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3-Amino-2-phenylthietanes as potential MAO inhibitors 1977

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3-AMINO-2-PHENYLTHIETANES AS POTENTIAL MAO INHIBITORS M.Sc. Seoul National University, Seoul, Korea, 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Division of Medicinal Chemistry of the Faculty of Pharmaceutical Sciences We accept . this thesis as conforming to the required standard by Gun-II Kang THE UNIVERSITY OF BRITISH COLUMBIA April, 1977 In presenting th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho la r ly purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l ica t ion of th is thes is fo r f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of Pharmaceutical Sciences The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 A p r i l 14, 1411 i i ABSTRACT 3 - A m i n o - 2 - p h e n y l t h i e t a n e d e r i v a t i v e s were c o n s i d e r e d as a u s e f u l t o o l t o e l u c i d a t e t h e mechanism of i n h i b i t i o n o f MAO by t r a n y l c y p r o m i n e - t y p e i n h i b i t o r s . The s y n t h e s i s o f 3- a m i n o - 2 - p h e n y l t h i e t a n e s a p p e a r e d w o r t h w h i l e from the s y n t h e t i c p o i n t o f view s i n c e no s u c c e s s f u l p r e p a r a t i o n has been r e p o r t e d f o r t h i s t y p e of compound. I t was c o n s i d e r e d t h a t t h e r e a c t i o n between s u i t a b l e 1, 3 - d i h a l ogeno a l k a n e s w i t h a l k a l i s u l f i d e would be t h e most e f f e c t i v e f o r the s y n t h e s i s o f 3 — a m i n o — 2 — p h e n y l t h i e t a n e d e r i v a t i v e s . P h e n y l s e r i n o l was p r e p a r e d by r e d u c i n g p h e n y l - s e r i n e e t h y l e s t e r u s i n g NaBH^. T r e a t m e n t of p h e n y l s e r i n o 1 w i t h t h i o n y l c h l o r i d e gave l - p h e n y l - l - c h l o r o - 2 - a m i n o p r o p a n e - 3 - o l . F u r t h e r c h l o r i n a t i o n o f t h e 3 - h y d r o x y l group was not s u c c e s s f u l . A t t e m p t s were p e r f o r m e d to s y n t h e s i z e 3-amino-2- p h e n y l t h i e t a n e v i a l - p h e n y l - l - t h i o c y a n a t o - 2 - a m i n o p r o p a n e - 3 - o l i n t e r m e d i a t e . 1 - P h e n y l - l - t h i o s u l f u r y l - 2 - a m i n o p r o p a n e - 3 - o l was o b t a i n e d by t r e a t i n g l - p h e n y l - l - c h l o r o - 2 - a m i n o p r o p a n e - 3 - o l w i t h t h i o s u l f a t e . l - P h e n y l - l - t h i o c y a h a t o - 2 - a m i n o p r o p a n e - 3 - o l was not o b t a i n e d from t h e r e a c t i o n of 1 - p h e n y l - l - t h i o s u l f u r y l - 2 - a m i n o p r o p a n e - 3 - o l w i t h sodium c y a n i d e , but 2- a m i n o - 4 - h y d r o x y l m e t h y l - 5 - p h e n y l - 2 - t h i a z o l i n e was i s o l a t e d . H y d r o l y s i s o f 1 - p h e n y l - l - t h i o s u l f u r y l - 2 - a m i n o p r o p a n e - 3 - o 1 gave l - p h e n y l - l - m e r c a p t o - 2 - a m i n o p r o p a n e - 3 - o l . i i i The u n s u c c e s s f u l attempt to prepare 1 - p h e n y l - 1 , 3 - d i c h l o r o - 2 - a m i n o p r o p a n e appeared due to the e l e c t r o n i c c h a r a c t e r of the pr imary amino group. Suppor t ing t h i s a s sumpt ion , 1 - p h e n y l - l , 3 - d i c h l o r o - 2 - b e n z o y l a m i n o p r o p a n e was s y n t h e s i z e d from N - b e n z o y l p h e n y l s e r i n o l . When an e t h a n o l s o l u t i o n of 1 - p h e n y l - l , 3 - d i c h l o r p - 2 - b e n z o y l a m i n o p r o p a n e was t r e a t e d w i t h sodium s u l f i d e , 2 - p h e n y l - 4 - b e n z y l i d e n e - 2 - o x a z o l i n e was i s o l a t e d i n s t e a d of 3 - N - b e n z o y l a m i n o - 2 - p h e n y l t h i e t a n e , i n - d i c a t i n g the ease of the e l i m i n a t i o n r e a c t i o n compared to r i n g f o r m a t i o n . The same r e s u l t was observed when 1-pheny l - 1 ,3-d ibromo-2-benzoylaminopropane s y n t h e s i z e d from c innamyl a l c o h o l was used . The r e d u c t i o n of the amide group of 1- p h e n y l - 1 , 3 - d i b r o m o - 2 - b e n z o y l a m i n o p r o p a n e u s ing d iborane was not s u c c e s s f u l . N , N - D i m e t h y l p h e n y l s e r i n o l was prepared for the purpose of s y n t h e s i z i n g 3 - N , N - d i m e t h y l a m i n o - 2 - p h e n y l t h i e t a n e v i a the i n t e r m e d i a t e of 1 - p h e n y 1 - 1 - t h i o c y a n a t o - 2 - N , N - d i m e t h y l a m i n o - p r o p a n e - 3 - o l . Synthes i s of l - p h e n y l - l - c h l o r o - 2 - N V - N - d i m e - t h y l a m i n o p r o p a n e - 3 - o l was not s u c c e s s f u l . 1 - p - N i t r o p h e n y l - l , 3-d ichloro-2-NV-N-dimethylaminopropane prepared from p- n i t r o p h e n y l s e r i n o l was t r e a t e d w i t h sodium s u l f i d e . I s o l a t i o n of the product as a h y d r o c h l o r i d e s a l t i n d i c a t e d the f o r m a t i o n of b i s ( l - p - n i t r o p h e n y l - 2 - N , N - d i m e t h y l a m i n o - 3 - c h l o r o p r o p a n e ) s u l f i d e h y d r o c h l o r i d e . 3 - H y d r o x y - 2 - p h e n y l t h i e t a n e prepared from 3 - c h l o r o - p r o p e n y l benzene was r e a c t e d w i t h b e n z y l s u l f o n y l c h l o r i d e or b e n z y l s u l f o n y l c h l o r i d e and sodium a z i d e . A l l at tempts u s i n g iv column chromatography to i s o l a t e products resulted in the i d e n t i f i c a t i o n of starting materials, revealing that the sulfonate or the azide might not be formed by the reaction probably because of the resistance of the hydroxyl group of 3-hydroxy-2-phenylthietane to the al k y l a t i o n . Discussions on the determination of the synthesized compounds using i r , nmr , uv, and gc-mass spectrometry are included. Recent concepts of the active sites of MAO and mechanisms of ir i h i t i b i o n of MAO ;. a r e . reviewed. Signature of Supervisor V TABLE OF CONTENTS Page ABSTRACT. i i LIST OF FIGURES................... x INTRODUCTION... 1 1. General description of the mechanism of action of tranylcypromine-type i n h i b i t o r s . . . . 1 2. A consideration of MAO and i t s i n h i b i t o r s . . . . 8 A. Active s i t e s of MAO 9 B. Relationships of flavoprotein with pargyline and hydrazine type i n h i b i t o r s . . 15 C. Multiple forms of MAO and their selective i n h i b i t o r s , 20 3. Thietane derivatives as potential MAO in h i b i t o r s 24 4. Synthetic routes to thietanes 26 DISCUSSION OF THE CHEMISTRY 34 1. Synthetic approach to 3-amino-2-pheny1- t h i e t a n e ( 2 2 ) v i a l-phenyl-l,3-dichloro-2- aminopropane . . 35 A. Phenylser in o l (55/, R( =R2 = R3 = H) 35 B. Threo-l-phenyl-l,3-dichloro-2-amino- propane (5_6 ,R^R^R^H) 39 2. Synthetic approach to 3-amino-2-pheny1- thietane (2_2) via a thiocyanate i n t e r - mediate 42 v i Page A. 2-Amino-4-hydroxy .methyl-5-phenyl- 2-thiazoline ( 7_4) . . 4 4 B. l-Phenyl-l-mercapto-2-aminopropane-3-ol (63) 50 3. Synthetic approach to 3-benzoylamino-2- pheny lthietane (2_3) 52 A. 1-Phenyl-l,3-dichloro-2-benzoylamino- propane (16_) 55 B. 1-Phenyl-l,3-dibromo-2-benzoylamino- propane (7_2) 59 C. 2-Phenyl-4-benzylidene-2-oxazoline (7_8) . . 61 D. 3-Benzylamino-2-phenylthietane (9_5) 72 4. Synthetic approach to 3-N,N-dimethylamino- 2-phenylthietane (2_4) and 3-N,N-dime- thylamino-2-p-nitrophenylthietane (2_6_) 74 A. N,N-D imethylpheny1ser ino1 (96) 76 B. Synthetic approach to 1-phenyl-l-chloro- 2-N,N-dimethylaminopropane-3-ol (111).... 76 C. l-p-Nitrophenyl-l,3-dichloro-2-N,N- dimethylaminopropane (.97) 78 D. Reactions of 1-p-nitrophenyl-1,3-dichloro- 2-N,N-dimethylamihopropane (97) with sodium s u l f i d e 82 5. Synthetic approach to 3-amino-2-phenyl- thietane (22) from 3-hydroxy-2-phenyl- thietane (100) 9 2 v i i Page ANALYTICAL METHODS 99 EXPERIMENTAL . 1. Synthesis of threo-phenylserine ethylester (108) 100 2. Synthesis of thr eo-phenylser i n o l (5_8) 101 3. Synthesis of threo-l-phenyl-l-chloro-2- aminopropane-3-ol HCl (6J)) 102 4. Attempted synthesis of 1-phenyl-l,3- dichloro-2-aminopropane (5_6_,R̂  = R2 = R2 = H) 103 5. Attempted synthesis of 1-phenyl-l- thiocyanato-2-aminopropane-3-ol (62) . . 103 6. Synthesis of 1-phenyl-l-thiosulfuryl-2- aminopropane-3-?ol(^l, Bunte salt) 104 7. Attempted synthesis of 1-phenyl-l-; thiocyanato-2-aminopropane-3-ol (6_2) via the Bunte salt (Synthesis of 2-amino-4- hydroxyimethyl-5-phenyl-2-thiazoline ,_74_) 104 8. Synthesis of l-phenyl-l-mercapto-2-amino- propane-3-ol HCl (6_3) ...... 105 9. Synthesis of threo-N-benzoyl-phenylserinol (65) 105 10. Synthesis of 1-phenyl-l,3-dichloro-2- benzoylaminopr opane ( 66 ) 106 11. Synthesis of 1-phenyl-l,3-dibromo-2- benzoylaminopropane (_72) 107 v i i i Page 12. Attempted synthesis of 3-benzoylamino- -2-phenylthietane (2_3) (Synthesis of 2- phenyl-4-benzylidene-2-oxazoline , 7_8) 108 13. Attempted synthesis of 1-pheny1-1,3- dibromo-2-benzylaminopropane (94 ) 110 14. Synthesis of l-phenyl-2-N,N-dimethylamino- propane-1, 3-d i o l (96_) I l l 15. Attempted synthesis of l-phenyl-l-chloro-2- N ,N-dimethylaminopropane-3-ol ( 111 ) . I l l 16. Synthesis of N,N-dimethyl-p-nitro- phenylserinol (110) 113 17. Attempted synthesis of 1-p-nitrophenyl-l- chloro-2-N,N-dimethylaminopropane-3-ol HCl (112) 114 18. Synthesis of 1-p-nitrophenyl-l,3-dichloro-2- N ,N-dimethylaminopropane HCl (9_7) 114 19. Attempted synthesis of 2-p-nitrophenyl-3- N ,N-dimethylaminothietane (2_6 ) ( Synthesis of bis (l-p-nitrophenyl-2-N,N-d imethy1amino-3- chloropropane) sul f i d e HCl (123) ... 115 20. Synthesis of 3-chloro-l-phenylpropylene oxide-1, 2 (107) 117 21. Synthesis of ^ - toluenesulf onylchloride 117 ix Page 22. Synthesis of 3-hydroxy-2-pheny1thietane(100).. 118 23. Attempted synthesis of 3-azido-2-phenyl- thietane (103) 118 24. Attempted synthesis of 2-phenyl-3-thietanone (104) 120 BIBLIOGRAPHY 121 r X TABLE OF FIGURES Figure Page 1. Ir spectrum of ,2-amino-4-hydroxy. methyl-5- phenyl-2-thiazoline (7 4) 47 2. Ir spectrum of 1-phenyl-l-mercapto-2-amino- propane-3-ol (6_3) - .53 3. Molecular structure of 2-phenyl-4-benzylidene- 2-oxazoline (7_8) 63 4. Ir spectrum of 2-phenyl-4-benzylidene-2- oxazoline (7_8) 64 5. Uv spectrum of 2-phenyl-4-benzylidene- 2-oxazoline ( 7_8) ....... 65 6. Nmr spectrum of 2-phenyl-4-benzylidene- 2-oxazoline (7_8) 68 7. Mass spectrum of N,N-dimethylphenylserinol (£6) 7 7 8. Ir spectrum of 1-p-nitrophenyl-1,3- . dichloro-2-N , N-dimethylaminopropane (9_7) 80 9. Nmr spectrum of the products from the reaction of 1-p-nitrophenyl-l,3-dichloro-2-N,N-dime- thylaminopr opane (9 7, 1 mol) with sodium su l f i d e (2.25 mol) g5 10. Nmr spectrum of bis (l-p-nitrophenyl-2-N, N-dimethylamino-3-chloropropane)sulfide (122) . 89 LIST OF ABBREVIATIONS bp - b o i l i n g point DCC - dicyclohexylcarbodiimide Diglyme - diethyleneglycol dimethyl ether DMF - dimethy1formamide DMSO - dimethyl sulfoxide EtOH - ethanol MeOH - methanol FAD - f l a v i n adenine dinucleotide FMN - f l a v i n mononucleotide gc - gas (liquid) chromatography HMPT - hexamethylphosphoric triamide 1,-Q - i n h i b i t o r concentration for 50% inhib i tion ' o f'• the enzyme a c t i v i t y i r - infrared (spectroscopy) Km - Michaelis constant MAO - monoamine oxidase mp - melting point nmr - nuclear magnetic resonance (spectroscopy) SAR - s t r u c t u r e - a c t i v i t y relationship THF - tetrahydrofurane t i c - thin layer chromatography uv - u l t r a v i o l e t (spectroscopy) ACKNOWLEDGEMENTS The author is indebted to Dr. Frank S. Abbott for his advice and encouragement throughout the course of this work. The author would l i k e to thank the Dean of the Faculty, Dr. B. E. Reidel, for giving him a chance to study in Canada. \ 1 INTRODUCTION 1. General description of the mechanism of action of tranylcypromine-type i n h i b i t o r s . Tranylcypromine (1)» because of i t s r e l a t i v e l y r i g i d and simple structure and unique geometry, has been extensively studied as a model compound to elucidate the mechanism of i n - h i b i t i o n of MAO. It is now known that MAO i s a flavoprotein and that two other important i n h i b i t o r s , phenylhydrazine (2̂ ) and pargyline (_3) e l i c i t their i n h i b i t o r y action on MAO by interaction with the f l a v i n moiety of the enzyme. Most of the SAR studies of tranylcypromine derivatives as MAO i n - h i b i t o r s were published before 1965. The relationship of tranylcypromine with active sites of MAO has not been dealt with the same d e t a i l as have recent studies of phenylhydrazine and pargyline 4 Only a broad outline of the interaction has been described. Therefore, i t appears worthwhile to generalize recent concepts of the active sites of MAO.and i t s mechanism of i n h i b i t i o n emphasizing the f l a v i n moiety of the receptor 2 and to establish the right direction for studies of tranylcy- promine-type i n h i b i t o r s . One of the important methods to estimate whether any in h i b i t o r s d i r e c t l y combine with active s i t e , or whether they complex with the enzyme at, possibly, a l l o s t e r i c sites and thereby lead to i n h i b i t i o n of binding of substrates i s based on k i n e t i c studies using Lineweaver-Burk plots. Maass and Nimmo (1) reported a non-competitive i n h i b i t i o n of rat brain MAO by tranylcypromine with respect to substrate, serotonin. According to Belleau and Moran (2) using kynura- mine as the substrate, the mechanism of MAO i n h i b i t i o n by tranylcypromine was cle a r l y of the competitive type. Zeller and Sarkar (3) observed that when tyramine and tranylcy- promine were added simultaneously to the enzyme preparation, the degree of i n h i b i t i o n was decreased with increasing sub- strate concentrations. On the other hand, no effect on i n h i b i t i o n was observed when tyramine was added 15-30 minutes after i n h i b i t o r s . Tranylcypromine is d i f f i c u l t to remove by d i a l y s i s from MAO (4). However, 40 per cent reversal of i n - h i b i t i o n was shown by d i a l y s i s against substrate (5). A long duration of action of tranylcypromine similar to that of a non-competitive i n h i b i t o r i s due to i t s high binding a f f i n i t y for an enzyme. Although a report was published that i n h i b i - tion by tranylcypromine can be reversed readily by the com- pe t i t i v e substrate, 4-phenylbutylamine (3),,this evidence was refuted by later work (7). At present, most of the evidence 3 points to the fact that tranylcypromine is a competitive i n - hibitor, having high a f f i n i t y binding and thus e l i c i t s i t s action by direct interference of the binding of substrate at the active s i t e s . It was postulated by Belleau and Moran (2) that the chemical role of the cyclopropane ring could consist in pro- 2 moting charge-transfer complex formation through i t s SP - l i k e electrons with the f l a v i n cofactor. Burger (8) also suggested that i t should be possible to draw a picture of tight chemical binding of tranylcypromine to protein-bound r i b o f l a v i n . When li m i t i n g amounts of varying mixtures of pargyline , phenylhydrazine , and tranylcypromine were incubated with the enzyme, the nature of the i n h i b i t i o n observed was additive $5). Benzylamine treatment protected MAO from i n h i b i t i o n by these i n h i b i t o r s , implying that these i n h i b i t o r s act independently, but at the same s i t e , le. the c a t a l y t i c s i t e of the enzyme. The puzzling f a c t , however, is that pargyline and phenylhydrazine reduce f l a v i n while tranylcypromine does not. Although f l a v i n reduction by benzylamine (9) is an att r a c t i v e fact which might demonstrate the c a t a l y t i c mechanism of MAO, evidence providing confirmation of the p a r t i c i p a t i o n of the f l a v i n moiety in the oxidation of substrate has not been published. The reduction of f l a v i n by inh i b i t o r s i s monitored by the increase in absorbance at 412 nm. The elaboration of better methods to monitor the i n t e r - actions of drugs or substrates with f l a v i n might reveal some forms of intereactions between them which have not yet been detected. 4 Possible interaction of tranylcypromine with f l a v i n was favored by the analogy that t r i c y c l i c psychoactive drugs (chlorpromazine 4-, amitriptyline .5, and chlorprothixene 6) i n - h i b i t MAO and those having a double bond between the ring moiety and the a l i p h a t i c side chain were the most ef f e c t i v e i n h i b i t o r s of type B MAO (10). It has been shown by Karreman et a l . (11) R _ C H 2 - C H — N ( C H 3 ) 2 4_ -R = S R — R3 = N—CH 2 R . = Cl 4 R — C H 2 C H 2 R ̂ — ̂  — ^ — ^ ̂ R1 = S R2 '̂ C—CH R. = H 4 R = Cl 4 and Yagi (12) that chlorpromazine forms charge-transfer com- plexes with r i b o f l a v i n and FAD, respectively. Molecular o r b i t a l calculations have indicated that chlorpromazine i s an extemely good electron donor (13). These data suggest that i t may be electron donating properties of the t r i c y c l i c moieties of these drugs which influence their a b i l i t y to i n - h i b i t flavoproteins such as MAO. Lineweaver-Burk plots showed mixed (amitriptyline) or competitive type (chlorprothixene) i n h i b i t i o n . Pure beef plasma MAO was inhibited by tranylcypromine (14) This i n h i b i t i o n is not related to the f l a v i n moiety since beef plasma MAO i s a diamine oxidase enzyme which i s copper- pyridoxal dependent. It has also been reported that pargyline showed i n h i b i t i o n of beef plasma MAO (15). Three hypotheses have been published on the binding mode of tranylcypromine to MAO. These hypotheses can ^ be reconciled and complemented each other since they emphasize dif f e r e n t points of structure requirement for MAO i n h i b i t o r s . Z i r k l e et a l . (16) postulated that three st r u c t u r a l requirements, a cyclopropane ring, an amino group attached d i r e c t l y to the cyclopropane ring, and a 2-substituent contain- ing aromatic moiety are necessary for potent in vivo MAO in h i b i t o r y a c t i v i t y . Emphasizing the possible stereochemical factors that may be involved in the binding process, they sug- gested that in the enzyme-inhibitor complex the tranylcy- promine derivatives do not assume a conformation in which the phenyl and amino groups are nearly coplanar but one in which these groups l i e in d i s t i n c t l y different planes. The good a c t i v i t y of MAO i n h i b i t i o n of the r i g i d l y b u i l t 1-amino- cycloprop (a) indane (_7) .supports this suggestion since in this derivative the benzene ring can barely attach to the same enzyme surface as the amino group. f̂ Ti T>- n h2 6 On the basis of isotope effects observed in the MAO- catalyzed oxidation of deuterium-labeled kynuramine, Belleau and Moran (17) concluded that in the oxidation t r a n s i t i o n state the carbon atoms of the amine substrate acquire double bond character and thereby approach the trigonal state. The high a f f i n i t y of tranylcypromine for the enzyme could thus be due to the fact that in ground state this amine resembles, e l e c t r o n i c a l l y and s t e r i c a l l y , the t r a n s i t i o n state of the amine substrate during the oxidation process. This hypothesis suggests that substrates and i n h i b i t o r s bind to the same active s i t e s . Their idea, however, that the N-C^-C2 atoms of the cyclopropylamine are e s s e n t i a l l y coplanar in the enzyme- in h i b i t o r complex seems unlikely as c r i t i c i z e d by Zir k l e et a l . (16), who emphasized the importance of stereochemical factors for the binding of MAO i n h i b i t o r s to the receptor s i t e ( s ) . To investigate the importance of the electronic properties of a cyclopropane ring to the binding of i n h i b i t o r s to the enzyme and to explain that the poor i n h i b i t o r y action of 2- phenylcyclobutylamine (̂ 5) i s due to i t s electronic properties rather than s t e r i c factors, 3-amino-2-phenylazetidine (9̂ ) was synthesized (18). 