UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Investigation of homo- and hetero-aromatic analogues of cyclohexyl substituted Antergan for pharmacological… Wang, Stanley Yih Song 1972

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1972_A1 W36.pdf [ 7.55MB ]
Metadata
JSON: 831-1.0101515.json
JSON-LD: 831-1.0101515-ld.json
RDF/XML (Pretty): 831-1.0101515-rdf.xml
RDF/JSON: 831-1.0101515-rdf.json
Turtle: 831-1.0101515-turtle.txt
N-Triples: 831-1.0101515-rdf-ntriples.txt
Original Record: 831-1.0101515-source.json
Full Text
831-1.0101515-fulltext.txt
Citation
831-1.0101515.ris

Full Text

INVESTIGATION OF HOMO- AND HETERO-AROMATIC ANALOGUES OF CYCLOHEXYL SUBSTITUTED ANTERGAN FOR PHARMACOLOGICAL ACTIVITIES by STANLEY YIH SONG WANG B.S.P., The National Taiwan University, 1964 M.S.P., University of B r i t i s h Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Division of Medicinal Chemistry of the Faculty of Pharmaceutical Sciences We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1972 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y of B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department The U n i v e r s i t y o f B r i t i s h C olumbia Vancouver 8, Canada i ABSTRACT In the present study seven t e r t i a r y ethylenediamine d e r i v a t i v e s were synthesized. They had the basic structure of N.N-dlmethyl-N'-^-M'-Rg-ethylenediamine where B 1 = cyclohexy 1 -methyl and = 2-pyridyl, 2-pyrlmidyl, 2-pyrazyl, 1-naphthyl, 5 - q u l n o l y l ; and R 1 = cyclohexanecarbonyl and Hg = 2-pyrimidyl, 2-pyrazyl. Two oxetane d e r i v a t i v e s , 2 , 2-diphenyl - 3-chloro-oxetane and 2 , 2-diphenyl - 3-(pyrrolidln - 2-one-l-yl)oxetane, have also been synthesized as intermediates f o r further i n -v e s t i g a t i o n . , The general reaction sequence followed was to s t a r t with the appropriate primary aromatic amine and b u i l d up to a secondary amine v i a a cyclohexanecarboxamide. The desired secondary amine was reacted with ^-dimethylaminoethy1 c h l o r i d e . The t e r t i a r y diamine r e a d i l y formed i n good y i e l d s . In cases where the primary aromatic amines could not be b u i l t up to a secondary amine v i a the above route, the t e r t i a r y diamine de-r i v a t i v e was obtained by two condensation reactions, i . e . the primary aromatic amine was f i r s t condensed with ^-dimethyl-aminoethyl chloride to form the desired secondary amine which was then condensed with cyclohexylmethyl bromide to produce the t e r t i a r y diamine d e r i v a t i v e . The o v e r a l l y i e l d s were lower than those of the f i r s t route. The oxetane de r i v a t i v e s were obtained by photocyclo-ad d l t i o n of benzophenone to e i t h e r v i n y l chloride or N - v i n y l -2-pyrrolidinone. 11 The antihistaminic a c t i v i t y and the possible a n t i -c h o l i n e r g i c a c t i v i t y of the t e r t i a r y diamine derivatives were studied and compared with those of diphenhydramine, atropine, and the cyclohexy1 analogue of Antergan. These compounds ex-h i b i t e d n e g l i g i b l e a n t i c h o l i n e r g i c a c t i v i t y . However, t h e i r r e l a t i v e l y potent antihistaminic a c t i v i t y was found to give more evidence In d i r e c t support of Nauta*s antihistaminic receptor theory. Signature of Examiners H i ACKNOWLEDGEMENT My sincere gratitude i s extended to my major professor, Dr. T. H. Brown, f o r professional guidance, understanding and encouragement during the course of t h i s study. I must express my thanks to Dr. K. Y. Chang of the Department of E l e c t r i c a l Engineering, U. B. C. f o r the techni c a l assistance i n p l o t t i n g the cumulative log dose-response curves using the IBM 360/67 Computer. F i n a n c i a l support by the National Research Council and by a U. B. C. Fellowship i s g r a t e f u l l y acknowledged. Iv TABLE OF CONTENTS PART Page Abstract 1 Acknowledgement i i i L i s t of Tables v i i L i s t of Figures v l l l I, INTRODUCTION 1 A. Biogenesis and Physiology of Histamine and Anaphylaxis 3 B. Histamine Receptors 22 C. Antihistamines and Antihistamine Receptor... 29 I I . DISCUSSION OF THE CHEMISTRY 36 I I I . ANALYTICAL METHODS 62 IV. EXPERIMENTAL 63 A. Synthesis of N,N-Dimethyl-N'-Cyclohexyl-methyl-N * - 2-Pyridylethylenediamine 63 1. Cyclohexanecarbonyl Chloride 63 2 . N-2-Pyridyl-Cyclohexanecarboxamlde, 63 3 . 2-Cyclohexylmethylamlno pyridine 64 4 . N,N-Dimethyl-N'-Cyclohexylmethyl-N ' - 2-Pyridylethylenediamine 65 B. Synthesis of N,N-Dimethyl-N'-Cyclohexyl-methyl-N' -2-Pyrimidylethylenedlamine........ 67 1. Cyclohexy lmethylamlnopyrlmidlne 67 2. N,N-Dimethyl-N * - 2-Pyrimldylethylene-diamine 68 3 . N,N-Dimethyl-N'-Cyclohexanecarbonyl-N' - 2-Pyrimldyle thylenedlamlne 70 4 . N,N-Dimethyl-N'-Cyclohexylmethyl-N' - 2-Pyrimidyle thylenedlamlne 71 V PART Page C. Synthesis of N,N-Dimethyl-N--Cyclohexyl-methyl-N' - 2-Pyrazylethylenedlamlne 73 1. N-2-Pyrazyl-Cyclohexanecarboxamlde. 73 2 . N,N-Dimethyl-N'-2-Pyrazylethylenediamine.. 74 3 . N,N-Dlmethyl-N'-Cyclohexanecarbonyl-N ' - 2-Pyrazylethylenediamine 75 4 . N,N-Dimethyl-N'-Cyclohexylmethyl-N* - 2-Pyrazylethylenediamlne 76 D. Synthesis of N,N-Dimethyl-N'-Cyclohexyl-methyl-N'-1-Naphthylethylenediamine 78 1. N-l-Naphthyl-Cyclohexanecarboxamide 78 2 . N-Cyclohexylmethyl-N'-Naphthylamine 79 3 . N,N-Dimethyl-N'-Cyclohexylmethyl-N' -1-Naphthylethylenediamine 81 E. Synthesis of N,N-Dimethyl-N'-Cyclohexyl-methyl-N* - 5-Quinolylethylenediamine 82 1. N-5-Quinolyl-Cyclohexanecarboxamide 82 2 . 5-Cyclohexylmethylaminoquinollne 83 3 . N,N-Dimethy1-N--Cyclohexylmethyl-N' - 5-Quinolylethylenediamlne 84 F. Synthesis of N,N-Dimethyl-N'-Cyclohexyl-methyl-N ' - 3-Pyridazylethylenediamine 86 1. 3-Amino -6-Chloropyridazine 86 2 . 3-Aminopyridazine 87 3 . N,N-Dimethyl-N' - 3-Pyridazyl-ethylenediamine 88 G. Synthesis of 2 ,2-Diphenyl-4-Dimethy1-amlnomethyloxetane 89 1. 2 , 2-Dlphenyl - 3-Chlorooxetane 89 2 . 2 , 2-Diphenyl - 3-(Pyrrolidin - 2-one-1-yl) oxetane 90 v l PART Page V. PHARMACOLOGICAL TESTING AND ANTIHISTAMINIC RECEPTOR STUDIES 92 A. Introduction...... 92 B. Experimental 93 C. Results and Discussion 96 D. Antihistaminic Receptor Studies 105 VI. SUMMARY 137 VII. APPENDICES 141 VIII. BIBLIOGRAPHY 15^ Biographical Information 162 v i i LIST OF TABLES TABLE Page 1. Pharmacologic Actions of Mediators of Anaphylactic Shock 17 2. B i o l o g i c a l l y Active Substances i n Mast C e l l s 18 3 . Agents That Release Substances from Mast C e l l s . . . . . 19 4. Agents That I n h i b i t Histamine Release and Mast C e l l Degranulation Induced by Antigen and 48/80.... 19 5* Cyclohexanecarboxamides 40 6. Secondary Aromatic Amines of Cyclohexylmethyl Derivative ^3 7. T e r t i a r y Ethylenediamine Derivatives (XII) 48 8 . Secondary Aromatic Amines of ^-Dimethylamino-ethyl Derivative (XIV) 50 9 . T e r t i a r y Ethylenediamine Derivatives (XV) 53 10. T e r t i a r y Cyclohexanecarboxamides (XVI) 5^ 11. Some Solvents Which Have Been Used Successfully for the Photocycloaddition Reaction • 58 12. Results for Antihistaminic and A n t i c h o l i n e r g i c Tests 98 13. Competitive Antagonists......... 152 14. Non-competitive Antagonists. 153 v l i i LIST OF FIGURES FIGURE Page 1. General Mechanism of Histamine Release from Mast Cells 21 2 . Schematic Representation of Receptor for Histamine in Ileum of the Guinea-pig 23 3 . Schematic Representation of the Forces Involved in the Interaction of Histamine with Its Hypothetical Receptor Site 25 4. Schematic Representation of the Binding of a Metal to One Molecule of Histamine 27 5 . Intermolecular Hydrogen Bonding between Two Molecules of Histamine 28 6. NMR Spectrum of N,N-Dimethyl-N'-3-Pyridazyl-ethylenediamlne 51 7. NMR Spectrum of N,N-Dimethyl-N'-2-Pyrazyl-e thylenedlamlne 5 2 Cumulative Dose-Response Curves of: 8 - 1 0 . N,N-Dimethyl-N•-CyclohexyImethyl-N'-Fhenylethylenedlamine (Ila) vs Histamine..... 118 11 - 13. N,N-Dimethyl-N'-Cyclohexylmethyl-N'-2-Pyridylethylenediamine (lib) vs Histamine.. 119 14-16. N,N-Dimethyl-N'-CyclohexyImethyl-N'-2-Pyrlmidylethylenediamine (lie) vs Histamine 121 17 - 19. N,N-Dimethyl-N'-Cyclohexylmethyl-N*-2-Pyrazylethylenedlamine (lid) vs Histamine.... 122 20 - 2 2 . N,N-Dlmethyl-N'-Cyclohexylmethyl-N'-l-Naphthylethylenediamine (lie) vs Histamine... 124 23 - 25 . N,N-Dimethyl-N'-Cyclohexylmethyl-N'-5-Quinolylethylenedlamine (I l f ) vs Histamine... 125 26 - 2 9 . N,N-Dimethyl-N'-Cyclohexylmethyl-N'-Phenylethylenediamine (Ila) vs Acetylcholine 127 i x FIGURE Page Cumulative Dose-Response Curves of: 30 - 32. N,N-Dlmethyl-N'-Cyclohexylmethyl-N'-2-Pyridylethylenedlamlne ( l i b ) vs Acetylcholine 129 33 - 35. N,N-Dlmethyl-N'-Cyclohexylmethyl-N'-2-Pyrlmldylethylenediamine ( l i e ) vs Acetylcholine 130 36 - 38. N,N-Dimethyl-N*-Cyclohexylmethyl-N'-2-Pyrazylethylenediamlne ( l i d ) vs Acetylcholine 132 39 - 42. N,N-Dimethyl-N'-Cyclohexylmethyl-N'-1-Naphthylethylenediamine ( l i e ) vs Acetylcholine 133 43 - 45. N,N-Dlmethyl-N'-Cyclohexylmethyl-N'-5-Quinolylethylenediamine ( I l f ) vs Acetylcholine 135 D E D I C A T I O N niy wife Sarko. PART I INTRODUCTION I n a s e a r c h t o u n d e r s t a n d t h e r e c e p t o r s f o r e t h y l e n e -d i a m i n e - t y p e a n t i h i s t a m i n e s , o u r l a b o r a t o r y has shown t h a t one o f t h e a r o m a t i c r i n g s i n A n t e r g a n (I) c o u l d be r e p l a c e d by an a l i -c y c l i c s y s t em ( e . g . c y c l o h e x y l , I l a ) w i t h no l o s s i n a n t i h i s -t a m i n i c a c t i v i t y (1, 2 ) . T h i s was i n c o n t r a s t t o t h e c o n v e n -t i o n a l s t r u c t u r e a c t i v i t y r e l a t i o n s h i p w h i c h a p p e a r e d t o ( I ) r e q u i r e t h e p r e s e n c e o f b o t h a r o m a t i c r i n g s i n o r d e r t o have h i g h a c t i v i t y . Rep l a cemen t o f b o t h a r o m a t i c r i n g s by c y c l o a l i -p h a t i c r i n g s g r e a t l y d e c r e a s e d t h e a c t i v i t y . Thus , i t was a p -p a r e n t t h a t t h e a c t i v i t y was due m o s t l y t o t h e r e m a i n i n g a r o m a t i c m o i e t y . I t was t h e pu rpose o f t h i s s t u d y t o i n v e s t i g a t e how v a r i a t i o n s i n s i z e and s t r u c t u r e o f t h e a r o m a t i c m o i e t y (Rg g r oup i n I I ) i n t h i s a l i c y c l i c s u b s t i t u t e d sys tem ( I I ) wou l d a f f e c t t h e a c t i v i t y a t t h e r e c e p t o r s i t e s . A t t e m p t s have a l s o been made t o s y n t h e s i z e o xe t ane d e r i v a t i v e s ( I I I o r I V ) w h i c h were s t r u c t u r a l a n a l o g u e s o f d i phenhyd r am ine ( V ) , however , s y n t h e t i c d i f f i c u l t i e s p r e v a i l e d and t h e r e s u l t s were u n s u c c e s s f u l . A l l t h e compounds s y n t h e s i z e d i n t h e ( I l ) - s e r i e s have been f o u n d t o be u s e f u l i n f u r t h e r d e f i n i n g t h e s t r u c t u r e -2. ( I D R t = CH 2» R 2 = (a) phenyl, (b) 2-pyridyl, (c) 2-pyrimidyl, (d) 2-pyrazyl, (e) 1-naphthyl, ( f ) 5-quinolyl. R. = -C-; R~ = (g) 2-pyrimidyl, (h) 2-pyrazyl. l it (V) a c t i v i t y r e l a t i o n s h i p s of antihistamines. The r e s u l t s obtained from pharmacological t e s t i n g of these compounds give more e v i -dence i h d i r e c t support of the histamine/antihistamine receptor model (3) and the mode of binding of diphenhydramine (V) to the receptor as proposed by Nauta ( 3 , 4) recently. Also, since the a c t i v i t y of antihistaminic agents overlaps with that f o r ant i a c e t y l c h o l i n e agents, these new compounds have also been investigated for t h e i r c h o l i n o l y t i c a c t i v i t y . A. BIOGENESIS AND PHYSIOLOGY OP HISTAMINE AND ANAPHYLAXIS U n t i l about i 9 6 0 i t was believed that a l t e r a t i o n s i n the histamine content of tissues, the release therefrom, and i t s excretion, would r e f l e c t the p a r t i c i p a t i o n of histamine i n normal reactions. Schayer (5) showed that i n normal and patho-l o g i c a l reactions great changes i n the rate of endogenous histamine formation d i d occur without any corresponding changes i n tissue histamine content. From then on, the concepts of what are referred to as "non-mast-cell histamine" ( 6 , 7) and "histamine forming capacity (HFC)" (6) have r e a d i l y become accepted. The l a t t e r term (HFC) ref e r s to the rate at which histamine i s formed i n minced tissues, c e l l suspensions, or the whole body; i t denotes h i s t i d i n e decarboxylase a c t i v i t y i n numerical terms ( 6 ) . Non-mast-cell histamine indicates the histamine formed i n c e l l s other than mast c e l l s ; i n non-mast c e l l s the HFC can be s t r i k i n g l y high and the r e s u l t i n g histamine i s often "nascent" i n nature ( 7 ) . The nascent h i s -tamine i s seemingly involved i n c e r t a i n kinds of rapid tissue growth. Its action appears to be linked to the very process of i t s formation. The action of nascent histamine i s presum-ably not achieved by injected histamine (exogenous histamine), and i t s action i s not prevented by antihistamines. The biogenesis and physiology of histamine has r e -cently been extensively reviewed ( 8 , 1 3 ) . The p r i n c i p a l s i t e of histamine synthesis i s the mast-cell system ( 9 ) . In the r a t the evidence (8) appears overwhelming i n support of the 4 . view that the histamine contained i n tissues i s also formed therein. This was concluded from an experiment showing that germ-free r a t s , neither i n any p a r t i c u l a r tissue examined, nor i n the whole animal, contained le s s histamine than non-germ-free ones. Indeed, the uniformity i n tissue histamine content between the two groups should be taken as conclusive evidence that even i n non-germ-free rats fed on a histamine free d i e t , the histamine contained i n the various tissues i s endogenous i n o r i g i n . Also mast c e l l s were counted i n the mesentery, and no difference i n t h e i r number was found between germ-free and ordinary r a t s . In f a c t , every mammalian tissue that contained histamine, inclu d i n g white blood c e l l s , appeared capable of manufacturing the amine from h i s t i d i n e . I t thus seems l i k e l y that the mast c e l l s form h i s -tamine and store i t i n granules, from where i t can be released into the surrounding tissues when a suitable stimulus i s r e -ceived by the c e l l (11). Such pre-formed histamine appears to be the main source of histamine released i n the body, but Schayer (12) produced evidence that histamine can also be r e a d i l y formed i n response to p h y s i o l o g i c a l demand. This newly-formed histamine i s apparently not produced i n the mast c e l l s and has variously been c a l l e d non-mast c e l l histamine, induced histamine and nascent histamine ( 1 3 ) . Mast c e l l s are connective-tissue c e l l s widely d i s -tributed throughout the body, p a r t i c u l a r l y i n the lungs and l i v e r (14), although there are places where they are not found such as the wall of the stomach, human-epidermis and 5. the CNS. Histamine i s also present i n substantial amounts i n such nonmast-cell s i t e s . Besides histamine v i r t u a l l y a l l endogenous heparin was located i n the mast-cell system. Ro-dents' mast-cell also contained 5-hydroxytryptamine (5-HT) ( 9 ) . At l e a s t two d i f f e r e n t enzymes are able to form histamine from h i s t i d i n e ( 1 5 ) . One i s a nonspecific L-aminoacid decarboxylase (dopa decarboxylase), and the other i s a s p e c i f i c h i s t i d i n e decarboxylase. The former acts on many aromatic amino acids, such as 5-hydroxytryptophan and 3 ,^-dihydroxyphenylalanine. The l a t t e r ( s p e c i f i c decarboxy-lase) acts only on h i s t i d i n e , and i t i s t h i s enzyme that i s found i n the stomach, mast c e l l s , and f e t a l l i v e r of the r a t s ; t h i s enzyme i s the p r i n c i p a l enzyme involved i n vivo r e q u i r i n g pyridoxal - 5-phosphate as a cofactor. The enzyme can be i n h i b -i t e d by ot-methylhistidlne as well as by 4-bromo-3-hydroxy-benzyloxyamlne. Furthermore, t h i s enzyme i s inducible and the histamine-forming capacity at the nonmast-cell s i t e s i s sub-j e c t to regulation by various p h y s i o l o g i c a l and other f a c t o r s ( 1 3 ) . Present evidence (35) indicates that nonspecific aro-matic L-aminoacid decarboxylase does not p a r t i c i p a t e i n h i s t a -mine synthesis i n vivo. In mast c e l l s (rat) histamine i s stored i n basophil granules consisting mainly (over 95$) of a heparln-proteln complex. This heparln-proteln complex i s water insoluble and i s held together by the strong a t t r a c t i v e forces between two macromolecules with opposite charges, the acid heparin and the basic*: protein, ( .16 )•.„,- The sulphuric',?.ci:*'.groupsk o.f t h e , : .heparin" are e s s e n t i a l for the formation of the heparln-proteln complex, the desulphated heparin lacking complex forming capacity ( 1 7 ) . The histamine binding s i t e s i n the granules are carboxyl groups belonging to the protein part of the heparln-proteln complex ( 1 6 ) . Recent observations ( 1 8 ) also Indicate that carboxyl groups, and not sulphuric a c i d groups, are the most l i k e l y i o n i c binding s i t e s f o r histamine i n the mast c e l l granules. Uvnas ( 1 9 ) suggested a possible gross structure of the granular heparln-proteln-histamlne complex (VI): i n the i n t a c t complex at pH 7 the acid groups of heparin should be linked to the amino (lmino) groups of the basic protein, thus leaving only the COO~-group of the protein a v a i l a b l e f o r histamine binding. In t h e i r binding of histamine the granules show the properties of a weak cation exchange material, allowing an exchange with other cations (e.g. an equivalent exchange between histamine and sodium). Thus on degranulation the mast c e l l s discharge mechanism allows an immediate e x t r a c e l l u l a r release of h i s t a -mine from discharged granules ( 1 6 ) by exchanging histamine Heparin Protein Histamine CH (VI) histamine-containing granules and the weak cation exchange 7. with the cations present In the e x t r a c e l l u l a r medium. Histamine i s also formed i n the nervous system. Within the central nervous system, except f o r r e s t r i c t e d areas ( i . e . pineal body, portions of the hypophysis and choroid plexus), mast c e l l s are absent and the histamine there must be held In neural c e l l s , or supporting c e l l s , or both (20, 2 1 ) , It i s of in t e r e s t that the pattern of d i s t r i b u t i o n of histamine i n the br a i n i s much l i k e that of the other amines- norepine-phrine, dopamine, and 5-HT; the concentration i s highest i n the hypothalamus (where indeed, there i s twice as much h i s t a -mine as 5-HT), intermediate i n the thalamus and midbrain, and lowest i n the cerebral cortex and white matter (20, 2 1 ) . In the peripheral nerves histamine content i s grossly c o r r e l a t i b l e with mast c e l l count (22, 2 3 ) . Recent i n vivo investigations (24, 25, 26) have shown that histamine i n the brain i s formed by the act i o n of a s p e c i f i c h l s t l d i n e decarboxylase. Campos and Jurupe (27. 28) suggest the involvement of a choliner g i c mechanism i n h i s -tamine synthesis. They found that e l e c t r i c a l stimulation of rat paws depleted b r a i n acetylcholine and concomitantly i n -creased brain histamine l e v e l s . Intraperitoneal i n j e c t i o n s of cholinesterase i n h i b i t o r s (physostlgmine s a l i c y l a t e 0 .2 mg./kg. or an equimolar dose of parathlon) also increased brain acetylcholine and histamine. The brain acetylcholine released during central nervous stimulation apparently t r i g -gered a mechanism to accelerate brain histamine synthesis i n vivo (27, 28). 8 . In the peripheral nerve i t i s l i k e l y that at l e a s t some of the decarboxylase i s i n the associated mast c e l l s and therefore i s a s p e c i f i c h i s t i d i n e decarboxylase (20). Ryan and Brody (29) studied the r o l e of histamine as a sympathetic neurotransmitter released during active r e f l e x v a s o d i l a t i o n i n the canine autonomic nervous system and found supportive e v i -dence that histamine i s l i b e r a t e d from sympathetic h i s t a -minergic f i b e r s during r e f l e x v a s o d i l a t a t i o n . The neurogenic stores of histamine appear to be non-mast c e l l i n nature. There i s s t i l l no certainty, however, that t h i s histamine was intimately r e l a t e d to axons, and the existance of such h i s t a -minergic nerves i s quite uncertain (21). To what compound or compounds histamine i s bound i n p a r t i c u l a t e material of brain i s not known (20). Histamine forms complexes with a c i d i c l i p i d s and other a c i d i c substances, including sulfomucopolysaccharides to which histamine i n mast c e l l s i s believed to be bound (30). Like histamine,~sulfo-mucopolysaccharides are present i n gray matter and i n the micro-somal f r a c t i o n of b r a i n (30). In the mast c e l l s found i n nerve f i b e r s and i n structures associated with brain, i t might be assumed that histamine i s bound as i n other mast c e l l s (19). Most knowledge about catabolism of histamine was learned from the various C- l a b e l l e d catabolites excreted i n the urine a f t e r the i n j e c t i o n of a small amount of C-histamine. Investigations of the urinary pattern of radioactive histamine catabolites have given r e s u l t s which are d i f f i c u l t to i n t e r -pret, and perhaps, do not r e f l e c t p l a i n l y p h y s i o l o g i c a l events 9 . (8). Studies with injected histamine! (exogenous histamine) cannot reveal p r e c i s e l y what i s happening to endogenously formed histamine ( 1 5 ) . I t i s assumed, although i t i s d i f f i c u l t to prove, that small amounts of exogenous histamine are catabolized i n the same way as endogenous histamine. Otherwise t h e i r functional status i s perhaps not the same i n that endogenous histamine seems to exert functions which can not he reproduced by exog-enous histamine. In man and most laboratory animals there are two major pathways of histamine .catabolisin ( 2 1 ) . The more important pathway involves r i n g N-methylation and i s catalyzed by the enzyme histamine-N-methyl-transferase which methylates h i s t a -mine without a f f e c t i n g other imidazoles or aromatic amines ( 3 1 ) . Most of the product, methylhistamine, i s converted by MAO to raethylimidazole a c e t i c a c i d . R e i l l y and Schayer ( 3 2 ) showed methylhistamine i n h i b i t s histamine methylation i n vivo. In the other pathway histamine undergoes oxidative deamination catalyzed by the enzyme known as histaminase, with the produc-t i o n of imidazole a c e t i c a c i d and i t s r i b o s i d e . I t i s not agreed that histaminase i s the same as diamine oxidase ( 1 5 , 2 0 ) . The various metabolites are then excreted i n the urine. The r e l a t i v e r o l e s of these enzymes i n the metabolism of endogenous histamine have not yet been established, nor i s i t clear why histaminase a c t i v i t y of plasma r i s e s sharply during pregnancy ( 1 3 ) . As mentioned above, the catabolism of endogenous h i s t a -mine, remains a topic for further study (8). Ring, methylation, i s a. major pathway of, histamine-10 . catabolisra i n brain ( 3 3 ) . The brain contains N-methyl-transferase and monoamine oxidase but does not contain diamine oxidase ( 2 0 ) . A f t e r perfusion of cerebral v e n t r i c l e s i n cats with histamine, both methylhistamine and methyliraidazole a c e t i c a c i d are detected. The regional d i s t r i b u t i o n of histamine methyltransferase i n monkey brain, measured i n homogenates, was shown as follows: The hypothalamus, which i s e s p e c i a l l y r i c h i n histamine, showed high a c t i v i t y . The hypophysis, which has l e s s histamine than the hypothalamus, had the greatest a c t i v i t y of a l l the areas measured ( 2 0 ) . There are numerous reasons f o r supposing that h i s t a -mine has important functions i n the body's economy. Histamine not only i s an extremely a c t i v e substance, capable of mimicking a v a r i e t y of p h y s i o l o g i c a l and pathological phenomena, but also occurs n a t u r a l l y i n tissues throughout the body, most of which contain the enzymes that both form i t and ina c t i v a t e i t ( 2 1 ) . Although some of the most f a m i l i a r hypotheses on the function of histamine have been concerned with pathological physiology and, i n p a r t i c u l a r , with anaphylaxis, a l l e r g y , i n j u r y , and shock, i n d i c a t i o n s of a normal p h y s i o l o g i c a l function are accumulating r a p i d l y with the development of highly r e f i n e d methods of study. For example, there i s now impressive evidence that endogenous histamine i s the f i n a l common mediator of gas-t r i c secretory responses, and there are some grounds for sup-posing i t to be involved i n t i s s u e growth and r e p a i r , i n regu-l a t i o n of the m i c r o c i r c u l a t i o n , and i n the functioning of the CNS'(3, 1 3 0 . 