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Part 1: Design, synthesis and biological activities2-(4 of -isothiocyanatobenzyl)imidazoline analogues in rat and bovine tissues. Part 2: Design and synthesis of selective 2-amino-3-(3 -hydroxy-5f -methylisoxazol-4 t -yl)propanoic acid (AMPA) receptor antagonists for potential use in drug abuse
Slavica, Meri, Ph.D.
The Ohio State University, 1994
Copyright ©1994 by Slavica, Meri. All rights reserved.
UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 Part 1: Design, Synthesis and Biological Activities of
2-(4’-lsothiocyanatobenzyl)imidazoline Analogues in
Rat and Bovine Tissues Part 2: Design and Synthesis of Selective 2-Amino-3-(3’-hydroxy 5’-methylisoxazol-4’-yl)propanoic Acid (AMPA) Receptor
Antagonists for Potential Use in Drug Abuse
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate School of the Ohio State University
by
Meri Slavica, B.S. Pharm.
*****
The Ohio State University 1994
Dissertation Committee: Duane D. Miller, Ph.D. Robert W. Brueggemeier, Ph.D. Duane D. Miller, Ph.D.,co-advisor
Larry W.Robertson, Ph.D. Dennis R. Feller, Ph.D. Robert W. Brueggemei&^Ph.D., co-advisor College of Pharmacy Copyright by Meri Slavica 1994 DEDICATION
To my Mom and Dad
& ACKNOWLEDGEMENTS
I would like to express my sincere thanks to the following individuals:
Dr. Duane D. Miller for his guidance, friendship and never ending enthusiasm throughout my graduate career, and especially for his encouragement and support after moving to UT-Memphis.
The members of my dissertation committee Dr. Robert Brueggemeier, Dr. Dennis Feller and Dr. Larry Robertson for their help and advice in preparing this manuscript.
Longping Lei (M.S.), Dr. Popat Patil and Dr. Dennis Feller (fax-pals) for their patience and persistence in obtaining the pharmacological data for IBI analogues in rat aortic tissues. Also for their good sense of humor that was a necessity in a process of solving the IBI mystery.
Dr. Norman Uretsky, Dr. Lane Wallace and Ms. Kara Brooks for their work on the biological evaluation of the AMPA antagonists.
Dr. Norman Uretsky for his comments and help in preparing the second part of this document.
Dr. Paul Ernsberger for the pharmacological data of IBI and its analogues in the bovine ventrolateralmedulla.
Dr. Jeff Herndon for his invaluable help and advice on molecular modeling aspects of this work and for his friendship.
John Miller for his help in running the NMR (specially one at the College of Pharmacy, UT Memphis), obtaining mass spectra of my compounds and for his support and friendship.
Ms. Kathy Brooks for all her help and assistance with administrative problems, for securing my paychecks and specially for helping me meet the graduation requirements and deadlines.
Ms. Stella Bain (UT-Memphis), Ms. John Dandrea (OSU) and Ms. Carol Settlers for all their help and assistance throughout my stay at The Ohio State University and University of Tennessee-Memphis.
The faculty members of the Division of Medicinal Chemistry and Pharmacology at The Ohio State University: Dr. Robert Brueggemeier, Dr. Robert Curley, Dr. Raymond Doskotch, Dr. Duane Miller, Dr. Carter Olson, Dr. Larry Robertson, Dr. Albert Soloway and Dr. Donald Witiak for giving me an opportunity to study in the USA, for sharing their, knowledge, and for providing me with excellent environment to learn and work. I would also like to take this opportunity and thank them for awarding me with Hoechst-Roussel Graduate Student Award in Medicinal Chemistry.
My friends and lab-mates: Kim, Hong, Mustapha, Yasser, Kazu, Yoshiya, Ron, Jeff, Pat, Sun, Joe, Pam, Lakshmni and Sophie for their friendship, advice, help, support and tolerance.
The Ohio State University and National Institute of Health for their financial support.
Most of all to John H. Sheley for his love, friendship, support and tolerance. Teddy Bear, I wouldn’t be able to make it without you! VITA
June 1,1965 Bom-Sibemk, Croatia (Yugoslavia) Jun, 1987 B.S. Pharmacy, College of Pharmacy, University of Zagreb Zagreb, Croatia(Yugoslavia)
Jan. 1 9 8 8 -Sept. 1989 Graduate Teaching Assistant College of Pharmacy University of Zagreb Zagreb, Croatia(Yugoslavia) Sept. 1989-July 1991 Graduate Teaching Assistant College of Pharmacy, The Ohio State University Columbus, Ohio
July 1991 - Oct. 1992 Graduate Research Associate College of Pharmacy, The Ohio State University Columbus, Ohio
Oct. 1992-present Graduate Research Associate College of Pharmacy, University of Tennessee-Memphis
PUBLICATIONS 1. Rendic, S.; Slavica, M. Urinary Excretion and Metabolism of Orally Administered Mefenorex. In Recent Advances in Doping Ana/ys/s-Proceedings of the 11th Cologne Workshop on Dope Analysis 7th-12th March 1993. Eds. M. Donike, H. Geyer, A. Gotzmann, U. Mareck-Engelke, S. Rauth, Sport und Buch Strau(3, Koeln 1994.
2. Rendic, S.; Slavica, M. Urinary Excretion and Metabolism of Orally Administered Mefenorex. Eur. J. Drug Metab. Pharmacokin. (accepted for publication)
3. Slavica, M.; Lei, L.; Patil, P.; Kerezy, A.; Feller, D.; Miler, D. Synthesis and Biological Activities of a New Set of Irreversibly Acting 2-(4’-lsothiocyanatobenzyl)imidazoline (IBI) Analogues in Rat Thoracic Aorta. J. Med. Chem. (in press)
FIELDS OF STUDY Major Reid: Pharmacy Studies in Synthetic Medicinal Chemistry
v TABLE OF CONTENTS
PAGE DEDICATION ...... i
ACKNOWLEDGEMENTS ...... iii VITA ...... v
LIST OF FIGURES...... Ix
LIST OF TABLES...... xii
LIST OF SCHEMES...... xiv Part 1 Design, Synthesis and Biological Activities of 2-(4’-lsothiocyanatobenzyl) imidazoline Analogues in Rat andBovine Tissues ...... 1
CHAPTER I INTRODUCTION ...... 2 1.1 The Autonomic Nervous System ...... 2 1.2 The Adrenergic Nervous System ...... 3 1.2.1 Subclassification of a-Adrenergic Receptors ...... 4 1.2.2 Subclassification of a-i-and ar Adrenergic Receptors ...... 7 1.2.3 Molecular Biology of a-Adrenergic Receptors ...... 10 1.2.3.1 Purification, Cloning and Expression of ar AR — ...... 10 1.2.3.2 Purification, Cloning and Expression of 0 2 -AR ...... 11 1.2.4 a-Adrenergic Receptor Structure ...... 13 1.2.5 The Role of Affinity and Photoaffinity Labeling Techniques and Site Directed Mutagenesis in Molecular Characterization ofa-AR ...... 16 1.2.6 a-Adrenergic Receptors and their Signal Transduction Pathways 18 1.3 Calcium Channels ...... 20 1.3.1 Classification of Calcium Channels ...... 21 1.3.2 Drugs Active at Calcium Channels ...... 23 1.3.2.1 Calcium Channel Antagonists ...... 23 1.3.2.2 Calcium Channel Agonists(activators) ...... 27 1.3.3 Structure of the Calcium Channels ...... 28 1.3.4 The Role of Affinity and Photoaffinity Labeling in the Identification of Calcium Channel Antagonist Binding S ites ...... 30 1.4 Imidazoline Receptors ...... 32 1.4.1 Phenethanolamines vs Imidazolines ...... 32 1.4.2 Identification of non-adrenergic Imidazoline Binding Sites ...... 34 1.4.3 Endogenous Ligand for Imidazoline Receptor ...... 36 1.4.4 Classification of Imidazoline Receptors ...... 37 1.4.5 Isolation and Characterization of Imidazoline Receptor ...... 39
vi CHAPTER II STATEMENT OF PROBLEMS AND OBJECTIVES...... 42
CHAPTER III RESULTS AND DISCUSSION ...... 47 3.1 Chemistry ...... 47 3.2 Biology ...... 56 3.2.1 Effects of PBZ Pretreatment on the Contractile Activities of IBI Analogues on Rat A o rta ...... 56 3.2.2 Effect of Imidazoline Ligand (idazoxan, cirazoline) Pretreatment on Stimulatory Activities of IBI and Analogues 6Z - ZQ on Rat Thoracic Aorta ...... 61 3.2.3 Effects of IBI and its Analogues 65. f>Z, 68, and 70 on Saturation Binding Kinetics of [i25|]p-lodoclonidine at ^-Imidazoline and o^-AR in the Bovine VLM ...... 63 3.2.4 Effect of Ca2+-Channel blocker (Nifedipine, Verapamil) Pretreatment on the Stimulatory Activities of IBI and Analogues 67 - 70 on Rat Thoracic Aorta ...... 65 3.3 Molecular Modeling ...... 68 3.4 Summary ...... 75
CHAPTER IV EXPERIMENTAL ...... 76
Part 2 Design and Synthesis of Selective 2-Amino-3-(3’-hydroxy-5’-methyl- isoxazol-4’-yl)propanoic Acid (AIUIPA) Receptor Antagonists for Potential Use in Drug Abuse ...... 108
CHAPTER V INTRODUCTION...... 109 5.1 L-Glutamate-Primary Excitatory Neurotransmitter ...... 109 5.2 Excitatory Amino Acid Receptors ...... 110 5.2.1 NMDA Receptors ...... 111 5.2.1.1 Agonists at NMDA Receptors ...... 112 5.2.1.2 Competitive antagonists at NMDA receptors ...... 115 5.2.1.3 Noncompetitive NMDA receptor antagonists ...... 116 5.2.2 AMPA receptors ...... 116 5.2.2.1 AMPA receptor agonists ...... 118 5.2.2.2 AMPA receptor antagonists ...... 120 5.2.3 Kainate receptors ...... 121 5.2.4 AP4 receptors ...... 123 5.2.5 ACPA(Metabotropic) receptors ...... 123 5.3 Molecular biology of EAA receptors ...... 124 5.4 Role of Excitatory Amino Acids in Drug Abuse ...... 127 5.4.1 Cocaine and Amphetamine Abuse: History and Epidemiology ...... 127 5.4.2 Mechanism of Action of Psychostimulant Drugs ...... 129 5.4.3 Dopaminergic and Glutaminergic Systems in Nucleus Accumbens (NAc) and Actions of Psychostimulant Drugs ...... 130 5.4.4 Current Pharmacotherapies for Cocaine and Amphetamine A buse 133
CHAPTER VI STATEMENT OF PROBLEMS AND OBJECTIVES...... 136
vii CHAPTER VII RESULTS AND DISCUSSION ...... 143 7.1 Chemistry ...... 143 7.1.1 Synthesis of o-Tyrosine Derivatives ...... 143 7.1.2 Synthesis of Quinoxaline-2,3-dione Amino Acids ...... 161 7.2 Biology ...... 168 7.3 Summary ...... 171 CHAPTER VIII EXPERIMENTAL...... 172
BIBLIOGRAPHY...... 192
viii LIST OF FIGURES
FIGURE PAGE 1. a!-Adrenergic Receptor Agonists and Antagonists ...... 5
2. c^-Adrenergic Receptor Agonists and Antagonists ...... 6
3. Transmembrane Topology of the a1B-Adrenoceptor ...... 15
4. Affinity and Photoaffinity Ligands for a-A R ...... 17
5. Cross Section Arrangement of Transmembrane Helices of a-AR and Interaction with Norepinephrine ...... 18
6 . Proposed Interaction between a-AR, G-Protein and Calcium Channel . . . 20
7. Calcium Channel Antagonists ...... 24
8. New Structural Classes of Calcium Channel Antagonists ...... 26
9. Calcium Channel Agonists ...... 27
10. Schematic Representation of L-Type Calcium Channel ...... 29
11. Representative Affinity and Photoaffinity Probes for Calcium Channels .. 31
12. Schematic Representation of ar Subunit of the L-Type Calcium Channel and Location of the Binding Sites for Phenylalkylamines (A) and 1,4-Dihydropyridines (B) ...... 32
13. Schematic Representation of Easson-Stedman Hypothesis for Norepinephrine ...... 33
ix 14. Imidazoline Receptor Ligands ...... 38
15. The Action of IBI on Rat Thoracic Aorta in Physiological Salt Solution (PSS) as a Control and in Ca2+ Defficient B uffer ...... 66
16. Superimposition of IBI (yellow) and Nifedipine (multicolor) ...... 70
17. Superimposition of IBI (yellow) and Bay K 8644 (m ulticolor) ...... 71
18. Superimposition of §7 (purple) and Nifedipine (m ulticolor) ...... 72
19. Superimposition of §Z (purple) and Bay K8644 (multicolor) ...... 73
20. Superposition of 68 (red) and Nifedipine (multicolor) ...... 74
21. Compounds Used to Classify EAA Receptor Subtypes 111
22. Schematic Representation of EAA Receptor Subtypes ...... 113
23. Some NMDA Receptor Agonists ...... 114
24. Competitive NMDA Receptor Antagonists ...... 115
25. Noncompetitive Antagonists of NMDA Receptor Acting at the PCP and Glycine Site ...... 117
26. AMPA Receptor Agonists ...... 119
27. AMPA Receptor Antagonists ...... 121
28. Kainate Receptor Agonists ...... 122
29. AP4 Receptor Agonists and Antagonists ...... 123
30. ACPD (Metabotropic) Receptor Antagonists ...... 124
31. Schematic Representation of Glutamate Receptor Subunit ...... 126
x 32. Cocaine Reinforcing Action ...... 130
33. Relationship between Dopaminergic and Glutaminergic Neurones ... 132
34. HPLC Chromatogram of Derivatized D-DNoTyr with Marfy’s Reagent .. 155
35. HPLC Chromatogram of Derivatized L-DNoTyr with Marfy’s Reagent .. 157
36. HPLC Chromatogram of o-Tyr, DNoTyr and N-Acetyl-DNoTyr ...... 158
37. Progress of L-DNoTyr Formation from N-Acetyl-DNoTyr Catalyzed by Aspergillus Acylase I...... 159
38. Mass Spectrum of Compound 207 ...... 166
39. Effect of Different DNoTyr and o-Tyrosine Analogues on PH] AM PA Binding to Brain Membranes ...... 169
40. Inhibition of Specific pH]AMPA Binding by Compounds 164 and 16g ... 170
xi LIST OF TABLES
TABLE PAGE 1. Classification and Characteristics of a r A R ...... 12
2. Classification and Characteristics of 0 2 -AR ...... 14
3. Classification of Voltage-Gated Ca2+ Channels ...... 22
4. Current and Potential Uses of Calcium Channel Antagonists ...... 24
5. Classification and Characteristics of Imidazoline Receptors ...... 40
6. Comparison of Maximal Contractile Tension Changes and Potencies of IBI and its Analogues 61. §2. 65,66 and 71 with and without PBZ Pretreatment on Rat Thoracic Aorta Strips ...... 57
7. Comparison of Maximal Contractile Tension Changes and Potencies of IB! Analogues 68 and 78 with and without PBZ Pretreatment on Rat Thoracic Aorta Strips ...... 58
8. Comparison of Maximal Contractile Tension Changes and Potencies of IBI Analogues 69 and 70 with and without PBZ Pretreatment on Rat Thoracic Aorta Strips ...... 59
9. Effect of Imidazoline Ligands on Contractile Responses of IBI and its Analogues 6Z - 70 in Rat Thoracic Aorta ...... 62
10. Summary of Binding Properties of IBI and its Analogues 65,87, 68 and 70 at h-lmidazoline and a2-AR Binding Sites in Bovine V L M 64
11. Effects of Various Treatments on the Concentration Dependent
xii Responses to IBI and Selected IBI Analogues fiZ - ZQ in Rat Thoracic Aorta ...... ; ...... 67
12. Excitatory Amino Acid Receptor Fam ily ...... 125
13. Medication Under Investigation for Treatment of Drug Abuse ...... 135
xiii LIST OF SCHEMES
SCHEME PAGE I Proposed IBI Related Analogues for Studying the Structural Requirements for the Interaction with a- and non-a-AR ...... 45
II Synthesis of 2-(4’-Nitrobenzyl)imidazoline Z4 ...... 47
III Synthesis of Imidazolines 60,65,66 and 71 ...... 48
IV Synthesis of 2-(4’-Methylbenzyl)imidazoline 62 ...... 50
V Synthesis of Imidazolines 67 and gg ...... 51
VI Attempted Dehydrogenation of 74 to g g ...... 52
VII Synthesis of 2-(4’-lsothiocyanatobenzyl)imidazole 69 ...... 53
VIII Synthesis of N.N’-Dimethyl^-^’-lsothiocyanatophenylJethylamineTQ .. 54
IX Synthesis of 2-(3’-lsothiocyanatobenzyl)imidazoline 6 4 ...... 55
X Attempted Synthesis of 2-(2’lsothiocyanatobenzyl)imidazoline g g 55
XI Asymmetric Synthesis of D-3,5-Dinitro-o-Tyrosine 1 6 7 ...... 144
XII Synthesis of N-(Phenylsulfonyl)-L-Serine 175 ...... 145
XIII Formation of oc-Amino Ketone from N-(Phenylsu!fonyl)-L-Serine and Grignard Reagent 1 8 7 ...... 146
XIV Synthesis of Ketone 178 from N-(Phenylsulfonyl)-L-Serine and Grignard Reagent 1 8 7 ...... 147
xiv XV Reduction of Carbonyl Group of 178 to Methylene in 179 or to sec-Alcohol in 188 ...... 148
XVI Reactions Attempted to Reduce sec-Alcohol 188 to Methylene Group in order to Obtain 1 7 9 ...... 149
XVII Modifications of Reduction Reaction with EtaSiH/TFA ...... 150
XVIII Attempted Resolution of D-3,5-Dinitro-o-Tyrosine from the Racemic Mixture using L-Amino Acid Oxidase ...... 153
XIX Chemo-enzymatic Synthesis of D-3,5-Dinitro-o-Tyrosine 167 Utilizing L-Amino Acid Oxidase ...... 154
XX Chemo-enzymatic Synthesis of L-3,5-Dinitro-o-Tyrosine Utilizing Aspergillus Acylase I...... 156
XXI Synthesis of 3.5-Dinitro-2-methoxyphenylalanine 169 ...... 160
XXII Synthesis of 1 7 0 ...... 160
XXIII Derivatization of 3,5-Dinitro-o-Tyrosine with Marfy’s Reagent 1 9 Z ...... 162
XXIV Synthesis of Quinoxaline-2.3-dione Amino Acid 171 ...... 163
XXV Proposed Asymmetric Synthesis of L-QXAA173 ...... 164
XXVI Synthesis of Serine-p-Lactone 203 ...... 164
XXVII Reaction of Phenylenediamine with Serine-p-lactone 203 ...... 165
XXVIII Fragmentation of 207 ...... 167
xv Part 1
Design, Synthesis and Biological Activities of
2-(4’-lsothiocyanatobenzyl)imidazoline Analogues
in Rat and Bovine Tissues
1 CHAPTER I
INTRODUCTION
1.1. THE AUTONOMIC NERVOUS SYSTEM
The nervous system is divided into the central nervous system (CNS) and the peripheral nervous system (PNS). The PNS is further divided into somatic and autonomic nervous systems. The autonomic nervous system is widely distributed throughout body and controls the so called automatic or vegetative functions. This includes the regulation of the circulation, respiration, digestion, body temperature, carbohydrate and fat metabolism and the secretion of certain endocrine glands. In general, the function of the autonomic nervous system is to maintain the homeostasis of the internal environment and this is achieved through neurohumoral transmission, i The concept of neurohumoral transmission is that nerve impulses elicit responses in smooth, cardiac and skeletal muscles, exocrine glands and postsynaptic neurones through release of specific chemicals called neurotransmitters. Norepinephrine (noradrenaline) and acetylcholine have been established as neurotransmitters in the autonomic nervous system. Furthermore, the autonomic nervous system is divided into sympathetic (adrenergic) and parasympathetic
(cholinergic) systems according to which neurotransmitter is released at the junction between neurones and the peripheral effectors
2 1.2. THE ADRENERGIC NERVOUS SYSTEM
The adrenergic nervous system is that part of the autonomic nervous system where norepinephrine (noradrenaline, 1) is the neurotransmitter between the postganglionic nerve ending and the effector organ. Epinephrine (adrenaline, 2) is a hormone secreted from adrenal medulla cells, which are embryologicaily analogous to postganglionic sympathetic neurones. These endogenous catecholamines, epinephrine 2 and norepinephrine 1, exert their physiological effects on target tissues through adrenergic receptors.3 The adrenergic receptors were initially divided by Ahlquist -» into two general classes, termed alpha (a) and beta (13), based on their relative responses to the series of sympathomimetic amines: epinephrine 2, norepinephrine 1, phenylephrine 2, and isoproterenol 4. For a adrenergic receptors (a-AR) the rank order of potency of sympathetic agents was epinephrine > norepinephrine > phenylephrine > isoproterenol.
In contrast, 13-adrenergic receptors (B-AR) exhibited a different set of responses: isoproterenol > epinephrine > norepinephrine > phenylephrine. Functionally the a-AR were shown to be associated with most of the excitatory functions (vasoconstriction, stimulation of the uterus and pupil dilatation) and inhibition of the intestine. On the other hand the B-AR were implicated in vasodilation, bronchodilation and myocardial stimulation.-*
r 1=r 2=o h , r 3=h NOREPINEPHRINE 1
NHR, r 1=r2=o h , r 3=c h 3 EPINEPHRINE 2
R,=OH, R2=H, R3=CH3 PHENYLEPHRINE3
R,=R2=OH, R3=CH(CH3)2 ISOPROTERENOL 4
The development of highly selective and potent agonists and antagonists for each receptor class led to further subclassification into subtypes. For example, B-AR were subdivided into Bi and B 2 based on their affinities for isoproterenol 4, epinephrine 2 and norepinephrine l.s 4
1.2.1. SUBCLASSIFICATION OF g-ADRENERGIC RECEPTORS
a-AR were first classified based on their anatomical location as pre- and postsynaptic.e Langere proposed that the postsynaptic a-AR that mediates the responses of the effector organ be termed a-i-AR, while the presynaptic a-AR that regulates release of the neurotransmitter, through a negative feedback mechanism, be called a2-AR. Berthelsen and Pettinger suggested a functional classification of a-AR, which was based on the type of function mediated by the receptor subtype. They proposed that the a-ARs that mediate excitatory responses are a1t while a2-ARs are those that mediate responses inhibitory in nature. However, neither of these methods proved reliable for classification of a-AR into subtypes. Development of highly selective and potent antagonist and to some extent agonists, as well as the development of the radioligand binding techniques, led to the pharmacological classification of a-AR. Subclassification of a-AR by this method was based on the relative potencies of a series of agonists and antagonists. Thus, Stark proposed division of a-AR into ap and a2-AR based on their affinities for the antagonists prazosin and yohimbine, respectively^
Some selective a-AR agonists and antagonists that are commonly used in the subclassification of ap and a2-AR are shown in Figures 1 and 2. a 1-AR selective agonists include methoxamine 5, cirazoline £, ST 587 7, amidephrine 8 and Sgd 10175 9, while selective antagonists for ar AR are prazosin 10, doxazosin H and coryanthine 12. On the other side a-AR activated by clonidine 13, guanabenz 14, UK 14305 15, BHT 920 15 and competitively blocked by rauwolscine 17, yohimbine 18. and idazoxan 19, are classified as an a2-AR.s.io Norepinephrine 1, the natural occurring adrenergic neurotransmitter is a relatively nonselective a-AR agonist, while the adrenal hormone, epinephrine 2, elicits slight selectivity for a^AR. 5
OCH, OH ci NH. » = o CH. H,C OCH3 Methoxamine 5 Cirazoiineg ST 587 Z
H OH N = < h 3c o 2s h n v _ / ^ J X /n h c h 3 N' H
I CH3
Amidephrine fi Sgd 101752 ^VVJTO
Prazosin 10
H3COOC' OH ^ nY nWv^N-C-C°X) / 11 0 ^ ^ Coryanthine 3 2
Doxazosin 11
Figure 1. a-|-Adrenergic Receptor Agonists and Antagonists 6
ci ci NH / ~ i _ // r V- c h =n n h - cn NH2 C f N^ M Cl ci Clonidine 13 Guanabenz 14
: N Br H2C=HCH2C - n
UK 1430515 BHT92015
h3c o o c ’
OH OH
Rauwolscine 1 Z Yohimbine 18
Idazoxan 19
Figure 2. a 2 *Adrenergic Receptor Agonists and Antagonists 1.2.2. SUBCLASSIFICATION OF aL AND a2 ADRENERGIC RECEPTORS
The radioligand binding assays and functional studies revealed that both ar AR and cc2-AR could be subdivided into at least two additional subtypes. The existence of two populations of postsynaptic ar AR: a1A and a1B was first suggested by McGarth in
1982J1 His studies with the rat anococcygeus muscle have shown that phenylethanolamines produce dose response curve with a “shoulder” indicating two binding sites.”
Morrow and Creese^ found that displacement of [3H]prazosin at Of-AR binding sites in rat cerebral cortex with antagonists WB4101 20 and phentolamine 21 is biphasic in nature. They suggested that the high affinity site in the rat cerebral cortex be termed a1A-AR, while the low affinity site be termed the a1B-AR. While [3H]prazosin had equal affinity for both ar AR subtypes phentolamine for example had 23 times higher affinity to a 1A than for a1B subtype.’2
0 . CH2NHCH2CH;P
WB 4101 20 Phentolamine 21
In 1987, Johnson and Minneman suggested the existence of two types of ai-AR in the rat cerebral cortex that differed in sensitivity toward chlorethylclonidine (CEC, 22).’3
They found when membranes of cerebral cortex were pretreated with CEC 22, only about half of the total binding sites labeled with p25|]BE 2254 2 3 could be inactivated. However, CEC 22 did not inactivate any [125|]BE 2254 23 binding sites in hippocampus.
Han et aM* extended the studies with CEC 22 to various tissues in order to determine whether selective inactivation might reveal the presence of discrete subtypes of ar AR. It was found that site directed alkylating agent CEC inactivates 70-80% of a-i-AR binding sites in rat liver and spleen, 25% in neocortex, but it had no effect on a r AR in hippocampus, heart, vas deferens and caudal artery.™
OH
CIHaC Cl
Clorethylclonidine 22 [125I]BE 2254 23
Minneman et aljs had shown that subtypes of arAR inactivated by CEC 22 were equivalent to those with low affinity for WB4101 22- They concluded that aiA subtype has a relatively high affinity for WB4101 23 and it is not inactivated by CEC 22. while subtype has a low affinity for WB4101 23 and is CEC 22 sensitive.^
As for arAR, pharmacological and functional studies have suggested that o^-AR are not a homogeneous class of receptors but can be subdivided into subtypes. In 1985,
Bylund suggested a subclassification of a2-AR, based on the relative potency of prazosin 10 .16 The human platelet was the prototype tissue for the a^-AR, which had a low affinity for prazosin. In contrast, neonatal rat lung that was the prototype for a 2B-AR subtype had a high affinity for prazosin 1 0 .1 6 Approximately at the same time another group, Nahorski et alJ? independently proposed a2A and o^b adrenergic receptor subtypes based on differences in affinity of a variety of antagonists such as yohimbine
18, rauwolscine 17, prazosin 10, and indoramine 24.
Bylund and co-workers^ compared the affinities of 34 adrenergic antagonists for a2-AR in binding assays within tissues and cell lines that contained only one subtype. As a source of a^-AR they used human platelet, HT29 cell line and human cerebral cortex, Indoramine 24 Oxymetazoline 25
while neonatal rat lung and NG108-15 cell line were source of a2B-AR. While oxymetazoline 25 was selective for a^-AR, prazosin was cx 2b -AR selective. Furthermore, three new a2B subtype selective drugs were identified: ARC-239 26, chlorpromazine 27.
7-hydroxychlorpromazine 28.i8.is A third a2-AR subtype had been described in the opposum kidney derived OK cell line.zo This receptor was very similar to the cc2b -AR subtype having relatively high affinity for prazosin 10, chlorpromazine 27 and ARC-239
26 and low affinity for oxymetazoline 25. However, comparison of the pKj values of variety of drugs for the a2-AR in OK cells with those for a2A and o^b subtypes gave relatively poor correlation so that a 2-AR in OK cells was designated as the a2c-AR subtype.
h 3c c h .
R=H Chlorpromazine 27 ARC-239 26 R=OH 7-Hydroxychlorpromazine 2fi 10
Bovine pineal and rat submaxillary gland a2-AR had been designated as a fourth subtype: the a2o.21 This receptor had high affinity for phentolamine 21 and lower affinity for rauwolscine 17 and SKF104078 29 than the three other subtypes.
ci
SKF-104078 29
1.2.3. MOLECULAR BIOLOGY OF a-ADRENERGIC RECEPTORS
Definitive evidence for existence of a-AR subtypes was obtained through molecular cloning studies. 1.2.3.1 .Purification, cloning and expression of ot^-AR
Complete purification of intact ar AR was a necessary prerequisite to explore their molecular biology. The (immobilized prazosin analog), wheat germ lectin chromatography and size exclusion H P L C .2 2 The purified peptide Mp=80,000, showed all the characteristics of an a-i-AR. The purified receptor was then cleaved at methionine residues by CNBr, and three peptides were resolved by reverse-phase H P L C .2 2 On the basis of the sequence of one of the peptides an oligonucleotide probe was constructed, radiolabeled and used to screen a hamster genomic library. The cDNA encoding for the c^-AR expressed in the Syrian hamster was obtained. The deduced amino acid sequence encoded for a protein of 515 amino acids with a calculated Mr of 56,000 daltons. These results suggest that 30% of the Mr of the purified ar AR is carbohydrate. Two other distinct a-pAR were subsequently isolated: one from bovine b ra in z 3 and another one from human hippocampus.24 In order to confirm that isolated cDNA encodes the protein of interest (in this case ar AR) the cDNA from Syrian hamster and cDNA from bovine brain were inserted into the expression vector pBC12BI (separately) and used to transfect COS-7 cells. ar ARs expressed in COS-7 cells were able to bind the ai adrenergic receptor antagonists [125|]HEAT (also known as [i25|]BE-2254, 23) with high specific activity and affinity. On the basis of the ligand binding properties of the three apARs after expression in COS-7 cell it become clear that the receptor isolated from hamster appears to have characteristics of the pharmacologically defined ocib subtype,*2 while the receptor cloned from rat brain appears to be the subtype.zs The bovine cDNA clone expressed in COS-7 cells shows similar pharmacological binding properties to one described for gcia receptors: high affinity for WB4101 20 and phentolamine 21 and the agonist oxymetazoline 25- However, while ar AR are insensitive to CEC 22 bovine ar AR are not .23 It was therefore proposed that bovine ar AR be termed the a1c-AR. Important features of ar AR subtypes are summarized in Table 1 .Z6 1,2.3.2.Purification. cloning and expression of ctg-AR The strategy for cloning of DNA encoding the ot2-ARs was similar to the one described above for ar ARs. The a2-ARs of human platelets had been purified to apparent homogeneity by a combination of affinity chromatography over SKF 101253 Sepharose CL-6B, heparin agarose and WGA-agarose chromatography.z7 The purified a2-AR shows a single major band of Mr=64,000 on SDS-PAGE, and pharmacology characteristic for 0 2 receptors.Z7 Chemical cleavage with CNBr and enzymatic cleavage (protease) of the purified receptor gave 4 peptides which are separated on HPLC. On the basis of sequence of one of these peptides two overlapping oligonucleotide probe 12 Table 1. Classification and Characteristics of ai-AR (Modified from Lomasney, 26) a l AB C Localization on 5 human chromosome 5 8 Amino acids 560(rat) 515(rat) 466(bovine) Giycosylation sites yes yes yes Tissue localization cerebral cortex, cerebral cortex, rabbit liver hippocampus, liver, heart vas deferens, aorta P.ha^acplpjgxpf ai;AR Agonist Norepinephrine> Oxymetazoline> Oxymetazoline» Epinephrine> Norepinephrine> Epinephrine> Phenylephrine> Epinephrine> Norepinephrine> Oxymetazoline Phenylephrine Phenylephrine Antagonist Prazosin> Prazosin» Prazosin> WB4101» WB4101> WB4101> Phentolamine Phentolamine Phentolamine were constructed, radiolabeled and used to screen human genomic library. This led to isolation and identification of the first o^-AR gene, the one encoding for the human platelet receptor.28 Somatic cell hybrid analysis revealed that this gene resides on human chromosome 10, so the receptor was termed a2C10. Southern blot analysis of human genomic DNA suggested the possible existence of two additional a2-AR residing on chromosomes 2 and 4. Subsequently these additional genes were isolated. Regan and co-workersz® used the 0.95 kb Pstl restriction fragment of the a2-AR gene for the human platelet as a probe to screen human kidney cDNA library. They 13 cloned a distinct a2-AR subtype whose gene resides on chromosome 4, and it was termed Lomasney et al .26 using different approach, based on polymerase chain reaction, isolated the third human c^-AR gene located on chromosome 2, which they termed CX 2C2 . When each of these three a2-AR genes were expressed in COS-7 cells, separately, the receptors displayed unique pharmacologies. The platelet a2C10 subtype showed relatively low affinity for prazosin I f l and high affinity for oxymetazoline 25 . In contrast, oxymetazoline 25 bound to a2C2 AR with low affinity while prazosin 10 showed relatively high affinity. Finally the kidney 0^04 adrenoceptor had a relatively intermediate affinity for prazosin 10. Oxymetazoline 25 seem to be the most useful drug to discriminate between these three receptor subtypes having approximately a 10-fold difference in affinity for each receptor. Bylund et a l .30 compared the pharmacological characteristics of the pharmaco logically defined a2-AR subtypes with the characteristics of the receptors identified by molecular cloning and expressed in COS 7 cells. The major conclusion of their study was that according to the correlation analyses of the pK| values a2A subtype (from HT29 cell line), corresponds to the cloned human o^CIO, ot^ receptor of the neonatal rat lung can be assigned to a2C2 clone and a2c subtype represents clone . 30 Table 2 summarizes important features of the proposed a2-AR sub typ es .26 1.2.4. a-ADRENERGIC RECEPTOR STRUCTURE Based on the characterization of purified protein? the a-i and a2-AR have been identified as glycoproteins with a molecular weight of 80,000 and 64,000 daltons, respectively. The a-ARs are members of large family of receptors (I3-AR, dopamine, muscarinic receptors, 5HT, rhodopsin) that mediate their effects through G-protein. The primary structure of these proteins share remarkable similarity. Hydrophobicity analysis of the identified amino acids sequence of a-AR reveled seven hydrophobic clusters, each Table 2. Classification and Characteristics of 0C2-AR (Modified from Lomasney, 26) 02 AB C D Localization on human chromosome 10 2 4 ? Amino acids 450(human) 450(human) 461 (human) ? Glycosylation sites yes no yes ? Tissues localization cerebral cortex, liver, cerebral cortex rat submaxiliary, human platelet kidney, bovine pineal neonatal lung . Rharmacology o f 0 2 -AR. Agonist Oxymetazoline» Epinephrine> Oxymetazoline= Oxymetazoline» Epinephrines Norepinephrines Epinephrines Norepinephrine Norepinephrine Oxymetazoline Norepinephrine Antagonist Phentolamine= Phentolamines Rauwolscine> Rauwolscine> Rauwolscine»> Rauwolscine» Phentolamine> Prazosin Prazosin Prazosin Prazosin of which comprises 20-25 hydrophobic residues, separated by stretches of hydrophilic residues. It has been proposed that hydrophobic regions represent seven transmembrane-spanning domains that are connected by 3 extracellular and 3 intracellular loops, with NH2 terminus being extracellular and COOH terminus intracellular.27 Figure 3 presents deduced amino acid sequence and putative topography of the hamster a 1B-A R .2 2 .2 & t 2 6 .31 The proposed arrangement of the protein in the membrane is based primarily on the high resolution diffraction studied of the membrane protein, bacteriorhodopsin, the purple membrane protein of Halobacterium Halolium.32.a1 u c x ^ -N H 2 o ;o m o Extracellular Membrane Intracellular £)©©©00000* rt0©GG0O0000O®®®®00®®^^ HOOC-O®®®6^ Figure 3. Transmembrane Topology of the a^-Adrenoceptor (Modified from Lomasney, 26) 16 The most conserved regions in the structure of a-AR are the putative transmembrane domains. On the other hand the amino and carboxyl terminus as well as third intracellular loop differ the most in amino acid composition and length. For example the COOH terminus is the longest in the a-pAR while the third cytoplasmic loop is longest in the ct2-AR (~150 amino acids ).22 Each of the receptors contains putative glycosylation sites in the amino-terminal region. apAR are glycosylated with complex type N-linked oligosaccharides at Asp residues. These oligosaccharides may be required to protect the amino terminus from protease attack. The a2-AR contains an essential sulfhydryl residue.^ Multiple serine and threonine residues found in intracellular loops may be sites for phosphorylation by regulatory kinases such as protein kinase C and A.22 it has been postulated that major function associated with the cytoplasmic domains of ai and a2-AR is coupling to guanine nucleotide regulatory proteins (G-proteins) while conserved transmembrane domains may be involved in ligand bindings 1.2.5. THE ROLE OF AFFINITY AND PHOTOAFFINITY LABELING TECHNIQUES AND SITE DIRECTED MUTAGENESIS IN MOLECULAR CHARACTERIZATION OF g-AR Affinity and photoaffinity probes have become one of the key approaches for studying the receptor binding sites. Examples of compounds that have been used either for molecular identification of a-AR or to identify a ligand binding region of a-AR are given in Figure 4. Leeb-Lundberg and co-workers described the synthesis of a high affinity radioiodinated a r AR photoaffinity probe [125|]APDQ 30.33 They used this probe to identify and label apAR in variety of mammalian tissues. This probe labeled an apAR peptide of Mr=85,000 in rat hepatic plasma membrane. Endogenous proteolysis of this peptide generated smaller ligand binding fragments (Mr=52,000, 42,000, and 32,000 daltons). 17 o n h 2 125l APDQ30 n3 Phenoxybenzamine 31 SKF102229 32 p-Azidoclonidine 33 Figure 4. Affinity and Photoaffinity Ligands for a-AR Using the affinity label, phenoxybenzamine 3L Regan et al. showed that the ligand binding site of the human platelet a 2-AR is located on a peptide with a molecular mass of Mf=61,000 daltons.w Studies conducted by Matsui et al. implicated the fourth transmembrane domain in the receptor-ligand interactions of the human platelet a 2-AR. Using two specific photoaffinity ligands [3H]SKF 10229 32 (an antagonist) or p-azido[3H] clonidine 31 ( an agonist) and peptide mapping they were able to isolate peptide of Mp=2400 which was part of the fourth transmembrane domain. In contrast, Wang et al. using different approach, site directed mutagenesis, identified Aspi*3 in transmembrane domain III and Cys2oi and Ser?-04 in transmembrane domain V as potential sites of interaction with the N moiety and the catechol hydroxyl group, respectively of the phenylethylamine class of compounds (Figure 5).3s 18 III II Asp113 IV OH VII HO OH SH OH VI Figure 5. Cross Section Arrangement of Transmembrane Helices of a-AR and Interaction with Norepinephrine. (Modified from Wang, 35) Furthermore Suryanarayana and co-workers had shown that replacement of a single amino acid Phe412-Asn in the seventh transmembrane domain of the 0 2 -AR reduces affinity for yohimbine by 350 fold, and increases affinity for B-blocker alprenolol by 3000 fold. These results imply that the seventh transmembrane domain is important for ligand binding.