Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~37~26
I~lis application is a divisional of our copending Canadian Paten~
Application Serial No. 085,973 filed June 19, 1970.
This invention is ~oncerned with improvements in relation to
pharmaceutical preparations having anaesthetic activi~y, and more particular-
ly aqueous preparations suitable for intravenous injection.
It has long been known that a number of steroids giY0 rise to
profound depression of the central nervous system and act pharmacodynamically
as anaesthetics or hypnotics. Such compounds have been the subject of con-
siderable study in an attemp~ to find anaesthe~ics to replace such substances
as thiopentone sodium normally used but well known to be accompanied by some
degree of hazard or disadvantage. The literature shows that very many steroid
compounds have been studied in this regard. Reviews and discussions of some
of the work carried out are to be found> for example, in "Methods in Hormone
Research" (~dited by Ralph I. Dorfman, Vol. III, Part A, Academic Press London
and New York 1964, pages 415-475); }1. Witzel, Z. Vitamin Hormon-Ferment~orsch
1959, 10, 46-74; H. Selye, Endocrinology, 1942, 30, 437-453; S.K. Figdor et
al,, J. Pharmacol. Exptl. ~lerap., 1957, 119 299-309 and Atkinson et al.,
J. Med. Chem. 1965, 8, 426-432.
A thorough review of the literature indicates that anaesthetic
steroids generally possess poor activity and/or long induction periods. With
such compounds a variety of undesired side effects such as paraesthesia and
thrombophlebitis and vein damage have been noted. Many steroid compounds
having anaesthetic action are also of poor solubility and thus much research
has hitherto been directed to the introduction of solubilising groups into
such steroids, e.g. by the formation of partial esters with di- or polybasic
acids; such work has hitherto not resulted in ~he discovery of a satisfactory
anaesthetic steroid compound. Anaesthetic steroids are generally relatively -
simple pregnane derivatives, often hydroxylated in the 3-position, the general
trend having been in the latter case to study 3~-hydroxy compounds in pre- :
ference to 3d-hydroxy compounds. ~ -
~'~ ' '"' ''
: ... .
C~ .' ~''
~, .
,. . .
~. ~, . . . , - ~ . . . . ... . .. . . . . .. .
~LCI 37~Z6
As a result of prolonged study of numerous steroids exhibiting
anaesthetic activity we have found that 3~-hydroxy-5c-pregnane-11,20-dione
(hereinafter called "steroid I") has quite remarkable properties as an
anaesthetic. This substance has but poor solubility in water and although
~ , ~era~re
referred to in the above cited i~ t~ as having anaesthetic properties,
has apparently been rejected by reason of its poor solubility and the
difficulty to be anticipated in its use by intravenous administration arising
therefrom.
We have found however that steroid I can be brought into aqueous
solution with the aid of certain non-ionic surface active agents to yield
solutions of a sufficient concentration for injection, the resulting solutions
when injected showing excellent anaesthetic properties. These solutions also ~
contain one or more 21-acyloxy-3~-hydroxy-5a-pregnane-11,20-diones whic'h act ' '
as solubility promoters for steroid I.
Thus the aqueous solutions of steroid I according to the invention
induce anaesthesia and possess short induction periods, the anaesthetic action
at suitable doses being indeed instantaneous; the solutions are thus excellent
anaesthetics or inducing anaesthesia which is to be maintained e.g. by an
inhalation anaesthetic such as ether, halothane, nitrous oxide, trichloro-
ethylene etc. The solutions are however capable of maintaining anaesthesia
; and analgesia to a sufficient degree to enable various surgical operations to
be conducted without the aid of an inhalation anaesthetic, the required degree
of anaesthesia being maintained if necessary by repeated administration ~or
even continuous administration). Recovery from anaesthesia (where this is
induced only by the solutions of this invention) is excellent, the patient ~ '
exhibiting a feeling of well-being in distinction to the unpleasant after
effects generally associated with conventional anaesthetics. Moreover,
anaesthetic solutions in accordance with the invention in general give rise ~" '
to none o the undesired side-effects hither to associated with steroidal
, ~
. ,:
- 30 anaesthetics.
,
~ ' .
~)37~26
~ ccording to the invention therefore we provide an anaesthekic com-
position suitable for use by parenteral administration of 3~hydroxy-5c~rpreg-
nane-11,20-dione in solution in an aqueous medium in an amount of at least
1 mg/ml of said medium, said medium also con~aining at least one further
steroid of the formula ~
CH20R , ~
C = O
O ~ ' "
I l (II)
"~ I ' , ~ ~,, ' ,. -'~",
H0 H
.. ..... ..
wherein R is selected from the group consisting of lower alkanoyl groups, the
hemisuccinyl group and the benzoyl group, said further steroid increasing the
solubility of 3~-hydroxy-5~-pregnane-11,20-dione in said medium; and said
medium also containing at least 1% by weight of a parenterally acceptable non-
ionic surface active agent having an HLB value of at least 9.
The non-ionic surface active agents used for the purpose pf this
invention are generally those of the water soluble type, preferably having an
HLB value of from 9 to 18, ~ore preferably at least about 12, and advantageous-
ly at least about 13. Preferably the HLB value of the surface active agent is
not greater than about 15. The surface active agent must naturally be one
which is physiologically compatible, i.e of itself give rise to no physiolog-
ically unacceptable side effects in the dosages employed in the intended
species to be treated (man or animal). Surface active agents for use in ~ : -
.
accordance with the invention are for example to be found among the following
non-ionic surfactants and classes of surfactants:- Polyoxyethylated derivat-
ives of fatty ~C12-C20) glyceride oils, e.g. castor oil, containing from 35
to 45 oxyethylene groups, per mole of fatty oil. Polyoxyethylene ethers
~containing from 10 to 30 polyoxyethylene groups) of long chain alcohols ~con-
~: .",' ' .i ' '
3 _ ~--
,: ., . :
; -:
~037~G
taining for example from 12-18 carbon atoms).
Polyoxyathylene-polyoxypropylene ethers containing from 15 to 35 and
from 15 to 30 oxyethylene and oxypropylene groups respectively. Polyoxy-
ethylene ethers (containing from 6 to 12 oxyethylene groups) of alkyl phenols
the alkyl groups of which preferably contain 6-lO carbon atoms.
