CHEMICAL AND SPECTRAL STUDIES IN MODIFIED STEROIDS

DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE OF

fS^^ttx of ^U^iovHip IN

TO THE ALIQARH MUSUM UNIVERSITY, ALIOARH

SUHAIL AHMAD

DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY

AUGARH (INDIA)

1986 ^ ^

DS899

* w -»rTc^A^ ? •-.^^^UM^UHixtr-^*

Tel : 5515

mmxh MwiUm Winiiytxsitp ALIGARH 202001 U. P. INDIA

•Ixeac'er

S t e r i o J R e r a t o r u

of CUmiftT-Li H) cite 15 , January , 1987.

The d i s s e r t a t i o n e n t i t l e d 'Chemical and

S p e c t r a l s t u d i e s *in modif ied s t e r o i d s by

Mr, Suha i l Ahmad i s s u i t a b l e f o r t h e degree of

Mas te r of Ph i losophy i n Chemist ry .

( Dr. Shafiiillah ) ^ ^

ACKNOWLEDGEMENT

I am very grateful to Prof.M.Shahabuddin Ahmad,

Chairman, Department of Chemistry, for h\3 valuable

guidance and providing necessary research facilities.

I am also thankful to Dr. Shafixillah, for his generous

help and constant encouragement.

I would like to exr)ress my thankfulness to my

research colleagues and friends for their cooperation.

( Suhail Ahmad )

CONTENTS

Syntheses of Steroidal Tetrazoles Pa^e

Introduction 1

Theoretical 3

Discussion 19

Experimental 33

References 43

INTRODUCTION

Steroids are widely distributed in nature and play

an important role in the vital activity of living organisms.

They constitute a family of the substances critically important

to plant and animal life. The discovery of the cholestrol, the

first steroidal compound to be known, was reported as early as

1812 by M.Eo Cherve\il, the dramatic expansion of steroidal

Chemistry came with the discovery of sex hormones in 1929-35.

Steroids include the ademal cortical hormones, the sex hormones,

some of the vitamines, plants and animal sterols.

In recent past, the chemistry of steroids has provided

one of the most interesting research area for organic chemists.

The first phase steroidal research was mainly concerned with the

isolation of steroids from natural sources, their structural

elucidation and bio-chemical studies. These researches resiilted

in the realization of the facts that naturally occuring oxa -

and azasteroids, such as steroidal alkaloids are endowed with

prono\mced and specific biological activities. This prompted

the organic chemists to undertake synthetic modifications of

natural steroids to enhance selectivity in certain parameters of

their biological activity. Synthesis oi the new steroidal

analogues and their pharmacological testing have now become a

- 2 -

major preoccupation with organic chemists and continue to

fascinate them the world over.

Our laboratory, concerned mainly with the synthesis of

steroidal compounds and their identification and characteriza-

tion by chemical and spectral studies. The characterization of

the compoimds has been done by chemical as well as the modem

instrumental methods. The sjmthesis of a large number of oxa-

and azasteroids, mainly from cholestane and stigmastane series,

has been reported. Beckmann rearrangement, schmidt reaction

and Baeyer villiger oxidation have been extensively used for

these syntheses.

In the present work an attempt has been made for

tetrazoles synthesis from steroidal olefins and epoxide^

THEORETICAL

The doubly unsaturated five raembered ring system with

four nitrogen and one carbon atom are Icnovm as tetrazoles.

aiadin ' reported the first tetrazole (I) formed

by the treatment of dicyanophenyl hydrazine with nitrous

acid. Tetrazoles have found various biological as well as

nonbiological applications. , Pentaraethylene tetrazole (II)

is a potent stimulant of the central nervous system and is

used clinically to counter act intoxication due to over

dosage of barbiturates .

NO G=N

(I) ( II )

Stimulant and depressant effects are exhibited by

disubstituted tetrazoles carrying alkyl, cycloalkyl, aryl,

4-7 amino and amide groups; Analgesic and sedative activities o

are shown by 5-mono-substituted tetrazoles . Anticonvulsant

activity in mice is exhibited by l-(m-aminophenyl)-5-ethyl-

tetrazole ( I I I ) , Hypotensive activity is found in 5-(2-

dimethylaminoethoxymethyl)-l-phenyltetrazole (IV), the ef"^ect Q

being directly on cardiac and vascular muscle . Various

- 4 -

biological activities such as antiallergic, anticonvul-

sent, antiulcer, antibacterial, antiviral, anti-

fungal, antiinflammatory, ^ analgesic, and antihyper-

tensive have recently been reported for tetrazoles, Tetra-

zoles have been applied in explosives and in propellants.

17 Tetracene has been used as an initiator. Applications in

photography * have also been reported.

CH.

Ht-C I ' '"a> n.. "-T'

( I I I )

( IV )

Tetrazoles were formed by ( i ) addition of hydrazoic

acid to the compoxindshaving carbon-nitrogen unsaturation,

such as n i t r i l e s , cyanates, thiocyanates, isocyanates, carbo-

mides and isothiocyanates ( i i ) displacement reaction between

imide chloride and hydrazoic acid ( i i i ) hydrazine-ni tr i te

reactions (iv)) acylhydrazine diazonium reaction, (v) hydra-

zone-azide reaction, (vi) rearrangments and (v i i ) reaction of

ketones with hydrazoic acid.