3-Amino-2- phenylazetidine has a unique structure which can maintain the electron density of the ring with similar s t e r i c properties to those of 2-phenylcyclobuty- lamine. The fact that 3-amino-2-phenylazetidine was 62 percent as active as iproniazid by in v i t r o testing could be used as evidence which supports Belleau and Moran's theory. 7 H 8 9 Paget and Davis (19) reported MAO i n h i b i t i o n a c t i v i t y of phenyldiaziridines (_10) , which implies that a free amino group i s not necessary for MAO i n h i b i t i o n . The synthesis of phenylaziridines (1_1_) by Wells et a l (20) helps confirm C H 3 _ N — NT-H // \\_CH NR \ , ' R = H, CH, \ / R = CH(CH ) , Y V • C H2 \\ \ R C H2 C H2 C6 H5 10 11 this evidence. These compounds are unique in that the amine nitrogen i s an i n t e g r a l part of the ring system. Similar compounds having mesoionic structures were synthesized by Wiseman and Cameron (21,22). They suggested that the TT electron system of the anhydrothiadiazolium compunds (12)and N-arylsydnones (3J3) mimics that of the cyclopropyl ring by binding to a JT area on the enzyme. / 8 Benzooxadiazoles such as substituted furoxanobenzofuroxan (14) showed in v i t r o i n h i b i t i o n of MAO (23). This evidence may further support the theory that a high electron conjugation system as well as an aryl group are requirements for e f f e c t i v e MAO i n h i b i t i o n in cyclopropylamines and s t r u c t u r a l l y similar compounds. 2. A consideration of MAO and i t s i n h i b i t o r s Amine oxidases could be c l a s s i f i e d into three groups, MAO, diamine oxidase, and diamine oxidase-related MAO. MAO contains f l a v i n as a prosthetic group. Diamine oxidase and diamine oxidase-related MAO are probably copper-pyridoxal phosphate proteins. These differences in cofactor require- ments explain the fact that diamine oxidase and diamine oxidase-related MAO accept only primary amines, whereas MAO oxidizes secondary and t e r t i a r y amines as well as primary amines (24). A recent review a r t i c l e by Youdim (25) defined MAO as the enzyme which is responsible for the oxidative deamination of such amines, as adrenaline, noradrenaline, isopropylamine , 3 , 4-dihydro;X'-yphenylethylamine , tyramine, and tryptamine. MAO (monoamine: 0^ oxidoreductase (deaminating) EC 1,4,3,4) is the name given by the Enzyme Commission of the International Union of Biochemistry. A. Active sites of MAO (1) Metal Ions The presence of metal ion at active sites is derived from the observation that chelating agents i n h i b i t mitochondrial MAO (26). Galay and Valcourt (27) even proposed that the + 2 amino group of tranylcypromine participates in an MAO-Cu (tranylcypromine)2 complex which leads to an i n h i b i t i o n of MAO. Their postulation was challenged by the fact that trans-2 phenylcyclopropylcarbinol, which is also able to form a complex with Cu ion, showed a 400- and 1500-fold decrease in MAO i n h i b i t o r y a c t i v i t y from that of tranylcypromine with human and beef, mitochondrial MAO (28). With the a v a i l a b i l i t y of highly p u r i f i e d preparations of the enzyme, metal contents were determined. Nara et a l . (29) published the presence of 0.07 % copper in preparations of mitochondrial MAO from bovine l i v e r . Youdim and Sourkes (30) reported 0.03 % of copper and 0.12 % of iron in highly p u r i f i e d mitochondrial MAO pre- parations from rat l i v e r . According to Oreland (31), copper, manganese, and molybdenium were found in n e g l i g i b l e amounts but the content of iron was 0.5-2 moles per mole of f l a v i n in pig l i v e r mitochondrial MAO. Treatment of the preparations with a strong iron chelator 1,10-phenanthroline did not decrease the enzyme a c t i v i t y , which 10 implies that iron does not play a part in the c a t a l y t i c function of the enzyme. From experiments designed to prove the r e l a t i o n - ship of chelating agents and their i n h i b i t i o n of MAO, Severina and Shermetevskaya (32,33) reported that MAO i n h i b i t o r y action of 8-hydroxyquinoline i s not due to i t s chelate formation with metal ions but to i t s a b i l i t y to bind to a hydrophobic and a polar region in the active s i t e of mitochondrial MAO. However, support for the role of iron in f u l l functional a c t i v i t y of MAO has been published from studies of iron deficiency in rats. Symes et a l . (34) reported that copper deficiency in rats does not affect enzyme a c t i v i t y but s i g n i f i - cantly lower a c t i v i t y was observed with iron deficient rats. Iron deficiency also leads to a large decrease in the rate at 14 > 14 which the animals metabolize C pentylamine to CO2• Youdim et a l . proposed that iron may be necessary at some stage in the biosynthesis of the apoenzyme. Recent studies on p l a t e l e t MAO from iron-deficient subjects indicate that this may indeed be so (34). Although the Km of p l a t e l e t MAO from iron deficient subjects is similar to the Km of the enzyme from normal subjects using the substrates dopamine, serotonin, and kynuramine, the maximum velocity is s i g n i f i c a n t l y reduced. Evidence that iron-deficient p l a t e l e t MAO i s much more sensitive to heat 14 inac t i v a t i o n and binds s i g n i f i c a n t l y less C deprenil \ per mg. p l a t e l e t protein than pl a t e l e t s from normal subjects can be explained in terms of implicating iron in the bio- synthesis of MAO protein. Youdim (36) has published a review a r t i c l e on metals and rat l i v e r mitochondrial MAO. 11 (2) H y d r o p h o b i c and P o l a r r e g i o n The p r e s e n c e of a h y d r o p h o b i c r e g i o n a t t h e a c t i v e c e n t e r of MAO i s b a s e d on the f a c t t h a t a l i p h a t i c rapnQarad-ties must have a minimum c h a i n l e n g t h i n o r d e r t o have s i g n i f i c a n t a f f i n i t y f o r MAO. S h o r t c h a i n a l i p h a t i c d i a m i n e s a r e not o x i d i z e d by MAO. I t i s o n l y the l o n g c h a i n a l i p h a t i c d i a m i n e s t h a t a r e o x i d i z e d by MAO ( 3 7 ) . McEwen e t a l (56) p e r f o r m e d k i n e t i c s t u d i e s u s i n g MAO of human l i v e r m i t o c h o n d r i a w i t h s u b s t r a t e a n a l o g s and s u g g e s t e d t h a t an e l e c t r o p h i l i c b i n d i n g s i t e o c c u p i e s a d e f i n i t e p o s i t i o n a d j a c e n t to the h y d r o p h o b i c r e g i o n of t h e enzyme a c t i v e s i t e as shown by the f a c t t h a t jg - n a p h t h o l ,but n o t o L - n a p h t h o l i s a p o t e n t c o m p e t i t i v e i n h i b i t o r of t h e enzyme. Some e v i d e n c e was p r o p o s e d t h a t t y r a m i n e b i n d s to the n u c l e o p h i l i c p o l a r r e g i o n and t h a t e l e c t r o p h i l i c p o l a r r e g i o n i n t h e immediate v i c i n i t y of some h y d r o p h o b i c r e g i o n i s e s s e n t i a l f o r t h e b i n d i n g of s e r o t o n i n ( 3 3 , 3 5 ) . (3) S u l f h y d r y l group E a r l y a s s u m p t i o n s of t h e i m p o r t a n c e of a s u l f h y d r y l group stemmed from the f a c t t h a t a l k y l a t i n g a g e n t s w h i c h r e a c t w i t h s u l f h y d r y l groups cause a d e c r e a s e i n e n z y m a t i c a c t i v i t y ( 3 9 ) . E r w i n and H e l l e r m a n (40) p u b l i s h e d e v i d e n c e t h a t s u l f - h y d r y l g r o u p s might be d i r e c t l y i n v o l v e d i n c a t a l y s i s b a s e d on the p r o p o r t i o n a l i t y between the o b s e r v e d a c t i v i t y of b e e f k i d n e y MAO and measured s u l f h y d r y l group c o n t e n t . C o n t r a r y f i n d i n g s were p u b l i s h e d by Gomes e t a l . (41) t h a t s u l f h y d r y l 12 groups do not play a c a t a l y t i c role but probably only a str u c t u r a l role. It is well known that protein sulfhydryl groups play a role in s t a b i l i z i n g t e r t i a r y structure through hydrogen bonding and hydrophobic int e r a c t i o n . Even sulfhydryl groups which are not present in the c a t a l y t i c s i t e react with alkylating agents to affect conformational s t a b i l i t y and cause a decrease in enzyme a c t i v i t y (42). Beef l i v e r mitochondrial MAO contains seven sulfhydryl groups per 100,000 grams of MAO (41). Seven sulfhydryl groups were also reported by Erwin and Hellerman for beef kidney MAO (40). When MAO and methyl- mercuric chloride are reacted, a l l seven sulfhydryl groups are t i t r a t e d , whereas, when harmaline or benzylamine are added before the methylmercuric chloride, only five sulfhydryl groups are t i t r a t e d . It was postulated therefore that two sulfhydryl groups are near the active s i t e of MAO which is present in a hydrophobic region and that the other five sulfhydryl groups are presumably on the surface of the enzyme playing a role in conformational s t a b i l i t y -(43) . The roles of two sulfhydryl .groups near the active s i t e have not been explained. Treatment of highly p u r i f i e d MAO with oxidizing agents not only decreased the rate of deamination of monoamines by MAO but also induced the a b i l i t y to catalyze deamination of histamine. This treatment also caused a reversible decrease in the content of sulfhydryl groups (38). These facts may suggest that oxidizing agents affect sulfhydryl groups which result in the conformational change re l a t i n g to the hydro- phobic region and induce new a c t i v i t y of deaminating diamines. 13 (4) Flavin moiety Before 1965 when MAO could not be isolated in a p u r i f i e d form, an indirect method was used to demonstrate the presence of f l a v i n . Studies of r i b o f l a v i n - d e f i c i e n t rats by Hawkins and Sourkes (44) showed that there was a decrease in hepatic MAO a c t i v i t y a r i s i n g from the deficiency. After that, the presence of f l a v i n was confirmed by absorption spectrum data, fluorescence data and evidence from thin layer chromatography of the pu r i f i e d enzymes isolated from beef l i v e r (45), bovine kidney (40), and pig brain (47). Trichloroacetic acid treatment of the enzyme does not release the f l a v i n moiety and substantial amounts were released only when the enzyme was treated with proteolytic enzyme. It was suggested, therefore, that f l a v i n may be covalently bound to the enzyme. In 1971 the structure of the f l a v i n peptide from MAO was determined (48,49,50) and authenticated through synthesis (51). f R l CH-CH— S — H2C C0R2 15 R - rest of FAD in native enzyme or rest of FMN in pure peptide R1= serylglycylglycine R2= tyrosine It was shown that pure f l a v i n pentapeptide isolated from hepatic MAO contains 1 mole each of serine and tyrosine, 14 2 moles of glycine, and a cysteine which i s covalently linked to C-8 of r i b o f l a v i n (15), Tipton (52) reported that the enzyme isolated from pig brain contains, in contrast, f l a v i n in a non-covalent linkage. Salach et a l . (53) followed his procedures, but confident conclusion on the non-covalently bound f l a v i n could not be made since a l l samples of the brain enzyme con- tained some acid-extractable f l a v i n . Any confirming evidence of the p a r t i c i p a t i o n of f l a v i n in the c a t a l y t i c action of MAO have not been published. The only evidence of f l a v i n p a r t i c i p a t i o n in the oxidation of 14 monoamines was from the study using C labeled benzylamine. One mole of benzaldehyde was produced per mole of FAD in the enzyme. Spectral changes which denote reduction of f l a v i n were observed, but no semiquinone has yet been detected. The existence of semiquinone was i n d i r e c t l y shown by the procedure of Massey and Palmer ( 9 ) . A plausible mechanistic hypothesis for the reduction of the f l a v i n molecule by a substrate such as benzylamine was proposed (54). This .hypothesis described covalent bonding formation of substrate with f l a v i n molecule, reduction of f l a v i n , and formation of imine which is hydrolyzed to the corresponding aldehyde and ammonia. This hypothesis, however, was based on very l i t t l e experimental data. Mechanisms explaining the oxidation of amines by MAO propose that oxidation of amine occurs v i a a dehydrogenation step to y i e l d a s c h i f f base. This idea i s based on the very firm evidence that aldehyde and ammonia, in the case of a primary amine, are products of the enzyme reaction. 15 E + RCH 2—— NH2 — -> EH 2 + RCH = N'H RCH = NH + H20 i> RCHO + NH^ , EH 2 + 0 2 > E + H 20 2 This does not give a satisfactory description of the molecular mechanism in terms of a f l a v i n moiety or any other c a t a l y t i c s i t e alth ough i t seems real that f l a v i n plays a role in that function. If t e r t i a r y amines are substrates for MAO, the formation of a s c h i f f base seems unlikely since such amines do not have a hydrogen atom attached to the nitrogen. Previous attempts to explain this anomaly seem unsatisfactory, either invoking a protonated substate (6) or postulating hydride ion abstract- ion (46) from the oi-carbon atom. Williams (55) hypothesized the presence of a lysine residue at the active center of MAO and proposed a plausible dehydrogenation mechanism applicable to primary, secondary, and t e r t i a r y amines. This report, however, did not show how an oi -proton is abstracted and any involvement with f l a v i n . The presence of a, polar region whether i t i s lysine or not seems r e a l . B. Relationships of flavoprotein with pargyline and hydrazine type i n h i b i t o r s . (1) Pargyline type i n h i b i t o r s According to Hemmerich (59), the overlap between f l a v i n and s u b s t r a t e 7 T - o r b i t a l s within the c a t a l y t i c a l l y active 16 complex can hardly be the mode of the f l a v i n substrate contact since for e f f i c i e n t 7T -overlap a rather large area of contact is needed, and this requires less s t e r i c r e s t r i c t i o n s than obviously exist. "Sigma character was expected for f l a v i n - substrate contact at the two acceptor s i t e s , ;C(4a) and N(5)>. In 1972 a covalent cycloaddition product of flavoquinone and an acetylenic enzyme i n h i b i t o r was prepared and elucidated by i r and nmr spectroscopy. The addition of 2-propynylamine occurred to the C(4a)=N(5) azomethine grouping of oxidized enzyme (58). The same procedure of using a flavoquinone model system was applied to pargyline and i t was found that a covalent complex which shows disappearance of peaks represent- ing the quinonoid structure was formed and that the t r i p l e bond was replaced by an o l e f i n i c group which was a part of an enamine system (60). MAO from beef l i v e r was p u r i f i e d . Addition of pargyline to this p u r i f i e d MAO in the absence of l i g h t induced the appearance of a 410 nm peak with disappearance of the 460 nm peak which denotes reduction of the f l a v i n s moiety. Several important facts were observed (60). Optical changes at 410 nm closely paralleled the degree of i n h i b i t i o n . The number of t i t r a t a b l e sulfhydryl --groups did not change after i n a c t i v a - tion of the enzyme. Pu r i f i e d MAO does not respond by display- ing the 410 nm peak when i t i s treated with substrate or with tranylcypromine and i p r o n i a z i d . This implies that the mechanism of catalysis and i n h i b i t i o n of MAO by tranylcypromine or 17 iproniazid seems to be quite different from that of acetylenic amines. By using MAO of bovine kidney, i d e n t i c a l results were reported (61). Spectroscopical data that showed loss of long wave- length absorption (450-500nm) and development of a new band at 410 nm was also observed by experiments using 3-dime- thylamino-1-propyne and bovine l i v e r MAO (62). Instead of C(4a)=N(5) adduct formation N(5) adduct formation,was pro- posed. This result is different from the observation when lactate 2-monooxygenase is treated with the appropriate acetylenic substrate analog. In this case, the inactivator becomes attached to both N-5 and C-4a of the f l a v i n as i s the pattern of flavoquinone and 2-propynyl amine interaction (16). The mechanism by which adduct formation occurs -at N-5 (17.) i s not known. R: 16 17 Adduct formation with the f l a v i n was also reported in the case of in h i b i t o r of type B MAO (63). of l i v e r deprenil MAO at position 5 which i s a selective 18 Williams and Lawson (64) observed that a compound such as N-(2,4-dichlorobenzyl)-N-methylpropargylamine (18) i s a much better i n h i b i t o r than i t s non-halogenated analog, pargyline and that the l a t t e r i s more ef f e c t i v e than i t s desmethyl derivative, N-benzylpropargylamine (19). 18 19 Differences in the p a r t i t i o n c o e f f i c i e n t s among pargyline derivatives showed a close relationship to the effectiveness of i n h i b i t i o n of mitochondrial MAO as measured by I^Q values. This implies that the ease of penetration through a l i p i d barrier attached to the enzyme is an important factor determining the i n h i b i t i o n by pargyline derivatives. 