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Synthesis and biological results of compounds acting on alpha-adrenergic receptors
Hong, Seoung Soo, Ph.D.
The Ohio State University, 1991
Copyright ©1991 by Hong, Seoung Soo. All rights reserved.
UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106
SYNTHESIS AND BIOLOGICAL RESULTS OF COMPOUNDS ACTING
ON ALPHA-ADRENERGIC RECEPTORS
DISSERTATION
Presented in Partial Fulfillment of the Requirement for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By Seoung Soo Hong/ B.S., M.S.
* * * * ★
The Ohio State University
1991
Dissertation Committee: Approved by
Duane D. Miller, Ph.D.
Robert W. Brueggemeier, Ph.D.
Larry W. Robertson, Ph.D.
Dennis R. Feller, Ph.D. Duane D. Miller, Ph.D., Advisor College of Pharmacy Copyright by Seoung Soo Hong 1991 DEDICATION
To my parents, wife, children, and family
ii ACKNOWLEDGEMENTS
I would like to express my sincere thanks to the following individuals:
Dr. Duane D. Miller, my adviser, for his guidance, unending encouragement, and understanding throughout my graduate career, making this dissertation possible.
Drs. Dennis R. Feller, Karl J. Romstedt, for the pharmacological data and their help in the interpretation of the data.
Carol Settles, for her friendship and greately appreciated assistance throughout my graduate career.
The falculty, staff and my fellow graduate students, Ron Hill, Kimberly Markovich, Kazu Matsumoto, for their friendship and support during my graduate study, especially Ron for help preparing this manuscript.
Drs. C. George, Judy Flippen-Anderson at the Naval Research Laboratory, for their assistance for X-ray analysis.
Dr. Fu-Lian Hsu at the U.S. Army Chemical Research, for his invaluable discussion on the synthesis of medetomidine analogs.
Jack Fowble and John Miller, for their assistance in obtaining and interpreting the NMR and mass spectra.
Financial supports from U.S. Army CRDEC and the National Institute of Health are gratefully acknowledged.
iii VITA
August 18, 1955 Born - Taegu, Korea
1981 B.S. Pharmacy, Yeungnam University, Taegu, Korea
1984 M.S. Pharmacy, Yeungnam University, Taegu, Korea
1986 - present Graduate Research Associate, College of Pharmacy, The Ohio State University
PUBLICATIONS
1. See-Ryun Chung, Seoung-Soo Hong, and Kyung Hee Jeune- Chung, "Isolation and Purification of Lectin from Phaseolus radiatus", Yakhak Hoeji 1983, 2J_, 221.
2. Yoshia Amemiya, Seoung S. Hong, Burrah V. Venkataraman, Popat N. Patil, Gamal Shams, Karl Romstedt, Dennis R. Feller, Fu-Lian Hsu, and Duane D. Miller, "Synthesis and a-Adrenergic Activities of 2- and 4-Substituted Imidazoline and Imidazole Analogs", J. Med. Chem., in press.
3. S.S. Hong, D.D. Miller, K. Matsumoto, K.L. Romstedt and D.R. Feller, "Synthesis, Isolation, Characterization and a2~Adrenergic Activity of the Optical Isomers of 4-[l-(l- Naphthyl)Ethyl]-1H-Imidazole", Proceedings of the 1991 U.S. Army CRDEC, in press. 4. K. Matsumoto, S.S. Hong, K.L. Romstedt, D.R. Feller and D.D. Miller, "a2-Adrenergic Activity of a New Series of Analogs of 4-[l-(1-Naphthyl)Ethyl]-lH-Imidazole", Proceedings of the 1991 U.S. Army CRDEC, in press.
FIELDS OF STUDY
Major Field: Pharmacy
Studies in Synthetic Medicinal Chemistry
v TABLE OF CONTENTS
PAGE
D e d i c a t i o n ...... ii
Acknowledgements ...... iii
VITA ...... iv
List of Fi g u r e s ...... viii
List of Tables ...... x
List of Schemes ...... xi
CHAPTER
I INTRODUCTION ...... 1 1.1 The Nervous System ...... 1 1.2 The Adrenergic Neuronal System ...... 3 1.2.1 Biosynthesis of Catecholamines • • • 4 1.2.2 Catecholamine Storage ...... 6 1.2.3 Catecholamine Release ...... 7 1.2.4 Catecholamine Metabolism and Reuptake ...... 7 1.3 Subclassification of a-Adrenergic R e c e p t o r s ...... 11 1.4 Signal Transduction of a-Adrenergic R e c e p t o r s ...... 24 1.5 Structural Properties of a-Adrenergic Receptors ...... 30 1.6 Structure Activity Relationships of a-Adrenergic Agonists ...... 33 1.7 Structure Activity Relationships of a-Adrenergic Antagonists ...... • 45 1.8 Physiological Functions of a-Adrenergic Agonists and Antagonists • • * 49
II STATEMENT OF PROBLEMS AND OBJECTIVES ...... 53 2.1 Medetomidine Analogs ...... 53 2.2 Phentolamine Analogs ...... 61 Ill RESULTS AND DISCUSSION...... 66 3.1 Chemistry ...... 66 3.1.1 Synthesis of Naphthalene Analogs of Medetomidine...... 66 3.1.2 Resolution of 4-[1-(1-naphthyl) ethyl]imidazole enantiomers * • • • 75 3.1.3 Synthesis of Indole Analogs of Medetomidine ...... 82 3.1.4 Synthesis of Phentolamine Analogs • 90 3.2 Biology ...... 99 3.3 Summary ...... 107
IV EXPERIMENTAL...... 109
BIBLIOGRAPHY ...... 154
vii LIST OF FIGURES
FIGURE PAGE
1. Subdivision of the nervous system ...... 2
2. Biosynthesis of catecholamines...... 5
3. Norepinephrine and epinephrine metabolism ...... 9
4. The formation of an aldehyde by M A O ...... 10
5. c^-Adrenoceptor agonists and antagonists ...... 13
6. a2-Adrenoceptor agonists and antagonists...... 14
7. a2B~Adrenergic receptor selective compounds ...... 22
8. Modern classification of adrenoceptors ...... 24
9. Signal transduction mechanism activated by a^-adrenoceptor ...... 26
10. G protein coupled signal transduction ...... 29
11. Signal transduction mechanism activated by presynaptic a2-adrenoceptors ...... 29
12. Topography of the human a2ft-adrenergic receptor • • • 31
13. Proposed conformation of norepinephrine and clonidine for interaction with a-adrenergic receptors ...... 35
14. Schematic representation of the Easson-Stedman hypos i s ...... 36
15. Aromatic ring substituted clonidine-like compounds • 42
16. Imidazoline modified clonidine-like compounds • • • • 44
17. Prazosin derivatives as c^-adrenergic antagonists ...... 45 viii 18. Angles between naphthalene, indole rings and a carbon bridge containing imidazole r i n g ...... 56
19. HPLC chromatogram for the racemic mixture of 4-[1-(1-naphthyl)ethyl]imidazole ...... 79
20. HPLC chromatograms for each enantimer of 4-[l-(1-naphthyl)ethyl]imidazole ...... 80
21. ORTEP representation of (S)-4-[l-(1-naphthyl) ethyl]imidazole ...... 81
22. Resonance hybrids of indole ...... 82
23. ORTEP representation of Z-102 ...... 98.
ix LIST OF TABLES
TABLE PAGE
1. c^-Adrenergic Receptor Subtype Characteristics • • • 20
2. Differences between Phenethylamines and Imidazolines for Agonist Activity ...... 34
3. Physiological Functions of a-Adrenoceptors ...... 52
4. c^/c^-Selectivity of Medetomidine and its Optical Isomers with Reference Compounds in Rat Brain Membranes ...... 55
5. Comparison of Medetomidine and its Optical Isomers with Reference Compounds in Mouse vas deferens ...... 58
6. Functional Groups used as Isosteric Replacements* • 63
7. Comparison of Adrenergic Activities of Medetomidine (51) and Analog S4 in Rat Aorta and Human Platelets ...... 100
8. Inhibition of Epinephrine-Induced Human Platelet Aggregation by Compounds j54, S(+)-52, and R( -) -57 • 102
9. Comparison of Adrenergic Activities of Compounds 54, R(-)-57, S(+)-57, and Reference Compounds ...... 103
10. Inhibition of Epinephrine-Induced Human Platelet Aggregation by Analogs of 54 105
11. Inhibition of Epinephrine-Induced Human Platelet Aggregation by Phentolamine (1JJ) and Analogs • • ■ 106
x LIST OF SCHEMES
SCHEME PAGE I. Synthesis of 4-[1-(1-naphthyl)ethyl] imidazole hydrochloride (54J 68
II. Synthesis of 4-[1-(1-naphthyl)methyl] imidazole hydrochloride (!58)...... 69
III. Synthesis of 4-(N-triphenylmethyl) imidazole carboxaldehyde (68) 69
IV. Synthesis of 4-[1-(1-naphthyl)hydroxymethyl] imidazole hydrochloride (59^)...... 70
V. Synthesis of 4-[(1-naphthyl)carbonyl] imidazole oxalate (6_0) ...... 71
VI. Attempted synthesis of 4-[1-(naphthyl) methoxymethyl ] imidazole hydrochloride (6_1) • • • 72
VII. Attempted synthesis of 4-[1-(naphthyl)- 2,2,2-trifluoromethyl]imidazole hydrochloride ( 6 2 J ...... 73
VIII. Attempted methods to remove a tertiary hydroxy group ...... 74
IX. Resolution of 4-[1-(1-naphthyl)ethyl] i m i d a z o l e ...... 78
X. Attempted synthesis for C-3 alkylated indole ...... 84
XI. Another approach for C-3 alkylated indole ...... 85
XII. Synthesis of ethyl 1-tritylimidazle 4-carboxylate ( 7 8 ) ...... 86 XIII. Attempted synthesis for C-3 alkylated indole with a haloalkyl imidazole ...... 86
XIV. Synthesis of a bromoethyl imidazole ...... 87
XV. Synthesis of C-3 alkylated indole ...... 88
XVI. Proposed synthetic scheme for 5 5 ...... 89
XVII. Attempted reduction of amide 7£ to amine • * * * 90
XVIII. Synthesis of 2-[2-(3-hydroxyphenyl)-2- (4-methylphenyl)ethyl]imidazole hydrochloride ( 6 3 ) ...... 91-92
XIX. Attempted conversion of nitrile £4 to an iminoacetate...... 94
XX. Attempted synthesis to introduce nitrile and ester functional groups ...... 95
XXI. Synthesis of Z-£4 and E - 6 £ ...... 97
xii CHAPTER 1
INTRODUCTION
1.1 THE NERVOUS SYSTEM
The nervous system can be subdivided into the central nervous system (CNS) and the peripheral nervous system (PNS) as shown in Figure 1. The PNS can be further subdivided into the autonomic and somatic nervous systems. The autonomic nervous system is largely independent and under unconscious control. It is concerned primarily with visceral functions - cardiac output, blood flow to various organs, digestion, and elimination. The other major division of the nervous system, the somatic nervous system is concerned with consciously controlled functions such as locomotion, respiration, and posture. The nervous system relies primarily on rapid
electrical transmission of information over nerve fibers.
However, between nerve cells, and between nerve cells and
their effector cells, signals are usually carried by chemical
rather than electrical impulses. This chemical transmission
takes place through the release of small amounts of
transmitter or "neuromediator" substances from the nerve
1 NERVOUS SYSTEM
PERIPHERAL CENTRAL
AUTONOMIC SOMATIC
CHOLINERGIC ADRENERGIC (PARASYMPATHETIC) (SYMPATHETIC)
a-ADRENERGIC B-ADRENERGIC RECEPTORS RECEPTORS
Figure 1^: Subdivision of the nervous system. terminals into the synapse. The chemical transmitter crosses
the synaptic cleft by diffusion and activates (or inhibits)
the postsynaptic cell by binding to a specialized receptor
molecule.
1.2 THE ADRENERGIC NEURONAL SYSTEM
The branch of the autonomic nervous system in which
norepinephrine is the neurotransmitter between the nerve
ending and the effector muscle is known as the adrenergic
nervous system. It is involved in the homeostatic regulation
of a wide variety of functions, including cardiac contraction,
vasomotor tone, blood pressure, bronchial airway tone, and
carbohydrate and fatty acid metabolism. Because of the diverse
functions that are mediated or modified by the adrenergic
nervous system, agents that mimic or alter its activity are useful in the treatment of several clinical disorders,
including hypertension, shock, cardiac failure and
arrhythmias, asthma, allergy, and anaphylaxis [1]. The first
attempts to find a chemical mediator resulted in the isolation
of epinephrine, the hormone produced by the adrenal medulla.
Because its biologic response was similar to that resulting
from stimulation of adrenergic nerves, it was believed to be
the neurotransmitter. Further investigation, however, showed
that it was formed by the metabolism of the true
neurotransmitter, norepinephrine. Because epinephrine was originally called adrenalin or sympathin, the terms sympathetic and adrenergic nervous system have been applied to this part of the autonomic nervous system [2].
1.2.1 BIOSYNTHESIS OF CATECHOLAMINES
The biosynthetic pathway to dopamine, norepinephrine, and epinephrine illustrated in Figure 2 was originally suggested by Blaschko [3] in 1939 and by Holtz [4] in 1939 following the discovery of DOPA decarboxylase and has been confirmed experimentally by many authors [5]. The precursor of the noradrenergic neurotransmitter norepinephrine, i.e. the amino acid L {—) -tyrosine, is taken up into the axoplasm by an active transport process. The intraneuronal synthesis of norepinephrine begins with conversion of L(-)-tyrosine to L(-
)-3,4-dihydroxyphenylalanine (L-DOPA). This reaction is the rate-limiting step in NE synthesis and is catalyzed by the enzyme tyrosine hydroxylase, which requires a tetrahydrofolate coenzyme, 02, and Fe++. This enzyme is stereospecific and shows a fairly high degree of substrate specificity. L(-)-DOPA is then converted to the corresponding amine, dopamine, by DOPA decarboxylase. This enzyme acts on all aromatic amino acids and requires pyridoxal phosphate (vitamine B6) as a cofactor.
Dopamine, synthesized in the axoplasm, is actively taken up into storage granules and is converted into (-)-NE by dopamine
II-hydroxylase (DBH). DBH is a Cu++-containing protein using 02 and ascorbic acid. DBH does not show a high degree of substrate specificity and acts in vitro on a variety of 5
NHj L-Tyrosine if H^CO„H HO
Tyrosine hydroxylase
HO NH, DOPA H C02H HO
Dopa decarboxylase
HO,1 NH. Dopamine ' HO'CT'
Dopamine B-hydroxylase
H OH
Norepinephrine
Phenylethanolamine N-methyltransferase
H. OH NHCH, Epinephrine
Figure 2: Biosynthesis of catecholamines. substrates besides dopamine, oxidizing almost any phenylethylamine to its corresponding phenylethanolamine [6].
Finally, NE is converted to epinephrine by the enzyme phenylethanolamine N-methyltransferase (PNMT) which utilizes
S-adenosyl-methionine as a methyl donor. The concentration of
PNMT is highest in the adrenal medulla, which is the main site of epinephrine synthesis, but PNMT activity is also found in the brain. PNMT shows low substrate specificity and will transfer methyl groups to the nitrogen atom on a variety of R- hydroxylated amines [6]
1.2.2 CATECHOLAMINE STORAGE
The catecholamines synthesized or taken up by the nerve terminals are stored in specialized subcellular vesicles in sympathetic nerve endings and chromaffin cells. It is generally believed that the vesicles are formed in the cell body and subsequently transported to the nerve terminal region
[6,7] . The vesicles are different sizes depending on location.
The largest ones (up to 120 nm) are formed in the adrenal medulla and are called chromaffin granules. These vesicles contain adenosine triphosphate (ATP) in a molar ratio of catecholamine to ATP of about 4:1, in association with the acidic protein chromogranin. This complex keeps the neurotransmitter in a hypoosmotic form even though its concentration is very high (up to 2.5 M ) , and also prevents it from destruction by the intraneuronally located mitochondrial 7 enzyme monoamine oxidase (MAO) [7].
1.2.3 CATECHOLAMINE RELEASE
The mechanism of catecholamine release has been studied mainly in the adrenal medulla. In the medulla, a neuronal impulse releases acetylcholine. This allows the inflow of Ca++, which triggers fusion of the chromaffin cell membrane with the secretory vesicle, resulting in exocytosis of the entire vesicle contents (chromogranin, ATP, DBH, and catecholamines)
[7,8]. It is unclear whether neurotransmitter release in sympathetic nerve endings follows the same mechanism. The freshly synthesized or recycled norepinephrine may be released first. These facts suggest that norepinephrine exists in more than one pool in the sympathetic neuron. The release and turnover of catecholamines are subject to complex regulation.
The most important type is modulation of release by presynaptic receptors which respond to catecholamine in the synapse (high concentrations inhibiting release and low concentrations augmenting release). Prostagrandins of the E series inhibit the stimulation-evoked release of norepinephrine from several sympathetically innervated tissues and their actions appear to be dissociated from any interaction with presynaptic receptors[7,9]
1.2.4 CATECHOLAMINE METABOLISM AND REUPTAKE
The actions of norepinephrine, epinephrine, and dopamine 8 are terminated by (1) reuptake into nerve terminals (2) dilution by diffusion out of the junctional cleft and uptake at extraneuronal sites and (3) metabolic transformation. The two major mammalian enzymes which carries out the metabolic inactivation of catecholamines are monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT), as shown in Figure 3
[1,10]. MAOs are flavoprotein enzymes which convert catecholamines, presumably via an intermediate imine, to their corresponding aldehydes as shown in Figure 4 [11]. The aldehyde product may be reversibly reduced to the alcohol or oxidized to the acid metabolite. MAOs are intraneuronal enzymes which are mainly localized in the outer mitochondrial membrane. They exist in at least two types, designated type A and type B based on substrate specificity and sensitivity to inhibition by selected inhibitors [6]. A-type enzyme has a substrate preference for norepinephrine and serotonin. B-type enzyme has a preference for fl-phenylethylamine and benzylamine as substrates [6].
COMT, a magnesium-dependent enzyme discovered by Axelrod in
1957, is relatively nonspecific and catalyzes the transfer of methyl group from S-adenosylmethionine to the m-hydroxy group of catecholamines and various other catechol compounds [6,11].
It is found in the cytoplasm of most cells and has been suggested to function largely extraneuronally.
Conjugating enzymes are important enzymes for deactivating catecholamines. Phenolsulfotransferases (PST) are enzymes 9
OH OH c h 3o CH ALP DEHYD HO xrV- :xfr\ Glucuronide or Tcomt 4 Sulfate I OH f Conjugate
HO"xA ^
Tal d IDEHYD OH OH NHCH, HO MAO
HO IxrV x^ ALD COMT COMT RED i' OH IOH iOH c h 3o CH.° v^X^^NHCH, H0 HO HO XT^ HO xx^ COMT 1OH CH OH MAO ' ° r r ^ MAO HO
ALD t RED ... Glucuronide Glucuronide CH. or or " f v V " Sulfate o Sulfate Conjugate HO Conj ugate
Figure 3: Norepinephrine and epinephrine metabolism, (modified from [1,10]) ALD DEHYD: aldehyde dehydrogenase ALD RED: aldehyde reductase 10 responsible for sulfation of 3-methoxy-4-hydroxy-phenylglycol
(MHPG) as the major metabolite of norepinephrine metabolite and other phenols [11].
HO R-CH2-NH2 R-CH=NH ► RCHO + NH3
MAO h 2o O 2
Figure 4: The formation of an aldehyde by MAO.
Phosphoadenosinephosphosulfate (PAPS) is the sulfate donor for sulfations catalyzed by PST [11]. Glucuronide formation is also an important means of deactivating catecholamines.
Uridine diphosphoglucuronyl transferase (UDPGT) is responsible for the glucuronide formation, where activated glucuronic acid, uridine diphosphoglucuronic acid, is transfered to the phenolic hydroxide of catecholamine metabolites [11].
The principal mechanism for the deactivation of released catecholamines is not enzymatic destruction but reuptake into the nerve terminals [7]. J. H. Burn [6] in 1932 suggested that exogenous norepinephrine might be taken into peripheral storage sites. The uptake of catecholamines is a Na+-dependent active-transport system as it proceeds against a concentration gradient. There are at least two types of uptake. Uptake 1, with high stereospecificity for {-)-norepinephrine, normally operates after nerve transmission [2]. Circulating amines can 11
also be taken up by this mechanism. Extraneuronal tissues also
take up catecholamines. This process, uptake 2, is less
stereospecific and operates when higher concentrations of the
neurotransmitter or structurally related compounds are present
at the receptor [2]. Once catecholamines have been taken up in
either nerves or smooth muscle, they may be inactivated
enzymatically.
