FIELD OF THE INVENTION:
The present invention relates to efficient process of reduction of steroidal trans diols. In particular the present invention relates to an efficient process of conversion of hydroxvl Estrone to 17p- Estratriol substantially free of 17a-alpha isomer.
BACKGROUND OF THE INVENTION
17J}-Estratriol ((16a, 17P)-estra-l ,3,5(10)-triene-3,16,17-triol) (17P-Estriol) is a steroidal
sex hormone and is the major estrogen in humans. 17p-Estriol (also Oestriol) is one of
the three main estrogens produced by the human body in significant amounts during
pregnancy as it is made by the placenta.
The structure of 17p-Estriol is given below;

17P-Estriol

17a-Estriol [a carcinogenic impurity]
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The epimer of Estriol, referred to as 17a-EstrioI has the following structure which is known to be having carcinogenic activity:

Estriol was first prepared from Estrone by Huffman (Journal of Biological Chemistry page 167, 1947) wherein Estrone is first methylated to produce 3- methy! ether and the latter was nitrosated to give 16-oximino derivative. This 16-oximino compound was carried through a Stodola reduction to yield crude a-ketol. This a-ketol was reduced using sodium amalgam at room temperature to yield alpha- glycol I two isomers. The estriol-3-methyl ether was obtained by fractional crystallization which was then acetylated, demthylated and finally saponified to produce estriol. The overall yields from Estrone to Estriol were 22-26%.
A process is disclosed in PCT patent WO 98/04577 for preparing Estriol from 16 a-hydroxy-estra-1,3,5(10)-triene derivative wherein an Estra-l,3,5(l 0), 16-tetraene derivative is converted by means of peracids into a 16a ,17a-Epoxide, which is split by means of mineral acids or converted by means of a reagent which supplies bromine cations into the corresponding 16a-Bromine derivative, which is converted into the 16p -Bromine derivative. The bromine is replaced by an hydroxy! group and if required the thus obtained 16a-Hydroxy-l,3,5(10)triene-17-one derivative is reduced by means of sodium borohydride in the presence of a buffer solution with a pH value from 5 to 6.5 into the corresponding 16 a ,17p -Trihydroxy-estra-1,3,5(10)-triene derivative.
Conventionally the Estriol derivatives were prepared by converting the Estrone derivative into an enol acetate by reaction with isopropenyl acetate. The corresponding epoxide compound was obtained by treatment with peracid. This epoxide derivative was then reduced to give the corresponding Estriol using lithium aluminum hydride or sodium borohydride as described in US patent 3679716 and DD 253249, as depicted in Scheme-1
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J 70-EstriaJ J 7a- Atrial
Scheme-1
However the above methods resulted in two isomers namely 17a and 17p-isomers. It has been found that 17a- estriol (Formula III) is a potential carcinogen and hence its impurity levels in the Estriol needs to be monitored stringently.(Science , 279, 1631 -2, 1998)
The conventional reduction using sodium borohydride giive rise to 17a-Estriol impurity to the tune of 8-10%. The removal of this impurity in Estriol by crystallization techniques is difficult due to the low solubility of Estriol in most of the solvents and little solubility difference between the 17a-Estriol impurity and 17p-Estriol. This necessitated the use of chromatographic separations thus reducing the yields of the final product.
Recently M. Giannageli has disclosed in E? 1149838 that use of B- ipc- 9-BBN can
effect the desired reduction to yield the Estriol without alpha estriol impurity. However
the cost of the reductant poses a limitation on its industrial feasibility so also the purity
obtained which is 99%, as efforts to improve the purity would result in further loss in
yield.
Thus it is known from the prior art that 17a-Estriol is usually formed as an impurity
during the reduction of Estrone to 17p-Estriol. Both 17a-EstrioI and 17p-Estriol have
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similar solubility profile and thus there is need for the preparation of 17p-Estriol free from 17a-Estriol.
Looking into the need for an efficient reducing agent for selectively obtaining 17p-Estriol in high yields, the present invention has focused on using an achiral and less expensive reducing agent to obtain pure crude 17p-Estriol without the need of recrystallisation. Further the present invention also aims to provide a reducing agent suitable for stereoselective reduction of transdiol compounds.
OBJECT OF THE INVENTION
It is the object of the present invention to provide a method for 1, 2-asymmetric reduction of compounds wherein 1,2-trans diol is present.
It is the object of the present invention to provide a process for preparation of 17p-Estriol and its derivatives from corresponding Estrone.