14 Using C labelled pargyline and clorgyline i t was shown that in v i t r o , these substances f a i l to bind to proteins other than MAO, and that, a number of other inhibitors'., such as iproniazid and tranylcypromine prevent pargyline from binding to this enzyme (65). The precise mode of i r r e v e r s i b l e i n - h i b i t i o n of plasma MAO by pargyline i s not known. It is assumed that the enzyme mediated - double bond migration precedes the actual i r r e v e r s i b l e i n h i b i t i o n so that the unreactive acetylene is f i r s t converted into the highly reactive allene (15). There- fore, b e f b r e attacking the f l a v i n moiety or unknown group of plasma MAO, electron migration to the allene i s a pre- 19 requisite for acetylenic amine-induced i n h i b i t i o n . It i s also possible that other i n h i b i t o r s such as iproniazid and tranylcypromine may prevent the activation or positioning of the acetylenic amines prior to secondary attack of the enzyme This positioning s i t e may also be important for the c a t a l y t i c action of MAO. (2) Hydrazine type i n h i b i t o r s In 1972 i t was reported that hydrazine type i n h i b i t o r s are also oxidized by MAO following the same pattern as substrates. It was reported that the immediate product of this oxidation i s the hydrazone and that this hydrazone i s responsible for the i r r e v e r s i b l e i n h i b i t i o n of the enzyme. Phenylethylidene hydrazine (2_0, R= CgH,_CH2)was isolated and presented as an i n h i b i t o r (66). E + RCH2NH NH2 _ > EH 2 + RCH== NNH"2 , 2_0 EH 2 + RCH 2N= NH 21 EH 2 + 0 2 > E + H 20 2 It was not shown, however, what part of the enzyme molecule is responsible for this conversion. Phenylethylidene hydrazine (20, R = C,HCCH •••)) was also found to be a time dependent i n -6 -> 2 hi b i t o r of MAO (67). , Evidence that the f l a v i n moiety of MAO plays a part in the action of hydrazine type i n h i b i t o r s was reported (68). It was suggested that the oxidation of the hydrazine proceeds 20 with formation of the corresponding diazene (21) and this diazene i s responsible for the i n h i b i t i o n of MAO by forming an adduct with FAD. However, in the case of phenylethylhydrazine, phenylethylidene hydrazine was iso l a t e d , which may result from rearrangement of the diazene or perhaps from a different oxidation route. C. Multiple forms of MAO and their selective i n h i b i t o r s . I n i t i a l evidence on the presence of multiple forms of MAO was derived from the fact that c h a r a c t e r i s t i c bands of multiple forms were obtained by polyacrylamide gel e l e c t r o - phoresis of pu r i f i e d s o l u b i l i z e d enzyme. The same pattern was observed from tissues of s p e c i f i c o r i g i n regardless of the methods of s o l u b i l i z a t i o n employed although the number of such bands detected is variable depending on the tissue and species used. I n i t i a l work done by Youdim et a l . (69) showed 5 bands of MAO isolated from rat l i v e r . MA0-l,MA0-2, and MAO-3 were more sensitive to i n h i b i t i o n by clorgyline than MAO-4 and MAO-5 (70). Bands, MAO-4 and MAO-5 contain lower phospholipid than MAO 1-3 (7.1). However, a l l forms of rat l i v e r mitochondrial MAO have the same absorption spectra c h a r a c t e r i s t i c of the f l a v i n moiety and have similar molecular weight (72,73). Objection to the concept of multiple forms of MAO was that bands on the gel electrophoresis might be a r t i f a c t s from the s o l u b i l i z a t i o n of MAO since MAO i s bound to the outer mitochondrial layer so t i g h t l y that drastic condition have to be employed to s o l u b i l i z e and iso l a t e the enzyme. 21 It was shown that bands on the gel electrophoresis are due to the varying phospholipid content and that a homogenous band was observed after treatment of the enzyme(s) by a chaeotropic agent ( 7 4 ) . Houslay and Tipton also (74) suggested that the degree of i n h i b i t i o n by clorgyline and the double sigmoid curve obtained by clorgyline depend solely on the associated phospholipid membraneous material. Removal of phospholipid material by chaeotropic agents results in the disappearance of substrate-selective i n h i b i t i o n and the double sigmoid curve. Two sigmoidal curve was usually employed to explain multiple forms of MAO. The f i r s t curve designates i n h i b i t i o n by type A MAO, and the second by type B MAO. It i s now generally admitted that this difference in the phospholipid content of MAO is not due to an a r t i f a c t in the process of s o l u b i l i z a t i o n but constitutes important character- i s t i c s of MAO in v i t r o and in vivo. MAO exists in the c e l l strongly bound to different amounts of phospholipid. Thus, this association may in turn account for observed differences in substrate and i n h i b i t o r s p e c i f i t i e s and also explain con- formational differences between the multiple forms of MAO. Heat treatment influences the a c t i v i t i e s of MAO in the extent of oxidation of substrate and i n h i b i t i o n by i n h i b i t o r s ( 7 5 ) . A plausible explanation would be that the conformation of MAO in the s p e c i f i c environment of lipoprotein could be affected by heat treatment and thus show dif f e r e n t enzymatic a c t i v i t y . It was also reported that thermostability of MAO preparations 22 was dependent on the presence of phospholipid (76). MAO B which contains high phospholipid is more "sensitive to heat than MAO A. The effic a c y of pargyline derivatives in i n h i b i t i n g MAO is to some extent related to l i p o p h i l i c character (64). This apparent relationship between l i p o p h i l i c i t y and inhi b i t o r y potency of pargyline derivatives suggest the importance of the l i p i d environment of MAO. Yang and Neff (77) reported the existence of two types of MAO in vivo by using selective i n h i b i t o r s clorgyline and pargyline. Youdim et a l . (78) administered clorgyline or deprenil to two different groups of rats in an attempt to demonstrate the in vivo existence of type A and type B enzymes. Analysis of urine samples, however, showed inconclusive r e s u l t s . Type A MAO s e l e c t i v e l y deaminates serotonin and nor- epinephrine and is inhibited by harmine and clo r g y l i n e . Type B MAO s e l e c t i v e l y deaminates benzylamine and phenethylamine and i s inhibited by pargyline and deprenil. Tyramine, dopamine, and tryptamine are common substrates for both types and i n h i b i t o r s such as tranylcypromine and phenylethylhydrazine i n h i b i t both types to a similar extent. Serotonin, nor- i epinephrine, and clorgyline which interact with MAO A have more polar aromatic rings than benzylamine, phenylethylamine, and deprenil which interact with type B MAO. In general, adding a polar hydroxyl group to phenylethylamine (tyramine) or removing one from serotonin (tryptamine) produces a common substrate. The enzyme formed with the lower phospholipid 23 content, type A appears to be more sensitive to i n h i b i t i o n by•clorgyline since penetration through l i p i d associated with MAO may be a factor in the s e n s i t i v i t y of clorgyline towards the multiple forms of the enzyme (77). Some evidence was reported that mitochondria are pro- bably not homogenous. The multiple forms of s o l u b i l i z e d MAO, therefore, may result from dif f e r e n t preparations of mitochondria (79). However, i t was found that there i s no obvious correlation between heterogeneity of mitochondrial MAO and the ele c t r o p h o r e t i c a l l y separable multiple forms of enzyme (80). M u l t i p l i c i t y of MAO i s not universal. MAO from sources of pig brain, monkey small intenstine, and human pl a t e l e t s are homogenous. For example, MAO from human pl a t e l e t s consists of only type B MAO. Human pl a t e l e t MAO studies should not be used to i n d i r e c t l y evaluate MAO levels in the brain. Peripheral sympathetic nerves mainly show type A MAO and antihypertensive action is produced by blocking MAO in peripheral neurons. It is also generally recognized that the blockade of MAO metabolism of transmitter amines such as dopamine, norepinephrine, and serotonin in brain leads to the a l l e v i a t i o n of depression. Type A MAO i s related to these substrates. Therefore, i t seems that type A MAO i s the primary consideration for both antidepressant therapy and a n t i - hypertensive treatment. Thus in order to design a n t i - hypertensive drugs, the drug structure should provide limited penetration into the brain and accumulate in sympathetic neurons. One attempt to achieve this selective action has been 2 4 reported (81). It is also emphasized that depression i s related to the deficiency of s p e c i f i c biogenic amines at sp e c i f i c sites of the brain. This was i l l u s t r a t e d by post- mortem studies of patients who had been treated with MAO in h i b i t o r s (82). Different MAO inh i b i t o r y patterns were observed at different sites of the brain. For example, MAO of the pineal body was the most sensitive to i n h i b i t i o n by clorgyline which i s a type A MAO i n h i b i t o r . Conclusive data for these studies, however, i s yet to be obtained since such specimens are d i f f i c u l t to obtain. 3. Thietane derivatives as potential MAO i n h i b i t o r s . It has been considered in this research project that 3-amino-2-phenylthietane devivatives ( 2_2_ - _2_6_ ) could be a useful tool to elucidate the mechanism of i n h i b i t i o n of MAO by tranylcypromine-type derivatives. 22 R±= H R 2 ' R 3 = H 23 R = H R = H R = COC^H I 2 3 6 5 24 R = H R2,R3= CH 3 25 R = N0'2 R 2 ' R 3 = H _2_6 R 1 = N0 2 R2,R3= CH3 25 Relative to tranylcypromine, 2-phenylcyclobutylamine is only 1/1000 times as active by in v i t r o testing. This difference in a c t i v i t y is presumably due to the loss of electronic delocalization and geometrical factors. Con- tr i b u t i o n of geometrical factors seems to be minimally since 3-amino-2-phenylazetidine (18) and 2-benzyl-3-dimethyl amino- thietane (83) showed MAO-inhibitory a c t i v i t y . Although above successful examples may give an evidence on the importance of electronic property of cyclopane ring for the tranylcypromine derivatives, synthesis of 3-amino-2-phenyl thietane derivatives could provide a confirming proof of Belleau and Moran's hypothesis ( 1 7 ) . Sulfur i s known to part i c i p a t e in conjuga- tion and thus may enhance electron density of the ring so that attachment to the active sites of MAO could be strengthened, resulting in strong i n h i b i t o r y a c t i v i t y . Besides, special attention has been drawn from the fact that azetidine has a similar conformation to the thietanes shown by unpublished X-ray work ( 8 4 ) . 3-Amino-2-phenyl thietane 1,1-dioxide derivatives synthesized (85) proved to be poor i n h i b i t o r s of MAO. Some correlation could be drawn from the fact that chloropromazine sulfoxide does not i n h i b i t MAO ( 1 0 ) . Electron abstraction from sulfoxide i s more d i f f i c u l t and this might lead to the lack of a c t i v i t y of this compound, or s t e r i c factors may come into play. Mono—and di-substitution of the amino group of tranylcy- promine with methyl decreases a c t i v i t y only s l i g h t l y . N-Carbethoxy (27) and N-Carbobenzoxy show a f a i r degree of a c t i v i t y (16). (2 8) compounds also NHCOX 2 7 X =0C2H5 28 X = 0CH„C,.H 2-Carbobenzoxy derivative gave twice the in vivo a c t i v i t y as tranylcypromine. It was postulated that the a c t i v i t y of the acyl derivative i s due to their hydrolysis in vivo to the parent amine. In this regard, synthesis of compound 2_3 was considered to be useful. The synthesis of 3-amino-2-phenylthietane ; derivatives appeared Worthwhile from the synthetic point of view. No successful preparation has been reported. Wells and Abbott (85) attempted to reduce 3-dimethylamino-2-phenylthietane 1,1-dioxide and 3-morpholino-2-phenylthietane 1,1-dioxide with LiAlH^, only i s o l a t i n g unidentified material resulting from ring cleavage.. Modification of a hydroxyl group of 2- phenylthietanol en route to azide formation and reduction to the amino group was not successful (83). 4. Synthetic routes to thietanes. The purpose of this section is not to'give a com- prehensive description of synthetic routes to thietanes but to explain why the reaction of 1,3-dihaloalkanes with a l k a l i sulfides i s the most prospective synthetic route to 3- a.mino-2-phenylthietane derivatives. Major synthetic routes, therefore, are b r i e f l y mentioned. Several review a r t i c l e s have been published on thietane chemistry (102, 103, 104, 105). Reduction of thietane 1,1-dioxide results in thietane. Treatment of 2_9 with LiAlH^ gave _30 (86). The a p p l i c a b i l i t y of this method is favored by the fact that thietane 1,1-dioxide -s.o2 1 R=C1, 0C0CH , CH S 3 3 29_ 3C2 is r e l a t i v e l y more stable than thietane, and modifications of oxide derivatives are more easily accomplished. Moreover, cycloaddition of enamines with sulfene provides a convenient entry to substituted thietane 1,1-dioxides which can be re- duced to thietanes having structures necessary for study as potential MAO i n h i b i t o r s . For example, 3_3 was synthesized by reducing 32 prepared by reacting an a c e t o n i t r i l e solution N(CH,).. 33 28 of 31 and triethylamine with methane sulfonylchloride at 0-5° (83). Limitations of the reduction of thietane 1,1- dioxides to thietanes were observed in the case of 2-phenyl substituted derivatives . LiAlH^ reduction of 3h_ was attempted but offerred a complex mixture of products from which only diethylamine was i d e n t i f i e d (87). Reduction of 3_5 with N(Et-)2 , N ( C H 3 ) 2 LiAlH^ } however, i s readily accomplished to give 3-(N,N- dimethylamino) thietane (88). Wells and Abbott (85) sug- gested that theo^ proton on 2-phenyl substituted thietane 1,1-dioxides may be too ac i d i c for hydride reduction of the sulfone. Base catalysed ring opening of the 2-phenyl sub- stituted thietane 1,1-dioxides was demonstrated. By reacting chloromethylthiirane (^6) with either alkalicor a l k a l i phenoxides thietane (_3_7) was formed (89). Dittmer and Christy (90) prepared 3-thietanol by the exposure CH Cl S ArO / vj, ^ - c „ 2 C 1 * **~»^ 2 ArO 36 3_7 of 3-chloropropylene oxide-1,2 to hydrogen sul f i d e and barium hydroxide. 3-Hydroxy-2-phenylthietane (39) was prepared by 29 treating the oxide (_3_8) with hydrogen sul f i d e (83). Attempts to modify the hydroxyl group to an amino group proved un- successful (83). -CH2C1 38 3_9 Direct cycloaddition of thioketene with certain olefins ̂ gave thietanes (91). For example, bis(trifluoromethy1) ( C F 3 ) 2 C = C = S 40 CH30-<V —CK = CH2 ( C F 3 ) 2 C - OCH. 41 42 thioketene 4_0 reacted with p-methoxystyrene to give the thietane 4_2. The a p p l i c a b i l i t y of this reaction to the synthesis of 3-amino-2-phenylthietane derivatives seems limited. Thietane 44 was obtained by heating the c y c l i c carbonate of the 1,3-diol 43 with a l k a l i thiocyanate (92). The high C—0 \/ \ c c=o " W SCN ^C—SCN -CO c—0 x 2 \ / i^i -> C C^=N / \ / Cr—0 -> , C C=.N c — o - > / G Y — n -0CN 44 30 s t e r e o s p e c i f i c i t y supports the postulated mechanism of suc- cessive Ŝ 2 displacements (93). The 1,3-dioxane-2-ones were prepared readily from the corresponding 1,3-diols by ester exchange with ethyl carbonate. The advantage of this method compared to the reaction of 1,3-dibromide with sodium s u l f i d e is the greater convenience in converting 1,3-diols into carbonate ester rather than 1,3-dibromides. This process may have application to the preparation of 2-phenyl-3-amino- thietane derivatives. 1,2-Dithiolanes undergo f a c i l e desulfurization with t r i s (diethylamino)phosphide to give thietanes. By this method, the tetrahydropyranyl ester of c^--lipoic acid (4_5_) afforded (after hydrolysis) thietane 2-valeric acid (4_6/) (94). An attempted preparation of benzothiete (4_7) by this method f a i l e d . 31 47 P h o t o c y c l o a d d i t i o n of t h i o c a r b o n y l s w i t h a l k e n e s g i v e s t h i e t a n e s (4_8) and 1 , 4 - d i t h i a n s (4J9) ( 9 5 ) . The ( n , j T * ) t h i o c a r - b o n y l e x c i t e d s t a t e r e a c t s w i t h e l e c t r o n r i c h a l k e n e s to g i v e P h 2 c = s Ph CR=CH, hV (5 89 nm) Vh P*h Ph -> Ph Ph Ph R P,h-^ P h 7 ^ s Ph 48 Ph R 49 b o t h p r o d u c t s and the r a t i o depends on s t e r i c f a c t o r s and on the c o n c e n t r a t i o n o f t h l o k e t o n e . A h i g h e r e n e r g y s t a t e of t h i o b e n z o p h e n o n e r e a c t s w i t h e l e c t r o n d e f i c i e n t a l k e n e s s u c h as d i c h l o r o e t h y l e n e (5jO) to g i v e a t h i e t a n e (5_1) , s t e r e o s p e c i f i c a l l y ( 9 6 ) . Ph C = S 2 + c i C l P.h- 51 50 Ph T h i e t a n e s a r e formed by i n t r a m o l e c u l a r c y c l i z a t i o n r e - a c t i o n s from t h i o c y a n a t e w i t h sodium h y d r i d e , b u t i n t h i s c a s e , b o t h c i s - and t r a n s - p r o d u c t s a r e formed. T h i o c y a n a t e i s p r e p a r e d f r o m t h e r e a c t i o n of cyanogen b r o m i d e on: t h i o l or 32 f r o m n u c l e o p h i l i c r e a c t i o n of p o t a s s i u m t h i o c y a n a t e on h a l o g e n . NaH NaH HS OH Br CN NCS OH Tr i g l y m e NaS OCN N.CS ONa T r o s t e t a l . (97) p r e p a r e d c i s - a n d t r a n s - 2 , 4 - d i m e t h y l t h i e t a n e and 2 , 2 , 4 - t r i m e t h y l t h i e t a n e from t h i o c y a n a t e and sodium h y d r i d e . L i m i t a t i o n s of t h i s r e a c t i o n f o r t h e s y n t h e s i s of 3 - a m i n o s u b s t i t u t e d t h i e t a n e s a'.r;e due t o the f a c t t h a t t h e p r i m a r y amino group may r e a c t w i t h . e i t h e r cyanogen b r o m i d e o r sodium h y d r i d e and t h a t f o r m a t i o n of a t h i a z o l i n e may be p o s - s i b l e by r e a c t i o n between t h e p r i m a r y amino and the t h i o c y a n a t e g r o u p . U s i n g t h i s a p p r o a c h , the f o l l o w i n g s e q u e n c e s of r e a c t i o n might be a p p l i e d to the s y n t h e s i s of 3 - a m i n o - 2 - p h e n y l t h i e t a n e d e r i v a t i v e s . C o n t r o u l i s e t a l . (100) have r e p o r t e d t h e s y n t h e s i s o f 2 - n i t r o - l , 3 - p r o p a n e d i o l ( 5 2 ) . NO, '/ Vs_ "»̂ 2 — * // W T " z ^// V ^>— C H — C H — C H „ 0 H < ^ >— CH—CH—CH nH(' > OH NO, CH-CH— CH 20H SCN One of t h e common methods of p r e p a r a t i o n of t h i e t a n e s i s to r e a c t 3 - h a l o g e n a t e d t h i o l a c e t a t e , t h i o l , o r t h i o c y a n a t e (5 3) w i t h a l k a l i . 