxetine; 5-HT1A agonists; SSRIs; Atypicals; Atypicals; NMDA antagonists; NMDA antagonists 5-HT agonists; Ca2+ channel antagonists; 1A NMDA antagonists; PCPA Ca2+ channel antagonists

Misses Clomipramine; ? 5-HT1A agonists Fluvoxamine

1.5.2 CHRONIC MODELS FOR DEPRESSION

The major disadvantage of the acute models for depression, is that the antidepressant activity is detected following acute administration, whereas in clinical depression, several weeks elapse before a therapeutic effect is observed. This consideration argues strongly for the use of chronic models of depression. These models should not only be of demonstrable validity, but should also maintain an abnormal state for a prolonged period, during which therapy may be administered. Models that meet these criteria are of value not only for drug development, but also as experimental tools for investigating the physiological mechanisms underlying the depression-like behavior and the therapeutic action of antidepressant drugs. Because these models would simulate depression in a relatively realistic and valid manner, the conclusions of such studies are likely to generalize to the clinic. And while the traditional screening tests are limited in that they only detect antidepressant action, the chronic models can also be used to discover new antidepressants with a shorter onset of action.

CHRONIC MILD STRESS

In the chronic mild stress (CMS) model, rats or mice are exposed sequentially to a variety of extremely mild stressors (e.g. overnight illumination, cage tilt, change of cage mate), which change every few hours over a period of weeks or months. This procedure causes a decrease in sensitivity to rewards.216-218 It should be noted that the variety in stressors is essential.219 CMS-induced behavioral deficits may be maintained for several months. However, chronic treatment with tricyclic or atypical antidepressants during continued application of CMS, will restore normal behavior. A variety of neurochemical systems have been examined as a potential basis for the behavioral changes following CMS. Although the 5-HT and NA systems are traditional starting points for thinking about antidepressants, it seems logical to focus

34 also on the mesolimbic DA system, because it has been the major focus for studies concerning the responsiveness to rewards.220 Both pre- and postsynaptic markers indicate a decrease in DA transmission in animals exposed to CMS, specific to the nucleus accumbens.221, 222 In animals successfully treated with antidepressants, behavioral recovery is reversed by acute administration of D2/D3 receptor 223-225 antagonists. It has been suggested that sensitization of D2/D3 receptors may be responsible for the therapeutic action of antidepressants in this model. Hence, the mesolimbic DA system might be a potential target for antidepressant drug development. DA reuptake inhibition is actually a feature of several recent antidepressants, like (1.78), buproprion, (1.79) and minaprine (1.80). However, these drugs have not yet been tested in the CMS model. Another antidepressant candidate, tolcapone (1.81) - which raises synaptic levels of DA by inhibition of the catabolic enzyme COMT - does show antidepressant-like activity in 226 the CMS model. The novel D2/D3 receptor agonist pramipaxole (1.82) also reversed the effects of CMS, exactly as observed with conventional antidepressants. Pramipaxole is currently evaluated in Phase 3 trials as an antidepressant. Besides the effects of CMS on the mesolimbic DA system, several interactions with the 5-HT system have been described. It has been found that CMS increases tissue levels of 5-HT and its metabolite 5-HIAA in the nucleus accumbens, but not in the dorsal striatum.227 Receptor binding studies revealed that CMS increased the density of both 5-HT1A and 5-HT2 receptors in the cerebral cortex. Chronic treatment with imipramine decreased the density of the 5-HT2 receptors in control animals and normalized the 5-HT2 receptor density in animals exposed to CMS. However, in the 228 case of the 5-HT1A receptor, imipramine failed to normalize the effect of CMS.

The full 5-HT1A receptor agonist 8-OH-DPAT and partial 5-HT1A receptor agonist (1.83), also did not reverse the CMS-induced adhedonia. However, the 5-

HT1A receptor antagonist WAY100635 shows antidepressant-like activity in the CMS model.229 Studies of the interactions between the NA system and CMS have been less extensive than those of the DA and 5-HT system. It has been shown that CMS increases the density of -adrenergic receptors in the cerebral cortex and also that the cAMP response to NA in cortical slices is increased. Chronic treatment with imipramine reversed these effects of CMS.228, 230

OLFACTORY BULBECTOMY

A second chronic animal model for depression is the olfactory bulbectomized (OB) rat. This model differs from the acute and CMS models for depression, in that it is not based on stress. In the OB rat model, both main olfactory bulbs are removed, without disturbing the anterior olfactory nucleus.231, 232 The OB rat model is capable of detecting antidepressant activity following chronic, but not acute treatment with anitdepressants.231, 233, 234 Following bilateral olfactory bulbectomy, several behavioral

35 changes have been observed, e.g. hyperactivity in the open-field test, enhanced nocturnal activity, deficits in memory and changes in food motivated and conditioned taste aversion behavior.235-241 Removal of both olfactory bulbs also leads to alterations in noradrenergic, serotonergic, cholinergic, GABA-ergic and glutamatergic neurotransmitter systems.234, 242-245 Immune changes in the OB rat model include reduced neutrophil phagocytosis, lymphocyte proliferation, thymus and spleen weight 242, and increased monocyte proliferation, neutrophils and α1-acid glycoprotein levels. 246, 247 The most commonly employed indicator of antidepressant activity is attenuation of the OB-related hyperactivity in the open-field. TCAs (amitriptyline, desipramine), atypical antidepressants (mianserin, mirtazapine, nomifensine, trazodone), SSRIs (paroxetine, sertraline, fluvoxamine, but not fluoxetine), RIMAs (moclobemide), as well as putative antidepressants such as

5-HT1A agonists (ipsapirone, 8-OH-DPAT), noncompetitive NMDA antagonists

(1.84, MK-801) and 5-HT2 antagonists (ketanserin) have showed antidepressant activity in the OB rat model.231, 248-251 Many of the changes observed in the OB rat are qualitatively similar to those observed in depressed patients. Hence, the OB rat model is not only an animal model for detecting antidepressant activity, but also a model for exploring the links that exist between the neurotransmitter, behavioral and endocrine and immune systems. The OB rat model might also be of use in detecting antidepressant therapies with early onset of action, since it is only capable of detecting antidepressant action following chronic administration of the current (putative) antidepressants.

Figure 1.8 Brief outline of the connections of the main and accessory olfactory bulbs. MOB, main olfactory bulb; AOB, accessory olfactory bulb; AON, anterior olfactory nucleus; APir, anterior pyriform cortex; Pir, pyriform cortex; DOT, dorsal olfactory tract; Ent, entorhinal cortex; me, medial nucleus of the amygdala; PLCo, posterolateral cortical amygdaloid nucleus; ACo, anterior cortical amygdaloid nucleus; BNST, bed nucleus of the stria terminialis; PmCo, periamydaloid cortex; VNO, vomeronasal organ.252

36 NH2 O CH N 3 CH3 N O 1.78 HN N 1.80 (CH ) H 2 6 N N N 1.79 OH H N N N O S N Pr O O NH OH 2 S (CH ) N N 2 4 1.83 1.82 H C OH O 3 1.81 S CH3 NO2 S

HN N N CH3

1.85 CH3 1.84

Chart 1.9 Chemical structure of nomifensine (1.78), amineptine (1.79), minaprine (1.80), tolcapone (1.81), pramipaxole (1.82), ipsapirone (1.83), MK-801 (1.84), methiothepin (1.85).

1.6 DEVELOPMENT OF A THIRD GENERATION OF ANTIDEPRESSANTS ; FUTURE PHARMACOLOGICAL APPROACHES

The principal advantage of the second generation of antidepressants is an improved side-effect profile compared to the classical tricyclic antidepressants and MAOIs. In order to achieve a clear improvement over current antidepressants, however, two major therapeutic objectives must be met. First, compared to other treatments for neuropsychiatric disorders, the efficacy of currently available antidepressant drugs is relatively low. There are no antidepressants that are effective in all depressed patients. In general, only ~70% of patients exhibit some response to any given antidepressant, with merely one-half of these experiencing a full response.253 Combination therapies administering, for instance, TCAs with non-selective inhibitors of MAO have been used to improve antidepressant efficacy, but controlled studies indicate that there is no significant therapeutic advantage over treatments with a single antidepressant drug.254 Second, a reduction (and in the ideal, elimination) of the ‘therapeutic lag’ encountered with the traditional antidepressants. The time between the start of medication and the clinical onset-of-action remains one of the great enigmas of antidepressant therapy. Since it has been estimated that, world wide, about 340 million people suffer from depression and that the current cost (from absenteeism, lost productivity, lost earnings, treatment and rehabilitation) exceeds $40 billion yearly in the United States alone, this objective is both worth and timely.255 Thus there is a need for a new generation of antidepressants that have higher efficacy and that retain the gains

37 already achieved in terms of fewer side effects with the second generation of antidepressant drugs.

1.6.1 REUPTAKE INHIBITION COMBINED WITH 5-HT AUTORECEPTOR BLOCKADE

Given the ability of SSRIs to selectively inhibit the 5-HT transporter thereby increasing the synaptic concentration of 5-HT, their clinical actions can be attributed to an enhanced activation of one or several postsynaptic 5-HT receptors. This hypothesis is supported by clinical data showing that the administration of tryptophan- free amino acid mixtures to recovered major depressive patients receiving either SSRIs or MAOIs transiently abolishes their antidepressant effect.256 In rats that are chronically treated with the SSRI fluvoxamine, this procedure causes a very marked reduction of neuronal 5-HT release, thus supporting the association between recovery from depression and enhancement of 5-HT activity.257 There is evidence that hippocampal postsynaptic 5-HT1A receptors participate in the action of several types of antidepressant drugs.258-260 However, given the abundance of symptoms exhibited by depressed patients, it is likely that the effects of antidepressants involve the activation of receptors in more than one brain structure. Despite the ability of the SSRIs to block the 5-HT transporter soon after their administration, significant clinical improvement of depressed patients requires prolonged administration. This suggests the existence of neurobiological adaptive mechanisms responsible for their clinical action. This delay cannot be attributed to a downregulation of the cortical - adrenoceptor-coupled cAMP generating system, because most SSRIs do not induce such effects after chronic treatment.261-264 More likely, the slow onset of clinical action and the limited efficacy of antidepressant drugs (less than two-thirds of patients usually respond to the first drug administered) may be partly ascribed to the inhibition of 5-HT release by forebrain serotonergic nerve terminals after the administration of drugs that inhibit 5-HT uptake or MAO activity.265, 266

This negative feedback involves stimulation of the 5-HT1A and 5-HT1B autoreceptors on the serotonergic neurons. Early in the course of treatment, SSRIs fail to elevate 5- HT release in the forebrain. However, as treatment continues, cell body autoreceptors desensitize, firing rate increases, and 5-HT release in the forebrain normalizes; in these circumstances, blockade of reuptake by an SSRI now causes the expected increase in levels of extracellular 5-HT. Thus, the slow onset of action of SSRIs may reflect the time necessary to cause a desensitization of 5-HT autoreceptors (see figure 267 1.9). Activation of the somatodendritic 5-HT1A receptors is also responsible for the attenuation of cell firing observed after a single administration of antidepressant 268, 269 drugs. Further attempts to reveal the desensitization of somatodendritic 5-HT1A autoreceptors have yielded somewhat contradictory results, although the most recent data give additional support to this hypothesis.270-278 The loss in efficacy of the 5-

HT1A autoreceptors does not appear to be accounted for by a reduction in their 279 number. Hence, the changes in the sensitivity of 5-HT1A (auto)receptors - revealed

38 by behavioral, neurochemical and electrophysiological means - must be accounted for by other factors, for example, variations in the efficacy of the receptor-effector coupling.

Figure 1.9 Schematic representation of the effects of 5-HT uptake inhibitors on 5-HT neurons. The blockade of the 5-HT transporter at the level of the raphé nucleus leads to activation of the somatodendritic 5-HT1A receptors. This causes a reduction in 5-HT release by axon terminals, the opening of K-channels and a subsequent reduction in the firing rate of the 5-HT neuron. The terminal 5-HT1B receptors exert a local control of 5-HT synthesis and release. Activation of these autoreceptors, upon the administration of SSRIs, reduces the efficiency of released 5-HT on postsynaptic receptor sites. Heteroregulation of the activity of serotonergic neurons is exerted by afferents at somatodendritic and terminal levels.

In view of the above observations, it has been put forward that the treatment with SSRIs (or MAOIs) combined with a 5-HT autoreceptor antagonists would accelerate and enhance the antidepressant effects of the former.266 The normalization of cell firing and release produced by 5-HT1A (auto)receptors antagonists would enable SSRIs to increase the 5-HT levels to a greater extent than when administered alone. The accumulated experimental evidence fully supports this working hypothesis. Early experiments indicated that in the dorsal raphé nucleus of the nonselective 5-HT1 antagonist methiothepin (1.85) enabled a low dose of citalopram to significantly increase the release or 5-HT in the frontal cortex.280 Further reports using selective 5-

HT1A antagonists, such as WAY100635, or a 5-HT1B/1D , such as

GR127935, or a 5-HT1B inverse agonist, such as SB224289, have documented greater increments of 5-HT release when concurrently administered with SSRIs.281, 282 The further enhancement in 5-HT release after the combination of the SSRI citalopram and the antagonist GR127935, e.g., is accounted for by the simultaneous blockade of both reuptake sites and autoreceptors of the 5-HT neuron (see figure 1.10). These greater effects on 5-HT release of antidepressants in combination with a 5-HT autoreceptor antagonist, demonstrate the importance that self-inhibition of 5-HT neurons have in their mechanism of action.

39 5-HT % Basal * * * * 1200 * * GR127935 + * citalopram 1000

800

600 citalopram

400

200 GR127935

0 -100 -50 0 50 100 150 Time (min)

Figure 1.10 Effects of GR127935 (1 µM/kg sc.), citalopram (10 µM/kg sc.) and the co- administration of GR127935 with citalopram (1 + 10 µM/kg sc.) on 5-HT release in the ventral hippocampus of the freely moving rat (mean ± SEM, n = 5, *p < 0.05 vs. control).

If the hypothesis that enhancement of serotonergic activity leads to clinical improvement in major depression is correct, pharmacological interventions increasing terminal 5-HT release are advisable. It is therefore important to obtain a deeper understanding of the mechanisms that determine extracellular 5-HT concentrations at the somatodendritic and terminal levels, encompassing both self-control mechanisms (i.e. transporter and autoreceptors) and heterocontrol mechanisms exerted by excitatory and inhibitory afferents at somatodendritic and terminal levels. Another aspect that deserves attention are the postsynaptic changes elicited by enhanced 5-HT levels after chronic SSRI treatment, and the identification of the receptors involved. The existence of adaptive changes in second and third messengers by chronic antidepressant treatments in target neurons has been documented. 283-285 However, it is uncertain if the clinical improvement is the immediate consequence of an enhanced activation of certain 5-HT receptor subtypes, or whether postsynaptic adaptive changes are required. In the first case, only the presynaptic component would be responsible for the delayed onset of action.

1.6.2 OTHER STRATEGIES

5-HT-MODULINE

The existence of an endogenous ligand able to specifically interact with 5-HT1 receptors was suspected for a long time.286-288 Purification from brain tissue and protein microsequencing resulted in the characterization of a short neuropeptide, 5- HT-Moduline, consisting of four amino acids: Leu-Ser-Ala-Leu. The principal

40 -10 property of this peptide is its high affinity for 5-HT1B receptors (IC50 ~ 10 M) and 3 289 its capacity to effectively antagonize the binding of [ H]-5-HT to 5-HT1B receptors. 5-HT-Moduline appears to regulate 5-HT activity, particularly its release. Interaction of this peptide with the 5-HT1B autoreceptor leads to an increase in 5-HT activity. This effect is shared with a variety of antidepressant drugs, in particular SSRIs.

Hence, compounds that could interfere with the 5-HT-Moduline/5-HT1B receptor interactions, may have interesting therapeutic properties.

CALCIUM CHANNEL ANTAGONISTS

Calcium channel antagonists are similar to lithium in their pharmacodynamic profile of psychotropic activity, but differ in their mechanism of action at the cellular level. They first emerged as a therapeutic group in the 1970s. Calcium channel antagonists share a common mechanism of action, but are chemically very heterogeneous. In general, three main classes can be distinguished: 1,4-dihydropyridines, phenylalkylamines and benzothiazepines. The prototypes of these classes are nifedipine (1.86), verapamil (1.87) and diltiazem (1.88), respectively. The first two were developed as coronary vasodilators.290-292 Diltiazem, however was developed as a potential psychotropic agent, with an antidepressant/anxiolytic profile. Only later studies revealed its marked cardiovascular activity and calcium channel blocking properties.293 The most consisting animal data support potential antidepressant and antimanic activity of calcium channel antagonists.55, 56 On a clinical level, the evidence for a potential use of these drugs in mood disorders is not conclusive. The data obtained in bipolar disorder, however, may be viewed as encouraging.57 TCAs have calmodulin- blocking properties and appear to inhibit voltage-activated calcium and calcium- activated potassium channels at clinically relevant concentrations.58-60 ECT inhibits calcium transport form cerebrospinal fluid to the brain, decreases protein kinase C activity and, depending on the brain area, either decreases or increases the density of dihydropyridine recognition sites.61-65 Chronic treatment with the SSRI citalopram, 66 leads to an increase in receptor density (Bmax) of dihydropyridine recognition sites. The evidence that calcium channel antagonists score positive in several animal models of depression as well as in screening tests for antidepressant-like properties (e.g., the forced-swim test) and that current antidepressant therapies influence cellular calcium trafficking, together with the important role of intracellular calcium in neuronal processes and the promising results from preliminary clinical studies give reason to scrutinize more carefully the possible role of (a new generation) of calcium channel antagonists in the treatment of depression and bipolar mood disorders.

41

NMDA-ANTAGONISTS

The NMDA receptor is a fast acting, use-dependent, ligand-gated ion channel that mediates glutamatergic neurotransmission under conditions of strong depolarization. The influx of calcium through the NMDA subtype of glutamate receptors initiates long-term modifications of synaptic and cellular responses that regulate neural plasticity.295, 296 The competitive NMDA antagonist 2-amino-7-phosphonoheptanoic acid (1.89, AP- 7), the NMDA receptor-coupled channel blocker MK-801 and the partial agonist at the -insensitive glycine modulatory site of the NMDA receptor 1- aminocyclopropanecarboxylic acid (1.90, ACPC) all reduced the duration of immobility in the forced-swim test with efficacies comparable to TCAs. These results support the hypothesis that NMDA receptor activation is involved in the behavioral deficits observed after inescapable stressors.297-300 Although the pharmacological side-effect profile of competitive NMDA antagonists restricts their potential clinical use, partial agonists and antagonists at the glycine site of the NMDA receptor appear to be devoid of neurotoxic, ataxic, amnesic and psychomimetic side effects.301 While the potential absence of undesirable side effects by glycine/NMDA antagonists predicted in animal models needs to be substantiated by clinical experience, there is already evidence from Phase 1 clinical trials indicating that ACPC lacks psychomimetic-like actions.302 In addition, ACPC shows good oral efficacy and a high therapeutic index. Thus, functional NMDA antagonists at the glycine site may constitute a novel class of antidepressants.

SIGMA RECEPTORS

Sigma (σ) receptors have been implicated in several behavioral and biochemical effects. For example, it has been shown that the σ-receptors are involved in the motor side effects of neuroleptics, have been implicated in psychotic disorders and may play a role in neuroprotective mechanisms.303-306 Based on specific ligand and biochemical 307 studies, the σ-receptors have been classified in two main types, σ1 and σ2. The σ-receptors are unique in that drugs form different therapeutic groups show high affinity for this receptor. For example, the antipsychotics (1.91) and chlorpromazine bind to σ-receptors. However, this is not a common property of most neuroleptics.308 In addition, some antidepressants, including the MAOIs bind to σ- receptors.309-311 Recent experimental studies have shown that NPY - an endogenous ligand for the σ-receptors - may play a role in the mechanism of action of antidepressant drugs and in the pathology of mood disorders. Several clinical studies have implicated a dysfunctional NPY system in the etiology of depression.312-316 The functional modulation of σ-receptors by NPY suggests that these receptors may play an important role in the pathogenesis of depression.

42 Using electrophysiological studies in rats, it has been shown that the administration of the SSRI sertraline selectively potentiates the effect of NMDA on pyramidal neurons in the CA3 region in the hippocampus. This potentiation was reversed by the σ- receptor ligands haloperidol, pentazone (1.82) and N-allyl-normethazocine (1.93).317, 318 The modulation of the glutamate-NMDA receptor complex by antidepressant action on σ1 receptors may open up new possibilities to the understanding of the mechanism of action of antidepressants and the development of novel therapeutic agents.

H MeO OMe H3C N CH3 O CH3 H C O O CH 3 3 S O H N O O 2 OH NO O 2 N CH 1.89 H C N O O 3 1.86 3 1.88 N H3C CH3 H3C

NH2 NC O CH3 Cl F

O CH3 MeO N P OH OMe 1.87 OH O OH 1.91 OH H H 1.90 CH3

N N CH2 H CH3 H CH CH HO 3 HO 3 1.92 1.93 H C 3 H3C

Chart 1.11 Chemical structure of nifedipine (1.86), verapamil (1.87), diltiazem (1.88), AP-7 (1.89), ACPC (1.90), haloperidol (1.91), pentazone (1.92) and N-allyl- normethazocine (1.93).

1.7 SCOPE OF THE THESIS

As described in this chapter, the 5-HT system is believed to play an important role in the pathogenesis and treatment of depression. Today, the antidepressants form the 3rd largest therapeutic area, with a current annual growth of 20%. Two-thirds of the prescribed antidepressants are reuptake inhibitors. Today’s’ antidepressants suffer from poor response, late clinical onset-of-action and side effects - nausea and - that seriously affect patience compliance. Most antidepressants (TCAs, MAOIs and SSRIs) elevate synaptic levels of 5-HT and their clinical action may very well be attributed to the enhanced activation of postsynaptic 5-HT receptors that modulate GABA, glutamate and ACh release.

43 It has been postulated that the 5-HT autoreceptors are an important factor in the delay in time-of-onset of therapeutic action of the first and second generation of antidepressants.

Chapter 2 focuses on the 5-HT1A antagonist WAY100635 (1.69). The synthesis of several O-substituted , N-substituted 4-N-(o-methoxyphenyl)- aminopiperidine and benzamide analogues of WAY100635 is described, together with their in vitro and in vivo profile at both pre and postsynaptic 5-HT1A receptors. In Chapter 3, the in vitro and in vivo profile of WAY100635 and the para- substituted benzamide analogues both pre- and postsynaptic 5-HT1A receptors is studied in more detail. Dose-response curves vs 8-OH-DPAT (0.2 mg/kg sc) are recorded, measuring the antagonism of induced hypothermia and the depressed 5-HT release (microdialysis in ventral hippocampus, presynaptic model).

Chapter 4 focuses on the 5-HT1B/1D antagonist GR127935 (1.71) and the 5-HT1B inverse agonist SB224289 (1.70). Several close analogues are evaluated in vitro for their affinity for the 5-HT autoreceptors and the 5-HT transporter. Of these, the in vitro most potent ligands are evaluated in vivo for their potential to elevate 5-HT release alone and upon co-administration with the SSRI citalopram. Chapter 5 and 6 cover the in vitro and in vivo profile of 6-methoxymianserin. In chapter 4, the tetracyclic azepin is used to replace the 2-methoxyphenyl moiety of GR127935. The fact that this azepin is structurally very close to the atypical antidepressants mianserin (1.32) and mirtazapine (1.33) justifies an elaborate study of this new tetracyclic compound itself. In Chapter 5, the synthesis and in vitro binding profile of several analogues of 6-methoxymianserin is described, including the two probable metabolites 6-hydroxymianserin and N-desmethyl-6-methoxymianserin. The ability of 6-methoxymianserin to influence the release of NA and 5-HT in the ventral hippocampus at dosages similar as used for mianserin and mirtazapine, is studied. Chapter 6 deals with the separation of the enantiomers of 6-methoxymianserin on chiral HPLC and the determination of the absolute configuration of the enantiomers. The in vitro binding profiles of the enantiomers are assessed and they are both evaluated in vivo for their effects on the release of NA, DOPAC, 5-HT and 5-HIAA.

To summarize, this thesis deals with the synthesis and preliminary in vitro and in vivo evaluation of new chemical entities that are of possible use in the treatment and/or the study of the pathogenesis of depression. Further evaluation in animal models of depression and farmacodynamic, farmacokinetic and toxicology studies are required to determine the therapeutic potential of these new compounds.

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[282] Liao, Y.; Böttcher, H.; Harting, J.; Greiner, H.; Van Amsterdam, C.; Cremers, T.I.F.H.; Sundell, S.; März, J.; Rautenberg, W.; Wikström, H.V. New selective and potent 5-HT(1B/1D) antagonists: chemistry and pharmacological evaluation of N-piperazinylphenyl biphenylcarboxamides and biphenylsulfonamides. J. Med. Chem. 2000, 43, 517-525.

[283] Nestler, E.J.; Terwilliger, R.Z.; Duman, R.S. Chronic antidepressant administration alters the subcellular distribution of cyclic AMP-dependent protein kinase in rat frontal cortex. J. Neurochem. 1989, 53, 1644-1647.

[284] Popoli, M.; Vocaturo, C.; Pérez, J.; Smeraldi, E.; Racagni, G. Presynaptic Ca2+/calmodulin- dependent protein kinase. II: Autophosphorylation and activity increase in the hippocampus after long-term blockade of serotonin reuptake. Mol. Pharmacol. 1995, 48, 623-629.

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[288] Hoyer, D.; Clarke, D.E.; Fozard, J.R.; Hartig, P.R. Martin, G.R.; Mylecharane, E.J.; Saxena, P.R.; Humphrey, P.P.A. International union of pharmacology. Classification of receptors for 5-hydroxytryptamine (serotonin). Pharmacol. Rev. 1994, 46, 157-203.

[289] Fillion, G.; Sequin, L.; Massot, O.; Rousselle, J.C.; Fillion, M.P.; Cloëz-Tayarani, I.; Grimaldi, B.; Sezned, J.C.; Prodhomme, N. 5-HT-Moduline. Ch.3 from Antidepressants: New Pharmacological Strategies (1997). Ed. P. Skolnick, Humana Press Inc., Totowa, NJ, USA.

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[291] Kazda, S. The story of nifedipine, in Adalat (Lichtlen, P.R. and Reale, A. eds.), 1991, Springer, Berlin, Germany, 9-26.

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[294] Rafterty, E.B. Cardiovascular drugs with withdrawal syndromes. A potential problem with calcium antagonists? Drugs, 1984, 28, 371-374.

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[300] Marvizon, J.C.G.; Lewin, A.; Skolnick, P. 1-Aminocyclopropanecarboxylic acid: a potent and selective ligand for the glycine modulatory site of the N-methyl-D-aspartate receptor complex. J. Neurochem. 1989, 52, 992-994.

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66 [314] Gjerris, A.; Widerlöv, E.; Werdelin, L.; Ekman, R. Cerebrospinal fluid concentrations of NPY in depressed patients and control. J. Psychiatr. Neurosci. 1992, 17, 23-27.

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67

68

STRUCTURAL ANALOGUES OF THE 5-HT1A ANTAGONIST 2 WAY100635: SYNTHESIS AND IN VITRO AND IN VIVO * FUNCTIONAL STUDIES

2.1 Introduction 69 2.2 Chemistry 70 Synthesis of o-Substituted Phenylpiperazine, Benzamide and N-substituted 4-N-(o-Methoxyphenyl)aminopiperidine analogues of WAY100635 2.3 Pharmacology 73

Receptor Binding, Determination pA2, Ultrasonic Vocalization, Rat Hypothermia, Rat Gross Behavioral Studies and Biochemistry 2.4 Results and Discussion 75 2.5 Experimental Section 80 2.6 References 88

ABSTRACT

WAY100635 (2.2), N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide, is a silent serotonin 5-HT1A antagonist, which is now widely used to study the 5-HT1A receptor both in vivo and in vitro. In this chapter, we describe the synthesis and in vitro (5-HT1A-affinity and pA2 values at guinea pig ileum strips) and in vivo pharmacology (hypothermia, 5-HT syndrome and ultrasonic vocalization) at the serotonin 5-

HT1A receptor of several closely related analogues of WAY100635. Introduction of a bromine atom para of the methoxy group of WAY100635, compound 2.5, resulted in a sharp decrease in affinity for the 5-HT1A receptor and a loss of antagonistic activity, both in vitro as in vivo. This is also the case when the arylpiperazine moiety of WAY100635 is replaced by an arylaminopiperidine moiety (2.15, 2.16). The exchange of the cyclohexane ring of WAY100635 for a p-substituted phenyl ring (2,11, 2.12) provides close analogues of p-MPPI (2.3, a SPECT radio ligand) and p-MPPF (2.4, a PET radioligand). In vitro, the benzamide analogues show nanomolar affinity for the 5-HT1A receptor. Conflicting results are obtained with the in vitro determination of the pA2 values. In vivo, the do not block the 8- OH-DPAT (1 mg/kg sc) induced hypothermia or the ‘5-HT syndrome’(gross behavioral studies) at 0.1, 1.0 and 10.0 µmol/kg sc. Substitution of the o-methoxy group of WAY100635 by larger fluoroalkoxy or sulfonyloxy substituents does not significantly alter the in vitro or in vivo pharmacology. The O-desmethyl analogue 2.6, is the most potent antagonist in the whole series in blocking the 8-OH-DPAT induced hypothermia and the ‘5-HT syndrome’.

* A modified version of this chapter has been published by Mensonides-Harsema, M.M.; Liao, Y.; Böttcher, H.; Bartoszyk, G.D.; Greiner, H.E.; Harting, J.; De Boer, P.; Wikström, H.V. in J. Med. Chem. 2000, 43, 432-439.

69 2.1 INTRODUCTION

As described in Chapter 1, the serotonin (5-hydroxytryptamine, 5-HT) receptor 1 family consists of seven distinct classes (serotonin 5-HT1-7). The serotonin 5-HT1 receptors, based on considerations of structural features, functional pharmacology and secondary messenger coupling systems, have been further divided into five subtypes.

They have been classified as serotonin 5-HT1A, 5-HT1B, 5-HT1D, 5-ht1E, and 5-ht1F receptor, and are all negatively coupled to adenylyl cyclase.2-5

The serotonin 5-HT1A receptors are present throughout the whole body, including the central nervous system (CNS). In the CNS, the serotonin 5-HT1A receptors are located both presynaptically on the cell body of the serotonergic neurons in the raphé nucleus - where they control the firing of the serotonin 5-HT neuron via auto-inhibition - as well as postsynaptically in the projection areas such as the dentate gyrus of the hippocampus (CA1 region), frontal and neocortex, amygdala and lateral septum.6-11 These brain areas have frequently been associated with mood control and the serotonin 5-HT1A receptor is generally considered to be an important target for the treatment of mood disorders like anxiety and depression.12-14 Furthermore, it has been postulated that the somatodendritic 5-HT1A autoreceptors are involved in the late onset of clinical action of the several classes of antidepressants. Acute administration of selective serotonin reuptake inhibitors (SSRIs) leads to attenuation of cell firing of the serotonergic neuron. As treatment continues, the cell firing rate comes back to normal frequencies. This effect is due to desensitization of the somatodendritic 5- 15-27 HT1A autoreceptors.

Activation of serotonin 5-HT1A receptors with agonists, like 8-OH-DPAT (2.1), leads to a number of physiological changes that can easily be quantified in several in vitro and in vivo assays. In rats, the administration of serotonin 5-HT1A agonists results in a rectal temperature decrease, a characteristic set of motor changes collectively known as the serotonin motor syndrome (both models for postsynaptic 5-

HT1A receptor activation), inhibition of synthesis rate of serotonin 5-HT in the terminal areas, inhibition of ultrasonic vocalization and inhibition of neuronal firing 28-33 activity (all models for presynaptic 5-HT1A receptor activation). Positron emission tomography (PET), using effective positron-emitting radioligands, provides a means to visualize 5-HT1A receptor densities in living subjects and to monitor inhibition of receptor binding with unlabelled ligands (both endogenous and exogenous).34-36 Agonists, like 8-OH-DPAT, however, are limited in their use for imaging in vivo, possibly because they bind to the high-affinity agonist state of the receptor only, which quickly reverts to a low-affinity state and a fast dissociation of radioligand.37-39 Antagonists bind to receptors both in their high- and low-affinity state. WAY100635 (N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl)- cyclohexane carboxamide, 2.2) is known to be one of the first potent, silent and selective antagonist for serotonin 5-HT1A receptors at both somatodendritic and postsynaptic receptor sites.40-42 The [11C-carbonyl]-labeled O-desmethyl analogue 2.6

70 of WAY100635 has proven to be a successful radioligand for studies of central 43 serotonin 5-HT1A receptors in rat, monkey and human brain using PET. Two other

5-HT1A receptor antagonists that are used to study the distribution of 5-HT1A receptors are the p-iodo (p-MPPI, 2.3) and p-fluoro (p-MPPF, 2.4) benzamide analogues of WAY100635.44 In this chapter the synthesis of several o-substituted phenylpiperazine analogues of WAY100635 (2.5 – 2.10), some p-substituted benzamide analogues of p-MPPI and p-MPPF (2.11 – 2.14) and two arylaminopiperidine analogues (2.15 and

2.16) is described. The affinity and selectivity at the serotonin 5-HT1A receptor is determined as well as the in vitro antagonistic properties (pA2 values). Furthermore, the ability to counteract, in vivo, the 8-OH-DPAT-induced inhibition of ultrasonic vocalization, hypothermia and the ‘5-HT syndrome’ in rats is studied for several of the test compounds. Finally, the potential agonistic properties of 2.2 its triflate analogue 2.7 are assessed, using an in vivo biochemistry assay in which the accumulation of 5-hydroxytryptophan (5-HTP) is used as an indicator for the 5-HT turnover.45-47 R

N OH N N N N O O N O O N 2.1 2.2 N N 2.3 R = I 2.4 R = F

Chart 2.1 Chemical structure of 8-OH-DPAT (2.1), WAY100635 (2.2), p-MPPI (2.3) and p- MPPF (2.4)

2.2 CHEMISTRY

SYNTHESIS OF O-SUBSTITUTED PHENYLPIPERAZINE ANALOGUES OF WAY100635.

WAY100635 (2.2) was prepared as previously described.40 2-Aminopyridine is coupled to chloroacetyl chloride in dichloromethane in the presence of triethylamine, yielding N-2-(2-chloroethyl)amidopyridine (2.2a) (CAUTION: SENSITIZING). This intermediate is reacted with 1-(2-methoxyphenyl)piperazine in DMF at 60 ˚C, in the presence of potassium carbonate, yielding N-2-[2-[4-(2-methoxyphenyl)-1- piperazinyl]ethyl]amidopyridine (2.2b), which is subsequently reduced to

N-[2-[4-(2-hydroxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)amine, using LiAlH4 in THF (2.). This intermediate is coupled to cyclohexanecarbonyl chloride in dichloromethane in the presence of triethylamine, yielding N-[2-[4-(2-methoxy- phenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide (WAY100635, 2.2) Bromination of WAY100635 was effected by reacting with bromine in a solvent mixture of EtOH/MeOH and dichloromethane at room temperature, yielding

71 N-[2-[4-(3-bromo-5-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (2.5) (Scheme 2.1).

N H3C O O N a H b H NH2 N Cl N N N N 2.2b O 2.2a

H C N H3C N 3 O O N N d e c H N N N N O 2.2c 2.2

H3C N O N N N O Br 2.5

SCHEME 2.1 Reagents and conditions: (a) chloroacetyl chloride, TEA, dichloromethane, room temperature; (b) o-methoxyphenyl piperazine, K2CO3, DMF, 60 ˚C; (c) LiAlH4, THF, room temperature; (d) cyclohexanecarbonyl chloride, chloroacetyl chloride, TEA, dichloromethane, room temperature; (e) Br2, EtOH/MeOH, dichloromethane, room temperature.

The O-demethylation of WAY100635 could not be effected with refluxing HBr

(48%), with or without acetic acid (partial conversion), or by employing BBr3 in

CH2Cl2 (no conversion). The reaction was, however, carried out smoothly and in high yield upon treatment with 6 equivalents of AlCl3 refluxing in freshly distilled benzene under a nitrogen atmosphere, yielding N-[2-[4-(2-hydroxyphenyl)-1- piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (2.6).48 Derivatisation of the hydroxyl group of compound 2.6 was accomplished either with the appropriate alkylbromides in the presence of Cs2CO3, yielding N-[2-[4-(2-(2- fluoroethoxy)phenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (2.7) and N-[2-[4-(2-(3-fluoropropoxy)phenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (2.8), or with methanesulfonyl chloride in the presence of triethylamine, yielding N-[2-[4-[2-[methanesulfonoxyl]phenyl]-1-piperazinyl]ethyl]- N-(2-pyridinyl) cyclohexane carboxamide (2.9) or with N-phenyl-trifluoromethane sulfonimide using phase-transfer-conditions with 10% NaOH (aq) and CH2Cl2, in the presence of the phase-transfer-catalyst tetrabutylammonium iodide, yielding N-[2-[4- [2-[[(trifluoromethyl)sulfonoxyl]phenyl]-1-piperazinyl]ethyl]-N-(2- pyridinyl)cyclohexane carboxamide (2.10) (Scheme 2.2).

72

N N

abN N 2.2 O R O OH N O N 2.6 2.7, R = (CH ) F N N 2 2 2.8, R = (CH2)3F 2.9, R = SO2CH3 2.10, R = SO CF 2 3 SCHEME 2.2 Reagents and conditions: (a) aluminum chloride, benzene, ∆; (b) fluoroethyl / fluoropropyl bromide, cesium carbonate, acetonitrile, ∆ (2.7/2.8); methanesulfonyl chloride, TEA, CH2Cl2, room temperature (2.9); N-phenyltrifluoromethane sulfonimide, tetrabutylammonium iodide, 10% NaOH(aq)/CH2Cl2, room temperature (2.10).

SYNTHESIS OF BENZ(SULFON)AMIDE ANALOGUES OF WAY100635

N-acylation of the intermediate 2.2c was accomplished using 4-acetoxybenzoyl chloride in dichloromethane in the presence of triethyl amine, yielding 4-(2’- methoxyphenyl)-1-[2’-N-(2’’-pyridinyl)-p-acetylbenzamido]-ethyl]-piperazine (2.11, p-MPPOAc). Mild basic conditions, potassium carbonate in aqueous MeOH, were used for the de-acetylation of p-MPPOAc - in order to prevent hydrolysis of the amide-bond – providing 4-(2’-methoxyphenyl)-1-[2’-N-(2’’-pyridinyl)-p- hydroxylbenzamido]-ethyl]-piperazine (2.12, p-MPPOH). Triflation of p-MPPOH with N-phenyltrifluoromethane sulfonimide using phase-transfer conditions with 10% aq NaOH and CH2Cl2 in the presence of tetrabutylammonium iodide (PTC), provided N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)-4-trifluorosulfonxy- phenyl carboxamide (2.13, p-MPPOTf). For the preparation of the sulfonamide, the intermediate 2.2c was reacted with pipsyl chloride in refluxing pyridine, yielding 4- (2’-methoxyphenyl)-1-[2’-N-(2’’-pyridinyl)- N-(p-iodobenzyl)sulfonamido]-ethyl]- piperazine (2.14) (Scheme 2.3).49

SYNTHESIS OF ARYLAMINOPIPERIDINE ANALOGUES OF WAY100635

For the synthesis of 4-N-(o-methoxyphenyl) analogues, 2-methoxyaniline was coupled to 1- benzyl-4-piperidone in refluxing in the presence of p- toluenesulfonic acid, hydrogenated using Adam's catalyst, and debenzylated by applying H2 (60 psi) in the presence of palladium hydroxide on carbon in a mixture of MeOH/THF, yielding 4-N-(2-methoxyphenyl)amino piperidine (2.15a). This intermediate was then coupled to 2-chloro-N-pyridin-2-yl acetamide27 in refluxing acetonitrile in the presence of triethyl amine, potassium carbonate and tetrabutylammonium iodide, yielding 2-[4-N-(o-methoxyphenyl)amino-piperidinyl]- N-pyridin-2-yl-acetamide (2.15b). N-Methylation of 2.15b was achieved with reductive amination in a Parr-shaker using formaldehyde in EtOH in the presence of

73 palladium hydroxide (20 %) on carbon, yielding 2-[4-N-(o-methoxyphenyl)-N- methylamino-piperidinyl]-N-pyridin-2-yl-acetamide (2.15c). The acetamide was reduced in ether by using DIBAL-H, yielding 2-[4-N-(o-methoxyphenyl) -N- methylamino -piperidinyl]-N-pyridin-2-yl-amine (2.15d), which was subsequently coupled to cyclohexane carboxylic acid chloride in CH2Cl2 in the presence of triethylamine yielding 2-[4-N-(o-methoxyphenyl) -N-methylamino -piperidinyl]-N-(2- pyridinyl) cyclohexane carboxamide (2.15). Reduction of intermediate 2.15b in ether by using DIBAL-H, yielded 2-[4-N-(o-methoxyphenyl)amino-piperidinyl]-N-pyridin- 2-yl-amine (2.16a). Reacting 2.16a with cyclohexane carboxylic acid chloride in

CH2Cl2 in the presence of triethylamine yielded [4-N-(o-methoxyphenyl) -N- cyclohexanecarboxamidepiperidinyl]-N-(2-pyridinyl) cyclohexane carboxamide (2.16) (Scheme 2.4).

