Note: Descriptions are shown in the official language in which they were submitted.
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Microbiological Processes for the Production of 7 alpha-Substituted 11 alpha-
Hydroxy Steroids
Description:
The invention relates to microbiological processes for the production of 7a-
substituted 11 a-hydroxy steroids, 7a,17a-substituted 11 P -halogen steroids
that can be
produced therefrom, production processes for the latter compounds as well as
their use
and pharmaceutical preparations that contain these compounds. In addition, the
invention
relates to other 7a-substituted I 1 f3-halogen steroids, namely 7a-substituted
estra-
1,3,5(10)-trienes that can be obtained from the 7a-substituted l la-hydroxy
steroids.
For treatment of male menopause and for development of male sexual organs as
well as for male birth control, androgens, especially testosterone, are used.
In addition,
these hormones also have partial anabolic active components, which promote,
i.a., muscle
growth.
Male menopause is characterized by an age-related reduction in the endogenous
androgen production, such that hormone replacement is carried out for
treatment thereof
(HRT: hormone replacement therapy).
In addition to a reduction in spermatogenesis, the LH-RH administration for
male
birth control also results in the release of LH and in the dropping of
testosterone levels
and libido, which are compensated for by administering testosterone
pharmaceutical
agents (D. E. Cummings et al., "Prostate-Sparing Effects of the Potent
Androgen 7a-
Methyl-19-Nortestosterone: A Potential Alternative to Testosterone for
Androgen
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Replacement and Male Contraception," Journal of Clinical Endocrinology and
Metabolism, Vol. 83, No. 12, pages 4212-4219 (1998)).
A combination therapy with the administration of androgens and a
gestagenically
active component can be used for control of male fertility (see, for example,
WO
01/60376 A as well as the documents cited therein).
In the case of a treatment with testosterone, it has been shown that side
effects
develop, especially an enlargement of the prostate owing to an increase in the
number of
cells and glands of the stroma (BPH: benign prostate hyperplasia). In the
metabolism of
testosterone that is mediated by 5a-reductase, dihydrotestosterone (DHT) that
can result,
i.a., in the occurrence of BPH is produced (Cummings et al., ibid.; WO
99/13883 Al).
The inhibition of the 5a-reductase is therefore used for treating BPH in
clinical practice
(finasterides).
The quick metabolism of the androgenic steroid testosterone in the human body
further results not only in the formation of undesirable DHT, but also in that
an oral
administration of higher doses is necessary to reach the desired effect level
of
testosterone. Alternative forms for dispensing, such as i.m.-injections or
large patches,
are therefore necessary.
To replace testosterone in the above-mentioned indication areas, 7a-methyl-19-
nortestosterone (MeNT) was proposed which has, on the one hand, a higher
biological
effectiveness as testosterone, since it has a higher binding affinity to the
androgen
receptors. On the other hand, because of a steric inhibition by the 7a-methyl
group, it
presumably resists metabolization by 5a-reductase (Cummings et al., ibid., WO
99/13883
Al, WO 99/13812 Al, US-A-5,342,834).
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During metabolism of testosterone, a smaller portion of this compound is also
reacted by aromatization of ring A of the steroid system to form estradiol,
especially in
the brain, in the liver and in the fatty tissue. With respect to the total
action of the
testosterone and its metabolites, estradiol is substantially responsible for
sex-specific
behavior and gonadotrophin regulation. Therefore, its action just like that of
testosterone
for the adult male is regarded as advantageous (Cummings et al., ibid.).
It has been shown, however, that the pharmacokinetics of testosterone is not
satisfactory. In particular in the case of oral dispensing, testosterone is
quickly excreted
again, so that the effectiveness and the duration of action of the thus
produced
pharmaceutical agents is unsatisfactory. Other testosterone derivatives were
therefore
also synthesized. Such derivatives are described in, i.a., US-A-5,952,319, in
particular
7a-,1 l (3-dimethyl derivatives of 19-nortestosterone, namely 7a, I I [3-
dimethyl-17(3-
hydroxyestr-4-en-3-one, 7a, 11 [3-dimethyl-17(3-heptanoyloxyestr-4-en-3-one,
7a,II(3-
dimethyl-17 I -[[(2-cyclopentylethyl)-carbonyl]-oxy]-estr-4-en-3-one, 7a, I I
[3-dimethyl-
17(3-(phenylacetyloxy]-estr-4-en-3-one, and 7a,11 [3-dimethyl-17(3-[[(trans-4-
[n-
butyl] cyclohexyl)-carbonyl ] -oxy] -estr-4-en-3 -one.
The above-mentioned 7a,11(3-dimethyl derivatives have the above-mentioned
advantages, like MeNT, including an improved pharmacokinetics, i.e., their
effectiveness
and duration of action are improved relative to testosterone. These
derivatives, however,
can be produced only via an expensive synthesis method.
A synthesis of steroids in the microbiological method is described in EP 0 900
283 B 1. It is indicated there that estr-4-ene-3,17-dione and canrenone can be
transformed
with use of a microorganism that is selected from the group that comprises
Aspergillus
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nigricans, Rhizopus arrhizus and strains of Pestelotia into the corresponding
11 a-'
hydroxy analog. In the introduction of the description, however, reference is
also made
to Shibahara et al., Biochim. Biophys. Acta, 202 (1970), 172-179, who reported
that the.
microbiological 11 a-hydroxylation reaction in steroids can be unpredictable.
The problem on which this invention is based is therefore to find derivatives
of
testosterone that are not sensitive to reduction with 5a-reductase, that also
have an
improved pharmacokinetics, and that are especially easy to produce. A very
significant
aspect of this invention consequently consists in finding a process for better
accessibility
of the initial products, with which the initial products are easy to produce.
The problem on which this invention is based is solved by the
microbiologicrall.
processes described herein for the production of 7a-substituted steroids and
7a, 1 7a-
substituted 11(3-halogen steroids. Also provided are pharmaceutical
preparations that
contain these 7a, 1 7a-substituted 11(3-halogen steroids, as well as 7a-
substituted 11 R-
haloestra-1,3,5(10)-trienes, and uses of such compounds.
Definitions:
The definitions below relate to all portions of the description and the claims
as
well as to Diagram I that is attached:
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All groupings, radicals or other structural units can in each case be varied
independently of one another within the indicated areas of meaning.
All alkyl, alkylene, alkenyl, alkenylene, alkinyl, and alkinylene groups can
be
either straight-chain or branched. For example, a propenyl group can be
described by one
of the chemical structures below: -CH=C-CH3, -CH2-C=CH2, or -C(CH3)=CH2. Thus,
for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-
pentyl, i-pentyl,
t-pentyl, neo-pentyl, n-hexyl, 1-methyl-n-pentyl, 2-methyl-n-pentyl, 3-methyl-
n-pentyl,
4-methyl-n-pentyl, 1-ethyl-n-butyl, 2-ethyl-n-butyl, etc., fall under C1- to
Cog-alkyl.
Alicyclic alkyl is either a cycloalkyl or a cycloalkyl that is substituted
with one
alkyl group or several alkyl groups, which is bonded directly via the
cycloalkyl ring or
via one of the alkyl groups.