^ 1.2.6. a-ARs AND THEIR SIGNAL TRANSDUCTION PATHWAYS ar ARs produce change in target cells by increasing the concentration of intracellular Ca 2+.26 There is a large body of evidence suggesting that all three arARs (a1A, a1Band a1c) are coupled to phospholipase C via pertussis toxin-insensitive GTP-binding protein (G-protein). Activation of ar AR by an agonist causes dissociation of GDP from G-protein (a subunit), and association of GTP. Activated G-protein, stimulates 19 phospholipase C activity, which initiate hydrolysis of phosphatidylinositol bisphosphate (a membrane phospholipid) to produce two second messengers: diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). IP3 then mediates the release of Ca2+ from intracellular stores (endoplasmatic reticulum). DAG the other second messenger activates the phospholipid-sensitive protein kinase C, which phosphorylates a variety of specific proteins.37 In addition to PLC signalling, pathways activated by ar AR may include phospholipase A2 and phosphatidylcholine-specific phospholipase D.^ It has been shown that some o^-AR may couple directly to influx of extracellular Ca 2+ through receptor operated Ca2+ channels.se-io Traditionally, a2-ARs are coupled, via pertussis toxin sensitive Gr protein to inhibition of adenylyl cyclase. So the activation of G -protein. 40,41 in this case, the a2-AR mediated vasoconstriction is dependent on translocation of extracellular calcium through dihydropyridine calcium channels. Figure 6 depicts the proposed model of interaction between a^- and og-AR, G- proteins and Ca2+ mobilization in vascular smooth muscle, according to Nichols and Ruffolo .42 20 HO, NHCH) contractions Figure 6. Proposed Interaction between a-AR,G-Protein and Calcium Channel. (Modified from Nichols, 42) 1.3. CALCIUM CHANNELS The cell usually maintains a very low concentration of ionized calcium, approximately 10-8 M, however during the excitation this rises to the level of 10-6 - 1 0 -7 M. Increase in intracellular calcium concentration can be induced in two different ways: chemically or electrically. Example of chemically induced increase in the intracellular Ca2+ concentration is activation of a r or a2-AR (as previously described). In contrast some cells respond to membrane depolarization with an increase in the concentration of intracellular Ca2+ and this is an example of voltage-dependent Ca2+ mobilization. Calcium may enter the cytoplasm from axoplasm through Ca2+ channels located in the plasmalemmal membrane, or it could be released from intracellular stores (sarcoplasmic reticulum) through intracellular calcium channels. Calcium channels may be described as membrane components whose function is to respond to chemical and/or electrical signals and allow translocation of Ca2+ ions, that sen/e as second messengers, activating a great variety of cellular processes. The physiological functions of Ca2+ channels include excitation-contraction coupling (specially in the smooth muscle), control of secretion, and nerve conduction and excitation. 1.3.1. CLASSIFICATION OF CALCIUM CHANNELS There are two different types of calcium channels in the cell membrane that allow translocation of the extracellularly derived Ca2+ into the cell. 1.) The potential-dependent calcium channels (PDC). also known as voltaae-aated calcium channels fVGCL are channel population that is responsive to changes in membrane potential. They open every time when potential across the membrane is reduced, as a consequence of electrical or chemical stimulation.^,'w .45 pdc have been subdivided into four distinct classes the L-, N-, P- and T-type Ca2+ channels according to their localization, function and electrophysiological properties. L-, N-, and P-type Ca2+ channels are defined as high voltage activated channels. They can be found in neuronal and non-neuronal cells, and they are usually activated at high membrane potential and inactivate slowly. T-type Ca2+ channels are also known as low voltage activated channels since they are activated with small amount of depolarization and inactivate rapidly. L-type Ca2+ channels are expressed in cardiac, smooth and skeletal cells as well as in neuronal and endocrine cells. They play the critical role in excitation-contraction coupling in the smooth muscle and are necessary for generation and propagation of electrical impulses and initiation of contraction in heart. It seems that L-channels help control release of some neurotransmitters and h o r m o n e s /6,47.48 The N-type Ca2+ channels are found mainly if not almost exclusively in neuronal cells were their main rote is to control release of neurotransmitters. The third type of high threshold Ca2+ channels has been named P-type since they were first observed in Purkinje cells. Their predominant function is regulation of neurotransmitter release. The T-type channels are found in wide variety of 22 excitable and non excitable cells. Their most important function is support of pacemaker activity. In addition they may play an important role in aldosterone secretion and they have some developmental role since they are the most abundant cell line in embryotic cells. The VDC can be distinguished based on their sensitivity toward different pharmacologic agents, for example 1,4-dihydropyridines (DHP), co-conotoxin, funnel web spider toxin and amiloride. The characteristics and properties of VDC are summarized in Table 3. T a b le 3 Classification of voltage gated Ca2+ channels (modified from Ref. 50) Property L T N P Conductance.pS: 25 8 12-20 10-12 Activation treshold: high low high moderate Inactivation rate: slow fast moderate rapid Permeation: Ba2+>Ca2+ Ba2+=Ca2+ Ba2+>Ca2+ Ba2+>Ca2+ Pharmacological sensitivity: 1,4-Dihydropyridines (Agonists/antagonists) Phenylalkylamines Benzothiazepines Sensitive Insensitive Insensitive Insensitive m-Conotoxin Insensitive Insensitive Sensitive Insensitive Octanol, amiloride Insensitive Sensitive Insensitive ? Funnel web spider toxin Insensitive Insensitive Insensitive Sensitive 2. The receptor-operated calcium channels (ROC) are ion channels controlled or operated by a receptor. The ROC may be sometimes potential dependent and sometimes potential independent.43 There are several possible organizations of ROC. The channel may be composed of the subunits of the receptor and this receptor/channel complex is permeable to Ca2+ ions. There are three examples of receptor gated channels: N-methyl-D-aspartate (NMDA) channel in central neurons, the ATP channels in smooth muscle and the nicotinic channel at the neuromuscular junction.43 Some recent findings suggest another possible organization of ROC where receptor directly controls voltage dependent or independent Ca2+ channels via guanine nucleotide binding proteins (G-proteins).44-43^ Examples of this kind of ROC are a1Aand <% AR ( as previously described).33 The third type of ROC are channels activated by receptors indirectly through specific receptor initiated biochemical signals such as formation of cyclic AMP, DG or IP3. There is a variety of receptor types that are indirectly connected with calcium channels, for example ar and az~ AR, I3-AR, muscarinic receptors, 5HT receptors and polypeptide hormones. 1.3.2. DRUGS ACTIVE AT CALCIUM CHANNELS 1.3.2.1 .Calcium channel antagonists From the chemical point of view one can distinguish three main classes of calcium channel antagonists: 1,4-dihydropyridines (DHP), phenylalkylamines (PAA) and benzothiazepines. Representative examples for each of these classes of compounds are shown in Figure 7. Verapamil 34. diltiazem 35 and nifedipine 36 are now established agents for treatment of number of cardiovascular disorders. Table 4 summarizes current and potential uses of Ca2+ channel antagonists. so,5i,46 The introduction of nifedipine, the lead compound of 1,4 dihydropyridine, was followed by number of structurally similar compounds. The first generation of nifedipine analogs was concerned with variation of ester moieties in position C-3 and C-5 of the DHP ring. Second generation of 1,4-DHP antagonists includes bicyclic substitution in C-4 position of DHP ring, modification of 24 CH. CN OCH; H3CO OCH. Verapamil 34 OCH, NOz COOCH. o c o c h . CH. Diltiazem 35 Nifedipine 32 Figure 7. Calcium Channel Antagonists Table 4. Current and Potential Uses of Calcium Channel Antagonists Current uses Potential Uses Arrhythmias Pulmonary hypertension Angina Asthma Hypertension Premature labor Atrial fibrillation Epilepsy Cerebral vasospasm Glaucoma Alzheimer’s disease Dysmenorrhea Cardiac prevention methyl groups in position C-2 of DHP ring and N substitution. Finally, the third generation includes compounds where DHP ring is exchanged for other heterocyclic systems such as pyrimidine.52 Structure activity relationship studies of 1,4-DHP antagonists have revealed some basic structural requirements for antagonist activity of this type of compounds. First of all the 1,4-DHP ring is essential. Oxidation to pyridine is detrimental, while reduction to tetra- or hexahydropyridine is tolerated. The NH group of 1,4-DHP is essential for optimum activity, the reason being that it probably serves as a hydrogen bond donor at the binding site. The substituent in the C-2 and C-6 positions of the 1,4-DHP ring should be lower alkyl, however one NH 2 group can be tolerated. Ester groups in the C-3 and C-5 positions are optimum. Replacement with -CN or -COCH3 greatly reduces activity. Substitution of the C-4 position of the DHP ring with a phenyl or substituted phenyl increases activity. Substitution in the ortho or meta position on phenyl ring increases activity, however any substituent in the para position reduces activity. 52,51,44 The structure activity relationship for verapamil and its analogs have been determined by M a n n h o ld .52 The tertiary amino group plays a very important role; exchange by sulphur or methylene is detrimental for activity. Quaternary ammonium derivatives of verapamil are inactive. Exchange of the -CN group reduces potency, while the isopropyl group can be changed into n-hexyl and the analog still has the same activity as verapamil. Aromatic substituents strongly influence potency, with small electronegative substituents in meta position being most potent. Conformationally restricted analogs of verapamil have been studied. 53,54 In order to determine the structure activity relationship for calcium channel blockers related to diltiazem, an extensive series of benzazepine analogs had been prepared and studied.s5.56,s7 Replacement of the sulphur atom of the benzothiazepinone nucleus with a methylene group gave compounds with similar potency in vitro. Two pharmacophores were important for the Ca2+ channel blocking activity: a basic amino group attached to the N atom in benzoazepinone ring, and the 4’ methoxy substituent on the 4 aryl ring. It appears that a protonated amino group that serves as a hydrogen bond donor moiety 26 and the hydrogen bond acceptor capability of 4’-OCH3 provide critical binding interactions at the receptor binding site. Substitution of the fused aryl ring in position 6 with electron withdrawing -CF3, -CN or N02 groups gave some of the most potent compounds.55,56 New structural classes of Ca2+ channel antagonists have been reported (for review see reference 50) for example benzothiazines (SD3211 37), benzothiepins (AJ 2615, 38). CH. CH3 SD 3211 37 NHCO(CH2)3 - H3C00l C00(CH2)3— N BMY 20064 39 Figure 8. New Structural Classes of Calcium Channels Antagonists Since it is known that combination drug therapy can be beneficial in the treatment of various cardiovascular disorders, hybrid molecules combining Ca2+ channel blocking activity and a-pAR antagonism have been developed, such as BMY 20064 39 (Figure 8). 27 1.3.2.2.Calcium Channel Agonists (activators) Certain 1,4-DHP namely Bay K 8544 4Q, H 160/51 41 and 202-791 42, have shown increase of Ca2+ ions influx into the cell (Figure 9).50 They behave as C a2+ channel agonists (activators) increasing contractions of muscles, secretion of hormones and neurotransmitters and excitation of cardiac and nerve cells. CF- NO- NO, HoC' CH BAY K 8644 40 202-791 42 COOCH; CH. NH, H 160/51 41 FPL 64176 4£ Figure 9. Calcium Channel Agonists Structurally there are very similar to calcium channel antagonists. The only difference is that antagonists have ester functionality in both C-3 and C-5 positions on the 1,4-DHP ring while the known agonists have one ester combined with a non ester functionality in these positions. This results in an asymmetric center in position C-4, and existence of 2 enantiomers. It has been shown that Bay K 8644 40 exhibits remarkable enantiomeric selectivity: the S- enantiomer acts as an agonist (causing a concentration dependent 28 vasoconstriction) while antagonism is associated with R-enantiomer .59 The structural basis for the opposing pharmacology of Bay K 8644 vs nifedipine is not completely understood, however there are some arguments that the pharmacological properties of agonists and antagonists may be based on differences in hydrogen bonding pattern at the active site . 44 Other interpretations are also possible, one of them is that there are discrete activator and antagonist binding sites on the same protein.^ Recently a new benzoylpyrroles class of Ca2+ channel activators has been reported.so FPL 64176 43 is one representative example of this class, which promotes influx of Ca2+ into cells and causes positive inotropic effects. Calcium channel agonists could serve as a base for development of various analogues for treatment of heart failure. However their clinical application depends on resolving vasoconstricting effects from the cardiostimulating properties. 1.3.3. STRUCTURE OF THE CALCIUM CHANNELS The voltage gated L-type Ca2+ channel was first isolated from skeletal muscle and since then it has been used as an model for studying the structure of the Ca2+ channels. The purified C a 2+ channel is a complex composed of five protein subunits: a i , 0 .2 , B , y and 8 60 as shown in Figure 10. Tanabe and co-workersei determined the primary structure of the ai subunit (Mr=170 kD) of the C a 2+ channel from rabbit skeletal muscle by cloning and sequencing DNA complementary to its mRNA. Based on the hydrophobicity analysis it has been predicted that a-i subunit has transmembrane topology with four homologous repeats each containing six helical membrane spanning segments labeled S1-S6. The fourth segment in each repeat (S4) contains positively charged residues at every third or fourth position, and it is believed to form part of voltage-sensitive machinery. The ai subunit of the C a 2+ channel from skeletal muscle bears resemblance to Na+ and K+ channels. It also contains binding sites for all known calcium channel blockers (DHP, PAA and benzothiazepines).M.48, 47,62 29 Figure 10. Schematic Representation of L-Type Calcium Channel. (Modified from Catteral, 60) The skeletal muscle a2 subunit (143 kD) is linked via a disulfide bridge to the 5 subunit (27 kD). This a2 5 complex is heavily glycosylated transmembrane protein primarily exposed at the extracellular surface.62 The 8 subunit is associated with ai subunit. It is an intracellularly located membrane protein without any glycosylation sites (Figure 10).eo,48 The remaining y subunit is peripherally associated with the ai subunit. This is also an integral membrane protein. Its determined amino acid sequence contains four putative transmembrane domains and two extracellularly located glycosylation sites as shown in Figure lo .66^.62 The ai subunit expressed in Xenopus oocyte by itself yields voltage activated Ca2+ currents. However, the conductance of these channels is much lower as well as the number of high affinity binding sites. Coexpression of other 4 subunits together with ai subunit significantly improves the amount of Ca2+ current 6°. 30 1.3.4. THE ROLE OF AFFINITY AND PHOTOAFFINITY LABELING IN THE IDENTIFICATION OF CALCIUM CHANNEL ANTAGONIST BINDING SITES In addition to radioligand binding studies, studies with a variety of affinity and photoaffinity ligands have been carried out in order to identify ligand binding sites on calcium channel. The structure of affinity and photoaffinity probes utilized in these studies are shown in Figure 11. Compound 44 was prepared from verapamil and it showed irreversible antagonism to pHJgallopamil binding in myocardial membrane homogenates “ Striessnig and co-workers used a photoaffinity probe, [3H] ludopamil 4f>, to label the phenylalkylamine binding site on purified skeletal muscle calcium channels. Initially they discovered that ludopamil incorporates into a 155 kDa peptide which was subsequently identified as the ai subunit of the skeletal Ca2+ channel. Specifically labeled a 1 subunit was submitted to exhaustive digestion with proteolytic enzymes, and photoaffinity labeled regions were identified by mapping of labeled peptide fragments with antipeptide antibodies.es Based on these studies it has been proposed that phenylalkylamine binding site is located in segment S6 of domain IV and beginning of the C terminal cytoplasmic loop as shown on Figure 12“ Basically, the same antibody mapping procedure was used to identify the DHP binding site on oq subunit of Ca2+ channel 67.es The subunit was first labeled with either azidopine 4§ or diazipine 47. Analyzing results it has been concluded that site of action of DHP is located to segment S6 of domain III and IV from the extracellular surface C a2+ channel as shown in Figure 12.“ The binding site for benzothiazepines has not been completely identified. 31 c h 2n c s ? H; h 3c o OCH. OCH. 44 Haco CH. CN H3CO Ludopamil 45 CF: COOCHoCHpNHCi CH Azidopine 46 N = N FaC‘ CF. H,C'CH. [3H] Diazipine 47 Figure 11. Representative Affinity and Photoaffinity Probes for Calcium Channels Figure 12. Schematic Representation of ai-Subunit of the L-Type Calcium Channel and Location of the Binding Sites for Phenylalkylamines (A) and 1,4- Dihydropyridines (B). (Modified from Rampe, 50) 1.4. IM1PAZQLINE.RECEPTQRS 1.4.1. PHENETHANOLAMINES VS. IMiDAZOLtNES There are two main chemical classes of compounds that interact with a-AR: the phenethylamines, such as norepinephrine 1, phenylephrine 2, and the imidazolines, such as cirazoline 6, clonidine 12, idazoxan 12 and oxymetazoline 25- These two classes of direct acting a-sympathomimetic drugs do not interact with a-AR in an identical manner. One of the first indications for a different mode of interaction at the a-AR was a lack of cross desensitization between a-AR agonists of the imidazoline and phenylethylamine classes.69 The major difference between these two classes of compounds was the applicability of the Easson -Stedman hypothesis.™ According to this hypothesis there is a three point attachment involved in the binding of an optically active sympathomimetic 33 amine to the adrenoceptor. The three functional groups for phenethylamines were the hydroxy groups of the catechol, benzylic hydroxyl group and the basic nitrogen atom.7° Figure 13 depicts the interaction of the above mentioned groups to the adrenoceptor. (R)-(-)-Norepinephrine (S)-(+)-Norepinephrine Desoxy-norepinephrine (Dopamine) Figure 13. Schematic Representation of the Easson-Stedman Hypothesis for Norepinephrine. (Modified from Nichols, 42) Whereas optically active phenethylamines follow the Easson-Stedman hypothesis and interact with the a-AR via three point attachment with relative order of potency R(-)-isomer > S(+)-isomer = desoxy analog, the optically active imidazolines do not follow the Easson-Stedman hypothesis (the order of potency being desoxy analog > R(-)-isomer > S(+)-isomer) and interact with the receptor by only a two point attachment.7*^ Furthermore it has been shown that presence of the benzylic hydroxyl group at the B-carbon atom of the phenethylamines increases a-AR agonist activity by at least 100 fold, while hydroxylation of the carbon bridge in imidazolines decreases activity by up to 10-fold 7V3 Thus, it has been documented that structure-activity relationship for agonist affinity at a-AR differ significantly between the imidazolines and phenetylamines.72 These finding suggest the presence of different interacting sites on the a-AR or distinct receptors for compounds of the imidazoline and phenylethylamine classes. 34 1.4.2. IDENTIFICATION OF NQN-ADRENERGIC IMIDAZOLINE BINDING SITES. Clonidine 13 has been intensively used in the therapy of hypertension for the last 20 years, and is one of the most potent hypotensive drugs. Clinical usefulness of this imidazoline 13 has been attributed to its action within the central nervous system. It has been proposed that clonidine 1 3 interacts with central a 2-AR to decrease sympathetic outflow to periphery.74 However, it is very unlikely that clonidine 1 3 interacts with the presynaptic a2-AR, since neither destruction of noradrenergic nerve terminal with 6-hydroxydopamine or neurotransmitter depletion with reserpine, significantly attenuated hypothensive response to clonidine.™ Bousquet et al.™.77 implicated the nucleus reticularis lateralis (NRL), a part of the rostral medulla oblongata, as a site of hypotensive action of clonidine. They have shown that very low doses of clonidine administered directly to the NRL of anesthetized cats produced significant drop in arterial pressure. Destruction of the ventral surface of the brain stem abolishes hypotensive action of clonidine .™,77 Bousquet and co-workers™ and Emsberger et al.™ observed functional differences between a-adrenergic substances bearing catecholamine structure (a-methylnorepi- nephrine) and a series of a-AR agonists with an imidazoline ring as the core structure. Injection of a-methylnorepinephrine (selective c^-AR agonist) into cat’s NRL was unable to significantly affect arterial pressure while cirazoline 6 and ST 587 7 produced dose dependent hypotensive effect.™ They proposed that these imidazoline compounds produce hypotensive effects by interacting with imidazoline-preferring sites in the NRL. Binding studies conducted by Ernsberger and co-workersao showed that [3H]p- aminoclonidine ([3H]PAC) labels two distinct populations of binding sites in the bovine ventrolateral medulla. While clonidine displaced the total population of the [3H]PAC labeled sites only 70% of the sites labeled with [3H]PAC could be displaced by norepinephrine and other phenethylamines, even when they are used in mM concentrations. The remaining norepinephrine-insensitive binding sites were sensitive to clonidine. The 35 imidazoline sensitive sites seems to be restricted to ventrolateral medulla, since they are not found in frontal cortex. Autoradiographic and functional studies in rat brain carried out by Boyajian and Leslie^ >82 showed that PHJidazoxan, a selective a2~AR antagonist, labels additional population of sites that are not labeled by [3H]rauwolscine (selective a2-AR antagonist). Coupry et al.,B3 using [3H]idazoxan and [3H]rouwolscine, identified two populations of sites in the basolateral membranes from rabbit kidney. [3H]Rauwolscine binds only to a2-AR, while idazoxan labels a2-AR and non-adrenergic binding sites, presumably imidazoline-preferring receptors, ss Existence of “specific” [3H]idazoxan binding sites that do not recognize catecholamines was established in rat and human kidney, human platelets, myometrium and erythroleukemia cells.** Bricca et al. used [3H]clonidine to characterize imidazoline sensitive and catecholamine-insensitive receptors in bovine and human NRL.85.86 They found that 25% of the binding sites in bovine NRL were insensitive to displacement by an excess of norepinephrine, in contrast human NRL contains only imidazoline-preferring sites, and no a-AR could be detected. Only imidazoline containing compounds like cirazoline & idazoxan 19, oxymetazoline 25, ST587 7 were able to displace [3H]clonidine in human NRL. Phenethanolamines like norepinephrine 1, and adrenaline 2 have virtually no effect. Recently, Tibirica et al.,87 using in vivo electrochemical studies in normotensive rats, concluded that the hypotensive effect of clonidine is a consequence of its interaction with imidazoline preferring receptors in the ventrolateral medulla oblongata. On the other hand, the sedative effect of clonidine is due to its interaction with a2-AR in the locus ceruleus87. Other approaches have been used to distinguish the [3H]idazoxan binding site from the a2-AR, which includes the studies on COS-7 cells expressing cloned c^-AR subtypes.88 The results indicate that the imidazoline binding sites are not co-expressed with a2-AR. Non-adrenergic, imidazoline preferring sites have been described in a variety 36 of tissues and species such as rat lung membranes, 89 rat liver c e lis ^ human kidneys and rabbit cerebral cortex mitochondria.^ 1.4.3. ENDOGENOUS LIGAND FOR IMIDAZOLINE RECEPTOR identification of the imidazoline binding sites in the brain and other tissues demonstrated existence of a novel ligand binding site. However, an evidence that the imidazoline binding site is a functional receptor was demonstrated by the hypotensive effect in cats upon application of imidazolines to the NRL, while no catecholamine was active. Existence of an endogenous ligand for the imidazoline receptor is an additional requirement for the definition of a new receptor. Isolation and partial purification of an endogenous ligand for the imidazoline receptor has been reported in 1984 by Atlas 92.93 and in 1986 by Meeley .94 The endogenous ligand isolated from bovine and rat brain displaced pHjdonidine from rat brain, hence it was named “clonidine-displacing substance” (CDS ) . 92 This ligand does not bind to a-i-AR or I3-AR; however, it has affinity for 012-AR in human platelets, where it displaces pHjyohimbine and [3H]rouwolscine.95 Subsequently it has been discovered that CDS, like clonidine, also binds to a nonadrenergic binding site, the imidazoline receptor in bovine ventrolateral medulla Direct injection of CDS in cat’s NRL causes a significant increase in the arterial pressure, contrary to clonidine. 96 However when Meeley and co-workers injected the CDS in the rostral ventrolateral medulla of the rat it produced a reversible dose-dependent fall in arterial pressure and heart rate. 94 Recently there were some attempts to explain these contradictory results. It was suggested that one of the CDS preparations was contaminated with GABA or glutamate which resulted in their opposite effects on the blood p re ss u re . 97 in the periphery, CDS mimics the action of clonidine causing the inhibition of norepinephrine induced platelet aggregation.98 37 CDS is not a peptide since it retains its activity after prolonged incubation at pH-2.0 at room temperature or 5h at 23°C at pH 10.5. The molecular weight of CDS, determined by gel filtration (size-exclusion chromatography), is 500±50 Da92 and was confirmed by plasma desorption mass spectrometry, 1^587,812." Although the isolation of CDS was reported in 1984 its structure was established only recently." The CDS was purified and determined by mass spectroscopy to be agmatine (decarboxylated arginine, 48)." Arginine decarboxylase, the enzyme synthesizing agmatine has been found in rat brain. Agmatine shares properties of CDS: it binds to o^-AR and imidazoline receptors, and has following selectivity l1>a2-AR>l 2 .97'99 It also stimulates release of catecholamines from adrenal chromaffin cells." NH H Agmatine 48 These observations support the hypothesis that agmatine 48 is the endogenous ligand for imidazoline receptors. 1.4.4. CLASSIFICATION OF IMIDAZOLINE RECEPTORS * Distinct pharmacological characteristics have been observed among the imidazoline receptors isolated from different species, or using different radioligand label. For example, imidazoline sites in bovine brain labeled with [3H]PAC and described for the first time by Ernsberger and collegues" exhibited high affinity for many imidazoline compounds, such as p-aminoclonidine 48, cimetidine 50 (an H 2-histamine antagonist) and imidazole-4-acetic acid §1.79 These sites are not recognized either by catecholamines, guanidino or benzazepine compounds.™ 38 In contrary, imidazoline sites labeled with [3H]idazoxan in rat kidney 100 or rat livers have high affinity for some imidazolines (tolazoline 52), but very low affinity for clonidine 13. In addition these sites showed low affinity for amiloride 53 and very high affinity for guanidino compounds (e.g. guanobenz U , guanoxan 54).89 The [3H]idazoxan labeled sites from rabbit tissuesea.ioi have no affinity for catecholamines, but have high affinity for all imidazolines (even clonidine), for guanidino compounds and for amiloride § 3 . Structures of different compounds that bind to the imidazoline receptors are summarized in Figure 14. H h 3c n NCN H3CHN- C - NHCH2CH2SCH2 H p-Aminoclonidine 49 Cimetidine 5Q o NH c h 2c o o h lmidazole-4-aceticacid51 Tolazoline 52 Amiloride 53 ci Guanoxan 54 Rilmenidine 55 Moxonidine 55 Figure 14. Imidazoline Receptor Ligands The heterogeneity of imidazoline receptors has been noticed for some time .102 in this regard, subclassification of imidazoline receptors into \-\ and I 2 was proposed during the 1st International Symposium on Imidazoline Preferring Receptors.97 Designation “I" includes not only compound with imidazoline , imidazole or imidazolidine ring as a core structure but also other potential ligand at imidazoline sites bearing the oxazoline ring (e.g. rilmenidine 55) or the guanidino moiety. Ii sites are labeled at nM level by pHJclonidine or pHJPAC and display the following rank order of potency for competing agents: phentolamine 21 > para-aminoclonidine 49 > clonidine 13 > idazoxan IS > cimetidine 5Q > imidazole-4-acetic acid 5 1 » cirazoline 6 » amiloride 53. On the other hand l2 sites have pM affinity for clonidine, but nM for pHJidazoxan, and show high affinity for guanidines. The rank order of potency for displacement of pHJidazoxan is: cirazoline fi > idazoxan 19 > amiloride 53 > clonidine 13 > imidazoline-4-acetic acid 51-103 Table 5 summarizes the classification and characteristics of imidazoline receptors. Recently a new highly selective compound for ^ has been reported by Ernsberger, moxonidineSfi (clonidine derivative), i°4 It is a centrally active hypotensive drug with attenuated sedative effects due to its higher affinity for imidazoline receptors than for aAR in the VLM. In light of the most recent studies, some additional heterogeneity among ^ and l2 types has been noticedio5.ioe.io7 however, a subclassification of these types is considered premature. 1.4.5. ISOLATION AND CHARACTERIZATION OF IMIDAZOLINE RECEPTOR Parini and co-workers reported the partial purification and characterization of an imidazoline/guanidinium receptive site (IGRS) from rabbit renal cortex. ioa They found that IGRS is an intrinsic membrane protein that can be physically separated from Table 5. Classification and Characteristics of Imidazoline Receptors Imidazoline Parameter h b Pharmacological clonidine>moxonidine> idazoxan=cirazoline selectivity idazoxan»cirazoline» amiloride>c!onidine amiloride Molecular weight unknown 60-70 kDa Glycosylation unknown no Sensitivity to GTP sensitive insensitive nonhydrolyzable analogs Cellular distribution plasma membrane plasma membrane, outer mitochondrial membrane Tissue localization ventrolateral medulla rat liver, rat lung, oblongata, rat kidney, human kidney, brain, human platelets, bovine bovine adrenal chro adrenomedullar maffin cells, adipo chromaffin cells. cytes, urethral smooth muscle. Function 1.involved in control of Implicated in Na+/H+ blood pressure in the VLM exchange in renal 2.stimulate insulin release proximal tubules from pancreatic B cells 3.may mediate Na+ exchange in the kidney 41 were identified by pHjidazoxan binding that was not inhibited by epinephrine. However cirazoline 6, idazoxan IS, guanabenz 14 and tolazoline §2 competed for pHJidazoxan binding sites.ioa In addition to plasma membrane, IGRS was found in high density in the outer mitochondrial membrane.^ Lemon et al. have reported isolation of mitochondrial IGRS from rabbit kidney.P ure homogeneous IGRS was obtained by two step purification procedure using chromatophocusing and hydroxyapatite-agarose chromatography. SDS-PAGE of the purified protein revealed only one band with an apparent Mr= 60 kDa.no Wang et al .m isolated and partially purified an imidazoline receptor protein from the bovine adrenal chromaffin cells by affinity chromatography using imidazoline agents (e.g. idazoxan or p-aminoclonidine) as ligands. Molecular weight of the purified receptor protein was determined to be around 70 kDa, by SDS-PAGE. The imidazoline-binding protein had affinity for the imidazolines cirazoline 6, idazoxan 19 and clonidine 1£ and no affinity for the catecholamine epinephrine 2 and o^-AR antagonist rauwolscine 1 7 .m Lanier and co-workersii2 have recently prepared the photoaffinity label 2-[3-azido-4-[i25|]iodophenoxy]methylimidazoline 52, a cirazoline analog and it has been used to covalently label the binding subunits of the IGRS in various tissues (kidney, brain, liver). This photoaffinity label was found to be incorporated into two major peptides with the apparent molecular weight of 5 5 and 61 kD a.112 2-3-Azido-4-[125l]iodophenoxy methylimidazoline §7 CHAPTER II STATEMENT OF PROBLEMS AND OBJECTIVES There are several lines of evidence that suggest that imidazolines and phenethanolamines interact differently with a-AR (see Chapter I). Wang et al. ™ have proposed that similar to phenethanolamines, the positively charged imidazoline ring of 2-substituted imidazolines interact with Aspua jn transmembrane domain III of the a-AR (see Figure 7). However, the remaining functional groups of imidazolines are thought to interact with a-AR in a different m a n n e r s Designing affinity and photoaffinity label probes of the imidazoline type for the a-AR could provide useful information about the interaction of imidazolines with a-AR. Indeed several affinity and photoaffinity labels of the imidazoline type have already been reported in the literature and include previously mentioned chlorethylclonidine (CEC, 22), p-azidoclonidine 33 as well as p-isothiocyanato clonidine 58 and p-methylisothiocyanato clonidine 5 9 .113.114,115 Cl Cl Cl Cl p-lsothiocyanato clonidine 58 p-Methylisothiocyanato clonidine 59 42 CEC 22 turns to be very useful alkylating agent for distinguishing between ar AR subtypes (see page 8), p-isothiocyanatoclonidine covalently labelled o^-AR in the central nervous system and periphery m and p-azido-[3H]clonidine labeled the fourth transmembrane domain of the (X 2-AR as one bearing the ligand binding site for imidazoline type of compounds, us To extend these studies a selective a-adrenergic site directed affinity probe 2-(4’-isothiocyanatobenzyl)imidazoline (IBI, 6ft) was synthesized. The structure of IBI is based on tolazoline 52. an o^-AR antagonist, and ar AR partial agonist. The objective of this study was to find out if 2-benzvl substituted imidazolines behave in a manner similar to 2-aminoimidazolines like clonidine 13. S=C=N IBI 60 Interestingly, IBI 6ft produced a slow onset and sustained contractions of rat thoracic aorta, with an ED50 value of 1.63x10-5 m. However, these IBI dependent effects were not blocked by ar AR blockers (phentolamine 2 1, prazosin 1 ft), an a2-AR blocker (yohimbine 18) or irreversibly acting a-AR blockers (phenoxybenzamine (PBZ, 31) or CEC 2 2 ).h a ii 8 In contrary, Decker and co-workers have shown that contractions of rat aorta induced by p-isothiocyanato clonidine 58 could be blocked by pretreatment with prazosin 1ft, yohimbine 1ft phentolamine 2 1 or phenoxybenzamine 3 1 . These results suggest that IBI 6Q does not interact with a-AR in rat aorta, and mediates irreversible contractions of rat aorta through a non-a-AR mechanism. In this regard, preliminary experiments conducted by Hussain et al. indicated that IBI competes with pHjidazoxan for ofe-AR and imidazoline 44 preferring non-a-AR sites of guinea pig cortex membrane. 