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Synthesis and detection of potential N-and α-C- oxidized metabolites of methadone and recipavrin Slatter, John Gregory 1983

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SYNTHESIS AND DETECTION OF POTENTIAL N- AND o-C- OXIDIZED METABOLITES OF METHADONE AND RECI PAVRIN By JOHN GREGORY SLATTER B S c , Lakehead U n i v e r s i t y , 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Faculty of Pharmaceutical Sciences D i v i s i o n of Pharmaceutical Chemistry We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1983 © John Gregory S l a t t e r I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t 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 agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t 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 o f Pharmaceutical Sciences The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date August 5, 1983 DE-6 (3/81) i i ABSTRACT This t he si s describes the attempted c h a r a c t e r i z a t i o n of a new metabolite of methadone which was observed by GCMS f o l l o w i n g B-glucuronidase h y d r o l y s i s of conjugated metabolites in the b i l e o f methadone dosed r a t s . Kang (1) proposed a methylene n i t r o n e s t r u c t u r e f o r t h i s metabolite and suggested that i t arose from a glucuronide conjugated secondary hydroxylamine. The ni t r o n e s t r u c t u r e was also assigned to a chemical o x i d a t i o n product o f methadone's major metabolite EDDP which had i d e n t i c a l GCMS c h a r a c t e r i s t i c s to the metabolite ( 1 ) . This t h e s i s shows that the chemical o x i d a t i o n product was i n fac t an o x a z i r i d i n e which, under GC c o n d i t i o n s , isomerizes to a formamide, with mass spectrum and r e t e n t i o n time i d e n t i c a l to the observed metabolite. Attempts to synthesize the p o t e n t i a l l y thermol a b i l e methyl ene nitrone s t r u c t u r e proposed by Kang f a i l e d due to i n s t a b i l i t y o f key intermediates or by f a c i l e i s o m e r i z a t i o n to the formamide. A model compound, r e c i p a v r i n , which lacks the ethyl ketone side chain o f methadone, was selected to c h a r a c t e r i z e p o t e n t i a l N- and a-C-oxidized m e t a b o l i t e s and to e s t a b l i s h whether formation o f the formamide-1 ike metabolite was general to the many drugs containing the 4 ,4-diphenyl -N,N-dimethyl -2-butyl amine s t r u c t u r e . The n i t r o n e , o x a z i r i d i n e and formamide were synthesized as po t e n t i a l N-and a-C o x i d i z e d metabolites o f r e c i p a v r i n and were c h a r a c t e r i z e d by GCMS, LCMS, NMR, IR and UV spectroscopy. By exact analogy to methadone, r a t s tr e a t e d with r e c i p a v r i n produced a conjugated m e t a b o l i t e , which upon B-glucuronidase h y d r o l y s i s had GCMS c h a r a c t e r i s t i c s i d e n t i c a l to that of the sy n t h e t i c o x a z i r i d i n e and formamide. LC and LCMS were not s e n s i t i v e enough to determine whether the formamide or a thermol a b i l e precursor was respo n s i b l e for the observed m e t a b o l i t e . i i i While i t was not p o s s i b l e to determine which precursor was res p o n s i b l e for the formamide observed by GCMS, there i s a good p o s s i b i l i t y that i t i s a formamide a r i s i n g from a t e r t i a r y formamide carbinolamine glucuronide. iv TABLE OF CONTENTS Page ABSTRACT 11 LIST OF FIGURES xi LIST OF ABBREVIATIONS xvi I . INTRODUCTION 1 1. Observation of a new methadone metabolite 1 2. P o t e n t i a l s i g n i f i c a n c e of the new metabolite 2 3. Methadone, r e c i p a v r i n and r e l a t e d compounds 3 4. Major metabolic pathways of methadone and r e c i p a v r i n 4 5. Metabolism of t e r t i a r y a l i p h a t i c Amines 7 A. N - d e a l k y l a t i o n 7 B. Stable carbinolamines 9 C. Carbinolamine glucuronide conjugates 10 D. Factors i n f l u e n c i n g carbinolamine s t a b i l i t y 11 E. N-Formyl metabolites 12 i ) Formyl conjugates 12 i i ) Formyl metabolites of t e r t i a r y a l i p h a t i c amines 13 i i i ) P o t e n t i a l s i g n i f i c a n c e of the formamide metabolite 15 6. Metabolic N-oxidation 16 A. Substrates and mechanisms 16 B. Metabolic f a t e and d e t e c t i o n of N-oxidized metabolites 16 i ) Source of methylene nitrones 16 i i ) P o t e n t i a l sources of methylene o x a z i r i d i n e s 18 7. T h e r m o l a b i l i t y and i n t e r c o n v e r s i o n of p o t e n t i a l metabolites 19 during GCMS a n a l y s i s 8. Synthesis of N-oxidized and r e l a t e d drug metabolites 21 V TABLE OF CONTENTS (CONT'D) Page I I . EXPERIMENTAL 22 A. REAGENTS AND MATERIALS 22 B. INSTRUMENTAL 25 1. Nuclear magnetic resonance spectra 25 2. Infrared spectra 25 3. Gas chromatography mass spectrometry 26 4. Liq u i d chromatography mass spectrometry 27 5. U l t r a v i o l e t spectrometry 28 6. Melting points and Elemental analyses 28 7. Metabol ism experiments 28 a) C e n t r i f u g a t i o n 28 b) Tissue homogenization 28 c) L y o p h i l i z a t i o n 28 C. IN VIVO METABOLISM 29 1. B i l e duct cannulation and dosages 29 2. E x t r a c t i o n of conjugated and non conjugated 29 metabolites from b i l e D. IN VITRO METABOLISM 30 1. Preparation of microsomes 30 2. Metabol i c procedure 31 E. GENERAL . 31 1. Flash chromatography 31 F. SYNTHESIS OF POTENTIAL N-OXIDIZED METABOLITES 32 OF METHADONE v i TABLE OF CONTENTS (CONT'D) Page 1. Synthesis of 2,2-diphenylpent-4-ene n i t r i l e (38) 32 2. Synthesis of 3,3-diphenyl-5-methyltetrahydro-2- 32 furanone imine (40) 3. Synthesis of 2,2-diphenyl-4-val erol actone (41_) 33 4. Synthesis of l.l-diphenyl-2-butanone (42) 34 5. Synthesis of 4,4-diphenyl-2,5-heptanediol (43) 34 6. Synthesis of 4,4-diphenyl-2,5-heptanedione (45) 36 7. Synthesis of 2-(4 ' ,4'-diphenylheptan-5'-one-2'yl) 37 - o x a z i r i d i n e (5) 8. Synthesis of 2-(N-formyl)-4,4-diphenyl-5-heptanone (31) 38 G. DIKETONE REACTIONS 39 1. Synthesis of 4,4-diphenyl-2,5-heptanedione-2-oxime (46) 39 2. Sodium cyanoborohydride reduction of 4,4-diphenyl- 40 2,5-heptanedione-2-oxime 3. Reaction with N-methyl hydroxy 1 amine hydrochloride 40 H. REACTIONS OF METHADONE OXAZIRIDINE (5) 41 1. Reduction with l i t h i u m aluminum hydride 41 2. Reduction with sodium cyanoborohydride 42 I. SYNTHESIS OF RECIPAVRIN RELATED COMPOUNDS 42 1. Synthesis of V-phenyl-N,N-a-trimethyl benzenepropanamine 42 perc h l o r a t e (6) 2. Synthesis of 1,1-diphenyl-3-butanone (5J_) 43 3. Synthesis of 1,1-diphenyl-3-butanone oxime (52) 44 4. Synthesis of a-methyl - y -phenyl benzenepropanamine (53) 45 V l l TABLE OF CONTENTS (CONT'D) 5. Synthesis of N,a-dimethyl- Y-phenyl benzene-propanamine (54) 6. Synthesis of N-hydroxy-a-methyl -y -phenyl benzenepropanamine (55) 7. Synthesis of N, a-dimethyl-N-hydroxy-y -phenyl benzenepropanamine (57) 8. Synthesis of N-methyl i d e n e - l , l - d i p h e n y l - 3 -aminobutane (59) 9. Synthesis of 1,1-diphenyl-3-nitrosobutane (61) 10. Synthesis of N-formyl-a-methyl-y -phenyl benzene propanamine (15) 11. Synthesis of 2-(4 1,4'-diphenyl-but-2'-yl) o x a z i r i d i n e (14) 12. Synthesis of a-methyl-(N-methylene)-Y-phenyl benzenepropanamine N-oxide (13) 13. Synthesis of 1,1-diphenyl-3-butanone-0-methyloxime (64) 14. A l k a l i n e o x i d a t i o n of N, a-dimethyl-N-hydroxy-y -phenyl benzenepropanamine (57) J . ATTEMPTED CHARACTERIZATION OF THE RECIPAVRIN METABOLITE BY LCMS K. ATTEMPTED GC CIMS CHARACTERIZATION OF THE RECIPAVRIN METABOLITE v i i i TABLE OF CONTENTS Page I I I . RESULTS AND DISCUSSION 56 1. CHEMISTRY: Synthetic methods and an a l y s i s 56 A. Synthetic Pathways 56 B. O x a z i r i d i n e s 60 i . Synthesis and p r o p e r t i e s 60 i i . Detection and i s o l a t i o n 62 i i i . Reactions of methadone o x a z i r i d i n e 63 i v . *H and 1 3C NMR of o x a z i r i d i n e s 6 4 v. Infrared spectra 70 v i . U l t r a v i o l e t spectra 70 v i i . Thermol a b i l i t y and mass sp e c t r a l a n a l y s i s 70 C. FORMAMIDES 73 i . Synthesis and NMR spec t r a 73 i i . I nfrared spectra 78 i i i . Mass spectra 81 D. NITRONES 87 i . Attempted synthesis of the methadone n i t r o n e v i a 87 the primary hydroxyl amine i i . Attempted synthesis of the methadone nit r o n e v i a 90 the secondary hydroxyl amine (3_) i i i . Other attempts to obtain the methadone n i t r o n e 90 i v . Synthesis of the r e c i p a v r i n n i t r o n e 91 a. NMR spectra 91 b. Infrared and UV spec t r a 96 c. GCMS and mass spectra 96 i x Page I I I . RESULTS AND DISCUSSION (CONT'D) E. KETONES 99 i . NMR spectra 99 i i . I n f r a r e d spectra 102 i i i . Mass spectra 102 i v . Problems i n the diketone synthesis 102 F. OXIMES 112 G. AMINES 112 H. HYDROXYLAMINES 113 I. SYNTHESIS OF THE IMINE (59) 115 J. SYNTHESIS OF THE NITROSO COMPOUND 115 K. PEROXIDATION OF EM DP 116 2. METABOLISM 118 A. METHADONE METABOLISM 118 B. RECIPAVRIN METABOLISM 118 i . In v i t r o metabolism 118 i i . In v i v o metabolism 125 a. Non conjugated metabolites 125 b. Conjugated metabolites 125 1. Detection of the formamide metabolite 125 2. Hydroxy1 amines as precursors of the 135 observed r e c i p a v r i n metabolite C. NORRECIPAVRIN METABOLISM 139 i . Conjugated metabolites 139 X Page I I I . RESULTS AND DISCUSSION (CONT'D) D. METABOLISM OF THE METHADONE ANALOGUE 142 i . Non conjugated f r a c t i o n 142 i i . Conjugated metabolites 146 i i i . In v i t r o metabolism 146 E. FINAL COMMENTS ON THE UNKNOWN METABOLITE 151 i . Problems in the a n a l y s i s of thermolabile drug 151 metabolites i i . P o s s i b l e pathways to glucuronide conjugated 152 N-formyl me t a b o l i t e s of methadone and r e c i p a v r i n i i i . Other p o t e n t i a l sources o f a formamide 153 metabolite IV. SUMMARY AND CONCLUSIONS 154 V. REFERENCES 155 VI. APPENDIX 161 A. GC TOTAL ION CURRENT TRACES 161 B. INFRARED SPECTRA 164 C. MASS SPECTRA 179 i . S ynthetic compounds 179 i i . M e t a b o l i t e s 214 D. MASS SPECTRA AND MASS CHROMATOGRAMS (CAPILLARY GCMS) 221 E. NMR SPECTRA .221 F. UV SPECTRA 222 xi LIST OF FIGURES Page 1. Major metabolic pathways of methadone 5 2. N - d e a l k y l a t i o n of t e r t i a r y amines, showing proposed 7 r e a c t i v e intermediates 3. Stoichiometry of cytochrome P-450 catalyzed a-C o x i d a t i o n 8 4. Mechanism of t e r t i a r y amine o x i d a t i o n v i a an iminium ion 8 5. I n s t a b i l i t y of carbinolamines i n acid s o l u t i o n 10 6. P o s s i b l e metabolic pathways to a formyl o x i d a t i o n product 14 of aminopyrine 7. Metabolic N-oxidation of primary, secondary and t e r t i a r y 17 amphetamines 8. Attempted synthesis of the methadone nitrone (2) 57 9. Synthesis of the methadone o x a z i r i d i n e {5) and formamide (3JJ 58 10. Synthesis of p o t e n t i a l N and a-C o x i d i z e d metabolites 59 of r e c i p a v r i n 11. Mechanism f o r the formation of a methylene o x a z i r i d i n e 61 from EDDP 12(a) 1H and * 3C NMR r e s u l t s f o r methadone and r e c i p a v r i n 65 o x a z i r i d i n e s compared to those of 2-t-butyl o x a z i r i d i n e (b) 400 MHz 1H NMR of 2-(4',4'-diphenylheptan-5'-one'2'-yl) 66 o x a z i r i d i n e major diastereomer (5) (c) 400 MHz *H NMR of 2-(4'4'-diphenyl-but-2'-yl) o x a z i r i d i n e 67 major diastereomer (14) (d) SF0RD 400 MHz l 3C NMR of 2-(4' ,4 '-diphenylheptan-5 '-one 68 -2'-yl) o x a z i r i d i n e major diastereomer (5_) (e) SF0RD 400 MHz 1 3C NMR of 2-(4 ' ,4'-diphenyl-but-2'-yl) 69 o x a z i r i d i n e major diastereomer (14) 13(a) I n f r a r e d spectrum ( f i l m ) of 2-(4',4'-diphenylheptan-5'-one- 71 2'-yl) o x a z i r i d i n e (5J (b) I n f r a r e d spectrum ( f i l m ) of 2-(4' ,4'-diphenyl-but-2'-yl) 72 o x a z i r i d i n e (14) 14(a) Comparison of *H NMR r e s u l t s f o r r e c i p a v r i n and methadone 74 formamides with those of isopropyl formamide xii LIST OF FIGURES (CONT'D) Page 14(b) 400 MHz 1H NMR of 2-(N-formyl)-4,4-diphenyl-5-heptanone 75 (c) 400 MHz *H NMR of N-formyl-a-methyl- Y-phenyl benzene- 76 propanamine (15) (d) 400 MHz  l H NMR of N-formyl-a-methyl-Y-phenyl benzene- 77 propanamine c o l l e c t e d o f f a GC column 15(a) I n f r a r e d spectrum ( f i l m ) of 2-(N-formyl)-4,4-diphenyl-5- 79 heptanone (31) (b) I n f r a r e d spectrum ( f i l m ) of N-formyl-a-methyl- Y-phenyl 80 benzenepropanamine (15) 16(a) Mass spectrum (GCMS) of N-formyl-a-methyl- Y-phenyl 82 benzenepropanamine (1_5) ( i d e n t i c a l to o x a z i r i d i n e (14) and the r e c i p a v r i n metabolite (b) Mass sp e c t r a l fragmentation pathway of N-formyl-a-methyl 83 - Y-phenyl benzenepropanamine (15) 17. Mass spectrum (GCMS) of N, a-dimethyl-N-formyl-Y -phenyl 84 benzenepropanamine (63) 18(a) Mass spectrum (GCMS) of 2-(N-formyl)-4,4-diphenyl 85 -5-heptanone (31) ( i d e n t i c a l to o x a z i r i d i n e (5J and the methadone metabolite) 18(b) Mass s p e c t r a l fragmentation of methadone formamide 86 19(a) Comparison of *H NMR r e s u l t s f o r r e c i p a v r i n n i t r o n e 92 (13) with a l i t e r a t u r e compound (67) (b) 13Q NMR 0 f the r e c i p a v r i n n i t r o n e 92 (c) 400 MHz *H NMR of a-methyl-(N-methylene ) - T-phenyl 93 benzenepropanamine N-oxide (13) (d) SF0RD 400 MHz 1 3 C NMR of a-methyl-(N-methylene) 94 -y-phenyl benzenepropanamine N-oxide (13) 20. Infrared spectrum ( f i l m ) of a-methyl-(N-methylene) 95 - Y-phenyl benzenepropanamine N-oxide (13) - y-phenyl-benzenepropanamine N-oxide (13) (b) Fragmentation pathways of the r e c i p a v r i n n i t r o n e (13) by analogy to Coutts et al (97) 22(a) 80 MHz  l H NMR of 4,4-diphenyl-2,5-heptanedione (45) 98 100 x i i i LIST OF FIGURES (CONT'D) Pa°e. 22 ( b ) 1 0 0 MHz *H NMR of 1,1-diphenyl-3-butanone ( 5 1 ) 1 0 1 (c) Broad band decoupled 2 0 MHz 1 3 C NMR of 4,4-diphenyl- 1 0 3 2,5-heptanedione ( 4 5 ) 2 3 . Infrared spectrum (nujol mull) of 4,4-d ipheny l-2,5- 1 0 4 heptanedione ( 4 5 ) 2 4 . Electron impact mass fragmentography of the ketones ( 4 2 _ ) 1 0 5 ( 4 5 ) and ( 5 J J 2 5 . NMR resu l ts for the diketone synthetic pathway 1 0 6 26 ( a ) Electron impact mass fragmentography of the THF imine 1 0 7 (b) Compounds which fragment via the m/z 2 0 7 , 1 2 9 series 1 0 8 (c) Fragmentation of di ol ( 4 3 ) and ethylidene tetrahydrofuran 1 1 0 ( 4 4 ) (d) Fragmentation of 2-ethyl idene-l,4-dimethyl-3,3-diphenyl- 1 1 1 2,3-dihydropyrol le ( 7 0 ) compared to EDDP (66) 2 7 . Comparison of NMR resu l ts for the primary and secondary 1 1 4 hydroxylamines and the corresponding amines 2 8 . Thermal rearrangement and fragmentation of the EMDP 1 1 7 oxaz i r id ines ( 7 2 ) and ( 7 3 ) v ia the ketones ( 7 4 _ ) and ( 7 5 ) 2 9 ( a ) Mass spectrum (GCMS) of the methadone formamide metabolite 1 1 9 in the conjugated f rac t ion of b i l e from a methadone dosed rat (b) Mass spectrum (GCMS) of the 2 H I Q methadone formamide 1 2 0 metabolite from the conjugated f ract ion of b i l e from a methadone dosed rat (c) Mass spectrum (GCMS) of the methadone formamide metabolite generated in v i t r o 1 2 1 3 0 . TIC of in v i t ro methadone metabolic extract 1 2 2 3 1 . GCMS of an in v i t r o rec ipavr in metabolic extract showing 1 2 3 metabolites ( 7 6 ) ( 7 7 ) ( 7 8 ) 3 2 . Mass chromatograms of ions present in the non conjugated 1 2 5 f rac t ion of b i l e from a rec ipavr in dosed r a t , showing dealkyl and deaminated metabolites 3 3 ( a ) GCMS of the conjugated f rac t ion of b i l e from a recipavr in 1 2 6 dosed rat xiv LIST OF FIGURES (CONT'D) Page 33(b) Mass spectrum (GCMS) of the r e c i p a v r i n formamide 127 metabolite in the conjugated f r a c t i o n of b i l e from a r e c i p a v r i n dosed r a t 34(a) LCMS o f ni t r o n e (13_) with some formamide present as 129 a decomposition product (b) LCMS o f formamide (15.)) 130 (c) LCMS of o x a z i r i d i n e (14) 131 35(a) C a p i l l a r y CI GCMS SIM of the r e c i p a v r i n n i t r o n e showing 133 thermal breakdown products A (imine (59J), B (oxime (52)) and C (formamide (15)) (b) SIM o f the same ions to detect r e c i p a v r i n metabolites from 133 the conjugated f r a c t i o n o f r a t b i l e 36. Mass chromatograms showing metabolites i n the TMS 134 d e r i v i t i z e d conjugated f r a c t i o n of b i l e from a r e c i p a v r i n dosed rat 37. GCMS o f the conjugated f r a c t i o n o f blank r a t b i l e spiked 136 with the secondary hydroxylamine (57_). Formamide present at scan (63) 38. SIM of the m/z 146 peak of the r e c i p a v r i n 0-TMS secondary 137 hydroxylamine (58) and the BSTFA d e r i v a t i z e d r e c i p a v r i n m e tabolites from the conjugated b i l e f r a c t i o n , showing s l i g h t l y d i f f e r e n t r e t e n t i o n time 39(a) HPLC (UV detection) of a mixture of r e c i p a v r i n nitrone 138 (13) (17.2 min), formamide (15) (24.41 min) and o x a z i r i d i n e (14J (40.87 min) 39(b) HPLC o f secondary hydroxyl amine (57_) a f t e r treatment 138 with a l k a l i , showing n i t r o n e (17.32 min) as a major product, with unreacted secondary hydroxylamine at 54.37 min (attenuated peak) 40(a) m/z 253 mass chromatogram and mass spectrum of the 140 formamide metabolite from the conjugated f r a c t o n of b i l e from a n o r r e c i p a v r i n dosed rat (GC c o n d i t i o n s f i i ) 40(b) P e t h i d i n e , i t s m e t a b o l i t e norpethidine and a N-formyl 141 compound which a r i s e s during workup by r e a c t i o n with chloroform contaminants 41. TIC o f methadone analogue (86_) non conjugated b i l i a r y 143 metabolites 42(a) Fragmentation of BDDP 144 XV LIST OF FIGURES (CONT'D) Page 42(b) Mass spectrum (GCMS) of methadone analogue metabolite 145 buty l i d e n e dimethyldiphenyl p y r o l l i d i n e (BDDP) (87) 43(a) TIC of methadone analogue (86) conjugated b i l i a r y 147 metabolites (b) Mass spectrum (GCMS) of a formamide l i k e conjugated 148 methadone analogue me t a b o l i t e (E) 44(a) TIC showing in v i t r o metabolites of the methadone 149 analogue (86) (b) Mass spectrum (GCMS) of the Cope e l i m i n a t i o n product 150 of methadone analogue N-oxide from an in v i t r o metabolic e x t r a c t 45. P o s s i b l e mechanism f o r the formation of formamide 152 metabolites of methadone and r e c i p a v r i n Xvi LIST OF ABBREVIATIONS Page Ac Acetyl Ar Aryl BDDP 2-butylidene-N,5-dimethyl-3,3-diphenyl p y r o l l i d i n e BMDP 2-butyl-5-methyl-3,3-diphenyl p y r o l l i d i n e BSTFA N , 0 - b i s - ( T r i m e t h y l s i l y l ) T r i f l u o r o a c e t a m i d e CAS Chemical a b s t r a c t s s e r v i c e CI GCMS Chemical i o n i z a t i o n gas chromatography mass spectrometry DMCS Dimethyl c h l o r o s i l a n e d doublet dd doublet of doublets DPAN d i p h e n y l a c e t o n i t r i l e EDDP 2-ethylidene-N,5-dimethyl-3,3-diphenyl p y r o l l i d i n e EI E l e c t r o n impact EMDP 2-ethyl-5-methyl-3,3-diphenyl p y r o l l i d i n e EtMgBr Ethyl magnesium bronide GCMS Gas chromatography mass spectrometry GCQ Gas chrom Q GLC Gas l i q u i d chromatography GLU Glucuronic Acid H-Bond Hydrogen bond HPLC High performance l i q u i d chromatography IR Infrared LAH Lithium aluminum hydride LC L i q u i d chromatography LCMS L i q u i d chromatography mass spectrometry x v i i LIST OF ABBREVIATIONS (CONT'D) m medium ( I R ) , m u l t i p l e t (NMR) M + molecular ion NIC Mass chromatogram MCPBA metachloroperbenzoic a c i d MeOH methanol MOX 0-methyl hydroxyl amine hydrochloride MS mass spectrometry MIZ mass to charge r a t i o NADP Nicotinamide adenine d i n u c l e o t i d e phosphate NOE Nuclear Overhauser E f f e c t MDP 5-methyl-3,3-d iphenyl pyrol1 id ine NMR Nuclear magnetic resonance DDP N ,5-dimethyl-3,3-d iphenyl pyrol 1 id ine pet.ether petroleum ether (30°-60°) ppm parts per mil 1 ion py p y r i d ine q quadrupl et r f TLC m o b i l i t y r e l a t i v e to solvent f r o n t s s i n g l e t (NMR), strong (IR) SIM selected ion monitoring S t r s t r e t c h TMAH Tr i m e t h y l a n i l inium hydroxide UV u l t r a v i o l e t xvi i i ACKNOWLEDGEMENT The a u t h o r wishes t o thank D r . F.S. A b b o t t f o r h i s g u i d a n c e and s u p p o r t t h r o u g h o u t t h i s program. Thanks a l s o go t o Roland B u r t o n f o r a b l e a s s i s t a n c e i n GCMS and LCMS a n a l y s i s , S h e i l a F e r g u s o n f o r a s s i s t a n c e w i t h s u r g e r y , M a r g a r e t Heldman and t h e s t a f f o f t h e NMR l a b i n t h e Department o f C h e m i s t r y f o r f a s t and e x c e l l e n t NMR s e r v i c e , G e o f f Sunahara and Ahmad Fawzi f o r d i s c u s s i o n and encouragement, and L i s a Wong f o r p r e p a r a t i o n o f the m a n u s c r i p t . L a s t l y the f i n a n c i a l s u p p o r t p r o v i d e d by the F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s , and t h e U n i v e r s i t y G r a d u a t e F e l l o w s h i p Committee a r e g r a t e f u l l y acknowledged. xix DEDICATION To my family and f r i e n d s - 1 -I. INTRODUCTION 1. OBSERVATION OF A NEW METHADONE METABOLITE In h i s PhD t h e s i s , Kang (1) r e c e n t l y described an unknown metabolite of methadone (1_). The meta b o l i t e was c h a r a c t e r i z e d f o l l o w i n g the g-glucuronidase h y d r o l y s i s of the conjugated f r a c t i o n of b i l e from methadone dosed r a t s . He proposed the n i t r o n e s t r u c t u r e {2), and presumed that i t arose from the secondary hydroxyl amine (_3)» as a r e s u l t of o x i d a t i o n during a l k a l i n e workup. This was s i m i l a r to the in v i t r o formation and e x t r a c t i o n of secondary hydroxylamine metabolites of amphetamines described by Beckett I d e n t i f i c a t i o n of the meta b o l i t e (2) was made on the basis of a m-chloroperbenzoic acid o x i d a t i o n of EDDP (4J the major metabolite of methadone. The EDDP o x i d a t i o n product had the same GC r e t e n t i o n time and mass spectrum, as the metabolite and was a l s o assigned the ni t r o n e s t r u c t u r e . Kang alluded that the o x i d a t i o n product could also be the o x a z i r i d i n e [5), i f thermal i s o m e r i z a t i o n to the nitr o n e i n the GC i n l e t occurred. - 2 -In a c o n t i n u a t i o n of t h i s work, t h i s t h e s i s examines the p o s s i b i l i t y of an N- or a-C- o x i d a t i v e metabolic pathway f o r methadone (1_) and the s t r u c t u r a l l y r e l a t e d model compound, r e c i p a v r i n , (6) i n r a t s . The major o b j e c t i v e s were f o u r f o l d : 1) To i d e n t i f y and synthesize the methadone metabolite proposed by Kang. 2) To synthesize and screen f o r p o t e n t i a l N- and a-C- o x i d i z e d metabolites of r e c i p a v r i n , and thus e s t a b l i s h whether the pathway i s general to compounds co n t a i n i n g the 4,4-diphenyl-2- (N,N-dimethylamino) butane s t r u c t u r e . 3) To determine the s t a b i l i t y of p o t e n t i a l N and a-C o x i d i z e d metabolites of methadone and r e c i p a v r i n t o GLC a n a l y s i s . 4 To i n v e s t i g a t e the NMR spectra of methylene nitrones and o x a z i r i d i n e s as an aid to s t r u c t u r e e l u c i d a t i o n of t h e r m o l a b i l e m e t abolites. 2. P o t e n t i a l S i g n i f i c a n c e of The New M e t a b o l i t e The s t r u c t u r e e l u c i d a t i o n of new metabolites of drugs has t h e o r e t i c a l j u s t i f i c a t i o n i n the need f o r b e t t e r understanding of the pharmacokinetics and t o x i c i t i e s of the drug. 4 5 6 - 3 -Methadone i s a potent analgesic but i t s use i s viewed with some t r e p i d a t i o n in c e r t a i n countries due to i t s a f f i l i a t i o n with the treatment of heroin a d d i c t i o n . I t s other use as a narc o t i c analgesic mostly involves pain r e l i e f in terminal cancer p a t i e n t s . Thus although the metabolic pathway i s novel, and the metabolites p o t e n t i a l l y t o x i c or c a r c i n o g e n i c , p u b l i c sympathy does not l i e with heroin a d d i c t s , and long term t o x i c i t y i s c e r t a i n l y not a concern of terminal cancer p a t i e n t s . Methadone i s however being reviewed i n B r i t a i n as a post operative a n a l g e s i c , and i t s b a s i c s i d e chain i s a component of many other drugs. Thus the g e n e r a l i t y of a novel metabolic pathway, and the p o s s i b i l i t i e s that t o x i c or a c t i v e metabolites are formed i s worth i n v e s t i g a t i n g . The t o x i c i t y of aromatic amine N-oxidized metabolites i s well documented (3). Several workers have t r i e d to i m p l i c a t e covalent binding of N-oxidized metabolites of various a l i p h a t i c amines in t h e i r t o x i c i t y and c a r c i n o g e n i c i t y and N-oxidized metabolites have been postulated as p o t e n t i a l mediators in the pharmacology of endogenous amines (4) and as the cause of the undesirable s i d e e f f e c t s of amphetamine abuse (5). From a t h e o r e t i c a l standpoint, the mechanism of N - d e a l k y l a t i o n , and the trapping of r e a c t i v e intermediates i n N-dealk y l a t i o n r e a c t i o n s may be f u r t h e r e l u c i d a t e d . 