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The investigation of dispiro and fused ring tricyclic analogues of ethylenediamine type antihistaminics Stephanson, Lawrence Gerald 1972

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THE INVESTIGATION OF DISPIRO AND FUSED RING TRICYCLIC ANALOGUES OF ETHYLENEDIAMINE TYPE ANTIHISTAMINICS by LAWRENCE GERALD STEPHANSON B.S.P., U n i v e r s i t y of B r i t i s h Columbia, 1967 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the D i v i s i o n of M e d i c i n a l Chemistry of the F a c u l t y of Pharmaceutical Sciences We accept t h i s t h e s i s as conforming to the r e q u i r e d 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 in 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 o f B r i t i s h C o l u m b i a , I a g r e e tha t the 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 purposes 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 tha 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 of M e d i c i n a l Chemistry F a c u l t y of Pharmaceutical Sciences The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date September, 1972 to Anne "...and with you there to help me then i t probably w i l l . " - Ian Anderson i i i ABSTRACT The s y n t h e s i s and pharmacological t e s t i n g of two s e r i e s of compounds f o r use i n the i n v e s t i g a t i o n of the s t e r i c and e l e c t r o n i c nature of the a n t i h i s t a m i n i c receptor i s d e s c r i b e d . The dimethylaminoacetyl d e r i v a t i v e s of 7-amino-14-azadispiro[5.1.5 .l\ -pentadecan-15-one and 8-amino-16-azadispiro[6.1.6.2 ] heptadecan-17-one and the methiodide s a l t s of these compounds were s y n t h e s i z e d . The d i -methylaminoacety1 d e r i v a t i v e s of the reduced r i n g systems, 7-amino-14-a z a d i s p i r o [ 5 .1.5 .2] pentadecane and 8-amino-16-azadispiro [6 .1.6 .2] hepta-decane, and the dimethiodide s a l t s were al s o s y n t h e s i z e d . S u i t a b l e com-pounds i n t h i s s e r i e s were test e d f o r l o c a l a n e s t h e t i c a c t i v i t y by the guinea p i g intradermal wheal method and for a n t i h i s t a m i n i c and antimus-c a r i n i c a c t i v i t y i n i s o l a t e d guinea p i g ileum. S e v e r a l of the compounds i n t h i s s e r i e s were a l s o screened f o r general a c t i v i t y and acute t o x i c -i t y . The compounds i n t h i s s e r i e s were i n a c t i v e or showed only a very low order of a c t i v i t y . The 2-dimethylaminoethy1 d e r i v a t i v e s of c a r b a z o l e , 1 , 2 , 3 , 4 - t e t r a -hydrocarbazole, dodecahydrocarbazole, l , 2 , 3 , 4 - t e t r a h y d r o c y c l o p e n t [ b ] -i n d o l e , dodecahydrocyclopent [b] i n d o l e , 5,6,7,8,9,10-hexahydrocyclohept-[b] i n d o l e , tetradecahydrocyclohept[b] i n d o l e , diphenylamine, d i c y c l o h e x y l -amine, f l u o r e n e , and 1,2,3,4,4a,9a-hexahydrofluorene were s y n t h e s i z e d . The bis ( 2 - d i m e t h y l a m i n o e t h y l ) d e r i v a t i v e of f l u o r e n e was a l s o obtained. A l l of these compounds were tested f or a n t i h i s t a m i n i c and antimus-c a r i n i c a c t i v i t y i n i s o l a t e d guinea p i g ileum. The compounds showed only non-competitive a n t i m u s c a r i n i c a c t i v i t y which was probably due to a n o n - s p e c i f i c t o x i c e f f e c t . A l l of the compounds showed competitive i v a n t i h i s t a m i n i c a c t i v i t y . The f u l l y hydrogenated compounds and the d i -s u b s t i t u t e d f luorene d e r i v a t i v e showed non-competitive a n t i h i s t a m i n i c a c t i v i t y , again probably due to a n o n - s p e c i f i c t o x i c e f f e c t , as w e l l as weak competitive antagonism. The seven aromatic or i n d o l i c compounds showed a high degree of com p e t i t i v e a c t i v i t y . These compounds, followed by the pA2 v a l u e s , were: 5-(2-dimethylaminoethy1)-5,6,7,8,9,10-hexahydrocyclohept[b] i n d o l e (7.94), 9-(2-dimethylaminoethyl)-l,2,3,4,4a,9a-hexahydrofluorene (7.45), 9-(2-dimethylaminoethyl)-l,2,3,4-tetrahydrocarbazole (6.90), N-(2-di-methylaminoethyl)diphenylamine (6.67), 9-(2-dimethylaminoethyl)carbazole (6.38), 9-(2-dimethylaminoethy1)fluorene (6.12), and 4-(2-dimethylamino-e t h y l ) - l , 2 , 3 , 4 - t e t r a h y d r o c y c l o p e n t [ b ] i n d o l e (6.12). The pA£ value f o r diphenhydramine was found to be 7.75. The r e s u l t s are discussed i n terms of the s t e r i c nature and pos-s i b l e b i n d i n g modes of the a n t i h i s t a m i n i c r e c e p t o r . S ignatures of Examiners V TABLE OF CONTENTS Page ABSTRACT i i i LIST OF TABLES x LIST OF FIGURES x i LIST OF CHARTS x i i INTRODUCTION 1 Histamine „ 5 A n t i h i s taminics 11 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 s 13 L o c a l A n e s t h e t i c s 18 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 s 19 A n t i m u s c a r i n i c s . . . . . 22 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 s . . . . 25 Drug Receptor I n t e r a c t i o n s 30 DISCUSSION OF THE CHEMISTRY PART ONE: DERIVATIVES AND ANALOGUES OF 7-AMIN0-14-AZADISPIRO [5.1.5.2]PENTADECAN-15-0NE „ 38 PART TWO: DERIVATIVES AND ANALOGUES OF CARBAZOLE 60 ANALYTICAL METHODS . 87 EXPERIMENTAL PART ONE: SYNTHESIS OF DERIVATIVES AND ANALOGUES OF 7-AMINO-14-AZADISPIRO [5.1.5.2]PENTADECAN-15-ONE 1. 14-Hydroxy-14-azadispiro [5.1.5.2]pentadec-9-en-7,15-dione 7-oxime 32 89 2. 7-Amino-14-azadispiro [5 .1.5.2]pentadecan-15-one 4 . . . . 89 3. 7-Chloroacetamido-14-azadispiro [5.1.5.2] pentadecan-15-one 22 90 4. 7-Dimethylaminoacetamido-14-azadispiro [5.1.5.2]-pentadecan-15-one Z3 v i Page A. w i t h benzene as sol v e n t 91 B. w i t h ethanol (100%) as sol v e n t 5. 7-(2-Dimethylaminoethylaraino)-14-azadispiro-[5 .1.5 .2]pentadecane (attempted) > : 6 . 7-Dimethy laminoacetamido- 14-azadispiro [5.1.5.2]-pentadecan-15-one methiodide 24 >L 7. 7-Dimethylaminoacetamido-14-azadispiro [ 5.1.5 . 2 ] -pentadecane dimethiodide 2b. • '4 8. 16-Hydroxy-16-azadispiro [6 .1.6.2]heptadec-10-en-8,17-dione 8-oxime J38 25 9. 8-Amino-16-azadispiro [6 .1.6 .2] heptadecan-17-one .5 . . . . 95 10. 8-Chloroacetamido-16-azadispiro [6 .1.6.2]heptadecan-17-one 27 96 11. 8-Dime thy laminoacetamido- 16-azadispiro [6 .1.6 .2] -heptadecan-17-one 2j8 97 12. 8-(2-Dimethylaminoethy1amino)-16-azadispiro-[6.1.6.2]heptad ecane (attempted) A. w i t h l i t h i u m aluminum hydride -i B. w i t h diborane C. by r e d u c t i o n over copper chromium oxide 100 13. 8-Dimethylaminoacetamido-16-azadispiro [6.1.6 .2] -heptadecan-17-one methiodide 29. 1C 14. 8-Dimethylaminoacetamido-16-azadispiro [6.1.6.2] -heptadecane dimethiodide 3_1 10. 15. 8-Dimethylaminoacetamido-16-azadispiro [6 .1.6.2]-heptadecane methyl s u l f a t e (attempted) . . . . . . . . . 102 MISCELLANEOUS REACTIONS 1. Diborane i n te t r a h y d r o f u r a n 103 2. Copper chromium oxide c a t a l y s t 103 3. 8-Amino-16-azadispiro [6 .1.6.2]heptadecane (attempted) . . 104 v i i Page 4. 7-Amino-14-azadispiro [5 .1.5.2]pentadecane (attempted) . . 105 5. H y d r o l y s i s of 8-dimethylaminoacetamido-16-azadispiro-[6 .1.6.2]heptadecane (attempted) 105 PART TWO: SYNTHESIS OF DERIVATIVES AND ANALOGUES OF CARBAZOLE 1. 9-(2-Dimethylaminoethyl)carbazole _52 . . . 107 2. 9-(2-Dimethylaminoethyl)-l,2,3,4-tetrahydro-carbazole 53 A. 1,2,3,4-tetrahydrocarbazole 64 108 B. 9-(2-di m e t h y l a m i n o e t h y l ) - l , 2 , 3 , 4 - t e t r a -hydrocarbazole . 109 3. 9-(2-Dimethylaminoethyl)dodecahydrocarbazole j>4 A. dodecahydrocarbazole 49 i . from c y c l o h e x y l i d e n e cyclohexanone (attempted) a. c y c l o h e x y l i d e n e cyclohexanone I l l b. dodecahydrocarbazole 112 i i . from carbazole 112 B. 9-(2-dimethylaminoethyl)dodecahydrocarbazole . . 114 4. 4-(2-Dimethylaminoethy1)-1,2,3,4-tetrahydrocyclopent-[b] i n d o l e 55 A. 1,2,3,4-tetrahydrocyclopent [b ] i n d o l e 66 116 B. 4-(2-dimethylaminoethy1)-1,2,3,4-tetra-hydrocyclopent [b] i n d o l e i . u s i n g sodium amide as the base (attempted) . 116 i i . u s i n g sodium hydride as the base (attempted) 117 i i i . w i thout using a base (attempted) 118 i v . u s i n g sodium metal as the base 118 5. 4-(2-Dimethylaminoethyl)dodecahydrocyclopent [b] i n d o l e _56 v i i i Page A. dodecahydrocyclopent [b] i n d o l e _7_5 120 B. 4-(2-dimethylaminoethyl)dodecahydrocyclopent-[b] i n d o l e i . u s i n g sodium metal as the base (attempted) . 121 i i . w ithout u s i n g a base 122 6. 5-(2-Dimethylaminoethy1)-5,6,7,8,9,10-hexahydro-cyclohept[b] i n d o l e 5_7 A. 5,6,7,8,9,10-hexahydrocyclohept[b] i n d o l e 68 . . 125 B. 5-(2-dimethylaminoethyl)-5,6,7,8,9,10-hexahydrocyclohept [b ] i n d o l e 125 7. 5-(2-Dimethylaminoethy1)tetradecahydrocyclohept [b]-i n d o l e 58 127 8. 9-(2-dimethylaminoethyl)fluorene _59 A. u s i n g potassium _t-butoxide as the base 128 B. u s i n g sodium hydride as the base (attempted) . . 130 C. using potassium metal as the base 130 D. u s i n g sodium amide as the base 133 . E. u s i n g sodium amide as the base 134 F. u s i n g potassium metal as the base „ , . . . . . 136 9. 9-(2-Dimethylaminoethyl)dodecahydrofluorene and 9,9-bis-(2-dimethylaminoethyl)dodecahydrofluorene (attempted) . . 138 10. N-(2-Dimethylaminoethyl)diphenylamine 62 . „ 139 11. N-(2-Dimethylaminoethyl)dicyclohexylamine 63_ A. without using a base (attempted) 141 B. u s i n g potassium metal as the base (attempted) . 142 C. 0 C-chloro-N,N-dicyclohexylacetamide 142 D. o*--dimethylamino-N,N-dicyclohexylacetamide . . . 143 E. N-(2-dimethylaminoethyl)dicyclohexylamine . . . 144 Page MISCELLANEOUS REACTIONS 1. 2-Dimethylaminoethy1 c h l o r i d e 146 2. 2-Dimethylaminoethy1 bromide hydrobromide 146 3. P u r i f i c a t i o n of dioxane 147 PHARMACOLOGICAL TESTING PART ONE: DERIVATIVES AND ANALOGUES OF 7-AMINO-14-AZADISPIRO-[5.1.5.2]PENTADECAN-15-ONE A. L o c a l A n e s t h e t i c A c t i v i t y 149 B. A n t i h i s t a m i n i c A c t i v i t y 150 C. A n t i m u s c a r i n i c A c t i v i t y 152 D. General A c t i v i t y and Acute T o x i c i t y 152 PART TWO: DERIVATIVES AND ANALOGUES OF CARBAZOLE A. E v a l u a t i o n of Drug Parameters i . Agonists 154 i i . A ntagonists 157 B. Cumulative Dose-Response Curves 161 C. Experimental Procedure 163 D. M a n i p u l a t i o n of the Data 166 DISCUSSION OF THE RESULTS . 177 SUGGESTIONS FOR FUTURE WORK 187 BIBLIOGRAPHY 190 APPENDICES Appendix I I n f r a r e d Spectra 198 Appendix I I Manufacturers and Grades of Reagents, S o l v e n t s , and Gases 206 Appendix I I I Computer Programs 21.1. Appendix IV Nuclear Magnetic Resonance Spectra 225 X LIST OF TABLES Table Page I Representative A n t i h i s t a m i n i c s . 14 I I R e p r e s e n t a t i v e L o c a l A n e s t h e t i c s . 20 I I I Belladonna a l k a l o i d s and semisynthetic d e r i v a t i v e s . 23 IV Representative A n t i m u s c a r i n i c s . 24 V A z a d i s p i r o compounds synthesized i n t h i s work. 39 VI D e r i v a t i v e s and analogues of carbazole synthesized i n t h i s work. 61 V I I M e l t i n g and b o i l i n g p o ints of dodecahydrocarbazole and i t s d e r i v a t i v e s obtained by v a r i o u s workers. 65 V I I I Y i e l d s of c y c l o h e x y l i d e n e cyclohexanone obtained under v a r i o u s r e a c t i o n c o n d i t i o n s . 68 IX Sequence of doses to be added to a 25 ml organ bath f o r cumulative dose-response curves. 162 X Mean values of c o n t r o l responses f o r complete sets of experiments. 170 X I Drug parameters c a l c u l a t e d from the experimental data. 175 X I I Data f o r the cumulative dose-response curves obtained i n a t y p i c a l experiment; s p e c i f i c a l l y , f i r s t experiment us i n g apparatus 1. the 216 x i LIST OF FIGURES Fi g u r e Page 1 The s t e r i c r e l a t i o n s h i p between the s i d e chain and the a l i c y c l i c r i n g s i n d i a z a d i s p i r o and a z a d i s p i r o compounds. 1 2 D i a z a d i s p i r o compounds. 2 3 A z a d i s p i r o compounds. 3 4 H y p o t h e t i c a l h e p a r i n - p r o t e i n - h i s t a m i n e b i n d i n g . 6 5 Conformations of est e r and alkylamine a n t i m u s c a r i n i c s . 28 6 Receptor model f o r the a n t i h i s t a m i n i c s . 33 7 Dose-response curves f o r agonists having d i f f e r e n t a f f i n i t i e s and i n t r i n s i c a c t i v i t i e s . 156 8 T h e o r e t i c a l dose-response curves f o r an agonist i n the presence of constant, g e o m e t r i c a l l y i n c r e a s i n g doses of a n t a g o n i s t s . 158 9 Responses of guinea p i g ileum to cumulative doses of histamine. 168 10 Average dose-response curve f o r the s t i m u l a t i o n of guinea p i g ileum w i t h histamine. 171 11 Dose-response curves i n the presence and i n the absence of 10 M 5-(2-dimethylaminoethy1)tetra-decahydrocyclohept[b] i n d o l e h y d r o c h l o r i d e . 172 12 Comparison of the pD£ values f o r i n h i b i t i o n of responses to histamine and a c e t y l c h o l i n e . 181 x i i LIST OF CHARTS Chart Page 1 Determination of the f u n c t i o n a l groups present i n the 2:2 condensation product of cyclohexanone and nitromethane. 44 2,3 Degradation experiments on the 2:2 condensation product of cyclohexanone and nitromethane. 46 4 An a l t e r n a t e s y n t h e s i s of the reduced 2:2 condensation product of cyclohexanone and nitromethane. 48 5 A proposed mechanism f o r the formation of the 2:2 condensation product of cyclohexanone and nitromethane. 48 6 Mechanism of the F i s c h e r i n d o l e s y n t h e s i s . 73 x i i i ACKNOWLEDGEMENTS F i n a n c i a l support from the U n i v e r s i t y of B r i t i s h Columbia and the Medic a l Research C o u n c i l of Canada i s g r a t e f u l l y acknowledged. The author wishes to express h i s thanks to Dr. L. Weiler of the Department of Chemistry, U.B.C., f o r a s s i s t a n c e i n determining r e l a t i v e e l e c t r o n d e n s i t i e s . A great d e a l of c r e d i t i s due Mr. Glenn Morgan f o r i n v a l u a b l e a s s i s t a n c e i n w r i t i n g the computer program. The author i s indebted to Dr. T. H. Brown f o r e n l i g h t e n i n g and f r u i t f u l d i s c u s s i o n s d u r ing the course of t h i s work. 1 INTRODUCTION I t i s g e n e r a l l y accepted ( 1 , 2 ) t h a t , i n order to o b t a i n good a n t i -h i s t a m i n i c a c t i v i t y i n ethylenediamine compounds (see Table I , p 14), N^ " must be s u b s t i t u t e d w i t h two aromatic f u n c t i o n s or w i t h one aromatic and one aromatic methyl f u n c t i o n . However, work i n t h i s l a b o r a t o r y (3,4) has shown that at l e a s t one of these f u n c t i o n s may be r e p l a c e d w i t h an a l i c y c l i c f u n c t i o n and a reasonably high degree of a c t i v i t y i s r e t a i n e d . With t h i s i n mind, work was s t a r t e d on a s e r i e s of d i a z a d i s p i r o d e r i v a -t i v e s which, i t was hoped, would allow f u r t h e r e l u c i d a t i o n of the s t r u c -t u r a l requirements f o r a c t i v i t y at the a n t i h i s t a m i n i c r e c e p t o r . These compounds are unique i n that the two a l i c y c l i c r i n g s attached to the c e n t r a l lactam r i n g are held perpendicular to the s i d e chain which i s s u b s t i t u t e d on the apex of the lactam r i n g . F i gure 1 shows the s t e r i c r e l a t i o n s h i p of the a l i c y c l i c r i n g s and the s i d e c h a i n . Due to t h i s r e -l a t i o n s h i p , the d i s p i r o r i n g system should be a h i g h l y e f f i c i e n t b l o c k i n g group i f i t i s i n c o r p o r a t e d i n t o a molecule which w i l l b i n d at a r e c e p t o r . F i g u r e 1: The s t e r i c r e l a t i o n s h i p between the side chain and the a l i -c y c l i c r i n g s i n d i a z a d i s p i r o (a) and a z a d i s p i r o (b) compounds. 6 , 1 2 - d i a z a d i s p i r o [4.1.4.2] tridecan-13-one _1 H 7 , 1 4 - d i a z a d i s p i r o [5.1.5.2]pentadecan-15-one 2 8 , 1 6 - d i a z a d i s p i r o [6 .1.6.2] heptadecan-17-one 3_ Fi g u r e 2: D i a z a d i s p i r o compounds 3 Substitution of a 2-dimethylaminoethy1 group, which i s t y p i c a l of a n t i -h i stamines, on the secondary amino group of the lactam ri n g was there-fore attempted (5,6). Probably because of the large s t e r i c effect of the a l i c y c l i c rings, this could not be accomplished i n 2 (Figure 2). However, the 6-chloracety 1 derivative of 1. was successfully prepared (7). Compound 3_ could not be prepared (6) . Because of these d i f f i c u l t i e s , attention was focussed on azadispiro compounds (Figure 3) where the amino function has been removed from the lactam r i n g . A number of derivatives of these compounds were prepared 7-amino-14-azadispiro [5.1.5.2]pentadecan-15-one 4 H \ /NH. / ° 8-amino-14-azadispiro[6.1.6.2] heptadecan-17-one 5 Figure 3: Azadispiro compounds 4 and t e s t e d f o r pharmacological a c t i v i t y . I t was r e a l i z e d at the outset that the a z a d i s p i r o compounds rep r e -sent a major divergence from the t y p i c a l ethylenediamine a n t i h i s t a m i n i c s . However, i t was a l s o f e l t that the r e s u l t s would be of s u f f i c i e n t i n t e r -est to warrant c o n t i n u a t i o n of the p r o j e c t . These compounds a l s o bear a resemblance to the ethanolamine a n t i h i s t a m i n i c s (see Table I) where the oxygen atom has been replaced by n i t r o g e n . Although the 2-dimethylaminoethy1 side chain i s t y p i c a l of a n t i -h i s t a m i n i c agents there i s c o n s i d e r a b l e overlap of a c t i v i t i e s w i t h l o c a l a n e s t h e t i c s and a n t i m u s c a r i n i c s . Since only minor changes i n the s i d e chain are r e q u i r e d to make these l a t t e r a c t i v i t i e s predominant, compounds were a l s o prepared i n the a z a d i s p i r o s e r i e s which i t was hoped would show these a c t i v i t i e s . P r e l i m i n a r y pharmacological t e s t i n g of the r e s u l t a n t compounds i n d i c a t e d a very low order of a c t i v i t y . A s e r i e s of d e r i v a t i v e s and analogues of carbazole was then synthe-s i z e d . I t was f e l t that t h i s approach o f f e r e d a greater chance of suc-cess i n o b t a i n i n g compounds which could be used i n the study of the e l e c t r o n i c and s t e r i c requirements of the a n t i h i s t a m i n i c r e c e p t o r . 5 HISTAMINE Histamine 1_ ( 2 - ( 4 - i m i d a z o l y l ) e t h y l a m i n e ) was f i r s t synthesized i n 1907 as a chemical c u r i o s i t y . Soon a f t e r , i t s potent smooth muscle s t i m u l a n t and intense depressor e f f e c t s were dis c o v e r e d . However, i t was not u n t i l 1927 that i t was une q u i v o c a l l y demonstrated to be a con-s t i t u e n t of normal mammalian t i s s u e ( 2 ) . Histamine i s synthesized i n the body by d e c a r b o x y l a t i o n of the amino a c i d h i s t i d i n e 6. o -CH — C H — N H 2 , 2 COOH -CO, H This r e a c t i o n may be mediated by a s p e c i f i c h i s t i d i n e decarboxylase or by n o n - s p e c i f i c aromatic amino a c i d decarboxylase which i s r e s p o n s i b l e f o r the d e c a r b o x y l a t i o n of dihydroxyphenylalanine and 5-hydroxytrypto-phane (5-HTP) to norephinephrine (NE) and s e r o t o n i n (5-HT) ( 8 ) . The r a t e at which d e c a r b o x y l a t i o n occurs i s dependent on the species and t i s s u e s t u d i e d . I t appears that histamine i s continuously synthesized i n the body. Although there i s some c e l l u l a r uptake of exogenous h i s -tamine, notably a f t e r a b s o r p t i o n from the gut of histamine formed by b a c t e r i a l a c t i v i t y , t h i s seems to be simply a means of r a p i d l y reducing blood l e v e l s of c i r c u l a t i n g histamine and i s followed by r a p i d catabo-l i s m of the exogenous amine (20). Once formed, histamine may be c a t a b o l i z e d or stored i n e i t h e r of two s i t e s : i n mast c e l l s , or i n non-mast c e l l s i t e s . Mast c e l l s are l a r g e , u s u a l l y ovoid c e l l s , 8-20j+ i n l e n g t h , which c o n t a i n a large number of dark s t a i n i n g granules. They are w i d e l y d i s -t r i b u t e d i n the connective t i s s u e s and large concentrations of these c e l l s are found around the small blood v e s s e l s . These c e l l s are a l s o found i n l a r g e numbers i n the mesentery, lung p l e u r a , thymus, scrotum, and uterus of mammals, but are absent from the c e n t r a l nervous system. The number of mast c e l l s i s increased by chronic inflammation or by other c o n d i t i o n s which r e s u l t i n increased l o c a l n u t r i t i o n (9). Histamine i n the mast c e l l s i s p r i m a r i l y stored i n granules which are separated from the cytoplasm by a p e r i g r a n u l a r membrane ( 10) . The granules c o n s i s t of a p r o t e i n - h e p a r i n complex to which the histamine i s e l e c t r o s t a t i c a l l y bound, as represented i n Figure 4. The turnover r a t e H-:oo - N S 0 l \)S0~ H 3N H3N"1 + P-COO H 3 N - C H 2 — C H 2 ^ N3N \ F i g u r e 4: H y p o t h e t i c a l h e p a r i n - p r o t e i n - h i s t a m i n e b i n d i n g . H = heparin; P = p r o t e i n (from r e f . 10) of histamine i n mast c e l l s t o r e s i s very slow. The p r o t e i n p o r t i o n of the granule c o n s i s t s of a number of enzymes which have protease a c t i v i t y (10) . Zinc has a l s o been i m p l i c a t e d i n the bi n d i n g (15). In response to a number of s t i m u l i , the mast c e l l granules are 7 r e l e a s e d from the c e l l i n t o the surrounding t i s s u e . This r e a c t i o n may be e l i c i t e d by an antigen-antibody r e a c t i o n on the surface of the c e l l , or by a number of chemical substances which may be a p p l i e d l o c a l l y or s y s t e m i c a l l y . One of the most potent of these histamine r e l e a s i n g chemicals i s compound 48/80, a s y n t h e t i c amine polymer. The a n a p h y l a c t i c response i s due to the massive r e l e a s e of histamine and other p h y s i o l o g i c a l l y a c t i v e substances from the mast c e l l (12). Release of l e s s e r q u a n t i t i e s of these substances i n a l o c a l i z e d area probably accounts for other a l l e r g i c r e a c t i o n s (10). The r e l e a s e of histamine from the mast c e l l i n v o l v e s : a) a t t a c h -ment of the r e l e a s i n g agent (antigen or 48/80) to the c e l l membrane, b) s e l e c t i v e and extremely r a p i d degranulation of the c e l l by an energy r e q u i r i n g " e x o c y t o s i s " , l i b e r a t i n g only the histamine c o n t a i n i n g gran-ules without d i s r u p t i o n of the c e l l membrane, and c) l i b e r a t i o n of h i s -tamine from the h i s t a m i n e - h e p a r i n - p r o t e i n complex by a process of ion exchange w i t h c a t i o n s of the e x t r a c e l l u l a r f l u i d (18). Some evidence has been presented that adenosine 35 1-monophosphate i s i n v o l v e d i n preventing the r e l e a s e of histamine from the mast c e l l i n response to dextran or 48/80 (18,19). The r e l e a s e of mast c e l l granules can be accomplished w i t h adenosine 5 1 -triphosphate (ATP) and i s dependent on the presence of calcium ion (11). On r e l e a s e from the c e l l , the granule loses i t s membrane and h i s -tamine i s r e l e a s e d from the complex by exchange w i t h (probably) sodium-io n s . The encapsulated granules are pharmacologically i n e r t . Anticoag-u l a n t and chymase a c t i v i t y i s a l s o observed and i s dependent on the r a t e 8 at which the granule d i s s o l v e s (10). In c o n t r a s t to the mast c e l l s tores of histamine, there are other s t o r e s which have a very f a s t turnover r a t e and which are not r e l e a s e d by 48/80. For example, human epidermis, which i s almost devoid of mast c e l l s , contains large q u a n t i t i e s of histamine ( 2 ) . The h a l f - l i f e of hypothalamic histamine i n the r a t was found by r a d i o t r a c e r s t u d i e s (8) to be about f i v e minutes. A l l mammalian t i s s u e contains s t o r e s of histamine not depleted by 48/80 (13) . I t i s these stores of histamine which have been i m p l i c a t e d i n a u t o r e g u l a t i o n of body f u n c t i o n s (13,14,20). Considering the high r a t e of s y n t h e s i s at these s i t e s , the s o - c a l l e d nascent histamine may account f o r the major p o r t i o n of histamine i n the body. I n j e c t i o n of sma l l amounts of t r i t i a t e d histamine i n d i c a t e d that the mast c e l l s do not take up exogenous histamine but i t i s incorporated i n t o other s t o r e s which have a h a l f - l i f e of 1-3 hours (13) . The same study a l s o i n d i c a t e d that some exocrine gland s e c r e t i o n s are c o n t r o l l e d by the r e l e a s e of nascent histamine. L o c a l production and r e l e a s e of histamine has a l s o been suggested as a c o n t r o l mechanism f o r the m i c r o c i r c u l a t o r y system. From t h i s p o s t u l a t e Schayer (14) has developed u n i f i e d t h e o r i e s which account f o r the g l u c o c o r t i c o i d a c t i v i t y of the s t e r o i d hormones and f o r the a c t i v i t y of the t h y r o i d hormone. The main pharmacological e f f e c t s of r e l e a s e d histamine are on the v a s c u l a r system, smooth muscles, and exocrine glands ( 2 ) . The most important v a s c u l a r e f f e c t i n man i s a powerful d i l a t a t i o n of c a p i l l a r i e s and venules, which i s accompanied by a weak c o n s t r i c t o r e f f e c t on the la r g e r v e i n s . The a r t e r i o l e s are a l s o d i l a t e d . This d i l a t a t i o n i s 9 caused by a d i r e c t e f f e c t on the v e s s e l s , i s r e s i s t a n t to a t r o p i n e , and i s only p a r t i a l l y prevented by the a n t i h i s t a m i n i c s although i t can be reversed by sympathomimetic amines. P a r t i c u l a r l y w i t h l a r g e r doses, the d i l a t a t i o n i s accompanied by an increase i n p e r m e a b i l i t y of the v e s s e l w a l l s , r e s u l t i n g i n edema due to exudation of plasma f l u i d and p r o t e i n s i n t o the e x t r a c e l l u l a r spaces. Two commonly described phenomena a t t r i b -u t a b l e to t h i s d i l a t o r y e f f e c t are the t r i p l e response and histamine headache. Histamine has no d i r e c t e f f e c t on the heart i n the i n t a c t animal although r e f l e x a c t i o n s increase the c a r d i a c output. With l a r g e doses, shock occurs. Histamine causes c o n s t r i c t i o n of smooth muscle t i s s u e to a degree which i s dependent on the species and p a r t i c u l a r t i s s u e s t u d i e d . Although i n normal man b r o n c h o c o n s t r i c t i o n due to endogenous histamine i s not severe, i t i s i n those s u f f e r i n g w i t h asthma and other pulmonary d i s e a s e s . This e f f e c t i s r e a d i l y countered by aminophylline, epinephrine, or i s o -p r o t e r e n o l w h i l e a n t i h i s t a m i n i c s are l e s s e f f e c t i v e and slower i n a c t i o n . The c o n t r a c t i l e e f f e c t of histamine i s dependent on the presence of cal c i u m i o n (2). The exocrine glands, p a r t i c u l a r l y those of the stomach, are stimu-l a t e d to increased s e c r e t i o n by histamine. Actions on s a l i v a r y , pancre-a t i c , i n t e s t i n a l , b r o n c h i a l , and l a c h r i m a l glands are much l e s s prominent and appear to have a c h o l i n e r g i c component. Metabolism of histamine (16,17) i s mediated by histaminase, which may be the same as diamine oxidase, to give imidazole 4 - a c e t i c a c i d 8 v i a the acetaldehyde 9_. The a c i d i s excreted f r e e or as the r i b o s i d e . Other metabolites are 2- ( l - m e t h y l - 4 - i m i d a z o l y l ) e t h y l a m i n e 10, formed by 10 the action of histamine N-methyl transferase, and (1-methy 1-4-imidazoly1)-acetic acid 11. Very l i t t l e unmetabolized histamine i s excreted. 10 11 11 ANTIHISTAMINICS As previously stated, the release of histamine from the mast c e l l response to an antigen-antibody reaction i s associated with the a l l e r g i c response and, i n i t s most severe form, the anaphylactic syndrome. Although usually not of a severe nature, the a l l e r g i c response i s a nuisance and for this reason, drugs to counteract the effect of endogen-ously released histamine were sought. These effects may be reversed by administration of physiological antagonists such as ephedrine or epine-phrine which have an effect diametrically opposed to that of histamine. This i s the treatment of choice i n anaphylactic reactions. However, antihistaminics are usually defined (2,21) as drugs which antagonize some of the effects of histamine by a mechanism other than the production of a physiological antagonism. Antihistaminics do not act by a physical or chemical i n a c t i v a t i o n of histamine, and they do not prevent the synthesis of histamine nor i t s release from the mast c e l l s i t e s (22). In fact some antihistaminics cause the release of histamine. Antihistaminics w i l l diminish, i n varying degree, the bronchiolar and i n t e s t i n a l spasm, increased c a p i l l a r y permeability, s a l i v a t i o n , cutaneous wheal, and release of epinephrine from the adrenals which are caused by histamine. These compounds have no effect on ga s t r i c secretion induced by histamine. A new class of compounds which competitively i n h i b i t actions of histamine on gastr i c secretion has recently been reported (101). Antihistaminics i n h i b i t the effects of histamine by competing with the histamine molecule for the s p e c i f i c receptor at which histamine exerts i t s e f f e c t . This implies that a given dose of histamine w i l l be countered by a s u f f i c i e n t l y high dose of antihistaminic, and vice versa. For a further discussion of drug antagonism, see Pharmacological Testing, 12 P a r t Two, p 157. Other e f f e c t s of a n t i h i s t a m i n i c s i n c l u d e c e n t r a l nervous system e f f e c t s ( e i t h e r depression or e x c i t a t i o n ) , l o c a l anesthesia when a p p l i e d t o p i c a l l y , and a n t i m u s c a r i n i c a c t i v i t y ( 2 ) . A n t i h i s t a m i n i c s are used i n the treatment of a la r g e number of mi l d to severe a l l e r g i c c o n d i t i o n s . They are of value i n hay f e v e r , p a r t i c -u l a r l y at the s t a r t of the season when exposure to the a l l e r g e n i s l i m -i t e d , and i n s k i n r e a c t i o n s such as drug e r u p t i o n s , p r u r i t i s , contact d e r m a t i t i s , a t o p i c d e r m a t i t i s , and u r t i c a r i a . The drugs may be used s y s t e m i c a l l y or a p p l i e d l o c a l l y , although there i s a r i s k of a l l e r g i c r e a c t i o n to t o p i c a l l y a p p l i e d a n t i h i s t a m i n i c s ( 2 , 2 1 ) . These drugs are u s e f u l i n serum sickness but play a minor adjuvant r o l e i n an a p h y l a c t i c r e a c t i o n s . Some a n t i h i s t a m i n i c s are u s e f u l i n motion sickness and other c o n d i t i o n s causing nausea and vo m i t i n g , such as pregnancy and r a d i a t i o n therapy. This e f f e c t i s probably the r e s u l t of a c e n t r a l a c t i o n of the compounds. A number of si d e e f f e c t s are common to the a n t i h i s t a m i n i c s . There i s a great v a r i a t i o n i n the s e v e r i t y and occurrence of these w i t h the i n d i v i d u a l p a t i e n t and drug. The most frequent side e f f e c t i s se d a t i o n , although c e n t r a l s t i m u l a t i o n i s sometimes seen. Other c e n t r a l e f f e c t s are d i z z i n e s s , i n c o o r d i n a t i o n , b l u r r e d v i s i o n , d i p l o p i a , t i n n i t i s , l a s -s i t u d e , f a t i g u e , nervousness, tremors, and euphoria. G a s t r i c e f f e c t s ranging from nausea and vomi t i n g through d i a r r h e a or c o n s t i p a t i o n are a l s o seen. Drying of the mucous membranes of the mouth and r e s p i r a t o r y system i s due to the a n t i m u s c a r i n i c e f f e c t s of the a n t i h i s t a m i n i c s . 13 STRUCTURE ACTIVITY RELATIONSHIPS (1,2,21) Innumerable compounds of exceedingly d i v e r s e s t r u c t u r e s have been te s t e d f o r a n t i h i s t a m i n i c a c t i v i t y . From these s t u d i e s has come a f a i r l y w e l l d efined idea of the molecular s t r u c t u r e r e q u i r e d to a t t a i n a c t i v i t y . A general formula f o r a n t i h i s t a m i n i c s may be represented as where X may be carbon, n i t r o g e n , or an oxy ether to carbon ( ^ CH-O). The use of a t h i o ether reduces a c t i v i t y (23). The nature of X i s com-monly used to c l a s s i f y a n t i h i s t a m i n i c s , where R3 i s ethylene, as p r o p y l -amine, ethylenediamine, or ethanolamine d e r i v a t i v e s . Two subclasses of the ethylenediamines are the p i p e r a z i n e and phenothiazine d e r i v a t i v e s . Representative examples of each c l a s s are given i n Table I. I t i s g e n e r a l l y accepted that R^ and R 2 must be aromatic or hetero-aromatic r i n g s , one of which may be separated from X by a methylene group. R^ and R 2 may be l i n k e d i n the ortho p o s i t i o n s by Y, a methylene, a heteroatom, or a methylene-heteroatom f u n c t i o n . Except i n the cases where Y i s present, R-j must be ethylene to a t t a i n good a n t i h i s taminic a c t i v i t y . Lengthening or branching the chain markedly decreases a c t i v i t y although R 3 may be incorporated i n t o a n i t r o g e n c o n t a i n i n g r i n g as i n the p i p e r a z i n e d e r i v a t i v e s . For good a n t i h i s t a m i n i c a c t i v i t y , R-^  and R 2 should not be coplanar. That i s , the two r i n g s should not be d i r e c t l y ortho, connected.. 14 D i p h e n h y d r o m i n e ( 8 e n a d r y l ) B r o m o d i p h e n h y d r a m i n e HCI ( A m b o d r y l ) D i m e n h y d r i n a t e ( C r a v o l ) C a r b i n o x o m i n e m a f e a f e ( C l i j t i n ) C y c l i i i n e HCI U.S .P . ( M a r z i n e ) O — C H r - C H j — N i C H i ) , C h l o r c y c l i z i n e HCI U.S .P . ( P e r o z i l ) O—CH,—CH,—N(CH»}j CH C H , C H , CH ; I +/ C H , N H \ CH. M e c l i z i n e HCI U .S .P . ( B o n o m i n e ) C H I C — C H j — C H j — N !CHj) j M e t h o p y r i l e n e HCI ( H i j l a d y l ) C H — N N — C H , /—V C H — N N — C H j P i p e r a z i n e s S t CH, CHr—CH,—N[CHS)» D o x y l a m i n e succinate ( O e c a p r y n ) D J p h e n y l p y r o t i n e HCI O — C H , — C H r - N ( C H , 1 , CH I O — ^ N — C H , P y r i l a m i n e m o l e a t e ( N e o - A n t e r g o n ) Tr ipelenrvamine HCI ( P y r i b e n r o m i n e ) o x : H , — C H r - N [ C H A OCH, o a i r : H r — C H r — N ( C H , V . E t h o n o l o m i n e s £ t r i y t e n e d i a m i n e s \ c / C H — 0 — C H 0 — C H 0 — N / }\ R 1 \ 1 N / R/ C H „ — C H „ — N Table 1: Representative Antihistaminics 15 P h e n i r o m i n e m a l e a f e N .F . (Tr imeron) C H I C H r — C H j — N ! C H J , C h l o r p h e n i r a m i n e mateo te U .S .P . <Ch io r -T r ipo lon ) B r o m p h e n i r a m i n e m a l e a t e N .F . ( D i m e t a n e ) * N C H I C H j — C H j — N ( C H i ) : C H I C H j — C H j — N t C H J s P romethaz ine HCI U .S .P . ( P h e n e r g a n ) I C H t — C H — N ( C H j J i I C H , D i m e t h i n d e n e m a l e a t e N . N . D . ( Fo rh i s ta l ) C H ; CH^-CH»—NlCHJ, o T r i m e p r a z i n e ta r t ra te N . N . D . (Paneety l ) C H j — C H — C H j — N ( C H i ) , I C H j T r i p r o l i d i n e HCI N . N . D . ( A c t i d i l ) • ^ 7 1 — C H , C H — C H j — [ M e t h d i l a z i n e HCI N . N . D . ( D i l o s y n ) -Q-CH, Pyr robuto f f l ine p h o s p h a t e N . N . D . (Py ro f l t l ) C H j C C M - C H r - N ~ J P r o p y l a m i n e s P h e n o t h i o t i n e s J v 1 2-^ R4 S N - R - j - N ^ // W R4 \ ( / C H — CH„ / C H 2 — N \ Table I ( c o n t ' d ) : R e p r e s e n t a t i v e A n t i h i s t a m i n i c s 16 Where Y i s present, may be branched (X to the n i t r o g e n atom, as i n promethazine, or a propylene group may be used. In both cases, a c t i v -i t y i s increased over the analagous compound c o n t a i n i n g an ethylene group. In g e n e r a l , R^ are methyl groups, although -N(R^)2 may be incorpo-r a t e d i n t o a small r i n g system. Use of e t h y l groups for R^ decreases the a n t i h i s t a m i n i c a c t i v i t y and increases the a n t i m u s c a r i n i c a c t i v i t y . A d i s t a n c e of 5-6 A from the amino n i t r o g e n to the centre of one of the aromatic r i n g s gives the strongest competitive a c t i v i t y . S u b s t i t u t i o n of one of the aromatic r i n g s w i t h a methyl or a c h l o r o group g e n e r a l l y increases a c t i v i t y . Ortho s u b s t i t u t i o n on both r i n g s decreases a c t i v i t y and has l i t t l e e f f e c t i f only one of the r i n g s i s s u b s t i t u t e d . Nauta (24a) has explained t h i s r e l a t i o n s h i p i n a l k y l sub-s t i t u t e d diphenhydramine d e r i v a t i v e s by proposing that one of the aro-matic r i n g s i n t e r a c t s w i t h the oxygen lone p a i r e l e c t r o n s to decrease the e l e c t r o n d e n s i t y of the oxygen atom. Para a l k y l s u b s t i t u t i o n enhances the overlap i n t e r a c t i o n w h i l e or tho s u b s t i t u t i o n decreases the i n t e r a c t i o n . In o p t i c a l l y a c t i v e compounds the dextro isomer i s g e n e r a l l y more a c t i v e than the levo isomer although the r e v e r s e i s true i n some cases. Few s t u d i e s have been done on compounds whose absolute c o n f i g u r a t i o n s are known. S t e r e o s e l e c t i v i t y i s observed only when the asymmetric carbon atom i s oc to the aromatic r i n g system. Since histamine i t s e l f i s o p t i -c a l l y i n a c t i v e , t h i s i n f o r m a t i o n may i n d i c a t e that an asymmetric area i s adjacent to the histamine r e c e p t o r , or that an asymmetric conformer of histamine i s the a c t i v e s p e c i e s . F a i l u r e to r e l a t e p h y s i c a l p r o p e r t i e s such as s o l u b i l i t y , r e l a t i v e s u rface a c t i v i t y , and i o n i z a t i o n constants to a c t i v i t y of a n t i h i s t a m i n i c s , 17 as w e l l as isomer s t e r e o s e l e c t i v i t y , indicates that s t e r i c factors are very important i n determining the a c t i v i t y of these compounds. 18 LOCAL ANESTHETICS (25,26) L o c a l a n e s t h e t i c s are drugs which block nerve conduction when ap-p l i e d to nerve f i b r e s . A l l nerve f i b r e s , sensory or motor, myelinated or unmyelinated, are a f f e c t e d by these agents. The a c t i o n of l o c a l an-e s t h e t i c s i s i d e a l l y completely r e v e r s i b l e so that there i s no permanent damage to the nervous t i s s u e . L o c a l a n e s t h e t i c s may be a p p l i e d t o p i c a l l y to whole or abraded s k i n , i n j e c t e d i n t r a - or subcutaneously i n t o the area to be anesthetized ( i n f i l t r a t i o n a n e s t h e s i a ) , or i n j e c t e d adjacent to nerve trunks and t h e i r branches or i n t o the e p i d u r a l or subarachnoid spaces (nerve b l o c k and s p i n a l a n e s t h e s i a ) . The f i n e nerve f i b r e s and endings are blocked i n the r e g i o n of the i n j e c t i o n by i n f i l t r a t i o n an-e s t h e s i a w h i l e nerve impulses to and from areas remote from the i n j e c t i o n are h a l t e d by nerve b l o c k and s p i n a l a n e s t h e s i a . The e f f e c t s of these drugs are sensory impairment and muscle r e l a x -a t i o n . Their systemic e f f e c t s are of no ther a p e u t i c v a l u e . The mechanism of a c t i o n i s unknown although i t appears that the s i t e of a c t i o n i s the c e l l membrane w i t h no e f f e c t on the axoplasm. Shanes (27a,b) has suggested that these compounds increase the surface pressure of the nerve membrane l i p i d s , thus c l o s i n g o f f the pores through which ions move i n the propagation of the a c t i o n p o t e n t i a l . The drugs act by preventing the large t r a n s i e n t i n c r e a s e , caused by a s l i g h t d e p o l a r i z a -t i o n of the membrane, i n p e r m e a b i l i t y of the membrane to sodium i o n s . The r e s t i n g p e r m e a b i l i t y to potassium ions i s a l s o reduced. The c a t i o n i c form of the molecule i s r e s p o n s i b l e f o r the a c t i v i t y at the nerve mem-brane . The d u r a t i o n of a c t i o n of l o c a l a n e s t h e t i c s i s l a r g e l y determined 19 by the r a t e at which the drug i s absorbed i n t o the blood stream. In order to prolong a n e s t h e s i a , v a s o c o n s t r i c t o r substances are f r e q u e n t l y added to preparations f or i n j e c t i o n . This has the added advantage of reducing the incidence and s e v e r i t y of systemic s i d e e f f e c t s s i n c e the r a t e of metabolism of absorbed compound i s not a f f e c t e d . Most of the s y n t h e t i c l o c a l a n e s t h e t i c s are e s t e r s of aromatic c a r b o x y l i c acids and are metabolized by plasma or l i v e r e s t e r a s e s . The t o x i c e f f e c t s of l o c a l a n e s t h e t i c s are caused by systemic extension of t h e i r t h e r a p e u t i c p r o p e r t i e s . Thus, there i s decreased e l e c t r i c a l e x c i t a b i l i t y and conduction i n the myocardium. The c e n t r a l nervous system i s both depressed and e x c i t e d , although i t i s thought that the e x c i t a t i o n i s due to a p r e f e r e n t i a l depression of i n h i b i t o r y f i b r e s . C o n t r a c t i o n s of smooth muscle are a l s o diminished. In r a r e cases, h y p e r s e n s i t i v i t y occurs and may appear as a d e r m a t i t i s , an a s t h -matic a t t a c k , or an a n a p h y l a c t i c r e a c t i o n . A d d i t i o n of a v a s o c o n s t r i c -tor may r e s u l t i n t i s s u e n e c r o s i s , edema, or delay i n wound h e a l i n g , p a r t i c u l a r l y when sympathomimetic amines are used. L o c a l s i d e e f f e c t s are n e u r o l y s i s , and subsequent slough of the t i s s u e , and n e c r o s i s of surrounding t i s s u e s . Some examples of s y n t h e t i c l o c a l a n e s t h e t i c s are given i n Table I I . 