119 Based upon these results, as well as the structural similarity between IB) 6Q and imidazoline receptor ligands such as idazoxan 19, clonidine 13 and cirazoline 6, there is a possibility that the non-a-adrenergic activity of IBI 60 in rat thoracic aorta was related to the interaction of IBI 66 with the imidazoline receptor site. To determine the structural requirements for the interaction with a and non-a- adrenergic receptors in rat aorta a number of IBI 68 related analogs were proposed (see Scheme 1). Three types of modifications were proposed: 1) Modifications of the electrophilic group Analogs 65 and 66 carrying cholacetamido, 65, or iodoacetamido group, 6§, in place of isothiocyanato group, were designed to examine the importance of the isothiocyanato group. Furthermore, we were curious if replacement of this electrophilic group with -CH3 group in analog 62 or nucleophilic NH 2 group in compound 61 would still produce sustained contractions of rat aortic tissues. Isomers of IBI, compounds 63 and 64, were proposed to see if the position of isothiocyanato group, relative to the imidazoline ring, is critical. 2) Modifications of the distance between the aromatic and imidazoline rings. In compounds 67 and 68 the effect of an elongation of the bridge between the aromatic and imidazoline rings will be examined. These two analogs incorporate structural features of the imidazoline receptor ligands, idazoxan and cirazoline. 3) Modifications of the imidazoline ring were imidazoline ring was replaced with imidazole ring in compound 69 and with tertiary amino group in analog 70. These modifications should give us information about the significance of the imidazoline ring vs. the positive charge or hydrogen bonding interactions at the active site of the receptor. 45 Scheme I Proposed IBI Related Analogues for Studing the Structural Requirements for the Interaction with a- and non-a-AR. SCN SCN SCN R =NH2 61 R =CH3 62 67 69 CHq / c h 3 SCN SCN R =2'-NCS 63 R =3'-NCS 64 68 70 X = Cl 65 X = I 66 46 Compound Z1 was designed as a potential radioaffinity label. Isotopic labeling of this ligand with 125l may be used for studying the non-a-adrenergic receptor binding site in rat aorta. L> r v sN SCN U H 71 CHAPTER III RESULTS AND DISCUSSION 3.1. CHEMISTRY The synthesis of 2-(4’-nitrobenzyl) imidazoline 74, shown in Scheme II, has been previously reported by Cavallini et a l .120 Using the Pinner conditions^ commercially available 4-nitrophenylacetonitrile 72 in the presence of an anhydrous EtOH and HCI gas, was converted to the corresponding ethyl imide ester 72. Due to the instability of imidate 73 its isolation should be performed quickly. Imidate 73 was then refluxed with ethylenediamine in dry EtOH, overnight to obtain imidazoline 74. Acid base extraction prove to be a method of choice for the isolation of imidazoline 74 from the reaction mixture, in a pure form. The imidazoline 74 was then used as an intermediate to obtain a variety of 2-benzyl substituted imidazolines as shown in Scheme III. Scheme II Synthesis of 2-(4'-Nitrobenzyl)imidazoline 74 CN EtOH/HCI 47 Scheme Ilf Synthesis of 6Q, 65, 66 and 71 (XCH2C0)20 ■N. HN- HN- HN- HCI HCI SCN HCI Z4 SI 1.TICI3i Nal CH3COONa (pH=4.1) 2. HCI gas HN- HN 2HCI SCN HCI Z5 Z1 £ 49 The synthesis of our lead compound 2-(4’-isothiocyanatobenzyl) imidazoline (IBI, 60 is outlined in Scheme III. Catalytic hydrogenation (10% palladium on carbon in methanol) of imidazoline 74 afforded 2-(4’-aminobenzyl) imidazoline 61 as a monohydrochloride salt, which was then treated with thiophosgene (CSCI2) to give IBI 60.122 Stirring compound 61 in acetonitrile with chloroacetic or iodoacetic anhydride resulted in formation of 2-(4’-chloroacetamidobenzyl) imidazoline 65 or 2-(4’-iodo- acetamidobenzyl) imidazoline 66- While 2-(4’-chloroacetamidobenzyl) imidazoline 65 was isolated as a monohydrochloride salt, its iodo analog 65 according to the elemental analysis, crystallized as monohydroiodide salt probably as a result of exchange of Cl from HCI (starting material 61 comes in form of HCI salt) and I from iodoacetic anhydride. lodination of 61 was performed according to the procedure for iodination of p-(4- aminophenyl) ethylamine described by Ruoho .123 Heating a mixture of imidazoline 61, thallium trichloride and sodium iodide in sodium acetate buffer (pH=4.1) gave the desired 2-(4,-amino-3’-iodobenzyl) imidazoline 75 (See Scheme III). Use of sodium acetate buffer for the control of pH in this reaction is mandatory since the protonation of aniline amino group would stop the reaction or direct iodination to the meta position (in regard to amino group) on the aromatic ring. Treatment of 2-(4’-amino-3’-iodobenzyl) imidazoline 75 with thiophosgene under conditions described by Rice et al. gave 2-(3’-iodo-4’- isothiocya- natobenzyl) imidazoline Z l .122 Compound 62,2-(4’-methylbenzyl) imidazoline, was synthesized according to the procedure previously described by Sonn and it is shown in Scheme IV.124 We wanted to obtain this compound, 62., for our biological testing. Commercially available 4-methylphenylacetonitrile 76 was converted into imidate 77 under Pinner conditions in dry ethanol in the presence of HCI gas .121 The imidazoline ring was built by refluxing imidate 77 with ethylenediamine, giving rise to 2-(4'-methylbenzyl) imidazoline 62. 50 Scheme IV Synthesis of 2-(4'-Methylbenzyl)imidazoline 62 QN EtOH/HCI gas 1. H2NCH2CH2NH2 The synthesis of compounds 67 and 63. is outlined in Scheme V. p- Nitrobenzylacetonitriie was synthesized according to the modified procedure for synthesis of 2 -nitrobenzylacetonitriie.i 2s Hence, treatment of 4-nitrophenylethylbromide 78 with NaCN in ethanol gave crude nitrile 80. Nitrile 31 was obtained by refluxing 4-nitrophenol 79, K2C03 and bromoacetonitrile in acetone for 20 h .126 Using the Pinner method, 121 nitrile 80 and 3 1 in the presence of anhydrous EtOH and HCI gas were converted to the corresponding imidates 32 and 33, respectively. Refluxing the imidates, 32 and 32 with ethylenediamine in ethanol overnight, gave imidazolines 34 and 35 respectively which were then converted into monohydrochloride salts by treatment with HCI gas. Reduction of the nitro group of 34 and Sfi by catalytic hydrogenation afforded 2-(4’-aminophenethyl) imidazoline 36 and 2-(4’-aminophenoxymethyl) imidazoline 3Z. respectively. Treatment of 86 and 87 with thiophosgene gave the final products 67 and 33, respectively. Compound 68 has been reported in the literature as an insecticide and a c a r ic id e .127 In addition to the above described procedure for synthesis of 2-substituted imidazolines, where readily available nitriles were converted to imidates which were then reacted with ethylenediamine to obtain the desired imidazolines, there are other available procedures found in the literature. For example, if nitriles are not readily available acids or esters have been reported to be converted to the imidazolines on the treatment with 51 Scheme V Synthesis of imidazolines §Z and 68 EtOH/HCI gas OCH2CH3 BrCH2CN H2,10% P d /C N H 2. HCI gas, MaOH HCI HCI x =c h 2 g g CSCI. x=o fiZ N HCI SCN X=CH2 fiZ X =0 62 52 trimethylaluminum ethylenediamine complex (TMA-EDA), in refluxing toluene. 120,129 However, the method utilizing nitriles has the advantages due to the easier workup procedure. An attractive route to imidazoles is the dehydrogenation of the 2-imidazolines. However, this dehydrogenation of readily available 2-imidazolines to the corresponding resonance stabilized imidazoles can be sometimes quite difficult. Amemiya et al. successfully applied this methodology for conversion of 2-[1-(1-Naphthyl)ethyl] imidazoline to 2-[1-(1-Naphthyl)ethyl] imidazole.i30.i3i Furthermore, Hughey, et al reported conversion of some unactivated 2-imidazolines to imidazoles with barium manganate (BaM n04)J32 Attempted dehydrogenation of 2-(4’-nitrobenzyl) imidazoline Z4. with palladium on carbon under reflux failed to give desired 2-(4’-nitrobenzyl) imidazole fig (Scheme VI). Scheme VI Attempted dehydrogenation of 74 to fifi An alternative route to imidazole 69 is outlined in Scheme VII. Reaction of 4’- nitrobenzylacetonitrile 72 with anhydrous EtOH and HCI gas provided the imidate Z3.121 which was then treated with 2-aminoacetaldehyde dimethylacetal to afford amidine §§.133,134 Addition of acetic acid and HCI gas to 89 resulted in the formation of imidazole 9Q which precipitated from the reaction mixture. In this particular case the amount of HCI gas added does not make a significant difference, because of the electron withdrawing nature of the nitro group in the para position on aromatic ring. However it was found that compounds with an electrondonating group (for example OH, NH 2 or OCH3) para to the point of cyclization gave aminobenzazepines if enough acid is present . ^ 4 Catalytic hydrogenation ( 1 0 % Pd-C in methanol) of ££ afforded § 1 which was treated with CSCI 2 to give imidazole § 9.122 Scheme VII Synthesis of 2-(4'-lsothiocyanatobenzyl)imidazole §£ EtOH/HCI HC| NH2CH2CH(OCH3)2 | A N. .CHfOCHaJj 1.CSCI2 2.NaHCO; The synthesis of N,N-dimethyl-2-(4’-isothiocyanatophenyl)ethytamine Zfi. is shown in Scheme VIII. N,N-dimethyl-2-(4-aminophenyl)ethylamine was synthesized as previously described from 4-nitro-p-haloethylbenzene 78 and 40% aq. dimethylamine in ethanol, to give N,N-dimethyl-2-(4-nitrophenyl)ethylamine £2. which upon hydrogenation gave corresponding amine 93.. An alternative route to N,N- dimethyl-2-(4-aminophenyl)ethylamine 94 is Eschweiler-Clark methylation of 2-(4’- nitrophenyl)ethylamine and subsequent hydrogenation to afford 93-135 Treatment of N,N- dim ethyl^-^’-aminophenyOethylamine 93 with CSCI 2 in acetone afforded amine ZQ-122 Scheme VIII Synthesis of N,N -Dimethyl-2-(4-lsothiocyanatophenyl)ethylamine ZQ N(CH3)2 1.40% aq. NH(CH 3)2 • HCI N(CH3)2 N(CH3)2 •HCI •HCI Scheme IX outlines the synthesis of IBI isomer 2-(3’-isothiocyanatobenzyl) imidazoline M - We started synthesis by converting 3-nitrobenzylbromide 94. into 3- nitrophenylacetonitrile 9 9 that could easily be converted to imidazole 9Z. Indeed conversion of 95. to the imidazole 9Z has been previously reported by Cavallini.120 Catalytic hydrogenation of QZ gave 2-(3’-aminobenzyl) imidazoline 9 5 which upon reaction with thiophosgene afforded £4- Attempted synthesis of yet another IBI isomer 2-(2’-isothiocyanatobenzyl) imidazoline £3 is described in Scheme X. It follows previously described synthesis of IBI and its isomer 64* However we were unable to isolate 2-(2’-isothiocyanatobenzyl)imidazoline 63- Characterization of the isolated product pH NMR, 13C NMR, IR, MS and elemental analysis), proved our suspicion that under the 55 Scheme IX Synthesis of 2“(3'-lsothiocyanatobenzyl)imidazoline 54 Br NaCN HCI EtOH/HQ gas n h 2c h 2c h 2n h 2 OCH2CH3 34 35 10% Pd/C, H j Scheme X Attempted Synthesis of 2-(2'-lsothiocyanatobenzyl)imidazoline 55 NO- NO: CN EtOH/HCI gas NH2CH2CH2NH2 m NCS 10% Pd/C, Hj, CSCI2 HN HCI m SB m 56 existing reaction conditions the nitrogen atom in the imidazoline ring, in close proximity to highly reactive isothiocyanato group reacts with it giving rise to benzodiazepine 103. 3.2. BIOLOGY 3.2.1. Effects of PBZ pretreatment on the contractile activities of IBI analogues on rat aorta. All IBI analogues produced concentration-dependent contractile responses on rat thoracic aorta similar to that of IBI which were characterized by a slow onset and long duration of action. Tables 6, 7 and 8 summarize the effect of PBZ pretreatment on the action of the IBI analogues in rat thoracic aorta. Pretreatment of rat thoracic aorta with 30 pM PBZ alkylates a-AR and the preparation was insensitive to a-AR ligands.ise With the exception of 2-(4’-methylbenzyl) imidazoline §2, 2-(4’-aminobenzyl) imidazoline 61 and 2-(3'-iodo-4’-isothiocyanatobenzyl) imidazoline H the stimulatory activities of the remaining IBI analogues were unaffected by PBZ pretreatment (Tables 6, 7 and 8). These results suggest that a non-a-AR mediated mechanism must be involved for initiation of contractile responses by all analogues other than 61, §2 and Zl- With the latter analogues, the replacement of 4’-NCS group in IBI with 4’-amino group 61 or 4’- methyl group 62 altered the stimulatory activity of IBI from a non-a-AR to an a-AR mediated mechanism (Table 6). Introduction of an iodo atom in the 3’ position of the aromatic ring of IBI led to analog 71; however, this compound shows mixed activity by interacting with both a and non-a-AR sites in rat aorta. Thus, it appears that 71 will not be useful as a potential radioaffinity label for studying the non-a-AR receptor sites in rat aorta. Lanier et al .112 have recently prepared the photoaffinity label 2-[3-azido-4- [i25|]jodophenoxy]methyl imidazoline, (§7 a cirazoline analog) and used it to covalently label the binding subunits of IGRS in various tissues (kidney, brain, liver). This photoaffinity label was found to be incorporated into two major peptides with apparent molecular weights of 55 and 61 kD .112 57 Table 6. Comparison of Maximal Contractile Tension Changes and Potencies of IBI and its Analogues 61. 62, §5, fig and 71 with and without Phenoxybenzamine (PBZ, 30 nM for 20 min) Pretreatment on Rat Thoracic Aorta Strips.a %Emaxb EC5o(pM)c Compound R1 R2 control +PBZ control +PBZ IBI(60) HNCS 118±12 100±7 48±7 52±11 f i l H n h 2 69± 4 9±3 3±1 31±20 62 H c h 3 14± 3 5±6 57±5 d 65 H n h c o c h 2c i 41 ±14 32 ±1 116±9 129±39 66 H n h c o c h 2i 25+ 6 13±7 47±3 - d Z l I NCS 31 ± 6 28 ±6 10 ±6 58±5e a Tissues were pretreated with PBZ (3x10-8M). Analogue induced responses were monitored between 20-30 min after addition. b%Emax = percent of maximal contractile response to IBI analogue expressed relative to the maximal contraction produced by 30 pM phenylephrine e Data represent the mean ± S.E.M. of n=4-7. EC50value=drug concentration to produce 50% of maximal response (pM). dNot calculated. Maximal response was about 5-13% of the control preparation, and the EC50 in PBZ pretreated preparation could not be determined. eMeans are statistically different from the corresponding control value (IBI analog alone) at p<0.01. 58 Table 7. Comparison of Maximal Contractile Tension Changes and Potencies of IBI Analogues 67 and 68 with and without Phenoxybenzamine (PBZ, 30 nM for 20 min) Pretreatment on Rat Thoracic Aorta Strips* EC5o(pM)c Compound X control +PBZ control +PBZ 67 c h 2 107112 10217 291 7 2911 68 0 10817 117113 69141 4 7 1 8 a Tissues were pretreated with PBZ (3x10-8M). Analogue induced responses were monitored between 20-30 min after addition. b%Emax = percent of maximal contractile response to IBI analogue expressed relative to the maximal contraction produced by 30 pM phenylephrine c Data represent the mean ± S.E.M. of n=4-7. EC50 value=drug concentration to produce 50% of maximal response (pM). 59 Table 8. Comparison of Maximal Contractile Tension Changes and Potencies of IBI Analogues S3 and ZQ with and without Phenoxybenzamine (PBZ, 30 nM for 20 min) Pretreatment on Rat Thoracic Aorta Strips.** %Emaxb ECS0(pM)c Compound n R control +PBZ control +PBZ €9 1 70 ±6 68 ±3 31 ±8 36±12 <0N'----- H 70 2 N(CH3)2 98±2 113±10 41 ± 4 33±6 a Tissues were pretreated with PBZ (3x10-8M).Analogue induced responses were monitored between 20-30 min after addition. b%Emax = percent of maximal contractile response to IBI analog expressed relative to the maximal contraction produced by 30 pM phenylephrine c Data represent the mean ± S.E.M. of n=4-7. EC50 value = drug concentration to produce 50% of maximal response (pM). 60 Changing the electrophilic group from an isothiocyanato to a chloroacetamido or iodoacetamido gave compounds §5 and 66 respectively. They produced very weak contractile responses alone (£5 ECs0=116 pM with maximal response only 40% relative to that caused with 30 pM phenylephrine); however, the affinity of 65 and §6 was not significantly affected by PBZ pretreatment (Table 6). A possible explanation for the low activity of compounds §5. and ££ is that the highly reactive electrophile is needed for activity or that the distance between the aromatic ring and the electrophile is important. In a search for the optimum distance between the imidazoline ring and the aromatic ring we prepared 2-(4’-isothiocyanatophenethyl)imidazoline 67 and 2-(4’-isothio- cyanatophenoxymethyl) imidazoline §g. These compounds incorporate structural features of IBI 60, idazoxan 19 and cirazoline 6 and they produced IBI like responses which were not significantly blocked by PBZ pretreatment of rat aortic tissues (Table 7). It appears that modification of the bridge does not greatly improve the non-a-AR activity of IBI. To examine the significance of the imidazoline ring on the action of IBI in rat aorta, the imidazoline was replaced with an imidazole ring in 2-(4’-isothiocyanatobenzyl) imidazole 69 and with tertiary amino group in N,N-dimethyl-2-(4’-isothiocyantophenyl) ethylamine 70. Both compounds produced concentration-dependent response similar to that of IBI in rat aorta and their responses were not affected by the PBZ pretreatment (Table 8). This finding suggests that the non-a-AR mediated contractile responses in smooth muscle are not solely due to the presence of an imidazoline ring, but rather the positive charge or potential of H-bonding is important for the production of contraction by IBI analogues in aortic smooth muscle. Furthermore, the data indicate that the maximal response but not potency of analog 69 is less than that of IBI (Tables 6 and 8). This may be due to a conformational and/or an electronic factor. First, the imidazole ring is flat relative to the imidazoline ring due to presence of an additional double bond, and secondly, the pKa of the imidazole group is lower than the pKaof the imidazoline, resulting f* in a smaller proportion of the protonated form of 69 at physiological pH. 3.2.2. Effect of imidazoline liaand fldazoxan. cirazolinel pretreatment on stimulatory activities of IBI and analogues 67-70 on rat thoracic aorta. Since Hussain et al.ns have shown that IBI competes with [3H]-idazoxan for imidazoline preferring non-a-AR sites of guinea-pig cortex membrane, we presumed that the non-a-AR activity of IBI in rat aorta might be related to the interaction of IBI with the IGRS. To test this hypothesis idazoxan or cirazoline were added to PBZ pretreated rat aortic tissues, prior to the addition of IBI. In neither case, did idazoxan (at 0.1 pM or 1 pM) or cirazoline (3 pM), affect the contractile activity of IBI on rat aorta in PBZ pretreated tissues (Table 9). Furthermore, Table 9 summarizes the effect of cirazoline and idazoxan on action of selected analogues 6Z, 69, 69, and 70 in rat aorta. These four compounds were chosen since they showed an afffinity and intrinsic activity similar to IBI in PBZ treated aortic strips (see tables 6,7 and 8). For these compounds no significant change in the concentration-response curve was observed in PBZ pretreated tissues in the presence of cirazoline or idazoxan. These results show that non-a-AR stimulatory activities of IBI and analogues 67, 68, 69, and 70 in rat aorta are not related to the activation of IGRS. Binding studies carried out using pH]-p-aminoclonidine, [3H]-idazoxan and pH]- clonidine have shown the presence of putative IGRS in different tissues and species with the highest abundance in the brain, liver and kidney. 80.109.137 Tesson et aljse have studied the distribution of the IGRS in different rabbit and human tissues. They could not detect any [3H]idazoxan binding in the heart homogenate, neither in rabbit or human heart.138 Only a very low density of IGRS was detected in mitochondrial fraction from rabbit and human h e a rts We conclude that rat heart is deficient of IGRS. If rat thoracic aorta also lacks IGRS, then this is the reason why our compounds can not exert their contractile action through activation of IGRS. So we decided to test our compounds in tissues rich with IGRS, bovine ventrolateral medulla. 62 Table 9. Effect of Imidazoline Ligands (Cirazoline and Idazoxan) on Contractile Responses of IBI and its Analogues 67-70 in Rat Thoracic Aorta. Treatment n EC5o(pM)c %Emaxd IBI 4 45.0 ± 3.0 100 ± 4 IBI+PBZ&CIRa 4 43.0 ± 6.0 94 ± 3 IBI+PBZ&IDA(0.1pM)a 3 54.0 ± 3.0 104 ± 6 IBI+PBZ&IDA(1pM)a 3 54.0 ± 6.0 105 ± 9 SZ 4 18.3 ± 0.7 100 ±11 PBZ+CIR+67b 3 14.9 ± 1.3 100 ±13 PBZ+IDA+67b 3 18.1 ± 0.6 114 ±13 68 3 17.2 ± 0.9 100 ±12 PBZ+CIR+gfib 3 16.4 ± 1.3 117 ±21 PBZ+IDA+g8b 3 16.1 ± 1.1 110 ±29 69 3 28.6 ±11.7 100 ±23 PBZ+CIR+S9b 3 25.5 ± 8.6 102 ±32 PBZ+IDA+69b 3 40.9 ± 9.5 79 ±13 70 4 16.8 ± 0.2 100 ± 9 PBZ+CIR+70b 3 16.9 ± 0.0 91 ± 6 PBZ+IDA+70b 3 16.3 ± 0.6 94 ± 9 a Tissues were pretreated as follows: 30 nM PBZ for 20 min with washout followed by 3 pM cirazoline (CIR) for 1 h; 30 nM PBZ for 20 min washout followed by 0.1 or 1 pM idazoxan (IDA) for 1 h, as compared to 30 pM phenylephrine (100% maximal response) which was obtained prior to these treatments. IBI induced responses were monitored between 20-30 min after addition. b Tissues were pretreated with PBZ (10-8 m , 20 min) with washout followed by CIR (10-6M, 60 min) or IDA (10-6 m , 60 min). Analogue-induced responses were monitored between 35-45 min after each addition, c Data represent the mean ± S.E.M. of n=3-4. EC50 value =drug concentration to produce 50% of maximal response (pM). d %Emax = percent of maximal contractile response to compound expressed relative to the maximal contraction produced by the compound alone. 63 3.2.3. Effects of IBI and its analogues 65.67.68 and 70 on saturation binding kinetics of f32S|]p-iodoclonidine at ^-imidazoline and otg-AR in the bovine VLM. To date there is no selective radioligand that will label exclusively imidazoline binding sites. [3H]Clonidine and [3H]p-aminoclonidine have been used for labeling of both a2-adrenergic and imidazoline i-i receptors. In order to establish that the subpopulation of the specific sites labeled by [3H]clonidine corresponds to the imidazoline sites, a masking technique can be used to selectively block the a2-AR. Recently, another radioligand, [i25|]p-jodoclonidine 104. has been described that shows a significantly higher affinity for both lrimidazoline and a2-ARsJ°4 It has been proposed that higher affinity of 104. probably due to its lipophilicity, might improve the detection of lrimidazoline sites. 104 Ernsberger et al. have shown that two-thirds of the sites labeled by 0.5 nM [i25|]p-j0doclonidine in the bovine VLM are a2-AR (since epinephrine at 0.1 mM inhibited only 67% of the specific binding). 104 The remaining are lrimidazoline binding sites, since imidazole-4-acetic acid and cirazoline (ligands that selectively bind at the imidazoline site) inhibited about 33% of the binding sites labeled with 1 0 4 .w ci [125l]p-lodo Clonidine 104 Table 10 summarizes the binding properties of IBI and analogues §5,67, 68 and 70 to the lrimidazoline and a2-AR binding sites in the bovine VLM. According to the results IBI and tested analogues inhibited [i25|]p-jodoclonidine binding to both lrimidazoline and a2-AR of bovine brainsteam giving a rank order of potencies ( ICS0, pM): 68 (5.81) > §7 (5.5) > IBI (5.13) > 70 (5.10) > 6§ (4.79). Pretreatment of the bovine VLM with PBZ and N-ethylmaleimide (to mask the cts-AR) did not alter the order of 64 Table 10. Summary of Binding Properties of IBI and its Analogues 65, 67, 68 and 70 at ^-Imidazoline and c^-AR Binding Sites in Bovine VLM a x^, V R w n plCsob Compound R X n control +PBZc 70 NCS N(CH3)2 CH2 1 5.10± 0.05 5.38± 0.05 65 NHCOCH2CI CH2 0 4.79± 0.05 5.08± 0.03 oN 60 NCS CH2 0 5.13± 0.02 5.63±0.02d ON' H 67 NCS CH2 1 5.5 ± 0.04 5.47± 0.05 ON' H 68 NCS 1 5.81±0.05 6.38± 0.05d N H aAssays were conducted by increasing conc. of competing drug with BVLM and 0.2 nM [i25|]p.jodoclonidine. bData are plC50 values (-log M) for 4 experiments, each conducted in triplicate. ^Treated BVLM were preincubated with 0.1 pM phenoxybenzamine and 0.25 mM N-ethylmaleimide to irreversibly inactivate a majority of the a2-AR. ^Significant effect of PBZ pretreatment, p<0.05. 65 potency. However, PBZ pretreatment increased the affinity of IBI and 68 indicating a modest selectivity of these two analogues for lrimidazoline over a2-AR binding sites. From these results we can recognize the significance of the isothiocyanato group as an electrophilic group, since all compounds with a NCS group show higher affinity for both lrimidazoline and a2-AR. This could be due to the higher reactivity of the isothiocyanato group vs. chloroacetamido group (compound 65 has the lowest affinity among tested analogues) or the distance between the electrophile and the aromatic ring plays a significant role. Although compounds 67,68 and IBI (comprised of an intact imidazoline ring) have higher affinity for imidazoline sites than analog 7fi, it appears that probably the positive charge, associated with the imidazoline ring or tertiary amine, is primarily responsible for interaction with the lrimidazoline binding site. Finally, it appears that increasing the distance between the aromatic and imidazoline ring from one carbon atom in IBI to two atoms in gZ or 6§ increases the affinity not only for imidazoline but also for a2-AR. Another yet interesting finding is that exchange of single carbon atom in 67 with oxygen in gfi significantly increases affinity and selectivity for imidazoline receptor over a2-AR sites (see Table 10). In conclusion, our assumption that IBI and its analogues bind at imidazoline receptors proved to be true. However, they have low potency and selectivity for lrimidazoline receptor, so the search for selective and potent irreversible ligand at this new site continues. Furthermore, we are planing to test these compounds in kidney tissue that is rich in lrimidazoline sites. 3.2.4. Effect of Ca2± channel blocker (nifedipine, verapamih pretreatment on the stimulatory activities of IBI and analogues 67-70 on rat thoracic aorta. Although, we have shown that IBI and its analogues interact with the imidazoline receptors in the brain we were still curious as to the mechanism of action of IBI and its analogues in the heart. First, we tried unsuccessfully to block the activity of IBI and its 66 analogues in smooth muscle preparations (rat aorta, guinea pig ileum) with variety of compounds including cinanserin (5-HT2 blocker), cimetidine (H2-histamine blocker), pyrilamine (Hrhistamine blocker), 4-aminopyridine (K+ channel blocker). Pretreatment of rat aortic tissues with nifedipine (10*7M) or verapamil (1(HM) shifted the concentration response curve of IBI as well as its analogues fiZ. fifi, £9 and ZO to the right (Table 11)."? It appears that the contractile effects of IBI and analogues 67-70 depends on a calcium sensitive mechanism. Furthermore, when IBI was incubated with rat aortic strips in Ca2+ deficient media, the concentration response curve was shifted to the right with a marked reduction of the maximal response (35% of control, Table 11, Figure 15). This is an indication that the contractile action of IBI in rat aorta is mainly dependent on the extracellular calcium, and translocation of extracellular calcium through a voltage-dependent Ca2+ channel. □ IBI (n=4) ■ Ca2+ deficient buffer + IBI (n=4) -7 -6 i -5 Log [Drug] (M) Figure 15. The Action of IBI on Rat Thoracic Aorta in Physiological Salt Solution(PSS) as a Control (□) and in Ca2+ Deficient Buffer (■). 67 Table 11. Effect of Various Treatments on the Concentration Dependent Responses to IBI and Selected IBI Analogues ££-70 in Rat Thoracic Aorta.* Treatment n EC50(pM)b %Emaxc IBI 4 5.8 ± 1.9 100 ± 9 Verapamil+IBI 4 21.4 ± 9.7d 48 ± 10d Nifedipine+IBI 3 19.8 ± 8.7d 42 ± 12d Ca2+ deficient buffer + IBI 4 12.7 ± 0.3d 34 ± 9d 67 3 18.3 ± 0.7 100 ±11 Nifedipine+6Z 3 67.0 ±16.5d 21 ± 6d 68 3 17.2 ± 0.9 100 ± 1 2 Nifedipine+§8 3 52.1 ± 0.0d 11 ± 6d m 3 28.6 ±11.7 100 ± 23 Nifedipine+69 3 38.9 ±13.2 35 ± 5d ze 4 16.8 ± 0.2 100 ± 9 Nifedipine+70 3 48.5 ± 6.1d 29 ± 9d * Tissues were pretreated with verapamil (10-6 M, 60 min), nifedipine (10-e M, 60 min) or calcium-deficient PSS. Analogue-induced responses were monitored between 35-45 min after each addition. b Data represent the mean ± S.E.M. of n=3-4. EC50 value = drug concentration to produce 50% maximal response (pM). c%Emax= percent of maximal contractile response to IBI analogue, expressed relative to the maximal contraction produced by the compound alone. d Means are statistically different from the corresponding control value (IBI analogue alone) at p < 0.05 (paired observations). Does IBI activate Ca2+ channels directly or does it act via receptors coupled to membrane C a2+ channels? In this regard, a*- and a2-AR are examples of receptor systems coupled to membrane Ca2+ channels, and activation of these receptors involve the translocation of extracellular Ca2+ through Ca2+ channels.*** However, in our case pretreatment of the smooth muscle tissues with variety of different receptor blockers still failed to block the effect of IBI and its analogues 67-70. At the moment we are exploring a possibility that IBI and its analogues exert their action, in rat aortic tissue, by activating C a 2 + channels directly. We compared the structure of IBI, and its analogues £7 and £g with C a 2+ channel blocker, nifedipine and with Ca2+ channel activator (agonist) Bay K 8644 using molecular modeling (Sybyl). The reason for the selection of the 1,4-DHP system was that Bay K 8644 and closely related molecules were the only activators of C a 2+ channels and IBI also act as an activator of Ca2+ channel. Very recently a new set of competitive Ca2+ channel agonists have been reported. «9 In the past several groups have reported on the action of irreversible C a2+ channel antagonists^ 3.3. MOLECULAR MODELING Nifedipine 36 and Bay K 8644 40 are both members of 1,4-dihydropyridine family of compounds that interact with the Ca2+ channel.** However, while nifedipine acts as an antagonist, Bay K 8644 acts as an agonist at the 1,4-dihydropyridine binding site of the Ca2+ channel.** Furthermore, Bay K 8644 exhibits remarkable enantiomeric selectivity: the S-enantiomer acts as agonist (causing concentration-dependent vasoconstriction) and antagonism is associated with R-enantiomer.59 Structures of 1,4-dihydropyridines (nifedipine and Bay K 8644) and IBI are chemically quite different. However, certain structural similarities are noted: all compounds are comprised of an aromatic ring, and a proton carrying N atom either as a part of 1,4-dihydropyridine ring or the imidazoline ring. Structure-activity relationship data for nifedipine show that the presence of the N1-H is essential for activity, probably due to its role as a hydrogen bond donor at the 1,4-dihydropyridine binding site of Ca2+ channel. Other basic structural requirements for 69 activity are a phenyl or substituted phenyl ring in 4 position of 1,4-dihydropyridine ring and ester groups in the C3 and Cs positions.51 It is generally agreed that the active conformation of 1,4-dihydropyridines include a quite flattened boat conformation of 1,4-dihydropyridine ring with the phenyl ring in a pseudoaxial position.44 The structural basis for the opposing pharmacology of Bay K 8644 vs nifedipine is not completely understood, however there are some arguments that the pharmacological properties of agonists and antagonists may be based on differences in hydrogen bonding patterns at the active site.44 Other interpretations are also possible, one proposal is that there are discrete activator and antagonist binding sites.44 IBI and analogues H a n d f»8 were superimposed with (S)-Bay K 8644 and nifedipine, in order to find out if there is any possibility that IBI, IZ and S I can accommodate the 1,4 dihydropyridine active site of the Ca2+ channel. Nifedipine and Bay K 8644 are quite constrained molecules with the distance between the center of the aromatic ring and N atom of 1,4-dihydropyridine ring of 4.8 A. We decided to use this distance to constrain the systematic search for analogues 67 and 68 so that distance between the aromatic ring of 67 and one of the N atoms in the imidazoline ring is between 4.55-5.05 A. The results of superposition indicate that the aromatic rings of Bay K 8644 or nifedipine are in the same plane with aromatic ring of IBI or that of 67 and 68 (Figures 16, 17,18,19 and 20) . Another striking feature is the overlay of hydrogen bond donor group N1-H in dihydropyridines, with one of N-H group in the 2 substituted imidazolines. The only feature that is missing in IBI and analogues 67 and 68 that is present in both nifedipine and Bay K 8644 is hydrogen bond acceptor group (ester or nitro group). In contrast IBI and analogues IZ/68 have an additional hydrogen bond donor group (that is another N-H group). These results (Figures 16,17,18,19 and 20) depict that IBI and its analogues @7 and 61 are able to mimic, to a reasonable extent, spacial orientation of calcium channel ligands (Bay K 8644 as well as nifedipine), and therefore there is a distinct possibility that IBI and its analogues exert their action in rat aorta by increasing the influx of extracellular Ca2+ through the activation of Ca2+ channels. Figure 16. Superimposition of IBI (yellow) and Nifedipine (multicolor). 71 Figure 17. Superimposition of IBI (yellow) and Bay K 8644 (multicolor). 72 Figure 18. Superimposition of 67 (purple) and Nifedipine (multicolor). 73 Figure 19. Superimposition of 67 (purple) and Bay K 8644 (multicolor). Figure 20. Superimposition of 68 (red) and Nifedipine (multicolor). SUMMARY The synthesis of IBI, jBO and its meta analog §4 was successfully completed in four steps starting from the corresponding nitrobenzylonitrile. The ortho isomer 52 could not be isolated due to the intramolecular reaction between the highly reactive isothiocyanato group and imidazoline nitrogen that led to the formation of 103. The synthesis of affinity label analogs of IBI 5 5 - 2 1 was successfully completed. Compound 69 could not be obtained by dehydrogenation procedure of imidazoline intermediate Z 4 . All IBI analogues produced concentration-dependent contractile responses on rat thoracic aorta in a manner similar to IBI characterized by a slow onset and long duration of action. The analogs 5 1 , 62 and Z1 produce the contractions of rat thoracic aorta by activating ar AR in this tissue. However, all other analogs produce the stimulatory actions in this tissue through the non-ai-AR mediated mechanism. According to some pharmacological data the functional effect of IBI and analogs 67 - 7Q in rat thoracic aorta depends upon calcium sensitive mechanism. The molecular modeling results depict that IBI and its analogs 5Z and 5 8 are able to mimic, to a reasonable extent, spacial orientation of calcium channel ligands, and therefore there is a distinct possibility that IBI and its analogs exert their action in rat aorta by increasing the influx of extracellular Ca2+ through the activation of voltage dependent Ca 2+ channels. The binding studies of IBI and 65,67, 6 8 and 70 to the bovine VLM show that these compounds bind to both a2-AR and 11-imidazoline receptors, and that IB I and 58 show a modest selectivity for lrimidazoline binding sites. CHAPTER IV EXPERIMENTAL Melting points were determined on a Thomas-Hoover capillary melting point apparatus and are uncorected. Infrared data were collected on an Analect RFX-40 FTIR spectrophotometer (The Ohio State University - College of Pharmacy) or on an Perkin Elmer 2000 FT-IR spectrophotometer (University of Tennessee - College of Pharmacy). The NMR spectra were obtained either on an IBM AF-250 FTNMR spectrometer (250Hz)(The Ohio State University - College of Pharmacy) or on an Bruker ARX 300 FT NMR spectrometer (300 MHz) (The University of Tennessee - College of Pharmacy) and are reported in parts per million. Mass spectra were obtained at the College of Pharmacy by use of a Kratos MS25 RFA mass spectrophotometer or at the Ohio State University Campus Chemical Instrumentation Center by use of a VG 70-250S or a Kratos MS-30 mass spectrometer. Elemental analyses were performed by Galbraith Laboratories, Inc., Knoxville, TN or by Atlantic Microlab, Inc., Norcross, GA and were within ±0.4% of the theoretical values for the elements indicated. Anhydrous ethanol was prepared by carefuly warming the mixture of magnesium turnings, sodium iodide and ethanol until a vigorous reaction occured. After the reaction subsided heating was continued until all the magnesium was converted into magnesium ethoxide. At this point more ethanol was added, refluxed for one hour and distilled off. Anhydrous benzene was produced by refluxing with and distillation from sodium. Anhydrous dichloromethane was produced by refiuxing with and destilation from calcium hydride. 