U.S. patent application number 10/534079 was filed with the patent office on 2006-09-21 for synthesis of estetrol via estrone derived steroids.
Invention is credited to Thomas Koch, Mark Theodoor Verhaar, Erwin Gerardus Jacobus Warmerdam.
Application Number | 20060211669 10/534079 |
Document ID | / |
Family ID | 32309415 |
Filed Date | 2006-09-21 |
United States Patent
Application |
20060211669 |
Kind Code |
A1 |
Verhaar; Mark Theodoor ; et
al. |
September 21, 2006 |
Synthesis of estetrol via estrone derived steroids
Abstract
A process is provided for the making of estetrol starting from a
3-A-oxy-estra 1,3,5(10),15-tetraen-17-one, wherein A is a
C.sub.1-C.sub.5 alkyl group, preferably a methyl group, or a
C.sub.7-C.sub.12 benzylic group, preferably a benzyl group. This
process is particularly suitable to industry.
Inventors: |
Verhaar; Mark Theodoor;
(Groningen, NL) ; Koch; Thomas; (Groningen,
NL) ; Warmerdam; Erwin Gerardus Jacobus; (Weert,
NL) |
Correspondence
Address: |
The Webb Law Firm
436 Seventh Avenue
700 Koppers Building
Pittsburgh
PA
15219-1818
US
|
Family ID: |
32309415 |
Appl. No.: |
10/534079 |
Filed: |
November 7, 2003 |
PCT Filed: |
November 7, 2003 |
PCT NO: |
PCT/NL03/00782 |
371 Date: |
November 14, 2005 |
Current U.S.
Class: |
514/182 ;
552/612 |
Current CPC
Class: |
C07J 21/00 20130101;
C07J 1/0059 20130101; C07J 1/007 20130101; C07J 1/0066
20130101 |
Class at
Publication: |
514/182 ;
552/612 |
International
Class: |
C07J 1/00 20060101
C07J001/00; A61K 31/56 20060101 A61K031/56 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 8, 2002 |
EP |
02079676.9 |
Claims
1-31. (canceled)
32. A process for the preparation of
estra-1,3,5(10)-trien-3,15.alpha.,16.alpha.,17.beta.-tetraol (1),
comprising the steps of: 1) converting estrone (7) into
3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6), wherein A is a
protecting group; 2) reduction of the 17-keto group of
3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6) to
3-A-oxy-estra-1,3,5(10),15-tetraen-17.beta.-ol (5); 3) protection
of the 17-OH group of
3-A-oxy-estra-1,3,5(10),15-tetraen-17.beta.-ol (5) to
3-A-oxy-17-C-oxy-extra-1,3,5(10),15-tetraene (4), wherein C is a
protecting group; 4) oxidizing the carbon-carbon double bond of
ring D of 3-A-oxy-17-C-oxy-estra-1,3,5(10),15-tetraene (4) to
protected estetrol (3); and 5) removing the protecting groups,
wherein protecting group A is removed first to form 17-OC protected
estetrol (2) and subsequently protecting group C is removed to form
estetrol (1); wherein the protecting group A is selected from the
group consisting of a C.sub.1-C.sub.5 alkyl group and a
C.sub.7-C.sub.12 benzylic group and the protecting group C is
selected from monofunctional aliphatic hydroxyl protecting
groups.
33. The process according to claim 32, wherein the protecting group
is a C.sub.7-C.sub.12 benzylic group.
34. The process according to claim 32, wherein the protecting group
is a benzyl group.
35. The process according to claim 32, wherein the protecting group
C is selected from monofunctional aliphatic hydroxyl protecting
groups.
36. The process according to claim 35, wherein the monofunctional
aliphatic hydroxyl protecting group is acetyl.
37. The process according to claim 32, wherein the reduction of the
carbonyl group is carried out using a reducing agent selected from
the group of metal hydride compounds.
38. The process according to claim 37, wherein the metal hydride
compound is selected from the group consisting of LiAlH.sub.4,
NaBH.sub.4, NaBH(OAc).sub.3, ZnBH.sub.4, and
NaBH.sub.4/CeCl.sub.3.
39. The process according to claim 38, wherein the metal hydride
compound is NaBH.sub.4 in combination with CeCl.sub.3 hydrate.
40. The process according to claim 32, wherein the oxidation of the
carbon-carbon double bond in ring D is carried out with an
oxidizing agent comprising osmium tetroxide.
41. The process according to claim 40, wherein the oxidizing agent
is osmium tetroxide immobilized on PVP (OsO.sub.4--PVP).
42. The process according to claim 32, wherein the oxidization of
the carbon-carbon double bond in ring D is carried out with a
catalytic amount of OsO.sub.4--PVP.
43. The process according to claim 42, wherein the OsO.sub.4--PVP
is used in combination with a co-oxidant.
44. The process according to claim 43, wherein the co-oxidant is
selected from the group consisting of trimethylamine-N-oxide,
N-methyl morpholine-N-oxide and hydrogen peroxide.
45. The process according to claim 44, wherein the co-oxidant is
trimethylamine-N-oxide.
46. The process according to claim 32, wherein the protective
C.sub.7-C.sub.12 benzylic group is removed by catalytic
hydrogenation conditions.
47. The process according to claim 46, wherein the catalytic
hydrogenation conditions comprise a hydrogenation reaction using Pd
on activated carbon under a hydrogen atmosphere.
48. The process according to claim 32, wherein the protective
C.sub.1-C.sub.5 alkyl group is removed by using BBr.sub.3.
49. A process for the preparation of
3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6), comprising the steps
of: (a1) converting the 3-OH group of estron (7) into a 3-AO group
to form 3-A-oxy-estra-1,3,5(10)-trien-17-one (8); (b1) converting
the 17-keto group of 3-A-oxy-estra-1,3,5(10)-trien-17-one (8) into
a protected keto group to form 3-A-oxy-17-D-estra-1,3,5(10)-triene
(9); (c1) halogenating C.sub.16 of
3-A-oxy-17-D-estra-1,3,5(10)-triene (9) to form
3-A-oxy-16-X-17-D-estra-1,3,5(10)-triene (10), wherein X is a
halogen atom selected from the group consisting of chloride,
bromide and iodide; (d1) dehalogenating
3-A-oxy-16-X-17-D-estra-1,3,5(10)-triene (10) to
3-A-oxy-17-D-estra-1,3,5(10),15-tetraene (11); and (e1)
deprotecting the protected keto group of
3-A-oxy-17-D-estra-1,3,5(10),15-tetraene (11) to form
3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6); wherein A is
selected from a C.sub.1-C.sub.5 alkyl group or a C.sub.7-C.sub.12
benzylic group and wherein D is ethylene dioxy.
50. The process according to claim 49, wherein the halogen atom is
bromide.
51. The process according to claim 49, wherein A is a methyl
group.
52. The process according to claim 49, wherein A is a benzyl
group.
53. A process for the preparation of
3-A-oxy-estra-1,3,5(10),15-tetraene-17-one (6), comprising the
steps of: (a2) converting the 17-keto group of estron (7) into a
protected keto group to form 17-D-estra-1,3,5(10)-trien-3-ol (12);
(b2) converting the 3-OH group of 17-D-estra-1,3,5(10)-trien-3-ol
(12) into a 3-AO group to form
3-A-oxy-17-D-estra-1,3,5(10)-trien-17-one (9); (c2) halogenating
C.sub.16 of 3-A-oxy-17-D-estra-1,3,5(10)-triene (9) to form
3-A-oxy-16-X-17-D-estra-1,3,5(10)-triene (10) wherein X is a
halogen atom selected from the group consisting of chloride,
bromide and iodide; (d2) dehalogenating
3-A-oxy-16-X-17-D-estra-1,3,5(10)-triene (10) to
3-A-oxy-17-D-estra-1,3,5(10),15-tetraene (11); and (e2)
deprotecting the protected keto group of
3-A-oxy-17-D-estra-1,3,5(10),15-tetraene (11) to form
3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6); wherein A is
selected from a C.sub.1-C.sub.5 alkyl group or a C.sub.7-C.sub.12
benzylic group, and wherein D is ethylene dioxy.
54. The process according to claim 53, wherein the halogen atom is
bromide.
55. The process according to claim 53, wherein A is a methyl
group.
56. The process according to claim 53, wherein A is a benzyl
group.
57. The process according to claim 49, wherein the protected keto
group D is formed by converting the 17-keto group with ethylene
glycol.
58. The process according to claim 53, wherein the protected keto
group D is formed by converting the 17-keto group with ethylene
glycol.
59. The process according to claim 49, wherein steps (e1) and (e2)
are carried out in the presence of a component selected from the
group consisting of p-toluenesulfonic acid, pyridinium
p-toluenesulfonate and pyridinium chloride.
60. The process according to claim 49, wherein steps (e1) and (e2)
are carried out in the presence of p-toluenesulfonic acid.
61. The process according to claim 49, wherein steps (e1) and (e2)
are carried out in the presence of p-toluenesulfonic acid
monohydrate using aqueous acetone as solvent.