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 s Most of the s t r u c t u r e s which have been test e d f o r l o c a l a n e s t h e t i c a c t i v i t y may be r e l a t e d to a general r e p r e s e n t a t i o n as l i p o p h i l i c p o r t i o n - i n t e r m e d i a t e c h a i n - h y d r o p h i l i c p o r t i o n o r , more s p e c i f i c a l l y , 20 (AROMATIC \ / INTERMEDI-RESIDUE j y ATE CHAIN AMINOS GIOVT) (AROMATIC \'/INTERMEDI-\ /AMINO \ RES1DLTE j j[ ATE CHAIN J ^GROfP / I h ^ ° \ i T Benoxinote j H,N - O C H j C H i — U \ Procoine C o c a i n e ^ ~ ^ C O r - O C H C H j C H C,H, CHJ •CH C H J C O O C H , ) H j N ^ ^ C O p - O C H ^ H , -Butethomine ! k CHjCHjay, C O — O C H j C H , — N C , H , C , H , C O . — N H C H z O ^ - J - N D ibuca ine CH, CjH, ^ ^ O - j O C H f C H j r C H : N ^ — ^ Hexyloaine I I I I C H , • IkJocoine x > N H [ - C O C H 2 J N ^ W " P r C H , I I I I I CzHs C , H , j C H , H { — C O -Meptvoco ine I I r*1 i ^ ~ ^ c 4 - O C H , C H 2 C H , — ^ • i Prperocome I I I I 1 I I ! ' H \ r O T - O C H 2 C H i - i N ^ „ . W I ! X c l H u N o e p a i n e | I I C j H j O — ^ ^ N 4 q C H 3 ) Phenocoine 1 • I OC2Hj V - N j I , C , H , l ,N f V o r - O C H z C H i - r N ; \ = / J | \ , H S HrCjO, Proporocoine j o I CH» O j j I ^ ^ C O ' p C H t C H ^ H t j - N ^ y O/ctomel f r /come I I O C H z C H , C H , — ^ > Pramoxine Dimefhisoquia Tel rocoine Table I I : R e p r e s e n t a t i v e L o c a l A n e s t h e t i c s 21 aromatic r e s i d u e - i n t e r m e d i a t e chain-amino f u n c t i o n A balance between l i p o p h i l i c and h y d r o p h i l i c nature i s necessary, as i n a l l drugs, to a t t a i n d i s t r i b u t i o n through the e x t r a c e l l u l a r (aqueous) phase and the l i p o i d a l membranes. The amino group i s u s u a l l y t e r t i a r y but may be secondary. Increas-ing the s i z e of the N - a l k y l s u b s t i t u e n t s increases a c t i v i t y and t o x i c i t y of the compound. The N - a l k y l s u b s t i t u e n t s may be the same or d i f f e r e n t . The amino f u n c t i o n may a l s o be incorporated i n t o a r i n g system such as piper i d i n o or morpholino. The intermediate chain i s u s u a l l y an e t h y l group which i s l i n k e d to the aromatic r e s i d u e v i a an e s t e r . Amides, imides, e t h e r s , and ketones may a l s o be used. Lengthening of the chain increases a c t i v i t y and tox-i c i t y w i t h the p r o p y l group being o p t i m a l . The nature of the l i n k to the aromatic f u n c t i o n l a r g e l y d i c t a t e s the d u r a t i o n of a c t i v i t y s i n c e i t i s t h i s l i n k which i s hydrolyzed i n the body. Thus 2 , 6 - d i s u b s t i t u t i o n on the aromatic r i n g i s o f t e n seen. G e n e r a l l y , the aromatic r e s i d u e i s a benzoic a c i d d e r i v a t i v e , although a l a r g e number of other f u n c t i o n s can be used. S u b s t i t u t i o n of a para-amino group on benzoic a c i d increases a c t i v i t y . Subsequent a l k y l a t i o n increases a c t i v i t y f u r t h e r although t o x i c i t y a l s o increases w i t h the s i z e of the a l k y l group. A para-alkoxy a l s o increases a c t i v i t y and t o x i c i t y i n p r o p o r t i o n to the s i z e of the group. 22 ANTIMUSCARINICS (28,29) The a n t i m u s c a r i n i c s are drugs which b l o c k the a c t i o n s of a c e t y l -c h o l i n e (ACh) at s t r u c t u r e s which are innervated by p o s t g a n g l i o n i c para-sympathetic ( c h o l i n e r g i c ) f i b r e s . They a l s o b l o c k the e f f e c t on smooth muscles which respond to ACh but l a c k c h o l i n e r g i c innervation.. A n t i -muscarinics g e n e r a l l y have no e f f e c t on other a c t i o n s of ACh but at high doses t r a n s m i s s i o n at autonomic g a n g l i a and neuromuscular j u n c t i o n s may be blo c k e d . The a n t i m u s c a r i n i c s exert t h e i r e f f e c t s by a competitive blockade of ACh at the muscarinic r e c e p t o r . These drugs are used i n c o n d i t i o n s c h a r a c t e r i z e d by gla n d u l a r hyper-s e c r e t i o n and spasm or h y p e r m o t i l i t y of smooth muscle. They are w i d e l y used i n ophthalmology to produce mydriasis and c y c l o p l e g i a . They are used as p r e a n e s t h e t i c medication to depress s a l i v a r y and r e s p i r a t o r y t r a c t s e c r e t i o n s , and they are used i n the treatment of g a s t r i c and duo-denal u l c e r s . They may a l s o provide symptomatic r e l i e f of acute r h i n i t i s i n hay fever and c o l d s . A l l of the a n t i m u s c a r i n i c s have c e n t r a l e f f e c t s which vary w i t h the p a r t i c u l a r drug used. Only the belladonna a l k a l o i d s , a t r o p i n e and scopolamine (the prototype compounds), and t h e i r semisyn-t h e t i c d e r i v a t i v e s are c l i n i c a l l y u s e f u l i n t h i s r e s p e c t . These compounds are shown i n Table I I I . These a l k a l o i d s are used i n the treatment of parkinsonism, and scopolamine i s one of the more u s e f u l drugs for prevention of motion s i c k n e s s . The sedative and t r a n q u i l i z i n g e f f e c t s of scopolamine have been used i n other s t a t e s , i n c l u d i n g l a b o r , t o x i c psychoses, and d e l i r i u m tremens. The a n t i m u s c a r i n i c s are a l s o used i n the treatment of poisoning w i t h organophosphate i n s e c t i c i d e s and the r a p i d type of mushroom poison-23 -0+-I C H j O H N - C H , C H - O - C O - c / \ « I I H W / H j C - 1 C H C H j r; Atrop ine C H , C H , I 0 ) O H C H , — N C H - O - C O - c / \ N O , H2C-- - C O - C ^ -CH- - C H , A t rop ine Merfiylnitrote H2C CH CH, N-CH, CH-C HrC CH CH, O H C H , H - O - C O - o / " " \ I I H\—/  C H , Hornotroptne H C ^ C H - C H j C H , O H N - C H , C H - O - C O - c / - \ H C — C H C H , - C , Scopolamine - C H i -CH a F T 0M2OH C H , — * / ' C H - O - C O - C | X C H , I » \ = - C H - -CH, Scopolamine Mefhylbromtde - C H - - C H , Br I OH CH,—tf' CH-0-CO-C/~~\ H,C-^CHJ CH CH, H \ — / Homatropine Methy lbromide Table I I I : A n t i m u s c a r i n i c Belladonna A l k a l o i d s and Semisynthetic D e r i v a t i v e s . i n g due to Amanita s p e c i e s . Examples of s y n t h e t i c a n t i m u s c a r i n i c s are giv e n i n Table IV. The s u s c e p t i b i l i t y of the parasympathetic n e u r o e f f e c t o r j u n c t i o n to the a n t i m u s c a r i n i c s v a r i e s . The order of s e n s i t i v i t y to the v a r i o u s drugs v a r i e s l i t t l e although some compounds have a degree of s p e c i f i c i t y . A n t i m u s c a r i n i c agents, when administered s y s t e m i c a l l y , tend to i n h i b i t s a l i v a t i o n and sweating most r e a d i l y . This i s f o l l o w e d , at i n c r e a s i n g doses, by i n h i b i t i o n of the responses of the i r i s and c i l i a r y body, v a g a l c o n t r o l of c a r d i a c r a t e , g a s t r o i n t e s t i n a l and u r i n a r y bladder tone and m o t i l i t y , and f i n a l l y g a s t r i c s e c r e t i o n . The s i d e e f f e c t s of these drugs are r e l a t e d to blockade of responses at organs other than the t a r g e t of therapy. Side e f f e c t s are seldom 24 KOfritOMJETAftY KAMI T1AM M A M E , NO>?*O^IUtlA»V N A M E T*ATX K A V X i otnnouL STXUCTTUS Qaasermaj Ammomlu Dibuiiae CM, Prop-art I hriroc Pre-BmlKnr it c - o o w y A » , DipbezBsml Pantal TxicycUact Horiae Tncoioii Gjuiyymjliie Xoccaal TrirfiVifthyi V " / C H j C H , Hgxocyv'Tnna X n l § > ~ o C M a d CH, C r t Dirbid CH, Other SymlJ*ikAjam AiaiaopmfimwV K t H - C C CHjCH-N(Obi 0 Bx&Onae H. .C—OCMjCMJ*—CH, 0 0 0 " Cydopeatotal£ Pjdogyl Mepiperpbemdol Duitioe OH CH—CH, CHj CH, 1 C M A Oayphcaomam Aotrenyl / y/^ccoyti^CH, \ / I CVH Oxyphcocydiiiiine Duicoa Ykwhrae Q v W O j — . <*, ^ ^ - C - C - O O V H j N ^ C H , KpmtWifc D i a l ( X i . CiH, Rpul HO—C—C—O—t 1 Ftocyi&cfinc PoUOne Nictoa Tnxaulc Q 0 Table IV: Re p r e s e n t a t i v e A n t i m u s c a r i n i c s 25 s e r i o u s but l i m i t therapy because of s u b j e c t i v e unpleasantness. Dryness of the mouth, b l u r r e d v i s i o n , photophobia, and t a c h y c a r d i a are the most common si d e e f f e c t s . I n h i b i t i o n of sweating may cause heat i n t o l e r a n c e i n some i n d i v i d u a l s i n hot c l i m a t e s . Acute glaucoma may be p r e c i p i t a t e d by l o c a l or systemic use and i t i s of p a r t i c u l a r importance that care be taken i n the use of these drugs i n p a t i e n t s over 40 years of age. C o n s t i p a t i o n i s not uncommon and complete blockage may occur. Urinary r e t e n t i o n a l s o occurs and may be aggravated by p r o s t a t i c hypertrophy. Impotence i s a l e s s common s i d e e f f e c t of compounds having some g a n g l i -onic b l o c k i n g a c t i v i t y . Reduction of the volume of b r o n c h i a l s e c r e t i o n s may r e s u l t i n the formation of v i s c i d plugs i n the b r o n c h i a l t r e e which may be p a r t i c u l a r l y dangerous i n i n d i v i d u a l s w i t h chronic r e s p i r a t o r y d y s f u n c t i o n . H y p e r s e n s i t i v i t y r e a c t i o n s , manifested u s u a l l y as s k i n rashes which may proceed to e x f o l i a t i o n , are r a r e occurrences. The belladonna a l k a l o i d s have a greater tendency to produce b l u r r e d v i s i o n than s i d e e f f e c t s r e l a t e d to the reduced tone and m o t i l i t y of smooth muscle w h i l e the converse i s true of s y n t h e t i c agents which are capable of producing c o n s i d e r a b l e g a n g l i o n i c blockade. Tolerance to the s i d e e f f e c t s i s not uncommonly developed, although a p a r a l l e l r e d u c t i o n i n t h e r a p e u t i c e f f e c t i s u s u a l l y seen. 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 s Because of the d i f f i c u l t y i n s e p a r a t i n g the e f f e c t s of the a n t i -m u s c a r i n i c s , vast numbers of compounds have been synthesized. The chemical s t r u c t u r e s vary a great deal and t h i s has given r i s e to a h i g h l y complex set of 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 s which can be 26 only s u p e r f i c i a l l y reviewed here. Even though a great v a r i e t y of com-pounds has been s y n t h e s i z e d , few of these have anything to o f f e r i n the way of increased s p e c i f i c i t y , w i t h respect to the s i t e of a c t i o n , over the prototype a l k a l o i d s . A general formula f o r the a n t i m u s c a r i n i c s may be represented as R, R. C R/ 1 + U s u a l l y , R^ i s an aromatic f u n c t i o n f u l f i l l i n g c e r t a i n s p a t i a l requirements, R 2 i s a normal a l k y l or a l i c y c l i c f u n c t i o n , and R^ i s a group capable of hydrogen bonding, a s m a l l a l k y l , or hydrogen. The group, R^, l i n k i n g the c y c l i c s u b s t i t u e n t to the c a t i o n i c head i s gen-e r a l l y an a l k y l e s t e r of a s p e c i f i c l e n g t h . The one fe a t u r e which i s common to n e a r l y a l l e f f e c t i v e antimus-c a r i n i c s i s the c a t i o n i c head. I t i s t h i s group which i s thought to be most important i n the i n i t i a t i o n of complex formation between the muscarinic receptor and the antagonist. This f u n c t i o n i s a l s o common to the muscarinic a g o n i s t s . U s u a l l y the c a t i o n i c head i s a quaternary ammonium group, altho.igh t e r t i a r y amines having a pK^ such that a large p o r t i o n of the dose used i s protonated at p h y s i o l o g i c a l pH may a l s o be used. Sulfonium and phosphonium groups are used i n f r e q u e n t l y . High b a s i c i t y of the c a t i o n i c f u n c t i o n increases the s t a b i l i t y of the drug-receptor complex and thus 27 increases a c t i v i t y . However, s t e r i c f a c t o r s are a l s o important. The best a c t i v i t y i s seen w i t h d i - i s o - p r o p y 1 - or diethylamino and t r i m e t h y l -ammonium f u n c t i o n s . Larger and smaller groups show a decrease i n a c t i v -i t y . The c a t i o n i c head may be incorporated i n t o a s u i t a b l e r i n g system. The c a t i o n i c charge i s not a b s o l u t e l y necessary to a c t i v i t y , s i n c e the trimethy 1 ammonium group may be r e p l a c e d by t^-butyl to give a c t i v e agon-i s t s and a n t a g o n i s t s . The c y c l i c s u b s t i t u e n t s , and R 2, are most commonly phenyl r i n g s . They may be i n c o r p o r a t e d i n t o a p o l y c y c l i c system such as f l u o r e n e , or h e t e r o c y c l i c systems may be used. Fre q u e n t l y , one of the r i n g s i s a l i -c y c l i c C-5 or C-6 or the corresponding unsaturated c a r b o c y c l i c system. A normal a l k y l s u b s t i t u e n t may a l s o be used but t h i s decreases a c t i v i t y . The g r e a t e s t a c t i v i t y i s obtained i n compounds w i t h two c y c l i c s u b s t i t -uents; a t h i r d decreases a c t i v i t y . Large c y c l i c groups such as naphthyl and b i p h e n y l a l s o decrease a c t i v i t y . One c y c l i c group w i l l confer ac-t i v i t y provided an a l k y l or hydroxy1 group i s a l s o present. Compounds w i t h d i f f e r i n g c y c l i c groups are u s u a l l y more a c t i v e and l e s s t o x i c . Normal or branched a l k y l s u b s t i t u e n t s are o f t e n l o c a t e d at the same c a r -bon atom as one or two c y c l i c groups. These groups, R^, R 2, and R^, are f e l t to c o n t r i b u t e to receptor b i n d i n g by hydrophobic or van der Waals b i n d i n g and thus increase the a f f i n i t y for the r e c e p t o r . I f the groups are s u f f i c i e n t l y l a r g e , they may overlap w i t h adjacent r e c e p t o r s and thus give a greater degree of a c t i v i t y provided s t e r i c i n t e r a c t i o n does not prevent b i n d i n g to the r e c e p t o r . The c a t i o n i c head and the c y c l i c moiety are connected by a c h a i n , 28 R^, that must meet certain requirements. For compounds containing an ester function, f i v e atoms must be contained in the chain for maximum a c t i v i t y . These are usually esters of substituted phenylacetic acids with trialkylammoniumethanol. Where the chain consists of an a l k y l group, maximum a c t i v i t y i s attained with the n-propyl function. I t i s thought that the ester compounds interact with the receptor in a skewed s p a t i a l conformation which has a structure similar to that of muscarine 12, while the alkylamines interact in an extended conformation. The distance from the cationic head to the carbon atom on which the c y c l i c function i s substituted i s the same i n both cases. This conformational representation i s shown i n Figure 5. Figure 5: Active conformations of muscarine 12^ , acetylcholine and a n t i -muscarinics (ester (a) and alkylaraine (b) types). (from r e f . 29 Branching of the chain decreases a c t i v i t y . The e s t e r f u n c t i o n i s not necessary for a c t i v i t y although i t may increase b i n d i n g by i n t e r a c t -i n g w i t h the r e c e p t o r . I t may a l s o i n f l u e n c e the conformation of the compound and ..thus i n f l u e n c e the degree of i n t e r a c t i o n of the e s s e n t i a l groups w i t h the r e c e p t o r . The presence of a hydroxyl group on the chain j3 to the carbonyl group of the e s t e r increases a c t i v i t y . Compounds w i t h an cc-hydroxy f u n c t i o n are a l s o a c t i v e . In the aminoalcohols ( i e R^ i s an a l k y l group), optimal a c t i v i t y i s a t t a i n e d when the hydroxy i s on the t h i r d carbon atom from the n i t r o g e n . G e n e r a l l y , hydroxy f u n c t i o n s are l o c a t e d at the same, or the carbon adjacent t o , carbon to which the c y c l i c s t r u c -tures are attached. The hydroxy group may be r e p l a c e d by cyano or p r i -mary carboxamide, but a c t i v i t y i s decreased. Alkoxy and acetamido groups a l s o lower a c t i v i t y . Where a centre of asymmetry i s present, the levo isomer g e n e r a l l y has more a c t i v i t y than the dextro isomer. Although absolute c o n f i g u r a -t i o n s are not g e n e r a l l y known and no s p e c i f i c i n f erences can be drawn from the data, i t does p o i n t out that the muscarinic receptor i s s t e r e o -s e l e c t i v e . 30 DRUG-RECEPTOR INTERACTIONS The mechanism by which homeostasis i s maintained i s dependent on a d e l i c a t e balance between the e f f e c t s of the a c t i v e humoral agents normally present i n the body. In disease s t a t e s t h i s balance i s d i s -rupted and drugs are administered which may mimic or antagonize the i e f f e c t s of one or more of the n a t u r a l agents i n an attempt to r e a t t . i i n . t the n a t u r a l balance. I t i s g e n e r a l l y assumed, f o r drugs which e i t h e r d i r e c t l y mimic or c o m p e t i t i v e l y i n h i b i t the a c t i o n s of normal c o n s t i t -uents of the body, that the drugs act at a " r e c e p t o r " at which the humoral agents are normally a c t i v e . The mechanism by which drugs exert t h e i r e f f e c t i s f o r the most part unknown, although a great num-ber of t h e o r i e s have been proposed. No d i r e c t evidence on the nature of the p o s t u l a t e d r e c e p t o r s i s a v a i l a b l e , but by i n d i r e c t evidence obtained c h i e f l y by the study of the r e l a t i o n s h i p of chemical s t r u c t u r e to a c t i v i t y i t i s p o s s i b l e to des c r i b e the s t e r i c and e l e c t r o n i c requirements of the re c e p t o r s and to t h e o r i z e on the mechanism of drug a c t i o n . Nauta and Harms (30) have noted that r i g i d and s e m i - r i g i d s t r u c -tures such as phenindamine 1_3 and c h l o r c y c l i z i n e 1_4 are h i g h l y a c t i v e a n t i h i s t a m i n i c s , w h i l e ortho s u b s t i t u t e d benzhydryl ethers 1_5 are nea r l y i n a c t i v e as a n t i h i s t a m i n i c s but a n t i m u s c a r i n i c a c t i v i t y i s increased. They have proposed that the a c t i v e conformation of the a n t i -h i s t a m i n i c s i s one i n which the si d e chain i s folded as i n 16. Or tho s u b s t i t u t i o n i n h i b i t s f o l d i n g of the side c h a i n . Studies of models show that n e a r l y a l l of the p r a c t i c a l l y u s e f u l a n t i h i s t a m i n i c s can assume t h i s conformation. They have a l s o proposed that the extended conformer 31 H—0—CH 2—CH 2—N(CH 3) 2 Substitution at 2 2,2' 2,6 2,6,2' 2,6,2'6' 15 17 i s the in h i b i t o r y species at the muscarinic receptor. The distance from the amino nitrogen to the central carbon atom i n 1_7 f a l l s within 32 the range common to the a n t i m u s c a r i n i c s . The concept of a c l o s e f i t at a receptor i s strengthened when the r e l a t i v e a c t i v i t i e s of o p t i c a l antipodes i s considered. Ham (31) has r e c e n t l y shown that appreciable q u a n t i t i e s of the gauche conf ormers are present i n aqueous solutions.- of the s a l t s of s ome an t i h i s t a m i n i cs. Through a s e r i e s of papers, Nauta ejt al_ (24b) have f u r t h e r d e v e l -oped t h e i r receptor theory to the p o i n t where s p e c i f i c anchoring groups have been proposed. Their s p e c u l a t i v e receptor model i s shown i n F i g u r e 6. The minimum requirements of a receptor surface for a n t i h i s -t a m i n i c s , and t h e r e f o r e for histamine, would appear to be an a n i o n i c s i t e to accommodate the b a s i c centre which i s protonated at p h y s i o l o g -i c a l pH, and a f l a t r e g i o n , at a more or l e s s f i x e d d i s t a n c e from the a n i o n i c s i t e , which i s capable of van der Waals b i n d i n g w i t h one of the aromatic r i n g s . The two b i n d i n g s i t e s must be approximately co-planar. Other b i n d i n g models which may be important at the receptor s u r f a c e are London f o r c e s , hydrogen bonds, and hydrophobic bonds. Throughout the work of Nauta et al the concept of complementarity of the h i s t a m i n i c and muscarinic r e c e p t o r s appears. They have found that i n n e a r l y every case an increase i n a n t i h i s t a m i n i c a c t i v i t y i s accompanied by a decrease i n a n t i m u s c a r i n i c a c t i v i t y (23) . This e f f e c t extends to the stereoisomers of the compounds they have prepared such t h a t , i n a s e r i e s of or tho s u b s t i t u t e d compounds, one set of antipodes i s predominantly a n t i h i s t a m i n i c w h i l e the other set i s predominantly a n t i m u s c a r i n i c (32). A s i m i l a r e f f e c t i s seen i n the para s u b s t i t u t e d compounds although the p a t t e r n i s not as r i g i d as i n the or tho s u b s t i t u t e d a b c a) oC-Helix receptor model showing three anchorage s i t e s : 1) s e r i n e -OH; 2) h i s t i d i n e -N 3) phenylalanine -phenyl group. Small c i r c l e s a H; large shaded c i r c l e s • C; large open c i r c l e s a 0; centred c i r c l e s ° N; broken l i n e s • H-bonds. b) Receptor model accommodating histamine. c) Receptor model accommodating 4-methyIdiphenhydramine. (from r e f . 24) 34 s e r i e s . I t appears that Nauta has now abandoned the concept of f o l d e d and extended conformers i n favour of a receptor theory which embodies the concept of complementarity between the a n t i h i s t a m i n i c and a n t i m u s c a r i n i c r e c e p t o r s (32). The e l e c t r o n d e n s i t y on the oxygen atom i s the most c r i t i c a l f a c t o r i n determining the type of a c t i v i t y demonstrated w i t h a p a r t i c u l a r compound and i s i n f l u e n c e d by the manner i n which the aro-matic r i n g s are presented to the receptor s u r f a c e . I t was pointed out that t o p o l o g i c a l l y i d e n t i c a l r e c e p t o r s are not i m p l i e d i n t h i s theory. I t i s f e l t that both r e c e p t o r s have no b u l k t o l e r a n c e to accommodate p r o t r u d i n g para s u b s t i t u e n t s at the b i n d i n g s i t e f o r the aromatic r i n g w h i l e the r e c e p t o r s w i l l accommodate ortho s u b s t i t u e n t s . S u b s t i t u e n t s on the unbound aromatic nucleus a l t e r the e l e c t r o n d e n s i t y on the oxy-gen atom and thus a l t e r the a c t i v i t y of the compound. A balance between a n t i h i s t a m i n i c and a n t i m u s c a r i n i c a c t i v i t y i s thus obtained. In the case of para a l k y l s u b s t i t u e n t s a p o s i t i v e mesomeric e f f e c t increases the overlap i n t e r a c t i o n r e s u l t i n g i n decreased e l e c t r o n d e n s i t y about the oxygen atom and increased a n t i h i s t a m i n i c potency. The ortho a l k y l s u b s t i t u e n t s s t e r i c a l l y i n h i b i t the i n t e r a c t i o n causing decreased a n t i -h i s t a m i n i c e f f e c t w i t h a concomitant increase i n the a n t i m u s c a r i n i c e f f e c t . The p o s i t i v e i n d u c t i v e e f f e c t of or tho a l k y l groups tending to increase the oxygen e l e c t r o n d e n s i t y must a l s o be considered. This concept i s at odds w i t h the p r e v i o u s l y mentioned theory f o r which the receptor s i t e shown i n Figure 6 was proposed. As the e l e c -t r o n d e n s i t y about the oxygen atom decreases, the tendency to form a hydrogen bond would a l s o be expected to decrease, thus lowering the 35 t o t a l b i n d i n g energy of the drug-receptor complex which would r e s u l t i n a decrease i n a c t i v i t y . Nauta's work has been p r i m a r i l y concerned w i t h the e f f e c t of a l k y l s u b s t i t u t i o n i n the aromatic r i n g s of diphenhydramine and i t i s d i f f i -c u l t to determine the a p p l i c a b i l i t y of h i s t h e o r i e s to other s e r i e s of a n t i h i s t a m i n i c s . I t was p r e v i o u s l y s t a t e d that the aromatic r i n g s of a n t i h i s t a m i n i c s should not be coplanar. This c o n c l u s i o n has been reached from s t u d i e s of dimethylaminoethy1 9 - f l u o r e n y l ether _18, which shows only 1% of the a c t i v i t y of diphenhydramine, and of a number of compounds where the two aromatic r i n g s are ortho l i n k e d by one or more atoms, which demonstrate good a n t i h i s t a m i n i c a c t i v i t y . However, 9-(2-dimethylaminoethy1)fluorene 19, synthesized i n t h i s work, shows s p e c i f i c a c t i v i t y of a f a i r l y high order. Two other compounds synthesized i n t h i s work, 9-(2-dimethy1-aminoethyl)carbazole 2_0 and N-(2-dimethylaminoethyl)diphenylamine 21 show comparable a c t i v i t i e s . The c o n t r i b u t i o n to receptor b i n d i n g f o r a f i v e , s i x , or seven-membered a l i c y c l i c r i n g i s expected to be l e s s than that of an aromatic r i n g . The f a c t o r s which i n f l u e n c e the degree to which a r i n g i s bound are the s i z e and p l a n a r i t y of the r i n g and the angle between the sub-s t i t u e n t c h a i n and the plane of the r i n g . In the a z a d i s p i r o compounds synthesized i n t h i s work, the two outec a l i c y c l i c r i n g s are held perpendicular to the c e n t r a l r i n g and can assume a p o s i t i o n coplanar w i t h the s u b s t i t u e n t chain amino f u n c t i o n only at the expense of c o n s i d e r a b l e non-bonded i n t e r a c t i o n . In t h i s coplanar c o n f i g u r a t i o n the i n t e r a t o m i c d i s t a n c e from the terminal n i t r o g e n to 37 the apex carbon of the lactam r i n g i s about 4-4.5 A, which i s i n c l o s e agreement w i t h the corresponding d i s t a n c e i n c h l o r c y c l i z i n e and phenind-amine. The d i s t a n c e from the t e r m i n a l n i t r o g e n to the centre of one of the coplanar r i n g s i s about 5 A w h i l e the d i s t a n c e to the centre of the other r i n g i s about 7 A. Corresponding dis t a n c e s to the centre of e i t h e r r i n g i n c h l o r c y c l i z i n e are 5.5 A and i n phenindamine are 5.5 A. A s i m i l a r d i s t a n c e i s noted i n the folded conformer of the carbazole analogues which were s y n t h e s i z e d . I t would appear from a study of models of a c t i v e a n t i h i s t a m i n i c s 9 and the compounds proposed i n t h i s work that the two s e r i e s of compounds would be u s e f u l t o o l s w i t h which to i n v e s t i g a t e the s t e r i c and e l e c t r o n i c requirements of the a n t i h i s t a m i n i c r e c e p t o r . 3 8 DISCUSSION OF THE CHEMISTRY PART ONE DERIVATIVES AND ANALOGUES OF 7-AMINO-14-AZADISPIRO [5 .1 .5 .2]PENTADECAN-15-ONE The compounds synthesized i n t h i s work are shown i n Table V. Those teste d f or pharmacological a c t i v i t y were 23^, 2 A , 25_, . 2 6 , 2 8 , and 3 0 . The general r e a c t i o n sequence used to synthesize the compounds i s shown i n Scheme I , using the 1 4 - a z a d i s p i r o [ 5 . 1 . 5 . l \ p e n t a d e c a n - 1 5 - o n e s e r i e s as an example. Synthesis of the corresponding 1 6 - a z a d i s p i r o [ 6 . 1 . 6 . 2 ] -heptadecan-17-one d e r i v a t i v e s was accomplished by replacement of c y c l o -hexanone w i t h cycloheptanone i n the f i r s t step of Scheme I . The i n i t i a l d i s p i r o product, 1 4 - h y d r o x y - 1 4 - a z a d i s p i r o [ 5 . 1 . 5 . 2 J -p e n t a d e c - 9 - e n - 7 , 1 5 - d i o n e 7-oxime 31, was f i r s t prepared, although not i d e n t i f i e d , i n 1 9 4 1 by N i g h t i n g a l e , jet al ( 3 3 ) i n 8 . 3 7 , y i e l d by heating nitromethane and cyclohexanone at 1 0 5 ° i n the presence of p i p e r i d i n e or di-n-propylamine c a t a l y s t . Analogous s o l i d products were a l s o ob-tained from 4-methyl- and 3-methylcyclohexanone, but not from 2-methyl-cyclohexanone ( 3 4 ) . Lambert and Lowe ( 3 9 ) obtained the same product us i n g diethylamine as the c a t a l y s t and assigned the formula C - ^ ^ Q ^ O - J . They a l s o i s o l a t e d 1 - n i t r o m e t h y l c y c l o h e x a n o l 3 3 , 1-nitromethylcyclohexene 3 4 , and 1 , l - b i s ( n i t r o m e t h y l ) c y c l o h e x a n e 3 5 from the r e a c t i o n . \ / C H 2 — N02 CH 2 N 0 2 \ ' C H 2 — N 0 2 .J A CHo — N O ' 3 3 3.4... 3 5 39 (CH 2) (CH ) 2 n R l = " N H 2 R 2 = -NH-C0-CH2C1 R 3= -NH-CO-CH 2-N(CH 3) 2 R 4= -NH-CO-CH 2-N(CH 3) 2. MeI Compound No. nucleus 22 23 24 27 28 29 Y Compound Name R^ 7-amino-14-azadispiro [5.1.5 . 2 ]pentadecan-15-one R 2 7-chloroacetamido-14-azadispiro [5.1.5 . 2 ] -pentadecan-15-one R-j 7-dimethylaminoacetamido-14-azadispiro-[ 5 . I . 5 . 2 ] pentadecan-15-one R^ 7-dimethylaminoacetamido-14-azadispiro-[5.1.5 .2]pentadecan-15-one methiodide R^ 8-amino-16-azadispiro [6 .1.6 .2] heptadecan-17-one Ro 8-chloroacetamido-16-azadisp i r o [6.1.6 . 2 ] -heptadecan-17-one R 3 8-dimethylaminoacetamido-16-azadispiro-[6.1.6 .2]heptadecan-17-one R^ 8-dimethylaminoacetamido-16-azadispiro-[6.1.6 .2] heptadecan-17-one methiodide Table V: A z a d i s p i r o compounds synthesized i n t h i s work. 40 (CH,) 2>n 25 B 1 7-dimethylaminoacetamido-14-azadispiro-[5 .1.5 .2] pentadecane 26 C 1 7-dimethylaminoacetamido-14-azadispiro-[5.1.5.2]pentadecane dimethiodide 30 B 2 R3 8-dimethylaminoacetamido-16-azadispiro-[6.1.6.2] heptadecane 31 C 2 R^ 8-dimethylaminoacetamido-16-azadispiro-[ 6.I . 6 . 2 ]heptadecane dimethiodide Table V ( c o n t ' d ) : A z a d i s p i r o compounds synthesized i n t h i s work. 41 Scheme I : Synthesis of d e r i v a t i v e s of 7-amino-14-azadispiro[5.1.5.2]-pentadecan-15-one NOH 1. ^ ^J, benzene 0 + ^ CH 3-N0 2 ^ ( a q ) > H /RaNi V-> Et0H(1007 o) C1CH2-C0C1 H x benzene 22 HN(CH 3) 2 benzene NH—C \ L i A l H 4 * X N THF H 23 N(CH ) 3 2 NH—C \ 25 I 2 N ( C H 3 ) 2 Mel benzene 2/Me I benzene 24 26 42 N i g h t i n g a l e , e_t al_ (34) obtained low y i e l d s of 32. from 2l3i and from 34 i n the presence of secondary amine c a t a l y s t s . Increased y i e l d s (147o) of 3_2 from cyclohexanone were obtained by a d d i t i o n of benzene to the r e a c t i o n mixture to allow a z e o t r o p i c removal of the water formed i n the condensation r e a c t i o n . Nitroethane, nitropropane, and p h e n y l n i t r o -methane d i d not give a s o l i d product w i t h cyclohexanone and no s o l i d was obtained w i t h the use of sodium ethoxide as the c a t a l y s t i n a r e a c t i o n w i t h nitromethane. Cyclopentanone gave an analogous s o l i d product w i t h nitromethane (34). A thorough i n v e s t i g a t i o n i n t o the e f f e c t of the amount and type of c a t a l y s t used was c a r r i e d out by N i g h t i n g a l e , e_t <il_ (35,36,40). In g e n e r a l , p i p e r a z i n e was found to be the best c a t a l y s t f o r the s y n t h e s i s of 3_2, i t s 3 , 1 1 - d i a l k y l d e r i v a t i v e s 3_6, and the analogous compounds (3_7,38_, and 39_ r e s p e c t i v e l y ) prepared from cyclopentanone, cycloheptanone, and cyclooctanone. R = Me Et NOH n-Pr n-Bu s-Bu i - P r t-Bu 36 43 37 38 39 Other c a t a l y s t s used i n c l u d e p i p e r i d i n e , morpholine, 2-methylpiperazine, p y r r o l i d i n e , hexamethylenamine, and diethylamine. Methylamine, t r i e t h y l -amine, tetramethy1ammonium hydroxide, and sodium ethoxide do not c a t a l y z e the r e a c t i o n . A study of the r e a c t i o n s of 3_2 l e d N i g h t i n g a l e , e_t al_ (34) to pro-pose the p a r t i a l s t r u c t u r e (C^H^gNO) -CONHOH, a hydroxamic a c i d . They a l s o suggested the presence of non-conjugated I ^ C — C d and —N- func-t i o n s . The s t r u c t u r e of 32^  was determined i n 1962 by Noland and Sundberg (37) by f u n c t i o n a l group determination and degradation experiments. These experiments are summarized i n Charts 1,2, and 3. These Charts are copied d i r e c t l y from (37a); compounds I and I I i n the Charts correspond to 3_2 and 4, r e s p e c t i v e l y , i n t h i s t h e s i s . In N i g h t i n g a l e ' s proposed formula, h y d r o l y s i s of the hydroxamic a c i d should lead to a c a r b o x y l i c a c i d (C._H. oN0)-C00H. F a i l u r e of a c i d 13 l o c a t a l y z e d e s t e r i f i c a t i o n of the h y d r o l y s i s product IV from I (32) and the low frequencies of i r a b s o r p t i o n bands p o s s i b l y a t t r i b u t a b l e to a c a r b o x y l f u n c t i o n c a s t doubt on the presence of hydroxamic and c a r b o x y l i c a c i d g r o u p s . Chart 1: Determination of the f u n c t i o n a l groups present i n the 2:2 condensation product of cyclohexanone and nitromethane (from (37a)) 4 5 Low pressure hydrogenolysis of the a c i d i c hydroxyl group of IV and the r e l a t e d methoxyl group of IVa were incompatible w i t h the f o r m u l a t i o n of IV as a c a r b o x y l i c a c i d and of IVa as i t s methyl e s t e r . However, the data were c o n s i s t e n t w i t h the presence of a five-membered r i n g N-hydroxy-lactam which was r e s i s t a n t to h y d r o l y s i s ; . An oximino group' would then account f o r the remaining N and 0 atoms i n I and the h y d r o l y s i s i n a c i d to IV. The i r s p e c t r a of I,IV,IVa, and XV a l l were c o n s i s t e n t w i t h the hypothesis that a ketoxime i n I had undergone h y d r o l y s i s to a f i v e mem-bered r i n g ketone. Attempts to convert IV back to I gave i n s t e a d the syn or a n t i stereoisomer Ie. I could be p a r t i a l l y isomerized to Ie by r e f l u x i n g i n xylene and by incomplete o x i d a t i o n of I w i t h aqueous KMnO^. C a t a l y t i c hydrogenation of I and Ie gave I I . M e t h y l a t i o n of I and Ie gave isomeric d e r i v a t i v e s Id and I f r e s p e c t i v e l y which both gave IVa on h y d r o l y s i s , p r oving that the d i f f e r e n c e between I and Ie was i n the stereochemistry of the oxime. In some hydrogenation experiments, r e d u c t i o n of I stopped at an iminolactam ^ ^ 2 2 ^ 0 , I l g , which could be reduced over f r e s h c a t a l y s t to I I . H y d r o l y s i s of I l g gave XV, as d i d o x i d a t i o n of I I . XV could be converted to I I by oximation followed by c a t a l y t i c hydrogenation. This data confirmed that I was an unsaturated, five-membered r i n g ketoxime N-hydroxy lactam. Degradation experiments were then c a r r i e d out on the primary amino-lactam I I (Chart 2) and; on the hydroxyamine V (Chart 3) . Attempted Hofmann degradation of I I , v i a l i d and l i e , l ed instead to a product XIV where hydroxyl had replaced the trimethylammonium group. A s i m i l a r CHART 2 Charts 2 and 3: Degradation experiments on the 2:2 condensation product of cyclohexanone and nitromethane (from (37a)) •e'-er. 47 displacement product I l f was obtained from l i e when the r e a c t i o n was run i n ethylene g l y c o l . Production of the displacement products r a t h e r than o l e f i n i c e l i m i n a t i o n products i n d i c a t e d that the carbon bearing the amino group i n I I was attached to carbons bearing no hydrogen atoms. Hofmann degradation of V,. v i a Vb and X I , gave an epoxydimethylamine XXX v i a an i n t r a m o l e c u l a r displacement r e a c t i o n which i s c h a r a c t e r i s t i c of & -hydroxyamine methiodides. Consequently, the hydroxyl group i n V, which corresponds to the amino group of I I , was assigned a p o s i t i o n b e t a to the amino n i t r o g e n . Assignment of s t r u c t u r e XXX to the epoxy-amine C ^ H ^ Q N O permitted the assignment of s t r u c t u r e s to i t s precursors and the key compounds I I and XV. The s t r u c t u r a l assignment was v e r i -f i e d by the s y n t h e t i c pathway shown i n Chart 4. The s y n t h e t i c sample of XV was i d e n t i c a l to that obtained p r e v i o u s l y from I . As XV had been converted to I I , and since I I was d e r i v a b l e from I by low pressure c a t a l y t i c hydrogenation under m i l d c o n d i t i o n s , i t was assumed that the atomic s k e l e t o n proved to be present i n I I was a l s o present i n I . A c t i o n of strong a l k a l i at 200° on IV gave eyelohexane c a r b o x y l i c a c i d . This cleavage of the p-ketolactam and subsequent h y d r o l y s i s of the N-hydroxyamide proved that the o l e f i n i c double bond was not i n the a l i c y c l i c r i n g attached oC to the carbonyl carbon of the lactam but that i t was i n one of two p o s i t i o n s i n the r i n g attached <X to the lactam n i t r o g e n . Nmr s p e c t r a of IVa and the 3,11-dimethyl analogue showed c l e a r l y that the double bond was i n the 9,10-position. On completion of the s t r u c t u r a l assignment to 32, Noland and Sundberg proposed the mechanism given i n Chart 5 for i t s formation from the s t a r t i n g m a t e r i a l s . The key intermediate LXXI, they f e e l , could H XVe X V Chart 4: An a l t e r n a t e s y n t h e s i s of the reduced 2:2 condensation product of cyclohexanone and nitromethane (from (37a)) Chart 5: A proposed mechanism f o r the formation of the 2:2 condensation product of cyclohexanone and nitromethane (from (37a)) 49 be formed e i t h e r from LXX, which has been i s o l a t e d from a r e a c t i o n where I a l s o formed, or from LXLX. From LXXI there would f o l l o w a s e r i e s of steps, p o s s i b l y i n the order shown, i n v o l v i n g i n t r a m o l e c u l a r oxygen t r a n s f e r , dehydration, and r e d u c t i o n - o x i d a t i o n l e a d i n g to the second key intermediate LXXVII which could isomerize to I . House and Magin (38) independently a r r i v e d at the same atomic s k e l -eton by a d i f f e r e n t and complementary degradative procedure. In t h i s work 32 was prepared i n 47% y i e l d , M.P. 272-274°. The max-imum reported y i e l d was 67%, M.P. 273-274° ( 3 5 ) . A value of 47%, M.P. 263-265.5°, not r e c r y s t a l l i z e d , has a l s o been reported ( 4 0 ) . The c y c l o h e p t y l analogue 3_8 was probably f i r s t s ynthesized by E c k s t e i n , e_t aj. (41) i n 1957. A c r y s t a l l i n e by-product i n the r e a c t i o n of cycloheptanone and nitromethane i n the presence of p i p e r i d i n e c a t a -l y s t was noted, but no p h y s i c a l constants, y i e l d , or elemental a n a l y s i s was r e p o r t e d . N i g h t i n g a l e , et a l (34) attempted to prepare t h i s compound but found that no s o l i d p r e c i p i t a t e d when p i p e r i d i n e or di-n-propylamine were used as the c a t a l y s t . I t was l a t e r found (35) that the compound had been formed but had remained i n s o l u t i o n . A d d i t i o n of a c i d to the r e a c t i o n mixture p r e c i p i t a t e d 3_8 i n 46% y i e l d , M.P. 247-249°. In t h i s work, the use of p i p e r a z i n e as the c a t a l y s t r e s u l t e d i n the p r e c i p i t a -t i o n of the p i p e r a z i n i u m s a l t of 3J3 which was e x t r a c t e d w i t h hot d i l u t e HCI (1:1) to give 3J3 i n 417« y i e l d . The l i t e r a t u r e value u s i n g p i p e r a z i n e as the c a t a l y s t i s 44.37» of the p i p e r a z i n i u m s a l t . The hydrogenation of 3_2 has been reported u s i n g Raney n i c k e l at 2900 p s i , heated to 160° (4240 p s i ) , to give 92% of 4 and u s i n g copper chromium oxide at 70-90° and 3400 p s i to give 85% of 4 ( 3 4 ) . Noland 50 and Sundberg (37a) reported t h i s r e d u c t i o n at two atmospheres (ca 30 p s i ) and room temperature f o r 67 hours over Raney n i c k e l to give 957. of 4. However, they s t a t e d that i n s e v e r a l instances the r e d u c t i o n stopped p a r t i a l l y or completely before s a t u r a t i o n of the carbon-nitrogen double bond, g i v i n g an iminolactam ( I l g i n Chart 2) which could be r e -duced to 4 over f r e s h c a t a l y s t . In t h i s work, the r e d u c t i o n was c a r r i e d out at room temperature over 24 hr at 400-500 p s i to give 867o of 4, M.P. 192-193°. Lower pressures almost always gave the iminolactam. A commercial a c t i v e n i c k e l c a t a l y s t (W.R.Grace, Raney n i c k e l No. 28 a c t i v e c a t a l y s t i n water) was used which may not have been as a c t i v e as the p r e p a r a t i o n used by Noland and Sundberg. The commercial c a t a l y s t was purchased f o r the r e d u c t i o n of a n i t r i l e compound i n other work and was claimed to be l e s s a c t i v e than the usual Raney n i c k e l c a t a l y s t s . J a n i s (6) r e p o r t s the hydrogenation of 32_ over f r e s h l y prepared Raney n i c k e l W-4 at room temperature and 30 p s i to give 4 i n 45.57, y i e l d , M.P. 192-193°. In some instances the iminolactam was obtained. The r e d u c t i o n of 38_ was c a r r i e d out over Raney n i c k e l at 400 p s i and room temperature f o r 24 hr to give 897, of 5, M.P. 188-190°. The chloroacetamido- d e r i v a t i v e s 22 and 2_7 were prepared by adding c h l o r o a c e t y l c h l o r i d e to a benzene s o l u t i o n of 4 or 5 r e s p e c t i v e l y . The r a t i o of a c i d h a l i d e to amine was 1:2. The y i e l d s were 807,, M.P. 286-287° f o r 22 and 847=,, M.P. 210-212° f o r 27. Jan i s (6) r e p o r t s a low y i e l d of 2_7, M.P. 210-212°, us i n g a 1:1 r a t i o of a c i d h a l i d e and amine w i t h an excess (257> over theory) of potassium carbonate as the HCI ac-ceptor. For the r e a c t i o n w i t h 5_, the excess of amine i n the r e a c t i o n mixture could be recovered as the p r e c i p i t a t e d h y d r o c h l o r i d e s a l t . 