FIELD OF THE INVENTION
The present invention relates to a synthetic process for the preparation of
chenodeoxycholic acid of Formula I, a precursor used for preparation of
Obeticholic acid, \ '

Formula I
wherein compound of Formula I can be used for preparation of Obeticholic acid.
The present invention further provides highly pure chenodexycholic acid comprising less.than about 0.15%w/w of each of the impurities such as lithocholic acid, deoxycholic acid, nutriacholic acid or mixture thereof,

Lithocholic acid, and

Htf H
Deoxycholic acid,

HOx*

Nutriacholic acid.

BACKGROUND OF THE INVENTION
Obeticholic acid is a.semi-synthetic bile acid analogue which has the chemical structure, 6a-ethyl-chenodeoxycholic acid, as represented below, and is used for treating primary biliary cholangitis (PBC). The Obeticholic acid is generally prepared from a precursor i.e. chenodeoxycholic acid wherein chenodeoxycholic acid is in itself is a bile acid. The Chenodeoxycholic acid occurs as a white crystalline substance insoluble in water but soluble in alcohol and acetic acid. Chenodeoxycholic acid is naturally synthesized in the liver from cholesterol by a process which involves several enzymatic steps.

HOx%
Obeticholic acid
Obeticholic acid is known from US patent 7,138,390. Said patent also discloses a process for preparation of obeticholic acid by using 3a-hydroxy-7-keto-5P-cholan-24-oic acid as represented below, which is generally prepared by chenodeoxycholic acid.

HOxl
There are several patents/patent applications that discloses the method of separation or extraction of chenodeoxycholic acid from bile.
CN105646631 discloses extraction method for chenodeoxycholic acid, and relates to the field of biological engineering. The method comprises the following steps:

subjecting fresh or frozen poultry bile to saponification so as to obtain crude bile acid or crude bile acid calcium salt, then subjecting a crude bile acid ester solution and an organic nitrogen-containing compound so as to remove most of hydrophilic impurities in the crude bile acid by utilizing an organic nitrogenTContaining compound, then subjecting the obtained crude bile acid ester solution and another organic nitrogen-containing compound to a reaction so as to obtain a precipitate, and carrying out steps like filtering, decolorizing and refining so as to obtain chenodeoxycholic acid with a purity of about 95%.
CN105418716 discloses a method for synthesizing chenodeoxycholic acid by using duck cholic acid extracted from duck bile. The method is characterized in that a duck cholic acid crude product is extracted from the duck bile, then methyl - esterification and acetylation are carried out on the duck cholic acid crude product, and then a Huang Minglong reduction is used for preparing chenodeoxycholic acid.
CN103360454 provides a method for separating and purifying chenodeoxycholic acid from goose bile.
There are several references known in the art for extraction and purification of chenodexoycholic acid wherein the process involves biological material and biological extraction processes. However, these processes are highly tedious, lengthy and uneconomical processes. Further, the biological process requires biological materials like bile acid which is highly uneconomical and are not environmentally friendly. Moreover, biological processes involves production of large number of endotoxin impurities which are required to be purified making process more tedious.
There are several patents and patent applications that, discloses the process of preparation of Obeticholic acid by using chenodeoxycholic acid, however, there are very few literature available in prior published references that discloses the chemical synthesis of chenodeoxycholic acid.

One such disclosure is Journal of Medicinal Chemistry, 2007, 50, 6048-6058 which discloses a process for preparation of chenodeoxycholic acid as mentioned in the scheme-1.

HO'
OH
Scheme-1
■" ^\ o EtPPh 3Br/t-BuOK/THF - *, --'>^. * ^ AC2O/DMAP/
pyridine


O
*OH
HO*
X
XI

XII

methyl propiolate, Et2AICI

Pd/C, H2 O
A

The major drawback of the literature available in the prior published references is development of chenodeoxycholic acid by using complex biological processes. Taking in consideration of the drawbacks of the prior art processes, the present invention is focused towards the production of chenodeoxycholic acid through synthetic method wherein the method is simple, economic and environmental friendly. Further the process developed by the inventors of the present invention is

easy to handle and involves less to negligible side products and hence very few purification steps are required, making the process more economical.
OBJECT OF THE INVENTION
The main aspect of the present invention is to develop a synthetic method for the production of chenodeoxychoHc acid.
/.
Another aspect of the present invention is to develop an ecofriendly and simple process for production of chenodeoxychoHc acid.
Another aspect of the present invention is to prepare chenodeoxychoHc acid with high purity and yields.
Another aspect of the present invention is to prepare chenodeoxychoHc acid substantially free of endotoxin impurities.
SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide an improved process for the preparation of chenodeoxychoHc acid of Formula I,