76 77 2-(4,-NItrobenzynim[dazoline hydrochloride 74. To a cold suspension of 4-nitrophenylacetonitrile (16.20 g, 100 mmol) in CH2 Cl2/C 6 H6 (125 mL/125 mL) was added anhy. EtOH (5.00 g, 100 mmol) and the mixture was bubbled with anhy. HCI gas (6.00 g, 150 mmol), while cooling and stirring in an ice-water bath. The resulting mixture was kept in a refrigerator for 3 days, poured into anhy. Et20 (500 mL) to give white imidate 75 2 1 .43 g (8 8 %, lit. 96%): mp 195-197°C (lit. mp 195°C). To a suspension of imidate 7£ (20.60 g, 84 mmol) in EtOH (200 mL) was added ethylendiamine (5.11 g, 90 mmol) in EtOH (50 mL) via cannulation over period of 30 min. The resulting mixture was refluxed overnight, cooled to r.t., acidified with conc. HCI (to pH * 2 ) and the solvent removed in vacuo to give brown residue. The residue was dissolved in water (100 mL) and washed with CH 2CI2 (2 x 50 mL). The water layer was then alkalized with 10% NaOH (pH = 10) and extracted with CH 2CI2 (3 x 50 mL). Combined organic extracts were washed with brine, dried over Na 2S04, filtered and solvent removed under reduced pressure on rotavapor to give 74 as a free base in the form of yellow solid 15.61 g (90.3%). Solution of free base (15.61 g, 80 mmol) in EtOH (100 mL) was bubbled with HCI gas (4.20 g, 130 mmol), and then poured into EtOAc (100 mL) to give yellow precipitate. Recrystallization from MeOH/Et20 gave slight yellow crystals of 74 13.16 g (72%, lit. 30%), mp 254-255°C (lit. mp 244-246°C); 1H NMR (CD3 OD, 250 MHz) 5 8.28-8.25 (d, JAB= 8 .8 Hz, 2H, ArH, ortho to N 02), 7.64-7.60 (d, Jab= 8 . 8 Hz , 2H, ArH, meta to N02), 4.07 (s, 2H, CH2), 3.95 (s, 4H, 2 x CH 2 imidazoline). 78 2-(4,-Aminobenzyl)imidazoline hydrochloride 61. HN A mixture of 2-(4’-nitrobenzyl)imidazoline hydrochloride 74 (5.00 g, 21 mmol) and 10% Pd/C (0.50 g) in MeOH (50 ml) was hydrogenated using a Parr apparatus at room temperature, 40 psi for 4 h. The resulting mixture was filtered over Celite, and evaporated to give white residue, which was recrystallized from MeOH/Et20 to afford 4.09 g (93%) of 01 as colorless crystals: mp 217-218°C; 1H NMR (CD 3OD, 250 MHz) 8 7.06-7.02 (d, Jab=8.5 Hz, 2H, ArH), 6.72-6.69 (d, JAB=8.5 Hz, 2H, ArH), 3.89 (s, 4H, 2 x CH2), 3.71 (s, 2H, CH2); IR (KBr, cm-1) 3400 (N-H stretching), 3320 (N-H stretching), 1605 (N-H bending); Anal. Calcd for C 10H14CIN3 x 0.5 H20 : C, 54.41; H, 6.85; N, 19.04. Found: C, 54.81; H, 6 .8 6 ; N, 19.22. 79 2-(4,-lsothiocvanatobenzvMmidazoline hydrochloride (IBh. 60. HN HCI SCN To a solution of CSCI 2 (5.43 g, 0.047 mol) in acetone (30 mL) was added 2-(4’- aminobenzyl)imidazoline 61 (1.00 g, 5 mol) in H20 (10 mL) dropwise over 10 minutes, while cooling in an ice-water bath. The stirring was continued at room temperature for additional 1.5 h. Removing the solvent in vacuo at room temperature gave a brown solid residue which was taken into CH 2CI2 (50 mL), washed with H 20 , (10 mL) and dried over Na2 S04. The resulting CH 2 CI2 solution was concentrated under reduced pressure to afford a viscous oil which was crystallized with acetone. Recrystallization from acetone/Et20 gave white crystals of 6 Q 0.51 g (43%): mp 153-155°C; 1H NMR (CDCI3, TMS, 250 MHz) 5 7.67-7.64 (d, JAB=8.2 Hz, 2H ArH), 7.11-7.08 (d, JAB=8.2 Hz, 2H, ArH), 4.10 (s, 2H, CH2), 3.87 (s, 4H, 2 x CH 2 imidazoline); IR (KBr, cm-1) 3060 (N-H stretching), 2175, 2104 (-N=C=S), 1618 and 1595 (C=C and C=N stretching); Anal. Calcd. for CnH^CINgS: C, 52.08; H, 4.77; N, 16.57. Found: C, 52.29; H, 4.63; N, 16.72. 80 2-f4,-Chloroacetamidobenzv0imidazoline hydrochloride 65. HCI CIH2 C0CHN To a suspension of 2-(4,-aminobenzyl)imidazoline hydrochloride f i l (0.20 g, 0.9 mmol) in CH3CN (15 mL) was added a solution of chloroacetic anhydride (0.19 g, 1.1 mmol) in acetonitrile (5 mL). The mixture was stirred overnight at room temperature and then concentrated under reduced pressure yielding white solid. Recrystallization from MeOH/Et20 afforded §5 0.19 g (73%) as white needles: mp 214.6-216°C; 1H NMR (CD3OD, 250 MHz) 5 7.64-7.61 (d, JAB= 8 .6 Hz, 2H, ArH), 7.33-7.30 (d, JAB= 8 . 6 Hz, 2 H, ArH), 4.18 (s, 2H, CH2), 3.91 (s, 4H, 2 x CH2), 3.86 (s, 2H, CH 2CI); IR (KBr, cm-1) 3113 (CONJH stretching), 3051-2975 (imidazoline N-H stretching), 1683 (CONH. stretching), 1613 and 1596 (C=C and C=N stretching); MS m/z 251/253 (M+-HCI), 250 (base); Anal. Calcd. for C 12H15C|2N30 : C, 50.02; H, 5.25; N, 14.58. Found: C, 50.15; H, 5.23; N, 14.51. 81 2-f4’-lodoacetamidobenzvhimldazoline hvdroiodide 66. HI ih2coch To a suspension of 2-(4,"aminobenzyl)imidazoline HCI f il {0.53 g, 2 mmol) in CH3CN (20 mL) was added a solution of iodoacetic anhydride (1.00 g, 2 mmol) in CH3CN (5 mL). The mixture was stirred overnight at room temperature and than concentrated under reduced pressure yielding yellow solid. Recrystallization from Me0H/Et20 gave @6 0.186 g (20%) as yellow crystalls: mp 180°C dec.; 1H NMR (CD 3 OD, 250 MHz) 5 7.61-7.57 (d, JAB= 8 .6 Hz, 2H, ArH), 7.32-7.28 (d, J= 8 .6 Hz, 2H, ArH), 3.92 (s, 4H, Cidfe! and CHg), 3.86 (s, 4H, 2 x CH2 imidazoline); IR (KBr, citH ) 3129 (COM! stretching), 1652 fCONH streatching), 1599 (C=C and C=N stretching), 1458 (aromatic C=C stretch); MS-FAB 344 (MH+-HI); Anal. Calcd. for C 12H15l2 N30 : C, 30.62; H, 3.21; N, 8.93. Found: C, 30.72; H, 3.23; N, 8.92. 82 2-f4,-Amino-3,-iodobenzyl)imidazoline dihydrochloride 75. HN H2N To a solution of Nal (2.16 g, 14 mmol) and 2-(4’-aminobenzyl)imidazoline HCI f i l in sodium acetate buffer (0.1 M, pH=4.1) was added a solution of thallium trichloride TICI 3 (5.78 g, 17 mmol) in H20 (50 mL), over a 30 min period. The brown mixture was heated on a oil bath for 2 h under argon. The reaction mixture was stopped by the addition of Na2 S 0 3 (1.79 g, 14 mmol) in H20 (20 mL). After the mixture had cooled to room temperature, the solution was alkalized (pH=9) by adding Na 2C0 3 , and then extracted with CHCI 3 (4x20 mL). The organic extracts were combined, washed with brine, dried over Na 2S04 and concentrated under reduced pressure to give Z§. as a free base, 3.56 g (83%) as yellow solid. Imidazoline Z5, free base, (3.4 g, 1 1 mmol) was dissolved in MeOH (50 mL) and saturated with HCI gas. The mixture was than concentrated under reduced pressure to give yellow solid which was recrystallized from MeOH/Et20 to give 2-(4’-amino-3’-iodobenzyl)imidazoline dihydrochloride 75 3.31 g (78%): mp 209-210°C; 1H NMR (DMSO-d6, 250 MHz) 5 10.27 (s, 1H, =NH+), 7.69-7.68 (d, J=2 Hz, 1H, ArH), 7.22-7.18 (dd, J=2 Hz, J = 8 Hz, 1H, ArH), 6.92-6.89 (d, J = 8 Hz, 1H, ArH), 5.66 (br.s, 3H, ArNH3+) 3.77 (s, 4H, 2 x CH 2 imidazoline), 3.7 (s, 2H, CH2); IR (KBr, cm-1) 3135 (NH3+, N-H stretching), 1612 (C=C and C=N stretching), 1488 (aromatic C=C stretching); MS m/z 301 (M+, base), 174 (M+-I); Anal. Calcd. for C io ,H 14C I2 IN 3 : c, 32.11; H, 3.77; N, 11.23. Found C, 32.40; H, 3.84; N, 11.10. 83 2-(3’-lodo-4’isothiocvanatoberizvnimidazoline hydrochloride 71. HN HCI SCN To a cold solution of CSCI 2 (1.15 g, 10 mmol) in acetone (20 mL) was added a solution of NaHC0 3 (0.10 g, 1 mmol) and imidazoline 75 (0.40 g, 1 mmol) in H20 (7 mL). The mixture was stirred at room temperature for 2 hrs and than solvent removed in vacuo to give residue which was taken in CH 2CI2 to afford pink solid. Recrystallization from MeOH/Et20 gave 71 as white crystalls 0.22 g (54%): mp 218-220°C; 1H NMR (DMSO-de, 250 MHz) 8 10.25 (s, 1H, =NH+), 7.99-7.98 (d, J=1.6 Hz, 1H, ArH), 7.54- 7.45 (m, 2H, ArH), 3.88 (s, 2H, CH2), 3.79 (s, 4H, 2 x CH 2 imidazoline); IR (KBr, cm-1) 3272 (N-H stretching), 2189, 2094 (-N=C=S), 1639 (C=C stretching); MS m/z 343 (M+ -HI), 342 (base), 216 (M+-I), 284 (M+-NCS); Anal. Calcd. for: CnHnCIINgS: C, 34.80; H, 2.92; N, 11.06. Found: C, 34.63; H, 2.99; N, 10.95. 84 4-Nitrophenylpropylonitrile 80. To a solution of 4-nitrophenethylbromide (5.00 g, 22 mmol) in DMF (20 mL) was added NaCN (1.30 g, 26 mmol). The mixture was stirred at r.t. for 1.5 h, and then heated on an oil bath at 70-80°C. After 4 h, the mixture was cooled to r.t. poured into H20 (20 mL) and extracted with CH 2CI2 (3 x 25 mL). Combined organic extracts were washed with brine, dried over anhy. Na 2S04l filtered and solvent removed under reduced pressure on rotavapor to give brown oil. Trituration with H20 gave brown solid 3.4 g ( 8 8 %). Recrystallization from acetone/H20 affored yellow crystals (needles) of nitrile 3 Q 2.5 g (65%) mp 78.5-80°C; 1H NMR (CDCI3i 250 MHz) 8 8.24-8.21 (d, JAB = 8 .6 Hz, 2 H, ArH ortho to N 0 2), 7.45-7.42 (d, J= 8 . 6 Hz, 2H, ArH meta to N02), 3.11-3.06 (t, J=7.2 Hz, 2H, CH2), 2.73-2.67 (t, J= 7.2 Hz, 2H, CH2); IR (KBr, cm-1) 311,3083 (aromatic C-H stretch), 2243 (-C=N stretch), 1609 (C=C stretch), 1513 (asymmetric N0 2 stretching), 1340 (symetric N0 2 stretching). 85 Ethyl-S-^’-nitrophenyQiminopropionate hydrochloride 82. OCH2CH3 To a cold solution of p-nitroacetonitrile (3.06 g, 17 mmol) in benzene/CH 2CI2 ( 1 0 m L /10 mL) was added anhy. EtOH (0.80 g, 17 mmol) and the mixture was bubbled with HCI gas (=0.5 g) while cooling in an ice-water bath. The mixture was kept in a refrigerator for 3 days, poured into Et20 (100 mL) to afford imidate 3 2 4 . 1 2 g (92%) mp 119-121 °C as white solid.iH NMR (DMSO-d 6 , 250 MHz) S 8.19-8.16 (d, Jab= 8 .8 Hz, 2H, ArH ortho to N 02), 7.57-7.54 (d, JAB= 8 . 8 Hz, 2H, ArH meta to N02), 4.42-4.33 (q, J=7 Hz, 2H, -OCHoCH'al. 3.12-3.01 (m, 4H, CH2-CH2), 1.32-1.26 (t, J=7 Hz, 3H, -OCHoCH^. 86 2-(4,-Nitrophenethyl)imidazoline hydrochloride 84. HCI To a slurry of imidate §2 (3.80 g, 15 mmol) in EtOH (40 mL) was added ethylenediamine (0.93 g, 16 mmol) and the mixture was refluxed overnight, cooled to r. t. acidify with conc. HCI and EtOH removed in vacuo to give brown solid. This residue was dissolved in H20 (50 mL) washed with CH 2CI2 (20 mL). The water layer was alkalized with 10% NaOH (pH= 9-10) and extracted with CH 2CI2 (3 x 20 mL). Organic extracts were combined washed with brine, dried over Na 2 S0 4 , filtered and concentrated on rotavapor to give 2-(4’-nitrophenethyl)imidazoline 84 as a free base 2.75 g (85%). The free base was converted into HCI salt by passing HCI gas throught the solution of 84 in MeOH. Recrystallization from MeOH/Et20 gave yellow crystals of §4 as HCI salt 2 . 0 2 g (79%) mp 182-184°C; 1H NMR (DMSO-d6, 250 MHz) 5 8.21-8.17 (d, JAb= 8 . 8 Hz , 2 H, ArH ortho to N 02), 7.56-7.53 (d, J=8.7 Hz, 2H, ArH meta to N 02), 3.77 (s, 4H, 2 x CH2 imidazoline), 3.16-3.10 (t, J=7.8 Hz, 2H, CH2), 2.89-2.83 (t, J=7.7 Hz, 2H, CH2); IR (KBr, cm-i) 3069-2957 (N-H stretching), 1601 (C=C stretching), 1343 (symmetric N0 2 stretching) ; MS m/z 219 (M+-HCI), 218 (base), 172 (M+-HCI, N02); Anal. Calcd. for ChH 14CIN30 2: C, 51.67; H, 5.52; N, 16.43. Found: C, 51.72; H, 5.50; N, 16.60. 87 2-f4,-AminoDhenethv0imidazoline dihvdrochloride 86. A mixture of 2-(4’-nitropnenethyl)imidazoline hydrochloride 84 (1.50 g, 6 mmol) and 10% Pd/C (=0.15 g) in MeOH (20 mL) was hydrogenated using a Parr hydrogenation apparatus at r. t . , 40 psi for 3 h. The resulting mixture was filtered over Celite, and evaporated to give yellow oil. Crystallization from MeOH/Et20 gave slight yellow long needls of imidazoline 8 6 1.27 g (95%) mp 168-169°C as a monohydrochloride salt. To a solution of monohydrochloride salt of (0.2 g, 0.9 mmol) in MeOH was bubbled HCI gas to to affored imidate as dihydrochloride salt. Solvent was removed on rotavapor and recrystallization from MeOH/Et 2 0 gave white chrystalls 0.17g (73%) mp >250°C, dec.; 1H NMR (DMSO-d6, 250 MHz) 5 10.26 (s. 2 H, NH2), 7.35-7.26 (m, 4H, ArH), 3.77 (s, 4H, 2 x CH2 imidazoline), 2.98-2.95 (t, J = 8 Hz, 2H, CH2), 2.81-2.79 (t, J = 8 Hz, 2H, CH2).; IR (KBr cm-i) 2969 NH3+ N-H stretching), 1588 (C=C stretching); MS m/z 189 (M+-2HCI, base); Anal. Calcd. for CnH^CfeNs: C, 50.39; H, 6.54; N, 16.03. Found: C, 50.44; H, 6.53; N, 16.04. 88 2-f4,-lsothiocvanatophenethvhimidazoline hydrochloride 67. * HCI, 1/2H20 SCN To a cold solution of thiophosgen (0.304 g, 3 mmol) in acetone (20 mL) was added solution of 2-(4’-aminophenethyl)imidazoline hydrochloride 8 6 (0.5 g, 0.002 mol) in H 2O (5 mL), over period of 15 min, while cooling in an ice water bath. After addition was completed the mixture was stirred overnight at r.t., solvent removed in vacuo to give yellow oil. The oil was dissolved in CH 2CI2 (50 mL), and washed with brine ( 1 0 mL). The water layer was then extracted with CH 2CI2 (3 x 50 mL). All organic extracts were combained, dried over Na 2S04, filtered, and solvent removed under reduced pressure on rotavapor at r. t. to give imidazoline as white solid 0.47 g (80%). Recrystallization from MeOH (cold)/ Et20 gave white crystalls 0.29 g (49%) mp 170-171 °C; 1H NMR (DMSO-de, 250 MHz) 5 10.27 (s, 1H, =NH+), 7.41-7.31 (q, J=8.5 Hz, 4H, ArH), 3.76 (s, 4H, 2 x CH2 imidazoline), 3.02-2.96 (t, J=7.7 Hz, 2H, CH2), 2.82-2.76 (t, J=7.6 Hz, 2H, CH2); IR (KBr, cm-1) 3098 (N-H stretching), 2179, 2120 (-N=C=S), 1600 (C=C stretching); MS m/z 231 (M+-HCI, base) 230 (M+-HCI.H); Anal. Calcd. for C 12H i4CIN3S x 1/2H20 : C, 52.07; H, 5.46; N, 15.18. Found: C, 51.94; H, 5.44; N, 15.11. 4-Nitrophenoxyacetonitrile 81. To a solution of 4-nitrophenol ( 1 0 .0 0 g, 72 mmol) in acetone (50 mL) was added bromoacetonitrile (8.60 g, 72 mmol) and Na 2C0 3 (7.60 g, 72 mmol). The mixture was refluxed on an oil bath for 42 h, cooled to r. t., and solvent removed on rotavapor to give brown residue. The residue was taken into H20 (50 mL), and extracted with EtOAc (3 x 50 mL). Organic extracts were combined, washed with 2N NaOH (50 mL), H20 (50 mL), brine (50 mL), and dried over Na 2 S04. Solvent was then removed under reduced pressure to give brown solid. Recrystallization from hot MeOH affored collorless crystals 10.28 g (80%, lit. 58%), mp 73-75°C (lit. 68-70°C); 1H NMR (acetone-d6, 250 MHz) 8 8.32-8.28 (d, JAB = 9.3 Hz, 2 H, ArH), 7.33-7.29 (d, J=9.3 Hz, 2H, ArH), 5.31 (s, 2 H, OCH2); IR (KBr, cm-1) 3116, 3088 (aromatic C-H stretching), 2264 (-C=N stretching), 1333 (asymmetric N0 2 stretching), 1241 (C-O-C stretching). 90 Ethyl^-^’nitrophenoxv) iminoacetate hydrochloride 83. H • HCI 0CH2 CH3 To a cold solution of p-nitrophenoxyacetonitrile (9 g, 51 mmol) in CH 2CI2/benzene (25 mL/ 25 mL) was added anhy. EtOH (2.33 g, 51 mmol) and the mixture was bubbled with HCI gas (=2.2 g, 61 mmol) while stirring and cooling in an ice-water bath. The mixture was kept in a refrigerator overnight poured into Et20 (150 mL) and filtered to give 12.3 g (93%) of white imidate S3 : mp 114-115°C (decomp.); 1H NMR (DMSO-d6l 300 MHz) 5 8.22-8.18 (m, 2 H, ArH), 7.16-7.12 (m, 2H, ArH), 4.97 (s, 2H, OCH2), 4.61 (s, 1H, =NH), 4.20 (q, J=7.1 Hz, 2H, OCtbCHg), 1.22 (t, J=7.1 Hz, 3H, OCH 2CM3); IR (KBr, cm-1) 3162 (N-H stretching), 1756 (C=N stretching), 1501 (asymmetic N0 2 stretch), 1338 (symmetric N0 2 stretch), 1252 (C-O-C stretching). 91 2-M,-Nitrophenoxvmethvnimidazoline hydrochloride 85. • HCI 0 2 N" Imidate 8 3 (12 g, 46 mmol) was suspended in EtOH (100 mL) and reacted with ethylendiamine (2.79 g, 47 mmol). Workup was followed according to the procedure described for compound 7 4 to afford 8.9 g (87%) 8 3 mp 219-220°C; 1H NMR (DMSO-d6, 250 MHz) 8 10.56 (s, 1H, =NH+), 8.29-8.25 (d, J=9.2 Hz, 2H, ArH), 7.26-7.22 (d, J=9.2 Hz, 2 H, ArH), 5.28 (s, 2H, OCH2), 3.89 (s, 4H, 2 x CH2 imidazoline); IR (KBr, cm*i) 3422-2451 (N-H stretching); 1587 (C=C stretching), 1504 (asymmetric N0 2 stretching), 1337 (symmetric N0 2 stretching), 1251 (C-O-C stretching); Anal. Gated, for C10H12CIN3O3 X 1/2H20 : C, 45.04; H, 4.91; N, 15.76. Found: C, 45.02; H, 4.91; N, 15.87. 92 2-f4,-Aminophenoxvmethvhimidazoline dihydrochloride 87. • 2HCI H A mixture of 2-(4,-nitrophenoxymethyl)imidazoline HCI 85 (5.00 g, 19 mmol), and 5% Pd/C (0.7 g) in MeOH (200 mL) was hydrogenated using a Parr apparatus at r. t., 40 psi for 3 h. The resulting mixture was filtered over Celite to give almost collorless solution. The solvent was removed in vacuo to give white residue. Recrystallization from MeOH/Et 2 0 afforded 87 as monohydrochloride salt 4.11 g (93%) in form of white needles: mp 184-186° C. Sample for analysis was prepared by dissolving monohydrochloride salt 57 in MeOH and passing HCI gas to afford 8 Z as white powder mp > 250°C. 1H NMR (DMSO-d6,250 MHz) 8 6.75-6.72 (d, J=9 Hz, 2H, ArH), 6.55-6.50 (d, J=9 Hz, 2 H, ArH), 4.90 (s, 2H, OCH2), 3.85 (s, 4H, 2 x CH2 imidazoline), 3.15 (s, 2 H, NH2); IR (KBr, c i t H ) 3138-2597 (N-H stretching),1603 (C=C stretching), 1261 (C-O-C stretch); MS m/z 191 (M+-HCI); Anal. Calcd. for C 10H15CI2 N3 O: C, 45.47; H, 5.72; N, 15.91. Found: C, 45.46; H, 5.67; N, 15.84. 93 2-M,-lsothiocvanatophenoxvmethvnimidazoline hydrochloride 68. SCN To a solution of CSCI 2 (0.76 g, 7 mmol) in acetone, was added a solution of imidazoline 8 7 (1.00 g, 4 mmol) in 10 mL of H 20/acetone (1/1). the mixture was stirred for 1.5 h at room temperature, acetone removed under reduced pressure. The H20 layer was alkalyzed with Na 2C03, and extracted with EtOAc (3x70 mL). Organic extracts were combined washed with brine (2x50 mL), dryed over Na 2S04, and poured into Et20 saturated previously with HCI gas. Precipitate was collected and recrystallized from MeOH/Et20 to give yellow crystalls of fig. as hydrochloride salt 0.4 g (37%): mp 138-140°C; 1H NMR (DMSO-d6, 250 MHz) 8 10.4 (s, 1H, =NI+), 7.48-7.44 (d, Jab= 8 .9 Hz, 2H, ArH), 7.09-7.06 (d, JAB=8.9 Hz, 2H, ArH), 5.13 (s, 2H, OCH2), 3.88 (s, 4H, 2 x CH2 imidazoline); IR (KBr, cnrH) 3353-3110 (N-H stretching), 2084 (-N=C=S), 1610 (C=C stretching), 1508 (N-H bending), 1444 ( aromatic C=C stretching); MS m/z 233 (M+-HCI, base); Anal. Calcd. for CnH^CINaOS x H20 : C, 45.91; H, 4.90; N, 14.60. Found: C, 45.90; H, 4.96; N, 14.50. 94 2-(4’-Nitrobenzyl)imidazole hydrochloride 90. HCI To a suspension of imidate 7£ (11.20 g, 46 mmol) in glyme (25 mL) was added dropwise aminoacetaldehyde dimethylacetale (4.84 g, 46 mmol), while cooling in an ice- water bath. The resulting mixture was stirred at room temperature overnight, acidified by adding AcOH (25 mL) and bubbled with HCI gas (=1.7 g). The solution was then heated at 50°C on an oil bath for 3 days, cooled down, poured into ether (100 mL), and filtered to give brown solid. Recrystallization from MeOH/Et20 gave 5.21 g (47%) of brown crystalls of SO: mp 209-211°C; 1H NMR (CD 3OD, 250 MHz) 8 8.28-8.25 (d, J= 8 . 8 Hz, 2H, ArH), 7.57-7.53 (d, J= 8 . 6 Hz, 2 H, ArH), 7.50 (s, 2H, 2 x CH imidazole), 4.51 (s, 2H, CH2); IR (KBr, cm-1) 3127 (N-H stretching), 1526 ( asymmetric N 0 2 stretching), 1344 (N 0 2 symmetric stretching); MS m/z 203 (M+-HCI) 156 (M+-HCI,N02), 55 (base); Anal. Calcd. for C 10H10CIN3O2: C, 50.12; H, 4.20; N, 17.53. Found: C, 50.00; H, 4.21; N, 17.37. 95 2-(4’-Aminobenzyhimidazole Hydrochloride 91. HCI A mixture of 2-(4’nitrobenzyl)imidazole hydrochloride 9Q (2.30 g, 10 mmol) and 10% Pd/C (0.23 g) in MeOH (10 mL) was hydrogenated using a Parr hydrogenation apparatus at r.t., 40 psi for 4 h. Filtration over Celite gave collorless solution. The solvent was removed under reduced pressure on rotavapor to give white residue. Recrystallization from MeOH/Et20 afforded 1.75 g (87%) of imidazole 9 1 : mp 225-227°C; 1H NMR (CD3OD, 250 MHz) 8 7.39 (s, 2H, 2 x CH imidazole), 7.03-7.00 (d, J= 8 . 6 Hz, 2 H, ArH), 6.74-6.70 (d, J=8.5 Hz, 2 H, ArH), 4.17 (s, 2 H, CH2); IR (KBr, cm-1) 3346-3153 (NH3+, N-H stretching), 1624 (C=C and C=N stretching); MS m/z 173 (M+-HCI), 55 (base); Anal. Calcd. for C 10H12CIN3: C, 57.28; H, 5.76; N 20.04. Found: C, 57.14; H, 5.79; N, 20.03. 96 2-f4’-lsothiocvanatobenzvnimidazole 69. 1/4H20 SCN To a cold solution of thiophosgene (5.48 g, 48 mmol) in acetone (30 mL) was added 2-(4’-aminobenzyl)imidazole hydrochloride S I (1.00 g, 5 mmol) in H20 (10 mL), via cannulation over period of 30 min while stirring and cooling the mixture in an ice water bath. Resulting mixture was stirred at r.t. for 1.5 h, cooled and alkalized with sat. NaHC03. This afforded white crystals of imidazole 63 0.71 g (69%): mp 201.5-203°C; 1H NMR (CD 3OD, 250 MHz) 8 7.27-7.18 (m, 4H, ArH), 6.94 (s, 2H, 2 x CH imidazole), 4.05 (s, 2H, CH2); IR (KBr, cm-1) 2189,2140 (-N=C=S), 1453 aromatic C=C stretching); MS m/z 215 (M+, base); Anal. Calcd. for CnHgN3S x 1/4 H 2 0 : C, 60.13; H, 4.13; N, 19.12. Found: C, 60.28; H, 4.31; N, 19.14. 97 N.N-Dimethyl-2-(4’-nitrophenvnethvlamine hydrochloride 92. ,CH3 / sch 3 HCI 0 2 To a cold solution of 40% aq. NH(CH 3 ) 2 ( 1 0 mL) was added 4- nitrophenylethylbromide 78 (2.00 g, 9 mmol) and EtOH (15 mL) The mixture was stirred at r.t. for 4 h, EtOH removed under reduced pressure on rotavapor. The water layer was alkalized with 10% NaOH (pH=10) and extracted with Et20 (3x15 mL). Extracts were combined, dried over Na 2 S04, filtered and solvent removed on rotavapor to give a free base of amine as a brown redish oil 1 .6 g (95%). The oil was dissolved in Et20 (10 mL) and bubbled with HCI gas to obtain yellow solid. Recrystallizarion from MeOH/Et20 gave amine § 2 as monohydrate salt 1.51 g (80%) in form of yellow needless:mp 179- 181°C; 1H NMR (DMSO-d 6 , 250 MHz) 5 8.22-8.19 (d, J Ab = 8.7 Hz, 2H, ArH), 7.59-7.56 (d, JAB=8.7 Hz, 2H, ArH), 3.32-3.27 (t, J = 3.8 Hz, 2H, CH2), 3.22-3.19 (t, J=3.8 Hz, 2H, CH2) 2.78 (s, 6 H, 2 x CH3). 98 N.N-Dimethvl^-M'-aminophenyhethvlamine hydrochloride 93. • HCI A mixture of N,N-dimethyl-2-(4’-nitrophenyl)ethylamine hydrochloride 22 (1.45 g, 6 mmol) and 10% Pd/C (0.15 g) in MeOH (30 mL) was hydrogenated in a Parr apparatus at r.t., 40 psi for 3 h. The resulting mixture was filtered over Celite to give collorless solution. The solvent was removed on rotavapor to give yellow oil. Crystallization from MeOH/Et20 gave yellow long needles of 93 1.12 g (89%): mp. 162-164°C; 1H NMR (DMSO-d6 , 250 MHz) 8 6.91-6.87 (d, JAB = 8.3 Hz, 2H, ArH), 6.52-6.49 (d, JAB=8.2 Hz, 2H, ArH), 3.15-3.11 (m, 2H, CH2), 2.83-2.79 (t, J =4.5 Hz, 2H, CH2), 2.74 (s, 6 H, 2 x CHa). 99 N.N-Dimethvl^-W-isothiocvanatoDhenvnethylamine hydrochloride 70 / h3 Procedure same as for §7: from 93 (0.5 g, 3 mmol) and CSCI2 (2.9 g 30 mol) was obtained 0.24 g (52%) of 70 as hydrochloride salt in form of white fluffy crystals: mp 174-175°C; 1H NMR (DMSO-d6, 250 MHz) 8 7.42-7.33 (m, 4H, ArH), 3.27-3.21 (m, 2H, CH2), 3.07-3.00 (m, 2H, CH2), 2.76 (s, 6H, 2 x CH3); IR (KBr, cm-1) 2042 (-N=C=S); MS m/z 206 (M+), 58 (base); Anal. Calcd. for CnHigCINjjS: C, 54.42; H, 6.23; N, 11.54. Found: C, 54.28; H, 6.21; N, 11.43. 3-Nitrophenethylacetonitrile 95. Sodium cyanide (2.14 g, 44 mmol) was dissolved in H20 (15 mL) and placed in 250 mL three necked round bottomed flask, equiped with condenser and dropping funnel. m-Nitrobenzyl chloride (5 g, 29 mmol) was dissolved in EtOH and added by cannulation via dropping funnel over period of 30 min. The mixture was then heated at 70°C on an oil bath. Progress of the reaction was monitored by TLC (Rf= 0.56 on silica on glass, 100 Hex/EtOAc (1/1) as solvent system). Reaction was completed after 22 h, cooled to r.t. and organic solvent removed under reduced pressure on rotavapor. Water layer was extracted with CH2CI2 (3x40 mL). Organic extracts were combined, washed with brine, dried over Na2S 0 4 filtered and filtrat concentrated in vacuo to give orange oil 3.6 g (77%). The product was crystallized by tritturation with Hex. Recrystallization from Et20/Hex gave 55 as yellow crystals 3.1 g (66%): mp 60-61 °C (lit. mp 61-62°C); 1H NMR (CDCI3, 250 MHz) 5 8.21-8.19 (m, 2H, ArH ortho to N02 ), 7.72-7.69 (d, J=7.7 Hz, 1H ArH para to N02), 7.62-7.56 (m, 1H, ArH), 3.88 (s, 2H, CH2); IR (KBr, cm-1) 3102 and 3086 (aromatic C-H stretch), 2256 (-C=N stretch), 1519 (N02 asymmetric stretching), 1346 (N02, symmetric stretching). 2-(3’-Nitrobenzv0imidazoline hydrochloride 97. HN HCI Imidazoline 97 was synthesized by the procedure previously described for 2- (4’-nitrobenzyl)imidazoline 74: from 3’-nitrophenylacetonitrile (3.00 g, 19 mmol) in CH2CI2/benzene (5mL/ 20 mL), EtOH (0.85 g, 19 mmol) and HCI gas, was obtained imidate 9g 4.06 g (90%, lit. 76% ): mp 119-120°C (lit. mp 120°C) as a white solid. Imidate 96 was then reacted with ethylenediamine (0.78 g, 13 mmol) and the crude product was treated in the same manner as described for 73 to afford 74 in form of HCI salt as yellow crystalls 2.0 g (75%, lit. 47%): mp 215-216°C (lit. 212°C); 1H NMR (DMSO-d6, 250 MHz) 5 8.37 (s, 1H, ArH, ortho to N 02), 8.19-8.16 (d, J=8 Hz, 1H, ArH, ortho to N 0 2), 101 7.98-7.95 (d, J=8 Hz, 1H, ArH para to N 02), 7.71-7.64 (t, J=8 Hz, 1H, ArH) 4.09 (s, 2H, CH2), 3.80 (s, 4H, 2 x CH2 imidazoline). a-fS’-Aminobenzynimidazoline dihydrochioride 98. 2H C I A mixture of 97 (1.50 g, 6 mmol) and 10% Pd/C (0.15 g) in MeOH (50 mL) and conc. HCI (0.9 mL) was hydrogenated using a Parr hydrogenation apparatus at r.t., 40 psi for 3.5 h. The resulting mixture was filtered over Celite, filtrate was concentrated under reduced pressure on rotavapor to give white solid 1.52 g (99%) which was recrystallized from MeOH/Et20 to afford 1.3 g (84%, lit 45%) of 98 as slight yellow crystals: mp>265°C (lit. mp 253°C); 1H NMR (DMSO-d6, 250 MHz) 8 11.02 (s, 1H, NH), 7.92-7.87 (m, 4H, ArH), 4.40 (s, 2H, CH2), 4.27 (s, 4H, 2 x CH2 imidazoline). 102 2-(3’-lsothiocvanatobenzyl)imidazoline hydrochloride 64. SCN HN HCI Thiophosgene (0.56 g, 5 mmol) was dissolved in acetone (20 mL) and cooled to 0-5°C, while stirring in an ice-water bath, in a hood. To the cold solution of thiophosgene in acetone a solution of 9S (1.00 g, 4 mmol) and NaHC03 (0.4 g, 5 mmol) in H20 (10 mL) was added over period of 15 min, and stirring was continued overnight at r.t. The solvent was then removed in vacuo to give yellow oil which was taken into CH2CI2 (50 mL) and washed with brine (10 mL). CH2CI2 layer was then dried over Na2S04l filtered and solvent removed under reduced pressure to give yellow oil. Trituration with acetone gave white precipitate. Recrystallization from cold MeOH/Et20 afforded white crystals of M 0.56 g (55%): mp 166-167°C; 1H NMR (DMSO-d6, 300 MHz) 67.52-7.38 (m, 4H, ArH), 3.91 (s, 2H, CH2), 3.80 (s, 4H, 2 x CH2 imidazoline): IR (KBr, cm-1) 3063 (N-H stretching), 2121 (-N=C=S stretching), 1602 (C=C and C=N stretching): MS m/z 217 (M+-HCI), 216 (M+-H.HCI, base); Anal. Calcd. for C^H^ClNaS: C, 52.07; H, 4.76; N, 16.56. Found: C, 51.94; H, 4.74; N, 16.55. 103 Ethvl-2-f2,-n8troDhenvniminoacetate hydrochloride 100. HCI 0CH2CH3 To a cold solution of 2-nitrophenylacetonitrile (4.00 g, 25 mmol) in benzene/CH 2 CI2 (60 mL/ 40 mL) was added anhy, EtOH (1.14 g, 38 mmol). This mixture was then cooled to 0-5°C and bubled with HCI gas (=1.4 g) while cooling in an ice-water bath. The resulting mixture was kept in refrigerator for 4 days and then poured into Et20 (200 mL) to give white precipitate. Filtration and drying in vacuo afforded white imidate lO fl4.77 g (78%): mp 107-108.5°C; 1H NMR (DMSO-d6, 250 MHz) 8 8.18-8.15 (d, J=8 Hz, 1H, ArH ortho to NOs ), 7.83-7.77 (t, J=7.4 Hz, 1H, ArH), 7.68-7.63 (t, J=7 Hz, 2H, ArH), 4.44-4.37 (m, CH2 and OCH,CH*). 1.19-1.13 (t, J=7 Hz, 3H, OCH?CH2): IR (KBr, cm-1) 3399 (N-H stretching), 1654 (C=N stretching), 1522 (N02 asymmetric stretching), 1345 (N02 symmetric stretching): Anal. Calcd. for C10H13CIN2O 3 : C, 49.09; H.5.36; N, 11.45. Found: C, 49.16; H, 5.40; N, 11.53. 104 2-f2’-Nitrobenzyl)imidazoline hydrochloride 101 N 02 According to the same procedure as described for compound 74: imidate 1QQ (4.2 gr 17 mmol) was then reacted with ethylenediamine (1.1 g, 20 mmol) to give imidazole 101 2.94 g (81%): mp 246-247°C; 1H NMR (DMSO-d6, 250 MHz) 8 10.22 (s, 2H, NH), 8.19-8.15 (d, J=8 Hz, 1H, ArH ortho to N 02), 7.83-7.77 (t, J=7.2 Hz, 1H, ArH para to N 02), 7.70-7.63 (t, J=8.3 Hz, 2H, ArH meta to N 0 2), 4.26 (s, 2H, CH2), 3.80 (s, 4H, 2 x CH2 imidazoline); IR (KBr, cm-1) 3629-2672 (N-H stretching), 1606 (C=C and C=N stretching), 1578 (N02 asymmetric stretching), 1343 (N02 symmetric stretching); MS m/z 205 (M+-HCI), 159 (M+-N02, base); Anal. Calcd. for C 10H12CIN3O2: C, 49.69 ; H, 5.00 ; N, 17.39. Found: C, 49.80; H, 5.01; N, 17.47. 105 2-(2’-Aminobenzyl)imidazoline dihydrochloride 102. NH2 A mixture of I f l l {1.5 g, 6 mmol) and 10% Pd/C (0.15 g) in MeOH (50 mL) and conc. HCI (0.34 g, 0.95 mL) was hydrogenated using a Parr apparatus at r. t . , 40 psi for 3.5 h. The resulting mixture was filtered over Celite, filtered concentrated under reduced pressure to give white residue. Recrystallization from H20 / acetone afforded 1£2 as white crystals 1.1 g (71%): mp >270°C (decomp); 1H NMR (D20, 300 MHz) 8 7.35-7.26 (m, 4H, ArH), 3.89 (s, 2H, CH2), 3.75 (s, 4H, 2 x CH2 imidazoline); IR (KBr, cm-i)3281-2589 (br. N-H stretch in NH3+), 1597 (C=C and C=N stretching); MS m/z 175 (M+-2HCI), 117 (M+-2HCI,-NHCH2CH2NH, base); Anal. Calcd. for CioHi5C|2N3: C, 48.40; H, 6.09; N, 16.93. Found: C, 48.42; H, 6.07; N, 16.97. 106 Imidazolino f2.1-c)-5.10-dihvdro-4H-1.3-benzodiazeDin-4-thione 103. N— Thiophosgen (0.39 g, 3.4 mmol) was dissolved in acetone (20 mL) and cooled to 0-5°C, while stirring in an ice-water bath, in a hood. To the cold solution of thiophosgen was added a solution of 102 and NaHC03 (0.29 g, 3.4 mmol) in water over period of 15 min and stirring was continued under argon atmosphere at r.t. overnight. The solvent was then removed in vacuo to give yellow oil which was taken into CH2CI2 (100 mL) and washed with brine (10 mL). Water layer was extracted with CH2CI2 (3 x 50 mL), and all organic extracts were combnined, dried over Na 2 S0 4 filtered and removed under reduced pressure to give oil. Addition of acetone afforded beige precipitate 0.18 g, (25 %):mp 201-203°C; 1H NMR (CD3OD, 300 MHz) 87.48- 7.31 (m, 4H, ArH), 4.56-4.49 (t, J=9.8 Hz, 2H, CH2 imidazoline), 4.15 (s, 2H, CH2), 3.96-3.89 (t, J=9.8 Hz, 2H, CH2 imidazoline); 13C (CD3OD, 300 MHz) 8 178 (£=S), 167,138.9,131,130,129,125.8,122.6,54.5,44.5; IR (KBr, cm*i) 3440-2601 (broad N- H stretching), 1647 (C=S stretching); MS m/z 217(M+, base), 192,177; Anal. Calcd. for ChH12CIN3S: C, 52,07; H, 4.77; N, 16.56. Found: C, 52.03; H, 4.77; N, 16.59. 107 Computational analyses. Molecular modeling was performed using the Sybyl software (Tripos Associates Inc.) on an IRIS workstation. Structure of nifedipine was build from X ray data.^o Bay K 8644 was obtained by modifing the structure of nifedipine. IBI and its analogs £7 and ££ were build from standard fragments within Sybyl. Atomic charges were computed using the Gasteiger-Huckel method. Each molecule was minimized using the Tripos force field, prior to subjecting it to a systematic conformational analysis to derive all its minimum-energy conformations. Three rotatable bonds were defined for IBI (bond 1 and 2 were searched in 3° increment whereas bond 3 had 10° increment), while 4 rotatable bonds were defined for analogs 67 and 68 (bond 1, 2 and 3 were searched in 3° increment, and bond 4 in 10° increment). Systematic search for compounds £7 and ££ was set up with distance constrains between “dummy atom”, center of the aromatic ring, and one of N atoms in imidazoline ring to be between 4.55-5.05 A. This coresponds to the distance between the “dummy atom” and N in dihydropyridine ring of nifedipine. Low energy conformers were overlap with the use of FIT ATOM or MULTIFIT comand within Sybyl. Part 2 Design and Synthesis of Selective 2-Amino-3-(3,-hydroxy- 5’-methylisoxazol-4’-yl)propanoic Acid (AMPA) Receptor Antagonists for Potential Use in Drug Abuse 108 CHAPTER V INTRODUCTION 5.1. L-GLUTAMATE- PRIMARY EXCITATORY NEUROTRANSMITTER As early as 1954, glutamate 105 was shown to have an excitatory effect on neurons. Hayashi, studying epileptic phenomena, observed that application of high concentration of sodium glutamate into the cerebral cortical cells of dogs, monkeys and humans generates convulsions.ni In the early 1960’s Curtis and Watkins, using microelectrophoretic techniques, demonstrated that the acidic amino acids, L-glutamate and L-aspartate, were powerful neuroexcitants.H 2.143 Furthermore, their extensive investigation of related amino acids led to the establishment of the structural requirements for the excitatory action of these amino acids. 142.143 Since glutamate was biologically active, it was postulated to act as a neurotransmitter. However, the ubiquitous presence of L-glutamate in substantial amounts in virtually every cell type, as well as its involvement as a key intermediate in a variety of important metabolic pathways, seriously complicated studies designed to demonstrate that glutamate is a neurotransmitter. In addition to its role as a building block of peptides and proteins, glutamate contributes to the regulation of osmolarity and ammonia levels (glutamine), serves as a precursor for the inhibitory neurotransmitter 4-aminobutanoic acid (GABA) and it is a key component of glutathione. 144 Furthermore, glutamate can be easily oxidized to a-ketoglutarate, an important intermediate for the Krebs cycle.144 Several studies have provided evidence that glutamic acid is a neurotransmitter:(1) it is stored in vesicles in presynaptic terminalHs.ne; (2) it is released 109 110 from presynaptic terminals in a Ca2+-dependent fashionn6,i47; (3) it activates postsynaptic receptors when applied exogenouslyi«; (4) its excitatory signal is terminated due to uptake of glutamate by high-affinity transporter located on presynaptic terminal and surrounding astrocytes^.^. In addition to the role of glutamate in standard fast excitatory synaptic transmission, the glutamate is involved in even more complex neuronal processes such as synaptic plasticity,^ and long term potentiation (LTP),iso.i5i and may be involved thus in learning and memory. Glutamate at high concentrations produces excitatory effects. EAA “excitotoxicity” seems to be associated with a wide variety of neurodegenerative diseases such as ischemia, anoxia, stoke, hypoglycemia, epilepsy, Huntington’s disease, neurolathyrism and Alzheimer’s disease.152.153 5.2. EXCITATORY AMINO ACID RECEPTORS. EAAs exert a strong excitatory action on most neurons in the mammalian CNS via excitatory amino acid receptors. In analogy to other neurotransmitters, heterogeneity among EAA receptors has been observed. The EAA receptors have been classified into subtypes based on electrophysiological,is4 radioligand bindings and molecular biology studiesJS6 There are at least five subtypes: (1) NMDA (N-methyl-D-aspartic acid, 106): (2) AMPA((R,S)-2-amino-3-(3’-hydroxy-5’- methylisoxazol-4’-yl)propanoic acid, 107); (3) KAIN (kainic acid, 1081: (4) AP4 (L-2-amino-4-phosphonobutanoic acid, 1091: and (5) ACPD (trans-1-aminocyclo- pentane-1,3-dicarboxylate, 1101 receptors. Each of these receptor subtypes was named after selective agonist (Figure 21) for a given receptor sub typ e .157 Based on the transduction mechanism the EAA receptors can be classified into two groups: I lonotropic receptors (include NMDA, AMPA, KAIN and AP4 receptors) and II Metabotropic receptors (ACPD receptor), isa Activation of ionotropic receptors causes depolarization of the postsynaptic membrane, allowing an influx of Na+ and an efflux of K+ ions through ion channels associated with the receptor. The ion channel associated with 111 NMDA receptor appears to allow influx of Ca2+ ions. 157 Metabotropic receptors are coupled to Q-protein and their activation leads to an increase in intramolecular level of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DG) followed by mobilization of intracellular calcium and phosphorylation of proteins.^ HO © _© o HO' CHg L-GIutamate 105 NMDA 106 L-AMPA107 J302H J COzH O KAIN 108 AP4109 ACPD 110 Figure 21. Compounds Used to Classify EAA Receptor Subtypes. 5.2.1. NMDA RECEPTORS. The NMDA receptor is the best characterized EAA receptor subtype, primarily due to an early discovery of potent and selective agonists (such as NMDA, 106) and antagonists (such as AP5,120) for this subtype of receptors. The discovery that various classes of compounds can inhibit or selectively potentiate NMDA receptor mediated responses has led to the discovery of several distinct binding sites on the NMDA 112 receptor-channel complex. In addition to the agonist and competitive antagonist binding sites on the NMDA receptor-ion channel complex the following binding sites have been identified: (1)a binding site for an inhibitory cation McP+ located inside the channel; (2) modulatory site that binds co-agonist glycine, that selectively potentiates NMDA-induced responses; (3) a site within the channel that binds noncompetitive antagonists such as phencyclidine, 125 and MK-801.128: (4) a modulatory polvamine binding site: and (5) a binding site for Z n 2+. inhibitory modulator, located near the mouth of the ion channel.160-161.157 A schematic representation of a NMDA receptor-ion channel complex with binding sites for various agonists and antagonists is shown in Figure 22A. In contrast to the other EAA ionotropic receptors, the activity of the NMDA receptor-ion channel is voltage dependent. This phenomenon is a consequence of the NMDA receptor blockade by Mg?