Description:Title of the Invention

“PROCESS FOR THE PREPARATION OF 12-KETO OBETICHOLIC ACID”

This application is a divisional application of Indian Patent application number 202241018789 dated on 30th March, 2022.

Field of the Invention
The present invention related to an improved process for the preparation of Obeticholic acid having the chemical structural Formula I.

Background of the Invention
Obeticholic acid is a semi-synthetic bile acid analogue. It is an agonist of farnesoid X receptor (FXR) ligand. Obeticholic acid is indicated for the treatment of primary biliary cirrhosis (PBC). Obeticholic acid (OCA) or 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oic acid or compound of formula I in the present invention is a 6α-ethylated derivative of bile chenodeoxycholic acid (CDCA). The chemical structure of Obeticholic acid is shown below.

PCT patent application WO 2002072598 A1 (hereinafter referred as the WO'598 application) discloses Obeticholic acid and process for preparation thereof. The synthetic route comprises: the protection of the hydroxyl group at position C3 of 3α-hydroxy-7-keto-5β-cholan-24-oic acid with a tetrahydropyranyl group to yield 3α-tetrahydropyranyloxy-7-keto-5β-cholan-24-oic acid, alkylation of the carbon at position C6, and esterification of the carboxylic group with ethyl bromide and deprotection of the tetrahydropyranyl group to yield ethyl 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oate, reduction of the ketone group at position C7 to hydroxyl with sodium borohydride to yield ethyl 3α, 7α-dihydroxy-6α-ethyl-5β-cholan-24-oate, and finally deprotection of the ester group to yield Obeticholic acid.

The problem with this synthetic route is the low yield (3%), and that it furthermore involves multiple steps of purification by column chromatography, which hinders putting it into practice on an industrial scale.

Zampella et al. [J. Med. Chem., 2012, 55, 84-93] disclose another obeticholic acid synthesis route comprising the oxidation of chenodeoxycholic acid (CDCA) with a solution of sodium hypochlorite/NaBr and tetrabutylammonium bromide in a mixture of methanol/acetic acid/water/ethyl acetate as solvent, followed by benzylation of the carboxylic acid at position C24, to yield the benzyl ester of 7-ketolitocholic acid. Next, the silyl enol ether is generated followed by aldol addition with acetaldehyde in the presence of BF3•OEt2 to yield ethyl 3α- hydroxy-6α-ethylidene-7-keto-5β-cholan-24-oate. Then, selective reduction of ketone at position C7 with NaBH4/CeCl3 in a mixture of THF/methanol and after that hydrogenation of the exocyclic double bond, together with the removal of the benzyl protecting group, are carried out to yield obeticholic acid. The yield of this synthesis route is 32%. Despite having improved the yield, this synthetic route still involves various steps of purification by column chromatography, rendering it unsuitable for industrial implementation.

Several processes for preparation of Obeticholic acid of Formula I and its intermediates have been disclosed in US7994352B2,US 8338628 B2, CN 107400154 A and CN 106589039 A. Nevertheless, these synthetic routes are includes several steps of purification by column chromatography, rendering its unsuitable for industrial implementation.

Obeticholic acid is synthetic bile acid and hence achieving ICH grade of purity is known to be difficult. Moreover, the starting material for the synthesis of Obeticholic acid is chenodeoxycholicacid which is obtained from animal origin. Hence, the purification of Obeticholic acid is essential for the use of it as drug. Hence, there remains a need for an alternate commercially feasible process for the preparation of pure Obeticholic acid which is suitable to be used as drug.

In view of the importance of treating and preventing disorders associated with farnesoid X receptor (FXR) ligand for the treatment of primary biliary cirrhosis (PBC), it would be desirable and of paramount importance to have a process for the preparation of Obeticholic acid of Formula I, by employing inexpensive, readily available, easy to handle reagents. It would also be desirable to have a process that can be readily scaled up and which does not require a special purification step, thereby making it more it more suitable for industrial scale preparation.

Summary of the Invention
The present invention provides a cost effective, novel and an efficient process for the preparation of Obeticholic acid compound of Formula I with higher yields and better purity.

In one aspect, the present invention provides an improved process for the preparation of Obeticholic acid having the structural Formula I,

which comprises:
i. oxidation of 12-hydroxy Obeticholic acidwith sodium hypobromite or sodium hypochlorite solution or O-iodoxybenzoic acid

in presence of suitable base and solvent or mixture of organic solvents to obtained 12-keto obeticholic acid having the structural Formula II;
ii. reduction of 12-keto Obeticholic acid having the structural Formula II

in presence of suitable base and solvent or mixture of organic solvents to obtained Obeticholic acid having the compound of Formula I.