- 5 -

Has«ner e't a l ^^ reported one of the most

valuable method for the preparat ion of 1 ,5 -d i subs t i tu t ed

t e t r a z o l e s i s the reac t ion "between compoiind having a t l e a s t

one carbon-carbon double bond with halogen and sodium

azide using n i t r i l e as a solvent , and proposed the follow-

ing mechanism.

(X = halogen, R = n i t r i l e )

Xo

( V )

x\x I^ NaN,

]Sr=C—R +

OH

21

( VI ) ( VII )

L e s t e r e t a l r e p o r t e d t h e format ion of 1 , 5 - d i s u b -

s t i t u t e d t e t r a z o l e from n i t r i l i u m s a l t and sodium az ide

and proposed the fo l lowing mechanism.

- 6 -

[R'-d=N-R] BF " + NaN,

(VIII)

-• [R'-G = N-R] N" + NaBF.

^ ^'-if - -N-R N N̂

(IX)

22 Barnes et al were the first to report the formation

of tetrazoles in steroid and triterpenoid systems. Treatment

of 7,ll-dioxolano3t-8-en-3p-yl acetate (X) with hydrazoic acid

yielded a tetrazole (Vl^a or VI-b) in addition to two isomeric

monolactams (XII and XIII).

AcO

( X ) ( XIa ) ( Xlb)

^y

- 7 -

B i o l o g i c a l l y a c t i v e s t e r o i d a l t e t r a z o l e s were

r e p o r t e d by Mechoiilam. When 5 a - o h o l e s t a n - 3 - o n e (XIV)

and 17p-hydroxy-5a -andros t an -3 -one (XV) were s u b j e c t e d t o

Schmidt r e a c t i o n , i somer ic t e t r a z o l e s (XVI - XIX) were

o b t a i n e d .

R

^V^ 'cfX^r^Xy

(XIV)

(XV)

R

^8^17

OH

(XVI)

(XVIIi)

R

^A7 OH

a

(XVII)

(XIX)

R

°8^17

OH

Cervantes e t a l r e p o r t e d the fo rmat ion of r i n g - D

fused t e t r a z o l e (XXI) from t h e r e a c t i o n of 17-ketoxime(XX)

wi th an excess of sodium az ide i n t h e p resence of HpSO..

The fozmation of n l t r i l * ' ^ X X I I ) and lac tam (XXIII) were a l so

r e p o r t e d .

- • 8 -

^2^°4 H^CO

' ( XX ) ( XXI ) (XXII),.^ +

( XXIII )

Singh e t a l ' have r e p o r t e d t h a t a n d r o s t - 4 - e n e - 3 ,

17-dione (XXIV) on t r e a t m e n t w i th excess of hydrazo ic a c i d -

B F , - e t h e r a t e i n chloroform y i e l d e d t h e expected 3 , 1 7 a - d i a z a -

A,D-bishomoandrost -4a-eno [ 3 , 4 - d ] [ l 7 a , 1 7 - d ] b i s t e t r a z o l e

(XXV) and an unusua l p r o d u c t , 13 ,17-seco-13a-az ido-A-homo-

a n d r o s t - 4 a - e n o [ 3 , 4 - d ] t e t r a z o l - 1 7 - n i t r i l e (XXVI). The a z i d o -

n i t r i l e c y c l i z e d on hea t ing t o g ive (XXIX).

(XXIV) (XXV) (XXVI)

- 9 -

To account f o r t h i s novel c leavage of 17-oxo-

s t e r o i d (XXIV) t o az ido n i t r i l e (XXVIII) and i t s s u b s e -

quent cyGliza"cion t o t e t r a z o l e (XXIX) t h e fo l lowing mech-

anism was proposed .

N — N S N

(XXIV)

= N

'NiatN

-H +

|^'"^vx^^ (m The rn a l

\ \ ^ ^ ' \ ^ G y l i z a t i o n

(XXVIII)

( XXIX )

10 -

When 3p-acetoxypregn-5-ene-7,20-dione (XXX) was

t r e a t e d with hydrazoic acid-BF^ e the ra t e in chloroform 27 yielded 17p-(5-niethyltetrazole-l-yl)-7a-aza-B-hoinoandro3t

-5-eno[7a ,7-d] t e t r azo l -5P -o l (XXXI) as major product f o l l -

owed by other t e t r a z o l e s (XXXII - XXXIV) .

OCH.

AcO HO

(XXX) (XXXI)

IHAc

(XXXIY) OGĤ (XXyjI)

(XXXIII)

- 11 -28 Ring-G fused t e t r a z o l e (XXXVI) was r e p o r t e d

thro\igh a s i m i l a r r e a c t i o n from (XXXV). Various s t e r -

o i d a l t e t r a z o l e s have been r e p o r t e d »-̂ t o be formed

by the r e a c t i o n of excess of h y d r a z o i c ac id w i th 6-oxo

and 7-0X0 s t e r o i d a l compounds. In most of t h e cases

l ac t ams have b«en r e p o r t e d as one of t h e p r o d u c t s .

AcO H

(xx:iV) H (XXXVI)

I r i s m e t o v e t al ' ' '^ r e p o r t e d t h a t Schmidt r e a c t i o n of

5a and 5 p - s p i r o s t a n o n e s (XXXVII) gave t h e l a c t a m s (XXXVIII)

and t e t r a z o l o s p i r o s t a n s (XXXIX).

N—N

N—N

( XXXVII ) ( XXXVIII ) ( XXXIX )

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DISCUSSION

Steroidal tetrazoles have become of interest in

recent past, because of the discovery of biological

activity associated with a number of tetrazoles and also

because of their uses as potential drugs, AS a result

of this realization, synthesis of steroidal tetrazoles

become a matter of interest and consequently a n\imber of

papers appeared describing the preparation of tetrazoles

from various steroidal ketones.