1.3 SUBCLASSIFICATION OF g-ADRENERGIC RECEPTORS
The adrenergic receptors have been classified
pharmacologically based on the relative potencies of selective
agonists and antagonists. The initial classification of
adrenergic receptors into the a and 13 subtypes was suggested
by Ahlguist [12] in 1948 based on their relative sensitivity
to a series of sympathomimetic amines and their responses in
a variety of tissues. For the a-adrenergic receptor the order
of potency is norepinephrine > epinephrine > phenylephrine >
isoproterenol for vasoconstriction, excitation of the uterus,
contraction of the nictitating membrane, dilation of the
pupil, and inhibition of gut. In contrast, for the 13-
adrenergic receptor the order is isoproterenol > epinephrine
> norepinephrine > phenylephrine for vasodilation, inhibition
of the uterus, and myocardial stimulation. Subsequently, 13-
adrenergic receptors were subdivided into J31 and 132 subtypes based on the relative potencies of agonists in several
isolated organ systems (Lands and co-workers, 1967 [13]). 12
Similarly, a-adrenergic receptors were subdivided into and a2 subtypes by Langer [14] in 1974 based on presumed anatomical localization, o^-receptors being postsynaptic and a2~receptors being presynaptic. Although most presynaptic a- adrenergic receptors are of the a2 subtype, some of the postsynaptic adrenergic receptors have the pharmacological properties of a2-receptors. Therefore, the classification of a-adrenergic receptors by anatomical localization is not a useful definition of subtypes. This resulted in the proposal by Berthelsen and Pettinger [15] in 1977 that a-adrenergic receptors should be classified on the basis of their pharmacology, and this method is now widely accepted method for subclassification of the receptors.
Some selective a1~ and a2~adrenergic receptor agonists and antagonists are shown in Figures 5 and 6 [16-19]. ax~
Adrenergic receptors are generally defined as receptors which are selectively stimulated by the agonists phenylephrine (.1), methoxamine (2), cirazoline (^) # and amidephrine (4), potently blocked by the competitive antagonist prazosin (5) and irreversibly blocked by the alkylating agent phenoxybenzamine
(8). Clonidine (9.), UK 14,304 (1.0), guanabenz (.11.), and BHT
920 (12) selectively stimulate the a2-adrenergic receptors.
Yohimbine (^3), rauwolscine (ljl), idazoxan (L5), and piperoxan
(16) are selective antagonists of the a2-adrenergic receptors.
It is now accepted that a1~ and a2~adrenergic receptors coexist postjunctionally in the vasculature of many mammalian 13
Agonists:
OH OCH, OH NH CH CH
OH OCH 3
H OH H H3C00 'S^NV XT ^ Ss i,^ >s^'NnCH
Antagonists:
CH,0 O CH COJ NH, OCH,
.Cl oneH,C X) OH
Figure 5: aA-Adrenoceptor agonists and antagonists. Agonists:
ocro
Figure 6: a 2-Adrenoceptor agonists and antagonists 15
species and mediate vasoconstriction. In vivo, postjunctional
vascular a2-adrenergic receptors are located predominantly
extrajunctionally, while postjunctional vascular c^-adrenergic
receptors exist primarily at the vascular neuroeffector
junction [20]. Stimulation of prejunctional a2-adrenergic
receptors by endogenously released norepinephrine inhibits the
release of the neurotransmitter into the synapse in response
to nerve stimulation by a negative feedback mechanism [21,22].
A pharmacological differentiation between pre- and
postjunctional a2~adrenergic receptors both in vivo and in
vitro has been demonstrated by Ruffolo and co-workers [23,24] using SK&F 104078 (12). SK&F 104078 (22) is a potent and highly selective antagonist at postjunctional a2-receptors but
is at least 100-fold less active at most prejunctional receptors.
From recent experimental findings, ^-adrenergic receptors can be further subdivided pharmacologically into distinct
subtypes. This subdivision is based on several criteria, such as sensitivity of a1-adrenergic receptor-mediated response to blockade by prazosin (5), phenoxybenzamine (8), and other a- adrenergic receptor antagonists; differing interactions between a1-adrenergic receptor agonists and vasodilators; and different requirements of a^-adrenergic induced responses for extracellular calcium [25].
There has been a recent proliferation of further subtypes proposed by several groups. In 1982, Coates and co-workers [26] proposed alphalB as a subset of a1-adrenergic receptors which were activated by Sgd 101/75 (indanidine, 18), particularly sensitive to phenoxybenzamine (8), and separate from other ^-adrenergic receptors where norepinephrine was active.
In 1982, McGrath [27] suggested that alpha-1 mediated responses in the rat anococcygeus muscle result from activation of two receptor populations, termed alpha-la and alpha-lb. Low concentrations of phenethylamine agonists activate the alpha-la first, followed by the alpha-lb as the concentration is increased. His hypothesis would explain the
"shoulder" on the norepinephrine dose response curve in this tissue, indicative of two binding components. However, imidazolines produce monophasic curves since they cannot activate the alpha-lb population. McGrath and co-workers
[28,29] later found that the proposed ala-subtype was susceptible to Ca2+ antagonists and thus attributable to opening of Ca2+ channels, while the proposed alfc-subtype releases Ca2+ from intracellular stores. 17
In 1986, Flavahan and Vanhoutte [30] noted the wide variation In affinity for prazosin (5J and yohimbine (13) reported in functional studies in the vascular smooth muscle of blood vessels from rodents and rabbits. They proposed that there were two distinct subtypes of ax-adrenergic receptors, one subtype with high affinity for both prazosin (5) and yohimbine (JL3) and a second subtype with low affinity for both antagonists.
In 1986, Morrow and Creese [31] suggested that the two types of a1-adrenergic receptor binding sites should be called au and a1B based on differences in the relative potencies of antagonists, particularly phentolamine (.19) and WB4101 (6), at
[3H]prazosin binding sites. The a^-adrenergic receptor was the highest affinity for phentolamine (1JJ), while the lowest affinity for phentolamine (^9J was the a1B-adrenergic receptor.
The rank order of antagonist affinity for the proposed subtype was WB4101 > prazosin > phentolamine > indoramine > dihydroergocryptine. In contrast, the rank order for ou was prazosin > indoramine > dihydroergocryptine > WB4101 > phentolamine.
Using a different approach, Johnson and Minneman [32] noted differences in the sensitivity of c^-adrenergic sites labeled with [ I ]BE 2254 (20) and by the alkylating derivative of clonidine, chlorethyl clonidine (CEC, 2_1). They noted that pretreatment of rat cerebral cortex with CEC (21) inactivated only half of the o^-adrenergic receptor binding sites, whereas 18
CEC (21) pretreatment of hippocampus membranes did not inactivate any c^-adrenergic receptor binding sites. This suggests that there are two types of ^-adrenergic receptor binding sites in rat cerebral cortex. Furthermore, Han et al.
(33] showed that pretreatment of membranes from rat liver or spleen with CEC (21) caused a 70 to 80% loss of specific
[1Z5I]BE 2254 (20) binding sites, whereas [125I]BE 2254 binding in membranes from rat hippocampus or vas deferens was not affected. These results suggest that rat liver and spleen contain mainly "CEC-sensitive" a^-adrenergic receptors, while hippocampus and vas deferens contain "CEC-insensitive" a1- adrenergic receptors.
12 5.
19 20
In 1987, Han et al. [34] provided functional evidence for ala and alb subtypes by establishing a correlation with differences in agonist and antagonist potency at the muscle contraction level. Alpha la subtype, having high affinity for
WB4101 (1), causes contractions by allowing the influx of extracellular Ca2+ through dihydropyridine-sensitive channels 19 via an unknown mechanism and does not stimulate inositol phosphate formation. Alpha lb subtype, having low affinity for
WB4101 (6.), stimulates inositol phosphate formation which causes the release of intracellular Ca2+ and causes contractions which are independent of extracellular Ca2+.
In 1988, Bevan et al. [35] proposed a "variable receptor affinity hypothesis" based on variations of norepinephrine affinity for a-adrenergic receptors in different tissues. They suggested that variation in affinity can explain variation in sensitivity and this does not provide a basis for receptor type subdivision. They also found that adrenergic antagonist affinity can vary significantly in tissues. The factors affecting this variation include differences in receptor chemical structure, in receptor microenvironment, and in intracellular processes [36].
In 1988, Cotecchia and co-workers [37] cloned the cDNA of the syrian hamster a1~adrenergic receptor and found these clones to have properties completely consistent with an alb subtype. These properties are low affinity for WB4101 (6) and phentolamine (.19), and coupling to inositol phospholipid metabolism. In 1990, rat liver and brain membrane ax- adrenergic receptors were purified by Terman and co-workers
[38] using successive chromatographic steps. The a^selective ligand CEC irreversibly inactivated 100% of [3H]prazosin binding sites in partially-purified preparations of rat liver
(alb), while only 50% of the brain receptors were irreversibly inactivated (crla) . The CEC-insensitive sites in brain were
resistant to photoaffinity labeling by [ 125I Jazidoprazosin.
These results provide additional evidence that there are structurally distinct forms of the receptor subtype in membrane preparations, and that functional variations are not due to differences in membrane components that interact with the receptor. A number of distinct characteristics of ola and
«lb-adrenergic receptor subtypes are summarized in Table 1.
Table 1
c^-Adrenerqic Receptor Subtype Characteristics
Subtype
Characteristics l a a i b
Tissue distribution brain/aorta/ liver/spleen vas deferens
Ligand binding
WB 4101 high affinity low affinity
Phentolamine high affinity low affinity
CEC sensitivity - + + +
PI turnover* - + + +
Ca2+ influx + + + -
[ 1 2 5 I] Azidoprazosin • + + + photolabling
phosphatidylinositol turnover 21
Recently, Schwinn and co-workers [39] cloned a novel ax~ adrenergic receptor subtype from a bovine brain cDNA. The analysis of human chromosome provides evidence that the bovine a^-adrenergic receptor (localized to chromosome 8) is distinct from the hamster cr^-adrenergic receptor (localized to chromosome 5). Bovine a1-adrenergic receptor subtype shows a binding profile consistent with the subtype (high affinity for the c^-adrenergic antagonists WB4101 and phentolamine and the agonist oxymetazoline) but is relatively susceptible to chlorethylclonidine inactivation. Furthermore, a lack of expression in tissues where the subtype exists suggests that this receptor may represent a novel a^-adrenergic receptor subtype.
ci
cl
21 22
Alpha2-adrenergic receptors were also subdivided into a2A and a2B subtypes by Bylund [40] and Nahorski et al. [41] independently. The human platelet is the prototype tissue for the which has a low affinity (K± = 240 nM) for prazosin
(5), whereas the neonatal rat lung has a high affinity binding site (1^ = 5 nM) for prazosin (5) and is the prototype tissue for the a2fl subtype. Oxymetazoline (22) is selective for alpha-
2A receptors, whereas prazosin (5) is alpha-2B selective.
Also, in the aa subtype the pharmacological profile is of yohimbine (.13) having equal affinity to rauwolscine (14), whereas the a2B subtype has different antagonist properties, rauwolscine (.1£) is more potent than yohimbine (.13). To provide a more definitive characterization of or2-adrenergic receptor subtypes, Bylund and co-workers [42] compared the potency of antagonists in binding assays with tissues and cell lines that contain only one subtype. They used the human platelet, HT29 cell line, and human cerebral cortex for aa and neonatal rat lung and NG108-15 cell line for the a2H~adrenergic receptor subtype. Three new a2B subtype selective compounds relative to aa subtype were identified (Figure 7): ARC-239
(23), chlorpromazine (24), and 7-hydroxychlorpromazine (25) are 100-, 18-, and 17-fold selective, respectively.
Figure Z : a 2 B“Adrenergic receptor selective compounds Bylund et al. [43] also proposed a third a2-adrenergic receptor subtype as a result of their characterization of a2~ adrenergic receptors in OK cells, an opossum kidney-derived cell line. This receptor has a unique pharmacological profile which is similar to the a2B; it has a relatively high affinity for prazosin (_5), but the K± ratio of prazosin (5) to yohimbine (1^) is closer to that of the a [44,45]. Thus, the a2~receptor on the OK cell was called a2c. The same research group recently identified a fourth a2-adrenergic receptor, which has been termed alpha-2D, in the bovine pineal gland based on the potencies of six antagonists [46]. Especially,
SK&F 104078 (.17) can differentiate the a2D subtype from the other three subtypes. This compound has similar affinities for the A, B, and C subtypes, but a much lower affinity for the cr2D subtype.
Kobilka et al. [47] cloned the genes encoding a2~adrenergic receptors from the human platelet. This receptor has high and equal affinity for yohimbine (ljl) and rauwolsine (.14) and lower affinity for prazosin (5) and is thus classified as an a2A-adrenergic receptor. The receptor from the human platelet derives from chromosome 10 and is referred to as the a2-C10 receptor [45]. Regan et al. [48] cloned the cDNA of the human kidney a2~adrenergic receptor. The kidney receptor is called a2-C4 because the gene coding for it is on chromosome 4. The a2~C4 has high affinity for prazosin (5) and low affinity for oxymetazoline (22) as compared with a2~C10. The a2_C4 also 24
shows significantly higher affinity for rauwolscine (,14) as
compared with a2-C10. The a2-C4 receptor is clearly different pharmacological profiles from the a2-C10 receptor and was
tentatively identified as aa . However, when correlating drug
potencies between the expressed a2-C4 receptor and cell lines
containing the a2A, a2B, and a2c receptors, the a2~C4 receptor
is neither an a2B nor an a2c and thus may represent a fourth
subtype [45]. Figure 8 shows the modern classification of
adrenergic receptors [45].
ADRENOCEPTORS
p-ADRENOCEPTOR a2-ADRENOCEPTOR -ADRENOCEPTOR
P1 P2 P 3 a2A a 2B a 2 c a2D alA a iB °1C
Figure 8: Modern classification of adrenoceptors.
1.4 SIGNAL TRANSDUCTION OF cr-ADRENERGIC RECEPTORS
Cells communicate with each other by a variety of signal molecules, such as neurotransmitters, hormones, and a variety of regulatory and growth-promoting factors, that are detected by specific receptors on the plasma membrane of the corresponding cell. Stimulation of a receptor by its agonist initiates a cascade of biochemical processes that produce an 25 intracellular signal, which causes a change in the behaviour of the cell, such as secretion of an enzyme, contraction or initiation of cell division [49].
c^-Adrenergic receptors are involved in a variety of physiological processes, including control of blood pressure, appetite, and mood. These receptors initiate signals in their target cells by increasing the concentration of intracellular free Ca2+ (Figure 9) [50]. This results from the activation of membrane bound phospholipase C via a guanine nucleotide regulatory protein (G protein). Activated phospholipase C catalyzes the hydrolysis of phosphatidylinositol 4,5- bisphosphate (PIP2) into two intracellular second messenger molecules, inositol 1,4,5-triphosphate (IP3) and 1,2- diacylglycerol (DG) [17]. Phosphatidylinositol 4-phosphate
(PIP) and PIP2, collectively referred to as polyphosphoinositides (PPI), are plasma membrane phospholipids and have more rapid metabolic turnover than any other membrane phospholipid. IP3 then mediates the release of Ca2+ from intracellular stores, such as the endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR) in muscle, which is responsible for the initial contraction [50]. A specific receptor exists for IP3 on the ER which mediates the opening of a Ca2+ channel in the ER membrane. The action of IP3 is terminated by the combined action of two separate pathways.
IP3 can be dephosphorylated via an inositol triphosphatase to give 1,4-inositol bisphosphate (IP2) which has no Ca2+ 26
C a2+
pip pip
PLC (+) IP
2 + Ca Diacylglycerol
(+)
Protein kinase C
2 + 2 + Ca Ca Phosphorylation of specific proteins 2 + Ca / calmodulin kinases Selected responses substratesI ► Integrated responses Selected responses
Figure 9: Signal transduction mechanism activated by ax-adrenoceptor. (modified from [50]) 27 mobilizing activity, or phosphorylated via an inositol triphosphate kinase to inositol 1,3,4,5-tetrakisphosphate
(1,3,4,5-IP4), which may function as a second messenger to regulate the entry of Ca2+ across the membrane [50]. The free cytosolic Ca2+ binds to the regulatory protein calmodulin and the Ca2+-calmodulin complex, in turn, activates a variety of enzymes and other cellular proteins altering their activities and leading to various physiological responses: in the case of smooth muscle in many tissues, a^-adrenergic receptors cause contraction via activation of myosin light chain kinase by the
Ca2+-calmodulin complex, which then phosphorylates the light chain myosin. Phosphorylation of myosin is required for actin activation of myosin ATPase activity and the formation of cross bridges between actin and myosin results in contraction
[47]. Diacylglycerol, the other second messenger molecule, activates the phospholipid-sensitive protein kinase C to phosphorylate a variety of specific proteins. The DG/protein kinase C pathway is proposed to modulate various aspects of the Ca2+ signalling pathway to give an integrated and highly versatile receptor mechanism which controls many cellular processes including secretion, metabolism, contraction, neuronal excitability, and cell growth.
Guanine nucleotides influence agonist binding to a2~ and 15- adrenergic receptors and play a fundamental role in the coupling of these receptors to adenylate cyclase. The effects of these nucleotides are mediated by the two specific guanine nucleotide binding proteins termed G±, which mediates the effects of inhibitory agonists on adenylate cyclase resulting in inhibition of cAMP production, and Ga, which mediates the effects of stimulatory agonists on the enzyme resulting in cAMP production [51]. Both G proteins are cytosolic, membrane- bound heterotrimers composed of an a, fl, and y subunit [52].
G± has been shown to have three subtypes of the a-subunit which are responsible for the inhibition of adenylate cyclase and the activation of cardiac K+ channels [53]. The a-subunit of Ga contains the binding site for guanine nucleotides, shows
GTPase activity, and is subject to ADP-ribosylation under the influence of specific bacterial toxins. The R- and y-subunits appear to be the same for G. and G with M of 35 KDa and 8 1b r KDa, respectively. However, the a-subunit shows polymorphism: in G,1 it has an MT of around 41 KDa and in G 8 it has a Mr 45 - 52 KDa [51]. Pertussis toxin abolishes the inhibitory effect of a2-adrenergic agonists on adenylate cyclase because it catalyzes the ADP-ribosylation on the a-subunit of G±. This interferes with the coupling of G^ to the receptor, resulting in increased intracellular cAMP levels [54].
The general mechanism of G protein coupled signal transduction is shown in Figure 10 [49]. From the basal state
(a), agonist binds to the receptor resulting in receptor coupling to the G protein. This causes a conformational change in the receptor-G protein complex (b) which allows GTP, in the presence of Mg2+, to replace GDP on the a subunit. The fly 29
a
GDP
Et factor
GDP—i
GTP GDP,
E ffector
< s >
Figure 10: G protein coupled signal transduction. Ca Voltage-dependent (N-type) Ca2+ channel
-Q AC
ATP cAMP 2 + Ca
cAMP-dependent protein kinase
Figure 11: Signal transduction mechanism activated by presynaptic a2-adrenoceptors. (modified from [49]) 30 subunit dissociates, and the activated a-GTP subunit interacts with the effector, adenylate cyclase. The intrinsic GTPase activity of the a subunit hydrolyses GTP to GDP, releasing inorganic phosphate (PA), and the a-GDP complex recombines with the iiy subunit to end the activation cycle (c).
Stimulation of presynaptic a2-adrenergic receptors leads to inhibition of neurotransmitter release by decreasing the cAMP dependent-enhancement (phosphorylation) of the opening of N- type slow Ca2+ channels (Figure 11) [50]. Whereas stimulation of presynaptic a2-adrenergic receptors is to limit the availability of extracellular Ca2+, activation of postsynaptic a2-adrenergic receptors in vascular smooth muscle promotes the influx of Ca2+ ions via specific receptor-operated Ca2+ channels without mobilization of intracellular Ca2+ through breakdown of PI. However, the vasoconstrictor effects of a2-adrenergic agonists are sensitive to attenuation by pertussis toxin, suggesting participation of Gt in this process. At present, there is no evidence showing that a2-adrenergic receptor stimulation affects cAMP levels in vascular smooth muscle.
1.5 STRUCTURAL PROPERTIES OF tt-ADRENERGIC RECEPTORS
The a-adrenergic receptors are composed of a single polypeptide and are integral membrane glycoproteins [55].
These receptors are homologous proteins; a1-receptors consist of 515 amino acids [37], while a2~receptors consist of 450 amino acids [47]. The topography includes a-helical transmembrane regions joined by intracellular and extracellular loops as shown in Figure 12 for a2R-adrenergic receptors [56,57]. The less hydrophobic amino acids of a helices likely project toward the interior of the molecule, while the more hydrophobic residues may form a boundary with the plasma membrane [58].
n h 2
EXTRACELLULAR
INTRACELLULAR
COOH
Figure 12: Topography of the human a 2A-adrenergic receptor, (modified from [6]) 32
The hydrophobic domains of the a-adrenergic receptors may form a pocket in the plasma membrane for binding ligands [55]. The most conserved regions in the structures of a-adrenergic receptors are the putative transmembrane regions. The amino- terminal regions, the third cytoplasmic loops, and the carboxy-terminal regions show less similar sequences than the membrane spanning domains, perhaps indicative of less stringent structural requirements [55]. Each of the receptors contains putative glycosylation sites in the amino-terminal region which contains oligosaccharides. These oligosaccharides may be required to protect the amino-termini from protease attack. A major function associated with the cytoplasmic domains is coupling to effector guanine nucleotide regulatory proteins. The sequences in the intermediate and C-teminal portions of the third intracellular loop, as well as in the N- terminal segment of the cytoplasmic tail are important for coupling of the a^adrenergic receptor to phosphatidylinositol
(PI) hydrolysis. In addition, Ala293 and Lys290 in the C- terminal portion of the third intracellular loop increase the affinity of norepinephrine binding and its potency for stimulating PI hydrolysis [56]. The C-terminus of the a^ adrenergic receptor beyond Arg368 is not required for either ligand binding or effector activation. The a2-adrenergic receptor can be phosphorylated in an agonist-dependent fashion by A-adrenergic receptor kinase (AARK) and then receptor down regulation (desensitization) occurs [55]. The serine and 33
threonine residues in the third intracellular loop may serve
as substrates for DARK. The a2-adrenergic receptor possesses a short C-terminus devoid of serine and threonine residues.