HO' Formula I,
wherein said process comprising of:
a) keto protection of 4-androstene-3,17-dione to get compound of Formula II;

Formula II,
b) oxidation .n of compound of Formula II at C-7 in presence of alkyl peroxide to give compound of Formula III;

Formula III,
c) hydrogenation of compound of Formula III to get compound of Formula IV;

Formula IV,
d) keto reduction of compound of Formula IV in presence of metal borohydride to get compound of Formula V;

O Formula V,

e) hydrolysis of compound of Formula V in presence of formic acid to get compound of Formula VI;
O

Formula VI
f) C3-keto reduction of compound of Formula VI to get compound of Formula VII;

HO' Formula VII,
g) conversion of compound of Formula VII to VIII in presence of ethyltriphenylphosphonium bromide at ambient temperature;

HO ^ v OH Formula VIII,
h) hydroxyl protection and condensation of compound of Formula VIII with methyl propiolate in presence of halogenated solvent to get compound of Formula IX;

PO* Formula IX,
wherein P is a protecting group;
i) hydrogenation of Formula IX in presence of palladium catalyst to get compound
of Formula X;

PO' Formula X,
and
j) de-protection of compound of Formula X to get chenodeoxycholic acid of
Formula I.

The present invention further provides substantially pure chenodeoxycholic acid comprising either of the impurities such as lithocholic acid, deoxycholic acid, nutriacholic acid, or, mixture thereof, wherein each impurity is less than about
0.15%w/w;

Lithocholic acid, and

Deoxycholic acid,

C02H

Nutriacholic acid. ^
DETAILED DISCRIPTIQN
The present invention will now be explained in details. While the invention is susceptible to various modifications and alternative forms, specific embodiment thereof will be described in details below. It should be understood, however that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the scope of the invention as defined by the appended claims.
The steps of a method may be providing more details that are pertinent to understanding the embodiments of the present invention and so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

Further characteristics and advantages of the process according to the invention will
result from the description herein below of preferred exemplary embodiments,
which are given as indicative and non-limiting examples. /
The main aspect of the present invention is to develop a synthetic method for the production of chenodeoxycholic acid.
Accordingly, in one embodiment the present invention provides an improved process for the preparation of chenodeoxycholic acid of Formula I,

HO' Formula I,
wherein said process comprising of:
a) keto protection of 4-androstene-3,17-dione to get compound of Formula II;

Formula II,
b) oxidation of compound of Formula II at C-7 in presence of alkyl peroxide to give compound of Formula III;

Formula III,
c) hydrogenation of compound of Formula III to get compound of Formula IV;

Formula IV,
d) keto reduction of compound of Formula IV in presence of metal borohydride to
get compound of Formula V;

Formula V,
e) hydrolysis of compound of Formula V in presence of formic acid to get compound of Formula VI;
O

Cr ^ ^ X>H Formula VI
f) C3-keto reduction of compound of Formula VI to get compound of Formula VII;
O
/
HO' Formula VII,
g) conversion of compound of Formula VII to VIII in presence of ethyltriphenylphosphonium bromide at ambient temperature;

HO' v ^ X>H Formula VIII,
h) hydroxyl protection and condensation of compound of Formula VIII with methyl propiolate in presence of halogenated solvent to get compound of Formula IX;.,.,,

PO Formula IX,
wherein P is a protecting group;
i) hydrogenation of Formula IX in presence of palladium catalyst to get compound
of Formula X;

j) de-protection of compound of Formula X to get chenodeoxycholic acid of
Formula I. . '
In other embodiment, the alkyl peroxides used in step b) is selected from the group comprising of tert butyl hydroperoxide and benzoyl peroxide. More preferably, alkyl peroxide selected is tert butyl hydroperoxide. Other reagents that can be used in step b) are oxidizing agent such as mixture of chromium trioxide and pyridine.

