Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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8(9~-DEHYDROESTRADIOL DERIVATIVES
BACKGROUND OF THE INVENTION
The use of naturally occurring esrrogenic compositions of substantial purity
and low toxicity such as PRE1~~IARIN (conjugated equine estrogens) has became
a
preferred medical treatment for alleviating the symptoms of menopausal
syndrome,
osteoporosis/osteopenia in estrogen deficient women and in other hormone
related
disorders. The estrogenic components of the naturally occurring estrogenic
compositions have been generally identified as sulfate esters of estrone,
equilin,
equilenin, 17-~i-estradiol, dihydroequilenin and 17-~i-dihydroequilenin (U.S.
Patent
2,834,712). The estrogenic compositions are usually buffered or stabilized
with
alkali metal salts of organic or inorganic acids at a substantially neutral pH
of about
6.5 to 7.5. Urea has also been used as a stabilizer (U.S. 3,608,077). The
incorporation of antioxidants to stabilize synthetic conjugated estrogens and
the
failure of pH control with tris(hydroxymethyl)aminomethane (TRIS) to prevent
hydrolysis is discussed in U.S. 4,154,820.
8,9-Dehydroestrone is a known compound useful as an intermediate in the
synthetic production of estrone by isomerixation to 9,11 unsaturation (U.S.
Patent
3,394,153) and as an intermediate in the production of 3-cyclopentyloxy-17-
ethynyl
derivatives of the hormone (U.S. Patent 3,649,621). In addition, 8,9
dehydroestrone is known to possess estrogenic activity and to lower blood
lipid
levels (tJ.S. Patent 3,39I,169). The alkali metal salts of 8,9-dehydroestrone,
8,9
dehydroestrone-3-sulfate ester and its alkali metal salts, and their use in
estrogen
replacement therapy, atherosclerosis, and senile osteoporosis are disclosed in
U. S .
Patents 5,210,081 and 5,288,717.
DESCRIPTION OF THE INVENTION
In accordance with this invention, there are provided 17a,08,9-
dehydroestradiol or a pharmaceutically acceptable salt of its 3-sulfate ester,
and
17 j3,8,9-dehydroestradiol or a pharmaceutically acceptable salt of its 3-
sulfate ester.
These are collectively referred to as the compounds of this invention. The
structures of
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17a,08,9-dehydroestradiol and 17(3,08,9-dehydroestradiol are shown below as
compounds I and II, respectively.
I II
The compounds of this invention can also be named 17a,08,9-dehydroestrone
and 17(3,A8,9-dehydroestrone; and 17a-estra-1,3,5( 10),8-tetraene-3,17-diol
and 17 Ji-
estra-1,3,S(10),8-tetraene-3,17-diol, depending on which nomenclature system
is used.
Pharmaceutically acceptable salts of 17a,08,9-dehydroestradiol 3-sulfate ester
or 17J3,08,9-dehydroestradiol 3-sulfate ester include, but are not limited to)
the alkali
metal salts, alkaline earth metal salts, ammonium salts) allcylammonium salts
containing
1-6 carbon atoms or dialkylammonium salts containing 1-6 carbon atoms in each
alkyl
group) and trialkylammonium salts containing 1-6 carbon atoms in each alkyl
group.
17a,08,9-dehydroestradiol-3-sodium sulfate and l7Ji,08,9-dehydroestradiol
3-sodium sulfate are metabolites of D8,9-dehydroestrone, that are formed
following the
administration of 08,9-dehydroestrone. This invention therefore also provides
I7a,08,9-dehydroestradiol or a pharmaceutically acceptable salt of its 3-
sulfate ester in
greater than one percent purity, and 17 J3,08,9-dehydroestradiol or a
pharmaceutically
acceptable salt of its 3-sulfate ester in greater than one percent purity.
As used in accordance with this invention, treating covers treatment of an
existing condition, ameliorating the condition, or providing palliation of the
condition
and inhibiting includes inhibiting or preventing the progress or development
of the
condition.
The compounds of this invention can be prepared either by the in vivo
metabolism of D8,9-dehydroestrone, as shown in Example 5, or can be prepared
synthetically from 08,9-dehydroestrone as outlined in Schemes I and II.
Scheme I outlines the preparation of 17a,08,9-dehydroestradiol and salts of
its
3-sulfate ester beginning with the reduction of the 17-ketone of D8,9-
dehydroestrone
with a suitable reducing agent) such as sodium borohydride to produce 17i-08,9
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dehydroestradiol. Other hydride reducing agents can be readily used such as
sodium
cyanoborohydride or lithium aluminum hydride. The 3-hydroxyl group can be
selectively acylated with a suitable acylating agent) such benzoyl chloride or
acetyl
chloride. Inversion of stereochemistry at the 17-position can be accomplished
using a
S Mitsunobu reaction. Hydrolysis of the acyl moieties with a suitable aqueous
base,
such as sodium hydroxide gives 17a,8,9-dehydroestradiol. To form the 3-sulfate
ester, the diol is acylated with a suitable acylating reagent such as acetic
anhydride, and
the 3-acyl group is selectively cleaved using mild basic conditions, such as
potassium
carbonate in methanol. Formation of the 3-sulfate ester can be accomplished
with a
sulfur trioxide -ammonia, -alkylamine) -dialkylamine, or -trialkyamine
reagent, such as
sulfur trioxide-triethylamine or sulfur trioxide-pyridine. The resultant
ammonium,
monoalkylammonium, diallcylammonium or trialkylammonium salt of the 3-sulfate
ester
can be converted into another salt by exchange of cations, optionally via the
acid. For
instance) conversion to the 3-sulfate metal salt can be accomplished with a
metal
hydroxide solution. The preparation of l7oc,08,9-dehydroestradiol and the
sodium
and triethylammonium salts of its 3-sulfate ester is provided in Example 1.
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O OH
NaBH4
w
HO ~ HO
NOZ
PhCOCI
.OC
NOZ
3,S~irtitrobenmic acid
Ph3P
PhCOz
1. NaOH
2. Ac20 / pyridi
K2C0~ / MeOH
~OAc
Et3N-SO~
HO
,OH NaOH
Na03S
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Similarly, 17~i,~8,9-dehydroestradiol and salts of its 3-sulfate ester can be
prepared as shown in Scheme Ii, starting from D8,9-dehydroestrone, except that
the
inversion of stereochemistry at the 17-position is not needed. Using the
process
outlined in Scheme II, 17(3,D8,9-dehydroestradiol and the sodium and
triethylammonium salts of its 3-sulfate ester are produced. Other salts of the
3-sulfate
ester can be formed by varying the bases used. The preparation of 17~3,08,9-
dehydroestradiol and the sodium and triethylacnmonium salts of its 3-sulfate
ester is
provided in Example 2.
O OH
NaB H4
/ ~ \ / ~ \
H \ HO \
Ac20
pyridine
K2C03
MeOH / CH2C12
S03-Et3N
NaOH
The compounds of Examples 1 and 3 were found to degrade over time. The
preparation of TRIS stabilized complexes of the compounds of Examples 1 and 3
are
shown in Examples 2 and 4, respectively.