However/ the c^-adrenergic receptor is not a substrate for the
DARK [59]. Major determinants of a2~adrenergic receptor agonist and antagonist ligand binding specificity are contained within the seventh membrane-spanning domain [58].
Furthermore, several of first five hydrophobic domains may also contribute to agonist binding specificity. The a2~ adrenergic receptor has the longest third intracellular loop and shortest C-terminus compared to other receptors [60].
1.6 STRUCTURE-ACTIVITY RELATIONSHIPS OF g-ADRENERGIC AGONISTS
The vast majority of a-adrenergic receptor agonists can be divided into two main classes, the D-phenethylamines and imidazolines. Although many notable exceptions to this generalization exist, as a general rule the D-phenethylamines are either nonselective or show selectivity for aj-adrenergic receptors. In contrast, the imidazolines are either nonselective or show selectivity for the a2~adrenergic receptor [18,61]. In addition, the D-phenethylamines are, with
few exceptions, full agonists, whereas the imidazolines are generally partial agonists [62]. The reason is that D- phenethylamines have low affinity and high intrinsic activity
(efficacy), while the imidazolines have high affinity and low intrinsic activity. Although structure-activity relationships 34
in ctj- and a2-adrenergic receptor agonists are largely unexplored, the imidazolines and fl-phenethylamines have marked differences in their structural requirements for a-adrenergic
receptor agonist activity. Table 2 summarizes differences between ii-phenethylamines and imidazolines interacting with a-
adrenergic receptors [61].
Table 2
Differences between Phenethylamines and Imidazolines for Agonist Activity
Phenethylamines Imidazolines
Easson-Stedman hypothesis applicable not applicable
Stereochemical demands high low
Aromatic hydroxyl substitution increase affinity increase efficacy
Benzylic hydroxyl substitution increase affinity decrease affinity
N-methyl substitution increase activity decrease activity
Subtype selectivity ai °2 Intrinsic efficacy high low
Agonist profile full agonists partial agonists
Calcium utilization intra- and extracellular extracellular
Cross-desensitization no yes with oxymetazoline
Time course of response rapid slow 35
The most active enantiomer of norepinephrine and other fl- phenethylamines at a%- and a2~adrenergic receptors is the R(-)- isomer. The relative positions in space of the three important functional groups (aromatic ring, fl-hydroxy group, and aliphatic nitrogen atom) of a sympathomimetic amine when bound to the a-adrenergic receptor are obtained from an analysis of the comformational demands made by the receptors. Theoretical calculations show that the preferred conformation of R(-)- norepinephrine in solution is the extended trans conformation in which the amine and phenyl groups are at a dihedral angle of 180° [18]. It has been reported that the imidazoline clonidine prefers the conformation in which the phenyl and imidazoline rings assume a perpendicular arrangement similar to that of norepinephrine. Figure 13 shows proposed conformations of norepinephrine and clonidine interacting with a-adrenergic receptors [8].
H
< ► 5.1-5.2 A 5.0-5.1 A
Figure 13: Proposed conformations of norepinephrine and clonidine for interaction with a-adrenergic receptors. (modified from [8]) 36
The fl-phenethylamines and imidazolines show a major difference when applying the Easson-Stedman hypothesis [63].
It was proposed that a three-point attachment (includes three functional groups) was involved in the binding of fi- phenethylamines possessing an asymmetric carbon atom to the a- adrenergic receptor. This hypothesis explains why the R(-)- phenethylamines are more active than the enantiomeric S( + )- enantiomer or corresponding desoxy derivative, which are equally active to each other. Only the R(-)-isomer has the ideal stereochemical configuration, so all three functional groups may interact with appropriate groups on the o- adrenergic receptor. However, the S(+)-enantiomer and the R- desoxy derivative, either has wrong configuration or an appropriate group is absent for binding to the receptor.
Therefore, only a two-point attachment is possible with the
S(+)-isomer and desoxy derivative, which are less potent relative to the R(-)-compound. This hypothesis predicts the rank order of potency for /J-phenethylamines: R(-) > S( + ) = desoxy. Figure 14 shows the schematic representation of the
Easson-Stedman hypothesis for norepinephrine [63].
However, optically active imidazolines do not follow the
Easson-Stedman hypothesis. Using isomers of 2-(3,4,a- trihydroxybenzyl)imidazoline (.26), and the corresponding desoxy derivative, Ruffolo et al. [64] found that the rank order of potency at a1~ and a2~adrenergic receptors was desoxy
> R(-) > S (+). 37
OH OH OH OH OHOH P P
OH- NH H NH
R(-) S(+) Desoxy
Figure 14: Schematic representation of the Easson-Stedman hyposis. A - amine binding site, H - hydroxyl binding site, P - phenyl binding site
OH
26
From this data, the imidazolines may bind to the a-adrenergic receptor via a two-point attachment, the phenyl ring and nitrogen atom of the imidazoline nucleus. The structure- activity relationships of fl-phenethylamines will be discussed mainly for four areas of structural modification: a) substitution at the aromatic ring; b) substitution at the A- 38 carbon atom; c) substitution at the ct-carbon atom; and d) subsitution at the aliphatic nitrogen [65].
C— C-l-N— R ■ : i R R : R
a i b j c I d
Phenolic groups on the phenyl ring of A-phenethylamines significantly affect the a-adrenergic agonist activity. The
3,4-dihydroxy substituted derivatives are the most potent at- and a2~adrenergic receptor agonists. The rank order of potency for a:- and a2-adrenergic receptor agonists is 3,4-dihydroxy
> 3-hydroxy > 4-hydroxy > nonphenolic [65]. The aromatic hydroxyl substitution of A-phenethylamines affects affinity for a1-adrenergic receptors, but decreases intrinsic activity for a2-adrenergic receptors. Sulfonamido and halogen subsituents have been investigated as hydroxy group replacements on the aromatic ring. When the 3-hydroxyl group of epinephrine or norepinephrine is replaced with a methanesulfonamido group, a1-adrenergic agonist activity is retained, but replacement of the 4-hydroxy group with a methanesulfonamido group abolishes activity [65]. Increasing the bulk of the sulfonamido group at the 3 position to the n- butylsulfonamido group decreases agonist activity, and activity is abolished when an arylsulfonamido group is 39
introduced. Replacing the catechol of epinephrine with 3,4-
dichloro or 3,4-difluoro substituents reduces a^-agonist
activity. Kirk et al. [66-68] prepared a series of ring
fluorinated norepinephrine analogs with which exert marked differences in adrenergic activities. 2-Fluoronorepinephrine
(27) is a selective A-adrenergic agonist, whereas 6-
fluoronorepinephrine (29) is a pure a-adrenergic agonist that
is completely devoid of A-adrenergic agonist activity (a
selective a2~adrenergic agonist [69]), and 5-
fluoronorepinephrine (28) possesses both A- and a-adrenergic
agonist activity.
F
F
27 28 29
Substitution at the a-carbon can produce significant effects in the a: vs. a2-adrenergic receptor selectivity of the
A-phenethylamines. 2S(+)-a-Methyl dopamine was found to be highly selective for the a2-adrenergic receptor, whereas 2R(-
)-a-methyl dopamine is nonselective [70]. These results suggest that the a2~adrenergic receptor can recognize methyl substituents at the a-carbon atom of A-phenethylamines when correctly oriented. Thus, it has been proposed that a2- 40 adrenergic receptors, but not a1~adrenergic receptors, have an additional a-methyl recognition site.
Substitution at the nitrogen atom of fl-phenethylamines can affect ctj-agonist activity. As the nitrogen atom is substituted with larger normal and branched alkyl groups, a1- adrenergic agonist activity is decreased and abolished with N- butyl and larger substitutions. Agonist activity is however regained with dobutamine (30) bearing a phenyl-containing group.
30
In addition to the fi-phenethylamines, another important class interacting with the a-adrenergic receptor is the imidazoline derivatives. This discussion of structure-activity relationships of imidazolines will focus on the three areas: a) modification of the aromatic ring; b) substitution at the nitrogen or carbon bridge; and c) substitution at the imidazoline ring [8,65]. The phenolic hydroxyl groups of imidazoline analogs evidentally interact with a-adrenergic receptors in a similar manner to those of the Ji- phenethylamines. So, the rank order of potency observed for 41 the imidazolines is same as that of fl-phenethylamines on both errand a2~adrenergic receptors.
X - C or N R H
a b c
The aromatic hydroxyl groups of the imidazolines predominantly affect intrinsic activity, but not affinity, which is different from the fl-phenethylamines. The activity of phenylaminoimidazolines (clonidine-like imidazolines) at a2~ adrenergic receptor is increased by the addition of a chlorine atom at either the 2, 3, or 4 position of the phenyl ring, with the greatest activity observed for the 2-chloro derivative [65]. The agonist activity is further enhanced by the addition of a second chlorine atom; the greatest activity is obtained with the 2,6-dichloro derivative as clonidine (£), although the 2,3-, 2,4-, and 2,5-dichloro derivatives also exhibit increased activity. Although a significant decrease in activity has been observed when both chlorine atoms of clonidine are replaced by fluorine or bromine, the 2-fluoro,
6-bromo and 5-fluoro derivatives of clonidine (£) retain the same activity as clonidine. A methyl and ethyl group can be substituted for the halogen with only little losses of potency
[65]. The various halogen- and alkyl-substituted derivatives show pronounced differences in centrally mediated hypotensive 42
effects, although no marked changes In agonist activity are
observed at the receptor level. This is due to the marked
differences in distribution properties which depend on the
lipophilicity. The replacement of the 2,6-dichlorophenyl
moiety of clonidine, as in tiamenidine (31J, reduces o-
adrenergic agonist activity. However, replacing the phenyl
ring with a pyrimidine system, as in moxonidine (32),
possesses a potent a-agonist activity. An aromatic ring is not
prerequisite for activating a-adrenergic receptors because the
oxazolidine S3341 (rilmenidine, 33), was found to be a non-
selective a-adrenergic agonist and was recently marketed in
France as Hyperium" (Figure 15) [8].
31 32 33
Figure 15: Aromatic ring substituted clonidine-like compounds.
Optimum agonist activity at a2-adrenergic receptors requires
a nitrogen atom separating the phenyl and imidazoline rings
[65]. Replacing this nitrogen atom with either carbon or
sulfur slightly decreases a2-agonist activity, whereas
replacement with an oxygen atom abolishes activity. Allyl or
cyclopropylmethyl substituents on the nitrogen atom abolish a- 43
agonist activity. Extension of the bridge length between
phenyl and imidazoline rings decreases agonist activity. This
effect is especially great when the ring junction exceeds
three atoms [8]. However, an exception is the non-selective
agonist lofexidine (34), which is as potent as clonidine (9).
When tolazoline-like imidazolines (benzylimidazolines) are
hydroxy-substituted at the methylene bridge, a significant
reduction in a-adrenergic agonist activity results. This is in
contrast to the fi-phenethylamines, as mentioned above.
Unsubstituted the imidazoline ring gives optimum affinity for
a2-adrenergic receptor.
34
Furthermore, additional unsaturation at the 4,5 positions to
give imidazoles decreases activity [65]. Opening of the
imidazoline ring to give guanidine analogs also decreases activity. Replacing one of the imidazoline nitrogen atoms with a carbon (pyrrolidine), oxygen (oxazolidine) or sulfur
(thiazolidine) atom reduces a2~adrenergic agonist activity, with the greatest reduction resulting from the oxygen atom replacement. Enlargement of the five-membered imidazoline ring 44 into a six-, seven-, or eight-membered ring produces a dramatic decrease in a-adrenergic agonist activity. Different five-membered mono- or bicyclic heterocyclic systems provide very potent a-adrenergic agonists, as in 44-549 (35J# ICI
106270 (.36), Bay c 6014 (37), whereas a six-membered heterocyclic ring as in xylazine (38) reduces activity (Figure
16) [8]. In addition, it has been reported that medetomidine,a new imidazole compound, possesses potent and selective a2~ adrenergic agonist activities which will be discussed later.
Cl F
35 36
CH 3 CH 3
37 38
Figure 16: Imidazoline modified clonidine-like compounds. 45
1.7 STRUCTURE-ACTIVITY RELATIONSHIPS OF g-ADRENERGIC
ANTAGONISTS
Structure-activity relationships for at- and a2-adrenergic
antagonists are difficult to interpret because of the
diversity of chemical structures capable of blocking a-
adrenergic receptors. Thus, only selected ax- and a2~
adrenergic antagonists will be discussed below. Prazosin (5),
a quinazoline derivative, is the prototype a1-adrenergic
antagonist, possessing the highest potency as well as
selectivity. It is used in the management of hypertension and
heart failure. Modification of the basic structure of prazosin
(5) produces other selective a^adrenergic antagonists, such
as doxazosin (39) and trimazosin (40) (Figure 17) [8].
c h 3 o - k A f 1»
nh2
39 40
Figure 17: Prazosin derivatives as 0^ -adrenergic antagonists.
Doxazosin (39j has a longer half-life than that of prazosin
(5) . Trimazosin (40) has lower affinity for ctj-adrenergic
receptors than prazosin (5), but it also blocks
phosphodiesterase, thus enhancing its functional potency. The 46 presence of the 4-amino group is important for its o^- adrenergic receptor affinity because replacing it with a 4- methyl group causes a loss in affinity. WB4101 ( , a benzodioxane derivative, is also a potent and selective o^- adrenergic antagonist. Replacing the diortho methoxy groups with methyl groups decreases affinity to approximately that of the non-methoxylated molecule, while repositioning a methoxy group to the meta or para position reduces potency.
Introduction of a hydroxyl group increases potency [8],
Replacing the oxygen atom at the 4-position in the benzodioxane moiety with a sulfur atom yielded a more potent and selective o^-adrenergic antagonist. Alkylation of the secondary amine in the side chain causes a loss affinity for
^-adrenergic receptors. AR-C 239 (41) and SGD-1534 (42), phenylpiperazine derivatives, possess peripheral a^-adrenergic antagonist activity. SGD-1534 (42) is a more potent and more selective o^-adrenergic antagonist than prazosin [8].
h3c ch3 H
41 42
Phenoxybenzamine (8), a fl-haloalkylamine, is an irreversible a1-selective antagonist. It is thought to form the 47 corresponding aziridinium ion which then reacts with a receptor nucleophile to form a covalent bond with the a%- receptor [18]. Benextramine (43), a tetramine disulfide, forms a covalent bond by disulfide interchange with thiol groups on the a-adrenergic receptor [18,62].
OCH
CH2NH(CH2 )6NHCH2CH2S
2
43
The structure-affinity relationships for the indole alkaloid yohimbine (T3) and related diastereomers suggest that the indole nucleus, the nitrogen atom N-4, the carbomethoxy substituent at C-16, and the planarity of rings A, B, C, and
D are important for a-adrenergic receptor affinity [72].
Yohimbine preferentially antagonizes a2~adrenergic receptors and the diastereomer rauwolscine (JJl) also shows selectivity for a2~adrenergic receptors. The C-16 carbomethoxy substituent is important for a1/a2 selectivity: the C-16 axially subsituted corynanthine (2) gives ax-adrenergic receptor selectivity, whereas the equatorially substituted isomers, yohimbine (13) and rauwolscine (24), give a2-adrenergic receptor selectivity
[73]. In addition, the c-17 hydroxy group plays an important role in determining a2-selectivity [74]. The substituted benzoguinolizines, such as Wy 25309 (44), Wy 26392 (.45), and
Wy 26703 (46), are structurally related to yohimbine (22) and 48 are more potent and selective as a2~antagonists [8].
0 < 0 H
N h 3c ' 'so2ch2ch2oh OH
R = isobutyl R - n-propyl R = methyl
Combining structural features of yohimbine (£3) and benzoguinolizines have led to the discovery of L-654,284 (£7) .
More recently a berbane derivative CH-38083 (48) has been described as a potent and highly selective a2-antagonist. It is a more potent a2~adrenergic receptor antagonist than idazoxan (£5) and is more potent and a2-selective than Wy
26703 (£6) or yohimbine (£3)• The combination of the structural features of piperoxan (£6), a potent but non- selective a-adrenergic antagonist, and the related compound fenmetazole (£9), weak a2~adrenergic antagonist, led to the design of both idazoxan (£5) and imiloxan (50) [74]. Idazoxan
(15) is one of the most potent and selective a2-adrenergic antagonists. The modification of the benzodioxane and the imidazoline ring results in a reduction or loss of both selectivity and potency [74]. The selectivity and potency of idazoxan analogs are enhanced by certain substituents, such as 49 a methoxy group located at the 2-position of the benzodioxane ring [8,16]. In addition, phentolamine (^9), the classical competetive a-adrenergic antagonist, is potent but non- selective.
Cl
49 50
1.8 PHYSIOLOGICAL FUNCTIONS OF q-ADRENERGIC AGONISTS AND
ANTAGONISTS
The diverse physiological functions mediated by a- adrenergic receptor activation are summarized in Table 3
[8,75]. a-Adrenergic receptors exist in many organs of the body and play a pivotal role in the regulation of a variety of physiological processes, particularly within the cardiovascular system. Both ax~ and a2-adrenergic receptors are located postsynaptically in vascular smooth muscle and stimulation of these receptors by agonists mediates vasoconstriction [8]. The postsynaptic vascular a^receptors are proposed to reside at the neuroeffector junction (i.e., junctional receptors) and interact with endogenous norepinephrine liberated from sympathetic nerves. Therefore, postsynaptic junctional a1-adrenergic receptors can contribute to the maintenance of peripheral arterial tone. In contrast, 50 the postsynaptic vascular a2-receptors are proposed to be located away from the neuroeffector junction (i.e., extrajunctional receptors) and may respond to circulating epinephrine acting as a blood-borne hormone. Thus, postsynaptic vascular a2~adrenergic receptors may play a role in the increased peripheral resistance seen in hypertension disease states in which circulating plasma epinephrine concentrations are elevated at rest and during stress [8,76].
Selective blockade of peripheral vascular postjunctional a1- adrenergic receptors leads to an effective antihypertensive action with a decrease in peripheral resistance, prazosin (5) being the most common drug used for this effect. The myocardial ax-adrenergic receptors mediate a positive inotropic response with little effect on cardiac rate. The mechanism by which cardiac a1~adrenergic receptors increase the force of contraction is not completely understood [77].
The neuronal control of renal excretory functions is mediated via a-adrenergic receptors: renal nerve stimulation potentiates tubular water and sodium reabsorption via innervated a1-adrenergic receptors, whereas a2-adrenergic receptors mediate the opposite effects [8]. In the urogenital system, a1-adrenergic receptors mediate bladder contraction and a mixed population of otj/a., receptors can contract the urethra. In the prostate, aj-adrenergic receptors mediate contraction. The antihypertensive action of a2~adrenergic receptor agonists, such as clonidine (9J, guanfacine and 51
azepexole is supposed to result from stimulation of
postsynaptic a2-adrenergic receptors in the brainstem [78].
Thus, stimulation of postsynaptic a2~adrenergic receptors
gives rise to a decrease in peripheral sympathetic outflow
resulting in a reduction of vascular sympathetic tone and a
reduction in arterial pressure. Central a2~adrenergic receptor
stimulation can also enhance parasympathetic outflow. The
peripheral prejunctional action of a2-adrenergic receptor
agonists does not make a major contribution to the
antihypertensive activity. Centrally-acting a2~adrenergic
receptor agonists produce sedation as a prominent side effect
[8]. The involvement of central a2-adrenergic receptors in
these sedative effects has been proposed because the sedative
action of a2-adrenergic agonists such as clonidine (£),
detomidine (52) was inhibited by a2~antagonists but not a1-
adrenergic antagonists [79-82]. In addition, the duration of
sedation was dose-dependent. Presynaptic a2-adrenergic
receptors are involved in the regulation of transmitter (e.g.,
norepinephrine) release through a negative feedback mechanism
mediated by the neurotransmitter itself. a2~Adrenergic
receptors are also present on platelets, and a2 agonists
induce aggregation [76]. In addition, a2~adrenergic receptors mediate inhibition of insulin release from pancreatic islets and lipolysis in fat cells. While there is clearly a therapeutic role for a2-adrenergic agonists, clinical applications of a2-adrenergic antagonists have not been 52 established. Two major therapeutic areas, depression and diabetes, are currently under investigation [74].