1 1 . The mast-cell histamine has an important.role i n the pathological physiology of tissue responses to anaphylaxis, i n j u r y , and c e r t a i n drugs (3^)« The histamine stored i n mast c e l l s i s not a c t i v e l y metabolized, and turnover rate i s slow. When t i s s u e s r i c h i n these c e l l s are depleted of t h e i r h i s t a -mine stores, i t might take weeks before they are returned to normal. On the other hand histamine i n non-mast c e l l s i t e s undergoes a b r i s k turnover and the histamine synthesized i s released at once rather than stored. This histamine c o n t r i -butes importantly to the d a i l y excretion of histamine and i t s metabolites i n the urine. The histamine-forming capacity of such non-mast c e l l s i t e s i s subject to regulation by various p h y s i o l o g i c a l f a c t o r s (13» 3*0. I t i s t h i s non-mast c e l l h i s -tamine that was important i n various p h y s i o l o g i c a l processes which are discussed here. Endogenous histamine i s believed to be the f i n a l common stimulator of the p a r i e t a l c e l l of the g a s t r i c mucosa and therefore i s the chemical mediator f o r g a s t r i c secretion. Feeding i s the only hitherto recognized p h y s i o l o g i c a l circum-stance i n which preformed t i s s u e histamine has been shown to be mobilized ( 8 ) . In sections of the stomach the number of acid-secreting c e l l s p a r a l l e l the amount of histamine, which i s held i n enterochromaffin-like c e l l s i n the g a s t r i c mucosa ( 1 5 ) . Furthermore, the output of histamine into the g a s t r i c juice p a r a l l e l s the volume of hydrochloric a c i d secretion, whether t h i s secretion i s induced by the i n j e c t i o n of g a s t r i n , by the ingestion of food,, or by the. i n j e c t i o n of cholinergic; 1 2 . drugs ( 1 3 ) . This output i s p a r a l l e l e d by an increased syn-the s i s of histamine i n the stomach, which suggest a kind of feedback control ( 1 3 ) » Moreover, the demonstration of a lowered mucosal histamine content associated with e x c i t a t i o n of a c i d secretion i s taken as strong evidence that endogenous h i s t a -mine i s the chemical mediator f o r g a s t r i c secretion ( 8 ) . Schayer ( 3 6 ) recently pointed out that histamine ap-pears to be the most important amine i n microcirculatory r e -gu l a t i o n . An e a r l i e r view that bradykinin was the mediator of fu n c t i o n a l v a s o d i l a t a t i o n has recently been rendered doubtful. The c o r r e l a t i o n between changes i n h i s t i d i n e decarboxylase a c t i v i t y and c i r c u l a t o r y changes was taken by Schayer ( 3 7 ) as a basis f o r the hypothesis that induced histamine serves as a governor of the f u n c t i o n a l state of the terminal vessels, i n -duced histamine being formed at a rate required to maintain homeostasis. For example, induced histamine serves as an i n t r i n s i c mediator of vascular d i l a t a t i o n whose u t i l i z a t i o n rose during excercise (3<3)» The i n t r i n s i c m icrocirculatory d i l a t o r e f f e c t of histamine i n d i r e c t l y influences every aspect of body economy ( 3 8 ) . A moderate histamine excess increases n u t r i t i v e blood flow and permits c e l l s to reach t h e i r f u l l p o t e n t i a l i t i e s . Conversely, a histamine i n s u f f i c i e n c y leads to impaired c e l l n u t r i t i o n and to development of abnormalities i n c e l l chemistry and function. The f i r s t r e a l i n d i c a t i o n that histamine might be involved i n human reproduction was the discovery that the h i s -taminase a c t i v i t y of the placenta increases gr e a t l y during 1 3 . pregnancy (8). The enzyme i s produced under the influence of progesterone and can be measured i n the plasma of pregnant women ( l l ) . Determination of maternal histaminase has been used as a c l i n i c a l guide i n cases of threatened abortion, as i t has been shown that plasma histaminase a c t i v i t y increasing according to the normal pattern indicates a good prognosis, whereas a f a l l below the normal range presages spontaneous abortion (39). The meaning of the histaminase increase i n human pregnancy i s open to question. Smith (40) has demon-strated that placental histaminase i s capable of o x i d i z i n g a number of amines, several a l i p h a t i c diamines are oxidized even f a s t e r than histamine. This implies a more fundamental s i g -n i f i c a n c e of histaminase i n c e l l metabolism. Much evidence indicates that a conspicuously high histamine-forming capacity i s present i n many tissues under-going r a p i d growth or r e p a i r , such as embryonic t i s s u e , regen-erating l i v e r , bone marrow, wound ( 15» ^1) and granulation t i s s u e , and malignant growths i n various species, p r i n c i p a l l y r a t s (8, 1 3 ) . This implies the nascent histamine has a r o l e i n anabolic processes. Other supporting evidence (42, 43, 44) that neonatal rat brain contains a considerably higher concen-t r a t i o n of histamine than adult r a t brain and has both h i s t a -mine synthesizing and ca t a b o l i z i n g enzymes as early as 3 days a f t e r b i r t h , suggest a function f o r histamine i n modulating the growth processes of the neonatal brain. F i n a l l y , the possible r o l e of endogenous histamine i n the nervous system has been investigated. The e f f e c t of 14. histamine on neural tissues has recently been reviewed ( 2 0 ) . S p e c i f i c methods f o r measuring brain histamine show that some psychotropic drugs cause changes i n histamine l e v e l s . Reser-pine i n the dose range 0 . 5 to 10 mg./kg. does not a f f e c t h i s t a -mine l e v e l i n the hypophysis but lowers i t i n other areas, i n hypothalamus to about hQ%$ i n thalamus to 64$ of control ( 13» 2 0 ) . Chloropromazine, when given three times at a dose of 50 mg./kg., increases histamine l e v e l s i n hypothalamus by about $0% and i n the medial thalamus by 32%, by i n h i b i t i n g histamine methyltransferase ( 1 3 , 2 0 ) . The phenothiazines f a i l to r a i s e histamine l e v e l s i n the hypophysis, perhaps because most of the histamine i s i n the mast c e l l s which do not me-thylate histamine at a measurable rate ( 4 5 ) . Also, i t i s per-tinent that several antihistamines had central actions. Fur-thermore, histamine has a va r i e t y of central e f f e c t s , including stimulation of sympathetic centers and convulsions when i n t r o -duced d i r e c t l y into the cerebral v e n t r i c l e s or into the brain t i s s u e ( 2 1 ) . A l l t h i s evidence hints that histamine has impor-tant functions within the bra i n . In the peripheral nervous system, i t has been sug-gested that the va s o d i l a t a t i o n that follows elevation of blood pressure might be due to a l i b e r a t i o n of histamine from the sympathetic nerves ( 2 0 ) . Ryan and Brody ( 2 9 ) recently provided supportive evidence for the postulate that histamine was l i b -erated from sympathetic histaminergic f i b e r s during r e f l e x v a s o d i l a t a t i o n . Studies with the exogenous histamine, however, were i n accord with the idea that histamine was the vasodi l a t o r . Thus, Beck et a l (46) observed that norepinephrine-induced r e -f l e x d i l a t a t i o n resulted from the increased neural release of histamine into the venous effluent of dog g r a c i l i s muscle. Green (20) suggested that some of the histamine that appeared to function i n peripheral nerves might derive from mast c e l l s . These observations lead to the conclusion that histamine i s a neuromediator f o r the baroreceptor depressor r e f l e x . The h i s -tamine l i b e r a t e d by c e l l i n j u r y has a r o l e to play i n the i n i -t i a t i o n of sensory impulse evoking pain and i t c h . Also, the analgesic e f f e c t of l o c a l l y administered antihistamine drugs and of compound 48/80 (potent histamine l i b e r a t o r ) suggest that histamine i s the mediator f o r cutaneous pain. In conclusion, the p r i n c i p a l actions of histamine, i n the ranking as we see them at present, namely the part played i n g a s t r i c secretion and i n metabolic processes of t i s s u e growth and protein synthesis, are not obviated by the common a n t i -histamines. Recently Black, et a l (129) have found that burimamlde competitively antagonizes histamine-stimulated g a s t r i c secretion. Endeavours for decades have not succeeded i n demonstrating histamine release i n any purely p h y s i o l o g i c a l event. Now, at l a s t , by sheer chance, a case of p h y s i o l o g i c a l histamine r e -lease has been shown to occur, namely a lowering of the g a s t r i c mucosal histamine content on feeding a rat deprived of food. Although the ph y s i o l o g i c a l r o l e of histamine i s s t i l l poorly understood, there i s enough evidence to j u s t i f y further inves-t i g a t i o n into the r o l e of histamine i n p a r t i c i p a t i n g i n every aspect of body economy ( 3 8 ) . 16. In the following discussion the now-classical involve-ment of histamine release from mast c e l l s i n various patholog-i c a l processes, p a r t i c u l a r l y i n anaphylaxis, w i l l be considered. Histamine release i n anaphylaxis has recently been extensively reviewed (4?, 48, 49, 50, 51 and 5 2 ) . In b r i e f , the anaphy-l a x i s picture which emerges i s that of a system activated by contact with the antigen to which the animal had been s p e c i f i -c a l l y s e n s i t i z e d by previous exposure. An antigen might be a polysaccharide or protein or a simple compound that i s covalently bound to a protein (53)* As a defense against the s p e c i f i c antigen, the mammalian organism manufactures a s p e c i -f i c antibody from the g l o b u l i n f r a c t i o n of the blood. Renewed contact of antigen and antibody at a l a t e r date i n i t i a t e s a chain of events r e s u l t i n g i n release of histamine, serotonin, a l i p i d slow-reacting substance (SRS), bradykinin (polypeptides) and other compounds from mast c e l l , basophilic leucocytes, and p l a t e l e t s (53)» There i s no general requirement f o r hemolytic complement i n t h i s r eaction ( 5 0 ) . The r e a c t i o n involves at l e a s t two steps, i . e . , f i x -a t i o n of bivalent immunoglobulin and a c t i v a t i o n of processes leading to release of vasoactive mediators from the c e l l (Table 1) ( 5 2 ) . The actions of various mediators, and thus the prominence of these manifestations of shock, varies from one animal species to another ( 5 0 ) . For example, i n dogs, the more pronounced e f f e c t s of anaphylactic shock are hypotension, p o r t a l venous c o n s t r i c t i o n , and hemorrhage of the G-I t r a c t . In guinea-pigs, bronchoconstriction i s most prominent, and 17. anaphylaxis i n man might be manifested by a combination of u r t i c a r i a , hypotension, and bronchoconstriction (50, 5 2 ) . TABLE 1 PHARMACOLOGIC ACTIONS OF MEDIATORS OF ANAPHYLACTIC SHOCK Agent Hypotension Increased C a p i l l a r y Broncho-Permeability c o n s t r i c t i o n Histamine + Serotonin + or -Slow-reacting substance (SRS) Bradykinin + + + + + or + + + + Note i + = present} - = absent Some pharmacologically active agents that are stored i n t i s s u e mast c e l l s have been implicated i n anaphylactic shock. Mast c e l l s contain p r o t e o l y t i c enzymes and have the capacity to form or l i b e r a t e "slow-reacting substance"(SRS) (Table 2) ( 5 2 ) . The amines are bound i n in a c t i v e form to a complex of heparin and protein i n mast c e l l granules (see previous d i s -cussion and L i t . ( 1 6 ) ) . SRS, a l i p i d spasmogen, has not been completely characterized, but i t i s d i s t i n c t from prostaglan-dins (50 , 5 2 ) . Release of histamine and other substances from mast c e l l s can be induced by antigen-antibody reactions, by inj u r y , or by various chemical agents (Table 3)» The antigen-antibody i n t e r a c t i o n require f i x a t i o n of "anaphylactic" (Immunoglobulin E, IgE) antibodies ( 5 0 ) , but do not require complement ( 5 2 ) . TABLE 2 BIOLOGICALLY ACTIVE SUBSTANCES IN MAST CELLS Granules Particulate Cytoplasm C e l l Membrane Histamine Serotonin (rat, mouse) Dopamine (hamster, ungulates) Heparin Chymotrypsin (rat) Trypsin (man, dog) Leucineaminopeptidase (man, rat) Esteroprotease (mouse) Acid phosphatase Alkaline phosphatase Succinic dehydrogenase Amine oxidase Fumarase ATP Lacti c dehydrogenase Dopa decarboxylase H i s t i d i n e decarboxylase 5-Hydroxytryptophan decarboxylase Sulfurylase Sulfokinase ATP ATPase Phospholipase A SRS (?) 19. TABLE 3 AGENTS THAT RELEASE SUBSTANCES FROM MAST CELLS Selective Non-selective Undetermined Antigen-antibody Tissue i n j u r y Anaphylatoxin Cationic proteins P r o t e o l y t i c enzymes Bradykinin Amines Venoms Endotoxin (?) d-Tubocurarine Stilbamidine Morphine Polymyxin B Compound 48/80 The re a c t i o n i s s e l e c t i v e i n that constituents of the granules can be released without apparent damage to the c e l l membrane (60, 6 l ) . The release reaction can be blocked by low tempera-tures, by anoxia, and by a vari e t y of other i n h i b i t o r s of c e l l metabolism (Table 4) (5^, 55)• This has been taken as evidence TABLE 4 AGENTS THAT INHIBIT HISTAMINE RELEASE AND MAST CELL DEGRANULATION INDUCED BY ANTIGEN AND 48/80 SH group NH2 group Inhi b i t o r s of oxi - „, i n h i b i t o r s i n h i b i t o r s dative metabolism lemperature Iodoacetate Ninhydrin Anoxia Cold (0-20°C) p-Chloromercuri- D i n i t r o f l u o r o - Cyanide Heat (45°C) benzoate benzene D i n i t r o -N-ethylmaleimide Phenyliso- phenol o-Iodosobenzoate cyanate 2 0 . for an enzymatic process, but s p e c i f i c enzymatic pathways r e -main u n i d e n t i f i e d ( 5 2 ) . The most popular mechanism for release of histamine and other amines from mast c e l l s by antigen has been proposed by Uvnas ( 5 6 , 57 and 58) and has received supporting comments from other workers ( 2 1 , ^ 8 , 51 and 5 2 ) . Release of amines from mast c e l l s involves at l e a s t two d i s t i n c t steps, i . e . , exocytosis of the granules and displacement of amines from heparin-protein matrix ( 1 6 , 5 6 ) . In the re a c t i o n of mast c e l l s with antigen, peri-granular membranes become fused with each other and with the c e l l membrane so that the granule matrix i s excluded from the i n t e r i o r of the c e l l ( 5 0 , 5 9 ) . Once the granules are exposed to the external medium through channels formed by fused membranes or by extrusion to the surface of the c e l l s , the amines are r a p i d l y displaced by cations from the e x t r a c e l l u l a r f l u i d ( F i g . 1) ( 1 6 , 19 , 5 2 , 5 6 ) . The fundamental biochemical changes that induce t h i s r e a c t i o n are s t i l l obscure ( 5 2 ) . The experimental f i n d -ings so f a r have indicated that the biochemical mechanism i s heat-sensitive, requiring calcium and narrow pH range f o r ac-t i v i t y , i s i n h i b i t e d by SH reagents and other enzyme i n h i b i t o r s , and lacks a requirement of complement (48). Probably defor-mation of the mast c e l l membrane by reaction of antigen with f i x e d antibody induce physical changes i n the c e l l that stim-ulate rearrangement of i n t r a c e l l u l a r membranes through one or more enzymatic steps, e.g. cleavage of ATP by membrane ATPase and l i b e r a t i o n of calcium or other " i n t r a c e l l u l a r messengers" ( 5 2 ) . 2 1 . tigen Formation of SRS Histamine Serotonin Dopamine (?) FIG It General Mechanism of Histamine Release from Mast C e l l s . Antigen i n t e r a c t s with s p e c i f i c immunoglobulins associated with mast c e l l membranes. Exocytosis (Step 1) exposes the mast c e l l granules to the external medium and r e s u l t s i n f o r -mation of SRS. Amines are displaced from the granule matrix (Step 2 ) by cations i n e x t r a c e l l u l a r f l u i d . 2 2 . B. HISTAMINE RECEPTORS L i t t l e i s known of the nature of the "receptors" for histamine i n the d i f f e r e n t t i s s u e s , but two points are clear from studies of the s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p and the e f f e c t of s p e c i f i c blocking agents ( 2 1 ) . F i r s t , histamine receptors, whether on smooth muscles, c a p i l l a r y endothelium, nerves, ganglion c e l l s , chromaffin c e l l s , or g a s t r i c glands, are d i s t i n c t from each of the various "receptors" activated by other autacoids such as the catecholamines, 5-HT, ACh, and the polypeptides bradykinin and angiotensin. Second, i t i s obvious that there i s more than one pharmacologically i d e n t i f i a b l e histamine receptor. Ash and S c h i l d ( 6 2 ) d i f f e r e n t i a t e the histamine receptors into at l e a s t two classes. The s p e c i f i c antagonism of some actions of histamine by low concentrations of antihistamine drugs characterize one type of histamine r e -ceptor for which they suggest the symbol H^. Such receptors occurr i n guinea-pig ileum and bronchi. Several other actions of histamine, for example, stimulation of g a s t r i c a c i d secre-t i o n , i n h i b i t i o n of r a t uterus and stimulation of i s o l a t e d a t r i a , can not be antagonized by common antihistamines ( 1 2 9 ) . These actions are l i k e l y to be--mediated .by-.non-H- receptors. Histamine receptor was f i r s t envisaged by Rocha e S i l v a ( 6 3 , 6 4 ) . He proposed a model for histamine receptor i n the ileum of the guinea-pig. The model was constructed on the basis of the following ideas then acceptedi (a) the active form of histamine under p h y s i o l o g i c a l conditions was that i n which hydrogen bonding was possible between the amine nitrogen 2 3 . (N +) of the side chain and the pyridine nitrogen (N) of the imidazole r i n g ; (b) the secondary anchorage group of histamine to f i t i t s receptor s i t e , could be the imine (=NH) r a d i c a l of the imidazole r i n g and a carbonyl group i n a polypeptide chain; (c) the pK of the receptor s i t e was found to be around pH = 7.10-7.00 ( t h i s pH range was l a t e r confirmed by Ariens (81) and Gero (82)), thus suggesting a h i s t i d i n e moiety at the receptor s i t e . The function of histamine would be to protonate the py-r i d i n e nitrogen of the receptor h i s t i d i n e , s h i f t i n g the double bond and re l e a s i n g a high energy r a d i c a l h y pothetically bound to the ''pyrrole"1 nitrogen of the h i s t i d i n e moiety, as shown i n F i g . 2. FIGURE 2 Schematic representation of the receptor f o r histamine i n the ileum of the guinea p i g. P However, t h i s model did not include the pyridine nitrogen of histamine which was important f o r H^ histamine ac-t i v i t y (3» 6 2 ) . Kier ( 6 5 ) also pointed out i n his molecular o r b i t a l c a l c u l a t i o n s that the preferred conformations of 24. histamine could not form an intramolecular hydrogen bond be-tween the side chain N+-H and the pyridine nitrogen (N) because of an unfavorable geometry and l e s s than minimal energy consid-er a t i o n . K i e r (65) further proposed a dual a c t i v i t y of h i s t a -mine to be a consequence of the existence of the two preferred conformations i n equilibrium. One of these conformations placed the quaternary nitrogen of the side chain and the p y r i -o dine nitrogen of histamine 4 . 5 5 A apart, which was quite com-o parable to the 4.8 A estimated f o r the internltrogen distance i n the antihistaminic t r i p r o l i d l n e . Therefore, t h i s was the one s p e c i f i c f o r the histamine receptor. The second pre-o f e r r e d conformation with an internitrogen distance of 3.60 A was responsible f o r a c t i o n other than those on the receptor such as stimulation of g a s t r i c a c i d secretion. For d i s t i n c t i o n , K i e r designated the l a t t e r receptor as H^. I t needs a s p e c i f i c antagonist to define the s t r u c t u r a l c h a r a c t e r i s t i c s of H 2 re-ceptor. The recent f i n d i n g of burimamlde (129), which can competitively antagonize, the actions of histamine on r a t uterus and stomach, may help to unravel the histamine H receptor. In 1969 Eocha e S l l v a ( 6 6 ) improved h i s o r i g i n a l H 1 receptor ( 6 3 , 64) based on the findings by K i e r ( 6 5 ) . The model then suggested that histamine was attracted to the spe-c i f i c receptor s i t e (H^) by: (a) strong e l e c t r o s t a t i c i n t e r -a ction between the pyridine (N~) nitrogen of the h i s t i d i n e moiety and the strongly charged protonated nitrogen (N +) of the histamine cation, and (b) the r e c i p r o c a l l y inverted dipoles i n the peptide l i n k of the receptor and the carbon ( C + ) -25. pyridine nitrogen (N~) of the imidazole r i n g of the agonist ( F i g . 3 ) ' These anchorage s i t e s were almost the same as those proposed by Nauta et a l ( 3 ) . FIGURE 3 Schematic representation of the forces involved i n the i n t e r a c t i o n of histamine with i t s hypothetical receptor s i t e . (E) e l e c t r o s t a t i c s (D-D) dipole-dipole i n t e r a c t i o n s . Protonation of the pyridine (-N=) nitrogen of the h i s t i d y l moiety of the r e -ceptor would s h i f t double bond to -C=N- p o s i t i o n . I - C - N - H i - N - ' X C - N * I I • O H - Receptor 6- &+ + s i t e __ i / 2 ^  N -< N— Histamine cation ( D - D ) (E) The French worker A l l a i n (67) also proposed that histamine promoted the a c t i v a t i o n of a receptor by a transfer of a proton, very l i k e l y by accepting a proton from the r e -ceptor or, i n a few cases, by gi v i n g a proton to the receptor, The analysis of the physico-chemical properties of histamine showed that i t could exist i n the body i n tautomeric, i o n i c , and conformational forms (67). This fact might explain why histaminic receptors could not be absolutely s i m i l a r . A l l a i n ' s view gave some support f o r Kier's dual a c t i v i t y of 26. histamine at the receptors. Paiva et a l (63) found, i n aque-ous solutions of histamine, that no intramolecular hydrogen bond was possible between the side chain amino group and an imidazole nitrogen atom. However, Kier's work ( 6 5 ) has recently been se-verely c r i t i c i z e d by Green et a l ( 6 9 ) . They ( 6 9 ) commented that Kier's work ( 6 5 ) was based on the extended Hiickel method which has been described as givin g "hopelessly inaccurate r e -s u l t s " and "worthless as a procedure for predicting the struc-tures or chemical behavior of molecules". Also, i n Kier's work the dihedral angle was rotated every 6 0 ° , a procedure that could miss important conformers. In t h e i r studies the INDO (Intermediate Neglect of D i f f e r e n t i a l Overlap) molecular o r b i t a l method was used. The t o t a l energy of the molecule was minimized as a function of two dihedral angles, Q/^p a n < * 6 ^ - ^ for histamine (VII). N^CH 2-CH 2-NH 2 a ( V I I ) The angles were rotated at JO - i n t e r v a l s and when necessary every 15°» The r e s u l t f o r histamine free base ap-peared that approximately 9&% of the histamine would be i n the configuration Q^_Q= 1 5 0 ° and 6 ^ . ^ = 3 3 0 ° ' This conformation 2 7 . would appear to be s a t i s f a c t o r y for metal binding ( F i g . k) and, moreover, r o t a t i o n of one of the side-chain amino FIGURE k Schematic representation of the binding of a metal to one molecule of histamine. hydrogens revealed that a suitable bond distance was a t t a i n -able between t h i s hydrogen and the N-3 of the imidazole r i n g to permit hydrogen bonding. An analysis of the histamine cation showed that one conformer had a s u b s t a n t i a l l y lower p o t e n t i a l energy, namely, where = 1 8 0 ° ( i n the plane of the imidazole ring) and O^-p = 30° or 330° ( the energy of the molecule was the same whether the quaternary nitrogen was 30° above or below the plane of the imidazole r i n g ) . The quaternary nitrogen was close to N-3 of the imidazole r i n g to permit hydrogen bonding. This r e s u l t was confirmed by Pullman et a l ( 7 0 ) who reported that they found at p h y s i o l o g i c a l pH, i n the c a t i o n i c form, the folded structure of histamine seemed to be more stable; i t included an intramolecular hydrogen bond. Green et a l ( 6 9 ) also found that the high f r o n t i e r electron density of C - 5 might permit i n t e r a c t i o n with an electron acceptor, e.g., the hydroxyl groups of heparin, an 28. OH- j z .interaction. Moreover, N-3 of the imidazole r i n g , being a locus of negative charge, might hydrogen-bond to N-1 of another imidazole r i n g (intermolecular hydrogen bond) ( F i g . 