33 X SR X S X 53 R= COCH , H, CN By t h i s method, 3 - h y d r o x y t h i e t a n e was p r e p a r e d from 2- h y d r o x y - 3 - c h l o r o - p r o p a n t h i o l (101) . The e a r l i e s t and so f a r the most g e n e r a l method f o r the s y n t h e s i s o f t h i e t a n e s c o m p r i s e s t h e r e a c t i o n of 1,3- d i h a l o a l k a n e s w i t h a l k a l i s u l f i d e s or t h i o u r e a f o l l o w e d by a l k a l i n e telle?a;v'ag"e of t h e t h i o u r o n i u m s a l t ( 5 4 ) . 54 34 DISCUSSION OF THE CHEMISTRY It~'was considered that the reaction between suitable 1, 3-dihalogenoalkanes (56) and a l k a l i s u l f i d e would be the most eff e c t i v e for synthesis of 3-amino-2-phenylthietane derivatives (_57_) V Ring formation at the last step might overcome the possible i n s t a b i l i t y problem of thietanes, which i s usually encountered when attempting modification of the thietane structure. In addition, since the most favorable route i . e . reduction of thietane 1,1-dioxides from cycloaddition reactions f a i l e d to give thietanes especially in the case of 2-phenyl derivatives, i t appeared desirable to adopt the most common and c l a s s i c a l method for the synthesis of 3-amino-2-phenyl- thietane derivatives (Scheme 1). Scheme 1. Proposed Synthetic routes to 3-Amino-2- phenylthietanes (5_7) . N 55 56 R2— H > CĤ 57 35 1. Synthetic approach to 3-amino-2-phenylthietane (22) A. Phenylserinol .(5 5 , R1= R-, = R3 = H) Extensive studies have been done on the synthesis of phenylserinol (l-phenyl-2-amino-l,3-propanediol) which i s a key intermediate for this synthetic approach. This i s due to the fact that phenylserinol is one of the important intermediates for the synthesis of chloramphenicol. Three main methods of synthesis of phenylserinol have been published starting from glycine and benzaldehyde (106,107), acetophenone (108), and cinnamyl alcohol (109). The various steps are shown in methods, 1-3. Method 1 NH-CH.COOH NH0 2 2 ,2 C,H —CHO TS* C rH— CHOHCH— COONa > C^H— CH—CH—COOH 6 5 6 5 | 6 5 I C.H _CH=N ' OH 6 5 NH.HC1 NH„ l 2 I 2 ~> C,H-— CH—CH—C00C H >C,H— CH— CH— CH-OH 6 5 | 2 5 6 5 | 2 OH OH 108 58 Method 2 C-H— COCH. > C,H— COCH.Br —> C,H_—COCH. (C.H. _N. )Br 65 3 65 2 65 2 6 12 4 NHC0CH„ i 3 -> C,H—C0CH„NHo —> C-H — COCH.NHCOCH,, ;> C-H=- COCH—CH„0H o 5 1 1 6 5 2 3 65 2 NHC0CH'o NH„ I -> I 2 -> C,H—CH—CH CĤ OH > C,H— CH—CH—CH„0H o 5 j 2. 6 5 j I OH OH 36 Br -Method 3 Br > C,H_CH— CH— CHo0H 6 5 | 2 \ > C,H— CH 6 5 \ OH 0 -> C,H— CH 6 5 v CH Methods 2-3 show parts of the route representing an economical synthesis of chloramphenicol. The hydroxyl groups of phenylserinol are protected by acetylation and the t r i - acetyl derivative i s nitrated with fuming n i t r i c acid to give 1-(p-nitrophenyl)-1,3-diacetyl-2-acetylaminopropane. Acid hydrolysis, o p t i c a l p u r i f i c a t i o n , and reaction with methyldich- loroacetate give chloramphenicol. Method 1 was followed with some modification in the reduction step for the synthesis of phenylserinol used in this project. Although method 3 could be a convenient route to phenylserinol, the ease of acquiring reagents and references was considered in favor of method 1. The main product from the condensation of benzaldehyde and glycine under alkaline condition i s or d i n a r i l y threo- phenylserine. However, some erythro-compound i s also formed (106). Shaw and Fox (107) isolated each of the diastereomers from the condensation mixture by using selective r e c r y s t a l - l i z a t i o n or by selective p r e c i p i t a t i o n of the stable addition compound of the erythro form with dioxane. They also reported that the ratio of each diastereomer is dependent on the con- densation time and thus found that a 24 hours reaction time results exclusively in the threo form. Thus, following the 37 procedure of Shaw and Fox, threo-phenylserine was prepared. The orignal work by Shaw and Fox u t i l i z e d LiAlH^ to reduce phenylserine ethylester. In this experiment, NaBH^ was used for the reduction process. Synthesis of o p t i c a l l y - active^- aminoalcohols by the reduction of^-amino acid esters with NaBH^ was published by Seki et a l . (110). They found that the reduction i s accomplished in high y i e l d with more than four moles of NaBH, to one mole of ester in a 50 % ethanol 4 solvent after s t i r r i n g at 0-10° for 2 days. Application of this process to phenylserine ethylester or i t s hydrochloride salt showed that continuous extraction was necessary to obtain a high y i e l d but i t does ensure easier handling compared to us ing LiAlH^. ' Since threo-phenylserinol i s a key intermediate in this research project, several methods were used to confirm i t s structure. Melting points of threo-phenylserinol and i t s oxalic and benzoic acid salts were i d e n t i c a l with the reported values (111,112). Suzuki and Shino (113) reported a single absorption in the region of 950-1000 cm ^ in the threo- compound and 900-950 cm ^ in the erythro compound possibly due to the C-H bond of the assymetric carbon atom. Their published i r spectrum in the region of 850-1100 cm ^ was i d e n t i c a l with synthesized threo-phenylserinol. An nmr spectrum was not obtained, but Koya and Yamada (114) reported 5.16"C for threo- and 4.86~c for the erythro-benzylic proton of phenylserinol using acetic acid as solvent. The mass spectrum of threo- 38 Scheme 2. Proposed Fragmentation Pattern of threo- Phenylserinol (58) in the Mass Spectrum / NVCH-i-CH-^CH„OH OH M+"m/e 167 (2) r / ~ ^ H - C H - N H 2 OH m/e 136 (19) ^ v VCH=:H m/e 107 (26) -H« // \X + CH= 0" m/e 106 (57) + U&zz C~ NH„ m/e 118 (60) NH^CH—CH OH m/e 60 (100) -OH* + NH— CH—CH • m/e 43 (100) -H« + NH^=rC=CH m/e 42 (100) -H« A c , : (80) m/e 105 C6 H5 + ^ C4 H3 + m/e 77 (100) m/e 51 (58) 39 phenylserinol was studied. The molecular ion was observed and a fragmentation pattern consistent with the peaks observed is presented in jcheme 2. B . Threo-l-phenyl-1.3-dichloro-2-aminopropane (56, R. R, R3 = H) L l "2 According to Ikuma (115), both the threo-(5 8) and erythro- phenylserinol (5_9) give r i s e to threo-l-phenyl-l-chloro-2- aminopropane-3-ol(60) when treated with thionyl chloride. This reaction has been studied extensively by the fact that inversion of the erythro compound to threo form could be applied in the synthesis of chloramphenicol which requires a threo form. The following mechanism was suggested to explain the Walden Inversion (115). P a r t i c i p a t i o n of the amino group in the substitution of the hydroxyl group by 'HO— C- H I H— C— NIL CH20H 58 H— C —( -OH I H— C— NH„ CH20H " 59 vr c i o s o - C—H H— ;C —NH I CH20H CH20H I C l ^ | * K—C—0S0C1 H—C —0 I . > H—C— NH„ I 2 | H2 CHlOH 1 2 CH20H H— c— i r * •s—oci Xll-r C —H CL- C —H H-C—NH^HCl CH2C1 H—C-NH2 HC1 CH2OH 60 40 chlorine when treated with thionyl chloride was well known. Threo-l-phenyl-l-chloro-2-aminopropane-3-ol (^0) was synthesized by treating phenylserinol hydrochloride with thion chloride. A major difference in the i r spectrum of the product was observed in the range of 1200-1000 cm ^. In case of phenylserinol, two strong absorptions were assigned to the secondary alcohol at 1070 cm and primary alcohol at 1040 cm The chlorination product showed primary alcohol absorption at 1050 cm ^. However, the fact that reference data assigned to primary and secondary alcohol are 1075-1010 cm and 1120-1100 cm respectively (116) and that structure deter- mination performed in 1950's might be based on f r i a b l e data prompted confirmation of this structure by mass spectrometry. The mass spectrum c l e a r l y complied with the C-l chloro compound as shown in Scheme 3. Fragmentation at m/e 60 and m/e 154 implied the presence of primary alcohol and C-l chloro substitution. Threo-phenylserinol hydrochloride was treated with an excess amount of thionyl chloride in dry chloroform for the purpose of obtaining threo-l-phenyl—.'il- „3--dichlorb-3-amino- propane (56_, R̂  = R̂  = R̂  = H) . Only monochloro compound (60) was isol a t e d . The treatment of 6_0 with thionyl chloride under rigorous conditions led to i s o l a t i o n of the st a r t i n g material. A possible reason for the resistance of the primary alcohol group to chlorination by thionyl chloride is not known but may result from some p a r t i c i p a t i o n of the neighbouring amino group. Scheme 3. Proposed Fragmentation Patten of 1-Phenyl-l-chloro- r2h.aminoprc),pane-3-ole (60) i n the Mass Spectrum. ~1 + • IH- CH—CH 20H C l (M+2)+* m/e 187 (1) — H0CH„ (/ y-CH-CH=NH 2 C l m/e 154 (2) - C l - + = NH„ m/e 119 (53) 1 C l + N H ^ CH—CH 20H m/e 60 (100) -OH- -H« ^ ~ ^ - C H = C = NH2 m/e 118 (61) -HCN ft M NH 2~ CH— CH2* m/e 43 (5) r-H' + NH= C=CH 2 m/e 42 (6) m/e 91 (7) 42 2. Synthetic approach to 3-amino-2-phenylthietane via a thiocyanate intermediate. , This f a i l u r e to prepare threo-l-phenyl-1,3-dichloro-2- aminopropane led to a new synthetic scheme.using threo-1- pheny1-1-chloro-2-aminopropane-3-o1. Scheme 4. Proposed synthetic route to thietanes via a thiocyanate intermediate 22 This scheme,however, contains several problems. Reaction of a thiocyante group with a neighbouring amino group i s well documented as shown by the synthesis of thiazoline or thiazole derivatives (117). Rachlin and Enemark (118) observed that 3-chloro-(3,4-dihydroxy phenyl)ethylamine hydrochloride r : ; d ^ (64) reacts with potassium thiocyanate to form the thiazoline (6̂ 5) . 43 HO- / / \ V-CH—:CH—NH2 HC1 HO HO - V V—CH — CH. Cl 64 NHn 65 Sodium hydride is a useful reagent to convert an amine to i t s sodio derivative. Therefore, the amino, as well as the hydroxyl group may react with sodium hydride under the reaction conditions causing formation of side products. Conversion of the t h i o l group to a thiocyanate group using cyanogen bromide may-.: also involve reaction of the cyanogen bromide with the amino group. OH BrCN 66 67 An example of this possible side reaction i s i l l u s t r a t e d by the reaction of cyanogen bromide with the 1,2-amino alcohol (66) to form the t r i c y c l i c product 67 (119). In spite of the above weak points, i t was strongly .pro- posed that the product (2_2) could be synthesized via Scheme 4 through con t r o l l i n g the reaction conditions. The assumption was that the reaction of.': thiocyanate with amino group may be avoided by reacting at low temperature.. For example, the reaction of cyanogen bromide with the sodium salt of the t h i o l - type thiamine (68) at ice-cooling temperatures afforded the 44 CH, 'NH2 .CHO L .SNa CH; NH„ ,CHO CH2CH2OH / V CH3 CH2CH2OH 68 69 cyanothiamine (<6_9) (98) . Dissolving the cyanothiamine in H20 followed by treatment of a l k a l i gave the thietane (70) (99). A. 2-Amino-4-hydroxy . methyl-5-phenyl-2-thiazoline (74) Thiocyanate anion is a strong nucleophile, but usually substitution of chlorine by potassium thiocyanate requires refluxing temperatures (120). To ensure that the reaction would proceed at low 'temperature, dicyclohexyl-18-crown-6 (]_3^) was used. These polyether complexes of sodium, potassium, and related cations by neutral molecules lead-- to s o l u b i l i z a t i o n of the salt in aprotic solvent and increasing dissociation of the ion pairs provides highly reactive, unsolvated anions (121). The hydrolysis of s t e r i c a l l y hindered esters (71) is greatly accelerated in the presence of the appropriate polyether (122). The reaction of benzylchloride with potassium thiocyanate in the presence of Kryptofix 222 in chloroform for 6 days at room temperature gives an 80 % yi e l d of benzylthiocyanate (72). 45 C02CH3 COOH H C CH„ ' Dicydohexyl- , C R 18 — crown - 6 KOH 71 C H 2 C 1 KSCN Kryptofix 222 CH2SCN 72 73 The potassium thiocyanate complex of dicyclohexyl-18- crown-6 was prepared by the method of Pederson (123) . Monochlof.d compound (60) dissolved in methanol was treated with polyether-potassium thiocyanate complex at room tem- perature. However, attempts to separate reaction products from the polyether complex were not successful. While polyether e f f i c i e n t l y activates the anionic function in aprotic solvent, 60_ was only soluble in pr o t i c solvents. Polyether (_7_3) i s a mixture of 2 stereoisomers and i s soluble in both protic and aprotic solvents. Therefore, separation i s a problem unless the product is precipitated out or can be d i s t i l l e d . It was not possible to confirm the formation of any thiocyanate product by i r spectroscopy of the reaction mixture. 46 Another route to synthesize thiocyanate compounds instead of direct reaction with potassium thiocyanate i s by using organic thiosulfate (Bunte s a l t ) . As shown in Scheme 4, the Bunte salt (61) reacts with a l k a l i cyanide forming the thiocyanate (6 2) at room temperature. The reaction i s usually completed in half an hour (124). Treatment of 60^ with sodium thiosulfate gave 1-phenyl-l-thiosulfuryl-2- aminopropane-3-ol (6>_1) . The i r spectrum showed strong absorption at 1200 and 1230 cm ^ indicating the -S02~ group. Presence of a strong band at 640 cm further proved absorption by the -SO2O- group. It is evident that a strong band at 640 cm ^ could be used s p e c i f i c a l l y to d i f f e r e n t i a t e -SO2CI from -SO2OH of i t s hydrolyzed product as shown by the example of benzylsulfonic acid and benzylsulfonylchloride. I d e n t i f i - cation by mass spectrometry was not successful because of i t s high melting point. The Bunte salt (6_1) was dissolved in water by adding sodium carbonate and s t i r r e d at room temperature with an equimolar amount of sodium cyanide. Product precipitated out which appeared to be a thiazoline on the basis of a strong band at 1640 cm ^ of the i r spectrum (Fig.l) . In thiazoline, a band near 1640 cm has been signed as the C = N stretching vibration in the absence of external conjugation (125) . The formation of thiazoline (7_4) was further confirmed by mass spectrometry. The mass spectrum reported for  48 2-aminothiazoline (75) shows three f i s s i o n s across the ring as described below (126). Synthesized thiazoline (7_4) gave the molecular ion and also fragmented by a_ and b_ f i s s i o n . m/e 56 m/e 45 Scheme 5. Proposed Fragmentation Pattern of 2-Amino-4- hydr.bxy:';methyl-5-phenyl-2-thiazoi.ii.f1]e (74) ^ \ j - C - C H 2 0H~| + ' \// ' a C M — HOCtL NH2 + . m/e 208 (3) Y NH„ m/e 177 (100) b H H // W c - i + + HC= S m/e 135 (25) m /e 45 (12) H H c= c —CH2OH1 "* m/e 134 H H + m/e 117 (11) + ^ V>^H=C=CH c = s m/e 121 ( 9 V * _ „ + C6 H5 m/e 77 (15) + C4 H3 m/e 51 (11) . m/e 115 (12) / f + il m/e 91 (36) 50 The f a c i l e formation of thiazoline '(74) even at room temperature indicates the ease of reaction of the thiocyanate group with the neighbouring amino group in this case and con- sequently this approach to the thietanes proved disappointing. B. l-Phenyl-l-mercapto-2-aminopropane-3-ol hydrochloride (63). An alternative approach using the compounds in Scheme 4 was investigated in a preliminary fashion. This stemmed from the fact that 1-mercapto derivatives may be of interest for the synthesis and testing of chloramphenicol derivatives and adrenergic compounds. One possible modification of the , . NH„HC1 CH„C,H.S0oCl , , NH„ (or NH-S0o C,H.CH.) /7~\ l 2 3 6 4 2 / — \ | 2 2 6 4 3 (' X)-CH- CH—CH— OH ^ (/ X)-CH—CH-CH— 0S0„ C,H.CH0 \=J k 2 V=L/ J H 2 2 6 4 3 — NH (or-NH—S0o C£H.CH„) | 2 2 6 4 3 — < g > - C / ) c H 2 s 1-mercapto compound 6_3 to thietanes could not be ruled out as shown above. The r e a c t i v i t y of tosylchloride might provide selective tosylation of the primary hydroxyl group. Tosylamide may be cleaved after formation of the thietane by alkaline conditions or by reductive cleavage. At least, tosyl sub- stituted thietane would be worthwhile to test for pharma- cological a c t i v i t y as a MAO i n h i b i t o r . However, considering the time used for this research, i t was determined to r e s t r i c t this part of the work to the synthesis of the 1-mercapto compound 51 Scheme 6. Proposed Fragmentation Pattern of 1-Phenyl-l-mercapto- 2-aminopropane-3-ol (63) in the Mass Spectrum. a b i NH • (/ y-CH-r CH-}-CH2OH + • // \\ ! + • \ V~CHrCH=NH0 \=y I i 2 SH m/e 152 (8) HS- O r N J+ SH m/e 123 (4) -H« <0̂ ch= m/e 122 (7) -H* SH (M+l)+* m/e 184 (04) CH - CH=NH2 m/e 119 (18) -H< + ^~^_CH— C—NH2 m/e 118 (26) m/e 91 (43) + C = S m/e 121 (14) G'6H5 m/e 77 (22) C4 H3 + m/e 51.(18) + NH = CH—CH20H m/e 60 (100) - HO- + NH = CH—CH^ m/e 43 (18) - H' + N H Z = C = C H 2 5 2 i t s e l f . A c i d — c a t a l y z e d hydrolysis of S-alkyl and S-aryl thiosulfate is a useful method of preparing t h i o l s ( 1 2 7 ) . Hydrolysis of Bunte salt are known A-l processes. The hydrolysis i s usually performed in s i t u without i s o l a t i o n + f a s t + - i R - S - S 0 3 + H ^ R - S - S 0 3 S ± ° ™ - > - R S H + S C > 3 using s u l f u r i c or hydrochloric acid . Concentrated hydrochloric acid was used to prepare 6 _ 3 from the Bunte salt ( 6 1 ) . The i r spectrum showed a t y p i c a l band at 2 5 2 0 cm ^ indicating the mercapto group (Fig. 2 ) . A molecular ion was observed in the mass spectrum. Fragmenta- tion patterns for 63 which account for observed peaks are shown in Scheme 6 . 3 . Synthetic approach to 3-benzoylamino -2-phenylthietane ( 2 3 ) . The approach proposed for the synthesis of.3-benzoylamino- 2-phenylthietane ( 2 _ 3 ) considered as a potential MAO i n h i b i t o r in this research project i s o u t l i n e d i n Scheme 7 .  54 Scheme 7. Proposed s y n t h e t i c r o u t e to 3 - b e n z o y l a m i n o - 2 - p h e n y l t h i e t a n e (̂ _3) . NHCOCgH -> // \\-CH- CH— CH_OCOC-.Hc 2 6 5 OCOĈ Hc 6 5 64 NHCOĈ Hr NHCOĈ Hr CH-CH—CH„OH > // \yCH—CH—CH0C1 66 NHCOĈ Hr 6 5 23 A l t h o u g h 1-pheny 1-2-aminopr opane-1 , 3 - d i o l (5_8) f a i l e d to g i v e 1 - p h e n y l - l , 3-dichloro-2-aminopropane (56, R^R^R^H) , the s y n e t h e s i s of 6_6 from 6_5 appeared p o s s i b l e . The u n s u c c e s s f u l attempt to prepare 1 - p h e n y l - l , 3 - d i c h l o r o - 2 - a m i n o p r o p a n e appeared due to the e l e c t r o n i c c h a r a c t e r of the p r i m a r y amino group. A decrease of e l e c t r o n d e n s i t y as i n the amino f u n c t i o n of the amide compound (6_5) might a l l o w c h l o r i n a t i o n of the pri m a r y a l c o h o l group. 