Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
Methods And Compositions For Inducing Sleep
25 Background
The present invention is directed to methods for
interacting with the gamma-aminobutyric acid receptor-
chloride ionophore complex ("GABAA receptor complex") to
induce sleep in humans. More particularly, the present
30 invention relates to the use of certain 3a-hydroxy-5-
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reduced steroids and derivatives thereof in the treatment
of insomnia.
The relationship between steroids, the GABAA receptor
complex, and brain excitability has previously been
described in U.S. Patents Nos. 5,120,723 and 5,232,917.
The inventors have found that certain steroids,
called neuroactive steroids, acting at the steroid site on
this receptor complex can be used to induce sleep and in
particular, treat insomnia. For example, the inventors
have found that 3a-hydroxy-5f3-pregnan-20-one
("pregnanolone"), can induce sleep when administered
orally. Arafat and co-workers had established a
correlation between the progesterone metabolites
pregnanolone and 3a-hydroxy-5a-pregnan-2 0 -one and sleep
following administration of progesterone in humans.
Arafat, E.S., Hargrove, J.T., Maxson, W.G., Desiderio,
D.M., Wentz, A.C., and Anderson, R.N. "Sedative and
Hypnotic Effects of Oral Administration of Micronized
Progesterone May Be Mediated Through Its Metabolites" Am.
J. Obstet. Gynecol. 159:1203-1209 (1988). The oral
activity of pregnanolone is surprising since Laszlo
Gyermek found that it was almost inactive orally in
"Pregnanolone: a Highly Potent, Naturally Occurring
Hypnotic-Anesthetic- Agent" Proc. Soc. Exp. Biol. Med.
1967, 125:1058-1062.
No incentive existed, however, to make an orally
active formulation of pregnanolone because earlier
investigations indicated that the neuroactive steroids
acted like the barbiturates, which exhibit the undesirable
side effect of anesthesia when used to induce sleep, a low
therapeutic index, abuse potential, and adverse side
effects such as rebound insomnia. Majewska, M.D.;
Harrison, N.L.; Schwartz, R.D.; Barker, J.L.; and Paul,
S.M. "Steroid Hormone Metabolites Are Barbiturate-Like
WO 94/27608 2163 7 4 8 PCT/US94/05820
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Modulators of the GABA Receptor" Science 232:1004-1007,
1986.
It has been known since 1967 that progesterone, an
anesthetic steroid, when administered at subanesthetic
doses could induce sleep in the cat. Heuser, G.
"Induction of Anesthesia, Seizures and Sleep by Steroid
Hormones" Anesthesiology 28:173-183 (1967). Heuser
concluded that progesterone produced a "natural" sleep in
the cat because REM sleep was not suppressed during
administration of the drug, nor was REM sleep increased
upon withdrawal of the drug. Significantly, Heuser found
that not all anesthetic steroids were capable of producing
this "natural" sleep.
The National Commission on Sleep Disorders Research
estimates that 40 million Americans suffer from chronic
sleep disorders such as insomnia, narcolepsy, and sleep
apnea. It is estimated that 20-30 million Americans
experience intermittent sleep-related problems. "Wake Up
America: A National Sleep Alert" Vol. 1, p. vi; Draft
Report of the National Commission on Sleep Disorders
Research 1992. The large incidence of insomnia and other
sleep problems is not surprising when one considers that
normal sleep-wake patterns can be interrupted by emotional
distress, drugs, jet lag, shift work, illness, and strong
sensory stimuli such as pain or hunger.
The social costs of this high incidence of sleep
disorders and sleepiness are significant. These costs
include reduced productivity, lowered cognitive
performance, increased likelihood of accidents, higher
morbidity and mortality risk, and decreased quality of
life. Id. The Commission estimated that in 1990 alone,
sleep disorders and sleepiness cost the United States at
least $15.9 billion in direct costs alone, which does not
include indirect costs from sleep related tragedies such
as the grounding of the Exxon Valdez. Id. Society
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desperately needs safe and effective treatments for sleep
disorders.
Currently, insomnia is treated with a variety of
drugs, all of which have certain drawbacks. A common
treatment is the use of the drug Triazolam, known by the
tradename Halcion''" Its drawbacks include rebound insomnia
and limited efficacy in increasing sleep. Rebound
insomnia is a return of abnormal sleep patterns as the
drug wears off or as a result of either tolerance to the
drug or withdrawal symptoms when the treatment is stopped.
Zolpidem, a new insomnia drug, also tends to cause rebound
insomnia.
The compounds of the present invention have superior
characteristics to currently available drugs. The present
invention provides an insomnia treatment with rapid onset
of sleep, high potency and efficacy, and minimal rebound
insomnia.
To aid in fully understanding the present invention,
a brief outline of sleep and how it is studied follows.
Sleep is often defined using two sets of criteria: 1)
electrophysiological; and 2) behavioral. The electro-
physiological criteria can include electroencephalograms
(EEGs), electro-oculograms (EOGs), and electromyograms
(EMGs). The most common criterion used for studying sleep
is the EEG which is a recording of the electrical activity
of the brain. By studying cortical EEGs of sleeping
individuals, scientists have found that there are two
major stages of sleep manifested by the production of
different electrical wave patterns. These two stages are
called non-REM (NREM) and REM. NREM sleep is conven-
tionally divided into four stages in which the EEG pattern
is commonly described as synchronous, with characteristic
waveforms such as sleep spindles, K complexes and high-
voltage slow waves. Kryger, M.H.; Roth, T.; and Dement,
W.C. Principles and Practice of Sleep Medicine, p.3, 1989
(hereinafter "PPSM"). REM sleep generally shows greater
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EEG activation, muscle atonia, and episodic periods of
rapid eye movements. Id.
When an individual closes his or her eyes in
preparation for sleep, the cortical electrical rhythms
5 synchronize to a rhythm of 8-13 Hz. This is known as an
a-rhythm. An EEG taken in the first stage of sleep shows
a slower rhythm (4-6 Hz) and a lower amplitude. Bowman,
W.C., and Rand, M.J. Textbook of Pharmacolocr , p 6.24, 2nd
ed. 1980 (hereinafter Bowman and Rand). In the second
stage of sleep, more irregular and slower EEG waves of 1-5
Hz with a larger amplitude appear. These irregular, slow
waves are interspersed with bursts of faster waves called
sleep-spindles. In this second stage of sleep there are
also K-complexes which consist of a small sharp wave,
followed by one or two larger waves and then by faster
waves of about 12 Hz. Id. Stage 2 accounts for 45-55
of sleep time. PPSM at p. 9.
Stage 3 sleep is characterized by high voltage, slow
EEG wave activity accounting for 20-50 % of the EEG
activity in this stage. PPSM p. 7. In stage 4, high
voltage, slow wave activity accounts for >50% of the EEG,
and K-complexes cannot be evoked by stimuli such as
calling the subject's name. PPSM at p. 7; Bowman and Rand
p. 6.24.
After about an hour of sleep the EEG pattern speeds
up to a 4-10 Hz rhythm of low amplitude waves that are
desynchronized. This is REM sleep and is often referred
to as paradoxical sleep because the EEG resembles that of
wakefulness, yet it is very deep sleep. Bowman and Rand
p. 6.24. The biological significance of REM sleep is not
yet known. However, alcohol's strong inhibition of REM
sleep has been associated with sleep disruption.
Triazolam and other insomnia drugs reduce REM sleep,
whereas compounds of the present invention have the
further advantage of only minimally disrupting REM sleep.
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Summary of the Invention
The present invention is directed to methods for inducing sleep in human
beings. The invention relates to a method for inducing sleep without inducing
hypnosis or anaesthesia, comprising administering orally to a patient in need
of
such treatment an amount of 3a-hydroxy-5p-pregnan-20-one effective to induce
sleep without inducing hypnosis or anaesthesia. More particularly the
invention
relates to the use of certain 3a-hydroxylated-5-reduced steroid derivatives
and
prodrugs thereof which act at a recently identified site on the GABAA receptor
complex to treat insomnia.
Experiments by the inventors have established that the compounds used
when practising the invention give a rapid onset of sleep and have potency and
efficacy. They also cause less rebound insomnia and fewer effects on body
temperature that known hypnotic drugs used to treat insomnia.
Brief Description of The Drawings
These and other features, aspects, and advantages of
the present invention will become better understood from
the following description, the appended claims, and the
accompanying drawings, where:
FIGS. 1A and 1B are plots of the percentage of NREM
sleep per hour in rats vs. clock time for 30mg/kg and 3 &
l0mg/kg of pregnanolone, respectively, administered at
circadian time 18 hours (CT-18);
FIG. iC is a plot of the percentage of NREM sleep per
hour in rats vs. clock time for 10mg/kg of pregnanolone,
administered at CT-5.
FIGS. 2A and 2B are plots of the minutes spent sleep-
ing vs. clock time when 3omg/kg and 3 & 10mg/kg of pregna-
nolone, respectively, administered at CT-18;
FIG. 2C is a plot of the minutes spent sleeping vs.
clock time for 10mg/kg of pregnanolone administered at CT-
5.
FIGS. 3A and 3B are plots of the mean deviation in
body temperature vs. clock time for 30mg/kg and 3 &
10mg/kg of pregnanolone, respectively, administered at CT-
18;
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FIG. 3C is a plot of the mean deviation in body
temperature vs. clock time for 10mg/kg of pregnanolone
administered at CT-5.
FIGS. 4A and 4B are plots of counts of movement per
hour (locomotor activity) vs. clock time for 30mg/kg and
3 & 10mg/kg of pregnanolone, respectively, administered at
CT-18;
FIG. 4C is a plot of counts of movement per hour
(locomotor activity) vs. clock time for 10mg/kg of pregna-
nolone, administered at CT-5.
FIGS. SA and 5B are plots of the percentage of REM
sleep per hour vs. clock time for 30mg/kg and 3 & 10mg/kg
of pregnanolone, respectively, administered at CT-18;
FIG. 5C is a plot of the percentage of REM sleep per
hour vs. clock time for 10mg/kg of pregnanolone, adminis-
tered at CT-5.
FIGS. 6A, 6B, and 6C are plots if the percentage of
NREM sleep per hour vs. clock time for 0.4 mg/kg of
Triazolam administered at CT-5, for 0.4 mg/kg administered
at CT-18, and for 0.4 mg/kg and 1.6 mg/kg of Triazolam
administered at CT-18, respectively.
FIG. 6D is a plot of the minutes spent sleeping vs.
clock time for 0.4 mg/kg and 1.6 mg/kg Triazolam adminis-
tered at CT-18.
FIG. 6E is a plot of the mean deviation in body
temperature vs. clock time for 0.4 mg/kg and 1.6 mg/kg
Triazolam administered at CT-18.
FIG. 6F is a plot of the counts of body movement per
hour (locomotor activity) vs. clock time for 0.4 mg/kg and
1.6 mg/kg Triazolam administered at CT-18.
FIG. 6G is a plot of the percentage of REM sleep per
hour vs. clock time for 0.4 mg/kg and 1.6 mg/kg of Triazo-
lam administered at CT-18.
FIGS. 7A and 7B show the EEG pattern during NREM
sleep for 10 mg/kg of pregnanolone. FIG. 7B is an
expanded view of part of FIG. 7A.
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FIGS. 7C and 7D show the EEG pattern during NREM
sleep for 30 mg/kg Zolpidem. FIG. 7D is an expanded view
of part of FIG. 7C.
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FIGS. 8A, BE, and 8C are plots of the per- centage of
NREM sleep per hour vs. clock time for 10 mg/kg of Zolpi-
dem administered at CT-5 and CT-18, respectively, and for
30 mg/kg of Zolpidem administered at CT-18.
FIG. 8C is a plot of the minutes spent sleeping vs.
clock time for 10 mg/kg and 30 mg/kg Zolpidem administered
at CT-18.
FIG. 8D is a plot of the mean deviation in body
temperature vs. clock time for 10 mg/kg and 30 mg/kg of
Zolpidem administered at CT-18.
FIG. 8E is a plot of the counts of body movement per
hour (locomotor activity) vs. clock time for 10 mg/kg and
30 mg/kg Zolpidem administered at CT-18.
FIG. BF is a plot of the percent REM sleep per hour
vs. clock time for 10 mg/kg and 30 mg/kg Zolpidem adminis-
tered at CT-18.
FIGS. 9A and 9B are plots of the percentage of NREM
sleep per hour vs. clock time for 40 ug/kg of dexmedetomi-
dine administered at CT-5 and CT-18, respectively.
FIGS. 10A, 10B, and IOC are plots of the percentage
of total power (activity during NREM sleep) vs. minutes
after treatment for pregnanolone, Zolpidem, and Triazolam,
respectively, administered at CT-5.
FIGS. 11A, 11B, and 11C are plots of the percent of
total power (activity during NREM sleep) vs. frequency for
pregnanolone, Zolpidem, and Triazolam, respectively,
administered at CT-5.
FIG. 12 is a bar graph showing the change from base-
line in locomotor activity for the first hour after
treatment with various drugs. Locomotor activity is
measured in counts per hour.
FIG. 13 is a bar graph depicting the change in REM
sleep 2-3 hours after treatment with various drugs.
FIGS. 14A and 14B show plots of counts of movement
per 5 minutes vs. minutes after treatment for pregnanolone
and Zolpidem administered at CT-18, respectively.
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FIGS. 15A-15H are plots of human plasma concentration
of pregnanolone over time for Formulation 1, which is 5%
pregnanolone, 94% polyvinylpyrrolidone, and 1% sodium
laurel sulfate. Arrows indicate time points when sleep
was observed.
FIGS. 16A-16H are plots of human plasma concentration
of pregnanolone over time for Formulation 2, which is an
inclusion complex of 12.3% pregnanolone and 87.7% /3-
cyclodextrin. Arrows indicate time points when sleep was
observed.
FIGS. 17A-17H are plots of human plasma concentration
of pregnanolone over time for Formulation 3, which is 99%
pregnanolone nanosized by supercritical fluid nucleation
with 1% sodium laurel sulfate. Arrows indicate time
points when sleep was observed.
FIGS. 18A-18H are plots of human plasma concentration
of pregnanolone over time for Formulation 4, which is 99%
micronized pregnanolone from AKZO with 1% sodium laurel
sulfate. Arrows indicate time points when sleep was
observed.
FIG. 19 is a plot of serum concentration in human
subjects of pregnanolone versus time for all four formula-
tions.
FIG. 20 is a table showing human mean plasma concen-
trations of pregnanolone from two formulations at half-
hour intervals.
FIG. 21 is a table showing the mean time awake in
male human subjects during the 5 minute polygraphic
recording sessions at half-hour intervals from two
formulations of pregnanolone.
FIG. 22A is a graph plotting the number of sleep
attempts by male human subjects verses plasma concentra-
tion of pregnanolone.
FIG. 22B is a table showing that the number of sleep
attempts by male human subjects during a study day is
related to the plasma concentration of pregnanolone.
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FIGS. 23A-C are tables showing the mean change in
NREM sleep, the mean change in REM rebound, and the mean
change in REM sleep, respectively in rats receiving 3a-
hydroxy-3/i-trifluoromethyl-5,6-pregnan-20-one 21-phosphate
disodium salt, 3/3-ethynyl-3a-hydroxy-5/3-pregnan-20-one,
3a-hydroxy-30-methyl-5a-pregnan-20-one, 3x,21-dihydroxy-
30-trifluoromethyl-50-pregnan-20-one 21-hemisuccinate
sodium salt, 3a-hydroxy-3g-ethynyl-5a-pregnan-20-one,
3a,21-dihyrdoxy-3/-ethynyl-50-pregnan-20-one 21-hemisuc-
cinate, 3a-hydroxy-30-trifluoromethyl-l9-nor-5Q-pregnan-
20-one, 3a-hydroxy-21-(pyrid-4-ylthio)-5$-pregnan-20-one,
3a-hydroxy-39-trifluoromethyl-50-19-nor-pregnan-20-one 21-
phosphate disodium salt, or controls.
FIGS. 24A and 24B are plots of the percentage of NREM
sleep and REM sleep, respectively, per hour in rats vs.
clock time for 18 mg/kg of 33-ethynyl-3a-hydroxy-5Q-
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pregnan-20-one (CCD-3093), administered at circadian time
18 hours (CT-18).
FIG. 25 is a plot of the minutes spent sleeping vs.
clock time for 18 mg/kg of 3p-ethynyl-3a-hydroxy-50-
5 pregnan-20-one administered at CT-18.
FIGS. 26A and 26B are plots of the mean deviation in
body temperature vs. clock time and of counts of movement
per hour (locomotor activity) vs. clock time, respective-
ly, for 18 mg/kg of 30-ethynyl-3a-hydroxy-50-pregnan-20-
10 one administered at CT-18.
Description
The compounds used in this invention are 3a-hydroxy-
lated-5-reduced steroids which interact with the GABAA
receptor complex. These compounds include various 3a-
hydroxylated-5-pregnan-20-ones; 3a-hydroxylated-5-androst-
anes; and 3a,21-pregnanediol-20-ones; and ketone, ester,
ether, sulfonate, sulfate, phosphonate, phosphate, oxime,
and thiazolidine derivatives thereof, which derivatives
are referred to as prodrugs. The expression "prodrug"
denotes a derivative of a known direct acting drug, in
this case a drug used in the invention, which derivative
has enhanced delivery characteristics and therapeutic
value as compared to the drug, which is transformed into
the active drug by an enzymatic or chemical process in the
patient's body; see Notari, R.E., "Theory and Practice of
Prodrug Kinetics," Methods in Enzymology, 112:309-323
(1985); Bodor, N., "Novel Approaches in Prodrug Design,"
Drugs of the Future, 6(3):165-182 (1981); and Bundgaard,
H., "Design of Prodrugs: Bioreversible Derivatives for
Various Functional Groups and Chemical Entities," in
Design of Prodrugs (H. Bundgaard, ed.), Elsevier, New York
(1985). It should be noted that some of the synthetic
derivatives useful in the method of the present invention
may not be true prodrugs because, in addition to the above
characteristics, they
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also possess intrinsic activity. However, for present
purposes they will be referred to as prodrugs.
Among the 3a-hydroxy pregnanes and 3a-hydroxy andro-
stanes useful in the method of this invention are 3a-
hydroxy-30-R-5a or 5Q-19-R'-173-R2-pregnanes and 3a-
hydroxy-30-R-5a or 5/3-19-R'-17$-R2-androstanes having the
structural formula I:
Rz
R'
R
HO
wherein:
R is hydrogen, a lower alkyl group, e.g., methyl and
methoxymethyl, a lower alkynyl group, e.g., ethynyl,
4-hydroxypent-1-ynyl and (4'-acetylphenyl)ethynyl, a lower
trihaloalkyl group, e.g., trifluoromethyl, a lower
monohaloalkyl group, e.g. chioromethyl, a lower alkenyl
group, e.g., ethenyl and 2-phenylethenyl, an aryl group,
e.g., phenyl, or an aralkyl group, e.g., benzyl;
R' is hydrogen or methyl;
R2 is methylene (=CH2), cyano, hydroxymethyl, methoxy-
methylene (=CHOCH3), acetyl, 2'-hydroxyacetyl, 2'-
hydroxyacetyl acetate, 1-hydroxyethyl, 2'-hydroxyacetyl
hemisuccinate, 2'-methoxyacetyl, pyrid-4-ylthioacetyl,
ethylene (=CHCH3), propylene (=CHCH2CH3), 2'-hydroxyacetyl
hemisuccinate sodium salt, 1-hydroxybutyl, and 1-hydroxy-
1-methylethyl, 1-hydroxypropyl, 1-propionyl, 3-
methoxypropionyl, ethynyl, 2'-mesyloxyacetyl, or 1'-
(ethylenedioxy)ethyl;
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and pharmaceutically acceptable 3-esters, 20-esters,
21-esters, 3,20-diesters, and 3,21-diesters, thereof;
with the proviso that the 3a-hydroxy-5u-pregnanes do
not have a 21-hydroxy substituent.
A preferred group of compounds includes those in
which R is not hydrogen, the compound has an ICs0 , value of
< 300nM as an allosteric modulator of ['SS] TBPS binding,
and the compound is orally active.
Another preferred group of compounds which can be
used in the method of this invention are 3a-hydroxy-3f3-R-
5a or 5/3-19-R1-21-R2 -pregnan-20-ones having the structural
formula II:
0
R2
Ri
R
Hd H
wherein:
R is a lower alkyl group, e.g. methyl; a lower alkynyl group, e.g. ethynyl,
optionally
substituted by a single aryl, hydroxy, halo or keto group; or a lower
trihaloalkyl group e.g.
trifluoromethyl.
R1 is hydrogen or a lower alkyl group;
R2 is hydrogen, hydroxy, a pyrid-4-ylthio group, a
hemisuccinoyloxy group, or a phosphoryloxy group and the
sodium salts thereof; with the provisos that:
(1) when R is hydrogen, R' is a pyrid-4-ylthio Vroup;
(2) when R1 is hydrogen, R is a trihalo(lower) alkyl
group; and
(3) when R2 is other than hydrogen or a pyrid-4-
ylthio group, R is a trihalo(lower) alkyl group.
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A preferred group of compounds of Formula II is where
R is a lower alkynyl group or a trihalo (lower alkyl)
group.
Another preferred group of compounds of Formula II is
where R2 is a hemisuccinoyloxy group or a phosphoryloxy
group, or sodium salts thereof.
The preferred neuroactive steroids of the present
invention include 3a-hydroxy-5p-pregnan-20-one
(pregnanolone) (available from Diosynth); 3a-hydroxy-3p-
trifluoromethyl-50-pregnan-20-one 21-phosphate disodium
salt; 3(3-ethynyl-3a-hydroxy-5p-pregnan-20-one;3a-hydroxy-
3S-methyl-5a-pregnan-20-one; 3a,21-dihydroxy-3Q-
trifluoromethyl-5(3-pregnan-20-one 21-hemisuccinate sodium
salt; 3a-hydroxy-3p-ethynyl-5a-pregnan-20-one; 3a,21-
dihydroxy-3/3-ethynyl-53-pregnan-20-one 21-hemisuccinate;
3a-hydroxy-30-trifluoromethyl-19-nor-5p-pregnan-20-one;
3a-hydroxy-21(pyrid-4-ylthio)-5P-prenan-20-one; and 3a-
hydroxy-3Q-trifluoromethyl-50-19-norpregnan-20-one 21-
phosphate disodium salt.
In accordance with an aspect of the present
invention, there is provided a method of inducing sleep
comprising administering to an animal subject an amount
of a 3a-hydroxy-30-R-5a or 5(3-19-R1-17(3-R2-pregnane or
androstane that will increase the duration of
uninterrupted NREM sleep, and will maintain the level of
locomotor activity found in normal sleep, said pregnane
or androstane having the structural formula I:
R2
R'
R
~= H
i
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wherein:
R is hydrogen, a lower alkyl group, a lower alkynyl
group, a lower trihaloalkyl group, a lower monohaloalkyl
group, a lower alkenyl group, and aryl group, or an aralkyl
group;
R1 is hydrogen or methyl;
R2 is methylene (=CH2), cyano, hydroxymethyl, methoxy-
methylene (=CHOCH3), acetyl, 2'-hydroxyacetyl, 2'-
hydroxyacetyl acetate, 1-hydroxyethyl, 2'-hydroxyacetyl
hemisuccinate, 2'-methoxyacetyl, pyrid-4-ylthioacetyl,
ethylene (=CHCH3), propylene (=CHCH2CH3), 2'-hydroxyacetyl
hemisuccinate sodium salt, 1-hydroxybutyl, and 1-hydroxy-1-
methylethyl, 1-hydroxypropyl, 1-propionyl, 3-
methoxypropionyl, ethynyl, 2'-mesyloxyacetyl, or 1'-
(ethylenedioxy)ethyl;
and pharmaceutically acceptable 3-esters, 20-esters,
21-esters, 3,20-diesters, and 3,21-diesters, thereof;
with the proviso that the steroid is not 3a,21-
dihydroxy-5a-pregnan-20-one.