5). FIGURE 5 Intermolecular hydrogen bonding between two molecules of histamine. -N N — H N N — H From the above evidence ( F i g . k) Green suggested that histamine was stored i n the mast c e l l as a metal complex because of the fac t that high concentration of zinc and i r o n had been found i n the mast c e l l s (compared with the previous discussion on histamine storage i n mast c e l l s , structure VI and L i t . (19)). In conclusion, much work has to be done i n the future to c l a r i f y the structure requirements at the histamine receptor s i t e s . As to the mechanism for the stimulant effect of histamine on smooth muscle, i t has now been f i r m l y established that an action on the c e l l membrane to f a c i l i t a t e calcium entry did explain the stimulant e f f e c t of histamine and other autacoids on smooth muscle contraction (21). Histamine 29. promoted calcium i n f l u x by actions on the membrane that r e -sulted i n increased permeability to ions (including calcium). The i n f l u x of calcium ions provided the immediate i n t r a c e l l u l a r stimulus f o r a c t i v a t i o n of the actinmyosin system i n smooth muscle((71 ) . Likewise, one could at present only speculate on the mode of action of histamine and other autacoids to produce smooth muscle rel a x a t i o n , including v a s o d i l a t a t i o n . One permissible conjecture was that these agents somehow lowered calcium i o n concentration, perhaps by diminishing membrane permeability and spontaneous i n f l u x , and thus r e l a x a t i o n (21). C. ANTIHISTAMINES AND ANTIHISTAMINE RECEPTOR Antihistamines are drugs with the a b i l i t y to an-tagonize i n varying degree most, but not a l l , of the pharma-cologic actions of histamine ( 7 2 ) . They f a l l into that large group of pharmacological antagonists that appear to act by occupying the receptor s i t e s on the e f f e c t o r c e l l s to the exclusion of the agonist ( 7 3 ) . Apparently they bind with the histamine receptor without i n i t i a t i n g a response. Most a n t i -histamines act as competitive antagonists to histamine ( 7 3 ) . Antihistaminics seem to act as a n t i a l l e r g e n i c agents by more than one mechanism. The b e l i e f that they are antidotes to factors other than histamine i n the a l l e r g i c syndrome i s supported by the overlapping of antihistaminic 3 0 . and antiacetylcholine properties i n many of these agents. Serotonin and bradykinin, known to be released during anaphy-l a x i s i n small animals, are also antagonized by some a n t i h i s -taminics ( 5 3 ) . However, the value of antihistamines i n systemic a l l e r g i e s i s variable ( 8 , 2 1 ) . The discovery that i n anaphylaxis increased histamine formation takes place i n a l l t issues investigated, and that the elevated HFC p e r s i s t s f o r a long time a f t e r histamine release has ceased, appear h e l p f u l i n explaining the f a i l u r e of histamine antagonists to a f f o r d protection i n the l a t e r stage of anaphylaxis ( 8 ) . Histamine antagonists do not i n t e r f e r e with actions exerted by histamine formed and acting within the c e l l s . Moreover, i n anaphylaxis agents other than histamine (e.g. SRS released i n human lung) are believed to account for the symptoms which are not a l l e v i a t e d by histamine antagonists ( 8 , 2 1 , 5 3 ) ' Con-sequently, drugs such as theophylline, epinephrine and isopro-terenol are necessary, a c t i n g . d i r e c t l y to convert the broncho-c o n s t r i c t i o n to bronchodilatation ( 7 3 ) . Antihistamines act on the CNS to produce either a stimulant or depressive e f f e c t . Depression i s the most common and i s frequently used for i t s sedative e f f e c t as an adjunct to h y p e r s e n s i t i v i t y therapy and or insomnia. Use of a n t i h i s t a -mines i n parkinsonism has led to lessened r i g i d i t y and improved spontaneous movement and speech. Theoretical discussions were presented recently (7*0 regarding the s t r u c t u r a l requirement for anticonvulsant a c t i v i t y i n antihistaminic drugs. The ef f e c -tiveness of d i f f e r e n t antihistamines i n t h i s disease roughly 3 1 . p a r a l l e l e d t h e i r effectiveness i n motion sickness. The a n t i -motion sickness action of certa i n antihistamine drugs appears to be more re l a t e d to central a n t i c h o l i n e r g i c a c t i v i t y than to peripheral antihistamine a c t i v i t y ( 7 3 ) . Maximum antihistaminic a c t i v i t y i s found i n the following structure (VIII), where and R 2 are aromatic or heteroaromatic rings, one of which may be separated from X by ( V I I I ) a methylene groupj however, work i n t h i s laboratory ( 1 , 2) has shown that one of these aromatic rings may be replaced by an a l i c y c l i c system (e.g. cyclohexyl) and the a c t i v i t y i s retained. X i s CO, N, or CHj R^ i s generally an ethylene group or a two-carbon fragment of a nitrogen heterocyclic system, and branching decreases a c t i v i t y . The aromatic rings R^ and R 2 may or may not be ortho connected by Y, where Y = CH2» a heteroatom, or CH"2-heteroatom. R^ and R^ are methyl groups, but a small planar c y c l i c group may be employed advanta-geously. The t e r t i a r y nitrogen i s necessary since primary and secondary compounds are i n a c t i v e and quaternary compounds tend to be a n t i c h o l i n e r g i c . The antihistamine receptor was f i r s t proposed by Nauta et a l ( 3 ) i n 1966 a f t e r t h e i r extensive studies on 3 2 . a l k y l s u b s t i t u t i o n s a t p h e n y l g r oups i n d i phenhyd ram ine f o r a n t i h i s t a m i n i c and a n t i c h o l i n e r g i c a c t i v i t i e s ( 7 5 ) . They showed t h a t one o f t h e a r y l g roup p a r t i c i p a t e d i n an o v e r l a p i n t e r a c t i o n w i t h t h e e t h e r oxygen and a l s o t h a t i t was t h i s a r y l g r oup t h a t was assumed t o be more o r l e s s c o p l a n a r w i t h t h e C - 0 i n o r d e r t o have h i g h a n t i h i s t a m i n i c a c t i v i t y . S u p p o r t i n g e v i d e n c e was t h e p o t e n t a n t i h i s t a m i n i c t r i p r o l i d i n e ( I X ) i n w h i c h t h e p y r i d y l r i n g was c o p l a n a r w i t h t h e o l e f i n i c bond (65)1 c o p l a n a r i t y was a l s o known i n t h e 2 - ( d i m e t h y l a m i n o ) -e t h y l e t h e r o f t h e r i g i d 1 0 , l l - d i h y d r o d i b e n z o - ( a , d ) -c y c l o h e p t e n - 5 - o l (X ) wh i c h was 2-4 t i m e s more a c t i v e t h a n d i phenhyd ram ine ( 7 6 ) . To a c c o u n t f o r t h e i r f i n d i n g s Nau ta ( I X ) CH CH 3 3 (X) 33. proposed an antihistamine receptor showing three anchorage sites to accommodate histamine or the diphenhydramine molecule-(i) Histidine-N^ in the receptor site was hydrogen-bonded with the protonated NH2 and N(CH^)2 groups of the respective side chains; ( i i ) Serine-OH was hydrogen-bonded with the atom of the histamine and the oxygen atom of the ether, respectivelyi and ( i i i ) Phenylalanine-phenyl group was inju-complexation with the imidazole ring of the histamine and one of the two aryl rings of the ether, respectively. Recently, Kutter and Hansch (77) concluded through use of thermodynamically derived steric parameters (Es) and hydrophobic constants (TC) that substituents in the ortho and meta positions of the more highly substituted ring of diphen-hydramine had parallel deactivating effects and substituents in the ortho and meta positions of the less substituted ring had l i t t l e effect. Mono-para substitution had an activating effect up to an optimum size and then a deactivating effect; a second para substituent appeared to have a deactivating effect. These conclusions were in agreement with those of Nauta's ( 3 ) . Later, Nauta's research group (4) studied the thioether analogs of substituted diphenhydramine and found the in vitro antihistaminic a c t i v i t i e s of the thioether analogs were less than those of the corresponing 0 analogs, whereas 3 * . the a n t i c h o l i n e r g i c a c t i v i t i e s were greater. The decreased a c t i v i t y of the t h i o compounds may have two causes i ( i ) Since the free electrons of s u l f u r showed l i t t l e or no tendency to form associative structures, i t was doubtful whether an overlapping i n t e r a c t i o n between these free electrons and the ^.-electrons of one of the aromatic rings, such as postulated i n d i -phenhydramine, was a r e a l p o s s i b i l i t y , here. I f i n t e r a c t i o n did occur there were sound reasons for suspecting s t e r i c interference (bulk of the s u l f u r atom compared with oxygen) so as to lower the ac^ t i v i t y i n the t h i o analogs, ( i i ) Because hydrophilic bonds were less r e a d i l y formed by s u l f u r than by oxygen, the thio compounds would be more weakly bound to the receptor than the ethers. The differences i n a c t i v i t y found i n t h i s s e r i e s c l e a r l y supported the concept- that i n the diphenhydramines antihistaminic and a n t i c h o l i n e r g i c a c t i v i t y was more or l e s s complementaryi factors that increased the antihistaminic a c t i v i t y lowered the a n t i c h o l i n e r g i c e f f e c t , and vice versa. This complementarity between these two a c t i v i t i e s was also extendible to the chemical s h i f t value of the central hydrogen atom (78) and to the configuration of the active o p t i c a l isomers (79) of substituted diphenhydramines. The former (78) was shown by the fact that high antihistaminic a c t i v i t y was coupled with low s h i f t value, whereas high a n t i c h o l i n e r g i c a c t i v i t y was accompanied by a high s h i f t value? and the l a t t e r 3 5 . (79) by the fact that, i n ortho-substituted compounds, the highest a c t i v i t i e s always resided i n i d e n t i c a l configurations, one predominantly antihistaminic and i t s antipode c h i e f l y a n t i -c h o l i n e r g i c , whereas i n the para derivatives, the same antipode was the more active one i n both respects. The f a c t that ste r e o s e l e c t i v e a c t i v i t y was observed only when the asymmetric carbon atom was oc to the aromatic r i n g system indicated that regions adjacent to the histamine receptor (H^) might also be asymmetric. Further, antihistamines may be antagonists through a r e v e r s i b l e a l l o s t e r i c type of molecular perturbation mecha-nism and may not be blocking by binding to the same s i t e which binds histamine. Using bovine serum albumin (BSA) as a model i n v i t r o system the binding of antihistamines and C o r t i s o l have been studied by employing "coupled" ion-exchange membrane electrodes (80). The data indicated that at appropriate con-centrations antihistamines (e.g. diphenhydramine HCI) induced changes i n BSA which prevented binding of histamine. I t r e -mained open to question how f a r BSA could be compared to the p h y s i o l o g i c a l receptor. PART II DISCUSSION OF THE CHEMISTRY The p r e s e n t s t u d y i s c once rned w i t h t h e s y n t h e s i s o f s e ven e t h y l e n e d i a m i n e d e r i v a t i v e s ( I ) and two oxe t ane com-pounds ( I I and I I I ) . The e t h y l e n e d i a m i n e d e r i v a t i v e s ( I ) have been i n v e s t i g a t e d a s p o t e n t i a l a n t i h i s t a m i n i c s and p o s s i b l e a n t i c h o l i n e r g i c a g e n t s . The g e n e r a l r e a c t i o n sequence f o r CH -5I-N-CH2-CH2-NCch3 R 2 3 ( I ) R x = C H 2 , R 2 = ( a ) 2 - p y r l d y l . (b) 2 - p y r i m i d y l , ( c ) 2 - p y r a z y l , (d) 1 - n a p h t h y l , ( e ) 5 - q u l n o l y l . R-i = - C - , R~ = ( f ) 2 - p y r l m l d y l , (g) 2 - p y r a z y l . 1 II * 0 (II) (III) t h e p r e p a r a t i o n o f t h e e t h y l e n e d i a m i n e d e r i v a t i v e s ( I ) s t a r t e d w i t h t h e a p p r o p r i a t e p r i m a r y homo- and h e t e r o c y c l i c -a r o m a t i c a m i n e s . The p r i m a r y amines were r e a c t e d w i t h c y c l o -h e x a n e c a r b o n y l c h l o r i d e t o fo rm t h e a m i d e s . L i t h i u m a luminum 37. hydride was used to produce the desired secondary amines by reduction of the amides. In cases where the primary amines did not form the amides or where the amides could not be reduced to the corresponding secondary amines, the primary aromatic amines were condensed with £-dimethylaminoethyl chloride to form the desired secondary amines. In the former case, the appropriate secondary amine was condensed with dimethylaminoethyl chloride to obtain the f i n a l ethylenediamine d e r i v a t i v e s j while i n the l a t t e r case, cyclohexylmethyl bro-mide was used to condense with the appropriate secondary amine to form the f i n a l products. Generally speaking, the reaction sequence involving reduction of the amides appeared to give the better y i e l d s . Except f o r 3-aminopyridazine, the s t a r t i n g primary aromatic amines were a l l commercially a v a i l a b l e (e.g. 2-amino-pyridine, 2-aminopyrimidine, 2-aminopyrazine, 1-naphthylamine, and 5-aminoquinoline). 3-Aminopyridazine was unstable as a free base but the mono-hydrochloride or mono-picrate was stable ( 8 3 ) . Therefore, 3-arainopyridazine was prepared from 3 , 6-dichloropyridazine according to the methods of Steck and co-workers ( 8 4 ) . Reaction of 3 , 6-dichloropyridazine (IV) with ammoniacal ethanol at 125-130°C. gave 3-amino - 6-chloropyridazine (V) ( t h i s r e a c t i o n was performed i n a pressure reaction appa-ratus ( i . e . a bomb) instead of a shaking autoclave which was s p e c i f i e d i n Steck's paper ( 8 4 ) ) . Hydrogenolysis of (V) with palladium-charcoal c a t a l y s t produced 3-aminopyridazine as the mono-hydrochloride s a l t (VI). The s a l t (VI) was converted to 38. the free base and purified just before using for the next reaction because of i t s unstable nature. A lower yield was obtained when using 3,6-dibromopyridazine instead of IV (83). (IV) (V) (VI) After obtaining the appropriate primary aromatic amine, the next step was to react the amine with cyclohexane-carbonyl chloride. Cyclohexanecarboxylic acid was commercially available and was used to prepare the acid chloride. Three reagents are commonly used for this purpose« thionyl chloride, SOClgj phosphorus trichloride, PCl^j and phosphorus penta-chloride, PCl^. R-CO-OH + S0C1 2 >- R-C0-C1 + S0 2 + HCI 3R-C0-0H + PCl^ *- 3R-C0-C1 + H^ PO-j R-CO-OH + PCl^ — R - C 0 - C 1 + HCI + POCl-^ Thionyl chloride was chosen as the reagent for the preparation of cyclohexanecarbonyl chloride not only because the products formed besides the acid chloride were gases and thus easily separated from the acid chloride, but because the acid chloride formed was found to have higher boiling points than this reagentj any excess of the low-boiling thionyl chloride (b.p. 79°C, 1 atm.) was easily removed by d i s t i l l a t i o n . 39. Two precautions were taken f o r running t h i s r e a c t i o n . The f i r s t was to protect the reac t i o n and the acid chloride from moist air? the second was to avoid high temperatures which may cause p y r o l y s i s of the aci d chloride by using a water bath during the d i s t i l l a t i o n . 2-Aminopyridine, 2-aminopyrazlne, 1-naphthylamlne, and 5-aminoquinoline r e a d i l y formed amides with cyclohexane-carbonyl chloride i n the presence of pyridine. Pyridine was added to neutrali z e the hydrogen chloride formed during the reac t i o n . The cyclohexanecarboxamides thus formed were found to be water-insoluble, and on completion of the reaction, water was added to wash away the pyridine hydrochloride. In the case of 5-aminoquinoline i t was found that the product amide could also abstract the hydrogen chloride through i t s N^-quinoline. This meant that the N - 5-quinolyl-cyclohexane-carboxamide-HCl s a l t and pyridine-HCl were both present i n the water washings. The aqueous so l u t i o n was, therefore, made basic with excess 10$ NaOH solution, the crude amide (base) was p r e c i p i t a t e d out and could be separated from the pyridine. A l l the amides r e a d i l y formed i n good y i e l d s (Table 5 ) . + Pyridine-HCl 40. TABLE 5 CYCLOHEXANECARBOXAMIDES R MELTING POINT (°C.) $ YIELD 2-Pyridyl 9 1 . 0-92 . 5 9 3 . 9 (New compd.) 2-Pyrazyl 1 6 5 . 9 - 1 6 6 . 8 9 1 . 5 (New compd.) 1-Naphthyl 1 8 9 . 6 - 1 9 0 . 8 9 0 . 5 ( L i t . ( 8 5 ) , 188°C.) 5-Quinolyl 202 .9-204 .0 8 6 . 3 (New compd.) N-l-Naphthyl-cyclohexanecarboxamide had "been prepared by Gilman and Furry ( 8 5 ) by reacti n g cyclohexylmagnesium bromide and 1-naphthyl isocyanatei N=C=0 The r e a c t i o n between 2-aminopyrimidine and cyclo-hexanecarbonyl chloride was unsuccessful. However, some s o l i d p r e c i p i t a t e s were obtained a f t e r washing away the pyridine-HCl. The crude s o l i d p r e c i p i t a t e s were found to contain most of the unreacted s t a r t i n g 2-aminopyrimidine and some other prod-ucts. To get r i d of the s t a r t i n g material, the s o l i d precip-i t a t e s were suspended i n 1000 ml. of water and the mixture was brought to pH 8 - 9 with d i l u t e ammonium hydroxide s o l u t i o n ( 8 6 ) . The insoluble product was again c o l l e c t e d by f i l t r a t i o n , 41. washed with cold water and was p u r i f i e d by r e c r y s t a l l i z a t i o n from n-hexane to constant melting point (120.5-122.3°C.). The IR of the p u r i f i e d product showed two carbonyl peaks* one at 1680 cmT 1 corresponding to the secondary amide carbonyl band, and the other at 1740 cmT 1 corresponding to the ketone car-bonyl peak. The elemental analysis r e s u l t suggested that i t may have the following s t r u c t u r a l formulai Calcd. for CjgH^N^Ogi C, 6 8 . 5 ^ 1 H, 7.99; N, 1 3 - 3 3 . Found; C, 6 8 . 6 6 ; H, 7.81; N, 1 3 . 3 9 . The r e a c t i o n between 3-affiinopyridazine and cyclo-hexanecarbonyl chloride was not attempted, because some of the nitrogen-containing aromatic amides when reduced with lithium aluminum hydride did .not form the corresponding sec-ondary amine (see l a t e r discussion) (87). In order to obtain the secondary aromatic amine, the corresponding cyclohexanecarboxamide was reduced with lithium aluminum hydride. The reagent discovered by Finho l t , Bond and Schlesinger (88) i n 19*+7 (4LiH + A1C1 3 ether^ LiAlH^ + 3LiCl) has proved to be a remarkable reducing agent for the carbonyl group i n amides and s i m i l a r carbonyl compounds (89). Amides were not r e a d i l y reducible to pure amines by other chemical methods. Hydrogenation with a catalyst at high temperatures and pressures could be accomplished, but 42. usually resulted i n a mixture of products. The powdered LiALH^ can be safely handled, even i n very humid a i r , probably because of the formation of a pro-t e c t i v e coating of aluminum hydroxide (88). It i s generally used i n soluti o n or suspension i n dry ether ( 2 5 - 3 0 g« s o l i d hydride dissolves i n 100 g. ether at 2 5 ° C ) . In the normal procedure the substance to be reduced i s added to an ethereal s o l u t i o n or s l u r r y of the hydride. I f the substance to be reduced i s an ether-soluble l i q u i d or s o l i d , the soluti o n i s added dropwise to produce gentle r e f l u x . For moderately soluble materials, a Soxhlet extractor or a continuous-return type of extractor i s used. In the reduction of the amides, an excess (2- to 3-times the st o i c h i o m e t r i c a l quantities) of LiALH^ was used. Water was then added to destroy the excess hydride with the C. + LiAlH. >- 2 ( V-CHo-NHR + L1A10„ NHR 4 \ / 2 2 evolution of hydrogen, and the p r e c i p i t a t i o n of li t h i u m -and aluminum-hydroxide. LiAlH^ + 4H 20 LiOH + A l ( 0 H ) 3 + 4H 2 f As the amine was ether soluble, the mixture was treated with strong hydroxide s o l u t i o n to dissolve the pr e c i p i t a t e d alumina. This allowed a clear-cut separation of phases on centrifugation. N-2-Pyridyl-cyclohexanecarboxamide, N - l -naphthyl-^3. cyclohexanecarboxamide and N-5-quinolyl-cyclohexanecarboxamide were found to be r e a d i l y reduced to the corresponding secondary amine (Table 6 ) . However, when N - 2-pyrazyl-cyclohexanecar-boxamide was reduced with excess lithium aluminum hydride, TABLE 6 SECONDARY AROMATIC AMINES OF CYCLOHEXYLMETHYL DERIVATIVE. R MELTING POINT °C BOILING POINT °C. (mm.Hg.) % YIELD 2-Pyridyl 9 1 . 2 - 9 2 . 2 98 .2 (New compd.) 1-Naphthyl 149-152 (0 . 2 5 ) 86.4 (New compd.) 5-Quinolyl — - 186-190 (0 . 7-0 . 7 5 ) 8 3 . 3 (New compd.) instead of the corresponding secondary amine a mixture of cyclohexylcarbinol and 2-aminopyrazine was obtained. Similar reactions were found i n the following three examples ( 8 7 ) t C H 3 - 0 " S " i J 0 ( c o n t r l e d ^ ^ " ^ - j r * amount) Excess _ „„ / \ LiAlH^ * CH 3 -^J>~CH 2-0H 2) 0 N LiAlHi (controlled amount) C-H II 0 •N H-N 3) o II 'CH-C-N' LiAlHi (controlled amount) 'CH-C-H + H-N; II 0 Upon obtaining the appropriate secondary aromatic amines (Table 6 ) , the next portion of the molecule to be attached was the £-dimethylaminoethyl side chain to completely form the f i n a l t e r t i a r y ethylenediamine products. Our previous work ( 2 ) had shown that the c y c l o a l k y l analogues of Antergan could be synthesized i n good y i e l d through the following reac-t i o n pathways. .CH2-NH + CH2C1-C0C1 E t h e r (VII) i c e - s a l t b a t h N {100%) NaOH HCI 2 NH(CH 3) 2 e t h e r , i c e s a l t - b a t h (83#) CH„-N-C-CH 0-N C II * 0 •CH -N-C-CH_C1 2 II 2 0 + •CH2-NH.HC1 (CH3)2NH'HC1 45. LIAIH^ > (93%) The secondary aromatic amines we obtained i n Table 6 were s i m i l a r i n structure to (VII) except that the aromatic group was d i f f e r e n t . Except for the 1-naphthyl deriv a t i v e , a l l the others contained nitrogen heteroatom(s) i n the aromatic r i n g An attempt was made to get 2-cyclohexylmethylaminopyridine to re act with chloroacetyl chloride under the same conditions as (VII) i n the above scheme. However, a t a r r y residue soon res u l t e d a f t e r the a d d i t i o n of chloroacetyl chloride with no desired product formationi CH2C1-C0C1 >• No product I c e - s a l t bath This could be due to the extra nitrogen present i n the aromatic r i n g (pyridine-N^ atom) v/hich may serve as a hydrogen chloride acceptor or may react with chloroacetyl chloride. Leung ( 1 ) had succeeded i n obtaining the t e r i a r y (VIII) 77% y i e l d from (VII) 46. diamine (VIII) by condensing the secondary amine (VII) with ^-dimethylaminoethylbromide HBr i n the presence of sodamlde but the y i e l d was very poor (21.8%). ^CH 2 NaNH •CH2-NH + Br-CH2-CH2-N ^  -'•HBr CH^ Dry xylene or toluene (VII) / \ /CH 3 < >CH2-N-CH2-CH2-N^ J (VIII) 21.8% y i e l d The poor y i e l d could be due to the unstable character of the free base of ^ -dlmethylaminoethylbromlde i n the presence of the strong base sodamlde. Huttrer and co-workers (90) i n t h e i r o r i g i n a l syn-thesis of Neo-Antergan (X) used a small excess of l i t h i u m amide to react with p-methoxybenzylaminopyridlne (IX) f i r s t , and a few hours l a t e r the free base of ^ -dlmethylamlnoethyl chloride equal to the sto i c h i o m e t r i c a l quantity of l i t h i u m amide was added to the reaction mixture. The condensation effectiveness was gr e a t l y increased and t h e i r y i e l d f o r Neo-Antergan was ex-ce l l e n t (81%), Other l a t e r workers (91 , 92) also employed the free base of ^ -dimethylamlnoethyl chloride to synthesize the secondary aromatic amines bearing the pyridine (91) and pyrimidine rings ( 9 2 ) , and t h e i r r e s u l t s were a l l better than that of Leung's ( 1 ) . 47 . N H — N - C H „ - C H 0 - N C 3 / = \ CI-CH 2-CH 2-N(CH_) / = \ 3 (IX) (X) The free base of ^-dimethylaminoethyl chloride was unstable In the a i r and dlmerlzed e a s i l y to become a p r e c i p i -tate which made the c l e a r base solu t i o n cloudy. However, i f the free base was f r e s h l y d i s t i l l e d from i t s hydrochloride s a l t and f l a k e sodium hydroxide (93) and stored i n an ice-bath or i n the r e f r i g e r a t o r , i t was quite stable f o r some time. For a l l the reasons c i t e d above, the secondary aro-matic amines (Table 6) were condensed with the f r e s h l y d i s - . t i l l e d ^-dimethylaminoethy1 chloride using sodamide as conden-sing agent. Huttrer's (90) and Adams* methods (92) were f o l -lowed and a stream of dry nitrogen gas was bubbled through the reac t i o n mixture so as to prevent the sodamide from reacting with atmospheric carbon dioxide and more important to drive away the ammonia gas formed In favor of the desired r e a c t i o n . / \ N 2 gas, r e f l u x / \ Na + \ >-CH2-NH-B + NaNHp W V C H ? - N - H I + NH 0| \ / * i n dry benzene \ / * _ * 3 1 or toluene (XI) The sodium s a l t s of the secondary amines (XI) were Insoluble i n dry benzene or toluene and could be i s o l a t e d from the so-l u t i o n i f adequate precautions were taken to prevent contact 4 8 . from the moist a i r (e.g. worked up i n a dry box under dry nitrogen atmosphere). For the present purpose, the sodium s a l t s did not have to be i s o l a t e d , the f r e s h l y d i s t i l l e d p-dimethylaminoethyl chloride i n a l i t t l e dry benzene or dry toluene was added to the reaction mixtures (at room temperature) and then refluxed for 24 hours. The t e r t i a r y amines (XII) were d i s t i l l e d at reduced pressure and obtained as a l i g h t yellow or yellow o i l i n r e l a t i v e l y good y i e l d (Table 7 ) . Na •CH2-N-R + C1-CH 2-CH 2-N^ CH. •CH 2-N-CH 2-CH 2-<J R 3 (XII) TABLE 7 TERTIARY ETHYLENEDIAMINE DERIVATIVES (XII) R BOILING POINT °C. (mm.Hg.) % YIELD 2-Pyridyl 1 4 5 - 1 4 6 . 5 ( 1 . 0 - 1 . 1 ) 8 3 . 1 ( L i t . ( 9 4 ) , 1 6 0 - 1 6 5 (13 mm.)) 1-Naphthyl 1 8 8 - 1 9 0 ( 1 . 5 ) 7 1 . 2 (New compd.) 5-Quinolyl 164 -168 ( 0 . 1 5 - 0 . 2 ) 6 0 . 9 (New compd.) Kyrides and co-workers ( 9 4 ) had obtained the t e r -t i a r y diamine, N,N-dimethyl-N'-cyclohexylmethyl-N' -2-pyridyl-ethylenediamine ( i . e . XII, R= 2 - p y r i d y l ) , as a trihydrochloride s a l t . However, t h e i r elemental analysis r e s u l t showed only 4 9 . the chloride percentage, they did not mention how to obtain the trihydrochloride s a l t and the solvent used for r e c r y s t a l -l i z a t i o n , and t h e i r s t a r t i n g materials were d i f f e r e n t . They used N,N-dimethyl-N'-2-pyridylethylenediamine (XIII) to con-dense with cyclohexylmethyl bromide, a method which was found to give poor y i e l d s (usually 10-40$) i n a s i m i l a r reaction ( see l a t e r discussion). Also, i t was found that the hydro-chloride s a l t of the t e r t i a r y diamine was too hygroscopic to handle and a suitable solvent or solvent pairs for r e c r y s t a l -l i z a t i o n could not be found. Therefore, the d i p e r c h l o r i c a c i d s a l t derivative was prepared. secondary aromatic amines of cyclohexylmethyl derivative ( Table 6 ) , usually two successive condensation reactions using Adams* method ( 9 2 ) were required to obtain the desired t e r -t i a r y diamine products from the primary aromatic amines. The o v e r a l l r e s u l t s obtained showed much lower y i e l d s than the above synthetic method (Table 5 , 6 and 7 ) . CR- or LINH For those other compounds that could not form the The appropriate primary aromatic amines were reacted with f r e s h l y prepared ^-dimethylaminoethy1 chloride using 5 0 . sodamide as condensing agent. The secondary amines were ob-tained as a yellow o i l (Table 8 ) . CH NaNH2 H C H R-HH_ + C1-CH 9-CH 0-N^ 3 ^ B-N-CH0-CH0-N^ 3 2 2 CH 3 2 2 ^CH 3 (X IV) TABLE 8 SECONDARY AROMATIC AMINES OF £-DIMETHYLAMINOETHYL DERIVATIVES (XIV) R BOILING POINT °C. (mm.Hg.) % YIELD 2-Pyrimidyl 94 (0.9) 37.1 ( L i t . ( 9 0 ) , 85-90 (0.02)j ( L i t . ( 9 0 ) , 24.0; (92), 90-95 (3.0)) ( 9 2 ) , 29.4) 2-Pyrazyl 105-106 (0.95) 32.4 (New compd.) 3-Pyridazyl 107-108 (1.1) 0 c. 1 /New compd, \ 0 , x K unstable ; The N,N-dimethyl-N ' - 3-pyridazylethylenediamine (XIV, R = 3 -p y r l d a z y l ) was not stable i n the a i r , but the NMR of f r e s h l y d i s t i l l e d product ( F i g , 6 ) looked s i m i l a r to that of N,N-dimethyl-N*-2-pyrazylethylenediamine ( F i g . 7). The -NH-proton of either amine was exchangeable with deuterium when D 20 was added. How-ever, when e f f o r t s were made to p u r i f y the compound by column chromatography ( s i l i c a g e l ) , the amine was decomposed. The unstable character of 3-aminopyridazine had been described ( 8 3 ) 80 7-0 60 50 40 30 2 0 10 0 PPM (8) FIG. 6 i NMR spectrum of freshly d i s t i l l e d N,N-dimethyl-N'-3-pyridazylethylenediamtne dissolved i n C D C 1 « ( 1 0 % ) . The integration was done a f t e r Do0 added. 5 3 . and i t s secondary amine derivative (XIV, R = 3-pyridazyl) ap-peared to be even more unstable. In order to obtain the f i n a l t e r t i a r y diamine prod-ucts, another condensation reaction was required. The appro-pr i a t e secondary amine (Table 8 ) was condensed with cyclohexyl-methyl bromide i n the presence of sodamide. The yellow o i l y l i q u i d t e r t i a r y diamines (XV) were obtained by vacuum-distil-l a t i o n (Table 9 ) . H / CH- / \ NaNH/> R-N-CH2-CH2-N ^  J + Br-CH 2-/ \ — > (X IV ) CH^ •CH2-N-CH2-CH2-N^ 3 R C H 3 (XV) TABLE 9 TERTIARY ETHYLENEDIAMINE DERIVATIVES (XV) R BOILING POINT °C. (mm.Hg.) fo YIELD 2-Pyrimidyl 129-130 ( 0 . 5 ) 10 (New compd.) 2-Pyrazyl 126-136 ( 0 . 2 5 - 0 . 3 5 ) 3 8 . 6 (New compd.) Owing to the low y i e l d s of the t e r t i a r y diamines (XV, Table 9 ) e f f o r t s were made to increase the y i e l d s by other synthetic routes. Since the aromatic secondary amines of cyclohexylmethyl derivative (Table 6 ) seemed to condense better with ^-dimethylaminoethyl chloride (Table 7 ) , 2-amino-pyrimidine condensed f i r s t with cyclohexylmethyl bromide and 5 4 . then with ^-dimethylaminoethyl chloride. However, the r e s u l t from the reaction between 2-aminopyrimidine and cyclohexyl-methyl bromide also gave a low y i e l d of product ( 2 0 $ ) . The secondary aromatic amines (Table 8 , XIV, R= 2-pyrimidyl, and 2-pyrazyl) were reacted with cyclohexane-carbonyl chloride and then the amides were reduced to produce the corresponding t e r t i a r y diamine products as l i s t e d i n Table 9 . I t was found that the amides (XVI) r e a d i l y formed i n moderate y i e l d s (Table 1 0 ) . H ^ CH~ / \ pyridine /CH_ R-N-CH0-CH0-N J + ( V C - C l - ^ ^ >• R-N-CH9-CH5-N ^  J CH- \ / II I CH_ 3 0 C=0 3 (X IV ) (XV I ) TABLE 10 TERTIARY CYCLOHEXANECARBOXAMIDES (XVI) BOILING POINT °C. (mm.Hg.) $ YIELD 2-Pyrimidyl 2-Pyrazyl 137-141 (o . 