U.S. patent application number 12/929644 was filed with the patent office on 2011-06-02 for method and intermediates for preparing 2-alkoxy and 2-aryloxy estrogen compounds.
This patent application is currently assigned to NAXOSPHARMA S.R.L.. Invention is credited to Franco Buzzetti, Paolo Lombardi.
Application Number | 20110130581 12/929644 |
Document ID | / |
Family ID | 41606628 |
Filed Date | 2011-06-02 |
United States Patent
Application |
20110130581 |
Kind Code |
A1 |
Lombardi; Paolo ; et
al. |
June 2, 2011 |
Method and intermediates for preparing 2-alkoxy and 2-aryloxy
estrogen compounds
Abstract
The present invention relates to 2-alkoxy and 2-aryloxyestrogen
compounds, and the intermediate compounds prepared during the
preparation thereof, which intermediate compounds are useful
intermediates in the preparation of certain physiologically active
compounds.
Inventors: |
Lombardi; Paolo; (Cesate,
IT) ; Buzzetti; Franco; (Monza, IT) |
Assignee: |
NAXOSPHARMA S.R.L.
|
Family ID: |
41606628 |
Appl. No.: |
12/929644 |
Filed: |
February 7, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12285901 |
Oct 16, 2008 |
7901904 |
|
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12929644 |
|
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Current U.S.
Class: |
552/518 ;
552/614 |
Current CPC
Class: |
C07J 41/0005 20130101;
C07J 1/0059 20130101; C07J 1/007 20130101 |
Class at
Publication: |
552/518 ;
552/614 |
International
Class: |
C07J 41/00 20060101
C07J041/00; C07J 1/00 20060101 C07J001/00 |
Claims
1. A compound of general formula (III) ##STR00020## wherein R.sub.a
is H, lower alkyl, alkenyl or alkynyl radical.
2. A compound of general formula (IV) ##STR00021## wherein X is a
member of the group consisting of O and ##STR00022## wherein
R.sub.a is H, lower alkyl, alkenyl or alkynyl radical; and R is: 1)
a linear, branched or cyclic alkyl radical containing from 1 to 8
carbon atoms, optionally substituted with 1-3 substituents selected
from the group consisting of (a) halogen; (b) OR.sub.b, wherein
R.sub.b is H, lower alky or aryl; (c) (C1-C6) cycloalkyl,
optionally substituted with 1-3 substituents selected from the
group consisting of halogen, lower alkyl, aryl and OR.sub.b,
wherein R.sub.b is H, lower alkyl or aryl; (d) (C6-C10) aryl,
optionally substituted with 1-3 substituents selected from the
group consisting of halogen, lower alkyl, aryl and OR.sub.b,
wherein R.sub.b is H, lower alkyl or aryl; (e) (C6-C10) heteroaryl,
optionally substituted with 1-3 substituents selected from the
group consisting of halogen, lower alkyl, aryl and OR.sub.b,
wherein R.sub.b is H, lower alkyl or aryl; or 2) an aryl radical
containing from 6 to 10 carbon atoms, optionally substituted with
1-3 substituents selected from the group consisting of (a) halogen;
(b) OR.sub.b, wherein R.sub.b is H, lower alkyl or aryl; (c)
(C1-C6) linear, branched or cyclic alkyl, optionally substituted
with 1-3 substituents selected from the group consisting of
halogen, lower alkyl, aryl and OR.sub.b, wherein R.sub.b is H,
lower alkyl or aryl; with the proviso that when X represents O, R
is different from an alkyl radical containing from 1 to 6 carbon
atoms.
3. A compound of general formula (IVb) ##STR00023## wherein X is a
member of the group consisting of O and ##STR00024## wherein
R.sub.a is H, lower alkyl, alkenyl or alkynyl radical; and R is: 1)
a linear, branched or cyclic alkyl radical containing from 1 to 8
carbon atoms, optionally substituted with 1-3 substituents selected
from the group consisting of (a) halogen; (b) OR.sub.b, wherein
R.sub.b is H, lower alkyl or aryl; (c) (C1-C6) cycloalkyl,
optionally substituted with 1-3 substituents selected from the
group consisting of halogen, lower alkyl, aryl and OR.sub.b,
wherein R.sub.b is H, lower alkyl or aryl; (d) (C6-C10) aryl,
optionally substituted with 1-3 substituents selected from the
group consisting of halogen, lower alkyl, aryl and OR.sub.b,
wherein R.sub.b is H, lower alkyl or aryl; (e) (C6-C10) heteroaryl,
optionally substituted with 1-3 substituents selected from the
group consisting of halogen, lower alkyl, aryl and OR.sub.b,
wherein R.sub.b is H, lower alkyl or aryl; or 2) an aryl radical
containing from 6 to 10 carbon atoms, optionally substituted with
1:3 substituents selected from the group consisting of 2) an aryl
radical containing from 6 to 10 carbon atoms, optionally
substituted with 1-3 substituents selected from the group
consisting of (a) halogen; (b) OR.sub.b, wherein R.sub.b is H,
lower alkyl or aryl; (c) (C1-C6) linear, branched or cyclic alkyl,
optionally substituted with 1-3 substituents selected from the
group consisting of halogen, lower alkyl, aryl and ORb, wherein Rb
is H, lower alkyl or aryl.
4. A compound of general formula (I) ##STR00025## wherein X is a
member of the group consisting of O and ##STR00026## wherein
R.sub.a is H, lower alkyl, alkenyl or alkynyl radical; and R is: an
aryl radical containing from 6 to 10 carbon atoms, optionally
substituted with 1-3 substituents selected from the group
consisting of (a) halogen, (b) OR.sub.b, wherein R.sub.b is H,
lower alkyl or aryl, (c) (C1-C6) linear, branched or cyclic alkyl,
optionally substituted with 1-3 substituents selected from the
group consisting of halogen, lower alkyl, aryl and OR.sub.b.