Polyoxyethylated ~containing from 15 to 30 oxyethylene groups) fatty
acid (e.g. C12-18) esters of sugar alcohol anhydrides e.g. sorbitan or
mannitan. Long-chain (e.g. Cl0-16) alkanoyl mono- and di-alkanolamides (the
alkanol portions of which for example contain l 5 C atoms) for example
lauroyl mono- and di-ethanolamides~ Polyethylene glycol esters (containing
from 6 to 40 ethylene oxide units) of long chain fatty acids (containing for
example 12-22 C atoms) e.g. polyethyleneglycol mono-oleate (containing for
example 8 ethylene oxide units).
Examples of non-ionic surface active agents, of the foregoing types,
useful in accordance with the invention include:-
Cremophor (trade mark) EL, a polyoxyethylated castor oil containingabout 40 ethyleneoxide units per triglyceride unit;
Tween (trade mark) 80, polyoxyethylene sorbitan monooleate containing
about 20 ethylene oxide units.
~0 Tween (trade mark) 60, polyoxyethylene sorbitan monostearate contain-
ing abo~t 20 ethylene oxide units; and ~
Tween ~trade mark) 40, polyoxyethylene sorbitan monopalmitate con- ~ -
taining about 20 ethyleneoxide units. -
The expression "solutions" is used herein to denote liquids which -
; have the appearance of true solutions and are thus optically clear and cap- --
~able of passage, or example, through a micro-porous filter, irrespectlve of
whether such solutions are true solutions in the classical chemical sense and
irrespective of whether they are stable or metastable. Thus it may be that
the steroid is associated with micelles. The solutions of this invention,
irrespective of their precise physical nature, behave as true solutions for
j
- 4 -
. , '
~ ' ,
- :
~37~DZ6
the practical purpose of intravenous injection.
As stated above, we have found that the total amount of anaesthetic
steroid which may be dissolved in accordance with the invention may be sub-
stantially increased by the presence of a steroid of general formula:
CH20R
C - O .... ..
II
I J
",~
H0 H
wherein R is a lower alkanoyl group having a straight or branched chain (con-
taining for example 2-4 carbon atoms) 9 the hemisuccinyl group or the benzoyl
group. Such steroids of formula II thus act as solubility promoters for the
steroid I and are capable of substantially increasing the amount of the latter
which may be dissolved in the compositions of this invention. Solutions con-
taining steroid I together with a steroid of formula II may thus be prepared ~ :
having a substantially greater anaesthetic potency than a solution of steroid -I alone, due to the increased amount of steroid I which can be dissolved and
the anaesthetic action of the steroid of formula II per se. -
Compounds of formula II above are new compounds, and constitute a
further feature of the invention. :'
Preferred co~pounds of formula II are those in which R is an acetyl,
hen~;suc c~/
propionyl, isobutyryll, or benzoyl group.
The proportion of surface active agent to be used in the compositions
of this invention depends upon its nature and upon the concentration of ster-
: ,
oid deslred in the final composition.
In preferred compositions according to the invention the proportion
of surfactant is preferably at least 5% by weight and advantageously above ;
10% by weight. A very convenient proportion of surfactant has been found to ~ ;
,
~(137~6
be 20% by weight but 30% and up to 50% may be used. The proportions of sur-
factant are expressed by weight in relation to the total volume of the composi-
tion in metric units.
The compositions according to this invention may contain up to 40
or even 50% by weight of the total steroid of a compound of formula II; as
little as 5% of the total steroid present of a compound of formula II provides
useful solubility promotion. For practical purposes it is preferred that of
the total steroid at least 10% by weight, and preferably not more than 30%
by weight of the total steroid consists of a compound of formula II. Very
satisfactory results have been achieved with a mixture of steroid I with a
steroid of formula II in which the proportion ~the steroid of formula II is
about 25%,
As will be clear, the proportion of steroid I in the aqueous solut-
ion according to the invention deperdsupon the nature and amount of surface
active agent used and also upon the amount of steroid o formula II present
(if used). The composition will contain at least l mg/ml of steroid I and
solutions can be made containing for example 2 and ~ mg/ml; using a steroid of
formula II as solubilisar the amounts of total steroid can be increased up to
50 mg/ml although less than 30 mg/ml total steroid and generally less than
20 mg/ml will usually be satisfactory. A composition comprising 9 mg/ml of
steroid I to 3 mg/ml of a compound of formula II in which R is acetyl has been
found to be very satisfactory.
In all cases, as stated above the relative proportion of the various `~
components are adjusted to give a clear solution.
~ ~ According to a ~urther feature of the invention, the anaesthetic :~
solutlons are prepared by mixing steroid I with one or more steroids of
',.~ :
formula II and then dissolving the steroid mixture in the selected surfactant
for example, with heating and mechanical agitation and the resulting solution
dissolved in water. Alternatively the steroid mixture may be dissolved in a
volatile organic solvent advantageously having a boiling point o less than
-- 6 -
:
' ; ~,.,', ''
-
~;37~Z6
about 80C which is miscible wi~h the surface active agent such as a volatilelower aliphatic ketone e.g. acetone or methyl ethyl ketone or a volatile halo-
genated hydrocarbon e.g. chloroform or methylene chloride. Acetone is
particularly preferred for this purpose. The surface active agent is then
added to this solution, the organic solvent removed by evaporation, for example
by passing a stream of an inert gas through the solution e.g. nitrogen~ and the
resulting solution of steroids in surfactant is mixed with water. By these
procedures in general it is possible to dissolve increased amoun~s of steroid
I as compared with other methods.
Thus according to ano~her feature of this invention there is provided:
a process for preparing an anaesthetic composition suitable for use by
parenteral administration in the form of a solution in an aqueous medium con-
taining at least 1 mg/ml of 3~-hydroxy-5~-pregnane-11,20-dione wherein the
solubility of this substance in water has been increased by the presence of
one or more steroids of the formula:
CH2R
C=O
~b
H0
wherein R is selected from the group consisting of lower alkanoyl groups, th0
;hemisuccinyl group and the benzoyl group and wherein said medium also co~ains
~ at least 1% by weight of a parenterally acceptable non-ionic surface active
2~ agent having an ~ILB value of at least 9. -
The solutions may aIso be prepared by shaking the steroid mixture
with an aqueous solution of the surface active agent.
, ~ .
In all cases simple tests enable one to determine the reIative pro-
portions of surface active agent required.
The anaesthetic solutions accorcling to the invention are generally
'
.:.- :
:~ ~ '.''''
