The present invention relates to a biocatalytic method for the preparation of ursodeoxycholic acid. Present invention further relates to chemical-biocatalytic method for preparation of chenodeoxycholic acid (CDCA) and use of said CDCA in preparation of ursodeoxycholic acid.

BACKGROUND OF THE INVENTION
Bile acids are synthesized in the liver from cholesterol and are essential to normal digestive and liver functions. The biological and chemical properties of bile acids have led to their use as therapeutic agents in the treatment of liver disease, cancer and for dissolving gallstones. Ursodeoxycholic acid (UDCA) has been shown to be an effective therapeutic agent in the treatment of primary biliary cirrhosis.

UDCA is known as a product of considerable interest in human therapy, in which it is used for its multiple functions such as solubilising bile calculi, lowering the percentage of cholesterol in the blood, lowering glycaemia, as a diuretic, and as an accelerant for the lipid metabolism.

The first conventional process known for preparing UDCA was extraction method involving direct extraction from the bear bile. Another process known in the prior published references are synthesis of UDCA through chenodeoxycholic acid through the complex chemical processes. This suffers from drawbacks such as multi-step method, long production times and high costs. Later after the 1980s, enzymology, microbial fermentation method were developed for the preparation of UDCA.

Helvetica. Chimica Acta. 1984, 67 (2) 612, discloses nine step preparation of UDCA starting from androstenedione. The major drawback of starting from androstenedione is the step of introduction of the side chain which is the most tedious and lengthy process.

J. Org. Chem. 1982. 47 (2). 2331 discloses process of preparing UDCA by using long eight-step reactions taking 4-pregnene-3, 20-dione (progesterone) as starting material.

WO2018036982, discloses a process for the preparation of UDCA through chemical oxidation to 3,7-diketo-ursodeoxycholic acid and after subsequent reaction with 7ß-hydroxysteroid dehydrogenase from C. aerofaciens and 3a-hydroxysteroid dehydrogenase from C. testosteroni. It also discloses Wolff-Kishner reduction of 12-keto UDCA wherein said 12-keto UDCA is synthesized through cholic acid that is used as starting material. WO’982 further discloses preparation of UDCA through dehydrocholic (DHCA), which upon two reductive steps converts to 12-keto-UDCA, by the action of stereoselective HSDHs (3a- and 7ß-HSDHs).

Biotechnology Letters 1992, 14 (12), 1131-1135, Carrea et al. describe a method for the production of UDCA from CA with the use of an enriched 7ß-HSDH and a 3a-HSDH. However, a disadvantage in this method is the use of a 7ß-HSDH from the pathogenic microorganism Clostridium absonum and need to purify the enzyme from the bacterial extract.

Similarly, US 2016/0194615 discloses 7ß-hydroxysteroid dehydrogenases which are obtained from bacteria of the genus Collinsella, especially of the strain Collinsella aerofaciens, and the sequences encoding said enzymes. US’615 further discloses use of said enzymes in the enzymatic conversion of cholic acid compounds, and especially in the production of ursodeoxycholic acid. The major drawback of this method is the preparation of an isolated recombinant 7ß-hydroxysteroid dehydrogenase which adds to the number of steps for preparing UDCA.

There are several known processes for preparing ursodeoxycholic acid. Majority of the known processes have the disadvantage of leading to the production of a mixture of ursodeoxycholic, chenodeoxycholic, lithocholic, cholic acid and isoursodeoxycholic acid in variable amounts from process to process. Other known processes involves preparation or isolation of recombinant enzymes from different strain which adds to the total number of steps for the preparation of UDCA that makes the process quite lengthy.

As it is already known from the literature that the oxidoreduction reactions catalyzed by hydroxysteroid dehydrogenases are reversible, hence the present invention is focussed towards the cofactor regeneration system coupled with the oxidation reaction not only to reduce the amounts of NAD(P)+ to be used, but also to push the reaction equilibrium towards high conversion percentages.

Moreover, as compared to the processes known from the literature, the process of the present invention is simpler and economically advantageous, as the protection of the hydroxyl groups are not required due to the use of absolute regioselectivity of hydroxysteroid dehydrogenase. Further, the process can be carried out in mild reaction conditions, which brings both economical and environmental advantages.

OBJECT OF THE INVENTION
The main object of the present invention is to develop a simple cost effective process for the preparation of ursodeoxycholic acid.

Another object of the present invention is to develop chemical-biocatalytic method that involves simple and easily available enzymes for selective reduction and oxidation during production of ursodeoxycholic acid.

SUMMARY OF THE INVENTION
In one aspect, the present invention provides a process for the regioselective biocatalytic oxidation at position 7 and /or 12 of Formula VII, or salts thereof,
,
wherein
R is selected from a-OH, ß-OH, -CO;
R1 is selected from H, -CO, a-OH, ß-OH; and
R2 is selected from H, straight or branched chain alkyl;
comprising reacting compound of Formula VII with NAD(P)+ dependent hydroxysteroid dehydrogenase in the presence of suitable dehydrogenase and ester selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain.