H C 3 N O O R a N 2.11, R = COCH 2.2a N 3 b N 2.12, R = H O c d 2.13, R = SO2CF3

H3C O N I N N N S O O 2.14

SCHEME 2.3 Reagents and conditions: (a) 4-acetoxybenzoyl chloride, TEA, CH2Cl2, room temperature; (b) K2CO3, MeOH/H2O, room temperature; (c) N-phenyltrifluoromethane sulfonimide, NaOH (10% aq), tetrabutylammonium iodide, CH2Cl2, room temperature;(d) pipsyl chloride, pyridine, reflux.

2.3 PHARMACOLOGY

The affinity of test compounds 2.5-2.12 and 2.15 and 2.16 for the 5-HT1A receptor was established by measuring their ability to displace in vitro the radioligand [3H]-8- 50 OH-DPAT. In addition, the pA2-values of test compounds 2.5-2.12 and 2.15 and 2.16 were determined from their ability to antagonize the 8-OH-DPAT-induced contraction of a strip of guinea pig ileum smooth muscle. (Table 2.1). 51, 52

To assess presynaptic activity at the serotonin 5-HT1A receptors in vivo, compounds 2.5-2.9 and 2.15 and 2.16 were evaluated in a ultrasonic vocalization assay for their ability to antagonize the 8-OH-DPAT-induced inhibition of footshock-evoked ultrasonic vocalization in rats (Table 2.2).31, 32, 53

To evaluate postsynaptic activity at the serotonin 5-HT1A receptors in vivo, the effects of compounds 2.5-2.12 and 2.14 on 8-OH-DPAT-induced hypothermia in rats (Table

74 2.3), and 8-OH-DPAT-induced ‘5-HT syndrome’, including on-set of flat body posture (FBP), locomotor activity (LA) and lower lip retraction (LLR) were measured (Table 2.4).29, 54-56 For clarity, the deviations of core temperature from those of the respective placebo-treated control groups are presented instead of the original temperature curves. Both WAY100635 and its triflate analogue 2.9 were evaluated for agonistic properties an in vivo biochemical assay, in which the accumulation of 5-hydroxytryptophan (5-HTP) is used as an indicator for the 5-HT turnover (Figure 2.1).45-47

H O O O N N a NH b N O NH N N 2 H H 2.15a 2.15b

c d

H H O N N O N N N N O N N 2.15c H 2.16a

d e

N H O N N O N N N O N N 2.15d 2.16 O e

N

O N N O N 2.15

SCHEME 2.4 Reagents and conditions: (a) i: p-TsOH, toluene, reflux; ii: H2 (50 psi), PtO2, o o toluene, 60 C; iii: H2 (60 psi), 20% Pd(OH)2/C, MeOH/THF, 60 C; (b) 2-chloro-N-pyridin-

2-yl acetamide, K2CO3, tetrabutylammonium iodide, acetonitrile, reflux; (c) HCHO, H2 (60 o o psi), 20% Pd(OH)2/C, EtOH, 60 C; (d) DIBAL-H, Et2O, 0 C to room temperature; o (e) cyclohexanecarboxylic acid chloride, TEA, CH2Cl2, 0 C to room temperature.

75 2.4 RESULTS AND DISCUSSION

Structure-affinity relationships (SARs) were determined from affinity values obtained using radioligand binding for the 5-HT1A (Table 2.1). None of the intermediates showed affinity for this receptor subtype. The affinity of the o- substituted phenylpiperazine analogues, in which the methoxy group of WAY100635 has been replaced by a hydroxy (2.6), a fluoroalkoxy (2.7, 2.8) a methanesulfonoxy (2.9) or the even stronger electron withdrawing trifluoromethanesulfonoxy group

(2.10) for the serotonin 5-HT1A receptor remains in the nanomolar range. Compared to WAY100635, test compounds 2.6-2.9 are ca. ten-fold less potent in displacing the 3 [ H]-8-OH-DPAT from the 5-HT1A receptor. The decrease in in vitro affinity for the

5-HT1A receptor of the triflate analogue 2.10, as compared to WAY100635, is a hundred-fold. These binding results suggest that the serotonin 5-HT1A receptor site can accommodate larger substituents than the methoxy group. Thus, the fluoroalkyloxy analogues offer the opportunity to introduce 18F, resulting in PET- 11 ligands with a much longer half-life than C (t1/2 of 109 min vs. 20 min. respectively).57, 58 The aryl-triflate, which is known to make the phenyl ring less prone to metabolic degradation, can serve as an alternative to aromatic methoxy substituents, present in several compounds with CNS activity, improving their oral 59-62 availbility. The 5-HT1A receptor affinity of the brominated analogue 2.5 is a hundred-fold less as compared to WAY100635. Because of the use of different radioligands and binding assays, it is difficult to compare the affinities of the benzamides p-MPPI (2.3) and p-MPPF (2.4) with

WAY100635 and test compounds 2.11 and 2.12 for the 5-HT1A receptor. The affinity of the p-acetyl and p-hydroxyl analogues 2.11 and 2.12, respectively, appears to be in the same range as the o-substituted analogues 2.6-2.9, i.e. ca ten-fold less potent than WAY100635. Despite the facts that the affinity of p-MPPI and p-MPPF is determined by their ability to displace the radioligand [125I]-8-OH-PIPAT and that the values are given as Ki (in nM), it appears that their affinity for the 5-HT1A receptor is in the same range as test compounds 2.11 and 2.12.

In vitro functional data (pA2 values) were obtained using the guinea pig ileum assay, in which the ability of the test compounds (2.5-2.12 and 2.15 and 2.16) to antagonize the 8-OH-DPAT-induced contractions of a strip of ileum tissue was measured (Table 2.1).49 The data obtained with this model show that the o-substituted analogues 2.6- 2.9 are potent antagonists. The triflate analogue 2.10 is a ten-fold less potent, while the brominated analogue 2.5 is unable to antagonize the 8-OH-DPAT-induced contractions at reasonable concentrations. The pA2 values of the p-acetyl and p- hydroxyl benzamides 2.11 and 2.12 are conflicting. From these in vitro data it should be concluded that the p-acetyl analogue 2.11 is a potent antagonist, while the p- hydroxyl analogue 2.12 is not. The aminopiperidines 2.15 and 2.16 showed no affinity for the serotonin 5-HT1A receptor and are not able to antagonize the 8-OH-DPAT induced contractions in ileum tissue (pA2).

76

TABLE 2.1 In vitro affinities at 5-HT1A receptors (IC50 + SEM in nM, n = 3) and a pA2-values.

b compound 5-HT1A pA2 WAY100635 0.37 + 0.02 8.7 2.5 50d <7 2.6 2.10 + 0.5 8.0 2.7 1.55 + 0.6 8.5 2.8 6.05 + 0.2 8.3 2.9 7.25 + 2.7 8.6 2.10 36.0 + 13.5 7.7 p-MPPI 2.6 + 0.7c NTe p-MPPF 3.3 + 0.8c NT 2.11 6.0d 8.2 2.12 4.7 + 1.8 <7 2.15 1290 + 1330 <7 2.16 500 + 266 <7 a b 3 c vs 8-OH-DPAT (guinea pig ileum); [ H]-8-OH-DPAT (rat hippocampus); Ki values in nM from ref. 44 vs [125I]-8-OH-PIPAT; d n = 1; e NT means not tested.

TABLE 2.2 Inhibition of ultrasonic vocalization after administration of 8-OH-DPAT (0.1 mg/kg sc) alone, or in combination with WAY100635 or test compounds 2.5 - 2.10 and 2.16 (0.3 mg/kg sc).

treatment (n = 4-8) vocalization time (s + SEM) control 148 + 11 8-OH-DPAT 4.9 + 4.1 WAY100635 158 + 11* 2.5 7.0 + 6.3§ 2.6 166 + 11* 2.7 164 + 8* 2.8 171 + 4* 2.9 167 + 3* 2.10 146 + 12* 2.16 7.4 + 5.8§ * p < 0.05 vs 8-OH-DPAT; § p < 0.05 vs control.

In vivo functional data of the test compounds were obtained using the rat ultrasonic vocalization model (Table 2.2), the rat hypothermia model and gross behavioral changes in the 8-OH-induced ‘5-HT syndrome’ (Tables 2.3 and 2.4). Inhibition of ultrasonic vocalization by serotonin 5-HT1A receptor agonists results from activation of presynaptic (somatodendritic) receptors, because local (as well as systemic) injections inhibit firing of raphé nuclei neurons and reduce ultrasonic vocalization

77 even after hippocampal lesions.63-65 WAY100635, as well as compounds 2.6 - 2.10, did antagonize the 8-OH-DPAT-induced inhibition of ultrasonic vocalization at 0.3 mg/kg sc. test compound vs. 0.1 mg/kg agonist, demonstrating their ability to act as autoreceptor antagonists in vivo. This suggests that the aryl piperazine analogues are all able to cross the blood brain barrier and exert their effect at the somatodendritic 5-

HT1A receptors in the raphé nuclei. The brominated analogue 2.5 and the aminopiperidine 2.16, however, were not able to counteract the agonist-induced inhibition at the doses tested. This is in line with the in vitro obtained pA2 values.

TABLE 2.3 Effects of WAY100635 and test compounds 2.5 - 2.12 and 2.14 on 8-OH-DPAT (1 mg/kg sc)-induced hypothermia (n = 4).

decrease in body temperature (oC) compound 0.1 (µmol/kg) 1.0 (µmol/kg) 10.0 (µmol/kg) WAY100635 1.5 + 0.2 0.9 + 0.1* 0.9 + 0.0* 2.5 NT NT 2.3 + 0.1 2.6 0.6 + 0.0* 0.2 + 0.1* 0.3 + 0.1* 2.7 1.8 + 0.1 1.1 + 0.1 0.4 + 0.2* 2.8 2.4 + 0.1 0.6 + 0.1* 0.5 + 0.2* 2.9 1.1 + 0.3 0.5 + 0.2* 0.4 + 0.0* 2.10 1.5 + 0.2 0.0 + 0.1* 0.3 + 0.1* 2.11 2.6 + 0.3 2.6 + 0.1# 1.7 + 0.1 2.12 2.2 + 0.2 2.5 + 0.2 1.9 + 0.1 2.14 2.5 + 0.0# 2.1 + 0.1 2.1 + 0.1 control 0.0 + 0.1 8-OH-DPAT 2.0 + 0.1 * p < 0.05 vs control; # p < 0.05 vs 8-OH-DPAT

The decrease in body temperature, as observed in rats treated with serotonin 5-HT1A receptor agonists, is postsynaptically mediated, because hypothermia is still present following depletion of endogenous 5-HT.29, 66-68 In the hypothermia assay, a high dose of 8-OH-DPAT (1 mg/kg; 3.5 µmol/kg sc.) is chosen, to ensure full agonist receptor occupancy. The O-desmethyl aryl piperazine analogue 2.6 proved to be the most potent functional postsynaptic antagonist in this model. It significantly antagonized the 8-OH-DPAT-induced hypothermia at the lowest dose tested (0.1 µmol/kg). WAY100635 and the fluoropropyl (2.8), the mesylate (2.9) and the triflate (2.10) analogues are equipotent in this assay. They were all able to significantly block the decrease in body temperature at 1.0 µmol/kg sc. Of the o-substituted , the fluoroethyl analogue 2.7 was the weakest antagonist in this post-synaptic 5-HT1A activation model. It only blocked the decrease in body temperature significantly at the highest dose tested (10 µmol/kg sc.). The bromine analogue 2.5, as can be expected from the in vitro obtained pA2 value, does not inhibit the 8-OH-DPAT induced decrease in core temperature. The benz(sulfon)amide

78 analogues 2.11, 2.12 and 2.14 also do not block the induced temperature decrease. In fact, the appear to decrease the body temperature of rats even further, thus acting as an agonists at the postsynaptic 5-HT1A receptors. The 5-HT syndrome, of which lower lip retraction (LLR), a flat body posture (FBP) and locomotor activity (LA) are well-known features, is induced through activation of 52-54 postsynaptic 5-HT1A receptors. The findings in the temperature assay are also reflected in the behavioral changes, where the 8-OH-DPAT-induced LLR, FBP and LA are fully antagonized by doses of test compounds that are able to significantly block the induced hypothermia. In this assay, O-desmethyl analogue 2.6 is again the most potent antagonist, followed by the mesylate analogue 2.9, which is able to antagonize the induced LA at the lowest dose tested. Both the fluoroethyl analogue 2.7 and the triflate analogue 2.10 are able to fully antagonize the ‘5-HT syndrome’ at 1.0 µmol/kg. WAY100635 and the fluoropropyl analogue antagonize all three features of the 5-HT syndrome at the highest dose of 10 µmol/kg. Of the three markers of the 5-HT syndrome, the locomotor activity is the most sensitive towards the antagonistic actions of the test compounds, while the lower lip retraction appears to be the most sensitive towards the agonistic actions of 8-OH-DPAT.

TABLE 2.3 Effects of WAY100635 and test compounds 2.6 - 2.12 and 2.14 on 8-OH-DPAT (1 mg/kg sc)-induced ‘5-HT syndrome’ (n = 4) within 5 min after administration of the agonist.

0.1 µmol/kg 1.0 µmol/kg 10.0 µmol/kg compound LLR FBP LA LLR FBP LA LLR FBP LA control − − − − − − − − − 8-OH-DPAT + + + + + + + + + WAY100635 + + + 1/4 − − − − − 2.6 + 3/4 − − − − − − − 2.7 + + + 2/4 2/4 - − − − 2.8 + + + − − − − − − 2.9 + + − − − − − − − 2.10 + + 1/4 − − − − − − 2.11 + + + + + + 1/4 1/4 − 2.12 + + + + + + + + + 2.14 + + + + + + + + + N.B. − = 0/4; + = 4/4

In literature, both p-MPPI and p-MPPF are described as antagonists at both pre- and postsynaptic 5-HT1A receptors. The ID50 values of p-MPPI and p-MPPF in 8-OH- DPAT induced hypothermia (0.5 mg/kg ip) are approximately 9 and 7 µmol/kg.44, 69-73 Surprisingly, at the highest dose of 10 µmol/kg, neither the p-acetyl and p-hydroxyl benzamides 2.11 and 2.12, nor the p-iodosulfonbenzamide 2.14 were able to significantly antagonize the 8-OH-DPAT (1.0 mg/kg sc) induced hypothermia. In the

79 gross behavioral studies, only the p-acetyl benzamide analogue 2.11, at 10 µmol/kg, is able to fully block the induced LA and partly antagonize the induced FBP and LLR. It has been claimed in literature, that WAY100635 is a full, silent antagonist.42 To establish the absence of partial agonistic activity, the effect of both WAY100635 and its o-triflate analogue 2.10 were evaluated using a supersensitive in vivo biochemical 5-HTP measurement assay. Compound 2.10 is selected for this assay for, from the in vitro pA2 value (7.7) and the in vivo effects in the pre- and postsynaptic assays, it appears to be the weakest, but still a full antagonist. The 5-HTP assay utilizes the well-established phenomenon of autoreceptor-mediated inhibition of the serotonergic neuron. The synthesis rate of 5-HT is inhibited by directly acting 5-HT1A receptor agonists. 5-HTP accumulation, following decarboxylase inhibition by 3- hydroxylbenzyl hydrazine (NSD 1015), is used as an indicator for the 5-HT turnover in the different brain areas (Figure 2.1). Rats are pre-treated with reserpine to induce supersensitivity of the system.55, 56 WAY100635, at 10 µmol/kg sc., appears to have a weak inhibitory effect on the 5-HT turnover in the nucleus accumbens, the striatum and tubercule (olfactory structure, significant). The triflate analogue 2.10 had no effect on the 5-HTP accumulation in any of the brain areas. These results indicate that WAY100635, despite previous claims, is not a full antagonist.

FIGURE 2.1 Effects of WAY100635 (2.2) and test compounds 2.10 on 5-HTP accumulation in different brain areas of the rat. The animals received reserpine 24 h before the test drug (10 µmol/kg sc.), which was given 60 min before decapitation, and NSD 1015 (30 min before decapitation).

0.75 control 5-HTP ( 2.2

µ 2.10 gram/gram wet tissuue wet gram/gram 0.50 *

0.25

0.00 accumbens striatum tubercule hippocampus cortex

The in vivo results clearly show that the in vitro data obtained are not able to discriminate the most potent serotonin 5-HT1A receptor antagonist in vivo. The subnanomolar affinity of WAY100635 for the serotonin 5-HT1A receptor binding site, combined with the highest pA2, value do not ensure that in vivo, it is indeed the most potent serotonin 5-HT1A antagonist. In fact, in vivo in the post-synaptic 8-OH-DPAT- induced hypothermia assay, WAY100635 is equipotent to the triflate analogue 2.10,

80 which, in vitro, is the weakest aryl piperazine. The discrepancy between the in vitro and in vivo data and results may be due to differences in pharmacokinetic, pharmacodynamic and/or metabolic profiles of the different test compounds and WAY100635.

To summarize, a small series of structural analogues of WAY100635 have been synthesized and their in vitro and in vivo pharmacology has been evaluated. The substituent in the ortho position of the phenyl piperazine can be both smaller and larger than the original methoxy substituent of WAY100635 and have strong electron withdrawing properties (2.6-2.10). Bromination of WAY100635 para of the methoxy substituent (2.5) reduces the affinity for the 5-HT1A receptor significantly, and the antagonistic properties are fully abolished. In the 8-OH-DPAT-induced hypothermia assay, WAY100635 is equipotent to the fluoropropyl analogue 2.7 (a possible [18F]PET ligand), and the triflate analogue 2.10. However, the O-desmethyl analogue 2.6, which has been shown to be a very promising radioligand in PET studies,44 is the functionally most potent serotonin 5-HT1A antagonist at the postsynaptic receptor sites.

The benzamide analogues 2.11 and 2.12 show a similar binding profile for the 5-HT1A receptor as the o-substituted analogues. The pA2 values of the p-acetyl and p-hydroxyl benzamides, however, are conflicting. It is expected that these benzamides are antagonists, for their structural analogues p-MPPI and p-MPPF are reported to be full antagonists at both pre- and postsynaptic 5-HT1A receptors. In vivo, however, in the 8- OH-DPAT-induced hypothermia and gross behavioral studies, neither the p-acetyl and p-hydroxyl benzamides 2.11 and 2.12 nor the sulfonbenzamide analogue 2.14 are able to fully antagonize the effects of the agonist 8-OH-DPAT - bar the highest dose of 2.11 in the 5-HT syndrome. Because both p-MPPI and p-MPPF are used as antagonists in visualization studies of the 5-HT1A receptor, a more elaborate in vivo study of the antagonistic profile of the benzamides vs WAY100635 both in vitro and in vivo appears to be justified. Substitution of the arylpiperazine for an arylaminopiperidine (2.15 and 2.16) sharply reduces the affinity for the 5-HT1A receptor while antagonistic properties, both in vitro

(pA2) and in vivo (ultrasonic vocalization) are fully abolished. Finally, the results in the sensitive biochemistry assay show that in brain areas as the nucleus accumbens, the striatum and the tubercule, WAY100635 is not a full antagonist.

2.5 EXPERIMENTAL

CHEMISTRY Melting points were determined with a Electrothermal digital capillary melting point apparatus and are uncorrected. NMR spectra were acquired on a Varian Gemini-200 spectrometer at

200 MHz (proton) and 50.3 MHz (carbon). CDCl3 was employed as the solvent unless otherwise stated. Chemical shifts are given in δ units (ppm) and relative to tetramethylsilane or deuterated solvent. Infrared spectra were recorded with a ATI-Mattson spectrometer. Elemental analyses were performed

81 in the labs of Merck KGaA Darmstadt (D) or in the Microanalytical Department of the University of Groningen (NL) and were within + 0.4% of theoretical values unless otherwise indicated. GC-MS (EI and CI) mass spectra were recorded on a Unicam610/ Automass 150 GC/MS system (70 mV). HRMS- spectra were performed in the Microanalytical Department of the University of Groningen (NL), and were recorded on a JEOL The MS Route JMS 600H. For column chromatography silica gel 60 (70 - 230 mesh) (Merck) was used. Reactions were carried out under a nitrogen atmosphere, solvents used were distilled and/or dried by standard techniques immediately prior to use and in case of an aqueous work-up, magnesium sulfate mono-hydrate was used as the drying agent.

N-2-(2-Chloroethyl)amidopyridine (2.2a) 2-Aminopyridine (1.00 g, 10.6 mmol), chloroacetyl chloride (1.80 g, 15.9 mmol) and triethyl amine (1.61 g, 15.9 mmol) are dissolved in CH2Cl2 (100 mL). The reaction mixture is stirred at room temperature for 17 h, after which the organic phase is washed with an aqueous solution of NaHCO3 (10%). The organic layer is dried and concentrated in vacuo. The residue is purified using flash chromatography (CH2Cl2), yielding 1.50 g (83%) of 2.2a as a pinkish 1 solid (CAUTION, IRRITATING); H NMR δ 8.24 (d, J = 4.89, 1H), 8.15 (d, J = 8.30, 1 H), 7.77- 13 7.68 (dt, J1 = 7.51, J2 = 1.90, 1 H), 7.11-7.04 (dt, J1 = 7.39, J2 = 0.92, 1 H), 4.16 (s, 3 H); C NMR δ 165.0, 150.1, 147.4, 138.8, 120.4, 114.2, 42.5; IR (NaCl) 1720 cm-1; HRMS Calcd (Obsd) for

C7H7N2OCl: 170.025 (170.025).

N-2-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]amidopyridine (2.2b) A suspension of 2.2a (1.20 g, 7.00 mmol), 1-(2-methoxyphenyl)piperazine (1.35 g, 7.00 mmol) and K2CO3 (2.5 g, 18.0 mmol) in

DMF (50 mL) is stirred at 60 ˚C for 2.5 h. The reaction is quenched with H2O (200 mL) and extracted with EtAc (3 x 150 mL). The combined organic layers are washed with brine, dried and removed in vacuo. The residue is purified using gradient flash chromatography (hexane to EtAc), yielding 1.90 g (83%) of 2.2b as a light yellow oil; 1H NMR δ 8.32-8.23 (m, 2H), 7.71-7.70 (m, 1 H), 7.07-6.84 (m, 5 H), 3.85 (s, 3 H), 3.22 (s, 2H), 3.19-3.14 (m, 2H), 2.86-2.79 (m, 6H); 13C NMR δ 169.2, 152.1, 150.9, 147.9, 140.8, 138.2, 123.1, 120.9, 119.7, 118.2, 113.7, 111.0, 62.0, 55.2, 53.6, 50.4; IR (NaCl) 1696 -1 cm ; HRMS Calcd (Obsd) for C18H22N4O2: 326.174 (326.174).

N-[2-[4-(2-Hydroxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)amine (2.2c) A suspension of 2.2b

(1.75 g, 5.40 mmol) and LiAlH4 (0.61 g, 16.10 mmol) in THF (100 mL) is stirred at room temperature for 2 h. The reaction is quenched by adding sequentially, 1 x H2O (0.6 mL), 1 x NaOH (10% aq, 0.6 mL) and 3 x H2O (0.6 mL), after which the mixture is stirred for another 30 min at room temperature. After filtration, the solvent is dried and concentrated in vacuo, yielding 1.60 g (95.5%) of 2.2c as a colorless oil, which is used without further purification; 1H NMR δ 8.33-8.10 (m, 1H), 7.48-7.39 (m, 1 H), 7.08-6.87 (m, 4 H), 6.62-6.55 (m, 1H), 6.45 (d, J = 8.54, 1H), 3.88 (s, 3 H), 3.48-3.39 (m, 2H), 3.19-3.12 (m, 4H), 2.78-2.72 (m, 6H); 13C NMR δ 151.4, 148.0, 137.3, 135.6, 128.1, 122.9, 120.9, 118.1, 112.6, 111.0, 107.1, 56.6, 55.1, 52.9, 50.3, 38.2; IR (NaCl) 1600 cm-1; HRMS Calcd (Obsd) for

C18H24N4O: 312.195 (312.196).

N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (WAY100635, 2.2) A solution of 2.2c (1.5 g, 4.9 mmol), cyclohexanecarbonyl chloride (1.1 g, 7.3 mmol) and TEA (0.7 g, 7.3 mmol) in CH2Cl2 (50 mL) is stirred at room temperature for 17 h, after which the organic phase is washed with an aqueous solution of NaHCO3 (10%). The organic layer is dried and concentrated in vacuo. The residue is purified using flash chromatography (CH2Cl2), yielding 1.42 g (69%) of 2.2 as a colorless oil, which was converted to the oxalate salt and recrystallized from 1 EtOH; mp 188-191 ˚C; H NMR δ 8.53-8.49 (dd, J1 = 5.25, J2 = 1.60, 1 H), 7.92-7.71 (dt, J1 = 7.72, J2 = 2.00, 1 H), 7.31-7.19 (m, 2 H), 7.02-6.81 (m, 4 H), 3.98 (t, J = 6.96, 2 H), 3.82 (s, 3H), 2.98 (m, 4 H), 2.64-2.57 (m, 6 H), 2.28-2.15 (m, 1 H), 1.76-1.42 (m, 7 H), 1.26-0.98 (m, 3 H); 13C NMR δ 176.3, 155.8, 152.2, 149.2, 141.2, 138.1, 122.8, 122.3, 122.2, 120.9, 188.0, 111.2, 56.1, 55.3, 53.3, 50.5, 45.1,

82 -1 +1 42.3, 29.5, 25.6; IR (NaCl) 1659 cm ; MS (CI with NH3) m/z 423 (M ); HRMS Calcd (Obsd) for

C24H32N4O2: 422.268 (422.269); Anal (C25H34N4O2.C2H2O4) C, H, N.

N-[2-[4-(3-Bromo-6-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (2.5) To a solution of 2.2 mono-oxalate (0.10 g, 0.24 mmol) in a mixture of

EtOH/MeOH (1/1 v/v, 6 mL) is added a solution of bromine (0.39 g, 0.24 mmol) in CH2Cl2 (6 mL). The reaction is stirred for 24 h at room temperature, after which the solvent is removed in vacuo. The residue was purified by flash chromatography (CH2Cl2 with 10% MeOH), converted to the oxalate salt and recrystallized from EtOH to yield 56 mg (49%) of 2.5 as white crystals: ° 1 mp 156-159 C; H NMR (CD3OD) δ 8.54-8.51 (dd, J1 = 4.88, J2 = 0.98, 1 H), 7.93 (m, 1 H), 7.57-7.53 (m, 1 H), 7.44-7.36 (m, 1 H), 7.23 (d, J = 5.37, 2H), 7.09 (s, 1H), 4.06-4.00 (t, J = 5.74, 2 H), 3.85 (s, 3H), 3.79-3.38 (m, 10 H), 1.71-1.42 (m, 1 H), 1.28-0.96 (m, 7 H); HRMS Calcd (Obsd) for

C25H33N4O2Br: 500.179 (500.176); Anal.( C25H33N4O2Br.C2H2O4) C, H, N.

N-[2-[4-(2-Hydroxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (2.6) A suspension of WAY100635 (50.0 mg, 118 µmol) and AlCl3 (95.0 mg, 700 µmol) in benzene (5 mL) was refluxed for 2 h. The reaction was quenched with H2O (5 mL) and neutralized with solid NaHCO3.

The mixture was extracted with CH2Cl2, (3 x 20 mL) and the combined organic layers were washed with brine, dried and concentrated in vacuo, to yield 45 mg (93%) of a colorless oil, which was converted to the oxalate salt and recrystallized from i-PrOH yielding 43 mg (73%) of 2.6 as white ° 1 crystals; mp 204-207 C; H NMR δ 8.56-8.53 (dd, J1 = 4.96, J2 = 1.78, 1 H), 7.84-7.75 (dt, J1 = 7.75, J2 = 2.01, 1 H), 7.31-7.23 (m, 2 H), 7.14-7.03 (m, 2 H), 6.96-6.81 (m, 2 H), 4.03-3.96 (t, J = 6.93, 2 H), 2.82-2.78 (m, 4 H), 2.67-2.60 (m, 6 H), 2.31-2.18 (m, 1 H), 1.79-1.57 (m, 7 H), 1.26-0.98 (m, 3 H); 13C NMR δ 176.1, 151.5, 149.3, 138.9, 138.0, 126.4, 122.1, 119.9, 114.0, 100.0. 56.1, 53.8, 52.5, 45.1, -1 +1 42.4, 29.5, 25.6; IR (NaCl) 3299, 1659 cm ; MS (CI with NH3) m/z 409 (M ); HRMS Calcd (Obsd) for C24H32N4O2: 408.253 (408.253); Anal (C24H32N4O2.C2H2O4.0.5 H2O) C, H; N.

N-[2-[4-(2-(2-Fluoroethoxy)phenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide (2.7) To a suspension of 2.6 (0.10 g, 0.25 mmol) and Cs2CO3 (0.60 g, 1.84 mmol) in acetonitrile (30 mL), fluoroethyl bromide (0.05 g, 0.40 mmol) was added. After refluxing for 3 h, the solvent was removed in vacuo. The residue was redissolved in H2O and extracted with CH2Cl2. The combined organic layers were washed with brine, dried and concentrated in vacuo. The oil obtained was converted to the oxalate salt and recrystallized from EtOH, yielding 60 mg (45%) of 2.7 as white o 1 crystals; mp 207 - 209 C; H NMR δ 8.55-8.52 (dd, J1 = 5.25, J2 = 1.59, 1 H), 7.81-7.72 (dt, J1 = 7.70,

J2 = 2.06, 1 H), 7.32-7.21 (m, 2 H), 6.99-6.82 (m, 4 H), 4.88 (t, J = 4.11, 1 H), 4.64 (t, J = 4.13, 1 H), 4.31 (t, J = 4.09, 1 H), 4.17 (t, J = 4.13, 1 H), 3.99 (t, J = 6.96, 2 H), 3.10-2.95 (m, 4 H), 2.65-2.58 (m, +1 6 H), 2.24-2.18 (m, 1 H), 1.89-0.83 (m, 10 H); IR (KBr) 1658; MS (CI with NH3) m/z 455 (M );

HRMS Calcd (Obsd) for C26H35N4O2F1: 454.274 (454.274); Anal. (C26H35N4O2F1.C2H2O4.0.25H2O) C, H, N.

N-[2-[4-(2-(2-Fluoropropoxy)phenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide (2.8). To a suspension of 2.6 (0.02 g, 0.05 mmol) and Cs2CO3 (0.08 g, 0.25 mmol) in acetonitrile (4 mL), fluoropropyl bromide (0.08 g, 0.06 mmol) was added. After refluxing for 2 h, the solvent was removed in vacuo. The residue was redissolved in H2O and extracted with CH2Cl2. The combined organic layers were washed with brine, dried and concentrated in vacuo. The oil obtained was converted to the oxalate salt and recrystallized from EtOH yielding 17 mg (62%) of 2.8 as white ° 1 crystals; (mono-oxalate salt) mp 181 - 183 C; H NMR δ 8.55-8.52 (dd, J1 = 5.22, J2 = 1.61, 1 H),

7.81-7.72 (dt, J1 = 7.70, J2 = 1.93, 1 H), 7.32-7.21 (m, 2 H), 7.02-6.84 (m, 4 H), 4.78 (t, J = 5.82, 1 H), 4.55 (t, J = 5.80, 1 H), 4.12 (t, J = 6.63, 1 H), 4.00 (t, J = 6.96, 1 H), 3.00 (m, 4 H), 2.71-2.58 (m, 6 H), 2.27 (t, J = 5.96, 1 H), 2.14 (t, J = 5.96, 1 H), 1.93 (m, 1 H), 1.79-1.52 (m, 7 H), 1.26-0.97 (m, 3 H);

83 13C NMR δ 176.2, 156.0, 149.2, 141.5, 138.0, 131.0, 122.7, 122.3, 122.2, 121.3, 118.2, 82.5, 79.3 ,

63.8, 63.7, 56.2, 53.4, 50.6, 45.1, 42.4, 30.9, 30.5, 29.5, 25.6; IR (KBr) 1656; MS (CI with NH3) m/z +1 469 (M ); HRMS Calcd (Obsd) for C26H35N4O2F1: 468.290 (468.290); Anal.

(C26H35N4O2F1.C2H2O4.0.5H2O) C, H, N.

N-[2-[4-[2-[Methanesulfonoxyl]phenyl]-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide (2.9) To a cooled solution (-78 oC) of 2.6 (0.05 g, 0.12 mmol) and triethylamine (10 µL) in CH2Cl2 (2 mL), methanesulfonyl chloride (0.02 g, 0.18 mmol) was slowly added. The reaction was allowed to warm to room temperature, after which it was stirred for another 6 h. The mixture was quenched with H2O and extracted with CH2Cl2. The combined organic layers were washed with brine, dried and concentrated in vacuo. The residue was purified by chromatography (CH2Cl2 with 3% MeOH), converted to the oxalate salt and recrystallized from EtOH to yield 30 mg (43 %) of 2.9 as o 1 white crystals: mp 234-237 C; H NMR δ 8.55-8.52 (dd, J1 = 5.49, J2 = 1.93, 1 H), 7.82-7.73 (dt, J1 =

7.71, J2 = 2.00, 1 H), 7.32-7.20 (m, 4 H), 7.08-7.00 (m, 2 H), 3.98 (t, J = 6.89, 2 H), 3.15 (s, 3 H), 3.02- 2.98 (m, 4 H) 2.65-2.58 (m, 6 H), 2.32-2.15 (m, 1 H), 1.77-0.96 (m, 10 H); 13C NMR δ 176.3, 155.8, 149.3, 144.7, 142.9, 138.1, 128.2, 124.6, 123.3, 122.2, 122.1, 119.8, 56.1, 53.4, 51.0, 45.0, 42.3, 38.3,

29.5, 25.5; IR (KBr) 1657, 1366, 1157; HRMS Calcd (Obsd) for C25H34N4O4S1: 486.230 (486.230);

Anal. (C25H34N4O4S1.C2H2O4.0.5H2O) C, H, N.

N-[2-[4-[2-[[(Trifluoromethyl)sulfonoxyl]phenyl]-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (2.10) To a suspension of 2.6 (0.10 g, 0.20 mmol) and tetrabutylammonium iodide (10 mol%) in CH2Cl2 (3.0 mL) and a 10% solution of NaOH (aq) (1.5 mL), N-phenyl-trifluoromethane sulfonimide (0.36 g, 1.0 mmol) was added. The reaction was stirred at room temperature for 36 h, after which it was quenched with H2O (10 mL) and extracted with CH2Cl2. The combined organic layers were washed with brine, dried and concentrated in vacuo. The residue was purified by flash chromatography (CH2Cl2 with 5% MeOH), converted to the oxalate salt and recrystallized from EtOH to yield 46 mg (36%) of 2.10 as white crystals: mp 182-184 °C; 1H NMR δ

8.55-8.52 (dd, J1 = 5.55, J2 = 2.04, 1 H), 7.82-7.73 (dt, J1 = 7.72, J2 = 1.91, 1 H), 7.36-7.25 (m, 4 H), 7.22-7.03 (m, 2 H), 4.04-3.97 (t, J = 6.89, 2 H), 3.04-2.94 (m, 4 H), 2.76-2.64 (m, 6 H), 2.31-2.20 (m, 1 H), 1.88-1.46 (m, 7 H), 1.27-0.96 (m, 3 H); IR (KBr) 1665, 1414, 1207 cm-1; MS (EI) m/z 540 (M+);

HRMS Calcd (Obsd) for C25H31N4O4S1F3: 540.201 (540.201); Anal.(C25H31N4O4S1F3.C2H2O4) C, H, N.

N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)-4-acetoxyphenyl carboxamide

(2.11) To a solution of 2.2c (500 mg, 1.6 mmol) and TEA (243 mg, 2.4 mmol) in CH2Cl2 (10 mL), a solution of 4-acetoxy benzoyl chloride (476 mg, 2.4 mmol) in CH2Cl2 (7 mL) was added drop-wise. The reaction was stirred at room temperature for 5 days, after which it was quenched with a 10% solution of Na2CO3 (aq). After separation, the organic layer was dried and concentrated in vacuo. The residue was purified by flash chromatography (CH2Cl2 with 3% methanol ) to yield 540 mg (80 %) of 2.11 as a light yellow oil, from which 100 mg was converted to the oxalate salt and recrystallized from ethanol to give 102 mg (86%) as white crystals: (mono-oxalate salt) mp 193-195 oC; 1H NMR δ 8.45- 8.42 (m, 1H), 7.46-7.33 (m, 3H) 7.06-6.75 (m, 8H), 4.29 (t, J = 6.70, 2H), 3.84 (s, 3H), 3.00-2.85 (m, 4H), 2.75 (t, J = 6.7, 2H), 2.66-2.60 (m, 4H), 2.26 (s, 3H); 13C NMR δ 169.3, 168.8, 156.4, 152.2, 151.8, 148.6, 141.3, 137.1, 133.5, 130.1, 122.9, 122.8, 121.1, 120.9, 118.0, 111.1, 56.3, 55.28, 53.3, -1 +1 50.6, 45.5, 21.1; IR (KBr) 1647 cm ; MS (CI with NH3) m/z 475 (M ); Anal.

(C27H30N4O4.C2H2O4.0.5H2O) C, H, N.

N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)- 4-hydroxyphenyl carboxamide

(2.12) To a solution of 2.11 (250 mg, 0.55 mmol) in methanol (5 mL), a solution of K2CO3 (80 mg, 0.6 mmol) in H2O (5 mL) was added. The reaction was stirred at room temperature for 20 h, after which methanol was removed in vacuo. The aqueous residue was extracted with CH2Cl2, and the combined

84 organic layers were washed with brine, dried and concentrated in vacuo. The residue was purified by flash chromatography (ethyl acetate) to yield 210 mg (92%) of 2.12 as white crystals: mp 76-80 °C; 1H

NMR δ 8.41-8.36 (dd, J1 = 5.25, J2 = 1.60, 1H), 7.44-7.35 (dt, J1 = 7.72, J2 = 2.00, 1H), 7.13 (d, J = 8.33, 2H), 7.04-6.73 (m, 6H), 6.54 (d, J = 8.33, 2H), 4.28 (t, J = 6.96, 2H), 3.82 (s, 3H), 3.00-2.86 (m, 4H), 2.79-2.60 (m, 6H); 13C NMR δ 170.8, 158.6, 152.0, 148.4, 147.7, 137.2, 130.8, 126.7, 123.2, -1 +1 122.7, 120.9, 118.2, 115.0, 111.0, 105.6; IR (KBr) 1645, 1237 cm ; MS (CI with NH3) m/z 433 (M );

HRMS Calcd (Obsd) for C25H28N4O3 432.216 (432.216); Anal. (C25H28N4O3.H2O) C, H, N.

N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)- 4-trifluorosulfonxyphenyl carboxamide (2.13) To a suspension of 2.12 (75 mg, 0.17 mmol) and tetrabutylammonium iodide (10 mol%) in CH2Cl2 (2 mL) and a 10% solution of NaOH (aq) (1 mL), N-phenyl-trifluoromethane sulfonimide (180 mg, 0.50 mmol) was added. The reaction was stirred at room temperature for 36 h, after which it was quenched with H2O (10 mL) and extracted with CH2Cl2. The combined organic layers were washed with brine, dried and concentrated in vacuo. The residue was purified by flash 1 chromatography (CH2Cl2 with 5% EtOH) to yield 80 mg (82%) of 2.13 as colorless oil:; H NMR δ

8.42-8.38 (dt, J1 = 4.88, J2 = 0.98, 1H), 7.45(d, J = 0.74, 2H), 7.08 (d, J = 0.73, 2H), 7.13-6.82 (m, 7H), 4.40 (m, 2H), 3.84 (s, 3H), 3.16 (m, 4H), 2.94 (m, 6H); 13C NMR δ 175.1, 167.6, 154.0, 150.6, 148.7, 147.4, 136.2, 134.7, 129.3, 121.9, 121.3, 120.2, 119.5, 116.8, 109.7, 54.3, 53.8, 51.7, 47.9; MS (CI +1 with NH3) m/z 565 (M ).

