FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES , 2003
COMPLETE SPECIFICATION
(See Section 10; rule 13)


PROCESS FOR THE PREPARATION OF ESTRADIOL AND ITS
DERIVATIVES
RELIANCE LIFE SCIENCES PVT.LTD an Indian Company having its Registered Office at Dhirubhai Ambani Life Sciences Centre, R-282, TTC Area of MIDC, Thane Belapur Road, Rabale, Navi Mumbai - 400 701 Maharashtra India.

The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:-


TECHNICAL FIELD
The present invention relates to a process for reducing steroidal ketones. In particular, the present invention relates to an efficient process for reducing estrone and its derivatives to Estradiol and its derivatives.
BACKGROUND
Estradiol (estra-1, 3, 5(10)-triene-3, 17p-dioI or 17p-Estradiol) is a steroidal sex hormone and is the major estrogen in humans. Estradiol has not only a critical impact on reproductive and sexual functioning, but also affects other organs including bone structure. Estradiol and its derivatives are commonly administered in hormone replacement therapy regimens and are also indicated in treating climacteric complaints and the treatment of osteoporosis. Estradiol has the following structure:

17Estradiol
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17p-Estradiol The epimer of estradiol, referred to as 17cc-Estradiol, has the following structure.


This epimer is less potent than the 17p form. In certain cell tines, the 17a form has been found to be only one tenth as potent as the 17{3 form (see, for example, Edwards DP & McGuire WL, Endocrinology, 1980, Vol. 107, 884-891).
Estradiol is known to form several crystal structures. The most stable form of estradiol is estradiol hemihydrate. Two anhydrous structures have also been reported in the literature. Estradiol also forms solvates with organic solvents such as methanol and ethanol.
A common synthetic preparation of Estradiol reported in the art is the reduction of estrone (3(3-Hydroxyestra-l, 3, 5(10)-trien-17-one) to the 17-hydroxy equivalent (estradiol). Reducing agents used in the prior art include Raney Nickel, Alumiminum isopropoxide, lithium aluminum hydride, sodium borohydride and catalytic reduction using Na.
US patent 2,623,886 describes a method of reducing of Estrone using methanolic sodium borohydride at room temperature. However this process involves high volumes of solvent and yields up to 2% of 17a-Estradiol.
The reduction of Estrone described in the prior art has mainly resulted in a mixture of the 17a and 17p forms. Repeated solvent recrystallisations and chromatography techniques have been employed to yield 17[3-Estradiol with high purity.
The prior art processes also result in incomplete conversion of Estrone to Estradiol. Due to their structural similarity and similar properties, the separation of Estrone and Estradiol is also a challenge.
There is therefore a need for a process that results in efficient conversion of Estrone and its derivatives to 17p-Estradiol and its derivatives, substantially free of Estrone and 17a-Estradiol impurities.
It has now been surprisingly found that 17p-Estradiol and its derivatives can be prepared by a new process that avoids many of the problems described in the prior art. Furthermore,
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an increase both in yield and purity of the 17(3 product can be achieved. In contrast to the processes described in the prior art, the process of the present invention involves reducing estrone and its derivatives in a specific temperature range using a borohydride.
Reduction of Estrone to 17p-Estradiol is advantageously performed at low temperature. This low temperature is thought to favour the kinetic 17p product over the thermodynamic 17a product. Reduction to 17p is faster than reduction to 17a, so a lower temperature favours reduction to the 17{3 product.
Further, it has surprisingly been found that an almost complete conversion of estradiol to the 17(3 product is achieved if the reduction is carried out first at a low temperature, followed by a maintenance step at a higher temperature. This two-step process has the advantages both of a very high yield of Estradiol and also of a very high purity of the 17p product.
SUMMARY OF THE INVENTION
The present invention therefore provides a process for the preparation of a compound of formula I

Formula I
wherein R1 represents a hydrogen atom, halogen atom, cyano group, hydroxyl group, straight or branched C1-C10 alkyl group, straight or branched C2-C10 alkenyl group or C6-C10 aryl group; or a -OR2, -O(CO)R2, or -R2-OH group, wherein R2 is a straight or branched C1-C6 alkyl group or a straight or branched C2-C6 alkenyl group, which process comprises treating a compound of formula II
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Formula II wherein Ri is as defined above, with a compound of formula III
M(BH4) Formula III
wherein M is a monovalent metal ion, in a solvent at a temperature of from -10 to 10°C.
Also provided is a compound of formula (I) obtained by the process of the present invention as defined above.
Also provided is a compound of formula (I), which is substantially free of a compound of formula (IV)


Formula IV DETAJ1EJ) DESCRIPTION OF THE IJVVEJVTJOJV
Scheme 1 below shows an embodiment of the process of the invention, in which the compound of formula (I) is (3-Estradiol.