~L~37~26
administered by intravenous injection although as is ~nown in the anaesthetic
art in certain cases, e.g. with young children intramuscular injection might
be preferred.
As is usual in the case of anaesthetics, the quantity of steroid I
used to induce anaesthesia depends upon the weight of the individual to be
anaesthetised. For intravenous administration in the average man a dose of
from 0.45 to 3.S mg/Kg will in general be found to be satisfactory to induce
anaesthesia9 the preferred dose being within the range of from 0.7 to 2.5 mg/Kg.Generally a dose of about 1.35 mg/Kg is very satisfactory. The dose will
naturally vary to some extent dependent upon the physical condition of the
patient, and the degree and period of anaesthesia required, all as is well
known in the art. It is thus possible by adjustment of the dose to achieve
durations of anaesthesia varying from about 10 minutes to up to an hour or
more. If it is desired to maintain prolonged anaesthesia, repeated doses of
the solutions of this invention may be used, such repeated doses being -
generally either of the same order or lower than the original dose. Alternat~
ively continuous administralion may be undertaken at for example a rate of
0.09-1.8 mg/Kg/Min.
Where the anaesthetic solutions are administered intramuscularly,
naturally higher doses are generally necessary.
3~-Hydroxy-5-pregnane-11,20-dione (steroid I) can be prepared in any
con~enient manner for example as described by Camerino et al, Helv. Chim. Acta.,1953, 36, 1945 or by Nagata et al., Helv. Chim. Acta 1959, 42, 1399. In apply-
ing the last-mentioned process we have encountered various difficulties at
several stages due to side reactions, which we have found ~o be due to epimer-
; isation at position 17. As a result of further work we have developed a new
.
synthesis of steroid I based essentially upon the synthesis of Nagata et al but
working essentially with intermediates having ~16-unsaturation. Our new synthe-
.: .
sis provides generally better yields and affords greater ease of working and
purity of product, due essentially to the impossibility of epimerisation at
position 17 in compounds of the Q16 series.
'
- 8 -
~a~;37~z~
Thus in accordance with the new synthesis 3~-hydroxy-5a-pregnane-
11~20~dione is prepared by hydrogenation of 3~-hydroxy-5~-pregn-16-ene-11,20-
dione.
The hydrogenation may be conveniently effected in an organic solvent,
for example an alkanol or an ether e.g. methanol, ethanol, propanol, diethyl
ether or tetrahydrofuran. A catalyst is preferably used to effect the hydro-
genation for example palladised charcoal, Raney nickel, platinum catalysts
and the like.
3~-Hydroxy-5~-pregn-16-ene-11,20-dione may, for example,.be prepared
,
by solvolysis of the 3~-acyloxy group of a 3~-acyloxy-5~-pregn-16-ene-11,20-
dione. The acyloxy group at the 3-position may, for example, be a formyloxy,
. . . .
acetoxy, propionyloxy, butyryloxy or benzoyloxy group.
The new synthesis of 3~-hydroxy-5~-pregnane-11,20-dione is described
in more detail in our copending Canadian Application Serial No. 085,973 filed
June 19, 1970.
As stated above, s~eroids of general formula II above are new com-
pounds and constitute another feature of.the invention. The preferred com-
pounds of formula II for use as solubilising agents in pharmaceutical pre-
parations are those in which the 21-acyloxy group is a 21-acetoxy, 21-
propionyloxy, 21-isobutryloxy or 21-benzoyloxy group.
This invention also r01ates~ to a process for increasing the
solubility in an aqueous medium of 3~-hydroxy-5~-pregnane-11,20-dione and
thereby preparing therefrom as anaesthetic composition suitable for use by
parenteral administration which comprises incorporating in a suitable aqueous ;
medium at least 1 mg/ml of 3~-hydroxy-5~-pregnane-11,20-dione toge~her with
one or more steroids of the formula ll20R
C=O .,
O ~ , ''~''' '. ' .,
,~ ,.. .
H0
' '
H
'~~9~
~al3~2~
wherein R is selected from the group consisting of low0r alkanoyl groups,
the hemisuccinyl group and the benzoyl group, the said steroid being
prepared by a process as defined below or by an obvious chemical equivalent
thereof, and including at least 1% by weight of a parenterally acceptable
non-ionic surface active agent having an HLB value of at least 9.
The 21-acyloxy compounds of formula II above are prepared according
to known processes for the preparation of analogous compounds. Thus this
invention relates to a process for preparing a steroi.d of the formula (IIJ:-
CH20R ,... ..
C = O : .~
J~,,, '' '
~i0 1 ~ ''
H ' .
wherein R is selected from the group consisting of lower alkanoyl groups~ the -
hemisuccinyl group and the benzoyl group, which comprises ~a) reacting a
compound of the formula
,, , . ::
1 3
C = O , :.
O I ',',, ,'' " .
:~ H - :
~ -9a
~L03~ 6
in which Rl represents a hydroxy or protected hydroxy group with a lead te~ra-
acylate of the formula:
Pb(OR)4
wherein R is as defined above and when Rl is a protected hydroxy group con-
verting it into a hydroxy group; or (b) reducing a corresponding 21-acyloxy-
5~- pregnane-3,11,20-trione of the formula:
C~120R
C = O
J~
~ '
,'.
wherein R is as defined above; or (c) reacting a 21-halide of the formula:
FH2X ~ :
C= O ' ' '. .
' ~ ':.
~10' , " ' ,
whereln X is chlorine, bromine or iodine, with a salt of an acid of the
formula: -
R.OH
wherein R is as defined above or td) acylating a compound of the formula:
. IH2~ ; .
CO
o~J~ ' '' '
2' ~ -
R
.. ;, . . :
~, - '10 -
1~37~
wherein R represents a protected hydroxy group so as to replace the hydroxy
group by the group RO- and converting the group R2 into a hydroxy group. In
process variant (a) a compound of formula:
COCH3
\ ~III)
H
(in which Rl represents a hydroxy or protected hydroxy group e.g. a nitrate,
trimethylsilyloxy or trichloroethoxycarbonyloxy group~ with a lead tetraacylate,
preferably in the presence of a Lewis acid, and, where Rl represents a protec-
ted hydroxy group, subsequent conversion of said group to a hydroxy group. :
Protection of the 3a-hydroxy group of the compound of formula III however, is
not essential before the acyloxylation reaction. `
.