51 Compound 27_ could be r e c r y s t a l l i z e d from benzene w h i l e 22 was much l e s s s o l u b l e i n r e f l u x i n g benzene and was r e c r y s t a l l i z e d from e t h a n o l . F i l -t r a t i o n of the r e a c t i o n mixture from .22 gave a f i l t e r cake c o n t a i n i n g a mixture of 4 (as the hydrochloride) and 22^. This was e x t r a c t e d w i t h water i n a mortar and p e s t l e and the aqueous suspension was f i l t e r e d . The s o l i d 22 was r e c r y s t a l l i z e d from ethanol w h i l e 4 was recovered by p r e c i p i t a t i o n w i t h NaOH. The recovery was only 50-757, of the excess of 4 used, due i n part to mechanical l o s s e s . S y n t h e s i s of the dimethylaminoacetamido d e r i v a t i v e 23^  was accom-p l i s h e d by adding an excess of dimethy lamine to a suspension of 22^ i n benzene. The y i e l d was 44.57,, M.P. 203-204°, and 53.57, of 22 was r e -covered. The low y i e l d of Z3 i s probably a t t r i b u t a b l e to low s o l u b i l -i t y of 22 i n the r e a c t i o n s o l v e n t . A longer r e a c t i o n time may have markedly improved the y i e l d . This r e a c t i o n was a l s o attempted w i t h ethanol (1007,) as the s o l v e n t . The y i e l d was 537, and no 22^  was recovered. Synthesis of the analogous ^8 was accomplished by a d d i t i o n of an excess of dimethy lamine to a benzene s o l u t i o n of 27_. The y i e l d was 827,, M.P. 173.5-174.5°. The methyl i o d i d e s a l t s of 23_ and 28 were prepared by a d d i t i o n of methyl i o d i d e to a benzene s o l u t i o n of each of the compounds. Compound 24, the methyl i o d i d e s a l t of 23_, was r e c r y s t a l l i z e d from g l a c i a l a c e t i c a c i d , M.P. 160-161°. R e c r y s t a l l i z a t i o n of 29, the methyl i o d i d e s a l t of 2Q, from ethanol (1007,)/anhydrous ether gave two c r y s t a l l i n e forms which could be separated manually. R e c r y s t a l l i z a t i o n of each of the dimorphs gave a mixture of the dimorphs. The next compounds proposed f o r s y n t h e s i s were 7-(2-dimethylamino-52 e t h y l a m i n o ) - 1 4 - a z a d i s p i r o [ 5.1 . 5 . 2 ]pentadecane 40 and 8- ( 2-dimethylamino-ethylamino)-16-azadispiro [6.1.6 .2 ]heptadecane 41. 40 41 However, r e d u c t i o n of 22. w i t h l i t h i u m aluminum hydride gave a par-t i a l l y reduced m a t e r i a l whose i r spectrum showed one carbonyl group. Elemental analyses were c o n s i s t e n t w i t h the isomeric s t r u c t u r e s 42 and 25. 2 Reduction of 28 w i t h l i t h i u m aluminum hydride gave a s i m i l a r compound w i t h elemental analyses c o n s i s t e n t w i t h the isomers 43 and 30. 53 2 43 30 Reduction of ^ 8 w i t h diborane i n te t r a h y d r o f u r a n a l s o gave the p a r t i a l l y reduced compound. Attempted r e d u c t i o n of the p a r t i a l l y reduced compound 43 or 30 over copper chromium oxide at 1320 p s i and 220° was un s u c c e s s f u l Although N i g h t i n g a l e , et_ a l (34,35,36), Noland and Sundberg (37) and House and Magin (38) make reference to l i t h i u m aluminum hydride r e -du c t i o n of 44 and/or 45 to give the aminoalcohol 4£, they do not mention attempted r e d u c t i o n of 4. 54 H OH 46 Reduction of 4 and 5_ was attempted i n t h i s work to determine whether the lactam or the a c y c l i c amide had been reduced i n the sets of isomers 42-25 and 43-30- A s o l u t i o n of U was added to a suspension of l i t h i u m aluminum hydride i n t e t r a h y d r o f u r a n and was r e f l u x e d f o r 48 hours. The expected 7-amino-14-azadispiro[5.1.5.2j pentadecane 47 was not obtained. L i k e w i s e , a s o l u t i o n of 5_ added to a suspension of l i t h i u m aluminum hydride i n t e t r a h y d r o f u r a n and r e f l u x e d f o r 40 hours d i d not g i v e the expected 8-amino-16-azadispiro[6.1.6.2 ]heptadecane 48. Only s t a r t i n g m a t e r i a l was i s o l a t e d from these r e a c t i o n s . 47 48 55 H y d r o l y s i s of the remaining amide bond i n the isomer p a i r 43-30 was then attempted i n 30% s u l f u r i c a c i d . Only s t a r t i n g m a t e r i a l was r e -covered from t h i s r e a c t i o n . Noland and Sundberg (37a) r e p o r t the h y d r o l y s i s of 7-imino-14-azadispiro [5 .1.5.2]pentadecan-15-one 49 i n s u l f u r i c acid:ethanolrwater (1:2:3) f o r 20 hr to g i v e the 7-keto com-pound 44 i n 48% y i e l d . They do not mention formation of a product where the lactam bond had hydrolyzed. N H 49 On the b a s i s of t h i s evidence, and a degree of b i a s on the part of the experimenter, the p a r t i a l l y reduced compounds were assigned s t r u c t u r e s 42 and 43. The methyl i o d i d e and hydr o c h l o r i d e s a l t s of these two compounds were formed and pharmacological t e s t i n g was c a r -r i e d out. However, during the w r i t i n g of t h i s t h e s i s , a review of the spec-t r a l data i n d i c a t e d that the two compounds were 7-dimethylaminoacet-amido-14-azadispiro [5 .1.5 .2] pentadecane 2_5 and 8-dimethy laminoacet-amido- 16-azadispiro [ 6 . I . 6 . 2 ] heptadecane 30. The presence of an i r absorption band at 1510-1520 cm which may be an Amide I I band, i n d i c a t e d that the products of the r e d u c t i o n r e a c t i o n s were Z5 and 30 s i n c e lactams c o n t a i n i n g l e s s than nine atoms 56 i n the r i n g do not show an Amide I I band (42). The p o s i t i o n f o r the Amide I band of a five-membered lactam i s c_a 1700 cm ^ w h i l e that f o r an a c y c l i c secondary amide i s c_a 1680 cm ^ i n the f r e e s t a t e and ca 1655 cm ^ i n the a s s o c i a t e d s t a t e (42) . The spectrum of 23_ has absorp-t i o n bands at 1660 and 1700 cm ^ w h i l e that of ^8 has a b s o r p t i o n bands at 1680 and 1695 cm ^. In the reduced products, the one a b s o r p t i o n band remaining i n t h i s area i s at 1680 cm ^ f o r the product from 23 and at 1670 cm ^ f o r the product from 2*3. This data a l s o i n d i c a t e d that the lactam r e d u c t i o n products Z5 and 30 had been obtained. The nmr spectrum of 2JS_ has two downfield s i g n a l s ( = 7.78 and 6.92) corresponding to the two amide protons Hfl and r e s p e c t i v e l y . The s i g n a l at £ = 7.78 i s a broad 1:1 doublet, J = 10Hz. There i s a 1:1 doublet, J = 11Hz, at & = 4.34 corresponding to H c, and sharp s i n g l e t s at & = 3.06 (H d) and at 6 = 2.38 (N-methyl p r o t o n s ) . 2 An envelope band centred at 6 = 1.59 accounts for the protons i n the a l i c y c l i c r i n g s . A f t e r a d d i t i o n of D^O the spectrum was e s s e n t i a l l y the same except that the 1:1 doublet at S = 4.34 became a 1:0.75:0.85 t r i p l e t , J = 5.5Hz, centred at 5 = 4.34. I t appears that p a r t i a l 57 exchange of the amide protons (H f l and H^) has occurred, g i v i n g r i s e to a mixture of two species. In that species where H has exchanged, H appears as a s i n g l e t which appears at the same chemical s h i f t as does the doublet due to the species where H a has not exchanged. The s i g n a l to noise r a t i o i n the s p e c t r a d i d not allow q u a n t i t a t i o n of the degree of exchange but the i n t e g r a l over the re g i o n i n which Hfl and absorb had decreased by approximately 50%. The nmr spectrum of the r e d u c t i o n product has a poorly d e f i n e d s i g n a l at 6 = 7.3 to 7.7, and a 1:1 doublet at <5 = 3.85, J = 11Hz, i n i t i a l l y assigned to Hfl and r e s p e c t i v e l y i n 43. 2 43 30 Sharp s i n g l e t s at cS = 3.00 and 2.74 were assigned to the two p a i r s of ethylene protons, and sharp s i n g l e t s at S - 2.36 and 2.10 were assigned to the N-methyl protons and H r e s p e c t i v e l y . A f t e r a d d i t i o n of D„0, the s i n g l e t at 6 = 2.10 disappeared and the doublet at 0 = 3.85 c o l -lapsed to a s i n g l e t at S = 3.76. The e n t i r e spectrum had s h i f t e d up-f i e l d by 5-6Hz so that the chemical s h i f t of t h i s s i n g l e t was a c t u a l l y 3.85 which corresponds to the value of the doublet i n the spectrum be-58 f o r e exchange. No e x p l a n a t i o n was o f f e r e d for the l a c k of a t r i p l e t p a t t e r n for the two s i g n a l s assigned to the ethylene protons. The re-examination of the s p e c t r a which revealed the u p f i e l d s h i f t a l s o i n d i c a t e d that the amide proton had exchanged. The s i g n a l to n oise r a t i o i n the r e g i o n of absorption of the amide proton d i d not a l l o w q u a n t i t a t i o n of t h i s exchange although the a b s o r p t i o n s i g n a l was much reduced. The s p e c t r a were f u r t h e r confused by the presence of an ab-s o r p t i o n s i g n a l at 8 = 7.3 (±0.05) due to chloroform impurity present i n the deuterchloroform used as s o l v e n t . Although amide protons do not undergo r a p i d chemical exchange (77a) no other hypothesis explained a l l of the data. The s p e c t r a were then r e a d i l y assigned to _30. The poorly defined downfield s i g n a l was seen to be a doublet, J = 8-12Hz, and was assigned to H^, the doublet at c5 = 3.85 to H c, the two sharp s i n g l e t s at & = 3.0 and 2.74 to H g and r e s p e c t i v e l y , and the s i n g l e t at & -2.10 to the amine proton H . r a The exchange of the amide protons i n 23 and J25 was found to be time dependent. Immediately a f t e r a d d i t i o n of D2O, the protons were about 50% exchanged. The a c y c l i c amide proton i n 23_ was more r e a d i l y exchanged than the lactam proton. A f t e r 16 hr at room temperature ex-change of the three amide protons i n 23^ and Z5 was n e a r l y complete. In the r e d u c t i o n of a carbonyl group by l i t h i u m aluminum hydride, a hydride ion i s t r a n s f e r r e d to the carbonyl carbon (43). I t i s there-f o r e p o s s i b l e to account f o r the l a c k of r e d u c t i o n of 4 and 5 w i t h l i t h i u m aluminum hydride, w h i l e 4_5 does reduce, on the b a s i s of s u b s t i t -uent e f f e c t s . The amino group ( o-^  = 0.10) i s much l e s s e l e c t r o n w i t h -drawing than the hydroxyl group ( Ot = 0.25) (44) and t h e r e f o r e w i l l 59 not s t a b i l i z e the intermediate formed i n the r e d u c t i o n of the carbonyl group i n 4 and j> as w e l l as the hydroxyl group i n 45. A c e t y l a t i o n of e i t h e r f u n c t i o n a l group increases the e l e c t r o n withdrawing e f f e c t of the s u b s t i t u e n t . The value for the acetamido group i s cT^.=0.28 which i s i n the range of the value for the hydroxyl group (44). I t would there f o r e be expected that the e f f e c t of the acetamido and, by exten-s i o n , the dimethylaminoacetamido, f u n c t i o n would be s i m i l a r to that of the hydroxyl group, i e the r e a c t i o n would proceed to give the secondary amine from the lactam. No e x p l a n a t i o n f o r la c k of r e d u c t i o n or h y d r o l y s i s of the a c y c l i c amide comes to mind. 60 PART TWO DERIVATIVES AND ANALOGUES OF CARBAZOLE The compounds which were synthesized i n t h i s s e r i e s are shown i n Table V I . A l l of the compounds i n Table VI were tested f o r a n t i h i s t a -minic and a n t i m u s c a r i n i c a c t i v i t y . A t y p i c a l s y n t h e s i s i s shown i n Scheme I I . The i n i t i a l o b j e c t of t h i s work was the s y n t h e s i s of a p p r o p r i a t e l y s u b s t i t u t e d d e r i v a t i v e s of dodecahydrocarbazole 49 and the compounds wherein the two a l i c y c l i c r i n g s fused to the a z o l i d i n e r i n g had been v a r i e d to c o n t a i n f i v e and seven carbon atoms, as i n 50 and 5_1. Com-pounds c o n t a i n i n g i s o l a t e d and conjugated double bonds were a l s o to be syn t h e s i z e d . H H H 49 50 51 A search of the l i t e r a t u r e revealed only one method of p r e p a r a t i o n of 49 which may have been a p p l i c a b l e to the sy n t h e s i s of 50 and 51. Jager, et. a_l (45) have reported a product, which they have i d e n t i f i e d as 49, from the r e a c t i o n of cy c l o h e x y l i d e n e cyclohexanone w i t h form-amide i n ethylene g l y c o l . This s y n t h e s i s i s an example of the Leuckart-Table VI: D e r i v a t i v e s and Analogues of Carbazole synthesized i n t h i s work. R = -CH 2-CH 2— N(CH 3) 2 Compound Compound Number Name 52 9-(2-dimethylaminoethyl)carbazole 53 9-(2-dimethylaminoethy1)-1,2,3,4-tetrahydrocarbazole 54 9-(2-dimethylaminoethyl)dodecahydrocarbazole 55 4-(2-dimethylaminoethyl)-l,2,3,4-tetrahydrocyclopent[b]indole 56 4-(2-dimethylaminoethyl)dodecahydrocyclopent[b]indole 57 5-(2-dimethylaminoethy1)-5,6,7,8,9,10-hexahydrocyclohept[b]indole 58 5-(2-dimethylaminoethyl)tetradecahydrocyclohept [b]indole 59 9-(2-dimethylaminoethy1)fluorene 60 9-(2-dimethylaminoethy1)-1,2,3,4,4a,9a-hexahydrofluorene 61 9,9-bis(2-dimethylaminoethyl)fluorene 62 N-(2-dimethylaminoethyl)diphenylamine 63 N-(2-dimethylaminoethyl)dicyclohexylamine Table VI: (cont'd) D e r i v a t i v e s and Analogues of Carbazole synthesized i n t h i s work. S i Scheme I I : Synthesis of 9-(2-dimethylaminoethy1)-1,2,3,4-tetrahyd ca r b a z o l e . 64 Wallach r e a c t i o n , which i n v o l v e s the r e d u c t i v e a l k y l a t i o n of ammonia or primary or secondary amines w i t h carbonyl compounds i n the presence of formic a c i d which acts as the reducing agent. In a review, Moore (46) s t a t e s that the method i s u n s u i t a b l e f or a p p l i c a t i o n to OC,p -un-saturate d ketones, because of the formation of resinous by-products. The l i q u i d product, b 1 2 130-132°, obtained by Jager, et_ a l (45) i n 32% y i e l d , formed a h y d r o c h l o r i d e , M.P. 265-268°; a p i c r a t e , M.P. 186°; an e t h y l i o d i d e , M.P. 187°; and a benzoyl d e r i v a t i v e , M.P. 179°. Elemen-t a l analyses were c a r r i e d out on the h y d r o c h l o r i d e ( C I ) , the e t h y l i o -dide ( I ) , and the benzoyl d e r i v a t i v e (C,H,N). The values obtained by v a r i o u s workers f o r the m e l t i n g and b o i l i n g p o i n t s of dodecahydrocarbazole and some of i t s d e r i v a t i v e s are shown i n Table V I I . Adkins and Coonradt (47) had reported the high pressure c a t a l y t i c hydrogenation of c a r b a z o l e , o b t a i n i n g 83-87%, y i e l d of 49 as a l i q u i d , b^Q 124-125°, or as a s o l i d , M.P. 73-74.5°, w i t h the same b o i l i n g range The p i c r a t e of the s o l i d was prepared, M.P. 167-168°. Elemental a n a l -yses (C,H) were c a r r i e d out on the h y d r o c h l o r i d e s , M.P. 208-209°. P e r k i n and Pla n t (48) had p r e v i o u s l y reported the s y n t h e s i s of 49, M.P. 65°, i n "very good" y i e l d by the e l e c t r o l y t i c r e d u c t i o n of 1,2,3,-4,5,6,7,8-octahydrocarbazole. Elemental analyses (C,H) were c a r r i e d out on the s o l i d f r e e base. The p i c r a t e was a l s o prepared, M.P. 187°. Masamune, e_t a_l (49) hydrogenated cis-hexahydrocarbazole and o c t a -hydrocarbazole over platinum i n a c e t i c a c i d to give 49, b^ ,_ 134-136°; h y d r o c h l o r i d e , M.P. 209-211°, and 49; h y d r o c h l o r i d e , M.P. 208-210° r e s p e c t i v e l y . The p i c r a t e was a l s o prepared, M.P. 175-177°. 65 dodecahydro-carbazole h y d r o c h l o r i d e p i c r a t e benzamide Jager, et a l (45) b 130-132° 265-268° 186° 179° Adkins and Coonradt (47) b 1 Q 124-125° M.P. 73-74.5° 208-209° 167-168° P e r k i n and Pl a n t (48) M.P. 65° 187° Masamune, et a l (49) b 134-136° 209-211° 208-210° 175-177° This work a b b ~ „ 62° 0.2 M.P. 38.5-39° 255-257°d 175-176° M.P. 75.5-76.5° 211.0-212.0° 168-168.5° 130.5-131.7° a - constants f o r the u n i d e n t i f i e d product obtained by the method of Jager, £t al_ (45). b - constants f o r the product obtained from high pressure hydrogenation of c a r b a z o l e . Table V I I : M e l t i n g and b o i l i n g p o i n t s of dodecahydrocarbazole and i t s d e r i v a t i v e s obtained by v a r i o u s workers 66 A f t e r a c o n s i d e r a t i o n of the above data i t seemed d o u b t f u l that Jager, e_t a_l had obtained the reported compound. However, a number of ge o m e t r i c a l isomers of 49 e x i s t w i t h respect to the c o n f i g u r a t i o n about the bonds l a b e l l e d a, b, and c. There are two i n t e r n a l l y compensated isomers: c i s - s y n - c i s and t r a n s -syn-trans and four p a i r s of d i a s t e r e o i s o m e r s : t r a n s - s y n - c i s ; c i s - a n t i -c i s ; t r a n s - a n t i - c i s ; and t r a n s - a n t i - t r a n s . G e n e r a l l y , c a t a l y t i c reduc-t i o n s occur to give the c i s o r i e n t a t i o n about the reduced bond, although trans hydrogenation does occur (50) . The expected products from the c a t a l y t i c hydrogenation of carbazole are t h e r e f o r e the c i s - s y n - c i s i s o -mer and a l e s s e r q u a n t i t y of the c i s - a n t i - c i s isomer. The e l e c t r o l y t i c r e d u c t i o n of P e r k i n and P l a n t and the s y n t h e t i c route of Jager, -et a l would be expected, from f i r s t c o n s i d e r a t i o n s , to lead predominantly to the t r a n s - a n t i - t r a n s isomer. However, a study of models i n d i c a t e s that the c i s - s y n - c i s or the c i s - a n t i - c i s isomers are l e s s s t r a i n e d than the expected t r a n s - a n t i - t r a n s isomer. An i n v e s t i g a t i o n of the isomers of l,2,3,4,4a,9a-hexahydrocar-bazole 6!? (51) revealed that r e d u c t i o n of 1,2,3,4-tetrahydrocarbazole 64 w i t h t i n and h y d r o c h l o r i c a c i d gave l e s s than 2% of the trans isomer. The s t r u c t u r a l assignment was not r i g i d l y proven but was made on the b a s i s of a c o n s i d e r a b l e amount more s t r a i n i n the trans than the c i s 67 isomer, as demonstrated w i t h the a i d of models. 65 66 Other examples of s t r a i n e d trans fused r i n g systems were mentioned by the authors (51). A s i m i l a r r e l a t i o n s h i p may be noted i n models of 1,2,3,4,4a,5,6,7,8,9a-decahydrocarbazole, the expected intermediate i n the e l e c t r o l y t i c r e d u c t i o n of octahydrocarbazole, as w e l l as i n dodeca-hydrocarbazole. Booth, e_t al (52) a l s o suggest the c i s - f u s e d r i n g s are present i n the dihydrogenated products f o l l o w i n g r e d u c t i o n of t e t r a h y -drocarbazole and l , 2 , 3 , 4 - t e t r a h y d r o c y c l o p e n t [ b ] i n d o l e j56 over Raney n i c k e l . During the course of t h i s work, s e v e r a l attempts were made to pre-pare 49 by the method of Jager, e_t al (45). Cyclohexylidene cyclohex-anone was prepared by the method of Gault, e_t a l (53) to give a l i q u i d , b l 5 1 0 5 - 1 0 8 3 ( l i t e r a t u r e b l g _ 2 o 150-155°) i n v a r i a b l e y i e l d depending on the r e a c t i o n c o n d i t i o n s . The y i e l d r e p o r t e d i n the l i t e r a t u r e was 837» based on the amount of cyclohexanone consumed i n the r e a c t i o n . The y i e l d s of c y c l o h e x y l i d e n e cyclohexanone obtained under v a r i o u s c o n d i t i o n s are summarized i n Table V I I I . The low y i e l d s obtained may have been due to the use of a somewhat too concentrated s u l f u r i c a c i d 68 Reaction Weight (g) cyclohexanone Reaction Temperature Reaction Time (hr) Y i e l d <%) A 100 R.T. 24 27 5 B 500 R.T. 24 7 4 C 100 R.T. 5 38 D 100 0° a 24 35 6 E 165.5 b R.T. 3.5 45 F 100 C R.T. 5 36 G 200 d R.T. 5.5 30 Table V I I I : Y i e l d s of c y c l o h e x y l i d e n e cyclohexanone obtained under v a r i o u s r e a c t i o n c o n d i t i o n s . Reaction A was separated by e x t r a c t i o n w i t h ether and the ether s o l u t i o n was washed w i t h water. Reaction B was steam d i s t i l l e d a f t e r d i l u t i o n w i t h two volumes water. Reactions C-G were quenched by the a d d i t i o n of two volumes water and were e x t r a c t e d w i t h ether. a a f t e r 24 hours very l i t t l e upper layer had separated; the r e a c t i o n was allowed to warm to room temperature over seven hours and was worked up as above. b cyclohexanone recovered from previous r e a c t i o n s . c two p o r t i o n s of 50 g each were c e n t r i f u g e d at 1200 rpm, mixed, and worked up as above, d two p o r t i o n s of 100 g each were c e n t r i f u g e d at 1400 rpm, mixed, and worked up as above. 69 s o l u t i o n , s i n c e Gault, e_t a l s t a t e d that concentrations i n excess of 607» caused a decrease i n y i e l d , w h i l e more d i l u t e a c i d s o l u t i o n s (20, 30,40 and 507») possessed only " f e e b l e " c a t a l y t i c a c t i v i t y . Jager (54) prepared c y c l o h e x y l i d e n e cyclohexanone i n an average 707. y i e l d by r e f l u x i n g cyclohexanone w i t h p_-toluenesulfonic a c i d i n benzene and removing water produced i n the r e a c t i o n by a z e o t r o p i c d i s -t i l l a t i o n . He s t a t e d that the compound r e a d i l y isomerized to 2 - ( c y c l o -hexen-l-yl)cyclohexanone which does not r e a c t i n the same manner as the d e s i r e d compound. Attempts to condense c y c l o h e x y l i d e n e cyclohexanone w i t h formamide i n ethylene g l y c o l gave only u n i d e n t i f i e d products. A s o l i d , M.P. 38.5-39°, was obtained from the d i s t i l l a t e , bg ^ 62°, of the product from the r e a c t i o n . The Hinsberg procedure i n d i c a t e d that the s o l i d was a secondary amine. A s o l u t i o n of the s o l i d i n acetone spontaneously isomerized to a mixture of two m a t e r i a l s . The i s o m e r i z a t i o n could be followed by vapour phase chromatography. On evaporation of the acetone the s o l i d , contaminated w i t h a small amount of the other component of the mixture, could be recovered. The nmr spectrum of the s o l i d m a t e r i a l had a s i g n a l at 6* = 5.40 which may have been due to a v i n y l i c proton, and a broad, poorly defined a b s o r p t i o n s i g n a l centred at <S = 1.16. The spectrum showed no exchangeable proton although the amine proton s i g n a l may have been l o s t i n the <S = 1.16 s i g n a l . The h y d r o c h l o r i d e s a l t , M.P. 255-257°d, and the benzoyl d e r i v a t i v e , M.P. 175-176°, were prepared. No u s e f u l data could be d e r i v e d from the nmr i n t e g r a t i o n curve or the i r spectrum. Elemental analyses c a r r i e d out on the s o l i d and the 70 hydrochloride indicated a mixture of products even through vapour phase chromatography of the s o l i d i n i t i a l l y showed only one peak. The mass spectrum also indicated a mixture of products, one of which may have had a molecular weight of 179 or 180. The l i q u i d d i s t i l l a t e from the reaction was a less pure f r a c t i o n of the same material. Although the i r spectra of the s o l i d and l i q u i d materials were i d e n t i c a l , they varied markedly from the spectrum of a reference sample of 4_9 prepared by high pressure hydrogenation of car-bazole. Following f a i l u r e to obtain 49 by a chemical synthesis, the approach to the problem v i a derivatives of 49, 5_0, and 51_ was abandoned i n favour of an approach v i a the indole derivatives 6_7, where x = 1, 2, or 3. The f u l l y hydrogenated derivatives of these compounds, as w e l l as the CH 2 CH, I N ( C H 3 ) 2 67 aromatic and s a t u r a t e d carbazole and fluorene analogues, were a l s o to be s y n t h e s i z e d . Four of the object compounds--52, 53, 5_7, and 62--as w e l l as the i n d o l e bases 64, j36, and 68 have been p r e v i o u s l y r e p o r t e d , p r i m a r i l y i n the patent l i t e r a t u r e . T e s t i n g of the reported compounds has been f o r psychotropic a c t i v i t y and. f.o.r a n t i h i s t a m i n i c . a c t i v i t y . 71 The s y n t h e s i s of 9-(2-dimethylaminoethyl)carbazole 5_2_ was accom-p l i s h e d by the r e a c t i o n of 2-dimethylaminoethyl c h l o r i d e h y d r o c h l o r i d e w i t h the carbazole anion, prepared by a d d i t i o n of a s o l u t i o n of carba-z o l e i n dimethylformamide to an equimolar q u a n t i t y of sodium methoxide i n anhydrous methanol. The y i e l d a f t e r p u r i f i c a t i o n by reduced pressure s u b l i m a t i o n was 35%, M.P. 40.5-41°. This compound has been reported i n the patent l i t e r a t u r e (55) i n 36% y i e l d from the r e a c t i o n of carba-z o l e w i t h 2-dimethylaminoethy1 c h l o r i d e i n dimethyl s u l f o x i d e using aqueous sodium hydroxide (50%) as the base. The m e l t i n g p o i n t of the compound was reported to be 240°; t h i s was o b v i o u s l y the h y d r o c h l o r i d e s a l t which was found to melt at 243-244.5° i n t h i s work. Another r e -ported (56) s y n t h e s i s of J32 i n v o l v e d the r e a c t i o n of carbazole w i t h sodium amide i n dry benzene fo l l o w e d by the a d d i t i o n of 2-dimethylamino-e t h y l c h l o r i d e to g i v e 60% of 52, b^_ 6 190-210°, hy d r o c h l o r i d e M.P. 240-242°d. At that time the product was t e s t e d f o r a n t i h i s t a m i n i c a c t i v i t y and was found to be i n e f f e c t i v e i n guinea pigs when administered i n t r a -p e r i t o n e a l l y i n doses of 25 mgm/kgm. This compound was a l s o found to be i n a c t i v e i n preventing dextran-induced edema or d e p o s i t i o n of I n d i a i n k carbon p a r t i c l e s at s i t e s of edema caused by histamine or dextran i n r a t s (57,58). Tetrahydrocarbazole 64, 5,6,7,8,9,10-hexahydrocyclohept rb]indole 68, and l , 2 , 3 , 4 - t e t r a h y d r o c y c l o p e n t [ b ] i n d o l e 66^  were prepared by the F i s c h e r i n d o l e s y n t h e s i s . The F i s c h e r i n d o l e s y n t h e s i s i n v o l v e s the e l i m i n a t i o n of ammonia from the arylhydrazone of a ketone or aldehyde w i t h formation of an i n d o l e nucleus. The r e a c t i o n i s c a r r i e d out i n the presence of a 72 64 66 68 c a t a l y s t , which may be an a c i d or a metal or metal s a l t . I t i s not necessary to i s o l a t e the arylhydrazone. The mechanism of the r e a c t i o n , shown i n Chart 6, may be considered to c o n s i s t of three e s s e n t i a l l y sep-arate stages: a) hydrazone-enehydrazine e q u i l i b r i u m ( 6 9 5 ^ 7 0 ) ; b) f o r -mation of the new carbon-carbon bond ( 7 0 — ^ 7 1 ) ; and c) e l i m i n a t i o n of ammonia by e i t h e r of two p o s s i b l e routes to give the i n d o l e compound 72. There i s good experimental evidence, which has been reviewed (59, 60), to support steps a and b of the above mechanism and the e l i m i n a t i o n 2 of N of the arylhydrazone, although the mechanism of e l i m i n a t i o n i s s t i l l i n doubt. Further d e t a i l e d k i n e t i c s t u d i e s of the F i s c h e r i n d o l e s y n t h e s i s , under v a r i o u s c o n d i t i o n s of c a t a l y s i s , are needed to f u r t h e r i n v e s t i g a t e the mechanism and to determine the r a t e c o n t r o l l i n g step which may vary w i t h the experimental c o n d i t i o n s (60). Tars are o f t e n formed i n the F i s c h e r i n d o l e s y n t h e s i s and i t has been suggested (59) that i n some cases these m a t e r i a l s a r i s e from the para-rearrangement of the s u b s t i t u t i o n on the a r y l nucleus since the products.so formed would be unstable under the- r e a c t i o n c o n d i t i o n s . 73 Chart 6: Mechanism of the F i s c h e r i n d o l e s y n t h e s i s (from r e f . 59) 74 In the synt h e s i s of 64 and 681, r e f l u x i n g the appropriate a l i c y c l i c ketone w i t h an equimolar q u a n t i t y of phenylhydrazine i n g l a c i a l a c e t i c a c i d gave 66%, M.P. 113-117°, and 74%, M.P. 142.5-143.5°, r e s p e c t i v e l y , of the d e s i r e d compounds. The l i t e r a t u r e values for j64 are 827», M.P. 116-117° (61) and for 68 are 74%, M.P. 142-144° (62). Synthesis of ji6 was accomplished by s t i r r i n g cyclopentanone phenyl-hydrazone, prepared from equimolar q u a n t i t i e s of cyclopentanone and phenylhydrazine, i n d i l u t e aqueous s u l f u r i c a c i d w h i l e heating below the b o i l i n g p o i n t of the mixture. I t was not necessary to p u r i f y the phenylhydrazone before c a r r y i n g out the c y c l i z a t i o n . The y i e l d was 41.5%, M.P. 106.5-107.5°. The l i t e r a t u r e values were 45%, M.P. 108-109° (63). Both cyclopentanone phenylhydrazone and 66 were unstable i n a i r and l i g h t , the r a t e of decomposition being increased by heat. The use of s t r o n g l y a c i d i c media i n the c y c l i z a t i o n step of the r e a c t i o n r e s u l t e d i n extensive degradation of the compound to t a r s . P e r k i n and P l a n t (63) s t a t e that the use of h y d r o c h l o r i c or a c e t i c a c i d s , or s u l -f u r i c a c i d s o l u t i o n s more concentrated than 20 ml i n 360 ml water leads to c o n s i d e r a b l e h y d r o l y s i s of the hydrazone and there i s a tendency to the formation of much t a r . Witkop, e_t a_l (64) found that 66 was unstable i n polar s o l v e n t s , decomposing to the lactam 7_4 presumably v i a the 8b-hydroperoxide 73. 0 H H 73 74 75 The s i x - and seven-membered r i n g homologues 6^4 and 68^ were s t a b l e i n s o l u t i o n but homologous products were obtained when the s o l u t i o n s were c a t a l y t i c a l l y oxygenated (64). The 4a-hydroperoxide of 64 has been i s o l a t e d . The t e s t compounds _53, 5_5, and 5_7 were then prepared by r e a c t i o n of the anions of 64, 6_6, and _68, r e s p e c t i v e l y , w i t h 2-dimethylaminoethyl bromide hydrobromide or w i t h 2-dimethylaminoethyl c h l o r i d e . A d d i t i o n of 64- i n dimethyIformamide to a s o l u t i o n of sodium ethoxide i n ethanol (1007.) and subsequent a d d i t i o n of 2-dimethylaminoethyl bromide hydro-bromide, d i s s o l v e d i n dimethyIformamide, gave 36.5% of 53, b^ 2 155-160°, hy d r o c h l o r i d e M.P. 244.5-246.0°d. The r a t i o of £4 to the bromide s a l t was 2:1 and 707o of the excess of 64 was recovered. The l i q u i d product p a r t i a l l y c r y s t a l l i z e d on standing and was p u r i f i e d by reduced pressure s u b l i m a t i o n , M.P. 47.5-48.5°. Compound 53 has been p r e v i o u s l y s y n t h e s i z e d , the h y d r o c h l o r i d e r e -ported as M.P. 243-244° (66,67) and 248-250° (68). Y i e l d s were not quoted. No b i o l o g i c a l data were reported although the two patents (66, 67) c l a i m t h i s compound, among o t h e r s , to be u s e f u l as an intermediate i n the manufacture of dyes and pharmaceuticals, p a r t i c u l a r l y a n t i h i s t a -minics and antispasmodics. A d d i t i o n of 618, d i s s o l v e d i n benzene, to a suspension of sodium amide i n benzene and subsequent a d d i t i o n of 2-dimethylaminoethyl c h l o r i d e gave 597, of 57, b Q 5 154-158°, h y d r o c h l o r i d e M.P. 226.5-228.5°. The r a t i o of j>8 to h a l i d e was 2:1 and 407, of the excess was recovered. Compound 5_7 has been p r e v i o u s l y synthesized (69) from the r e a c t i o n of £8 w i t h 2-dimethylaminoethyl c h l o r i d e i n dimethyIformamide using sodium 76 hydride. No y i e l d was quoted, 131-136°. The patent claims a s e r i e s of compounds f o r use i n the treatment of depression although no s p e c i f i c b i o l o g i c a l data was gi v e n . Although 64 and 618 f a i r l y r e a d i l y formed the anion necessary f o r the above r e a c t i o n s to occur, the s y n t h e s i s of 5_5 was much more d i f f i -c u l t due to the inherent i n s t a b i l i t y of 66_. Compound 66^ was reasonably s t a b l e i n the c r y s t a l l i n e s t a t e , although decomposition was a c c e l e r a t e d i n a i r and probably by l i g h t . R e c r y s t a l l i z a t i o n of the compound could be accomplished from petroleum ether, a non-polar s o l v e n t , but heating a s o l u t i o n of 66^  i n a p o l a r s o l v e n t l e d to extensive degradation. The sodium d e r i v a t i v e of 66^ was prepared by very vigourous s t i r r i n g of sodium metal w i t h a xylene/ether (4:1) s o l u t i o n of 66. A d d i t i o n of dimethyl-aminoethyl bromide hydrobromide i n dimethy I f or mamide gave 17.57, of 55, b Q 5 136.5-139.5°, p i c r a t e M.P. 198.0-198.5°. The h y d r o c h l o r i d e was unstable i n ethanol s o l u t i o n and could not be s u c c e s s f u l l y r e c r y s t a l -l i z e d . The y i e l d might have been improved i f 2-dimethylaminoethy1 c h l o r i d e had been used as the a l k y l a t i n g agent, e i t h e r neat or i n a non-polar s o l v e n t . The r a t i o of ji6 to h a l i d e s a l t was 2:1. None of the excess s t a r t i n g m a t e r i a l could be recovered. High pressure hydrogenation of carbazole over 57, rhodium on alumina c a t a l y s t gave 36.57, of 49, M.P. 75.5-76.5°, h y d r o c h l o r i d e M.P. 211.0-212.0°. The p i c r a t e and N-benzoyl d e r i v a t i v e s were a l s o prepared, M.P. 168-168.5° ( l i t e r a t u r e (47) M.P. 167-168°) and 130.5-131.7°, r e s p e c t i v e l y . Adkins and Coonradt (47) obtained 83-877, of 49, M.P. 73-74.5°, hydro-c h l o r i d e M.P. 208-209°, by r e d u c t i o n of carbazole at 220-260° over Raney n i c k e l or copper chromium oxide c a t a l y s t s at pressures of 3750 to 4500 77 p s i . The equipment a v a i l a b l e i n t h i s l a b o r a t o r y d i c t a t e d an upper l i m i t of 1800 p s i and ther e f o r e the h i g h l y a c t i v e rhodium c a t a l y s t was used. This c a t a l y s t i s i n h i b i t e d by the presence of b a s i c m a t e r i a l s (70) and i t was t h e r e f o r e necessary to add a c e t i c a c i d i n s u f f i c i e n t q u a n t i t y to n e u t r a l i z e the amine products. The nature of the c a r r i e r on which the c a t a l y s t i s supported has some e f f e c t on the r e a c t i o n s , c h a r c o a l being the p r e f e r r e d support f o r hydrogenations w h i l e alumina i s favored f o r hydrogenolyses (70). The low y i e l d s obtained i n some of the reac-t i o n s c a r r i e d out using t h i s c a t a l y s t may i n part be due to hydrogenol-y s i s . Although an upper l i m i t of 1800 p s i was o b t a i n a b l e , most of the hydrogenations i n t h i s work were c a r r i e d out at pressures between 1000 and 1500 p s i . At these pressures d i f f i c u l t y was experienced i n main-t a i n i n g an e f f e c t i v e s e a l i n the apparatus due to leakage about the s t i r r e r s h a f t . The apparatus used (Paar 4511) was designed f o r use at pressures l e s s than 1000 p s i . Q u a n t i t i e s of hydrogen taken up during the v a r i o u s r e a c t i o n s could not be c a l c u l a t e d w i t h any degree of accu-r a c y . The course of the r e a c t i o n c o u l d , however, be followed by w i t h -drawal of a l i q u o t s of the r e a c t i o n mixture. Hydrogenation of 6j> under c o n d i t i o n s s i m i l a r to those used f o r the hydrogenation of J54 gave 847, of dodecahydrocyclopent[b] i n d o l e _7_5, b n „ 64-66°, h y d r o c h l o r i d e M.P. 241.5-243.0°. H 75 78 Reaction of 4 9 w i t h sodium amide i n benzene followed by a d d i t i o n of dimethylaminoethy 1 bromide i n benzene gave 507, of 5 4 , b^ ^ 115 -116° , h y d r o c h l o r i d e M.P. 2 46 - 247° . The r a t i o of 4 9 to bromide was 1 0 : 6 and 5 2 . 5 7 , of the excess s t a r t i n g m a t e r i a l was recovered. The a d d i t i o n of _75 to m e t a l l i c sodium i n dioxane gave no r e a c t i o n . However, a d d i t i o n of 2-dimethylaminoethyl c h l o r i d e to 7_5 i n benzene gave 577, of 5 6 , b Q 9 7 - 100° , h y d r o c h l o r i d e M.P. 2 0 9 - 2 1 0 . 7 ° . The r a t i o of _75 to h a l i d e was 2 : 1 . The recovery of excess s t a r t i n g m a t e r i a l was not c a l c u l a t e d . I t i s d o u b t f u l that the i n c l u s i o n of sodium amide i n the r e a c t i o n of 4£ w i t h dimethylaminoethy1 bromide served any u s e f u l purpose. I t was necessary to use a s t r o n g l y b a s i c substance to i o n i z e the i n d o l i c and aromatic compounds because the n i t r o g e n atom i s very weakly b a s i c due to the i n d u c t i v e e f f e c t of the aromatic r i n g s . In the f u l l y hydro-genated bases, the n i t r o g e n atom i s much more b a s i c and i s capable of r e a c t i n g w i t h the a l k y l h a l i d e s provided that s t e r i c e f f e c t s do not i n -h i b i t the r e a c t i o n . Since 7_5 was not s u f f i c i e n t l y a c i d i c to r e a c t w i t h sodium metal i t i s d o u b t f u l that 4 9 reacted w i t h sodium amide. High pressure hydrogenation of 5_7 over the rhodium c a t a l y s t gave 777, of 5 8 , b x 2 1 2 6 - 1 2 7 . 5 , p i c r a t e M.P. 2 0 4 . 0 - 2 0 5 . 