The present work describes the preparation of

tetrazoles from steroidal olefins such as cholest-5-ene

(LVII), 3p-chlorocholest-5-ene (LVIII), 3p-acetoxycholest-5-

ene and--S"&eroidal et)oxide such as •3p-acetoxy-5,6a-eTooxy-

5a-cholestane(LXVI).

QeHiT

AcO

(LVII)

(LVIII)

(LIX)

i t

H

01

OAc

O-'

(LXVI)

- 20 -

A.Reaction of chole8t-5-ene (LYII) with bromine«acetoni t r i le

arid sodium azide - AlOl^

Gholest-5-ene (LVII) was t r ea t ed with bromine in the

presence of anhydrous AlCl, (as a t rap ing agent ) , using

a c e t o n i t r i l e as solvent and to t h i s so lu t ion sodium azide

was added. After the usual wrok up of the r eac t ion mixtiore

and column chromatography over s i l i c a ge l , provided the com-

pound m.p. 172° and 156°C respec t ive ly .

CpH 8^17

^8^17

(LVII)

Charac ter iza t ion of the compound m.p. 172" as 3--bromo-6tj-

acetylamino-3a-cholestage (LX )

The compound m.p. 172° was analysed for C q̂Ht-QNOBr

(pos i t i ve Be i l s t ieln t e s t ) . The I .R. spectrum showed bands

at 3420(-NH), 1670(-C-NH), 730cm"-'-(C-Br). I t s n .m. r . spectrum

exhibi ted a broad s ing le t at 6 3.2 (IH, Wi-=4.5Hz; e q u i t o r i a l )

- 21 -

and was a s s i g n e d t o C6-aH. A sha rp s i n g l e t a t 6 2 .1 f o r

CH,-C-NH and a broad s i n g l e t f o r NH were o b t a i n e d a t 6 5 .8 .

The a n g u l a r methyl p ro tons were observed a t 6 1.2(C10-CH,),

0.72(C13-CH^), 0 . 9 and 0 .85 ( f o r remaining methyl p r o t o n s ) .

On t h e b a s i s of t h e s e ev idences t h e compound (LX) was c h a r a -

c t e r i z e d as 5 -b romo-6p-ace ty l2amino-5a -cho le s t ane .

C h a r a c t e r i z a t i o n of t h e compo-und. m.p. 156^ as 5a--bromocho-

l e s t a n - 6 B - ( l ' ) ~ 5 * " m e t h y l t e t r a z o l e ^(LXI>

The compound, m.p, 156 was ana lysed c o r r e c t l y fo r

CpqH.QN^Br. I t s I . E . spectrum e x h i b i t e d peaks a t 1530,1460

and 1580cm""' '(C^, Vi=^)^ *^ i n d i c a t i n g t h e p re sence of t e t r a -

z o l e moiety i n t h e compound, and 740cm~ (C-Br ) , p o s i t i v e

B i e l s t i e n t e s t confirms t h e p resence of bromine . The n . m . r .

spectrum e x h i b i t e d a broad s i n g l e t a t 6 3 . 1 (Wl=5Hz) i n t e g r a -

t i n g f o r one p r o t o n , was a s s i g n e d t o C6-offl,. A sharp s i n g l e t

a t 6 2 ,5 i n t e g r a t i n g f o r t h r e e p ro tons a s c r i b a b l e t o methyl

(-N-C=Jf-) group a t t a c h e d to t h e t e t r a z o l e moie ty was o b t a i n e d .

GH -3 The a n g u l a r methyl p ro tons were observed a t 6 1.02

(C10-CH-), 0.7(C13-CH^), 0 . 9 1 and 0 . 8 1 ( f o r remaining methyl

protons ) . On t h e b a s i s of above d i s c u s s i o n t h e compound (LXJ)

was c h a r a c t e r i z e d as 5 a - b r o m o c h o l e s t a n - 6 p ( l ' ) - 5 * - m e t h y l t e t r a -

z o l e .

- 22 -

The formation of the compoimd (LXl)was also explained on the

ba3is of reaction mechanism given in sbheme ~ I.

Scheme-I

qsHiv

N N

( LXf)

- 2-5 '

Reaction of 3B-chlorochole3t~5-ene (LYIII) with bromine«

a c e t o n i t r i l e and sodium azide-AlQl-r

3p-chlorocholest-5-ene (LVIII) was t r e a t e d with bromine

in the presence of anhydrous AlCl, (as t rap ing agent) using

a c e t o n i t r i l e as a so lvent , to t h i s so lu t ion sodium azide was

added. After usual work up of the reac t ion mixture and column

chromatography over s i l i c a ge l , two o i ly compounds were obtained

^,8%7

(LVIII)

^8^17

/

It

(

- c ^ W N

LXIII

2!H'7

)

(LXII)

- 24 -

Gharacter iza t ion of the o i l y coapound as 5B~chloro-5-bromo-

6B-'acetylanilno~3tt~cholestane (LXII)

The o i l y compound (LXII) was analysed for Q^gE.JSOBrOl

(pos i t i ve B i e l s t i e n t e s t ) . I t s I .R, spectrum showed bands at

3410(irH), 1660(amide), 740, 715cm"-'- (C-Cl, C-Br). I t s n .m. r .

spectrum showed a broad peak at 6 3.76 (W^ = 4.5 Hz; e q u a t o r i a l ) ,

integ-rating for one proton assigned to (C6-o^) and a broad

mul t ip le t a t 4.41 (W^ = 16 Hz, a x i a l ) , a s sc r ibab le to (C3-aH).