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As used herein, the term "Estradiol" refers to 17p-Estradiol including all polymorphs, solvates, esters and salts thereof, for example, Estradiol hemihydrate.
Some compounds of the present invention may exhibit polymorphism. The scope of the present invention includes all polymorphic forms of the compounds according to the invention, which forms the further aspect of the invention. It is to be understood that the present invention encompasses all polymorphic forms or mixtures thereof which form or forms possess properties useful in the treatment of the conditions for which they are indicated.
As used herein, the term halogen atom refers to chlorine, fluorine, bromine or iodine atoms typically a fluorine, chlorine or bromine atom, most preferably chlorine or fluorine. The term halo when used as a prefix has the same meaning.
As used herein, the term C1-C10 alkyl includes both saturated straight chain and branched alkyl groups. Examples of C1-C10alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl. Typically, the Ct-C10 alkyl group is a C2-C6alky! group, more preferably a C1-4halky! group.
As used herein, the term C2-C10 alkenyl refers to groups containing one or more carbon-carbon double bonds, which group may be straight or branched. Typically, the C2-C10 alkenyl group is a C2-C6 alkenyl group, preferably a C2-C4 alkenyl group. Most typically, the C2-C10 alkenyl group is a vinyl, allyl or crotyl group, most preferably an allyl group.
As used herein, the term C6-C10 aryl refers to a 6-10 membered aromatic carbocyclic group. It may be monocyclic or polycyclic, for instance monocyclic or bicyclic. Examples of a C6-C10 aryl group include phenyl and naphthyl. Phenyl is preferred.
As used herein, the term C1-C6 alcohol means a compound of formula R-OH, wherein R is a C1_C6 alkyl group as defined herein. Examples of a C1-C6 alcohol include methanol,
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ethanol, n-propanol, i-propanol, n-butanol, i-butanol, s-butanol, t-butanol, pentanol and hexanol. Typically, the C1-C6 alcohol is a C1-4 alcohol, preferably a C1-2 alcohol. Generally, the C1-C6 alcohol is methanol or ethanol, preferably methanol.
Typically, R1 is a hydrogen atom or a group -OR2.
Typically, a group -OR2 is a methoxy, ethoxy, propoxy, isopropoxy, butyloxy, pentyloxy or hexyloxy group. Preferably, a group -OR2 is a methoxy or ethoxy group, more preferably a methoxy group.
Preferably, Rl is a hydrogen atom.
Typically, M is a monovalent metal ion of a Group F metal. For example, M is Li, K, Na or Rb. Preferably, M is Na or K, more preferably Na.
Typically, the process of the invention is a process for producing a compound of formula I, as defined herein, wherein less than 2%, preferably less than 1%, more preferably less than 0.7% of compound of formula IV, as defined herein, is produced.
Typically, the process of the invention is a process for producing a compound of formula I, as defined herein, in a purity of higher than 98%, preferably higher than 99%, more preferably higher than about 99.4%.
Typically, the process of the invention is conducted at a temperature of from -10 to less than 0°C, preferably from -10 to -3°C, more preferably from -10 to -5°C, most preferably from -7 to -5°C.
Typically, the solvent used in the process of the present invention is an aqueous organic solvent, preferably an aqueous C1-6 alcohol. Generally, the solvent used in the process of the present invention comprises methanol. Preferably, the solvent is aqueous methanol or ethanol, more preferably aqueous methanol.
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Typically, the process of the present invention is conducted under alkaline conditions. Methods of achieving alkaline conditions will be well known to the skilled person. Alkaline conditions are suitably achieved using an aqueous alkali. Suitable examples of an aqueous alkali include an aqueous solution of a compound of formula M(OH), wherein M is as defined above.
Most preferably, the solvent used in the process of the present invention is alkaline aqueous C1-6alcohol, typically alkaline aqueous methanol.
Typically, the ratio of compound of formula (III) to compound of formula (II) is from 0.5:1 to 6.5:1, preferably from 0.5:1 to 2.5:1, more preferably from 0.5:1 to 0.8:1.
Typically, the compound of formula (III) is present as an alkaline solution in a solvent, as defined herein.
In the process of the present invention, the reaction mixture is typically maintained at the required temperature for a period of from 1 to 4 hours, preferably 1.5 to 2.5 hours, more preferably about 2 hours.
In one aspect, the process of the present invention comprises:
(i) preparing an alkaline solution of a compound of formula (II), as defined above; and (ii) adding to the alkaline solution prepared in step (i) an alkaline solution of a compound of formula (III), as defined in claim 1, and maintaining the resulting reaction mixture at a temperature as defined above.
Typically, step (i) comprises adding aqueous alkali to a solution of a compound of formula (II) in a solvent.
Alternatively, step (i) comprises preparing a solution of a compound of formula (II), as defined above, in a solvent and adding the thus obtained solution to an aqueous alkali.
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The aqueous alkali is typically as defined above. Preferably, the aqueous alkali is aqueous sodium hydroxide.
Typically, in step (ii), the alkaline solution of a compound of formula (III) is added to the alkaline solution of a compound of formula (II) over a period of 1 to 4 hours, preferably 1.5 to 2.5 hours.
Compounds of formula (III) are typically commercially available or can be prepared by known methods.
Compounds of formula (II), where Ri is a hydrogen atom, i.e. estrone, are commercially available.