N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)- 4-iodophenyl sulfoncarboxamide (2.14) A solution of 2.2c (460 mg, 1.60 mmol) and pipsyl chloride (1.47 g, 4.80 mmol) in pyridine (10 mL) is refluxed for 2 h, after which the solvent is removed in vacuo. The residue was purified by flash chromatography (CH2Cl2 with 10% EtOH) yielding 200 mg (30 %) of 2.14 as a 1 yellow oil. Of the starting material, 100 mg is recovered; H NMR δ 8.22-8.20 (dd, J1 = 4.76, J2 = 1.47,

1H), 7.67 (d, J = 8.42, 2H), 7.44-7.35 (dt, J1 = 7.69, J2 = 1.95, 1H), 7.53 (d, J = 8.79, 1H), 7.23 (d, J = 8.42, 2H), 7.07-7.03 (m, 1H), 6.90-6.71 (m, 4H), 3.85 (t, J = 6.78, 2H), 3.72 (s, 3H), 2.82 (m, 4H), 2.49-2.47 (m, 6H); 13C NMR δ 149.9, 149.7, 145.7, 138.7, 135.8, 135.6, 135.3, 126.4, 120.8, 120.4, 119.4, 118.4, 115.6, 108.6, 97.9, 54.5, 52.8, 50.7, 48.0, 42.9.

4-N-(2-Methoxyphenyl)amino piperidine (2.15a) A solution of o-anisidin (3.00 g, 25.0 mmol) and N- benzyl-4-piperidone (3.80 g, 20.0 mmol) and p-toluenesulfonic acid (0.10 g) in toluene (50 mL) was refluxed under Dean-Stark conditions for 3 h. After cooling to room temperature, PtO2 (0.10 g) was o added and the mixture was hydrogenated in a Parr-shaker with H2 (60 psi) for 2 h at 60 C. A solution of 25% ammonia (aq) (5 mL) was added and the mixture was hydrogenated further for another 1 h (50 psi) at room temperature. The reaction mixture was filtered over Celite and the solvent removed in vacuo. The residue was purified by gradient flash chromatography (hexane to EtOAc), yielding 2.7 g (46 %) of the intermediate as a brown-yellow oil. For the debenzylation, this intermediate (2.7 g, 9.1 mmol) was dissolved in THF (10 mL) and MeOH (190 mL), 20% Pd(OH)2/C (200 mg) was added and o the mixture was hydrogenated in the Parr-shaker with H2 (60 psi) for 2 h at 60 C. The reaction mixture was filtered over Celite and the solvent removed in vacuo. The residue was recrystallized from hexane, yielding 1.7 g (91 %) of 2.15a as white crystals: mp 107-109 oC. 1H NMR δ 6.89-6.60 (m, 4 H), 3.83 (s, 3 H), 3.39-2.70 (m, 4 H), 2.3-2.08 (m, 2 H), 1.51-1.10 (m, 4 H), 0.88 (m, 1 H); 13C NMR δ 145.3, 135.2, 119.7, 114.8, 108.7, 108.1, 53.9, 48.1, 43.7, 31.9; MS (EIPI) m/z 206 (M+); HRMS Calcd (Obsd) for C12H18N2O1: 206.14 (206.14).

2-[4-N-(o-Methoxyphenyl)amino-piperidinyl]-N-pyridin-2-yl-acetamide (2.15b) A suspension of compound 2.15a (0.25 g, 1.20 mmol), 2-chloro-N-pyridin-2-yl acetamide (0.20 g, 1.17 mmol), triethylamine (2 mL), K2CO3 (0.10 g, 0.72 mmol) and tetrabutylammonium iodide (0.05 g) was refluxed for 18 h, after which the solvent was removed in vacuo. The residue was purified by flash

85 chromatography (hexane with 20 % EtOAc to pure EtOAc), yielding 270 mg (66 %) of 2.15b as a brown-yellow oil. The oil was redissolved in CH2Cl2, converted to the HCl salt and recrystallized from o 1 MeOH/Et2O: mp 156 C. H NMR δ 9.65 (b, 1 H), 8.32-8.22 (m, 2 H), 7.73-7.63 (m, 1 H), 7.06 (m, 1 H), 6.85-6.59 (m, 4 H), 4.15 (b, 1 H), 3.83 (s, 3 H), 3.35 (m, 1 H), 3.16 (s, 2 H), 2.87 (m, 2 H)2.40 (m, 2 H), 2.10 (m, 2 H), 1.60 (m, 2 H); 13C NMR δ 169.0, 105.9, 147.9, 146.8, 132.2, 136.6, 121.0, 119.7, 116.3, 113.6, 110.2, 109.5, 62.0, 55.2, 52.8, 48.5, 32.3; MS (EI) m/z 340 (M+); HRMS Calcd (Obsd) for

C19H24N3O2: 340.19 (340.19).

2-[4-N-(o-Methoxyphenyl)-N-methylamino-piperidinyl]-N-pyridin-2-yl-acetamide (2.15c) A mixture of compound 2.15b (0.50 g, 1.47 mmol), formaldehyde (10 mL) and 20% Pd(OH)2/C (0.10 g) o in EtOH (100 mL), was hydrogenated with H2 (60 psi) for 18 h at 60 C. The residue was purified by gradient flash chromatography (CH2Cl2 with 10-20 % EtOH), yielding 400 mg (77 %) of 2.15c as a o 1 viscous brown oil. The oil was redissolved in Et2O and converted to the HCl salt: mp 180 C. H NMR δ 9.60 (b, 1 H), 8.32-8.21 (m, 2 H), 7.69-7.65 (m, 1 H), 7.06-6.85 (m, 5 H), 3.84 (s, 3 H), 3.22 (m, 1 H), 3.11 (s, 2 H), 2.97 (m, 2 H), 2.74 (s, 3 H), 2.24 (m, 2 H), 1.82 (m, 2 H), 1.76 (m, 2 H); 13C NMR δ 169.5, 153.1, 150.9, 147.8, 140.9, 138.2, 122.8, 121.3, 120.4, 119.7, 113.8, 111.1, 62.1, 58.4, 55.2, + 53.9, 34.2, 28.2; MS (EIPI) m/z 354 (M ); HRMS Calcd (Obsd) for C20H26N3O2: 354.21 (354.21).

2-[4-N-(o-Methoxyphenyl) -N-methylamino -piperidinyl]-N-pyridin-2-yl-amine (2.15d) To an ice cooled solution of compound 2.15c (0.30 g, 0.85 mmol) in Et2O (15 mL), a 1.0 M solution of DIBAL- H in toluene (6 mL) was added drop-wise. The mixture was stirred for 18 h at room temperature after which H2O (3 drops) and acetone (1 mL) was added. The solvent was removed in vacuo. The residue was purified by gradient flash chromatography (EtOAc, THF and then CH2Cl2 with 10-25 % EtOH and

2 % aq ammonia), yielding 260 mg (90 %) of 2.15d as a yellow oil. The oil was redissolved in Et2O and converted to the HCl salt: mp 165 oC. 1H NMR δ 8.09 (m, 1 H), 7.41 (m, 1 H), 7.02-6.81 (m, 4 H), 6.57-6.37 (m, 2 H), 5.13 (b, 1 H), 3.84 (s, 3 H), 3.32 (m, 1 H), 3.18-2.99 (m, 2 H), 2.73 (s, 3 H), 2.57 (m, 2 H), 1.97-1.73 (m, 8 H); 13C NMR δ 153.5, 148.0, 137.2, 137.1, 122.6, 121.3, 120.4, 120.3, 112.5, 111.1, 106.8, 59.2, 56.4, 55.2, 53.1, 38.6, 33.9, 28.1; MS (EIPI) m/z 340 (M+); HRMS Calcd (Obsd) for

C20H28N3O1: 340.23 (340.23).

2-[4-N-(o-Methoxyphenyl) -N-methylamino -piperidinyl]-N-(2-pyridinyl) cyclohexane carboxamide (2.15) To an ice-cooled solution of 2.15d (0.17 g, 0.50 mmol) and triethylamine (2 mL) in CH2Cl2 (10 mL), a solution of cyclohexanecarboxylic acid chloride (1 mL, 7.5 mmol) in CH2Cl2 (10 mL) was added drop-wise. The reaction mixture was stirred at room temperature for 2 h, after which the solvent was removed in vacuo. The residue was purified by gradient flash chromatography (CH2Cl2 with 5-15 % EtOH), yielding 140 mg (62 %) of 2.15 as a yellow oil. The oil was redissolved in Et2O and converted to the HCl salt: mp 130-135 oC. 1H NMR δ 8.50 (m, 1 H), 7.78 (m, 1 H), 7.26 (m, 2 H), 7.00-6.82 (m, 4 H), 4.11 (m, 2 H), 3.84 (s, 3 H), 3.28 (m, 3 H), 2.90 (m, 2 H), 2.68 (s, 3 H), 2.15-0.93

(m, 17 H); Anal. (C27H38N4O2.2.4HCl.4.6H2O) C, H, N, Cl.

2-[4-N-(o-Methoxyphenyl)amino-piperidinyl]-N-pyridin-2-yl-amine (2.16a) To an ice-cooled solution of 2.15b (0.27 g, 0.80 mmol) in Et2O (15 mL), a 1.0 M solution of DIBAL-H in toluene (6 mL) was added. The reaction was stirred for 18 h at room temperature. The excess of DIBAL-H was destroyed with H2O (3 drops) after which the solvent was removed in vacuo. The residue was purified by gradient flash chromatography (EtOAc to THF to CH2Cl2 with 10-25 % EtOH and 2 % aq ammonia), yielding 200 mg (77 %) of 2.16a as a yellow oil. The oil was redissolved in Et2O and converted to the HCl salt: mp 122 oC. 1H NMR δ 8.09 (m, 1 H), 7.26 (m, 1 H), 6.99-6.38 (m, 6 H), 5.16 (b, 1 H), 4.18 (b, 1 H), 3.84 (s, 3 H), 3.33 (m, 3 H), 2.88 (m, 2 H), 2.66 (s, 3 H), 2.33-2.17 (m, 4 H), 1.43 (m, 2 H); 13C NMR δ 158.7, 148.0, 146.7, 137.2, 136.8, 121.1, 116.0, 112.5, 110.1, 109.4, 106.9,

86 + 56.2, 55.2, 52.0, 49.3, 38.6, 33.2; MS (EIPI) m/z 326 (M ); HRMS Calcd (Obsd) for C20H26N3O2: 326.23 (326.22).

[4-N-(o-Methoxyphenyl) -N-cyclohexanecarboxamide -piperidinyl]-N-(2-pyridinyl) cyclohexane carboxamide (2.16) To an ice-cooled solution of 2.16a (0.20 g, 0.62 mmol) and triethylamine (2 mL) in CH2Cl2 (10 mL), a solution of cyclohexane carboxylicacid chloride (2 mL, 15.0 mmol) in CH2Cl2 (10 mL) was added drop-wise. The reaction mixture was stirred at room temperature for 2 h, after which the solvent was removed in vacuo. The residue was purified by gradient flash chromatography

(EtOAc to CH2Cl2 with 10 % EtOH), yielding 140 mg (62 %) of 2.16 as a yellow oil. The oil was o 1 redissolved in Et2O, converted to the HCl salt: mp 137-140 C. H NMR δ 8.40 (m, 1 H), 7.39 (m, 1 H), 7.26-6.88 (m, 6 H), 4.48 (m, 1 H), 3.81 (m, 2 H), 3.72 (s, 3 H), 2.88 (m, 2 H), 2.41 (m, 2 H), 2.20-1.96 (m, 4 H), 1.74-1.40 (m, 18 H), 1.06-0.81 (6 H); 13C NMR δ 176.7, 176.1, 156.4, 155.8, 148.9, 137.8, 131.1, 129.3, 127.7, 122.0, 120.3, 116.0, 57.9, 55.7, 54.9, 52.3, 45.1, 42.1, 42.0, 30.7, 29.6, 29.2, 28.6,

28.5, 25.4, 25.3; Anal. (C33H46N4O3.1.9HCl.3.3H2O) C, H, N, Cl.

BIOLOGICAL ASSAYS.

ANIMALS. For the guinea-pig ileum assay male Dunkin Hartley guinea-pigs (300-400 g) were used. The hypothermia experiments were performed using male Wistar rats (200-300 g). The animals were housed in groups (four rats per cage) in a temperature- and humidity-controlled colony room (20 + 2 °C; 50-60 %) on a natural day-night cycle (light period 6:30 - 17:00 h). Food and water were available ad libitum except during the actual experiments. Testing was done between 10 h and 16 h during the light phase of the day-night cycle. For the ultrasonic vocalization experiments Sprague-Dawley rats (290-390 g) were used. Animals were housed in groups (two rats per cage) in a temperature- and humidity-controlled colony room (29 + 2 °C; 50-60 %) under a natural day-night cycle (light period 6:00 - 18:00 h) with food and water ad libitum except during the actual experiments. Procedures were conducted in accordance with guidelines published in the NIH Guide for the Care and Use of Laboratory Animals.

RECEPTOR BINDING. Affinities of compounds were determined using competition binding assays to determine IC50 values at the various receptors. Affinities at the 5-HT1A receptors were measured as the displacement of [3H]-8-OH-DPAT (rat frontal cortex)46,47

38 GUINEA PIG ILEUM ASSAY FOR DETERMINATION OF PA2-VALUE. The animals were stunned and exsanguinated. After removal of the ileum, segments of about 1 cm long were dissected 10 cm from the ileocaical junction. After clearance of the mesenteric connective tissue and removal of the luminal contents, each ileal segment was mounted between platinum stimulation electrodes in a 50 mL, two- chambered organ bath with an internal circulation. The segments were bathed in a modified Krebs

Henseleit solution (in mmol: NaCl 118; NaHCO3 25; KH2PO4 1.19; CaCl2 1.25; KCl 4.7; MgSO4 0.6; 10). Ketanserin (0.3 µmol ) and (3 µmol) were added to block 5-HT2, 5-HT3 and 5- o HT4 receptors. The solution, maintained at 35 C, was gassed and continuously mixed by bubbling with

95% O2/ 5% CO2. Contractions were recorded isometrically under a load of 1 g. After an equilibration for 20 min the preparations were stimulated continuously with square-wave pulses (0.05 Hz, 1 ms, 40 V) until the contractions reached a constant level. The preparations were washed and then stimulated with trains of 6 stimulations at intervals of 7 min, until the mean responses were comparable. Cumulative concentration-response curves were constructed with an exposure period of 7 min with 8- OH-DPAT, followed by 6 electrical stimulations, after which the next higher concentration would be applied. Controls received the corresponding volume of the vehicle only. For examining antagonistic activity, the compounds were applied at fixed concentrations 60 min before starting the cumulative concentration-response curve of the agonist. Responses were measured as an increase in the isometric

87 tension and expressed as a percentage of the mean effect of the last 6 stimulations before the start of the experiment. Because the 5-HT1A -agonist 8-OH-DPAT decreases the electrically-induced contractions maximally by about 40%, EC20 values were determined graphically form the mean effect of 4-6 experiments. Apparent pA2 values were calculated according to Mac Kay: pA2 = -log[antagonist] + 56 (DR-1), where DR is the ratio of EC20 values in the presence and absence of the antagonist.

ULTRASONIC VOCALIZATION18,19,45 Ultrasonic vocalization was measured in a sound-attenuated test chamber (W 24 cm, L 22 cm, H 22 cm) with a grid floor for delivery of footshocks (scrambled chock of 1.8 mA for 0.3 s, shocker Getra BN 2002). Ultrasonic vocalization was recorded (microphone 4004, Bruel and Kjaer) and processed by an interface (developed at Merck KGaA) to select 22 + 4 kHz signals and to digitize the resulting signals for automatic processing in a personal computer. In a priming phase, each rat was placed in the test chamber. After 2 min, a series of at most ten shocks (trials), 1.8 mA for 0.3 s, separated by 20 s shock-free intervals, was delivered via the grid floor of the test chamber. In the shock-free intervals the occurrence of ultrasonic vocalization was automatically recorded, and the duration of ultrasonic vocalization was calculated immediately. The priming session was terminated either when the rat constantly vocalized at least for 10 s on three consecutive trials or after the tenth trial. Rats that did not respond with ultrasonic vocalization were excluded from the test. In the actual test performed the next day, each rat received five initial shocks (1.8 mA for 0.3 s, separated by 20-s shock-free intervals) in the test chamber, and the duration of ultrasonic vocalization was recorded after the last shock for 3 min Antagonists or vehicle were given 40 min and 8-OH-DPAT or vehicle 30 min before start of ultrasonic vocalization test. Means and SEM values were calculated for each group and compared to the data for the respective placebo group by one-way analysis of variance (ANOVA) followed by Dunnett's t-test.

8-OH-DPAT-INDUCED HYPOTHERMIA.16 Animals were treated with 1 mg/kg 8-OH-DPAT 30 min prior to measurement; test compounds/saline were administered 45 min prior to measurement. The core temperature was determined by insertion of a digital temperature probe (CMA/150 Temperature Controller, CMA/Microdialysis Sweden) 4-5 cm into the rectum for 60 s until a stable reading was obtained, to the nearest 1/10th °C. For each independent experiment new control and 8-OH-DPAT studies were conducted and changes in temperature due to test compounds were assessed using a one- way analysis of variance (ANOVA) with post hoc analysis carried out using Dunnett's t-tests against their own control data.

8-OH-DPAT-INDUCED BEHAVIOUR.51-53 Animals were treated with 1 mg/kg 8-OH-DPAT 30 min prior to measurement; test compounds/saline were administered 45 min prior to measurement. LLR, FBP and LA were scored positive if the onset-of-action was within 5 min after administration of 8-OH- DPAT.

54,55 5-HTP MEASUREMENTS. Rats were reserpinized (5 mg/kg) 24h prior to administration of the test compound. The test compound was administered 15 min prior to the administration of 100 mg/kg NSD. After 30 min the rats were decapitated and the brain quickly dissected upon ice. Striatum, accumbens, frontal cortex and hippocampus were stored at - 70 °C until analysis. Before analysis, the samples were homogenized in TCA and centrifuged. The supernatant was analyzed by means of HPLC with electrochemical detection for 5-HTP. 5-HTP accumulation was expressed as [5-HTP] in µg/g wet tissue.

88 ACKNOWLEDGEMENTS Dr. Y. Liao is thanked for synthesizing the arylaminopiperidine analogues 2.14 and 2.15. Drs. H.E. Greiner, J. Harting and G.D. Bartoszyk are acknowledged for performing the 5-HT1A binding, pA2 and ultrasonic vocalization studies, respectively. Dr. P. de Boer is thanked for his assistance with the temperature and behavioral studies.

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[2] Leonhardt, S.; Herrick Davis, K.; Titeler, M. Detection of a novel serotonin receptor subtype

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94

IN VIVO PHARMACOLOGICAL EVALUATION OF p-SUBSTITUTED 3 BENZAMIDE ANALOGUES OF WAY100635 AT PRE- AND POST- * SYNAPTIC 5-HT1A RECEPTORS

3.1 Introduction 96 3.2 Pharmacology 97 Receptor Affinity and Efficacy, Rat Hypothermia and Microdialysis in Rat Ventral Hippocampus 3.3 Results and Discussion 97 3.4 Experimental 102 3.5 References 103

ABSTRACT

The serotonin 5-HT1A receptor agonist 8-hydroxy-(di-N-propylamino)tetralin (8-OH-DPAT; 3.1; 0.2 mg/kg sc) decreases the rat core body temperature by 1.5 °C at 45 min after administration of the agonist. This effect is antagonized dose-dependently by the 5-HT1A receptor antagonist WAY100635 (3.2; ED50 ca. 0.01 µmol/kg sc) and its benzamide analogue N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl)-p-hydroxyphenyl carboxamide

(p-MPPOH; 3.6; ED50 ca. 4 µmol/kg sc). In microdialysis, 8-OH-DPAT (0.2 mg/kg sc i.e. 0.7 µmol/kg sc) induces a decrease in 5-HT release in the rat ventral hippocampus to ca. 40% of basal values, an effect which is antagonized dose-dependently by both WAY100635 and p-MPPOH. The ED50 values derived from the dose-response curves at 60 min after administration of the agonist, are ca. 0.02 µmol/kg sc and 0.4 µmol/kg sc, respectively. The difference in potency ratio’s of p-MPPOH and WAY100635 in the pre- (hypothermia) and the postsynaptic (microdialysis) assays is 20 and 400, respectively. Furthermore, the 5-HT1A receptors located postsynaptically appear to be more sensitive to WAY100635 then those located presynaptically (pre/post ratio ca. 80), while the opposite is true for the benzamide analogue p-MPPOH (pre/post ratio ca. 0.5). Therefore, the benzamide analogue p-MPPOH is suggested to have a slight preference for the presynaptic 5-HT1A receptors and to be a more effective 5-HT1A autoreceptor antagonist than WAY100635.

* Parts of this chapter will be described in: p-MPPOH, a new 5-HT1A antagonist, shows a preference for

5-HT1A autoreceptors in the raphé nuclei. Mensonides-Harsema, M.M.; Cremers, T.I.F.H.; Damhof, M.A.; Eggink, M.; Motzen, E.; Timmerman, W.; Wikström, H.V. (manuscript in preparation).

95 3.1 INTRODUCTION

As described in the previous chapters, the serotonin 5-HT1A receptor is present both 1-6 pre- and postsynaptically. The presynaptic 5-HT1A autoreceptors are located on the cell bodies of the serotonin neurons in the raphé nucleus and control the firing rate of the neuron via auto-inhibition. Microdialysis measurements of 5-HT release in the ventral hippocampus reflects the presynaptic serotonergic activity.7-9 The 5-HT neurons project to different brain regions, where 5-HT1A receptors are located on postsynaptic structural elements. Hypothermia in rat following the administration of a 10- 5-HT1A agonist, like 8-OH-DPAT (3.1), reflects postsynaptic serotonergic activity. 17 The 5-HT1A receptor antagonist WAY100635 (3.2) and several o-phenyl substituted analogues have been evaluated for their suitability as radioligands in PET studies. Two other antagonists, which are currently being evaluated for their use in visualizing the bio-distribution the 5-HT1A receptors in the brain, are the p-iodo and p-fluoro benzamide analogues of WAY100635, p-MPPI (3.3) and p-MPPF, respectively (3.4).18-23 In the in vivo hypothermia and gross behavioral studies described in Chapter 2, it became apparent that the p-acetyl (p-MPPOAc; 3.5) and p-hydroxyl (p-MPPOH; 3.6) benzamide analogues of WAY100635 at doses up to 10 µmol/kg sc were not able to block the 8-OH-DPAT- induced (1 mg/kg sc) decrease in core body temperature and 5-HT syndrome. This is surprising, since both p-MPPI and p-MPPF are known to be potent and full 5-HT1A receptor antagonists, with ID50 values of 9 and 7 µmol/kg vs 0.5 mg/kg ip 8-OH-DPAT induced hypothermia, respectively.20 While both benzamides showed high affinity for the 5-HT1A receptor (Chapter 2, table 2.1), the in vitro pA2 values of p-MPPOAc (8.2) and p-MPPOH (<7) suggested that only p-

MPPOAc is a potent 5-HT1A receptor antagonist. These conflicting data justify a more detailed in vitro (h5-HT1A binding and cAMP study) and in vivo study (temperature and microdialysis) of the actions of the benzamides at the 5-HT1A receptors, using a low dose of 8-OH-DPAT (0.2 mg/kg i.e. 0.7 µmol/kg sc). R

OH N N N N N O O H3C H3C O N O N 3.1 3.2 3.3 R = I N N 3.4 R = F 3.5 R = OAc 3.6 R = OH 3.7 R = OTf Chart 3.1 Chemical structure of 8-OH-DPAT (3.1), WAY100635 (3.2), p-MPPI (3.3), p-MPPF (3.4), p-MPPOAc (3.5), p-MPPOH (3.6) and p-MPPOTf (3.7).

96 In this chapter, the in vitro affinity and efficacy of the benzamides 3.5 - 3.7 at the

5-HT1A receptor subtype is described. To study the in vivo efficacy of WAY100635 and p-MPPOH - the benzamide analogue with the highest affinity at h5-HT1A receptors and efficacy in cAMP assay - at somatodendritic (microdialysis) and postsynaptic (hypothermia) 5-HT1A receptors, dose-response curves vs 0.2 mg/kg 8- OH-DPAT are obtained.

3.2 PHARMACOLOGY

WAY100635 and test compounds 3.5 - 3.7 were evaluated for their ability to 3 displace in vitro the radioligand [ H]-5-CT at the h5-HT1A receptor which was expressed in HeLa cells.24 The efficacy of WAY100635 and compounds 3.5 - 3.7 in antagonizing 5-HT-mediated inhibition of forskolin-stimulated cAMP formation was measured as well as the intrinsic activity of these compounds in this assay.25

To evaluate postsynaptic activity at the serotonin 5-HT1A receptors in vivo, the effects of WAY100635 and benzamide analogue p-MPPOH were measured in a non-invasive hypothermia assay vs a low dose of 8-OH-DPAT (0.2 mg/kg sc).16 For clarity, the deviations of core temperature from those of the respective placebo-treated control groups are presented instead of the original temperature curves. To assess presynaptic activity at the serotonin 5-HT1A receptors in vivo, WAY100635 and p-MPPOH were evaluated in a microdialysis study, measuring the 5-HT levels in the ventral hippocampus of the awake rat after administration of 0.2 mg/kg sc 8-OH-DPAT and different doses of the respective antagonist.

3.3 RESULTS AND DISCUSSION

SAR relationships were determined from affinity values and activity values at cloned h5-HT1A receptors in permanently transfected HeLa cells (Table 3.1). The affinity of the benzamide analogues of WAY100635 remains in the nanomolar range. Compared to WAY100635, p-MPPOH is six-fold less potent in displacing [3H]-5-CT from the h5-HT1A receptor. The benzamide analogues with a larger substituent in the para position of the phenyl ring, p-MPPOAc and p-MPPOTf, are ca. 30 times less potent. 26, 27 The pA2 values obtained from the guinea pig ileum assay (Ch2, table 2.1) are conflicting, suggesting that in this tissue preparation, p-MPPOAc is a potent antagonist while p-MPPOH is either a weak antagonist or a (partial) agonist. In literature, the p-substituted benzamides p-MPPF and p-MPPI are both described as 18, 20 potent antagonists at the 5-HT1A receptor. In the in vitro activity assay, neither WAY100635 nor the benzamides analogues 3.5 - 3.7 were able to inhibit forskolin- induced cAMP formation in doses up to 1000 nM, whereas they antagonized the

97 5-HT-induced inhibition. The difference in the antagonistic potency between the tested benzamide analogues and WAY100635, was 60 for p-MPPOH, ca 130 for p- MPPOTf and 400 for p-MPPOAc. From these potency data it may be concluded that the pA2 value obtained for p-MPPOH in the guinea pig ileum assay is incorrect. Furthermore, it appears from the cAMP assay that the large, strong electron- withdrawing triflate substituent in the para position of the phenyl ring is more favorable for antagonistic activity at the 5-HT1A receptor than the ester substituent.

TABLE 3.1 In vitro affinity and antagonistic efficacy at the 5-HT1A receptor (Ki in nM).

a c compound 5-HT1A affinity 5-HT1A efficacy WAY100635 0.1 0.03 p-MPPI 2.6b NTd p-MPPF 3.3b NT p-MPPOH 0.6 1.80 p-MPPOAc 3.6 12.0 p-MPPOTf 3.1 3.80 a [3H]-5-CT; b [123I]-8-OH-PIPAT18; c blockade of 5-HT-induced inhibition of cAMP release; d not tested.

In Chapter 2, it was shown that neither p-MPPOAc nor p-MPPOH were able to antagonize 1.0 mg/kg sc 8-OH-DPAT-induced hypothermia at 10 µmol/kg sc. However, their structurally close related benzamide analogues p-MPPI and p-MPPF are reported to act as full antagonists in 8-OH-DPAT-induced hypothermia.20 In table 3.2, the effects of WAY100635 and the benzamide analogue p-MPPOH on 0.2 mg/kg sc 8-OH-DPAT-induced decrease in core temperature at 15 and 45 min after administration of the agonist are presented. Both compounds are able to completely counteract the agonist-induced fall in temperature, and thus act as full antagonists. In figure 3.1 the hypothermia data are plotted and from the dose-response curves the respective ED50 values of both antagonists at 15 and 45 min after administration of 8- -3 OH-DPAT can be obtained. At t = 15 min, the ED50 values are ca 5 x 10 µmol/kg and 2 x 100 µmol/kg for WAY100635 and p-MPPOH, respectively, and at 45 min they are ca 1 x 10-2 µmol/kg and 4 x 100 µmol/kg. These data imply that the antagonist

WAY100635 is about 400 times more potent at postsynaptic 5-HT1A receptor sites than p-MPPOH.

98 TABLE 3.2 Effects of WAY100635, p-MPPOAc and p-MPPOH on 8-OH-DPAT (0.2 mg/kg sc)-induced hypothermia (n = 6).

Decrease in body temperature (oC) at compound dose (µmol/kg) t = 15 min t = 45 min 8-OH-DPAT 1.7 + 0.2 * 1.5 + 0.2 * WAY100635 1 x 10-3 1.5 + 0.2 * 1.3 + 0.2 * 3 x 10-3 1.3 + 0.0 * 0.7 + 0.1 * a 1 x 10-2 1.0 + 0.1 * 0.6 + 0.1 a 3 x 10-2 0.6 + 0.1 0.5 + 0.1 1 x 10-1 0.4 + 0.2 0.3 + 0.1 3 x 10-1 0.2 + 0.1 0.0 + 0.1 p-MPPOH 1 x 10-1 1.8 + 0.1 * 1.2 + 0.1 * 3 x 10-1 1.9 + 0.3 * 1.2 + 0.2 * 1 x 100 1.2 + 0.2 * 1.0 + 0.2 * 3 x 100 0.7 + 0.1 * 0.7 + 0.1 * 1 x 101 0.7 + 0.1 * 0.6 + 0.0 3 x 101 0.4 + 0.2 0.3 + 0.2 * p < 0.05 vs control (n = 7); a n = 8

FIGURE 3.1 Effects of WAY100635 (15 min: –■–; 45 min: –□–) and p-MPPOH (15 min: –●–; 45 min: –○– ) on 8-OH-DPAT-induced hypothermia.

∆ T oC

1.5

1.0

0.5

0.0 10-5 10-4 10-3 10-2 10-1 100 101 102 103 µ Dose antagonist ( mol/kg sc)

99 FIGURE 3.2 Effects of WAY100635 and p-MPPOH on 5-HT release in the rat ventral hippocampus after co-administration of 8-OH-DPAT (0.2 mg/kg sc) versus 8-OH-DPAT (0.2 mg/kg sc) alone.

175

150

125

100

75

% of basal 5-HT levels 5-HT basal % of 50

25

0 -60-300 306090120150Time (min)

8-OH-DPAT was administered at t = 0 min, while test compounds or vehicle were administered 15 min prior to the agonist; 8-OH-DPAT (–■–; n = 7); 10 nmol/kg WAY100635 (–●–; n = 4); 100 nmol/kg WAY100635 (–▲–; n = 6); 1 µmol/kg p-MPPOH (–○–; n = 6); 10 µmol/kg p-MPPOH (–∆–; n = 3);

FIGURE 3.3 Effects of WAY100635 (–■–) and p-MPPOH (–●–) on 5-HT release in the rat ventral hippocampus at 60 min. after administration of 8-OH-DPAT (0.2 mg/kg sc)

% Basal 5-HT 100

75

50

10 -3 10 -2 10 -1 10 0 10 1 Dose antagonist (µmol/kg sc.)

100

The results of the microdialysis study with WAY100635 and p-MPPOH are depicted in figures 3.2 and 3.3. Administration of 8-OH-DPAT (0.2 mg/kg sc) alone decreased the 5-HT release to about 40% of basal values. This effect is fully antagonized dose- dependently by both WAY100635 and p-MPPOH (Figure 3.2). In figure 3.3, the percentage basal 5-HT levels at 60 min after administration of the agonist, are plotted, together with their proposed full dose-response curves (dotted lines). For the calculation of these curves, the 40% level is assumed to be the maximum effect of the agonist, while the 100% level is assumed to be the maximum effect of the antagonist. This latter assumption is supported by the facts that (i) in the in vitro cAMP assay, neither of the antagonists were able to inhibit the forskolin-induced cAMP production and (ii) both ligands act as full antagonists in the hypothermia assay. The results obtained in the microdialysis assay show that at 60 min after -2 administration of 8-OH-DPAT, the antagonist WAY100635 (ED50 ~ 2 x 10 -1 µmol/kg) is about 20 times more potent than p-MPPOH (ED50 ~ 4 x 10 µmol/kg).

In summary, the benzamides all show nanomolar affinity for the 5-HT1A receptor, with p-MPPOH being the most potent test compound in this series. This benzamide analogue of WAY100635 is also the most potent antagonist in blocking the 5-HT inhibition of forskolin-induced cAMP formation. None of the benzamides showed any agonistic properties in this cAMP assay. p-MPPOH is a full antagonist in both the microdialysis assay - which reflects the antagonistic activity at presynaptically located

5-HT1A autoreceptors in the raphé nucleus - and the hypothermia assay - which reflects the antagonistic activity at postsynaptically located 5-HT1A heteroreceptors in the 5-HT projection areas. At the presynaptic 5-HT1A receptors, WAY100635 is 20 times more potent than p-MPPOH. At the postsynaptic 5-HT1A receptors, WAY100635 is 400 times more potent than p-MPPOH. However, the difference in in vivo antagonistic activity at pre- vs postsynaptic 5-HT1A receptors is ca 80 for WAY100635 and 0.5 for p-MPPOH. This means that WAY100635 is a postsynaptic- selective antagonist, while p-MPPOH is a presynaptic-selective antagonist. It has been shown, that there is a large receptor reserve for the somatodendritic

5-HT1A receptor-mediated inhibition of 5-HT synthesis in the rat cortex and hippocampus, measured as the accumulation of 5-hydrosytryptophan (5-HTP) after inhibition of 5-HTP decarboxylase. Thus, 8-OH-DPAT, a full 5-HT1A agonist, produced maximal effects in the ventral hippocampus at about 30-40% receptor occupancy in the cell body region.28, 29 This phenomenon explains the difference in efficacy of WAY100635 at the pre- and postsynaptic 5-HT1A receptor sites. However, the selectivity of the benzamide p-MPPOH for the presynaptic 5-HT1A receptor must come from other differences in pharmacokinetic, pharmacodynamic and/or metabolic profiles at the distinct brain areas. It has recently been published that both WAY100635 and its benzamide analogue p-MPPF are substrates for the

101 P-glycoprotein (Pgp), a transmembrane protein which functions as an ATP-dependent efflux pump for lipophilic compounds, thus preventing them from passing the blood- brain barrier.23 Experiments in which a Pgp antagonist like cyclosporine A is used or in Pgp knock-out mice show, that the uptake of WAY100635 is affected to a lesser extent than the uptake of p-MPPF.23, 30 The higher density of Pgp in the brain areas where postsynaptic 5-HT1A receptors are located might be responsible for the difference in efficacy of the benzamides in the 8-OH-DPAT-induced hypothermia assay as compared to WAY100635 and for the higher efficacy of p-MPPOH at the presynaptic 5-HT1A receptor sites in the raphé nucleus. Future studies at both pre- and postsynaptic receptor sites, e.g. electrophysiological, ultrasonic vocalization, voltammetric and behavioral assays will be necessary to confirm the selectivity of the benzamides found for the 5-HT1A autoreceptors.

The clinical implication of an autoreceptor selective antagonist could be, that the latency in the therapeutic onset-of-action of the current antidepressants, which is attributed to the necessary development of desensitization of presynaptic 5-HT1A receptors, would be abolished, while the required activation of postsynaptic 5-HT1A receptors via the enhanced 5-HT release would not be hindered.

3.5 EXPERIMENTAL

BIOLOGICAL ASSAYS

ANIMALS. For the hypothermia and microdialysis experiments male Wistar rats (200-300 g) were used. The animals were housed in groups (four rats per cage) in a temperature- and humidity-controlled colony room (20 + 2 °C; 50-60 %) on a natural day-night cycle (light period 6:30 - 17:00 h). Food and water were available ad libitum except during the actual experiments. Testing was done between 10 h and 16 h during the light phase of the day-night cycle. Procedures were conducted in accordance with guidelines published in the NIH Guide for the Care and Use of Laboratory Animals.

RECEPTOR BINDING AND ANTAGONISTIC POTENCY . Affinities and antagonistic potency at the 3 3 h5-HT1A receptors were measured as the displacement of [ H]-5-CT or [ H]-5-HT at human cloned 5-

HT1A receptors expressed in HeLa-cells as described in the references 24 and 25.

8-OH-DPAT INDUCED HYPOTHERMIA.16 Animals were treated with 0.2 or 1 mg/kg sc 8-OH-DPAT 30 min prior to measurement; test compounds/saline were administered 45 min prior to measurement. The core temperature was determined by insertion of a digital temperature probe (CMA/150 Temperature Controller, CMA/Microdialysis Sweden) 4-5 cm into the rectum for 60 s until a stable reading was obtained, to the nearest 1/10th °C. For each independent experiment new control and 8- OH-DPAT studies were conducted and changes in temperature due to test compounds were assessed using a one-way analysis of variance (ANOVA) with post hoc analysis carried out using Dunnett's t- tests against their own control data.

31 MICRODIALYSIS. The rats were anaesthetized with / (50/8 mg/kg ip) and atropine (0.1 mg/kg sc) after a premedication of (5 mg/kg sc). Preceding insertion of the microdialysis probe, rats were placed in a stereotaxic frame (Kopf, USA). Following local

102 application (10%), a home-made I-shaped probe,34 fabricated from polyacrylonitrile/sodium methyl sulfonate copolymer dialysis fiber (i.d. 0.22 mm, o.d. 0.31 mm, AN 69, Hospal, Italy) was implanted in the ventral hippocampus (coordinates: IA: +3.7 mm, lateral : +4.8 mm, ventral: -8.0 mm from the Dura Mater, Paxinos and Watson, 1982) and secured with stainless steel screws and dental cement. The exposed length of the membrane was 4 mm. After surgery, rats were allowed a recovery period of at least 24 h before starting the experiments. Probes were perfused with artificial CSF containing 147 mM

NaCl, 3.0 mM KCl, 1.2 mM CaCl2, and 1.2 mM MgCl2, at a speed of 1.5 µL/min (Harvard apparatus, South Natick, Ma., USA). Samples were collected on-line in a 20 µL loop and injected automatically onto the column every 15 min. 5-HT was analyzed using HPLC pump (Shimadzu LC-10 AD liquid chromatograph) in combination with a reversed phase column (phenomenex hypersil 3 C18, 100 X 2.0 mm, 3 µm, Bester, Amstelveen, the Netherlands) and an EC detector with a carbon electrode (Intro, Antec Leyden, Leiden, the Netherlands), working at a potential of 500 mV vs. Ag/AgCl reference electrode. The mobile phase consisted of 5.0 g/L (NH4)2SO4, 0.5 g/L EDTA, 50.0 mg/L heptasulfonic acid and 30 µL/L triethylamine, at pH 4.65 and 30 oC. The flow of the mobile phase was set at 400 µL per min. The detection limit of 5-HT was 0.5 fmol per sample of 20 µL (signal to noise ratio 2). Four consecutive microdialysis samples with less then 20 % difference were considered as control and set at 100 %. Data are presented as percentages of control level (mean + SEM). Statistical analysis was performed using two-way ANOVA for repeated measurements, followed by Student Newman Keuls post hoc analysis. The significance level was set at p<0.05.