3. Methadone, R e c i p a v r i n and Related Compounds Methadone (1_) was f i r s t synthesized by Bockmuhl i n the 1940's ( 6 ) . The (-)-isomer had analgesic potency equal to morphine and t h i s observation prompted the synthesis and t e s t i n g of several hundred diphenylpropyl amine analogues ( 7 ) , among them, the analgesics methadol (7_)> 1-acetylmethadol (8J, and propoxyphene (9_). - 4 -Because of i t s long h a l f l i f e (25 hours) r e l a t i v e to morphine and a l e s s severe abstinence syndrome, methadone i s c u r r e n t l y used in the treatment of heroin a d d i c t i o n . R e c i p a v r i n [6) was synthesized by the Wellcome company i n 1949 ( B r i t i s h Patent 624117) (8). I t was found to be i n a c t i v e as an a n a l g e s i c , but i t s a n t i h i s t a m i n i c and a n t i c h o l i n e r g i c p r o p e r t i e s r e s u l t e d in i t s use as an antispasmodic under the trade names Robetyl and Preparyl (9) during the l a t e 1950's. In t h i s t h e s i s r e c i p a v r i n was chosen as a model compound to determine whether other t e r t i a r y 4,4-diphenyl-2-butylamines undergo N-oxidation, i n a manner s i m i l a r to that described by Kang (1). The pka value, (9.48) i s s i m i l a r to that of methadone, (8.99) (4 ) , and i t s metabolism although not p r e v i o u s l y i n v e s t i g a t e d , was expected to be s i m p l e r , due to the lack of an ethyl ketone side c h a i n . This precludes the formation of p y r o l l i d i n e s l i k e EDDP, the major metabolite of methadone (Figure 1) (10). 4. Metabolic Pathways of Methadone and R e c i p a v r i n As o u t l i n e d i n Figure 1, N-demethylation of methadone, r e s u l t s i n spontaneous c y c l i z a t i o n of normethadone to EDDP (£), which may be i n t u r n - 5 -Figure 1. Major metabolic pathways of methadone - 6 -be r i n g hydroxylated (10), f u r t h e r dealkylated to EMDP ( U J , o x i d i z e d t o DDP (12), or excreted i n t a c t i n b i l e and u r i n e . The s t r u c t u r e e l u c i d a t i o n of these metabolites was performed by Beckett et al (11) and S u l l i v a n and Due (12). B i l i a r y metabolism has been i n v e s t i g a t e d by Kreek et a l (13) and Roerig (14). A t e r t i a r y N-oxide, has been described by Beckett (15), and i s observed by GCMS at short r e t e n t i o n time as the Cope e l i m i n a t i o n , product 4,4-diphenyl-5-hept-2-enone. The remaining metabolites a r i s e from o x i d a t i o n of the ethyl ketone side chain or by r i n g h y d r o x y l a t i o n . The new metabolite observed by Kang was not r i n g hydroxylated, since a d m i n i s t r a t i o n of phenyl r i n g l a b e l l e d ^W\Q - methadone r e s u l t e d i n t o t a l r e t e n t i o n of deuterium. This suggested glucuronidation on or near the nitrogen atom, since the keto f u n c t i o n was i n t a c t i n the chemical o x i d a t i o n product of EDDP. The long GC r e t e n t i o n time of the metabolite suggested a very polar compound, ther e f o r e the n i t r o n e s t r u c t u r e was a good proposal i n l i g h t of the nitrone and hydroxylamine metabolites of N-methyl amphetamine observed in v i t r o by Coutts et al (16). There are problems with the proposal however. F i r s t l y , attempts to detect the secondary hydroxylamine were unsuccessful, and secondly, there are few examples i n the l i t e r a t u r e of the in vivo N-oxidation of a l i p h a t i c amines (17), and no examples of a l i p h a t i c secondary hydroxylamine N-O-glucuronides (18). However, glucuronide conjugates of primary a l i p h a t i c hydroxylamines (19, 20), aromatic hydroxylamines and a r y l hydroxamic acids are well known (18). L a s t l y , the r e q u i s i t e secondary amine precursor (normethadone) i s not a s t a b l e compound, and EDDP d i d not produce the r e q u i r e d metabolite when administered to r a t s ( 1 ) . - 7 -To f u r t h e r e l u c i d a t e the s t r u c t u r e of the metabolite observed by Kang, the well documented t h e r m o l a b i l i t y of N-oxidized metabolites prompted attempts to synt h e s i z e the r e c i p a v r i n n i t r o n e (13) and o x a z i r i d i n e (21). The thermal rearrangement of o x a z i r i d i n e to amides observed by Emmons (21) i n d i c a t e d t h a t the formamide (1_5) could also account f o r the observed met a b o l i t e . This i n t r o d u c t i o n w i l l review the l i t e r a t u r e precedents f o r p o t e n t i a l N and a-C ox i d i z e d metabolites and t h e i r glucuronide precursors. 5. Metabolism of t e r t i a r y a l i p h a t i c amines A. N-Dealkylation I t i s g e n e r a l l y held that carbinolamines formed metabolic al 1 y from t e r t i a r y amines decompose to formaldehyde and the corresponding desalkyl amine as o u t l i n e d i n Figure 2 (22). / v R - N - ( C H 3 ) 2 — v A + R - N - ( C H J 9 R - N - CH90H 1 l C H 3 R -.N = CH •y - 2 CH, R - NHCH. H2C0 Figure 2. N - d e a l k y l a t i o n of t e r t i a r y amines showing proposed r e a c t i v e intermediates. - 8 -The intermediates a r i s e by a cytochrome P-450 mediated oxene [:6:] i n s e r t i o n mechanism, or by dehydrogenation of the amine to an iminium i o n . The stoichiometry and the mechanism of o x i d a t i o n are o u t l i n e d i n Figure 3 and 4 (23). T e r t i a r y N-oxides, once thought to be the r e a c t i v e intermediates i n N- d e a l k y l a t i o n , are now thought to be excreted i n t a c t , or reduced back to the parent t e r t i a r y amine p r i o r to N-d e a l k y l a t i o n (24). 3. R2N-CH2R' + NADPH + 0 2 + H + ^ R2N"CH0H-R' + NADP++ H 20 4-. R2NCH2R' + Fe 3 +:0 — > R2NCH2R' + Fe 3 +:Q-Figure 3. Stoichiometry of Cytochrome P-450 catalyzed a-C o x i d a t i o n . Figure 4. Mechanism of t e r t i a r y amine o x i d a t i o n v i a an iminium i o n . - 9 -B. S t a b l e carbinolamines There are a number of examples of weakly basic drugs, whose carbinolamine i n vivo metabolites are s t a b l e enough to i s o l a t e . These i n c l u d e N-Methyl Carbazole (25), Monuron (16, 17J, (26) N-methyl benzamide (26), c l e b r o p r i d e (27) and hexamethylmelamine (26). 16 17 The existence of l e s s s t a b l e carbinolamines has been i n f e r r e d from the dis c o v e r y of a-oxo metabolites such as the lactams" formed metabol i c a l l y from n i c o t i n e , (18,',>-l-9r~20), (28) p r o l i n t a n e (29), medazepam (30) and methyprylon (31). The preference f o r dehydrogenation to the lactam over d e a l k y l a t i o n to open chain compounds i s d i f f i c u l t to r a t i o n a l i z e i n l i g h t of the basic nature of the nitrogen atom i n these compounds. 18 19 20 - 1 0 -In each of these three examples the c a r b i n o l group i s adjacent to a basic nitrogen atom, t h e r e f o r e even m i l d l y a c i d i c c o n d i t i o n s , should d e a l k y l a t e the intermediate by the mechanism shown in Figure 5 ( 2 2 ) . + R 2 - N - C H 0 H - R ' R2NH-CrHOH -R' —> R2NH + RC ( = 0)H Figure 5 . I n s t a b i l i t y of carbinolamines i n acid s o l u t i o n . C. Carbinolamine Glucuronide Conjugates In the metabolism of benzyl-N-benzylcarbethoxy hydroxamate ( 3 2 ) , N-Methyl-a-phenyl-a-ethyl g l u t a r i m i d e ( 3 3 ) , diphenamid ( 3 4 ) , N-methyl ca r b a z o l e ( 2 5 ) and various d i m e t h y l a r y l t r i a z e n e s ( 2 6 ) ( 2 1 , 2 2 , 2 3 ) , semi s t a b l e c a r b i n o l amine metabolites are trapped by g l u c u r o n i d a t i o n p r i o r to dealkylation. 2 1 2 2 2 3 The anticonvulsant N-2- L5-m-chlorophenyl)-l,2,4-oxadiazol-3-yl] ethyl-N-methyl acetamide (oxadiazol) (24), i s metabolized to a glucuronide conjugate of the carbinolamine (25), which a f t e r enzymatic h y d r o l y s i s l o s e s formaldehyde and i s analyzed as the corresponding secondary amide (26) (35). This may be the mechanism whereby a formamide metabolite of methadone or r e c i p a v r i n could a r i s e , concomitant upon i n i t i a l a-C o x i d a t i o n of a t e r t i a r y N-methyl to a formamido group. 2 4 2 5 2 6 D. Factors i n f l u e n c i n g carbinolamine s t a b i l i t y A l l of the above carbinolamines are s t a b i l i z e d by conjugation to a - a r y l , or a-carbonyl groups. These s t a b i l i z i n g groups lower the pKa value of an amido nitrogen to one or l e s s ( 3 6 ) , a s a r e s u l t of d e l o c a l i z a t i o n of and non bonding e l e c t r o n s in the e q u i l i b r i u m : 0 H R — C — N — RT — ) « 2 R — 0- R i / / C = N + \ R 2 o-c — / N — R\ « 2 - 12 -The resu l tant planar sp2 geometry at nitrogen contrasts the pyramidal geometry of amines, and precludes oxidat ion of the nitrogen s ince the lone p a i r is not ava i l ab le fo r bond formation with e l e c t r oph i l e s . This evidence supports carbinolamine intermediacy in the N-dealkylation of t e r t i a r y amides (37). E. N-Formyl metabolites i ) Formyl conjugates Just as N - a c e t y l a t i o n of primary amines i s known to be a metabolic pathway, there are examples ( a l b e i t few) of N-formyl metabolites. In the metabolism of v i l o x a z i n e (27_) (38), 2-aminoanthraquinone (28J (39) 2-aminonaphthalene (29) (40), and c a f f e i n e (41), the N-formyl group i s a conjugate supplied by the organism. Sante et al (42) have postulated a tr a n s f o r m y l a t i o n mechanism i n v o l v i n g the enzyme kynurenine formidase to account f o r these metabolites. - 13 -i i ) Formyl metabolites of t e r t i a r y a l i p h a t i c amines There i s one example of a N-formyl major metabolite a r i s i n g from the t e r t i a r y dimethyl amine, aminopyrine (30) (43) ( f i g u r e 6 ) . By analogy, a formamide metabolite of methadone (31) or r e c i p a v r i n (15) can be proposed. The pathway i s unusual i n l i g h t of the f a c t that carbinolamines of aminopyrine are thought to decompose spontaneously (44) (as should those of methadone and r e c i p a v r i n ) . In f a c t , aminopyrine has long been used as a s u b s t r a t e to measure N-demethylase a c t i v i t y i n microsomal preparations by q u a n t i f y i n g the amount of formaldehyde produced versus time (45, 46). The authors demonstrated that (1 3CH3)2-N-1abel1ed aminopyrine retained the 1 3C l a b e l on the formyl carbon a f t e r metabolism by r a t s . The appropriate M+l ions in the mass spectrum and a formyl resonance at 161.3 ppm in the * 3C NMR of a u r i n e e x t r a c t were present. Pathway B ( f i g u r e 6) i s supported by SIM d e t e c t i o n of the formamide a r i s i n g i n v i t r o from the d e s a l k y l compound (32) (47). In vi v o metabolism of the didesmethylaminopyrine (33) f a i l e d to produce any formamide; t h i s was f u r t h e r evidence against a formyl conjugative pathway. A f r e e r a d i c a l mechanism may be r e s p o n s i b l e , as proposed f o r the synthesis of formamides from t e r t i a r y amines (48), and the cumene hydroperoxide mediated N - d e a l k y l a t i o n of aminopyrine (49). Proof that the 31 15 - 14 -CH3 Figure 6. P o s s i b l e metabolic pathways to a formyl o x i d a t i o n product of aminopyrine f u r t h e r o x i d a t i o n of a carbinolamine i s enzymatic i s only a v a i l a b l e f o r those carbinolamines which form lactams. Schwartz and K o l i s (50) have shown that the microsomal o x i d a t i o n of 2-hydroxymedazepam (34) to diazepam (35) 34 35 - 15 -involves a NAD+ dependent dehydrogenase, and r e c e n t l y , Brandange and Lindblom (51) have suggested that an aldehyde oxidase (EC 1.2.3.1) was res p o n s i b l e f o r the o x i d a t i o n of n i c o t i n e to c o t i n i n e . They claim that an iminium ion i s a more l i k e l y substrate f o r the enzyme than the carbinolamine. On the basis of the observations of Nod a et al and i n l i g h t of thermal i s o m e r i z a t i o n s of N-oxidized compounds observed by Emmons (21). The synthesis of the methadone and r e c i p a v r i n formamides was necessitated to compare to the unknown metabolite. i i i ) P o t e n t i a l s i g n i f i c a n c e of formamide metabolites Gescher et al (26) have r e c e n t l y discovered that carbinolamines of intermediate s t a b i l i t y , such as hydroxymethyl pentamethylmel amine are p o s i t i v e i n the a n a l y s i s of formaldehyde by the Nash method ( r o u t i n e l y used to measure metabolic N-demethyl a t i o n (52). He i n f e r s that s t a b l e c a r b i n o l amines may have been overlooked in the past and s t a t e s : "Oxidative metabolism of the N-methyl moiety i n xenobiotic molecules i s more complex than simply a pathway leading to the desmethyl compound. Depending on as yet unknown f a c t o r s associated with the s t r u c t u r e of the molecule, N-hydroxymethyl groups can be generated with widely d i f f e r e n t s t a b i l i t i e s . " ( 2 6 ) He postulates that carbinolamines of intermediate s t a b i l i t y may l i b e r a t e formaldehyde e x t r a h e p a t i c a l l y , or react with t i s s u e nucleophiles such as c y s t e i n e or h i s t i d i n e . The carbinolamine, hydroxymethyl penta-methylmel amine i s a c y t o t o x i c major metabolite of hexamethylmel amine and i s a c t i v e as an antitumor agent (26). - 16 -6. Metabolic N-Oxidation A. Substrates and mechanisms B i o l o g i c a l o x i d a t i o n of nitrogen in a l i p h a t i c amines has been an a c t i v e area of research f o r the past 13 years (17, 18). The metabolic products are determined by the s u b s t i t u t i o n on n i t r o g e n , (primary, secondary or t e r t i a r y ) , and by the a v a i l a b i l i t y of a-hydrogen atoms. The products formed by r e p r e s e n t a t i v e amphetamines, which have a v a i l a b l e a-hydrogen atoms are o u t l i n e d in Figure 7. While t h i s pathway i s well known in v i t r o , N-oxidation products of secondary or primary a l i p h a t i c amines are r a r e l y c o n c l u s i v e l y demonstrated i n v i v o , p o s s i b l y because of f a c i l e reduction to the precursor amines or by i n s t a b i l i t y to GC a n a l y s i s . The mechanism of N-oxidation of secondary a l i p h a t i c amines proposed by Beckett (53) i n v o l v e s intermediate N-hydroperoxides which decompose to imines, or n i t r o n e s , or a reduced f l avoprotein:C>2 complex which loses water to form a secondary hydroxylamine. B. Metabolic Fate and Detection of N-Oxidized Metabolites i ) Source of Methylene Nitrones Besides a r i s i n g from unstable N-hydroperoxides, nitrones may also form by condensation of a primary hydroxylamine with formaldehyde, or by o x i d a t i o n of a secondary hydroxyl amine i n a l k a l i as occurs during the e x t r a c t i o n of N-hydroxy amphetamine metabolites (54). Condensation with formaldehyde i s a fundamental re a c t i o n i n endogenous metabolism (55) and i n drug metabolism (56). Coutts has presented evidence that the methylene n i t r o n e rather than the isomeric methanimine N-oxide i s the i n v i t r o m e t a b o l i t e of methamphetamine d e s p i t e s i m i l a r i t i e s in t h e i r a n a l y s i s by GCMS - 17 -0 " Figure 7. Metabolic N-oxidation products of t e r t i a r y , secondary and primary amphetamines. - 18 -(57). Non enzymatic o x i d a t i o n and h y d r o l y s i s of secondary hydroxylamines has been implicated i n the r e l a t i v e l y r a p i d N - d e a l k y l a t i o n of secondary amines, p a r t i c u l a r l y i n l i g h t of the f a c i l e demethylation of secondary hydroxylamines (58), but c o n c l u s i v e evidence that a l i p h a t i c n i t r o n e s are formed enzymatical l y rather than chemically i s l a c k i n g . The nature of the glucuronide precursor l i m i t s the source of the methylene n i t r o n e to a 2° hydroxyl amine. There are several examples of t e r t i a r y amines such as cyproheptadine which when glucuronidated, e x i s t as quaternary amine i n t e r n a l s a l t s (36) (59, 60), and i t i s known that glucuronides are formed at e l e c t r o n dense c e n t r e s , but there are no known examples of glucuronide conjugates of nitrones with the s t r u c t u r e (37). 36 37 i i ) P o t e n t i a l metabolic sources of methylene o x a z i r i d i n e s There are no references to o x a z i r i d i n e drug metabolites i n the l i t e r a t u r e . This might be due to the unstable nature of the o x a z i r i d i n e r i n g , which decomposes under mild c o n d i t i o n s . Condensation of a primary amine with formaldehyde, followed by pe r o x i d a t i o n (as i n s y n t h e t i c procedures) or the p e r o x i d a t i o n of iminium d e a l k y l a t i o n intermediates could r e s u l t i n the required s t r u c t u r e . The s t r u c t u r e s of the glucuronides - 19 -precursor i s l i m i t e d to a N-glucuronidated imine which are only known to occur i n heterocycles l i k e s u l f i s o x a z o l e . By analogy, i f an imino glucuronide of methadone were present, h y d r o l y s i s with g-glucuronidase would probably r e s u l t in c y c l i z a t i o n to EDDP or i t s unsaturated analogues (see sy n t h e t i c s e c t i o n ) . 7. T h e r m o l a b i l i t y and i n t e r c o n v e r s i o n of p o t e n t i a l metabolites during GCMS  a n a l y s i s Studies by Emmons (21) have shown that o x a z i r i d i n e s thermally isomerize to both nitrones and amides, depending on the p y r o l y t i c c o n d i t i o n s , according to the pathway (61): 0 " N i t r o n e s , known to produce the higher energy o x a z i r i d i n e s by p h o t o l y s i s , are s u s c e p t i b l e to a v a r i e t y of thermal and chemical rearrangements. These include amide formation (under the i n f l u e n c e of a l k a l i or a c y l a t i n g agents), and base catalyzed double bond m i g r a t i o n ; - 20 -as well as thermal oxime 0-ether formation (62): or a l t e r n a t i v e l y , desoxygenation (61): - 21 -L a s t l y , even amides are known to a u t o x i d i z e thermally i n t o N-formylamides and N-acylamides by bond s c i s s i o n (63): 0 0 There are several p o s s i b l e s t r u c t u r e s a v a i l a b l e f o r the metabolite which could a r i s e both by chemical changes during workup and by thermal changes i n the GC. Therefore synthesis was centred on a v a r i e t y of p o t e n t i a l metabolites and t h e i r most probable glucuronide precursors. 8. Synthesis of N-Oxidized and r e l a t e d drug metabolites The s y n t h e t i c methods used i n t h i s t h e s i s p a r a l l e l those developed f o r N-oxidized amphetamine metabolites by Morgan and Beckett (64), Beckett et al (65) and Coutts et al (57). Carbon-13 and *H NMR i n v e s t i g a t i o n s of the n i t r o n e and o x a z i r i d i n e s synthesized i n t h i s study were c a r r i e d out as an aid to s t r u c t u r e e l u c i d a t i o n , because no examples of * 3C NMR spectra of methylene o x a z i r i d i n e s c o n t a i n i n g a-protons e x i s t i n the l i t e r a t u r e , and as a p o t e n t i a l means of i d e n t i f y i n g the metabolites of ( 1 3CH3)2-N-l a b e l l e d r e c i p a v r i n by 1 3 C NMR. - 22 -I I . EXPERIMENTAL A. REAGENTS AND MATERIALS Chemicals were reagent grade and purchased from the f o l l o w i n g sources: 1. A l d r i c h Chemical Co. (Milwaukee, Wisconsin) A l l y ! c h l o r i d e , Aluminum c h l o r i d e (anhydrous), Benzal acetone, Calcium c h l o r i d e (anhydrous), Deuterochloroform (gold l a b e l ) , Diethylene g l y c o l , D i p h e n y l a c e t o n i t r i l e , Ethylbromide, Lithium aluminum hydride, Magnesium t u r n i n g s , N-methylhydroxylamine hydrochloride, Sodium Cyanoborohydride, Sodium hydride (50% o i l d i s p e r s i o n ) . 2. A l l i e d Chemical, (New York, New York) Sodium acetate, Ferrous s u l f a t e 3. American S c i e n t i f i c and Chemical Co. ( S e a t t l e , Washington) Formaldehyde (38% aqueous), Hydrochloric a c i d , Potassium hydroxide, Sodium hydroxide, S u l f u r i c a c i d . 4. Applied Science Laboratories (State C o l l e g e , Pennsylvania) De x s i l 300 5. J.T. Baker L t d . ( P h i l i p s b u r g , New Jersey) S i l i c a Gel f o r Flash Chromatography, S i l i c a Gel precoated p l a t e s , Sodium metal. - 23 -6. B r i t i s h Drug Houses (Poole, U.K.) A c e t o n i t r i l e , Acetone, Benzene, Calcium carbonate, 2,4-dinitrophenylhydrazine, Ethyl formate, Ethyl phthalate, Hydroxylamine hy d r o c h l o r i d e , L i g r o i n e , Magnesium s u l f a t e , Petroleum ether (30°-60°), Potassium c h l o r i d e , Propylene g l y c o l , P y r i d i n e , Sodium c h l o r i d e , Toluene. 7. Brinkman Instruments (Toronto, Ontario) Dragendorf's Reagent. 8. Caledon Laboratories (Georgetown, Ontario) Chloroform ( d i s t i l l e d in glass grade), Ether, Ethyl acetate ( d i s t i l l e d i n g l a s s ) , Methanol ( d i s t i l l e d i n glass grade), Methanol (HPLC grade), Water (HPLC grade). 9. Eastman Kodak Co. (Rochester, New York) Methyl amine 40% Aqueous 10. F i s h e r Chemical, ( F a i r l a w n , New Jersey) Benzophenone, Chromium t r i o x i d e , Magnesium c h l o r i d e , Sodium bicarbonate. 11. Linde Co. (Union Carbide, Vancouver, B.C.) Molecular s i e v e s , Nitrogen gas. 12. M a l l i n c k r o d t (St. L o u i s , M i s s o u r i ) Sodium b i s u l f a t e , Sodium s u l f a t e , Xylene, 60% P e r c h l o r i c A c i d . - 24 -13. Matheson Limited (Edmonton, A l b e r t a ) Gaseous hyd r o c h l o r i c A c i d . 14. Matheson, Coleman and B e l l (Norward, Ohio) Dimethyl s u l f o x i d e , Disodium hydrogen phosphate, Methyl a c r y l a t e , Potassium carbonate (anhydrous), Propylene oxide, Sodium dihydrogen phosphate. 15. Merck Co. (Rahway, New Jersey) Yellow mercuric oxide 16. Merck Sharp and Dohme (Isotopes) (Montreal, Quebec) Deuterium oxide. 17. P i e r c e Chemical Co. (Rockford, I l l i n o i s ) BSTFA, TMAH, MOX. 18. Sigma Chemical Co. ( S t . Louis M i s s o u r i ) Glucose-6-phosphate, Glucose-6-phosphate dehydrogenase, Glucurase, Glusulase, NADP. 19. Stanchem L t d . (Winnipeg, Manitoba) 95% Ethanol. 20. Supelco L t d . (Bel 1 anfonte, Pennsylvania) DMCS 3% 0V-17 on GCQ - 25 -21. Synthesized in our laboratory (Abbott et a l . ( 6 6 ) ) 2,2-Diphenyl-4-dimethylami no v a l e r o n i t r i 1 e E thylidene dimethyl diphenyl p y r o l l i d i n e p e r c h l o r a t e . Ethyl methyl diphenyl p y r o l l i d i n e . Methadone hydrochloride. Methadone-^Hio h y d r o c h l o r i d e . 22. Terochem, (Edmonton, A l b e r t a ) 85% M-chloroperbenzoic a c i d . 23. Ventron (Beverly, Massachusetts) Sodiim borohydride. B. INSTRUMENTAL 1. Nuclear Magnetic Resonance Spectra Routine proton NMR spectra were recorded on a Bruker WP-80 or a Varian XL-100 spectrometer. Decoupling and high r e s o l u t i o n experiments were performed on a Bruker WP-400 spectrometer. 1 3C NMR spectra were recorded on a CFT-20 and Bruker WP-400 spectrometers. A l l high r e s o l u t i o n SFORD experiments were performed on the 400 MHz instrument. Spectra were recorded in CDCI3 w i t h TMS as an i n t e r n a l standard. A l l spectra are included in the Appendix. A l l NMR spectra (save one) were recorded at the NMR f a c i l i t y i n the Department of Chemistry, U.B.C. The m i c r o - c e l l 400 MHz spectrum of eluted GC peaks was recorded at the NMR f a c i l i t y at Simon Fraser U n i v e r s i t y . 2. I n f r a Red Spectra were obtained with sodium c h l o r i d e disks e i t h e r as l i q u i d f i l m s , nujol m u l l s , or i n 0.1 mm path length s o l u t i o n c e l l s using - 26 -CHC13 as a s o l v e n t , on an Unicam SP-1000 spectrometer. A l l spectra are included i n the Appendix. 3. Gas Chromatography Mass Spectrometry GCMS a n a l y s i s was performed on a Hewlett Packard 5700A gas chromatograph i n t e r f a c e d to a Varian Mat - I l l Mass Spectrometer v i a a v a r i a b l e s l i t separator. E l e c t r o n impact spectra were recorded at 80eV, ion source pressure 8.0 x 10"^ t o r r , emission current 300 uA. D i r e c t probe experiments were performed on the Mat - I l l . Computerized background s u b t r a c t i o n s were made to p l o t mass s p e c t r a . The scan range was 4 to 500 with one scan every f i v e seconds. TIC p l o t s were based on m/z 50 to 500. Mass chromatograms were p l o t t e d i n scan mode. The data was processed by an on l i n e Varian 620 L computer system. Chemical i o n i z a t i o n , c a p i l l a r y GCMS was performed on a Hewlett Packard 5987A instrument using methane as reagent gas. Gas Chromatographic Conditions (a) 3% 0V-17 on 80/100 mesh Chromosorb W-HP 200°-280° at 8°/minute, Helium flow 20 ml/min. Column length 2 m, column i n t e r n a l diameter 2 mm. (b) 3% 0V-17 on 100/120 mesh Gas Chrom Q 150-280° at 4°/minute, helium flow 20 ml/minute. Column le n g t h 2 m, column i n t e r n a l diameter 2 mm. (c) 3% 0V-17 on 100/120 mesh Gas Chrom Q. 200-280° at 4°/minute, helium flow 20 ml/minute. Column length 2 m, column i n t e r n a l diameter 2 mm. - 27 -(d) 3% Dexil 300 on Chromosorb W-AW. 200-280° at 8°/minute. Helium flow 20 ml/minute. Column length 2 m, column i n t e r n a l diameter 2 mm. (e) 2.3% Dexil 300 on 80/100 Gas Chrom Q. 200-280° at 8°/minute. Helium flow 20 ml/minute. Column length 2 m, column i n t e r n a l diameter 2 mm. ( f ) C a p i l l a r y Column: Cross l i n k e d Dimethyl S i l i c o n e . (Hewlett Packard part number 19091-60312) Length 12.5 metres. Internal diameter 0.2 mm. Column Temperature: 50-200° at 30°/minute. Then ( i ) 10 or ( i i ) 5°/minute to 280° (g) 3% 0V-17 on 100/120 mesh Gas Chrom Q. 150°-280° at 2° per minute. Helium flow 20 mL/minute. Column length 2 m, diameter 2 mm. In a l l of the above, the GC i n l e t l i n e , i n j e c t i o n port and separator temperature were 250°. I n j e c t i o n s were i n a s p l i t l e s s mode. 4. L i q u i d Chromatography Mass Spectrometry L i q u i d Chromatography was performed on a Hewlett Packard 1082B L i q u i d Chromatograph e i t h e r with UV d e t e c t i o n at 254 nm or i n t e r f a c e d to a Hewlett Packard 5987A Mass Spectrometer. A Hewlett Packard RP.8, 10 cm le n g t h , 4 mm diameter reversed phase column (Cat.A79918B) equipped with a C^g guard column was run at a flow rat e of 1 mL/minute and column pressure 7 bar. Using a mixture of 2 solvents (a) 50% water 50% methanol and (b) 100% methanol, the f o l l o w i n g solvent program gave s a t i s f a c t o r y r e s o l u t i o n of n i t r o n e s , o x a z i r i d i n e s and formamides. - 28 -Time (minutes) % B %A 0.0 0.0 100 30.0 0.0. 