Another aspect of the present invention provides a process for preparing ursodeoxycholic acid of Formula I, wherein said process comprising the steps of:

a) selectively oxidizing position 12 of cholic acid of Formula II to give 12-keto cholanic acid of Formula III, in presence of a 12a-hydroxysteroid dehydrogenase and a cofactor regeneration system, comprising NAD(P)+ dependent dehydrogenase and ester selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain,
; and
b) converting compound of Formula III to ursodeoxycholic acid.

Another aspect of the present invention provides a chemical-biocatalytical method for preparation of chenodeoxycholic acid of Formula IV,
,
wherein said process comprising the steps of:
a) selectively oxidizing position 12 of cholic acid of Formula II to give 12-keto cholanic acid of Formula III, in presence of a 12a-hydroxysteroid dehydrogenase and a cofactor regeneration system, comprising NAD(P)+ dependent dehydrogenase and ester selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain,
; and
b) chemically reducing compound of Formula III to chenodeoxycholic acid of Formula IV,
,
wherein intermediate of Formula IIIa is optionally isolated.

Another aspect of the present invention provides a biocatalytical process for preparing ursodeoxycholic acid of Formula I,

wherein said process comprising the steps of:
a) selectively oxidizing the chenodeoxycholic acid of Formula IV to 3a-hydroxy-7-ketocholanic acid (7-keto CDCA) of Formula V, in presence of at least one hydroxysteroid dehydrogenase and a cofactor regeneration system, comprising NAD(P)+ dependent dehydrogenase and ester selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain,
; and
b) converting compound of Formula V to ursodeoxycholic acid.

Another aspect of the present invention provides a biocatalytical process for preparing ursodeoxycholic acid of Formula I,

wherein said process comprising the steps of:
a) selectively oxidizing the chenodeoxycholic acid of Formula IV to 3a-hydroxy-7-ketocholanic acid (7-keto CDCA) of Formula V, in presence of at least one hydroxysteroid dehydrogenase and a cofactor regeneration system, comprising NAD(P)+ dependent dehydrogenase and ester selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain,
; and
b) selectively reducing position 7 of 7-keto-CDCA of Formula V in presence of NAD(P)+ dependent hydroxysteroid dehydrogenase to get ursodeoxycholic acid.

Another aspect of the present invention provides process for the purification of ursodeoxycholic acid wherein said process comprising the steps of:
a) treating ursodeoxycholic acid with base to give ursodeoxycholic acid base adduct of Formula VI in presence of suitable solvent,
;
b) converting compound of Formula VI to ursodeoxycholic acid;
c) treating with suitable solvent; and
d) isolating ursodeoxycholic acid having purity of 99.0% and above.

Another aspect of the present invention provides ursodeoxycholic acid base adduct of Formula VI,
.

Another aspect of the present invention provides pure ursodeoxycholic acid substantially free from chenodeoxycholic, lithocholic acid, cholic acid and other related impurities wherein each impurity is less than about 0.2% w/w.

DETAILED DESCRIPTION
Definitions:
According to the present invention, a “pure form” or a “pure” or “essentially pure” enzyme is an enzyme with a purity of more than 80, preferably more than 90, in particular more than 95, and above all more than 99 wt. %, based on the total protein content, determined by means of normal protein estimation methods, such as for example the biuret method or the protein estimation after Lowry et al. (see description in R. K. Scopes, Protein Purification, Springer Verlag, New York, Heidelberg, Berlin (1982)).

A “redox equivalent” is understood to mean a small molecule organic compound usable as an electron donor or electron acceptor, such as for example nicotinamide derivatives such as NAD+ and NADH+ or the reduced forms thereof NADH and NADPH respectively. NAD(P)+ here stands for NAD+ and/or NADP+ and NAD(P)H here stands for NADH and/or NADPH.

In main embodiment, the present invention provides a process for the regioselective biocatalytic oxidation at position 7 and /or 12 of Formula VII, or salts thereof,
,
wherein
R is selected from a-OH, ß-OH, -CO;
R1 is selected from H, -CO, a-OH, ß-OH; and
R2 is selected from H, straight or branched chain alkyl;
comprising reacting compound of Formula VII with NAD(P)+ dependent hydroxysteroid dehydrogenase in the presence of suitable dehydrogenase and ester selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain.

In another embodiment, the ester used as co-substrate along with a suitable dehydrogenase is selected from, but not limited to, the group comprising of ethyl acetoacetate, propyl acetoacetate, n-butyl acetoacetate, isopropyl acetoacetate, isobutyl acetoacetate, amyl acetoacetate, isoaamyl acetoacetate, t-butyl acetatoacetate, and the like.