Table 3
Physiological Functions of g-Adrenoceptors
- Smooth muscle contraction - Hypotensive effect via central mechanism - Increased cardiac contractility - Inhibition of postganglionic - Decreased cardiac automaticity norepinephrine release decreasing in the geriatric (presynaptic) increasing in the young - Arterial constriction - Inhibition of renin secretion - Stimulation of platelet - Increased secretion of adrenocor aggregation ticotropic hormone (ACTH) from p ituitary - Inhibition of insulin release from pancreatic islet cells - Bladder contraction - Inhibition of lipolysis - Prostate contraction - Ocular hypotension - Renal nerve stimulation water and sodium reabsorption CHAPTER II
STATEMENT OF PROBLEMS AND OBJECTIVES
2.1 MEDETOMIDINE ANALOGS
Medetomidine (5_1), a new imidazole drug, was synthesized by
Farmos Group Limited (1981) in Finland as part of a research project investigating the pharmacological properties of non- phenethylamine substances acting on the a-adrenergic receptors. Medetomidine is one of the most selective and potent a2-adrenergic agonists known. It has an a2/a1-receptor binding ratio of 5060 as compared to clonidine (£) with a ratio of 969. Oj/a^Selectivity in vitro was studied in receptor binding experiments using rat brain membranes (Table
4) [83]. Medetomidine (jU), a bridge-methylated derivative of detomidine (DomosedanR) (52), is currently used in
Scandinavian countries as a veterinary sedative-anesthetic m drug with analgesic properties [84-87]. Furthermore, it has hypotensive, bradycardic, hypothermic, and mydriatic effects
[84]. Human phase 1 studies have recently been initiated with medetomidine [88,89].
53 IS - T IS-P 55
Table 4
ot/q^Selectivity of Medetomidine and its Optical Isomers with Reference Compounds in Rat Brain Membranes
3H-Clonidine 3H-Prazosin Ct /ct “ displacement8 displacement11 Selectivity IC50, nM IC50, nM
Medetomidine (51) 3.3 16700 5060 d-Enantiomer (d-51) 1.2 55019 45849
1-Enantiomer (1-51) 46 189975 4129
Detomidine (52) 3.7 242 65
Clonidine (£) 6.4 6200 969
“a2-Adrenergic receptor binding assay. a1-Adrenergic receptor binding assay.
The structure of medetomidine (5JL) was modified in the phenyl
ring, the carbon bridge joining the phenyl and imidazole
rings, and imidazole ring. From this structure-activity study,
optimum a2-adrenergic activity was obtained with a 2,3-
dimethylated phenyl ring, methyl substitution at the carbon
bridge between the phenyl and imidazole rings, and an
unsubstituted imidazole ring [90,91].
The first objective of this research was to design and
synthesize a series of medetomidine analogs and compare their 56 relative biological activities to the parent molecule. The proposed analogs would further elucidate the basic structural requirement for a2-adrenergic receptors. The replacement of the 2,3-dimethylphenyl ring system with a naphthyl ring was initially carried out to see if the receptor could tolerate the larger group and if this group might confer increased affinity through enhanced n-n interactions. The naphthyl ring system was also chosen because it was present in naphazoline
(52), a potent a-adrenergic agonist. The replacement of the naphthalene ring system with an indole was proposed to see if such a bioisosteric replacement of a benzene ring was possible.
The angles between naphthalene, indole rings and a carbon bridge containing imidazole ring were determined by molecular modeling after energy minimization using SybylR (Tripos)
(Figure 18).
H H
A: 120.89° A: 127.18° A: 126.22° B: 117.17° B: 105.68° B: 104.19° C: 121.93° C: 127.14° C: 126.16°
Figure 18: The angles between naphthalene, indole rings and a carbon bridge containing imidazole ring using SybylR (Tripos). 57
Thus, the target compounds were 4—[1—(1— naphthyl)ethyl]imidazole hydrochloride (54)# 4-[l-(3- indo1y1)ethy1]imidazo1e (55) and 4-[l~(N- indolyl)ethyl]imidazole (56).
To determine the stereochemical requirement for binding to the a2-adrenergic receptor, the optical isomers of medetomidine were separated [83]. Both d- and 1-enantiomers of medetomidine (51.) are selective and potent a2-adrenergic agonists. Table 5 shows pD2 values of a2-agonists which are able to block electrically-induced muscle contractions by activating the presynaptic a2-adrenergic receptors and thus diminishing the secretion of neurotransmitter [83]. From these experiments, it was shown that the pharmacological activity resided primarily in the d-enantiomer (d-51). The d-enantiomer
(d-51) gives enhanced in vivo a2-selectivity and potency compared to the racemic mixture [92].
The complementarity required between a receptor and a drug molecule may result in a clear distinction between the biological activity of the enantiomers of a chiral drug molecule [93]. Since the d-isomer of medetomidine (d-51) was more potent than 1-isomer (1-51) [83], the second objective of this study was to separate and evaluate the biological activities of (+)-4-[1-(1-naphthyl)ethyl]imidazole tartaric acid (S-57) and (-)-4-[l-(1-naphthyl)ethyl]imidazole tartaric acid (R-JT7) • The absolute configurations of S-57 and R-57 were also determined. 58
Table 5
Comparison of Medetomidine and its Optical Isomers with Reference Compounds in Mouse vas deferens
a2-agonism in vitro
PD2
Medetomidine (51) 9.0
d-Enantiomer (d-51) 9.3
1-Enantiomer (1-51) 6.0
Detomidine (52) 8.5
Clonidine (5?) 8.5
pD2: negative logarithm of the molar concentration of compound producing 50% of maximal inhibition.
CH, JTn> C 0 ,H CH3 U ' > C 0 ,H N N h " T H ■—1— OH H'V H H HO H HO ■ H OH 1 1 +1 H c o 2 h 4C0,H
S-57 R-57 59
A third goal of this research was to begin a structure- activity relationship study of the naphthalene compound 54 due to its potent and selective a2-adrenergic activity. Since the methyl group added to the benzylic carbon (detomidine to medetomidine) gave an enhanced activity, and since we have observed large differences in activity between enantiomers, we have chosen to extensively investigate the carbon bridge between the naphthalene and imidazole rings. A variety of functional groups were placed at this position to find optimum activity. Modifications to 54 included the removal and replacement of the methyl group with methoxy and more polar hydroxyl group, conversion of the benzylic position to a ketone. The methyl group was also replaced with the more lipid-soluble and electron withdrawing trifluoromethyl group.
It is known that the addition of fluorine substituents has a dramatic effect on adrenergic agonists [67-69]. The introduction of a trifluoromethyl group might increase CNS vs. peripheral adrenergic activity. The trifluoromethyl group is the highly electron withdrawing group: the am and ap values for a methyl substituent are -0.07 and -0.17, respectively, whereas for the trifluoromethyl group, the om and ap values are
0.43 and 0.54, respectively [94]. Thus, the trifluoromethyl substituent 62 may lower the pKn of imidazole ring, causing it to become less ionized as compared to the methylated compound
54. Furthermore, the trifluoromethyl group (rcCF3 = 0.88) is more lipophilic than the methyl (n^ = 0.56) [95]. Thus, 62 60 should penetrate the blood brain barrier more readily than the parent compound 54. The proposed target compounds were 4-[(l- naphthyl)methyl]imidazole hydrochloride (58), 4—[(1— naphthyl)hydroxymethyl]imidazole hydrochloride (59), 4—[(1— naphthyl)carbonyl]imidazole oxalate (£0), 4—[1—(1— naphthyl )methoxymethyl] imidazole hydrochloride (61.), and 4 — [ 1—
(1-naphthyl)-2,2,2-trifluoroethyl]imidazole hydrochloride
(62).
i/2 (COOH) 2
62 61
2.2 PHENTOLAMINE ANALOGS
Phentolamine (1£) was discovered during a search for an
antihypertensive "a-blocker" by Urech et al [96]. It is a
member of the imidazoline class of adrenergic drugs.
Phentolamine (_19) is known primarily as an antagonist of o-
adrenergic receptors and is thought to be equipotent on ax-
and a2-adrenergic receptors. Over the years it has been used
in a number of clinical situations and is now used in patients
with pheochromocytoma or in some patients to avoid tissue
slough in instances of inadvertent infiltration of
norepinephrine [97]. Pheochromocytoma is a tumor usually found
in the adrenal medulla that releases a mixture of epinephrine
and norepinephrine [98]. Patients with a catecholamine excess
show many signs including hypertension, tachycardia, and
arrhythmias. Phentolamine (lj)) is not useful in treatment of
systemic hypertension and in patients with myocardial
infarctions since it induces cardiac stimulation. This side
effect is thought to be caused by presynaptic a2~blockade, which results in enhanced neuronal release of norepinephrine.
The released norepinephrine activates a-receptors in the heart
producing cardiac stimulation [97,99]. Having a non-selective
a-adrenergic antagonist, it was our goal to see if isosteric analogs of phentolamine in which a nitrogen atom was replaced by carbon would give increased selectivity. Such isosteres would also allow us to prepare optical and geometrical isomers. 62
The term "bioisosterism" has been widely used to describe the selection of structural components whose steric, electronic and solubility characteristics make them more or less interchangeable in drugs of the same pharmacologic class.
The idea of isosterism was introduced by Langmuir [100] in
1919 as an explanation for similarities in chemical and physical properties of nonisomeric molecules [102]. Isosteres were described as molecules or molecular fragments containing the same number of atoms and valence electrons. Isosteres which are isoelectric (containing the same total charge as well as same number of electrons) often possess similar physical properties. For example, N2 and CO, each with 14 electrons and no net charge, have similar physical properties.
The term "bioisosterism" was introduced by Friedman in 1951 to describe the phenomenon of structurally similar compounds found to produce similar or antagonistic biological properties
[102]. The broadest definition of bioisosteres may be used with functional groups or molecules which have chemical and physical similarities producing similar biological properties
[103]. There are two types of isosteric substitutions; classical and non-classical isosteres. Classical isosteres have similar peripheral electronic arrangements and size.
Table 6 shows examples of classical isosteres. 63
Table 6
Functional Groups Used as Isosterlc Replacements
—N= - o - -F
“CH= -NH- -OH
-CHr -n h 2 -s- -CH.
"Non-classical isosteres" are functional groups that are not structurally or electronically similar. In making a non- classical isosterlc replacement, consideration must be given to the functional group parameters that are being changed during isosteric substitution. These include, size, shape
(bond angles, hybridization), electronic distribution
(polarizability, inductive effects, charge, dipoles), solubility (lipid, water), pKa, chemical reactivity (including metabolism), and hydrogen bonding capacity [102]. It is unlikely that any isosteric replacement will leave all of these parameters unchanged. The isostere that affects the functional group parameters in a desirable fashion in a drug's activity will be the most effective isostere. Usually, the way in which an isosteric substitution will affect a drug's activity is not predictable. The determination of an 64 isostere's effect on biological activity will be part of the structure-activity study. Consequently, isosteric substitutions are used to develop more potent and less toxic drugs, to develop enzyme inhibitors from known substrates, and receptor antagonists from known agonists. Furthermore, it can be used to improve selectivity, absorption, and duration of action.
The isosteric analogs of proposed herein should provide knowledge as to stereochemical demands for the interaction of antagonists with o^- and a2-adrenergic receptors. A better understanding of the structural requirements for the subtypes of a-receptors could result in more selective drugs treating nasal decongestion, hypertension, hypotension, hyperglycemia, depression, liver cell proliferation and hyperaggregability of platelets. The investigation of the stereoselectivity or stereospecificity of phentolamine (^9) analogs at a-adrenergic receptors has been rather limited. This is due in part to the fact that a number of a-adrenergic antagonists do not possess chiral centers. Thus, we proposed the preparation of the following compounds: 2-[2-(3-hydroxyphenyl)-2-(4- methylphenyl)ethyl]imidazoline hydrochloride (63), 2-[2-(3- hydroxyphenyl)-2-(4-methylphenyl)-(Z)-ethenyl]imidazoline hydrochloride (Z—64), and 2-[2-(3-hydroxyphenyl)-2-(4- methylphenyl)-(E)-ethenyl]imidazoline hydrochloride (E-64). 65
Z-64 E-64 CHAPTER III
RESULTS AND DISCUSSION
This chapter is divided into four parts. The first part involves a discussion of the synthesis of naphthalene analogs of medetomidine. The second part involves a discussion of the resolution of the 4-[1-(1-naphthyl)ethyl]imidazole enantiomers. The third section is a discussion of the synthesis of indole analogs of medetomidine. The fourth part involves a discussion of the synthesis of phentolamine analogs.
3.1 CHEMISTRY
3.1.1 Synthesis of naphthalene analogs of medetomidine
There are a variety of synthetic methods to form an imidazole ring, few of which have wide applicability, and in many instances the mechanism is poorly understood. For example, some syntheses almost certainly involve concerted cycloaddition reactions, but evidence for this process is lacking. In addition, numerous examples have appeared of conversions of other heterocyclic compounds into imidazoles.
66 These synthetic methods require specific functional groups and
other heterocyclic rings, such as furan, which can be
converted to an imidazole ring system. Generally, these methods include many steps and result in a poor overall yield.
One method used to introduce the imidazole ring system into a molecule is to generate an anion on the imidazole ring and allow it to carry out a nucleophilic reaction on compounds containing electrophilic functional groups. These methods use
fewer steps and provide relatively good overall yields as compared to methods requiring the construction of an imidazole ring. Recently, Karjalainen et al [90] used TMS-imidazole in the presence of titanium tetrachloride in a reaction with halogenated compounds to introduce an imidazole ring system.
This reaction provides low yields, but the imidazole ring is introduced in only one step and ambiguity in assigning the substitution pattern is eliminated. Another approach is to use an imidazole ring system which contains an electrophilic functional group such as an aldehyde, or ester. These two methods were used to prepare the 4-substituted imidazole target compounds.
The 4-[1-(1-naphthyl)ethyl]imidazole hydrochloride (54) was synthesized as shown in Scheme I. 1-Naphthylaldehyde (65) was treated with methylmagnesium iodide in diethyl ether to give an alcohol in 98% yield followed by the treatment of thionyl chloride in toluene to give the crude chloro compound
66 in 97% yield [104,105]. The chloro compound 66 was treated 68 with TMS-imidazole in the presence of titanium tetrachloride using chloroform as the solvent to provide the 4-substituted
imidazole [87]. Treatment of the imidazole with dilute HC1 in methanol gave the desired imidazole hydrochloride salt 54.
Scheme I
Synthesis of 4-f1-(1-naphthyl)ethyl1 imidazole hydrochloride (54)
1) TMS-imidazole 1) MeMgl TiCl CHC1.
2) S0C1 2) dil. HC1 toluene
65 66 54
The synthetic scheme leading to the synthesis of the 4 — [ 1 —
(naphthyl)methyl]imidazole hydrochloride (58) is shown in
Scheme II. For the synthesis of 58, a new reaction scheme was used. 1-Bromonaphthalene (67J was treated with magnesium turnings to form a Grignard reagent which was condensed with imidazole carboxaldehyde 68 to give alcohol 6£ in 75% yield
from the aldehyde. In our research, the condensation reaction was unsuccessful with an unprotected 4-imidazole carboxaldehyde. Furthermore, when scale-up was attempted, the yield of product 69 was low and the separation was difficult.
The best reaction conditions for this step used 4.14 mmol of 69
68, 3 equivalents of 1-bromonaphthalene (ST), and 3.1 equivalents of magnesium turnings. Compound 68 was synthesized by the method of Kelly et al (Scheme III) [106].
Scheme II
Synthesis of 4-[1-(1-naphthyl)methyl1 imidazole hydrochloride (58)
1) EtgSiH I I CFgCOOH H CH0C1, ^ |] • HC1
CPh3 68
Scheme III
Synthesis of 4-(N-triphenylmethyl)imidazole carboxaldehyde (6 8 )
PhgCCl 0
H 0 " Y n> Et3N 2 N DMF ^*N dioxane H • HCl c?h3 CPh3
70 71 68 70
The triphenylmethyl group in 6jJ was assigned to the nitrogen atom across the ring from the carbonyl group using the method of Matthews et al [107]. The aromatic protons of 1,4- and 1,5- disubstituted imidazoles show the protons on the imidazole coupled to each other across the ring. These cross-ring coupling constants are designated J2 5 and J2 4, respectively.
J25 was larger than J24 and measured in the range of 1.1-1.5
Hz, while J, . measured in the range of 0.9-1.0 Hz [107]. The 2 14 imidazole 68 was accordingly assigned as a 1,4-disubstituted imidazole because the NMR signals at a 7.61 (doublet) and 7.53
(doublet) were coupled with J = 1.2 Hz. The hydroxy imidazole
69 was treated with triethylsilane and trifluoroacetic acid in methylene chloride to give the dehydroxylated and N- deprotected imidazole as a free base after sodium bicarbonate workup in 65% yield, which was treated with dilute HC1 in methanol to give the HC1 salt 58 in 60% yield [108].
Direct deprotection of £9 also gave 4—[1—(1— naphthyl)hydroxymethyl]imidazole hydrochloride (5SJ) (Scheme
IV). Treatment of 69 with 2N hydrochloric acid at refluxing temperature for lh followed by basic workup afforded the free base in 50% yield, which was converted to the hydrochloride salt 59 in 57% yield by treatment with dilute HC1 in methanol.
Synthesis of 4-[(1-naphthyl)carbonyl]imidazole oxalate (60) is outlined in Scheme V. The imidazole alcohol 69_ was treated with manganese oxide in methylene chloride to give oxidized imidazole 72. in 82% yield, which was further treated with 2N HC1 followed by base work up to give the free base in 54% yield. The free base was converted to the oxalate salt 60 in
82% yield.
Scheme IV
Synthesis of 4-[1-(1-naphthyl)hydroxymethyl1 imidazole hydrochloride (59)
CPh, N /> HO HO N
1) 2N HC1 • HC1 2) dil. HC1
69 59
Scheme V
Synthesis of 4-f(1-naphthyl)carbonyl1 imidazole oxalate (60)
CPh, ?Ph2 N /> HO N MnO 1) 2N HC1 ------p CH-Cl 2) (COOH). (COOH)
69 72 60
Scheme VI outlines the synthesis of 4—[1—(1— naphthyl)methoxymethyl]imidazole hydrochloride (61,). In earlier attempts, treatment of imidazole 59, as a free base, 72 with thionyl chloride and methanol gave extensive decomposition. In addition, when the imidazole was treated with HC1 gas or a concentrated HC1 solution in CH3OH, three unidentified products were evident by TLC. However, the imidazole hydrochloride salt 59 reacted with thionyl chloride in chloroform to give a chlorinated compound which was treated with methanol without further purification to give methoxy imidazole 61.
Scheme VI
Attempted synthesis and synthesis of 4 - f 1 - (1 -naphthyl) methoxymethyl]imidazole hydrochloride (61)
HO
1) S0C1. decomposition N 2) CH,OH
CHgOH HC1 gas 3 products or CH3OH C-HCl
N '> "> HO N CH-0 N H H 1) S0C1. • HC1 HC1 2) CH-OH
59 61 73
The attempted synthesis of the trifluoromethyl-substituted analog 62 is outlined in Scheme VII.
Scheme VII
Attempted synthesis of 4-fl-(l-naphthyl)-2,2,2-trifluoroethyl1 imidazole hydrochloride (62)
CPh CPh,
N TMS-CF, 73 H HC1
72 74 62
1) 2N HC1 2) dil. HC1
HC1
75
The imidazole ketone 72 was treated with TMS-CF3 in dry tetrahydrofuran in the presence of tetrabutylammonium fluoride to give 21 in 75% yield. TMS-CF3 1_3 was made by the method of
Olah et al [109,110]. A large amount of tetrabutylammonium fluoride (almost 8 equivalents) was needed to obtain complete reaction. Reduction of the tertiary alcohol geminal to the trifluoromethyl group in 21 has not been successful with 74 triethylsilane in trifluoroacetic acid, hydrogenation under a variety of conditions, NaBH4, or Li/NH3 (Scheme VIII).
Scheme VIII
Attempted methods to remove a tertiary hydroxy group
CPh,
HO
74 H2, Pd/C
CF3COOH
H2, Pd black CF3COOH
H 2/Pt02 CF3COOH -► decomposition
'> N 4 HO NaBH H CF,COOH
Li/NH, -► decomposition 75
Under these conditions, only the triphenylmethyl protecting
group was removed or decomposition was evident. Both steric
and electronic effects may prevent the reduction of tertiary
alcohol 74. A method for the reduction of tertiary alcohols
geminal to trifluoromethyl groups has not been reported in the
literature. The imidazole hydrochloride 75 is being examined
for its biological effects.
3.1.2 Resolution of 4-[l-(l-naphthyl)ethyl]imidazole
enantiomers.
