Part I. the Reactions of 2-Oximino-Cholesta-4, 6-Diene-3

This dissertation has been microfilmed exactly as received ® 7-2401 AHMED, Quazi Anwaruddin, 1938- PART I. THE REACTIONS OF 2-OXIMINO-CHOLESTA-4,6-DIENE-3-ONE. PART n. THE ALKALOIDAL CONSTITUENTS OF TABERNAEMONTANA RIGIDA. The Ohio State University, Ph.D„ 1966 Chemistry, organic

University Microfilms, Inc., Ann Arbor, Michigan PART I THE REACTIONS OP 2-0XIMIN0-CH0LESTA-^,6-DIENE-3-0NE

PART I I THE ALKALOIDAL CONSTITUENTS OP TABERNAEMONTANA RIGIDA

DISSERTATION Presented in Partial Pulfillment of the Requirements for the Degree of Philosophy in the Graduate School of the Ohio S tate U niversity

By Quazi Anwaruddin Ahmed, B .Sc..(Honours), M.Sc,

******

The Ohio State U niversity 1966

Approved by

Adviser Department of Chemistry ACKNOWLEDGMENTS

I wish to express my sincere thanks and appreciation to Professor Michael P. Cava for the guidance and encourage ment received during the course of these problems' and to Dr. G. Praenkel for acting as a temporary adviser. I am indebted to Dr. B. H. Bhat for many helpful dis cussions both in and outside the laboratory. I also thanlc Dr. ,M. J. Mitchell, Dr. K. V. Rao and.Dr. K. Bessho for their helpful suggestions. Lastly, I owe a debt of gratitude to my parents-and most esp ecially to my wife Verena,.

. l i VITA

September 1938 Born - Rajshahi, East Pakistan November, 1958 B. Sc. (Hons.) in Chemistry, Dacca Uni versity, Dacca, East Palcistan November,, 1959 M. Sc. (Thesis Gr.) in Chemistry, Dacca University, Dacca, East Pakistan

1961-1963 . . . . Teaching Assistant, Department of Chemis try , The Ohio S tate U niversity, Columbus, Ohio 1961+-1966 . . . . Research Assistant, Department of Chemis try , The Ohio State U niversity, Columbus, Ohio

FIELDS OP STUDY

Major Field; Organic Chemistry

i l l CONTENTS ■ Pap:e Acknowledgmants ...... i l V ita ...... ; ...... i l l Part I - The Reactions of 2-oximino-choiesta- l+,6-diene-3-one Introduction and Statement of Problem ...... 1 Historical ...... 5 D isc u ss io n...... 38 Experimental ...... 77 Cholesta-^,6-diene-3-one (CXXII) ...... 77 2-Oximino-cholesta-^H,6 -diene-3-one (CXXIII) ...... 78 Beckmann rearrangement of 2-oximino-cholesta- . ^-,6-diene-3-one (CXXIII) ...... 79 Alkaline degradation of the Beclonann.rearrange ment product CXXIV ...... 80 Estérification of the acidic degradation product CXXVI ...... 81 Methyl 2,3-secocholesta-^-,6-diene-2-nitrile- 3-oate (CXXV) ...... 84 3-Hydroxy-3,3-diphenyl-2,3-secocholesta- 4.6-diene-2-nitrile (CXLVI) ...... 84 3-Hydroxy-3,3-dimethyl-2,3-secocholesta- 4.6-diene-2-nitrile (CXLVII) ...... 86 2,3“Secocholesta-4,6-diene-2-nitrile-3-oic acid-3-amide (CXLV) ...... 87 2,3-Secocholesta-4,6-diene-2-nitrile-3-oic acid-3-N-phenylamide (CXLVIII) ...... 87 Attempted hydrolysis of methyl 2,3-secocholesta- 4 .6 -d io n e -2 -n itrile -3 -o a te (CX:^) ...... 88

i v CONTENTS (contd.) P a_ge

Estérification of 2 , 3-secocholesta-^, 6 -diene- 2.3-dioic acid-2-amide (CXLIX) ...... 90 2 .3-S.ecocholesta-^, 6 -d ien e- 2- n i t r i l e - 3-oio acid (CXXVI) ...... 91 Conversion of 2, 3- s e co choie s t a-4-, 6 -d i ene- 2- nitrile-3-oic acid (CXXVI) to 2,3-secocholesta- ^,6-diene-2-nitrile-3-oic acid-3-aiïiide (CXLV).. 91 Attempted isolation of 3-imino-A-homo-cholesta- ^-a,6-diene-^-one-2-methyl ether (CXXIX) ...... 92 2.3-Secocholesta-^,6“diene-2,3-dinitrile (CLII).. 92 Attempted preparation of 2-oximino-3“-phenyl- cholesta— 6 —diene—3^—ol (CXL) ...... 93 Beckmann rearrangement of 2-oximino-3a-phenyl- cholesta-^j6-diene-3i8-ol (CXL) ...... 9^- 2- 0ximino-3a-methylcholesta-^, 6 -d ien e- 3;8-o l (CXXXVIII)...... 95 Beclcmann rearrangement of 2-oximino-3»-methylcholesta-^,6-diene-3i8-ol (CXXXVIII) ...... 96 2- Diazo- choie s ta-^-, 6-diene-3-one (CLV) ...... • 96 Photolysis of 2-diazo-cholesta-l+,6-diene-3-one (CLV) ...... 97 Attempted lithium and liquid ammonia reduction of 2-oxim ino-cholesta-^,6-diene-3-one (CXXIII) . . . 99 ; Catalytic hydrogenation of 2-oximino-cholesta- 4 ,6-diene-3-one (CXXIII) ...... 100 Catalytic hydrogenation of methyl 2.3-secocholesta-'+,6-diene-2-nitrile-3-oate (CXXV) .... 103 Methyl 2,3“Seco-5®-cholestan-2-nitrile>^3-oate (CLXVIII) ...... 10^- Catalytic hydrogenation of 2,3-secocholesta- lf,6-diene-2,3-dinitrile (CLII) ...... 10^-

V CONTENTS (contd.) Pa^e 2,3“Seco-5“-cholestan-2~nitrile-3-oic acid- 3-amide (CLXX) ...... 105 2,3-Seco-5®“Cholest9n-2,3-dlnitrile (CLXIX) .... 106

Part II - Tlie Alkaloidal Constituents of Tabernaemontana' Rigida Intro d u ctio n and Statement ofProblem ...... 107 Discussion ...... '...... 120 Experimental ...... 132 Basic hydrolysis of dl-vincamine (XVIII) to dl-vincaminic acid (XIX) ...... 13^ Silver oxide oxidation of dl-vincaminic acid (XIX) to dl-eburnamonine (XX) ...... •...... 135 Dehydration of dl-vincamine (XVIII) to dl-apovincamine (XXI) ...... 136 Sodium borohydride reduction of dl-vincamine (XVIII) to dl-vincam inol (XXII) ...... 137 % Attempted preparation of the quarternary salt XXIII ...... • 138 Isolation of alkaloids from the crude amorphous tertiary bases B ...... 138 Isolation of alkaloids from P-la ...... 137 Isolation of alkaloids from F-lb ...... I^Ai- Chromatography of pH 6,6 fraction of F-lb ...... 1,^5 Chromatography of pH 6.0 fraction of F-lb 1^ Chromatography of pH 5.0 fraction of F-lb 1^7

V i CONTENTS (contd.).

Appendix Page I. Infrared Spectra ...... 1^9 II. Ultraviolet Spectra ...... 159 III. Nuclear Magnetic Resonance Spectra ...... 172

v ii Charts

P art I I Tentative structure of the dimeric product CXXIV ■...... ^5

P art I I I Alkaloids isolated from Tabernaemontana-... 116 II KnoTOi structures of alkaloids isolated from Tabernaemontana ...... ' 118 III Plow sheet for the extraction of the total alkaloids ...... 131

v i i i . PART I

INTRODUCTION

• Steroids are organic molecules ^lÆiich have in common a perhydrocyclopentanophenanthrene nucleus (Fig. 1).

Fig. 1

They are so named because they are related to, and in most cases derived from, sterols which are found abundantly in nature, usually in the non-saponifiable fraction of ani mal and plant fats."* The commonest example of a steroid is cholesterol, first isolated from human gallstones, which occurs practically in all animal tissue. It has been found, for example, in beef brain and spinal cord or sheep wool grease. Sitosterol and,

"*N. A. Applezweig, "Steroid Drugs," McGraw-Hill Book Co., Inc., New York, N.Y,, 1962, p. 9* 2 stigmasterol, found in vegetable oils, and ergosterol, derived from yeast and other microbiological sources, are all higher alcohols of cyclopentanophenanthrene, having a hydrox yl group at the 3 position"and differing primarily in the side chain at 17. In addition, these naturally occurring ste rols have a double bond between positions 5 and 6. Among other important steroids of natural origin are testosterone, progesterone and estrone. Most of these hormones have a car bonyl group at position 3 and a double bond between positions and 5» There is also a hydroxyl group or side chain at po s itio n 17 and another hydroxyl group or carbonyl group at . position 11 in the case of cortisol-and cortisone respectively. The structure of an insect hormone, ecdysone, has been deter mined and it. was found to be a complex cholesterol derivative. Recently, Kerb and his co-workers^ have synthesized this hormone from a rea d ily av ailab le ste ro id d e riv a tiv e . In recent years, many.authors have reported the prepa ration of modified steroids which have biological activities greater than those of naturally occurring steroids. In these studies, modifications of pharmacological activity resulting from the introduction of halogen atoms, double bonds, hydrox-

^U. Kerb, P. Hocks, and R. Wiechert, Tetrahedron L e tte rs, I 387 (1966 ). 3 yl group,3 methyl groups,^.cyano groups,^ thiocyano groups^ as well as combinations of these functions^ have been exam ined. The continuing search for modified steroids with hormonal or antihormonai activity is currently emphasizing structures vri.th hetero atoms such as nitrogen incorporated in the polycyclic nucleus,^ Although some work has been done on the synthesis of aza-steroids by the Beckmann rearrange ment of simple saturated and unsaturated ketoximes,^ a lit-

' 3n . a . Steinberg, R. Hirschmann, and J. M. Chemuda, Chem. Ind. (London),, 975 (1958). ^H. L. Herzog, A. Nobile, S. Tolksdorf, W. Charney, E. B. Hersberg, B. L. Perlman, and M. M. Pecket, Science 121. 176 (1955). 5j, C. Orr, A. D. L. Roz, and A. Bowers, J. Org.. Chem.. 22, 3300 (196>+); A. Bowers, ib id ., 26, 20^3 (1961); W. Naga- ta, S. Hirai, H. Itazaki, and K. Take da, ibid., 26, 2^+13 (1961 ); H. M. Kissman, A. S. Hoffman, and M. J. Weiss, ibid. 26, 2610 (1961 ), 22, 3168 (1962); A. Bowers., E. Denot, M. B. Sanchez, L. M. Sanchez-Hidalgo, and H. J. Ringold, J. M. Chem. Soc., 8l_, 5233 (1959); J. A. Zderic, 0. Halpern, H. . Carpio, A. Ruiz, D. C. Limon, L. Magana, H. Jimenez, A. Bowers, and J. Ringold, Chem. Ind. (London), 1625 (I960); P. Borrevang, Acta Chem. Scand., % 587 (1955). ^ I. Kitagawa, Y. Heda, T. Kawasaki, and E. M osettig, J. Org. Chem., 28, 2228 (1963). "^8. Bernstein, R. H. Lenhard, W. S. Allen, M. Heller, R. Littell,- S. M. Stolar, L. I. Feldman, and R.H. Blank,' J. Am. Chem. Soc., 28, 5693 (1956). ^R. H. Mazur, J. Am. Chem. S oc., 8l_, 1^+5*+ (1959); H. ÏÏ, Mazur, ib id ., 82, 3992 (I 96 O); T. L. Jacobs and R. B. Broimfield', ibid., 82, 4033 (I960); M. Out and M. Uskokovic, J . Org. Chem., 26, T ^ 3 (1961); N. J. Dorenbos and C. L. Huang, ib id ., 2'5T ^106 ( I 96 I ). ^For a review on the preparation ofaza-steroids, see the historical part of this dissertation, p. 5. erature survey shows that the Beckmann rearrangement of ste roids containing the a-oximinoketone function has been very little investigated up to the present time. The primary subject of the research in this disserta tion was the reactions of 2-oximinocholesta-^+,6-diene-3-one, v/ith a special emphasis on the Beckmann rearrangement of this ketone and the determination of the structures of the re sulting products. HISTORICAL

The rearrangement of a ketoxime to an isomeric amide was discovered by E. Beclcmann 10 in 1886 and is known as the Beckmann rearrangement, Beckmann treated benzophenone oxime with phosphorous pentachloride and found that the product of the reaction, after hydrolysis, was benzanilide.

NOH O

Since the discovery of this reaction, numerous publi cations have appeared which deal with the mechanism of the reaction, the determination of the stereochemical configu ration of the oximes employed and the synthetic applications

of the reaction. Blatt,H J o n e s ,12 Knunyants, ^^ and more

IOe. Beckmann, B er., 12, 988 (1886); 20, 1^0? (1887).

11a . H. Blatt, Chem. Rev., 12, 21? (1933). I^B. Jones, Chem. Rev., H , 335 (19^)» 18l. L. Knunyants and B. P. Fabrichnyi, Uspekhi Khim., .18, 633 (1949) C. A., 41, 6172 (1951) . recently, Heldt^^ have summarized the published literature concerning the Beckmann rearrangement up to 1960. The more common rearranging agents are p-toluenesulfonyl chloride, thionyl chloride, phosphorous pentachloride, p-acetylaminobenzenesulfonyl chloride, benzenesulfonyl chloride, acetic anhydride, concentrated sulfuric acid and polyphosphoric acid . . ' A b rie f review of work done by d iffe re n t authors w ith the object of achieving syntheses of aza-steroids using the Beclonann rearrangement of steroidal ketoximes is given below:

1 ) A-ring aza-steroids. Hara"*^ obtained lactams II iQ3%) and III (1^) by treating methyl 3-oximino-A-norchola- nate (I) with p-toluenesulfonyl chloride in pyridine.

I IIIII

Z, Heldt, "Organic Reactions," Vol. 11, John Wiley and Sons, Inc., New York, N.Y., I960, p.' 1. ^^8. Hara, Chem. Pharm. Bull, (Tokyo), 2, 209 (1955)* A similar treatment of methyl 3-oximino-oholanate (IV) gave a mixture of lactams V (80^) and VI (3^)»

+ HI

IV VI Furthermore, Hara prepared 2-azaoholanes by a Beckmann rearrangement of methyl 2-oximino-A-nor-5/8-cholanate and was able to separate the lactams formed by chromatography. Shoppee and Sly^^ prepared 3-aza-A-homo-5®- and 5/3- cholestan-4-ones (VIII) from 5® and 5/3-cholestan-3-ones oxime (VII) by treatment with thionyl chloride and reported that in both the cases they isolated a single oxime from each re spective ketone.

S o cij.

VII = 5® or VIII = 5® or 5/3

W. Shoppee and J . C. P. Sly, J. Chem. Soc., 3^i-58 (1958). 8

Later, after preparing the lactam 3-aza-A-homo-^a- cholestan-*+-one by a more rigorous route, Shoppee"'7 recog nized that his original product was an inseparable mixture of 3-aza-A-homo- ^a-cholestan-^-one and the isomeric Beclonann rearrangement product 4—aza-A-homo-^*-cholestan-3-one. Shoppee tre a te d 5^“-androstan-3-one oxime (IX) and 17^- hydroxy-5“-androstan-3-one oxime (X) with thionyl chloride and was able to isolate in each case both' the isomeric 3-aza and aza-A-homo ste ro id s.

R-

HoH IX R=H XI R=H XIII R=H X R=OH XII R=OH XIV R=OH

In order to explain the formation of isomers, Shoppee postulated that the oximes of the saturated steroid'ketones- may have been mixtures of syn- and, anti-isomers although they were crystalline compounds of relatively sharp melting point and appeared to' be homogeneous.

1 7 '0, W, Shoppee, A. Krueger, and R, N, Mirrington, J . Chem. Soc., 1050 (1962). Shoppee et al.^^ also prepared 3-aza-A-homo-androst- ^a-ene-^-one (XVI) by treating non-crystalline oxime of androst-^-en-3-one XV with thionyl chloride at -15°.

^ amorphous oxime

- o- XVI

. The Beckmann rearrangement of a ketoxime with thionyl chloride has been postulated as proceeding by mechanism (A);' this ordinarily involves hydroxyl ions or water in the last stage,and it has been sho-vm,^® by the use of isotopically labelled water for decomposition of the product from benzophenone oxime and phosphorous pentachloride that there is complete loss of the original oxime-oxygen. Recently, however, another mechanism (B) has been invoked^l whereby rearrange ment may occur in the complete absence of water; this mecha nism requires there to be complete retention of the original oxime-oxygen. . "

l8c, W, Shoppee and A, Krueger, J. Chem, Soc., 1050 (1962 ). B. Lampert and F. G, Bord we 11, J . Am, Chem. S oc,, 21, 2369 (1951). 20a , E, Brodsky and G, P, Micliichin, Compt, rend. Acad, Sci. U.S.S.R.,32, 558 (19^1); Acta Physiochim, U.S.S.R., 16, 63 (19)+2). . • ~ Stephen and B. Staskun, J. Chem. Soc,, 980 (1956). 10

Mechanism (A)

+ C -R " c | - o s o - c —r !' h o — c— r " j^-H ^ ^ -M ^ ^

0 = C - R " -N h

Mechanism (B)

^)C=NOH — >[l^feNH-ÛM-C^ïï]cl

— >RWci^~ON=c.^------> (\^N=C^)jP------

r n = c c 1r !' - h i^ h h c o r !'

Shoppee^ examined the Beckmann rearrangement of the oxime of cholest-4—en-3-one. This oxime has been reported to exist in two forms, regarded as geometrical isomerides desig-. nated as the B-form, m.p. 1 5 2 - 1 and .the A-form, double m.p. 65°/l52°, both of which possess the same ultraviolet absorption spectra in acetic acid and in ethanol; the two ■ forms are interconvertible by crystallization from appropri ate solvents and the A-form gives the B-form when heated above 85°. The A- and B-forms are designated as the anti- and syn- 11 oxiraes by Shoppee on the basis of the fa c t th a t the syn- oxime XVIII of cholest-^-ene-3-one reacts \d.th an excess of thionyl chloride to giye S-aza-A-homo-cholest-^+a-ene-^-one (XIX) whereas similar treatment of the anti-oxime XVII failed to give a rearrangement product even under more vigorous conditions; in benzene, under conditions in which the anti- is converted into the svn-form, the product was the same lactam XIX (13^ crude).

XVIII XIX

AcNIi^SOf I XIX "SiVvofeBw. a t a.o° 'y^ 0\\

>-Soc\, %.k0H -> p-Aminobenzene- sulfonate derivative of the. oxime No reaction 12

Thus, the partial conversion of pure anti-oxime XVII via the syn-oxime XVIII to give XIX can be visualized as follow s

OH XIX •M XVII ■ XVIII

■ The failure of the anti-oxime XVII to undergo rear rangement under ordinary condition has been explained by the possibility that conjugation via the six-membered ring leading to some degree of double bond character in the 3»^- bond of the anti-oxime XVII may be responsible for resistance to the migration of that bond, which would be involved in à Beckmann rearrangement. When the oxime XX of cholest-5-ene- 3-one-was treated \d.th thionyl chloride at -10°, a single lactam XXI was obtained. The oxime XX, unlike XVIII, thus appears to have the hydroxyl group anti to the 5?6-double bond.

OH > 0

XX XXI

22c, W, Shoppee, R. E. Lack, and B, C. Newman,. J , Chem. Soc., 3388 (196^). 13

The anti- and. syn-lsomers of the oximes derived from a,yS-unsaturated ketones in the steroid series could be distin guished by nuclear magnetic resonance spectroscopy.23 A single lactam, ^-aza-^jS-cholestan-3-one (XXIII) was obtained by Beckmann rearrangement^' of A -nor-cholestan-3- one oxime (XXII) with e ith e r p-acetylam inobenzenesulfonyl chlorideor thio n y l chloride.^5

HON' .XXII

I t is in te re s tin g to know th a t'th e oxime I (see page 6) however, afforded two isomeric lactams on treatment with p-toluenesulfonyl chloride in pyridine. Doorenbos and Havranek26 obtained a mixture of 2-aza- 5»-cholestan-3-one- (XXV) and 3-aza-5»-cholestan-2-one (XXVI)

23g, Slomp and W. J. Wechter, Chem. Ind. (London), ^1 (1962 ). T. Edwards and P. F. Morand, Can. J . Chem., 31, 1316 (I960). , ^^C. W, Shoppee, R. Killick, and G, Krueger, J. Chem. Soc., 2275 (1962). 26n . J. Doorenbos and R. E, Havranek, J, Org. Chem., 20, 247)+ (1965). by treating A-nor-^a-oholestan-2-pne oxime (XXIV) with poly phosphoric acid. The product appeared to exist as a uniform, homogeneous m ateria l, m.p., 220-221°, a fte r p u rific a tio n . Although their presence was established by the preparation and separation of some derivatives, all attempts to separate, isomers XXV and XXVI from this mixture were unsuccessful.