100 35.0 25.0 75 50.0 25.0 75 51.0 100.0 0 60.0 stop 5. U l t r a v i o l e t Spectrometry UV spe c t r a were recorded in methanol on a Beckman Model 24 U V - v i s i b l e spectrometer i n 1 cm path length c e l l s . 6(a) M e l t i n g Points M e l t i n g points were determined on a Thomas-Hoover C a p i l l a r y melting point apparatus and are uncorrected. 6(b) Elemental Analyses Elemental analyses were performed by the Canadian M i c r o - A n a l y t i c a l S e r v i c e L t d . 5704 U n i v e r s i t y Blvd. Vancouver, B.C. V6T 1K6. 7. Metabolism Experiments (a) C e n t r i f u g a t i o n An I n t e r n a t i o n a l Equipment Co. Model B-20 c e n t r i f u g e and a Beckman L5-50 u l t r a c e n t r i f u g e were employed i n the microsomal s t u d i e s . (b) Tissue Homogenization This was performed on a F i s h e r Dynamix t i s s u e homogenizer. (c) L y o p h i l i z a t i o n A V i r T i s L y o p h i l i z e r (Gardiner, N.J.) was used. - 29 -(C) IN VIVO METABOLISM 1. BILE DUCT CANNULATION Sprague-Dawley r a t s of e i t h e r sex weighing 200-250 grams were anaesthetized with ether during surgery. A m i d l i n e abdominal i n c i s i o n was made and the common b i l e duct was i s o l a t e d and cannulated w i t h poiyethyl ene-10 tub i n g . The abdomen was closed with i n t e r r u p t e d sutures and the r a t was placed in a r e s t r a i n t cage. A f t e r recovery from anaesthesia, each r a t was administered subcutaneously one of the f o l l o w i n g drugs i n 0.1 ml of v e h i c l e . DRUG DOSAGE VEHICLE Methadone HC1 20 mg/kg s t e r i l e water Methadone ^H10.HC1 20 mg/kg s t e r i l e water R e c i p a v r i n ( f r e e base) 20 mg/kg propylene g l y c o l N o r r e c i p a v r i n HC1 20 mg/kg propylene g l y c o l Methadone analogue HC1 20 mg/kg water B i l e was c o l l e c t e d f o r 18 hours, w i t h a second dose of drug administered a f t e r 12 hours. 2. EXTRACTION OF CONJUGATED AND NONCONJUGATED METABOLITES FROM BILE Following the method of Kang (1) w i t h m o d i f i c a t i o n s , each b i l e sample (10-15 ml) was d i l u t e d w i t h an equal volume of water, and adjusted to pH 9 with 0.01 M sodium hydroxide. Non conjugated metabolites were extracted by g e n t l e mixing with t h r e e , f i f t y mL a l i q u o t s of chloroform. The organic phase was d r i e d over potassium carbonate and evaporated. The residue was d i s s o l v e d i n 20-50 uL of methanol and 5 yL analyzed by gas chromatography mass spectrometry. - 30 -The aqueous phase, containing the conjugated metabolites was l y o p h i l i z e d , the pH adjusted to 5 with d i l u t e a c e t i c acid and the residue taken up in 0.1 M sodium acetate buf f e r (pH 5). F i v e thousand u n i t s of g-glucuronidase (as Glusulase or Glucurase) was added to the buffered s o l u t i o n . A f t e r 12 hours incubation at 37°C, the s o l u t i o n was adjusted to pH 8-9 w i t h 0.01 M sodium hydroxide, then extracted gently with f o u r , f i f t y ml a l i q u o t s of chloroform, d r i e d over potassium carbonate, f l a s h evaporated, and the residue r e d i s s o l v e d i n 20 uL of methanol. Five to ten uL were i n j e c t e d i n t o the GCMS. (D) IN VITRO METABOLISM A f t e r Beckett (67), Gorrod (68), and Coutts (69). 1. PREPARATION OF MICROSOMES Sprague-Dawley rats of e i t h e r sex, weighing 200-250 grams, were stunned and then k i l l e d by d e c a p i t a t i o n . The peritoneum was opened and the l i v e r perfused with cold 1.15% potassium c h l o r i d e v i a the portal v e i n . The l i v e r was removed and placed i n cold 1.15% KC1. Five g p o r t i o n s , i n c l u d i n g the main lobe were homogenized i n 20 mL of cold 1.15% KC1, then centrifuged at 4°C f o r ten minutes at 10,000 r.p.m. Surface l i p i d was removed with a cotton swab and 8 ml of supernatant (equal to 2 g l i v e r ) was pipetted i n t o an u l t r a c e n t r i f u g e tube, and spun at 100,000 times g r a v i t y f o r 65 minutes at 4°C. The tubes were placed on i c e , the supernatant decanted, and the remaining microsomal p e l l e t s washed with 3 f i v e mL a l i q u o t s of pH 7.2, 0.1 M phosphate b u f f e r . The p e l l e t s were resuspended i n 4 mL of phosphate b u f f e r . - 31 -2. METABOLIC PROCEDURE A f t e r ten minutes e q u i l i b r a t i o n at 37°C, 4 mL c o f a c t o r s o l u t i o n and 40 ^mol of drug were added to the microsomal suspension and vortexed b r i e f l y . A f t e r incubation with gentle a g i t a t i o n f o r 2 hours at 37°, the tubes were placed on i c e and adjusted to pH 8 w i t h 0.1 M NaOH. Each tube was extracted gently with three, f i f t y mL a l i q u o t s of chloroform. The combined extracts were d r i e d over K2CO3 evaporated, then r e c o n s t i t u t e d i n 20 uL of methanol and 5 ul i n j e c t e d i n t o the GCMS. Cofactor S o l u t i o n MgCl 2 8.34 mM 4 mL NADP 1.32 mM 30.6 mg GIucose-6-phosphate 13.32 mM 112.8 mg GIucose-6-phosphate Dehydrogenase 200 u n i t s 800 uL T r i s b u f f e r pH 7.5 1.29 mM 11.2 mL E. GENERAL 1. FLASH CHROMATOGRAPHY ( A f t e r S t i l l et al (70)) A s i x inch bed of s i l i c a gel ( f l a s h chromatography grade) was packed over a 0.5 cm bed of s i l i c a sand. The column was topped with an ad d i t i o n a l 0.5 cm of s i l i c a sand. Eluent, proven by TLC to have p o l a r i t y s u f f i c i e n t to move the desired component to an Rf of 0.3 to 0.4, was forced through the column with nitrogen gas u n t i l no more a i r was evident i n the column. The sample was placed on column i n 0.5 mL of eluent and 5 mL f r a c t i o n s c o l l e c t e d . Nitrones and formamides were eluted with ethyl acetate i n 10 mL f r a c t i o n s . - 32 -F. SYNTHESIS OF POTENTIAL N-OXIDIZED METABOLITES OF METHADONE 1. SYNTHESIS OF 2,2-DIPHENYLPENT-4-ENE NITRILE (ALLYL DIPHENYL  ACETONITRILE) (38) Following a modified method of Wilson (71), diphenyl a c e t o n i t r i l e (21.6 g, 0.11 mol) was treated with a suspension of 5.25 g (0.11 mol) of sodium hydride ( p r e v i o u s l y washed f r e e of o i l with dry benzene) i n 90 mL of dry benzene c o n t a i n i n g 2 drops of d i m e t h y l s u l f o x i d e . A l l y ! c h l o r i d e (8.42 g, 0.11 mol) i n 10 mL dry benzene was added dropwise. The s o l u t i o n was refluxed f o r 2 hours and l e f t s t i r r i n g overnight protected from moisture with a CaCl2 guard tube. The s o l u t i o n was f i l t e r e d throught s i n t e r e d g l a s s , and washed twice w i t h 10 mL 1 N HC1, once with 20 mL NaHC03 and twice with 10 mL H2O, then dried over CaCl2 and f l a s h evaporated. The product (C17H15N, MW 233.32) d i s t i l l e d as a c l e a r l i q u i d at 170°/1.0 mm ( l i t e r a t u r e bp 162/1.5 mm) (71). Mass Spectrum: m/z 233 (M + 12%), 192(100), 165(68). Reaction of the product with ethylmagnesium bromide afforded 4,4-diphenyl-hepten-5-one (3_9) (GCMS evidence). Mass Spectrum: m/z 129(100), 91(85), 207(40) 57(10), 264(3). 2. SYNTHESIS OF 3.3-DIPHENYL-5-METHYL TETRAHYDR0-2-FURAN0NEIMINE (40) (CAS 17279-17-3) Following the method of Easton et al (72) but s u b s t i t u t i n g sodium hydride f o r sodium, amide. Diphenyl a c e t o n i t r i l e (1.0 g, 4.7 x I O - 3 mol) in dry benzene was added slowly to a s t i r r e d s o l u t i o n of 0.3 g (6.3 x 1 0 - 3 mol) of sodium hydride (washed f r e e of 50% o i l d i s p e r s i o n with benzene) i n 20 mL dry benzene which contained a drop of dimethyl s u l f o x i d e . A f t e r warming the s o l u t i o n g e n t l y , 300 mg (5.2 x 10~ 3 mol) of propylene - 33 -oxide i n 5 mL dry benzene was added to the green s o l u t i o n . A f t e r a gentle r e f l u x overnight, followed by suction f i l t r a t i o n through s i n t e r e d g l a s s , the s o l u t i o n was poured slowly i n t o 100 mL c o l d H2O. The benzene was separated, d r i e d over MgSO/j, and evaporated, y i e l d i n g 0.7 g (54%) of a t h i c k l i q u i d , d i s t i l l i n g at 180°/0.5 mm. The product s o l i d i f i e d on t r i t u r a t i o n with petroleum ether. A n a l y s i s For: C 1 7 H 1 8 NO, (Molecular Weight 251.33). Mass Spectrum: m/z 251 (M + 30%), 129(100), 91(85), 207(30), 210(30), 165(20). j_R ( m u l l ) : v m a x 3400 cm"1 (Sym. N-H s t r ) ; 1670s, 1590(m, N-H Bend), 1360(m) 910(m) 685(s). The hydrochloride s a l t was p r e c i p i t a t e d from dry benzene with gaseous HC1. M e l t i n g Point 222°-decomposes ( l i t e r a t u r e 220-222° (72)). 3. SYNTHESIS OF 2,2-DIPHENYL-4-VALER0LACT0NE (41_) The lactone was synthesized by a modified method of Attenburrow et al (73), with sodium hydride s u b s t i t u t e d f o r sodamide. D i p h e n y l a c e t o n i t r i l e (75 g, 0.4 mol) was added to a s t i r r e d suspension of 18.66 g (0.4 mol) sodium hydride (washed f r e e of o i l with dry benzene) i n dry benzene c o n t a i n i n g 5 drops of DMSO. The s o l u t i o n was warmed gently on a steam bath u n t i l green. Then 23.2 g (0.4 mol) propylene oxide was added dropwise, and the s o l u t i o n was r e f l u x e d overnight, protected with a CaClg guard tube. A f t e r c o o l i n g and f i l t r a t i o n through s i n t e r e d g l a s s , the s o l u t i o n was poured slowly i n t o 100 mL of i c e cold 2N HC1. The l a y e r s were separated and the organic phase extracted with two f u r t h e r 25 mL a l i q u o t s of 2N HC1. The imine was hydrolyzed by r e f l u x i n g the HC1 s o l u t i o n f o r 2 hours on a steam bath. The cooled s o l u t i o n was extracted with ether, washed with water, d r i e d over CaS0 4, f i l t e r e d , and evaporated. Following - 34 -r e c r y s t a l l i z a t i o n from benzene and l i g r o i n e , 77.8 g (79%) of white c r y s t a l s m e l t i n g at 111°C ( l i t e r a t u r e 115-116°(73)) were recovered. A n a l y s i s For: C i 7 H 1 6 0 2 , (Molecular Weight 252.315) Mass Spectrum: m/z 252 (M + 8%), 115(100), 208(90), 193(78), 130(75). NMR (100 MHz): 6 ppm, 1.48 doublet (3H, -CH 3); 4.5 m u l t i p l e t (IH, CH); 2.45-2.75, dd(lH, CH 2); 2.95-3.2, dd(lH, CH 2). JR ( m u l l ) : vmax, 1745 cm" 1, ( s , C=0 s t r ) , 1480(m), 1440(s), 1370(m), 1330(m), 1170 ( s , C-0 s t r ) , 960(m), 690(s). 4. SYNTHESIS OF 1,l-DIPHENYL-2-BUTAN0NE (42) (CAS 6336-52-3) Diphenyl a c e t o n i t r i l e (10 g, 5.2 x 1 0 - 2 mol) d i s s o l v e d i n 30 mL dry toluene and added to a Grignard reagent of 1.9 g (8 x 10"2 mol) Mg tur n i n g s and 8.7 g (8 x I O - 2 mol) ethyl bromide in dry ether. Ether was d i s t i l l e d o f f u n t i l the f l a s k temperature was 96°C. A f t e r r e f l u x i n g overnight, protected with a C a C l 2 guard tube, the f l a s k was cooled and the r e a c t i o n mixture decomposed by the slow a d d i t i o n of 200 mL 2N HC1, followed by 2 hours s t i r r i n g on a steam bath. The toluene l a y e r was removed, d r i e d over C a C l 2 , evaporated and the residue d i s t i l l e d . The ketone d i s t i l l e d at 152-155° (0.7 mm), y i e l d i n g 7.23 g (61.8%) of a c l e a r 1 i q u i d . A n a l y s i s For: C^H^O, (Molecular Weight 224.305). Mass Spectrum: m/z 224 (M + 2%), 167(100), 165(23), 57(22). 5. SYNTHESIS OF 4,4-DIPHENYL-2,5 HEPTANE DIOL (43) Following the method of Wilson (71), 50 g (0.2 mol) 2,2-diphenyl-4-v a l e r o l a c t o n e i n 25 mL toluene was added dropwise to a f r e s h l y prepared Grignard reagent c o n t a i n i n g 7.23 g (0.3 mol) of magnesium turnings and - 35 -32.7 g (0.3 mol) ethyl bromide i n dry ether. The s o l u t i o n was l e f t s t i r r i n g o vernight. Water was added dropwise with c o o l i n g . The s o l u t i o n was a c i d i f i e d w i t h 2N HC1 and extracted w i t h three f i f t y mL a l i q u o t s of benzene. The organic phase was d r i e d over CaCl2 and evaporated. T r i t u r a t i o n of the residue w i t h pet. ether, and r e c r y s t a l l i z a t i o n from benzene/1 i g r o i n e (60-80°) gave 36.6 g (65%) of white c r y s t a l s melting at 121° ( l i t e r a t u r e M.P. 124-125° (71)). A n a l y s i s For: C19H24O2, (Molecular Weight 284 .4) . Mass Spectrum: m/z 264(M + -20, 5%), 208(100), 193(82), 103(80), 130(55), 115(50), 91(42), 181(40), 45(30). NMR (400 MHz): 6ppm, 0.82 m (IH, CH 3-CHaH b); 0.97 t(2H, CH3-CH 2); 1.12 d(3H, CH3-CH); 1.67 m (IH, CH 3 C H ^ ) ; 2.27 dd(lH, CH3-CHaHb-CH); 2.44 dd(lH, C^-CH^a-CH) ; 3.72 m (IH, CH0H-CH3) ; 4.38 dd(lH, CH0H-CH2); 7.2 (10H, Arom). IR_ ( m u l l ) : vmax 3150 cm" 1, (broad, s, OH S t r ) , 1580(m) 1440(s) 1360(s) 1330(m, 0-H Bend), 1160(s) ( s , C-0 S t r ) , 750 ( s , Ar) , 700 ( s , A r ) . A petroleum ether wash of the crude product afforded 10 g (17.8%) of 3 , 3-diphenyl - 5-methyl - 2-ethylidene tetrahydrofuran (44) (CAS 17494-37-0) as a pal e y e l 1 ow o i l . A n a l y s i s For: C19H20O, (Molecular Weight 264.37). Mass Spectrum: m/z 264 (M+ 100%), 91(45), 179(40), 105(32), 115(25), 131(22), 43(22), 207(20), 222(14), 249(12). NMR (100 MHz): 6ppm, 1.38 d(3H, CH3~CH=); 1.68 d(3H, CH3-CH); 2.6 dd(2H, CH 2); 3.92 q (1H ,=CH-CH3); 4.12 m(lH, CH 3CH-CH 2); 7.3 (10H, Aromatic). 2 ! ( f i l m ) : vmax 1680 cm" 1, ( s , C=C S t r , v i n y l e t h e r ) , 1590(m) , 1490(s), 1440(s), 1360(m), 1290(m) , 1200-1000 ( s , C-0 S t r ) , 740 ( s , Ar) , 700 ( s , A r ) . - 36 -6. SYNTHESIS OF 4,4-DIPHENYL-2,5 HEPTANEDIONE (45) Jones reagent was prepared by d i s s o l v i n g 500 mg of CYO3 i n a s o l u t i o n o f 0.46 mL H2SO4 and 0.8 mL water then d i l u t i n g to 2 mL with water. The Jones reagent was added dropwise to 150 mg o f 4,4-diphenyl-2,5-heptanediol i n acetone, on an ice bath. The a d d i t i o n was discontinued when the red brown c o l o r p e r s i s t e d f o r 15 minutes. The s o l u t i o n was d i l u t e d with water and isopropanol and extracted with ether. The ether phase was washed with d i l u t e NaHC03 s o l u t i o n , then with water, d r i e d over K2CO3 and the solvent evaporated. The yel l o w o i l c r y s t a l i z e d upon t r i t u r a t i o n with petroleum ether (30-60°). F r a c t i o n a l c r y s t a l i z a t i o n from ether/petroleum ether (30°-60°) affo r d e d 5% diketone (tedious procedure), with the major product being 2,2-diphenyl-4-valerolactone (41). A n a l y s i s For: Cigr^oPz' (Molecular Weight 280.37) Elemental A n a l y s i s : C a l c u l a t e d C, 81.397%; H, 7.19%; 0, 11.43% Found C, 81.14%; H, 7.1%; 0, 11.76% Mass Spectrum: m/z 262 (M +-18, 2%), 43(100), 57(15), 223(12), 181 (10), 206(8), 29(10). lH MR (80 MHz): 5 ppm, 0.94 t (3H, CH3-CH2); 2.0 s (3H CH3 -C=0); 2.39 q (2H, CH3-CH2); 3.58 s (2H, CH2-C -Ar2) 7.3 (10H, Aromatic). IR_ (m u l l ) : vmax 1710 cm-1, ( s , C-0 S t r ) , 1490(m) 1460(s, CH3 bend) 1370, 1360 (m s,CH-C(=0 )-CH) 1180 (m, C-C(=0)-C bend) 1120(m) 700(s,Ar). 13CNMR (20 MHZ): & ppm, 8.8 (CH 3-CH 2); 31.24 (-CH2-CH3); 33.04 (CH 3-C(=0)); 52.6 (CAr-CH2-C(=0)); 63.92 (Ar 2C_R 2); CO not observed. - 37 -7. SYNTHESIS "OF 2-(4',4'-DIPHENYLHEPTAN-5'-ONE-2'YL)-OXAZIRIDINE (5) A m o d i f i c a t i o n of Kang's (1) method: To a s o l u t i o n of 100 mg (2.6 x 10~ 4 mol) ethylidene dimethyl diphenyl p y r o l l i d i n e p e r c h l o r a t e in 5 mL of CHC13 at 0°C was added 100 mg (5.2 x IO" 4 mol) of metachloroper-benzoic acid i n 5 mL CHC13. A f t e r s t i r r i n g overnight at 0°C, the s o l u t i o n was f i l t e r e d and washed with two ten mL a l i q u o t s of 1.5 N NaOH, then twice w i t h water, d r i e d over MgS04 and the solvent f l a s h evaporated. The residue was f l a s h chromatographed using 75% petroleum ether (30°-60°) 25% ethyl ether as eluent ( a l t e r n a t i v e l y 10% ethyl acetate i n petroleum ether 30°-60°) under standard c o n d i t i o n s . The product o x a z i r i d i n e , as a mixture of diastereomers was eluted in the 15-25 mL f r a c t i o n s . The product was detected on t h i n l a y e r chromatograms as a p a i r of p a r t i a l l y resolved black spots when sprayed with Dragendorf's reagent. Y i e l d a f t e r chromatography, 30 mg (36%). On long standing i n CDCI3 i n the f r e e z e r , l a r g e cubic c r y s t a l s were deposited, melting at 80° and decomposing at 150-160°. The c r y s t a l s were s t a b l e at 0°. An a l y s i s For: C 2 0H 2 3N0 2, (Molecular Weight 309.411). Elemental A n a l y s i s : %C c a l c u l a t e d 77.64; found 77.61 %H c a l c u l a t e d 7.49; found 7.45 %N c a l c u l a t e d 4.52; found 4.52 %0 c a l c u l a t e d 10.34; found 10.42 NMR (400 MHz): 5 ppm (major diastereomer) 0.85, t(3H, CH3-CH2); 0.84 d (3H, CH3-CH); 1.8, m (1H,-CH-CH 3); 2.27, q buried (2H,-CH2-CH3); 2.32, dd ( lH , C H ^ - C H ) ; 2.80, dd(lH CH^Hg); 3.17, d ( lH ; o x a z i r i d i n e r i n g ) ; 3.62, d ( lH , o x a z i r i d i n e r i n g ) ; 7.3 (10H, Aromatic). (100 MHz) 6 ppm (minor diastereomer) 0.56, d(3H, CH3-CH-N), 0.85, t(3H, CH3- CH 2); 1.8, m(lH, CH3-CH-); 2.45, q buried (2H, -CH 2-CH 3); - 38 -2.35, dd(lH,-CHaHb-CH); 3.2 dd ( lH , CH^-CH), 3.42, d ( lH , o x a z i r i d i n e r i n g ) , 3.92, d ( lH , o x a z i r i d i n e r i n g J=10Hz), 7.3, (10H, Aromatic). IR ( f i l m ) : v max 3000 cm" 1, (m), 1710 ( s , C=0 s t r ) , 1600(m-w), 1500(s), 1450(m), 1380 (w-m) o x a z i r i d i n e , 1350 (w-m, -C=0 S t r ) , 1250(m), 1150(w-m, C-C=0-C, S t r . and Bend), l l l O(s-m), 1050(m), 950(w); 770(s, A r ) , 710(s, A r ) . UV (Methanol ) : X max 296 mu (e=502); 265(e=548); 25.45 (e=480, TT-TT * A r ) ; 207.5 (e=21, 168 TT-TT *Ar) 13CNMR: (Broad Band and SF0RD 400 MZ) <5 ppm 9.18, q(CH 3-CH 2); 21.29, q(CH 3-CH); 33.04, t(CH 2-CH 3); 211.26, s(weak C=0); 65.46, s(weak) ( R 2 - C - A r 2 ) ; 42.6, t(CH 2-CH); 64.00, d(CH-CH 2); 72.54, t (CH 2-oxazi r i d i n e ) Mass Spectrum: By GCMS; m/z 309 (M+3%), 72(100), 207(72), 73(60), 208(50), 44(46), 253(42), 129(30), 57(22), 291(8). By D i r e c t I n l e t : (Source Temp. 100°) m/z 56(100), 72(78), 57(60), 207(40), 42(37). 8. SYNTHESIS OF 2-(N-F0RMYL)-4,4-DIPHENYL-5-HEPTAN0NE (31) 2-(4',4'-diphenylhept-5 ,-one-2 ,-yl) o x a z i r i d i n e (100 mg, 3.2 x IO" 4 mol) was added to 10 mL dry oxygen f r e e , m-xylene, and re f l u x e d under nitrogen overnight in the dark. The pale y e l l o w s o l u t i o n was f l a s h evaporated and f l a s h chromatographed. A f t e r a 50 mL prerun o f 50% petroleum ether (30-60°) i n ethyl acetate, the amide was eluted with pure ethyl acetate in the 15-25 mL f r a c t i o n . Twenty mg (20%) of product was obtained. The column was s t r i p p e d w i t h 25 mL methanol to i s o l a t e the isomeric n i t r o n e , but none was detected. Mass Spectrum: By GCMS: I d e n t i c a l to s t a r t i n g m a t e r i a l . - 39 -NMR (400 MHz): Major Component 6 ppm 0.85, t(3H, CH 3-CH 2); 1.11, d(3H, CH3-CH); 2.1-2.3, q buried (2H, CH2-CH3); 2.4-2.5, dd buried (IH, CH aH b-CH); 2.8-2.9, dd( l H , CH^HgCH); 3.25, m(lH, CH 2-CH-CH 3); 5.98, b s ( l H , NH); 7.1-7.4, m(10H, Ar) 7.75, s ( l H , C(=0)H). Minor Component: 6 ppm 0.85, t(3H, CH3-CH2); 1.12, d(3H, CH3-CH); 2.14-2.3, q buried (CH 3-CH2-C(=0)); 2.3-2.4, dd( l H , CH aH b-CH 2-); 2.7-2.8, dd( l H , CH bH a-CAr 2); 2.9-3.0, m(CH 3-CH-CH 2); 5.52, b s ( l H , NH); 7.1-7.4, S(10H, A r ) ; 7.45, d ( l H , C(=0)H). IR ( f i l m ) : v max: 3360 cm" 1, (m, sho u l d e r ) , 3260 (m, broad), 1735(w-m sh o u l d e r ) , 1710(s, C=0 S t r ) , 1670(s), 1535(m), 1495(m), 1445(m), 1380(mw), 1140(mw), HOO(wm-doublet), 1035(2m), 915(w-m broad), 755(m), 730(m), 700(s). G. DIKETONE REACTIONS 1. SYNTHESIS OF 4,4-DIPHENYL-2,5-HEPTANEDI0NE-2-0XIME (46) 4,4-Diphenyl-2,5-heptanedione, 50 mg (1.78 x 10"^ mol) i n 5 mL of methanol was added to a s o l u t i o n of 13 mg (1.9 x 10-4 m o i ) hydroxylamine hydrochloride i n 2 mL H 20, preadjusted to pH 9.5 wi t h 0.1 M NaOH. A f t e r s t i r r i n g at room temperature o v e r n i g h t , the s o l u t i o n was d i l u t e d w i t h water, saturated w i t h NaCl and extracted with ether. The ether l a y e r was separated, d r i e d over C a C l 2 and evaporated to g i v e a ye l l o w o i l which s o l i d i f i e d on t r i t u r a t i o n w i t h petroleum ether (30-60°). R e c r y s t a l 1 i z a t i o n from ether afforded 25 mg (50%) of product. A n a l y s i s For: C i g H 2 i N 0 2 , (Molecular Weight 295). NMR (80 MHz): 6 ppm 0.78, t(3H, CH3-CH 2); 1.8, broad s i n g l e t (3H, CH3-N=0H); 2.1, broad quadruplet (2H, -C=0-CH2-CH3); 3.3, broad s i n g l e t (2H, C(=N0H)CH2), 7.3 (10H, Ar) IR ( f i l m ) : v max 1620 cm" 1, ( s , C=N S t r ) , 1710(C=0 S t r ) , 1440(s), 1370(m), 1320(m), 1180(s), 1130(m). - 40 -Mass Spectrum: m/z 279 (M +-16,4%), 42(100), 91(70), 57(65), 29(40), 206(50), 220(55), 238(25), 246(23), 261(20). 2. SODIUM CYANOBOROHYDRIDE REDUCTION OF 4,4-DIPHENYL-2,5-HEPTANEDI0NE  2-OXIME Attempted synthesis of 4,4-diphenyl-2-(N-hydroxylamino)-5-heptanone (47). The previous product (46) (1.4 x I O - 4 mol) i n 10 mL methanol was adjusted to pH 4-5. NaBH3CN (10 mg, 2.1 x IO" 4 mol) was added with s t i r r i n g . 2N HC1 was added dropwise to maintain pH 4-5. When the pH remained constant, the s o l u t i o n was s t i r r e d f o r one hour, then adjusted to pH 1. Gas ev o l u t i o n was allowed to subside. A f t e r adjusting to pH 8, the s o l u t i o n was saturated with sodium c h l o r i d e , extracted with ether, dri e d over CaCl2» and evaporated, leaving an amber l i q u i d , p o s s i b l y 4,4-diphenyl -2-(N-hydroxylamino)-5-heptanol. A n a l y s i s For: C19H25NO2, (Molecular Weight 299 (expected f o r the heptanol (48)). Mass Spectrum: m/z 279(M +-20,35%), 91(100), 105(50), 115(40), 250(40), 129(30), 193(30), 208(30), 280(10), 281(2 (M-18)). NMR (80MHz) (impure): 6 ppm 0.7, t(3H CH3-CH2); 1.8, broad(2-3H, CH3-CH2-); 1.5, d(3H, CH3-CH); 1.2, s(2H, NH), 2.4 m(-CH2-CH(-N)); 3.0, m(lH-CH 2-CH(NHOH)-CH3); 4.0, t ( l H , -CH(OH)- CH2); 7.1-7.3-(10H Aromatic). 3. REACTION OF DIKETONE WITH N-METHYLHYDROXYLAMINE HYDROCHLORIDE Attempted synthesis of 4,4-diphenyl-2-(N-hydroxy-N-methylamino) -5-heptanone (3_) - 41 -To a s o l u t i o n of 20 mg (5 x 10" 4 mol) N-methylhydroxy!amine hydrochloride i n 1 mL H 20 was added 70 mg (2.5 x 10"^ mol) diketone (45) i n 10 mL methanol. A f t e r adjusting to pH 6 w i t h 5% KOH, NaBH3CN 30 mg (5 x IO" 4 mol) was added, and the pH maintained below 6 by dropwise a d d i t i o n of 5% HC1. When pH was s t a b l e , the s o l u t i o n was a c i d i f i e d to pH 1, with 5N HC1. Gas bubbles were allowed to subside. A f t e r adjusting to pH 8 w i t h 1 N NaOH, the s o l u t i o n was saturated with sodium c h l o r i d e , extracted w i t h ether, and the ether phase drie d over C a C l 2 . A f t e r evaporation, GCMS revealed a mixture of products. Treatment of t h i s mixture with yellow mercuric oxide f a i l e d to produce the methadone n i t r o n e , 1-methyl-(N-methylene)-3,3-diphenyl-4-oxohexanamine-N-oxide (2_) as determined by GCMS. The products were not f u r t h e r c h a r a c t e r i z e d . Mass Spectrum: Major Product: m/z 276(8%), 199(100), 200(20), 183(10), 42(8), 247(5), 262(4), 220(4). H. REACTIONS OF METHADONE OXAZIRIDINE {S) I. REDUCTION WITH LITHIUM ALUMINUM HYDRIDE Methadone o x a z i r i d i n e (10 mg) was s t i r r e d over excess LAH i n dry ether f o r 12 hours. A f t e r f i l t r a t i o n and a water wash, the ether s o l u t i o n was d r i e d over C a C l 2 and evaporated. A n a l y s i s For: DIN0RMETHAD0L (49): C 2 0H 2 7N0. (Molecular Weight 297.44). Mass Spectrum: m/z 222(2%) 44(100), 58(82), 91(20), 208(18), 193(15), 115(15), 179(10). - 42 -NMR: (impure sample) 6 ppm 0.88, t(3H, CH3-CH2); 1.4 m, 1.25 m(2H, CH2-CH3), 4.04, dd ( lH , CH-OH), 1.07, d(3H, CH3-CH); 2.68, m(lH, CH-CH3); 2.2, dd, 2.4, dd(2H, CAr 2-CH2-CH); 1-2, bs(2H, NH 2); 7.2-7.5, (10H, Aromatic). IR ( f i l m ) : v max 3400 cm - 1, (w, broad, NH or OH S t r ) , 2970 (m, OH i n t r a m o l e c u l a r H-bond), 1600(w-m), 1490(m, s CH 2), 1440(m), 1260(2), 1120(w, C-N S t r ) , 750(m), 730(m), 700(s). 