Compounds of formula (II), where Ri is not a hydrogen atom are commercially available, or may be prepared by known methods, for example as described in WO-A-06013196, the entirety of which is incorporated herein by reference.
Typically, after the reaction has proceeded substantially to completion, the temperature of the reaction mixture is maintained at a temperature of from 0 to 10°C, typically at 5 tolO°C, preferably at 8 toI0°C, more preferably at about 10°C. The chosen temperature is typically maintained for a period of from 0.5 to .1.5 hours, preferably 0.8 to 1.2 hours, more preferably for about 1 hour. Generally, HPLC is used to determine that the reaction has proceeded substantially to completion.
In one embodiment, after the reaction has proceeded substantially to completion, the reaction mixture is maintained at a temperature of from 0 to I0°C for a period of from 0.5 to 1.5 hours.
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In a particularly preferred embodiment, the invention provides a process for the preparation of a compound of formula I, as defined herein, which process comprises the sequential steps of:
(a) treating a compound of formula II, as defined herein, with a compound of formula III, as defined herein, at a temperature of from -10 to less than 0 °C for a period of from 1 to 4 hours; and
(b) after the reaction has proceeded substantially to completion, maintaining the reaction mixture at a temperature of from 0 to 10°C for a period of from 0.5 to 1.5 hours.
Typically, the temperature employed in step (a) is from -10 to -3°C, preferably from -10 to -5°C, more preferably from -7 to -5°C.
Typically, the temperature employed in step (b) is from 5 tol0°C, preferably from 8 tolO°C, more preferably about 10°C.
Typically, the period of time employed in step (a) is from 1.5 to 2.5 hours, preferably about 2 hours.
Typically, the period of time employed in step (b) is from 0.8 to 1.2 hours, preferably about 1 hour
As used herein, the term "substantially to completion" means that generally greater than 70%, preferably greater than 75%, more preferably greater than about 80% of the Estrone or Estrone derivative has been reduced to estradiol or estradiol derivative.
Typically, completion of the reaction is monitored by HPLC.
Typically, after the reaction has proceeded substantially to completion, the compound of formula (I) is isolated. Typically, isolation of the compound of formula I is effected by acidifying the reaction mixture (for example, so that the pH = 2) with dilute aqueous acid
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(for example, dilute HC1), stirring at room temperature and recovering the crude product by filtration. The filtered product is typically washed free of acid and dried.
When the compound of formula (I) is 17j3-Estradiol, the overall yield of crude product is typically quantitative, for example 99% or higher. The crude Estradiol typically has a purity greater than 99%, for example 99.14%. The crude Estradiol typically has less than 0.65% 17a-estradioI as impurity, for example 0.64%.
Typically, the crude product is recrystallised from aqueous isopropyl alcohol.
When the compound of formula (I) is 17p-Estradiol, the overall yield of recrystallised product is typically 82 to 85%, for example, 82.2%. The purity of the recrystallised product is typically 99.5% or greater, preferably 99.75% or greater, for example 99.8%. The recrystallised estradiol typically has less than 0.075% 17a-estradiol as impurity, for example 0.07%.
A compound of formula (I) substantially free of a compound of formula (IV) typically contains less than 0,65% compound of formula (IV), preferably less than 0.3%, more preferably less than 0.1%o, even more preferably less than 0.075%, for example 0.7% of compound of formula (IV).
As mentioned above, the process of the present invention typically takes place under alkaline conditions. The reaction goes to completion under alkaline conditions and the best results are observed when an alkaline sodium borohydride solution is used.
As mentioned above, a methanol solvent is preferred. Thus, reductions were carried out using a variety of solvents such as methanol, THF, alkaline THF and alkaline Methanol. It was found that the best results were obtained with alkaline methanol.
The reaction time was varied from 4- 16 hours and it was observed that estrone was reduced efficiently within 4 hours.
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The effect of the temperature of the reaction is critical in determining the amounts of the 17a- and 17p- forms of estradiol produced. To this end, reactions were carried out at temperature from -10 to 55°C. It was observed that when the reduction was carried out at -10 to -5°C and the reaction mixture was then maintained at 0-10°C, l7p-estradiol was obtained in an amount of approximately 99%.
As mentioned above, the crude product is typically recrystallised from aqueous isopropyl alcohol. Thus, recrystallisation of crude 17(3-estradiol were carried out using various solvents such as methanol, Isopropyl alcohol and 5% aqueous solutions of these solvents. It was observed that 5% aqueous isopropyl alcohol was effective in increasing the purity of crude estradiol from 99% to 99.84% without greatly compromising the yields.
EXAMPLES
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 illustrated in the above scheme I, suitable substitutions can be easily bemade to provide a variety of estradiol derivatives. Conventional methods and/or techniques of separation and purification known to one of ordinary skill in the art can be used to isolate & 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
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adsorbents such as silica gel, and thin-layer chromatography), recrystallization, and differential (i.e., liquid-liquid) extraction techniques.