Description
[0001] This is a divisional of the U.S. application Ser. No.
12/285,901 filed on Oct. 16, 2008.
[0002] The subject of the present invention is a method for
preparing 2-alkoxy and 2-aryloxyestrogen compounds, and the
intermediate compounds prepared during the use of this method,
which intermediate compounds are useful intermediates in the
preparation of certain physiologically active compounds.
BACKGROUND OF THE INVENTION
[0003] 2-alkoxyestrogen derivatives are steroid compounds which
have or might have therapeutic value. In this respect,
2-methoxyestradiol, a physiological urinary metabolite of
endogenous estradiol, has been reported to be an anticancer agent
of high clinical relevance for many tumour types.
[0004] In U.S. Pat. No. 5,504,074, continuation U.S. Pat. No.
5,661,143 and divisional U.S. Pat. No. 5,892,069 there are claimed
methods of treating mammalian diseases characterized by undesirable
angiogenesis and abnormal mitosis, respectively, by administering
2-methoxyestradiol.
[0005] In U.S. Pat. No. 5,643,900 there is claimed a method of
suppressing the growth of solid tumours sustained by angiogenesis
in mammals comprising administering 2-methoxyestradiol.
[0006] In U.S. Pat. No. 5,958,892 there is claimed a method of
treating a patient having a tumour comprising the step of
administering a p53 gene in combination with
2-methoxyestradiol.
[0007] In WO02/42319 there are claimed compositions and methods for
treating mammalian diseases characterized by undesirable
angiogenesis by administering 2-methoxyestradiol.
[0008] 2-Methoxyestradiol is reported to have multiple mechanisms
of action, to be orally available, non-toxic, rapidly excreted,
non-estrogenic. For a short but comprehensive review, see for
example Pharmacotherapy, 23, 165, (2003).
[0009] In J. Med. Chem. 40, 2323, (1997), 2-ethoxy and
2-(2,2,2-trifluoroethoxy) estradiol analogues with enhanced
biological effects are disclosed.
[0010] In J. Med. Chem. 47, 5126, (2004), certain 17 alkyl
derivatives of 2-methoxyestradiol with enhanced metabolic stability
are reported.
[0011] Processes for the preparation of 2-methoxyestradiol are
known, and all refer to two general methodologies for the
preparation of 2-alkoxyestrogens from estradiol or estrone as
starting materials.
[0012] One methodology entails generally the halogenation at C-2 of
a suitably protected estradiol or estrone followed by a nuclophilic
displacement of the halogen atom by the alkoxy moiety. A variety of
conditions have been employed. See, for example, U.S. Pat. No.
6,051,726; U.S. Pat. No. 6,054,598; Hunan Daxue Xuebao, Ziran
Kexueban 24, 40, (1997) (C.A. 128:180572); Sichuan Daxue Xuebao,
Ziran Kexueban 27, 106, (1990) (C.A. 114:6922); Youji Huaxue 9,
266, 1989 (C.A. 111:195225); Steroids 471, 63-6 (1986); Sichuan
Daxue Xuebao, Ziran Kexueban 114, (1986) (C.A. 107:176307); J.
Chem. Res., Synop. 348, (1985); J. Chem. Soc., Chem. Commun. 533,
(1983); Synthesis 168, (1977). Besides the need of
protection/deprotection steps, the aromatic halogenation reaction
provides also varying amounts of 4- and 2,4-halogenated
intermediates in addition to the desired 2-halogen regioisomer,
which consequently should be carefully separated from the unwanted
regioisomers before proceeding in the synthesis.
[0013] The second methodology entails generally the aromatic
acylation at C-2 of a suitably protected estradiol or estrone
obtained by either a Friedel Craft-type direct acylation or the
above mentioned haloderivative. In both cases, mixture of
regioisomers may be obtained. A Baeyer-Villiger oxidation follows
to provide a 2-acyloxy derivative, which is hydrolized and the
resulting phenolic moiety is etherified with a suitable alkylating
agent. A variety of conditions have been employed. See, for
example, U.S. Pat. No. 6,051,726; U.S. Pat. No. 6,054,598; Synth.
Commun. 28, 4431, 1998; Bioorg. Med. Chem. Lett. 4, 1725, (1994);
J. Am. Chem. Soc. 80, 1213, (1958). The desired product is obtained
after a deprotection step.
[0014] Neither 2-aryloxyestrogen compounds nor their methods of
preparation have been previously disclosed.
[0015] Since estradiol and 19-nortestosterone (nandrolone) are
commercially available bulk materials which are similarly quoted on
the global market, we addressed the practical issue of using
19-nortestosterone as an alternative starting material in lieu of
estradiol/estrone, and have now found a process route to 2-alkoxy
and 2-aryloxy derivatives of estradiol and estrone starting from
19-norsteroid derivatives. The instant route, entailing
advantageously a straightforward aliphatic chemistry with no use of
any protecting group, implies the aromatization of the ring A of
the 2-alkoxy (aryloxy)-19-norsteroid derivative as the ultimate
step. In addition, certain of the process intermediates may have
therapeutic applications or are physiological precursors to
therapeutic agents.