In the same way, an alicyclic alkenyl is either a cycloalkenyl or a
cycloalkenyl or
cycloalkyl that is substituted with one or more alkenyl groups or with one or
more
alkenyl groups and alkyl groups or with one or more alkyl groups, which is
bonded
directly via the cycloalkenyl ring or via one of the alkenyl groups or
optionally alkyl
groups, whereby at least one double bond is contained in the alicyclic
alkenyl.
On the one hand, aryl can be phenyl, but also 1-naphthyl or 2-naphthyl. In
principle, aryl also includes heteroaryl, especially 2-, 3- and 4-pyridinyl, 2-
and 3- furyl-,
2- and 3-thienyl, 2- and 3-pyrrolyl, 2-, 4- and 5-imidazolyl, pyridazinyl, 2-,
4- and 5-
pyrimidinyl as well as 3- and 4-pyridazinyl.
Halogen is fluorine, chlorine, bromine or iodine.
Pharmaceutically compatible addition salts are salts of the corresponding
compounds with inorganic or organic acids, for example with hydrochloric acid,
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hydrobromic acid, hydroiodic acid, acetic acid, citric acid, oxalic acid,
tartaric acid and
methanesulfonic acid. The esters can be formed in particular with succinic
acid.
Superscript numbers on symbols R, for example R13, designate their position on
the steroid ring skeleton, whereby the C atoms in the steroid ring skeleton
are numbered
according to IUPAC nomenclature. Superscript numbers on symbols C, for example
C10,
designate the position of the respective carbon atom in the steroid ring
skeleton.
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Description of the Invention:
Novel microbiological processes are used for the production of 7a-substituted
I 1 a-hydroxy steroids with general formula 4,B:
R13 O
HOi~~
Rio
O / R7
4,B
in which
R7 is the grouping P-Q, whereby
P represents C1- to C4-alkylene and Q represents a hydrogen,
Cl- to C4-alkyl- or C1- to C4-fluoroalkyl (alkyl that is partially or
completely fluorinated), and grouping P-Q is bonded via P to the
steroid skeleton,
R10 stands for H, CH3 or CF3, and
R13 is methyl or ethyl.
In a first process variant for the production of these substances, a suitable
7a-
substituted steroid with general formula 3,A:
AMENDED
SHEET
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R13 OH
R10
0 R7
3,A
in which R7, R10 and R13 have the same meanings as indicated for the compounds
with
general formula 4,B,
is hydroxylated and oxidized in one process step with use of a microorganism
that is
selected from the group that comprises Aspergillus sp., Beauveria sp.,
Glomerella sp.,
Gnomonia sp., Haplosporella sp. and Rhizopus sp. Especially preferred are
Aspergillus
awamori, Aspergillusfischeri, Aspergillus malignus, Aspergillus niger,
Beauveria
bassiana, Glomerella cingulata, Gnomonia cingulata, Haplosporella hesperedica
and
Rhizopus stolonifer, whereby in particular Aspergillus awamori (CBS),
Aspergillus
fischeri (ATCC 1020), Aspergillus malignus (IMI 16061), Aspergillus niger
(ATCC
9142), Beauveria bassiana (ATCC 7159), Glomerella cingulata (CBS 15226, CBS
23849, CBS 98069, ATCC 56596, ATCC 64682, IFO 6425), Gnomonia cingulata (CBS
15226), Haplosporella hesperedica (CBS 20837) and Rhizopus stolonifer (ATCC
15441)
are used.
As an alternative, this microbiological production process can also be
performed
in two stages, whereby the hydroxylation reaction and the oxidation reaction
take place in
sequential reaction steps. The course of the reaction can be controlled via
the reaction
period: by the reaction being interrupted, for example, after a certain
reaction time, the
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hydroxylated, but still not oxidized, species can be isolated. Both process
steps can
therefore be performed separately or in a mixed fermentation:
To this end, the compound can be hydroxylated in 11-position with general
formula 3,A in a first microbiological process step with use of a first
microorganism,
selected from the group that comprises Aspergillus sp., Beauveria sp.,
Gibberella sp.,
Glomerella sp., Gnomonia sp., Metarrhizium sp., Nigrospora sp., Rhizopus sp.
and
Verticillium sp., whereby a 7a-substituted steroid is formed with a hydroxy
group in 11 a-
position. This compound has general formula C:
R13 OH
HO~,~
R10
O / //// R7
C
in which R7, R10 and R13 have the same meanings as indicated above for the
compounds
with general formula 4,B. Used in particular are Aspergillus malignus,
Aspergillus
melleus, Aspergillus niger, Aspergillus ochraceus, Beauveria bassiana,
Gibberella
fujikuroi, Gibberella zeae, Glomerella cingulata, Glomerellafusaroides,
Gnomonia
cingulata, Metarrhizium anisopliae, Nigrospora sphaerica, Rhizopus oryzae,
Rhizopus
stolonifer and Verticillium dahliae. In this connection, especially
Aspergillus malignus
(IMI 16061), Aspergillus melleus (CBS), Aspergillus niger (ATCC 11394),
Aspergillus
ochraceus (NRRL 405, CBS 13252, ATCC 46504), Beauveria bassiana (ATCC 7159,
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IFO 5838, ATCC 13144, IFO 4848, CBS 11025, CBS 12736), Gibberellafujikuroi
(ATCC 14842), Gibberella zeae (CBS 4474), Glomerella cingulata (ATCC 10534,
CBS
23849, CBS 23749, ATCC 16646, ATCC 16052, IFO 6459, IFO 6425, IFO 6470, CBS
98069, IFO 7478, IFO 5257, ATCC 64682, ATCC 15470), Glomerellafusaroides
(ATCC 9552), Gnomonia cingulata (CBS 15226), Metarrhizium anisopliae (IFO
5940),
Nigrospora sphaerica (ATCC 12772), Rhizopus oryzae (ATCC 4858, ATCC 34102,
ATCC 34102), Rhizopus stolonifer (ATCC 6227b, ATCC 15441) and Verticillium
dahliae (ATCC 11405) are used for the hydroxylation.
Intermediate product C is then oxidized in a second microbiological process
step
with use of a second microorganism that is selected from the group that
comprises
Bacillus sp., Mycobacterium sp., Nocardia sp. and Pseudomonas sp., with the
formation
of 7a-substituted l la-hydroxy steroids with general formula 4,B. Used in
particular are
Bacillus lactimorbus, Bacillus sphaericus, Mycobacterium neoaurum,
Mycobacterium
smegmatis, Nocardia corallina, Nocardia globerula, Nocardia minima, Nocardia
restrictus, Nocardia rubropertincta, Nocardia salmonicolor and Pseudomonas
testosterone, whereby in particular Bacillus lactimorbus (ATCC 245), Bacillus
sphaericus
(ATCC 7055), Mycobacterium neoaurum (ATCC 9626, NRRL B-3683, NRRL B-3805),
Mycobacterium smegmatis (ATCC 14468), Nocardia corallina (ATCC 31338),
Nocardia
globerula (ATCC 9356), Nocardia minima (ATCC 19150), Nocardia restrictus (NCIB
10027), Nocardia rubropertincta (ATCC 14352), Nocardia salmonicolor (ATCC
19149)
and Pseudomonas testosterone (ATCC 11996) are used.