4. REDUCTION WITH SODIUM CYANOBOROHYDRIDE To a s o l u t i o n of 5 mg (1.6 x 10"^ mol) methadone o x a z i r i d i n e (j>) i n 5 mL methanol was added 2 mg (3.2 x 10"^ mol) NaBH3CN. The pH was maintained at 6-7 by the dropwise a d d i t i o n of 0.1 M methanolic HC1. When pH remained constant the s o l u t i o n was a c i d i f i e d to pH 1 and s t i r r e d u n t i l gas e v o l u t i o n ceased. A f t e r a d j u s t i n g to pH 8 w i t h 0.1 N NaOH, the s o l u t i o n was d i l u t e d with water, saturated with sodium c h l o r i d e and extracted with chloroform. A f t e r drying and evaporation the product was analysed i n methanol by GCMS. The diketone and a product which by GCMS appeared to be an EDDP analog (50) were present. The product did not react with BSTFA i n CH3CN and was not f u r t h e r c h a r a c t e r i z e d . A n a l y s i s : MASS SPECTRUM OF MAJOR PRODUCT: m/z 280(1%), 99(100), 84(40), 250(22), 208(20), 193(18), 42(18), 115(15), 130(13), 251(5), 279(3). I. SYNTHESIS OF RECIPAVRIN AND RELATED COMPOUNDS 1. SYNTHESIS OF Y-PHENYL-N,N,g-TRIMETHYL BENZENEPROPANAMINE PERCHLORATE (6) (CAS 13957-55-6) Following the procedure o u t l i n e d by May and Mossetig (74), 2 - d i m e t h y l a m i n o - 4 , 4 - d i p h e n y l v a l e r o n i t r i l e (1 g, 3.73 x 10" 3 mol) - 43 -was d i s s o l v e d , i n a s o l u t i o n of 1 g KOH i n 10 mL diet h y l e n e g l y c o l (bp 250°) and r e f l u x e d overnight. When c o o l , the r e a c t i o n mixture was d i l u t e d with 20 mL H 20, extracted with ether, d r i e d over CaS04 and concentrated to 10 mL. The pe r c h l o r a t e s a l t was p r e c i p i t a t e d by adding 60% p e r c h l o r i c acid to the ether s o l u t i o n . A f t e r two r e c r y s t a l l i z a t i o n s from ethanol 0.73 g (81%) white/beige c r y s t a l s were obtained. A n a l y s i s For: C18H24NO4CI, (Molecular Weight 353.84). Me l t i n g P o i n t: 160°C (decomposes). Mass Spectrum: m/z 253(4%), 72(100), 73(12), 167(12), 44(8), 238(1). IR (nujol m u l l , p e r c h l o r a t e s a l t ) : v max 1600 cm - 1 ( s ) , 1500(m), 1470(s), 1380(s), 1150-1000(broad, s t r o n g ) , 950(m), 810(m), 770(m), 750(m), 670(s). A n a l y s i s For: C T ^ H ^ N , (Molecular Weight 253.39) ( f r e e base). NMR: (80MHz)6ppm 0.90, d(3H, CH3CH); 2.0-2.5, m (3H, CH2-CH); 2.20, s (6H, N - ( C H 3 ) 2 ) ; 4.2, t ( l H , Ar 2-CH-); 7.25 (10H, A r ) . 2. SYNTHESIS OF 1,l-DIPHENYL-3-BUTAN0NE (51) (CAS 5409-60-9) The synthesis followed was that of Burckhalter et al (75). In a one l i t r e , three neck round bottom f l a s k f i t t e d with a sealed s t i r r e r , thermometer, and 500 mL dropping f u n n e l , were placed 300 mL dry benzene and 29 g (0.223 mol) A l C l 3 . A f t e r c o o l i n g to 10°C on an i c e bath, the suspension was maintained below 20° during the dropwise a d d i t i o n of a s o l u t i o n of 25 g (0.112 mol) benzalacetone i n 60 mL dry benzene (approximately 30 minutes r e q u i r e d ) . The i c e bath was removed and s t i r r i n g continued overnight. The dark brown s o l u t i o n was decanted i n t o 160 mL of 0.8 M HC1, and f i l t e r e d w i t h s u c t i o n . The benzene l a y e r was separated, washed twice with water, dri e d over CaS04 and evaporated. - 44 -D i s t i l l a t i o n afforded 28.5 g (74%) diphenylbutanone b o i l i n g at 125° (0.3 mm) ( L i t e r a t u r e 164.5-168° at 4.5 mm (75)). The c l e a r d i s t i l l a t e s o l i d i f i e d i n the r e c e i v e r . A n a l y s i s For: C I ^ H T ^ O , (Molecular Weight 224.305). M e l t i n g Point: 46° ( l i t e r a t u r e 46°) (75). Mass Spectrum: m/z 224 (M+ 32%), 43(90) 167(100), 103(65), 165(38), 181(35), 152(22), 77(20). NMR (100 MHz) 6ppm 2.08, s(3H, CH 3); 3.22, d(2H, CH 2); 4.63 t ( l H , CH), 7.2 (10H, Aromatic). JR ( m u l l ) : vmax 3425 cm"1, (w), 1712 ( s , C=0 S t r ) , 1440(m), 1236(m) 1162(s). 3. SYNTHESIS OF 1,1-DIPHENYL-3-BUTAN0NE OXIME (52) (CAS 36317-57-4) To a methanol i c s o l u t i o n of 0.403 g (5.8 x IO" 3 moL) hydroxylamine hydrochloride (adjusted to pH 8.5 w i t h 2N NaOH) was added, 1 g (4.46 x 10~ 3 moL) 1,1-diphenyl-3-butanone i n methanol. A f t e r s t i r r i n g overnight at room temperature, the sample was d i l u t e d with water, extracted w i t h CHC13, d r i e d over K2CO3 and evaporated. The residue d i s t i l l e d at 193° (0.06 mm). A n a l y s i s For: C 1 6H 1 7N0, (Molecular Weight 239.319). Mass Spectrum: m/z 239 (M+ 8%), 167(100), 165(25), 152(15), 103(18), 42(12), 118(11), 181(11), 220(6). NMR (100 MHz): 2:1 mixture of syn-and anti-oximes SYN: « ppm 1.8, s(3H, CH 3); 2.97, d(2H, CH 2); 4.35, t ( l H , CH), 7.3, (10H, A r ) . ANTI: 6 ppm 1.57, s(3H, CH 3); 3.15, d,(2H, CH 2); 4.4, t ( l H , CH); 7.3 (10H, A r ) . IR ( f i l m ) : v max 3280 cm - 1, ( s , broad OH-internal H bond), 3050(s), 2910 ( s ) , 1665(w-m), 1610(m, C=N S t r ) , 1595(w shoulder), 1500(s), 1460(s), - 45 -138(m), 1270(m), 1100(w), 1050 (w-shoulder), 1040(m), 970(m), 940(w-m sho u l d e r ) , 760(s), 710(s). 4. SYNTHESIS OF g-METHYL-Y-PHENYL BENZENEPROPANAMINE-(DINORRECIPAVRIN) (53) (CAS 29869-77-0) A s o l u t i o n of 2 g (8.9 x I O - 3 mol) 1,1-diphenyl-3-butanone, 6.8 g (8.9 x 10~2 mol) ammonium acetate, and 0.78 g (1.25 x 10~ 2 mol) NaBH3CN i n 50 mL methanol were s t i r r e d overnight at room temperature. The s o l u t i o n was adjusted to pH 2 w i t h 5N HC1. A f t e r bubbles had subsided, the methanol was evaporated and the residue taken up i n 50 mL 2N NaOH. The amine was extracted with chloroform. The amine s a l t was extracted i n t o 2N HC1. The HC1 e x t r a c t was made a l k a l i n e with 5N NaOH and the f r e e base extracted with ether, d r i e d over CaS04 and evaporated to give 0.6 g (29%) product. A l t e r n a t i v e l y , a f t e r e x t r a c t i o n from 2N NaOH with chloroform, drying and evaporation y i e l d s 1.4 g (70%) crude amine, which s o l i d i f i e s upon t r i t u r a t i o n with petroleum ether. This product may be r e c r y s t a l 1 i z e d from benzene: l i g r o i n e . The HC1 s a l t was p r e c i p i t a t e d from dry ether w i t h gaseous HC1. A n a l y s i s For: C 1 6H 1 9N, (Molecular Weight 225.34). M e l t i n g Point: 172°C (HC1 s a l t ) . Mass Spectrum: m/z 225 (M+ 2%), 44(100), 208(12), 167(10), 165(5), 58(5), 115(4), 193(3). NMR (100 MHZ): ( f r e e base) <S ppm 1.1, d(3H, CH 3); 1.24, s(broad) (2H, NH 2); 2.06, m(2H, CH 2); 2.76, m(lH, CH-CH 3), 4.11, t(CH-CH 2), 7.3 (10H, Aromatic). - 46 -5. SYNTHESIS OF N, g-DIMETHYL-v-PHENYL BENZENEPROPANAMINE (NORRECIPAVRIN) (54) (CAS 29869-78-1) To a s o l u t i o n of 4.15 g (0.133 mol) of methylamine (11.25 g of 4 0 % aqueous s o l u t i o n ) i n 50 mL methanol, was added 1.63 g (4.5 x 10"2 mol) of h y d r o c h l o r i c acid i n 10 mL methanol followed by 5 g (2.2 x 10"2 mol), l,l-diphenyl-3-butanone and 2.1 g (3.35 x I O - 2 mol) NaBH3CN. The mixture was s t i r r e d over molecular sieve f o r 48 hours. The s o l u t i o n was f i l t e r e d and a c i d i f i e d to pH one. A f t e r bubbles subsided, the s o l u t i o n was f i l t e r e d and the methanol removed by f l a s h evaporation. The residue was d i s s o l v e d i n 25 mL water, adjusted to pH 8 w i t h 6N NaOH, saturated with sodium c h l o r i d e , extracted with ether, d r i e d over CaS04, and the hydrochloride s a l t p r e c i p i t a t e d with gaseous HC1. Product, 4.25 g (67%) of b l u i s h granules, was r e c r y s t a l 1 i z e d from ethyl acetate and a c e t o n i t r i l e . A n a l y s i s For: C17H22NCI, (Molecular Weight 275.82). M e l t i n g Point: 125°. A n a l y s i s For: C 1 7 H 2 i N , (Molecular Weight 239.363). Mass Spectrum: m/z 239 (M+ 3%), 58(100), 167(8), 59(5), 115, 134, 152, 193, 208(2). NMR (80 MHZ): 6ppm 1.06, d(3H, CH3-CH); 1.36, bs(NH, H bonded), 1.87-2.5, m(3H CH3-CH-CH2), 2.34, s(3H, N-CH3); 4.04, t(lH,-CH-Ar2); 7.1(10H, A r ) . IR ( f i l m ) : vmax 3300 cm" 1, (b, weak, NH S t r ) , 3000(s), 1615(m, NH bond), 1505(s), 1460(s, CH 2), 1380(m), 1160(m), 1050(m, C-N S t r ) , 760(m-s doublet, NH wag), 705(s). - 47 -6. SYNTHESIS OF N-HYDROXY-g-METHYL- "Y-PHENYL BENZENEPROPANAMINE (55) To 2 g (8.9 x 10~ 3 mol) 1,1-diphenyl-3-butanone in methanol, was added 0.8 g (1.1 x 10~ 2 mol) hydroxylamine hydrochloride. The s o l u t i o n was adjusted to pH 6 w i t h 6N KOH and 0.72 g (1.1 x 10~ 2 mol) NaBH3CN was added. The pH was maintained at 5-6 by dropwise a d d i t i o n of 4N HC1. When pH remained constant, the s o l u t i o n was s t i r r e d f o r an a d d i t i o n a l hour then adjusted to pH 1 w i t h 4N HC1. A f t e r gas evo l u t i o n ceased, the s o l u t i o n was d i l u t e d w i t h water, and adjusted to pH 8 wi t h 6N KOH, saturated with NaCl, and extr a c t e d 6 times with ether. The ether e x t r a c t was d r i e d over MgS04 and evaporated. T r i t u r a t i o n with petroleum ether (30°-60°) and r e c r y s t a l 1 i z a t i o n from benzene / 1igroine gave 0.59 g (30%) of a white s o l i d . A TMS d e r i v a t i v e was prepared f o r GCMS by t r e a t i n g the hydroxylamine with BSTFA i n CH3CN at room temperature. A n a l y s i s For: C 1 6H 1 9N0, (Molecular Weight 241.335). Combustion A n a l y s i s : C a l c u l a t e d : C, 79.63; H, 7.94; N, 5.8; 0, 6.63 Found: C, 78.93; H, 8.42; N, 5.45; 0, 7.20 Mass Spectrum: Decomposes on column to D i n o r r e c i p a v r i n (80). TMS D e r i v a t i v e (56): ( C 1 9 H 2 7 N 0 S i ) : m/z 313(M+ 8%), 132(100), 44(52), 116(50), 75(30), 167(30), 118(20), 91(10), 208(10), 298(8), 223(4). NMR (100 MHz): s ppm 1.1, d(3H, CH 3); 2.4 and 1.96, m(2H, CH 2); 4.1, t d i s t o r t e d (-CH-Ar 2); 2.9, m(lH, CH 2-CH-CH 3); 7.1-7.4 (10H, Aromatic), NH, OH not observed. IR (nujol m u l l ) : v max: 3250 cm" 1, (s(N-H/0H S t r ) , 2858(b,s), 1696(w-m), 1610(m, NH bend), 1505(s), 1460(s, -CH 2), 1373(N-0 S t r ) , 1230(s), 1108, 1031, 750-780(s), 710(s) - 48 -7. SYNTHESIS OF N, g-DIMETHYL-N-HYDROXY-y -PHENYL BENZENEPROPANAMINE (57) To a s o l u t i o n of 0.42 (5 x I O - 3 mol) N-methylhydroxylamine hydrochloride i n 0.5 mL H 20 was added 1.0 g (4.4 x 10* 3 mol) 1,1-diphenyl-3-butanone (51) i n 26 mL methanol. The s o l u t i o n was adjusted to pH 6 and 0.35 g (5.5 x IO" 3 mol) NaBH3CN was added. HC1 (5%) was added dropwise to maintain pH 5-6 u n t i l pH remained constant. A f t e r s t i r r i n g f o r an a d d i t i o n a l hour, the s o l u t i o n was adjusted to pH 1 w i t h 6N HC1. A f t e r gas bubbles subsided the s o l u t i o n was d i l u t e d w i t h 10 mL water, washed with ether, and adjusted to pH 8 w i t h 5% KOH. A f t e r s a t u r a t i n g with NaCl and e x t r a c t i o n with ether, the ether was drie d over CaS04> and evaporated. T r i t u r a t i o n with petroleum ether afforded 0.24 g (50%) of a white s o l i d , which melts at 115°C. A TMS d e r i v a t i v e was prepared f o r GCMS wi t h BSTFA i n CH3CN at room temperature. A n a l y s i s For: C 1 7H 2 1N0, (Molecular Weight 255.357). Combustion A n a l y s i s C a l c u l a t e d : C:79.96%, H:8.29, N:5.48, 0:6.27. Found: C:79.67%, H:8.44, N:5.47, 0:6.42. Mass Spectrum: Decomposes to N-methyl-1,1-diphenyl-3-butylamine i n the GC. TMS D e r i v a t i v e (58): ( C 2 0 H 2 g N 0 S i ) , (Molecular Weight 327.36). m/z 327(M+ 4%), 146(100), 58(28), 167(15), 132(5), 73(5), 165(5), 208(3), 312(3). NMR (100 MHZ): 6ppm 1.06, d(3H, CH3-CH); 2.56, s(3H, CH3-N); 4.1, t ( l H , CH-Ar 2) 2.0, m(lH,N-CH-CH 3); 2.4-2.8, m buried (2H, -CH-CI^-CH); 7.1-7.4 (10H, Aromatic); 9.7, (OH). Inf r a r e d (nujol m u l l ) : v max: 3200 cm" 1, (m broad N-H:0H S t r ) , 1950(w), 1590(m, N-H Bend), 1580(m), 1490(s, sharp), 1450(s broad -CH 2), 1370(s, N-0 S t r ) , 1360(m s h o u l d e r ) , 1220, 1190, 1160, 1130(m), 1060, 1045, 1030(m), 930(w), 910(w), 800(m), 780, 750, 730(m-s), 700(s). - 49 -8. SYNTHESIS OF N-METHYLIDENE-1,1-DIPHENYL-3-AMINOBUTANE (59) A s o l u t i o n of 1.0 g (4.18 x 10~ 3 mol) 1,1-diphenyl-3-aminobutane (53) and 0.51 mL (6.25 x 10~ 3 mol) of 38% aqueous formaldehyde s o l u t i o n i n 20 mL methanol was s t i r r e d over molecular s i e v e at room temperature f o r 2 days. A f t e r f i l t r a t i o n , GCMS revealed the c o r r e c t molecular weight f o r the imine, which probably e x i s t s as the t r i a z i n e (60). A n a l y s i s For: C^H^oN, (Molecul ar Weight 237.35). Mass Spectrum: m/z 237(5%), 57(100), 222(20), 56(15), 91(10), 58(9), 167(9), 165(9), 44(5), 152(5). INFRARED ( f i l m ) : v m a x 2950 cm"l, (m(broad)), 1600(w-m, C=N S t r ) , 1490(m-s, C=N S t r ) , 1450(m-s), 1380(w-m broad), 1150(m-s), 1070-1100(m-broad), 910(w-m), 740(s), 700(s). 9. SYNTHESIS OF 1,1 DI PHENYL-3-NITR0S0BUTANE (61) 1,1-Diphenyl-3-aminobutane (1.0 g (4.4 x 10" 3 mol)) (53_) i n a minimum of CH2CI2 was tr e a t e d w i t h 1.06 g (5.33 x 1 0 - 3 mol) 85% metachloroperbenzoic acid at 0° f o r 24 hours. The s o l u t i o n was f i l t e r e d and the residue p u r i f i e d by f l a s h chromatography i n 98% petroleum ether 12% ethyl acetate. Product, 100 mg (9.3%) was recovered as a white waxy s o l i d . The product v i s u a l i z e d as black spots on TLC pl a t e s sprayed with Dragendorf's reagent. The product probably e x i s t s as the dimer (62). A n a l y s i s For: CigH 1 7N0, (Molecular Weight 239.319). GCMS: Decomposes on column. NMR - 6ppm:1.43, d(3H, CH 3); 2.0-2.4, m(lH, H, CH aH b); 2.45-2.8, m(lH, CH^Hg); 3.2-3.4, q-m(lH, CH 3-CH-CH 2); 4.13, t ( l H , Ar 2CH-CH 2). - 50 -IR (nujol m u l l ) : vmax: 1602 cm -*, (w-m) (N-0 monomer S t r e t c h (or A r y l ) ) , 1378(m, c i s n i t r o s o dimer), 787(m, N-0 S t r e t c h ) , 760(m), 738(m), 702(s). 10. SYNTHESIS OF N-FORMYL-g-METHYL-y-PHENYL BENZENEPROPANAMINE (15) 1,1 Diphenyl-3-aminobutane (53) was r e f l u x e d i n ethyl formate f o r 4 days. A f t e r f l a s h evaporation, the residue was taken up i n 1 mL 20% ethyl acetate i n petroleum ether (30°-60°) and f l a s h chromatographed with t h i s solvent on a 10 cm by 1 cm column. Forty mg (18%) of product was recovered. A n a l y s i s For: C 1 7H 1 9N0, (Molecular Weight 253.346). Mass Spectrum: (GCMS) m/z 253(25%), 73(100), 208(42), 167(40), 165(28), 193(27), 130(37), 181(23), 72(23), 115(18), 58(12), 44(10). (IDENTICAL TO OXAZIRIDINE Q4)). NMR: (400 MHz) Mixture of 2 Compounds. Chemical s h i f t v a r i e s with r e l a t i o n to nitrogen lone p a i r . A. (major) <5ppm 1.19, d(3H, CH 3); 2.25-2.35, m(lH, CH aH b); 2.0-2.1, m(lH, CHi)Ha) 4.02, t ( A r 2 - C H ) ; 4.02, m buried (IH CH2-CH-CH3) 5.16, s, broad (NH); 7.1-7.3 (10H, A r ) ; 8.05, s(C(=0)H). B. (minor) 1.24, d(3H, CH 3); 2.1-2.2, m(lH, CH aH b); 2.25-2.35, m buried (IH, C H ^ g ) ; 3.3-3.4, m(lH, CH 2-CH-CH 3); 4.04, t buried (1H.-CH- A r 2 ) ; 5.35 broad s ( l H , NH); 7.1-7.3 (10H, Aromatic) 7.82; broad doublet (IH, C(=0)H). Infrared ( f i l m ) (poor spectrum): vmax: 3350 cm" 1, (broad m, N-H s t r ) , 1730(m, (C=0 contaminant formate e s t e r ) , 1675 (m shoulder C=0 Stretch-Amide I band), 1460(m), 1390(m), 1265(w s h o u l d e r ) , 1190(broad m-s formate e s t e r contaminant), 1050(w), 750, 770(w), 710(m). - 51 -The sample (pure by GCMS) was d e r i v a t i z e d on column w i t h TMAH as described i n the P i e r c e catalogue (76). The formamide (1 mg i n 10 yL CH3CN), was treated w i t h 20 yL TMAH and 5 yL was i n j e c t e d i n the GCMS. The product, eluted at longer r e t e n t i o n time, had a mass spectrum c o n s i s t e n t w i t h the t e r t i a r y amide, N, a-dimethyl-N-formyl -y-phenylbenzenepropanamine (63). Mass Spectrum: m/z 267 (M+, 10%), 87(100), 85(70), 58(28), 72(25), 167(22), 165(20), 208(18), 193(17). 11. SYNTHESIS OF 2-(4',4'-DIPHENYL-BUT-2'-YL) OXAZIRIDINE (14) Following a m o d i f i c a t i o n of the method of Krimm (87), 500 mg (1.9 x 10~ 3 mol), of D i n o r r e c i p a v r i n (80) i n 10 mL water was cooled to 0°. Aqueous formaldehyde (38%) 0.4 mL (4 x 10~ 3 mol) and 50 mL of CHC13 were added. To the s t i r r e d mixture was added 0.79 g (3.9 x 1 0 - 3 mol) MCPBA (85% pure). A f t e r 2 hours, 10 mL of 1.1 m CaC03 s o l u t i o n was added dropwise with s t i r r i n g . The CHCI3 phase was separated, d r i e d over potassium carbonate, and evaporated. Flash chromatography of the residue i n 9:1 petroleum ether (30°-60°): ethyl acetate afforded 165 mg (34%) mixture of 2 diastereomeric o x a z i r i d i n e s which gave black spots when v i s u a l i z e d by Dragendorf's reagent on TLC p l a t e s . The chromatography was repeated and the major isomer i s o l a t e d i n pure form f o r NMR a n a l y s i s . Samples were stored i n the f r e e z e r but were unstable. A n a l y s i s For: C17H19NO, (Molecular Weight 253.346). Mass Spectrum: 1. GCMS: i d e n t i c a l to N-formyl-1,1-diphenyl-3-aminobutane. 2. D i r e c t I n l e t : m/z 253(M+, 4%), 139(100), 156(90), 111(50), 167(48), 141(32), 158(30), 75(20), 50(18), 57(18), 208(12), 224(6), 237(2). - 52 -NMR (400 MHz): Major Isomer 6ppm 1.2, d(3H, CH 3); 1.86, ( s e x t u p l e t ) (IH, CH 3-GH); 2.34, dd(lH,-CH aH b-CH, J A B=18 Hz); 2.21, dd(lH CHbHa-CH); 3.82, d ( l H , o x a z i r i d i n e H a, JAB=10 Hz); 3.28, d ( l H , o x a z i r d i n e H b) 4.01, t ( l H , Ar2-CH-) 7.1-7.4 (10H, Aromatic). Minor Isomer (100 MHz): 6 p p m 1.12, d(3H, CH 3); 1.85, m(lH, CH 3-CH-CH 2); 2.24, dd( l H , CH-CH^HB-CH, JAB=18 HZ); 2.64, dd(lH CH-CHnHA-CH); 3.46, d ( l H , o x a z i r i d i n e ( H A J A B=10 HZ); 3.95, d ( l H , o x a z i r i d i n e H B ) ; 4.25, t(Ar 2-CH-CH 2); 7.1-7.4 (10H, Aromatic). 1 3C NMR (BB and SFORD): Major Isomer: 6 p p m : 19.73(q, CH 3); 40.12 ( t , -CH2-CH); 65.09 (d, CH 3-CH-CH 2); 48.23 (d, CH-Ar 2); 71.97 ( t , o x a z i r i d i n e CH 2). JR ( f i l m ) : v max 3100-2900cm" 1, (m), 1750(w), 1610(m), 1590(w s h o u l d e r ) , 1520(s), 1480(s), 1390(m, o x a z i r i d i n e ) , 1270(m-s), 1170(w-m), 1120(w-m), 1080(w-m), 1050(w-m), 970(w-m), 770(m-s), 700(s). SYNTHESIS OF g-METHYL-(N-METHYLENE)~T ~PHENYLBENZENEPROPANAMINE N-OXIDE (Rec i p a v r i n Nitrone) (13) Aqueous formaldehyde 38% w/v (0.4 mL, (5 x 10" 3 mol)) i n 20 mL dry benzene was re f l u x e d f o r 2 hours i n a Dean and Stark apparatus. Then 0.5 g (2.1 x 10" 3 mol) N-hydroxy-1,1-diphenyl-3-aminobutane i n benzene was added slowly w i t h s t i r r i n g . The apparatus was flushed w i t h N 2 and r e f l u x e d f o r 20 minutes. A f t e r c o o l i n g , the benzene was evaporated and the product f l a s h chromatographed i n ethyl acetate. The p o l a r product (Rf. 0.2) was eluted slowly (100-150 mL) y i e l d i n g 250 mg (47%) of a c l e a r t h i c k 1 i q u i d . A n a l y s i s For: C i 7 H 1 9 N 0 , (Molecular Weight 253.346). - 53 -Mass Spectrum: GCMS: Decomposes on column to N-methylidene-1,1-diphenyl-3-aminobutane (major) (59), 1,1-diphenyl-3-butanone oxime (j>2), and N-formyl-1,1-diphenyl-3-aminobutane (minor) (15). D i r e c t I n l e t : m/z 253 (M+, 2%) 56(100), 236(15), 208(25), 91(40), 222(4). !H NMR: 6 p p m , 1.42, d(3H, CH 3-); 2.23, dd(l H , CH-Ch^HB-CH); 2.74, dd(lH C H - C H R H A - C H - ) ; 3.79, m(lH, CHAHB-CH J A B=18 Hz); 5.98, d ( l H , N=CH AH B); 6.41, d ( l H , N = C H R H A ) ; 7.1-7.4 (10H, Aromatic). 1 3C NMR (Broad Band andSFORD): 6 p p m 19.92(q, CH 3); 39.41 ( t , CHAr 2-CH 2-CH); 47.74(d,-CHAr 2); 69.03(d, CH 3-CH-); 122.29(t, N=CH2). Infrared ( f i l m ) : v max 3395 cm - 1, (weak, broad) 1566(C=N), 3100-2900(m), 1580(w-m), 1490(s), 1450(s), 1296 (m), 1200(w), 1060(s), 920(w), 800(w), 760(doublet(m)), 710(s). U l t r a v i o l e t : (methanol) Amax 235 my(e=6210), 220(£=10,120). 13. SYNTHESIS OF 1,1-DIPHENYL-3-BUTAN0NE-0-METHYL OXIME (64) Following the procedure i n the Pierce Catalogue (76). To 50 mg (2.23 x 10~ 4 mol) 1,1-diphenyl-3-butanone i n 0.1 mL p y r i d i n e was added 1.0 mL M0X. A f t e r heating f o r 3 hours at 60° i n a r e a c t i v i a l w i t h a t e f l o n l i n e d cap, the s o l u t i o n was d i l u t e d w i t h 4 mL water, and extracted w i t h three 10 mL a l i q u o t s of benzene. The organic phase was washed w i t h IN HC1 and 1% NaHC0 3, then d r i e d over MgS04, and concentrated to a volume of 0.5 mL. Two yL was i n j e c t e d i n t o the GCMS. Mass Spectrum: m/z 253 (M+, 8%) 167(100), 165(22), 152(12), 103(12), 118(12), 42(10), 77(9), 181(8), 222(3). NMR (80 MHZ): Mixture of syn and a n t i oximes (2:1)- impure sample - 54 -A: 6ppm 1.72, s (3H, CH3-C=N); 2.9, d(2H, CH_2-C=N); 3.76, s(3H,-0-CH3); 4 .35 , t ( l H , Ar 2-CH-); 7.25, s(10H, A r ) . 14. ALKALINE OXIDATION OF N, g-DIMETHYL-N-HYDROXY- Y-PHENYL- BENZENEPROPANAMINE (57) A. B i l e from a r a t was worked up by the procedure o u t l i n e d e a r l i e r . A f t e r the e x t r a c t i o n of non-conjugated m e t a b o l i t e s , 10 mg of the secondary hydroxylamine was added to the conjugated f r a c t i o n , and the sample work up continued. GCMS a n a l y s i s of the conjugated f r a c t i o n i n d i c a t e d the presence of a compound with mass spectrum i d e n t i c a l to N-formyl-a-methyl-y-phenyl benzenepropanamine (15). B. Secondary hydroxylamine (42 mg) was treated with e t h a n o l i c NaOH f o r one week. The sample was d i l u t e d w i t h water, extracted w i t h CHC13, d r i e d over K2CO3, evaporated and examined by HPLC. J . ATTEMPTED CHARACTERIZATION OF THE RECIPAVRIN METABOLITE BY LCMS Li q u i d chromatographic c o n d i t i o n s were determined, to o p t i m a l l y separate the n i t r o n e (_13), amide (JL5) and o x a z i r i d i n e (14). A sample of the conjugated f r a c t i o n of r a t b i l e from a r e c i p a v r i n dosed r a t was examined, but concentrations were too low f o r d e t e c t i o n by se l e c t e d ion monitoring of the m/z 254 (M+l) i o n . LCMS Mass Spectrum: N-formyl-a-methyl-Y-phenyl benzenepropanamine (15) (tR=24.41 min) m/z 254 (M++1, 100%); 255(M ++2, 20%) Mass Spectrum: 2-(4',4'-Diphenyl-but-2'-yl) o x a z i r i d i n e (14) (t R=40.87 min) m/z 254 (M++1, 100%); 238 (20); 255 (20); 226(15); 73(4) - 55 -Mass Spectrum: a-methyl-(N-methyl ene)-y-phenyl benzenepropanamine N-oxide (II). (tR=17.20 min) m/z 254 (M ++l, 100%), 238 (M+l-16, 20%), 74(20). K. ATTEMPTED GAS CHROMATOGRAPHY/ CHEMICAL IONIZATION MASS SPECTROMETRY  CHARACTERIZATION OF THE RECIPAVRIN METABOLITE The r e c i p a v r i n n i t r o n e and o x a z i r i d i n e CI mass spectra and c a p i l l a r y GC chromatograms were obtained. The conjugated f r a c t i o n of b i l e from a r e c i p a v r i n dosed r a t was examined. Chemical I o n i z a t i o n O x a z i r i d i n e (14) same as formamide (15): 254(M + +1, 100%); 255(M + +2, 20%); 282(M + +29, 20%), 57(10), 176(8). - 56 -I I I . RESULTS AND DISCUSSION 1. CHEMISTRY: SYNTHETIC METHODS AND ANALYSIS A. S y n t h e t i c Pathways In the s y n t h e t i c portion of t h i s t h e s i s , the diketone (45) was a key intermediate i n the attempted synthesis of the methadone n i t r o n e (2_). The diketone was obtained in low y i e l d v i a the s y n t h e t i c pathway: — 68 45 — 1. a) NaH, C5H5, DMSO 3. a) EtMgBr/Ether/toluene/A b) Propyleneoxide b) H30 + 4. a) Cr03/H2S04/H20/Acetone 2. H 3 O V A b) Isopropanol - 57 -In Figure 8 the attempted synthesis of the methadone n i t r o n e {2} i s o u t l i n e d . The oxime (46) was synthesized but was unstable even when stored at 0°. Attempts to s e l e c t i v e l y reduce the oxime p o r t i o n of 46 w i t h NaBH3CN r e s u l t e d i n a mixture of p y r o l l i d i n e l i k e products or concomittant reduction of the keto group. Since the desired primary hydroxylamine (47) was u n a v a i l a b l e , attempts to synthesize the methadone n i t r o n e (2_) were di s c o n t i n u e d . 