U.S. patent application number 11/141029 was filed with the patent office on 2005-12-15 for method for the preparation of 21-hydroxy-6,19-oxidoprogesterone (21oh-6op).
This patent application is currently assigned to APPLIED RESEARCH SYSTEMS ARS HOLDING N.V.. Invention is credited to Burton, Gerardo, Lantos, Carlos P., Veleiro, Adriana Silvia.
Application Number | 20050277769 11/141029 |
Document ID | / |
Family ID | 8169774 |
Filed Date | 2005-12-15 |
United States Patent
Application |
20050277769 |
Kind Code |
A1 |
Burton, Gerardo ; et
al. |
December 15, 2005 |
Method for the preparation of 21-hydroxy-6,19-oxidoprogesterone
(21OH-6OP)
Abstract
A 21-Hydroxy-6,19-oxidoprogesterone derivative of Formula (1): 1
where R is --C(O)--CH.sub.2CH.sub.3,
--C(O)--(CH.sub.2).sub.2--COOH,
--C(O)--(CH.sub.2).sub.7--CH.dbd.CH--(CH.sub.2).sub.7--COOH, or
--C(O)--PO.sub.3Na.sub.2.
Inventors: |
Burton, Gerardo; (Provincia
de Buenos Aires, AR) ; Lantos, Carlos P.; (Buenos
Aires, AR) ; Veleiro, Adriana Silvia; (Martinez,
AR) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
APPLIED RESEARCH SYSTEMS ARS
HOLDING N.V.
Curacao
NL
|
Family ID: |
8169774 |
Appl. No.: |
11/141029 |
Filed: |
June 1, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11141029 |
Jun 1, 2005 |
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10380167 |
Jul 18, 2003 |
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10380167 |
Jul 18, 2003 |
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PCT/EP01/10734 |
Sep 17, 2001 |
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Current U.S.
Class: |
540/81 |
Current CPC
Class: |
C07J 71/0005 20130101;
C07J 71/00 20130101 |
Class at
Publication: |
540/081 |
International
Class: |
C07J 071/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 18, 2000 |
EP |
00119494.3 |
Claims
1-13. (canceled)
14. A 21-Hydroxy-6,19-oxidoprogesterone derivative of Formula (1):
11wherein R is selected for the group consisting of
--C(O)--CH.sub.2CH.sub.3, --C(O)--(CH.sub.2).sub.2--COOH,
--C(O)--(CH.sub.2).sub.7--CH.dbd.CH--(CH.sub.2).sub.7--COOH, and
--C(O)--PO.sub.3Na.sub.2.
15. The 21-hydroxy-6,19-oxidoprogesterone derivative of claim 14,
wherein R is C(O)--(CH.sub.2).sub.2--COOH.
16. A composition comprising the 21-hydroxy-6,19-oxidoprogesterone
derivative of claim 14 and a pharmaceutically acceptable
carrier.
17. A method of preparing the composition of claim 16 for the
treatment of at least one disorder associated with a glucocorticoid
imbalance comprising incorporating and mixing the
21-hydroxy-6,19-oxidoprogesterone derivative into the carrier to
form the composition.
18. A method for treating a disease comprising a glucocorticoid
imbalance comprising administering the
21-hydroxy-6,19-oxidoprogesterone derivative of claim 14, in an
amount sufficient to treat the disease, to a patient in need
thereof.
19. The method of claim 18, wherein the glucocorticoid imbalance is
an excess of glucocorticoids.
20. The method of claim 18, wherein the disease is selected from
the group consisting of Cushing's syndrome, iatrogenic
hypercoticolism, and depression.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a novel method of preparing
21-hydroxy-6,19-oxidopro-gesterone (21OH-6OP) and/or its
21-acetate, 21-propionate, 21-hemisuccinate, 21-phosphate and
21-oleate derivatives. 21OH-6OP and its esters are
antiglucocorticoids for the treatment or prophylaxis of diseases
associated with a glucocorticoid imbalance, in particular for
treating Cushing's syndrome or depression.
BACKGROUND OF THE INVENTION
[0002] Corticosteroides are steroid hormones related structurally
to cholesterol. These hormones are synthesized in the adrenal
cortex and include the glucocorticoids (e.g. cortisol), the
mineralocorticoids (e.g aldosterone) as well as weak androgens and
estrogens. The adrenal function, like that of the thyroid gland, is
under the control of the hypothalamus (HPT) and the pituitary
(PIT). When cortisol (the naturally-occuring glucocorticoid) levels
drop below a setpoint, the hypothalamus releases CRH (corticotropin
releasing hormone), which stimulates adrenocorticotropic hormone
(ACTH) release from the pituitary. ACTH is a tropic hormone which
stimulates
[0003] the synthesis and secretion of cortisol (it has minimal
effects on aldosterone synthesis/secretion), and
[0004] the growth of the adrenal gland. When cortisol levels
increase, this shuts off CRH and ACTH secretion (cf. FIG. 1).
[0005] Cortisol is characterized by its properties related to the
biosynthesis and metabolism of glucose and propeties related to
non-specific as well as specific immunity. Due to their effects on
the glucose metabolism, cortisol and natural or synthetic analogues
thereof are usually named glucocorticoids. They bind to the
glucocorticoid receptor (GR).