According to an additional embodiment of the
invention, there is provided the aforementioned method,
wherein the level of REM sleep is maintained compared to
that found in normal sleep.
According to a further embodiment of the invention,
there is provided the aforementioned method, wherein
substantial rebound insomnia in not induced.
20 Definitions
In accordance with the present invention and as used
herein, the following terms are defined with the following
meanings, unless explicitly stated otherwise..
The term "alkyl-" refers to saturated aliphatic groups
25 including straight chain, branched chain, and cyclic
groups with i to 10 carbon atoms. Alkyl includes
substituted lower alkyl.
The term "alkynyl" refers to unsaturated groups which
contain at least one carbon-carbon triple bond and
includes straight chain, branched chain, and cyclic
groups. Alkynyl includes substituted lower alkynyl.
The term "trihaloalkyl" refers to alkyl groups
substituted with three halogen atoms, F, Br, Cl or I.
Preferred trihaloalkyl groups include trifluoromethyl and
2',2',2'-trifluoroethyl.
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The term "lower" referred to herein in connection
with organic radicals or compounds defines such as up to
and including 8, preferably up to and including 6, and
advantageously one, two, three, or four carbon atoms.
The term "alkenyl" refers to unsaturated groups which
contain at least one carbon-carbon double bond and
includes straight chain, branched chain, and cyclic
groups. Alkenyl is meant to include the allenyl group,
which possesses two consecutive double bonds, ~.
~c=c=c\
Alkenyl may be substituted with aryl.
The term "aryl" refers to aromatic groups which have
at least one ring having a conjugated pi electron system
and includes carbocyclic aryl and biaryl groups, all of
which may be substituted.
"Carbocyclic aryl" groups are groups wherein the ring
atoms on the aromatic ring are carbon atoms.
The term "biaryl" represents aryl groups substituted
with other aryl groups.
The term "substituted lower alkyl" refers to lower
alkyl groups substituted with an azido, cyano, or a lower
alkoxy group.
"Alkoxy" refers to an alkyl group linked to a carbon
atom through an oxygen ether linkage.
The term "monohaloalkyl" refers to an alkyl group
substituted with one halogen atom, F, Br, Cl, or I.
The term "substituted aryl" refers to aryl groups
substituted by one to five substituents, independently
selected from lower alkyl, aryl, hydroxy, lower alkoxy,
amino, halo, lower acyl, nitro, lower trihaloalkyl, cyano,
carboxy and lower acyloxyacetyl.
The term "amino" refers to -NRR' where R and R' are
independently selected from hydrogen or lower alkyl, or it
may refer to a morpholino group.
The term "halo" refers to the halogen atoms F, Br,
Cl, and 1.
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The term "acyl" refers to -C(O)R where R is lower
alkyl.
The term "carboxy" refers to the group -C(O)OH.
The term "acyloxyacetyl" refers to -C (O) CH2OC (O) R
5 where R is lower alkyl.
The term "substituted lower alkynyl" refers to lower
alkynyl groups substituted by one substituent selected
from the group consisting of aryl, hydroxy, halo, and
keto.
10 The term "aralkyl" refers to an alkyl group
substituted with an aryl group. Suitable aralkyl groups
include benzyl and may be substituted on the aryl.
The term "pharmaceutically acceptable" refers to
esters or salts derived from the combination of a steroid
15 used in this invention and an organic or inorganic acid.
Preferred esters of hydroxy groups at positions 3, 20
and/or 21 are those described by their corresponding
acids: acetic, propionic, maleic, fumaric, ascorbic,
pimelic, succinic, glutaric, bismethylene-salicylic,
methanesulfonic, ethane-di-sulfonic, oxalic, tartaric,
salicylic, citric, gluconic, itaconic, glycolic, p-
aminobenzoic, aspartic, glutamic, gamma-amino-butyric, a-
(2-hydroxyethylamino) -propionic, glycine and other a-amino
acids, phosphoric, sulfuric, methane sulfonic, glucuronic,
and 1-methyl-1,4-dihydronicotinic. Especially preferred
are esters of acetic, succinic, and phosphoric acids.
It is believed that the substituent at the 3a-
position can be either hydroxyl or any ester due to the
fact-that the ester will be cleaved off as the prodrug is
converted to drug form. These are referred to herein as
cleavable esters.
The following synthetic methods and examples are
directed to the preparation of compound used in the
present invention.
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Synthetic methods
The compounds according to the invention may be
prepared by any convenient method, e.g. using conventional
techniques such as are described in "Steroid Reactions"
Djerassi, published in 1963 by Holden-Day, Inc., San
Francisco or "Organic Reactions in Steroid Chemistry",
Fried and Edwards, published in 1972 by Van Nostrand-
Reinhold Co., New York.
The compounds of the invention may be prepared by any
suitable technique known in the art or henceforth
developed.
GENERAL METHODS
20-hydroxy pregnanes were prepared by the reduction
20-keto pregnanes with conventional reducing agents.
21-hemisuccinates were prepared from pregnan-20-one
derivatives which were first brominated with molecular
bromine to obtain the corresponding 21-bromo pregnanes.
The bromo compounds were then reacted with various dioic
acids, such as succinic acid, in the presence of an amine
to yield 21-hydroxy esters. The resulting esters from the
dioic acids were then converted to their sodium salts by
conventional means.
The esters may be formed using reactions well known
in the art between the hydroxyl group on the compounds
discussed above with an organic and inorganic acid, acid
halide, acid anhydride, or ester.
38-Substituents
Halomethyl
The 30-monohalomethyl compounds of this invention may
be prepared by treatment of a 3-spiro-2'oxirane steroid
with a source of halide ions in an inert solvent, such as
tetramethylammonium halide in toluene, and preferably a
proton source, such as acetic acid.
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Saturated or unsaturated alkyl
Other 3-substituted steroids may be prepared by the
addition of an organometallic reagent to a 3-ketosteroid
in which other reactive functional groups may be protected
as necessary. Thus, 3-alkynyl compounds may be prepared
by the use of an lithium acetylide in inert solvents or
with a reagent prepared in situ from 1,2-dibromoethylene
and butyl lithium as the organometallic reagent.
Likewise, compounds of Formula I in which R is alkenyl
group may be prepared by the reaction of a 3-ketosteroid
with a vinyl organometallic reagent such as vinyl
magnesium bromide. Compounds in which the unsaturation is
removed from the site of reaction, such as a 2-propenyl
group, may also be prepared with reagents such as allyl
magnesium bromide. Similarly, the use of an alkyl
Grignard such as methyl magnesium iodide will lead to 3-
alkyl compounds.
Trifluoromethyl
The-trifluoromethyl group may be prepared by reaction
of a 3-ketosteroid with trimethyltrifluoromethylsilane
catalyzed by fluoride ion.
21-Oxygenated compounds
Lead tetraacetate oxidation of 21-methyl
Various compounds of this type may be prepared by a
reaction sequence in which a pregnan-20-one is oxidized
with lead tetraacetate to give a 21-acetoxy derivative,
hydrolysis of the acetate to give a 21-alcohol. The 21-
alcohol may then be acylated with an appropriate
carboxylic acid derivative, such as an acid anhydride or
acid chloride or other reagent capable of replacing the
hydrogen of the hydroxyl group, such as methanesulfonyl
chloride.
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Bromination of C-21
Alternatively, a pregnan-20-one may be treated with
a brominating agent such as bromine in methanol to give a
21-bromopregnan-20-one. The bromide may then be displaced
with an oxygen nucleophile, such as succinate anion, to
give other 21-substituted pregnan-20-ones.
Pregnan-17-enes
These may be formed by the reaction of a 17-
ketosteroid with a Wittig reagent such as the ylide
derived from treatment of n-propyltriphenylphosphonium
bromide with a strong base such as potassium t-butoxide.
3,20-Diols
A pregnan-3,20-diol may be formed by addition of a
borane hydride such as diborane to the double bond of a
pregnan-17-ene followed by oxidation of the thus formed
organoborane with, for example, hydrogen peroxide, to give
the 20-hydroxy. Alternatively, pregnan-3,20-diols may be
formed by the reduction of a 20-one group to a 20-hydroxy
group. Suitable reagents are hydride reagents such as
sodium borohydride or dissolving metals such as sodium in
n-propanol and the like.
EXAMPLE 1
a. 3a-Hydroxy-5(3-Pregnan-20-one cyclic 20-(1,2-
ethanediyl ketal)
A mixture of 3a-hydroxy-50-pregnan-20-one (10.8 g, 34
mmol), ethylene glycol (45 mL), and triethyl orthoformate
(30 mL) was stirred at r.t. for 5 min. p-Toluene sulfonic
acid (pTSA) (200 mg) was then added and the stirring was
continued at r.t. for 1.5 hr. The resulting thick paste
was poured into a sat. NaHCO3 solution (250 mL). The
precipitated solid was collected by filtration, washed
thoroughly with cold water and dried. This semi-dried
product was dissolved in CH2C12 (350 mL) and dried over
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anhyd. K2CO3. The solution of the ketal was then filtered
and used as such for the next step.
b. 58-Pregnan-3,20-dione cyclic 20-(1,2-ethanediyl
ketal)
The above solution of 3a-hydroxy-50-pregnan-20 -one
cyclic 20-(1,2-ethanediyl acetal) in CHzCl2 was stirred
with N-methylmorpholine-N-oxide (8.8 g, 75 mmol), and
powdered 4A molecular sieves (58 g) under N2 for 15 min.
Tet rapropyl ammonium perruthenate (400 mg) was then added
and the stirring was continued at r.t. for 2 hr. The
resulting dark green mixture was passed through a short
column of Florisil and eluted with CH2C12. The fractions
containing the product (by TLC) were combined and evapora-
ted. The crude product was then crystallized from a
mixture of EtOAc:Hex (1:1) to yield the title compound
(10.3 g) as long rods.
An analogous method was used to prepare 5a-pregnan-
3,20-dione cyclic 20-(1,2,ethanediyl ketal).
c. 3 a- hydroxy- 3 B-phenylethynvl - 5 (3 -preanan- 2 0 -one
A solution of 50-Pregnan-3,20-dione cyclic 20-(1,2-
ethanediyl acetal) (180 mg, 0.5 mmol) in dry THE (15 mL)
was treated with lithium phenylacetylide (1M in THF, 1.5
mmol, 1.5 mL) at -70 . After stirring the mixture at this
temp. for 1 h and then at r.t. for 2 h, it was quenched
with 2N HC1 (1 mL). The solvent was removed and the
residue was dissolved in acetone (20 mL) . After adding iN
HC1 (5 mL) the solution was stirred at r.t. for 15 h. The
solvents were removed and the residue was extracted with
CHzCl2. The organic layer was washed with water, dil.
NaHCO3 soln., water, and brine. After drying over anhyd.
MgSO4 the solution was filtered and evaporated to yield the
crude product (300 mg). This crude product was then dis-
solved in a small amount of CH2C12 and poured on a column
of silica gel. On elution with toluene:acetone mixture
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(95:5) gave 3a-phenylethynyl-3Q-hydroxy-5/3-pregnan-20-one
(70 mg) as a first fraction. Further elution with the
same solvent mixture yielded 30-phenylethynyl-3a-hydroxy-
50-pregnan-20-one (160 mg), m.p. 188-190 C.
EXAMPLE 2
a. 3a-hydroxy-3I3-(2'-phenylethyl)-5a-pregnan-20-one
A solution of 3a-hydroxy-30-phenylethynyl-50-pregnan-
20-one (44 mg) was dissolved in EtOAc (12 mL), Pd/C (5%,
12 mg) was added and the mixture was hydrogenated at 400
Kpa pressure overnight at r.t. Filtration of the catalyst
followed by evaporation of the solvent yielded the crude
product, which was purified by chromatography over silica
gel to isolate the pure title compound (33 mg); mp 153-
154 C; TLC Rf (hexane:acetone 7:3) = 0.4.
EXAMPLE 3
a. 38-(3',4'-Dimethoxyphenylethynyl)-3a-hydroxy-5a-
pregnan-20-one
A solution of 2,2-dibromo-l-(3',4'-dime thoxyphenyl)
ethene (prepared by the Wittig reaction of 3,4-dimethoxy-
benzaldehyde with carbon tetrabromide in the presence of
triphenyl phosphine) (966 mg, 3 mmol) in dry THE (15 mL)
was treated under N2 with n-BuLi (2.5M in THF, 6 mmol, 2.4
mL) at -78 . The mixture was stirred at this temp. for 2
hours and a solution of 50-pregnan-3,20-dione cyclic 20-
(1,2-ethanediyl acetal) (720 mg, 2 mmol) in dry THE (10
mL) was added dropwise over a period of 30 min. After
stirring the resulting mixture at -78 C for 2 hr, the
cooling bath was removed and the stirring was continued at
r.t. for another hr. It was then quenched with 2N HC1
solution (1 mL) at -10 C. The solvent was removed and the
residue was then dissolved in acetone (25 mL). After
adding 2N HC1 (10 mL) the solution was stirred at r.t. for
2 h. A saturated NaHCO3 soln. was added to neutralize the
acid. The solvents were removed and the residue was
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extracted with EtOAc. The organic layer was washed with
water, dil. NaHCO3 soln., water, and brine. After drying
over anhyd. MgSO4 the solution was filtered and evaporated
to yield the crude product (1.2 g). This crude product
was then dissolved in a small amount of CH2C12 and poured
on a column of silica gel. Elution with toluene:acetone
mixture (96:4) gave a phenylacetylene compound, which was
not characterized. Further elution with the same solvent
yielded 3a-(3',4'-dimethoxyphenylethynyl)-3$-hydroxy-5$-
pregnan-20-one (120 mg) as a first fraction, and 3$-
(3',4'-dimethoxyphenylethynyl)-3a-hydroxy-5/3-pregnan-20-
one as a second fraction (430 mg); mp 82-88 C.
An analogous method was used to prepare: 3$- (4' -
methoxyphenylethnyl)-3a-hydroxy-5$-pregnan-20-one; 3$-(4'-
Chlorophenylethynyl)-3a-hydroxy-5$-pregnan-20-one; 3$-(2'-
Methoxyphenylethynyl)-3a-hydroxy-5$-pregnan-20-one; 3$-
(4' -biphenylethynyl)-3a-hydroxy-5$-pregnan-20-one; 3$-(4'-
dimethylaminophenylethynyl)-3a-hydroxy-5a-pregnan-20-one;
3$-(4'-Cyanophenylethynyl)-3a-hydroxy-5$-pregnan-20-one
EXAMPLE 4
3(3-(3' 4'-Dimethoxyphenylethyl)-3a-hydroxv-5(3-Arecman-20-
one
A mixture of Pd/C (5%, 28 mg) and EtOAc (12 mL) was
presaturated with hydrogen by stirring it under hydrogen
for 10 min. A solution of 3$-(3',4'-dimethoxyphenyl-
ethynyl)-3a-hydroxy-59-pregnan-20-one (185 mg) in EtOAc (5
mL) was then added and the mixture was hydrogenated at 300
Kpa pressure overnight at r.t. Filtration of the catalyst
followed by evaporation of the solvent yielded the crude
product, which was purified by chromatography over silica
gel (hexane:acetone 4:1) to isolate the pure title
compound (135 mg); TLC Rf (hexane:acetone 4:1) = 0.14.
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EXAMPLE 5
38-(4'-Nitrophenylethynyl)-3a- ydroxy-58-pregnan-20-one
A solution of 2,2-dibromo-l-(4-nitrophenyl)ethene
(prepared by the Wittig reaction of 4-nitrobenzaldehyde
with carbon tetrabromide in the presence of triphenyl
phosphine) (296 mg, 1 mmol) in dry THF (20 mL) was treated
under N2 with n-BuLi (2.5M in THF, 2 mmol, 0.8 mL) at -95 .
The mixture was stirred at -80 to -100 C for 0.5 hr. and
then a solution of 50-pregnan-3,20-dione cyclic 20-(1,2-
ethanediyl acetal) (720 mg, 2 mmol) in dry THF (10 mL) was
added dropwise over a period of 10 min. After stirring
the resulting mixture at -80 C for 1 hr, and then at 0 C
for 1 more hr. it was quenched with NH4C1 solution (3 mL).
The solvent was removed and the residue was then dissolved
in acetone (25 mL) . After adding 2N HC1 (10 mL) the
solution was stirred at r.t. for 1 h. Saturated NaHCO3
soln. was added to neutralize the acid. The solvents were
removed and the residue was extracted with CH2C12. The
organic layer was washed with water, and brine. After
drying over anhyd. MgSO4 the solution was filtered and
evaporated to yield the crude product (400 mg). This
crude product was then dissolved in a small amount of
CH2C12 and poured on a column of silica gel. Elution with
toluene:acetone mixture (96:4) gave the title compound as
a brown solid (70 mg); TLC Rf (toluene: acetone 95:5) _
0.18.
EXAMPLE 6
3a-hvdroxy-38-phenyl-58-pregnan-20-one
A solution of 5f3-pregnan-3,20-dione cyclic 20-(1,2-
ethanediyl acetal) (720 mg, 2 mmol) in dry THF was treated
with Phenyl magnesium bromide (3M in THF, 6 mmol, 2 mL) at
-70 C. After stirring the mixture at this temp. for 3 h
and then at r.t. for 2 h, it was quenched with 2N HC1 (10
mL). The solvent was removed and the residue was dis-
solved in acetone (20 mL). After adding IN HC1 (5 mL) the
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solution was stirred at r.t. for 15 h. The solvents were
removed and the residue was extracted with CH2C12. The
organic layer was washed with water, dil. NaHCO3 soln.,
water, and brine. After drying over anhyd. MgSO4 the
solution was filtered and evaporated to yield the crude
product (1.3 g). This crude product was then dissolved in
a small amount of CH2C12 and poured on a column of silica
gel. Elution with toluene:acetone mixture (95:5) gave 3a-
phenyl-3/3-hydroxy-5a-pregnan-20-one (420 mg) as a first
fraction. Further elution with the same solvent mixture
yielded 3/3-phenyl-3a-hydroxy-5(3-pregnan-20-one (185 mg),
m.p. 182-184 .
EXAMPLE 7
3a-hydroxy-38-benzyl-53-pregnan-20-one
A solution of benzyl magnesium bromide (2M in THF, 2
mmol, 1 mL) was diluted with THE (15 mL) and was treated
dropwise with a solution of 50-pregnan-3,20-dione cyclic
20-(1,2-ethanediyl acetal) (360 mg, 1 mmol) in dry THE (15
mL) at -60 . After stirring the mixture at this tempera-
ture for 1 h and then at r.t. for 15 h, it was quenched
with 2N HC1 (10 mL). The solvent was removed and the
residue was dissolved in acetone (20 mL). After adding iN
HC1 (5 mL) the solution was stirred at r . t . for 30 min.
It was neutralized with 2N NaOH. The precipitated solid
was collected by filtration, washed with water and dried
to yield 3/3-hydroxy-3a-benzyl-50-pregnan-20-one (238 mg).
The filtrate was extracted with EtOAc. The organic layer
was washed with water, dil. NaHCO3 soln., water, and brine.
After drying over anhyd. MgSO4 the solution was filtered
and evaporated to give the crude product (160 mg). This
crude product was then dissolved in a small amount of
CH2C12 and poured on a column of silica gel. Elution with
toluene:acetone mixture (95:5) gave 3/3-hydroxy-3a-benzyl-
50-pregnan-20-one (40 mg) as a first fraction. Further
elution with the same solvent mixture yielded 3a-hydroxy-
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3/3-benzyl-5,6-pregnan-20-one (30 mg), which was crystal-
lized from hexane:CH2C12 (4:1) as colorless rods (15 mg)
m.p. 133-141 C; TLC Rf (toluene: acetone 9:1) = 0.5.
EXAMPLE 8
3j3- (1-Heptynyl) -3a-hydroxy-5(3-preanan-20-one
A solution of 1-heptyne (0.327 mL, 2.5 mmol) in dry
THF (15 mL) was treated with n-BuLi (2.5M in THF, 2.5
mmol, 1 mL) at -78 C. After stirring the mixture at this
temperature for 1 hr, a solution of 56-pregnan-3,20-dione
cyclic 20-(1,2-ethanediyl acetal) (360 mg, 1 mmol) in dry
THF (15 mL) was added and the mixture was stirred at -78 C
for 1 hr. It was then quenched with 2N HC1 solution (1
mL). The solvent was removed and the residue was then
dissolved in acetone (10 mL). After adding 2N HC1 (10 mL)
the solution was stirred at r.t. for 1 hr. Saturated
NaHCO3 soln. was added to neutralize the acid. The
solvents were removed and the residue was extracted with
EtOAc. The organic layer was washed with water, dil.
NaHCO3 soln., water, and brine. After drying over anhyd.
MgSO4 the solution was filtered and evaporated to yield the
crude product (400 mg). This crude product was then
dissolved in a small amount of CH2C12 and poured on a
column of silica gel. Elution with toluene:acetone
mixture (93:7) gave 3.0-(1-heptynyl)-3a-hydroxy-50-pregnan-
20-one (260 mg) as a colorless solid; mp 121-123 C.
An analagous method was used to prepare: 30-(1-
Hexynyl)-3a-hydroxy-5f3-pregnan-20-one; 3/3-(1-Octynyl)-3a-
hydroxy-59-pregnan-20-one; 3a-Hydroxy-3$-[6-oxo-1-
heptynyl)-50-pregnan-20-one, using 1-heptyne-6-one cyclic
(1,2-ethanediyl acetal).
EXAMPLE 9
3- [3' (R/S) -Hvdroxybutynyll -3a-hydroxy-5a-nrecrnan-20-one
A solution of 3(R/S)-hydroxybutyne (0.470 mL, 6 mmol)
in dry THF (15 mL) was treated with n-BuLi (2.5M in THF,
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12 mmol, 4 mL) at -70 C. After stirring the mixture at
this temperature for 0.5 hr, a solution of 50-pregnan-
3,20-dione cyclic 20-(1,2-ethanediyl acetal) (1.08 g, 3
mmol) in dry THE (30 mL) was added and the mixture was
5 stirred at -78 C for 1 hr. The cooling bath was removed
and the mixture was stirred at r.t. for another 1.5 hr.
It was then quenched with NH4C1 solution (3 mL) . The
solvent was removed and the residue was then dissolved in
acetone (10 mL) . After adding 2N HC1 (5 mL), the solution
10 was stirred at r.t. for 1 hr. Saturated NaHCO3 soln. was
added to neutralize the acid. The solvents were removed
and the residue was extracted with EtOAc. The organic
layer was washed with water, dil. NaHCO3 soln., water, and
brine. After drying over anhyd. MgSO4 the solution was
15 filtered and evaporated to yield the crude product (1.4
g). This crude product was then dissolved in a small
amount of CH2C12 and poured on a column of silica gel.
Elution with toluene:acetone mixture (85:15) gave 3(3-
[3'(RS)-hydroxy-butynyl]-3a-hydroxy-5(3-pregnan-20-one (145
20 mg) as a colorless solid; TLC Rf (toluene:acetone 4:1) _
0.24.