Acknowledgements. Dr. Wia Timmerman is greatly acknowledged for useful suggestions for conducting a more detailed study of the behavior of benzamides at both pre- and postsynaptic 5-HT1A receptor subtypes. Thomas Cremers and Michiel Damhof are thanked for conducting the microdialysis experiments. Matthijs Eggink is thanked for his efforts in the hypothermia experiments (Neurochemistry, Groningen). Dr. Ejner Moltzen is greatly acknowledged by providing the affinity and activity data (Lundbeck A/S, Copenhagen, Denmark).

3.6 REFERENCES

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103 Autoradiographic mapping of serotonin-1 receptors. Neuroscience. 1987, 21, 97-122.

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[8] Sprouse, J.S.; Aghajanian, G.K. Electrophysiological responses of serotoninergic dorsal raphe neurons to 5-HT1A and 5-HT1B agonists. Synapse 1987, 1, 3-9.

[9] Westerink, B.H.C. and Timmerman, W. Do neurotransmitters sampled by brain microdialysis reflect functional release. Analytica Chimica Acta 1999, 379, 263-274.

[10] O’Connell, M.T.; Sarna, G.S.; Curzon, G. Evidence for postsynaptic mediation of the

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[13] O’Connell, M.T. and Curzon, G. A comparison of the effects of 8-OH-DPAT pretreatment on different behavioural responses to 8-OH-DPAT. Eur. J. Pharm. 1996, 312, 137-143.

[14] Hillegart, V. Effects of local application of 5-HT and 8-OH-DPAT into the dorsal and median raphe nuclei on motor activity in the rat. Physiol. Behav. 1990, 48, 143-148.

[15] Tricklebank, MD.; Forler, C.; Fozard, J.R. The involvement of subtypes of the 5-HT1 receptor and of catecholaminergic systems in the behavioural response to 8-hydroxy-2-(di-n- propylamino) tetralin in the rat. Eur. J. Pharmacol. 1984, 106, 271-282.

[16] Berendsen, H.H.G.; Jenck, R.; Broekkamp, C.L.E. Selective activation of 5-HT1A receptors induces lower lip retraction in the rat. Pharmacol. Biochem. Behav. 1989, 33, 821-827.

[17] Molewijk, H.E.; Van der Heyden, J.A.M.; Olivier, B. Lower lip retraction is selectively mediated by activation of the 5-HT1A receptor. Eur. J. Neurosci. 1989, 214, 121-125.

[18] Zhuang, Z.P.; Kung, M.P.; Kung, H.F. Synthesis and evaluation of 4-(2’-methoxyphenyl)-1-[2’-

[N-(2’’-pyridinyl)-p-iodobenzamido]ethyl] piperazine (p-MPPI): A new iodinated 5-HT1A ligand. J. Med. Chem. 1994, 37, 1406-1407.

[19] Kung, H.F.; Stevenson, D.A.; Zhuang, Z.P.; Kung, M.P.; Frederick, D.; Hurt, S.D. New 5-HT1A receptor antagonist: [3H]p-MPPF. Synapse, 1996, 23, 344-346.

[20] Thielen, R.J.; Fangon, N.B.; Frazer, A. 4-(2’-Methoxyphenyl)-1-[2’-[N-(2’’-pyridinyl)-p- iodobenzamido]ethyl] piperazine and 4-(2’-methoxyphenyl)-1-[2’-[N-(2’’-pyridinyl)-p-

fluorobenzamido]ethyl] piperazine, two new antagonists at pre- end postsynaptic serotonin-1A

104 receptors. J. Pharmacol. Exp. Ther. 1996, 277, 661-670.

[21] Shiue, C.Y.; Shiue, G.G.; Mozley, P.D.; Kung, M.P.; Zhuang, Z.P.; Kim, H.J.; Kung, H.F. 18 p-[ F]-MPPF: a potential radioligand for PET studies of 5-HT1A receptors in humans. Synapse 1997, 25, 147-154.

[22] Le Bars, D.; Lemaire, C.; Ginovart, N.; Plenevaux, A.; Aerts, J.; Brihaye, C.; Hassoun, W.; Leviel, V.; Mekhsian, P.; Weissmann, D.; Pujol, J.F.; Luxen, A.; Comar, D. High-yield radio synthesis and preliminary in vivo evaluation of p-[18F]MPPF, a fluoro analog of WAY-100635. Nucl. Med. Biol. 1998, 25, 343-350.

[23] Passchier, J.; Van Waarde, A.; Doze, P.; Elsinga, P.H.; Vaalburg, W. Regional distribution of

two 5-HT1A receptor ligands in rat brain and effect of PGP modulation, J. Nucl. Med. 1999, 40, 263P.

[24] Harrington, M.A.; Shaw, K.; Zong, P.; Ciaranello, R.D. Agonist-induced desensitization and loss of high-affinity binding sites of stably expressed human 5-HT1A receptors. J. Pharmacol. Exp. Ther. 1994, 268, 1098-1106.

[25] Pauwels, P.J.; Van Gompel, P.; Leysen, J.E. Activity of serotonin (5-HT) receptor agonists,

partial agonists and antagonists at cloned human 5-HT1A receptors that are negatively coupled to adenylate cyclase in permanently transfected HeLa cells. Biochem. Pharmacol. 1993, 45, 375-383.

[26] Kobilka, B. Adrenergic receptors as models for G protein-coupled receptors. Annu. Rev. Neurosci. 1992, 15, 87-114.

[27] Tricklebank, M.D.; Forler, C.; Fozard, J.R. The involvement of subtypes of the 5-HT1 receptor and of catecholaminergic systems in the behavioural response to 8-hydroxy-2-(di-n-propyl- amino)tetralin in the rat. Eur. J. Pharmacol. 1984, 106, 271-282.

[28] Meller, E.; Goldstein, M.; Bohmaker, K. Receptor reserve for 5-hydroxytryptamine1A-mediated inhibition of serotonin synthesis: possible relationship to anxiolytic properties of 5-hydroxy-

tryptamine1A-agonists. Mol. Pharmacol. 1990, 37, 231-237.

[29] Blier, P.; Serrano, A.; Scatton, B. Differential responsiveness of the rat dorsal and medial raphe

5-HT systems to 5-HT1 receptor agonists and p-chlroramphetamine. Synapse, 1990, 5, 120-133.

[30] Sugiyama, Y.; Kusuhara, H.; Suzuki, H. Kinetic and biochemical analysis of carrier-mediated efflux of drugs through the blood-brain and blood-cerebrospinal fluid barriers: importance in the drug delivery to the brain. J. Controlled Release 1999, 62, 179-186.

[31] Santiago, M. and Westerink B.H. Characterization of the in vivo release of dopamine as recorded by different types of intracerebral microdialysis probes. Naunyn-Schmiedebergs Arch. Pharmacol. 1990, 342, 407-414.

105

106

STRUCTURAL ANALOGUES OF THE 5-HT1B/1D ANTAGONIST 4 GR127935 AND 5-HT1B INVERSE AGONIST SB224289: SYNTHESIS * AND IN VITRO AND IN VIVO FUNCTIONAL STUDIES

4.1 Introduction 108 4.2 Chemistry 110 Synthesis of ‘Biphenyl Tails’, 2,6-Dimethoxy-, 6-Methoxymianserin- and 2-Chromane Analogues of GR127935 and of Spirocyclic Piperidine and Indoline Analogues of SB224289 4.3 Pharmacology 115 Receptor Binding, 5-HT Reuptake Inhibition and Microdialysis in Rat Ventral Hippocampus 4.4 Results and discussion 115 4.5 Experimental 120 4.6 References 127

ABSTRACT

A series of analogues of N-[4-methoxy-3-(4-methyl-piperazin-1-yl]2’-methyl-4’-(5-methyl-

1,2,4-oxadiazol-3-yl)-biphenyl-4-carboxamide (GR127935, 4.1), a 5-HT1B/5-HT1D partial antagonist, and 1’-methyl-5-[[2’-methyl-4’-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4- yl]carbonyl]-2,3,6,7-tetrahydro-spiro[furo[2,3-f]indole-3,4’-piperidine] (SB224289, 4.2) was prepared. In this chapter, the synthesis and in vitro (affinity for the rat 5-HT1B and calf 5-HT1B receptors and the 5-HT reuptake site) and in vivo pharmacology (microdialysis in the ventral hippocampus of the freely moving rat) of these new compounds is described. In vivo, both GR127935, the spiropiperidine SB222553 (4.10), and the 6-piperazinoindoline analogue 4.17, (1 µmol/kg sc.), caused a rapid, long-lasting augmentation of the citalopram-induced (4.4, 10 µmol/kg sc.) increase of 5-HT levels up to 1100% above baseline. The piperazino-amidine analogues of both GR127935, compound 4.18, and SB22553, compound 4.11, failed to further enhance the citalopram-induced increase in 5-HT release.

The in vitro affinity for the r5-HT1A receptor and in vivo microdialysis study show that an oxygen substituent in the central phenyl ring of GR127935 and SB224289, despite previous claims, is not essential for antagonist function at terminal 5-HT autoreceptors in vivo.

* Parts of this chapter will be described in : Structural analogues of the 5-HT1B/1D partial antagonist GR127935 and 5-HT1B inverse agonist SB224289: synthesis and in vitro and in vivo functional studies. Wikström, H.V.; Liao, Y.; Mensonides-Harsema, M.M.; Cremers, T.I.F.H.; Damhof, M.; Vermeulen, E.; Matzen, L.; Devant, R.; Van Amsterdam, C.; Greiner, H.E.; Rautenberg, W. and Böttcher, H. (manuscript in preparation).

107 4.1 INTRODUCTION

As described in Chapter 1, the release of serotonin is subject of regulation by a negative feedback system, mediated by autoreceptors located on the cell bodies 1-11 (5-HT1A and 5-HT1D) and neuronal terminals (5-HT1B). The 5-HT1B receptor is of special interest, since it has been shown that the majority of terminal 5-HT 12-14 autoreceptors in rat, guinea pig, and human are of the 5-HT1B receptor subtype. In

1996, a small neuropeptide, 5-HT-Moduline, with nanomolar affinity for the 5-HT1B receptor was isolated.15-18

The 5-HT1B/1D receptor antagonist GR127935 (4.1) has been reported to increase extracellular 5-HT when perfused into the frontal cortex of the guinea pig.19-21 The 5-

HT1B receptor inverse agonist SB224289 (4.2) has been reported to antagonize sumatriptan-induced inhibition of 5-HT release in the frontal cortex of the guinea pig and to increase the 5-HT levels in the guinea pig dentate gyrus.22-25 Co-administration of the SSRI citalopram with GR127935 further elevates the release of 5-HT by administration of citalopram alone. It has been hypothesized that co- administration of a SSRI with an autoreceptor antagonist will instantaneously mimic desensitization of the autoreceptors, a necessary step for onset of action of 26-30 antidepressants. Recent studies have also suggested that supersensitive 5-HT1B/1D receptors may be involved in the pathophysiology of obsessive compulsive disorders

(OCD), and thus 5-HT1B/1D antagonists may prove to be useful in the treatment of OCD.31 Me N

N O N O F Me N N O H O Me Me 4.1 N N N H Me O O N N O O 4.3

N N Me N Me O Me 4.2 O N Me

4.4

F

Chart 4.1 Chemical structure of GR127935 (4.1), SB224289 (4.2), ketanserin (4.3) and citalopram (4.4)

108 In this chapter, the synthesis of several analogues of GR127935 and SB224289 is described. To study the structure affinity relationships (SAR) of GR127935, (i) a second methoxy group was introduced in the phenylpiperazine moiety (4.6), (ii) the piperazine moiety was incorporated into a tetracyclic azepin (4.8) or (iii) ‘incorporated’ a chromane (4.9). Furthermore, the effect of the substitution of the 1,3,4-oxadiazole for a 1,2,4-oxadiazole or methyl-, propyl- or piperazino-amidine in both GR127935 (4.7) and SB222553 32 (4.10 - 4.13) analogues and the effect of placing the piperazine or amino-alkoxy substituent in the 5 or 6 position of an indoline analogue of GR127935 (4.14 - 7.17) is studied (Figure 4.1). The in vitro affinity for the different species autoreceptor subtypes was established using tissue binding of the rat 5-HT1B receptors (frontal cortex) and calf 5-HT1B receptors

(striatum), which will reflect affinity for the human 5-HT1B receptor. Binding at rat frontal cortex and calf striatum tissue is specific for 5-HT1B receptor affinity, since ketanserin (4.3), a non-specific antagonist with high affinity for cloned 5-HT1D 33-35 receptors and low affinity for 5-HT1B receptors, does not bind to these tissues. In a microdialysis study, the effect of administration of the arylpiperazines GR127935 (4.1) and 4.18, the spiropiperidines SB222553 (4.10) and 4.11 and the 6- piperazinoindoline 4.17 alone and combined with the SSRI citalopram on 5-HT release in the rat ventral hippocampus is measured.36

Figure 4.1 Incorporation of arylpiperazine of GR127935 in either a tetracyclic azepin or chromane structure and alteration of arylpiperazine of GR127935 to spiropiperidine-indoline structure of SB224289, to different spiropiperidine and 5- and 6-substituted indolines.

GR-tail HN biphenyl-tail GR-tail N NH

O N

O O N N

SB224289 GR-tail HN GR-tail HN

O O N and N N N O O N N GR-tail GR-tail N N GR127935

N

N N N GR-tail N N GR-tail

109 4.2 CHEMISTRY

SYNTHESIS OF BIPHENYL-TAILS 4.5a-4.5e.

The 1,2,4-oxadiazole acid biphenyl tail 4.5a is synthesized using a Suzuki coupling of 2-(4’-bromo-3’-methylphenyl)-5-methyl-1,2,4-oxadiazole and 4-carboxybenzene- boronic acid in a mixture of dimethoxyethane/H2O, in the presence of sodium carbonate and tetrakis(triphenylphospine)palladium(0). To prepare the acid chloride intermediate 4.5b, the 1,2,4-oxadiazole acid intermediate 4.5a was refluxed in thionyl chloride. The N-methyl-piperazinyl and N-alkyl acid biphenyl tails 4.5c-4.5e were synthesized by coupling 4-bromo-3-methyl- to N-methyl-piperazine, N- methylamine or N-propylamine in dichloromethane in the presence of triethyl amine. These amidine intermediates, using a Suzuki coupling, are converted to the biphenyl building blocks by reacting them with 4-carboxybenzene-boronic acid in a mixture of dimethoxyethane/H2O, in the presence of sodium carbonate and tetrakis(triphenylphospine)palladium(0) (Scheme 4.1).

N O N O O a O Br + (HO) B 2 b H C N OH H C N OH Cl 3 3 4.5b CH3 CH3 4.5a

O (HO) B O O a; 2 O O c OH Br Br R N OH HO R2N 2 CH CH3 CH3 3 4.5c; N-Me-piperazinyl 4.5d; NH(Me) 4.5e; NH(n-Pr) SCHEME 4.1 Reagents and conditions: (a) Pd(PPh3)4, Na2CO3, DME/H2O (1:1 v/v), ∆; (b)

SOCl2, ∆; (c) N-Me-piperazine (4.5c)/ N-methylamine (4.5d)/ N-propylamine (4.5e), triethyl o 37 amine, CH2Cl2, 0 C to room temperature.

Cl N + C N N

P

Chart 4.2 Chemical structure of P-EDC.39

110 SYNTHESIS OF 2,6-DIMETHOXY ANALOGUES OF GR127935 (4.6 AND 4.7).

2,6-Dimethoxybenzoic acid chloride was subject to a Curtius rearrangement, yielding 2,6-dimethoxy aniline (4.6a). Refluxing the aniline in chorobenzene with mechlorethamine.HCl (CAUTION) yielded the required phenyl piperazine 4.6b which was nitrated with potassium nitrate in concentrated H2SO4 (4.6c) and subsequently hydrogenated to 4-(1-amino-3,5-dimethoxyphenyl)-1-methylpiperazine 38 (4.7). The aniline was coupled to building block 4.5b in CH2Cl2 at room temperature in the presence of triethylamine yielding the desired product 4.6, or to building block 4.5c) on P-EDC (Chart 4.2) in chloroform/THF at room temperature yielding 4.8 (Scheme 4.2).

H3C H3C H3C O O O

O N CH3 a b c NH2 N OH O O 4.6a O 4.6b

H3C H3C H3C

H3C H3C O O N CH N CH 3 d 3 N N

4.6c O2N O H2N O 4.6d

H3C H3C e

O NO R CH3 CH N O 4.6 R = N 3 H CH3

O H3C O N

4.7 N R = N N CH 3 CH 3 SCHEME 4.2 Reagents and conditions: (a) (i) thionyl chloride, reflux; sodium azide, anisol, o o 160 C; (ii) benzyl alcohol, 100 C; (iii) acetic acid, HCl (36%), H2O, reflux; (b) o mechlorethamine HCl, benzene, reflux; (c) sodium nitrate, H2SO4 (96%), 15 C; (d) H2, Pd/C

(10%), EtOH, 3 bar, room temperature; (e) 4.5b, triethyl amine, CH2Cl2, room temperature

(4.6)/ 4.5c, P-EDC, CHCl3/THF, room temperature (4.7).

SYNTHESIS OF 6-METHOXYMIANSERIN ANALOGUE OF GR127935 (4.8).

Details of the synthesis of 9-amino-6-methoxy-mianserin will be described in

Chapter 5. This building block is reacted with 4.5b in CH2Cl2 at room temperature in the presence of triethylamine yielding the desired product 4.8 (Scheme 4.3).

111 NH 2 N O O CH3 H C N N O 3 H CH N 3 O N H3C N 4.8 N CH CH3 3

SCHEME 4.3 Reagents and conditions: 4.5b, triethyl amine, CH2Cl2, room temperature.

SYNTHESIS OF 2-CHROMANE ANALOGUE OF GR127935 (4.9).

4-Chromanone was converted to 4.9a, by a Wittig-Horner reaction with triethylphosphonoacetate, which was reduced by catalytic hydrogenation. Hydrolysis by 10% NaOH (aq) afforded intermediate 4.9b, which was treated with refluxing thionyl chloride and was subsequently reacted with dimethylamine to 4.9c.

O O

O O CH3 OH

a b,c d, e

O O O 4.9a 4.9b O O

CH CH CH3 3 3 N N N CH3 CH3 CH3 H2N H2N f, g h

O O O 4.9c 4.9d 4.9e i

O N CH3 N H C 3 N CH H 3 N

CH3 O O 4.9

SCHEME 4.4 Reagents and conditions: (a) triethylphosphonoacetate, NaOEt, benzene, room temperature; (b) H2 (g), Pd/C (10%), EtOH; (c) 10% NaOH (aq); (d) SOCl2, ∆; (e) 40% o o NH(CH3)2, CH2Cl2, 0 C to room temperature; (f) Cu(NO3)2, Ac2O, 0 C to room temperature;

(g) Pd/C (10%), H2 (g), EtOH, room temperature; (h) LiAlH4, THF/Et2O, room temperature;

(i) 4.5a, P-EDC, CHCl3/THF, room temperature

Compound 4.9c is nitrated in the 7 position of the chromane ring system, using

Cu(NO3)2 in acetic anhydride and subsequently hydrogenated with H2 gas in the presence of 10% Pd/C in ethanol to 4.9d. The amide function of 4.9d was reduced using LiAlH4 in THF/ether, yielding 4.9e. This aniline was directly coupled to

112 building block 4.5a on P-EDC in chloroform/THF at room temperature, yielding the desired product 4.9 (Scheme 4.4).

SYNTHESIS OF SPIROCYCLIC PIPERIDINE ANALOGUES OF SB224289 (4.10-4.13).

The ether 4.10a was prepared from 2-iodophenol using Mitsunobu conditions. Cyclization to the spiropiperidine 4.10b was carried out using tributyltin hydride in benzene in the presence of α, α’-azobisisobutyronitrile (AIBN). Nitration with copper nitrate in acetic anhydride gave 4.10c, which was reduced by catalytic hydrogenation to the aniline 4.10d.32 The spirocyclic piperidine was then directly coupled to the different biphenyl building blocks 4.5a, 4.5c-e using P-EDC in CHCl3 with 20% THF, yielding the desired products 4.10 – 4.13 (Scheme 4.5).

I I CH3 a b N O N OH CH3 O 4.10a 4.10b

O N CH3 H N CH3 2 N 2 N c d

O O CH 4.10c 4.10d 3 e N R O NO CH H 3 N 4.10 R = CH 4.12 R = N N 3 H O O O CH 3 CH3 4.11 R = N N 4.13 R = N

O

o SCHEME 4.5 Reagents and conditions: (a) DEAD, PPh3 , THF, 0 C to room temperature; (b) o Bu3SnH, AIBN, benzene, ∆; (c) Cu(NO2)2, Ac2O, 0 C to room temperature; (d) H2, Pd/C (10%), EtOH; (e) biphenyl carboxylic acid building block 4.5a (4.10), 4.5c (4.11), 4.5d

(4.12), 4.5e (4.13), P-EDC, CHCl3/THF, room temperature.

SYNTHESIS OF INDOLINE ANALOGUES OF SB224289 (4.14-4.17).

1,5 SUBSTITUTED INDOLINES Catalytic hydrogenation of 1-acetyl-5-nitroindoline gave 1-acetyl-5-aminoindoline (4.14a) quantitatively. The formation of a piperazine ring with mechlorethamine could not be effected in refluxing acetonitrile in the presence o of diisopropylethylamine or Cs2CO3 (no conversion), or in DMF at 130 C using pyridine (low yields). The reaction, however, ran smoothly and in reasonable yields in refluxing benzylchloride to 4.14b. De-acetylation was effected by refluxing in concentrated hydrochloric acid, yielding 4.14c. The 5-piperazino indoline was then

113 directly coupled to the different biphenyl building blocks 4.5a, using P-EDC in CHCl3 with 20% THF, yielding the desired products 4.14 (Scheme 4.6).

CH NO NH 3 2 2 N N ab c N N N

H3C H3C 4.14a H3C O O O 4.14b

CH CH N 3 N 3 N N d O N N N H H C N O 3 4.14 4.14c CH3

SCHEME 4.6 Reagents and conditions: (a) H2, Pd/C (10%), EtOH, 3 bar, room temperature; (b) mechlorethamine.HCl, benzylchloride, 130 oC; (c) HCl (36%), 110 oC; (d) 4.5a, P-EDC,

CHCl3/THF, room temperature.

Via a Sandmeyer reaction, 1-acetyl-5-amino indoline (4.14a) is converted to 1-acetyl- 5-hydroxy indoline (4.14d). This compound is subsequently alkylated with either 2- dimethylaminoethyl chloride or 3-dimethylaminopropyl chloride in refluxing acetonitrile in the presence of Cs2CO3 gave the corresponding aminoethyl and aminopropyl indolines 4.15a and 4.16a. De-acetylation was effected by refluxing in concentrated hydrochloric acid, yielding 4.15b and 4.16b. The indolines are were coupled to biphenyl building block 4.5b in CH2Cl2 at room temperature in the presence of triethylamine yielding 4.15 and 4.16 (Scheme 4.7).

NH2 OH O CH3 abn N N CH N N 3

H3C H3C 4.14a O 4.14d H3C O O 4.15a, n = 2 4.16a, n = 3

O CH3 c n N CH N 3 H d O 4.15b, n = 2 CH3 n N 4.16b, n = 3 N O N CH3 N O 4.15, n = 2 H3C CH 4.16, n = 3 3 o SCHEME 4.7 Reagents and conditions: (a) H2SO4, NaNO2, H2O, 0 C/ CuSO4, H2O, ∆; (b) 2- dimethylaminochloroethane HCl (4.15a)/3-dimethylaminochloropropane HCl (4.16a), cesium o carbonate, acetonitrile, reflux; (c) HCl (36%), 110 C; (d) 4.5b, triethyl amine, CH2Cl2, room temperature.

114 1,6 SUBSTITUTED INDOLINES For the synthesis of 6-piperazino indoline 4.17a, the same chemistry was used as described for 5-piperazino indoline 4.14c, using 1-acetyl-6- nitroindoline as the starting material instead. Compound 4.17a was then directly coupled to biphenyl building block 4.5a, using P-EDC in CHCl3 with 20% THF, yielding compound 4.17 (Scheme 4.8).

a N NO N N 2 H N 4.17a CH H3C 3 O b

O N N N N N O CH H3C 4.17 3 CH3

SCHEME 4.8 Reagents and conditions: (a) (i)H2, Pd/C (10%), EtOH, 3 bar, room temperature; (ii) mechlorethamine.HCl, benzylchloride, 130 oC; (iii) HCl (36%), 110 oC; (b) 4.5a, P-EDC,

CHCl3/THF, room temperature.

4.3 PHARMACOLOGY

Compounds 4.6-4.18 were evaluated for their in vitro binding affinity at the 125 serotonin r-5-HT1B receptor, using [ I]-cyanoiodopindolol (frontal cortex), c-5-HT1B 3 3 receptors, using [ H]-5-HT (striatum) and 5-HT1A receptors, using [ H]8-OH- DPAT.40-42 In addition, the ability to block the 5-HT re-uptake site was evaluated (Table 4.1).43 Intracerebral microdialysis in freely moving rats was used to establish the effects of GR127935 (4.1), SB222553 (4.10), test compounds 4.11 and 4.17 and the N-Me- piperazino-amidine analogue of GR127935, compound 4.18 (Chart 4.3) on serotonergic transmission in the ventral hippocampus, upon co-administration with the selective serotonin re-uptake inhibitor citalopram (Figures 4.2 – 4.6).44

4.4 RESULTS AND DISCUSSION

All intermediates and the final products 4.8, 4.14, and 4.15 were inactive in the different binding assays. None of the final products had affinity for the 5-HT1A receptor. Only the 6-piperazino-indoline analogue 4.17, showed high affinity for the calf 5-HT1B/1D receptor. The chromane analogue 4.9, the spiropiperidine 4.10 and the 6-piperazino-indoline analogue 4.17, displayed a weak affinity for the 5-HT re-uptake site (Table 4.1).

From the in vitro structure-activity relationships (SARs) at the rat 5-HT1B receptor of GR127935, SB224289 and the final products, the following can be concluded. Introduction of a second methoxy substituent in the phenyl piperazine part (4.6),

115 induces selectivity for the r5-HT1B receptor, while the affinity for this receptor subtype vs GR1`27935 is slightly diminished. Replacement of the oxadiazole ring by a piperazino-amidine (4.7), leads to a further decrease in affinity for the r5-HT1B receptor. Incorporation of the arylpiperazine in a tetracyclic azepin structure (4.8) leads to a complete loss in affinity, while substitution of the o-methoxyphenyl piperazine for a chromane ringstructure (4.9) also leads to a considerable loss in affinity for the r5-HT1B receptor. Replacement of the o-methoxyphenyl piperazine for a spiropiperidine, proves more successful. The spiropiperidine analogues 4.10 – 4.13 all display good affinity for the r5-HT1B receptor. Compounds 4.11 – 4.13, in which the oxadiazole has been replaced by a piperazino-, methyl- or propyl-amidine, were ca. ten times less potent as SB222553 (4.10). In the series in which the o- methoxyphenyl piperazine is replaced by a 5-substituted-indoline, only the propyl analogue (4.16), has affinity for the r5-HT1B receptor. Substitution of the o- methoxyphenyl piperazine for a 6-piperazino-indoline (4.17) resulted in a ligand with equipotent affinity for the r-5-HT1B receptor than the reference compounds GR127935 and SB224289. Thus, the substituent carrying the basic nitrogen should be in the 6- and not in the 5-position of the indoline.

TABLE 4.1 In vitro affinities at rat 5-HT1D, calf 5-HT1B/1D, 5-HT1A and 5-HT re-uptake sites of GR127935, SB22489, and test compounds (IC50 in nM).

‡ ‡ compound r-5-HT1B c-5-HT1B 5-HT RU GR127935 1.6 + 0.7 52 + 13 SB224289 1.0 4.6 63.3 ± 0.7 4.7 900 4.9 443 ± 72 71 4.10 5.0 ± 2.0 250 4.11 40 4.12 50 4.13 40 4.16 800 4.17 3.7 ± 1.2 300 310 4.18 3 100

‡ IC50 > 1 µM, unless indicated differently.

Microdialysis studies have shown that administration of GR127935 alone does not effect the 5-HT release in the frontal cortex.32 Administration of GR127935 (1 µmol/kg sc.) alone did not alter the 5-HT release in the rat ventral hippocampus either (Figure 4.2). Administration of 10 µmol/kg sc. citalopram alone, increases the 5-HT levels to ca. 500 % above baseline. Combined administration of GR127935

116 (1 µM/kg sc.) with citalopram (10 µmol/kg sc.) doubled the citalopram-induced increase [F(1.140) = 9.95, p < 0.05 at t = 45-150 min].

FIGURE 4.2 Effects of GR127935 (1 µmol/kg sc.), citalopram (10 µmol/kg sc.) and GR127935 + citalopram (1 + 10 µmol/kg sc.) on 5-HT release (mean ± SEM, n = 5, *p < 0.05 vs. control).

GR127935+ citalopram 5-HT H3C % Basal O * * * * N N CH * * 1200 3 * O N 1000 H

800

CH3 600 citalopram N O N 400

CH3 200 GR127935

0 -100 -50 0 50 100 150 Time (min)

FIGURE 4.3 Effects of SB222553 (1 µmol/kg sc.), citalopram (10 µmol/kg sc.) and SB222553 + citalopram (1 + 10 µmol/kg sc.) on 5-HT release (mean ± SEM, n = 5, *p < 0.05 vs. control).

5-HT SB222553 + % Basal O * citalopram 1200 * * O N * CH * * 1000 N 3 * H *

800

CH 600 3 citalopram

N 400 O N

CH 200 3 SB222553

0

-100 -50 0 50 100 150 Time (min)

117 The spiropiperidine SB222553 and its analogue 4.11 (1 µmol/kg sc.) did not alter basal 5-HT levels alone. Co-administration of 1 µmol/kg sc. SB222553 with citalopram (10 µmol/kg sc.) again doubled the citalopram-induced increase [F(1.136) = 8.00, p < 0.05 at t = 45-150 min] (Figure 4.3).

FIGURE 4.4 Effects of 4.11 (1 µmol/kg sc.), citalopram (10 µmol/kg sc.) and 4.11 + citalopram (1 + 10 µmol/kg sc.) on 5-HT release (mean ± SEM, n = 5, *p < 0.05 vs. control).

5-HT % Basal O O 1000 N H H3C N N 800 N CH3 CH3 O 600

citalopram 400 4.11 + citalopram 200 4.11

0 -100 -50 0 50 100 150 Time (min)

FIGURE 4.5 Effects of 4.18 (1 and 10 µmol /kg sc.), citalopram (10 µmol/kg sc.) and 4.18 + citalopram (1 + 10 µmol/kg sc.) on 5-HT release (mean ± SEM, n = 5, *p < 0.05 vs. control).

5-HT CH3 % Basal O O 1000 N N H H C N 3 N CH 800 3 N CH3 O citalopram 600

400 4.18 + citalopram 200 4.18

0 -100 -50 0 50 100 150

Time (min)

118

Surprisingly, co-administration of 1 µmol/kg sc. of the N-Me-piperazino-amidine analogues 4.11 and 4.18 with citalopram (10 µmol/kg sc.), did not elevate the citalopram-induced increase in 5-HT levels (Figure 4.4 and 4.5). After administration of the 6-piperazinoindoline analogue 4.17 (10 µmol/kg sc.) alone, a tendency to increase the levels of 5-HT relative to baseline values is seen (Figure 4.6). However, this increase is not significant. Co-administration of 4.17 (1 µmol /kg sc.) and citalopram (10 µmol /kg sc.), resulted in an 100% augmentation of the citalopram -induced increase in 5-HT levels [F(1.130) = 8.39, p < 0.05 at t = 45-150 min].

FIGURE 4.6 Effects of 4.17 (1 and 10 µmol/kg sc.), citalopram (10 µmol/kg sc.) and 4.17 + citalopram (1 + 10 µmol/kg sc.) on 5-HT release (mean ± SEM, n = 5, *p < 0.05 vs. control).

5-HT 4.17 + citalopram % Basal N N CH3 * * * * N * 1200 * O * *

1000

CH3 800 N O N 600 citalopram H3C

400 4.17 (10 µM/kg) 200 4.17 (1 µM/kg) 0 -100 -50 0 50 100 150 Time (min)

To summarize, several structural analogues of GR127935 and SB224289 have been prepared. The SAR study for the GR127935 analogues show that incorporation of the arylpiperazine in an tetracyclic azepin ringsystem (4.8) or substitution for a chromane structure (4.9) is not favorable for affinity for the r5-HT1B or c5-HT1B receptors. Introduction of a second o-methoxy substituent in the arylpiperazine in GR127935, compound 4.6, induces selectivity for the r5-HT1B receptor, while the affinity for this receptor subtype is decreased by a factor ten. Substitution of the oxadiazole for an N- Me-piperazino-amidine further decreases the affinity with another factor ten. The SAR study of the SB224289 analogues show that, the spiropiperidine SB222553

(4.10) is equipotent and selective at the r5-HT1B receptor as the spiropiperidine indoline SB224289. Substitution of the oxadiazole for N-alkyl-amidines (4.11 - 4.13) results in a decrease for this receptor subtype with a factor ten. Furthermore it is shown in the 5- and 6-substituted indolines 4.14 - 4.17, that the 5-position is not favorable for the basic nitrogen carrying substituent. However, in vitro, introducing

119 the piperazine ring in the 6-position of the indoline (4.17) results in equipotency to SB224289. In vivo, the N-Me-piperazino-amidine analogues of GR127935, compound 4.18, and

SB222553, compound 4.11, despite high affinities for the r5-HT1B receptor, are not able to further enhance the citalopram-induced increase in 5-HT release in the rat ventral hippocampus. The citalopram-induced increase in 5-HT is enhanced by GR127935, SB222553 and the 6-piperazinoindoline 4.17, the maximum augmentation being approximately 11 times the initial, pre- levels and 2.2 times the 5-HT levels after administration of citalopram alone. The in vitro and in vivo results with 4.17 prove, that the ether oxygen substituent in GR127935 or SB224289 (Chart 4.3), despite previous claims,45 is not necessary for antagonism of the terminal 5-HT1B autoreceptor function.

N not carbonyl needed needed basic N N needed oxadiazole O N necessary for R = any lipophilic R in vivo substituent aromatic antagonistic N z needed functionality group H

H not substituent needed not needed

Chart 4.3 Claimed 5-HT1B structure-affinity relationships in GR 127935/SB224289 and result from presented in vitro and in vivo studies.45

4.5 EXPERIMENTAL

CHEMISTRY For general remarks see Chapter 2, Section 2.5. Compounds 4.5a – 4.5d, 4.6b and 4.10a - 4.10 were prepared as earlier described.32, 37, 46 Compounds 4.5a - 4.5e and 4.18 were synthesized by Dr. Y. Liao; compounds 4.10b and 4.14d were synthesized by Dr. L. Matzen.

1-Methyl-(2,6-dimethoxy-3-nitrophenyl)piperazine (4.6c) To an ice-cooled solution of 4.6b (350 mg; 1.6 mmol) in concentrated sulfuric acid (96 %; 4 mL), potassium nitrate (150 mg; 1.6 mmol) was added in small portions. The reaction mixture was stirred at room temperature for 17 h, after which it was again cooled on ice and basified with an aqueous solution of sodium hydroxide (4 N) to pH=8. The aqueous phase was extracted with CH2Cl2 and the organic layer was subsequently washed with brine, dried and removed in vacuo. The residue was purified by chromatography (CH2Cl2 with 5% methanol) yielding 220 mg (53%) of 4.6c as a yellow oil: 1H NMR δ 7.76-7.73 (d, J = 9.16, 1H), 6.76-6.73 (d, J = 9.16, 1H), 4.05 (s, 3H), 3.98 (s, 3H), 3.31-3.28 (m, 4H), 2.64-2.61 (m, 4H), 2.45 (s, 3H); 13C NMR δ 158.9, 148.7, 135.3, 131.9, 118.7, 103.1, 59.7, 52.9, 52.8, 47.0, 43.4; MS (EIPI) m/z (M+1).

1-Methyl-(3-amino-2,6-dimethoxyphenyl)piperazine (4.6d) A solution of 4.6c (110 mg; 0.4 mmol) in ethanol (20 mL) was hydrogenated over 10% Pd/C (10 mol%) in a Parr apparatus under a H2 pressured of 4 atm. for 1.5 h. The catalyst was removed by filtration through Kieselguhr and the solvent removed in vacuo, yielding 100 mg (102 %) of 4.6d as a colorless oil, which was used without further

120 purification: 1H NMR δ 5.88 (s, 1H),3.73 (s, 6H), 3.15-3.10 (m, 4H), 2.56-2.51 (m, 4H), 2.35 (s, 3H); 13C NMR δ 158.4, 144.4, 120.8, 92.9, 92.0, 56.0, 55.8, 50.1,46.1; MS (EIPI) m/z (M+1).

2’-Methyl-4’-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [2,4-dimethoxy-3-(N- methyl) piperazin-1-yl)-phenyl]-amide (4.6) To an ice-cooled solution of 4.6d (100 mg; 0.4 mmol) and triethyl amine (90 µL) in THF (10 mL) a cold solution of 4.5b (0.5 mmol) in THF (5 mL) was added. The reaction mixture was stirred at room temperature for 17 h, after which the solvent was removed in vacuo. The residue was purified by gradient chromatography (hexane to ethyl acetate to THF) yielding 4.6 as a colorless oil. This was converted to the oxalate salt and recrystallized from EtOH to give 200 mg (80 %) as white crystals: (mono-oxalate) mp 214-217 oC; 1H NMR δ 8.53 (s, 1H) 8.27-8.17 (m, 1H), 8.02-7.94 (m, 3H), 7.50-7.34 (m, 4H), 6.71-6.66 (d, J = 9.03, 1H), 3.97 (s, 3H), 3.81 (s, 3H), 3.36 (m, 4H), 2.80 (m, 4H), 2.67 (s, 3H), 2.53 (s, 3H), 2.36 (s, 3H); 13C NMR δ 164.6, 146.3, 144.5, 143.4, 136.0, 135.7, 133.9, 130.1, 129.4, 129.2, 128.6, 126.8, 125.4, 124.8, 116.4, 107.2, 61.7, +1 55.6, 55.2, 49.1, 45.2, 20.2; MS (EIPI) m/z (M ); Anal. (C30H33N5O4.0.5 H2O) C, H, N

1-[2’-Methyl-4’-(N-methyl-piperazinamido)-biphenyl-4-carboxyl acid [2,4-dimethoxy-3-(N- methyl) piperazin-1-yl)-phenyl]-amide (4.7) A mixture of 4.6d (70 mg; 0.3 mmol), 4.5c (140 mg; 0.4 mmol) and P-EDC (900 mg) in CHCl3 (5 mL) and THF (1 mL) were shaken for 17 h. The reaction mixture was filtrated and the solvent removed in vacuo. The residue was purified by chromatography 1 (CH2Cl2 with 10% EtOH and 1% NH4OH) yielding 94 mg (60%) of 4.7 as a brown oil/solid: H NMR δ 8.52 (s, 1H) 8.06-8.01 (d, J = 8.31, 1H), 7.93-7.89 (m, 1H), 7.39-7.23 (m, 3H), 7.07 (s, 1H), 6.81 (s, 1H), 3.81 (s, 3H), 3.74 (s, 3H), 3.53-3.39 (m, 6H), 3.07 (m, 4H), 2.73-2.72 (m, 4H), 2.41 (m, 2H), 2.34 (s, 3H), 2.24 (s, 3H), 2.17 (s, 3H); 13C NMR δ 166.0, 165.2, 144.6, 143.4, 136.2, 135.9, 130.6, 129.5, 129.0, 127.1, 126.8, 125.4, 124.3, 123.5, 115.2, 107.0, 97.1, 96.8, 55.7, 54.9, 47.7, 45.8, 44.1, 20.1; MS (FAB) m/z 572 (M+1).