2 3. NaBH3CN/MeOH/pH6 4. H 2C0/C 6H 6 5. Yellow HgO/Acetone Figure 8: Attempted syntheses of the methadone n i t r o n e [2). - 58 -The methadone o x a z i r i d i n e (5) was made by a m o d i f i c a t i o n of Kangs method f o r the o x i d a t i o n of EDDP. The formamide (31) was obtained by thermal i s o m e r i z a t i o n of the o x a z i r i d i n e (Figure 9). ^ CH C N CH3 i . C - 0 CH, 4 CH. CH, I 2 3 CH 2 CH, '</ CH2 CH3 1. a) MCPBA/CHCI3 b) IN NaOH 2. Toluene/A ^ ^ w Figure 9. Synthesis of methadone o x a z i r i d i n e (j>) and formamide (31). Since the methadone n i t r o n e (2_) proposed by Kang was not s u c c e s s f u l l y s y n t h e s i z e d , r e c i p a v r i n was chosen as a model f o r sy n t h e t i c and metabolic s t u d i es. In the r e c i p a v r i n s e r i e s , the N and a-C oxidised p o t e n t i a l metabolites were obtained by the reactions o u t l i n e d in Fi g u r e 10. The stem compound diphenyl butan-3-one (51) was synthesized by the method of Burckhalter (75). Re c i p a v r i n was synthesized by the a l k a l i n e h y d r o l y s i s and decarboxylation of methadone n i t r i l e as described by May and Mossetig (74). A low y i e l d of the r e c i p a v r i n formamide was obtained by r e f l u x i n g the primary amine (53) i n ethyl formate. - 59 -55 11 59 15 1 2 ,H N 1 3 C 6H 5 CH3 I H . N XX 13 14 63 \ c / H fl 0 l a . C6HA, AlCT 3 b) H 3 0 + 2. CH3NHOH, NaBHjCN, CH3OH, pH5 3. CH3NH2, NaBH 3CN, CH3OH, Molecular Sieve 4. CH30NH2, P y r i d i n e 5. NH20H, MeOH, pH8 6 . N H 4 A C , MeOH, NaBH3CN 7. NaBH3CN, MeOH, pH6 8. H2CO, MeOH 9. MCPBA, CH 2C1 2 10. Ethyl formate 11. H2C0, C&s 12. MCPBA, CHC13A 13. TMAH, C5H5N L F i g u r e 10. Synthesis of N and a-C Oxidized P o t e n t i a l M e t a b o l i t e s of Re c i p a v r i n . - 60 -Since we were concerned with compounds which appeared by GCMS to be i d e n t i c a l to the unknown metabolites of methadone and r e c i p a v r i n , the a n a l y s i s of these o x a z i r i d i n e s , formamides and nitr o n e are discussed f i r s t . The r e l a t e d N-oxidized compounds and t h e i r s y n t h e t i c precursors are discussed i n subsequent s e c t i o n s . B. OXAZIRIDINES i ) Synthesis and pr o p e r t i e s O x a z i r i d i n e s are three membered heterocycles containing oxygen ( p o s i t i o n 1), nitrogen ( p o s i t i o n 2) and carbon ( p o s i t i o n 3). They were f i r s t described by Kn'mm (77) and Emmons (21) i n the l a t e 1950's. The r e c i p a v r i n and methadone o x a z i r i d i n e s 5) were synthesized by per o x i d a t i o n of the imine _59 (which probably e x i s t s as the t r i a z e n e ) and the p y r o l l i d i n e EDDP (4_) r e s p e c t i v e l y . A mechanism f o r the pe r o x i d a t i o n of EDDP to o x a z i r i d i n e (5J which also accounts f o r the presence of EMDP (11.), and the diketone (£5), can be proposed based on the i n v e s t i g a t i o n s of M i l l i e t et al (78) in t o peracid o x i d a t i o n of p y r o l l i d i n e a l k a l o i d s a l t s (Figure 11). I n i t i a l p e r oxidation of the immonium s a l t i s followed by two p o s s i b l e proton a b s t r a c t i o n s A and Figure 1 1 . Mechanism f o r the formation of a methylene o x a z i r i d i n e from EDDP - 62 -B. Pathway A leads to consumption of a second mole of peroxide and r i n g opening to a keto o x a z i r i d i n e . M i l l i e t said pathway B leads to the desalkyl product ( i n our case EMDP). However, by f o l l o w i n g a pathway analogous to A, the methylene o x a z i r i d i n e could be obtained. Two equivalents of MCPBA are required i n the o x i d a t i o n (1). The y i e l d of o x a z i r i d i n e was s u b s t a n t i a l l y improved, and the amount of byproducts reduced by hydrolyzing the perester r e a c t i o n products w i t h IN NaOH ra t h e r than by Kang's procedure using aqueous NaHC03 or NaHS03. Kang had proposed a mechanism supporting a nitr o n e s t r u c t u r e f o r t h i s compound. NMR, LCMS and GC data presented here prove that the corresponding o x a z i r i d i n e i s the c o r r e c t s t r u c t u r e . The synthesis of the r e c i p a v r i n o x a z i r i d i n e s followed the method of Krimm (77), who found that although methylene imines e x i s t i n the t r i m e r i c ( t r i a z e n e ) form (60), they undergo p e r o x i d a t i o n to o x a z i r i d i n e s i n a manner s i m i l a r to secondary imine monomers. Emmons states that the a c i d i c reagent apparently depolymerizes the t r i a z e n e to the imine which i s then oxidized (21). i i ) Detection and i s o l a t i o n Almost a l l the reactions of o x a z i r i d i n e s i n v o l v e f i s s i o n of the r i n g . The f a c t that they were assayed i o d o m e t r i c a l l y by Emmons showed that they o x i d i z e i o d i d e to i o d i n e . Thus o x a z i r i d i n e s are e a s i l y detected as black spots when TLC plates were sprayed with Dragendorf's Reagent. The non po l a r nature of o x a z i r i d i n e s r e l a t i v e to nitrones and amides was r e f l e c t e d by t h e i r TLC m o b i l i t y (Rf=0.3-0.4) i n 15% ethyl acetate in petroleum ether. P u r i f i c a t i o n by f l a s h chromatography (70) allowed the p a r t i a l p u r i f i c a t i o n of two diastereomeric forms of each o x a z i r i d i n e s i m i l a r to r e s u l t s obtained - 63 -by Morgan and Beckett (64). Diastereomers of o x a z i r i d i n e s can be i s o l a t e d as a r e s u l t of two contiguous asymmetric c e n t r e s . These are the methine carbon a to the n i t r o g e n , and the pseudoasymmetric centre of n i t r o g e n , which a r i s e s from the high i n v e r s i o n b a r r i e r (138 Kj/mol at 120°) (79) of the o x a z i r i d i n e r i n g . Emmons sta t e s that methylene o x a z i r i d i n e s c o n t a i n i n g a-protons, f o r example 2 - n - b u t y l o x a z i r i d i n e , are the l e a s t s t a b l e (21). This was t r u e of the r e c i p a v r i n o x a z i r i d i n e s which decomposed at 0° i n a matter of weeks to the primary amine (53) and methylene imine (59) as determined by GCMS and the formamide (5) as observed by LCMS. The methadone o x a z i r i d i n e deposited cubic c r y s t a l s from deuterochloroform that were s t a b l e i n d e f i n i t e l y at 0°. Sol u t i o n s tended to decompose to EMDP. i i i . Reactions of methadone o x a z i r i d i n e Reduction wit h excess l i t h i u m aluminum hydride produced dinormethadol (49) q u a n t i t a t i v e l y . Primary Amine 3400(m,broad), 1590(m) 740(s) Secondary Alcohol:1110 cm _ 1(s) No C=0 s t r e t c h m/z 44 (100%) Primary Amine 49 I t was hoped that reduction w i t h NaBr^CN at pH 6-7 would s e l e c t i v e l y reduce the o x a z i r i d i n e r i n g . Two equivalents of reducing agent were required f o r complete r e a c t i o n . The product d i d not react w i t h BSTFA, t h e r e f o r e i t was not the expected secondary hydroxylamine. By GCMS the 1 .Am 1.25m*% 0.88t r-^S. CH,CH, IR: ^ CHr—CH-NH2buried /* I ^ 2.2dd C H> 2 - 6 8 m 2.Add 1.07d MS: - 64 -product fragmented s i m i l a r to ethyl dimethyl diphenyl p y r o l l i d i n e (molecular weight 279). i v ) *H and 1 3 C NMR of o x a z i r i d i n e s The *H and 1 3C NMR of the major diastereomers and s t r u c t u r e s showing *H NMR chemical s h i f t values f o r major and minor diastereomers of methadone and r e c i p a v r i n o x a z i r i d i n e s are o u t l i n e d in Figure 12. Spectra from decoupling experiments and the *H NMR of minor isomers are included in the Appendix. Chemical s h i f t values of three to four ppm ( f o r the o x a z i r i d i n e r i n g proton doublets) agree with those observed by C r i s t et al (80) f o r 2-t-butyl o x a z i r i d i n e . Jordan and C r i s t (81) have observed that the proton trans to the lone p a i r resonates at higher f i e l d . The coupling constant (J/\g = 10 Hz) and the chemical s h i f t value of the a-methine proton (1.8 ppm) were c o n s i s t e n t between methadone and r e c i p a v r i n o x a z i r i d i n e s . However, the o x a z i r i d i n e r i n g has marked e f f e c t s on e s u b s t i t u e n t s , and d i f f e r e n c e s in chemical s h i f t between isomers demonstrated long range e f f e c t s on the protons a- to the carbonyl group in the case of the methadone o x a z i r i d i n e . The r e s u l t s shown f o r the major diastereomer c o r r e l a t e s with the 100 MHz r e s u l t s of Kang, chemical s h i f t s , are more acc u r a t e l y determined here as a r e s u l t of high r e s o l u t i o n , b e t t e r sample p u r i f i c a t i o n and decoupling experiments. SFORD and broad band decoupled * 3C NMR were obtained f o r the major diastereomers of each o x a z i r i d i n e ( f i g u r e 12). The o x a z i r i d i n e carbon was observed as a broad t r i p l e t at 72 ppm i n the SFORD sp e c t r a , as compared to 65.5 ppm in 2 - t - b u t y l o x a z i r i d i n e (80). The la r g e coupling constant i n d i c a t e s adjacent e l e c t r o n e g a t i v e atoms. These are the f i r s t examples of 1 3 C NMR of methylene o x a z i r i d i n e s with protons a to the nitrogen atom. - 65 -H <— 4.25d / 1.85m v // H H CH, 95d J=10Hz 3.46d ' A . O l t C/ H 1.86m 3.82d ' \ I /H C -CH- N— C J=10Hz / \ I I / v H H H CH, 0 2.34m f 1 > 2 9 d 2.21m 0 I 0.85t ll I ^ C-CHjCH, . ' 1.8m / \ - C H ^ N - C \ /) / \ CH3 0 ^ He Hd | r 2.35dd 0.56d 3.2dd 3.28d Major diastereomers 2f> 0 .83. ^C-CH 2CH, rj • 8m \ C -CH- N— C He Hd A Ha 3.62d J=10Hz Hb 3.17d 2.80dd 84d 2.32dd 25.1 Ha / 3.76 * 1.05 65.5 / / Hb Hb / 3.66 33.04t 1 ^ 9 . 1 8 q ^ ^ 48.23d ^ V Z I I ^ C H , C ^ H 65.09d 7 1 Q 7 \JL > C = 0 \ / 7 K 9 7 t , T \ ^ W - 0 0 d 7 2 . 5 4 t <f t ^ C H r - C H - N - C H . 6 5 -J° / \ 7. / \\ /J ' * ' A / ^ C H ^ C H - N - C H 2 19.73q 42.6t| 21.29q Figure 1 2 ( a ) . *H and 1 3 C NMR r e s u l t s f o r methadone and r e c i p a v r i n o x a z i r i d i n e s compared to those of 2 - t - b u t y l o x a z i r d i n e . ~~i 1 1 1 1 r 5 4 3 2 1 0 Figure 1? (b) 400 MHz lH NMR of 2-(4' ,4'-diphenyl heptan-5 '-one'2 ' - y l ) o x a z i r i d i n e major diastereomer (5) - 67 -Hi 2300 JL 33.04t 1 ^ 9 . 1 8 q , c . , V ' 64.00d 72.54t 65.46 ^ ^ 42 .6t | 21.29q — r ~ 100 150 0 50 Figure 12 (d) SFORD 400 MHz 1 3C NMR of 2-(4\4'-diphenylheptan-5 ,-one -2 '-yl) o x a z i r i d i n e major diastereomer (j>) Figure 12(e) SFORD 400 MHz 1 3C NMR of 2-(l',1'-diphenyl-but-2'-yl) o x a z i r i d i n e major diastereomer (14) - 70 -v) Infrared spectra By comparison of i n f r a r e d spectra of the o x a z i r i d i n e s with the diketone and diphenylbutanone s p e c t r a , a number of bands appear to be associated with the o x a z i r i d i n e r i n g . An unusual medium i n t e n s i t y absorption at 1730 cm - 1 i n the r e c i p a v r i n o x a z i r i d i n e , u s u a l l y associated with a strong carbonyl absorption of esters i s present. The band i s very strong i n poorly p u r i f i e d samples and probably i s a contaminant, perhaps an unhydrolyzed N-3-chlorobenzyl ester of MCPBA as described by Beckett (64) i n the MCPBA ox i d a t i o n of secondary amines or po s s i b l y a decomposition product of the formamide (15). A sharp medium band at 1600 cm - 1 i s s i m i l a r to an amide band. The band at 1250 cm'1 i s l i k e l y an N-0 (or C-N) s t r e t c h s i m i l a r to that of an a l i p h a t i c n i t r o s o dimer. Bands at 1035, 935 cm"1 are l i k e l y associated with the C-0 bond. The i n f r a r e d spectra of methadone and r e c i p a v r i n o x a z i r i d i n e s are shown i n Figure 13. v i ) U l t r a v i o l e t spectra In the u l t r a v i o l e t spectrum of methadone o x a z i r i d i n e two bands at 296.5 my (e=502) and 265.5 my (c=548) were observed in add i t i o n to aromatic bands at 254 and 207 my. These are po s s i b l y N - 0 t r a n s i t i o n s of each heteroatom i n the o x a z i r i d i n e r i n g . v i i ) T h e r m o l a b i l i t y and mass sp e c t r a l a n a l y s i s The thermal i s o m e r i z a t i o n of o x a z i r i d i n e s to amides has been well documented by Emmons (21). This study shows that both methadone and r e c i p a v r i n o x a z i r i d i n e s isomerize i n the GC to the re s p e c t i v e formamides (5) and (15_). S t . C l a i r e Black et al (82) have indicated that o x a z i r i d i n e s thermally isomerize to nitrones at temperatures below 200° and to amides above 200°. Thermal i s o m e r i z a t i o n of methadone o x a z i r i d i n e under r e f l u x in Figure 13(a) Infrared spectrum ( f i l m ) of 2-(4',4'-diphenyl heptan-5'-one-2'-yl) o x a z i r i d i n e (ji) Figure 13 (b) Infrared spectrum ( f i l m ) of 2-(4' ,4'-diphenyl-but-2 ' - y l ) o x a z i r i d i n e (14) - 73 -m-xylene under nitrogen afforded the formamide (68) but no n i t r o n e . The methadone o x a z i r i d i n e i s non v o l a t i l e and t h e r m o l a b i l e . Thus even moderate heating of the source in d i r e c t probe mass spectral a n a l y s i s produces ions c h a r a c t e r i s t i c of the formamides. The only d i f f e r e n t ions i n the d i r e c t probe mass spectrum occur at m/z 42 (C3H5) and 56 (C4H8), these suggest i s o m e r i z a t i o n to the nitrone upon gentle heating of the source (see d i s c u s s i o n of the r e c i p a v r i n nitrone mass spectrum). The marked s i m i l a r i t y o f the b e n z y l i d i n e n i t r o n e s and o x a z i r i d i n e s studied by Morgan and Beckett (64) were a t t r i b u t e d to thermal i s o m e r i z a t i o n of o x a z i r i d i n e to n i t r o n e . While t h i s i s p o s s i b l e , e s p e c i a l l y at the lower GC temperatures required to elute these compounds, GC r e t e n t i o n times and GCMS were not d e t a i l e d , and a s i m i l a r i s o m e r i z a t i o n to formamides or benzamides i s p o s s i b l e . C. FORMAMIDES i ) Synthesis and NMR spectra The r e c i p a v r i n formamide (15_) was synthesized i n low y i e l d by r e f l u x i n g the primary amine (53_) i n ethyl formate for several days. A f t e r p u r i f i c a t i o n by f l a s h chromatography, peak areas in the NMR spectrum of the product showed that two species were present i n a r a t i o of 8:6. Comparison with the NMR of isopropyl formamide (83) showed that the r e l a t i o n o f s u b s t i t u e n t groups to the nitrogen lone pair had marked e f f e c t s on chemical s h i f t s of s u b s t i t u e n t s r e s u l t i n g i n two d i s t i n c t spectra. The NMR of major and minor components of the methadone and r e c i p a v r i n formamides and isopropyl formamide are shown and summarized in Figure (14a). Decoupled spectra are included i n the appendix. Decoupling by the m u l t i p l e t at 4.05 ppm of the r e c i p a v r i n formamide c o l l a p s e d the methylene protons - 74 -5.16 (bs) f\ S 0 A t / SrSs (s> w / 5;35 ( b s ) C " " ~ 3.3-3.4m^ „ _ 7 . 8 2 b d \ ^ / * f C H 3 ° \ = / H A H B ^ MINOR 2. 25-2. 35m/ 1 MAJOR 2.25-2.35m ^ 1.24d 2.0-2.lm 1 , 1 9 d 2.1-2.2m 2.1-2.3q ( b u r i e d ) \ 0.85t C —CH,CH, _ _„ / 2 3 0 J.78 C 3-i5m . . ' II / C - C H - N— CH N-HaOu CH 3 ^ 5 - 9 8 b s ^ Hb 2 - 4 - 2 - 5 d d I . . i n t i . : 2.8-2.9dd MAJOR 2.14-2.3q 0 J 0.85t .C-CHjCHj" 2.9-3.0m 0 7.45d \ V -C - C H - N - C H \t Ha^' CH 3 H < - 5 . 5 2 ( b s ) v ~ — H b K 2 - 3 " 2 ^ d d \ l . \ 2 d 2.7-2. MINOR MAJOR CH j C H 3 T 1.17 4.2 CH 3" 8.05 H ^ 5 - 7 b r o a d 5-7 b r o a d ^ CH CH 3 ^ I f 3.66(m) 1.24d N 1 1 8.05 MINOR Figure 14(a) Comparison of NMR r e s u l t s f o r r e c i p a v r i n and methadone formamides with those of isopropyl formamide Figure 14- (b) 400 MHz *H NMR of 2-(N-formyl)-4,4-diphenyl-5-heptanone II \ 4.04t ' * / 5.35 (bs) v * / ^ „ H 3. 3-3.4m fl 7 C v V ^ N N . H ^ 7 ' 8 2 b d 5.16 (bs ) // \ /\ \ o / 8.05 ( s ) \___/ H A Hg C H ^ MINOR / \ \ ^ i </.,o-2.35m \ 1 .24d C CH^ M ^ l f 2 .1 - 2 . 2m ' \ I 11 f \ / - ° 2 t C v ' 5 4 3 2 Figure 14 (c) 400 MHz *H NMR of N-formyl-a-methyl-Y-phenyl benzene-propanamine (15) propanamine c o l l e c t e d o f f a GC column - 78 -i n both diastereomers as well as reducing the N-H peak i n t e n s i t y and c o l l a p s i n g the methyl doublet i n the major diastereomer. Thus, the proton a to the nitrogen o f the major diastereomer was hidden by the di phenylmethine protons o f major and minor diastereomers. The s i n g l e t formyl resonance at 8.05 ppm appears u p f i e l d at 7.82 as a broad doublet i n the minor diastereomer o f the r e c i p a v r i n formamide. The corresponding peak in the methadone formamide occurred u p f i e l d at 7.78, p o s s i b l y due to i n t e r a c t i o n of the formyl and keto side chains. Since the observed metabolite of methadone and r e c i p a v r i n had an i d e n t i c a l GC r e t e n t i o n time and mass spectrum to the o x a z i r i d i n e and the formamide of each drug, i t was important to know that the formamide survived GC a n a l y s i s . The r e c i p a v r i n formamide was i n j e c t e d into the GC and the peaks were c o l l e c t e d i n a cold c a p i l l a r y tube. The NMR spectrum (obtained in a 1 mm NMR tube on 500 yg of m a t e r i a l ) , o f the eluted peaks were i d e n t i c a l to that o f the formamide ( f i g u r e 14d), the r e f o r e although the s t r u c t u r e of the metabolite remains unclear, t h i s i s evidence that i t i s observed by GCMS as the formamide. i i ) I n f r a r e d Spectra The i n f r a r e d spectrum of the formamides (15_) and (3JJ (Figure 15) have a broad hydrogen bonded NH s t r e t c h at 3200-3300, a strong secondary amide I band at 1670-1660 cm"1, a weak amide II band at 1605 cm"1, amide C-N s t r e t c h i n g at 1385 cm - 1 and a p a r t i a l l y resolved amide peak below 800 cm - 1 which obscured by phenyl r i n g bending (84). The r e c i p a v r i n formamide sample saved f o r IR was weak and contaminated with an e s t e r (1730 cm" 1, 1175 cm" 1), p o s s i b l y methyl formate, however the low i n t e n s i t y peaks of the weak sample c o r r e l a t e d with those observed for the methadone formamide. Figure 15(a) Infrared spectrum ( f i l m ) of 2-(N-formyl)-4,4-diphenyl-5-heptanone (31) benzenepropanamine (15) impure sample - 81 -i i i ) Mass spectra The mass spectra of the formamides ( i d e n t i c a l to the metabolites of methadone and r e c i p a v r i n ) each show a molecular ion m/z 309 and m/z 253, wi t h base peaks at e i t h e r m/z 72 or 73. LCMS and CI GCMS revealed a M ++l base peak at m/z 254 f o r r e c i p a v r i n . The mass spectrum and p o s s i b l e fragmentation pathways of the r e c i p a v r i n formamide are shown i n Figure (16). Beta cleavage accounts f o r s e r i e s derived from m/z 208 as described by Abbott et al (66). A l l other major fragments can be r a t i o n a l i z e d as a r i s i n g from Y-cleavage of the formamide. Me t h y l a t i o n on the GC column using trimethy'lanilinium hydroxide (TMAH) produced the corresponding t e r t i a r y amide (63) (Figu r e 16,17) with a molecular ion at m/z 267 and base peak at m/z 87. This compound was synthesized as a p o t e n t i a l metabolic precursor of the r e c i p a v r i n formamide me t a b o l i t e . o lb 63 The methadone formamide (Figure 18) can undergo two p o s s i b l e McLafferty rearrangements. The f i r s t (pathway A) r e s u l t s in a neutral mass 264 fragment (not observed), which, i f cleaved a to the carbonyl group, could give r i s e to m/z 57, and the m/z 207, 129 s e r i e s c h a r a c t e r i s t i c of both t h i s compound and the s y n t h e t i c diphenylheptenone (39). The strong ion at m/z 253 c o n s i s t a n t with r e c i p a v r i n appears to a r i s e by a proton t r a n s f e r process which we have t e n t a t i v e l y shown as a 7 ce n t r e rearrangement. - 82 -fl I I NT - '* t»7« -* 4 " ••Ii -fc ifc m ...ft. «« MM* » »v UM . • » ll«t.- >«»• Figure 16(a) Mass spectrum (GCMS) of N-formyl-a-methyl- Y-phenyl benzenepropanamine (15) ( i d e n t i c a l to o x a z i r i d i n e (14) and the r e c i p a v r i n m e t a b o l i t e - 83 --(C 6H 5^CH 2 m/z 180 H » \ "H* 72 m/z V 1+) C I t» CH CH, H m/z 73 \ r- ' ^ • + ~-CH2 CH3 / H ' r H / ^ / N ^ H - ( C 6 H 5 ) 2 C H / \ C L I C H H I I H C H 3 -(HC(=0)NH2) C 6H 5 m/z 208 s e r x e s -CH, 0 CH, H """" C t ~ ~ ~ C H ~ C H 3 H -CO ^ m/z 86 CH-H2N CH2 m/z 44 t -CO 0 » l H m/z 72 ^ C H 2 X H-N CH CHn + 6 m/z 58 + (C 6H 5) 2C-CH 2 A 6H 5 C 6H 5 » m/z 115 C 7 H 7 Q 1 m/z 91 m/z 193 Fiqure 16 ( b ) M a s s spect ra l fragmentation pathway of N-formyl-a-methyl -Y -phenyl benzenepropanamine (15) 10* I ** I I I IM I I I * |"* / • n l i"%i M i " t i l iT- , . .»; i i l i l>i;» .Ill__ i l l i f 1ir j . - J r n » „ l i it «t k k ii« ilt 1)1 ib lit ik ib iJt fit ik * >lt m • I I K . I * » *r»» I » « K P • »» r»i. * - i * iim.» M 0 J « FlguVe 17. Mass spectrum (GCMS) of N, a-dimethyl -N-formyl- Y -phenyl benzenepropanamine (63) - 85 ->!«..».4. t m * • MMI « «• CM.. • II l l i n . * | « 4 | 7 C * to to * to to to to to to to to to-I 1 M J * 1 4 4 €*•• *» RMkCC • 4 » C M . • I I tt«T. -> I M I T il • .J.L-|-Ji'..-iJ •-* •* * * * ,n ...ii •fc -A •*• a fr I" i l l l l , m . ..II *: Figure 18(a) Mass spectrum (GCMS) of 2 - ( N-formyl)-4,4-diphenyl -5-heptanone (31) ( i d e n t i c a l to o x a z i r i d i n e (5) and the methadone metabolite m/z 72 Figure 18(b) Mass s p e c t r a l fragmentation o f methadone formamide - 87 -The m/z 253 fragment then leads to a l l ions present i n the r e c i p a v r i n spectrum. The base peak at m/z 72 or 73 can be r a t i o n a l i z e d on the basis of s c i s s i o n of the carbonyl group of the formamide. While Y s c i s s i o n of carbonyl groups with or without proton rearrangement does not normally produce intense peaks (85), the f a c t that m/z 178, 180 fragments are of high i n t e n s i t y in a l l compounds s t u d i e d , i n d i c a t e s t h a t , f o r s t e r i c or e l e c t r o n i c reasons, the phenyl rings d i r e c t fragmentation at t h i s bond. Gamma cleavage of the ethyl group of sec-butylacetamide (86) by t h i s mechanism gives r i s e to a 30% r e l a t i v e i n t e n s i t y peak at m/z 86. In the methadone formamide, t h i s bond i s y to both carbonyl groups and may be doubly a c t i v a t e d . The methadone ethyl ketone chain does not normally give r i s e to McLafferty g-cleavage (66), f u r t h e r s u b s t a n t i a t i n g t h i s s c i s s i o n pathway. D. NITRONES i ) Attempted synthesis of the methadone n i t r o n e v i a the primary  hydroxylamine (47) Since the methylene ni t r o n e (2J was proposed by Kang as a p l a u s i b l e s t r u c t u r e f o r the unknown metabolite of methadone, several attempts to synthesize i t , using the diketone (45) as a s t a r t i n g material were t r i e d . The pathway o u t l i n e d e a r l i e r v i a the oxime (46), and hydroxylamine (47) to the n i t r o n e (2_) appeared most promising. - 88 -CH,- C=N- OH C H r C=N- OH 52 syn 52 a n t i CHj-C=NOH 2.0s \ / / 3 .3bs CH3 1.8bs 45 46 NMR (80MHz) r e s u l t s showed broad bands which when compared with the NMR of syn and anti r e c i p a v r i n oximes (j>2) and diketone (45), support the keto oxime s t r u c t u r e (46). The i n f r a r e d spectrum of the keto oxime (46) had a strong C=N s t r e t c h at 1620 cm - 1, a broad i n t e r m o l e c u l a r hydrogen bonded OH band at 3200 cm - 1 and medium bands at 1025 and 950 cm - 1, i n common wi t h the r e c i p a v r i n oxime (52). The C=0 doublet as seen i n the i n f r a r e d of the diketone remained as a s i n g l e peak at 1710 cm"1 i n the keto oxime. In the mass spectrum of the oxime (65), the a l k y l fragment m/z 42 (100%) i s also found i n the diphenyl butanone oxime mass spectrum ( n e i t h e r compound - 89 -has the T protons required f o r a McLafferty rearrangement) and m/z 29, 57 and 239, o - s c i s s i o n products of an ethyl ketone are evident. The highest mass evident i s 279 (M+-16), and the s e r i e s m/z 206, 191, 128, 115, 91 analogous to the 208 s e r i e s of EMDP-like compounds are prominent. The high mass fragments (279, 261, 246, 206) may a r i s e from d e a l k y l a t i o n of p y r o l l i d i n e type fragments. The reduction of the keto oxime was attempted using sodium cyanoborohydride at c o n t r o l l e d pH. One equivalent of reducing agent should have s e l e c t i v e l y reduced the oxime at pH 5, however complete conversion of the s t a r t i n g material required two moles of reducing agent suggesting concomittant reduction of the carbonyl group. A NMR t r i p l e t at 4.04 ppm (-CHJ0H)-) of the crude product i n d i c a t e d that both groups were reduced. The product i s thought to be the dinormethadol primary hydroxylamine (47). Subsequent re a c t i o n s with formaldehyde to produce a n i t r o n e gave r i s e to mixtures of products by GCMS and were not pursued. I t i s p o s s i b l e that the methadol n i t r o n e (6>5) or i t s acetal could be i s o l a t e d using t h i s procedure. 47 65 - 90 -i i . Attempted synt h e s i s of the methadone n i t r o n e v i a the secondary  hydroxyl amine (3_) The attempted synthesis of the methadone secondary hydroxylamine ( 3 J as a precursor of the methadone n i t r o n e , by re d u c t i v e N-methyl hydroxyl-amination of the diketone gave products, which were shown by i n f a r e d spectrometry to have l o s t the ketone f u n c t i o n a l group. The mass spectrum resembled that of EDDP i n that l o s s of a phenyl r i n g i s a major fragmentation pathway (66). However, the peaks were one mass u n i t l e s s and had d i f f e r e n t r e l a t i v e i n t e n s i t i e s ( i . e . ) m/z 276 (5%) and 199 (100%) versus 277 (M + 100%) and 200 (15%) f o r EDDP. The even numbered high mass fragment implies that m/z 276 i s not the molecular i o n . I t i s p o s s i b l e t h a t the compound was the methadol secondary hydroxyl amine (J66). Because of the low y i e l d of the diketone and because y e l l o w mercuric oxide o x i d a t i o n f a i l e d to give the desired keto product, t h i s s y n t h e t i c pathway was not pursued. Further work should give access to the methadol n i t r o n e . I t w i l l be important not to r e l y h e a v i l y on GC a n a l y s i s since the intermediates are unstable and GCMS r e s u l t s are misleading. i i i . Other attempts t o obta i n the methadone n i t r o n e Thermal i s o m e r i z a t i o n of the methadone o x a z i r i d i n e (j>) under nitrogen in xylene (the b o i l i n g point of xylene approximates the temperature at which the methadone o x a z i r i d i n e v i s i b l y decomposes) gave the formamide (68) as a major product, plus a t r a c e of material at short r e t e n t i o n time which, by c C H 5 - C H - N — CH 3 I I CH, OH 66 - 91 -GCMS had a molecular weight of 309 a.m.u. with a base peak at m/z 222 (M +-87). This product was minor, and was not i s o l a t e d . Flushing the f l a s h chromatography column with methanol followed by NMR a n a l y s i s of the concentrated eluate f a i l e d to detect any n i t r o n e type compounds. Treatment of crude o x a z i r i d i n e thermal i s o m e r i z a t i o n products with methyl a c r y l a t e , i n an attempt to trap any n i t r o n e produced as an isoxazol i d i n e 1,3 a d d i t i o n product (57) did give r i s e to a minor long r e t e n t i o n time product. No molecular ion was present i n the mass spectrum and the product was not f u r t h e r c h a r a c t e r i z e d . i v ) Synthesis of the r e c i p a v r i n n i t r o n e (13) The r e c i p a v r i n n i t r o n e was synthesized i n good y i e l d from the corresponding primary hydroxylamine (55_). The polar product (Rf 0.05 by TLC i n ethyl acetate) was p u r i f i e d by f l a s h chromatography and examined by NMR IR, UV, GC and LCMS. a) NMR Spectra The *H NMR spectrum was comparable to r e s u l t s obtained f o r N - ( l - ( 3 ' ,4'-dimethoxyphenyl) prop-2-yl n i t r o n e (J57) (64) as shown i n Figure 19. The methylene protons of the nitrones are well downfield i n accord w i t h t h e i r o l e f i n i c nature. Any d i f f e r e n c e in the chemical s h i f t of the methine proton can be a t t r i b u t e d to the i n s e r t i o n of an extra carbon between i t and the aromatic portion of the molecule . No comparable l i t e r a t u r e values f o r 1 3C NMR were a v a i l a b l e , however the l a r g e downfield s h i f t of the azomethine carbon (122.29 ppm) r e l a t i v e to 72 ppm of the o x a z i r i d i n e r i n g carbon i s i n accord with the o l e f i n i c nature of t h i s atom. SFORD revealed that t h i s was a broad t r i p l e t , as in the r e c i p a v r i n o x a z i r i d i n e (14). - 92 -(a) 3.96dd / H 3.79m 5 9 8 d C \ Ha \ \ \ • . I H B 2.23m 2.74m 13 ,42d (a) O C H 3 O C H 3 2.73 3.18 0 H a / / 6.24 « / X H GH 3 5.95m <\ ^ 1.47d J 8 H z (b) 47. 