XXIV

Collvlîwû ^ No CXXIX l^edcV'tcrvu

Treatment of the mixture XXV and XXVI with phosphorous pentachloride yielded a mixture of dichloro lactams which were separated into two fractionsm.p. 227-229° and 209- 211° in a 1:1 ratio. The carbonyl absorption in these products was shifted to 0,l8p toward a smaller wave length as for a^a- dichloroamides. The higher melting fraction yielded ^-chloro- 2-aza-^-cholesten-3-one (XXIX) on treatment with collidine establishing that it was *+,>+-dichloro-2-aza-5«-cholestan-3- one (XXVII). The lower melting fraction, 1,1-dichloro-3-aza- 1?

5«-cholestan-2-ona (XXVIII), which is unable to ëlimlnate HCl, was unchanged by this treatment. Shoppee et a l.^7 obtained approximately equal amounts of the normal rearrangement product 1-aza-A-homo-5“-cholestan 2-one (XXXI) and the abnormal’feecond-order"Beckmann cleavage product, 1,1-seco-1-cyano-compound XXXII by treating 5“-cholestan-1-one-oxime—(XXX) with thionyl Chloride at -20°.

HON LAH

XXXI ■ XXXIII XXX CH.

The £ -lactam ex hibits ^ max 3320cm“’' (N-H), 1652cm“1 [CO(NH)] . The 1,1-seco-compound XXXII exhibits infrared maxima at • 2237cm"" 1 (C=N) and 1631cm""', 895cm"’' (C=GH 2). The structure of.the above £-lactam as XXXI is supported by the preparation

(page 17) , of 2-aza-A-homo-5«-cholestan (XL) which is differ-

27c, W. Shoppee, R. E. Lack, and S. K. Roy, j. Chem. Soc. 3767 (1963). 16 ent from 1-a za-A-homo- - ohole s tan (XXXIII). Further, XXXIII and XL gave different N-acetyl derivatives, m.p. 80° and , 1^2-1 respectively when treated with acetic anhydride in pyridine. On treatment with thionyl chloride at -20°, A-nor-5“- cholestan-1-one oxime (XXXIV) similarly gave the normal Beclcmann product, 1 -aza-^a-cholestan-2-one (XXXV) (35%) ^ accompanied by the 1-cyano-1,1-seco-compound XXXVI (50%). A-nor-5a-cholestan-1-one oxime thus has the configuration XXXIV (OH a n ti to 10/8-Me).

H o N

I XXXIV

Although ^a-cholestan-2-one oxime (XXXVII) appeared to be homogeneous with a sharp m elting point, unaltered on re crystallization or on repeated chromatography, it gave a mix ture of 3-aza-A-homo-5“-cholestan-^2-one (XXXVIII) and 2-aza- A-homo-^a-choiestan-3-one (XXXIX) with thionyl chloride at -.20°, indicating that the solution of the oxime in thionyl" chloride must, therefore, contain the.syn- and■anti-forms. 17

Ç o c i

x m x

H c o ç L M -1

A similar treatment of the oxime XLI and syn-oxime XLII^S. yielded ^+a-aza“A-hcmo-5®-cholestan-*+“one (XLIII) and

3 - 0 X 0 - A“homo-5“-choleist-1-ene (XLIV) resp ectiv ely , as the only reaction products.

■o

• XLIII

W, Shoppee, R. Lack, R. N. Mirrington, and C, R, Smith, J. Chem. Soc., ^868 (1965). 18

Ho> XLII XLIV The formation of lactams and w-cyano-olefins in the Beckmann rearrangement■of ketoximes appears to correspond to two types of heterolysis of the bond C-R:29

M- -R HI B o T

N ITNH c

Since oxime geometry determines the product in normal Beckmann rearrangements, the covalency changes (a) must be concerted. In the formation of w-cyanorolefins, the electron displacement (b) may be concerted or consecutive. If the displacement (b) is concerted, the stereoelectronic require ment should be a trans-coplanar arrangement of the G-H bond, broken (in R) to the G-C bond cleaved. In 2,2-dimethyl cyclo-

P. P e rris , J . Org. Ghem., 2^, 12 (I960). 19 hexanone oxime (XLV) the equatorial' C^-hydrogen atom s a t is fies this requirement, but in -cholestan-1-oxime XXX only the axial 5® and 9®-hydrogen atoms, and in j8-amyrenone oxime (XLVI) only an axial 5«-hyd.rogen atom, are available, so that trans elimination of a proton from an axial methyl group is p referred .

— Chi

XLV XXX

XLVI

If, on the other hand, the displacement (b) is a consecutive process, the samem-cyano-olefin should arise with equal ease from both geometrical isomers of a ketoxime, since this stereochemical distinction between syn- and anti- forms should disappear in the intermediate iminium cation; there appears to be no suitable example recorded in the li terature with which to test the matter. 20

Further, Shoppee and Roy30 obtained a nearly q u a n tita tive yield of the 5-oxo-!+,5-seoocholestano-6-nitrile (XLVIII) by tre a tin g 5-hydroxy-5“-cholestan-'+-one oxime (XLVII) w ith thionyl chloride or ethereal hydrogen chloride.

XLVIIIXLVII

Thus, the formation of cyano-olefiris appears to parallel the ready formation of aldehydes or ketones and cyanides from anti-g-hydroxy oximes (1) and of acylium cations and cyanides from anti-g-ketoximes (2) by "second order" Beckmann rear- rangements30,31,32 T^hile syn-hydroxy oximes and syn- -ketoximes yield aldehydes, ketones or acylium ions, and isocyanides (3 ).32,33

30c, W. Shoppee and S. K. Roy, J . Chem. Soc.-, 377^ (1963). 3^A. Werner and T. Detszheff, Ber., ^3, 69 (1905).

32a , H. Blatt and R. P. Barnes, J. Am. Chem. Soc., 11^-8 (193^) . ■ 33a. Werner and A. Piguet, Ber, ^ 37, *+295 (190^). 21

RCN

NyOH ■ W i' ^ o

2 . R -^ Y ^ ^ JlaS^ R,co,h ' ■■■ RCN

Ho ,- H-

H O % S ^ _ , M-R -^+^

Mazur3^ made the surprising observation that the Beckmann product is not necessarily related configurational- ly to the starting oxime according to the accepted mechanism of the rearrangement. Ha o’btained only one lactam (the S-aza- ^'^-^--ketone) by treating testosterone propionate oxime (XLIX) (containing only about 10^ syn-form) and 17o-methyltestoster- rone acetate oxime (L) (pure a n ti- compound) resp ectiv ely with thionyl chloride in dloxane. The above lactam is the iso mer expected from the syn-oxime. This result is explained as the product of thermodynamic control for the a,/8-unsaturated

3^R. Mazur, J . Org. Chem., 28, 2^-8 (1963). 22 oximes under the reaction conditions employed. OR)

XLIX R =NOH LI R'»=C2H5C0,R’'=H , R»=C2H5C0,R"=H

L ■ R =NOH LII R'=CH3G0,R"=CH3 R'=CH3G0,R"=CH3

2) B-ring aza-steroids. Knof35 prepared the lactams IV and LVI by treating 3/8-acQtoxy-choiestan-6-ketoxime (LIII) and 3j8-acetoxy-cholestan-7-ketoxime (LIV) vath thionyl chlo rid e .

Knof, Ann, Ghem,, 6J+2, 19^ (1961). 23

S O Cl,

LIV LVI Shoppee ejb al.36 obtained 7a-aza-B-homo-5«-cholestan- 7-one (LIX) and 7a->aza-B-homo-choiest-5-ene-7-one (LX) respectively as the only product of reactions of oxime LVII and syn-oxime LVIII w ith thionyl chloride.

LIX ■ LVII

LVIII LX

36c, W, Shoppee, M. I, Akhtar, and R, E, Lack, Jr. Chem. Soc., 3392 (1964-). 2%+

The lactam LXIII and thew-cyano ketone LXIV are re spectively obtained as the only product of reaction of the oxime LXI^^ and LXII^O with thionyl chloride as shoi-m below.

LXI b LXIII

LXII

Hara37 prepared 7-oxo-7a-aza-B-homo-cholanate (LXVI), an oil, by heating a pyridine solution of methyl-7-oximino cholanate (LXV) with p-toluene'sulfonyl chloride to 100° for 1 hr. Lithium aluminum hydride reduction of this product afforded the crystalline 2k-hydroxy-7-oxo-7a-B-homo-cholane,

Hara, J . Pharm, Soc, (Japan), 209 (1955)» 25

m LXVI LXV

1H4-

Barton at a2.38 prepared the lactams LXIX and LXX, LXXI by treating 7j11“diketolanostan-2-yl acetate monoxime (LXVII) and 7,11-d ik eto lan o st-8 -en e-2 -y l acetate monoxime (LXVIII) respectively, with phosphorous pentachloride in benzene.

'NOH LXVII LXIX

38p, H. Barton, C. S. Barnes, J. S. Fawcett, and B. R. Thomas, J . Chem. Soc., 2339 (1952)., - 26

P O c

LXVIII •

LXXI

3) C-ring aza-steroids. Mazui*39 prepared 3i8-acetoxy- 12a-aza-G-lioino-5“-22a-spirostan-12-one (LXXIII) by heating (95- 105° fo r ^ h r. ) 3i8-acetoxy-5«—22a-spirostan-12-one oxime (LXXII) with p-toluenesulfonyl chloride in benzene.

LXXII

39r. h . Mazur, J . Am. Chem. Soc., 1^-5^ (1959)» 27

Zderic and Iriarte^^ obtained 9a-azar-C-bomo-txcogenin

(LXXVI) and 9a-aza-C-homo-5“-pregnane-3/3,17»jS I-trlo l-l 1,20- dlone-BMDu-3-acetate (LXXVII) by treating the oxime LXXIV and

LXXV respectively \fith phosphorous oxychloride in pyridine.

LXXVI LXXIV O

R o c

LXXVII

Bladon and McMeekin^”* obtained homo-$'a-2^D-spirost-9(T1 )-ene-12-one (LXXIX) by Beckmann re arrangement of 9(11)-dehydrohecogenin acetate oxime LXXVIII with p-toluenesulfonyl chloride.

1+0 J . A.-Zderic and J . I r i a r t e , J . Org. Chem., 2%, 1756 (1962 ).

^•1P. Bladon and W, McMeekin, J . Ghem. Soc,, 35Q1+ (1961) 28

■LXXIX LXXVIII A,

D~ring aza-steroids. Kaufmaim*^'^ prepared d e hydro- isoandrololactam acetate (LXXXIII), testololactam (LXXXIV) and estrololactam benzoate (LXXXV) by treating the ,17-oximes of dehydroisoandrosterone acetate LXXX, A^androstene-3,7- dione LXXXI and estrone benzoate LXXXII ifith p-acetylaminobenzenesulfonyl chloride in pyridine.

AlOH

LXXX 4 OH

h2 S, Kaufmann, J . Am. Chem. Soc,, 21» 1779 (1952). 29

LXXXVI LXXXVII H

LXXXI o LXXXIV

NOH

AcNH 0 5 0 2 = 1 ^ » \ o LXXXV LXXX7III R=OH'

In the case of LXXI, however, it was necessary to pro tect the 3-keto group temporarily by preparing its enol ether so that only the 17-keto group was converted into the oxime. In order to have the reaction proceed normally ifith 3-hy- , droxy steroids, it was necessary to esterify the 3-hydroxy group. The D-ring lactams are very stable and attempts to open the lactam ring with alkali or acids were unsuccessful. 30 In all cases only one lactam was isolated from the reaction mixture and no indications of the presence of an isomer was noted. In order to establish the possition of the N-H group, the lactam LXXXVI was ,dehydrogenated with selenium at 350°. A small amount of crystalline material isolated from the acid- soluble fraction of the dehydrogenation mixture proved to be identical in all respects with an authentic sample of 1- azachrysene (LXXXVII). Hence, the lactams obtained from the three above listed compounds are considered to have structures LXXXIII, LXXXIV and LXXXV respectively. The oxime LXXX has been rearranged to the lactam LXXXIII by catalysts such as thionyl chloride in dioxane,^^ p-acetylaminobenzenesulfonyl chloride in pyridine,while its Si^-cbloro-derivative LXXXIX has also been arranged to the corresponding 3/8-chlorolactam XG by thionyl chloride in dioxane.^^

^'■3b. M. Regan and P. N. Hayes, J. Chem. Soc., 639 (1956). ^R« Anliker, M. Muller, J, Wohlfahrt, and H, Heusser, . Helv. Ghim. Acta, ]8, 1^0^+ (1955).

*^5r, Anliker, M, Muller, M. Perelman, J. Wohlfahrt, and H. Heusser, ibid., j+2, 1071 (1959) » ^^R. T. Blickenstaff and E, L. Poster, J, Org, Ghem.', 5029 (1961). 31

cl cl LXXXIX x c

Heusser et a l.^7 obtained' both isomeric lactams XCII and XCII I by treating the oxime of A^-3i8-acetoxy-17a-keto- D-homo-androstene XCI >ri.th p-acetylaminobenzenesulfonyl chlo ride in pyridine.

NOH

AcÛ XCI XCII XCIII

Tsuda and Hayatsu^'^ separated two stereoisomeric oximes XCV and XCVI by chromatography. These oximes XC? and XCVI were produced by the reaction of 16 -ke t o- cho le s ta n -3j8-o l- benzoate (XCI?) with hydroxylamine hydrochloride in the ratio 7:1. Treatment of the, pyridine solution of XCV with p-toluene-

Heusser, J. .Wohlfahrt, M. Muller, and R. Anliker, Helv. Chim. Acta, 3 8, 1399 (1955). Tsuda and R. Hayatsu, J. Am. Chem. Soc., 78, 1+107 .(1956). . 32 sulfonyl chloride gave lactam XCVII; XCVI afforded the ether soluble lactam XCVIII, and the ether insoluble oxime tosylate

XCIX. The lactam XCVII was also obtained by a different route from the oxime of l6-keto-/^-cholestenyl benzoate C upon

Beclcmann rearrangem ent, followed by hydrogenation.

f9H\7

XCVII HHnOH

XCIV

XCVI XCVIII

XCIX

XCVII BO A, 33 Although the oxime of l5-keto-A'‘**‘'^-cholesten-3;S-ol acetate Cl I afforded 15-aza-l6-keto-^^'*'-D-homo-cholesten- 3^-ol acetate (GUI) by Beckmann rearrangement, a similar rearrangement of the oxime. CIV, which has no double in ring C, gave only the oxime to sy late CV.

1=0

T scI

CXI c m

NOH 'N -O S 0 - BP CIV CV Hassner and P o m e r a n t obtained 3)8-acetoxy-l6-acetoximino-5-a'ndrostene-17-one (C V II) by treating lô-oxlmlno-^- androstene-3^-ol-17“One (C V I) with acetic anhydride in pyri dine fo r 2h hr. The product C V II is readily cleaved by aqueous acid or base, or by heating with aqueous solvents. Attempted recrystallization from aqueous methanol of the crude product from the preparation of C V II gave 3^-acetoxy-16,17-seco-5~ androstene-l6-nitrile-17-oic acid ( C V I I I ) . Treatment of the product C V II with 0,25 N KOH at room temperature afforded 3)8-hydroxy-16,17-seco-5-androstene-l6-nitrile-17-oio acid

(CIX).

■H '"OH OAc

CVI CVII

CIXCVIII

Lq ■ •^A. Hassner and I. H. Pome rant z, J. Org, Chem,, 27, 1760 (1962 ), 35

The easy conversion of CVII to cyano acids CVIII and CIX is explained as a solvolysis reaction passing through an intermediate state of the type CX followed by the reaction with water or hydroxide ion.

CVII CX

CVIIICIX

Hassner arid P o m e r a n t found that the cyano acid CIX could be caused to undergo p a rtia l conversion to the amide acid CXI by refluxing the former with potassium hydroxide in 95^ ethanol. The resistance of the nitrile function in CIX to hydrolysis in basic solution was•explained by proximi ty effect exerted by the- carboxylate ion. The cyano acid was also unaffected by alkaline hydrogen peroxide.

OH ÇNH, eroH

CXI CIX 36

It has been reported that the saturated analog of CVII is converted to CXI I \fith acetic anhydride in pyridine?*^ by standing overnight. However, the correct stru c tu re of the reaction product was found recently by Hassner and Pome- r a n t2^9 to be CXIII.

A CXIII CXII

,5) With di- and tri-ketoximes. 8ohenck^1 applied the classical rearrangement conditions ( 15-20 min., heating at 95° with 90^ sulfuric acid) to prepare lactams from CXI? and CXV, however, the structures of the products were not firmly established, and they were not well characterized.

03.H

HOM CXI?

D. Heard, M, T, Ryan, and H. I. BoIker, J; Org, Chem., 172 (1959). 51M, Schenck, Z. Physiol. Chem., 360 (191^)- 37 Singh and Parashar^^ have recently prepared the com- ponnd CXVII, the first diazasteroid of definitely established stru c tu re by trea tin g the oxime of 17a-aza-D-homo-androst- ^-ene-Sjl7-dione (CXVI) with thionyl chloride.

HON CXVI CXVII

Singh and V. V. Parashar, Tetrahedron Letters, 983 (1966 ). . DISCUSSION

A comprehensive literature survey shows that several authors have reported the Beclcmann rearrangement of ketozimes involving different rings of steroids and in most cases the structure of the product (or products) has been successfully established. Beckmann rearrangement of 2-oximino-choles'ta-^,6“diene-3-one and determination of the structures of the rearranged products would, there fore, complement' the past research in this field of che mistry. This oximino ketone which had previously been made, by Glamkowski^^ \^as similarly prepared as outlined in the following,sequence of reactions:

CXVIII By CXIX

CXXI CXXII

014

CXXIII

^•^E. Glamkowski, Ph.D. d is s e rta tio n , The Ohio S tate University, 1$63. 38 39 -

It was found that 30 min. wore needed for the complete oxidation of the dibronide CXIX to CXX with sodium dichromate in acetic acid. Glamkowski, however, reported that 10 min. were required for the above oxidation. The conversion of cholest-5-2ne-3-one (CXXI) to cholesta-^,6-diene-3-one (CXXII) was carried out by refluxing CXXI \d.th chloranil in tertiary butyl alcohol; 3 hr. were required for complete dehydroge nation. Glamlcowski, however, carried out the dehydrogenation fo r 1 hr. I t was proven by Glamkowski, using various methods, th a t the oximino hydroxy group in 2-oximino-cholesta-V,6-diene- 3-one (CXXIII) was trans vri.th respect to the carbonyl group in the molecule.^3 It has been mentioned before (see page 3^) that the saturated five membered D-ring acetoximino ketones are very unstable and easily undergo "second order" Beckmann cleavage by aqueous acid or base, or even on attempted recrystalliza tion from aqueous methanol. Glamkowski prepared 2-acetoximino- cholesta-4-,6-diene-3-one by treating the oximino ketone CXXIII with acetic anhydride in pyridine. This compound was found stable enough to be recovered unchanged after one day in aci dic acetone solution. It failed to .give any normal or "second order" Beckmann rearranged product even after refluxing in aqueous acetone. The product, however, could be hydrolyzed back to the starting oximino ketone by allowing it to stand ■ ho in concentrated, hydrochloric acid solution at 25° fo r 2h h r. Glamlcowski?3 iso la te d a c ry s ta llin e Beckmann rearrangement product, m.p. 116° by treating CXXIII vath p-toluenesulfonyl chloride in pyridine at room temperature for 3 days. This product showed infrared absorption at 5.71yd, 5.96ju, 6.l8ji, 6.2hp. and 6.32yU and had the follovâng elemental analysis; C, 78.25; H. 9.50; N, 3.77; S, 0.00; other data such as the Ultraviolet absorption maxima or the optical rotation were not given. No further work was done vâth this Beckmann pro duct. We treated 2-oximino-cholesta-^,6-diene-3-one (CXXIII) with p-toluenesulfonyl chloride in pyridine at room tempera ture fo r 2h hr. and after work up a crystalline material CXXIV, m.p. 197- 198° was obtained in 61^ yield. The infrared spectrum of the product showed two carbonyl absorptions at 5. 7O/1, 5.97yd and bands a t 6.1*;^, 6.23yd, 6 .3 ^ probably due to double bonds and C=N groups in the molecule. The ultravio

let absorption spectrum showed a shoulder at.220nyi (.g. 20, 900)

and a maximum a t 311i^ (& 55?^60). - Meisenheimer^ determined that the migrating group in the Beckmann rearrangement approaches the nitrogen atom from the side opposite to the departing oxygen atom. Beckmann re arrangement in the present case should therefore, involve

