FORM 2
THE PATENTS ACT 1970 (Act 39 of 1970)
&
THE PATENTS RULE 2003 (SECTION 10 and rule 13)
PROVISIONAL SPECIFICATION
"EPIMERIZATION BY STEREOSELECTIVE SYNTHESIS OF VITAMIN D ANALOGUES"
Glenmark Pharmaceuticals Limited
an Indian Company, registered under the Indian company's Act 1957
and having its registered office at
Glenmark House, HDO - Corporate Bldg, Wing -A,
B.D. Sawant Marg, Chakala, Andheri (East), Mumbai - 400 099
THE FOLLOWING SPECIFICATION DESCRIBES THE NATURE OF THE NVENTION
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FIELD OF THE INVENTION
The present invention relates to novel epimerization process of C-24 ketones to desired C-24 alcohols by stereo selective reduction using chiral borane reducing agents for the preparation of calcipotriene.
BACKGROUND OF THE INVENTION
Calcipotriol or calcipotriene (structure I) shows a strong activity in inhibiting undesirable proliferation of epidermal keratinocytes [F.A.C.M. Castelijins, M. J. Gerritsen, I. M. J. J. van Vlijmen-Willems, P. J. van Erp, P. C. M. van de Kerkhof; Acta Derm. Venereol. 79, 11, 1999]. The efficiency of calcipotriol in the treatment of psoriasis was shown in a number of clinical trials [D. M. Ashcroft et al.; Brit. Med. J. 320, 963-67, 2000] and calcipotriol is currently used in several commercial drug formulations.

In the preparation of calcipotriol, the specific stereochemistry for the hydroxyl group at C-24 is necessary for full expression of the biological activity. Under current methodology, the required stereochemistry is introduced by the following methods:
Non distereoselective reduction of C-24 keto triens and then chromatographic separation of diastereomeric mixtures of C-24 (S) and C-24 (R) hydroxy epimers along with satureated alcohol disclosed in US 4866048 and M. J. Calverley; Tetrahedron, 43 (20), 4609-19, 1987. This process is most widely used process, but separation of desired C-24 (S) hydroxy epimer from such a mixture by chromatography method is very difficult on large production scale.
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M. J. Calverley, Synlett, 157-159, 1990 disclosed a process for the preparation of C-24 (S) hydroxy epimer by condensing an enantiopure C-24-hydroxyl carrying side chain to the vitamin D skeleton. This process is an unfavourable for scale up due to its multi-step nature and production cost.
WO 2003/060094 disclosed method of selectively enzymatically esterifying and selectively enzymatically solvolyzing epimers at C-24 of analogs of vitamin D and esters using enzymes. The enzymatic esterification synthesis is not commercially favourable since it requires costly enzymes and depending on the selectivity of enzyme requires additional reaction steps involved.
WO 2005/087719 disclosed a process for preparation of diastereomerically enriched C-24 hydroxyl epimers of calcipotriene derivatives by stereo selective reduction of labile triene system which is protected as sulfur dioxide adduct. This process gives, 1:1 or more of desired 24 (S)-hydroxyl epimer. This process requires protection of C-24 keto triene compound by sulfur dioxide and deprotection of sulfur dioxide adducts of C-24 (S) and (R) hydroxyl mixtures followed by separation of desired (S)-epimer from the forced epimer mixtures. This would results in reduced yields, impure products and tedious work-up procedures, especially on large scale.
It would be desirable to provide a novel epimerization process of C-24 ketones to desired C-24 alcohols for preparing intermediates of Calcipotriene and their use in the preparation of Calcipotriene in a convenient and cost efficient manner and on a commercial scale.
SUMMARY OF THE INVENTION
In accordance with a first aspect of the present invention, a method of reducing a compound of general structure II,
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H,G
R,0

II
Wherein R) and R2 may be the same or different and represent hydrogen, or a hydroxy protecting group, in an inert solvent with a borane reducing agent in the presence of a chiral auxiliary, wherein the chiral auxiliary is (R)-2-methyl-CBS-oxazaborolidine to give a mixture of compounds of general structure Ilia and IIIb,