In another embodiment, the suitable dehydrogenase used along with co-substrate is selected from, but not limited to, the group comprising of lactate dehydrogenase, alcohol dehydrogenase, glucose dehydrogenase and the like.

In one another embodiment, the present invention provides a process for preparing ursodeoxycholic acid of Formula I by regioselective biocatalytic oxidation of Formula VII, or salt thereof, wherein said oxosteroid of Formula VII is provided in the reaction at a concentration less than 50 g/l.

In another embodiment, the present invention provides a process for preparing ursodeoxycholic acid of Formula I,

a) selectively oxidizing position 12 of cholic acid of Formula II to give 12-keto cholanic acid of Formula III, in presence of a 12a-hydroxysteroid dehydrogenase and a cofactor regeneration system, comprising NAD(P)+ dependent dehydrogenase and ester selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain,
; and
b) converting compound of Formula III to ursodeoxycholic acid.

In another embodiment, the 12a-hydroxysteroid dehydrogenase is selected from 12a-hydroxysteroid dehydrogenase of E. coli strain which is a derivative of E. coli K12.

In another embodiment, the suitable NAD(P)+ dependent dehydrogenase used along with co-substrate is selected from, but not limited to, the group comprising of lactate dehydrogenase, alcohol dehydrogenase, glucose dehydrogenase and the like.

Moreover, the oxidation at position 12 of cholic acid of Formula II is carried out in presence of base selected from organic and inorganic base such as alkali and alkaline earth metal phosphates, hydroxides, carbonates, bicarbonates and the like.

In further embodiment, the regioselective biocatalytic oxidation at position 12 of cholic acid of Formula II is carried out in presence of enzyme such as NADP(+) dependent 12a-hydroxysteroid dehydrogenase (EC 1.1.1.176) of E. coli strain which is a derivative of E. coli K12. The oxidation is performed in presence of regeneration system of the co-factor NADP(+) comprising atleast one alcohol dehydrogenase ADH (EC 1.1.1.1) along with ester reagent selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain. Here the enzyme can be a natural or recombinantly produced enzyme. The enzyme in principle be present mixed with cellular, such as for example protein impurities, but preferably in pure form.

In another embodiment, the 12-keto cholanic acid of Formula III so obtained by the process of the present invention is optionally purified or crystallized before proceeding to chemical reduction to chenodeoxycholic acid.

In another embodiment, the present invention provides a chemical-biocatalytical method for preparation of chenodeoxycholic acid of Formula IV,
,
wherein said process comprising the steps of:
a) selectively oxidizing position 12 of cholic acid of Formula II to give 12-keto cholanic acid of Formula III, in presence of a 12a-hydroxysteroid dehydrogenase and a cofactor regeneration system, comprising NAD(P)+ dependent dehydrogenase and ester selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain,
; and
b) converting compound of Formula III to chenodeoxycholic acid of Formula IV through Wolff-Kishner reduction of the ketone group in position 12 in presence of hydrazine hydrate and glycol,
,
wherein intermediate of Formula IIIa is optionally isolated.

In another embodiment, the present invention provides a biocatalytical process for preparing ursodeoxycholic acid of Formula I,

wherein said process comprising the steps of:
a) selectively oxidizing the chenodeoxycholic acid of Formula IV to 3a-hydroxy-7-ketocholanic acid (7-keto CDCA) of Formula V, in presence of at least one hydroxysteroid dehydrogenase and a cofactor regeneration system, comprising NAD(P)+ dependent dehydrogenase and ester selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain,
; and
b) converting compound of Formula V to ursodeoxycholic acid.

In another embodiment, the selective oxidation at position 7 of CDCA is performed by reacting with stereoselective 7a-hydroxysteroid dehydrogenase of E. coli strain which is a derivative of E. coli K12. Here the enzyme can be a natural or recombinantly produced enzyme. The enzyme in principle be present mixed with cellular, such as for example protein impurities, but preferably in pure form.

In another embodiment, the 3a-hydroxy-7-ketocholanic acid of Formula V so obtained by the process of the present invention is optionally purified or crystallized before converting to UDCA.

In another embodiment, the present invention provides a biocatalytical process for preparing ursodeoxycholic acid of Formula I,

wherein said process comprising the steps of:
a) selectively oxidizing the chenodeoxycholic acid of Formula IV to 3a-hydroxy-7-ketocholanic acid (7-keto CDCA) of Formula V, in presence of at least one hydroxysteroid dehydrogenase and a cofactor regeneration system, comprising NAD(P)+ dependent dehydrogenase and ester selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain,
; and
b) selectively reducing position 7 of 7-keto-CDCA of Formula V in presence of NAD(P)+ dependent hydroxysteroid dehydrogenase to get ursodeoxycholic acid.