3 5 ) 130 ( 0 . 2 0 ) 5 0 . 1 (New compd.) 4 7 . 6 (New compd.) 55. However, when the amides (XVI) were reduced with excess lithium aluminum hydride, the same thing happened as when N-2-pyrazyl-cyclohexanecarboxaraide was reacted (see previous discussion) and a mixture of cyclohexylcarbinol and the respective secondary amine (XIV) was obtained instead of the corresponding t e r t i a r y diamines (Table 9 ) . R-N-CH2-CH2-NV C=0 (XVI) /CH 3 E x c e s s CH- LiAlH, H •CHgOH + R-N-CH2-CH2-Nv CH. (XIV) F i n a l l y , the two oxetane derivatives (II and III) synthesized for the present study were two intermediates and not the f i n a l products for antihistaminic study purpose. The desired products (XVII and XVIII) were oxetane analogues of the potent antihistamine, diphenhydramine (XIX). (XVII) (XVIII) 56 Aromatic and a l i p h a t i c ketones and aldehydes may form oxetanes on i r r a d i a t i o n i n the presence of o l e f i n s (95). Although the y i e l d s of oxetanes are generally only about 5 -10?o and the re a c t i o n i s very slow, the s i m p l i c i t y of the method makes i t u s e f u l . In addition, photocycloaddition i s frequently the method of choice for the preparation of oxetanes since they can be d i f f i c u l t to prepare by more c l a s s i c a l methods. The possible mechanism for the photochemical syn-t h e s i s of oxetanes has been discussed but not systematically studied. Buchi has suggested a step-wise process, i n i t i a t e d by photoactivation of the carbonyl compound to the d i r a d i c a l n,jc* t r i p l e t state (96). Subsequent addition to the o l e f i n would give 1,4-diradical intermediates, followed by r i n g closure. As i t seems reasonable that the most stable d i r a d i c a i would be that with the unpaired electrons on the most sub s t i -tuted carbon atoms ( i n other words, the addition reaction took place with the oxygen atom becoming attached to the l e s s -substituted of the doubled bonded carbon atoms), t h i s mechanism 5 7 . appears to be i n agreement with the structures of the oxetanes obtained (Notei there are, however, exceptions). The i n i t i a l e x c i t a t i o n i s of the carbonyl compound, rather than the o l e f i n (97)* since the r e a c t i o n can be brought about by i r r a d i a t i o n i n a region where only the carbonyl com-pound absorbs. The simple o l e f i n s are e s s e n t i a l l y transparent at wavelengths longer than 250 mu. In order to excite the carbonyl compound exclusively without a f f e c t i n g the o l e f i n molecule, a pyrex f i l t e r was used to f i l t e r out a l l l i g h t below 290 mu (=2900 £ ) . In a d d i t i o n to the usually desired c h a r a c t e r i s t i c s , solvents f o r the photocycloaddition reaction should be (a) transparent to the wavelengths used (usually > 300 mu), (b) i n e r t (no e a s i l y abstractable hydrogen atoms), and (c) have no quenching properties. Table 11 indicates the v a r i e t y of solvents that have been used suc c e s s f u l l y f o r the photocycloaddition r e a c t i o n . 5 8 . TABLE 11 SOME SOLVENTS WHICH HAVE BEEN USED SUCCESSFULLY FOR THE PHOTOCYCLOADDITION REACTION Benzene Pyridine A c e t i c a c i d A c e t o n i t r i l e Excess o l e f i n (2-n$ethyl -2butene, cyclohexene, etc.) Excess carbonyl compound (acetone, perfluorocarbonyl compds,) Saturated hydrocarbons (hexane, pentane, etc.) Ethers (dioxane) In the present study, two oxetane derivatives ( H a n d III) have been synthesized ( 1 3 0 ) . 2 , 2~Diphenyl - 3-chlorooxetane (II) was obtained by photolyzing the benzophenone (through a pyrex f i l t e r ) i n the presence of v i n y l chloride using benzene as solvent. The process of the reaction was followed by TLC analysis of aliquots and by IR which indicated the decreased benzophenone carbonyl absorption band at 1670 cm."1. The formation of the oxetane r i n g was indicated by the presence of a strong absorption band at 980 cm."*1. Guepet and co-workers ( 9 8 ) have shown that the bands corresponding to the symmetrical and anti-symmetrical vibrations of the oxetane r i n g , a f f e c t i n g the C-O-C bond, have a constant frequency within the l i m i t s 960 - 980 cm. - 1, regardless of the substituents on the r i n g . The isomer obtained was assigned the structure (II) on the basis of NMR data. High chemical s h i f t values for the C^-methylene hydrogens (i 4.80-5.02 ppm) i s i n accord with being adjacent to the ether oxygen ( 9 9 ) . •59. (II) New Compd. 14 . 8$ y i e l d The same method was used to i r r a d i a t e benzophenone i n the presence of N-vinyl - 2-pyrrolidinone and 2 , 2-diphenyl-3 -(pyrrolidin - 2-one-l-yl)oxetane (III) was formed i n 1 . 3 $ y i e l d . Although oxetane (III) was synthesized independently i n our laboratory, Ogata and co-workers ( 9 9 ) published the Ogata ( 9 9 ) 6 0 . In order to obtain the desired oxetane end products (XVII and XVIII), a number of photosynthetic reactions were attempted. However, due to the easy ring-opening of the oxe-tanes by common chemical reactions and to the major d i f f i c u l t y which was usually encountered during e f f o r t s to i s o l a t e and pur i f y the products, the r e s u l t s were unsuccessful. The un-successful reactions are l i s t e d i n the following equations for the -reference,of future workers. 1) l T T T Neat > N H ^ ^ Ring opened „ _ In xylene + NaNH, >• Ring opened Grignard Mg, dry ice — — N o reaction reaction .CH_ hv 2) (CvH.)9C=0 + CH?=CH-CH0-N 3  D c c * ^CH^ i n AcOH 3) (C 6H 5) 2C=O + CH 2=CH-CH 2-N' 4) H (C 6H 5) 2C=O + CH 2=CH-CH 2-CI hv H i n AcOH hv Seemed to have oxetane forma-nt ions but could not i s o l a t e them. i n Benzene /H hv 5) (C 6H 5) 2C= 0 + H 2C=C X >- No oxetane Br i n Benzene f o r m a t l o n 6 1 . o II c hv 6) (C 6H 5) 2C= 0 + CH2=CH-CH2-N C II 0 In Benzene No oxetane formation H hv 7) (C 6H )2C=0 + 0=C N(CH ) 2 i n Benzene >• No reaction In conclusion, the compounds i n Table 5 - 10 as well as two oxetane compounds (II and III) were synthesized i n the present study. I f the compounds were s o l i d , pure and r e c r y s t a l l i z e d substances were analyzed f o r percentage com-po s i t i o n of carbon, hydrogen, nitrogen and halogen. No de-r i v a t i v e s were made. For those l i q u i d compounds, hydrochloride-, p i c r a t e - or perchlorate-derivatives were made f o r elemental microanalysis. I t was found that most of the perchlorate d e r i -vatives had a large melting point range (more than 2°C.) but nevertheless, they were pure compounds according to the elemental analysis r e s u l t s . To supplement the a n a l y t i c a l r e s u l t s of a derivati v e , an i n f r a r e d spectrum and NMR spectrum were also taken on the synthesized compound. PART III ANALYTICAL METHODS Melting points were determined using a Thomas-Hoover C a p i l l a r y Melting Point Apparatus (Arthur H. Thomas Co., PA., U.S.A.). A l l melting points and b o i l i n g points are re-ported uncorrected. A Beckman IR-10 Infrared Spectrophotometer (Beckman Instruments, Inc., C a l i f o r n i a , U.S.A.) was used to record the i n f r a r e d spectra. The NMR spectroscopy was performed by Miss P h y l l i s Watson of the Department of Chemistry, U.B.C., using a Varian A - 6 0 , T - 6 0 or XL -100 Spectrometer. The concentration of solutions was about 10$ and tetramethylsilane served as the i n t e r n a l standard. Solvents are s p e c i f i e d . Elemental microanalyses were performed by A l f r e d Bernhardt, Mikroanalytisches Laboratorium, 5251 Elbach liber Engelskirchen, Fritz-Pregl-StraBe 14-16, West-Germany. Cumulative log dose-response curves were plotted by using a Calcomp P l o t t e r with the a i d of an IBM 360/67 Computer (see Appendix A f o r an example fo r p l o t t i n g 3 f i g u r e s , and the p l o t t i n g routine, Superplot, which was used f o r p l o t -t i n g ) . The V®2' P A 2 a n d v a l u e s were calculated by using the WANG'S advanced programming calculator (600 series) (see Appendix B f o r Program T i t l e t Mean, Variance, Standard Deviation (Ungrouped Data)). PART IV EXPERIMENTAL^ A. Synthesis of N.N-Dimethyl-N'-Cyclohexylmethyl-N'-2- Pyridylethylenediaminet 1. Cyclohexanecarbonvl Chloride i Cyclohexanecarboxylic a c i d (128 g. f 1 .0 mole) was placed i n a 1 - l i t e r , three-necked f l a s k f i t t e d with a r e f l e x condenser and drying tube, a 250 ml.-dropping funnel and a mechanical s t i r r e r . Thionyl chloride (238 g., 2 . 0 moles) was added dropwise with s t i r r i n g . The f l a s k was placed on a heat ing mantle and heated at r e f l u x for 1 . 5 - 2 hours. After that time, the. mixture was d i s t i l l e d , and the crude product c o l -lected from 160-135°C.. This f r a c t i o n was r e d i s t i l l e d under reduced pressure and the f r a c t i o n b o i l i n g at 8 2 - 8 5°C. (14-15 mm.) was c o l l e c t e d to y i e l d 118 g. ( 0 , 8 mole, 80.5%) of prod-uct ( L i t . ( 100),b.p. 67-67.5°C. (14 mm.), 81%; ( 1 0 1 ) , b.p. 76°C. (1? mm.)). 2. N-2-Pyridyl-Cyclohexanecarboxamide1 A mixture of 2-aminopyridine ( 7 5 . 3 g.» 0 , 8 mole), pyridine ( 6 3 . 2 g., 0 . 8 mole), and dry benzene (120 ml.) was placed i n a 1 - l i t e r three-necked f l a s k f i t t e d with a mechani-c a l s t i r r e r , a r e f l u x condenser (drying tube) and a dropping funnel (250 ml.). The mixture was cooled i n an ice-bath and cyclohexanecarbonyl chloride (118 g., 0 . 8 mole) was added (a) A table of a l l the chemicals and reagents used for the EXPERIMENTAL PART together with t h e i r grades and company names was l i s t e d i n Appendix C, 6 4 . dropwise with s t i r r i n g . A f t e r the addition was complete, the react i o n was refluxed with s t i r r i n g for 1 hour, cooled and washed with water (150 ml.). The s o l i d which p r e c i p i t a t e d was suction f i l t e r e d and the layers were separated. The aqueous phase was extracted with two 100 ml. portions of ether. The ether extracts were combined with the benzene layer, and dried over anhydrous sodium s u l f a t e . The solvents were removed by f l a s h evaporation to recover more of the amide i n a t o t a l y i e l d of 1 5 3 . 3 g. ( 0 . 7 5 mole, 93.9$). The amide was r e c r y s t a l l i z e d from benzene-petroleum ether (b.p. 3 0 - 6 0°C.) mixtures; m.p. 9 1 . 0 - 9 2 . 5 ° C Infrared spectrum of the s o l i d amide (KBr) showed a strong C-0 stretching band at 1675 cm."1 and the character-i s t i c cyclohexyl C-H stretching v i b r a t i o n at 2750 cm.""1 and 2820 cm.-1- also the absence of N-H stretching for 2-amino-pyridine at 3300 cm."1 and 3440 cm."1 indicated the amide structure. NMR signals ( 6 0 MHz, CDCl^h £ 0 . 9 0 - 2 . 5 0 ppm ( mult i p l e t , 11 H, C^H^), 6.82-8.40 ppm (multiplet, 4 H, C ^ N ) and 8 . 7 0 ppm ( s i n g l e t , 1 H, NH). Anal. Calcd. for c 1 2 H l 6 N 2 0 , C ' 7 0 t , 5 5 * H » 7 .90} N, 1 3 . 7 2 . Foundi C, 7 0 . 7 4 ; H, 7 . 7 5 ; N, 1 3 . 9 1 . 3» 2-Cyclohexylmethylaminopyridine 1 To a 2 - l i t e r three-necked f l a s k f i t t e d with a dropping funnel ( 5 0 0 ml.), a r e f l u x condenser (drying tube) and a mechanical s t i r r e r was placed 1 l i t e r of dry ether. Lithium aluminum hydride (38 g., 1 mole) was added to the ether, and the mixture was gently refluxed with s t i r r i n g for 6 5 . 4 hours. The mixture was cooled to room temperature and a solu t i o n of N-2-pyridyl-cyclohexanecarboxamide ( 102 g., 0 . 5 mole) i n dry ether (700 ml.) was added from the dropping funnel at such a rate as to maintain gentle r e f l u x . A f t e r the addi-t i o n was complete, the mixture was s t i r r e d and refluxed for 3 days. At the end of t h i s time, the reaction was cooled i n an ice-bath and 100 ml. of water was added slowly to decompose the excess hydride; then s u f f i c i e n t 40% NaOH sol u t i o n was added to allow complete separation. The ethereal layer was separated by decantation and excess benzene solvent (800 ml.) was used to extract more of the amine from the white s o l i d residues. The organic layers were combined and dried over anhydrous sodium sul f a t e and reduced i n volume to y i e l d 9 3 . 3 g« ( 0 . 4 9 mole, 9 8 , 2 % ) of product. The crude product was r e -c r y s t a l l i z e d from n-hexane; m.p. 9 1 , 2 - 9 2 . 2°C.. Infrared spectrum for the s o l i d amine (KBr) showed the absence of the C=0 band at 1675 cm.""1 i n d i c a t i n g complete reduction of the amide. NMR signals (60 MHz, CDCl^)! £ 0 . 6 0 -2 . 0 0 ppm (multiplet, 11 H, C^H^), 2 . 9 0 - 3 . 2 0 ppm ( t r i p l e t , 2 H, CH 2), 4 . 5 0 ppm ( s i n g l e t , 1 H, NH), and 6 . 1 0 - 6 . 6 0 ppm, 7 . 1 0 - 7 . 5 0 ppm and 7 . 9 5 - 8 . 1 0 ppm (multiplet, 4 H, C ^ N ) . Anal. Calcd. for C 1 2H QN 2 1 C, 7 5 . 7 4 ; H, 9 . 5 3 ? N, 14 , 7 3 . Foundt C, 7 5 - 5 8 ; H, 9 . 4 5 ; N, 14 .62 4 , N,N-Dimethyl-N'-Cyclohexylmethyl-N'-2-Pyridylethvlene- diaminet A mixture of 2-cyclohexylmethylaminopyridine (38 g., 0 . 2 mole), sodium amide ( 1 1 , 7 g., 0 . 3 mole) and dry benzene 6 6 . ( 3 0 0 ml.) was placed i n a 0 . 5 - l i t e r three-necked f l a s k f i t t e d with a mechanical s t i r r e r , r e f l u x condenser (drying tube), a dropping funnel ( 2 5 0 ml.) and a dry nitrogen gas i n l e t tube. The mixture was bubbled with dry nitrogen gas and refluxed f o r two hours. The mixture was then allowed to cool and the n i -trogen was discontinued. A soluti o n of ^ -diraethylaminoethyl chloride ( 3 2 . 3 g., 0 . 3 mole) f r e s h l y d i s t i l l e d from i t s hydrochloride s a l t and flake sodium hydroxide ( L i t . ( 9 3)j (102), b.p. 109°C. at 750 mm.), i n 70 ml. of dry benzene was added from the dropping funnel to the mixture which was then r e -fluxed for another 24 hours. The mixture was cooled and f i l -tered and the solvent was removed by f l a s h evaporation. The residue was fractionated under reduced pressure. The t e r t i a r y amine was obtained as a l i g h t yellow o i l , b.p. 145-146.5°C. at 1 . 0 - 1 . 1 mm., y i e l d 4 3 . 4 g. (0.17 mole, 8 3 . 1 $ ) . ( L i t . ( 9 0 ) ; ( 9 4 ) , b.p. 160-165°C. at 13 mm.). A t e r t i a r y amine (neat) was indicated from the IR spectrum by the absence of N-H stretching band at 3250 cm. - 1 and the presence of the c h a r a c t e r i s t i c C-H stretching for R-N(CH 3) 2 at 2840 cm."1, 2790 cm."1, 2750 cm."1. NMR signals ( 6 0 MHz, CDCl^ J i £ 0 .40 - 1 . 8 0 ppm (multiplet, 11 H, CgHjj), 2 . 2 2 ppm ( s i n g l e t , .6 H, N(CH^)2)., 2.4.0^2^55 ppm (doublet, 2 H, CH 2), 3 . 2 0 - 3 . 7 8 ppm (two t r i p l e t , 4 H, ( C H 2 ) 2 ) , and 6 . . 3 0 - 6 . 5 0 , 7 . 2 0 -7 . 5 0 and 8 . 0 5 - 8 . 2 0 ppm (multiplet, 4 H, C ^ N ) . Di-perchlorate Derivative! A solution of the t e r t i a r y amine ( 2 . 6 1 g., 0 . 0 1 mole) i n 15 ml. of 95$ ethanol was treated with a so l u t i o n of 6 7 . 70% perchloric acid ( 1 . 4 4 g.) i n 5 ml. of 95% ethanol ( 1 0 3 ) . The clear reaction mixture was saturated with excess dry ether to force the perchloric acid s a l t to p r e c i p i t a t e out. After standing overnight i n the r e f r i g e r a t o r , the mixture was f i l -tered and the s a l t r e c r y s t a l l i z e d from dry ethanol and dry ether (using a water bath at 6 5 ° C ) ; m.p. 8 7 - 1 1 0 ° C . Anal_ Calcd. for C ^ H ^ C l g O g , C f 4 l # 5 6 | H > 6 > 3 2 | N, 9 . 0 9 * C l 1 5 . 3 4 . Foundi C, 41 . 7 6 ; H, 6 . 5 0 ; N, 8 . 8 9 ; C l 1 5 . 1 0 . B i . Synthesis of N, N-Dimethyl-N' -Cyclohexylmethyl-N' - 2 - Pyrimidylethylenediamine 1 1. 2-Cyclohexylmethylaminopyrimidine1 A mixture of 2-aminopyrimidine (9«5 g.» 0 . 1 mole), sodium amide ( 5 . 8 5 g » i 0 . 1 5 mole) and dry toluene ( 150 ml.) was placed i n a 250-ml. three-necked f l a s k f i t t e d with a me-chanical s t i r r e r , r e f l u x condenser (drying tube), a dropping funnel ( 125 ml.) and a dry nitrogen gas i n l e t tube. The mix-ture was bubbled with dry nitrogen gas and refluxed at 125°C. i n an o i l - b a t h for 10 hours. The mixture was cooled and the n i -trogen flow discontinued.A s o l u t i o n of cyclohexylmethyl bromide ( 1 7 . 7 g « t 0 . 1 mole) i n 20 ml. of dry toluene was added from the dropping funnel. The mixture was refluxed and s t i r r e d for 16 hours, cooled and washed with 100 ml. of water. The toluene layer was separated and the water layer saturated with potassium carbonate and extracted three times (3 x 75 ml.) with ether. The combined extracts and the toluene layer were d i s t i l l e d through a Claisen f l a s k to get r i d of the excess 2 -aminopyrimidine and the residue fractionated under reduced 6 8 . pressure to y i e l d 3 .82 g. ( 0 . 0 2 mole, 20.0%) of product, b.p. 1 1 2 - l l 6°C. at 0.75 mm. ( L i t . ( 9 D ) . A secondary amine (neat) was shown from the IR spec-trum by the presence of one sharp peak at 3260 c m . a l s o the presence of the c h a r a c t e r i s t i c cyclohexyl C-H stretching bands -1 -1 at 2910 cm. and 2850 cm. indicated the amine structure. NMR signals (60 MHz, CDCl^Ji j l . 1 0 - 1 . 9 0 ppm (multiplet, 11 H, C 6 H n ) , 3 . 1 5 - 3 . 3 5 ppm ( t r i p l e t , 2 H, CH 2), 5-55 ppm ( s i n g l e t . 1 H, NH), and 6 . 3 5 - 6 . 5 5 ppm and 8 . 2 0 - 8 . 3 0 ppm ( t r i p l e t and doublet respectively, 3 H, C^H^Ng). Mono-picrate Derivative> A sample of the secondary amine ( 0 . 5 g.) was added to 95% ethanol (10 ml.). This s o l u t i o n was then added to 10 ml. of a saturated s o l u t i o n of p i c r i c a c i d i n 95% ethanol, and was heated to b o i l i n g . The solu t i o n was allowed to cool slowly, and the bright yellow c r y s t a l s of the picr a t e were i s o l a t e d by suction f i l t r a t i o n . The s o l i d was then r e c r y s t a l -l i z e d from 95% ethanol; m.p. l 6 l - l 6 3 ° C . Anal. Calcd. f o r c 1 7 H 2 o N 6 ° 7 J C » ^Q*57'> H » 4 » 8 0 ; N, 2 0 . 0 0 . Found; 4 8 . 4 8 ; H, 4 . 8 7 ; N, 1 9 . 8 0 . 2. N,N-Dimethyl-N'-2-Pyrimidylethyienediamine1 A mixture of 2-aminopyrimidine ( 3 3 . 9 g.» 0 . 3 6 mole), sodium amide ( 1 5 . 6 g., 0 . 4 mole) and dry toluene ( 2 8 0 ml.) was placed i n a 0 . 5 - l i t e r three-necked flask f i t t e d with a mechanical s t i r r e r , r e f l u x condenser (drying tube), a dropping funnel (250 ml.) and a dry nitrogen gas i n l e t tube. The mix-ture was bubbled with dry nitrogen gas and refluxed at 125°C. 6 9 . i n an o i l - b a t h for 12 hours. The mixture was cooled and the nitrogen flow discontinued. A solution of /3-dimethylaminoethy1 chloride (43 g., 0 . 4 mole), fr e s h l y d i s t i l l e d from i t s hydro-chloride s a l t and flake sodium hydroxide ( L i t . ( 9 3 ))• i n 50 ml. of dry toluene was added from the dropping funnel to the mixture which was then refluxed another 24 hours. At the end of t h i s time, the mixture was cooled, f i l t e r e d , and the f i l -t r a t e dried over anhydrous potassium carbonate and kept i n the r e f r i g e r a t o r overnight. The cooled mixture was f i l t e r e d and the solvent removed from the f i l t r a t e by f l a s h evaporation. The concentrated l i q u i d mixture was heated under reduced pressure (70°C. at 0 . 9 mm.) to get r i d of more of the s o l i d s t a r t i n g amine by sublimation. The l i q u i d residue was f r a c -tionated under reduced pressure and the f r a c t i o n b o i l i n g at 94°C. ( 0 . 9 mm.) was c o l l e c t e d to y i e l d 2 2 . 0 g. ( 0 . 1 3 mole, 3 7 . 1 $ ) ( L i t . ( 9 0 ) , b.p. 8 5 - 9 0°C. at 0 . 0 2 mm., 24$; ( 9 2 ) , b.p. 9 0 - 9 5 ° C at 3 mm., 2 9 . 4 $ ) . The in f r a r e d spectrum (neat) showed the c h a r a c t e r i s t i c C-H stretching for R-NtCH^g at 2830 cm."1, 2780 cm."1 and 2750 cm."1. The N-H stretching v i b r a t i o n with one sharp peak at 3260 cm. - 1 was c h a r a c t e r i s t i c of a secondary amine. NMR signals (60 MHz, CS 2)i £2.20 ppm ( s i n g l e t , 6 H, U(CH^)2)t 2 . 3 0 - 2 . 5 0 ppm ( t r i p l e t , 2 H, CH 2N), 3 - 3 0 - 3 . 5 5 ppm (quartet, 2 H, CH2NH), and 6 .25-6.40 ppm and 8 . 1 0 - 8 . 1 5 ppm ( t r i p l e t and doublet respectively, 4 H, NH-C^H-N,,), 7 0 . 3 . N,N-Dimethyl-N'-Cyclohexanecarbonyl-N'-2-Pyrimidyl- ethylenediamine i A mixture of N,N-dimethyl-N'-2-pyrimidylethylene-diamine ( 8 . 4 g., 0 . 0 5 mole), pyridine ( 7 . 9 g.» 0 . 1 mole) and dry benzene ( 2 0 ml.) was placed i n a 100-ml. three-necked f l a s k f i t t e d with a mechanical s t i r r e r , a r e f l u x condenser (drying tube) and a dropping funnel ( 5 0 ml.). Freshly pre-pared cyclohexanecarbonyl chloride ( 8 . 8 g., 0 . 0 6 mole) was added dropwise to the s t i r r e d mixture i n the fl a s k (cooled i n an ice-bath). Af t e r the addition was complete, the reaction was refluxed with s t i r r i n g f o r 2 hours, cooled and washed with two 30 ml. portions of 2 0 $ NaOH. The layers were sepa-rated and the aqueous layer was extracted with two 60 ml. portions of ether. The ethereal extract was combined with the benzene layer and dried over anhydrous sodium s u l f a t e . The solvents were flashed o f f and the concentrated l i q u i d fractionated under reduced pressure to y i e l d 7 . 0 g. ( 0 . 0 2 5 mole, 5 0 . 1 $ ) of product, b.p. 1 3 7 - l 4 l ° C . at 0 . 3 5 mm.. Infrared spectrum of the product (neat) indicated an amide by the absence of the N-H stretching v i b r a t i o n band at 3 2 6 0 cm. - 1, and by the presence of a strong carbonyl ab-sorption at I67O cm. . NMR signals ( 6 0 MHz, CCl^)- £ o . 9 0 -1 . 9 0 ppm (multiplet, 11 H, C^H^), 2 . 2 0 ppm ( s i n g l e t , 6 H, N(CH 3) 2), 2 . 3 0 - 2 . 5 0 ppm ( t r i p l e t , 2 H, CH 2), 3 . 9 2 - 4 . 1 5 ppm ( t r i p l e t , 2 H, CH 2), and 6 . 8 5 - 7 . 0 0 and 8 . 5 0 - 8 . 6 0 ppm ( t r i p l e t and doublet respectively, 3 H, C^H^N2). 71 . Mono-perchlorate Derivative» The t e r t i a r y amine ( 2 . 8 g., 0 . 0 1 mole) i n 15 ml. of 95% ethanol was treated with a s o l u t i o n of 70% perchloric acid ( 1 . 4 4 g.) i n 5 ml. of 95% ethanol and worked up as de-scribed f o r the N,N-dimethyl-N'-cyclohexylmethyl-N* -2-pyridyl-ethylenediamine d i - p e r c h l o r i c acid d e r i v a t i v e . The white t e r t i a r y amine mono-perchlorate was r e c r y s t a l l i z e d from dry ethanol; m.p. 1 5 3 - 1 5 5 . 5°C.. Anal. Calcd. for C ^ H ^ N ^ C l C y C, 47.80; H, 6 . 6 9 * N, 14 . 8 7 ; C l , 9 . 4 l . Found; C, 4 7 . 7 7 « H, 6 . 7 6 ; N, 14 . 6 7 ; c i , 9 . 3 2 . 4 . N.N-Dimethvl-N'-Cyclohexy lmethyl-N ' - 2-Pyrirriidyl- ethylenediamine; A mixture of N,N-dimethyl-N' -2-pyrimidylethylene-diamine (16.6 g., 0.1 mole), sodium amide ( 4 . 3 g., 0 .11 mole) and dry toluene ( 8 0 ml.) was placed i n a 150-ml. three-necked fl a s k f i t t e d with a mechanical s t i r r e r , r e f l u x condenser (dry-ing tube), a dropping funnel ( 5 0 ml.) and a dry nitrogen gas i n l e t tube. The mixture was bubbled with dry nitrogen gas and refluxed at 125°C. i n an o i l - b a t h for 15 hours. The re-action mixture was cooled and the nitrogen discontinued. A so l u t i o n of cyclohexylmethyl bromide (35«4 g., 0 . 2 mole) i n 10 ml. of dry toluene was added from the dropping funnel to the mixture, which was then refluxed another 48 hours. At the end of t h i s time, the mixture was cooled, f i l t e r e d and the f i l t r a t e fractionated under reduced pressure a f t e r f l a s h evaporation of the solvent. The t e r t i a r y amine was obtained 7 2 . as a yellow o i l , b.p. 129-130°C. at 0 . 5 mm., y i e l d 2 . 6 g. ( 0 . 0 1 mole, 1 0 $ ) . A t e r t i a r y amine (neat) was indicated from the IR spectrum by the absence of the sharp secondary amine N-H stretching v i b r a t i o n band at 3260 cm. - 1 and the presence of the c h a r a c t e r i s t i c cyclohexyl C-H stretching bands at 2920 cm."1 and 2850 cm."1. NMR signals ( 6 0 MHz, CCl^ and trace of CDCl^)' £ 1 . 0 0 - 1 . 7 5 ppm (multiplet, 11 H, C 6 H U ) , 2 . 2 5 ppm ( s i n g l e t , 6 H, H(CK^)2)t 2 . ^ 3 - 2 . 6 0 ppm and 3 . 3 5 - 3 . 7 5 ppm (mul-t i p l e t , 6 H, ( C H 2 ) 2 and CHg), and 6 . 2 5 - 6 . 5 0 and 8 . 1 5 - 3 . 2 2 ppm (quartet and doublet respectively, 3 H, C^Hy",,). Because a trace of the s t a r t i n g secondary amine ( b.p. 9*+°C. at 0 . 9 mm.) was carr i e d over during the d i s t i l l a t i o n of the t e r t i a r y amine, i t was necessary to p u r i f y the t e r t i a r y amine by column chromatography. A suspension of 80 g. s i l i c a gel ( s p e c i f i c a t i o n MIL-D-3716, Davision Commercial Grade, Fisher S c i . Co.) i n petroleum ether (b.p. 30-60°C.) was packed into a pyrex-glass column (3 x 60 cm.) i n v e r t i c a l p o s i t i o n . When the suspension s e t t l e d down i n the column, the height of the s i l i c a g e l was about two-thirds of the t o t a l column length. A solut i o n of 2 g. of the impure t e r t i a r y amine i n petroleum ether was poured into the column and eluted with benzene, benzene and chloroform ( 1 * 1 ) , and chloroform. The eluate was received into a 2 . 5 x 20 cm. test tube (about 70 ml. i n volume) and checked by both TLC using sublimed iodine as the spotting agent and by IR spectrum. The pure t e r t i a r y amine came out i n the chloro-form f r a c t i o n s . 7 3 . Di-perchlorate Derivative; The white d i - p e r c h l o r i c acid s a l t of the p u r i f i e d t e r t i a r y amine was prepared as described for the N,N-dimethyl-N'-cyclohexylmethyl-N' - 2-pyridylethylenediamine d i - p e r c h l o r i c acid derivative; m.p. 182.5-184°C.. Anal. Calcd. f o r c1£i28l*kC1208l C ' 3 8 . 8 8 ; H, 6 . 0 9 ; N, 1 2 . 1 0 ; C l , 1 5 . 3 1 . Found; C, 3 8 . 9 9 l H, 6 . 2 3 ; N, 1 1 . 9 1 ; C l , 1 5 . 2 6 . C. Synthesis of N.N-Dimethyl-N'-Cyclohexylmethvl-N'-2- Pyrazylethylenediamine t 1. N-2-Pyrazyl-Cyclohexanecarboxamidet A mixture of 2-aminopyrazine (4?.6 g., 0 . 5 mole), pyridine ( 3 9 . 5 g«» 0 . 5 mole), and dry benzene (250 ml.) was placed i n a 0 . 5 - l i t e r three-necked f l a s k f i t t e d with a me-chanical s t i r r e r , a r e f l u x condenser (drying tube) and a dropping funnel (250 ml.). To the s t i r r e d mixture i n the f l a s k (cooled i n an ice-bath) was added f r e s h l y prepared cyclo-hexanecarbonyl chloride ( 7 3 * 3 g», 0 . 5 mole) dropwise. A f t e r the addi t i o n was complete, the reaction was refluxed with s t i r r i n g for 1 .5 hours, cooled and washed with water ( 2 0 0 ml.). The s o l i d which p r e c i p i t a t e d was suction f i l t e r e d and the layers were separated. The aqueous phase was extracted with two 200 ml. portions of ether. The ethereal extracts were combined with the benzene layer, and dried over anhydrous sodium s u l f a t e . The solvents were flashed o f f to recover more of the amide i n a t o t a l y i e l d of 9 3 . 9 g. (0.46 mole, 9 1 . 5 % ) . The amide was r e c r y s t a l l i z e d from benzene; m.p, 165.9-166.8°C.. 7 4 . The IR spectrum of the s o l i d amide (KBr) showed a strong C-0 stretching band at 1700 cm."1 and the character-i s t i c cyclohexyl C-H stretching v i b r a t i o n at 2870 cm."1 and 2940 cm,"1; also the absence of N-H stretching for 2-amino-pyrazine at 3160 cm."1 and 3340 cm."1 indicated the amide structure. NMR signals (60 MHz, CDCl^); i 1 . 