The compounds of this invention are estrogenic, as shown in an in viv
standard pharmacological test procedure which measured uterine growth in
immature
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female mice and ovariectomized rats as an evaluation of estrogenicity.
l7a,e8,9-
Dehydroestradiol-3-sodium sulfate and 17i,O8,9-dehydroestradiol-3-sodium
sulfate
were evaluated as representative compounds of this invention. Estrone and e8,9-
dehydroestrone were also evaluated for the purpose of comparison. The
compounds to
be evaluated were prepared as suspensions in corn oil. Corn oil alone was used
as a
control.
The following procedure describes the evaluation of estrogenicity in immature
mice. Intact immature Charles River CD female mice (23 days of age) were used.
The
compounds to be evaluated were administered subcutaneously or orally at total
doses
administered over 3 days of 1 to 1000 p.g subcutaneously and 3 to i000 ltg
orally.
Estrone was administered subcutaneously at total doses of 0.03 to 1 pg over 3
days.
The mice were sacrificed approximately 24 hours after the last dose was
administered,
and the uteri was removed and weighed. The results obtained are summarized in
the
following table.
ESTROGENIC
ACTIVITY
- MOUSE UTERINE
GROWTH
Treatments Route Minimal Effective Doubling
Doseb Doses
Estrone s.c. 0.06-0.1 0.1-0.2
e8,9 s.c. 6.2 i4.8
17a s.c. 1.75 7.61
17i s.c. 0.24 5.0
Estrone p.o. 5.9 25.9
e8,9 p.o. 2.6 26.7
17a p.o. 2.84 6.67*
17i p.o. 3.0 18.8
a D8,9 = D8,9-dehydroesrrone; 17a = I7a,e8,9-dehydroestradiol; 173 = 17~i,e8,9-
dehydroestradiol.
b Minimal effective dose given over 3 days required to produce a significant
increase in
uterine weight over that of vehicle.
c Total dose given over 3 days required to produce a 100% increase in uterine
weight
over that of vehicle.
* A value of dose of 16.6 ~.g would be obtained if the results from an
isolated strong
uterine response to the 10 ~g dose are deleted.
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The following procedure describes the evaluation of estrogenicity in
ovariectomized rats. Immature Charles River CD rats were ovariectomized at 30
days
of age and allowed 12 days for uterine regression. Administration of compounds
to be
evaluated was initiated 13 days post ovariectomy and was continued for ? days.
The
compounds to be evaluated were administered at daily subcutaneous or oral
doses of 3
to 1000 p.g/ratlday. The rats were sacrificed approximately 24 hours after the
last dose
was administered. The uteri were removed and weighed. The results obtained are
summarized in the following table.
ESTROGENIC
ACTTVITY -
MOUSE UTERINE
GROWTH
Treatments R_o~te Minimal Effective Doubling
Doseb Doses
Estrone s.c. 0.05-0.1 0.12-0.7
O8,9 s.c. 0.3 1.2
17a s.c. 10.4 171.4
17i s.c. 0.8 2.9
Estrone p.o. 10.6 22
e8,9 p.o. 3.3 21.5
17a p.o. 67.1 116.0
173 p.o. 12.0 14.5
a O8,9 = O8,9-dehydroestrone; 17a = 17a,8,9-dehydroestradiol; 17i = 17~i,08,9-
dehydroestradiol.
b Minimal effective dose given over 7 days required to produce a significant
increase in
uterine weight over that of vehicle.
c Total dose given over 7 days required to produce a 100~lo increase in
uterine weight
over that of vehicle.
The compounds of this invention are antioxidants. The antioxidant properties
of
l7oc,08,9-dehydroestradiol and l7(3,08,9-dehydroestradiol were established in
a
standard pharmacological test procedure that measured the its ability to
inhibit the
formation of oxidatively modified low density lipoprotein (LDL) induced by
exposure
to either Cu++ ions or cultured endothelial cells (Parthasarathy S) Proc Natl
Acad Sci
USA 86:1046-l050 (1989)) by the TBARS (thiobarbituric acid reactive
substances)
method for analysis of free aldehydes (Magi K., Biochem Med 15:212-216
(1976)).
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The results obtained in this standard pharmacological test procedure
demonstrate
that 17a,08,9-dehydroestradiol and 17(3,08,9-dehydroestradiol are potent
inhibitors of
LDL oxidation, inhibiting the process by up to 96%. ICsps of 0.19 EtM was
obtained
in the Cu++ mediated oxidation for both 17a,08,9-dehydroestradiol and 17
ji,08,9-
dehydroestradiol. ICsps of 0.26p.M and 0.I71tM were obtained in the porcine
aortic
endothelial cell mediated oxidations, respectively. By comparison, an ICsp of
0.56 E,iM
was obtained for estrone in the porcine aortic endothelial cell mediated
oxidation test
procedure.
To further demonstrate that the antioxidant properties of 17a,08,9-
dehydroesti adiol and 17 j3,08,9-dehydroestradiol, two additional standard
pharmacological test procedures were conducted using cells in culture. In the
first test
procedure) radiolabeled-LDL (125I_LDL) (McFarlane AS, In: Munro HN, Allison
JB,
eds. Mammalian Protein metabolism, Vol. 1. New York: Academic Press 297-341
( I964)) was modified by exposure to Cu++ in the presence and absence of 17
a,08,9-
dehydroestradiol or 17(3,08,9-dehydroestradiol. Next, J774 macrophages, which
express scavenger lipoprotein receptors which bind oxidatively modified-LDL)
were
exposed to the treated 125I-LDL. The results of this experiment demonstrate
that
binding of the LDL that was oxidized in the presence of 2.Sp.M concentration
of
17a,08,9-dehydroestradiol or I7(3,08,9-dehydroestradiol, was reduced by 48%
and
54%, respectively, and at a concentration of 0.25 uM was reduced by 34% and
28%,
respectively. By comparison, the same concentrations of estrone reduced the
binding
of LDL that was oxidized by 39% and 0%, respectively. Since binding and
metabolism
of oxidized LDL by macrophages is though to contribute strongly to the
development of
foam cells and therefore, atherosclerotic plaque, this effect of reducing LDL
oxidation
and subsequent binding to scavenger receptors is thought to be of significant
benefit.
In the second test procedure, porcine aortic endothelial cells (PAEC) were
exposed to LDL that had been modified as above, by exposure to Cu++ in the
presence
and absence of 17a,8,9-dehydroestradiol or 17 ji,08,9-dehydroestradiol,.
Oxidized
LDL has been demonstrated to be cytotoxic to endothelial cells, and this
process has
also been strongly implicated in the atherogenic process. Subsequent to a 24
hr
incubation of the cells with the treated LDL, an MTT assay was performed to
assess
cytotoxicity (Hansen MB, J Immu Methods 119:203-210 ( 1989)). This test
procedure
assesses the percent of cells that are viable (live) in a given assay. In the
assay,
following exposure to 25 p.g/ml LDL oxidized in the absence of compound, only
2% of
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the cells remained viable. In contrast, the percent live cells following
exposure to LDL
Cu++ treated in the presence of I7a,~8,9-dehydroestradiol or 17 (3,d8,9-
dehydroestradiol (2.SN.M) was I00% or greater. Other compounds tested in this
same
assay had minimal effects on protection of PACE ( 17(3-estradiol = 11 %
living, Equilin
S = 4% living; Estrone = 37% living. The results of this test procedure
demonstrate that
LDL modified in the presence of 17a,08,9-dehydroestradiol or 17~3,48,9-
dehydroestradiol was not cytotoxic, and therefore, the data is in agreement
with the
inhibition of oxidative modification by 17a,08,9-dehydroestradiol or 17~3,~8,9-
dehydroestradiol as demonstrated by the TBAR5 method above.
