PARKER, Roger Alan, 1943- the SYNTHESIS and BIOCHEMICAL PHARMACOLOGY of CHOLESTANE-3 5«/, 6/3.-TRIOL and RELATED AMINO ANALOGS AS HYPOCHOLESTEROLEMIC AGENTS

This dissertation has been microfilmed exactly aB received JQ _ (jg47

PARKER, Roger Alan, 1943- THE SYNTHESIS AND BIOCHEMICAL PHARMACOLOGY OF CHOLESTANE-3 5«/, 6/3.-TRIOL AND RELATED AMINO ANALOGS AS HYPOCHOLESTEROLEMIC AGENTS.

The Ohio State University, Ph.D., 1969 Chemistry, pharmaceutical

University Microfilms, Inc., Ann Arbor, Michigan THE SYNTHESIS AND BIOCHEMICAL PHARMACOLOGY OP

CHOLESTANE-3y0, $Gf, ^J-TRIOL AND RELATED AMINO

ANALOGS AS HYPOCHOLESTEROLEMIC AGENTS

DISSERTATION

Presented in Partial Fulfillment of the Requirements of the Degree Dootor of Philosophy in the Graduate Sohool of the Ohio State University

t>y Roger Alan Parker, B, S, Pharm,

The Ohio State University 1969

Apppwad by

Advisor Department of Pharmaoy Division of Medioinal Chemistry ACKNOWLEDGMENTS

I would like to express my personal gratitude and very sincere appreciation to my adviser, Professor

Donald T. Witiak, for his guidance and encouragement throughout this study.

I am indebted and deeply thankful to Professor

William E. Connor of the Department of Internal Medicine,

University Hospitals, The University of Iowa for his interest, encouragement and biological evaluation of our compounds•

I am indebted to Professor Mary E, Dempsey of the

Department of Biochemistry, The University of Minnesota for her interest and the in vitro biological studies of our compounds.

I wish to thank Professor Lester A. Mltscher for many helpful suggestions.

I gratefully acknowledge The Dow-Pltman-Moore

Company, Indianapolis, Indiana and the National Institutes of Health, Bethesda, Maryland for their financial support through predootoral fellowships.

Last, but not least to my wife, Carolyn, who provided love and encouragement throughout my graduate studies. ii VITA

January 31, 19^3...... Born- West Union, Iowa

1965 ••••B.S. Pharmacy, College of Pharmacy, The University of Iowa, Iowa City, Iowa

1965-196 6 ...... *...... •Dow-Pitman-Moore Graduate Fellow

1966-1969 U.S. Public Health Service Predoctoral Fellow

FIELDS OF STUDY

Major Field: Medicinal Chemistry

ill CONTENTS Page ACKNOWLEDGMENT • il

VITA ...... , , , , , t lii

FIGURES t » * .» * v

TABLES vi

INTRODUCTION • • » . . ,»»,*»••••• 1

HISTORICAL ...... 3

RESULTS AND DISCUSSION 12

EXPERIMENTAL , , , * ...... 67

SUMMARY 98

REFERENCES 101

iv FIGURES

Figures Page

lp The effect of oholestane-3i£L» , 6^-triol in preventing the usual oholesterol-induaed hyperoholesterolemia in rabbits ,,••••», 15

2, The effeot of oholestane-3^, 5 OCt 6^-triol in reduoing oholesterol induced hypercholesterol emia in rabbits. Regression studies , , , , , 19

3* The effeot of oholestane-3^2, 5_oC, 6^-triol In individual rabbits on the absorption of orally administered Li-C^-oholesterol , , , , , , , , 26

l|. The effeot of stigmoatane-Vi, !?_££» f$.-trlol on serum oholesterol levels of oholesterol fed rabbits 39

5t Chair oonformation of ring A of lanosterol , , 1|1

6, Boat oonformation of ring A of lanosterol , , , I|1

7, Flattened ohair oonformation of ring A of lanosterol »»,»••»»»»•,,,»,,• i|3

8, Conformation of the A-B rings of 5oC-aminooholestane-3£-ol ,»»»»»•»» t » 1|3

9, Proposed allyllo rearrangement of the 3 cxf-thlo- cyanate, the 3i*-azido, and the 3 OC-oyanamlde of J^-oholestene t , , ,,•»»,,»»•,, , 7

10, Optical rotatory dispersion curves of oholestane- 3/7. 5of-diol-6~one and 6-oximlnooholestane-3£, 3-oC -dlol »»,»»»•»•••,•»•,,,, 61|

11, Optioal rotatory dispersion curves of oholestane- 5j2?-6^-diol-3-one-6-aoetate and 3-oximino- aholestane-^oc ,6/#-diol-6-aoetate ,,,,,,, 65

12, Optioal rotatory dispersion ourves of oholestane- 3, 6-dione-5>oC-ol and 3, 6-dioximinooholestane- $oC-ol, 66

v TABLES

Table Page

1, Dietary regimens of rabbits fed oholestane-3#* SOC ,6|0-triol ».•••••••••••••»»» Ik

2, The effeot of Gholestane-3J?#$_0£,6^triol on serum and tissue oholesterol concentrations and aortio atherosolerosis • »»»••••••••» 16

3* Cholestane-3^ ,!?.££., 6^,-triol oontent of various tissues in rabbits fed both oholesterol and oholestane-3^ , 5>0£,“6$-triol 21

Ij, The effeot of oholeatane-3yff «5>oC.6^-triol on the feoal sterol exoretion in cholesterol fed rab bits ...... 23

5, The effeot of oholestane-3^,^^*6^-triol on the intestinal absorption of oholesterol 28

6, The absorption of Ij-G^-oholestane-3,#,E>oC,65- triol , , • , , . T . 7 . , 30 7» The effeot of Vo days ingestion of oholestane- 3#.l?oc. 6y3-triol and related compounds with onolesterol on serum oholesterol levels in rab bits , ...... 37

vl INTRODUCTION

At the present time, no single factor can be iso

lated as exclusive in the etiology of atherosclerosis.

The implication of elevated serum lipids as a factor in the development of atherosclerosis has focused atten tion on chemical agents that will lower the concentra tion of circulating lipids, Particular attention has been given to cholesterol for several reasons. It is generally accepted that hypercholesterolemia is one of the predisposing factors contributing to the genesis of atherosclerosis, A connection between the two seems definite, but the extent of this relationship, the ques tion of endogenous versus exogenous oholesterol and the pathology of the disease are all germane to the problem.

The most cogent argument for a relationship between cholesterol and atherosclerosis is that this sterol is always present and is one of the first lipids to accumu late in the plaque. There is a statistical correlation between serum oholesterol levels and the incidence of coronary artery disease. Further, it is generally agreed that metabolism of oholesterol is the slowest of all the components of the plaque, While It Is still not certain that depressing oholesterol concentration in serum has a beneficial therapeutic effeot in human atherosclerosisf basic research in the general areas of hypooholesterolemic compounds, cholesterol biosynthesis and oholesterol metabolism and exoretion should provide much evidence relevant to the problem.

Compounds designed to prevent or treat hyper cholesterolemia may exert their action in a variety of ways, If absorbed Into the body, they may suppress the biosynthesis of oholesterol In various tissues or enhance the catabolism of oholesterol to bile acids In the liver.

Another mechanism of activity may involve the prevention of intestinal absorption of oholesterol (or bile acids) and thereby promote their exoretion from the body. Our program is designed to study the synthesis of a number of compounds whioh may effeot oholesterol absorption and/ or metabolism as well as to evaluate their effectiveness as hypocholesterolemio agents. HISTORICAL

A large number of oompounds have been investigated

in reoent years for their effeot on oholesterol metabo

lism, Inhibition of both endogenous and exogenous sup

plies of this mammalian sterol has been the subject of

extensive Investigations during the past two decades,1

While a majority of the active oompounds investigated

have not significantly reduced blood oholesterol levels,

or have not been of value in the treatment of athero

sclerosis, investigations of their effect on the metabo

lism of oholesterol have yielded much basic information oonoerning sterol biogenesis, Even highly toxic drugs,

later shown not to be of value In the treatment of

diseased states have yielded considerable Information oonoerning the nature of intermediates in the biosynthesis

and degradation of oholesterol.

The development of a procedure for the synthesis of oholesterol In oell-free homogenates^, the observed transformation of squalene to lanosterol and oholesterol^, and the disoovery of mevalonic aoid as an effective pre- ourser to oholesterol led to a rapid elucidation of the U 5 major steps in the biosynthesis of this sterol ,

3 The biosynthesis of cholesterol may be arbitrarily divided into two phases. The first stage is a reductive

phase^ whioh involves the condensation of two aotivated aoetate units to form aoetoaoetyl CoA, Aoetoaoetyl CoA condenses with a third mole of aotivated acetyl CoA affording^-hydroxy-^-methylglutaryl CoA (HMG-CoA), The oarboxy group of HM3-CoA then undergoes reduotion to an alcohol with the loss of CoA by the oonoomidant consump tion of two moles of NADPH; this results in the forma tion of mevalonio aoid. This C-6 unit (mevalonate) undergoes phosphorolation followed by loss of water and carbon dioxide affording isopentenyl pyrophosphate,

Isopentenyl pyrophosphate is the active isoprene unit whioh undergoes an enzyme aatalyzed rearrangement to dimethylallyl pyrophosphate, Subsequent condensation of these two isoprene units yields geranyl pyrophosphate.

Condensation of this C-10 unit (geranyl pyrophosphate) with another isoprene unit affords farnesyl pyrophos phate, two moles of whioh condense to form squalane thereby terminating the early phase of oholesterol * biosynthesi s,

The second stage Is initiated by oxidative oyollza- tion of squalene. First, squalene undergoes epoxida- tion yielding the corresponding 2,3-epoxide (2), The

2,3-epoxide undergoes oonoerted oyolization and rearrangement to form lanosterol (3j> • Subsequent to the oyolization of squalene oxide the pathway is less well £ Q defined * , Conversion of lanosterol to oholesterol (6)

Involves the following changes: (a) removal of the two gem-dlmethyl groups at position Ij. and the single methyl group at position llj, (b) removal of the 8,9-double bond, Insertion of a double bond into the 5,6 position,

and (0 ) reduction of the side chain.

Many investigators have presented evidence for the

sequence by whioh the methyl groups of lanosterol are removed^, It has been suggested that the ltj of-methyl group is first oxidatively removed; this is followed by removal of the two gem-dimethyIs at position 1*, Recently

Sharpiess-*-® and co-workers have suggested that the ti OC- methyl is oxidatively removed first. After oxidative removal of the methyl groups the enzymatic process for the isomerization of the 8,9 double bond has not been well characterized. Presently all evidence suggests Q p| the •* 4 diene (zymosterol) is first converted to a

A 7 ' 21* diene whioh undergoes dehydration affording a

/§,!, 21* triene. Subsequent reduction of the 7,8-double bond affords desmosterol (JJ5J, Oxygen is required for this process and NADPH is required for reduction of the

7,8 double bond11. Reduction of the 2l|,25 double bond in the sterol side chain may ooour at almost any stage 12 between lanosterol and desmosterol , It has been sug gested that the pathway from lanosterol to oholesterol is a non-fixed process with either early or late satura tion as the preferred routes1-^. 6

8 = HO HO

HO HO

A direct attack on the problem of reduoing serum oholesterol would appear to be through Inhibition of endogenous cholesterol synthesis since there appears to be a lack of negative "feed-back" control between exo genous and endogenous oholesterol supplies. In the human, endogenous synthesis adds considerably more oholesterol to body stores dally than does dietary intake1^. Two types of inhibitors may be considered: (l)

inhibition prior to oyolization of squalene and (2)

Inhibition after oyolization of squalene. Inhibition

prior to the formation of squalene would probably also

inhibit the synthesis of ubiquinone side-ohains and

other known or unknown polylsoprenoids, Blocking the

synthesis after oyolization of squalene epoxide might

result in accumulation of sterols other than oholesterol

whioh may be atherogenic themselves. On this basis, the

ideal plaoe to blook biosynthesis seems to be either dur

ing oyolization of squalene or in the early stages of

steroid ring formation1"’,

While numerous oompounds have been investigated for

their ability to inhibit oholesterol biosynthesis this

report considers only those drugs related to inhibitors

prepared in our laboratories. Recently, Corey and oo-

workers1^ have prepared 2,3-iminosqualene {/£), While

this oompound blocks oyolization of 2,3-oxidosqualene

to lanosterol, 3^-ainino-8,25J-lanostadiene (jBj and

amino-8-lanostene (9) showed little inhibition. Prior

to these reports Counsell and oo-workers1^ investigated

diaza-sterol (10) as an inhibitor of oholesterol bio-

synthesis. The authors proposed that this oompound,

whioh simulates oholesterol* would bind more readily to

a negative feedbaok enzyme and thereby inhibit oholes terol biosynthesis, However, reoent biologioal evalua tion revealed that these azasteroids blook oholesterol 8

biosynthesis at a late reductive stage A large number

of A- and B- ring modified azaoholesterols and oholes

terol analogs with oxa- and oxa-aza- side chains have

also been reported^. These oompounds also generally

blook the /ffi reduotase step^.

H2N

Other approaches to lowering blood oholesterol

levels may involve the use of oompounds whioh stimulate

oholesterol metabolism and/or excretion* These oompounds

generally oause fewer side effects than those whose meoh-

anism involves inhibition of oholesterol biosynthesis*

Suoh drugs oan be arbitrarily divided into two categor

ies: (1) those whioh operate by inoreasing the metabolism

and excretion of oholesterol (e*g,, throxine and related 21 analogs ) and (2) those whioh exert their influenoe in

the intestine by sequestering bile aoids* Cholestyramine,

an anion exchange resin works by the later mechanism;

1*0*, by sequestering bile aoids from the intesting ?2 thereby stimulating oholesterol degradation * An example of another agent somewhat related to the oompounds we will disouss in this thesis is an azaderlvative of 23 oholesterol known as azacholestane (11), This oompound also lowers serum oholesterol levels by the sequestering mechanism.

^-Sitosterol ^ (12), has been shown to exert a hypo oholesterolemio effeot by still a different meohanismj

_i,e,, through inhibition of oholesterol absorption in the gut, However, this oompound is not particularly effeotive as a hypooholesterolemio and anti-atherogenio agent. Other analogs of oholesterol suoh as oholestanol

(13) (dihydrooholesterol) and 2l|-methylene oholesterol

(III), when fed orally to experimental animals, also show oholesterol lowering aotivity2^. These analogs of oholesterol are absorbed into the body and are therefore P A athrogenio themselves^ , 10

Recently, Aramaki and co-workers*^ reported that

oholaatane-3^,!^oCt6y£triol {l£J has hypocholesterolemio

activity in various experimental animals. These workers

report that triol l£ not only lowers the cholesterol

level in blood and liver, but also has a normalizing

effeot on serum phospholipid and triglyceride concentra

tions, In addition to the parent triol a number of 3,6-

diasters 16 exhibited good activity.

R^=bisuoo in ate ; R2=H T, Rl=bisuooinate=bisuoo inat ; R2=C0CH3 d_, R 1=303Na; R2=H

These investigators showed that 3^, £_Of,6^-trihydroxy-

oholanio aoid {17}, the main metabolite of triol l£ was biologically inactive, Rosooe and co-workers have confirmed the metabolic fate of triol 1? in the rat, pn Other studies by the Japanese workers were oarried out to study the meohanism of action, and toxioity of triol l£. Their studies show triol 1^ to be relatively nontoxio in experimental animals. Preliminary results

suggested the oompound to work by inhibition of oholes terol absorption, but the meohanism was not well estab lished, These investigators conoluded that the oompound did not inhibit oholesterol biosynthesis. 11

H' RESULTS AND DISCUSSION

To further study the meohanism of action, long

term toxloity and struotural requirements for biological

activity in the oholestanetriol series and to explore

the effeot of amino analogs on in_ vitro inhibition of

oholesterol biosynthesis, a number of compounds related

to triol 15j were synthesized and subjected to blologioal

evaluation. The results and discussion which follow

first consider oholestanetriol meohanism of aotion and

effectiveness as a hypooholesterolemic and antiathero

genic agent in rabbits,

Cholestane-3y3.!? oC.6y£-triol (15) was prepared utiliz

ing the classical synthesis of Pieser and Rajagopalan^O,

This involves treatment of cholesterol formate (l8j with

hydrogen peroxide in formic aaid followed by subsequent

hydrolysis of the 3,6-diformate 19, affording oholestane-

3^,5oC,6^.triol (l£),

> HgO

OCH We investigated the hypooholesterolemio activity of

triol l£ in the rabbit, The intestinal absorption of both ^-C^-oholestane-3^,^_2Sr, 6^-trlol and -a hole sterol 12 were measured• Presently, studies are being oonduoted utilizing guinea pig, White Carneaux pigeon and Rhesus monkey.