5 ° d . The hydro-c h l o r i d e p r e c i p i t a t e d as an o i l which could not be r e c r y s t a l l i z e d . The analogous N - (2-dimethylaminoethyl)diphenylamine 6i2 and N - ( 2 -d ime thy laminoethy l ) d i c y c l o h e x y lamine 6^3, i n which the two r i n g s attached to the n i t r o g e n atom are not or_tho-linked, were a l s o s y n t h e s i z e d . Re-a c t i o n of diphenylamine w i t h sodium amide i n benzene and subsequent ad-d i t i o n of 2-dimethylaminoethyl bromide hydrobromide i n dimethylformamide 79 gave 227= of 62, b. Q 127-130°, hyd r o c h l o r i d e M.P. 255.0-256.5°. The U. o r a t i o of diphenylamine to h a l i d e s a l t was 2:1 and 73.57o of the d i p h e n y l -amine was recovered. This compound had p r e v i o u s l y been reported (71) i n 367» y i e l d , h y d r o c h l o r i d e M.P. 244-247°d, from r e a c t i o n i n the s o l i d s t a t e between diphenylamine and 2-dimethylaminoethyl c h l o r i d e hydro-c h l o r i d e . Other reported values f o r the hy d r o c h l o r i d e are M.P. 246-247° (66) and 252-254° (69). B i o l o g i c a l data are not reported (69,71) although the patent (66) claims t h i s compound, among ot h e r s , f or a n t i -h i s t a m i n i c and antispasmodic a c t i v i t y . Another r e p o r t (56) gives a y i e l d of 807, from the r e a c t i o n of diphenylamine w i t h sodium amide f o l -lowed by a d d i t i o n of 2-dimethylaminoethy1 c h l o r i d e . The product was found to be about one-tenth as a c t i v e as diphenhydramine when i n j e c t e d subcutaneously i n guinea p i g s . An attempt to form the anion of dicyclohexylamine by r e f l u x i n g w i t h m e t a l l i c potassium i n dioxane was u n s u c c e s s f u l , due to the very low a c i d i t y of the amine. Attempted condensation of dicyclohexylamine w i t h 2-dimethylaminoethy1 c h l o r i d e , i n the absence of a b a s i c reagent, was a l s o u n s u c c e s s f u l . This was probably because of s t e r i c i n h i b i t i o n of the a l k y l a t i o n r e a c t i o n due to the large b u l k of the c y c l o h e x y l r i n g s . Although the r a t e of the r e a c t i o n w i t h dicyclohexylamine would be slow, the a l k y l a t i n g agent would be q u i c k l y removed from s o l u t i o n by d i m e r i z a -t i o n and no appreciable q u a n t i t y of the d e s i r e d product could be formed. This s t e r i c i n h i b i t i o n of r e a c t i o n would not be a large f a c t o r i n the r e a c t i o n w i t h the hydrogenated c y c l o a l k [ b ] i n d o l e s 64 and 66 sin c e the r i n g s are r i g i d l y held to one si d e of the n i t r o g e n atom. Reaction of dicyclohexylamine w i t h c h l o r o a c e t y l c h l o r i d e , followed 80 by dimethylamination and subsequent r e d u c t i o n of the amide gave 60.5% o v e r a l l y i e l d of 63, b Q 94-98°, hy d r o c h l o r i d e M.P. 216.8-217.5°. This s y n t h e t i c approach would probably have given improved y i e l d s of 54, 56, However, i t was f e l t that the saving i n time a t t a i n e d by u s i n g the 2-dimethylaminoethy1 h a l i d e route overshadowed c o n s i d e r a t i o n s of y i e l d . R eaction of c h l o r o a c e t y l c h l o r i d e w i t h the aromatic and i n d o l i c compounds would very l i k e l y have been u n s u c c e s s f u l . The r e a c t i o n of f l u o r e n e w i t h s o l i d potassium t.-butoxide i n anhy-drous benzene followed by a d d i t i o n of 2-dimethylaminoethy1 bromide hydrobromide i n dimethylformamide gave only 12% of 5_9, ^ 140-145°, p i c r a t e M.P. 179.5-180.3°. Scherf and Brown (72) have reported the p r e p a r a t i o n of 9 - f l u o r e n y l potassium by r e a c t i o n of fluorene w i t h m e t a l l i c potassium i n dioxane. The product was s u f f i c i e n t l y s t a b l e to be i s o l a t e d as a reddish-brown s o l i d , r a p i d l y decomposed by moisture and oxygen i n the a i r . I t appears that the m e t a l l a t e d d e r i v a t i v e i s formed v i a a r a d i c a l anion which i s s t a b l e below -50°C i n t e t r a h y d r o f u r a n s o l u t i o n (73). An attempt to increase the y i e l d of 59 u t i l i z i n g * t h i s procedure w i t h e q u i v a l e n t amounts of f l u o r e n e , potassium, and 2-dimethylaminoethy1 c h l o r i d e i n p u r i f i e d dioxane led to a mixture of the mono- and d i s u b s t i -tuted f luorenes 5_9 and 6_1. Separation of the two components could not be accomplished w i t h the equipment a v a i l a b l e i n t h i s l a b o r a t o r y . Scherf and Brown (72) have reported that s i x absorption peaks i n the i r spectrum of f l u o r e n e are a t t r i b u t a b l e to the methylene group and they have used i r s p e c t r a to i d e n t i f y mono- and d i s u b s t i t u t e d d e r i v a t i v e s and 58 from 49, 7_5, and r e s p e c t i v e l y . 81 of f l u o r e n e . None of these bands was of any use i n t h i s work. The s p e c t r a of 5_9 and 6_1 were i d e n t i c a l as t h i n f i l m s and as s o l u t i o n s i n carbon t e t r a c h l o r i d e . S t r u c t u r a l assignment was made on the b a s i s of the nmr s p e c t r a of the p r e v i o u s l y prepared 5_9 and pure 6_1 prepared l a t e r , as w e l l as the elemental analyses of h y d r o c h l o r i d e s and p i c r a t e s . R e a c t i o n of the sodium d e r i v a t i v e of f l u o r e n e , prepared by r e f l u x -ing e q u i v a l e n t q u a n t i t i e s of fluorene and sodium amide i n p u r i f i e d d e c a l i n and suggested (72) as a h i g h l y s a t i s f a c t o r y method of preparing 9-monosubstituted f l u o r e n e s , w i t h an e q u i v a l e n t amount of 2-dimethyl-aminoethyl c h l o r i d e a l s o gave a mixture of 59 and 61. R e p e t i t i o n of t h i s r e a c t i o n w i t h f l u o r e n e , sodium amide, and 2-dimethylaminoethyl c h l o r i d e i n the r a t i o 25:20:7.5 gave only the d i s u b s t i t u t e d d e r i v a t i v e i n 727o y i e l d . The d e s i r e d compound was f i n a l l y s ynthesized i n 307, y i e l d from the r e a c t i o n i n dioxane s o l u t i o n of f l u o r e n e , potassium, and 2-dimethylaminoethy1 c h l o r i d e i n the r a t i o 3:2:1. The presence of l a r g e q u a n t i t i e s of 6_1 i n the r e a c t i o n products i s d i f f i c u l t to r a t i o n a l i z e . Reduction of f l u o r e n e to hydrofluorenes i n the presence of a l k a l i metals i n e t h e r e a l s o l v e n t s has been noted. This r e d u c t i o n may occur by a b s t r a c t i o n by the r a d i c a l anion from the 9 - p o s i t i o n of f l u o r e n e or from the s o l v e n t , or by l i b e r a t i o n of hydrogen r a d i c a l s from the r a d i c a l anion during the m e t a l l a t i o n process. E i s c h and Kaska (74) reported that a p o r t i o n of the s t a r t i n g m a t e r i a l was reduced during the r e a c t i o n of f l u o r e n e w i t h l i t h i u m i n t e t r a h y d r o f u r a n . The s t o i c h i o m e t r y of the r e a c t i o n r e q u i r e s that one e q u i v a l e n t of f l u o r e n e r e a c t w i t h 0.80 e q u i v a l e n t of l i t h i u m to give r i s e to 0.80 e q u i v a l e n t of 9 - f l u o r e n y l l i t h i u m and t e t r a h y d r o f l u o r e n e . The experimental values 82 (74) showed that one e q u i v a l e n t of fluorene reacted w i t h 0.83 e q u i v a l e n t of l i t h i u m to give 0.83 e q u i v a l e n t of m e t a l l a t e d product plus a n e u t r a l o i l which was determined to be a mixture of t e t r a - and hexahydrofluorene. The use of more than the c a l c u l a t e d amount of metal i n the r e a c t i o n could t h e r e f o r e r e s u l t i n the formation of b i - m e t a l l a t e d f l u o r e n e i n the reac-t i o n mixture, l e a d i n g to the formation of _6_1. The presence of r e d u c t i o n products may have accounted f o r the d i f f i c u l t y encountered i n p u r i f y i n g f l u o r e n e recovered from these r e a c t i o n s although no r e d u c t i o n products were i s o l a t e d . The r a t i o of products (59:61) obtained i n the r e a c t i o n using equi-molar q u a n t i t i e s of f l u o r e n e , potassium, and 2-dimethylaminoethy1 c h l o r i d e i n dioxane i n d i c a t e d that approximately 25% (10 g) of the f l u o r e n e would have been reduced i f t h i s mechanism were o p e r a t i v e . I t i s h i g h l y un-l i k e l y that t h i s amount of r e d u c t i o n products would have been overlooked. Furthermore, Scherf and Brown (72) reported that the r e a c t i o n of fluorene w i t h potassium i n dioxane was accompanied by the e v o l u t i o n of hydrogen gas and not by r e d u c t i o n . I t i s a l s o u n l i k e l y that the f l u o r e n y l potassium r e s u l t i n g from the r e a c t i o n of equimolar q u a n t i t i e s of f l u o r e n e and potassium was a c t u a l l y a mixture of f l u o r e n e w i t h the mono- and dipotassium d e r i v a t i v e s (72). The monopotassium compound has been i s o l a t e d and i s reasonably s t a b l e i n the absence of a i r and moisture w h i l e the product of the r e -a c t i o n of two e q u i v a l e n t s of potassium w i t h one e q u i v a l e n t of f l u o r e n e was too h i g h l y r e a c t i v e to be i s o l a t e d (72). Greenhow, et. a_l (75), Scherf and Brown (72), and other authors ( l o c . c i t . ) have a l l reported the s y n t h e s i s of monosubstituted f l u o r e n e s 83 from the r e a c t i o n of equimolar q u a n t i t i e s of fluorene w i t h b a s i c r e t a l -i a t i n g reagents and an a l k y l a t i n g agent. Weissgerber obtained 9,9-di-benzyIfluorene from the r e a c t i o n of equimolar q u a n t i t i e s of f l u o r e n y l potassium and benzyl c h l o r i d e i n toluene (76,75). The f l u o r e n y l potas-sium was prepared by f u s i o n of flu o r e n e w i t h potassium hydroxide at 280°, a procedure found u n s a t i s f a c t o r y by other workers (72). The temperature and the nature of the sol v e n t are described as c r i t i c a l (75) i n the m e t a l l a t i o n of fluorene w i t h sodium amide, r e f l u x -i n g d e c a l i n at 180° being the suggested system. The 9 - f l u o r e n y l sodium which p r e c i p i t a t e d i n good y i e l d d u r i ng the described work (75) was a brownish y e l l o w powder and an acceptable value f o r sodium was found on elemental a n a l y s i s . Other authors (72) have not been able to d u p l i c a t e the good y i e l d and high q u a l i t y obtained i n (75), the product (72) being d e s c r i b e d as a b l a c k amorphous s o l i d adhering to the lower p o r t i o n of the r e a c t i o n f l a s k . In work c a r r i e d out i n t h i s l a b o r a t o r y the p r e c i p -i t a t e was a b l a c k gummy mass which s o l i d i f i e d on c o o l i n g i n t o a substance resembling bitumen. I t i s p o s s i b l e that the sodium amide, being of unknown v i n t a g e , had decomposed to the po i n t where unexpected r e a c t i o n s occurred. How-ever, the m a t e r i a l was not v i s i b l y d i f f e r e n t from a r e c e n t l y purchased and f r e s h l y opened sample of the same reagent. I t i s more l i k e l y that the gummy mass p r e c i p i t a t e d i n the r e a c t i o n entrapped a p o r t i o n of the sodium amide which was i n s o l u b l e i n the s o l v e n t , thus preventing the r e a c t i o n from going to completion w i t h respect to f l u o r e n e . Removal of the solvent and subsequent washing of the p r e c i p i t a t e would then r e -move flu o r e n e from the r e a c t i o n . A d d i t i o n of the a l k y l a t i n g agent and 84 s t i r r i n g would then break up the matrix of the p r e c i p i t a t e , f r e e i n g any entrapped sodium amide to r e a c t w i t h _59 formed i n the r e a c t i o n . Subse-quent r e a c t i o n of m e t a l l a t e d _59 would give r i s e to 6_1. Although Greenhow, jet al (75) d i d not rep o r t the presence of d i s u b s t i t u t e d f l u o r e n e s , Scherf and Brown (72) had to separate 9,9-dimethylfluorene from the 9-methylfluorene prepared by t h i s method. The r e s u l t s of the r e a c t i o n s i n v o l v i n g 9 - f l u o r e n y l potassium (A) may perhaps be best explained by p o s t u l a t i n g an e q u i l i b r i u m r e a c t i o n between A and _59 to give f l u o r e n e and 9-(2-dimethylaminoethyl)fluoren-9-yl potassium (C). Although such an e q u i l i b r i u m would be i n favour of A and 5_9, the removal of C by i r r e v e r s i b l e r e a c t i o n w i t h the a l k y l -a t i n g agent would r e s u l t i n the accumulation of an appreciable q u a n t i t y of j5_l. C would be expected to r e a c t w i t h the a l k y l a t i n g agent at a r a t e comparable to that of A. The r e a c t i o n of the a l k y l a t i n g agent w i t h 9-f l u o r e n y l potassium was slow enough to allow such an e q u i l i b r i u m to occur s i n c e only a 307» y i e l d of ^ 9 was obtained over three hours when the a l k y l a t i n g agent was added over a pe r i o d of approximately one min-ute. Dropwise a d d i t i o n of the a l k y l a t i n g agent to the r e a c t i o n mixture would be expected to increase the p r o p o r t i o n of 6_1 obtained and t h i s was i n f a c t observed. This mechanism would a l s o account f o r the presence of dide u t e r a t e d f l u o r e n e i n monodeuterated fl u o r e n e prepared by t h i s method (72). The r e a c t i o n would of course not be i r r e v e r s i b l e i n the case of deuterated fluorene and the mixture obtained may represent an e q u i l i b r i u m mixture. Scherf and Brown (72) suggested that the presence of di d e u t e r a t e d fluorene was due to isotope exchange w i t h excess mediated by KOD. I t i s 85 d o u b t f u l however that t h i s base i s a strong enough reagent to cause t h i s to occur under the c o n d i t i o n s used. Attempted hydrogenation of a mixture of .59 and 6_1 gave the s t a r t -i n g m a t e r i a l s as w e l l as 517„ of 9-(2-dimethylaminoethyl)-l,2,3,4,4a,9a-hexahydrofluorene 60, b Q Q 5 99°, h y d r o c h l o r i d e M.P. 190.2-191.5°. Several other compounds were present i n small q u a n t i t i e s , as determined from vapour phase chromatography of the p a r t i a l l y p u r i f i e d r e a c t i o n mixture. The safe l i m i t s of the r e a c t i o n apparatus were exceeded during t h i s u n s u c c e s s f u l attempt to o b t a i n the f u l l y hydrogenated d e r i v a t i v e s of 5_9 and 6_1 so no f u r t h e r attempts were made. The minor products from the r e a c t i o n were probably p a r t i a l l y hydrogenated d e r i v a t i v e s of 59 although some 61 may have been p a r t i a l l y reduced. No attempt was made to i s o l a t e or i d e n t i f y these minor c o n s t i t u e n t s . By analogy w i t h the r e d u c t i o n products of c a r b a z o l e , the expected c o n f i g u r a t i o n of 60 i s as shown. Since H a and are c i s o r i e n t e d on a cyclopentane r i n g the d i -h e d r a l angle approaches zero degrees and the c o u p l i n g constant f o r these protons i s expected to be about 8 cps (77) . A s i m i l a r r e l a t i o n s h i p holds between and H c > U n f o r t u n a t e l y , the nmr spectrum of 6_0 was not w e l l enough r e s o l v e d to support t h i s assignment. Other s p l i t t i n g 86 patterns g r e a t l y confuse the spectrum. A study of models r e v e a l s no s t e r i c reasons f o r f a i l u r e of reduc-t i o n of the remaining aromatic r i n g . The absence of a p p r e c i a b l e q u a n t i t i e s of r e d u c t i o n products of 6_1 may be explained on the b a s i s of s t e r i c i n t e r a c t i o n s of the p r o t r u d i n g dimethylaminoethy1 groups on both sides of the plane of the r i n g s pre-v e n t i n g complex formation w i t h the c a t a l y s t s u r f a c e . 87 ANALYTICAL METHODS M e l t i n g p o i n t s were determined, unless otherwise i n d i c a t e d , i n open c a p i l l a r i e s i n a Thomas-Hoover Unimelt apparatus. Some me l t i n g p o i n t s were determined on a M e t t l e r SP2 microscope hot stage and are i n d i c a t e d as such. A l l m e l t i n g points are reported uncorrected. I n f r a r e d ( i r ) s p e c t r a were determined on a Beckmann IR-10 spec-trophotometer. L i q u i d samples were scanned as t h i n f i l m s between NaCl p l a t e s and s o l i d s were incorporated i n KBr d i s c s . Nuclear magnetic resonance (nmr) spec t r a were recorded by Miss P. Watson, Department of Chemistry, U.B.C., on a Var i a n A s s o c i a t e s A-60 or T-60 instrument. S o l u t i o n s were about 107» and so l v e n t s are s p e c i f i e d . T e t r a m e t h y l s i l a n e (IMS) was used as an i n t e r n a l standard. Peaks i n the nmr sp e c t r a are reported according to the f o l l o w i n g f o r -mat: chemical s h i f t from IMS (peak m u l t i p l i c i t y , number of protons, c o u p l i n g constant where a p p l i c a b l e ) . The f o l l o w i n g a b b r e v i a t i o n s are used f o r the peak m u l t i p l i c i t y : s, s i n g l e t ; d, doublet; t , t r i p l e t ; m, m u l t i p l e t ; e, envelope - a wide i n d i s t i n c t band, covering up to 1 ppm. Vapour phase chromatography (vpc) was c a r r i e d out on a Micro-Tek Model 220 instrument equipped w i t h a flame i o n i z a t i o n d e t e c t o r . Peak areas were measured by a Model 222 Disc I n t e g r a t o r . Microanalyses were performed by A l f r e d Bernhardt M i k r o a n a l y t i s c h e s Laboratorium, West Germany. Benzene sulfonamide d e r i v a t i v e s were o f t e n formed to determine the presence of primary and secondary amines i n d i s t i l l a t i o n f r a c t i o n s . The presence of halogen was determined by burning a sample of the com-pound under t e s t on a copper w i r e i n a bunsen burner. 88 Y i e l d s are based on the amount of p u r i f i e d compound i s o l a t e d . 89 EXPERIMENTAL PART ONE SYNTHESIS OF DERIVATIVES AND ANALOGUES OF 7-AMINO-14-AZADISPIRO [5.1.5.2] PENTADECAN-15-ONE 1. 14-Hydroxy-14-azadispiro[5.1.5.2] pentadec-9-en-7,15-dione 7-oxime 32^  This compound was prepared e s s e n t i a l l y by the method used by N i g h t i n g a l e , e_t al (35). A mixture of cyclohexanone (196 g,2 mole), nitromethane (122 g,2 mole), and anhydrous p i p e r a z i n e (172 g,2 mole) i n 400 ml anhydrous benzene was r e f l u x e d f o r f i v e days i n a one l i t r e b o i l i n g f l a s k f i t t e d w i t h a Dean-Stark tube, r e f l u x condenser, and dry-ing tube. During the r e f l u x p e r i o d , 75 ml water was c o l l e c t e d . The r e a c t i o n mixture was f i l t e r e d and the s o l i d m a t e r i a l ( p i p e r -azinium s a l t of the d e s i r e d compound) was washed w i t h hot benzene. The s o l i d was then e x t r a c t e d w i t h 500 ml hot, d i l u t e HCI (1:1) f o r 40 min. The r e s u l t a n t s o l i d was f i l t e r e d o f f , washed w i t h water, d r i e d in vac, and r e c r y s t a l l i z e d from ethanol to give c o l o u r l e s s c r y s t a l s , M.P. 272-274°. Y i e l d 124 g (47%) ( l i t (35) M.P. 273-274°, 67%) i r (KBr); 3110,2930,2680,1700,1650,1440,1415,1350,1160,1045,1000,980, 935,920,815 cm" 1 2. 7-Amino-14-azadispiro [5.1.5.2] pentadecan-15-one 4 14-Hydroxy-14-azadispiro [5.1.5.2]pentadec-9-en-7,15-dione 7-oxime (44 g,0.17 mole) was d i s s o l v e d i n 400 ml ethanol (1007«) i n a Paar 4511 pressure r e a c t i o n apparatus. Raney n i c k e l No. 28 (W.R.Grace) (4 tsp) was washed w i t h ethanol (100%) to remove water and added to the s o l u -t i o n . The r e a c t i o n mixture was s t i r r e d at room temperature under 500 90 p s i hydrogen for 48 hr and f i l t e r e d , and the c a t a l y s t was washed w i t h hot ethanol. The solvent was removed on a r o t a r y evaporator to give a white s o l i d r e s i d u e . This was r e c r y s t a l l i z e d from d i l u t e ethanol to g i v e c o l o u r l e s s c r y s t a l s , M.P. 192-193°. Y i e l d 34 g (86%). ( l i t . (37a) M.P. 193-195°, 95%) i r (KBr); 3180,2940,2860,1690,1450,1405,1260,810 cm" 1, nmr (CDC1 3); cf 7.20 ( s , l ) lactam proton; 2.76 ( s , l ) C-7 proton; 1.60 (e,22) A f t e r a d d i t i o n of D 20 the spectrum was as above except 6 7.20 absent. 3. 7-Chloroacetamido-14-azadispiro [5 .1.5 . 2] pentadecan-15-one 2^2 A s o l u t i o n of c h l o r o a c e t y l c h l o r i d e (11.3 g,0.10 mole) i n 50 ml anhydrous benzene was added dropwise over 1.5 hr to a g e n t l y r e f l u x i n g s o l u t i o n of 7-amino-14-azadispiro [5.1.5.2] pentadecan-15-one (47.3 g, 0.20 mole) i n 500 ml anhydrous benzene i n a one l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and dry-ing tube. The r e a c t i o n mixture was r e f l u x e d f o r a f u r t h e r 2.5 hr and s t i r r e d at room temperature f o r 12 hr. The r e a c t i o n mixture was then f i l t e r e d and the s o l i d was washed w i t h benzene. The solvent was removed on a r o t a r y evaporator to give a white s o l i d which was r e c r y s t a l l i z e d from ethanol to give c o l o u r l e s s c r y s t a l s , M.P. 286-287°. The p r e c i p i t a t e d s o l i d which had been f i l t e r e d from the r e a c t i o n mixture was e x t r a c t e d w i t h water i n convenient p o r t i o n s i n a mortar and p e s t l e . The aqueous suspension was f i l t e r e d and the s o l i d m a t e r i a l was r e c r y s t a l l i z e d from ethanol to give c o l o u r l e s s c r y s t a l s , M.P. 286-287°. T o t a l y i e l d 25 g (807,, based on c h l o r o a c e t y l c h l o r i d e ) , i r (KBr); 3375,3210,2960,2780,1695,1670,1550,1270,790 cm" 1. 91 M.W. 312.85 CI,11.33; N,8.95 CI,11.31; N,8.79 The f i l t e r e d aqueous s o l u t i o n was made b a s i c w i t h NaOH (407,) and the r e s u l t a n t p r e c i p i t a t e was f i l t e r e d o f f , d r i e d in vac and r e c r y s -t a l l i z e d from ethanol to give c o l o u r l e s s c r y s t a l s of 7-amino-14-aza-d i s p i r o [5 .1.5 .2] pentadecan-15-one . Recovery ranged from 50-757, of the excess used. 4. 7-Dimethylaminoacetamido-14-azadispiro [5.1.5.2] pentadecan-15-one 23 A. With benzene as sol v e n t Dimethylamine (100 ml,1.66 mole) was c o l l e c t e d from a compressed gas c y l i n d e r i n a graduated c y l i n d e r i n an acetone/dry i c e bath and was d i s s o l v e d i n 100 ml anhydrous ether. This s o l u t i o n was then added drop-wise to a suspension of 7-chloroacetamido-14-azadispiro[5.1.5.2]penta-decan- 15-one (82.7 g,0.27 mole) i n 1500 ml anhydrous benzene i n a two l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and d r y i n g tube. The r e a c t i o n mixture was r e f l u x e d f o r seven hr and s t i r r e d at room temperature f o r 15 hr. Since c r y s t a l -l i n e m a t e r i a l was s t i l l present i n the r e a c t i o n , a f u r t h e r 50 ml p o r t i o n of dimethylamine was added and the r e a c t i o n mixture was r e f l u x e d f o r 18 h r . The r e a c t i o n mixture was f i l t e r e d and so l v e n t s were removed on a r o t a r y evaporator to give a white s o l i d . The r e s i d u e and the s o l i d m a t e r i a l from the r e a c t i o n were suspended i n 300 ml HCI (57,) and f i l -t ered to recover s o l i d unreacted 7-chloroacetamido-14-azadispiro[5.1.5.2] -pentadecan-15-one (recovery 43.3 g,53.57,). The a c i d i c f i l t r a t e was made A n a l y s i s f o r C J ^ ^ I J C I ^ C ^ c a l c u l a t e d C , 6 1 . 4 3 ; H . 8 . 0 6 ; found C , 6 1 . 6 2 ; H , 8 . 2 0 ; 92 b a s i c w i t h NaOH (50%) and p r e c i p i t a t e d s o l i d was f i l t e r e d o f f . The s o l i d m a t e r i a l was d r i e d in vac and r e c r y s t a l l i z e d from benzene/hexane to g i ve c o l o u r l e s s c r y s t a l s , M.P. 203-204°. Y i e l d 38 g (44.5%) i r (KBr); 3290,3200,3100,2940,2870,2840,2780,1695,1660,1560,1460,1410, 1165,1055,870 cm" 1, nmr (CDCl-j); c?7.64(d,l,J = 11Hz) amide proton; 7.45(s,l) lactam pro-ton; 4 . 3 2 ( d , l , J = 11Hz) C-7 proton; 3.05(s,2) methylene protons; 2.35(s,6) N-methyl protons; 1.57(e,20) A f t e r a d d i t i o n of D 20 the spectrum was as above except 6^7.64 absent, c£4.32 became 4 . 3 2 ( s , l ) . A n a l y s i s f o r C l g H 3 1 N 3 0 2 M.W. 321.47 c a l c u l a t e d C,67.30; H,9.72; N,13.07; 0,9.95 found C,67.48; H,9.61; N,12.92 B. With ethanol (100%) as s o l v e n t Dimethylamine (90 ml,1.5 mole) was c o l l e c t e d i n a graduated c y l i n d e r i n an acetone/dry i c e bath and d i s s o l v e d i n 100 ml ethanol (100%). This s o l u t i o n was then added dropwise to a suspension of 7-chloroacetamido-14-azadispiro[5.1.5.2]pentadecan-15-one (40 g,0.128 mole) i n 1000 ml ethanol (100%) i n a two l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and d r y i n g tube. The r e a c t i o n mixture was then r e f l u x e d f or 15 hr, cooled to room tem-perature, and the solvent was removed on a r o t a r y evaporator to give a white s o l i d . The res i d u e was d i s s o l v e d i n 500 ml HCI (5%) and the s o l u t i o n was f i l t e r e d . The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH (507o) and the s o l i d p r e c i p i t a t e was f i l t e r e d o f f . R e c r y s t a l l i z a t i o n 93 from benzene/hexane gave c o l o u r l e s s c r y s t a l s , M.P. 202-204°. Y i e l d 21.8 g (53%). 5. 7-(2-Dimethylaminoethylamino)-14-azadispiro[5.1.5.2] pentadecane (attempted) L i t h i u m aluminum hydride (9.5 g,0.25 mole) was suspended i n 1000 ml anhydrous t e t r a h y d r o f u r a n i n a two l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , Soxhlet e x t r a c t o r , condenser, and d r y i n g tube. 7-Dimethylaminoacetamido-14-azadispiro[5.1.5.2j pentadecan-15-one (38 g,0.12 mole) was conti n u o u s l y e x t r a c t e d i n t o the hydride sus-pension over 16 hr. The r e a c t i o n mixture was then cooled and the excess hydride was destroyed by dropwise a d d i t i o n of 10 ml NaOH (10%) fo l l o w e d by an excess of water. The r e a c t i o n mixture was f i l t e r e d , the s o l i d m a t e r i a l was washed w i t h ether, and the s o l v e n t s were removed from the f i l t r a t e on a r o t a r y evaporator to give a s l i g h t l y grey-brown o i l which c r y s t a l l i z e d a f t e r d r y i n g in vac. This m a t e r i a l was r e c r y s t a l l i z e d from hexane to give c o l o u r l e s s c r y s t a l s , M.P. 110-111°, and was subse-quently i d e n t i f i e d as 7-dimethylaminoacetamido-14-azadispiro[5.1.5.2]-pentadecane 25. Y i e l d 21.5 g (60%) i r (KBr); 3375,3140,2930,2860,2790,1685,1515,1460,1410,1345,1280,1180, 1155,1105,1050,870,540 cm" 1. nmr (CDC1 3); <£ 7.30(d,1,J = 11Hz) amide proton; 3 . 7 6 ( d , l , J = 11Hz) C-7 proton; 3.01(s,2) methylene protons; 2.90(s,l) and 2.88(s,l) C-15 protons; 2.35(s,6) N-methyl protons; 2.05(s,l) amine proton; 1.5(e,20) A f t e r a d d i t i o n of D 20 the spectrum was as above except ($7.30 and 2.05 absent, 3.76 became 3.76(s,l) A n a l y s i s f o r C 1 QH„»N_0 M.W. 307.48 c a l c u l a t e d 0,70.38; H.10.74; N,13.67; 0,5.21 found C,70.13; H,10.65; N.13.35 A second r e a c t i o n w i t h 7-dimethylaminoacetamido-14-azadispiro-[5.1.5.2]pentadecan-15-one (2.05 g,0.0064 mole) and l i t h i u m aluminum hydride (0.57 g,0.015 mole) i n te t r a h y d r o f u r a n was r e f l u x e d f o r s i x days and gave only the p a r t i a l l y reduced compound. 6. 7-Dimethylaminoacetamido-14-azadispiro[5.1.5.2] pentadecan-15-one methiodide 24 To a s o l u t i o n of 7-dimethylaminoacetamido-14-azadispiro[5.1.5.2J -pentadecan-15-one (10 g,0.03 mole) i n 100 ml anhydrous benzene i n a 250 ml three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and d r y i n g tube, was added dropwise a s o l u t i o n of methyl i o d i d e (8.5 g,0.06 mole) i n 25 ml anhydrous benzene. The r e -a c t i o n mixture was r e f l u x e d f o r two hr a f t e r a d d i t i o n was completed. The mixture was cooled and f i l t e r e d to give a y e l l o w , amorphous pow-der, which on r e c r y s t a l l i z a t i o n from g l a c i a l a c e t i c a c i d gave dark y e l l o w c r y s t a l s , M.P. 160-161°d. Y i e l d not c a l c u l a t e d , i r (KBr); 3230,3180,2935,2860,1665,1560,1450,1280,930 cm" 1. A n a l y s i s f o r C 1 9 H 3 4 I N 3 0 2 M.W. 434.61 c a l c u l a t e d 0,49.28; H,7.34; 1,27.40; N,9.07; 0,6.91 found C.49.00; H,7.22; 1,27.41; N,8.97 7. 7-Dimethylaminoacetamido-14-azadispiro[5.1.5.2]pentadecane dimethiodide 26 To a s o l u t i o n of 7-dimethylaminoacetamido-14-azadispiro[5.1.5.2] -95 pentadecane (10 g,0.03 mole) i n 100 ml anhydrous benzene i n a 250 ml three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and d r y i n g tube, was added dropwise a s o l u t i o n of methyl i o d i d e (8.5 g,0.06 mole) i n 25 ml anhydrous benzene. The r e a c t i o n mixture was r e f l u x e d for two hr and f i l t e r e d to give an orange amorphous pow-der. A s u i t a b l e solvent f o r r e c r y s t a l l i z a t i o n could not be found, i r (KBr); 3440,3260,2940,2860,1680,1545,1455,1025cm" 1. 8. 16-Hydroxy-16-azadispiro(j>.1.6.2] heptadec-10-en-8,17-dione 8-oxime A mixture of cycloheptanone (112 g , l mole), nitromethane (61 g , l mole) and anhydrous p i p e r a z i n e (43 g,0.5 mole) i n 150 ml anhydrous benzene was r e f l u x e d f o r four days i n a one l i t r e b o i l i n g f l a s k f i t t e d w i t h a Dean-Stark t r a p , condenser, and d r y i n g tube. During the r e f l u x p e r i o d 40.5 ml water was c o l l e c t e d . The r e a c t i o n mixture was cooled and the s o l i d p i p e r a z i n i u m s a l t was f i l t e r e d o f f , washed w i t h ether, then w i t h hot benzene, and d r i e d i n vac. The s a l t was then e x t r a c t e d w i t h hot d i l u t e HCI (1:1) f o r 40 minutes and the crude b u f f - c o l o u r e d product was f i l t e r e d o f f . Recrys-t a l l i z a t i o n from ethanol gave c o l o u r l e s s c r y s t a l s , M.P. 246-249°d. Y i e l d 54.5 g (41.2%) ( l i t (35) M.P. 250-251°d, 51.5%) i r (KBr); 3400,3120,2940,2870,1690,1655,1470,1425,1190,1175,945,920, -1 690 cm . 9. 8-Amino-16-azadispiro[6.1.6 .2] heptadecan-17-one 5_ 16-Hydroxy-16-azadispiro[6.1.6.2]heptadec-10-en-8,17-dione 8-oxime (40 g,0.14 mole) was d i s s o l v e d i n 400 ml ethanol (100%) i n a Paar 4511 pressure r e a c t i o n apparatus. Raney n i c k e l No. 28 (W.R. Grace) (4 tsp) 96 was washed w i t h ethanol (1007.) to remove water and added to the s o l u -t i o n . The r e a c t i o n mixture was s t i r r e d at room temperature under 400 p s i hydrogen f o r 24 hr and f i l t e r e d , and the c a t a l y s t was washed w i t h hot ethanol. The sol v e n t was removed on a r o t a r y evaporator to give a white s o l i d which was r e c r y s t a l l i z e d from d i l u t e ethanol to g i v e c o l o u r l e s s c r y s t a l s , M.P. 188-190°d. Y i e l d 32.2 g (897.) i r (KBr); 3200,3080,2940,2865,1690,1465,1390,1350,925,860 cm" 1, nmr (CDCl-j); r3*7.45(s,l) lactam proton; 2.14(s,l) C-8 proton; 1.63 (e,26) A f t e r a d d i t i o n of D 20, Ci7.45 absent, 1.63 became 1.63(e,24). 10. 8-Chloroacetamido-16-azadispiro[6.1.6 .2]heptadecan-17-one 27_ A s o l u t i o n of c h l o r o a c e t y l c h l o r i d e (2.5 g,0.02 mole) i n 50 ml anhydrous benzene was added dropwise to a ge n t l y r e f l u x i n g s o l u t i o n of 8-amino-16-azadispiro[6.1.6.2]heptadecan-17-one (10.6 g,0.04 mole) i n 100 ml anhydrous benzene i n a 250 ml three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and d r y i n g tube. The r e a c t i o n mixture was r e f l u x e d f o r f i v e hr a f t e r a d d i t i o n was completed, and the p r e c i p i t a t e d s o l i d was f i l t e r e d o f f and washed w i t h hot acetone. The s o l v e n t s were removed from the f i l t r a t e on a r o t a r y evaporator to gi v e a white s o l i d r e s i d u e which was r e c r y s t a l l i z e d from benzene to gi v e c o l o u r l e s s c r y s t a l s , M.P. 210-212°. Y i e l d 6.2 g (847.) i r (KBr); 3330,2940,2870,1695,1670,1550,1470 cm" 1 nmr (CDCl^); r£6.90(s,l) lactam proton, o v e r l a p p i n g 6 . 9 6 ( d , l , J = 9.5Hz) amide proton; 4 . 3 3 ( d , l , J = 10Hz) C-8 proton; 4.15(s,2) methylene protons; 2.6(e,24) There was no change on a d d i t i o n of D 20. 97 The excess of 5 was recovered from a f i l t e r e d aqueous s o l u t i o n of the p r e c i p i t a t e from the r e a c t i o n by a d d i t i o n of NaOH (40%) and c o l l e c -t i o n of the r e s u l t a n t p r e c i p i t a t e by f i l t r a t i o n , f ollowed by d r y i n g i n  vac. Recovery was 907» of the excess used. 11. 8-Dimethylaminoacetamido-16-azadispiro[6.1.6.2]heptadecan-17-one 2Ji Dimethylamine (66 m l , l mole) was c o l l e c t e d i n a graduated c y l i n d e r i n an acetone/dry i c e bath and was d i s s o l v e d i n 100 ml reagent ether. This s o l u t i o n was then added dropwise to a s o l u t i o n of 8-chloroacetamido-16-azadispiro[6.1.6.2]heptadecan-17-one (51 g,0.15 mole) i n 1000 ml r e -agent benzene i n a two l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and dr y i n g tube. The r e a c t i o n mix-ture was r e f l u x e d f o r three hr and s t i r r e d at room temperature f o r 34 hr. The r e a c t i o n was then f i l t e r e d and sol v e n t was removed on a r o t a r y evaporator to g i v e a s l i g h t l y grey-green s o l i d . The r e s i d u e was d i s -solved i n 500 ml benzene and 800 ml HCI (5%) was added. The white pre-c i p i t a t e was f i l t e r e d o f f and the aqueous phase was separated from the benzene s o l u t i o n . The white p r e c i p i t a t e was suspended i n the aqueous phase and s u f f i c i e n t NaOH (40%) was added to give a p e r s i s t e n t b a s i c r e a c t i o n . The b a s i c suspension was f i l t e r e d , the s o l i d was washed w i t h water, d r i e d i n vac, and r e c r y s t a l l i z e d from benzene/hexane to give c o l o u r l e s s c r y s t a l s , M.P. 173.5-174.5°. Y i e l d 43 g (82%) i r (KBr); 3330,3190,3080,2935,2860,2795,1695,1680,1530,1470,1390,1350, 1055 cm"1. nmr (CDC1 3); £ 7 . 7 9 ( d , l , J - 10Hz) amide proton; 6.93(s,l) lactam pro-ton; 4 . 3 5 ( d , l , J = 11Hz) C-8 proton; 3.05(s,2) methylene protons; 2.38(s,6) N-methyl protons; 1.59(e,24) 98 A f t e r a d d i t i o n of D^O, p a r t i a l exchange of <S 7.79 occurred so that 6*4.35 became 4.35(d,J = 11Hz) overlapped w i t h 4.35(s), t o t a l 1 pro-ton . A n a l y s i s f o r C20H35N3O2 M.W. 349.52 c a l c u l a t e d 0,68.75; H,10.09; N,12.02; 0,9.13 found C,69.12; H,10.36; N,11.70 12. 8-(2-Dimethylaminoethy1amino)-16-azadispiro[6.1.6.2]heptadecane (attempted) A. With l i t h i u m aluminum hydride A s o l u t i o n of 8-dimethylaminoacetamido-16-azadispiro[6.1.6.2]-heptadecan-17-one (27 g,0.08 mole) i n 600 ml anhydrous t e t r a h y d r o f u r a n was added dropwise to a suspension of l i t h i u m aluminum hydride (5.7 g, 0.15 mole) i n 600 ml anhydrous t e t r a h y d r o f u r a n i n a two l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and d r y i n g tube. A f t e r a d d i t i o n was completed, the r e a c t i o n mixture was r e f l u x e d f o r 42 hr and cooled, and excess hydride was destroyed by dropwise a d d i t i o n of 10 ml NaOH (57.) and 10 ml water. The mixture was f i l t e r e d , the s o l i d was washed w i t h ether, and the combined f i l t r a t e s were d r i e d over MgSO^. The s o l u t i o n was f i l t e r e d and so l v e n t s were removed on a r o t a r y evaporator to give a white s o l i d which was r e c r y s -t a l l i z e d from hexane to give c o l o u r l e s s c r y s t a l s , M.P. 104-105°, sub-sequently i d e n t i f i e d as 8-dimethylaminoacetamido-16-azadispiro[6.1.6.l\-heptadecane 30. Y i e l d 19.5 g (757,). i r (KBr); 3350,3250,2920,2850,2780,1675,1520,1465,1335,1275,1150,1055, 965,925,885,860,550 cm" 1 nmr (CDClo); (S 7.52(d,1,J = 10Hz) amide proton; 3 . 8 5 ( d , l , J = 11Hz) 99 C-8 proton; 3.00(s,2) methylene protons; 2.75 (s,2) C-17 protons; 2.36(s,6) N-methyl protons; 2.10(s,l) amine pro-ton; 1.52(e,24) A f t e r a d d i t i o n of D2O, the spectrum was as above except 6 7.52 and 2.10 absent, 3.85 became 3 . 8 5 ( s , l ) . A n a l y s i s f o r C^H^N-jO M.W. 335.54 c a l c u l a t e d C,71.57; H,11.14; N,12.52; 0,3.77 found C.71.48; H,11.17; N,12.29 B. With diborane (78) A s o l u t i o n of 8-dimethylaminoacetamido-16-azadispiro[6.1.6.2] -heptadecan-17-one (1.4 g,0.004 mole) i n 35 ml anhydrous t e t r a h y d r o -furan was added dropwise, over one hour at 0°C, to a s o l u t i o n of d i -borane i n anhydrous t e t r a h y d r o f u r a n (30 ml, approximately 0.3M i n d i -borane) i n a 100 ml four-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and d r y i n g tube. A f t e r a d d i t i o n was com-p l e t e d , the r e a c t i o n mixture was r e f l u x e d f o r two hr and cooled to room temperature, and 25 ml HCI (10%,) was added dropwise to decompose excess reagent. Tetrahydrofuran was removed from the r e a c t i o n s o l u -t i o n on a r o t a r y evaporator, and the aqueous r e s i d u e was made b a s i c w i t h NaOH (40%) and then e x t r a c t e d w i t h 3x75 ml ether which was d r i e d over MgSO^ .. The ether s o l u t i o n was f i l t e r e d and solvent was removed on a r o t a r y evaporator to give a g r a y i s h o i l . R e c r y s t a l l i z a t i o n from hexane was u n s u c c e s s f u l . The crude o i l was d i s t i l l e d at reduced pres-sure to give a c o l o u r l e s s o i l , bg 3 175-180°, which s o l i d i f i e d on standing. The product was found to be 8-dimethylaminoacetamido-16-azadispiro[6.1.6.2]heptadecane. Y i e l d 0.7 g (52%). 100 C. By r e d u c t i o n over copper chromium oxide (78,79) Copper chromium oxide c a t a l y s t (2.6 g) was added to a s o l u t i o n of 8-dimethylaminoacetamido-16-azadispiro[6.1.6.2] heptadecane (3.1 g, 0.0093 mole) i n 75 ml p u r i f i e d dioxane i n a Paar 4511 pressure r e a c t i o n -apparatus. The r e a c t i o n mixture was s t i r r e d at 220°C and 1320 p s i hy-drogen f o r 22 hr. The r e a c t i o n mixture was cooled and f i l t e r e d , and the s o l i d m a t e r i a l was washed w i t h ether. Solvents were removed from the f i l t r a t e on a r o t a r y evaporator to give an o i l y r e s i d u e which was d i s s o l v e d i n 100 ml ether. The ether s o l u t i o n was e x t r a c t e d w i t h 3x50 ml HCI ( 5 % ) . The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH (40%) and was e x t r a c t e d w i t h 3x75 ml ether. The f i n a l ether e x t r a c t was d r i e d over MgSO^, f i l t e r e d , and solvent was removed on a r o t a r y evaporator to give a l i g h t brown o i l which p a r t i a l l y s o l i d i f i e d on standing. Only s t a r t i n g m a t e r i a l could be recovered from t h i s r e s i d u e . 13. 8-Dimethylaminoacetamido-16-azadispiro[6.1.6.2] heptadecan-17-one methiodide 29 Methyl i o d i d e (4.0 g,0.028 mole) was added dropwise to a magnet-i c a l l y s t i r r e d s o l u t i o n of 8-dimethylaminoacetamido-16-azadispiro[6.1.6 heptadecan-17-one (5 g,0.014 mole) i n 100 ml anhydrous benzene i n a 250 ml erlenmeyer f l a s k . The y e l l o w amorphous p r e c i p i t a t e was f i l t e r e d o f f and r e c r y s t a l l i z e d from ethanol (1007.)/anhydrous ether to give two d i s -t i n c t c r y s t a l l i n e forms, which could be manually separated. R e c r y s t a l l i z a t i o n of each c r y s t a l l i n e form, a f t e r manual s e p a r a t i o n gave a mixture of both forms. A) l i g h t y e l l o w , f i n e , broken needles, M.P. 169.1-170.0° ( M e t t l e r 101 microscope hot stage). B) darker y e l l o w , s p h e r i c a l clumps of needles (a hedgehog appear-ance), M.P. 239.3-239.4° ( M e t t l e r SP 2 microscope hot st a g e ) . Y i e l d not c a l c u l a t e d . Each of the two c r y s t a l l i n e forms' gave a s i n g l e spot w i t h the same R.£ val u e when chromatographed on s i l i c a g e l u s i n g ethanol as the de v e l -oping s o l v e n t . A n a l y s i s for polymorphism by d i f f e r e n t i a l scanning c a l o r i m e t r y gave no thermal peak f o r e i t h e r c r y s t a l l i n e form, probably because the com-pounds melt w i t h decomposition. I r s p e c t r a f o r both c r y s t a l types were i d e n t i c a l , i r (KBr); 3230,3080,2930,2860,1680,1660,1545,1465,1280,1240,980,935, 910,760 cm"1 A n a l y s i s f o r C o lH O QIN„0„ M.W. 492.51 ci. Jo j Z c a l c u l a t e d C,51.21; H,7.79; 1,25.77 A) found C,50.91; H,8.22; 1,25.89 B) found C,51.05; H,7.92; 1,25.85 N,8.53; 0,6.70 N,8.33 N,8.35 14. 8-Dimethylaminoacetamido-16-azadispiro[6.1.6.2] heptadecane dimethiodide 3^1 Methyl i o d i d e (0.85 g,0.006 mole) was d i s s o l v e d i n 25 ml anhydrous benzene and was added dropwise to a r e f l u x i n g s o l u t i o n of 8-dimethyl-aminoacetamido-16-azadispiro[6.1.6.2] heptadecane (1 g,0.003 mole) i n 50 ml anhydrous benzene i n a 250 ml three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping funnel and d r y i n g tube. The r e a c t i o n mixture was r e f l u x e d f or one hr and was s t i r r e d at room temperature f o r 14 hr. The dark y e l l o w p r e c i p i t a t e from the r e a c t i o n was r e c r y s t a l l i z e d -102 from g l a c i a l a c e t i c a c i d to give yellow-orange c r y s t a l s , M.P. 186.3° ( M e t t l e r S P 2 ) • Y i e l d not c a l c u l a t e d . i r (KBr); 3420,3260,2930,2860,1690,1540,1465,1235cm" 1 A n a l y s i s for C o-H / cI oN.0 M.W. 605.43 23 45 2 J c a l c u l a t e d C,43.61; H,7.16; 1,40.07; N.6.63; 0,2.53 found C.41.23; H,7.05; 1,40.10; N,6.67 15. 