CA1113086A

CA1113086A - 25-alkylcholesterol derivatives

Info

Publication number: CA1113086A
Application number: CA310,200A
Authority: CA
Inventors: Robert J. Chorvat
Original assignee: GD Searle LLC
Current assignee: GD Searle LLC
Priority date: 1977-08-29
Filing date: 1978-08-28
Publication date: 1981-11-24
Also published as: ES472877A1; DE2837414C2; GB2009180B; JPS6259720B2; FR2401935B1; IT1106121B; AU3931878A; CH643566A5; JPS5448751A; GB2009180A; DE2837414A1; IT7850879A0; FR2401935A1; AU520334B2

Abstract

Abstract of the Disclosure The present invention relates to the prepartation of derivatives of 25-alkylcholesterol which display valuable pharmacological properties in that they inhibit the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, an enzyme which controls the rate at which cholesterol is synthesized in mammalian liver (one of the two principal sources of serum cholesterol). .

Description

~l3~86 . . :.

The present invention relates to a group of 25-alkylcholesterol.derivatives of the formula ` "
.
Rl ., ~ CH3 RO

wherein R2 represents alkyl having 1-4 carbon atoms, R . . !1 represents hydrogen, alkanoyl containing 2-7 carbon atoms or a radical of the formula O o '.
Il 11 ~O-C ~-- CH2 '~` C -- ~ -.

¦~ in which n represents a positive integer less than 4;
Y represents methylene or carbonyl; and whenever Y is ~: carbonyl, Rl represents hydrogen, and whenever Y is ~: -methylene, Rl represents OX wherein X represents hydrogen, alkanoyl containing 2-7 carbon atoms, or a radical of ~ the formula :
O o 11 11 .
HO--CtCH2 tn~C , "

.

.

.

:
in which n represents a positive integer less than 4;
and the wavy line represents R or S stereochemistry.

. . .
An embodiment of this invention are compounds of the formula OX
H3C~ ~ CH3 ., ~ .

Ro , wherein R2 represents alkyl having 1-4 carbon atoms;
R and X each represent hydxogen, alkanoyl containing 2-7 carbon atoms or a radical of the formula o o HO-C~CH2~ C--. .
in which n represents a positive integer less than 4;
: and the wavy line represents R ~r S stereochemistry.
' `10 Another embodiment of this invention are compounds of the formula ~ ~ CH

~ ~ C~3 RO O

;.

.: , - ' ~-. :, .

`
wherein R2 represents al~yl having 1-4 carbon atoms;
Rl represents hydrogen; R represents hydrogen', alkanoyl ' containing 2-7 carbon atoms or a radical of the formula O O
Il 11 HO-C ~ C~2 ~ C -in which n represents a positive integer less than 4;
and the wavy line represents R or S stereochemistry. ~'~
'' The compounds of the present invention are useful by reason of their valuable pharmacological properties.
Thus, for example, they inhibit the activity,of ~-hydroxy-~-methylglutaryl coenzyme A ~MG CoA) reductase, an enzyme which controls the rate at which eholesterol is synthesized in mammalian liver (one of the two prineipal sources of , serum cholesterol).
~' A major risk in the davelopment of atherosclerotic '~
disease and associated clinical conditions is the level ~15 of circulating serum total cholesterol. As the level of , serum total cholesterol rises above 180 mg~ml the risk of atherosclerosis also increases~ The low density lipoproteins whieh are rich in cholesterol have been implicated as the ` primary vehicle for carrying cholesterol which will be ,20 deposited in tissues.
, ' ' ' .

The major source of arterial cholesterol in the ~' atherosclerotic patient appears to be of endogenous ~
origin. A reduction in the rate of eholesterol biosynthesis , will lead to a lowering of serum cholesterol levels.
.~ . ' ' -The rate limiting step in the biosynthesis of cholesterol is the enzymatic reduction of ~-hydroxy-~-methylglutaryl CoA (HMG CoA) to mevalonic acid (MVA) by the enzyme ~MG CoA reductase. Therefore, the regulation of cholesterol biosynthesis by suppressing the activity of HMG CoA
reductase will lead to a lowering of serum cholesterol levels.
.
Compounds of the present invention inhibit the activity o~ HM~ CoA reductase. This type of activity should ~10 be particularly useful in controlling type II hypercholesterol-emia, an inherited condition caused by an autosomal dominant mutation of a single gene locus [Brown and Goldstein, Science 191, 150 (1976)].

; Reduction of HMG CoA reductase activity by the present compounds is demonstrated by the following assay.
Male rats of the CD strain from Charles River weighing 180-250 g. initially, being kept on a regular laboratory diet, are used. The rats are maintained in reverse light cycle room for 3-6 days. 20,25-Diaæacholesterol is admin-. . . _ ~20 istered for a total of 6 days at~a dose of 5mg/kg/day (IG).
The last 3 days the test compound is administered along with the 20,25-diazacholesterol. Both compounds are given

2 hours prior to the test on last day. The~rats are ` anesthetized with ether, sacrificed, and the livers .
;25 re ved. Liver microsomes are collected by differential centrifugation after homogenation. Liver microsomes are used as the source of the HMG CoA reductase. Details of the '' .' .
'.

_5_ .. , ~, ~.