^ J . Meisenheimer, P. Zimmermann, and U. Kummer, Ann., h h e . 205 ( 1926 ). 5+1 the migration of the carbonyl carbon atom to the nitrogen atom to give the expected aza-homo-steroid A, although the possibility of migration of the C-1 carbon atom to the nitrogen atom could not be completely discounted as some ex ceptions have been reported in the literature^2 (see page 12). On the basis of the above information the likely pathways for the formation of Beckmann rearranged products from CXXIII may be depicted as follows:

oT< OH

CXXIII

X ^2 Surprisingly, however, the elemental analysis of the product CXXIV (C, 80.^; H, 9.91; N, 3.42; 8, 0.00) was far from what was expected for the isomers of

formula A or B, namely C27H41O2N (C, 78. 78; ÏÏ, 10.04; N, 3.40; 8, 0.00) but agreed with that expected for a dimeric product C^Iiiî 8o03N2. The confirm ation of the- latter formula accrued from a. molecular weight determina tio n of the compound CXXIV in chloroform by the osmo-

metric method (Calcd. for C 5I1.H80O3N2: 804; found 795). In order to test the presence- of reducible carbonyl group- in the product CXXIV it was treated with sodium borohydride in methanol at room temperature. After 24 hr. -the reaction mixture was worked up and found to contain two products, as evidenced by spots on s ilic a th in layer chromatograms. On preparative thin layer chromatography, the reaction mixture afforded two crystalline materials of m.p. 167° and 237°. The product CXXV, m.p. 167°,dis played infrared absorption at 4.4(^ (probably a nitrile - group), 7. 7^ (probably an ester carbonyl group) and 6.1 ^-Kiu, 6 .2 ^ (probably double bonds), no absorption attributable to a hydroxy group and analyzed for C 28H1+3O2N. The W.M.R. spectrum showed absorption at 6.27?, charac teristic of a methoxyl group and 3 •29-4.31’’ -indicative of olefinic protons. The product of melting point 237° was in all respects identical vâth the original oximino ketone CXXIII. 43 Isolation of the product CXXV suggested that sodium borohydride in methanol acted simply as a source of methoxide ions. This view was substantiated by treating CXXIV v/ith sodium methoxide in anhydrous methanol at room temperature for 24 hr. Work up' of the reaction mixture, followed by separation on preparative thin layer chroma tography, afforded CXXV and CXXIII, identical in all respects v/ith the respective authentic samples. Next, a so lu tio n of the Beckmann product CXdV in methanol was stirred \fith sodium hydroxide at room tempe rature for 24 hr., which gave, after the usual work up, an acidic and a neutral crystalline material. The acidic product CXXVI, m.p. 21:8°, showed infrared absorption at 4.4Qh (characteristic of a nitrile group), 3 »8C^ (bonded OH of CO2H), ^ .8^ (COgH) and 6.1(^, 6,2^ (double bonds) and analyzed for Cg^H^lOzN. The infrared spectrum of the above neutral product was superimposable with that of the oximino ketone CXXIII and a mixed melting point was un depressed. The cyano acid CXXVI readily reacted with an ethe real. solution of diazomethane to give a cyano methyl ester, m.p. 167°, which analyzed as C 28HI+3O2N''. This pro duct was identified as CXXV by its infrared spectrum-,, rotation, Rf value and undepressed mixed melting point. When the product CXXIV was refluxed with potassium hydroxide in methanol for 24 hr., the solution turned deep' red and after work up, the same acidic compound CX]{VZ was obtained which afforded the same cyano methyl ester CXXV vriLth diazomethane but the brovna red neutral substance could not be. crystallized and appeared polymeric in nature. The oximino ketone was also found to give a similar polymeric material when refluxed v/ith a base. On the basis of the above chemical and physical data, a tentative structure of the dimeric product CXXIV was derived as sho\m in Chart I (page '+5). Next it was deemed essential to verify the struc ture of the cyano acid CXXVI and the cyano methyl ester CXXV by an independent synthesis. The oximinoketone was dissolved in anhydrous methylene chloride and treated with thionyl chloride at room temperature for 2*+ hr. The brown crude substance obtained after removal of the thionyl chloride and the solvent under vacuum had infra red absorption at l+.l+Sju (characteristic of a cyano group) and (indicative of a carbonyl group of an unsatu rated acid chloride) and no hydroxy group; it showed an ultraviolet maximum at 297^^ in dioxane. This substance was treated separately with water and sodium methoxide to give the cyano acid CXXVI and the cyano methyl ester CXXV in 66% and 77% yield respectively. A likely mechanism proceeding,through a cyano acid Chart I Tentative structure of the dimeric product CXXIV.

CXXIV

9sM

CXXIII

CXXVI CXXIV

CXXIII k-6 chloride CXKSfll intermediate as evidenced by the shift of the carbonyl absorption to lower wavelength in the in frared compared to the carbonyl group in the parent oximino ketone, can be postulated as follows: ■

O S ocl OH i©

CXXIII

c x x y i i

CXXV o CXXVI

This mechanism explains the role of water for the ready formation of the cyano acid from the intermediate cyano acid chloride CXXVII and is further strengthened by the fact that an attempted trapping of the other possible h-7 intermediate CXXVIII as an imino-ether C3CXIX by reaction \d.th anhydrous methanol did not materialize; the reaction afforded only the cyano methyl ester CXXV in yield.

O'- CXXIII

o©

N e el C 6 CXXVII c x x c x ^8 Cleavage reactions of this type are sometimes re ferred to as "second-order"Beclmann rearrangement:^9,31-33 The different behavior of oximinoketone CXXIII on treat ment \'âth thionyl chloride and tosyl chloride In pyridine Is not surprising, as It Is knomi that catalysis of the rearrangement Is often specific. Thus, phosphorous-penta- chlorlde rearranges dlbenzalacetone oxlme CXXX to N-sty- rylclnnamamlde (CXXXI), but concentrated sulfuric acid causes cycllzatlon to the Isoxazollne (CXXXII).^^ This behavior Is fairly general for oxlmes of a,0-unsaturated ketones.^3

CXXXI c= M O H % CXXX

C X X X II ^

Auwers: and H. Brink, J'. Prakt. Chem., (2), 221} (1932 ). H« Blatt and J. P. Stone, J. Am. Chem. Soc., 2 1 , 1133 (1931); Ibid., 21, ^13^ (1931). ^-9 An example of a cyano acid chloride formed as a result of the Beckmann rearrangement of an oximinoketone is reported by Borsche and Sander.. These authors isolated o-cyanocinnamoyl chloride (CXXXIV) by treating 1-nitroso-2-naphthol (CXXXIII) with phosphorous penta- ch lo rid e.

NOW •OH pel.

CXXXIII

CXXXIV

• Borsche and W. Sander, Ber., 2815 (191^)»' 50 A suitable mechanism for the formation of the di meric product CXXIV may be postulated as follows:

ÇH

CXXIII

CXXIV

Y = CXXIII 51 The presence of an imine intermediate (step II) in the Beclonann rearrangement was demonstrated by Kuhara and his co-workers who showed that N-phenylbenzimide- benaenesulfonate (CXXXVI) was formed as a result of the rearrangement of diphenyl ketoxime benzenesulfonate (CXXXV).

C. // } NOH

If water is replaced by a solvent which can act as a nucleophile the rearrangement is arrested at the imine s ta te .

Kuhara, K. Matsumiya. and N. Ma tsunami, Mem. C oll. Sci. Kyoto Imp. Univ., 1, 105 (19lW C.A., 1613 (1915)' .

59w. Z. Heldt, J. Am. Chem. Soc. 80, 5880 (1958). 52 The,existence'Of an imine intermediate was further indicated by the isolation of imine derivative CXXXVII.

NOSO^ ^ I — s ^ O S % 0

CXXXVII

The sulfonyl ester group in CXXXVII can be displaced by phenol, ethanol or amines to give the respective imino-ether or amidine in high yields. Hill^”' treated the ketoxime (C) with p-toluenesulfonyl chloride in pyridine and isolated in high yield an unexpected product'; which on the basis of chemical and physical data was assigned the structure (D). The mechar nism postulated by him is as follows:

GOp. Oxley and W. P. Short, J . Chem. Soc., 1.51^ (19^4-8). K. H ill, J . Org. Chem., 2£, 30 (1962). 53

■NOH 0 c

NOH

CN ©

Isolation of such a dimeric compound by Hill at once suggested that a. compound of structure (E) iso meric with CXXIV can similarly arise as shoi/m below:

CXXIII

O-—ç 5^

Such an isomeric compound will show cyano absorp tion which, however, was absent in the infrared spectrum of the dimeric.product CXXIV. In order to obtain further evidence in favor of the structure CXXIV for the dimeric product, it was de cided to synthesize the compound (E). The cyano acid CXXVI obtained by the basic hydrolysis of CXXIV was re fluxed vath thionyl chloride for ^5 min. and the excess; of the thionyl chloride removed under vacuum. The in fra red and the ultraviolet spectra of the crude cyano acid chloride were superimposible vâth that of the cyano acid chloride CXXVII obtained from the oximinoketone. A solution of the oximinoketone in dry pyridine was added to an equimolar amount of the cyano acid chlo ride and the mixture was stirred at room temperature for 2^- hr. The usual work up; gave a mixture of compounds as .shoim. by thin layer chromatography. The presence of the cyano acid CXXVI and the oximinoketone CXXIII in the mixture-^were readily detected by spotting a plate w ith the mixture and with authentic samples of CXXIII, CXXIV and CXXVI, the plate being developed with chloro form - ether (1:1). No trace of a spot corresponding to compound CXXIV was detected in the m ixture, shovrlng th at the isomeric compound (E) was not formed at all. The appearance of the cyano acid can be explained on the

basis of reaction of the unreacted cyano acid chloride 55 with water during work up conditions, which involved pouring the reactio n mixture in to w ater. Thus the possibility of the alternative isomeric structure (S) is completely ruled out due to the failure of the oximinoketone to react with the cyano acid chloride in pyridine. The assignments' of the two- carbonyl absorptions in the infrared spectriun of the compound CXXIV at and 5*9'^ can now be rationalized, unambiguously, each absorption being assigned its particular chromophore on the basis of the observed carbonyl absorption of the parent oximinoketone CXXIII which absorbs at 5.9^* Again its ester derivative 2-acetoximino-cholesta--^,6- diene-3-one shows the absorption of the C-3 carbonyl group: at almost the same place (5-96ji). The higher wavelength carbonyl band of dimer CX}[IV (5*9^.) is therefore attributable to the carbonyl group in the six membered oximinoketone part of the molecule. The remaining maximum a t 5* 70ju must be due to the carbonyl absorption in the seven membered ring as a s h ift to lower wave length can be observed when an atom (such as nitrogen) with electron withdrawing properties is attached to a carbonyl.' This requires a higher force constant or carbonyl strechlng frequency. 56

PCOCM.

CXXIII 2-acetoxiiiiino-cholesta- ^!-,6-diene-'3-one

— o-

CXXIV

With a view to investigating further the chemical reactions of the oximinoketone C X X I I I , it was treated with methyl Grignard, which afforded the expected a-hydroxyoxime C X X X V III in crystalline form. The elemen tal analysis and the infrared spectrum supported the stru c tu re of th is product as C^OCWIII. The compound

O C X X V III gave a facile ring, cleavage product CXXXIX, m.p. 138°. when treated with p-toluenesulfonyl chloride in pyridine at room temperature: for 2h hr. The infrare;d spectrum of this compound displayed absorption at ^-.^-2ju (nitrile), 5.9Qn (carbonyl), 6.1^, 6,3^ (double bonds) and the u ltra v io le t spectrum showed a maximum a t' 288mju ' (6 7,688). 51 Similarly, the oximinoketone was treated with phenyl Grignard reagent to give:: an oil which after re peated chromatography failed to crystallize. The infra red spectrum of this oil showed absorption at 2.9^ (OH) and no carbonyl absorption which indicated that it may have the structure CXL. This compound, however, under went rearrangement to a crystalline compound C]{LI, m.p, 101°, on treatment with p-toluenesulfonyl chloride in pyridine. The structure of CXLI was in accord vriLth its infrared spectrum and elemental analysis.

pl -1 OH

GXXXVIII R=CH] CXXIII ■ CXL R=C6 H^

o=c

^ CXXXIX R=CH' CXLI R=C6 H5 58 Ample analogies are found in the literature for the above transformations. Thus Forster^^ obtained the alcohol CXLIII by rea ctio n of 2-oximinocamphor (CXLII) with methyl .Grignard.

NOH NOH CXLII • C X L I I I

Blatt and Barnes^^ obtained acetophenone and benzo- nitrile as a result of second order Beckmann rearrangement by treating methyl benzoine oxime CXLIV with benzenesulfonyl chloride in pyridine.

C -> C h. Pv OH N—Oh rl- CXLIV CN

^2m. 0 . F orster and K. A. N., Rao, J. Chem.' Soc., 2670 (1926 ). .5 9 Although.the preparation of several cyahosteroids have been reported,^’^3-65 cyano-A-nor steroids have not been extensively investigated. Therefore a synthesis of this type of compound would be of interest for reasons of biological testing. The cyano acid chloride CXXVII was' chosen as the starting material because of its ready a v a ila b ility from oximinoketone CXXIII. The cyano acid chloride CXXVII was found to react with a. wide variety of nucleophiles and the following cyano-A-nor steroid derivatives have been prepared.

63#. E. Wolff and T.Jen, J . Med. Chem., 6 , 726(19630.. Jen and M. E. Wolff, J. Org. Chem., 28, 1573 (1963). &5j. Fishman and H. Guzik, Tetrahedron Letters, 14Ü3 (1966). 60

0 O K CXXVII CXLV R=m2-C- OH CXCiVI R=0-G- h OH CXLVII R=CH3-Ç- CH3

CXLVIII R=0im-C- 8

CXXV r=ch 3 -o- c-

CXXVI R = H O -C - . 0

The consiclerahle degree of steric hindrance of the n i t r i l e group in these compounds was illu s tr a te d by hydro lysing methyl 2, 3-secocholesta-^-, 6 -d ien e- 2- n i t r i l e - 3~oate (CXXV) to the corresponding amide acid CXLIX by refluxing with potassium hydroxide in ethylene glycol, for 5 h r .,, no dicarboxylic acid was obtained. The infrared spectrum of the amide acid CXLIX displayed absorption at 3*10ju (N-H),

3 . 7^ ; 3 . 89/1 (OH of COgH), 5. 72/1 (COgH) and 6 . 05/1 (COM2). 61

The structure of the amide acid was in accord \ri.th its con version to methyl 2, 3-secocholesta-*+, 6 -d ie n e - 2-oic a c id - 2- amide-3-oate (CL) by. treatment with an ethereal solution of diazomethane. This ester CL load infrared absorption at

2.91/1, 3.23/i (N-H), 5.71/1 (CO 2CH3) and 6 . 01/ 1.

■ KOH

Hydrolysis of the cyano methyl ester CXXV with po tassium hydroxide in 95% ethanol or water - ethylene glycol mixture led only to the isolation of 2, 3-secocholesta-^,6-diene-2-nitrile-3“Oic acid (CXXVI), the nitrile function being completely unattacked by the base in these

, reactions..It can be mentioned in this connection.that Ireland and Chaykovsky'^^ recovered the sta rtin g d in itr ile

E. Ireland and M. Chaykovsky, J . Org. Chem., 748 (1963 ). 62

CLI whGii attempting to hydrolyze it by alkali

CLI ■

Similarly, the resistance of the nitrile function in 3^-hydroxy- 16 , 17-se co -5-androstene- 16 - n i t r i l e -1 7-o ic acid ( 01%) to hydrolysis in basic solution was observed by Hassner and Pomerantz (see page 35)* The cyano amide GXLV was dehydrated by refluxing with thionyl chloride, affording crystalline 2 , 3-secocholesta-^,6-diene-2,3-dinitrile (GLII) which analyzed as expected for G 27H^QN2. The dinitrile was characterized also by i t s in frared spectrum, which showed absorption a t k,h3p. (unconjugated GN), h,^2jx (conjugated,CN) as well as 6.l4^ and 6 . 33^^ (double bonds).

SOCI

GXLV GLII 63 A number of ring contracted stero id s have been syn thesized by the photochemical Wolff rearrangement of cyclic • steroidal a-diazoketones.^7 For example, photolysis of l6-diazo-estrone-3-methyl ether (CLIII) afforded the D- norc'arboxylic acid CLIV.^^

CLIII GLIV

Glamlcowski^3 synthesized 2-diazo-cholesta-)+,6-diene- 3-one (CIV) from oximinoketone CXXIII by reaction with chloramine, and he decomposed it photolytically in seve ral different solvents. He claimed to have isolated an amorphous acidic fraction which was not further character ized. It seemed desirable to reinvestigate this photolysis in somewhat greater detail. It was hoped that an unsatu-

6?J. L. Mateos and 0. Chao, Bol. In st. Quim (Mexico), 12.5 3 (1961)5 J . Meinwald, G. G. C urtis, and P. G. Gassman, J. Am. Chem. Soc., 8]+, 116 (1962): G. M uller, C. Huynlc, and J. Mathieu, Bull. Soc. Chirn. Prance, 296 (1962)5 A. Hassner, A. W. Coulter, and W. S. Seese, Tetrahedron Letters, 759 ( 1962)5 J. L. Mateos, 0. Chao, and H. R. F lores, T etra hedron, 1^, 1051 (1963)5 M. P. Cava, P. M. Weintraub and E. G. Glamkowski, J. Org. Chem., 3I 5 201.5 (1966). P. Cava and E. Moroz, J. Am. Chem. Soc., 8^-, 115 ( 1962 %. , - - 6^ rated acid CLVII might be formed by way of a ketocarbene CLVI intermediate as illustrated below:

pH \\v

CXXIII CLV

CLVI

CLVII

A solution of the diazoketone CLV in tetrahydrofuran and sodium- bicarbonate solution was irradiated with ultraviolet light for ^5 min., after which gas evolution ceased and the solution was red. The solution was extracted ülth ether and the ether evaporated under reduced pressure, giving a deep red gum which showed no diazo absorption in the infrared. This material was chromatographed over silica but no crystalline substance 6 5 was obtained. The aqueous bicarbonate extract on acidifi cation I’ri-th dilute hydrochloric acid gave no precipitate. Extraction of the above acid^ied solution with ether followed by evaporation left no residue, showing.that no acidic product was formed in the photolysis’. The result of th is experiment w arrants some comments: unlike Glamkowski we were unable to isolate any acidic product even though the photolysis was carried out essentially following his procedure. ■ All attempts to isolate pure crystalline components from the neutral gum formed in the photolysis of diazo ketone CLV failed, suggesting that this gum may contain a complex mixture of products. Some idea of the types of compounds present in th is mixture may be obtained from the brief literature review given below. Rubin et reported the isolation of a photodi- meric product CLVIII of androsta-^,6-diene-3-one-1% 8-ol propionate by exposing a benzene - petroleum ether solution of the above compound to the light of wave length SOOcyi although they reported that there was the possibility of formation of twenty dimeric products.

B. Rubin, G. E. Hipns, and D. Glover, J. Org. Chem., 22, 68 i^96k). 66

. CLVIII

That the formation of dimeric product is not an un usual occurrence in the photolysis of steroids is also strengthened by e a rlie r observation of Ushakov and Koshe leva 7® who obtained a compound of molecular formula CjkH3L.02’ by irradiation of choies ta--:-, 6-diene-3-o]ie in hexane by a Hg lamp in a quartz vessel. Later, in 1962, Jeger at irradiated a solution of cholesta-^,6-diene- 3-one in ethanol in a pyrex vessel equipped with a centrally located high pressure Hg lamp and, was able to is o la te a dimeric compound for which stru ctu re CLVIII was ■oronosed.

. 70W. l i Ushakov and K. F, Kosheleva, J, Gen. Chem. ('USSR), ilf, 1138 (19^-) C.A. 1+0; 1+071 (1946) . 71--!.. ?. Throndsen, G. Cainelli, D. Afigoni, and 0. Jeger, Helv. Chim. Acta, 234-2 (1962). 67 Again, dimerization may not be the only side reaction in the photolysis of the diazoketone. Instead of ring contraction, a hydride migration'^^ also possible lea ding to the intermediate CLIX, the A-ring of which will lead to the 1,^-diene-3-one CLX. Irradiation of an analogue of CLX in the androstane series has been found to afford a complex mixture of four phenolic and five ketonic photoisomers.73

ST 9 9 GLV CLIX

CLX

Although the preparation of 2-oximino-5“-cholestan- 3-one has been reported, ^.3 a. literature survey shows that its epimer, 2-oxim ino-5;8-cholestan- 3-one has not been

?^Por examples of hydride migration when a-diazokstones undergo Wolff rearrangement, see V. Pranzen, Ann., 602, 199 (1957). Duttler, C. Ganter. H. Ryf, E. C. Utzinger, K.- Weinberg, K, Schaffner, D. Arigoni, and 0. Jeger, Helv. Chim. Acta, 23^6 (1962)5 see also C. Ganter, E. C, Utzinger, K. Schaffner, D. Arigoni,' and 0. Jeger, ibid., . 6 ^, 2403 (1.962),' 6 8 synthesized so far. We, therefore, investigated the, reduc tion of 2-oximinocholesta-^,6-diene-3-one with lithium in liquid ammonia in order to determine if either or both of the corresponding saturated oziminoketones would be pro duced. The reduction of the oziminoketone CXXIII was first carried out for a period of about $0 min. and the excess of lithium was decomposed by addition of ethanol. The ■product was chromatographed over neutral alumina and three different colored fractions were collected. None of the fractions gave any crystalline material. Unexpectedly these fractions did not show carbonyl absorption in the infrared although they showed a band at 2.7^ suggesting the presence of a hydroxyl or amino group. Fukushima and Da urnisolated a considerable amount (72/0 of the alcohol CLXII when attempting to reduce CLXI by lithium in liquid ammonia in tetrahydrofuran, after quenching the reaction with ethanol.