There are two principle methods used to separate
enantiomers: conventional fractional crystallization and chiral chromatography. In the separation of the 4-[l-(l- naphthyl)ethyl]imidazole enantiomers, it was possible to use either a fractional crystallization or chiral high performance
liquid chromatography (HPLC). Fractional crystallization was chosen because of the large quantities of each isomer needed
for in vivo testing. Also, the free base of the imidazole was poorly soluble in the mobile phase required by the preparative chiral HPLC column, and the method was therefore not suitable for preparation of large quantities of material. HPLC using a chiral analytical column (Chiralcel 0DH) was used for the determination of optical purity of each enantiomer. Racemic
4-[1-(1-naphthyl)ethyl]imidazole was separated into the enantiomers by conversion of the racemate to a mixture of diastereoisomeric salts using the isomers of tartaric acid and separating them by fractional crystallization. The salt formed
by the addition of 1.02 molar equivalents of ( + )-tartaric acid
to a solution of the racemic mixture in methanol (Scheme IX)
gave (+)-enantiomer after 14 crystallizations from methanol
and a mixture of methanol/ethanol 50 : 50. The remaining salt
was converted back to the free base by treatment with sodium
hydroxide followed by extraction with methylene chloride. The
other diastereomeric salt was formed with (-)-tartaric acid,
and (-)-enantiomer was also isolated through 11
crystallizations.
The second approach to the separation of enantiomers of 54 was a chromatographic separation using HPLC. The racemic mixture was separated using an analytical Chiralcel OD*
(Diacel) column. This chiral stationary phase (CSP) is a cellulose tris (3,5-dimethylphenylcarbamate)polymer. The chromatographic separation of enantiomers on Chiral HPLC columns involves the formation of diastereomeric complexes between the enantiomers and the CSP [111,112]. For the enantiomers to be resolved, there must be a difference in the stability of the resultant diastereomeric complexes [111,112].
The Chiralcel ODR column was chosen because a naphthalene- containing compound, propranolol, has been resolved on this same column [113]. Using a mobile phase of hexane/isopropanol
90 : 10, the first enantiomer (+ isomer) was eluted at 9.87 min and the other enantiomer (- isomer) was eluted at 16.69 min (Figure 19). The optical purity of each enantiomer was 77
determined using the same conditions described above.
Chromatograms of each enantiomer are shown in Figure 20, the
other enantiomer was not detected. To confirm the absolute
configuration of the isomeric imidazoles (+)-57 and (-)-57# x-
ray analysis of the tartaric salt of (+)-57 was first
attempted. This salt did not provide suitable crystals for x-
ray analysis; however, suitable crystals for x-ray analysis
were obtained with the (+)-dibenzoyl-D-tartaric acid salt of
( + )~52_' Based on the known absolute configuration of ( + )-
dibenzoyl-D-tartaric acid, with both asymmetric centers having
the S absolute configuration, it was determined that the
asymmetric ring junction between naphthalene and imidazole
rings of (+)-57 had the S configuration as shown in Figure 21.
Thus, the (-)-enantiomer should have the R configuration. Scheme IX
olution of 4-[1-(1-naphthyl)ethyl 1 imidazole
c o 2h
H —t -OH HO — |— H (+)-tartaric acid N CO-H '> N t S-57, [a]D +56.6C H CH3OH
ch3 II CO_H HO — I— H (-)-tartaric acid H — I— 0]OH CO„H
R-57, [a]n -56.0°
'> c. o 2.h N H PhOCO -J — H H — I — OCOPh
c o 2h
76, [a]D +105.2° 79
CH
CH
S(+)-isomer 9.87
16.69 R(-)-isomer
0 10 20 30
retention time (min)
Figure 19: HPLC chromatogram for the racemic mixture of 4-[1-(1-naphthyl)ethyl]imidazole. 80
CH.
9.87
S(+)-isomer
CH.
16.69
R(-)-isomer
0 10 20 30
retention time (min)
Figure 20: HPLC chromatograms for each enantiomer of 4- [1-(1-naphthyl)ethyl]imidazole. Figure 21: ORTEP representation of (S)-4-[l-(l-naphthyl) ethyl]imidazole. oo 82 3.1.3 Synthesis of Indole analogs of medetomidine
The molecular orbital description of indole is very similar to that of pyrrole, the only added feature being the distribution of 10 n-electrons over the cyclic framework instead of the six. From the resonance hybrids (Figure 22), structures a-c are more important than d and e in which benzenoid resonance has been destroyed and charge separation is large [114].
Figure 22: Resonance hybrids of indole. 83
The increased electron density at the 3-position of indole may
be contrasted with the increased negative character at the 2-
position of pyrrole. From theoretical considerations, the
electrophilic substitution of indole should occur at the 3- position and this is generally observed.
Nucleophilic reactions of indoles have been little studied and in general are not well understood. The N-H proton of
indole, being appreciably acidic, is readily abstracted by
such reagents as metallic sodium, potassium hydroxide at
elevated temperatures, n-butyllithium, and Grignard reagents.
The reactivity of the resulting anionic substances appears to vary with the nature of the metal ion, the experimental conditions, and the nature of the co-reactant. Indole derivatives react at the 1- and/or 3-positions. As a general rule, sodium, potassium, and lithium salts yield predominantly
N-alkylation products, whereas magnesium salts of indole lead to substitution at the 3-position, although many exceptions are known [114]. Recently, Saulnier et al [115] reported halogen-metal interconversion reactions at the indole 3- position. They used 1-(phenylsulfonyl)-3-iodoindole to avoid competitive N- and C-3-substituted products.
Scheme X shows an attempted C-3 alkylation of indole.
Indole (7J7) in diethyl ether treated with methylmagnesium bromide and then quenched with aldehyde 6J5 yielded only the starting materials. In this reaction, a new product was evident by TLC. However, aqueous workup converted this product 84
back to the starting materials.
Scheme X
Attempted synthesis for C-3 alkylated indole
CH3MgBr 00 '> MgBrO 00 * N (c2h5)2o H CPh, Cl CPh.
77 68
0
77 68
This synthetic scheme was designed using the knowledge of
indole chemistry previously mentioned: magnesium salts of
indole generally lead to substitution at the 3-position. In
this case, however, reacting the magnesium salt of indole with
aldehyde 6jJ apparently leads to substitution at the 1-position
instead of the 3-position. For further research into indole
substitution patterns, the imidazole ester 78 was allowed to
react with indole (Scheme XI). Indole (77) in diethyl ether was treated with methylmagnesium bromide to form
indolemagnesium bromide, and then quenched with imidazole 85 ester 78. The product was found to be the N-acylated compound
79. Under these experimental conditions, the magnesium salt of indole provided substitution at the 1-position instead of the
3-position. The imidazole ester 78 was synthesized by the method of Cohen et al (Scheme XII) [116].
Another approach to the N-l alkylation was to use a haloalkyl imidazole (Scheme XIII). Indole (22.) in diethyl ether was treated with methylmagnesium bromide and then treated with the bromoethyl imidazole 84 which was prepared from alcohol j[3 with triphenylphosphine and carbon tetrabromide. Alcohol 83 was synthesized by the method of
Kelly et al (Scheme XIV) [106]. Under these conditions, the bromoethyl imidazole 84 was converted through elimination to conjugated imidazole 85, which was confirmed by lH-NMR.
Unreacted indole was also isolated.
Scheme XI
Another approach for C-3 alkylated indole
77 78 79 86
Scheme XII
Synthesis of ethyl 1-tritylimidazole 4-carboxylate (78)
HOOC COOH HOOC EtOH Et02C f = \ i) Ac,o r = \ h 2so4 f = \ .N-H ------► .N*H------► m *H V 2) H-0 V V
80 81 82
Ph,CCl Et02C y = \ N ^ N - C P h 3 DMF
78
Scheme XIII
Attempted synthesis for C-3 alkylated indole with a haloalkyl imidazole 00 •'*0-S?G0 H CPh3 H CPh3
71 84 77 85 87
Scheme XIV
Synthesis of a bromoethyl imidazole
OH Ph3P Br H CH3MgBr CBr, ► \ N> \T-> THF .'Vs^ XT V:N (C2H 5)20 "Y< N CPh, CPh, CPh,
68 83 84
Scheme XV shows the synthesis of the desired C-3 alkylated
indole. l-(Phenylsulfonyl)-3-iodoindole (86) was synthesized
by the method of Saulnier et al [115]. The iodoindole 86. in
dry tetrahydrofuran was treated with t-butyllithium at -78°C
for lithiation at the C-3 position. The 3-lithio-l-(phenyl-
sulfonyl) indole was quenched with the imidazole aldehyde 6£ to
give the c - 3 alkylated indole 82 in 61% yield from the
aldehyde. The indole alcohol was oxidized with manganese
oxide in methylene chloride to give ketone 88 in 85% yield.
The proposed remaining steps to the desired product 55 are shown in Scheme XVI. 88
Scheme XV
Synthesis of C-3 alkylated indole
CPh,
1) t-BuLi 1) n-BuLi, I2 1 THF ^ ^ \ \ THF ► l ^ N> --- :7*°c » N 2) PhS02Cl ^ 2) o H LDA S02Ph S02Ph
N 77 86 CPh, 87
68 CPh,
MnO.
CH2C12 SO-Ph
88
Attempts at preparing the nonmethylated analog 89 rather then the target compound also failed. In the attempted reductions of the ketone 79 using either borane or LiAlH4, only unreacted starting material was isolated (Scheme XVII).
The ketone imidazole 90 was obtained and submitted for biological testing. 89
Scheme XVI
Proposed synthetic scheme for 55
CPh CPh, HO CH k 2c o 3 CH3MgBr
c h 3oh S02Ph SOjPh
1) h 2, Pd/C CF.COOH
55 90
Scheme XVII
Attempted reduction of amide 79 to amine
BH. • THF, il ' ) - 3 / ► N or LiAlH CO
» ■no N 79 CPh3 CPh3
1) 2N HC1 1) 2N HC1 2) dil.HCl 2) dil. HC1 CO CO
!-> H •HC1 H HC1
90 89
3.1.4 Synthesis of phentolamine analogs
The initial synthesis of the carbon analog 63 of phentolamine (JL9) began with esterif ication of 3- hydroxybenzoic acid (91^) with methanol in the presence of excess HC1 gas to give ester 92 in 90% yield (Scheme XVIII)
[117]. The ester !J2 was treated with potassium carbonate and benzyl chloride in acetone to give a benzylated £3 in 89% yield which was treated with potassium hydroxide in water to give benzoic acid 94 in 96% yield [118,119]. The benzoic acid 91
Scheme XVIII
Synthesis of 2-f2-(3-hydroxyphenyl)-2-(4-methylphenyl) ethyl]imidazoline hydrochloride (63)
CH_OH PhCH2Cl OCH„Ph pr HC1 gas pr" KjCO3 * pr COOH COOCH3 acetone COOCH
91 92 93
KOH OCH2Ph soCl. OCH-Ph H„0 pr CC1, pr COOH COC1
94
MgBr OCH2Ph
(EtO)2POCH2CN CH- NaOEt CHCN THF
CH
95 96 92
Scheme XVIII
Synthesis of 2-[2-(3-hydroxyphenyl)-2-(4-methylphenyl) ethyl 1 imidazoline hydrochloride (63) (continued)
OH
H2, Pd/C CHCN CHCN CHCN EtOH Acetone
CH 3 ch3 CH3
96 97 98
OCH.Ph
CHgOH NH-HC1 HC1 gas >-OCH CH-Cl
ch2ci2 2) (COOH) • (COOH)
CH 3 CH3
99
OH
1) H 2 ,Pd/C CH-OH ► 2) sat. NaHC03 • HC1 3) HC1 gas
CH3
63 93
94 in carbon tetrachloride was treated with thionyl chloride
in the presence of dimethyl formamide to give the acyl
chloride, which was condensed with p-tolylmagnesium bromide in
tetrahydrofuran to give ketone £5 in 66% yield. The ketone 95
was treated with diethylcyanomethyl phosphonate in the
presence of sodium metal in absolute ethanol to give nitrile
96 in 97% yield [120]. Selective reduction of the double bond
in £6 using a sodium borohydride was not successful. Catalytic
hydrogenation of £6 gave benzyl-deprotected saturated compound
97 in 70% yield. Phenol £7 was treated with potassium
carbonate and benzyl chloride in acetone to give the benzyl-
protected £8 in 82% yield. Conversion of nitriles to
imidazolines via the Pinner reaction has been well described
by Bristow [121] and Miller et al [122-124]. The nitrile £8 was next converted to the iminoacetate with methanol and HC1
gas in 71% yield by Pinner reaction. The iminoacetate
contained some impurites and without further purification the
iminoacetate was allowed to react with ethylene diamine to yield the imidazoline (75% yield), which was converted to the
oxalic acid salt ££ in 81% yield. The benzyl protecting group was removed by catalytic hydrogenation to give phenolic
imidazoline in 76% yield which was converted to the
hydrochloride salt £3 in 88% yield.
For the synthesis of geometric isomers, Z-64 and E-64, the
nitrile intermediate £6 (Scheme XVIII) was used. The nitrile
96 could not be converted to the iminoacetate under Pinner 94 reaction conditions (Scheme XIX).
Scheme XIX
Attempted conversion of nitrile 94 to an iminoacetate
OCH2Ph
CHgOH HC1 gas CHCN no reaction (C2H5)20
CH 3
96
In another approach, the Reformatsky reaction using activated zinc and bromoacetonitrile gave a product mixture which was difficult to separate (Scheme XX). Similarly, in the third approach using a modified Wittig reaction, triethyl phosphonoacetate in the presence of NaH gave a mixture of the starting material and product which was also difficult to separate (Scheme XX). However, the Reformatsky reaction using bromoethylacetate with activated zinc in benzene was successful and gave the hydroxylated ester 100 in 84% yield
(Scheme XXI) [125]. Several methods are used to prepare activated zinc metal for use in a Reformatsky reaction. Zinc dust was activated by washing with 2% hydrochloric acid, then with water, methanol, acetone, and absolute ether. The treated zinc was dried in a vacuum for 30 min and used immediately
[126,127]. Scheme XX
Attempted synthesis to introduce nitrile and ester functional groups
BrCH2CN Zn ------► separation problem benzene with low yield
95
(EtO)2POCH2COOEt NaH " ► separation problem THF
Hydroxylated ester 100 was treated with p-toluenesulfonic acid monohydrate in benzene to give the unsaturated ester 101 as a mixture of geometric isomers. The unsaturated ester 101 was treated with trimethylaluminum and ethylenediamine in toluene to give the imidazoline as the free base in 70% yield, which was converted to the oxalate salt [128]. A mixture of geometric isomers was obtained and separated by fractional crystallization to give Z-102 and E-102 isomers in 34% and 31% yield, respectively. The absolute configuration of one isomer
(Z-102) was determined by X-ray crystallography (Figure 23).
The Z-102 was treated with 50% hydrochloric acid solution in methanol to give the imidazoline hydrochloride salt Z-6£ in
84% yield. However, the benzyl protecting group of the other isomer (E-isomer) was not removed under these same conditions. The E-102 was treated with 10% sodium bicarbonate solution to give the free base which was treated in turn with boron trichloride to give the debenzylated hydrochloride salt E-64 in 65% yield. 97
Scheme XXI
Synthesis of Z-64 and E-64
OCH2Ph OCH-Ph OCH,Ph
BrCH-COOEt CH OH COOEt benzene COOEt
CH CH
95 100 101
OCH_Ph 1)H2N NH. (Me)3Al" NH toluene • (COOH)
2) (COOH), • (COOH) NH
CH CH
Z-102 E-102
1) 10% NaHCO;
HC1/CH-OH 2) BC13 ch2ci2
OH OH
NH
• HC1 NH
CH CH
Z-64 E-64 032
Figure 23: ORTEP representation of Z-102 ot> 99
3.2 BIOLOGY
Medetomidine (51.) and 4-[l-(1-naphthyl)ethyl]imidazole hydrochloride (54) have been evaluated for comparative activities on a1- and a2-adrenergic receptors in rat aorta and human platelets, respectively (Table 7). Complete experimental details are reported elsewhere [129,130]. For studies of a 2~ adrenoceptor antagonism, analogs were incubated with human platelets in the presence of an optimal concentration of epinephrine [e.g., 10 j/M (-)-epinephrine] which gave a maximal primary wave aggregation response. In all experiments, platelets were pretreated with aspirin (1 mM) in order to study the effects of the drugs on the primary wave (a2- receptor mediated phase) of aggregation.
On rat aorta, the percentage of agonist response shown in
Table 7 is calculated relative to the maximum response produced by the reference agonist, phenylephrine. Medetomidine
(51) is a partial agonist with a maximum response of 42% and a pEC50 value of 6.51, while medetomidine analog 54 is also a partial agonist with a maximum response of 27% and a pEC50 value of 6.67. On the c^-adrenergic receptor (rat aorta), the pEC50 values of S4 and medetomidine (51) are very similar; however, the intrinsic activity is different. Analog 54 only produces a 27% maximal contraction whereas 51^ produces a 42% maximal effect. Using human platelets as an a2~adrenoceptor system, neither 51^ nor 54 produced a detectable increase in aggregation response at concentrations of up to 300 jjM (Table Table 7
Comparison of Adrenergic Activities of Medetomidine (51) and Analog 54 in Rat Aorta and Human Platelets
Rat Aorta (ax) Human Platelets (a2)
Potency Potency Compound Maxima (%)b pIC50 + SEMd PEC50 + SEMa Ratioc Ratio®
Medetomidine 6.51 + 0.004 (4) 42 ± 7 1.0 5.48 ± 0. 20 (4) 1.0 (51)
54 6.67 ± 0.141 (4) 27+3 1.4 5.47 + 0.07 (4) 0.97
apECS0 = -log ECS0 where EC_0 is equal to the molar concentration of compound which produces 50% of maximum response. bData are expressed as the percent maximal analog response relative to phenylephrine (100%). °Potency ratio = EC50(medetomidine)/EC50(analog). dpIC50 = -log IC50 where IC50 is equal to the molar concentration of compound which inhibits the response of epinephrine-induced aggregation by 50%. Inhibitor was added 1 min prior to epinephrine (10 pM) in human platelet-rich plasma. Aspirin (1 mM) was included for determination of primary wave aggregation. ePotency ratio = IC50 (medetomidine)/ IC5 0 (analog). fValues in parentheses indicate the number of experiments (n). 101
7). However, both medetomidine (51) and analog 54 are concentration-dependent inhibitors of human platelet aggregation induced by epinephrine. Medetomidine (51) and analog 54 gave pIC50 values of 5.48 and 5.47, respectively.
These results indicate that replacement of the dimethylphenyl group with a naphthalene ring may lead to a loss of a1- adrenoceptor agonist activity (rat aorta) whereas these compounds retain comparable a2-adrenergic antagonist activity
(human platelets).
The enantiomers of 54 [S(+)-57 and R(-)-571 were evaluated for their a2~adrenergic activities in human platelets and compared to that for the enantiomers of medetomidine (51)
(Table 8). The pICS0 values found for S(+)-57 and R(-)-57 are
5.21 and 6.04, respectively, whereas the corresponding values for ( + )- and (-)-medetomidine are 5.65 and 5.44, respectively.
Thus, R(-)-57 is about 9-fold more potent than S(+)-57 as an inhibitor of epinephrine-induced platelet aggregation. In contrast, (+)-medetomidine is only about 2-fold more potent than (-)-medetomidine.
In another set of experiments, we also evaluated analog 54 and enantiomers [S(+)-57 and R(-)-571 for ax- and a2-adrenergic functional activities in guinea pig ileum preparations (Table
9). Oxymetazoline and phenylephrine were employed as reference compounds. Complete experimental details are reported elsewhere [131-133]. Racemic 54 and enantiomers [S(+)-57 and
R(-)-57] do not show high intrinsic activities as agonists of Table 8
Inhibition of Epinephrine-Induced Human Platelet Aggregation by Compounds 54, S(+)-57, and R(-)-57
Platelets (a2)
Compound P I C 50 ± SEM"
54 5.27 + 0.22 <6)b
S(+)-57 5.21 ± 0.32 (5)
R(-)-57 6.04 + 0.20 (5)
(+)-Medetomidine (d-51) 5.65 ± 0.12 (5)
(-)-Medetomidine (1-51) 5.44 + 0.07 (5)
ttpIC50 = -log IC where IC is equal to the molar concentration of compound which inhibits the response by 50%. Inhibitors were added 1 min prior to epinephrine (10 pM) in human platelet-rich plasma. Aspirin (1 mM) was included for determination of primary wave aggregation. Values in parenthesis indicate the number of experiments (n).
^-adrenergic receptors in guinea pig ileum. In functional
assays on electrically stimulated guinea pig ileum (a2-
adrenoceptor model), R(-)-57 shows no intrinsic activity and
S(+)-57 shows enhanced a2-agonist activity compared to the racemic 54.