Based on the results of these standard pharmacological test procedures)
17a,08,9-dehydroestradiol or a pharmaceutically acceptable salt of its 3-
sulfate ester,
and 17 j3,08,9-dehydroestradiol or a pharmaceutically acceptable salt of its 3-
sulfate
ester are useful in replacement therapy in estrogen deficiency. The compounds
of this
1 S invention are therefore useful in providing estrogen replacement therapy
following
ovariectomy or menopause, and in relieving symptoms related to estrogen
deficiency,
including vasomotor symptoms, such as hot flushes, and other menopausal
related
conditions, such as vaginal atrophy, vaginitis, and atrophic changes of the
lower
urinary tract which may cause increased urinary frequency, incontinence, and
dysuria.
The compounds of this invention are useful in preventing bone loss and in the
inhibition
or treatment of osteoporosis. The compounds of this invention are
cardioprotective and
they are useful in the treatment of atherosclerosis. These cardiovascular
protective
properties are of great importance when treating postmenopausal patients with
estrogens
to prevent osteoporosis and in the male when estrogen therapy is indicated.
The
compounds of this invention are also antioxidants are therefore useful in
treating or
inhibiting free radical induced disease states. Specific situations in which
antioxidant
therapy is indicated to be warranted are with cancers, central nervous system
disorders,
Alzheimer's disease, bone disease, aging, inflammatory disorders, peripheral
vascular
disease, rheumatoid arthritis, autoimmune diseases, respiratory distress,
emphysema,
prevention of reperfusion injury, viral hepatitis, chronic active hepatitis,
tuberculosis,
psoriasis) systemic lupus erythematosus, adult respiratory distress syndrome,
central
nervous system trauma and stroke. Additionally, the compounds of this
invention are
useful in the suppression of lactation, and in the prophylaxis and treatment
of mumps
orchitis.
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The compounds of this invention can be formulated neat. More preferably one
may prepare a pharmaceutical composition comprising a compound of this
invention in
association or combination with a pharmaceutically acceptable carrier. The
proportion
of the pharmaceutical carrier may be determined by the solubility and chemical
nature of
the compound, chosen route of administration and standard pharmacological
practice.
The pharmaceutical carrier may be solid or liquid.
A solid carrier can include one or more substances which may also act as
flavoring agents, lubricants, solubilizers, suspending agents, fillers,
glidants)
compression aids, binders or tablet-disintegrating agents; it can also be an
encapsulating
material. In powders, the carrier is a finely divided solid which is in
admixture with the
finely divided active ingredient. In tablets, the active ingredient is mixed
with a carrier
having the necessary compression properties in suitable proportions and
compacted in
the shape and size desired. The powders and tablets preferably contain up to
99% of
the active ingredient. Suitable solid carriers include, for example, calcium
phosphate,
magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin)
cellulose, methyl
cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine) low melting
waxes
and ion exchange resins.
Liquid carriers are used in preparing solutions, suspensions, emulsions)
syrups, elixirs and pressurized compositions. The active ingredient can be
dissolved or
suspended in a pharmaceutically acceptable liquid carrier such as water, an
organic
solvent, a mixture of both or pharmaceutically acceptable oils or fats. The
liquid carrier
can contain other suitable pharmaceutical additives such as solubilizers,
emulsifiers)
buffers, preservatives, sweeteners, flavoring agents, suspending agents,
thickening
agents, colors, viscosity regulators, stabilizers or osmo-regulators. Suitable
examples
of liquid carriers for oral and parenteral administration include water
(partially
containing additives as above, e.g. cellulose derivatives, preferably sodium
carboxymethyl cellulose solution), alcohols (including monohydric alcohols and
polyhydric alcohols, e.g. glycols) and their derivatives, Iethicins, and oils
(e.g.
fractionated coconut oil and arachis oil). For parenteral administration, the
carrier can
also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile
liquid carriers
are useful in sterile liquid form compositions for parenteral administration.
The liquid
carrier for pressurized compositions can be halogenated hydrocarbon or other
pharmaceutically acceptable propellant.
Liquid pharmaceutical compositions which are sterile solutions or suspensions
can be utilized by, for example, intramuscular, intraperitoneal or
subcutaneous
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injection. Sterile solutions can also be administered intravenously. The
compounds of
this invention can also be administered orally either in liquid or solid
composition form.
The compounds of this invention may be administered rectally or vaginally in
the form of a conventional suppository. For administration by intranasal or
intrabronchial inhalation or insufflation, the compounds of this invention may
be
formulated into an aqueous or partially aqueous solution) which can then be
utilized in
the form of an aerosol. The compounds of this invention may also be
administered
transdermally through the use of a transdermal patch containing the active
compound
and a carrier that is inert to the active compound) is non toxic to the skin,
and allows
delivery of the agent for systemic absorption into the blood stream via the
skin. The
carrier may take any number of forms such as creams and ointments, pastes,
gels, and
occlusive devices. The creams and ointments may be viscous liquid or semisolid
emulsions of either the oil-in-water or water-in-oil type. Pastes comprised of
absorptive powders dispersed in petroleum or hydrophilic petroleum containing
the
active ingredient may also be suitable. A variety of occlusive devices may be
used to
release the active ingredient into the blood stream such as a semipermiable
membrane
covering a reservoir containing the active ingredient with or without a
carrier, or a
matrix containing the active ingredient. Other occlusive devices are known in
the
literature.
In addition, the compounds of this invention may be employed as a solution,
cream, or lotion by formulation with pharmaceutically acceptable vehicles
containing
0.1 - 5 percent, preferably 2%, of active compound which may be administered
to a
fungally affected area.
The dosage requirements vary with the particular compositions employed, the
route of administration, the severity of the symptoms presented and the
particular
subject being treated. Based on the results obtained in the standard
pharmacological test
procedures, projected daily dosages of active compound would be 0.02 pg/kg -
500
p.g/kg. Treatment will generally be initiated with small dosages less than the
optimum
dose of the compound. Thereafter the dosage is increased until the optimum
effect
under the circumstances is reached; precise dosages for oral) parenteral,
nasal, or
intrabronchial administration will be determined by the administering
physician based
on experience with the individual subject treated. Preferably, the
pharmaceutical
composition is in unit dosage form, e.g. as tablets or capsules. In such form,
the
composition is sub-divided in unit dose containing appropriate quantities of
the active
ingredient; the unit dosage forms can be packaged compositions, for example,
pocketed
powders, vials) ampoules, prefilled syringes or sachets containing liquids.
The unit
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dosage form can be, for example, a capsule or tablet itself, or it can be the
appropriate
number of any such compositions in package form.
The following provides the preparation of representative compounds of this
invention.
Exam 1R a 1
17a-Estra-1.3.S(10),8-tetraene-3.17-diol 3 sulfate. sodium alt
A. 17~~-Estra-1.3.5(10).8-tetraene-3.I7-diol
To a stirred suspension of 3-hydroxyestra-1,3,S(10)-8-tetraene-17-one (24.13
g, methanol (18S mL) and methylene chloride (138 mL) was added sodium
borohydride (6.92 g) portionwise over 25 minutes while maintaining the
temperature at
28-30'. To the slightly cloudy solution was added distilled water (555 mL) in
aliquots.