.:

assay are reported in L. W. White and H. Rudney, Biochemistry 9, 2713 (1970); Brown et al., J. Biol. Chem. 248, 4731 (1973); and C. A. Edwards, Biochem. Biophys. Acta. 409, 39 (1975). The percent change in formation of [14C]-S mevalonic acid from [14C]-HMG CoA is used as a measure of enzyme activity for treated groups versus control groups of rats. If the treated groups have less activity, and the decrease is statistically significant at P < 0.05, the compound is rated active.

The compounds 25-methylcholest-5-en-3~,22-diol and 3~-hydroxy-25-methylcholest-5-en-7-one of the present invention were found active by the above test at doses of 5mg/kg (IG). The corresponding compounds of the natural series lacking the 25-methyl group, cholest-5-en-3~,22-diol lS and 3~-hydroxycholest-5-en-7~one, were both found inactive by the above test at doses of 30mg/kg (IG and SC). The explanation of the difference in biological activities of the synthetic vs the natural series is probably due to the diffexence in the rate of metabolism of the two series;
.
sea Counsell et al., J. of Lipid Research, 18, No. 1, 24-31, (1977). These results are surprising in view of M. S. Brown and J. L. Goldstein, J. of Biol, Chem., 249, `~ No. 22; 7306-7314, 7308 (1974) which stated that the addition of a methyl group or ethyl group to~position 24 :25 of cholesterol significantly reduced its HMG CoA reductase inhibitory activity.

.` `

~ . . ...

f ~

The compound 25-methylcholest-5-en-3~-ol~3~-acetate of the present invention has been disclosed in Elser, W. et al., Mol. Crys. and Liq. Crys. 13 p 255-_ .
270 (1971). The article teaches the mesomorphic behaviGr of modified cholesterols and makes no mention of pharma-ceutical utility or pharmaceutical preparations.
~, '.
The compounds herein described are combined by - art recognized techniques with pharmaceutically acceptable carriers to provide novel pharmaceutical compositions. The concentration of active ingredient in the composition is not critical, but is preferably 1-80~. These compositions can be administered orally, suitable forms for such admin-istration including tablets, lozenges, capsules, dragees, pills, powders, solutions, suspensions and syrups.
Acceptable pharmaceutical carriers axe exemplified by gelatin capsules: ~ugars such as lactose or sucrose;
starches such as corn starch or potato starch; cellulose derivatives such as sodium carboxymethyl cellulose, ethyl ~ - cellulose, methyl cellulose, or cellulose acetate phthalate;
;20 gelatin; talc; calcium phosphates such as dicalcium phosphate or tricalcium phosphate; sodium sulfate; calcium ~; sulfate polyvinyl pyrrolidone: acacia; polyvinyl alcohol;
stearic acid; alkaline earth metal stearates such as magnesium stearate; oils ~uch as peanut oil, cottonseed oil, sesame oil; olive oil, corn oil, oil of theobroma; wat~r;
agar; alginic acid; and benzyl alcohol, as well as other . :
'~ non-toxic compatible substances used in pharmaceutical .
i formulations.

'`: , ;

In addition to the above utility, compounds of the present invention are also useful as antiprotozoal agents, particularly anti-Trichomonas vaginalis.
.
Compounds of the present invention are obtained by processes originating with the compound 6~-methoxy-3~, 5-cyclo-5a-23,24-bisnorcholan-22-al lSteroids, 15, 113 (1970)] having the formula ''' ' ' H3C \ CHO

I

~;~ OCH3 .~ .

.
`~ 6~-methoxy-3a,5-cyclo-5~-23,24-bisnorcholan-22-al is alkylated by the addition of a 3,3-dimethylalkyl magnesium halide having the formula ~:~ CH3 __ i I `
, ~ X - Mg-cH2 - c~2 - f-R2 ?i;~ CH3 ` wherein X is a halogen and R2 is an alkyl c~ntaining 1-4 `
carbon atoms. The resultant i-steroid alcohol of the j~ formula .;' , :
'.' ' ' ', ' ~' . . .

~, : ~ ~ ....
:' ' ' . ' : .
~. :

3 ~ R

II

.

.~ is rearranged by heating in aqueous dioxane with an acid : such as 4-methylbenzenesulfonic acid monohydrate, giving rise to a diol of the invention, having the formula ~ III ; -.~ HO ~ :

..- .:
. . . .
: ~ Heating a compound of formula II with an alkanoic .~S acid affords a 3-ester of the invention having the formula , . .
'' . :
. :, ::
~ . ' ;'~'.','.' ' ': :' . .

OH . .
:~ H3C

H3C ~ R2 IV : :
alkyl-CO
.~
~: :
, >
.. . .
., .
:, ~
'~ whereas heating a compound of formula III in pyridine with ~ -an alkanoic acid anhydride or chloride affords a mixture of esters of the invention having the formulas alkyl-CO ~ ~

N3C ~h ~ ~ :
`` ` ~ ~ CH3 .

VI
: alkyl-CO
i . , .

~ separable via chromatography on silica gelj using ~ . .
.. . . .
5 ~ : methylbenzene and mixtures thereof with increasing amounts of ethyl acetate as developing solvents. Similarly, heat-ing a compound o formula III in pyridine with a methyl Q-chloro-Q-oxoalkanoate affords a mixtura of mixed esters .~ ~ having the formulas ~ .
-10~
- . . . .