SUMMARY OF THE INVENTION
[0016] The subject of the invention is a method for preparing
compounds of general formula (I):
##STR00001##
wherein X is a member of the group consisting of O and
##STR00002##
wherein R.sub.a is H, lower alkyl, alkenyl and alkynyl radical; and
R represents: a linear, branched or cyclic alkyl radical containing
from 1 to 8 carbon atoms, which may be further optionally
substituted with 1-3 substituents chosen among the group consisting
of
[0017] (a) halogen,
[0018] (b) OR.sub.b, wherein R.sub.b represents H, lower alkyl, and
aryl,
[0019] (c) (C1-C6) cycloalkyl, optionally substituted with 1-3
substituents such as halogen, lower alkyl, aryl, or OR.sub.b,
[0020] (d) (C6-C10) aryl, optionally substituted with 1-3
substituents such as halogen, lower alkyl, aryl, or OR.sub.b,
[0021] (e) (C6-C10) heteroaryl, optionally substituted with 1-3
substituents such as halogen, lower alkyl, aryl, or OR.sub.b, an
aryl radical containing from 6 to 10 carbon atoms, which may be
further optionally substituted with 1-3 substituents chosen among
the group consisting of
[0022] (a) halogen,
[0023] (b) OR.sub.b, wherein R.sub.b represents H, lower alkyl, and
aryl,
[0024] (c) (C1-C6) linear, branched or cyclic alkyl, optionally
substituted with 1-3 substituents such as halogen, lower alkyl,
aryl, or OR.sub.b, comprising the following steps: [0025] (1)
subjecting a compound of formula (II)
##STR00003##
[0025] wherein X being as defined above, with the proviso that X
does not represent O, and R.sub.c represents: [0026] H; [0027]
trifluoromethyl; [0028] phenyl, optionally substituted with fluoro,
chloro, nitro, methoxy; or (lower)alkoxycarbonyl; to a diazo
transfer reaction with a sulfonyl azide derivative so as to obtain
a compound of formula (III)
##STR00004##
[0028] wherein X being as defined above, with the proviso that X
does not represent O;
[0029] (2) treating the compound of formula (III) with a transition
metal compound, salt or complex, such as a compound, salt or
complex of, for example, Rh, Cu, or Ru in presence of a hydroxy
compound of formula ROH, R being as defined above, so as to obtain
a compound of formula (IV)
##STR00005##
wherein R being as defined above, and X represents the group
##STR00006##
; and, if desired, the compound of formula (IV) so obtained in step
(2) when R.sub.a represents hydrogen may be optionally oxidized
into a compound of formula (IV) wherein X represents O;
[0030] (3) the compound of formula (IV), wherein R and X being as
defined by all the meanings above is treated with an aromatization
agent so as to obtain a compound of formula (I), R and X being as
defined above;
and, if desired, as an optional part of the step (3), a compound of
formula (I), R being as defined above and X represents the
group
##STR00007##
[0031] wherein R.sub.a represents hydrogen, may be oxidized into a
compound of formula (I) wherein X represents O, and this compound
of formula (I) so obtained wherein X represents O, may be
transformed into a compound of formula (I), X being as defined by
the group
##STR00008##
R.sub.a being as defined above.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0032] As a non-limitative example of lower alkyl, alkenyl or
alkynyl radicals which R.sub.a may represent, there may be
mentioned methyl, ethyl, ethenyl and ethynyl radicals.
[0033] As a non-limitative example of a linear, branched or cyclic
alkyl radical containing from 1 to 8 carbon atoms which R may
represent, there may be mentioned methyl, ethyl, propyl, butyl,
pentyl, hexyl and octyl radicals, the branched isomers of these
radicals isopropyl, isobutyl, isopentyl, neopentyl, isohexyl,
3-methylpentyl, sec-butyl, tert-butyl and tert-pentyl, and the
cyclic isomers of the radicals cyclopropyl, cyclobutyl, cyclopentyl
and cyclohexyl.
[0034] As a non-limitative example of a linear, branched or cyclic
alkyl radical containing from 1 to 8 carbon atoms which may be
further optionally substituted with 1-3 substituents chosen among
the groups (a)-(e) as previously indicated which R may represent,
there may be mentioned: [0035] (a) 2,2,2-trifluoroethyl; [0036] (b)
2-methoxyethyl, 2-phenoxyethyl; [0037] (c) cyclopropylmethyl,
cyclopropylethyl, cyclohexylmethyl, (4-methylcyclohexyl)methyl,
(4-chlorocyclohexyl)methyl, (4-fluorocyclohexyl)methyl,
(4-methylcyclohexyl)methyl, (4-phenylcyclohexyl)methyl,
(4-methoxycyclohexyl)methyl, (4-phenoxycyclohexyl)methyl; [0038]
(d) benzyl, o-, m-, and p-chlorobenzyl, o-, m-, and p-fluorobenzyl,
4-methylbenzyl, 4-(o-, m-, and p-methoxyphenyl)benzyl, -o-, m-, and
p-methoxybenzyl, 3,4-dimethoxybenzyl, 3,4,5 trimethoxybenzyl,
1-naphthylmethyl, 2-naphthylmethyl; [0039] (e) pyridinemethyl,
furanmethyl, N-(loweralkyl)pyrrolemethyl, oxazolemethyl,
benzofuranmethyl.
[0040] As a non-limitative example of an aryl radical containing
from 6 to 10 carbon atoms, which R may represent, there may be
mentioned phenyl, 1-naphthyl, 2-naphthyl radicals.
[0041] As a non-limitative example of an aryl radical containing
from 6 to 10 carbon atoms, which may be further optionally
substituted with 1-3 substituents chosen among the groups (a)-(c)
as previously indicated which R may represent, there may be
mentioned: [0042] (a) (o-, m-, and p-chloro)phenyl, (o-, m-, and
p-fluoro)phenyl; [0043] (b) (o-, m-, and p-methoxy)phenyl,
3,4-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, (o-, m-, and
p-phenoxy)phenyl; [0044] (c) o-, m-, and p-tolyl, (o-, m-, and
p-ethyl)phenyl, (o-, m-, and p-trifluoromethyl)phenyl,
4-(2-methoxyethyl)phenyl.