The Effeot of Cholestant-3/3,5 PC,6#trlol (l5J on Serum and Tissue Cholesterol Levels and on Atherosolerosls,

The dietary regimen of five groups of rabbits is presented in Table 1, The triol prevented the usual hyper cholesterolemia whioh ooaurs from feeding 0,5# choles terol to rabbits (Figure 1), The wide differences in serum cholesterol levels of animals treated with l£ com pared with those fed oholesterol alone are illustrated in

Table 2, In Group III rabbits fed oholesterol and triol

(l^n for 12 weeks, six rabbits developed only slight hypercholesterolemia. The other five exhibited no change despite large amounts of oholesterol in the diet. The average serum oholesterol rose from an initial value of

37 to 118 mg per cent at the end of 12 weeks (Figure 1),

In contrast, Group II animals fed 0,5# oholesterol for the same time period had an average final serum oholes terol level of 1773 mg per oent. The rabbits (Group I) fed neither oholesterol nor triol (15) maintained oonstant serum oholesterol concentrations, averaging 1|3 mg per cent.

The tissue oholesterol concentrations provided key information about the possible site of action of oholestane triol (l£) in preventing hypercholesterolemia in oholesterol fed rabbits (Table 2), The Intestinal oholesterol TABLE 1

DIETARY REGIMENS OF RABBITS FED CHOLESTANE-3^,S OC, 6^-TRIOL ( )

DIET Group Number of Period Rabbits Cholesterol 0,5% Cholesterol 0,5% Cholesterol and free in 2,5% peanut oil 0,5% Oholestanetriol in 2,5% peanut oil

Control

I > »

II • . Experimental

III • . IV , , 2 . . V , , 2 , • 3 • .

•p- SERUM CHOLESTEROL (mg/100 ml) 100 150 200 50 preventing the usualcholesterol-induced hypercholesterol iue1 The effectFigure of 1. dholeatane-3^. emia in rabbits. WEEKSOF STUDY 6jft.tr!

61 In 12

15 TABLE 2

THE EFFECT OF CHOLESTANE-3$ 5& 65.-TRIOL ON SERUM AND TISSUE CHOLESTEROL CONCENTRATIONS AND AORTIC ATHEROSCLEROSIS

Rabbit Dietary Cholestane- Terminal Degree of Tissue Cholesterol Levels Group Cholesterol triol Serum Cholesterol Atherosclerosis (mg/gm dry weight) (mg per cent) Grade 0-4 Intestine Liver Aorta

I 0 0 43+8 0 8.10+2.2 10.1+0.2 4.1+2

II 0.52 0 1733+248 2.48+0.22 a.4+1.4 130+11.9 69.2+16.4

i n 0.52 0.52 118+31 0.08 19.1+4.4 17.0+14.4 3.3+0.5

IV 0.52 0.52 43+10 0.74 16.0+4.1 12.1+4.9 6.1+2.6

V 0.52 0.52 173488 0.76 19.3+3.5 19.1+8.4 4.5+1.2

Fed in variable sequence (See Table 1).

Mean values with standard deviations. was inoreased in all animals fad cholesterol (Groups II-

V) whether or not triol 15 was given concurrently. On

the other hand, liver and aortia oholesterol concentra

tions were much lower in rabbits treated with oholestane

triol. These differences suggest that triol l£ blooked

the usual absorption of oholesterol into the blood and

extra-intestinal tissues, but did not prevent its

accumulation in the intestine,

Triol l£ especially retarded the usual development

of aortio atherosclerosis in the oholesterol-fed rabbit.

Atherosclerosis was measured utilizing two criteria:

gross lesions of the aorta and the aortic oontent of

oholesterol. Atherosclerotic lesions were evaluated on

a grading system of 0 to Ij, The animals fed triol 1$

(Group III) had no or slight lesions with an average

grade of 0,08 compared to a rather severe grade of 2,1+8

in the Group II animals not given the triol. Similarly,

the aortic oholesterol oontent did not increase to any

appreciable extent In the rabbits receiving triol l£.

In those not receiving the triol the level rose greatly

(3*3 versus 69*2 mg/gm of dried tissue).

Regression Studies,

Group IV and Group V rabbits were given different

schedules of oholesterol feeding, with and without triol

1£, in order to study the effeot of this oompound upon

already existing hyperoholesterolemla, Feeding oholes terol alone for three weeks resulted In a rise of serum 18

oholesterol levels in Group IV from 37 to 1030 mg per

oent (Figure 2), The addition of triol V~> to the diet

for the next 18 weeks oaused a gradual decline in the

serum oholesterol oontent to a low of 32 mg per oent.

These animals were aotually normo-oholesterolemio for

the last seven weeks of this regression period. The

oholesterol oontent of the tissues was respectively:

liver 12,0, intestine 16,0 and aorta 6,1 mg/gm of dried

tissue (Table 2), These values were somewhat higher

than those of the animals not fed any oholesterol (Group

I), but were muah lower than the animals fed only oholes

terol and not given any of oompound l£ (Group II), The

serum oholesterol levels of Group V rabbits continued

to average $0 mg per oent (Figure 2), Upon withdrawal

of triol l£ from the diet, the serum oholesterol levels

began to rise, Four weeks later the serum oholesterol

level reached 759 mg per oent. In previous experiments

the feeding of 0 , $ $ oholesterol for four weeks Inoreased

the oholesterol level considerably above 759 mg per oent.

When the oholesterol oontalning diet of Group IV was

again supplemented with triol l£ for the next II4 weeks,

the serum oholesterol values were lowered to 175 fig per

oent, This deoline substantiates the hypooholesterolemio

aotion of this oompound in the faoe of oontinued ohol

esterol feeding. The tissue oholesterol oontent at the

end of this period was somewhat higher than in both

Group III and IV (Table 2) and refleoted the longer SERUM CHOLESTEROL (mg/100 ml) 1000 1100 1200 100 300 200 500 600 800 700 900 rabbits. Regression studies. in Regression hypercholesterolemia rabbits. induced cholesterol reducing Figure 2. The effect of cholestane-3£,5gf»6^&.trlol in in cholestane-3£,5gf»6^&.trlol of effect The 2. Figure *o- GROUP 17 GROUP WEEKS OF STUDY OF WEEKS v ---- X + Cholesterol + Cholestane- 2&5SC$6-triol Cholestane- 2&5SC$6-triol Cholesterol 19 20

period of "unproteoted" cholesterol feeding.

Both the amount of atherosoleroais and aortio

oholesterol content appeared slightly increased in

Group IV and V when compared to Groups III and I, It

appears that the effeots of even a short term exposure

to excessive dietary oholesterol was not completely

obliterated by the subsequent administration of triol 15,

Tissue Content of Cholestane-3^,5 oC,6jfl-trlol ( ^ ) » The triol 15 content of the tissues of rabbits fed

this compound is shown in Table 3, Both liver and aorta contained relatively small amounts of triol 15 as com-

pared with the amount found in whole intestine.

The intestinal mucosa was analyzed separately for both oholesterol and triol l£ content in 1+ rabbits in

Group IV, Immediately after removal at autopsy the

intestine was divided into I; equal portions from the duodenum to the aeoum. The intestinal segments were opened, washed, and the muoosa oolleoted by gentle scrap ing with a glass slide, The mean oholesterol content of eaoh of the 1+ samples (proximal to distal segments) was

11,91, 12,11, 13,50 and 12,lil+ mg/gm of dried muoosa, respectively. The corresponding amounts of triol 15, present were i+,87, 6,1+0, 7,39 and 5.69 mg/gm of dried muoosa. The total muoosal oholesterol was 1+9,95 nig and the total muoosal triol l£ was 21+,35 mg. The muoosal oholesterol to cholestane-3^£»!?£C,6^2-triol ratio was 2:1,

This ratio was low when compared to the ratio of oholesterol TABLE 3 CHOLESTANE-3^,£_^,6^-TRIOL CONTENT OP VARIOUS TISSUES IN

RABBITS FED BOTH CHOLESTEROL AND CHOLESTANE-3^. 5

Cholestane-triol mg/gm dry weight GROUP Intestine Liver Aorta

III 7,06^2,921 0,1+13-0,11+2 0,365-0,059

IV 1+,91*2.1+31 0,51+6*0,260 0.506*0,135

V 1+.1+7*3.351+ 0,303-0,096 0 ,1+02*0,11+8

ro 22

of triol 15 obtained in the livers and aortas in this

same group of rabbits. These ratios were 30il and 12:1,

respectively, In the whole intestine, the ratio dropped

to 3 H * These results indicate that most of the absorbed

triol remains in the intestine, primarily In the mucosa.

Fecal Steroid Exoretion After C h o l e s t a n e - 3 ^ , 5 S ^ t r l o l

Administration,

The triol l£ increased the fecal excretion of neutral

steroids, reported as the sum of cholesterol and its bac

terial derivatives, coprostanol and coprostanone (Table

Ij), The excretion of bile acids, on the other hand,

increased as the triol 15 was withdrawn. This refleots rW inoreased intestinal oholesterol absorption and subse quent catabolism to bile acids.

However, several problems arose in the interpreta

tion of these excretion data. The consumption of food

varied considerably at times, A larger intake of food containing oholesterol would, In itself, oause an inoreased

fecal excretion of neutral sterols when it is considered that some of the dietary oholesterol is not absorbed.

Furthermore, it Is not known whether the intestinal baoteria in rabbits are capable of destroying the steroid nuoleus. The ratio of fecal "oholesterol" sterols to fecal plant sterols was used In order to judge If changes in fecal steroids were affected by the presence of triol

15 in the diet. The use of this ratio would oorreot for both (1 ) the possible degradation of the steroid nuoleus TABLE

THE EPPEGT OF CHOLESTANE-3£,5_0C,6^-TRIOL

ON THE FECAL STEROL EXCRETION IN CHOLESTEROL-FED RABBITS

(Group V)

Katlo oi' Chol'esL tarol Neutral RABBIT Cholesterol Plant Sterols Sterols to Plant Fecal Bile Acids Neutral Sterols Sterol3

Given Not Given G iven Not Given G iven Not Given Given Not Givt

1 199 122 1*8 71 I*.11* 1.72 30 82 2 279 208 60 83 1*.65 2,51* 163 201

3 279 198 1*8 85 5 *82 2.33 91 11*0

U 338 206 6$ 95 5.20 2.17 Ik 119

172 179 55 70 3.33 2,56 75 150

6 207 103 l*o 61 5.17 1.69 36 76

Means and 21*6*26 169-19 53*1* 78+5 U.72io.36 2 ,17*0.16 78+20 128+19 Standard Errors

Loi\> (since all of these steroids should be degraded to approximately the same degree) and (2 ) variations in dietary intake and the daily output.

When both oholesterol and the triol were fed, the mean ratio of feoal "oholesterol" sterols to feaal plant sterols was ^,72 (Table ^), The ratio deoreased to 2,17 when oholesterol alone was administered, This suggests far less oholesterol absorption in the presence of triol l£, The dlfferenoe between these two ratios was signifi cant, Also, with regard to this ratio, the individual responses to the oholesterol triol diet seemed to vary more than the individual responses to oholesterol feed ing, There appeared to be a rough inverse relationship between the height of the serum oholesterol level and this feoal sterol excretion ratio. The response of rab bit 5 was especially noteworthy 3lnoe the serum oholes terol changed little (from l6l| to 182 mg per aent with out triol (15), The feoal ratio of steroids similarly showed little change (3»33 versus 2,56 respectively).

The Intestinal Absorption of -Cholesterol During

Administration of C? hole at an e-3^, 5 o£, 6^ t r l o l (lgj ,

Further explanation for the mechanism of action of triol was provided by studies of oholesterol absorption in which a test meal of 1|-C^^-cholesterol was administered.

The serum and feoal radioactivities were subsequently determined, The serum radioaotivity reached a maximum

2\\ to lj.8 hours after the isotope was given and decreased with time thereafter (Figure 3 K A difference in serum

radioaotivity was observed between the rabbits receiv

ing a i|-C^-oholesterol and triol 1^ in the diet and

those receiving a similar diet deficient In the triol.

Thirteen to 22 per cent of the administered label appeared

in 100 ml of serum after 2lj. hours in the animals fed 0,5#

oholesterol alone, but only 2-k per cent of the label was

present in the animals given 0,$$ oholesterol plus 0,5#

triol l£; 1,9,, the absorption of radio-labeled oholes

terol is reduced by concomitant administration of triol

The suppression of oholesterol absorption by triol

1^ was substantiated by feoal analysis. The inclusion of triol l£ in the diet, with or without dietary oholesterol,

promoted the mean excretion of 38 par oent of the dose as

C*^ neutral sterols during the six days after cholesterol administration (Table 5), In oontrast, when the triol was excluded only 12 per oent of the adminis tered dose was exoreted as C-^ neutral sterols. The feoal radloaotivity remained somewhat higher in the stool through out the entire eight days of study in the treated rabbits.

Although the radioactivity found in the bile acid fraction was low for all groups, some interesting dif ferences ooourred (Table 5)» The lowest amount of bile aoid was found In the group receiving on only the triol. Without triol 15> in the diet the radloaotivity of the total bile aoid fraction was two times greater. Per cent of Administer Dose in 100 ml Serum 22 12 iue3 Teefc fcoetn-_( r_ cholestane- of effect The _ ( 3. Figure absorption of orally administered ^-C^- administered orally of -cholesterol absorption Days After Isotope Administration Isotope After Days & 3 - ' 5s^-triol on individual rabbits on the rabbits on individual 5s^-triol on individual 0.5% Cholesterol + Cholesterol 0.5% Diet 0.5% Cholestane-^,5i£6£-triol 0.5% rabbits on the Apparently more oholesterol was available for oonversion

to bile aoids when the triol was exoluded from the diet.

The distribution of radloaotivity in the different

neutral sterol fractions of stool was determined after

thin layer chromatography. The radloaotivity in the

neutral sterol fraotlon during the first two days of

stool collection in triol fed rabbits was oontained

largely in the oholesterol band. By the eighth day of

feoal collection, however, the radloaotivity was contained

mostly in the ooprostanol band with a corresponding dim

inution of the radloaotivity in the oholesterol band,

Slnoe the triol causes oholesterol to remain longer in

the Intestine, one would expeot that essentially all of

the radloaotivity should eventually appear in the oopro

stanol and ooprostanone fractions, Suoh was the oase dur

ing days 6-8 ,

Three rabbits were also given ii-C^-oholestane-

3^,5 oC,6^-trlol orally while fed the oontrol diet alone.

This was done in order to determine the amount of absorp

tion of the radiolabel as well as its rate of exoretion.

The results are included In Table 6 , Within 2\\ hours

after dosage only 0,5-1*9 per oent of the label was pres ent in 100 ml of serum, In the stool only between 5 and

16 per oent oould be aooounted for as radio labeled triol.