62. The process according to claim 53, wherein steps (e1) and (e2)
are carried out in the presence of a component selected from the
group consisting of p-toluenesulfonic acid, pyridinium
p-toluenesulfonate and pyridinium chloride.
63. The process according to claim 53, wherein steps (e1) and (e2)
are carried out in the present of p-toluenesulfonic acid.
64. The process according to claim 53, wherein steps (e1) and (e2)
are carried out in the presence of p-toluenesulfonic acid
monohydrate using aqueous acetone as solvent.
65. A pharmaceutical composition, comprising a carrier and the
product obtainable by the method of claim 32.
66. A method of hormone replacement therapy, of treating vaginal
dryness, of contraception, of enhancing libido, of treating skin,
of promoting wound healing or of treating or preventing an
autoimmune disease, breast tumours or colorectal tumours,
comprising administering the pharmaceutical composition of claim 65
to a subject in need thereof.
67. A cosmetic method of treating skin, comprising adminstering
topically the product obtainable by the process of claim 32.
68. A compound according to formula 5, wherein A is a
C.sub.7-C.sub.12 benzylic group.
69. A compound according to formula 4, wherein A is a
C.sub.7-C.sub.12 benzylic group and C is selected from
monofunctional aliphatic hydroxyl protecting groups.
70. A compound according to formula 3, wherein A is selected from a
C.sub.1-C.sub.5 alkyl group or a C.sub.7-C.sub.12 benzylic group
and C is selected from mono functional aliphatic hydroxyl
protecting groups.
71. A compound according to formula 2, wherein C is selected from
monofunctional aliphatic hydroxyl protecting group.
72. A compound according to formula 10, wherein A is a
C.sub.7-C.sub.12 benzylic group, D is ethylene dioxy and X is
halogen.
73. A compound according to formula 11, wherein A is a
C.sub.7-C.sub.12 benzylic group and D is ethylene dioxy.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to the synthesis of estetrol
[estra-1,3,5(10)-trien-3,15.alpha.,16.alpha.,17.beta.-tetraol; CAS
Nr. 15183-37-6; for convenience, this compound is referred to in
this patent application as "estetrol"] via estrone derived
steroids, preferably to the synthesis of estetrol which is obtained
with high yield and high purity.
BACKGROUND OF THE INVENTION
[0002] Estrogenic substances are commonly used in methods of
Hormone Replacement Therapy (HRT) and methods of female
contraception. These estrogenic substances can be divided in
natural estrogens and synthetic estrogens. Examples of natural
estrogens that have found pharmaceutical application include
estradiol, estrone, estriol and conjugated equine estrogens.
Examples of synthetic estrogens, which offer the advantage of high
oral bioavailability include ethinyl estradiol and mestranol.
[0003] Recently, estetrol has been found effective as an estrogenic
substance for use in HRT, disclosure of which is given in the
Applicant's co-pending application WO 02/094276. Estetrol is a
biogenic estrogen that is endogeneously produced by the fetal liver
during human pregnancy. Other important applications of estetrol
are in the fields of contraception, therapy of auto-immune
diseases, prevention and therapy of breast and colon tumors,
enhancement of libido, skin care, and wound healing as described in
the Applicant's co-pending applications WO 02/094276, WO 02/094279,
WO 02/094278, WO 02/094275, EP 2077272.9, EP 2077273.7, WO
03/041718, WO 03/018026, EP 2077812.2, and EP 2077322.2.
[0004] The synthesis of estetrol and derivatives thereof on a
laboratory scale basis is known in the art: Fishman J., Guzik H.,
J. Org. Chem. 33, 3133-3135 (1968); Nambara T. et al., Steroids 27,
111-121 (1976); or Suzuki E. et al., Steroids 60, 277-284
(1995).
[0005] Fishman J., Guzik H., J. Org. Chem. 33, 3133-3135 (1968)
discloses a successful synthesis of estetrol from an estrone
derivative (compound (III); cf. for a synthesis of compound (III)
Cantrall, E. W., Littell, R., Bernstein, S. J. Org. Chem 29,
214-217 (1964)). In a first step, the carbonyl group at C.sub.17 of
compound (III) was reduced with LiAlH.sub.4 to
estra-1,3,5(10),15-tetraene-3,17-diol (compound VIa) that was
isolated as the diacetate (compound VIb). Compound VIb was
subjected to cis-hydroxylation of the double bond of ring D by
using OsO.sub.4 which resulted into the formation of
estra-1,3,5(10)-triene-3,15.alpha.,16.alpha.,17.beta.-tetraol-3,17-diacet-
ate (compound Ib) that under heating with K.sub.2CO.sub.3 in
methanol produces estetrol (Scheme 1). ##STR1##
[0006] The overall yield of this three step process is, starting
from estrone derivative III, only about 7%. It is worth noting that
the protected derivative
17,17-ethylenedioxyestra-1,3,5(10),15-tetraene-3-ol-3-acetate
(compound IV) could be cis-hydroxylated to its
15.alpha.,16.alpha.-diol derivative (compound Va), but that
thereafter the dioxolane group could not be removed (p-toluene
sulfonic acid in acetone at room temperature) or that the
hydrolysis (aqueous sulfuric acid in warm dioxane) of the dioxolane
group resulted in a mixture containing a multitude of products
(Scheme 2). ##STR2##
[0007] Nambara T. et al., Steroids 27, 111-121 (1976) discloses
another synthesis of estetrol wherein estrone is the starting
material. The carbonyl group of estrone is first protected by
treatment with ethylene glycol and pyridine hydrochloride followed
by acetylation of the hydroxy group at C.sub.3. The next sequence
of steps involved a bromination/base catalyzed dehydrobromination
resulting into the formation of
17,17-ethylenedioxyestra-1,3,5(10),15-tetraene-3-ol (compound IVa).
This compound IVa was subsequently acetylated which produced
17,17-ethylenedioxyestra-1,3,5(10),15-tetraene-3-ol-3-acetate
(compound IVb). In a next step, the dioxolane group of compound IVb
was hydrolysed by using p-toluene sulfonic acid to compound Vb,
followed subsequently by reduction of the carbonyl group at
C.sub.17 (compound Vc) and oxidation of the double bond of ring D
thereby forming
estra-1,3,5(10)-triene-3,15.alpha.,16.alpha.,17.beta.-tetraol-3,17-diacet-
ate (compound VIb). See Scheme 3.
[0008] Suzuki E. et al., Steroids 60, 277-284 (1995) also discloses
the synthesis of estetrol by using compound Vb of Nambara T. et al.
as starting material. The carbonyl group at C.sub.17 of this
compound was first reduced followed by acetylation yielding
estra-1,3,5(10),15-tetraene-3,17-diol-3,17-diacetate (compound 2b).
The latter was subjected to oxidation with OsO4 which provided
estra-1,3,5(10)-triene-3,15.alpha.,16.alpha.,17.beta.-tetraol-3,17-diacet-
ate (compound 3b) in 46% yield. ##STR3##
[0009] According to the Nambara T. et al. and Suzuki E. et al., the
synthesis of estetrol can be performed with a yield of
approximately 8%, starting from estrone.
[0010] Poirier D., et al., Tetrahedron 47, 7751-7766 (1991)
discloses the following compounds which were prepared according to
methods that have been used to prepare similar compounds:
##STR4##
[0011] Dionne, P. et al., Steriods 62, 674-681 (1997) discloses the
compound shown above wherein R is either methyl or
t-butyldimethylsilyl.
[0012] Magnus, P. et al., J. Am. Chem. Soc. 120, 12486-12499 (1998)
discloses that the main methods for the synthesis of
.alpha.,.beta.-unsaturated ketones from saturated ketones are (a)
halogenation followed by dehydrohalogenation, (b) utilising sulphur
or selenium derivatives, (c) DDQ and (d) utilizing palladium(II)
complexes.
[0013] Furthermore, it has also been found that by following the
prior art methods mentioned above, estetrol of high purity was
obtained only in low yield when using an acetyl group as a
protecting group for the 3-hydroxy group of
estra-1,3,5(10),15-tetraen-3-ol-17-one, in particular because its
sensitivity to hydrolysis and solvolysis. In particular, the
lability of the acetyl group lead not only to an increased
formation of byproducts during the reactions, but also during
chromatography and crystallisation for purification of intermediate
products when protic solvents such as methanol were used.
Therefore, it is difficult to isolate purified estetrol and
intermediates thereof in good yield.
[0014] Additionally, the reduction of the carbonyl group at
C.sub.17 with LiAlH.sub.4 proceeds with a low selectivity since
various amounts of .beta.-estradiol
(estra-1,3,5(10)-trien-3,17.beta.-diol) are obtained as well.
Obviously, the formation of such a by-product reduces the yield as
well as the purity of the desired product which requires additional
purification steps.
[0015] The prior art methods also employ stoichiometric amounts of
OsO.sub.4 in the oxidation step that is known to be a toxic and
expensive compound. Consequently, the use of such a reagent is
undesired in view of safety and operational costs.