The lead tetra-acylate used may be for example lead tetraacetate. The
Lewis acid may for example be boron trifluoride, conveniently used as its ;
etherate. We have found, for example that the presence of boron trifluoride :
improves the rate of reaction and can enable lower temperatures of reaction to
be employed, thus~ in many cases, the reaction proceeds satisfactorily at
:: l
ambient temperature, i.e. in the absence of applied heat.
The yield obtained by this process is frequently better than that
.
obtained at an elevated temperature in the absence of boron trifluoride and the
fact that the reaction rate is faster and thus lower temperatures can often be
: ~
~used means that this process possesses economic advantages on the large s~e.
~ The ability to operate at lower temperatures also means that there may be less
, .
; likellhood oP undesired side reactions taking place. - ;
The acyloxylation of compounds of the formula III may be carried out ~ ~
in a solvent medium comprising a mixture of a hydrocarbon solvent and an ~ -
alcohol. Suitable hydrocarbon solvents are, for example, benzene or toluene
- 1 1 -
'
~ ~ .
~37~2~i
and the alcohol may, for example, be methanol. Advantageously the solvent
comprises a mixture of benzene and methanol in the ratio of 19:1.
The acyloxylation is especially suitable for acetoxylation using
lead tetraacetate but other lead acylates, e.g. lead ~etrapropionate may, of
course, be used with the formation of the corresponding 21-propionyloxy com-
pound.
The 3~-trimethylsilyloxy compounds of formula III (Rl representing a
trimethylsilyloxy group) may, for example, be prepared by the reaction of the
parent 3u-hydroxy compound of formula III tR' representing a hydroxy group)
with a trimdhylsilyl halide e.g. trimethylchlorosilane or hexamethyl disilazane
in the presence of a tertiary base e.g. pyridine and, if desired, in the pre-
sence of a solvent e.g. a halogenated hydrocarbon such as methylene chloride
or tetrahydrofuran. The reaction may conveniently be effected at room tempera-
ture or i desired, at lower tamperatures e.g. 0C. The trimethylsilyloxy pro- :-
~ tecting group is generally automatically removed during the acyloxylation.
j The 3~-trichloroethoxycarbonyloxy compound of formula III may be pre-
.
pared by reaction of the parent 3~-hydroxy compound with an alkylchloroformate,
e.g. trichloroethyl chloroformate, preferably in the presence of an acid bind-
ing agent e.g. a tertiary amine such as pyridine, conveniently in a solvent
such as dioxan or t~trahydrofuran. The trichloroethoxy carbonyl protective
group may subsequently be removed by reduction for example using a metal/acid
system such as zinc and acetic acid.
The 3~-nitrate of formula III may be prepared by reaction of the
1 . - .
parent 3-hydroxy compound with fuming nitric acid and acetic anhydride pre-
ferably in the presence of a solvent e.g. chloroform. The nitrate protection
group may subsequently be remoyed by reduction, for example, using a metal
acid system such as zinc and acetic acid or catalytic hydrogenation using for
example, palladium on charcoal as catalyst.
~; Other methods for the preparation of the 21-acyloxy compounds of
,
~ 30 ormula II above can also be used. Thus according to process variant (b) a
1:~ .
~ - 12 -
~037~26
21-acyloxy-5~-pregnane-3,11,20-trione is reduced, for example, using an
enzymatic method such as reduction with brewers yeast (Saccharomyces cerevis-
l ). This method is convenient for the preparation of 21-alkanoyloxy com-
pounds such as the 21-acetoxy compound.
According to process variant (c) compounds of formula II are also
prepared via the corresponding 21-chloro, 21-bromo or 21-iodo compounds. We
prefer to proceed via the 21-bromo intermediate which may be prepared from
5~-pregnan-3~-ol-11,20-dione. The bromination is effected for example using
molecular bromine, in a solvent such as methanol or ethanol advantageously
at a temperature of from -10 to +30C. The reaction is preferably conducted
in the presence of a catalyst such as acetyl chloride or hydrogen bromide in
acetic acid. The 21-halogeno compound may then be converted into the desired
21-acyloxy compound by reac~ion with the salt of corresponding carboxylic
acid, such as an alkali metal salt e.g. the potassium salt or a tertiary amine
salt conveniently N-methylmorpholine or N-ethylpiperadine or a trialkyl-
ammonium salt e.g. the triethylammonium salt. The reaction is preferably
carried out in a solvent for example acetone or methanol, advantageously under
anhydrous conditions. -
An alternative method is process variant (d), which particularly con-
venient for the preparation of compounds of the formula II in which R is other
than an acetyl group, comprises acylating a compound of ~ormula~
COCH2OH ;
~ ~ IV
2 "'
R
(wherein R represents a protected hydroxy group) and deprotecting the 3~-
.
hydroxy group of the 21-acyloxy derivative of the compound of formula IV
produced. The compound of formula IV is conveniently prepared by the deacyla-
tion of a 21-acyloxy derivative of a compound of formula IV, for example a
- 13 -
,: ,' :'
~ ' '
~0~3l7~ 2~i
21-acetoxy derivative thereof, and provides a method for converting one com-
pound of formula II for example in which R is acetyl to another compound of
formula II in which R is a different acyl group. The 21-acetyl compound of
formula II may thus, for example, be converted into another 21-acyl compound
of formula II by firstly protecting ~he 3~-hydroxy group with a protecting sub-
stituent which may be rernoved under acidic, reductive or other conditions but
is stable under alkaline conditions, such as a 3~-ether substituent e.g. a
tetrahydropyranyl or triphenylmethyl substituent or preferably a 3~-nitrate
ester, hydrolysing the 3a-protected compound to yield the corresponding 21-
hydroxy compound, under basic conditionsJ preferably in the presence of
potassium or sodium hydrogen carbonate, conveniently in the presence of a
solvent e.g. methanol, ethanol or tetrahydrofuran, reesterifying the resultant
product and removing the 3~-protecting substituent.
The reesterification is preferably effected using the anhydride or
chloride of the desired acid preferably in the presence of a tertiary amine
(e.g. pyridine, collidine, or dimethylaniline) which may also serve as solvent
for the reaction.
The protecting group at position 3 may be removed in conventional
manner, conditions may be chosen which will not effect the rest of the molecule. -
Thus for example when the 3~-hydroxy group of the compound of ~ormula II is
protected by the formation of a nitrate ester, the nitrate group may be removed
by acid hydrolysis of the compound for example using aqueous mineral acid, or
by reduction using, for example zinc and acetic acid or by catalytic hydrogena-
tion using, for example, ~lladium on charcoal as catalyst. ~
The following Examples are given by way of illustration only, all
temperatures being in degrees Centigrade.