OH


Ilia IIIb
which is enriched with Ilia, wherein Rj and R2 are as defined above.
In accordance with a second aspect of the present invention, a process for preparing calcipotriene, comprises the following steps
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a. reducing a compound of general structure II,


II
Wherein Ri and R2 may be the same or different and represent hydrogen, or a hydroxy protecting group, in an inert solvent with a borane reducing agent in the presence of a chiral auxiliary, wherein the chiral auxiliary is (R)-2-methyl-CBS-oxazaborolidine to give a mixture of compounds of general structure IIIa and III b,

which is enriched with Ilia, wherein Ri and R2 are as defined above.
b. isolating the compound of formula-IIIa and converted to calcipotriene.
In accordance with a third aspect of the present invention, a pharmaceutical composition is provided comprising at least one pharmaceutically acceptable excipient and calcipotriene obtained through the process of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
As used herein a "hydroxy protecting group" is any group which forms a derivative that is stable to the projected reactions wherein said hydroxy protecting group can be selectively removed by reagents that do not attack the regenerated hydroxy group. Silyl derivatives, such as tert-butyldimethylsilyl forming silyl ethers are examples of hydroxy protecting groups. Silyl chlorides such as tert-butyldimethylsilyl chloride (TBSCI), trimethylsilylchloride, triethylsilylchloride, diphenylmethylsilylchloride, triisopropylsilylchloride, and tert-butyldiphenylsilylchloride are examples of hydroxy protecting agents. Ethers or esters are also used as hydroxy protecting groups for example ethers such as tetrahydropyranyl (THP)
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ether, including alkoxyalkyl ethers (acetals), such as methoxymethyl (MOM) ether, esters such as chloroacetate ester, trimethylacetate, acetate or benzoate ester.
As used herein "borane reducing agent" is a borane containing compound which is capable of enantioselectively or diastereoselectively reducing, the C24 keto group of formula-II to alcohol of formula-IIIa and Mb.
As used herein, "chiral auxiliary" means any chiral compound or optically active catalyst, e.g. a compound comprising asymmetrically substituted carbon atoms or axially chiral compounds, or mixtures of chiral compounds and/or optically active catalysts, which will improve the yield of a compound of general structure Ilia with respect to its epimer (increase the molar ratio Ilia: Mb) in the reduction of a compound of general formula II with the reducing agent.

A mixture of compounds of general structure Ma and Mb, which is enriched with Ma,
means a mixture, were the molar ratio (diastereomer ratio) of Ma/Mb is one (50: 50) or larger than one, thus that the mixture contains at least 50% of the compound of general structure Ma (containing 50% or less of the compound of general structure Mb). Where compound of general structure Ma means C24 (S)-hydrocy epimer of calcipotriene intermediate and general structure Mb means C24 (R)-hydroxy epimer of calcipotriene intermediate.
As used herein "separating a compound" means the purification and/or isolation of a compound more than 90% purity, such as at least 95% purity. It also means increasing the concentration of required isomer in a mixture of compounds.
In one embodiment, the process for preparing compounds of formula-Ma and/or Mb includes
a) reducing a compound of the formula-II
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H,C
R,0

II
Wherein Ri and R2 may be the same or different and represent hydrogen, or hydroxy protecting group, with a borane reducing agent in the presence of a chiral auxiliary in an inert solvent to form a compounds of Formula-IIIa and III b