A sharp s i n g l e t a t 6 2.01 and a broad s i n g l e t a t 5.8 were

in teg ra ted for th ree methyl protons and one amide (H^

proton, the angular methyl protons appeared at 1.26(C10-CH,),

0.68(C13-CH,), '0.9 and 0.83 ( fo r remaining methyl p ro tons ) .

On the bas is of above evidences the cortpoimd (LXII) was chara-

c te r ized as 3p-ohloro-5~bromo-6p-acetylamino--5a-cholestane.

Oharacter iza t ion of the o i ly compound as 36-chloro-5a-bromo-

choles tane-6B( l ' ) -5 ' -methy l t e t r a z o l e (LXIII)

The o i l y compotind showed the molecular composition

CpoH.gKT.BrCl ( p o s i t i v e B e i l s t e i n t e s t ) . I t s I .R. spectrum

exhibi ted bands at 1525, 1460, 1375(0=^, N=N) ind ica t ing the

presence of t e t r a z o l e moiety in the compound and 740, 720cm~

for carbon halagen bonds (C-Cl, C-Br). The n .m. r . spectrum

- 25 -

exhibi ted a broad s igna l at 6 3.95 (W^ = l6Hz ; a x i a l ) ,

i n t eg ra t ing for one proton was ascr ibable to C3-aH, a x i a l ) .

A broad s ing le t a t 6 3.6 (W-̂ = 4Hz) i n t eg ra t i ng fo r one proton

was assigned to C6-aH. A sharp s ing le t appeared at 6 2.52

i n t e g r a t i n g for th ree protons asc r ibab le t o (-N-C=N-) methyl

protons . The angiilar methyl protons were observed at 6 1.2

(ClO-GH,), 0.7(C13-CH_), 0.93 and 0.83 ( fo r remaining methyl

protonsK On the bas i s of forgoing f a c t s the compound (LXIII)

confirmed as 3p-chloro-5a-bromocholestan-6p(I»)-5 '-methyl

t e t r a z o l e .

Reaction of 36~acetoxychole3t~5-ehe (LIX) with bromine, ace to -

n i t r i l e and sodixun azide-AlOl-?

3p-acetoxycholest-5-ene (LIX) when t r e a t e d with bromine

and sodium azide in the presence of anhydrous A1C1-, (as trapin^

agent) , using a c e t o n i t r i l e as solvent afforded two compotinds

(LXV), m.p. 178° 0 and (LXlV)m.p. 159°C.

- 26

3

(LIX) (LXIV) (LXV)

C h a r a c t e r i z a t i o n of t h e compoiHid m.p. 159 0 as 3-bromo-6t3-

a c e t . y l a m i n o - 5 a - c h o l e s t - 3 - e n e (LXIV)

-1

The compound m.p. 159 was showing molecular compositi-

on as CpgH^gNOBr (positive Bellstai^n test). Its I.R, spectrum

exhibited bands at 3430(NH), 1670(amide), 1625(C=C) and 730cm

(C-Br), Its n.m.r. spectrum showed a broad band at 6 3.2

(Ŵ - = 5Hz; equatorial), integrating for one proton was assigned

to C6-aH. A broad multiplet between 5.5-5.41 integrating for

two protons was assigned to C3 and C4 vinylic protons. A sharp

singlet at 6 2.1 integrated for three methyl protons (CH^-C-FH)

and one amide proton as a broad singlet at 6 6.2CCH2G-NH;

exchangeable with D ). The angular methyl protons were

appeared at 6 1.2 (CIO-CH3), 0.7 (CI3-GH3), 0.92 and 0.8 (for

- 27 -

remaining methyl p ro tons ) . On the bas i s of above ana ly t i c a l

and s p e c t r a l evidences the compound (LXIV) was character ized

as 5-bromo-6p-acetylamino-5a-cholest-3-ene.

Charac ter iza t ion of the compoimd m.p. 178° as 3a-bromochole3t-

3-en-6B(I ' )~5 '-methyl t e t r a z o l e (LXV)

The compotind m.p. 178° was analysed for CpqH.JT.Br

(pos i t i ve B i e l s t i e n t e s t ) . I t s I .R . spectrum exhibi ted bands

a t 1520, 1465, 1580 (C=^, N=^), 1620 (C=C), 750cm~-̂ ( C-Br).

In i t s n .m. r . spectrum a broad s ing le t at 6 3.06(¥-i-=4.5Hz;

e q u a t o r i a l ) , i n t eg ra t ing fo r one proton was assinged to C6-aH

therefore t e t r a z o l e moiety at tached to C-6 was p-or ien ted . A

broad mul t ip le t between 6 5.53-5.45 in t eg ra t i ng for two protons

was assigned to C3 and 04 v iny l i c protons , A sharp s ing l e t

at 6 2.56 in t eg ra t ing fo r th ree protons was ascr ibable to

methyl protons (-ir-C=iN-) of t e t r a z o l e moiety. The angular

methyl protons were appeared at 6 1.15 (ClO-CH^), 0 .7l (C13-

CH,), 0.91 and 0.83 ( for reamining methyl p ro tons ) . On the

bas i s of above ana ly t i c a l and spec t r a l evdiences the compotmd

(LXV) was charac ter ized as 5a -b romocho les t -3 -en -6p- ( I ' ) -5 ' -

methyl t e t r a z o l e .