2’-Methyl-4’-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [1,2,3,4,10,14b- hexahydro-6-methoxy-2-methyldibenzo[c,f]pyrazino[1,2-a]azepin]-amide (4.8) To an ice-cooled solution of 4.5b (138 mg; 0.47 mmol) in THF (7 mL), 9-Amino-1,2,3,4,10,14b-hexahydro-6-methoxy- 2-methyldibenzo[c,f]pyrazino[1,2-a]azepin* (91 mg; 0.29 mmol) in THF (3 mL) and triethyl amine (80 µL) were added. The reaction mixture was stirred at room temperature for 17 h, after which the solvent was removed in vacuo. The residue was purified by gradient chromatography (hexane to ethyl acetate to THF) yielding 15 mg (9 %) 4.8 as a colorless solid: mp 144-148 oC; 1H NMR δ 8.16-8.10 (m, 3H), 7.79 (s, 1H), 7.68-7.65 (d, J = 7.69, 1H), 7.53-7.50 (d, J = 7.69, 1H), 7.26-7.20 (m, 5H), 6.90-6.87 (d, J = 8.79, 1H), 4.90-4.86 (m, 1H), 4.76-4.73 (m, 1H), 4.22 (m, 1H), 3.92 (s, 3H), 3.74-3.69 (m, 1H), 3.40- 3.56 (m, 1H), 3.21-3.05 (m, 2H), 2.82 (s, 3H), 2.61 (s, 3H), 2.52 (s, 3H), 2.67-2.48 (m, 2H); MS (FAB) m/z 586 (M+1).

4-Ethoxycarbonylmethylenyl-chromane (4.9a) To a solution of 4-chromanone (15.0 g, 98.8 mmol) in benzene (120 mL) triethylphosphonoacetate (20 mL, 1 equivalent) was added. To this reaction mixture a solution of freshly prepared solution of NaOEt (2.8 g Na(s) in 110 mL EtOH) was added drop-wise. The reaction was stirred at room temperature for 60 h and quenched with ice (100 mL). The aqueous layer was extracted with CH2Cl2. The combined organic layers washed with brine, dried and removed in vacuo, yielding 22.8 g (103 %) of 4.9a as a dark brown oil: MS (EIPI) m/z 218 (M+).

4-Carboxylmethyl-chromane (4.9b) A solution of 4.9a (25.0 g; 144.7 mmol) in MeOH (120 mL) was hydrogenated over 10% Pd/C (10 mol%) in a Parr apparatus under a H2 pressured of 45 psi at room temperature for 16 h. The reaction was filtrated through Celite and the solvent removed in vacuo. The

* For the synthesis of 9-amino-6-methoxy-mianserin, see Chapter 5.

121 residue was recrystallized from MeOH, yielding 22.3 g (89 %) of 4-ethoxycarbonylmethyl-chromane as white crystals: mp = 176-180 oC; 1H NMR δ 8.33, 7.56 (m, 1H), 7.12-6.57 (m, 3H), 4.47-4.08 (m, 4H), 2.71-2.31 (m, 3H), 1.39-1.30 (m, 5H); IR (KBr): 1725 cm-1; MS (EIPI) m/z 220 (M+). A solution of 4-ethoxycarbonylmethyl-chromane (20.1 g, 91,2 mmol) in 2N NaOH (aq) (100 mL) was stirred at 40 oC for 19 h. The reaction was quenched with ice-cooled 4N HCl (aq) (60 mL) and extracted with

CH2Cl2. The combined organic layers were washed with brine, dried and removed in vacuo, yielding 18.0 g (100 %) of 4.9b as a brown oil: IR (KBr): 1713 cm-1; MS (EIPI) m/z 192 (M+).

4-(N,N-Dimethylaminocarbonylmethyl)-chromane (4.9c) A solution of 4.9b (8.0 g; 42 mmol) in

SOCl2 (30 mL) was refluxed for 30 min. The excess of SOCl2 was removed in vacuo and the residue (dark brown oil) used without further purification. The acid chloride intermediate (8.8 g; 42 mmol)was dissolved in CH2Cl2 (75 mL) and added drop-wise to an ice-cooled 40 % (aq) solution of dimethylamine (100 mL). The reaction mixture was stirred at room temperature for 1 h. The layers were separated and the aqueous layer washed with CH2Cl2 (3 x 50 mL). The combined organic layers were washed with 2 N (aq) NaOH (3 x 50 mL) and brine (3 x 15 mL), dried and removed in vacuo. The residue was purified by gradient chromatography (hexane with 25 % EtOAc to 100 % EtOAc), yielding 1.81 g (23 %) of 4.9c as a light yellow oil.

6-Amino-4-(N,N-dimethylaminocarbonylmethyl)-chromane (4.9d) To an ice-cooled solution of

7.15 (1.81 g; 8.34 mmol) in acetic anhydride (50 mL) Cu(NO3)2 (2.41 g; 9.98 mmol) was added in small portions. The reaction was stirred at 0 oC for 2 h and at room temperature for 1 h, after which the reaction mixture was poured on ice (1 L). Potassium carbonate was added in small portions to pH ~ 8. The aqueous layer was extracted with EtOAc (3 x 200 mL). The combined organic layers were washed with brine, dried and removed in vacuo. The residue was purified by gradient chromatography (hexane with 25 to 50 % EtOAc), yielding 1.26 g (57 %) of 4-(N,N-dimethylaminocarbonylmethyl)-6-nitro- chromane as a red-brown oil: IR (NaCl): 1644, 1513, 1343 cm-1. A suspension of the nitro intermediate

(1.26 g; 4.8 mmol) and 10 % Pd/C (10 mol%) in EtOH (200 mL) were hydrogenated under a H2 atm (45 psi) at room temperature for 3 h, using a Parr-shaker. The mixture was filtrated and the solvent removed in vacuo. The residue was purified by chromatography (EtOAc with 25 % hexane), yielding 0.86 g (77 %) of 4.9d as a yellow-brown oil: 1H NMR δ 6.71-6.50 (m, 3H), 4.24-4.16 (m, 2H), 3.72 (b, 2H), 3.43-3.40 (m, 1H), 2.96-2.94 (m, 6H)*, 2.72-2.70 (m, 1H), 2.57-2.50 (m, 1H), 2.29-2.10 (m, 1H), 1.86-1.78 m, 1H); 13C NMR δ 171.3, 135.1, 125.1, 120.0, 118.1, 113.0, 63.2, 39.9, 37.0*, 35.3, 29.9, 27.4; IR (NaCl) 3455, 3347, 1635 cm-1; MS (EI) m/z 234 (M+).

6-Amino-4-(N,N-dimethylaminoethyl)-chromane (4.9e) To an ice-cooled solution of 4.9d (0.58 g;

2.49 mmol) in THF (60 mL) LiAlH4 ( 285 mg; 7.50 mmol) was added in small portions. The reaction mixture was stirred on room temperature for 2 h, after which the excess of LiAlH4 was destroyed with

H2O (1 x 0.3 mL), 10 % (aq) NaOH (1 x 0.3 mL) and H2O (3 x 0.3 mL). The mixture was filtrated and the solvent removed in vacuo. The residue was purified by chromatography (EtOAc with 20 % EtOH and 1 % TEA), yielding 0.33 g (60 %) of 4.9e as a colorless oil: 1H NMR δ 6.70-6.67 (m, 1H), 6.59- 6.55 (m, 2H), 4.26-4.21 (m, 2H), 3.72 (b, 2H), 2.98-2.80 (m, 1H), 2.47-2.40 (m, 2H), 2.30 (s, 6H), 2.10-1.97 (m, 2H), 1.84-1.75 (m, 2H); 13C NMR δ 135.3, 119.9, 118.2, 112.7, 63.4, 56.9, 45.1, 33.7, 31.3, 26.9; IR (NaCl) 3448, 3365, 3189 cm-1; MS (EI) m/z 220 (M+).

2’-Methyl-4’-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid 6’-[4’-(N,N- dimethylaminoethyl)-chromane]-amide (4.9) A suspension of 4.9e (125.0 mg; 0.57 mmol), 4.5a

(335.0 mg; 1.14 mmol) and P-EDC (2 g) in CHCl3 (8 mL) and THF (2 mL) were shaken for 48 h. The mixture was filtrated and the solvent removed in vacuo. The residue was purified using gradient

* both cis and trans conformers of the amide are present in a 1:1 ratio

122 chromatography (CH2Cl2 with 7 to 10 % EtOH), yielding 30 mg (11 %) of 4.9 as a colorless solid: mp 130-135 oC; 1H NMR δ 8.56 (s, 1H), 8.40-8.36 (m, 1H), 8.00-7.94 (m, 3H), 7.85-7.31 (m, 3H), 6.95- 6.92 (m, 2H), 4.36-4.30 (m, 2H), 2.98-2.90 (m, 1H), 2.98 (s, 3H), 2.68 (s, 3H), 2.61-2.57 (m, 2H), 2.43 (s, 6H), 2.35-2.30 (m, 3H), 2..16-2.10 (m, 2H), 1.93-1.86 (m, 2H); 13C NMR (125 MHz) δ 176.4, 167.9, 164.7, 145.3, 144.4, 143.4, 143.2, 143.1, 136.6, 135.9, 134.0, 133.8, 130.1, 130.0, 129.4, 129.2, 129.1, 129.0, 128.5, 127.0, 127.0, 126.8, 126.0, 124.7, 124.7, 123.4, 123.3, 120.2, 118.6, 117.8, 88.8, 64.1, 56.4, 54.1, 44.5, 31.2, 29.5, 26.7, 20.2, 12.3; MS (FAB) m/z 497 (M+1).

N-(2,3-Dihydro-1'-methylspiro[benzofuran-3,4'-piperidine]-5-yl)-2'-methyl-4' (5-methyl-1,2,4- oxadiazol-3-yl)- biphenylcarboxamide (4.10) A mixture of 4.10d (218 mg; 1.0 mmol), 4.5a (590 mg;

2.0 mmol) and P-EDC (3 g) in CHCl3 (32 mL) and THF (8 mL) were shaken for 72 h. The reaction mixture was filtrated and the solvent removed in vacuo. The residue was purified by chromatography

(CH2Cl2 with 10% EtOH and 1% NH4OH) yielding 220 mg (45%) of 4.10 as a brown oil/solid: mp = 106-110 °C1H NMR δ 8.22 (s, 1H) 7.97-7.91 (m, 3H), 7.54-7.26 (m, 4H), 6.76-6.72 (m, 2H), 4.35 (s, 2H), 2.92-2.89 (m, 4H), 2.65 (s, 3H), 2.35 (s. 3H), 2.26 (s, 3H), 2.18-2.02 (m, 2H), 1.77-1.70 (m, 2H); 13C NMR δ 156.3, 144.4, 143.5, 135.9, 135.0, 133.7, 131.1, 130.2, 130.0, 129.5, 129.2, 128.9, 127.2, 127.0, 126.0, 124.7, 121.7, 116.5, 109.5, 80.5, 52.5, 45.7, 43.7, 35.5, 20.1, 12.1; MS (FAB) m/z 497 (M+1).

N-(2,3-Dihydro-1'-methylspiro[benzofuran-3,4'-piperidine]-5-yl)-2'-methyl-4'-(N- methylpiperazinamido)biphenylcarboxamide (4.11) A mixture of 4.10d (55 mg; 0.3 mmol), 4.5c

(120 mg; 0.4 mmol) and P-EDC (800 mg) in CHCl3 (5 mL) and THF (1 mL) were shaken for 17 h. The reaction mixture was filtrated and the solvent removed in vacuo. The residue was purified by chromatography (CH2Cl2 with 10% EtOH and 1% NH4OH) yielding 51 mg (38%) of 4.11 as a brown oil/solid: 1H NMR δ 8.71 (s, 1H) 7.97-7.93 (d, J = 8.30, 1H), 7.52-7.45 (m, 2H), 7.30-7.17 (m, 2H), 6.78-6.73 (d, J = 8.55, 1H), 4.37 (s, 2H), 3.78 (m, 2H), 3.50 (m, 2H), 2.88 (m, 4H), 2.45 (m, 2H), 2.35 (s, 3H), 2.31 (s, 3H), 2.22 (s, 3H),2.09-2.04 (m, 4H), 1.78-1.73 (m, 2H); 13C NMR δ 170.1, 165.4, 156.2, 144.1, 142.3, 135.8, 135.0, 134.9, 133.6, 131.4, 129.5, 128.9, 127.2, 124.3, 121.7, 116.6, 109.4, 80.5, 52.7, 46.0, 45.8, 43.8, 35.8, 20.1; MS (FAB) m/z 539 (M+1).

N-(2,3-Dihydro-1'-methylspiro[benzofuran-3,4'-piperidine]-5-yl)-2'-methyl-4'-(N- methylamido)biphenylcarboxamide (4.12) A mixture of 4.10d (131 mg; 0.6 mmol), 4.5d (322 mg;

1.2 mmol) and P-EDC (2 g) in CHCl3 (8 mL) and THF (2 mL) were shaken for 72 h. The reaction mixture was filtrated and the solvent removed in vacuo. The residue was purified by chromatography

(CH2Cl2 with 10% EtOH and 1% NH4OH) yielding 20 mg (7%) of 4.12 as a brown solid: mp = 220 - 225 oC; 1H NMR δ 8.28 (s, 1H) 7.96-7.92 (m, 2H), 7.69-7.45 (m, 2H), 7.35-7.19 (m, 2H), 6.78-6.43 (m, 3H), 4.37 (s, 2H), 3.03-2.89 (m, 4H), 2.42 (s, 3H), 2.25 (s. 3H), 2.15-2.03 (m, 4H), 1.42 (s, 3H); 13C NMR δ 183.1, 172.7, 168.0, 166.0, 156.3, 155.6, 144.2, 144.0, 143.7, 140.2, 135.6, 134.0, 133.7, 132.0, 131.2, 139.6, 129.1, 129.0, 127.0, 125.1, 124.1, 122.3, 121.8, 119.3, 116.5, 11.7, 109.6, 80.4, 79.3, 60.3, 52.5, 45.6, 43.6, 40.4, 35.4, 31.1, 29.4, 26.6, 25.9, 20.1, 19.7, 14.3; MS (FAB) m/z 470 (M+1).

N-(2,3-Dihydro-1'-methylspiro[benzofuran-3,4'-piperidine]-5-yl)-2'-methyl-4'-(N- propylamido)biphenylcarboxamide (4.13) A mixture of 4.10d (55 mg; 0.3 mmol), 4.5e (120 mg; 0.4 mmol) and P-EDC (800 mg) in CHCl3 (5 mL) and THF (1 mL) were shaken for 17 h. The reaction mixture was filtrated and the solvent removed in vacuo. The residue was purified by chromatography 1 (CH2Cl2 with 10% EtOH and 1% NH4OH) yielding 40 mg (32%) of 4.13 as a brown oil/solid: H NMR δ 8.33 (s, 1H) 7.98-7.94 (d, J = 8.30, 1H), 7.70-7.23 (m, 5H), 6.81-6.76 (d, J =9.03, 1H), 6.32 (t, J =

5.37, 1H), 4.39 (s, 2H), 3.49-3.39 (dd, J1 = 13.67, J2 = 6.67, 2H), 3.07-3.02 (m, 2H), 2.47 (s, 3H), 2.29

(s, 3H), 2.23-2.10 (m, 2H), 1.87-1.81 (m, 2H), 1.75-1.57 (sextet, J1 = 14.65, J2 = 7.32 , 2H), 1.25-0.96

123 (t, J = 7.4, 3H); MS (FAB) m/z 498 (M+1); HPLC-MS: purity 96%, peak at RT 3.02 min, MS (APCI) m/z 498 (M+1).

1-Acetyl-5-amino indoline (4.14a) A suspension of 1-acetyl-5-nitro indoline (5.0 g; 24.3 mmol) and

10 % Pd/C (10 mol%) in EtOH (500 mL) is hydrogenated under H2-atmosphere (3 bar) at room temperature, using a Parr-shaker. The reaction mixture was filtrated and the solvent removed in vacuo 1 yielding 4.1 g (95 %) of 4.14a: H NMR δ 7.73 (d, J = 8.5, 1H), 6.45 (m, 1H), 6.33 (dd, J1 = 8.5, J2 = 2.4, 1H), 4.81 (s, br, 2H), 3.96 (t, J = 8.4, 2H), 2.99 (t, J = 8.4, 2H), 2.07 (s, 3H); MS (EIPI) m/z 176 (M+1).

1-Acetyl-5-(N-methyl)piperazinyl indoline (4.14b) A suspension of 4.14a (1.8 g; 10 mmol) and mechlorethamine HCl (2.9 g, 15 mmol) and triethylamine (4 mL) is refluxed in chlorobenzene (50 mL) for 3 days. The solvent is removed in vacuo. The residue is purified by flash chromatography (ethyl 1 acetate with 25 % ethanol), yielding 1.2 g (45 %) of 4.14b as a pinkish solid: H NMR (CD3OD) δ 7.95 (d, J = 8.79, 1H), 6.86-6.72 (m, 2H), 4.01 (t, J = 8.47, 2H), 3.15-3.11 (m, 6H), 2.61-2.56 (m, 4H), 2.33 13 (s, 3H), 2.15 (s, 3H); C NMR (CD3OD) δ 168.9, 148.2, 135.8, 133.0, 116.8, 114.6, 112.9, 54.4, 49.1, +1 48.4, 44.5, 27.3, 22.0; MS (EIPI) m/z 259 (M ); Anal. (C15H21N3O1.½ H2O) C, H, N: found 15.7 (calc. 15.1).

5-(N-Methyl)piperazinyl indoline (4.14c) A solution of 4.14b (120 mg, 0.46 mmol) in conc. HCl (5 mL) is refluxed for 17h. The reaction mixture is quenched with ice and basified with a 4N NaOH (aq) solution. The aqueous phase is extracted with CH2Cl2 (3 x 15 mL). The combined organic layers are washed with brine, dried and concentrated in vacuo, yielding 70 mg (70 %) of 4.14c as a brown oil which is used without further purification: 1H NMR δ 6.85-6.84 (m, 1H), 6.70-6.57 (m, 2H), 3.56-3.47 (m, 4H), 3.10-2.95 (m, 4H), 2.62-2.58 (m, 4H), 2.36 (s, 3H); 13C NMR δ 145.7, 145.0, 130.6, 116.4, 115.3, 109.9, 55.1, 51.0, 47.4, 45.8, 30.1; MS (EIPI) m/z 217 (M+); MS (FAB) 217, 218 (M+1).

1-[2’-Methyl-4’-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxyl]-5-[(N-methyl) piperazino] indoline (4.14) A suspension of 4.14c (100 mg, 0.45 mmol), 4.5a (200 mg, 0.67 mmol) and P-EDC

(1.0 g) in CHCl3 (10 mL) and THF (1 mL) is shaken at room temperature for 48 h. The reaction mixture is filtrated and the solvent removed in vacuo. The residue is purified using gradient chromatography (CH2Cl2/EtOH with 1 % NH4OH) yielding 110 mg (50 %) of 4.14 as a light-brown solid: 1H NMR δ 8.00-7.94 (m, 2H), 7.65-7.54 (m, 3H)7.44-7.34 (m, 3H), 6.85 (m, 2H), 4.13 (b, 2H), 3.21-3.07 (m, 6H), 2.68 (s, 3H), 2.66-2.64 (m, 4H), 2.39 (s, 3H), 2.36 (s, 3H); 13C NMR δ 144.0, 136.0, 133.8, 131.6, 130.1, 129.2, 127.1, 125.8, 124.7, 54.8, 49.5, 45.7, 20.2, 12.2; MS (FAB) m/z 494 (M+1);

Anal. (C30H31N5O2. C2H5OH) C, H, N.

1-Acetyl-5-dimethylaminoethoxy indoline (4.15a) To a suspension of 1-acetyl-5-hydroxyindoline

(177 mg, 1.0 mmol) and CsCO3 (1.6 g; 5.0 mmol) in acetonitrile (10 mL), 2-dimethylamino-1- chloroethane.HCl (180 mg, 1.2 mmol) is added. The reaction mixture was refluxed for 3 h and filtrated after which the solvent is removed in vacuo. The residue is redissolved in H2O and basified with solid

NaHCO3 to pH ~ 8. The aqueous layer is extracted with CH2Cl2 (3 x 15 mL). The combined organic layers are washed with brine, dried and concentrated in vacuo. The residue is purified by flash chromatography (EtOAc with 20 % ethanol) yielding 140 mg (56 %) of 4.15a as an off-white solid: 1H NMR (DMSO) δ 8.03 (d, J = 10.27, 1H), 6.92-6.78 (m, 2H), 4.30 (t, J = 5.02, 2H), 4.09 (t, J = 8.49, 2H), 3.54 (t, J = 5.01, 2H), 3.15 (t, J = 7.61, 2H), 2.91 (s, 6H) 2.14 (s, 3H); IR (KBr) 1644 cm-1; MS (EIPI) m/z 248 (M+1).

1-Acetyl-5-dimethylaminopropoxy indoline (4.16a) Procedure 4.15a: 1-acetyl-5-hydroxyindoline

(350 mg, 2.0 mmol) and CsCO3 (3.3 g; 10.0 mmol) in acetonitrile (50 mL) and 3-dimethylamino-

124 chloropropane.HCl (300 mg, 2.5 mmol); yield 360 mg (69 %) of 4.16a as a colorless oil; 1H NMR δ 8.09 (d, J = 8.30, 1H), 6.73-6.67 (m, 2H), 4.05-3.93 (m, 4H), 3.13 (t, J = 8.42, 2H), 2.45 (t, J = 7.33, 2H), 2.25 (s, 6H) 2.18 (s, 3H), 1.93 (p, 2H); 13C NMR δ 167.8, 155.5, 136.5, 132.5, 117.3, 122.6, 111.2, 66.4, 56.2, 48.7, 45.2, 27.9, 27.3, 23.7; MS (EIPI) m/z 262 (M+1).

5-Dimethylaminoethoxy indoline (4.15b) Procedure 4.14c: 4.15a (200 mg, 0.8 mmol) and conc. HCl (5 mL); yield 130 mg (80 %) of 4.15b as a brown oil: 1H NMR δ 6.77-6.76 (m, 1H), 6.63-6.53 (m, 2H), 4.00 (t, J = 5.74, 2H), 3.52 (t, J = 8.30, 2H), 2.99 (t, J = 8.30, 2H), 2.73 (t, J =5.74, 2H), 2.36 (s, 6H), 1.93 (p, 2H); 13C NMR δ 152.7, 145.2, 130.9, 112.9, 112.2, 109.9, 66.9, 56.3, 47.6, 45.2, 30.1, 27.4; IR (NaCl) 1493 cm-1; MS (EIPI) m/z 206 (M+1).

5-Dimethylaminopropoxy indoline (4.16b) Procedure 4.14c: 4.16a (300 mg, 1.2 mmol) and conc. HCl (5 mL); yield 150 mg (60 %) of 4.16b as a brown oil: 1H NMR δ 6.77-6.75 (m, 1H), 6.58 (m, 2H), 3.93 (t, J = 6.47, 2H), 3.52 (t, J = 8.42, 2H), 2.99 (t, J = 8.30, 2H), 2.47 (t, J = 7.45, 2H), 2.27 (s, 6H), 1.93 (p, 2H); 13C NMR δ 152.7, 145.2, 130.9, 112.9, 112.2, 109.9, 66.9, 56.3, 47.6, 45.2, 30.1, 27.4; MS (EIPI) m/z 220 (M+1).

1-[2’-Methyl-4’-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxyl]-5-dimethylamino-ethoxy indoline (4.15) A solution of 4.5a (215 mg, 0.73 mmol) is refluxed in SOCl2 (3 mL) for 30 min. The solvent is removed in vacuo, after which 4.5b is redissolved in CH2Cl2 (10 mL). This solution is than added drop-wise to an ice-cooled solution of the 4.15b (100 mg, 0.49 mmol) and triethyl amine (0.1 mL) in CH2Cl2 (10 mL). The reaction is stirred at room temperature for 17 h, after which the solvent is removed in vacuo. The residue is purified using gradient flash chromatography (CH2Cl2/EtOH with 1 1 % NH4OH), yielding 150 mg (64 %) of 4.15as an off-white solid: H NMR δ 8.00-7.93 (m, 3H), 7.65- 6.61 (m, 2H), 7.44-7.34 (m, 4H), 6.82 (s, 1H), 4.10 (m, 4H), 3.11 (t, J = 8.06, 2H), 2.82 (t, J = 5.49, 2H), 2.68 (s, 3H), 2.42 (s, 6H), 2.35 (s, 3H); 13C NMR δ 176.5, 168.1, 144.0, 136.0, 130.12, 129.2, 129.1, 17.0, 125.9, 124.7, 112.7, 111.5, 65.8, 57.8, 45.4, 20.2, 12.2; MS (FAB) m/z 483 (M+1).

1-[2’-Methyl-4’-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxyl]-5-dimethylamino-propoxy indoline (4.16) Procedure 4.15: 4.5a (150 mg, 0.55 mmol), 4.16b (100 mg, 0.45 mmol); yield 150 mg (67 %) of 7.41 as an off-white solid: 1H NMR δ 8.00-7.93 (m, 3H), 7.65-6.61 (m, 2H), 7.44-7.27 (m, 3H), 6.81-6.80 (m, 2H), 4.13 (m, 2H), 4.00 (t, J = 6.23, 2H), 3.11 (t, J = 8.18, 2H), 2.68 (s, 3H), 2.49 (t, J = 7.21, 2H), 2.35 (s, 3H), 2.28 (s, 6H), 1.96 (p, 2H); 13C NMR δ 176.5, 168.1, 143.7, 136.0, 130.1, 129.2, 129.1, 127.0, 125.9, 124.7, 112.6, 111.4, 66.4, 56.1, 45.2, 27.2, 20.2, 12.2; MS (FAB) m/z 497 (M+1).

6-(N-Methyl)piperazinyl indoline (4.17a) Procedure 4.14a: 1-acetyl-6-nitro indoline (1.0 g, 4.9 mmol), 10 % Pd/C (10 mol%) in EtOH (125 mL); yield 0.8 g (94 %) of 1-acetyl-6-amino indoline as a 1 colorless oil: H NMR δ 7.67-7.66 (d, J = 1.95, 1H), 6.94-6.90 (d, J = 7.81, 1H), 6.37-6.32 (dd, J1 = 13 7.81, J2 = 2.20, 1H), 4.00 (t, J = 8.43, 2H), 3.49 (b, 2H), 3.06 (t, J = 8.43, 2H), 2.19 (s, 3H); C NMR δ 146.0, 143.7, 124.7, 120.8, 110.1, 104.3, 49.3, 27.0, 24.1; MS (EIPI) m/z 176 (M+1). Procedure 4.14b: 1-acetyl-6-amino indoline (1.0 g, 5.7 mmol), TEA (10 mL) and mechlorethamine.HCl (1.6 g, 8.5 mmol) in chlorobenzene (100 mL); yield 0.7 g (50 %) of 1-acetyl-6-(N-methyl)piperazinyl indoline as 1 an off-white solid: H NMR δ 7.97 (d, J = 2.20, 1H), 7.03 (d, J = 8.30, 1H), 6.60-6.55 (dd, J1 = 8.18, J2 = 2.32, 1H), 4.02 (t, J = 8.30, 2H), 3.21 (t, J = 4.88, 4H), 3.09 (t, J = 8.35, 2H), 2.57 (t, J = 5.01, 4H), 2.35 (s, 3H), 2.20 (s, 3H); 13C NMR δ 168.7, 151.2, 143.8, 124.4, 122.1, 111.2, 105.5, 54.9, 49.3, 45.8, 26.9, 24.2; MS (FAB-MS) 260 m/z (M+1); HPLC-MS 260 (M+1). Procedure 4.14c: 1-acetyl-6-(N- methyl)piperazinyl indoline (150 mg, 0.58 mmol) and conc. HCl (5 mL); yield 68 mg (48 %) of 4.17a as a dark brown oil: 1H NMR δ 7.02-6.97 (m, 1H), 6.34-6.23 (m, 2H), 3.53 (t, J = 8.18, 2H), 3.16-3.11

125 (m, 4H), 2.94 (t, J = 8.18, 2H), 2.58-2.53 (m, 4H), 2.34 (s, 3H); 13C NMR δ 152.5, 151.6, 124.6, 124.2, 121.0, 106.9, 98.5, 55.0, 49.7, 47.6, 45.9, 28.9; IR (NaCl) 1617 cm-1; MS (EIPI) m/z 217 (M+); MS (FAB) 218(M+1).

1-[2’-Methyl-4’-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxyl]-6-[(N-methyl)piperazino] indoline (4.17) Procedure 4.14: 4.17a (44 mg, 0.20 mmol), 4.5a (120 mg, 0.41 mmol) and P-EDC (0.8 1 g) in CHCl3 (5 mL) and THF (1 mL); yield: 28 mg (28 %) of 4.17 as a light-brown solid: H NMR δ 8.01-7.94 (m, 2H), 7.66-7.26 (m, 6H), 7.12-7.08 (m, 1H), 6.34 (m, 1H), 3.27-3.18 (m, 2H), 3.10-3.02 (m, 4H), 2.68 (s, 3H), 2.55 (m, 4H), 2.36 (s, 3H), 2.35 (s, 3H); 13C NMR δ 176.5, 143.7, 135.9, 133.9, 130.1, 129.5, 129.2, 129.1, 128.9, 127.1, 124.7, 54.8, 49.1, 45.8, 20.2, 12.2; MS (FAB) m/z 494 (M+1);

Anal. (C30H31N5O2) C, H, N.

BIOLOGICAL ASSAYS

ANIMALS. For the microdialysis experiments male Wistar rats (280-350 g) were used. The animals were housed in groups (four rats per cage) in a temperature- and humidity-controlled colony room (20 + 2 °C; 50-60 %) on a natural day-night cycle (light period 6:30 - 18:30 h) until surgery. Food and water were available ad libitum at all times. Testing was done between 10:00 h and 18:00 h during the light phase of the day-night cycle. Procedures were conducted in accordance with guidelines published in the NIH Guide for the Care and Use of Laboratory Animals and protocols were approved by the Groningen University Institutional Animal Care and Use Committee.

RECEPTOR BINDING.40-43 Affinities of compounds were determined using competition binding assays to determine IC50 values at the various receptors. Affinities were measured from the displacement of 3 3 [ H]-8-OH-DPAT from 5-HT1A receptors (rat frontal cortex), [ H]-5-HT from 5-HT1B/1D receptors (calf striatum) in the presence of 8-OH-DPAT and to mask the 5-HT1A and 5-HT2C binding 125 3 sites, [ I]- from 5-HT1B/1D receptors (rat frontal cortex) and inhibition of [ H]-5-HT re-uptake.

MICRODIALYSIS.44 Rats were anaesthetized with ketamine/xylazine (50/8 mg/kg ip) and atropine (0.1 mg/kg sc) after a premedication of midazolam (5 mg/kg sc). Preceding insertion of the microdialysis probe, rats were placed in a stereotaxic frame (Kopf, USA). Following local lidocaine application (10%), a home-made I-shaped probe, fabricated from polyacrylonitrile/sodium methyl sulfonate copolymer dialysis fiber (i.d. 0.22 mm, o.d. 0.31 mm, AN 69, Hospal, Italy) was implanted in the ventral hippocampus (coordinates: IA: +3.7 mm, lateral : +4.8 mm, ventral: -8.0 mm from the Dura Mater, Paxinos and Watson, 1982) and secured with stainless steel screws and dental cement. The exposed length of the membrane was 4 mm. After surgery, rats were allowed a recovery period of at least 24 h before starting the experiments. Probes were perfused with artificial CSF containing 147 mM

NaCl, 3.0 mM KCl, 1.2 mM CaCl2, and 1.2 mM MgCl2, at a speed of 1.5 µL/min (Harvard apparatus, South Natick, Ma., USA). Samples were collected on-line in a 20 µL loop and injected automatically onto the column every 15 min. 5-HT was analyzed using HPLC pump (Shimadzu LC-10 AD liquid chromatograph) in combination with a reversed phase column (phenomenex hypersil 3 C18, 100 X 2.0 mm, 3 µm, Bester, Amstelveen, the Netherlands) and an EC detector with a carbon electrode (Intro, Antec Leyden, Leiden, the Netherlands), working at a potential of 500 mV vs. Ag/AgCl reference electrode. The mobile phase consisted of 5.0 g/L (NH4)2SO4, 0.5 g/L EDTA, 50.0 mg/L heptasulfonic acid and 30 µL/L triethylamine, at pH 4.65 and 30 oC. The flow of the mobile phase was set at 400 µL per min. The detection limit of 5-HT was 0.5 fmol per sample of 20 µL (signal to noise ratio 2). Four consecutive microdialysis samples with less then 20 % difference were considered as control and set at 100 %. Data are presented as percentages of control level (mean + SEM). Statistical analysis was

126 performed using two-way ANOVA for repeated measurements, followed by Student Newman Keuls post hoc analysis. The significance level was set at p<0.05.

ACKNOWLEDGEMENTS. Drs. Thomas Cremers and Michiel Damhof are gratefully acknowledged for their generous assistance with the microdialysis experiments. Drs. H.E. Greiner, W. Rautenberg and C. van Amsterdam are thanked for conducting the binding assays (Merck KGaA, Darmstadt).

4.6 REFERENCES

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blockade of 5-HT1A and 5-HT1B receptors with WAY100,635 and GR127,935. J. Neurochem. 1997, 68, 1159-1163.

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The selective 5-HT1B receptor inverse agonist 1’-methyl-5-[[2’-methyl-4’-(5-methyl-1,2,4- oxadiazole-3-yl)biphenyl-4-yl]-carbonyl]-2,3,6,7-tetrahydrospiro[furo[2,3-f]indole-3,4’- piperidine] (SB 224289) potently blocks terminal 5-HT autoreceptor function both in vitro and in vivo. J. Med. Chem. 1998, 41, 1218-1235.

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130

6-METHOXYMIANSERIN AND STRUCTURAL ANALOGUES: 5 SYNTHESIS AND IN VITRO AND IN VIVO PHARMACOLOGICAL * EVALUATION

5.1 Introduction 132 5.2 Chemistry 133 Synthesis of 6-Methoxymianserin and of O-Substituted, N-Desmethyl- and 9-Substituted Analogues 5.3 Pharmacology 135 Receptor Binding and Microdialysis in Rat Ventral Hippocampus 5.4 Electrochemical Oxidation 135 5.5 Results and Discussion 136 5.6 Experimental 143 5.7 References 148

ABSTRACT

The two tetracyclic atypical antidepressants mianserin (5.1) and mirtazapine (5.2) are thought to elicit their clinical effect through different pathways. Mianserin is a moderate noradrenaline (NA) reuptake inhibitor and thus elevates NA levels, whereas mirtazapine, an

α2-receptor antagonist, elevates both NA and serotonin (5-HT) levels. In this chapter the synthesis and in vitro affinity relationships of some analogues of mianserin are described. In vitro, the test compounds showed reasonable to high affinity for the 5-HT2A/2C receptors, with 6-methoxymianserin (5.8) being the most potent. None of the test compounds displayed affinity for the NA reuptake site. In vivo, in a microdialysis study in the ventral hippocampus of freely-moving rats, 5 mg/kg sc. of 6-methoxymianserin, caused a rapid, long-lasting increase of NA and dihydroxyphenylacetic acid (DOPAC) levels up to 100% and 55% above baseline, respectively, and a concurrent transient increase in 5-HT levels, reaching up to 100% above baseline after 60 min. In a preliminary electrochemical oxidation assay it was shown, that (i) chemical oxidation of mianserin generates oxidation products with the same molecular weights as those of known metabolites of mianserin, (ii) produces very similar oxidation products for both mianserin, 6-methoxymianserin and several of the tetracyclic analogues, and (iii) the triflate analogue 5.10 needs a higher potential than is required for the oxidation of mianserin or 6-methoxy- mianserin.

* A modified version of this chapter by Mensonides-Harsema, M.M.; Cremers, T.I.F.H.; Böttcher, H.; Greiner, H.E.; Leibrock, A.; Seifried, C.; Rautenberg, W.; Wikström, H.V., has been accepted by the J. Bioorg. Med. Chem.

131 5.1 INTRODUCTION

As described in chapter 1, depression has since long been associated with decreased levels of both noradrenaline (NA) and serotonin (5-HT).1, 2 The marketed antidepressants mianserin (5.1, Tolvon®) and mirtazapine (5.2, Remeron®) both possess a tetracyclic structure in which an arylpiperazine moiety is incorporated.3, 4 The mechanisms of action of mianserin and mirtazapine, however, are believed to be different. The increase in hippocampal NA levels after administration of mianserin is associated with its relatively weak affinity for NA re-uptake sites.5 Mirtazapine is an 6 α2 adrenoceptor antagonist, which elevates both hippocampal NA and 5-HT levels.

The increase in NA release is believed to be due to blockade of α2-autoreceptors that are located on the NA nerve terminals. In the raphé nucleus, the serotonergic cell bodies receive an excitatory noradrenergic input from the locus coeruleus. As a consequence, the cell firing of 5-HT neurons is under a positive α1-mediated noradrenergic control and under a negative 5-HT1A-mediated serotonergic control. In the ventral hippocampus, α2-adrenoceptors are co-located with 5-HT1B/1D autoreceptors on the 5-HT nerve terminals, where they both control the 5-HT release via a negative feedback mechanism. Mirtazepine has a preferential affinity for presynaptic α2-adrenoceptors and low affinity for the α1-adrenoceptors. The increase in 5-HT release is believed to be due to indirect stimulation of α1-adrenoceptors at the level of the serotonergic cell bodies and/or by blockade of α2-heteroreceptors present on the of serotonergic nerve terminals.7-10 We hypothesized that a methoxy substituent in the 6-position of mianserin might mimic the aromatic nitrogen atom in mirtazapine. Furthermore, the o-methoxyphenyl piperazine moiety is also present in several compounds with high affinity for the serotonergic autoreceptors, e.g. WAY100635 (5.3), a 5-HT1A antagonist, and

GR127935 (5.4), a 5-HT1B/1D antagonist. In this chapter the synthesis of 6- methoxymianserin (5.8), its 6-desmethyl (5.9), 6-triflate (5.10) and 6-tosylate (5.11) analogues is described, together with the synthesis of its N-desmethyl (5.12) and 9- bromo (5.13), 9-nitro (5.14) and 9-amino (5.15) analogues. The in vitro affinity of these compounds for the different adrenergic, serotonergic and histaminergic receptor subtypes has been determined. The effects of 5 mg/kg sc 6-methoxymianserin in the rat ventral hippocampus on the release of NA, dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA) and 5-HT were measured and compared to those after the administration of similar doses of both mianserin and mirtazapine (data from literature).10 Metabolism studies in laboratory animals and man have indicated that both mianserin and mirtazapine are largely metabolized by hydroxylation, N- demethylation, N-oxidation and C4-oxidation (Phase I) and excreted in the urine in their conjugated forms (Phase II).11-13

132 Electrochemical oxidation on-line with mass spectrometry (EC-MS and MS-MS) was applied as a quick and convenient way to mimic the oxidation by enzymes of mianserin, 6-methoxymianserin and 6-triflate-mianserin. This method has shown promising results in providing preliminary information about metabolic oxidation reactions. Since the method is purely instrumental, it is quick and easy to perform, and as such has advantages over in vitro studies with purified enzymes or liver slices at the early stages of drug development.14-20

CH3 N N N N N N N CH CH O 3 3 CH 5.1 5.2 3 N

N O H3C O N N N N O CH O 3 5.4 5.3 N

H3C

Chart 5.1 Chemical structure of mianserin (5.1), mirtazapine (5.2), WAY100635 (5.3) and GR127935 (5.4)

5.2 CHEMISTRY

SYNTHESIS OF 6-METHOXYMIANSERIN AND o-SUBSTITUTED ANALOGUES

2-Methylaminoethanol and styrene oxide were reacted at 130 oC, yielding β- hydroxy-N-methyl-N-hydroxyethylphenyl ethylamine (5.5), which was converted to the chlorinated phenylethyl amine 5.6 with thionyl chloride in refluxing chloroform. 2-Amino-3-methoxybenzoic acid was reduced to the corresponding alcohol in THF using lithium aluminum hydride, and subsequently coupled to 5.6 in refluxing acetonitrile in the presence of diisopropylethylamine, yielding 1-methyl-3-phenyl-4- (2-methoxy-6-hydroxyethyl)phenyl piperazine HCl (5.7). Ring closure of 5.7 was accomplished by heating in PPA at 100 oC, yielding the desired 6-methoxymianserin

(5.8). O-demethylation of 5.8 could neither be effected by employing BBr3 in CH2Cl2 nor AlCl3 in ethyl mercaptane (no conversion). Partial conversion could be achieved upon treatment with 6 equivalents of AlCl3 in freshly distilled, refluxing benzene, providing 6-hydroxymianserin (5.9). Triflation and tosylation of 5.9 was

133 accomplished with trifluoromethanesulfonyl anhydride of methylsulfonyl chloride in

CH2Cl2 in the presence of triethyl amine, yielding 6-triflate-mianserin (5.10) and 6-tosyl-mianserin (5.11) (Scheme 5.1).