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In another process variant, the compounds with general formula 4,B can be
produced in a microbiological reaction from 7a-substituted steroids with
general formula
D:
O
APR'
Rio
O R
D in which R7, R10 and R13 have the same meanings as indicated in the
compounds with
general formula 4,B. This reaction is performed with use of a microorganism
that is
selected from the group that comprises Aspergillus sp., Beauveria sp.,
Curvularia sp.,
Gibberella sp., Glomerella sp., Gnomonia sp., Haplosporella sp., Helicostylum
sp.,
Nigrospora sp., Rhizopus sp. and Syncephalastrum sp., whereby the steroid
skeleton is
hydroxylated in 11 a-position and thus the 7a-substituted 1 I a-hydroxy
steroid with
general formula 4,B is produced. Aspergillus alliaceus, Aspergillus awamori,
Aspergillus
fischeri, Aspergillus malignus, Aspergillus melleus, Aspergillus nidualans,
Aspergillus
niger, Aspergillus ochraceus, Aspergillus variecolor, Beauveria bassiana,
Curvularia
lunata, Gibberella zeae, Glomerella cingulata, Glomerellafusaroides, Gnomonia
cingulata, Haplosporella hesperedica, Helicostylum piriformae, Nigrospora
sphaerica,
Rhizopus oryzae and Syncephalastrum racemosum are preferred, whereby in
particular
Aspergillus alliaceus (ATCC 10060), Aspergillus awamori (CBS),
Aspergillusfischeri
(ATCC 1020), Aspergillus malignus (IMI 16061), Aspergillus melleus (CBS),
Aspergillus
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nidualans (ATCC 11267), Aspergillus niger (ATCC 9142, ATCC 11394), Aspergillus
ochraceus (NRRL 405, ATCC 13252, ATCC 46504), Aspergillus variecolor (ATCC
10067), Beauveria bassiana (IFO 5838, ATCC 13144, IFO 4848, CBS 11025, CBS
12736, ATCC 7159), Curvularia lunata (IX3), Gibberella zeae (CBS 4474),
Glomerella
cingulata (ATCC 10534, CBS 23849, CBS 23749, ATCC 16646, IFO 6459, IFO 6425,
IFO, 6470, ATCC 15093, ATCC 10529, IFO 5257, ATCC 56596, ATCC 64682),
Glomerellafusaroides (ATCC 9552), Gnomonia cingulata (CBS 15226),
Haplosporella
hesperedica (CBS 20837), Helicostylum piriformae (ATCC 8992), Nigrospora
sphaerica
(ATCC 12772), Rhizopus oryzae (ATCC 4858) and Syncephalastrum racemosum (IFO
4827) are used.
Especially suitable are processes in which 7a-substituted 11a-hydroxy steroids
with general formula 4,B are produced, in which, independently of one another,
R7 stands
for CH3 and/or R10 stands for H and/or R13 stands for CH3.
The process is carried out in the usual way. To this end, typically first a
sterilized
nutrient solution is produced for the strain, and this nutrient solution is
then inoculated
with the culture solution of the strain to cultivate the strain. The
preculture that is
produced in this way is then added to a fermenter that is optionally coated
with a suitable
nutrient solution. Preferably after a growth phase for the culture of the
strain, the starting
substance is then added to the fermenter, in this case either a compound with
general
formula 3,A or a compound with general formula D, so that the reaction
according to the
invention can proceed. After the reaction has ended, the mixture of substances
is purified
in the usual way to isolate the desired 7a-substituted I Ia-hydroxy steroid.
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From the thus obtained compounds with general formula 4,B, other compounds
according to the invention can be synthesized with production processes also
according to
the invention. In particular, the 7a, 1 7a-substituted 11 n-halogen steroids
with general
formula 8, 10, 12:
R17
R13 = OR17'
R11
R10
V. "/R7
8,10,12
in which
U-V-W-X-Y-Z stands for one of the ring structures C'-C2-C3-C4=C5-C10,
C1-C2-C3-C4-CS=C10 or C'-C2-C3-C4-C5-C10, whereby in this case, an oxo
group (=O) is bonded to W (=C3), or for the ring structure C'=C2-C3=C4-
C5=C6, whereby in this case radical OR3 is bonded to W (=C3),
R3 stands for H, CI- to C4-alkyl, Cl- to C4-alkanoyl or a cyclic C3- to C7-
ether with the O-atom of the OR3 radical,
R7 is the grouping P-Q, whereby
P represents a C1- to C4-alkylene and Q represents a hydrogen,
AMENDED
SHEET
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Cl- to C4-alkyl- or C1- to C4-fluoroalkyl (alkyl that is partially or
completely fluorinated), and the grouping P-Q is bonded via P to
the steroid skeleton,
R10 can be in a- or R-position and stands for H, CH3 or CF3, and is present
only if X-Y-Z is not C4-C5=C10,
R" is a halogen,
R13 is methyl or ethyl,
R'7 stands for H, C1- to C18-alkyl, alicyclic C1- to C18-alkyl, C1- to C18-
alkenyl, alicyclic C1- to C18-alkenyl, C1- to C18-alkinyl, C1- to C18-
alkylaryl, C1- to C8-alkylenenitrile or for the grouping P-Q, whereby the
grouping P-Q has the above-mentioned meaning,
R17' stands for H, C1- to C18-alkyl, alicyclic C1- to C18-alkyl, C1- to C18-
alkenyl, alicyclic C1- to C18-alkenyl, C1- to C18-alkinyl or C1- to C18-
alkylaryl, whereby R'7' also can be bonded via a keto group to the 17R-
oxy group, and whereby R17' also can be substituted in addition with one
or more groups NR'8R'9 or one or more groups SOXRZ0, whereby x = 0, 1
or 2, and R'8, R19 and R20 in each case independently of one another can
have the same meaning as R17,
as well as their pharmaceutically compatible addition salts, esters and
amides, produce
advantageous active ingredients. These compounds that can be obtained by
additional
process steps from the 7a-substituted 11 a-hydroxy steroid with general
formula 4,B are
valuable active ingredients with strong androgenic action without the above-
mentioned
side effects. These compounds are suitable for the production of
pharmaceutical agents
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and especially of effective contraceptive agents and active ingredients for
hormone
replacement therapy (HRT).
If, in addition, R17' is substituted with a group NR'8R19, this can be a
methylamino, dimethylamino, ethylamino, diethyl amino, cyclohexylamino,
dicyclohexylamino, phenylamino, diphenylamino, benzylamino or dibenzylamino
group.
Especially suitable 7a, 17a-substituted 11(3-halogen steroids with general
formula
8, 10, 12 are compounds in which U-V-W-X-Y-Z stands for the ring structure C'-
C2-C3-
C4=C5-C10, C'-C2-C3-C4-CS=C10 or C'=C2-C3=C4-C5=C'0.
In the first case, (U-V-W-X-Y-Z = C'-C2-C3-C4=C5-C10), these are steroids with
general formula 10:
R17
R13 = ORi7'
R11
R10
In the second case (U-V-W-X-Y-Z = C'-C2-C3-C4-C5=C10), these are steroids
with general formula 12:
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R17
R13 = OR1T
R"
O R7
12
Compounds with general formulas 10 and 12 are androgenic compounds.