It is the object of the present invention to provide a process for the preparation of 17p-Estriol using an achiral reductant and using 16a- hydroxyl moiety to induce asymmetric induction in the reduction process.
It is the object of the present invention to provide a process specific for stereochemical reduction resulting predominantly the corresponding beta isomer of Estriol.
It is the object of the present invention to provide a process for pure Estriol without the need of recrystallisation.
SUMMARY OF THE INVENTION
The present invention discloses the process of preparation of 17p-Estriol. In particular the present invention provides a process for stereochemical reduction of the Estrone to
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predominantly 17p-isomer of estriol. In particular the present invention provides 170-Estriol substantially free of 17a-Estriol
In one aspect, the present invention provides a process for selective reduction of the Estrone and its derivatives to its corresponding 17p-Estriol. By using a achiral reducing agent. In one preferred embodiment, the present invention employs an achiral reducing agent such as sodium triacetoxyborohydride.
In one aspect, the present invention provides a process for preparation of 17p-Estriol using solvents that will form a homogenous mixture so as to direct the completion of reduction.
In one preferred embodiment the present invention employs Dioxane and methanol mixture to homogenize the reaction and the product is precipitated out as it is formed. Thus the resulting pure 17p-Estriol is easier to obtain as the reagents and the starting material are soluble in the said solvents.
In one aspect, the present invention provides a process for the preparation of 17p-Estriol in high yields with high purity without necessity of further purification.
DETAILED DESCRIPTION OF THE INVENTION
Definitions:
The term "Estrone" as used herein refers to Estrone and includes all derivatives of estrone
with substitution.
The term "Estriol" as used herein refers to 17p-Estriol and its derivatives.
The term "STAB" as used herein refers to sodium triacetoxyborohydride
The present invention provides a convenient synthesis of 17p-Estriol in high yield and purity. The present invention discloses the process for diastereoselective reduction of C-17-ketone of 16a-hydroxyestrone to yield Estriol hVhigh purity of 99%. Typically the
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process of the present invention provide 17p-Estriol wherein the impurities are less than 1%, preferably less than 0.6% of compound of 17a-Estriol.
The present invention has provided the conversion of Estrone to 17p-Estriol in a purity of higher than 98% and preferably higher than 99%, more preferably higher than 99.3%. The overall yield of the crude 17p-Estriol is almost quantitative for example 90% or higher. The crude 17p-Estriol has typically a purity greater than 99%, for example 99.3%.
In this respect the present invention has developed a method wherein Sodium triacetoxy borohydride is used as a reductant
The present invention has developed a process for obtaining "trans" diol in high yields and purity if the alpha hydroxy! ketone is obtained in good optical purity. The present invention provides methods for 1, 2- asymmetric reduction of other compounds to obtain the 1,2- trans diol in high stereoselectivity.
The process of the present invention uses the 16a-hydroxyl moiety to direct the reduction of C-17 ketone thus resulting in high diastereoselectivity. It was reasoned that if the reductant is coordinated or bonded with C-I6 hydroxyl moiety then the attack would happen from the alpha face resulting in C-17 beta hydroxyl moiety in a stereoselective or specific way thus limiting /eliminating the impurity.
The present invention has found that sodium triacetoxy borohydride helps in directing the CI6- hydroxyl moiety to stereoselective reduction to C17p-hydroxyl group. Although the exact mechanism is not possible to predict, the following possibility appears most probable. The reductant, sodium triacetoxy borohydride is known to exchange with alcohols and one could expect an equilibrium as shown in the below Scheme-2 to exist.
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Scheme- 2
This sodium triacetoxyborohydride [STAB reagent] can exchange with OH functionality of C-16 hydroxy group in 2 thus enabling the reduction in the desired manner.
The effect of solvent in the reduction process was also studied. It was indeed found that the reaction was sluggish [~7 hours] when THF ( as previously used in other reductions with sodium or lithium hydride) was used as a solvent.
When Dioxane-Methanol solvent combination was used with STAB on 2, the reaction proceeded in three hours and exclusively and only Estriol was formed with <0.05% of the unwanted impurity 1795% yield.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Although specific starting materials and reagents are depicted in the Scheme-2 , the suitable substitution can be easily made to provide a variety of derivatives. Conventional methods and/or techniques of separation and purification known to one of ordinary skill in the art can be used to isolate and purify the compounds of the present invention. Such techniques will be well known to one of ordinary skill in the art and may include, for example, all types of chromatography (high pressure liquid chromatography (HPLC), column chromatography using common adsorbents such as silica gel, and thin-layer chromatography), recrystallization, and differential (i.e., liquid-liquid) extraction techniques.