IIIa

Wherein Ri and R2 may be the same or different and represent hydrogen, or hydroxy protecting group, preferably Ri and R2 are both tert-butyl dimethyl silyl group.
The compound of Formula II is known in the art and can be prepared by any known method, for example, a compound of Formula II can be synthesized by the process mention in US 4866048 and "MJ Calverley, tetrahedron 43(20), 4609-4619, 1987, the contents of each of which are incorporated herein by reference.
A suitable inert solvent for use herein may be, for example any organic solvent compatible with said reducing agent under the reaction conditions employed, or mixtures of such solvents. Non limiting examples of inert solvents include hydrocarbons, such as toluene and
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the like, and ethers, such as tert-butyl methyl ether, diisopropyl ether or tetrahydrofuran and the like.
Reduction reaction of a prochiral ketone is usually carried out in a temperature interval between -50 and 50°C, preferably between -20 and 20°C, more preferably between -10 and 10°C, most preferably between 0 and 5°C. The temperature of the reduction reaction will depend on the specific reaction conditions and reagents used.
The reducing agent, optionally dissolved or mixed with an inert solvent, may be added to the compound of general structure II optionally dissolved or mixed with an inert solvent, e.g. under an inert atmosphere, such as nitrogen. Alternatively the compound of general structure II, optionally dissolved or mixed with an inert solvent, may be added to the reducing agent, optionally dissolved or mixed with an inert solvent. The sequence of addition of reducing agent and/or compound of general structure II is not particularly critical.
The "Borane reducing agent" for use herein may be, for example borane or any borane derivatives, such as borane complexes with amines or ethers. Non-limiting examples of borane reducing agents e.g. include but are not limited to N, N-diethylaniline-borane, borane-tetrahydrofuran, 9-borabicyclononane (9-BBN), or borane dimethylsulfide. Preferably N, N-diethylaniline-borane is used as borane reducing agent.
The "chiral auxiliary" for use herein may be, for example chiral oxazaborolidine reagents
selected from but are not limited to (lR,2S)-cis-l-amino-2-indanol, (lS,2R)-cis-l-amino-2-
indanol, (S)-prolinol, (R)-prolinol or B-(3-pinanyl)-9-borabicyclo [3.3.2] nonane (alpine-
borane), or e.g. 5, 5-diphenyl-2-methyl-3,4-propano-l,3,2-oxazaborolidine, (S)-2-methyl-
CBS-oxazaborolidine, (R)-2-methyl-CBS-oxazaborolidine. (R)-2-methyl-CBS-
oxazaborolidine is prefered as chiral auxiliary.
The reducing agent may be used about 2.0 to 3.5 mol equivalents to the starting prochiral ketone compound of formula-II, preferably 2.2 to 2.6 mol equivalents and most preferably
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2.4 mol equivalents. The chiral auxiliary may be present in catalytic amounts, such as substoichiometric, or equimolar or in molar excess referring to a prochiral ketone compound of formula-II. Eg. The ratio of chiral auxiliary and prochiral ketone compound of formula-II may be 1:1.5 mol equivalents, preferably 1:1.1 mol equivalents. The ratio of chiral auxiliary to the reducing agent may be 0.3 to 0.8 mol equivalents, preferably 0.5 mol equivalents.
After completion of the reduction reaction N,N-diethyl aniline formed in reaction byproduct may be recovered by conventional techniques for example extraction of reaction mixture with aqueous acid solution. Aqueous acids are selected from acetic acid, hydrochloric acid, hydrobromic acid and the like and mixtures thereof, preferably hydrochloric acid is used.
The compound of formula-IIIa and IIIb may be obtained by the present invention with the ratio of 60:40, preferably with the ratio of 65:35. The separation, isolation and purification methods of the present invention include, but are not limited to solvent crystallization, distillation, chromatography method such as adsorption chromatography (including column chromatography and simulated moving bed (SMB)). The separation, isolation and purification methods may be used subsequently and in combination.
In another aspect of the present invention, compounds IIIa and IIIb thus obtained may be further purified by dissolving the crude mixture in an organic solvent selected from the group consisting of ethers such as diisopropyl ether, diethyl ether, Methyl tert-butylether, and the like and mixtures thereof at ambient temperatures. Concentrating the formed solution by any known method for example concentration under vacuum to get the residue. The residue so obtained may be further isolated directly by any known method or may be adding second solvent selected from the group consisting of CM alcohols for example methanol, ethanol, propanol, isopropanol, butanol and the like and mixtures thereof, preferably methanol is used as second solvent. A compound of general structure Ilia can be recovered by, for example, crystallizing the solution; and isolating the crystals by techniques known in the art, e.g., filtration. If desired, the isolated crystals can then be dried. The temperature during stirring
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can range from about 0°C to about 25°C, preferably at temperature from about 10 to about 15°C.
The process of the present invention advantageously provide general structure Ilia in relatively high purity, e.g., a purity of greater than or equal to about 90%, preferably greater than or equal to about 95%, and more preferably greater than or equal to about 97.5%.
In another aspect of the present invention, compounds of general structure IIIa and IIIb,