In another embodiment, the suitable NAD(P)+ dependent dehydrogenase used along with co-substrate is selected from, but not limited to, the group comprising of lactate dehydrogenase, alcohol dehydrogenase, glucose dehydrogenase and the like.

In another embodiment, reduction of the 3a-hydroxy-7-ketocholanic acid of Formula V is carried out in presence of 7ß-hydroxydehydrogenase and formate dehydrogenase. The cofactor NADPH or NADH can be regenerated by an NAD(+) dependent alcohol dehydrogenase or formate dehydrogenase.

In another embodiment, the present invention provides a process for the preparation of ursodeoxycholic acid of Formula I,

wherein said process comprising the steps of:
a) selectively oxidizing position 12 of cholic acid of Formula II to give 12-keto cholanic acid of Formula III, in presence of a 12a-hydroxysteroid dehydrogenase and a cofactor regeneration system, comprising NAD(P)+ dependent alcohol dehydrogenase and ester selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain,
;
b) converting compound of Formula III to chenodeoxycholic acid of Formula IV through Wolff-Kishner reduction of the ketone group in position 12 in presence of hydrazine hydrate and glycol,
,
wherein intermediate of Formula IIIa is optionally isolated;
c) selectively oxidizing the chenodeoxycholic acid of Formula IV to 3a-hydroxy-7-ketocholanic acid (7-keto CDCA) of Formula V, in presence of at least one hydroxysteroid dehydrogenase and a cofactor regeneration system, comprising NAD(P)+ dependent alcohol dehydrogenase and ester selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain,
;
d) selectively reducing position 7 of 7-keto-CDCA of the Formula V to ursodeoxycholic acid in presence of 7ß-hydroxydehydrogenase, and regeneration system comprising NADP(+) dependent formate dehydrogenase; and
e) optionally purifying the ursodeoxycholic acid.

In a preferred embodiment, the reactions (oxidations of (12 and/or 7 position of cholic acid or chenodeoxycholic acid) is carried out for a period of time ranging from 2 to 20 h. The oxidation reaction is carried out in presence of potassium phosphate buffer biphasic or monophasic system using cofactor regeneration system.

In another embodiment, the present invention provides process for the purification of ursodeoxycholic acid wherein said process comprising the steps of:
a) treating ursodeoxycholic acid with base to give ursodeoxycholic acid base adduct of Formula VI in presence of suitable solvent,
;
b) converting compound of Formula VI to ursodeoxycholic acid;
c) treating with suitable solvent; and
d) isolating ursodeoxycholic acid having purity of 99.0% and above.

In one another embodiment, the present invention provides process of purification of ursodeoxycholic acid wherein said process comprising the steps of:
a) treating ursodeoxycholic acid with base to give ursodeoxycholic acid base adduct of Formula VI in presence of suitable solvent,
;
b) acidifying compound of Formula VI in presence of an acid to give solid mass;
c) filtering and treating the solid mass with polar solvent to give precipitates;
d) filtering the precipitates and washing with polar solvent; and
e) drying the precipitates to give pure ursodeoxycholic acid.
In another embodiment, the base used for purification and preparation of ursodeoxycholic acid base adduct of Formula VI is selected from, but not limited to, organic and inorganic base such as metal salts, primary, secondary and tertiary amines.

In another embodiment, the suitable solvent used for purification of ursodeoxycholic acid is selected from, but not limited to, methyl isobutyl ketone, diethyl ketone, water, acetone, methyl ethyl ketone, ethyl acetate, isopropyl acetate, t-butyl acetate, isobutyl acetate, n-propyl acetate, n-butyl acetate, pentyl acetate, tetrahydrofuran, methyl-tetrahydrofuran, dioxane, diethyl ether, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, 2-nitroethanol, 2-fluoroethanol, 2,2,2-trifluoroethanol, hexafluoroisopropyl alcohol, ethylene glycol, 1-propanol, methanol, ethanol, 2-propanol (isopropyl alcohol), 2-methoxyethanol, 1-butanol, 2-butanol, t-butyl alcohol, 2-ethoxyethanol, diethylene glycol, polyethylene glycol, 1-, 2-, or 3-pentanol, neo-pentyl alcohol, t-pentyl alcohol, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethyl ether, diisopropyl ether, methyl t-butyl ether, glyme, diglyme, cyclohexanol, phenol, glycerol, toluene, glycols, methyl t-butyl ether, diisopropyl ether, acetonitrile, propionitrile, butanenitrile, xylene, cyclohexane, n-Heptane, methyl acetate, hexane, anisole, pentane and mixture thereof.