* ions, which is released only under certain conditions, when neurons are partially depolarized. At the normal resting potential (=70mV), physiological concentrations of Mg2+ ions and low frequency stimulation, the NMDA receptor action is suppressed. However, once the membrane potential moves from resting potential to » -20mV-0mV due to abundant transmitter release or high frequency stimulation, Mg2+ blockade is released allowing further depolarization, leo.iei in order to further understand the function of the NMDA receptors, it is important to observe that NMDA receptor channels are permeable to C a 2+, Na+ and K+ ionsJ57 Two properties, voltage dependance and Ca2+ permeability of NMDA receptors are involved in processes like long-term potentiation (LTP) synaptic plasticity and thus in learning and m em o ry .151 According to radioligand binding studies, NMDA receptors are localized in the cerebral cortex, hippocampus, striatum, septum, and amygdala.1^ 5.2.1.1 .Agonists at NMDA receptors. In addition to previously established NMDA receptor agonists: L-Glu 105. and NMDA Iflg a number of various potent agonist of NMDA receptors have been identified. Some of the most potent and selective NMDA receptor agonists are shown in Figure 23. Na+ 113 A Ca2+ NMDA Na+(Ca24) / CNQX.DNQX AMPA.KAIN Modulatory protein «P3 Ca2+ **' Figure 22. Schematic Representation of EAA Receptor Subtypes: A) NMDA Receptor B) Non-NMDA Receptor C) Metabotropic Receptor. 114 HO C 0 2H CO? h 2 a ,NH3 '3 trans-2,3-PDA H I IBP 112 cis-1R,3R-ACPD m L-HCA114 C 0 2H N = N c o 2h / i N m t x !>> c o 2h V/ © CO? T * « « H3N ^ C O ? H ^T 'COz © trans-ACBD 115 T Z G 1 1 6 2R.3S.4S-CCG H Z 2S,3R,4S-CCG H 8 Figure 23. Some NMDA Receptor Agonists. Most of these agonists are conformationally restricted analogues incorporating structural elements of glutamic or aspartic acid. In ibotenic acid (IBO, 1121 a natural product isolated from the mushroom Amanita muscariaw 3-hydroxy isoxazole serves as a bioisosteric replacement for carboxyl group as a tetrazole ring (pKa =4.9) presumably does in (±)-(tetrazol-5-yl)glycine (TZG, 1 1 6 1 157 Search for the active conformation of glutamate at NMDA receptors led to the following analogues: trans-2t3-piperidine dicarboxylic acid (trans-2,3-PDA, 1111. (1R,3S)cis-1-amino-cyclopentane-1,3-dicarboxylic acid (cis-ACPD, 1131. trans-1-amino cyclobutane-1,3-dicarboxylate (trans-ACBD, 115). and 2R,3S,4S,a-carboxycyclo-propaneglycine (CCG, 117) and 2S,3R,4S,a-carboxy- cyclopropaneglycine (CCG, 1181.is7.i63.i64 Numerous reviewsi65.i63.i6o have summarized structural requirements for agonist activity at NMDA receptors and they are as follows: (1) two negative and one positive charge, where two negatively charged groups are 2-3 atoms apart (like in Asp, Glu or in L-homocysteic acid L-HCA, 114): (2) different acidic 115 groups in co-position can be tolerated with order of potency C00H >S03H>P03H; (3) substitution in the chain can be tolerated; N-alkylation usually causes a decrease in potency except in NMDA; (4) the bioactive conformation of glutamate at NMDA receptor seems to be a folded one with two carboxyl groups in close proximity; and (5) L-configuration at the a carbon atom carrying an amino group is preferred, however the D-configuration is also tolerated. 5.2.1,2.Competitive antagonists at NMDA receptors. Examples of some of the most potent NMDA receptor antagonists are given in Figure 24. Structural features that distinguish NMDA receptor antagonists from agonists are: (1) a longer chain length between two acidic groups, preferably 4 or 6 atoms; (2) antagonist activity resides exclusively in D-enantiomers; and (3) replacement of the ©-carboxyl group with the phosphono acidic group increases potency and selectivity 160 , 165 i 163 *, HO OH OH OH D-AA 119 D-AP532Q D-AP7121 N COOH COOH H H2N COOH H CPP122 CGP 39653123 CGS-2-ene 124 Figure 24. Competitive NMDA Receptor Antagonists. 116 5.2.1.3.Noncompetitive NMDA receptor antagonists. In addition to divalent cations Mg2+ and Zn2+, which under physiological conditions noncompetitivelly block the NMDA receptor, there are at least two other distinct classes of noncompetitive NMDA receptor antagonists. Examples of such antagonists are: (1) dissociative anesthetics phencyclidine (PCP, 125). and ketamine 126: (2) benzomorphans such as N-allyl-normetazocine 127: (3) MK-801 128. The site where these compounds exert their action is known as phencyclidine or PCP receptor and it is located within the ion channel coupled to the NMDA receptor (Figure 2 2 A ).^ The action of noncompetitive antagonists is also called “use dependenf since the binding of PCP ligands is enhanced by the presence of NMDA receptor agonist. In addition to the PCP binding site antagonism of strychnine-insensitive glycine site is another way of modulating NMDA receptor activity.1^ Some selective glycine site non-competitive NMDA antagonists are shown in Figure 25. 5.2.2. AMPA RECEPTORS The AMPA receptor-ion complex was originally named the quisqualate receptor, since it was activated by quisqualate (QUIS, 1321. a natural product isolated from plant seeds of the Quisqualis fructus.168 However, it become apparent that quisqualic acid is not a selective ligand for QUIS/AMPA receptor, since it also exhibits high affinity for other EAA receptors such as kainateies and metabotropic receptors. Therefore, the more selective, synthetic agonist AMPA 107. became a ligand of choice for studying these receptors consequently termed AMPA receptors. While the structure of NMDA receptor has been well characterized (Figure 22A), the structure of the other ionotropic receptors, including the AMPA receptors are not very well defined. Figure 22B shows schematic representation of a non-NMDA receptor. In addition to the agonist/antagonist binding site, modulatory sites for arthropod toxins have been proposed, since toxins from Argiope and Joro spiders reduce the effects of exogenously administered glutamate and quisqualateJ&6 Barbiturates are also known to HO. NHCH3 HN PCP 125 Ketamine 126 N-allylmetazocine (SKF 10047) 12Z MK-801 128 COOH HN Cl COOH Cl N '"COOH COOH H 129 13Q 1 3 1 Figure 25. Noncompetitive Antagonists of NMDA Receptors Acting at the PCP and Glycine site. 118 block the effect of various agonists at this receptor subtype. The receptor appears to be influenced by a modulatory protein. 170 in contrast with the NMDA receptor class the AMPA receptors are voltage independent, permeable to Na+ (K+the being counterion) and responsible for mediating fast excitatory postsynaptic potential (EPSP) at AMPA synapses.171 Radioligand binding studies using pHjAMPA have shown that AMPA receptors are localized primarily in telencephalic regions, with high levels in hippocampus, cortex, lateral septum, striatum and the molecular layer of cerebellum.162 5.2.2.1 .AMPA receptor agonists. Structures of the selective AMPA receptor agonists are shown in Figure 26. The heterocyclic ring of the naturally occurring amino acid, QUIS 132. is fully ionized at physiological pH (the pKa of the N-H proton being 4.2) and it serves as a bioisosteric replacement of the distal carboxyl group of glutamate.167 Another naturally occurring heterocyclic amino acid willardiine appears to selectively activate the AMPA subtype of EAA receptors.172 B-N-Oxalyl-a,(3-diaminopropanoic acid (ODAP, 1341 yet another naturally occurring AMPA receptor agonist is found in Lathyrus sativus. This compound is neurotoxic and is believed to be the cause of human neurolathyrism.176 Elongation of the side chain of I BO 112 (non-selective EAA receptor agonist) by an additional methylene group and introduction of different substituents produced isoxazole amino acids such as 4-bromohomoibotenic acid 138, and 4-methyl-homoibotenic acid 139.174'176 Modifications of the AMPA structure gave D,L-a-amino- 3-hydroxy-5-tert-butyl-4-isoxazolepropanoic acid (ATPA, 136).176-177 All the above mentioned compounds are selective AMPA receptor agonists with little effect on NMDA or KAIN receptors.166 Structure activity relationship studies of AMPA receptor agonists have led to a hypothetical model of AMPA receptor, which, in addition to recognition sites for two 0o m l i r - H 0 t-N h H3N o O ' ^ © pKa = 4.2 QUIS 132 WILLARDIINE 13a HO HQ HO. .0 0© ,0 NH3 © R = CH2Br ABPA 135 Y = Br BrHIBO 13fi P-ODAP 134 R = C(CH3)3 ATPA1SS R = Ph APPA137 Y = CH3 MeHIBO 139 HO I HO HO O© 5-HPCA140 7-HPCA141 4-HPCA142 Figure 26. AMPA Receptor Agonists. negative and one positive charge on the molecule, also contains a pocket that can accommodate bulky substituents in position 4 (such as t-butyl group in ATPA, 136 or phenyl group in APPA, 13Z)-178 In order to gain further insight into the active conformation of these compounds at the AMPA receptor site, bicyclic analogues 3-hydroxy-4,5,6,7-tetrahydroisoxazole[5,4-c] pyridine-7-carboxylate (7-HPCA, 141). 3-hydroxy-4,5,6,7-tetra-hydroisoxazole[5,4-c] 120 pyridine-5-carboxylate (5-HPCA, 140) and a-amino-3-hydroxy-7,8-dihydro-6H[1,2-d] isoxazole-4-propanoic acid (4-AHCP, 142) were prepared.179,176 While IBO 1 1 2 . is a relatively nonspecific NMDA receptor agonist, its conformationally restricted analog 7-HPCA 1 4 1 , does not interact with the NMDA receptor but is a specific agonist at AMPA receptors. 179.176 Like 7-HPCA 141. the conformationally restricted analog of AMPA 5-HPCA 140. is also a very potent and specific AMPA receptor agonist.176 Molecular modeling studies together with 1H-NMR spectroscopy and X-ray crystallography have shown that both compounds 141 and 14Q adopt almost identical conformations with the six member ring in a half-chair conformation and the carboxyl group in pseudoequatorial position.i78.iao Conformations of these compounds may reflect the bioactive form of AMPA. 4-AHCP 1£2 is yet another homolog of AMPA and it is also an AMPA receptor selective agonist in vivo, equipotent with AMPA .179 To determine the stereochemical requirements for activation of AMPA receptors S and R isomers of AMPA 107,4-bromohomoibotenic acid 138 and 5-HPCA 140 have been synthesized using chemoenzymatic procedures. 181,182,160 |n all cases the neuroexcitatory effects reside in the S-enantiomers of the afore mentioned compounds. 5.2.2.2.AMPA receptor antagonists. The development of selective AMPA receptor antagonists until recently has been very slow (Figure 27). One of the first antagonists reported at this receptor site were glutamate diethyl ester (GDEE, 143) and y-D-glutamylaminomethylsulfonic acid (GAMS, 144). However, these antagonists couldn’t distinguish between AMPA and KAIN r e c e p to r s .183 More recently a new group of highly potent AMPA receptor antagonists has been described: the quinoxalinediones such as 6,7-dinitro quinoxaline-2,3-dione (DNQX, 145). 6-cyano-7-nitro quinoxaline-2,3-dione (CNQX, 146) and 2,3-dihydroxy-6-nitro- 7-sulfamoylbenzo(F) quinoxaline NBQX 147.184.185 While NBQX 147 is exclusively selective for AMPA receptors (IC50=0.15 pM), DNQX 14§ (IC50=0.15 pM) and CNQX 146 (IC5o=0.30 pM) are approximately one-fifth as effective at KAIN receptor and they 121 also bind to the strychnine insensitive glycine site of NMDA receptors.184,185 Ohmori et al. reported another very selective AMPA receptor antagonist of quinoxalinedione type 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalindione 148 (IC5o=0.084 pM) showing that 1H-imidazol-1-yl moiety can serve as an efficient bioisostere for the cyano and nitro groups in compounds binding to the AMPA receptors.186 e,o^ ^NH3 oei O h3n XXX H © R1=R2 = N02 DNQX 145 GDEE 143 GAMS 144 R i = CN, R2 =N02 CNQX H S H 4 ' H n\ ^ S 5 5 ^ N ^ O N ^ O NBQX H Z 148 Figure 27. AMPA Receptor Antagonists. 5.2.3. KAINATE RECEPTORS Considerable amount of evidence has been gathered over last couple of years supporting the hypothesis that KAIN and AMPA receptors are two distinct entities. For example, physiological studies have shown that there are significant differences with respect to receptor desensitization (while AMPA receptors desensitize rapidly, KAIN receptor does so very slowly).i57 Binding studies with pH]KAIN have localized KAIN receptors in hippocampal area CA3, cortex, and lateral septum. 162 in addition, molecular 122 biology studies have provided additional evidence that KAIN receptors are distinct from AMPA receptors (vida infra). Nevertheless, KAIN receptors show a number of features that are characteristic to AMPA receptors (see chapter 5.2.2. and Figure 22B) The most selective KAIN receptor agonists are shown in Figure 28. The prototypic agonist at this site, a-kainic acid 108. was isolated from the seaweed Digenea simplex.™ Domoic acid 149 from the seaweed Chondria armataw and acromelic acid A 150 and B 151 from the poisonous mushroom Clitocybe acromelalga 155 are even more potent agonists than kainic acid at this receptor site.157 H .N„ R = c o 2h ■co2h ACROMELIC ACID B150 CO, N HOOC*^ H2 H H CH3 H02C, R = DOMOIC ACID H 2 ACROMELIC ACID A 351 Figure 28. Kainate Receptor Agonists A common structural feature of the KAIN receptor agonists seems to be the presence of a rt-electron system in the 4-position of pyrrolidine ring. It seems this unsaturation is important for high activity since the reduction of the isopropenyl side chain diminishes agonist activity.157 In contrast to a number of selective and potent KAIN receptor agonists, there are no selective antagonists and the search for selective and potent antagonists continues. 123 5.2.4. AP4 RECEPTORS In the early 1980’s it has been reported that L-AP4 (L-a-amino-4- phosphonobutanoic acid, 1091 attenuates the electrically evoked synaptic response, presumably acting as an antagonist at one of the EAA receptor subtypes; however, it did not antagonize the effect of EAA agonists applied directly to the preparations. ia>,i9i At the present time the most probable explanation for this behavior is that AP4 acts as agonist at presynaptic receptors and controls the release of L-glutamate. Figure 29 shows some selective agonists and antagonists at this presynaptic receptor. Agonists (R =) Antagonists (R =) •COOH -P03H2 -S 0 3H O - S\ r « H,N CH, nr - n V « - < N H Figure 29. AP4 Receptor Agonists and Antagonists. 5.2.5. ACPD (METABOTROP1C1 RECEPTORS. The discovery that trans-ACPD (1S.3R-ACPD, 1101 stimulates hydrolysis of phosphatidylinositol, without activating NMDA, AMPA or KAIN receptors, led to the hypothesis that it might selectively activate a new distinct subtype of EAA receptor. The characterization of this most recent identified EAA receptor is in its early stage. In addition to 1 S.3R-ACPD HQ other agonists may exert their effects through this receptor subtype, such as quisqualate 132 and 2S.3S.4S-CCG HZ- Although several putative antagonists have been identified for this receptor site, such as AP3152, L-aspartate-13-hydroxamate 153 and y-glutamylglycine 154 (Figure 30), they are not very specific, so the search for 124 selective agonists and antagonists continues.192 Structural characteristics of the ACPD receptor are depicted in Figure 22C. p o 3h 2 r NHOH h3n*x ^ co2 CO. AP3 J§2 L-Aspartate-p-hydroxamate 153 y-GIutamylglycine 154 Figure 30. ACPD (Metabotropic) Receptor Antagonists 5.3 MOLECULAR BIOLOGY OF EAA RECEPTORS Hollmann eta U ^ reported isolation of the first cDNA clone (now called GluR1 subunit) for the AMPA receptor, by screening a rat forebrain cDNA library. These workers used a new cloning technique that was successfully pioneered by Nakanishi and co-workers and does not require purified and partially sequenced receptor (channel) protein.is4 Since the landmark cloning of the first receptor subunit by Hollmann, additional 16 channel subunits have been cloned using different methods, such as polymerase chain reaction (PCR) or cross-hybridization screening.^, 196,197 Expression cloning in the Xenopus oocyte has also led to the molecular characterization of the principal NMDA receptor subunit. 19b Table 12 summarizes all cloned subunits to date, together with their properties. All subunits are comprised of approximately 900 amino acid residues, with molecular weight -100 kDa. According to the hydropathy analysis each subunit contains four putative transmembrane-spanning regions following a large extracellular NH2-terminal domain (Figure 31)J56 The precise topology of these receptors still has to be determined; however, the structure of EAA receptors is usually discussed based on the comparison with the other ligand-gated ion channels like nicotinic acetylcholine receptor.^ 125 Table 12. Excitatory Amino Acid Receptor Family. EAA receptor Subunit Characteristics -subunits form homo- or heteromeric oligomers -GluR2 subunit determines AMPA GluR1-GluR4 the channel conductance and (or GluRA-GluRD) Ca2+ permeability -each subunit occurs in two major forms, named “flip” and “flop”, due to alternative splicing. -subunits can form homo- or heteromeric subunits KA GluR5-GluR7 and KA-1, KA-2 -heterogeneity among subunits noticed due to RNA editing (posttranscriptional modifications) 7 isoforms generated by alternative splicing of NMDAR1 (NMDAR1A NMDAR1G). 4 subtypes of NMDAR2 NMDA NMDARIand NMDAR2 NMDAR1 essential for the activity, NMDAR2 potentiates NMDA receptor activity. mGluRI and mGluR5 catalyze formation of IP3 and DG Metabotropic mGluRI- receptors mGluR6 mGluR2-mGlu4 and mGluR6 coupled to inhibitory G|. 126 membrane HOOC Figure 31. Schematic Representation of Glutamate Receptor Subunit AMPA receptor subunits can form homomeric or heteromeric oligomers. The stoichiometry is still unknown; however, it has been proposed that up to five subunits could be involved in forming the receptor-ion channel. Numerous combination of subunits GluR1 through GluR4 give rise to AMPA receptors that differ in Ca2+ permeability and channel conductance. It seems that subunit GluR2 plays a key role in keeping AMPA receptors essentially impermeable to Ca2+ ions. Site directed mutagenesis studies have shown that a single amino acid residue determines the C a2+ permeability through AMPA receptors. While GluR2 has an amino acid Arg in the putative transmembrane segment TM2 and is essentially impermeable to Ca2+, GluR1, G!uR3 and GluR4 subunits all have Glu in place of Arg and are permeable to Ca2+.i99, 200.201 High affinity KAIN receptors can be obtained from subunits GluR5-GluR7 and KA1 or KA2.202.197 Molecular heterogeneity was noticed among GluR1-GluR7 (each subunit exists in two major forms named flip and flop). This phenomena has been explained by genetic mechanisms such as alternative splicing and RNA editing.^?, 203,156 The primary structure of the NMDAR1 subunit revealed structural similarities with AMPA and KAIN receptors: for example, both have four transmembrane spanning 127 domains. Its high Ca2+ permeability might be attributed to the amino acid residue, Asp, located at a position occupied by Arg or Glu in the transmembrane domain TM2 of AMPA and KAIN receptor subunits. It has been shown that replacement of Asp by Glu or Arg in NMDAR1 leads to a decrease in Ca2+ permeability.w.ise NMDAR1 subunit serves as a basic subunit of the NMDA receptor complex and it can form a homo oligomeric receptor-ion channel complex. In contrast to the NMDAR1 subunit the individual NMDAR2 subunit can not function as receptor. However, co expression of NMDAR2 subunits with NMDAR1 subunit markedly potentiates NMDA receptor activity.^ Six different subtypes of metabotropic receptors have been cloned. All six different mGluR are significantly larger that the other members of G-protein coupled receptors; however they share same structural characteristics: seven transmembrane spanning domains, extracellular NH2-terminus and intracellular COOH term inus.^ While mGluRI and mGluR5 catalyze phosphatidylinositol 4,5-diphosphate hydrolysis, all other mGluR subtypes inhibit accumulation of cAMP, indicating that they are coupled to a inhibitory Gi protein.iM .159 Site directed mutagenesis experiments in a putative agonist binding region of the AMPA receptor, conducted by Uchino et a l .204 have implicated Glu 398, Lys 445 and Arg 481 as residues involved in selective interaction with AMPA receptor agonist. 5.4. ROLE OF EXCITATORY AMINO ACIDS IN DRUG ABUSE 5.4.1. COCAINE AND AMPHETAMINE ABUSE: HISTORY AND EPIDEMIOLOGY. Cocaine 155 is an alkaloid that was isolated from the leaves of South American plat Erythroxylon coca in the 1860’s, by German chemist Albert Nieman. However the stimulating properties of the coca leaf have been known to humans for at least 1500 years. The Incas named the plant “gift of the Sun God\2°5 The physiological effects of cocaine are numerous: powerful vasoconstriction (used during surgery when control of bleeding was required), increase in blood pressure, 128 and positive inotropic effects. 206,207 Cocaine’s early legitimate use in medicine was due to its local anesthetic properties. In contrast to cocaine, amphetamine, 156 a synthetic compound, does not have significant local anesthetic properties. The major medical uses of the amphetamines is in the treatment of attention deficient disorders (ADD) in children and narcolepsy. Structural analogues of amphetamine are used for weight reducing. h3c N COOCH; COCAINE 155 AMPHETAMINE 155 Cocaine and amphetamine are the most powerful positive reinforcers known to man .208 The ability of these drugs to produce euphoria and their extremely powerful reinforcing properties made them one of the most popular drugs of abuse in The United States over the past decade.205,209 Today's epidemic is the third for cocaine, the two previous ones occurring in 1890 and the late 1920’s.209 According to the surveys of The National Institute of Drug Abuse (NIDA) taken in the mid 1980s and the beginning of 1990s, it has been .estimated that 3 million people abuse cocaine regularly, more than once a week.209,210 The epidemic has also been characterized by increase in emergency room visits attributed to cocaine abuse, as well as increase in number of cocaine related deaths. Due to the magnitude of today’s epidemic and the realization of the medical and social problems associated with drug abuse, research on the treatment of drug abuse (particularly cocaine and amphetamine abuse) has become more active. 129 5.4.2. MECHANISM OF ACTION OF PSYCHOSTIMULANT DRUGS Cocaine 155 and amphetamine 155 have complex effects on the central neurotransmitter system; they affect dopaminergic, noradrenergic, serotonergic and cholinergic systems . 211 It is generally accepted, however, that the increase in dopamine neurotransmission in the brain reward centers is responsible for the euphoric effects of amphetamine and cocaine. Di Chiara and Imperato found that amphetamine and cocaine increase synaptic dopamine concentrations in the nucleus accumbens septi (limbic area) and the caudate nucleus (motor a re a ).2 i2 Several lines of evidence suggest that amphetamine and cocaine block the dopamine reuptake by binding to the dopamine transporter .213 In addition, amphetamine causes dopamine release from nerve terminals. Ritz et al. have shown a significant correlation between the binding of a variety of cocaine like compounds to the dopamine transporter and the potencies of the same compounds in eliciting self administration behavior .214 Additional evidence has been accumulating which suggests that reward behavior is mediated primarily by specific pathways in the nucleus accumbens (NAc) involving dopamine. Lesions of the ventral tegmental areas and NAc with the neurotoxin, 6- hydroxydopamine, abolishes not only locomotor stimulation produced by the psychostimulants, amphetamine and cocaine.215 but also disrupts the self administration of cocaine.216 Furthermore, it has been shown that selective blockade of the dopaminergic receptors attenuates amphetamine euphoria, and blocks the rewarding action of intravenous cocaine.217 While acute administration of cocaine and amphetamine produces a temporary increase in dopamine neurotransmission that may be responsible for binges (“rush”) felt by cocaine users, chronic administration of cocaine and amphetamine appears to cause prolonged dopamine reduction.2o7. 2ie it is believed that this reduction of dopamine is the cause of anhedonia seen in withdrawal in chronic users of psychostimulants and is primarily responsible for craving phenomena (“the dopamine depletion hypothesisn).2ie,2i9 Figure 32 schematically describes a hypothesis of cocaine reinforcing action. 130 Postsynaptic Neurone Presynaptic Neurone 0 ^ I. Rest State III. Reuptake II. Stimulated IV. Cocaine Inhibition of Reuptake Figure 32. Cocaine Reinforcing Action (Modified from Carroll, 206) 5.4.3 DOPAMINERGIC AND GLUTAMINERGIC SYSTEMS IN NUCLEUS ACCUMBENS fNAcl AND ACTIONS OF PSYCHOSTIMULANT DRUGS The NAc has been implicated in locomotor activity, rewarding and affective behavior elicited by psychostimulant drugs, such as cocaine and amphetamine. 220 This forebrain region functions as a link between limbic and the motor systems and contains nerve terminals of the mesolimbic dopamine projection.221 In addition to dopaminergic projections the NAc also receives glutamatergic projections from several brain areas 131 including the cerebral cortex, amygdala and the hippocampus.222.223.224 Due to this anatomical arrangement several studies have suggested an interaction between glutamatergic and dopaminergic neurones in the NAc. Biochemical studies in vitro have shown that L-Glu releases dopamine from accumbal s lic e s .^ Cheramy et al. have shown a stimulatory effect of L-Glu on dopamine release in vivo.&* Direct injection of EAA receptor agonists QUIS, kainic acid and NMDA stimulated locomotor activity in rats that could be blocked by either systemic or intra-accumbens injection of dopaminergic antagonists. 227 These results suggest that the hypermotility produced by EAA is mediated through release of dopamine and the subsequent stimulation of dopamine receptors in the NAc.227 Furthermore, it has been shown that the motor stimulant effects produced by the intra-accumbens administration of glutamatergic agonists can be inhibited by specific antagonists of EAA receptor subtypes.228.229.23o These data suggest that the dopaminergic system within the nucleus accumbens is under glutamatergic control via EAA receptors (NMDA, AMPA, and KAIN). The detailed mechanism by which the glutamate neurotransmission may modulate dopamine function is not known. However, there are two different mechanisms by which this could be accomplished. One explanation is the existence of presynaptic EAA receptors on dopamine nerve terminal that could directly stimulate dopamine release (see Figure 33). Another possibility is that glutamate-dopamine interaction takes place on postsynaptic level, where postsynaptically located EAA receptors are able to augment the effects of dopamine receptor stimulation (Figure 3 3 ).22s Electrophisiological s tu d ies 23i and release studies of pHjdopamine performed on striatal dopaminergic neurones have provided some evidence for existence of AMPA and NMDA receptors on these dopaminergic neurons.232 All these data taken together suggest that both dopaminergic and glutamatergic system in NAc might be involved in psychostimulant induced motility and positive reinforcement. Indeed, several groups have shown that hypermotility response to GLUTAMATERGIC PROJECTION Glu Glu Glu AMPA NMDj STRIATAL DOPAMINERGIC NEURON __ 0 ) @ DA Glu Glu DA AMPA NMDA Figure 33. Relationship between Dopaminergic and Glutamatergic Neurons 133 psychostimulant drugs, such as cocaine and amphetamine, can be modulated by intra- accumbens administration of either EAA receptor antagonists or dopamine antagonists. Pulvirenti et al. have demonstrated that hyperactivity induced by amphetamine or cocaine can be reduced by glutamic acid diethyl ester and 2-amino-5-phosphonovaleric acid, respectively.233.234 Willins et al. have shown that AMPA antagonists DNQX and GAMS were able to inhibit the locomotor stimulation produced by amphetamine .235 Furthermore, it has been demonstrated that EAA receptor antagonists are able to block cocaine induced convulsions and death in rodent models. 236 .237.238 Kaddis et al .239 found that the intra-accumbens administration of DNQX or GAMS inhibits the locomotor stimulation produced by cocaine. They also investigated the possible location of AMPA receptors with respect to the dopamine nerve terminal. Since the locomotor stimulation produced by dopamimetic agents in reserpine treated animals was still antagonized by DNQX it was suggested that AMPA receptors are located postsynaptically where they can modulate the effects produced by the direct activation of dopaminergic receptors (Figure 33). 5.4.4. CURRENT PHARMACOTHERAPIES FOR COCAINE AND AMPHETAMINE ABUSE. While the FDA has approved use of methadone 15Z and naltrexone 158 for the treatment of opiate withdrawal, no such drug yet bears FDA labeling for the treatment of cocaine/amphetamine addictions COCHoCH CH2CH(CH3)N(CH3)2 METHADONE 157 NALTREXONE 158 134 The medication that is currently under investigation can be divided into three major classes:(1) drugs for the treatment of co-existing psychiatric disorders; (2) cocaine antagonists; and (3) anticraving agents.^ Since it appears that almost half of cocaine/amphetamine dependent patients have coexisting psychiatric disorders, such as depression or attention deficient disorder, it has been suggested that treatment of original problems might be beneficial in fighting drug addiction. It has been reported that use of tricyclic antidepressant desipramine 159 can reduce cocaine craving and promote retention of cocaine users in treatment 242. However, it is frequently very difficult to make an accurate diagnosis when the patient is using psychoactive drugs, due to the withdrawal symptoms that include anhedonia and depression. CH2CH2CH2NHCH3 DESIPRAMINE 159 METHYLPHENIDATE W. Methylphenidate 160 (an dopamimetic agent) was effective only in patients with ADD. If given to patients that do not suffer from ADD, it may even increase cocaine craving .211 Use of cocaine antagonists (dopamine receptor blockers) such as haloperidol 161. chlorpromazine 2Z, fluphenazine 163) was rationalized in the following way: if a drug can block the euphoric effect of cocaine, the desire for psychostimulant drug would d e c re a s e .241 However, only few studies have been conducted using these agents. Without any control studies the available results are inconclusive. The majority of drugs that are under investigation for treatment of cocaine withdrawal and craving include dopamimetic agents (dopamine agonists): bromocriptine, 135 F l /---- \ CH2CH2CHr __^N— CH2CH2OH HALOPERIDOL 161 FLUPHENAZINE 162 L-dopa/carbidopa, amantadine, pergolid mesylate, tyrosine and tryptophan, and carbamazepine. Since, in the post withdrawal, dysphoria may be the result of an impairment in dopamine neurotransmission, it was thought that drugs capable of increasing dopamine activity should alleviate cocaine craving and anhedonia. Bromocriptine gave some promising results; however, most studies had to be discontinued due to the side effects: nausea, headache, and orthostatic hypotension. The results of trials with amantadine (an indirect dopamine agonist) have been mixed. Most of these compounds have not shown any significant benefit in reducing craving and withdrawal symptoms.24i.242,210 Table 13 gives an overview of the medication under investigation for the treatment of drug abuse. Table 13. Medication Under Investigation for Treatment of Drug Abuse Class Drugs Indications Antidepressants Desipramine to reduce cocaine craving Imipramine to treat coexisting psychiatric disorders Cocaine antagonists Haloperidole to reduce cocaine craving (dopamine receptor Chlorpromazine blockers Fluphenazine Dopamimetic agents Methylphenidate ADD treatment (dopamine receptor Amantadine to reduce cocaine craving agonists) Bromocriptine Pergolid mesilate CHAPTER VI STATEMENT OF PROBLEM AND OBJECTIVES Excitatory amino acid (EAA) neurotransmission plays an important role in a variety of physiological processes as mentioned in the previous chapter. However, excessive activation of EAA receptors has been implicated in a number of neurodegenerative disorders such as ischemia, stroke, epilepsy as well as dementia of the Alzheimer type, Huntington’s disease, parkinsonism and psychosis .153,152 Since the role of EAA in a wide range of neurodegenerative processes has been recognized, there has been considerable interest directed toward the development of EAA receptor antagonists as therapeutic agents .243 Competitive and noncompetitive NMDA receptor antagonists have been at the center of attention because of their availability. The NMDA antagonists have shown cerebroprotective effects in a focal ischemia model, but they may be ineffective in severe global ischemia . 244 in addition, questions have been raised concerning safety of noncompetitive NMDA antagonists such as MK-101 12S, PCP 125 and ketamine 126. In addition to their psychotomimetic action, non-competitive NMDA antagonists produce pathomorphological changes in specific population of brain neurons, and this may limit their use as therapeutic agents. 245,240 On the other hand competitive NMDA receptor antagonist AP5 120 has been shown to cause a selective impairment of learning by suppressing the long term potentiation (LTP).247 These disclosures have shifted attention toward the non-NMDA receptor sites, and the search for selective AMPA and kainate 136 137 receptor antagonists have begun in order to explore the role of these receptors in a number of CNS disorders and to address their therapeutic applications. In addition to competitive AMPA receptor antagonists such as CNQX 14§, DNQX 145 and NBQX 147. 2,3-benzodiazepine (GYKI52466, 1631 has been identified as a potent and selective noncompetitive antagonist at AMPA/kainate receptors . 243 Except for NBQX 147, all these antagonists lack selectivity for AMPA receptor vs. kainate and/or NMDA receptors.184-161 Nevertheless, these compounds have shown neuroprotective activity in a variety of brain ischemia. They also act as anticonvulsants.243 .N h2n o 2s H2N R1 =R 2 = N 0 2 DNQ X 145 NBQX 147 R-l = CN, R2 =N02 CNQX 146 GYKI52466 163 Our biological studies have been directed toward the possible role of AMPA receptors in the nucleus accumbens (NAc) and the ventral pallidus in the rewarding and motor stimulating effects of psychostimulant drugs such as amphetamine and cocaine. As discussed in the previous chapter, psychostimulant drugs (amphetamine and cocaine) are believed to produce their behavioral stimulation through activation of dopaminergic neurotransmission in NAc.212 However, it has been shown that EAA antagonists, within the NAc, modulate the psychomotor activation induced by cocaine and amphetamine, presumably by regulating the release of dopamine.286 These results suggest that in addition to the dopaminergic component, activation of the glutaminergic component in the NAc has a role in hypermotility and possibly in rewarding effects of psychostimulant drugs. 138 AMPA receptors seem to be particularly important for the effects of cocaine and amphetamine since intraccumbens injection of DNQX 145 and GAMS 144 blocks the hypermotility caused by systemic administration of amphetamine and/or cocaine.^, 239 Furthermore, it has been shown that administration of DNQX 145 into the NAc before amphetamine inhibited the development of a conditioned place preference's This experiment strongly suggests that activation of the AMPA receptors in the NAc is necessary for production of the rewarding effect of amphetamine and that blockade of this © 0 0 © 0 NH- CH NO; DNoTyr 1 5 4 G lul05 AMPA 107 receptor attenuates the development and expression of reward.248 Hence, the development of selective AMPA antagonists might provide a new pharmacological strategy for treating the problems associated with drug addiction. In order to extend the structure activity relationship studies for AMPA receptor agonists, 3,5-dinitro-o-tyrosine (DNoTyr, 164) was synthesized. The rationale behind this design was that the phenolate anion (pKa=3.4) in 164 might serve as a bioisoster for the y-carboxyl group in glutamate 105. as the 3-hydroxyisoxazole anion in AMPA 107 presumably does. Surprisingly, DNoTyr 164 was an antagonist at AMPA receptors, inhibiting the locomotor stimulation produced by amphetamine. Although DNoTyr 164 is less potent than AMPA 107 in inhibiting specific pHjAMPA binding in rat brain homogenate, it shows much higher selectivity for AMPA vs kainate receptors (approximately 20 fold more selective for AMPA than for kainate receptors).249 DNoTyr 164 did not antagonize the specific binding of [3H]NMDA, pH]MK-801 or [3HJPCP 139 indicating further selectivity for AMPA receptors .249 Additional significance of DNoTyr lies in the fact that this is one of the first amino acids AMPA antagonist reported to date. Two other compounds namely 2-amino-3-[3-(carboxymethoxy)-5*methyl- isoxazol-4-yl]propionic acid (AMOA, 1651 and 2-amino-3-[2-(3-hydroxy-5-methyl- isoxazol-4-yl-methyl-3-oxoisoxazolin-4-yl)]propionic acid (AMNH, 1661 have been reported to be weak AMPA antagonists. While AMNH 166 was a moderately potent inhibitor (IC5o=12pM), AMOA 165 was a much weaker antagonist (IC50=88pM ) .250 © 0 0 0 OH AMOA 165 AMNH1M The AMPA receptor shows a pronounced stereoselectivity toward the L- enantiomer of agonists: for example L-AMPA shows a 4000 fold higher affinity for the AMPA receptor than D-AMPAJeo The neuroexcitatory effects of the two additional AMPA agonists: 4-bromohomoibotenic acid 13fi and 5-HPGA 14Q also reside only with the S-enantiomers. While NMDA receptors tolerate L- and D- configurations of agonists, only D-isomers of NMDA receptor antagonists show activity.