OH
H3C~
H C ~ I `~1 L ~ R2 . ~ CH3 :O O ~ VII 11 l ll alkylene-COCH
CH30C-alkylene-C0 1 3 C=O

j CH3 Il Il .
CH30C-alkylene-CO
VIII

separable via chromatography on silica gel as aforesaid; .
and heating an ester of formula VI or VII with lithium : . .
iodide in pyridine, 2,6-dimethylpyrid.ine, or 2,4,6-trimethylpyridine affords an ester of the invention ~5 having the formula OH
: H3C
CH3 :::

CH

HOC-alkylene-CO IX
,alkylene-COH. : :-Ç=O
., , . ;.: , .

11 11 .' ~OC-alkylene-CO X ~

- -11- .
:. .
' ' - ~: . ' ~ :
. - ~,. .
' : ' ' ' ' ' respectively. Heating a compound of formula VI or X
with sodium bicarbonate in aqueous ethanol affords a 22-ester of the invention having the formula OZ
H3C~, ~ CH3 ~ ~ C~3 HO

.

wherein Z represents l~oxoalkyl or Q-carboxy.-l-oxoalkyl, respective~y. Finally, heating a compound of formula IX
in pyridine with an alkanoic acid anhydride or chloride affords a mixed ester of the invention having the formula o 11 : .
OC-alkyl Il 11 HOC-alkylene-CO XII

As an exception to the foregoing procedure, a . compound of formula IX wherein the esterifying moiety is 3-carboxy-1-oxopropyL is preferably prepared by heating a ~ -compound of formula III with butanedioic acid anhydride in :

_ . . .. _ pyridine. In each of formulas I through XII hereinbefore, R2 represents alkyl preferably containing fewer than 5 : carbons, and the wavy line represents R or S stereochemistry.
' Optionally, alkylation of the compound of formula I may be accomplished by the addition of a 3,3-dimethyl-alkyl triphenyl phosphonium iodide to give compounds of the formula .. '` - .

~; ~ CH3 OCH3 . XIII .. ~.:

~', ' which is hydrogenated over 5% palladium/carbon to give the `::
` compound CH` ~ :

OCH3 XIV `

.' ' . .

.

~ -13-..
,: ' .. . . . . . ..
.

which may be rearranged by heating with an alkanoic acid to give the compounds of the formula ~-- ' H3C :.

~ -- R2 O XV
alkyl- CO

~:

which when contacted with chromium oxide and pyridine in dichloromethane under nitrogen affords the corresponding 7-one of the invention, havlng the ormula H3C ~ ~ R2.
y 3 O ~ ~ XVI
alkyl- CO O _.

` , . ' ~
:~ i .
: Heating an ester of formula XVI with sodium ... .
bicarbonate in aqueous ethanol affords the corresponding ; ' , ' 3~-hydroxy compound of the invention, having the formula :

C~13 fH3 : H ~ C ~ CH2CH2CH2C R `:

HO O XVII

:. Heating a compound of formula XVII in pyridine with a methyl Q-chloro-Q-oxoalkanoate and heating the . mixed ester thus obtained ~ .

~ CH3 - CH30C-alkylene-CO . o XVIII
'`: ;'~."'' ' ' ~

: ' ~ ' '' .:
.~5 with li~hium iodide in pyridine, 2,6-dimethylpyridine, : -.: or 2,4,6-trimethylpyridine affords an ester of the invention ~ ~ having the formula .: .

. .
. ' .

:` :

~`

CH
O O
HOC-alkylene-CO . O XIX
., ;' '~ .

: As an exception to the foregoing procedure, a compound of fo.rmula XIX wherein the esterlfying moiety is 3-carboxy-1-oxopropyl is preferably prepared by heating a .: compound of formula XVII with butanedioic acid anhydride in pyridine.
. .
.

~ In each o formulas XIII through XIX hereinbefore . .
;
R2 represents alkyl containing 1-4 carbon atoms,~ and~the .: ~ wavy line represents R or S stereochemistry~
,; ~ ~ .:
. - The following examples describe in detail 10;~ ; com~ounds illus~rative of the present invention and methods which have been devised for thelr.preparation. It will be .~ apparent to those skilled in the art that many modifications, both of materials and of methods, ma~.y be practiced without - departing from the:purpose and intent of this disclosure.
`15 Throughout the examples hereinafter set forth, temperatures ~ . are given~in degrees.centigrade (C) and relative amounts of materials in parts by weight unless parts by volume is specified. The relationship between parts by welght and ' ~

parts by volume is the same as that existing between grams and milliliters.

' :

:` :

EXAMæLE 1 -50 Parts by volume of 1.35 M 3,3-dimethylbutyl `~
magnesium chloride solution was added dropwise to a solution of 13.4 parts 6~-methoxy-3,5-cyclo-5~-23,24-bisnorcholan-22-al in 100 parts by volume tetrahydrofuran under nitrogen at a temperature of 0-5C. After the addition was completed, -the cooling bath was removed and the reaction mixture was stirred at room temperature for about one hour. 100 Parts by volume of saturated ammonium chloride solution was then added to the mixture. 100 Parts by volume of ether was added to the hydrolyzed reaction solution and the layers were separated. ~he aqueous phase was extracted with an additional portion of ether and the combined extracts were washed with saturated salt solution and dried over sodium sulfate. Removal of the solvent gave the product 6~-methoxy-25-methyl-3a,5-cyclo-5a-cholestan-22-ol as an oil which was used in its crude form for the subsequent reactions.