Essentially no counts were found in other bands on the thin layer plate, In contrast to the distribution of label after oholesterol administration, where at the TABLE 5

THE EFFECT OF CHOLESTANS-3 f*>,$QC, 6ff-TRIOL UPON THE INTESTINAL ABSORPTION OF CHOLESTEROL

Feoa'l l*-(jil+ Cholesterol RABBIT DIET TREATMENT Ingested ,, (Total Neutral Group and with Choles- Cholesterol-l*-^) Sterol-6 day's Per Cent Number tanetriol --- (apm STIUDV ' ' stool Absorption

Group I Cholesterol free 31 None 2,68 0,39 85 32 2,69 0.61* 76 Group II 36 0,5# Cholesterol None 1.59 0,10 91* 36 1,80 0.05 97

Group III 0,5# Cholesterol o,5# 26 2.97 1.58 1*7 29 2.56 0.75 71 30 2,70 0.96 61* Group IV Cholesterol free o,5# 39 2,13 0,88 59 1*0 2.15 0,71 67

Mean Per Cent Absorption in Rabbits Without Treatment 88 1 9,5

Mean Per Cant Absorption in Rabbits Given Cholestanetriol 62 t 9 ,3* ttSienii'ioaht dirierenoe rroin non-treatment c’rouD. d O.Oi . S -h. = standard dBviatinn n the mean. 29

most 10 per oent of the administered doss was found in

the bile aoid fraction, 10-30 per oent of the dose was

found in this fraction after triol was given, An

additional 5-9 per oent remained unextraotable by

organio solvents. No counts remained when U -C^-o holes -

terol was given (Table 5)* No oounts were detected In

the neutral sterol fraatlon after the sixth day; but the

aounts in the bile acid fraction persisted and aould be

detected even after 10 days, Aramake and co-workers2? have also reported that the triol is metabolized at a

much higher rate than is oholesterol. One of the products

results from apparent oxidative removal of oarbon atoms

25, 26 and 27 and conversion of C-2lj to C02H group, The relatively large amount of radio-label found in the bile

acid fraction as well as those unextraotable by organio

solvents is most likely a reflection of the metabolism

of triol 1 5 .

These studies confirm the observation of Aramaki

and co-workers that the triol prevents the usual hyper cholesterolemia which occurs from feeding oholesterol to rabbits2?, Cholestane-3^3,5_2P,6^-triol (15,) dramatically retarded the usual development of aortio atherosclerosis

and reduced total oholesterol concentration of the aorta.

Tissue and feoal oholesterol concentration studies reveal

that this compound blocks the usual absorption of oholes terol Into the blood and extra-intestinal tissues. This meohanism was substantiated by the observation that there TABLE 6

ABSORPTION OF l+X^-CHOLESTANE 3£,5_^, 6^-TRIOL

Rabbit Per Cent of Administered Dose Group Ingested and i4-C J-4-Tr iol Number Diet (dmp x lu6 J In iuu ml Serum In Tea as of O-Day Collection Neutral Bile Residue Total Day Sterols Acid 1 2 3 7 Group 5 28 Choles terol free 2.56 0.1+5 0.25 0,26 0 16,62 0,90 9,0 56.02 CO rn o 33 • 1,86 0,1+7 0.53 0,22 1+.98 26,15 5.1+ 3 6.53

3k 3.05 0.70 0,50 0.1+3 0.29 5.09 10,20 8.6 73.89

VjJ o 31

is a decrease of 80 to 8£ per oent ii-C^-oholesterol

absorption with concomitant administration of triol 1 $ 9

Regression studies show that the triol not only

prevents hyperoholesterolemia and atherosohlerosls, hut

also oan return a hyperoholesterolemia rabbit to normal

even while administering oholesterol in the diet. Under

suoh experimental conditions liver, intestine and aorta cholesterol concentrations returned to levels only

slightly higher than normal, This would be expeoted if oontinued oholesterol absorption were prevented and normal mechanisms were operable to effeat metabolism and excre tion of the excess oholesterol in the animal.

The sterol balance investigations with radio-labeled and non-radio-labeled triol 1^ show that only negligible amounts of this oompound is absorbed Into the blood and extra-intestinal tissues. Therefore, the triol is not replacing oholesterol in the hyper- or hypooholesterolomlo rabbit. Although substantial amounts of lj-0^-oholestane-

3^.5 CX.6^-trlol was recovered in the feces, all of the radio-label was not accounted for. Experiments utilizing non-labeled triol do show that considerable amounts of the oompound are found In the intestinal muoosa. Appar ently, insertion of two diaxlal hydroxyl groups into the oholesterol molecule at the 5 and 6 positions renders the oompound less readily absorbable and a good blocker of oholesterol absorption. These data, however, do not pre clude the possibility that the triol may also exert an 32

effect by Inhibiting steroidogenesis in the, intestinal

wall. In this regard, however, Imai and ao-workers ^7b

have shown that triol 1|? administered orally enhanoes

oholesterol-genesls in both liver slioes and the liver of

intact rats suggesting the triol does not exert its effect

by Inhibition of cholesterol biosynthesis. However, the

effect of inoreased hepatic oholesterogenesis is probably

a reflection of maintainanoe of homeostasis of the choles

terol level. In previous experiments, we have shown that

triol l£ is absorbed to some extent in the intestinal

wall. In addition, it Is known that a substantial amount

of oholesterol biosynthesis takes place in the intestine^.

For these reasons, it seemed to us that Investigation of

the effoot of triol l£ or in vitro oholesterol biosyn- — ■ ■ — — thesis may be of value in eluoldating in vivo mechanism

of action. In collaboration with Dr, Mary E, Dempsey

(University of Minnesota) studies on various stages of

this biosynthetic pathway are presently being carried out.

Preliminary results show that triol l£ exhibits a compet

itive Inhibition of the conversion of ^-aholestenol to ^ ’^-oholestadienol. Cholesterol Itself does not

inhibit this reaction. In addition, there is some evidence that triol If? Inhibits the reductive step from /^»

oholestadienol to oholesterol. Additional data indicates

that triol 1^> also inhibits oholesterol biosynthesis at

some point after mevalonate, This results in accumula tion of an unidentified oompound which is slightly more 33 polar than lanosterol, The accumulation of this oompound is a vary consistent finding*^, Structural Identifica tion of this moleoule is presently underway in Dr,

Dempsey’s laboratory. The preliminary findings described above suggest that the hypooholesterolemlo activity of triol 15 may also be due to an interference with oholes- /V terol biogenesis in the intestinal wall,

At the present time, we oannot be certain of the exact meohanism of action of triol 15* Long term toxioity studies of this oompound whioh are presently being oarrled out in Dr, Connor's laboratory indicate that triol 15 may be of value in treating hypercholesterol emia and atherosclerosis in humans^ #

Although triol 15, shows potential as an important hypooholesterolemlo agent very little is known about the structural requirements for biological activity. We previously pointed out that a number of 3 ,6-diesters of triol l£ show equal aotivity to the parent compound^.

We, therefore, set out to synthesize other structural analogs of triol 15, and to investigate lri vivo and In vitro aotivity,

Triol upon reaction with aoetio anhydride in pyridine, afforded the 3,6-diaoetate 2 0^ , Aoid oatalized aoetylation of the diaoetate afforded the known triacetate derivative £ 1 ^ * Synthesis of the 6-ketone 22, was carried out by Pieser’s method^ whioh involved N-bromosuaoini- mide oxidation of triol 15* The corresponding 3,6-dione 3b 23 was prepared by ohromio aoid oxidation of either the

6-one 22 or triol Seleotive hydrolysis of the 3,6-

diaoetate 20 as described by Ellis and Petrow-^ afforded

the 6 raonoaoetate 2 k , Chromio aoid oxidation of the

aoetate 2)j gave rise to the 3 -keto analog of oholestane-

trlol 2$,

15 Ac Ao OH, OAc OAc OAc 20 21

15 15 HO OH OH 22 23

20 HO 0 OAc 25 Cholestane-3^£,5>.oC-dlol 28 was prepared by lithium alumi-

num hydride reduotion of oholestane-3/3,5 0C. 6y3^.trlol-3-

ethyl-oa^bonate-6-methanesulfonate (£2-)* Compound 2£ was synthesized by treatment of triol l£ with ethyl- ohloroformate in pyridine^? affording the 3^-oathylate

(26) whioh was subsequently oonverted to 2 ^ through the use of methanesulfonylohloride in pyridine. c2H5dgo

OH SO2CH3

The corresponding 3yff.5cc. 6j6-trlol of ^-sitosterol

(stigmastane -3^,^,6^-triol) (£2) was prepared from

purified ^.-sitosterol (^2,) using a procedure similar to the synthesis of triol l£ from cholesterol.

In vivo aotivity of the above compounds was deter mined by the same method used to evaluate the parent triol

15, Preliminary results are shown in Table 7, The 3,6- /— diformate 16a has been shown by Aramaki and co-workers^ to have hypoaholesterolemio aotivity. Our results show that the triacetate 21 exhibited little biological aotivity indicating the free 5 QC-hydroxyl funotion to be important; apparently hydrolysis of the ^oC-acetate does not ocour in the gut. In addition it Is a possibility 36 that this oompound is absorbed* While we anticipated that the 6-keto analog 22 would be hypooholesterolemio, in vivo experiments showed only a small oholesterol lowering ability; the 6£-0H function or a prodrug (1.0*, the corresponding ester of the 6j3-aloohol) seems to be a requirement for aotivity. However, another possible reason for the inactivity of the 6-one analog may be beoause of the conformational ohange in the struoture of the steroid when going from a 6-tetrahedral oarbon to a trigonal oarbonyl system* In addition, relief of the

6 ,19 interaction should effeot a conformational ohange; this may interfere with receptor interaction. The

5>PC-dlol 28, was synthesized to test this hypothesis; the oompound is presently undergoing biological evalua tion, As expected from data utilizing the 6-keto analog, the 3 ,6-dlketone 23 also showed little aotivity in rab- /V bits. Presently, we have no information oonoernlng the biologioal importance of the 3^,-aloohol funation. The

3-keto derivatlng 2£ is presently being investigated bio logically.

The plant sterol, y£-sitosterol (12,) > 1193 been used for some time as a hypooholesterolemio agent2**, Like triol l£, ^-sitosterol is not absorbed and acts by block ing oholesterol absorption. This compound is only one third as effeotive as triol in lowering serum oholes terol levels^, v/e anticipated that the 3y8.E> PC.6ff-trl- hydroxy derivative of-^-sitosterol 29 might also exhibit TABLE 7

THE EFFECT OF 15 DAYS INGESTION OF CHOLESIAHE-3£, 5*. ^-TRIOL AND

RELATED COMPOUNDS 'WITH CHOLESTEROL ON SERUM CHOLESTEROL LEVELS IN RABBITS

Diet Contents; No. of Initial serum 15 days feeding Cholesterol (0,j$) with animals cholesterol serum cholesterol equal vrts. of the tested rag./ % “g./ % following compounds

Control 27 37 971

Cholestane-3^,5®4 6$-triol (15 ) 17 31 63

Cholestane-3A 52* triol A 23 655 triacetate (^21)

Cholestane-3*6-dione-5cf*ol 2 A3 553 t » )

Cholestane-3^P 5^diol-6-one 5 5A 631 (22.)

5 <£■ azido ch ole stane-3^, 65-diol 2 50 366

VjJ —0 38 exoellent aotivity. Surprisingly, this oompound was not vary active, A oomparison with triol l£ is shown in figure 1|, The only difference between triol 1 ^ and stlgmastane-3^*!? OC.6ft-triol (2£) is the presenoe of a

2l|-ethyl group in the later oompound. It Is interesting that this slight ohange at a point distant from the A-B ring of the steroid (i,e,, the presumed important part of the steroid ring for hypooholesterolemlo aotivity) oauses suoh a large ohange In biological aotivity. Appar ently, the oholestane struoture itself as well as the polyfunotional A-B ring is important for a strong inter action with the in vivo receptor site.

In addition to ijn vivo evaluation of the above analogs of triol l£, studies on Iji vitro oholesterol bio synthesis are being carried out by Dr, Dempsey, While all of these compounds have not been tested preliminary results show that the 3 ,6-dione inhibits the conversion

^-oliolestandienol to oholesterol. This oompound

(23)/"■w also inhibits oholesterol biosynthesis at some point between mevalonate and squalene^.

Our studies with simple structural modifioations of oholestanetriol on iji vivo biologioal aotivity suggested the free 5.2C-hydroxyl to be important for lowering serum oholesterol levels. We therefore proposed that isosterio replacement of the 5-hydroxyl by an amino group might render the moleoule more active than the parent oompound

(15) by virtue of the high electron density on nitrogen and inoreased ability to reaot with an In vivo reoeptor site. Serum cholesterol ( 1200 1000 800 400 600 200 4 weeks, 0.5$ cholesterol. Each value represents the average results of 6 rabbits, 6 of results average the represents value Each cholesterol. 0.5$ weeks, 4 Figure 4 . The effect of stigmostane-3&5c£»6&*triol on serum cholesterol levels cholesterol serum on stigmostane-3&5c£»6&*triol of effect The . 4 Figure 0 cholesterol; 2 and 3 weeks, 0.5$ cholesterol and 0.5$ stigmostane-3^,5££»triol; 0.5$ and cholesterol 0.5$ and 3 weeks, 2 cholesterol; fcoetrlfdrbis Deaypros 0we uiaco; ek 0.5$ 1 week, chow; Purina 0 week periods; Dietary rabbits. fed cholesterol of ^ m ^ i n ___ 1 V 4 1 s _ L isa . j X nostane-3^5o£t&&*triol on serum choiesterox . choiesterox serum on nostane-3^5o£t&&*triol Weeks Wddlcs 2 ----- A *m I 1 uaeV ft V e a u 1 aI« * a m A . »_ 3

VjJ vO bo

In addition to our interest in the E> oC-amlno system

for in vivo biologioal evaluation we envisioned that

suoh insertion of the angular amino function may render the oompound a potent inhibitor of oholesterol biosyn thesis, This proposal is related to the suggestion by 17 Counsell that oholesterol may be piotured as bound to the surface of the feedbaok-inhibition enzyme through van der Waals foroes and hydrogen bonding. Counsell suggested that oholesterol biosynthesis may be inhibited through the Incorporation of nitrogen atoms into the sub strate moleoule. These hetero atoms would allow for the involvement of muoh stronger ionio foroes and therefore bind more strongly to the feedback site. However, we

Invisloned the blook with our 5OC.amino oompounds would take plaoe at a different point in the biosynthetio scheme,

For stereoohemioal reasons, we anticipated that £.Q£- amlnocholestane-3^ , 6^-diol (^0) or fjj2&-aminooholestane-

3^-ol (^1J would blook the biosynthesis of oholesterol at those points where squalene Q J Is oonverted to lanosterol (JQ and subsequently to zymosterol U[J • hi

Conformational analysis of ring A of lanosterol indioates

that if ring A ware in the ohalr conformation (Figure 5)

a 1 ,3 -diaxial interaotion would exist between the kfe-

methyl group and the angular methyl group at C-10, Such

an interaotion requires more than 1|,$ koal per mole.

This is generally thought to be sufficient to cause ring

A to exist in the boat conformation-^ (Figure 6 ),

CH CH H OH CH CH,

Figure £, Chair conformation Figure 6 , Boat oonfor- for ring A of lanosterol, mation of ring A of lanosterol,

In the pure boat conformation a l,i|-interaotion

between the 3^£-hydroxyl group and the angular methyl

results, To alleviate this interaotion, it is expeated

that ring A does not exist as a pure boat, but rather

that it reverts to a preferred twisted conformation^.

However, in the case of lanosterol, twisting the number

3 carbon away from the reader (Figure 6 ) recreates the undesirable 1 ,3-diaxial interaotion between the i^-methyl group and the angular methyl group at C-10, Twisting the

number 3 carbon towards the reader is difficult because of the strain introduced between the bonds of A-B ring

system. On the basis of dipole moment and rotary dis

persion studies for a series of 3-ketosterolds containing 2 ,2- or 2+,I4 — gem dimethyl groups it was proposed that the

flattened ohair structure is preferred^0, Other workers

have interpreted their data as indicating an equilibrium

between boat and flattened chair forms^, Suoh a flat

tened ohair conformation also appears to represent the

preferred form for ring A of lanosterol since the 1,3-

interaction between the 1^3-methyl and the angular methyl

group is relieved as well as the interaction between the

3^S-ol and the angular methyl whioh would be present in

the boat form. Another possibility is that an equilib

rium exists between the flattened ohair and the strained

twisted boat forms. In either of the two latter possi

bilities the h OC-methyl group assumes a position nearly

axial to the ring. This is illustrated with figure 7 in

whioh ring A is pictured in the flattened chair conforma

tion, In this conformation (Figure 7) the 3^L-hydroxyl

group assumes a quasi-equatorial position having a topog

raphy similar to the hydroxyl group of oholestane-3p-ol

in which ring A exists in the ohair conformation. In

addition, the UoC-methyl group beoomes quasl-axial and

is displaced to a position which is oloser to the position

occupied by the 5>oC-hydrogen of cholestane-3^rOl, Drled-

ing molecular models^ indioate that the distanoe between

the 3^-hydroxyl group and the U.OC-methyl group in the

flattened ohair conformation is fairly close to the dis

tanoe between the 3/2-hydroxyl group and the ^ QC-amlno group in 'd flC-amlnooholestane~3^-ol (Figure 8 ), Assuming the stereoohemioal interpretation of

lanosterol to be oorreot and also to be a requirement

for enzymatic demethylation we anticipated that suoh as 5 oC-amlnooholestane-3^-ol (Figure 8 ) or the

aminodiol-analog (^0 ) to exhibit a specific blook at this stage in the biosynthesis of cholesterol. If squalena oxide (2 ) assumes a preferred conformation prior to cyolization, we thought it also possible that the

5 OC-amlnosterols would also exhibit a biosynthetio blook during oonaerted oyclization sinoe the gem-dimethyl groups may have a similar stereoohemioal relationship as is found in lanosterol, CH3

H NH2

Figure 7* Flattened ohair Figure 8 , Conformation of conformation of ring A the A-B rings of 5 OC-amlno- of lanosterol, cholestane-3^-ol.