1S The temperature rose to 30-31~. The resulting slurry was stirred at 25-30~
for 0.5 hour
and then cooled to O.S~. The product was collected on a filter and washed with
water
(40 x 40 mL). Drying the initially white, wet cake (29.66 g) in a vacuum
desiccator
over phosphorus pentoxide for 4 days provided 23.54 g (96.83~l0) of yellow
product
having appropriate spectral (ir and pmt) data.
B . 173-Estra-1.3,5( 10),8-tetraene-3,17-diol 3-benzoate
A 2 L mufti-neck flask was equipped with a mechanical stirrer, a condenser
with
a nitrogen inlet, a pressure equalizing graduated addition funnel and a
thermometer.
The flask was charged with I7~-estra-1,3,5(10),8-tetraene-3,17-diol (60.00 g,
0.2219
mol.), tetrahydrofuran (S00 mL) and triethylamine (46.4 mL, 0.3329 mol., 1.5
molar
excess) and the mixture was stirred under nitrogen until a solution was
obtained. The
addition funnel was charged with benzoyl chloride (30.98 mL, 0.2669 mol.,
1.203
molar excess) and tetrahydrofuran up to the 100 mL mark. The benzoyl chloride
solution was added dropwise over 25 minutes, under nitrogen, while maintaining
the
reaction temperature between 15 and 20~ using a water/ice bath. The cooling
was
removed and the stirred suspension (EtgN.HCI ppt) was allowed to warm to room
tempetature (23~). Three and a half hours after completion of the addition of
the
benzoyl chloride solution, the reaction mixture was chilled in: ice and a
mixture of
saturated brine (204 mL)) water (68 mL) and concentrated hydrochloric acid (34
mL)
was added rapidly. The temperature rose from 23~ to 26~. After stirring 5
minutes,
solid precipitated in the lower layer. The aqueous phase was separated from
the organic
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phase and extracted with tetrahydrofuran (1 x 200 mL and 1 x 100 mL). The
combined
organic phases were washed with saturated brine (2 x 100 mL) and thrice with a
mixture of saturated brine (70 mL) and 5 % sodium bicarbonate (30 mL). A
further
wash with saturated brine ( 100 mL) did not completely remove residual benzoyl
chloride and so the solution was dried by magnetic stirring with anhydrous
magnesium
sulfate for 15 minutes. The drying agent was removed by filtration and the
filter pad
washed with teuahydrofuran. The filtrate and washings were evaporated at 30~
to a dry
solid. The solid was stirred magnetically with heptane (400mL) for 3 1j2
hours. The
resulting white solid was collected on a filter and washed with chilled
heptane (0-5~, 2 x
75 mL, 1 x 100 mL). The wet cake ( 108.09 g) was dried in a vacuum oven at
room
temperature for 3 days to provide a quantitative yield (83.35 g, 100.3%) of
crude 17~i-
estra-1,3,5(10),8-tetraene-3,l7-diol 3-benzoate. This material was suitable
for use in
the next step.
The crude benzoate (5.0 g) was stirred and heated in 95% ethanol (65 mL) to
give a solution which was filtered through a fluted, fast filter paper. The
filter was
rinsed with hot 95% ethanol and the solution was distilled to 50 mL volume.
The
solution was allowed to cool and the resulting slurry of crystals was
refrigerated. After
chilling to -10~ the product was collected on a filter and washed with cold (-
10~) 95%
ethanol. Drying in a vacuum oven at 65~ overnight gave purified product (3.82
g,
76.40%). Spectral (ir, pmr & ms) data were appropriate and elemental analyses
were
acceptable.
C. 17a-Estra-1.3.5_(10).8-tetraene-3.l7-diol 3-benzoate 17-(3.5-
dinitrobenzoatel
To a 1 L multi-neck flask equipped with a mechanical stirrer, a reflux
condenser
with a nitrogen inlet and a thermometer was added, under nitrogen, 17~-estra-
1,3,5(10),8-tetraene-3, 17-diol 3-benzoate (73.42 g) 0.196l mol.),
triphenylphosphine
(66.85 g, 0.2549 mol.), 3,5-dinitrobenzoic acid (54.06 g, 0.2549 mol.) and
toluene
(530 mL). To the stirred suspension was added a solution of diethyl
azodicarboxylate
(40 mL, 0.2549 mol.) and toluene ( 10 mL) from a 50 mL pressure equalizing
dropping
funnel) over a period of 7 minutes. A 10 mL rinse of toluene was used to
complete the
addition (total toluene 550 mL). The stirred suspension was slowly heated to
?0~ and
held at that temperature for 2.5 hours. (After 1/2 hour of heating at 70~
crystallization
began.)
The slurry of crystals was allowed to cool to 43~ and then cooled to -10~. The
crystals were collected on a filter and washed with cold (-10~) toluene (2 x
80 mL) cold
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(-10') heptane-toluene (7:3) and cold (-10') heptane (2 x 80 L). Drying the
wet cake
( 118.79 g) in a vacuum oven overnight at room temperature gave 107.56 g of
crude
product contaminated with 1,2-dicarbethoxyhydrazine.
To a 1 L Erlenmeyer was added the crude product (I07.56 g) and methanol
(310 mL). The mixture was stirred magnetically for 5 minutes and the product
was
collected on a filter. After washing with methanol (2 x 60 mL) the wet cake
(83.23 g)
was dried at 65' in a vacuum oven overnight. The yellow crystalline product
(75.65 g,
67.87%) had appropriate spectral (ir) pmr & ms) data and acceptable elemental
analyses.
D. 17a-Estra-Z.3,5(10).8-tetraene-3.I7-diot 3.17-diacetate
To 17a-es~a-1,3,5(10),8-tetraene-3, 17-diol 3-benzoate 17-(3,5-dinitro
benzoate) (71.00 g, 0.1249 mol), in a 3 L mufti-neck flask, was added
tetrahydrofuran
(630 mL) and 320 mL of 2N sodium hydroxide. The mixture was stirred at room
temperature (23-27~)) under nitrogen, for 24 hours. 2N Hydrochloric acid (196
mL)
and ethyl acetate (880 mL) were added rapidly. The resulting layers were
separated.
The lower aqueous phase was washed with ethyl acetate (3 x 200 mL). The
combined
organic extracts were washed with saturated brine (2 x 300 mL) pH of last wash
7).
The solution was dried by stirring magnetically with anhydrous magnesium
sulfate for
15 minutes.
Evaporation gave a red solid (54.08 g) which was subjected to an oil pump
vacuum for a few minutes. Pyridine (250 mL) and acetic anhydride (250 mL) were
added and the stoppered reaction flask was swirled to achieve solution. The
solution
was Ieft overnight at room temperature and poured onto ice. The volumn was
made up
to 2.5 L with water and the resulting suspension was stirred until a
manageable red
solid was obtained. The solid was collected on a filter and then blended with
300 mL
of water. The product was collected and the filter cake was washed with water
(total -
2 L). Drying over phosphorus pentoxide in a vacuum desiccator over a weekend
gave a
pink solid (44.23 g). It was stirred magnetically in methanol ( 100 mL) for a
few
minutes and collected on a filter. The filter cake was washed thrice with
methanol (total
50 mL) and dried in a vacuum oven at room temperature overnight. The pink
material
weighed 38.60 g (87.21%).