21-Acetoxy-3~-hydroxy-5~-pregnane-11,20-dione
Boron trifluoride etherate (37.~ ml.) was added to a stirred solution
of 3~-hydroxy-5~-pregnane-11,20-dione (6.64 g., 20 mmole) and lead tetraacetate
(10.1 g., 22 xmole) in dry benzene (280 ml.) and methanol (15.1 ml.) at room
.~: .
14 -
~37~26
temperature. After 2 hours the mixture was poured into water
(2 1.) and extracted with ether (1 1.). The combined ether extracts
were washed successively with sodium bic~rbonate solution and water,
dried over magnesium sulphate, and concentrated in vacuo to ~ive
a white crystalline mass. Four recrystallisations from acetone-
petroleum ~bp. 40-60) gave 21-acetoxy-3~ -hydroxy-S~ -pregnane-
11,20-dione as fine needles (4.22 g., 54%) m.p. 172-173, ~]D
~ 104 (c 1-0- CHC13) (Found: C,70.8; ~, 8.9; C23H3405 requires C, ~ -
70.8; ~, 8.8%).
~ ' '
,
21-Acetoxy-3~ -hydroxy So~-yreg~ane-11,20-dione via a trimethyl-
silyl derivative
,, ,
Trimethylchlorosilane (0.33 ml, 1.5 equiv) and pyridine
C0~485 mlJ 2 equiv) were added to a solution of ac~-hydroxy-5~ - - ;
pregnane~ 20-dione (l.Og.,) in methylene chloride (16.6 ml) at
room temperature. After 16 hours at 0 more trimethylchlorosilane
(0.33 ml~ 1 equiv) was added and after a further 1 1/2 hour the
solvent was removed in vacuo. Ether was added to the residue and
the white solid was filtered off. The ether was removed in vacuo
and the residue triturated with a little petroleum (bp 40-60)
to give 3~ -trimethyl-siloxy-S~ -pregnane-11,20-dione (0.2 g.,)
m.p. 95-96. This compound was also prepared
' , , .
,
,
~)37026
by the use of hexamethyldisilazane and trimethylchlorosilane in tetrahydro-
furan.
?~ The 21-acetoxylation was carried out as described in Example ~ one
crystallisation from acetone-petroleum (bp 40-60) afforded the 21-acetate
m.p. 170-173.
Example 3
21-Acetoxy-3 ~hydroxy-5~-pregnane-11,20-dione via a trichloroethoxycarbonyl
derivative.
,
2,2,2-Trichloroethylchloroformate (1.1 ml.) was added to a cooled,
stirred, solution of 3~-hydroxy-5~-pregnane-11,20-dione in pyridine (0.74 ml.)
and dioxan (5.6 ml.). After 10 minutes dilute hydrochloric acid (14.5 ml.)
was added and the solution was heatedat 100 for 30 minutes. The resulting
oil crystallised on cooling to give 3~-(2,2,2-trichloroethoxycarbonyloxy)-5x
pregnane-11,20-dione (1.45 g.,) m.p. 130-131, [~]D + 56 (c 1.0, CHC13)
(Found: C, 56.0; H, 6.5; Cl, 21.1; C23H33C1305 requires C, 55.6; H, 6.6;
Cl. 20.7%).
21-Acetoxylation of this compound (0.5 g.,) using the procedure
described in Example 1 gave a syrupy residue that crystallised on the addit-
ion of ether and petroleum. Recrystallisation from ether-petroleum (bp 40-60)
gave 21-acetoxy-3~- ~2,2,2-trichloroethoxycarbonyloxy)-5~-pregnane 11,20-dione
~0.325 g.,) m.p. 143-146 (decomp), [~]D ~ 75.2 (c 1.0, CHC13) (Found:
C, 55.1; H, 6.0; Cl, 18.6. C25H35C1307 requires C, 54.3; H, 6.4; Cl, 19.2%).
Removal of the 2,2,2-trichloroethoxycarbonyl group was effected by -
stirr m g a solution of the steroid ~0.10 g.,) in ethanol (2 ml.~ and glacial
acetic acid (2.5 ml.) for 5 hours with addition of zinc dust ~0.3 g.) during
~`~ this period. The solids were filtered off, washed with ethanol and the combin-
ed fi]trate was reduced in volume in vacuo. Addition of water to the residual
solution gave 21-acetoxy-3~-h~oxy-5~-pregnane-11,20-dione (0.036 g.,) m.p.
168-172.
,; : .
Example 4
21-Acetoxy-5o~-Eegnan=3a-ol-ll~2o-dione .,,.,~
Dried yeast (Sa_charomyces cerevisiae 60 g.) was stirred at room
- 16 -
- , .:
7~Z6
temperature with tap water (2 1.) containing sucrose (200 g.) and di~ammonium
hydrogen phosphate (4 g.). After two hours 21-acetoxy-5a-pregnane-3,11,20-
trione (1 g.) (Mancara et al., J. Amer. Chem. Soc., 1955, 77 5669) in ethanol
(100 ml.) was added. The fermentation was stirred at room temperature for a
further 24 hours, the yeast was removed by fil~ration, partially dried at the
pump and extracted with hot chloroform ~4 x 250 ml.). The extracts were
shaken with the filtrate, washed with water, dried (Na2SO4) and evaporated
to a brown gum (634 mg.). This was subjected to preparative thin-layer
chromatography which yielded two pure substances. Of these the less polar was
the starting material 21-acetoxy-5~-pregnane-3,11,20-trione, (68 mg.); which
was recrystallised from acetone/hexane as colourless needles, m.p. 169-172;
[~]D + 127.5, (c 0.87 CHC13). Gas-liquid chromatography confirmed ~hat this
was identical with the starting material.
The more polar substance (70 mg.) was recrystallised from acetone/
hexane to afford 21-acetoxy-5~-pregnan-3~-ol-11,20-dione (57 mg.) as colourless
needles, m.p. 179-182; [~]D + 107, (c, 109 CHC13). Gas-liquid chromatography
confirmed the identity of this material.