[0006] The glucocorticoid receptor is a member of a protein super
family of closely related intracellular receptors which function as
ligand-activated transcription factors. Other members of this super
family are the mineralocorticoid receptor (MR) and the progesterone
receptor (PR). MR and GR have shown to be highly homologous, thus
natural and even synthetic steroids exhibit cross-reaction between
these receptors. With respect to PR, its natural ligand
progesterone also cross-reacts with MR and GR.
[0007] Cushing's syndrome is a disorder resulting from increased
adrenocortical secretion of cortisol. Hyperfunction of the adrenal
cortex may be ACTH-dependent or it may be independent of ACTH
regulation, e.g. production of cortisol by an adrenocortical
adenoma or carcinoma. The administration of supraphysiologic
quantities of exogenous cortisol or related synthetic analogs
suppresses adrenocortical function and mimics ACTH-independent
glucocorticoid hyperfunction. ACTH-dependent hyperfunction of the
adrenal cortex may be due to hypersecretion of ACTH by the
pituitary, secretion of ACTH by a nonpituitary tumor such as small
cell carcinoma of the lung (the ectopic ACTH syndrome), or
administration of exogenous ACTH. While the term "Cushing's
syndrome" has been applied to the clinical picture resulting from
cortisol excess regardless of the cause, hyperfunction of the
adrenal cortex resulting from pituitary ACTH excess has frequently
been referred to as Cushing's disease, implying a particular
physiologic abnormality. Patients with Cushing's disease may have a
basophilic adenoma of the pituitary or a chromophobe adenoma.
Microadenomas can usually be visualized by CT or, preferably, MRI
scan, using a high-resolution technique augmented by gadolinium.
Some micro-adenomas are difficult to visualize even with these
modalities. In some cases, no histological abnormality is found in
the pituitary despite clear evidence of ACTH overproduction.
[0008] Reference to Cushing's syndrome is herein intended to mean
the clinical picture resulting from cortisol excess regardless of
the cause, which may be also iatrogenic, both by the injection of
ACTH or by the direct administration of cortisol or synthetic
analogs such as prednisone, prednisolone, dexamethasone or others
that are widely used in various types of diseases including
alergic, asthmatic, inflammatory or immunologic. Cushing's syndrome
includes in addition adrenal tumours secreting corticoids, ectopic
ACTH production and Cushing's disease.
[0009] Clinical manifestations include rounded "moon" faces with a
plethoric appearance. There is truncal obesity with prominent
supraclavicular and dorsal cervical fat pads ("buffalo hump"); the
distal extremities and fingers are usually quite slender. Muscle
wasting and weakness are present. The skin is thin and atrophic,
with poor wound healing and easy bruising. Purple striae may appear
on the abdomen. Hypertension, renal calculi, osteo-porosis, glucose
intolerance, reduced resistance to infection, and psychiatric
disturbances are common. Cessation of linear growth is
characteristic in children. Females usually have menstrual
irregularities. An increased production of androgens, in addition
to cortisol, may lead to hypertichosis, temporal balding, and other
signs of virilism in the female.
[0010] Although development of antihormonal agents related to the
estrogen and androgen receptors has been successful, the search for
selective anti-corticoids is more restricted.
[0011] Known agents suppressing the synthesis of steroid hormones
at various levels (i.e.inhibitors of enzymes which catalyze various
stages of the synthesis of steroid hormones) are reviewed in
J.Steroid Biochem., vol. 5, p. 501(1974) and include the
following:
[0012] a) derivatives of diphenylmethane, e.g. amphenon B (which
suppresses the synthesis of steroid hormones at stages 11-beta-,
17- and 21- of hydroxylase);
[0013] b) derivatives of pyridine (SU-c series), e.g. metirapon
(which suppresses synthesis at stage 11-beta of hydroxylase);
[0014] c) substituted alpha, alpha-glutaramides, e.g.
aminoglutetimide (which impedes the synthesis of pregnenolone from
cholesterol through suppression of 20-alpha-hydroxylase and
C.sub.20, C.sub.22-liase;
[0015] d) steroid substances e.g. trilostan(3 beta-substituted
steroid-3 beta-hydroxy-5-androsten-17-one), which suppresses 3 beta
-desoxysteroidhydrogenase-5.4-isomerase (Steroids, vol. 32, p.
257).
[0016] e) steroids of the spironolactone family which are used as
rapidly dissociating anti-Mineralocorticoids (PNAS USA 71(4) p.
1431-1435 (1974).
[0017] f) a synthetic steroid described as an
anti-Mineralocorticoids, ZK91587, showing specific binding
properties for the kidney (Z.Naturforsch., 45b, p. 711-715 (1990))
and hippocampus type I MR (Life Science, 59, p. 511-21 (1996)), but
not for type II GR. It may therefore be conveniently useful as a
tool in the investigation of MR function in tissues containing both
receptor systems.
[0018] Agents that specifically suppress the interaction of
glucocorticoid hormones with hormone receptors are:
[0019] a) Mifepriston
(11.beta.,17.beta.)-11-[4-(Dimethylamino)phenyl]-17--
hydroxy-17-(1-propynyl)estra-4,9-dien-3-one, which acts on
receptors of glucocorticoid hormones to form a complex incapable of
initiating mechanisms leading to glucocorticoid effect (Annals of
New-York Academy of Science, vol. 761, p. 5-28 (1995)).