Our first approaoh to the synthesis of the E>oC- amino system involved 3^2’ addition to azide ion to I4- aholestene-3 -one (^2)t Nagata and co-workers^ have described a similar addition of cyanide to this unsatu rated ketone. However, all attempts using this approaoh failed, ^ kk Another possible way to insert a 5 OC-amino funotion is by rearrangement of the oarbamate of l*-oholestene-

3 OC-ol (3^), Suoh a reaction is patterned after other angular alkylation reaotions of an intramolecular L3 .iih nature , It was antieipated that l|-oholestene-3- one oould be oonverted to the oorresponding 3QgT-oI 33, whioh upon reaation with phosgene followed by ammonia may afford the 3QC-oarbamate 3U»

nh2 +co,

However, reduction of the 3-keto compound 32 with lithium aluminum hydride afforded mainly the 3^-aloohol 3!?^*

Purification of [|-aholestene-3^fi.-ol was most conveniently accomplished via the benzoate ester 36 of the crude reduotion product; this method was more facile than pre viously published chromotographio procedure. Hydrolysis of the reorystallized ester followed by one reorystallization of the resulting 3^-ol ^ afforded very pure material in exoellent yields.

HO 35 While the 3oC~ol may be synthesized by other methods and

separated from the 3^£-ol through the use of digitonin,

the yields are very low^, For these reasons we first

examined the proposed synthesis utilizing the 3^-ol

isomer. Reaction of the 3^?-ol ^ with phosgene affords,

under very mild conditions, 3^-ohloro-i+-oholestene (^8,)*

This must have occurred through a rapid Sjji rearrange

ment of the intermediate allylic ohloroformate 37* Such

reaotions with allyl alcohols and phosgene are well doc

umented in the literature^, however, in all other oases

the ohloroformate oould be isolated prior to rearrange

ment ,

C-Cl 35 cr

Several attempts were made to trap the ohloroformate with ammonia to obtain the presumed more stable oarbamate

(1^), Again, in this case only the 3^-ohloro compound

a n d ^ ,5-cholestadiene (3 9 ) were isolated. Other

approaches to the synthesis of the allylic oarbamate (J4O)

involving transesterlfiaation procedures were not suc cessful and we therefore abandoned this approach. 1(6

2 5 > 3A

The reaction of !4-cholestene-3.^-ol with phosgene repre sents an excellent approach to the synthesis of ¥- ohloro-li-oholestene (^i* This reaction has many advan- J /i* tages over use of thionyl ohloride since the reaotion was faster, the work-up was easier and a higher yield of the pure 3^5 - ohloro compound was obtained* 3^3-Chloro-U- oholestene prepared utilizing either phosgene or thionyl ohloride was unstable at room temperature in chloroform.

The optical rotation of a ohloroform solution increased from [ccj0 +32*50 to +103,75°. This instability of the ^-ohloro oompound will be investigated at another time.

Preliminary studies involving the conversion of

ohloro-^-oholestene (^,8) to the 3 -thiooyanate, 3- oyanamide and 3-azido (Figure 9) were carried out. How ever, the produots of these reactions could not be crys tallized, We attempted to prepare the 3 oC-thiooyanate and the 3 OC-azide of ij-oholestene using another approach,

Reoently Lang and Sykes reported that treatment of k- cholestene-3^-01 with £-toluenesulfonyl ohloride in pyridine does not yield the corresponding 3 -tosylatej rather a mixture of ^ 3 ,5 -ohole3tadiene, 3jtX,5>-oyolo-

5of-cholest-L|.-ene and N-(U-oholestane-3 oC-vl) pyridinium hi

tosylate was obtained, We confirmed this observation

and also found that the 3^-ro0thanesufonata oould not

be isolated, Slnoe the tosylate or mesylate of 35> is

unstable several attempts were made to prepare the cor

responding 3_OC-thiooyanate and 30C-azide by _in situ

reaction with the sulfonate ester of The oompounds

isolated from the reaation mixture again could not be

crystallized even after chromatography. Infrared speotra

of these products were identical to the speotra obtained

from displacement reactions of the 3^.-°hloro oompound.

Attempted allylic rearrangement of these derivatives

afforded products whioh have not been fully characterized

and will be the subject of a future investigation,

x-y=z XEY-Z (a) X=S, Y=C, Z=N (b) X=NH, Y=C, Z=N (a) X=N, Y=W, Z=N

Figure 9, Proposed allyllo rearrangement of (a) the 32G-thiooyanate, (b ) the 3.2f-azldo, and (o) the 3 c?C- cyanamide of Ij-ohoiestene,

Another approach to the synthesis of the 5oC~arcino

system Involves addition of IOCN to the double bond of

oholesterol acetate (1| 1) or lj-oholestene-3-one (32), A

number of attempts were made with this reaction, however, only starting material oould be recovered, Hassner and oo-workers^ carried out a similar reaction with oholesterol 1+8

and also found that only starting material oould be reoovered* Apparently the double bond of oholesterol

and oholestenone is too sterioally hindered for attaok by this pseudo halogen, Reoently, Ponsold and oo-worlcers

desoribed the addition of another pseudohalogen, CIN^, to

cholesterol, progesterone and testosterone.

Ac or AcO AcO

32 > 0 0

Sinoe these methods were not suooessful, we investi. gated the synthesis of ^QC-amlnooholestane-ffi.^3-diol

(JO) from the 6yfi-epoxlde of oholesterol aooordlng to the method of Ponsold^, Cholesterol was oonverted by two different methods to the correspondingy^-spoxide jj^.

The procedure of Levine, erb involved diaxial addi tion of bromonium aoetate to the double bond of oholes terol acetate affording the 5QC-bromo-lygff^-dlaoetoxy compound (^)» Treatment with base afforded the 5jg,6£- epoxide 1^3, in low yield, A substantial amount of 1:1 U9 crystalline oomplex of the jgC and epoxides of oholes terol was obtained. This may be a result of the presence of 6^-bromo-3^,^oS-diaoetoxyohole3tane^ in the starting material,

Sinoe it was desirable to have good yields of the

pure^?-epoxlde, the second method of Davis and Petrow described by Fieser was employed^. This involved treat- pi ment of oholestane-lffif^

41 AcO Ac OAc Br OAc Br

21 HO HO

gl A modified method of Ponsold involving sodium azide in DWSO and sulfuric a d d was used in the conver sion of oholesterol-epoxlde (^3 ) to *■>OC-azldooholestane-3^,6^-diol Our first attempts with this prooedure resulted in very low yields of the 5 OCazldo 5o

aompound Higher yields were obtained simply by

raising the temperature of the reaotlon to 120°, Reduo-

tion of It 6 with lithium aluminum hydride afforded 5 oC-

amlnooholestane-3^?,6^-diol (^0)* Several derivatives

of the azido steroid U6 were synthesized as desorlbed

below. Following the modified prooedure of Ponsold

5°C-azldoQhole3tane-3y?«^-dlol-3-acetate (^8) was prepared

from the ^-epoxide of oholesterol acetate Oxidation

of the aoetate [j^ with chromio aaid afforded the cor

responding 6-keto derivative Compound ^ 9 was also

synthesized by seleotlve oxidation of the azidodiol 1^6

with chromio acid and subsequent aoetylation, In a

related system, Ellis and Fetrow^ and other workers^0* ^

have previously desorlbed the selective oxidation of the

axial 6^Lhydroxyl of triol l£, Another derivative, 3^3-

aoetoxv-5QC-azido-6^-ohlorooholestane £0 was prepared by

treatment of ^8 with thionyl ohloride. After this work

was complete Snatzke"^ reported the preparation of this

compound by a similar method. Several attempts were made

to prepare the 6 ]?-toluene sulfonate of 1^8, however, no reaction ooourred. This was presumably due to sterio hlnderanoe to approach to this axial hydroxyl group.

However, the corresponding 6-mesylate 5l was synthesized 56 from the azide 1^8 , We found* that it was Important to carefully dry the starting material in this reaction.

Lithium aluminum hydride reduction of 51 affords 5 oc,6QC-

iminoaholestane-3y£-ol (52)• Si

43 HO OH 46 30

AcO AcO

HO AcO

48 AcO 3 Cl

AoD HO 3 OMs

52 /V51 $2

Feeding experiments were carried out with two of the

above compounds in order to determine their in. vivo activ

ity, Only a few seleoted oompounds oan be evaluated for

in vivo activity sinoe a large amount of the compound

(20 g) is required, 5 cC-Azidooholestane-3ygf6|g-dlol (^60

showed some hypooholesterolemlo activity In rabbits

(Table 7), This compound showed greater activity than the 6-keto analog 22 or the 3»6-diketo derivative 23; however, none of these analogs were as active as trlol

l£, In_ vivo studies were attempted with 0,5# 5 PC.aminooholestane-3^,6^-diol (^0,) in the diet. Unfortunately, we were unable to determine whether this compound has any l_n vivo aotivlty. The rabbits refused to eat the ahow containing this aminosterol and nearly died of star vation, Additional experiments will be carried out by administering this oompound through the use of a stomach tube,

In v3tro experiments with the 5 oC-aminosterol as well as with a number of the other oompounds are being oarried out in order to test our proposal that these oom pounds may block oholesterol biosynthesis between squalene and zymosterol, Preliminary results from Dr, Dempseyfs laboratory show that this aminosteroid (30) does blook conversion of^ ,7-oholestadlenol to oholesterol. In faot, this amino analog appears to be even more effective than trlol 15 as an inhibitor. In addition, it appears that $ o C - a m lnoaholestane-3^, 6£- diol blooks the biosynthetic pathway at some point between mevalonate and squalene.

We had previously shown that the and 6^-hydroxy groups of triol may also effeot l_n vivo and iJi vitro activity. The 3,6-dlester has the same activity as cholestanetriol itself; suoh compounds may undergo rapid in vivo hydrolysis to the triol. To further examine structural requirements for iji vivo and iji vitro activity, especially in light of the high iji vitro activity observed for the 50C-amino analog, we synthesized the 3ft- amino- lsostere as well as some other amino analogs,

3^,-Aminooholestane-5!.£C,6^-diol was synthesized by lithium aluminum hydride reduotion of 3-oxlminooholestane-

6JLdiol-6-aoetate (53)* This oxime (£3) was obtained in good yields from oholestane-5

(3^3) amine 5U, This assignment of configuration is based on work described by Shoppee-^ in the lithium aluminum hydride reduotion of 6-oximlno-5LgC-hydroxyoholestane, Further proof of configuration for the 3^-assignment was obtained by comparison with the 3 QCamlno isomer syn thesized by another route. 25 HON OAo

AoN lAc

The 3^9-arol nosteroid 5^ orystallizes only with difficulty.

Since purification of the amine 5i| was difficult, ohar-

aoterization was accomplished by conversion of the amine

to the corresponding 3-aoetamido-6-aoetate 55* Reaotion of 51} with acetic anhydride in pyridine at room temper-

ature afforded the amide which was easily purified by crystallization*

In addition to the 3^2-amlno analog of oholestanetriol

[$l[), the axial 3^-amlno derivative 5j was synthesized in order to determine whether or not the^-J-equitorial con figuration at oarbon 3 was essential for maximum biolog ical aotivity; arguments similar to those presented in the proposal for anticipated in vitro biologioal aotivity of the % c C -amino analog are also appllable to the 3 oc isomer. The 3 DC-amino analog of oholestanetrlol was prepared using several different approaches. One method involved

the synthesis of oholestane-3^,5 QC>6j3-trlol-3-P-toluene-

sulfonate by treatment of oholestanetrlol with £-toluene- 58 sulfonylohloride in pyridine"^ , Subsequent reaction of

the tosylate £ 6 with ammonia afforded the 3 °C-ami no com

pound 57* "Poor yields of 57 were obtained. This most

likely was a result of the neoessity to oarry out the

displacement reaotion at 100° and at high pressures,

Under these reaotion conditions a number of unidentified

produots were obtained. One compound isolated from this reaotion was characterized as 3 PC.5 QC-oxldooholestane-

6^-ol (£8)* We expected to obtain the oxido compound

since Clayton and oo-workers^ reported the synthesis of

3 oc.5 oxldooholestane (£0) by treatment of oholestane-

3^,5 OC-dlol-3-g-toluenesulfonate { ^ ) with strong base.

Since the 3 0^.5 QC-oxlde is a possible intermediate whan

^6 undergoes reaotion with ammonia, we may also expect to obtain the 3,^3-ami no sterol 51+* While the 3^-aminodlol

5^ may also be present in the reaotion mixture (because of this possible anohimerio assistance of the 5_2?-hydroxyl group) thus far, we have only isolated the 3,oC-amIno com pound 57* This aminodiol (57) was found to orystallize only with difficulty, therefore, characterization was oarried out on the amide 61, Treatment of 57 with aoetio anhydride in pyridine afforded 3£?-aoetamldo-6^-aoetoxy- cholestane-5j2£-01 (61) which was easily purified by orys- talllzation. 56

OAo

OH CH

Displacement of the toayl group of 15 with azide ion in DMF afforded 3<3C-azidoohol9stane-5 (%- 6|S-dlol (6£)t Compound 62 was converted to the corresponding 6- aoetate (63,) for characterization purposes,

A better synthesis for the 3PC-amino analog involved the preparation of oholestano-3^.5(X.6{3-trlol-3-methanesulfonate-6-aoetate, Reaotion of cholestanetriol-6- aoetate (2t|) with methanesulfonyl ohloride in pyridine affords the 3-mesylate 6i|, Mesylate 6k was more readily purified than the corresponding 3-tosylate, Displacement of the methanesulfonate 61j. with azide ion afforded the

3 OC-azldo steroid 63 in good yield. 57

CH^SC^O OAc

To establish the configuration of the 3-azic3o group, the following approach was employed: treatment of triol l£ with methanesulfonyl ohloride in pyridine fol lowed by reorystalllzation afforded Sj2Ct6£C-epoxy-3^- oholestanol mothanesulfonate (65),

CH^SOg

65 66

Compound 6£ was also prepared by treatment of oholesterol

Of-epoxide (66) with methanesulfonylohloride in pyridine,

Reaotion of 65 with azide ion in DMF afforded 3 OC-azldo- 58

5 OC, 60£-epoxyoholestane (67). In this reaotion anohlm- 1 n JU- erio assistance was not possible and the azide funatlon 51 should have the 3 QC-conflguratlon, Ponsold has shown

epoxide opening with sodium azide requires strongly aoidio

oonditions; l,e,, generation of the 5,6-diol as an inter

mediate whioh oould anohimerio assist is unlikely in this

reaotion. Treatment of 3OC-azldo-5

with periodic aoid in aqueous acetone afforded 3 OC-azldo-

oholestane-^Q^.6^-dlol (62,), Compound 62 was subsequently

oonverted to the corresponding 6-aoetate 63, The azido

steroid 6j synthesized in this manner was found identical

in all respects with the produat obtained by azido dis

placement of the 3-tosylate or 3-mesylate of oholestane-

triol-6-acetate,

Reduotion of the 3-azido group of the above oompounds to the corresponding amine was carried out by two different methods. Lithium aluminum hydride reduotion of the azide

6£ or the aoetate azide 6^ afforded 3 of-amlnooholestane-

5-2f*6^3-diol, This produot was similarly oharaoterized by conversion to the 3 PC-aoetamldooholestane-5 cC.6^-dlol-6- aoetate. Amide 61 was found to be identical in all respects 5>9 to the amide obtained from ammonia displacement of the

3^3-tosylate of trlol 1? followed by aoetylation, This establishes the 3jXconfiguration for the azido and amino analogs. In addition, this information substantiates our

3^-amino assignment of the amine ^ obtained by hydride reduotion of the oxime $3* A more convenient method for reducing the azido function to the amine Involved the E>1 hydrazine-Raney nickel prooedure employed by Ponsold in the reduotion of azidosteroids, Compound 6^ when treated with hydrazine hydrate and Raney nickel in ethanol afforded

3 QC-amlnooholastane-^

AcN"

We thought it of interest, for reasons previously discussed, to prepare the 6^-amino isostere of triol l£.