.
;- ~
: 17.2 Parts of the compound prepared in Example 1 was added to 75 parts by volume glacial acetic acid and was heated on a steam bath for 3 hours. The reaction mixture was cooled and 300 parts by volume water wa~s~added, forming ` an oily solidO The oily solid was extracted by ether and etheral extracts were washed with 5% sodium bicarbonate ~25 solutions until the aqueous extracts remained basic. The , . .

:` :

,' :

~ .c ~

organic phase was then dried over sodium sulfate and the sol-vent was removed. The solid product was recrystallized from acetone to give the product in two crops. Chroma-tography of the mother liquors provided an additional portion of the desired product. An additional recrystallization from acetone gave the analytical compound 25-methylcholest- ~ -5-ene-3~,22-diol-3 acetate which melts at 185-187C.

~'' ' - .

0.1 Part tosyl acid hydrate was added to a solution of 2.5 parts 6~-methoxy-25-methyl-3~,5-cyclo-5~-cholestan-22-ol in 60 parts by volume dioxane and 20 parts by volume water and the reaction mixture was heated on a steam bath for 5 hours. Water was then added to tha reaction mixture and the solution was extracted with ether. The combined ether extracts were washed with a saturated salt solution and dried over sodium sulfate. The so:lvent was removed to give an oil~ which upon trituration with ethanol afforded a solid product. Recrystallization from aqueous ethanol afforded the pure product 25-methylcholest-S-ene-3~,22-diol -which melts at 191-193C. Alternatively, 25-methylcholest-5-ene-3~,22-diol was prepared in the following manner. `
5 Parts by volume 5~ sodium hydroxide was added to a solution of 1.0 part 25-methylcholest-5-ene-3~,22-diol-3-acetate in 20 parts by volume ethanol and the reactlon ,' ' ' .

' . ~ ' ~L .

mixture was refluxed for 1 1/2 hours. The hot reaction mixture was filtered and a small amount of water was ...
added to the filtrate. Upon cooling, the product resulted in two crops. Recrystallization from ethanol gave the pure compound melting at 187-191C.

To 1.6 parts 25-methylcholest-5-ene-3~,22-diol in 25 parts by volume pyridine was added 1.0 part succinic anhydride and the reaction mixture was heated on the steam bath for 18 hours. Water was then added to the cooled reaction mixture and an oil separated, which solidified upon stirring. The solid was recrystallized from aqueous ethanol then ethyl acetate/hexane to afford 25-methylcholest-5-ene 3~,22-diol-3~-hemisuccinate which melks at 190-193C.
~ ' .

.
To 2.5 parts 25-methylcholest-5~ene-3~,~2-diol-3~-acetate in 20 parts by volume pyridine was added 10 parts by volume acetic anhydride, and the reaction mixture was left standing at room temperature for 5 hours. The ~;
reaction mixture was then warmed on a steam bath for one hour. Ater cooling, 200 parts by volume of water was ; ,' ~: ' . .

. .. . . . .... ..

added to the reaction mixture. The solid which formed was recrystallized from ethanoL to give the product 25- -methylcholest-5-en-3~,22-diol-3~,22-diacetate which melts at 175-177C.

. ' , .
To 200 parts by volume methylene chloride containlng 15.8 parts pyridine under a nitrogen atmosphere was added in portions, at room temperature 10 parts chromic acid, previously dried over phosphorous pentoxide. The burgundy solution was stirred for 30 minutes and then 2.7 parts 25-methylcholest-5-ene-3~,22-diol-3B,22-diacetate in 15 parts by volume methylene chloride was rapidly added.
The reaction mixture was stirred at room temperature for 20 hours and then decanted from the insoluble residue.
The residue was wash~d with several portions of ether, and ? the ether washings were combined with the decantate. ~he :; etheral-decantate solution was treated with Florex and , .
~iltered through diatomaceous earth. ~he filtrate was washed with 1 N hydrochloxic acïd and then saturated salt ~20 solution, dried over sodium sulfate treated with activated charcoal and filtered. The solvent was removed and the crude solid product was recrystaLlized from ethanol to afford the pure compound 3~,22-dihydroxy-25-methylcholest-5-ene-7-one-3~,22-diacetat& melting at 260-262C.
,:

* Trade Mark , .` ,~.
- , . ' ~$~;

To 1.3 parts 3~,22-dihydroxy-25-methylcholest-5-ene-7-one-3~,22-diacetate in 130 parts by volume ethanol was added 13 parts by volume of 5~ sodium bicarbonate S solution and the reaction mixture was refluxed for 2.5 hours. After cooling, the orange solution was acidified with a small amount of acetic acid and 200 parts by volume of water was added. The precipitate which was formed was recrystallized from ethanol in two crops. These were combined and recrystallized twice from ether/hexane to afford 3~,22-dihydroxy-25-methylcholest-5-en-7-one-22-acetate meltiIig at 217-218C.