[0020] b) non-steroid substances (J:Steroid Biochem., vol. 31, p.
481-492 (1988)) e.g. drotaverina hydrochloride (a derivative of
isoquinoline-1-(3.4dietoxibene
zilidene)-6.7-dietoxy-1,2,3,4-tetrahydrizo- quinoline) or
acetylsalicic acid (Moskovskaya Meditsina, 1990, "Receptor
mechanisms of the glucocorticoid effect" by V. P. Golikov).
[0021] To-date, the only therapeutical application for
antiglucocorticoids (e.g. Mifepristone) that has been attempted in
a clinical setting is to treat inoperable cases of nonpituitary
Cushing's syndrome. In the case of Mifepristone (both an
anti-progesterone and an anti-glucocorticoid), high doses (up to
800 mg per day) are required.
[0022] Employing a systematic application of strategies to increase
activity and decrease cross-reactivity and undesirable side
effects, progress has been reported in the development of
antihormonal agents with greater potency and selectivity,
especially in the antiestrogen and antiandrogen fields.
[0023] In EP-903'146 the synthetic steroid,
21-hydroxy-6,19-oxidoprogester- one (21OH-6OP) of formula I, 2
[0024] is disclosed as a selective antiglucocorticoid which does
not substantially cross-react with uterus-PR or kidney-MR. Said
21-hydroxy-6,19-oxidoprogesterone antiglucocorticoid could be used
in the treatment of diseases associated with an excess of
glucocorticoids, where an anti-glucocorticoid virtually lacking
mineralocorticoid or glucocorticoid properties as well as affinity
for MR or PR is desirable.
[0025] The synthesis of 21-hydroxy-6,19-oxidoprogesterone (1) and
its 21-acetate (2) was first accomplished by Deghenghi in 1966 as
intermediate in a synthesis of 19-hydroxy-desoxy-corticosterone,
starting from 21-hydroxypregnenolone diacetate. The procedure
summarized in Scheme 1 involves the use of a "hypoiodite type"
reaction with lead tetraacetate (Pb(AcO).sub.4). Compound 1 was
neither isolated nor characterized, but acetylated in situ to
acetate 2. Overall yield of partially purified 2 was only 8.3%.
Further to the low yield, this method of preparation is generally
perceived as being difficult to reproduce notably due to the
formation of the chlorohydrin. 3
[0026] In a more recent synthesis of 19-hydroxydeoxycorticosterone,
Kirk and Yeoh (J. Chem. Soc. Perkin Trans. I, 2945 (1983)) prepared
acetate 2 as an intermediate. This procedure starting from
pregnenolone acetate is depicted in Scheme 2. Although full details
of the first 3 steps are not given in the experimental section of
their publication, according to the literature cited the yield for
these steps may be estimated as ca. 34-37% giving an overall yield
of acetate 2 of ca. 12% from pregnenolone acetate. 4
[0027] According to a further method, depicted in Scheme 3, the
remote functionalization reaction with Pb(AcO).sub.4 and iodine
under thermal or photochemical conditions is replaced with the more
reproducible and "milder" HgO/iodine system under photochemical
conditions and the 21-hydroxylation step is carried out with a
hypervalent iodine compound (see A. S. Veleiro, M. V. Nevado, M. C.
Montesern a and G. Burton, Steroids, 60, 268-272 (1995); M. Akhtar
and D. H. R. Barton, J. Am. Chem. Soc., 86, 1528-1534 (1964); R. M.
Moriarty, L. S. John and P. C. Du, J. Chem. Soc. Chem. Commun.,
641-642 (1981). 5
[0028] The above procedure starts also from pregnenolone acetate
which is first converted into 21-hydroxypregnenolone diacetate. In
the best case, overall yield of 1 is ca. 26%; however this product,
although apparently pure according to TLC and NMR, could not be
crystallized, and thus required additional purification steps by
chromatography; yield of pure crystalline 1 is about 13% (19% from
21-hydroxypregnenolone diacetate). Although the acetate 2 is an
intermediate in this synthetic route, complete purification could
only be achieved after deacetylation (i.e. on compound 1).
Furthermore, although this procedure allows the synthesis of small
amounts of 1 for the initial biological tests carried out in 1996,
attempts to scale up the procedure failed. Although, the currently
known methods provide 21-hydroxy-6,19-oxidoprogesterone (1), they
consistently give poorer yields as well as byproducts which are
difficult to eliminate.
DESCRIPTION OF THE INVENTION
[0029] It is an object of the invention to provide a new method for
preparing 21-hydroxy-6,19-oxidopro-gesterone (21OH-6OP) and/or its
esters.
[0030] It is a further object of the invention to provide
21-hydroxy-6,19-oxidoprogesterone and/or its esters.