The following examples illustrate the embodiments of the present invention. It is to be understood, however, that the embodiments of the invention are not limited to the specific details of these Examples, as other variations thereof will be known, or apparent in light of the instant disclosure, to one of ordinary skill in the art.
a) All operations carried out at room or ambient temperature were in the range of 18-25 degree Centigrade unless otherwise specified.
b) The course of the reaction was monitored by thin layer chromatography (TLC) and reaction times are given for illustration only.
c) Melting points are uncorrected, the melting points are given for the materials prepared as described, polymorphism may result in isolation of materials with different melting points in some preparations.
d) The structure and purity of all final products were assured by the following techniques: TLC, NMR (nuclear magnetic resonance) spectroscopy, mass spectroscopy and microanalysis.
e) When given, NMR data is in the form of delta (5) values for major diagnostic protons given in parts per million (ppm) relative to tetramethylsilane (TMS) as internal standard determined at 300 MHz or 400 MHz using the indicated solvent.
f) Chemical symbols have their usual meanings: the following abbreviations have also been used : v (volume), w (weight), Bp. (boiling point), Mp. (melting point), L ( litres) , ml (milliliters), gms (grams), mg (milligrams), mol(moles) mmole (milimoles), eq (equivalents), °C (degree centigrade), cone, (concentrated), Si02 (silicon dioxide), Na2SO4 (sodium sulphate).
g) The starting materials used in the following examples have been sourced from Sigma.
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EXAMPLE 1: Process for the preparation of estradiol from estrone
Sodium hyroxide solution (7.32 gm in 36 ml water) was added to a stirred solution of estrone (45 gm) in Methanol (225ml) at room temperature until a clear solution was achieved. This solution is cooled to -10°C after which an alkaline sodium borohydride solution (4.4 gm sodium borohydride in 3.328 gm of NaOH dissolved in 18 ml water) was added to the Estrone solution at -JO to -5°C over 2 hours. The progress of the reaction was monitored by HPLC. After the reaction had gone to completion, the reaction mixture was raised to 10°C and stirred for 1 hour. The reaction mixture was thenacidified to pH 2 using 10% diltute HC1 solution (90m!) and stirred at room temperature till until precipitation occurred. The crude estradiol precipitate was then filtered off, washed free of acid and dried.
The crude estradiol (45 gm) obtained was found to have 99.14% purity with an amount of 17a-Estradiol of 0.64% as confirmed by HPLC, with moisture content of 3.5%. The crude estradiol (45 gm) was then recrystallised by dissolving in 540 ml of 5 % aqueous isopropyl alcohol with heating under reflux conditions. The product was obtained by cooling to 0°C and filtering off the precipitate. After drying, the purified product weighed 42 gm (yield 82.2%) with a purity of 99.8 % with an amount of 17a-Estradiol of 0.07% and moisture content of 3.3 8%.
Yield = 82-85%
Purity passes Pharmacopoeia! standard Individual impurity not more than 0.3% Total impurities not more than 0.5%
EXAMPLE 2: Effect of reaction conditions on the amount of 17cc-Estradiol produced a) Reducing agent
A reduction of estrone to Estradiol was carried out using reduced alumina in THF as described in the prior art. It was observed that at a reaction temperature of from 0-5°C 17p-Estradiol was obtained in 96.85% purity with an amount of 17a-Estradiol of 1.71%.
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Further it was observed that alkaline sodium borohydride solution was more effective in reducing estrone as compared to sodium borohydride reagents.
b) Solvent
Sodium borohydride reductions of estrone to Estradiol were carried out using methanol, tetrahydrofuran (THF), alkaline methanol and alkaline THF as solvents. It was observed that alkaline methanol was effective in complete dissolution of estrone and conversion to Estradiol within 4 hours whereas it took about 12- 16 hours with methanol and THF. With alkaline THF the purity of 17(3-Estradiol obtained was 97% as compared to 99% with alkaline methanol.
c) Reaction temperature
Sodium borohydride reductions of Estrone to Estradiol were carried at a variety of temperatures. It was found that the reduction of estrone to 17p-Estradiol was most favoured at -5 to -10°C. The optimum reaction conditions for reduction reaction involved addition of sodium borohydride at a temperature of from -5 to -7°C followed by maintenance at 10°C after the reaction had proceeded substantially to completion. These specific conditions gave 17p-Estradiol of 99% purity.
d) Recrystallisation solvents
Recrystallisations of crude Estradiol were carried out using methanol, aqueous methanol, isopropyl alcohol (IPA) and aqueous IPA. For methanol and aqueous methanol, it was observed that although the moisture content and the amount of 17a-Estradio! were reduced following recrystallisation, a drop in yield was also observed as compared to recrystallisation from aqueous IPA. 5% aqueous IPA was found to be the most suitable solvent for recrstallising crude Estradiol. This gave 17-Estradiol with a purity of 99.84% and moisture content of 3.32% without resulting in a significant lowering of the yield.
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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.
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Dated this 5TH DAY OF JUNE , 2009
For Reliance Life Sciences Pvt Ltd
K. V. Subramaniam President
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CLAIMS
1. A process for the preparation of a compound of formula I
OH