The product (38.60 g) was dissolved in hot methanol (120 mL) and the hot
solution was heated briefly with charcoal. The charcoal was removed by
filtration
through Celite and the filter pad was washed with hot methanol. The product
started to
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crystallize in the filtrate. The filtrate and washings were reheated to
dissolve the
product and the solution was distilled to approximately 120 mL volume. The
solution
was allowed to cool to near room temperature and the resulting slurry of
crystals was
chilled to 0-5~. The crystals were collected on a filter and washed with cold
(0-5~)
methanol (2 x 35 mL). Drying in a vacuum oven at room temperature gave the
title
compound (31.86 g, 82.39%).
E. 17a-Estra-1-3-5( 10).$-tetraene-3,17-diol 17-acetate
To 17a-estra-1,3,5(10),8-tetraene-3,17-diol 3,17-diacetate (30.00 g) in
dichloromethane (67 mL) and methanol ( 182 mL) at 0~, under nitrogen, was
added
dropwise saturated methanolic potassium carbonate (136 mL) while maintaining
the
temperature at 0~ or below. The reaction mixture was stirred at 0~ (inside
temperatwe)
using a thermostated cooling bath for 1 1/4 hours and then examined by TLC on
silica
gel with dichloromethane-ethyl acetate ( 19:1 ). No starting material was
observed. The
reaction mixture was distilled under oil pump vacuum while maintaining the
reaction
flask in the cooling bath. This produced an inside temperature significantly
below 0~
(e.g. -7 to -10~). To the resulting thick paste as added rapidly 430 mL of 5%
(v/v)
aqueous acetic acid and the mixture stirred until a filterable solid was
obtained. The
solid was collected on a filter and then blended with 70 mL of 5% acetic acid
and an
appropriate volume of water in a blender. The crude product was collected on a
filter
and washed well with water. Drying in a vacuum desiccator over phosphorus
pentoxide for 3 days provided 26.19 g (99.04%) of crude product.
Crude product (26.19 g) was magnetically stirred in methanol (270 mL)
containing a few drops of glacial acetic acid (pH of sol. = 4) and heated to
achieve
solution. The solution was filtered through a fluted) fast filter paper and
rinsed through
with 20 mL of hot methanol. The solution was distilled to a volume of 175 mL
and
allowed to cool. Cooling in ice and scratching enhanced crystallization. After
cooling
in the freezer (-20~) for 1 hour) the crystals were collected on a filter and
washed with
cold (-20~) methanol (3 x 20 mL). Drying at 60~ in a vacuum oven provided
purified
product (22.72 g, 86.75%) which had appropriate spectral (ir, pmr & ms) data
and
acceptable elemental (C & H) analyses.
A second crop (172 g, 6.57%) was obtained from the mother liquor. This was
combined with material remaining from the first crop (22.27 g) and the total
(22.99 g)
was stirred magnetically and heated in methanol (230 mL) to produce a yellow
solution.
The solution was filtered through a fluted, fast filter paper and rinsed
through with 20
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mL of hot methanol. The solution was distilled to a volume of 125 mL, allowed
to cool
to room temperature and then refrigerated. The resulting crystals were
collected on a
filter and washed with cold (0~) methanol (3 x 15 ml). Drying at 60' in a
vacuum oven
for 20 hours provided l8.79 g {81.73% recryst. step, 7I.06% overall) of white
product
having appropriate spectral (ir, pmr & ms) data and acceptable elemental (C &
H)
analyses.
F. 17n-Estra-1 3 5(l0) 8-tetraene-3 17-diol 3-sulfate 17-acetate
triethvlammonium
To a stirred solution of 17a-estra-1,3,5(10),8-tetraene-3,17-diol 3,17-acetate
( 17.50 g, 0.0560 mol.) in tetrahydrofuran (204 mL) at room temperature) under
nitrogen, was added triethylamine-sulfur trioxide complex (20.30 g, 0.1120
mol., 2
molar excess). The solid dissolved rapidly and the solution was stirred 4
hours at room
temperature under nitrogen. TLC on silica gel with dichloromethane-ethyl
acetate
(19:1) had showed no starting material after 3.7S hours. Anhydrous ether (612
mL)
was added and an oil formed. Shortly afterwards) the oil crystallized. The
mixture was
stirred for 1/2 hour longer and the crystals were collected on a filter. The
crystals were
washed with ether (2 x 125 mL). Drying the wet crystals (43.31 g) in a vacuum
oven
at room temperature overnight gave 28.90 g (104.51%) of product contaminated
with
excess reagent.
To a stirred solution of the crude product (28.90 g) in methanol (70 mL) was
carefully added triethylamine (2-3 mL) to bring the pH to 7-8. Ether (800 mL)
was
added rapidly in aliquots. Again, an oil formed which quickly crystallized
Stirring
was continued for 15 minutes and the slurry of crystals was cooled to -15~.
The
crystals were collected on a filter and washed with ether (3 x 80 mL). Drying
in a
vacuum oven at room temperature overnight gave 25.09 g (90.74%) of white
material
containing some solidified lumps.
To a stirred solution of the above material (25.09 g) in methanol (40 mL) at
room temperature was carefully added triethylamine (a few drops) to adjust the
pH to 7.
The solution was filtered into a 1 L mufti-neck flask and the flter was washed
with
methanol (20 mL). To the mechanically stirred solution was added ether (600
mL.).
Once again, the product formed as an oil which quickly crystallized. The
mixture was
stirred vigorously for 1/2 hour and then cooled to -10~. The white crystalline
product
was collected on a filter and washed with ether (3 x 60 mL). Drying at room
temperature in a vacuum oven provided title product (23.64 g, 94.22% and
85.49%
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overall) having appropriate spectral (ir, pmr & ms) data and acceptable
elemental (C, H
& N) analyses.
G. 17a.-Estra-1.3.5(107.8-tetraene-3.17-diol 3-sulfate sodium salt
To a solution of 17a-estxa-1,3,5( 10),8-tetraene-3, I7-diol 3-sulfate 17-
acetate
triethylammonium salt (22.50 g, 0.04558 mol.), in methanol (144 mL), under
nitrogen,
was added 144 mL of 1.6N sodium hydroxide and the mixture stirred magnetically
for
4.5 hours. The resulting yellow solution was evaporated at room temperature
until 150
mL of distillate was collected. n-Butanol (450 mL) was added and the mixture
was
stirred until the sticky white crystals dissolved and two phases were
obtained. The
mixture was transferred to a separatory funnel with a mixture of 20 mL n-
butanol, 10
mL of saturated brine and 10 mL of water. The layers separated slowly. The
upper
organic phase was washed consecutively with saturated brine (120 mL, 2 x 100
mL, 3
x 75 mL and 1 x SO mL) until the pH of the final wash was 7. The cloudy
organic
phase was filtered through a small pad of solka Floc (prewashed with n-
butanol) on a 7
cm Buchner funnel and rinsed through with n-butanol (2 x 2S mL). The solution
was
transferred to a 2 L round bottom flask with a 50 mL rinse of n-butanol. The
solution
was evaporated at 35-40~ under oil pump vacuum until a thick slurry of
crystals was
obtained and 300 mL of distillate had been collected. Ether (500 mL) was added
and
mixed with the slurry. The white crystals were collected on a 10 cm filter and
washed
with ether (4 x 80 mL). The filter cake was transferred to a 1 L Erlenmeyer
and
magnetically stirred with 400 mL of ether for 5 minutes. The crystals were
collected
once more and washed with ether (5 x 70 mL). The wet cake was dried in a
vacuum
oven with a nitrogen bleed for 6 days to give 17.17 g (96.49% based on a
hydrate) of
white solid.