[0031] In a first aspect, the present invention provides a new
method of preparing 21-hydroxy-6,19-oxidoprogesterone (1) or its
esters, for example carboxylate, phosphate or sulfate esters. In
preferred embodiments, the invention provides a new method for
preparing 21OH-6OP and its 21-acetate, 21-propionate,
21-hemisuccinate, 21-phosphate and 21-oleate derivatives. The
method of the invention avoids essentially all of the hitherto
known inconveniences. The method of the invention produces
21-hydroxy-6,19-oxidoprogesterone (1) in good yield, having an
acceptable degree of purity, and avoids heavy metals as reagent.
The method of the invention is suitable for large scale industrial
preparation of 21-hydroxy-6,19-oxido-progesterone (1) and its
esters.
[0032] The objects of the invention are met according to the main
claim. Preferred embodiments are set out within the dependent
claims which are incorporated herewith.
[0033] The novel synthetic procedure according to the present
invention comprises or consists of the following consecutive basic
steps:
[0034] a) Providing 21-acetoxypregnenolone (3) (which is actually a
commercial product);
[0035] b) Transforming the C-3 hydroxy group of
21-acetoxypregnenolone into a labile ester, preferably a formate
ester,
[0036] c) Obtaining the bromohydrin product from the protected 21
-acetoxypregnenolone, said bromohydrin resulting from the addition
of a bromine and a hydroxy group onto the double bond of position
of C5-C6;
[0037] d) Performing an intramolecular cyclisation with the C19
atom with the "Suarez-reagent" (diacetoxyiodobenzene; see Armas et
al., J.Chem.Soc. Perkin I, 405 (1989)) and iodine under
irradiation, thus obtaining the 6,19 oxido-bridge within the
scaffold;
[0038] e) Performing a selective hydrolysis, preferably with
HCl/MeOH/dichloromethane, followed by an oxidation, preferably with
pyridinium chlorochromate (PCC), thus obtaining a bromoketone;
[0039] f) Performing a hydrolysis of the bromoketone resulting from
step e) to obtain 21-hydroxy-6,19-oxidoprogesterone (1), and
optionally
[0040] g) Acylating 21-hydroxy-6,19-oxidoprogesterone (1), to
afford the 21-ester (the acetate is shown as 2);
[0041] Preferred esters are C1-18 acyl esters (optionally
substituted with COOH, 1 or 2 times) and phosphate esters. Acyl
esters may be obtained by reacting
21-hydroxy-6,19-oxidoprogesterone (1) with an organic acid, in the
presence of a coupling agent (for example
N,N'-dicyclohexylcarbodiimi- de), or with an activated organic
ester (for example, a nitrophenol ester), or with an acyl halide
(for example, an acyl chloride), or with an acyl anhydride.
Phosphate esters may be obtained by reacting
21-hydroxy-6,19-oxidoprogesterone with a phosphorylating agent (for
example phosphorus oxychloride, followed by basic hydrolysis).
[0042] The 21-propionate or 21-hemisuccinate, and 21-oleate and
derivatives are obtained by esterifying
21-hydroxy-6,19-oxidoprogesterone (1) of step f) with propionic
acid, succinic or oleic acid, their anhydrides, activated esters or
acyl chlorides.
[0043] A preferred synthetic procedure according to the present
invention is depicted in Scheme 4. 6
[0044] According to the preferred method illustrated in Scheme 4, a
formate group is introduced as protecting group for position 3.
Formates have the advantage that they may be hydrolized under
relatively mild acid conditions (HCl/MeOH-dichloromethane) in which
primary acetates and even .alpha.-acetoxy-ketones are stable. The
introduction of a formate moiety is preferably carried out under
very mild conditions using preferably mixed acetic-formic anhydride
(prepared in situ from formic acid and acetic anhydride). The
bromo-hydrin formation, is carried out with N-bromnoacetamide in
THF yielding 80% of the desired bromohydrin 5 and about 20% of the
5.alpha.-HO-6.beta.-Br isomer. The mixture may be separated by
crystallization or Thin Layer Chromatography. For the
intramolecular cyclisation involving C-.sub.19, the "Suarez
reagent" (diacet-oxyiodobenzene (DAIB)) and iodine is employed as
the "hypoiodite" as generating system under suitable
irradiation--preferably with a standard tungsten lamp.
[0045] Quite surprisingly, it turns out that upon performing the
intramolecular cyclisation step in di-chloromethane
(CH.sub.2Cl.sub.2), the reaction is not only rapid (complete
conversion in less than 1 hour) and clean, but also excellent
yields could be obtained.
[0046] Temperature control (25.degree. C.) of the cyclisation step,
achieved by using a glass jacketed reactor with water circulation,
increases the yield and diminishes excessive oxidation by-products.
Furthermore, epoxide formation is eliminated, the isomeric
bromohydrin is inert to the reagent and could be easily separated
(together with the iodobenzene side product) from the desired
bromoether 6 by vacuum filtration through a silica gel column
(VFC).
[0047] The selective hydrolysis of the formate ester, is preferably
performed with HCl in MeOH-dichloromethane, followed by an
oxidation, preferably with PCC, thus affording the bromoketone 8.
This product partially eliminates HBr to give the unsaturated
ketone (2) when subjected to VFC (very short silica gel column)
purification, however this poses no problem as the unsaturated
product is the desired product of the following step, in which the
treatment with a base deacetylates position 21 and completes the
elimination of the C-5 bromine thus giving the desired pro-duct
(1).