.
5.0 Parts by volume of 5% sodium bicarbonate solution was added to 50 parts by volume ethanol containing 1.0 part 25-methylcholest-5-ene-3~,22-diol-3~,22-diacetate ~ -and refluxed for 2.5 hours. Water was added and the solid product formed was collected and recrystallized from ethanol to afford 25-methylcholest-5-ene-3~,22-diol-22-acetate which melts at 210-211.5Co ` '. .

. '.:

To 0.9 part 25-methylcholest-5-ene-3~-,22-diol-3~-acetate suspended in 15 parts by volume acetone at room `~ `
temperature was added 0.35 part o~ ~ones reagent and the reaction mixture was stirred for 30 minutes~ The excess :
____ .... r reagent was then destroyed with a small amount of iso-propanol and the reaction mixture was decanted from the insoluble residue. Addition of water to the decantate caused formation of a solid which was collected and re-crystallized from ethanol to afford 3~-hydroxy-25-methyl-cholest-5-en-22-one-3~-acetate which melts at 187-187.5C.

, To 1.3 parts 3~-hydroxy-25-methylcholest-5-en-22-one-3~-acetate in 4~ parts by volume ethylene glycol `10 was added 7.0 parts hydraæine hydrate (65%.hydrazine) and 10 parts by volume ethanol and the reaction mixture was refluxed for 1 hour, after which time it beca~e homogeneaus.
3.0 Rarts potassium hydroxide pelIets were added and the i solutlon was heated to 190C. and maintained at that ~15 temperature for 16 hours, under nitrogen. Another 3.0 parts potassium hydroxide was then added and heating was i continued for another 24 hours. Water was added to the cooled solution and the precipitate was collected. The precipitate was dissolved in 1:1 ether/pPntane solution and the solution was filtered through a cake of magnesium sulfate. The solvent was removed from the filtrate and the solid product was recrystalli~ed from acetone, then methanol, to afford 25-methylcholest-5-en-3~-ol which melts at 153-156C.
;,'~ ' .
. EXAMPLE 11 ~' '' ' ' . '' To 0.9 part 25-methylcholest-5-en-3~-ol in ~--23- ~

. .

12 parts by volume pyridine was added 5.0 parts by volume acetic anhydride and the reaction mixture was allowed to stand for 16 hours. Water was then added and the precipi-tate which was collected was recrystallized from methanol to afford 25-methylcholest-5-en-3~-ol-3~-acetate which melts at 146-148C.

To 70 parts by volume of methylene chloride containing 7.8 parts pyridine in a nitrogen atmosphere was added S.0 parts chromic oxide, in portions, at room temp-erature. Aftex stirring the burgundy solution for 1/2 hour, ...
.1.0 part 25-methylcholest-5-en-3~-ol-3~-acetate in 5 .
parts by volume methylene chl`oride was added and the .
reaction mixture was stirred at room t~emperature for 16 15 hours. Three volumes of ether were addea to the reaction : .
: . .
mixture and the solution was decanted from the insoluble . residue. Florex was added to the solution, which was : then filtered through diatomaceous earth. The filtrate :.;
was washed with 1 N hydrochloric acid,_then 5% sodium .
.20 bicarbonate, and then a saturated salt solution. The solution .~
was dried over sodium sulfate and the solvent was removed .....
to give an.oily residue. Crystallization from ethanol containing a slight amount of water afforded.3~-hydroxy-25-methylcholest-5-en-7-one-3~-acetate which melts at 183-185C. ~

: .

* Trade Mark . ~ 1 ' -24- .

EXAMPLE`13 To 0.48 part 3~-hydroxy-25-methylcholest-5-en-7-one-3~-acetate in 40 parts by volume ethanol was added

4.0 parts by volume 5% sodium bicarbonate solution and the reaction mixture was refluxed for 3 hours. After cooling, a small amount of acetic acid was added to neutralize the base, followed ~y the addition of 50 parts by volume of water which caused a precipitate to form. The precipitate ~..
was collected and chromatographed over Woelm silica gel using ehtyl acetate in benzene as the eluent. Recrystalli-zation from aqueous ethanol afforded 3~-hydroxy-25-methylcholest-5-en-7-one which melts al: 176-178 C.

, ~i ' ' ' `' .
To 0.2 parts 3~-hydroxy-25-methylcholest-5-en-15~ 7-one in 6 parts by volume pyridine was added 0.5 part succinic anhydride and the reacticn mixture was heated ~ ~ .
on a steam bath for 18 hours. Water was added to the cooled reaction mixture, followed by the addition of 1 N
hydrochloric acid. The solid was collec~ed and dissolved in ether. The etheral solution was washed with 1 N
hydrochloric acid, saturated salt solution, and then dried over sodium sulfate and ~iltered. q~he solvent was removed and the product was recrystallized from methanol ~ ~ -`: :

.... _ .. . .. .... _ .. . __ _. _ ,.. _ _ _ _ .

to afford 3~-hydroxy-25-methylcholest-5-en-7-one-3~-hemisuccinate which melts at 211-212C.