Examp~e_5
21-Acetoxy-5u-pregnan-3~-ol-11~20-dione
(a) ?1-Bromo-5~-pregnan-3a-ol-11,20-dione
5~-Pregnan-3~-ol-11,20-dione (1 g., 3 mmoles) in stirred methanol
(7 ml.) at 30 was treated with acetyl chloride ~1 drop). After two minutes ~
bromine (0.19 ml., 3.52 mmoles) in me~hanol (4.5 ml.) was added dropwise, the ; ;
solution being allowed to decolourise between the addition of each drop. The
resu~ing clear solution was poured into chloroform (100 ml.), washed with
water ~3 x S0 ml), dried (Na2SO4) and evaporated to a white froth ~1.40 g.).
Preparative thin-layer chromatography afforded 21-bromo-5~-pregnan-3~-ol-11,20- ;
dione (715 mg.) which crystallised from chloroform/ether as clusters of colour-
less needles, m.p. 160-163; [~D + 109 (c 0.82, CHC13); (Found: C, 61.0;
H, 7.9; Br, 19.7; C21H31BrO3 requires C, 61.3; H, 7.6; Br, 19.45%).
,
- 17 -
...
- . -1 . ~
~370;~ ~
A second, unstable substance (415 mg.) isolated from this reaction
was examined spectroscopically and iden~ified as 17 ~bromo-5 ~pregnan-3 ~ol-
11,20-dione.
(b) 21-Acetoxy-5~-pregnan-3~-ol-11,20-dione
5~-Pregnan-3-ol-llJ20-dione (lg. 3 mmoles) was brominated in the
manner described above. The total product from the reaction (1.38 g.) was
refluxed with stiring with dry acetone (25 ml.) and anhydrous potassium
acetate (2 g). After four hours the reaction mixture was poured into chloro-
form (100 ml.), washed with water (3 x 50 ml) dried (Na~S0~) and evaporated to
a white froth (1.19 g). Preparative thin-layer chromatography afforded 21-
acetoxy-5~-pregnan-3~-ol-11,20-dione (634 mg) as a white froth which on cry-
stallisation from acetone/ether gave colourless needles, m.p. 177-180;
[~]D + 102.5, (c 0.95, CHC13). Gas-liquid chromatography confirmed the
identity of this material.
Example 6
21-Acetoxy-5a-pregnan-3a-ol-11,20-dione
5a-Pregnan-3a-ol-11,20-dione (5 g., 15.0 mmoles) in stirred methanol
(35 ml) at 0 was treated with 5 drops of a 50% w/v solution of hydrogen
bromide in glacial acetic acid. After several minutes, bromine (0.95 ml.,
2.815 g 17.6 mmoles) in methanol (22.5 ml) was added portionwise over ca.
10 minutes. The solution was left stirring until the colour disappeared and ;
then poured into water. The product was isolated by filtration, washed with
water and dried in vacuo at 40 for five hours. Final drying was over phos-
~phorus pentoxide in a desiccator tovernight). Yield: approximately quantita-
; tive.
The crude bromo-intermediate was dissolved in acetone (100 ml, dried
ovir~potassium carbonate) and refluxed in the presence of potassium acetate
(10 g) for four hours.
~ ,. ,
~ The reaction mixture was poured into water ~1 1.) and extracted with
~-
ether ~300 ml and 250 ml). The ether was dried over anhydrous magnesium sul-
, ~ :
.' . . . ": -
- 18
~, ' ',, '
~370;26
phate, filtered and evaporated under reduced pressure.
The residual foam was recrystallised from acetone-petroleum ether to
give 21-acetoxy-5a-pregnan-3a-ol-11,20-diolle as white needles. Yield, 63%;
m.p. 173-175; [a]D + 109, (c 1.0, CHC13)-
Example 7
21-Propionyloxy-5a-pregnan-3a-ol-11,20-dione
5a-Pregnan-3~_ol-11,20-dione (2 g., 6 mmoles) in methanol (14 ml.) at
30 was ~reated with acetyl chloride t 2 drops). The solution was stirred for
two minutes and was treated dropwise with bromine (0.38 ml., 7 mmoles) in
methanol (9 ml.) in the manner described above. The reac~ion mixture was
poured into stirred water (250 ml.). The product was isolated, washed and
dried at the pump and redried over phosphorus pentoxide in vacuo to afford a
white powder ~2.49 g.). This was refluxed with propionic acid (11.1 ml.,
0.15 mmoles) and triethylamine (7.25 ml., 0.1 moles) in dry acetone (60 ml.). -
After five hours the solution was poured into chloroform (300 ml.)~ washed
with aqueous potassium hydrogen carbonate (2 x 200 ml.) and with water ~2 x
200 ml.) dried (Na2S0~) and evaporated to a white froth (2.59 g.). Preparat-
ive thin-layer chromatography afforded material which on recrystallisation ~;
from chloroform/ether/hexane gave 21-propionyloxy-5a-pregnan-3a-ol-11,20-
dione ~589 mg.) as colourless irregular prisms, m.p. 159-170; [~D + 99'
(c 0-92, CHC13); (Found: C, 70.5; H, 8.9. C24H3605.1/4 H20 requires C, ~ -
70.45; H, 9.0%)
Example 8
21-Isobutyryloxy-5a-pregnan-3a-ol-11,20-dione
a-Pregnan-3~-ol-11,20-dione ~2 g., 6 mmoles) in methanol (14 ml.) at
.
;; ~ 30 was treated with acetyl chloride (2 drops). The solution was stirred for
~two minutes before being treated dropwise with bromine (0.38 ml., 7 mmoles)
in methanol (9 ml.) in the manner described above. When the reaction was com-
plete the solution was poured into stirred water (250 ml.). The product was
isolated, washed and dried at the pump. A final drying over phosphorus pent-
oxide~in vacuo afforded a white powder (2.5 g.). This was re~luxed with iso-
- 19 -
z~
butyric acid (14.1 ml., 0.15 moles) and triethylamine (7.25 ml., 0.10 moles)
in dry acetone (60 ml.). After four hours the solution was poured into chloro-
form (300 ml.), washed with aqueous potassium hydrogen carbonate ~2 x 200 ml.),
wi~h water (2 x 200 ml.), dried (Na2S04) and evaporated to a white froth
(2.54 g.). Preparative thin-layer chromatography afforded 21-isobutyryloxy-
5a-pregnan-3a-ol-11,20-dione (1.45 g.) as a white froth, [a]D * 93.5, (c 0.66,
CHC13), (Found: C, 70.4; H, 8.8.C25H3805, 1/2 H20 requires C, 70.25; ~l, 9.2%).