[0048] Purification of 1 may be achieved with a fast VFC through a
short silica gel column to afford chromatographically pure product
in over 31% yield. This may be crystallized from absolute ethanol
to yield crystalline 1 in 27% yield.
[0049] The 21-acetate (2) is prepared by standard acetylation of 1
with acetic anhydride in pyri-dine. The acetate is recrystallized
from absolute ethanol.
[0050] The novel synthesis according to the present invention has
the following advantages:
[0051] a) It eliminates the need of the selective acetylation step
or any need to differentiate the hydroxyl groups at C-21 and
C-3.
[0052] b) It modifies the hypoiodite reaction so that no heavy
metals are required (lead, silver or mercury based reagents) and it
proceeds under homogeneous conditions, thus allowing a scale up to
multigram and eventually kilogram amounts.
[0053] c) It reduces the secondary products formed at different
stages (specially 5,6-epoxide formation) so as to minimize the
purification steps, and specially to avoid or minimize the need of
column chromatography on silica gel, as bromoethers, compound 1 and
2 are intrinsically unstable under these conditions.
[0054] d) It reduces reaction volumes throughout to allow scale
up.
[0055] Starting from compound 1, synthesis of acyl esters, such as
21-propionate (2a), and 21-hemisuccinate (2b) and 21-oleate (2c)
-6,19-oxidoprogesterone derivatives can be performed as illustrated
in Scheme 5.
[0056] Also shown in Scheme 5 is the preparation of the
21-phosphate derivative (2d). 78
[0057] The compounds synthesised according to the present invention
are for use as a medicament, alone or in combination with
pharmaceutically acceptable carriers and/or excipients. Such
medicament is notably suitable in the manufacture of a medicament
for the treatment or prophylaxis of diseases associated with an
excess of glucocorticoids, e.g. for the treatment of Cushing's
syndrome, iatrogenic hypercortisolism or depression.
[0058] The invention will now be described, by way of illustration
only, with reference to the following examples:
EXAMPLES
MATERIALS AND METHODS
[0059] Melting points were taken on a Fisher-Johns apparatus and
are uncorrected. IR spectra were recorded in thin films using KBr
disks on a Nicolet Magna IR 550 FT-IR spectrometer. .sup.1H and
.sup.13C NMR spectra were measured in Bruker AC-200 or AM-500 NMR
spectrometers in deu-teriochloroform (using TMS as internal
standard). The J values are given in Hz. Spectra were assigned by
analysis of the DEPT, COSY 45 and HETCOSY spectra and by comparison
with those of progesterone.
[0060] The electron impact mass spectra (EI) were measured in a VG
Trio 2 mass spectrometer at 70 eV by direct inlet. FAB mass spectra
and electron impact high resolution mass spectra (HRMS) were
obtained in a VG ZAB BEQQ mass spectrometer. All solvents used were
reagent grade. Solvents were evaporated at ca. 45.degree. C. under
vacuum. Zinc dust was activated by suspending in 1M HCl, washing
with water, absolute ethanol and diethyl ether and drying 2 h at
120.degree. C. The homogeneity of all compounds was confirmed by
thin layer chromatography.
Large Scale Synthesis of 21-hydroxy-6,19-oxidoprogesterone (1)
[0061] Structures of starting material, intermediates and final
products (1) and (2): 910
3.beta.-Formyloxy-21-acetyloxy-5-pregnen-20-one (4)
[0062] Acetic anhydride (13.4 ml) is added dropwise to formic acid
(6.6 ml) at 0.degree. C., the solution is heated at 50.degree. C.
for 15 min and cooled rapidly to 0.degree. C. The resulting
acetoformic anhydride solution is added dropwise to a stirred
suspension of 21-acetoxypregnenolone (3, 8.0 g) in dry pyridine
(20.8 ml) at 0.degree. C., and stirring is continued at that
temperature for 2 h. The reaction product is poured over cold
saturated aqueous sodium bicarbonate solution, filtered and the
solid is washed with saturated aqueous sodium bicarbonate solution,
water and 1N HCl and water (until neutral) rendering formate 4 (8.0
g); .sup.1H NMR (200.13 MHz) .delta..sub.H 0.70 (3H, s,
13-CH.sub.3), 1.02 (3H, s, 10-CH.sub.3), 2.16 (3H, s,
21-CH.sub.3CO), 2.53 (1H, t, J=8.0 Hz, 17-H), 4.50 (1H, d, J=17.0
Hz, 21a-H), 4.70 (1H, d, J=17.0 Hz, 21b-H), 5.32 (1H, m, 3-H), 5.38
(1H, d, J=3.0 Hz, 6-H), 8.02 (1H, s, HCOO).