The known ^6-amino analog of oholestanetrlol (£0) was synthesized by two different methods. The first approach involved the synthesis of the oxime of oholestane 60

diol-6-one (69) bv a method similar to that reported by r— Shoppes^0 and Gunther^, Lithium aluminum hydride reduo

tion of the 6-oxime affords 6|%-aminooholestane-3^«5 0C-

diol, Purification of this amino steroid was also dif

ficult, Treatment of aholestane-3^ f£jx;,6jg-triol-3-athyl-

oarbonate-6-methanesulfonate (2J) with sodium azide in

DMP gave excellent yields of 6y9-azidooholestane-3^.^(X-diol-

3-ethyloarbonate (£L), The configuration of the 6^3-azldo

group was proved by hydrolysis followed by aoetylation

affording the known 6j(ft-azldooholestane-3/3,.[3 OC-diol-3-

aoetate (12), Reduotion of azide Jg with Raney nlokel and 91 hydrazine as described by Ponsold afforded 6yS-amino-

cholestane-3^,5_oC-diol-3-aoetate (73)» Basio hydrolysis

of the acetate ester 73 yielded the aminodiol 70 which was /V- identical in all respeots with the amino steroid obtained by the reduction of the 6-oxime,

HO HO OH OH NE

AcO OH

Ac OH

71 72 Isosterio replacement of both the 3- and 6-hydroxyl

groups of triol l£ with NH2 was also successful,

Gholestane-3,6-dione-HjOC-ol (23) was treated with hydroxyl-

amine affording the 3,6-dioxime 7h in excellent yields.

Lithium aluminum hydride reduotion of 7U yielded the

3,6-diamino oompound 75,, Again, this oompound was found

difficult to purify. The 3^?,6£-diaoetamidooholastane-

$ j £ - o l derivative (76) was prepared by aoetylation in

pyridine, purified by recrystallization from ether, and

subjected to elemental analysis. Theoretical and calcu

lated analysis were in exoellent agreement; based on the

results obtained during lithium aluminum hydride reduotion

of oompounds containing either the 3 or 6 oxime we tenta

tively assigned the 3^,6^-oonfiguration to the amino func

tions of oompound 75,

In vitro biological evaluation of the amino analogs

is presently under investigation in Dr, Dempsey’s labora tories ,

NHAc 62

During the course of these investigations, we measured

the optical rotary dispersion (ORD) curves of the 3 and

6 monoketones plus the 3*6-diketo derivatives as wall as

their corresponding oximes, The ORD aurves for the 6-one

22 and the corresponding 6-oxime 69 are shown in figure

10, The sign of the Cotton curve remained negative in

these two oompounds. However, the trough shifted to lower

wavelength by 9? m^t in going from the ketone to the oxime.

This followed the corresponding shift in the ultraviolet

of a^nax of 300 m £ for ketone 22 to lower wavelength (end

absorption) for the oxime 69, In addition, the molecular

rotation <1>) of the trough increased threefold in going

from ketone 22 to the oxime 69* The ORD curves of the

3-one 2£ and its oximino derivative 5^ are shown in figure

11, As expected, the sign of the Cotton aurvs is positive

for these two analogs. In this case, the shift of the peak

to shorter wavelength was only 25> mjA and the Intensity

remained about the same, Again, this paralleled the ultra

violet absorption of the ketone { X max> 272, MEOH) to end

absorption for the oxime derivative. The ORD curves of

the 3»6-diketone 23 and the 3,6-dioxime 7k (figure 12)

again showed a large shift (95> m^O of the trough to shorter wavelength. In addition, a 2,7 fold inorease in molecular rotation of the trough was observed.

The results of the ORD ourves of this series of oom

pounds seems to indicate that an adjaoent group (5gC-hydroxyl) 63 to the oxime ohromophore oauses a large shift (95 m/*) of the Cotton curve to lower wavelength. In addition, this interaction results in a large increase in the moleaular rotation of the first extremum. This effect was not seen in the case of the 3-oximino oompound 53 which y*'-' does not possess an adjacent axial hydroxyl group. At the present time, we are not oertain of the reason for this observation. Clearly, other steroidol ketones and their corresponding oximes should be studies in order to determine the effect of adjacent groups on the ORD curves of the oxlmino function. [ijjlx 10- -120 Figure 10. Optical rotatory dispersion curves. A. Cholestane-^S,5ftdiol-6-one (C, 0.100, MeOH, Cholestane-^S,5ftdiol-6-one A. curves. dispersion rotatory Optical Figure 10. -100 5) B 6Oiancoetn-l§5f-3o C 010 eH 2°. o>> 6-Oxiiainocholestane-3li§,5cfc-<3iol B. 25°). ~ 25°).(C, MeOH, 0.100, +40 +60 +20 -6o -40 -20 -80 200 (mg) 250 0

Q 300 O H 8 «■ h o n 350 +30

+20

+10

-20

-30 \/ -40

200 300 350 (mii) Figure 11 . Optical rotatory dispersion curves. A. Ch.olostane-5aC,^-diol-3-one-6-acetate (C, 0.100, MeOH, 25°). B. 3-Qxiininocholestane-5a;6£-diol-6-acetate (C, 0.025, MoOH, 25°). +80

+60

+40

+20

-20

-40

-60

-80

- 1 0 0 1 1 1 200 250 300 350 Figure 12 . Optical rotatory dispersion curves. A. Cholestane-3,6-dione-5X-ol (C, 0.050, MeOH, EXPERIMENTAL62

Cholestane-3^,5 gC,6^6-trlol (15J was prepared by the q n method of Fieser and Rajogopalan^ * In order to prepare the large amounts of o.holestanetriol needed the follow

ing modified prooedure was used. Cholesterol (200 g,

0,^2 mole) and 2 1, of 90$ formio aoid were plaoed in a 6 1, flask and heated on a steam bath with oooasional stirring for approximately one hour. An oily layer of the corresponding formate ester separated. The reaotion mixture was oooled on an ioe bath with agitation in order to obtain a fine white preoipitate, Hydrogen peroxide

(200 oo of 30$ solution, 1 *U7 mole) was added and reao tion shaken oooasionally and allowed to stand at room temperature overnight. Care must be taken to prevent the reaotion from heating above lj50C • The reaotion was allowed to continue until a olear faintly blue solution was obtained,

Bolling water (3*5 1*) was added to the reaotion with aon- stant stirring to deoompose the excess hydrogen peroxide and preoipitate the reaction produot. The mixture was allowed to oool in a refrigerator and the white precipi tant oolleoted and dried. The preoipitant was transferred to a 6 1, flask and heated to reflux with 5 1* methanol.

Aqueous NaOH (25$, 200 ml) was slowly added to the refluxing

67 68

solution, After addition the reaotion mixture was refluxed

for an addition l£ to 2 hours. Concentrated HC1 was added

with stirring to neutralize the solution, Upon oooling

triol l|p orystallizes as white needles, Reorystallization

from methanol affords pure oholestane-3^,5 oC, 6^-triol,

The mother liquor may be heated and water added to the

oloud point to obtain more oholestatriol, The oombined

orystallized triol totaled 200 g (92$), mp 21+2—2UU°»

lit,30 mp 2i|l|°,

JC^-Cholastane-3^,5_OC,6^3-trlol was prepared utiliz

ing the same prooedure starting with i|-C^-oholesterol,

ll-C^-Trlol was purified by thin layer ohromatography on

silioa-gel,

Cholestane-3^,5 flC,6^-trlol-3,6-dlaoetate ( 20,) was

prepared from triol 1^ by the usual methods employing

excess aoetio anhydride in pyridine at room temperature,

mp 16£-166°, lit ,3^ tnp 166° ^

Chol9stane-3/g,5 OC, 6yg-trlol trlaoetate (21),- The

diaoetate 20 was oonverted to the triacetate 21 by an

acid aatallzed aoetylation as desorlbed by Davis and

Petrow3'’, mp ll|8-lif9°f lit,3^ mp 1^9°»

Cholestane-3yg,5 gC-dlol-6-one (£2.),- Triol l£ was

oxidized with N-bromosuooinimlde affording the correspond

ing 6-one 2^2 aocording to the method of Plaser and

Rajogopalan30, mp 232-233°, lit,30 mp 232°,

Cholestane-3 ,6-dlone-5 gC-ol ( ^ 3 . ) Trlol ^ was oonverted to the 3,6-dlone 23 as described by Prelog and 69

Tagmann36, mp 239° dec,, lit,36 232° dec,

Cholestane-3^3,9 OC,6^-trlol-6-ao9tate (gl+J was pre pared by seleotive hydrolysis of the 3 ,6-diaoetate £0 as described by Ellis and Petrow3^, mp ll+3-ll+l+° lit,3^ mp

11)4°.

Cholestane-9 oc, 6/9.dlol-6-aoetate-3-on9 ( 29.) was pre pared by the method of Ellis and Petrow3^, mp 161-162°, lit ,3^ mp 161-162°,

Chol9stane-3jjg>9QC-6jg-triol-3-ethyl oarbanate (26,),-

Triol 19 (1+0,0 g, 0,099 mole) in 1+00 ml, pyridine was oooled on an ioe bath. Ethyl ohloroformate (88,0 g, 0,81 mole) was added dropwise with stirring. The reaction mix ture was allowed to stand at room temperature for six hours, poured into HpO and extracted with ether, The dried ether layer (Na^SO^) was evaporated under reduced pressure,

Reorystallizatlon from methanol-aoetone (1:1) afforded 1+0 g (89$) oholestanetrlol-3-oathylate, mp 189-188°, lit,3? mp 188°,

Cholestana-3yg,9 ag^yS-triol-B-oathylate-b-mesylate ( 2.7.) »- Gathylate 26 (36,0 g, 0,099 mole) in 120 ml dry pyridine was oooled on an loe bath, Methanesulfonylohloride (60,0 g, 0,929 mole) in 90 ml pyridine was added dropwise with stirring and the reaotlon was allowed to stand at room temperature overnight. The reaotion mixture was poured into ioe water and extracted (ether). The dried ether layer (NapSO^) was evaporated under reduced pressure afford ing 36,0 g orystalline produot, Reorystallizatlon from 70

aoetone afforded 29.0 g (70$) 27, mp 174-176° dea ,

Cholg3tane-3^,5 cC-diol (28),- Cholestane-^?,5.oc;,(^3-

triol-3-ethyloarbonate-6-methanesulfonate (2,00 g, 0,0035

mole) in 75 ml of ether was added dropwise with stirring

to LiAlH^ (2,00 g, 0,050 mole) at 0°, The reaotion was

allowed to stir at room temperature overnight. The reaa-

tion mixture was poured into ioe water and extracted

(Et^O), The ether layer was washed with water, dried

(Na2$0^), filtered and removed under reduoed pressure

affording 1,1+0 g white orystalline residue, Reorystalliza-

tlon from aoetone-methanol (1:1) gave 1,35 g (95$)

aholestane-3^,5_0C-diol, mp 224-225°, lit,63 mp 225°,

Stigmastane-3^, 5 PC, (g9j ,- ^-sitosterol (12)

(50 g, 0,124 mole) suspended in 500 ml 90$ formio acid was heated on a steam bath with oooaslonal stirring for

1 hour. The mixture was allowed to oool at room tempera ture and 80 ml of 30$ hydrogen peroxide was added. The reaotion mixture was stirred for 72 hours at room tempera ture, Boiling water (3 It) was added with stirring; the mixture was oooled filtered. The dried filtrate was heated to reflux in 3 1, of methanol. An aqueous solution of

NaOH (25$, 80 ml) was added and the reaotion was refluxed for 1 hour, neutralized with 10$ H31, and allowed to oool to room temperature, Stigmastane 6^-triol orystallized affording white needles, Reorystallizatlon from methanol afforded 54 g (94$) 29, mp 242-245°, lit,6^ mp 248-2500, 71

Anal, Calod for C 29H£2^3 : C,77»^5j H, 11,66,

Pound C, 77.93 ; H,11,614,

Attempted reaotion of ^-oholaateng-Q-one (3_2) with azide Ion,- Several attempts ware made to carry out an SNg1 addition of azide ion to l4-cholestene-3-one, The proced ure was patterned after the S ^ ' addition of cyanide ion to ^2, described by Nagata and oo-workers^, The reaotion was carried out utilizing lj-oholestene-3-one and a large excess (20 equivalents) of sodium azide under a variety of reaotion conditions. Solvents employed in the reaction were methanol-water, ethanol-water, DMF, DMSO, pyridine, acetic anhydride, and glacial acetio aoid. In addition, a number of strong Br^nsted and Lewis acids' were used in the reaotion prooedure, Only starting material and traoe amounts of unidentified products were obtained.

Attempted reaotion of U-oholestene-3-ona (j2j with acetio anhydride and azide ion,- Several reactions were carried out in hopes of synthesizing 3-aoatoxy-5^P-azido- oholest-3-one via aoetylation of the enol of oholest-l;- ene-3-one in the presence of azide ion, Acetio anhydride,

DMSO, DMF and glacial aoetio aoid were used as solvents.

In addition, catalysts such as HC1, p-toluenesulfonio aoid and _p-toluene sulfonylahloride were employed. All attempts afforded only starting material,

il-Cholestene-3^-01 (35)»- Lithium aluminum hydride reduction of l4-oholestene-3-one was oarried out as described by MoKennis and Gaffney^8 , However, poor yields (30$) 72 of l*-ohole3ten-3.^-ol were obtained by this method, l|.-

Cholestene-3jOC-ol oould not be isolated from this reaotion.

The method of Wheeler^15 was employed, again low yields (30%) of oholest-l4-ene-3^£-ol were obtained.

In order to obtain higher yields of the 3^-ol 3$ a modified procedure of B r o w n ^ 0 was oarrled out. Lithium aluminum hydride (Lj,0 g, 0,10£ mole) in a £00 ml flask equipped with a dropping funnel and drying tube was stirred with 100 ml freshly purified THF on an ioe bath, t-Butanol

(U0 ml) was added dropwise and the reaotion mixture was stirred for 30 minutes, l*-Cholestene-3-one (£,0 g, 0,013 mole) in 7? ml THF was added dropwise. The reaotion was allowed to warm to room temperature and was refluxed for at least 2 hours. The reaotion mixture was poured into exoess loe water 10# HC1 (1:1) and extracted (EtpO), The ether layer was washed several times with water, dried

(Na2S0[j) and removed under reduoed pressure, A white crys talline produat (3*8 g) was obtained, Purifioation was readily aooomplished by column ahromatography on aluminum oxide (£0 g) with benzene-hexane (1:1) and ethanol, Dianes and other by-produots were eluted with benzene-hexane; elution with ethanol afforded pure 35, Reorystallizatlon from aoetone-methanol (1:1) afforded 3,0 g (£9#) white needles of 3$, mp 129-131°, lit,**6 mp 132°; \oc] / ' - 1 f— - 1 0 +1*2,£° (CHC1 J ') lit,**6 ULOT J Q +l*£°. Pure ^35 may also be obtained without column chromatography after four to six reorystallizatIons from aoetone-methanol (1:1) 73

Chromatography on silioia aoid with chloroform afforded mainly^J,5-oholestadiene, mp 78-79°, llt,^ mp 80°; [ ^ ] 0

-120° (CHOI3 ), litt6^[pc]D -123°.