An analogous method was used to prepare: 3/3-
[4'(R/S)-hydroxypentynyl]-3a-hydroxy-5(3-pregnan-20-one.
EXAMPLE 10
25 3- (4'-Acetylphen ly ethynyl)-3a-hydroxy-5a-pregnan-20-one
A solution of 4-iodoacetophenone (95 mg, 0.39 mmol),
3(3-ethynyl-3a-hydroxy-5a-pregnan-20-one (106 mg, 0.3 mmol)
in dry degassed pyrrolidine (3 mL) was stirred under argon
at r.t. Bis(triphenylphosphine)palladium chloride (5 mg)
and CuI (5 mg) were added and the mixture was stirred at
r.t. for 15 hr. The TLC showed 100% conversion of the
starting material, hence, the mixture was quenched with
NH4C1 solution (15 mL) and was extracted with EtOAc. The
organic layer was washed with water, dil. NaHCO3 soln.,
water, and brine. After drying over anhyd. MgSO4 the
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solution was filtered and evaporated to yield the crude
product (150 mg). This crude product was then dissolved
in a small amount of CH2C12 and poured on a column of
silica gel. Elution with hexane:acetone mixture (4:1)
afforded 313-(4'-acetylphenylethynyl)-3a-hydroxy-5a-
pregnan-20-one (35 mg) as a colorless solid; TLC Rf
(hexane:acetone 7:3) = 0.4.
An analogous method was used to prepare: 3a-Hydroxy-
31-(4'-trifluoromethylphenylethynyl)-5a-pregnan-20-one;
3a-Hydroxy-3/3-(4'-methylphenylethynyl)-5$-pregnan-20-one;
3a-Hydroxy-3(3-(4'-acetoxyacetylphenylethynyl)-5,6-pregnan-
20-one; 3a-Hydroxy-3(3-(pentafluorophenyl)ethynyl-5(3-
pregnan-20-one; 3/3-(2'-Hydroxyphenyl)ethynyl-3a-hydroxy-
50-pregnan-20-one; 3/3-(3'-Hydroxyphenyl)ethynyl-3a-
hydroxy-5/3-pregnan-20-one; and 3a-Hydroxy-3/3-(4'-
carboxyphenylethynyl)-50-pregnan-20-one, ethyl ester.
EXAMPLE 11
3a-Hydroxy-38-methoxymethyl-5a-pregnan-20-one
The 3(R)-epoxy-5a-pregnan-20-one was prepared by the
reaction of 5a-pregnan-3,20-dione with trimethylsul-
foxonium methyliodide in the presence of a strong base,
such as potassium t-butoxide, in THF.
To a solution (there were few undissolved solid
particles) of 3(R)-epoxy-5a-pregnan-20-one (5.145 g, 15.57
mmol) in CH3OH (600 mL) was added freshly cut Na (- 600
mg). CAUTION: H2 gas evolution. After all Na had
reacted, a clear solution resulted. The reaction solution
was stirred overnight. Since TLC (3:1 hexane/acetone)
indicated mainly the starting material, the mixture was
heated (close to boiling point) for 8 h. and then left at
room temperature overnight. TLC still showed a small
amount of the starting material, besides two new, less-
polar spots. Therefore, the mixture was heated for
another 8 h. at which time there was no unreacted starting
epoxide. After the reaction solution had come to room
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temperature, glacial acetic acid (3 mL) was added drop-
wise. The solvent was then removed under reduced
pressure, and CH2C12 and water were added. The aqueous
layer was back-extracted with CH2C12. The combined
organics were washed with saturated NaHCO3, dried (MgSO4),
filtered, and evaporated under reduced pressure to give a
white solid (5.55 g), which was found to be a 84:16
mixture of 3a-hydroxy-30-methoxymethyl-5u-pregnan-20 -one
and its 17-epimer on the basis of its 1H NMR. R-
ecrystallization from hot 1:1 hexane/acetone furnished
only 3a-hydroxy-30-methoxymethyl-5a-pregnan-20-one (3.270
g, 58%) as white crystals. mp 163-4 *C. As expected, the
mother liquors contained both 17-epimers in approximately
equal amounts.
An analogous method was used to prepare: 3a-hydroxy-
30-ethoxymethyl-5a-pregnan-20-one; 3a-hydroxy-3f3-
propoxymethyl- 5a-pregnan-20-one;
EXAMPLE 12
3a-hydroxy-38-cyanomethyl-5a-pregnan-20-one
Potassium cyanide (210mg; 3.2mmol) and 20,20-
ethylenedioxy-3R-spiro-2'-oxirane-5a-pregnan-20-one (1.0
g; 2.7mmol) were stirred in ethanol (175mL) for 2 days,
refluxed for 5 hours and then allowed to cool overnight.
Ether (100mL) and brine (100mL) were added to the reaction
mixture. The separated aqueous phase was washed with
ether and the combined organic phases were washed with
brine. Aqueous HC1 (1M; 6mL) and THE (15mL) were added to
the -organic phases and the solution stirred overnight.
Aqueous NaHCO3 (20mL) was added and then water (50mL) . The
separated aqueous phase was washed with ether and the
combined organic phases were dried over MgSO4 and
evaporated. The white solid residue was purified by flash
column chromatography (40:20:1 CH2C12/hexane/acetone) to
give a white solid (493mg; 52%); mp 218.5 - 220 C.
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EXAMPLE 13
3a-Hydroxy-3a-azidomethyl-5a-pregnan-20-one
To a solution of 3(R)-epoxy-5a-pregnan-20-one (1.012
g, 3.06 mmol) in dry DMF (Aldrich; 50 mL) was added NaN3
(226 mg, 3.48 mmol), all of which did not dissolve. Then
glacial acetic acid (1.05 mL, 17.5 mmol) was added, and
the resulting mixture was heated a little bit with a heat
gun in order to dissolve all the solid. The mixture was
stirred for a while, giving a clear solution. TLC (100:1
CH2C12/acetone or 3:1 hexane/acetone) after stirring the
mixture for 4 h still indicated the presence of starting
material. The reaction mixture was then heated with
stirring at 35-50 'C for 3 days. By TLC the reaction was
complete. CH2C12 and water were added. The aqueous layer
was back-extracted with CH2C12. The combined organics were
washed with saturated NaHCO3, dried (MgSO4), filtered, and
concentrated on rotary evaporator to give a liquid con-
taining a lot of DMF. Further Evaporation by distillation
under high vacuum provided a solid (1.09 g). 1H NMR of
this solid revealed the presence of mainly the desired 3a-
hydroxy-3R-azidomethyl-5a-pregnan-20-one and almost none
of its 17-epimer. Purification by flash column chroma-
tography using CH2C12 as eluant afforded 3a-hydroxy-30-
azidomethyl-5a-pregnan-20-one (834 mg, 73%) as a white
solid; mp 147-8'C.
EXAMPLE 14
3a,20a-Dihydroxy-5a-19-nor-oregnane
-To a solution of 3a-hydroxy-5a-19-nor-17(Z)-pregn-
l7(20)-ene (232 mg, 0.81 mmol) (prepared from the
androstane-17-one with the Wittig reagent as described
below) in THE (20 mL) was added diborane THE complex (1M
solution in THF, 3.0 mL) dropwise at 0 C. The reaction
was allowed to warm to 25 C for 2.5 h. Then a solution of
sodium hydroxide (2N, 15 mL) was added very slowly at 0 C
followed by an addition of hydrogen peroxide (30%, 8 mL).
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Water then was added and THE layer was separated by
separatory funnel. The aqueous layer was extracted with
ethyl acetate (2x40 mL). The organic solution was dried
over potassium carbonate and the solvent removed in vacuo.
A column chromatography using acetone and methylene
chloride in hexane (8:8:84) resulted in a small amount of
pure product (15mg).
EXAMPLE 15
3x,21-hydroxy-38-methyl-5a-pregnan-20-one, 21-hemi-
succinate
A mixture of 3x,21-hydroxy-39-methyl-5a-pregnan-20-
one (600 mg, 1.72 mmol) and succinic anhydride (388 mg,
3.88 mmol) in 12 mL of pyridine was allowed to stir at rt
for 23h. The reaction was concentrated in vacuo and the
residue was partioned between EtOAc and an aqueous HOAc
solution (pH 4) . The organic layer was separated and
washed with a sat. NaCl solution and concentrated. The
crude hemisuccinate was purified by flash chromatography
(12 cm of silica in a 3.5 cm dia. column, eluted with 400
mL of 45/55 EtOAc/CH2C12 and 200 mL each of 50/50 and 60/40
EtOAc/CH2C12. Recrystallization from MeOH/water gave 346 mg
(45%) of the hemisuccinate as a white solid.
The sodium salt of the hemisuccinate was prepared by
dissolving the acid.in methanol and adding one equivalent
of an aqueous solution of sodium bicarbonate. The solvent
was removed in vacuo and the residue was triturated with
ether/pet. ether to give a white solid.
EXAMPLE 16
a. Triphenylprop-2-ynylstannane
Propargyl bromide (80 w% solution in toluene; 1.7 mL,
11.43 mmol) was added to a mixture of Mg (471 mg, 19.38
mmol) and dry Et20 (25 mL). HgCl2 (a few crystals) was
then added. The mixture was stirred for -5 min. Since no
reaction occurred, more HgC12 (15 mg) was added. The
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resulting mixture was first stirred at room temperature
and then warmed briefly. The mixture became turbid, and
the reaction ensued. The mixture was stirred at <20 C
(cold water bath) for -1 h after which time most of Mg had
5 reacted. The amount of the unreacted Mg remained
unchanged over a period of -30 min. Therefore, propargyl
bromide (80 w% in toluene; 0.4 mL, 2.69 mmol) was added.
The mixture was stirred at <20 C for -1 h. Subsequently,
a suspension of triphenyltin chloride (95%; 5.045 g, 12.43
10 mmol) in Et20 (50 mL) was added dropwise through a cannula
to the above solution of propargylmagnesium bromide. The
resulting mixture, which contained a white solid, was
stirred at room temperature for -3 h. TLC (3:1 hexane/
acetone) showed complete consumption of the starting tin
15 chloride. The mixture was quenched with saturated aqueous
NH4C1 (10 mL). A white solid was formed. The organic
layer was separated from the aqueous layer that contained
the white solid. Finally, the organic phase was dried
(MgSO4) , filtered, and evaporated under reduced pressure to
20 give a white solid (4.78 g) . Recrystallization of the
solid from hot hexane (50 mL) furnished triphenylprop-2-
ynylstannane as white plate-shaped crystals (3.92 g, 81%)
b. 3a-Hydroxv-3Q-(butadi-2',3'-enyl)-5a-pregnan-20-one
(Reference: Baldwin, J. E.; Adlington, R. M.; Basak,
25 A. J. Chem. Soc., Chem. Commun. 1984, 1284)
A mixture containing 33-iodomethyl-3a-hydroxy-5a-
pregnan-20-one (234 mg, 0.51 mmol), triphenylprop-2-
ynylstannane (795 mg, 2.04 mmol), azobisisobutylnitrile
(18 mg, 0.11 mmol), and dry benzene (2 mL) was degassed
30 for -10 min. The above mixture was then refluxed for 2 h.
As indicated by TLC (100:1 CH2C12/acetone) , there was still
some (--33%) unreacted starting material, the amount of
which remained unchanged over a period of 2 h. TLC
showed, besides the starting material, two spots in -1:1
ratio, the less-polar spot being a new spot while the
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other corresponding to 3$-methyl-3a-hydroxy-5a-pregnan-20-
one. The reaction mixture was cooled to room temperature.
After removing the solvent, the residue was subjected to
flash chromatography to afford 3a-hydroxy-39-(butadi-
2',3'-enyl)-5a-pregnan-20-one as a white solid (20 mg,
11%)
EXAMPLE 17
3a-Hydroxy-38-(prop-2'-ynyl)-5a-pregnan-20-one
To a mixture of Mg (268 mg, 11.02 mmol), propargyl
bromide (80 w%; 1.5 mL, 10.09 mmol) and dry Et20 (15 mL)
was added HgC12 (10 mg). The resulting mixture was
slightly warmed in a water bath in order to initiate the
reaction. The mixture became turbid, and the reaction
ensued. The mixture was stirred at <20 C until all Mg
reacted (for -.1 h).
A solution of 20,20-ethylenedioxy-5a-pregnan-3-one
(711 mg, 1.97 mmol) in dry Et20 (20 mL) and dry THE (10 mL)
was cooled to -78 C. Some of the starting material
precipitated on cooling. Subsequently, propargylmagnesium
bromide solution (-0.6 M; 6.5 mL), prepared above, was
added dropwise at -78 C. The resulting mixture was
stirred at -78 C for -1 h. The mixture was then allowed
to warm to room temperature where it was stirred for 1 h.
1N HC1, p-toluenesulfonic acid monohydrate, and acetone
were added in order to hydrolyze the ketal function. The
two-phase mixture was stirred at room temperature over-
night. TLC (7:1 hexane/acetone) showed completion of the
reaction. Et20 and water were added. The aqueous layer
was back-extracted with Et20. The combined organics were
washed with saturated aqueous NaHCO3 and brine, dried
(MgSO4), and evaporated under reduced pressure to give a
white solid (690 mg) . 1H NMR of the crude solid indicated
it to be a 1:2 mixture of the desired 3a-hydroxy-33-(prop-
2'-ynyl)-5a-pregnan-20-one and its 3-epimer, respectively.
The mixture was flash-chromatographed twice with 10:1
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hexane/acetone to provide 3a-hydroxy-30-(prop-2'-ynyl)-5a-
pregnan-20-one as a white solid (70 mg, 11%). m.p. 220-
225 C (dec.).
EXAMPLE 18
3a-Hydroxy-313-(but-3'-enyl)-5a-pregnan-20-one
(Reference: Keck, G. E.; Yates, J. B. J. Am. Chem.
Soc. 1982, 104, 5829)
Dry benzene (3 mL) was degassed for 15 min by
bubbling Ar through it. Next were added successively 30-
iodomethyl-3a-hydroxy-5a-pregnan-20-one (263 mg, 0.574
mmol), allyltributyltin (97%; 0.5 mL, 1.56 mmol), 1,2'-
azobisisobutyronitrile (20 mg, 0.122 mmol). The resulting
mixture was refluxed for 3.5 h. TLC (100:1 CH2C12/acetone)
showed complete consumption of the starting iodide deriva-
tive, with the appearance of two new spots in -1:1 ratio,
the more-polar spot corresponding to 30-methy_l-3a-hydroxy-
5a-pregnan-20-one. The reaction mixture was cooled to
room temperature. After removing the solvent, the residue
was subjected to flash chromatography twice, with CH2C12 and
7:1 hexane/acetone. 3a-Hydroxy-30-(but-3'-enyl)-5a-
pregnan-20-one was obtained as a white solid (75 mg, 35%).
m.p. 140-141 C.
EXAMPLE 19
a. 3a-Hydroxy-5l3-pregnan-20-one, 20-ketal
A mixture of 3a-hydroxy-5(3-pregnan-20-one (10.8 g, 34
mmol), ethylene glycol (45 mL), and triethyl orthoformate
(30 mL) was stirred at r.t. for 5 min. p-toluenesulfonic
acid (200 mg) was then added and the stirring was con-
tinued at r.t. for 1.5 hr. The resulting thick paste was
poured into a sat. NaHCO3 solution (250 mL) . The precipi-
tated solid was collected by filtration, washed thoroughly
with cold water and dried. This semi-dried product was
dissolved in CH2C12 (350 mL) and dried over anhyd. K2CO3'
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The solution of the ketal was then filtered and used as
such for the next step.
b. 58-Pregnan-3,20-dione, 20-ketal
The above solution of 3a-hydroxy-50-pregnan-20-one,
20-ketal in CH2C12 was stirred with N-methylmorpholine-N-
oxide (8.8 g, 75 mmol), and powdered 4A molecular sieves
(58 g) under N2 for 15 min. Tetrapropylammonium perru-
thenate (400 mg) was then added and the stirring was
continued at r.t. for 2 hr. The resulting dark green
mixture was passed through a short column of Florisil and
eluted with CH2C12. The fractions containing the product
(TLC) were combined and evaporated. The crude product was
then crystallized from a mixture of EtOAc:Hx (1:1) to
yield the title compound (10.3 g) as long rods.
c. Preparation of the lithium reagent from 1,2-
dibromoethylene
A 100 mL three neck flask equipped with a N2 gas
bubbler, a thermometer, and a dropping funnel was charged
with 1,2-dibromoethylene (cis/trans mixture, 98t, Aldrich,
0.164 mL, 2 mmol, mw = 186, d = 2.246) . Dry THF (15 mL)
was added and the solution was cooled to -78 C in a dry
ice-acetone bath. n-BuLi (2.5M in THF, 1.6 mL, 4 mmol) was
added dropwise over a period of 10 min. The mixture was
stirred at this temperature for 1 hr and the resulting
reagent was used immediately for the next step.
d. 36-Ethynyl-3a-hydroxy-58-pregnan-20-one
The above solution of the reagent in THF, which was
maintained at -78 C, was treated dropwise with a solution
of 50-pregnan-3,20-dione, 20-ketal (180 mg, 0.5 mmol) in
THF (15 mL). The temperature was maintained below -70 C
during the addition. The stirring was continued at this
temperature for 15 min. (100% conversion as detected by
TLC). The cooling bath was removed and the resulting
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solution was quenched with 2N HC1 (pH 6) The solvent was
removed and the residue was dissolved in acetone (10 mL).
After adding 2N HC1 (4 mL) the solution was stirred at
ambient temperature for 0.5 h. The mixture was neutral-
ized with dil. NaHCO3 solution. The precipitated solid
(158 mg, 9396) was collected by filtration, washed with
water, and dried. The crude product is then purified by
crystallization from EtOAc or from a mixture of acetone-
hexane to yield the title compound; mp 196-197 C, TLC-R,
0.45 (hexane:acetone 7:3).
e. 3 -Ethyl-3a-hydroxy-5l3-precrnan-20-one
A mixture of Pd/C (50, 24 mg) and MeOH (12 mL) was
presaturated with hydrogen by stirring the mixture under
hydrogen for 10 min. A solution of 3f3-ethynyl-3a-hydroxy-
5,6-pregnan-20-one (155 mg) in MeOH (5 mL) was then added,
and the mixture was hydrogenated for 1.5 h. under 200 Kpa
pressure at r.t. Filtration of the catalyst followed by
evaporation of the solvent yielded the title compound,
which was purified by crystallization from hexane:acetone
(9:1) yield : 105 mg; mp 115-117 C. TLC Rf (hexane:acetone
7:3) = 0.48.
f. 3 -Ethyl-3a-hydroxy-5aõprecrnan-20-one:
To a mixture of 570 mg (3 mmol) of cuprous iodide and
10 ml of dry ether cooled to -60 C under argon was added
4 ml of 1.5M methyl lithium. The reaction mixture was
warmed to 0 C, stirred until a colorless solution was
obtained and then recooled to -60 C. An additional 0.5-1
ml of methyl lithium solution was added to keep the
solution colorless. A cooled (0 C) solution of 0.5 g (1.5
mmol) of 3R- spiro-2' -oxirane- 5a-pregnan- 20 -one in 20 ml of
ether was added slowly to the above reaction mixture while
maintaining the temperature at -60 C. An additional 0.2
ml of methyl lithium was added and the reaction mixture was
stirred at -60 C for 1 hr. It was then quenched by the
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addition of saturated ammonium chloride solution, allowed
to warm to room temperature and diluted with ether and
ammonium chloride solution. The organic phase was
separated. The aqueous phase was extracted with ether.
5 The combined organic extracts were dried and concentrated
to leave 450 mg of crude product. Chromatography on silica
gel using 5% acetone/hexane gave 356 mg of product.
EXAMPLE 20
Disodium 3a,208-dihydroxy-58-pregnane, bis(hemisuccinate)
10 A suspension of 3u,200-dihydroxy-53-pregnane (Stera-
loids; 250 mg, 0.78 mmol) in 5 mL of dry pyridine was
treated with succinic anhydride (200 mg, 2.0 mmol). The
mixture was heated at 100 C for a total of 10 h. An
additional 6 mmol of succinic anhydride was added in three
15 portions as the reaction was heated. The dark mixture was
concentrated (0.05 mmHg, 30 C) to remove the solvent and
then heated at 90 C (0.05 mmHg) to remove excess succinic
anhydride. The residue was recrystallized from ether/
hexanes giving a solid that consisted mainly of succinic
20 acid. The mother liquor was concentrated and subjected to
column chromatography (flash silica gel, eluted with
95/5/0.1 CH2C12/MeOH/HOAc) giving a white solid which was
recrystallized from ether/hexane. The bis(hemisuccinate),
mp 81-90 C was carried on to the bis(sodium salt).
25 The bis(hemisuccinate) (100 mg, 0.192 mmol) was
dissolved in a minimum volume of methanol. A solution of
NaHCO3 (2 eq, 33 mg, 0.393 mmol) in 0.6 mL of water was
added dropwise. After 3h, the solution was concentrated in
vacuo to give a white solid.
30 EXAMPLE 21
a. 3a-Hydroxy-3(3-methyl-5a-androstanel713-carboxylic acid
Bromine (2.0 mL, 38.8 mmol) was added dropwise to an
aqueous solution of NaOH (3.746 g in 30 mL of water) at
0 C. Subsequently, a solution of 3a-hydroxy-30-methyl-5a-
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pregnan-20-one in dioxane (250 mL) and water (30 mL) was
added to the above solution at 0 C over a period of 1 h.
The resulting yellow mixture that contained some solid was
allowed to warm to room temperature. After some time all
S the solid dissolved. The reaction solution was stirred
overnight at which point it turned colorless. Then CH2C12
(150 mL), water (100 mL) and IN HC1 (50 mL) were added.
Brine was also added to facilitate the separation of the
two liquid phases. The aqueous layer was separated and
back-extracted with CH2C12 (200 mL) . The two organic phases
were combined and washed with water, dried (MgSO4),
filtered, and evaporated under reduced pressure to afford
a white solid (3.851 g). Recrystallization of this solid
from hot 1:1 hexane/acetone (500 mL) gave 3a-hydroxy-30-
methyl-5a-androstane 170-carboxylic acid as a white solid
(1.838 g, 50%). m.p. 222.5-224 C. Evaporation of the
mother liquor followed by recrystallization from hot
methanol furnished more product as white crystals (1.50 g,
40%). m.p. 223-225 C.
20.b. 178-(Hydroxvmethyl)-38-methyl-5a-androstan-3a-ol
To a stirred suspension of LiAlH4 (95%; 546 mg, 13.67
mmol) in dry THE (30 mL) at 0 C was added a solution of 3a-
hydroxy-30-methyl-5a-androstane 170-carboxylic acid (1.633
g, 4.88 mmol) in dry THE (50 mL + 10 mL for the rinse)
through a pressure-equalizing funnel over a period of 30
min. The resulting mixture was then allowed to warmed to
room temperature. The stirring was continued overnight.
TLC (7-:1 hexane/acetone) showed completion of the reaction.