7^0. ,K. Fukushima and S. Da urn, J , Org, Chem.,. 26, 69 c

Th p

CLXI CLXII

It is also well documented in the literature that reduction of à ,/3-unsaturated ketones can be stopped at the saturated ketone stage by addition of reagent such as ammonium chloride.-In an attempt to avoid overreduction, o the oziminoketone CXXIII was reduced during periods of 2, 10, I5j 20 and 30 min. in separate experiments, the reduction mixtures being quenched by the addition of solid ammonium chloride. The mixtures of products obtained were separated by preparative thin layer chromatography over silica to give the starting material and two other unidentified ma terials which resisted all attempts at crystallisation. The infrared spectrum of the non-crystallizable materials did not show'carbonyl absorption. Moreover, it was found that as the duratipn of the reduction time increased there was

75c, Djerassi, "Steroid Reactions", Holden-Day, Inc., San Francisco, 1963;, p. 300. 70 a decrease in the starting material and the proportion of non-crystallizable materials increased. After liaving thus failed to obtain any positive results from the reduction of the oziminoketone CXXIII uith lithiu m in liq u id ammonia, we next turned our a tte n tio n towards achieving the preparation of 2-o;>cimino-5i3~cholestan- 3-one by catalytic hydrogenation..The oximinoketone CXXIII was hydrogenated in tetrahydrofuran over 5/" palladised charcoal. The products were: successfully separated on pre parative thin layer chromatography on silica, affording two new c ry s ta llin e compounds-CLXIII (Rf O. 87), m .p .-I 9O- 192° and CLXIV (Rf O. 7O),. m.p. 27^° in 2% and 20^ yield re sp ec tiv e ly . The in fra re d spectrum of CLXIII had bands a t 2,89ju (OH), 5’90p. (carbonyl), 9 ‘ 72ji and 9»Q5p- while that of CLXIV had bands a t 3*l8/a (OH), 5 . 8I/1 (carbonyl) and 10.2%u. Further, the ultraviolet absorption spectra of CLXIII and CLXIV in neutral solution showed identical maximum at 2^6mju which changed with alkali to 303mja in both the cases. The above bathochromic shift in alkaline solution confirms the anti-configuration of the oximino hydroxyl group with respect to the carbonyl group at the 3-position of the ste ro id nucleus. Such a bathochromic s h ift seems to be gen eral for a-oximinoketones in which the oximino hydroxyl

group is anti to the keto g r o u p . 76 purther evidence for the

H. R. Barton and J. Beaton, J. Jim. Chem. Soc., 8]_, 4083(1961 ). 71 anti-configuration of the hydroxyl group in CLXIII and CLXIV stemmed from the fa c t th a t these compounds formed complexes . with Ni++, Co++, Cu++, a behaviour consistent only with the anti isomers. Sidgwick?? depicted a mechanism of this colored complex formation with the oxime group in the ni- trone form as shovm below;

. + 4- 4- M

.© + -I

iln oximinoketone in which the oxime hydroxyl group is syn to the carbonyl group apparently cannot form such a complex. Both the compounds CLXIII and CLXIV analyzed for C27%5'02N. Further, CLXIV was found identical in all respects with an authentic sample of 2-oximino-5a-cholestan-3-one which has been prepared by the direct oximination of • cholestan-3-one with one equivalent of 2-octyl nitrile. 53

77n . V. Sidgwick, "The Organic Chemistry of Nitrogen," Oxford University Press, Oxford, 1937, p. 195-196. . 72

On the basis of the above physical and chemical data the compound CLXIII th erefo re, must be the ^-epim er.

H CLXII1 CXXIII

CLXIV

It is well knomi that alkali may alter the stereo chemical course of the reduction of a,y5-unsaturated k eto n es.F o r example, Wilds, Jolinson and Sutton?^ pre pared trans-16-equilenone (CLXVI) in 8k-fo y ield by hydro genation of the unsaturated ketone CLXV over palladium- on- char coal in dioxane while a reduction of the same compound CLXV in alkaline solution led to the formation of cis-16-equilenone (CLXVII) in 89% yield,.

A. Weidlich, Chemie., ^8, 30 (19^5).

^^A. L. Wilds, J'. A. Johnson, Jr., and R. E. Sutton, J . Am. Chem. Soc., ^^2^- (1950). 73

— —$ >

CLXVI

I t seemed of in te r e s t to see whether the ad dition of alkali would increase the ratio of ^/8-to isomer. It was found indeed that a higher ratio of the 50-isomer was formed under these conditions; the 50-isomer was obtained in y i e l d ._ Finally, a brief investigation was made of the cata lytic reduction of several 2,3-secocholesta-*+,6-diene deriv a tiv e s . Methyl 2,3-secocholesta-^,6-diene-2-nitrile-3-oate (CXXV) was catalytically hydrogenated in ethyl acetate over palladium-on-charcoal (10/0. The reaction mixture showed two spots on th in layer chromatography. Separation of this mixture was carried out on preparative thin layer plates on silica which,afforded a crystalline compound 7*+

CLXVIII (Rf 0.85), m.p. 91:0 and an oil (Rf 0.11) in 8l^ and 9/j yield respectively. The oil was not further in- .vestigated due to lack of material. The success of the reduction in OCCF was .evidenced by a shift of ester carbonyl absorption of the crystalline reduction product from 5-78/1 to lower wavelength at 5-7^ in the infrared due to the reduction of the double bonds. Similarly, it showed only a maximum at 206mju in the ultra- ' violet as compared to a maximum at 270mjj. in the spectrum of the starting material. Furthermore, its N.M.R. spectrum showed n o .o le fin ic protons. The stereochem istry of th is product CLXVIII at C-5 was established by an independent synthesis. A solution of 2-oximino-5“-cholestan-3-one (CLXIV) was treated with thionyl chloride in methylene chloride for 2k hr.. Excess of thionyl chloride and the sol vent were removed under, vacuum and the crude cyano acid chloride (identified by infrared and ultraviolet spectra) was treated with sodium methoxide. A colorless crystalline product was obtained in 93^ yield, the infrared spectrum of which was superimposable with that of the product prepared by the catalytic hydrogenation of CXXV. A mixed melting point determination further confirmed the identity of these compounds and estab lish ed the a-configuration fo r the hydrogen at C-5. 7?

o CLXVIII

CLXIV

Similarly, hydrogenation of 2,3-secocholGsta-^-,6- diene-2,3-dinitrile (CLIX) over palladium-on-charcoal (10/?

N c Nc

CLIX CLXXX

CLXXV

NH.3 SOCL

CLXXX EXPERIMENTAL

All melting points were taken on a Pisober-Johns apparatus and are uncorrected. Infrared absorption spectra were determined with a Perkin-Slmer model 137 recording spectrophotometer using potassium bromide disks unless. , otherwise specified. Ultraviolet absorption spectra were measured ^-ri.th a model202 Perkin-Elmer spectracord using 95/^ ethanol solutions unless otherwise noted. Optical ro tations were taken on a Perkin-Elmer model 1^'r1 polarimeter in chloroform solutions unless otherwise indicated. The nuclear magnetic absorption spectra were run on a Varian A-60A in deuteriochloroform with tetramethylsilane serving: as internal standard. Elemental analyses were carried out by Dr. A. Bernhardt, Mulheim, Germany, and by Midwest Micro- lab, Inc., Indianapolis, Indiana.

Cholesta-^,6-diene-.3-one. (GXXII). Cholesta-V,6-diene- 3-one (CXXII) was prepared by the method of Glamkowski^3 except that 3 hr. of reflux was required to complete the dehydrogenation of cholest-^-ene-3-one (CXXI) vâth chloranil •in t-butyl alcohol and that the yellow-brown residue (ob-' tained after filtration of excess of chloranil and evapora tion of the solvent) was purified by chromatography on a tall

77 78. colmin of basic Alcoa Alumina (700 g .); the product was eluted with benzene until the eluant was no longer colored. Evaporation of the solvent left an oil which solidified on standing. Crystallization from methanol afforded CXXII in 50?j yield as faintly yellow needles, m.p. 8 2 - 8 2 .(reported^3 m.p. 81-82°. Infrared spectrum: ^ 6.0^, shoulder at 603/1 (3- ketone), 6 .I 8/1 and 6 . 3Q/1 (double bonds) reported 6.01ju, shoulder at 6 . 03,u (3-ketone), 6.1S^u and 6 . 31/i (double bonds) . Ultraviolet spectrum: 283m/i, logs ^-.39 (reported ■ 283m/i, (logs ^.*+0),

2-0%imino-cholesta-)f .6-diene-3-one (CX]{III). 2-Oximi- no-cholesta-'+,6-diene-3-one (CXXIII) was prepared from CX)CEI by the procedure of G1 amlcowski^3in 61/5 yield.. It crystallized as pale yellow-green flakes, m.p. 235-237° dec. (reported m.p. 23^ - 236 ° d e c.). In frared spectrum: ^ 3.08/1 (oximino-OH), 5.93/1 (3- ketone), and 6.19/1, 6 . 28/1 (conjugated double bonds) repor

ted 3 . 09/1, 5. 9I/1, 6 . 20/1 and 6.2^u . “ultraviolet spectrum: StOH g-;(g. 28,800);

29921/1 (€ 25, 910) and 3^2yi (& 11,129). [re-,

p o r t e d X ^^utral, StOH ^lOm/i (e 29,000); X 299m/i

(e ca. 2 6 , 000) and 3^2nyi (e 12,000) . 79 Beckmann rearrangement of 2-oxlmino-cholesta-^i-,6-cllene- 3-one (CXXIII). A solution of the oximinoketone CXXIII 3.0 g ., 7.31 mmoles) and p-toluenesulfonyl chloride ('+.50 g.) in 50 ml. of dry pyridine was stirred for 2'+ hr. at room temperature. The reaction mixture was then poured in 500 ml. of ice-cold water whereupon a yellow colored precipitate separated. The product was extracted t\hLce with 350 ml. of ether and the combined ether extracts were washed +/ith satu rated sodium bicarbonate solution (2x300 m l.) water (2:c500 ml.), and saturated sodiuKi chloride solution and then dried over anhydrous sodium sulfate. The solvent was evaporated in a ro tary evaporator and the re su ltin g brown gum was d is solved in a mixture of ether and methanol (1:1). The bro+m solution on concentration deposited I .9 0 g, ( 63/0 of pro duct, m.p. 195-197° as pale greenish yellow crystals. Two additional crystallizations from the same solvent mixture afforded I .83 g. (61fO of pure CILXIV, m.p. 197-198°. In frared spectrum: ^max 5.7Qn (C=0.in the 7-membered rin g ), 5.97/1 (0=0 in the 6-membered rin g ), 6.17/ij 6 . 23ju and 6 . 3O/1 (C=N and double bonds).

- 131° (0 2.62). Ultraviolet spectrum: X 220mp, (sh.£ 20,900) and • 311m/i (c 55,460). ' Anal. Calcd. for C^goO^Ng: 0, 80.59; H, 9.95; N, 3.^85 S, 0.00. Found: C, 80.5^; H, 9.91; N, 3.42; S, 0.00. 8 0

The combined sodium bicarbonate washings from the above work-up were acidified with 6 N HGl and extracted several times with ether. The ether extracts were combined, washed with water, dried over anhydrous sodium sulfate and evaporated, giving a few rag. of a brown smudge. Thus, no acidic product was formed in the reaction.

Alkaline degradation of the Beclcraann rearrangement nroduct CXXIV. i) A mixture of the Beclcraann product CICKI? (1.0 g . , 1.2>+ mmoles) and potassium hydroxide (1.0 g.) in 50 ml. of methanol was heated under reflux for 24 hr. during which'the solution turned deep brown. The reddish brov/n residue obtained by evaporation of the solvent was treated with 50 ml. of water and extracted thrice with 100 ml. of ether. The combined brovrnish ether extracts were washed twice with water, saturated sodium chloride solution and dried over anhydrous sodiuri sulfate. Evaporation of the ether in a rotary evaporator gave a red gum (0.63 g.) which resisted all attempts at crystallization from'a variety of solvents. The aqueous alkaline extract was acidified with 6' N HGl to give a yellovash guimray precipitate. The mixture was extracted thrice with 100 ml. of ether, washed with water, saturated sodium chloride solution and dried over anhydrous sodium sulfate. The solvent was removed in a rotary evapo rator leaving a browish gum which yielded an almost color- 81 less precipitate on treatment with petroleum ether (Skelly Solve P) and weighed 0,20 g. (39/^), m.p. 105-110°. T\fo crystallisations of this material from ethanol afforded 0.175 g. (31$) of acid CXX^/I as colorless needles, m.p. 218 °. . In frared spectrum: ^ i-^a:c 3 .80/i (bonded OH), ^.'h-Oju (ON), 5. 89/u (0=0), 6.10/:, 6,22/: (conjugated double bonds), [j-^J -32,3° (c 1.12; tetrahedrof uran). Ultraviolet spectrum: ^ ^ax 266m/: (c 2,897). Anal.Calcd, fo r 027®-;--,OpN: G, 76.78; ÏÏ, 10.0^-; N, 3,^.'-0. Pound: 0, 78. 8 3; H, 10.15; N, 3.37.

Estérification of the acidic degradation product OXZfPI. A solution of the acidic product CXJCVI (0.10 g ., 0.244 mmole) in 10 ml, of ether was added to an ethereal solution of diasomethane maintained at 0°. The reaction was over in hr. (checked by thin layer chromatography) and the crystalline material obtained by slow evaporation of the solvent was filtered, washed with petroleum ether and dried to give

0 .0 8 g. (77$) of OXXP as colorless needles, m.p, 165°. -An analytical sample, m.p. 166.5° was obtained by crystalliza tion from ether - methanol (1:1). Infrared spectrum: ^maz 4-.4-4^. (ON), 5.78/: (0=0), 6 ,1 ^ , 6,25/: (conjugated double bonds). [ o ( J - 3 . 30° (c 1,44). Ultraviolet spectrum: ^ max 270:/: (6 4-9,790). 82

Anal.Calcd. for C28% 302N: C, 79.01 5 H, 10.18; N,

3 . 29. Found: C, 79-01; H, 10.37; N, 3.25. II) To a solution of the Beclcnann product CXXIV (0.10 g.,- 0.12^ i-mole) In 50 ml, of ether - methanol (1:1), there was added 15 ml. of methanollc sodium hydroxide solution and the mixture was stirred at room temperature for 20 hr. Removal of the solvents In a ro tary evaporator gave a brown residue which was treated with 20 ml. of water and extracted thrice with 20 ml. of ether. The combined ether extracts were washed with water several times, then dried over an hydrous sodium sulfate. The solvent was removed In a rotary evaporator leaving an almost colorless residue (0.0^1 g., h-O/i), m.p. 221- 231°, which crystallized from ethanol to furnish CXXIII (0.035 g., 3^:-/^'), m.p. 235° dec.. Identical In all respects with an authentic sample of 2-oxlmlno-cholesta-)-i-,6-dlene-3-one (CX](III). The alkaline aqueous extract on acidification with 6 N HCl gave a browilsh precipitate which after the usual work-up afforded 0.0^ g. (VO/o) of C3CXVI, m.p. 218°, Identi

cal In all respects with a sample of CjyXVI. III) To a solution of CXXIV (0.10 g ., 0.12^ mmole) In

50 ml. of anhydrous ether - methanol (1:1), there was added sodium methoxide (O.OI 3 g . , 0.248 mmole) and the mixture • was stirred at room temperature In an atmosphere of nitrogen • for 20 hr. The solvents were removed In a rotary evaporator 03 to give a yellow solid which was treated with ^0 nl. of water and extracted thrice with 50 rnl. of ether. The combi ned ether extracts were washed with water, saturated sodium chloride solution and' dried over anhydrous sodium sulfate. The ether extracts on evaporation gave a yellow residue (0.091 g.) which showed two spots on thin layer chromatogra phy. The mixture was separated by preparative silica gel thin layer chromatography using benzene - ethyl acetate

(1:1) as the eluant. The faster moving band of Hf 0 . 9I was separated and extracted well with chloroform. A crystalline substance (0.0^5 g., ^7#), m.p. 166°, was obtained by slow evaporation from a mixture of ether - methanol (1:1); it was identified as ester C3CCV by mixture melting point, in

frared spectrum and Rf value. The oximinoketone CXXIII was obtained from the slower moving band of Rf 0,59 as yellow green flakes which weighed 0.035 S* (3^/j). There was no de pression in the mixture molting point with an authentic

sample of 2-oximino-cholesta-^-,6-diene-3-one (CÜOCIII) and the infrared spectra were also found identical. The original alkaline aqueous solution on acidification gave no precipi tate and so there was no acidic product formed in this reac tio n .

i v ) t o a s o l u t i o n - of CXXIV ( 0 .1 0 g ., 0.1 mmole) in

50 ml. of ether - methanol (1:1), there was added sodium

borohydride (0.10 g.) and the mixture Was stirred at room temperature for 2^:- hr. The products were worked up exactly in the same way as described in the previous experiment, giving C]{XIII and CXXV in y]$ and yield respectively.

Meth?/1 2. i-secocholesta-!-!-.6-diene-2-nitrilc-l-oato (CXXV). A solution of the oximinoketone CXXIII (0.20 g., O.l+BB mrfiole) in 5 ml. of dry methylene chloride was treated with ^ ml. of thionyl chloride and the mixture was stirred a t room temperature fo r 2^ hr.- The brown amorphous cyano acid chloride CXXVII, obtained after the complete removal of thionyl chloride and methylene chloride in a rotary eva porator, was treated with a solution of sodium methoxide (0.10 g.) in 10 ml. of anhydrous methanol. The resulting suspension was stirred for Ip min., diluted with 10 ml. of water, filtered and washed free of sodium hydroxide. The precipitate was crystallized from ether - methanol (1:1) to afford 0.173 g. (9&0) of CXXV as colorless needles, m.p. 157-16'+°. Tvro additional crystallizations from the same mixed solvents gave 0,16 g. (77^) of CX/CV as colorless needles, m.p. 167°, identical in all respects v/ith an authen tic sample of CXXV (see page 81).

3-Hydroxy-3.3-di'ohenyl-2.3-secocholesta-'+.6-diene-2- nitrile (CD3.VI). A solution of the oximinoketone OIXIII

(0.20 g . , O.ÎtBB mmole) in 5 ml of dry methylene chloride was treated with '+ ml. of thionyl chloride and the mixture was 85 stirred at room temperature for 24 hr. The cyano acid chlo ride CXXVll obtained after the removal of thionyl chloride and methylene chloride under vacuum was dissolved in 10 ml. of anliydrous ether and treated vâth an ethereal solution of phenyl magnesium bromide (0.265 S., 1.465 nmioles); The reaction mixture was refluked on a steam bath for 45 min. The solution was then poured into a 50 ml. saturated ammo nium chloride solution and the mixture was stirred at room temperature for 2 hr. The precipitate was extracted well with ether. The organic extract was washed with water, saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The solvent was removed in a rotary evapo rator leaving a residue which crystallized from ether - pe troleum ether (1:1) to yield 0.16 g. (62/'S) of material, m.p. 205-210°. By recrystallization from the same solvent pair ' 0.15 g. (56/j) of white rosettees of C3CLVI were obtained, m.p. 215°. Infrared spectrum: ^i^ax 2.88,u (OH), 4.43jJ. (OH), 6.10/1, 6.25ju (double bonds), 13*38/u, 13.5^/1, l4.17/i and 14.38^. +7 0 .9 (c 1.185 tetrahydrofuran). Ultraviolet spectrum: 206mjU (€ 20,660) and 249ii^ (e 20,640).

A n a l. C a lc d , fo r C39H51OU: c, 85.19; H, 9.35; ^4 2.55.

Found: C, 84.99; H, 9.09; N, 2.64. 8 6 3-IIydro:-:y-3, 3-dim ethyl-2,3-secocholesta~^, 6-dien@-2- n l t r i l e ( CXLVII). The crude cyano acid chloride CXXVII (0 ,2 0 g.j 0.463 rffiiole) vas treated vith an ethereal solu tion of methyl magnesium bromide ( 0 .160 g . , 1.35 mmoles). The yellow suspension which formed immiediately was stirred at room temperature for -g- hr. and after addition of 10 ml. of anhydrous ether, the stirring was continued for one more hour. The mixture was poured into a 25 ml. saturated solution of ammonium chloride and stirring of the mixture was continued for 1 hr. The products were extracted twice

w ith 30 ml, of ether, washed with water, saturated sodium chloride solution and dried over anhydrous sodiuu sulfate. Evaporation of the ether in a rotary evaporator gave a brown gum. Treatment of this material with petroleum ether (Skelly Solfe P) afforded 0.07 g. ( 36 /3) of a light yellow precipitate, m.p, 121-129°. Chromatography of this product over neutral alumina (activity IV, 1.0 g.) using ether as eluant afforded 0 ,056 g ( 3O/3) of colorless crystals, m.p, 145°. Infrared spectrum: X 2,91/j (OH), 4.43ju (ON),- 6-.06ju, 6.1 lju (double bonds). +7 ,2 (c 1.42), Ultraviolet spectrum: X 24liiyi (6 16,360), Anal,Calcd. for C29H47ON: C, 81.82; H, 11,13; N, 3.29. Pound: C, 82.02; H, 11.19; N, 3.32. 87 2^3-Secocholesta-^!-.,6-diene-g-nitrile-3-oic acld-3- amlde (ŒCLV). The cyano acid chloride CXX^/II (0.20 g., 0.463 mmole) was dissolved in 20 ml. of anhydrous ether and cooled to 0° in a ice water bath. Anhydrous aimaonium gas was bubbled through the solution with occasional shaking when a white precipitate separated. The passing of aimnonia was stopped after 10 min..and 20 ml. of water were added to the flask. The precipitate was extracted thrice with 20 ml. of e th e r. The combined ether ex tracts were washed v/ith wa t e r , dried over anhydrous sodium su lfa te and the solvent removed in a rotary evaporator to give 0.131 g. ( 69 /i) of a crystalline residue, m.p. 192-197°. Further recrystalliza tion of this material from petroleum ether (Skelly Solve P) afforded 0.12 g. (65%) of CjQLV as colorless crystals, m.p. 209°. Infrared spectrum: X 3.01/a, 3.15/a (N-K) and 5.94/a, 6.05/a (CONE2). - 3 . 2° (c 3.63). Ultraviolet spectrum: 205m,u (6 19,700) and (6 20,650 ).