8-Dimethylaminoacetamido-16-azadispiro[6.1.6.2] heptadecane methyl s u l f a t e (attempted) Dimethyl s u l f a t e (2.52 g,0.02 mole) was added to a s o l u t i o n of 8-dimethylaminoacetamido-16-azadispiro[6.1.6.2]heptadecane (6.7 g,0.02 mole) i n 45 ml anhydrous benzene i n a 250 ml erlenmeyer f l a s k . The r e a c t i o n mixture was heated on a steam bath f o r three hr and allowed to stand f o r 16 hr. During t h i s i n t e r v a l a gummy t r a n s l u c e n t m a t e r i a l separated. The benzene s o l u t i o n was decanted and the gum was washed w i t h ether which was decanted i n t o the benzene s o l u t i o n . The s o l v e n t s were evaporated on a steam bath and the r e s i d u a l syrup and the o r i g i n a l gum were d r i e d i n vac to give hygroscopic g l a s s e s . A s u i t a b l e s olvent f o r r e c r y s t a l l i z a t i o n could not be found. 103 MISCELLANEOUS REACTIONS 1. Diborane i n tetrahydrofuran (78) Sodium borohydride (17.1 g,0.45 mole) was d i s s o l v e d i n 340 ml anhydrous diglyme ( p r e v i o u s l y d i s t i l l e d from l i t h i u m aluminum h y d r i d e ) , and the cloudy s o l u t i o n was f i l t e r e d . Boron t r i f l u o r i d e etherate (42.6 g,0.30 mole) was d i s s o l v e d i n 300 ml anhydrous diglyme i n a one l i t r e b o i l i n g flask, f i t t e d w i t h a dropping f u n n e l , a gas i n l e t tube, and a gas o u t l e t tube leading through a mer-cury s a f e t y trap to a 500 ml erlenmeyer f l a s k c o n t a i n i n g 250 ml anhydrous t e t r a h y d r o f u r a n . The o u t l e t tube was p o s i t i o n e d about f i v e mm from the bottom of the erlenmeyer. The erlenmeyer was f i t t e d w i t h a d r y i n g tube and was cooled i n i c e water. The system was flushed w i t h a stream of dry n i t r o g e n f o r one hour previous to and during the course of the r e a c t i o n . The borohydride s o l u t i o n was added dropwise to the r e a c t i o n f l a s k w h i l e s t i r r i n g mag-n e t i c a l l y . When a d d i t i o n was completed the r e a c t i o n mixture was sl o w l y heated to b o i l i n g to d r i v e o f f d i s s o l v e d diborane. I t was necessary to add i c e to the i c e water c o o l i n g bath s e v e r a l times during the course of the r e a c t i o n . Y i e l d of diborane: 2 g increase i n weight of e r l e n -meyer and contents (50%) 0.072 mole i n 250 ml, 0.29M. 2. Copper Chromium Oxide c a t a l y s t (79,80) Cupric c h l o r i d e d ihydrate (91.5 g,0.5 mole) and barium n i t r a t e (15.5 g,0.06 mole) were d i s s o l v e d i n water and the s o l u t i o n was made up to a t o t a l volume of 450 ml. This s o l u t i o n was heated to 80°C and poured i n a t h i n stream i n t o a room temperature aqueous s o l u t i o n 104 c o n t a i n i n g sodium dichromate (VI) dihydrate (89 g,0.3 mole) and ammonium hydroxide (28%) (112.5 ml) i n a t o t a l volume of 450 ml. The mixture was s t i r r e d by hand during a d d i t i o n and f o r ten minutes a f t e r a d d i t i o n was completed. The orange-brown p r e c i p i t a t e was f i l t e r e d o f f at a water a s p i r a t o r u n t i l as dry as p o s s i b l e . The s o l i d was then d r i e d f o r two hours i n a vacuum oven at 110° and 31 inches vacuum. The product, i n lump form, was d i v i d e d i n t o two equal p o r t i o n s i n evaporating dishes and was decomposed f o r one hour at 350-400°C i n a m u f f l e furnace. The now black, c a t a l y s t was powdered and leached w i t h 500 ml a c e t i c a c i d (10%) f o r 30 minutes. The suspension was f i l t e r e d and the crude product was washed w i t h 4x100 ml d i s t i l l e d water. The s o l i d was f i l t e r e d o f f at a water a s p i r a t o r u n t i l as dry as p o s s i b l e and d r i e d in vac (110°, 31 inches vacuum). The c a t a l y s t was s t o r e d i n a screw cap j a r . Y i e l d 85.2 g. 3. 8-Amino-16-azadispiro[6.1.6.2]heptadecane (attempted) A s o l u t i o n of 8-amino-16-azadispiro[6.1.6.2]heptadecan-17-one (5.28 g,0.02 mole) i n 60 ml anhydrous t e t r a h y d r o f u r a n was added drop-wise to a suspension of l i t h i u m aluminum hydride (1.52 g,0.04 mole) i n 20 ml anhydrous t e t r a h y d r o f u r a n i n a 100 ml four-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and d r y i n g tube. The r e a c t i o n mixture was r e f l u x e d f o r 40 hr, excess hydride was destroyed by a d d i t i o n of 1.5 ml NaOH (10%,) and an excess of water, and the r e a c t i o n mixture was f i l t e r e d . The s o l i d was washed w i t h ether and the combined f i l t r a t e s were d r i e d over MgSO^. The s o l u t i o n was f i l t e r e d and sol v e n t s were removed on a r o t a r y evaporator to give a white s o l i d r e s i d u e . Re-c r y s t a l l i z a t i o n from ethanol gave c o l o u r l e s s c r y s t a l s of the s t a r t i n g 105 m a t e r i a l , M.P. 187-190°d. The i n f r a r e d spectrum showed no changes from the s t a r t i n g m a t e r i a l and the mixed mel t i n g p o i n t w i t h authentic s t a r t -ing m a t e r i a l was not depressed. 4. 7-Amino-14-azadispiro[5.1.5.2]pentadecane (attempted) A s o l u t i o n of 7-amino-14-azadispiro[5.1.5.2]pentadecan-15-one (2.36 g,0.01 mole) i n 100 ml anhydrous t e t r a h y d r o f u r a n was added drop-wise to a suspension of l i t h i u m aluminum hydride (0.38 g,0.01 mole) i n 50 ml anhydrous te t r a h y d r o f u r a n i n a 250 ml three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and d r y i n g tube. The r e a c t i o n mixture was r e f l u x e d f o r 48 hr and cooled to room temper-ature . Excess hydride was destroyed by the dropwise a d d i t i o n of 3 ml water and 3 ml NaOH ( 5 % ) . The mixture was f i l t e r e d and the s o l i d was washed w i t h ether. The combined f i l t r a t e s were d r i e d over MgSO^, f i l -t e r e d , and the solve n t s were removed on a r o t a r y evaporator to give a white s o l i d . R e c r y s t a l l i z a t i o n from ethanol gave c o l o u r l e s s c r y s t a l s of s t a r t i n g m a t e r i a l , M.P. 191-193°'. 5. H y d r o l y s i s of 8-dimethylaminoacetamido-16-azadispiro[6.1.6.2]-heptadecane (attempted) A s o l u t i o n of 8-dimethylaminoacetamido-16-azadispiro[6.1.6.2]-heptadecane (1.25 g,0.004 mole) i n 20 ml H 2S0 4 (30%), i n a 50 ml b o i l -i n g f l a s k f i t t e d w i t h a condenser, was r e f l u x e d f o r four hr, cooled, and made b a s i c w i t h NaOH (40%.) . The a l k a l i n e suspension was e x t r a c t e d w i t h 3x100 ml ether and the s o l u t i o n was d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and solvent was removed on a r o t a r y evaporator to g i ve a c o l o u r l e s s o i l which c r y s t a l l i z e d on standing. R e c r y s t a l -106 l i z a t i o n of the re s i d u e from hexane gave c o l o u r l e s s c r y s t a l s of s t a r t i n g m a t e r i a l , M.P, 109-110°. 107 PART TWO SYNTHESIS OF DERIVATIVES AND ANALOGUES OF CARBAZOLE 1. 9-(2-Dimethylaminoethyl)carbazole 52 A s o l u t i o n of carbazole (33.4 g,0.2 mole) i n 200 ml dimethylform-amide was added over ten min to a s o l u t i o n of sodium methoxide prepared by r e a c t i n g f r e s h l y cleaned sodium metal (4.6 g,0.2 g atom) w i t h 50 ml anhydrous reagent methanol i n a one l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and d r y i n g tube. The r e a c t i o n mixture was r e f l u x e d f or ten min and a s o l u t i o n of 2-dimethyl-aminoethyl c h l o r i d e h y d r o c h l o r i d e (14.4 g,0.1 mole) i n 500 ml dime t h y l -formamide was added dropwise to the r e a c t i o n mixture. The r e a c t i o n mixture was r e f l u x e d f o r e i g h t hr, cooled, and f i l t e r e d . The solvent was removed on a r o t a r y evaporator and the s o l i d r e s i d u e was suspended i n 800 ml HCI (2%) and f i l t e r e d to remove the excess of ca r b a z o l e . The a c i d i c s o l u t i o n was e x t r a c t e d w i t h 3x150 ml ether, was made b a s i c w i t h NaOH (30%), and was then e x t r a c t e d w i t h 3x150 ml ether. The l a t t e r ether e x t r a c t was d r i e d over MgSO^ and f i l t e r e d , and the sol v e n t was removed on a r o t a r y evaporator to give a brown s o l i d . A f t e r s e v e r a l u n s u c c e s s f u l attempts to r e c r y s t a l l i z e the r e s i d u e , reduced pressure s u b l i m a t i o n gave c o l o u r l e s s c r y s t a l s , M.P. 40.5-41°. Y i e l d 8.3 g (35%). i r (KBr); 3045,2970,2940,2815,2760,1585,1475,1445,1340,1320,1250,1220, 1175,1145,845,820 cm"1 nmr (CC1 4); S 7.93(disturbed d o u b l e t , 2 , J = 6.5Hz); 7.25 (m,6); 4.25 ( t , 2 , J = 8Hz); 2.55(t,2,J= 8Hz); 2.22(s,6) There was no change a f t e r a d d i t i o n of DO. 108 A n a l y s i s f o r C.,H 1 0N 0 M.W. 238.33 1 0 1 0 i. c a l c u l a t e d C.80.63; H,7.61; N.11.75 found C,80.45; H.7.41; N,12.04 A p o r t i o n of the c r y s t a l s was d i s s o l v e d i n ether, the s o l u t i o n was d r i e d over MgSO^ and f i l t e r e d , and the s a l t was p r e c i p i t a t e d by addi-t i o n of HCI gas. R e c r y s t a l l i z a t i o n from ethanol (1007o) /anhydrous ether gave c o l o u r l e s s c r y s t a l s , M.P. 243-244.5°. i r (KBr); 3050,3010,2940,2650,2580,2520,2485,1595,1485,1455,1355,1330, 1235,1160,1020,975,755,730 cm" 1 2. 9-(2-Dimethylaminoethyl)-l,2,3,4-tetrahydrocarbazole 53_ A. 1,2,3,4-Tetrahydrocarbazole 64 A s o l u t i o n of cyclohexanone (78 g,0.79 mole) i n 270 ml g l a c i a l a c e t i c a c i d i n a one l i t r e three-neck f l a s k , f i t t e d w i t h a mechanical s t i r r e r , dropping f u n n e l , and condenser, was heated to r e f l u x tempera-t u r e , heat was removed, and f r e s h l y d i s t i l l e d phenylhydrazine (81 g, 0.75 mole) was added at a r a t e s u f f i c i e n t to maintain r e f l u x i n g . The r e a c t i o n mixture was r e f l u x e d f or one hr f u r t h e r and was then poured i n t o a beaker and s t i r r e d v i g o u r o u s l y w h i l e c o o l i n g to 8°C i n i c e . The s l u r r y was f i l t e r e d and the red-brown s o l i d was washed w i t h 75 ml water, suspended i n 200 ml water, and f i l t e r e d . The f i l -t r a t e s were d i s c a r d e d . The s o l i d was washed w i t h 100 ml etha n o l , sus-pended i n 200 ml etha n o l , and f i l t e r e d . The combined e t h a n o l i c f i l -t r a t e s were d i l u t e d to 1500 ml w i t h water, allowed to stand for one hr, and f i l t e r e d . The combined s o l i d m a t e r i a l s were a i r d r i e d (12 hr) and r e c r y s -t a l l i z e d from methanol, a f t e r d e c o l o u r i z i n g w i t h c h a r c o a l , to give 109 s l i g h t l y y e l l o w c r y s t a l s , M.P. 113-117°. Y i e l d 89 g (66%) ( l i t (61) M.P. 116-117°, 82%) i r (KBr); 3400,3065,2940,2865,1470,1450,1440,1370,1330,1305,1240,1150, 745,640,570,485 cm" 1 nmr (CDCl-j); ($7.44(m,2); 7.16(m,3); 2.66(m,4); 1.86(m,4) There was no change a f t e r a d d i t i o n of D2O. B. 9-(2-dimethylaminoethy1)-1,2,3,4-tetrahydrocarbazole F r e s h l y cleaned sodium metal (4.6 g,0.2 g atom), cut i n t o small p i e c e s , was reacted w i t h 50 ml ethanol (100%) i n a one l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and dr y i n g tube. When most of the sodium had r e a c t e d , a s o l u t i o n of 1,2,3,4-tet r a h y d r o c a r b a z o l e (34.2 g,0.2 mole) i n 130 ml dimethyIformamide was added over a pe r i o d of ten min. The r e a c t i o n was r e f l u x e d for 30 min and a s o l u t i o n of 2-dimethylaminoethy1 bromide hydrobromide (23.2 g,0.1 mole) i n 300 ml dimethyIformamide was added dropwise to the r e f l u x i n g r e a c t i o n mixture. The r e a c t i o n mixture was r e f l u x e d f o r 12 hr a f t e r a d d i t i o n was completed. The r e a c t i o n mixture was cooled to room temperature and f i l -t e r e d , and so l v e n t s were removed on a r o t a r y evaporator. The s e m i s o l i d r e s i d u e was suspended i n four l i t r e s HCI (5%) and was e x t r a c t e d w i t h 4x500 ml ether which was then d r i e d over MgSO^. The excess of s t a r t i n g m a t e r i a l was recovered from t h i s s o l u t i o n (707o) . In s o l u b l e m a t e r i a l suspended i n the a c i d i c phase was f i l t e r e d o f f and p a r t i t i o n e d between 200 ml ether and 50 ml NaOH (30%) on a mag-n e t i c s t i r r e r . The ether s o l u t i o n was e x t r a c t e d w i t h 2x150 ml water and was d r i e d over MgS04. 110 The a c i d i c phase was made b a s i c w i t h NaOH (307») and was ex-t r a c t e d w i t h 3x400 ml ether which was then d r i e d over MgSO^. The two ether s o l u t i o n s were f i l t e r e d ( s e p a r a t e l y ) and the so l v e n t s were removed on a r o t a r y evaporator to give brown o i l s . I n f r a -red s p e c t r a i n d i c a t e d that the two residues were the same. The com-bined residues were d i s t i l l e d at reduced pressure to g i v e a l i g h t y e l -low o i l , bl 2 155-160°. Y i e l d 6.25 g (36.5%). i r ( l i q u i d f i l m ) ; 3055,2930,2845,2780,1460,1370,1315,1175,1150,1040, 740 cm" 1 nmr (CC1 4,50%); r$7.25(m,l); 6.96(m,3); 3.66(t,2,J= 7Hz); 2.48(e,4); 2.20(t,2,J = 7Hz); 2.00(s,6); 1.70(e,4). There was no change a f t e r a d d i t i o n of D2O. A p o r t i o n of the d i s t i l l a t e was d i s s o l v e d i n ether, the s o l u -t i o n was d r i e d over MgSO^ and f i l t e r e d , and the s a l t was p r e c i p i t a t e d by a d d i t i o n of HCI gas. R e c r y s t a l l i z a t i o n from ethanol (100%)/anhydrous ether gave c o l o u r l e s s c r y s t a l s , M.P. 244.5-246.0°d. i r (KBr); 3420,2930,2850,2660,2585,2480,1455,1370,1230,1150,1010,965, 735 c m " 1 A n a l y s i s f o r C..H 0 0ClN„ M.W. 278.83 lb 23 2 c a l c u l a t e d C,68.92; H,8.32; CI,12.72; N,10.05 found C,68.77; H,8.19; CI,12.67; N,10.14 The remaining d i s t i l l a t e p a r t i a l l y c r y s t a l l i z e d on t r a n s f e r r i n g the d i s t i l l a t e to a v i a l f o r storage. Reduced pressure m i c r o s u b l i m a t i o n of t h i s m a t e r i a l gave a l i q u i d and a s o l i d , M.P. 47.5-48.5°. The i r s p e c t r a of both s o l i d and l i q u i d were i d e n t i c a l w i t h that of the d i s -t i l l a t e . Hydrochloride s a l t s of the s o l i d and l i q u i d were prepared. I l l Their m e l t i n g p o i n t s were the same as that of the s a l t of the d i s t i l -l a t e . Mixed m e l t i n g p o i n t s of a l l combinations of the three s a l t s showed no depression from the mel t i n g p o i n t of the s a l t of the d i s t i l l a t e . 3. 9-(2-Dimethylaminoethyl)dodecahydrocarbazole 54 A. Dodecahydrocarbazole 49 i ) from 2-cyclohexylidene cyclohexanone (attempted) a) 2-Cyclohexylidene cyclohexanone (53) A s o l u t i o n of cyclohexanone (100 g,1.01 mole) i n 100 g H^SO^ (60%) i n a 500 ml erlenmeyer f l a s k was t i g h t l y stoppered and allowed to stand at room temperature f o r 24 hr. The two l a y e r s were separated and 148 ml water was added to the aqueous (lower) l a y e r , r e s u l t i n g i n the s e p a r a t i o n of more organic l a y e r . The combined organic m a t e r i a l was washed w i t h 5x35 ml saturated Na2S0^ s o l u t i o n . Since t h i s proce-dure gave a poor s e p a r a t i o n , the combined aqueous phases were ext r a c t e d w i t h 3x150 ml e t h e r , and ether was added to the organic phase to f a c i l -i t a t e s e p a r a t i o n of the aqueous phase. The combined ether s o l u t i o n s were d r i e d over MgSO^ and f i l t e r e d , and s o l v e n t was removed on a r o t a r y evaporator to g i v e a r e d d i s h o i l . Unreacted cyclohexanone was recovered from the r e s i d u e by d i s t i l l a t i o n at atmospheric pressure. The r e s i d u e was d i s t i l l e d at reduced pressure to give a y e l l o w o i l , b^ ^ 105-108°. Y i e l d 24.5 g (27.5%) ( l i t (53) b ? 6 0 155-158°, 76-83% based on c y c l o -hexanone consumed i n the r e a c t i o n ) . This r e a c t i o n was repeated s e v e r a l times us i n g d i f f e r e n t r e -a c t i o n times and temperatures, and was a l s o run i n a c e n t r i f u g e f o r f i v e hr. The best y i e l d obtained was 58.570, based on the amount of c y c l o -hexanone consumed i n the r e a c t i o n (Table V I I I , p 68). 112 b) Dodecahydrocarbazole (45) 2-Cyclohexylidene cyclohexanone (24.5 g,0.137 mole) was mixed w i t h formamide (36 g,0.8 mole) and 25 ml ethylene g l y c o l i n a 100 ml four-neck r e a c t i o n f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, and d r y i n g tube. The r e a c t i o n mixture was ge n t l y r e f l u x e d at 120° f o r eig h t h r , then at 180° for two hr. The ethylene g l y c o l and excess formamide were then d i s t i l l e d out of the r e a c t i o n mixture at reduced pressure to give a t h i c k brown o i l . The r e s i d u e was washed by decan-t a t i o n w i t h an equal volume (35 ml) of water. An equal volume of HCI (38%) was added and the mixture was r e f l u x e d f o r three hr. V o l a t i l e m a t e r i a l was then removed on a r o t a r y evaporator, two volumes (50 ml) of water were added to the dark brown r e s i d u e , and the mixture was ex-t r a c t e d w i t h 4x25 ml ether. The aqueous phase was then made b a s i c w i t h NaOH (30%) and e x t r a c t e d w i t h 3x100 ml ether, which was d r i e d over K2CO3. The ether s o l u t i o n was f i l t e r e d and s o l v e n t was removed on a r o t a r y evaporator to give a brown o i l . D i s t i l l a t i o n of the r e s i d u e at ' reduced pressure gave an u n i d e n t i f i e d product, b 2 5 96-100°. ( l i t (45) b 1 2 130-132°, 32%, h y d r o c h l o r i d e , M.P. 265-268°, a n a l y s i s f o r CI only; A d i f f e r e n t s y n t h e s i s (47) gives b 1 Q 124-125°, h y d r o c h l o r i d e M.P. 208-209°, a n a l y s i s for C 1 2 H 2 2 C 1 N (C,H). i i ) from carbazole (47,70) G l a c i a l a c e t i c a c i d (17.0 g,0.283 mole), and 5%, rhodium on alumina c a t a l y s t (10 g) were added to a s o l u t i o n of carbazole (47 g, 0.28 mole) i n 450 ml tet r a h y d r o f u r a n i n a Paar 4511 pressure r e a c t i o n apparatus. The r e a c t i o n mixture was s t i r r e d at room temperature under 1060 p s i hydrogen. A f t e r 14 hr the pressure was 900 p s i . The pressure 113 was increased to 1040 p s i and the r e a c t i o n was heated to 125°. A f t e r e i g h t hr the pressure was 520 p s i at room temperature. The pressure was increased to 1040 p s i and the r e a c t i o n was s t i r r e d at room temper-ature f o r 14 h r , a f t e r which time the pressure was 1000 p s i . The r e -a c t i o n mixture was f i l t e r e d and the s o l i d m a t e r i a l was washed w i t h ether. The solve n t s were removed on a r o t a r y evaporator to give a l i g h t y e l l o w o i l . The s o l i d m a t e r i a l was suspended i n 500 ml water, the re s i d u e from the evaporation was added to the aqueous suspension, and the sus-pension was made a c i d i c w i t h HCI (57o) . The a c i d i c suspension was f i l -t ered through dry k e i s e l g u h r and the f i l t r a t e was e x t r a c t e d w i t h 3x300 ml ether. The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH (307.) and was ex t r a c t e d w i t h 300 ml ether. The aqueous phase was f i l t e r e d and the g e l which had formed at the i n t e r f a c e was f i l t e r e d through dry k e i s e l -guhr w i t h the e t h e r e a l phase. The g e l was washed w i t h ether and t h i s was added to the f i l t r a t e . The aqueous phase was then e x t r a c t e d w i t h 2x300 ml ether. The combined ether e x t r a c t s were d r i e d over MgS0 4 and f i l t e r e d , and the sol v e n t was removed on a r o t a r y evaporator to give a s l i g h t l y y e l l o w s o l i d . The re s i d u e was sublimed in vac and r e c r y s -t a l l i z e d from hexane to give c o l o u r l e s s c r y s t a l s , M.P. 75.5-76.5°. Y i e l d 18.1 g (36.57»). ( l i t (47) 73-74.5°, 83-877.) i r (KBr); 3250,2930,2860,1450,1095,955,905,845 cm" 1 nmr ( C C l ^ ) ; c? 3.1(e); 2.32(s); 1.50(e) The i n t e g r a t i o n curve could not be i n t e r p r e t e d . A f t e r a d d i t i o n of D.O, the spectrum was as above except 2.32 absent. 114 A n a l y s i s f o r C H N M.W. 179.31 c a l c u l a t e d C,80.38; H.11.81; N.7.81 found C,80.35; H,11.64; N.7.79 A p o r t i o n of the s o l i d was d i s s o l v e d i n ether, the s o l u t i o n was d r i e d over MgSO^ and f i l t e r e d , and the s a l t was p r e c i p i t a t e d by a d d i t i o n of HCI gas. R e c r y s t a l l i z a t i o n from ethanol (100%)/anhydrous ether gave c o l o u r l e s s c r y s t a l s , M.P. 211.0-212.0°. ( l i t (47) 208-209°) i r (KBr); 2950,2880,2750,2720,2545,1580,1445,1065,1040,1010,980,935 cm B) 9-(2-Dimethylaminoethy1)dodecahydrocarbazole A s o l u t i o n of dodecahydrocarbazole (18.1 g,0.1 mole) i n 250 ml reagent benzene was added to sodium amide (3.9 g,0.1 mole) i n a one l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and d r y i n g tube, and the r e a c t i o n mixture was r e f l u x e d f o r two hr. 2-Dimethylaminoethy1 bromide hydrobromide (23.3 g,0.2 mole) and f l a k e sodium hydroxide (16.0 g,0.4 mole) were placed i n a 250 ml three-neck f l a s k f i t t e d w i t n a mechanical s t i r r e r , C l a i s e n s t i l l head, and a condenser set f o r d i s t i l l a t i o n i n t o a ta r e d , i c e - c o o l e d r e c e i v e r . This apparatus was connected to a water a s p i r a t o r and the pressure was reduced to about 35 mm Hg. The s o l i d r e a c t a n t s were slowly mixed t o -gether, t u r n i n g the s t i r r e r s h a f t by hand. When the r e a c t i o n was pro-ceeding smoothly, the motor of the s t i r r e r was s t a r t e d . Reaction of the product occurred during the d i s t i l l a t i o n , as evidenced by p r e c i p i -t a t i o n of a f i n e white s o l i d i n the r e c e i v e r . Benzene (100 g ) , pre-v i o u s l y cooled i n i c e , was added to the d i s t i l l a t e and the suspension was f i l t e r e d i n t o a t a r e d , i c e - c o o l e d f l a s k . Y i e l d 9.3 g (0.061 mole, 115 39%) of 2-dimethylaminoethy1 bromide. The s o l u t i o n was used immediately. A s o l u t i o n of 2-dimethylaminoethy1 bromide (9.3 g,0.061 mole) i n 100 g benzene was added dropwise to the r e f l u x i n g dodecahydrocarbazole r e a c t i o n mixture and the mixture was r e f l u x e d f o r 24 hr. The r e a c t i o n mixture was cooled and f i l t e r e d , and the solvent was removed on a r o t a r y evaporator to give an orange s o l i d . R e c r y s t a l l i z a t i o n of the r e s i d u e from hexane gave c o l o u r l e s s c r y s t a l s of dodecahydrocarbazole, y i e l d 3.7 g (52.57« of excess used). The amorphous gray s o l i d which was f i l t e r e d out of the r e a c t i o n mixture was decomposed by c a u t i o u s l y adding the s o l i d to 700 ml water. The s t r o n g l y b a s i c s o l u t i o n was e x t r a c t e d w i t h 4x200 ml ether and the combined e x t r a c t s were d r i e d over MgSO^. The ether s o l u t i o n was f i l -t ered and the so l v e n t was removed on a r o t a r y evaporator to g i v e a y e l -low o i l which was d i s t i l l e d at reduced pressure to give a c o l o u r l e s s l i q u i d , b Q ? 115-116°. Y i e l d 10 g (507,). i r ( l i q u i d f i l m ) ; 2940,2870,2830,2780,1455,1360,1270,1170,1160,1130, 1110,1050,1035,865,840 cm"1 nmr (CDCI3) ; c^2.4(m,4); 2.13(s,6); 1.40(e,20) There was no change a f t e r a d d i t i o n of D2O. A p o r t i o n of the d i s t i l l a t e was d i s s o l v e d i n ether, the s o l u -t i o n was d r i e d over MgSO^ and f i l t e r e d , and the s a l t was p r e c i p i t a t e d by a d d i t i o n of HCI gas. R e c r y s t a l l i z a t i o n from ethanol (1007=,)/anhydrous ether gave c o l o u r l e s s c r y s t a l s , M.P. 246-247°. i r (KBr); 2950,2870,2660,2490,1635,1450,1425,1375,1275,1000 cm"1 A n a l y s i s f o r C n,H 0 0 C 1 0N„ M.W. 323.36 io JZ Z z c a l c u l a t e d C.59.43; H,9.98; CI,21.93; N,8.66 found C.59.19; H,9.72; CI,21.80; N,8.84 116 4. 4-(2-Dimethylaminoethy1)-1,2,3,4-tetrahydrocyclopent[b]indole 55 A. 1,2,3,4-Tetrahydrocyclopent[b] i n d o l e 6j5 A mixture of cyclopentanone (42 g,0.5 mole) and f r e s h l y d i s -t i l l e d phenylhydrazine (54 g,0.5 mole) was shaken i n a 250 ml e r l e n -meyer f l a s k and allowed to stand f o r 20 minutes, during which a h i g h l y exothermic r e a c t i o n occurred. The mixture was then heated on a steam bath f o r ten min and the red syrup was poured i n t o a s o l u t i o n of H^SO^ (58 ml) i n water (1039 ml). This mixture was heated for 45 min w h i l e s t i r r i n g v i g o u r o u s l y , and was then cooled i n i c e . The red s o l i d was f i l t e r e d o f f and d r i e d in vac over s u l f u r i c a c i d . R e c r y s t a l l i z a t i o n of the dry product from petroleum s p i r i t (60-80°) gave s l i g h t l y pink c r y s t a l s , M.P. 106.5-107.5°. Y i e l d 32.5 g (41.5%) ( l i t (63) 108-109°, 45%) . i r (KBr); 3400,3060,2980,2950,2770,1580,1470,1450,1370,1320,1300,750, 640 cm nmr (CC1 4); C$7.20(m,2); 6.97(m,3); 2.63(m,6) There was no change a f t e r a d d i t i o n of D2O. B. 4-(2-Dimethylaminoethy 1)-1,2,3,4-tetrahydrocyclopent[b] i n d o l e i ) u s i n g sodium amide as the base (attempted) A s o l u t i o n of 1,2,3,4-tetrahydrocyclopent[b] i n d o l e (20.2 g, 0.129 mole) i n 250 ml dimethylformamide was r e f l u x e d w i t h sodium amide (6.6 g,0.17 mole) for four hr under a dry n i t r o g e n atmosphere i n a one l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , d r y i n g tube, and gas i n l e t tube. The dark brown sus-pension was cooled to room temperature and 2-dimethylaminoethyl c h l o r i d e (18.5 g,0.17 mole) was added dropwise. The r e a c t i o n mixture was r e f l u x e d 117 f o r three hr and s t i r r e d at room temperature f o r 24 hr. The r e a c t i o n mixture was c a u t i o u s l y d i l u t e d to two l i t r e s w i t h water and f i l t e r e d , and the r e s u l t a n t b a s i c s o l u t i o n was e x t r a c t e d w i t h 3x300 ml ether. The ether s o l u t i o n was ex t r a c t e d w i t h 3x200 ml HCI (37o) and was d r i e d over MgSO^. No l,2,3,4-tetrahydrocyclopent[b] i n d o l e could be recovered from t h i s ether s o l u t i o n . The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH (307.) and was ex t r a c t e d w i t h 3x200 ml ether which was d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and the sol v e n t was removed on a r o t a r y evapora-tor to gi v e a small amount of n e a r l y b l a c k resinous m a t e r i a l , from which none of the d e s i r e d product could be i s o l a t e d . i i ) u s i n g sodium ethoxide as the base (attempted) F r e s h l y cleaned sodium (2.9 g,0.125 g atom), cut i n sm a l l p i e c e s , was reacted w i t h 50 ml ethanol (1007.) i n a 500 ml three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and d r y i n g tube. The apparatus was wrapped i n aluminum f o i l to p r o t e c t the i n d o l e from l i g h t . A s o l u t i o n of 1,2,3,4-tetrahydrocyclopent[b]-i n d o l e (19.7 g,0.125 mole) i n 90 ml ethanol (1007.) was added dropwise to the slo w l y r e f l u x i n g s o l u t i o n of ethoxide. A s o l u t i o n of 2-dimethyl-aminoethyl bromide hydrobromide (14.5 g,0.063 mole) i n 200 ml warm ethanol (1007=) was then added dropwise, over 2.5 hr, to the gen t l y r e -f l u x i n g r e a c t i o n mixture. The r e a c t i o n mixture was s t i r r e d at room temperature f o r 12 hr and f i l t e r e d , and the sol v e n t was removed on a r o t a r y evaporator. The re s i d u e was suspended i n 600 ml water and the b a s i c suspension was e x t r a c t e d w i t h 3x150 ml ether. The e t h e r e a l s o l u -t i o n was e x t r a c t e d w i t h 3x150 ml HCI (37=) and d r i e d over MgSO/ . None 118 of the s t a r t i n g m a t e r i a l could be recovered from t h i s s o l u t i o n . The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH (30%) and was ex t r a c t e d w i t h 3x150 ml ether which was d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and sol v e n t was removed on a r o t a r y evaporator to give a small amount of b l a c k o i l from which none of the d e s i r e d product could be i s o l a t e d . i i i ) without using a base (attempted) A s o l u t i o n of 2-dimethylaminoethy1 bromide hydrobromide (11.65 g,0.05 mole) i n 200 ml ethanol (100%) was heated to a slow r e f l u x i n a 500 ml three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and d r y i n g tube. The apparatus was wrapped i n alumi-num f o i l to p r o t e c t the i n d o l e from l i g h t . A s o l u t i o n of 1,2,3,4-tetrahydrocyclopent[b] i n d o l e (7.85 g,0.05 mole) i n 80 ml ethanol (100%) was added over a p e r i o d of three hr to the r e a c t i o n f l a s k . The reac-t i o n mixture was r e f l u x e d f o r 12 hr, cooled to room temperature, and the s o l v e n t was removed on a r o t a r y evaporator. The s o l i d r e s i d u e was suspended i n 250 ml ether and was e x t r a c t e d w i t h 3x90 ml HCI ( 5 % ) . The ether s o l u t i o n was d r i e d over MgSO^. None of the s t a r t i n g m a t e r i a l could be recovered from t h i s s o l u t i o n . The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH (307o) and was ex t r a c t e d w i t h 3x100 ml ether which was d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and the solvent was removed on a r o t a r y evapora-tor to give a brown l i q u i d r e s i d u e , which was not i d e n t i f i e d . None of the d e s i r e d product could be i s o l a t e d from the r e s i d u e , i v ) u s i n g sodium metal as the base F r e s h l y cleaned sodium metal (3 g,0.13 g atom) was very f i n e l y 119 d i s p e r s e d i n 150 ml xylene, p r e v i o u s l y d r i e d over sodium w i r e , i n a one l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r (Hershberg typ e ) , dropping f u n n e l , condenser, and d r y i n g tube. The apparatus was wrapped i n aluminum f o i l to p r o t e c t the i n d o l e from l i g h t . A s o l u t i o n of 1,2,3,4-tetrahy d r o c y c l o p e n t [b] i n d o l e (32.5 g,0.15 mole) i n 100 ml xylene/anhydrous ether (1:1) was added to the r e a c t i o n f l a s k over 2.5 hr, w h i l e s t i r r i n g very v i g o u r o u s l y . A s o l u t i o n of 2-dimethylaminoethyl bromide hydrobromide (16.3 g,0.07 mole) i n 200 ml dimethylformamide was then added dropwise to the v i g o u r o u s l y s t i r r e d r e a c t i o n mixture and the r e a c t i o n was s t i r r e d f o r 14 hr at room temperature. The r e a c t i o n mixture was f i l t e r e d , the p r e c i p i t a t e was washed w i t h benzene, and the so l v e n t s were removed from the combined f i l t r a t e s on a r o t a r y evaporator. The re s i d u e was d i s s o l v e d i n 500 ml ether and was e x t r a c t e d w i t h 3x150 ml water which had been made s l i g h t l y b a s i c w i t h NaOH ( 5 7 o ) . The ether s o l u t i o n was then e x t r a c t e d w i t h 3x150 ml HCI ( 3 7 o ) and was d r i e d over MgSO^. None of the s t a r t i n g m a t e r i a l could be recovered from t h i s s o l u t i o n . The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH ( 3 0 7 , ) and was ex-t r a c t e d w i t h 3x150 ml ether which was d r i e d over MgSO^. The ether s o l u -t i o n was f i l t e r e d and sol v e n t was removed on a r o t a r y evaporator to give a brown o i l . D i s t i l l a t i o n of the resi d u e at reduced pressure gave a s l i g h t l y y e l l o w o i l , b Q 5 5 136.5-139.5°. Y i e l d 5.97 g ( 1 7 . 5 7 , ) . i r ( l i q u i d f i l m ) ; 3050,2950,2860,2830,2780,1460,1380,1355,1295,1175, 1150,1035,745 cm" 1 nmr (CC1 4); £ 7.23(m,1); 6.99(m,3); 3.85(t,2,J= 7.5Hz); 2.65 overlapped w i t h 2.42 (e and t r e s p e c t i v e l y , t o t a l 8, J of t = 7Hz); 120 2.12(s,6) A p o r t i o n of the d i s t i l l a t e was d i s s o l v e d i n ether, the s o l u -t i o n was d r i e d over MgSO^ and f i l t e r e d , and the s a l t was p r e c i p i t a t e d by a d d i t i o n of HCI gas. The mauve p r e c i p i t a t e was r e c r y s t a l l i z e d from ethanol (1007=)/anhydrous ether to give l i g h t purple c r y s t a l s , M.P. 220-226.5°d. This s a l t decomposed on r e c r y s t a l l i z a t i o n . The p i c r a t e was prepared and r e c r y s t a l l i z e d from.ethanol to give y e l l o w c r y s t a l s , M.P. 198.0-198.5°. i r (KBr); 2940,2860,2640,1630,1560,1460,1430,1365,1315,1270,1165,1085, 745 cm A n a l y s i s f o r C ^ H ^ N ^ M.W. 457.44 c a l c u l a t e d C,55.14; H,5.07; N,15.31; 0,24.48 found C,55.14; H,5.13; N,15.26 5. 4-(2-Dimethylaminoethyl)dodecahydrocyclopent[b] i n d o l e 5>6 A. Dodecahydrocyclopent [b] i n d o l e 7j) G l a c i a l a c e t i c a c i d (15.0 g,0.25 mole) and 57= rhodium on a l u -mina c a t a l y s t (10 g) were added to a s o l u t i o n of 1,2,3,4-tetrahydro-cyclopent[b] i n d o l e i n 550 ml anhydrous t e t r a h y d r o f u r a n i n a Paar 4511 pressure r e a c t i o n apparatus. The r e a c t i o n mixture was s t i r r e d at room temperature under 1300 p s i hydrogen f o r 24 hr. The apparatus was then heated to 130° f o r 16 hr. A f t e r a f u r t h e r 20 hr at room temperature, the r e a c t i o n mixture was f i l t e r e d and the s o l i d was washed w i t h ether. The solv e n t s were removed from the combined f i l t r a t e s on a r o t a r y evaporator to give a gray s o l i d r e s i d u e . The re s i d u e was sus-pended i n 250 ml ether and was e x t r a c t e d w i t h 3x125 ml HCI (37=,). The ether s o l u t i o n was then d r i e d over MgS04» No s t a r t i n g m a t e r i a l could 121 be recovered from t h i s s o l u t i o n . The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH (30%) and was ex t r a c t e d w i t h 3x100 ml ether, which was then d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and solvent was removed on a r o t a r y evap-or a t o r to g i v e a mobile brown l i q u i d . The res i d u e was d i s t i l l e d at reduced pressure to g i v e a c o l o u r l e s s l i g h t o i l , b^ g 64-66°. Y i e l d 28.5 g (84%). i r ( l i q u i d f i l m ) ; 3275,2920,2855,1450,1403,1335,1280,1235,1210,1115, 1100,945,865,850,715 cm" 1 nmr ( n e a t ) ; s e v e r a l envelope bands i n the r e g i o n 1.0 to 3.8. One sharp peak at 1.70 which disappeared a f t e r a d d i t i o n of D^ O. The i n t e g r a t i o n curve was i n d e c i p h e r a b l e . A p o r t i o n of the d i s t i l l a t e was d i s s o l v e d i n ether, the s o l u -t i o n was d r i e d over MgSO^ and f i l t e r e d , and the s a l t was p r e c i p i t a t e d by the a d d i t i o n of HCI gas. R e c r y s t a l l i z a t i o n from benzene gave c o l o u r -l e s s , feathery c r y s t a l s , M.P. 241.5-243.0°. i r (KBr); 2940,2870,2760,2715,2560,1580,1450,1420,1390,1070,945,585, 470 cm" 1 A n a l y s i s f o r C U H 2 0 C 1 N M.W. 201.74 c a l c u l a t e d C,65.49; H,9.99; CI,17.58; N,6.94 found C.65.66; H,9.76; CI,17.50; N,7.12 B. 4-(2-Dimethylaminoethyl)dodecahydrocyclopent[b] i n d o l e i ) u s i n g sodium metal as the base (attempted) A s o l u t i o n of dodecahydrocyclopent [b] i n d o l e (24.8 g,0.15 mole) i n 200 ml p u r i f i e d dioxane was added, under a n i t r o g e n atmosphere, to f r e s h l y cleaned sodium metal (3.4 g, 0.15 g atom), cut i n t o small 122 p i e c e s , i n a 500 ml three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , d r y i n g tube, and gas i n l e t tube. The reac-t i o n was r e f l u x e d f or 70 hr, during which time only a small amount of the sodium had re a c t e d . An excess of ethanol (100%) was c a u t i o u s l y added to the cooled r e a c t i o n mixture to destroy the unreacted sodium and the mixture was d i l u t e d to one l i t r e w i t h water. The r e s u l t a n t suspension was f i l t e r e d and the f i l t r a t e was e x t r a c t e d w i t h 3x250 ml ether. The ether s o l u t i o n was e x t r a c t e d w i t h 3x150 ml HCI (3%,) and dis c a r d e d . The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH (30%) and was ex t r a c t e d w i t h 3x100 ml ether which was then d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and the sol v e n t was removed on a r o t a r y evaporator to give a l i g h t brown o i l . D i s t i l l a t i o n of the re s i d u e at reduced pressure gave 927, recovery of dodecahydrocyclopent[b] i n d o l e . i i ) without u s i n g a base a) A s o l u t i o n of 2-dimethylaminoethyl bromide hydrobromide (2.3 g,0.01 mole) i n 20 ml ethanol ( 1 0 0 7 c . ) was heated to r e f l u x i n a 50 ml b o i l i n g f l a s k f i t t e d w i t h a condenser, dropping f u n n e l , and d r y i n g tube. The s o l u t i o n was s t i r r e d m a g n e t i c a l l y . A s o l u t i o n of dodeca-hydrocyclopent [b] i n d o l e (1.65 g,0.01 mole) i n 10 ml ethanol (100%,) was added dropwise to the r e a c t i o n f l a s k , g i v i n g an immediate white pre-c i p i t a t e . The r e a c t i o n mixture was r e f l u x e d f o r two hr a f t e r a d d i t i o n was completed and was then cooled to room temperature and f i l t e r e d . The f i l t r a t e was d i l u t e d w i t h 250 ml water and was made a c i d i c w i t h HCI ( 5 7 o ) . The a c i d i c s o l u t i o n was ex t r a c t e d w i t h 3x50 ml ether which was d i s c a r d e d . 123 The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH (30%) and was ex t r a c t e d w i t h 3x50 ml ether which was d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and solvent was removed on a r o t a r y evaporator to g i ve a c o l o u r l e s s o i l . D i s t i l l a t i o n of the res i d u e at reduced pressure gave 73% recovery of dodecahydrocyclopent[b] i n d o l e . The amorphous white s o l i d which p r e c i p i t a t e d during the r e -a c t i o n had a me l t i n g p o i n t i n excess of 303° and contained halogen ( B r ) . I t was i n s o l u b l e i n ether, but was f r e e l y s o l u b l e i n water. No pre-c i p i t a t e was formed when the aqueous s o l u t i o n was made b a s i c w i t h NaOH ( 5 % ) . The i n f r a - r e d spectrum (KBr) showed very weak NH s t r e t c h i n g and di d not have the c h a r a c t e r i s t i c f i n g e r p r i n t r e g i o n which i s common to the saturated c y c l o a l k [ b ] i n d o l e s . I t was t e n t a t i v e l y i d e n t i f i e d as the dimer (or polymer) of 2-dimethylaminoethy1 bromide hydrobromide. b) A s o l u t i o n of dodecahydrocyclopent[b] i n d o l e (1.6 g,0.01 mole) i n 20 ml ethanol (1007*) was heated to r e f l u x i n a 100 ml four-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and dr y i n g tube. A s o l u t i o n of 2-dimethylaminoethy1 bromide hydrobromide (2.33 g,0.01 mole) i n 50 ml ethanol (100%) was added dropwise to the r e a c t i o n mixture and the mixture was r e f l u x e d for one hr and s t i r r e d at room temperature f o r 42 hr. The r e a c t i o n was worked up as i n a) above to give 46% recovery of dodecahydrocyclopent [ b ] i n d o l e . c) A suspension of 2-dimethylaminoethy1 c h l o r i d e (6.5 g,0.06 mole) i n 100 ml anhydrous benzene was added dropwise to a r e f l u x i n g s o l u t i o n of dodecahydrocyclopent [ b j i n d o l e (20 g,0.12 mole) i n 50 ml anhydrous benzene i n a 250 ml three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and d r y i n g tube. The r e a c t i o n mixture was 124 r e f l u x e d f o r 34 hr, cooled, and d i l u t e d to 500 ml w i t h ether. The suspension was then e x t r a c t e d w i t h 3x250 ml water, adding s u f f i c i e n t HCI ( 5 7 o ) to each e x t r a c t i o n to give an a c i d i c r e a c t i o n to l i t m u s . The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH (30%) and was ex t r a c t e d w i t h 3x125 ml ether which was d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and sol v e n t was removed on a r o t a r y evaporator to give a l i g h t y e l l o w o i l . Attempted d i s t i l l a t i o n of the re s i d u e at atmospheric pressure r e s u l t e d i n the p r e c i p i t a t i o n of a white s o l i d . The r e s i d u e was r e f l u x e d f o r three hr and the s o l i d was f i l t e r e d o f f and washed w i t h ether. The i n f r a - r e d spectrum and melti n g p o i n t of the s o l i d i n d i c a t e d that i t was dodecahydrocyclopent[b]indole hydro-c h l o r i d e . The combined f i l t r a t e s were d i s t i l l e d at atmospheric pressure to remove the ether wash solvent and then at reduced pressure to gi v e a c o l o u r l e s s o i l , b Q 5 97-100°. Y i e l d 8.1 g (49.5%). Recovery of dodecahydrocyclopent [b] i n d o l e 12.5 g (62.5%,) i r ( l i q u i d f i l m ) ; 2940,2865,2830,2780,1455,1275,1220,1155,1110,1055, 950,860 cm" 1 The nmr spectrum (neat) showed s e v e r a l envelope bands i n the re g i o n 0.8-3.0 w i t h the c h a r a c t e r i s t i c dimethylamino band (sharp s i n -g l e t ) at 2.20. There was no change a f t e r a d d i t i o n of D 20. The i n t e -g r a t i o n curve was i n d e c i p h e r a b l e . A p o r t i o n of the d i s t i l l a t e was d i s s o l v e d i n ether, the s o l u -t i o n was d r i e d over MgSO^ and f i l t e r e d , and the s a l t was p r e c i p i t a t e d by a d d i t i o n of HCI gas. The p r e c i p i t a t e was r e c r y s t a l l i z e d from eth-anol (100%,)/anhydrous ether to give c o l o u r l e s s f l a t t e n e d needles, M.P. 125 209.0-210.7°. i r (KBr); 3460,3390,2930,2860,2600,2470,1635,1450,1410,1260,1070,1045, 1005 cm"1 A n a l y s i s f o r C ^ H ^ C l ^ M.W. 309.33 c a l c u l a t e d C,58.24; H,9.78; CI,22.93; N,9.06 found C,58.60; H,9.41; CI,22.87; N,9.07 6. 