In preferred embodiment, the polar solvent used for recrystallization of ursodeoxycholic acid is selected from, but not limited to, the group comprising of acetone, methyl isobutyl ketone, ethyl methyl ketone, ethyl acetate, isopropyl acetate, t-butyl acetate, isobutyl acetate, n-propyl acetate, n-butyl acetate, pentyl acetate, methanol, ethanol, isopropanol, butanol, t-butanol, glycols, water, methyl t-butyl ether, diisopropyl ether, acetonitrile, xylene, cyclohexane, n-Heptane, methyl acetate, and mixture thereof.

In one another embodiment, the present invention provides ursodeoxycholic acid base adduct of Formula VI,
.

In a preferred embodiment, the base in ursodeoxycholic acid base adduct is selected from triethyl amine, dicyclohexyl amine, diisopropyl ethyl amine, diisopropyl amine, methyl amine, sodium metal, potassium metal, calcium metal, magnesium metal, barium metal, cesium metal, lithium metal, ethanolamine, meglumine, piperidine, benzyl piperazine, diethyl amine, methyl benzyl amine, morpholine, N, N-dibenzyl ethylene diamine and the like.

In another embodiment, the present invention provides substantially pure ursodeoxycholic acid substantially free from chenodeoxycholic, lithocholic acid, cholic acid and other related impurities wherein each impurity is less than about 0.2% w/w.

In another embodiment, the present invention provides substantially pure ursodeoxycholic acid substantially free from one or more impurities of Formulae II, IV, V, VIII, IX, X, XI and XII, wherein each impurity is less than about 0.2% w/w,
, , , , , ,
, and .

In a preferred embodiment, the present invention provides a stable and substantially pure ursodeoxycholic acid substantially free of impurities wherein each impurity is less than about 0.1%w/w and wherein total impurity is less than about 0.15% w/w.

In another embodiment, the ursodeoxycholic acid is having the purity of 99.0% and more by HPLC, preferably, 99.5% and more by HPLC, and most preferably 99.9% and more by HPLC.

In another embodiment, the ursodeoxycholic acid is characterized by the particle size distribution wherein, d90 is between 0.1µm to 200µm. More preferably, the ursodeoxycholic acid is characterized by particle size distribution wherein, d90 is between 2.0 µm to 150µm.

In another embodiment, the present invention provides a process for the preparation of an amorphous solid dispersion of ursodeoxycholic acid, comprising the steps of:
a) adding ursodeoxycholic acid in a suitable solvent to get a mixture;
b) providing a solution of atleast one pharmaceutically acceptable carrier in a suitable solvent and adding to the mixture obtained in step a), wherein said suitable solvent is optionally similar to the solvent used in step a); and
c) isolating to get amorphous solid dispersion of ursodeoxycholic acid.

In another embodiment, pharmaceutically acceptable carrier used for preparing solid dispersion may include, but not limited to, an inorganic oxide such as SiO2, TiO2, ZnO2, ZnO, Al2O3 and zeolite; a water insoluble polymer is selected from the group consisting of cross-linked polyvinyl pyrrolidinone, polyvinyl pyrrolidone, cross-linked cellulose acetate phthalate, hydroxypropyl methyl cellulose acetate succinate, microcrystalline cellulose, polyethylene glycol, polyethylene/polyvinyl alcohol copolymer, polyethylene/polyvinyl pyrrolidinone copolymer, cross-linked carboxymethyl cellulose, sodium starch glycolat, and cross-linked styrene divinyl benzene, polyvinylpyrrolidone vinylacetate, co-povidone NF, polyvinylacetal diethylaminoacetate (AEA®), polyvinyl acetate phthalate, polysorbate 80, polyoxyethylene–polyoxypropylene copolymers (Poloxamer® 188), polyoxyethylene (40) stearate, polyethyene glycol monomethyl ether, polyethyene glycol, pluronic F-68, methylcellulose, methacrylic acid copolymer, hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, Soluplus® polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG)), gelucire 44/14, ethyl cellulose, D-alpha-tocopheryl polyethylene glycol 1000 succinate, cellulose acetate phthalate, carboxy methyl ethyl cellulose and the like; cyclodextrins, gelatins, sugars, polyhydric alcohols, and the like; water soluble sugar excipients, preferably having low hygroscopicity, which include, but are not limited to, mannitol, lactose, fructose, sorbitol, xylitol, maltodextrin, dextrates, dextrins, lactitol and the like; polyethylene oxides, polyoxyethylene derivatives, polyvinyl alcohols, propylene glycol derivatives, and mixture of any above mentioned carriers.

In still another embodiment, the present invention provides a pharmaceutical composition comprising ursodeoxycholic acid along with atleast one pharmaceutically acceptable excipients.

The present invention is explained below by way of examples. However, the examples are provided as one of the possible way to practice the invention and should not be considered as limitation of the scope of the invention.