3.beta.-Formyloxy-5.alpha.-bromo-6.beta.-hydroxy-21-acetyloxypregnan-20-on-
e (5)
[0063] Formate 4 (8.0 g), is dissolved in diethyl ether (100 ml)
and THF (37.2 ml) and cooled to 10.degree. C. To the stirred
solution at 10-15.degree. C.--which protected from light--7.5%
perchloric acid (11.88 ml) is added, followed by N-bromoacetamide
(4.75 g) in 8 portions over a 25 min period. Stirring is continued
for 45 min at 25.degree. C. and the reaction is stopped by addition
of 10% aqueous sodium thiosulfate solution until complete
decoloration. The reaction mixture is then extracted with
dichloromethane/methanol 10:1 and the organic layer, is washed with
water, dried with anhydrous sodium sulfate and the solvent is
evaporated to afford bromo-hydrin 5 (10.4 g, containing about 20%
of the 5.alpha.-hzydroxy-6.beta.-bromo isomer as determined by
.sup.1H NMR).
3.beta.-Formyloxy-5.alpha.-bromo-21-acetyloxy-6,19-oxidopregnan-20-one
(6)
[0064] Nitrogen is bubbled for 5 min through a solution of
bromohydrin compound 5 (10.4 g, containing about 20% of the
5.alpha.-hydroxy-6.beta.-- bromo isomer) in freshely distilled
dichloro-methane (723 ml) contained in a 1 liter glass vessel
fitted with an external cooling jacket with circulating water at
25.degree. C. and magnetic stirrer.
[0065] Diacetoxyiodobenzene (Suarez reagent, 7.66 g) and iodine
(5.46 g) are successively added with stirring. The vessel is
exposed to two 300 Watt tungsten lamps (5000 lm each) and vigorous
stirring is continued for 1 h at 25.degree. C. Irradiation is
turned off and a saturated aqueous solution of sodium thiosulfate
is added until complete decoloration. The organic layer is
separated, dried with anhydrous sodium sulfate and the solvent
evaporated. The resulting solid is dissolved in dichloro-methane (8
ml) and applied to a silicagel G-60 column (12 cm diameter.times.8
cm height) previously flushed with hexane; successive elution
(applying vacuum to the outlet) with hexane-ethyl acetate 9:1 (1100
ml), 8:2 (700 ml), 7:3 (700 ml) and 6:4 (600 ml) affords
31.times.100 ml fractions. Fractions are analyzed by TLC and those
containing bromoether 6 are pooled and evaporated to dryness to
afford 6 (6.8 g). .sup.1H NMR (200.13 MHz) .delta..sub.H 0.70 (3H,
s, 13-CH.sub.3), 2.16 (3H, s, 21-CH.sub.3CO), 2.52 (1H, t, J=8.8
Hz, 17-H), 3.73 (1H, d, J=8.4 Hz, 19a-H), 3.94 (1H, d, J=8.4 Hz,
19b-H), 4.08 (1H, d, J=4.2 Hz, 6-H), 4.50 (1H, d, J=16.8 Hz,
21a-H), 4.71 (1H, d, J=16.8 Hz, 21b-H), 5.34 (1H, m, 3-H), 8.02
(1H, s, HCOO).
3.beta.-Hydroxy-5.alpha.-bromo-21-acetyloxy-6,19-oxidopregnan-20-one
(7)
[0066] A stirred solution of the bromoether 6 (6.8 g) obtained
above, is dissolved in dichloro-methane (45.7 ml) and methanol
(154.7 ml) and is cooled to 0.degree. C. in an ice bath and water
(10.9 ml) while conc. HCl (23.0 ml) is added. After about 30 min of
vigorous stirring at 0.degree. C. (disappearance of the starting
material is monitored by TLC) the reaction mixture is neutralized
with 20% aqueous sodium hydroxide and extracted with
dichloromethane. The organic layer is dried with anhydrous sodium
sulfate and the solvent evaporated to afford the alcohol compound 7
(6.5 g); .sup.1H NMR (200.13 MHz) .delta..sub.H 0.69 (3H, s,
13-CH.sub.3), 2.16 (3H, s, 21-CH.sub.3CO), 2.52 (1H, t, J=8.5 Hz,
17-H), 3.62 (1H, d, J=8.5 Hz, 19a-H), 3.92 (1H, d, J=8.5 Hz,
19b-H), 4.07 (1H, d, J=4.0 Hz, 6-H), 4.15 (1H, m, 3-H), 4.51 (1H,
d, J=17.0 Hz, 21a-H), 4.70 (1H, d, J=17.0 Hz, 21b-H).
5.alpha.-Bromo-21-acetyloxy-6,19-oxidopregnane-3,20-dione (8)
[0067] A suspension of pyridinium chlorochromate (12.1 g), barium
carbonate (5.0 g) and 3 .ANG. molecular sieves (9.60 g), in dry
dichloromethane (480 ml) is stirred under nitrogen for 10 min. To
the resultant orange slurry a solution of bromoether 7 (6.5 g)
obtained above in dry dichloromethane (324 ml) is added and
stirring is continued for about 90 min, until the starting material
(TLC) has disappeared. The reaction mixture is percolated through
a-short silicagel G 60 column (12 cm diameter.times.8 cm height)
washed with diethyl ether (2.times.150 ml) and hexane-ethyl acetate
1:2 (3.times.150 ml). Fractions containing the product are pooled
and evaporated to dryness affording 5.5 g of ketone 8 (containing
about 10% of .DELTA..sup.4-3-ketone (2); .sup.1H NMR (200.13 MHz)
.delta..sub.H 0.70 (3H, s, 13-CH.sub.3), 2.16 (3H, s,
21-CH.sub.3CO), 2.51 (1H, t, J=8.5 Hz, 17-H), 2.85 (1H, d, J=16.0
Hz, 4a-H), 3.40 (1H, d, J=16.0 Hz, 4b-H), 3.90 (1H, d, J=9.0 Hz,
19a-H), 4.07 (1H, d, J=4.0 Hz, 6-H), 4.15 (1H, d, J=9.0 Hz, 19b-H),
4.50 (1H, d, J=17.0 Hz, 21a-H), 4.71 (1H, d, J=17.0 Hz, 21b-H).