3^-Benzoxy-ij.-ohol93ten9 (.36), - Impure 14-aholestene-

3^3-ol (1,00 g, 0,0026 mole) in 5 ml pyridine was oooled on an ioe bath. Benzoyl ohloride (1,50 g, 0,0011 mole)

in 5 ml pyridine waa added dropwise with stirring and the reaotion mixture was allowed to stand at room temperature overnight, The reaotion was extraoted (Et,,0) and washed several times with water followed by aqueous sodium bicar bonate, The dried ether layer (Na2S0j^) was evaporated under reduoed pressure affording 1,22 g orystalline residue, Reorystallizatlon from aoetone affords 1,10 g

(87$) benzoate £6, mp 125-126°: lit,^ mp 125-128°,

Anal, Calod for C ^ H ^ q O^ G, 83,21; H, 10,27,

Pound: C, 82,68; H, 10,07,

ll-Cholestene-3^-01 (35.) from S^-benzoxy-^-oholestene

(3.6),- Benzoate 3^6 (1,00 g, 0,0021 mole) was heated to reflux in 100 ml ethanol, 10# aqueous NaOH (15 ml) was added and the reaotion refluxed for five hours. The reao tion was allowed to oool and neutralized with 10# aqueous

HC1 and extracted with ether. The ether layer washed with water and 10# adqeous NaHCO^, dried (Na2S0^), filtered and evaporated under reduoed pressure affording 0,77 g white residue, Reorystallizatlon from aoetone-methanol (1:1) affords 0,70 g (89#) 35 as white needles, mp 131-132°, lit,**6 mp 132°, ih

3^-Chloro-jj.-oholgst8ne (3j),) from I|.-ohol9stene-3j3-ol

(15.) flnd phosgene,- l*-Gholestene-3^-ol (2 g, 0,00^2 mole)

In 50 ml, ether was oooled on an loo bath, Phosgene gas

was slowly added for 20 minutes to tho oold solution. The

athar and exoess phosgene ware removed undar reduoed press

ure without heat affording a white orystalling produat

(2,1 g), Reorystallizatlon from ether aoetonltrlle ether

(1:1) affords 1,7 g (Big) £8, mp 118-120°, lit,1+7 mp

116-120°; [oc]D +32,5° (OHCl^) lit,1*7 [c<]0 +26°,

Anal, Calad for C 27^ $ ^» 80,05; H, 11,20; ol, 8,75, Pound: G, 79.97; H, 10,58; Cl, 8,16,

Several attempts were made to Isolate the correspond

ing ohloroformate 37 of l|-°holestene-3y§-ol, Ether, benzene,

pyridine and THF were used as solvents and the reaotion

was oooled on a dry ioe-aoetone bath. In addition,

pyridine, trimethylamine, triethylamine and sodium carbonate

were used to trap the HC1 generated in the reaotion, How

ever, under all reaotion oondltions, only 3^-ohloro-I^-

oholestene and ^3 ,5-oholestadiene ( ^ ) were isolated,

3t#-Chloro-l|-ohole3tene (3j3) from U-oholestene-lyS-ol

(^5) and thlonyl ohlorlde,- l^-Chloro-lt-oholestene was

prepared from !4-oholestene-3^3-ol by the method of Young,

et al,^7*5 using thlonyl ohloride, first attempts using this

prooedure resulted in poor yields (15$ - 30$), Reorys

tallizatlon of the ohloro oompound from ether aoetonltrlle

(1:1) afforded J8, mp 116-120°, lit,**? mp 116-120°;

+32,5° (CHCI3 ) lit,^7 [oc]D +26°, 75

It waa observed that the optioal rotation of ohloro-l^-oholestene upon standing in ohloroform at room temperature increased from fpc]D +32,5° to +103,75°*

Attempted conversion of 3^-obloro^-oholesteng (1 8 ) to l|-oholestene-30C-ol (33)*- The procedure of Young and ------■ - —------7“— — co-workers^was utilized, however, 1 flC-ao e toxv-b- aholestene oould not be obtained by this method,

^ } , 5>-Gholestadiene, Was isolated from ahromatography on neutralized aluminum with pentane as the eluant, mp 78-79°, lit,65 mp 80°,

Attempted synthesis of i|-oholestene-3y0-ol carbamate

(llPJ »- Several attempts were made to prepare the oarbamate of i[-oholestene-3^2,-ol by trapping the intermediate ohloroformate with exoess of ammonia. All attempts were unsuc cessful and only 3/2-ohloro-ij-aholestene and ,5-oholestadiene were obtained,

Reaotion of l;-oholestene-3jQ-ol (35) with NaH and subsequent addition of phosgene and ammonia, Ij-Cholestene-

3^9-ol (0,5 g, 0,0013 mole) in I|0 ml ether was plaoed in a dry 2^0 ml flask and flushed with NaH (0,035 g »

0t00l£ mole) was added and the suspension stirred at 0° for

2l\ hours, The ether removed under reduoed pressure and the solid obtained was suspended in 80 ml hexane. The hexane suspension was added dropwise with stirring to a solution of exoess phosgene in 100 ml hexane on a dry ioe acetone bath. The reaotion was allowed to oontinue for 3 hours at 0°, The exoess phosgene was removed under reduoed pressure without heat and ammonia gas added to the solu tion for 30 minutes. The reaotion was allowed to warm up to room temperature and extracted with ether, 3^1-Ohloro-

^-oholestene and ^ ,5-oholestadiene were obtained from this reaotion.

The reaotion was carried out as described above except

NaNHjp was used in place of NaH, Again, 3^-obloro-^- oholestene and ,5-oholestadlene were isolated,

Reaotion of 4-oholestene-3yg-ol (35J with ethyl oarbamateSeveral attempts were made to synthesize the carbamate of i;-oholestene-3^-ol via transester if lo at ion reaotion with ethyl oarbamate, Several solvent systems were employed and aluminum isopropoxide was used as a catalist. However, only starting material and trace amounts of unidentified products were obtained.

Attempted synthesis of 3 oC-azldo-U-oholestene from

3>ff-ohloro-[{-oholestene (38),- 3^3-Chloro-lj-oholestena

(0,150 g, 0,00037 mole) in 10 ml ether was slowly added to a stirred suspension of NaN^ (0,300 g,, 0,00i|6 mole) in 50 ml methanol. The reaotion mixture was stirred at room temperature overnight, poured into ioe water, and extracted (Et20), The ether layer was washed several times with water, dried (Na2S0^), filtered and removed under reduoed pressure affording 0,110 g of a oolorless oil, ir

(CHCl^) 2100 (N3 ), 1655 om"^ (C=C), All attempts to crys tallize this product failed even after ahromatography on silioio aoid with CHCl^, 77

Attempted synthesis and rearrangement of 3 oC-thlooyano-l+-oholestene from 3J/$-ohloro-[|-ohole3tene (3.8),-

3^>-JChloro-i|.-ohol03tene (0,5 g, 0,0012 mole) in 30 ml

ether was slowly added to a stirred solution of KSCN

(1,0 g, 0,010 mole) in 50 ml acetone, The reaction mix ture was allowed to stir overnight at room temperature,

poured into ioe water and extracted (Et20), The ether layer was washed several times with water, dried ( N a ^ O ^ , filtered and removed under reduoed pressure affording

0,503 g of white oil, ir (CHCl^) 2160 (SON), l6£0 cm"1

(C=C), All attempts to crystallize this oil even after chromatography on silicic aoid with CHCl^ failed.

Several attempts were made to carry out a thermo rearrangement of the 3-thiooyano oompound. However, all attempts afforded starting material and unidentified prod ucts, Another approach involved a base oatalized rearrangement^*?, 3 thiooyanate (1,80 g, 0,00[j.2 mole) in 100 ml methanol was flushed with N 2 and stirred at room temperature. Aqueous K^CO^ (10 ml, 10# solution) was added dropwise and the reaotion mixture was stirred at room temperature for 2\\ hours, neutralized with glaoial acetio aoid, poured into ioe water and extracted (Et20),

The ether layer was washed with water, dried (Na2S0^), filtered and removed under reduoed pressure affording 1,67 g of a white oil, ir (CHCI3 ) 3i|10, 3380, 1700, 1655, 1$00 om--1, All attempts to orystallize this rearrangement product failed even after chromatography on silicic acid with chloroform. 78

Several attempts were made to hydrolize the above product, However, all attempts afforded only starting material.

Attempted synthesis of 3 oC-oyanamldo4+-oholestene from 3p-ohloro-Ij.-Qholestene (3.8)*- 3^rChloro-l+-oholestene

(0,1+90 g, 0,0012 mole) in 3 0 ml ether was stirred at room 68 temperature. Aero oyanamide 50 (30 ml) was added. The suspension was stirred overnight at room temperature, poured into ioe water and extracted (Et£0), The ether layer was washed several times with water, dried (Na2S0+|), filtered and evaporated under reduoed pressure affording

0,1+90 g white residue, ir (CHGl^) 3580 (OH), 3380 (NH),

2220 (NGN), 16^0 om"1 (C=C), Reorystallizatlon from aoetone afforded 0,200 g white needles, mp 137 - li+0°, ir (CHGl^) 3580 (OH), 1655 om"1 (C=C), All attempts to crystallize the mother liquor which still showed presence of -NHCN (31+00, 2220 om”1) failed even after chromatog raphy on silioia aoid with CHCl^,

Attempted synthesis of 3 oC~azido-l+-oholestene from i+-oholestene-3y^-ol (3l5)» - il-Cholestene-S^-ol (0,500 g, 0,0013 mole) and NaN^ (0,750 g, 0,0115 raole) were suspended in 10 ml dry pyridine. The reaotion mixture was stirred and oooled on an ioe bath, Tosyl chloride (0,500 g,

0,00261+ mole) in 5 ral dry pyridine was added dropwise to the suspension, The reaction was allowed to warm up to

0° and stirred overnight, poured into ioe water and extraoted with ether. The ether layer was washed with aqueous NaHCO^, then water, dried (Na2S0^), filtered and removed under reduoed pressure affording 0,1|10 g white oil ir (CBCI3 ) 3^80 (OH), 2120 (N3 ), 1655 om-1 (C^C), Again, all attempts to orystallize this oil failed.

Attempted synthesis of 3 QC-thlooyano-U-oholestene from li-oholestene-Q^-ol (3l5)»- l+-Gholestene-3^rOl (0,500 g

0,0013 mole) and KSGN (1,5 Zt 0,0151; mole) were stirred with 20 ml dry pyridine. The reaotion mixture was stirred and oooled on an ioe bath. Methanesulfonyl chloride

(0,75 g, 0,00655 mole) in 5 ml dry pyridine was slowly added to the reaotion mixture. The reaotion was allowed to warm up to 1|0° and was stirred overnight. The reaotion mixture was poured into ioe water and extraoted (EtgO),

The ether layer was washed several times with water, dried

(Na2S 0jj), filtered and removed under reduoed pressure affording 0,^50 g light yellow oil, lr (CHCI3) 2160 (SON),

1650 orn-1 (C=C ) * All attempts to orystallize this oil failed,

Attempted reaotion of 3B- aoetoxy-5-oholestene (£i3>) with AgOCN and Ip,- Several attempts were made to reaot oholesterol acetate with I0CN, Procedures similar to those desoribed by Hassner and Heathooolr^ in other unsaturated systems were employed. All attempts failed and only starting material was recovered.

Attempted reaotion of lj.-oholestene-3-one (32) with ...... , . ,. . , — — ...... 1 .1 . ^ .. , . AgOCN and I?,- Several reaotions were oarried out to add

I0GN to the double bond of lj.-oholestene-3-one, However, 80 in all oases only starting material was recovered.

5> oC-Bromo-3^,^-dlaoetoxyoholestane (Ii2) The syn thesis of from oholesterol aoetate was oarried out as described by Levine and co-workers*2 , However, longer reaotion times were employed (up to three hours) in order to obtain a better yield of j?OC-bromo-3yff«6^-dlaoetoxy- aholestane. In all oases this prooedure afforded very impure product whloh was difficult to purify by reorystallization, Reorystallizatlon four to six times from methanol afforded crystals (10-20$), mp 87-90°, lit,*2 mp 89-91°.

iffiffi-Epoxyooprostane^^-ol ( ) from $ oc-bromo- 3ft t6^?-diaoetoxyoholestane (1^2,) .- The reaotion was oarried out by the method of Levine and co-workers*2, However, due to the slight impurity of the starting material this prooedure afforded low yields of pure £jf?-epoxlde (30$), mp 130-131°, lit,*2 mp 132°,

A substantial yield (£0$) of a crystalline complex of and the corresponding 5 PC. 6 QC-epoxlde of oholes terol was obtained, mp 108-109°, lit,^^ mp 107-108°;

-12° (OHBlj), lit.69 M o -15°.

5ff,60-Epoxyooprostane-3^-ol (|+3.) from oholestanetrlol trlaoetate (2_1).- Cholesterol^-oxide was prepared from oholestane-3jB.^ trlol triacetate using a method similar to that of Davis and Petrow in the androstone series as described by Fieser*-^, However, when the reaotion was soaled up to larger quantities of the 81 triaoetate 21 (20 g) the reflux time was increased to 8 hours, Reorystallizatlon from methanol afforded aholes- terol^S,-epoxide (80#), mp 130-131°, lit,^^ mp 131°;

[oc], +8° (CHCI3 ), lit,^2 [<*]D +10°,

3^-Aoetoxy-^,^)9-epoxyoopro3tane iltf) ,- Cholesterol

^-epoxide (Ij^) (25 g, 0,062 mole) in 1E>0 ml dry pyridine was stirred at room temperature, Aoetio anhydride (75 St

0,73 mole) in 75 ml dry pyridine was added and the reao tion mixture allowed to stand at room temperature overnight, poured into ioe water and extracted (Et20), The ether layer was washed several times with water, dried (Na2S0^)f filtered and removed under reduoed pressure affording

27 g white residue, Reorystallizatlon from methanol afforded 25 g (91#) IjJ, mp 112-lli;0, lit ,^2 mp 113°.

5 oC-Azldooholestane-3y& 6jg-diol (lt.0.) was synthesized by the method of Ponsold^-1-, The following modified pro oedure afforded the best yield of the azidosteroid,

5^,64-Epoxyooprostane-3^-ol (2,0 g, O.OOJpO mole) and sodium azide (6,0 g, 0,092 mole) were suspended in

150 ml dry DMSO and the reaotion mixture was stirred and heated to 120° on an oil bath, Sulfurio aoid (2 g, 100#) in 10 ml dry DMSO was added dropwise and the reaotion was heated with stirring for an additional 36 hours. The reaotion mixture was allowed to oool and poured into salt water and extraoted (Et^O), The ether layer washed several times with water, dried (Na2S0^), filtered and removed under reduoed pressure affording after three 82

reorystallizations from methanol, 0,80 g (36$) 5QC-

azidooholestane-3^, 6^3-diol, mp 170-172°, lit,'*1 mp

171-172°.

5 OC-Azldoohol93tanQ-3^f 6fl-dlol-6-aoetate (1^8) wa3

synthesized starting with oholesterol ^?-epoxide acetate

(lj/7) using the same reaction conditions as described

above. Epoxide hi afforded 0,95 6 (h3%) nip l8!|-l88°,

lit,^1 mp 188°; [oc]B _22° (CHCl^), lit 0 -25°,

5 0C-Aminoohol9 3tane-3y3,6fl-dlol (3JD) >- The synthesis

of £ 2 from either the azidodiol or the 3^-aoetate

was oarried out as described by Ponsold 1 , Purification

of the amine was accomplished by formation of the amine

hydrochloride in ether, filtration and neutralization of

the hydroahloride in methanol followed by ether-water

extration. The amine hydroohloride was again prepared and

separated from the ether layer. The hydroohloride was dis

solved in hot methanol and the solution was neutralized with 10# aqueous NaOH, Water was added to the point of turbidity, and the solution allowed to oool, 5 OC-amlno-

cholestane-3^?,6^-dlol crystallizes as white needles, mp

2lj.l-2i|3°, lit,51 mp 2i|2-2l|3°.