' To 7.8 parts 3,3-dimethylbutyltriphenyl phos-phonium iodide in 75 parts by volume tetrahydrofuran under nitrogen was added 9.4 parts by volume of 1.75 molar phenyllithium solution over a 5 minute period, and the -`
orange-red solution was stirred at room temperature for 15 minutes. After cooling the solution to -70C, 4.0 parts 6~-methoxy-3a,5-cyclo-Sa-23,24-bisnorcholan-22-al in 25 parts by volume tetrahydrofuran was added over a 5 minute period and the reaction miXture was stirred at -70C for 30 minutes be~ore removing the cooling bath and letting the reaction warm to room temperature. Saturated ammonium chloride solution was then added, followed by the addition ; of ether, and the layexs were separated. ~he aqueous ~ ;
phase was extracted with additional ether and the combined extracts were washed with a saturated salt solution and dried over sodium sulate. Removal of the solvent gave an oil which was taken up into pentane and after stirring several minutes at room temperature the precipitate which formed was removed by filtration. The volume of the filtrate was reduced to one-half the original and the precipitate which again formed was removed by filtration~

.. .

The precipitate was triphenylphosphene oxide. Removal of the solvent from the filtrate gave the product 6~-methoxy-25-methyl-3a,5-cyclo-5a-cholest-22-ene as an oil, which was used in its crude form for subsequent reactions.

4.8 Parts 6~-methoxy-25-methyl-3~,5-cyclo-5a-cholest-22-ene in 200 parts by volume ethanol was hydrogenated at 35 psi at room temperature over 4.8 parts

5% palladium/caxbon until an equivalent of hydrogen was consumed. The catalyst was removed from the solution by filtration, and the solvent was removed from the iltrate to give the product, 6~-methoxy-25-methyi-3a,5-cyclo-5a-cholestane.

, lS 4.3 Parts crude 6B-methoxy-25-methyl-3a,5-cyclo-S~-cholestane in 30 parts by volume glacial acetic acid was heated on a steam bath for 2 hours. 30 Parts methanol was added to the solution and, upon cooling, the crystalline product was formed~ The solvent was removed and the product was recrystallized from ether/

methanol to afford the pure product 25-methylcholest-5-en-3~-ol-3~-acetate which melts at 152-153C.

`

~3~

.
To a solution of 4 parts 25-methylcholest-5~
ene-3~,22-diol in 25 parts of pyridine was added a solution of 1 part of acetyl chloride in 5 parts of pyridine. The resultant mixture was stirred at room temperature for

6 hours, then diluted with an equal volume of water. The ~ -mixture thus obtained was extracted with ether. The extract was consecutively washed with 5% hydrochloric acid and water, then stripped of solvent by vacuum distillation. -....
The residue was taken up in methylbenzene; and the methyl-benzene solution chromatographed on silica gel, using methylbenzene and mixtures thereof with increasing amounts of ethyl acetate as developing solvents. From eluates selected via thin layer chromatography and stripped of lS solvent by vacuum distillation, 25-methylcholest-5-ene-3~, 22-diol 3-acetate and 25-methylcholest-5-ene-3~,22-diol 3,22-diacetate were isolated as residues which, recrystallized . . .
from ethanol, melt at 185-187 and 175-177 respectively.
~The diester, being the less polar, was eluted first.

A. To a solution of 4 parts of 25-methylcholest-5-ene-3~,22-diol in 25 parts of pyridine was added a solution ~- of 2 parts of 3-methoxy-3-oxopropanoyl chloride in 5 parts .. ...
~ of pyridine. The resultant mixture was stirred at 90-95 for 3 hours, then diluted with an equal volume of water.
The mixture thus obtained was extracted with ether; and :. ....
the extract was consecutively washed with 5% hydrochloric . .. ....

..~ ' .

~$~ S

acid and water, then stripped of solvent by vacuum distillation. The residue was ta~en up in methyl~enzene;
and the methylbenzene solution chromatographed on silica gel, using methylbenzene and mixtures thereof with in-creasing amounts of ethyl acetate as developing solvents.
From eluates selected via thin layer chromatography and stripped of solvent by vacuum distillation, 25-methyl-cholest-S-ene-3~,22-diol 3-(3-methoxy-3-oxopropanoate) and 3,22-bis(3-methoxy-3-oxopropanoate) were isolated as the residues. (The diester, being the less polar, was eluted first.) ;' ' ` ,.
B. A mixture of 4 parts of 25-methylcholest-5- `
ene-3~,22-diol 3,22-bis(3-methoxy-3-oxopropanoate), 12 parts o lithium iodide, and 30 parts of 2,6-dimethyl-pyridine was heated at the boiling point under reflux with ~ `
stirring overnight, whereupon an equal volume of water was -added and the solid precipitate which eventuated was filter-ed off, washed with water, and dried in air. The product `
~ thus isolated was 25-methylcholest-5-ene-3~,22-diol 3-`20 (hydrogen propanedioate).
:. " ' A mixture of l part of 25-methylcholest-5-; ene-3~,22-diol-3~-(hemisuccinate), lO parts of pyridin , .
~ and 5 parts of acetic anhydride was stirred at room ``25 temperature overnight, then diluted with an equal volume , ~ ` .

.

\

:: :
of water. The solid precipitate which eventuated was filter~
ed off, washed with water, and dried in air. The product thus isolated was 25-methylcholest-5-ene-3~,22-diol 22-acetate-3~-hemisuccinate.