Anal.Calcd. fo r C2y%20N2: 0 , 78.97; H, 10.31; N, 6.82. Found: C, 78. 78; H, 10.35; U, 6 . 8I.

2,3-Secocholesta-4,6-diene-2-nitrile-3"Oic aoid-3-N- phenylamide (C^OLVIIX) . The cyano acid chloride C}DCVII

(0 .2 0 g., 0.463 mmole) was treated with 2 ml. of pure ani 8 8 line with stirring at room temperature for i- hr. The reac tion mixture was then heated for 1 5 min. on a steam bath, cooled and diluted with 10 ml. of water. The precipitate was filtered, washed with water and dried to give 0.15 g* (66/j) of material, m.p. 192-197°. The product was crystal lized from acetone, giving 0.13 g. (60)^) of OILVIII as shiny colorless crystals, m.p. 215°. Infrared spectrum: X 3.20/a (N-E), (ON), 5.97M (COM), 6.15/i, 6.25/1, 6.51/U 13.29/1 and ll+.^-9/i. [ck] p -li'.o° (c 1 .^3).

Ultraviolet spectrum: 20)-!.m/i (€ 12,230),

255m/i (6 7,36^), 286nyi (C 21,030); 223:^

(C Ik-,350) and 290m/i (€ 21,800). Anal.Calcd. for.C33ïïk|-60ÏÏ2: 0, 81.43; H, 9.53; N, 5.76. Pound: 0, 81.44; H, 9.56; N, 5 . 7 8.

Attempted hydrol 7/"sis of methyl 2.3-secocholesta-4,6- diene-2-nitrile-3-oate (CXXV). i) A solution of methyl 2,3-secocholesta-4,6-diene-2-nitrile-3-oate (CXXV) (0.05 g ., 0.0118 miaole) in 1 ml, of water and 4 ml. of ethylene glycol containing 1.8 g. of potassium hydroxide was refluxed for 48 hr. The mixture was then poured in 10 ml. of water 'and acidified with 6 N HCl. The resulting precipitate-was extracted ti/àce with 30 ml. of ether. The combined ether extracts were washed vâth water, dried over anhydrous sodium sulfate and the solvent removed in a rotary evaporator 8 9 leaving a residue'which crystallized from ethanol to give 0 .0 2 9 g. (610) of material, m.p. 218°. The product was identical by infrared and raixture melting point with a sample of cyano acid-CXXVI. i i ) A solutio n of the same amount of CSCV in 2 ml, of of water and 2 ml., of ethanol containing 1.0 g. of potas sium hydroxide was refluxed for 5 hr. After the usual work up, CX}C\ri was obtained in 89 0 y ield . iii) A solution of CXXV (0.10 g., 0.236 mmole) in 6 ml.

6 ml. of glacial acetic acid, 0.5 ml. of concentrated sul furic acid and 0.5 ml. of water was refluxed for 2-j5 h r. du ring which the color of the solution turned dark brovna. After the usual work-up, a red gum ,(0.09 g.) was obtained which resisted all attempts at crystallization. The gum was dissolved in tetrahydrofuran and treated with an ethereal solution of diasomethane at 0°. Work-up in the usual way failed to give a crystalline product even after chromatogra phy on n eu tral aluraina- (a c tiv ity I I , 0.50 g.). iv) A solution of CXXV (0,20 g., 0.4^2 mmole) in 6 ml, of ethylene glycol containing 0.40 g. of potassium hydroxide was reflux-ced fo r 5 hr. The resu ltin g yellow solution was poured into 50 ml. of water and cooled. Acidification with 6 N HCl gave a precipitate which was extracted thrice with 50 ml. of ether. The' combined ether extracts were washed with water and dried over sodium sulfate. The solvent was ' 90 removed in a Notary evaporator leaving a brownish residue which crystallized from methanol to give 0,103 g . (51^) of

2,3-secocholesta-^-,6-diene-2,3-dioic acid-2-amide (CXLIX), m.p. 155“173°‘ Three additional crystallizations from methanol gave 0.078 g. (.39%) of an analytically pure sample, m.p. 290°,

Infrared spectrum; ^max 3*10ju (N-H), 3.7^» 3*89/1 (OH),

%72}X (0=0), 6.05/i(C0NH2). -9.60 (c 1.07).

Ultraviolet spectrum: ^ 207m;i (6 5,291).

Anal.Calcd. for CgyH^gOgN: 0, 75.48; H, 10.09; N, 3.26.

Pound: G, 75*53, H, 10.22; N, 3*4l.

Estérification of 2.3-secocholesta-4.6-diene-2.3-di-

oic acid-2-amide (CXLIX). A solution of 2,3-secocholesta- .

l+,6-diene-2,3-dioic acid-2-amide (CXLIX) (0.05 g., 0.116

mmole) in 10 ml. of dry tetrahydrofuran was treated with an

excess of ethereal solution of diasomethane as.described in

the preparation, of CXXV from CXXVI. Slow evaporation of the

solvent gave 0.037 g. (73%) of methyl 2,3-secocholesta-4,6-

diene-2-dic acid-2-amide-3-oate (CL), m.p. 160°.

Infrared spectrum: ^max 2.92/1, 3*23/1 (N-H), 5*71/i (C=0), ‘6 . 00/1.

[c3k]g^ -20.9° (o 1.37).

Ultraviolet spectrum: ^max 207mju (e 5,426).

Anal.Calcd. for C28Hli.^02N: C, 75*80; H, 10.22; N, 3*16.

Pound: C, 76.00; H, 10.27; N, 3*09* 91

2,3“SecQcholesta--^.6~dlene-2-nltr'llQ-Volc acid

(CXXVI). The cyano acid chloride CXXVII (0.20 g., 0.463 mmole) was treated with 10 ml. of water and stirred for

5 hr. whereupon a light brown precipitate separated. Fil

tration gave 0.T5 g. (76^) of product, m.p. 10^-112°. Two

crystallizations of this material from ethanol afforded

0,12 g. (66%) of pure cyano acid CXXVI, m.p. 218°, identi

cal in all respects with a sample obtained by hydrolysis

of the Beckmann product CXXIV with sodium hydroxide.

Conversion of 2.3-secocholesta-4.6-diene-2-nitrile-

3-oic acid (CXXVI) to 2.3-secocholesta-4.6-diene-2-nitrile-

3-oic acid-3-amide (CXLV). A solution of 2,3~secocholesta-

4,6-diene-2-nitrile-3-oic acid (CXXVI) (0.030 g., 0.073

mmole) in 1 ml. of thionyl chloride was refluxed on a

steam bath for hr. Excess of thionyl chloride was removed

under vacuum and the brownish residue was treated with

ammonia excactly the same way as described for the prepa

ration of CXLV from CXXVII. Crystallization of the brovm

precipitate was effected from petroleum ether to give

0.013 g. (h6%) of product, m.p. 208°. This, product was iden

tical by infrared, rotation, and mixture melting point

with an authentic sample of 2,3-secocholesta-4,6-(iiene-2-’

nitrile-3-oic acid-3-amide (CXLV). 92

Attempted isolation of ^-Imino-A-homo-cholesta-^a.6- dlene-^-one~2 methyl ether (CXXIX) from the oxlmlnoketone

CXXIII. The same experiment was repeated with the oximinoketone CXXIII as for the synthesis of CXXV except that the brownish amorphous substance obtained after the complete removal of thlonyl chloride under vacuum was treated with

15 ml. of anhydrous reagent grade methanol. A crystalline compound identical in all respects with an authentic sample of methyl 2,3-secocholesta-^,6-dlene-2-nitrile-3-oate (CXXV) was only obtained in 87^ yield.

2.3-8ecocholesta-^-.6-diene-2.3-dinitrile (CLIP. A solution of 2,3-secocholesta-)+;6-diene-2-nitrile-3-oic acid-

3-amide (CXLV) (0.20 g., 0.488 mmole) in 0.6 ml. of thionyl chloride was heated to reflux on a steam bath for 1 hr.

Excess thionyl chloride was removed under vacuum and the bro^m gum was taken up in 30 ml. of ether. The organic ex tract was washed with water and dried over anhydrous sodium sulfate. The solvent was removed in a rotary evaporator leaving a brown residue which gave an almost colorless pre

cipitate on treatment with petroleum ether. The material weighed O.O8O g. (42#) and had am.p. of 105-110°. Two crys

tallizations of this material from ether - petroleum ether

(1:1) afforded 0.077 g. (40#) of CLII, m.p. 116°

Infrared spectrum: ^max 4.43^ (uncohjugated CN), 4.5^^

(conjugated CN), 6.l4/i, 6.33/1 (conjugated double bonds). 9 3 +6.5° (o 0.65). Ultraviolet spectrum: 265nyi (e. ^0,560).

Anal.Calcd. for C27% o N 2 : C, 82.59; H, 10.27; N, 7.1^.

Pound: C, 83.0^; H, 10.15; N, 7.01.

Attempted preparation of 2-oximlno-3a-Dhenvlchoiesta-

^,6~dlene~3/3»ol (CXL). i) To a stirred solution of 2-oximino-cholesta-^,6-diene-3“One (CXXIII) (0.20 g., 0.^88 mmole) in 10 ml. of dry tetrahydrofuran at room temperature, there was added an ethereal solution of phenylmagnesium bromide

(0.265 g., 1.465 mmoles)' and after 1 hr, the solution was added to a diluted hydrochloric acid - ice mixture. The mixture was extracted well with 100 ml. of ether. The ether was washed with water, saturated sodium bicarbonate solution, once again with water, then dried over anhydrous sodium

sulfate. Evaporation of the solvent gave a brown residue which crystallized from ethanol to give back the starting material.

ii) A solution of 2-oximino-cholesta-4,6-diene-3-one (CXXIII)

(0.4o g., 0.976 mmole) in 12 ml. of dry. tetrahydrofuran

was refluxed with an ethereal solution of phenylmagnesium

bromide (0.530 g., 2.930 mmoles) on a steam bath for 3 hr.

•The solvent was removed in a rotary evaporator and the gummy

material was stirred with a saturated solution of ammonium

chloride for 15 min. The mixture was extracted thrice with

50 ml. of ether and the ether extracts were washed with 9 4 water, saturated sodium chloride solution and then dried over anhydrous sodium sulfate. The solvent was evaporated in a rotary evaporator to give a brown gum which resisted all attempts at crystallization. The material was then chro matographed on neutral alumina (activity III, 10.0 g.).

Elution with methylene chloride gave 0.20 g. (39^) of a

pale yellow semi-solid as a chromatographycally homogene

ous product GXL (one spot on thin layer chromatography on

silica gel). The infrared spectrum of this material displayed

bands at 2.9?/i (OH) and 1 ^ . 3 ^ (aromatic).

Beckmann rearrangement of 2-oximino-la-nhenvlcholesta-

^.6-diene-3^ol (GXL). A solution of amorphous GXL (0.20 g.,

0.385 mmole) in 2 ml. of dry pyridine was treated with

p-1oluenesulfony1 chloride (0.370 g., 1.93 mmoles) and the

solution was stirred at room temperature for 2^ hr. The

solution was then poured into a 6 N HCl - ice mixture and

extracted twice with 100 ml* of ether. The combined extracts

were washed with water and dried over anhydrous sodium sul

fate. Evaporation of the solvent in a rotary evaporator gave

. a brown residue (Ô.I89 g.) which was chromatographed oh

neutral alumina (activity III, 0.50 g.). Elution with methy

lene chloride and subsequent crystallization from methanol

yielded O .050 g. (27.5%) of 3-ketb-3-phenyl-2,3-secocholesta-

^,6-diene-2-nitrile as pale yellow crystals, m.p. 101°.

Infrared spectrum; ^ ^ax (ON), 6 .03/1 (G=0), 6 .19/1, 9 ^

6 .22/1 and 6 .39/1.

[d] ^ +36.80 (c 1.11).

Ultraviolet spectrum: ^ max 205n^ (e 12,050), 220m/i

(e 11,820), 2»4-8m/i (e 19,320).

Anal. Calcd. for Cg^H^^ON: C, 84^02; H, 9.62; N, 2.97.

Pound: C, 84.09; H, 9.67; N, 2.87.

2-0xlmlno-3a-methylcholesta-4,6-dlene-3^-bl (CXXXVIII).

A solution of 2-oximino-cholesta-4,6-diene-3-oné (CXXIII)

.0.20 g., 0.488 mmole) in 20 ml* of dry tetrahydrofuran and an ethereal solution of methyl magnesium bromide (0.175 Sr

1.47 mmoles) were heated under reflux on a stem bath for

1i- hr. The solvents were evaporated in a rotary evaporator and the resulting brown gum was stirred with a saturated

solution of ammonium chloride for 2 hr. The usual work-up

gave a brown material which upon scratching with petroleum

ether afforded 0.13 S* (60% of product, m.p. l4l-l47°. Two

additional crystallizations from petroleum ether - methylene

chloride (3:1) yielded 0.10 g. (50%) of pure CXXXVIII as

white fluffy crystals, m.p. 153°.

Infrared s p e c t r u m : 2 ,91/1 (OH), 6 . 0 ^ , 6.09/1.

[o>J +4.4° (c 0.66).

Ultraviolet spectrum: ^max 230m/i (sh.6 26,190), 237nyi

(e 29,240) and 246m/i (6 20,040).

Anal. Calcd. for C28%502N: C, 78.63; H, 10.61; N, 3.28.

Pound: C, 78.39; H, 10.57; N, 3.17* 96 .

Beckmann rearrangement of 2-o%lmlno-la-methyloholesta-

1+,6-dlene-l^-ol (CXXXŸIII). A solution of 2-oximlnor3«-methy1- cholesta-^,6 -diene-3/8-ol (0,060 g., 0 ,1^0 mmole) in 5 ml. of dry pyridine was treated with p-toluenesulfonyl chloride

(0.160 g., 0.837 mmole). After stirring the mixture at room temperature for 2h hr., the solution was worked up as usual to give a red gum which failed to crystallize. The product was then chromatographed on neutral alumina (activity III,

1 .0 g.) and eluted first with 25 ml. of hexane which on evaporation gave a brown gum (0.015 g.) and could not be crystallized. The column was further eluted with 15 ml. of benzene and the solvent evaporated in a rotary evaporator

to give a light brown residue which crystallized from etha nol to afford 0.010 g. (17^) of 3-keto-3-methyl-2 ,3*-seco- ’

cholesta-)+,6 -diene-2-nitrile (GXXXIX), m.p. 133°.

Infrared spectrum: ^ ig^x (CN), 5.90jU (0=0), 6.1^,

6,32p. (conjugated double bonds).

Ultraviolet spectrum; ^ max 288mju (6 7,688).

Anal. Calcd.for C28H43ON: 0, 82.09; H, 10.58.

• Found: 0, 81.8^; H, 10.50.

2-Diazo-chole8ta-!+.6-diene-3-one (CLV). The bright

yellow diazoketone CLV, m.p. 104-105° was prepared from the

oximinoketone CXXIII in 50^ yield by following the method .

of Glamkowski^3 (reported m.p. 105-106°)*

Infrared spectrum: ^max ^•77p (diazo group), 6 . 0 ^ 97 (3-ketona), 6,h3)x (conjugated double bonds); [reported

^.ySjU (diazo-group),6.l5)u (3-ketone), 6A3jU (conjugated double bonds^.

Ultraviolet spectrum: 23^mji (6 7,9^1), 288n|U

(e 19,^80) and 3^5m|U (e 10,609) ; ^reported X 23*+nyi

(6 7,950), 288mju (£ 19,600) and 3^5nju (e 10,600^.

Photolysis of 2-dlazo-chole8ta-M-.6-diene-3-one (CLV). i) in THF A solution of diazoketone CLV (0,50 g., 1.225 mmoles) in 50 ml. of redistilled tetrahydrofuran and 50 ml. of water containing 1.0 g, of sodium bicarbonate was irra diated with a low pressure argon (Hanovia) lamp. Evolution of nitrogen stopped after ^5 min. during which the yellow solution of diazoketone changed to dark red. This was ex tracted thrice with 100 ml. of ether, washed with water and dried over magnesium sulfate. The solvent was removed in a rotary evaporator under low pressure to give a dark

red gum (0.50 g.) which showed no diazo absorption in the •

infrared and failed to give a crystalline substance from

different solvents. This neutral material was dissolved in

■ benzene and eluted through a column containing silica gel

(20.0 g.). None of the fractions afforded crystalline mate

rial. The aqueous extract on acidification with 6 N HCl gave

no precipitate showing that no acidic product was formed in

the photolysis. , . , • , 9& ii) In benzene A well stirred solution of diazoketone CLV

0,20 g., 0 .1+90 mmole) in 100 ml. of reagent grade benzene was irradiated (G.E.Sunlamp) through a soft glass filter for 5 hr. Work-up in the usual way gave back the starting material in almost quantitative yield. iii) In benzene To a yellow solution of diazoketone CLV

(0.50 g., 1.225 mmoles) in 100 ml. of reagent grade benzene was added 100 ml. of water containing 1.0 g. of sodium bi

carbonate. The mixture was irradiated with a low pressure

argon (Hanovia) lamp and light of wave lengths below 300AP

was filtered out through pyrex. Irradiation was stopped

immediately after cessation of nitrogen evolution 0+^ min.).

The red colored benzene solution was separated from the

aqueous solution in a separatory funnel, washed with water

and dried over magnesium sulfate. The solvent was removed

in a rotary evaporator to give a reddish brown material

(0.50 g.) which showed no diazo absorption in the infrared

spectrum. Thin layer chromatography showed it to bo a mix

ture of at least five compounds of the following Rf: 0 .09 ,

0.24-, 0.4-6, 0 .70, 0.93» The substance was dissolved in •

benzene and repeatedly chromatographed over neutral alumina

(activity I, II, III, 3 .0 g. each time). None of the frac

tions afforded a crystalline substance. The aqueous extract

combined with the benzene washings gave no precipitate on

acidification with 6 N HCl. 99 Attempted lithium and liquid ammonia reduction of 2- oximlno-cholesta-k-.6-dlene-l-one (CXXIII). i) The oxlmlno-. ketone CXXIII (1*0 g., 2.MfO mmoles) vasr dissolved in 50 ml.

of dry tetrahydrofuran and 250 ml, of liquid ammonia was

added with stirring. Lithium (1.50 g.) was added in small

pieces during 5 min. when the solution turned h lu e .^ O

After a further 15 minutes' stirring absolute ethanol was

added dropwise so that the solution became colorless after

35-^0 min. The solvents were evaporated and water was added

to the residue. Extraction \fith ether and evaporation of

the solvent left a red sticky solid (0.90 g.). The material

was dissolved in 6 ml. of ether and chromatographed over

neutral alumina (activity III, 15.0 g.). Three red colored

bands were separated by eluting with benzene. None of the

fractions gave any crystalline material nor showed carbonyl

absorption in the infrared spectrum.

ii) A solution of the oximinoketone CXXIII (0.20 g., 0.^88

mmoles) in 5 ml. of dry tetrahydrofuran was added with stir

ring to a solution of lithium (0.10 g.) in 50 ml. of liquid

ammonia and after 1 min. the blue color was discharged .with

solid ammonium chloride.The residue obtained after evapo

ration of the ammonia was treated with water and the resul

ting mixture was extracted with ether, washed with water

and dried over anhydrous sodium sulfate. The solvent on

^9a, p. Daglish, J. Green, and V. D. P o o le, J. Chem.. Soc., 2627 (195^).

81r . e .. Schaub and M. J. Weiss, Chem.& Ind., 2003 (1961). 100 ,

evaporation gave a gum (0,161 g.) which showed 3 spots on

thin layer chromatography on silica gel. The mixture was

separated by preparative thin layer chromatography (silica

gel), giving the starting oximinoketone CXXIII (0.060 g.)

and two other gummy materials which failed to crystallize.

The same experiment was run for 2j 10, 15» 20 and 30 min.

and after the usual work-up the unreacted oximinoketone

CXXIII and two gummy materials were obtained in different

proportions. These materials showed no carbonyl absorption

in the infrared spectrum and all attempts at crystallization

proved futile.

Catalytic hydrogenation of 2-oximino-cholesta-^.6-

diene-3-one (CXXIII). i) In basic solution A mixture of

the oximinoketone CXXIII (0.10 g., 0.2M+ mmole) and 10^

palladium-on-charcoal catalyst (0,10 g.) in 20 ml. of 9?^

ethanol, 7 ml. of water and 3 ml. of 10^ sodium hydroxide

was hydrogenated with vigorous stirring at atmospheric pres

sure. At the end of 6 min. approximately 2 equivalents of

hydrogen were absorbed. The catalyst was removed by filtra-

• tion and the resulting yellow solution was acidified with

6 N HCl which gave an immediate precipitate. The mixture was

'diluted with 5 ml. of water and extracted twice vri.th 50 ml.

of ether. The combined ether extracts were washed with water

and dried over anhydrous sodium sulfate. The solvent was

removed in a rotary evaporator leaving a residue (0,10 g.) 101 which showed 3 spots on thin layer chromatography on silica gel. The residue (0.10 g.), m.p. 160-210° was dissolved in

^ ml. of tetrahydrofuran and eluted by benzene - ethyl ace tate (1:1) on a silica gel preparative thin layer plate.

Three Separate bands were distinguishable with a U.V.Lamp.

The fastest moving band of Rf 0.95 was extracted id.th etha nol to give a brown gum (0.025 g.) which failed to crystal lize and was rejected. The fraction with Rf 0.87 was extrac ted repeatedly with tetrahydrofuran - methanol (1:1) and the extract was filtered and the filtrate evaporated to dryness leaving a residue (0.05 g., 50#), m.p. 178-186°.