21-Hydroxy-6,19-oxido-4-pregnene-3,20-dione (1)
[0068] The ketone 8 (5.5 g) obtained from the preceding step is
dissolved in methanol (263.8 ml) and dichloromethane (13.2 ml). To
the stirred solution 14% methanolic KOH (53.4 ml) is added and
stirring is continued at room temperature for about 15 min, until
the starting material (TLC) has disappeared. The reaction mixture
is neutralized with 1N HCl and extracted with dichloromethane. The
organic layer is dried with anhydrous sodium sulfate and the
solvent is evaporated thus yielding crude
21-hydroxy-6,19-oxidoprogesterone (1, 4.6 g). The solid is
dissolved in dichloromethane (5 ml) and sent through to a silicagel
G-60 column (8.5 cm diameter.times.5 cm height) previously flushed
with hexane-ethyl acetate 7:3; successive elution (applying vacuum
to the outlet) with hexane-ethyl acetate 6:4 (1350 ml) and 1:1 (900
ml) affords 30 fractions. The fractions are analyzed by TLC and
those con-taining 1 are pooled and evaporated to dryness to afford
21-hydroxy-6,19-oxidoproges-terone (1, 2.3 g). .sup.1H NMR (200.13
MHz) .delta..sub.H 0.74 (3H, s, 13-CH.sub.3), 2.45 (1H, t, J=8.5
Hz, 17-H), 3.51 (1H, d, J=8.8 Hz, 19a-H), 4.18 (3H, s,
21-CH.sub.3), 4.20 (1H, d, J=8.8 Hz, 19b-H), 4.69 (1H, d, J=5.0 Hz,
6-H), 5.82 (1H, s, 4H).
[0069] Re-crystallization from absolute ethanol affords a first
crop of crystalline 1 (1.27 g), mp 165-166.degree. C. The mother
liquor is concentrated to yield a second crop of 1 (0.68 g).
21-Acetyloxy-6,19-oxido-4-pregnene-3,20-dione (2)
[0070] Crude 21-hydroxy-6,19-oxidoprogesterone (1, 2 g before
chromatographic purification) was dissolved in dry pyridine (15.6
ml) and acetic anhydride (15.6 ml) added. The solution was stirred
for 90 min at 25.degree. C., poured over 1M HCl and filtered
(alternatively the solid may be extracted with dichloromethane).
The solid was washed with water (until neutral), dried, dissolved
in dichlorometane (2 ml) and applied to a silicagel G-60 column (7
cm diameter.times.5 cm height) previously flushed with hexane-ethyl
acetate 7:3; succesive elution (applying vacuum to the outlet) with
hexane-ethyl acetate 7:3 (700 ml) and 6:4 (700 ml) afforded 20
fractions. Fractions were analyzed by TLC and those containing 2
pooled and evaporated to dryness to afford
21-acetyloxy-6,19-oxidoprogesterone (2, 1.12 g). Recrystallization
from absolute ethanol (with drops of methanol) afforded crystaline
2 (0.72 g), mp 190-191.degree. C. .sup.1H NMR (200.13 MHz)
.delta..sub.H 0.76 (3H, s, 13-CH.sub.3), 2.17 (3H, s,
21-CH.sub.3CO), 2.51 (1H, t, J=8.5 Hz, 17-H), 3.51 (1H, d, J=8.2
Hz, 19a-H), 4.20 (1H, d, J=8.2 Hz, 19b-H), 4.50 (1H, d, J=16.7 Hz,
21a-H), 4.69 (1H, d, J=5.0 Hz, 6-H), 4.72 (1H, d, J=16.7 Hz,
21b-H), 5.82 (1H, s,4-H).
21-Propanoyloxy-6,19-oxido-4-pregnene-3,20dione (2a)
[0071] Crude 21-hydroxy-6,19-oxidoprogesterone (1, 0.2 g before
chromatographic purification) was dissolved in dry pyridine (0.28
ml) and propanoic anhydride (0.2 ml) added. The solution was
stirred for 1 h at 25.degree. C., methanol was added to destroy
excess anhydride and the solution concentrated in vacuo. The
residue was diluted with dichloromethane, washed with 1M HCl and
water, and evaporated to dryness (0.233 g). Purification as above
afforded 21-propanoyloxy-6,19-oxidoproge- sterone (2a, 0.116 g).