74d 67 122.29t Figure 19(a) Comparison of iH NMR r e s u l t s f o r r e c i p a v r i n n i t r o n e (13) with a l i t e r a t u r e compound (67) (b) 1 3C NMR of the r e c i p a v r i n n i t r o n e J a. OEM 2«» J U U U Zoca l(MO 3.96dd / H 3.79m. 5 > 9 8 d Aid J=8 2.23m 2.74m ,42d 5 4 3 2 Figure 19 ( c) 400 MHz *H NMR of a-methyl-(N-methyl ene)-T -phenyl benzenepropanamine N-oxide (13) ——rmr M / 1 VM : LB .1 I HJO SO 1500 . ' U U U I U U 0 , A7.7Ad »,C J 39.Alt J J J 122. 29t 0 CH<— 69.03d | ^,19.92q CH 3 r 180 170 160 150 1A0 130 120 110 100 90 80 70 60 Figure 19 (d) SFORD 400 MHz 1 3C NMR of a-methyl-(N-methylene) 50 An i n 10 Y-phenyl benzenepropanamine N-oxide (13) Figure 20. Infrared spectrum (f i lm) of a-methyl-(N-methylene) -T-phenyl benzenepropanamine N-oxide (13) - 96 -b) Infrared and UV spectra The i n f r a r e d spectra (Figure 20) also compared well with Beckett's n i t r o n e (67) i n that they both had a C=N s t r e t c h at 1566 cm - 1. UV spectra revealed an absorption at 233 my ( e= 8160) as compared to 235 my ( e= 6210) f o r compound (67) probably a ir—ir* t r a n s i t i o n of the azomethine bond. Additonal bands were observed at 282 my( e = 2170) and 340 my ( e=870). c) GCMS and mass spectra The n i t r o n e decomposed during GCMS to produce three breakdown products. The major compound was the imine (84), with l e s s e r amounts of the oxime (79) and l a s t l y , a small amount of the formamide (15J. This c o r r e l a t e s well with work by Beckett on the methylene ni t r o n e of N-methyl-3 ',4'- dimethoxy-amphetamine (67) which, on GLC a n a l y s i s "behaved anomalously and produced an oxime and an u n i d e n t i f i e d product". The formamide i n our case i s not always evident as a sharp peak, unless a f r e s h l y packed column i s used, sometimes i t i s necessary to monitor the m/z 73 and 253 peaks to detect i t . The is o m e r i z a t i o n to the amide may not take place i n Beckett's case since the more v o l a t i l e amphetamine d e r i v a t i v e s r e q u i r e GLC column temperatures between 100 and 200°. This i s much l e s s than the temperatures required to el u t e or produce the r e c i p a v r i n formamide. The imine i s the deoxygenated product of the nitr o n e ( i n accord with l i t e r a t u r e reports on thermal deoxygenation (61)). L i t e r a t u r e precedent f o r thermal production of the oxime e x i s t s only i n Becketts work (67). The absence of the former two components i n the GCMS of conjugated r e c i p a v r i n metabolites of rat b i l e w i l l be demonstrated i n the metabolism s e c t i o n , as evidence against the nitr o n e as the s t r u c t u r e of the metabolite. The s y n t h e t i c n i t r o n e chromatographed at short r e t e n t i o n time by reversed phase HPLC, and LCMS revealed the - 97 -M M * • • « • .• I 1**7 n **—s ft a ft; ft" !!:"l.!!.J ... . i.i'i;.<* m } ro ••IB! . I C I It* -tt tt iii —I*—•Hi1'' m—m—in i i i — I F — * — * — * — * • s , t c - 4 c » i t . j « ?4 *("•«• • * %m** • • « r » L . • «* l i m . * 11**7 i ^ - J - i l l m v * " * * ^ T ^ " * * * * * * * * * * * * * * " Figure 21(a) Mass spectrum ( d i r e c t i n l e t ) of a-methyl-(N-methylene) -Y-phenyl-benzenepropanamine N-oxide (13) - 98 -H c H f » t ' < C — CH L H : I CH 3 m/z 253 0 CH2NOH -(C 6H 5) 2CH m/z 167 -0 CH, CH N — CH„ + 2 m/z 56 C 6H 5 C 6H 5 C SH 5 C 6H 5 4s m v /z 208 x 4 ^ C H , m/z 236 C 6H m/z 180 CH 2 N -CH CH^ m/z 73 - 0 * C H 2 = r N CH 2 CH 3 m/z 57 m/z 193, 130, 115, 91 series Figure 21 ( b) Fragmentation pathways of the r e c i p a v r i n n i t r o n e (13) by analogy to Coutts et al (57) - 99 -M ++l ion at m/z 254. The d i r e c t i n l e t mass spectrum had strong peaks at m/z 56 and m/z 91 (C6H5 - C H 2 ) + c o n s i s t e n t with the d i r e c t i n l e t mass spectrum of a-methyl- (N-methylidene) benzene ethanamine N-oxide reported by Coutts et a l . The M+-15 (m/z 238) fragment was l e s s important i n the mass spectrum of the r e c i p a v r i n n i t r o n e but a prominent M+-17 (m/z 236) was present. As in the o x a z i r i d i n e , the m/z 208 s e r i e s of ions was prominent. Based on the mass spectra of deuterated methamphetamine n i t r o n e s , the fragmentation pathway proposed by Coutts applies to the r e c i p a v r i n n i t r o n e (Figure 21a). As the d i r e c t probe of the mass spectrometer i s heated, the ions c h a r a c t e r i s t i c of the formamide become prominent in the d i r e c t i n l e t mass spectrum of both the r e c i p a v r i n nitrone and o x a z i r i d i n e . In l i g h t of the occurrence of m/z 56 and 42 fragments of the d i r e c t probe mass spectrum of the methadone o x a z i r i d i n e which appear p r i o r to heating the probe, the evidence here supports i s o m e r i z a t i o n of o x a z i r i d i n e s to nitrones at low temperatures and of the o x a z i r i d i n e s to formamides as the source i s heated, s i m i l a r to r e s u l t s reported by St. C l a i r e Black (82). E. KETONES The ketones 1,1-diphenyl-2-butanone (42) (CAS 6336-52-3), 1,1-diphenyl-3-butanone (51) (CAS 5409-60-9) and 4,4-diphenyl-2,5-heptanedione (45) (the l a t t e r described by Kang as a byproduct i n the peroxidation of EDDP) were made f o r use as s y n t h e t i c intermediates. Compounds (4j>) and (51_) were c h a r a c t e r i z e d in d e t a i l as they are p o t e n t i a l metabolites of methadone and r e c i p a v r i n r e s p e c t i v e l y . i ) NMR Spectra !H NMR r e s u l t s , presented i n Figure 22 are i n accord with the ketone f u n c t i o n a l groups present. Of a l l the compounds s t u d i e d , only the ketones Figure 22(a) 80 MHz L H NMR of 4,4-diphenyl-2,5-heptanedione (45) - 101 -- 102 -and oximes (with double bonds) had equivalent chemical s h i f t s f o r the methylene group a- to the methyl ketone. The 20 MHz 1 3C NMR spectrum of compound (45) ( r e s u l t s shown i n Figure 22c) supports the diketone s t r u c t u r e when compared to r e s u l t s f o r the methadone o x a z i r i d i n e and the chemical s h i f t s of a l i p h a t i c ketones. The carbonyl carbons were not observed, s i n c e they lack nuclear overhauser enhancement and were l o s t in the b a s e l i n e . i i ) I n f r ared spectra In the i n f r a r e d spectra both ketones exhibited strong C=0 s t r e t c h e s , the dione as a doublet at 1710 cm"1 and 1695 cm"1 (Figure 23) and the 3-butanone as a s i n g l e peak at 1710 cm"1. i i i ) Mass spectra Mass spectra showed weak molecular ions (m/z 224, M+) plus a - s c i s s i o n ions of the carbonyl group at m/z 43 and 57 i n diphenylbutanones (51) and (42) r e s p e c t i v e l y . Both these ions plus weak M + -18 s e r i e s peaks occurri n g at m/z 262, 223, 206, were present in the diketone. The mass sp e c t r a l fragmentation pathways are o u t l i n e d i n Figure (24). i v ) Problems i n the diketone synthesis Much time was spent on the s y n t h e t i c pathway s t a r t i n g from diphenyl a c e t o n i t r i l e to the lactone (4_1), d i o l (43_) and diketone (45). A l l these compounds except the diketone were i n v e s t i g a t e d i n the 1950's and l i t t l e data besides combustion a n a l y s i s was a v a i l a b l e f o r them. The lactone was obtained i n good y i e l d v i a the in s i t u acid h y d r o l y s i s of the THF imine (40) as described by Attenburrow et al (73). NMR r e s u l t s are summarized i n Figure 25. The i n f r a r e d had a strong S -lactone C=0 s t r e t c h at 1750 cm-1 and the mass spectrum showed a weak 4dbo Hi jJoo •JLo T T T 16 I 2 9 • 3 4 I 9 I • t I I 7 I 7 a t 8 I 3 1 7 1 8 4 6 . 9 2 9 8 1 . 1 2 9 6 4 . 8 2 5 4 1 . 5 I 9 7 1 . 4 I 9 4 I . I 1 9 * 9 . 2 I 3 7 8 . 4 1 0 9 2 . I • 6 0 . • • 2 4 . 8 1 7 6 . 0 0 . 0 1 4 2 I 2 * I 2 8 I 27 7 8 7 7 7 8 • 1 8 2 3 1 31 8 0 . 1 4 . I • . 2 3 . 0 7 . • 7 . 0 9 . 4 9 . 9 2 . 6 0 . 0 4 . 24 . 8 0 . 0 0 I • I • I • I • I • I 1 31-24 8 < 8 CH,CH, CHj—C = 0 33.04 CDC1 J L J L I ' I ' I 1 T - T ' M i ' T - r - r ' I 1 I ' I 1 I 1 I 200 1 90 180 170 1 60 150 14 0 1 30 1 20 110 100 90 80 70 60 50 40 30 20 10 0 Figure 22 (c) Broadband decoupled 20 MHz 1 3 C NMR of 4,4-diphenyl-2,5-heptanedione (45) Wav«numbei Figure 23. Infrared spectrum (nujol mull) of 4,4-diphenyl-2,5-heptanedione (45) - 105 -<-6n5 M* 224 (4) 51 C 6H 5 M T 224 (5) 42 C 6 H 5 ^ / C H 2 CH 3 m/z 43 (90) C 6 H 5 " 4 ^m/z 181 (35) 0 + -R mfe 57 (20) •R' 167 .(100) V 165 (35) I 152 (25) + 167 (100) 6 n 5 N x ^ g n 5 i H 165 (25) 152 (15) C 6H 5 C 6H 5 0 M + 280 (not observed) 45 m/z 262 m/z 57 (15) m/z 43 (100) m/z 223 (12) C f i H s ^ ^ \5H C 6H 5 v , C 6H 5 m/z 206 (8) 167 m/z "j Figure 24. E lec t ron impact mass fragmentography of the ketones (42) (45) and (51) - 106 -2.6dd l.5d 41 3.92q CH CH, l.38d CH, CH A. 12m CH, 1.68d 0.97t 4.38dd ^3.72m CH2—CH-OH 1.12d Figure 25. NMR r e s u l t s f o r the diketone s y n t h e t i c pathway - 107 -/z 207 (32) m/z 129 (100) m/z 167 Figure 26(a) E l e c t r o n impact mass fragmentography of the THF imine - 108 -molecular ion at m/z 252 (8%) which loses CO2 and fragments v i a the m/z 208 s e r i e s described e a r l i e r . In the intermediate THF imine (40) hydrogen bonding (NH) i n the 3400 cm - 1 region and a C=N s t r e t c h at 1670 cm"1 were present i n the i n f r a r e d spectrum. The intermediate THF imine (70) had the mass sp e c t r a l fragmentation pathway o u t l i n e d in Figure (26a). The ions at m/z 207 and 129 only occur i n compounds which appear to e l i m i n a t e nitrogen to form a double bond in the amino side chain. This pathway i s also important i n the fragmentation of 4,4-diphenyl-5-hept-2-enone (39) and the methadone formamide (3_1_). The m/z 207, 129 s e r i e s i s also d i a g n o s t i c of the Cope e l i m i n a t i o n products of t e r t i a r y N-oxide metabolites, which a r i s e i n the GC during the a n a l y s i s of these l a b i l e compounds (Figure 26b). Figure 26 ( b ) . Compounds which fragment v i a the m/z 207, 129 s e r i e s . - 109 -Reaction of the lactone (41J w i t h one equivalent o f EtMgBr was expected to g i v e the l a c t o l (68) as described by Wilson ( 7 1 ) . Two equivalents o f EtMgBr were required f o r the complete conversion of the lactone to a product c o r r e c t l y ascribed the d i o l s t r u c t u r e (43) by Craig et al ( 8 7 ) . The ethyl idene tetrahydrofuran (44) was a major byproduct ( 1 0 % ) . The d i o l e x h i b i t e d a strong broad 0-H i n t r a m o l e c u l a r hydrogen bonding band at 3200 cm - 1 and a C-0 s t r e t c h c h a r a c t e r i s t i c of saturated secondary a l c o h o l s at 1120 cm - 1. No l a c t o l (68) was obtained. In the mass spectrometer the d i o l fragmented v i a the 208 (base peak) s e r i e s , with a minor pathway v i a the e t h y l i d e n e tetrahydrofuran (M +-20) as o u t l i n e d i n Figure 25. The major fragmentation pathway of the e t h y l i d e n e THF (44) (base peak M + 264) i s l i k e l y v i a the open chain compounds A and B ( F i g u r e 26c) s i n c e the fragments m/z 43, 57 and the 207 s e r i e s are r e l a t i v e l y important. Attempts to o x i d i z e the d i o l with Jones reagent gave very low y i e l d s (5%) and required a tedious f r a c t i o n a l c r y s t a l l i z a t i o n procedure to p u r i f y the minor product. The major product was the lactone (41). Syntheses using p y r i d i n i u m chiorochromate, sodium dichromate, and a number of other oxidants were a l l u n s u c c e s s f u l . An attempt to synthesize the o x a z i r i d i n e intermediate (69) by r e a c t i n g the diketone (45) w i t h methyl amine i n methanol produced a major product which by GCMS was assigned the d i h y d r o p y r o l l e s t r u c t u r e (70), based on i t s s i m i l a r i t y to the fragmentation of EDDP (66). ( F i g u r e 26d) 69 70 - 110 -C 6 H 5 £2^ > C 6 H 5 m/z 282 (not observed) m/z 264 d i o l (5%) ene (100%) As-m/z 249 d i o l (< 1) ene (12) CcH 6 n 5 C 6 H 5 S C 3 H 5 O C 6 H 5 C 6 H 5 ~ 2 64 d i o l (5) ene (100) C 6 H CrH in/z 43 C 2 H 3 G 6 n 5 m/z 57 (15) C 6 H 5 C 6 H 5 m/z 129 (20) /z 207(20^1 C 6 H 5 0 0 + m/z 249 ene (10) m/z 222 v m/z 208 d i o l (100%) ene (5%) I m/z 235 m/z 193, 130, 115, 91 Figure 26 (c) Fragmentation of diol (£3) and ethyl idene tetrahydrofuran (44) - I l l -m/z 122 (18) Figure 26 (d) Fragmentation of 2-ethylidene-1,4-dimethyl-3,3-diphenyl-2 , 3 - d i h y d r o p y r o l l e (70) compared to EDDP (66) - 112 -F. OXIMES The oximes (52) and (64) were synthesized i n good y i e l d from 1,1-diphenyl-3-butanone. NMR and IR r e s u l t s f o r (52) have already been discussed by comparison with the keto oxime ( 4 6 ) . L i k e the oxime (52) the NMR of the 0-methyloxime ( 6 4 ) , (isomeric w i t h the formamides, nitrones and o x a z i r i d i n e s ) shows a 2:1 mixture of syn and anti isomers (shown below). The mass spectra are f a i r l y simple, since no Y hydrogens are present to allow McLafferty rearrangement. Both oximes have molecular ions and e x h i b i t g-cleavage to give the diphenyl methane base peak m/z 167 (100%) with a m/z 42 (10%) a l k y l fragment present. Alpha-cleavage i s a minor pathway i n both compounds g i v i n g ions at m/z 181 and 103 ( 1 0 % ) . The m/z 207 and 220, 206 s e r i e s (a major pathway in the keto oxime (46) fragmentation pathway) are present to the extent of 5% i n (64) and (52) r e s p e c t i v e l y . 3s 64 syn 64 a n t i G. AMINES The amines, r e c i p a v r i n (6J (CAS 13957-55-6), n o r r e c i p a v r i n (54) (CAS 29869-78-1) and d i n o r r e c i p a v r i n (53) (CAS 29869-77-0) were synthesized i n good y i e l d , from methadone n i t r i l e i n the case of r e c i p a v r i n ( 7 4 ) , or by r e d u c t i v e amination of 1,1-diphenyl-3-butanone (51_) i n the l a t t e r two cases. NMR r e s u l t s f o r the primary and secondary amines are o u t l i n e d i n the - 113 -f o l l o w i n g s e c t i o n . The NMR r e s u l t s f o r r e c i p a v r i n compare fa v o r a b l y with those obtained by Beckett and Casy (88). Mass spectra e x h i b i t base peaks at m/z 72, 58, and 44 r e s u l t i n g from a-cleavage of the t e r t i a r y , secondary and primary amines r e s p e c t i v e l y . Weak molecular i o n s , diphenylalkane residues and evidence of the m/z 208 s e r i e s were also present to a minor extent. 2.0-2.5m 0.9d 6 H. HYDROXYLAMINES The hydroxyl amines (55_) and (_57_) were obtained by red u c t i v e hydroxyl-amination or r e d u c t i v e N-methylhydroxylamination of diphenyl butanone (51). They are both p o t e n t i a l metabolites and s y n t h e t i c precursors of the methylene n i t r o n e . The NMR r e s u l t s shown below (Fig u r e 27) show the d e s h i e l d i n g e f f e c t s of the hydroxyl group on one of the a-methylene protons, and on the amino methine proton. A s i m i l a r downfield s h i f t of 0.2 ppm was observed f o r the a-methyl ene and N-methyl protons of the secondary hydroxylamine. - 114 -54 2. 6m l.ld 57 1.2s dNcH-—CH «-2.8m t I 2.06m CH3 l . l d 53 4.06t H 2.9m OH 2.4m 1.96m CH, 1 .Id 55 H 1.3b Figure 27. Comparison of NMR results for the primary and secondary hydroxylamines and the corresponding amines. In the infrared spectrum of the hydroxylamines the weak N-H stretch band at 3300 cm - 1 of the secondary amine i s replaced by a strong intermol ecular ly hydrogen bonded 0-H stretch at 3150 cm" 1 . Two C-N stretches occurring between 1200 and 1000 cm" 1 ( character is t i c of secondary a l ipha t i c amines) are also present in the hydroxylamines. A medium intens i ty doublet at 1220 and 1190 cm - 1 could be a N-0 stretch s im i l a r to that of an a l iphat i c nitroso dimer. Morgan and Beckett (64), who have synthesized the corresponding amphetamine secondary hydroxylamines, - 115 -c h a r a c t e r i z e d oxalate s a l t s of these compounds, making comparison to t h e i r data d i f f i c u l t . ° The secondary hydroxylamines decomposed i n the GC i n l e t , to the corresponding amines. When d e r i v a t i z e d with BSTFA, each showed a weak molecular ion (m/z 327 and 313 r e s p e c t i v e l y ) and base peaks at m/z 146 and 132 r e s p e c t i v e l y a r i s i n g from a-cleavage of the -N(R)-0TMS group. This i s c h a r a c t e r i s t i c of a l i p h a t i c hydroxylamines (53). These ions were used to search f o r any hydroxylamine metabolites i n d e r i v a t i z e d b i l e e x t r a c t s . I. SYNTHESIS OF THE IMINE (59) The imine (59) as noted p r e v i o u s l y , probably e x i s t s as the t r i a z e n e (60). A weak molecular ion at m/z 237 (monomer) appeared to fragment by a McLafferty rearrangement to the base peak at m/z 57 as o u t l i n e d below. Minor amounts of a-cleavage to m/z 222 leading to the m/z 208 s e r i e s was observed. J . SYNTHESIS OF THE NITR0S0 COMPOUND Treatment of the primary amine (53) w i t h MCPBA produced a th e r m o l a b i l e n i t r o s o compound (61) which by i n f r a r e d spectrometry was c o n s i s t e n t with a dimeric s t r u c t u r e . - 116 -As i n the r e c i p a v r i n n i t r o n e , N-methine proton was deshielded (3.3 ppm), s i g n i f y i n g an e l e c t r o n withdrawing s u b s t i t u e n t . A sharp i n f r a r e d band at 1380 cm"1 w i t h a shoulder at 1365, i s c h a r a c t e r i s t i c of a l i p h a t i c c i s - n i t r o s o dimers. l.Ad 61 K. PEROXIDATION OF EMDP Treatment of EMDP base with 2 moles of MCPBA gave r i s e to 5 major products by GCMS. The l a s t three peaks eluted from the GC column may be i d e n t i f i e d based on the mechanistic schemes of M i l l i e t et a l (78). Peak th r e e (Appendix, page 163) i s l i k e l y 4-methyl-2,2-diphenyl p y r o l l i d o n e (71). The s t r u c t u r e proposed i s based on i t s mass spectral s i m i l a r i t y to DDP (12) since i t has a strong molecular ion (M"*251) which fragments by the m/z 208 s e r i e s . The l a s t two peaks, appear as a po t e n t i a l methyl ketone, and an ethyl ketone as shown by base peaks at m/z 43 and 57 r e s p e c t i v e l y . Knowing that thermal i somen z a t i o n o f o x a z i r i d i n e s to amides i n the GC i n l e t i s p o s s i b l e , a l l o w s us to p r e d i c t the EMDP o x a z i r i d i n e s t r u c t u r e s (72_), (73J f o r peak 4 and 5 based on t h e i r long r e t e n t i o n time and the observed molecular ions ( F i g u r e 28) . These compounds could form the ketones (74J and (75) i n the GC and fragment by a - s c i s s i o n i n the mass spectrometer since no T protons are present. - 117 -113 M* 277 114 Mt 279 -(C 6H 5) 2CH t m/z 234 -OH + m/z 43 (100%) m/z 250 m /z 112 Figure 28. Thermal rearrangement and fragmentation of the EMDP o x a z i r i d i n e s (72) and (73) v i a the ketones (74) and (75) - 118 -2. METABOLISM A. METHADONE METABOLISM The GCMS experiments performed by Kang demonstrating the formamide i n the conjugated f r a c t i o n of b i l e from ^ H \ Q - a r , d * H10~ methadone dosed rats were repeated. The spectra of the formamide metabolites observed at the c o r r e c t r e t e n t i o n time are shown i n Figure 29a and b. The formamide was also formed to a minor extent i n v i t r o as shown by the mass spectrum (Figure 29c) and the t o t a l ion current trace i n Figure 30. Two other u n i d e n t i f i e d minor metabolites ( i n c l u d i n g a compound resembling a methadone nonanone analogue non conjugated metabolite) as well as EDDP, EMDP and DDP were also observed i n the i n v i t r o experiments. The i n v i t r o metabolism of EDDP produced EMDP and DDP but no detectable formamide. B. RECIPAVRIN METABOLISM i ) In V i t r o Metabolism Both nor- and dinor- r e c i p a v r i n were present as i n v i t r o metabolites but were not completely resolved by packed column GLC ( F i g u r e 31). Mass spectra of the p a r t i a l l y resolved secondary amine metabolite (M + m/z 239) and of the overlapping t e r t i a r y secondary and primary amine metabolites are included i n the Appendix. The peaks at longer r e t e n t i o n time than the parent drug are l i k e l y the phenol (76_) and catechol (77_) plus an u n i d e n t i f i e d compound (78) . D e r i v a t i z a t i o n and CI MS were not performed so these proposals are made s o l e l y on the basis of the m/z 72 base peak which i n d i c a t e s that the t e r t i a r y amino group i s i n t a c t . Compounds (77_) and (78) both had detectable m/z 269 i o n s , corresponding to the molecular ions of 72 ,44 253 178 100 207 150 f i - V - t - l I J I I l I I J ?00 250 300 Figure 29(a) Mass spectrum ( G C M S ) of the methadone formamide metabolite in the conjugated f r a c t i o n of b i l e from a methadone dosed rat - 120 -i ii h i " : !:l Hi I | !|;.,„m„lj lii i . l i . i j.lllllii 11 I" ill HIIHiil in lim nil ' i f f ? Tf ii i! r ii iii 7> * jr. . i T t r i t 9\%tM* r:.r>I LAkht ;:,*] I It *N > - M i l . 11 r t fit fl* * •* * * * * I ib •> A * *-Figure 29 (b) Mass spectrum (GCMS) o f the 2 H I Q methadone formamide me t a b o l i t e from the conjugated f r a c t i o n o f b i l e from a methadone dosed rat - 121 -...ti*.:i . - • • • r : - T _ I E M R I . • - • • T : . r * » • « ; . « : . **1 207 253 j j r 7 5 0 300 •• ^2 44 1 4- 11 fr3 129 - * * ± ft rr-207 167 129 ! Ml 150 200 50 100 Figure 29 (c) Mass s p e c t r i n (GCMS) o f the methadone formamide met a b o l i t e generated i n v i t r o - 122 -EDDP METHADONE l Y r- r f . . 1....I• I I . . I I I I I I I I I i P t T i 1 1 1 1 r r r i 1 1 1 1 1 1 1 cA. Scan 100 200 300 400 500 600 Figure 30. TIC o f i n v i t r o methadone metabolic e x t r a c t GC c o n d i t i o n s ( g ) w i t h 150° isothermal f o r 10 minutes before temperature programing a t 2 /minute to 280 - 1 2 3 -Scan 31. GCMS of an i n v i t r o r e c i p a v r i n metabolic e x t r a c t showing m e t a b o l i t e s (76) (77 ) (78) GC c o n d i t i o n s ( g ) w i t h 150° isothermal f o r 10 minutes before temperature programing - 1 2 4 -phenolic metabolites ( a l l metabolite mass spectra are included i n the Appendix). i i ) R e c i p a v r i n i n vivo metabolism a) Non conjugated metabolites The mass chromatograms i n Figure 32 demonstrate the presence of the de s a l k y l m e t a b o l i t e s nor- and d i n o r r e c i p a v r i n as for the in v i t r o experiments. The d e s a l k y l metabolites are not resolved by packed column GLC. No other metabolites were detected. b) R e c i p a v r i n conjugated m e t a b o l i t e s 1. D e t e c t i o n of the formamide metabolite One of the r i s k i e r hypotheses of t h i s t h e s i s was that r e c i p a v r i n would undergo metabolism to a metabolite analogous to that observed f o r methadone. For t u n a t e l y t h i s turned out to be the case. The experiment was repeated w i t h three separate r a t s and the fbrmamide was present at the c o r r e c t r e t e n t i o n time by GCMS a n a l y s i s of the conjugated f r a c t i o n i n a l l three cases (Figure 33a and b). OH HO 76 77 - 1 2 5 -Scan A/1 m/z 224 (Diphenylbutanone) (51) m/z 225 (Primary Amine) (53) m/z 253 (Recipavrin) (6) m/z 239 (Secondary Amine (54) I I and Oxime (52)) I UAt I I L 1 1 1 — I 60 120 F i g u r e 32. Mass chromatograms o f i o n s p r e s e n t i n the non c o n j u g a t e d f r a c t i o n o f b i l e from a r e c i p a v r i n dosed r a t , showing d e a l k y l and deaminated m e t a b o l i t e s (GC c o n d i t i o n s ( d ) ) - 1 2 6 -Figure 33(a) GCMS of the conjugated f r a c t i o n of b i l e from a r e c i p a v r i n dosed r a t (GC c o n d i t i o n s ( d \ i n j e c t i o n delayed 15 seconds) n •IMtl.M».l|. M l — * -50 1 1 -•1 n> t 1(1 n 1 i n i ,1, k II II i l w 1 r i l i hH - T S — * i c I ! .in 100 -nr nr-150 rtr 200 I INI* II r b k A A l h/iMi • All I Al . • IO I ( H I . r.? ........iii'. if I.r" ^ ••ij, i I, i • V' ..I,." _L. ..I I II I M I * 1*9 i fc 250 300 Figure 33(b) Mass spectrum (GCMS) of the rec ipavr in formamide metabolite in the conjugated f rac t ion of b i l e from a rec ipav r in dosed rat - 1 2 8 -The synthetic samples have demonstrated the minor d i f f e r e n c e s i n the mass spectra of the methadone and r e c i p a v r i n formamides. The long r e t e n t i o n time and thermal conversion of the precursor o x a z i r i d i n e s and nitrones to the formamides make them r e l a t i v e l y easy to detect even through present i n very small q u a n t i t i e s . The fact that r e c i p a v r i n i s metabol ized to a compound s i m i l a r to the metabolite observed by Kang supports the hypothesis that other diphenyl-2-aminobutane and diphenyl -2-ami no heptane r e l a t e d compounds, w i l l undergo the same metabolic pathway. LCMS experiments to deduce which thermolabile precursor was responsible f o r the formamide observed by GCMS were performed. LCMS o f the synthetic n i t r o n e , formamide and o x a z i r i d i n e (Figure 34 a-c) revealed that a M ++l base peak at m/z 254 occurred i n a l l three compounds with s i g n i f i c a n t m/z 238 (20%) fragments only o c c u r r i n g in the mass spectra of the nitr o n e and o x a z i r i d i n e . The nitrone was unique in that i t also had a strong m/z 74 ion (75%). The LCMS mass chromatograms show that both the nitr o n e and o x a z i r i d i n e decompose to the formamide on standing. Unfortunately, the small amount of t h i s metabolite i n r a t b i l e and the 2% s p l i t of sample entering the LCMS combined to put the metabolite below the l e v e l of d e t e c t i o n . A selected ion monitoring c a p i l l a r y GC chemical i o n i z a t i o n GCMS experiment on the conjugated metabolite f r a c t i o n of r e c i p a v r i n did i n d i c a t e t h a t i t was not the ni t r o n e . As shown in Figure (35a), monitoring the t o t a l ion current o f s i x r e p r e s e n t a t i v e ions r e s u l t e d in three peaks corresponding to the imine (59_) oxime (52J and formamide (15) , a l l decomposition products o f GC a n a l y s i s of the synthetic nitrone (13). When the same ions were monitored during the a n a l y s i s of r e c i p a v r i n - 1 2 9 -M » « M *•*> « M "•>• »«*->< e H T 1 1 • I • I ' I • fi 12 lb --«• n i t »i »» •« KJOTAVKIN NITRONE V*-97 238 \ i t ? . . . i s . \ _> LI 2 W . W 3 8 5 412 . V *8 •ft 2* 12 •29 -16 i t 14 ! i 1» w Figure 34(a) LCMS o f n i t r o n e (13J w i t h some formamide present as a decomposition product - 1 3 0 ->>i«- . i t Sc*r. 57* ] M4js.eE • i n , E H 85 N lib I3i 1J t 122 / 151 l «•*> .it" 1 4 * I t * 167" / T 176 / M 7 I S * • 1 ' 2*e 2r* 2Jtf> / Z9* 27* \ 24© 26* 2*€> : 4 e * * -1 S***-:;««<»•»• :e**w-?et>*-• * 2? •} .T-2S4 .? *a>u . I M P T P P 4*»« 1 1 . 