In another embodiment, the metal borohydride used in step d) is selected from the group comprising of sodium borohydride, sodium cyanoborohydride, lithium borohydride, lithium tri butoxyaluminium hydride and aluminium borohydride. In preferred embodiment, the borohydride used in step d) is sodium borohydride, lithium borohydride and lithium tributoxyaluminium hydride. Most preferably, sodium borohydride is used as a source of reducing agent in step d).
In one another embodiment, the keto reducing agent used for reducing compound of formula VI in step f) is selected from lithium trimethoxyalumimum hydride and lithium tri-butoxyaluminium hydride.
In further embodiment, the protecting group of compound of Formula IX and X is
selected from the group comprising of acetyl, benzyl, benzoyl, di-tert-butyl
j
dicarbonate, tetrahydropyranyl, methoxy ethoxy methyl chloride, trityl and silyl ethers.
In preferred embodiment, the protecting group selected is acetyl group and benzyl group and most preferably, hydroxy 1 groups are protected by acetyl group.
In further embodiment, the halogenated solvent as used in step h) is selected from the group comprising of dichloromethane, chloroform, chlorobenzene, dichloroethane tetrachloromethane and mixture thereof.
In further embodiment, the palladium catalyst used in step i) is selected from palladium hydroxide, Pd/C and platinum oxide.
In yet another embodiment, the chenodeoxycholic acid obtained as per the process of the present invention can be used for the preparation of semi-synthetic bile acid analogue i.e. obeticholic acid by using any of the known methods.

Also, the starting material i.e. 4-androstene-3, 17-dione as used in step a) may be obtained from the commercial sources available in the market.
In still another embodiment, the compound of formula IV can be prepared as per the disclosure of publication, Journal of medicinal chemistry, 2012, 55,1334-i345. The said publication discloses process of preparation of steroid intermediates (represented in Scheme 2) wherein the process involves conversion of androstenedione compound to compound of Formula III in presence of p-toluene sulfonic acid and t-butyl hydroperoxide which is then converted to compound of Formula IV via hydrogenation of compound of Formula III.
Scheme-2

pTSA/ethylele glycol
t-BuOOH
Androstene dione

Formula III

Pd/C, H2

Formula IV

In further embodiment, the chenodeoxycholic acid prepared as per the process of the present invention is isolated with purity of 98% and above.
In preferred embodiment, the chenodeoxycholic acid prepared as per the process of the present invention is isolated with purity of 99% and above, and most preferably with purity of 99.5% and above.
In further embodiment, the chenodeoxycholic acid prepared as per the process of the present invention is substantially free of one or more of impurities selected from lithocholic acid, deoxycholic acid, and nutriacholic acid;

HO''

HO ^Ha
T CH3 >

Lithocholic acid, and

Deoxycholic acid,

C02H
HO'1 Nutriacholic acid.

In other embodiment, the present invention provides substantially pure chenodeoxycholic acid comprising less than about 0.15%w/w of either of the impurities lithocholiCacid, deoxycholic acid, nutriacholic acid or mixture thereof.
In preferred embodiment, the present invention provides substantially pure chenodeoxycholic acid comprising either of the impurities lithocholic acid, deoxycholic acid, nutriacholic acid, or mixture thereof, wherein each impurity is about 0.05%w/w or less.
In one more embodiment, the chenodeoxycholic acid prepared as per the process of the present invention is converted to obeticholic acid by known prior published processes.
In another preferred embodiment, the obeticholic acid is characterized by the particle size distribution cbo in the range of 0.1 ^im to 200|im, wherein said obeticholic acid is prepared by using chenodeoxycholic acid of Formula I which is prepared as per the process of the present invention.