OH CH3 Cl CH3 O abN N c OH Cl

5.5 5.6

OH

de f N N N H C O OH H C O 3 3 N N N CH3 CH3 5.7 CH3 5.8 5.9

N 5.10, R = SO2CH3, O R 5.11, R = SO2C6H4-(p)CH3 N CH 3 o SCHEME 5.1 Reagents and conditions: (a) 2-methylaminoethanol, 130 C; (b) SOCl2, CHCl3, reflux; (c) 2-amino-3-methoxybenzyl alcohol, N,N-diisopropylethylamine, acetonitrile, reflux; o (d) PPA, 100 C; (e) AlCl3, benzene, reflux; (f) trifluoromethanesulfonyl anhydride (5.10)/4- o toluene sulfonyl chloride (5.11), CH2Cl2, TEA, -78 C to room temperature.

ab N N N H C O H C O O 3 3 CH H C O 3 3 N N NH CH3 5.8 5.12 O Cl

c/d

R NH2

e/f N N O O H3C H3C N N CH3 CH3 5.13, R = Br 5.15 5.14, R = NO 2 SCHEME 5.2 Reagents and conditions: (a) 1-chloroethylchloroformate, EtCl2, ∆, (b) MeOH,

∆, (c) bromine, ethanol, CH2Cl2, room temperature (5.13); (d) KNO3, trifluoroacetic acid, 0

°C to room temperature (5.14); (e) i: n-BuLi, tosylazide, THF, room temperature; ii: H2, 10%

Pd/C, THF, 40 psi, 60 °C; (f) H2, 10% Pd/C, EtOH, 40 psi, room temperature.

134 SYNTHESIS OF N-DESMETHYL AND 9-SUBSTITUTED ANALOGUES OF 6- METHOXYMIANSERIN

The N-demethylation was effected via a -intermediate: treatment of 5.8 with 1-chloroethyl chloroformate in ethylene chloride yielded the carbamate intermediate, which was isolated using flash chromatography. The intermediate was subsequently refluxed in methanol, yielding the N-desmethyl analogue 5.12.

Bromination of the 9 position of 5.8 with bromine in mixture of ethanol and CH2Cl2 selectively yielded 9-bromine-6-methoxymianserin (5.13). Introduction of an azide in the 9 position of was accomplished by treatment of 5.13 with n-butyllithium and tosylazide in THF. The azide intermediate 5.14 was then hydrogenated with H2 gas in the presence of 10% Pd/C in ethanol, yielding 9-amino-6-methoxymianserin (5.15). For nitration at the 9 position of 6-methoxymianserin (5.8), the compound was treated with potassium nitrate in either trifluoroacetic acid at 0 - 15 oC, yielding the mono- nitrated product 5.16. Hydrogenation of this compound with H2 gas in the presence of 10% Pd/C in THF, provided the corresponding amine 5.15 (Scheme 5.2).

5.3 PHARMACOLOGY

Compounds 5.7-5.15 were all evaluated for their ability to displace in vitro 3 3 3 the radioligands [ H] (α1), [ H]idazoxan (α2), [ H]ketanserin (5-HT2A), 3 3 3 [ H]mesulergine (5-HT2C), [ H]-8-OH-DPAT (5-HT1A), [ H]-5-HT (5-HT1B/1D) 3 21-27 and [ H]mepyramine (H1) were assessed (Table 5.1). Furthermore, mianserin, mirtazapine, 6-methoxymianserin (5.8) and 6-hydroxymianserin (5.9) were evaluated in the functional, in vitro [3H]-NA uptake model.28 Intracerebral microdialysis in freely moving rats was used to establish the effects of 5 mg/kg sc 6-methoxy- mianserin on the noradrenergic and serotonergic transmission in the ventral hippocampus, by monitoring the release of NA, DOPAC, 5-HT and 5-HIAA (Figures 5.1-5.4).29

5.4 ELECTROCHEMICAL OXIDATION

Mianserin and test compounds were oxidized in a flow through an electrochemical cell, using 1 mM HAc as a supporting electrolyte. The potential was ramped from 0 to +1800 mV in steps of 10 mV during 15 min. The oxidation products were monitored with a triple quadropole mass spectrometer. Full scan spectra could be taken for any given potential and the signal from the different oxidation products extracted from the full scan data file and plotted against the cell potential (Figure 5.6).

135 5.5 RESULTS AND DISCUSSION

The structure-activity relationships (SARs) are presented in Table 5.1. None of the intermediates displayed affinity for any of the receptor subtypes tested. Furthermore, despite the introduction of an ortho methoxy group in the phenylpiperazine moiety of mianserin, present in the well-known 5-HT autoreceptor antagonists WAY100635 and

GR127935, none of the final products 5.8 - 5.15 showed affinity for the 5-HT1A or

5-HT1B/1D receptors. Mianserin itself is thought to elicit its anti-depressant action through inhibition of NA at the NA reuptake site. Neither 6-methoxymianserin nor 6- hydroxymianserin displayed functional activity at the NA reuptake site, however.

Only 6-methoxymianserin exhibits increased affinity for the 5-HT2A receptor vs both mianserin and mirtazapine and is equipotent at the 5-HT2C receptor to mirtazapine. The O- and N-desmethyl analogues of 6-methoxymianserin, compounds 5.9 and 5.12 are also equipotent to mirtazapine at the 5-HT2C receptor subtype. The 9-Br (5.13), 9-

NO2 (5.14) and 9-NH2 (5.15) substituted analogues of 6-methoxymianserin all show high to good affinity for the 5-HT2A receptor subtype.

The new tetracyclics all have reduced affinity for the H1 receptor, no affinity for the

α1 and weak, 6-methoxymianserin and its 9-bromo analogue 5.13, to no affinity for the α2 adrenoceptors. Furthermore, it should be noted that the 6-triflate (5.10) and 6- tosylate (5.11) analogues show remarkable selectivity for the 5-HT2A receptor subtype.

TABLE 5.1 In vitro affinities at α1, α2, 5-HT2A, 5-HT2C, and H1 receptors of mianserin, * mirtazapine, and test compounds 5.8-5.15 (IC50 in nM).

‡ ‡ ‡ ‡ compound α1 α2 5-HT2A 5-HT2C H1 mianserin 800 90 3.6 20 0.56 mirtazapine 200 24 2 0.33 5.8 1000 600 1.4 2 23 5.9 1000 1000 12 2 21 5.10 1000 277 5.11 76 1000 5.12 26 8 44 5.13 730 5.3 40 79 5.14 59 8 153 5.15 25 10 355 ‡ IC50 > 1000 nM

* The receptorbinding assays were run in the laboratories of Merck KGaA in Darmstadt, Germany

136

6-Methoxymianserin showed the most favorable in vitro binding profile at the 5-HT2 receptors and was therefore evaluated in vivo.

FIGURE 5.1 Effects of 6-methoxymianserin (5 mg/kg sc.) on NA release in the ventral hippocampus of the freely moving rat (mean ± SEM, n = 5, *p < 0.05 vs control).

240 Noradrenaline % of basal * * 220 * * * * * 200 * * 180 160 6-MeO-mianserin 140 120 100 80 60 control 40 20 0 -100 -50 0 50 100 150 Time (min)

FIGURE 5.2 Effects of 6-methoxymianserin (5 mg/kg sc.) on DOPAC release in the ventral hippocampus of the freely moving rat (mean ± SEM, n = 5, *p < 0.05 vs control).

250 DOPAC % of basal

200 * * * * * * * 150 6-MeO-mianserin

100

control

50

0 -100 -50 0 50 100 150 Time (min)

137 A microdialysis study in the rat ventral hippocampus showed, that administration of 6-methoxymianserin (5 mg/kg sc) caused an increase in NA levels up to 100% above base-line [F(1,98) = 11.68, p < 0.05 at t = 45-150 min] (Figure 5.1) and in DOPAC levels up to 60% above base-line [F(1,98) = 14.20, p < 0.05 at t = 60-150 min] (Figure 5.2), and a concurrent, transient increase of 5-HT levels up to 100% above base-line [F(1,131) = 2.31, p < 0.05 at t = 60 min] (Figure 5.3). The 5-HIAA release did not change after treatment with 6-methoxymianserin (Figure 5.4).

FIGURE 5.3 Effects of 6-methoxymianserin (5 mg/kg s.c.) on 5-HT release in the ventral hippocampus of the freely moving rat (mean ± SEM, n = 5, *p < 0.05 vs control).

250 5-HT % of basal * 6-MeO-mianserin

200

150

100

control

50

0 -100 -50 0 50 100 150 Time (min) In a previous microdialysis study in the rat ventral hippocampus, it was shown that mianserin (5 mg/kg sc) causes a gradual increase in DOPAC levels up to 60% above base-line (t = 150 min), but has no effect on 5-HT release. This study also showed that mirtazapine (5 mg/kg sc) causes an increase in DOPAC levels up to 100% above base-line (90-150 min) and a transient increase in 5-HT levels up to 80% after 60 min.10 The increase in NA levels upon administration of 6-methoxymianserin cannot easily be explained by adrenergic stimulation alone, due to its low affinity for the α2

(auto)receptor. There is, however, substantial evidence for the involvement of 5-HT2 receptors in the regulation of the NA release in the different brain areas. Both 5-HT2A and 5-HT2C receptors are believed to have a tonically inhibitory influence upon adrenergic transmission.30-33 Although DOPAC is a metabolite formed both in dopaminergic and noradrenergic neurons, in brain areas with little dopaminergic innervation, like the locus coeruleus and hippocampus, it reflects the noradrenergic activity.34, 35 The increase in DOPAC levels upon administration of 6-methoxy- mianserin, therefore, is believed to be due to the enhanced noradrenergic activity in

138 these brain regions. The noradrenergic cell bodies in the locus coeruleus project to the serotonergic cell bodies in the raphé nucleus and terminals in the hippocampus. It has been suggested that endogenous noradrenaline exerts a direct tonic stimulatory control on the release of serotonin through α1 adrenoceptors that are located on the serotonergic cellbodies.36-38

FIGURE 5.4 Effects of 6-methoxymianserin (5 mg/kg s.c.) on 5-HIAA release in the ventral hippocampus of the freely moving rat (mean ± SEM, n = 5, *p < 0.05 vs control).

250 5-HIAA % of basal

200

150 6-MeO-mianserin

100 control

50

0 -100 -50 0 50 100 150 Time (min)

The enhancement of serotonergic transmission after administration of 6- methoxymianserin is, therefore, probably due to the stimulation of raphé cell firing in response to an increase of noradrenergic activity. The weak affinity of 6- methoxymianserin for the α2 adrenoceptor may contribute to the increase in 5-HT release via blockade of the tonic activation of inhibitory α2 adrenoceptors located on the 5-HT terminals.10, 39 The metabolism of mianserin has been studied extensively.31-33 The proposed sites for phase I bio-transformation of mianserin are N-demethylation, N-oxidation, C4- oxidation and C8-hydroxylation. These oxidative products are conjugated with sulphuric or glucuronic acid during phase II bio-transformation and excreted in the urine. Non-conjugated phase I metabolites of mianserin have also been isolated from faeces (Figure 5.5). In Figure 5.6, the EC-MS of mianserin from 0 mV to +1800 mV is presented. At a low potential, mianserin is oxidized to ‘metabolites’ with molecular weights corresponding with N-desmethyl-mianserin (m/z 251/249, M+1/M+), N-desmethyl-N-oxide-mianserin (m/z 265, M+) at a very low concentration, and 4- keto-mianserin (m/z 279, M+1). As the potential is increased, an oxidation product with a molecular weight corresponding with and 4-keto-N-oxide-mianserin is formed (m/z 295, M+) (Chart 5.2). The spectrum of the oxidation products of mianserin does

139 not show the molecular weights that would be corresponding with the expected hydroxylation metabolites 8-hydroxymianserin (m/z 282, M+1) or 8-hydroxy-N- desmethylmianserin (m/z 268, M+1). However, the oxidation products formed with the molecular weights 265 and 295 as opposed to being the N-oxides, might be hydrolyzed in the 8 position. By studying the MS-MS patterns of mianserin and the oxidation products, it can be ascertained whether these metabolites are indeed hydrolyzed in the phenyl ring (fragments m/z +16 or –18) of the tetracyclic structure, or whether they show the same fragmentation products as mianserin.

FIGURE 5.5 Proposed in vivo biotransformation pathways for mianserin in man (■) and rat (○).a, b

9 8 10 11 12

7 5a 14a 13 N 6 N 14b 14 5 4 1 + N 2 N Glu 3 CH CH3 3

HO

N N desmethyl N 8-hydroxy N-oxide (MW=251) (MW=281) (MW=281) N CH N N 3 CH H 3 O

Glu HO3S HO O O HO

N N 8-hydroxy N N N 8-hydroxy N desmethyl N-oxide N N (MW=267) (MW=297) N N N N CH3 O Glu SO H 3 H CH CH 3 3 O

Glu Glu SO3H O O O

N N N O N N N H H H a Redrawn from ref. 13; b Numbering as allocated in ref. 3.

140 FIGURE 5.6 Extracted [M(+H)]+ ions of mianserin and the oxidation products plotted against the applied cell potential.

Intensity (cps) ---m/z 265

---m/z 249 3e6

m/z 279----

-----m/z 295

0 0 200 400 600 800 1000 1200 1400 potential (mV)

The MS-MS study showed fragmentation products with m/z 206, 192, 179 and 165 for mianserin and the oxidation products that would correspond with N-desmethyl- mianserin, N-oxide-mianserin and 4-keto-N-oxide-mianserin. The molecular weight of these fragments fit with the molecular weight of two tricyclic azepin fragments, bond breaking between N2-C1 and C4-N5 (m/z 206) and bond breaking between C1-C14b and C4-N5 (m/z 192), and two fragment where the bonds are dissolved between either C14b-N5 and C14b-C14 (m/z 179) or between C5a-N5 and C14b-C14 (m/z 165). The results of the MS-MS experiments indicates that the electrochemical oxidation method used, does not generate hydroxylation products. The MS-MS experiments also demonstrate that the oxidation product with m/z 279 is actually dibutylphtalate. Electrochemical oxidation of 6-methoxymianserin results in a similar oxidation pattern as seen for mianserin, with ∆ m/z +30, which corresponds with the molecular weight of the methoxy group, as do the N-desmethyl (5.12, ∆ m/z +16) and the 9- bromo analogue of 6-methoxymianserin (5.13, ∆ m/z +110). The oxidation of 6- triflate analogue of mianserin, compound 5.10 to an oxidation product with a molecular weight corresponding with the N-desmethyl-6-triflate-mianserin metabolite (m/z 399, M+1) only starts at +300 mV. From +800 mV and up, some oxidation product corresponding with 4-keto-N-oxide-6-triflate-mianserin (m/z 443, M+1) can be detected (< 1% of cps m/z 397). The aryl-triflate is known to be chemically stable under solvolytic conditions and to make the phenyl ring less prone to metabolic degradation. It has been shown that it can serve as an alternative to aromatic methoxy substituents.34-37

141

Since the actions of cytochrome P450 and other oxidation enzymes often involve regio-selective reactions, it is difficult to fully simulate biological reactions with this instrumental method. However, the results of the electrochemical oxidation of the known antidepressant mianserin and its analogue 6-methoxymianserin may point in the direction of the possible metabolites to look for, both in in vivo and in in vitro experiments.

N N N M+1 = 251 M+ = 265 N N N H CH3 H 5.1 O

HO

N N N M+1 = 279 M+ = 295 M+1 = 295 O O O N N N CH3 CH3 CH3 O

Chart 5.2 Proposed chemical structure of oxidation products of mianserin, after electrochemical oxidation.

To summarize, all of the new compounds showed a diminished affinity for the H1 receptor of a factor 5-100. This is favorable since the sedative effects and weight gain in patients using the antidepressants mianserin or mirtazapine are associated with the 40 postsynaptic receptor blockade of histaminergic H1 receptors. The affinity of 6- methoxymianserin (5.8) for the 5-HT2A and the 5-HT2C receptors is equipotent to mirtazapine, while the affinity for the α2 adrenoceptor is diminished. In vivo, 5 mg/kg sc of 6-methoxymianserin exhibited the same effects on both noradrenergic and serotonergic activity as has been described for mirtazapine.10 The in vitro binding profile of 6-methoxymianserin and the in vivo microdialysis results suggest, that the inhibitory tone on NA release in the ventral hippocampus might be blocked via

5-HT2A/2C receptors. Such a mechanism of action would be similar as the one expected for antidepressant nefazodone (1.38), a 5-HT2A antagonist which has only weak affinity for the NA and 5-HT reuptake sites. The elevated NA levels, in turn, stimulate the firing of the 5-HT neurons via the α1 adrenoceptors on the cell bodies of the 5-HT neurons in the raphé nucleus, which results in an increase in hippocampal 5-HT release. Thus, 6-methoxymianserin, like mianserin and mirtazapine, may have antidepressant properties, and possibly exhibit fewer of the H1-related side effects.

142 5.6 EXPERIMENTAL

CHEMISTRY For general remarks see Chapter 2, Section 2.5.

β-Hydroxy-N-methyl-N-hydroxyethylphenylethylamine (5.5) 2-Methylaminoethanol (4.90 g, 65.3 mmol) was heated to 100 oC. To this, styrene oxide (5.30 g, 44.2 mmol) was added drop-wise. The temperature was raised to 130 oC and the mixture was stirred for 24 h, after which it was purified by 1 flash chromatography (CH2Cl2 with 10 % EtOH) yielding 7.5 g (88 %) of 5.5 as a light yellow oil: H

NMR δ 7.40-7.16 (m, 5H), 4.80-.4.73 (dd, J1 = 9.64, J2 = 3.79, 1H), 3.69-3.60 (m, 2H) 3.50 (broad peak, OH), 2.79-2.41 (m, 4H), 2.38 (s, 3H); 13C NMR δ 142.2, 128.3, 127.5, 125.9, 70.4, 61.4, 59.3, 54.9, 42.3; MS (EIPI) m/z 195 (M+).

β-Chloro-N-methyl-N-chloroethylphenylethylamine (5.6) To an ice cooled solution of 5.5 (1.30 g,

6.70 mmol) in CHCl3 (20 mL) a solution of thionyl chloride (10 mL) in CHCl3 (20 mL) was slowly added. The reaction mixture was refluxed for 2 h, after which it was quenched with H2O (10 mL). The aqueous layer was basified with a 2N NaOH (aq) solution and extracted with CHCl3. The combined organic layers were washed with brine, dried and concentrated in vacuo yielding 1.00 g (67 %) of 5.6 as a brown oil. The intermediate was used without further purification: 1H NMR δ 7.43-7.32 (m, 5H), 4.93 (t, J = 7.08, 1H), 3.48 (t, J = 7.10, 2H), 3.18-2.94 (m, 2H), 2.83 (t, J = 7.05, 2H), 2.38 (s, 3H); 13C NMR δ 140.2, 128.6, 128.4, 127.3, 65.6, 60.8, 59.3, 42.6, 41.3; MS (EIPI) m/z 231 (M+).

2-Amino-3-methoxybenzyl alcohol To an ice-cooled solution of 2-amino-3-methoxybenzoic acid (2.50 g; 15.0 mmol) in THF (150 mL) lithium aluminum hydride (2.30 g; 60.5 mmol) was added in small portions. The reaction mixture was stirred at room temperature for 17 h. The reaction was quenched with subsequently H2O (2.3 mL), a solution of 10 % NaOH(aq) (2.3 mL) and three more portions of H2O (2.3 mL). The mixture was stirred for 2 h at room temperature after which the solvent was removed in vacuo. The residue was purified using flash chromatography (CH2Cl2 with 2.5 % methanol) yielding 1.90 g (82 %) of 2-amino-3-methoxybenzyl alcohol (dark brown solid): mp 38 oC; 1H NMR δ 6.81-6.68 (m, 3H), 4.60 (s, 2H), 3.86 (s, 3H); 13C NMR δ 147.5, 135.5, 121.2, 117.4, 110.2, 63.7, 55.7; IR (NaCl) 3367 (OH); MS (EIPI) m/z 153 (M+).

1-Methyl-3-phenyl-4-(2-methoxy-6-hydroxylethyl)phenyl piperazine (5.7) To a solution of 2- amino-3-methoxybenzyl alcohol (2.70 g; 11.6 mmol) in acetonitrile (20 mL) was added a solution of 5.6 (1.20 g; 7.80 mmol) in acetonitrile (10 mL). The reaction was stirred at room temperature for 30 min. N,N-diisopropylethylamine (1.00 g; 7.80 mmol) was added and the reaction mixture was refluxed for 7h, after which it was stirred at room temperature for 16 h. The formed hydrochloride salt was filtered and recrystallized from acetonitrile to give 1.80 g (74 %) of 5.7 as white crystals (HCl-salt): mp °o 1 222 C; H NMR (300MHz, CD3OD) δ 6.71-6.32 (m, 7H), 4.47 (s, 3H), 4.42-4.38 (m, 1H), 3.96-3.91 (m, 1H), 3.48 (m, 1H), 3.35 (s, 2H), 2.92-2.85 (m, 1H), 2.72-2.68 (m, 1H), 2.50-2.45 (m, 1H), 2.35- 13 2.32 (m, 1H); C NMR (CD3OD) δ 157.8, 140.7, 137.0, 133.2, 128.0, 127.8, 127.5, 126.9, 119.6, + 110.0, 60.8, 60.5, 59.2, 53.9, 53.8, 48.5, 42.2; MS (EIPI) m/z 312 (M ); Anal. (C19H24N2O2.HCl.

¼H2O) C, H, N, Cl.

1,2,3,4,10,14b-Hexahydro-6-methoxy-2-methyldibenzo[c,f]pyrazino[1,2-a]azepin (5.8) Compound 5.7 (1.30 g; 4.20 mmol) was suspended in polyphosphoric acid (14 g) and stirred at a 100 oC for 2h.

The reaction was quenched with a ice (140 mL), after which CH2Cl2 (140 mL) was added. The mixture was basified with a 2N NaOH(aq) solution. The organic layer was separated from the aqueous layer which was subsequently extracted with CH2Cl2. The combined organic layers were washed with brine, dried and concentrated in vacuo. The residue was purified by flash chromatography (CH2Cl2 with 5 %

143 EtOH), yielding 0.89 g (73 %) of 5.8 as a pinkish solid (mp 115 oC). Of the obtained product, 0.29 g was converted to the oxalate salt and recrystallized from ethanol, yielding 0.31 g (80%) of 5.8 as off- o 1 white crystals; mp 205 C; H NMR (300MHz, CD3OD) δ 6.77-6.68 (m, 4H), 6.36-6.21 (m, 3H), 4.47 (s, 3H), 4.44-4.39 (m, 1H), 4.20-4.10 (m, 1H), 3.75-3.65 (m, 1H), 3.02-2.68 (m, 4H), 2.91(s, 3H), 2.43 13 (s, 2H); C NMR (CD3OD) δ 154.0, 143.3, 140.4, 137.0, 128.7, 127.8, 126.6, 126.1, 123.3, 118.6, 111.2, 63.4, 62.9, 55.8,54.5, 50.9, 48.2, 47.8, 44.5, 38.2; MS (EIPI) m/z 294 (M+); Anal.

(C19H22N2O.C2H2O4. ½H2O) C, H, N.

1,2,3,4,10,14b-Hexahydro-6-hydroxy-2-methyldibenzo[c,f]pyrazino[1,2-a]azepin (5.9) To a solution of 5.8 (300 mg, 1.02 mmol) in freshly distilled benzene (30 mL) aluminum trichloride (1.00 g, 7.59 mmol) was added. The reaction was refluxed for 48 h, after which the solvent was removed in vacuo. The residue was purified by flash chromatography (CH2Cl2 with 5% ethanol basified with 25 %

NH4OH (aq)), yielding 150 mg (53 %) of 5.9 as a white foam and 100 mg (33 %) starting material: mp o 1 195-197 C; H NMR (300MHz, CD3OD) δ 6.89-6.28 (m, 7H), 4.69-4.23 (m, 3H), 3.44 (s, 3H), 3.32- 2.84 (m, 4H), 2.58 (s, 2H); 13C NMR δ 129.1, 127.9, 125.6, 118.9, 116.6, 63.0, 61.6, 56.3, 49.5, 44.4, + 39.7; MS (EIPI) m/z 280 (M ); Anal. (C18H20N2O.2H2O. ¼NH3) C, H, N.

1,2,3,4,10,14b-Hexahydro-2-methyl-6-trifluoromethanesulfonoxydibenzo[c,f]pyrazino[1,2- a]azepin (5.10) A solution of 5.9 (150 mg, 0.530 mmol) and triethylamine (1 mL) in CH2Cl2 (15 mL) is cooled to -78 oC, after which trifluoromethanesulfonic anhydride (90 µL) is added. The reaction mixture is subsequently stirred at room temperature for 4 h. The solvent is removed in vacuo and the residue purified by flash chromatography (CH2Cl2 with 5% ethanol), yielding 220 mg (100 %) of 5.10 1 as a brown solid: H NMR δ 7.28-6.82 (m, 7H), 4.72-4.48 (dd, J1 = 0.18, J2 = 0.06, 2H), 3.74 (t, J =

0.05, 1H), 3.49 (d, J = 0.07, 1H), 3.15 (d, J = 0.06, 1H), 2.94-2.73 (dd, J1 = 0.15, J2 = 0.06, 2H), 2.55- 2.25 (m, 2H), 2.36 (s, 3H); 13C NMR δ 128.2, 126.5, 126.1, 125.7, 125.2, 121.1, 119.6, 57.9, 54.5, 50.1, + 44.5, 37.7; MS (EIPI) m/z 412 (M ); HRMS Calc (Obsd) for C19H19N2O3F3S: 412.107 (412.108).

1,2,3,4,10,14b-Hexahydro-2-methyl-6-p-toluene-sulfonoxydibenzo[c,f]pyrazino[1,2-a]azepin

(5.11) A solution of 5.9 (150 mg, 0.530 mmol) and triethylamine (1 mL) in CH2Cl2 (15 mL) is cooled to -78 oC, after which trifluoromethanesulfonic anhydride (90 µL) is added. The reaction mixture is subsequently stirred at room temperature for 4 h. The solvent is removed in vacuo and the residue purified by flash chromatography (CH2Cl2 with 5% ethanol), yielding 220 mg (100 %) of 5.11 as a white solid: 1H NMR δ 7.62-6.70 (m, 11H), 4.68-4.62 (m, 1H), 3.98-3.68 (m, 2H), 3.37-3.16 (m, 2H), 2.91-2.62 (m, 2H), 2.43 (s, 3H), 2.38 (m, 1H), 2.33(s, 3H), 1.25 (t, J = 6.96, 1H); 13C NMR δ 128.3, 127.0, 126.4, 125.8, 125.0, 123.8, 120.9, 120.7, 62.2, 62.0, 54.6, 50.6, 44.4, 37.6, 20.3; MS (EIPI) m/z + 435 (M ); HRMS Calc (Obsd) for C25H26N2O3S: 434.166 (434.166).

1,2,3,4,10,14b-Hexahydro-6-methoxybenzo[c,f]pyrazino[1,2-a]azepin (5.12) A solution of 5.8 (110 mg, 0.34 mmol) and 1-chloroethylchloroformate (100 mg, 0.70 mmol) in dichloroethane (10 mL) were refluxed for 18 h, after which the solvent was removed in vacuo. The residue was purified by flash chromatography (CH2Cl2), yielding 120 mg (81%) of the carbamate intermediate as an off-white solid: o 1 mp 264-267 C; H NMR (300MHz, CD3OD) δ 8.12 (d, J = 3.66, 1H), 7.19-7.02 (m, 4H), 6.91-6.72

(m, 3H), 4.80-4.70 (m, 1H), 4.30 (d, J = 10.74, 3H), 4.11 (dd, J1 = 14.28, J2 = 7.20, 1H), 3.77 (s, 3H), 3.68-2.91 (m, 7H), 2.58; MS (EIPI) m/z 398 (M+). The carbamate intermediate (120 mg, 0.30 mmol) is redissolved in MeOH (10 mL) and the solution is refluxed for 18 h, after which the MeOH was removed in vacuo. The residue was purified by gradient flash chromatography (CH2Cl2 with 5-10% EtOH), yielding 80 mg (95%) of 9 as colorless crystals: mp 131-134 oC; 1H NMR δ 7.10-7.03 (m, 4H), 6.92-6.74 (m, 3H), 4.87-4.81 (m, 1H), 4.42 (m, 1H), 3.82 (m, 1H), 3.76 (s, 3H), 3.58-3.03 (m, 6H),

144 2.58; 13C NMR δ 175.2, 126.7, 125.7, 125.2, 122.2, 117.7, 110.1, 54.2, 50.945.2, 37.7, 28.2; MS (EIPI) + m/z 280 (M ); HRMS Calc (Obsd) for C18H20N2O1: 280.158 (280.157).

9-Bromo-1,2,3,4,10,14b-hexahydro-6-methoxy-2-methylbenzo[c,f]pyrazino[1,2-a]azepin (5.13) To a solution of 5.8 (100 mg, 0.34 mmol) in ethanol (10 mL), a solution of bromine (17.5 µL) in CH2Cl2 (10 mL) was added drop-wise. The reaction was stirred at room temperature for 17 h, after which the solvent was removed in vacuo. The residue was recrystallized from acetonitrile yielding 40 mg (31 %) of 5.13 as white crystals: mp 171-173 oC; 1H NMR δ 7.07 (m, 4H), 6.90 (s, 2H), 6.82 (s, 2H), 4.81-4.75 (m, 1H), 4.43-4.36 (m, 1H), 3.82-3.75 (m, 1H), 3.73 (s, 3H), 3.27-3.04 (m, 2H), 2.87-2.73 (m, 2H), 2.33 (s, 3H), 2.30-2.21 (m, 2H); 13C NMR δ 155.6, 144.0, 139.4, 137.4, 134.2, 129.3, 128.2, 127.0,

126.7, 122.0, 115.3, 114.6, 64.0, 62.8, 56.2, 55.7, 51.2, 45.9, 38.7; Anal. (C19H21BrN2O) C, H, N.

9-Azido-1,2,3,4,10,14b-hexahydro-6-methoxy-2-methyldibenzo[c,f]pyrazino[1,2-a]azepin To a solution of 5.13 (100 mg, 0.30 mmol) in THF (2 mL) that is cooled to - 78 oC, a 2.5 M solution of n- butyllithium in THF (0.25 mL, 2 eq) is added. The reaction is stirred for 15 minutes, after which a solution of tosylazide (160 mg, 0.81 mmol) in THF (1 mL) is added. The reaction is stirred at room temperature for 17 h. The solvent is removed in vacuo, after which the residue is redissolved in

CH2Cl2. The organic layer is washed several times with H2O, dried and removed in vacuo, yielding 47 mg (52 %) of the aza-intermediate: 1H NMR δ 7.74-7.70 (d, J = 8.30, 1H), 7.23-7.02 (m, 3H), 6.92- 6.71 (m, 2H), 4.83-4.77 (m, 1H), 4.52-4.46 (m, 1H), 4.05-3.93 (m, 1H), 3.74 (s, 3H), 3.35-3.01 (m, 4H), 2.91-2.78 (m, 2H), 2.66 (s, 3H); IR (KBr) 2941, 2840, 2799, 1453, 1262, 1150, 1074 cm-1; MS (EIPI) m/z 335 (M+).

1,2,3,4,10,14b-Hexahydro-6-methoxy-2-methyl-9-nitrodibenzo[c,f]pyrazino[1,2-a]azepin (5.14) To an ice cooled solution of 5.8 (200 mg, 0.68 mmol) in trifluoroacetic acid (5 mL) sodiumnitrate (115 mg, 0.70 mmol) is added in small portions. The reaction is stirred at 0 oC for 1 h after which it is allowed to reach room temperature. The reaction mixture is stirred at room temperature for an additional 30 minutes and then quenched with ice (20 mL) and basified with a 4 N NaOH (aq) solution.

The aqueous layer is extracted with CH2Cl2, the combined organic layers are washed with brine, dried and the solvent is removed in vacuo. The residue is purified by gradient chromatography (CH2Cl2 with 2% to 5% EtOH) yielding 127 mg (55 %) of the mono nitrated compound 5.14 as a yellow solid: mp 198-200 oC; 1H NMR δ 7.56 (d, J = 9.28, 1H), 7.72-7.37 (m, 1H), 7.15-6.99 (m, 3H), 6.72 (d, J =

9.28,1H), 4.74 (d, J=12.21, 1H), 4.54-4.48 (dd, J1 = 10.38, J2 = 2.57, 1H), 4.18 (d, J=12.21, 1H), 4.04- 3.97 (m, 1H), 3.83 (s, 3H), 3.16-2.82 (m, 3H), 2.39 (s, 3H), 2.37-2.24 (m, 2H); 13C NMR δ 158.8, 138.5, 137.9, 135.7, 135.2, 129.4, 129.0, 127.6, 127.3, 121.2, 109.7, 62.2, 61.7, 55.6, 55.5, 50.4, 44.9, -1 +1 32.1; IR (KBr) 1514, 1275 cm ; MS (CI with NH3) m/z 340 (M ); Anal. (C19H21N3O3.0.4H2O) C, H, N.

9-Amino-1,2,3,4,10,14b-hexahydro-6-methoxy-2-methyldibenzo[c,f]pyrazino[1,2-a]azepin (5.15) i. Hydrogenation of a suspension of 9-azido-1,2,3,4,10,14b-hexahydro-6-methoxy-2- methylbenzo[c,f]pyrazino[1,2-a]azepin (40.0 mg, 0.119 mmol) and 10% Pd/C (3 mg) in EtOH (5 mL) + with H2 (g) (3 bar) for 4 h at room temperature, using a Parr-shaker. MS (EIPI) m/z 309 (M ). ii. A suspension of 5.14 (100 mg, 0.29 mmol) and 10 % Pd/C (10 mol%) in ethanol (10 mL) was hydrogenated under a H2-atmosphere (3 bar) for 4 h at room temperature, using a Parr-shaker. The reaction mixture was filtrated ad the solvent removed in vacuo. The residue was purified by gradient chromatography (ethyl acetate to EtOAc/THF 1:1 v/v), yielding 90 mg (99%) of 5.15 as a colorless oil: 1H NMR δ 7.27-7.20 (m, 4H), 6.73 (d, J = 8.42, 1H), 6.48 (d, J = 8.42, 1H), 4.83 (d, J=12.81, 1H), 4.63-4.59 (m, 2H), 4.13-4.05 (m, 1H), 3.83 (s, 3H), 3.56-2.33 (m, 2H), 3.11-2.97 (m, 2H), 2.54 (s, 3H), 2.37-2.24 (m, 2H); 13C NMR δ 158.8,; IR (KBr) 3431, 3394 cm-1; MS (EIPI) m/z 309 (M+); HRMS

Calc (Obsd) for C19H23N3O: 309.184 (309.184).

145 ELECTROCHEMICAL-MASSSPECTROMETRY14, 18, 19

GENERAL . A Series200 micro LC pump (Perkin Elmer, Norwalk, CT, USA) delivered a flow of 50 µL/min. The analytes were injected with a 1 mL injection loop and passed through an ESA Coulochem 5011 analytical cell (ESA Inc., Bedford, Mass., USA) connected to a home made potentiostat. A MacLab system with Chart 3.5.7 software (AD Instruments, Castle Hill, NSW, Australia) was used to control the potentiostat and to apply the desired potential over the electrochemicalcell. The potential ran from 0 to +1800 mV in steps of 10 mV during 15 min. The cell current was continuously registered by Chart and full scan spectra were taken continuously with an API 3000 triple quadropole mass spectrometer (PE-Sciex, Concord, Ontario, Canada) equipped with a TurboIonSpray interface. The MS was operated at such a low orifice voltage that ‘up front’ collision induced dissociation did not take place. The delay between the electrochemical cell and the mass spectrometer was determined as follows. At a continuous flow of analyte, a potential step from 0 to +1000 mV was performed. The time between the potential step and the appearance of the oxidation product at the mass spectrometer was measured by selected ion monitoring of the main oxidation product. With the delay between the electrochemical cell and the mass spectrometer determined to 45 sec, full scan spectra can be shown for any given potential and the signal from the different oxidation products can be extracted form the full scan data file and plotted against the potential. All compounds were tested in 1 mM acetic acid (p.a. MERCK) in ultra pure water (Maxima Ultrapure water system ELGA, High Wycombe, Bucks, U.K.) which was sonicated for about 15 min before use.

EXPERIMENTAL. EC-MS data (+600 mV) 5.1: m/z 249, 251, 265, 277, 279, 295, 317; 5.8: m/z 277, 281, 295, 307, 325, 347; 5.10: m/z 397, 399, 413; 5.12: m/z 265, 267, 281, 293, 311, 333; 5.13: m/z 358, 360, 374, 386, 404, 426. MS-MS data of 5.1 and its oxidation products 5.1: m/z 250, 234, 222, 208, 193, 179, 166, 161, 146, 130, 118, 106; 249: m/z 233, 206, 192, 179, 165, 99, 57; 265: m/z 249, 235, 206, 192, 178, 165, 145, 103, 91, 58; 279: m/z 149, 135, 121, 117, 99, 93, 57; 295: m/z 249, 236, 218, 206, 192, 165, 115, 99, 86, 58; 317: m/z 233, 206, 197, 121, 99.

BIOLOGICAL ASSAYS

ANIMALS. For the microdialysis experiments male Wistar rats (280-350 g) were used. The animals were housed in groups (four rats per cage) in a temperature- and humidity-controlled colony room (20 + 2 °C; 50-60 %) on a natural day-night cycle (light period 6:30 - 18:30 h) until surgery. Food and water were available ad libitum at all times. Testing was done between 10:00 h and 18:00 h during the light phase of the day-night cycle. Procedures were conducted in accordance with guidelines published in the NIH Guide for the Care and Use of Laboratory Animals and protocols were approved by the Groningen University Institutional Animal Care and Use Committee.

RECEPTOR BINDING.21-27 Affinities of compounds were determined using competition binding assays to determine IC50 values at the various receptors. Affinities were measured as the displacement of 3 3 3 [ H]prazosin from α1 receptors (rat cortex), [ H]idazoxan from α2 receptors (rat cortex), [ H]-8-OH- 3 DPAT from 5-HT1A receptors (rat frontal cortex), [ H]-5-HT from 5-HT1B/1D receptors (calf striatum) in the presence of 8-OH-DPAT and mesulergine to mask the 5-HT1A and 5-HT2C binding sites, 3 3 [ H]ketanserin from 5-HT2A receptors (rat cortex), [ H]mesulergine from 5-HT2C receptors (pig plexus 3 choridus), [ H]mepyramine from H1 receptors (guinea pig cerebellum).

29 SYNAPTOSOMAL NA-UPTAKE. Crude synaptosomal fraction (P2 fraction) of rat cerebral tissue was prepared according to Whittaker, giving a suspension enriched in synaptosomes of 3 mg protein/mL.

146 The synaptosomal uptake was performed in a total volume of 570 µL, which contained 10 nmol/L [3H]- noradrenaline. Incubation was performed at 37 ˚C for 4 min. in Krebs-Ringer buffer (126 mmol/L

NaCl, 1.4 mmol/L MgCl2, 4.8 mmol/L KCl, 15.8 mmol/L Na2HPO4, 11 mmol/L glucose, 0.9 mmol/L

CaCl2, pH 7.4, 346 mosmol). Unspecific uptake was determined in sodium-free Krebs-Ringer buffer

(252 mmol/L sucrose, 15.8 mmol/L Tris, 11 mmol/L glucose, 1.4 mmol/L MgCl2, 4.8 mmol./L KCl,

0.9 mmol/L CaCl2, pH 7.4, 346 mosmol).