Example 9
21-Benzoyloxy-5~-pregnan-3a-ol-11,20-dione
5a-Pregnan-3a-ol-11,20-dione (2 g., 6 mmoles) in methanol (14 ml.)
was treated with acetyl chloride (2 drops) and bromine (0.38 ml., 7 mmoles)
as described above. Isolation of the crude bromination product in the usual
manner afforded a white powder (2.498 g.). This was refluxed with benzoic
acid (18.3g., 0.15 moles) and triethylamine (7.25 ml., 0.1 moles) in dry ace-
tone (100 ml.). After five hours the solution was poured into chloroform
(500 ml.), washed with aqueous sodium hydrogen carbonate (2 x 200 ml.) and
with water (3 x 200 ml.), dried (Na2S04) and evaporated to a white froth.
Preparative thin-layer chromatography afforded 21-benzoyloxy-5a-pregnan-3a-ol-
11,20-dione (1.27 g.) as a white froth, [a]D + 104, (c 0.65, CHC13), (Found:
C~ 72'6; H~ 7-7- C28H3605 l/2 H20 requires C, 72.85; H, 8.05%).
Example 10
21-Isobutyryloxy-5a-pregnan-3~-ol-ll~2o-dione 3-nitrate
i) Fuming nitric acid (1.3 ml.) was added to stirred acetic ~ -~
anhydride (5 ml.). The solution was cooled to -5 and 21-acetoxy-5a-pregnan-
3-ol-11,20-dione (500 mg. 1.28 mmoles) in chloroform (2.5 ml.) was added.
The resulting solution was stirred at 5 for an hour before being poured into - ;
dilute aqueous sodium hydroxide tlO0 ml.), extracted with chloroform ~2 x 75 ~-
ml.) and the extract washed with aqueous sodium bicarbonate (50 ml.), with
water (3 x 50 ml.), dried (Na2S04) and evaporated to a gum (527 mg.). Crystàl-
lisation from ether/acetone afforded 21-acetoxy-5~-pregnan-3a-ol-11,20-dione
,~."' :,
- 20 -
,:
".., ~..,
-
1~371a~Z6
3-nitrate (386 mg.) as colourless needles, m.p. 1~7~ ; [a]D + 103 ~c 1.06
CHC13); (Found: C, 63.6; H, 7.5; N, 3.1. C23H33NO7 requires C, 63-45;
H, 7.65; N, 3.2%).
ii) 5~-Pregna-3a,21-diol-11,20-dione 3-nitrate
~a) 21-Acetoxy-5~-pregnan-3~-ol-11,20-dione 3-nitrate ~837 mg~, 2 mmoles) in
ethanol ~42 mg.) and tetrahydrofuran ~42 ml.) was stirred with 10% aqueous
potassium hydrogen carbonate ~4 ml.) and 2N aqueous sodium hydroxide ~2 ml.).
After three hours glacial acetic acid ~1 ml.) was added, the solution was
poured into water ~400 ml.) and extracted with chloro~orm ~3 x 120 ml.). The
extracts were washed with water ~2 x 50 ml.), dried ~MgSO4) and evaporated to
a froth. Preparative thin-layer chromatography afforded pure material which
on recrystallisation from ether/acetone gave 5~-pregna-3~?2l-diol-ll~2o-dione
3-nitrate ~219 mg.) as colourless irregular prisms, m.p. 172-176; [~]D ~ 91
~c 0.97, CHC13) ~Found: C, 63.9; H, 7.8; N, 3.5. C21H31NO6 requires
C, 64.1; H, 7.95; N, 3.55%).
~b) 21-Acetoxy-5~-pregnan-3~-ol-11,20-dione ~5 g., 13.3 moles) in chloroform
~25 ml.) was added to a stirred solution of fuming nitric acid ~13 ml.) in
acetic anhydride ~50 ml.) at -5. The reaction mixture was stirred at -5 for
an hour, poured into sarred aqueous sodium hydroxide ~ 1 l.) and extracted with
chloroform ~2 x 200 ml.). The extract was washed with aqueous sodium hydrogen
carbDnate ~100 ml.) and with water ~2 x 100 ml.), dried ~Na2S04) and evaporat-
ed to a white froth. This was dissolved in methanol ~500 ml.), the solution
flushed with nitrogen and stirred with 10% aqueous potassium hydrogen carbon-
ate (17.5 ml.) for Eour hours, Glacial acetic acid (3 ml.) was added, the
solution evaporated to small bulk, poured into water ~1 1.) and extracted with
chloroform ~2 x 200 ml.). The extract was washed with water ~2 x 100 ml.),
dried ~Na2SO4) and evaporated to a white froth. Crystallisation from acetone/
ether gave 5~-pregna-3~,21-diol-11,20-dione 3-nitrate ~4.31 g.) as colourless
irregular~prisms, m.p. 174-181.
iii) 21-Isobutyryloxy-5~-pregnan-3~-ol-11,20-dione 3-nitrate
- 21 -
:' ' , '
- . . - .. ~ . . , ~ . . . ., . . -, ~ . . ; . . . . . .
37~2~;
S~-Pregna-3a,21-diol-11,20-dione 3-nitrate (2.0 g., 5.08 mmoles~ in
dry pyridine (20 ml.) was treated with isobu~yryl chloride (2 ml.). The pink
solution was allowed to stand at room temperature for 20 hours before being
poured into stirred water and acidified with dilute hydrochloric acid. The
product was isolated, washed thoroughly with water and dried at the pump.
Recrystallisation from acetone/hexane afforded 21-isobutyryloxy-5a-pregnan-3a-
ol-11,20-dione 3-nitrate (1.01 g.) as colourless needles, m.p. 167-169;
[a]D + 97 5~ (c 1.48, CHC13), (Found: C, 64.5; H, 7.8; N, 3.2. C25H37NO7
requires C, 64.8; H, 8.05; N, 3.0%).
iv) 21-Isobutyryloxy-5a-pregnan-3a-ol-11,20-dione
21-Isobutyryloxy-5a-pregnan-3a-ol-11,20-dione 3-nitrate (1.35 g.,
2.91 mmoles) in glacial acetic acid (35 ml.) was stirred for an hour at room
temperature with zinc powder (4 g.). The zinc was removed, washed with hot
chloroform (200 ml.) and the combined washings and filtrates were washed with
water ~4 x 100 ml.) dried (Na2SO4) and evaporated to a white froth. Preparat-
ive thin-layer chromatography afforded 21-isobutyryloxy-5a-pregnan-3a-ol-11,20-
dione as a white froth, [a]D t 97.5 (c 1.05, CHC13). Analytical thin-layer
chromatography and gas-liquid chromatography indicated that this material was ;
identical with a sample prepared by the method described in Example 12.
xample 11
5~-Pregna-3a,21-diol-11,20-dione 21-hemisuccinate :
. . .