EXAMPLES

EXAMPLE 1: Preparation of 12-keto cholanic acid of Formula III:
Charged 300 ml of DM water and 8.0g of dipotassium hydrogen phosphate into a round bottom flask at 20-30oC. Added 100g of cholic acid and adjusted the pH to 6.5 by addition of solution of 20% sodium hydroxide in water followed by addition of 0.20g of magnesium chloride hexahydrate. To another round bottom flask was added 300ml of DM water and 10.32g of dipotassium hydrogen phosphate and adjusted the pH to 6.5 by 10% potassium dihydrogen phosphate (0.3g) followed by addition of magnesium chloride hexahydrate (0.2g) to make a buffer solution. Took 50% of the above buffer solution in another vessel for enzyme slurry and remaining was kept for flushing. Charged 5.0g of 12a-hydroxysteroid dehydrogenase, 5.0 g of alcohol dehydrogenase and 500 mg of NAD and stirred the enzyme slurry for 30 min. Charged the enzyme slurry to the reaction mass containing cholic acid and added 100.0 ml isopropyl acetoacetate at 20-30oC. Flushed the enzyme vessel with remaining 50% buffer solution and added to the reaction mass. Maintained the reaction at 20-30oC for 10h. Cooled the reaction mass and adjusted the pH to 1.0 by hydrochloric acid solution. Stirred the reaction mass for 30 min. Filtered the reaction mass and washed the wet cake with DM water. Dried the wet cake to get 200gm of compound of Formula III.

EXAMPLE 2: Preparation of 12-keto cholanic acid of Formula III:
Charged 300 ml of DM water and 8.0g of dipotassium hydrogen phosphate into a round bottom flask at 20-30oC. Added 100g of cholic acid and adjusted the pH to 6.5 by addition of solution of 20% sodium hydroxide in water followed by addition of 0.20g of magnesium chloride hexahydrate. To another round bottom flask was added 300ml of DM water and 10.32g of dipotassium hydrogen phosphate and adjusted the pH to 6.5 by adding 10% potassium dihydrogen phosphate (0.3g) followed by addition of magnesium chloride hexahydrate (0.2g) to make a buffer solution. Took 50% of the above buffer solution in another vessel for enzyme slurry and remaining was kept for flushing. Charged 5.0g of 12a-hydroxysteroid dehydrogenase, 5.0 g of alcohol dehydrogenase and 500 mg of NAD and stirred the enzyme slurry for 30 min. Charged the enzyme slurry to the reaction mass containing cholic acid and added 100.0 ml Ethyl acetoacetate at 20-30oC. Flushed the enzyme vessel with remaining 50% buffer solution and added to the reaction mass. Maintained the reaction at 20-30oC for 10h. Cooled the reaction mass and adjusted the pH to 1.0 by hydrochloric acid solution. Stirred the reaction mass for 30 min. Filtered the reaction mass and washed the wet cake with DM water. Dried the wet cake to get 150gm of compound of Formula III.

EXAMPLE 3: Preparation of chenodeoxycholic acid (CDCA) of Formula IV:
Charged 150 ml of DM water and 200gm of potassium hydroxide in round bottom flask and stirred till clear solution is obtained. Charged 130 gm of 12-keto cholanic acid followed by addition of 500 ml of ethylene glycol/triethylene glycol. Stirred the reaction mass and added hydrazine hydrate 80% solution (400 ml) and stirred the reaction mass for 10-15 min. Heated the mass to 105-115oC for 60-70 min. Distilled the water from the reaction mass and cooled to 10-20oC. Added 900 ml of water to the reaction mass and cooled the reaction mass to 10-20oC and adjusted the pH to 1.0 by hydrochloric acid solution. Stirred the reaction mass for 60-70 min. Filtered the mass and washed with DM water to get wet cake of 500gm of CDCA of Formula IV.

EXAMPLE 4: Preparation of 3a-hydroxy-7-ketocholanic acid (7-keto CDCA) of Formula V:
Charged 700ml of DM water and 20.0 gm of dipotassium hydrogen phosphate in round bottom flask and adjusted the pH of the buffer solution to 7.0 by 10% potassium dihydrogen phosphate (0.42g) at 20-30oC. Charged magnesium chloride hexahydrate (0.2g) and took 400ml of above buffer solution in another round bottom flask. Added chenodeoxycholic acid wet cake (400gm) to above 400ml of buffer solution. To 150ml of remaining buffer solution was added 5.0g of 7a-hydroxysteroid dehydrogenase, 5.0g of alcohol dehydrogenase and 500.0 mg of NAD and stirred the enzyme slurry so obtained for 30 min. Charged enzyme slurry to the reaction mass containing chenodeoxycholic acid and added 50.0 ml of acetone. Flushed the enzyme round bottom flask with remaining 150 ml of buffer solution and added to the reaction mass. Stirred the reaction mass for 2-3 at 15-25oC. Distilled out the acetone under vacuum at 40-50oC. Cooled the reaction mass at 10-20oC and adjusted the pH of the reaction mass to 1.0 by hydrochloric acid solution. Stirred the reaction mass for 30 min. Filtered the reaction mass and washed the wet cake with DM water and dried to get 100gm of 7-keto chenodeoxycholic acid of Formula V.