[0016] Accordingly, it is an object of the present invention to
provide a synthesis route for estetrol whereby high yields and high
purities of estetrol are obtained.
[0017] Still accordingly, there is a need for a synthesis of
estetrol wherein the production of by-products is limited. i.e.
preferably less than its detection level.
[0018] It is a preferred object of the invention to provide a
synthesis of estetrol wherein good yield and good purity of
estetrol are provided.
[0019] By a good yield, it is meant a yield of at least 10%,
preferably higher than 10%, more preferably of at least 12.5%,
starting from estrone (100%).
[0020] By a good purity, it is meant a purity of at least 97%,
preferably of at least 98%, more preferably of at least 99%.
Preferably, single impurities are not allowed to exceed 1%. Also
preferred is that .beta.-estradiol is not allowed to exceed the
detection level.
[0021] For the purpose of the present invention, determination of
purity is made by HPLC-MS. The following conditions are used:
[0022] HPLC-MS is performed using a Hewlett Packard 1100 series:
[0023] Column: Discovery C18 (150.times.4.6 mm) Supelco [0024]
Mobile phase: Solution A:Solution B=70:30 (5 min).fwdarw.(10
min).fwdarw.10:90 (5 min) [0025] Flow: 1 mL/min [0026] UV: 280 nm
[0027] Temp: 22.degree. C. [0028] MS: API-ES negative [0029]
Solution A: 9.65 g NH.sub.4OAc, 2250 mL H.sub.2O, 150 mL MeOH, 100
mL CH.sub.3CN [0030] Solution B: 9.65 g NH.sub.4OAc, 250 mL
H.sub.2O, 1350 mL MeOH, 900 mL CH.sub.3CN
[0031] It has now been found that protecting the 3-OH group of
estra-1,3,5(10),15-tetraen-3-ol-17-one by an C.sub.1-C.sub.5 alkyl
group, preferably a methyl group, or a C.sub.7-C.sub.12 benzylic
group, preferably a benzyl group, fulfils such a need. Indeed, it
has been found that the use of a more stable protective group such
as a C.sub.1-C.sub.5 alkyl group, preferably a methyl group, or a
C.sub.7-C.sub.12 benzylic group, preferably a benzyl group, on the
3-OH group is not cleaved at an undesired stage of the synthesis.
Therefore the formation of by-products is limited and the
purification of intermediates is more practical.
[0032] In this patent application the term "alkyl" includes linear,
branched and cyclic alkyl groups such as methyl, ethyl, n-propyl,
i-propyl, c-propyl, n-butyl, s-butyl, t-butyl, c-butyl, n-pentyl,
s-pentyl, t-pentyl, c-pentyl and methylcyclobutyl. Additionally,
the C.sub.7-C.sub.12 benzylic group has to be understood as a
benzyl group that may be substituted with one or more substituents
at the ortho, meta and/or para position of the aromatic nucleus,
wherein the substituents are aliphatic groups, optionally
substituted by one or more heteroatoms and/or halogen atoms that do
not adversely interfere with the synthetic process. As is obvious
to a skilled person in the art, the alkyl and benzylic groups are
intended as a protecting group and these groups must therefore be
relatively easy to add and relatively easy to remove under such
conditions that do not have an adverse effect on the molecular
structure of the estrone derived steroid molecules.
[0033] Because of the selected protecting groups which are used and
the yield and purity obtained, it appeared that the synthesis
disclosed in this patent application can be suitably transposed to
an industrial scale. This represents a particular advantage in
comparison to the current lab-scale syntheses which have been
disclosed in the prior art and which hamper from several
disadvantages as disclosed above. Indeed, a problem with industrial
syntheses are the quantities of chemicals as well as the toxicity
and hazardous properties thereof which are involved, thus making
the prior art lab-scale methods not transposable to an industrial
scale. The reason behind such impossible replication is that
usually the known method either does not provide a sufficient
yield, i.e. at least 10% to be considered economically feasible
from an industrial point of view and/or produce by-product(s) which
necessitates at least a subsequent purification step, thus raising
the cost of the method.
[0034] Accordingly, it is also another preferred object of the
invention to provide a method which is suitable for use in
industry.
SUMMARY OF THE INVENTION
[0035] Accordingly, in one aspect of the present invention, a
process is provided for the obtainment of
1,3,5(10)-estratrien-3,15.alpha.,16.alpha.,17.beta.-tetraol which
comprises the steps of: [0036] 1)
3-A-oxy-estra-1,3,5(10),15-tetraen-17-one wherein A is a protecting
group; [0037] 2) reduction of the 17-keto group; [0038] 3)
protection of the reduced carbonyl function of the
3-A-oxy-estra-1,3,5(10),15-tetraen-17-one; [0039] 4) oxidizing the
alkene bond of the cyclopentenol moiety of the acetylated
3-A-oxy-estra-1,3,5(10),15-tetraen-17-ol; and [0040] 5) removing
the protecting groups; wherein the protecting group A is selected
from an C.sub.1-C.sub.5 alkyl group, preferably a methyl group, or
a C.sub.7-C.sub.12 benzylic group, preferably a benzyl group.
[0041] Hence, according to this first aspect of the invention, a
process is provided for the preparation of
estra-1,3,5(10)-trien-3,15.alpha.,16.alpha.,17.beta.-tetraol (1)
which comprises the steps of: [0042] 1) converting estrone (7) into
3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6), wherein A is a
protecting group; [0043] 2) reduction of the 17-keto group of
3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6) to
3-A-oxy-estra-1,3,5(10),15-tetraen-17.beta.-ol (5); [0044] 3)
protection of the 17-OH group of
3-A-oxy-estra-1,3,5(10),15-tetraen-17.beta.-ol (5) to
3-A-oxy-17-C-oxy-estra-1,3,5(10),15-tetraene (4), wherein C is a
protecting group; [0045] 4) oxidizing the carbon-carbon double bond
of ring D of 3-A-oxy-17-C-oxy-estra-1,3,5(10),15-tetraene (4) to
protected estetrol (3); and [0046] 5) removing the protecting
groups, wherein preferably protecting group A is removed first to
form 17-OC protected estetrol (2) and subsequently protecting group
C is removed to form estetrol (1); wherein the protecting group A
is selected from an C.sub.1-C.sub.5 alkyl group, preferably a
methyl group, or a C.sub.7-C.sub.12 benzylic group, preferably a
benzyl group, and the protecting group C is selected from
monofunctional aliphatic hydroxyl protecting groups, said
monofunctional aliphatic hydroxyl protecting groups being
preferably selected from the group consisting of C.sub.1-C.sub.5
carboxylates wherein the alkyl group of the carboxylates is as
defined above, said protecting group C being most preferably
acetyl.
[0047] The process according to this first aspect of the invention
is shown in Scheme 4. ##STR5##
[0048] In another aspect of the invention, there is provided the
use of the obtained compound as estrogenic substance, preferably
for cosmetic and/or therapeutic applications selected from hormone
replacement therapy, contraception, therapy of autoimmune diseases,
prevention and therapy of breast and colon tumors, enhancement of
libido, skin care, and wound healing.
DETAILED DESCRIPTION OF THE INVENTION
[0049] According to one aspect of the invention, a process is
provided for the obtainment of
1,3,5(10)-estratrien-3,15.alpha.,16.alpha.,17.beta.-tetraol. The
invention process comprises the steps of: [0050] 1) providing a
3-A-oxy-estra-1,3,5(10),15-tetraen-17-one, wherein A is a
protecting group selected from an C.sub.1-C.sub.5 alkyl group,
preferably a methyl group, or a C.sub.7-C.sub.12 benzylic group,
preferably a benzyl group.
[0051] This may be achieved by methods known in the art for making
such compounds such as given in J. Am. Chem. Soc. 79, 2005-2009
(1957), "14-Isoestrone Methyl ether and its identity with totally
synthetic material" by W. S. Johnson and W. F. Johns (A is methyl);
Biosci. Biotech. Biochem. 60, 411-414 (1996), "Synthesis of
(14.beta.,17.alpha.)-14-Hydroxy- and
(14.beta.,17.alpha.)-14-Dihydroxyestradiols and Their Activities"
by M. Sakakibara and A. O. Uchida.
[0052] Still, another process of obtainment has been found
effective for providing the
3-A-oxy-estra-1,3,5(10),15-tetraene-17-one (6) wherein A is a
protecting group selected from a C.sub.1-C.sub.5 alkyl group,
preferably a methyl group, or a C.sub.7-C.sub.12 benzylic group,
preferably a benzyl group. This process comprises the steps of:
[0053] Step i)--protecting the phenol function of estrone by
alkylation to obtain a protected estrone; [0054] Step
ii)--protecting the carbonyl function of the protected estrone
obtained in step a), [0055] Step iii)--forming an alkene bond in
the C.sub.15-C.sub.16 position of the protected estrone obtained in
step b); and [0056] Step iv)--deprotecting the carbonyl
function.