In a preferred embodiment^ the obeticholic acid is characterized by the particle size distribution d9o in the range of 2.0 |im to 150|im, wherein said obeticholic acid is prepared by using chenodeoxycholic acid of Formula I which is prepared as per the process of the present invention.
In still another embodiment, the present invention provides a pharmaceutical composition comprising obeticholic acid and at least one of the pharmaceutical acceptable excipients, wherein said obeticholic acid is prepared by using chenodeoxycholic acid of Formula I which is prepared as per the process of the present invention.
Now, the present invention will be explained in details through experimentations. 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 androst-5-ene-3,17-dione, cyclic bis(l,2-ethanediyl acetal);
Compound of Formula-H
To a solution of 50g of androstenedione in 50 ml of tetrahydrofuran was added 21.5 g of ethylene diol (2 mol equivalents) and a catalytic amount of para toluene sulphonic acid. The reaction mixture was stirred at 70-80°C till completion of reaction. Cooled the reaction mass to room temperature and diluted with 500 ml of dichloromethane. Extracted the compound with 250 ml water to remove inorganic impurities. Distilled out the dichloromethane to obtain an oil that was proceeded to next step without purification.
EXAMPLE 2

Preparation of androst-5-ene-3,7,17-trione, cyclic 3,17-bis(l,2-ethanediyl acetal); Compound of Formula III
To the crude androst-5-ene-3,17-dione, cyclic bis(l ,2-ethanediyl acetal) as obtained from example 1 was added 500 ml of ethyl acetate and 78 g of 70% tert butyl hydro peroxide (3.5 mol eq). Added 74.4 g of 10% sodium hypo chlorite drop wise within 7 hr duration at 0- 5°C. The reaction mixture was stirred at 60°C on molecular sieves for 16 hr till completion of reaction. Cooled the reaction mass to room temperature and diluted with 500 ml of ethyl acetate. Washed with 250 ml of water to remove inorganic impurities.,Distilled out the ethyl acetate to obtain an oil that,was proceeded to next step without purification.
EXAMPLE 3
Preparation of androstane-3,7,17-trione, cyclic 3,17-bis(l,2-ethanediyl acetal)-
(5a); Compound Formula IV
Dissolved the crude androst-5rene-3,7,17-trione, cyclic 3?17-bis(l,2-ethanediyl acetal) as obtained from example 2 in ethyl acetate and added 0.5 g of 10% Pd/C. The resulting mixture was hydrogenated with hydrogen gas in a Parr apparatus (50psi) at 50°C for 16 hrs. After completion of reaction, cooled the reaction mass to room temperature and filtered through small plug of celite. Distilled out the solvent under vacuum to obtain semi solid material that was proceeded to next step without purification.
EXAMPLE 4
Preparation of androstane-3,17-dione, 7-hydroxy-, cyclic 3917-bis(l,2-
ethanediyl acetal)- (5a,7a); Compound of Formula V
Dissolved androstane-3,7,17-trione, cyclic 3,17-bis(l,2-ethanediyl acetal)-(5a) as obtained from example 3 in 200ml of tetrahydrofiiran and added 12 g of sodium

borohydride portion wise. The reaction mixture was stirred at ambient temperature for 10 hrs. After completion of reaction, the reaction mixture was diluted by 500ml of dichloromethane. Washed the compound with water to remove inorganic impurities. The dichloromethane layer was dried over MgS04, filtered and distilled . out under vacuum to obtain crude alcohol. The crude product was purified by column chromatography in ethyl acetate/hexane to obtain 34.8 g pure product. -
EXAMPLES'"
Preparation of androstane-3,17-dione, 7-hydroxy-, (5a,7a); Compound of Formula VI
Dissolved 34.5g of androstane-3,17-dione, 7-hydroxy-, cyclic 3,17-bis(l,2-ethanediyl acetal)- (5a,7a) in 150ml of tetrahydrofuran followed by addition.to 10 ml of 5% aqueous solution of formic acid. The reaction mixture was stirred at ambient temperature for 12hrs. After reaction completion, a mixture of IN HC1 and ethyl acetate was added. The two phases were separated and aqueous layer was extracted twice with 100 ml of ethyl acetate. The combined organic layer was washed with saturated brine solution and dried over NaiSCU, filtered and distilled out under vacuum to obtain 22.7g of an oily material.
EXAMPLE 6 (
Preparation of androstan-17-one, 3, 7-dihydroxy-(3p, 5a, 7a); Compound of
Formula VII
Dissolved 22.5g of androstane-3,17-dione, 7-hydroxy-, (5a,7a) as obtained from example 5 in tetrahydrofuran and cooled at - 40°C and added drop wise solution of 1.05 mol eq of lithium tri-butoxyaluminium hydride in an inert atmosphere. The reaction mixture was stirred for 2 hrs. After completion of reaction, the reaction mixture was quenched by adding a mixture of IN HC1 and ethyl acetate. The two phases were separated and aqueous layer was extracted with ethyl acetate. The