.
EXAMPLE 21 ~;~

:
A. To a solution of 4 parts of 3~-hydroxy-25-methylcholest-5-en-7-one in 25 parts of pyridine was added a solution of 2 parts of 3-methoxy-3-oxopropanoyl chloride in 5 parts of pyridine. The resultant mixture was stirred at 90-95 for 3 hours, then diluted with an equal volume of water. The mixture thus obtained was extracted with ether; and the extract was consecutively washed with 5%
hydrochloric acid and water, then stripped of solvent by vacuum distillation. The residue was 3~-hydroxy-25-methylcholest-5-en-7-one 3-(3-methoxy-3-oxopropanoate).

.. . .
B. A mixture of 4 parts of 3~-hydroxy-25-methylcholest-5-en-7-one 3-(3-methoxy-3-oxopropanoate), 12 parts of lithium iod.ide, and 30 parts of 2,6-dimethylpyridine -~
was heated at the boiling point under reflux with stirring overnight, whereupon an equal volume of water was added and the solid precipitate which eventuated was filtered off, washed with water, and dried in air. The product thus isolated was 3~-hydroxy-25-methylcholest-5-en-7-one 3-(3-hydrogen propanedioate).

.;~ ' , ' ` .

:: `
: ' .

"

EXA~LE 22 Pharmaceutical formulations were prepared in the following manner with amounts indicàting the relative amounts per 1000 tablets, capsules, suppositoxies .
or parenteral product.
TABLETS
500 Grams of a representative compound, e.g., `
25-methylcholest-5-ene-3~,22-diol was combined with 265 grams of lactose, 200 grams of corn starch, and 30 grams of polyvinylpyrrolidone, mixed thoroughly, and passed through a 40 mesh screen. The mixture was then granulated with isopropyl alcohol, spread on trays, and `
dried at 49C for 16 hours. The dried granualtion was then screened. The granules were mixed thoroughly with 5 grams of magnesium stearate and the mixture compressed into tablets of the appropriate size. There was thus obtained :
a batch of 1000 tablets having a concentration of active ingredients of 500 mg/tablet.
C~PSULES
:
500 Grams of 25-methylcholest-5-ene-3~,22-diol ~- was mixed thoroùghly with 87.5 grams of corn starch and 87.5 grams of lactose, screened through a 40 mesh screenj and .
remixed. 75 Grams of talc was added and the mixture was thoroughly mixed and filled into a No. 00 size hard ~25 gelatin capsule by hand or machine using 750 mg. fill per capsule to give a final product containing 500 mg. of .

.

_ .
.- ' ~, ~

active ingredient per capsule.
"

In the preparation of tablets and capsules from the compounds of the present invention, a variety of excipients can be used. These are summarized as follows:
Sugars such as lactose, sucrose, mannitol, or sorbitol;
starches such as corn starch, tapioca starch, or potato starch; cellulose derivatives such as sodium carboxymethyl cellulose, ethyl ceIlulose, or methyl cellulose; gelatin, calcium phosphates such as dicalcium phosphate or tricalcium phosphate; sodium sulfate, calcium sulfate; polyvinyl-pyrrolidone; polyvinyl alcohol; stearic acid; alkaline earth metal stearates such as magnesium stearate; stearic acid vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn ~il; surfactants (nonionic, cationic, anionic); ethylene glycol polymers; bèta-cyclodextrin; fatty alcohols; hydrolyzed cereal solids;
as well ~s other non-toxic compatible fillers, binders, ; disintegrants, and lubricants commonly used in pharma-: . , .
ceutical formulations.
SUPPOSITORIES
; 500 Grams of cocoa butter was melted into a water or steam bath to avoid local overheating, then 500 grams of 25-methylcholest-5-ene-3~,22~diol was either ` emulsified or suspended in the melt. Finally the mass was poured into cooled metal moldsj which were chrome plated, and the suppository was readily solidified. There was tbus -3~-: : . . . -- : ~

obtained a batch of 1000 suppositories having a concentra-~` tion of activa ingredient of 500 mg. per suppository.
, ' ' ' '' In the preparation of suppositories from compounds of the present invention a variety of vehicles and bases can be used. These are summarized as follows: tri-glycerides of oleic, palmitric, and stearic acids ~Cocoa butter), partially hydrogenated cottonseed oil, branched saturated fatty alcohols such as suppository base G, -hydrogenated coconut oil triglycexides of C~2-C18 fatty ~10 acids, water dispersible vehicles such as the polyethyl-ene glycols, glycerin, gelatin, polyoxyl 40 stearates, and polyethylene-4-sorbitan monostearates, and materials ; which can raise the melting point o the suppository base, suc~ as beeswax, spermaceti, etc.
PARENTERAL PRODUCTS
. . .
10 Grams of 25-methylcholest-5-ene-3~,22-diol was dissolved in 1000 milliliters of ethyl alcohol and sesame oil was added to give a total volume of 5000 milliliters. Tha mixture was filtered and filled into ampuls and sealed. The ampuls were then sterilized by an appropriate procedure. There was thus obtained a ~ ~ .
batch of 1000 ampuls having a concentration of active ` ingredient of 10 mg/5ml. per ampul.
' ' .

In the preparation of parenteral products from -25 thè compounds of the present invention a variety-of ' . .

3~

vehicles and solubilizers can he used. These are summa-rized as follows: vegetable oils such as peanut, corn, .
cottonseed~ sesame oil, benzyl alcohol, saline, phosphate buffer, water, ethylene glycol polymers, urea, dimethyl ::
` S acetamide, triton, dioxolanes, ethyl carbonate, ethyl lactate, glycerol formal, isopropyl myristate, surfactants (nonionic, cationic, anionic), polyalcohols, and ethanol.