[0057] This process for the preparation of
3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6) comprises a third
embodiment of the invention and comprises the following steps:
[0058] (a1) conversion of the 3-OH group of estrone (7) into a 3-AO
group to form 3-A-oxy-estra-1,3,5(10)-trien-17-one (8); [0059] (b1)
conversion of the 17-keto group of
3-A-oxy-estra-1,3,5(10)-trien-17-one (8) into a protected keto
group to form 3-A-oxy-17-D-estra-1,3,5(10)-triene (9); [0060] (c1)
halogenation of C.sub.16 of 3-A-oxy-17-D-estra-1,3,5(10)-triene (9)
to form 3-A-oxy-16-X-17-D-estra-1,3,5(10)-triene (10) wherein X is
a halogen atom selected from the group chloride, bromide and iodide
and wherein X is preferably bromide; [0061] (d1) dehalogenation of
3-A-oxy-16-X-17-D-estra-1,3,5(10)-triene (10) to
3-A-oxy-17-D-estra-1,3,5(10),15-tetraene (11); and [0062] (e1)
deprotection of the protected keto group of
3-A-oxy-17-D-estra-1,3,5(10),15-tetraene (11) to form
3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6), wherein A is
selected from an C.sub.1-C.sub.5 alkyl group, preferably a methyl
group, or a C.sub.7-C.sub.12 benzylic group, preferably a benzyl
group, and wherein D is ethylene dioxy.
[0063] The process according to this third embodiment of the
invention is depicted in Scheme 5. ##STR6## Step (a)
[0064] Estrone (7) is a product which is commercially available
from Acros, Aldrich under the tradename estrone. Other suppliers of
estrone are Andard-Mount Company Ltd., Diosynth B. V., Productos
Quimicos Naturales S. A. de C. V.-Proquina, Schering A G,
Mistsubishi Chemical Corporation.
[0065] The protection of the 3-OH group by alkylation is typically
carried out by reacting estrone with a component selected from an
alkylating agent, preferably a C.sub.1-C.sub.5 alkyl halogenide,
preferably a methyl halogenide, or a C.sub.7-C.sub.12 benzylic
halogenide, preferably benzyl halogenide. Preferably, the halogen
atom of the alkylating agent is bromide, chloride or iodide, most
preferably bromide or iodide. According to the present invention,
the most preferred alkylating agent is benzyl bromide or methyl
iodide, wherein benzyl bromide is even more preferred than methyl
iodide. According to the invention, however, it is possible to use
a dialkyl sulphate instead of a C.sub.1-C.sub.5 alkyl halogenide,
wherein the alkyl groups contain 1-5 carbon atoms and wherein the
alkyl groups are preferably methyl (i.e. that the preferred dialkyl
sulphate is dimethyl sulphate). Nevertheless, according to this
embodiment of the invention, the most preferred alkylating agent is
benzyl bromide.
[0066] According to this third embodiment of the present invention,
it is in particular preferred to first suspend estrone (7) and
potassium carbonate in a mixture of dichloromethane (DCM)/methanol.
A 1:1 mixture of DCM/methanol is preferred. The alkylating agent
C.sub.7-C.sub.12 benzylic halogenide, preferably benzyl bromide, is
added and the resulting mixture is refluxed for a period of 8-16
hours. It is preferred to reflux the mixture for 16 hours. The
reaction mixture is then cooled to Room Temperature (RT). The
product is isolated by filtering off the solids. The filter cake is
washed with a protic solvent, preferably methanol. The filtrate is
concentrated to give a suspension which is filtered and washed with
heptanes to give the product as a white solid. The product can be
purified by recrystallisation from a mixture of DCM and MeOH to
obtain a white crystalline solid, wherein the preferred ratio of
DCM:MeOH is 1:2.
[0067] In particular, it is preferred to first suspend estrone (7)
and potassium carbonate in DMF. The C.sub.1-C.sub.5 alkyl
halogenide, preferably methyl iodide, is added with cooling,
keeping the temperature between 18.degree. and 22.degree. C. The
resulting mixture is stirred for a period of time at RT, preferably
for 5 days. The reaction mixture is poured into water and stirred
for 2 hours. The product is collected by filtration and washed with
water. The product is dried to give a white crystalline solid.
Step b)
[0068] The protection of the 17-keto group is preferably carried
out by reacting 8 with ethylene glycol using an acid catalyst such
as p-toluene sulfonic acid, HCl pyridine, sulfuric acid or acetic
acid and a solvent selected from dimethoxyethane, toluene, benzene,
trimethyl orthoformate or triethyl orthoformate. More preferably
the reaction is performed with ethylene glycol, triethyl
orthoformate and p-toluenesulfonic acid.
[0069] In particular, it is preferred to suspend 8 in a mixture of
triethyl orthoformate and ethylene glycol in a preferred volume
ratio of 4:3, more preferably 2:1. Subsequently, p-toluenesulfonic
acid is added and the reaction mixture is stirred for a period of
time at 35.degree. C. Preferably, after 1-16 hours, preferably
about 3 hours, the mixture is poured into a mixture of ice/water
and pyridine. After stirring for 1 h the product is collected by
filtration. It is washed with water and dried to yield the product
as a white solid.
[0070] Alternatively, it has also been found that steps (a) and (b)
can advantageously be performed simultaneously or sequentially
without the need for purification and/or isolation of the
intermediate products whilst still providing an end-product with
good yield and purity. This is particularly advantageous for use in
industry where the reduction of the number of process step provides
both an economical advantage and a simplification of the process by
eliminating the need for an additional step like purification
and/or isolation between the two steps.
[0071] If the process is made sequentially, the order for carrying
the synthesis is preferably by having first the protection of the
3-OH group (step (a)) and then protection of the 17-keto group
(step (b)). Still, it is preferred to first have step (b) and then
step (a) carried out. Indeed, by use of this order, the formation
of by-products has been found reduced upon industrial process.
[0072] Hence, according to a fourth embodiment of the present
invention, there is provided a process for the obtainment of a
3-A-oxy-estra-1,3,5(10),15-tetraen-17-one wherein A is the
protecting group selected from an C.sub.1-C.sub.5 alkyl group,
preferably a methyl group, or a C.sub.7-C.sub.12 benzylic group,
preferably a benzyl group, which process comprises the steps of:
[0073] Step ia) protecting the carbonyl function of estrone to
obtain a protected estrone; [0074] Step iia) protecting the phenol
function of the protected estrone obtained in step ia) by
alkylation, [0075] Step iii) forming an alkene bond in the
C.sub.15-C.sub.16 position of the protected estrone obtained in
step iia); [0076] Step iv)--deprotecting the carbonyl function;
wherein steps ia) and iia1) are performed simultaneously or
sequentially without purification and/or isolation of the obtained
intermediate product.
[0077] This process for the preparation of
3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6) comprises a fourth
embodiment of the invention and comprises the following steps:
[0078] (a2) conversion of the 17-keto group of estrone (7) into a
protected keto group to form 17-D-estra-1,3,5(10)-trien-3-ol (12);
[0079] (b2) conversion of the 3-OH group of
17-D-estra-1,3,5(10)-trien-3-ol (12) into a 3-AO group to form
3-A-oxy-17-D-estra-1,3,5(10)-trien-17-one (9); [0080] (c2)
halogenation of C.sub.16 of 3-A-oxy-17-D-estra-1,3,5(10)-triene (9)
to form 3-A-oxy-16-X-17-D-estra-1,3,5(10)-triene (10) wherein X is
a halogen atom selected from the group chloride, bromide and iodide
and wherein X is preferably bromide; [0081] (d2) dehalogenation of
3-A-oxy-16-X-17-D-estra-1,3,5(10)-triene (10) to
3-A-oxy-17-D-estra-1,3,5(10),15-tetraene (11); and [0082] (e2)
deprotection of the protected keto group of
3-A-oxy-17-D-estra-1,3,5(10),15-tetraene (11) to form
3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6), wherein A is
selected from an C.sub.1-C.sub.5 alkyl group, preferably a methyl
group, or a C.sub.7-C.sub.12 benzylic group, preferably a benzyl
group, and wherein D is ethylene dioxy.
[0083] Preferably, this is achieved by stirring a mixture of
estrone (7), ethylene glycol and triethyl orthoformate to which is
then added a catalytic amount of acid, preferably p-toluene
sulfonic acid. The reaction temperature is then raised to between
about 40.degree. and about 60.degree. C., preferably to about
45.degree. C. The slurry is stirred at that same temperature until
completion of the reaction, i.e. protection of the carbonyl
function of estrone. The conversion is checked with HPLC. To the
slurry, a solution of base, preferably sodium methoxide in methanol
is added resulting in a clear yellow solution. By use of such a
base, the 3-OH group is completely deprotonated which
advantageously allows the use of the less reactive but more
economical C.sub.7-C.sub.12 benzylic chloride, preferably benzyl
chloride, in the alkylation process. The temperature is raised to
65.degree. C. This high temperature further enables a good
crystallisation of the product. Although lower temperatures such as
down to 20.degree. C. can be used, it is believed that the low
temperature would incur a lower reactivity, thus longer reaction
times and probably incomplete conversions. C.sub.7-C.sub.12
benzylic chloride, preferably benzyl chloride, is then added over a
few minutes, such as 5 minutes, upon which the solution becomes
turbid and slowly thickens into a slurry. After 1.5 hours the
conversion is checked with HPLC, usually a conversion of >95% is
observed, which is sufficient for further processing.