In the third case (U-V-W-X-Y-Z = C 1=C2-C3=C4-C5=C6), these are steroids
with general formula 8:
R17
R13 = OR17'
R"
R3O R7
8
These compounds are estrogens (estrogen rezeptor-affine compounds).
In all three cases, radicals R3, R7, R10, R", R13, R'7 and R17' have the same
meanings as the corresponding radicals in general formula 8, 10, 12.
Independently of one another, R' preferably stands for H and/or R7 stands for
CH3 and/or R" stands for fluorine and/or R13 stands for CH3 and/or R17 stands
for H,
CH3, Ci- to C18-alkinyl, in particular ethinyl, CH2CN or CF3, and/or R17'
stands for H.
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7a,17a-Substituted 11(3-halogen steroids with general formula 8, 10, 12 that
are
especially suitable according to the invention are:
17a-Ethinyl-11(3-fluoro-17(3-hydroxy-7a-methylestr-4-en-3-one (Formula 10)
I7a-Ethinyl-II(3-fluoro-17(3-hydroxy-7a-methyl estr-5(10)-en-3-one (Formula
12)
17a-Ethinyl-110-fluoro-7a-methylestra-1,3,5(10)-triene-3,173-diol (Formula 8).
For the production of these compounds, the following production methods can be
adopted:
For the production of 7u, 1 7u-substituted 11 P-halogen steroids with general
formula 10, in which U-V-W-X-Y-Z stands for the ring structure C1-C2-C3-C4=C5-
C10,
the 7a-substituted 11 a-hydroxy steroids with general formula 4,B that are
obtained with
the microbiological production process according to the invention are used as
starting
substances.
In a first synthesis step, these thus obtained 7a-substituted I I a-hydroxy
steroids
are converted by nucleophilic substitution with a halodehydroxylating reagent
into the
corresponding 7a-substituted 11 [3-halogen steroids 5:
Rt3 O R13 O
HO ///"", RII
[Halo-
R10 dehydroxylierung] R10
O R7
O R7
4,B 5
[Key: Halodehydroxylierung = halodehydroxylation]
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As halodehydroxylating reagents, all compounds that are commonly used for this
purpose are suitable, for example, hydrofluoric acid, hydrochloric acid,
hydrobromic acid
or hydroiodic acid, thionyl chloride or thionyl bromide, phosphorus
pentachloride,
phosphorus oxychloride, N-chlorosuccinimide, triphenylphosphine/carbon
tetrachloride,
HF/pyridine or diethylaminosulfur trifluoride or preferably nonaflyl
fluoride/1,5-diazabi-
cyclo[5.4.0]undecene.
Compound 10 is then produced from 5 by selective alkylation on C17 of the ring
skeleton (see Diagram 1 in this respect). For selective alkylation, common
alkylating
reagents can be used, for example Grignard compounds and organometallic
compounds,
especially alkyllithium compounds. For example, ethinylmagnesium bromide can
be
used as an alkylating agent for the production of the corresponding 17a-
ethinyl-17(3-
hydroxy-estr-4-en-3-one from estr-4-ene-3,1 7-di one.
For the production of 7a, 17a-substituted 11(3-halogen steroids, in which
U-V-W-X-Y-Z stands for the ring structure C'-C2-C3-C4-C5=C10 and which have
general
formula 12, the compounds with general formula 10 are used and isomerized,
such that
the A4-double bond is isomerized into a A5(10)-double bond. To protect the 3-
keto group,
first a cyclic ether is formed in 3-position for this purpose. Then, the A4-
double bond is
isomerized into the A5(10)-double bond, whereby the 7a,17a-substituted 11 [3-
halogen
steroid with general formula 12 is formed, and the protective group is cleaved
again.
For the production of additional 7u, I 7a-substituted 11 [3-halogen steroids
with
general formula 8, in which U-V-W-X-Y-Z stands for C'=C2-C3=C4-C5=C10, the
procedure is as follows:
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First, as already described above, the corresponding 11(3-halogen steroid with
general formula 5 is formed from the 7a-substituted l la-hydroxy steroid,
obtained by
microbiological hydroxylation and oxidation, with general formula 4,B by
halodehydroxylation in a nucleophilic substitution reaction.
From the latter, a 7a-substituted estra-1,3,5(10)-triene with general formula
6 is
then formed by oxidation, for example with a copper(II) salt:
R13 O
R11
R3O R7
6
in which R3, R7, R" and R13 have the same meanings as designated above. If R3
stands
for H, these compounds can be synthesized directly. If another radical is to
stand as H
for R3, the corresponding ethers or esters must be formed in the known way,
after the
1,3,5(10)-triene ring has been formed by oxidation.
The 7a-substituted 113-haloestra-1,3,5(10)-trienes with general formula 6 as
well
as pharmaceutically compatible addition salts, esters and amides thereof are
also new and
are therefore claimed as intermediate products in the synthesis of 7u, 1 7a-
substituted II 3-
halogen steroids with general formula 8 also according to the invention.
CA 02492079 2005-01-12
An especially preferred 7a-substituted 11(3-haloestra-1,3,5(10)-triene with
general
formula 6 is 11(3-fluoro-3-hydroxy-7a-methylestra-1,3,5(10)-trien-l7-one.
The 7u, 1 7u-substituted 11 P-halogen steroid with general formula 8 according
to
the invention can be formed from the 7a-substituted 11(3-haloestra-1,3,5(10)-
triene with
general formula 6 in the same way as is previously described for the synthesis
of the
compound with general formula 10 by selective alkylation at C17 of the ring
skeleton.
In addition, 7a-substituted 11 n-halogen steroids with general formula 9 can
also
be produced from the substances with general formula 4,B that are obtained by
microbiological hydroxylation and oxidation from the 7a-substituted steroids
with
general formula 3,A or D, and said 7a-substituted 11(3-halogen steroids also
have
androgenic action:
13 OH
R11
O j R7
9
in which R7, R" and R13 have the same meanings as indicated above. An
especially
preferred compound is 11(3-fluoro-173-hydroxy-7a-methylestr-4-en-3-one. The
compounds with general formula 9 as well as the pharmaceutically compatible
addition
salts, esters and amides thereof also have androgenic action.
CA 02492079 2005-01-12
21
For the production of compounds with general formula 9, estr-4-ene-3,17-dione
5
is reduced to 17(3-hydroxy-estr-4-en-3-one 9, for example with a boron
hydride.
In addition, the compounds with general formula 9 can be converted into the
corresponding 7a-substituted II P-haloestra-5(10)-enes:
R13 OH
R11
O R7
whereby R7, R10, R" and R'3 have the meanings as in general formula 8, 10, 12.
For this
purpose, the compounds with general formula 9 are isomerized by alteration of
the A4-
double bond into a A5(10'-double bond. To protect the 3-keto group, first a
cyclic ether in
3-position is formed for this purpose. Then, the A4-double bond is isomerized
into the
A5110)-double bond, whereby the above-indicated 7a-substituted 11(3-halogen
steroid is
formed, and the protective group is then cleaved again.