Crystallization of the residue from tetrahydrofuran - me thanol afforded O.O^fO g. (4o#) of pure 2-oximino-5)3-cholestan-3-one (CLXIII), m.p. 192°.

Infrared spectrum: \ 2.90yu (oximino-OH), 5.9Qh

(0=0), 6.20/1 (C=N), 9.7^, 9.85/1, 10.06/1, 12.60/1 and 13.59/1.

[dv]^^+2.5® (c 1.26; tetrahydrofuran).

Ultraviolet spectrum: 246:^ (e. 9,221);

^ma?°’ 303m/i (£19,9^0). Anal.Calcd. for CgyHi^^OgN: C, 78.02; H, 10.91; N," 3.3%

Found: C, 78.18; H, 10.76; N, 3.7^.

The slowest moving band of Rf 0.70 was exhaustively

extracted with 60 ml. of tetrahydrofuran - methanol (1:1)

and. the extract was filtered and the filtrate bn evaporation

gave 0.081 g. (16#) of 2-oximino-^a-oholestan-3-one (CLXIV), 102 m .p. 250- 261 ° . Two additional crystallizations of this ma terial from tetrahydrofuran - methanol afforded 0,012 g.

(12^) of pure CLXIV, m.p. 27^°’ (reported^3 m.p. 270°). Infrared spectrum: ^max 3.18/1 (oximino-OH), 5.8lju

(0= 0), 6.20jU (C=N), 10.00/U, 10.27yU, 10.50;u, 12.26/1.

[o(J +50.7° (o O . M ; tetrahydrof uaran), Ultraviolet spectrum: X EtOH 246m/i (& 9,877);

303m/i (6 16,250).

Anal. Calcd. for C27B 1.5O2N: C, 78.02; H, 10,91.

Found: C, 78,21; Hj 10.98.

The epimers CLXIII and CLXIV both underwent colored complex formation as studied by adding one drop of a 5^ aqueous solution of the metal cation. M(OAc) 2 to 1 ml. of a

solution of the oximinoketones (0*001 g.) in tetrahydrofuran.

Tlie following complexes were observed: a yellow-brown colo

ration was produced with Co++, while Ni++ afforded a light

green complex and Cu++ gave a green complex,

ii) In neutral solution The hydrogenation of the oximino

ketone CXXIII (0,10 g., 0,244 mmole) with 5^ palladized

charcoal (0,10 g.) in 25 ml, of dry purified tetrahydrofu

ran at atmospheric pressure was complete after 11 min.

Filtration followed by the evaporation of the solvent gavé

a residue. (0,10 g.), m,p, 1711-214°. This material afforded

0 .0 3 5 g. of CLXIII, m.p. 192° and 0,025 g. (25^) of ' CLXIV, m.p. 272.5° respectively after preparative thin layer; 103 chromatography on silica in the usual way. Both the isomers exhibited the same color complexes with various metals.

Catalytic hydrogenation of methyl 2.3-seoocholesta- .

^-46-diene-2-nitrile-3-oate (CXXV). A solution of methyl

2,3-secocholesta-^,6-diene-2-nitrile-3-oate (0.195 g., 0.460 mmole) in 30 ml. of reagent grade ethyl acetate was hydro genated over 10^ palladium-on-charcoal catalyst (0.10 g.) at atmospheric pressure. The experiment was terminated at the end of 7 min. during which approximately 2 molar equi valents of hydrogen absorption took place. Filtration fol lowed by evaporation of the solvent gave a viscous oil which solidified upon addition of a little methanol. A thin layer chromatography of this material on silica; showed two spots. The separation of the mixture was achieved by pre parative thin layer chromatography on silica, using benzene - methanol (1:1) as the eluant. After the usual work-up, the band of Rf 0.85 afforded 0.16 g. (8l^) of chromatographycally pure methyl 2,3-seco-5a-cholestan-2-nitrile-3-oate (CLXVIII)

as white rosettees, m.p. 91°. The slowest moving band of

,Rf 0.11 after the usual work-up gave 0.017 g« (9^) of an

oil which failed to crystallize and was not.further inves

tigated.

Infrared spectrum: \ 4.42^ (CN), 5 . 7 ^ (0=0).

[o^]|^+23.6° (o 1.9).

Ultraviolet spectrum: ^ ^ax 206mja ( 6 1,630). 10^

Anal. Calcd « for Ç28% 702N: C, 78.27; H, 11.03; N, 3.26.

Pound; C, 78.50; H, 10.70; N, 3.l4\

Methyl 2.3- s e oo- -choie s tan- 2-nl trile-3-oa t e (CLXVIII).

A solution of 2-oximino-5»-oholestan-3-one (CLXIV) (0,20 g.,

0 .^ 8 2 mmole) in ^ ml. of thionyl chloride and 5 ml. of methylene chloride was stirred at room temperature’ for 2^ hr, ,

After the usual work-up, the amorphous substance was treated with sodium methoxide (0.10 g.) dissolved in 10 ml. of an hydrous methanol. The precipitate was filtered, washed with water and dried to give 0.195 g. (9^^) of material, m.p.

78- 82° . Two crystallizations of this product from methanol - ether afforded 0.176 g. (85^) of methyl 2,3-seco-5®-cholestan-

2-nitrile-3-oate (CIXVIII), m.p. 91-91.5°. This substance was identical in all respects with a sample obtained by hy

drogenation of methyl 2,3-secocholesta-^,6-diene-2-nitrile-

3-oate (CXXV).

Catalytic hydrogenation of 2.’3-secocholesta-^.6-diene-

2.3-dinitrile (CLII). A solution of 2,3-secocholesta-^+,6-

diene-2,3-dinitrile (CLII) (0.093 g., 0.238 mmole) in 20 ml.

of reagent grade ethyl acetate was hydrogenated over 10^

palladium-on-charcoal catalyst (0.05 g.) at a’bmospheric

pressure. Hydrogenation was stopped at the end of 23 min.

when approximately 2 equivalents of hydrogen absorption was

observed. The.catalyst was removed by filtration and the 105 solvent removed in a rotary evaporator to give an oil which solidified on standing ^fith a little methanol. A thin layer ■ chromatography of this product on silica showed two spots.

The separation of the mixture was carried out by preparative thin layer chromatography on silica gel using benzene - ethyl acetate (1:1) as the eluant. After the usual work-up, the band corresponding to Rf 0.90 afforded 0.067 g. (71^) of 2,3-seco-5»-obolestan-2,3-dinitrile (GLXIX), m.p. 116P.

Infrared spectrum: X g^ax (CN), 6.8lyU , 6.91ju, 7»20yU, 7»2^/U.

[ca]§^ +19.9° (C 1.0).

Anal. Calcd. for C27Hl{i^N2: C, 81.75; H, 11.18; N, 7.06.

Found: C, 81.87; H, 11.25; N, 6.9^.

The band corresponding to Rf o.12 gave o.ol g. (11^) of an oil which was not further investigated.

2,3-Seco-5n-cholestan-2-nitrile-3-oic acid-3-amide

(GLXX). A solution of 2-oximino-5«i-cholestan-3-one (GLX7)

(0.20 g., O.J+82 mmole) in ml. of thionyl chloride and

5 ml. of methylene chloride was stirred at room temperature

,for 2^ hr. After the usual work-up the cyano acid chloride

was dissolved in 20 ml. of dry methylene chloride and

•saturated with ammonia gas at 0° for 10 min. The organic ex

tract was then washed with water and dried over anhydrous

sodium sulfate. Evaporation of the solvent left a brown

residue which was crystallized from petroleum ether - chlo 106 roform (1:1) to give 0.17 g. (86 ^) of GLXX as shiny color less crystals, m.p. 261 °.

Infrared spectrum: 2.90^, 3.0(^, 3.l8/a (N-H),

^.^•2)1 (CN), 6.0^ (CONH2).

[a] §6 +33.60 (0 1.03 )

Ultraviolet spectrum: X max 20^m/i (6 2,532).

Anal. Calcd, for C27HJ+6 ON2 : C, 78.20; H, 11,18; N, 6 .76 .

Found: C, 78.30; H, 11.02; N, 6 .80.

2.3-Seco-5a+cholestan-2.3-dinitrile (CLXIX). A solu

tion of 2 ,3-seco-5“-cholestan-2-nitrile-3“Oic acid-3-amide

(CLXX) (0.10 g., 0.21+0 mmole) in 3 ml. of thionyl chloride

was refluxed for 18 hr. Thionyl chloride was removed in a

rotary evaporator to give a brown material, which wa crystal

lized from petroleum ether to give 0.06 g. (66 ^) of 2 ,3-

seco-5“-cholestan~2,3-dinitrile (CLXIX), m.p. 118-119°.

This product was identical in all respects with a sample

obtained by the catalytic hydrogenation of 2 ,3-secocholesta-

1+,6 -diene-2,3-dinitrile (CLII). PART II

INTRODUCTION

The history of Tabernaemontana Linn. (Family Apo- cynaceae) is exceedingly complex. At various times, botanists have split other genera from it (e.g. Voacanga. Ervatamia. .

Gabunia. Conopharvngia) leaving a residue of synonymy of genera and/or species within the group. With regard to South

American representatives, botanical opinion concerning their classification has varied widely. Markgraf"* has separated and readjusted the genus Tabernaemontana L. into nine genera, preserving the original name for species found in the Antil les, Central America, and parts of northwestern South America, while maintaining Feschiera A. DC. for those found in Brazil.

Woodson,2 however, does not share this opinion.

The plant under present investigation was given its present name of Tabernaemo'ntana rigida (Miers) by Woodson and R o d rig u es.3 In i860, Mueller^ gave the name T. mac-

^F. Markgraf, "Die Amerikanischen Tabernaemontanao- ideen", Notizld. XIV, No. 121, 1^1 (1938). % . E. Woodson, Jr., "North American Flora: Asclepia- dales - Apocynaceae" 29, II, 103 (1938). ^Dr. W. Rodrigues, private communication. The reclassi fication of this species does not appear to have been pub lished, probably because of the death of Dr. Woodson.

^J. Mueller, in Martius, Fl. Bras.- 6 (1): 7? (i860).

,1 0 7 108 rophylla to this plant. But in I817, Poiret^ had already called a different plant T, macrophylla; therefore Muell.

Arg. T. macrophylla was an illegitimate name since two different plants cannot have the same scientific name. In

1878, Miers^ called the plant under investigation Phrisso-»

carpus rigidus. thus rigidus is the first legitimate speci fic epithet to be applied. Later, Markgraf? decided that

this plant belonged to still another genus, Anacampta. It can be noticed here that Markgraf used Miers epithet rigida

(rigida instead of rigidus to agree with the gender of the

genus). However, according to Woodson the species rigida

really belongs in the genus Tabernaemontana. Since we already

know that T. macrophvlla is illegitimate and therefore can ■

not be used, we must look for first legitimate epithet,

which is rigida.

Tabernaemontana rigida (Miers) is a tree native to

Brazil. It usually grows up to the height of about 5 meters.

The leaves are subsessile, oblong-elliptic, obtuse or shortly

subacuminate at apex. The cyme is very compact with about

12 flowers; peduncles are four times as long as petioles, and

denseley pubescent. Pedicels are shorter than calyx. The

?J. L. M. Poiret em Encyc. Meth. Bot., Supl. V (1), 276 (18,17). •

Miers, in Apocyn, S. Am., 72 (I878).

?F. Markgraf, Notlzblatt XIV,'Ilf6 (1938). 109 . caly x a re 5-parted to the middle and the corolla about five times as long as the calyx. The ovaries are ovoid and four to five times shorter than the style.^ All the alkaloids so far isolated, from the genus Tabernaemontana have in common the indole nucleus. They dif fer, however, in skeletal structures as well as in the nature of the substituents. Two crystalline compounds,® tabernaemontanine (I), m.p.

208- 210°, and a substance of unknown structure, coronarine (II), a yellow compound, m.p. 196-198°? were isolated from T. corohariai in India. A more recent investigation^ yielded only tabernaemontanine (I) of.m.p. 217- 218° . Since the shrub T. coronaria (Svn; Ervatamia coronaria) a ls o grows in F lo rid a , U .S .A ., Gormon e t al.^® made a chemi cal investigation of the plant to determine whether its alkaloidal constituents would be of the same nature as those found

in Indian m aterial. A small amount of the alkaloids dregamine (III) and non crystalline coronaridine (IV) were isolated from this plant; the latter compound was characterized as its

• ®A. N. Ratnagiriswaran and K. Venkatochalom. Quart. J. Pharm. and Pharmacol., 12, 17^ (1939) C.A., 2^, 83 >6 (1939) . . 9s, A, Warsi and B. Ahmed, Pakistan J. Sci., 1, 128 (1949). - Gorman, N. Neuss, N. J. Cone, and J. A..Deyrup, J . Am. Chem. S o c., 1l42 (1960). 110 crystalline hydrochloride, m.p. 235°- Another alkaloid, ta bernaemontanine (I), m.p. 217- 218° was also isolated from the same plant. Tabernaemontanine (I) and dregamine (III) are isomeric,

2-acylindole alkaloides.^^ iheir formula (C21H26 O3N2 ) correspond to that, of a dihydrovobasine, while infrared and ultraviolet data indicate a sim ilarity to vobasine (V). Their exact relationship was shown by a series of hydrogena tion and epimerization experiments. The hydrogenation of. vobasine over Raney nickel in ethanol gives a mixture of dregamine (predominant) and tabernaemontanine (minor). Hydro genation over platinum in ethanol yields tabernaemontanine as the only isolable product. As the ultraviolet and infra red spectra of these compounds show retention of the 2- acylindole and carbomethoxyl functions, the reduction was interpreted on the basis of the conversion of the ethylidene group to an ethyl group. The N.M.R. ■ spectra of the isomers show that in both instances the carbomethoxyl group has re tained its orientation toward the aromatic system, thus re quiring that the epimeric relationship reside at the asym metric center created, during ethylidene reduction. A determi nation of the stereochemistry of the ethyl group was made ■

Renner, p. A. Prins, A. L. Burlingame, and K. Biemann, Helv. Chim. Acta, if 6 , 2186 (1963). A, W eisbach and B. D ouglas, Chem. In d .(L o n d o n ), 623 (1965). Ill as follows: under alkaline conditions, tabernaemontanine is isomerized to the corresponding epi compound, while dregamine is recovered unchanged. The resistance of dregamine to iso merization is a result of the severe steric forces which are introduced by the interaction of the cis ethyl moiety with the carbon bridge upon which the carbomethoxyl resides. On this basis, tabernaemontanine (I) and dregamine (III) were assigned the formula I and III respectively.

I , R=H; R-jsCgH^

I I I , R=C2H^; R i =H

Gorman et. all® further investigated three species of Tabernaemontana namely T.' und'ulata, T. psychotrifolia and T. oppositifolia collected on Trinidad and neighboring is lands where they are found in natural and cultivated states. Coronaridine (IV), voacangine (VI), voacamine (VII) and a • small amount of a yellow crystalline alkaloid olivacine (VIII) were isolated from T. psychotrifo lia . Chromatography of the alkaloidal fraction prepared from benzene extract of 112 the root of T, oroosltlfolla yielded ihogamine (IX), coro naridine (IV), voacangine (VI) and finally voacamine (VII), The alkaloidal fraction obtained from T. undulata showed the presence of tree components in the paper chromatography. Repeated attempts to get crystalline material from the above fraction proved to be unsuccessful. The alkaloids voacangine (VI) and voacamine (VII) -were also isolated from T, australis, collected in northeastern Argentina. The occurrence of the three alkaloids coronaridine (IV), voacangine (VI) and ibogamine (IX) in the same plant species is suggestive of a close biogenetic relationship among these compounds. Buchi et a l.^3 have elucidated the structure of voaca mine (VII) and it is found to be a dimeric bisindole alkaloid. These authors also synthesized it by acid-catalyzed conden sation of voacangine with vobasinol. Thomas and S tarm er’'^ re p o rte d th e is o la tio n o f conopharyngine (X) from the plant T, nachsinhon vàr. cumminisl collected from Mpraiso district, Ghana. Coronaridine (IV) and tabernaemontanine (I) are obtained from the bark of the

^^G. Buchi, R. E. Manning, and S. A, Monti, J. Am. 'Chem. Soc., 86, 1+631 (196*+).

Thomas and G. A. S tarm er, J . Pharm. and Pharm acol., Ii, (7), ^87 (1963). 113 species T. miicronata'.^ ^ Aquilaar-Santos and his co-workers^^ reported the isolation of coronaridine (IV) from the plant T, pandacaaui. Tabersonine (XI), a member of the distinct group of aspidopermine-type alkaloids, has been isolated from T, alba by Collera and his co-workers.^ 7,1 8 Cava et a l.^9 reported the isolation of six bases of the iboga group from the bark of the Jamaican species T, lau- rifo lia.These are isovoacangine (XII), tabemanthine (XIII), ibogamine (IX), iboxygaine (XIV), coronaridine (IV) and isovoacristine (XV). The compound XV is an amorphous base and crystallizes only as a dimethyl sulfoxide solvate, m.p. 104- 107°.

The genus T, hevneanai. was investigated by Govindachari and his co-workers.^0 From the bark of this plant was iso lated the base heyneanine (XVI). The structure of this

15a . C. Santos, G. A. Santos, and L. L. Tibayan, Anales Real Acad. Farm. (Madrid), 21, 3 (1965). Aquilar-Santos, A, C. Santos, and L. M. Jason, J. Philipini Pharm. Assoc., ^0, 321; 333 (l96'+), 17o. Collera, F. Walls, A. Sandoval, F* Garcia, J. Herran, and M. C, Perezamador, Bol, Inst. Quim. Univ. Nac. Auton, Mexico, Jit, 3 (I962 ). ^®R. H. F. Manske, "The alkaloids", VIII. The Academic Press, New York, 1965, p. My. I^M. P. Cava, S. K. Mowdood, and J . L. B eal, Chem. Ind. (London), 2Q(h (1965). 20rp. R. Govindachari, B. S. Joshi, A. K. Saksena, S. S. S ath e, and N. V isw anathan, Chem. Commun., 97 ( 1966 ). 11k- compound is based on N.M.R. and mass spectral analysis and finally confirmed by its transformation to ibogamine. In addition to this, coronaridine (IV) was isolated as its crystalline hydrochloride, m.p. 232-233° (dec.),

Heyneanine (XVI) was also isolated by Kupchan et al.21 and Hootele et al.^^ from Ervatamia. dichotoma and Cono-

•pharvngia .iollvana respectively. The former workers also

independently proved its structure. In conclusion it may be noted that although indole alkaloids appear to occur in all Tabernaemontana.species, alkaloids of the ibogamine structural type(a)have been iso lated most frequently in addition to a few alkaloids of the vobasine structural type(b). Only two alkaloids, olivacine (VIII) and tabersonine (XI) have so far been reported which do not belong to either of these above mentioned structural gro u p s.

21s, M. Kupchan, J. M. Cassady, and S.- A. Telang, Tetrahedron Letters, 1251 (1966). 22c, Hootele, A. McCormick, J. Pecher, and R. H. Mar tin , "Symposium uber Chemie und Stereochemie der Steroid und Indolalkaloide',' Sraolenice, Czechoslovakia, Sept. Ik—18, 1965} Abstracts, p. 10,. 11?

In view of the confusion still’ existing in the bota nical literature concerning the classification of Tabernae montana species, the alkaloidal examination of further member of this genus seems to be particularly desirable from a chemotaxonomic point of view.