Example 1: Preparation of A1,3,5'16-Estratetraene-3,17-diol diacetate To a three necked flask(500ml) equipped with a distillation assembly, thermometer and an addition funnel, was charged isopropenyl acetate(250ml) followed by Estrone(125gm; 463m.mole) and heated to 70-75°C. A solution of pTSA(22.0gm;l 16m.mole) in isopropenyl acetate(lOOml) was charged in to the addition funnel and was added drop wise to the reaction mixture. After approximately 5-10 minutes, distillate started coi/ecting. The distillate was collected for 35-40 minutes and (he voiume of Che cotfected distillate was ~100ml.
At this point, another 100ml of isopropenyl acetate was charged into addition funnel and this was added drop wise to the reaction mixture while collecting the distillate for 35-40 minutes when another lot of distillate (MOOml) had been collected.
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The above sequence of operation was repeated two more times after which, a solution of triethylamine (20ml) in ethyl acetate( 100ml) was added to the reaction mixture to keep the medium basic(pH was between 8-9). This reaction mass was transferred to a 5.01itre flask and ethyl acetate(3.501itres) was added . This solution was taken in a separatory funnel and was washed with saturated sodium bicarbonate solution (3X200ml) followed by a brine wash (200ml).
The organic layer was dried over sodium sulphate and the solvent was removed in-vacuo to obtain a syrupy mass which was triturated with methanol (100ml) to obtain a slurry which was filtered off and dried to obtain the product as a colourless to pale yellow solid. Yield: 140gm(84%); MP-. 149-150°C
]H NMR)(400MHz; CDC13): 0.92(3H, s), 1.40-3.00(13H, m), 2.17(3H,s), 2.27(3H,s), 5.19(1H, dd, J= 1.6Hz and 3.2Hz), 6.79(1 H,dJ=2.4Hz), 6.84(1H, dd, 8.4Hz and 2.4Hz), 7.25(lH,d, 8.4Hz).
EXAMPLE 2: Preparation of 16a, 17a-Epoxy-AI,3,5,-estratriene-3,17-diol diacetate
To a 500ml three necked round bottom flask was added ethyl acetate (100ml) followed by A1,3'5'I6-Estratetraene-3,17-diol diacetate (50gm,141.24 mmole) and the solution was cooled to 7-10°C. mCPBA (65.60gm, 65%, 247mmole) was dissolved in ethyl acetate (250ml), washed with saturated sodium bicarbonate solution (3 X 100ml) and the ethyl acetate layer was separated. This solution of mCPBA in ethyl acetate was added drop-wise to the above solution maintained temperature 7-10°C over an hour. The reaction mix was gently brought to room temperature and stirred for six hours after which a 20% solution of Sodium thiosulphate (200ml) was added and stirred vigorously. The ethyl acetate layer was separated and washed with thiosulphate solution (3 X 100ml) and brine (1 X 100ml).The ethyl acetate layer was dried over anhydrous Sodium sulphate and the solvent was removed in-vacuo at room temperature. The semi solid material thus obtained was triturated with methanol (100ml) which precipitated a colourless solid. The solid was filtered off and dried in-vacuo to yield a dry colourles solid .
11

Yield : 45gm (86%).