It may be possible to differentiate subtypes of a 2~ adrenergic receptors using enantiomers of 54 because two different systems (guinea pig ileum and human platelets) show different biological properties. On human platelets R(-)-57 has a much higher affinity than S(+)-57, whereas only S(+)-57 Table 9
Comparison of Adrenergic Activities of Compounds 54, R(-)-57, S(+)-57, and Reference Compounds
Ileum (ai) Ileum (a2)
Compound EC50 (nM)a E max (%)b' ' ECSO W (%)"
54 15982 24 9 73
R(-)-57 10814 25 c c
S(+)-57 10635 50 11 84
Oxymetazoline 2028 55 125 87 (22) Phenylephrine 11788 88 62940 62 (1) aEC50 is the concentration which produces 50% of maximum response. ba1-Receptor efficacy was determined in isolated guinea pig ileum (contractile response) using (-)-epinephrine as a control. a2-Receptor efficacy was studied in field-stimulated guinea pig ileum model (supra-maximal voltage, 0.1 Hz, 0.5 msec) using UK-14304 as a control. cNo activity up to 100 pM. 104 has intrinsic activity in guinea pig ileum. The analogs of 54, which were synthesized as part of a structure-activity relationship study, were tested for their ct2-adrenergic activities in human platelets and compared with racemic 54
(Table 10). Most imidazolines and imidazoles such as medetomidine possess inhibitory rather than stimulatory activities for platelet adrenergic receptor sites (a2A subtype)
[45]. With the exception of the desmethyl compound 58, none of the other analogs of 54 had enhanced a2-adrenergic activity in the structure-activity relationship study. An interesting result from these studies is that the activity of the desmethyl compound 58 against a2-adrenoceptor-mediated aggregation in platelets is enhanced 3-fold relative to racemic 54. This result is in contrast to biological results previously reported for medetomidine (51^) [83] using rat vas deferens (a2); in this case, a non-methyl containing compound
52, detomidine, showed less activity than the racemic mixture of medetomidine (51j.
In these studies, the naphthalene analogs of medetomidine
(51) do not follow the same biological activity patterns as medetomidine (51): 1) R(-)-57 is more potent than S(+)-57 on human platelets (a2A system); 2) the desmethyl compound 58 is more potent than racemic 54 on human platelets; and 3) there is a large difference in the intrinsic activities of the two enantiomers on electrically-stimulated guinea pig ileum (an a2~adrenoceptor system). 105
Table 10
Inhibition of Epinephrine-Induced Human Platelet Aggregation by Analogs of 54
N '>
N» H . HC1
Platelets (a2)
Compound R r ' PIC50 ± SEMa
54 ch3 H 5.27 ± 0.22 (6 )b
58 H H 5.62 ± 0.22 (3)
59 OH H 4.31 ± 0.21 (3)
60 0= 3.98 ± 0.40 (3)
61 och3 H 3.55 + 0.07 (3) 75 cf3 OH c
90 c
*pIC50 = -log IC where IC50 is equal to the molar concentration of compound which inhibits the response by 50%. Inhibitors were added 1 min prior to epinephrine (10 uM) in human platelet-rich plasma. Aspirin (1 mM) was included for determination of primary wave aggregation. ‘Values in parentheses indicate the number of experiments (n). cNo activity up to 300 /iM. 106
Analogs of phentolamine (19.) were tested for their relative
a2-adrenergic activities in human platelets (Table 11). They were concentration-dependent inhibitors of epinephrine induced
aggregation in aspirin-treated platelets with pIC50 values of
3.93, 4.16, and 3.95 for 63, Z-64, and E-64, respectively;
these analogs were about 66-, 39-, and 63-fold less potent as
inhibitors than phentolamine (.19). These analogs of phentolamine will also be tested for a1-adrenergic activities
in rat aorta.
Table 11
Inhibition of Epinephrine-Induced Human Platelet Aggregation by Phentolamine (19) and Analogs
Platelets (a2)
Compound pIC50 ± SEM“
Phentolamine (19) 5.75 + 0.20 (3)b
63 3.93 ± 0.11 (3)
Z-64 4.16 ± 0.07 (3)
E-64 3.95 ± 0.04 (3)
apIC50 = -log IC where IC is equal to the molar concentration of compound which inhibits the response by 50%. Inhibitors were added 1 min prior to epinephrine (3-7 pM) in human platelet-rich plasma. Aspirin (1 mM) was included for determination of primary wave aggregation. bValues in parentheses indicate the number of experiments (n) 107
3.3 SUMMARY
1. The naphthalene analog 54 of medetomidine was
synthesized, and was equipotent to medetomidine (S_1) as
an antagonist of a2-adrenergic activity in human
platelets (ot2A).
2. The replacement of the dimethylphenyl group of 51 with a
naphthalene ring, as in analog 54, did not diminish a2-
adrenergic activity while leading to a loss of c^-
adrenergic activity.
3. The enantiomers of compound 54 were separated using
fractional crystallization and isolated as tartaric acid
salts.
4. Enantiomeric purities were determined using an analytical
Chiralcel ODR HPLC column, and the absolute configuration
of S(+)-57 was determined by X-ray crystallography.
5. R(-)-57 was 9-fold more potent than S(+)-57 as an
inhibitor of epinephrine-induced human platelet primary
wave aggregation.
6 . From functional assays using guinea pig ileum, R(-)-57
exhibited no intrinsic activity, whereas S(+)-57 showed
enhanced a2~agonist activity as compared to the racemic
54.
7. A variety of functional groups were introduced at the
carbon bridge between the naphthalene and imidazole rings
of compound 54 for a study of structure-activity
relationships. 108
8. The desmethyl analog 58 was 3-fold more potent than the
racemic mixture of 54 in human platelets (at^).
9. With the exception of the non-methyl containing compound
58/ none of the other analogs of 5£ had enhanced a2~
adrenergic activity in the structure-activity
relationship study.
10. Several approaches to the synthesis of indole analogs of
medetomidine were unsuccessful, but one promising
synthetic path has been partly completed.
11. Three analogs of phentolamine were synthesized, and the
absolute configuration of Z-64 was determined by X-ray
crystallography.
12. Compared to phentolamine, the isosteric analogs of
phentolamine exhibited a dramatically reduced inhibitory
potency against epinephrine-induced human platelet
aggregation. CHAPTER IV
EXPERIMENTAL
Melting points were determined on a Thomas-Hoover capillary melting point apparatus and are uncorrected. Infrared spectra were obtained with an Analect RFX-40 FTIR spectrometer. The
NMR spectra were obtained on either an IBM AF-250 FTNMR spectrometer (250 MHz) or an IBM AF-270 FTNMR spectrometer
(270 MHz) and are reported in parts per million. Mass spectra were obtained with either a Kratos MS25RFA mass spectrometer at The Ohio State University College of Pharmacy or at the
Ohio State University Chemical Instrumentation Center with a
Kratos MS-30 mass spectrometer. Optical rotations were measured on a Perkin-Elmer 241 polarimeter. Elemental analyses were performed by Galbraith Laboratories, Inc., Knoxville, TN.
All analytical results for the indicated elements were within
± 0.4% of the theoretical values. Anhydrous THF was produced by refluxing with and distillation from calcium hydride. THF was kept refluxing with sodium using benzophenone as an
indicator for dryness. Anhydrous benzene was produced by refluxing with and distillation from sodium. Toluene was dried by refluxing with and distillation from calcium hydride.
109 110
Methanol was distilled over magnesium methoxide, generated in
situ from magnesium, iodine, and methanol.
4-Tl-(1-Naphthyl)ethyl1imidazole hydrochloride (54)
N
| ■ HC1
A solution of methylmagnesium iodide in 100 mL of diethyl ether was prepared from dry magnesium turnings (1.08 g, 44.4 mmol) and methyliodide (6.27 g, 44.2 mmol) under an argon atmosphere. To this was added a solution of 1-naphthylalde’hyde
(65) (5.75 g, 36.8 mmol) in 10 mL of diethyl ether with cooling in an ice-water bath, and the resulting reaction mixture was stirred for 2 hours at room temperature. The reaction mixture was treated with 25 mL of 2N HC1, and the organic layer was separated from the aqueous layer. The organic layer was washed with brine (1 X 50 mL) and dried over sodium sulfate. Evaporation of the solvent under reduced pressure gave 6.20 g (97%) of the alcohol as a viscous oil. A solution of the alcohol (6.34 g, 36.8 mmol) and thionyl chloride (8.76 g, 73.6 mmol) in 65 mL of toluene was refluxed for 4 hours. The reaction mixture was evaporated under reduced pressure to give the residue as an oil. The resulting residue was dissolved in 150 mL of ethyl acetate, washed successively
with water (1 X 50 mL), saturated sodium bicarbonate solution
(1 X 50 mL) and brine (1 X 50 mL), and dried over sodium
sulfate. The solvent was removed under reduced pressure to
give 6.83 g (97%) of the chloride 66^ as an oil. To a solution
of TiCl4 (10.8 g, 72.0 mmol) in 70 mL of dry chloroform was
added a solution of TMS-imidazole (10.0 g, 71.6 mmol) with
cooling in an ice-water bath for 30 minutes under an argon
atmosphere. The resulting orange colored mixture was stirred
for additional 30 minutes, and then a solution of chloride 6(5
(6.83 g, 36.8 mmol) in 35 mL of dry chloroform was added to
the reaction mixture with cooling in an ice-water bath. The
reaction mixture was stirred overnight at ambient temperature.
Water (150 mL) was added, the reaction mixture was extracted with methylene chloride (3 X 100 mL), and then 2N NaOH (150
mL) was added to make aqueous layer basic. The aqueous layer was extracted with chloroform (3 X 100 mL). The combined
organic extracts were washed with brine (3 X 100 mL), dried
over sodium sulfate, and evaporated under reduced pressure to
give 1.56 g (19.6%) as a solid. A solution of the free base
(0.23 g, 1.03 mmol) in 3 mL of methanol was treated with 1.1 mL of IN HC1 in methanol. Evaporation of the solvent gave the
solid which was recrystallized from methanol/diethyl ether to
give 0.20 g (76%) of the imidazole hydrochloride 54: mp 120.0-
123.5°C; IR (KBr, cm"1); 3174 (NH); XH NMR (CD3OD) 6 8.81 ( s,
1H, imidazole), 8.16 (d, 1H, J = 8.3 Hz, ArH), 7.0-7.42 (m, 112
7H, ArH and imidazole), 5.15 (q, 1H, J = 7.1 Hz, CH3), 1.80
(d, 3H, J = 7.1 Hz, CH); MS m/z 222 (M+ - HCl), 207 (base).
Analysis for C1SH15C1N2; calculated: C, 69.63; H, 5.84; N,
10.83; found: C, 69.73; H, 5.89; N, 10.88
4-[(1-Naphthyl)hydroxymethyl1-N-triphenylmethyl imidazole (69)
C P h , ft ^ N
N
Dry magnesium turnings (312 mg, 12.82 mmol) were covered with 34 mL of dry tetrahydrofuran and 1-bromonaphthalene (67)
(2.57 g, 12.42 mmol) was added under an argon atmosphere.
After the addition was complete, the reaction mixture was refluxed for about 1 hour until the magnesium turnings were consumed. The reaction mixture was cooled to about 40-45°C and then a solution of aldehyde 6J) (1.4 g, 4.14 mmol) in 20 mL of dry tetrahydrofuran was added dropwise. After the addition was complete, the reaction mixture was allowed to attain room temperature, stirred for an additional 30 minutes, cooled in an ice-water bath, and treated with 20 mL of 2N HCl with agitation. The stirring was continued for an additional 15 minutes, and the mixture was then extracted with ethyl acetate
(2 X 30 mL). The combined organic layers were washed with 113 brine (1 X 30 mL), dried over sodium sulfate, and evaporated under reduced pressure to give yellow solids, the solids were
collected, and washed with ethyl acetate (1 X 10 mL) to give
1.45 g (75% from the aldehyde) of 69 as a white solid: mp 154-
155°C; IR (KBr, cm'1) 3248 (OH); XH NMR (CDC13/TMS) 6 8.18 (s,
1H, imidazole), 7.86-6.93 (m, 23H, ArH and imidazole), 6.73
(s, 1H, CH), 6.10 (s, 1H, OH); MS m/z 466 (M+), 243 (base).
Analysis for C33H26N20; calculated: C, 84.95; H, 5.62; N, 6.00;
found: C, 84.75; H, 5.85; N, 6.12.
4-T(1-Naphthyl)methyl)imidazole hydrochloride (58)
A stirred solution of 6J9 (0.7 g, 1.5 mmol) in 20 mL of
methylene chloride was treated with triethylsilane (1.4 g, 12
mmol) and trif luoroacetic acid (5.47 g, 48 mmol). The
resulting clear solution was stirred at room temperature
overnight and 20 mL of water were then added. The reaction
mixture was made basic with solid sodium bicarbonate. The
organic layer was separated, washed with water (1 X 10 mL),
dried over sodium sulfate, and concentrated under reduced 114
pressure to give an oil which was crystallized from methylene
chloride to give 0.204 g (65%) of the free base as a peach-
colored solid. A solution of this free base (0.53 g, 2.54
mmol) in 5 mL of methanol was treated with 2.7 mL of IN HC1 in
methanol. Evaporation of the solvent under reduced pressure
gave a red-orange solid which was recrystallized from methanol/diethyl ether to give 0.37 g (60%) of 58 as a white
solid: mp 205-206°C; IR (KBr, cm'1) 3506 (NH); *H NMR (CD3OD) 5
8.78 (d, 1H, J = 1.3 Hz, imidazole), 7.54-7.42 (m, 7H, ArH),
7.19 (d, 1H, J = 1.3 Hz, imidazole), 4.55 (s, 2H, CH2); MS m/z
208 (M+ - HC1, base). Analysis for C14H13C1N2; calculated: C,
68.71; H, 5.35; N, 11.45; found: C, 68.56; H, 5.54; N, 11.56.
4-(N-Triphenylmethyl)imidazolylmethanol (71)
CPh 3
A reaction mixture of 4-hydroxymethylimidazole hydrochloride (7j)) # 30 mL of dry N,N-dimethylformamide, and triethylamine (7.26 g, 71.7 mmol) was treated dropwise with a solution of triphenylmethyl chloride (8.5 g, 30.5 mmol) dissolved in 110 mL of dry N,N-dimethylformamide under an argon atmosphere. After 30 minutes an additional portion of 115
triethylamine (0.73 g, 7.21 mmol) and triphenylmethyl chloride
(0.85 g# 3.0 mmol) in 25 mL of N,N-dimethylformamide was
added. After an additional 30 minutes, the reaction mixture
was poured over 500 g of crushed ice, the solids were
collected and washed with water (1 X 100 mL). The crude
product was digested with 100 mL of dioxane, cooled, collected
by filtration, and washed with diethyl ether to give a solid which was recrystallized from dioxane to give 8.82 g (88%) of
71 as a white solid: mp 228-229°C (lit. mp [106] 228-230°C);
*H NMR (CD3OD) 6 7.42-7.13 (m, 17H, ArH, imidazole), 4.48 (s,
2H, CH2OH) .
4-(N-Triphenylmethy1)imidazolecarboxaldehyde (68)
o
N^-N*cph3
A mixture of 21 (8.3 g, 24.4 mmol) and manganese oxide
(21.2 g, 243.8 mmol) in 230 mL of dioxane was stirred and refluxed for 4 hours. The hot reaction mixture was filtered through a Celite pad and the solids were washed with 250 mL of hot dioxane. The combined filtrate and washings were evaporated under reduced pressure, and the white solid was 116 dried under reduced pressure at 110°C to give 7.5 g (90%) of
68 as a white solid: mp 196-198°C (lit. mp [106] 196-197°C) XH
NMR (CDCI3/TMS) 6 9.88 (s, 1H, HC=0), 7.61 ( d, 1H, J = 1.2
Hz, imidazole), 7.53 ( d, 1H, J = 1.2 Hz, imidazole), 7.38-
7.09 (m, 15H, ArH).
4 — T(l-Naphthyl)hydroxymethyl]Imidazole hydrochloride (59)
HO
• H C 1
The suspension of 6jJ (1 g, 2.14 mmol) in 20 mL of 2N HC1 was refluxed for 1 hour. After cooling to room temperature, 20 mL of methylene chloride were added to dissolve precipitated materials (triphenylmethyl alcohol). The organic layer was discarded. The water layer was made basic with saturated sodium bicarbonate solution and extracted with methylene chloride (2 X 20 mL) . The organic layers were washed with brine (1 X 20 mL), dried over sodium sulfate, and concentrated under reduced pressure to give 0.24 g (50%) of the free base as a white solid. A solution of this free base (0.65 g, 2.9 mmol) in 5 mL of methanol was treated with 3 mL of IN HC1 in 117
methanol. The mixture was concentrated under reduced pressure
to give an oil which was crystallized from methanol/diethyl
ether to give 0.432 g (57%) of 59 as a white solid: mp 162-
165°C; IR (KBr, cm"1) 3259 (OH, NH); XH NMR (CD3OD) 6 8.83 (S,
1H, imidazole), 8.1-7.48 (m, 7H, ArH), 7.17 (s, 1H,
imidazole), 6.63 (s, 1H, CHOH); MS m/z 244 (M+ - HC1), 205
(base). Analysis for C14H13C1N20; calculated: C, 64.50;H, 5.03;
N, 10.74; found: C, 64.25; H, 5.02; N, 10.71.
4-T(1-Naphthyl)carbonyl1-N-(triphenylmethyl)imidazole (72)
C P h 3
N
N
Activated manganese oxide (9.21 g, 105.88 mmol) was added to a solution of 6S) (4.94 g, 10.59 mmol) in 100 mL of methylene chloride. The reaction mixture was refluxed for 4 hours, cooled to the room temperature, filtered through a
Celite pad, and the solids were washed with methylene chloride
(1 X 20 mL). The combined filtrate and washing were evaporated under reduced pressure to give 4.04 g (82%) of 72 as a white solid: mp 159-161°C; IR (KBr, cm'1) 1646 (C=0); XH NMR
(CDC13/TMS) 6 8.32-7.12 (m, 21H, ArH), 7.94 (d, 1H, J = 8.2 118
Hz, ArH), 7.63 (d, 1H, J = 1.4 Hz, imidazole), 7.53 (d, 1H, J
= 1.4 Hz, imidazole); MS m/z 464 (M+), 243 (base). Analysis for C33H24N20; calculated: C, 85.32; H, 5.21; N, 6.03; found: C,
85.52; H, 5.17; N, 5.99.
4-T(1-Naphthyl)carbonyl)imidazole oxalate (60)
A suspension of 72 (1 g, 2.15 mmol) in 20 mL of 2N HC1 was refluxed for 1 hour. After cooling to room temperature, the precipitated materials were removed by filtration. The water layer was made basic with solid sodium bicarbonate, and extracted with methylene chloride (2 X 20 mL). The organic layers were washed with brine (2 X 20 mL), dried over sodium sulfate, and concentrated under reduced pressure to give 0.26 g (54%) of the free base as a white solid. A solution of the free base (0.25 g, 1.12 mmol) in 5 mL of methanol was treated with a solution of oxalic acid dihydrate (0.15 g, 1.18 mmol) in 5 mL of methanol. The mixture was concentrated under reduced pressure to give a solid which was recrystallized from methanol to give 0.244 g (82%) of 6_0 as white crystals: mp 119
218-221°C; IR (KBr, cm'1) 3415 (NH), 1608 (C=0); NMR (CD3OD)
6 8.26 (s, 1H, Imidazole), 8.20-7.51 (m, 5H, ArH), 8.09 (d,
1H, J = 8.3 Hz, ArH), 7.83 (dd, 1H, J = 7.1 and 1.2 Hz, ArH),
7.7 (s, 1H, imidazole); MS m/z 222 (M+ - CH02), 221 (base).
Analysis for C14H10N2O. 1/2 H2C204; calculated: C, 67.40; H,
4.15; N, 10.49; found: C, 67.45; H, 4.29; N, 10.56.
4-fl-(1-Naphthyl)methoxymethvl1imidazole hydrochloride (61)
■ H C 1
A suspension of 5?) (0.93 g, 3.57 mmol) in 10 mL of chloroform was treated dropwise with 1.1 mL of thionyl chloride (14.8 mmol) at room temperature. The resulting mixture was refluxed overnight and then the solvent removed under reduced pressure to give a solid. The solid was dissolved in 20 mL of methanol and refluxed for 5 hours. The resulting solution was stirred an additional 1 hour at room temperature and the solvent was removed under reduced pressure to give a solid which was recrystallized from methanol/ethyl acetate to give 0.73 g (75%) of 6_1 as light yellow crystals: mp >260°C with decomposition; IR (KBr, cm"1) 3174 (NH); XH NMR 120
(CD3OD) 6 8.87 ( d, 1H, J = 1.3 Hz, imidazole), 7.97-7.12 (m,
7H, ArH), 7.11 ( d, 1H, J = 1.3 Hz, imidazole), 6.16 (s, 1H,
CH), 3.46 (s, 3H, OCH3); MS m/z 238 (M+ - HC1), 207 (base).
Analysis for C15H15C1N20; calculated: C, 65.57; H, 5.50; N,
10.20; found: C, 65.22; H, 5.61; N, 10.00.