0 0 - 2 . 2 0 ppm ( mult i p l e t , 11 H, CgH^), 8 . 10-8.40 ppm (multiplet, 4 H, C^H^N2), and 9 . 6 5 ppm ( s i n g l e t , 1 H, NH). Anal. Calcd. for ^ H^N^O; C, 64 . 3 6 ; H, 7 . 3 7 ; N, 20.48. Found; C, 64 . 5 2 ; H, 7 . 3 3 ; N, 2 0 . 6 3 . 2 . N,N-Dimethyl-N'-2-Pyrazylethylenediamine1 A mixture of 2-aminopyrazine ( 4 3 . 4 g., 0.46 mole), sodium amide ( 1 9 . 5 g«» 0 . 5 mole) and dry toluene (320 ml.) was placed i n a 0 . 5 - l i t e r three-necked f l a s k f i t t e d with a mechanical s t i r r e r , r e f l u x condenser (drying tube), a dropping funnel (250 ml.) and a dry nitrogen gas i n l e t tube. The mix-ture was bubbled with dry N 2 gas and refluxed at 125°C. i n an o i l - b a t h for 24 hours. The reaction mixture was then cooled and the nitrogen discontinued. A s o l u t i o n of ^-dimethylamino-ethyl chloride ( 5 3 * 8 g » i 0 . 5 mole), f r e s h l y d i s t i l l e d from i t s hydrochloride s a l t and flake sodium hydroxide ( L i t . (93) ) , i n 30 ml. of dry toluene was added from the dropping funnel to the mixture, which was then refluxed another 24 hours. At the end of t h i s time, the mixture was cooled and f i l t e r e d and the f i l t r a t e concentrated by f l a s h evaporation. The concen-trated l i q u i d was kept i n the r e f r i g e r a t o r overnight. The cooled mixture was f i l t e r e d and the f i l t r a t e heated under 7 5 . reduced pressure (90°C. at 0 . 9 5 mm.) to get r i d of more of the s o l i d s t a r t i n g amine by sublimation. The l i q u i d residue was fractionated under reduced pressure to y i e l d 24 ,6 g. ( 0 . 1 5 mole, 3 2 . 4 $ ) , b.p. 105-106°C. at 0.95 mm. ( L i t . (92)). Infrared spectrum (neat) showed the c h a r a c t e r i s t i c C-H stretching for R-N(CH 3) 2 at 2820 cm."1, 2780 cm."1 and 2730 cm."1. N-H stretching v i b r a t i o n with one broad peak at 3300 cm."1 (conjugated NH) indicated the presence of a sec-ondary amine. NMR signals (60 MHz, CCl^ ) t ^ 2 . 2 0 ppm ( s i n g l e t , 6 H, N(CH 3) 2), 2 . 3 5 - 2 . 5 5 ppm ( t r i p l e t , 2 H, CH 2), 3 . 2 0 - 3 . 5 0 ppm (quartet, 2 H, CH2-NH), 5*30 ppm ( s i n g l e t , 1 H, NH), and 7 . 6 5 - 7 . 9 0 ppm (multiplet, 3 H, C ^ H ^ ) . Mono-nicrate Derivative 1 The bright yellow p i c r i c acid s a l t was prepared as described f o r the 2-cyclohexylmethylaminopyrimidine mono-p i c r i c acid d e r i v a t i v e . The N.N-dimethyl-N*-2-pyrazylethylene-diamine mono-picrate was r e c r y s t a l l i z e d from reagent acetone* m.p. 1 5 8 - 1 5 9 . 5 ° C . Anal. Calcd. for C ^ H ^ O ^ i C, 42 . 5 3 ; H, 4.34; N, 24.81. Foundi C, 42 . 3 7 ; H, 4 . 4 5 ; N, 2 5 . 0 0 . 3 . N.N-Dimethyl-N'-Cyclohexanecarbonyl-N'-2-Pyrazyl- ethylenediamine1 A mixture of N,N-dimethyl-N'-2-pyrazylethylene-diamine ( 4 . 1 g., 0 . 0 2 5 mole), pyridine ( 3 . 2 g., 0.04 mole) and dry benzene (20 ml.) was placed i n a 100-ml. three-necked f l a s k f i t t e d with a mechanical s t i r r e r , a r e f l u x condenser (drying tube) and a dropping funnel (50 ml.). To the s t i r r e d 7 6 . mixture i n the flask (cooled i n an ice-bath) was added f r e s h l y prepared cyclohexanecarbonyl chloride ( 5 . 9 g.» 0.04 mole) dropwise. A f t e r the addition was complete, the reaction was refluxed with s t i r r i n g for 2 hours, cooled and washed with 20 ml. of 20$ NaOH. The layers were separated. The aqueous layer was extracted with two 30 ml. portions of ether. The ethereal extracts were combined with the benzene layer and dried over anhydrous sodium s u l f a t e . The solvents were r e -moved by f l a s h evaporation to y i e l d 3.24 g. ( 0 . 0 1 2 mole, 4 7 . 6 $ ) of product, b.p. 130°C. at 0 o 2 0 mm.. The amide structure (neat) was indicated by the presence i n the IR spectrum of a strong carbonyl absorption band at 16?0 cm."1. NMR signals ( 6 0 MHz, CCl^)- £ 1 . 0 0 - 1 . 8 0 ppm (multiplet, 11 H, C^H.^), 2 . 1 5 ppm ( s i n g l e t , 6 H, N(CH 3) 2), 2 , 3 0 - 2 . 5 2 ppm ( t r i p l e t , 2 H, CHg), 3 . 8 6 - 4 . 0 0 ppm ( t r i p l e t , 2 H, CH 2), and 8 . 3 5 and 8 . 6 5 (two s i n g l e t , 3 H, <V*3N2). Mono-picrate Derivative! The bright yellow p i c r i c a c i d s a l t was prepared as described for the 2-cyclohexylmethylaminopyrimidine mono-p i c r i c acid d e r i v a t i v e , and was r e c r y s t a l l i z e d from water? m.p. 1 5 8 - 1 5 9 . 5 °C . . Anal. Calcd. for C ^ H ^ N ^ i C, 4 9 . 8 9 ? H, 5 . 3 8 ? N, 19.40. Found- C, 4 9 . 7 3 ? H, 5 .45? N, 1 9 . 2 1 . 4 . N,N-Dimethyl-N'-Cyclohexylmethyl-N'-2-Pyrazyl-ethylenediaroinet 7 7 . A mixture of N,N-dimethyl-N'-2-pyrazylethylene-diamine ( 11.5 g., 0 . 0 7 mole), sodium amide ( 3 . 9 g.» 0 . 1 mole) and dry toluene ( 1 0 0 ml.) was placed i n a 2 5 0-ml. three-necked f l a s k f i t t e d with a mechanical s t i r r e r , r e f l u x condenser (dry-ing tube), a dropping funnel ( 1 2 5 ml.) and a dry nitrogen gas i n l e t tube. The mixture was bubbled with dry nitrogen gas and refluxed at 1 2 5°C. i n an o i l - b a t h for 2 2 hours. The re-a c t i o n mixture was cooled and the nitrogen discontinued. A so l u t i o n of cyclohexylmethyl bromide ( 1 7 . 7 g.» 0 . 1 mole) i n 10 ml. of dry toluene was added from the dropping funnel to the mixture, which was then refluxed another 24 hours. At the end of t h i s time, the mixture was cooled, f i l t e r e d and the f i l t r a t e fractionated under reduced pressure a f t e r removal of the solvent by f l a s h evaporation. The t e r t i a r y amine was obtained as a yellow o i l , b.p. 1 2 6 - 1 3 6°C. at 0 . 2 5 - 0 . 3 5 mm., y i e l d 7 . 0 g. ( 0 . 0 2 6 mole, 3 8 . 6 % ) . A t e r t i a r y amine (neat) was indicated by the ab-sence i n the IR spectrum of the N-H stretching v i b r a t i o n at 33 ° 0 cm."1 and the presence of the c h a r a c t e r i s t i c cyclohexyl C-H stretching bands at 2 9 2 0 cm."1 and 2 8 5 0 cm."1. NMR s i g -nals ( 6 0 MHz, C C 1 ^ ) « £ 0 . 9 0 - 1 . 7 0 ppm (multiplet, 11 H, C 6 H n ) , 2 . 1 5 ppm ( s i n g l e t , 6 H, n(CK^)2)* 2 . 3 0 - 2 . 5 0 ppm and 2 . 9 0 -3.55 ppm (multiplet, ( C H 2 ) 2 and CH 2), and 7 . 5 0 - 7 . 7 5 ppm mult i p l e t , 3 H, C ^ H ^ ) . Because a trace of the s t a r t i n g secondary amine ( b.p, 1 0 5 - 1 0 6°C, at 0 . 9 5 mm.) was brought over during the d i s -t i l l a t i o n of the t e r t i a r y amine, i t was necessary to pu r i f y 7 8 . the t e r t i a r y amine by column chromatography. The t e r t i a r y amine was p u r i f i e d by s i l i c a g e l column as described for N,N-dimethyl-N'-cyclohexylmethyl-N'-2-pyrimidylethylene-diamine. The pure N,N-dimethyl-N'-cyclohexylmethyl-N'-2-pyrazylethylenediamine came out i n the benzene-chloroform (2«8) f r a c t i o n s . Di-perchlorate Derivative! The light-yellow d i - p e r c h l o r i c acid s a l t of the p u r i f i e d t e r t i a r y amine was prepared as described for the N,N-dimethyl-N'-cyclohexylmethyl-N'-2-pyridylethylenediamine d i - p e r c h l o r i c acid derivative, and was r e c r y s t a l l i z e d from dry ethanol and dry ether (using a water bath at 6 0 ° C ) ; m.p. 1 5 0 - 1 5 7°C . Anal. Calcd. for Cj^HggN^ClgOgi C, 33.88; H, 6 . 0 9 ; N, 1 2 . 1 0 ; C l , 1 5 . 3 1 . Found; C, 3 3 . 9 9 ? H, 6 . 1 9 ; N, 1 1 . 9 4 ; ci, 1 5 . 0 7 . D. Synthesis of N.N-Dimethyl-N'-Cyclohexylmethyl-N'-1- Naphthylethylenediamine; 1. N-l-Naphthyl-Cyclohexanecarboxamide 1 A mixture of 1-naphthylamine ( 4 3 . 0 g., 0 . 3 mole), pyridine ( 2 3 . 8 g. F 0 . 3 mole), and dry benzene ( 2 0 0 ml.) was placed i n a 0 . 5 - l i t e r three-necked f l a s k f i t t e d with a me-chanical s t i r r e r , a r e f l u x condenser (drying tube) and a dropping funnel (250 ml.). Freshly prepared cyclohexane-carbonyl chloride ( 4 9 . 5 g«» 0 . 3 4 mole) was added dropwise and with s t i r r i n g to the reaction f l a s k cooled i n an ice-bath. A f t e r the addition was complete, the reaction was refluxed 7 9 . with s t i r r i n g for 1 hour, cooled and washed with 200 ml. of water. The solid which precipitated was suction f i l t e r e d and the layers were separated. The aqueous phase was extracted with two 200 ml. portions of ether. The ether extracts were combined with the benzene layer, and dried over anhydrous sodium sulfate. The solvents were removed by flash evapora-tion to recover more of the amide in a total yield of 6 8 . 6 g. ( 0 , 2 7 mole. 9 0 . 5 $ ) • The amide was recrystallized from 9 5 $ ethanol- m.p. 1 8 9 . 6 - 1 9 0 . 8°C. ( L i t . ( 8 5 ) , m.p. 1 8 8 ° C ) . Infrared spectrum of the solid amide (KBr) showed a strong C-0 stretching band at 1660 cm."1 and the character-i s t i c cyclohexyl C-H stretching vibration at 2860 and 29^0 cm." 2 . N-Cyclohexylmethyl-N-l-Naphthylaminei In a 1 - l i t e r three-necked flask equipped with a mechanical s t i r r e r , a Soxhlet extractor (drying tube) and a dropping funnel (250,ml.) was placed lithium aluminum hydride (11.4 g., 0 . 3 mole) in dry ether ( 7 0 0 ml.). The mixture was stirred with gentle reflux on a heating mantle for 4 hours. After this time, N-l-naphthyl-cyclohexanecarboxamide (30.4 g., 0 . 1 2 mole) was packed into the Soxhlet extractor whose bottom was lined with glass wool and a f i l t e r paper to prevent the blockage of the siphon arm. Three glass rods were inserted into the powder as channels for the extracting solvent. Re-fluxing was continued u n t i l a l l the amide had been carried into the flask. The reaction was then refluxed for 3 days. The reaction was then worked up with water (50 ml.) and suf-ficient 40$ NaOH solution for complete separation. The 80. ethereal layer was separated by decantation and dried over anhydrous sodium s u l f a t e . The solvent was removed by f l a s h evaporation to y i e l d 24.8 g. ( 0 . 1 mole, 86.4$) of product, b.p. 149-152°C. at 0 . 2 5 mm.. The l i g h t yellow o i l s o l i d i f i e d a f t e r standing overnight. The secondary amine (neat) was indicated i n the IR spectrum by the sharp N-H stretching v i b r a t i o n at 3440 cm. and the absence of a strong carbonyl absorption at 1660 cm."1 NMR signals (60 MHz, CCl^); <£o.90-1.90 ppm (multiplet, 11 H, C 6 H 1 1 ) , 2 . 9 2 - 3 . 0 2 ppm (doublet, 2 H, CH 2), 4 . 15 ppm ( s i n g l e t , 1 H, NH), and 6 . 3 5 - 6 . 5 0 ppm and 7.00-7.80 ppm (multiplet, 7 H C 1 0 H ? ) . Mono-hydrochloride Derivativei Dry hydrogen chloride was passed from a cylinder into a s o l u t i o n of the secondary amine (1 g.) i n dry ether ( 5 0 ml.). When p r e c i p i t a t i o n was complete, the s o l i d was sue t i o n f i l t e r e d under a stream of dry nitrogen gas to prevent i t from contacting a i r moisture and was washed with a small amount of dry ether. The pale-brown HCI s a l t was r e c r y s t a l -l i z e d from dry ethanol and n-pentane (using a water bath at 65°C.)j m.p. 165-178°C. Anal. Calcd. for C ^ H ^ N C l t C, 74 .02} H, 8.04-N, 5 . 0 8 ; CI, 1 2 . 8 6 . Foundi C, 7 3 . 9 6 ; H, 7.90; N, 5 . 2 1 ; CI, 1 2 . 7 7 . 81. 3 . N,N-Dimethyl-N'-Cyclohexylmethyl-N'-1-Naphthyl- ethylenediamine i A mixture of N-cyclohexylmethyl-N-l-naphthyl-amine (12.0 g., 0 . 0 5 mole), sodium amide (2.3 g., 0.06 mole) and dry toluene (80 ml.) was placed i n a 150-ml. three-necked f l a s k f i t t e d with a mechanical s t i r r e r , r e f l u x condenser (dry-ing tube), a dropping funnel ( 5 0 m l . ) and a dry nitrogen gas i n l e t tube. The mixture was bubbled with dry nitrogen gas and refluxed at 125°C. i n an o i l - b a t h for 12 hours. The mixture was cooled and the nitrogen flow discontinued. A solut i o n of/S-dimethylaminoethyl chloride (6 . 5 g., 0 . 0 6 mole), f r e s h l y d i s -t i l l e d from i t s hydrochloride s a l t and flake sodium hydroxide ( L i t . (93)), i n 30 ml. of dry toluene was added from the dropping funnel to the mixture, which was then refluxed an-other 24 hours. At the end of t h i s time, the mixture was cooled, and f i l t e r e d and the f i l t r a t e fractionated under reduced pressure a f t e r removal of the solvent by f l a s h evap-oration. The t e r t i a r y amine was obtained as a yellow o i l , b.p. 188-190°C. at 1 . 5 mm., y i e l d 11.1 g. (O .036 mole, 71.2%). A t e r t i a r y amine (neat) was indicated i n the IR spectrum by the absence of the sharp secondary amine N-H stretching v i b r a t i o n band at 3440 cm. - 1 and the presence of the c h a r a c t e r i s t i c C-H stretching for R-N(CH 3) 2 at 2820 cm."1' 2780 cm."1 and 2730 cm."1. NMR signals (60 MHz, CC l^ ) i ^0 . 9 0-1 . 9 0 ppm (multiplet, 11 H, C^H^), 2.10 ppm ( s i n g l e t , 6 H, N(CH.j) 2), 2.20-2.50 ppm and 2 . 9 5-3 . 3 0 ppm (multiplet, 6 H, (CH-)- and CH-), and 7.00-7.80 ppm and 8.20-8.35 ppm 82. (multiplet, ? H, C 1 Q K ^ ) , Because a trace of the s t a r t i n g secondary amine (b.p. l49 - 1 5 2°C. at 0 . 2 5 mm.) was brought over during d i s t i l -l a t i o n of the t e r t i a r y amine, i t was necessary to pur i f y the t e r t i a r y amine by column chromatography. The t e r t i a r y amine was p u r i f i e d on a s i l i c a gel column as described for the N,N-dimethyl-N'-cyclohexylmethyl-N'-2-pyrimidylethylene-diamine. The pure N,N-dimethyl-N'-cyclohexylmethyl-N-l-naph-thylethylenediamine came out i n the chloroform f r a c t i o n s . Di-perchlorate Derivative 1 The grayish white d i - p e r c h l o r i c acid s a l t of the p u r i f i e d t e r t i a r y amine was prepared as described for the N,N-dimethyl-N'-cyclohexylmethyl-N'-2-pyridylethylenediaraine d i - p e r c h l o r i c a c i d derivative, and was r e c r y s t a l l i z e d from dry ethanol and dry ether; m.p. 106.5-108.5°C.. Anal. Calcd. f o r ^ i ^ V ^ S 1 C* ^ 9 . 3 2 | H, 6 . 3 I ; N, 5.48; CI, 13.87. Found; C, 49.14; H, 6.46; N, 5 . 6 2 ; CI, 1 3 . 7 3 . E. Synthesis of N,N-Dimethyl-N'-Cyclohexylmethyl-N'-5- Quinolylethylenediamine t 1• N-5-Quinolyl-Cyclohexanecarboxamide t A mixture of 5-aminoquinoline (30 g., 0 .21 mole), pyridine (17.4 g., 0.22 mole), and dry benzene (100 ml.) was placed i n a 250-ml. three-necked f l a s k f i t t e d with a mechani-c a l s t i r r e r , a r e f l u x condenser (drying tube) and a dropping funnel ( 125 ml.). To the s t i r r e d mixture i n the flask (cooled i n an ice-bath) was added dropwise fr e s h l y prepared cyclo-8 3 . hexanecarbonyl chloride ( 3 2 . 3 g., 0 . 2 2 mole). The reaction was fluxed for two hrs., cooled and made basic with 100 ml. of 10% NaOH so l u t i o n . The crude amide was pr e c i p i t a t e d as a yellow-i s h white s o l i d , which was c o l l e c t e d by suction f i l t r a t i o n and washed with water u n t i l the water washing was neutral to litmus paper, y i e l d 4 5 . 7 g. (0.18 mole, 8 6 . 3 % ) . The amide was r e c r y s t a l l i z e d from 95% ethanol, m.p. 202.9-204.0°C.. Infrared spectrum of the s o l i d amide (KBr) showed a strong C -0 stretching band at 1650 cm."1 and the character-i s t i c cyclohexyl C-H stretching v i b r a t i o n bands at 2750 cm."1 and 2820 cm."1} also the absence of N-H stretching for 5-aminoquinoline at 3180 cm."1 and 3330 cm."1 proved the amide structure. NMR signals ( 6 0 MHz, CDCl^Ji 1 . 0 0 - 2 . 6 0 ppm ( mult i p l e t , 11 H, C^H^), 7 . 2 0 - 8 . 2 5 ppm (multiplet, 6 H, C 9H 6N), and 8 . 9 0 ppm (doublet, 1 H, NH)• Anal. Calcd. for C^H^ONgi C, 7 5 . 5 6 } H, 7 . 1 3 ; N, 1 1 . 0 2 . Foundi C, 7 5 . 5 0 } H, 7 . 0 2 } N, 1 1 . 2 0 . 2 . 5-CyclohexylmethylaminoQuinoline x In a 1 - l i t e r three-necked f l a s k equipped with a mechanical s t i r r e r , a Soxhlet extractor (drying tube) and a dropping funnel ( 2 5 0 ml.) was placed lithium aluminum hydride ( 9 . 5 g.» 0 . 2 5 mole) i n dry ether ( 6 0 0 ml.). The mixture was s t i r r e d with gentle r e f l u x f o r 4 hours. After t h i s time N-5-quinolyl-cyclohexanecarboxamide ( 2 5 . 5 g . i 0 . 1 mole) was packed into the Soxhlet extractor as described for the reduc-t i o n of N-l-naphthyl-cyclohexanecarboxamide under section PART IV D - 2 . Refluxing with s t i r r i n g was continued u n t i l a l l 84. the amide had been dissolved. The reaction was refluxed for 3 days and then worked up with water (50 ml.) and s u f f i c i e n t 40$ NaOH soluti o n to give complete separation. The separated organic layer was dried with anhydrous sodium s u l f a t e and r e -duced i n volume to y i e l d 2 0 . 0 g. ( 0 . 0 8 mole, 8 3 . 3 $ ) of 5-cyclohexylmethylaminoquinoline, b.p. 186-190°C. at 0.70-0.75 mm.. The l i g h t yellow o i l s o l i d i f i e d a f t e r standing overnight. The infrared spectrum (neat) indicated complete reduction by the absence of the carbonyl absorption band at 1650 cm,"1 and by the s h i f t of the N-H stretching v i b r a t i o n to 3320 cm."1, NMR signals (60 MHz, CDCl^): £ 1.10-1.90 ppm (multiplet, 11 H, O^K^), 3 . 0 5 - 3 . 1 5 ppm (doublet, 2 H, CH 2), 4 . 8 0 ppm ( s i n g l e t , 1 H, NH), and 6 . 5 0 - 6 . 6 5 ppm, 7.15-7.70 ppm, 8 . 1 5-8 . 3 2 ppm and 8 . 8 0-8 . 8 5 ppm (multiplet, 6 H, C ^ N ) . Mono-hydrochloride Derivative! The blood red c r y s t a l s of the secondary amine mono-hydrochloride s a l t were prepared as described for the N-cyclohexylmethyl-N-l-naphthylamine mono-HCl derivative; m.p. 240-242°C. (decomposed). Anal. Calcd. f o r CjgH^NgCli C, 69.42; H, 7 . 6 5 ; N, 1 0 . 1 2 ; CI, 12.81. Foundi C, 6 9 . 5 6 ; H, 7 . 7 2 ; N, 1 0 . 0 1 ; c i , 1 2 . 6 5 . 3 . N,N-Dimethyl-N'-Cyclohexylmethyl-N'-5-Quinolylethylene- diamine1 A mixture of 5-cyclohexylmethylaminoquinoline ( 12.0 g., 0 . 0 5 mole), sodium amide (2,4 g., 0 . 0 6 mole) and dry toluene ( 1 0 0 m l . ) was placed i n a 150-ml. three-necked f l a s k 85. f i t t e d with a mechanical s t i r r e r , r e f l u x condenser (drying tube), a dropping funnel (50 ml.) and a dry nitrogen gas i n l e t tube. The mixture was bubbled with dry nitrogen gas and re-fluxed at 125°C. i n an oil - b a t h for 20 hours. The reaction was cooled and the nitrogen flow discontinued. A solution of -dimethylaminoethyl chloride (6.5 g . i 0.06 mole), f r e s h l y d i s -t i l l e d from i t s hydrochloride s a l t and flake sodium hydroxide ( L i t . (93)), i n 20 ml. of dry toluene was added from the dropping funnel to the mixture, which was then refluxed an-other 24 hours. At the end of t h i s time, the reac t i o n was cooled and f i l t e r e d and the f i l t r a t e fractionated under re-duced pressure a f t e r f l a s h evaporation of the solvent. The t e r t i a r y amine was obtained as a yellow o i l , b.p, 164-168°C. at 0,15-0.20 mm., y i e l d 9.5 g. (0.03 mole, 60.9%). A t e r t i a r y amine (neat) was indicated by the absence of the secondary amine NH stretching v i b r a t i o n band at 3320 cm."1 i n the i n f r a r e d spectrum and the presence of the charac-t e r i s t i c G-H stretching v i b r a t i o n for R-N(CH.j)2 at 2820 cm."1, 2730 cm."1 and 2730 cm."1. NMR signals (60 MHz, CDCl^Ji ^0.90-1.80 ppm (multiplet, 11 H, C^H^), 2.10 ppm ( s i n g l e t , 6 H, N(CH 3) 2), 2.24-2.50 ppm and 2.90-3.30 ppm (multiplet, 6 H, ( C H 2 ) 2 and 0H 2), and 7.10-7.85 ppm and 3.55-8.85 ppm (multiplet, 6 H, C ^ N ) . Because a trace of the s t a r t i n g secondary amine (b.p. 186-190°C. at 0.70-0.75 mm.) was carr i e d over during d i s t i l l a t i o n of the t e r t i a r y amine, i t was necessary to puri f y the t e r t i a r y amine by column chromatography. The t e r t i a r y 8 6 . amine was p u r i f i e d on a s i l i c a gel column as described for the N.N-dimethyl-N'-cyclohexylmethyl-N*-2-pyrimidylethylene-diamine. The pure N.N-dimethyl-N*-cyclohexylmethyl-N'-5-quinolylethylenediamine came out i n the chloroform-rcethanol ( 9 11) f r a c t i o n s . Di-hvdrochloride Derivative! The orange-red c r y s t a l s of the p u r i f i e d t e r t i a r y amine di-KCl s a l t were prepared as described for the N-cyclo-hexylmethyl-N-1-naphthylamine mono-KCl derivative! m.p. 147-150°C. (decomposed). Anal. Calcd. for C ^ H ^ N y ^ i C, 6 2 . 4 9 , H, 8 .13? N, 1 0 . 9 3 ; C l , 18 . 4 5 . Foundi C, 6 2 . 5 5 ? H, 8 .33J N, 10 .75? C l , 1 8 . 3 0 . F . Synthesis of N.N-Dimethyl-N'-Cyclohexylmethyl-N'-3-Pyridazylethylenediamine,! 1 # 3-Amino-6-Chloror>yridazine! A mixture of 3 » 6-dichloropyridazine ( 3 0 . 0 g., 0 . 2 mole), absolute ethanol (400 ml.) and l i q u i d ammonia ( 3 5 . 3 g. 2 . 0 8 mole, trapped from an ammonia cylinder into a f l a s k cooled i n an acetone-dry ice bath) was heated and s t i r r e d for 10 hrs. i n a pressure reaction apparatus (Parr Instrument Co., Inc., Moline, 111., U.S.A., Pat. No. 2 6 2 5 2 9 6 ) . The s t a r t i n g pressure b u i l t up insi d e the apparatus was around 160 p s i and at the end of 10 hours the pressure went down to about 40 p s i . The reaction was cooled and the solvent and excess ammonia removed by f l a s h evaporation. The brownish residue ( 3 ^ . 4 g.) was then extracted i n a Soxhlet extractor v.'ith ethyl acetate 8 7 . solvent ( 3 5 0 ml.) overnight. C r y s t a l l i z a t i o n from the ex-t r a c t i o n solvent gave 1 3 . 0 g. ( 0 . 1 mole, 5 0 . 0 $ y i e l d ) of 3 -amino - 6-chloropyridazine, m.p. 206-208°C. (decomposed). ( L i t . (84), m.p. 210-212°C. decomposed, 70$ y i e l d ) . A primary amine (KBr) was indicated i n the IR spectrum by the presence of N-H stretching v i b r a t i o n at 3160 cm. - 1, 3300 cm."1 and 3340 cm. - 1- also the absence of C-Cl absorption band at 790 cm.~z indicated the su b s t i t u t i o n of 3-chloro group by an amino group. 2 . 3-Aminopyridazine i A suspension of 3-amino - 6-chloropyridazine ( 2 0 . 8 g. t 0 . 1 6 mole), sodium hydroxide ( 6 . 4 g., 0 . 1 6 mole) and 2 . 4 g. of 7$ palladium-charcoal ca t a l y s t i n 600 ml. of absolute ethanol was subjected to hydrogenation at 3 atmospheres pres-sure ( i . e . 45 psi) i n a pressure reaction apparatus (Parr Instrument Co., U.S.A.) s t i r r i n g was continued for 4 days. After t h i s time, the mixture was warmed, f i l t e r e d and then excess hydrogen chloride was added before concentration of the f i l t r a t e s . The solvent was flashed o f f and the yellowish white residue r e c r y s t a l l i z e d from ethanol-pentane produced 1 3 . 7 g. (0.144 mole, 90 . 0 $ y i e l d ) of 3-aminopyridazine as white microcrystals, m.p. 174.0-176.0°C.. ( L i t . (84), m.p. 1 7 5 . 5 - 1 7 6 . 5°C., 9 1 . 5 $ y i e l d ) . The hydrochloride was converted to the base by sodium bicarbonate and the crude product c r y s t a l l i z e d from ethyl acetate. Transparent blades were obtained, m.p. l 6 9 - 1 7 1°C. ( L i t . (84), m.p. 170-171°C.j (104) 1 6 8 - 1 7 0 ° C . J ( 1 0 5 ) 1 7 2 ° C ) . 88. 3 . N , N-Dimethyl - N ' - 3-Pyridazylethylenediamine i A mixture of 3-arcinopyridazine ( 1 2 , 3 g.» 0 , 1 3 mole), sodium amide (5.46 g., 0.14 mole) and dry toluene (110 ml.) was placed i n a 150 ml. three-necked f l a s k f i t t e d with a me-chanical s t i r r e r , r e f l u x condenser (drying tube), a dropping funnel (50 ml.) and a dry nitrogen gas i n l e t tube. The mix-ture was bubbled with dry nitrogen gas and refluxed at 125°C. i n an o i l - b a t h for 20 hours. The reaction was cooled and the nitrogen discontinued. A so l u t i o n of ^ -dimethylaminoethyl chloride ( 1 5 . 0 6 g., 0.14 mole), which v/as fre s h l y d i s t i l l e d from i t s hydrochloride s a l t and flake sodium hydroxide ( L i t . (93)» i n 20 ml. of dry toluene was added from the dropping funnel to the reaction mixture, which was then refluxed an-other 24 hours. At the end of t h i s time, the reaction was cooled and f i l t e r e d and the f i l t r a t e , a f t e r removal of solvent by f l a s h evaporation was fractionated under reduced pressure to y i e l d 5.4 g. ( 0 . 0 3 mole, 2 5 . 1 $ ) of N , N - d i m e t h y l - N * - 3 -pyridazylethylenediamine, b.p. 10?-108 oC. at 1.1 mm.. A secondary amine (neat) was indicated i n the I R spectrum by the presence of a broad N - K stretching v i b r a t i o n band (conjugated) at 3280 cm."1 and by the presence of the c h a r a c t e r i s t i c C-H stretching v i b r a t i o n for R - N ( C H 3 ) 2 at 2820 cm."1, 2780 cm."1 and 2730 cm."1. NiYiR signals (60 KHz, CDCl.j)i ^ 2 . 3 0 ppm (s i n g l e t , 6 H, N(CH^) 2), 2.60-2.80 ppm ( t r i p l e t , 2 H, CH 2), 4 . 0 5-4 . 3 0 ppm ( t r i p l e t , 2 H, CH 2), 5 . 5 5 ppm (s i n g l e t , 1 H, N H ) , and 6 .55-6.60 ppm and 7.25-7.35 ppm (multiplet, 3 H, C^H.Ng). 8 9 . The secondary amine was not stable and decomposed a f t e r standing overnight. Column chromatography ( s i l i c a gel) was t r i e d to p u r i f y the secondary amine but the e f f o r t s were i n vain. G, Synthesis of 2,2-Diphenyl-4-Dimethylaminomethyloxetane» 1. 2 , 2-Diphenyl - 3-Chlorooxetanet Benzophenone ( 2 . 0 0 g., 0 . 0 1 1 mole) was dissolved i n 150 ml, of reagent benzene i n a 200-ml. photochemical r e -action vessel (Hanovia, U.S.A.). A quartz immersion well was f i t t e d i n t o the reac t i o n vessel, and v i n y l chloride was bub-bled from a v i n y l chloride cylinder i n t o the sol u t i o n through a small diameter PVC tube, u n t i l the volume reached the max-imum, l e v e l ( t o t a l volume 200 ml, which contained about 40 ml. of v i n y l c h l o r i d e ) . The gas was turned o f f and the soluti o n was cooled with running water and i r r a d i a t e d with a 450-w. mercury lamp (Hanovia, U.S.A.) through a pyrex f i l t e r tube (which f i l t e r e d out a l l l i g h t below 2900 £ wave length) f o r 2\ hours.. The benzophenone carbonyl absorption band at 1670 cm."1 i n IR was almost gone at the end of t h i s time. The s o l u t i o n was then suction f i l t e r e d and the f i l t r a t e con-centrated by f l a s h evaporation. The yellowish white residue was extracted several times with excess hot petroleum ether (b.p B 3 0 - 6 0°C.). The petroleum ether extracts were concen-trated and c r y s t a l l i z e d from hexane to give 400 mg. ( 0 . 0 0 1 6 mole, 14 .8% y i e l d ) of the adduct, m.p. 8 5 . 5 - 8 7 . 0 ° C o . Infrared spectrum of the s o l i d oxetane (KBr) showed a strong absorption band at 980 cm."1 which was c h a r a c t e r i s t i c 90. of an oxetane r i n g . NMR signals (60 MHz, CDCl^)- £ 4 . 5 0 - 4 . 7 2 ppm and 4.80-5.02 ppm (two t r i p l e t , 2 H, CH 2), 5 . 3 0 - 5 . 5 0 ppm ( t r i p l e t , 1 H, CHC1), and 7 . 2 . 0 - 7 . 5 5 Ppm (multiplet, 1 0 H, CgH^-C-C^H^). The low chemical s h i f t values of CH2 proved the structure. Anal. Calcd. f o r C ^ H ^ O C l i C, 7 3 o 6 2 ? H, 5 * 3 5 * CI, 14 . 4 9 . Foundt C, 7 3 . 7 0 ? H, 5 . 5 1 ; CI, 14 . 4 3 . . . 2 . 2 , 2-Diphenyl - 3 - (Pyrrolidin - 2-one-l-yl)oxetane: Benzophenone ( 2 . 