The reaction mixture was cooled to 0 C, and IN HC1 (25 mL)
was added dropwise. Subsequently, Et20 (100 mL), water (25
mL) , and conc. HC1 (5 mL) were added. The aqueous layer
was still not completely clear. The two phases were
separated. Conc. HC1 (15 mL) was added to the aqueous
layer which was then extracted with Et20 (100 mL). The two
organic layers were combined, washed with saturated aqueous
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NaHCO3, dried (MgSO4), filtered, and evaporated under
reduced pressure to afford 17(3- (hydroxymethyl) -3(3-methyl-
5a-androstan-3a-ol as a white solid (1.605 g). 1H NMR of
the product showed it to contain some (4%) unreacted
starting material and THE (4%).
c. 178-Formyl-38-methyl-5a-androstan-3a-ol
To a solution of 170-(hydroxymethyl)-30-methyl-5a-
androstan-3a-ol (96% pure; 1.58g, 4.73 mmol) in dry CH2C12
(90 mL) at room temperature was added pyridinium chloro-
chromate (98%; 1.115 g, 5.07 mmol). The resulting mixture
was stirred at room temperature for 7 h. TLC (7:1 hexane/
acetone) showed complete consumption of the starting acid.
The mixture was then filtered through neutral florisil and
successively washed with CH2C12 (250 mL) and 5:1 hexane/
acetone (100 mL) (The filtrate was checked by TLC for the
presence of the product.) The solvents were removed to
give a light yellow solid (1.390 g) which was purified by
flash chromatography employing CH2C12 as eluent to furnish
173-formyl-30-methyl-5a-androstan-3a-ol as a white solid
(1.081 g, 72%) . m.p. 156-166 C.
d. 178-cyano-3a-hydroxy-5a-androstane
To a solution of 3a-hydroxy-5a-androstan-l7-one (1.511
g, 5.20 mmol) in 135 mL of 1,2-dimethoxyethane (DME) was
added a 1M solution of potassium tert-butoxide in THE (52
mL, 52 mmol). The resulting cloudy orange solution was
treated over 2.5 h. with a solution of 2.06 g (10.6 mmol)
of p-toluenesulfonylmethyl isocyanide in 30 mL of DME.
After an additional 2.5 h., the reation was diluted with
100 mL of ether and 100 mL of water was added. The aqueous
layer was extracted with ether (2 x 50 mL) and the pooled
organic layers were washed with a brine solution, dried
(MgSO4) and concentrated. Column chromatography on silica
gel (100% of CH2C12 as eluent) gave 860 mg of slightly
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impure product. Recrystallization from EtOAc gave 300 mg
(19%) of pure nitrile, mp 159-1600C.
EXAMPLE 22
3a-hydroxy-513-pregn-17(20)-ene
To the ylide prepared from ethyltriphenylphosphonium
bromide (Aldrich; 1.606 g, 4.33 mmol) and solid potassium
tert-butoxide (475 mg, 4.23 mmol) was added 3a-hydroxy-50-
androstan-17-one (Steraloids; 503 mg, 1.73 mmol) in one
portion. The red-orange mixture was heated at reflux for
103.5 h and then allowed to cool. It was further cooled to
0 C and then added to an ice-cold sat. NH4C1 solution/ether
mixture. The aqueous layer was separated and washed twice
with ether. The ether layers were then extracted with a
sat. NaCl solution, dried (MgSO4) and concentrated. Flash
chromatography (17.5 cm of silica in a 4 cm dia. column,
eluted with 400 mL of 2% acetone/CH2C12 and 200 mL of 2.5%
acetone/CH2C12) afforded 530 mg (100%) of the alkene as a
white solid. By 1H NMR the compound was an 8:1 mixture of
Z- and E-isomers.
EXAMPLE 23
3a-hydroxy-38-ethenyl-53-pregnan-20-one
A solution of 5,8-pregnan-3,20-dione, 20-ketal (1.18g,
3.3 mmol) in dry THE (20 mL) was treated with vinyl
magnesium bromide (1M in THF, 3.7 mmol, 3.7 mL) at -70 .
After stirring the mixture at this temp. for 5 min. and
then at r.t. for 2.5 h, it was quenched with sat. NH4C1
solution (10 mL). The solvent was removed and the residue
was extracted with EtOAc. The organic layer was washed
with water, dil. NaHC03 soln., water, and brine. After
drying over anhyd. MgS04 the solution was filtered and
evaporated to yield the crude product (1.2 g). This crude
product was then dissolved in acetone (20 mL). After
adding 1N HC1 (10 mL) the solution was stirred at r.t. for
15 h. The solvents were removed and the residue was
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extracted with CH2C12. The organic layer was washed with
water, dil. NaHCO3 soln., water, and brine. After drying
over anhyd. MgSO4 the solution was filtered and evaporated
to yield the crude product (890 mg). This crude product
was then dissolved in a small amount of CH2C12 and poured on
a column of silica gel. Elution with toluene:acetone
mixture (94:6) gave 3a-ethenyl-3S-hydroxy-5(3-pregnan-20-one
(126 mg) as a first fraction. Further elution with the
same solvent mixture yielded 3$-ethenyl-3a-hydroxy-53-
pregnan-20-one (189 mg), m.p. 113-116 .
EXAMPLE 24
a. 3a-Hydroxy-5a-androstan-17-one
To a solution of 3a-hydroxy-5a-androstan-l7-one (6g)
and diethyl azodicarboxylate (5.04 g) in THE (50 mL) was
added trifluoroacetic acid (3.3 g) and the mixture became
yellow. Then triphenylphosphine (7.6 g) was added. The
reaction turned to colorless and heat was given off.
Sodium benzoate was added after 5 min and then water (100
mL) was added. The mixture was extracted with methylene
chloride (3x80 mL) and organics was dried over magnesium
sulfate. The solvent was removed in vacuo and the crude
product was hydrolyzed with potassium hydroxide (10%, 10
mL) in methanol (150 mL) for 1 h. Most of the methanol was
then removed in vacuo and the remaining material was
partitioned between methylene chloride and ammonium
chloride. The product (4.7 g, 78%) was purified by
chromatography (CH2C12: acetone=9:1).
b. 3a-Hydroxy-21-methyl-5a-pregn-17(Z)-ene
3a-Hydroxy- 5a-androstan- 17 -one (2 g, 6.9 mmol) was
added to Wittig reagent produced from propyltriphenyl-
phosphonium bromide (13.3 g) and potassium t-butoxide (3.9
g) in THE (20 mL). The reaction was refluxed for 12 h and
cooled to 25 C. Then a solution of ammonium chloride (60
mL) was added and the organic layer was separated by
WO 94/27608 216 3 7 4 8 PCT/US94/05820
separatory funnel. The aqueous layer was extracted with
methylene chloride (2x50 mL) . The organic solution was
dried over potassium carbonate and the solvent removed in
vacuo. The crude product was purified by chromatography
5 (acetone:methylene chloride:hexane = 1:2:7) to produce 0.84
g of product (39%) It was mixture of Z and E isomers
(Z:E=13:1).
An analogous procedure was used to prepare 3a-hydroxy-
17-methylene-5a-androstane using the Wittig reagent
10 produced from methyltriphenylphosphonium bromide.
c. 3a-t-Butyldimethylsiloxy-21-methyl-5a-precrn-17(Z)-ene
The mixture of 5a-3a-hydroxy-21-methyl-pregn-17 (Z) -ene
(0.84 g, 2.66 mmol), TBDMSC1 (1.2 g, 8.0 mmol) and
imidazole (0.91 g, 13.3 mmol) in methylene chloride (10 mL)
15 and DMF (30 mL) was stirred for 12 h and then ammonium
chloride was added. It was then extracted with methylene
chloride (3x40 mL) and washed with brine (50 mL). The
organic solution was dried over potassium carbonate and
then solvent removed and it was found that there was still
20 some DMF. It was then redissolved in ether and washed with
brine (2x50 mL) and then dried over potassium carbonate.
Chromatography with hexane resulted 1.14 g of the pure
product (100%).
d. 3a-t-Butyldimethylsiloxy-20a-hydroxy-21-methyl-5a-
25 pregnane
To a solution of 3a-t-butyldimethylsiloxy-21-methyl-
5a-pregn-17 (Z) -ene (1.14 g, 2.66 mmol) in THE (30 mL) was
added diborane THE complex (1M solution in THF, 5.3 mL)
dropwise at 0 C. The reaction was allowed to warm to 25 C
30 for 1 h. Then a solution of sodium hydroxide (20%, 10 mL)
was added very slowly at 0 C followed by an addition of
hydrogen peroxide (30%, 10 mL). Aqueous ammonium chloride
then was added and THE layer was separated by separatory
funnel. The aqueous layer was extracted with ether (2x40
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41
mL) The organic solution was dried over potassium carbon-
ate and the solvent removed in vacuo. The pure product was
obtained by column chromatography (0.52 g, 44%).
e. 3a,20a-Dihydroxy-2l-methyl-5a-gregnane
A solution prepared by mixing HF (48%, 5 mL) and CH3CN
(30 mL) was added to a flask containing 3a-t-butyldimethyl-
siloxy-20a-hydroxy-21-methyl-5a-pregnane (0.51 g) and a
white precipitate appeared. The reaction was stirred for
1 h and then filtered. The white solid was washed with
ether for three times and it gave satisfactory analytical
results (0.3 g, 79%). m.p. 227-231 C.
EXAMPLE 25
3a 21-Dihvdroxy-3a-trifluoromethyl-58-19-nor-pregnan-20-
one
To a solution of 3a-hydroxy-30-trifluoromethyl-50-19-
nor-pregnan-20-one (300 mg, 0.87 mmol) in toluene (15 mL)
was added MeOH (1 mL) and BF3SOEt2 (1.4 mL, 11.3 mmol). The
resulting mixture was cooled to 0 C and Pb(OAc)4 (0.54 g,
1.21 mmol) was added. The reaction was warmed to 25 C with
stirring for 45 min and then NaHCO3 solution (sat., 30 ml)
was added, and the mixture was stirred for 1 h. It was
poured into a separatory funnel containing water (50 mL)
and extracted with ether (3x40 mL). The ethereal solution
was washed with brine (50 mL) and then dried over K2CO3. The
crude product obtained by removal of solvent was dissolved
in MeOH (25 mL) and K2CO3 solution (sat., 8 mL) was added.
The reaction was stirred for 5 h and then the reaction
mixture was poured into a separatory funnel containing 50
mL of water. It was then extracted with CH2C12 (3x30 mL).
The combined extracts were dried over K2CO3 and the crude
material obtained was purified by chromatography to give
the product (160 mg) along with 21-methoxy by-product (40
mg) The product was further purified by recrystallization
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from 10% acetone in hexane to give 88 mg of the pure
product (28%) as a white solid, m.p. 140-142 C.
EXAMPLE 26
a. 3a-Hydroxy-38-methyl-58-19-nor-pregn-(Z)17(20)-ene:
To a solution of 5/3-19-nor-pregn-17(z)-en-3-one (600
mg, 2.1 mmol) in THE (20 mL) at -78 C was added methyl-
magnesium bromide (3 M in ether, 1 mL, 3 mmol). The
reaction was warmed to 25 C. Ammonium chloride solution
(50 mL) was added and the mixture was extracted with ether
(3x30 mL) . The product (577 mg, 91%) purified by chromato-
graphy (10% acetone in hexane) was obtained as a white waxy
solid.
b. 3a-Hydroxy-3a-methyl-58-19-nor-pregnan-20-one:
To a solution of 3a-hydroxy-30-methyl-50-19-nor-17(z)-
15pregn-17(20)-ene (0.57 g, 1.9 mmol) in THE (30 mL) was
added BH3=THF solution (1 M in THF, 3.8 mL, 3.8 mmol) at
25 C. The reaction was completed in 30 min and NaOH
solution (20%, 8 mL) was added very cautiously followed by
a addition of hydrogen peroxide solution (30%, 8 mL). The
mixture was stirred for 20 min and NH4C1 solution was then
added. The mixture was extracted with CH2C12 (3x30 mL) and
the combined extracts were dried over K2CO3. Removal of the
solvent left the crude product (760 mg) which was dissolved
in CH2C12 (40 mL) . 4-Methylmorpholine N-oxide (0.6 g, 4.75
mmol) and freshly ground molecular sieves (4A, 5 g) were
added and the resulting mixture was stirred for 20 min. The
catalyst tetrabutylammonium perruthenate (100 mg) was
added. The oxidation was complete within 40 min and the
reaction mixture was filtered through a pad of Florisil
which was rinsed with a 2:1 mixture of ether and CH2CI2 (60
mL). The crude material (0.62 mg) obtained by the removal
of the solvent was purified by chromatography (20% EtOAc in
hexane) to give the product (53 mg, 9%) as a white solid.
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EXAMPLE 27
38-Ethvnyl-3a,20a-dihvdroxv-58-pregnane and 38-ethynyl-
3cx,20a-dihydroxy-58-pregnane
To a solution of 33-ethynyl-3a-hydroxy-5$-pregnan-20-
one (0.31 g, 0.91 mmol) in methanol (20 mL) was added
sodium borohydride (200 mg, 5.3 mmol) and it was stirred at
25 C for 1 h. Ammonium chloride (50 mL) solution was then
added and the mixture was extracted with CH2C12 (3x30 mL).
A chromatography (EtOAc:Hex=3:7) gave 30-ethynyl-3a,20$-
dihydroxy-5$-pregnane (200 mg, 65%) as major product: m.p. ,
221-223 C. The minor product, 3$-Ethynyl-3a,20a-dihydroxy-
5$-pregnane was further purified by another chromatography
(25-30% ethyl acetate in hexane, 16 mg, 5%): m.p., 187-
188 C.
EXAMPLE 28
a. 3a,21-dihydroxy-38-ethynyl-58-pregnan-20-one
To a solution of 3x,21-dihydroxy-3$-ethynyl-50-
pregnan-20-one 21-acetate (725 mg, 1.81 mmol) in 45 mL of
methanol at 0 C was added an aqueous 10% K2CO3 solution
(3.75 mL). After stirring for 30 m at rt, the mixture was
recooled to 0 C and a 2N aqueous HOAc solution (1.8 mL) was
added. The reaction was added to an EtOAc/water mixture.
The aqueous layer was extracted twice with EtOAc and the
pooled organic layers were extracted with a brine solution,
dried (Na2SO4) and concentrated. Purification by flash
chromatography (20 cm of silica gel in a 4 cm dia. column,
eluted with 2 liters of 20% acetone/hexane) gave 582 mg
(90%) of the diol as a white solid, mp 155.5-157 C.
b. 38-Ethynvl-3a-hydroxy-21-methoxy-58-pregan-20-one
A solution of 3$-ethynyl-3a-hydroxy-5$-pregan-20-one
(503 mg, 1.47 mmol) in methanol (4 mL) and toluene (17 mL)
was cooled in an ice/water bath and neat BF3-Et20 (2.9 mL,
3.35 g, 23.6 mmol) was added followed by 1.0 g (2.25 mmol)
of solid lead tetraacetate. The mixture was allowed to
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warm to r.t. and then stirred for 80 min. The reaction was
then added to 25 mL of an ice-cold sat. NaHCO3 solution.
The aqueous layer was extracted with EtOAc (2 x 10 mL) and
the combined organic layers were extracted with a sat.
NaCl solution, dried (Na2SO4) and concentrated. Two flash
columns (silica gel, eluted with 2.5% acetone/CH2C12)
afforded 148 mg (27%) of the title compound as a white
solid, mp 198.5-199.5 C.
EXAMPLE 29
Preparation of (3-R)-20,20-ethylenedioxy-5a-pregnane-spiro-
2'-oxirane
A mixture of trimethylsulfoxonium iodide (Aldrich;
8.79 g, 39.9 mmol) and solid potassium tert-butoxide
(Aldrich, 95%; 4.3 g; 36.6 mmol) in 125 mL of dry THE was
heated at reflux for 1.5 h. The reaction was allowed to
cool to r.t. and 20,20-ethylenedioxy-5a-pregnan-3-one (12.0
g, 33.3 mmol) was added as a solid in one portion. The
resulting mixture was stirred at r.t. for 20h. and then
concentrated in vacuo. The residue was dissolved in CH2C12
and extracted with water. The aqueous layer was back
extracted with CH2C12 and the combined organic layers were
washed with a saturated NaCl solution, dried (MgSO4) and
concentrated. The white solid obtained (12.0 g, 96%, mp
160-165 C) was recrystallized from 60/40 acetone/methanol.
EXAMPLE 30
3a.20-dihvdroxy-38.20-dimethyl-5a-Dregnane
5a-Pregnan-3,20-dione (1.5 g, 4.7 mmol) was dissolved
in 250 mL of dry benzene and 15 mL of 3M methylmagnesium
bromide was added. The reaction mixture was stirred at room
temperature for 30 min. The excess methyl magnesium bromide
was decomposed by adding 100 mL of sulfuric acid dropwise
at 0 C. The product solution was extracted with 250 mL of
diethyl ether. The organic extract was combined, dried
over magnesium sulfate, and evaporated to give a 60:40
WO 94/27608 2163748 PCT/US94/05820
mixture of 30-methyl and 3a-methyl isomers. The product
mixture was separated by flash chromatography on silica gel
using a 70:30 (hexane:ethyl acetate and 1% methanol).
elution to give 0.476 g (24% yield) of the desired product.
5 EXAMPLE 31
3a,21-dihvdroxy-38-trifluoromethyl-58-pregnan-20-one
A solution of 3a,21-dihydroxy-30-trifluoromethyl-5$-
pregnan-20-one, 21-acetate (1.36g, 3.06mmol) in MeOH (75mL)
was cooled to 0 C. A solution of K2CO3 (10% aqueous,
106.45mL, 4.67mmol) was then added dropwise. After stirring
for 1.5 hours at 0 C, a solution of acetic acid (2N
aqueous, 2.5mL, 5.Ommol) was added dropwise and the mixture
was allowed to warm to room temp. EtOAc, CH2C12 and water
(100mL each) were added and thoroughly mixed. The organic
15 phase was isolated, washed with aqueous NaHCO3 and NaCl
solutions, dried over MgSO4 and evaporated in vacuo. The
residue was purified by flash column chromatography
(hexane/EtOAc 3:1) to yield a white solid (973mg, 79%)
mp. 148-150 C.
20 EXAMPLE 32
a. 3a-Hydroxy-5a-2l-ethyl-Rrecrn-17(z)-ene
To a Wittig reagent prepared by stirring the mixture
of n-butyltriphenylphosphonium bromide (3.9 g, 9.66 mmol)
and potassium t-butoxide (1.1 g, 9.66 mmol) in THE (10 mL)
25 for 30 min was added 3a-hydroxy- 5a-androstan- 17 -one (0.7 g,
2.4 mmol) and the mixture was heated to reflux for 16 h.
The reaction was then quenched with ammonium chloride
solution and extracted with CH2C12 . The pure product (230
mg, 29%) was obtained by chromatography (10% acetone in
3 0 hexane) .
b. 3a-t-Butyldimethylsiloxy-5a-21-ethyl-pregn-17(z)-ene
The mixture of t-butyldimethylsilylchloride (0.32 g,
2.1 mmol) imidazole (0.24 g, 3.5 mmol) and 3a-hydroxy-5a-
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21-ethyl-pregn-17(z)-ene (0.23 g 0.7 mmol) was stirred for
12 h at 25 C. Ammonium chloride solution was then added and
extraction was done with ether (3x30 mL) . The obtained
extract was washed with brine (3x30 mL). The pure product
(0.27 g, 87%) was then obtained by chromatography (Hexane)
c. 3a,20a-Dihvdroxy-5a-21-ethylpregnane
To a solution of 3a-t-butyldimethylsiloxy-5a-21-ethyl -
pregn-17(Z)-ene (0.27 g, 0.6 mmol) in THE (20 mL) was added
BH3=THF (1M in THF, 1.2 mL) at 25 C. Then sodium hydroxide
(20%, 5 mL) was added cautiously after the reaction was
stirred for 1 h. Then hydrogen peroxide solution (30%, 5
mL) was added and the mixture was stirred for another hour.
Ammonium chloride solution was then added and it was
extracted with CH2C12. The 20a-hydroxy-3a-silyl ether
compound was obtained by chromatography (5% acetone in
hexane). 60 mg of this compound was desilylated using
hydrofluoric acid in acetonitrile. Chromatography (20-30%
ethyl acetate in hexane) gave 11 mg of the pure title
compound (m.p., 229-232 C, Rf=0.28, EtOAc:Hex=3:7).
EXAMPLE 33
38-Fluoromethvl-3a-hydroxy-5a-gregnan-20-one
A mixture of n-Bu4NF=xH2O (7.873 g) and benzene (50 mL)
was refluxed with a Dean-Stark trap overnight. The
mixture, which was not a clear solution, was then concen-
trated (normal pressure) to -10 mL and allowed to cool to
room temperature. A solution of 3(R)-5a-pregnan-3-spiro-
2'oxir.ane-20-one ethyleneketal (2.55 g, 6.81 mmol) in dry
benzene (15 mL + 5 mL for the rinse) was added, using a
double-ended needle, to the above concentrated solution.
The resulting solution was concentrated, using a short-path
distillation apparatus, to -10 mL and refluxed for 15 min.
Since it was very difficult to spot the concentrated
reaction solution on the TLC plate, dry benzene (5 mL) was
added. TLC (100:1 CH2C12/acetone or 3:1 hexane/acetone)
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showed some unreacted starting material, besides two new
less-polar spots. The reaction mixture was again concen-
trated and then refluxed for 30 min; TLC (after diluting
the mixture with benzene) now showed almost complete
consumption of the starting epoxide. As before, the
mixture was concentrated and refluxed for a while. It
must be noted here that this reaction takes place only when
the mixture is highly concentrated. After the mixture had
come to room temperature (giving a light-yellow solid),
ether and water were added. Since all the solid did not
dissolve, CH2C12 was added. The aqueous layer was back
extracted with CH2C12. The combined organic extracts were
washed with water (x2), dried (Na2SO4), filtered, and
evaporated under reduced pressure to provide a white solid
(3.33 g) whose 1H NMR showed it to be a 4:1 mixture of 30-
fluoromethyl- 3a-hydroxy-5a-pregnan-20-one ethyleneketal and
3a-fluoro-33-hydroxymethyl-5a-pregnan-20-one ethylene
ketal.
In order to hydrolyze the ketal, acetone (100 mL),
water (5 mL), and p-TsOH=H20 (143 mg, 0.752 mmol) were added
to the above solid. The pH was adjusted by adding in HC1
until slightly acidic. The mixture was heated with a heat
gun for a while in order to get a clear solution, which was
stirred at room temperature for 2 h. The mixture became
turbid, so CH2C12 was added to obtain a clear solution. TLC
indicated completion of the reaction. The solvent was
removed under reduced pressure, giving a white solid to
which were added CH2C12 and water. The aqueous layer was
back-extracted with CH2C12. The combined organics were
washed with saturated NaHCO3, dried (MgSO4), filtered, and
evaporated under reduced pressure to furnish a white
crystalline solid (2.5 g). Flash column chromatography of
mecrude product with CH2C12 as eluent provided the desired
3jS-fluoromethyl-3a-hydroxy-5a-pregnan-20-one (1.41 g, 59%).
mp 201-3 C.