Finally, the corresponding 7a-substituted 11(3-haloestra-5(10)-enes can also
be
converted from the compounds with general formula 5 by isomerization of the A4-
double
bond into the A5(1 )-double bond:
CA 02492079 2005-01-12
22
R13 O
R"
O R
whereby R7, R10, R" and R' 3 have the meanings as in general formula 8, 10,
12. For this
purpose, the compounds with general formula 5 are isomerized by alteration of
the A4-
double bond into a A5(10)-double bond. To protect the 3-keto group, in turn
first a cyclic
ether is formed in 3-position for this purpose. Then, the A4-double bond is
isomerized
into the A5 ' '-double bond, whereby the 7a-substituted 11(3-halogen steroids
that are
indicated above are formed, and the protective group is finally cleaved again.
All above-mentioned compounds can also be further esterified or etherified if
corresponding hydroxy groups are present in 3- or 173-position. For example,
compound
9 can be converted into a corresponding 170-ether or 17(3-ester. A preferred
compound is
11(3-fluoro-173-(4-sulfamoylbenzoxy)-7a-methylestr-4-en-3-one. As substituents
on the
oxy-oxygen atom at C17, basically the same radicals as are also indicated for
R'7' are
suitable.
In particular, the 7a, 17a-substituted 11 n-halogen steroids with general
formula 8,
10, 12 are suitable for the production of pharmaceutical agents. This
invention therefore
also relates to the use of the above-mentioned compounds with general formula
8, 10, 12
for the production of pharmaceutical agents as well as pharmaceutical
preparations that
CA 02492079 2005-01-12
23
contain at least one of the above-mentioned compounds with general formula 8,
10, 12 as
well as at least one pharmaceutically compatible vehicle.
The 7a, l 7a-substituted 11(3-halogen steroids with general formula 10, 12
according to the invention are compounds with strong androgenic action without
the
above-mentioned side effects, for example stimulation of the prostate
(especially no
benign prostate hyperplasia). The compounds are easy to synthesize. It has
been shown
that the compounds according to the invention with general formula 10 or 12
can be used
not only for male HRT, but these compounds, even without additional
administration of
other active ingredients, are also suitable as effective male contraceptive
agents, if a
sufficient metering is done to adequately reduce the blood level of LH, of
testosterone
that is produced in the body as well as of FSH (follicle-stimulating hormone).
This
depends on the 1 l (3-halogen steroids according to the invention inhibiting
the release of
LH and FSH. LH stimulates the Leydig cells, so that testosterone is secreted.
If the blood
level of LH is kept low, the release of endogenous testosterone also drops.
Testosterone
is required for spermatogenesis, while FSH stimulates the germ cells.
Sufficiently high
FSH and LH blood levels are therefore necessary for an effective
spermatogenesis,
whereby a sufficiently high LH blood level results in the testosterone release
that is
necessary for spermatogenesis.
Since a treatment exclusively with the 7a, 17a-substituted 11 n-halogen
steroids
can already result in effective male contraception without additional active
ingredients
for sterilization, the administration of a pharmaceutical agent that is
suitable for this
purpose can be significantly simplified, and the costs of use can be
considerably lowered.
CA 02492079 2005-01-12
24
The 7a,17a-substituted l 1 [3-halogen steroids according to the invention can
also
be used in combination with a gestagen to control male fertility.
Moreover, the 7a, l 7a-substituted 11(3-halogen steroids according to the
invention
effectively inhibit 5a-reductase and the steroid- l l -hydroxylase [CYP 11 B
(P450c11), G.
Zhang, W. L. Miller, Journal of Clinical Endocrinology and Metabolism, Vol.
81, pages
3254-3256 (1996)], such that, for example, the stimulation of the prostate is
selectively
avoided, and these compounds have an improved pharmacokinetics. The inhibition
of the
l 1-hydroxylase results in a reduced deactivation of the androgenic compounds
and in
their reduced excretion from the human body. As a result, the effectiveness
and the
duration of action of these compounds compared to known compounds are improved
especially after oral administration.
For the reasons above, these compounds are suitable especially for use in male
birth control as well as for androgen replacement therapy with a reduced
tendency toward
5a-reduction with simultaneously obtained aromatizability to form estrogenic
steroids
and an advantageous influence on serum lipids and the central nervous system.
The androgenic action and the observation that the above-mentioned side
effects
do not occur were determined with a seminal vesicle test for the compounds
according to
the invention with general formulas 10 and 12. The effectiveness of the
compounds with
general formula 8 according to the invention was checked for estrogenic action
with a
uterus growth test.
The 7a,1 7a-substituted 11 n-halogen steroids with general formula 10 or 12
according to the invention or the pharmaceutical preparations according to the
invention
that contain these compounds are extremely well suited for treating non-
sterile male
CA 02492079 2005-01-12
patients as well as basically also male mammals. An application for male
contraception
results in that the male patients are only temporarily sterile. After the
application of the
active ingredients according to the invention or the pharmaceutical
preparations is
completed, the original state is produced again, so that the male patient is
no longer
sterile, and the spermatogenesis takes place again to the original extent. To
keep the
condition of temporary sterility constant over a desired period, the
administration of the
active ingredient or the preparation is to be performed continuously, whereby
the
administration, depending on the form of administration, is to be repeated
daily, at a
shorter interval or else periodically at a longer interval. After the one-time
or repeated
administration of the active ingredient or the preparation is completed, the
non-sterile
condition of the male patient optionally is not immediately restored but
rather only
slowly restored, whereby the time span that is necessary for this purpose
depends on
various factors, for example the dosage, the body constitution of the patients
and the
parallel administration of other pharmaceutical agents.
If the purpose of administration consists in contraception, the dosage of the
7a,17a-substituted 11(3-halogen steroids must be set high such that the blood
levels of
LH and FSH in each case are at most 2.5 I.E./ml (I.E.: International Units),
especially at
most 1.0 I.E./ml, and the blood level of testosterone is at most 10 nmol/l,
especially at
most 3 nmol/l.
If the 7a, 1 7a-substituted 11(3-halogen steroids according to the invention
are to
be used for HRT without a contraception being achieved, the dosage is set
lower. For
this case, an attempt is made to achieve effect levels that make possible the
blood levels
CA 02492079 2005-01-12
26
for LH and FSH of respectively more than 2.5 I.E./ml and for testosterone of
more than
nmol/l.
The dosages of the 7a, l 7a-substituted 11 n-halogen steroids with general
formula
10 or 12 according to the invention that are required to set the blood level
of LH, FSH
and testosterone depend on a number of factors and must therefore be
determined in an
administration-specific manner. First, the dosage is naturally dependent on
the type of
therapy. If the compounds are to be used for male contraception, significantly
higher
doses must be given than in the case of a use for HRT. The dosage also depends
on the
type of 7a,17a-substituted l 1(3-halogen steroid and its bio-availability. The
type of
administration is also essential for the amount to be administered. Finally,
the dosage
also depends on the body constitution of the patient to be treated and other
factors, for
example the state of whether other pharmaceutical agents are provided in
parallel.
The compounds can be administered orally and parenterally, for example i.p.