.sup.1H NMR (200.13 MHz) .delta..sub.H 0.76 (3H, s, 13-CH.sub.3),
1.18 (3H, t, J=7.6 Hz, 21-CH.sub.3CH.sub.2CO), 2.46 (2H, q, J=7.6
Hz, 21-CH.sub.3CH.sub.2CO), 2.51 (1H, t, J=8.0 Hz, 17-H), 3.51 (1H,
d, J=8.2 Hz, 19a-H), 4.20 (1H, d, J=8.2 Hz, 19b-H), 4.50 (1H, d,
J=16.8 Hz, 21a-H), 4.70 (1H, d, J=5.0 Hz, 6-H), 4.73 (1H, d, J=16.8
Hz, 21b-H), 5.82 (1H, s, 4H). EIMS m/z 400 (17) [M].sup.+, 342 (5),
313 (24), 285 (23), 267 (10), 57 (100).
21-Succinoyloxy-6,19-oxido-4-pregnene-3,20dione (2b)
[0072] 21-hydroxy-6,19-oxidoprogesterone (1, 0.75 g) was dissolved
in dry dichloromethane (37.5 ml) and pyridine (1.9 ml) and succinic
anhydride (0.75 g) added. The solution was stirred for 4 h at
25.degree. C., a second portion of succinic anhydride (0.375 g)
added and stirring continued for 6 h (until disappearance of
starting material). The reaction mixture was concentrated, and the
residue extracted with diethyl ether. The ethereal solution was
washed with 1M HCl and extracted with aqueous 10% sodium carbonate.
The aqueous layer was acidified with conc. HCl to pH=3 and
extracted with dichloromethane. Washing with water and evaporation
to dryness afforded crude 21-succinoyloxy-6,19-oxidoprogester- one
(2b, 0.823 g). The solid was dissolved in dichloromethane (1 ml)
and applied to a silicagel G-60 column (6 cm diameter.times.4 cm
height) previously flushed with hexane-ethyl acetate 2:8; succesive
elution (applying vacuum to the outlet) with hexane-ethyl acetate
2:8 (200 ml) and ethyl acetate (500 ml) afforded 14 fractions.
Fractions were analyzed by TLC and those containing 2b pooled and
evaporated to dryness to afford
21-succinoyloxy-6,19-oxidoprogesterone (2b, 0.455 g). .sup.1H NMR
(200.13 MHz) .delta..sub.H 0.75 (3H, s, 13-CH.sub.3), 2.52 (1H, t,
J=8.0 Hz, 17-H), 2.75 (4H, m, 21-HCOOCH.sub.2CH.sub.2CO), 3.51 (1H,
d, J=8.2 Hz, 19a-H), 4.20 (1H, d, J=8.2 Hz, 19b-H), 4.54 (1H, d,
J=16.9 Hz, 21a-H), 4.70 (1H, d, J=5.0 Hz, 6-H), 4.75 (1H, d, J=16.9
Hz, 21b-H), 5.82 (1H, s, 4-H). EIMS m/z 444 (3) [M].sup.+, 344
(24), 313 (55) 285 (44), 267 (17), 91 (60), 79 (54), 55 (100).
21-Oleoyloxy-6,19-oxido-4-pregnene-3,20dione (2c)
[0073] 21-hydroxy-6,19-oxidoprogesterone (1, 0.052 g) was dissolved
in dry dichloromethane (1 ml) and pyridine (0.12 ml) and oleoyl
chloride (0.1 ml) added. The solution was stirred for 24 h at
25.degree. C., diluted with dichloromethane, washed with 1M HCl and
water, and evaporated to dryness. Purification by preparative TLC
afforded 21-oleoyloxy-6,19-oxido- progesterone (2c, 0.075 g).
.sup.1H NMR (200.13 MHz) .delta..sub.H 0.76 (3H, s, 13-CH.sub.3),
0.88 (3H, t, J=7.0 Hz, CH.sub.3--CH--CO), 2.34 (2H, t, J=7.7 Hz,
CH--CH.sub.2CO), 2.50 (1H, t, J=8.5 Hz, 17-H), 3.51 (1H, d, J=8.3
Hz, 19a-H), 4.20 (1H, d, J=8.3 Hz, 19b-H), 4.49 (1H, d, J=16.9 Hz,
21a-H), 4.70 (1H, d, J=4.5 Hz, 6-H), 4.73 (1H, d, J=16.9 Hz,
21b-H), 5.34 (2H, m, CH--CH.dbd.CH--CH--CO), 5.82 (1H, s, 4-H).
21-Phosphate-6,19-oxido-4-pregnene-3,20dione (2d)
[0074] 21-hydroxy-6,19-oxido-4-pregnene-3,20dione (1, 0.052 g) was
dissolved in dry dichloromethane (1 ml) and pyridine (0.12 ml), and
phosphochloridic acid diallyl ester (0.024 g, 1 eq.) was added
dropwise at 0.degree. C., over 30 minutes. The mixture was allowed
to warm to room temperture and stirred overnight. The mixture was
washed three times with 5% NaHCO.sub.3, the organic layer was dried
and evaporated in vacuo without heating. To the resulting syrup was
added a solution of NaOH (2.2 equivalents) in water (3 ml). The
solution was brought slowly to reflux, and gently refluxed for 12
hours. Lyophilization yielded the crude phosphate disodium salt,
which was recrystallized from ethanol. Alternatively, the crude
disodium salt may be dissolved in water, and the pH adjusted with
HCl, to cause precipitation of the free phosphate.
* * * * *