[0045] The subject of the invention is a method for preparing, as
defined above, compounds of general formula (I), more particularly
in which R represents CH.sub.3, CH.sub.3CH.sub.2 or
CF.sub.3CH.sub.2, X being as defined by the group
##STR00009##
, most particularly for preparing the antitumour agent
2-methoxyestradiol.
[0046] According to the stepwise method of the present invention,
in the step (1) a compound of formula (II)
##STR00010##
R.sub.c and R.sub.a being as defined above, is subjected to a diazo
transfer reaction with a sulfonyl azide derivative so as to obtain
a compound of formula (III)
##STR00011##
R.sub.a being as defined above.
[0047] The diazo transfer reaction is a standard reaction which is
carried out according to methods known to persons skilled in the
art. As for the steroid field, the diazo transfer reaction has been
disclosed for instance in U.S. Pat. No. 4,317,817 for the obtaining
of a 4-diazosteroid. 21-diazosteroid derivatives are disclosed in
U.S. Pat. No. 2,832,772 and a 16-diazoestrone is disclosed in J.
Org. Chem. 38, 3525 (1973). 2- and 4-diazosteroid analogues have
been reported also in J. Org. Chem. 32, 2644 (1967), but obtained
with a different procedure.
[0048] As for the sulfonyl azide reagent, any sulfonyl azide
derivative known in the art and/or commercially available can be
used. As illustrative, non-limitative examples the following
benzenesulfonyl azide, methanesulfonyl azide, 4-toluenesulfonyl
azide, 4-dodecylbenzenesulfonyl azide, 4-acetamidobenzenesulfonyl
azide, 2,4,6-triisopropylbenzenesulfonyl azide,
trifluoromethanesulfonyl azide, 4-carboxybenzenesulfonyl azide,
4-nitrobenzenesulfonyl azide and imidazole-1-sulfonyl azide may be
cited.
[0049] A compound of formula (IIa) is easily prepared starting from
a suitable steroid by a Claisen-type acylation reaction. Preferred
starting steroids are 19-nortestosterone (nandrolone),
17alpha-ethyl-19-nortestosterone (norethandrolone),
17alpha-methyl-19-nortestosterone (normethandrone),
17alpha-ethynyl-19-nortestosterone (norethyndrone). The
Claisen-type acylation reaction is a standard reaction which has
been carried out in the steroid field according to methods known to
persons skilled in the art, for instance as described in J. Med.
Chem. 37, 4227 (1994), and in J. Am. Chem. Soc. 82, 2840
(1960).
[0050] Any suitable acylating agent may be used. As acylating
agents, preferred are the lower alkyl esters of formic, benzoic,
p-nitrobenzoic, oxalic acids, most preferred are ethyl formate and
diethyl oxalate. Claisen-type acylations are promoted by bases,
such as the lower alkoxides of alkali metals, for instance sodium
methoxide or sodium ethoxide, and are generally carried out in
apolar solvents, from where the product separates as a precipitate
in the form of the enolate salt. Since the above diazo transfer
reaction has to be carried out in basic media, an advantageous
aspect of the subject step of the present process provides the
isolation and purification of the compound of formula (II) from the
Claisen-type acylation in the form of the precipitated di-salt of
formula (IIa), M.sup.+ being the cation of an alkali metal, for
instance sodium,
##STR00012##
and the direct reaction of the compound salt of formula (IIa) so
obtained with the sulfonyl azide. The choice of the solvent is not
critical. There can be used lower alkanols, linear and cyclic alkyl
ethers, chlorinated solvents, water, and the mixtures thereof.
[0051] According to the stepwise method of the present invention,
in the step (2) the diazosteroid of formula (III), R.sub.a being as
defined above, is exposed to the catalysis of a transition metal
compound, salt or complex, such as a compound, salt or complex of
Rh, Cu or Ru, in presence of a hydroxy compound of formula ROH, R
being as defined above, in a suitable inert solvent. This results
in the decomposition of the diazo functional group which leaves the
reaction media as gaseous nitrogen, with insertion of the RO
moiety, so as to obtain a compound of formula (IVa)
##STR00013##
[0052] Although this type of intermolecular C--O bond formation is
documented in the scientific literature (reviewed for example in
Tetrahedron, 51, 10811, (1995)), and both in Helv. Chim. Acta, 33,
417, (1950), for example, and in the above cited U.S. Pat. No.
2,832,772 the insertion of an oxy group following the decomposition
of a primary diazo group in the side chain of a steroid is
disclosed, there is no knowledge or teaching about the
intermolecular RO-- insertion onto a secondary and cyclic carbon
following decomposition of the corresponding diazo group. In the
documents referring to the disclosure of 2- and 4-diazosteroids,
previously cited, there is no mention of further chemical
transformation of the diazo groups.
[0053] As for the transition metal compound, salt or complex such
as of, for example, Rh, Cu or Ru, the following non-limitative
derivatives may be cited: dirhodium tetracarboxylates (e.g. the
tetraacetate, tetraoctanoate, tetraperfluoropropionate, for a
review see Tetrahedron, 47, 1765, (1991)); certain dirhodium
tetraprolinates (Tet. Lett. 37, 4129 & 4133, (1996)); dirhodium
tetrakis(acetamide); copper bis(acetylacetonate) (but also other
compounds of copper, nickel and lead, as reported in the above
cited Tetrahedron, 51, 10811, (1995)); ruthenium dichloride
tris(triphenylphosphine) (Tet. Lett. 37, 8815, (1996)). Such
derivatives are either commercially available or easily prepared,
and the reaction is carried out in a suitable inert solvent at a
temperature which generally may range from -78.degree. C. to
100.degree. C. for from few minutes to 3 days. If desired, an
organic amine such as, for example, triethyl amine,
diisopropylethyl amine, pyridine, quinoline, and the like may be
added to the reaction mixture as an adjuvant.