In a second aspect, the present invention relates to 12-keto obeticholic acid having the structural Formula II

In a third aspect, the present invention relates to 12-keto obeticholic acid having the structural Formula II using for the preparation of compound of Formula I.

Detailed Description of the Invention

Accordingly, the present invention provides an improved process for the preparation of Obeticholic acid having the compound of Formula I.

The main aspect of the present invention provides an improved process for the preparation of Obeticholic acid having the compound of Formula I, which is outlined below in Scheme-II.

Step i) oxidation of 12-hydroxy Obeticholic acid with sodiumhypobromite or sodiumhypochlorite solution or O-iodoxybenzoic acid in presence of suitable base and solvent or mixture of organic solvents to obtained 12-keto Obeticholic acid having the structural Formula II.
Step ii) reduction of 12-keto obeticholic acid having the structural Formula II in presence of suitable base and solvent or mixture of organic solvents to obtained Obeticholic acid having the compound of Formula I.

Solvent is selected from the group consisting of ethylene glycol, diethylene glycol, and triethylene glycol, acetone, diethyl ketone, methyl ethyl ketone, methyl isobutyl ketone and methyl propyl ketone, cyclobutanone, cyclopentanone, cyclohexanone, methanol, ethanol, n-propanol, isopropanol, n-butanol, pentanol, isobutanol, tertiary butanol, cyclopropanol, cyclobutanol, cyclopentanol, cyclohexanol or mixtures.

Base is selected from the group consisting of alkali metal hydroxides, alkali metal carbonates, alkali metal bicarbonates or organic bases

EXPERIMENTAL PORTION
The details of the invention are given in the examples provided below, which are given to illustrate the invention only and therefore should not be construed to limit the scope of the invention.
Example-1: Process for the preparation of 7-Ketocholic acid methyl ester
12-Hydroxy-7-Ketolithocholic acid (10.0 grams) was taken in Methanol (50 mL) at ambient temperature. Added slowly Trimethylsilyl chloride, stirred for 2-3 hours and monitored the un-reacted 12-Hydroxy -7-Ketolithocholic acid by TLC.Added 8% aqueous sodium bicarbonate solution (10 mL) followed by added dichloromethane (50 mL). Stirred, settled and separated the organic layers .Re extracted aqueous layer with dichloromethane (30 mL) and separated the organic layers. Combined organic layer distilled and degas completely to 7-Keto cholic acid methyl ester
Yield: 10.00grams.

Example-2: Process for the preparation of 3,7,12-tri-(trimethylsilyloxy) compound of 7-ketocholic acid methyl ester.
12-Hydroxy Obeticholic acid methyl ester (10.0 grams) was taken in Toluene (100 mL) at ambient temperature followed by stirring to obtained clear solution. Charged sodium iodide (17.82 grams) and charged acetonitrile (50 mL) followed by triethylamine (13.6 grams) at ambient temperature then after added slowly trimethylsilyl chloride (11.88 grams) at ambient temperature and heated to 50-55 0C and maintained for 4-6 hours at the same temperature. Monitored the unreacted 12-Hydroxy7-ketolithocholic acid methyl ester by TLC. Cooled the reaction mass to 10 oC and added 120 ml aqueous sodium thiosulphate and sodium bicarbonate solution (120 mL) to the reaction mass. separated the organic layers and extraction, combined organic layer distilled out under vacuum below 55 oC to obtained residue of3,7,12-tri-(trimethylsilyloxy) compound of 7-ketocholic acid methyl ester, which is processed for the next step.
Yield: 15.00 grams.

Example-3: Process for the preparation of Olefinic compound.
3,7,12-Tri-(trimethylsilyloxy) compound of 7-ketocholic acid methyl ester (15.0 grams) was taken in dichloromethane (210 mL) and cooled to -30 0C. Added anhydrous acetaldehyde (3.10 grams) and further cooled to - 70 0C and added slowly Boron trifluoride diethyl etherate (11.22 grams) at -70 to -65 0C and maintained the same temperature for 2-3 hours after then raised the temperature to 20-250C and maintained for 2-3 hours. Monitored unreacted 3,7,12-tri-(trimethylsilyloxy) compound by TLC. Cooled to 10 0C and added 8% aqueous sodium bicarbonate solution (200 mL) at 10-20 0C and separated the organic layer and washed the aqueous layer with dichloromethane (50 mL). Combined the organic layer and distilled out completely under vacuum to obtained title compound as a residue.
Yield: 13.00 grams.