11 1 1 1 1 1 ?e «4 i i " 4 32'' at ' 46 ' 44 ' 46 Figure 34. (b) LCMS o f formanide (15)) - 131 -_ „ t f « r « * * * *** «*.?• min. J J rs 113 1 » 1C7 i»» r > :»*>*>*-l ;••***-1 • » . « • - 3 * 6 * « * u 7 » 12 ' I T I 1 12 Figure 34. (c) LCMS o f o x a z i r i d i n e (14J - 1 3 2 -m e t a b o l i t e s , the formamide and oxime peaks were present i n roughly the r i g h t i n t e n s i t y , but the imine peak (formerly the l a r g e s t ) was v i r t u a l l y absent (Figure 35b). The imine (78J could account for the 7.4 minute peak i n Figure 35b since the metabolite does not decompose to the same products as the s y n t h e t i c methylene n i t r o n e . The oxime (52_) i s e i t h e r a metabolite o f r e c i p a v r i n or p o s s i b l y a decomposition product of the endo n i t r o n e ( 7 9 ) . The i n a b i l i t y to c o n c l u s i v e l y demonstrate the secondary hydroxylamine and the absence of the h y d r o l y s i s product, 1,1 - d i phenyl-3-butanone i n the conjugated f r a c t i o n does not favor the endo nitrone s t r u c t u r e . Attempts to detect the t e r t i a r y amide (63_) i n the conjugated f r a c t i o n were unsu c c e s s f u l . o 79 The r e c i p a v r i n m e t a b o l i t e s from the conjugated b i l e f r a c t i o n were d e r i v a t i z e d with BSTFA and observed by GCMS as shown in Figure 36. - 133 -U . I " »»*•*•«• 59 I » # » € > | no**-52 15 A i L i l TS ' >•'.• ' ««•• >»•• . • • • • • ' il>*>«»: 59 52 _15 v;» ' - i . i l . 1 1 • n.» " »*'•• '. 1 3 • • F i q u r e 35(a) C a p i l l a r y CI GCMS SIM o f t h e r e c i p a v r i n n i t r o n e showing t h e r m a l breakdown p r o d u c t s A ( i m i n e (59J), B (oxime (52J) and C (formamide (15J) (GC c o n d i t i o n s f i ) (b) SIM o f the same i o n s t o d e t e c t r e c i p a v r i n m e t a b o l i t e s from t h e c o n j u g a t e d f r a c t i o n o f r a t b i l e - 134 -72 M/Z - I 253 M/Z 1 i A I . I 1 1 I 1 I 1 60 120 Figure 36. Mass chromatograms showing me t a b o l i t e s i n the TMS d e r i v a t i z e d conjugated f r a c t i o n of b i l e from a r e c i p a v r i n dosed r a t (GC c o n d i t i o n s e) - 1 3 5 -Metabolite A i s l i k e l y a phenolic secondary amine, while B and C are the phenolic and catechol metabolites of r e c i p a v r i n with base peaks at m/z 72. 2) Hydroxylamines as precursors of the observed r e c i p a v r i n metabolite Beckett has observed that secondary hydroxylamines are converted to nitro n e s i n a l k a l i n e s o l u t i o n (54) . To determine whether o x i d a t i o n of the secondary hydroxylamine (57J during e x t r a c t i o n from a l k a l i n e s o l u t i o n could account f o r the observed m e t a b o l i t e , a sample of blank r a t b i l e was extracted and l y o p h i l i z e d by the normal procedure. A f t e r r e c o n s t i t u t i o n with water, 10 mg of secondary hydroxylamine (57_) (proven formamide and nitro n e f r e e by GCMS) was added p r i o r to b a s i f i c a t i o n and e x t r a c t i o n . GCMS of the e x t r a c t revealed the formamide (m/z 73,253) at the c o r r e c t r e t e n t i o n time. Oxidation to the nitrone had occurred to a minor extent with the bulk of the un d e r i v a t i z e d hydroxylamine chromatographing as the secondary amine (54) ( F i g u r e 37, m/z 239). The i m p l i c a t i o n i s that detectable amounts of precursor hydroxylamine should be i d e n t i f i a b l e i n the ra t b i l e e x t r a c t . In the case of methadone, GCMS a n a l y s i s of a methadone hydroxylamine should p r i m a r i l y give r i s e to EDDP or l e s s probably a secondary amine (base peak m/z 58 w i t h an intense 57 f o r the carbonyl fragment) n e i t h e r of which were detected i n the conjugated f r a c t i o n . D e r i v a t i z a t i o n of r e c i p a v r i n metabolites from the conjugated f r a c t i o n w i t h BSTFA d i d produce a compound with a m/z 146 peak ( c h a r a c t e r i s t i c of a TMS secondary hydroxylamine) that was not observed i n blank samples. However, the r e t e n t i o n time was d i f f e r e n t by a small but s i g n i f i c a n t amount i n c a p i l l a r y and packed column GCMS, as shown i n Figure 38. The experiment was performed by ion monitoring, thus no mass spectrum was a v a i l a b l e to compare to the sy n t h e t i c sample. - 136 -Scan Figure 37. GCMS o f the conjugated f r a c t i o n of blank rat b i l e spiked with the secondary hydroxyl amine (57J. Formamide present at scan (63J (GC condit ions(d)) Figure 38. SIM of the m/z 146 peak of the rec ipav r in 0-TMS secondary hydroxylamine (58) and the BSTFA de r i va t i zed r e c ipav r i n metabol i tes from the conjugated b i l e f r a c t i o n , showing s l i g h t l y d i f f e r en t retent ion time (GC conditions(d), onescan/second) - 138 -M CD to V B CM Figure 39(a) HPLC (UV de t e c t i o n ) of a mixture o f r e c i p a v r i n n i t r o n e (13) (17.2 min), formamide (15) (24.41 min) and o x a z i r i d i n e (IT) (40.87 min) Figure 39(b) HPLC o f secondary hydroxylamine (57J a f t e r treatment with a l k a l i , showing n i t r o n e (17.32 min) as a major product, with unreacted secondary hydroxylamine at 54.37 min (attenuated peak) - 139 -To ensure that the product o f a l k a l i n e o x i d a t i o n was the ni t r o n e and not the formamide or o x a z i r i d i n e , the secondary hydroxyl amine was treated fo r one week with ethanol i c NaOH, and then compared with HPLC reference standards of the r e c i p a v r i n n i t r o n e , formamide and o x a z i r d i n e . The chromatograms shown in Figure (39) i n d i c a t e that the ni t r o n e (tR, 17.32 minutes) i s the major ox i d a t i o n product, in accord the r e s u l t s of amphetamine hydroxyl amine o x i d a t i o n performed by Beckett and Bel anger (54). C. NORRECIPAVIN METABOLISM i ) Conjugated metabolites T h i s experiment was perfomed to see whether the secondary amine was also a source o f the formamide metabolite. Rats dosed with n o r r e c i p a v r i n also produced the formamide metabolite in the conjugated f r a c t i o n , (tp 11.4 minutes,GC conditions ( f i i ) ) a s shown by the mass chromatogram and mass spectrum in Figure 40a, b: Compounds resembling a conjugated primary amine (80), and secondary amine (8J_) phenolic m e t a b o l i t e s , the 2° amine catechol (82), a short r e t e n t i o n time oxime l i k e metabolite and two other u n i d e n t i f i e d compounds were also present. As i n Noda's experiments (43), the formamide metabolite of the secondary amine was present only at the l i m i t o f d e t e c t i o n . This r e s u l t may be due to an e r r o r i n the sample workup (the pH was i n c o r r e c t during h y d r o l y s i s with g-glucuronidase) and the experiment should be repeated. An a l t e r n a t i v e explanation f o r the decreased amount o f fbrmanide observed may be the r a p i d d e a l k y l a t i o n of the secondary amine to d i n o r r e c i p a v r i n , or a concerted mechanism for the metabolism of the t e r t i a r y amine to the formamide. The primary amine (53) was not administered to r a t s . The observations - 140 -i i) • • * • • •• * • • • • * •' • • 1 * 4 4 4 * » t > l ?••«»-_ 4 n • • • • • • m/z 253 —rr*—^ r».» "•»•• »»•• »*••• •••• r 11 * -t'.Ci tt • B e B 1 9 E 3 1 4 7 Ic »r. 4C3 I I . 3 7 t i n . ~1 ) J 3 l 171 V 1EE / 25.3 j l l j A i l j l l l . M l >*7 V'V 4 * F i g u r e 4 0 ( a ) m/z 2 5 3 mass chromatogram and mass s P ^ r £ ° ^ h * formamide m e t a b o l i t e from t h e c o n j u g a t e d f r a c t o n o f b i l e from a n o r r e c i p a v r i n dosed r a t (GC c o n d i t i o n s f n ) - 1 4 1 -of Noda e t a l ( 4 3 ) s u g g e s t t h a t a f o r m y l c o n j u g a t i o n pathway i s n o t a s o u r c e of the f o r m y l m e t a b o l i t e s of a m l n o p y r i n e . However, f o r m y l m e t a b o l i t e s a r e known to a r i s e as a r t i f a c t s d u r i n g a l k a l i n e workup by r e a c t i o n w i t h s o l v e n t c o n t a m i n a n t s . S t i l w e l l e t a l (52) have shown t h a t a f o r m y l m e t a b o l i t e of p e t h i d i n e , a r i s e s from c o n d e n s a t i o n of c h l o r o f o r m c o n t a m i n a n t s w i t h n o r p e t h i d i n e v i a a d i c h l o r o c a r b e n e i n t e r m e d i a t e ( F i g u r e 4 0 ) . O x i d a t i o n of the methyl group of p e t h i d i n e was r u l e d o u t f o l l o w i n g t h e a d m i n i s t r a t i o n of ^ Q ^ - N and 2H3C-N p e t h i d i n e when the formyl m e t a b o l i t e d i d not r e t a i n the l a b e l . Oddly enough, t o l u e n e , benzene and e t h y l a c e t a t e e x t r a c t i o n r e s u l t e d i n lower but s t i l l d e t e c t a b l e amounts of the same m e t a b o l i t e . The c o n j u g a t e d m e t a b o l i t e of methadone would have t o h y d r o l y z e to a p r i m a r y amine f o r t h i s pathway to be r e l e v a n t . In our case the p r i m a r y amine dinormethadone would c y c l i z e s p o n t a n e o u s l y to EMDP i n a l k a l i n e s o l u t i o n . F i g u r e 4 0 . P e t h i d i n e , i t s m e t a b o l i t e n o r p e t h i d i n e and a N-formyl compound which a r i s e s d u r i n g workup by r e a c t i o n w i t h c h l o r o f o r m c o n t a m i n a n t s . - 142 -E. METABOLISM OF THE METHADONE ANALOGUE The nonanone analogue of methadone (86) was a v a i l a b l e i n our l a b , so attempts to detect the corresponding formamide metabolite i n r a t b i l e and 1n v i t r o were undertaken, but were not c o n c l u s i v e . i ) Non Conjugated F r a c t i o n The major b i l i a r y m e t a b o l i t e , as i n methadone, a r i s e s from N-demethylation and c y c l i z a t i o n of the nor compound to 2 - b u t y l i d e n e - l , 5 --3,3-dimethyldiphenyl p y r o l l i d i n e (BDDP) (87) (peak C Fi g u r e 41), which i s chemically or m e t a b o l i c a l l y converted to DDP (12_) and BMDP (88) (peak A) BNDP had an i d e n t i c a l mass spectrum but d i f f e r e n t t ^ than EMDP ( 1 1 ) . 86 87_ 88 BDDP has a strong molecular ion (m/z 305) (20%) and fragments v i a the m/z 275 pathway o u t l i n e d i n Figure 42a. Fiqure 41. TIC of methadone analogue (86) non conjugated b i l i a r y metabolites (GC con d i t i o n s g). A 7 BMDP (.88), B Tunknown 315, m/z 291, 262. C. BDDP (87) D.Unknown E. Unknown M 315 (2%) m/z 72 (100). - 144 -C 6 H5 C 6 H 5 M T 305 (20) ~ C 6 H 5 m/z 290 m/z 276 (100) Figure 42a. Fragmentation of BDDP. A t e r t i a r y amine metabolite had a mass spectrum and tft i d e n t i c a l to r e c i p a v r i n (peak A Figure 41) but masses above m/z 73 are of low i n t e n s i t y and therefore u n r e l i a b l e . Two long r e t e n t i o n time compounds, a t e r t i a r y amine (D) and a secondary amine (E) (m/z 72 and m/z 58 base peaks r e s p e c t i v e l y ) are p o s s i b l y the r e s u l t of metabolic reduction of the keto group to the corresponding alcohol since they have not c y c l i z e d . A novel m e t a b o l i t e (B) ( s i m i l a r to another observed i n the i n v i t r o metabolism of methadone) appears to fragment v i a a cyclopentenone pathway s i m i l a r to that observed f o r (89). - 145 -i 1 " T* * •*• >h <* ftr ••" •** • i in • • •M "Hi * * «Sr 200 50 100 150 •*til' 11«• ' K 7 - . T r . " ' r i l M T t*»S !fc * r -r tr. rr. •+ A. iiU fit r> fir 400 250 300 350 F i g u r e 42(b) Mass sp e c t r u m ( G C M S ) o f methadone analogue m e t a b o l i t e b u t y l i d e n e d i m e t h y l d i p h e n y l p y r o l l i d i n e (BDDP) (87) - 146 -0 89 i i ) Conjugated metabolites  In the conjugated b i l e f r a c t i o n one compound at long r e t e n t i o n time (peak E Figure 43) did have some mass ion fragments c h a r a c t e r i s t i c of the formamides of methadone and r e c i p a v r i n ( ( i e ) m/z 73, 57, 129, 207). The r e t e n t i o n time was shorter than expected with the compound present i n small q u a n t i t i e s and no syn t h e t i c standards were a v a i l a b l e f o r comparison. Oddly enough, the other observed metabolites were not derived from BDDP s i n c e the mass spectra i n d i c a t e i n t a c t t e r t i a r y and secondary amino groups, with few other intense peaks. The observed t e r t i a r y amine and secondary amines could be the phenolic m e t a b o l i t e s . i i i ) In v i t r o metabolism The major metabolite i n v i t r o was BDDP (peak A Figure 44a). Other observed compounds were DDP (peak C) BMDP and the N-oxide, which by GCMS appeared as the Cope e l i m i n a t i o n product, 4,4-diphenyl-5-non-2-enone a t very short r e t e n t i o n time (Figure 44b). No formamide type compounds were detected. - 147 -Figure 43(a) TIC of methadone analogue (86) conjugated b i l i a r y metabolites (GC c o n d i t i o n s gT,A. m/z 72 (100?) r e c i p a v r i n l i k e compound.. B. 72 m/z (100%). C. U n i d e n t i f i e d D. 72 m/z (100"). E. m/z 73, 129 compound. F. Secondary amine l i k e (m/z 58 (100°O): 57 lllllfl III I7« mijlllitji. »7 ft tVT Hriwnt .nw.is. 7i« ; r - r : ; . , » -BlUi . t " 0 » I P O H . . M » „ . IT . • rr,o v . E ^ f w - I I I I " , I I - / H I K C M I . < « : • » RAM" A I 1 111 , = 1 > .1 • .111,101 114 i:r. I M i n i I l r,7 ;n'^ I I i l l l I i HiLtfliT.HWS.ii: w« : 11 - „ „ . . - TUMI r l W l ? l W l W ! l ' C . Figure 43 (b) Mass spectrum (GCMS) of a formamide l i k e conjugated methadone analogue metabol i te (E) (Analogue (86) A BQDP A Scan Figure 4 4 ( a ) TIC showing i n v i t r o m e t a b o l i t e s o f t h e methadone ana l o g u e (86) Ml 7»» •M IM JW IM o|l ! . ! : i . . 57 7/ "fa-II II Mil « tM"' in , , C 7 | , ! , , 3 . . " ' . i l l .i:w , i " 171 .11 172 . n i l 2 ISI;M.t l'..9 » -BKO.» 137 7 ("ROM. MASS. 13 » 2*.0 V.n.TrtlW 1 7 S P E C - . I P H H . I I vi scut t:-,91 H O K H F ir,?; if . - ! H t I M T . • » m i 2 1 * : r : i : 7 * nn **' JOT. 311 ,31' ,B* 1 i 1 " • . ^ • ^ • ^ • " d * ? ) e ! " : - ^ " - ! - ^ ^•••'•••JH ,»» rf.1 rf* rf* rft rf* ^ * > » f o H I P j T * I M 5 i t l 5 i JT3 Figure 44 ( b ) Mass spectrum (GCMS) of the Cope e l iminat ion product of methadone analogue N-oxide from an in v i t r o metabolic extract - 151 -E. FINAL COMMENTS ON THE UNKNOWN METABOLITE i ) Problems i n the a n a l y s i s of thermolabile drug metabolites The chemical r e s u l t s presented i n t h i s t h e s i s have shown that the metabolite u l t i m a t e l y leaves the gas chromatograph as a formamide. Two compounds, the nitrone and o x a z i r i d i n e i n the case of r e c i p a v r i n , and the o x a z i r i d i n e i n the case of methadone were shown to produce the corresponding formamide upon GCMS a n a l y s i s . The actual s t r u c t u r e of the metabolite or i t s glucuronide i s s t i l l unknown. Comparison of ^ C NMR peaks of a l l p o t e n t i a l l y thermolabile compounds with the spectrum of a metabolic e x t r a c t of b i l e from a (^CH3)2 N - r e c i p a v r i n dosed r a t should solve the problem. This experiment worked well f o r Noda et al i n the study of aminopyrine metabolism (43) but i t s success depends on r e t e n t i o n of the l a b e l . An a l t e r n a t i v e experiment involves HPLC a n a l y s i s of b i l e , development of a successful p r e l i m i n a r y sample cleanup and many r a t s , since the major problem here i s the s e n s i t i v i t y of the HPLC and the minute q u a n t i t i e s of these m e t a b o l i t e s . GCMS a n a l y s i s i s of no use i n e l u c i d a t i n g the s t r u c t u r e when so many thermolabile precursors are p o s s i b l e . I t should be stated that t h i s i s a minor m e t a b o l i t e . Successful d e t e c t i o n by packed column GC requires about half of the e x t r a c t of conjugated metabolites from one r a t . In t h i s study the recovery i s v a r i a b l e , and the metabolite i s best observed by selected ion monitoring. The 2 Hio metabolite required several experiments before i t was observed i n v i v o , and attempts to demonstrate i t i n v i t r o f a i l e d e n t i r e l y . - 152 -i i ) P o s s i b l e pathways t o g l u c u r o n i d e c o n j u g a t e d N-formyl m e t a b o l i t e s o f  methadone and r e c i p a v r i n I f the o b s e r v a t i o n s of Noda e t a l (43) s u p p o r t i n g a formamide m e t a b o l i t e o f a m i n o p y r i n e are c o u p l e d w i t h those of A l l e n e t a l (44) where an amide c a r b i n o l a m i n e g l u c u r o n i d e h y d r o l y z e s and d e a l k y l a t e s to the amide ( s e e i n t r o d u c t i o n ) , a mechanism f o r the f o r m a t i o n of formamide m e t a b o l i t e s of methadone and r e c i p a v r i n may be p o s t u l a t e d ( F i g u r e 4 5 ) . F i g u r e 4 5 . P o s s i b l e mechanism f o r f o r m a t i o n of formamide m e t a b o l i t e s of methadone and r e c i p a v r i n . The i n i t i a l s t e p , o x i d a t i o n by an unknown mechanism to a formamide, d e c r e a s e s the b a s i c i t y of the n i t r o g e n and s t a b i l i z e s the subsequent - 153 -f o r m a t i o n of a c a r b i n o l a m i n e . T h i s a l l o w s g l u c u r o n i d a t i o n of the c a r b i n o l a m i n e , w h i c h , d u r i n g ^ - g l u c u r o n i d a s e h y d r o l y s i s and workup, decomposes to the formamide p l u s f o r m a l d e h y d e (Pathway B ) . S i n c e any b a s i c d e s a l k y l m e t a b o l i t e of methadone would c y c l i z e s p o n t a n e o u s l y , a mechanism wherein the n i t r o g e n l o n e p a i r i s d e l o c a l i z e d p r i o r t o N - d e a l k y l a t i o n i s n e c e s s a r y . The l a c k of formamide i n the non c o n j u g a t e d f r a c t i o n r e f l e c t s e i t h e r a c o n c e r t e d mechanism, o r h y d r o l y s i s of non c o n j u g a t e d formamide t o EMDP v i a d i n o r m e t h a d o n e . The f a c i l e d e f o r m y l a t i o n under m i l d l y a l k a l i n e c o n d i t i o n s of the f o r m y l c a f f e i n e m e t a b o l i t e o b s e r v e d by Tang e t a l (41) s u p p o r t s the l a t t e r p r o p o s a l . The e x i s t e n c e of N - g l u c u r o n i d e c o n j u g a t e s of a l i p h a t i c amides such as s u l f a d i m e t h o x i n e s u g g e s t t h a t a formamide N - g l u c u r o n i d e (pathway C, F i g u r e 8) i s a l s o a p o s s i b l e p r e c u r s o r of the observed m e t a b o l i t e . i i i ) O t h e r p o t e n t i a l s o u r c e s of a formamide m e t a b o l i t e Hydroxamic a c i d s such as (90) a r e w e l l known as g l u c u r o n i d e c o n j u g a t e d m e t a b o l i t e s o f a r y l a m i n e s and amides (88) and have been i m p l i c a t e d i n t h e c a r c i n o g e n i c i t y of s e v e r a l compounds ( 8 9 ) . The non b a s i c n a t u r e of an a l i p h a t i c formamido n i t r o g e n may make i t amenable t o N - h y d r o x y l a t i o n and g l u c u r o n i d a t i o n , but o n l y one r e f e r e n c e , w i t h o u t c i t a t i o n o r s t r u c t u r e to a l i p h a t i c hydroxamic a c i d m e t a b o l i t e s was found i n the l i t e r a t u r e ( 3 7 ) . None of the r e a r r a n g e m e n t p r o d u c t s of t e r t i a r y amine N - o x i d e s ( i e . P o l o n o v s k i , Meisenheimer and Cope Rearrangements) a r e formamides o r t h e i r p r e c u r s o r s ( 9 0 ) . H n ,° 90 - 154 -IV. SUMMARY AND CONCLUSIONS During the course o f t h i s t h e s i s , the EDDP oxi d a t i o n product was c o r r e c t l y assigned the o x a z i r i d i n e s t r u c t u r e (5J and was shown to thermally isomerize to the formamide (31_). GCMS behavior of both compounds was i d e n t i c a l to the b i l i a r y metabolite observed by Kang in r a t s . The corresponding o x a z i r i d i n e (14J and formamide (15J of r e c i p a v r i n were synthesized and proven i d e n t i c a l by GCMS to a conjugated b i l i a r y m e t a b o l i t e of r e c i p a v r i n i n r a t s . The p o s s i b i l i t y of a novel metabolic pathway of a l i p h a t i c t e r t i a r y amines was discussed in l i g h t of these observations. During GCMS a n a l y s i s the r e c i p a v r i n n i t r o n e (13J decomposed to the imine (59J oxime (52) and a trace of formamide (16J i n accord with l i t e r a t u r e r e p o r t s . The decomposition product imine (5 J J was not present by GCMS o f b i l i a r y e x t r a c t s , suggesting that the ni t r o n e i s not the precursor of the observed formamide m e t a b o l i t e . The diketone (45J described by Kang was synthesized in low y i e l d and cha r a c t e r i z e d by *H and 1 3 C NMR. It i s a po t e n t i a l synthetic precursor o f methadone and the N-oxidized congeners of the drug methadol. The 1 3C NMR r e s u l t s f or the o x a z i r i d i n e s (5_) and (14J and the ni t r o n e (13) are the f i r s t r e p o r t s of chemical s h i f t values f o r methylene o x a z i r i d i n e s and nitrones which have a hydrogen atoms. The synthesis and c h a r a c t e r i z a t i o n of ten new compounds; the ni t r o n e ( 1 3 ) , o x a z i r i d i n e (14_), formamide ( 1 5 J , hydroxyl amines (55J and ( 5 7 J , t h e i r TMS d e r i v a t i v e s (56) and ( 5 8 J , the imine ( 5 9 J , n i t r o s o compound (6JJ and oxime (64J as well as d e t a i l e d c h a r a c t e r i z a t i o n of the known amines ( 6 J , (53_), ( 5 4 J , ketone (51_) and oxime (52J provide the a n a l y t i c a l basis f or f u r t h e r i n v e s t i g a t i o n o f a novel in vivo metabolic pathway. - 155 -V. REFERENCES 1. Kang, G.I. 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Carbon-13 Nuclear Magnetic Resonance Spectra of Isomeric O x a z i r i d i n e s . E f f e c t s of the Nitrogen Lone P a i r on Carbon-13 Chemical S h i f t s . Org. Mag. Res. 9_: 322 (1977 ). 82. Bapat J.B. and D.S.C. Black. Nitrones and Oxazirans, Aust. J. Chem., 21: 2521 (1968). 83. Sadtler Co. Lt d . Reference *H NMR Spectrin 29952M. 84. S i l v e r s t e i n , R.M., et a l . Spectrometric I d e n t i f i c a t i o n of Organic Compounds. (4th ed.). J.Wiley and Sons, New York (1981). 85. Budzikiewicz, H., et a l . Mass Spectrometric of Organic Compounds. Holders Day, San Francisco (1967). 86. McLafferty, F.W. I n t e r p r e t a t i o n of Mass Spectra, Univ. Science Books, M i l l s V a l l e y Ca. p.22 4 (1980). 87. C r a i g , R.N., et a l . The Reaction of Grignard Reagents with Lactones. I. Ethyl and Methyl Grignard Reagents with 2,2-Diphenyl-4-Butanol actones. J. Am. Chem. Soc. 75_: 4731 (1953). 88. Hinson, J.A. and J.R. M i t c h e l l . N-Hydroxylation of Phenacetin by Hamster L i v e r Microsomes. Drug Metab. Disp., 4_: 430 (1976). 89. Belman, S., et a l . C a r c i n o g e n i c i t y o f Aryl Hydroxamic A c i d s . Cancer Res., 28j 535 (1968). 90. Oae S., and K. Ogino. Rearrangements o f t-Amine Oxides. Heterocycles. 6_: 583 (1977). - 1 6 1 -APPENDIX GC TOTAL ION CURRENT TRACES Page 1. TIC of GCMS of EMDP ox idat ion products (GC condi t ions c) 162 2. TIC of GCMS of EDDP oxidat ion products (GC condi t ions b) 163 - 163 -- 164 -APPENDIX INFRARED SPECTRA Page 1. Infrared spectrum (nujol mull) of 3,3-diphenyl-5-methyl 165 tetrahydro-2-furanone imine (40) 2. Infrared spectrum (nujol mull) of 2,2-diphenyl-4-val ero- 166 la c t o n e (4_1) 3. Infrared spectrum (nujol mull) of 4,4-diphenyl-2,5-heptane 167 d i o l (43) 4. Infrared spectrum of 3,3-diphenyl-2-ethyl idene-5-methyl- 168 tetrahydrofuran (44) 5. Infrared spectrum ( f i l m ) of 4,4-diphenyl-2,5-heptanedione- 169 2-oxime (46). 