55 A. 1-Phenyl-l,3-dichloro-2-benzoylaminopropane (66). Several methods for the synthesis of N-benzoylphenyl- ser i n o l (^4) have been published. The direct Schotten-Baumann reaction does not guarantee selective benzoylation of the amino group. Therefore, a possible route was to use the tribenzoyl compound (6̂ -) and to hydrolyze i t by using sodium hydroxide (128) . Another procedure i s to start from cinnamyl alcohol as shown below (128). M-CH=CH— CH„OH > (/ V~CH—CH—CH„0H \ — / 1 1  N ' Br Br 68 NH / v NHCOCH, // \\ " // \\ t 6 5 {' N)-CH— CH— CĤ -0-C-Ĉ Hr (' /— CH —CH— CH„ OH ^ X / I I 2 6 5 \ / I 2 Br Br N ' OH 69 « In this research project, N-benzoylphenylserinol (̂ _5) was prepared by selective hydrolysis of the tribenzoyl compound. The tribenzoyl compound was prepared by the Schotten-Baumann reaction using an excess amount of benzoyl chloride. The i r spectrum of synthesized N-benzoylphenylserinol showed amide bands but no ketone band thus implying the success of selective hydrolysis. N-benzoylphenylserinol was refluxed with thionyl chloride and work up gave crystals which showed a s l i g h t ketone band and no hydroxyl bands in the range of 1200 - 1000 cm ^ in the i r spectrum. Benzene was an e f f i c i e n t r e c r y s t a l i z a t i o n solvent to remove the contaminating iv.ketone compound. 1-Phenyl-l, 3-dichloro-2-benzoylaminopropane (66) showed a mp of 148-150. 5 6 A report has been published on the synthesis of 6̂6_ from 70 by treating with thionyl chloride (129). A mp of 131-132 was reported. No spectroscopic data was mentioned. This difference in melting point of synthesized compound from the reported one prompted confirmation of the structure of 6_6 by using spectroscopic data and elemental analysis. Two amide bands appear in the i r spectrum. Hydroxyl bands are no longer present. In the ...nmr spectrum, two ortho-phenyl protons to- the carbonyl group have signals in the 7.50-7.73^ region. The remaining eight phenyl protons appeared at 7.60-7.37 & as a multiplet. A doublet appeared at 5.32 S i s due to the methine proton. Methylene protons attached to chlorine were shown at 3.50^ ,-.a;s a multiplet. This multiplet nature seems due to the coupling not only with the neighbouring methine but also with phenyl protons. The broad bands centered at 6.40^ were assigned to the proton in the amide group. Mass data was collected for 66 and compared with the star t i n g material, N-benzoylphenylserinol (65). The fragmen- tation of N-benzoylphenylserinol is shown in Scheme 8. The base peak at m/e 164 corresponds to the m/e 60 fragment of phenylserinol. A peak at m/e 240 was observed with high 70 66 Scheme 8. Proposed Fragmentation Pattern of N-Benzoyl- phenylserinol (65) i n the Mass Spectrum 0 i l NHC Cr^r I i 6 5 CH—CH-i-CHo0H I : ' 2 OH ft A - C H — C H — N H C C . H - 6H 6 : 5 m/e 2 4 0 ( 4 5 ) -OH- (I X>-CH— CH— NHC C,.HC. o 5 m/e 2 2 3 ( 2 7 ) hH- a b i/e 2 7 1 + 7 \\_CH=0. m/e 1 0 6 ( 9 5 ) M/ + m/e 1 0 5 ( 1 0 0 ) q N H C 1 C , H C I 6 5 + C H — C H 2 0 H m/e 1 6 4 ( 1 0 0 ) -HO- NH-Cr' C6 H5 +CH m/e 147 (100) 0 ^ N^-CH=^C-NHCI C,HC 6 5 m/e 2 2 2 ( 4 5 ) -CO C 6 H 5 m/e 7 7 ( 1 0 ) 58 Scheme 9. Proposed Fragmentation Pattern of 1-Phenyl-l, 3-dihalo-2-N-benzoylaminopropane in the Mass Spectrum. 0 Il I NHC C . H C | | ; 6 5 / / > \ _ C H J - C H - ^ C H „ X L T I : 2 N — ' X , NHC C^H r 1 6 5 +CH—CH2X a b N 0 I I CH- CH—CH. X X = Cl m/e 182, 184 X = Br m/e 226, '.228 * > C 6 H 5 I I +CH —CH„ f./e 146 + C,HC— C = 0 6 5 m/e 105 C6H5 + i/e 77 C4H3 m/e 51 X = C1 m/e 271 CHr: C—CH. m/e 235 H N / T V A \ / / \ \ -CH=C— CH m/e 130 + CH m/e 103 m/e 91 59 intensity by b_ f i s s i o n . As shown in the mass spectra for 66^ in Scheme 9, m/e 271 and 235 showed the rearrangement of N- benzoyldichloro compound (6 6) to the oxazoline structure. Fragmentations by ID f i s s i o n were not observed. Fragmentations by a. f i s s i o n showed ratios of natural isotopic abundances at m/e 182 and 184 for the chlorocompound 66^ and at m/e 226 and 228 for the bromocompound 7 2. 1 Elemental analysis supported these spectroscopic data. B. 1-Phenyl-l,3-dibromo-2-benzoylaminopropane (72). The ring closure reaction to thietane was i n i t i a l l y performed using the N-benzoyl-dichloro compound (6^6). As research progressed, several problems were encountered con- cerning the preparation of 66^. Rearrangement to the ester (10 9) caused a low y i e l d . The rearrangement of 65 to 109 has been well described (130) . NHCOC6H5 S0C12 / _ _ . NH2 HCl [ ' \ \ - C H ^ C H - C H 2 0 H > / / \ V C H - C H - C H 2 0 C 0 C 6 H 5 zu O H \ — / c i 65 109 Moreover, preparation of 66^ s t a r t i n g from glycine and benzaldehyde was considered a time consuming process. There- fore, 1-phenyl-l, 3-dibromo-2-benzoylaminopropane (_7_2) was synthesized to substitute for the dichloro compound. The synthesis of 72 star t i n g from cinnamyl alcohol (67) has been reported in the patent l i t e r a t u r e (131,132). i 60 The cinnamyl alcohol, mp 30-33, has been assigned the trans H H H HCl HN C=C—CH„ OH H 6 ? 1 ' B r B r 68 ^ Br " 7 1 " 5 q . . H II HN . H ^ C6H5 I' \\-C-C — CH— O-C — C.H^s- (' \\—C— C—CH„Br NV-  — C — CH-— 0 — C— C.H \>— C— C—CH.I •s ' Br Br x ' Br H . 6 9 72 configuration. Assuming that halogens, as i s generally recognized, add p r e f e r e n t i a l l y to o l e f i n i c double bonds in a trans fashion (133) , the dibromide (6J5) may be assigned the erythro configuration. A dry ether solution of 6 8 and benzonitrile upon saturation with dry hydrogen chloride and standing in the cold gave 7_1 which was then converted to i t s base by treatment with sodium carbonate. The y i e l d was dependent upon anhydrous conditions of the reaction mixture in the condensation step. Compound 6̂9 was c a r e f u l l y dried before use. Refluxing 6̂9 dissolved in dry toluene gave an i n i t i a l p r e c i p i t a t e , the i r spectrum of which was i d e n t i c a l to the hydrobromide of 6_9_ reported by Taguche et al (130) . Further refluxing and evaporation of the solvent under reduced pressure resulted in p r e c i p i t a t i o n of the N-benzpyl dibromo compound (_72_) • The i r and nmr spectra of 7_2 were i d e n t i c a l to those of the N-benzoyl dichloro compound (6j6) . The mass spectrum also showed the same pattern of fragmentation as that of the N-benzoyldichloro compound (Scheme 9 ) . A high intensity 61 peak at m/e 235 was s i m i l a r l y i n d i c a t i v e of formation of an oxazoline. C. 2-Phenyl-4-benzylidene-2-oxazoline (78) When an ethanol solution of 1-phenyl-l,3-dichloro-2- benzoylaminopropane (66) (or 1-phenyl-l,3-dibromo-2-benzoy- laminopropane (7_2)) was treated with sodium s u l f i d e , the solution turned to a yellow color. After refluxing for 2 hours and d i s t i l l i n g the solvent, the residue was dissolved in water and extracted with chloroform. A viscous residue was obtained after removing the chloroform. TIC of this residue on s i l i c a gel plates showed 4 spots with R^sO, 0.2, 0.5, and 0.7. The major spot was at R̂  0.7. Preparative column chromato- graphy using chloroform as eluant was successful in i s o l a t i n g each of the components. Components of R^s 0, 0.2, and 0.5 showed amide bands in their i r spectra. Amide bands were not present for the major component (Fig. 4). Since components with R̂  0.2 and 0.5 were isolated in very small amounts, further attempts to determine their structures were not undertaken. The main ef f o r t was concentrated on determining the structure of the major component at R̂  0.7. Absence of amide bands in their spectrum did not support formation of a thietane. Extensive p u r i f i c a t i o n of the s o l i d from hexane using a dry ice-acetone bath elevated the melting point from 60-80° to 90-93° and showed no s i g n i f i c a n t changes in the i r pattern. The yellow coloration denotes high conjugation of this compound. 62 Several possible reactions leading to the absence of amide and a high degree of conjugation were examined and are i l l u s t r a t e d in Scheme 10. Scheme 10. Proposed products from the side reactions of dibromocompound (72) with sodium s u l f i d e . The f i r s t clue to the solution of this problem was obtained by the results of elemental analysis. The a n a l y t i c a l data was consistent with either structures _7_5 or. 7_8. If the major component i s r e a l l y 7 5 or 7 8, i r is not an e f f e c t i v e tool to s e l e c t i v e l y i d e n t i f y this compound1. The i d e n t i f i c a t i o n of C = N absorption in conjugated c y c l i c systems i s rendered Fig. 3 Molecular structure of 2-Phenyl-4-benzylidene-2-oxazoline (78).   66 d i f f i c u l t by the interaction with other double bonds. Specific assignments of C = N frequency have not been possible in tetrazoles, benzthiazoles, and thiazoles. The absorption bands found in the 1650-1500 cm \ region in such compounds can only be associated with the entire ring system (116). The uv absorption of the major component showed three 4 4 absorption maxima at 201 nm (£2.4x10 ), 214 (6 1.9x10 ), and 4 344 (£ 1.6x10 ) (Fig.5). Ethanol was used as a solvent. Absorption at 344 nm indicated the presence of extensive con- jugated double bonds. According to the absorption data on oxazole derivatives (134), highly conjugated 2,4,5-tri- phenyloxazole (7_9_) showed absorption at 306 nm, whereas 2,4- diphenyloxazole (8_0) and 2,5-diphenyloxazole (8J-) showed absorption at 280 and 275 nm respectively. 79 80 81 Considering these oxazole examples and the fact that compound 78 i s more extensively conjugated than isomer 15_, the uv data was considered to favor structure 78. Mass spectrum data showed a and b f i s s i o n of the ring (Scheme 11). Molecular ion was observed at m/e 235. Base peak was m/e 105. 6 7 Scheme 11. Proposed Fragmentation Pattern of 2-Phenyl-4- benzylidene-2-oxazoline (78) in the Mass Spectrum. b m/e 51 (11) m/e 91 (23)  69 The molecular structure for 7_8 with the configuration shown in Fig. 3 was constructed to f i t the observed nmr data (Fig.6). The proton a is equidistant to the equivalent protons b of the ring. The peaks observed were a doublet at 5.05<S(H. ) and a t r i p l e t at 5.73^(H.„) with the long range coupling constant a' being 3 Hz. The low f i e l d bands for the aromatic protons are not as easily explained but were given the following interpre- tation. After substracting the contribution of solvent (CHCl^) from the band at 7.10-7.40^, the band centered at 7.20 ^ represents 6 protons while the band centered at 7.80 S\ represents 4 protons. The lower f i e l d band at 7.8<$~was assigned to the c , d, e, and f protons, the deshielding ; of these ortho aromatic protons occurring because of the paramagnetic influence of nitrogen and oxygen atoms in the ring. In addition to the major compound 7 8, the compound with R̂ =0 was isolated as a small amount of s o l i d . Amide bands (amide -1 ' -1 -1 I, 1650 cm , amide II, 1540 cm ),-CH— (1420 cm ), and primary OH (1050 cm were observed in the i r spectrum. A proposed struc- ture 82_ was supported by mass spectral data in which such ions as m/e 148 and m/e 222 were observed (Scheme 12). The formation and i s o l a t i o n of the oxazoline 7_8 in this work i s not without precedent. Amides (8 3) enter into reactions especially displacement reactions, because of their n u c l e o p h i l i c C— N— < C— NH— < > — C=NH 84 ' 8_3 85 character. Displacement by amide groups can occur either through the intermediate 8_5 or under strongly basic conditions, through the amide anion 84- (135) . This is i l l u s t r a t e d by the following set of reactions. Under strongly 70 Scheme 12. Proposed Fragmentation Pattern of l-Phenyl-2- benzoylamino-l-propene- o l (82) i n the Mass Spectrum. NHCO C.HC i 6 5 < ^ ~ ^ - CH= C —CH 20H + NH - C^H CO 6 5 ft M - CH=C— CH20H m/e 148 (10) NM // \\_ / \ '' N>-CH=C CHOH m/e 147 (9) + V NH H= C CH m/e 130 (4) // W c H = CH m/e 103 (17) m/e 91 (9) 0 NHC C.HC I 6 5 (V Vw-CH=C+ m/e 222 (3) -C.H CO 6 5 + \V CH— C - NH m/e 117 (6) + '/ \\_C=0 m/e 105 (99) C 6 H 5 + m/e 77 (45) C 4 H 3 m/e 51 (11) 71 a l k a l i n e c o n d i t i o n s t h e T - b r o m o b u t y l a m i n e (86) forms t h e p y r r o l i d o n e J3_9_ v i a t h e amide a n i o n . H e a t i n g or e t h a n o l y s i s 86 88 A //^N R Br 0 H KOH fusion Br N X ) 0 NR Br N x0 8.7 89 of ^6^ g i v e s the i m i n o v a l e - r o l a c t o n e h y d r o b r o m i d e (8_7) v i a t h e i n t e r m e d i a t e 85^. The f o r m a t i o n o f t h e o x a z o l i n e 9JL by m o l e c u l a r s u b s t i t u t i o n from t h e r e a c t i o n of N - 2 - b r o m o e t h y l b e n z a m i d e (90) w i t h m e t h o x i d e i o n has been p u b l i s h e d ( 1 3 7 ) . A s i m i l a r r e a c t i o n i s i l l u s t r a t e d by t h e c o n v e r s i o n of o i - b e n z o y l a m i n o - p - r - / C,HCC-N-CH0 CH„Br + 0 CH 0 — > C,HCC ̂  6 5 2 2 3 6 5 0-CH„ + CH30H + Br 90 N-CHr 91 c h l o r o p r o p i o n i c a c i d (91) to 2 - p h e n y l - 4 - c a r b o x y o x a z o l i n e (93) i n a p p r o x i m a t e l y 50 % y i e l d i n sodium b i c a r b o n a t e s o l u t i o n (138) 72 H CH CH-COOH CH. CH" COOH I I ^ I I Cl NH > 0 ,N C = 0 C / I Rh RH 92 93 It appears therefore that In this work the reaction of the 1,3-dibromobenzamido compound (_7_2) with the mildly alkaline sodium sul f i d e favors formation of the oxazoline 7_8 via an intermolecular substitution reaction. The pr e f e r e n t i a l formation of the str u c t u r a l isomer 78 and the i s o l a t i o n of the unsaturated compound 8_2 would indicate that elimination of the 1-bromo group occurs prior to ring formation. One can not rule out a thietane structure as a possible intermediate to the oxazoline (̂ 7J3) , but the absence of appreciable amounts of any organic sulfur compound in the reaction products makes this unlikely. (4) 3-Benzylamino-2-phenylthietane (95) The reduction of the amide carbonyl group of 7_2 was attempted to remove the p o s s i b i l i t y of oxazoline formation due to the amide group. The end product would then be the benzylaminothietane 9_5. A number of methods have been published 73 NHC C H NHCH CH, v CH—CH—CH^Br I 2 Br 72 94 95 on the reduction of the amide functional group. Methods using sodium acyloxyborohydride (139) and sodium borohydride (140) are examples. Alkaline conditions and any reducing agents causing hydrogenolysis of the carbon-halogen bonds, however, could not be applied in this procedure. Diborane has been successfully u t i l i z e d for the reduction of halogen-substituted amide derivatives to the corresponding halogen-substituted amines (141,142,143,144). Zweifel and Brown (145). After reaction, excess diborane and diborane adduct were destroyed by adding absolute ethanol. Hydrogen chloride solution was avoided to prevent the addition of any possible water which might cause hydrolysis or re- arrangement. Dry.hydrogen chloride gas was used since i t was f e l t that the reduction product 9_4 should be isolated as the hydrochloride s a l t to avoid possible formation of an aziridinium compound. Work up gave a residue. The i r showed a. strong ketone band which might have resulted from rearrangment of the starting material under the acidic conditions. The end result implied that amide 7 2 i s reluctant to reduction under the conditions employed. Further attempts at the reduction of 7_2 were not performed. Diborane was generated externally by the method of 7 4 4. Synthetic approach to 3 N,N-dimethy1amino-2-phenyl- thietane (2_4) and 3-N,N dimethylamino-2-p^nit>r-o - phenylthietane (26). A further consideration of Scheme 4 suggested that thietane synthesis may be possible by this route i f the amino group of phenylserinol was replaced by a dimethylamino group. This stemmed from the fact that the formation of thiazoline which was encountered in the case of primary amine would be alkyla t i o n with formaldehyde and formic acid derivatives (Clarke- Eschweiler method) has proved to be a useful method for the pre- paration of methylated amines. According to a report (147) which described the application of the Clarke-Eschweiler method to the synthesis of N,N-dimethylephedrine, formaldehyde, formic acid, and sodium formate (1:1:1) gave high y i e l d s . The marked reduction of carbonyl side product by the addition of sodium formate was considered to presumably occur through enhancement of the reduction step of the methylation sequence avoided by using a t e r t i a r y amine group (Scheme 13). Reductive (148) . Scheme 13. Proposed synthetic scheme to 3-N,N- dimethylamino-2-phenylthietane (24) and 3-N,N-dimethylamino-2-p-nitro- phenylthietane (26) NH„ 58,R=.H 117,R=N02 R-(^ ^-CH-CH-CH^H OH 96,R=H 110,R=N02 R- N(CH ) I 3 2 -CH-CH-CH2OH OH N(CH3)2 N(CH3)2 R-( / M-CH-CH-CH2OH 113 R=H 114, R=NO, U5_,R=H 116,R=N02 R- -CH-CH-CH OH SCN N(CH3)2 ,58,R=H 26,R=NQ2 R-V ^-CH CH. 76 A. N , N - ( D i m e t h y l p h e n y l s e r i n o l (96) . N , N - d i m e t h y l p h e n y l s e r i n o l was p r e p a r e d u s i n g t h e C l a r k e - E s c h w e i l e r method. Sodium f o r m a t e was n o t r e q u i r e d t o a c h i e v e a h i g h y i e l d . N , N - d i m e t h y l p h e n y 1 s e r i n o 1 was e a s i l y s o l u b l e i n c h l o r o f o r m , whereas p h e n y l s e r i n o l was n o t . The nmr s p e c t r u m c l e a r l y showed d i m e t h y l p r o t o n s a t 2.5 S . The b e n z y l i c p r o t o n a p p e a r e d a t 4.37 & i n d i c a t i v e o f t h e t h r e o c o n f i g u r a t i o n . As a l r e a d y m e n t i o n e d (114), t h r e o p h e h y 1 s e r i n o 1 showed t h e b e n z y l i c p r o t o n a t 4.863 , t h e e r y t h r o a t 5.14 &. S i m i l a r l y , t h e v a l u e 4.67 cT was a s s i g n e d to t h e b e n z y l i c p r o t o n o f t h r e o - 2 - a m i n o - 1 - p h e n y l - l - p r o p a n o l w h i l e t h e e r y t h r o compound showed the b e n z y l i c p r o t o n a t 5 . 2 0 ^ . In t h e mass s p e c t r u m of N,N- d i m e t h y l p h e n y l s e r i n o l ( F i g . 