Trifluoromethyltrimethylsilane (73)
C H 3 CH3-Si-CF3 ch3
Bromotrifluoromethane (ca. 50 mL) was condensed into a 250
mL three-necked flask equipped with a Dewar condenser filled with dry ice-acetone mixture. A solution of
chlorotrimethylsilane (21.4 g, 197 mmol) in 50 mL of
benzonitrile was added dropwise over 30 minutes to the
bromotrifluoromethane stirred at -78°C under an argon
atmosphere. A white precipitate formed during the addition.
After stirring for an additional 30 minutes at the same
temperature, a solution of hexaethylphosphoroustriamide (50 g,
202 mmol) in 30 mL of benzonitrile was added dropwise over 30 minutes. The reaction mixture was allowed to warm to room
temperature over a three hour period. The precipitate
dissolved during this period, resulting in a homogeneous
yellow solution. Volatile materials were collected into 121 another distillation flask cooled at -78°C under reduced pressure using aspirator vacuum at room temperature for 2 hours. The resulting liquid was distilled at atmospheric pressure to afford 22.2 g (79%) of 73 as a colorless liquid: bp 53-57°C (lit. bp [110] 55-55.5°C). *H NMR(CDC13/TMS) : 6 0.25
(s , 9H, SiMe3). 13C NMR (CDC13) : 6 131.7 (q, JCF3 = 321.9 Hz,
CF3): -5.2 (s, SiMe3) .
4-f1-(1-Naphthyl)-2,2,2-trifluoroethylhydroxyl- N-
(triphenylmethyl)imidazole (74)
C P h - i 3 N
N
A solution of 72 (1 g, 2.15 mmol) and TMS-CF3 (0.86 mL,
6.02 mmol) in 10 mL of dry tetrahydrofuran was cooled in an ice-water bath for 30 minutes, and tetrabutylammonium fluoride
(4.3 g, 16.58 mmol) was added under an argon atmosphere. An orange color developed instantaneously and the reaction mixture was brought to room temperature and stirred overnight.
The reaction mixture was treated with 30 mL of water and stirred for 30 minutes, then extracted with methylene chloride
(2 X 50 mL). The organic extracts were washed with water (1 X
50 mL) and brine (1 X 50 mL), and dried over sodium sulfate. 122
The solvent was removed under reduced pressure to give a solid which was recrystallized from methylene chloride/hexane to
give 0.86 g (75%) of 74 as a white solid: mp 231-232°C; IR
(KBr, cm'1) 3300-3000 (OH), 1152 (CF3); *H NMR (CDC13/TMS) 5
8.13 (d, 1H, J = 8.7 Hz, ArH), 7.81-6.94 (m, 21H, ArH), 7.49
(d, 1H, J = 1.3 Hz, imidazole), 6.34 (d, 1H, J = 1.3 Hz,
imidazole), 5.3 (s, 1H, OH); MS m/z 534 (M+), 243 (base).
Analysis for C34H25F3N20; calculated:: C, 76.39; H, 4.71; N,
5.24; found: C, 76.37; H, 4.92; N, 5.24.
4-T1-(1-Naphthyl)-2,2,2-trifluoroethylhydroxyl imidazole hydrochloride (75)
N
A solution of 74 (0.7 g, 1.31 mmol) in 30 mL of methylene chloride was treated with triethylsilane (1.24 g, 10.48 mmol) and trifluoroacetic acid (4.74 g, 41.92 mmol). The reaction mixture was stirred overnight and 20 mL of water were then added. The solution was basified by adding solid sodium bicarbonate and extracted with methylene chloride (3 X 20 mL) .
The extracts were washed with water (2 X 20 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure 123 to give a solid which was recrystallized from methanol to give
0.35 g (97%) of the free base as a white solid. A solution of the free base (0.46 g, 1.6 mmol) in 5 mL of methanol was treated with 1.7 mL of IN HC1 in methanol. Evaporation of the solvent under reduced pressure gave an oil which was crystallized with methanol/diethyl ether to give 0.36 g (68%) of 75 as a white solid: mp 258~260°C with decomposition; IR
(KBr, cm'1) 3163 (OH, NH), 1162 (CF3) ; XH NMR (CD3OD) : 6 8.94 ( d, 1H, J = 1.3 Hz, imidazole), 8.10 (d, 1H, J = 8.5 Hz, ArH),
8.01 (d, 1H, J = 8.3 Hz, ArH), 7.94-7.34 (m, 5H, ArH), 7.28 ( d, 1H, J = 1.3 Hz, imidazole); 13C NMR BB (CD3OD) 6 126.13 (q,
JCF3 = 287.4 Hz, CF3), 77.34 (q, = 30.0 Hz, ring junction
C bonded to CF3), 136.54, 136.11, 134.63, 132.46, 132.35,
131.69, 130.36, 127.35', 127.28, 126.91, 126.86, 125.48,
120.07; 19F NMR (CFC13 = 0 ppm external reference) 6 -74.77; MS m/z 292 (M+ - HC1), 95 (base). Analysis for C15H12C1F3N20; calculated: C, 54.81; H, 3.68; N, 8.52; found: C, 55.08; H,
3.87; N, 8.44. 124
Resolution of 4-F1~(1-Naphthyl)ethyl 1Imidazole to give (+)-4-
l-( 1-Naphthyl) ethyl] imidazole tartaric acid (S~57) and (-)-4-
fl-(l-(1-Naphthyl)ethyl1 imidazole tartaric acid (R-57)
S - 5 7 R - 5 7
The racemic mixture of 4-[l-(1-naphthyl)ethyl]imidazole
(9.34 g, 42 mmol) in 200 mL of hot methanol was combined with
( + )-tartaric acid (6.44 g, 43 mmol) in 100 mL of hot methanol.
The resulting solution was allowed to stand at room temperature for 50 hours and filtered to give 5.85 g of the tartaric acid salt which was recrystallized ten times from a minimum amount of methanol, and three times from methanol/ethanol 50:50 to give 2.54 g (16%) of the (+)-isomer
(S-57) as a cotton type solid which was dried overnight at 5 mmHg: mp 197-198°C; [a]D +56.6° (1% solution in methanol). The mother liquor solutions from each recrystallization procedure were combined and evaporated, and dried under reduced pressure to give 9.49 g of the imidazole tartaric acid salt which was treated with 6 g of sodium hydroxide in 400 mL of water and extracted with methylene chloride (2 X 150 mL). The organic layers were dried over sodium sulfate and concentrated under 125 reduced pressure to give the free base (4.94 g). The free base was dissolved in 100 mL of hot methanol and combined with (-)- tartaric acid (3.402 g, 22.7 mmol) in 100 mL of hot methanol.
The solution was kept at room temperature for 52 hours to yield 5.2 g of tartaric acid salt which was recrystallized eight times from methanol and two times from methanol/ethanol
50:50 to give 1.57 g (10%) of the (-)-isomer (R-57) as a cotton type solid which was dried overnight at 5 mmHg: mp 197-
198°C; [a]D -56.0° (1% solution in methanol).
Separation and determination of optical purity of the enantiomers of 4-\1-(1-Naphthyl)ethyl1 imidazole by HPLC
The free base of 4-[l-(1-naphthyl)ethyl]imidazole was resolved on an analytical Chiralcel ODR (Daicel) HPLC column
(4.6 X 250 mm). The mobile phase was hexane/isopropanol 90:10 and the flow rate was 1 mL/min. The separation was performed using a Beckman System Gold Chromatograph using UV detection at 280 nm: k/ = 0.48 ; k2' = 1.51 ; a = 3.15; Rs = 6.5; ( + )- isomer: Retention time = 9.87 minutes; (-)-isomer: Retention time = 16.69 minutes. The optical purities of the each enantiomer as a free base were determined on the same system as above. 126
4-(N-Indolylcarbonyl)-N-(triphenylmethyl)imidazole (79)
CPh3
A solution of indole (77) (1.06 g, 9.04 mmol) in 15 mL of
diethyl ether was cooled in an ice-water bath for 30 minutes
and treated with a solution of methylmagnesium bromide (3.0 M
in diethyl ether, 3.11 g, 9.04 mmol) in 15 mL of dry
tetrahydrofuran under an argon atmosphere. After the addition was complete, the reaction mixture was allowed to attain room
temperature, stirred for additional 30 minutes, and 40 mL of
dry methylene chloride were added to the reaction mixture in
order to bring the complex into solution. The mixture was
cooled again in an ice-water bath, and then a solution of T6
(2 g, 8.2 mmol) in 40 mL of dry methylene chloride was added
dropwise. The mixture was stirred for an additional 1 hour in
the ice-water bath, and then overnight at room temperature.
Ammonium chloride (0.476 g, 8.9 mmol) in 10 mL of water was
added with cooling in an ice-water bath. The organic layer was
separated, and the water layer was extracted with methylene
chloride (2 X 15 mL). The extracts were washed with brine (2 127
X 10 mL), dried over sodium sulfate, and the solvent was removed under reduced pressure to give an oil. Flash column chromatography using a gradient solvent system of methylene chloride/hexane 60:40 to 100% methylene chloride yielded a solid which was recrystallized from ethylacetate to give 2.74 g of 79 (74% from the ester) as a white solid: mp 185-186°C;
IR (KBr, cm'1) 1658 (C=0); NMR (CDC13/TMS) 6 9.06 (d, 1H, J
= 3.8 Hz, CHCH), 8.54 (d, 1H, J = 7.9 Hz, indole), 7.89 ( d,
1H, J = 1.2 Hz, imidazole), 7.53 ( d, 1H, J = 1.2 Hz, imidazole), 7.59-7.14 (m, 18H, ArH and indole), 6.63 (d, 1H,
J = 3.8 Hz, CHCH); MS m/z 453 (M+), 243 (base); Analysis for
C3iH23N30; calculated:C, 82.10; H, 5.11; N, 9.26; found: C,
82.32; H, 5.14; N, 9.12.
Imldazole-4-carboxyllc acid (81)
HOOC
N ^ N - H
A suspension of imidazole-4,5-dicarboxylic acid (80) (20 g,
128 mmole) in 750 mL of acetic anhydride was stirred and refluxed for 6 hours until solution was nearly complete (the solution turned brown). A small amount of gray solid was removed by filtration and the light brown filtrate was 128
evaporated to dryness. Water (300 mL) was added to the
residual material, and this mixture was stirred overnight and
then heated on a steam bath for 1 hour. Ethanol (300 mL) and
decolorizing charcoal (Norit) were added, and the mixture was
heated on a steam bath for 30 minutes and filtered. The
filtrate was refrigerated overnight, and pale yellow crystals
were collected and dried under reduced pressure to give 9.85
g of 81. The mother liquor was concentrated to dryness, the
residual material was dissolved in 100 mL of 50% aqueous
ethanol, and the solution was chilled overnight to give an
additional 1.02 g of 81 as a white solid, for a total yield of
76%: mp 278-279°C with decomposition (lit. mp [116] 279-281°C
with decompossition); *H NMR (DMS0-dg) 5 7.76 (s, 1H,
imidazole), 7.68 (s, 1H, imidazole).
Ethyl lmldazole-4-carboxylate (82)
N,n .N-H
A suspension of 8JL (10 g, 89.2 mmol) in 180 mL of absolute
ethanol was treated with 10 mL of concentrated sulfuric acid.
The mixture was refluxed for 40 hours under an argon
atmosphere. The solution was cooled in an ice-water bath and made basic to around pH 8-9 with 40 mL of 5N sodium hydroxide. 129
The solvent was removed under reduced pressure and the
residual material was dissolved in a minimum volume of boiling water (around 80 mL) and kept at room temperature overnight.
The solids were collected and concentration of the mother
liquor gave an additional crop for a total yield of 11.2 g
(90%) of 82 as a white solid, mp 155-157°C (lit. mp [116] 159-
160°C); *H NMR (D20) 6 7.71 (s, 1H, imidazole), 7,66 (s, 1H, imidazole), 4.19 (q, 2H, J = 7.2 Hz, OCH2CH3) , 1.20 (t, 3H,
J=7 .1 Hz, OCH2CH3).
Ethyl l-triphenylmethylimidazole-4-carboxylate (78)
N^N-CPh3
A solution of 8£ (4.2 g, 30 mmol) in 100 mL of dry N,N- dimethylformamide was treated with triphenylmethyl chloride
(8.5 g, 30.6 mmol). When solvation was complete, 5 mL of triethylamine were added. The reaction mixture was stirred overnight at room temperature and the solvent was removed under reduced pressure to give a solid which was dissolved in a mixture of 50 mL of saturated sodium bicarbonate solution and 50 mL of chloroform. The aqueous layer was extracted with chloroform (3 X 20 mL), the combined organic extracts were 130
washed with brine (1 X 50 mL) and dried over sodium sulfate.
The solvent was removed under reduced pressure, the residual
solid was dissolved in 100 mL of boiling ethanol, and 20 mL of
water were added to the mixture. The mixture was heated on the
steam bath until it became a clear solution and then allowed
to cool to room temperature to give 10.7 g of 78.
Concentration of the mother liquor and addition of 15 mL of
water gave an additional 0.4 g of 78 as a white solid, for a
total yield of 97%: mp 164-166°C (lit. mp [116] 165-167°C); *H
NMR (CDCI3/TMS) 6 7.58 ( d, 1H, J = 1.4 Hz, imidazole), 7.44
( d, 1H, J = 1.4 Hz, imidazole), 7.37-7.10 (m, 15H, ArH), 4.33
(q, 2H, J = 7.1 Hz, OCH2CH3), 1.37 (t, 3H, J = 7.1 Hz, OCH2CH3) .
1-f4-(N- Triphenylmethyl)imidazolyl1 ethanol (83)
OH
CPh3
To an ice-bath cooled solution of methylmagnesium bromide
(3.0 M in diethyl ether, 0.52 g, 1.48 mmol) was added a solution of aldehyde 68 (0.25 g, 0.74 mmol) in 4 mL of tetrahydrofuran under an argon atmosphere. After 1.5 hours at room temperature a solution of ammonium chloride (0.1 g, 1.85 mmol) in 5 mL of water was added to the reaction mixture. The mixture was stirred for 1 hour and filtered and the solids were washed with tetrahydrofuran. The combined filtrate and washings were washed with water (1 X 10 mL) and brine (1 X 10 mL)f dried over sodium sulfate, and evaporated under reduced pressure to give a white powder which was recrystallized from chloroform/hexane to give 0.21 g (80%) of 83 as a white solid: mp 155-156°C (lit. mp [106] 156-157°C); *H NMR (CDC13/TMS) 6
7.36 (s, 1H, imidazole), 7.34-7.09 (m, 15H, ArH), 6.70 (s, 1H, imidazole), 4.84 (q, 1H, J = 6.5 Hz, CH), 1.47 (d, 3H, J = 6.5
Hz, CH3)
l-(Phenylsulfonyl)-3-iodoindole (86)
A solution of indole (77) (6 g, 51.22 mmol) in 70 mL of dry tetrahydrofuran was cooled to -78°C under an argon atmosphere, and n-BuLi (2.5M in hexane, 14.5 g, 52.24 mmol) was added via syringe over 5 minutes. The resulting milky white suspension was warmed to -10°C over 1.5 hours and then slowly added by syringe into a -78°C solution of iodine (13.26 g, 52.24 mmol) in 70 mL of dry tetrahydrofuran over 15 minutes. This dark- colored solution was warmed to 0°C over 2 hours and then 132 cooled to -78°C over 30 minutes. The resulting solution of 3- iodoindole was then treated at -78°C over 2 minutes with a solution of LDA prepared from diisopropylamine (5.3 g, 52.24 mmol) and n-BuLi (2.5M in hexane, 14.21 g, 51.22 mmol) in 20 mL of dry tetrahydrofuran. The resulting light-orange reaction mixture was stirred at -78°C for 25 minutes and then treated with benzenesulfonyl chloride (9.55 g, 53.78 mmol) via syringe over 1 minute. After warming to room temperature overnight, the reaction mixture was cooled to 0°C, poured into 500 mL of
2% aqueous sodium bicarbonate solution, and extracted with diethyl ether (2 X 200 mL). The combined extracts were washed with 3% aqueous sodium bisulfite solution (1 X 300 mL), water
(2 X 200 mL), and brine (2 X 200 mL), and dried over sodium sulfate. The solvent was removed under reduced pressure to afford wisky color crystals. These crystals were dissolved in
20 mL of methylene chloride, and chromatography on Florisil using diethyl ether/hexane 50:50 gave a solid which was recrystallized from methylene chloride/hexane to give 16.9 g
(86% from the indole) of 86 as a white solid: mp 124-126°C
(lit. mp [115] 125-127°C); *H NMR (CDC13/TMS) 6 8.0-7.26 (m,
9H, ArH, indole), 7.71 (s, 1H, indole). 133
4 -{ f 3-(N-Phenylsulfonyl)lndolyl1hydroxymethy1}-N-
(triphenylmethyl)Imidazole (87)
C P h ,
HO
9 S 0 2 P h
A magnetically-stirred solution of 86 (2.27 g, 5.91 mmol) in 50 mL of dry tetrahydrofuran under an argon atmosphere was cooled to -78°C. This solution was treated rapidly via syringe with t-BuLi (1.7M in pentane, 4.6 q, 11.84 mmol) and stirred at -78°C for 20 minutes, and then treated via syringe with aldehyde 6£( (1 q, 2.96 mmol) in 25 mL of dry tetrahydrofuran.
The reaction mixture was warmed slowly to room temperature over 6 hours and stirred overnight. The resulting solution was treated with 100 mL of water and 100 mL of methylene chloride.
The organic layer was separated and the aqueous layer was made acidic with 2N HC1, treated with 50 mL of brine, and extracted with additional methylene chloride (3 X 50 mL). The combined organic layers were washed with water (1 X 100 mL) and brine
(2 X 100 mL), dried over sodium sulfate, and concentrated under reduced pressure to give a solid which was washed with ethylacetate (1 X 30 mL) to give 1.08 g (61% from the aldehyde) of 87 as a slightly yellow solid: mp 224-227°C; IR 134
(KBr, cm'1) 3149 (OH); XH NMR (CD3OD) 6 7.93 (d, 1H, J = 8.3 Hz,
Indole), 7.85-7.07 (m, 26H, indole, imidazole, and ArH), 6.55
(S, 1H, CHOH) , 5.97 (s, 1H, OH); MS m/z 578 (M+ - H20) , 165
(base); Analysis for C37H2gSN303; calculated: C, 74.60; H, 4.91;
N, 7.05; found: C, 74.35; H, 5.04; N, 7.17.
4 - { f 3-(N-Phenylsulfonvl)indolyllcarbonyl}-N-
(tripher/lmethyl)imidazole (88)
C P h 3
S O „ P h
A solution of alcohol 87 (1.86 g, 3.13 mmol) in 50 mL of methylene chloride was treated with manganese oxide (2.72 g,
31.3 mmol) and then refluxed for 4 hours. The reaction mixture was cooled to room temperature and filtered through a Celite pad and the solids were washed with methylene chloride (1 X 20 mL). The combined filtrate and washings were evaporated under reduced pressure to give an oil which was crystallized from ethyl acetate/hexane to give 1.57 g (85%) of as a white cotton type solid: mp 205-206°C; IR (KBr, cm"1) 1650 (C=0); ^
NMR (CDC13/TMS) 6 8.11-7.14 (m, 25H, indole and ArH), 7.74 (d,
1H, J = 1.3 Hz, imidazole), 7.54 (d, 1H, J = 1.3 Hz, 135 imidazole); MS m/z 593 (M+), 243 (base); Analysis for
C37H27SN303; calculated: C, 74.85; H, 4.58; N, 7.08; found: C,
74.88; H, 4.62; N, 7.19.
4-(N-Indolylcarbonyl)Imidazole hydrochloride (90)
A suspension of 79 (2 g, 4.41 mmol) in 10 mL of 50% glacial acetic acid was heated on a steam-bath for 20 minutes. After cooling to room temperature, the reaction mixture was filtered to remove precipitated materials (triphenylmethyl alcohol).
The filtrate was extracted with methylene chloride (2 X 50 mL), removed solvent under reduced pressure to give 0.53 g
(57%) of the free base as a white solid. A solution of this free base (0.5 g, 2.37 mmol) in 5 mL of methanol was treated with 2.5 mL of IN HC1 in methanol. The mixture was concentrated under reduced pressure to give a solid which was recrystallized from methanol/diethyl ether to give 0.45 g
(77%) of 90 as a white solid: mp 240-242°C; IR (KBr, cm"1) 3451
(NH), 1697 (C=0); XH NMR (CD3OD) 6 8.97 ( d, 1H, J = 1.1 Hz, imidazole), 8.40 (dd, 1H, J = 8.7 Hz and 1.0 Hz, indole), 8.29
( d, 1H, J = 1.1 Hz, imidazole), 7.98 (d, 1H, J = 3.8 Hz, 136
CHCH), 7.64-7.31 (m, 3H, indole), 6.80 (d, 1H, J = 3.8 Hz,
CHCH); MS m/z 211 (M+ - HCl), 117 (base): Analysis for
C12H10ClN3O; calculated: C, 58.19; H, 4.07; N, 16.97; found: C,
58.36; H, 4.15; N, 16.97.