EXAMPLE 5: Preparation of 3a-hydroxy-7-ketocholanic acid (7-keto CDCA) of Formula V:
Charged 700ml of DM water and 20.0 gm of dipotassium hydrogen phosphate in round bottom flask and adjusted the pH of the buffer solution to 7.0 by 10% potassium dihydrogen phosphate (0.42g) at 20-30oC. Charged magnesium chloride hexahydrate (0.2g) and took 400ml of above buffer solution in another round bottom flask. Added chenodeoxycholic acid wet cake (400mg) to above 400ml of buffer solution. To 150ml of remaining buffer solution was added 5.0g of 7a-hydroxysteroid dehydrogenase, 5.0g of alcohol dehydrogenase and 500.0 mg of NAD and stirred the enzyme slurry so obtained for 30 min. Charged enzyme slurry to the reaction mass containing chenodeoxycholic acid and added 50.0 ml of Ethyl acetoacetate. Flushed the enzyme round bottom flask with remaining 150 ml of buffer solution and added to the reaction mass. Stirred the reaction mass for 2-3 at 15-25oC. Distilled out the acetone under vacuum at 40-50oC. Cooled the reaction mass at 10-20oC and adjusted the pH of the reaction mass to 1.0 by hydrochloric acid solution. Stirred the reaction mass for 30 min. Filtered the reaction mass and washed the wet cake with DM water and dried to get 100gm of 7-keto chenodeoxycholic acid of Formula V.

EXAMPLE 6: Preparation of ursodeoxycholic acid of Formula I:
Charged 800ml of DM water and 20.0gm of dipotassium hydrogen phosphate in round bottom flask. Adjusted the pH of the buffer solution to 7.0 by 10% potassium dihydrogen phosphate (0.50g). Charged magnesium chloride hexahydrate (0.4g) and took 400ml of above said buffer solution into reactor and added 7-keto chenodeoxycholic acid (100gm) at 20-30oC. Took 200ml of the remaining buffer solution into another vessel for enzyme slurry and rest 200ml was kept for flushing. Charged 5.0gm of 7ß-hydroxysteroid dehydrogenase, 5.0gm of formate dehydrogenase and 200mg of NAD and stirred the resultant enzyme slurry for 30 min at 15-25oC. Prepared sodium formate 50% solution and added the said sodium formate solution to the reaction mass and flushed sodium formate vessel by DM water (100ml) at 20-30oC. Charged enzyme slurry to the reaction mass and added 200ml of methyl isobutyl ketone to the reaction mass and then flushed the enzyme round bottom flask with 200ml of buffer solution and charged into reaction mass. Stirred the reaction mass for 25-30 hrs at 20-30oC. Cooled the reaction mass at 10oC and adjusted the pH of the reaction mass to 1.0 by hydrochloric acid solution. Stirred the reaction mass for 30 min at 10-20oC and then filtered and washed the wet cake with DM water (500ml). Charged acetone (300ml) to the wet cake in a round bottom flask and heated the mass to reflux for 45-60 min at 50-60oC and then added 1000ml of DM water and stirred the mass for 60-45 min at 50-60oC. Cooled the mass to 15-22oC. Filtered the mass and washed the wet cake with DM water to get 130gm of ursodeoxycholic acid.

EXAMPLE 7: Purification of ursodeoxycholic acid:
Charged 5.0 V of methyl isobutyl ketone to 130 gm of ursodeoxycholic acid and heated the reaction mass to 60oC and slowly added triethyl amine (0.29 times of ursodeoxycholic acid) and stirred the reaction mass for 30-45 min at 60oC. Cooled the mass to 20-25oC and stirred for 90-120 min. Filtered the mass and washed with methyl isobutyl ketone. Charged the UDCA-TEA into 10.0 V of DM water and heated the mass to 40-50oC. Adjusted the pH to 1.0 by hydrochloric acid and stirred at 40-50oC. Cooled the slurry to 20-30oC and stirred for 30-45 min at 20-30oC. Filtered the mass and washed the wet cake with DM water. Charged methyl isobutyl ketone (5.0 V) to the wet cake and heated the mass to 85-90oC and then cool to 0-10oC. Stirred the reaction mass for 45-60 mins at 0-10oC. Filtered the mass and washed the wet cake with methyl isobutyl ketone and dried to get 80gm of pure ursodeoxycholic acid.