(intraperitoneally), i.v. (intravenously), i.m. (intramuscularly) or
percutaneously. The
compounds can also be implanted in the tissue. The amount of the compounds to
be
administered can fluctuate within a wide range if an effective amount is
administered.
Based on the condition to be treated and the type of dispensing, the amount of
administered compound can vary within a wide range. In humans, the daily dose
is in the
range of 0.1 to 100 mg. The preferred daily dosage in humans is 0.1 to 10 mg.
The
duration of administration depends on the purpose to be achieved.
Capsules, pills, tablets, coated tablets, creams, ointments, lotions, liquids,
such as
syrups, gels, injectable liquids, for example for i.p., i. v., i.m. or
percutaneous injection,
etc., are suitable for use, whereby the individual forms for dispensing
release the
CA 02492079 2005-01-12
27
compounds according to the invention to the body gradually or in the entire
amount
within a short time depending on the type thereof.
For oral administration, capsules, pills, tablets, coated tablets and liquids
or other
known oral forms for dispensing are used as pharmaceutical preparations. In
this case,
the pharmaceutical agents can be formulated in such a way that they release
the active
ingredients either in a short time and deliver them to the body or they have a
depot action,
so that a prolonged, slow feed of active ingredient to the body is achieved.
In addition to
the 7x, 17a-substituted 11(3-halogen steroid, the dosage units can contain one
or more
pharmaceutically compatible vehicles, for example substances for adjusting the
rheology
of the pharmaceutical agent, surfactants, solubilizers, microcapsules,
microparticles,
granulates, diluents, binders, such as starch, sugar, sorbitol and gelatin,
also fillers, such
as silicic acid and talc, lubricants, dyes, perfumes and other substances.
In particular, the 7a,17a-substituted 11 n-halogen steroids according to the
invention can also be formulated in the form of a solution that is intended
for oral
administration and that in addition to the active I 1 R-halogen steroid
contains as the
following components: a pharmaceutically compatible oil and/or a
pharmaceutically
compatible lipophilic surfactant and/or a pharmaceutically compatible
hydrophilic
surfactant and/or a pharmaceutically compatible water-miscible solvent. In
this respect,
reference is also made to WO-A-97/21440.
To achieve better bio-availability of the steroid, the compounds can also be
formulated as cyclodextrin clathrates. For this purpose, the compounds are
reacted with
a-, 3- or y-cyclodextrin or derivatives thereof.
CA 02492079 2005-01-12
28
If creams, ointments, lotions and liquids that can be applied topically are to
be
used, the latter must be constituted in such a way that the compounds
according to the
invention are fed to the body in a sufficient amount. In these forms for
dispensing,
adjuvants are contained, for example substances for adjusting the rheology of
pharmaceutical agents, surfactants, preservatives, solubilizers, diluents,
substances for
increasing the permeability of the steroids according to the invention through
the skin,
dyes, perfumes and skin protection agents, such as conditioners and
moisturizers.
Together with the steroids according to the invention, other active
ingredients can also be
contained in the pharmaceutical agent.
For parenteral administration, the active ingredients can be dissolved or
suspended in a physiologically compatible diluent. As diluents, very often
oils with or
without the addition of a solubilizer, a surfactant, a suspending agent or
emulsifier are
used. Examples of oils that are used are olive oil, peanut oil, cottonseed
oil, soybean oil,
castor oil and sesame oil. To formulate an injectable preparation, any liquid
vehicle can
be used in which the compounds according to the invention are dissolved or
emulsified.
These liquids frequently also contain substances to regulate viscosity,
surfactants,
preservatives, solubilizers, diluents and other additives, with which the
solution is set to
isotonic. Other active ingredients can also be administered together with the
7a, I 7a-
substituted 1 1(3-halogen steroids.
The 11 3-halogen steroids according to the invention can be administered in
the
form of a depot injection or an implant preparation, for example
subcutaneously, which
can be formulated in such a way that a delayed release of active ingredients
is made
possible. To this end, known techniques can be used, for example depots that
dissolve or
CA 02492079 2005-01-12
29
that operate with a membrane. Implants can contain as inert materials, for
example,
biodegradable polymers or synthetic silicones, for example silicone gum. The I
I (3-
halogen steroids according to the invention can also be incorporated in, for
example, a
patch, for percutaneous administration.
The examples that are indicated below are used for a more detailed explanation
of
the invention:
CA 02492079 2005-01-12
A. Microbiological Synthesis:
11a-Hydroxy-7a-methyl-estr-4-ene-3,17-dione (Compound 4,B):
Example 1:
OH O
Mikrobiologische - HO,,,
H Hydroxylierung H
and Oxidation
OZ 0 A B
[Key: Mikrobiologische Hydroxylierung and Oxidation = Microbiological
Hydroxylation and Oxidation]
A 2 1 Erlenmeyer flask that contained 1000 ml of a nutrient solution,
sterilized for
30 minutes at 121 C in an autoclave and consisting of 3% by weight of
glucose, I% by
weight of corn steep liquor, 0.2% by weight of NaNO3, 0.1 % by weight of
KH2PO4, 0.2%
by weight of K2HPO4, 0.05% by weight of KCI, 0.05% by weight of MgSO4,7H20 and
0.002% by weight of FeSO4 7H2O (pH 6.0) was inoculated with a slant rod
culture of the
strain Gnomonia cingulata (CBS 15226) and shaken for 72 hours at 28 C in a
rotary
shaker at 165 rpm. With this preculture, a 201 fermenter that was coated with
19 1 of
sterile medium of the same final composition as described for the preculture
was
CA 02492079 2005-01-12
31
inoculated. In addition, before the sterilization, another 1.0 ml of silicone
oil and 1.0 ml
of synperonic (oxoalcohol ethoxylate) were added for foam abatement. After a
growth
phase of 12 hours at 0.7 bar of overpressure, a temperature of 28 C, an
aeration of 20
1/minute and a stirring speed of 250 rpm, a solution of 4.0 g of 17(3-hydroxy-
7a-
methylestr-4-en-3-one in 40 ml DMF was added. Stirring was continued, and it
was
aerated. After 135 hours, the culture broth was harvested and extracted for 12
hours with
1 of methyl isobutyl ketone and for 5 hours with 5 1 of methyl isobutyl
ketone. The
combined organic phases were evaporated to the dry state. The silicone oil was
washed
out with hexane. After chromatography on silica gel with a gradient that
consists of
hexane and ethyl acetate, 1.64 g (39%) of I1 a-hydroxy-7a-methylestr-4-ene-
3,17-dione
was isolated.