[0084] The mixture is allowed to cool to 20.degree. C. while
stirring, and then the solid product is isolated by filtration. The
solid is then washed and dried.
[0085] According to the present invention, the fourth embodiment is
more preferred than the third embodiment.
Step 3)
[0086] The formation of the carbon-carbon double bond in ring D is
preferably carried out by steps (c2) and (d2) defined above.
[0087] The halogenation is carried out with a halogenating agent.
Preferred halogenating agents are selected from bromine,
phenyltrimethylammonium perbromide or pyridinium bromide
perbromide. A more preferred halogenating agent for use herein is
pyridinium bromide perbromide. The solvent is selected from
CHCl.sub.3, dioxane, dimethoxyethane, ethylene glycol or THF. The
preferred solvent is THF without any co-solvent.
[0088] In particular, it is preferred to dissolve the previously
obtained compound 9 in dimethoxyethane, which is subsequently added
to a solution of the brominating reagent in a mixture of ethylene
glycol and dimethoxyethane. The resulting mixture is stirred until
completion of the reaction. Preferably after 16 hours the product
is isolated. A solution of sodium thiosulfate pentahydrate in water
is added to the reaction mixture. The product is extracted with an
organic solvent, preferably dichloromethane. The extract is dried
using sodium sulphate and the solvents are evaporated to obtain a
sticky oil which can advantageously be used without further
purification.
[0089] However, it is even more preferred to dissolve compound 9 in
pure THF and to perform the reaction at room temperature for less
than two hours, followed by removal of the THF by distillation and
adding a solvent that is essential not miscible with water,
preferably toluene. Water can then be removed from the product 10
by azeotropic distillation. Before the next step is performed, the
toluene solution of 10 is concentrated to dryness and the solvent
to be used in the next step is added.
[0090] The dehydrohalogenation reaction is carried out by using a
base selected from potassium tert-butoxide, DBU
(1,8-diazabicylo[5.4.0]undec-7-ene) or potassium hydroxide and is
preferably potassium tert-butoxide. The solvent is selected from
benzene, xylene, methanol or DMSO. The more preferred base and
solvent for use in this step are respectively potassium
tert-butoxide and dimethyl sulfoxide (DMSO).
[0091] In particular, it is preferred to add a suspension of the
previously obtained 10 compound in DMSO to a solution of potassium
tert-butoxide in DMSO. The resulting mixture is then stirred until
completion of the reaction. Preferably after about 2 hours the
reaction mixture is poured into a mixture of ice and water. The
product is extracted with an organic solvent, preferably DCM. The
extract is dried using sodium sulfate and the solvents are
evaporated to obtain a sticky oil which can be used without further
purification.
[0092] However, it is even more preferred to perform the
dehydrohalogenation step for less than one hour and to perform the
extraction with toluene at about 60.degree. C. Furthermore, the
toluene solution of compound 11 is preferably dried by azeotropic
distillation before the next step is carried out.
Step 4)
[0093] Deprotection of the carbonyl function is preferably carried
out by a component selected from p-toluenesulfonic acid, pyridinium
p-toluenesulfonate, and pyridinium chloride, preferably
p-toluenesulfonic acid monohydrate. More preferably, the
deprotection is performed using p-toluenesulfonic acid monohydrate
in the presence of aqueous acetone as solvent.
[0094] In particular, it is preferred to add p-toluenesulfonic acid
monohydrate to a solution of the previously obtained compound in
aqueous acetone, preferably with 10-20% water. The mixture is
stirred until completion of the reaction. Preferably after about 3
hours, DCM and saturated aqueous sodium bicarbonate are added.
After separating the layers, the aqueous layer is extracted with
DCM. The combined extracts are washed with brine and concentrated
to give a suspension. The product is collected by filtration and is
washed with organic solvents, preferably with cold acetone and
heptane. The product can be purified by recrystallization.
[0095] However, it is even more preferred to perform this
deprotection step in aqueous acetone (water content about 10%)
during which the product 6 crystallises from the solution. To
enhance the crystallisation process, water is added after
completion of the reaction which provides nice crystals that are
easily collected by filtration and which eliminates the necessity
of further purification, e.g. by recrystallisation.
[0096] The obtained 3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6)
is advantageously used in the process for the obtainment of
estetrol.
2) Reduction of the 17-keto Group
[0097] Reduction of the 17-keto group is preferably performed by
reacting 3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6) with a
reducing agent selected from the group of metal hydride compounds,
said group of metal hydride compounds preferably comprising
LiAlH.sub.4, AlH.sub.3, NaBH.sub.4, NaBH(OAc).sub.3, ZnBH.sub.4,
and NaBH.sub.4/CeCl.sub.3. Most preferably the metal hydride
compound is NaBH.sub.4/CeCl.sub.3. More preferred reducing agents
for use herein are those that will provide a chemo- and
stereo-selective reduction of the 17-keto group in favour of the P
position. For that reason, the most preferred chemo- and
stereo-selective reducing agent for use herein is NaBH.sub.4 in
combination with CeCl.sub.3 hydrate, preferably the
heptahydrate.
[0098] In particular, it is preferred to suspend
3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6) and CeCl.sub.3
heptahydrate in a mixture of a protic solvent, preferably MeOH and
THF and to stir the mixture for about 1 h at room temperature. A
preferred volume ratio of MeOH to THF is 2:1 to 4:1. Then the
mixture is cooled, preferably to 0.degree.-5.degree. C., and
NaBH.sub.4 is added in small portions maintaining the temperature
below 8.degree. C. After a period of time, preferably 2 hours, 1 N
NaOH and DCM are added. After 30 minutes of stirring, the layers
are separated and the aqueous layer is extracted with DCM. The
combined organic extracts are dried with sodium sulphate and
concentrated to give the product as a white solid.
[0099] However, it is even more preferred to quench the reaction
mixture with an acid, preferably 2 N HCl, to remove the solvents by
distillation under vacuum at about 30.degree. to about 40.degree.
C. and to add toluene. Preferably, the temperature is then raised
to about 70.degree. C. to induce phase separation. The organic
phase is then separated, washed with an aqueous solution of
Na.sub.2CO.sub.3 and water. The final organic phase is dried by
azeotropic distillation, cooled to about 50.degree. C. and used for
the next step.
[0100] 3) Protecting the reduced carbonyl function of the
3-A-oxy-estra-1,3,5(10),15-tetraen-17-one, i.e. protection of the
17-OH group of 3-A-oxy-estra-1,3,5(10),15-tetraen-17.beta.-ol (5)
to form 3-A-oxy-17-C-oxy-estra-1,3,5(10),15-tetraene (4), wherein C
is a protecting group.
[0101] The 17-OH group is protected by preferably selected by
acetylation using a reagent selected from acetic anhydride or
acetyl chloride. Preferably, acetic anhydride is used.
[0102] In particular, it is preferred to treat a solution of the
compound in pyridine with acetic anhydride and
4-dimethylaminopyridine. The mixture is stirred for a period of
time. Preferably after 2 hours at room temperature the volatiles
are removed. The residue is dissolved in ethyl acetate (EtOAc) and
the resulting solution is washed with water and brine. The solution
is dried using sodium sulphate and concentrated to give the crude
product. Recrystallization from a mixture of organic solvents,
preferably ethyl acetate, heptane and ethanol gives the product as
a white solid.
[0103] However, since 4-dimethylaminopyridine is toxic and
difficult to remove by distillation, it is more preferred to
perform the reaction with a trialkylamine, preferably
triethylamine, and an acetyl halide (about two equivalents),
preferably acetyl chloride (about 1.5 equivalent) in toluene at
about 25.degree. to about 60.degree. C., preferably about
40.degree. to about 50.degree. C. The work up is then performed by
washing with water, aqueous acid and aqueous base. Purification of
the product is then achieved by crystallisation, i.e. by removing
the toluene by distillation, dissolving the crude product in ethyl
acetate and heating this solution to about 70.degree. to about
80.degree.. To this heated solution, small portions of ethanol are
added to induce crystallisation (preferred ratio of ethyl acetate
to ethanol is about 1 to about 8).
4) Oxidizing the alkene bond of the cyclopentenol group of the
acetylated 3-A-oxy-estra-1,3,5(10),15-tetraen-17-ol, i.e. oxidizing
the carbon-carbon double bond of ring D of
3-A-oxy-17-C-oxy-estra-1,3,5(10),15-tetraene (4) to protected
estetrol (3)
[0104] The oxidation of carbon-carbon double bond inn ring D is
carried with an oxidising agent providing selective
cis-hydroxylation of the carbon-carbon double bond. Preferably, the
oxidising agent is osmium tetroxide and more preferably the
oxidising agent is osmium tetroxide immobilized on PVP
(OsO.sub.4--PVP) that is used in a catalytic amount (cf. G.