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EXAMPLE 34
3a-hydroxy-38-methyl-5a-precrn-17(20)-ene
To a solution of 5a-pregn-17 (20) -en-3 -one (207 mg,
0.689 mmol) in 8 mL of dry THE at -30 C (At -78 C, not all
of the steroid dissolved) was added a 3M solution of MeMgBr
in ether (Aldrich; 0.34 mL, 1.02 mmol) dropwise via
syringe. After stirring at -30 C for 4.75 h, the reaction
was added to a sat. NH4C1 solution/ether mixture. The
aqueous layer was extracted with ether (3x30 mL) and the
organic layers were combined and washed with a brine
solution, dried (MgSO4) and concentrated at reduced
pressure. The crude material was dissolved in CH2C12 and
added to 32 cm of flash silica gel in a 2 cm column. The
column was eluted with 100% CH2C12 until unreacted ketone
(8.7 mg, 4%) had eluted. Elution with 100mL each of 2.5 and
5% acetone/CH2C12 gave 38 mg (18%) of 3a-hydroxy-3/3-methyl-
5a-pregn-17 (20) -ene (Rf 0.35, 100% CH2C12) and 3(3-hydroxy-3a-
methyl-5a-pregn-17 (20) -ene (Rf 0.16, 100% CH2C12) EXAMPLE 35
203x-hydroxy-l7Z- and 17E-methoxymethylene-58-androstane
Solid 3a-hydroxy-5g-androstan-l7-one (504 mg, 1.74
mmol) was added in portions to the ylide prepared from
methoxymethyltriphenylphosphonium chloride (Aldrich; 1.484
g, 4.33 mmol) and potassium tert-butoxide (479 mg, 4.27
mmol). This mixture was ref luxed for 7h and then cooled to
0 C and added to an ice-cold sat. NH4C1 solution/ether
mixture. The aqueous layer was extracted twice with ether
and the combined organic layers were washed with a sat.
NaCl solution, dried (MgSO4) and concentrated. The crude
alkene was dissolved in CHzClZ and added to 13 cm of flash
silica in a 3.5 cm dia. column. The column was eluted with
400 mL of 5% acetone/CH2C12 and 200 mL of 7.5%
acetone/CH2C12. The desired alkene was isolated as a 1:1
mixture of Z- and E-isomers (158 mg, 28%) along with
recovered 17-one (220 mg, 44%). The mixture of alkene
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isomers was resubjected to flash chromatography (15 cm of
silica in a 3.5 cm dia. column, eluted with 500 mL of 4%
acetone/toluene and 200 mL each of 5,6, and 7% acetone/
toluene) without good separation. Preparative HPLC (21.4
mm id x 25 cm Microsorb 5m column; solvent system 2.5%
acetone/CH2C12, 10 mL/min; RI detection) allowed the
separation of Z-isomer, mp 150-152 C, (more polar) and E-
isomer, mp 151-153 C (less polar).
EXAMPLE 36
103x.208-Dihvdroxy-30-methyl-5a-pregnane and 3a,20a-
Dihydroxy-3a-methyl-5a-pregnane
Reference: House, H. 0.; Muller, H. C.; Pitt, C. G.;
Wickam, P. P. J. Org. Chem. 1963, 28, 2407.
To a mixture of freshly cut sodium (250 mg, 10.87
mmol) and boiling toluene (5 mL) were added dropwise over
a period of 30 min a solution of 3a-hydroxy-39-methyl-5a-
pregnan-20-one (340 mg, 1.022 mmol) in isopropyl alcohol (3
mL, 39.18 mmol) and toluene (4 mL + 2 mL for the rinse) .
The resulting mixture was refluxed for 2.5 h at which point
TLC (3:1 hexane/acetone) indicated completion of the
reaction and displayed two new spots that had lower Rf's
than the starting ketone and had approximately same
intensity. The reaction solution was allowed to come to
room temperature and then was cooled in an ice-bath. iN
HC1 (10 mL) was added dropwise. After diluting the mixture
with CH2C12 (-50 mL) , the aqueous layer was separated and
back-extracted with CH2C12 (x2). The combined organics were
washed with saturated NaHCO3, dried (MgSO4), filtered, and
evaporated under reduced pressure to give a white solid
(354 mg). Purification of these two products was
accomplished by flash chromatography employing 15:1 and
10:1 hexane/acetone as eluant.
Evaporation of the early fractions provided 3a,20/-
dihydroxy-39-methyl-5a-pregnane (80 mg) as a white solid.
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'H NMR and capillary GC were employed to determine the
purity.
Further elution provided a 9:1 mixture (on the basis
of GC) of 3a,20a-dihydroxy-3/6-methyl-5a-pregnane and
5 3a, 20g-dihydroxy-3/3-methyl-5a-pregnane (total 248 mg) . This
mixture was subjected to recrystallization from hot ethanol
(-10 mL); No crystals were obtained. Attempted next was
hot 5:1 hexane/acetone (-50 mL), which led to white
crystals (110 mg). According to GC, these crystals had a
10 purity of only 96%. Finally, pure 3a,20a-dihydroxy-3,3-
methyl-5a-pregnane (80 mg) could be obtained by another
recrystallization from hot ethanol.
EXAMPLE 37
3a-Hydroxy-3/3-iodomethyl-5a-pregnan-20-one
15 To a solution of 3[R]-spiro-2'-oxirane-5a-pregnan-20-
one (40 mg, 0.12 mmol) in acetone (3 mL) was added NaI (39
mg, 0.26) . The resulting solution was stirred at room
temperature for -4 h. TLC (3:1 hexane/acetone) indicated
consumption of only a small amount of the starting
20 material, with appearance of a new, slow-moving spot; the
relative intensities of these two spots did not change with
time, suggesting presence of an equilibrium between the
starting material and the iodomethyl product (as alkoxide).
Subsequently acetic acid (-0.02 mL) was added to the above
25 reaction solution. The mixture was stirred at room
temperature overnight. TLC now showed complete dis-
appearance of the starting epoxide. The reaction mixture
contained some white crystals (sodium acetate). The
solvent was removed under reduced pressure, and then ether
30 and water were added. The aqueous phase was separated and
back-extracted with ether. The combined ether solutions
were successively washed with water and brine, dried
(MgSO4), filtered, and evaporated under reduced pressure to
give 3a-hydroxy-33-iodomethyl-5a-pregnan-20-one as white
35 solid (51 mg, 92%)
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- ~l
EXAMPLE 38
a. 3,3-Ethylenedioxy-58-prean-17(20)-ene
5Q-3-Ethylenedioxy-androstan-l7-one (6 g) was added to
Wittig reagent produced from ethyltriphenylphosphonium
bromide (15 g) and potassium t-butoxide (4.5 g) in THE (15
mL). The reaction was refluxed for 2 h and cooled to 25 C.
Then methylene chloride (80 mL) and a solution of ammonium
chloride (60 mL) was added and the organic layer was
separated by separatory funnel. The aqueous layer was
extracted with methylene chloride (2x50 mL). The organic
solution was dried over potassium carbonate and the solvent
removed in vacuo. Most of the phosphoroxide was removed by
washing with hexane. The obtained product (6 g) was
dissolved in acetone (100 mL), and hydrochloric acid (2 N,
10 mL) added. The crude product (5.5 g) obtained by a
basic work-up followed by methylene chloride extraction was
purified by chromatography to produce 4.5 g of product
(83%). The pure stereoisomer was obtained by repeated
recrystalization from hexane.
b. 3a-Hvdroxy-38-trifluoromethyl-5B-precrn- (Z) 17 (20) -ene
To a solution of 59-pregn- (Z) -17 (20) -en-3-one (950 mg,
3.17 mmol) in THE (15 mL) was added trifluoromethyl-
trimethylsilane (0.5 M solution in THF, 9.5 mL) at 0 C.
The solution turned to brown gradually and the reaction was
finished in 30 min. Water (30 mL) was added and organic
layer collected. The aqueous layer was extracted with
ether (3x50 mL) and organic solution was dried over
potassium carbonate. The pure product (680 mg, 58%) was
isolated by column chromatography using ethyl acetate and
hexane (1 : 9) as eluent.
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EXAMPLE 39
a. 3a,21-Dihvdroxv-38-ethynyl-55-pregnan-20-one 21-
acetate
A suspension of 3(3-ethynyl-3a-hydroxy-5/6-pregnan-20-
one (1.00 g, 2.92 mmol) in 35 mL of toluene was treated
with 2 mL of methanol. The resulting solution was cooled
in an ice/water bath and neat BF3-Et20 (Aldrich; 5.8 mL,
5.02 g, 35.4 mmol) was added. Solid lead tetraacetate
(Aldrich; 1.96 g, 4.42 mmol) was added in a few portions.
A light purple solution formed initially and became light
brown as stirring continued at 0 C. The mixture was
stirred at r.t. for 3 h. and then recooled to 0 C. The
cold reaction was added to a mixture of 52 mL of a sat.
NaHCO3 solution, water and crushed ice. The resulting
mixture was extracted with EtOAc (2 x 75 mL). The combined
organic layers were extracted with a sat. NaCl solution,
dried (Na2SO4) and concentrated. The crude product was
purified by column chromatography (25 cm of flash silica
gel in a 5 cm dia. column eluted with 3 liters of 20%
acetone/hexane) affording 749 mg (64%) of the acetate, mp
196-198 C.
b. 3x,21-Dihvdroxv-3/3-etVinyl-56-pregnan-20-one 21-
hemisuccinate
To a stirred solution of 21-bromo-39-ethynyl-3a-
hydroxy-5$-pregnan-20-one (1.5g, 3.56 mmol) in 50 mL of
acetone was added succinic acid (2.1g, 17.8 mmol) and
triethylamine (3mL, 21.4 mmol). The reaction was heated to
ref lux. for 3.5 h. then cooled to r.t. before partitioning
between ethyl acetate and dilute aq. HC1. The organic
layer was washed with sat. NaCl, dried with Na2SO4 and
concen-trated in vacuo to give a crude solid. The solid
was triturated with dichloromethane to remove succinic acid
as a solid, and the liquid was concentrated in vacuo to
give a foam (1.36g). Flash chromatography on 6 in. of
silica gel in a 3cm column collecting 50 mL fractions and
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eluting with 1:3 -> 3:1 ethyl acetate : hexane with a
gradual adjustment of the gradient to give 830mg (51%) of
the title compound.
c. Sodium 3a,21-dihydroxv-313-ethynyl-58-pregnan-20-one
21-hemisuccinate
The hemisuccinate, 633 mg, mp 62-68 C, was dissolved
in methanol and an aqueous solution of 116 mg (0.253 mmol)
of NaHCO3 was added. After stirring for 3.5 h, the solvent
was removed under reduced pressure and the residue was
triturated with an ether/hexane mixture. The light yellow
solid obtained, weight 616 mg, was soluble in water at > 20
mg/mL.
EXAMPLE 40
3a,21-dihydroxy-3Q-trifluoromethyl-58-pregnan-20-one, 21-
acetate
Under an anhydrous argon atmosphere, 3a-hydroxy-39-
trifluoromethyl-59-pregnan-20-one (1.94g, 5.02mmol) was
dissolved in toluene (86mL) and MeOH (5.2mL). Boron
trifluoride etherate (10.4mL, 84.3mmol) was added via
syringe. Lead tetraacetate (2.89g, 6.51mmol) was then
added. The mixture was stirred for 70min, poured into
water and extracted three times with CH2C12. The organic
phases were combined,, washed with aqueous NaHCO3 and NaCl
solutions, dried over MgSO4 and evaporated in vacuo to give
a pale yellow solid (2. 18g) . This solid was purified by
flash column chromatography (CH2C12/EtOAc 150:1 and
hexane/EtOAc 4:1) to give a white solid (1.54g; 69%). mp.
167-168.5 C.
EXAMPLE 41
3!3-Chloromethyl-3a-hydroxy-5a-pregnan-20-one
A mixture of 3[R]-spiro-2'-oxirane-5a-pregnan-20-one
(46 mg, 0.14 mmol), tetramethylammonium chloride (25 mg,
0.23 mmol) and dry DMF (Aldich; 5 mL) was heated at -130 C.
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A clear solution was obtained after some time. TLC (7:1
hexane/acetone) showed, besides the starting material, a
new spot that moved slower than the starting epoxide. There
was no further progress in the reaction after 2 h. More
tetramethylammonium chloride (-25 mg) was added to the
reaction mixture. The resulting mixture was stirred at 130
- 150 C for 1 h. Not all the solid dissolved. There was
no change in TLC. Presumably there is an equilibrium
between the chloromethyl product (as alkoxide) and the
starting epoxide. Acetic acid (0.1 mL, 1.75 mmol) was then
added to the reaction mixture while the mixture was still
very hot. All the solid particles that were present in the
mixture now dissolved, and a clear solution resulted. This
solution was heated at --100 C for 2 h at which point TLC
indicated completion of the reaction, displaying only one
spot. The solvent was removed by distillation under
vacuum. To the resulting solid were added CH2C12 and water.
The aqueous layer was back-extracted with CH2C12. The
combined organics were dried (MgSO4), filtered, and
evaporated under reduced pressure to give a solid (46 mg).
'H NMR of this solid showed only the desired regioisomer
(i.e., 3g-chlororomethyl-3a-hydroxy-5a-pregnan-20-one).
Finally, flash chromatography employing 9:1 hexane/acetone
as eluant provided the title compound (35 mg, 68%) as a
white solid. m.p.209-10 C (dec.).
EXAMPLE 42
a. 3a-(3'-Bromo-l-propynyl)-3a-hydroxy-58-pregnan-20-one
A solution of propargyl bromide (80% soin. in toluene,
0.4 mL, 3.6 mmol) in dry THE (7 mL) was treated with n-
BuLi (2.5M in THF, 3 mmol, 1.2 mL) at -78 . After stirring
the mixture at this temp. for 10 min. a solution of 5J3-
pregnan-3,20-dione, 20-ketal (360 mg, 1 mmol) was added and
the mixture was stirred at -78 C for 2 hr. It was then
quenched with sat. NH4C1 solution (1 mL) The solvent was
removed and the residue was then dissolved in acetone (40
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mL). After adding 2N HC1 (10 mL) the solution was stirred
at r.t. for 3.5 h. Sat. NaHCO3 soln. was added to neutralize
the acid. The solvents were removed and the residue was
extracted with CH2C12. The organic layer was washed with
S water, dil. NaHCO3 soln., water, and brine. After drying
over anhyd. MgSO4 the solution was filtered and evaporated
to yield the crude product (360 mg). This crude product
was then dissolved in a small amount of CH2C12 and poured on
a column of silica gel. Elution with hexane:acetone
10 mixture (90:10) gave 3a-(3'-bromo-l-propynyl)-30-hydroxy-
5,6-pregnan-20-one (50 mg) as a first fraction. Further
elution with the same solvent mixture yielded 30-(3'-bromo-
1-propynyl)-3a-hydroxy-5,3-pregnan-20-one (181 mg).
EXAMPLE 43
15 3a-Bromomethyl-3a-hydroxy-5a=pregnan-20-one
A mixture of 3[RI-spiro-21-oxirane-5a-pregnan-20 -one
(362 mg, 1.10 mmol), tetramethylammonium bromide (322 mg,
2.16 mmol), acetic acid (1.0 mL, 17.5 mmol), and dry DMF
(Aldich; 30 mL) was heated at -130 C. There was still some
20 undissolved tetramethylammonium bromide present in the
mixture, which was stirred at 110 - 120 C. TLC (3:1
hexane/acetone) after heating for 30 min at 110 -120 C
showed, besides the starting material, a new, more-polar
spot. There were also two other very light spots that
25 moved slower than the major product spot; the intensity of
these two very light spots increased with time. After 3 h
at 110 -120 C, TLC displayed three spots of almost equal
intensity, besides the spot corresponding to the unreacted
starting material whose amount did not change with time.
30 The solvent was removed by distillation under vacuum. To
the resulting solid were added CH2C121 water, and brine (to
prevent the emulsion formation) . The organic layer was
washed with saturated NaHCO3, dried (MgSO4) , filtered, and
evaporated under reduced pressure to give a solid (380 mg).
351H NMR of this solid indicated presence of three products,
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namely, 30-bromomethyl-3a-hydroxy-5a-pregnan-20-one, 3-
hydroxymethyl-5a-pregn-2-en-20-cne,and 3/3-(acetoxymethyl)-
3a-hydroxy-5a-pregnan-20-one and the unreacted starting
material. Finally, flash chromatography employing 9:1
hexane/acetone as eluent provided pure 30-bromomethyl-3a-
hydroxy-5a-pregnan-20-one (98 mg, 22%) as a white solid.
m.p. 196- 97 C (dec.).
EXAMPLE 44
a. 3a-Hydroxy-19-nor-5a-17-methoxymethleneandrostane
To a solution of 19-nor-5cx-17-methoxymethleneandro-
stan-3-one (200 mg, 0.65 mmol) in THE (20 mL) was added K-
selectride (1 M in THF) by a syringe at -78 C and the
reaction was stirred at this temperature for 4.5 h. Then
mixture was poured into a separatory funnel containing
ammonium chloride solution (saturated, 20 mL) and methylene
chloride (40 mL) . The aqueous layer was extracted with
methylene chloride (2x15 mL). The organic solution was
dried over potassium carbonate and the solvent removed in
vacuo. A column chromatography (4% acetone, 4% methylene
chloride in hexane) resulted 130 mg product (64%).
b. 3a-Hvdroxy-l9-nor-5a-androstan-17/3-carboxaldehyde
To a solution of 3a-hydroxy-19-nor-5a-i7-methoxy-
methleneandrostane (130 mg, 0.43 mmol) in THE (10 mL) was
added a solution of hydrochloric acid (1N, 1 mL) at 25 C
and the reaction was stirred for 6 h. Then sodium bicar-
bonate solution (NaHCO31 saturated, 20 mL) and methylene
chloride (30 mL) were added. The organic layer was
separated and aqueous layer extracted with methylene
chloride (2x20 mL). The organic! solution was dried over
potassium carbonate and the solvent removed in vacuo. The
pure product (80 mg, 65%) was isolated by column chromato-
graphy using acetone and hexane (1:4) as eluent.
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EXAMPLE 45
3a,21-Dihydroxy-38-methyl-5a-pregnan-20-one 21-Disodium
phosphate.
To a solution of 21-bromo-3a-hydroxy-3(3-methyl-5a-
5pregnan-20-one(1.0 g, 2.43 mmol) in 10 mL THE at RT was
added dibenzyl hydrogen phosphate(2.1 g, 7.3 mmol) and
triethylamine (1.085 mL, 7.53 mmol) with stirring. The
reaction was then heated to reflux for 4.5 h and then
cooled to RT. Dichloromethane(25 mL) was then added and
the solution transferred to a separatory funnel, washed
with 1N HCl, sat. aq. NaHCO3, dried with MgSO4 and
concentrated in vacuo to give the dibenzylphosphate as a
crude oil (1.205 g). The dibenzylphosphate (790 mg, 1.3
mmol) was dissolved in 2 : 1 EtOH : THE (30 mL) with a few
drops of sulfuric acid, charged with 5% Pd/C (180 mg, 200
by weight) and subjected to 50 psi of H2 at rt until the
reaction was complete by TLC. The catalyst was removed by
filtration and the filtrate was concentrated. The residue
was dissolved in 4 : 1 MeOH : water (10 mL) and titrated to
pH 11 with 1M NaOH. (The solution was cloudy) The
solution was treated with acetone until a readily filter-
able solid precipitated after which the mixture was cooled
to 0 C and filtered to isolate a crude solid 260 mg. The
solid was dissolved in 20 mL water forming a cloudy
solution that was filtered and then concentrated to give
220 mg of the title compound as a white solid.
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EXAMPLE 46
a. 3a,21-dihvdroxy-38 -trifluoromethyl-58-precrnan-20-one
21-hemisuccinate
Succinic acid (20 g, 170 mmol) and triethylamine (19
mL, 140 mmol) were added to a solution of crude 21-bromo-
3a-hydroxy-33-trifluoromethyl-50-pregnan-20-one (5.36 g; s
10.9 mmol) in acetone (200 mL). The mixture was refluxed
for 1.75 hr. The residue was poured into ice water (350
mL). More water (100 mL) was then added. The mixture was
stirred and then extracted three times with CH2C12 (200 mL,
200 mL and 100 mL). The combined organic phases were dried
over MgSO4 and evaporated in vacuo to give a semi-solid
residue (5.71 g) which was purified by flash column
chromatography (CH2C12/MeOH; gradient 90:1 to 20:1) to give
a white solid (3.63 g, 66% over 2 steps). mp: dec. (over
broad range).
b. 3x,21-dihvdroxy-38-trifluoromethyl-56-precrnan-20-one,
21-hemisuccinate, sodium salt
3a,21-dihydroxy-30-trifluoromethyl-50-pregnan-20-one,
21-hemisuccinate (859mg, 1.71mmol) was dissolved in MeOH
(35mL). NaHCO3 (143mg, 1.70mmol) in water (10mL) was then
added dropwise. More MeOH (10mL) was added. After stir-
ring for 2.5 hours, the solvent was evaporated. Toluene
was added and evaporated in vacuo. The residue was treated
with ether/hexane and then with hexane to give a white
solid. McOH was added and removed. Heptane was added and
removed in vacuo. The residue was treated with hexane and
dried.in vacuo to give a white solid (878mg, 98%).
EXAMPLE 47
a. 21-(N,N-dimethylamino)methyl-3a-hydroxy-38-methyl-5a-
pregnan-20-one
A solution of 3a-hydroxy-30-methyl-5a-pregnan-20 -one
(3.32 g, 10 mmol) and N,N-dimethylmethyleneammonium chlor-
ide (1.6 g, 17 mmol) in acetonitrile (15 mL) was refluxed
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for 2.5 hr. After cooling the solvent was removed and the
residue was made basic by adding sat. NaHCO, soln. The
mixture was then extracted with CH2C121 washed with brine,
dried over anhyd. MgSO4, and evaporated to yield the crude
product. This product was crystallized from acetone to
give 21-(N,N-dimethylamino)methyl-3a-hydroxy-3,3-methyl-5a-
pregnan-20-one as colorless rods (550 mg).
The mother liquor was evaporated to dryness and the
residue was chromatographed over silica gel. Elution with
hexane: acetone (2:3, 1:4) afforded 21-(N,N-dimethylamino)
methyl-3a-hydroxy-30-methyl-21-methylene-5a-pregnan-20-one
as a colorless solid (200 mg). Further elution with
acetone yielded more 21-(N,N-dimethylamino)methyl-3a-
hydroxy-3f3-methyl-5a-pregnan-20-one as colorless solid
(1 g) .
b. 3a-hvdroxv-38-methyl-21-methylene-5a-pregnan-20-one
A mixture of 21-(N,N-dimethylamino)methyl-3a-hydroxy-
3Q-methyl-5a-pregnan-20-one (500 mg) and MeI (4 mL) was
stirred at r.t. for 1.5 hr. 4 mL of CH2C12 was added and
the stirring was continued for another 15 hr. MeI and CH2C12
were removed and the residue was taken up in CH2C12 (20 mL)
and was treated with sat. NaHCO3 solution (15 mL). After
stirring at r.t. for 2.5 hr the organic layer was
separated, washed with brine, dried over anhyd. MgSO4, and
evaporated. The residue (500 mg) was chromatographed over
silica gel. Elution with CH2C12:acetone (95:5) gave 3a-
hydroxy-3(3-methyl-21-methylene-5a-pregnan-20-one (300 mg).
c. 3a-hydroxy-3!3.21-dimethyl-5a-pregnan-20-one
A solution of 3a-hydroxy-30-methyl-21-methylene-5a-
30pregnan-20-one (250 mg) in EtOAc (30 mL) was hydrogenated
(2.5 atm of H2) in the presence of Pd/C (27 mg, 5 s) at r.t.
for 1 hr. After filtration and then removal of the solvent
gave the title compound as colorless solid (198 mg); mp
190-192 C.
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EXAMPLE 48
a. 21-(N,N-dimethylamino)methyl-3a-hydroxy-5,6-pregnan-20-
one
A solution of 3a-hydroxy-50-pregnan-20-one (318 mg, 1
5 mmol) and N,N-dimethylmethyleneammonium chloride (100 mg,
1.1 mmol) in acetonitrile (3 mL) was refluxed for 2 hr.