Attempted oxidation of 5 oC-azldooholestane-3J<3,^-

dlol-3-aoetate (j^8.) with N-Bromosuooinimide•- Several

attempts were oarried out to oxidize to the corresponding ■a rj 6-one, The prooedure of Pleser-3 desoribed in the oxida tion of oholestane-3ftf 6^-trlol was used. However, in all oases only starting material was recovered. 83

5 oC-Azldo-3j8-aoetoxyoholestans-6-on9 (1^9.),- Chromlo

anhydride (0,176 g, 0,0057 mole) in aoetio aoid (10 ml,

97,5$) was slowly added to 1^8 (1,000 g, 0,0021 mole) in

100 ml aoetio aoid at room temperature. After addition

was oomplete the reaotion mixture was diluted with exoess

water and extraoted (Et£0), The ether layer was washed

with water, filtered and evaporated under reduoed pressure

affording 0,9lf g of white residue, Reorystallizatlon from

methanol afforded 0,70 g (70$) lj^9, mp 186-188°, lit,^ mp

188,5-189°; [cc], _U5° (CHClj), lit,55 M 0 _-l;7.20.

Anal, Calod for G 2gHj|giN^0^! C, 71,90; H, 9,96;

N, 8,65. Pound: C, 71,76; H, 9.5^; N, 8,1*1,

5 OC--Azldo-3Jj6-ao9toxyohole3tane-6-one (1^9.) from

5 oC-azldooholestane-3^,6/S-dlol (1*6),- Chromio anhydride

(0 ,3^0 g, 0,011 mole) in aoetio aoid (30 ml, 95$) was

added dropwise (1 drop every 15 seconds) with stirring to

the azidodiol Ij^ (2,000 g, 0,001*2 mole) in aoetio aoid

(150 ml, 95$) at room temperature. After addition was

oomplete the reaotion mixture was poured into ioe water

and extraoted (Et^O), The ether layer washed several times

with water, dried (Na^SO^), filtered, and removed under reduoed pressure affording 1,9 g white residue. The produot was aoetylated without further purification, Aoetio

anhydride (5 ml) in 5 ml pyridine was added to the oxida

tion product In 50 ml pyridine. The reaotion mixture was

allowed to stand at room temperature overnight, poured into

ioe water and extraoted (Et20), The ether layer was washed 81+

with water, dried (Na2S0^) and evaporated under reduced

pressure affording 2,0 g white orystalllne residue,

Reorystallization twice from methanol afforded 1,5 g

(68$) lj9, mp 187-189°, lit,^ mp 188,5-189°. The product

was identioal In all respects with the product obtained

by oxidation of 5jfi?-azidoaholestane-3^3,6_^rdlol-3-acetate,

3j3-A°etoxy-5 oC-azldo-6^-chlorooholestane (5p) *-

The azido steroid lj^8 (1,00 g, 0,002 mole) in 10 ml chloro

form was stirred at 0° and excess thlonyl chloride (3 ml)

was added, The reaotion was allowed to warm up to room

temperature and was refluxed for 6 hours. The solvent

and exoess thionyl ohloride were removed under reduoed

pressure affording an 1,00 g of an oil whioh did not orys

tallize, Chromatography on 1+0 g silioia acid with

chloroform and ohloroform-methanol (95i5 ) afforded 0,850

St 3y%-aoetoxv-5 pC.azldo-6^-ohlorooholestane (£0,)* Crys

tallization from methanol yielded 0,700 g (67$) 50, mp

120-121°, llt,^ mp 120-122°,

Attempted reaotion of 5 oC-azidooholestane-3yfl,

dlol-3-aoatate (ljJ3.) with p-toluene sulfonyl ohloride,-

Several attempts were made to prepare the 6-tosylate of

5J2£-azidooholestane-3.^,6£-diol-3-aoetate using a wide vari ety of reaotion conditions. The reaotion was oarried out with a twenty molar exoess of p-toluene sulfonyl ohloride in pyridine at room temperature for up to four days. In

addition, the reaotion mixture was heated on a steam bath for up to twelve hours. In all oases only starting material was isolated, 5oC-A z idoohole at ane-3^, ^Q-dlol-3-ao at ate-6-methane

sulfonate ($1) ■ - 5.fiC-Azidooholestan9-3^,6y(^-diol-3-aoetat9

was oonverted to the 6-ma3ylat9 derivative by the method

of Ponsold^, We found it was important to aarefully dry

the starting azide. This was accomplished by azeotroping

the water present with benzene. The 6^-mesylate ^1 was

obtained, mp 125-126°, lit,^6 mp 125-126°,

5 PC? 6 QC-Iminooholest ane-3yff-ol (^?) »- 5 OC-Azidooholes- tane-3^ , 6Jg-dlol-3 -aoetat9-6-methanesulfonate (£l) (1,100 g,

0,00177 mole) in 50 ml dry THP was added dropwise with

stirring to LiAlH^ (2,00 g, 0,0525 mole) in 50 ml THP cooled on an ioebath. The reaotion mixture was allowed

to warm to room temperature and stirred for an additional

21* hours, The exoess LiAlH^ was decomposed by slow addi

tion of 2 ml aqueous 10$ NaOH and 8 ml water to the oold

reaotion mixture, The precipitate was filtered and washed

twice with THP, Evaporation of the combined filtrate

afforded 0,700 g white residue, Reorystallizatlon twioe

from methanol yielded 0,1*00 g (5l$) 52, mp 21l*-2l5°, lit,^6 mp 211*0,

3-Oximlnooholestane-5cx:,6j/^-dlol-6-aostate (£3 )»-

Gholestane-5^’,6^-dlol-6-acetate-3-one (2,1* g, 0,0052 mole), hydroxy lamina hydroohloride (3.0 g, 0,01*32 mole) and NaOAo *3H20 (1*,5 E» 0,031*6 mole) were stirred with

100 ml absolute ethanol. The reaction mixture was refluxed for 8 hours, poured into ioe water and extraoted (Et^O),

The ether layer was washed with water, dried (Na£So^) 86

filtered and removed under reduoed pressure affording

2,5 g white product, Rearystallization three times from

ether-aoetonitrile afforded l.l^g (£6$) 53, softening

point 115°, 200° (deo,).

Anal, Calod for : C } 73,22; H, 10,38;

N, 2.9U. Found: C, 73.37; H, 10,07; N, 2,93.

3^-Amlnooholestane-5o^,6^)S-dlol (E&.)»- The 3-oxime

53 (2 ,l| g, 0,0052 mole) in 50 ml anhydrous ether was added

dropwise with stirring LiAlH^ (2,5 g, 0,066 mole) in 50 ml ether at room temperature. The reaotion mixture was

allowed to stir at room temperature for an additional Lj8 hours. The exoess LIAIH^ was deoomposed by dropwise addi tion of 2,5 ml 10$ NaOH and 10,0 ml water. The filtrate was collected and the precipitate extraoted twice with

THF, The combined filtrate was evaporated under pressure affording 2,0 g (86$) white residue. Crystals formed from methanol, mp 175-178°,

Anal, Calod for C ^ H ^ O ^ 0, 77.25; H, 11,75;

N, 3,35. Found: C, 76,35; H, 11,52; N, 3.18,

3^-Aoetamldooholestane-5 QC,6^-diol-6-aoetate (55J >- Aoetio anhydride (10 ml) in dry dyrldlne was added to the 3y3-amine 5U (2,0 g, 0,00l|8 mole) in 50 ml dry pyridine.

The reaotion mixture was allowed to stand at room tempera ture overnight, poured into ioe water and extraoted (Et20),

The ether layer washed several times with water, dried

(Na2S0|^) and evaporated under reduoed pressure yielding

2,20 g white residue, Reorystallizatlon three times from acetone afforded l*6g (67$) white crystals mp 205-207 •

Anal, Calod for G3iH53N0^ : G » 73*91; H, 10,61;

N, 2,78, Found: C, 73.5*4 J H, 10,48; N, 2 ,86,

Chol9stana-3j3,5 oC,6j6-trlol-3-p-toluenesulfonate (56),

Cholestanetriol (10,0 g, 0,0238 mole) was dissolved in

200 ml of dry pyridine, jp-Toluenesulfonyl ohloride

15*0 g (0,0785 mole) in 100 ml pyridine was added dropwise with stirring to the reaotion mixture at room temper ature, The reaotion was allowed to stand at room tempera ture overnight then poured into ioe water and extraoted

(Et20), The ether layer was washed with 10$ aqueous

NaHCO^ and water, dried (Na2S0[j) ancj removed under reduoed pressure affording 13,0 g of crystalline product. The orude oholestane-3y3.9 cC. 6yfl-trlol-3-tosylate was reorystallized twloe from aoetone affording, 8,5 g (62$) £6, mp li|8-l50° deo,, lit,^8 166° deo,

Anal, Calod for C ^ H ^ O ^ S : C, 70,91; H, 9,45;

S, 5,57, Found: C, 71,44; H, 9,73; S, 5,66,

Reaotion of oholestana-3y&5 oC»6/?-trlol-3-p-toluanesulfonate (56) with ammonia,- Several attempts were made — — - 1 ■ fN H I.... 1 — — ... to displace the 3^-tosylate of oholestane-^,5^S*,6^-triol with ammonia. All attempts failed with exoess ammonia in a oitrate bottle at room temperature. Only starting mate rial was recovered,

Cholestane-3^,5_^£, 6^-triol-3-p-toluenesulfonate (10 g, 0,0174 mole) was placed in a glass lined stainless steel bomb. The reaotion bomb was oooled in a dry ioe-aoetone bath for five minutes and exoess liquid

ammonia added (approximately 100 oo), The bomb was

sealed and heated in an oven at 100° for l|8 hours. The

exoess ammonia was allowed to esoape at room temperature.

The residue was extraoted with ether and water, evapora

tion of the dried (Na2S0j^) ether layer afforded 7.3 g

residue. Attempts to orystallize this product failed,

The residue was dissolved in ether, HC1 added and the

preoipltate oolleoted. The ether layer was removed under

reduced pressure affording a white residue whioh crys

tallized from ethanol affording 2,1 g (30$) white crystals

of 3j2£>!^OC-oxidocholestane.-6^-ol, mp 130-131°,

Anal, Calod for CgyHi^Og: C, 80,51]; H, 11,52,

Pound: C, 80,66; H, ll,i|0.

The amine hydroohloride was neutralized and extraoted

(Et20), The dried ether layer (Na2S0^) removed under

reduoed pressure affording L[,0 g yellow residue, All

attempts to orystallize this product failed, Aoetylation was oarried out with aoetio anhydride (10 ml) in $ 0 ml

dry pyridine affording 1],1 g (lj7$) of a white produot.

Chromatography on 100 g sllloio aoid with chloroform and ohloroform-methanol (95:5) afforded 3jX-aoetamidooholes- / tane-5 QC* 6jg-dlol-6-aoetate (£l) • Reorystallizatlon from aoetone yielded white needles, mp 126-127° (foams),

Anal, Calod for C ^ H ^ N O ^ : C, 73.91; H, 10,61;

N, 2,78, Pound: C, 7^,23; H, 10,75; N, 2,99, 89

3 CC-Azldooholestane-5 6^-dlol-6-aoetate ( 63.) from oholestane-3 ^, 5 gC-6#-triol-3-tosylate (£6 ),- Cholestane-

triol-3-P-toluanesulfonate (0,500 g, 0,0087 mole) and

2 g (0,031 mole) sodium azide were suspended in £0 ml

BMP, The reaotion was heated and stirred at 100° for 10 hours, The reaction mixture was poured into salt water-

ice, extracted (Et^O) and washed several times with water.

Evaporation of the dried (Na2S0j^) ether layer afforded

0,375 gm (96$) d e a r oil. Chromatography on [|0 g silica gel with ohloroform as the eluent afforded 0,350 g azidosteroid whioh failed to crystallize, Azidosteroid

62^ was aoetylated by treatment with 2 ml acetic anhydride in 10 ml dry pyridine at room temperature overnight. The reaotion mixture was poured into ioe-water and extraoted

(Et20), The ether layer washed several times with water, dried (Na2S0^), filtered and removed under reduced pressure affording 0,3^0 g white product, Reorystallizatlon from aoetonemethanol (1 :1 ) afforded 0,250 g (17$) 63, mp 77-78°,

Anal, Calod for C 29Hlj9rT3 °3 : c > 71 »Ul; H, 10,13; tf, 8,62, Pound: C, 71,51; H, 10,13; N, 8 ,73,

Cholestane-3^,5 PC, 6yg-trlol-3-methanesulfonate-6- aoetate (61j),- Cholestane-3^3.5 PC. 6^-trlol-6-aoetate (2ij,)

(0,832 g, 0,0018 mole) in 50 ml dry pyridine was stirred and oooled on an ioe bath, Methanesulfonylchloride (2 ml) in 5 nil dry pyridine was added dropwise. The reaotion mixture was allowed to stand at $°C overnight, poured into ioe water and extraoted (Et20), The ether layer was washed 90 with water, dried (Na^SO^), filtered and removed under reduoed pressure affording 0,8lL|. g white solid* Reorys-

tallization from acetone afforded 0,670 g (69$) 61+, mp

155-156° dec.

Anal* Galod for C^ qH^O^S: ^6,38; 9*66;

S, 5.91. Pound: C, 67.17; H, 9.73; S, 5.93.

3 oC-Azidooholestane-5 C£,6yg-dlol-6-aoetate (ffi.) from oholestane-3^,5 PC, 6yg-trlol-3-methanesulfonate-6-aoetate

(6Ij.),- Mesylate 61^ (0,550 g, 0,00102 mole) and 1,0 g

(0,015 mole) sodium azide were stirred with 30 ml DMP,

The reaotion was heated to 90° for 20 hours then poured into salt-ioe water and extraoted (Et20), The ether was washed several times with water, dried (Na^O^), filtered and removed under reduoed pressure affording 0,^50 g arystalline product, Reorystallizatlon from aoetone- methanol (1:1) yielded 0,310 g (63#) 63, mp 78-79°* This produot was identioal in all respeots with the azide obtained by displaoement of the 3-tosylate followed by aoetylatlon,

5 QC,6oC-Epoyy-3^-oholestanol methanesulfonate (6gJ from oholestane-3y8,5 PC, 6yg-trlol (l5.)»- Cholestane-3^.5 oC 6^.-trlol (5,0 g, 0,012 mole) in 100 ml dry pyridine was oooled on an ioe bath, Methanesulfonyl ohloride (10 ml) in 20 ml dry pyridine was added dropwise with stirring.

The reaotion was allowed to stand at room temperature over night, poured into ioe water and extraoted (Et20), The ether layer was washed with water, filtered, and evaporated 91 under reduoed pressure affording 5 t0 g white residue,

Reorystallization twioe from aoetone affords 3.5 g (51$) white crystals, mp II46-I500 (deo, 167°),

Anal, Calod for C jsH^q O^S: C, 70,00; H, 10,07;

S, 6,67. Found: C, 69.27; H, 9,63; S, 6,5^1.

5 PC,6 OC-~Bpoxyoholestane-3yS-ol ( 66),- Cholestane-3^3, 5_2C, 6^-triol-3-ethyloarbonat e-6-methane sulfonate (20 g,

0,035 mole) was dissolved in 250 ml hot ethanol, Alooholio

KOH (200 ml, 10$) was added and the reaotion mixture refluxed for 2 hours then allowed to oool to room tempera ture, The reaotion mixture was neutralized with glaoial aoetio aoid and extraoted (BtgO), The ether layer washed several times with water, dried (Na^O^) and removed under reduoed pressure affording 13,0 g white residue, Reorys tallization twioe from methanol yielded 9.5 g (6l|$) 66, mp 1^1-11*2°, lit,70 mp li|2,5°.

(0,500 g, 0,0012 mole) in 25 ml dry pyridine was oooled on an ioe-salt water bath, Methanesulfonyl ohloride

(0,5 ml) in 5 ml dry pyridine was added dropwise with stir ring, The reaotion mixture was allowed to stand at -5° overnight, poured into ioe water and extraoted (Et20), The ether layer was washed several times with water, dried

(Na^Ojj), filtered and removed under reduoed pressure affording 0,550 g white product, Reorystallization twioe from aoetone yielded 0,200 g 65, mp li|.l|-ll|80 (deo, 165°), This produot was Identical in all respects with the methanesulfonate 65 obtained from triol 15.