- 2B -

B. Reaction of 56-acetoxy-5.6g--epoxy-5a-chole3tane ( LXVI )

with a o e t o n i t r i l e , sodiiun azide -• AlCl^

When 3p-acetoxy-5,6a-epoxy-5a-cholestane( LXVI ) when

t r ea t ed with a c e t o n i t r i l e sodiumazide-AlCl^ (as c a t a l y s t )

provided th ree compounds m.p. l80° , 198° and 132°c.

^ 8 % 7

( LXVI )

N (LXIX)

Characterization of the compound m,p,180°as 3B-acetoxy-3-

hydroxy-5a-cholestan--6-one(LXVIl2

The compound m.p.iso^ was analysed correctly for

- 29 -

O^QB-AQOA' 1*3 1,R» spectrum exhibi ted bands at 3375(0H),

1735 (CH^-C-), 1700(-C-) and 1250cm"-^ (C-0) . The n .m. r .

spectrum of the compound exhibi ted a mul t ip le t centered at

6 5.16 which was assingned to C3-aH (Wi = 16 Hz)"^^. A

sharp s i n g l e t appeared at 6 2.01 in t eg ra t i ng for th ree

protons for CH^-C-0- . OH proton appeared at 6 1.6 , exch-

angeable with deuterium. The angular methyl protons appeared

at 6 1.2 (ClO-CH^), 0.7l(C13-CH^), 0.91 and 0.82 ( f o r remai-

ning methyl p ro tons ) . On the bas i s of above discussion the

compound m.pl80° was character ized at 3p-acetoxy-5-hydroxy-

5a-cholestan-6-one CLXVII )

Character iza t ion of the compotind m.p. 198 as 33-acetoxy-5-

hydroxy-63~acetylamino-5a-cholestane(LXVIII )

The compound m.p. 198*^ C was analysed for G^nHc-rNO..

The I .R. spectrum showed bands at 3450(OH), 3400(NH),

1715(CH,-C-0-), l655cm~-'' (amide). The n .m. r . spectrum of

the compound exhibi ted a mul t ip le t centred at 6 5.2 which was

asstoged to G3-aH (Wi = 16 Hz)^^. A broad s ing l e t appeared

at 6 3.54 in t eg ra t i ng for one protons fo r C6-aH(Wi=3Hz) and

a sharp s i n g l e t at 6 2.01 i n t e g r a t i n g for s ix protons fo r

- 30 -

CH^-C-0- and -NH-C-CH^. The OH proton appeared at 6 1.46

and the NH proton at 6 5.8 both were exchangeable with

deuterium. The angtilar and s ide chain methyl T)rotons appe-

ared at 6 1.2 (ClO-CH^) 6 0.71(C13-CH,), 0.91 a»d 0 .83 . On

the "basis of above evidences the compound (LXVIlt)wa3 chara-

c te r ized as 3p-acetoxy-5-hydroxy-6p-acetylamino-5a-cholestane

Charac te r iza t ion of the compound m.p. 132° as 3B-acetoxy-4-

aza-A-homochole3tan-4a-one-6B(I ')-5'-methyl t e t r a z o l e ( LXIX)

The compound m.p. 132 was analysed for 03iHc-iNc0.z.

The I .R. spectrum of the compound showed bands a t 3450(SH),

1720(^^000) , l 665 ( -g -0 - ) , 1525, 1470, 1380 (C=N, N=N)^'^'^^

which i nd i ca t e - the presence of t e t r a z o l e moiety. The n .m. r .

spectrum of the compound exh ib i t s a broad peak at 6 5.78 which

was assinged to (NH, exchangeable with deuterium), A broad

mti l t iplet centred at 6 5.15 was assinged to C3-a^ (W-2-=16 Hz)

and a s igna l at 6 4.1 for C6-aH(W-i-=5Hz). A sharp s ing le t

appeared at 6 2.1 i n t eg ra t i ng fo r s ix protons was assinged to

(a|,-COO and -N=C-N-). The angular and s ide chain methyl CH-T

protons appeared at 6 1.1 (ClO-CH^), 6 0.68(C13-CH^), 0.90 and

- 31 -

0 . 8 . On the b a s i s of above observat ions the compound

(LXIX) was character ized BL;J 3p-acetoxy-4--aza-A-homocholestan.-

4a-one-6p-( I ' )--5*-methyl t e t r a z o l e s .

The formation of t h i s compound (LXIX) was explained

by the following mechanism schem'e-II,

CgH-LY

(LXVI )

AcO

/ N GH N ,G^ ^ II // IT IT

/ II N

NaN3

H - S o l . AcO

/H, C

II N

- • 5 2 -

AcO AcO

/-o/°='' II

N

(IXIX)

Reactions of 5,6a-epoxy-5a-cholestane .and i t s 3P-chloro

and 3p-hydroxy-analogues with ace toni t r i le> sodiumazide-AlCl,

are tinder progress .