EXAMPLE 8: Preparation of solid dispersion of ursodeoxycholic acid with HPMC:
Charged 5.0V of methanol to ursodeoxycholic acid (1.0g) and added a mixture of HPMC (1.0g) in acetone. Stirred the solution so obtained at room temperature and then filtered to get the desired compound.

CLAIMS:WE CLAIM
1. A process for the regioselective biocatalytic oxidation at position 7 and /or 12 of Formula VII, or salts thereof,
,
wherein;
R is selected from a-OH, ß-OH, -CO; R1 is selected from H, -CO, a-OH, ß-OH; and R2 is selected from H, straight or branched chain alkyl;
comprising reacting compound of Formula VII with NAD(P)+ dependent hydroxysteroid dehydrogenase in the presence of suitable dehydrogenase and ester selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain.

2. The process as claimed in claim 1, wherein said compound of Formula VII is provided in the reaction at a concentration less than 50 g/l.

3. A process for preparing ursodeoxycholic acid of Formula I,

a) selectively oxidizing position 12 of cholic acid of Formula II to give 12-keto cholanic acid of Formula III, in presence of a 12a-hydroxysteroid dehydrogenase and a cofactor regeneration system, comprising NAD(P)+ dependent dehydrogenase and ester selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain,
; and
b) converting compound of Formula III to ursodeoxycholic acid.

4. A biocatalytical process for preparing ursodeoxycholic acid of Formula I,

wherein said process comprising the steps of:
a) selectively oxidizing the chenodeoxycholic acid of Formula IV to 3a-hydroxy-7-ketocholanic acid (7-keto CDCA) of Formula V, in presence of at least one hydroxysteroid dehydrogenase and a cofactor regeneration system, comprising NAD(P)+ dependent dehydrogenase and ester selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain,
; and
b) converting compound of Formula V to ursodeoxycholic acid.

5. The process as claimed in claims 3 and 4, wherein said process further comprising the steps of:
a) selectively oxidizing position 12 of cholic acid of Formula II to give 12-keto cholanic acid of Formula III, in presence of a 12a-hydroxysteroid dehydrogenase and a cofactor regeneration system, comprising NAD(P)+ dependent alcohol dehydrogenase and ester selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain,
;
b) converting compound of Formula III to chenodeoxycholic acid of Formula IV through Wolff-Kishner reduction of the ketone group in position 12 in presence of hydrazine hydrate and glycol,
,
wherein intermediate of Formula IIIa is optionally isolated;
c) selectively oxidizing the chenodeoxycholic acid of Formula IV to 3a-hydroxy-7-ketocholanic acid (7-keto CDCA) of Formula V, in presence of at least one hydroxysteroid dehydrogenase and a cofactor regeneration system, comprising NAD(P)+ dependent alcohol dehydrogenase and ester selected from CH3COCH2COOR3 wherein R3 is selected from C2-C5 alkyl chain,
;
d) selectively reducing position 7 of 7-keto-CDCA of the Formula V to ursodeoxycholic acid in presence of 7ß-hydroxydehydrogenase, and regeneration system comprising NADP(+) dependent formate dehydrogenase; and
e) optionally purifying the ursodeoxycholic acid.

6. A process for the purification of ursodeoxycholic acid wherein said process comprising the steps of:
a) treating ursodeoxycholic acid with base to give ursodeoxycholic acid base adduct of Formula VI in presence of suitable solvent,
;
b) converting compound of Formula VI to ursodeoxycholic acid;
c) treating with suitable solvent; and
d) isolating ursodeoxycholic acid having purity of 99.0% and above.

7. The process as claimed in claim 6, wherein said base in ursodeoxycholic acid base adduct is selected from triethyl amine, dicyclohexyl amine, diisopropyl ethyl amine, diisopropyl amine, methyl amine, sodium metal, potassium metal, calcium metal, magnesium metal, barium metal, cesium metal, lithium metal, ethanolamine, meglumine, piperidine, benzyl piperazine, diethyl amine, methyl benzyl amine, morpholine, and N, N-dibenzyl ethylene diamine.

8. The process as claimed in claim 6, wherein said ursodeoxycholic acid isolated in step (d) is substantially free from chenodeoxycholic, lithocholic acid, cholic acid and other related impurities wherein each impurity is less than about 0.2% w/w.

9. A process for the preparation of an amorphous solid dispersion of ursodeoxycholic acid, comprising the steps of:
a) adding ursodeoxycholic acid in a suitable solvent to get a mixture;
b) providing a solution of atleast one pharmaceutically acceptable carrier in a suitable solvent and adding to the mixture obtained in step a), wherein said suitable solvent is optionally similar to the solvent used in step a); and
c) isolating to get amorphous solid dispersion of ursodeoxycholic acid.

10. Composition comprising ursodeoxycholic acid and atleast one pharmaceutically acceptable excipient wherein said ursodexycholic acid is prepared as per the process claimed in any of the preceding claims.