0 0 g. f 0.011 mole) and 5 ml. of N-vinyl - 2-pyrrolidinone (about 5 g.» 0.047 mole) were dissolved i n 180 ml, of reagent benzene i n a 2 0 0-ml. photochemical r e -ac t i o n v e s s e l (Hanovia, U.S.A.). A quartz immersion well was f i t t e d i n t o the r e a c t i o n v e s s e l , and the solution was bubbled with dry nitrogen gas f o r 1 0 minutes. The gas flow was turned o f f and the s o l u t i o n was cooled with running water and i r r a d i a t e d with a 4 5 0 - w . mercury lamp (Hanovia, U.S.A.) through a pyrex f i l t e r tube (which f i l t e r e d out a l l l i g h t below 2 9 0 0 J wave length) f o r 3 hours. The benzophenone was almost gone by the end of t h i s time (IR showed the benzophenone carbonyl absorp-t i o n band at I67O cm."x had almost disappeared). The solvent was flashed o f f g i v i n g 6 . 5 g. of an o i l residue. The o i l was extracted by shaking several times v/ith hexane and decanting the hexane soluble portions. The hexane insoluble residue was dissolved i n a small amount of ethanol. Water was then added to the ethanolic s o l u t i o n g i v i n g a p r e c i p i t a t e which was c o l l e c t e d by suction f i l t r a t i o n , ; The p r e c i p i t a t e was separated on 4 TLC plates ( 2 0 x 2 0 cm.) using s i l i c a gel G (Art, 7 7 3 1 , . E . Merck, Germany) and solvent system CHClo(98$)-9 1 . MeOH (2%), and a l a r g e band was c o l l e c t e d ( R f = 0 , 7 , 170 mg.) and c r y s t a l l i z e d f rom e t h e r - h e x a n e m i x t u r e g i v i n g 42 mg. ( 0 .00014 m o l e , 1.3% y i e l d ) o f 2 , 2 - d i p h e n y l - 3 - ( p y r r o l i d i n - 2 - o n e -l - y l ) o x e t a n e , m.p . 140-142°C. ( L i t . ( 9 9 ) . m.p. 141,5- l43°C.) . I n f r a r e d spe c t r um o f t h e s o l i d o xe t ane ( K B r ) showed t h e s t r o n g o xe t ane r i n g a b s o r p t i o n band a t 980 c m . " 1 } a l s o t h e s t r o n g amide c a r b o n y l a b s o r p t i o n band a t I 6 7 O c m . " 1 i n d i -c a t e d t h e a d d u c t f o r m a t i o n . NMR s i g n a l s (60 MHz, CDC l^J i ^1 .30-3.40 ppm ( m u l t i p l e t , 6 H, C^HgNO), 4 . 7 5 - 5 . 0 2 ppm (two t r i p l e t , 2 H, C H 2 ) , 5 . 6 5 - 5 . 9 0 ppm ( t r i p l e t , 1 H, CHN), and 7 . 2 0 - 7 . 8 0 ppm ( m u l t i p l e t , 10 H, C A H < - C - C A H < ) . PART V PHARMACOLOGICAL TESTING AND ANTIHISTAMINIC RECEPTOR STUDIES A. INTRODUCTION Formal analysis of the dose-response r e l a t i o n s h i p s i s a fundamental tool i n receptor studies; one should remem-ber, however, that the information so obtained shows only how clo s e l y r e l a t i o n s h i p s proposed on the basis of a c e r t a i n theo-r e t i c a l concept are able to mimic the r e a l s i t u a t i o n ( 1 0 6 ) . Dose-response curves of various drugs and of combinations of drugs i n i s o l a t e d organs have provided a deeper in s i g h t into the mode of action of various classes of drugs ( 1 0 7 , 1 0 8 ) . For example, most antihistamines act competitively and antag-onize the action of histamine on guinea-pig ileum by means of a r e v e r s i b l e union with a common receptor s i t e . This could be studied by making dose-response curves and by character-i z i n g t h e i r maximum height, the slope of the curves and t h e i r positions on the dose-axis ( 1 0 8 ) . Previous work i n t h i s laboratory ( 1 , 2 ) had shown that, i n the ethylenedlamine-type antihistaminic receptor, one of the aromatic rings i n Antergan (I) could be replaced by an a l i c y c l i c system (e.g. cyclohexyl, Ila) and the a n t i -histaminic a c t i v i t y was f u l l y retained. Therefore, i t was of i n t e r e s t to study how v a r i a t i o n s i n size and structure of the aromatic moiety (R 2 i n II) i n t h i s a l i c y c l i c substituted system (II) would a f f e c t the a c t i v i t y at the receptor s i t e . 93. v\ >CH2-N-CH2.CH2-N /C H 3 (I) R„ = CH 2' -C-; II 0 R~ = R2 3 (II) (a) phenyl, (b) 2-pyrldyl, (c) 2-pyrlmidyl, (d) 2-pyrazyl, (e) 1-naphthyl, and (f) 5 - q u i n o l y l . (g) 2-pyrlmidyl, and (h) 2-pyrazyl, B. EXPERIMENTAL Drug-receptor interactions were studied and evalu-ated according to the k i n e t i c methods of van Rossum ( 1 0 7 . 1 0 8 ) and the technique f o r the making of cumulative dose-response curves was followed ( 1 0 8 ) . Histamine acid phosphate ( B r i t i s h Drug Houses, Poole, England) and acetylcholine bromide (Eastman Kodak, U.S.A.) were used as the reference agonists f o r study-ing the antihistaminic and a n t i c h o l i n e r g i c a c t i v i t i e s , respec-t i v e l y , of a series of eight ethylenedlamine-type a n t i h i s t a -minic compounds, I.e. (Ila) and ( I l f ) as mono- and di-hydro-chloride s a l t s respectively, (Ilg) and (Ilh) as mono-perchloric 9 4 . acid s a l t s , and ( l i b ) , ( H e ) , ( l i d ) and (He) as d i - p e r c h l o r i c acid s a l t s . Diphenhydramine hydrochloride (Sigma Chem. Co., Missouri, U.S.A.) and atropine s u l f a t e ( B r i t i s h Drug Houses, London, England) were used as a standard antihistaminic and an t i c h o l i n e r g i c agent, r e s p e c t i v e l y . A l l compounds tested ( i . e . agonists and antagonists) were fre s h l y prepared i n nor-mal saline solutions and calculated as molar concentration of the respective s a l t i n terms of the s a l t used. A l l donor guinea-pigs were fasted f o r 24-48 hours p r i o r to s a c r i f i c e . S t r i p s of guinea-pig's terminal ileum 2 - 4 cm. long were aerated with a mixture of 95% oxygen and $% carbon dioxide i n a 25-c.c. bath containing Tyrode s o l u t i o n (8 . 0 g. NaCl, 0 . 2 g. KC1, 0 . 1 g. CaCl 2, 0.1 g. MgCl 2, 0 . 0 5 g . NaH2P0^, 1.0 g. glucose, and 1 .0 g. NaHCO^, water to 1000 ml.). The bath f l u i d was kept at a constant temperature (37 t 0 . 5 ° C ) by pumping water from a thermostatically-controlled r e s e r v o i r through a jacket sur-rounding the bath. The lo n g i t u d i n a l contractions of the i n -testine were recorded on a sooted drum with the aid of a l i g h t i s o t o n i c lever ( i . e . a kymograph). Cumulative dose-response curves were obtained by stepwise addition of agonist (histamine or acetylcholine) to the organ bath, such that the next dose of agonist was added just as the gut s t r i p had reached a steady maximum contraction due to the previous dose. Doses were i n -cremented at half log units f o r histamine and at log units f o r acetylcholine, i . e . histamine was added so that the t o t a l bath concentration was 1 0 " 8 M/l, 3 x 1 0 ~ 8 M/l, 1 0 ~ 7 M/l, 3 * 1 0 " 7 - 4 M/l, 3 x 10 M/l without washing the preparation u n t i l 9 5 . maximum contraction was reached; while the t o t a l hath concen-—8 "7 "5 t r a t i o n for acetylcholine was 10~ M/l, 10 M/l, ...... 10~J M/l. The height of contraction f o r every cumulative dose was measured i n mm. and calculated i n percentages of the maximum height of the reference compound. The percentages were plotted on a l i n e a r scale as mm. (100% was 100 mm.) on the ordinate while the doses were plotted on a logarithmic scale by using 30 mm. f o r every log 10 i n t e r v a l on the abscissa (see Appendix A f o r the computer program, Superplot, which was used f o r p l o t t i n g the log dose-response curves). Cumulative dose-response curves f o r the agonist were made u n t i l at l e a s t two consecutive, reproducible dose-response curves had been obtained. A 1 5 - 2 0 min. wash period was established between each curve u t i l i z i n g three 100 ml. washings of fresh perfusate. Antagonistic e f f e c t s were then determined by allowing the antagonist to incubate with the ileum f o r 15 min. (115) p r i o r . t o documenting the cumulative log dose-response curve f o r the agonist In i t s presence. The in t e s t i n e was then washed out with four 100 ml. of fresh Tyrode solu t i o n during a 2 5 - 3 5 min. Interval f o r complete recovery. In t h i s manner the cumulative dose-response curve of the reference agonist was made, a l t e r n a t i v e l y i n the pres-ence and i n the absence of a constant concentration of the test compound (antagonist), and the procedure was repeated three times. A fresh piece of gut was used f o r each concen-t r a t i o n of the test compounds. Each compound was tested at three dose concentrations (generally \ log unit apart) and 96. each concentration was tested i n four separate organ prepara-t i o n s . The dose-response curves a f t e r adding the test com-pounds were compared to the control curves, and from the d i s -placement and / or depression of the test curve from the control, values f o r the pA 2 and pD^ were calculated (for det a i l e d procedures see Results and Discussion). Values f o r the pD 2 of the agonists were determined from the control curves. C. RESULTS AND DISCUSSION Within the concentrations tested, the a n t i h i s t a -minic a c t i v i t y of the test compounds ( I l a - f ) were shown to exhibit two types of antagonistic actions, i . e . compounds (Ila-d) exhibited pure competitive antagonism (Fig. 8 - 1 9 , page 1 1 8 - 1 2 3 ) while compounds ( I l e - f ) exhibited mixed competitive-noncompetitive a c t i v i t y (Fig. 2 0 - 2 5 , page 124-126). At high concentrations most c l i n i c a l l y used antihistamines become non-competitive antagonists of histamine. For example, diphen-hydramine hydrochloride i s usually thought to be a highly potent competitive antagonist of histamine, but when the con-centration i s equal to and higher than 1 x 10"^ M/l, i t becomes a noncompetitive antagonist of histamine on i s o l a t e d guinea-pig ileum ( 1 0 9 ) . With respect to a n t i c h o l i n e r g i c a c t i v i t y on the Isolated guinea-pig ileum, a l l compounds tested ( I l a - f ) d i s -played noncompetitive antagonism of acetylcholine (Fig. 2 6 - 4 5 , 9 7 . page 1 2 7 - 1 3 6 ) . Compounds (Ilg) and ( I l h ) , however, were found to be Inactive In both antihistaminic and a n t i c h o l i n e r g i c tests even when the concentration used was as high as 1 x 10"* ^  M/l. The a c t i v i t y of any p a r t i c u l a r drug w i l l depend on i t s a f f i n i t y f o r the receptor upon which i t acts and on i t s i n t r i n s i c a c t i v i t y at that receptor ( 1 1 0 , i l l ) . I n t r i n s i c a c t i v i t y i s defined as the r a t i o between the maximum e f f e c t of a drug and the maximum possible e f f e c t i n that system with any other reference compound. Both h i s t a -mine and acetylcholine have i n t r i n s i c a c t i v i t y equal to unity. The a f f i n i t y of an agonist i s determined as the negative logarithm of the dose which produces a response equal to 50% of the maximal response and has been c a l l e d the pD 2 value (106, 108). pD 2 = - log A 5 Q ( i ) where i s the agonist concentration which produces a 50% e f f e c t ( E ^ 0 ) . The a f f i n i t y pD 2 of histamine can be estimated from the log dose-response curve (e.g. F i g . 8 and 9 ) by draw-ing a v e r t i c a l from E ^ Q to the abscissa to obtain the £A^ Q } and. then applying the above equation ( 1 ) . Because the s e n s i -t i v i t y of the gut s t r i p s varied a c e r t a i n amount even i n d i f -ferent segments of the same gut ( 1 1 2 ) , the pD 2 values obtained from F i g . 8 and 9 also varied. However, t h i s could be over-come by repeating the experiment and by random s e l e c t i o n of 9 8 . experimental animals ( 112) . Therefore, 222 control plots have been performed i n the present study to obtain the average pD 2 value for histamine, i . e . pD 2 = 6 . 4 l (Table 1 2 ) . TABLE 12 RESULTS FOR ANTIHISTAMINIC AND ANTICHOLINERGIC TESTS Histamine t pD £ = 6 . 4 l a ( 0 . 0 2 ) c , L i t . value = 6 .60 (108) Acetylcholine t pD 9 = 6 . 7 0 b ( 0 . 0 3 ) , L i t . value = 7 .00 (113) Compounds pA 2 vs Histamine** pD 2 vs Histamine 6 f pD 2 vs Ach. Ila-monoHCl 8.46 (0.04) c 5.55 ( o . 0 5 ) c IIb-diHC10^ 7.77 (0 . 0 3 ) 5.01 ( 0 . 0 5 ) IIc-diHC10^ 7.24 (0 . 0 5 ) 4 .56 (0.02) Ild-diHClO^ 6.37 (0.02) 4 .45 (0.04) Ile-diHClO^ 7.19 ( 0 . 0 3 ) 6.04 ( o . o 6 ) c 6.18 ( 0 . 0 5 ) I l f - d i H C l 7 .23 ( 0 . 0 3 ) 5.96 (0.04) 5.11 (0.02) Diphen- l h hydramine 8.27 6(0 .03) 6 .58 1 ^ (0.02) Atropine 8.92 1 t l i(0.04) at average of 222 control p l o t s . bt average of 265 control p l o t s . ct numbers i n parentheses are standard error. di mean of 30 to 48 values. ei mean of 24 values. f i mean of 30 to 48 values (Ach. = Acet y l c h o l i n e ) , gi L i t e r a t u r e value 7.7 (108), 8.0 (112). hi pA 2 vs Acetylcholine. i i L i t e r a t u r e value 8.9 (rat i n t e s t i n e ) (108), 8 .6 (guinea-pig ileum) (112). 9 9 . The pDg value f o r acetylcholine bromide (e.g. F i g . 26) was obtained i n the same way as f o r histamine acid phos-phate, the value being pD 2 = 6.70 ( L i t . (113), Acetylcholine pD 2 = 7 .0) (Table 1 2 ) . It should be noted that the parameters so obtained were empirical and not corrected f o r a possible n o n - l i n e a r i t y between stimulus and e f f e c t and that they therefore might deviate from the exact values. To f i n d the exact value would be an enormously laborious procedure (108). Pure competitive antagonists are Inactive as agonists. They have a f f i n i t y towards the s p e c i f i c receptors but no i n t r i n s i c a c t i v i t y and :;hus have no a b i l i t y to generate a stimulus. Independently of the r e l a t i o n s h i p between stimulus and e f f e c t , the dose-response curves of the agonist i n the presence of a constant concentration of the competitive drug have a shape e s s e n t i a l l y i d e n t i c a l with the curve when the antagonist i s absent (106, 108., 110) . A s i m i l a r e f f e c t as f o r instance, 50%, i s reached only at higher doses. The curves are p a r a l l e l l y s h i f t e d to the r i g h t on the log dose axis (e.g. F i g . 8 - 1 0 ) . The extent of s h i f t i n g of the dose-response curve of a stimulant drug can be used f o r determining the a f f i n i t y of the antagonist (111). The pA 2 value was introduced by Schil d (112, 114) to measure the a f f i n i t y of the competitive drug and was defined as the negative logarithm of the molar concentration of antagonist which caused a s h i f t of a value 2. A ce r t a i n dose of the antagonists causes a s h i f t x (e.g. x^, x 2, and x~ i n F i g . 8 , 9 , and 10, respectively) and i t s 100 . negative logarithm i s denoted as pA x where the r e l a t i o n s h i p pA x and pA 2 i s as follows: pA 2 = pA^ . + log (x - 1) ( i i ) The molar concentration of the antagonist (e.g. B x 10 ^  M/l) tested i n the experiment was used to calculate the pA^ value, thus pA^ = p - log B. The distance i n mm. between the $0% e f f e c t point i n the control curve and that i n the test curve was denoted as x (e.g. x ^ x 2, and x^ i n F i g . 8, 9, and 10 respectively) and by the a i d of Table 13 (see Appendix D, from reference 108) x was converted into log (x - 1 ) . The pA 2 value was then read according to the above equation ( 1 1 ) . However, i t was pointed out by S c h l l d (112) that when the a c t i v i t y of the antagonist had to be defined i n terms of some other drug, the r e s u l t s were not equally r e -producible since the apparent a c t i v i t y varied i n successive experiments, even though conditions were kept as constant as possible, This was what was found f o r the r e s u l t of diphen-hydramine. I t s pA 2 value was equal to 8 .27 while the l i t e r a -ture value was 7 .7 (108) and 8 . 0 (112) (Table 1 2 ) . To ob-t a i n a representative value of pA 2 f o r a given ti s s u e , and an antagonistic drug, the mean of a random sample of determinations on d i f f e r e n t i n d i v i d u a l animals must be determined. Therefore, pA 2 i s a s t a t i s t i c a l constant ( 1 1 2 ) . V a r i a b i l i t y may be reduced by comparing the a c t i v i t y of one 10-1. antagonist with that of another ( 112) . For t h i s reason the experimental conditions were kept as constant as possible and the pA 2 values of a series of antagonists were compared with that of diphenhydramine hydrochloride. The potency i n de-creasing order of the compounds tested (pA^ value In parenthe-s i s ) was as follows: H a (8.46) > Diphenhydramine (8.27) > l i b (7.77) > H e (7.24) > l i d (6.37). The pAg values f o r both diphenhydramine and a t r o -pine (Table 12) as antagonists f o r acetylcholine i n guinea-pig ileum were determined i n the same way as that of diphen-hydramine vs histamine ( i . e . used equation i i ) . Ralna and Das (125) showed that diphenhydramine was 56-times more act i v e i n antagonizing histamine than a c e t y l c h o l i n e . This was s i m i l a r to the present r e s u l t s which Indicated that diphenhydramine was 49-times as e f f e c t i v e i n antagonizing histamine than ace t y l c h o l i n e . A pure noncompetitive antagonist, l i k e a competi-t i v e antagonist, does not produce a response i n the t i s s u e . I t has no a f f i n i t y f o r the s p e c i f i c receptor of the agonist but in t e r a c t s with d i f f e r e n t receptors and so influences stimulus formation or stimulus e f f e c t u a t i o n . Therefore, i t has i n t r i n s i c a c t i v i t y with a negative sign. The a f f i n i t y of the noncompetitive antagonist f o r i t s receptors can be calculated from the depression of the maximal response to 102. the agonist i n the presence of the antagonist. The pD 2 value i s defined as the negative log of the molar concentration of antagonist which causes a depression of the maximal response to the 50% l e v e l . From the depression (x) caused by a c e r t a i n dose of the non-competitive antagonist the pD^ value can be calculated as follows: pD 2 = pD^ + log (x - 1) ( i i i ) -p From the molar concentration of the antagonist B x 10 the pD' value i s calculated as pD* = p - log B. The maximum ef f e c t of the test curve i s expressed i n percentage of the maximum e f f e c t of the contr o l . With the a i d of Table 14 (see Appendix E, from Reference (108)) the percentage i s converted into log (x - 1). The pD 2 value i s e a s i l y found according to equation ( i i i ) . The r e s u l t s f o r the non-competitive action of com-pounds H a - f against acetychollne were shown i n Table 12 and F i g . 26-4-5. These compounds merely caused a depression of the dose-response curves without s h i f t i n g which indicated that they were pure non-competitive antagonists. It i s possible to get dualism of antagonism, i . e . a drug may act as both a competitive and a non-competitive antagonist. In t h i s case both components show t h e i r own c h a r a c t e r i s t i c e f f e c t : the competitive i n t e r a c t i o n s h i f t s the dose-response curve to the r i g h t i n r e l a t i o n to pA 2, the non-competitive antagonist component depresses the maximum i n 103. r e l a t i o n to pD 2 (106). Thus, both pA 2 and pD 2 values may be calculated, provided that the competitive antagonism i s of greater order than the non-competitive (108). Compounds (He) and ( H f ) were found to exhibit dualism of antagonism (Fig. 20-25 and Table 12). At concen-t r a t i o n l e s s than 1 x 10"7 M / l , both (He) and ( H f ) displayed competitive antagonism towards the s p e c i f i c histamine receptor; -7 while at concentration equal to or higher than 1 x 10 M / l , they antagonized histamine non-competltlvely. In f a c t , t h i s type of i n t e r a c t i o n could often be seen, as a large number of competitive antagonists produced non-competitive depressive a c t i v i t y when used i n very high concentrations (106, 108, 109, 110, 112). Recently several workers have studied some of the variables which might influence the experimental r e s u l t s i n the guinea-pig ileum preparation. Bruno and co-workers (116) reported that a f t e r prolonged contact with the ileum the a c t i v i t y of diphenhydramine (as hydrochloride and pamoate) decreased. S c h i l d (112) i n h i s o r i g i n a l paper also found that the pA 2 values were d i f f e r e n t between the 2 min. and Ik min. contact times of the antagonist with the ileum. In the present study the contact time was set at 15 min. as suggested by Reuse (115). However, as mentioned by Reuse (115) the procedures Involved the assumption that a f t e r 15 min. the response to the agonist would remain unchanged. The f a c t that t h i s i s not c e r t a i n may account f o r some of the v a r i a t i o n s i n the r e s u l t s ; nevertheless l f a l l 104. antagonists tested were incubated with the ileum f o r the same period of time, the pA 2 values so obtained s t i l l were valuable f o r comparing the r e l a t i v e a c t i v i t y of each antago-n i s t towards the histamine receptor s i t e s . Rocha e S l l v a (11?) have found that the recovery of the histamlne-induced c o n t r a c t i l e response of Isolated guinea-pig following i n h i b i t i o n of t h i s response with diphen-hydramine (2-3 ug/ml.) was f a s t e r at 4°C. than at 3 7 ° C . In the present study the ileum was washed with Tyrode solu t i o n warmed through a 37°C.-jacket and found that a f t e r 25-35 min. of r e s t i n g period most i l e a could repeat the o r i g i n a l response before adding any antagonists. Clr s t e a (118) reported that the contractions induced by histamine and acetylcholine i n the guinea-pig i n -t e s t i n a l musculature were lar g e r and equal respectively at 2 7°C, as compared to 3 7 ° C . He concluded that the unex-pected e f f e c t of low temperature on the contraction Induced by histamine was due to a functional blocking of some i n h i -b i t o r y ganglion c e l l s from the enteric nervous plexuses. Cortese ( 1 1 9 ) mentioned that the s u r v i v a l time of Isolated guinea-pig i n t e s t i n e showed c h a r a c t e r i s t i c differences according to the parameters or function used f o r the study. Thus frequent washings with fresh Tyrode so l u t i o n which supplied constantly the ions and n u t r i t i o n needed f o r the ileum could make the i n t e s t i n e l i v e longer. In the present work i t was found that although the i l e a were immersed i n Tyrode s o l u t i o n and kept i n the r e f r i g e r a t o r , they could not function properly 10 hours a f t e r s a c r i f i c i n g 1 0 5 . the animal. For t h i s reason a l l the i l e a were used within 8 hours a f t e r s a c r i f i c i n g the animal. Tamarit (120) reported that the dose-effect of acetylcholine and histamine on the i s o l a t e d guinea-pig ileum were represented by a sigmoid-type cure. He used d i f f e r e n t equations from those of van Rossum's (108) to measure the r e l a t i v e a c t i v i t y f o r histamine and acetylcholine acting on the ileum; the r e s u l t s were that acetylcholine was found to be about 1.8-f o l d more ac t i v e than histamine i n the ileum. This agreed with the present r e s u l t where i t was found that acetylcholine was I.9-fold more active than histamine i n the ileum. D. ANTIHISTAMINIC.RECEPTOR STUDIES The most comprehensive study on the antihistaminic receptor was that c a r r i e d out by Nauta and co-workers (3» 4, 75# 78, 79). They proposed an antihistaminic receptor i n the smooth muscle c e l l s of ileum to accommodate both histamine and diphen-hydramine molecules. The high antihistaminic a c t i v i t y of diphen-hydramine was a t t r i b u t e d to the f a c t that f i r s t l y , one of the a r y l groups p a r t i c i p a t e d i n an overlap i n t e r a c t i o n with the ether oxygen; secondly, i t was also t h i s a r y l group that was assumed to be more or l e s s coplanar with the C-0; t h i r d l y , one of the a r y l groups was i n TC-complexation with the phenyl group of phenyl-alanine i n the polypeptide chain;.and f i n a l l y , the protonated-NtCH^Jg group interacted with the anionic s i t e on the receptor through a pot e n t i a l hydrogen-bond between i t s e l f and the N«-1 0 6 . atom of histidine residue. The fact that compound (Ila) i s highly potent (1.5-times more active than diphenhydramine) made us believe that the phenyl group of (Ila) participates in an overlap interaction with the amino nitrogen, i s co-planar with C-N, and was in %-complexation at the anchorage site in the receptor. The overlap interaction i s far more important due to the fact that 4-methyldlphenhydramlne (Neo-benodine, III) enhanced the overlap interaction through the positive mesomerlc effect Induced by the k-CKj group and therefore (III) was 10-fold more potent than diphenhydramine i t s e l f ( 3 ) ; also, i t i s . relevant that the thio ether analogues of substitued diphenhydramine exhibited less antihistaminic a c t i v i t y due to the fact that the free electrons of sulfur are not as available as those of oxygen for overlapping interaction with the ^-electrons of one of the aromatic rings and the steric interference of the bulkier sulfur atom also interfered with this interaction ( 4 ) . As a result, any factors which interfered with this overlap interaction would decrease the antihistaminic a c t i v i t y . This was further supported by compounds ( H e ) and ( l i d ) HC-0-CH2-CH2-N ( I I I ) 107. which were 1 0 .7-fold, and 79.4-fold, respectively, l e s s potent than diphenhydramine. The reason f o r less a c t i v i t y was due to the f a c t that both compounds ( l i e ) and ( l i d ) contain two electron-withdrawing centers ( i . e . two electro-negative nitrogens) In compound (Lie), the carbon flanking the two nitrogens ( i . e . C 2-atom) i n the pyrimidlne r i n g i s highly p o s i t i v e l y charged (IV) ( 1 2 1 ) . This p o s i t i v e l y charged C^-atom a t t r a c t s the lone + . 0 5 4 + .007 \ N-.0948 +.0746 N' -.0828 f ^+.0414 +.0164 V +.0034 +.037 (IV) (V) (VI) p a i r electrons from the amino nitrogen i n the ethylenediamine chain towards i t s e l f so that the overlapping i n t e r a c t i o n be-tween the ^ - e l e c t r o n s of the pyrimidine r i n g and the lone p a i r electrons of the amino nitrogen i s greatly reduced. Consequently, the observed antihistaminic a c t i v i t y was decreased. Compound ( l i d ) i s l e s s a c t i v e than ( l i e ) while the Cg-atom i n (V) i s less p o s i -t i v e l y charged than that of (IV). This i s probably because the ac-t i v i t y of (V) i s d i f f e r e n t from that of (IV) a t the receptor s i t e s . Compound (lib) contains only one electron-withdrawing center i n the pyridine r i n g such that the Cg-atom of the pyridine i s the l e a s t p o s i t i v e l y charged (VI) as compared with C^-atoms 108. of pyrlmldine (IV) and pyrazine (V) r i n g s . Thus ( l i b ) was approx-imately as active as diphenhydramine (although the pA^ value c a l c u l a t i o n showed that ( l i b ) was 3 . 