• . The work described in this portion of this disserta tion is concerned with the alkaloids of the previously un- inve.stigated species? Tabernaemontana rigida. Chart I AIJCALOIDS ISOLATED FROM TABERIUEMGNTANA

Alkaloids Formula Specie Melting {*1; R eferences P o in t

Tabernaemontanine C21H26 O3N2 T, coronaria 208- 210° -57°(c) 8, 9, 10, (I) 217 - 219 ° 15 C oronarine Cl{lfH5606N^ T. c o ro n a ria 8 (II)

Dregamine C21H26 O3N2 T. coronaria 196 - 198 ° 10 (III)

Coronaridine ^21^2602^^2 T. coronaria amorph. B.HCl 10 (IV) T. psychotrifolia -8°(m ) 10 T, oppositifolia 10 T. mucronata T. pandacaqui ÎI T, laurifolia 19 T. heyneana 20 Voacangine C22H28O3N2 T. psychotrifolia 137-138° -42°(c) 10 (VI) T. oppositifolia 10 T. australis 10

Voacamine 01^.505606 % T. psytrofolia 223° - 52° ( c ) 10 (Voacanginine) T. oppositifolia 10 (VII) T. australis 10

O liv acin e Ci 1^02 T. psychotrifolia 318- 32^ ° q 10 (VIII) o\ Chart I (continued)

A lk a lo id s Formula Specie M elting [® ]d R eferences P o in t

Iho gamine ^19^24^2 . T. oppositifolia I 62 -I 6 V0 -5 ^ 0 (a) 10 (IX) T. l a u r i f o l i a 19

Conopharyngine C23H30OMÎ2 T. pachsiphon -510(c) Ilf (X)

Tabersonine C21H26 O2M2 T. a lb a 1960 -3 1 0 (c ) 1 7, 18 (XI)

Isovoacangine C22H28G3N2 T. l a u r i f o l i a 156-1570 - 520(c) 19 (XII)

Tabernanthine C20H26 ON2 T. l a u r i f o l i a 211 -2120 - 350(c ) 19 (XIII)

Iboxygaine C20H26 O2M2 T. laurifolia 2340 - 50(c) 19 (XIV)

Isovoacristine 022^ 270^^2 T. laurifolia amorph. -1 9 .6 o ( c) 19 (XV)

Heyneanine C21H25O3N2 I. heyneana 105-1070 20 (XVI) 160-1620

a, CH3 CH2 OH5 c, CHCI3 ; m, CH3 OH 118

Chart I I

Ibogamine (IX) R=Ri=R 2 =H Tabernanthine (XIII) R=R 1=H, R2=CH30 Ib o x ig ain e (XIV) R=CR^O, R-]=H, RgsQH

Cû^CH,

Coronaridine (IV) R=R^ =R 2=H Voacangine (VI) RsCH^C, R^=R 2=H Conopharyngine (X) R=Ri=CHgO, R 2=H Isovoacangine (XII) RsR 2=H, Rj=CH^O Is o v o a c ris tin e (XV) R=H, R^sCR^O, R2=0H Heyneanine (XVI) R=R-]=H, R2=0H 119 Chart II (continued)

CH..

voacamine (VII)

ollvacine (VIII) •tabersonine (XI) . • DISCUSSION

The plant material used in this investigation was the bark of Tabernaemontana rigida, obtained near Manaus, Brazil. The bark was dried and extracted \fith ethanol. The alcoholic concentrate on treatment with ethyl acetate gave an insolu ble residue designated as C. The ethyl acetate soluble frac tion was acidified and the neutral material was removed by benzene. The aqueous acidic fraction was then made alkaline with ammonium hydroxide and the precipitated alkaloid mix ture was extracted with chloroform. The chloroform extract on concentration afforded an almost colorless precipitate A, the filtrate from which on evaporation gave the remaining tertiary bases as a gum B, The fractions A, B and C were provided by Dr. J. A. V/eisbach of Smith, Kline and French laboratories, Pennsylvania. The work described in this dissertation was carried out with the fractions A and B; the minor basic portion of fraction C showed the same spots on thin layer chromatography as fraction B and was, therefore, not further investigated. A detailed description of the iso lation of the different fractions mentioned above is given in Chart III, and in the experimental section, page 132 .As

A was an almost white powder, it was decided to investigate • this fraction first.

120 121

The white powder A, see Chart III (page 131) was crys tallized thrice from a methanol - chloroform mixture to give a colorless crystalline compound Ai, m.p. 227-228°. Its elemental analysis corresponded to the formula 021^2603^2" The infrared spectrum of this compound in Nujol displayed bands at 7*+2cm“^, 720cm"’' (indicative of 1,2-disubstituted benzene ring), 1757cm"'' (prob ably a s a tu ra te d e s te r carb o n y l) and 1073cm"’' (for C-OH group), and no band around 3320cm"’', characteristic of N-H group.23 The a lk a lo id belongs on th e

basis of its ultraviolet spectrum, 226nyi (loge >+.13) and 279mjU (loge 3.59) to the indole group of alkaloids.2^+ The above • physical data, seem to be qiuite in agreement with the properties of d-vincamine, C 21H26 O3N2 (XVII) j^eported25>26

m .p. 232- 233°, I.R. in Nujol: 7'+7cm“’’, 727cm"’' ( 1 , 2- d i s u b s t i tuted benzene ring), 1756 cm”"' (saturated ester) and 107^cm’"^ (C-OH); Ü.V. in methanol; 225mp (loge 'f.l^-) and 278mja ( lo g e 3 . 61 ^ . A remarkable difference was observed however in the optical rotation of this compound Ai compared to that repor ted for d-vincamine. d-Vincamine (XVII) has a rotation of [o(j ^ +'+1° ±4-° (pyridine)25 whereas the compound A-| has a

23j. R, Dyer, "Applications of Absorption Spectroscopy o.f Organic Compounds", Prentice-Hall, Inc., Englewood, 1965, p . 3 0 . 2'+A. W, G angster and K. L. S tu a r t, Chem. R ev., 69 (1 965). ■' 25j, Trojanek, 0. Strouf, J. Holubek, and Z. Cekan, Tetrahedron Letters, 20, 702 (I 96 I). Trojanek, 0. Strouf, J. Holubek, and Z. Cekan, C o ll. Czech. Chem. Comm., 2£, 4-33 (19649. 122 rotation of only[c^J§^ +*+°' (pyridine). This observation immediately led us to think that Ai could be a mixture of d- and dl-vinoamine. Such an example of a d- and dl-alkaloid mixture is not without precedence in the literature. Smith and Wahid ^7 reported the isolation of a mixture of d- and dl-vincadifform ine from Rhazya s t r i c t a and separated the dl-vincadifformine from the mixture by fractional crystal liz a tio n . With this information in hand we next proceeded to attempt the separation of the possible d-form from the d- and dl-vincamine mixture A-]. Repeated crystallization (seven times) of 12 g, of Ai from methanol - tetrahydrofuran (3:1) afforded 9 g. (75^) of pure crystalline dl-vincamine (XVIII), m.p. 235-236°, 0°. The accumulated mother liquors on

concentration afforded 1.80 g. ( 15/^) of pure d-vincamine (XVII), m.p. 232^233°, +40° (pyridine). Finally, d- vincamine was identified by mixed melting point, Rf and infrared spectra with an authentic sample supplied by Dr. Kompis, Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Czechoslovakia. There are a few characteristic differences in the infrared spectra of d- and dl-vincamine in KBr. The former shows bands at 17^8cm",^ (ester carbonyl group) and 675cm“'^ which are shifted to 1736cm"1 (ester car-

27g , F.' Smith and M, A. Wahid, J. Chem. Soc., ^002 (1963). 123 bonyl group) and 686cm“^ in the case of the latter. These differences, however, disappeared when the spectra were taken in chloroform solution. d-Vincamine has been isolated previously from Vinca minor L.28 (Apocynaceae), Vinca manor Vinca difformis Pourr^O and Vinca erecta The occurrence of vincamine in Tabernaemontana rigida during this investigation marked the first recorded isolation of an alkaloid of this structu ral type in this genus. Moreover, there is no previous report in the literature of the presence of dl-vincamine in any"plant.

Schlittler and A. Purlenmeier, Helv. Chim. Acta, 36. 2017 (1953); M. Pailer and L. Belohlav, Monatsh., 1055 (195^); P. E. King, J. H. Gilks, and M. W. Partridge, J, Chem. Soc., 1+206 (1955); K. Szasz, L. Szporny, S, B itt ner, I. Gyenes, E, Havel, and E,- Mago, Magyar Kem. Polyoirat, 61+, 296 (1958); J. Trojanek, J. Hoffmannova, 0. Strouf, and Z. Cekan, Coll. Czech. Chem. Comm., - 526 (1959); J. Troja^ nek, K. itavkova, 0, Strouf, and Z. Cekan, ibid., 26, 867 (1961); J. Trojanek, 0, Strouf, K. Kavkova, and Z, Cekan, ib id ., 22., 2801 (19b2); Z. Cekan, J. Trojanek, 0. Strouf, and K. Kavkova, Pharm. Acta Helv., 25, 96 (19o0); 0. Bauer- ova, J. Mokry,'I. Kompis, 8. Bauer, and J. Tomko, Chem. zv esti 523 ( 196 I); J. Trojanek, 0. Strouf, K. Kavkova, and Z. Cekan, Chem. Ind. (London), 790 (1961).

29n . r. Farnsworth, P. J. Draus, R. W. Sager, and J. A.Bianculli, J. Am. Pharm. Assoc. Sci. Ed., j+2; 5o9 (I960).

30m. M. Janot, J. LeMen, and Ch.,Pan, Ann. pharm. .franc. Ü , 513 (1957). 31p. Ch. Juldasev, Izv. Akad. Nauk SSSR, 188 (1953); ' S. J. Junusov, P. Ch. Juldasev, and N. V. Plechanova, Dokl. Akad. Nauk Uzbek. SSR, 2, 13 (1956); 8. J. Junusov and P. Ch. Juldasev, Z. Obsc. Chim., 2%, 2015 (1957). . . 12^

Further proof of the identity of dl-vincamine (XVIII) was established by its chemical transformation into several known vincamine transformation products. Thus XVIII afforded dl-vincaminic acid (XIX), m.p. 263-26^+° (lit.^5 m.p, 262- 263° fo r the d-corapound) in 80fo yield when refluxed \fith potassium hydroxide for 3 hr. The ultraviolet spectrum of this acid was identical with that reported in the literature?^ Acid XIX, on oxidation with ammonical silver oxide 32 solution, gave a yield of dl-eburnamonine (XX), m.p. 196° (lit.^3m.p. 203-2Ql+° for synthetic ,dl-eburnamonine). The infrared spec trum of this compound in chloroform was superimposable with that of eburnamonine recorded in the literature;3^

I I

XVIII

32o, Strouf and J. Trojanek, doll. Czech. Chem. Comm., 22, h h 7 (19649.

33m. p . Bartlett and W..I. Taylor, J. Am. Chem. Soc., 82, 594-1 (1960). ’ 34-physical data of indole and dihydroindole alkaloids, Eli Lilly and Company, Indianapolis, Indiana (1962). 12?

dl-Vincamine (XVIII) was then dehydrated to dl-apovincamine (XXI) by refluxing it id.th formic acid for 1 hr.3? or by treatment with 11 N HCl at room temperature.^^ The infrared spectrum of this compound XXI in chloroform dis played a shift of ester carbonyl group to shorter frequency 17^-7cm- 1 against Ai which has the ester carbonyl absorption a t 1757cm"^. The appearance of two more bands a t 16l8cm”^ and 16^2cm"’^ re s u lts from the extended conjugation in XXI. The infrared (in chloroform solution) and the ultraviolet spectra of this compound were also in accord with those mentioned in the literature for the d-compound.^5

RCOgH

c o m —. XVIII XXI

Although the reduction of d-vincamine to d-vincaminol by lithium aluminum hydride has been reported by several

a u t h o r s , 37,38 ^ literature survey shows that the sodium boro-

350. Glauser, K. Gesztes, and K. Szasz, Tetrahedron Letters, 1l4? (1962). ----- 3% , P la t, D. D. Manh, J . Le Men, M, M. Janot, H. Bud- zikiewicz, J. M. Wilson, L. J. Durham, and C, Djerassi, Bull. Soc. Chim, France, IO 82 ( 1962 ). 37p, I , Major and I . E, Kholy, J . Org. Chem., 28, ^91 (1963 ). 3% , P la t, R, Lemay, J , Le Men, M. 'M. Janot, C. Djerassi, and H, Budzikiewicz, Bull. Soc. Chim. Prance, 2^+97 (19651. 126 hydride reduction of this compound has not been reported up to the present time. It was, therefore, of interest to know if the reaction of sodium borohydride with dl-vinca mine would occur; it is knovm that esters may be reduced to the corresponding primary alcohols by this reagent.39 It has again been reported that such an abnormal reduction is facilitated by the presence of neighboring functional groups. Schenker^^ implies that these groups may in some way take part in the reduction, although no suggestion was made as to the mechanism of this effect, dl-Vincamine (XVIII) was dissolved in a mixture of hot methanol - tetrahydrofuran (3:2) and an excess of sodium borohydride was added over 10 min. to this hot solution and the .mixture, was s tirr e d a t room temperature fo r 6-%- h r. Work-up in the usual way followed by crystallization from methanol affor ded dl-vincam inol (XXII) in 51^ y ie ld .

XVIII XXII ■

39h, Rapoport and M. S. Bro\m, J. Org. Chem., 3261 ( 1963 ); V. Boekelheide and R. J, Windgassen, Jr., J . Am, Chem. Soc., 8l_, l4^6 (1959). Ln E, Schenker, Angew. Chem., 23.» 81 (1961). 127

Janot e t a l . 36 reported the conversion of d-vincamine to the corresponding quarternary salt XXIII by treatment \’ri.th 11 N HCl based on the u ltra v io le t spectrum vhich showed maxima.at 233nyi (loge 3-95)» 27%yi (loge^3.93)j 317i^ (loge 3 . 83) and 360m^ (loge 3.85). We considered this inter mediate quarternary salt XXIII to be a potentially interesting starting material for further reactions of vincamine not pre viously recorded. Surprisingly all attempts to convert dl- vincamine (XVIII) into XXIII by treatment with 11 N HCl proved to be unsuccessful as shovm by the ultraviolet spectrum which had absorption maxima characteristic of only apovincamine 5 no maximum around 360nyi was found. Next, we repeated the same experiment x-âth an authentic sample of d-vincamine supplied to us by Dr. I. Kompis but we were unable to observe any maximum around 360mjU, instead the ultraviolet spectrum was identical with that of apovincamine. This result suggested that the maximum at 360nyi.as reported by Janot et al,^^ must have been due to some sort of impurity in the sample.

IlNHCl

OTIII XXIII VJ,

XXI 128

The amorphous crude tertiary base fraction B, see Chart III (page 131 ) was first separated from remaining neu tral impurities by dissolving it in aqueous tatraric acid, followed by exhaustive extraction of the. aqueous solution with ether. The aqueous phase on basification %-n.th ammonium hydroxide solution ( 10^) a t 0° precipitated the purified alkaloid mixture. This product was extracted into ether, any residual alkaloids in the solution being then removed by chloroform extraction. The ether extract P-la (33.8 g.) was chromatographed over neutral alumina and the fraction 1 obtained by eluting with benzene gave crystalline material. This product (1.5 g.) was found to be d-apovincamine, m.p. 160 - 162 ° ( l i t . 25^ m.p. 159-161° for d-apovincamine). The infrared and ultraviolet spectra of this compound were iden tical ifith those described in the literature. Fraction 2 and 3 gave a mixture of d- and dl-vincamine which was char acterized in the usual manner. No crystalline material was obtained from fractions ^i- and 6 on repeated chromatography over alumina of different activities. Fraction .5 was rechro matographed over neutral alumina whereupon only fraction 2, among others, afforded 0.015 g. and 0.025 g of two crystal lin e compounds , XXIV and XXV resp ectiv ely . The compound XXIV, m.p. 209- 211°, +1.8 (chloroform), revealed bands in the infrared spectrum at 175^cm“ '' (carbonyl), 108lcm”’*, 758cm"’' and 7^+1 cm"’'. The compound XXV had a m elting p o in t' of 186- 187°, -5° (chloroform) and showed the follow ing 129 bands in the infrared spectrum at (carbonyl), 1079om""^,, 762cm"^ and 7^3cm“ '^. The compounds XXIV and XXV are almost certainly indole alkaloids as sho\m by their ultraviolet spectra: 22$^a (loge, ^.69), 278in)u (loge and 226mju (loge 3.58), 277iyi (loge 3.03) resp ectiv ely . Although only very weak N.M.R. spectra of XXIV and XXV could be obtained due to the small quantities of pure mate rial available, peaks at 6.31? in XXIV and 6A1t in XXV, c h a ra c te ristic of a OCHg.group was found to be present in ■ their respective spectrum. Structural studies could not be performed for lack of materials at this time. As the original chloroform extract (F-1b) showed over lapping spots on thin layer chromatography over alumina it was thought that a pH fractionation of this mixture might aid in the separation of the alkaloidal mixture. In order to test the effectiveness of this method about. 1.0 g, of the mixture was first exhaustively and successively extracted with citric acid - sodium hydrogen phosphate buffer solutions of pH 6.6, 6.0, 5.0, V.O, 3.6, 3.0 and 2.2. As most of the material came into the 6.6, 6.0 and 5.0 pH fractions, the remaining chloroform extract (1^.^ g.) was then extracted successively only vri.th buffer solutions of pH 6.6, 6.0 and 5.0. While pH fractions 6.6 and 5.0 failed to give any crystalline material on repeated chromatography, the pH 6.0 fraction, however, afforded 0.028 g. of a crystal line material XXVI, m.p. 207-209°, +2.2 ±2. The infra- 130 • red and the u ltra v io le t spectra of th is compound showed ab sorptions at 176lcm"1 (carbonyl), 108$cm-1, , 753cm" 7^1 cm"1 and 22kmp. (loge 277myi (loge ^,10) respective ly. The N.M.R. spectrum of this compound could not be run because of the low solubility in deuterio-chloroform, deute- riated dimethyl sulfoxide", and carbon tetrachloride. Further characterization was not carried out due to lack of material. ,131-

chart in

Flow Sheet for the Extraction of the Total Alkaloids

Alcoholic Concentrate from 23 kgs of Bark.

CH3CO2C2H5

Tarry substance CH3CO2C2H5

Gl- HO Ac 5% H2SO4 15 liters dilute with HgO I______to 5% and filter acidic aqueous CH3CO2C2H5 fraction 5% H2SO4 Residue Filtrate OgHg, DNA I H2SO4 CH3CO2C2H5 NH4OH OgHg H2SO4

CHCI3 DNA NH4OH. pH-10

Aqueous CHCI3 CHCI3 OgHg 45.0 g. Evaporate CfiHgng H2SO4 pH2.5withHCl Residue to near dryness C then filter. Add Ammonium 4 1 NH OH Reineckate I I pH-10 25.0 g. CHCI3 • Aqueous Residue colorless 67.5 g. B CIÏCIJ, 3 DNA 1 2 0 . 0 g. powdery pre cipitate A Reineckate Aqueous CHCI3

Salt DNA 1 .6 8 g.

Combined with B

DNA: Discarded Non-Alkaloidal. EXPERIMENTAL

The plant Tabernaemontana rigida was botanically Identified by Dr. W. Rodrigues of the Centro de Pesquisas P lo re sta is, In s titu te Nacional de Pesquisas; de Amazonia, Brazil and registered under herbarium No. 93/2. The bark of T. rig id a was collected in the month.of february, 1965 near Manaus, Brazil. The dried bark was extracted with ethanol by Dr. A. I. Rocha, Dept, of Chemistry, Institute Nacional de Pesquisas de Amazonia, B razil. The extracted plant material was further processed under the supervision of Dr. J. A. Weisbach of Smith, Kli ne and French Laboratories, Philadelphia, Pennsilvania. The alcoholic concentrate (from 23 Kgs of dried bark) on treat ment vri.th ethyl acetate gave an ethyl acetate soluble fraction and a residue. The residue was designated as C. The ethyl acetate soluble fraction was made acidic with 5^ sulfuric acid and the mixture was extracted vâth benzene to remove the non-alkaloidal.material, which was discarded.. The aqueous acidic solution was then basified with ammonium hydroxide to pH 10 when the alkaloids precipitated. The pro duct was extracted with chloroform and the extract was con- • centrated to near dryness, affording 20 g. of an almost colorless; powdery precipitate A. The filtrate from A on eva

poration gave 67.5 g. of non-quarternary bases designated

132 133 as B. All of the fractions A, B and C were found to give positive tests for alkaloids when treated vâth Mayers and Dragendorffs' reagents. The almost white powder A showed two spots on th in layer chromatography over alumina using chloroform - metha nol (1:1) as the developing solvent. An analytical sample A i, m.p. 227- 228° was prepared by three crystallizations of the material A from chloroform - methanol (1:1). Infrared spectrum in Nujol: 7^2cm"^ (1,2-disubstituted . benzene ring), 1730cm"^ (saturated ester carbonyl), and 1073cm"'' (for C-OH). [o(] (c 1. 0 ; pyridine). Ultraviolet spectrum: 226raji (lpg<£ ^.13) and

279m)i (lo g e 3 .59). Anal.found: C, 71.1'B; H, 7.39; N, 7.90. The above physical data, with the sole exception of the rotation, appear to correspond with those of d-vincamine^^ C21H26 O3N2 ' (0, 71.16; H, 7.39; N, 7.90), m.p. 232-233°, [a] +^1° îl+° (pyridine). Hence it was thought that the crystalline material Ai could have been a mixture of d- and dl-vincamine. A further purification of this material A| was therefore deemed necessary to separate dl-vincamine from d-vincamine present in the mixture. In order to accomplish this object the method of Smith and Wahid27 was employed,. Repeated crys

tallizations (seven times) of Ai ( 12.0 g.) from tetrahydro- 1 3 ^

furan - methanol (3*. 1) afforded 9.0 g (75^) of pure dl-vin camine, m.p. 235-236°. Infrared spectrum: 1736cm“1 (ester carbonyl), 1075cm"^ (C-OH) and 686cm"^.

[o .] d ^ 0° (c 1.0; pyridine). Ultraviolet spectrum: 226mja (loge ^.26) and 279mju (loge 3 . 72). The combined mother liquors gave 2.0 g. (15^) of ma terial, m.p. 221-229°. Two additional crystallizations of this material from tetrahydrofuran - methanol (1:1) yielded 1.8 g. ( 15#) of pure crystalline d-vincamine, m.p. 232-233°. Infrared spectrum: '17^8cm’"1 (ester carbonyl), 107^cm-1 (c-OH) and 675om-1. +^0° (c 1.0; pyridine) [lit.25 +lfio + 1^.0 (pyridine^ The infrared spectrum of this material was superimpos able with that of the authentic sample of d-vincamine. There was no depression in the mixture melting point.

Basic hydrolysis of dl-vincamine (XVIII) to dl-vinca minic acid.(XIX). The procedure employed was essentially that bf Trojanek^G A solution of dl-vincamine (XVIII) (0.20 g., 0.056 mmole) in 30 ml. of 1 N methanolic potassium hydroxide was heated to boiling fo r 3 h r. Most of the methanol was re- • moved under reduced pressure, 3 ml. of water were added and the methanol removed completely under reduced pressure. The residual qaueous solution was acidified i^rith 1 N HCl to pH 135 5 - 6 and set aside overnight in the refrigerator. The crys tals which had separated were filtered under suction, washed with water and dried, affording 0.1 g. (75^) of dl-vin caminic acid (XIX), m.p. 263-264-° ( l i t .^ ^ m.p. 262-263° fo r d-vincaminic acid). In frared spectrum; 3390'cm“ 1 (OH), 2500cra~1 (bonded OH of carboxyl), I695cia” ^ (carboxyl). U ltra v io le t spectrum; MeOH 231mji (logé 4-.4-1 ) and 282nyi (logé 3 -79); j l i t . ^eOE 230mji (logé4.44) and 28lmji (logé 3 . 86^ .