6. Infrared spectrum ( f i l m ) of 4,4-diphenyl-5-hydroxy-2-amino- 170 heptane (dinormethadol) (49) 7. Infrared spectrum (nujol mull) of 1,1-diphenyl-3-butanone (j>l_) 171 8. Infrared spectrum ( f i l m ) of 1,1-diphenyl-3-butanone oxime (52) 172 9. Infrared spectrum ( f i l m ) of N,a-dimethyl -Y-phenyl - 173 benzenepropanamine (54) 10. Infrared spectrum (nujol mull) of N-hydroxy-a-methyl-Y -phenyl 174 benzenepropanamine (55) 11. Infrared spectrum (nujol mull) of N,a-dimethyl-N-hydroxy -Y- 175 phenyl-benzenepropanamine (57) 12. Infrared spectrum ( f i l m ) of a-methyl-N-methylidene - Y-phenyl- 176 benzenepropanamine (59) 13. Infrared spectrum (nujol mull) of 1,1-diphenyl-3-nitroso 177 butane (61) 14. Infrared spectrum (nujol mull) of Y-phenyl-N,N,a-trimethyl 178 benzenepropanamine perchl o rate {§) 1. Infrared spectrum (nujol mull) of 3,3-diphenyl-5-methyl tetrahydro-2-furanone imine (40) 2. Infrared spectrum (nujol mull) of 2,2-diphenyl-4-val ero-lactone (41J 3. Infrared spectrum (nujol mull) of 4,4-diphenyl-2,5-heptane d i o l (43) W r i v e n umber 4. Infrared spectrum of 3,3-diphenyl-2-ethy"l idene-5-methyl -tetrahydrofuran (44) Wavelength fjm Wsvemin-1---5. Infrared spectrum ( f i l m ) of 4,4-diphenyl-2,5-heptanedione-2-oxime (46). Wavunumbcr Infrared spectrum ( f i l m ) of 4,4-diphenyl -5-hydroxy-2-amino-heptane (dinormethadol) (49) WarvnwnMr Infrared spectrum ( f i l m ) of 1,1-diphenyl-3-butanone oxime (52) 9. Infrared spectrum (film) of N,a-d imethyl- y-phenyl-benzenepropanamine (54) W n v e n u m b e i 10. Infrared spectrum (nujol mull) of N-hydroxy-a-methyl -y-phenyl benzenepropanamine (55) 1 1 . Infrared spectrum (nujol mull) of N,a-dimethyl -N-hydroxy-Y-phenyl-benzenepropanamine (57) W*v«l«nQth M m w/avttiMtml'i't . Infrared spectrum (f i lm) of a-methyl -N-methyl idene-y-phenyl-benzenepropanamine (59) V.'. v e n M i n h r f 13. Infrared spectrum (nujol mull) of 1,1-diphenyl-3-nitroso' butane (61) 14. Infrared spectrum (nujol mull) of Y-phenyl-N.N.a-trimethyl benzenepropanamine perchlorate (6) - 179 -APPENDIX MASS SPECTRA Page A. SYNTHETIC COMPOUNDS 1. Mass spectrum (direct in le t ) of 2-(4 1 ,4'-diphenylheptan-5'- 182 one-2'-yl) oxaz i r id ine {5) 2. Mass spectrum (GCMS) of Y -phenyl-N,N-a-trimethyl 183 benzenepropanamine {6) 3. Mass spectrum (d i rect i n l e t ) of 2-(4' ,4'-diphenyl-but-2'-yl) 184 oxaz i r id ine (14) 4. Mass spectrum (GCMS) of 2,2-diphenyl-pent-4-enenitril e (38) 185 5. Mass spectrum (GCMS) of 4,4-diphenyl-hepten-5-one (39) 186 6. Mass spectrum (GCMS) of 3,3-diphenyl-5-methyl tetrahydro- 187 furanone imine (40) 7. Mass spectrum (GCMS) of 2,2-diphenyl-4-val erol actone (41) 188 8. Mass spectrum (GCMS) of 1,1-diphenyl-2-butanone (42) 189 9. Mass spectrum (GCMS) of 4,4-diphenyl-2,5-heptane diol (43) 190 10. Mass spectrum (GCMS) of 3,3-diphenyl-2-ethylidene-5- 191 methyl-tetrahydrofuran (44) 11. Mass spectrum (GCMS) of 4,4-diphenyl-2,5-heptanedione (45) 192 12. Mass spectrum (GCMS) of 4,4-diphenyl-2,5-heptanedione-2- 193 oxime (46) 13. Mass spectrum (GCMS) of 4,4-diphenyl-5-hydroxy-2-amino 194 heptane (dinormethadol) (49) 14. Mass spectrum (GCMS) of 1,1-diphenyl-3-butanone (51) 195 15. Mass spectrum (GCMS) of 1,1-diphenyl-3-butanone oxime (52_) 196 16. Mass spectrum (GCMS) of ot-methyl- Y-phenyl-benzenepropanamine (53)197 17. Mass spectrum (GCMS) of -N, a-dimethyl-Y-phenyl 198 benzenepropanamine (54) 18. Mass spectrum (GCMS) of N-hydroxy-a-methyl-Y-phenyl 199 benzenepropanamine-TMS-ether (56) 19. Mass spectrum (GCMS) of N,a-dimethyl-N-hydroxy-Y- phenyl 2 0 0 benzenepropanamine-TMS-ether (58) - 180 -APPENDIX MASS SPECTRA Page 20. Mass spectrum (GCMS) of a-methyl-N-methylidene-y-phenyl - 201 benzenepropanamine ( t r i m e r ) (59) 21. Mass spectrum (GCMS) of 1,1-diphenyl-3-butanone oxime- 202 0-methyl ether (64) 22. Mass spectrum (GCMS) of EMDP o x i d a t i o n product MDP (71J 203 23. Mass spectrum (GCMS) of EMDP o x i d a t i o n product G (72) 204 24. Mass spectrum (GCMS) of EMDP o x i d a t i o n product E (73) 205 25. Mass spectrum of cyclopentenone product of a l k a l i n e treatment of 206 diketone (89) 26. Mass spectrum of NaBh^CN reduction product of the 207 methadone o x a z i r i d i n e (j>) 27. Mass spectrum (GCMS) of ethyl methyl diphenyl p y r o l l i d i n e 208 l i k e r e ductive hydroxylamination product of diketone 28. Mass spectrum (GCMS) of product of r e a c t i o n of diketone and 209 methyl amine (Dimethyl diphenyl ethyl idene 2 ,3-dihydro p y r r o l e 29. Mass spectrum (GCMS) of EDDP l i k e major product r e d u c t i v e N-methyl hydroxylamination of diketone 210 30. Mass spectrum (GCMS) of long r e t e n t i o n time product of 211 r e c i p a v r i n n i t r o n e and methyl a c r y l a t e . 31. Mass spectrum (GCMS) of long r e t e n t i o n time product of 212 r e a c t i o n with thermally isomerized (120 ° ) methadone o x a z i r i d i n e 32. Mass spectrum (GCMS) of the by product of thermal 213 i s o m e r i z a t i o r of methadone o x a z i r i d i n e (5J B. METABOLITES 214 1. Mass spectrum (GCMS) of a novel EDDP l i k e i n v i t r o methadone 215 m e t a b o l i t e 2. Mass spectrum (GCMS) of a novel i n v i t r o methadone 216 m e t a b o l i t e , also found as a non conjugated methadone anal ogue metabol i t e 3. Mass spectrum (GCMS) of i n v i t r o r e c i p a v r i n metabolite (76) 217 - 181 -APPENDIX Page 4. Mass spectrum of t e r t i a r y amine in v i t ro recipavrin 218 metabolite (77) 5. Mass spectrum of t e r t i a r y amine in v i t ro recipavrin metabolite 219 (78) 6. Mass spectrum of overlapping dealkylated recipavrin 220 in v i t r o metabolites (Nor (81), and Dinor (80) Recipavrin) - 182 -S P F t - 4 0 ( I N I * B 5 Bi:«« 1 A K G 4 1 4 I I X T . " 72 r mMst.ws.is. it» J M l , l , « , [ ,1111 , | , i i . i in , III';",,,!!!!,'* \\\\{,\\M to w lit lit i*t Ii" w lit ite lit ilr m lit I i 1 " Ml , i . l l l i . m at >lt i FRO".MASS. ft » 1 0 0 U.E«F»Mn ) » B f F r . - 4 0 U N . 1 4 RT> S C H 4 MS B A h G • 14 H A L . - 7 I I H T . " J 7 0 » I l,„ I f f . II II I I . IH III ^ m m i». i >i°: i»«i ..^ jj..!...^ .. i i f 1. Mass spectrum ( d i r e c t i n l e t ) of 2-(4',4'-diphenylheptan-5 '-one-2'-yl) o x a z i r i d i n e (5>) - 183 -v . r lOCt T: K* tit W» to* 1* ?*• 10* 44 n M — ± if—:Ji • '"' '''to I J ' * _J_ . i :S , i7t , i t e a r * : . * ; — r j . oo i M. . • 144 I i r o i fc RK •. i ;•; i HA i . i i f l — r t t rt M»l : r«fk - R 1 1HT . rin rfr- -i» rtt rfc !*{ * sh • t 14 J* 11 At- •=4 * r 3kr >W"- iV A r — •<kr >k iJr ilr Jlr «V 2. Mass spectrum (GCMS) of y -phenyl-N,N - a-trimethyl benzenepropanamine {§) - 184 -S F F T - 4 0 UN.lt fl.t f HRA 1 J H A K C • i l C A I . 1 M N T . - 2 5 V 7 ,ll» NiyCST.MS&.lE. 7U \ .il,iiJl111 - ,„,!"" [rill'",." „.:». JU i rill,™ « » * i •« i* it« is* ik its iii itt I 4 ,f\ rr / Z\ fSr,N.» 1 3 » -B K G . # .1 i FfcQM.HA^S. ) 5 » 2 0 0 V . F X P A N D 1 » 5 S P F f ; -40 I I N J * fl:* r ft ft A 1 1 B A K G » a C A L , - 1 M M T , * 2 3 9 7 «2N;:«:?l?»s*& * & * * * .<« &• & & & & * * * 3. Mass spectrum ( d i r e c t i n l e t ) of 2-(4',4'-diphenyl-but-2'-yl) o x a z i r i d i n e (14) If* I ft* Mt . . 1.1 111 . M l t l l l l l i l l ||l,l I l l | • it • I M M . M C t . l l . ?ff -r%-t r F f : - < 0 . . . . » W > J I P S I J I I M'AI i 0 1 , M „ ; .WM,.. „ 7 J L ' f e . , ; ; : ^ ' ^ 1 A— — a - — — it. 4. Mass spectrum (GCMS) of 2,2-diphenyl-pent-4-enenitrile (38) - 186 -k k k i : X co QJ c o I i n i c (D +-> Q. Ol C T3 I is :5 I SS i « !1§S . r> c u • • oc fc. : c -3: i i o 3 S_ +-> o a. 01 «3 I • Jit• .,1,1 "H <e k—h to • I d r i i . i K S . i l . ju J M : I » > r" M 111111 iini"- mi — 1 » ro 1*» t •C ..II..1 -Jir * fV-M M . I . .M:I;I . :J» vvriifti..:. r.'i.; r. 1.1. • ^ u t u . ' ? t » / 'Mr * * *r — " * r * i h +> •** •»• "* >*-6. Mass spectrum (GCMS) of 3,3-diphenyl-5-methyl t e t r a h y d r o -furanone imine (40) - 188 -_ c o o 'Jo > i ^-i >> c: cu •o i (XI _ _ O OJ Q . </> (/I 100* W t sot 49* jot lot 37 43 .115 . n " . l > ...» . . P . ,)/ , ifffffi. , . 132 ,133 I IBCH.» 96 T -BKB.• 93 ? FROM .KABB i 13 T 230 V.EXPAND t ? SPEC 3B I I N J * 2V»Sr;M» VAIBAKB* V3fTERR* 9IIHT." 3326 t ' I NIC 8. Mass spectrum (GCMS) of 1,1-diphenyl-2-butanone (42) I. i« !u-K1»CS1.MSS.II. !15 \ ill, "III, I I I " ,1111 I, « ,„ « | | IM |.m MI ),iu II , . i In i' | J » \ K l i t l i t ' l l f . I 1 M J « 3D S C N » 6 0 » « K 0 • 5 3 C « l . « - 3 3 , I N , . « 3 9 7 ? 1% 9. Mass spectrum (GCMS) of 4,4-diphenyl-2,5-heptane d i o l (43) - 191 -10. Mass spectrum (GCMS) of 3,3-diphenyl-2-ethylidene-5-methyl-tetrahydrofuran (44) I / I, " k It it it it U tt fc lit lit ifc tit lU t i t i U ifc i b i f c r f « i h iW K I B C f T . M S S . I S . HI \ 11. Mass spectrum (GCMS) of 4,4-diphenyl-2,5-heptanedione (45) 12. Mass spectrum (GCMS) of 4,4-diphenyl-2,5-heptanedione-2-oxime (46) s r r ( . -40 I — l SM " k U U b U J » b b ib i b \ W i b i b - i b i b \W i b i b > b > U > b " n w i i j H B . i l . m \ I,N.I* » m'M» All RrtKH » T.A T A I . !% i , M T . 1 7 1 * _ & ' ' j f e * 13. Mass spectrum (GCMS) of 4,4-diphenyl -5-hydroxy-2-amino heptane (dinormethadol) (49) BPrC -40. MM , U M . I » A4 f t ( : N » 17 B A K R » IT) Cf>l . •' - A H N T . ' 1834 m m IM 41 ,1*1 U I,» 4,uV\,IL s» it to IM 1»9 ,115 ."I ,147 , | | I .!» . .Ml. •iw* \to w »*»•»»' in IM .154 IM in IB ,m \to ilv rtl !*»•••"• str sJ*M 14. Mass spectrum (GCMS) of 1,1-diphenyl-3-butanone (51) - 196 -7\ isr .N . i 1? ' .17 -I-KT,.* IS •* 33 t-kOH.KAr.fi. i s * v.F.xfAwri i T SrFL-40 M i l l 97 sr.M# 37 NAM. • 33 CAL. - -ft I1MT.-- ' i - i * . $ MlGtCM.MSS.IS. 239 \ .1. i " , n " . 1 1 . " . . ° . ! , " . r. I " ' i r - I i i1 M In r-'iiim IIIIIII r - M i n i k l i t l i t l i t i f a I * t l i t I f c i i i } t t » .IIN.I. 97 SCH. .W HAKG . 13 CAL. - -B I1N1.- ?614 •'-•jit <U > i t > l t T H & > » t l i t l i t l 7 t l i t l i t l i t l i t l i t i n s t t t i t l i t l i t l i t " K l f t f S l . M S S . l S . 7T* \ 15. Mass spectrum (GCMS) of 1,1-diphenyl-3-butanone oxime (52) F R O f t . H A S R . 24 T 1 5 T 2 5 -»*,n.» 2 1 f T r k R A i ' t H A M i • 2 1 H A L . - I A » 1 H r . » 2 5 1 B 2 IMt M -•St Mt m SM Mt Mt I M it; m u V; mWSI.M5S.lt. ?» \ « 31 1 1 w 1 7 7 — ( 4 101 ('" j'" ' l i t l i t I W ' - 9* , _ R . I-I U •I»t- "1" -J*'• Ar-16. Mass spectrum (GCMS) of ct-methyl - Y-phenyl-benzenepropanamine (53) r - H K f l . t * J t T 2 2 0 W . I X P A M I • 1 r u m ? « fcAKti • J J TAI 8 I I M t . - ? • • » ! r * _ »•• l b rU _ ) III' . . . . * ) ' tte _»•• - i b i b ' _ r i b ' tk •b1 « b i\ mm.* ; t t l c O I I . » A S i . I!> T - K M - . . • 21 ' T U . m f O K O 1 T • f u l l * ? A U t l • 7 ] C M • I 1 M I . - 2*481 M H -f i t m m 4flt •M «M IM n* IM H u l " f . i • U - l l . H _ . i l . M \ , 1 . " ) , ••_ -il 1 • i l 1 •it " * l i t l i t l<« . . . . • l i t l i t , « }. l i t l i t 1, I t - lb - i b i b » b m • i t t t b -17. Mass spectrum (GCMS) of -N, a-dimethyl-Y-phenyl benzenepropanamine (54) /\ ,si:».t I t ' il -mn.t u ' t«8«.mn». 1 3 T w.i«f»«ri i i 11 N.I* 4.1 S C N t .17 K K H 4 ? V C«l . I I I 1 N 1 . " 4 2 4 4 W U I r t r W I t It l i t l i t ' " U M I I . M t . l l . I l l \ \ i s m I it ' - H K f i . t :'v ' ',1 i r . , r i " i t , f . . / lit lit i V i l r > W i W i » . I I N I t 41 SI H t 17 * » M . • ? ' . " I f N I . • I I » I N I . * 6^44 : « i l i t 'I.:-., * ,,-Au- » * * * * * nr ,v * * * " a i i « i i . i M i . n ! in - ' 18. Mass spectrum (GCMS) of N-hydroxy-a-methyl-y-phenyl benzenepropanamine-TMS-ether (56) - 200 -fRQl.BASS. 15 * .I1N.I# 44 fitM» *!S M f c b < ? S C * l . . - 14 ( I N T . * 11761 ID I K ,» i~ UN," .III , II .IJ", . . . . J " m l . . * .Ill, l . » .HH" • . j- ' " . . " . : " !,,, A ! ' ••••Jo _ it i t i t - I t i t - t o lit ( I t l i t f i t l i t r f t l i t i k l i t l«t Jit !l« Si . I W —SS.Il. I7t \ b. t C - 4 0 .....MJ* 44 S C . * * S 4 r .», . - 14 . . N T . - . 3 7 * . A - l i t t U A •»/«'••-•* ' t i t » i « _ « i U A _ J • < _ 19. Mass spectrum (GCMS) of N,a-dimethyl-N-hydroxy-y- phenyl benzenepropanamine-TMS-ether (58) - 201 -7\ i ^ r w . t ?6 7 -bKn . o n1 * SFFr.-40 I I N . I * V H K C U A ?« nflur, • 2 1 C A I . . « -V I I H 1 . ' 1776B . _ • , 57 1 0 * TO* JO* ...... ' i - vii J , 1 1 ~ j ' " , | , iji ,"i , i n " ; HIIII'- . ii™ . iiiii1" M ' " i , , j .11 . I l l I I S 117 maffsi.Atss.is. m \ 7\ ISr.N.O ?t 7 -BKfi.O 21 7 FfcHH. MA5>5>. 15 T 225 V.F.XPANn I 7 SFfl • A O tN.lt »B SHNO 2A m i * 71 I.AI . . -V (tut... 17748 • , :!7 ••sb-& & sb >b sb •M, & i b & i b & l b \\ i b >b «b «1t ab «b" 20. Mass spectrum (GCMS) of«-methyl-N-methyl idene-T-phenyl-benzenepropanamine (tri m e r ) (59) . ( I H . l t J Hi: Nt IM B»KS t III C M . • l » l l « T . - I K « 7 « i i • i i • i» i u » iL"ii"irn ii!* \,<» ii* .nt.itt i , n „ i . i i i i i M . . IS ( I I C » . » >J T -•«(!.• I I T F R O H . H M i t l . IS T M O ».Hr»»B I T l . l» > HUHt Z J M K B • l « C M . » l » M i l t . . I ! l « 7 » 21. Mass spectrum (GCMS) of 1,1-diphenyl-3-butanone oxime-0-methyl ether (64) - 203 -22. Mass spectrum (GCMS) of EMDP o x i d a t i o n product - 204 -spectrum (GCMS) of EMDP ox i d a t i o n product G - 205 -24. Mass spectrum (GCMS) of EMDP o x i d a t i o n product E - 206 -. I 1 N J 4 7 S C N t 5 3 » « * G « 4 8 C A L . - - 3 6 t l N T . * 2 . 3 B 1,1 i l . ' iii^ jii; iiiir „II,": iin i III! Ml i l l " . ! i i i: • — IIM;II- • 111; r I 211 ,247 I V ' f f ' " , ' & at ; U At- "• • • • -Av tfrAr iW */ i t i H i t * ' * r »ta to- >5t »k-25. Mass spectrum of cyclopentenone product of a lka l ine treatment of diketone (89) 26. Mass spectrum of NgBri^CN reduction product of the methadone o x a z i r i d i n e (J5) 27. Mass spectrum (GCMS) of ethyl methyl diphenyl p y r o l l i d i n e l i k e r e d u c t i v e hydroxylaminat ion product of diketone - 209 -t C « « St » » » 0 * 4 » C * L . • -I* f I « T . . « l < 28. Mass spectrum (GCMS) of product of re a c t i o n of diketone and methyl amine (Dimethyl diphenyl ethyl idene 2,3-dihydro p y r r o l e •w-41.. M l . . M M 4 HNI IIS I t l l N U L . < -v nm. - mi 3M JH I I . '.'] I.» ' i l l -rtr- Lia mtt « ,i*A I ON 1(2 LM I -.4 . £ . • III «W Ilk £ Ir r t =, , , , , , , . . , . Mo A* Jto **> »«> JJO Uu 3* yfc 4 X < 29. Mass spectrum (GCMS) of EDDP l i k e major product r e d u c t i v e N-methyl hydroxylamination of diketone S S r.NI 1 4 7 H A * 0 » 14<> DAI < ( 0 I 1 N T , -" ^ ii k HlMI1.M8l.il. U3 \ S P F C - 4 0 .1 Jl I . Ill* i " ! i .ii< !<J .b'^ir-"'.!. ill if- .ir . U N I . .1 S U M I4» N A M ) t M i ' ':»'-• • , 1 H I ' ; imil-jr. •»U A" 30. Mass spectrum (GCMS) of long r e t e n t i o n time product of r e c i p a v r i n nitrone and methyl a c r y l a t e . I N * I N . l t < M ' • • M » il , i h i t k l . » i 1n I A l . ..".i/V I,.. "fc fc fc • , f c " " " l t , " " " l l l ill Ill Ifc Ill Ifc 1*1 A * fc b-irni.nmi.n. m \ S P F r : - 4 0 M N . I f I R A T M * M A R A K R I M O CAI . r r III * • • I M K T . " 7 3 3 0 •\t»' iW iW Ar •ill- lW >V •fc" «J»-31. ' Mass spectrum (GCMS) of long r e t e n t i o n time product of reaction with thermally isomerized (120°) methadone o x a z i r i d i n e I I M . I I M 1 I RHA K A K i : • J R C M . • IV I I N T . - I B I S I to I ••« _• , t i n i II J trartTjHa.n. stv \ J \ 4 ? T " - P M ' . . » S B » r M M . I M M I . IS T 2 ? 3 V . F X P A H B I T I, III I I I H IIS m b- 1 -th-lit I N • • • I t ) i l l " I . II.. •Hi"Il t , . iir r * fc-i i ! * v > J» «(« i , I 1 M I I M 4 F k R A K A K i : I .HI VM. . •. I ? I l » 1 . • 1 B I S •fe- •*» : • J» ill >fe * r >to Ar »!»• »<r i i i " 32. Mass spectrum (GCMS) of the by product of thermal isom e r i z a t i o n of methadone o x a z i r i d i n e (5_) - 214 -APPENDIX B. METABOLITES 1. Mass spectrum (GCMS) of a novel EDDP l i k e i n v i t r o methadone meta b o l i t e 2. Mass spectrum (GCMS) of a novel i n v i t r o methadone m e t a b o l i t e , also found as a non conjugated methadone anal ogue metabol i t e 3. Mass spectrum (GCMS) of i n v i t r o r e c i p a v r i n metabolite (76) 4. Mass spectrum of t e r t i a r y amine i n v i t r o r e c i p a v r i n m e t a b o l i t e (77) 5. Mass spectrum of t e r t i a r y amine i n v i t r o r e c i p a v r i n metaboli (78) 6. Mass spectrum of overlapping d e a l k y l a t e d r e c i p a v r i n i n v i t r o metabolites (Nor (81), and Dinor (80) Recipavrin) - 215 -i iN . i t D r . i : : r m .in:-it.*.':r>» 1 • •!• I n l l l l i l i l l i r l i h l i l i : " ' ! ! . ! ii K C : . . W . 3 . IM s r E r - 3 B i m j t i b t s r . M « 3 H 7 H A I ; B » J t s i T E M " 7 ; i f»T - i l l I S * I K -tti * tr Tfc * 1»! & A * rt«— 1. Mass spectrum (GCMS) of a novel EDDP l i k e i n v i t r o methadone metabolite - 216 -:T:.-3* 7 t < .; I :i : . tu;:..:...: ff.-r.V s r E r . - i E . . ...! i!! O I , B |.'T> ii ,i is: | mihlLlliiHii ,Ji.!!,t;: M ; i , ! i ' 'I ! I! i!!!!.Lr!..!! IX t,r.ISCM» .1 —A I BRKtil r j A l U B K :f.f ;:_ Ill _ * tit iv A - * a r -2. Mass spectrum (GCMS) of a novel i n v i t r o methadone me t a b o l i t e , also found as a non conjugated methadone analogue metabolite - 217 -KM.)* »:»C«f 20»J!Tl**< MI...—— t , tt i r -A— -ffi & 1* *~™rti * * A -Si—Sr-~* 8r- - f t — 4 r * * * * *~ 3. Mass spectrum (GCMS) of i n v i t r o r e c i p a v r i n metabolite (76) - 2 1 8 -ruon-.-.-iss-C>tr . I1H.J* » tB- !<- ' _ . ' M - » I B » & * ho*.****. I S * = « v.ft_»M» 1 * g P C C . M I 1 N J » » 1 « M . M l i A K M 7 _ f » T F r - - - I M I P t . - * W 7 •ib- it *t i f c - A " ^ *• A- * * • A A r A -Mass spectrum of t e r t i a r y amine in v i t r o r e c i p a v r i n m e t a b o l i t e (77) - 219 -n i f i - . ^ f . t f " ait " r i B j t *t:;r.n» 2«?t»f.no» 2«mT*:»« - i r : i n i wcr. 5. Mass spectrum of t e r t i a r y amine i n v i t r o r e c i p a v r i n m etabolite - 220 -1 i I 77 TI . • » , ! » f" U/1" I 1 " <m . if" it II N.I* ? : ! X H » -..ir.trft*?! l . ! ( i l H . 6 > - K . MNT. 137*4 6. Mass spectrum of overlapping d e a l k y l a t e d r e c i p a v r i n i n v i t r o m e tabolites (Nor (81), and Dinor (80) Recipavrin) - 2 2 1 -APPENDIX MASS SPECTRA AND MASS CHROMATOGRAMS (CAPILLARY GCMS) Page 1. a) C a p i l l a r y CI GCMS of r e c i p a v r i n formamide (15_) 223 b) C a p i l l a r y EI GCMS o f r e c i p a v r i n formamide (15_) 224 *H NMR SPECTRA 1 a) 400 MHz lH NMR of methadone o x a z i r i d i n e (5) 225 b) Decoupled at 2.3 ppm c) Decoupled at 1.8 ppm 2. 100 MHz *H NMR of 2 - ( 4 ' , 4 '-diphenylheptan - 5'-one - 2'-yl ) 226 o x a z i r i d i n e (5J minor diastereomer 3. 100 MHz JH NMR of Y-phenyl-N,N, a-trimethyl-benzene- 227 propanamine (Recipavrin) (6) 4. a) 400 MHz *H NMR of Recipavrin n i t r o n e (13) 228 b) Decoupled at 3.96 ppm 5. a) 400 MHz *H of 2 - ( 4 ' , 4 '-diphenyl -but - 2 ' - y l ) 229 o x a z i r i d i n e (major diastereomer) (14) b) Decoupled at 4.01 ppm 6. 100 MHz lH NMR of 2 - (4 ' , 4'-diphenyl-but - 2 ' - y l ) 230 o x a z i r i d i n e (minor diastereomer) (14) 7. a) 400 MHz !H NMR of N-formyl-a-methyl-Y-phenyl-benzene 231 propanamine (15) b) Decoupled at "4T1 ppm 8. a) 400 MHz lH NMR of 2-(N-formyl ) - 4 , 4-diphenyl- 232 5-heptanone (31) b) Decoupled at 2.3 ppm 9. 100 MHz *H NMR of 2 , 2-diphenyl-4-valerolactone (41) 2 3 3 10. a) 400 MHz *H NMR of 4 , 4-diphenyl - 2 , 5-heptanediol (43) 234 b) Decoupled at 2.44 and 2.27 ppm c) Decoupled at 3.72 ppm 11. 400 MHz *H NMR of 4 , 4-diphenyl - 2 , 5-heptanediol (43) 235 - 222 -APPENDIX iH NMR SPECTRA Page 12. 100 MHz *H NMR of 3,3-diphenyl-5-methyl-2-ethyl idene 2 3 6 tetrahydrofuran (44) 13. 80 MHz lH NMR of 4,4-diphenyl-2,5-heptanedione-2-oxime (46) 2 3 ? 14. 80 MHz lH NMR of keto oxime (46) reduction product 2 3 8 15. 400 MHz *H NMR of 4,4-diphenyl-2-amino-5-heptanol (49) 2 3 9 16. 100 MHz lti NMR of syn and ant i 1,1-diphenyl-3-butanone oximes 2 4 0 17. 100 MHz *H NMR of «-methyl-Y-phenyl-benzenepropanamine 2 4 1 ( D i n o r r e c i p a v r i n ) (53) 18. 100 MHz lH NMR of N, a-dimethyl - Y-phenyl-benzene- 2 4 2 propanamine (Norrecipavrin) (54) 19. 100 MHz lH NMR of -N-hydroxy-a-methyl -Y-phenyl-benzene- 2 4 3 propanamine (55) 20. 100 MHz *H NMR of N, a-dimethyl-N-hydroxy - Y-phenyl- 2 4 4 benzenepropanamine (57) 21. 100 MHz lH NMR of 1,1-diphenyl-3-nitrosobutane-dimer (61) 245 22. a) 80 MHz lH NMR of syn and anti 1,1-diphenyl-3-butanone- 246 0-methyl oxime (64) 1 3 C NMR SPECTRA 23. Broad band decoupled 400 MHz 1 3C NMR of 2-(4' ,4'-diphenyl 247 heptan-5'-one-2'-yl) o x a z i r i d i n e (major diastereomer) (j>) 24. Broad band decoupled 400 MHz 1 3 C NMR of a-methyl- 2 4 8 (N-methylene)-y-phenyl-benzenepropanamine N-oxide (13) 25. Broad band decoupled 400 MHz 1 3C NMR of 2 - ( l ' , l ' - d i p h e n y l - 249 but-2'-yl) o x a z i r i d i n e (major diastereomer) (14) ULTRAVIOLET SPECTRA 1. U l t r a v i o l e t spectrum of Re c i p a v r i n n i t r o n e Q3_) i n methanol 250 2. U l t r a v i o l e t spectrum of methadone o x a z i r i d i n e (5_) i n 251 methanol (A) 3.54 x IO" 3 M (B) 3.54 x IO" 5 M - 223 -' i i * > . 'S fc.29 S . . 3 9 8 . 4 9 fc.fcft 8.68 «f.7» 8 .98 t . 9 9 9 . 9 8 9 . 1 * 9 .28 9 . 3 8 9 . 4 9 f tea 189 i*» •* ? ? ; j r » 9 E l .SJM»U. i : - 9 1 « 9 1 5 9 •SMI* . ; » » » * : :19994V : i * * 9 « ^ 1 9 8 8 8 8 -. 9 9 9 9 9 - ) tsee»-4 9 9 9 9 -i999**= f : . 2 9 -. . 5 9 S 4 9 6 .S .9 . 6 9 6 . 7 9 * . 6 9 6 . 9 8 ? . 9 9 9 . 1 9 9 . 2 9 9 . 3 9 9 . 4 9 9 . S 9 - c « r . c"-. 9 * • t r.. 28-19-9-67 72 66 1 9 5 131 / I 1 4 8 T — " 6 9 176 / 1 9 5 128 1 6 9 288 226 282 2 6 8 2 9 4 1 8 9 2 4 9 2 8 9 • : - J 2 1 C . 5 . 9 - 2 S 3 . S - » u . 1. C a p i l l a r y CI GCMS of r e c i p a v r i n formamide (15) - 224 -Ce68&-148888-128888-• 188888-seeee-oaeee-48888^ ;i'8888->CS68 9999.8-8.8 a « u . see 4ee 6 0 0 I I ' ' I ' l l I... 1 1 • I 1 1 1 1 1 1 l l - . 388 leee i2.ee . 1 4 8 * i ' r i ^ i 18 14 lb •1 • I ' I 28 £2 F i l e -GSfcO Bpk nt. 1 7 S 3 4 1 88H 98H T3 / an 4SE F. I E n r . . 88H C.8H sen 4«H 3 H 2eH 167 \ 1 3 8 115 4 4 18 91 i I 4 8 liLAJi 88 128 253 181 ?88 JUL J L l 168 ? I ' ' • I • 1 * I ' • • 1 • • • i • • • i • - • i • - • i 88 248 288 328 C a p i l l a r y GC and mass spectrum of the re c i p a v r i n formamide (15) - 225 -J i I ! J L J i \ w . A A J L . _J 1 u, ; J r • • i i 1 J \,A i — i --5 4 3 2 1 0 1 a) 400 MHz *H NMR of methadone o x a z i r i d i n e (_5) b) Decoupled at 2.3 ppm c) Decoupled at 1.8 ppm 2. 100 MHz lH NMR of 2-(4',4'-diphenyl heptan-5'-one-2 '-yl) o x a z i r i d i n e (5_) minor diastereomer 1 1 " - 1 rf M r l 1 1 T w tf M I * to • f I ••• — 1 1 0 1 1 1 • 0 • 1 I M to 10 i n M 11 / V i —-<{Z J i i 1 1 1 1 1 1 1 t i l l I 1 3. 100 MHz lH NMR of y-phenyl-N,N, a-trimethyl-benzene-propanamine (Recipavrin) (6J 4. a) 400 MHz *H NMR of Recipavrin n i t r o n e (13) b) Decoupled at 3.96 ppm JK. iJtjiJLJLJl JulJj L Jtfl 5. a) 400 MHz *H of 2-(4',4'-diphenyl-but-2'-yl) o x a z i r i d i n e (major diastereomer) (14) b) Decoupled at 4.01 ppm 7. a) 400 MHz *H NMR of N-formyl-a-methyl-Y-phenyl-benzene propanamine (15) b) Decoupled at 4~7l ppm 8. a) 400 MHz *H NMR of 2-(N-formyl)-4,4-diphenyl-5-heptanone (31) b) Decoupled at 2.3 ppm 9 . 100 MHz 1H NMR of 2 , 2-diphenyl - 4 - v a l erol actone ( 4 1 ) - 234 -10. a) 400 MHz XH NMR of 4,4-diphenyl-2,5-heptanediol (43) b) Decoupled at 2.44 and 2.27 ppm c) Decoupled at 3.72 ppm - 235 -- 2 3 6 -C 0) "O • 1 — f — >> 4-> <u 1 CM , >> -C +-> Oi E | i n i *>> c 0) JCZ CL •r— | CO —* CO «d-««- - — -o c >o SI s_ •z. 4-O 1—1 S_ T3 IM >•> :r -C 2: <o s_ 0 +-> 0 o> +-> - 237 -14. 80 MHz 1H NMR of keto oxime (46) reduction product 16. 100 MHz lH NMR of syn and anti 1,1-diphenyl-3-butanone 17. 100 MHz XH NMR of a-methyl-Y-phenyl-benzenepropanamine ( D i n o r r e c i p a v r i n ) (53) 18. 100 MHz *H NMR of N, a-dimethyl-Y-phenyl-benzene-propanamine (Norrecipavrin) (54) 4-20. 100 MHz lH NMR of N, a-dimethyl -N-hydroxy-Y-phenyl benzenepropanamine (57) 21. 100 MHz *H NMR of 1,l-diphenyl -3-nitrosobutane-dimer (61) -| r r L i i I I I I 1 L. 22. a) 80 MHz lH NMR of syn and anti 1,1-diphenyl-3-butanone-0-methyl oxime (64) -rmr 20UU W I MO 1000 5 U U O J M O •Ml I . * f t l I H - l.OOW * CUt* SOW F K O INTCCWM. IHTCHSITV 1 2214 21279.434 211.29*9 .94* 1.261 i 4601 14294.467 141.4991 .679 1.943 3 4812 14*39. 142 141.9*27 .730 1.794 4 7414 13014.269 1*9. 3467 9.189 94.073 9 7417 13011.467 1*9.J230 7.993 29.0*1 6 7447 I**~J4.6JB 1*8.9992 . 16.291 26.000 7 77 3* 126*4. f>4* 127.4t>40 4.69* 10. 091 8 774* l * 6 * U . * 4 * 127.4*0* 3.934 9.964 » 1 104*. 7779.397 77.3193 10. 390 14.099 10 110.7 7747.3^4 77 .0013 10.6*4 14.794 I I 11068 7719.4*8 74.b8.i9 1t.4*6 14.790 1? 113*1 660 72.9493 4.671 12.367 13 t i 8 * e 6964.Id8 49.4«.u0 .»79 2.700 14 114*4 64 i * . 7 2 * 64.0044 3.6*0 11.969 19 13339 4*8*.9*7 4*.4037 4. k(i* 10.942 14 13*49 33*4.412 33.0433 3.459 7.796 1? 14740 214*.41* 21.2939 4.464 9.949 !• 1933* 9*3.600 9.1814 2.31* 6.347 ( S O "yi<^<Ntf^tt^*»HAv*A>^lif>»i|»^H«)i< U 1 turn, M»yryiy^ytvJ ~ ~ i — 100 5 0 o 23. Broad band decoupled 400 MHz 1 3 C NMR of 2-(4',4'-diphenyl heptan-5'-one-2'-yl) o x a z i r i d i n e (major diastereomer) [5) tisr i i . ir-I4MM . I I4JXJ f-PH IMItwKm- Itt l t N l l T ' i . * 1 irt '. 741* '.4111 1. ?t>\ 4. u*4 i. J4£ 00 180 170 160 150 140 130 120 110 100 90 80 70 60 50 AO 30 20 10 24. Broad band decoupled 400 MHz 1 3 C NMR of a-methyl-(N-methylene)-y-phenyl-benzenepropanamine N-oxide (13J -249-l 3 ^ * 3 T s — — • • T I J " 3 ^ - T ' p " u 3 o » -» i** )» _ - c -I ; 3 - = . t...\jr. »S ' - i / 4 i 4 PS — '.j f •. 3 "r ' . *> T » A T- '"'J ?• •>''••''?> ** * B S / " » » ^ . .O - ' . /> >J /> 0 = 3 = I . T - B * . ' m < \ 6 <"t •) * 0 3 4 t t 3^ .1,1 £ - r. j — a _ • •. f - .j _ 3 — .f ,-, « xi T. j% » Vt fv. X ''J • * J"I 2 A O .ij -jj »| 3 j) ,y ij ^ J. — * «/>1\l-.Hl3»t - '\| • t n t i i l i s j . ' \ ' i . ' 4 - i i j s > * * M •* « i M y w v ii y ^ . • * • — 1 • = 0 * •» a i j> . 3 . T v / . ij * — . 9 ! - 4 >• it '• • 'I 4 J i| i) 4 P . .'. t M t l t \ » > l l s * j -<3^^V •".•» — * s s f ^ ^ * ^ IJ -3 — 3 . * ^ ' | — S ,> T — 3 V L . « * — 4 * • Jt »• T. f- IJ JJ •. . , . 0 .-. V s. V 2 3 * 5 - t f a - • ii r 'J u s - 'j IJ r /> >. r i , i p f t 4 » ll j ( 4 4 t'- . J 9 S D 0 3 3 3 3 V O F ^ ^ K » — y -» » ^ « -v » * 3 — XI •» - - - - - - - - - - . j i , MM 1 Jv — - 250 -- 251 -2. U l t r a v i o l e t spectrum of methadone o x a z i r i d i n e (5J i n methanol (A) 3.54 x IO" 3 M (B) 3.54 x IO" 5 M 

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