After cooling, the solvent was removed and the residue was
made acidic by adding 5 mL of 2N HC1. The mixture was then
extracted with EtOAc. The aqueous layer was separated and
10 neutralized with 2N NaOH. The precipitated solid was
extracted in CH2C12. The organic layer was separated,
washed with brine, dried over anhyd. MgSO4, and evaporated
to yield the crude 21-(N,N-dimethylamino)methyl-3a-hydroxy-
50-pregnan-20-one as a colorless solid (200 mg), which was
15 used as such for the next step.
b. 3a-hvdroxy-2l-methylene-58-pregnan-20-one and 3a-
hydroxy-21-methoxymethyl-58-pregnan-20-one
A mixture of 21-(N,N-dimethylamino)methyl-3a-hydroxy-
50-pregnan-20-one (550 mg) , CH2C12 (2 mL) , and MeI (4 mL)
20 was stirred at r.t. for 1 hr. MeI and CH2C12 were removed
and the residue was refluxed in MeOH for 72 hr. After
cooling, the mixture was treated with ice-water and
extracted with EtOAc. The organic layer was separated,
washed with brine, dried over anhyd. MgSO4, and evaporated.
25 The residue (500 mg) was chromatographed over silica gel.
Elution with CH2C12:acetone (95:5) gave 3a-hydroxy-21-
methylene-50-pregnan-20-one (150 mg). Further elution with
the same solvent afforded 3a-hydroxy-21-methoxymethyl-50-
pregnan-20-one as a colorless solid (120 mg).
30 EXAMPLE 49
a. 3a,21-dihydroxy-36-trifluoromethyl-5/3-pregnan-20-one
21-phosphate disodium salt
3a,21-dihydroxy-30-trifluoromethyl-5/3-pregnan-20-one
21-dibenzylphosphate (712 mg, 1.07 mmol) and palladium on
35 activated carbon (5% Pd, 180 mg) in ethanol (20 mL) were
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put under a 50 psi atmosphere of hydrogen (on the Parr
apparatus) for 2 hours. The palladium on carbon was
removed by filtration and the filtrate was evaporated
giving an oil. This oil was dissolved in methanol/water
4:1 (lOmL) . 1.00 M NaOH (1.97 mL) was added dropwise to
bring the pH to 11 ( 1). A small amount of solid was
removed by filtration and most of the solvent from the
filtrate was removed by evaporation. The remainder (mostly
water) was removed by freeze-drying to give a white solid
(494 mg, 880). mp: 224 C dec. 1H NMR available.
b. 3a,21-dihydroxy-3a-trifluoromethyl-5/3-precgnan-20-one
21-dibenzylphosphate
3a-dihydroxy-30-trifluoromethyl-21-bromo-50-pregnan-
20- one (1.20 g, 2.58 mmol), dibenzylphosphate (2.15 g,
7.74 mmol) and triethylamine (1.08 mL, 7.74 mmol) were
stirred in THE (10 mL) and then heated to ref lux for 3
hours. After cooling to room temp., EtOAc and 0.5 M HC1
were added. The organic phase was isolated and washed with
aqueous NaHCO3 and NaCl, dried over MgSO4 and evaporated in
vacuo to give a brown oil. This oil was purified by flash
column chromatography (hexane/acetone 4:1) to give a clear
oil (712 mg, 42k).
c. 3a-hvdroxy-38-trifluoromethyl-21-bromo-5(3-precrnan-20-
one
Hydrobromic acid (48%, 2 drops) was added to a stirred
solution of3a-hydroxy-3$-trifluoromethyl-59-pregnan-20-one
(4.20 g, 10.9 mmol) in methanol (100 mL) . A solution of
bromine (2.17 g, 13.6 mmol) in methanol (60 mL) was then
added dropwise over a period of 10 min. After stirring for
30 min., solid NaHCO3 was added (until all hydrobromic acid
was neutralized) and the solvent was evaporated. The resi-
due was partitioned between CH2C12 and water. The organic
phase was isolated, washed with brine, dried over MgSO4 and
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evaporated in vacuo to give a yellow semi-solid residue
(5.36 g) which was used without further purification.
d. 3a-hvdroxy-38-trifluoro-58-pregnan-20-one
(Reference: Krishnamurti, R.; Bellew, D.R.: Surya
Prakash, G.K. J. Org. Chem. 56:984 (1991))
To a solution of 5f3-pregnan-3,20-dione 20-ethylene-
ketal (60 mg, 0.166 mmol) in dry THE (3 mL) was added 0.5
M F3CSi(CH3)3 in THE (0.5 mL, 0.25 mmol). The resulting
colorless solution was cooled to 0 C. , and n-Bu4NFxH2O (few
crystals) was added. The cooling bath was removed, and the
mixture was allowed to warm to r.t. Unlike the same reac-
tion involving 5a-pregnan-3,20-dione 20-ethyleneketal, the
above reaction mixture did not turn yellow and there was no
gas generation. More 0.5 M F3CSi (CH3) 3 in THE (0.5 mL, 0.25
mmol) was added. The resulting mixture was stirred at room
temperature for a few minutes. TLC (3:1 hexane/acetone)
displayed a new spot with an Rf close to 1, but there was
still some unreacted starting material present. Therefore,
more 0.5 M F3CSi(CH3)3 in THE (0.5 mL, 0.25 mmol) was added.
The mixture was again stirred at room temperature for a
while. No unreacted starting material was found. iN HC1
(-3 mL) was added, and the resulting two-phase mixture was
stirred at room temperature overnight. The spot that was
formed as a result of trifluoromethylation had now
completely disappeared, and there were two new, less polar
spots, the lower one being the major product. The mixture
was then diluted with ether and water. The aqueous layer
was separated and extracted with ether. The combined
organice layers were washed with saturated NaHCO3 and brine,
dried (MgSO4), filtered, and evaporated under reduced
pressure to give a white crystalline (foamy) solid. The
two epimers were separated by flash chromatography with
15:1 hexane:acetone. Evaporation of the early fractions
gave the minor isomer. Further elution of the column gave
50 mg of the title compound.
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EXAMPLE 50
a. 3a-Trifluoromethvl-3a-hydroxv-2l-bromo-58-19-nor-
pregnan-20-one
To a flask containing a solution of 3a-hydroxy-30-
trifluoromethyl-50-19-nor-pregnan-20-one (0.65 g, 1.75
mmol) in methanol (10 mL) was added a solution of bromine
(0.15 mL) in methanol (10 mL) dropwise in such a rate to
maintain the brown color the bromine until this color was
persistent. Then water (50 mL) was added the mixture was
extracted with CH2C12 (3x4OmL). The combined extracts were
dried over K2CO3. Removal of the solvent resulted in the
product as a foamy white solid (0.805 g).
b. 38-Trifluoromethyl-3a-hydroxy-513-19-nor-pregnan-20-
one-21-dibenzy1phosphate
To a solution of 3/3-trifluoromethyl-3a-hydroxy-21-
bromo-5$-19-nor-pregnan-20-one (0.805 g) in THE (10 mL) was
added dibenzylphosphate (1.48 g, 5.32 mmol) and
triethylamine (538 mg, 5.32 mmol) and the mixture was
heated to ref lux for 1.5 h. HC1 (0.5 N, 40 mL) was added
and it was extracted with CH2C12 (3x40 mL) . The combined
extracts were dried over Na2SO4. Removal of the solvent
resulted in the crude material which was purified by
chromatography to give 733 mg product (65%).
c. 3a-Hvdroxv-3,3-trifluoromethyl-58-19-nor-pregnan-20-
one-21-phosphate, disodium salt
A mixture of 3/3-trifluoromethyl-3a-hydroxy-5/3-19-nor-
pregnan-20-one-21-dibenzylphosphate (733 mg) and Pd/C (5%,
200 mg) in ethanol (20 mL) was attached to a Parr
hydrogenator. The hydrogenolysis was done within 30 min.
under about 50 psi and the resulting mixture was filtered
through #5 filter paper to remove the catalyst. A white
foamy solid (494 mg) was obtained after the solvent was
removed. This solid was dissolved in methanol (10 mL) and
a solution of NaHCO3 (190 mg) in water (8 mL) was added at
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0 C. The methanol was then removed in vacuo and the
resulting aqueous solution was freeze-dried to give the
product (530 mg, 92%).
EXAMPLE 51
53a-Hydroxy-2l-(pyrid-4-ylthio)-5Li-pregnan-20-one.
To a solution of 21-bromo-3a-hydroxy-5/3-pregnan-20-
one(2. 8 g, 7.05 mmol) and 4-mercaptopyridine(940 mg, 8.46
mmol) in 30 mL dry THE at 25 C was added triethylamine
(1 mL) dropwise and the mixture heated to 60 C and stirred
for 1.5 h. The solution was then partitioned between
ethylacetate and 1:1 sat. NaHCO3 and water. The organic
layer was then washed with sat. NaCl, dried with MgSO4and
concentrated in vacuo to a crude solid weighing 3.19g.
Flash chromatography on 6 in. of silica gel in a 5.5 cm
column collecting 50 mL fractions and eluting with 10%-15%
acetone : CH2C12 resulted in 2.246g(75%) of the title compound
as a yellow solid. MP 192-4 C.
EXAMPLE 52
a. 3(3-Ethynyl-3a-hydroxy-5a-pregnan-20-one, 20-ketal
A 250 mL three neck flask equipped with a gas inlet,
a thermometer and a condenser was charged with lithium
acetylide-EDA complex (2.75 g, 90%, 27.5 mmol). Dry
benzene (60 mL) was added and acetylene gas was bubbled
through the mixture at a moderate rate. The mixture was
then heated to 50-55 C in an oil bath and treated in parts
with 5a-pregnan-3,20-dione, 20-ketal (9 g, 25 mmol). The
stirring was continued at this temp. for 5 h. and then at
r.t. for another 17 h. The resulting suspension was cooled
to 10 C and was treated with sat. NaCl soln. (5 mL). The
solvent was removed and water was added to the residue.
The water insoluble product was collected by filtration,
washed with water, and dried under vacuum. This crude
product was then crystallized from EtOAc to yield 3cr-
ethynyl-30-hydroxy-5a-pregnan-20-one, 20-ketal (3.35 g).
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The mother liquor was evaporated to dryness and the residue
was purified by column chromatography over silica gel.
Elution with toluene:acetone mixture (92:8) gave the
unreacted starting ketone (1.3 g) followed by 30-ethynyl-
53a-hydroxy-5a-pregnan-20-one, 20-ketal (1.3 g) as a second
fraction. Further elution with the same solvent mixture
yielded the more polar 3a-ethynyl-30-hydroxy-5a-pregnan-20-
one, 20-ketal (270 mg).
b. 3!3-Ethynyl-3a-hydroxy-5a-pregnan-20-one
10 30-Ethynyl-3a-hydroxy-5a-pregnan-20-one, 20-ketal(550
mg) was dissolved in a mixture of acetone (20 mL) and 2N
HC1 (10 mL) and the mixture was stirred at r.t. for 15 h.
The solvents were removed and the residue was extracted
with CH2C12. The organic layer was washed with water, dil.
15 NaHCO3 soln., water, and brine. After drying over anhyd.
MgSO4 the solution was filtered and evaporated to yield the
crude product (414 mg). This crude product was then
dissolved in a small amount of CH2C12 and poured on a column
of silica gel. Elution with toluene:acetone mixture (92:8)
20 gave the title compound (280 mg), mp 175-177 C.
c. 3a-ethynyl-32-nitrooxy 5a-pregnan-20-one
A solution of 3a-ethynyl-33-hydroxy-5a-pregnan-20-one,
20-ketal (2.15 g) in CHC13 (45 mL) was cooled to -20 C and
treated with acetic anhydride ( 20 mL). Fuming nitric acid
25 ( 4 mL) was then added and the mixture was stirred at this
temp. for 45 min. After warming to -5 C, the yellow
solution was poured into a mixture of 2N NaOH (70 mL) and
water (150 mL) to yield the resultant solution of pH 3-4,
which was then extracted with CHC13, washed with water, sat.
30 NaHCO3 solution, brine, dried (MgSO4) and evaporated to
yield the title compound as a viscous material (3 g), which
was used as such for the next step.
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d. 38-Ethvnvl-3a-hydroxy-5a-pregnan-20-one
Crude 3a-ehtynyl-3/3-nitrooxy-5a-pregnan-20-one (3 g)
from the above step was taken in a mixture of THF and water
(30 mL, 1:1) . AgNO3 (516 mg) was added. After stirring at
r . t . for 15 h. the solvents were removed and the residue
was extracted with CH2C12. The organic layer was washed
with water, dil. NaHCO3 soln., water, and brine. After
drying over anhyd. MgSO4, the solution was filtered and
evaporated to yield the crude product (2 g). This crude
product was then dissolved in a small amount of CH2C12 and
poured on a column of silica gel. Elution with
toluene:acetone mixture (93:7) gave 3/3-ethynyl-3a-hydroxy-
5a-pregnan-20-one (550 mg) as a first fraction.
EXAMPLE 53
a. Preparation of the lithium reagent from 1,2-
dibromoethylene
A 250 mL three neck flask equipped with a N2 gas
bubbler, a thermometer, and a dropping funnel was charged
with 1,2-dibromoethylene (cis/trans mixture, 98%, Aldrich,
2.1 mL, 26 mmol, mw = 186, d = 2.246). Dry THF (40 mL) was
added and the solution was cooled to -78 C in a dry ice-
acetone bath. n-BuLi (2.5M in THF, 20 mL, 50 mmol) was
added dropwise over a period of 35 min. The mixture was
stirred at this temperature for 40 min. and the resulting
reagent was used immediately for the next step.
b. 36-Ethynvl-3a-hydroxy-5a-precrnan-20-one 20-ketal
The above solution of the reagent in THF, which was
maintained at -78 C, was treated dropwise with a solution of
5a-pregnan-3,20-dione, 20-ketal (4.68g, 13 mmol) in THF (50
mL) over a period of 1 hr. The temperature was maintained
below -70 C during the addition. The stirring was continued
at this temperature for 15 min. (100% conversion as
detected by TLC). The cooling bath was removed and the
resulting solution was quenched with 2N HC1 (pH 6-7). The
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solvent was removed and the residue was extracted with
chloroform, the organic layer was separated, washed with
water, and dried over anhydrous MgSO4. Removal of the
solvent gave the title product (3.9 g) as an epimeric
mixture with the corresponding 30-hydroxy epimer (85:15).
EXAMPLE 54
3a-Hydroxy-38-trifluoromethyl-5a-19-nor-pregnan-20-one
To a solution of 3a-hydroxy-3(3-trifluoromethyl-5(3-19-
nor-pregn-17(20)-ene ( 2.6 g, 7.3 mmol) in THE (80 mL),was
added diborane THE complex (1M solution in THF, 22 mL)
dropwise at 25 C. The reaction was complete in 1 h. and
then a solution of sodium hydroxide (20%, 50 mL) was added
very slowly at 0 C followed by an addition of hydrogen
peroxide (30%, 30 mL). Water was then added and the THE
layer was separated by separatory funnel. The aqueous
layer was extracted with methylene chloride (2x40 mL) . The
organic solution was dried over potassium carbonate and the
solvent removed in vacuo. A quick column (hex:acetone= l:1)
gave 1.8 g of product which was subjected to PCC oxidation
(PCC, 2.1 g, 9.6 mmol; sodium acetate, 0.8 g, 9.6 mmol) .
The pure product (700 mg, 26%) was purified by column
chromatography using ethyl acetate and hexane (15:85) as
eluent; m.p. 151.5-153.0 C.
EXAMPLE 55
a. Synthesis of benzyl phenyl sulfoxide
To a solution of benzyl phenyl sulfide (Aldrich; 1.068
g, 5.3-3 mmol) in 25 mL of CH2ClZ at -78 C was slowly added
a solution of m-chloroperbenzoic acid (Aldrich, 50-60%; 760
mg, 2.64 mmol if 60%) in 10 mL of CH2ClZ. After warming to
room temperature and stirring overnight, the solution was
added to 20 mL of a saturated NaHCO3 solution. The aqueous
layer was separated and extracted with CH2ClZ (2 x 10 mL).
The pooled organic layers were then dried (MgSO4) and
concentrated. The residue was subjected to flash column
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chromatography (silica gel, 10% acetone/hexane and 15%
acetone/hexane) affording the sulfoxide (632 mg, 55%) as a
white solid, mp 123-126 C.
b. 20,20-ethylenedioxy-3a-hydroxy-3a-[[2-(phenyl-
sulfinyl)-2-phenyllethyl]-5a-pregnane
A solution of diisopropylamine (Aldrich, freshly
distilled from CaH2; 0.5 mL, 361 mg, 3.57 mmol) in 2 mL of
dry THE was cooled to -10 C and treated with a 1.6 M
solution of n-BuLi in hexanes (Aldrich; 1.0 mL, 1.6 mmol)
added dropwise via syringe. After 10 min., the reaction
was cooled to -75 C and a solution of benzyl phenyl
sulfoxide (347 mg, 1.60 mmol) in 5 mL of dry THE was added
dropwise via syringe over 30 min. To the resulting deep
yellow solution was added 297 mg (0.79 mmol) of solid
1520,20-ethylenedioxy-3(R)-5a-pregnan-3-spiro-2'-oxirane.
The reaction was allowed to warm to r.t. and then warmed to
50 C. After 5 h, the reaction was allowed to cool to r.t.
and added to 30 mL of ice-cold water. The resulting
mixture was extracted with EtOAc (3 x 20 mL). The combined
EtOAc layers were back extracted with a sat. NaCl solution,
dried (Na2SO4) and concentrated. The residue was purified
by flash chromatography (silica gel, gradient from 100%
CH2C12 to 20% acetone/CH2C12) affording the sulfoxide (405
mg, 86%) as a mixture of two diastereomers. This mixture
was carried on to the elimination step.
c. 3a-hydroxy-30-12-(E)-phenylethenyll-5a-oregnan-20-one
A. suspension of the sulfoxide (200 mg, 0.339 mmol) in
1.5 mL of p-isopropyltoluene containing 0.2 mL of 2,4,6-
collidine (distilled from CaH2) was heated in an oil bath at
135 C for 60 min. After cooling to r.t., the solution was
allowed to stir overnight. A white precipitate formed and
was isolated and washed with p-isopropyltoluene (3 x 1mL).
By 1H NMR and TLC, the precipitate (69 mg, 44%) was the
desired20,20-ethylenedioxy-3a-hydroxy-2-(E)-phenylethenyl-
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5a-pregnane. A solution of the ketal in acetone at 00 C
was treated with a 1M HC1 solution and stirred cold for one
hour. The reaction was poured into an EtOAc/water mixture
and the organic layer was washed with water and a sat. NaCl
solution. After drying (Na2SO4), the solvent was removed in
vacuo and the residue was purified by flash chromatography
(silica gel, eluted with it acetone/CH2C12)
EXAMPLE 56
3a-hydroxy--38- (2' -propenyl) -5(3-precrnan-20-one
A solution of 50-pregnan-3,20-dione, 20-ketal (180 mg,
0.5 mmol) in dry THF (20 mL) was treated with allyl
magnesium bromide (2M in THF, 1 mmol, 0.5 mL) at -70 C.
After stirring the mixture at this temp. for 5 min. and
then at r.t. for 1.5 h, it was quenched with 2N HC1 (1 mL).
The solvent was removed and the residue was dissolved in
acetone (15 mL). After adding Dowex-40 resin (2 g) the
solution was stirred at r.t. for 20 min. The resin was
filtered off and the solvent was removed. The residue was
extracted with CH2C12. The organic layer was washed with
water, dil. NaHCO3 soln., water, and brine. After drying
over anhyd. MgSO41 the solution was filtered and evaporated
to yield the crude product (250 mg). This crude product
was then dissolved in a small amount of CH2C12 and poured on
a column of silica gel. Elution with toluene:acetone
mixture (95:5) gave 3a-(2'-propenyl)-3$-hydroxy-5f3-pregnan-
20-one (45 mg) as a first fraction. Further elution with
the same solvent mixture yielded 3(3-(2'-propenyl)-3a-
hydroxy-5(3-pregnan-20-one (85 mg), m.p. 119-120 C.
EXAMPLE 57
a. 3a-Hydroxy-17b-ethvnyl-5a-androstane
3a-tert-butyldimethylsilyloxy-17b-ethynyl-5a-
androstane (100 mg, 0.24 mmol) was dissolved in
acetonitrile/THF (30 mL, 2:1 v/v) and cooled to 00 C.
Aqueous hydrofluoric acid (48t, 2 mL, 60 mmol) was added
WO 94/27608 216 3 7 4 8 PCT/US94/05820
dropwise and the reaction solution was allowed to warm to
room temperature. After 2 hours, the reaction solution was
poured into water and extracted with CH2C12. The organic
phase was washed with aqueous sodium bicarbonate, dried
5 over MgSO4 and evaporated in vacuo to give a white solid (69
mg). This solid was purified by flash column chromato-
graphy (hexane/acetone 12:1) to give a white solid (59 mg,
82%); mp: 158 - 160 C.
b. 3a-tert-butyldimethylsilyloxy-l7b-ethynyl-5a-
10 androstane
Under a dry argon atmosphere, diisopropylamine (125
mL, 0.95 mmol, distilled from CaH2) was dissolved in THE (2
mL) and the solution was cooled to 0 C. n-Butyllithium
(2.5 M in hexane, 380 mL, 0.95 mmol) was added dropwise.
15 After stirring at this temperature for 5 min., the solution
was cooled to -78 C. 3a-tert-butyldimethylsilyloxy-5y-
pregnan- 20-one 20-enol diethylphosphate (164 mg, 0.288
mmol) was dissolved in THE (1mL) and this solution was
added dropwise to the previously prepared solution of
20 lithium diisopropylamide (still at -78 C). The reaction
mixture was allowed to warm to room temperature over 2.5
hours. Water (1 mL) was added, then the mixture was
diluted with 75 mL ether. The organic phase was washed
with 1 M HC1, water and aqueous sodium bicarbonate, dried
25 over MgSO4 and evaporated in vacuo to give a semi-solid
residue (100 mg, 83%) which was used without further
purification.
c. 3a-tert-butyldimethylsilyloxy-5a-pregnan-20-one 20-
enoldiethylphosphate
30 Under a dry argon atmosphere, lithium bis(trimethyl-
silyl) amide (1.0 M solution in THF, 1.02 mL, 1.02 mmol)
was diluted with THE (1 mL). The orange solution was
cooled to -78 C. 3a-tert-butyldimethylsilyloxy-5a-
pregnan-20-one (400 mg, 0.92 mmol) was dissolved in THE (1
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mL) and added dropwise to the previously prepared solution
of lithium bis(trimethylsilyl) amide (still at -78 C).
After stirring at this temperature for 45 min., diethyl--
chlorophosphate (140 mL, 0.97 mmol) was added dropwise and
the resulting solution was allowed to warm to room
temperature. Water (1 mL) was added and then ether. The
organic phase was washed with 1N HC1, water and aqueous
sodium bicarbonate, dried over MgSO4 and evaporated in vacuo
to give a yellow oil (394 mg) which was purified by flash
column chromatography (hexane/acetone 10:1) to give a clear
oil (164 mg, 31t).
d. 3a-tert-butyldimethylsilvloxy-5a-precrnan-20-one
Under a dry argon atmosphere, 3a-hydroxy-5a-pregnan-
20-one (1.1 g, 3.5 mmol) was dissolved in anhydrous DMF (17
mL). Imidazole (0.94 g, 13.8 mmol) was added and the
mixture was stirred until all dissolved. tert-Butyldi-
methylsilylchloride (1.04 g, 6.91 mmol) was then added and
the reaction mixture was stirred overnight. Ether (approx.