MICRODIALYSIS.29 The rats were anaesthetized with ketamine/xylazine (50/8 mg/kg ip) and atropine (0.1 mg/kg sc) after a premedication of midazolam (5 mg/kg sc). Preceding insertion of the microdialysis probe, rats were placed in a stereotaxic frame (Kopf, USA). Following local lidocaine application (10%), a home-made I-shaped probe [41], fabricated from polyacrylonitrile/sodium methyl sulfonate copolymer dialysis fiber (i.d. 0.22 mm, o.d. 0.31 mm, AN 69, Hospal, Italy) was implanted in the ventral hippocampus (coordinates: IA: +3.7 mm, lateral : +4.8 mm, ventral: -8.0 mm from the Dura Mater, Paxinos and Watson, 1982) and secured with stainless steel screws and dental cement. The exposed length of the membrane was 4 mm. After surgery, rats were allowed a recovery period of at least 24 h before starting the experiments. Probes were perfused with artificial CSF containing 147 mM

NaCl, 3.0 mM KCl, 1.2 mM CaCl2, and 1.2 mM MgCl2, at a speed of 1.5 µL/min (Harvard apparatus, South Natick, Ma., USA). Samples were collected on-line in a 20 µL loop and injected automatically onto the column every 15 min. 5-HT was analyzed using HPLC pump (Shimadzu LC-10 AD liquid chromatograph) in combination with a reversed phase column (phenomenex hypersil 3 C18, 100 X 2.0 mm, 3 µm, Bester, Amstelveen, the Netherlands) and an EC detector with a carbon electrode (Intro, Antec Leyden, Leiden, the Netherlands), working at a potential of 500 mV vs. Ag/AgCl reference electrode. The mobile phase consisted of 5.0 g/L (NH4)2SO4, 0.5 g/L EDTA, 50.0 mg/L heptasulfonic acid and 30 µL/L triethylamine, at pH 4.65 and 30 oC. The flow of the mobile phase was set at 400 µL per min. The detection limit of 5-HT was 0.5 fmol per sample of 20 µL (signal to noise ratio 2). NA, DOPAC and HIAA were analyzed using an HPLC pump (Shimadzu LC-10 AD liquid chromatograph) in combination with a reversed phase column (C18, 15 X 0.46, 3 m, Supelco, Zwijndrecht, the Netherlands). Quantification was performed using an electrochemical detector (ESA, Bedford, MA, USA). Upon oxidation at 175 mV, molecules were reduced at -250 mV. The mobile phase consisted of 4.1 g/L sodiumacetate, 140.0 mg/L octanesulfonic acid, 50.0 mg/L EDTA and 7 % methanol (v/v/ (pH = 4.4.). At a flow of 1 mL/min, the detection limit of the assay was 2 fmol/sample (signal to noise ratio 2). Four consecutive microdialysis samples with less then 20 % difference were considered as control and set at 100 %. Data are presented as percentages of control level (mean + SEM). Statistical analysis was performed using two-way ANOVA for repeated measurements, followed by Student Newman Keuls post hoc analysis. The significance level was set at p<0.05.

Acknowledgements. Thomas Cremers is thanked for conducting the microdialysis study and Ulrik Jurva for performing the electrochemical oxidation experiments (Dept. of Med. Chemistry, Groningen). Drs. Greiner, Leibrock, Seyfried and Rautenberg are acknowledged for performing the binding assays and 5-HT uptake studies (Merck KGaA, Darmstadt, Germany).

5.7 REFERENCES

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[12] Delbressine, L.P.; Moonen, M.E.G.; Kaspersen, F.M.; Jacobs, P.L.; Wagenaars, G.L. Biotransformation of mianserin in laboratory animals and man. Xenobiotica, 1992, 22, 227-236.

[13] Sandker, G.W.; Vos, R.M.E., Delbressine, L.P.C., Sloofs, M.J.H.; Meijer. D.K.F.; Groothuis, G.M.M. Metabolism of three pharmacologically active drugs in isolated human and rat hepatocytes: analysis of interspecies variability and comparison with metabolism in vivo. Xenobiotica, 1994, 24, 143-155.

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[19] Regino, M.C.S.; Brajter-Toth, A. An electrochemical cell for on-line electrochemistry/mass spectrometry. Anal. Chem. 1997, 69, 5067-5072.

[20] Volk, K.J.; Yost, R.A.; Brajter-Toth, A. On-line mass spectrometric investigation of the peroxidase-catalyzed oxidation of uric acid. J. Pharm. Biomed. Anal. 1990, 8, 205-215.

[21] Klockow, M.; Greiner, H.R.; Haase, A.; Schmitges, C.J.; Seyfried. C. Studies on the receptor profile of . Arzneim.-Forsch/Drug Res. 1986, 36, 197-200.

[22] See ref. 21, with the modification that 1 mg protein/mL of rat cortical membranes were incubated with 0.4 nM [3H]-idazoxan.

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[24] Seyfried, C.A.; Greiner, H.E., Haase, A.F. Biochemical and functional studies on EMD 49,980: a potent, selectively presynaptic D-2 dopamine agonist with actions on serotonin systems. Eur. J. Pharmacol. 1989, 160, 31-41.

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[26] Hoyer, D.; Waeber, D.; Schoeffter, P.; Palacios, J.M.; David, A. 5-HT1C receptor-mediated stimulation of inositol phosphate production in pig choroid plexus. A pharmacological characterization. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1989, 339, 252-258.

[27] Korner, M.; Bouthenet, M.L.; Ganellin, C.R.; Garbarg, M.; Gros, C.; Ife, R.J.; Sales, N.; 125 Schwartz, J.C. [ I]-Iodobolpyramine, a highly sensitive probe for histamine H1-receptors in guinea-pig brain. Eur. J. Pharmacol. 1986, 120, 151-160.

[28] Whittaker, V.P. The synaptosome. In Handbook of Neurochemistry, Vol. 2, (Lajtha, A. ed.) 1969, Plenum, London and New York, 327-364.

[29] Santiago, M. and Westerink B.H. Characterization of the in vivo release of dopamine as recorded by different types of intracerebral microdialysis probes. Naunyn Schmiedebergs Arch. Pharmacol. 1990, 342, 407-414.

[30] Done, C.J.; Sharp, T. Evidence that 5-HT2 receptor activation decreases noradrenaline release in rat hippocampus in vivo. Br. J. Pharmacol. 1992, 107, 240-245.

149 [31] Done, C.J.; Sharp, T. Biochemical evidence for the regulation of central noradrenergic activity

by 5-HT1A and 5-HT2 receptors: microdialysis studies in the awake and anaesthetized rat. Neuropharmacology 1994, 33, 411-421.

[32] Millan, M.J.; Dekeyne, A.; Gobert, A. Serotonin (5-HT)2C receptors tonically inhibit dopamine (DA) and noradrenaline (NA), but not 5-HT, release in the frontal cortex in vivo. Neuropharmacology 1998, 37, 953-955.

[33] Gobert, A.; Millan, M.J. Serotonin (5-HT)2A receptors tonically inhibit dopamine (DA) and noradrenaline (NA), but not 5-HT, release in the frontal cortex in vivo. Neuropharmacology 1999, 38, 315-317.

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[36] Clement, H.W.; Gemsa, D.; Wesemann, W. The effect of adrenergic drugs on serotonin metabolism in the nucleus raphé dorsalis in the rat, studied by in vivo voltammetry. Eur. J. Pharmacol. 1992, 217, 43-48.

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inhibitory action of endogenous noradrenaline on α2-adrenergic heteroreceptors of 5-hydroxytryptamine terminals in the rat hippocampus. Naunyn-Schmiedeberg’s Arch. Pharmacol.1993, 347, 266-272.

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150

RESOLUTION AND IN VITRO AND IN VIVO PHARMACOLOGICAL * 6 EVALUATION OF THE ENANTIOMERS OF 6-MEO-MIANSERIN

6.1 Introduction 152 6.2 Chemistry 153 Synthesis of (R)-Mianserin from (-)-6-Methoxymianserin Synthesis of 6-Methoxymianserin from (R)-Styrene Oxide 6.3 Resolution and Optical Rotation 154 6.4 Pharmacology 154 Receptor Binding and Microdialysis in Rat Ventral Hippocampus 6.5 Results and Discussion 155 6.6 Experimental 161 6.7 References 162

ABSTRACT

The optically pure enantiomers of 6-methoxymianserin (6.3) were obtained by means of chiral HPLC over a cellulose-based OD column. Both enantiomers showed high affinity for the 5-HT2A and 5-HT2C receptor subtypes and no affinity for the 5-HT3 receptor or NA and 5-

HT reuptake sites. The (S)-(+)-enantiomer showed high selectivity for the 5-HT2 receptors.

The (R)-(-)-enantiomer, however, also showed good affinity for the D2 receptor and a favorable ‘Meltzer ratio’ (5-HT2/D2) over the atypical neuroleptic clozapine (6.6). The absolute configuration was determined indirectly, via O-demethylation of (-)-6- methoxymianserin, triflation and subsequent reduction to (R)-(-)-mianserin (6.1). In vivo, in a microdialysis study in the ventral hippocampus of rats, 2.5 mg/kg sc. of either (-)- or (+)-6- methoxy-mianserin caused a rapid and long-lasting increase in the levels of NA, DOPAC (not significant for the (+)-enantiomer), 5-HT (transient) and 5-HIAA (significant for the (-)- enantiomer).

* A modified version of this chapter by Mensonides-Harsema, M.M.; Cremers, T.I.F.H.; Moltzen, E.K.; Arnt, J.; Wikström, H.V., has been accepted by the J. Med. Chem.

151 6.1 INTRODUCTION

In Chapter 5, the synthesis of several analogues of mianserin (Tolvon®, 6.1) and mirtazapine (6-azamianserin, Remeron®, 6.2) is described. The lead compound of this series, 6-methoxymianserin (6.3) has been evaluated in a microdialysis study. In vivo, 6-methoxymianserin (5 mg/kg sc.) elicits a long-lasting increase of noradrenaline (NA) and dihydroxyphenylacetic acid (DOPAC) levels and a transient increase in the serotonin (5-HT) release. Similar effects on NA, DOPAC and 5-HT release have been reported for mirtazapine.1 Administration of mianserin, however, only elevates the NA and DOPAC release, but does not effect the 5-HT levels.1

The α2-blocking effect and the affinities for the 5-HT2A and 5-HT2C receptors of mianserin reside in the (S)-(+)-enantiomer.2, 3 The (R)-(-)-enantiomer shows selectivity for the 5-HT3 receptor. For mirtazapine, the same stereoselectivity at the

α2-adrenoceptors is found. The stereoselectivity for the serotonergic receptors, however, is considerably less profound.4, 5 Neither mianserin nor mirtazapine show stereoselectivity for the histamine H1 receptor, and both compounds have negligible 6, 7 affinity for the dopamine D2 receptors. In this chapter the direct enantiomeric separation of 6-methoxymianserin, 6-hydroxymianserin (6.4) and 6-trifluoromethylsulfonyloxymianserin (6.5) by means of chiral HPLC is reported. The absolute configuration of the (+)- and (-)-enantiomers of 6-methoxymianserin is determined and the in vitro binding profile of both enantiomers is assessed. The effects of these two enantiomers (2.5 mg/kg sc.) in the rat ventral hippocampus on the release of NA, DOPAC, 5-HT and 5- hydroxyindoleacetic acid (5-HIAA) were measured. Furthermore, an attempt is made to directly synthesize the different enantiomers starting from commercially available (S)-(+)- or (R)-(-)-styrene oxide.

H N

Cl R N N N N CH3 6.1, R = CH N 6.2, R = N 6.6 CH 6.3, R = COCH3 3

Chart 6.1. Chemical structure of mianserin (6.1), mirtazapine (6.2), 6-methoxymianserin (6.3) and clozapine (6.6).

152 6.2 CHEMISTRY

SYNTHESIS OF MIANSERIN FROM (-)-6-METHOXYMIANSERIN .

O-demethylation of the (-)-enantiomer 6-methoxymianserin was achieved upon treatment with 6 equivalents of AlCl3 in freshly distilled, refluxing benzene, providing (-)-6-hydroxymianserin (6.4). Triflation of (-)-6-hydroxymianserin was accomplished with trifluoromethanesulfonyl anhydride in CH2Cl2 in the presence of triethyl amine, yielding (-)-6-trifluoromethylsulfonyloxymianserin (6.5). The triflate substituent was removed by reduction using Pd(OAc) in the presence of PPh3 in refluxing CH2Cl2, yielding (R)-(-)-mianserin (Scheme 6.1).8

ab c N N N O H OH H O O H H3C S N N O N CH CH CH 3 3 F F 3 (-)-6.3 (-)-6.4 F (-)-6.5

N H N CH3 (-)-6.1

SCHEME 6.1 Reagents and conditions: (a) AlCl3, benzene, reflux; (b) trifluoromethane- o sulfonyl anhydride, CH2Cl2, TEA, -78 C to room temperature; (c) Pd(OAc)2, PPh3, TEA, HCOOH, MeOH, reflux.

SYNTHESIS OF 6-METHOXYMIANSERIN FROM (R)-STYRENE OXIDE.

2-Methylaminoethanol and (R)-styrene oxide were reacted at 130 oC, yielding (R)- β-hydroxy-N-methyl-N-hydroxyethylphenyl ethylamine (6.7), which was converted to the chlorinated phenylethyl amine 6.8 with thionyl chloride in refluxing chloroform. This substitution reaction, however, is not stereoselective. The optical rotation of the intermediates 6.8 and 6.9 and the final product 6-methoxymianserin is zero (Scheme

6.2). Replacement of the solvent CH2Cl2 by pyridine also leads to the loss of optical activity of 6.8, as is the case when the reaction is carried out with POCl3 in pyridine/pentane.9 - 12

153

OH CH3 Cl CH3 O abN N C OH Cl H H H (S)-(+)-sterene oxide (R)-(-)-6.7 (+)-6.8

OH

N H N H H C O O 3 H3C N N

(+)-6.9 CH3 (+)-6.3 CH3

o SCHEME 6.2 Reagents and conditions: (a) 2-methylaminoethanol, 130 C; (b) SOCl2, CH2Cl2, o o o 0 C to reflux or SOCl2, pyridine, 0 C to reflux or POCl3, pyridine, pentane, -20 C to reflux.

6.3 RESOLUTION AND OPTICAL ROTATION

The enantiomers of mianserin, 6-methoxymianserin, 6-hydroxymianserin and 6- trifluoromethylsulfonoxymianserin were separated over a semi-preparative, chiral (cellulose-based) stationary phase The chromatographic data were calculated as follows: the selectivity α = k2’/k1’, the capacity factor of the least and of the most retained enantiomers (k1’ and k2’, respectively) with k1’ = (t1-t0)/t0 and the dead time 13 t0 = tsolvent front - tinjection (Table 6.1 and Figure 6.1). The optical rotation of the individual enantiomers of compounds 6-methoxymianserin and 6-trifluoromethyl- sulfonoxymianserin were determined according to the following equation:

20 [α]D = 100.α / l.c, where α is the rotation measured, l is the length of the cuvette (in dm) and c is the concentration of the individual enantiomers (in MeOH). The enantiomeric purity of the respective enantiomers were determined from the HPLC data of their combined fractions.

6.4 PHARMACOLOGY

The ability of 6-methoxymianserin, 6-trifluoromethylsulfonyloxymianserin and their enantiomers to displace the respective radioligands at the dopamine D2, D3, D4, serotonin 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2C, 5-HT3, noradrenaline α1, α2, and histamine H1 and H2 receptors and at the 5-HT and NA re-uptake sites were determined (Table 6.3).14-23 The extracellular levels of NA, DOPAC, 5-HT and 5- HIAA in the ventral hippocampus were measured by in vivo microdialysis after

154 administration of 5 mg/kg sc. of the racemate of 6-methoxymianserin or 2.5 mg/kg sc. of either of the enantiomers, respectively. Levels were measured every 15 min. for a period of 2.5 h after administration of the test compound. Before administration of test compounds, four consecutive samples are collected (base-line). For clarity, the deviations in the extracellular levels are presented as area under the curves (figure 6.2).24

6.5 RESULTS AND DISCUSSION

The stereochemical course of conversion to haloalkanes is normally inversion for the

SN2 reaction of the secondary (inter molecular). However, when optically active 2-octanol is reacted with thionyl chloride in ether, the configuration is retained. When the solvent is pyridine, the stereochemical pathway is conversion. The initial intermediate formed in the thionyl chloride reaction is an alkyl chlorosulfite, to create a better leaving group. In ether, most of the HCl generated during chlorosulfite formation is not present as dissociated ions. Chloride for the substitution step comes from decomposition of the chlorosulfite. As the sulfur dioxide departs, the chloride is oriented at the same side of the reacting carbon atom. In the nonpolar ether, charge separation is unfavorable and the chloride and carbocation are attracted to each other as an ion pair, that is, a positive and negative ion held close to each other. Collapse of the ion pair to product takes place with retention of configuration. This uncommon reaction in which part of the leaving group attacks the substrate, detaching itself from the rest of the leaving group in the process, is termed substitution nucleophilic internal

(SNi). The phenomenon is similar to the neighboring-group mechanism, which consists essentially of two SN2 substitutions. In the first step the neighboring group acts as a nucleophile, causing an inversion. In the second step an external nucleophile displaces the neighboring group by a backside attack, so the net result is retention of configuration (anchimeric assistance). When pyridine is added to the reaction, + – ROSON C5H5 supposedly is formed before anything further can take place. The Cl freed in this process now attacks from the rear, which leads to inversion of the configuration (Scheme 6.3).

R R

H C OH H2C O O 2 C + SOCl2 C S + HCl Ph Ph H H Cl

R R R SO H2C O O Ph + 2 H C Cl C S C 2 C Ph Ph Cl H Cl H H

9, 10 SCHEME 6.3 Intra-molecular substitution via the SNi mechanism.

155

However, when the carbon at the β-position of the secondary alcohol is saturated, as is the case for the phenyl substituent, again both reaction mechanisms take place. Thus, it is explained that the enantioselective synthesis of 6-methoxymianserin from optically pure styrene oxide, under the conditions tested, proved to be unsuccessful.

Table 6.1. The selectivity (α) and capacity factors (k’) of mianserin (6.1), 6-methoxy- mianserin (6.3), 6-hydroxymianserin (6.4) and 6-trifluoromethyl-sulfonyloxymianserin (6.5).

Test 20 20 compound α k1’ [α]D EE (%) k2’ [α]D EE (%) 6.1 1.38 0.72 1.00 6.3 1.63 0.94 -403.0 >99.5 1.53 +407.6 >99.7 6.4 1.49 2.31 3.44 6.5 3.11 0.37 -184.4 >99.6 1.15 +181.0 >99.9

Under the conditions chosen,13 reasonable quantities, 50 mg/mL, of the racemic mixture of 6-methoxymianserin could be base-line separated in a short time (Figure 6.1A). Both the selectivity α and the capacity factor of the second eluted enantiomer k2’ are improved, as compared to mianserin. 6-Hydroxymianserin exhibited a decrease in selectivity and considerable tailing, which is due to the increased interactions with the supporting silica gel. In contrast, 6-trifluoromethylsulfonyloxy- mianserin showed a strongly improved separation on the cellulose selector compared to 6-methoxymianserin. Injection of 140 mg/mL of the racemic compound (14 mg/injection) still resulted in baseline separation on the semi-preparative column (Figure 6.1B). For each compound the (-)-enantiomer eluted first. The optical rotation values of the (-)- and (+)-enantiomers and the enantiomeric purity (ee) of the different enantiomers of 6-methoxymianserin and 6-trifluoromethylsulfonoxylmianserin, which were determined with HPLC for the combined fractions collected of the different enantiomers. Because no suitable crystals for X-ray crystallography could be obtained, the absolute configuration was determined indirectly, via the synthesis of mianserin from 6-methoxymianserin. O-Demethylation of 6-methoxymianserin and subsequent triflation and reduction to mianserin, proved that the (-)-enantiomer of 6-methoxy- mianserin has the (R)-configuration. The product of the synthesis presented in Scheme 6.1 elutes at the same time as does the (R)-(-)-enantiomer of mianserin (Figure 6.3).

156

Figure 6.1. Semi-preparative separation on Ciralcel OD (250 x 10 mm I.D.) of racemic 6-methoxymianserin (A; 5 mg/injection) and 6-trifluoromethyl- sulfonoxylmianserin (B; 14 mg/injection)

Figure 6.3. Elution of (R)-(-)-mianserin (A), (S)-(+)-mianserin (B), (-)-6-trifluoromethylsulfonyloxymianserin (C), reaction mixture after reduction of (-)-6-trifluoromethylsulfonoxylmianserin to mianserin (D) on Ciralcel OD (250 x 10 mm I.D.).

Neither 6-trifluormethanesulfonyloxymianserin nor its enantiomers showed submicromolar affinity at any of the receptors tested (data not shown). The binding study of 6-methoxymianserin showed that it has high affinity for the 5-HT2A and 5-

HT2C receptors, which is found in both enantiomers, and no affinity for the 5-HT3 receptor. From literature it is known that both mianserin and mirtazapine have high 6, 7 affinity for the 5-HT3 receptor. It is therefore remarkable, that

157 6-methoxymianserin has no affinity for this receptor subtype. The selectivity of the (-)- and (+)- enantiomers of 6-methoxymianserin for both adrenergic receptors, is ca. two times for the α2 over the α1 adrenoceptor. 6-Methoxymianserin shows little stereoselectivity for the adrenoceptors and no stereoselectivity for the histamine H1 receptor. Like mirtazapine, 6-methoxy-mianserin is devoid of affinity for the NA reuptake site.

Table 6.2. In vitro binding profiles of mianserin (6.1), mirtazapine (6.2), and (+)-, (-)- and * (+)-6-methoxymianserin (6.3), (pKi’s), and clozapine (6.4) (IC50’s) in nM.

Receptor Test compounds subtype 6.1 6.2 6.3 (-)-6.3 (+)-6.3 6.6 a

D2 6.0 5.8 7.6 7.7 6.3 260 b D3 nt < 7.7 6.6 7.0 5.4 450

D4 nt < 7.6 7.0 7.3 6.8 52 5-HT1A 6.3 < 7.7 6.5 6.6 6.1 nt

5-HT1D nt nt nt 7.1 5.8 nt

5-HT2A 8.8 8.7 9.2 9.1 9.3 12

5-HT2C 8.9 8.2 9.7 9.4 9.8 11

5-HT3 7.1 nt 5.5 5.8 <5.3 nt

α1 7.6 6.0 7.1 7.1 6.7 9.2

α2 nt 7.3 nt 7.4 7.1 nt NA uptakec nt 260 1800 1200 2800 nt 5-HT uptakec 2900 >100 2900 1600 >10,000 nt

H1 8.3 8.3 nt 7.8 7.8 23

H2 nt nt 7.3 7.4 7.2 nt a b c data from reference 25; nt = not tested; IC50 values

Besides some affinity for the histamine receptor, the (S)-(+)-enantiomer of 6- methoxy-mianserin shows high selectivity for the 5-HT2 receptors (> 100 fold over the H1 receptor). The (R)-(-)-enantiomer of 6-methoxy-mianserin, on the other hand, also showed reasonable affinity for the 5-HT1B receptor subtype and the dopamine receptors. It should be noted, that the ‘Meltzer ratio’ (5-HT2A/D2 affinity), a marker which is used as to classify atypical neuroleptics like clozapine, is highly favorable.25, 26 Neuroleptics, which are used to treat schizophrenia are generally potent antagonists, especially at the D2 receptor. The typical antipsychotics, however, have a high propensity to induce acute or chronic motor disturbances (extrapyramidal side effects, EPS). The atypical nature of clozapine may be accounted -3 for with the Meltzer ratio (46 x 10 ). The fact that the affinity for both the 5-HT2 and the D2 receptor of the (R)-(-)-enantiomer of 6-methoxymianserin is about a factor 10 higher, plus an improvement in the ratio by a factor 2 (21 x 10-3), indicate that this enantiomer might be promising as an atypical antipsychotic. It would, however, be

* The receptorbinding assays were run in the laboratories of Lundbeck A/S in Copenhagen, Denmark

158 necessary to assess whether (R)-(-)-6-methoxymianserin acts as a D2 antagonist in a functional assay, e.g. by performing a microdialysis study in the rat striatum.

The microdialysis study in the ventral hippocampus of a freely moving rat showed that, administration of 6-methoxymianserin (5.0 mg/kg sc.) caused an increase in NA levels up to 100% and in DOPAC levels up to 60% above base-line. Furthermore, it induced a concurrent, transient increase of 5-HT levels up to 100% above base-line, with stabilization at 40% to 50% during the further course of the experiment. Administration of (-)- or (+)-6-methoxymianserin (2.5 mg/kg sc.) also caused an increase in NA levels up to 100%. The DOPAC levels were elevated up to 60% and 30% (not significant) above base-line for (-)- and (+)-6-methoxymianserin, respectively. Both enantiomers induced a transient, concurrent increase of the 5-HT release up to 100% above base-line. The 5-HIAA release was elevated up to 20% after the administration of the racemic mixture or either of the enantiomers. The increase, however, was only significant for the (-)-enantiomer. The area under the curves of NA, DOPAC, 5-HT and 5-HIAA release of 6-methoxymianserin and its enantiomers are presented in Figure 6.2.

AUC AUC * NA 10000 DOPAC * * 15000 *

10000 *

5000

5000 #

0 0 control 6.3 (-)-6.3 (+)-6.3 control 6.3 (-)-6.3 (+)-6.3

AUC * 5-HT * AUC 5-HIAA * 10000 4 000 *

5000 2000

0 0 control 6.3 (-)-6.3 (+)-6.3 control 6.3 (-)-6.3 (+)-6.3

Figure 6.2. Effects of 6-methoxy-mianserin (5 mg/kg, n = 4) and its enantiomers (2.5 mg/kg, n = 6) on NA and 5-HT transmission. Results are expressed as AUC and refer to cumulative percentage above base-line. Statistics: Mann-Whitney Rank Sum: * p < 0.05 as compared to control, # p < 0.05 as compared to (R)-(-)- 6-methoxy-mianserin.

159

The in vivo microdialysis study with the enantiomers of 6-methoxymianserin clearly shows, that the (R)-(-)-enantiomer is more potent than the (S)-(+)-enantiomer. This is especially profound for the release of DOPAC. It is difficult to account for the difference in potency from the in vitro binding results. To resolve the question whether the increased DOPAC levels originate from the increased noradrenergic or dopaminergic activity, another NA metabolite, 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), could be used as a marker for noradrenergic activity in the ventral hippocampus in a microdialysis study.27 - 30 If the increase in DOPAC in the ventral hippocampus is due to increased dopaminergic activity via D2 receptors, then this would raise the question why mianserin and mirtazapine, both devoid of D2 affinity, are able to induce significant elevation of DOPAC levels after their administration.

To summarize, the enantiomers of 6-methoxy-, 6-hydroxy- and 6-trifluromethyl- sulfonyloxymianserin have been directly separated by means of chiral HPLC. Using conditions described before, it was found that both the selectivity α and the capacity factor of the second eluted enantiomer are the most favorable for 6-trifluoromethyl- sulfonyloxymianserin.13 The absolute configuration of the (-)-enantiomer was determined indirectly via the synthesis of (-)-mianserin from the (-)-enantiomer of 6-methoxymianserin and was found to also be of the (R)-configuration. In vitro binding studies showed high affinity for the 5-HT2, reasonable affinity for both adrenoceptors (selectivity ca. 2.5) and the histamine receptors and no affinity for the

5-HT3 receptor or NA re-uptake sites. Furthermore, it was established that the (S)-(+)- enantiomer shows high selectivity for the 5-HT2 receptors, while the (R)-(-)- enantiomer also showed good affinity for the dopamine receptors and a favorable 25, 26 ‘Meltzer ratio’ (D2/5-HT2A). In vivo functional studies are necessary to asses whether (R)-(-)-6-methoxymianserin is a D2 agonist or antagonist. The microdialysis study showed a similar profile for both the racemic mixture and the different enantiomers of 6-methoxy-mianserin, with the (R)-(-)-enantiomer being the most potent. The fact that the (S)-(+)-enantiomer is not able to significantly increase the DOPAC release throws suspicion on the assumption that this metabolite stems from noradrenergic activation. It would therefore, be recommendable to use a different metabolite marker for NA activity, e.g. 3-methoxy-4-hydroxyphenylethylene glycol (MHPG).27 - 30 The fact that both enantiomers contribute to an increase in NA and 5-HT, encourages further research in the possible antidepressant properties of 6-methoxy- mianserin. Furthermore, the (R)-(-)-enantiomer of 6-methoxymianserin might even have potential as an atypical antipsychotic.

160 6.6 EXPERIMENTAL

CHEMISTRY For general remarks see Chapter 2, Section 2.5. For details of the synthesis of compounds 6.3 – 6.5, 6.7 - 6.9, see Chapter 5, Section 5.6.

Mianserin (6.1) To a suspension of 6.5 (50 mg, 0.121 mmol), triphenylphosphine (10 mol%), triethyl amine (10 mol%) and Pd(OAc)2 (10 mol%) in MeOH (5 mL) is refluxed for 48 h. The reaction mixture is filtrated and the solvent removed in vacuo. The residue is purified by flash chromatography (CH2Cl2 with 3% ethanol), yielding 10 mg (31 %) of 6.1 as a colorless oil. MS (EIPI) m/z 264 (M+).

HPLC The semi-preparative LC system consisted of an ISCO 2300 HPLC pump, equipped with a Rheodyne injection valve and a 100 µL injection loop. For the detection an ISCO V4 Absorbance detector with 5 µL flowcell (pathway 5 mm) and a Kipp and Zonen BD 40 two channel recorder were used (Beun de Ronde Amsterdam, NL and Delft, NL). The semi-preparative chiral stationary phase was a Chiralcel OD column (Daicel Chemical Industry, Ltd., 25 cm x 1.0 cm I.D. 10 µm particle size silica gel). The mobile phase consisted of 95 % n-hexane (HPLC grade, Labscan, Dublin, Ireland) and 5 % ethanol (LiChrosolv grade, Merck, Darmstadt, Germany). To the mobile phase was added 0.1 % triethyl amine (analytical grade, Janssen Pharmaceuticals, Beerse, Belgium), to prevent peak tailing. The flow-rate was 2.5 mL/min and the detector was set at λ 250 nm. The samples were dissolved in ethanol. Analyses were carried out at room temperature.

BIOLOGICAL ASSAYS

ANIMALS. For the microdialysis experiments male Wistar rats (280-350 g) were used. The animals were housed in groups (four rats per cage) in a temperature- and humidity-controlled colony room (20 + 2 °C; 50-60 %) on a natural day-night cycle (light period 6:30 - 18:30 h) until surgery. Food and water were available ad libitum at all times. Testing was done between 10:00 h and 18:00 h during the light phase of the day-night cycle. Procedures were conducted in accordance with guidelines published in the NIH Guide for the Care and Use of Laboratory Animals and protocols were approved by the Groningen University Institutional Animal Care and Use Committee.

RECEPTOR BINDING. 14 - 23 Affinities of compounds were determined using competition binding assays to determine IC50 values at the various receptors. The affinity for the D2 receptor subtype was measured 3 as the displacement of [ H]- (rat corpus striatum), for the D3 receptor subtype as the 3 displacement of [ H]-spiperone at human cloned D3 receptors expressed in CHO-cells and for the D4 3 receptor subtype as the displacement of [ H]YM-09151-2 at human cloned D4.2 receptors expressed in 3 CHO-cells. The affinity for the 5-HT1A receptor subtype was measured as the displacement of [ H]-5-

CT from human cloned 5-HT1A receptors expressed in HeLa-cells. The affinity for the 5-HT1B receptor 3 subtype was determined indirectly, as the displacement of [ H]-5-CT from human 5-HT1A receptors in synaptasomes. The cloned receptor was expressed in HeLa-cells. The affinity for the 5-HT2A receptor 3 subtype was determined from the displacement of [ H]-ketanserin (rat cortex) and for the 5-HT2C 3 receptors as the displacement of [ H]-mesulergine from rat cloned 5-HT2C receptors expressed in 3 NIH/3T3 cells. Affinity for the 5-HT3 receptor subtype was determined from the displacement of [ H]- LY-278584 from (rat brain). The affinity for the different adrenoceptor subtypes was determined from 3 3 the displacement of [ H]-prazosin (α1; rat brain tissue) and [ H]-RX-821002 (α2; rat brain tissue). Inhibition of 5-HT and NA uptake into rat brain synaptosomes was determined as previously described by K.P. Bøgesø (NA uptake 2.68; 5-HT uptake 2.04). The affinity for the H1 receptor subtype was 3 measured from the displacement of [ H]-pyrilamine in guinea pig lung tissue and for the H2 receptor subtype from the displacement of [3H]-APT from guinea pig striatum.

161

MICRODIALYSIS. 30 For procedures see Chapter 5, Section 5.6. Data are presented as area under the curve. Statistical analysis was performed with Mann-Whitney Rank Sum test. The significance level was set at p<0.05.

ACKNOWLEDGEMENTS. Special thanks to Drs. Ejner Knut Moltzen and Jørn Arnt from Lundbeck AS, Copenhagen, DK for providing the binding data on 6-methoxy- and 6-trifluoromethylsulfonyloxymianserin and their enantiomers and to Thomas Cremers and Nan Tran for performing the microdialysis experiments.

6.7 REFERENCES

[1] De Boer, T.; Nefkens, F.; Van Helvoirt, A; Van Delft, A.M.L. Differences in modulation of noradrenergic and serotonergic transmission by the alpha-2 adrenoceptor antagonists, mirtazapine, mianserin and idazoxan. J. Pharmacol. Exp. Ther. 1996, 277, 852-860.

[2] De Boer, T.; Maura, G.; Raiteri, M.; De Vos, C.J.; Wieringa, J.; Pinder, R.M. Neurochemical and autonomic profiles of the 6-aza-analogue of mianserin, Org 3770 and its enantiomers. Neuropharmacology, 1988, 27, 399-408.

[3] Kooyman, A.R.; Zwart, R.; Vanderheyden, P.M.L. Interaction between enantiomers of mianserin and Org 3770 at 5-HT3 receptors in cultured mouse neuroblastoma cells. Neuropharmacology, 1994, 33, 501-510.

[4] Fink, M. and Irwin, P. Pharmaco-EEG study of 6-azamianserin (ORG 3770): dissociation of EEG and pharmacologic predictors of antidepressant activity. Psychopharmacology, 1982, 78, 44-48.

[5] De Boer, T.; Maura, G.; Raiteri, M.; de Vos, C.J.; Wieringa, J. H.; Pinder, R.M. Neurochemical and autonomic pharmacological profiles of the 6-aza-analogue of mianserin, ORG 3770 and its enantiomers. Neuropharmacology 1988, 27, 399-408.

[6] De Boer, Th. The pharmacologic profile of mirtazapine. J. Clin. Psychiatry, 1996, 57, 19-25.

[7] Pinder, R.M. and Van Delft, A.M.L. The potential therapeutic role of the enantiomers and metabolites of mianserin. Br. J. Clin. Pharmac. 1983, 15, 269S-276S; Wood, M.D.; Thomas,

D.R. Watkins, C.J.; Newberry, N.R. Stereoselective interaction of mianserin with 5-HT3 receptors. J. Pharm. Pharmacol. 1993, 45, 711-714.

[8] Sonesson, C.; Lin, C.-H.; Hansson, L.; Waters, N.; Svensson, K.; Carlsson, A.; Smith, M.W.; Wikström, H. (S)-Phenylpiperidines as DA autoreceptor antagonists. J. Med. Chem. 1994, 37, 2735-2753.

[9] Pine, S.H. Organic Chemistry. Fifth Ed. Publ. McGraw-Hill Book Company 1987, 353-354.

[10] March, J. Advanced Organic Chemistry; reactions, mechanisms, and structure. Fourth Ed. Publ. John Wiley & Sons 1992, 326-327.

162 [11] Hoffman, H.M.R. Reaction kinetics and the Walden Inversion. Part X. A method for estimating the optical purity of certain secondary and primary alkyl halides. Application to 1-methylheptyl chloride, bromide, and iodide. J. Amer. Chem. Soc. 1964, 86, 1249-1251.

[12] Holland, H.L., Brown, F.M., Munoz, B., Ninniss, R.W. Side chain hydroxylation of aromatic hydrocarbons by fungi. Part 2. Isotope effects and mechanism. J. Chem. Soc. Perkin Trans. II 1988, 1557-1564.

[13] Selditz, U. Seperability of Racemates in Chiral Chromatography. Thesis. State University of Groningen, The Netherlands 1998, 58-72.

[14] Hyttel, J. and Larsen, J.J. Neurochemical profile of Lu 19-005, a potent inhibitor of uptake of dopamine, noradrenaline and serotonin. J. Neurochem. 1985, 4, 1615-1622.

[15] MacKenzie, R.G.; VanLeeuwen, D.; Pugsley, T.A.; Shih, Y.H.; Demattos, S.; Todd, R.D.; O’Malley, K.L. Characterization of the human dopamine D3 receptor expressed in transfected cell lines. Eur. J. Pharmacol. 1994, 266, 79-85.

[16] Harrington, M.A.; Shaw, K.; Zong, P.; Ciaranello, R.D. Agonist-induced desensitization and

loss of high-affinity binding sites of stably expressed human 5-HT1A receptors. J. Pharmacol. Exp. Ther. 1994, 268, 1098-1106.

[17] Heuring, R.E. and Peroutka, S.J. Characterization of a novel 3H-5-hydrosytrytamine binding site subtype in bovine brain membranes. J. Neurosci. 1987, 7, 894-903.

[18] Sánchez, C.; Arnt, J.; Dragsted, N.; Hyttel, J. Love-Lembol, H.; Meier, E.; Perregaard, J.; Skarsfelt, T. Neurochemical and in vivo pharmacological profile of , a limbic selective neuroleptic compound. Drug Dev. Res. 1991, 22, 239-250.

[19] Wong, D.T.; Robertson, D.W.; Reid, L.R. Specific [3H]LY278584 binding to 5-HT3 recognition sites in rat cerebral cortex. Eur. J. Pharmacol. 1989, 166, 107-110.

[20] Uhlen, S. and Wikberg, J.E. Rat spinal cord alpha 2-adrenoceptors are of the alpha 2A- subtype: comparison with alpha 2A- and alpha 2B-adrenoceptors in rat spleen, cerebral cortex and kidney using 3H-RX821002 ligand binding. Pharmacol. Toxicol. 1991, 69, 341-350.

[21] Dini, S.; Caselli, G.F.; Ferrari, M.P.; Giani, R.; Clavenna, G. Heterogeneity of [3H]- mepyramine binding sites in guinea pig cerebellum and lung. Agents Actions, 1991, 33, 181-184.

[22] Ruat, M.; Traiffort, E.; Bouthenet, M.L.; Schwartz, J.C.; Hirschfeld, J.; Buschauer, A.; Schunack, W. Reversible and irreversible labeling and autoradiographic localization of the cerebral using [125I]iodinated probes. Proc. Nat. Acad. Sci. USA 1990, 87, 1658-1662.

[23] Bøgesø, K.P. Neuroleptic activity and dopamine-uptake inhibition in 1-piperazino-3- phenylindans. J. Med. Chem. 1983, 26, 935-947.

163 [24] Santiago, M. and Westerink, B.H.C. Characterization of the in vivo release of dopamine as recorded by different types of intracerebral microdialysis probes. Naunyn Schmiedebergs Arch. Pharmacol. 1990, 342, 407-414.

[25] Liao, Y.; Venhuis, B.J.; Rodenhuis, N.; Timmerman, W.; Wikström, H.; Meier, E.; Bartoszyk, G.D.; Böttcher, H.; Seyfried, C.A.; Sundell, S. New (sulfonyloxy)piperazinyldibenzazepines as potential atypical antipsychotics: chemistry and pharmacological evaluation. J. Med. Chem. 1999, 42, 2235-2244.

[26] Meltzer, H.; Matsubara, S.; Lee, J.C. Classification of typical and antipsychotic drugs on the basis of dopamine D-1, D-2 and serotonin-2 pKi values. J. Pharmacol. Exp. Ther. 1989, 251, 238-246.

[27] Perry, K.W. and Fuller, R.W. Fluoxetine increases norepinephrine release in rat hypothalamus as measured by MHPG-SO4 and microdialysis in conscious rats. J. Neural Transm. 1997, 194, 953-966.

[28] Schulz, C. and Lehnert, H. Activation of noradrenergic neurons in the locus coeruleus by corticotropics in a microdialysis study. Neuroendocrinology, 1996, 63, 454-458.

[29] Sarre, S.; Michotte, Y.; Herregodts, P.; Deleu, D.; De Klippel, N.; Ebinger, G. High- performance liquid chromatography with electrochemical detection of levodopa, catecholamines and their metabolites in rat brain dialysates. J. Chrom. 1992, 575, 207-212.

164

SUMMARY

The research described in this thesis focuses on the use of aryl in antidepressant research.