Example 2:
OH OH
HO,,,
H Mikrobiologische H Mikrobiologische
Hydroxylierung Oxidation
0 0
A C
O
HO,,,
H
B
[Key: Mikrobiologische Hydroxylierung = Microbiological Hydroxylation
Mikrobiologische Oxidation = Microbiological Oxidation]
CA 02492079 2005-01-12
32
A 2 1 Erlenmeyer flask that contained 1000 ml of a nutrient solution,
sterilized for
30 minutes at 121 C in an autoclave and consisting of 3% by weight of
glucose, I% by
weight of corn steep liquor, 0.2% by weight of NaNO3, 0.1% by weight of
KH2PO4, 0.2%
by weight of K2HPO4, 0.05% by weight of KCI, 0.05% by weight of MgSO4"7H20,
and
0.002% by weight of FeSO4'7H20 (pH 6.0), was inoculated with a slant rod
culture of the
strain Glomerella cingulata (IFO 6425) and shaken for 72 hours at 28 C in a
rotary
shaker at 165 rpm. With this preculture, a 20 1 fermenter was inoculated, and
said
fermenter was coated with 19 1 of a sterile medium of the same final
composition as
described for the preculture. In addition, before the sterilization, another
1.0 ml of
silicone oil and 1.0 ml-of synperonic were added for foam abatement. After a
growth
phase of 12 hours at 0.7 bar of overpressure, a temperature of 28 C, an
aeration of 10
1/minute and a stirring speed of 350 rpm, a solution of 2.0 g of 17(3-hydroxy-
7a-
methylestr-4-en-3-one in 30 ml DMF was added. Stirring was continued, and it
was
aerated. After 19 hours, the culture broth was harvested and extracted for 16
hours with
20 1 of methyl isobutyl ketone and for 23 hours with 20 1 of methyl isobutyl
ketone. The
combined organic phases were evaporated to the dry state. The residue was
dissolved to
a large extent in methanol. The silicone oil was filtered off. It was
concentrated by
evaporation, and after chromatography on silica gel with a gradient that
consists of
dichloromethane and acetone, 1.55 g (73%) of 11a,I7(3-dihydroxy-7a-methylestr-
4-en-3-
one was isolated. After recrystallization from acetone/diisopropyl ether, 827
mg (39%)
of white crystals with a melting point of 163 C and ["ID = -16 (CHC13, c =
0.501) was
isolated.
CA 02492079 2005-01-12
33
A 2 1 Erlenmeyer flask that contained 500 ml of a nutrient solution,
sterilized for
30 minutes at 121 C in an autoclave and consisting of 0.5% by weight of
glucose, 0.5%
by weight of bacto-yeast extract, 0.1 % by weight of peptone, and 0.2% by
weight of corn
steep liquor (pH 7.5), was inoculated with four cryospheres from a culture of
the strain
Bacillus sphaericus (ATCC 7055) and shaken for 24 hours at 28 C in a rotary
shaker at
165 rpm. With this preculture, four 2 1 Erlenmeyer flasks that contained 500
ml of sterile
medium of the same composition as described for the preculture were inoculated
with
10% each of this culture broth. After a growth phase of 4 hours at a
temperature of 28 C
in a rotary shaker at 165 rpm, a solution of 50 mg of I 1 a,17(3-dihydroxy-7a-
methylestr-
4-en-3-one in 2.5 ml DMF was added to each flask. Shaking was continued for 48
hours.
The combined culture broths were extracted twice with 2 1 of methyl isobutyl
ketone.
The combined organic phases were dried on sodium sulfate and evaporated to the
dry
state. In this case, 630 mg of an oily-crystalline residue was obtained. After
recrystallization from acetone/diisopropyl ether, 103 mg (49.2%) of yellowish
crystals
with a melting point of 189 C and [a]D = +40.4 (CHC13, c = 0.529) was
isolated (direct
crystallization without previous chromatographic purification).
CA 02492079 2005-01-12
34
Example 3:
O o
HO,5H Mikrobiologische H
D B
[Key: Mikrobiologische Hydroxylierung = Microbiological Hydroxylation]
A 2 1 Erlenmeyer flask that contained 500 ml of a nutrient solution,
sterilized for
30 minutes at 121 C in an autoclave and consisting of 3% by weight of
glucose, 1 % by
weight of corn steep liquor, 0.2% by weight of NaNO3, 0.1 % by weight of
KH2PO4, 0.2%
by weight of K2HPO4, 0.05% by weight of KCI, 0.05% by weight of MgSO4,7H2O,
and
0.002% by weight of FeSO4'7H2O (pH 6.0), was inoculated with a half slant rod
culture
of the strain Aspergillus ochraceus (CBS 13252) and shaken for 72 hours at 28
C in a
rotary shaker at 165 rpm. With this preculture, a 10 1 fermenter was
inoculated, and said
fermenter was coated with 9.5 1 of a sterile medium of the same final
composition as
described for the preculture. In addition, before the sterilization, another
0.5 ml of
silicone oil and 0.5 ml of synperonic were added for foam abatement. After a
growth
phase of 6 hours at 0.7 bar of overpressure, a temperature of 28 C, an
aeration of 5
1/minute and a stirring speed of 350 rpm, a solution of 1.0 g of 7a-methylestr-
4-ene-3,17-
dione in 15 ml DMF was added. Stirring was continued, and it was aerated.
After 22
hours, the culture broth was harvested and extracted twice for 4 hours with 7
1 of methyl
isobutyl ketone. The combined organic phases were evaporated to the dry state.
The
CA 02492079 2005-01-12
residue was dissolved to a large extent in methanol. The silicone oil was
filtered off. It
was concentrated by evaporation, and after chromatography on silica gel with a
gradient
that consists of dichloromethane and acetone, 0.78 g (74%) of 11 a-hydroxy-7a-
methylestr-4-ene-3,17-dione was isolated. After recrystallization from
acetone/diisopropyl ether, 311 mg (29.6%) of white crystals with a melting
point of
200 C and [(X]D = +52 (CHC13, c = 0.5905) was isolated.
B. Chemical Production Process:
Example 4: Production of 11(3-fluoro-17 j3-hydroxy-7a-m ethylestr-4-en-3-one:
a) 11 [3-Fluoro-7a-methyl-estr-4-ene-3,17-dione:
11.5 ml of perfluorobutane-l-sulfonic acid fluoride was added in drops at 0 C
to a
solution of 13.08 g of l I a-hydroxy-7a-methyl-estr-4-ene-3,17-dione (produced
by means
of microbiological synthesis according to the invention [Part A]) in 250 ml of
toluene and
18.2 ml of 1,8-diazabicyclo[5,4,0]undec-7-ene. After 1 hour, it was
neutralized with 2 M
hydrochloric acid, added to water, extracted four times with ethyl acetate,
washed with
saturated sodium chloride solution, dried and concentrated by evaporation in a
vacuum.
After the crude product was chromatographed on silica gel with a hexane/ethyl
acetate
gradient, 8.7 g of 11(3-fluoro-7a-methyl-estr-4-ene-3,17-dione was obtained.
Melting
point: 101.4 C, [CL]D: +135.8 (CHC13).
CA 02492079 2005-01-12
36
b) 11(3-Fluoro-17(3-hydroxy-7a-methylestr-4-en-3-one:
A solution of 8.7 g of I 1 [3-fluoro-7a-methyl-estr-4-ene-3,17-dione in 148 ml
of
tetrahydrofuran was mixed drop by drop at 0 C with 29.5 ml of I M lithium
aluminium
tri-tert-butoxyhydride in tetrahydrofuran and stirred for 5.5 hours at 0 C.
Then, dilute
sulfuric acid was added at 0 C, and the reaction solution was added to ice
water,
extracted three times with ethyl acetate, washed neutral, dried on sodium
sulfate,
concentrated by evaporation in a vacuum and chromatographed on silica gel with
hexane/ethyl acetate. 5.8 g of 11(3-fluoro-17(3-hydroxy-7a-methylestr-4-en-3-
one with a
melting point of 143-144 C was obtained. [all) = +89.9 (CHC13).