7 ) , t h e m o l e c u l a r i o n (m/e 195) was p r e s e n t and t h e base peak a t m/e 88 was a t t r i b u t e d t o the f r a g m e n t (CH^) 2N = CH-CB.20H. A s e c o n d l a r g e peak o c c u r r e d + at m/e 58 ((CH^) ^ = Z Y i . , ^ ) w h i c h i s d i a g n o s t i c f o r d i m e t h y l a m i n o a l c o h o l s (149). O t h e r w i s e , t h e s p e c t r u m was v e r y s i m i l a r t o t h a t f o r p h e n y l s e r i n o l . B. S y n t h e t i c a p p r o a c h to l - p h e n y l - l - c h l o r o - 2 - d i m e t h y l a m i n o - p r o p a n e - 3 - o l (110). The h y d r o c h l o r i d e s a l t of J9_6 was t r e a t e d w i t h t h i o n y l c h l o r i d e i n o r d e r to o b t a i n t h e C - l monochloro compound (112) . U n i d e n t i f i a b l e gummy m a t e r i a l was i s o l a t e d . I t was c o n s i d e r e d t h a t t h e gummy m a t e r i a l may be due to e x i s t i n g i m p u r i t i e s r e - s u l t i n g from o x i d a t i o n of t h e amino group by t h i o n y l c h l o r i d e . 77 Fig. 7 Mass Spectrum of N,N-Dimethylphenylserinol (96) + N(CH3)2r-r CH2 C6 H5 + 58(14) 1 77.(7) « + 88 (100) N(CH 3) 2= CH— CH20H + / \Y_ C - E 0 oJUJ ll UJ CH= 0' 78 Hence, another chlorination method was used. Tertiary alcohols such as t-butyl alcohol easily react with : concentrated- - hydrochloric acid to give chloro products. Secondary alcohols such as oL-phenylethylalcohol give ^ - c h l o r o ethylbenzene with aqueous hydrochloric acid (150). Rachlin and Enemark (118) prepared 1-(3,4-dihydroxyphenyl)-1-chloro ethylamine by passing dry hydrogen chloride into the suspension of norepinephrine in dry dioxane. They found that this method gave a rather pure product compared to that prepared by thionyl chloride. When N,N-dimethylphenylser i n o l (9j>) was treated with concentrated hydrochloric acid or dry hydrogen chloride gas in dry dioxane or dry ether, the result was the same gummy material. Further attempts at p u r i f i c a t i o n of this material for i d e n t i f i c a t i o n were unsuccessful. Reactions with potassium thiocyanate or sodium thiosulfate using the gummy material resulted in un- i d e n t i f i a b l e products. C. 1-p-Nitr ophenyl-1, 3-dichloro-2-N ,N-dimethylaminopropane (9_7 ) . Because of the tedious and d i f f i c u l t process of preparing the dimethylamino phenylserinol (58) , attention was turned to p-nitrophenylserinol (117) as a starting material. p- Nitrophenylserinol i s an intermediate in chloramphenicol synthesis and was obtained from a pharmaceutical firm. The D(-) threo form was assigned to the p-nitrophenylserinol from melting point and i r spectrum data. Melting points of D(-) and L(+) threo forms are 164°, whereas those of DL-threo and 79 DL-erythro are 141.5-142.5° and 107-9° respectively (112,151). This i s a case where the racemic compound has a c r y s t a l structure quite different from those of the pure enantiomers and therefore d i f f e r s in melting point and s o l u b i l i t y . p-Nitro-N,N- dimethylphenylserinol (110) was prepared by the same method as for N ,N-dimethylphenylser i n o l (j^6) . Elemental analysis showed consistent values with theory. In the spectrum of 110, a doublet for the benzylic proton at 4.5 S indicated the threo form. The two hydroxyl protons centered at 3.1 S disappeared upon adding D^O. Two phenyl protons ortho to the nitro group appeared at 8.07^ , while the two meta phenyl protons appeared at 7.47^". Six protons at 2.5 & were assigned to N-methyl protons. Intergration showed p a r t i a l overlap of the methine proton with N-dimethyl protons. The mass data was in agree- ment with the required structure; m/e 88 ((CH3)2N=CH-CH2OH) was the base peak, m/e 58 ((CH„)„N=CH„) was next most intensive, + m/e 209 (N0o C .H, CH (OH)-CH = N (.CH_ ) _ ) was observed as was m/e Z D 4 J / + 1-832 (N0„C,H,,CH (OH) CH = NH„ as shown in the case of p-2 o 4 2 hitrophenylserinol. In the case of chlorination of threo-D(-)-p-hitro-N,N- dimethylphenylserinol, possible effects on the o p t i c a l purity by thionyl chloride was considered. This consideration is based on the fact that p a r t i a l racemization would lead to d i f f i c u l t y in separation and i d e n t i f i c a t i o n of the compound. A method using DMF-S0C12 to obtain o p t i c a l l y pure products was published (152,153,154). p-Nitro-N,N-dimethylphenylserinol  Scheme 13. Proposed Fragmentation Pattern of 1-p- Nitrophenyl-1,3Sdichloro-2-N,N-dimethylamino propane (97) in the Mass Spectrum. NO N(CH3)2 2 ^ / y-CH-CH—CH 2CI Cl M+* m/e 275 (0.2) N(CH3)2 CH i/e 159 NO — <^~^V- C H ~ CH—CH Cl m/e 240 -CH=C = NCCH ) CH ft +^)—CH-^CH m/e 191 (1) N ( C H 3 ) — CH_CH2C1 m/e 106 (100) m/e 108 (32) N(CH3)|^CH—CH2» N ( C H 3 ) p CH 2 i/e 58 (5) m/e 115 (4) m/e 71 (19) N(CH3) — C=CH2 m/e 70 (6) 82 (110) was treated with an excess amount of DMF-SOC^ reagent and heated for one hour. Work up gave a c r y s t a l l i n e product. Chlorination using thionyl chloride was also performed to find out any differences in the products formed or the separation problems encountered. It was found that the i r spectra of the two products isolated from each chlorination reaction were superimposable with each other. Therefore, further pre- parations of 1-p-nitropheny1-1,3-dichloro-2-N,N-dimethy1- aminopropane were made using thionyl chloride. Extensive p u r i f i c a t i o n was required to i d e n t i f y the structure. Elemental analysis was performed. Mass spectrum data were coll e c t e d . Fragmentation patterns of the molecule showed c l e a r l y the 1, 3-dichloro structure. This is based on the mass numbers such + as m/e 106, 108 (N(CH„)=CH-CH„Cl) and m/e 191 (N0_CcH -CH= 3 2 2 2 6 4 C=N(CH 3) 2) as shown in Scheme 13. Molecular ion was observed at m/e 2 75. D. Reactions of 1-p-nitrophenyl-l,3-dichloro-2-N,N- dimethylaminopropane (9_7) with sodium s u l f i d e . The 1,3-dichloro compound (97) was treated with sodium s u l f i d e . It was assumed that the reaction of 1,3-dichloro compound (_9_7) and sodium s u l f i d e would lead to the following reaction sequences. Aziridinium salt formation would give two nucleophilic attacking positions at C-l and C-j>. Q f 11G resulting in fW& possible end products , 2_6_9 T21 'or 9*8; '-"Under alkaline conditions, 9_8 and 012:1> wbuM prob'3b_£y: form _99 and 130 respectively. 83 , N ( C H 3 ) 2 H C 1 NO—<^J^)-CH— CH — CH 2C1 C l 97 N^CH 3) 2C1 '/ \V CH— CH— CH 2 + NO SNa 1/2 Na 2S — _ ^ Na 2S o r N(CH ) C l N 0 2 \ ) ~ C H — C H — C H 2 C 1 118 119 N ( C H 3 ) 2 N ( C H 3 ) 2 • C H — CH — C H „ C 1 SNa 120 N ( C H 3 ) 2 N 0 „ — V \)-CH—CH — C H „ 2 \ - / \ / 2 98 N(CH 3-) 2 NO 2 \ -/ \V-CH C H — C H „ N ( C H . ) 0 \ / 2 3 2 121 NO, CH —CH, 99 NOj-^_y-CH=r CH— CH 2N(CH 3) 2 130 To 1 mol o f 1 , 3 - d l c h l o r o compound, 2.25 mol (50 % e x c e s s ) o f sodium s u l f i d e was added. A f t e r r e f l u x i n g the r e a c t i o n m i x t u r e f o r 1 ho u r , t h e a s o l u t i o n was f i l t e r e d to g i v e a f i l t e r cake w h i c h was t r i t u r a t e d w i t h H 20. A brown c o l o r e d , c h l o r o f o r m s o l u b l e compound was o b t a i n e d . The same compound was o b t a i n e d f r o m the r e a c t i o n a t room t e m p e r a t u r e f o r 3 h o u r s . M e l t i n g p o i n t was b r o a d (60-110) . A t t e m p t s to p u r i f y the p r o d u c t by r e c r y s t a l l i z a t i o n were n o t s u c c e s s f u l . A CHC1 3 s o l u t i o n o f the compound was t r e a t e d w i t h d r y H C l gas s a t u r a t e d CHC1 3 to o b t a i n a h y d r o c h l o r i d e s a l t . Dark b l a c k - c o l o r e d m a t e r i a l r e s i s t a n t to f u r t h e r p u r i f i c a t i o n was p r e c i p i t a t e d o u t . 84 P u r i f i c a t i o n by forming picrate s a l t was performed. Although picrate could be isolated and r e c r y s t a l l i z e d , the melting point was not consistent, ranging 75-110° depending upon the^depth of coloration. Separation by column chloromatography was not successful. A l k a l i - f u s i o n tests revealed the presence of sulfur. The nmr (Fig.9) showed a mutliplet pattern and a decrease in the ratio of dimethylamino protons to phenyl protons. The ratio was around 4 : 4 while in the case of both the sta r t i n g material, 9_7 and the thietane, 2J>, the r a t i o i s 6 : 4. Possible elimination of the dimethylamino group during the reaction was further supported by the presence of protons at 6.40 J". An o l e f i n i c proton i s usually assigned at this range. To confirm this finding, gc-mass spectrometry was performed. The major fractions were observed at retention times, 8.9 (A) and 11.3 minutes (B). The compound A contained the dimethylamino group as indicated by strong m/e 58 fragment. Fragment 58 was not present in the mass spectrum of compound B. Major fragmentation for A agrees with a proposed frag- mentation pattern for 2-p-nitrophenyl-3-N,N-dimethylamino- thietane (Scheme 14). However, since mass data alone would not give an absolute determination of the structure, further attempts at the synthesis with the elaboration of reaction conditions to obtain a compound in p u r i f i e d form s t i l l remained. A r a t i o n a l explanation of the fragmentation pattern of B was not successful. To elucidate whether this elimination of the dimethylamino group is due to simple base-cat alyzed  86 Scheme 14 P r o p o s e d F r a g m e n t a t i o n P a t t e r n of 3-N,N-dimethylamino- 2 - p - n i t r o p h e n y l t h i e t a n e (26) i n the Mass Spectrum. N(CH.)„~1+-, 3 2 CH N (CH.)_- Jl 3 2 CH 2 2 m/e 84 NO —(/ V>- CH CH„ \ — / \ / \ \ s NO 2 \ / \ N ( C H j ~ 1 + " I J 2 CH / \ CH CH m/e 206 2 129 (CH 3) 2N=CH 2 m/e 58 N(CH 3) 2 , . ^CH [' + >Y-r:w r CH CH m/e 160 N(CH 3) 2 N O ^ r N)-CH—CH . m/e 205 "CH + N\_CH—CH m/e 116 f W + N0 2~\^_ /~CH= C= N(CH 3) 2 m/e 191 + . / r^\_CH=rC=N(CH 3) 2. m/e 145 N(CH. 3) 2 CH ^ + ^ - C H — \ l H - m/e 159 + CH—CH + N ( C H 3 ) 2 ~ C H - C H 2 * m/e 71 + N(CH 3) 2- C=CH 2 m/e 115 m/e 70 87 reaction or a sodium s u l f i d e - p a r t i c i p a t i o n reaction, the 1,3- dichloro compound was treated with NaOH. No prec i p i t a t e was formed except NaCl, which implies that sodium s u l f i d e plays a role in this elimination of the dimethylamino group. Further experiments to explain this elimination reaction were not performed. A f i l t e r e d solution of the reaction mixture was evaporated and extracted with chloroform. A dark residue was obtained after d i s t i l l i n g off the solvent. Gc-mass spectrometry showed at least 6 compounds. The largest gc peak, retention time 9.3 minutes showed similar mass fragmentation to those of compound A. This could be explained by the fact that some portion of compound A i s soluble in ethanol and was retained in the reaction solvent. Attempts to i s o l a t e each compound were not performed. An e a r l i e r assumption made was that the side products resulting from the elimination of the dimethylamine group may be due to the alkaline condition of the reaction mixture and probably from the excess amount of sodium sul f i d e present. To determine the effects of the concentration of sodium s u l f i d e , a reaction was performed using 1 mol of sodium s u l f i d e to 1 mol of dichloro compound (9_7) . A s o l i d was precipitated out when the reaction mixture was at neutral pH. T r i t u r a t i o n of the s o l i d with water gave a CHCl^-soluble yellow compound. P u r i f i c a t i o n by r e c r y s t a l l i z a t i o n was not successful. An a l k a l i - f u s i o n test showed that sulfur was p o s i t i v e , which implied that the 88 precipitate resulted from Na^S-participation in the reaction. This fact was further backed up by the observation that the reaction of 1 mol of dichloro compound with 2 mol of NaOH gave only a precipitate of NaCl. The nmr spectrum (Fig.10) of the yellow material showed dimethyl protons at 2.373 , 2 phenyl protons ortho to nit r o group at 8.10 & , 2 phenyl protons meta to nitro group at 7.33 <f, and 4 protons at 3.00-363 ̂ ". The yellow compound was dissolved in ether and dry HCl gas was introduced. A white c r y s t a l was formed. After f i l t r a t i o n , the f i l t e r cake was thoroughly washed with ether and r e c r y s t a l l i z e d from EtOH-ether. The hydrochloride salt was so hygroscopic that a d e f i n i t e melting point was not obtained. However, drying at 50 at 0.1 mm for 5 hours gave a c r y s t a l showing a d e f i n i t e melting point. Gc-mass spectrometry was performed on both the base and hydrochloride s a l t . The base contained at least 3 compounds. A major peak occurred at 9.3 minutes of retention time by gc and showed the same fragmentation pattern as that proposed in Scheme 14. The hydrochloride salt was r e l a t i v e l y pure and gave the same fragmentation pattern and gc retention time as those obtained for the major gc peak of the free base. The elemental analysis of the hydrochloride salt showed that the ratio of carbon, hydrogen, nitrogen, and sulfur i s 22 : 32 : 4 : 1. This ratio strongly suggested the formation of a dimer. A ra t i o n a l explanation for the formation of the dimer was at temp t ed . (Scheme 15).  Scheme 15. Proposed Mechanism for the Reaction of 1-p- Nitrophenyl-1,3-dichloro-2-N,N-dimethylamino- propane with Sodium Sulfide. 97 118 123 91 Evidence supporting structure 12 2 as the base and structure 12 3 as the hydrochloride salt was coll e c t e d . The assignment of the protons of the base was consistent with the nmr spectrum as shown in Fig. 10. The mass fragmentation pattern of 122 and 12 3 would be the same as the one proposed in Scheme 14 since 12 2 and 123 would give 12 9 as a i n i t i a l fragmentation product. This result implies that the compund A from treatment of 9J_ with an excess amount of sodium s u l f i d e i s the same compound as the base (12 2) as a comparison of gc retention time shows. The mass spectrum of the hydrochloride salt obtained by direct probe showed fragmentations at m/e 106 and 108 with the intensity r a t i o , 3 : 1 (Cl isotope r a t i o ) , + which implied the presence of N(CH^)^^^ CH-CH^Cl resu l t i n g from structure 12 3. The data from the elemental analysis was direct evidence supporting the structure 12 3. Formation of 118 from 9_7 could be ea s i l y explained by the ease of nucleophilic attack at C-l carbon because of the p-nitro function on the phenyl group. As shown in a report 107 S 124 100 (83), exclusive formation of 100 i s due to the favorable attack of sulfhydryl ion at the -carbon of 107. By analogy, sulfhydryl anion would favor attack at the C r l carbon of 118 to give 119 rather than 120 o . : 121. Besides t h i s , the p- 92 nitrophenyl group could also contribute to the exclusive formation of 119 by favoring nucleophilic attack at the benzylic carbon atom. One can use similar argument to support the forma- tion of the dimer (122). The formation of dimer indicated that nucleophilic attack of the sulfhydryl anion of 119 on a second molecule of 118 i s highly favored over the intramolecular c y c l i z a t i o n reaction to form the thietane. To determine any effect of the concentration of 118 r e l a t i v e to sodium s u l f i d e , 97 was added to an excess amount of sodium s u l f i d e . A yellow colored-material isolated was the same compound as obtained when sodium su l f i d e was added dropwise to a solution of 9 7. These results suggest that the reaction of sodium su l f i d e with 1,3-dichloro compound would not be an e f f e c t i v e method for the synthesis of 3-amino-2-phenylthietane derivatives. The reaction of sodium su l f i d e with 1,3-dichloro compounds which do not contain 2-amino group has been success- f u l l y applied to synthesize the intermediates for l i p o i c acid (155, 156) and the 3-chloromethyl-3-hydroxylmethylthietane (157). 5. Synthetic approach to 3-amino-2-phenylthietane (22) from 3-hydroxy-2-phenylthietane (100) . Modification of a hydroxyl group of 3-hydroxy-2- phenylthietane to synthesize 3-amino-2-phenylthietane as shown in Scheme 16 was published (83). The attempts at chlorination or oxidation of the hydroxyl group of 100 were not successful. 