.~.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for the preparation of compounds of the general formula wherein R2 represents alkyl having 1-4 carbon atoms; R
represents hydrogen or a radical of the formula in which n represents a positive integer less than 4;
Y represents methylene or carbonyl, and whenever Y is carbonyl, R1 represents hydrogen, and whenever Y is methylene, R1 represents OX wherein X represents hydrogen or a radical of the formula in which n represents a positive integer less than 4, and the wavy line represents R or S stereochemistry, which comprises (a) reacting a compound of the formula wherein R2 represents alkyl having 1-4 carbon atoms, with H2O in the presence of tosyl acid hydrate to produce the com-pounds of the formula wherein R2 is defined as above, optionally followed by heating with succinic anhydride or succinyl chloride in the presence of pyridine to produce the compounds of the formula wherein R2 is defined as above.
(b) heating a compound of the formula wherein R2 represents alkyl having 1-4 carbon atoms, Z
represents hydrogen or a radical of the formula and n is a positive integer less than 4 with lithium iodide in the presence of pyridine to give the compounds of the formula wherein R2 is defined as above and X' represents hydrogen or a radical of the formula and n is a positive integer less than 4; and, when X is a radical of the formula in which n is a positive integer less than 4, optionally followed by heating with sodium bicarbonate in aqueous alcohol to give the compounds of the formula wherein R2 is defined as above and X' represents a radical of the formula in which n is a positive integer less than 4.
(c) refluxing a compound of the formula wherein R2 represents alkyl of 1-4 carbon atoms with sodium hydroxide in aqueous alcohol to give the compounds of the formula wherein R2 is defined as above optionally followed by heating with a suitable acid anhydride or acyl chloride in the presence of pyridine to produce the compounds of the formula wherein R2 is defined as above and R represents a radical of the formula in which n represents a positive integer less than 4.
(d) refluxing a compound of the formula wherein R2 represents alkyl having 1-4 carbon atoms and R1 represents hydrogen with sodium bicarbonate in aqueous alcohol to give the compounds of the formula wherein R1 and R2 are defined as above optionally followed by heating with succinic anhydride or succinyl chloride in the presence of pyridine to give the compounds of the formula wherein R1 and R2 are defined as above.
(e) heating a compound of the formula wherein R2 represents alkyl having 1-4 carbon atoms, R1 represents hydrogen and Z represents a radical of the formula in which n represents a positive integer less than 4, with lithium iodide in the presence of pyridine, 2,6-dimethyl pyridine or 2,4,6-trimethyl pyridine to-produce the compounds of the formula wherein R2 and R1 are defined as above and R represents a radical of the formula in which n represents a positive integer less than 4.

2. The process according to Claim 1(a) for the preparation of 25-methylcholest-5-ene-3B,22-diol which comprises reacting 6B-methoxy-25-methyl-3a,5-cyclo-5a-cholestan-22-o1 with water in the presence of tosyl acid hydrate.

3. The process according to Claim 1(c) for the preparation of 25-methylcholest-5-ene-3B,22-diol which comprises refluxing 25-methylcholest-5-ene-3B,22-diol-3-acetate with sodium hydroxide in aqueous alcohol.

4. The process according to Claim 1(a) for the preparation of 25-methylcholest-5-ene-3B,22-diol-3B-hemisuccinate which comprises reacting 25-methylcholest-5-ene-3B,22-diol with succinic anhydride or succinyl chloride in the presence of pyridine.

5. The process according to Claim 1(b) for the preparation of 25-methylcholest-5-ene-3B,22-diol-3-(hydro-gen propanedioate) which comprises heating 25-methylcholest-5-ene-3B,22-diol 3,22-bis(3-methoxy-3-oxopropanoate) with lithium iodide in the presence of pyridine.

6. The process according to Claim 1(d) for the preparation of 3B-hydroxy-25-methylcholest-5-en-7-one-3B-hemisuccinate which comprises reacting 3B-hydroxy-25-methyl-cholest-5-en-7-one with succinic anhydride or succinyl chloride in the presence of pyridine.

7. A compound of the formula wherein R2 represents alkyl haviny 1-4 carbon atoms; R
represents hydrogen or a radical of the formula in which n represents a positive integer less than 4;
Y represents methylene or carbonyl, and whenever Y is car-bonyl, R1 represents hydrogen, and whenever Y is methylene, R1 represents OX wherein X represents hydrogen or a radical of the formula in which n represents a positive integer less than 4, and the wavy line represents R or S stereochemistry, when-ever prepared by the process of Claim 1.

8. 25-Methylcholest-5-ene-3B,22-diol whenever prepared by the process of Claim 2.

9. 25-Methylcholest-5-ene-3B,22-diol whenever prepared by the process of Claim 3.

10. 25-Methylcholest-5-ene-3B,22-diol-3B-hemi-succinate whenever prepared by the process of Claim 4.

11. 25-Methylcholest-5-ene-3B,22-diol 3-(hydrogen propanedioate) whenever prepared by the process of Claim 5.

12. 3B-Hydroxy-25-methylcholest-5-en-7-one-3B-hemisuccinate whenever prepared by the process of Claim 6.