50 mL) was added and the organic phase was washed with 1N
HC1, water and aqueous sodium bicarbonate, dried over MgSO4
and evaporated in vacuo to give a white solid (1.93 g).
This solid was purified by flash column chromatography
(100% CH2C12) to give a white solid (1.16 g, 78%)
EXAMPLE 58
3,21-hydroxy-35-methyl-5a-pregnan-20-one, 21-mesylate
To a solution of 3x,21-hydroxy-39-methyl-5a-pregnan-
20-one- (77 mg, 0.22 mmol) in pyridine (1 mL) at 0 C was
added 29 mL (42 mg, 0.37 mmol) of methanesulfonyl chloride.
After stirring cold for 2h. and 40 min., the reaction was
poured into water and the resulting precipitate was
collected. Flash chromatography (6" of silica in a 20 mm
dia. column, eluted with a gradient from 0 to 5% EtOAc/
hexane) gave 54 mg (57%) of the mesylate as a white solid.
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EXAMPLE 59
21-Bromo-3l3-ethvnyl-3a-hydroxy-5B-pregnan-20-one
To a stirred solution of 3/3-ethynyl-3a-hydroxy-5(3-
pregnan-20-one(2.5 g, 7.3 mmol) in 125 mL methanol at 00 C
was added a few drops of HBr followed by slow dropwise
addition of a solution of bromine (1.28g, 8.0 mmol) in 25
mL of methanol. The reaction was slowly warmed to 25 C,
and upon complete decolorization the solution was added to
ice water containing a small amount of NaHCO3. The
precipitate was filtered, dissolved in dichloromethane,
dried with MgSO4and concentrated in vacuo to yield a crude
foam. (2.837g) Flash chromatography on 6 in. of silica gel
in a 5.5 cm column collecting 50 mL fractions and eluting
with 1% acetone:CH2C12 resulted in 1.96g(64%) of the title
compound as a foam.
EXAMPLE 60
3,6-Chloroethynyl-3a-hydroxy-5a-pregnan-20-one
A solution of cis-1,2-dichloroethylene (194 mg, 0.16
mL, 2 mmol) in dry THE (7 mL) was treated under N2 with n-
20BuLi (2.5M in THF, 4 mmol, 1.4 mL) at -10 C. The mixture
was stirred at -30 C for 20 min and then at -5 C for 10
min. It was recooled to -30 C and a solution of 50-
pregnan-3,20-dione, 20-ketal (360 mg, 1 mmol) in dry THE
(10 mL) was added dropwise over a period of 10 min. The
cooling bath was removed and the mixture was stirred at
room temperature for 0.5 hr. NH4C1 solution (3 mL) was added
to quench the reaction. The solvent was removed and the
residue was then dissolved in acetone (25 mL). After
adding 2N HC1 (10 mL) the solution was stirred at r.t. for
1 h. Sat. NaHCO3 soln. was added to neutralize the acid.
The solvents were removed and the residue was extracted
with CH2C12. The organic layer was washed with water and
then brine. After drying over anhyd. MgSO4, the solution
was filtered and evaporated to yield the crude product (427
mg). This crude product was then dissolved in a small
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amount of CH2C12 and poured on a column of silica gel.
Elution with toluene:acetone mixture (95:5) gave the title
compound as a colorless solid (130 mg).
EXAMPLE 61
3a-hydroxy-38-methyl-5a-prectnan-20-one
To a solution (light yellow) of the (3R)-5a-pregan-3-
spiro-2'-oxirane-20-one (101 mg, 0.305 mmol) and NaI (115
mg, 0.767 mmol) in anhydrous 1,2-dimethoxyethane (DME)
(5mL) at room temperature was added rj-Bu3SnH (0.22 mL,
0.238 g, 0.818 mmol). The reaction solution became
colorless. Azobisisobutylnitrile (AIBN) (10 mg, 0.061
mmol) was then added. The resulting solution was refluxed
under a nitrogen atmosphere for 21 h. at which point TLC
(3:1 hexane/acetone) indicated completion of the reaction.
The reaction was quenched with methanol; the mixture was
stirred at room temperature for a while. The solvent was
removed in vacuo to give an oil which did not dissolve in
either. Addition of CH2C12 gave a solution which was washed
with water, IN HC1, and saturated NaHCO3. The organic layer
was dried (MgSO4), filtered, and concentrated under reduced
pressure to a white solid. Purification by gradient flash
chromatography (hexane, 7:1 hexane/acetone, 5:1 hexane/
acetone) furnished the titled compound (93mg, 920).
It will be obvious to one skilled in the art that the
above described compounds may be present as mixtures of
diastereomers which may be separated into individual
diastereomers. Resolution of the diastereomers may be
conveniently accomplished by gas or liquid chromatography
or isolation from natural sources. Unless otherwise
specified herein, reference in the specification and claims
to the compounds of the invention, as discussed above, is
intended to include all isomers, whether separated or
mixtures thereof.
Where isomers are separated, the desired pharma-
cological activity will often predominate in one of the
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diastereomers. As disclosed herein, the biological
activity of these compounds display a high degree of
stereospecificity.
TBPS binding assays were conducted according to the
following procedures, which are described in Gee, K.W.;
Lawrence, L.J.; and Yamamura, H. I., "Modulation of the
Chloride Ionophore by Benzodiazepine Receptor Ligands
Influence of Gamma-Aminobutyric Acid and Ligand Efficacy,"
Molecular Pharmacology, 30:218, 1986. Male Sprague-Dawley
rats (200-250g) were anesthetized with carbon dioxide and
decapitated. The rats' brains were removed and the
cerebral cortices were dissected and homogenized in 10
volumes of 0.32 M sucrose. Tissue was centrifuged at 1000
x g for 10 minutes and the resultant supernatant was
centrifuged at 9,000 g for 20 minutes. The resultant
pellet was resuspended in 10 volumes of 200 mM NaCl/50 mM
Na-K phosphate pH 7.4 buffer (binding buffer) and then
centrifuged at 9,000 x g for 10 minutes. This washing
procedure was repeated twice and the final P2 pellet was
resuspended in the binding buffer. For the binding assay,
[35S]TBPS (2 nM) was co-incubated in binding buffer with P2
membranes and 5 M GABA and either 1) no additions to
measure total binding; 2) 2 M unlabeled TPBS to measure
nonspecific binding; 3) the neurosteroid ranging in
concentration from 1 nM to 10 M. Following a 90 minute
incubation at 22 C, the suspension was filtered and the
bound radioactivity was determined by liquid scintillation
counting. The IC501 which is the concentration of the
neuron-teroid producing a 5011 inhibition of TBPS binding,
was calculated using the non-linear curve fitting routine
on Excel Solver by Microsoft. A lower IC50 indicates higher
affinity for the steroid site on the GABAA receptor-chloride
channel complex. Table 1 gives the IC50 values of
representative steroids used in the present invention.
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TABLE 1
Neuroactive Steroid IC50 nM)
3$-(4'-Acetylphenyl)ethynyl-3a-hydroxy-5a- 5
pregnan-20-one
5 3$-(4'-Carboxyphenyl)ethynyl-3a-hydroxy- 8
5$-pregnan-20-one ethyl ester
3$-(4'-Acetylphenyl)ethynyl-3a-hydroxy-5$- 14
pregnan-20-one
3a-hydroxy-21-(pyrid-4-ylthio)-5a-pregnan- 23
10 20-one
3a-hydroxy-50-pregnan-20-one 25
3$-(chloroethynyl)-3a-hydroxy-5$-pregnan- 26
20-one
3a-Hydroxy-3$-methyl-5$-19-nor-pregnan-20- 26
15 one
3a,20a-Dihydroxy-21-ethyl-5a-pregnane 27
3$- (4' -Dimethylaminophenyl) ethynyl-5$- 28
pregnan-20-one
3$-azidomethyl-3a-hydroxy-5a-pregnan-20- 34
20 one
3a,20a-Dihydroxy-21-methyl-5a-pregnane 35
3a-hydroxy-5a-pregnan-20-one 37
3$-ethynyl-3a-hydroxy-5$-pregnan-20-one 39
3a-hydroxy-3$-methoxymethyl-50-pregnan-20- 40
25 one
3$-(4'-biphenyl)ethynyl-3a-hydroxy-5$- 43
pregnan-20-.one
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Neuroactive Steroid ICs;, (nM)
3$-Ethynyl-3a-hydroxy-19-nor-50-pregnan- 44
20-one
3(3-(1-Hexynyl)-3a-hydroxy-5$-pregnan-20- 44
one
3$-ethenyl-3a-hydroxy-5$-pregnan-20-one 46
3a-Hydroxy-3$-(4'-nitrophenyl)ethynyl-5$- 46
pregnan-20-one
3a-Hydroxy-3$-(4'-methyoxyphenyl) ethynyl- 47
5$-pregnan-20-one
3$-(4'-Trifluoromethylphenyl)ethynyl-3a- 52
hydroxy-5$-pregnan-20-one
3a-hydroxy-3$-ethynyl-5a-pregnan-20-one 53
3a-Hydroxy-3$(E)-(2-phenylethenyl)-5a- 53
pregnan-20-one
3a-Hydroxy-3$-(2'-propynyl)-5a-pregnan-20- 56
one
30-(4'-Chlorophenyl)ethynyl-3a-hydroxy-5$- 58
pregnan-20-one
3a-Hydroxy-21-(pyrid-4-ylthio)-5$-pregnan- 59
20-one
3a-hydroxy-3$-methyl-5a-pregnan-20-one 62
3$-(1-Octynyl)-3a-hydroxy-5$-pregnan-20- 63
one
3$-(1-Heptynyl)-3a-hydroxy-5$-pregnan-20- 65
one
3a-hydroxy-3/3-methoxymethyl-5a-pregnan-20- 67
one
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Neuroactive Steroid IC5 (nM)
3a-hydroxy-17b-cyano-5a-androstane 67
3$-(4'-cyanophenyl)ethynyl-3a-hydroxy-5$- 73
pregnan-20-one
3x,21-dihydroxy-5a-pregnan-20-one 21- 75
acetate
3a-Hydroxy-3$-(2'-propenyl)-5$-pregnan-20- 75
one
3a-hydroxy-21-methyl-5$-pregnan-20-one 78
3a-Hydroxy-3$-(pentafluorophenyl) ethynyl- 79
5$-pregnan-20-one
3a-Hydroxy-3$-trifluoromethyl-19-nor-5$- 80
pregnan-20-one
3a-Hydroxy-3$-(4'-methylphenyl) ethynyl- 88
5$-pregnan-20-one
3a-hydroxy-3$,21-dimethyl-5a-pregnan-20- 89
one
3a-Hydroxy-3$-(6-oxo-l-heptynyl)-5$- 90
pregnan-20-one
3a-hydroxy-3$-methyl-5a-19-nor-pregnan-20- 91
one
3a-hydroxy-3$-chloromethyl-5a-pregnan-20- 93
one
3$-(3'-Bromo-l-propynyl)-3a-hydroxy-5$- 94
pregnan-20-one
3a-hydroxy-21-methoxymethyl-5$-pregnan-20- 96
one
3a,20a(S)-dihydroxy-5a-pregnane 97
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Neuroactive Steroid IC5 (nM)
3a-Hydroxy-30-(phenylethynyl)-50-pregnan- 98
20-one
3/i-Benzyl-3a-hydroxy-5/3-pregnan-20-one 109
30-(2,4-Difluorophenyl)ethynyl-3a-hydroxy- 109
50-pregnan-20-one
3x,21-dihydroxy-5a-pregnan-20-one 21- 117
hemisuccinate
3x,21-dihydroxy-5$-pregnan-20-one 21- 119
acetate
3a-Hydroxy-30-(2'-phenylethyl)-5/i-pregnan- 135
20-one
3f-ethyl-3a-hydroxy-5/3-pregnan-20-one 136
3a-Hydroxy-33-(3'-hydroxypropynyl)-50- 137
pregnan-20-one
3a-Hydroxy-30- [3' (RS) -hydroxybutynyl] -5a- 140
pregnan-20-one
3a,20-dihydroxy-20-methyl-5a-pregnane 151
3a-Hydroxy-30- [4' (R/S) -hydroxypentynyl] - 153
59-pregnan-20-one 4'(R/S)-hemisuccinate
sodium salt
30-(Ethoxymethyl)-3a-hydroxy-5a-pregnan- 154
20-one
3a-Hydroxy-3/3-methyl-50-19-nor-pregn- 154
17(Z) -ene
30-(5'-Cyanopentynyl)-3a-hydroxy-50- 158
pregnan-20-one
3a,20a-dihydroxy-30-methyl-5a-pregnane 166
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Neuroactive Steroid ICs. (nM)
3/3-(4'-Acetoxyacetylphenyl)ethynyl-3a- 171
hydroxy-5/3-pregnan-20-one
20,20-ethylenedioxy-3a-hydroxy-5a-pregnan- 176
20-one
3a-Hydroxy-3$-[4'(R/S)-hydroxypentynyl]- 178
5a-pregnan-20-one
3x,21-dihydroxy-50-pregnan-20-one (5/i- 180
THDOC)
3a-hydroxy-5/3-pregnan-cis(Z)-17(20)-ene 180
30-(Bromomethyl)-3a-hydroxy-5a-pregnan-20- 190
one
3/3-(2'-Hydroxyphenyl)ethynyl-3a-hydroxy- 192
50-pregnan-20-one
3a-hydroxy-3/3-fluoromethyl-5a-pregnan-20- 200
one
3a,20b-dihydroxy-50-pregnane 206
3a-Hydroxy-59-19-nor-pregn-17(z)ene 209
3a,21-dihydroxy-3/3-methyl-5a-pregnan-20- 211
one 21-Hemisuccinate, sodium salt
3a,21-Dihydroxy-3/3-ethynyl-50-pregnan-20- 216
one
3a-hydroxy-21-methyl-5a-pregnan-17(20)(Z)- 217
ene
3x,21-dihydroxy-50-pregnan-20-one 21- 224
hemisuccinate
3a-hydroxy-39-(2'-methoxyphenyl) ethynyl- 238
59-pregnan-20-one
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Neuroactive Steroid ICs, (nM)
3x,21-dihydroxy-30-methyl-5a-pregnan-20- 241
one 21-Hemisuccinate
30-(But-3-enyl)-3a-hydroxy-5a-pregnan-20- 242
one
5 3x,21-dihydroxy-5a-pregnan-20-one 21- 251
hemisuccinate, sodium salt
3a-Hydroxy-3/3-trifluoromethyl-19-nor-5a- 251
pregnan-20-one
3a,21-Dihydroxy-3/i-ethynyl-5/3-pregnan-20- 251
10 one, 21-acetate
3a-hydroxy-21-methoxy-3/3-trifluoromethyl- 252
50-19-nor-pregnan-20-one
3a-Hydroxy-5a-androstane 252
3a-Hydroxy-17b-ethynyl-5a-androstane 254
15 3a,21-dihydroxy-3/i-methyl-5a-pregnan-20- 255
one 21-mesylate
3a,20-dihydroxy-3/6,20-dimethyl-5a-pregnane 264
3a-hydroxy-3/3-ethyl-5a-pregnan-20-one 265
3x,21-Dihydroxy-30-trifluoromethyl-19-nor- 265
20 5/3-pregnan-20-one
30- [ (3' ,4' -Dimethoxyphenyl) ethynyl] -3a- 283
hydroxy-5/3-pregnan-20-one
3a,20a-dihydroxy-5/3-pregnane 292
3/3-(Buta-2,3-dienyl)-3a-hydroxy-5a- 347
25 pregnan-20-one
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Neuroactive Steroid ICS, (nM)
3a-hydroxy-17(Z)-methoxymethylene-19-nor- 350
Sa-androstane
3a-hydroxy-39-methyl-5a-19-nor-pregn-(Z)- 364
17(20) -ene
3a,21-Dihydroxy-30-trifluoromethyl-50- 370
pregnan-20-one, 21-acetate
3a-hydroxy-19-nor-5a-pregnan-cis-17(20)- 373
ene
3a-hydroxy-30-phenyl-50-pregnan-20-one 382
33-(3'-Hydroxyphenyl)ethynyl-3a-hydroxy- 407
59-pregnan-20-one
30-cyanomethyl-3a-hydroxy-5a-pregnan-20- 439
one
3a-hydroxy-17b-hydroxymethyl-5a-androstane 455
3f3-Ethynyl-3a-hydroxy-50-19-nor-pregn- 460
17(z) -ene
3a-Hydroxy-3$-trifluoromethyl-50-19-nor- 482
pregn-17(20)-ene
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The compounds of the invention may be prepared by
known techniques. For example, the naturally occurring
metabolites of progesterone may be extracted from various
animal excretion sources, e.g., urine, or extracted from
vegetable products like soy or yams. Such extractions are
conducted using the following steps: (i) hydrolysis with
HC1; (ii) extraction with toluene; (iii) removal of acidic
material from the toluene extract; (iv) elimination of
substances other than pregnanes from the neutral toluene-
soluble fraction by precipitations from ethanolic solution
with dilute NaOH and with water; and (v) weighing of the
purified pregnanes obtained. See Marrian et al., "The
Isolation of Pregnane-3a-of-20-one," Biochem., 40:376-380
(1947). These extracted compounds may then be chemically
altered to form the desired derivatives, or used directly.
The pharmaceutical compositions of this invention are
prepared in conventional dosage unit forms by incorporating
an active compound of the invention, or a mixture of such
compounds, with a nontoxic pharmaceutical carrier in
nontoxic amounts sufficient to produce the desired pharma-
codynamic activity in a subject, animal or human.
Preferably, the composition contains the active ingredient
in an active, but nontoxic amount, selected from about 5 mg
to about 250 mg of active ingredient per dosage unit. The
compositions and methods of this invention prevent insomnia
and produce sleep.
The pharmaceutical carrier employed may be, for
example, solids, liquids, or time release substances (see,
e.g., 'Remington's Pharmaceutical Sciences, 14th Edition,
1970). Representative solid carriers are lactose, terra
alba, sucrose, talc, gelatin, agar, pectin, acacia,
magnesium stearate, stearic acid, microcrystalline cellu-
lose, polymer hydrogels, and the like. Typical liquid
carriers are propylene glycol, aqueous solutions of ,6-
cyclodextrins, syrup, peanut oil, olive oil and like
emulsions. Similarly, the carrier or diluent may include
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any time-delay material well known to the art, such as
glycerol monostearate or glycerol distearate alone or with
wax, microcapsules, microspheres, liposomes, or hydrogels.
A wide variety of pharmaceutical forms can be
employed. Thus, when using a solid carrier, the prepara-
tion can be plain milled, micronized, in oil, tableted,
placed in a hard gelatin or enteric-coated capsule in
micronized powder or pellet form, or in the form of a
troche, lozenge, or suppository. When the compounds of the
invention are administered in the form of suppositories for
rectal administration, the compounds may be mixed in
material such as cocoa butter and polyethylene glycols or
other suitable non-irritating materials solid at room
temperature, but liquid at rectal temperature. When using
a liquid carrier, the preparation can be in the form of a
liquid, such as an ampule, or an aqueous or nonaqueous
liquid suspension. Liquid dosage forms will generally also
contain pharmaceutically acceptable preservatives and the
like. In addition, parental administration, nasal spray,
sublingual and buccal administration, and timed release
skin patches are also suitable pharmaceutical forms for
topical administration of the compounds of the invention.
The preferred formulations are for oral administra-
tion. The inventors have discovered that specific
formulations can significantly alter the activity of the
neuroactive steroid, which tends to be insoluble in water
and therefore poorly absorbed after oral administration.
For example, it was thought that pregnanolone was orally
inactive. L. Gyermek, "Pregnanolone: a Highly Potent,
Naturally Occurring Hypnotic-Anesthetic Agent" Proc. Soc.
Exp. Biol. Med. 125:1058-1062, 1967.
The inventors have discovered various formulations of
pregnanolone which the body can absorb through the gastro-
intestinal tract in sufficient quantities to induce sleep
after oral administration. The bioavailability of a drug
is quantified by measuring serum blood levels before and
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after administration. This method of quantification is
described by Robert H. Purdy et al. in "Radioimmunoassay
of 3a-hydroxy-Sa-pregnan-20-one in rat and human plasma"
Steroids 55:290-296 (1990) , which is incorporated by refer-
ence in its entirety. Generally, after a blood sample is
drawn, the serum is separated and then frozen. The serum
is chromatographed to remove competing materials including
endogenous steroids. Pregnanolone has a retention time of
19.0 minutes when it is chromatographed using HPLC
techniques on an 8 cm Radial-Pak-5- m silica column (Waters
Associates) using the solvent system 0.2% ethanol (95%) in
dichloromethane at a flow rate of 3 ml/min. Then, a
radioimmunoassay is run to quantify the serum blood levels
of the chromatographed material.
To demonstrate that pregnanolone, when administered
orally in certain formulations, is orally active to induce
sleep in humans, the inventors conducted a trial using 8
healthy male volunteers between the ages of 18 and 35. The
study was an unblinded, single dose crossover study in
which subjects were sequestered prior to and immediately
following drug administration.
Baseline medical history, laboratory values, drug and
alcohol screens were done for each subject. Throughout the
study the subjects remained in the clinical facility from
Day-1 until 48 hours post drug administration. While in
the clinic, the ingestion of caffeine, alcohol, or other
drugs was prohibited to the subjects.
Following an overnight fast, a defined high fat
breakfast was served to each subject and consumed within 15
minutes. Starting at 0900 GMT the subjects were
administered pregnanolone according to the randomization
defined in the protocol. Pregnanolone (500 mg; granulated
powder) was administered orally in one of four formula-
tions. Formulation 1 had the following ingredients:
pregnanolone (micronized, obtained from AKZO, Arnheim, The
Netherlands) (5%), polyvinylpovidone (polyvinylpyrrolidone
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or PDP) (Luviskol K-30, from BASF, Germany) (94%), and
sodium lauryl sulfate (SLS) (from Nomeco, Copenhagen,
Denmark) (1%). Formulation 2 contained preganolone
(12.3%), and 0-cyclodextrin (7 moles of H2O per 1 mole of
5 cyclodextrin; from Sigma, St. Louis) (87.7%). Formulation
3 contained pregnanolone (99%; nanosized), and sodium
lauryl sulfate (1%). Formulation 4 contained pregnanolone
(99%), and sodium lauryl sulfate (1%). The pregnanolone
used in the study was uniformly micronized (available from
10 AKZO) in all cases with the exception of Formulation 3
which was nanosized (submicron particle size). Access to
water ad libitum was allowed except during the hour
immediately following drug administration. Meals were
standardized in time throughout the study.
15 Formulation 1 was prepared by dissolving all
constituents in ethanol. The mixture was stirred and
heated until most of the ethanol had evaporated. The
product was dried to completion in vacuum for 24 hours and
ground in a mortar.
20 Formulation 2 was prepared by shaking 1.23 g. of
pregnanolone, 6.77 g. cyclodextrin in 50 ml. of water for
hours at 37 C. The complex was filtered, dried under
vacuum for 24 hours, and ground in a mortar.
Formulation 3 was prepared by dissolving the
25 constituents in carbon dioxide at an elevated temperature
and high pressure (up to 700 bar). The pressure is
released and the particles precipitate, which produced some
submicron particles.