Cainelli, M. Contento, F. Manesclachi, L. Plessi, Synthesis, 45-47
(1989)) in combination with a co-oxidant selected from
trimethylamine-N-oxide, N-methyl morpholine-N-oxide or hydrogen
peroxide, preferably trimethylamine-N-oxide. More preferably,
OsO.sub.4--PVP and trimethylamine-N-oxide are used with TBF as the
solvent.
[0105] In particular, it is preferred to add OsO.sub.4--PVP to a
heated solution of the compound prepared in the previous step in
THF. Preferably, the addition is performed at 50.degree. C.
followed by the addition of trimethylamine-N-oxide. Preferably, the
addition of trimethylamine-N-oxide is performed portion wise during
1 hour. The mixture is stirred at this temperature for a period of
time. Preferably, after 12 hours the mixture is cooled to room
temperature and filtered. The volatiles are removed and the residue
is dissolved in ethyl acetate and water is added. The aqueous layer
is acidified and the layers are separated. The aqueous layer is
extracted with ethyl acetate. The combined extracts are dried with
sodium sulphate and concentrated. The resulting residue is
triturated with heptanes and ethyl acetate to give the product as a
white precipitate that is filtered off. The product is purified by
recrystallization from a mixture of organic solvents, preferably
ethyl acetate, heptane and ethanol to give the product as a white
solid.
5) Removing the Protecting Groups A and C
[0106] Removal of the protecting groups is also an important aspect
of the present invention process. Indeed, it has been found that
not all protective groups can be removed without adverse effects on
the obtained product. Hence, where for example a methyl group is
used as the protective group for the 3-OH group, removal with
pyridine.HCl has been found to lead to decomposition of the
product.
[0107] Accordingly, it has been found that removal of the
protecting C.sub.1-C.sub.5 alkyl group is preferably performed
using BBr.sub.3 without leading to major decomposition of the
product.
[0108] Removal of the protective C.sub.7-C.sub.12 benzylic group is
preferably be performed using catalytic hydrogenation conditions
(Pd./H.sub.2) as is well known to the person skilled in the
art.
[0109] In particular, it is preferred to dissolve the protected
estrone (protected estetrol (3)) in a protic solvent, preferably
methanol. A catalytic amount of 10% Pd on carbon is added as a
preformed suspension in methanol and the mixture is placed under an
atmosphere of hydrogen, preferably 1 atmosphere. After stirring the
mixture for 3 hours at room temperature it is filtered over Celite.
The filtrate is concentrated to give 17-OC protected estetrol (2)
as a white solid.
[0110] Removal of protecting group C is effective using a protic
solvent such as methanol and a base, preferably K.sub.2CO.sub.3, to
yield estetrol.
[0111] In particular, it is preferred to dissolve the compound
obtained in the previous step in methanol. Potassium carbonate is
added and the mixture is stirred for 2 hours at room temperature.
Then the volatiles are evaporated and water and chloroform are
added. The mixture is neutralized with 0.1 N HCl and the product is
collected by filtration. It is then washed with water and dried to
give estetrol as a white solid.
[0112] Alternatively, the order of the two deprotection steps above
can be reversed. Thus, the complete deprotection can be
accomplished by first deprotection of protecting group C followed
by catalytic hydrogenation to remove protecting group A where A is
a protective C.sub.7-C.sub.12 benzylic group. The procedures are
identical to the ones described above. However, the first order of
the deprotection steps that is described hereinbefore is preferred
over the latter, i.e. that according to the invention it is
preferred to first remove protecting group A and subsequently
protective group C.
[0113] According to a most preferred embodiment of this step, the
deprotection reactions, i.e. the removal of A and C, are performed
in a single step if A is a protective C.sub.7-C.sub.12 benzylic
group. Preferably, compound 3 is dissolved in a C.sub.1-C.sub.3
alkyl alcohol, preferably methanol, and subjected to hydrogenation
at room temperature over night. Thereafter, the solution of
compound 2 is preferably used in the subsequent step, i.e. the
removal of C as described above. Work up of the reaction mixture is
then preferably carried out by concentrating the solution to about
20% of its original volume and by adding an amount of water
approximately equal to the volume of the concentrated solution.
Thereafter, concentrated acid, preferably concentrated HCl, is
added dropwise resulting in a white suspension which is filtered
off, washed with water and dried.
[0114] Compounds and intermediate products that are in particular
preferred according to the present invention are: [0115] the
compound according to formula 2, wherein C is acetyl. [0116]
compounds according to formula 3, wherein A is methyl or benzyl,
preferably benzyl, and wherein C is acetyl. [0117] compounds
according to formula 4, wherein A is methyl or benzyl, preferably
benzyl, and wherein C is acetyl. [0118] compounds according to
formula 5, wherein A is methyl or benzyl, preferably benzyl. [0119]
compounds according to formula 8, wherein A is methyl or benzyl.
[0120] compounds according to formula 9, wherein A is methyl or
benzyl, preferably benzyl, and D is ethylene dioxy. [0121]
compounds according to formula 10, wherein A is methyl or benzyl,
preferably benzyl, D is ethylene dioxy and X is bromo. [0122]
compounds according to formula 11, wherein A is methyl or benzyl,
preferably benzyl, and D is ethylene dioxy. Applications
[0123] In another aspect of the present invention is provided the
use of the product as obtainable by the invention process for the
manufacture of a pharmaceutical composition, preferably for use in
a method selected from a method of hormone replacement therapy, a
method of treating vaginal dryness, a method of contraception, a
method of enhancing libido, a method of treating skin, a method of
promoting wound healing, and a method of treating or preventing a
disorder selected from the group consisting of autoimmune diseases,
breast tumours and colorectal tumours.
[0124] In another aspect of the present invention is provided the
cosmetic/aesthetic use of the product as obtainable by the
invention process for treating skin.
EXAMPLES
The following are non-limited synthesis examples for the synthesis
of estetrol according to the invention:
The following methods and materials for determination were
used:
1H NMR spectra were recorded on a Varian 200 MHz apparatus in
CD.sub.3OD.
HPLC-MS was performed using a Hewlett Packard 1100 series:
Column: Discovery C18 (150.times.4.6 mm) Supelco
Mobile phase: Solution A:Solution B=70:30 (5 min).fwdarw.(10
min).fwdarw.10:90 (5 min)
Flow: 1 mL/min
UV: 280 nm
Temp: 22.degree. C.
MS: API-ES negative
Solution A: 9.65 g NH4OAc, 2250 mL H2O, 150 mL MeOH, 100 mL
CH3CN
Solution B: 9.65 g NH4OAc, 250 mL H2O, 1350 mL MeOH, 900 mL
CH3CN
DSC was measured using a Mettler Toledo DSC822 apparatus.
Example 1
3-Benzyloxy-estra-1,3,5(10)-trien-17-one (compound 6, A=benzyl)
[0125] To a suspension of estrone (7; 100 g, 0.370 mol) and
K.sub.2CO.sub.3 (160 g, 1.16 mol) in DCM/MeOH (800 mL, 1:1 v/v
ratio) at room temperature was added benzyl bromide (132 mL, 1.10
mol) in one portion. The resulting mixture was refluxed for 16 h
(50% conversion after 4 h according to TLC). The reaction mixture
was cooled to RT and solids were filtered off. The filter-cake was
washed with MeOH. The solution was concentrated (to a total volume
of ca. 300 mL). The precipitate that had formed was collected by
filtration and washed with heptanes to give a white solid. The
filtrate was concentrated further (to a total volume of 100 mL) and
triturated with heptane. The resulting precipitate was filtered off
and combined with the first batch of product. The product (153 g,
max 0.370 mol) still contained traces off benzyl bromide but was
used without further purification. The product can be purified by
recrystallization from DCM/MeOH (1/2). TLC: R.sub.f=0.5
(heptanes/ethyl acetate=4/1); HPLC-MS: 91%; .sup.1H-NMR (200 MHz,
CDCl.sub.3) .delta. 7.60-7.24 (m, 5H), 7.49 (d, 1H, J=8.4 Hz), 6.87
(dd, 1H, J.sub.1=2.6 Hz, J.sub.2=8.4 Hz), 6.82 (d, 1H, J=2.4 Hz),
5.12 (s, 2H), 3.05-2.90 (m, 2H), 2.66-2.01 (m, 5H), 1.77-1.47 (m,
8H), 0.99 (s, 3H) ppm.
Example 2
17,17-Ethylenedioxy-3-benzyloxy estra-1,3,5(10)-trien-17-one
(compound 4; A=benzyl, C=ethylene dioxy)
[0126] 3-Benzyl-estrone (compound 6, A=benzyl; 153 g (crude), max.