5-(2-Dimethylaminoethy1)-5,6,7,8,9,10-hexahydrocyclohept[b] -i n d o l e 5_7 A. 5,6,7,8,9,10-Hexahydrocyclohept[b]indole 68 A s o l u t i o n of cycloheptanone (16.8 g,0.15 mole) i n 55 ml g l a c i a l a c e t i c a c i d was heated to r e f l u x i n a 250 ml three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, and dropping f u n n e l . Phenylhydrazine (16.0 g,0.15 mole) was added dropwise to the r e a c t i o n f l a s k and the r e a c t i o n mixture was r e f l u x e d f o r one hr a f t e r a d d i t i o n was completed. The hot r e a c t i o n mixture was then poured i n t o a beaker and s t i r r e d v i g o u r o u s l y w h i l e c o o l i n g to 5° i n i c e . The s l u r r y was f i l t e r e d and the s o l i d m a t e r i a l was washed w i t h water. D e c o l o n i z a -t i o n and r e c r y s t a l l i z a t i o n from methanol gave c o l o u r l e s s c r y s t a l s , M.P. 142.5-143.5°. Y i e l d 20.5 g (74%) ( l i t (61) 142-144°, 74%). i r (KBr); 3400,3070,2920,2860,1470,1440,1430,1340,1320,1240,1190,1020, 960,750,590,510 cm"1 nmr (CDC1 3); <S 7.20(m,5); 2.76(e,4); 1.87(e,6) There was no change a f t e r a d d i t i o n of D^ O. B. 5-(2-Dimethylaminoethy1)-5,6,7,8,9,10-hexahydrocyclohept[b] -in d o l e 126 A s o l u t i o n of 5,6,7,8,9,10-hexahydrocyclohept [ b ] i n d o l e (19.4 g,0.105 mole) i n 250 ml benzene was added, under a n i t r o g e n atmosphere, to a suspension of sodium amide i n 50 ml of benzene i n a one l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , d r y i n g tube, and gas i n l e t tube. The r e a c t i o n mixture was r e -fl u x e d f o r e i g h t hr, g i v i n g a brown suspension. 2-Dimethylaminoethyl c h l o r i d e (11.3 g,0.105 mole) was added dropwise to the hot suspension and the r e a c t i o n mixture was r e f l u x e d f o r 12 hr. The mixture was cooled to room temperature, 500 ml water was c a u t i o u s l y added, the mix-ture was f i l t e r e d , and the phases were separated. The organic phase was e x t r a c t e d w i t h 2x250 ml water which was di s c a r d e d , and then w i t h 3x250 ml HCI ( 3 % ) . The organic phase was d r i e d over MgSO^ and f i l t e r e d , and solvent was removed on a r o t a r y evaporator to give a l i g h t brown s o l i d . The s o l i d was d e c o l o u r i z e d and r e c r y s t a l l i z e d from methanol to give 20% recovery of 5,6,7,8,9,10-hexahydrocyclohept Tb] i n d o l e . The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH (30%) and was ex t r a c t e d w i t h 3x250 ml ether which was then d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and sol v e n t was removed on a r o t a r y evap-o r a t o r to give a brown o i l . The re s i d u e was d i s t i l l e d at reduced pressure to give a l i g h t y e l l o w o i l , b Q 5 154-158°. Y i e l d 16.3 g (59%). i r ( l i q u i d f i l m ) ; 2920,2855,2830,2780,1465,1375,1320,1275,1210,1175, 1160,1050,1025,740 cm" 1 nmr (CC1 4) ; ($7.00(m,4); 4.08(t,2,J= 8Hz); 2.74(e,4); 2.40(t,2,J = 8Hz); 2.22(s,6); 1.83(e,6) There was no change a f t e r a d d i t i o n of DO. 127 A p o r t i o n of the d i s t i l l a t e was d i s s o l v e d i n ether, the s o l u -t i o n was d r i e d over MgSO^ and f i l t e r e d , and the s a l t was p r e c i p i t a t e d by a d d i t i o n of HCI gas. R e c r y s t a l l i z a t i o n from ethanol (1007„)/anhydrous ether gave c o l o u r l e s s c r y s t a l s , M.P. 226.5-228.5°. i r (KBr); 2935,2860,2660,2600,2480,1470,1380,1320,1220,1165,975,745 cm" A n a l y s i s for C 1 7 H 2 5 C 1 N 2 M.W. 292.86 c a l c u l a t e d C,69.72; H,8.61; CI,12.11; N.9.57 found C,69.52; H,8.50; Cl,12.30; N,9.47 7. 5-(2-Dimethy laminoethy 1) t e t r adecahydrocyc lohept [b] i n d o l e 5_8 G l a c i a l a c e t i c a c i d (3.6 g,0.06 mole) was added to a s o l u t i o n of 5-(2-dimethylaminoethyl)-5,6,7,8,9,10-hexahydrocyclohept [b]indole (8 g, 0.031 mole) i n 200 ml reagent methanol i n a Paar 4511 pressure r e a c t i o n apparatus. A suspension of 57, rhodium on alumina c a t a l y s t (3 g) i n 50 ml reagent methanol was added and the r e a c t i o n mixture was s t i r r e d under 900 p s i hydrogen f o r 20 min. The apparatus was heated to 150° and s t i r r e d f or 50 hr. The apparatus was then cooled to room temperature over 12 hr, at which time the pressure was 820 p s i . The suspension was f i l t e r e d and the s o l i d m a t e r i a l was washed w i t h ether and w i t h water. The combined f i l t r a t e s were d i l u t e d to 1500 ml w i t h water and were made b a s i c w i t h NaOH (57,) . The phases were sepa-r a t e d and the aqueous phase was e x t r a c t e d w i t h 3x200 ml ether, which was added to the organic phase from the f i l t r a t e . The combined e t h e r e a l s o l u t i o n s were d r i e d over MgSO^ and f i l t e r e d , and the solvent was removed on a r o t a r y evaporator to give a yellow o i l . The r e s i d u e was d i s t i l l e d at reduced pressure to give a l i g h t y e l l o w o i l , b 1 - 2 126-127.5°. Y i e l d 5.7 g (777,). 128 i r ( l i q u i d f i l m ) ; 2920,2850,1440,1355,1255,1140,1060,1040 cm"1 The nmr spectrum was very poorly d e f i n e d , w i t h a l l the absorption peaks ove r l a p p i n g i n the r e g i o n cfo.6-3.5. A p o r t i o n of the d i s t i l l a t e was d i s s o l v e d i n ether, the s o l u t i o n was d r i e d over MgSO^ and f i l t e r e d , and the s a l t was p r e c i p i t a t e d by the a d d i t i o n of HCI gas. The s a l t p r e c i p i t a t e d as an o i l which could not be r e c r y s t a l l i z e d . The p i c r a t e s a l t was formed and r e c r y s t a l l i z e d from ethanol to give dark y e l l o w c r y s t a l s , M.P. 204.0-205 .5°d. i r (KBr); 2925,2860,1625,1610,1565,1430,1365,1320,1270,1165,1080,920 cm A n a l y s i s for C„ H - N O , M.W. 722.66 29 38 8 14 c a l c u l a t e d C,48.20; H,5.30; N,15.51; 0,31.00 found C,48.34; H,5.38; N,15.36 8. 9-(2-Dimethylaminoethyl)f luorene _59 A. using potassium jt-butoxide as the base F r e s h l y cleaned potassium metal (10 g,0.25 g atom), cut i n t o s m a ll p i e c e s , was reacted w i t h 150 ml reagent _t-butyl a l c o h o l i n a one l i t r e b o i l i n g f l a s k f i t t e d w i t h a condenser, dropping f u n n e l , and dry-ing tube. The r e a c t i o n mixture was s t i r r e d m a g n e t i c a l l y . When a l l the potassium had reacted the excess s o l v e n t was d i s t i l l e d o f f at reduced pressure. A s o l u t i o n of fluorene (41.7 g,0.25 mole) i n 300 ml anhydrous benzene was then added to the s o l i d butoxide and the r e s u l t a n t s o l u t i o n was r e f l u x e d f o r ten min. A s o l u t i o n of 2-dimethylaminoethy1 bromide hydrobromide (28.8 g,0.125 mole) i n 300 ml dimethylformamide was then added dropwise to the r e f l u x i n g r e a c t i o n mixture. The mixture was r e -fl u x e d f or two hr a f t e r the a d d i t i o n was completed and s t i r r e d at room 129 temperature for 14 hr. The r e a c t i o n mixture was f i l t e r e d and the so l v e n t s were r e -moved on a r o t a r y evaporator. The r e d d i s h brown s o l i d r e s i d u e was suspended i n one l i t r e ether and was ex t r a c t e d w i t h 3x150 ml NaOH ( 5 % ) , which was di s c a r d e d . The ether phase was then e x t r a c t e d w i t h 3x150 ml HCI (57») and was d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and solvent was removed on a r o t a r y evaporator to gi v e a ye l l o w s o l i d r esidue of unreacted f l u o r e n e . This was d e c o l o u r i z e d and the excess fluorene was recovered by r e c r y s t a l l i z a t i o n from etha-n o l and reduced pressure s u b l i m a t i o n . The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH (257.) and was ex t r a c t e d w i t h 3x100 ml ether which was then d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and sol v e n t was removed on a r o t a r y evap-o r a t o r to gi v e a brown o i l . D i s t i l l a t i o n of the re s i d u e at reduced pressure gave a y e l l o w o i l , bp ^ 140-145°. Y i e l d 3.5 g (12.47,) . i r ( l i q u i d f i l m ) ; 3080,3050,3030,2950,2870,2830,2780,1460,1450,1265, 1145,1105,1045,850,745 cm" 1 nmr (CC1 4) ; <f 7 .24(m,8) ; 3 . 9 3 ( t , l , J = 5Hz) ; 2.09(strong s over l a p p i n g m,10) A p o r t i o n of the d i s t i l l a t e was d i s s o l v e d i n ether, the s o l u -t i o n was d r i e d over MgSO^ and f i l t e r e d , and the s a l t was p r e c i p i t a t e d by a d d i t i o n of HCI gas. The gummy brown s o l i d could not be r e c r y s t a l -l i z e d . The p i c r a t e was prepared and r e c r y s t a l l i z e d from ethanol to give feathery y e l l o w needles, M.P. 179.5-180.3°. i r (KBr); 3105,3060,2855,2780,1630,1620,1570,1495,1485,1440,1400,1370, 1335,1315,1270,1160,1145,1090,920,800,755,725 cm" 1 130 A n a l y s i s for C ^ H ^ N ^ M.W. 466.45 c a l c u l a t e d C.59.23; H.4.75; N,12.01; 0,24.01 found C,59.18; H,4.66; N,12.20 B. us i n g sodium hydride as the base (attempted) A s o l u t i o n of flu o r e n e (41.7 g,0.25 mole) i n 300 ml anhydrous benzene was added dropwise to a r e f l u x i n g suspension of sodium hydride (6.0 g,0.25 mole) i n 75 ml anhydrous benzene i n a one l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and dr y i n g tube. The r e a c t i o n mixture was then r e f l u x e d f o r f i v e days. A s o l u t i o n of 2-dimethylaminoethyl bromide hydrobromide (28.8 g,0.125 mole) i n 300 ml dimethylformamide was added dropwise to the r e f l u x i n g r e a c t i o n mixture. The mixture was r e f l u x e d for three hr a f t e r addi-t i o n was completed, then cooled and f i l t e r e d . The solve n t s were r e -moved from the f i l t r a t e on a r o t a r y evaporator to give a y e l l o w o i l which was d i s s o l v e d i n 1500 ml ether. The ether s o l u t i o n was e x t r a c t e d w i t h 3x500 ml water which was d i s c a r d e d . The ether s o l u t i o n was then e x t r a c t e d w i t h 3x100 ml HCI (57») and d r i e d over MgSO^ .. The ether s o l u t i o n was f i l t e r e d and solv e n t removed on a r o t a r y evaporator to give a dark y e l l o w r e s i d u e of unreacted f l u o r e n e which was d e c o l o u r i z e d and p u r i f i e d by sublima-t i o n . The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH (307>) and was ex t r a c t e d w i t h 2x100 ml ether which was d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and sol v e n t was removed on a r o t a r y evaporator to give a n e g l i g i b l e amount of yel l o w o i l . C. u s i n g potassium metal as the base (72,73) 131 F r e s h l y cleaned potassium metal (10 g,0.25 g atom), cut i n small p i e c e s , was suspended i n 100 ml p u r i f i e d dioxane i n a one l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , d r y i n g tube, and gas i n l e t tube. A s o l u t i o n of fluorene (41.7 g,0.25 mole) i n 350 ml p u r i f i e d dioxane was added and the r e a c t i o n mix-ture was r e f l u x e d under a dry n i t r o g e n atmosphere u n t i l a l l the potas-sium had reacted (about f i v e h r ) , g i v i n g a dark red-brown suspension. The r e a c t i o n mixture was then cooled to room temperature and a s o l u t i o n of 2-dimethylaminoethyl c h l o r i d e (27.0 g,0.25 mole) i n 100 ml p u r i f i e d dioxane was added dropwise to the r e a c t i o n mixture which was then r e -f l u x e d f o r 14 hr. The r e a c t i o n mixture was cooled and f i l t e r e d , the s o l i d was washed w i t h ether, and the so l v e n t s were removed from the combined f i l t r a t e s on a r o t a r y evaporator, g i v i n g a yellow-green o i l . The r e s i d u e was suspended i n 900 ml water and was e x t r a c t e d w i t h 4x200 ml ether. The ether s o l u t i o n was e x t r a c t e d w i t h 3x175 ml HCI (5%) and was d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and s o l v e n t was removed on a r o t a r y evaporator to give a dark y e l l o w s o l i d . Unreacted fluorene was recovered from the resi d u e by reduced pressure s u b l i m a t i o n a f t e r d e c o l o u r i z a t i o n . The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH (25%) and was ex t r a c t e d w i t h 3x200 ml ether which was then d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and solvent was removed on a r o t a r y evap-o r a t o r to gi v e an amber o i l . The res i d u e was d i s t i l l e d at reduced pressure to give two f r a c t i o n s , both y e l l o w o i l s , c o n t a i n i n g a mixture of the d e s i r e d compound w i t h 9,9-bis(2-dimethylaminoethy1)fluorene 61. F r a c t i o n A, 14 g, b Q 145-150 3, 65% of 59; 35% of 61, 132 from the nmr spectrum. F r a c t i o n B, 13.2 g, ^ 170-175°, n e a r l y pure 61, from the nmr spectrum, nmr ( C C l ^ , F r a c t i o n B) ; C^7.60(m,2); 7.34(m,6); 2.21 and 2.17 (over-lapped t,4 protons t o t a l , l a r g e i n t e n s i t y b u i l d u p , J = 6 and 5Hz); 1.93(s,12); 1.43 (overlapped t , 4 , J = 6,7) The i n f r a - r e d s p e c t r a (10% i n C C l ^ , 0.5 mm c e l l ) were i d e n t i -c a l f o r both f r a c t i o n s , except for s l i g h t v a r i a t i o n s i n i n t e n s i t y of s e v e r a l of the minor peaks. The s p e c t r a of both f r a c t i o n s as t h i n f i l m s , and as C C l ^ s o l u t i o n s , were the same as the spectrum obtained f o r the compound as p r e v i o u s l y synthesized (8,A). Separation of F r a c t i o n A was attempted u n s u c c e s s f u l l y by pre-p a r a t i v e vapour phase chromatography. The s t a t i o n a r y phase was UCON polar 50 HB660 (33% on u l t r a p o r t , 60/70 mesh) i n a 5 foot by 5/8 i n c h n i c k e l - c o a t e d brass column, i n a Beckmann GC-2 instrument. The c a r r i e r gas was helium at an i n l e t pressure of 40 p s i ; column temperatures were 160° and 190°. The d i s t i l l a t e was i n j e c t e d as a 50% s o l u t i o n i n etha-n o l . Only the s o l v e n t peak was detected. F r a c t i o n B p a r t i a l l y c r y s t a l l i z e d on standing at room temper-ature. R e c r y s t a l l i z a t i o n was attempted from a number of s o l v e n t s w i t h -out success. i r (KBr, F r a c t i o n B); 3070,3040,2995,2960,2940,2860,2820,2780,1460,1450, 1260,1180,1165,1150,1105,1045,865,785,750 cm"1 A p o r t i o n of F r a c t i o n B was d i s s o l v e d i n ethanol and the p i c r a t e was p r e c i p i t a t e d . R e c r y s t a l l i z a t i o n from ethanol gave very 133 f i n e , y e l l o w , matted needles, M.P. 230.0-231.5°. i r (KBr); 3030,2740,1635,1615,1570,1550,1440,1370,1325,1280,1170,1085, 925,755,725 cm" 1 A n a l y s i s f o r C^H,,. N o0. . M.W. 766.68 33 34 8 14 c a l c u l a t e d C,51.70; H,4.47; N,14.62; 0,29.22 found C,51.58; H,4.40; N,14.38 D. u s i n g sodium amide as the base (72,75) A s o l u t i o n of f l u o r e n e (41.7 g,0.25 mole) i n 400 ml d e c a l i n , p r e v i o u s l y d i s t i l l e d from sodium, was gently r e f l u x e d , under a dry n i t r o g e n atmosphere, w i t h sodium amide (10.0 g,0.25 mole) i n a one l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , d r y i n g tube, and gas i n l e t tube. A f t e r four hr a b l a c k , gummy mass had p r e c i p i t a t e d . This m a t e r i a l i n t e r f e r e d w i t h the s t i r r e r so s t i r r i n g was d i s c o n t i n u e d w h i l e the r e a c t i o n mixture was r e f l u x e d f o r one more hr. The mixture was then cooled and the s o l v e n t was removed through a s i n t e r e d g l a s s gas d i s p e r s i o n tube attached to an a s p i r a t o r . The b l a c k p a r t i a l l y c r y s t a l l i n e mass was washed twice, by d e c a n t a t i o n , w i t h 75 ml hexane p r e v i o u s l y d r i e d over sodium. The s o l i d was suspended i n 75 ml d r i e d hexane and broken up by s t i r r i n g v i g o u r o u s l y . A s o l u t i o n of 2-dimethylaminoethyl c h l o r i d e (29 g,0.25 mole) i n 100 ml d r i e d hexane was added dropwise and the mixture was r e f l u x e d f o r 15 hr. The r e a c t i o n mixture was cooled and 700 ml water was c a u t i o u s l y added. The r e s u l t a n t suspension was mixed w i t h 500 ml ether and then f i l t e r e d . The phases were separated and the ether s o l u -t i o n was washed w i t h 3x150 ml water which was added to the aqueous 134 phase. The s t r o n g l y b a s i c aqueous s o l u t i o n was then e x t r a c t e d w i t h 3x150 ml ether which was added to the ether s o l u t i o n . The ether s o l u t i o n was e x t r a c t e d w i t h 3x150 ml HCI (3%) and d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and so l v e n t was r e -moved on a r o t a r y evaporator to gi v e a dark y e l l o w r e s i d u e . A small amount of fl u o r e n e was recovered from the r e s i d u e by reduced pressure s u b l i m a t i o n a f t e r d e c o l o u r i z i n g . The a c i d s o l u t i o n was made b a s i c w i t h NaOH (307.) and was ex-t r a c t e d w i t h 3x150 ml ether which was d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and the solvent was removed on a r o t a r y evapora-tor to gi v e a dark brown o i l . The re s i d u e was d i s t i l l e d at reduced pressure to give two f r a c t i o n s , both y e l l o w o i l s . F r a c t i o n 1, 22.1 g b Q 2 5 Q 4 133-138°, 857, of 59 and 157, of j31 (from nmr) F r a c t i o n 2, 5.0 g, b Q 4 138-152°, 45% of 59 and 55% of 61 (from nmr) F r a c t i o n s 1 and 2 were combined w i t h F r a c t i o n s A and B from the r e a c t i o n of flu o r e n e u s i n g potassium metal (8,C) and the mixture was d i s t i l l e d at reduced pressure very slowly through a heated Vigreaux column. S i x f r a c t i o n s were c o l l e c t e d , a l l c o n t a i n i n g a mixture of the mono- and d i - s u b s t i t u t e d f l u o r e n e compounds. E. u s i n g sodium amide as the base A s o l u t i o n of flu o r e n e (41.7 g,0.25 mole) i n 400 ml anhydrous d e c a l i n was gently r e f l u x e d , under a n i t r o g e n atmosphere, w i t h sodium amide (8 g,0.2 mole) i n a one l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , d r y i n g tube, and gas 135 i n l e t tube. A f t e r f i v e hr the r e a c t i o n mixture was cooled and the ne a r l y c l e a r y e l l o w d e c a l i n s o l u t i o n was decanted from the r u s t coloured mass which had p r e c i p i t a t e d as a gum and s o l i d i f i e d on c o o l i n g . The p r e c i p i t a t e was washed twice w i t h 75 ml sodium d r i e d hexane and then broken up by s t i r r i n g very v i g o u r o u s l y w i t h 75 ml hexane. A s o l u t i o n of 2-dimethylaminoethyl c h l o r i d e (8 g,0.075 mole) i n 50 ml anhydrous hexane was added dropwise to the r e a c t i o n mixture and the mixture was r e f l u x e d f o r 14 hr. The r e a c t i o n mixture was cooled and 500 ml water was c a u t i o u s l y added. The mixture was s t i r r e d w i t h 250 ml ether and f i l t e r e d , and the phases were separated. The aqueous phase was e x t r a c t e d w i t h 2x150 ml ether which was added to the i n i t i a l ether phase. The combined ether s o l u t i o n was e x t r a c t e d w i t h 3x200 ml HCI (3%) and d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and sol v e n t was removed on a r o t a r y evaporator to give a dark y e l l o w s o l i d . Unreacted f l u o r e n e was recovered from the re s i d u e by reduced pressure s u b l i m a t i o n a f t e r d e c o l o u r i z i n g . The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH ( 3 0 7 o ) and was ex t r a c t e d w i t h 3x150 ml ether. The ether s o l u t i o n was d r i e d over MgSO^ and f i l t e r e d , and the solvent was removed on a r o t a r y evaporator to giv e a y e l l o w o i l which deposited cubic c r y s t a l s on standing. Recrys-t a l l i z a t i o n from hexane gave a c o l o u r l e s s amorphous powder, M.P. 93-100°. R e c r y s t a l l i z a t i o n from s e v e r a l s o l v e n t s d i d not improve the mel t i n g p o i n t . D i s t i l l a t i o n of the l i q u i d r e s i d u e at reduced pressure gave a l i g h t y e l l o w o i l bQ ^ 141-144°, which p a r t i a l l y s o l i d i f i e d on standing. I r and nmr s p e c t r a were c o n s i s t e n t w i t h the d i - s u b s t i t u t e d f l u o r e n e d e r i v a t i v e . Y i e l d 8.3 g (72% based on 2-dimethylaminoethyl 136 c h l o r i d e ) . The s o l i d was e v e n t u a l l y s u c c e s s f u l l y r e c r y s t a l l i z e d from d i l u t e acetone to give a c o l o u r l e s s amorphous powder, the main p a r t of which melted at 83.4-85.0°, but which had a m e l t i n g range of 79-89°. A n a l y s i s f o r C 2 1 H 2 g N 2 M.W 308.47 . c a l c u l a t e d C,81.78; H.9.15; N,9.08 found C,81.88; H,8.94; N,8.98 A p o r t i o n of the s o l i d m a t e r i a l was d i s s o l v e d i n eth e r , the s o l u t i o n was d r i e d over MgSO^ and f i l t e r e d , and the s a l t was p r e c i p i -tated by the a d d i t i o n of HCI gas. R e c r y s t a l l i z a t i o n of the gummy pre-c i p i t a t e from ethanol (1007»)/anhydrous ether gave c o l o u r l e s s c r y s t a l s , M.P. 238.0-239.2°. i r (KBr); 3015,2940,2670,2470,1470,1450,1420,1300,1165,1005,970,780, 745 cm A n a l y s i s f o r C 2 1 H 3 0 C l 2 N 2 M.W. 381.40 c a l c u l a t e d C,66.13; H.7.93; CI,18.59; N,7.35 found C,66.08; H,7.95; CI,18.74; N.7.21 F. u s i n g potassium metal as the base A s o l u t i o n of f l u o r e n e (50.0 g,0.3 mole) i n 500 ml p u r i f i e d dioxane was re a c t e d , under a n i t r o g e n atmosphere, w i t h f r e s h l y cleaned potassium (8.0 g,0.2 g atom) i n a one l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , d r y i n g tube, and gas i n l e t tube. The r e a c t i o n mixture was r e f l u x e d u n t i l no potassium was v i s i b l e (approximately three hr) and gave a deep red c o l l o i d a l suspen-s i o n . A s o l u t i o n of 2-dimethylaminoethy1 c h l o r i d e (10.7 g,0.1 mole) i n 100 ml p u r i f i e d dioxane was then run i n t o the cooled r e a c t i o n mixture 137 and the mixture was s t i r r e d at room temperature f o r three hr. Excess f l u o r e n y l potassium was destroyed by cautious a d d i t i o n of 100 ml water, s o l v e n t s were removed on a r o t a r y evaporator, and the r e s i d u e was par-t i t i o n e d between 1000 ml ether and 500 ml water. The phases were sep-arated and the ether s o l u t i o n was e x t r a c t e d w i t h 2x250 ml water. The ether phase was then e x t r a c t e d w i t h 3x150 ml HCI ( 3 7 o ) and d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and the solvent was removed on a r o t a r y evaporator to g i v e a y e l l o w s o l i d . Unreacted f l u o r e n e was recovered from t h i s r e s i d u e by s u b l i m a t i o n at reduced pressure a f t e r d e c o l o u r i z i n g . The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH ( 3 0 7 o ) and was e x t r a c t e d w i t h 3x150 ml et h e r , which was d r i e d over MgSO^ .. The ether s o l u t i o n was f i l t e r e d and solvent was removed on a r o t a r y evaporator to give a y e l l o w o i l . D i s t i l l a t i o n of the r e s i d u e at reduced pressure gave a l i g h t y e l l o w o i l , b^ 128°. I r and nmr s p e c t r a were c o n s i s t e n t w i t h the d e s i r e d compound. Y i e l d 7.0 g (30%, based on 2-dimethylamino-e t h y l c h l o r i d e ) . A p o r t i o n of the d i s t i l l a t e was d i s s o l v e d i n ether, the s o l u -t i o n was d r i e d over MgS04 and f i l t e r e d , and the s a l t was p r e c i p i t a t e d by a d d i t i o n of HCI gas. R e c r y s t a l l i z a t i o n from ethanol (1007)/anhydrous ether gave c o l o u r l e s s c r y s t a l s , M.P. 154.9-156.3°. i r ( K Br); 3060,3005,2930,2660,2570,2470,1470,1450,1420,1170,1160,1140, 1030,970,760,750 cm" 1 A n a l y s i s f o r c 1 7 H 2 0 c l N M - w - 273.81 c a l c u l a t e d C,74.57; H,7.36; Cl,12.95; N,5.12 found C,74.45; H,7.18; Cl.12.96; N,5.10 138 9. 9-(2-Dimethylaminoethyl)dodecahydrofluorene and 9,9-bis(2-dimethylaminoethyl)dodecahydrofluorene (attempted) An inseparable mixture (51.7 g) c o n t a i n i n g , as determined from the nmr spectrum, 9-(2-dimethylaminoethy1)fluorene (28.9 g,0.12 mole) and 9,9-bis(2-dimethylaminoethy1)fluorene (22.9 g,0.08 mole) was d i s s o l v e d i n 300 ml t e t r a h y d r o f u r a n i n a Paar 4511 pressure r e a c t i o n apparatus. G l a c i a l a c e t i c a c i d (17.2 g,0.28 mole) and 57» rhodium on alumina c a t a -l y s t (10 g) were added and the mixture was s t i r r e d under 1475 p s i hy-drogen and heated to 85°, when the pressure was 1650 p s i . A f t e r 20 hr the pressure had dropped to 1600 p s i . The apparatus was heated to 108° f o r 20 hr (1650 p s i ) and then to 120° f o r 24 hr (1700 p s i ) . The appa-r a t u s was cooled to room temperature. The pressure at t h i s time was 1190 p s i . The r e a c t i o n mixture was f i l t e r e d and the s o l i d s were washed w i t h 100 ml t e t r a h y d r o f u r a n . Fresh c a t a l y s t (3 g) was added and the mixture was s t i r r e d under 1500 p s i hydrogen. The apparatus was heated to 68° fo r 22 hr (1675 p s i ) , to 105° f o r 24 hr (1625 p s i ) , and to 110° f o r 24 hr (1535 p s i ) . A f t e r c o o l i n g to room temperature the pressure was 1125 p s i . A slow leak i n the apparatus was r e p a i r e d and the r e a c t i o n mixture was s t i r r e d under 1500 p s i hydrogen and heated to 85° f o r 44 h r , at which time the pressure was 1800 p s i . The apparatus was then cooled to room temperature (the pressure at room temperature could not be determined s i n c e the s e a l i n g cones on the s t i r r e r s h a f t of the appara-tus d i s i n t e g r a t e d during the c o o l i n g i n t e r v a l ) . The r e a c t i o n mixture was f i l t e r e d , the s o l i d was washed w i t h ether, and the s o l v e n t s were removed from the combined f i l t r a t e s on a r o t a r y 139 evaporator. The r e s i d u a l l i g h t brown o i l was suspended i n 500 ml water and the suspension was made a c i d w i t h d i l u t e HCI (1:1). The a c i d i c phase was e x t r a c t e d w i t h 3x150 ml ether which was di s c a r d e d . The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH (30%) and was e x t r a c t e d w i t h 3x150 ml ether which was then d r i e d over MgSO^. The ether s o l u t i o n was f i l -t ered and the sol v e n t was removed on a r o t a r y evaporator to give a n e a r l y c o l o u r l e s s o i l . The r e s i d u e was d i s t i l l e d at reduced pressure to give the two s t a r t i n g m a t e r i a l s (recovery of 9,9-bis(2-dimethylamino-e t h y 1 ) f l u o r e n e 14 g, 60%) and a s l i g h t l y y e l l o w o i l , b Q Q 5 99°. This was determined to be 9-(2-dimethylaminoethyl)-l,2,3,4,4a,9a-hexahydro-f l u o r e n e 60. Y i e l d 15 g (51%). i r ( l i q u i d f i l m ) ; 3065,3040,3020,2920,2850,2810,2760,1460,1445,1385, 1270,1180,1160,1140,1105,1045,1030,855,750 cm - 1 nmr ( C C l 4 ) ; £7.04(s,4); 3.02(e,3); 2.38(e,2); 2.20(s,6); 1.42(e,10) Se v e r a l other compounds were detected by vapour phase chromato-graphy as i m p u r i t i e s i n the d i s t i l l a t e f r a c t i o n s . These were not i d e n t i f i e d or i s o l a t e d . A p o r t i o n of the p a r t i a l l y reduced compound was d i s s o l v e d i n ether, the s o l u t i o n was d r i e d over MgSO^ and f i l t e r e d , and the s a l t was pre-c i p i t a t e d by a d d i t i o n of HCI gas. R e c r y s t a l l i z a t i o n from ethanol (100%)/ anhydrous ether gave c o l o u r l e s s c r y s t a l s , M.P. 190.2-191.5°. i r (KBr); 2920,2860,2670,1475,1460,1450,970,770,750cm" 1 A n a l y s i s f o r C 1 7H 2 6C1N M.W. 279.86 c a l c u l a t e d C,72.96; H,9.37; CI,12.67; N,5.01 found C.73.33; H,9.07; CI,12.51; N,5.16 10. N-(2-dimethylaminoethyl)diphenylamine 62 140 A s o l u t i o n of diphenylamine (68 g,0.4 mole) i n 200 ml benzene was r e f l u x e d f o r 24 hr under a n i t r o g e n atmosphere w i t h sodium amide (15.6 g,0.4 mole) i n a one l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , d r y i n g tube, and gas i n l e t tube. Heating was di s c o n t i n u e d and a s o l u t i o n of 2-dimethylaminoethy1 bromide hydrobromide (46.5 g,0.2 mole) i n 100 ml dimethyIformamide was added dropwise. The r e a c t i o n mixture was r e f l u x e d f o r 16 hr and f i l t e r e d , and the s o l v e n t s were removed on a r o t a r y evaporator to give a dark brown s o l i d . The r e s i d u e was suspended i n 750 ml water and the s t r o n g l y b a s i c suspension was e x t r a c t e d w i t h 3x250 ml ether. The ether s o l u t i o n was ex t r a c t e d w i t h 3x250 ml HCI (57,), g i v i n g a large amount of p r e c i p i t a t e i n the aqueous phase. This m a t e r i a l was f i l t e r e d o f f and washed w i t h ether. The ether s o l u t i o n s were combined and d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and sol v e n t was removed on a r o t a r y evapo-r a t o r to gi v e a brown s o l i d . The res i d u e was p u r i f i e d by s u b l i m a t i o n at reduced pressure to give 50 g (73.57, recovery) dipheny lamine. The a c i d i c s o l u t i o n and the p r e c i p i t a t e were combined and s t i r r e d m a g n e t i c a l l y w h i l e the suspension was made b a s i c w i t h NaOH (307,). The b a s i c suspension was then e x t r a c t e d w i t h 3x250 ml ether which was d r i e d over MgSO^. The ether s o l u t i o n was f i l t e r e d and sol v e n t was removed on a r o t a r y evaporator to give a dark brown o i l . The re s i d u e was d i s -t i l l e d at reduced pressure to give a s l i g h t l y y e l l o w o i l , b 127-130°. 0. o The d i s t i l l a t e p r o g r e s s i v e l y darkened through reddish-brown to n e a r l y b l a c k on exposure to l i g h t i n a d e s i c c a t o r . Y i e l d 10.5 g (227, based on 2-dimethylaminoethy1 bromide hydrobromide). 141 i r ( l i q u i d f i l m ) ; 2985,2960,2870,2835,2780,1590,1500,1465,1370,1255, 1185,1160,1050,760,705 cm" 1 nmr (CC1 4); £ 7.00(m,10); 3.77(t,2,J= 7.5Hz); 2.46(t,2,J= 7.5Hz); 2.18(s,6) There was no change a f t e r a d d i t i o n of D2O. A p o r t i o n of the d i s t i l l a t e was d i s s o l v e d i n ether, the s o l u t i o n was d r i e d over MgSO^ and f i l t e r e d , and the s a l t was p r e c i p i t a t e d by a d d i t i o n of HCI gas and r e c r y s t a l l i z e d from ethanol (1007»)/anhydrous ether to g i v e c o l o u r l e s s c r y s t a l s , M.P. 255.0-256.5°. i r (KBr); 2960,2680,2660,2595,2525,2490,1590,1500,1370,1270,1250,755, 710 cm" 1 A n a l y s i s f o r C 1 6 H 2 1 C 1 N 2 M.W. 276.81 c a l c u l a t e d C,69.42; H,7.65; CI,12.81; N,10.12 found C,69.21; H,7.45; CI,12.89; N,10.07 11. N-(2-dimethylaminoethyl)dicyclohexylamine 63 A. without u s i n g a base (attempted) A suspension of 2-dimethylaminoethy1 c h l o r i d e (5 g,0.047 mole) i n 50 ml anhydrous benzene was added dropwise to a s o l u t i o n of d i c y c l o -hexylamine (17.0 g,0.095 mole) i n 50 ml anhydrous benzene i n a 150 ml four-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and d r y i n g tube. The r e a c t i o n mixture was r e f l u x e d f or 68 hr during which a large amount of white s o l i d p r e c i p i t a t e d . The suspen-s i o n was cooled and f i l t e r e d and the solvent was removed by d i s t i l l a -t i o n . During the d i s t i l l a t i o n more white s o l i d p r e c i p i t a t e d . When a l l the solvent had been removed, the condenser was i n v e r t e d and the r e s i -due was r e f l u x e d f o r 16 hours. The r e a c t i o n mixture was f i l t e r e d and 1 4 2 the f i l t r a t e was d i s t i l l e d at reduced pressure to give d i c y c l o h e x y l -amine . The white s o l i d was found to be dicyclohexylamine hydrochlor-ide . B. using potassium metal as the base (attempted) F r e s h l y cleaned potassium ( 4 . 0 g , 0 . 1 g atom), cut i n t o s m a l l p i e c e s , was suspended i n 5 0 ml p u r i f i e d dioxane under a n i t r o g e n a t -mosphere i n a 1 5 0 ml four-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , d r y i n g tube, and gas i n l e t tube. A s o l u -t i o n of dicyclohexylamine ( 1 8 . 1 g , 0 . 1 mole) i n 5 0 ml p u r i f i e d dioxane was added and the r e a c t i o n mixture was r e f l u x e d f o r 2 0 hr. A f t e r t h i s time, a lar g e amount of potassium was s t i l l present. An a d d i t i o n a l 1 8 g dicyclohexylamine was added and the mixture was r e f l u x e d f o r 7 2 h r , a f t e r which only a small amount of potassium had r e a c t e d . The r e a c t i o n mixture was cooled to room temperature, excess potassium was destroyed by the caut i o u s a d d i t i o n of ethanol ( 1 0 0 7 . ) , and dicyclohexylamine was recovered ( 8 5 7 o ) by d i s t i l l a t i o n at reduced pressure. C. CX -chloro-N,N-dicyclohexylacetamide A s o l u t i o n of c h l o r o a c e t y l c h l o r i d e ( 1 1 . 3 g , 0 . 1 mole) i n 7 5 ml anhydrous benzene was heated to a slow r e f l u x i n a 2 5 0 ml three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and dr y i n g tube. Heat was removed and a s o l u t i o n of dicyclohexylamine ( 3 6 . 2 g , 0 . 2 mole) i n 7 5 ml anhydrous benzene was added at a r a t e s u f f i c i e n t to m aintain r e f l u x i n g . The r e a c t i o n mixture was s t i r r e d , without heat-i n g , f o r one hr more and f i l t e r e d . 143 The s o l i d m a t e r i a l was d i s s o l v e d i n 1600 ml water, to which 5 ml HCI (5%) had been added. The s o l u t i o n was e x t r a c t e d w i t h 3x100 ml ether which was d r i e d over MgSO^. The benzene s o l u t i o n ( f i l t r a t e ) was e x t r a c t e d w i t h 200 ml HCI ( 5 % ) , g i v i n g an i n t e r f a c i a l emulsion. The emulsion was drained o f f w i t h the aqueous phase and the benzene was e x t r a c t e d w i t h 2x200 ml HCI ( 5 7 o ) which was added to the emulsion phase. The benzene s o l u t i o n was d r i e d over MgSO^. The combined emulsion and a c i d i c s o l u t i o n was d i l u t e d to 1600 ml w i t h water and was e x t r a c t e d w i t h 2x200 ml ether which was then d r i e d over MgSO^. The three organic s o l u t i o n s were f i l t e r e d and the so l v e n t s were removed on a r o t a r y evaporator to give grey green s o l i d s , a l l c o n t a i n i n g halogen. The combined residues were r e c r y s t a l l i z e d from ethanol to g i v e c o l o u r l e s s c r y s t a l s , M.P. 112.6-113.2°. Y i e l d 19.3 g (75%). i r (KBr); 2940,2865,1635,1475,1460,1450,1380,1325,1140,715 cm" 1 nmr (CC1 4); £ 3 .90(s,2); 1.66(e,22) A n a l y s i s f o r C^H^ClNO M.W. 275.81 c a l c u l a t e d C,65.22; H,9.38; CI,13.75; N,5.43; 0,6.21 found C,65.46; H,9.51; CI,13.65; N,5.29 D. CT-Dimethy1amino-N,N-dicyclohexylacetamide Dimethylamine (33 ml,0.5 mole) was c o l l e c t e d i n a graduated c y l i n d e r i n an acetone/dry i c e bath and was d i s s o l v e d i n 50 ml anhydrous benzene. This s o l u t i o n was added dropwise to a s o l u t i o n of CC-chloro-N,N-dicyclohexylacetamide (16.7 g,0.065 mole) i n 150 ml anhydrous 144 benzene i n a 250 ml three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and d r y i n g tube. The r e a c t i o n mixture was s t i r r e d at room temperature for 68 hr, r e f l u x e d f o r f i v e h r, cooled to room temperature and f i l t e r e d . The s o l i d was washed w i t h benzene and the s o l v e n t was removed from the combined f i l t r a t e s on a r o t a r y evap-or a t o r to give a y e l l o w o i l which c r y s t a l l i z e d on standing. The r e s i -due was p u r i f i e d by s u b l i m a t i o n at reduced pressure to give c o l o u r l e s s c r y s t a l s , M.P. 58.5-59.5°. Y i e l d 16.6 g (96%). i r (KBr); 2925,2860,2820,2765,1630,1450,1440,1315,1130,1005,860 cm" 1 nmr (CC1 4) ; C>2.88(s,2); 2.60(e,2); 2.19(s,6); 1.5(e,20) A n a l y s i s f o r c l o H 3 o N 2 ° M.W. 266.43 c a l c u l a t e d C,72.13; H,11.35; N,10.51; 0,6.01 found 0,72.48; H,10.83; N,10.62 E. (2-Dimethylaminoethyl)dicyclohexylamine A suspension of l i t h i u m aluminum hydride (1.5 g,0.04 mole) i n 50 ml anhydrous ether was heated to r e f l u x i n a 150 ml four-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , condenser, dropping f u n n e l , and dry-i n g tube. Heat was removed and a s o l u t i o n of <X-dimethylamino-N,N-dicyclohexylacetamide (13.3 g,0.05 mole) i n 60 ml anhydr ous ether was added at a r a t e s u f f i c i e n t to maintain r e f l u x i n g . The r e a c t i o n mixture was r e f l u x e d f o r 19 hr a f t e r a d d i t i o n was completed, cooled to room temperature, and excess hydride was destroyed by the a d d i t i o n of 4.25 ml H 2S0 4 i n 25 ml water. The s o l u t i o n was decanted and the sludge was b a s i f i e d w i t h NaOH (57«) . The r e s u l t a n t g e l was f i l t e r e d i n four por-t i o n s and the s o l i d was m a g n e t i c a l l y s t i r r e d w i t h 300 ml ether f o r 15 minutes. The ether was decanted and the combined ether s o l u t i o n s were 145 washed w i t h 2x150 ml water and then e x t r a c t e d w i t h 3x100 ml HCI ( 3 % ) . The a c i d i c s o l u t i o n was made b a s i c w i t h NaOH (30%) and was ex t r a c t e d w i t h 3x150 ml ether. The ether s o l u t i o n was d r i e d over MfJO. and f i l t e r e d , and the solvent was removed on a r o t a r y evaporator to giv e a c o l o u r l e s s o i l . The re s i d u e was d i s t i l l e d at reduced pressure to give a c o l o u r l e s s o i l , b Q 1 94-98°. Y i e l d 10.7 g (84%). i r ( l i q u i d f i l m ) ; 2920,2840,2805,2760,1445,1370,1330,1265,1120,1070, 1045,1030,895,855 cm" 1 The nmr spectrum was poor l y r e s o l v e d w i t h a l l the ab s o r p t i o n s i g n a l s o v e r l a p p i n g i n the r e g i o n 1.0-2.7. A p o r t i o n of the d i s t i l l a t e was d i s s o l v e d i n eth e r , the s o l u -t i o n was d r i e d over MgSO^ and f i l t e r e d , and the s a l t was p r e c i p i t a t e d by the a d d i t i o n of HCI gas. R e c r y s t a l l i z a t i o n from ethanol (100%,)/ anhydrous ether gave c o l o u r l e s s c r y s t a l s , M.P. 216.8-217.5°. i r (KBr); 2940,2865,2560,2450,1480,1450,1025,980 cm" 1 A n a l y s i s f o r C 1 6 H 3 4 C 1 2 N 2 M.W. 325.38 c a l c u l a t e d C,59.06; H,10.53; CI,21.80; N,8.61 found C,59.06; H,10.33; CI,21.99; N,8.55 1 4 6 MISCELLANEOUS REACTIONS 1 . 2-Dimethylaminoethy1 c h l o r i d e A 2 5 0 ml three-neck f l a s k was f i t t e d w i t h a mechanical s t i r r e r , C l a i s e n s t i l l head, and a condenser set f o r d i s t i l l a t i o n i n t o an i c e cooled, tared r e c e i v e r . 2-Dimethylaminoethy1 c h l o r i d e h y d r o c h l o r i d e ( 4 8 . 0 g , 0 . 3 3 mole) was placed i n the r e a c t i o n f l a s k and f l a k e sodium hydroxide ( 2 7 . 0 g , 0 . 6 6 mole) was added. The pressure i n the system was reduced to about 3 5 mm Hg and the s t i r r e r s h a f t was slow l y r o t a t e d by hand. When the d i s t i l l a t i o n was proceeding smoothly, the s t i r r e r motor was s t a r t e d . When most of the s o l i d s had re a c t e d , the f l a s k was heated on a steam bath u n t i l no more d i s t i l l a t e was c o l l e c t e d . Y i e l d 3 1 . 2 g ( 8 8 7 o ) of c o l o u r l e s s , mobile l i q u i d . The product was g e n e r a l l y used immediately a f t e r d i s t i l l a t i o n , but could be stored i n a fr e e z e r f o r periods up to four weeks. 2. 2-Dimethylaminoethyl bromide hydrobromide ( 8 1 ) Hydrobromic a c i d ( 4 8 7 o ) ( 7 0 0 ml) was cooled i n i c e to between 0 ° and 5 ° i n a two l i t r e three-neck f l a s k f i t t e d w i t h a mechanical s t i r r e r , dropping f u n n e l , and a Vigreaux s t i l l head connected to a condenser s et fo r d i s t i l l a t i o n . 2-Dimethylaminoethanol ( 1 4 6 . 0 g , 1 . 