5a-Pregna-3a,21-diol-11,20-dione 3-nitrate 21-hemisuccinate (1.03
g,, 2.09 mmoles) and zinc powder (3 g.) were stirred with acetic acid (25 ml.)
for 90 minutes. The zinc was removed, washed with chloroform (100 ml.), 2N -
aqueous hydrochloric acid (200 ml.) and chloroform (100 ml.). The combined
filtrates were equilibrated and the organic phase was washed with water (3 x
100 ml), dried (Na2SO4) and evaporated to a white froth. Crystallisation from
benzene/hexane/chloroform gave 5~-pregna-3a,21-diol-11,20-dione 21-hemisuccin-
ate (8C0 mg.) as colourless needles, m.p. 166-168; [a]D + 90.5, (c 1.32 in
CHC13~; (Found: C, 66.9; H, 8Ø C25H36O7 requires C, 66.95; H, 8.1%).
The starting material for this preparation was obtained from 5a- ~;
- 22 -
~ ~'''"','
~0370Z~i
., ~oii
C pregna-3a,21-diol-11,20-dione 3-nitrate (see Example ~t~) as described below:-
5~-Pregna-3~,21-diol-11,20-dione 3-nitrate (2g., 5.0~ mmoles) and
succinic anhydride (2g. 20 mmoles) in dry pyridine (20 ml.) were allowed to
stand at room temperature for 18 hours. The solution was poured into stirred
water (300 ml.) which was then acidified. After two hours, the precipitate
was collected, washed well with water and dried. Recrystallisa~ion from
benzene/hexane gave 5~-pregna-3~,21-diol-11,20-dione 3-nitrate 21-hemisuccinate
(1.51 g.) as colourless micro-needles,; m.p. 160-163; [~]D ' 89.5, (c 1.14,
CHC13), (Found C, 60.8; H, 7.3; N, 2.6. C25H35NOg requires C, 60.8;
H, 7~15; N, 2.85%).
Example 12
0.035 g of steroid I, finely divided in an air-attrition mill and
having an approximate mean particle diameter o~ 5~, was mixed with 0.035 g of
steroid II tR = CH3CO-) reduced in size as for steroid I. The steroids are
added to 10 ml of a 20% w/v solution of Cremophor EL containing 0.025 g of ;
sodium chloride and the mixture mechanically stirred for at least 24 hours.
Any traces of insoluble residue are filtered off using a sintered glass filter.
Example 13
13.5 g of ground steroid I and 4.5 g of steroid II ~R = CH3CO-) are
mixed by mechanical agitation with 300.0 g of Cremophor EL at 70C. in an
atmosphere of nitrogen. Heating and agitation are continued until the steroids
have completely dissolved. The mixture is diluted with sterile distilled
water containing 3.75 g of sodium chloride to give a final volume of 1500 ml.
Stirring is continued until the solution is homogeneous.
Example 14
0.045 g of steroid I and 0.015 g of steroid II ~R = CH3CO-) are
dissolved in 2 ml of acetone at 20C. The resultant solution is added to 1 g
of Cremophor EL at 20C, and stirred until homogeneous. The acetone is remov-
ed by a vigorous stream of nitrogen. The solution is diluted with sterile
distilled water containing 0.0125 g of sodium chloride to give a final volume
- 23 -
:.
of 5 ml. Similar solutions were prepared using chloroform or methylene
chloride in place o~ acetone.
Example 15
0.09 g of ground steroid I are mixed with 0.03 g of ground steroid
II (R = CH3CO-) and this added to 2.0 g of Tween 80. As in Example 14, the
mixture is mechanically agitated at 70C. in a stream of nitrogen until the
steroids have dissolved. The mixture is diluted with sterile distilled water
containing 0.025 g of sodium chloride to give a final volume of 10 ml.
Example 16
0.045 g of steroid I and 0.015 g of steroid II (R = (CH3)2CHCO-)
are formulated as in Example 14. Similar solutions are obtained using steroid
II wherein R = C6H5CO-J HO2CCH2CH2CO or C2H5-CO-.
Example 17
0.045 g of steroid I and 0.015 g of steroid II (R = CH3CO) are dis- ~ ;
solved in 2 ml of acetone at 20C. The resulting solution is added to 1 g of
Tween 40 at 20C and stirred until homogeneous. The acetone is removed by a
vigorous stream of nitrogen. The solution is diluted with sterile distilled -
water containing 0.025 g of sodium chloride ~o give a final volume of 10 ml.
Exam~le 18
0.036 g of ground steroid I are mixed with 0.012 g of ground
steroid II (R = CH3C0). This is added to 0.8 g of Tween 60. The mixture is
mechanically agitated at 70C in a stream of nitrogen until the steroids are
dissolved. The mixture is diluted with sterile distilled water containing -
0,025 g of sodium chloride to give a final volume of 10 ml. ;~
Example 19
16 mg of steroid I and 4 mg of steroid II (R = CH3CO) were dissolv-
ed in 0.2 ml of acetone, and 0.2 ml of Cremophor EL was mixed with this solu- -
tion. The acetone was removed by passing a stream of nitrogen through the
mixture. The resulting solution was mixed with 0.8 ml of sterile water.
', ~
` .
:..................................................................... ".
~137~26
Example 20
0.135 grams Steroid I and 0.045 grams of Steroid II, (R=CH3C0), are
dissolved in 2 mls of acetone at 20C. The resulting solution is added to 3
grams of Cremophor EL at 20C and s~irred until homogeneous. The acetone is
removed by a vigorous stream of nitrogen. The solution is diluted with sterile
distilled water containing 0.02S grams of sodium chloride to give a final
volume of 10 mls.
Example 21
0.045 grams of ground Steroid I are mixed with 0.012 grams of ground
Steroid II, (R=CH3C0)~ and the mixture added to 1 gram of Cremophor EL. The
mixture is mechanically agitated a~ 70C in a stream of nitrogen until the
steroids have dissolved. The mixture is diluted with sterile distilled water
containing 0.025 grams of sodium chloride to give a final volume of 10 mls.
- 25 - ~
~ :.- .'