0.370 mol) was suspended in a mixture of triethyl orthoformate (320
mL) and ethylene glycol (160 mL). p-TsOH monohydrate (5 g, 26.3
mmol) was added and the resulting pinkish suspension was stirred
for 3 h at 35.degree. C. (TLC indicated complete conversion after
1.5 h). The mixture was cooled to RT, poured into a mixture of
ice-water (2 L) and pyridine (40 mL). The resulting precipitate was
collected by filtration and washed with water (150 ml). The
remaining white solid was dried azeotropically by stripping with
toluene (2.times.200 mL) to afford the product (153 g, max. 0.370
mmol) as white crystalline material. TLC: R.sub.f=0.3
(heptanes/ethyl acetate=9/1); .sup.1H-NMR (200 MHz, CDCl.sub.3)
.delta. 7.60-7.24 (m, 5H), 7.29 (d, 1H, J=8.4 Hz), 6.86 (dd, 1H,
J.sub.1=2.6 Hz, J.sub.2=8.4 Hz), 6.80 (d, 1H, J=2.4 Hz), 5.11 (s,
2H), 4.03 (m, 4H), 3.05-2.90 (m, 2H), 2.46-1.28 (m, 13H), 0.96 (s,
3H) ppm.
Example 3
17,17-Ethylenedioxy-3-benzyloxy estra-1,3,5(10)-trien-17-one
(compound 4; A=benzyl, C=ethylene dioxy)
[0127] A reaction flask equipped with mechanical stirrer,
thermometer, nitrogen purge, condenser and dropping funnel was used
for the process. The flask was charged with 27 g (100 mmol) of
estrone, 50 ml (55 g, 9 equivalents) of glycol and 24 g of
triethylorthoformate. The resulting mixture was stirred. 0.5 g of
toluenesulfonic acid was added and the reaction temperature was
raised to 45.degree. C. At about 35-40.degree. C. an exothermic was
observed. The slurry is stirred for 1 hour at 45.degree. C. The
conversion is checked with LC. Usually after 1 hour almost complete
conversion is observed. To the slurry a solution of sodium
methoxide in methanol (30% wt.; 1.1 equivalents) is added from the
dropping funnel resulting in a clear yellow solution. The
temperature is raised to 65.degree. C. and 15 g of benzyl chloride
is added over 5 minutes. Within a few minutes the solution becomes
turbid and slowly thickens into a slurry. After 1.5 hours the
conversion is checked with LC, usually a conversion of >95% is
observed, which is sufficient for further processing.
[0128] The mixture is allowed to cool to 20.degree. C. while
stirring, and then the solid product is isolated by filtration. The
solid is washed with methanol (2*30 ml) and dried under atmospheric
conditions.
An amount of 33-34 g of product is obtained with an organic purity
of >97%.
Example 4
16-Bromo-17,17-ethylenedioxy-3-benzyloxy-estra-1,3,5(10)-triene-17-one
(compound 10, X=Br, A=benzyl, B=ethylene dioxy)
[0129] Pyridinium bromide perbromide (120 g, 375 mmol, 1.44 equiv)
was dissolved in a mixture of ethylene glycol (120 mL) and ethylene
glycol dimethyl ether (200 mL). 3-Benzyl-estrone ethylene glycol
acetal (compound 4; A=benzyl, C=ethylenedioxy; 153 g (crude), max.
0.370 mol) was dissolved in ethylene glycol dimethyl ether (400 mL)
and subsequently added to the brominating reagent within 5 minutes.
The mixture became yellow immediately and was stirred for 16 h at
RT (TLC showed the reaction to be converted to 50% after 2 h). A
solution of Na.sub.2S.sub.2O.sub.3.5H.sub.2O (205 g, 0.83 mol) in
water (700 mL) was added to the reaction mixture. DCM (1 L) was
added and the layers were separated. The aqueous layer was
extracted with DCM (2.times.200 mL). The combined organic layers
were washed with water (300 mL) and brine (300 mL), dried
(Na.sub.2SO.sub.4) and concentrated in vacuo to yield the
brominated product (180 g, max. 0.370 mol) as a yellow solid which
was used without further purification for the next step. TLC:
R.sub.f=0.25 (heptanes/ethyl acetate=9:1); HPLC-MS: 2 diasteromers
(together 85%) minor byproducts present; .sup.1H-NMR (200 MHz,
CDCl.sub.3) .delta. 7.60-7.20 (m, 5H), 7.27 (d, 1H, J=8.4 Hz), 6.85
(dd, 1H, J.sub.1=2.6 Hz, J.sub.2=8.6 Hz), 6.80 (d, 1H, J=2.4 Hz),
5.10 (s, 2H), 4.63 (m, 1H), 4.08 (m, 4H), 2.93 (m, 2H), 2.41-1.38
(m, 11H), 0.98 (s, 3H) ppm.
Example 5
17,17-Ethylenedioxy-3-benzyloxy estra-1,3,5(10),15-tetraene
(compound 11; A=benzyl, B=ethylene dioxy)
[0130] Potassium tert-butoxide (180 g, 1.6 mol) was dissolved in
DMSO (600 mL) and a suspension of
16-Bromo-17,17-ethylenedioxy-3-benzyloxy-estra-1,3,5(10)-triene-17-one
(compound 10, X=Br, A=benzyl, B=ethylenedioxy; 180 g (crude), max.
0.370 mol) in DMSO (600 mL) was added at RT within 5 min. The
temperature rose to 45.degree. C. during the addition. The colour
of the reaction mixture immediately changed to dark brown. The
reaction mixture was stirred for 2 h during which the temperature
fell to 25.degree. C. It was poured into ice/water (2 L) and
extracted with DCM (2.times.1 L, 2.times.300 mL). The organic
layers were combined, washed with water (300 mL) and brine (300 mL)
and dried with Na.sub.2SO.sub.4. The solution was concentrated in
vacuo to give the crude product (147 g, max. 0.370 mmol) as a brown
oil which was used without further purification for the next step.
TLC: R.sub.f=0.35 (heptanes/ethyl acetate=9/1); .sup.1H-NMR (200
MHz, CDCl.sub.3) .delta. 7.60-7.44 (m, 5H), 7.27 (d, 1H, J=8.4 Hz),
6.86 (dd, 1H, J.sub.1=2.6 Hz, J.sub.2=8.4 Hz), 6.80 (d, 1H, J=2.4
Hz), 6.33 (dd, 1H, J.sub.1=1.6 Hz, J.sub.2=7.4 Hz), 5.82 (dd, 1H,
J.sub.1=3.4 Hz, J.sub.2=6.0 Hz), 5.10 (s, 2H), 4.03 (m, 4H), 2.95
(m, 2H), 2.56-1.40 (m, 9H), 1.04 (s, 3H) ppm.
Example 6
3-Benzyloxy-estra-1,3,5(10),15-tetraen-17-one (compound 6;
A=benzyl)
[0131] To a solution of 17,17-Ethylenedioxy-3-benzyloxy
estra-1,3,5(10),15-tetraene (compound 11; A=benzyl,
B=ethylenedioxy; 147 g, max 0.370 mol) in acetone (0.9 L) and water
(100 mL) at RT was added p-TsOH monohydrate (4.8 g, 25 mmol). The
mixture was stirred for 3 h at RT (According to TLC the reaction
was complete after 1 h and a precipitate had formed). DCM (1.2 L)
and saturated aqueous NaHCO.sub.3 solution (300 mL) were added. The
mixture was stirred vigorously. The layers were separated and the
aqueous layer was extracted with DCM (300 mL). The combined organic
layers were washed with brine (300 mL) and concentrated until
precipitation started (volume of appr. 300 mL, T=50.degree. C.).
The precipitate was filtered off, washed with cold acetone and
hexanes to afford the product as an off-white solid which was
purified by recrystallization from acetone to give a white solid
(58 g, 0.162 mol, 44% over 5 steps). (purity according to HPLC-MS:
94%). The remaining mother-liquor still contained 40% of product
according to HPLC-MS. TLC: R.sub.f=0.3 (heptanes/ethyl
acetate=4:1); DSC: Mp. 161.9.degree. C. (purity 91.7%); .sup.1H-NMR
(200 MHz, CDCl.sub.3) .delta. 7.70 (dd, 1H, J.sub.1=1.6 Hz,
J.sub.2=6.0 Hz), 7.60-7.40 (m, 5H), 7.26 (d, 1H, J=8.8 Hz), 6.86
(dd, 1H, J.sub.1=2.6 Hz, J.sub.2=8.8 Hz), 6.84 (d, 1H, J=2.4 Hz),
6.17 (dd, 1H, J.sub.1=3.8 Hz, J.sub.2=6.6 Hz), 5.12 (s, 2H), 3.01
(m, 2H), 2.62-1.64 (m, 9H), 1.18 (s, 3H) ppm.