Chapter 1 gives a general introduction into the field of antidepressant research and serves as a background for chapters 2-6. In this chapter, the history of the pathogenesis of depression and the discovery of the 1st generation of antidepressants is reviewed. The currently used definition of depression is a syndrome consisting of affective, cognitive, motor and somatic signs and symptoms, which should be prominent, persistent and represent a change from previous function. The first antidepressant, imipramine (TCA), was marketed in 1952, followed by iproniazid (MAOI) in 1957 and the second TCA amitryptiline in 1961. It was in the early 1960s that a standard for the assessment of depression and of antidepressant effects, the Hamilton rating scale, was put forward. This scale, together with the first TCAs and MAOIs served as a blueprint in early antidepressant research. In 1965, Schildkraut put forward the catechol hypothesis of depression. Although he described the hypothesis to be ‘an oversimplification’ and ‘of heuristic value’ and despite several limitations and inconsistencies, it persisted for over two decades. Following the introduction of the first SSRIs in the early 1980s, the scientific discussion on the mechanisms of action of antidepressants and background of depression has been focussed more on the 5-HT hypothesis of depression. The first SSRI, zimeldine, was withdrawn after it had been associated with cases of Guillain- Barré syndrome. Several others, like fluoxetine (Prozac®) which had first been synthesized in 1972 and was marketed in 1987, proved quite capable of conquering the market for antidepressants. The need for this 2nd generation of antidepressants with more specific receptor targets, came mainly from the fact that the 1st generation suffered from serious adverse events and small therapeutic windows. The TCAs and MAOIs were associated with anticholinergic and cardiovascular side effects, sometimes life-threatening. The SSRIs, though not devoid of side effects, proved more tolerable and much safer in respect to overdosing. Other 2nd generation antidepressants include the atypical antidepressants, like the tetracyclics mianserin (a NA reuptake inhibitor) and mirtazapine (an α2 adrenoceptor antagonist), trazodone and nefazodone (5-HT2A antagonists) and buproprion (DA reuptake inhibitor). The classification, distribution and characterization of the 5-HT receptors is described in section 1.4. Based on the molecular biological properties and pharmacological profiles, seven distinct classes of 5-HT receptors are distinguished (5-HT1-7), several being further divided in different subtypes (e.g. 5-HT2A-2C). Bar the 5-HT3 receptor,

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which is a ligand-gated ion channel, the 5-HT receptors belong to the G-protein coupled receptor (GPCR) superfamily. Several animal models used in antidepressant research are described. These models are either acute (forced-swim test, learned helplessness and tail suspension) or chronic (chronic mild stress and olfactory bulbectomy). Despite the fact that all these animal models detect false positives and negatives, and although, in practice, they are used primarily to follow-up on leads, in theory they would require no preconceptions as to the mechanisms of action of new antidepressant compounds and thus could generate novel neurochemical hypotheses for depression. The last part of the chapter concerns the different strategies currently followed in the development of future, 3rd generation antidepressants. The principal advantage of the 2nd generation over the 1st was their improved side-effect profile. However, two major therapeutic objectives are still to be met: an improved overall and instant onset of clinical efficacy. One of the strategies involves the combination of SSRIs (or other classes of antidepressants) with a 5-HT autoreceptor antagonist. The justification of this combination comes from the hypothesis that desensitization of these autoreceptors is obligatory for the therapeutic efficacy of antidepressants. This hypothesis forms the justification of the scientific efforts described in chapters 2-4. Other strategies focus on the endogenous tetrapeptide 5-HT moduline, which shows high affinity and selectivity for the 5-HT1B (auto)receptors, calcium channel antagonists, NMDA antagonists, and the σ receptors and the role of its endogenous neuropeptide Y.

In Chapter 2, the synthesis and preliminary in vitro (5-HT1A affinity and pA2 values at guinea pig ileum tissue) and in vivo (ultrasonic vocalization, hypothermia and 5- HTP accumulation) pharmacology of several o-substituted aryl piperazine, N- substituted aminopiperidine and p-substituted (sulfon)benzamide analogues of WAY100635 is described. It is shown that the O-desmethyl analogue is the functional most potent 5-HT1A antagonist at postsynaptic 5-HT1A receptors. In a biochemical assay, it is demonstrated that the 5-HTP accumulation following the administration of WAY100635 in the nucleus accumbens and striatum (tendency), and in the tubercule (significant) is decreased. From these results in can be concluded that WAY100635 does not act as a full, silent antagonist in these brain areas. The aminopiperidine analogues display no in vitro affinity nor functional in vitro or in vivo antagonistic efficacy. The p-acetyl (p-MPPOAc) and p-hydroxyl (p-MPPOH) benzamide analogues show high affinity for the 5-HT1A receptor, mixed in vitro efficacy and no antagonistic potency in the rat hypothermia assay (10 µmol/kg sc. vs. 1 mg/kg 8-OH- DPAT sc.). This is inconsistent with previous obtained results with two other benzamide analogues, p-MPPI and p-MPPF (full antagonists with ED50 of 3 and 5 mg/kg vs. 0.5 mg/kg 8-OH-DPAT sc.). Chapter 3 describes a more detailed pharmacological evaluation of the para substituted benzamides. It is shown that the p-hydroxyl analogue, p-MPPOH, has the highest affinity for the h5-HT1A receptor and the highest antagonistic efficacy in the

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cAMP assay. In vivo, both WAY100635 and p-MPPOH act as full antagonists vs. 0.2 mg/kg 8-OH-DPAT sc. at pre- (microdialysis) and postsynaptically (hypothermia) located 5-HT1A receptor sites. In the microdialysis assay, WAY100635 is 20 times more potent and in the hypothermia assay 400 times more potent than p-MPPOH. The postsynaptic 5-HT1A receptors appear to be 80 times more sensitive to WAY100635 as compared to the 5-HT1A autoreceptors. This is probably due to the large receptor reserve for the somatodendritic 5-HT1A receptors. However, notwithstanding the presence of a somatodendritic receptor reserve, the 5-

HT1A autoreceptors are two times more sensitive to p-MPPOH than the postsynaptic receptor subtype. Such a selective antagonist would instantly mimic 5-HT1A autoreceptor desensitization, without interfering with the required activation of postsynaptic 5-HT1A receptors via the enhanced 5-HT release.

Chapter 4 deals with the synthesis and in vitro (r5-HT1B/c5-HT1B affinity and 5-HT uptake in synaptosomes) and in vivo (microdialysis in rat ventral hippocampus) pharmacology of structural analogues of the 5-HT1B/1D antagonist GR127935 (o-MeO- phenylpiperazine) and the 5-HT1B inverse agonist SB224289 (spiropiperidino indoline). It is shown that the incorporation of the arylpiperazine part of GR127935 in a tetracyclic azepine ringsystem or the substitution of the arylpiperazine for a chromane structure provides structural analogues devoid of 5-HT1B receptor affinity. Introduction of a second o-methoxy substituent in the arylpiperazine increases the selectivity for the r5-HT1B receptor. Furthermore, it is shown that the spiropiperidine SB222553 is equipotent to SB224289. Substitution of the spiropiperidinoindoline of SB224289 for a 6-piperazinoindoline moiety also provides structural analogues with nanomolar affinity for the r5-HT1B receptor. Substitution of the 5-membered oxadiazole ring for N-alkylamidines decreases receptor affinity by a factor 10. In vivo, GR127935, SB222553 and the 6-piperazinoindoline analogue of SB224289 all augmented the citalopram-induced increase in 5-HT release in the rat ventral hippocampus. Thus, despite previous claims, it is shown that the oxygen substituent in both GR127935 and SB224289 is not essential for antagonist functionality at terminal

5-HT1B receptors in vivo. The N-methyl-piperazinoamidine analogues of GR127935 and SB222553, notwithstanding their high r5-HT1B receptor affinity, failed to enhance the citalopram- induced increase in 5-HT release. In Chapter 5, the synthesis and preliminary in vitro (binding profile ) and in vivo (microdialysis) pharmacology of several 6- and 6,9-substituted analogues of mianserin are described. The currently marketed tetracyclics mianserin (NA reuptake inhibitor) and its successor mirtazapine (α2-antagonist) both act as antidepressants. Mianserin elevates the NA release, while mirtazapine elevates both NA and 5-HT release in the brain. Both antidepressants suffer from side effects that are related to their high affinity for the histamine H1 receptor (, weight gain). The new tetracyclics presented in chapter 5 all showed diminished affinity for the H1 receptor (5 - 100 times). 6-TfO- and 6-TsO-mianserin showed a remarkable selectivity for the

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5-HT2A receptor subtype. 6-MeO-mianserin showed the highest affinity for the 5-

HT2A/2C receptor. An electrochemical oxidation study of mianserin, showed the formation of oxidation products with molecular weights corresponding to those of known mianserin metabolites. MS-MS studies showed, that no hydroxylation products were formed and that in the conditions used, oxidation took place only at the 6- membered ring containing the basic nitrogen. Similar oxidation patterns were shown for 6-MeO-mianserin and several other tetracyclic analogues. 6-TfO-mianseirn required a higher oxidation potential then the other compounds. This is yet another indication that aryltriflates are metabolically more stable and that they can serve as an alternative for aromatic methoxy substituents. In vivo, 6-methoxymianserin elevates both NA and 5-HT release. The in vitro binding profile of 6-methoxymianserin suggests that the inhibitory tone on NA release might be blocked via 5-HT2A/2C receptors. The elevated NA levels, in turn, then stimulate the firing of 5-HT neurons via the α1 receptor, which results in an increase in hippocampal 5-HT release. The in vivo effects of 6-methoxymianserin and the diminished affinity for the H1 receptor suggests that 6-methoxymianserin might provide a new tetracyclic antidepressant with fewer of the H1-related side-effects. Chapter 6 describes the resolution and in vitro (binding profile) and in vivo (microdialysis in rat ventral hippocampus) pharmacological evaluation of the enantiomers of 6-methoxymianserin. An attempt to obtain the enantiomers via asymmetric synthesis failed, due to racemization during the conversion of a secondary alcohol into a chlorine atom (simultaneous SN2 and SNi substitution). The optically pure enantiomers were obtained by means of chiral HPLC over a cellulose-based OD column. The absolute configuration was determined chemically, via O-demethylation, triflation and subsequent reduction of (-)-6-methoxymianserin to (R)-(-)-mianserin.

The (S)-enantiomer displays higher affinity for the 5-HT2A/2C receptors than the (R)- enantiomer. The (S)-enantiomer also shows high selectivity for the 5-HT2A/2C receptors (>100 times). The (R)-enantiomer, on the other hand, also shows reasonable affinity for the α1- and α2-adrenoceptors (no selectivity), the 5-HT1D receptor and the dopamine D2 and D4,2 receptors. The (R)-enantiomer has a favorable ‘Meltzer’ ratio

(5-HT2A/D2) over the atypical neuroleptic clozapine. Therefore, if the (R)-(-)-6- methoxymianserin proves to be a D2 antagonist, it might also have potential as an atypical antipsychotic.

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SAMENVATTING

Het onderzoek dat in dit proefschrift beschreven wordt, richt zich op de toepassing van aryl piperazines in nieuw te ontwikkelen antidepressiva.

Hoofdstuk 1 vormt een algemene inleiding op het onderzoek naar depressie en antidepressiva en dient als achtergrondinformatie voor de hoofdstukken 2-6. In dit hoofdstuk wordt de geschiedenis van de pathogenese van depressie en de ontdekking van de eerste generatie antidepressiva behandeld. De huidige definitie van depressie luidt als volgt: depressie is een syndroom dat bestaat uit affectieve, cognitieve, motorische en somatische symptomen, welke prominent en hardnekkig zijn en die een duidelijke verandering vormen ten opzichte van het voorafgaand functioneren van de patient. Jaarlijks voldoet 5.8% van de Nederlanders aan de criteria van het ‘Diagnostic and statistical manual of mental disorders’ (DSM). Depressie wordt wel de verkoudheid van de psychiatrie genoemd. Net als bij verkoudheid, gaat ook een depressie vaak vanzelf over. Echter, de gemiddelde verkoudheid duurt zeven dagen. De gemiddelde depressie enkele maanden. En net als bij verkoudheid is er een kleine kans op een ernstig ziektebeloop. Bij verkoudheid betekent dat een longonsteking, of een hartprobleem. Bij een ernstige depressie betekent dat een ernstig verhoogd risico op zelfmoord. Het eerste antidepressivum, imipramine (TCA), is in 1952 op de markt gebracht, gevolgd door iproniazid (MAOI) in 1957 en amitryptiline in 1961. Begin jaren ‘60, is ook een nieuwe standaard, de ‘Hamilton scale’ geintroduceerd, waarmee de mate van depressie en het effect van de behandeling kan worden gemeten. Deze standaard, te zamen met de eerste antidepressiva, markeert het begin van het antidepressief onderzoek. In 1965, deponeerde Schildkraut de catechol hypothese van depressie. Deze hypothese is door hem beschreven als ‘een oversimplificatie’ en ‘van voorbijgaande aard’. Ondanks de vele beperkingen en inconsistenties van zijn hypothese, bleef deze meer dan twintig jaar overeind staan. Pas na de introduktie van de eerst heropname remmers, begin jaren tachtig, richtte de wetenschappelijke discussie over het werkingsmechanisme van antidepressiva en de dieperliggende achtergrond van het ziektebeeld depressie zich meer op een serotonine hypothese van depressie. De eerste heropname remmer, zimeldine, werd van de markt teruggetrokken, nadat het geassocieerd was met het Guillain-Barré syndroom. Vele anderen, zoals fluoxetine (Prozac®), dat overigens reeds in 1972 voor het eerst was gesynthetiseerd en pas in 1987 werd geïntroduceerd, hebben inmiddels aangetoond dat ze zeker in staat zijn een groot deel van de markt voor antidepressiva voor zich op te eisen. De behoefte aan een tweede generatie antidepressiva met een hogere selectiviteit, kwam voort uit het feit dat bij het gebruik van de eerste generatie antidepressiva veelvuldig anticholinerge, antiadrenerge en cardiotoxische

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bijwerkingen optraden. Verder leidde de interactie tussen MAO-remmers en sommige voedselbestanddelen (vooral tyramine) tot dodelijke hypertensieve crises. De heropname remmers, hoewel niet gevrijwaard van bijwerkingen (voornamelijk misselijkheid en sexueel dysfunctioneren), hebben aangetoond veiliger te zijn t.o.v. overdosering. Andere antidepressiva die tot de tweede generatie behoren zijn de atypische antidepressiva mianserine (noradrenaline heropname remmer) en mirtazapine (α2-antagonist), trazodon en nefazodon (5-HT2A antagonisten) en buproprion (dopamine heropname remmer). Tegenwoordig worden zeven verschillende klassen serotonine (5-HT) receptoren onderscheiden (5-HT1-7). Deze onderverdeling is gebaseerd of de moleculair biologische en farmacologische eigenschappen. De receptoren kunnen verder worden onderverdeeld in subtypen (bijvoorbeeld 5-HT2A-2C). Alle 5-HT receptoren behoren tot de G-eiwit gekoppelde receptor superfamilie, met uitzondering van de 5-HT3 receptor (ion kanaal). In hoofdstuk 1 worden ook enkele diermodellen beschreven die worden gebruikt in het onderzoek naar nieuwe antidepressiva. Deze modellen zijn dan wel acuut, dan wel chronisch. Hoewel al deze diermodellen vals positieve en vals negatieve resultaten geven - en ondanks het feit dat ze in de praktijk over het algemeen worden gebruikt om veelbelovende stoffen nader te onderzoeken op hun antidepressief potentieel - zouden ze in theorie kunnen worden gebruikt om volledig nieuwe antidepressiva op te sporen en aldus tot nieuwe hypotheses van depressie te komen. Het laatste deel van hoofdstuk 1, richt zich op de verschillende strategieën die worden gevolgd in het huidige antidepressief onderzoek. Het grootste nadeel van zowel de eerste als de tweede generatie antidepressiva is, dat deze niet in alle vormen van depressie effectief zijn. Een nog groter nadeel is dat ze pas na 3 weken aanslaan, terwijl de bijwerkingen vrijwel meteen hun intrede doen. Bovendien wordt de lethargie van een patient in deze eerste weken vaak wel al opgeheven, terwijl de patient zich geestelijk even miserabel blijft voelen. Dit verhoogdt het risico op zelfmoord pleging. Eén van de strategieën die momenteel wordt gevolgd is de combinatie van 5-HT autoreceptor antagonisten met een klassiek antidepressivum. De hypothese achter deze strategie is dat de autoreceptoren in aantal moeten afnemen voordat het klinische effect van het antidepressivum intreedt. Dit proces wordt ‘desensitisatie’ of ook wel ‘down-regulatie’ genoemd. Deze hypothese ligt ten grondslag aan het onderzoek dat wordt beschreven in de hoofdstukken 2-4. Andere strategieën richten zich op calcium blokkers, NMDA antagonisten, sigma receptoren en op het lichaam-eigen tetrapeptide

5-HT moduline. Dit peptide vertoont hoge affiniteit en selectiviteit voor de 5-HT1B (auto)receptor!

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In Hoofdstuk 2 worden de synthese en de voorlopige in vitro (5-HT1A affiniteit en pA2 bepalingen in cavia ileum weefsel) en in vivo (ultrasone vocalisatie, hypothermie en 5-HTP accumulatie) farmacologie van verscheidene o-gesubstitueerde arylpiperazine, N-gesubstitueerde aminopiperidine en p-gesubstitueerde (sulfon)benzamide analogen van WAY100635 beschreven. Aangetoond wordt, dat de

O-desmethyl analoog functioneel de meest potente 5-HT1A antagonist ter hoogte van de postsynaptische 5-HT1A receptoren is. Verder blijkt uit de resultaten met een biochemisch model dat de5-HTP accumulatie, volgend op de toediening van WAY100635, in de nucleus accumbens en in het striatum (beide tendens) en in de tuberculé (significant) is verlaagd. Hieruit kan worden afgeleid dat WAY100635, in tegenstelling tot voorafgaande stellingen, niet een volledige volle en stille antagonist is. De aminopiperidine analogen vertonen geen in vitro affiniteit en geen functioneel antagonisme noch in vitro noch in vivo. De p-acetyl (p-MPPOAc) en p-hydroxyl

(p-MPPOH) benzamide analogen hebben hoge affiniteit voor de 5-HT1A receptor. Ze vertonen echter geen antagonistische activiteit in het hypothermie model in de rat (10 µmol/kg sc. vs. 1 mg/kg 8-OH-DPAT sc.). Deze resultaten wijken sterk af van die die zijn verkregen met twee andere benzamide analogen, p-MPPI and p-MPPF (volle antagonisten met ED50’s van 3 en 5 mg/kg vs. 0.5 mg/kg 8-OH-DPAT sc.).

Hoofdstuk 3 beschrijft een gedetailleerde farmacologische studie van de in hoofdstuk 2 beschreven p-gesubstitueerde benzamides. Aangetoond wordt, dat de p- hydroxyl analoog, p-MPPOH, de hoogste affiniteit heeft voor de h5-HT1A receptor, en de grootste antagonistische activiteit vertoont in het 2nd messenger cAMP model. In vivo, profileren èn WAY100635 èn p-MPPOH zich als volle antagonisten vs. 0.2 mg/kg 8-OH-DPAT sc. terhoogte van zowel pre- (microdialysise) als postsynaptische

(hypothermie) 5-HT1A receptoren. In het microdialyse model is WAY100635 20 maal en in het hypothermie model 400 maal potenter dan p-MPPOH. De postsynaptische

5-HT1A receptors zijn 80 maal gevoeliger voor WAY100635 dan de 5-HT1A autoreceptoren. Dit fenomeen kan worden verklaard door de aanwezigheid van een grote autoreceptor reserve. Echter, de somatodendritische receptor is 2 maal zo gevoelig voor p-MPPOH dan de postsynaptische receptoren, ondanks de autoreceptor reserve. Een dergelijke selectieve antagonist zou theoretisch in staat moeten zijn de autoreceptor desensitisatie te fingeren, terwijl de postsynaptische receptoren vrij blijven om te profiteren van de verhoogde 5-HT afgifte.

Hoofdstuk 4 richt zich op de synthese en de in vitro (r5-HT1B/c5-HT1B affiniteit en 5-HT heropname) en in vivo (microdialysise in de ventrale hippocampus van de rat) farmacologie van analoge verbindingen van de5-HT1B/1D antagonist GR127935

(o-MeO-phenylpiperazine) en de 5-HT1B inverse agonist SB224289 (spiropiperidino indoline). Het ‘inbouwen’ van het arylpiperazine deel van GR127935 in a tetracyclisch azepine ringsysteem, of het substitueren van de arylpiperazine voor een chromaan struktuur, leiden tot een volledig verlies in affiniteit voor de r5-HT1B/

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c5-HT1B receptoren. De introduktie van een tweede o-methoxy substituent in de arylpiperazine verhoogd de selectiviteit voor de r5-HT1B receptor. Verder blijkt spiropiperidine SB222553 equipotent t.o.v. SB224289. Substitutie van de spiropiperidinoindoline struktuur van SB224289 voor een 6-piperazinoindoline resulteert in een analoog met een hoge affiniteit en selectiviteit voor de r5-HT1B receptor. Substitutie van de oxadiazol door N-alkylamidines verlaagd de receptor affiniteit met een factor 10. In vivo, verhogen GR127935, SB222553 en de 6-piperazinoindoline analoog van SB224289 de citalopram-geïnduceerde toename in de 5-HT afgifte in de ventrale hippocampus van de rat. Hiermee wordt aangetoond, dat het zuurstof atoom in GR127935 en SB224289 niet essentieel is voor in vivo antagonistische functionaliteit. De N-methyl-piperazinoamidine analogen van GR127935 en SB222553 - ondanks hun hoge affiniteit voor de r5-HT1B receptor - blijken niet in staat de citalopram- geïnduceerde toename in 5-HT afgifte verder te verhogen.

In Hoofdstuk 5, worden de synthese en de voorlopige in vitro (binding profiel) en in vivo (microdialysise) farmacologie van verscheidene 6- en 6,9-gesubstitueerde analogen van mianserine beschreven. De huidige tetracyclische antidepressiva mianserine - noradrenaline heropname remmer, verhoogd de noradrenaline afgifte - en diens opvolger mirtazapine - α2-antagonist, verhoogd zowel de noradrenaline als de serotonine afgifte - vertonen beide bijwerkingen die gerelateerd worden aan hun hoge affiniteit voor de histamine H1 receptor (o.a. sedatie en gewichtstoename). De nieuwe tetracyclische verbindingen hebben allen een verminderde affiniteit voor de H1 receptor (5 - 100 maal). 6-TfO- en 6-TsO-mianserine vertonen een opmerkelijke selectiviteit voor de 5-HT2A receptor. 6-MeO-mianserine heeft de hoogste affiniteit voor de 5-HT2A/2C receptoren. Een electrochemische oxidatie studie met mianserine laat zien dat de gevormde produkten een moleculaire gewicht hebben dat overeenkomt met bekende metabolieten van mianserine. Een MS-MS studie toont aan dat, onder de gebruikte condities, geen hydroxylerings produkten worden gevormd en dat de oxidatie slechts plaats vindt in de ring waarin zich het basische stikstof atoom bevindt. Vergelijkbare oxidatie patronen worden aangetoond voor 6-MeO-mianserin en verscheidene andere tetracyclish analogen. Voor de oxidatie van 6-TfO-mianserine is een hogere actie potentiaal nodig. Dit is een hernieuwde vingerwijzing dat aryltriflaten grotere metabole stabiliteit genieten en als alternatief kunnen dienen voor aromatische methoxy substituenten. In vivo, verhoogd 6-methoxymianserin zowel de noradrenaline als de serotonine afgifte. Het in vitro bindings profiel van 6- methoxymianserin suggereerd dat de inhiberende tonus op de noradrenaline afgifte wordt geblokkeerd door de 5-HT2A/2C receptoren. De verhoogde noradrenaline concentratie stimuleert op haar beurt de vuurfrequentie van de 5-HT neuronen via de

α1 receptor, hetgeen resulteert in een verhoogde 5-HT afgifte in de hippocampus.

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Het in vivo effect van 6-methoxymianserin te zamen met de verlaagde affiniteit voor de H1 receptor suggereren dat 6-methoxymianserin zou kunnen fungeren als een nieuw tetracyclisch antidepressivum met verminderde H1-gerelateerde bijwerkingen.

Hoofdstuk 6 beschrijft de resolutie en de in vitro (bindingsprofiel) en in vivo (microdialysise in de ventrale hippocampus van de rat) farmacologische evaluatie van de enantiomeren van 6-methoxymianserine. Een poging de beide verschillende enantiomeren via assymetrische synthese te verkrijgen, blijkt niet succesvol. Tijdens de substitutie van de secundaire hydroxyl groep voor een chloor atoom treedt racemisatie op (zowel SN2 als SNi substitutie). De beide enantiomeren worden verkregen door gebruik te maken van chirale HPLC. De absolute configuratie wordt chemisch bepaald, via O-demethylering, triflering en vervolgens reductie van (-)-6-methoxymianserine tot (R)-(-)-mianserine. De (S)-enantiomeer vertoond een hogere affiniteit voor de 5-HT2A/2C receptoren dan de (R)-enantiomeer. The (S)- enantiomeer is ook in hoge mate selectief voor de 5-HT2A/2C receptoren (>100 maal).

De (R)-enantiomeer vertoond redelijke affiniteit voor de α1- and α2-adrenoceptoren

(geen selectiviteit), de 5-HT1D receptor en de dopamine D2 en D4,2 receptoren. De (R)- enantiomeer heeft een ‘Meltzer’ ratio (5-HT2A/D2) die gunstiger is dan die van het atypische neurolepticum clozapine. De implicatie hiervan is, dat, vooropgesteld dat de

(R)-enantiomeer een D2 antagonist is, (R)-(-)-6-methoxymianserine een potentieel atypisch anxiolyticum is.

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PUBLICATIONS AND PRESENTATIONS

Mensonides, M.M. Synthese van de β3-adrenoceptor agonist BRL 35.135. P.W. 1995, 130, 1269-1270.

Mensonides, M.M.; de Boer, P.; Böttcher, H.; Barf, T.A.; Wikström, H.V. Synthesis and binding profiles of new analogues of the 5-HT1A receptor antagonist WAY100635. Groningen Guide Autumn Meeting. PWS suppl.0 1995, 17, 12.

Mensonides, M.M.; de Boer, P.; Böttcher, H.; Harting, J.; Greiner, H.E.; Wikström,

H.V. Two fluoroalkyl analogues of the 5-HT1A receptor selective antagonist WAY100635: synthesis and anti-serotonergic activity. Antidepressant therapy at the dawn of the 3rd Millenium, Castres, France, March 19-21, 1997.

Pike, V.W.; Halldin, C.; McCaron, J.A.; Lundkvist, C.; Hirani, E.; Olsson, H.; Hume, S.P.; Karlsson, P.; Osman, S.; Swahn, C.G.; Hall, H.; Wikström, H.; Mensonides, M.; Poole, K.G.; Farde, L. [carbonyl-11C]Desmethyl-WAY-100635 (DWAY) is a potent and selective radioligand for central 5-HT1A receptors in vitro and in vivo. Eur. J. Nucl. Med. 1998, 25, 338-346.

Mensonides-Harsema, M.M.; Cremers, T.I.F.H.; Böttcher, H.; Greiner, H.E.; van Amsterdam, C.; Rautenberg, W.; Seyfried, C.A.; Wikström, H.V. Synthesis and pharmacological properties of some related analogues of Mirtazapine. XVth International Symposium on Medicinal Chemistry, Edinburgh, UK, September 6-10, 1998.

Mensonides-Harsema, M.M.; Liao, Y.; Dijkstra, D.; Böttcher, H.; Matzen, L.; Greiner, H.E.; van Amsterdam, C.; Harting, J.; Seyfried, C.A.; Wikström, H.V.

Synthesis and pharmacological properties of several related analogues of 5-HT1B/1D selective antagonist GR127935. 28th Annual Meeting of the Society for Neuroscience, Los Angeles, USA, November, 7-12, 1998.

Mensonides-Harsema, M.M.; Liao, Y.; de Boer, P.; Böttcher, H.; Greiner, H.E.; Wikström, H.V. Synthesis and in vitro and in vivo functional evaluation of o-substituted phenylpiperazine- and N-substituted 4-N-(o-methoxyphenyl)- aminopiperidine analogues of the serotonin 5-HT1A antagonist WAY100635. NWO- Figon Symposium, Lunteren, Holland, April 10-11, 1999.

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Mensonides-Harsema, M.M.; Cremers, T.I.F.H.; Böttcher, H.; Greiner, H.E.; Wikström, H.V. Synthesis, resolution and pharmacological evaluation of 6-methoxymianserin. NWO-Figon Symposium, Lunteren, Holland, April 10-11, 1999.

Mensonides-Harsema, M.M.; Liao, Y.; Böttcher, H.; Bartoszyk, G.D.; Greiner, H.E.; Harting, J.; de Boer, P.; Wikström, H.V. Synthesis and in vitro and in vivo functional studies of o-substituted phenylpiperazine- and N-substituted 4-N-(o-methoxyphenyl)- aminopiperidine analogues of WAY100635. J. Med. Chem. 2000, 43, 432-439.

Mensonides-Harsema, M.M.; Cremers, T.I.F.H.; Moltzen, E.K.; Arnt, J.; Wikström, H.V. 6-Methoxymianserin enhances both noradrenaline and serotonin release in the rat ventral hippocampus. Synthesis, resolution and pharmacological evaluation of 1,2,3,4,10,14b-hexahydro-6-methoxy-2-methyldibenzo[c,f]pyrazino[1,2a]azepin and its enantiomers. J. Med. Chem. 2001 accepted for publication.

Mensonides-Harsema, M.M.; Cremers, T.I.F.H.; Böttcher, H.; Greiner, H.E.; Leibrock, A.; Seifried, C.; Rautenberg, W.; Wikström, H.V. 6-Methoxymianserin (1,2,3,4,10,14b-hexahydro-6-methoxy-2-methyldibenzo[c,f]pyrazino[1,2a]azepin) enhances noradrenaline and serotonin release in the rat ventral hippocampus. J. Bioorg. Med. Chem. 2001 accepted for publication.

176

DANKWOORD

Vandaag wordt ik bij gestaan door twee hele bijzondere vriendinnen: Fiona en Yuki. De afgelopen tijd heb ik vreselijk veel op jullie schouders mogen laden. Ik ben jullie beide heel erg dankbaar voor jullie vriendschap, kanttekeningen en organisatorische talenten. Het is me dan ook een waar genoegen dat jullie vandaag als mijn paranimfen willen aantreden.

NRC-Wetenschap en Onderwijs, 2 okt. 1999 (W. Köhler): ‘Gedachten aan de dood. Diepe wanhoop, het gevoel waardeloos te zijn, en schuldig. Verder: moe, moe, iedere dag doodmoe. Niet kunnen slapen. Angstaanvallen, paniek. Nergens belangstelling voor, ook nergens meer plezier aan beleven. Afgesloten, maar wel prikkelbaar, geagiteerd. Geen eetlust, of juist vreetbuien. Somber en terneergeslagen. Vertoon langer dan twee weken vijf van de negen bovengenoemde categorieën van symptomen en u lijdt aan een depressie.’ Vertoon alle van de bovengenoemde categorieën en u schrijft een dissertatie. Promotieonderzoek en het schrijven van een proefschrift is echter niet alleen maar kommer en kwel. In tegendeel. En je doet het ook zeker niet alleen. Bij de totstandkoming van dit proefschrift zijn vele mensen, in meer of mindere mate, betrokken geweest. Graag neem ik hier de gelegenheid te baat om ze, in enigszins chronologische maar verder volstrekt onlogische volgorde, te bedanken. Allereerst de heer Skolnik en zijn aanstekelijk enthousiasme voor scheikunde in de zesde van het VWO in Emmen. Vervolgens zijn dochter Meilan en haar aanstekelijke enthousiasme voor de studie farmacie. Twee absolute randvoorwaarden voor mijn gang, in 1988, naar de Faculteit der Farmacie aan de Rijksuniversiteit Groningen. Het door Prof. Dr. Ben Feringa gedemonstreerde paraplueffect (dat mij in hoofdstuk 6 nog flink heeft dwarsgezeten) en de ‘heldendaden’ van Dr. Ron Hulst tijdens het organisch chemisch practicum voor farmaceuten, inspireerden mij begin 1990 tot het aankloppen bij de heren Cor Grol, Hans Rollema en Ben Westerink, op dat moment de vakgroep farmacochemie. ‘Bolletjes’ als synthese, microdialyse, binding en moddeling, brachten me er vervolgens toe om in 1993 Prof. Dr. Håkan Wikström te vragen of hij me aan een stageplaats bij de farmaceutische industrie kon helpen. Het resultaat: zes maanden onder de vleugels van Dr. Beritte

Christensson bij Astra Hässle in Göteborg, Zweden, werken aan de synthese van een β3 agonist. Tack så mycket Beritte, och det är jätte kyl at jobba nu tilsammens på AstraZeneca! Na die zes maanden wist ik het zeker. Farmacochemicus was mijn beroepskeuze! Håkan, ik wil je heel hartelijk bedanken voor het feit dat je me de mogelijkheid hebt gegeven om in jouw vakgroep promotieonderzoek te doen. Ik heb de afgelopen jaren bijzonder veel geleerd. Ook wil ik je bedanken voor het feit dat je me de ruimte hebt gegeven om ‘naar buiten’ te gaan. Niet alleen om cursussen te volgen of congressen of symposia te bezoeken, maar ook om bijvoorbeeld drie maanden in de laboratoria van Dr. Henning Böttcher bij Merck KGaA in Darmstadt, Duitsland te werken. Henning und Christel, ich bin euch so viel verschuldet. Danke!

In Darmstadt bin ich dann auch einige Mädel begegnet, die mir den Rest meines Lebens besonders viel bedeuten werden. Frauentreff-mitglieder Soheila Anzali, Marina Lefort, Lisa Matzen und Karin Rölfing, und natürlich auch Werner, Michel, Kaj und Michael. Wir sehen uns bald mal wieder in ‘Madrid’. Het samenwerkingsproject met Merck KGaA hield in dat ik zij-aan-zij mocht werken met de post-doc Dr. Yi Liao. Yi, this not only meant that I had to speak English at any time of the day, but also that I had the possibility to turn to a very experienced chemist whenever I ran into difficulties with my syntheses. I am extremely grateful for all the practical chemistry tricks you have showed me through these years. Natuurlijk stonden we niet met z’n tweeën op het lab. Ten eerste was daar Pieter Tepper, die garant stond voor de GLP en die het lab in de praktijd draaiend hield. Verder heb ik de werkvloer de afgelopen jaren met zeer veel plezier gedeeld met o.a.: Kees, Gert, Evert, Tjeerd, Sander, Eytan, Wia, Hanneke, Jonas, Ulrike, Hiroshi, Paolo,Yuki, Olga, Durk, Sandrine, Erik, Mark, Bas-Jan, Nienke, Thomas, Peter, Geert Damsma (†) en Jan de Vries. En natuurlijk met vele studenten die door de jaren heen, ieder op eigen onnavolgbare wijze, het nodige leven in de brouwerij wisten te brengen. Michiel, ik ben ervan overtuigd dat je een geweldige apotheker wordt en ik wil je langs deze weg nogmaals bedanken voor het feit dat je je tijdens je keuze- en bijvakken hebt willen bezig houden met maken en testen van 5- HT antagonisten. De resultaten van jouw onderzoek duiken door vrijwel dit gehele proefschrift op gezette tijden op! Joke, het waren slechts 6 weken dat je je met de synthese van 6-methoxymianserine hebt bezig gehouden. Deze weken vormden echter wel de basis voor het onderzoek dat in hoofdstuk 6 staat beschreven. Ik wens je bijzonder veel succes met jouw promotie onderzoek bij de PET-afdeling van het Academisch Ziekenhuis in Groningen. Sinds mijn eerste aankloppen bij farmacochemie zijn er dingen gelijkgebleven en veranderd. Gelijkgebleven is mijn liefde voor de farmacochemie en dankbaarheid voor het welkom dat ik werd geheten op deze afdeling op een werkbespreking nu bijna 11 jaar geleden. Veranderd zijn de huidige posities en verblijfplaatsen van de drie heren van weleer. Hans, dank je dat ik bij Pfizer ‘op gesprek’ mocht komen. Oefening baart kunst en de concurrent is daar dan ook uiteindelijk voor gevallen. Ben, bedankt voor het ter beschikking stellen van de microdialytische infrastructuur en proficiat met het professoraat. Cor, heel erg bedankt! Farmacochemie is een onderdeel van de Faculteit Farmacie en de aanwezigheid van dit netwerk van farmaceutische kennis is mij tijdens het promotie onderzoek steeds bijzonder goed van pas gekomen. Immers, de synthese van nieuwe stoffen vormt slechts de basis van de ontwikkeling van nieuwe geneesmiddelen. Zodra een stof geïsoleerd is, worden het ‘chemische, biologische en farmaceutische profiel’ met hulp van de vele disciplines vertegenwoordigd in deze andere vakgroepen vastgesteld. Farmacie is gelukkig een klein wereldje waarin de deuren van iedereen altijd wijd open staan. Bovendien had ik het geluk dat de farmaceutische faculteit niet lang voor de start van mijn promotieonderzoek tot de onderzoeksschool GUIDE was toegetreden. Dit hield o.a. in dat er een boeiende verzameling cursussen werd aangeboden, waarvan ik gretig gebruik heb gemaakt. Verder betekende dit dat ik, dankzij de financiële ondersteuning van de onderzoeksschool, aan allerlei congressen

en symposia kon deelnemen. Vast aanspreekpunt binnen GUIDE was steeds Riekje Banus. Graag neem ik de gelegenheid te baat om haar, maar ook alle andere medewerkers en supporters van GUIDE, een hart onder de riem te steken. Buiten de zuurkast heb ik altijd veel support gekregen van de leden van mijn farmacie ‘mentorgroepje’. Daarmee bedoel ik natuurlijk Hedy, Heleen, Ineke, Iris, Karin, Klaske, Maaike, Marianne, Nicolien, Erik, Geert, Hans, Huub, Jeroen, Jan, Leon, Leonard, Maarten en Pieter. Dat wordt morgen weer ouderwets smikkelen en smullen! Halverwege mijn promotie onderzoek vlogen de verschillende apothekers van deze club echter uit over geheel Nederland (en Noorwegen). Dit betekende dat er een nieuwe Groningse sociale kring moest worden opgebouwd. Deze werd nogal internationaal van karakter. Ulrik en Jonas, het was me een waar genoegen mijn kledingkast met jullie te mogen delen! Ik koester de plaatjes en hoop jullie spoedig weer als collega’s te kunnen begroeten. Damon and Tina, meeting you guys was simply a treat! Working-out twice a week in the mornings would always give me an abundance in energy for the rest of the day and writing my thesis went at least three times as fast because of this. I hope I’ll be able to visit you in San Francisco any time soon. Vrienden vond ik echter ook veel dichter bij huis en wel in de Eerste en Tweede Spoorstraat, waarbij ik Cedric, Kari en Ronald met name wil noemen. Het was moeilijk afscheid nemen alle mensen en katten, maar dankzij het logeerbed op nummer 12, kan ik, wanneer overmand door heimwee, gelukkig altijd even terug. Verder van huis, maar na aan het hart; soms wat ongeduldig of in verwarring gebracht: mijn familie. Moeke, de basis voor dit promotieonderzoek ligt in het verleden. Van kinds af aan heb ik altijd de steun en ruimte gekregen om zelf keuzes te maken, een eigen leven op te bouwen en uit te groeien tot de persoon die ik nu ben. Hiervoor ben ik u heel erg dankbaar. Mijn persoonlijkheid is sinds mijn vroegste jaren natuurlijk ook gevormd (en vervormd) door mijn broers en zussen, Sibo en Lillian, Buddy, en Karina. Trots deel ik met jullie de naam Mensonides. De afgelopen jaren heb ik me verder mogen warmen aan de liefde en belangstelling (en de open haard in Roden) van mijn schoonfamilie. Agnes en Otto en Martine en Tom, dank jullie wel dat ik onderdeel van jullie familie mag uitmaken.

Tot slot wil ik mijn geliefde bedanken. Alexander, jouw liefde vormt al ruim 10 jaar mijn basis. Steeds samen hebben we de afgelopen jaren aan het verwezenlijken van elkaars dromen gewerkt. Onvermoeibaar heb je mij door de dalen geholpen en de kracht gegeven ook door die laatste fase te komen. Trots ben ik op MPT consultancy en blij met de nieuwe vestigingsplaats. Ik hou van jou.