[0054] As a person skilled in the art will appreciate, the
introduction of the RO-- moiety at C-2 of the steroid will generate
a new steric centre and two possible compounds (epimers) of formula
(IVa) may be obtained, namely with 2R and 2S configurations or, if
preferred, 2alfa and 2beta, with reference to the rule adopted with
naturally-occurring steroids. Indeed, in some instances, the two
possible epimers are obtained with varying 2R/2S ratios. After
having carried out extensive research, we have found that the 2R/2S
ratio is dependent on certain empyrical factors such as the nature
of the R group, the solvent used, the temperature at which the
reaction is performed, the type of transition metal salt or complex
used for the decomposition of the diazo group, the relative amount
of ROH in the reaction mixture, and the presence, if any, and the
nature of the adjuvant amine added.
[0055] Optionally, and if desired, after the completion of the
above described step (2) of the stepwise method of the present
invention, a compound of formula (IVa), wherein R.sub.a represents
hydrogen, may be oxidized into a compound of formula (IV), wherein
X represents oxygen. This oxidation reaction is a quite standard
reaction in the steroid field, which is carried out according to
methods known in the art for the oxidation of the 17-hydroxy group
to the 17-oxo group, as reported, for example, in U.S. Pat. No.
4,876,045.
[0056] According to the stepwise method of the present invention,
in the step (3) a compound of formula (IV), wherein R and X being
as defined by all the meanings above, is treated with an
aromatization agent so as to obtain a compound of formula (I), R
and X being as defined above.
[0057] The aromatization reaction may be carried out by either
chemical or biological agents, as known to a person skilled in the
art.
[0058] In a first aspect of the step (3) of the stepwise method of
the present invention, we have found that this aromatization may be
carried out as a dehydrogenation reaction catalyzed by Pd-, Pt-,
Rh-, Ru-, or Ni-based conventional hydrogenation catalysts in the
presence of suitable hydrogen acceptors such as cyclohexene,
cyclooctene, dialkyl maleates or any other commercially available
high boiling olefin, or nitrohydrocarbons, such as nitrobenzene, in
a suitable inert solvent at a temperature from 80.degree. C. to
180.degree. C., generally at the refluxing point of the appropriate
solvent/hydrogen acceptor mixture, for a time from 6 hours to 4
days. This catalyzed dehydrogenation reaction has been widely and
efficiently applied in the steroid field, as disclosed, for
example, in U.S. Pat. Nos. 3,44,9327, 3,458,502, 3,484,435 and
3,494,918.
[0059] In certain cases of this first aspect of the step (3) of the
invention, the catalyzed dehydrogenation reaction provides superior
yields when performed in presence of a base, such as aqueous sodium
or potassium hydroxides, carbonates and the like. Suprisingly, we
found that certain compounds of formula (IV), when exposed to the
action of the above mentioned bases, are transformed into compounds
possessing the "unnatural" stereochemistry at C-10 represented by
the formula (IVb)
##STR00014##
wherein R and X being as defined by all the meanings above, which
undergo the above mentioned catalyzed dehydrogenation reaction
rapidly and in high yield.
[0060] In a second aspect of the step (3) of the stepwise method of
the present invention, we have also found that this aromatization
may be carried out as a quinone-mediated dehydrogenation reaction,
for example a dehydrogenation by
2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) in an suitable inert
solvent under neutral or weakly acidic conditions, at a temperature
from 60.degree. C. to 120.degree. C., generally at the refluxing
point of the solvent, for a time from 1 to 24 hours. This
DDQ-mediated dehydrogenation reaction has been widely and
efficiently applied in the steroid field (see, for example, Chem.
Rev. 1967, 153).
[0061] In a third aspect of the step (3) of the stepwise method of
the present invention, we have also found that this aromatization
may be carried out by the oxidative action of selenium dioxide
(SeO.sub.2), a reagent widely used for the introduction of the
1,2-double bond in the A-ring of a steroid (U.S. Pat. Nos.
3,203,965 and 3,211,725).
[0062] In a forth aspect of the step (3) of the stepwise method of
the present invention, we have also found that this aromatization
may be carried out by the action of CuBr.sub.2--LiBr, as disclosed
in Tet. Lett. 10, 821 (1977) for the specific case of a
19-norsteroid.
[0063] In a fifth aspect of the step (3) of the stepwise method of
the present invention, we have also found that this aromatization
may be carried out by subjecting a compound of formula (IV) to the
action of enzymes from 1-dehydrogenating microorganisms. Methods,
enzymes and microorganisms useful to achieve this steroidal A-ring
1-dehydrogenation reaction leading to a 1,2-dehydrosteroid which,
as a person skilled in the art will appreciate, in the instant case
continues to undergo aromatization so that to obtain a compound of
formula (I) are described, for example, in U.S. Pat. Nos.
2,928,850, 3,047,469, 3,517,036, and 4,684,610; in J. Am. Chem.
Soc. 75, 5764, (1953), J. Biol. Chem. 234, 2009 & 2014, (1959),
Tetrahedron 18, 581 & 591, (1962), Biochem. 4, 2113, (1965), J.