HPLC Purity: 97.5%
'H NMR (400MHZ,CDC13)
0.93(3H,s),1.30-2.90(15Hlm),2.II(3H,s),2.27(3H,s),3.92(lH,s),6.77(lH,dJJ
=2.4Hz),6.83(lH,dd,J=8.4Hz and 2.4Hz), 7.25(lH,d,8.4H2-)-
Example 3: Hydrolysis to 16a- Hydroxyestrone:
In a three necked llitre round bottomed flask was charged dioxane (300ml) followed by 16a, 17a-Epoxy-AU'5,-estratriene-3) I7-dioldiacetate (40gm, 107.81 mmole). To this solution was added 2N sulphuric acid (200ml) slowly over a period of 45minutes maintaining the temperature between 10°C and 15°C and the mixture was brought to room temperature and stirred for 5 hours. The reaction mixture was concentrated in-vacws aud water (AQOml) was added.. The white precipitate obtained was filtered using a Buchner funnel, washed with water (3XI00ml), Ethyl acetate ( 100ml) and dried. Yield: 29gm (94%). HPLC Purity: 98.67% 'H NMR (400MHz, DMSO)
0.87(9H,s), 1.20-2.8(13H,m), 4.24(lH,dd,4.23(lHdd, J=5.6Hz and 8Hz), 5.37(lH,d,5.6Hz), 6.45(lH,d,2.4Hz), 6.51(lH,dd,J=2.4Hzand 8.4Hz), 7.04(lH,d,8.4Hz)
Example: 4: Reduction to 17p-estrioI:
Preparation of estra-l,3,5,(10)-trien-3,16a,17p -triol
To a 1:1 mixture of methanol: dioxane (500ml) in a one litre round bottomed flask, was added 3,16a -Hydroxy-estra-1,3,5 (10)-trien-17-one (22gm, 76.7mmole) and stirred to obtain homogeneous solution. At this point, sodium triacetoxyborohydride (16gm, 76mmole)was added slowly in lots so that the internal temperature does not exceed 20°C. The mixture was stirred for four hours at room temperature (23-25°C) after which another lot of reductant (25gm,l 17mmole)was added in four lots, each lot in one hour interval, after which the reaction mixture concentrated under reduced pressure and dil. HC1 (IN solution ,450ml) was added slowly to the residue and stirfed for 10 minutes vigorously. The precipitated solid was filtered and washed with water (2 X 110ml).The solid obtained
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was suspended in acetone (250ml) and stirred for 10 minutes and filtered to obtain the title compound.
Yield : 20.5gm (94%).
HPLC Purity: 99.53%
'H NMR (400MHZ,DMSO +CDCI3)
0.66(3H,s), 1.20-2.8(13H,m), 3.29(lH,m), 3.83(lH,m), 4.57(IH,dd,J=5Hz and 1.2Hz),
4.6(IH,dd,J=5Hz and 1.2Hz), 6.43(lH,s), 6.50(lH,d,J=8Hz), 7.03(lH,d,J=8.4Hz)
Thus, while we have described fundamental novel features of the invention, it will be understood that various omissions and substitutions and changes in the form and details may be possible without departing from the spirit of the invention. For example, it is expressly intended that all combinations of those elements and/or method steps, which perform substantially the same function in substantially the same way to achieve the same results, be within the scope of the invention.
Dated this 5th day of June 2009
For Reliance Life Sciences Pvt. Ltd
K. V. Subramaniam President
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r5JUN2009

Claims:
1. A process for the preparation of a 17p-Estriol from estrone which comprises reduction of 3,16 a -Hydroxy estra-1,3,5 (10) - triene-17-one with Sodium triacetoxyborohydride in a solvent to yield 17p-Estriol having less than 0.1% of 17 a-Estriol.
2. A process for an asymmetric reduction of a- hydroxyketone compounds , such as 2[Scherne-2], using sodium triacetoxyborohydride in an organic solvent or mixture of organic solvents.
3. A process according to claim 1, wherein the mixture of solvents is selected from ethereal solvents such as dioxane, THF and a C1-C4 alcohol..
4. A process according to any one of the preceding claims, wherein the solvent comprises mixture of dioxane and methanol, particularly in a ratio of 1: 1.
6. A process according to any one of the preceding claims, wherein the purity of 17p-Estriol not less than 99.3% with the carcinogenic impurity of 17 a-estriol less than 0.1%
8. A process for the preparation of 17p-Estriol from Estrone comprising steps of
a) Preparation of A1,3,5,16-Estratetraene-3,17-diol diacetate from estrone
b) Preparation of 16a, 17a-Epoxy-A1,3'5,-estratriene-3,17-diol diacetate from of A1'3,5'16-Estratetraene-3,17-diol diacetate
c) Hydrolysis of 16a, 17a-Epoxy-A1,3'5'-estratriene-3,17-diol diacetate in an acidic medium to yield 3,16 a-Hydroxy estra-1,3,5 (10)-triene-17-one
b) reduction of 3,16 a -Hydroxy estra-1,3,5 (10)-triene-17-one to give 17p-Estriol.
characterized in that the process gives 17p-Estriol substantially free of 17- alpha estriol.
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9. The process of preparation of 17p-Estriol as defined in any one of the preceding claims.
Dated this 5 day of June ,2009
For Reliance Life Sciences Pvt. Ltd
K. V. Subramaniam President
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-5JUN2009