1 - f o l d l e s s active than diphenhydramine, the f a c t that Bruno and co-workers (116) had shown that the a c t i v i t y of diphenhydramine pamoate was l e s s potent than the corresponding HCI s a l t supported the b e l i e f that because ( l i b ) was tested as the dl-HC10^ s a l t rather than the corresponding HCI s a l t , the larger s a l t d e r i v a t i v e had l e s s antihistaminic a c t i v i t y ) . Moreover, compound ( l i b ) resembled the structure of the potent antihistamine Tripelennamine (VII) except that the benzyl group of the l a t t e r was replaced by a cyclohexylmethyl group i n the former structure. not necessary to have both aromatic rings i n order to have high antihistaminic a c t i v i t y , yet the r i n g size of the a l i c y c l i c substituted system (VIII) was Important i n the i n t e r a c t i o n with the complementary groups on the receptor s i t e s . Cyclohexyl d e r i v a t i v e (VHIb = Ila) was the most active one, and cyclo-butyl (VHId), cyclopentyl (VIIIc), and cycloheptyl ( V i l l a ) ( V I I ) Although previous work (2) had shown that i t i s 1 0 9 . R-N-CH9-CH9-NS ^ * * CH (VIII) R = (a) cycloheptylmethyl, (b) cyclohexylmethyl (= I l a ) , (c) cyclopentylmethyl, (d) cyclobutylmethyl, (e) methyl, and ( f ) hydrogen. de r i v a t i v e s were 10.7-fold, 23.4-fold, and 2 7 . 5 - f o l d , respectively, l e s s a c t i v e than the cyclohexyl d e r i v a t i v e . Compounds (VIHe and f ) were i n a c t i v e . This could not be explained by the l i p o -p h i l i c character of the a l i c y c l i c substitutions since the highest r i n g s i z e cycloheptyl d e r i v a t i v e ( V i l l a ) was the l e a s t a c t i v e In the s e r i e s . I t was probably that the f i t of the cyclohexyl r i n g i n the complementary receptor s i t e s contributed most of the influence on the a c t i v i t y . This was further supported by the present study where compound ( l i b ) containing a cyclohexyl r i n g was more or le s s as ac t i v e as diphenhydramine (see above dis c u s s i o n ) . Moreover, l f the R group i n (VIII) was replaced with a cyclohexanecarbonyl group as In compounds (Hg) and (Hh ) , the antihistaminic a c t i v i t y was completely l o s t . The loss of a c t i v i t y i n compounds H g and I l h may have two causes: (a) the electron-withdrawing e f f e c t of both the s l i g h t l y p o s i t i v e carbonyl carbon atom (£*") and the p o s i t i v e l y charged C 2-atom 1 1 0 . In the respective pyrlmldlne and pyrazlne rings makes the lone p a i r electrons of the amino nitrogen even l e s s a v a i l a b l e f o r a t t a i n i n g the overlap i n t e r a c t i o n with the ^ - e lectrons of the hetero-aromatlc r i n g s ; (b) the b u l k i e r carbonyl group may show s t e r i c interference and thus reduce the " f i t " of the adjacent cyclohexy1 r i n g into the complementary receptor s i t e s . In ethylenedlamine-type antihistamines only one r i n g was found 4 . 8 - 6 . 0 % from the amino nitrogen, whereas the other o r i n g was 6 . 0 - 7 . 2 A away, depending on i t s conformation when the molecule was i n the trans form ( 5 3 , 1 2 2 ) . S i m i l a r l y i n the trans conformation of aminoalkyl ether-type antihistamines and o monoaminopropyl compounds a distance of 6 . 0 - 6 . 8 A ex i s t s ( 5 3 ) * It appeared that the strongest competitive antagonism occurred o when at l e a s t one r i n g was capable of assuming a pos i t i o n 5 - 6 A from the amino nitrogen. That only one r i n g may be necessary was evidenced by the high antihistaminic a c t i v i t y of 4- ( 2 -dlmethylaminoethylamlno)quinollne (IX) ( 1 2 3 ) , 3 - or 4 - ( 2 -(dlmethylamino)ethoxyjquinoline (X) (124, 125) and 4 - ( 2 - ( l -p y r r o l i d l n y l j e t h o x y j q u i n o l l n e (XI) ( 125) on the Isolated guinea-pig ileum. Compound (XI) was the most active one among the ( I X ) ( X I ) three qulnollne compounds and the i n v i t r o studies (guinea-pig ileum) indicated that (XI) was 4 times l e s s potent than diphenhydramine but the antagonism to histamine was more s p e c i f i c (125)» Compound (X) was 20 times l e s s active than diphenhydramine ( 1 2 5 ) . A l l t h i s evidence i s relevant to our present compounds (Ila) and ( l i b ) both of which contain only one aromatic r i n g and appeared to be capable of assuming o the antihistaminic distance ( 5 - 6 A). Thus (Ila) and ( l i b ) have been shown to be highly potent antihistamines. However, i f the remaining aromatic ri n g size i s l a r g e r than e i t h e r of the phenyl r i n g (Ila) or the p y r l d y l r i n g ( l i b ) , the r e s u l t i n g compounds (e.g. H e and H f ) exhibit mixed competltlve-noncompetitlve antagonism (Fig. 2 0 - 2 5 ) . At -7 concentrations l e s s than 1 x 10 M / l , both (He) and ( H f ) displayed competitive antagonism towards the H- receptor on 112. the ileum, while at concentrations equal to or lar g e r than 1 x 10""? M/l, they exhibited non-competitive antagonism. A s i m i l a r example was that compound (X) (side chain at 3-Position) antagonized histamine non-competitlvely (124). Both (He) and ( H f ) were found to be equipotent towards ei t h e r the s p e c i f i c or the non-competitive receptor (see Table 12 f o r pA 2 and pDg values). Their a c t i v i t i e s towards the spe-c i f i c histamine receptor were shown to be 11-12 times l e s s active than diphenhydramine. At higher concentrations they interacted with the non-specific receptor s i t e s and the a c t i v i t i e s were 170-200 times l e s s a c t i v e than diphenhydramine. Since the bulky,naphthaline and quinoline groups and hindrance from sur-rounding chemical groups i n the biophase could prevent s p e c i f i c receptor i n t e r a c t i o n , t h i s may account f o r the non-competitive antihistaminic a c t i v i t y of both ( H e ) and ( H f ) . Since the a c t i v i t y of antihistaminic agents overlaps with that f o r a n t i c h o l i n e r g i c agents, the compounds synthesized were also investigated f o r possible a n t i c h o l i n e r g i c a c t i v i t y . The s t r u c t u r a l requirement f o r a n t i c h o l i n e r g i c a c t i v i t y i n antihistaminic drugs has not been reported i n the l i t e r a t u r e . A l l that i s known i s that histamine and antihistamines and acetylcholine and c h o l i n o l y t i c drugs bear one thing i n common, i . e . c a t l o n i c groups at physiological pH. It seems probably that antihistamines compete with acetylcholine f o r the anionic s i t e on the receptor. But because the rest of the molecules 1 1 3 . In antihistamines could not f i t well Into the complementary groups on the receptor s i t e s , antihistamines show very weak and non-specific c h o l i n o l y t l c a c t i v i t y . Lands and Luduena ( 1 2 6 ) studied the c h o l i n o l y t l c action of a series of dlalkylamino-alkanes (XII, = H) and dlalkylamlno-alkanols (XII, = -OH) on Isolated segments of rabbit ileum. Shortening of the appendage ( X I I ) R t = H, or OH Rg = cyclohexyl, cyclopentyl, cyclopentenyl, phenyl, or a l k y l groups. side chain structure by one carbon atom resulted i n a marked reduction i n antispasmodic potency. Branching of the side chain also led to a d i s t i n c t reduction i n spasmolytic potency. These SAR r e l a t i o n s h i p s were same as those of antihistamines. A benzene r i n g may replace the cyclohexyl r i n g without changing the order of magnitude of c h o l i n o l y t l c a ction (note the s t r u c t u r a l s i m i l a r i t i e s with the present compounds ( I I ) ) . Also, cyclopentyl and cyclopentenyl s u b s t i t u t i o n provided compounds that were of comparable potency to those of the various cyclohexyl and 114. phenyl analogs. An increase i n the distance between the amino group and the terminal carbon by the addition of an ether oxygen between the second and terminal carbon (note the s t r u c t u r a l simi-l a r i t y with diphenhydramine - page 106 , III without 4-CH"3 group) provided compounds that were e s s e n t i a l l y equivalent i n spasmo-l y t i c potency to that of compounds of corresponding length of carbon side chain. Methyl quaternlzatlon of the amino side chain increased potency. However, the most s t r i k i n g difference i n spasmolytic potency resided on the terminal hydroxyl group. Without t h i s hydroxyl group (e.g. XII, R 1 = H) the potency drop-ped d r a s t i c a l l y to l e s s than 1% or 2-3% that of atropine s u l f a t e . With the hydroxyl group on the terminal carbon (e.g. XII, = OH), the potency was s i g n i f i c a n t l y increased to 2 0 - 9 0 % that of atropine. This suggested that the hydroxyl groups played an important r o l e , possibly by forming a second point of attachment to the receptor. In t h i s respect, they resembled the corresponding a n t i c h o l i n e r g i c esters. Since the present systems (compounds II) were without the hydroxyl group, they were there-fore very weak a n t i c h o l i n e r g i c agents (generally the potencies were 2 , 3 4 4 - 2 9 , 5 1 2 times l e s s than that of atropine except com-pound ( H e ) which was 550 times l e s s potent than atropine). They a l l displayed non-competitive antagonism against a c e t y l -choline on guinea-pig ileum. In compounds with the same aromatic r i n g size as that of phenyl group (e.g. compounds Ha-d), the a n t i c h o l i n e r g i c potency decreased i n the same d i r e c t i o n as that of antihistaminic a c t i v i t y , i . e . H a > l i b > H e > l i d 1 1 5 . i n decreasing potency. In compounds with l a r g e r aromatic r i n g size (e.g. compounds l i e and I l f ) , the spasmolytic potency did not follow the same pattern as that of antihistaminic a c t i o n . Compound ( l i e ) was more potent than ( I l f ) ( 1 2 times difference) as a spasmolytic agent, while as antihistaminic agents, they had the same potency. Compound ( l i e ) was the most potent a n t i c h o l i n e r g i c agent among the compounds tested but the reason was not known. Compounds (Ilg) and (Uh) were found to be i n a c t i v e . Lands and Lunduena ( 1 2 6 ) suggested the following c h o l i n o l y t l c receptor to accommodate the s e r i e s of compounds (XII) on the Isolated segments of rabbit ileum. Post-ganglionic parasympathetic f i b e r s of the autonomic nervous system supplied a single type of receptor which had the c h a r a c t e r i s t i c s of a crevice or trough-like structure i n which the active c h o l i n o l y t l c drug was held by e l e c t r o s t a t i c a t t r a c -t i o n to an anionic area by the c a t i o n i c head and by a second point of attachment to the receptor through ahydroxyl (polar) i o group, t h i s l a t t e r being at a distance of 4—7 A from the c a t i o n i c head. The o v e r a l l length of the reactive receptor surface was about 7-8 £, measured from the anionic s i t e . Quaternization of the amine with a methyl- or ethyl-halide increased c h o l i n o l y t l c potency, possibly by providing a more favorable f i t to the receptor s i t e and / or by increasing the e l e c t r o s t a t i c charge on the nitrogen. Thus, i t was apparent that our present compounds (II) with only one c a t i o n i c head but without both hydroxyl group and quaternization appeared 116 to have l i t t l e a n t i c h o l i n e r g i c a c t i v i t i e s on the guinea-pig ileum. F i n a l l y , where i s the s i t e of act i o n of histamine and / or acetylcholine on the guinea-pig ileum? Both a c e t y l -choline and histamine have a d i r e c t mode of act i o n on smooth muscle. Brownlee (127, 128) i n two successive papers des-cribed the evidence f o r the s i t e of action of histamine and acetylcholine on the guinea-pig ileum. Both histamine and acetylcholine could stimulate the intramural plexuses of the ileum as shown by t h e i r action on the c i r c u l a r muscle s t r i p . The l o n g i t u d i n a l muscle preparation was more sensi t i v e to the d i r e c t muscle stimulating a c t i o n of histamine and a c e t y l -choline than to t h e i r nerve plexus stimulating actions; t h i s made i t u n l i k e l y that any plexus stimulating a c t i o n would contribute to the response (128). 117. NOTE FOR FIGURE 8-4-5 (Cumulative Dose-Response Curves =  PRC); Since the figures on the following pages are-computer wrlte-outs, there i s i n s u f f i c i e n t space f o r complete captions f o r each f i g u r e . The following d e s c r i p t i o n i s there-fore necessary: Three or four figures are used to i l l u s t r a t e the a c t i v i t i e s of each te s t compound. In each figu r e the cumula-t i v e DRC f o r the agonist alone ( i . e . e i t h e r Histamine or Acetylcholine = Ach.) i s represented by the s o l i d l i n e (A A ) . The cumulative DRC f o r the agonist i n the presence of the tes t compound i s represented by dotted l i n e (x»»»x). The concen-t r a t i o n of the tes t compounds i s indicated by the numbers which are typed just beside each curve, f o r example, 0, 1, 3, 10, etc. : 0 means no t e s t compound present; 1 means 1 times the concentration of the tes t compound l i s t e d on each figure (e.g. 10-9 M/l); 3 means 3 times the concentration of the test compound l i s t e d on each fig u r e , etc.. HISTAMINE IL3G MOLRR CONCENTRATION) p. 119, HISTPMJNE CLOG MOLRR CONCENTRATION) HISTPMJNE [LOG MOLRR CONCENTRATION) F i g . 8 -10t E f f e c t of H a on DRC of histamine* note the progressive and p a r a l l e l s h i f t of DRC (competitive antagonist) See page 11? f o r d e t a i l . -a -7 -6 -5 -4 HISTAMINE [LOG MDLAR CONCENTRATION) F i g . H-131 E f f e c t o f l i b on DRC o f h i s t a m i n e , n o t e t h e p r o g r e s s i v e and p a r a l l e l s h i f t o f DRC ( C o m p e t i t i v e a n t a g o n i s t ) . See page 117 f o r d e t a i l . HISTAMINE ILOG MOLAR CONCENTRATION) ^ * p. 122, HISTAMINE CLOG MOLAR CONCENTRATION) l O O - i HISTAMINE CLOG MOLAR CONCENTRATION) F i g . l*+- l6 i E f f e c t of l i e on DRC of.histamine, note the progressive and p a r a l l e l s h i f t of DRC (competitive antagonist) See page 117 f o r d e t a i l . p. 123, HISTPHJNE CLOG MDLfiR CONCENTRATION) F i g . 17-19« E f f e c t of l i d on DRC of histamine, note the progressive and p a r a l l e l s h i f t of DRC (competitive antagonist) See page 117 f o r d e t a i l . -8 -7 -6 -5 - 4 HISTAMINE (LOG MOLAR CONCENTRATION) HISTAMINE CLOG MOLAR CONCENTRATION) Pig. 2 0 - 2 2 i E f f e c t of H e on DRC of histamine, showing a du-alism i n antagonism, note the combination of p a r a l l e l s h i f t (competition) and a reduction of the max. response (non-competitive antagonism). See page 117 for d e t a i l . p. 126, HISTAMINE CLOG MOLAR CONCENTRATION) 100 -i HISTAMINE CLOG MOLAR CONCENTRATION) F i g . 23-25i E f f e c t of I l f on DRC of histamine, showing a du-alism i n antagonism, note the combination of p a r a l l e l s h i f t (competition) and a reduction of the max. response (non-competitive antagonism). See page 117 f o r d e t a i l . lOO-i p. 127 ACETYLCHOLINE CLOG MOLPR CONCENTRPTIONJ lOO-i ACETYLCHOLINE CLOG MOLRR CONCENTRATION) -8 -7 -6 -5 -4 -3 ACETYLCHOLINE (LOG MOLAR CONCENTRATION J -8 -7 -6 -5 -4 -3 ACETYLCHOLINE (LOG MOLAR CONCENTRATION} F i g . 2 6 - 2 9 « E f f e c t of H a on DRC of Ach., note the pro-gressive depression of DRC (non-competitive antagonist). See page 1 1 ? for d e t a i l . ACETYLCHOLINE CLOG MOLPR CONCENTRATION) -a - 7 - 6 -s - 4 -a ACETYLCHOLINE (LOG MOLPR CONCENTRPTIONJ - 8 -1 - 6 - 5 - 4 ACETYLCHOLINE [LOG MOLRR CONCENTRPTIONJ F i g . 3 0 - 3 2 i E f f e c t of l i b on DRC of Ach., note the pro-gressive depression of DRC (non-competitive antagonist). See page 117 for d e t a i l . ACETYLCHOLINE (LOG MOLPR CONCENTRATION] 100-i 90-80-70-UJ C E £ 60 H CJ CC £ 50H z s P i g . 35« IO" 6 M/l 0 10 0 I I I I I I I I I I I I I I I I I I I I I I I » I X 1 I I I T I I I I I I I I I 1 I I I I I I I I -8 -7 -6 -5 -A -3 ACETYLCHOLINE CLOG MOLPR CONCENTRATION) F i g . 33-35« E f f e c t of H e on DRC of Ach., note the pro-gressive depression of DRC (non-competitive antagonist). See page 117 f o r d e t a i l . p. 132. ACETYLCHOLINE (LAG MOLAR CONCENTRATION) ACETYLCHOLINE CLOG MOLAR CONCENTRATION) - 8 - 7 - 6 - 5 - 4 - 3 ACETYLCHOLINE ILOG MOLAR CONCENTRPTIONJ 0 1 I 1 I I I I I I | 1 1 I I I I I I 1 | T T I I I I T I t ) I ! I T t I I I T | T I 1 T I I I I I | - 8 - 7 - 6 ^ 5 - 4 -3 ACETYLCHOLINE CLOG MOLAR CONCENTRPTIONJ F i g . 36-381 Effe c t of l i d on DRC of Ach., note the pro-gressive depression of DRC (non-competitive antagonist). See page 117 f o r d e t a i l . ACETYLCHOLINE (LOG MOLRR CONCENTRATION! -8 -7 -6 -5 -4 -3 ACETYLCHOLINE CLOG MOLAR CONCENTRATION) 0 — i i i i i i i i i | i i i i i i i i i | i i i i i i i i i | i i i i i i i i i | i i i i i i i i i | -8 -7 -6 -5 -4 -3 ACETYLCHOLINE [LOG MOLAR CONCENTRATION) F i g . 39-421 E f f e c t of H e on DRC of Ach., note the pro-gressive depression of DRC (non-competitive antagonist). See page 11? for d e t a i l . 0 j i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i r i i i r i i i r i i r i i r > i i - 8 - 7 - 6 - 5 - 4 - 3 ACETYLCHOLINE CLOG MOLPR CONCENTRATIONJ ACETYLCHOLINE CLOG HOLPR CONCENTRPTIONJ Fig. 4 3 - 4 5 i Effect of I l f on DRC of Ach.f note the pro-gressive depression of DRC (non-competitive antagonist). See page 1 1 7 for det a i l . PART VI SUMMARY Previous work ( 2 ) had shown that the cyclohexyl analogue of Antergan, N.N-dimethyl-N*-cyclohexylmethyl-N*-phenylethylenediamine ( I ) , was a very potent antihistaminic agent.. In order to gain more in s i g h t into the antihistaminic a c t i v i t y i n r e l a t i o n to the remaining aromatic r i n g , f i v e aromatic systems varying i n s i z e and structure were used i n the present study to substitute f o r the remaining phenyl r i n g . Seven t e r t i a r y ethylenediamine compounds were synthesized. They are as follows i (A) N,N-dimethyl-N*-cyclohexylmethyl-N' - 2-pyridylethylenediamine, (B) N,N-dimethyl-N'-cyclohexyl-raethyl-N'-2-pyrimidylethylenediaraine, (C) N,N-dimethyl-N*-cyclohexylmethyl-N* -2-pyrazylethylenediaraine, (D) N,N-dimethyl-N'-cyclohexylmethyl-N*-1-naphthylethylenediamine, (E) N,N-dimethyl-N'-cyclohexylmethyl-N' -5-quinolylethylenediamine, (F) N,N-dimethyl-N'-cyclohexanecarbonyl-N'-2-pyrimidyl-ethylenediamine, and (G) N,N-dimethyl-N'-cyclohexanecarbonyl-N' - 2-pyrazylethylenediamine• Two oxetane derivatives, 2 , 2 -diphenyl - 3-chlorooxetane and 2 , 2 - d i p h e n y l - 3 - ( p y r r o l i d i n - 2 -one-l-yl)-oxetane, were also synthesized as intermediates i n the present study, but the f i n a l products necessary for the antihistamine study could not be obtained. The general reaction sequence for the preparation of the ethylenediamine derivatives started with the appropriate primary homo- and heterocyclic-aromatic amine. The primary amine was reacted with cyclohexanecarbonyl chloride to form 138. the amide. Lithium aluminum hydride was used to produce the desired secondary amine. In cases where the primary aromatic amines did not form the amides or i f they did form the amides which could not be reduced to the required amines with LiAlH^ the primary aromatic amines were condensed with p-dimethy 1 -aminoethyl chloride to form the desired secondary amines. In the former case, the appropriate secondary amine was con-densed with yS-dimethy laminoethy 1 chloride to obtain the f i n a l t e r t i a r y diamine compounds- while i n the l a t t e r case, cyclo-hexylmethyl bromide was used to condense with the appropriate secondary amine to form the f i n a l products. Generally speaking, the products obtained by the f i r s t method appeared to give better y i e l d s than those of the second. These ethylenediamine derivatives were i s o l a t e d as the free base. The hydrochloride, p i c r a t e , and perchlorate s a l t s of these amines were prepared for elemental microanalysis. The two oxetane derivatives were obtained by photo-cycloaddition of benzophenone (a carbonyl compound) to either v i n y l chloride (an o l e f i n ) or N-vinyl - 2-pyrrolidinone (an o l e f i n ) . Benzene was used as the i n e r t solvent and a pyrex f i l t e r was used to f i l t e r out a l l l i g h t below 290 mu. A l l the intermediates synthesized were characterized through t h e i r physical constants, b o i l i n g point, melting point, i n f r a r e d and NMR spectra, and were v e r i f i e d by elemental microanalyses of the compounds (for s o l i d intermediates) or t h e i r hydrochloride, p i c r a t e , or perchlorate s a l t derivatives (for l i q u i d intermediates). 139. The antihistaminic a c t i v i t y and the possible a n t i -c holinergic a c t i v i t y of a l l the seven t e r t i a r y ethylenediamine compounds ( i . e . (A)-(G) above) were studied by the cumulative dose-response curves* method of van Rossum (107, 108). The dose-response curves of each compound so obtained were used to analyze t h e i r mode of action and t h e i r r e l a t i v e a n t i h i s t a -minic and a n t i c h o l i n e r g i c a c t i v i t i e s towards the respective receptor s i t e s . Their r e l a t i v e a c t i v i t i e s were compared with those of diphenhydramine, atropine, and the cyclohexyl analogue of Antergan ( I ) . It was found within the concentrations studied that compounds containing the aromatic r i n g s i z e com-parable to that of phenyl r i n g ( i . e . compounds (A), (B), (C), and (I)) displayed competitive antagonism towards the a n t i h i s t a -minic receptor, and compounds containing larger r i n g s i z e than phenyl group ( i . e . compounds (D) and (E)) exhibited mixed competitive-noncompetitive antagonism a c t i v i t i e s . The possible e l e c t r o n i c and s t e r i c e f f e c t s of these nitrogen-containing aromatic systems on t h e i r antihistaminic a c t i v i t y at the receptor s i t e s were explained. The overlapping i n t e r a c t i o n between the lone pair electrons of nitrogen and x^-electrons of the aromatic system seemed to be the determinant factor governing the antihistaminic e f f e c t of compounds (A), (B), (C) and ( I ) . The presence of two electron-withdrawing centers ( i . e . nitrogen atoms) i n the aromatic r i n g decreased t h i s overlap i n t e r a c t i o n and thus decreased t h e i r antihistaminic a c t i v i t y . This gave more evidence i n d i r e c t support of Nauta's antihistaminic receptor theory (3t 4 ) , Therefore, (I) was 140. shown to be the most active antihistaminic agent ( 1 . 5 - f o l d more ac t i v e than diphenhydramine), (A) was more or less as active as diphenhydramine, and (B) and (C) were les s a c t i v e . The bulky aromatic r i n g of compounds (D) and (E) seemed to impose s t e r i c hindrance more important than the overlapping i n t e r a c t i o n i n the biophase and thus prevents the receptor i n t e r a c t i o n . This i n turn may account for t h e i r non-competitive a c t i v i t y at concentrations ^ I O - 7 M/l and t h e i r r e l a t i v e l y l e s s antihistaminic a c t i v i t y at the receptor s i t e s . As to t h e i r a n t i c h o l i n e r g i c a c t i v i t y on the i s o l a t e d guinea-pig ileum, a l l compounds tested ( i . e . (A), (B), (C), (D), (E), and (I)) showed non-competitive antagonism towards the chol i n e r g i c receptor s i t e s . However, t h e i r a c t i v i t y was generally several thousand-fold less, active than atropine. Compounds (F) and (G) were i n a c t i v e i n both a n t i -histaminic and a n t i c h o l i n e r g i c t e s t s . j 141. PART VII APPENDICES A. SUPERPLOT - A GENERAL PURPOSE PLOTTING ROUTINE AND AN EXAMPLE FOR PLOTTING THREE FIGURES. B. PROGRAM TITLE : MEAN, VARIANCE, STANDARD DEVIATION (UNGROUPED DATA). C. CHEMICALS AND REAGENTS USED FOR THE EXPERIMENTAL PART (IV). D. TABLE 13 - COMPETITIVE ANTAGONISTS E. TABLE 14 - NON-COMPETITIVE ANTAGONISTS. FART VII APPENDICES A. SUPERPLOT - A GENERAL PURPOSE PLOTTING ROUTINE (By E.J. Tarnai) SUPERPLOT i s an object program which prepares a " p l o t f i l e " to draw l i n e s through sets of points. To Invoke i t $RUN TARN:SUPERPLOT 4=dataf.5=pagef.6=prlntf.o=plotf. Logical unit 4 contains data r e l a t i n g to the sets of points. It may be convenient to define It to be *SOURCE*. Logical unit 5 contains information relevent to the page s i z e , and the axes to which the l i n e s are to be drawn. If pagef. i s not s p e c i f i e d l o g i c a l unit 5 defaults to *SOURCE*. Logical unit 5 i s the f i l e on which error messages, etc., are written. Unit 5 i s usually l e t to default to *SINK*. Logical unit 9 i s the f i l e on which the " p l o t f i l e " i s written. The use of p l o t f i l e s i s described i n several UBC write-ups (e.g., PLOT, REPLOT, e t c . ) . A d e t a i l e d d e s c r i p t i o n of the input data-lines follows. Reading begins on l o g i c a l unit 5 and continues through s i x data l i n e s . These l i n e s are as follows: 1) PAGE (12), ORIENTATION (A4) PAGE i s an integer number (0 1 PAGE < 100) . For each new value of t h i s argument a new page i s started. I f the value i s the same as i t was at the l a s t read operation a new set of axes Is plotted on the same page. ORIENTATION i s Ignored i f new page i s not started. If a new page i s being started t h i s parameter may take on one of three values: 143. ORIENTATION HORI s p e c i f i e s an page. 1 1 " x 8 . 5 " ORIENTATION s p e c i f i e s an 8 . 5 " x 11 page. ORIENTATION NONE w i l l supress the drawing of page boundaries. 2) METHOD (A4). NARCS ( 1 2 ) , R (F10.0), IEND (12) METHOD may take one of the three values PARA, CIRC, EXPO. This argument s p e c i f i e s the type of c u r v e - f i t t i n g to be done. For d e t a i l s see UBC writeup SKETCH. If the f i e l d of t h i s argument i s l e f t blank the default value PARA i s assumed. NARCS s p e c i f i e s the number of arcs to be f i t t e d between consecutive data points. (0 < NARCS < 5 1 ) . I f NARCS i s outside the above range the number of arcs i s set to be 2 5 . Note: I f NARCS = 1 the l i n e segments Joining the points are s t r a i g h t . R i s the radius of curvature f o r method EXPO. (1.0 £ R < 2.0). Pox R outside t h i s range R i s . s e t to be 1 . 5 . For methods other than EXPO t h i s parameter i s not used, and need not be s p e c i f i e d . IEND s p e c i f i e s whether or not the f i r s t and l a s t l i n e segments are to be drawn. IEND = 0 end segments are l e f t unaltered. IEND = 1 end segments are straightened. IEND yt l or "0 end segments are dropped. 3) XOR (1) (G5.0), XOR (2) (G5.0), XLEN (G5.0), XMIN (G5.0), DX (G5.0), XTITLE ( 1 3 A 4 ) . XOR (1) & XOR (2) are the coordinates (in inches) of the s t a r t i n g point of the X-axis with respect to the lower l e f t corner of the page. The d e f a u l t l values are (1.0, 0 . 5 ) . (for Y0R (I) the default values are the coordinates XOR ( I ) ) . 1) Through-out t h i s writeup default value refers to that number which i s set equal to the variable which have input value s p e c i f i e d to be zero or l e f t blank. 