Silver oxide oxidation of dl-vincaminic acid (XIX) to dl-eburnamonine (XX). The method employed was that of

Trojanek.32 dl-Vincaminic acid (0.10 g. , 0.024-mmole) was treated with 15 ml. of an ammonical solution of freshly prepared silver oxide and the mixture was boiled for 20 min. (The s ilv e r oxide was prepared by p re c ip ita tin g 0.55 g. of silver nitrate with 10?a sodium hydroxide solution and washed \iâth water until neutral.) After 7 min. a silver mirror started to deposit on the walls of the flask. The cooled solution was filtered, the metallic silver was scratched and washed repeatedly with 6 N HCl until the reaction with ‘Mayers' reagent was negative, and the combined washings were added to the ammonical filtrate. The precipitated silver chloride was filtered and washed with 6 Kf HCl. Addition of concentrated ammonium hydroxide gave a precipitate which was 1 3 6 exhaustively extracted with methylene chloride arid crystal lized from ethanol to afford 0,035 g- (^0^) of dl-eburnamonine (XX), m.p. 196-198° (lit.33 m.p. 203-20^° for syn thetic dl-eburnamonine). , Infrared spectrum in chloroform: iyo4nm"1 (carboxyl), 163W -1, l4^3cm-1, 1381 cm-1, 1332cm"1 and 1139cra-1. Ultraviolet spectrum: 2)+3nyi (loge Î+.19), 269mji loge 10), 29^mju (loge 3.65) and 303m^ (loge 3 . 55); jlit.26 ^ max^ 2'+2myu (loge ^ . 28), 268myi (loge ^ .0 0 ), 29^nyu (log e 3 . 68 ) and 301 ryi (loge 3.68^. Anal.Calcd. for CigH220N2:'C, 77*525 H, 7.53* Pound:'C, 77*60; H, 7*66.

Dehydration of dl-vincamine (XVIII) to dl-anovincamine (]{XI). A solution of dl-vincamine (XVIII) (0.10 g., 0.286 mmole) in 2 ml. of formic acid was refluxed for 1 hr. and . cooled.85 The solution was then basified with a saturated solution of sodium bicarbonate to pH 12. The precipitate was extracted twice with 50 ml. of ether, washed with water and dried over anhydrous sodium sulfate. The solvent was removed in a rotary evaporator to give a colorless oil which solidi- ■ fied on scratching. Crystallization was effected from acetone to give 0.062 g. (65^) of crystalline dl-apovincamine (XXI), m.p, 158- 160 ° ( l i t . 25 m.p. 160-16,1°'for .d-apovincamine). Infrared spectrum in chloroform: 17^7cm“1 (ester carbo n y l), 1618cm" 1 and 16'+2cra“ 1 ; (lit.2 5 I7^6cm“ 1 (e s te r carbonyl). 137

. l6l8om"1 and 1638cm” '*). Ultraviolet' spectrum; 228m^ (logé-, 27^jyi (loge 3.98) and 31»+nyi (loge 3.75); [lit.2? 228m/i (loge

^ .^ 5)5 273nyi (logt^h.Ol+) and 313m/i (loge 3, 82^.

Sodium borohydride reduction of dl-vincamine (XVIII) to dl-vincaminol (XXII). dl-Vincamine (XVIII) (0.50 g ., 1.1+10 mmoles) was dissolved in 20 ml. of tetrahydrofuran and 30 ml. of methanol by heating. To this hot solution was added sodium borohydride ( 0.50 g.) over a period of 10 min. with stirring. A vigorous reaction started immediately and was complete after 6 hr. as checked by thin layer chromato graphy. The solvents were then removed in a rotary evapora tor to dryness and the white residue was dissolved in 50 ml. of water and acidified with 50^ acetic acid. The acidic solution was cooled in a ice-water bath and basified with concentrated ammonium hydroxide. The resulting precipitate was extracted thrice with 50 mli of ether. The combined ether extracts were washed with water and dried over anhy drous sodium sulfate. The solvent was removed in a rotary evaporator to give an almost colorless oil which crystallized from benzene to afford dl-vincaminol (XXII), m.p. 177° (lit. ( l i t . 37,38 in,p. l80° for d-vincaminol).- Crystallization of the oil from methanol,gave 0.258 g. (51^) of dl-vincaminol • with one mole of methanol of crystallization, m.p. 138°. 138

Infrared spectrum; 3322cm"-1 (probably the primary hydroxyl group resulting from reduction of carbomethoxy group) and 3030cm“"’ and 30^0cm""^. U ltra v io le t spectrum: 230myu (loge l+,5^) and 281 mjU (loge 3 . 88); jlit.^^ 232myu (logelf.^ 6 ) and 280iyi (log e 3 . 8^ . Anal.Calcd. for C2052602^2'CHgOH: C, 70.^0; H., 8.^5j N, 7.83. Found: 0, 70.31; H, 8. 86 ; N, 7.57. ,

Attempted nrenaration of the quarternary salt XXIII. dl-Vincamine (XVIII) (0.10 g., 0.286 mmole) was dissolved in 5 ml. of 11 N HCl. The ultraviolet spectrum of the so lu tio n showed maxima a t 228mu, 27^mu and 31^mu which were essentially identical with those of apovincamine. Excess of hydrogen chloride.was then blown off by nitrogen and the solution was cooled in a ice-water bath. Basification of this solution with ammonium hydroxide gave a precipitate. The product was extracted well with methylene chloride and crystallized from acetone to yield 0.073 g. ( 76 ^) of XXI, m.p. 159°. An infrared spectrum of this compound in chloroform was identical with that of d-apovincamine.

Isolation of alkaloids from the crude amorphous ter tiary bases B. The crude amorphous tertiary bases (67.5 &) see Chart III (page 131 ) were dissolved in 1200 ml. of 8% tartaric acid solution by heating on a steam bath. After 139 cooling the, dark bro’im solution was filtered through glass wool to remove insoluble m aterials. The so lu tio n was ex trac ted five times with ^00 ml. of ether to separate the neutral materials from the alkaloidal mixture. The aqueous acidic solution was then made alkaline vri.th cold aqueous ammonium hydroxide solution (10^) and the resulting precipitate was exhaustively extracted with 2,? 1. of ether. The ether ex tract was washed with water, dried over anhydrous sodium sulfate and evaporated to dryness. The aqueous alkaline so lution was further extracted with 1 1. of chloroform, washed with water, dried and the solvent evaporated. The above extracted fractions were then coded as shorn in the Table 1. Table 1 Purification of alkaloids. Code Weight neutral materials P-rln 3.00 g. ether soluble alkaloidal extract P -la 33.80 g. chloroform soluble alkaloidal extract• P-lb 15.^0 g.

Isolation of alkaloids from F-1a; As P-la contained the major portion, it was first worked up. As thin layer chromatography of this fraction over alumina showed three spots (using ethyl acetate - ethanol 1:2 as the developing solvent), it was considered to separate them by chromatogra phy over alumina. F-la (33.80 g) was dissolved in 100 ml. of benzene and was chromatographed on neutral alumina (ac- 1 ^ 0 tivity III, 860 g.) and the following fractions were col lected (Table 2).

Table 2

Fraction Flasks - Eluant Residue 100 ml. each

1 1-8 benzene 9.5 g.» mixture of two spots 2 9-29 benzene 3.0 g., crude mixture of vincamine 3 30-44 benzene - 1.5 g ., crude mixture ether (2:1) of vincamine 45-52 benzene - 3.0 g ., gummy mixture ether (1:1) 56-65 chloroform 8.0 g ., gummy mixture 6 66-71 methanol , 2.7 g., mixture

Thin layer chromatography on alumina, using ethyl acetate - ethanol (1:1) as the eluant was the basis; for com bining of fractions. i) Characterization of fraction 1. This fraction showed two spots on thin layer chromatography on alumina and was completely freed from benzene in a rotary evaporator under reduced pressure. On addition of acetone and scratching the walls, crystals (1.0 g.) finally deposited. Rechromatography of the gummy mother liquor on neutral alumina (activity II, 100.0 g.) yielded a further deposit (0.5 g.) of the same crystalline substance. Thus the total amount of crystalline substance obtained after drying was 1.5P g ., m.p. 158-159°. A recrystallization from chloroform - acetone gave an analytical sample, m.p. 159-160°. Infrared spectrum in chloroform; 17^-5cm""^ (carbonyl), I6l8cm” ^ and I6l+3cm“ ^. +118° (c 2.11; chloroform). Ultraviolet spectrum: 228mp. (loge, *+.39)? 27kr^ (logé *+.00) and 31^njh (loge 3.67). Anal.found: C, 7*+.62; H, 7.30; N, 8.I 6 . The above physical data appear to be in good, agreement with those of d-apovincainine C 21H2I+O2N2 (0, 7^.97; H, 7.19; N, 8. 33), m.p. 159-161°, +121° (chloroform) .35 The ' infrared spectrum in chloroform was identical with that of . dl-apovincamine prepared from dl-vincamine. The infrared and ultraviolet spectra of apovincamihe recorded previously in the literature are as follows;25 l7*+6cm“'', I6l8cm-1,

1638 cm- 1 and 22&mjx (lo g 64-.*+5), 273mju (loge *+.0*+), 313i^w (loge 3 . 82). Hence no further studies were continued on this m aterial. ii) Characterization of fraction 2. This fraction showed only two spots on thin layer chromatography on alumina .and afforded a crystalline compound (2.20 g.) on treatment with ethyl acetate, m.p. 227-228°. It was found to be a mixture of d- and dl-vincamine, +4# and was characterized in the usual way. iii) Characterization of fraction 3. This fraction also gave

d- and dl-vincamine ( 0^90 g.) on treatment with ethyl acetate. 1 ^ 2

[o^]ê^ +^0. Attempted purification of fractions ^ and 6 by chro matography over neutral alumina of different activities failed to give any crystalline material. iv) Characterization of fraction On thin layer chromato graphy on alumina, fraction 5 showed three spots. It was, therefore, thought that further chromatography might enable the alkaloids to be separated. For this reason it was chro matographed on neutral alumina (activity II, 200 g.) and the following fractions were collected (Table 3)* Table 3

Fraction Flasks - Eluant Residue 50 ml. each

1 1-100 benzene 0.7 g . , mixture 2 101-120 benzene - 1.3 g ., mixture chloroform (1:1) 3 121-150 chloroform 4-.0 g . , mixture 151-155 methanol 0.7 g ., mixture

The fractions were combined on the basis of thin layer chromatography on alumina using ethyl acetate - ethanol .(1:1,)as the eluant. The fractions 1,3 and ^ failed to give any crystal line material by repeated chromatography over alumina. ^^■3 i) Characterization of fraction 2. The mixture (1,3 g*) was dissolved in 20 ml. of acetone. Addition of petroleum ether to this solution deposited a crystalline material. After standing for -g- h r,, this product was filtered and crystal lized from ethanol - methylene chloride (3:1) to afford 0.015 g. of XXIV, m.p. 209-211°. Infrared spectrum; 175^cm-1 (carbonyl group), 108lcm“’' and 7^1 cm“’'. +1.8 (c 1 .^5) • Ultraviolet spectrum: ^^ax^ 225rjx (loge ^ . 69 ) and 278myU, (log<£ 1+.1^). ^ Anal, found: G, 71.05*, U, 7.^1; N, 7.35- The elemental analysis corresponds to the molecular formula C21H26 O3N2 (C , 71.16; H, 7.39; N, 7.90). The mother liquor on concentration,deposited white crystals which were filtered, washed with petroleum ether and dried to give 0.020 g. of XXVj m.p. 186-187°. A second crop of 0.050 g. was obtained on evaporation of the mother liq u o r. Infrared spectrum: 17^5cni“^ (carbonyl group), 1079om"^ and 74-3cm-"^. [o ] P -5° (c 2.69). Ultraviolet spectrum: ^^ax^ 226 mju (loge 3.58) and 277m^ (loge 3.03). Anal, found: 0, 70.82; H, 7.51; N, 7.^ 6 . The elemental analysis corresponds to the molecular . formula C21H26 O3N2 (C, 71.I 6 ; H, 7.39; N, 7.90).

Isolation of alkaloids from F-Tb. As the alkaloids of P-lh showed spots very close to each other on thin layer chromatography on alumina (using chloroform - methanol 1:1 as the developing solvent) it was decided to crudely sepa rate these alkaloids into pH fractions. P-Tb ( 1^.^ g.) was dissolved In 100 ml. of methylene chloride and then exhaustively and successively extracted with different buffer solutions (pH 6.6, 6.0 and 5.0) made up with citric acid - sodium hydrogen phosphate solutions. The buffered extracts were then cooled in a Ice-water bath and made alkaline with cold ammonium hydroxide solution (10^) and extracted exhaustively with methylene chloride.. ' The organic extracts were washed with water, dried over an hydrous sodium sulfate and solvents removed in a rotary eva porator to give substances whose weights are recorded in Table 5. Table 5

pH volume of b uffer methylene chloride gxtract ex tract

6.6 1000 ml. 6.23 g., mixture 6.0 1000 ml. 2.35g., mixture 5.0 1000 ml. . 1+.1.1 g . , mixture i) The pH 6.6 fraction (6.23 g.) was chromatographed over neutral alumina (activity III, 100 g.) and the following fractions were collected (Table 6). Table 6

F raction Flasks - Eluant Residue 2? 'ml. each

1 1-10 benzene 6.62 g., m ixture' 2 11-1? benzene - 1.15 g ., mixture chloroform (9:1) . 3 16-2? chloroform 3.77 g ., mixture 26-30 methanol 0.80 g.. mixture

Thin layer chromatography on alumina, using chloror- form - methanol (1:1) was the basis for combining of frac tio n s. Repeated attempts to purify the fractions 1 - (Table 6) by chromatography over neutral alumina (activity I, II, III, IV) proved futile as evidenced by very close Rf of substances on a thin layer plate. No further investigations were carried out with these fractions. ii) The pH 6.0 fraction (2.3? g.) was chromatographed over neutral alumina (activity III, ?0 g.) and the following fractions were collected (Table 7). 1 4 6 ♦

Table 7

Fraction Flasks - Eluant Residue 25 ml, each

1 1-6 benzene - 1.10 g ,. mixture methylene chloride 1: 1 2 7-10 methylene 0,50 g .. mixture chloride 3 11-15 methanol 0,30 g ,. mixture

Thin layer chromatography on alumina, using chloro form - methanol (1:1) was the basis for combining of frac tio n s . C haracterization of fra c tio n 1. This fra c tio n showed only two spots on thin layer chromatography. The residue obtained after, removal of the solvent was scratched in pre sence of a little acetone with a glass rod whereupon crystals deposited (0,020 g.)-, m,p, 207-208°, Further chromatography of the mother liquor on neutral alumina (activity III, 10 g.) afforded 0,000 g, of XXVT as colorless crystals, m.p, 207°. The total amount of crystalline material was 0,020 g. Infrared spectrum: 1761 cm"^ (carbonyl group;, 1O0$cm"1, 759cm"% 753cm"^ and 7^1 . +2,2 (c I.Olf), U ltra v io le t spectrum: 22^-mju (log6 l+,6^) and 277myu (lo g e 1+, 10), Anal, found: G, 70,26; H, 7.60; ÏÏ, 7.51. 1 ^ 7

The elemental analysis corresponds to the molecular formula C21H26 O3N2 (C, 70.1?; H, 7.6?; N, 8.18). None of. the fractions 2 - 3 afforded any crystalline material even on further chromatography on neutral alumina (activity I, II and III). iii) The pH ?.0 fraction (^+.11 g.) was chromatographed over neutral alumina (activity III, 100 g.) and the fol- lovd.ng fra c tio n s were collected (Table 8). Table 8

Fraction Flasks - Eluant Residue .2? ml. each

1 1-lf benzene - 0.8 g., mixture methylene chloride 1:1 2 ?-6 benzene - 1 *3 s* , mixture methylene chloride 1:2 3 7-iS methylene 1.0 g. , mixture chloride

9-10 methanol 0.3 g. , mixture

Thin layer chromatography on alumina, using chloro form - methanol (1:1) was the basis for combining of frac tio n s. None of these.fractions 1 - !+ afforded any crystalline material on repeated chromatography on neutral alumina (ac tivity I, II and IV). These fractions were not further in vestigated. i4a

As the fraction C showed the same spots ( R f 0.19,

0.78 and 0 .97) as those in the fraction B on thin layer chromatography on alumina, using ethyl acetate - ethanol as the developing solvent (1:2) no further work was carried out with it at the present time. APPENDIX I

INFRARED SPECTRA

149 1?0

CM' 4000 3000 2000 1500 1000 900 QOO 700 10O 0.0

Î60- i40-

20-

4000 3000 ZQfX) 1500 0.0

'60

i40

2 0 0 0 1500 iqoo 990 890 •00

i40

2 0 151

1500 ■il2QP0 III I IQOO 990 8Q0

|60

140

4000 3Q00 2QQ0 tSPQ IQOO IQQ. IIMil l U I I > Jk ■00

!60 i40

2 0

CM' 1500 1000 yoo 800 700 0.0

we 1.0 . 152

CM' 2000 1500 1000 900I 800 700 0.0

20 -

- 1.0

4 0 0 0 3 0 0 0 2 000 1500 [00-M"“ llll‘“ . •0.0

160 -2m i40 Î

% ' 6 S ' f ' 6 ^ ' lb ' WAVELENGTH (MICRONS)

4000, 3000 2000 1500 1000 900 800 700 IQQ. IIMUiU I I I I 0.0

-.35

- 1.0 153

CM' 4000 3000 2000 1500 1000 900 800 700 100 I-O.O

80-

60-

2 0 -

- 1.5

WAVELENGTH (MICRONS)

I5P0 800 _J700 ^0,0

-10

4000 3000 20 0 0

160

i40- •

. tSpO 990 890

^ ‘ ^ ' V aU ng A ici^ s) '■' & k ' k ' ' ^

^«»o.3qoq,,:..ay°. . iy°. . 19 °°. 89° . 89° . 7ço

CXLIX

’ ' ‘ ^ ^ ■WiiNOTH^aAs» :'' ■ i CM** ,j KP03%o , a y o . . Kpo . IQOO. 990 890 790 QP

V*—

i S ~ T 1??

400 0 3 0 0 0 2000 1500 100 00

i40

CiUI 'I'"' i.'" k ‘ |" ^ 4 - ' ' WAVELENGTH (MICROflS) CM-* 2000 1500 IQOO

160

2000 00

i40

20 :10

WAVE! 156

2000 1500 ■00

160-

:NGTH (MlCRl

4 0 0 0 5 0 0 0 2000 1500 IQQ. llllilliilu .il 0.0

160-

NC- 20 NC-

WAVELENGTH (MICROS) CM' 2000 1500 800 0.0

(60- -.21 .32 140-

20 157

4000 3U0Q 1000 900 100

_ Il II » J) 1 ko.

^wave Ængth’ imicr ^ ns) ” CM-' 1004000 3CpO , 2000. IQOO 990 890 790

à\~ XIK

^ ^ ‘ * W N G m L c R & a ''' ' ^ ' li* ■ > CM-' ,xm .3Ç p9,,...w .. .lyo IQOO 990 æ o 79a 158

2 000 1500

140

*.g=n

CM*' 4000 3000 lA- niiugji.Li.fcA rOO

160

140- Î 20-

6" ' ' ' ' & ' b ' jb ' WAVEŒNGTH (MICRONS)

160

140- -4 z APPENDIX I I

ULTRAVIOLET SPECTRA

1?9 160

0.0 0.1 0.2 0 .3 0 .4 0 .5 UO.6 ^0.7 SO-8 < 0 .9 1.0

CXXIII 1.4

300 390 V'AVaEMGTH ‘MIIUMICRONSI a b s o r b a n c e a b s o r b a n c e

fO eno

à B

À g I w I

w w ë

w U9

ON ABSORBANCE ABSORBANCE r P P P P P P P P P P CO ro — O 'O 00 N1 O' Oi ^ C O KO —• o o

(O TO o

ro g

o O

o\ ro ABSORBANCE Oi w ro o

fO OCn

; 5

l § o»‘

g o

ôt

o\ U> a b s o r b a n c e ABSORBANCE

i î I é I ï I I ço 5 5 s I

w OLn

"OW a b s o r b a k œ ABSORBANCE

? o

: w

Ut g-

o\ vn a b s o r b a n c e ABSORBANCE

I § I X ? g g - OCO

w o

o\ o\ ABSORBANCE ABSORBANCE Oi o

Kl fO o

(nK l Ch i i I § I w I

q Oiw

w W S

o\ ABSORBANŒ ABSORBANCE

to o

0=0

lO K> o OCn

z z i

w I O O

w O» s o

-sw o\ 03 ABSORBANCE ABSORBANCE — p p p p p P p p p p o o k) 03 N O' ü: — O

'O c

o\ vo ABSORBANŒ o o

8 o

0\fO

Ui o

o ABSORBANCE

o - o

N) Oi

I w APPENDIX I I I

NUCLEAR MAGNETIC RESONANCE

172 173

TT1S 17^