Org. Chem. 31, 2512, (1966), and Biochim. Biophys. Acta, 1038, 60,
(1990). As non-limitative examples of 1-dehydrogenating
microorganisms the following Bacterium cyclooxidans,
Corynebacterium simplex, Corynebacterium hoagii, Cylindrocarpon
radicicola, Mycobacterium rhodocrous, Nocardia restrictus, Nocardia
rhodocrous, Nocardia corallin, Nocardia coeliaca, Nocardia
globerula, Nocardia aurantia, Pseudomonas testosteroni, Septomyxa
affinis may be cited.
[0064] Optionally, and if desired, after the completion of the
above described step (3) of the stepwise method of the present
invention, a compound of formula (I), wherein X represents
##STR00015##
wherein R.sub.a represents hydrogen, may be oxidized into a
compound of formula (I), wherein X represents oxygen, and this
compound of formula (I) so obtained wherein X represents O, may be
transformed into a compound of formula (I), X being as defined by
the group
##STR00016##
, R.sub.a being as defined above. Such transformations require
quite standard reactions in the steroid field, which are carried
out according to methods well known in the art.
[0065] A relevant aspect of this invention is that we found that
certain of the compounds of formula (IV) and formula (IVb) are
aromatized by aromatizing enzymes of mammalian source. That
19-nortestosterone derivatives are readily aromatized in vivo and
that this aromatization is carried out by certain members of the
cytochrome P450 enzyme superfamily (CYP) which are expressed in
many tissues (including tumors) is a well known phenomenon. The
most relevant mammalian enzyme is the ubiquitous cytochrome P450
aromatase, which has been reported to process both C-19 and 19-nor
steroid substrates providing the aromatization of the A-ring
(Biochemistry 7, 33, (1968); Nippon Naibunpi Gakkai Zasshi 62, 18,
(1986); J. Biol. Chem. 262, 5717, (1987); J. Endocrinol. 120, 251,
(1989); J. Steroid Biochem. 32, 537, (1989); J. Steroid Biochem.
32, 729, (1989); J. Steroid Biochem. 33, 949, (1989); J. Steroid
Biochem. 48, 297, (1994); J. Endocrinol. 144, 517, (1995)).
However, the action of the cytochrome P450 aromatase may be not
necessary and the oxidative introduction of double bonds into the
A-ring of 19-nor steroids leading to the aromatization of A-ring
can be catalyzed by other cytochrome P450 enzymes, for example
hepatic cytochrome P450 enzymes (Endocrinol Jpn 33, 527, (1986);
Nippon Sanka Fujinka Gakkai Zasshi 40, 87, (1988); Climateric, 10,
344, (2007)).
[0066] The following examples are included for purposes of
illustration only and are not to be construed as limitations
herein.
EXAMPLES
Example 1
General Preparation of 2-acyl-19-Nortestosterone Derivatives
(II)
[0067] Following published procedures, by applying some
modifications, the compounds were prepared by combining the
19-nortestosterone derivative with 2.5 molar equiv. of freshly
prepared sodium methoxide in toluene under a nitrogen atmosphere.
After stirring at room temperature for 30 min, 2.5 molar equiv. of
the ester were added and the reaction mixture was allowed to stir
for additional 12 hours. The resulting suspension was diluted with
a mixture of diethyl ether-toluene, and filtered. The filter cake
was washed several times with the above solvent mixture, then with
diethyl ether, and dried under reduced pressure to give the enolate
salt which was stored to be directly used as such in the next
step.
Example 2
2-diazo-19-nortestosterone (III, R.sub.a=H)
[0068] 1.38 g (ca. 3.3 mmol) of crude
2-ethoxalyl-19-nortestosterone sodium enolate (or the molar
equivalent of 2-formyl-19-nortestosterone) from Example 1 was
dissolved in water (5 ml) and washed with dichloromethane
(2.times.3 ml) in order to extract any unwanted organic material.
The aqueous phase was transferred in a reaction vessel and added
with 1M KOH solution (3.3 ml) and tetrahydrofuran (15 ml). To the
stirred reaction mixture, 4-toluenesulfonyl azide (0.650 g, 3.3
mmol) dissolved in tetrahydrofuran (7 ml) was added dropwise. The
reaction mixture was stirred for 24 hours at room temperature,
after which time the precipitated 4-toluenesulfonyl acyl imide
sodium/potassium salt was filtered off and washed with diethyl
ether. The combined filtrate and washings were washed with 0.5 N
NaOH aqueous solution, brine, dried over sodium sulfate, filtered
and evaporated under reduced pressure to yield an oily residue
which was dissolved in dichloromethane and precipitated by adding
hexanes to furnish the title product as a light yellow solid (0.590
g).
[0069] IR (CHCl.sub.3, cm.sup.-1): 2100, 1650
[0070] NMR (200 MHz, d, CDCl.sub.3): 5.85 (1H, br s), 3.68 (1H, t),
2.98 (1H, dd), 2.61 (1H, dd), 2.55-0.9 (17H, m), 0.82 (3H, s).
Example 3
2-methoxy-19-nortestosterone (IVa, R=CH.sub.3, R.sub.a=H)
[0071] To dirhodium tetraacetate (0.0044 g) in dichlomethane (4 ml)
were added diisopropylethylamine (0.032 g) and methanol (0.032 g).