3 PC- A zldo-5 OCt 6flC-epoxyoholestane (&£)»- Mesylate ^

(2,00 g, 0,001^2 mole) and sodium azide (6,00 g, 0,091 mole) in 50 ml DMF were heated and stirred at 90° for 21+ hours. The reaotion mixture poured into salt-ioe water and extraoted with ether. The washed ether layer washed with water dried (Na^SO^), filtered and evaporated under reduoed pressure affording 1,62 g crystalline residue,

Reorystallization from aoetone gave 1,35 g (75$) needles,

3_QC-azldo-5<>Cf 6-OC.epoxyoholestane, mp 13l+-135° •

Anal, Calod for C27H1+5N3° ; c, 75*83; H, 10,61; N, 9,83, Found: C, 76,16; H, 10,28; N, 9,90,

3 of-Azldooholestane-5 PC, 6^dlol-6-aoetate ( £3.) from

(0,1+00 g, 0,00091+ mole) was refluxed in 30 ml aoetone.

Periodic aoid dehydrate (0,21*5 g, 0,00091+ mole) in 5 water was added, The reaotion mixture was refluxed for an additional 30 minutes, poured into ioe water and extraoted (EtpO), The ether layer washed with water, dried

(Na2S0[j) and evaporated under reduoed pressure affording

0,395 g (95$) white orystalline residue, Aoetylation of the residue was oarried out with 5 ml aoetio anhydride in

30 ml pyridine at room temperature overnight. The reaotion mixture was poured into loe water and extraoted (Et20),

The ether layer washed several times with water, dried

(Na^SOiJ and removed under reduced pressure affording 0,390 g white residue, Reorystallizatlon from aoetone- methanol (1:1) yielded 0,270 g (59^) 63, mp 79-80°, This

produot was identloal in all respeots with compound 6^ synthesized by the other methods described,

3 oC-Amlnooholestane-^QC,6^?-dlol (57.) from LiAlH[j reduction of 3 flC-azidooholsstane-5 OC, 6j$-dlol (&?.),-

Azidodiol 62 (0,250 g, 0,00056 mole) in 20 ml ether was /'s - y added dropwise with stirring to LiAlH^ (0,250 g, 0,0066 mole) in 50 ml ether at 0°, The reaotion mixture was allowed to stir at room temperature overnight. The exoess

LiAlHjj decomposed by slow addition of 0,25 ml aqueous NaOH

(1C$) and 1,0 ml water. The filtrate was oolleoted and the preoipitate extraoted twioe with THP, The combined filtrates were distilled under reduoed pressure affording

0,050 g (21%) yellow crystals, mp 120-132°,

Anal, Calod for C g y H ^ O g : C, 77,25; H, 11,75;

N, 3,35, Pound: C, 77,46? H, 11,55; N, 2,94.

The orude amine was oonverted to amide 61 by the aoetylation procedures previously described. Amide

(mp 125-127°) was identical in all respeots with the amide obtained by aoetylation of the displacement produot of tosylate 56 with ammonia,

3 OGAminooholestane-5 OC,6Jg-dlol (57.) from L1A1H)| reduotlon of 3 QC-azidooholestane-5oC,6yft-dlol-6-aoetate

(63),- Aminodiol 5 1. was synthesized from aoetate 63 via

LiAlH^ reduction as desorlbed above in 45# yield. Again the amine was characterized as the amide 61, 9h 3 QC-Aminoohole3tane-5 tX,6^3-dlol (5.7) from hydrazlne-

Raney nickel reduction of 3 OC-azidooholestane-5 0C,6^-dlol

(62),- Azidodiol p2 (0,300 g, 0,00067 mole), hydrazine hydrate (1 ml) and a small amount of W-2 Raney nickel were refluxed in 50 ml ethanol for one hour. The reaction mix ture was allowed to cool and 100 ml ether added. The mix ture was set aside until gas evaluation oeased (overnight).

The mixture was filtered, the ether layer washed with water, dried (Na2S0^) and distilled under reduced pressure affording 0,275 g (97%) white residue, This product was

aoetylated yielding the corresponding amide for character

ization purposes. The amide obtained was identioal in all respects with those prepared by the other methods, 3 oC-Amlnooholestane-^OC^^-diol-b-aoetate (frQj t_

Azide 63 (0,^00 g, 0,00082 mole) was oonverted to the amine

68 by the same prooedure as above, except 1,5 ml hydrazine hydrate was used. The reaction produot (0,370 g) would not orystallize. Analysis was carried out on the orude produot,

Anal, Calod for G 29h53n °3 : G, 75,^3; H, 11,15; N, 3.03, Pound: C, 714,59; H, 11,33; N, 2,72,

Amino aaetate 68 was treated with aoetio anhydride in pyridine under the usual reaction conditions affording amide ^ which was identioal in all respects with the amide prepared by other methods,

6-Oximinoohol9Stane-3/?,5oC-dlol (69.) was synthesized by the method of Shoppee^, mp 188-189°, lit,^° mp 170/190- 192° (double melting point), lit,61 21+8-250° p

6j|£-Amlnooholestane-3j|!3 0C-dlol #ZP.) from 6-oximlno-

Qhole3tane-3y3,5 oC-dlol ( 6 9 . ) Amine JO, was prepared by

LlAlHjj reduction as desorlbed by Shoppes60, However, the amine prepared by this method was very difficult to

orystallize, mp 196-199°. lit,60 mp 199-200°,

6jff-Azidooholestane-3&!?OC-dlol~3-ethyloarbcixylat9 ( 7JI.), Mesylate 2J (0,500 g, 0,000875 mole) and sodium azlde

(0,550 g, 0,0077 mole) In 50 ml DMP were heated with

stirring to 100° overnight. The reaction mixture was

poured into salt-ioa water and extracted (Et20), The ether layer washed with water, dried (Na2S0^), filtered and evaporated under reduced pressure affording 0,360 g white orystals, Reorystallization twice from methanol affords 0,290 g (62%) 71, mp 128-130°,

Anal, Calcd **or : C, 69.59; H, 9.93;

N, 8,12, Pound: C, 70,1;8; H, 9,77; N, 7.68,

6^-Azldooholestano-3/3,5 OC-dlol-3-aoQtate ( JgJ ,-

Cathylate 71 (2,80 g, 0,0051; mole) was dissolved in 100 oc hot ethanol. Aqueous NaOH (10 ml, 10^) was added and the reaction mixture refluxed for 2 hours, cooled, neutralized with aqueous HC1 (10%) and extraoted (Et20),

The ether layer was washed with water, dried (Na^SO^), filtered and removed under reduced pressure affording

2,1+0 g white residue. The reaction produot was aoetylated under the usual conditions of aaetlo anhydride in pyridine yielding 2,1+5 g 72, Reorystallization twice from methanol afforded 2,00 g (76%) 72, mp 151+-1550, lit,51 mp 151+°. 96

66-AminoQholestane-3/3,9 oC-diol-3-aoetate (73) from i t fTi ■ ■ — »i pi ■ » m ■ i — ■ i * / ■ ■ ■ — i ■ ■ ■■ i i, ^ i ■ ■ i 6^3-azldooholestan9-3/?, 5 oC-dlol-3-aoetat9 (72) ,- The method

of Ponsold^ was used to prepare 73, rap 190-191°, li t,^

mp 190-191°,

6J(g-Amlnoohol93tane-3^ >^ oC-diol (c^p) from 6^3-amino-

oholestane-3^,5 jXl-dlol-3-acetate (73.),- Amine £0 was

prepared by hydrolysis of aoetate 73 as described by

Ponsold^1, mp 198-199°, lit,^1 mp 199-200°,

3,6-Dioximlnooholestane-9 <%-ol (7U),- Cholestana- — . . ,. _.. . 3.6-dione-9 flf-ol (14,5 g, 0,0108 mole) and 10,0 g (0, II414

mole) hydroxylamine hydroohlorldo were suspended In 5>0

ml absolute ethanol and 20 ml dry pyridine, The reaction

mixture refluxed for seven hours, poured into ioe-water

and extracted with ether. The ether layer was washed with

water, dried (Na2S0^), filtered and evaporated under reduced pressure affording [4,7 g of a white residue,

Reorystallization twioe from methanol gives 3,6 g (7k%)

Ik, mp 188-190° dec, /N«* Anal, Galod for Cf 72,60; H » 10*38; N , 6,27, Pound: C, 72,58; H, 10,1+7; N, 6,18,

3yfl,6y$-Dlaminoohole3tane-5flC-ol (25.),- 3 ,6-Dioxlmlno- oholestane-^gC-ol (1,000 g, 0,0022 mole) in 79 ml ether was added dropwise with stirring to LiAlH^ (3,000 g,

0,029 mole) in £0 ml ether at 0°, The reaction mixture was allowed to stir at room temperature for 21+ hours.

The excess LIAIH^ was decomposed by dropwise addition of

1 ml aqueous NaOH (10$) and ij ml water. The filtrate was collected and the preoipitate extracted twiae with THP,

The combined filtrate was distilled at reduced pressure affording 0,$00 g white produot whioh orystallized with difficulty from methanol-water, affording the diamin

75, mp 1U5-1U80 •

Anal, Calod for G 77 ; H, 12* 0l| ;

N, 6,69, Pound: C, 77,69; H, 11,55; N, 6,10,

3fi,6^-Diaoetamidooholestan9-5 OC-ol (*7.6.)Aoetio anhydride (5 ml) In 5 tnl dry pyridine was added to the diamine 75 (0,010 g, 0,0015 mole) dissolved in 20 ml dry A/ pyridine. The reaction mixture was allowed to stand at room temperature overnight, poured into ioe-water and extraoted (Et20), The ether layer washed with water, dried (Na2S0^), filtered and removed under reduced press ure affording 0,560 g crystalline residue, Reorystalliza tion from ether afforded 0,310 g (l\2fo) dlamide 76, mp

119-120°,

Anal, Calod for G 31U ^ 203 : 0, 7^,05; H, 10,63;

N, 5,57, Pound: C, 7^,09; H, 11,08; N, U,99, SUMMARY

The observation that oholestane-3^,5 oCt 6yg-triol exhibits hypocholesterolemlo activity in experimental animals prompted investigation of this oompound in our laboratories for purposes of obtaining data which may help to determine meohanism of action and structural require ments for suoh aotivity, Cholestanetriol dramatically retarded the usual development of aortic atherosclerosis and reduced total cholesterol concentration of the aorta in rabbits. The serum cholesterol levels, degree of athero- sohlerosis and concentration of cholesterol and oholestanetriol was measured under a variety of feeding conditions.

Tissue and fecal cholesterol concentration studies were carried out. In vivo studies utilizing C^-oholesterol and C ^-oholestanetriol are also disaussed. Data show that oholestanetriol blocks the usual absorption of cholesterol into the blood and extra-intestinal tissues.

The sterol balance investigations with radio-labeled and non radio-labeled oholestanetriol show that only negligible amounts of the oompound is absorbed into the blood and extra-intestinal tissues. The triol does accumu late to some extent in the intestinal mucosa.

98 99

Preliminary in vitro investigations with oholestane

triol indioate that this sterol blooks the biosynthesis

of cholesterol at several stages between mevalonate and

cholesterol. The lack of toxioity and absorption of

oholestanetriol indioates that this oompound may be of

value in treating hyperaholesterolemia and atherosclerosis

in humans,

A number of compounds related to oholestanetriol were

synthesized and subjected to iji vivo and _in vitro biologioal

evaluation, The 3,6-diester of oholestane-triol has been

shown to have i_n vivo hypoaholesterolemlo aotivity. However,

the triacetate derivative, the 6-keto analog and the 3,6-

diketone exhibited little activity, In addition, the 3^,

6^-trihydroxy derivative of ^-sitosterol showed little

activity, In vivo evaluation of these compounds are presently being oarried out. The 3,6-dlketone derivative blooks oholesterol biosynthesis between mevalonate and squalene and

inhibits the conversion of ^-aholastadlenol to cholesterol.

In order to further study analogs of oholestanetriol and to test the proposal that the 5 gC-amino system may bloak certain steps in oholesterol biosynthesis (related to demethylation of lanosterol), we synthesized this oompound as well as a number of position Isomers, Several synthetic approaches used In the synthesis of the various amino analogs of oholestanetriol are lisoussed, 9 oC-Amlnooholestane-.

6^-diol, 3 ft -amlnooholastane-9 OC. 6yS-dlol. 3 QP-amlnooholestane-

!?0C-6jB-diol. 6^-amlnooholeatane-37^,£i£-dlol, 3^,^-dlamlno- oholestane-c? OC-ol as well as the corresponding amide and 10 0 aoetate derivatives were synthesized. The synthesis of several related analogs and intermediates are discussed.

The oximes of the 3- and the 6-keto oompound as well as the

3 ,6-diketo system, 5^0C-azldoaholestane-3^ , 6j(|-diol and derivatives, SaC, 6oC-lmlnooholestane-3^?-ol, 3 OC-azidooholestane-5!_2C,6^-diol-6-aoetate, 3 PC-azido-5>OC, 6_C>£-epoxyoholestane, ^-azidooholestane-3_^,5j2f“diol-3-oathylate, and related derivates were prepared.

In vitro biologioal evaluation of the E? (XT-amlno isotere show the oompound to inhibit the biosynthesis between oholestadienol and oholesterol as well as between mevalonate and squalene. In addition, this oompound appears to be a more effective inhibitor than oholestane-3^3,50C,6^-trlol,

Related position isomers are presently being investigated.

During the course of these investigations we measured the optioal rotatory dispersion (ORD) curves of the 3 and

6 monoketones plus the 3 ,6-dlketo derivative as well as their corresponding oximes. As expected conversion of the ketone to an oxime does not alter the sign of the Cotton effeot, However, in the oase of the 6-oxlme derivatives the ORD aurve shifted by 95 to lower wavelength and the moleoular rotation of the trough increased threefold. These results aided In characterization of the oximes; a physical interpretation of the results must await further work, REFERENCES

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(1+7) K, L, Oliver and W , G , Young, J, Am, Chem, Soo,, 81, $811 ( 1 9 $ 9 ) •

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(1+9) A, Hassner, M, L, Lorber and C, Heathoook, J, Org, Chem,, 32, $1+0 (1967), -106

(50) (a) K, Ponsold and D, Eiohhorn, Z, Chem,, 8, 59 (1968); (b) G, Drefahl, K, Ponsold, D, Eiohhorn, Ber,, 101, 1633 (1968),

(5D (a) K, Ponaold, Bar,, 95, 1727 (1962); (b)K, Ponaold, Ibid., 96, ll|TI (1963)*

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(53) L, F, Fieaer and M, Fieaer, Steroida, Reinhold, N, Y,, 1959, P, 198,

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(55) G, Snatzke and A, Veithen, Ann,, 703, 159 (1967),

(56) K, Ponaold, Ber,, 97, 352l| (1961;),

(57) C, W, Shoppee, P, Ram- S, K, Ray, J, Chem, Soo, (C), 1018, 1023 (1966),

(58) R, Bourdon and 3, Ranisteano, Bull, Soo, Chim, France, 1977, 1982 (I960),

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(60) C, W, Shoppee and S, L, Roy, ibid,, 3771; (1963),

(61) D, Gunther and K, Ponaold, Ber,, 91, 271 (1958),

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(63) PI, A, Plattner, Th, Petrzilka and W, Lang, Helv, Chim, Aota, 27, 513 W b b ) *

(61;) D, H, Coffey, I, M, Heilbron and F, 3, Spring, J, Chem, Soo,, 738 (1936),

(65) J, C. Eck, R, L, VanPeursem and E, W, Hollings worth, J, Am, Chem, Soo,, f 171 (1939), 107

(66) A, 3, Hallsworth, H, B, Henbest and T, I, Wigley, J , Cham, Soo,, 1969 (19$7).

(67) I, Kitagawa, Y, Ueda, T, Kawaski and E, Moaettlg, J, Org, Cham,, 28, 2 22 8 (1963), (68) American Cyanamid Co,, Industrial Chemical Division, Wayne, New Jersey,

(69) (a) J, Hatlori, J, Pharm, Sol,, Japan, 60, 12£ (191+0); (b) R, A, Baxter and P, S, Spring, J, Chem, Soa,, 613 (191+3).

(70) A, Furst and P, Koller, Helv, Chim, Aota, 30, ll+!?l+ (19U7)• “