wherein Ri represents a hydrogen atom, halogen atom, cyano group, hydroxyl group, straight or branched C1-C10 alkyl group, straight or branched C2-C10 alkenyl group or C6-C10 aryl group; or a -OR2, -0(CO)R2, or -R2-OH group, wherein R2 is a straight or branched C1-C6 alkyl group or a straight or branched C2-C6 alkenyl group, which process comprises treating a compound of formula II

wherein R1 is as defined above, with a compound of formula III
M(BH4) III
wherein M is a monovalent metal ion, in a solvent at a temperature of from -10 to 10°C.
2. A process according to claim 1, wherein R1 is a hydrogen atom.
3. A process according to claim 1 or claim 2, wherein the temperature is from -7 to -5°C.
4. A process according to any one of the preceding claims, wherein the solvent is methanol.
5. A process according to any one of the preceding claims, where process is conducted under alkaline conditions.
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6. A process according to any one of the preceding claims, wherein the ratio of compound of formula (III) to compound of formula (II) is from 0.5:1 to 0.8:1.
7. A process according to any one of the preceding claims, wherein the reaction mixture is maintained at -10°C to 0°C for 1-4 hours.
8. A process according to any one the preceding claims wherein, after the reaction has proceeded to > 97% of Estradiol with ~ 3% of Estrone remaining, the reaction mixture is maintained at a temperature of from Oto 10°C for a period of from 0.5 to 1.5 hours.
9. A process according to any one of the preceding claims, which process comprises: (i) preparing an alkaline solution of a compound of formula (II), as defined in
claim 1; and (ii) adding to the alkaline solution prepared in step (i) an alkaline solution of a compound of formula (III), as defined in claim 1, and maintaining the resulting reaction mixture at a temperature as defined in claim 1.
10. A compound of formula (I) as defined in claim 1, obtained by a process as defined
in any one of the preceding claims.
Dated this 5TH day of JUNE , 2009
For Reliance Life Sciences Pvt. Ltd
^ K. V. Subramaniam President
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