To the resulting stirred suspension was added under nitrogen at
0-5.degree. C. 2-diazo-19-nortestosterone (0.130 g) of Example 2
dissolved in dichlomethane (4 ml), dropwise in 15 min. The reaction
mixture was allowed to stir for further 2 hour, filtered on a pad
of celite which was extensively washed with dichlorometane. The
combined dichlomethane filtrate and washings were washed with
water, dried over calcium chloride, filtered and evaporated under
reduced pressure to give a solid residue which, as judged by TLC
(40: 1 dichloromethane/methanol), was composed of the two C-2
epimers of the title compound at Rf=0.30 and Rf=0.23, respectively,
in a 9:1 approx. ratio. The epimeric mixture was separated by
column chromatography on silica gel (1:1 hexane/ethyl acetate):
[0072] fast eluting epimer (Rf=0.30, 40:1
dichloromethane/methanol):
[0073] NMR (200 MHz, d, CDCl.sub.3): 5.78 (1H, t, J=1.5 Hz), 3.75
(1H, dd), 3.65 (1H, t), 3.56 (3H, s), 2.5-0.9 (17H, m), 0.80 (3H,
s);
[0074] slow eluting epimer (Rf=0.23, 40:1
dichloromethane/methanol):
[0075] NMR (200 MHz, d, CDCl.sub.3): 5.88 (1H, br s), 4.10 (1H,
dd), 3.65 (1H, t), 3.56 (3H, s), 2.5-0.9 (17H, m), 0.80 (3H,
s).
[0076] The combined total weight of the two epimers was 0.075
g.
[0077] By substituting dirhodium tetraoctanoate for dirhodium
tetraacetate and operating in the same way as above, the two
epimers were obtained in a (Rf=0.30)/(Rf=0.23)=approx 5:5.
Example 4
2-Methoxyestradiol by Catalytic Dehydrogenation
[0078] To 0.500 g of a mixture of the two C-2 epimers obtained as
in Example 3 dissolved in dioxane (30 ml) were added 10% NYC
catalyst (0.250 g) and cyclohexene (10 ml). Under nitrogen, the
resulting reaction mixture was vigorously stirred at reflux. The
reaction was monitored by TLC (40:1 dichloromethane/methanol) and
after 8 hours the analysis showed the disappearance of the epimer
at Rf=0.23 and the formation of a new product migrating at Rf=0.41,
which resulted positive to the ferric chloride/potassium
ferricyanide test. Since at this stage the reaction seemed to be
sluggish or not to proceed any further, potassium hydroxyde (0.6 ml
of a 1N aqueous solution) was added and the reaction mixture was
stirred and refluxed for additional 12 hours, after which time the
epimer at Rf=0.30 has disappeared and the product at Rf=0.41
increased. The cooled reaction mixture was neutralized by adding
hydrochloric acid (0.6 ml of a 1N aqueous solution), the catalyst
was filtered off and washed with dioxane. The filtrate was
evaporated to dryness under reduced pressure and the resulting
residue chromatographed on silica gel column (8:2 hexane/ethyl
acetate) to give 2-methoxyestradiol (0.300 g).
[0079] NMR (200 MHz, d, CDCl.sub.3): 6.79 (1H, s), 6.64 (1H, s),
5.40 (1H, s), 3.85 (3H, s), 3.70 (1H, t), 2.75 (2H, m), 2.25-1.1
(13H, m), 0.81 (3H, s).
Example 5
2-methoxy-10-epi-19-nortestosterone (IVb, R=CH.sub.3, X=
##STR00017##
[0081] This example was aimed at understanding the finding of
Example 4. 2-Methoxy-19-nortestosterone (epimer at Rf=0.30, 0.050
g) was refluxed in dioxane (5 ml) in presence of potassium
hydroxyde (0.06 ml of a 1N aqueous solution). After 3 hours, TLC
analysis (95:5 dichloromethane/ethanol) showed that the starting
material equilibrated (approx. 1:1) to a product migrating at a
slightly higher Rf. The reaction mixture was then cooled and
evaporated to dryness under reduced pressure, and the residue
chromatographed (8:2 hexane/ethyl acetate) to yield, as a first
eluting, a product to which was tentatively assigned the structure
of the C-10 epimer of the starting material based on its NMR
data.
[0082] NMR (200 MHz, d, CDCl.sub.3): 5.78 (1H, br s), 3.65 (1H, t),
3.50 (1H, dd), 3.38 (3H, s), 2.5-0.9 (17H, m), 0.78 (3H, s).
Example 6
2-Methoxyestradiol by Dichlorodicyanobenzoquinone Oxidation
[0083] 0.050 g of 2-methoxy-19-nortestosterone and 0.057 g of
dichlorodicyanobenzoquinone were refluxed in 10 ml of anhydrous
dioxane for about 15 hours. To remove the DDQ the suspension was
filtered through alumina. After evaporation of the solvent the
residue was dissolved in ethyl acetate, the organic layer washed
with water, dried over sodium sulfate and the solvent removed under
reduced pressure. The crude product was chromatographed as reported
in Example 4 to yield 0.020 g of 2-methoxyestradiol.
Example 7
2-Methoxyestradiol by Selenium Dioxide Oxidation
[0084] A mixture of 2-methoxy-19-nortestosterone (0.050 g),
selenium dioxide (0.050 g), tert-butanol (20 ml) and 0.1 ml
pyridine was heated at reflux under nitrogen for about 30 hours.
The cooled solution was filtered and then evaporated to dryness
under reduced pressure. The residue was taken up in ethyl acetate
(20 ml), treated with charcoal, filtered and washed with water,
ammonium sulfide aqueos solution, cold 17% ammonium hydroxide, cold
dilute hydrochloric acid, water, dried over sodium sulfate and
finally evaporated to dryness under reduced pressure. The crude
product was chromatographed as described in Example 4 to yield
0.025 g of 2-methoxyestradiol.