93 Scheme 16. Proposed s y n t h e t i c r o u t e s of 3-amino-2- p h e n y l t h i e t a n e (22; 104 105 Some evi d e n c e was g i v e n which suggested a s u c c e s s f u l s y n t h e s i s of b e n z y l s u l f o n a t e (10 2)- and a z i d e (10 3) but these s t r u c t u r e s were not c o n f i r m e d . A p l a n was made to i s o l a t e the b e n z y l - s u l f o n a t e (102) and a z i d e (10 3) which would g i v e c o n f i r m i n g e v i d e n c e of a s u c c e s s f u l s y n t h e s i s of these compounds. Oxida- t i o n of a h y d r o x y l group of 100 was a l s o performed u s i n g a newly e s t a b l i s h e d method (162). T h i e t a n e (100) was s y n t h e s i z e d from 10 7 w i t h a m o d i f i c a - t i o n of the p r e p a r a t i o n method f o r the epoxide 10 7. I n s t e a d of monoperphtha.lic a c i d , m-chloroperbenzo i c a c i d was used which r e s u l t e d .in a b e t t e r y i e l d of 10 7. The i r and m e l t i n g p o i n t of 94 100 were consistent with the reported values (83) . Assign- ments of phenyl protons, methine protons, and methylene protons were the same as those reported (83). A hydroxy proton appeared at 2.98cT which was exchanged with l^O* Modification to the chloro compound (101) was not at- tempted. No newly established methods for this reaction have been published in the last 2 years. It i s certain that direct amination of 101 to 2_2 would not be favorable because of the propensity for side reactions such as ̂  -elimination and ring f i s s i o n s (159). Almost a l l attempts to oxidize 100 to 104 were made by Haya (83). Oppenauer oxidation, Moffatt oxidation, and other methods using hydrogen abstractors were included in his at- tempts. Recently, several reports have been published on the oxidation of primary or secondary alcohols. However, most of them describe s l i g h t modifications of the Moffatt oxidation. Rao and F i l l e r (160) described the process of using a solution of sodium dichiromate and s u l f u r i c acid in DMSO to oxidize primary and secondary alcohols in high y i e l d . It was found that in those oxidations DMSO acts as a solvent and not as a reactant. Omura et a l . (161) proposed a DMSO- t r i f l u o r o a c e t i c anhydride as a new reagent for oxidation of alcohols. Albright (162) found that cyanuric chloride with DMSO in hexamethylphosphoramide at -20° oxidize primary and / 95 secondary alcohols in high y i e l d . By applying Moffatt oxidation with DMSO and DCC in the presence of a s u l f u r i c acid (163) or with DMSO acetic anhydride (164), Haya only isolated dark residues which do not show ketone bands in the i r spectrum. In this experiment, application of the method of Albright did not give a positive i d e n t i f i c a t i o n for ketone formation. The reaction of 100 with benzylsulfonylchloride was performed. Benzylsulfonylchloride which was prepared from benzylchloride using a known method (165) was used. The i r spectrum of the crude product obtained by work up showed 1185 and 1365 cm ̂  bands as mentioned by Haya. Contrary to his findings that the residue was not soluble in common solvents, the residue was found to be soluble in chloroform. Since the i r spectrum did not appear to give confirmation of formation of a sulfonate ester in the reaction mixture, p u r i f i c a t i o n of the residue using column chromatography was performed. Some question was raised as to the s t a b i l i t y of the possible sulfonate ester product (102) while on the column. However, column chromato- graphic techniques for the p u r i f i c a t i o n of sulfonates synthesized for use as leaving groups have been widely used (166,167,168). The reaction mixture product did not appear to be unstable. Tic showed 4 spots at R f S A (0), B (0.1), C (0.25), and D (0.8). The reaction mixture was partitioned between chloroform and water. The chloroform layer after reducing in volume was added to a column of s i l i c a gel and eluted using the same 96 solvents as used for t i c . The major component was D while B and C were isolated as gummy materials. B was similar to unreacted 3-hydroxy-2-phenylthietane by i r and values but determination of C was unsuccessful. A was a pre c i p i t a t e having' a melting point over 200° and was assumed to be a s a l t . Absence of a band at 1370 cm ^ in the i r spectrum ruled out the p o s s i b i l i t y of a sulfonate. D was p o s i t i v e l y i d e n t i f i e d as unreacted benzylsulfonylchloride by t i c , i r , and melting point. This result strongly suggests that the reaction did not proceed under the conditions used to form the sulfonate (10 2) although some precipitate of triethylamine hydrochloride was formed. To confirm this finding, the residue from the reaction of 3-hydroxyphenylthietane and benzylsulfonylchloride was d i r e c t l y treated with sodium azide in hexamethylphosphoramide. Work up gave a residue, which showed three spots by t i c . The residue gave a strong azide band at 2110 cm in the i r spectrum. This band, however, would not necessarily be an indication of the formation of 3-azido-2-phenylthietane (10 3) since even small contamination with an azide impurity could cause a strong band in the i r spectrum. Although azides are unstable and pot e n t i a l l y hazardous (169), the i s o l a t i o n of azide products has been described. Reckendork (170) isolated azide components having an amino sugar structure by column chromato- graphy using alumina. Alumina was also used to i s o l a t e cholesteryl azide (171) and s i l i c a gel chromatography was applied 9 7 to i s o l a t e an azide compound of a cephalosporin intermediate (172). The reaction mixture from the reaction of cholesteryl 3.(?-p-toluenesulf onate with azide ion was separated by chromatographic techniques using f l o r i s i l (173). Therefore, i t appeared worthy to is o l a t e the azide (to obtain positive evidence of the reaction. The residue was dissolved in a small amount of chloroform and fractionated on a f l o r i s i l column using n-hexane. One major compound A with R̂  0.8 by t i c was isol a t e d . Elution of the column with sether gave two fra c t i o n s , B of R̂ 0.4-0.7 ( t a i l i n g ) and C showing a baseline spot by t i c . Elution with methanol gave D which showed a baseline spot by t i c . I n i t i a l l y an azide band was observed in the i r spectrum of A, which disappeared after r e c r y s t a l l i z a t i o n . A was benzylsulfonylchloride. B and C were isolated as unidentified small amounts of dark residues. The:"±r spectrum of D showed bands at 650 cm and 3390 cm ^ indi c a t i v e of a sulfonic acid but confirming i d e n t i f i c a t i o n was not successful. Haya reported that the hydroxyl group of 100 was resistant to al k y l a t i o n using methods such as triethyloxonium tetrafluoroborate, Na-EtBr, and p-toluenesulfonylchloride. Two experimental results performed also revealed that the sulfonate (102) might not be formed by the reaction of 3- hydroxy-2-phenylthietane with benzylsulfonylchloride. A sug- gestion could be made that the use of appropriate selective leaving groups and reaction conditions would be necessary to confirm these findings. 98 Since unsuccessful formation of sulfonate (102) or azide (103) appeared clerao:,, further reduction of the re- action mixture was not performed. It is well known that azides can be reduced to amines by reductive methods such.as c a t a l y t i c hydrogenation (174), LiAlH^ (169), and sodium borohydride (175). It would be of interest to use ammonium su l f i d e for the reduction of azido compounds of the thietane ring since this reagent is widely used in recent semisythetic p e n i c i l l i n (176) and cephalosporin (172) chemistry. 99 ANALYTICAL METHODS Melting points were determined using Thomas-Hoover Capillary Melting Point Apparatus. A l l melting points and b o i l i n g points are uncorrected. A Beekman IR-10 Infrared Spectrophotometer was used to record a l l infrared spectra. 60 MHz nmr spectra were determined at the Department of Chemistry, U.B.C., using a Varian T-60 NMR Spectrometer. 100 MHz nmr spectra were determined at the Fisheries Research Laboratory, Vancouver, Canada, using Varian HA-100 NMR Spectrometer. Peak m u l t i p l i c i t i e s are abbreviated as follows: s ( singlet ), d ( doublet ), t ( t r i p l e t ), b ( broad ), and m ( multiplet ) . U l t r a v i o l e t spectra were obtained using Beekman Model 25 Spectrophotometer. Mass spectra and gc/mass spectral data were obtained using a Varian MAT-111 mass spectrometer. The ionizing voltage was 80 ev unless s p e c i f i e d . Microanalyses were performed by Alfred Bernhardt:J Mikroanalytisches Laboratorium, 5251 Elbach Uber Engelskirchen, Firtz-Pregle-Strasse 14-16, West Germany. 100 EXPERIMENTAL 1. Synthesis of threo-phenylserine ethylester (108) The methods reported by Shaw and Fox (106*107) were adopted with minor modification. A solution of glycine (30 g, 0.4 mol) and NaOH (24.0 g. 0.6 mol) in 100 ml of H20 was c h i l l e d to 15°. With cooling maintained at 15° on a water bath, and with rapid s t i r r i n g , benzaldehyde (84.9 g, 0.8 mol) was added a l l at once. The emulsified reaction mixture changed to paste but further s t i r r i n g gave di s s o l u t i o n . After 30 minutes, the reaction mixture became a l i g h t syrup which turned into a p r e c i p i t a t e , followed by rapid and complete s o l i d i f i c a - t i o n . After 24 hours at room temperature, the condensation cake was fragmented and concentrated HCl (50.0 ml, ca. 0.6 mol) was added dropwise during 30 minutes with cooling on a water bath at 15°. Mechanical s t i r r i n g was continued for one hour after addition of acid. The f i l t e r cakes obtained after a c i d i f i c a t i o n were thoroughly mixed with b o i l i n g EtOH (3x200 ml) and the r e s u l t i n g slurry was f i l t e r e d each time. Alcohol washed product was r e c r y s t a l l i z e d from H20 and dried to give threo-phenylserine monohydrate (44.6 g, 56 % ) . mp 192-193° ( l i t . (158), 193-194°). A vigorous stream of dry HCl gas was passed through a suspension of threo-phenylserine monohydrate (40 g, 0.2 mol) in absolute EtOH (250 ml). The solution was accompanied by evolution of s u f f i c i e n t heat to promote gentle r e f l u x i n g . 101 The solution was concentrated to 100 ml. Addition of ether (300 ml.) and overnight storage in the cold gave threo- phenylserine ethylester hydrochloride (39.5 g, 80 % ) . mp 137- 140° ( l i t . (106) , 140°) . Dry ammonia was passed through a suspension of threo- phenylserine ethylester hydrochloride (35 g, 0.14 mol) in ether (500 ml) for 15 minutes. The granular ammonium chloride was f i l t e r e d and washed with warm ether. Evaporation of the f i l t r a t e gave threo-phenylserine ethylester (27.8 g, 95 % ) . mp 79-80° ( l i t . (106), 82-83°) i r (KBr) 1740 cm"1 (carbonyl), 1040 (sec. OH), 1580 (NH 2). 2. Synthesis of threo-phenylserinol (58) . To an ice-cold solution of NaBH^ (37.8 g, 1 mol) in 70 % EtOH (200 ml) was added dropwise a solution of phenylserine ethylester (35.5 g, 0.17 mol) in 70 % EtOH (100 ml). The mixture was s t i r r e d with ice cooling for 3 days. The extent of reaction was monitored by t i c ( s i l i c a gel, iodine chamber, EtOH). R^s of ester and phenylserinol were on 0.8 and 0.3 respectively. The reaction mixture was f i l t e r e d and con- centrated to a volume of 20 ml. The pr e c i p i t a t e that deposited was f i l t e r e d off and the f i l t r a t e was extracted with ether (300 ml) u t i l i z i n g a continuous extraction apparatus. Drying the ether on anhydrous Na2S0^ and stripping off the solvent gave crystals (20.4 g, 71.8'%). mp 86-89 on r e c r y s t a l l i z a t i o n from ether-cyclohexane ( l i t . (106), 86-87 ). i r (KBr) 102 1070 cm ^, 1040 (—OH), 1580 (primary amine), no carbonyl; mass spectrum M-t'm/e 167(2), m/e 42 (100), m/e 60 (100), m/e 77 (100), m/e 105 (80), m/e 118 (19), m/e 136 (60). Treatment of a solution of phenylserinol (560 mg) in MeOH (5 ml) with oxalic acid (215 mg) in MeOH (5 ml) gave, after washing with water (1 ml), the oxalate (450 mg), mp 222- 223 ( l i t . ( I l l ) 215°). ' Phenylserinol (370 mg) in absolute EtOH (1 ml) was treated with benzoic acid (270 mg) dissolved in absolute EtOH (1 ml). Phenylserinol benzoate (300 mg) was collected. mp 159-161° ( l i t . (112), 162-163°). 3. Synthesis of threo-l-phenyl-l-ctiloro-2-aminopropane - 3-ol hydrochloride (60). Dry HC1 was passed through the solution of threo- phenylserinol (3 g, 0.018 mol) dissolved in absolute EtOH (15 ml). After d i s t i l l i n g the EtOH off, the viscous residue was suspended in dry CHCl^ (30 ml), followed by adding freshly d i s t i l l e d SOC^ (2.5 g, 0.02 mol). The mixture was s t i r r e d overnight at room temperature. The solution was evaporated in vacuo at room temperature to give a s o l i d . The s o l i d was dissolved in absolute MeOH and to this solution dry ether was added to give a s o l i d (mp 174-176°). P u r i f i e d product (1.95 g, 49 %) was obtained on second r e c r y s t a l l i z a t i o n from MeOH-ether. mp 193-194 ( l i t . (115), 192-193°); i r (KBr) -1 +* ' ' 1050 cm (-0H); mass spectrum (20 ev) (M+2) m/e 187(1), m/e 60 (100), m/e 118 (61), m/e 119 (53). 103 4. Attempted synthesis of 1-phenyl-l,3-dichloro-2-aminopropane. HG1 (.56, R1 = R2 = R3 = H) To l-phenyl-l-chloro-2-aminopropane-3-ol (0.22 g, 0.001 mol) in dry CHC13 (10 ml) was added freshly d i s t i l l e d S0C12 (2 g. 0.017 mol), Refluxing on a water bath for 2 hours and d i s t i l l a t i o n of the solvent gave a s o l i d . The s o l i d was dissolved in MeOH. Addition of ether gave c r y s t a l s . Mp (193-195°) and the i r spectrum showed unreacted st a r t i n g material. This unreacted material with added SOCI,, (10 g) was refluxed on a water bath for 2 hours. D i s t i l l a t i o n of S0C1 2 under reduced pressure gave a s o l i d . The s o l i d was redissolved in MeOH. Crystals obtained by adding ether showed s t a r t i n g material i d e n t i f i e d by i t s i r and mp. 5. Attempted synthesis of l-phenyl-l-thiocyanato-2- aminopropane-3-ol (62). A solution of KSCN (393 mg, 0.00405 mol) and dicyclohexyl- 18-crown-6 (1.49 g, C00004 mol) in MeOH (30 ml) was mixed with l-phenyl-l-chloro-2-aminopropane-3-ol hydrochloride (444 mg, 0.002 mol) in MeOH (10 ml). S t i r r i n g at room temperature for 4 hours and d i s t i l l a t i o n of the solvent gave a viscous l i q u i d . The residue was dissolved in H20 (10 ml) and extracted with CHC13 (2 x 100 ml) and thereafter with ethylacetate (2 x 100 ml). Tic ( s i l i c a gel, iodine chamber, ethylacetate) of the residues from CHC13 and ethylacetate showed the same pattern having more than 3 spots. The i r spectrum of ethylacetate and CHC1, ex- 104 tractions showed strong polyether (1100 cm ) and thiocyanate (2090 cm ^) absorptions. Attempts to find a developing solvent for t i c which would give clear separation of the spots were performed using ethylacetate, CHC1 3 > EtOH, CHCl 3~EtOH, hexane, and EtOH-hexane only to result in severe t a i l i n g or incomplete separat ion. 6. Synthesis of 1-phenyl-1-thiosulfuryl-2-aminopropane- 3-ol (61, Bunte salt) Na 2S 20 3.5H 20 (1.935 g, 0.078 mol) dissolved in H20 (15 ml) was added to the solution of 1-chloro compound (60) (1.68 g, 0.075 mol) in EtOH (45 ml). The mixture was refluxed for 5 hours on a water bath. After reducing the volume to 3 ml under reduced pressure, precipitated s o l i d was f i l t e r e d to give a Bunte salt (900 mg. 45.7 % ) . mp 246-249°; i r (KBr) 1200 cm"1, 1230, 640 ( S03H ). 7. Attempted synthesis of l-phenyl-l-thiocyanato-2- aminopropane-3-ol (6_2) via the Bunte salt (Synthesis of 2-amino-4-hydroxy.." methyl-5-phenyl-2-thiazoline 7 4). Bunte salt 61 (263 mg, 0.001 mol) was dissolved in Na 2C0 3 solution (53 mg, 0.0005 mol) in H20 (10 ml). A solution of NaCN (49 mg, 0.001 mol) in H20 (2 ml) was added at room temperature. A precipitate was observed after s t i r r i n g for one hour. After s t i r r i n g for one additional hour, white s o l i d was obtained by suction f i l t r a t i o n . R e c r y s t a l l i z a t i o n from 105 H20 gave a thiazoline 74 (100 mg, 48.0 %) mp 152-153.5°; -1 +' i r (KBr) 1640 cm (C = N); mass spectrum M m/e 208 (3), m/e 177i (100), m/e 91 (36), m/e 135 (25), m/e 45 (12), m/e 115 (12) . 8. Synthesis of 1-pheny1-1-mercapto-2-aminopropane-3-ol hydrochloride (63). Bunte salt 61 (300 mg, 0.0014 mol) with concentrated HC1 (5 ml) was s t i r r e d at 50-60° for 1.5 hours. The mixture changed to a clear solution. S t i r r i n g overnight at room temperature gave a white p r e c i p i t a t e . The s o l i d was re- c r y s t a l l i z e d from MeOH-ether. Yield 120 mg (48.1 % ) . mp 180-182°; i r (KBr) 2520 cm _ 1(-SH); mass spectrum (M+l) +* m/e 184 (0.4), m/e 60 (100), m/e 42 (78), m/e 91 (43), m/e 118 (26), m/e 77 (22), m/e 51 (18). 9. Synthesis of threo-N-benzoyl 'phenylserinol (65). To threo-phenylserinol (1.67 g, 0.01 mol) dissolved in 10 % NaOH (30 ml) was added benzoyl chloride (5.62 g, 0.04 mol) a l l at once. The mixture was shaken vigorously for 30 min. to give a s o l i d . The s o l i d was f i l t e r e d and r e e r y s t a l l i z e d from MeOH to give tribenzoylphenylserinol (4.17 g, 87 % ) . mp 193° ( l i t . (128), 194-195°). i r (KBr) 1640 cm"1, 1530 (amide),. 3360 ( NH) , 1720 (carbonyl). A solution of t r i - benzoylphenylserinol (2.4 g, 0.005 mol) and NaOH (0.4 g, 0.01 mol) in MeOH (200 ml) was refluxed for an hour. A clear 106 solution V7as observed. A s o l i d was obtained after concentra- tion of the reaction mixture in vacuo. The s o l i d was f i l t e r e d , washed with, saturated NaHC0_, and then Ho0, and dried ( mp 153-156°). R e c r y s t a l l i z a t i o n from ethylacetate gave colorless needles. Yield 1.0 g (74 % ) . mp 162° ( l i t . (128), 163-164°); i r (KBr) 1630 cm 1 , 1530 (amide), no carbonyl; mass spectrum m/e 105 (100), m/e 147. (100), m/e 164 (100), m/e 106 (95), m/e 240 (45), m/e 222 (45), m/e 223 (27). 10. Synthesis of 1-phenyl-l,3-dichloro-2-benzoylaminopropane(6^) . Threo-N-benzo.yl-phenylserinol (2.71 g, 0.01 mol) with freshly d i s t i l l e d S0C1 2(59.5 g, 0.5 mol) was heated on the water bath for 1.5 hours. After evaporating S0C1 2 under re- duced pressure, the resulting residue was treated with dry ether to obtain a s o l i d (2.89 g) . P u r i f i e d product was obtained on r e c r y s t a l l i z a t i o n from benzene (1.75 g, 56.8 % ) . mp 148- 150°; i r (KBr) 3330 cm"1 (NH), 1640,1530 (amide), no carbonyl; nmr (CDC1 )• 7.50-7.73 ̂ " (m, 2, protons ortho to carbonyl group), 7.60-7.37 (m, 8, phenyl protons), 6.40 (b, 1,-NH-C0-), 5.32 ( d, 1, CCH -CHC1- ), 4.43-5.00 ( m, 1, C,H -CH-CH- ), D J — 0 J 3.50 (m, 2, -CH^Cl ); mass spectrum m/e 105 (100), m/e 130 (100), m/e 146 (32), m/e 182 (30), m/e 235 (31). Anal. Calcd. for C, -H., CNC1„0: C, 62.33 ; H, 4.91; C l , 23.02 ; 16 15 I mol. wt.', 308.05. Found: C, 62.21; H, 4.89; C l , 23.20. 107 11. Synthesis of 1-phenyl-l,3-d ib romo-2-benzoylaminopropane(72) . A general procedure for this synthesis was taken from the l i t e r a t u r e (130,131,132). To a solution of trans-cinnamyl alcohol (10 g, 0.075 mol) in CC1 4 (100 ml), bromine (13.1 g, 0.082 mol) in CCl^ (10 ml) was added with s t i r r i n g at -5° for 35 minutes. The CC1. solution was washed with 10 % NaHS0„ 4 3 (15 ml), saturated NaHC03 (30 and 30 ml) and f i n a l l y with R̂ O (50 ml). The CC1 ' solution was dried over anhydrous Na„S0. 4 2 4 and concentrated in vacuo u n t i l a s o l i d appeared. Standing at -5° for 24 hours gave c r y s t a l s , which were r e c r y s t a l l i z e d from petroleum ether (30 to 60) to give colorless needles of erythro-3-phenyl-2 , 3-dibromopropameI—ll-ol (15 g, 68.2 % ) . mp 72-73° ( l i t . (130), 73-74°). A solution of dibromopropano1 (10 g, 0.034 mol) and benzonitrile (3.5 g, 0.034 mol) in dry ether (10 ml) was saturated with dry HCl gas at 0°. A s o l i d precipitated after standing in the cold for 7 days. The s o l i d was collected and washed with dry ether to give ery thro-3"-rphenyl-2 ,3- dibromopropyl benzimino ether HCl (5.2 g, 35.3 % ) . mp 147- 150° ( l i t . (130), 148-150°); i r (KBr) 3450 cm"1, 1640 \ (—NH.HC1). Benzimino ether HCl (5 g, 0.0115 mol) was ground with 10 % Na^CO^ solution (25 ml) in a mortar for 20 minutes and the so l i d was collected, washed with water and dried. Recrystal- l i z a t i o n from acetone gave colorless erythro-3-phenyl-2,3- 108 d i b r o m o p r o p y l b e n z i m i n o e t h e r (3.85 g, 8 4 . 1 % ) . mp 1 3 3 - 1 3 5 ° ( l i t . ( 1 3 0 ) , 1 3 3 . 