Example 7
3-Benzyloxy-estra-1,3,5(10),15-tetraen-17-ol (compound 5;
A=benzyl)
[0132] To a solution of 3-benzyl-dehydroestrone (compound 6;
A=benzyl; 58 g, 162 mmol) in a mixture of MeOH (900 mL) and THF
(200 mL) at room temperature was added CeCl.sub.3 heptahydrate
(66.4 g, 178 mmol). After stirring for 1 h the mixture was cooled
to 0-5.degree. C. using an ice/water bath. Then NaBH.sub.4 (12.2 g,
324 mmol) was added in small portions maintaining a temperature
below 8.degree. C. After stirring for 2 h at 0-5.degree. C. (TLC
showed the reaction to be complete) 1 N NaOH (300 mL) and DCM (1 L)
were added and the mixture was stirred for 1/2 h at room
temperature. The layers were separated and the aqueous layer was
extracted with DCM (200 mL). The organic layers were combined,
dried (Na.sub.2SO.sub.4) and concentrated in vacuo to give an
off-white solid (55.0 g, 152.8 mmol, 94%) TLC: R.sub.f=0.25
(heptanes/ethyl acetate=4:1); HPLC-MS: 93% .beta.-isomer, 2%
.alpha.-isomer; DSC: Mp. 149.7.degree. C., purity 96.6%;
.sup.1H-NMR (200 MHz, CDCl.sub.3) .delta. 7.48 (m, 5H), 7.27 (d,
1H, J=8.4 Hz), 6.85 (dd, 1H, J.sub.1=2.8 Hz, J.sub.2=8.6 Hz), 6.81
(d, 1H, J=2.4 Hz), 6.10 (d, 1H, J=5.8 Hz), 5.79 (dd, 1H,
J.sub.1=1.8 Hz, J.sub.2=3.4 Hz), 5.11 (s, 2H), 4.48 (d, 1H, J=7.6),
2.96 (m, 2H), 2.46-1.64 (m, 9H), 0.93 (s, 3H) ppm.
Example 8
17-Acetyloxy-3-benzyloxy-estra-1,3,5(10),15-tetraene (compound 4;
A=benzyl, C=acetyl)
[0133] A solution of 3-Benzyloxy-estra-1,3,5(10),15-tetraen-17-ol
(compound 5; A=benzyl; 55.0 g, max. 153 mmol) in pyridine (400 mL)
was treated with Ac.sub.2O (50 mL, 0.53 mol) and
4-dimethylaminopyridine (1.5 g, 12.3 mmol). The mixture was stirred
for 2 h at room temperature (TLC showed the reaction to be
complete). It was concentrated in vacuo. The residue was dissolved
in EtOAc (400 mL), washed with water (200 mL) and brine (150 mL),
dried (Na.sub.2SO.sub.4) and concentrated in vacuo to yield a
yellow solid (54.0 g, 49.8 mmol, 88%). The product was purified by
recrystallization from heptanes/EtOAc/EtOH (1:0.5:1) to afford a
white solid (45.0 g, 112 mmol, 73%) TLC: R.sub.f=0.6
(heptanes/ethyl acetate=4/1); HPLC-MS: 98% .beta.-isomer, 1%
.alpha.-isomer, 1.3% .beta.-estradiol; DSC: Mp. 122.8.degree. C.,
purity 99.8%; .sup.1H-NMR (200 MHz, CDCl.sub.3) .delta. 7.44 (m,
5H), 7.27 (d, 1H, J=8.4 Hz), 6.86 (dd, 1H, J.sub.1=2.6 Hz,
J.sub.2=8.4 Hz), 6.80 (d, 1H, J=2.6 Hz), 6.17 (d, 1H, J=5.8 Hz),
5.78 (dd, 1H, J.sub.1=1.4 Hz, J.sub.2=3.2 Hz), 5.45 (m, 1H), 5.11
(s, 2H), 2.96 (m, 2H), 2.40-1.54 (m, 10H), 2.18 (s, 3H), 0.93 (s,
3H) ppm.
Example 9
17-Acetyl-3-Benzyl estetrol (compound 3; A=benzyl, C=acetyl)
[0134] OsO.sub.4 on PVP (9 g, .about.5% w/w OsO.sub.4 on PVP,
prepared according to Cainelli et al. Synthesis, 45-47 (1989) was
added to a solution of
17-Acetyloxy-3-benzyloxy-estra-1,3,5(10),15-tetraene (compound 4;
A=benzyl, C=acetyl; 45 g, 112 mmol) in THF (450 mL) and the mixture
was heated to 50.degree. C. Trimethylamine-N-oxide dihydrate (24.9
g, 224 mmol) was added portion-wise over 2 h. After stirring for 36
h at 50.degree. C. (TLC showed the reaction to be complete) the
reaction mixture was cooled to room temperature. The solids were
filtered off, washed with THF (100 mL) and the filtrate was
concentrated. The residue was taken up in EtOAc (250 mL) and water
(250 mL) was added. The aqueous layer was acidified with 1 N HCl
(ca. 10 mL). The layers were separated and the aqueous layer was
extracted with EtOAc (150 mL). The organic layers were combined,
dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The residue was
triturated with heptanes/EtOAc (1:1, 100 mL), stirred for 2 h and
the resulting white precipitate was filtered off to give the
product as a white solid (41 g, 94 mmol, 84%). The product was
purified by recrystallization from heptanes/ethyl acetate/EtOH
(2:1:1) three times to afford a white solid (21 g, 48.2 mmol, 43%).
HPLC-MS: 99.5% .beta..alpha..alpha.-isomer; DSC: Mp. 159.3.degree.
C., purity 98.7%; .sup.1H-NMR (200 MHz, CDCl.sub.3) .delta. 7.49
(m, 5H), 7.27 (d, 1H, J=8.4 Hz), 6.84 (dd, 1H, J.sub.1=2.6 Hz,
J.sub.2=8.4 Hz), 6.81 (d, 1H, J=2.4 Hz), 5.11 (s, 2H), 4.45 (d, 1H,
J=4.4), 4.11 (m, 3H), 3.12 (m, 1H) 2.95 (m, 2H), 2.46-1.64 (m,
10H), 2.24 (s, 3H), 0.93 (s, 3H) ppm.
Example 10
17-Acetyl estetrol (compound 2; C=acetyl)
[0135] To a solution of 17-acetyl-3-benzyl estetrol (compound 3;
A=benzyl, C=acetyl; 21 g, 48.2 mmol) in MeOH (600 mL, HPLC-grade)
was added a preformed suspension of 10% Palladium on activated
carbon (2 g) in methanol (50 mL). The mixture was placed under an
atmosphere of H.sub.2 at 1 atm and stirred for 24 h (TLC showed the
reaction to be completed) at room temperature. It was filtered over
Celite.RTM. and the filter cake was washed with MeOH (200 mL). The
filtrate was concentrated in vacuo to give 17-acetyl estetrol as a
white solid (15 g, 43.4 mmol, 90%). TLC: R.sub.f=0.2
(heptanes/ethyl acetate=1/1); HPLC-MS: 99.2%, DSC: Mp.
212.2.degree. C., purity 98.9%; .sup.1H-NMR (200 MHz, CD.sub.3OD)
.delta. 7.14 (d, 1H, J=8.0 Hz), 6.60 (dd, 1H, J.sub.1=2.6 Hz,
J.sub.2=8.8 Hz), 6.56 (d, 1H, J=2.4 Hz), 4.81 (dd, 1H, J.sub.1=3.4
Hz, J.sub.2=6.4 Hz), 4.07 (m, 3H), 3.12 (m, 1H), 2.85 (m, 2H),
2.37-1.37 (m, 10H), 2.18 (s, 3H), 0.91 (s, 3H) ppm.
Example 11
Estetrol
[0136] 17-Acetyl-estetrol (compound 2; C=acetyl; 15 g, 43.4 mmol)
and K.sub.2CO.sub.3 (6 g, 43.4 mmol) were suspended in MeOH (500
mL, HPLC-grade) and stirred for 4 h at room temperature (TLC showed
the reaction to be complete). The solvents were evaporated in
vacuo. Water (200 mL) and CHCl.sub.3 (70 mL) were added and the
mixture was stirred and neutralized with 0.1 N HCl (50 mL). The
product was collected by filtration, washed with water (100 mL) and
CHCl.sub.3 (100 nL) to give estetrol as a white solid (12.2 g, 40.1
mmol, 92.5%, overall yield from estrone 10.8%) after drying at
40.degree. C. in an air-ventilated oven. TLC: R.sub.f=0.05
(heptanes/ethyl acetate=1/1); HPLC-MS: 99.1%, DSC: Mp.
243.7.degree. C., purity 99.5%; .sup.1H-NMR (200 MHz, CD.sub.3OD)
.delta. 7.14 (d, 1H, J=8.6 Hz), 6.61 (dd, 1H, J.sub.1=2.6 Hz,
J.sub.2=8.4 Hz), 6.56 (d, 1H, J=2.4 Hz), 4.83 (m, 1H), 3.93 (m,
3H), 3.50 (d, 1H, J=5.2), 3.38 (m, 2H), 2.84 (m, 2H), 2.32 (m, 3H),
1.97 (m, 1H), 1.68-1.24 (m, 5H), 0.86 (s, 3H) ppm.
* * * * *