6 4 mole) was then added dropwise, m a i n t a i n i n g the temperature at l e s s than 1 0 ° . The r e -a c t i o n mixture was then heated and 1 8 5 ml of d i s t i l l a t e was c o l l e c t e d . Heating was then decreased so that the mixture r e f l u x e d s l o w l y i n the Vigreaux column f o r one hr. A f u r t h e r 7 0 ml of d i s t i l l a t e was then c o l l e c t e d and the r e a c t i o n was again r e f l u x e d f o r one hr. This proce-dure was repeated, c o l l e c t i n g f r a c t i o n s of 8 5 , 3 0 , 1 0 , and 5 ml between 147 successive r e f l u x p e r i o d s . Each f r a c t i o n includes the amount of d i s -t i l l a t e c o l l e c t e d during the preceding r e f l u x period as w e l l as the volume c o l l e c t e d during a c t u a l d i s t i l l a t i o n . The r e a c t i o n mixture was then r e f l u x e d f o r four hr. The r e a c t i o n may be i n t e r r u p t e d at any stage up to t h i s p o i n t . F i n a l l y 230 ml of d i s t i l l a t e was c o l l e c t e d . The t o t a l volume of d i s t i l l a t e should be 615-630 ml. However, i f a f a i n t brown or v i o l e t c o lour appears i n the r e a c t i o n f l a s k , or i f white fumes are given o f f , the d i s t i l l a t i o n should be stopped immediately. The hot contents of the r e a c t i o n f l a s k were poured i n t o a one l i t r e erlenmeyer f l a s k and allowed to c o o l to about 80°. During t h i s i n t e r v a l , the r e a c t i o n f l a s k was r i n s e d w i t h 250 ml acetone which was then sl o w l y added to the s e m i - s o l i d mass i n the erlenmeyer. Acetone was then added to a t o t a l volume of about 400 ml acetone. The contents of the erlenmeyer were then w e l l mixed and cooled i n a r e f r i g e r a t o r . The c r y s t a l s were f i l t e r e d o f f , washed w i t h acetone u n t i l c o l o u r l e s s , and a i r d r i e d to remove acetone. The c r y s t a l s were stored i n a des-i c c a t o r . A f u r t h e r crop of c r y s t a l s was c o l l e c t e d by evaporating the acetone s o l u t i o n to about 100 ml and seeding the s o l u t i o n . The c r y s t a l s were r e c r y s t a l l i z e d from e t h y l a l c o h o l / e t h y l acetate (5:8) to give c o l o u r l e s s c r y s t a l s , M.P. 187-188.5°. Y i e l d 288.6 g (75.57,) ( l i t (81) 187-188°, 87.57»). i r (KBr); 3030,2950,2905,2680,2570,2495,1475,1325,1250,1020,965 cm" 1 3. P u r i f i c a t i o n of dioxane (82) Dioxane (2300 ml) was r e f l u x e d w i t h 32 ml concentrated HCI and 230 ml water i n a three l i t r e b o i l i n g f l a s k f o r e i g h t hr w h i l e a slow 148 stream of n i t r o g e n was bubbled through the r e a c t i o n . The s o l u t i o n was cooled to room temperature and KOH p e l l e t s were added u n t i l some remained u n d i s s o l v e d . The aqueous l a y e r was separated and the organic phase was d r i e d over f r e s h KOH p e l l e t s (200 g) for 12 hours. The r e a c t i o n mixture was then r e f l u x e d w i t h sodium metal (30 g) f o r ten hours and then d i s t i l l e d from the sodium to give a c o l o u r l e s s l i q u i d , b 7 f i 0 101° ( l i t (82) 101.5°). 149 PHARMACOLOGICAL TESTING PART ONE DERIVATIVES AND ANALOGUES OF 14-AZADISPIRO[5.1.5.2]PENTADECAN-15-ONE The s y n t h e s i s of the a z a d i s p i r o compounds was completed f a i r l y e a r l y i n t h i s work and these compounds were subjected to p r e l i m i n a r y t e s t s f o r pharmacological a c t i v i t y . The r e s u l t s d i d not i n d i c a t e that f u r t h e r i n v e s t i g a t i o n was warranted. As mentioned i n a previous s e c t i o n 7-dimethylaminoacetamido-14-a z a d i s p i r o [5 .1.5 .2] pentadecane 25. a n c* 8-dimethylaminoacetamido-16-azadispiro[6.1.6.2 ]heptadecane 30 were i n c o r r e c t l y i d e n t i f i e d as the isomeric products 7-(2-dimethylaminoethylamino)-14-azadispiro[5.1.5.2]-pentadecan-15-one 42 and 8-(2-dimethylaminoethylamino)-16-azadispiro-[6.1.6.2] heptadecan-17-one 43, r e s p e c t i v e l y . This e r r o r was not r e a l -i z e d at the time and the t e s t s f o r pharmacological a c t i v i t y were c a r -r i e d out as i f the l a t t e r compounds had been obtained. A. L o c a l A n e s t h e t i c A c t i v i t y The method used was e s s e n t i a l l y that of B u l b r i n g and Wajda (83). F u l l y grown guinea pigs of e i t h e r sex were used. The day previous to the experiment the h a i r on the animals' backs was c l i p p e d as short as p o s s i b l e using e l e c t r i c c l i p p e r s . This r e s u l t e d i n a degree of i n -flammation which subsided o v e r n i g h t . The compounds tested f o r l o c a l a n e s t h e t i c a c t i v i t y were 7-dimethyl-aminoacetamido-14-azadispiro [5 .1.5 .2] pentadecan-15-one 23_ and 8-dimethyl-aminoacetamido-16-azadispiro[6.1.6.2] heptadecan-17-one .28, as the hydro-c h l o r i d e s a l t s . The compounds were d i s s o l v e d i n 0.9% s a l i n e s o l u t i o n 150 at c o n c e n t r a t i o n s of 0.25, 0.5, 1.0, and 2.0%. S o l u t i o n s of 0.5 and 1.07o pontocaine h y d r o c h l o r i d e ( t e t r a c a i n e h y d r o c h l o r i d e U.S.P., Winthrop) were a l s o prepared. A constant volume of 0.25 ml of s a l i n e or of t e s t s o l u t i o n was i n j e c t e d i n t r a c u t a n e o u s l y i n t o the back of a guinea p i g , r e s u l t i n g i n the formation of a wheal approximately e i g h t mm i n diameter. This was d e l i n e a t e d w i t h i n k and a stimulus was a p p l i e d every f i v e min-utes f o r 30 minutes, and t h e r e a f t e r at 30 minute i n t e r v a l s f o r one hour. Stimulus was a p p l i e d w i t h a b i p o l a r e l e c t r o d e attached to a C.H.Stoelting e l e c t r o n i c s t i m u l a t o r d e l i v e r i n g 10 m i l l i s e c o n d pulses of 58 v o l t s at 60 pulses per second. The maximum stimulus time was three seconds. A f t e r f i v e minutes, there was no d i f f e r e n c e between the response to s t i m u l a t i o n of normal t i s s u e and s t i m u l a t i o n of a c o n t r o l wheal i n -duced by an i n j e c t i o n of s a l i n e . There was no decrease i n response to s t i m u l a t i o n of any of the wheals induced by i n j e c t i o n of the t e s t com-pounds. There was no response to s t i m u l a t i o n f o r one hour a f t e r 1.0% t e t r a c a i n e and f o r 30 minutes a f t e r 0.5% t e t r a c a i n e . B. A n t i h i s t a m i n i c A c t i v i t y (84) Compounds teste d f o r a n t i h i s t a m i n i c a c t i v i t y were 7-dimethylamino-acetamido-14-azadispiro [5 .1.5 .2] pentadecane 25_ and 8-dimethylaminoacet-amido-16-azadispiro[6.1.6.2 ] heptadecane 30, as the h y d r o c h l o r i d e s . S o l u t i o n s of agonist and t e s t compounds were prepared i n 0.9% s a l i n e . A s e c t i o n of guinea p i g ileum (described on p 163) was suspended i n a 50 ml i s o l a t e d organ bath c o n t a i n i n g Tyrode's s o l u t i o n maintained at 37°C by c i r c u l a t i n g water through the outer j a c k e t of the organ bath w i t h a thermostatic pump. The f l u i d i n the organ bath was oxygenated 151 w i t h 957, O2/57. CO2 • Cont r a c t i o n s of the i n t e s t i n e were a m p l i f i e d by means of a l i g h t i s o t o n i c lever w i t h a f r o n t a l w r i t i n g t i p t r a c i n g on a smoked kymograph paper. The lowest dose of histamine h y d r o c h l o r i d e which e l i c i t e d a maxi-mal response was then determined. The i n t e s t i n a l s t r i p was washed w i t h Tyrode's s o l u t i o n and allowed to e q u i l i b r a t e between each dose. A f t e r the c o n t r o l dose of histamine had been determined and s e v e r a l c o n t r o l responses recorded, the i n t e s t i n a l s t r i p was e q u i l i b r a t e d f o r 15 min-utes w i t h a dose of the t e s t compound. The c o n t r o l dose of histamine was then added to the bath and the response was recorded. The prepar-a t i o n was washed w i t h Tyrode's s o l u t i o n , allowed to e q u i l i b r a t e , and the c o n t r o l dose of histamine was added to determine that the prepar-a t i o n was s t i l l c o n t r a c t i n g maximally. I f the p r e p a r a t i o n showed signs of d e t e r i o r a t i o n a f r e s h s e c t i o n of i n t e s t i n e was suspended i n the bath and the c o n t r o l dose of histamine was again determined. This procedure was repeated f or a number of doses of each of the two t e s t compounds. At a c o n c e n t r a t i o n of 3.25xlO~^M f o r 25_» HCI, the response to h i s -tamine was reduced about 507c At t h i s c o n c e n t r a t i o n however, the response of the p r e p a r a t i o n became h i g h l y e r r a t i c . This was probably due to a n o n - s p e c i f i c t o x i c e f f e c t of the compound. -4 The maximum c o n c e n t r a t i o n tested f o r 3_0>HC1 was 3x10 M. There was no r e d u c t i o n i n the response to histamine at t h i s dose l e v e l , but there was a more r a p i d , e r r a t i c r e l a x a t i o n of the t i s s u e , compared w i t h the r e l a x a t i o n a f t e r a c o n t r o l , dose of histamine alone. Neither of the compounds e x h i b i t e d any a g o n i s t i c a c t i v i t y . 152 C. A n t i c h o l i n e r g i c A c t i v i t y (84) Compounds tested 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 were 7-dimethylamino-acetamido- 14-azadispiro [5.1.5.2] pentadecan-15-one methiodide 2A and 7-dimethylaminoacetamido-14-azadispiro [5.1.5.2] pentadecane dimethiodide 26. The heptadecane analogues 29_ and 3_1 were synthesized f o r t h i s pur-pose but lacked s u f f i c i e n t s o l u b i l i t y . The p r e p a r a t i o n and the procedure used were as described i n the t e s t f o r a n t i h i s t a m i n i c a c t i v i t y except that a c e t y l c h o l i n e bromide was used as the agonist i n place of histamine HCI. -4 The maximum c o n c e n t r a t i o n t e s t e d f o r both compounds was 2x10 M. There was no decrease of the maximal response at t h i s dose l e v e l . Com-pound 24 showed s l i g h t t r a n s i e n t a g o n i s t i c a c t i v i t y at high dose l e v e l s . D. General A c t i v i t y and Acute T o x i c i t y Screen (85) The compounds which were tested u s i n g t h i s procedure were 7-di-methylaminoacetamido-14-azadispiro [5.1.5.2]pentadecan-15-one 23 and 7-dimethylaminoacetamido-14-azadispiro [ 5.1.5.2 ]pentadecane Z5, as the hydrochlor i d e s , and 7-dimethylaminoacetamido-14-azadispiro [ 5.1.5.2 ] -pentadecane dimethiodide 26. S o l u t i o n s were prepared i n 0.9% s a l i n e . A constant volume of 0.5 ml was i n j e c t e d i n t r a p e r i t o n e a l l y i n t o a group of three ad u l t female mice (30-40 g ) . Doses of t e s t compound were i n -creased using a 1/2 l o g u n i t s c a l e , expressed i n mgm of t e s t compound (as the s a l t ) per kg body weight. Each group of animals was then housed i n a round glass o b s e r v a t i o n j a r c o n t a i n i n g a small amount of l i t t e r . A c o n t r o l group of animals was i n j e c t e d w i t h s a l i n e and used for comparison. The animals were 1 5 3 then observed f o r changes i n a la r g e number of p h y s i c a l responses which could i n d i c a t e pharmacological a c t i v i t y of the compounds under study. For example, c e n t r a l e f f e c t s may be i n d i c a t e d by tremors, aggres-s i v e behaviour, or other s i g n s . At the same time, p u r e l y p h y s i c a l responses such as d e f e c a t i o n , u r i n a t i o n , and changes i n heart and r e s -p i r a t o r y r a t e may be observed. Compounds 25_ and 26^ caused no observable changes i n the animals at doses which d i d not induce convulsions l e a d i n g to death. The com-pounds had an L D ^ Q of 2 3 0 and 5 7 mgm/kg r e s p e c t i v e l y . One animal sur-v i v e d convulsions induced by compound Z 5 . The animal showed no other e f f e c t s and i t s a c t i o n s were not n o t i c e a b l y abnormal f o r a p e r i o d of two weeks, a f t e r which i t was destroyed. Compound 23^ caused no observ-able changes i n the animals and had an L D i n excess of 5 0 0 mgm/kg. 154 PART TWO DERIVATIVES AND ANALOGUES OF CARBAZOLE A. E v a l u a t i o n of drug parameters (86,87,88,90) i . A gonists In order to a t t a i n a response of a b i o l o g i c a l system to a drug, a r e a c t i o n must take place between the drug molecule and a molecule i n the b i o l o g i c a l o b j e c t (the drug-receptor i n t e r a c t i o n ) . This i n t e r a c t i o n i s u s u a l l y represented as a bim o l e c u l a r r e a c t i o n R + A RA (1) where R i s the c o n c e n t r a t i o n of rec e p t o r s i n the b i o l o g i c a l o b j e c t , A i s the c o n c e n t r a t i o n of drug i n the biophase, and RA i s the c o n c e n t r a t i o n of the drug-receptor complex. The d i s s o c i a t i o n constant f o r the r e a c t i o n i s K^. The f r a c t i o n of rece p t o r s occupied, RA/R, i s dependent on A and on the a f f i n i t y of the drug f o r the receptor (1/K^). As a r e s u l t of rece p t o r occupation a stimulus i s b u i l t up. By d e f i n i t i o n , the stimulus i s d i r e c t l y propor-t i o n a l to the number of rece p t o r s occupied. SA/ Sm = cc RA/R (2) where the stimulus (S^) i s represented as a f r a c t i o n of the maximal stimulus ( S m ) which may be obtained i n a p a r t i c u l a r b i o l o g i c a l o b j e c t and oc i s the i n t r i n s i c a c t i v i t y of the drug, i . e . oc i s a measure of the a b i l i t y of the drug to cause a s t i m u l u s . The stimulus f i n a l l y g i v e s r i s e to an observable e f f e c t . This 1 5 5 may be represented by V E m * / < V S m > O ) where the e f f e c t of drug A (E^) i s given as a f r a c t i o n of the maximal e f f e c t (E m) which may be obtained w i t h the p a r t i c u l a r b i o l o g i c a l o b j e c t . This f u n c t i o n may be l i n e a r , n o n - l i n e a r , or a l l - o r - n o n e . I t should, however, be i n v a r i a n t i n the p a r t i c u l a r b i o l o g i c a l o b j e c t . The a f f i n i t y and i n t r i n s i c a c t i v i t y of an agonist may be deter-mined from the dose-response curve. The pD 2 v a l u e , which i s a measure of the a f f i n i t y , has been defined as pD 2 = - l o g A 5 Q (4) where A ^ Q i s the molar c o n c e n t r a t i o n of agonist which induces 507„ of the maximal response p o s s i b l e w i t h that a g o n i s t , i . e . the c o n c e n t r a t i o n f o r which E. /E. = 2. Am A The i n t r i n s i c a c t i v i t y i s determined by comparing the maximal e f f e c t of the agonist A ( E ^ ) w i t h the maximal e f f e c t o b t a i n a b l e i n the same b i o l o g i c a l object u s i n g a reference compound R (E^ )-0 0 " EAm / ERm <5> I t i s customary to use the endogenous a g o n i s t s , such as a c e t y l c h o l i n e , histamine, and a d r e n a l i n e , as the reference compounds. The choice of reference compound i s of course d i c t a t e d by the type of ac-t i v i t y which i s expected from the t e s t compound. C a l c u l a t e d dose-response curves f o r agonists w i t h v a r y i n g values of i n t r i n s i c a c t i v i t y and a f f i n i t y are shown i n Figure 7. 156 I ? I 10 I0 2 [A] F i g u r e 7: Dose-response curves f o r agon i s t s having d i f f e r e n t a f f i n i t i e s and i n t r i n s i c a c t i v i t i e s . Curves 1, 2, and 3 are for ag o n i s t s w i t h the same i n t r i n s i c a c t i v i t y ((X) but d i f f e r i n g a f f i n i t i e s (K ). Curves 4, 5, and 6 are f o r agonists w i t h the same a f f i n -i t y but d i f f e r i n g i n s t r i n s i c a c t i v i t i e s , (from r e f . 88, p 54) 157 i i . A ntagonists Antagonists are compounds which, when present i n a b i o l o g i c a l p r e p a r a t i o n , decrease or a b o l i s h the e f f e c t of an a g o n i s t . Antagonists may act i n a c o m p e t i t i v e or a non-competitive manner. A competitive antagonist i s a compound which, by v i r t u e of i t s a f f i n i t y f o r a r e c e p t o r , i n t e r a c t s w i t h the receptor and thus prevents the combination of the agonist w i t h that r e c e p t o r . The c o m p e t i t i v e antagonist has zero i n t r i n s i c a c t i v i t y and t h e r e f o r e does not i t s e l f g i v e r i s e to a stimulus on i n t e r a c t i o n w i t h the r e c e p t o r . For a pure co m p e t i t i v e a n t a g o n i s t , the maximal response of the t i s s u e to the agonist i s not a f f e c t e d ; however, higher doses of agonist are r e q u i r e d to a t t a i n responses e q u i v a l e n t to those a t t a i n e d i n the absence of the a n t a g o n i s t . The t h e o r e t i c a l dose-response curves of Figure 8a show the e f f e c t of i n c r e a s i n g c o n c e n t r a t i o n s of c o m p e t i t i v e a n t a g o n i s t . S c h i l d (87) has introduced.a s c a l e of drug antagonism, known as the pA s c a l e , which he has proposed as a common method of r e p o r t i n g r e s u l t s , thus a v o i d i n g d i f f i c u l t i e s which a r i s e i n r e p o r t i n g r e s u l t s of antagonist a c t i v i t y i n terms of another a n t a g o n i s t . The pA;^  v alue i s d e fined as the negative l o g a r i t h m of the molar c o n c e n t r a t i o n of a competitive antagonist which w i l l reduce the e f f e c t of a m u l t i p l e dose of an agonist to the l e v e l of e f f e c t of a u n i t dose of the agonist i n the absence of the a n t a g o n i s t . The values u s u a l l y reported are pA 2 and pA^Q, where the s u b s c r i p t r e f e r s to the m u l t i p l e dose of a g o n i s t . For example, i f p e t h i d i n e (1.6xlO~^M) reduces the e f f e c t of 2 y*% h i s -tamine to the l e v e l of e f f e c t of 1 / g histamine i n the absence of 158 F i g u r e 8a: T h e o r e t i c a l dose-response curves f o r an a g o n i s t (A) i n the presence of constant, g e o m e t r i c a l l y i n c r e a s i n g doses of a pure c o m p e t i t i v e a n t a g o n i s t ( B ) . b: T h e o r e t i c a l dose-response curves f o r an a g o n i s t (A) i n the presence of c o n s t a n t , but g e o m e t r i c a l l y i n c r e a s i n g doses of a pure non-competitive antagonist ( B ' ) . A b s c i s s a e are responses to A i n the presence of B or B 1 r e l a t i v e to the maximal response to A alone. The o r d i n a t e s are l o g A. (from r e f e r e n c e 86, p 311) 159 p e t h i d i n e , then the pA 2 p e t h i d i n e - h i s t a m i n e = 5.8. The constant d e r i v e d by t h i s method i s l i m i t e d to the p a r t i c -u l a r a g o n i s t - a n t a g o n i s t p a i r and to the b i o l o g i c a l o b j e c t used. A mean of s e v e r a l determinations should be r e p o r t e d . The pA values are depen-dent on the len g t h of time the antagonist i s i n contact w i t h the b i o -l o g i c a l o b j e c t , but are independent of the method of experimentation and of the c o n c e n t r a t i o n of agonist and antagonist used, provided the u n i t dose of agonist causes a sub-maximal c o n t r a c t i o n . The pA 2 v a l u e i s eq u i v a l e n t to the negative logarithm of the d i s s o c i a t i o n constant f o r the r e c e p t o r - a n t a g o n i s t complex (89). Since the method used by S c h i l d i s somewhat t e d i o u s , van Rossum (86) has adapted the c a l c u l a t i o n of pA 2 values so that dose-response curves may be used. A pure non-competitive antagonist has no a f f i n i t y f o r the par-t i c u l a r receptor w i t h which, the agonist i n t e r a c t s . Instead, the non-com p e t i t i v e antagonist i n t e r a c t s w i t h another receptor i n the b i o l o g i -c a l o b j e c t and i n t h i s way a f f e c t s e i t h e r stimulus formation or stimu-lu s e f f e c t u a t i o n , r e s u l t i n g i n a decrease i n the e f f e c t of the a g o n i s t . An increa s e i n the c o n c e n t r a t i o n of agonist cannot overcome the e f f e c t of a non-competitive a n t a g o n i s t , as i s shown by the t h e o r e t i c a l dose-response curves of Figure 8b. A non-competitive antagonist has an i n t r i n s i c a c t i v i t y w i t h a negative s i g n ( s i n c e i t must have some e f f e c t at i t s receptor to de-crease the response to the a g o n i s t ) . A measure of the a f f i n i t y (pD 2) of a non-competitive antagonist f o r the n o n - s p e c i f i c receptor may be c a l c u l a t e d from the decrease i n maximal height of the dose-response 1 6 0 curves. pD^ = - l o g B 5 Q ( 6 ) where B ^ Q i s the molar c o n c e n t r a t i o n of non-competitive antagonist which i s r e q u i r e d to cause a decrease of 5 0 7 o i n the maximal response to an a g o n i s t , i . e . the dose f o r which E /E D = 2. m mo The i n t e r a c t i o n s of competitive and non-competitive antagonists w i t h a receptor i n the presence of an agonist may be represented d i a -grammatically as f o l l o w s : E E R <- R ' A B' A = a g o n i s t ; B = com p e t i t i v e a n t a g o n i s t ; B ' = non-competitive a n t a g o n i s t ; E - e f f e c t ; R = s p e c i f i c receptor f o r A; R' = n o n - s p e c i f i c receptor f o r B' . In many cases, drugs are found to have m u l t i p l e a c t i o n s . For in s t a n c e , a p a r t i a l agonist (cC<^l) a l s o e x h i b i t s c o m p e t i t i v e antagon-ism i n the presence of a pure agonist ( OC = 1 ) . Dualism of antagonism, i e both c o m p e t i t i v e and non-competitive f a c e t s of antagonism, occurs when the antagonist has some a f f i n i t y f o r the s p e c i f i c receptor of the agonist as w e l l as a f f i n i t y f or some other r e c e p t o r ( s ) . This may be i l l u s t r a t e d diagrammatically by: 161 E In cases of m u l t i p l e a c t i o n , a l l of these drug parameters may be c a l c u l a t e d from the dose-response curves by the a p p l i c a t i o n of tech-niques d e s c r i b e d by van Rossum (86). Where there i s dualism of antag-onism, the pA 2 v a l u e may only be c a l c u l a t e d i f the c o m p e t i t i v e antago-nism i s of greater order than the non-competitive antagonism. B„ Cumulative Dose-Response Curves (86) Since i t i s very time consuming to determine a dose-response curve by measuring the e f f e c t of i n d i v i d u a l doses of a drug, a cumulative procedure was used. In t h i s procedure, the t o t a l dose of drug i n an organ bath i s incremented i n a stepwise f a s h i o n without washing out between doses. Dose-response curves prepared i n t h i s way are v i r t u a l l y i d e n t i c a l w i t h those prepared i n the c o n v e n t i o n a l manner. Table IX gives the volumes of agonist s o l u t i o n s which must be added to a 25 ml organ bath to give log 10 and 1/2 log 10 increments i n the t o t a l con-c e n t r a t i o n s . In t h i s work the dose response curves were always s t a r t e d at 10 and successive increments were added u n t i l there was no f u r t h e r contrac-t i o n of the i n t e s t i n a l s t r i p . The time taken to a t t a i n maximal contrac-t i o n i n response to each increment r a r e l y exceeded 20 seconds, and was log 10 to be added t o t a l cone. dose number ml of cone.(M) (M) 1/2 l o g 10 to be added ml of cone.(M) t o t a l cone. dose number (M) 0.1 25x10 10 0.1 25x10 10 -8 0.9 25x10 -7 10 -7 0.2 25x10 3x10 -8 0.09 25x10 10 -6 0.7 25x10 10 0.9 25x10 10 0.02 25x10 3x10 0.09 25x10 10 0.07 25x10 10 0.9 25x10 10 0.2 25x10 3x10 0.7 25x10 10 -5 0.2 25x10 0.7 25x10 3x10 10 -4 8 9 Table IX: Sequence of doses to be added to a 25 ml organ bath for cumulative dose-response curves. 163 u s u a l l y l e s s than 10 seconds. C. Experimental Procedure (84,86) Adul t guinea pigs of e i t h e r sex were used. The animals were f a s t e d f o r at l e a s t 12 but not more than 24 hours before use. A guinea p i g was stunned by a blow to the base of the s k u l l and exsanguinated. The abdomen was opened and the d i s t a l p o r t i o n of the small i n t e s t i n e (ileum) was removed and placed i n a beaker of c o l d Tyrode's s o l u t i o n . The upper end of the excised i n t e s t i n e was marked and s e c t i o n s 2-4 cm i n len g t h were cut from the lower p o r t i o n . I f necessary, the s e c t i o n s were allowed to r e l a x i n warm oxygenated Tyrode's s o l u t i o n and f e c a l m a t e r i a l was ge n t l y washed out w i t h a sy r i n g e f i l l e d w i t h t h i s s o l u t i o n . A s e c t i o n of the ileum was suspended i n each of four 25 ml organ baths ( l a b e l l e d 1, 2, 3, and 4) c o n t a i n i n g Tyrode's s o l u t i o n and bubbled w i t h 957, 02/57. C0 2. The temperature was maintained at 37°C by means of a thermostatic pump c i r c u l a t i n g water through the outer j a c k e t s of the baths. Record-ings were made on sooted paper on kymographs by means of a l i g h t i s o -t o n i c l e v e r f i t t e d w i t h a f r o n t a l w r i t i n g t i p . The p r e p a r a t i o n s were allowed to e q u i l i b r a t e f o r 10-20 minutes. P r e p a r a t i o n 1 was then s t i m u l a t e d w i t h a supramaximal dose of agonist and the agonist was washed out s e v e r a l times during a f i v e minute i n t e r -v a l . P r e p a r a t i o n 2 was s t i m u l a t e d w i t h the same dose of agonist f i v e minutes a f t e r 1. This was repeated at f i v e minute i n t e r v a l s f o r prep-a r a t i o n s 3 and 4, e s t a b l i s h i n g a c y c l e of twenty minutes d u r a t i o n . The c y c l e was repeated at l e a s t three times w h i l e a d j u s t i n g the r e c o r d i n g system. 164 When the responses to the agonist were c o n s i s t e n t w i t h i n each p r e p a r a t i o n , cumulative c o n t r o l responses to the agonist were recorded, m a i n t a i n i n g the 20 minute c y c l e . Generally three c o n t r o l responses f o r each p r e p a r a t i o n were recorded, although i n a large number of cases four were recorded. I f agreement between the c o n t r o l responses was p a r t i c u l a r l y good, only two responses were recorded. To maintain c o n d i t i o n s as constant as p o s s i b l e , the p r e p a r a t i o n s were not washed during the l a s t 15 minutes of the i n t e r v a l between c o n t r o l responses. While s t i l l m a i n t a i n i n g the e s t a b l i s h e d 20 minute c y c l e , a dose of t e s t compound was introduced i n t o the muscle bath c o n t a i n i n g prep-a r a t i o n 1 so that the p r e p a r a t i o n was i n contact w i t h the t e s t com-pound f o r 15 minutes before the cumulative t e s t response to the agonist was recorded. During t h i s 15 minute i n t e r v a l the f i n a l c o n t r o l responses were recorded f o r p r e p a r a t i o n s 2, 3, and 4, and the t e s t compound was introduced i n t o the muscle baths i n the same manner as f o r 1. The cumulative responses to the agonist i n the presence of t e s t compound were then recorded. At l e a s t two of the four preparations were t e s t e d f o r r e t u r n to c o n t r o l l e v e l s of response a f t e r the t e s t response was recorded. Using t h i s procedure data f o r four cumulative dose-response curves could be c o l l e c t e d i n about 3.5-4 hr. A f r e s h group of i n t e s t i n a l s t r i p s was used f o r each set of four experiments. S o l u t i o n s of agonists and antagonists were prepared i n 0.9% NaCl. Histamine stock s o l u t i o n s ( e i t h e r histamine d i h y d r o c h l o r i d e , N u t r i t i o n a l 165 B i o c h e m i c a l s , or histamine a c i d phosphate, B.P., B r i t i s h Drug Houses) were prepared every second day and d i l u t e d to the r e q u i r e d s t r e n g t h d a i l y . A c e t y c h o l i n e s o l u t i o n s ( a c e t y l c h o l i n e bromide, Eastman) were prepared d a i l y . Agonist s o l u t i o n s were stored i n a r e f r i g e r a t o r and 20 ml a l i q u o t s were withdrawn as needed. S o l u t i o n s of antagonists ( h y d r o c h l o r i d e s ) were prepared as needed and were kept i n a r e f r i g e r a -tor f o r a maximum of three days. (Two of the antagonists were not a v a i l a b l e as the h y d r o c h l o r i d e s . These were d i s s o l v e d i n a minimum volume of HCI (1%) and the r e s u l t a n t s o l u t i o n s were d i l u t e d to the c a l c u l a t e d volumes.) Standard antagonists used were diphenhydramine h y d r o c h l o r i d e (Parke, Davis & Co.) which was tested against histamine and against a c e t y l c h o l i n e , and a t r o p i n e s u l f a t e (B.P., B r i t i s h Drug Houses) which was tested against a c e t y l c h o l i n e . Each antagonist was t e s t e d at three doses, g e n e r a l l y 1/2 log u n i t apart. Each dose was t e s t e d i n four p r e p a r a t i o n s , f o r a t o t a l of 12 determinations of a c t i v i t y f o r each compound against each of the two a g o n i s t s . Tests f o r a n t i h i s t a m i n i c a c t i v i t y were c a r r i e d out using a 1/2 l o g 10 s c a l e f o r incrementing the dose of agonist w h i l e t e s t s 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 were c a r r i e d out using a l o g 10 s c a l e (see Table I X ) . Although a greater e r r o r was introduced i n p l o t t i n g the data 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 by u s i n g a log 10 s c a l e , i t was necessary to do so to prevent f a d i n g of the response at each increment of a g o n i s t . A f a i r l y l a r g e volume of agonist s o l u t i o n was added to the muscle baths during r e c o r d i n g of the responses (Table I X ) . Since a high degree of e r r o r at the higher dose ranges would have been introduced by adding 166 t h i s volume to 25 ml of Tyrode's s o l u t i o n i n the muscle baths, the baths were f i l l e d to a l i n e i n s c r i b e d at the 23.5 ml l e v e l . In t h i s way the e r r o r was d i s t r i b u t e d at the upper and lower dose l i m i t s and minimized at the more important middle dose range. D. M a n i p u l a t i o n of the Data Examples are given from the data c o l l e c t e d from t e s t s f o r a n t i h i s -taminic a c t i v i t y . Data c o l l e c t e d from t e s t s 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 were t r e a t e d i n the same manner. F i g u r e 9 i s a s c a l e drawing of the kymograph record obtained f o r one experiment. The height of c o n t r a c t i o n at each dose of agonist was measured and entered i n t o a form designed f o r the purpose (Form 1). When the data f o r a l l experiments had been c o l l e c t e d , a computer pro-gram was w r i t t e n to express the data as a percent of the maximal response. As w e l l , the average c o n t r o l response and standard e r r o r f o r each exper-iment, and over the complete set of experiments was c a l c u l a t e d at each dose of the two a g o n i s t s . The average c o n t r o l responses and the standard e r r o r s f o r the com-p l e t e sets of experiments are given i n Table X. From the data i n Table X, the mean dose-response curve f o r c o n t r o l responses was drawn and the pl>2 value f o r the agonist was c a l c u l a t e d by the method of van Rossum (86). pD 2 = - l o g A 5 Q From Figure 10, the dose of histamine which produces a 50% response i s 5.5x10 ^, g i v i n g pD 2 = 6.26. 167 P a i r s of dose response curves were then drawn, comparing the response i n the absence of antagonist to that i n the presence of antag-o n i s t f o r each experiment performed. See Figure 11. The d i s t a n c e from the 507o response l e v e l of the c o n t r o l curve to the 50% response l e v e l of the t e s t curve was measured and entered i n Form 2, which was adapted from the work of van Rossum. The percentage d i f f e r e n c e i n the maximum response between the c o n t r o l curve and the t e s t curve was entered i n Form 3, a l s o adapted from van Rossum. Values f o r the pA 2 and pD 2 of the antagonists were then c a l c u l a t e d , u s i n g t a b l e s of l o g ( x - l ) values given by van Rossum. Since log paper w i t h a c y c l e of 51.5 mm was used, i t was f i r s t necessary to express x i n terms of a c y c l e of 30 mm (as used by van Rossum). The r e s u l t s are given i n Table X I . F i g u r e 9: Responses of guinea p i g ileum to cumulative doses of histamine. The numbers r e f e r to dose of histamine, see Table IX. ' - A d d i t i o n of 3xlO~ 5M 5-(2-dimethylaminoethy 1)-t e t r a d e c a h y d r o c y c l o h e p t [ b j i n d o l e h y d r o c h l o r i d e to the muscle bath 15 minutes before the response was recorded. Compound: 5-(2-dimethylaminoethy1)tetradecahydrocyclohept[b]indole Dose: 3x10 M Agonist Dose Agonist: Histamine Con t r o l Contraction (mm/7o) Average of c o n t r o l response Sequence; Test C o n t r a c t i o n % of c o n t r o l No. 1 2 3 SEM mm (ave 107.3 1 0/0 — — 0.0 2 0.5/0.5 1.5/1.4 1/0.9 0.9 0.27 0 0 3 3.5/3.2 5/4.6 4.5/4.2 4.0 0.43 1 0.9 4 19/17.6 16/14.9 20/18.8 17.1 1.15 3.5 3.3 5 61/56.5 46.5/43.3 46/43.3 47.6 4.42 12.5 11.6 6 81/75 77.5/72.1 73/68.6 71.9 1.87 36 33.5 7 107.5/99.4 106/98.5 104/97.7 98.6 0.54 72.5 67.5 8 108/100 107.5/100 106/99.6 99.8 0.16 97 90.4 9 108/100 107.5/100 106.5/100 100 0.0 102 95 10 _ - _ 102 95 Form 1: Experimental Data Histamine A c e t y l c h o l i n e Agonist Dose Mean c o n t r o l response Mean c o n t r o l response % SEM % SEM 10" 8 1.10 0.09 0.98 0.07 3 x l 0 - 8 3.52 0.20 10" 7 12.02 0.46 13.76 0.55 3 x l 0 " 7 32.82 0.76 10" 6 66.76 0.77 55.04 0.97 -6 3x10 86.92 0.47 10" 5 98.22 0.12 86.07 0.45 3 x l 0 " 5 99.69 0.04 IO* 4 100 0.0 95.44 0.20 3 x l 0 ~ 4 IO" 3 100 0.0 T o t a l no. of observations 495 432 at each dose. Table X: Mean values of c o n t r o l responses for complete sets of experiments. Histamine Concentration ( M ) >J h-• Figure 10: Average dose-response curve for the s t i m u l a t i o n of guinea p i g ileum w i t h '>•••' ••.'•nine. The s i z e of the c i r c l e represents the standard error E i : ! i " o i i ' i i s the average of 495 observations. o o • _ Q ^ B Q _ : — — i o " 8 i o " 7 10" 6 I O - 5 I O - 4 Histamine Concentration (M) Figure 11: Dose-rcsponse curves i n the presence (-a o-) and i n the absence (-o o-) of l - ( 2 -dimethylaminoethyl)tetradecahydrocyclohept[b] indole (10"^M), showing the measure-ments needed for the c a l c u l a t i o n of pA2 and pD2 v a l u e s . Only the l i n e a r p o r t i o n of the curve i s shown. (x i s i n mm, x 1 i s i n 7o) 173 Competitive Antagonist ( B ) : 1-(2-Dimethylaminoethy1)tetradeca-h y d r o c y c l o h e p t [ b ] i n d o l e P A 2 4.75 ±0.04 Reference Agonis t : Histamine P D 2 = 6.26 Experiment EmB antagonist X l o g pA 2 no. 7, c o n c e n t r a t i o n x l 0 " 5 M PAx mm. (x-1) r e l a t i v e lA 95 3 4.52 14.3 0.30 4.82 2 A 97 3 4.52 17.6 0.45 4.97 3 A 100 3 4.52 16.4 0.41 4.93 4 A 94 3 4.52 13.5 0.26 4.78 h 95 10 4.00 32.8 1.06 5.06 2B 90 10 4.00 23.7 0.72 4.72 3 B 78 10 4.00 34.8 1.13 5.13 * B 71 10 4.00 26.4 0.82 4.82 ! C 69 30 3.52 37.5 1.22 4.74 2C 52 30 3.52 26.1 0.80 4.32 3C 17 30 3.52 25.5 0.78 4.30 4C 64 30 3.52 29.2 0.93 4.45 Average val u e 4.75 SEM 0.04 Form 2: Competitive Antagonists 174 Non-competitive Antagonist ( B ) : 1-(2-Dimethylaminoethy1)tetradeca-hydrocyclohept [ b ] i n d o l e pD£ = 3.46 ± 0.14 ierence a g o n i s t : Histamine PD 2 = 6.26 i e r iment EmB Antagonist 100-x' log pD' no. 7. c o n c e n t r a t i o n x l 0 " 5 M PD 2 7o ( x ' - l ) r e l a t i v e *A 95 3 4.52 95 2 A 97 3 4.52 97 3 A 100 3 4.52 100 4 A 94 . 3 4.52 94 h 95 10 4.00 95 2B 90 10 4.00 90 -0.96 3.04 3 B 78 10 4.00 78 -0.57 3.43 4 B 71 10 4.00 71 -0.39 3.61 1 C 69 30 3.52 69 -0.35 3.17 2c 52 30 3.52 52 -0.04 3.48 3 c 17 30 3.52 17 0.69 4.21 4C 64 30 3.52 64 -0.25 3.27 Average value 3.46 SEM 0.14 Form 3: Non-competitive Antagonists Compound pD 2 pA 2 vs Histamine pD 2 vs Histamine Histamine 6.26 a A c e t y l c h o l i n e 6.08 b 57 7.94 (0.0 4 ) C 60 7.45 (0.03) 53 6.90 (0.07) 62 6.67 (0.06) 52 6.38 (0.05) 59 6.12 (0.08) 55 6.12 (0.07) 4.29 (0.17) 58 4.75 (0.04) 3.46 (0.14) 63 4.54 (0.10) 3.88 (0.12) 61 4.42 (0.04) 3.67 (0.16) 56 4.38 (0.07) 2.85 (0.16) 54 4.29 (0.13) 3.56 (0.26) Diphenhydramine 7.75 (0.09) d Atropine — • Table X I : Drug parameters c a l c u l a t e d from the experimental data, (footnotes on page fol l o w i n g ) pD~ vs ACh. 4.54 (0.10) 4.67 (0.11) 4.28 (0.16) 4.38 (0.09) 4.17 (0.12) 4.22 (0.04) 3.92 (0.06) 3.24 (0.10) 3.91 (0.11) 3.42 (0.07) 3.11 (0.07) 3.28 (0.06) 6.57 ( 0 . 1 4 ) 6 9.55 ( 0 . 