Example-4: Process for the preparation of 12-Hydroxy beta ethynyl compound.
Olefinic compound (10.0 grams) was taken in Methanol (100 mL) at 25-30 0C in a clean and dry autoclave under nitrogen atmosphere. Charged 10% palladium carbon under nitrogen atmosphere. Closed the autoclave and flushed the nitrogen gas twice then filled the hydrogen gas 5-6 Kg/cm2. After then raised the temperature 50-55 0C and maintained the same temperature for 2-4 hours. Checked the unreacted staring compound content by TLC (limit NMT 5.0%). Cooled to ambient temperature filtered the catalyst and distilled the filtrate under vacuum completely till to obtained residue of 12-Hydroxy beta ethynyl compound obtained proceed in next step as such.
Yield: 10.00 grams.

Example-5: Process for the preparation of 12-Hydroxy alpha ethynyl compound.
Toluene (100 mL) in residue of 12-Hydroxy beta ethynyl compound (10 grams) charged under nitrogen then added slowly1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (10.20 grams) and heated the reaction mixture to 110-115 0C. Stirred the reaction mass 1320-1440 minutes at 110-115 0C. Checked the unreacted content by TLC (limit NMT 5.0%). Cooled the reaction mass at ambient temperature and added 20% aqueous Sodium chloride solution (100 mL). Separated the organic layer and re extracted aqueous layer with Toluene (50 mL). Combined organic layer then charged 10% aqueous Hydrochloric acid solution (50 mL) to toluene layers are charged 10 %aqueous Sodium bicarbonate solution (60 mL) to toluene layer, stirred and separated the layers. Product containing organic layerdistilled completely and added methanol (50 mL) then heated to 50-55 0C, maintained the same temperature for 1 hour and cooled to ambient temperature and filtered to obtained 12-Hydroxy alpha ethynyl compound.
Yield: 6.5 grams.

Example-6: Process for the preparation of 12-hydroxy acid.
Under Nitrogen charged 12-Hydroxy alpha ethynyl compound (10.0 grams) in methanol (50 mL) and added sodium hydroxide (2.67 grams) containing water (30 mL) at ambient temperature. Heated to 45-50 0C and maintained for 3-4 hours and checked unreacted 12-Hydroxy alpha ethynyl compound by TLC/HPLC (Limit NMT 1.0%). Distilled out methanol under vacuum, charged ethyl acetate (50 mL) and water 930 (30 mL) and adjusted pH 7.0 by hydrochloric acid. Separated the ethyl acetate layer and distilled out (30 mL) completely to obtained 7.0 grams residue of 12-Hydroxy acid.
Yield: 7.0 grams.

Example-7: Process for the preparation of 12-Hydroxy Obeticholic Acid.
Charged 12-Hydroxy acid (10.0 grams) under Nitrogen in methanol (50 mL) and cooled to 0-5 0C then added slowly sodium borohydride (1.74 grams) at controlled temperature 0-5 OC. Thanmaintained for 4 hours andchecked the unreacted 12-Hydroxy acid content by TLC (limit NMT -1.0 %). Added slowly acetic acid to the reaction mass and maintaining temperature 0-10 OC and raised the reaction mass temperature 45-50 OC and distilled the solvent methanol 80-90% under vacuum, cooled and charged ethyl acetate (50 mL) and 5% sodiumbicarbonate solution (50 mL).Stirred and separated the organic layer and aqueous layer again washed with ethyl acetate (30 mL) and collected both organic layer and distilled to obtained 12-Hydroxy Obeticholic acid.
Yield: 10.0 grams.

Example-8: Process for the preparation of 12-Keto Obeticholic Acid.
Process 1:
12-Hydroxy Obeticholic acid (5.0 grams) was taken in Methanol (15 mL) at ambient temperature followed by stirring to obtained clear solution. Charge acetic acid (10 mL) at ambient temperature to the solution added sodiumhypobromite solution (7.5 mL) slowly and maintained for 2-3 hours at temperature 25-30 OC. Then checked unreacted 12-Hydroxy Obeticholic acid content by TLC (Limit NMT 5.0%).Added 20% aqueous sodium bisulphite solution (12.5 mL) and stirred for 90 minutes then added water (75 mL) and stirred for 90-120 minutes (slurry mass obtained ) at 25-30OC. Filtered the solid under vacuum and washed the solid with water (50 mL). The white solid was dried under vacuum at60OC for 10-15 hours to obtained crude12-keto Obeticholic Acid,which was further purified by column chromatography ( Silica gel 60-120, Ethyl acetate/Hexane 20:80 ) to obtained pure 12-keto Obeticholic Acid.
Yield: 4.6 grams (crude) and 3.6 grams (pure).