Methyl 3-hydroxybenzoate (92)
OH
COOCH3
To a solution of 3-hydroxybenzoic acid (9_1) (20 g, 140 mmol) in 200 mL of methanol was added excess HCl gas and the mixture was refluxed overnight. The reaction mixture was then concentrated under reduced pressure and saturated potassium carbonate solution was added until the solution was basic (pH
9-10). The mixture was extracted with diethyl ether (3 X 100 mL). The combined organic layers were washed with water (1 X
100 mL), dried over sodium sulfate, and concentrated under reduced pressure to give 19.08 g (90%) of 92 as a white solid: mp 67-70°C (lit. mp[ 117 ] 68-69°C); *H NMR (CDCI3/TMS) 6 7.60
(dd, 2H, J = 7.1 and 1.3 Hz, ArH), 7.31 (t, 1H, J = 8.1 Hz,
ArH), 7.08 (ddd, 1H, J = 8.2, 2.5, and 1.1 Hz, ArH), 6.07 (s,
1H, ArOH), 3.92 (s, 3H, COOCH3) . 137
Methyl 3-benzyloxybenzoate (93)
COOCH
To a solution of 92 (19.08 g, 125 mmol) in 200 mL of acetone was added potassium carbonate (17.28 g, 125 mmol) and benzyl chloride (15.82 g, 125 mmol). The reaction mixture was refluxed overnight, cooled to room temperature, and concentrated under reduced pressure to give a solid. The residual solid was partitioned between 250 mL of water and 200 mL of diethyl ether. The organic layer was separated and the water layer was extracted with'diethyl ether (1 X 100 mL). The combined organic layers were washed with water (2 X 100 mL), dried over sodium sulfate, and then concentrated under reduced pressure to give a white solid which was recrystallized from methanol to give 27.04 g (89%) of 93 as a white solid: mp 70-
72°C (lit. mp [118] 68-70°C); NMR (CDC13/TMS) 6 7.66-7.14 (m,
9H, ArH), 5.10 (S, 2H, CH2) , 3.91 (s, 3H, CH3) . 138
3-Benzyloxybenzolc acid (94)
COOH
A solution of £3 (27.04 g, 111.6 mmol), potassium hydroxide
(12.52 g, 223.2 mmol) in 200 mL of water was heated under reflux overnight. The solution was made acidic with concentrated HCl and extracted with diethyl ether (3 X 150 mL). The combined organic layers were washed with water (2 X
150 mL), dried over sodium sulfate, and concentrated under reduced pressure to give a solid which was recrystallized from methanol to give 24.34 g (96%) of 94 as a white solid: mp 133-
135°C (lit. mp [119] 134°C); *H NMR (CDC13/TMS) 6 7.75-7.20 (m,
9H, ArH), 5.12 (s, 2H, CH2) . 139
3-Benzyloxyphenyl-4-methylphenyl ketone (95)
O C H 2 P h
To a solution of 94 (2 g, 8.7 mmol) in 30 mL of dry carbon tetrachloride was added 5 drops of dimethylformamide and freshly-distilled thionyl chloride (1.47 g, 12.4 mmol) under an argon atmosphere. The reaction mixture was heated to 40-
50°C in an oil bath. The temperature of the reaction mixture was maintained at ca. 50°C for 1 hour until the infrared absorption at 1665 cm"1 disappeared. The solution was cooled to room temperature and concentrated under reduced pressure to give a dark oil. Without purification, the oil was treated with p-tolylmagnesium bromide prepared by the following procedure: magnesium turnings (0.51 g, 20.88 mmol) were added to a solution of 4-bromotoluene (1.78 g, 10.4 mmol) in 30 mL of dry tetrahydrofuran, and the mixture was refluxed for 2 hours under an argon atmosphere. The phenylmagnesium bromide solution was cooled to room temperature and added dropwise to the acid chloride in 20 mL of dry tetrahydrofuran cooled to -
78°C in a dry ice-acetone bath. The reaction mixture was 140 brought to room temperature over 1 hour to give a clear solution, 30 mL. of water were added and the mixture was extracted with diethyl ether (2 X 30 mL). The ether extracts were washed with 30 mL of IN NaOH solution and brine (1 X 30 mL), and dried over sodium sulfate. The solvent was evaporated under reduced pressure to give a solid. Flash column chromatography using a solvent system of ethylacetate/hexane
2:98 afforded 1.74 g (66%) of 95 as a white solid: mp 86-87°C;
IR (KBr, cm'1) 1647 (C=0); XH NMR (CDC13/TMS) 6 7.71-7.16 (m,
13H, ArH), 5.10 (s, 2H, OCH2Ar), 2.43 (s, 3H, CH3); MS m/z 302
(M+), 91 (base); Analysis for C21H1802; calculated: C, 83.42; H,
6.00; found: C, 83.10; H, 6.10.
3-(3-Benzyloxyphenyl)-3-(4-methylphenyl)acrylonitrile (96)
CHCN
CH 3
A solution of Na metal (0.23 g, 10.1 mmol) in 20 mL of absolute ethanol was cooled in an ice-water bath for 30 min and treated with diethyl cyanomethylphosphonate (1.79 g, 10.1 mmol) under an argon atmosphere. The reaction mixture was stirred at room temperature for 10 minutes, 1.0 g (3.3 mmol) of 95 was added, and the mixture was stirred for 2 hours, heated at ca. 60°C for 30 minutes, and then concentrated under reduced pressure to give an oil. The oil was treated with 15 mL of water and extracted with diethyl ether (2 X 20 mL). The extracts were washed with water (1 X 20 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure to give 1.04 g (97%) of 96 as a pale yellow oil. The attempted separation of each isomer was not successful. IR (neat, cm’1)
2200 (CN); there was no C=0 stretch; XH NMR (CDC13/TMS) 5 7.43-
6.87 (m, 13H, ArH), 5.70 and 5.65 (s, 1H, C=CH, mixture of isomers), 5.06 and 5.03 (s, 2H, CH2Ar, mixture of isomers),
2.41 and 2.38 (s, 3H, CH3, mixture of isomers); MS m/z 325
(M+), 91 (base); Analysis for C23HigNO; calculated: C,84.89; H,
5.89; N, 4.30; found: C, 84.82; H, 5.80; N, 4.33.
3-(3-Hydroxyphenyl)-3-(4-methylphenyl)propionitrile (97)
CHCN
A solution of nitrile 96 (0.6 g, 1.8 mmol) was treated with 142
5% palladium on carbon (380 mg) in 15 mL of ethanol in a Parr hydrogenation bottle and hydrogenated at 40 p.s.i. overnight.
The catalyst was removed by filtration through a Celite pad.
The filtrate was concentrated under reduced pressure to give an oil. Flash column chromatography using a solvent system of ethylacetate/hexane 30:70 afforded 0.30 g (70%) of 97 as a white solid: mp 106-109°C; IR (KBr, cm'1) 3360 (OH), 2250 (CN) ? aH NMR (CDC13/TMS) 6 7.26-6.67 (m, 8H, ArH), 4.95 (s, 1H, OH),
4.28 (t, 1H, J = 7.7 Hz, CHCHZ), 2.99 (d, 2H, J = 7.7 Hz,
CHCH2), 2.32 (s, 3H, CH3); MS m/z 237 (M+) 69 (base); Analysis for C16H15NO; calculated: C, 80.98; H, 6.37; N, 5.90; found: C,
81.04; H, 6.48; N, 5.71.
3-(3-Benzyloxyphenyl)-3-(4-methylphenyl)propionitrile (98)
O C H - P h
CHCN
CH 3
A solution of nitrile 9J7 (3.48 g, 14.66 mmol) was dissolved in 30 mL of acetone was treated with potassium carbonate (2.03 g, 14.66 mmol) and benzyl chloride (1.86 g, 14.66 mmol). The 143 reaction mixture was refluxed overnight, cooled to room temperature, and concentrated under reduced pressure to give an oil. The oil was triturated with petroleum ether to give a solid which was recrystallized from methylene chloride/hexane to give 3.92 g (82%) of 98 as a white solid: mp 73-74°C; IR
(KBr, cm'1) 2250 (CN); XH NMR (CDC13/TMS) 6 7.42-6.82 (m, 13H,
ArH), 5.01 (S, 2H, CH2Ar), 4.29 (t, 1H, J = 7.7 Hz, CHCHj) ,
2.98 (d, 2H, J = 7.7 Hz, CHCHZ), 2.32 (s, 3H, ArCH3); MS m/z
327 (M+), 91 (base); Analysis for C23H21NO; calculated: C,
84.37; H, 6.46; N, 4.28; found: C, 84.01; H, 6.50; N, 4.20.
2-f 2-(3-Benzyloxyphenyl)-2-(4-methylphenyl)ethyl]imidazoline oxalate (99)
0 C H „ P h
• ( C 0 0 H ) 2
CH 3
Nitrile 98 (1.798 g, 5.5 mmol) was dissolved in 10 mL of dry methylene chloride and 23 mL of dry methanol (55 mmol), and HCl gas was bubbled into this solution with cooling in an ice-water bath until there was further no absorption of HCl gas by the reaction mixture (indicated by the change of moist pH paper to red). The mixture was allowed to stand at room temperature for 3 days. The mixture was poured into 50 mL of diethyl ether, collected a solid, washed with diethyl ether (2
X 20 mL), and dried under reduced pressure to give 1.52 g
(71%) of imidate as a white solid. This imidate was dissolved in 10 mL of dry methylene chloride, the solution was cooled at
0°C with stirring, and ethylene diamine (0.47 g, 7.76 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirring was continued for another 15 hours. After removing the solvent under reduced pressure, 20 mL of 10% sodium bicarbonate solution were added and this mixture was extracted with methylene chloride (3 X 20 mL). The combined organic layers were washed with water (1 X 20 mL), dried over sodium sulfate, and concentrated under reduced pressure to give 1.52 g (75% from the nitrile) of the free base. A solution of this free base (1 g, 2.7 mmol) in 10 mL of methanol was treated with a solution of oxalic acid dihydrate
(0.36 g, 2.84 mmol) in 10 mL of methanol. The mixture was concentrated under reduced pressure to give a solid which was recrystallized from methanol/diethyl ether to give 1.01 g
(81%) of 99 as a white solid: mp 162-165°C; IR (KBr, cm’1) 3096
(NH) 1606 (C=N) ; XH NMR (CD3OD) 6 7.40-6.84 (m, 13H, ArH), 5.04
(s, 2H, CH2Ph), 4.38 (t, 1H, J = 8.5 Hz, CHCHZ), 3.73 (s, 4H,
NCH2CH2N), 3.25 (d, 2H, J = 8.5 Hz, CHCH2), 2.29 (s, 3H, ArCH3);
MS m/z 370 (M+ - C2H204), 279 (base). Analysis for C27H20N2O5; 145
calculated: C, 70.42; H, 6.13; N, 6.08; found: C, 70.79; H,
6.20; N, 6.20.
2-F 2-(3-Hydroxyphenyl)-2-(4-methylphenyl)ethyl1imidazoline
hydrochloride (63)
OH
• H C l
A solution of the oxalate salt 9SJ (397 mg, 0.86 mmol) in 10 mL of methanol was treated with 5% palladium on carbon (80 mg) in a Parr hydrogenation bottle and hydrogenated at 40 p.s.i. for 4 hours. The catalyst was removed by filtration through a
Celite pad. The filtrate was concentrated under reduced pressure to give a white solid which was recrystallized from ethanol/diethyl ether to give 242 mg (76%) of the debenzylated product. A solution of the debenzylated product (242 mg, 0.65 mmol) in 10 mL of methanol was mixed with 10 mL of saturated sodium bicarbonate solution and extracted with methylene chloride (3 X 20 mL). The extracts were washed with water (1
X 20 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure to give a solid which was 146 dissolved in 10 mL of methylene chloride and excess HCl was bubbled into the solution to give a solid. Recrystallization from ethanol gave 0.183 g (88%) of £3 as a white solid: mp
197-198°C; IR (KBr, cm'1) 3100 (OH, NH) ; JH NMR (CD3OD) 6 7.18-
7.09 (m, 7H, ArH), 6.75 (d, 1H, J = 7.7 Hz, ArH), 4.38 (t, 1H,
J = 8.5 Hz, CHCH2), 3.74 (s, 4H, NCH2CH2N) , 3.22 (d, 2H, J =
8.5 Hz, CHCH2), 2.28 (s, 3H, ArCH3); MS m/z 280 (M+ - HCl), 279
(base). Analysis for C18H21C1N20- JH20; calculated: C, 66.53; H,
6.67; N, 8.63; found: C, 6 6 .8 8 ; H, 6.72; N, 8.62.
Ethyl 3 - ( 3-benzyloxyphenyl)- 3-hydroxy-3 -(4 - methylphenyl)propionate (100)
OH
C O O E t
CH 3
To a solution of 9j> (8.25 g, 27.3 mmol) and bromoethylacetate (1.67 g, 10 mmol) in 100 mL of dry benzene was added activated zinc (654 mg, 10 mmol) under an argon atmosphere. The reaction mixture was refluxed for 1 hour, poured into 100 mL of cold 20% sulfuric acid, and stirred at room temperature for 1 hour. The acid layer was drawn off and the organic layer was extracted with cold 10% sulfuric acid (2
X 50 mL). The organic layer was washed with a cold 10% sodium bicarbonate solution (1 X 100 mL), cold 5% sulfuric acid (1 X
100 mL), and water (2 X 100 mL). The combined acid fractions were extracted with diethyl ether (2 X 200 mL). The combined diethyl ether and benzene solutions were dried over sodium sulfate and concentrated under reduced pressure to give an oil. The resulting oil was purified by flash column chromatography using a solvent system of ethylacetate/hexane
5:95. The combined fractions from the column were evaporated under reduced pressure yielding 8.9 g (84%) of 100 as a white solid: mp 70-71°C; IR (KBr, cm 1) 3485 (OH) 1708 (C=0); XH NMR
(CDCI3/TMS) 6 7.41-6.80 (m, 13H, ArH), 5.03 (s, 1H, OH), 5.01
(s, 2H, OCHzPh), 4.09 (q, 2H, J = 7.1 Hz, CH2CH3) , 3.22 (s, 2H,
CH2COOEt), 2.3 (s, 3H, ArCH3), 1.16 (t, 3H, J = 7.1 Hz, CH2CH3);
MS m/z 390 (M+), 91 (base); Analysis for C25H2604; calculated:
C, 76.90; H, 6.71; found: C, 77.01; H, 6.79. 148
Ethyl 3-(3-benzyloxyphenyl)-3-(4-methylphenyl)-2-propenoate
(101)
C O O E t
CH
A solution of 100 (3.7 g , 9.5 mmol) in 20 mL of benzene was treated with p-toluenesulfonic acid monohydrate (180 mg, 0.95 mmol). The reaction mixture was refluxed for 1 hour, poured into 30 mL of saturated sodium bicarbonate solution, and extracted with diethyl ether (2 X 20 mL). The combined organic layers were washed with brine (1 X 20 mL), dried over sodium sulfate, and concentrated under reduced pressure to give 3.16 g (89%) of 101 as an oil. Separation of isomers was not successful: IR (Neat, cm"1) 1720 (C=0) 1600 (C=CH); XH NMR
(CDCI3/TMS) 6 7.43-6.80 (m, 13H, ArH), 6.33 and 6.30 (s, 1H,
C=CH, mixture of isomers), 5.03 and 5.00 (s, 2H, CH2Ph, mixture of isomers), 4.03 and 4.06 (q, 2H, J = 7.1 Hz,
COOCH2CH3, mixture of isomers), 2.38 and 2.34 (s, 3H, ArCH3, mixture of isomers), 1.14 and 1.10 (t, 3H, J = 7.1 Hz,
COOCH2CH3, mixture of isomers); MS m/z 372 (M+), 91 (base).
Analysis for C25H2403: calculated: C, 80.62; H, 6.49; found: C,
80.59; H, 6.45. 149
2 -f 2-(3-Benzyloxyphenyl)-2 -(4-methylphenyl)-(Z )-
ethenyll Imidazoline oxalate (Z-102) and 2-f2-(3-
Benzyloxyphenyl)-2-(4-methylpheny1)-(E )-ethenyl1Imidazoli ne
oxalate (E-102)
O C H - P h
NH • (C O O H )
• (C O O H ) NH
CH 3 CH 3
A stirred solution of trimethylaluminum (2.0 M solution in
toluene, 30 g, 74 mmol) in 100 mL of dry toluene with cooling
in an ice-water bath, and ethylenediamine (2.88 g, 48 mmol)
was added under an argon atmosphere with continued cooling.
After methane evolution ceased, 101 (10.65 g, 29 mmol) in 100
mL of dry toluene was added dropwise. The reaction mixture was
refluxed for 40 hours, cooled in an ice-water bath, treated with 30 mL of water dropwise, and diluted with 100 mL of
methanol and 100 mL of methylene chloride. The mixture was
refluxed for 30 minutes, dried over sodium sulfate, and
concentrated under reduced pressure to give an oil. The oil was suspended in 300 mL of ethyl acetate and refluxed for 30
minutes in order to remove traces of aluminum hydroxides from
the crude product. The reaction mixture was dried over sodium 150 sulfate and concentrated under reduced pressure to give 7.5 g
(70%) of the free base. A solution of the free base (7.5 g,
20.37 mmol) in 20 mL of methanol was treated with a solution of oxalic acid dihydrate (2.7g, 21.39 mmol) in 20 mL of methanol. The mixture was concentrated under reduced pressure to give a solid which was recrystallized from ethyl acetate
i /ethanol to give 3.21 g (34%) of the Z-102 as a white solid and 2.9 g (31%) of the E-102 as a white solid . Z-102: mp 200-
201°C; IR (KBr, cm'1) 3467 (NH); NMR (CD30D) 6 7.45-6.83 (m,
13H, ArH), 6.53 (s, 1H, C=CH), 5.11 (s, 2H, OCH2Ph), 3.75 (s,
4H, NCH2CH2N), 2.34 (s, 3H, ArCH3) ; MS m/z 368 (M+ - C2H204) , 277
(base). Analysis for C27H26N205*$H20; calculated: C, 69.36; H,
5.82; N, 5.99; found: C, 69.06; H, 6.20; N, 5.79. E-102: IR
(KBr, Cm'1) 3460 (NH); XH NMR (CD3OD) 6 7.35-6.86 (m, 13H,
ArH), 6.50 (s, 1H, C=CH), 5.03 (s, 2H, OCHzPh) , 3.80 (s, 4H,
NCH2CH2N), 2.42 (s, 3H, ArCH3); MS m/z 368 (M+ - C2H2C>4) , 277
(base). Analysis for C27H26N2005; calculated: C, 70.73; H, 5.72;
N, 6.11; found: C, 70.42; H, 5.97; N, 6.02. 151
2 - T 2 -(3-Hydroxyphenyl)- 2 -(4-methylphenyl)-(Z )- ethenyl1Imidazoline hydrochloride (Z-64)
OH
NH
• H C l
CH 3
A solution of Z-102 (0.9 g, 1.96 mmol) in 30 mL of
HCl/methanol (50:50) was refluxed for 4 hours and concentrated under reduced pressure to give an oil, which was crystallized from ethanol/diethyl ether to give 0.52 g (84%) of Z-64 as a tan solid: mp 223-225°C; IR (KBr, cm"1) 3255 (OH, NH); XH NMR
(CD3OD) 6 7.32 (t, 1H, J = 7.9 Hz, ArH), 7.23 (s, 4H, ArH),
6.96-6.65 (m, 3H, ArH), 6.49 (s, 1H, C=CH), 3.81 (s, 4H,
NCH2CH2N), 2.36 (S, 3H, ArCH3) ; MS m/z 278 (M+ - HCl), 277
(base). Analysis for C18HigClN20; calculated: C, 68.76; H, 6.10;
N, 8.92; found: C, 68.47; H, 6.14; N, 8.64. 152
2 ~T 2 -(3-Hydroxyphenyl)-2 -(4-methylphenyl)-(E ) - ethenyl1Imidazoline hydrochloride (E-64)
OH
• H C l NH
CH
The protected phenol E-102 (0.32 g, 0.87 mmol) was treated with 20 mL of a saturated sodium bicarbonate solution, extracted with methylene chloride (3 X 20 mL), and the extracts were dried over sodium sulfate. The solvent was removed under reduced pressure to give an oil. The oil was dissolved in 10 mL of methylene chloride and 1.7 mL of boron trichloride (1M solution in methylene chloride, 1.7 mmol) were added to the mixture under an argon atmosphere. The reaction mixture was stirred for 5 hours at room temperature, treated with 2 mL of methanol and stirred for an additional 1 hour.
The mixture was concentrated under reduced pressure to give an oil which was crystallized from methylene chloride/hexane to give 0.23 g (65%) of E-64 as a tan solid: mp 123-125°C; IR
(KBr, cm'1) 3255 (OH, NH); *H NMR (CD3OD) 6 7.32-6.72 (m, 8H,
ArH), 6.45 (S, 1H, C=CH), 3.81 (s, 4H, NCH2CH2N), 2.42 (s, 3H,
ArCH3); MS m/z 278 (M+ - HCl), 277 (base). Analysis for 153
Ci8Hi9ClN20? calculated: C , 68.76; H, 6.10; N, 8.92; found: C,
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