EX PERIMENTAL

All melting points are uncorrected I .R, spec t ra were

determined in KBr with a Perkin-Elmer P*?? infrared spec t ro -

photometer I .R. values are given in cm? N.M.R. sriectra were

run in CDCl̂ on a varian A60D instrument with Me.Si as the

i n t e r n a l standard and i t s values are given ppm (6) ( s , s i n g l e t ;

b r , broad and mc, mul t ip le t centred a t ) . Thin l a y e r chromato-

graphic (TLC) p l a t e s were coated with s i l i c a gel and sprayed

with 20 % aqueous so lu t ion of perch lor ic acid . S i l i c a gel

( '^ 20 gm) was uaed for each gram of the mater ia l to be separa-

ted in coltimn chromatograhy. Petroleum ether r e f e r s to a f r ac -

t ion of b . p . 60-80 . Soditim sulphate( anhydrous) was sued as

drying agent .

36-0hlorocholest~3-ene(LVIII)

Freshly pur i f ied th ionyl chlor ide (75 ml) was added

gradual ly to cho le s t ro l (100 gm)at room temperature. A

vigorous reac t ion ensued with the evolution of gaseo^is

products . When the reac t ion slackened, the mixture was gently

heated at a temperature of 50-60° on a water bath for 1 hr ,

and then poured onto crushed ice-water mixture with s t i r r i n g .

- 34 -

The yellow so l id thus obtained was f i l t e r e d under suct ion and

washed severa l times with ice cooled water and a i r d r ied . Re-

c r y s t a l l i z a t i o n of crude product from acetone gave 3p-chloro-

cholest-5-ene (LVIII) (95.5 gm), m.p. 96° (reported^^ 96-97°) .

I t gave pos i t i ve B e i l s t e i a t e s t .

Chole3t-5--ene (LVII)

3p-chlorocholest -5-ene (15.0 gm) was dissolved in warm

a«.yl alcohol (300ml) and sodium metal (35.0 gm) was added in

small por t ions t o the so lu t ion with continuous s t i r r i n g over

a period of e ight hours. The reac t ion mixture was heated now

and then during the course of reac t ion in order to keep the

sodium metal d issolved. The reac t ion mixture was poured in to

water, a c id i f i ed with HCl and allowed""to Staad overnight . A

white c r y s t a l l i n e so l id was obtained which was f i l t e r e d under

suct ion and washed throughly with water and a i r d r ied , Recryst-

a l l i z a t i o n of the crude mater ia l from acetone gave choles t -5-eae

(LVII) in cubes (10.6 gm), m.p. 93° (reported^^ m.p. 89 .5-91.2°) .

Reaction of chole3t-5-ene with bromine, a c e t o n i t r i l e and sodium

azide-AlQl^ ; 5-bromo-63-acetyl~amino-5a-cholestaiae (LX ) . 5a-

bigomocholestan-6e(l ')-5'"methyl t e t r a z o l e (LXI)

To a s t i r r e d so lu t ion of choles t -5-ene (LVII) (3 .0 gm) i»

- 35 -

acetonitrile (40 ml) was added aiihydrous AlCl, (3.5 gm),

bromine solution (0.8 gm) and sodium azide (2,0 gm). The

reaction mixture was fuarbher stirred for 7 hrs. and extracted

with chloroform. The chloroform layer was washed with water

and dried over sodium sulphate anhydrous. Removal of the

solvent \uider reduced pressured provided a viscous residue,

which was chromatographed over the column of silica gel.

Elution with petroletim ether : ether (5:1) afforeded a solid

compound (LX ) which,was recrystallized from acetone (0,8 gm)

m,p.l72°.

Analysis Pound : 0̂ 68,4 ; H, 9,9 ; N, 2.7

C2gH^0^0Br requires: C,68.^0;H, 9.84; N, 2.75 Z

^•S' • -s) ,nô 3420(NH), I670(amide), 730cm"-'-(C-Br).

-̂ H-NMH : 6 3.2 b r , s ( l H , C6-aH; Wi 4.5 Hz), 2,1 s(3H, -NH-G-GH^)

5.8 br ,s(-NH-, D exchangeable), 1.2(C10-CH™) 0.9

and 0.85 (remaining s ide chain methyl p ro tons ) .

Fur ther e lu t ion with petroleum e ther : e ther (3:1) yielded

5a-bromocholes tan-6p( l ' ) -5 ' -methyl t e t r a z o l e (LXI) R e c r y s t a l l i z -

ed from acetone (1,5 gm), m,p. i569

Analysis found : C, 65.3; H, 9,0; N, 10.4

G2gH4gN^Br requi res : G, 65,29; H, 9,19; N, 10,50 7. .

file:///uider

- 36 -

I«R- I S) m^^ 1530(0=^), 1460 and 1380 (N=N), and 740cni"^ maz. (C-Br).

I "3 •'-H-NMR : 6 3.1 br , s(lH, C6-aH, Wi=SHz), 2.5 s(3H, -N-C=N-),

1.02 (G10-Ca_), 0.7(C13-CH^), 0.91 and 0.81 (remaining

methyl p ro tons ) .

Reaction of 33-Qh.lorochole3t-3-3ne with bromine, a c e t o n i t r i l e

and aodlum azide-A101-^;3B--chloro-5~bromo-6g~acetylamino-5a~

cholestane (LXII ) 3B-chloro-5a-bromocholestan-66(l ' ) -5 ' -methyl

t e t r a z o l e ( L X I J H

To a well s t i r r e d so lu t ion of 3p-chlorocholest-5-ene(LVIII)

(3.0 gm) in a c e t o n i t r i l e (40 ml) was added anhydrous AlGl^(3.5gm),

bromine so lu t ion (0 .8 gm) and sodium azide (2.0 gm). The

reac t ion mixture was fu r the r s t i r r e d for 7 h r s . and worked up in

chloroform. The chloroform l a y e r was washed with water and dried

over anhydrous sodium su lpha te . Removal of solvent tinder reduced

pressure provided a viscous res idue , which was chromatographed

over the column of s i l i c a ge l , Blution with petroleum ether :

e the r (4:1) afforded 3p-chloro-5-bromo-6p-acetylamino-5oc-chole_

s tane (LXII ) as a nonc rys t a l l i z ab l e o i l .