Process 2:
12-Hydroxy Obeticholic acid (5.0 grams) was taken in Methanol (15 mL) at ambient temperature followed by stirring to obtained clear solution. Than charge acetic acid (10 mL)followed by slowly added aqueous sodium hypochlorite solution (10.53 mL) and maintained for 2-3 hours at temperature 25-30OC.Checked unreacted 12-Hydroxy Obeticholic acid by TLC (Limit NMT 5.0%).Added 20% aqueous sodium bisulphite solution (12.5 mL) and stirred for 90 minutes followed by added water (75 mL) and stirred for 90-120 minutes (slurry mass obtained ) at 25-30 OC. Filtered the solid under vacuum and washed with water (50 mL). The white solid was dried under vacuum at 60 OC for 10-15 hours to obtainedcrude 12-keto Obeticholic acid, which was further purified by column chromatography (Silica gel 60-120, Ethyl acetate/Hexane 20:80) to obtained pure12-keto Obeticholic acid.
Yield: 4.6 grams (crude) and 3.2 grams (pure).

Process 3:
Suspension ofO-iodoxybenzoic acid (2.20 grams) in Tertiary butanol (25 mL) was added to 12-Hydroxy Obeticholic acid (5.0 grams) then refluxed for 1 hour and reaction progress was monitored by TLC. After completion of reaction,solid were separated by filtration and washed with dichloromethane then combined filtrates, concentrated and residue was taken in dichloromethane (50 mL) and dichloromethane layer was washed with of 10% sodium bisulphate (15 mL) and brine solution (15 mL). Dichloromethanelayer was concentrated under reduced pressure to get crude product which was then purified by column chromatography (Silica gel 60-120, Ethyl acetate/Hexane 20:80) to obtained pure 12-keto Obeticholic acid.
Yield: 4.6 grams (crude) and 3.2 grams (pure).

Example-9: Process for the preparation of Obeticholic Acid.
12-Keto Obeticholicacid (10.0 grams), Potassium hydroxide flakes (5.0 grams), 20 g Triethylene glycol (20.0 grams)and 80% hydrazine hydrate (11.2 grams) were charged into a RB flask. The mass was heated to 120-130°C for 1 hour and the reaction mixture was subjected to distillation until a temperature of reaction mixture achieved between 200-210°C and maintained for 3-4 hours. Cooled the reaction mixture to 30-35 °C then slowly charged ice water (50 mL) and ethyl acetate (50 mL) to the reaction mass and acidifiedthe mixture by hydrochloric acid. Then ethyl acetate layer was separated and concentrated under reduced pressure to obtained residual solid. Whichwas then isolated in dichloromethane (25 mL) to obtained Obeticholic Acid.
Yield: 10.0 grams.

Example-10: Process for the preparation of Amorphous Form of Obeticholic Acid.
Obeticholic Acid (2.0 grams) was taken in Methanol (10 mL) at ambient temperature followed by stirring to obtained clear solution which is then filter. In another flask, water (30 mL micron filter) was taken and cooled to 0-5 OC and added to Methanol solution slowly at 0-5 OC stirred for 2 hours (slurry mass obtained) then filtered under vacuum and washed the solid with chilled Water (4 mL). The wet cake was dried under vacuum below 70OC for 10-15 hours to Amorphous Obeticholic Acid.
Yield: 1.8 - 1.9 grams Melting Point - 107.5 to 108.9 OC.

, Claims:1. A compound of 12-keto Obeticholic acid having the structural Formula II

2. The process for preparation of Formula II as claimed in claim 1, which comprises:
i. oxidation of 12-hydroxy Obeticholic acid with sodiumhypobromite or sodiumhypo chlorite solution or O-iodoxybenzoic acid

in presence of solvent to obtained 12-keto Obeticholic acid having the structural Formula II.
3. The process as claimed in claim 1, wherein solvent used are methanol or tertiary butanol.