organic layer was dried over Na2S04, filtered and distilled out under vacuum to obtain 19.25g of oily material.
EXAMPLE 7
Preparation of pregn-17(20)-ene-3,7-diol, (3a, 5p, 7a, 17Z)-(9C1); Compound
of Formula VIII
To the solution of potassium tert-butoxide in tetrahydrofuran (1M) was added dropwise a solution of ethyltriphenylphosphonium bromide (3 mol eq) in tetrahydrofuran in 30 minutes at ambient temperature. After stirring for 1 hour was added tetrahydrofuran solution of 19.0 g of androstan-17-one, 3, 7-dihydroxy-(3p, 5a, 7a) as obtained from example 6 within the time period of 30-40 minutes. Stirred the reaction mass for 5 hours at ambient temperature. After completion pf reaction, quenched the reaction mass in saturated ammonium chloride solution. Extracted the aqueous layer with ethyl acetate. The organic fractions were combined and concentrated to obtain crude solid (13.8g). The crude solid was taken to next step without purification.
EXAMPLE 8
Preparation of 17r((R,E)-5-methoxy-5-oxopent-3-en-2-yl)-10,13-dimethyI-
2,3,4,5,6,7,8,9,10,1142,13,14,15-tetradecahydro-lH-
cyclopenta[a]phenanthrene-3,7-diyl diacetate; Compound of Formula IX
13.4 g of Pregn-17(20)-ene-3,7-diol, (3a, 5p, 7a, 17Z)-(9C1) as obtained from example 7 was dissolved in dichloromethane and 4 mol. eq. of triethylamine, 0.2 mol. eq. of dimethyl amino pyridine and 3.0 mol. eq. of acetic anhydride were added sequentially at 25°C under inert atmosphere. The reaction mixture was" stirred at room temperature till completion of reaction. After reaction completion, the reaction mass was quenched in ice water. Separated the layers and extracted the aqueous layer with dichloromethane. The combined organic layer was concentrated

to obtain off white solid. The crude product was purified from isopropyl ether to obtain 14g of pure product.
To the solution of methyl propiolate (2.25 mol eq) in dichloromethane was added ethyl aluminum dichloride (3 mol eq) at 0°C and stirred for 30 minutes and then added above obtained product at 0°C. The reaction mass was stirred for 3 hours at 0°C and then at 25 °C for another 14 hours. Quenched the reaction mass in ice water mixture and separated the layers. The aqueous layer .was further extracted with ; dichloromethane. Concentrated the organic layer under vacuum.and resultant sticky compound was crystallized from methanol to get lO.lg of compound of Formula IX.
EXAMPLE 9
Preparation of cholan-24-oic acid, 3,7-bis(acetyloxy)-, methyl ester, (3a,5p,7a)
; Compound of formula X
To a solution of 10.Og of 17-((R,E)-5-methoxy-5-oxopent-3-en-2-yl)-10,13-dimethyl-233)4,5563758?9J10,ll312>13314,15-tetradecahydro-lH-cyclopenta[a]phenanthrene-3,7-diyl diacetate in ethyl acetate was added platinum oxide (0.5 mol eq) and the resultant slurry was hydrogenated in Parr apparatus (80 psi) for 10 hours till reaction completion. The mixture was filtered through celite bed and the solvent was removed under vacuum to obtain 9.0 g of off-white powder that was used in next step without purification.
EXAMPLE 10
Preparation of cholan-24-oic acid, 3,7-dihydroxy-, (3a,5p,); Compound of
Formula I
To the aqueous solution of 4.0 mol eq. of lithium hydroxide was added a solution of 8.5g of cholan-24-oic acid, 3,7-bis(acetyloxy)-3 methyl ester as obtained from example 9 in tetrahydrofuran. The reaction mass so obtained was stirred for 3-5 hours at 45°C. Upon reaction completion, the reaction mass was concentrated under