Analysis Found : C, 63.3 ; H, 9.1 ; N, 2.4

C2gH^9N0BrGl requ i res : G, 63.43 ; H, 9.04 ; N, 2.58 \

- 37 -

I . E . : -0 „ ^ 3410(NH), 1660(amide) , 740 and 715cm~^(C-Cl,C-Br).

•'•H-NMR: 6 4.41mClH, C3-oS, ¥i-=16Hz), 3.76 b r , s ( l H , C6-aH, Wi =

4 .5 Hz) , 5 .8 lDr,s(lH, -NH-C-), 2 .01 s(3H, -NH-8-CH,),

1.26 s(CIQ~GH,>,-0.6$ s(C13-GH_), 0 . 9 and 0 .83 ( remain ing

methyl p r o t o n s ) .

F u r t h e r e l u t i o n wi th pe t ro leum e t h e r : e t h e r ( 2 : 1 )

y i e l d e d 3 p - c h l o r o - 5 a - ' b r o m o c h o l e 3 t a n - 6 p ( l ' ) - 5 ' - m e t h y l t e t r a z o l e

(LXIII) ( 1 . 4 gm) as a n o n c r y s t a l l i z a b l e o i l .

A n a l y s i s found : C, 61 .2 ; H, 8.4 j N, 9 . 8

G^QE^^^BI:G1 r e q u i r e s : C , 61 .37 ; H,8 .57 ; N, 9 .88 */,

I . R . : ^ ^^^ 1525, 1460, 1375 (C=N, N=N), 740 and 720cm"^

(C-Cl , C-Br) .

•hi-NMR : 6 3.95 b r , s ( l H , C3-aH; Wi=l6Hz), 3 .6 b r , s ( l H , C6-aH;

Wi=4Hz), 2.52 s(3H, -N-C=N-), 1.2 s(C10-CH,) , 0 . 7 s

is, "̂ (C13-CH,), 0.93 and 0,83 (remaining methyl protons).

5B-Acetoxychole3t-5-ene (LIX)

A mixture of cholestrol (50 gm). Pyridine (75 ml) and

acetic anjiydride (50 ml) was heated on a water bath for two hrs.

The resulting brown solution was poured on to crushed ice-water

- 58 -

mixture with s t i r r i n g , A l i g h t brown sol id thus obtained was

f i l t e r e d under suction., washed with water and a i r d r i ed . The

crude product on r e c r y s t a l l i z a t i o n from acetone gave pure 3p-

acetoxycholest-5-ene (LIX) (45 gm), m.p. 1 1 4 - 1 1 5 ° ( l i t / " ' • l l 6 ° ) .

Reaction of 3B-acetoxychole3t-5-ene with bromine, a c e t o n i t r i l e

and sodium azide-AlOl-^ : 5-bromo-6B-acet.ylamino-5a~cholest~3-

ene (LXIY), 5a~bromocholest-3-en--6B^l')-5'-methyl tetrazole(LXV)

To a well s t i r r e d so lu t ion of 3p-acetoxychole3t-5-ene

(LIX) (3.0 gm) in a c e t o n i t r i l e (40 ml) was added anhydrous

AlCl, (3 .5 gm), bromine so lu t ion (0 ,8 gm) and sodium azide(2gm).

The reac t ion mixture was f u r t h e r s t i r r e d for 7 h r s . and worked

up in chloroform. The chlorofoinn l aye r was washed with water and

dr ied over anhydrous sodium su lpha te . Removal of the solvent

under reduced pressure provided a viscous res idue , which was

chromatographed over the column of s i l i c a g e l . Elut ion with

petroleiun e the r : e the r (3:1) afforded 5-bromo-6p-acetylamino-

5a-cholest -3-ene (LXIV) which was r e c r y s t a l l i z e d from acetone

(0 .7 gm), m.p. 159°

Analysis Found : G, 68.7 ; H, 9.4 ; N, 2.9

C2gH4QN0Br requ i res : G, 68.77 ; H, 9.46 ; N, 2.96 7,

I«a-s )̂ ™«̂ 3430(NH), 1670(amide)and 730cm"-'- (C-Br).

- 39 -

^H-NMR : 6 6.2 b r , s ( l H , NH-C-); 5 .5-5 .41 ,m(2H, C3-H, C4-H),

3 .2 b r , s ( l H , C6-aH ; Wi = 5Hz), 2 . 1 s(3H,CH^-C-NF-)

1.2 3(C10-CH,), 0 . 7 s(C13-CH^), 0 .92 and 0 . 8 ( remain ing

methyl p r o t o n s ) .

F u r t h e r e l u t i o n wi th only e t h e r p rovided 5a -b romocho les t -

3 - e i i - 6 p ( l ' ) - 5 ' - m e t h y l t e t r a z o l e (LXV) which was r e c r y s t a l l i z e d

from ace tone ( 1 . 4 gm) m«P« 178° .

Ana lys i s foiind : C, 65 .4 ; H, 8 .8 ; N, 1 0 . 4

O^^^rjE^Bx r e q u i r e s r C , 6 5 . 5 3 ; H, 8.85 ; N, 10 .54^

I«R» s S) n,o^ 1520, 1465, 1380 (C=N, N=N), 1620(C=C) and

730cm"-'-(C-Br).