The above crude product (17.17 g) was stirred magnetically in U.S.P. ethanol
(400 mL) until almost all the solid had dissolved. The mixture was filtered
and the
filtrate transferred to a 5 L mufti-neck flask with 20 mL of U.S.P. ethanol.
The
solution was stirred mechanically and ether ( 1.4 L) was added to cause
crystallization.
An additional 1.9 L of ether was used to complete the process. The slurry of
crystals
was cooled to -10~ and the crystals were collected on a filter. The filter
cake was
washed with ether (2 x 100 mL). Drying at room temperature in a vacuum oven
for
two days provided white crystals of title product ( I7.67 g, 86.34% based on
MW=448.989).
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Calc. for C1gH2105NaS. 0.9 C2HSOH. 1.3 H20. 0.2 NaCI (448.989): C, 52.97;
H, 6.51; Na, 6.14; C1, 1.58; EtOH, 9.23; H20) 5.22
Found: C, 52.78; H, 6.27; Na, 6.17; Cl, 1.70; EtOH, 9.12; H20, 4.01
~amQle 2
17a-Estra-1,3.5(10l.8-tetraene-3.17-dioI 3-sulfate. sodium salt.
with Tris (hydroxvmethYll aminomethane (TRIS)
To a solution of tris(hydroxymethyI)aminomethane (8.48 g) in distilled water
(I,400 mL) was added 17a-estra-1,3,5(10),8-tetraene-3,17a-diol 3-sulfate
(ester)
sodium salt ( 14.69 g, M W W-7332-3, P. R. 215-20) and the mixture was stirred
magnetically to dissolve the steroid. The solution was filtered and the filter
was washed
with water ( 100 L). The solution was transferred to a tray at -40~ in a large
Virtis
Freeze-dryer using a 500 mL water rinse. The solution was frozen and the solid
was
freeze dried for 5 days. The resulting white, soft, flaky material was
pressed, scraped,
and mixed well in a bottle. The material was dried for a further 4 days at
room
temperature in the freeze-drier. The white product weighed 20.93 g (98.54%).
ExamQle 3
A. I7~~-Estra-1.3g101,8-tetraene-3.l7-diol
To a stirred suspension of 3-hydroxyestra-1,3,S(10)-8-tetraene-17-one (24.13
g, methanol ( 185 mL) and methylene chloride ( 138 mL) was added sodium
borohydride (6.92 g) portionwise over 25 minutes while maintaining the
temperature at
28-30'. To the slightly cloudy solution was added distilled water (555 mL) in
aliquots.
The temperature rose to 30-31 ~. The resulting slurry was stirred at 25-30~
for 0.5 hour
and then cooled to 0.5~. The product was collected on a filter and washed with
water
(40 x 40 mL). Drying the initially white) wet cake (29.66 g) in a vacuum
desiccator
over phosphorus pentoxide for 4 days provided 23.54 g (96.83%) of yellow
product
having appropriate spectral (ir and pmr) data.
B . 171e-Estra-1,3.y10).8-tetraene-3 17-diol 3,17-diacetate
A mixture of 17~-estra-1,3,5(10)-8-tetraene-3, 71-diol (23,.09 g), was swirled
with a mixture of acetic anhydride (120 mL) and pyridine (120 mL) in a
stoppered 500
mL flask. The resulting yellow solution was left at room temperature
overnight. The
solution was added to crushed ice 0600 mL). A white precipitate formed. The
slurry
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was stirred magnetically and water (220 mL) was added. Stirring was continued
and
the temperature was allowed to rise to 18-20~. The product was collected on a
filter and
washed with water (100 mL). Drying the wet cake (90.6S g) over phosphorus
pentoxide in a vacuum desiccator gave 28.19 g (93.13%) opf white, crude
product.
To a 2 L multi-neck flask equipped with a mechanical stirrer, a thermometer
and
a one piece distillation apparatus, was added, under nitrogen, crude 17~-estra-
1,3,5( 10),8-tetraene-3) 17-diol 3,17-diacetate (28.19 g) and methanol (67S
mL). The
slurry was heated and the product dissolved at S9-60~ after the addition of a
further 200
mL of methanol. The solution was distilled to approximately 500 mL and 404 mL
of
distillate was collected. The resulting slurry of crystals was allowed to cool
to 3S~ and
then cooled to -10~. The crystals were collected on a filter and washed with
cold (-10~)
methanol (3 x SO mL). The wet crystals (30.l4 g) were dried in a vacuum oven
at 6S~
for 4 days to provide 25.1S g (89.24%) of white, crystalline 3) 17i-diacetate,
having
consistent spectral (ir, pmt and ms) data and acceptable elemental (C & H)
analyses.
1S
C. 17~~-Estra-1 (3.S110).8-tetraene-3.17-diol 17-acetate
To a stirred suspension of 173-estra-1,3,S(10),8-tetraene-3,Z7-diol 3,17
diacetate (24.16 g, 0.0682 mol. in methanol ( 1 SO mL) and dichloromethane (S6
mL)
cooled to -S~ under nitrogen in a constant temperature bath was added 113 mL
of
solution of saturated methanolic potassium carbonate over a period of 4S
minutes. (The
temperature of the reaction ranged between -3 and -S~ during the addition).
Stirring at -
S~ under nitrogen was continued for a total period of 22.S hours. A
precipitate
(product) formed after 1 3/4 hours. TLC on silica gel with dichloromethane-
ethyl
2S acetate (9.5:0.S) was used to judge reaction completion. The reaction flask
was
equipped with a one piece distillation apparatus. Distillation under
diminished pressure
(initially with an aspirator and later with an oil pump) while maintaining the
reaction
flask in the -S~ constant temperature batch was used to remove all of the
solvent. Five
percent acetic acid (3S0 mL) was added rapidly to the dry solid and the
resulting slurry
was stirred at -S to 0~ for 2 hours. The resulting pH was 4.5. The crude
product was
- collected on a filter and washed with 4 x SO mL of water. The white crude
material was
stirred magnetically with 300 mL of water and collected once more on a filter.
Washing
with water (9 x 40 mL0 removed traces of acid and the pH of the last wash was
S . S .
Drying the wet cake (37.06 g) in a vacuum desiccator over phosphorus pentoxide
3S overnight provided 20.02 g (94.03%) of crude 17~-monoacetate.