144. XLEN s p e c i f i e s the length of the X-axis i n inches. The default value i s 7.0 (or 10.0 fo r YLEN). XMIN s p e c i f i e s the minimum value to be assigned to the X-axis. The default value Is zero fo r l i n e a r axis or one f o r logarithmic a x i s . DX s p e c i f i e s the increment i n value by which the major scale d i v i s i o n s are to be l a -beled. The default value f o r DX i s 1.0. Note that f o r a logarithmic axis the i n -crement i s used as a multiplying f a c t o r so that the value of 10.0 i s assumed i f DX = 1.0 i s s p e c i f i e d . XTITLE i s the H o l l e r i t h l i t e r a l (not more than 52 characters) to be used as the t i t l e of the X-axis. 4) DIYX (P10.0), NTICS ( 1 3 ) , NDIGX ( 1 3 ) , NLABLEX ( 1 3 ) , PHIX (F10.0), LINEARX (I I ) , THETAX (F10.0), CSIZEX (F10.0), ISAX ( I I ) . DIVX s p e c i f i e s the length i n inches between the major scale d i v i s i o n of the X-axis. The default value i s 1.0. NTICS s p e c i f i e s the number of scale subdivisions and whether or not the l a b e l i n g i s done using a scale f a c t o r . For NTICS > 0 no scale f a c t o r i s used. NTICS = 0 defaults to the value 5 . If NTICS < 0 the absolute value i s used to determine the number of scale subdivisions and the l a b e l i n g i s done using 10 x scale factor, where x = NDIGX. NDIGX s p e c i f i e s the number of decimal d i g i t s to be used i n l a b e l i n g the major scale d i v i -sions of the X-axis. The default value fo r t h i s argument i s 2 . If NDIGX = -1 decimal d i g i t s and decimal point are sup-pressed, and i f NDIGX = - 2 the l a b e l i n g of the X-axis scale d i v i s i o n s i s sup-pressed . NLABLEX s p e c i f i e s the maximum number of characters, including negative sign and decimal point, i f applicable, i n the l a b e l i n g of the major scale d i v i s i o n of the X-axis. The 1 4 5 . default value for NLABLEX i s 6 . If NLABLEX < 0 the l a b e l i n g of the major scale d i v i s i o n s of the X-axis w i l l be suppressed. FHIX s p e c i f i e s the angle, i n degrees, of the l a b e l s on the major scale d i v i s i o n s of the X-axis. The default value i s 0 . 0 . (Note: the default value f o r PHIY i s 9 0 . 0 . If PHIY = 0 . 0 i s required, an a r b i t r a r y  negative number must be specifled.) LINEARX s p e c i f i e s l i n e a r scale d i v i s i o n f o r LINEARX = 0, or logarithmic scale incre-ment f o r LINEARX = 1 . THETAX s p e c i f i e s the angle of the X-axis i n de-grees. The default value i s 0 . 0 . (Note: the default value f o r THETAY i s 9 0 . 0 . I f THETAY = 0 . 0 i s required, a negative number must be s p e c i f i e d . ) CSIZEX s p e c i f i e s the height s i z e , i n Inches, of the characters used i n l a b e l i n g the X-axis. The default value i s 0 . 1 " . ISAX i s normaly 0 . I f ISAX = 1 the p l o t t i n g of the X-axis w i l l be suppressed. 5 ) & 6 ) contain Information regarding the Y-axis. The data on these l i n e s are arranged I d e n t i c a l l y as the data on l i n e s 3 and 4 . Reading continues on l o g i c a l unit 4 . Each l i n e contains information regarding one point. Reading con-tinues u n t i l an &ENDFILE i s reached. This terminates the set of points through which a l i n e i s being drawn. Reading con-tinues on t h i s same unit and other sets of points are deter-mined and l i n e s drawn u n t i l an &ENDFILE i s reached ( i . e . , two consecutive $ENDFILES) at which point reading continues on l o g i c a l unit 5« The datalines i n unit 4 are i n the following form: X (I) ( G 1 0 . 0 ) , Y (I) ( G 1 0 . 0 ) , SYMB ( 1 2 ) , LINET ( 1 2 ) , LSQ ( 1 3 ) . X (I) & Y (I) are the actual coordinates of the data points. These values w i l l be scaled by the program to the previously s p e c i f i e d axes. 146. SYMB s p e c i f i e s the type of symbol to be drawn at each data point SYMB < 0 s p e c i f i e s no symbol SYMB = 0 defaults to the most recent value of SYMB ( i n i t i a l l y set to be -1). SYMB = 1 s p e c i f i e s A SYMB = 2 s p e c i f i e s # SYMB = 3 s p e c i f i e s © SYMB = 4 s p e c i f i e s <$> SYMB = 5 s p e c i f i e s * SYMB = 6 s p e c i f i e s ^ LINET s p e c i f i e s the type of l i n e to be drawn through the points. The default value i s 1. The various l i n e s are as follows: LINET = 1 s p e c i f i e s LINET = 2 s p e c i f i e s LINET = 3 s p e c i f i e s LINET = 4 s p e c i f i e s * • * »**^»«« « mm LINET = 5 s p e c i f i e s LINET = 6 s p e c i f i e s LSQ s p e c i f i e s t i n g with whether or not le a s t square f i t -an orthogonal polynomial of de-gree M. I f LSQ = 0 no le a s t square f i t -t i n g w i l l be done. If LSQ i s a positi v e number between 1 and 30 M w i l l be set by the program to that value which w i l l give the best f i t such that M < LSQ i f LSQ < 0 M w i l l be set to ABS (LSQ) i f LSQ > - 3 0 . I f ABS (LSQ) > 30 M w i l l be set to 0 . Note: The parameters SYMB, LINET, LSQ are set by the datallne containing X (1) and Y (1). I.e. These parame-ters need to be s p e c i f i e d only once per l i n e to be drawn. 1 4 7 . I f reading returns to l o g i c a l unit 5 and the f i r s t data l i n e encountered i s an &ENDFILE It w i l l cause execution to terminate. «•#«•«»«•**«•*#* If the user requires c a l l i n g a subroutine during execution i n order to do more p l o t t i n g or to calculate the coordinates of the points on unit 4 , etc., he may add the subroutine RTWAIT (PAGE). E.g. $RUN TARN:SUFERPLOT+-LOAD# where -LOAD# contains the object deck containing subroutine RTWAIT. The MAIN routine c a l l s RTWAIT between the time read-ing i s completed on unit 5 and reading begins on unit 4 i f new page i s being started. I f the user does not supply t h i s routine, a UBC-library routine i s c a l l e d which i n batch mode does nothing and i n terminal mode does very l i t t l e (waits l e s s than 0.3 sec. i n r e a l time). *»*»**##•»••*## The wrlteup UBC-SKETCH describes a subroutine OUT which "draws" l i n e s through the points generated by SKETCH. Such a routine i s part of SUFERPLOT and may be used independ-ently by a program which c a l l s SKETCH. The l i n e type i s passed to OUT by the labeled common COMMON/LINETP/LINET f o r the exact l o c a t i o n of out within SUPERPLOT $RUN *0BJSCAN PAR=TARN:SUPERPLOT. Note t See an example for p l o t t i n g three figures on the next page. r 148. >- This i s an example for plotting three figures (i.e. Pig. 8, 9, and 10). -RFS _SS_I_G1NJ£1N_ IDFN T=1M PASSWORD $RUN -FORTRAN PAR=EXEC=0 DIMENSION I I (20 ,5 ), 1 2 ( 2 0 , 5 ) READ(5,10) I T , IMTD 10 FORMAT(15,A4) R FAD ( 5 . 1 1 ) 11.12 _ 11 13 12 FORMAT(20A4) IP=1 IC=0 . . WRITE(3,12) IP.IMTD FORMA T(12.AA) W R I T E O . l l ) I I I O IC+ 1 I F ( IC.GE . IT) GO TO 14 WRITE(3,12) IP.IMTO W R I T E ( 3 j l l ) 12 IP= IP+1 IC=IC+1 This fortran program is to generate control -cards—for—axe s T  IT = Total numbers of figures ( = 3 in th Is—example-)-. I F ( IC .GE.IT) GO TO 14 GO TO 13 14 WRITE(3,15 ) 15 FORM AT( '$ENDFIL E 1 ) STOP END SEND F I L E SRUiM -L0AD# 3 numbers of figures 2.50 6.00 4.75 -8.0 1.0HISTAMINE (LOG MOLAR CONCENTRATION) 1.18 10 -1 20 .0 QQ.O 2.50 6.00 4.00 0.3937 1 -1 0.0 10.00/0 CONTRACTURE 3 2 .50 1 .25 4 .75 -8.0 1 .0HISTAMINE (LUG MOLAR CONCENTRATION) 1.18 1 0 - 1 20.0 00.0 2.50 1.25 4.00 0.0 10.00/U CONTRACTURE 0.3937 1 - 1 3 $RUN USER:SUPERPLOT -7.52 4.08 . 1 1 -7.00 14.90 -6.52 39.80 -6 .00 69 . 39  5=-A 9=-PL0T 4=*SUUKCE* -5.52 91.84 -5.00 100 .0 SENDFILE (To be continued) 149. - 7 . 5 2 2.04 2 2 - 7 . 0 0 8.16 - 6 . 52 2 9 . 5 9  - 6 . 0 0 6 1 . 2 2 - 5 . 5 2 84 .69 - 5 . 0 0 9 5 . 9 2 - 4 . 5 2 100 .0 S E N D F I L E SFNDF TL F [ ' - 8 . 0 0 1 .56 1 1 - 7 . 5 2 3 . 4 1 -7 .00 1 6 . 5 9 - 6 . 52 4 6 . 3 4 - 6 . 0 0 75 .12 v-5.52 9 3 . 6 6 '"-5.00 1 0 0 . 0 S E N D F I L E -8 .00 0.49. 2 2 - 7 . 5 2 0.98 - 7 . 0 0 3 .90 - 6 . 52 22 .93 - 6 . 0 0 5 6 . 1 0 v. - 5 . 52 81 .46 - 5 . 0 0 9 5 .61 - 4 . 5 2 98 .05 S E N D F I L E SFNDFT I F - 8 . 0 0 2 .22 1 1 - 7 . 5 2 8.89 - 7 . 0 0 30 .56„ - 6 . 52 53 .33 - 6 . 0 0 7 9 .44 - 5 . 5? 91 .67 - 5 . 0 0 1 0 0 . 0 S E N D F I L E - 8 . 0 0 1.11 2 2 - 7 . 5 2 5.56 > - 7 . 0 0 1 6 . 6 7 - 6 . 5 2 28 .89 - 6 . 0 0 53 .33 - 5 . 5 2 7 7 . 7 8 - 5 . 0 0 9 6 . 1 1 - 4 . 52 100 .0 S E N D F I L E S E N D F I L E SRUN PLOT :Q PAR = -PLOT SS IGNOFF (End) 1 5 0 . PROGRAM TITLE: MEAN, VARIANCE, STANDARD DEVIATION (UNGROUPED DATA) No.: 1014-2-ST1 Programmer: E. Thlbeault Formulas Used: n X x 2 - n ( x ) 2 n - 1 where x^ = any of the data values being used ( x ^ n = number of values x = arithmetic mean of these values (f = variance (square of standard deviation) (f = standard deviation from the mean ( 68.27% of a l l values should f a l l within +1 deviation from the mean) Cf Then, Standard Error = 1 5 1 . C. CHEMICALS AND REAGENTS USED FOR THE EXPERIMENTAL PART (IV) Names Grades Company 2-Aminopyrazlne 2-Aminopyridine 2-Aminopyrimidine 5-Aminoqulnoline Benzophenone Cyclohexanecarboxyllc a c i d Cyclohexylmethyl bromide 3 , 6-Dlchloropyridazine Lithium aluminum hydride 1-Naphthylamine 70% p e r c h l o r i c acid S i l i c a gel (for column chromatography) P r a c t i c a l Reagent P r a c t i c a l Reagent A l d r i c h Chem. Co., Inc. Eastman Kodak A l d r i c h Chem. Co., Inc. n n Fisher S c i . Co. Eastman Kodak A l d r i c h Chem. Co., Inc. A l f a Inorganics, Ventron Corp. A l d r i c h Chem. Co., Inc. A l l i e d Chemical S p e c i f i c a t i o n Fisher S c i . Co. MIL-D-3716, Davision commercial grade. S i l i c a gel (for TLC) Art . 7731 Sodium amide V i n y l chloride N-Vlnyl - 2-pyrrolidinone E. Merck (West Germany). Fisher S c i . Co. Matheson of Canada Ltd. Matheson Coleman & B e l l , Div. of Matheson Co. Inc. 1 5 2 . D. TABLE 13 - from L i t . (108) COMPETITIVE ANTAGONISTS Relationship between x i n mm and log (x-1) to be used f o r the c a l c u l a t i o n of pA 2 values ac-cording to the equation: pA 2 = pA x + log (x - 1 ) mm. log log log mm • log (x - 1 ) mm. U - l ) mm. (x - 1 ) (x - 1 ) 0 .5 -1.41 2 0 . 5 O.58 40.5 1 .33 6 0 . 5 2 . 0 1 1 . 0 - 1 . 1 0 2 1 . 0 0 .60 41.0 1-35 6 1 . 0 2 . 0 3 1 .5 -0 .96 2 1 . 5 0.62 41 .5 I . 3 6 6 1 . 5 2 . 0 5 2 . 0 - 0 . 7 8 2 2 . 0 0.64 42 .0 1 .38 6 2 . 0 2 . 0 6 2 . 5 - 0 . 6 7 2 2 . 5 0 . 6 7 42.5 1.40 6 2 . 5 2 . 0 8 3 . 0 - 0 . 5 9 2 3 . 0 0 . 6 9 43.O 1.42 63.O 2 . 1 0 3 . 5 - 0 . 5 1 2 3 . 5 0 . 7 1 4 3 . 5 1 .43 6 3 . 5 2 . 1 1 4 . 0 - 0 . 4 5 24.0 0 . 7 3 4 4 . 0 1 .45 64 .0 2 . 1 3 4 . 5 -O.38 24.5 0 . 7 4 4 4 . 5 1 .47 64.5 2 . 1 5 5 . 0 - 0 . 3 3 2 5 . 0 O.76 4 5 . 0 1 .49 6 5 . 0 2 . 1 6 5 . 5 -0.28 2 5 . 5 0 . 7 8 4 5 . 5 1 .50 6 5 . 5 2 . 1 8 6 . 0 - 0 . 2 3 2 6 . 0 0.80 46 .0 1 .52 6 6 . 0 2 . 2 0 6 . 5 - 0 . 1 9 2 6 . 5 0.82 46.5 1 . 5 4 6 6 . 5 2 . 2 1 7 . 0 - 0 . 1 5 2 7 . 0 0.84 4 7 . 0 1 .55 6 7 . 0 2 . 2 3 7 . 5 - 0 . 1 1 2 7 . 5 0 . 8 6 4 7 . 5 1 .57 6 7 . 5 2 . 2 5 8 . 0 - 0 . 0 7 28 .0 0 . 8 8 48.0 1 .59 6 8 . 0 2 . 2 6 8 . 5 -0.04 2 8 . 5 0 . 9 0 48.5 1 .61 6 8 . 5 2 . 2 8 9 . 0 0 . 0 0 2 9 . 0 0 . 9 2 4 9 . 0 1 .62 69.O 2 . 3 0 9 . 5 0 . 0 3 2 9 . 5 0 . 9 4 4 9 . 5 1.64 6 9 . 5 2 . 3 1 1 0 . 0 0 . 0 6 3 0 . 0 0 . 9 5 5 0 . 0 1 .66 7 0 . 0 2 . 3 3 1 0 . 5 0 . 0 9 3 0 . 5 0 . 9 7 5 0 . 5 I . 6 7 7 0 . 5 2 . 3 5 1 1 . 0 0 . 1 2 3 1 . 0 0 . 9 9 5 1 . 0 I . 6 9 7 1 . 0 2 . 3 6 1 1 . 5 0 . 1 5 3 1 . 5 1 .01 5 1 . 5 1 .71 7 1 . 5 2 . 3 8 1 2 . 0 0 . 1 8 3 2 . 0 1 . 0 3 5 2 . 0 1 . 7 3 7 2 . 0 2.40 1 2 . 5 0 . 2 1 3 2 . 5 1 . 0 5 5 2 . 5 1 .74 7 2 . 5 2.42 1 3 . 0 0 . 2 3 3 3 . 0 1 . 0 6 5 3 . 0 I . 7 6 7 3 - 0 2 . 4 3 1 3 . 5 0 . 2 6 3 3 . 5 1 . 0 8 5 3 . 5 I . 7 8 7 3 . 5 2 . 4 5 14.0 0 . 2 9 3 4 . 0 1 .10 5 4 . 0 1 .79 7 4 . 0 2 . 4 7 14.5 0 . 3 1 3 4 . 5 1 .12 5 4 . 5 1.81 7 4 . 5 2.48 1 5 . 0 0 . 3 3 3 5 . 0 1 .13 5 5 . 0 I . 8 3 7 5 . 0 2 . 5 0 1 5 . 5 O.36 3 5 . 5 1 .15 5 5 . 5 1.84 7 5 . 5 2 . 5 2 1 6 . 0 0 . 3 8 3 6 . 0 1.17 56.O 1 .86 7 6 . 0 2 . 5 3 1 6 . 5 0 . 4 l 3 6 . 5 1 .19 5 6 . 5 1 . 8 8 7 6 . 5 2 . 5 5 1 7 . 0 0 . 4 3 3 7 . 0 1 .21 5 7 . 0 1 . 8 9 7 7 . 0 2 . 5 7 1 7 . 5 0 . 4 5 3 7 . 5 1 .22 5 7 . 5 1 .91 7 7 . 5 2 . 5 8 1 8 . 0 0 . 4 7 3 8 . 0 1.24 5 8 . 0 1 . 9 3 7 8 . 0 2 . 6 0 18.5 0 . 5 0 3 8 . 5 1 .26 5 8 . 5 1 .95 7 8 . 5 2 . 6 2 1 9 . 0 0 . 5 2 3 9 . 0 1.28 5 9 . 0 I . 9 6 7 9 . 0 2 . 6 3 1 9 . 5 0 . 5 4 3 9 . 5 1 . 3 0 5 9 . 5 1 .98 7 9 . 5 2 . 6 5 2 0 . 0 0 . 5 6 40 .0 1 .31 6 0 . 0 2 . 0 0 80 .0 2 . 6 7 1 5 3 . E. TABLE 14 - from L i t . (108) NON-COMPETITIVE ANTAGONISTS The r e l a t i o n s h i p between the maximal e f f e c t E AmB • » expressed as a percentage of the maximal e f f e c t (EAm / EAmB* = x a n d * = 1 0 ° / x ) a n d l o S ( x - 1 ) to be used f o r the c a l c u l a t i o n of pDg values according to the equation: pD^ = pD^ + l o g (x - 1) % log % l o g log % log % log (x - 1 ) % (x - 1 ) (x - 1 ) (x -1 ) (x - 1 ) 99 - 2 . 0 0 90 - 0 . 9 6 70 - 0 . 3 7 50 0 . 0 0 30 0 . 3 7 98 - 1 . 7 0 89 - 0 . 8 9 69 - 0 . 3 5 49 0 . 0 2 29 0 . 3 9 97 - 1 . 5 2 88 - 0 . 8 5 68 - 0 . 3 3 48 0 . 0 3 28 0.41 96 -1.40 87 -0.82 67 - 0 . 3 1 47 0 . 0 5 27 0 . 4 3 95 - 1 . 3 0 86 -0.80 66 - 0 . 3 0 46 0 . 0 7 26 0 . 4 4 94 - 1 . 2 2 85 - 0 . 7 7 65 - 0 . 2 7 45 0 . 0 9 25 0.48 93 - 1 . 1 5 84 - 0 . 7 2 64 - 0 . 2 5 44 0 . 1 0 24 0 . 5 0 92 - 1 . 1 0 83 - 0 . 7 0 63 - 0 . 2 3 43 0 . 1 2 23 0 . 5 3 91 - 1 . 0 5 82 - 0 . 6 6 62 - 0 . 2 1 42 0.14 22 0 . 5 5 81 -0.64 61 - 0 . 1 9 41 0 . 1 6 21 0 . 5 8 80 - 0 . 6 0 60 - 0 . 1 7 40 0 . 1 8 20 0 . 6 0 79 - 0 . 5 7 59 - 0 . 1 5 39 0 . 1 9 19 0 . 6 3 78 - 0 . 5 5 58 -0.14 38 0 . 2 1 18 0 . 6 6 77 - 0 . 5 2 57 - 0 . 1 2 37 0 . 2 3 17 0 . 6 9 76 - 0 . 4 9 56 - 0 . 1 0 36 0 . 2 5 16 0 . 7 2 75 -0.48 55 - 0 . 0 9 35 0 . 2 7 15 0 . 7 5 74 -0.46 54 - 0 . 0 7 34 0 . 2 9 14 0 . 7 9 73 - 0 . 4 3 53 - 0 , 0 5 33 0 . 3 1 13 0 . 8 3 72 -0.41 52 -0.04 32 0 . 3 3 12 0 . 8 7 71 - 0 . 3 9 51 - 0 . 0 2 31 0 . 3 5 11 0 . 9 1 70 - 0 . 3 7 50 0 . 0 0 30 0 . 3 7 10 0 . 9 5 PART VIII BIBLIOGRAPHY 1. F.Y.T. Leung, Synthesis of Cycloalkyl Analogues of Antergan, Master's Thesis, U.B.C., 1 9 6 4 . 2 . S.Y.S. Wang, Synthesis of Cycloalkyl Analogues of Antergan, Master's Thesis, U.B.C., 1 9 6 9 . 3 . W.Th. Nauta, R.F. Rekker and A.F. Harms, Proc. 3 r d Intern. Pharmacol. Meeting July 1966, i n Physicochemical Aspects of Drug Actions, £» 3 0 5 - 2 5 , Pergamon, Oxford, N.Y., 1 9 6 8 . 4 . H. Timmerman, R.F. Rekker, A.F. Harms and W.Th. Nauta, Arzueim.-Forsch. Q_, 1 2 5 8 - 9 ( 1 9 7 0 ) . 5 . R.W. Schayer, Physiol. Rev. 116-26 ( 1 9 5 9 ) . 6 . G. Kahlson, E. Rosengren and T. White, J . Physiol. 1 5 1 , 131-38 (I960). 7 . G. Kahlson, E. Rosengren and R. Thunberg, J . Physiol. 1 6 9 . 4 6 7 - 8 6 ( 1 9 6 3 ) . 8 . G. Kahlson and E. Rosengren, Biogenesis and Physiology of Histamine (Monographs of the Phy s i o l o g i c a l Society), E. Arnold Pub. Ltd., London, 318 pp ( 1 9 7 1 ) . 9 . M.J. Dallemagne, Fundam. Biochem. Pharmacol. 3 2 6 - 3 0 ( 1 9 7 1 ) , Ed. Z.M. Bacq, Pergamon Pressi Oxford, Engl.. 10. B. Gustafsson, G, Kahlson and E. Rosengren, Acta Physiol. Scand. 41, 217-28 ( 1 9 5 7 ) . 1 1 . R.G. M i t c h e l l and J.F. Porter, Biochem. Develop. 204-23 ( 1 9 7 1 ) , Ed. P. Benson, Spastics Int. Med. Publ.i London, Engl.. 1 2 . R.W. Schayer, Amer. J . Physiol. 2 0 2 , 6 6 - 7 2 ( 1 9 6 2 ) . 1 3 . G. Kahlson and E. Rosengren, Phy s i o l . Rev. 48, 155-96 ( 1 9 6 8 ) . 14. J.F. R i l e y and G.B. West, Handb. Exp. Pharmakol. 18 ( l ) , 116-35 ( 1 9 6 6 ) . 1 5 . J.P. Green, Medicinal Chemistry, 1633-42 ( 1 9 7 0 ) , Ed. A. Burger, 3 r d ed., Wiley-Intersci., N.Y.. 1 6 . B. Uvnas, Mem. Soc. Endocrin. 19.', 7 4 3 - 6 6 , Subcellular Organization and Function i n Endocrine Tissues, Proc, Symp. 1970, Ed. H. H e l l e r and K. Lederis, Cambridge, Univ. Press 1 9 7 1 . 1 5 5 . 17 . Y. Kobayshi, Arch. Biochem. £ 6 , 2 0 - 7 ( 1 9 6 2 ) . 1 8 . C H . Aborg, J . Novotny and B. Uvnas, Acta Physiol. Scand. 69_, 276-83 ( 1 9 6 7 ) . 1 9 . B. Uvnas, CH. Aborg and A. Bergendorff, Acta Ph y s i o l . Scand. 7_8» Suppl. 3 3 6 , 26 pp. ( 1 9 7 0 ) . 2 0 . J.P. Green, Handb. Neurochem. 4 , 2 2 1 - 5 0 ( 1 9 7 0 ) , Ed. A. Lajtha, Plenum Press, N.Y.. 2 1 . W.W. Douglas, The Pharmacol. Basis of Therapeutics, 4 t h ed., 6 2 1 - 4 5 ( 1 9 7 0 ) , Ed. L.S. Goodman and A. Gilman, MacMillan Co., N.Y.. 2 2 . A. Torp, Medicina Experimentalis 4 , 180-2 ( 1 9 6 1 ) . 2 3 . M.C French, G.N. Woodruff, G.A. Kerkut, Comp. Gen. Pharmacol. 2 ( 7 ) , 275-80 ( 1 9 7 1 ) . 24. K.M. Taylor, S.H. Snyder, J . Pharmacol. Exp. Ther. %22 ( 3 ) . 6 1 9 - 3 3 ( 1 9 7 1 ) . 2 5 . K.M. Taylor, S.H. Snyder, J . Neurochem. 1 £ ( 2 ) , 3 4 1 - 5 4 ( 1 9 7 2 ) . 2 6 . J . C Schwartz, C. Lampart, C. Rose, J . Neurochem. 1J9_ (3)» 801-10 ( 1 9 7 2 ) . 2 7 . H.A. Campos and H. Jurupe, Experientia 26 ( 6 ) , 6 1 3 - 4 ( 1 9 7 0 ) . 28. Ib i d . 26 ( 7 ) , 746-7 ( 1 9 7 0 ) . 2 9 . M.J. Ryan and M.J. Brody, J . Pharmacol. Exp. Ther. 174 ( 1 ) , 1 2 3 - 3 2 ( 1 9 7 0 ) . 3 0 . J.D. Robinson, J r . and J.P. Green, Yale J . B i o l . Med. 2$, 248-57 ( 1 9 6 2 ) . 31. A. Thithapandha, Biochem. Biophys. Res. Commun. 4£ ( 2 ) , 3 0 1 - 8 ( 1 9 7 2 ) . 3 2 . A.M. R e i l l y , R.W. Schayer, B r i t . J . Pharmacol. 38 ( 3 ) , 4 7 8 - 8 9 ( 1 9 7 0 ) . 3 3 . H.B. A n s t a l l , Introduction to Neuroscience, 231-2 ( 1 9 7 2 ) , Ed. J . Minckler, C V . Mosby Co., St. Louis. 3 4 . M. Rocha e S i l v a , Handb. Exp. Pharmacol. 18 ( l ) , 431-80 ( 1 9 6 6 ) . ~ 3 5 . R.J. Levine and W.W. N o l l , Ann. N.Y. Acad. S c i . 1 6 6 , 246-56 ( 1 9 6 9 ) . 1 5 6 . 3 6 . R.W. Schayer, Biogenic Amines Physiol. Regul., Symp. 239-51 ( 1 9 6 9 , Pub. 1 9 7 1 ) . Ed. J , J . Blum, Prentice-Hall Inc., N.J.. 37. R.W. Schayer, Fedn. Proc. Fedn. Am. Socs. Exp. B i o l . 24 ( 6 ) , 1295-7 ( 1 9 6 5 ) . 3 8 . R.W. Schayer, Ovum Implantation, Proc. 31-88 ( 1 9 6 7 , Pub. 1 9 6 9 ) , Ed. M.C. Shelesnyak and G.J. Marcus, S c i . Publ., N.Y.. 39. A.L. Southren and A.B. Weingold, Amer. J . Obstet. Gynec. 1 0 6 . 6 0 7 - 1 6 ( 1 9 7 0 ) . 40. J.K. Smith, Biochem. J . 103., 110-9 ( 1 9 6 7 ) . 41. J.J.L. Delso, Arch. Inst. Farmacol. Exp., Madrid 20 ( l ) , 51-73 ( 1 9 6 8 ) , through Chem. Abstr. 11* 43005e ( 1 9 7 0 ) . 42. A.B. Young, C.D. Pert, D.G. Brown, K.M. Taylor and S.H. Snyder, Science, 1£3_ (3993), 247-9 (1971). 4 3 . J.C. Schwartz, C. Lampart, C. Rose, M.C. Rehault, S. Bischoff and H. Pollard, J . Physiol. ( P a r i s ) , Suppl. 62 (3), 447 ( 1 9 7 0 ) , through Chem. Abstr. 7J>, l 4 9 5 0 3 x ( 1 9 7 1 ) . 44. J.C. Schwartz, C. Lampart, C. Rose, M.C. Rehault, S. Bischoff and H. Pollard, J . Neurochem. 18_ ( 9 ) , 1787-9 (1971). 45. A.V. Furano and J.P. Green, J . Physiol. 1 7 0 . 263-71 ( 1 9 6 4 ) . 46. L. Beck, D. Schon, A.A. P o l l a r d and D.G. Wyse, Res. Commun. Chem. Pathol. Pharmacol. 2 (4-5), 4 1 5 - 2 8 ( 1 9 7 1 ) . 47. B. Uvnas, A l l e r g o l . , Proc. Congr. Int. Ass. A l l e r g o l . 6 t h , 127-36 ( 1 9 6 7 , Pub. 1 9 6 8 ) , Ed. B. Rose, Excerpta Med. Found. Ameterdam, Neth. 48. H.0. S c h i l d , Biochem. Acute A l l e r g . React., Sympo. 99-118 ( I 9 6 7 , Pub. 1 9 6 8 ) , Ed. K.F. Austen and E.L. Becker, Blackwell S c i . Publ., Oxford. 4 9 . S.C. L a h i r i , Aspects of A l l e r g y and Applied Immunology, 2L% 143-46 ( 1 9 7 0 ) , Ed. R.K. Sanyal, S. Arora and P. Sen, New Heights Publ., Delhi, India. 5 0 . I. Broder, Inflammation Immunity and Hypersensitivity, 333-88 ( 1 9 7 1 ) , Ed. H.Z. Movat, Harper and Row Publ., N.Y.. 5 1 . S. Norn, Acta Pharmacol. Toxicol, JO Suppl. 1, 59 pp. ( 1 9 7 1 ) . 1 5 7 . 5 2 . A.R. Johnson, Advan. Exp. Med. B i o l . 2 J ( 5 ) , 3 6 5-79 ( 1 9 7 2 ) . 5 3 . D.T. Witiak, Medicinal Chemistry, 3 r d ed. 1643-68 ( 1 9 7 0 ) , Ed. A. Burger, Wiley-Intersci., N.Y.. 5 4 . N. Chakravarty, Acta Physiol. Scand. 48, 146-66 ( i 9 6 0 ) . 5 5 . J.L. Mongar and H.O. Sch i l d , Physiol. Rev. 42, 226-70 ( 1 9 6 2 ) . 5 6 . B. Uvnas, Ann. N.Y. Acad. S c i . 116, 880-90 ( 1 9 6 4 ) . 5 7 . B. Uvnas and I.L. Thon, Mech. of Release of Biogenic Amines, jj>, 367-70 ( 1 9 6 5 ) , Proc. Int. Wenner-Gren Symp., Stockholm, Pergamon Press, Oxford. 5 8 . I.L. Thon and B. Uvnas, Acta Physiol. Scand. 7_1» 3 0 3 - 1 5 ( 1 9 6 7 ) . 5 9 . G.D. Bloom and N. Chakravarty, Acta Physiol. Scand. 7 8 . 410-9 ( 1 9 7 0 ) . 6 0 . A.R. Johnson and N.C. Moran, Amer. J . Physiol. 216 ( 3 ) , 453-9 ( 1 9 6 9 ) . 6 1 . A.R. Johnson and N.C. Moran, C e l l . Humoral Mech. Anaphylaxis All e r g y , Proc. Int. Symp. Can. Soc. Immunol. 3 r d , 122-8 (1968 , PUD. 1 9 6 9 ) , Ed. H.Z.K. Movat, Basel, Switz.. 6 2 . A.S.F. Ash and H.O. Sch i l d , Br. J . Pharmac. Chemother. 22 , 4 2 7 - 3 9 ( 1 9 6 6 ) . 6 3 . M. Rocha e S i l v a , Chemotherapia J , 5 4 4 - 5 9 ( 1 9 6 1 ) . 64. M. Rocha e S i l v a , Handb. Exp. Pharmacol. 18 (1), 225-37 ( 1 9 6 6 ) . — 6 5 . L.B. Kier, J . Med. Chem. 11 , 441-5 ( 1 9 6 8 ) . 66. M. Rocha e S i l v a , J . Pharm. Pharmac. 2 1 , 778-80 ( 1 9 6 9 ) . 6 7 . P. A l l a i n , Therapie 24 ( 4 ) , 757 - 6 5 ( 1 9 6 9 ) ; through Chem. Abstr. 2i« H 0 9 l 4 z TT969). 6 8 . T.B. Paiva, M. Tominaga and A.CM. Paiva, J . Med. Chem. 11 ( 4 ) , 689 - 9 2 ( 1 9 7 0 ) . 6 9 . S. Margolis, S. Kang and J.P. Green, Int. Z. K l i n . Pharmakol. Ther. Toxikol. ^ ( 3 ) . 2 7 9 - 8 3 ( 1 9 7 1 ) . 7 0 . J.L. Coubeils, P. Courriere and B. Pullman, C. R. Acad. S c i . , Ser. D 2?2 ( 1 3 ) , I 8 1 3 - 6 ( 1 9 7 D * through Chem. Abstr. 2i» 1516*90 ( 1 9 7 1 ) . 1 5 8 . 7 1 . T. Nasu, H. Karaki, M. Ikeda and N. Urakawa, Jap. J . Pharmacol. 21 ( 5 ) . 5 9 7 - 6 0 3 ( 1 9 7 1 ) . 72. E.A. Hartshorn, Drug Intelligence, 2 , 1 9 8 - 2 0 1 ( 1 9 6 8 ) . 7 3 . J . Watt, Can. Pharm. J . 102 ( 9 ) . 275 -9 ( 1 9 6 9 ) . 74. V.C. Saxena, S.K. Bapat and B.N. Dhawan, Jap. J . Pharmac. 12, 477-84 (1969). 7 5 . A.F. Harms and W.Th. Nauta, J . Med. Pharm. Chem. 2 (1), 5 7 - 7 ? ( I 9 6 0 ) . 76. A.F. Harms, Chim. Ther. 2 7 7 - 9 ( 1 9 6 8 ) . 7 7 . E. Kutter and C. Hansch, J . Med. Chem. 1 2 , 647-52 (1969). 7 8 . R.F. Rekker and W.Th. Nauta, Arzeim.-Forsch. 20 ( 1 0 ) , 1 5 7 2 - 4 ( 1 9 7 0 ) . 7 9 . R.F. Rekker, H. Timmerman, A.F. Harms and W.Th. Nauta, Arzeim.-Forsch. 21 ( 5 ) . 6 8 8 - 9 1 (1971). 80. C. Botre, M. Marchetti, C. Del Vecchio, G. L i o n e t t i and A. Memoli, J . Med. Chem. 12 ( 5 ) , 8 3 2 - 6 ( 1 9 6 9 ) . 81. E.J. Ariens and A.M. Simonis, Arch. Int. Pharmacodyn. 141, 3 0 9 - 3 0 ( 1 9 6 3 ) . 82. A. Gero and M.T. Daniele, Arch. Int. Pharmacodyn. 1 8 3 , 315-9 (1970). 8 3 . T.L. Jacobs, Heterocyclic Compounds (Pyridazines ) , 6, 1 0 1 - 3 5 ( 1 9 5 7 ) , Ed. R.C. E l d e r f i e l d , N.Y., John Wiley and Sons, Inc.. 84. E.A. Steck, R.P. Brundage and L.T. Fletcher, J . Am. Chem. Soc. 7_6, 3225-6 ( 1 9 5 4 ) . 8 5 . H. Gilman and M. Furry, J . Am. Chem. Soc. j>0, 1214-6 ( 1 9 2 8 ) . 86. A.P. P h i l l i p s and J . Mentha, J . Am. Chem. Soc. 7 6 , 6200 -2 (1954). 8 7 . L.F. Fieser and M. Fieser, Reagents for Organic Synthesis, 5 8 1 - 5 9 5 ( 1 9 6 7 ) , John Wiley and Sons, Inc., N.Y.. 8 8 . A.E. Fi n h o l t , A.C. 3ond, J r . and H.I. Schlesinger, J . Am. Chem. Soc. 6 £ , 1 1 9 9 - 1 2 0 3 ( 1 9 4 7 ) . 8 9 . W.G. Brown, Organic Reaction 6 , 4 6 9 - 5 0 9 ( 1 9 5 D . 9 0 . C P . Huttrer, C. Djerassi, W.L. Beears, R.L. Mayer and CR. Scholz, J . Am. Chem. Soc. 6 8 , 1 9 9 9 - 2 0 0 2 ( 1 9 4 6 ) . 1 5 9 . 9 1 . F.C. Whitmore, H.S. Mosher, D.P.J. Goldsmith and A.M. Rytina, J . Am. Chem. Soc. 62, 3 9 3 - 5 (19^ 5 ) . 9 2 . R.R. Adams and F.C. Whitmore, J . Am. Chem. Soc. 6£, 7 3 5 - 3 (1945). 9 3 . R.R. Burtner, J . Am. Chem. Soc. 21* 2578 ( 1 9 4 9 ) . 9 4 . L.P. Kyrides, F.C. Meyer, F.B. Zienty, J . Harvey and L.W. Bannister, J . Am. Chem. Soc. 21» 745-8 ( 1 9 5 0 ) . 9 5 . R.O. Kan, Organic Photochemistry, 198-203 ( 1 9 6 6 ) , McGraw-Hill Book Co., N.Y.. 9 6 . S. Searles, J r . Oxetanes i n Heterocyclic Compounds with Three- and Four-Membered Rings, Part 2 , 983-1068 ( 1 9 6 4 ) , Ed. A. Weissberger, I n t e r s c i . Publ., N.Y.. 9 7 . D.R. Arnold, Advances i n Photochemistry, 6 , 301-423 (1968), Ed. W.A. Noyes, J r . , G.S. Hammond and J.N. P i t t s , J r . , I n t e r s c i . Publ., N.Y.. 9 8 . R. Guepet, J . Seyden-Penne, P. Piganiol and P. Chabrier, B u l l . Soc. Chim. Fr. 2081-3 (196l). 9 9 . M. Ogata, H. Matsumoto and H. Kano, Nippon Kagakukai, 22 ( 1 ) , 195-9 ( 1 9 6 9 ) . 100. W.F. Charnicki and J.B. Data, J . Am. Pharm. Assoc. S c i . M . 6 5 - 7 0 ( 1 9 5 6 ) . 101. I.M. Heilbron, "Dictionary of Organic Compounds", Oxford Univ. Press, New York, 2, 166 (1936). 102. K.H. S l o t t a and R. Behnisch, Ber., 68, 754-61 ( 1 9 3 5 ) . 1 0 3 . J.F. Stephen and E. Marcus, J . Org. Chem., 3k ( 9 ) , 2 5 3 5 -42 ( 1 9 6 9 ) . 104. G.W. Anderson, H.E. Faith, H.W. Marson, P.S. Winnek and R.O. Roblin, J r . , J . Am. Chem. S o c , 64, 2902-5 (1942). 1 0 5 . C. Grundmann, Chem. Ber., 81, 1-11 (1948). 1 0 6 . M. Wenke, Fundam. Biochem. Pharmacol. 367-410 ( 1 9 7 1 ) , Ed. Z.M. Bacq, Pergamont Oxford, Engl.. 1 0 7 . J.M. van Rossum and F.G. van den Brink, Arch. Int. Pharmacodyn. 14.2 (1-2), 240-6 ( 1 9 6 3 ) . 108. J.M. van Rossum, Arch. Int. Pharmacodyn. 143 (3-4), 2 9 9 - 3 3 0 ( 1 9 6 3 ) . 1 0 9 . R.W, T r o t t i e r , J r . and M.H. Malone, J . Pharm. S c i . 38 ( 1 0 ) , 1 2 5 0 - 3 ( 1 9 6 9 ) . 160. 110 . E.J. Arlens, Arch. Int. Pharmacodyn. 22* 3 2 - 5 0 ( 1 9 5 4 ) . 111 . E.J. Arlens and A.M. Slmonls, Acta Physiol. Pharmacol. Neerlandica 11, 151-72 ( 1 9 6 2 ) . 112 . H.O. Schild, B r i t . J . Pharmacol. 2 , 189-206 ( 1 9 4 7 ) . 1 1 3 . J.H. van Rossum, J . Pharm. Pharmacol. 1 £ , 2 8 5 - 3 1 6 ( 1 9 6 3 ) . 114. H.O. Schild, B r i t . J . Pharmacol. 4 , 277-80 ( 1 9 4 9 ) . 115 . J . J . Reuse, B r i t . J . Fharmacol. 3_. 1 7 4 - 8 0 ( 1 9 4 8 ) . 116 . Z. Bruno, Y. Kawase and M.M.S. Cunha, Rev. Fac. Farm. Bloqulm. Univ. Sao Paulo 7 ( 1 ) , 153-6" ( 1 9 6 9 ) ; through Chem. Abstr. £ 2 : 77153* U 9 7 0 ) . 117 . M. Rocha e S i l v a , F. Fernandes and A, Antonio, Eur. J . Pharmacol. 1 £ (3)» 333-40 (1972). 118. M. Cirstea, Stud. Cercet. F l z l o l . 1 £ ( 3 ) , 211-18 ( 1 9 7 0 ) ; through Chem. Abstr. 7 3 : 96879z ( 1 9 7 0 ) . 1 1 9 . I. Cortese, B o l l . Chlm. Farm. 110 ( 2 ) , 1 0 0 - 4 ( 1 9 7 1 ) ; through Chem. Abstr. £ i : l l 6 6 5 5 h ( 1 9 7 D . 1 2 0 . J . Tamarit, An. Inst, Farmacol. Espan. 1 £ , 291-11 ( 1 9 7 0 , Pub. 1 9 7 1 ) ; through Chem. Abstr. 21'- l 4 0 8 6 j ( 1 9 7 2 ) . 1 2 1 . D.J. Brown and S.F. Mason, Heterocyclic Compounds: The Pyrimidines, 1 - 2 9 ( 1 9 6 2 ) , I n t e r s c l . Pub., N.Y.. 1 2 2 . N.S. Ham, J . Pharm. S c i . 60 ( 1 1 ) , 1764-5 ( 1 9 7 1 ) . 1 2 3 . S.M. Deshpande and K. Nain, Indian J . Chem. 6 ( 1 1 ) , 6 2 8 - 3 0 ( 1 9 6 8 ) . 124. M.K. Raina and P.K. Das, Aspects A l l e r g y Appl. Immunol,, Proc. Conv., 147-50, 2nd ( 1 9 6 8 , Pub. I 9 6 9 ) , Ed. R. Viswanathan. 1 2 5 . M.K. Raina and P.K, Das, Indian J , Med. Res. ( 4 ) , 6 1 4 - 2 6 ( 1 9 7 D . 1 2 6 . M.M. Lands and F.P. Luduena, J . Pharmac. Exp. Ther. 116. 177-90 ( 1 9 5 6 ) . 1 2 7 . G. Brownlee and E.S. Johnson, B r i t . J . Pharmacol. 2 1 , 3 0 6 - 2 2 ( 1 9 6 3 ) . 128. G. Brownlee and J . Harry, B r i t . J . Fharmacol. 2_1, 5 4 4 - 5 4 ( 1 9 6 3 ) . 161. 129. J.W. Black, W.A.M. Duncan, C.J. Durant, C K . Ganellin and E.M. Parsons, Nature 2 j 6 , 385-90 (1972) . 130. I must express my thanks to Dr. G. Bauslaugh of the College of New Caledonia for designing the photochemical reactions. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0101515/manifest

Comment

Related Items