•••H-NMR : 6 5 . 5 3 - 5 . 4 5 , m(2H, C3-H, C4-H); 3.06 b r , s ( lH ,C6-aH ;

Wi=4.5 Hz) , 2.56 s(3H, -N-C=^- ) , 1.15 s(C10-CH,) ,

- 3

0 . 7 1 s(C13-CH,) , 0 . 9 1 and 0 .83 ( remain ing methyl

p r o t o n s ) .

- 40 -

3B--acetoxy-5t6a--epoxy-3g-cholestane (LXVI)

3p-acetoxycholest-5-ene (10 gm) in chloroform (35 ml)

was t r e a t e d with perbenzoic acid (7 gm) and l e f t at -8° for

24 h r s . The mixture was then washed with ice-co ld sodium

"bicarbonate soution (5 7, ) , sodium th iosu lpha te solut ion ( 5 7 . )

and water. I t was dried over anhydrous sodium sulphate .

Removal of the solvent gave an o i ly residue which was recirys-

t a l l i z e d from petroleum e ther to provide ( 7 gm) m.p. 96

reported ( a , p . 97°) .

Reaction of 33-acetoxy-5,6g-epoxy-5g-cholestane (LXVI) with

a c e t o n i t r i l e , sodium azide-AlGl-r (anhydrous) and few ml of

e t h e r solvent ; 36-acetoxy-^-hydroxy->5g-chole8tane-6-one

(LXVII)^ 36-acetoxy-5-hydroxy-6B-acetylamino-3g-cholestane

(LXVIII) t 3g-acetoxy-4-aza-A-homochole3tan-4a-'On-63(I' ) ~ 5 ' -

methyl t e t r a z o l e (LXIX)

To a s t i r r e d so lu t ion of 3p-acetoxy-5,6a-epoxy-5a-chol-

estane (3.0 gm) in a c e t o n i t r i l e (40 ml) and e the r solvent

- 41 -

(10 ml) was added AlCl, anhydrous (3.5 gm) and sodium azide

(2,0 gm). The reaction mixture was further stirred for 1 hr.

and extracted with chloroform. The chloroform layer was washed

with water and dried over sodium sulphate anhydrous. Removal

of the solvent under reduced pressure provided a viscous residue

which was chromatographed over the column of silica gel. Elution

with petroleum ether : ethyl acetate (20:1) afforded solid

compound (LX7II) which was recrystallized from petroleum ether

(0.7 gm), m.p.l80°

Analysis found : C, 75.6 ; H, 10.5

C2QH4gO^ requires : C, 75.65; H, 10.45 7,

I'S. : S) „«^ 3375("0H), 1735(GH,COO), 1700(00), 1250cm"^(C-0).

0

-••H-NMR : 6 5.16, m(lH, G3-aH; Wi l6Hz); 2.01, s(3H, CH^-C-0-);

1.6, b r , s ( l H , OH; exchangeable with D ) ; 1.2 s(G10-CH,); 0 .71 ,

s(C13-CH,); 0.91 and 0.82 ( for remaining methyl p ro tons ) .

Fur ther e lu t ion with petroleum e the r : e thyl ace ta te

(4:1) yielded 3p-acetoxy-5-hydroxy-6p-acetylamino-5a-cholestane

(LX7III) r e c r y s t a l l i z e d from petroleum e ther (0 .5 gm) m.p.l98°C

- A2 -

Analysis foimd i 0, 73.8 ; H, 10.5 ; N, 2.7

°31^53^°4 requires : C, 73.95; H, 10.53; N, 2.78/f

I»S' 5 N) mo^ 3450(0H); 3400(NH); 1715(CH,COO); and 1655cm"^

(amide).

•"•H-NMR : 6 5.8, br,3(lH, NH; exchangeable with D );

5.2, m(lH, C3-aH; Wi l6Hz); 3.54, br, s(lH, C6-aH;

Wi 3Hz); 2.01, s(6H, GH^-C-0- and -NH-C-CH^); 1.46,

br,s(lH, OH; exchangeable with D ); 1.2(C10-CH,);

0.71(G13-CH,); 0.91 and 0.83 (for remaining methyl

protons).

Further elution with petroleum ether : ethyl acetate

(1:1) afforded solid compound (LXIX) which was recrystallized

from petroleum ether (1.2 gm) m.p. 132°C

Analysis found : G, 68.7 ; H, 9.3 ; N, 10.9

Ĉ Ĥ̂ -̂ If̂ Ô requires: C, 68.76; H, 9.42; N, 11.09'/.

!•»• • S) ««^ 3450(NH); 1720(CH,C00); l665(lactam), 1525,

1470, 1380 cm"-̂ (C=Jf, lf=JI).

•̂ H-NMR : 6 5,78, br (1H,NH, exchangeable with D );5.5,m(lH,

C3-cffl;Wi-=l6Hz);4.1, br , s ( lH,C6-aH;Wi=5Hz); 2 . 1 , s ( 6H, GĤ COO and

-N=C-N-); 1 . 1 , 3 ( 0 1 0 - 0 2 3 ) ; 0 .68 , s (C13-CH3) ;e .9 and O.Rl ( f o r

CH-T

remaining methyl p r o t o n s ) .

REFERENCES

1 . B l a d i n , J . A . B e r . , 1 8 , 1544 , 2907 (1885 ) .

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