which is enriched with general structure Ilia having the ratio of about 97.5:2.5 may be
converted into calcipotriene by any known method, for example, as disclosed in US 4866048
and "MJ Calverley, tetrahedron 43(20), 4609-4619, 1987, the contents of each of which are
incorporated herein by reference.
The following examples are provided to enable one skilled in the art to practice the invention and are merely illustrative of the invention. The examples should not be read as limiting the scope of the invention as defined in the claims.
EXAMPLES
Example 1: Preparation of (la,3β,5E,7E,20R)-l,3-bis-(tert-butyldimethylsiloxy)-20-(-cyclopropyl-3S-hydroxy-prop-lE-enyl )-9, 10-Secopregna-5, 7, 10(19), triene (Structure Ilia, Where in Rj & R2 are tert- butyl dimethyl silyl) and (la ,3β,5E,7E,20R)-l,3-bis-(tert-butyldimethylsiloxy)-20-(-cyclopropyl-3R-hydroxy-prop-lE-enyl)-9,10-Secopregna-5, 7, 10(19), triene (Structure IIIb, Where in Ri & R2 are tert- butyl dimethyl silyl). (R)-2-methyl-CBS-oxazaborolidine (16.97 ml, 1M solution in toluene) was added to 200 ml of tetrahydrofuran at 0-5°C under nitrogen atmosphere and stirred for 15-20 minutes. Added 6.92 ml of N,N-diethylaniline borane at 0-5°C over a period of 20-30 minutes to the (R)-2-methyl-CBS-oxazaborolidine solution to form a chiral ligand mixture and maintained for 25-30 min at same temperature. Then solution of (la,3β,5E,7E,20R)-l,3-bis-(tert-butyl dimethylsiloxy)-20-(cyclopropyl-3-oxo-l-propenyl)-9, 10-Secopregna-5, 7, 10(19), triene
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(Structure II, Where in R\ & R2 are tert-butyl dimethyl silyl, prepared according to the method described by M.J. Calverley, Tetrahydron, Vol. 43, No. 20, pp 4609-4619,1987), 10 gm in 100 ml THF was added to the above obtained chiral ligand mixture over a period of 40-45 minutes at temperature 0-5°C and maintained the reaction at temperature 0-5°C for 60-90 minutes. Then added IN aqueous hydrochloric acid to the reaction solution at a temperature 0-5°C at pH 6-6.5 and extracted reaction mass with ethyl acetate (2X200 ml), washed the ethyl acetate layer with water (200 ml), 1% sodium bicarbonate solution (200 ml) followed by brine solution (200 ml). The organic phase was dried with over anhydrous sodium sulfate and concentrated under vacuum at below 40°C to give the oily residue of structural formula-IIIa and IIIb (HPLC purity: 65:35 ratio of Ilia and IIIb). The obtained oily residue (mixture of Ilia and IIIb) purified by passing through short path coloumn to remove the unwanted impurities to give the pure compound. Wt. of the residue: 11.0 gm HPLC purity (chiral): 65:35% ratio of Ilia and IIIb
Example 2: Purification of compound formula-IIIa and IIIb (Where in Ri & R2 are tert- butyl
dimethyl silyl).
11 gm of mixture of structure Ilia and IIIb (obtained from example-1) was dissolved in 11 ml
of di-isopropyl ether at temperature 40-45°C and added 110 ml of methanol. Cooled the
solution to 20-25°C and stirred for 5 hours again cooled the solution to 10-15°C, stirred for
30 minutes. Filtered the material and washed with chilled methanol to give the pure
compound, Ilia.
If desired, the above process may be repeated to improve chiral purity.
Wt. of the compound: 2.5-3.0 gm
HPLC purity (chiral): 97:3% ratio of Ilia and IIIb.
Example 3: Preparation of Calcipotriene
A solution of alcohol of structure Ilia with Ri = R2 =tert-butyl dimethyl silyl (10 gm), Anthracene (1.7 g), Triethyl amine (1.7 ml) and toluene (750 ml) contained in a photochemical reactor at 20-25°C was irradiated with light from a high pressure ultra violet
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lamp to completion. Cooled the reaction mixture and filtered the unwanted products and concentrated the toluene compoletely under vacuum to yield crude tert-butyl dimethyl silyl protected Calcipotriene. The tert-butyl dimethyl silyl protected calcipotriene was dissolved in 400 ml of Tetrahydrofuran (THF) and tetra-butyl ammonium fluoride (24 gm). The resulting mixture was heated at 60-65°C for 2 hours. Quenched the reaction mass into 2% sodium bicarbonate solution (1200 ml) and ethyl acetate (1200 ml) and stir for 15 minutes. Separated the ethyl acetate layer and washed with water followed by organic phase was dried over sodium sulfate. Concentrated the organic phase under vacuum at below 40°C to give the Calcipotrien as residue, which was crystallized from methyl formate to give pure Calcipotriene.
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FEATURES AND ADVANTAGES OF THE PRESENT INVENTION
A) A method of reducing a compound of general structure II,