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To a 500 mL round bottom mull-neck flask equipped with a mechanical stirrer,
a thermometer and a one piece distillation apparatus was added, under
nitrogen) crude
product (20.02 g), methanol ( 100 mL) and dichloromethane (200 mL). The pH
(wetted
pH paper) of the solution was 8; 5 drops of glacial acetic acid were added to
bring the
pH to 5. The solution was distilled at atmospheric pressure until
crystallization was
copious (volume of distillate 220 mL). The slurry was cooled to 40~ and then
to -10'
with a dry ice-acetone bath. The product was collected on a filter and washed
with cold
(-10') methanol (3 x 25 mL). The wet cake (21.65 g) was dried at 70' overnight
and
then at 85' for 5.5 hours in a vacuum oven. The resulting white crystalline
material
(18.07 g, 90.25%) had appropriate specral (ir, pmr and ms) data.
D. 173-Estra-1,3,5(10a8-tetraene-3.17-diol 3-Sulfate 17-acetate
Jriethylammonium salt
To a stirred solution of 17(3-estra-1,3,5(10),8-tetraene-3,17-diol 17-acetate
( 17.18 g, 0.05499 mol. in tetrahydrofuran (200 mL) udner nitrogen was added
sulfur
trioxide-triethylamine (i9.93 g, 0.1I0 mol.) and the solution was stirred for
4 hours at
room temperature. Reaction completion was judged by TLC on silica gel with
CH2C12:EtOAc:Me~H:NEt3 (3:3:1.5:2.5). Ether (600 mL) was added and the
resulting slurry was stirred for 30 minutes at room temperature. The crude
product was
collected on a filter and washed with ether (3 x 27 mL). Drying the wet cake
(36.S0 g)
for 2 days in a vacuum oven at room temperature afforded 30.13 g (100.98%) of
crude,
white product contaminated with excess reagent.
To a 1 L mufti-neck flask equipped with a mechanical stirrer, a reflux
condenser
with a nitrogen inlet and a thermometer was added, under nitrogen, crude
product
(30.13 g) and methanol (80 mL). The temperature dropped from 26 to 20~ and so
a
warm water bath was used to bring the temperature to 25-25~. Another 50 mL of
methanol was added to achieve solution at that temperature. The pH (wetted pH
paper)
of the solution was 3. The pH was carefully adjusted to pH 7-8 by the addition
of
triethylamine (2.5 mL).
To the clear solution was immediately added ether ( 1 L). The resulting slurry
was transferred to a 2 L Erlenmeyer with ether (2 x 75 mL) and stirred
magneticaly. Aa
additional 50 mL of ether (total 1,200 mL) was added and the resulting slurry
was
stirred at room temperature for 10 minutes. It was then cooled to -10~ with an
acetone-
dry ice bath. The product was collected on a fll ter and washed with ether (3
x 30 mL).
The wet cake (32.63 g) was dried overnight in a vacuum oven at room
temperature to
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give 23.2S g (85.64%) of purified material having appropraite spectral (ir,
pmr and ms)
data and acceptable elemental (C,H and N) analyses.
E. 17~~-Estra-l.3.5(10).8-tetraene-3.17-diol 3-sulfate S ium salt
To a solution of 17(3-estra-1,3,5( 10),8-tetraene-3,17-diol 3-Sulfate 17-
acetate
(diester) triethylammonium salt, (22.27 g, 0.09S 11 mol. in methanol ( 145 mL)
was
adced 145 mL of 1.6 N sodium hydroxide and the mixture was stirred at room
temperature (25~ until 140 mL of distillate had been collected. The white)
sticky solid
(which precipitated during the evaporation) was dissolved by stirring with n-
butanol
(150 mL)) under nitrogen, for 30 minutes. The mixture was transferred to a
separatory
funnel along with 20 mL of n-butanol, 10 mL of saturated brine and 10 mL of
water.
The separation of phases was slow so a further 60 mL of brine was added. The
lower
aqueous phase 0126 mL) was separated and the organic phase was washed with
saturated brine ( 1 x 40 mL, followed by 3 x 30 mL, final wash pH 13-14).
Solid
product started to form. n-Butanol (500 mL), saturated brine (100 mL) and
water (20
mL) were added. The product redissolved on mixing and the pH of the wash was
12.
The organic phase was washed further with saturated brine (6 x 100 mL) until
the final
wash had pH 7-8. The slightly cloudy organic phase was filtered through Sollca
Floc
and the filter pad was washed with n-butanol (50 mL). The solution was
transferred
with 50 mL of n-butanol to a round bottom flask and was evaporated under oiI
pump
vacuum at 35-38~ until crystallization occurred (volume of concentrate, 208
mL,
distillate volume 630 mL. ) To the stirred slurry was added ether (700 mL) and
the
slurry was stirred for 5 minutes at room temperature. The product was
collected on
filter and washed with ether (4 x 100 mL). The wet cake (39.99 g) was dried in
a
vacuum oven at room temperature (25~) to give 16.83 g off-white, crude
product.
To the above crude material (l6.83 g) was added 95% ethanol (165 mL) and the
mixture was stirred for 10 minutes to achieve a cloudy solution. The solution
was
filtered. To the magnetically stirred, clear filtrate was added ether (1,500
mL.)
(Crystallization was apparent after 500 mL had been added.)
The resulting slurry was stirred for 10 minutes at room temperature and then
filtered. The filter cake was washed with ether ( 5 x 100 mL.) The resulting
white,
powdery solid (17.28 g) still smelled of ethanol and so it was magnetically
stirred with
ether (300 mL) for 10 minutes and collected once more on a filter. The filter
cake was
washed with ether (4 x 50 mL) and the wet cake (25.18 g) was dried at room
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temperature overnight in a vacuum oven. The white product ( 15.$0 g, 86.S 1 %)
had
appropriate spectral (ir, pmr and ms) data.
$ ~',~,ample 4
l7j~Estra-1.3.y10).8-tetraene-3.17-diol 3-sulfate sodium salt
with tris !hy_,droxvmethyl)aminomethane (TRIS)
To a stirred solution of 17i-estra-1,3,S(10),8-tetraene-3,17-diol 3-sulfate
(ester)
sodium salt ( 13.11 g) in distilled water ( 1,400 mL) was added TRIS (7.56 g)
and the
solution was filtered. The filter was washed with 100 mL of distilled water.
The
filtrate and washing was transferred to a tray at -40~ using 500 mL of
distilled water.
The frozen solution was freeze dried over a period of 4 days. The resulting
white shiny
material (20.92 g) vsras pressed) mixed, placed in a preweighed bottle, and
dried a
further 4 days at room temperature (23~) in the freeze dryer. The material
weighed
19.69 g (97.SS%).
ExamBle 5
Preparation of I7a.08,9-deh~,droestradiol or 17f3.08.9-dehydroestradioI
by the in vivo metabolism of 08.9-dehvdroestrone
A. Dose administration and sample collection
Four female dogs, weighing 9 - 13 kg were given 1 mg/kg of ~g.9-
dehydroestrone, po in capsules. The dogs were housed throughout the study in
2S individual stainless steel metabolism cages. The dogs were fasted overnight
prior to
dosing and food was returned 4 hr afterwards. Water was provided ad libitum
throughout the study. Blood (8 ml) was collected at 0, O.S, 1, 2, 4, 6 and 8
hr post-
dose and plasma was obtained. Urine was collected for 24 hr post-dose over dry
ice.