Example 5: Production of 11[3-Fluoro-173-(4-sulfamoylbenzoxy)-7a-methylestr-4-
en-3-
one:
A solution of 500 mg of 11[3-fluoro-17(3-hydroxy-7a-methylestr-4-en-3-one in
7.5 ml of pyridine was mixed at room temperature with 750 mg of 4-
sulfamoylbenzoic
acid, 800 mg of N,N-dicyclohexylcarbodiimide as well as 125 mg ofp-
toluenesulfonic
acid and stirred for 8.5 hours. Then, it was added to sodium bicarbonate
solution,
extracted four times with dichloromethane, washed neutral, dried on sodium
sulfate,
concentrated by evaporation in a vacuum, and chromatographed on silica gel
with
dichloromethane/acetone. 302 mg of 11[3-fluoro-I7f3-(4-sulfamoylbenzoxy)-7a-
methylestr-4-en-3-one with a melting point of 232 C was obtained. [a]D =
+100.5
(CHC13).
CA 02492079 2005-01-12
37
Example 6: Production of 17a-Ethinyl-11 0-fluoro-170-hydroxy-7a-methylestr-4-
en-3-
one:
a) 11 0-Fluoro-3-methoxy-7a-methylestra-3,5-dien- l 7-one:
A solution of 2 g of 1 1(3-fluoro-7a-methylestr-4-ene-3,17-dione in 20 ml of
2,2-
dimethoxypropane was stirred with 200 mg of pyridinium tosylate for 6.5 hours
at 80 C.
Then, it was diluted with ethyl acetate, washed with sodium bicarbonate
solution and
sodium chloride solution, dried on sodium sulfate and concentrated by
evaporation in a
vacuum. 2 g of crude 11 [3-fluoro-3-methoxy-7a-methylestra-3,5-dien-17-one was
obtained.
b) 17a-Ethinyl-11 [3-fluoro- 17f-hydroxy-7a-methylestr-4-en-3-one:
A solution of 9.17 g of cerium(III) chloride in 60 ml of tetrahydrofuran was
mixed drop by drop at 0 C with 74.2 ml of an ethinylmagnesium bromide solution
(0.5 M
in tetrahydrofuran) and stirred for 1 hour at 0 C. Then, a solution of 2 g of
crude I1 [3-
fluoro-3-methoxy-7a-methylestra-3,5-dien-l7-one in 40 ml of tetrahydrofuran
was added
drop by drop and stirred for another 3.5 hours at 0 C. For working-up, a
saturated
ammonium chloride solution was added, added to water, extracted three times
with ethyl
acetate, washed with semiconcentrated hydrochloric acid, sodium bicarbonate
solution
and sodium chloride solution, dried on sodium sulfate, concentrated by
evaporation in a
vacuum and chromatographed on silica gel with hexane/ethyl acetate. 1.15 g of
pure
l 7a-ethinyl-11 f3-fluoro-17[3-hydroxy-7a-methylestr-4-en-3-one with a melting
point of
218-220 C was obtained. MD = +19.2 (CHC13).
CA 02492079 2005-01-12
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Example 7: Production of 17a-Ethinyl-1113-fluoro-I7(3-hydroxy-7a-methylestr-
5(10)-en-
3-one:
a) 3,3-Ethanediyldioxy- l 7a-ethinyl- 11(3-fluoro-7a-methylestr-5(10)-en-1713-
ol :
A solution of 700 mg of 17a-ethinyl-II P-fluoro-17(3-hydroxy-7a-methylestr-4-
en-3-one in 7 ml of dichloromethane and 4.7 ml of ethylene glycol was stirred
with 2.3
ml of trimethyl orthoformate and 30 mg ofp-toluenesulfonic acid hydrate for
6.5 hours at
room temperature. Then, it was added to sodium bicarbonate solution, extracted
three
times with ethyl acetate, washed neutral, dried on sodium sulfate,
concentrated by
evaporation in a vacuum, and chromatographed on silica gel with hexane/ethyl
acetate.
205 mg of 3,3-ethanediyldioxy-I7(x-ethinyl-11(3-fluoro-7a-methylestr-5(10)-en-
17(3-ol
was obtained.
b) 17a-Ethinyl-11(3-fluoro-17(3-hydroxy-7a-methylestr-5(10)-en-3-one:
A solution of 205 mg of 3,3-ethanediyldioxy-l7a-ethinyl-1113-fluoro-7a-methyl-
estr-5(10)-en-1713-ol in 27 ml of methanol and 3.6 ml of water was stirred
with 361 mg of
oxalic acid for 24 hours at room temperature. Then, it was added to sodium
bicarbonate
solution, extracted three times with ethyl acetate, washed neutral, dried on
sodium
sulfate, concentrated by evaporation, in a vacuum and chromatographed on
silica gel with
hexane/ethyl acetate. 95 mg of 17a-ethinyl- 1113-fluoro-17(3-hydroxy-7a-methyl
estr-
5(l0)-en-3-one with a melting point of 112-114 C was obtained.
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Example 8: Production of 17a-Ethinyl-113-fluoro-7a-methylestra-1,3,5(10)-
triene-
3,17(3-diol:
a) 11 Q-Fluoro-3-hydroxy-7a-methylestra-1,3,5(10)-trien- l 7-one:
A solution of 500 mg of 11(3-fluoro-7a-methylestr-4-ene-3,17-dione in 16.5 ml
of
acetonitrile was stirred with 400 mg of copper(II) bromide for 6.5 hours at 25
C. Then, it
was diluted with ethyl acetate, washed with sodium bicarbonate solution and
sodium
chloride solution, dried on sodium sulfate, concentrated by evaporation in a
vacuum and
chromatographed on silica gel with hexane/acetone. 280 mg of pure 11(3-fluoro-
3-
hydroxy-7a-methylestra-1,3,5(10)-trien-l7-one with a melting point of 185-186
C was
obtained.
b) 17a-Ethinyl-II(3-fluoro-7a-methylestra-1,3,5(10)-triene-3,17(3-diol:
A suspension of 2.03 g of cerium(Ill) chloride in 7.5 ml of tetrahydrofuran
was
mixed drop by drop at 0 C with 16.5 ml of an ethinylmagnesium bromide solution
(0.5 M
in tetrahydrofuran) and stirred for 0.5 hour at 0 C. Then, a solution of 280
mg of 11(3-
fluoro-3-hydroxy-7a-methylestra-1,3,5(10)-tri en-17-one in 2.8 ml
tetrahydrofuran was
added drop by drop and stirred for another 3.5 hours at 0 C. For working-up, a
saturated
ammonium chloride solution was added, added to water, extracted four times
with ethyl
acetate, washed neutral, dried on sodium sulfate, concentrated by evaporation
in a
vacuum and chromatographed on silica gel with hexane/ethyl acetate.
220 mg of 17a-ethinyl-1 1(3-fluoro-7a-methyl estra-1,3,5(10)-triene-3,173-diol
with a melting point of 115-117 C was obtained.