II
Wherein R\ and R2 may be the same or different and represent hydrogen, or a hydroxy protecting group, in an inert solvent with a borane reducing agent in the presence of a chiral auxiliary, wherein the chiral auxiliary is (R)-2-methyl-CBS-oxazaborolidine to give a mixture of compounds of general structure Ilia and IIIb,

which is enriched with Ilia, wherein Ri and R2 are as defined above.
B) The method as defined in (A) above, wherein the borane reducing agent is N,N-diethylaniline-borane, borane-tetrahydrofuran, 9-borabicyclononane, or borane dimethylsulfide.
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C) The method as defined in (A) above, wherein the inert solvent is toluene, tertahydrofuran, tert-butyl methyl ether or mixtures thereof.

D)

A process for preparing calcipotriene, comprises the following steps a. reducing a compound of general structure II,

II
Wherein R\ and R2 may be the same or different and represent hydrogen, or a hydroxy protecting group, in an inert solvent with a borane reducing agent in the presence of a chiral auxiliary, wherein the chiral auxiliary is (R)-2-methyl-CBS-oxazaborolidine to give a mixture of compounds of general structure Ilia and IIIb,

which is enriched with Ilia, wherein Ri and R2 are as defined above.
b. isolating the compound of formula-IIIa and converted to calcipotriene.

E) The method as defined in (D) above, wherein Rj and/or R2 represents tert-butyl dimethyl silyl.
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F) The method as defined in (D) above, wherein the method further comprises the steps
of
a) dissolving the mixture of compounds of general structure Ilia and Illb, which is enriched with Ilia in first organic solvent selected from diisopropyl ether, diethyl ether, methyl tert-butylether, and mixtures thereof,
b) combining the provided solution with second organic solvent Cj. 4 alcohols selected from methanol, ethanol, propanol, isopropanol, butanol and mixtures thereof,
c) crystallizing and isolating from the resulting suspention the compound of
general structure Ilia in chirally pure form.
G) The method as defined in (F) above, wherein the first organic solvent is diisopropyl
ether and the second organic solvent is methanol.
H) The method as defined in (F) above, wherein the crystallization is carried out at temperature between about 10 and about 15° C.

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ABSTRACT;
Provided is a method for epimerization process of C-24 ketones to desired C-24 alcohol by stereo selective reduction using chiral borane reducing agents in the presence of chiral auxiliary such as (R)-2-methyl-CBS-oxazaborolidine for the preparation of calcipotriene intermedites and its process to calcipotriene.
Dated this Third (03rd) day of August, 2007

(Signed)
MADHAVI KARNIK
DEPUTY GENERAL MANAGER - IPM
GLENMARK PHARMACEUTICALS LIMITED
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