Formulation 4 was prepared by dry blending the
30 micronized pregnanolone with sodium lauryl sulphate,
followed by sieving.
Serum samples for analysis of pregnanolone level were
obtained prior to and immediately following drug adminis-
tration and continued for 24 hours. Serum was separated,
35 frozen, and stored until analyzed by radioimmunoassay.
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Samples obtained during the study were analyzed for
pregnanolone using radioimmunoassays and the results
analyzed using appropriate non-compartmental pharmaco-
kinetic parameters. Relative bioavailability assessments
were made between formulations based on observed serum
concentration of pregnanolone.
Evaluation of clinical response to drug administra-
tion was obtained through observations carried out by the
clinical facility's staff and trained observers. Observa-
tions regarding onset of and apparent depth of sleep were
recorded over the course of the four hour treatment period
and later correlated with serum pregnanolone concentra-
tions. Duration of sleep was not evaluated as subjects
were frequently disturbed in the process of obtaining
vital signs and blood samples.
Each individual received the four formulations sepa-
rated by drug-free wash-out days during the three week
trial. No serious or unexpected adverse effects were
noted for any of the treatment groups. Body temperature
was closely monitored and recorded prior to drug adminis-
tration at 0.5, 1.0, 1.5, 2.5, 3, 4, 8, and 12 hours post
drug administration. No decrement in body temperature was
observed in any of the subjects during the course of the
study with any of the formulations.
After approximately one hour signs of drowsiness
and/or sleep was observed. Among the volunteers receiving
pregnanolone in 0-cyclodextrin (Formulation 2), seven of
eight subjects were observed to sleep with time of onset
ranging from <1 hour to 3 hours post drug administration.
Duration of sleep was impossible to evaluate as subjects
were awakened every 30 minutes for blood draws and vital
signs. Subjective evaluations indicated depth of sleep
ranging from light to difficult to arouse. Results are
summarized in Figures 16A-16H where arrows mark points at
which observations of sleep were recorded. Only one
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subject was observed to remain awake throughout the study
period after
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receiving pregnanolone formulated in 3-cyclodextrin.
Figures 16A-16H also show the plasma blood levels for this
formulation. Peak plasma levels for the 8 volunteers
ranged from about 40 to 120 nanograms per ml. Table 2
shows that the mean peak concentration for this formula-
tion was 61.7 nanograms per ml.
Table 2
Relative Absorption of Four CCD 3045 Formulations
CCD 3045 Formulation AUC Peak Concentration
(ng.h/mL) (ng/mL)
PVP Formulation 136.2 (61.1)* 60.9 (28.7)*
Beta CD Formulation 136.4 (53.8) 61.7 (28.5)
Nanosized Powder +1t SLS 59.8 (23.8) 22.0 ( 9.9)
Micronized Powder +1t SLS 117.1 (36.6) 59.4 (21.0)
*Mean (SD)
Similar findings were observed in subjects receiving
pregnanolone formulated with polyvinylpovidone and sodium
lauryl sulfate. All eight subjects were observed to
exhibit signs of hypnosis at time points ranging from <1
hour to three hours. Figures 15A-15H show that the peak
plasma levels for this formulation ranged from about 20 to
110 nanograms per ml. Table 2 shows that the mean peak
concentration for the PVP formulation was 60.9 nanograms
per ml.
Formulation 4 consisting of micronized pregnanolone
with 1% sodium lauryl sulfate produced peak plasma levels
ranging from about 40 to 110 nanograms per ml. Table 2
shows that the mean peak concentration for the micronized
formulation was 59.4 nanograms per ml.
Formulation 3 produced peak plasma levels ranging
from 10 to 35 nanograms per ml. Table 2 shows that the
mean
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peak concentration for the nanosized formulation was 22.0
nanograms per ml.
The assessments of relative absorption or bioavail-
ability demonstrate that all but the nanosized formulation
have comparable absorption both in terms of the rate and
extent of absorption. The serum samples were analyzed for
each subject over a 48 hour interval. Figure 19 illus-
trates the mean serum levels observed for the four differ-
ent formulations. Three of the formulations, PVP, Beta
CD, and the Micronized Powder + SLS all had comparable
mean serum concentration-time profiles, while the nanosiz-
ed pregnanolone formulation had much reduced levels, its
peak approximately 350 of the others. In addition to
examining the mean serum levels, relative assessment of
the peak and extent of absorption can be determined by
looking at the peak concentration and area under the serum
concentration-time (AUC) profile. Table 1 illustrates
those findings.
Figures 15A-H, 16A-H, 17A-H, and 18A-H are plots of
the serum concentration versus time for individual sub-
jects. At each point where sedative effects were ob-
served, an arrow is placed at the point in time. As can
be observed, higher serum concentrations, generally above
ng/ml, are related to observe sedative effects.
25 In another human study, plasma concentrations and
sleep propensity were measured in 18 young, healthy, male
volunteers for 2 hours after oral administration of single
doses of pregnanolane. Two different formulations were
tested. Formulation I used g-cyclodextrin as the excipi-
30 ent. Formulation II used sodium lauryl sulfate as the
excipient. Both /3-cyclodextrin and sodium lauryl sulfate
are known pharmaceutical excipients and were chosen to
enhance the absorption of pregnanolone without affecting
the toxicological profile of the compound.
Formulation I was a /6-cyclodextrin inclusion complex
prepared by suspending 15.Og of pregnanolone and 82.57 g
/(3-cyclodextrin in 610 mL distilled water in a 1000 mL
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Erlenmeyer flask. The suspension was stirred by magnetic
bar stirrer for 36 hours at 37 C. The suspension was then
filtered through a 0.22 m filter (Millipore, type GV) on
which the complex was collected. The resultant complex was
dried in a drying cupboard at 50 C for 12 hours after which
the mass was comminuted in a porcelain mortar, and the
granulate was passed through a 300 m sieve. The granulate
was dried in vacuo over P205 at 50 C for 24 hours. This
formulation was packaged in sachets containing 150 mg of
pregnanolone.
Formulation II was a 99:1 physical mixture of
pregnanolone and sodium lauryl sulphate prepared by mixing
49.50 g pregnanolone, which had been passed through a 125
m sieve, and 0.50 g sodium lauryl sulfate in a mortar and
pestle for 30 minutes. The mixture is then passed through
a 300 m sieve. This formulation was packaged as 250 mg
pregnanolone capsules and 500 mg pregnanolone sachets.
Prospective subjects were examined 21 days prior to
their first dose to determine medical histories and
baseline clinical laboratory values. The subjects were
told not to take any prescription or non-prescription drugs
before or during the study. Each subject participated
twice within a two-week period. Successive single dose
treatments were separated by a one-week interval.
Test subjects fasted overnight and reported to the
study center 3 hours prior to dose administration. A light
snack was given 2 hours prior to the dose administration.
A standard FDA-approved high-fat breakfast was given to the
subjects 20 minutes prior to dosing and was finished 5
minutes prior to dosing. After breakfast, the subjects
were given one of the two formulations in liquid yogurt.
The contents of a sachet was added to 100 mL of liquid
yogurt and stirred for 20-30 seconds. The subjects then
drank the yogurt. An additional 30 mL of yogurt was mixed
with the remnants in the cup and consumed assure full
administration. After drinking this, the sides and bottom
WO 94/27608 216 3 7 4 8 PCTIUS94/05820
of the cup were scraped and fed to the subject. No
liquids were allowed for the first hour after drug admin-
istration. Subjects had to remain sitting or standing for
the first hour after ingestion.
5 Mean plasma concentrations for the /3-cyclodextrin
formulation reached a peak value of 82.08 57.18 ng/mL at
1 hour post dosing. The mean plasma concentration was
79.33 49.71 ng/mL at 1.5 hours and was 74.21 37.21
ng/mL at 2 hours.
10 The sodium lauryl sulfate formulation reached its
peak mean plasma concentration later. The mean value was
64.06 54.91 ng/mL of 1.5 hours and was 66.63 49.60
ng/mL at 2 hours post dosing. These statistics are shown
in Figure 20.
15 Subjects were monitored by EEG, EOG, and EMG which
were recorded for 10 minutes in a darkened room with
closed eyes. The recordings were made for 5 minutes under
reaction time conditions. Subjects were supposed to
respond as quickly as possible to one of two different
20 tones presented in random sequence and intervals by press-
ing a button with their left or right thumb. The subjects
then were recorded for 5 minutes without interruption.
As shown in Figure 21 the time awake decreased post
dosing. The number of sleep attempts was also determined.
25 A sleep attempt was defined as a transition from wakeful-
ness to any sleep stage within the 5 minute recording
session. On each study day, a subject had five poly-
graphic recording sessions. Therefore, the maximum number
of sleep attempts could vary from 0 to S. A five indi-
30 cates that sleep was observed even during the 5 minute
pre-dose recording session. The number of sleep attempts
was plotted against the maximum plasma concentration (C
max) in Figure 22A. A low plasma concentration generally
coincided with few sleep attempts while high plasma
35 concentrations coincided with a high number of sleep
attempts. Figure 22B
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shows the trimean plasma concentration levels for different
numbers of sleep attempts.
The data from these human studies show: (1) that
pregnanolone can be formulated in such a way as to produce
suitable blood levels from oral administration; and (2)
that the blood levels of pregnanolone found in the volun-
teers correlated to the observation of drowsiness and/or
sleepiness.
To demonstrate the pharmacological properties of the
compounds used in the present invention, the inventors had
in vivo experiments run on rats in which the effects of
pregnanolone, 3a-hydroxy-30-trifluoromethyl-59-pregnan-20-
one 21-phosphate disodium salt, 3I3-ethynyl-3a-hydroxy-50-
pregnan-20-one, 3a-hydroxy-39-methyl-5a-pregnan-20-one,
153a,21-dihydroxy-30-trifluoromethyl-50-pregnan-20-one 21-
hemisuccinate sodium salt, 3a-hydroxy-3/3-ethynyl-5a-
pregnan-20-one, 3x,21-dihyrdoxy-3/3-ethynyl-50-pregnan-20-
one21-hemisuccinate,3a-hydroxy-30-trifluoromethyl-19-nor-
59-pregnan-20-one, 3a-hydroxy-21-(pyrid-4-ylthio)-5/3-
20pregnan-20-one, 3a-hydroxy-3Q-trifluoromethyl-5$-19-nor-
pregnan-20-one 21-phosphate disodium salt on brain EEG,
locomotor activity, and body temperature were measured.
Rats are a suitable model for human sleep because all
compounds that are hypnotics in man have hypnotic effects
25 in rats and vice versa. Edgar, D.M. "Drug Discovery and
Evaluation Using the Advanced Technology Sleep-Wake
Bioassay Laboratory at Stanford University" p. 4, December
1992. In addition, the rat model for human sleep has been
accepted by peer-review committees at NIH. at 5.
30 Furthermore, rat sleep and human sleep are fundamentally
the same. Id. at 4. For example, the proportion of time
spent in NREM versus REM sleep is about 4:1 in both rats
and humans. Id. at 5.
Rat preparation, monitoring, and analysis of the data
35 was conducted as described below. Adult, male Wistar rats
(275-350 g at the time of surgery, Charles River
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Laboratories) were anesthetized (Nembutal, 60mg/kg) and
surgically prepared with a cranial implant that permitted
intermittent EEG and EMG recording. Body temperature and
locomotor activity were monitored via a miniature trans-
mitter (Minimitter) surgically placed in the abdomen. The
cranial implant consisted of stainless steel screws (2
frontal [+3.9 AP form bregma, 2.0 ML] and two occipital
[-6.4 AP, 5.5 ML]) for EEG recording. Two Teflon-coated
steel wires were positioned under the nuchal trapezoid
muscles for EMG recording. All leads were soldered to a
miniature connector prior to surgery, and chemically
sterilized in Glutarex (3M Co.). The implant assembly was
affixed to the skull with dental acrylic. A minimum of
three weeks was allowed for recovery.
Each rat was permanently housed in its own individual
recording cage located within separate, ventilated compart-
ments of custom-designed stainless steel cabinets. Each
Nalgene microisolator cage, which is an individual record-
ing cage, was enhanced with a filter-top riser and swivel-
commutator. Food and water were available ad libitum. A
24 hour light-dark cycle (12 hours on, 12 hours off) was
maintained throughout the study using 4-watt fluorescent
bulbs 5cm from the cage. Animals were undisturbed for
three days both before and after treatments. In practice,
animals were undisturbed during the weekend. Treatments
were administered on Monday evening or Tuesday morning, and
the animals were again left undisturbed until Friday, when
cages were cleaned and the data copied off the computers.
The computers monitored the EEG, EMG, body temperature
and non-specific locomotor activity (LMA) via telemetry,
and drinking activity from 48 rats simultaneously. The
computer classified the arousal state every 10 seconds
using pattern-matching algorithms which included behavior-
dependent contextual rules. Drinking and LMA were recorded
every 10 seconds, while body temperature was recorded every
minute.
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As nocturnal creatures, rats are most active when the
lights are off, and like humans, are most sensitive to
hypnotic effects toward the end of their activity domi-
nated period. Therefore, time of treatment is important.
By administering the drug at CT-18, which is the peak of
the rat's activity dominated period, the natural tendency
for the rats to sleep at lights on (3:00 clock time or
Circadian Time, CT-0) cannot mask the hypnotic activity of
the drug. By administering the drug at CT-5 when the
propensity for REM sleep is greatest, the drug's effect on
REM sleep can be measured more accurately. The activity
dominated period begins when the lights are turned off at
CT-12 or 15:00 clock time on the Figures.
Compounds were suspended in sterile 0.25% methyl-
cellulose, 20-50% hydroxy propyl cyclodextrin, or water
depending on the physical properties of the steroid.
These vehicles provide adequate bioavailability for all
hypnotics tested to date. The drugs and controls were
injected intraperitoneally or administered orally in a
volume of 1 ml/kg or 10 mg/kg, respectively, under dim red
illumination for the CT-18 studies or under regular light
for the CT-5 studies.
A matched control group, N=20, was selected from a
pool of 60 available control candidates. These matched
control groups were constructed as follows. For each of
the variables (NREM, REM, LMA, body temperature, and sleep
bouts), a curve of hourly-averaged values was computed for
each candidate for the 24 hours preceding injection. The
20 candidates whose curve best fit (least squares) the
mean curve for the treatment group were selected. There-
fore, a matched-pairs procedure was not used, but parallel
treatment groups with closely comparable pre-treatment
baseline data were used.
Figures 1A, 1B, and 1C demonstrate that pregnanolone
administered at either CT-5 or CT-18, is effective in
promoting NREM sleep independent of the time at which it
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was administered. At both times, there is rapid onset and
high potency. At CT-5, the rat is more rested and it is
more difficult to induce sleep. These results indicate
that the neuroactive steroids of the invention are desired
hypnotic agents when sleep must be induced earlier than
usual, as may occur in shift work and jet lag. Further-
more, since a physician prescribing a hypnotic can never
be certain of the patient's sleep debt, it is advantageous
to have a hypnotic drug such as the present steroids with
a predictable hypnotic action independent of the patient's
sleep debt.
Figures lA-C also indicate that pregnanolone lacks a
rebound effect. Rebound effect is defined as the reduc-
tion of NREM sleep after the hypnotic action of the
treatment has returned to control levels. Pregnanolone
produced no rebound reduction of NREM sleep at CT-5 or
CT-18 for any dose. As shown by FIG. 24A, 39-ethynyl-3a-
hydroxy-50-pregnan-20-one also produced no rebound reduc-
tion of NREM sleep at CT-18. Triazolam showed a rebound
reduction of NREM sleep when administered at CT-18.
Figure 6B shows a star ("*") at the right hand side,
indicating a statistically significant reduction in NREM
sleep.
Pregnanolone is unique among hypnotic agents in being
both potent at both CT-5 and CT-18, and not producing
rebound wakefulness. While our studies indicate that
Zolpidem did not produce rebound insomnia, it was also
unable to effectively induce NREM sleep at CT-5, as shown
in Figure 8A. This feature could possibly imply that
pregnanolone or other neuroactive steroids which do not
produce rebound insomnia would produce less tolerance,
withdrawal, and rebound insomnia clinically compared to
current BZ-receptor ligands, which are their most
important limitations.
Figures 2A, 2B, 2C, and 25 demonstrates that admin-
istration of pregnanolone and the administration of
30-ethynyl-3a-hydroxy-50-pregnan-20-one causes a strong
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increase in the duration of uninterrupted NREM sleep or
"sleep bout length". Sleep bout length is of interest
because it may parallel the human tendency to awaken
periodically during the night, which has been shown to be
5 an important factor in determining the restorative value
of sleep in humans. This strong consolidation of sleep
has great potential in the treatment of age related
insomnia.
Figures 3A, 3B, and 3C indicate that pregnanolone
10 tends not to affect body temperature, contrary to the
teachings of Attallah Kappas et al. who found that intra-
muscular administration of pregnanolone caused a pyrogenic
effect in man. "Fever-Producing Steroids of Endogenous
Origin in Man" 105:68/701-68/708 A.M.A. Archives of
15 Internal Medicine 1960. Note in the hours prior to
injection, body temperature fluctuates in a near-sinusoi-
dal rhythm. Handling and injection cause a brief increase
in body temperature. Figure 26A also shows that 30-
ethynyl-3a-hydroxy-5g-pregnan-20-one has a minimal effect
20 on body temperature. Triazolam and Zolpidem affect body
temperature as revealed in Figures 6E and 8D, respec-
tively. A lack of temperature effect suggests that the
NREM inducing mechanism of pregnanolone and the neuroac-
tive steroids of the invention does not produce undesir-
25 able side-effects on homeostatic control functions.
Pregnanolone produces a moderately smaller reduction
in locomotor activity (LMA) over time at 30 mg/kg and no
reduction at 10 mg/kg as compared to Triazolam and Zolpid-
em, as illustrated in Figures 4A-C, 6F and BE. 30-Ethyn-
30 yl-3a-hydroxy-5f3-pregnan-20-one also produced a moderately
smaller reduction in LMA over time as illustrated in
Figure 26B. Both Triazolam and Zolpidem reduced LMA below
vehicle control levels when administered at CT-5 as
indicated in the bar graph of Figure 12, even though
35 Triazolam had no hypnotic effect at CT-5. Figures 14A and
14B demonstrate that during the effective period of
pregnanolone and Zolpidem when the rats are asleep, they
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are not moving. However, Zolpidem continues to depress
LMA even after the hypnotic action has worn off. Motor
incoordination appears to be comparatively reduced, which
is a valuable trait for hypnotics used with the elderly.
Figures 5A-5C and 24B indicate that pregnanolone and
3,6-ethynyl-3a-hydroxy-50-pregnan-20-one, respectively,
lack an inhibitory effect on REM sleep compared to other
hypnotics, such as the benzodiazepines, which reliably
reduce REM sleep. Figures 6F and 8F show that Triazolam
and Zolpidem, respectively, administered at CT-18 reduced
REM sleep in the first few hours after administration.
For example, the bar graph in Figure 13 demonstrates that
Zolpidem interferes with REM sleep at a dose administered
at CT-5 that induced significantly less NREM sleep than
pregnanolone.
Pregnanolone, 39-ethynyl-3a-hydroxy-50-pregnan-20-
one, and the other neuroactive steroids of the invention
are unique in that they combine a potent NREM-promoting
effect with slight REM-interference. Strong inhibition of
REM sleep in humans, such as alcohol and barbiturates
produce, has been associated with sleep disruption as the
drug effect diminishes and REM rebound sets in. Although
the biological function of REM sleep is unknown, the
finding that the neuroactive steroids of the invention
only slightly interfere with REM, while potently inducing
sleep, is a favorable attribute.
Pregnanolone produced a high-frequency, high -
amplitude EEG pattern during NREM sleep. This EEG pattern
is reproduced in Figures 7A-B. In contrast, deep normal
NREM sleep generally has a low frequency, high amplitude
EEG wave. Pregnanolone's effect on EEG spectral profiles
was closely similar after both CT-5 and CT-18 treatments.
This EEG pattern was distinct from the other hypnotics, as
shown in Figures 7C-D.
As illustrated in Figure 10A pregnanolone had charac-
teristic effects on NREM EEG delta activity (power).
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The "depth" of NREM sleep may be quantified as the propor-
tion of EEG activity that occurs at low (delta) frequen-
cies of 0.1-4.0 Hz. For each 10 second period of EEG
activity that was determined to be NREM sleep, the EEG
delta power was measured and averaged across 20 minute
intervals and then compared to vehicle controls. Figure
10A indicates that pregnanolone reduced the percentage of
EEG power in the low delta range for at least 5 hours
after administration. In contrast, Figures 10C and 10B
show that Triazolam and Zolpidem, respectively, increased
the percentage of EEG power in the delta range for at
least 1 hour after treatment.
It was also found that pregnanolone produced a
characteristic distribution of frequencies in the EEG
during the period of peak drug effect. The purpose of
determining a drug's "EEG spectral profile" is to
determine which frequencies have increased or decreased in
abundance following treatment. Figure 11A shows that
pregnanolone shifted the power from the lower frequencies
(0.1-8 Hz) to the higher frequencies (z10 Hz). In
contrast, Zolpidem and Triazolam increased the percentage
of EEG power in the lower delta frequency range. See
Figures 11B and 11C, respectively.
Using the same experimental design as used for
pregnanolone, nine other neuroactive steroids with various
structure modifications of the invention were tested on
rats. All nine synthetic neuroactive steroids produced
profiles similar to pregnanolone. The effects on REM,
NREM, rebound insomnia, locomotor activity, body tempera-
ture and power spectrum were similar to those described
above for pregnanolone and 30-ethynyl-3a-hydroxy-50-
pregnan-20-one. The changes in REM, rebound insomnia, and
NREM sleep in these experiments are summarized in Figures
23A-C, respectively. Like pregnanolone, all nine steroids
showed only slight REM interference, while producing a
potent NREM promoting effect. (Figs. 23A & C).
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Note also that these steroids produced little or no
rebound effects. See Fig. 23B.
In summary, pregnanolone and the nine other synthetic
neuroactive steroids strongly increased NREM sleep, but
did not cause any temperature drop, only slightly inter-
fered with REM sleep, did not significantly reduce locomo-
tor activity after the hypnotic action had worn off, and
was not followed by rebound wakefulness. By contrast,
Zolpidem and Triazolam reduced body temperature and loco-
motor activity after the hypnotic effects had worn off.
Zolpidem and Dexmedetomidine (an antihistamine which is a
class of drugs known to cause drowsiness) strongly inter-
fered with REM sleep. Dexmedetomidine produced strong
rebound effects. Taken together, these results show that
the neuroactivity steroids of this invention may have a
more specific mode of hypnotic action than BZ-receptor
ligands, thereby producing less impairment of other
important functions (i.e., body temperature, and possibly
motor coordination or motivation). Benzodiazepines, in
contrast, impair LMA after the hypnotic effects wear off,
a trait that is undesirable in a sleep inducing medica-
tion.
The method of producing sleep, in accordance with
this invention, comprises administering to a subject in
need of induced sleep, a compound of the invention,
usually prepared in a composition as described above with
a pharmaceutical carrier, in a nontoxic amount sufficient
to produce sleep. The preferred dose is 5 mg to 250 mg of
active compound.
While the preferred embodiments have been described
and illustrated, various substitutions and modifications
may be made thereto without departing from the scope of
the invention. Accordingly, it is to be understood that
the present invention has been described by way of illus-
tration and not limitation.
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