^ Our objective was to synthesize optical isomers of DNoTyr, D-DNoTyr 167 and L-DNoTyr Tgfi in order to compare the enantioselectivity of AMPA antagonists with that established for the AMPA agonists. This would be the first set of optical isomers synthesized and tested as antagonists of the AMPA receptors. 140 OH 0 OH 0 N 02 n o 2 D-DNoTvr 167 L-DNoTvr 168 In an attempt to further test our hypothesis that phenolate anion in DNoTyr 164 serves as a bioisoster of y-carboxyl group, compound 169 was designed, in which the phenolate is masked by converting a 2’-hydroxy to a 2’-methoxy group. An urgent need for compound 169 emerges from the fact that there are literature reports that the N02 group can serve as a bioisosteric replacement of y-carboxylate.zsi 169 Homologation is a classical approach used to convert agonist to antagonist. While in NMDA receptor agonists, two negative charges are 2-3 atoms apart, NMDA antagonists were obtained by increasing the chain length between two acidic groups, preferably to 4-6 atoms. It was therefore our objective to use o-Tyr (o-Tyr itself is unable to inhibit specific [3HJAMPA binding in rat forebrain homogenates) as a flexible template that could be converted into an antagonist. Compound 17Q was designed to probe the 141 distance between two carboxylic groups (6 atoms apart in comparison with distance in Glu lfl§ or AMPA 1QZ where they are only 3 atoms apart). OH In addition to substituted o-tyrosine, quinoxaline amino acids (QXAA, 171) have shown substantial activity in displacing the specific binding of [3H]AMPA and [3HJCNQX. This hybrid molecule, QXAA 171. combines features of the quinoxaline (CNQX, DNQX, NBQX ) AMPA antagonists and ODAP 134, an AMPA agonist (neurotoxin isolated from Lathyrus sativa). N ^ , 0 QXAA 171 QX 172 ODAP 134 While quinoxaline Q X 172 is essentially inactive at 100 pM, QXAA 1Z1 is quite active with (IC50=0.69 pM for the displacement of [3H]AMPA). We decided to develop a methodology for synthesis of optically active isomers of QXAA that could be utilized for a variety of other substituted QXAA (such as CNQX, DNQX od NBQX). Testing optically active 142 isomers of these compounds would give us further insight into enantioselectivity of interaction between AMPA receptor and AMPA receptor agonists and antagonists. OH NH; QXAA 174 CHAPTER VII RESULTS AND DISCUSSION 7.1. CHEMISTRY 7.1.1. Synthesis of o-tvrosine derivatives Asymmetric synthesis of D-DNoTyr 167. that utilizes N-phenylsulfonyl-L-serine 175 as the chiral adduct is outlined in Scheme XI. This methodology for synthesis of natural and unnatural D-amino acids has been developed by Rapoport et al. 252,253,254 Similarly, starting with D-serine, and following the same methodology, desired L-amino acid can be obtained. N-Phenylsulfonyl-L-serine 175 was synthesized according to the procedure of Maurer et al.252 as shown in Scheme XII. Influence of a variety of a-aminoprotecting groups on the addition of organometallic reagents to an amino acid has been studied.253.254 It has been shown that commonly used N-blocking groups carbobenzoxy (Cbz) and tert-butyloxycarbonyl (t-Boc) are not compatible with the reaction conditions under which amino acylation of organometallic reagent takes place: the former unstable to the reaction conditions and the latter gave lower yields. The N-blocking groups such as phenylsulfonyl, ethoxycarbonyl, acetyl and benzoyl can be used interchangeably.254 In the case where a carbamate or amide are used as N-protective groups, organolithium reagent may attack these positions in addition to the carboxylate.253 Furthermore removal of acetate and benzoate requires harsh conditions. On the other hand the ethoxycarbonyl protecting group wouldn’t probably survive the proposed 143 Scheme XI Asymmetric synthesis of D-3,5-Dinitrio-o-Tyrosine 16Z OCH3 OH 0 OCH3 och3 1 . n-BuLi, THF-78°C [ [| [ Et3SiH r B r ___ _ CF3COOHC6 Hso 2SHN 1ZZ c 6 h 5o 2 shnv 'h 178 (35-50%) 179 (59%) 175 CrO^^SCX* acetone r.t. OCH3 NO2 BF4 48% HBr CH3CN H^N" 'H phenol c 6 H50 2 SHNv *H 0-5°C no 2 167 (27%), 50%ee 181 (62%) 188 (67%) 145 Scheme XII Synthesis of N-(Phenylsulfonyl)-L-serine 17§ o h o NaHC03, H20 OH O OH OH c 6h 5s o 2ci H j N m C6H502SH n m m m reduction conditions of the carbonyl to the methylene (second step in Scheme XI) in which F3 COOH is used as solvent. These are the reasons, why we decided to use phenylsulfonyl as the amino protective group. o-Bromoanisol 177 was reacted with n-BuLi at -78°C to form the organolithium compound, which was then aminoacylated with the lithium carboxylate of 175. to give N- phenylsulfonyl-a-aminoketone 178 in 35-50% yield (method A).252 Since nucleophilic reagents are extremely basic one would expect to see racemization of the trisubstituted a-carbon atom to the carboxyl group. In contrast, it has been shown that this transformation can be carried out with complete retention of configuration .253,254,252 This becomes apparent from the mechanism of the reaction that is shown in Scheme XIII.254 At least four equivalents of the organolithium reagent are required for this reaction. Formation of intermediate 183 with two negatively charged centers adjacent to the chiral center, makes the abstraction of the a-methene proton most unlikely .254 Since the reaction with organolithium compound of 177 gave variable and poor yields of 178 we decided to improve yields by switching from an organolithium reagent to a Grignard reagent. So the Grignard reagent of o-bromoanisol 18Z was reacted with 183 in order to obtain ketone 18Z in higher yield (method B, Scheme XIV). It has been shown in the literature when a 146 Grignard reagent is added to an acid in addition to the corresponding ketone an equivalent or even greater amount of the corresponding tertiary alcohol is formed.254 Scheme XIII Formation of a-Amino Ketones from N-(Phenylsulfonyl)-L-Serine 1Z5 and Organolithium Reagents OH O RLi (1st eq) OH 0 RLi(2ndeq) OH O C6 H50 2 SHN" H C6 H502SHrr H C6 H50 2 SNv H 183 RLi ( 3 ^ ) a OH O H+, HzO RLi (4th eq) O O CeHsOpSHN' H C6 H50 2SNv H C6 H5O2 SN H © 185 184 This can be bypassed by preforming the lithium carboxylate from the amino acid with n-BuLi and then reacting 183 with the Grignard reagent. These results imply that the mixed lithium-magnesium salt of dianion 183 is more stable than just Mg salt . 254 However only 54% of 178 was obtained in the later reaction. Furthermore, reaction with Grignard reagent is significantly longer, 20-40 h at r.t., while the lithium reagent was aminoacylated in about 1.5 h at r.t. A variety of methods were attempted in order to reduce the carbonyl group in compound 1ZS to the corresponding methylene (Schemes XV, XVI and XVII). First, we tried the reduction with Et3SiH in trifluoroacetic acid (TFA).2S5 147 Scheme XIV Synthesis of Ketone 1Z8 from N-(Phenylsulfonyl)-L-Serine 175 and Grignard Reagent 187. 177 Magnesium tunings OCH: MgBr OH nBuLI (2 eq) 9 ^ is z O0 2.1NHCI © 183 128 Although the product was obtained the yield was very low( 25%). After hydrogenation under different conditions failed to provide the desired product 173 we decided to reduce the carbonyl in two steps: first forming an alcohol 18g and then reducing the alcohol to the methylene group( Schemes XV and XVI). While reduction with NaBH4 gave a secondary alcohol 188 in 96% yield, we were unable to further reduce the alcohol to the desired product 179. We then returned to the starting conditions Et3SiH in TFA and tried to improve the yield. It turned out that the reduction goes to completion.but the isolation of 179 is troublesome. TLC of the reaction mixture shows two spots: one belonging to JIS and another one with a higher Rf, presumably triethylsilyl ether of 179. Workup conditions (neutralization with NaHC03, and extraction with EtOAc) were not sufficient enough to quantitatively hydrolyze the triethylsilyl ether to 179. Adding tetrabutylammoniumfluoride 148 Scheme XV Reduction of carbonyl group of 178 to methylene group in 179 or to sec-alcohol in 188. OH OH Et3SiH 50°C 178 m H2 ,10%Pd/C Recovered starting material CH3 COOH, 22 h, 40 psi H2 i10% Pd/C Black tarr CF3 COOH, 10%HCIO4i 50CC, 40 psi DIBAL Five spots on TLC THF OH 9 H OCH3 NaBH4 EtOH C6H50 2SHN" H 188 (yield 96%) 149 Scheme XVI Reactions Attempted to Reduce sec-Alcohol in Compound 18g to Methylene Group in Order to Obtain 179. OH 9 H OCH3 H2 , 10% Pd/C Decomposition C6H50 2SHN HCI, 40 psi, 17 h 188 Et3 SiH, TFA Six spots on TLC 50°C H2 , 10% Pd/C No reaction TFA, 40 psi, 20 h H2 , 10% Pd/C Black tarr TFA, 10% HCIO4 50*0 (TBAF) to the reaction mixture after completion of the reaction and stirring for two hours hydrolysed the triethylsilyl ether giving only one spot on TLC. However, use of TBAF requires purification of the final product over a silicagel column (yield 46%). Alternatively, upon completion of the reaction, the reaction mixture was cooled to r.t., MeOH +H20 were added, stirring was continued overnight and the workup procedure performed the next day in order to obtain 179 in 63% yield, saving us purification over the silicagel column. 150 Scheme XVII Modifications of Reduction Reaction with Et 3 SiH/TFA OH 0 OCH3 OCH3 Et3SiH C6 H502SHN H CF3 COOH C6 H50 2 SHN^ *h 178 179 (yield 25%) 1. Et3 SiH, TFA 2. TBAF 3. Silicagel (yield 46%) column 1. Et3 SiH, TFA 2. MeOH, H 2 0, (yield 63%) stirr overnight 3. WU Inverse addition of an acetone solution of 179 to Jones reagent at 0°C gave acid 180 in 67% yield 256 D-o-Tyrosine (oTyr) 181 was obtained from Ififi by reflux with 48% HBr containing phen o l.257.253 Deprotection of the amino group in 48% HBrwas monitored with HPLC (ODS column; pBondpack, mobil phase: 15% MeOH in H 2O, UV detector ^ 2 5 4 nm, flow rate 2 mL/min). Nitration of 181 with N 0 2BF4 in CH3CN at 0-5°C gave DNoTyr 167.259 To make sure that nitration has gone to completion and that only the dinitro product is present, progress of the nitration was monitored with HPLC ( column: p.Bondpack C-18; k=254 nm; mobile phase 8 % acetonitrile in 0.1% TFA; flow rate 2 mL/min). The product was purified over an ion-exchange column (Dowex 50x8; mobile phase 0.3 M ammonium hydroxide). The enantiomeric excess of this product 18Z 151 surprisingly was only 50%ee. We suspect that harsh conditions employed for the deprotection of the amino group (step before the very last one) are responsible for the racemization and low optical purity. Although Maurer et al. reported deprotection of similar compounds under identical reaction conditions to precede under 30 min deprotection of 181 was completed after 2 h. The reason for a different time frame required for the cleavage of sulfonamide(s) is not known. Optically pure isomers of amino acids can be obtained either: a) from a natural source; b) through asymmetric synthesis or c) through resolution of a D.L-amino acids obtained by synthesis. The three major methods for resolution of D.L-amino acids that have been developed over many years are as follows:2^ \) Resolution of D.L-amino acids via diastereomeric salt formation. This classical method is tedious and time consuming but it is still successfully used in the amino acid field. The basic principal of this method is the formation of a pair of diastereomeric salts, separation of which is achieved through fractional crystallization. The diastereomeric salts can be formed between 1) a D.L-amino acid or D,L-amino acid esters and a optically active acid or 2) a N-protected-D,L-amino acid and a optically active base. An example of resolution of D.L-amino acid through diastereomeric salt formation is the separation of both enantiomers of racemic o-tyrosine using 1,1 ’-binaphthyl-2,2’-di-0- phosphoric acid as a resolving agent.2^ ihChromatoaraphic resolution of amino acid enantiomers using a chiral stationary phase. Unmodified a-amino acid racemates can be resolved by means of ligand exchange chromatography, where a chiral stationary phase is absorbed or covalently bound onto the surface of a conventional reverse phase packing and the mobile phase contains complexing metal ion ( usually Cu 2+).262 The resolution is based on the three point attachment between an amino acid, chiral phase (usually optically active 4-hydroxy- proline attached to the octadecyl groups of the support) and Cu2+ ions in the mobile phase. Gubitz et al.263 developed a method for HPLC separation of o-tyrosine enantiomers based on ligand exchange chromatography. However, the disadvantage of 152 this method is that it is primarily used for analytical purposes and it has not yet been used at preparative level by organic chemists. iih Use of enzvmes for resolution of D.L-amino acids. A variety of enzymes has been used for resolution of amino acid enantiomers: acylases, proteases, amino acid oxidases etc .260 The advantages of an enzyme-catalyzed reaction are high enantioselectivity for only one isomer, known absolute configuration of the recovered amino acid enantiomer and the procedure can be used for preparative scale resolution. Since de-novo synthesis of D-DNoTyr 167 as well as L-DNoTyr 168 gave only 50%ee, we decided to take a different approach in order to obtain the desired enantiomers with higher optical purity. We turned to biological resolution using known enzymes as the method to obtain our target(s). Hooker and SchellmanzM obtained D-o-Tyr by using the L-Amino acid oxidase, L-AAOx method of Parikh et al.zes for the resolution of the racemic mixture of amino acids. L-AAOx is a flavoenzyme that catalyzes the oxidation of L-amino acids utilizing FAD as its redox coenzyme. The resulting FADH 2 is reoxidized by 0 2. For each molecule of amino acid oxidized, 1 molecule of 0 2 is taken up, and 1 molecule of keto acid, NH3 and H20 2 are formed:2^ RCHNH2COOH + 0 2 = RCOCOOH + H20 2 +NH3 Phenylalanine is an excellent substrate for this enzyme, and examples from the literature show that even substituted phenylalanines (N02, F, OCH3, CH3, CF3) still undergo oxidation with L-AAOx.267 Kinetic resolution of D-3.5-dinitro-o-tyrosine 167 and D-o-tvrosine 191 bv L-amino acid oxidase fL-AAOx) Encouraged by literature reports we decided to directly produce D-DNoTyr 167 from the racemic mixture of DNoTyr 1§4 using L-AAOx as it is outlined in Scheme XVIII. However, we were unable to obtain the D-isomer in this way. We do not know the reason why the enzyme L-AAOx does not oxidize L-DNoTyr 153 Scheme XVIII Attempted Resolution of D-3,5*Dinitro-o-Tyrosine 167 from the Racemic Mixture Using L-Amino Acid Oxidaze. OH OH OH OH Nrt n o 2 N02 N0Z 164 167 189 into the p-ketoacid 189. however there are two possibilities: steric bulk or an electronic effect. Since this attempt failed we decided to resolve D-o-Tyr 181 from the racemic mixture of commercially available o-Tyr 19fi and nitrate it to obtain D-DNoTyr 1SZ as it is outlined in Scheme XIX. This was successful and 167 was obtained in 31 %yield (>99%ee, see Figure 34, for determination of optical purity vide infra). Pirrung et al.see have suggested that D-amino acid oxidase (DAAOx) could be used to obtain L-isomers of aromatic amino acids. However Parikh et al.ses have shown that attempts to prepare the L-isomers of phenylalanine, tryptophan, tyrosine and hydroxyproline using D-Amino acid oxidase D-AAOx led to products largely contaminated with respective D-isomer. Hellermann et al.2^ have shown that D-AAOx is considerably inhibited by many aromatic compounds such as benzoic acid, phenylpyruvic acid, and indole-2-carboxylic acid. Therefore, we predicted that the oxidation of D-o-Tyr 181 with the D-AAOx would be effectively hindered by the products of the reaction. Scheme XIX Chemoenzymatic Synthesis of D-3,5-Dinitro-o-Tyrosine 167 Utilizing L-Amino Acid Oxidaze. L-aminoacid oxidase p O2 , catalase, Tris-maleate buffer hT '""f 3 + 38°C, 72h 190 181 (44%) N 02BF4 CH3CN no2 167 (31%), >99%ee 155 Marfey*s reag. 0 .9 0 —1 p i r • 0.80 0.70 D-DN-o-Tyr 0.60 0.50 3 0.40 0.30 0.20 0.10 — 0.00 H in u te s Figure 34. HPLC chromatogram of derivatized D-DNoTyr 167 with Marfey’s reagent. Column: ODS (4.8 mm I. D. x 15 mm L); Eluent 24% CH3CN in acetate buffer (0.02 M, pH=4.0); Flow rate 1 mL/min; UV detector 1=340 nm. Sample injected 10 p i. Retention times: D-isomer (tR=12.433 min), 1- hydroxy 2,4-dinitrophenyl-5-L-alanine amide (tR=8.8 min). Retention time for L-DNoTyr IfiS under these conditions is tR=4.3 min. 156 Kinetic resolution of L-3.5-dinitro-o-tyrosine 168 by Aspergillus acylase I. Chemo-enzymatic synthesis of L-DNoTyr !£ £ is outlined in Scheme XX. Racemic o- tyrosine was nitrated with N02BF4in CH3CN to give racemic DNoTyr 164 which was then acylated under Schotten-Baumann conditions to give racemic N-acetyl-3,5-dinitro-o- tyrosine ( N-acetyl DNoTyr) 1 9 2 .27 o.27 i The method of W hitesides 272 was utilized to obtain optically active L-DNoTvr 168 in 41% yield, >99%ee(Figure 35). Scheme XX Chemoenzymatic Synthesis of L-3,5-Dinitro-o-Tyrosine 168 Utilizing A. Acylase. OH 1. (CH3C0)20, NaOH o2l 0© NH: 2. NaOH 2N, r.t. 30 min NHCOCH3 N0 2 164 192 (98%) A. Acylase, pH 7.5-8.0 40°C NHCOCH3 N0 2 168 (42%), >99%ee 193 (47%) 157 1.60 —i 1.40 — L-DN-o-Tyr 1.20 1.00 — Marfey's reag. a 0.80 0.60 — 0.40 — 0.20 — 0.00 Minutas Figure 35. HPLC chromatogram of derivatized L-DNoTyr 16g with Marfey’s reagent. Column: ODS (4.8 mm I. D. x 15 mm L); Eluent 24% CH3CN in acetate buffer (0.02 M, pH=4.0); Flow rate 1 mL/min; UV detector A^340 nm. Sample injected 10 pL. Retention times: L-isomer (tR=4.3 min), 1 -hydroxy- 2, 4-dinitrophenyl-5-L-alanine amide (tR=8.7 min). 158 Progress of the reaction of 132 with Aspergillus acyiase I (Sigma) was monitored by assaying aliquot for free amino acid with HPLC as shown in Figure 36. Figure 37 shows the progress of resolution of DNoTyr catalyzed by Aspergillus Acyiase I.The L-amino acid 133 was separated from nonhydrolysed N-acetyl-amino acid by extracting the later with EtOAc. An attempt was made to hydrolyze D-N-acetyl DNoTyr 193 and to obtain D- DNoTyr. However, hydrolysis in 2N HCI for 70 min gave D-DNoTyr in 80%ee. Basic hydrolysis (2.5N NaOH) destroyed the product. 0.40 0.35 0.30 0.25 o 4 0.20 0.15 0.10 0.05 0.00 10.00 Hinutea Figure 36. HPLC chromatogram of o-Tyr 130 (internal standard, tR=2.22 min), DNoTyr 164 (tR=4.32 min) and N-acetyl-DNoTyr 13J2 (tR=10.13 min). Column: ODS (4.8 mm I.D. x 15 mm L); Eluent: 8 % CH3CN in acetate buffer (0.02 M, pH=4.0);Flow rate 2 mL/min; Detector k=280 nm; Sample injected 10 pL. 159 60 e e v ► O13 0 5 10 15 20 25 30 tlme[b] Figure 37. Progress of L-DNoTyr 19B formation from N-acetyl-DNoTyr 192 catalyzed by Aspergillus acyiase I The synthesis of 3,5-dinitro-2-methoxy phenylalanine 199 is outlined in Scheme XXI. Treatment of DNoTyr 194 with di-tert-butyl dicarbonate under Schotten-Boumann conditions gave N-t-butyloxycarbonyl-(3,5-dinitro-2-hydroxy)phenylalanine 1 9 4 .z73 .zm Methylation of 19 4 with CH 3 I in the presence of K 2CO3 in DMSO gave compound 195. However, when a stronger base (e.g. NaH, KOH ) was used, methylation of protected amino group took place. Furthermore it has been reported that under harsh conditions even methylation of a-carbon atom can occur. The deprotection of the amino group of compound 195 was carried out in CF 3 COOH and purification over ion-exchange column afforded 169. 160 Scheme XXI Synthesis of 3,5-Dinitro-2-methoxy-phenylalanine 169 OH O0 ((CH3)3CC02)2 nh3 NHtBoc NaOH, H20 THF 164 194 (96%) OCHo 02N CH3I, k 2co3 0H CF3COOH NHtBoc DMSO 45 min, r.t. N02 195(72%) 199(40%) In Scheme XXII we showed the synthesis of 2-carbomethoxy phenylalanine 170. Reaction of o-Tyr 190 with di- terf-butyl dicarbonate gave compound 196.273 ,274 Treatment of 196 with ethylbromoacetate and K2C03 in acetone, followed by reflux in 2N HCI gave the final product, 2-carbomethoxy phenylalanine 170. Scheme XXII Synthesis of 170 ((CH3)3CC02)2O o h o 1.BrCH2COOEt pCH2COOH 0 K2C03, acetone o 'OH © NaOH, HaO rVAn 2 .2N HCI 3 THF NHtBoc NH2 *HCI 196 (91%) 170 (26%) 161 Optical purity. The optical purity of DNoTyr enantiomers 167 and 168 was established by diastereoisomer formation with Marfey’s reagent (1-fluoro-2,4-dinitro- phenyl-5-L-alanine amide, 197) as shown in Scheme XXIII and analysis by HPLC. 275 ,27 s Parallel reaction with the racemic DNoTyr allowed us to develop the HPLC method for separation of diastereoisomers. The blank probe (containing only Marfey’s reagent) allowed us to determine the retention time for the hydrolyzed reagent (1-hydroxy-2,4-dinitrophenyl-5-L-alanine amide). Use of Marfey’s reagent for derivati- zation and diastereoisomer formation has a number of advantages in comparison with other derivatizing agents that have been widely used for the same purpose. For example use of Mosher’s acid chloride (a-methoxy-a-trifluoromethyl)phenylacetyl chloride) or N-(phenylsulfonyl)prolyl chloride requires protection of the carboxyl group of amino acid, meaning an additional step for derivatization procedure.277 in contrary derivatization with Marfey’s reagent is simple, single step 90 min long. Furthermore high extinction coefficient of the formed diastereoisomer increases the sensitivity of the enantiomeric excess determination. Finally in comparison with chiral columns determination of %ee with Marfy’s reagent is quite inexpensive. 7.1.2. Synthesis of auinoxaline-2.3-dione amino acids. Compound 171 has been previously synthesized according to the procedure outline in Scheme XXIV .249 Quinoxaline-2,3-dione 172 was reacted under strong basic conditions (NaH) with bromodiethylacetal to form 2 0 1 . Acetal was then converted to aldehyde which under Strecker reaction conditions (KCN and NH 4 CI) gives amino acid 1 7 1 - However, this methodology gives extremely poor yields of the final product (overall yield <10%). In addition product is in a form of racemic mixture. Since one of our objectives is to study the enantioselectivity of receptor interaction we need to obtain enantiomers of H I in optically pure and if possible in higher yield. Scheme XXIII Derivatization of 3,5-Dinitro-o-Tyr 164 with Marfey's Reagent n o 2 OH N^.CONHa OzN A H CH3 + F MARFEY'S REAGENT D,L-3,5-Dinitro-o-Tyr 197 164 NaHCOa/acetone 60 min, 40°C N CONH; 0ZN HN, OH NO L,L-diastereoisomer L,D-diastereoisomer 198 199 163 Scheme XXIV Synthesis of Quinoxaline-2,3-dione Amino Acid 171 n h . (COOEt)2 BrCH2CH(OEt)z THF, Refl. 2M KOH, NH. 30% EtOH Refl. 2-3 weeks 200 201 OEt 1.2 M HCI OEt H 2. 7N HCI, Refl. H 171 202 We proposed to synthesize L-enantiomer 173 according to the Scheme XXV which makes use of methodology developed by Vederas. 270,279 Hence, N-protected-p-substituted D-and L-alanines can be obtained in stereochemically pure form by nucleophilic opening of the serine-p-lactone(s).27B, 279 N-(Tert-Butoxycarbonyl)-L-Serine-p-lactone 203 was synthesized from N-(tert-butoxycarbonyl)-L-serine 206 using modified Mitsunobu conditions (Ph3P, DEAD, in THF) as shown in Scheme XXVI, according to the procedure described by Vederas. 280,281 Lactone 203 was then reacted directly with excess of phenylenediamine 200 in THF, in an attempt to obtain 204. Progress of this reaction was monitored by FT-IR. Reaction was presumed to be completed when the peak at v=1847 cm-1 (C=0 group of p-lactone) completely disappears. Ring-opening of p-lactone can occur via two routes giving rise to two different products 201 or 2QZ as shown in Scheme XXVII (see 164 Scheme XXV Proposed Asymmetric Synthesis of L-QXAA173 NH; NH? 0 - / THF r.t. 48h NH n h 2 NHtBoc 200 2 0 3 204 OH NHtBoc (C00Et)z THF, reflux OH OH NHtBoc 205 m Scheme XXVI Synthesis of Serine-p-lactone 203 OH Ph3P, DEAD OH THF, -78°C "M il H H NHtBoc NHtBoc 206 203 165 Scheme XXVII Reaction of Phenylendiamine with Serine-p-lactone 203 Path A THF NH Empirical formula: r.t. 48h OH NHtBoc 2 0 4 + mi H NHZ NHtBoc NH; 200 2 0 3 THF Path B r.t. 48h NH OH Empirical formula: C 1 4 H 2 1 N 3 O 4 NHtBoc 2 0 7 Path A and B). According to the literature data “soft” nucleophiles (e.g. carboxylate, thiolate) usually attack the p-carbon, whereas “hard” nucleophiles (e.g. hydroxide, methoxide, organolithium compounds) tend to target the carbonyl group, Furthermore, it has been shown that ammonia in THF at 0°C attacks the p-position of N- carbobenzyloxy-L-serine p-lactone to give the diaminopropanoic acid, however, the same nucleophile in acetonitrile reacts with the carbonyl to produce serine amide. Although we expected to obtain compound 204 as a major product in the above reaction (Scheme XXVII) chemical shift of the methylene protons (CH 2) in 1H-NMR spectrum showed at 8 3.98-3.91 and 3.85-3.80 what corresponds more to the methylene group connected to OH, as in 207. then to the NH as in 204. The mass spectrum of the product 2Q7 (Figure 38) gave a definite answer as to its chemical structure, since the major fragments were m/z 108 [C6H4(NH2) 2] and 135 [C6H 4(NH2)NHCO]. If the isolated product had the structure of 204. the major fragments would probably be m/z 121 [C6H 4(NH2)NH=CH2j and 251 (M+- COOH). The fragmentation pattern of compound 20Z is given in Scheme XXVIII. Since we were not able to obtain the intermediate 204 synthesis of desired QXAA 1Z2 could not be accomplished according to the Scheme XXVI. However, it is possible that altering the conditions of the reaction between phenylenediamine 200 and lactone 203. such as changing the solvent from THF to acetonitrile, or using a different N-substituent (e.g. carbobenzyloxy, Cbz) could change the mode of addition in order to obtain the desired intermediate 204. 100" 80. 108 135 70- 60- 50- 40- 295 30- 20. 191 278 10 - mss 100 150 200 300 350 Figure 38. Mass Spectrum of Compound 207. 167 Scheme XXVIII Fragmentation of 207 n h 2 NH OH NHtBoc 207 t-e NH; NH. NH OH NH NHtBoc NHtBoc HO' OH NH. OH cxNHNH2 I C C NHtBoc NHtBoc > ••• ©u m/z 108 m/z 135 -e' © OH OH H NHtBoc ©NHtBoc m/z 160 m/z 187 168 7.2.BIOLOGY Racemic DNoTyr 164. D-DNoTyr 167. L-DNoTyr 168. 3,5-dinitro-2-methoxy / phenylalanine 169 and 2-carboxymethoxy phenylalanine 170 have all been examined for the inhibition of [3H]AMPA binding to the rat brain membranes. In addition to these analogues, intermediates with protected amino groups (N-acetyl-3,5-dinitro-o-tyrosine 192. N-tert-butyl-3,5-dinitro-o-tyrosine 194 and N-tert-butyl3,5-dinitro-2-methoxy phenyl alanine 1951. were examined for binding to the AMPA receptor. Reason for testing of the latter compounds was to examine the necessity of the free amino group in the binding to the AMPA receptors. Figure 39 summarizes the effect of different DNoTyr and o-Tyr analogues on the [3H]AMPA binding to brain homogenates. The most striking conclusion from this graph is that only one enantiomer of DNoTyr 164 namely L-DNoTyr 168 inhibited specific PH]AMPA binding to the rat brain membranes by 70 % at concentration of 10-4 M. The D-DNoTyr IfiZ does not displace [3HJAMPA at all at the same concentration, while the racemic mixture of DNoTyr 164 is active as expected; however, it is less then the L-isomer. As shown in Figure 40, the IC 50 values for racemic 164 and L-DNoTyr 168 have been determined to be 51 pM and 10 pM, respectively. Thus, the L-isomer is 5-fold more potent than the racemate. However, these are preliminary results (one experiment run in duplicate), and further testing, as well as functional studies are under way. The fact that L-isomer of DNoTyr 168. (racemate of which has been shown to behave like antagonist see reference 249) is more active follows the same trend as found for AMPA receptor agonists (L-AMPA and L-Glu) This could imply that antagonist binds in a similar way to agonist, but produces no intrinsic activity. Compound 1£9 (3,5-dinitro-2-methoxyphenylalanine) showed only 2 0 % inhibition of specific pHJAMPA binding. These results confirm that the phenolate anion is responsible for interaction with the receptor active site, since its masking results in compound 1 6 i with reduced ability to displace [3H]AMPA. The inhibition of binding by this compound may be attributed to one of the NO 2 groups, which, in the absence of phenolate anion, plays the role of bioisosteric replacement for a carboxyl group. PERCENT [ 3H ] —AMPA BOUND Figure 39. Effect of Different DN-o-Tyr and o-Tyr Analogs on pH]AMPA on Analogs o-Tyr and DN-o-Tyr Different of Effect 39. Figure 0 0 1 0 2 1 60 80 154 12 Z Binding to Brain Membranes. Brain to Binding ei 2 iq 192 izq 122 i le OC, 0 4M 10“ CONC., .V m i s i to O) 170 Somewhat surprisingly compound 170 (2-carboxymethoxyphenylaianine) did not show any inhibition of specific [3HJAMPA binding, even through we expected it to show antagonist activity due to its resemblance to compound 165. which was previously established to be weak AMPA receptor antagonist. Intermediates 192. 194 and 195. all of which have protected amino groups, did not displace [3HJAMPA at 10-^M. We conclude that the free amino group is necessary in order for these compounds to bind to the AMPA receptor. 120 100 < CL ST ■C n 1—> j— Ui o C£ 2 0 - LjJ CL 7 6 ■5 4 DRUG (log, mol/1) Figure 40. Inhibition of Specific PH]AMPA Binding by Compounds i£ 4 and 168. SUMMARY Attempted asymmetric synthesis of DNoTyr 164 enantiomers using optically active L- or D-serine as a chiral adduct gave 167 and 168 in poor optical purity (50%ee) and low overall yields. L-Amino acid oxidase was successfully used to obtain D-o-Tyr 181. from a racemic mixture of o-Tyr, which upon nitration with nitronium tetrafluoro borate gave D-DN-o-Tyr 167 in >99%ee. Selective hydrolysis of N-acetyl-DN-o-Tyr 192 with Aspergillus Acyiase I afforded exclusively L-DN-o-Tyr 1£8 in >99%ee. The determination of enantiomeric purity of DN-o-Tyr enantiomers 167 and 168 was accomplished by diastereoisomer formation with Marfey’s reagent and subsequent separation on a reverse phase HPLC column. Only one isomer, namely L-DN-o-Tyr 168 inhibited specific [3HJAMPA binding to brain membranes. Compound 169 inhibited only 20% of specific [3HJAMPA binding indicating that nitro group might be mimicking the carboxyl group to some extent. However, these results also show that phenolate anion rather then nitro group in compound 1§4 is more important for interaction with the active site of the AMPA receptor. Compound 170 did not show any inhibition of [3H]AMPA binding to brain membranes. Since compounds 192.194 and 195 , all of which have a protected nonbasic amino group, did not displace pH]AMPA, we concluded that the free amino group is necessary in order for these compounds to bind to the AMPA receptors. Reaction of phenylenediamine 200 and serine-p-lactone 203 did not give the desired intermediate 204 but rather 207. Due to this problem synthesis of QXAA 173 will have to be accomplished using a different approach. CHAPTER VIII EXPERIMENTAL General information concerning instrumentation, solvent preparation and elemental analysis may be found in the introduction of Chapter I. Additionally, THF was produced by refluxing with and distillation from sodium, using benzophenone as an indicator for dryness. Ion-exchange resine Dowex 50x8 200, as well as Dowex 1x8 200 were purified prior to use. Enzymes Acyiase I (EC 3.5.1.14) (0.47 units/mg solid), L-amino acid oxidase (EC 1.4.3.2) (0.53 units/mg solid), and catalase (EC 1.11.1.6) (1600 units/mg solid) were purchased from Sigma Chemical Co. Derivatizing reagent N,a-(2,4-Dinitro-5-fluorophenyl- L-alaninamide, 197 was purchased from TCI America, Inc. pH values were determined with Fisher Scientific Accumet pH meter 15. Optical rotations were recorded on an Antopol III at X= 589 nm at room temperature. High performance liquid chromatography was performed on a Waters HPLC system equiped with model Waters 510 pump or model Waters 501 pumps, U6K injector and a model Waters 486 detector. All solvents were HPLC grade. The chromatograph was operated isocratically. 172 173 N-Phenylsulfonvl-L-serine 175. OH O OH PhOaSHiM H To a solution of L-serine 17§ (10.0 g, 95 mmol) in H 2O (100 mL) was added Na 2C0 3 (28.0 g, 265 mmol), and benzene sulfonyl chloride (19.3 g, 109 mmol). The mixture was vigorously stirred overnight, H20 was added to dissolve the precipitated sodium salt. After filtration, the filtrate was acidified with conc. HCI (pH=2), and kept in refrigerator overnight. The product was collected and washed with H20, and EtOH, dried in vacuo to afford 5.1 g (22%): mp 222-224°C, dec. (lit. mp 223-225°C); 1H NMR (DMSO-d6, 300 MHz) 5 8.02-7.51 (m, 5H, ArH), 3.80-3.72 (m, 1H, CH), 3.50-3.48 (d, 2H, CH2); [a]D25= +9.20° (c2, MeOH) (lit. [a]D25=+9.25 (c2, MeOH)). N-Phenvlsulfonvl-D-serine 209. CH O N H SC ^Ph Procedure same as for 175 : from D-serine (20 g, 190 mmol) was obtained 31.2 g, 67% of 20§. as white fluffy powder: mp 222-224°C; 1H NMR same as for 175 described above; [a]D25= -9.3 0 (c2, MeOH). 174 (S)-a-(N-(Phenylsulfonvl)amino)-B-hydroxy-2-methoxy propiophenone 178. OH METHOD A 2-Bromoanisol 17Z (22.4 g, 120 mmol) was dissolved in THF (100 mL) with stirring under argon atmosphere, and the flask is then cooled to -78°C with a dry ice-acetone bath. n-BuLi (53 mL, 133 mmol) was added via syringe over period of 5 min, and stirring continued for additional 1 h at -78°C. The solution of N-phenylsulfonyl-L-serine 175 (4.9 g, 20 mmol) in THF (400 mL) was added via dropping funnel over 50 min at -78°C, the cooling bath was removed and the mixture was slowly wormed to room temp. The mixture was then poured into ice-cold 1.2N HCI (500 mL), and extracted with Et20 (3X300 mL). The organic extracts were combined, washed with saturated NaHC03 (300 mL), brine (300 mL), and dried over Na 2S0 4 . After filtration the organic solvent was concentrated under reduced pressure, and the product crystallized from EtOAc/Hex to give slight yellow crystals of 178 3.39 g, 51%: mp 109-110.5°C; 1H NMR (CDCI3, TMS, 250 MHz), 5 7.88-7.84 (dd, 2H, J=1.3, 8.1 Hz, ArH), 7.58-7.55 (dd, 1H, J=1.8, 7.75 Hz, ArH), 7.54- 7.40 (m, 4H, ArH), 7.00-6.90 (m, 2H, ArH), 6.17-6.14 (d, 1H, J=7 Hz, NH), 5.13-5.07 (m, 1H, CH), 3.99-3.94 (dd, 1H, Jvic=3.3 Hz, Jgem=11.6 Hz, CH2), 3.84 (s, 3H, OCH3), 3.74- 3.67 (dd, 1H, Jvic=4.3 Hz, CH2),2.32-2.27 (t, 1H, J=6.8 Hz, OH). FT-IR (KBr,cm-i) 3540 (OH stretching), 3270 (NH stretching), 1693 (C=0 stretching), MS(EI); m/z 336 (M+), 200 (M+-Ph(OCH3)CO), 135 (base). Anal. Calcd. for C 16H17N05S: C, 57.30; H, 5.11; N 4.18. Found: C, 57.25; H, 5.08; N, 4.17. [oc]D25 = +94.2° (c l, acetone). 175 METHOD B N-Phenylsulfonyl-L-serine 175 (2.45 g, 10 mmol) in freshly distilled THF (100 mL), was cooled to -78°C, in a dry ice-acetone bath, while stirring under nitrogen atmosphere. n-BuLi (8 mL, 20 mmol) was added dropwise via syringe, and stirring continued for additional 30 min at -78°C. The mixture was then treated with 2- methoxyphenylmagnesium bromide 187 in THF. Stirring was continued at room temperature for additional 40 h. It was then poured into 1N HCI (150 mL), while cooling in an ice-bath, and extracted with ether (3x100 mL). The combined extracts were washed with sat. NaHC03 (250 mL), brine (250 mL), dried over Na2S04, filtered and concentrated under reduced pressure on rotavapor. Crystallization of the residue from EtOAc/Hex gave 178 as white crystals 1.76 g, 53%: mp109-111°C. (RVa-(N-{Phenvlsulfonyl)amino)-B-hydroxy-2-methoxy propiophenone 210. CH According to the same procedure described as method A for compound 178: from N- phenylsulfonyl-D-serine 209 (4.91 g, 20 mmol) was obtained 2.7 g, 40% of 210 as beige crystals: mp 108-110°C; 1H NMR same as for 178 described above; [a b 25= -95.14° (c1, acetone). 