B . Determination of metabolite profiles by HPLC
1. UrinarX metabolite rn ofles
Urinary metabolites were determined by analyzing 0-24 hr samples after enzyme
hydrolysis. To hydrolyze the conjugates, an aliquot ( 1 ml) of urine was
combined with
3S I ml of 0.0S M sodium acetate buffer) pH 5.0, and incubated overnight at
37'C with
2000 units of Glusulase (Helix Pomatia, DuPont). The hydrolyzed samples were
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centrifuged at 2500 rpm for 10 min. S upernatants of hydrolyzed urine samples
were
extracted using C-18/N+ cartridges (Bifunctional cartridges containing C-18
and
quaternary amine sorbents from Chemical Separations, PA). The cartridge
exrractions
were performed by vacuum pull (Speed Mate 30 Applied Separations). The C-18/N+
cartridges were first conditioned with 2 x 1 m1 acetone) 2 x 1 m1 methanol) 2
x 1 m1
0.1 N acetic acid and 2 x 1 mI H20. The sample (supernatant) was applied and
the
cartridges were washed with 2 x 1 ml H20, 2 x 1 m1 0.1 N acetic acid, 2 x I ml
H20
and 2 x 1 m1 hexane. The metabolites were then eluted in 2 x 1 m1 acetone. The
extracts were dried under N2 and reconstituted in 1 m1 of mobile phase [60%
buffer
(0.05 M KH2 P04) pH 3.0), and 40% organic (2:1 acetonitrlie; methanol)].
Twenty-
five microliters of each extract were injected onto a C-6 Sp. HPLC column
(Alltech)
using an SP8780 autosampler (Spectra-Physics) and eluted at a flow rate of 1
nnl/min
with an ESA-420 pump. Metabolites were detected with an electrochemical
detector
(Coulochem II-ESA). The potentials for the detector were set at 0.75 V (guard
cell),
0.35 V (electrode 1) and 0.70 V (electrode 2) and the signals from electrode 2
were
recorded on a chart recorder (Kipp and Zonen BD 40).
2. Plasma metabolite profiles
Plasma samples were also analyzed by HPLC after hydrolysis. Hydrolysis of
the conjugates was achieved by incubating the samples with Glusulase as
described for
the urine samples above. Following centrifugation, the plasma samples were
extracted
using C-18/N+ cartridges by the above described procedure and analyzed by HPLC-
electrochemical detection.
3. Hvdrolvsis in the presence of enzyme inhibitor
The types) of conjugates present in the plasma and urine samples were
determined by conducting the hydrolysis of all samples in the presence or
absence of
200 u.M of saccharolactone, an inhibitor of ~-glucuronidase. The hydrolysis,
extraction
and HPLC analyses were performed as described above.
C. Identification of the metabolites by GC/MS anal, s~y_'s of ~-ydrolyzed
urine and
plasma sam lies
The structures of the metabolites were identified by EI-GC/MS analysis of TMS
derivatives of extracts of hydrolyzed urine and plasma samples. The structures
of the
metabolites were confirmed by analyzing and comparing the mass spectra of
authentic
reference standards.
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1. C~ S analysis of hvdrolyzed urine samples
Five ml of 0-24 hr urine from one dog was mixed with S ml of pH S.0 sodium
acetate buffer (0.05M) and 10,000 units of Glusulase. The mixture was
incubated
overnight at 37~C in a shaking water bath (Precision Scientific). The
following
morning) the hydrolysates were centrifuged at 2500 rpm for IO min. C-18/N+
cartridge
extraction was carried out as previously described. Following acetone elution
of the
metabolites, NZ was used to dry the extracts. Each extract or reference
standard was
then reconstituted in 80 ~1 of toluene; then 10 E.tl of BSTFA and 5 Etl of
pyridine were
added and reacted at 65 ~ C for 1 hr to form TMS derivatives of the
metabolites. The
derivatized samples were analyzed using a Finnigan-MAT 8230 high resolution
mass
spectrometer directly interfaced to a Varian 3400 gas chromatograph. The data
were
acquired on an S S-300 data system and printed on a Printronix. The source
ionization
mode was positive electron impact. The column used was a J&W DB-SMS 30 M x
0.32 mm m. The initial column was 80~C with a -1 minute hold programmed to
260~C
at 10~C/min. The injection temperature was 2S0~C. The injection volume was 2
~tl.
2. GC'-'.~N1S analysis of h~yzed plasma same
Two ml of plasma was mixed with 2 m1 of pH 5.0 sodium acetate buffer (0.05
M) and hydrolyzed with 2000 units Glusulase (DuPont). The mixture was
incubated
overnight at 37~C in a shaking water bath and extracted as described
previously.
Expermiental samples, control plasma and control plasma containing synthetic
standards were analyzed as described above.
D. Metabolite Profile
1. rive
The HPLC chromatograms of enzyme hydrolyzed 0-24 hour urine resulted in
three peaks which were not present in the chromatogram of an extract from
control dog
urine. The retention time of the third eluting peak was identical to that of
d8,9-dehydroestrone. Peaks 1 and 2 had the same retention times ( 17.4 min and
19.3
min, respectively), as l7p,e8,9-dehydroestradiol and 17a,08,9-
dehydroestradiol.
Also) co-chromatography with reference standards clearly indicated the
presence of
17~3,~8,9-dehydroestradiol and 17a,8,9-dehydroestradiol as peaks 1 and 2,
respectively. Analysis of samples that were extracted before hydrolysis did
not reveal
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-25-
any metabolites, suggesting that little of the metabolites exist in
unconjugated forms.
Samples from all dogs gave similar metabolite profiles.
2. la m
Analysis of plasma samples after hydrolysis also showed the presence of the
same three peaks as in the urine samples at all time points examined. In both
urine and
plasma sampes) addition of saccharolactone inhibited the hydrolysis of the
conjugates,
indicating that most of the metabolites were in glucuronide forms.
E. Identification of l7a,d8.9-dehydroestradiol
The mass spectrum of peak 2 in the GC/MS chromatograms displayed a
molecular ion m/z 414 suggesting that it is a diol. A weak M+ ion and a base
peak at
m!z 309 (M-I05 (CH3, TMS OH)] are characteristic of 17a-dihydro ring-B
unsaturated
estrogens. Other fragments at m/z 399 [M-15(CH3)]) 324 [M-90 (TMS)] and 283 (M-
13l] are also characteristic for 17-hydroxy estrogens. The mass spectrum of
peak 2
was identical to that of authentic reference 17a,08,9-dehydroestradiol. Based
on these
data the structure of this metabolite was identified as 17a.08,9-
dehydroestradiol.
E. Identification of 17Q,08.9-deh~droestradiol
The mass spectrum of peak 1 in the GC/MS chromatograms displayed a
molecular ion m/z 414 and fragment ions at m/z 399 (M-IS (CH3)], 324 (M-90
(TMS)], 309 (M-1Q5 (CHg, TMS)J and 283 (M-131) that are characteristic of ring-
B
unsaturated estrogen diols. Unlike 17a,08,9-dehydroestradiol, this compound
gave a
strong molecular ion with a relative intensity of about 95%, which is
characteristic of
17~-dihydro ring-B unsaturated estrogens. The mass spectrum of peak 3 was
identical
to that of reference 17¢,08,9-dehydroestradiol. Based on these data the
structure of
this metabolite was determined as 17¢,08,9-dehydroestradiol.