0 5 ) 6 Footnotes, Table X I . a average of 495 c o n t r o l expts; l i t e r a t u r e value 6.6 (86) b average of 432 c o n t r o l expts; l i t e r a t u r e value 7.0 (90) The d e v i a t i o n from the l i t e r a t u r e value i s probably due to the method of experimentation. No cumulative dose-response curves have p r e v i o u s l y been reported f o r t h i s a g o n i s t , c numbers i n parentheses are iS.E.M. d l i t e r a t u r e value 7.7 (86), 8.0 (87) e pA 2 value vs a c e t y l c h o l i n e f l i t e r a t u r e value 6.6 (87) g l i t e r a t u r e value 8.9 ( r a t i n t e s t i n e ) (86) 8.8 (guinea p i g i n t e s t i n e ) (87) 177 DISCUSSION OF THE RESULTS B i o l o g i c a l m a n i f e s t a t i o n s , no matter how complex, are the r e s u l t of chemical r e a c t i o n s and should t h e r e f o r e be subject to the laws of chemistry. The extreme complexity of even a simple b i o l o g i c a l event i s the reason f o r only being able to d e s c r i b e c h e m i c a l - b i o l o g i c a l r e l a -t i o n s h i p s i n almost a l l cases (91). This b a s i c premise u n d e r l i e s the concept of the drug-receptor i n t e r a c t i o n , although i t i s recognized that a r e a c t i o n i n v o l v i n g cova-l e n t bond formation does not n e c e s s a r i l y take place (and i n f a c t r a r e l y occurs) between the drug and the receptor s i t e . Although no d i r e c t evidence of the r e c e p t o r s i s a v a i l a b l e , they are g e n e r a l l y assumed to be d i s t i n c t areas of biopolymers (eg p r o t e i n s or enzymes) which may be l o c a l i z e d at c e l l u l a r or s u b c e l l u l a r membranes (91). In many cases drugs are known to act i n i n h i b i t i n g enzymes and p r o t e i n b i n d i n g i s a major c o n s i d e r a t i o n i n the e v a l u a t i o n of drug b i o - a v a i l a b i l i t y . The drug-receptor i n t e r a c t i o n has thus come to be considered as a p a r a l l e l of the i n t e r a c t i o n which occurs between the a c t i v e s i t e of an enzyme and i t s s u b s t r a t e . Other f a c t o r s such as decrease i n a c t i v i t y w i t h the i n t r o d u c t i o n of bulky groups i n t o a drug molecule and s t e r e o s e l e c t i v i t y where more than one isomer of a drug i s a v a i l a b l e have f u r t h e r s t r e n g t h -ened the analogy. The molecular shape and charge d i s t r i b u t i o n of a p r o t e i n i s d e t e r -mined by the t e r t i a r y and quaternary s t r u c t u r e . The a c t i v e s i t e i n an enzyme, and, by e x t e n s i o n , a receptor s i t e , c o n s i s t s of only a s m a l l number of amino acids which need not be adjacent i n the primary s t r u c -ture of the p r o t e i n - - t h e f o l d s and c o n v o l u t i o n s of the molecule b r i n g 178 the necessary p o r t i o n s i n t o c l o s e p r o x i m i t y (92,93,94a). In many cases a coenzyme or a p r o s t h e t i c group i s i n v o l v e d at the a c t i v e s i t e . X-ray c r y s t a l l o g r a p h y has revealed that enzymes have, on t h e i r roughly g l o b u l a r s u r f a c e s , pockets or c l e f t s i n t o which s u b s t r a t e s may f i t (94b). Extensive work on carboxypeptidase A (95) has shown that the a c t i v e s i t e c o n s i s t s of a pocket and a groove i n the surface of the molecule i n t o which the s u b s t r a t e f i t s . The amino acids present at t h i s s i t e have a l s o been determined. During the b i n d i n g of s u b s t r a t e to carboxypeptidase A, the phenolic -OH of a t y r o s y l r e s i d u e moves about 12 A nearer to the s u b s t r a t e . This and other conformational changes are concrete evidence to support the induced f i t theory of enzyme-su b s t r a t e and drug-receptor i n t e r a c t i o n . The a c t i v e s i t e of chymotrypsin has a l s o been revealed as a hole i n the surface of the g l o b u l a r enzyme (92). Using t h i s , and other, data c o l l e c t e d from X-ray c r y s t a l l o g r a p h i c s t u d i e s , s e v e r a l molecular mechanisms of enzyme a c t i o n have been pro-posed (92,95). There are c e r t a i n obvious analogies between the problems of i s o l a -t i o n of receptor m a t e r i a l and that of i s o l a t i o n and i d e n t i f i c a t i o n of the components of the a c t i v e s i t e s of enzymes. However, the problem of i s o l a t i o n of a receptor i s much more complex si n c e the p h y s i o l o g i c a l a c t i v i t y of receptor systems, an e s s e n t i a l c r i t e r i o n of t h e i r e x i s t e n c e , i s dependent on the i n t e g r i t y of the c e l l u l a r system or, at l e a s t , of i t s membrane components w h i l e the a c t i v i t y of an enzyme p r e p a r a t i o n may be more or l e s s r e a d i l y monitored during the e x t r a c t i o n and i s o l a t i o n procedures (96) . Two b a s i c techniques which have been used i n attempts to i s o l a t e receptor substances are measurements of the a b i l i t y of var-179 ious f r a c t i o n s of r e c e p t o r - c o n t a i n i n g t i s s u e s to r e v e r s i b l y bind ligands w i t h a f f i n i t i e s a p propriate to the receptor system and the use of agents which w i l l c o v a l e n t l y bind to the receptor and subsequent f r a c t i o n a t i o n of the t i s s u e s to o b t a i n l a b e l l e d m a t e r i a l . Neither of these approaches has l e d to the i s o l a t i o n of a pure m a t e r i a l which can be unambiguously s t a t e d to be the r e c e p t o r , or a p o r t i o n thereof, f o r a neuro t r a n s m i t t e r (96) although some progress has been made i n the i s o l a t i o n o f , f o r ex-ample, the c h o l i n e r g i c (96) and e s t r o g e n i c r e c e p t o r s (97). The work done i n t h i s l a b o r a t o r y has been d i r e c t e d not at the i s o -l a t i o n of an a n t i h i s t a m i n i c r e c e p t o r , but r a t h e r at the e l u c i d a t i o n of the s t e r i c and e l e c t r o n i c c h a r a c t e r i s t i c s of the r e c e p t o r . The eventual aim of work such as t h i s i s , of course, the proposal of s p e c i f i c b i n d -ing groups at the receptor s i t e and, i f p o s s i b l e , d e s c r i p t i o n of the molecular makeup of the receptor which would allow a b e t t e r understand-ing of the molecular b a s i s of drug a c t i o n . When tested f or a c t i v i t y i n s e v e r a l systems, the a z a d i s p i r o com-pounds were found to have a very low order of a c t i v i t y . This l a c k of a c t i v i t y i s probably due to the l a r g e b u l k of the a l i c y c l i c r i n g s pre-v e n t i n g f i t t i n g of the molecule i n t o a c l e f t on the receptor s u r f a c e . Although a great many drugs i n the pharmacological c l a s s e s for which a c t i v i t y was tested c o n t a i n bulky s u b s t i t u e n t s , these are a l l capable of f r e e r o t a t i o n about one or s e v e r a l bonds thus decreasing the amount of surface area which i s presented to the r e c e p t o r . The a z a d i s p i r o compounds are not capable of such conformational changes i n the r i n g system and thus would not f i t i n t o a narrow c l e f t . I t i s a l s o p o s s i b l e that the great degree of non-bonded i n t e r a c t i o n s between the si d e chain 180 and the a l i c y c l i c r i n g s prevented adoption of a conformation which could b i n d e f f e c t i v e l y to the r e c e p t o r . A l l of the compounds i n the carbazole s e r i e s e x h i b i t e d non-competitive i n h i b i t i o n when tested f o r a n t i m u s c a r i n i c a c t i v i t y . The f u l l y hydrogenated compounds 54, 56, 5_8, and 63, as w e l l as the disub-s t i t u t e d f l u o r e n e d e r i v a t i v e 61_ and the tetrahydrocyclopent[b] i n d o l e d e r i v a t i v e 55_, showed dualism of antagonism when test e d f o r a n t i h i s -taminic a c t i v i t y . The r e l a t i v e i n t e n s i t y of the non-competitive aspect of t h i s i n h i b i t i o n caused by these s i x compounds p a r a l l e l s the r e l a t i v e i n t e n s i t y of the non-competitive i n h i b i t i o n of a c e t y l c h o l i n e induced c o n t r a c t i o n by the same s i x compounds. This i s represented diagram-m a t i c a l l y i n F i g u r e 12. Except f o r 56, the l i n e s are p a r a l l e l w i t h i n the l i m i t s of slope d i c t a t e d by the S.E.M. This data may i n d i c a t e that e i t h e r a general t o x i c e f f e c t occurred or that the same non-competitive receptor s i t e was i n v o l v e d i n both i n h i b i t i o n s . The high doses at which these compounds were test e d i n d i c a t e that the former e f f e c t i s more l i k e l y . A l l of the compounds i n the carbazole s e r i e s e x h i b i t c o m p e t i t i v e i n h i b i t o r y a c t i v i t y a g a i n s t histamine. However, there i s a d i s t i n c t break i n the s e r i e s so that two groups of compounds are evident (Table X I , p 175). The f i r s t group contains a l l of the compounds which have an aromatic nucleus and show competitive a c t i v i t y of a f a i r l y high order. The second group contains a l l the compounds, except 55, which show dualism of antagonism. These compounds a l l show a low order of competitive a c t i v i t y - - t h e y are 1.4-3.6 l o g u n i t s l e s s a c t i v e than the compounds i n the f i r s t group. The com p e t i t i v e f a c e t of a c t i v i t y of 181 D' D' P 2 vs Histamine P 2 vs ACh (4.29) Figure 12: Comparison of the a f f i n i t i e s of compounds i n the carbazole s e r i e s for non-specific receptors in the guinea pig ileum which cause i n h i b i t i o n of responses to histamine (H) and acet y l c h o l i n e (ACh) 182 compounds i n the second group i s very l i k e l y due to the presence of the dimethylaminoethyl group which binds to the h i s t a m i n i c r e c e p t o r . How-ever, s i n c e no f l a t aromatic r i n g i s a v a i l a b l e to r e i n f o r c e the b i n d -i n g of 5_4, 56, _58, and 63, the drug-receptor complex i s unstable and the compounds are r e a d i l y r e p l a c e d by histamine, the n a t u r a l s u b s t r a t e . The s t e r i c e f f e c t of the second s u b s t i t u e n t i n the d i s u b s t i t u t e d f l u o r e n e 61 i s undoubtedly r e s p o n s i b l e f o r the i n s t a b i l i t y of ttie receptor com-pl e x formed w i t h t h i s compound. I t would seem wise to p o i n t out at t h i s time that although compet-i t i v e i n h i b i t i o n presupposes b i n d i n g at the same receptor s i t e as the a g o n i s t , i t i s not necessary that a l l the groups to which the agonist binds be covered by the i n h i b i t o r . For example, i f a three p o i n t b i n d -ing mechanism f o r histamine i s proposed to i n v o l v e a 7r'-complex w i t h the imidazole e l e c t r o n s , an i o n i c bond between the protonated amino group and an a n i o n i c centre of the r e c e p t o r , and a hydrogen bond i n v o l v -ing e i t h e r of the two p o s s i b l e s i t e s i n the imidazole r i n g , then b i n d -ing of an i n h i b i t o r to any of the three receptor b i n d i n g s i t e s would be expected to r e s u l t i n c ompetitive i n h i b i t i o n of the e f f e c t s of h i s -tamine. I t would be p o s s i b l e f o r the i n h i b i t o r to bind to any other convenient s i t e s around the s p e c i f i c histamine receptor and, provided a p o r t i o n of the receptor was covered, competitive a c t i v i t y would be observed. The most important assumption inherent i n the development of t h i s theory i s that histamine, being a n a t u r a l s u b s t r a t e , w i l l bind to the receptor more s t r o n g l y than an i n h i b i t o r . As a r e s u l t , the presence of a p a r t i a l l y bound histamine molecule at a receptor which i s p a r t i a l l y 183 occupied by an i n h i b i t o r w i l l decrease the b i n d i n g energy between the i n h i b i t o r and the receptor as a consequence of s t r a i n introduced by the s t e r i c i n t e r a c t i o n . B e l l e a u , i n h i s macromolecular p e r t u r b a t i o n theory (98), has suggested the involvement of p e r i p h e r a l groups i n the b i n d i n g of an i n h i b i t o r to a receptor s i t e . This i n t e r a c t i o n causes a non-s p e c i f i c conformational p e r t u r b a t i o n of the receptor p r o t e i n , as opposed to the s p e c i f i c conformational p e r t u r b a t i o n induced by b i n d i n g of an agonist to the r e c e p t o r . I t seems l i k e l y that t h i s occurs i n the case of the f u l l y hydro-genated d e r i v a t i v e s prepared i n t h i s work s i n c e the puckered a l i c y c l i c r i n g systems would not be expected to bind to a f l a t aromatic r e g i o n , but b i n d i n g to a hydrophobic group on the periphery of the receptor may be expected to occur. B i n d i n g of the histamine molecule to the recep-tor would d i s p l a c e the amino f u n c t i o n of the antagonist from the a n i o n i c s i t e , weakening b i n d i n g s u f f i c i e n t l y so that ready d i s s o c i a t i o n of the antagonist-macromolecule complex would occur. I t i s assumed that the same a n i o n i c s i t e i s i n v o l v e d i n b i n d i n g the antagonists and histamine. I t would seem reasonable, because of the l a r g e increase i n a c t i v i t y of those compounds c o n t a i n i n g an aromatic r i n g , to assume that the aro-matic r i n g of the antagonists binds at the same s i t e as does the imid-azole r i n g of histamine. The assumption i s made that at l e a s t two b i n d -i n g s i t e s f o r histamine--a f l a t area f o r ir'-complex formation or van der Waals b i n d i n g and an a n i o n i c s i t e f o r Coulombic i n t e r a c t i o n — e x i s t w i t h i n a c l e f t i n a p r o t e i n . Then the r e l a t i v e a c t i v i t i e s of the h i g h l y a c t i v e group of compounds may best be r a t i o n a l i z e d i f N-(2-dimethylamino-ethyl)diphenylamine 62 i s considered to be the parent of the s e r i e s . 184 A l l seven of these compounds are thought to i n t e r a c t w i t h the same b i n d i n g s i t e s . In a l l cases the protonated dimethylamino group i n t e r -a cts w i t h the a n i o n i c s i t e w h i l e the aromatic r i n g binds at the f l a t area of the r e c e p t o r . In 62^ , only one of the aromatic r i n g s i s i n -volved i n b i n d i n g , the second aromatic r i n g being n o n - e s s e n t i a l f o r b i n d i n g to the r e c e p t o r . This i s i n agreement w i t h Nauta's proposal (32) and w i t h the known a c t i v i t y of compounds c o n t a i n i n g only one aro-matic r i n g , eg N-phenyl-N,N',N'-triethylethylenediamine (21). Although the second r i n g i s n o n - e s s e n t i a l i t probably has a secondary r o l e i n receptor b i n d i n g due to hydrophobic bond formation and may be impor-tant i n determining the d i s t r i b u t i o n c h a r a c t e r i s t i c s of the compound. Three of the compounds i n t h i s group--9-(2-dimethylaminoethyl)-carbazole 5_2, 4-(2-dimethylaminoethyl)-l,2,3,4-tetrahydrocyclopent [b] -i n d o l e 55, and 9-(2-dimethylaminoethyl)fluorene 59--are l e s s a c t i v e than 62_. These compounds are a l l c h a r a c t e r i z e d by the presence of an e s s e n t i a l l y planar t r i c y c l i c r i n g system which suggests that a p o r t i o n of the receptor adjacent to the f l a t area protrudes from the f l o o r of the c l e f t and s t e r i c a l l y i n t e r f e r e s w i t h the t h i r d r i n g i n the system. A study of models shows that the dimensions of the cyclopentene r i n g i n 5_5, i n e i t h e r a planar or puckered conformation, d i f f e r only s l i g h t l y from those of the phenyl r i n g present i n 52 and 59. The three remaining compounds i n the series--9-(2-dimethylamino-e t h y l ) - ! , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e 53, 9-(2-dimethylaminoethyl)-l,2,3,4,4a,9a-hexahydrofluorene 6(), and 5-(2-dimethylaminoethyl)-5,6,7,8,9,10-hexahydrocyclohept [ b ] i n d o l e 57--show i n c r e a s i n g a c t i v i t y , i n the order g i v e n , over j62. In 53 and 5_7 the p a r t i a l l y saturated r i n g 185 may r e a d i l y assume a boat conformation i n which the r i n g i s tipped up-wards out of the plane of the aromatic r i n g and presumably away from the p r o t r u d i n g p o r t i o n of the receptor surface w h i l e i n 60 the probable c i s r i n g f u s i o n more e f f e c t i v e l y removes the saturated r i n g from the area of s t e r i c r e p u l s i o n . The assumption of a boat conformation i n these molecules i s e a s i l y accommodated by the induced f i t theory of drug-receptor i n t e r a c t i o n s . The increased a c t i v i t y of these compounds over 62 may be r a t i o n a l -i z e d on the b a s i s of hydrophobic bonding to the roof of the c l e f t or by assuming that the second aromatic r i n g of j6_2 s t e r i c a l l y i n t e r f e r e s w i t h r e c e p t o r b i n d i n g by i n t e r a c t i n g w i t h the r o o f of the c l e f t . A gr a d a t i o n i n the c o n t r i b u t i o n of hydrophobic b i n d i n g to t o t a l r e c e p t o r b i n d i n g must be invoked, i n the order 5_7>60>j>3, to account f o r the d i f f e r i n g a c t i v i t i e s of these compounds. The balance of the s t e r i c r e p u l s i v e f o r c e s and hydrophobic b i n d i n g f o r c e s w i l l determine the a c t i v i t y of each compound. The e l e c t r o n d e n s i t y about the n i t r o g e n atom bonded to the aro-matic r i n g system and about the corresponding carbon atom i n the f l u o r e n e d e r i v a t i v e s does not appear to be an important c o n s i d e r a t i o n i n deter-mining the a c t i v i t y of these compounds. Although no c a l c u l a t e d values are a v a i l a b l e f o r the s p e c i f i c compounds synthesized i n t h i s work a sequence of r e l a t i v e e l e c t r o n d e n s i t i e s may be determined by e x t r a p o l a -t i o n from compounds w i t h known values and by i n t u i t i v e reasoning. The c a l c u l a t e d v a l u e s of e l e c t r o n d e n s i t y about the n i t r o g e n atom of p y r r o l e , i n d o l e , and carbazole are 1.52, 1.57, and 1.67 r e s p e c t i v e l y (99). The presence of c y c l o a l i p h a t i c r i n g s fused to the in d o l e nucleus 186 would not be expected to have a s i g n i f i c a n t e f f e c t on the n i t r o g e n e l e c t r o n d e n s i t y and the e f f e c t of the dimethylaminoethyl s i d e chain may be neglected since i t i s present i n a l l of the compounds i n ques-t i o n (100) . C o n s i d e r a t i o n of the above values i n d i c a t e s that d e r e a l -i z a t i o n of the n i t r o g e n e l e c t r o n s decreases as aromatic character i n -creases over cyclopentadieny1 c h a r a c t e r . Therefore, the diphenylamine n i t r o g e n atom would be expected to have a higher e l e c t r o n d e n s i t y than those present i n i n d o l e and c a r b a z o l e . The fluorene d e r i v a t i v e s would be expected to have a much lower e l e c t r o n d e n s i t y at the nine p o s i t i o n than carbazole due to the l a c k of lone p a i r e l e c t r o n s . Of the two f l u o r e n e d e r i v a t i v e s the i n d u c t i v e e f f e c t of two aromatic r i n g s i n 59 would be expected to decrease the e l e c t r o n d e n s i t y more than would occur i n jp_0. The r e l a t i v e e l e c t r o n d e n s i t i e s at the p o s i t i o n considered would then be expected to f a l l i n the order 62>52>53 =55 = 57>60>59 which bears no resemblance to the a c t i v i t y s e r i e s . Further work i n t h i s area would seem necessary i n order to r e c o n c i l e these r e s u l t s w i t h the work of Nauta (32) on diphenhydramine d e r i v a t i v e s . Although the above s i m p l i s t i c d i s c u s s i o n i s of minimal use i n understanding the molecular b a s i s of a n t i h i s t a m i n i c a c t i v i t y , a much more d e t a i l e d study than was undertaken i n t h i s work would be necessary to propose f u r t h e r d e t a i l s of the receptor s i t e . 187 SUGGESTIONS FOR FUTURE WORK Although i t i s d o u b t f u l that other d e r i v a t i v e s of the a z a d i s p i r o compounds w i l l show app r e c i a b l e a c t i v i t y , the 2-dimethylaminoethyl d e r i v a t i v e s should be synthesized f or the sake of completeness. The success achieved w i t h the use of 2-dimethylaminoethyl c h l o r i d e i n the carbazole s e r i e s suggests that t h i s reagent would give the d e s i r e d com-pounds when reacted w i t h the 7- and 8-amino a z a d i s p i r o compounds 4 and 5. Using t h i s approach the a p p r o p r i a t e l y s u b s t i t u t e d lactam and r e -duced lactam (imino) compounds should be r e a d i l y a v a i l a b l e . Since the hydroxy d e r i v a t i v e s 45 and 16_ are r e a d i l y a v a i l a b l e the dimethylaminoethy1 e s t e r s should be e a s i l y s y n t h e s i z e d . E s t e r d e r i v a t i v e s are more commonly seen i n the a n t i m u s c a r i n i c s and l o c a l a n e s t h e t i c s than are amides. Furthur to t h i s , the e s t e r s of 45 and 76 would be the "reversed" e s t e r s . That i s , they are e s t e r s of a complex a l c o h o l w i t h a simple a c i d r a t h e r than e s t e r s of a simple a l c o h o l and a complex a c i d which are much more commonly seen i n com-pounds having a n t i m u s c a r i n i c and l o c a l a n e s t h e t i c p r o p e r t i e s . The c a r b o x y l i c a c i d d e r i v a t i v e s of the a z a d i s p i r o compounds should be 45 76 188 r e a d i l y d e r i v a b l e from 45 and _76 by replacement of the hydroxyl group w i t h halogen and subsequent carbonation of the Grignard reagent or l i t h i a t e d compound de r i v e d therefrom. In the carbazole s e r i e s , a great many compounds come r e a d i l y to mind as means of f u r t h e r r e f i n i n g the proposed receptor model. From the r e s u l t s obtained i n t h i s work i t appears that f u r t h e r work i n t h i s area should i n c l u d e only those compounds c o n t a i n i n g at l e a s t one aro-matic r i n g . Those compounds which would perhaps be most i n t e r e s t i n g are the indene analogues of the c y c l o a l k [b] i n d o l e s 5_3, 55_, and 5_7 synt h e s i z e d i n t h i s work. As w e l l , the dihydrogenated d e r i v a t i v e s of the i n d o l e s and the proposed indenes could be s y n t h e s i z e d . Extension of the s e r i e s to i n c l u d e 3-(2-dimethylaminoethyl)-l,2-dihydro-3H-cyclobut [bj i n d o l e , i t s indene analogue, and the r e s p e c t i v e dihydro d e r i v a t i v e s would a l s o be of i n t e r e s t as would the s y n t h e s i s of the higher homologue 5-(2-dimethylaminoethy1)-6,7,8,9,10,ll-hexahydro-5H-cyclooct [b] i n d o l e and i t s d e r i v a t i v e s . F urther i n f o r m a t i o n on the s t e r i c nature of the receptor may be o b t a i n a b l e from a study of 1-(2-dimethylaminoethy1)-2,3-dialkylindoles. The nature of the i n t e r a c t i o n w i t h the receptor of the second aro-matic r i n g of 62_ may be deducible from a study of the a c t i v i t y of N-phenyl-N-cyclohexyl-N',N'-dimethylethylenediamine and d e r i v a t i v e s con-t a i n i n g i s o l a t e d and conjugated double bonds i n the c y c l o h e x y l r i n g . By f a r the most c h a l l e n g i n g p o s s i b i l i t y based on t h i s work would be the s e p a r a t i o n of the R and S isomers of 60. In the a c t i v i t y s e r i e s determined i n t h i s work, 60 was found to be l e s s a c t i v e than 57 w h i l e , 189 on the b a s i s of a s t e r i c a l l y i n t e r f e r i n g group adjacent to and i n the same plane as the f l a t aromatic b i n d i n g area of the r e c e p t o r , 60 would be expected to be the most a c t i v e of the compounds s y n t h e s i z e d . Fur-ther to t h i s , the c o n f i g u r a t i o n of j50 prepared i n t h i s work, and assumed to be the c i s - s y n isomer, should be determined, perhaps by h i g h r e s o l u -t i o n or double resonance nmr s t u d i e s . The s y n t h e s i s of other isomers could a l s o be considered. Attempts to i s o l a t e a receptor substance are a l s o suggested. 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APPENDICES APPENDIX I INFRARED SPECTRA Spectrum 1 7-Amino-14-azadispiro [5.1.5.2]pentadecan-15-one (KBr d i s c ) Spectrum 2 7-Dimethylaminoacetamido-14-azadispiro[5.1.5.2 ]pentadecan-15-one (KBr d i s c ) Spectrum 3 7-Dimethylaminoacetamido-14-azadispiro[5.1.5.2Jpentadecane (KBr d i s c ) 199 IS 1 » 5 4 4 5 3 » * « * 5 7 7 J t • 10 II l> !« 1* 4 0 0 0 1 3 0 0 3 0 0 0 2 3 0 0 3 0 0 0 ItOO 1 4 0 0 1 4 0 0 I K O 1 0 0 0 0 0 0 4 0 0 W A V M J M I U C M ' W A V E L E N G T H «N M U C V O N S ' U 4 4 J f S-S * * J 7 7 5 • » 10 II t l 14 1* Spectrum 4 8-Amino-16-azadispiro[6.1.6.2 ] heptadecan-17-one (KBr d i s c ) Spectrum 5 8-Dimethylaminoacetamido-16-azadispiro[6.1.6.2]heptadecan-17-one (KBr d i s c ) Spectrum 6 8-Dimethylaminoacetamido-16-azadispiro [6.1.6.2]heptadecane (KBr d i s c ) 200 Spectrum 7 9-(2-Dimethylaminoethyl)carbazole (KBr d i s c ) Spectrum 8 9-(2-Dimethylaminoethyl)-l,2,3,4-tetrahydrocarbazole ( l i q u i d f i l m ) Spectrum 9 9-(2-Dimethylaminoethyl)dodecahydrocarbazole ( l i q u i d f i l m ) 201 Spectrum 10 4-(2-Dimethylaminoethy1)-1,2,3,4-tetrahydrocyclopent[b]indole ( l i q u i d f i l m ) Spectrum 11 4- (2-Dimethylaminoethyl)dodecahydrocyclopent|_b J i n d o l e ( l i q u i d f i l m ) Spectrum 12 5-(2-Dimethylaminoethy1)-5,6,7,8,9,10-hexahydrocyclohept[b]-i n d o l e ( l i q u i d f i l m ) 202 Spectrum 1 3 5 - ( 2 - D i m e t h y l a m i n o e t h y 1 ) t e t r a d e c a h y d r o c y c l o h e p t [ b J i n d o l e ( l i q u i d f i l m ) Spectrum 1 4 N- ( 2-Dimethylaminoethyl)diphenylamine ( l i q u i d f i l m ) Spectrum 1 5 N- ( 2-Dimethylaminoethyl)dicyclohexylamine ( l i q u i d f i l m ) 203 13 11 1} 1* I* Spectrum 16 9-(2-Dimethylaminoethyl)fluorene ( l i q u i d f i l m ) Spectrum 17 9-(2-Dimethylaminoethyl)fluorene (10% i n CC1, , 0.5 mm c e l l ) Spectrum 18 9,9-Bis(2-dimethylaminoethy1)fluorene (10% i n CC1,, 0.5 mm c e l l ) 204 1400 1300 WAVENUMftEf CM-* WAVfHENGTM IN M l C t O N S 9 10 II 1} 1 ' >6 ^ m j 5_ inn 3 ' U l /HI, 3 T - f l I Ir - t " b ^ f c = = » - j E T mm 20O0 mo 1400 1200 WAV&tfUMBE* C M - ' W*VflfNGT>* IN M l C t O N S « / 7.3 a 1 10 II 1J 1* 1* T P r-j-1 |t- It r- > --IU-1 |t=F*HsM f+ I! • 1 I i i A 4 4 iitt I I I I t : I l~~T l«O0 »?00 Spectrum 19 9,9-Bis(2-dimethylaminoethyl)fluorene (KBr d i s c ) Spectrum 20 9-(2-Dimethylaminoethy1)-1,2,3,4,4a,9a-hexahydrofluorene ( l i q u i d f i l m ) 205 APPENDIX I I MANUFACTURERS AND GRADES OF REAGENTS, SOLVENTS, AND GASES A. REAGENTS Chemical Manufacturer Grade or P u r i t y A c i d , hydrobroraic (487») Eastman A c i d , h y d r o c h l o r i c A l l i e d A.C.S. reagent A c i d , s u l f u r i c A l l i e d A.C.S. reagent Ammonium hydroxide A l l i e d A.C.S. reagent Barium n i t r a t e M a l l i n c k r o d t a n a l y t i c a l reagent Boron t r i f l u o r i d e e t h e rate J.T. Baker p r a c t i c a l Carbazole A l d r i c h 99% C h l o r o a c e t y l c h l o r i d e A l d r i c h reagent Cupric c h l o r i d e d i h y d r a t e M a l l i n c k r o d t a n a l y t i c a l reagent Cycloheptanone A l d r i c h Cyclohexanone A l d r i c h p u r i s s . Cyclopentanone A l d r i c h p u r i s s . Dicyclohexylamine A l d r i c h b.p. 255° Dimethyl s u l f a t e Eastman p r a c t i c a l Dimethylaminoethanol Eastman reagent Dime thylaminoethy1 c h l o r i d e h y d r o c h l o r i d e A l d r i c h 98% Diphenylamine F i s h e r p u r i f i e d Formamide Al d r i c h b 1 Q 109° Fluorene Eastman prac t i c a l L i t h i u m aluminum hydride A l f a Inorganics Magnesium s u l f a t e Methyl i o d i d e Nitromethane Phenylhydrazine P i c r i c a c i d P i p e r a z i n e (anhydrous) Potassium metal Potassium hydroxide Raney n i c k e l No. 28 5% Rhodium on alumina Sodium metal Sodium amide Sodium borohydride Sodium dichrornate d i h y d r a t e Sodium hydride Sodium hydroxide Sodium s u l f a t e B. SOLVENTS AND GASES Acetone A c i d , a c e t i c Benzene B u t a n o l , t e r t i a r y D e c a l i n Diglyme B r i t i s h Drug Houses Eastman A l d r i c h F i s h e r Merck A l d r i c h B r i t i s h Drug Houses A l l i e d W.R.Grace K & K Labs A l l i e d F i s h e r A l f a Inorganics Merck Du Pont B r i t i s h Drug Houses A l l i e d M a l l i n c k r o d t A l l i e d M a l l i n c k r o d t Matheson Eastman Eastman 208 anhydrous reagent C.P. reagent reagent c e r t i f i e d reagent N.F. p r a c t i c a l reagent A.C.S. reagent A.C.S. reagent reagent 98 +% Te c h n i c a l a n a l y t i c a l reagent A.C.S. reagent N.F. A.C.S. reagent reagent reagent p r a c t i c a l p r a c t i c a l 209 Dimethylamine Matheson Dime thy1formamide F i s h e r anhydrous reagent Dioxane J.T.Baker reagent Ethanol R e l i a n c e Chemical 95% Ethanol (100%) R e l i a n c e Chemical Ether F i s h e r N.F. Ether, anhydrous F i s h e r anhydrous reagent Ethylene g l y c o l A l l i e d reagent Hexane Matheson p r a c t i c a l Hydrogen Canadian L i q u i d A i r Hydrogen c h l o r i d e Matheson ---Methanol F i s h e r anhydrous reagent N i t r o g e n Canadian L i q u i d A i r G Petroleum s p i r i t (60-80°) B r i t i s h Drug Houses a n a l y t i c a l reagent Tetrahydrofuran B r i t i s h Drug Houses reagent Xylene M a l l i n c k r o d t reagent Where anhydrous s o l v e n t s are s p e c i f i e d i n experimental procedures, the s o l v e n t was f i r s t d r i e d over sodium w i r e . Exceptions are anhydrous methanol, which was used as s u p p l i e d , anhydrous ether used as s u p p l i e d as a s o l v e n t i n the r e c r y s t a l l i z a t i o n of h y d r o c h l o r i d e s a l t s , and an-hydrous t e t r a h y d r o f u r a n which was d i s t i l l e d from l i t h i u m aluminum hydride. In r e a c t i o n s which were run under a n i t r o g e n atmosphere, a c o n t i n -uous slow stream of n i t r o g e n was p u r i f i e d by bubbling the gas through concentrated s u l f u r i c a c i d and passing i t through a column loaded w i t h potassium hydroxide p e l l e t s and thence i n t o the r e a c t i o n f l a s k . 210 Other reagents were used as su p p l i e d except where noted i n the t e x t . APPENDIX I I I COMPUTER PROGRAMS 212 A. For i n t e r p r e t a t i o n of r e s u l t s from t e s t s f o r a n t i h i s t a m i n i c a c t i v i t y . Card number F o r t r a n statement 1 DIMENSION P(4,9,37,4),S(9),N(9),SS(9),NN(9),AVG(9) 2 DO 3 1=1,9 3 DO 2 M=l,4 4 DO 2 K=l,4 5 DO 2 J=l,37 6 P(M,I,J,K)=-1.0 7 2 CONTINUE 8 SS(I)=0.0 9 NN(I)=0 10 3 CONTINUE 11 DO 150 K=l,4 12 DO 4 L»1,9 13 S(L)=0.0 14 N(L)=0 15 SSQ(L)=0 16 4 CONTINUE 17 DO 100 J=l,37 18 READ (5,5) WMAX,XMAX,YMAX,ZMAX 19 5 FORMAT (4F10.2) 20 WRITE (6,6)J,K 21 6 FORMAT (/'SUBSET',13,', GROUP',12) 22 WRITE (6,8) 23 8 FORMAT (/10X,'WMAX',11X,'XMAX',11X,'YMAX',1IX,'ZMAX'/) 24 WRITE (6,9)WMAX,XMAX,YMAX,ZMAX 25 9 FORMAT (4F15.3) 26 WRITE (6,11) 27 11 FORMAT (IX) 28 WRITE (6,20) 29 20 FORMAT (5X,'NO',6X,'W",9X,'X',9X,'Y',9X,'Z1,9X,'A', 30 1 9X,'B',9X,'C,9X,'D',8X,'SUM',6X,'MEAN',5X,'SID ERR'/) 31 IF (ZMAX.NE.0.0) GO TO 70 32 IF (YMAX.NE.0.0) GO TO 50 33 DO 40 1=1,9 34 READ (5,31) W,X 35 31 FORMAT (2F10.2) 36 A=(W/WMAX)*100.0 37 ' P(1,I,J,K)=A 38 B=(X/XMAX)*100.0 39 P(2,I,J,K)=B 40 SUM=A+B 41 XMEAN=SUM/2 42 SE=SQRT(((A-XMEAN)**2+ (B-XMEAN)**2)/2) 43 N(I)=N(I)-f- 2 44 S( I ) = S ( I ) + SUM 45 WRITE (6,35)I,W,X,A,B,SUM,XMEAN,SE 46 35 FORMAT (I7,2(2F10.3,20X),3F10.3/) 213 47 40 CONTINUE 48 WRITE (6,11) 49 GO TO 100 50 50 DO 60 1=1,9 51 READ (5,51) W,X,Y 52 51 FORMAT (3F10.2) 53 A=(W/WMAX)*100.0 54 P(1,I,J,K)=A 55 B=(X/XMAX)*100.0 56 P(2,I,J,K)=B 57 C=(Y/YMAX)*100.0 58 P(3,I,J,K)=C 59 SUM=A+ B-r- C 60 XMEAN=SUM/3 61 SE=SQRT(((A-XMEAN)**2+(B-XMEAN)**24- (C-XMEAN)**2)/6) 62 N(I)=N(I)+3 63 S(I)=S(I)4-SUM 64 WRITE (6,55) I,W,X,Y,A,B,C,SUM,XMEAN,SE 65 55 FORMAT (17,2(3F10.3,10X),3F10.3/) 66 60 CONTINUE 67 WRITE (6,11) 68 GO TO 100 69 70 DO 80 1=1,9 70 READ (5,71) W,X,Y,Z 71 71 FORMAT (4F10.2) 72 A=(W/WMAX)*100.0 73 P(1,I,J,K)=A 74 B=(X/XMAX)*100.0 75 P(2,I,J,K)=B 76 C=(Y/YMAX)*100.0 77 P(3,I,J,K)=C 78 D=(Z/ZMAX)*100.0 79 P(4,I,J,K)=D 80 SUM=A-t-B + C + D 81 XMEAN=SUM/4 82 SE=SQRT(((A-XMEAN)**2 + (B-XMEAN)**2 -+• (C-XMEAN)**2 + 83 1 (D-XMEAN)**2)/12) 84 N ( I ) = N ( I ) + 4 85 S ( I ) = S ( I ) + SUM 86 WRITE (6,75)I,W,X,Y,Z,A,B,C,D,SUM,XMEAN,SE 87 75 FORMAT (17,11F10.3/) 88 80 CONTINUE 89 WRITE (6,11) 90 100 CONTINUE 91 DO 110 1=1,9 92 AVG(I)=S(I)/N(I) 93 SS(I)=SS(I) + S ( I ) 94 110 NN(I)=NN(I)+ N(I) 95 WRITE (6,115) 96 115 FORMAT (/20X,'VALUES FOR GROUP OF 37'//) 97 WRITE (6,118). 98 118 FORMAT (4X,'NO',4X,'NO OF OBS',11X,'SUM',11X,'MEAN', 214 99 1 9X, 1STD ERROR'/) 100 DO 130 1=1,9 101 DO 125 J=l,37 102 DO 120 M=l,4 103 IF (P(M,I,J,K).EQ.-1.0) GO TO 120 104 DIF=P(M,I,J,K)-AVG(I) 105 S S Q ( I ) = S S Q ( I ) + DIF**2 106 120 CONTINUE 107 125 CONTINUE 108 SE=SQRT(SSQ(I)/(N(I)*(N(I)-1))) 109 WRITE (6,128)I,N(I),S(I),AVG(I),SE 110 128 FORMAT (I6,I10,4X,2F15.3/) 111 130 CONTINUE 112 WRITE (6,11) 113 WRITE (6,11) 114 150 CONTINUE 115 WRITE (6,151) 116 151 FORMAT (//20X,'VALUES FOR COMPLETE SET'//) 117 WRITE (6,118) 118 DO 152 1=1,9 119 SSQ(I)=0.0 120 152 AVG(I)=SS(I)/NN(I) 121 DO 190 1=1,9 122 DO 180 K=l,4 123 DO 170 J=l,37 124 DO 160 M=l,4 125 IF (P(M,I,J,K).EQ.-1.0) GO TO 160 126 DIF=P(M,I,J,K)-AVG(I) 127 SSQ(I)=SSQ(I )+DIF**2 128 160 CONTINUE 129 170 CONTINUE 130 180 CONTINUE 131 SE=SQRT(SSQ(I)/(NN(I)*(NN(I)-1))) 132 WRITE (6,128)I,NN(I),SS(I),AVG(I),SE 133 190 CONTINUE 134 END The above program was designed around the format i n which data was c o l l e c t e d from the experiments f o r a n t i h i s t a m i n i c a c t i v i t y . In order to use the program the experiment must be set up i n the manner used i n t h i s work. T h i r t y - s e v e n separate experiments were run i n each of four sets of apparatus. Each experiment i n v o l v e d the determination of two, three, or four cumulative responses to histamine alone and one determination 215 of the cumulative response to histamine i n the presence of an an t a g o n i s t . Up to nine cumulative doses of histamine were used f o r each response. E s s e n t i a l l y , t h i s program computed the mean c o n t r o l response, w i t h i t s standard e r r o r , at each dose f o r each i n d i v i d u a l experiment, f o r each group of 37 experiments i n the same apparatus, and f o r the e n t i r e set (4x37) of experiments. Values f o r the t e s t responses were not computed but were c a l c u l a t e d w i t h a s l i d e r u l e . The job data deck c o n s i s t e d of four groups of cards and each group c o n s i s t e d of 37 subsets which each represented one experiment. Each subset c o n s i s t e d of 10 data cards. The f i r s t of these 10 cards c a r r i e d the values f o r the maximum response of the p r e p a r a t i o n , measured i n mm from the kymograph r e c o r d . The nine succeeding cards c a r r i e d the mea-sured responses at i n c r e a s i n g doses of histamine. I t was necessary to s t a r t each cumulative response at the same dose of histamine, p r e f e r a b l y one which e l i c i t s zero response. In t h i s work, 10"^M histamine was found to be a s a t i s f a c t o r y s t a r t i n g dose. The data c o l l e c t e d i n the f i r s t experiment c a r r i e d out using appa-ra t u s 1 i s shown i n Table X I I . I t i s necessary to have 10 cards i n each subset. Therefore the -4 responses at 10 histamine were assumed to be the same as those at 3x10' "5M. The data cards thus c a r r y the values l i s ted below Card 1 106.0 108 .0 114.0 117.0 Card 6 96.0 83.0 108 .0 108 .0 2 0.0 0, .0 0.0 0.0 7 95.0 91.0 108 .0 113 .0 3 1.5 1. .0 0.5 0.0 8 106.0 108.0 114 .0 117 .0 4 34.0 30. .0 34.0 23.0 9 106.0 108.0 114 .0 117 .0 5 53.0 74. .0 61.0 65.0 10 106.0 108.0 114 .0 117 .0 216 Histamine C o n t r a c t i o n (mm) dose (M) W X Y 10" 8 0.0 0.0 0.0 3 x l 0 " 8 1.5 1.0 0.5 10" 7 34.0 30.0 34.0 3 x l O " 7 53.0 74.0 61.0 10" 6 96.0 83.0 108.0 3 x l 0 ~ 6 95.0 91.0 108.0 10" 5 106.0 108.0 114.0 3 x l 0 - 5 106.0 108.0 114.0 IO" 4 ----- -C o n t r a c t i o n i n presence of Z Diphenhydramine (10 M) 0.0 0.0 0.0 0.0 23.0 0.0 65.0 16.0 108.0 46.0 113.0 68.0 117.0 103.0 117.0 105.0 107.0 Table X I I : Data f o r the cumulative dose-response curves obtained i n a t y p i c a l experiment; s p e c i f i c a l l y , the f i r s t experiment us i n g apparatus 1. 217 In the program, the values across card one correspond to WMAX, XMAX, YMAX, and ZMAX, i e WMAX = 106.0. The values across the remaining nine cards correspond to values W, X, Y, and Z used as operands i n the program. This data was reduced to a percentage of maximal response and the mean c o n t r o l response and standard e r r o r at each dose was computed. In the computer p r i n t - o u t , A represents the percentage values c a l c u l a t e d from W and B, C, and D s i m i l a r l y correspond to X, Y, and Z r e s p e c t i v e l y . The format of the p r i n t - o u t i s shown on page 218 f o r the data set given i n Table X I I . The values have been rounded o f f from the three decimal places given i n the p r i n t - o u t . "NO" i n the p r i n t - o u t r e f e r s to the dose -8 of histamine where 1 i s 10 M, pro g r e s s i n g i n 1/2 log u n i t steps (Table I X ) . The next 36 subsets i n group 1 were then p r i n t e d out i n the format given f o r subset 1, and t h i s was followed by: VALUES FOR GROUP OF 37 NO NO OF OBS SUM MEAN STD ERROR 1 123 244.589 1.989 0.249 2 123 650.111 5.285 0.484 3 123 1807.518 14.695 0.948 4 123 4217.223 34.286 1.461 5 123 8329.488 67.719 1.582 6 123 10811.082 87.895 0.798 7 123 12144.937 98.739 0.164 8 123 12266.863 99.731 0.059 9 123 12300.00 100.00 0.0 218 ai W O c o i ^ O r ^ o O O O O Q O O C N - c f c s i O J O O O H o co O N o CO o o o o O r~* vO o 1—1 o o o CM m cr, O N o o o o 0 0 CO vO o CM o o o o CM cr. o m o o o o CM v O o o o 1—1 CM CO CO <r <r O O VO CO vD O O O Q O O ON IO CM vD O O O r-1 m cr. Cr. O O O O <f 0 0 m o o o O O cr. CO o o o CM m O N O N o o o NJ o o O N 0 0 m <t CO o O o pa o o r-. 0 0 CM <r o O o r-1 CM vO 0 0 0 0 o O o t—1 r—4 r - l r - l o r-t o N O o o o < o r - l CM o O O N o o o CO m Cr. 0 0 o o o i-4 H r - l O <r o o o o O o o o o r - l r - l o o CO m 0 0 CO 1— 1— CM vO O <-4 r - l r-1 r - l x: a. O OS a H w o m o o o o o O o o >• o o CO r - l vO 0 0 o 0 0 o r - l <t r - l r - l 0 0 o t-4 o o o o o o o O o X o H o CO <r O N 0 0 r - l O N 0 0 O r - l 0 0 o r - l 0 0 o r - l o o m o O o o O o o v D o r - l DS o r - l co CO m vO O N m O N o r - l vO O H v O O r - l o z r 4 CM CO m N O 1 CO cr* 219 The values for groups 2, 3, and 4 were then p r i n t e d out i n the same format and were followed by: VALUES FOR COMPLETE SET NO NO OF OBS SUM MEAN STD ERR( 1 495 543.686 1.098 0.089 2 495 1743.600 3.522 0.197 3 495 5948.293 12.017 0.460 4 495 16244.668 32.818 0.755 5 495 33046.797 66.761 0.774 6 495 43027.152 86.924 0.468 7 495 48616.863 98.216 0.124 8 495 49345.602 99.688 0.041 9 495 49500.000 100.000 0.0 The values f o r the complete set were used to draw a c o n t r o l cumu-l a t i v e dose-response curve and from t h i s curve the pD 2 value f o r h i s t a -mine was c a l c u l a t e d . The values f o r the i n d i v i d u a l subsets were compared to the c o r r e s -ponding t e s t cumulative responses to determine pA 2 and pD 2 values f o r each of the t e s t compounds. 220 B. For i n t e r p r e t a t i o n of r e s u l t s from t e s t s f o r a n t i m u s c a r i n i c a c t i v i t y . Card number F o r t r a n statement 1 DIMENSION P(3,6,36,4),S(6),N(6),SS(6),NN(6),SSQ(6),AVG(6) 2 DO 3 1=1,6 3 DO 2 M=l,3 4 DO 2 K=l,4 5 DO 2 J=l,36 6 P(M,I,J,K)=-1.0 7 2 CONTINUE 8 SS(I)=0.0 9 NN(I)=0 10 3 CONTINUE 11 DO 150 K=l,4 f 12 DO 4 L=l,6 13 S(L)=0.0 : 14 N(L)=0 15 SSQ(L)=0 16 4 CONTINUE 17 DO 100 J=l,36 18 READ (5,5) WMAX,XMAX,YMAX,ZMAX 19 5 FORMAT (4F10.2) 20 WRITE (6,6) J,K 21 6 FORMAT (/'SUBSET',13,', GROUP',12) 22 WRITE (6,8) 23 8 FORMAT (/10X,'WMAX',11X,'XMAX',11X,'YMAX',1IX,'ZMAX'/)' 24 WRITE (6,9) WMAX,XMAX,YMAX,ZMAX 25 9 FORMAT (4F15.3) 26 WRITE (6,11) 27 11 FORMAT (IX) 28 WRITE (6,20) 29 20 FORMAT (5X,'NO',6X,'W',9X,'X',9X,'Y',9X,'Z',9X,'A' , 30 1 9X,'B',9X,'C',9X,'D',8X,'SUM',6X,'MEAN',5X,'STD ERR'/) 31 DO 80 1=1,6 32 READ (5,71) W,X,Y,Z 33 71 FORMAT (4F10.2) 34 A=(W/WMAX)*100.0 35 P(1,I,J,K)=A 36 B=(X/XMAX)*100.0 37 P(2,I,J,K)=B 38 C=(Y/YMAX)*100.0 39 P(3,I,J,K)=C 40 D=(Z/ZMAX)*100.0 41 SUM=A-f B-+-C 42 XMEAN=SUM/3.0 43 SE=SQRT(((A-XMEAN)**2-f (B-XMEAN)**2 + (C-XMEAN)**2)/6) 44 N(I)=N(I)-f- 3 45 S ( I ) = S ( I ) + SUM 221 46 WRITE (6,75) I,W,X,Y,Z,A,B,C,D,SUM,XMEAN,SE 47 75 FORMAT (17,11F10.3/) 48 80 CONTINUE 49 WRITE (6,11) 50 100 CONTINUE 51 DO 110 1=1,6 52 AVG(I)=S(I)/N(I) 53 SS(I)=SS(I) + S(I ) 54 110 NN(I)=NN(I )4- N(I) 55 WRITE (6,115) 56 115 FORMAT (/20X,'VALUES FOR GROUP OF 36'//) 57 WRITE (6,118) 58 118 FORMAT (4X,'NO',4X,'NO OF OBS*,11X,'SUM',11X, 59 1 'MEAN',9X,'STD ERROR'/) 60 DO 130 1=1,6 61 DO 125 J=l,36 62 DO 120 M=l,3 63 IF (P(M,I,J,K).EQ.-1.0) GO TO 120 64 DIF=P(M,I,J,K)-AVG(I) 65 SSQ(I)=SSQ(I) + DIF**2 66 120 CONTINUE 67 125 CONTINUE 68 SE=SQRT(SSQ(I)/(N(I)*(N(I)-1))) 69 WRITE (6,128)1,N(I),S(I),AVG(I),SE 70 128 FORMAT (I6,I10,4X,3F15.3/) 71 130 CONTINUE 72 WRITE (6,11) 73 WRITE (6,11) 74 150 CONTINUE 75 WRITE (6,151) 76 151 FORMAT (//20X,'VALUES FOR COMPLETE SET'//) 77 WRITE (6,118) 78 DO 152 1=1,6 79 SSQ(I)=0.0 80 152 AVG(I)=SS(I)/NN(I) 81 DO 190 1=1,6 82 DO 180 K=l,4 83 DO 170 J=l,36 84 DO 160 M=l,3 85 IF (P(M,I,J,K).EQ.-1.0) GO TO 160 86 DIF=P(M,I,J,K)-AVG(I) 87 SSQ(I)=SSQ(I) 4- DIF**2 88 160 CONTINUE 89 170 CONTINUE 90 180 CONTINUE 91 SE=SQRT(SSQ(I)/(NN(I)*(NN(I)-1))) 92 WRITE (6,128)I,NN(I),SS(I),AVG(I),SE 93 190 CONTINUE 94 END In the t e s t s f o r a n t i m u s c a r i n i c a c t i v i t y , four groups of 36 222 experiments were c a r r i e d out. Each experiment e n t a i l e d the r e c o r d i n g of three c o n t r o l cumulative responses and one t e s t cumulative response. Only s i x doses of agonist were used f o r each cumulative response. Minor a l t e r a t i o n s of Program A were necessary so that the data from these ex-periments could be i n t e r p r e t e d . The r e s u l t a n t Program B, above, p r i n t e d out the same data as was obtained from Program A. In a d d i t i o n , Program B reduced the measured t e s t responses to percentage v a l u e s . The data deck c o n s i s t e d of four groups, each c o n t a i n i n g 36 subsets. Each subset c o n s i s t e d of seven cards, each of which c a r r i e d four v a l u e s . An example of the data deck i s given below f o r subset 1, group 1. Card 1 120.5 125.0 124.0 123.2 2 0.0 0.0 0.5 0.0 3 6.0 7.5 8.5 7.5 4 72.5 76.0 58.5 40.0 5 110.0 115.0 108.5 84.0 6 117.5 122.0 118.5 101.0 7 120.5 125.0 124.0 103.5 The values across card 1 are WMAX, XMAX, YMAX, the measured maxima of the three c o n t r o l responses, and ZMAX, the average of WMAX, XMAX, and YMAX. A p o r t i o n of the p r i n t - o u t i s shown on page 223. The values have been rounded o f f from the three decimal places given i n the p r i n t -out. "NO" r e f e r s to the dose of agonist (1 = 10 - 8M a c e t y l c h o l i n e , l o g 10 increments). Columns W, X, and Y are measured values of the c o n t r o l responses, reduced to percentages i n columns A, B, and C r e s p e c t i v e l y . SUBSET 1, GROUP 1 WMAX 120.5 NO W 1 0.0 2 6.0 3 72.5 4 110.0 5 117.5 6 120.5 XMAX 125.0 X Y 0.0 0.5 7.5 8.5 76.0 58.5 115.0 108.5 122.0 118.5 125.0 124.0 YMAX 124.0 Z A 0.0 0.0 7.5 5.0 40.0 60.2 84.5 91.3 101.0 97.5 103.5 100.0 ZMAX 123.2 B C 0.0 0.4 6.0 6.9 60.8 47.2 92.0 87.5 97.6 95.6 100.0 100.0 D SUM 0.0 0.4 6.1 17.8 32.5 168.1 68.6 270.8 82.0 290.7 84.0 300.0 MEAN STD ERR 0.1 0.1 5.9 0.5 56.0 4.4 90.3 1.4 96.9 0.7 100.0 0.0 224 Column Z contains measured values f or t e s t responses i n the presence of antagonist w h i l e D i s the percentage values c a l c u l a t e d from Z. The agonist i n t h i s example i s 9-(2-dimethylaminoethyl)-l,2,3,4-tetrahydro--4 carbazole (10 M). Values f o r each group of 36 were p r i n t e d out as i n Program A. Values over the complete set are shown below. VALUES FOR COMPLETE SET NO NO OF OBS SUM MEAN STD ERROR 1 432 423.936 0.981 0.065 2 432 5945.629 13.763 0.554 3 432 23775.645 55.036 0.971 4 432 37180.230 86.065 0.449 5 432 41229.961 95.440 0.196 6 432 43199.914 100.000 0.002 These values were used to draw an average cumulative dose-response curve f o r a c e t y l c h o l i n e from which the pD 2 value was c a l c u l a t e d . The values f o r the i n d i v i d u a l subsets were compared ( t e s t vs average of c o n t r o l s ) to determine pD^ values f o r each of the t e s t compounds. C o n t r o l data f o r a t r o p i n e and diphenhydramine were c a l c u l a t e d by hand. APPENDIX IV NUCLEAR MAGNETIC RESONANCE SPECTRA 226 7-Dime thy lazminoacetaniido-L4-azadispiro. [ 5. .1..5..2] pentadecan- 15-oae (10%, CDC1 ) 227 7-D.lmethylaminoacetamido- 14-azadispiro [5.1. 5. 2 ]pentadecan-15-one (10%, CDC1 , DO added) 7-Dimethylaminoacetamido-l4-azadispiro[5.1.5.2]pentadecane (.10%,. CDC1 ) 

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