176 fS1-2-fN-fPhenylsulfonvnamino1-1.3-dihydroxv-3-f2,-methoxvDhenvl>Dropane 188. OH To a solution of 178 (0.50 g, 1.5 mmol) in ethanol (15 mL) was added solution of NaBH4 (0.07 g, 1.8 mmol) in ethanol (10 mL) via dropping funnel and the mixture was stirred at room temperature for 1 h. At this point TLC shows that the reaction has gone to completion (mobile phase CH2CI2/EtOH=9.7/0.3). Saturated solution of ammonium chloride was added to the reaction mixture until formation of bubbles couldn’t be observed anymore. Precipitated (NH4CI salt) was dissolved by addition of water and the mixture was that extracted with EtOAc (3x20 mL). Organic layers were combined, washed with brine, dried over sodium sulfate and concentrated in vacuo to give white solid 0.47 g (93%) of 188. According to the 1H NMR this is a mixture of diastereoisomers. 177 fR^-fN-fPhenylsulfonvllaminol-l-hydroxv-S-^^methoxvDhenvllpropane 179. OH To a solution of 1 7 g (5 g, 15 mmol) in CF3COOH (34 g, 298 mmol), was added triethylsilane (12.4 g, 6 6 mmol) via dropping funnel, while stirring and cooling in an ice- bath. The mixture was than stirred and heated on an oil-bath at 50-55°C for 2 h. After cooling the mixture to r. t., H20 (20 mL), and MeOH (20 mL) were added and stirring continued overnight. The mixture was neutralized with NaHC0 3 and extracted with EtOAc (3X100 mL). Combined organic extracts was washed with brine, dried over Na 2 S0 4 , concentrated under reduced pressure to give yellow oil. This residue was chromatographed on silicagel (mobile phase Hex/EtOAc 8 / 2 and then 1/ 1 ). The product 1 7 9 was crystallized from EtOAc/Hex in form of white crystals 2.84 g, 59%: mp 77-79°C; 1H NMR (CDCI3, 250 MHz) 8 7.63-7.59 (dd, 2H, J=1.3, 7.5 Hz, ArH), 7.49-7.44 (m, 1H, ArH), 7.36-7.30 (t, 2H, J=7.9, 8.2 Hz, ArH), 7.19-7.12 (td, 1H, J=,1.7, 7.7 Hz, ArH), 6.95-6.91 (dd, 1H, J=1.7, 7.4 Hz, ArH), 6.82-6.72 (q, 2H, J=7.4, 8.7 Hz, ArH), 5.36-5.33 (d, 1H, J=7 Hz, NH), 3.75 (s, 3H, OCH3), 3.55 (s, 2H, CH2), 3.49-3.39 (m, 1H, CH), 2.80-2.72 (t, 2H, J=5.8, 7.9, 6 .6 Hz, CH2OH), 2.67 (br.s, 1H, OH); FT-IR (KBr, cm-1) 3509.8 (OH stretching), 3299.6 (NH stretching); MS m/z 321 (M+), 290 (M+-OCH 3), 200 (M+-C6 H5(OCH3 )CH2), 77 (base)(C 6 H5). Anal. Calcd. for C 16 H19N 0 4S: C, 59.80; H, 5.96; N, 4.36. Found: C, 59.85; H, 5.92; N, 4.32. [ (S1-2-fN-(Phenvlsulfonvhaminol-1-hydroxv-3-f2’-methoxvDhenvhpropane 211. OCHa OH NHSOfcPh Prepared according to the same procedure discribed for 17§ only compund 2 1 0 was used as starting material. From 210 (2 g, 6.00 mmol) was obtained 1.2 g, 63 % of 211 in form of white needles: mp77-79°C; 1H NMR same as for 179: optical rotation [a]o=-66.0oC (c1, acetone). fR1-2-(N-fPhenylsulfonyl)amino)-3-(2,-methoxyphenyl)- propionic acid 180. NHS02Ph To a cold solution of Jones reagent (0.118 g of CrOa/mL of 1.5M H 2SO4) (12 mL), temp, between 5-10°C, was added a solution of 179 (1 g, 3.11 mmol) in acetone (30 mL) via dropping funnel over period of 30 min. The mixture was continued to stir at r.t. for 24 h, poured into Et 2 0 (70 mL) and extracted with brine (3X100 mL). The Et20 layer was then washed with 1N NaOH, combined basic extracts acidified with 10M H 2S0 4 and extracted with Et 2 0 (3X100 mL). The Et20 extracts were combined, washed with H20 (2X100 mL) brine (150 mL), dried over Na 2S0 4l concentrated under reduced pressure on a 179 rotavapor to give brown oil. The product 180 was crystallized from EtOAc/Hex in form of slight yellow crystals 0.62 g, 59 %: mp 146-147°C; 1H NMR (CDCI3i 250 MHz) 87.59- 7.56 (dd, 2H, J=1.4, 8.1 Hz, ArH), 7.50-7.44 (m, 1H, ArH), 7.44-7.30 (m, 2H, ArH), 7.22- 7.15 (td, 1H, J=1.6, 8.0, ArH), 6.99-6.95 (dd, 1H, J=1.7, 7.5, ArH), 6.83-6.71 (m, 2H, ArH), 5.39-5.35 (d, 1H, J=8.4 Hz, NH), 4.2-4.11 (m, 1H, CH), 3.69 (s, 3H, OCH3), 3.02-2.99 (d, 2H, J=6.8 Hz, CH2); FT-IR (KBr, cm-1) 3334 (OH stretching), 3004 (br. COOH stretching), 1727.9 (C=0 stretching); MS(EI) m/z 335 (M+), 290 (M+-COOH), 121 (base)(C6H 5 (OCH3)CH2),; Anal. Calcd. for C16H17N 0 5S: C, 57.25; H, 5.11; N, 4.18. Found: C, 57.30; H, 5.11; N, 4.20. [ oc] d = +38.9° (c1, acetone). (S)-2-(N-(Phenylsulfonynamino1-3-f2,-methoxvDhenvl1- propionic acid 212. o c h 3 NHSCbPh Prepared from compound 211 according to the same procedure as described for enantiomer 180 of this compound : 1 g, 3.11 mmol of 211 afforded 212 in form of beiae crystals 0.7 g, 67% : mp 146.5-147.5°C; 1H NMR same as for 18£) described above. [a]D= -36.0°C (c1, acetone). 180 D-o-TYROSINE 181. CH METHOD A A mixture of IfiQ (1.7 g, 4.7 mmol), phenol (1.8 g, 19 mmol) and freshly distilled 48% HBr (20.9 g, 258 mmol) was refluxed for 2.5 h. Progress of the reaction was monitored by assaying aliquots (0.5 mL) on HPLC (ODS column, mobil phase: 15% MeOH in H 20 , UV detector X=254 nm), every 30 min. The reaction mixture was cooled to room temp, diluted with H20 (50 mL) and extracted with EtOAc (50 mL). Water layer was purified over cation exchange column Dowex-50x8 (H+). The product was eluted from the column with 0.3M NH 4OH. Solvent was removed under reduced pressure to give 181 as white powder, 525 mg 62%: mp 248-250°C (lit 249-250°C); 1H-NMR (D 20, DSS, 300 MHz): 8 7.28-7.20 (m, 2H, ArH), 6.97-6.92 ( t , 2H, ArH), 4.07-4.03 (m, 1H, CH), 3.38-3.32 (dd, 1H, Jvic=5 Hz, Jgem=14 Hz 2, Chb), 3.08-3.00 (q, 1H, CH2); [cc]D25= +17.46° (c10, 1M HCI) [lit. [ METHOD B Preparation of D-o-Tyrosine 167 by enantioselective destruction using L-amino acid oxidase. A racemic o-Tyr ISO (2.72 g, 15 mmol) was suspended in Tris-maleate buffer (250 mL, 0.05 M, pH 7.8). To this mixture was added KCI (1.86 g, 25 mmol), followed by 40 mg of L-amino acid oxidase (Sigma, Type I, activity 0.53 units/mg) and 10 mg of catalase. The reaction mixture was vigorously stirred on a water bath at 35-40°C for 48 h. After 2 days the mixture was acidified with 1N HCI to pH 6-7, heated with Norite on a steam 181 bath, and filtered over Celite. The filtrate was alkalized to pH 10-11, and purified over anion exchange column Dowex 1x8 (OH-), eluting the amino acid from the column with 1M CH3COOH, followed by purification over cation exchange column Dowex 50x8(H+).The column was washed with H20 and EtOH, and the amino acid eluted with 0.3M NH4OH. The solvent was removed under reduced pressure and D-o-Tyr 181 obtained crystallization from water as white crystals 1.2 g, 88%: mp 249-251 °C; 1H NMR same as for 181 obtained by methode A; [a]p25= +31.5° (c 0.84,1M HCI) (lit. [a]D25=+25.0o (c 0.84, 1M HCI)). D-3.5-Dinitro-o-Tyrosine 167. CH A suspension of D-o-Tyrosine (obtained by Methode B) 181 ( 0.41 g, 2.3 mmol) in CH3CN (10 mL) was cooled in an ice-acetone bath to ~0-5°C, and N 02BF4 (0.8 g, 5.8 mmol) was added in small portions over period of 20 min, while the reaction mixture was continued to cool and stir. Progress of the reaction was monitored by assaying aliquots with HPLC (ODS column, mobile phase 8% CH3CN in 0.1% CF3COOH in H20, flow rate 2 mL/min, UV detector >.=254 nm). After 8 h reaction has gone to completion. The mixture was in water (120 mL), stirred overnight, and filtered over Celite. The filtrate was purified by cation exchange chromatography on Dowex 8x50 (H+). After loading the column and washing it with H20 (2L), and EtOH (500 mL), the amino acid was eluted with 182 0.3 M NH4OH. The eluent was concentrated under reduced pressure to 5 mL, pH adjusted to -3.S-4.2 with 1N HCI, to give 167 in form of yellow crystals 0.19 g, 31%: mp 235-240°C; 1H NMR (DMSO-d6, 300 MHz): 8 8.54-8.53 (d, 1H, J=3.1 Hz, ArH), 7.89-7.88 (d, 1H, ArH), 4.14-4.11 (m, 1H, CH), 3.12-3.06 (dd, 1H, JViC=3.8, Jgem= 14 Hz, CH2), 2.89-2.82 (dd, 1H, J ViC=7.4, CH2); FT-IR (KBr, cm-1): 3500-2500 (COOH,), 3278 (OH), 3083 (NH), 1637 (C=0); MS (FAB) m/z 272 (M++H); Analysis Calcd. for: C9H9N3O7 : C, 39.86; H, 3.34; N 15.49. Found: C, 39.74; H, 3.40; N, 15.40. [a ]D25= -5.66° (c2, 0.05M HCI). N-Acetyl-(D.L)-3.5-Dinitro-o-Tyrosine 192. CH CH NO*, A racemic DNoTyr 164 (3.3 g, 9.8 mmol) was dissolved in H20 (20 mL) and 2N NaOH (5 mL), and cooled in an ice-acetone bath to 0°C. Redistilled acetic anhydride (2 mL, 22 mmol), and 2N NaOH (24 mL) were added in small equal portions. After addition stirring was continued at room temp, for additional 40 min. The mixture was acidified with 10 N H2S0 4 , extracted with EtOAc (3x70 mL). Organic extracts were combined, washed with brine (100 mL), dried over Na2S04, filtered and concentrated under reduced pressure on rotavapor. Crystallization from acetone gave 192 as yellow powder 3 g, 98%: mp 174- 175°C;1H-NMR (acetone-d6, 300 MHz): 8 8.85-8.84 (d, 1H, J=3 Hz, ArH), 8.46-8.45 (d, 1H, ArH), 7.51-7.49 (br.d, 1H, J=8 Hz, NH), 4.93-4.85 (m, 1H, CH), 3.57-3.51 (dd, 1H, Jvic=5 Hz, Jgem=14 Hz, CH2), 3.17-3.09 (dd, 1H, Jvi0=9 Hz, CH2), 1.84 (s. 3H, CH3) ; 183 MS(EI) m/z 313 (M+), 268 (M+-COOH), 74 (base); Analysis Calcd. for C^H^NaOy: C, 42.18; H, 3.54; N, 13.42. Found: C, 42.19; H, 3.59; N, 13.36. (L)-3.5-Pinitro-o-Tyrosine 168. OH A racemic N-acetyl-3,5-dinitro-o-Tyrosine 1 9 2 (3.0 g, 9.6 mmol) was suspended in distilled H20 (50 mL) 2N KOH (10 mL) was added to make the final solution pH 7.5-8.0 and the solution was diluted with distilled H20 to 100 mL total volume. Aspergillus acylase I (Sigma, 0.47 units/mg, 40 mg) and CoCI 2 (15 mg) were added to the solution. The mixture was stirred on a water bath at 40°C. Progress of the reaction was monitored by assaying aliquots (0.5 or 0.25 mL) for free amino acid on HPLC (Figure 36) Resolution proceeded for 24 h, but reaction was stopped after 27 h. The pH of the reaction mixture was adjusted to =5 with 1N HCI, Norite (100 mg) was added and the mixture was stirred at 40°C for 30 min, filtered over Celite, cooled to r. t. and acidified to pH=1.5-2.0 with 1N HCI. The filtrate was then extracted with EtOAc (3x80 mL) to remove unhydrolyzed starting material (D-N- acetyl-3,5-Dinitro-o-Tyrosine). The aqueous layer was purified by cation exchange column Dowex 50x8 (H+). After loading sample, the column was washed with H20 (2L), and EtOH (500 mL), and the product eluted with 0.3M NH 4OH. The aqueous layer was concentrated under reduced pressure to 5 mL, pH adjusted to =3.5-4.2 , and the product 168 crystallized in form of yellow crystals 540 mg, 41%: mp 235°C decomp, starts, 243- 184 245°C rapid decomposition. 1H-NMR, FT-IR and elemental analysis data same as for 167. [a]D= +4.44°(c 1.92, 0.05 M HCI) N-t-Butyloxycarbonyl-(3.5-dinitro-2-hydroxv1 phenylalanine 194. CH CH To a solution of 164 (1.4 g, 4.2 mmol) in H20 (100 mL) was added 2 N NaOH (30 mL) and THF (150 mL). Di-t-butyldicarbonate (1.1 g, 4.6 mmol) was aded to a vigorously stirred mixture, and stirring continued for 2 h. TLC of the reaction mixture (R p 0.05 (starting material), Rp0.57 (product) on silicagel, on glass using 1% MeOH in CH 2CI2 + 2 drops of AcOH as solvent) shows that reaction has not gone to completion. Additional di-t- butyldicarbonate (2 g, 9 mmol) was added and stirring continued overnight. The mixture was then poured into H20 (200 mL) and extracted with n-hexanes to remove excess of di-t-butyldicarbonate. Water layer was then cooled in an ice-acetone bath (*0-5°C) acidified with 4N HCI to pH = 2 and extracted with EtOAc (3 x 100 mL). Organic extracts were combined, dried over Na 2 S0 4 , filtered and solvent removed under reduced pressure on rotavapor to give yellow powder. Recrystallization from EtOAc/Hex gave beige crystals of 194 1 .5 g (96%): mp 187-189°C; 1H NMR (acetone-d 6 , 300 MHz) 8 8.88-8.87 (d, 1H, J=2.6 Hz, ArH), 8.49-8.48 (d, J= Hz, 1H, ArH), 6.36-6.33 (br. d J=8.5 Hz, 1H, NH), 4.67-4.59 (m, 1H, CH), 3.62-3.56 (dd, J=4 Hz, J=14 Hz, CH2), 3.16-3.08 (m 1H, CH2), 1.27 (s, 9H, 3 XCH3); Analysis Calcd. for: C 14H17 N3O9: C, 45.29; H, 4.61; N, 11.32. Found: C, 45.46; H, 4.66; N, 11.31. 185 N-t-Butyloxycarbonyl-f3.5-dinitro-2-methoxy1 phenylalanine 195. O2N To a suspension of K2C03 (0.4 g, 2.7 mmol) in DMSO (5 mL) was added 194 (0.4 g, 1.1 mmol) followed immediatelly by methyliodide (0.42 g, 2.2 mmol). The mixture was vigorously stirred overnight. At this point TLC shows that reaction has gone to completion (Rp 0.5 (starting material) , R{=0.64 (product) on silica on glass using 1% MeOH in CH2CI2 +2 drops of AcOH as solvent). The mixture was poured into water (50 mL) acidified with 4N HCI while cooling in an ice-acetone bath (0:5°C) and extracted with EtOAc (3x50 mL). Organic extracts were combined, dried over Na2S04 and solvent removed under reduced pressure on rotavapor to give yellow residue. Recrystallization from acetone/hexane gave yellow crystalls 0.298 g (72%): mp 153-154°C;1H NMR (acetone-d6, 300 MHz) 5 8.88-8.87 (d, J=2.8 Hz, 1H, ArH), 8.47-8.46 (d, J=2.8 Hz, 1H, ArH)6.44-6.41 (br d, J=8.8 Hz, 1H, NH), 4.66-4.58 (m, 1H, CH), 3.72 (s, 3H, OCH3), 3.55-3.49 (dd, J=4.8 Hz, 1H, CH2), 3.16-3.80 (m, 1H, CH2), 1.27 (s, 9H, 3xCH3);Analysis Calcd. for C15H19N30 9: C, 46.76; H, 4.97; N, 10.90. Found: C, 46.86; H, 5.03; N, 10.83. 186 3.5-Dinitro-2-methoxy phenylalanine 195. OCH3 O P2N, (P NO2 Compound !3 § (0.6 g, 1.6 mmol) was dissolved in CF 3COOH and stirred at r.t. for 45 min. At this point TLC of the reaction mixture shows there is no starting material left [Rp 0.79 (starting material), Rp0.40 (product)] on siiica on glass using 5% MeOH in CH2CI2 + 2 drops of AcOH as solvent). The mixture was poured into H20 (100 mL) and extracted with EtOAc (3x50 mL). Organic extracts were combined dried over Na 2 S 0 4 filtered and concentrated in vacuo to =5 mL. Triethylamine was added to adjust pH«5 and addition of water afforded orange crystals 0.248 g, 56%: mp 227-228°C (dec.);1H NMR (DMSO-d6l 300 MHz) 8 8.59 (br s, 3H, NH3+), 8.54-8.53 (d, J=3.0 Hz, 1H, ArH), 7.87- 7.86 (d, J=3.0 Hz, 1H, ArH), 4.27 (s, 1H, CH), 3.66 (s, 3H, OCH3), 3.105-3.045 (dd, Jvic=4.2 Hz, J gem=14 Hz, 1H, CH2), 2.93-2.86 (m, 1H, CH2); 13C NMR (DMSO-d6, 300 MHz) 8 169.76 (COOH), 169.72 (ArC), 136.58 (ArC), 131.58 (ArC), 131.01 (ArC), 128.78 (ArC), 127.94 (ArC), 124.06 (ArC), 52.91 (OCH3), 52.19 (CHNH2), 33.47 (CH2); MS(EI) m/z 285 (M+), 8 8 (base). Anal. Calcd. for C^H^NaOy: C, 42.11; H, 3.89; N, 14.73. Found: C, 42.18; H, 3.93; N,14.63. 187 N-t-ButyloxycarbonyI-2-hydroxv phenylalanine 196. CH CH o-Tyrosine 190 (1g, 5.5 mmol) was dissolved in H20 (30 mL) containing 2N NaOH (10 mL), and THF (30 mL) was added. Di-t-butyldicarbonate (1.32 g, 6.1 mmol) was added to the vigorously stirred mixture. Stirring was continued for 5 h the mixture was poured into water (100 mL) extracted with n-hexanes to remove any excess of di-t- butyldicarbonate. Water layer was then acidified with 4N HCI while cooling in an ice- acetone bath (0-5°C) and extracted with EtOAc (3x50 mL). Organic extracts were combined, dried over Na 2 S 0 4 filtered and solvent removed on rotavapor to give white solid. Recrystallization from acetone/hexane gave white crystals of 196 1.41 g, 90.5%; 1H NMR (acetone-de, 300 MHz) 8 7.18-7.15 (dd, J=1.6 Hz, J=7.5 Hz, 1H, ArH), 7.08-7.03 (t, J=7.0 Hz, 1 H, ArH), 6.92-6.70 (m, 2H, ArH), 6.12-6.09 (br d, J=7.0 Hz, 1H, NH), 4.48-4.41 (m, 1 H, CH), 3.24-3.17 (dd, J=4.7, J=13.5 Hz, 1H, CH2), 2.98-2.90 (m, 1H, CH2), 1.33 (s, 9H, 3xCH3); 188 2-Carboxymethoxy phenylalanine 170. HO. • 0.9HCI Finelly powedered K 2CO3 (0.98 g, 7.1 mmol), N-t-butyloxycarbonyl-o-tyrosine 196 (0.8 g, 2.8 mmol) were stirred in acetone at 50°C on an oil bath for 1.5 h. Ethylbromoacetate (0.52 g, 3.1 mmol) was added to the mixture and stirring and heating continued for 3 h. TLC of the reaction mixture at this point shows three spots, one beeing starting material (RpO.17 on silica on glas, solvent Hex/EtOH (8/2)+ 2 drops of AcOH). Additional ethylbromoacetate (0.94 g, 5.6 mmol) was added and heating continued for next 4 h when all starting material dissipeard. The mixture was cooled to r.t. poured into 50 mL of water acidified with 4N HCI (pH=2) and extracted with EtOAc (3x50 mL) Organic extracts were combined concentrated under reduced pressure on rotavapor to give slightly yellow oil. This residue was suspended in 2N HCI (50 mL) refluxed for 24 hours, cooled to r.t. and solvent removed under reduced pressure to give white residue. Recrystallization from acetone/water/Et20 afforded white crystalls 0.203 g (26%): mp 203-204°C; 1H NMR 8.37 (br s 3H, NH3+), 7.26-7.17 (m, 2H, ArH), 6.93-6.85 (m, 2H, ArH), 4.72 (s, 2H, OCH2), 4.19-4.15 (t, 1H, J=6.5 Hz, CH), 3.24-3.17 (dd, Jvic=6.3 Hz, Jgem=14 Hz, 1H, CH2), 3.09-3.05 (m, 1H, CH2); 13C (DMSO-d6, 300 MHz) 8 170.8 (COOH), 170.6 (COOH), 156.2 (ArC), 131.6 (ArC), 129.0 (ArC), 123.5 (ArC), 121.2 (ArC), 111.9 (ArC), 64.9 (OCH2), 52.3 (£HNH2), 31.6 (CH2); MS(EI) m/z 221(M+-H20 ), 107 (base); Anal. Calcd. for CnH^NOsxO.gHCI: C, 48.57; H, 5.15; N, 5.14. Found: C, 48.53; H, 5.22; N, 5.13. 189 Derivatization and Determination of Enantiomeric Excess of L and D-3.5- Dinitro-o-Tyrosine (168 and 167). L-3,5-Dinitro-o-Tyrosine Ig g (100 p i, 5 jimol) was dissolved in 100 pL of 0.5 M NaHC03and 200 pL of 1% solution of Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-L- alanine amide) in acetone was added. The mixture was incubated at 40°C for 90 min, and after cooling 50 pL of 2N HCI was added, and sample allowed to degas. The sample was diluted with EtOH 3x and 10 pL aliquots were used for HLPC injection. The racemic DNoTyr was derivatized in a same fation, and used as a standard. The diastereoisomers were separated by reverse phase HPLC on an ODS column. N-(tert-Butoxycarbonyn-L-Serine-p-Lactone 203 V N H tBoc A three neck round bottomed flask (500 mL) equipped with a magnetic stirrer bar and low temperature thermometer was charged with THF and Ph3P (6.4 g, 24 mmol). After Ph3P has dissolved in THF the mixture was cooled to -78°C while stirring in a dry-ice- acetone bath, under argon atmosphere and DEAD (4.25 g, 24 mmlo) was added dropwise over period of 20 min, keeping the temperature of the mixture around -75°C. The resulting yellow solution was stirred at -75°C to -78°C for 30 min, until mixture become milky slurry. At this point solution of N-tBoc-L-Serine (5 g, 24 mmol) in THF was started adding from a droping funnel and addition continued for additional 60 min. After addition was complete the mixture was stirred at -75°C for 40 min and and then wormed to the r. t. over period of 3 h. Solvent was then removed in vacuo to give yellow oily residue which was suspended in hexane/EtOAc (85/15, 50 mL) to give white solid. This white precipitate (EtOOCNHNHCOOEt) was filtered off, the filtrate diluted with EtOAc (25 mL) and loaded on a silicagel column (120 g). The column was packed in hexane/EtOAc (85/15). The elution was began with hexane/EtOa (85/15) but after 500 mL it was changed to Hexane/EtOAc (65/35). Fractions # 38-48, 20 mL each were combined concentrated in vacuo to give white powder of lactone 203. 1.82 g (40%) m.p. 119-120°C (lit. m.p. 119.5-120.5°C); 1H NMR (CDCI3, 300 MHz) S 5.48-5.45 (br. d, J=7.45 Hz, 1H, NH), 5.13-5.07 (br. q, J 1=5.94 Hz, J«>=:12.4 Hz, 1 H, CH) 4.49-4.36 (m, 2H, CH2) 1.46 (s, 9H, 3 x CH3); FT-IR (KBr, cm-1) 3361 /NHCQ streching), 1838 ( lacton C=Q. streching), 1678 (NHCO. streching); [a]D25= -26.0° (c 1, CH 3CN) (lit. [a]D25= -26.2° (c 1, CH3CN). 2-(N-tert-butvloxvcarbonylamineV3-fN-(2,-aminophenvnamine1 ■REOPiPniC figiti.2P7 NHtBoc To a solution of 1,2-phenylendiamine (1.35 g, 12.5 mmol) in THF (25 mL) was added solution of p-lactone 203 (1.17 g, 6.24 mmol) in THF (25 mL) via dropping funnel over period of 45 min. The mixture was stirred at r.t. for 3 days (color of the reaction mixture changes from pale yellow to deep orange). Progress of the reaction was monitored by FT-IR spectrometry. After 3 days IR spectrum shows that all p-lactone 203 has been consumed (since peak at v=1845 cm-1 lactone C=0 group had desepeared). The solvent (THF) was removed on rotavapor to give yellow residue which was chromatographed on silicagel column (mobile phase 3% MeOH in CH 2CI2first 1 0 0 0 mL 191 and then 5% MeOH in CH 2CI2 aditional 1000 mL). Fractions # 43-74 containing product, each 20 mL, were combined solvent removed in vacuo to give 207. Recrystallization from MeOH/Et20 gave 0.84 g (46%) of 207 mp 157-158°C; 1H NMR (Acetone-d6, 300 MHz) 8 8.62 (s, 1H, NHCO), 7.23-7.19 (dd, 1H, ArH), 6.97-6.92 (m, 1H, ArH), 6.79-6.76 (dd, 1H, ArH), 6.62-6.57 (m, 1H, ArH), 6.15 (s, 1H, NHCOO), 4.55 (s, 2H, (J)NH2), 4.34-4.26 (m, 1H, NHCH), 3.98-3.91 (m, 1H, Ctfe), 3.85-3.80 (m, 1H, CH2), 1.43 (s, 9H, 3 x CH3); FT-IR (KBr, crrH) 3392 (N-H streching), 3247 (N-H streching), 1670 (C=0 streching), 1629 (C=0 streching); MS(EI) m/z 295(M+), 278(M+-OH), 135(C6H4(NH2)NHCO), 108 (C6 H4 (NH2)NH2+);Anal. Calcd. for C 14H21N30 4; C, 56.94; H, 7.17; N, 14.23; found: C, 56.90; H, 7.23; N, 14.14. BIBLIOGRAPHY (1) Lefkowitz, R. J.; Hoffmann, B. B.; Taylor, P. Neurohumoral Transmission: The Autonomic and Somatic Motor Nervous System. In The Pharmacological Basis of Therapeutics, 7th ed.;Gilman, A. G., Goodman, L. S., Rail, T. W., Murad, F. Eds.; Macmillan Publishing Co.: New York, 1985; pp 66-99. (2) Robinson, R. L. Introduction to the Autonomic Nervous System. In Modern Pharmacology, Craig, C. R., Stitzel, R. E., Eds.; Little, Brown and Company: Boston, 1982; pp 99-111. (3) Pepperl, D. J.; Regan, J. W. Adrenergic Receptors. In Handbook of Receptors and Channels: G-Protein Coupled Receptors; Peroutka, S. J., Ed.; CRC Press: Ann Arbor, 1994; pp 45-78. (4) Ahlquist, R. P. A Study of the Adrenotropic Receptors. Am. J. Physiol. 1948, 153, 586-600. (5) Lands, A. M.; Arnold, A.; McAuliff, J. P.; Luduena, F. P.; Brown, T. G. J. Differentiation of Receptor Systems Activated by Sympathomimetic Amines. Nature 1967, 214, 597-598. (6 ) Langer, S. Z. Presynaptic Regulation of Catecholamine Release. Biochem. Pharmacol. 1974, 23 ,1793-1800. (7) Berthelsen, S.; Pettinger, W. A. A Functional Basis for Classification of a- Adrenergic Receptors. Life Sci. 1977, 21,595-606. (8 ) Starke, K. a-Adrenoceptor Subclassification. Rev. Physiol. Biochem. Pharmacol. 1981, 8 8199-236. , (9) Vizi, E. S. Compounds Acting on Alphar and Alpha 2-Adrenoceptors: Agonists and Antagonists. Med. Res. Rev. 1986, 6 ,431-449. (10) Minneman, K. P. aiAdrenergic Receptor Subtypes, Inositol Phosphates and Sources of Cell Ca 2+. Pharmacol. Rev. 1988,4 0 , 87-119. (11) McGrath, J. C. Evidence for More then One Type of Postjunctional a- Adrenoceptor. Biochem. Pharmacol. 1982, 31,467-484. (12) Morrow, A. L.; Creese, I. Characterization of -Adrenergic Receptor Subtypes in Rat Brain: A Reevaluation of [3H]WB4101 and pHjPrazosin Binding. Mol. Pharmacol. 1986, 29,321-330. (13) Johnson, R. D.; Minneman, K. P. Differentiation of -Adrenergic Receptors Linked 192 193 to Phosphatidylinositol Turnover and Cyclic AMP Accumulation in Rat Brain. Mol. Pharmacol. 1987, 31,239-246. (14) Han, C.; Abel, P. W.; Minneman, K. P. Heterogeneity of apAdrenergic Receptors Revealed by Chlorethylclonidine. Mol. Pharmacol. 1987, 32,505-510. (15) Minneman, K. P.; Han, C.; Abel, P. W. Comparison of apAdrenergic Receptor Subtypes Distinguished by Chlorethylclonidine and WB4101. Mol. Pharmacol. 1988, 33,509-514. (16) Bylund, D. B. Heterogeneity of Alpha-2 Adrenergic Receptors. Pharmacol. Biochem. Beh. 1985, 22, 835-843. (17) Nahorski, S. R.; Barnett, D. B.; Cheung, Y. D. a-Adrenoceptor-Effector Coupling: Affinity States of Heterogeneity of the a 2-Adrenoceptor? Clin. Science 1985, (Suppl.10), 39S-42S. (18) Bylund, D. B.; Ray-Prenger, C.; Murphy, T. J. Alpha-2A and Alpha-2B Adrenergic Receptor Subtypes: Antagonist Binding in Tissues and Cell Line Containing Only One Subtype. J. Pharmacol. Exp. Ther. 1988, 245, 600-607. (19) Bylund, D. B. Subtypes of a 2-Adrenoceptors: Pharmacological and Molecular Biological Evidence Converge. TIPS 1988, 9 ,356-361. (20) Murphy, T. J.; Bylund, D. B. Characterization of Alpha-2 Adrenergic Receptors in the OK Cell, an Opossum Kidney Cell Line. J. Pharmacol. Exp. Ther. 1987, 244, 571-578. (21) Michel, A. D.; Loury, D. N.; Whiting, R. L. Differences Between the a 2- Adrenoceptor in Rat Submaxillary Gland and the a 2A- and a 2e-Adrenoceptor Subtypes. Br. J. Pharmacol. 1989, 9 8890-897. , (22) Cotecchia, S.; Schwin, D. A.; Randall, R. R.; Lefkowitz, R. J.; Caron, M. G.; Kobilka, B. K. Molecular Cloning and Expression of the cDNA for the Hamster ap Adrenergic Receptor. Proc. Natl. Acad. Sci. USA 1988, 85,7159-7163. (23) Schwin, D. A.; Lomasney, J. W.; Lorenz, W.; Szklut, P. J.; Fremean, R. T.;Yang- Feng, T. L.;Caron, M. G.; Lefkowitz, R. J.; Cotecchia, S. Molecular Cloning and Expression of the cDNA for a Novel a!-Adrenergic Receptor Subtype. J. Biol. Chem. 1990, 265, 8183-8189. (24) Bruno, J. F.; Whittaker, J.; Song, J.; Berelowitz, M. Moleculat Cloning and Sequencing of a cDNA Encoding a Human au-Adrenergic Receptor. Biochem. Biophys. Res. Comm. 1991, 179,1485-1490. (25) Lomasney, J. W.; Cotecchia, S.; Lorenz, W.; Leung, W. Y.; Schwinn, D. A.; Yang- Feng, T. L.; Brownstein, M.; Lefkowitz, R. J.; Caron, M. G. Molecular Cloning and Expression of the cDNAforthe apAdrenergic Receptor. J. Biol. Chem. 1991,266, 6365-6369. (26) Lomasney, J. W.; Cotecchia, S.; Lefkowitz, R. J.; Caron, M. G. Molecular Biology of a-Adrenergic Receptors: Implications for Receptor Classification and for Structure-Function Relationship. Biochem. Biophys. Acta 1991, 1095,127-139. (27) Regan, J. W.; Nakata, H.; DeMarinis, R. M.; Caron, M. G.; Lefkowitz, R. J. 194 Purification and Characterization of the Human Platelet (^-Adrenergic Receptor. J. Biol. Chem. 1986, 261,3894-3900. (28) Kobilka, B. K.; Matsui, H.; Kobiika, T. S.; Yang-Feng, T. L.; Francke, U.; Caron, M. G.; Lefkowitz, R. J.; Regan, J. W. Cloning, Sequencing and Expression of the Gene Coding for the Human Platelet a2-Adrenergic Receptor. Science 1987, 238, 650-656. (29) Regan, J. W.; Cobilka, T. S.; Yung-Feng, L. T.; Caron, M. G.; Lefkowitz, R. J.; Kobilka, B. K. Cloning and Expression of a Human Kidney cDNA for an a2- Adrenergic Receptor Subtype. Proc. Natl. Acad. Sci. USA 1988, 85, 6301-6305. (30) Bylund, D. B.; Blaxall, H. S.; Iversen, L. J.; Caron, M. G.; Lefkowitz, R. J.; Lomasney, J. W. Pharmacological Characteristics of a2-Adrenergic Receptors: Comparison of Pharmacologically Defined Subtypes with Subtypes Identified by Molecular Cloning. Mol. Pharmacol. 1992,4 2, 1-5. (31) Lefkowitz, R. J.; Caron, M. G. Adrenergic Receptors. J. Biol. Chem. 1988, 263, 4993-4996. (32) Dohlman, D. B.; Caron, M. G.; Lefkowitz, R. J. A Family of Receptors Coupled to Guanine Nucleotide Regulatory Proteins. Biochemisty 1987, 2 6 ,2657-2664. (33) Leeb-Lundberg, L. M. F.; Dickinson, K. E. J.; Heald, S. L.; Wikberg, J. E. S.; Hagen, P. O.; DeBernardis, J. F.; Winn, M.; Arendsen, D. L.; Lefkowitz, R. J.; Caron, M. G. Photoaffinity Labeling of Mammalian ai-Adrenergic Receptors. J. Biol. Chem. 1984, 259,2579-2587. (34) Regan, J. W.; DeMarinis, R. M.; Caron, M. G.; Lefkowitz, R. J. Identification of the Subunit-Binding Site of a2-Adrenergic Receptors Using [3H]Phenoxybenzamine. J. Biol. Chem. 1984, 259, 7864-7869. (35) Wang, C. D.; Buck, M. A.; Fraser, C. M. Site-Directed Mutagenesis of a 2- Adrenergic Receptors: Identification of Amino Acids Involved in Ligand Binding and Receptor Activation by Agonists. Mol Pharmacol. 1991, 4 0 , 168-179. (36) Suryanarayana, S.; Daunt, D. A.; Von Zastrow, M.; Kobilka, B. K. A Point Mutation in the Seventh Hydrophobic Domain of the a2-Adrenergic Receptor Increases its Affinity for a Family of (3-Receptor Antagonists. J. Biol. Chem. 1991,2 66,15488- 15492. (37) Summers, R. J.; McMartin, L. R.; Adrenoceptors and their Second Messenger Systems. J. Neurochem. 1993, 6 0 ,10-23. (38) Han, C.; Abel, P. W.; Minneman, K. P. -Adrenoceptor Subtypes Linked to Different Mechanisms for Increasing Intracellular Ca 2+ in Smooth Muscle. Nature 1987, 329,333-335. (39) Limbird, L. E. Receptors Linked to Inhibition of Adenylate Cyclase: Additional Signaling Mechanism. FASEBJ. 1988, 2 ,2686-2695. (40) Rosenthal, W.; Hescheler, J.; Trautwein, W.; Schultz, G. Contorl of Voltage- Dependent Ca2+Channels by G-Protein-Coupled Receptors. FASEBJ. 1988, 2, 2784-2790. 195 (41) Nichols, A. J.; Motley, E. D.; Ruffolo, R. R. J. Effect of Pertussis Toxin Treatment on Postjunctional Alpha -1 and Alpha-2 Adrenoceptor Function in the Cardiovascular System of the Pithed Rat. J. Pharmacol. Exp. Ther. 1989, 249, 203-209. (42) Nichols, A. J. a-Adrenoceptor Signal Transduction Mechanisms. In a- Adrenoceptors: Molecular Biology, Biochemistry and Pharmacology.; Ruffolo, R. R., Ed.; Karger: Basel, 1991; pp 44-74. (43) Bolton, T. B. Mechanism of Action of Transmitters and Other Substances on Smooth Muscle. Physiological Rev. 1979, 59, 606-718. (44) Triggle, D. J.; Langs, D. A.; Janis, R. A. Ca 2+ Channel Ligands: Structure-Function Relationships of the 1,4-Dihydropyridines. Med Res. Rev. 1989, 9 ,123-180. (45) Janis, R. A.; Silver, P. J.; Triggle, D. J. Drug Action and Cellular Calcium Reglation. Advances in Drug Res. 1987, 16,309-523. (46) Janis, R. A.; Triggle, D. J. Drugs Acting on Calcium Channels. In Calcium Channels: Their Properties, Functions, Regulation and Clinical Relevance.; Hurwitz, L., Partridge, L. D., Leach, J. K., Eds.; CRC Press Inc.: Ann Arbor, 1991; pp197-249. (47) Tsien, R. W.; Ellinor, P. T.; Home, W. A. Molecular Diversity of Voltage Dependent Ca2+Channels. TIPS 1991, 12,349-354. (48) Hullin, R.; Biel, M.; Flockerzi, V.; Hofmann, F. Tissue-Specific Expression of Calcium Channels. Trends Cardiovas. Med. 1993, 3 ,48-53. (49) MacDermott, A.; Miles, R. Calcium Channels and Patterns of Electrical Activity in Neurons. In Calcium Channels: Their Properties, Functions, Regulation and Clinical Relevance.] Hurwitz, L., Partridge, L. D., Leach, J. K., Eds.; CRC Press Inc.: Ann Arbor, 1991 ;pp126-135. (50) Rampe, D.; Triggle, D. J. New Synthetic Ligands for L-Type Voltage-Gated Calcium Channels. In Progress in Drug Research.; Jucker, E., Ed.; Birkhauser Verlag: Basel, 1993; pp 192-238. (51) Janis, R. A.; Triggle, D. J. New Developments in Ca2+ Channel Antagonists. J. Med. Chem. 1983, 26,775-785. (52) Mannhold, R. Molecular Pharmacology of Calcium Antagonists. In Recent Advances in Receptor Chemistry; MeTchiorre, C., Giannella, M., Ed.; Elsevier Science Publishers B. V.: Amsterdam, 1988; pp147-171. (53) Gualtieri, F.; Teodori, E.; Bellucci, C.; Pesce, E.; Piacenza, G. SAR Studies in the Field of Calcium (II) Antagonists. Effect of Modifications at the Tetrasubstituted Carbon of Verapamil like Compounds. J. Med. Chem. 1985, 2 8 ,1621-1628. (54) Dei, S.; Romanelli, M. N.; Scapecchi, S.; Teodori, E.; Chiarini, A.; Gualtieri, F. Verapamil Analogues with Restricted Molecular Flexibility. J. Med. Chem. 1991, 34, 2219-2225. (55) Floyd, D. M.; Kimball, S. D.; Krapcho, J.; Das, J.; Turk, C. F.; Moquin, R. V.; Lago, M. W.; Duff, K. J.; Lee, V. G.; White, R. E.; Ridgewell, R. E.; Moreland, S.; Brittain, R. J.; Normandin, D. E.; Hedberg, S. A.; Cucinotta, G. G. Benzazepinone Calcium Channel Blockers.2.Structure-Activity and Drug Metabolism Studies Leading to Potent Antihypertensive Agents. Comparison with Benzathiazepinones. J. Med. Chem. 1992, 35,756-772. Das, J. J.; Floyd, D. M.; Kimball, S. D.; Duff, K. J.; Vu, T. C.; Lago, M. W.; Moquin, R. V.; Lee, V. G.; Gongontas, J. Z.; Malley, M. F.; Moreland, S.; Brittain, R. J.; Hedberg, S. A.; Cucinotta, G. G. Benzazepinone Calcium Channel Blockers.3. Synthesis and Structure activity Studies of 3-Alkylbenzazepinones. J. Med. Chem. 1992, 35,773-780. Kimball, S. D.; Floyd, D. M.; Das, J.; Hunt, J. T.; Krapcho, J.; Rovnyak, G.; Duff, K. J.; Lee, V. G.; Moquin, R. V.; Turk, C. F.; Hedberg, S. A.; Moreland, S.; Britain, R. J.; McMullen, D. M.; Normandin, D. E.; Cucinotta, G. G. Benzazepinone Calcium Channel Blockers.4. Structure-Activity Overview and Intracellular Binding Site. J. Med. Chem. 1992, 35,780-793. Becham, M.; Hebisch, S.; Schramm, M. Ca 2+ Agonists: New Sensitive probes for Ca2+ channels. TIPS 1988, 9 ,257-261. Franckowiak, G.; Bechem, M.; Schramm, M.; Thomas, G. The Optical Isomers of 1,4-Dihydropyridine Bay K 8644 Show opposite Effects on Calcium Channels. Eur. J. Pharmacology 1985, 114, 223-226. Catterall, W. A. Functional Subunit Structure of Voltage-Gated Calcium Channels. Science 1991,253,1499-1500. Tanabe, T.; Takeshima, H.; Mikami, A.; Flockerzi, V.; Takanashi, H.; Kangawa, K.; Kojima, M.; Matsuo, H.; Hirose, T.; Numa, S. Primary Structure of the Receptor for Calcium Channel Blocker from Skeletal Muscle. Nature 1987, 328,313-318. Catterall, W. A.; Seagar, M. J.; Takahashi, M. Molecular Properties of Dihydropyridine-Sensitive Calcium Channels in Skeletal Muscle. J. Biol. Chem. 1988, 263, 3535-3538. Theodore, L. J.; Nelson, W. L.; Zobrist, R. H.; Giacomini, K. M.; Giacomini, J. C. Studies on Ca2+ Channel Antagonists. 5[(3,4-Dimethoxyphenethyl)rnethylamino]- 2-(3,4-dimethoxyphenyl)-2-isopropylpentyl Isothiocyanate, a Chemoaffinity Ligand Derived from Verapamil. J. Med. Chem. 1986, 2 9 ,1789-1792. Striessnig, J.; Knaus, H. G.; Grabner, M.; Moosburger, K.; Steitz, W.; Lietz, H.; Glossmann, H. Photoaffinity Labelling of the Phenylalkylamine Receptor of the Skeletal Muscle Transverse-Tubule Calcium Channel. FEBS Lett. 1987, 212,247- 253. Sieber, M.; Nastaiczyk, W.; Zubor, V.; Wernet, W.; Hoffmann, F. The 165 kDa Peptide of the Purified Skeletal Muscle Dihydropyridine Receptor Contains the Known Regulatory Site of the Calcium Channel. Eur. J. Biochem. 1987, 167,117- 122. Catterall, W. A.; Striessnig, J. Receptor Sites for Ca2+ Channel Antagonists. TIPS 1992, 13,256-262. Nakayama, H.; Taki, M.; Striessnig, J.; Grossmann, H.; Catteral, W. A.; Kanaoka, Y. Identification of 1,4-Dihydropyridine Binding Regions within the ai-Subunit of Skeletal Muscle Ca 2+ Channels by Photoaffinity Labeling with Diazepine. Proc. 197 Natl. Acad. Sci. USA 1991, 88,9203-9207. (6 8 ) Striessnig, J.; Murphy, B. J.; Catterall, W. A. Dihydropyridine Receptor of a-Type Ca2+ Channels: Identification of Binding domains for [3H](+)-PN 200-110 and pH]Azidopine within the ar Subunit. Procid. Natl. Acad. Sci. USA 1991, 88,10769- 10773. (69) Ruffolo, R. R. J.; Turowski, B. S.; Patil, P. N. Lack of Cross-desenzitization Between Structurally Dissimilar a-Adrenoceptor Agonists. J. Pharm. Pharmac. 1977, 2 9 ,378-380. (70) Easson, I. H.; Stedman, E. Studies on the Relationship between Chemical Constitution and Physiological Activity. Biochem. J. 1933,2 7 ,1257-1266. (71) Ruffolo, R. R. J.; Rice, P. J.; Patil, P. N.; Hamada, A.; Miller, D. D. Differences in the Applicability of the Easson-Stedman Hypothesis to the a! and ct 2-Adrenergic Effects of Phenethylamines and Imidazolines. Eur. J. Pharmacol. 1983, 86, 471- 475. (72) Nichols, A. J.; Ruffolo, R. R. J. Structure-Activity Relationships for a-Adrenoceptor Agonists and Antagonists. In a -Adrenoceptors: Molecular Biology, Biochemistry and Pharmacology; Ruffolo, R. R., Ed.; Karger: Basel, 1991; pp 75-114. (73) Ruffolo, R. R. J.; Yaden, E. L.; Waddell, J. E.; Dillard, R. D. Receptor Interactions of Imidazolines.VI. Significance of Carbon Bridge Separating Phenyl and Imidazoline Rings of Tolazoline-Like Alpha Adrenergic Imidazolines. J. Pharmacol. Exp. Ther. 1980, 214, 535-540. (74) Kobinger, W. Central a-Adrenergic Systems as Targets for Antihypertensive Drugs. Rev. Physiol. Biochem. Pharmacol. 1978, 81,40-100. (75) Bousquet, P. T.; Fedman, J.; Tibirica, E.; Bricca, G.; Molines, A.; Dontenwill, M.; Belcourt, A. New Concepts on the Central Regulation of Blood Pressure. Am. J. Med. 1989, 87(Suppl.3C), 3C-10S. (76) Bousquet, P.; Feldman, J.; Bloch, R.; Schwartz, J. The Nucleus Reticularis Latteralis: A Region Highly Sensitive to Clonidine. Eur. J. Pharmacol. 1981, 69, 389-392. (77) Bousquete, P.; Feldman, J.; Velly, J.; Bloch, R. Role of the Ventral Surface of the Brain Stem in the Hypothensive Action of Clonidine. Eur. J. Pharmacol. 1975, 34, 151-156. (78) Bousquet, P.; Feldman, J.; Schwartz, J. Central Cardiovascular Effects of Alpha Adrenergic Drugs: Differences between Catecholamines and Imidazolines. J. Pharmacol. Exp. Ther. 1984, 230,232-236. (79) Ernsberger, P.; Giuliano, R.; Willette, R. N.; Granata, A. R.; Reis, D. J. Hypothensive Action of Clonidine Analogues Correlates with Binding Affinity at Imidazole and not Alpha-2-Adrenergic Receptors in the Rostal Ventrolateral Medulla. J. Hyperten. 1988, 6(suppl 4.), S554-S557. (80) Ernsberger, P.; Meeley, M. P.; Mann, J. J.; Reis, D. J. Clonidine Binds to Imidazole Binding Sites as well as a2-Adrenoceptors in the Ventrolateral Medulla. Eur. J. Pharmacol. 1987, 134, 1-13. Boyajian, C. L.; Leslie, F. M. Pharmacological Evidence for Alpha-2 Adrenoceptor Heterogeneity: Differential Binding Properties of pHJIdazoxan in Rat Brain. J. Pharmacol. Exp. Ther. 1987, 241, 1092-1098. Boyajian, C. L.; Longhlin, S. E.; Leslie, F. M. Anatomical Evidence for Alpha-2 Adrenoceptor Heterogeneity: Differential Autoradiographic Distribution of PHjRauwolscine and pHJIdazoxan in Rat Brain. J. Pharmacol. Exp. Ther. 1987, 241, 1079-1091. Coupry, I.; Podevin, R. A.; Dausse, J. P.; Parini, A. Evidence for Imidazoline Binding Site in Basolateral Membranes from Rabbit Kidney. Biochem. Biophys. Res. Comm. 1987, 147, 1055-1060. Michel, M. C.; Regan, J. W.; Gerhardt, M. A.; Neubig, R. R.; Insel, P. A.; Motulsky, H. J. Nonadrenergic pHjldazoxan Binding Sites Are Physically Distinct from a2- Adrenergic Receptors. Mol. Pharmacol. 