5 - 1 3 5 ° ) . i r (KBr) 3340 cm" 1, 1640 ( = N H ) . A s o l u t i o n of ba s e (3.5 g, 0.0088 mol) i n t o l u e n e ( 35 m l , d r i e d on CaH 2 and d i s t i l l e d ) was r e f l u x e d on an o i l b a t h . A s o l i d was formed a f t e r 30 m i n u t e s w h i c h t h e n went i n t o s o l u t i o n . A f t e r r e f l u x i n g f o r 1.5 h o u r s , the s o l u t i o n was c o n c e n t r a t e d i n vacuo to g i v e a s o l i d w h i c h was r e c r y s t a l - l i z e d from e t h y l a c e t a t e . Y i e l d 1.4 g ( 40 %, l i t . ( 1 3 0 ) , 13 % ) , mp 1 3 2 - 1 3 4 ° ( l i t . ( 1 3 0 ) , 1 3 2 - 1 3 4 ° ) ; i r (KBr) 1650 cm" 1, 1530 ( a m i d e ) , 3320 ( NH) ; nmr ( C D C i p 7.6 -7.86<f ( m, 2, p r o t o n s o r t h o to c a r b o n y l g r o u p ) , 7.13-7.57 ( m,8, p h e n y l p r o t o n s ) , 6.50 ( b, 1, -NH-CO- ) , 5.38 ( d, 1, C,H C-CH-C1-), 4.63- 5.0 (m, 1, C^H -CH-CH-) 3.5 ( m, 2, - C H „ C 1 ) ; mass s p e c t r u m 6 5 — —2 m/e 105 ( 7 6 ) , m/e 77 ( 2 9 ) , m/e 146 ( 2 0 ) , m/e 103 ( 2 0 ) , m/e 235 (15) , m/e 82 (14) . 12. A t t e m p t e d s y n t h e s i s o f 3 - b e n z o y l a m i n o - 2 - p h e n y l t h i e t a n e (23) ( S y n t h e s i s of 2 - p h e n y l - 4 - b e n z y l i d e n e - 2 - o x a z o l i n e , ' 7 8 ) , To t h e dibromo compound (72) (397 mg, 0.001 mol) i n EtOH (10 m l ) , Na 2 S . 9 H 2 0 (360 mg , 0.0015 mol) i n EtOH (10 ml) was added a t room t e m p e r a t u r e o v e r 30 m i n u t e s . The m i x t u r e was r e f l u x e d f o r 1.5 h o u r s on a w a t e r b a t h . P r e c i p i t a t e d NaBr was f i l t e r e d and d i s t i l l i n g o f f t h e s o l v e n t a t r e d u c e d p r e s s u r e gave a r e s i d u e . A f t e r a d d i n g H 20 (10 m l ) , t h e m i x t u r e was e x t r a c t e d w i t h C H C l ^ (3 x 100 m l ) . D r y i n g o v e r a n h y d r o u s Na SO. and d i s t i l l a t i o n of CHC1 gave a y e l l o w v i s c o u s m a t e r i a l . 109 Tic ( s i l i c a gel, iodine chamber, CHCl^: cyclohexane 1:1) showed four spots at R s baseline (A), 0.2 (B) , 0.5 (C), and 0.7(D). Above viscous material was dissolved in ether (20 ml). A small amount of ether-insoluble crystals was collected and i d e n t i f i e d as l-phenyl-2-benzoylamino-l-propene-3-ol (82) . mp 162-167°; t i c Rf 0; i r (KBr) 1050 cm"1 (primary OH), 1540, 1650 (amide), 1420 (-CH=); mass spectrum m/e 105 (99), m/e 77 (45), m/e 103 (17), m/e 51 (11), m/e 148 (10), m/e 235 (2) . After f i l t e r i n g l-phenyl-2-benzoylamin 0-l-propene - 3-ol, the ether solution was concentrated to 2 ml and f r a c t i o n - ated on a column using s i l i c a gel and CHCl^ for elution. Fractions showing R̂  0.7 was i s o l a t e d . P a r t i t i o n on the column was monitored by i t s yellow band. D i s t i l l a t i o n of CHCl^ gave a yellow c r y s t a l of oxazoline (120 mg, 50.6 % ) . R e c r y s t a l l i z a t i o n from hexane on dry ice-acetone gave needle- l i k e yellow c r y s t a l s . mp 92-94°; uv max (EtOH) 210 (£ 24,000), 241 (£ 19 ,000), 344 (B 16,000); nmr (CDC13) 7.67-8.10<f (m, 4, 4 phenylprotons), 7.10-7.40 ( m, 6, 6 phenylprotons), 5.73 ( £, 1, C 6H 5CH=) , 5.05 ( d, 2, -CH_2-0) ; mass spectrum M+" m/e 235 (25), m/e 105 (100), m/e 77 (37), m/e 103 (31), m/e 91 (23). Anal.. Calcd. for C^H „N0 : C, 81.67; H, 5.57; N, 5.80; 16 13 mol. wt., 235.11. Found: C, 81.56; H, 5.65; N, 5.81. 110 B and C were obtained as viscous liq u i d s having amide bands at 1540 and 1650 cm 1 in the i r spectrum. Oxazoline 7j8 was s i m i l a r l y obtained from 1-phenyl-l,3-dichloro-2-benzoyl- aminopropane .-. . mp . 90-93°. The i r of the product was super- impos&ble with that from the 1,3-dibromo compound reaction. 13. Attempted synthesis of 1-phenyl-l,3-dibromo-2- benzylaminopropane (9h). Diborane prepared by the method of Zweifel and Brown (145) from NaBH^ (284 mg, 0.0075 mol) and borontrifluoride etherate (1.42 g, 0.01 mol) was passed in a stream of nitrogen during 1.5 hours through a s t i r r e d solution of the 1,3- dibromo compound (72.) (397 mg , 0.001 mol) in dry THF (20 ml, d i s t i l l e d from LiAlH^) at 0°. After refluxing for 2 hours, the reaction mixture was mixed cautiously with absolute EtOH (10 ml). Evolving S a s w a s observed. A stream of dry HCl gas was passed through the solution. It was then evaporated to dryness under reduced pressure to give a viscous residue. The i r spectrum showed a carbonyl band at 1720 cm 1 . ^ 0 (10 ml) was added to the viscous residue and a small amount of s o l i d was precipitated out. The i r spectrum of this s o l i d showed an amide band (1640 cm 1 , and 1520) and was i d e n t i c a l to that of the sta r t i n g material. The apeous layer was extracted with CHCl^. After pooling the CHCl^, addition of ether gave a.solid which soon changed to a gummy material. The i r spectrum showed a carbonyl band at 1730 cm 1 . I l l 14. Synthesis of l-phenyl-2-N,N-dimethylaminopropane-l, 3-diol(9j6) . Freshly d i s t i l l e d formic acid ( 85 %, 2.5 g, 0.046 mol) and formaldehyde (37 %, 5 g, 0.062 mol) were added to phenyl- seri n o l ( 2 g, 0.012 mol). The mixture was refluxed on a water bath for 20 hours. The reaction mixture was made alkaline to litmus using 50 % NaOH and extracted with ether ( 3 x 100 ml). After drying on anhydrous Na^SO^, d i s t i l l a t i o n of the solvent gave dimethylphenylserinol ( 1.85 g, 79 % ) . Re- c r y s t a l l i z a t i o n from hexane gave needlelike c r y s t a l s . mp 64-66; i r (KBr) disappearance of NH^ (1580 cm" 1); nmr (CDCl^) 7.3 S ( s , 5, phenyl protons) , . 4.37 ( d, 1, C_HC CHOH ), 3.43 ( t, 2, -CH20H), 2 . 97-3.33 ( b, 2 , hfy^pxy^. .) > 2.50-2.85 ( m, 7, -CH(NCH 3) 2 -CH 2); mass spectrum M+' m/e 195 (0.2), m/e 88 (100), m/e 58 (21), m/e 105 (7), m/e 77(12). Anal. Calcd. for C-^H^NO^ C, 67.64 ; H, 8.78; N, 7118; mol wt., 195 .15 Found: C, 67.69; H, 8.85; N, 7.01. 15. Attempted sy.hthesis of -l-phenyl-l-chloro-2-N ,N- dimethylaminopropane-3-ol (111) . Dimethylphenylserinol (1.95 g, 0.01 mol) was dissolved in CHCl^ (50 ml) and dry HCl gas was passed through for 5 minutes. D i s t i l l a t i o n of the solvent gave a p r e c i p i t a t e , which was r e c r y s t a l l i z e d from EtOH-ether to give pure dimethylphenylserinol hydrochloride (2.0 g, 86.6 %) mp 154- 112 156°. To dimethylphenylserinol hydrochloride (1.16 g, 0.0053 mol) in CHC13 (20 ml), freshly d i s t i l l e d S0C12 (0.89 g, 0.0075 mol) in CHCl^ (10 ml) was added dropwise with cooling in an ice bath during a period of 20 minutes. The mixture was s t i r r e d for 24 hours at room temperature. Solvent was removed under vacuum resulting in a brown viscous residue. Addition of ether gave a s o l i d which was i d e n t i c a l with unreacted hydrochloride as i d e n t i f i e d by the i r spectrum. The same procedure was followed except the mixture was refluxed for 2 hours instead of s t i r r i n g at room temperature for 24 hours. The resulting viscous residue dissolved in EtOH (30 ml) with Na 2S 20 3.5H 20 (1.24 g, 0.005 mol) in H20 (5 ml) was refluxed on a water bath for 5 hours. While d i s t i l l i n g the solvent o f f , a small amount of s o l i d was precipitated out. Mp ( 280) and i r (1230, 640 cm"1) indicated unreacted Na 2S 20 3. Stripping the solvent gave a viscous residue. Absolute EtOH (10 ml) was added to i s o l a t e a s o l i d , which was i d e n t i f i e d as NaCl. The EtOH solution was d i s t i l l e d to give a residue; i r (neat) -1 ' 1230, 640 cm . Positive i d e n t i f i c a t i o n of a Bunte salt was not successful. Dimethylphenylserinol (2.0 g) was dissolved in dry ether (100 ml). Dry HC1 gas was passed into the suspension of isolated dimethylphenylserinol hydrochloride for 5 hours. The mixture was f i l t e r e d . The f i l t e r cake and a s o l i d isolated from the f i l t r a t e after evaporating the solvent showed the same mp and i r spectrum as that for dimethyl- 113 phenylserinol hydrochloride. Dimethylphenylserinol (700 mg) was dissolved in dry dioxane (15 ml, dried on CaH^ and d i s t i l l e d ) . Dry HC1 gas was passed through the clear solution for 2 hours. D i s t i l l a t i o n of dioxane under reduced pressure gave a viscous brown residue. Positive i d e n t i f i c a t i o n of 111 was not successful. 16. Synthesis of N,N-dimethyl-p-nitrophenylserinol (110). D(-)-threo-p-nitrophenylserinol (6.36 g, 0.03 mol) with 37 % formaldehyde (12.5 g, 0.154 mol) and 85 % formic acid (7.5 g, 0.139 mol) was s t i r r e d at 80-100°for 13 hours. After making alkaline to litmus using 50 % NaOH, the mixture was extracted with ether (3 x 150 ml). Drying on anhydrous Na^SO^ and d i s t i l l a t i o n of the solvent gave a brown s o l i d . R e c r y s t a l l i z a t i o n from ether-hexane gave a white c r y s t a l (5.24 g, 72.18 % ) . mp 90-92°; i r (KBr) 1530 cm"1, 1360 ( -N0 2), disappearance of NH2 (1580); nmr (CDC13) 8Q07<$~( d, 2, ortho to nitro group), 7.47 (d, 2, meta to nitro group), 4.50 ( d, 1, N0„C,H CHOH-), 3.52 ( d, 2, -CH-OH), 3.10 ( b, 2 o 5 — — 2, hydroxyl OH, disappeared on addition of DO), 2.33-2.66 ( m, 7, -CHN(CH 3) 2); mass spectrum m/e 88 (100), m/e 58 (64), m/e 209 (6), m/e 163(6). Anal. Calcd. for C 1 1H 1,N o0. : C, 54.96 ; H, 6:.72; N, 11.67; 11 lo I 4 mo1. wt., 2 4 0.15. Found: C, 55.09; H, 6.71; N, 11.54. 114 17. Attempted synthesis of l-p-nitrophenyl-l-chloro-2- N,N-dimethylaminopropane-3-ol hydrochloride (112) . To p-nitro-N,N-dimethylphenylserinol (2.4 g) in dry ether (100 ml), HCl gas-saturated ether (200 ml) was added to obtain a s o l i d . F i l t r a t i o n and washing with ether gave a white HCl s a l t . This salt ( mp 116-123°) was hygroscopic and changed to a yellow color on standing. Freshly d i s t i l l e d S0C12 (262 mg, 0.0022 mol) in dry CHC13 (10 ml) was added to HCl salt (553 mg, 0,002 mol) in dry CHC13 (20 ml). The mixture was refluxed for 2 hours. D i s t i l l a t i o n of the solvent gave a brox/n s o l i d . R e c r y s t a l l i z a t i o n from EtOH-ether showed p- nitro-N,N-dimethylphenylserinol.HCl which was i d e n t i f i e d by superimposable i r spectrum. 18. Synthesis of 1-p-nit ropheny 1-1 , 3.-d i c h l o r o-2-N , N- dimethylaminopropane hydrochloride (.97.) . N,N-dimethy1-p-nitropheny1serino1 HCl (5.43 g, 0.02 mol) with freshly d i s t i l l e d thionyl chloride (50 ml) was heated at o 70-80 for 1.5 hours. D i s t i l l a t i o n of the thionyl chloride under reduced pressure gave a viscous residue. After d i s s o l v - ing the reside by adding EtOH (20 ml), ether (150 ml) was added to obtain a p r e c i p i t a t e . R e c r y s t a l l i z a t i o n from EtOH- ether gave a dichloro compound (4.73 g, 75.4 % ) . mp 161-163°; i r (KBr) 3420 cm"1 ( t e r t i a r y amine),'1530, 1350 (-N02), no strong absorption at 1200-1000; mass spectrum m/e 106 (100) , 115 m/e i m (32), m/e 71 (19), m/e 70 (6), m/e 58 (5). Anal. Calcd. for c 1 1 H i 5 N 2 ° 2 C 1 3 : C ' 4 2 - 1 0 ; H» 4.82; C l , 33.93; mol. wt. 313.52. Found: C, 42.10; H, 4.84; C l , 33.81. 19. Attempted synthesis of 2-p-nitrophenyl-3-N,N-dime- thylaminothietane (2 6) (Synthesis of bis (1-p- nitrophenyl-2-N,N-d imethy1amino-3-chloropropane) sulf i d e HC1, 123). 1) Reaction using excess amount of Na 2S.9H 20. Na2S.9 H20 (4.032 g, 0.0168 mol) dissolved in EtOH (50 ml) was added dropwise to the solution of 1-p-nitropheny1-1, 3-dichloro-2-N ,N-dimethylaminopropane H-C;l (2.34 g, 0.0075 mol) in EtOH (30 ml) during 10 minutes at room temperature. S t i r - ring was continued for 2 hours after addition of sodium sulf i d e at room temperature. The f i l t e r cake obtained after f i l t e r i n g the reaction mixture was t r i t u r a t e d with H20 to dissolve precipitated NaCl. Brown-colored crystals(1.22 g) were obtained. mp 60-110°; i r (KBr) 1520 cm"1, 1350 (-N02); gc-mass spectrum (3 % 0V-17 column, injector temperature 170° column temperature 80-300°, programming 20°/min.) Compound A ; retention time 8.9 minutes, m/e 70 (100), m/e 58 (55), m/e 206 (37), m/e 84 (30), m/e 115 (26). Compound B ; retention time 11.3 minutes, m/e 116 (74), m/e 115 (22), m/e 162 (14), m/e 227 (13), m/e 77 (11-). 116 2) Reaction using equimolar concentration of Na^S.^H^O (Synthesis of bis (1-p-nitrophenyl-2- N, N-dimethylamino-3-chloropropane) sul f i d e HC1.H 0 123) . Na2S.9H20 (1.441 g, 0.00 6' raol) dissolved in EtOH (30 ml) was added to the solution of 1-p-nitrophenyl-l,3-dichloro-2- N,N-dimethylaminopropane HCl (1.881 g, 0.006' mol) in EtOH (50 ml). The mixture was s t i r r e d for 3 hours at room temperature. The precipitated yellow crystals were f i l t e r e d and t r i - turated with H20. Yield 660 mg. mp. 115-135; a l k a l i - f u s i o n test sulfur, p o s i t i v e ; :nmr (CDCl^) 8.10 & (d, 2, phenyl protons ortho to nitro group), 7.33 (d, 2, phenyl protons meta to ni t r o group), 2.37 (s, 6, dimethyl protons), 3.00-3.63 (m, 4, (N0^C/.HcCHCHN(CHo)o CH„C1) 0S; gc-mass spectrum (3 % OV-17 Z O J — — J Z — Z Z column, injector temperature 170° column temperature 80-2 75°, programming 20°/min.) retention time 9.3 minutes, m/e 70 (61), m/e 58 (44), m/e 115 (19), m/e 84 (14), m/e 206 (12). The free base (600 mg) was dissolved in ether (200 ml). A white c r y s t a l was obtained by passing HCl gas through the ether solution. Yield 560 mg (overall y i e l d .15.4 %);mp 148 (dec.) ( r e c r y s t a l l i z a t i o n from EtOH-ether and drying at o 50/0.1 mm for 5 hours); a l k a l i - f u s i o n test sulfur, p o s i t i v e ; gc-mass spectrum (3 % OV-17 column, injector temperature 170° column temperature 80-275°, programming 20°/min.) retention time 9.3 minutes, m/e 70 (77), m/e 58 (55), m/e 84 (21), m/e 206 (18), m/e 115 (18). Anal. Calcd. for C 2 2 H 3 2 N 4 ° 5 S C 1 4 : C ' 4 3 - 5 5 5 H> 5.32; N, 9.24; S, 5.29; mol. wt. 606.20. 117 Found: C, 43.89 ; H, 5.24; N, 9.06; S, 4.98. 20. Synthesis of 3-chloro-l-phenylpropylene oxide-1,2(107) . Freshly d i s t i l l e d 3-chloropropenylbenzene (68-69°/0.1 mm, 15.2 g, 0.1 mol) was added to 300 ml of a chloroform solution of 85 % m-chloroperbenzoic acid (25.8 g, 0.12 mol). The solution was kept at 0° for 24 hours with frequent shaking, A precipitate (m-chlorobenzoic acid) was formed during this time. To the reaction mixture, 10 % Na^SO^ was added to destroy excess peracid u n t i l a test with starch-iodine paper was negative. The reaction mixture v/as f i l t e r e d into a 1000 ml separatory funnel and washed with saturated NaHCO^ (150 ml) u n t i l the washing solution remained basic. The .CHCl^ layer after washing with H„0 was dried over anhydrous Na.SO, in the 2 2 4 cold for 24 hours. The solvent was removed using a flash evaporator. The resu l t i n g yellow o i l (15.4 g) was d i s t i l l e d under reduced pressure u t i l i z i n g a vigreaux condenser to give 14.1 g (84 %) of a colorless l i q u i d . bp 74-75°/0.03 mm ( l i t . (83) 67/0.3 mm); i r (neat) 890, 940 cm"1 (epoxide ring) 21. Synthesis of oZ-toluenesulfonylchloride The preparation of oi-rtoluenesulfonylchloride was carried out according to a known procedure (165). 75.6 g (0.6 mol) of benzylchloride was used to obtain 103 g (90.3 %) of J. -toluenesulfonylchloride'... mp 89-91° ( l i t . (165), 88-91°). 118 22. Synthesis of 3-hydroxy-2-phenylthietane (100) . The method described by Haya (83) was used. 14.89 g (68 %) of 3-hydroxy-2-phenylthietane was obtained from 22.3 g (0.132 mol) of 3-chloro-l-phenylpropylene oxide-1,2. mp 54- 55° ( l i t . (83), 5 65-5 7.5) ; i r (KBr) 1065 , 3220 , 3310 cm"1 (-0H) ; nmr (CDC13) 2.95 <^(s, 1, OH, disappeared on addition of D 20), 3.10 ( d, 2, SCH 2), 4.60 (m, 2, ArCHCHOH), 7.33 ( m, 5, phenyl protons) 23. Attempted synthesis of 3-azido-2-phenylthietane (10 3) . This experiment was performed following known procedures (83). 3-Hydroxy-2-phenylthietane (4.6 g, 0.027 mol) was used to prepare 3-benzylsulfonoxyl-2-phenylthietane. THF was d i s t i l l e d over LiAlH.. Et„N was d i s t i l l e d and dried over K0H. 4 3 Half of the residue was used for determination of 3- berizylsulfonoxyl-2-phenylthietane. The attempted synthesis of 3-azido-2-phenylthietane was carried out using another half residue. Half of the residue was dissolved in CHCl^ and fractionated on column of s i l i c a gel (60-200 mesh, Davison Chemical). CHCl^ was used as eluent. As shown by preliminary t i c ( s i l i c a gel, iodine chamber, pet. etheriCHCl^ 1:1) separation of 4 compounds was achieved. values of the compounds were: A baseline spot, B 0.1, C 0.25, D 0.8. B and C were isolated as gummy materials. B showed the same R̂  values as 3- hydroxy-2-phenylthietane. D. showed a high melting point 119 (over 200°) and strong absorption at 3400 cm 1 in the infrared spectrum. D; mp 84-86° (89° on r e c r y s t a l l i z a t i o n from pet. ether-benzene), i r (KBr) 1175, 1370 cm"1 (S0 2); nmr (CDCl^ 4.83cT( s, 2, ArCH_2) , 7.43 (m, 5, Ar).' Half of the residue was dissolved in freshly d i s t i l l e d j HMPT (25 ml, 83°/0.9 mm). NaN (1.5 g, 0.023 mol) was added and the suspension was s t i r r e d on a cold water bath under N 2 atmosphere for 15 hours. The solution was added to 50 ml of H20 and extracted with ether ( 3 x 50 ml). . After drying with anhydrous Na2S0^, the ether extract was concentrated under vacuum. The residue showed a strong azide ( 2110 cm ^) band in the infrared spectrum. The residue was dissolved in a small amount of CHCl^ and fractionated on a column of F l o r i s i l (60-100 mesh, Fisher S c i e n t i f i c Company). Elution with n- hexane gave Fraction A (R^ 0.8, s i l i c a gel, iodine chamber, hexane : CHCl^ 3:1). Elution with ether gave two fractions ( B; R^0.4-0.7, t a i l i n g C; baseline spot), Elution with MeOH gave fr a c t i o n D which showed also baseline spot. A; mp 87-88.5° (on r e c r y s t a l l i z a t i o n from ether), i r (KBr) no azide band (azide band was observed before r e c r y s t a l l i z a t i o n ) , 1175, 1370 cm 1 (S0 2). B and C were isolated as small amount of dark residues having strong band at 2110 cm 1 . Tic of the fraction D showed a mixture of the several materials. After dissolving in MeOH, e t h e r was added resulting in pre- c i p i t a t i o n of a c r y s t a l , which showed strong band at 650 and 3390 cm 1 as well as at 2210 cm ^. ~ 120 24. Attempted synthesis of 2-phenyl-3-thietanone (104) . A solution of 3-hydroxy-2-phenylthietane (1.32 g, 0.008 mol) in freshly d i s t i l l e d HMPT (15 ml) and DMSO (6 ml) was cooled to -20° and cyanuric chloride (2.95 g, 0.016 mol) was added. After 5 hours at -20°, triethylamine (3.23 g, 0.032 mol) was added and the mixture was allowed to stand at room temperature for 10 minutes. The mixture was poured into ice-water (30 ml) and extracted with CHCl^ (3 x 150 ml). After drying over anhydrous Na^SO^, CHCl^ was d i s t i l l e d . Tic ( s i l i c a gel, iodine chamber, methanol) showed 2 spots at 0.8 and baseline. The residue was dissolved in a small amount of CHCl^ and fractionated on a s i l i c a gel column. Elution with CHCl^ gave a yellow o i l which showed a spot at 0.8 by t i c . 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