Note: Descriptions are shown in the official language in which they were submitted.
~l3~86
. . :.
25-ALKYLCHOLESTEROL DERIVATIVES
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 , "
.
.
--2--
.
:
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
R2
., ~ .
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
11 11
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.
.~ . ' '
--4--
-
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.
'`: , ;
-7-
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
.;' , :
'.' ' ' ', ' ~'
--8--
. . .
~, : ~ ~ ....
:' ' ' . ' : .
~. :
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
CH3
~ 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
., , . ;.: , .
R2
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
H3
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 :
-12-
_ . . .. _
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
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
; ' , '
-14-
3~-hydroxy compound of the invention, having the formula :
C~13 fH3
: H ~ C ~ CH2CH2CH2C R `:
CH3
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 ~ .
H3C
~ 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
.: .
. .
. ' .
-15-
:` :
~`
H3C
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
' ~
-16-
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.
EXAMPLE 2
.
;- ~
: 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.
EXAMPLE 3
~'' ' - .
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
,' ' '
.
--19--
' . ~ '
~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.
EXAMPLE 4
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.
~ ' .
EXAMPLE 5
.
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
; ,' ~: '
. .
-20-
. .. . . . .... ..
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.
EXAMPLE 6
. ' , .
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
,
-21-
.` ,~.
- , . '
~$~;
EXAMPLE 7
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.
EXAMPLE 8
.
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
` '. .
EXAMPLE 9
. '.:
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
-22-
:
____ .... 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.
EXAMPLE 1 0
,
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.
EXAMPLE 12
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.
,
EXAMPLE 14
~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 ~ ~ -
`: :
-25-
.... _ .. . .. .... _ .. . __ _. _ ,.. _ _ _ _
.
to afford 3~-hydroxy-25-methylcholest-5-en-7-one-3~-
hemisuccinate which melts at 211-212C.
EXAMPLE 15
'
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~
-26-
.. .
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.
EXAMPLE 16
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.
EXAMPLE 17
,
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.
-27-
`
~3~
EXAMPLE 18
.
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.
EXAMPLE 19
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
. .. ....
-28-
..~ ' .
~$~ 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).
:. " '
EXAMPLE 20
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
,
~ ` .
-29
.
\
:: :
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).
.;~ ' , ' ` .
:: `
: '
-30-
.
"
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
.
.
-31-
_ .
.- ' ~, ~
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
'
33-
. .
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.
-34-
.~.