1989, 37,65-68. Bricca, G.; Dontenwill, M.; Molines, A.; Feldman, J.; Belcourt, A.; Bousquet, P. Evidence for the Existence of a Homogenious Population of Imidazoline Receptors in the Human Brainstem. Eur. J. Pharmacol. 1988, 150,401-402. Bricca, G.; Dontenwill, M.; Molines, A.; Feldman, L.; Belcourt, A.; Bousquet, P. The Imidazoline Preferring Receptor: Binding Studies in Bovine, Rat and Human Brainstem. Eur. J. Pharmacol. 1989, 162,1-9. Tibirica, E.; Feldman, J.; Mermet, C.; Gonon, F.; Bousquet, P. An Imidazoline- Specific Mechanism for the Hypotensive Effect of Clonidine: A Study with Yohimbine and Idazoxan. J. Pharmacol. Exp. Ther. 1991,256,606-613. Michel, M. C.; Regan, J. W.; Gerhardt, M. A.; Neubig, R. R.; Insel, P. A.; Motulsky, H. J. Non-Adrenergic [3H] Idazoxan Binding Sites are Physically Distinct from a2- Adrenergic Receptors. Mol. Pharmacol. 1990, 37,65-68. Zonnenschein, R.; Diamant, S.; Atlas, D. Imidazoline Receptors in Rat Liver Cell: a Novel Receptor or a Subtype of c^-Adrenoceptors? Eur. J. Pharmacol. 1990, 190, 203-215. Lachaud-Pettiti, V.; Podevin, R. A.; Chretien, Y.; Parini, A. Imidazoline-Guanidinium and a2-Adrenergic Binding Sites in Basolateral Membranes from Human Kidney. Eur. J. Pharmacol. 1991,2 0 6 ,23-31. Tesson, F.; Parini, A. Identification of an Imidazoline-Guanidinium Receptive Site in Mitochondria from Rabbit Cerebral Cortex. Eur. J. Pharmacol. 1991,208,81-83. Atlas, D.; Burstein, Y. Isolation and Partial Purification of a Clonidine-Displacing Endogenous Brain Substance. Eur. J. Biochem. 1984, 144, 287-293. Atlas, D.; Burstein, Y. Isolation of an Endogenous Clonidine-Displacing Substance from Rat Brain. FEBS Lett. 1984, 170,387-391. Meeley, M. P.; Ernsberger, P. R.; Granata, A. R.; Reis D. J. An Endogenous Clonidine-Displacing Substance from Bovin Brain: Receptor Binding and Hypotensive Actions in the Ventrolateral Medulla. Life. Sci. 1986, 3 8 ,1119-1126. Atlas, D.; Diamond, S.; Fales, H. M.; Pannell, L. The Brain’s Own Clonidine: 199 Purification and Characterization of Endogenous Clonidine Displacing Substance from Brain. J. Cardiovas. Pharmacol. 1987, 10 (suppl. 12), S122-S127. (96) Bousquet, P.; Feldman, J.; Atlas, D. An Endogenous, Non-Catecholamine Clonidine Antagonist Increases Mean Arterial Blood Pressure. Eur. J. Pharmacol. 1986, 124, 167-170. (97) Michel, M. C.; Ernsberger, P. Keeping an Eye on the I Site: Imidazoline-Preferring Receptors. TIPS 1992, 13,369-370. (98) Atlas, D. Clonidine-Displacing Substance (CDS) and its Putative Imidazoline Receptor. Biochem. Pharmacol. 1991, 4 1 , 1541-1549. (99) Li, G.; Regunathan, S.; Barrow, C. J.; Eshraghi, J.; Cooper, R.; Reis, D. J. Agmatine: An Endogenous Clonidine-Displacing Substance in the Brain. Science 1994, 263, 966-969. (100) Coupry, I.; Atlas, D.; Uzilli, I.; Lanier, S. M.; Parini, A. Rabbit Renal Imidazoline/Guanidinium Receptive Substance (IGRS): Ligand Recognition Properties and Interaction with and Endogenous Displacing Substance. FASEB J. 1989, 2, A1187. (101) Coupry, I.; Atlas, D.; Podevin, R. A.; Uzielli, I.; Parini, A. Imidazoline-Guanidinium Receptive Site in Renal Proximal Tubule: Asymmetric Distribution, Regulation by Cations and Interaction with an Endogenous Clonidine Displacing Substance. J. Pharmacol. Exp. Ther. 1989, 252,293-299. (102) Michel, M. C.; Insel, P. A. Are there Multiple Imidazoline Binding Sites. TIPS 1989, 10, 342-344. (103) Reis, D. J.; Regunathan, S.; Wang, H.; Feinstein, D. L.; Meeley, M. P. Imidazoline Receptors in the Nervous System. Fund. Clin. Pharmacol. 1992, 6(Suppl.1), 23S- 29S. (104) Ernsberger, P.; Damon, T. H.; Graff, L. M.; Schafer, S. G.; Christen, M. O. Moxonidine, a Centrally Acting Antihypertensive Agent, is a Selective Ligand for l-i-lmidazoline Sites. J. Pharmacol. Exp. Ther. 1993,264, 172-182. (105) MacKinnon, A. C.; Stewart, M.; Olverman, H. J.; Spedding, M.; Brown, C. M. [3H]p-Aminoclonidine and pHJIdazoxan Label Different Population of Imidazoline Sites on Rat Kidney. Eur. J. Pharmacol. 1993,232,79-87. (106) Diamant, S.; Eldar-Geva, T.; Atlas, D. Imidazoline Binding Sites in Human Placenta: Evidence for Heterogeneity and a Search for Physiological Function. Br. J. Pharmacol. 1992, 106,101-108. (107) Mirrales, A.; Olmos, G.; Sastre, M.; Barturen, F.; Martin, I.; Garcia-Sevilla, J. A. Discrimination and Pharmacological Characterization of l2-lmidazoline Sites with [3H]ldazoxan and Alpha-2-Adrenoceptors with [3H]RX821002 (2-Methoxy Idazoxan) in the Human and Rat brains. J. Pharmacol. Exp. Ther. 1993, 264, 1187-1197. (108) Parini, A.; Coupry, I.; Graham, R. M.; Uzielli, I.; Atlas, D.; Lanier, S. M. Characterization of an Imdazoline/Guanidinium Receptive Site Distinct from the a2- Adrenergic Receptor. J. Biol. Chem. 1989, 264, 11874-11878. 200 (109) Tesson, F.; Prip-Buus, C.; Lemoine, A.; Pegorier, J. P.; Parini, A. Subcellular Distribution of Imidazoline-Guanidinium Receptive Sites in Human and Rabbit Liver. J. Biol. Chem. 1991,266,155-160. (110) Limon, I.; Coupry, I.; Lanier, S. M.; Parini, A. Purification and Characterization of Mitochondrial Imidazoline-Guanidinium Receptive Site from Rabbit Kidney. J. Biol. Chem. 1992,267,21645-21649. (111) Wang, H.; Regunathan, S.; Meeley, M. P.; Reis, D. J. Isolation and Characterization of Imidazoline Receptor Protein from Bovine Adrenal Chromaffin Cells. Mol. Pharmacol. 1992,42, 792-801. (112) Lanier, S. M.; Ivkovic, B.; Singh, I.; Neumeyer, J. L.; Bakthavachalam, V. Visualization of Multiple Imidazoline Guanidinium Receptive Sites. J. Biol. Chem. 1993,258,16047-16051. (113) Leclerc, G.; Rout, B.; Schwartz, J.; Velly, J.; Wermuth, C. G. Studies on some p- Substituted Clonidine Derivatives that Exhibit and a-Adrenoceptor Stimulant Activity. Br. J. Pharmacol. 1980, 71,5-9. (114) Atlas, D.; Plotec, Y.; Miskin, I. An Affinity Label for a2-Adrenergic Receptors in Rat Brain. Eur. J. Biochem. 1982, 126,537-541. (115) Decker, N.; Leclerc, G.; Quennedey, M. C.; Rouot, B.; Schwartz, J. Study of Two Alkylating Derivatives: the p-lsothiocyanato and the p-Methylisothiocyanato Clonidine. Eur. J. Pharmacol. 1983, 93,205-211. (116) Matsui, H.; Lefkowitz, R. J.; Caron, M. G.; Regan, J. W. Localization of the Fourth Membrane Spanning Domain as a Ligand Binding Site in the Human Platelet 0 2 - Adrenergic Receptor. Biochemistry 1989, 28,4125-4130. (117) Venkataraman, B. V.; Hamada, A.; Shams, G.; Miller, D. D.; Feller, D. R.; Patil, P. N. Paradoxical Effects of Isothiocyanate Analog of Tolazoline on Rat Thoracic Aorta and Human Platelets. Blood Vessels 1989, 26,335-346. (118) Lei, L. P.; Slavica, M.; Romstedt, K.; Patil, P. N.; Miller, D. D.; Feller, D. R. Non-a- Adrenergic Actions of 2-(4'-lsothiocyanatobenzyl)imidazoline (IBI) Analogs in Rat Aorta. Pharmacologist'll, 34,148. (119) Hussain, J. F.; Miall, F.; Patil, P. N.; Miller, D. D.; Kendall, D. A.; Wilson, V. G. Isothiocyanate Tolazoline Interacts with Non-Adrenoceptor and Imidazoline Binding Sites Labeled by PH]ldazoxan. Fund. Clin. Pharmacol. 1992, 6 ,54S. (120) Cavallini, G.; Milla, E.; Grumelli, E.; Massarani, E.; Nardi, D. Sopra Nuove 2- Benzil-lmidazoline Sostitute Farmacologicamente Attive. II Farmaco Ed. Sci. 1956, 11,633-661. (121) Nielson, D. G. In The Chemistry ofAmidines and Imidanes ; Patai, S., Ed.; John Wiley and Sons.: 1975; pp (122) Rice, K. C.; Brossi, A.; Tallman, J.; Paul, S. M.; Scolnick, P. Irazepine, a Noncompetitive, Irreversible Inhibitor of 3H-Diazepam Binding to Benzodiazepine Receptors. Nature 1979, 278,854-855. (123) Resec, J. F.; Ruoho, A. E. Photoaffinity Labeling the p-Adrenergic Receptor with an lodoazido Derivative of Norepinephrine. J. Biol. Chem. 1988, 2 6 3 ,14410- 14416. (124) Sonn, A. U.S. Patent 2161938. Chem. Abstr. 1939, 33 (125) Hach, V.; Protiva, M. Synthesis in the Estrogenic Hormone Group.XV. A Synthesis of 1,4-Hydrindandione. Chem. Abstr. 1958, 5 2 ,5310b (126) Grochowski, E.; Eckstein, Z. Synthesis of some 2,2,2-Trichloro-1,1-bis(N- Phenoxyacetamido)ethane Derivatives. Chem. Abstr. 1964, 60,2815e (127) Tetsuo, O.; Tsutomu, O.; Kinichi, I.; Takuji, S.; Yasuo, S.; Isao, A. Insecticides and Acaricides. Chem. Abstr. 1978, 88,100360c (128) Neef, G.; Eder, U.; Sauer, G. One Step Conversions of Esters to 2-Imidazolines, Benzimidazoles and Benzothiazoles by Aluminum Organic Reagents. J. Org. Chem. 1981, 46, 2824-2826. (129) Hiasta, D. J.; Luttinger, D.; Perrone, M. H.; Silbernagel, M. J.; Ward, S. J.; Haubrich, D. R. o^-Adrenergic Agonists/Antagonists: The Synthesis and Structure- Activity Relationship of a Series of lndolin-2-yl and Tetrahydroquinolin-2-yl Imidazolines. J. Med. Chem. 1987, 3 0 ,1555-1562. (130) Amemiya, Y.; Miller, D. D.; Hsu, F. L. Dehydrogenation of Imidazolines to Imidazoles with Pd-Carbon. Synth. Comm. 1990,2 0 ,2483-2489. (131) Amemiya, Y.; Hong, S. S.; Venkataraman, V.; Patil, P. N.; Shams, G.; Romstedt, K.; Feller, D. R.; Hsu, F. L.; Miller, D. D. Synthesis and a-Adrenergic Activities of 2- and 4-Substituted Imidazoline and Imidazole Analogues. J. Med. Chem. 1992, 35, 750-755. (132) Hughey, J. L.; Knapp, S.; Schughar, H. Dehydrogenation of 2-lmidazolines to Imidazoles with Barium Manganate. Synthesis 1980,489-490. (133) Robertson, D. W.; Krushinsky, J. H.; Beedle, E. E.; Leander, J. D.; Wong, D. T.; Rathbun, R. C. Structure-Activity Relationships of (Arylalkyl)imidazole Anticonvulsants: Comparison of the (Fluorenylalkyl)imidazoles with Nafimidone and Denzimol. J. Med. Chem. 1986, 2 9 ,1577-1586. (134) Robey, R. L.; Coupley-Merriman, C. R.; Phelps, M. A. Synthesis and Reactions of 2-Amino-7,8-Dimethoxy-1H-3-Benzazepines. Competitive Formation of 2- Amino-1H-3-Benzazepines vs. 2-Benzylimidazoles. J. Heterocyclic Chem. 1989, 2 6 ,779-783. (135) Bergel, F.; Everett, J. L.; Roberts, J. J.; Ross, W. C. J. Aryl-2- halogenoalkylamines. PartXV. Some Cationic and Basically Substituted Aryl Compounds. J. Chem. Soc. 1955,3835-3839. (136) Hoffmann, B. B.; Lefkowitz, R. J. Adrenergic Receptor Antagonists. In In The Pharmacological Basis o f Therapeutics, 8th ed.;Gilman, Rail, T. W., Nies, A. S., Taylor, P., Eds.; Pergamon Press: New York, 1990; pp 221-268. (137) Coupry, I.; Atlas, D.; Podevin, R. A.; Uzielli, I.; Parini, A. Imidazoline-Guanidinium Receptive Site in Renal Proximal Tubule: Asymetric Distribution, Regulation by Cations and Interaction with an Endogenous Clonidine Displacing Substance. J. 202 Pharmacol. Exp. Ther. 1990,252,293-299. 138) Tesson, F.; Limon, I.; Parini, A. Tissue-Specific Localization of Mitochondrial Imidazoline-Guanidinium Receptive Sites. Eur. J. Pharmacol. 1 9 9 2 ,219,335-338. 139) Baxter, A. J.; Dixon, J.; Ince, F.; Manners, C. N.; Teague, S. J. Discovery and Synthesis of Methyl 2,5-Dimethyl-3-carboxylate (FLP 64176) and Analogs: The First Examples of a New Class of Calcium Channel Activator. J. Med. Chem. 1993, 36,2739-2744. 140) Trigle, D. J.; Shefter, E.; Trrigle, A. M. Crystal Structures of Calcium Channel An tag onists:2,6-Di methyl-3,5-dicarbomethoxy-4-[2-n itro-3-cyano- 4(dimethylamino) and 2,3,4,5,6-pentafluorophenyl]-1,4-dihydropyridine. J. Med. Chem. 1 9 8 0 ,2 3 ,1442-1445. 141) Hayashi, T. Effects of Sodium Glutamate on the Nervous System. Keio J. Med. 1954, 3 ,183-192. 142) Curtis, D. R.; Watkins, J. C. The Excitation and Depression of Spinal Neurones by Structurally Related Amino Acids. J. Nuerochem. 1960, 6 ,117-141. 143) Curtis, D. R,; Watkins, J. C. Acidic Amino Acids with Strong Actions on Mammalian Neurones. J. Physiology. 1963, 166,1-14. 144) Voet, D.; Voet, J. G. Amino Acid Metabolism. In Biochemistry, John Wiley & Sons: New York, 1991; pp 678-729. 145) Maycox, P. R.; Hell, J. W.; Jahn, R. Amino Acid Neurotransmission: Spotlight on Synaptic Residues. 77A/S1990, 13, 83-87. 146) Fonnum, F. A Neurotransmitter in Mammalian Brain. J. Neurochem. 1984, 4 2 , 1-11. 147) Nicholls, D.; Attwell, D. The Release and Uptake of Excitatory Amino Acids. TIPS 1990, 77,462-468. 148) Taylor, R. Quick on the Uptake: Brain Glutamate Transporters Cloned. J. NIH Res. 1993, 5, 55-60. 149) Collingridge, G. L.; Singer, W. Excitatory Amino Acid Receptors and Synaptic Plasticity. TIPS 1990, 77,290-296. 150) Brown, T. H.; Chapman, P. F.; Kairiss, E. W.; Keenan, C. L. Long-Term Synaptic Potentiation. Science 1 9 8 8 ,242, 724-728. 151) Muller, D.; Joly, M.; Lynch, G. Contributions of Quisqualate and NMDA Receptors to the Induction and Expression of LTP. Science 1988, 242, 1694-1697. 152) Meldrum, B., S. Excitatory Amino Acid Receptor and Disease. Curr. Opin. Neurol. Neurosurg. 1992, 5 ,508-513. 153) Meldrum, B.; Garthwaite, J. Excitatory Amino Acid Neurotoxicity and Neurodegenerative Diseases. 7/PS 1990, 77,379-387. 154) Watkins, J. C.; Evans, R. H. Excitatory Amino Acid Transmitters. Ann. Rev. Pharmacol. Toxicol. 1 9 8 1 ,27,165-204. (155) Foster, A. C.; Fagg, G. E. Acidic Amino Acid Binding Sites in Mammalian Neuronal Membranes, Their Characteristics and Relationship to Synaptic Receptors. Brain Res. Rev. 1984, 7,103-164. (156) Seeburg, P. H. The TIPS/TINS Lecture: The Molecular Biology of Mammalian Glutamate Receptor Channels. TIPS 1993, 14,297-303. (157) Chamberlin, R.; Bridges, R. Conformationally Constrained Acidic Amino Acids as Probes of Glutamate Receptors and Transporters. In Drug Design for Neuroscience; Kozikowski, A. P., Ed.; Raven Press Ltd.: New, York, 1993; pp 231-259. (158) Farooqui, A. A.; Horrocks, L. A. Excitatory Amino Acid Receptor Neural Membrane Phospholipid Metabolism and Neurological Disorders. Brain Res. Rev. 1991, 16, 171-191. (159) Schoepp, D. D.; Conn, J. P. Metabotropic Glutamate Receptors in Brain Function and Pathology. TIPS 1993, 14, 13-20. (160) Hansen, J. J.; Krogsgaard-Larsen, P. Structural, Conformational and Stereochemical Requirements of Central Excitatory Amino Acid Receptors. Med. Res. Rev. 1990, 10, 55-94. (161) Monaghan, D. T.; Bridges, R. J.; Cotman, C. W. The Excitatory Amino Acid Receptors: Their Classes, Pharmacology and Distinct Properties in tne Function of the Central Nervous System. Ann. Rev. Pharmacol. Toxicol. 1989, 2 9 ,365-402. (162) Young, A. B.; Fagg, G. E. Excitatory Amino Acid Receptors in the Brain: Membrane Binding and Receptor Autoradiographic Approaches. TIPS 1990, 11,126-133. (163) Watkins, J. C.; Krogsgaard-Larsen, P.; Honore, T. Structure-Activity Relationships in the Development of Excitatory Amino Acid Receptor Agonists and Competitive Antagonists. 1990,25-33. (164) Watkins, J. C. Some Chemical Highlits in the Development of Excitatory Amino Acid Pharmacology. Can. J. Physiol. Pharmacol. 1991, 69,1064-1075. (165) Johnson, R. L.; Koerner, J. F. Excitatory Amino Acid Neurotransmission. J. Med. Chem. 1988, 31,2057-2066. (166) Lodge, D.; Johnson, K. M. Noncompetitive Excitatory Amino Acid Receptor Antagonists. TIPS 1990, 71,81-86. (167) Kemp, J. A.; Leeson, P. D. The Glycine Site of the NMDA Receptor Antagonists. 7/PS 1993, 74,20-25. (168) Takemoto, T.; Nakajima, T.; Arihara, S.; Koike, K. Studies on the Constituents of Quisqualis Fructus. II. Structure of Quisqualic Acid. J. Pharm. Soc. Jpn. 1975, 95, 326-329. (169) Slevin, J. T.; Collins, J. F.; Coyle, T. J. Analogue Interactions with the Brain Receptor Labeled by [3H]Kainic Acid. Brain Res. 1983, 265,169-172. (170) Barnes, J. M.; Henley, J. M. Molecular Characteristics of the Excitatory Amino Acid Receptors. Progress in Neurobiol. 1992, 3 9 ,113-133. 204 (171) Headley, P. M.; Grillner, S. Excitatory Amino Acid and Synaptic Transmission: the Evidence for a Physiological Function. TIPS 1990, 11,205-211. (172) Agrawal, S. G.; Evans, R. H. The Primary Afferent Depolarizing Action of Kainate in the Rat. Br. J. Pharmacol. 1986,87,345-355. (173) Ross, S. M.; Dwijendra, N. R.; Spencer, P. S. p-N-Oxalylamino-L-Alanine Action on Glutamate Receptors. Neurochem. 1 9 8 9 ,53,710-715. (174) Honore, T.; Lauridsen, J. Structural Analogs of Ibotenic Acid. Syntheses of 4- Methyl-homoibotenic Acid and AMPA, Including the Crystal Structure of AMPA, Monohydrate. Acta Chem. Scand. B 1980, 34, 235-240. (175) Krogsgaard-Larsen, P.; Honore, T.; Hansen, J. J.; Curtis, D. R.; Lodge, D. R. New Class of Glutamate Agonist Structurally Related to Ibotenic Acid. Nature 1980, 284, 64-66. (176) Krogsgaard-Larsen, P.; Brehm, L.; Johansen, J. S.; Vinzents, P.; Lauridsen, J.; Curtis, D. R. Synthesis and Structure-Activity Studies on Excitatory Amino Acids Structurally Related to Ibotenic Acid. J. Med. Chem. 1 9 8 5 ,28 ,673-679. (177) Lauridsen, J.; Honore, T.; Krogsgaard-Larsen, P. Ibotenic Acid Analogues. Synthesis, Molecular Flexibility and in Vivo Activity of Agonists and Antagonists at Central Glutamic Acid Receptors. J. Med. Chem. 1 9 8 5 ,28,668-672. (178) Brehm, L.; Jorgensen, F. S.; Hansen, J. J.; Krogsgaard-Larsen, P. Agonists and Antagonists for Central Glutamic Acid Receptors. Drug News Persp. 1988, 1,138- 144. (179) Krogsgaard-Larsen, P.; Nielsen, E. f.; Curtis, D. R. Ibotenic Acid Analogues. Synthesis and Biological and in Vivo Activity of Conformationally Restricted Agonists at Central Excitatory Amino Acid Receptors. J. Med. Chem. 1984, 27, 585-591. (180) Nielsen, E.; Madsen, U.; Schaumburg, K.; Brehm, L.; Krogsgaard-Larsen, P. Studies on Receptor Active Conformation of Excitatory Amino Acid Agonists and Antagonists. Eur. J. Med. Chem. 1 9 8 6 , 21,433-437. (181) Hansen, J. J.; Lauridsen, J.; Nielsen, E.; Krogsgaard-Larsen, P. Enzymic Resolution and Binding to Rat Brain Membranes of the Glutamic Acid Agonist a- Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid. J. Med. Chem. 1 9 8 3 ,2 6 , 901-903. (182) Hansen, J. J.; Nielsen, B.; Krogsgaard-Larsen, P.; Brehm, L.; Nielsen, E.; Curtis, D. R. Excitatory Amino Acid Agonists. Enzymic Resolution, x-Ray Structure, and Enantioselective Activities of (R)- and (S)-Bromohomoibotenic Acid. J. Med. Chem. 1989, 32,2254-2260. (183) Jones, A. W.; Smith, D. A. S.; Watkins, J. C. Structure-Activity Relations of Dipeptide Antagonists of Excitatory Amino Acids. Neurosci. 1984, 13,573-581. (184) Honore, T.; Davies, S. N.; Drejer, J.; Fletcher, E. J.; Jacobsen, P.; Lodge, D.; Nielsen, F. E. Quinoxalinediones: Potent Competitive Non-NMDA Glutamate Receptor Antagonists. Science 1 9 8 8 ,241, 701-703. (185) Sheardown, M. J.; Nielsen, E.; Hansen, A. J.; Jacobsen, P.; Honore, T. 2,3- 205 Dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline: A Neuroprotectant for Cerebral Ischemia. Science 1990,247, 571-574. (186) Ohmori, J.; Sakamoto, S.; Kubota, H.; Shimidzu-Sasamata, M.; Okada, M.; Kawasaki, S.; Hidaka, K.; Togami, J.; Furuya, T.; Murase, K. 6-(1 H-lmidazol-2-yl)- 7-nitro-2,3(1H,4H0-quinoxalinedione Hydrochloride (YM90K) and Related Compounds: Structure Activity Relationships for the AMPA-Type Non-NMDA Receptors. J. Med Chem. 1994, 37,467-475. (187) Ueno, Y.; Nawa, H.; Ueyanagi, J.; Morimoto, H.; Nakamori, R.; Matsuoka, T. Studies on the Active Components of Dignea Simplex.A J and Related Compounds I. Studies on the Structure of Kainic Acid. J. Pharm. Soc. Jpn. 1955, 75,807-812. (188) Takemoto, T.; Daigo, K.; Kondo, Y.; Kondo, K. Studies on the Constituents of Chondria Armata. VII. On the Structure of Domoic Acid. J. Pharm. Soc. Jpn. 1966, 86,874-880. (189) Konno, K.; Hashimoto, K.; Ohfune, Y.; Shirahama, H.; Matsumoto, T. Acromelic Acid A and B. Potent Neuroexcitatory Amino Acid Isolated from Clitocybe Acromealga. J. Am. Chem. Soc. 1988, 110,4807-4815. (190) Koerner, J. F.; Cotman, C. W. Micromolar L-2-Amino-4-phosphonobutyric acid Selectively Inhibits Perforant Path Synapses from Lateral Entorhinal Cortex. Brain Res. 1981 ,2 1 6 ,192-198. (191) Cotman, C. W.; Flatman, J. A.; Ganoug, A. H.; Perkins, M. N. Effects of Excitatory Amino Acid Antagonists on Evoked and Spontaneous Excitatory Potential in Guonea-Pig Hippocampus. J. Physiol. 1986, 378,403-415. (192) Schoepp, D.; Bockaert, J.; Sladeczek, F. Pharmacological and Functional Characteristics of Metabotropic Excitatory Amino Acid Receptors. TIPS 1990, 11, 508-515. (193) Hollmann, M.; O'Shea-Greenfield, A.; Rogers, S. W.; Heinemann, S. Cloning by Functional Expression of a member of the Glutamate Receptor Family. Nature 1989, 342, 643-648. (194) Nakaniski, S. Molecular Diversity of Glutamate Receptors and Implications for Brain Function. Science 1992,258,597-603. (195) Keinanen, K.; Wisden, W.; Sommer, B.; Werner, P.; Herb, A.; Verdoorn, T. A.; Sacmann, B.; Seeburg, P. H. A Family of AMPA-Selective Glutamate Receptors. Science 1990,249, 556-560. (196) Boulter, J.; Hollmann, M.; O'Shea-Greenfield, A.; Hartley, M.; Deneris, E.; Maron, C.; Heinemann, S. Molecular Cloning and Functional Expression of Glutamate Receptor Subunit Genes. Science 1990,249, 1033-1037. (197) Sommer, B.; Seeburg, P. H. Novel Properties and New Clones. TIPS 1992, 13, 291-296. (198) Kutsuwada, T.; Kashiwabuchi, N.; Mori, H.; Sakimura, K.; Kushiya, E.; Araki, K.; Meyuro, H.; Masaki, H.; Kumanishi, T.; Arahawa, M.; Mishina, M. Molecular Diversity of the NMDA Receptor Channel. Nature 1992, 358,36-41. (199) Mishina, M.; Sakimura, K.; Mori, H.; Kushiya, E.; Harabayashi, M.; Uchino, S.; Nagahari, K. A Single Amino Acid Residue Determines the Ca2+ Permeability of Commun. 1991, 180,813-821. (200) Hume, R. I.; Dingledine, R.; Heinemann, S. F. Identification of a Site in Glutamate Receptor Subunit that Controls Calcium Permeability. Science 1991, 2 5 3 ,1028- 1031. (201) Hollmann, M.; Hartley, M.; Heinemann, S. C a2+ Permeability of KA-AMPA-Gated Glutamate Receptor Channels Depends on Subunit Composition. Science 1991, 252,851-853. (202) Dingledine, R. New Wave of Non-NMDA Excitatory Amino Acid Receptors. TIPS 1991, 12,360-362. (203) Sommer, B.; Keinanen, K.; Verdoom, T. A.; Wisden, W.; Burnashev, N.; Herb, A.; Kohler, M.; Takagi, T.; Sakmann, B.; Seeburg, P. H. A Cell Specific Functional Switch in Glutamate-Operated Channels of the CNS. Science 1 9 9 0 ,2 4 9 , 1580- 1585. (204) Uchino, S.; Sakimura, K.; Nagahari, K.; Mishina, M. Mutations in a Putative Agonist Binding Region of the AMPA Selective Glutamate Receptor Channel. FEBS Lett. 1992, 308,253-257. (205) Kleber, H. D. Cocaine Abuse: Historical, Epidemiological and Psychological Perspectives. J. Clin. Psychiatry. 1988, 4 9 , 3-6. (206) Carroll, F. I.; Lewin, A. H.; Boja, J. W.; Kuhar, M. J. Cocaine Receptor: Biochemical Characterization and Structure-Activity Relationships of Cocaine Analogues at the Dopamine Transporter. J. Med. Chem. 1992, 35,969-981. (207) Skinner, M. H.; Thompon, D. A. Pharmacologic Considerations in the Treatment of Substance Abuse. Pharmacol. Subst. Abuse 1992, 8 5 ,1207-1219. (208) Johnson, C. E.; Fischman, M. W. Pharmacology of Cocaine Related to its Abuse. Pharmacol. Rev. 1989, 41, 3-52. (209) Gawin, F. H.; Ellinwood, E. H. Cocaine and Other Stimulants. New Eng. J. Med. 1988, 378,1173-1182. (210) Kaufman, E.; McNaul, J. P. Recent Developments in Understanding and Treating Drug Abuse and Dependence. Hospital Comm. Psych. 1992, 43, 223-236. (211) Gawin, F. H. Chronic Neuropharmacology of Cocaine: Progress in Pharmacotherapy. J. Clin. Psychiatry 1988, 49(Suppl.), 11-16. (212) DiChiara, G.; Imperato, A. Drugs Abused by Humans Preferentially Increase Synaptic Dopamine Concentrations in the Mesolimbic System of Freely Moving Rats. Proc. Natl. Acad. Sci. USA 1988, 85,5274-5278. (213) Giros, B.; Caron, M. G. Molecular Characterization of the Dopamine Transporter. TIPS 1993, 74,43-49. (214) Ritz, M. C.; Lamb, R. J.; Goldberg, S. R.; Kuhar, M. J. Cocaine Receptors on Dopamine Transporters are Related to Self-Administration of Cocaine. Science, 1987 ,237,1219-1223. 207 (215) Kelly, P. H.; Iversen, S. D. Selective 6-OH-Dopamine linduced Destruction of Mesolimbic Dopamine Neurones: Abolition of Psychostimulant Induced Locomotor Activity in Rats. Eur. J. Pharmacol. 1976, 40, 45-56. (216) Roberts, D. S. C.; Koob, G. F. Disruption of Cocaine Self Administration Following 6-Hydroxydopamine Lesions of the Ventral Tegmental Area in Rats. Pharm. Biochem. Behav. 1982, 17,901-904. (217) Wise, R. A.; Bozarth, M. A. Brain Reward Circuitry: Four Circuit Elements "Wired" in Apparent Series. Brain Res. Bull. 1984, 12,203-208. (218) Wyatt, R. J.; Karoum, F.; Suddath, R.; Hitri, A. The Role of Dopamine in Cocaine Use and Abuse. Psych. Ann. 1988, 18,531-534. (219) Dackins, C. A.; Gold, M. S. New Concept in Cocaine Addiction: The Dopamine Depletion Hypothesis. Neurosci. Biobehav. Rev. 1985, 9, 469-477. (220) Koob, G. F.; Bloom, F. E. Cellular and Molecular Mechanisms of Drug Dependence. Science 1988, 242, 715-723. (221) Mogenson, G. J.; Jones, D. L.; Yim, C. Y. From Motivation to Action: Functional Interface between the Limbic System and the Motor System. Progress in Neurobiol. 1980, 14, 69-97. (222) Fonnum, F.; Karlsen, R. L.; Malthe-Sorenssen, D.; Screde, K. K.; Walaas, I. Localization of Neurotransmitters, particularly Glutamate in Hippocampus, Septum, Nucleus Accumbens and Superior Colliculus. Progress in Brain Res. 1979, 51, 169-191. (223) Walaas, I. Biochemical Evidence for Overlapping Neocortical and Allocorticallutamate Projections to the Nucleus Accumbens and Rostal Caudatoputamen in the Rat Brain. Neurosci. 1981, 6 ,399-405. (224) Robinson, T. G.; Beart, P. M. Excitatory Amino Acid Projections from Rat Amygdala and Thalamus to the Nucleus Accumbens. Brain Res. Bull. 1988, 20,467-471. (225) Roberts, P. J.; Anderson, S. D. Stimulatory Effect of L-Glutamate and Related Amino Acids on [3H]Dopamine Release from Rat Striatum: An In Vitro Model for Glutamate Actions. J. Neurochem. 1979, 32,1539-1545. (226) Cheramy, A.; Romo, R.; Godechen, G.; Baruch, P.; Glowinski, J. In Vivo Presynaptic Control of Dopamine. Release in the Cat Caudate Nucleus.ll. Facilitatory or Inhibitory Influence of L-Glutamate. Neurosci. 1986, 19,1081-1090. (227) Donzanti, B. A.; Uretsky, N. J. Effects of Excitatory Amino Acids on Locomotor Activity after Bilateral Microinjection into the Rat Nucleus Accumbens: Possible Dependence on Dopaminergic Mechanisms. Neuropharmacol. 1983, 2 2 ,971-981. (228) Boldry, R. C.; Willins, D. L.; Wallace, L. J.; Uretsky, N. J. The Role of Endogenous Dopamine in the Hypermotility Response to Intra-Accumbens AMPA. Brain Res. 1991, 559,100-108. (229) Donzanti, B. A.; Uretsky, N. J. Antagonism of the Hypermotility Response Induced by Excitatory Amino Acids in the Rat Nucleus Accumbens. Naunyn- Schmiedeberg's Arch. Pharmacol. 1984, 325,1-7. 208 (230) Imperato, A.; Honore, T.; Jensen, L. H. Dopamine Release in the Nucleus Caudatus and in the Nucleus Accumbens is under Glutamatergic Control through Non-NMDA Receptors: a Study in Freely Moving Rats. Brain Res. 1990, 530, 223-228. (231) Wang, T.; French, E. D. Electrophysiological Evidence for the Existence of NMDA and Non-NMDA Receptors on Rat ventral Tegmental Dopamine Neurons. Synapse 1993, 13, 270-277. (232) Desce, J. M.; Godehen, G.; Galli, T.; Artand, F.; Cheramy, A.; Glowinski, J. Presynaptic Facilitation of Dopamine Release through D,L-a-Amino-3-hydroxy-5- methyl-4-isoxazole Propionate Receptors on Synaptosomes from the Rat Striatum. J. Pharmacol. Exp. Ther. 1991,259,692-698. (233) Pulvirenti, L.; Sweldrow, N. R.; Koob, G. F. Microinjection of a Glutamate Antagonist into the Nucleus Accumbens Reduces Psychostimulant Locomotion in Rats. Neurosci. Lett. 1989, 103,213-218. (234) Pulvirenti, L.; Swerdlow, N. R.; Koob, G. F. Nucleus Accumbens NMDA Antagonist Decreases Locomotor Activity Produced by Cocaine, Heroin or Accumbens Dopamine, but not Caffeine. Pharmacol. Biochem. Behav. 1991, 40, 841-845. (235) Willins, D.; Wallace, L. J.; Miller, D. D.; Uretsky, N. J. a-Amino-3-hydroxy-5- methylisoxazole-4-propionate/Kainate Receptor Antagonists in the Nucleus Accumbens and Ventral Pallidum Decrease the Hypermotility Response to Psychostimulant Drugs. 1992, 260,1145-1151. (236) Rochold, R. W.; Oden, G.; Ho, I. K.; Andrew, M.; Farley, J. M. Glutamate Receptor Antagonists Block Cocaine Induced Convulsions and Death. Brain. Res. Bull. 1991,27,721-723. (237) Witkin, J. M.; Tortella, F. C. Modulators of N-Methyl-D-Aspartate Protect Against Diazepam or Phenobarbital Resistant Cocaine Convulsions. Life. Sci. 1991, 48, PL51-PL56. (238) Karler, R.; Calder, L. D. Excitatory Amino Acids and the Actions of Cocaine. Brain. Res. 1992, 582,143-146. (239) Kaddis, F.; Wallace, L. J.; Uretsky, N. J. AMPA/Kainate Antagonists in the Nucleus Accumbens Inhibit Locomotor Stimulatory Response to Cocaine and Dopamine Agonists. Pharmacol. Biochem. Behav. 1993, 46, 703-708. (240) Wesson, D. R.; Ling, W. Medications in the Treatment of Addictive Disease. J. Psychoactive Drugs 1991,2 3 ,365-370. (241) Tutton, C. S.; Crayton, J. W. Current Pharmacotherapies for Cocaine Abuse: A Review. J. Addict. Dis. 1993, 12,109-127. (242) Rawson, R. A.; Obert, J. L.; Castro, F. G.; Ling, W. Cocaine Abuse Treatment: A Review of Current Strategies. 1991, 3 ,457-491. (243) Rogawski, M. A. Therapeutic Potential of Excitatory Amino Acid Antagonists: Channel Blockers and 2,3-Benzodiazepines. TIPS 1993, 14, 325-331. (244) Choi, D. W. Glutamate Neurotoxicity and Diseases of the Nervous System. 209 Neuron 1988, 1,623-634. (245) Koek, W.; Woods, J. H.; Winger, G. D. MK-801, a Proposed Noncompetitive Antagonist of Excitatory Amino Acid Neurotransmission, Produces Phencyclidine- Like Behavioral Effects in Pigeons, Rats and Rhesus Monkeys. J. Pharmaol. Exp. Ther. 1988, 245, 969-974. (246) Olney, J. W.; Labruyere, J.; Price, M. T. Pathological Changes Induced in Cerebrocortical Neurons by Phencyclidine and Related Drugs. Science 1989, 244, 1360-1362. (247) Morris, R. G. M.; Anderson, E.; Lynch, G. S.; Baudry, M. Selective Impairment of Learning and Blockade of Long Term Potentiation by an N-Methyl-D-aspartate Receptor Antagonist, AP5. Nature 1986, 319,774-776. (248) Layer, R. P.; Uretsky, N. J.; Wallace, L. J. Effects of the AMPA/Kainate Receptor Antagonist DNQX in the Nucleus Accumbens on Drug/Induced Conditioned Place Preference. Brain Res. 1993, 617,267-273. (249) Hill, R. A. Part 1. Design, Synthesis and Structure-Activity Studies of the Molecules with Activity at Non-NMDA Glutamate Receptors: Hydroxyphenylalanines, Quinoxalinediones and Related Moleculaes. Part 2. Computational Studies on the Interaction of the Dopamine Amino Group Replacements with Carboxylate A Model for Receptor Interactions., Ph.D. Thesis, The Ohio State University, 1991. (250) Krogsgaard-Larsen, P.; Ferkany, J. W.; Nielsen, E.; Madsen, U.; Ebert, B.; Johansen, J. S.; Diemer, N. H.; Bruhn, T.; Beattie, D. T.; Curtis, D. R. Novel Class of Amino Acid Antagonists at Non-N-Methyl-D-aspartic Acid Excitatory Amino Acid Receptors. Synthesis in Vitro and in Vivo Pharmacology, ana Neuroprotection. J. Med. Chem. 1991, 3 4 , 123-130. (251) Judice, K. J.; Gamble, T. R.; Murphy, E. C.; deVos, A. M.; Schultz, P. G. Probing the Mechanism of Staphylococcal Nuclease with Unnatural Amino Acids: Kinetic and Structural Studies. Science 1993,261,1578-1581. (252) Maurer, P. J.; Takahata, H.; Rapoport, H. a-Amino Acids as Chiral Educts for Asymmetric Products. A General Synthesis of D-a-Amino Acids from L-Serine. J. Am. Chem. Soc. 1984, 106,1095-1098. (253) Buckley, T. F. I.; Rapoport, H. a-Amino Acids as Chiral Educts for Asymmetric Products. Amino Acylation with N-Acylamino Acids. J. Am. Chem. Soc. 1981, 103, 6157-6163. (254) Knudsen, C. G.; Rapoport, H. a-Amino Acids as Chiral Educts for Asymmetric Products. Aminoacylation of Metallo Alkyls and Alkenyls. J. Org. Chem. 1983, 48, 2260-2266. (255) West, C. T.; Donnelly, S. J.; Kooistra, D. A.; Doyle, M. P. Silane Reduction in Acidic Media. II. Reduction of Aryl Aldehydes ana Ketones by Trialkylsilanes in Trifluoroacetic Acid. A Selective Method for Converting the Carbonyl Group to Methylene. J. Org. Chem. 1973, 38,2675-2681. (256) Millar, L. G.; Oehlschlager, A. C.; Wong, J. W. Synthesis of Two Macrolide Aggregation Pheromones from the Flat Grain Beetle, Cryptolestes pusillus. J. Org. Chem. 1983, 48, 4404-4407. (257) Snyder, H. R.; Heckter, R. E. A Method for the Rapid Cleavage of Sulfonamides. J. Am. Chem. Soc. 1952, 74, 2006-2009. (258) Weisblat, D. J.; Magerlein, B. J.; Myers, D. R. The Cleavage of Sulfonamides. J. Am. Chem. Soc. 1953, 75,3630-3632. (259) Kuhn, S. J.; Olah, G. A. Aromatic Substitution. VII. Friedel-Crafts Type Nitration of Aromatics. J. Am. Chem. Soc. 1961, 83,4564-4571. (260) Barret, G. C. Resolution of Amino Acids. In Chemistry and Biochemistry of the Amino Acids ; Barrett, G. C., Ed.; Oxford Polytechnic UK: Chapman and Hall, London,1991; pp 338-353. (261) Garnier-Suillerot, A.; Albertini, J. P.; Collet, A.; Faury, L.; Pastor, J. M. Spetrophotometric Studies of the Copper(ll)-D-o-Tyrosine Complex. Assignment of the 330-nm Dichroic Band in Copper(ll) and Iron(fll) Transferrins. J. Chem. Soc. (Dalton) 1981, 2544-2549. (262) Davankov, V. A.; Bochkov, A. S.; Kurganov, A. A., P.; Unger, K. K. Separation of Unmodified a-Amino Acids Enantiomers by Reverse Phase HPLC. Chromatographia 1980, 13,677-685. (263) Gubitz, G.; Juffmann, F.; Jellenz, W. Direct Separation of Amino Enantiomers by High Performance Ligand Exchange Chromatography on Chemically Bonded Chiral Phases. Chromatographia 1982, 16,103-106. (264) Hooker, T. M. J.; Schellman, J. A. Optical Activity of Aromatic Chromophores. I. o, m, and p-Tyrosine. Biopol. 1970, 9 ,1319-1348. (265) Parikh, J. R.; Greenstein, J. P.; Winitz, M.; Birnbaum, S. M. The Use of Amino Acid Oxidases for the Small-Scale Preparation of the Optical Isomers of Amino Acids. J. Am. Chem. Soc. 1958, 8 0 ,953-958. (266) Page, D. S.; VanEtten, R. L. L-Amino Acid Oxidase. III. Substrate Substituent Effects Upon the Reaction of L-Amino Acid Oxidase with Phenylalanines. Bioorg. Chem. 1971, 1,361-373. (267) Blanchardt, M.; Green, D. E.; Nocito, V.; Ratner, S. L-Amino Acid Oxidase of Animal Tissue. J. Biol. Chem. 1944, 155,421-440. (268) Pirrung, M. C.; Krishnamurthy, N. Preparation of (R)-Phenylalanine Analogues by Enantioselective Destruction Using L-Amino Acids Oxidase. J. Org. Chem. 1993, 58, 957-958. (269) Frisell, W. R.; Lowe, H. J.; Hellerman, L. Flavoenzyme Catalysis. Substrate- Competitive Inhibition of D-Amino Acid Oxidase. J. Biol. Chem. 1956,75-83. (270) DuVigneaud, V.; Meyer, C. The Racemization of Amino Acids in Aqueous Solution by Acetic Anhydride. J. Biol. Chem. 1932, 98,295-308. (271) Sealock, R. R. Preparation of D-Tyrosine, its Acetyl Derivatives, and D-3,4- Dihydroxyphenylalanine. J. Biol. Chem. 1946, 166,1-6. (272) Chenault, K. H.; Dahmer, J.; Whitesides, G. M. Kinetic Resolution of Unnatural and Rarely Occurring Amino Acids: Enantioselective Hydrolysis of N-Acyl Amino Acids Catalysed by Acylases I. J. Am. Chem. Soc. 1989, 111, 6354-6364. 211 (273) Moroder, L.; Hallett, A.; Wunsch, E.; Keller, O.; Wersin, G. Di-tert.- butyldicarbonate-ein vortrilhaftes Reagenz zur Einfuhrung der tert.- Butyloxycarbonyl-Schutzgruppe. Hoppe-Seyler's Physiol. Chem. 1976, 357, S.1651-1653. (274) Perseo, G.; Piani, S.; DeCastiglione, R. Preparation and Analytical Data of some Unusual t-Butyloxycarbonyl-amino Acids. Int. J. Peptide Protein Res. 1983, 21, 227-230. (275) Szokan, G.; Mezo, G.; Hudecz, F. Application of Marfey's Reagent in Racemization Studies of Amino Acids and Peptides. J. Chromatogr. 1988, 444, 115-122. (276) Aberhart, J. D.; Cotting, J. A.; Lin, H. J. Separation by High Performance Liquid Chromatography of (3R)- and (3S)-p-Leucine as Diastereomeric Derivatives. Anal. Biochem. 1985, 151,88-91. (277) Dale, A. J.; Dull, D. L.; Mosher, H. S. a-Methoxy-a-trifluoromethylphenylacetic Acid, a Versatile Reagent for the Determination of Enantiomeric Composition of Alcohols and Amines. J. Org. Chem. 1969, 34, 2543-2549. (278) Arnold, L. D.; Kalantar, T. H.; Vederas, J. C. Conversion of Serine to Stereochemically Pure ^-Substituted a-Amino Acids via p-Lactones. J. Am. Chem. Soc. 1985, 107,7105-7109. (279) Arnold, L. D.; May, R. G.; Vederas, J. C. Synthesis of Optically Pure a-Amino Acids via Salts of a-Amino -p-propiolactone. J. Am. Chem. Soc. 1988, 110,2237- 2241. (280) Pansare, S. V.; Arnold, L. D.; Vederas, J. C. Synthesis of N-tert-Butoxycarbonyl- L-serine p-lactone and the p-Toluenesulfonic Acid Salt of (S)-3-Amino-2- Oxetanone. Org. Synth. 1990, 7 0 ,10-17. (281) Ramer, S. E.; Moore, R. N.; Vederas, J. C. Mechanism of Formation of Serine p- lactone by Mitsunobu Cyclization: Synthesis and Use of L-Serine Stereospecifically Labelled with Deuterium at C-3. Can. J. Chem. 1985, 64, 706- 713. (282) Hashimoto, N.; Aoyama, T.; Shioiri, T. New Methods and Reagents in Organic Synthesis.14. A Simple Efficient Preparation of Methyl Esters with Trimethylsilyldiazomethane (TMSCHN2) and Its Application to Gas Chromatographic Analysis of Fatty Acids. Chem. Pharm. Bull. 1981, 29, 1475- 1478.