vacuum to obtain crude product, cooled to 0°C and added a solution of 1 N HCl to the crude product so obtained. The resultant precipitates were filtered and then washed with water. Dried the precipitates under vacuum to obtain 6.0g of Cholan-24-oic acid, 3,7-dihydroxy-, (3a,5p,).

WE CLAIM
1. A process for the preparation of chenodeoxycholic acid of Formula I,

HO' Formula I, i
wherein said process comprising of:
a) keto protection of 4-androstene-3,17-dione to get compound of Formula II;

O Formula II,

b) oxidation of compound of Formula II at C-7 in presence of alkyl peroxide to give compound of Formula III;

Formula III,
c) hydrogenation of compound of Formula III to get compound of Formula IV;

Formula IV,
d) keto reduction of compound of Formula IV in presence of metal borohydride to get compound of Formula V;

Formula V,
e) hydrolysis of compound of Formula V in presence of formic acid to get
compound of Formula VI;

Formula VI
f) C3-keto reduction of compound of Formula VI to get compound of Formula VII;

HO' Formula VII,
g) conversion of compound of Formula VII to VIII in presence of ethyltriphenylphosphonium bromide at ambient temperature;

HO v ^ OH Formula VIII,
h) hydroxyl protection and condensation of compound, of Formula yill with methyl ^ propiolate in presence of halogenated solvent to get compound of Formula IX;

PO' Formula IX,
wherein P is a protecting group;
i) hydrogenation of Formula IXin;presence of palladiumcatalyst to -getcompound
of Formula X;

PO' Formula X,
and
j) de-protection of compound of Formula X to get chenodeoxycholic acid of
Formula I.
2. The process as claimed in claim 1, wherein said alkyl peroxide used in step b) is selected from tert-butyl hydroperoxide and benzoyl peroxide.
3. The process as claimed in claim 1, wherein said metal borohydride used in step d) is selected from the group comprising of sodium borohydride, sodium cyanoborohydride, lithium borohydride, lithium tri butoxyaluminium hydride and aluminium borohydride.
4. The process as claimed in claim 1, wherein said keto reduction of compound of Formula VI is carried out in presence of reducing agent selected from the group comprising of lithium tributbxyaluminium hydride and lithium trimethoxyaluminium hydride.

5. The process as claimed in claim 1, wherein said hydroxyl protecting group of Formula IX and X is selected from the group comprising of acetyl, benzyl, benzoyl, di-tert-butyl dicarbonate, tetrahydropyranyl, methoxy ethoxy methyl chloride, trityl and silyl ethers.
6. The process as claimed in claim 1, wherein said halogenated solvent used in step
h) is selected from the group comprising of dichlorpmethane, chloroform,,/ ^ chlofobenzene, dichloroethane tetrachloromethane and mixture thereof.
7. The process as claimed in claim 1, wherein said palladium catalyst used for'
. hydrogenating compound of Formula IX in step i) is selected from palladium
hydroxide, Pd/C and platinum oxide.
8. The process as claimed in claim 1, wherein said chenodeoxycholic acid of
Formula I is used for the preparation of obeticholic acid.
9. A substantially pure chenodeoxycholic acid comprising of less than about
0.15%w/w of lithocholic acid, or deoxycholic acid, or nutriacholic acid, or mixture
thereof, wherein each of the impurity is less than about 0.15%w/w;

Lithocholic acid, and

Deoxycholic acid,

Nutriacholic acid.
10. A substantiallyp^echenodepxycholic acidcharacterized toy purity above 99%, wherein total amount of impurity is less than- about 1% w/w.