Example 8
2-Methoxyestradiol by Action of CuBr.sub.2--LiBr
[0085] A mixture of 2-methoxy-19-nortestosterone (0.050 g),
CuBr.sub.2 (0.135 g), LiBr (0.015 g) and acetonitrile (25 ml) was
heated at reflux under nitrogen for 30 min, after which time the
heating was stopped and cold water was added to the reaction
mixture. The cold reaction mixture was filtered, the organic layer
separated and the aqueous layer extracted twice with ethyl acetate.
The combined organics were washed with water and dried over sodium
sulfate and finally evaporated to dryness under reduced pressure.
The crude product was chromatographed as described in Example 4 to
yield 0.030 g of 2-methoxyestradiol.
Example 9
2-Methoxyestradiol by Microbiological Transformation
[0086] Following the procedure reported in U.S. Pat. No. 3,517,036,
2 mg of 2-methoxy-19-nortestosterone are combined with a cell-free
enzyme preparation from Corynebacterium simplex. At the end of the
incubation period, the mixture is extracted with methyl isobutyl
ketone. TLC analysis of the combined extracts (40:1
dichloromethane/methanol) showed the presence of
2-methoxyestradiol, confirmed against an authentic reference
standard.
Example 10
2-Methoxyestradiol by Placental Preparation (Aromatase)
[0087] Placental preparations equivalent to those obtainable from
30, 7 g of wet tissue, prepared and tested for aromatase activity
as described in J. Biol. Chem., 234, 268 (1959) and in J. Biol.
Chem., 249, 5364 (1974), respectively, were reconstituted in
aromatase phosphate buffer (pH 7.5, 10 mM potassium phosphate
buffer, 100 mM KCl, 1 mM EDTA and 1 mM dithiothreitol) and combined
with 0.5 mg 2-methoxy-19-nortestosterone, dissolved in propylene
glycol, and 25 umol NADPH in a final volume of 15 ml. The resulting
mixture was incubated in air for 3 hours at 37.degree. C. under
stirring, after which time was extracted with CHCl.sub.3
(4.times.10 ml) and the combined extracts were washed with water
and evaporated under reduced pressure. The residue, dissolved in
hexane (30 ml), was then extracted with 90% aqueous methanol
(3.times.25 ml) and the combined methanolic extracts were
evaporated under reduced pressure to leave a residue which was
taken up with toluene (70 ml). The toluene was extracted with 1N
NaOH (5.times.15 ml) and the combined basic extracts were adjusted
to pH 8.5 and extracted with ethyl ether. TLC analysis of the
ethereal solution (40:1 dichloromethane/methanol) showed the
presence of 2-methoxyestradiol, confirmed against an authentic
reference standard.
Example 11
2-(p-methoxyphenoxy)-19-nortestosterone (IVa,
R=p-(CH.sub.3O)--C.sub.6H.sub.4, R.sub.a=H)
[0088] The title compound was obtained from
2-diazo-19-nortestosterone, p-methoxyphenol and
[0089] dirhodium tetraacetate following the same procedure as in
Example 3.
[0090] NMR (200 MHz, d, CDCl.sub.3): 6.98 (2H, d), 6.70 (2H d),
5.80 (1H, br s,), 4.45 (1H, dd), 3.70 (3H, s), 3.65 (1H, t),
2.5-0.9 (17H, m), 0.81 (3H, s).
[0091] By substituting 3,4-dimethoxyphenol for p-methoxyphenol and
following the same procedure, there was obtained:
[0092] 2-(3,4-Dimethoxyphenoxy)-19-nortestosterone (IVa,
R=3,4-(CH.sub.3O).sub.2--C.sub.6H.sub.3, R.sub.a=H)
[0093] NMR (200 MHz, d, CDCl.sub.3): 6.51 (1H, d), 6.22 (1H s),
6.15 (1H, dd), 5.80 (1H, br s,), 4.45 (1H, dd), 3.70 (6H, s), 3.65
(1H, t), 2.5-0.9 (17H, m), 0.81 (3H, s).
Example 12
2-(p-methoxyphenoxy)estradiol (I, R=p-(CH.sub.3O)C.sub.6H.sub.4,
X=
##STR00018##
[0095] The title compound was obtained from
2-(p-methoxyphenoxy)-19-nortestosterone of Example 11 by catalytic
dehydrogenation as described in Example 4.
[0096] NMR (200 MHz, d, CDCl.sub.3): 7.35 (2H, d), 6.80 (2H, d),
6.79 (1H, s) 6.64 (1H, s), 5.40 (1H, s), 3.75 (3H, s), 3.70 (1H,
t), 2.75 (2H, m), 2.25-1.1 (13H, m), 0.81 (3H, s).
[0097] By substituting 2-(3,4-Dimethoxyphenoxy)-19-nortestosterone
(IVa, R=3,4-(CH.sub.3O).sub.2--C.sub.6H.sub.3, R.sub.a=H) for
2-(p-methoxyphenoxy)-19-nortestosterone (IVa,
R=p-(CH.sub.3O)--C.sub.6H.sub.4, R.sub.a=H) and following the same
procedure, there was obtained:
[0098] 2-(3,4-Dimethoxyphenoxy)estradiol (I,
R=(3-CH.sub.3O)(4-CH.sub.3O)C.sub.6H.sub.3, X=
##STR00019##
[0099] NMR (200 MHz, d, CDCl.sub.3): 6.79 (1H, s), 6.64 (1H, s),
6.51 (1H, d), 6.22 (1H s), 6.15 (1H, dd), 5.40 (1H, s), 3.75 (6H,
s), 3.70 (1H, t), 2.75 (2H, m), 2.25-1.1 (13H, m), 0.81 (3H,
s).
* * * * *