CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority from Italian patent application no. 102020000000328 filed on 10/01/2020, the entire disclosure of which is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a method for the preparation of a compound of formula (I), 3β-arachidylamido- 7α,12α-dihydroxy-5β-cholan-24-carboxylic acid, commonly known as Aramchol.

BACKGROUND ART 3β-arachidylamido-7α,12α-dihydroxy-5β-cholan-24- carboxylic acid (Aramchol) is derived from 3β-amino cholic acid and arachidic acid. Aramchol is molecule being developed by Galmed Pharmaceuticals for treatment of nonalcoholic steatohepatitis (NASH) which is a more advanced condition of non-alcoholic fatty liver disease (US6384024,

Hepatology, 38, 436442; Pathobiology 2002, 70,215-218: Biochem. Soc. Trans., 2004, 32, 131-133).

Aramchol affects liver fat metabolism and has been shown in a Phase Ila clinical study to significantly reduce liver fat content as well as to improve metabolic parameters associated with fatty liver disease. Furthermore, it has been shown to be safe for use, and with no severe adverse effects. It is also reported treating cholelithiasis through the delay of the crystalline growth velocity of cholesterol and the facilitation of the dissolution of formed cholesterol crystals.

Aramchol is being studied as an oral therapy for the treatment of nonalcoholic steatohepatitis (NASH) and fibrosis in Phase III/IV with a recommended daily dose of 600mg or 300mg twice. Phase II study showed good results from efficacy and safety data point of view.

The synthetic route of Aramchol disclosed in US6384024 (shown in Scheme 1) comprises the reaction of methyl 3β- amino-7α,12α-dihydroxy-5β-cholan-24-one reacted with arachidic acid chloride followed by hydrolysis using sodium hydroxide in methanol.

US20120277448 patent by Shangai Institute of Materai

Medica discloses another method for preparation of Aramchol shown in Scheme 2.

The method discloses the use of cholic acid without protection for the preparation of Aramchol. Cholic acid is acylated using mesyl chloride/tosyl chloride/trifluoro mesyl chloride to provide selectively 3-hydroxy protected compound. It is further reacted with sodium azide followed by reduction reaction using 10% Pd/C to yield 3β-amino cholic acid. The last reaction is the conjugation of 3β-amino cholic acid with arachidyl chloride to provide Aramchol. This synthetic route has poor selectivity in acylation reaction towards 3-hydroxy group of cholic acid using mesyl chloride/tosyl chloride/trifluoro mesyl chloride and leads to the formation of diacylated and triacylated impurities. During the purification steps, these impurities yield makes purification of intermediates and final active principle very difficult with simple solvent purifications. The presence of impurities affects also the overall yield of Aramchol and cost effectiveness of the process.

Pore V. S. and et al in Tetrahedron, 26, 2006, 11178-

11186 and Eui-Hyun Ryu and et al in Tetrahedron, 26, 2006, 6808-6813 disclose further procedures for 3-hydroxy acylation of cholic acid using mesyl chloride/tosyl chloride. Both of these procedures suffer selectivity issue toward the 3-hydroxy group that cause the formation of additional impurities and requires silica gel column purifications to get the desired pure product.

CN106496300 patent by Shangai Bozhi Yanxin Pharmaceutical Technology discloses another method for

Aramchol preparation (Scheme 3). This procedure discloses a new approach to prepare methyl 3β-amino cholic acid through a Mitsunobu reaction using pthalimide or succinimide and its use for Aramchol synthesis.

CN106496300 discloses that the purity of the prepared products can reach up to 98.00%. This clearly indicates that the procedure is not capable of producing pure Aramchol as per ICH Q3A guidelines. In addition, the overall yields based on examples provided are poor.

This procedure also exhibits disadvantages of poor selectivity of methyl cholate reaction with pthalimide/succinimide and results in the formation of di- and tri-substituted impurities. The presence of these impurities along with additional impurities generated during the deprotection of pthalimide/succinimide to form amine intermediate make this procedure difficult to produce Aramchol with a quality complying with ICH guidelines.

CN109503693 patent by Hefei University describes a method for the preparation of Aramchol as per the synthetic route given in Scheme 4.

This patent discloses the use of substituted p- toluenesulphonyl chlorides for the selective 3-OH protection of cholic acid. This procedure is not capable to provide good quality of intermediates and final product with simple solvent purifications. For almost every stage (stage 2, 3,

4, 5 and 6) silica gel column chromatography purification is required to get pure intermediates and final product as well which makes procedure too lengthy and not suitable for industrial production of Aramchol.

CN109503693 further describes a procedure using tosyl chloride for the selective 3-OH acylation of cholic acid which seems to be similar to the procedure disclosed in US20120277448 . The disadvantage of this procedure is that p- toluene sulfonyl chloride reacts both with the carboxyl group of the bile acid and the hydroxy group thus, the reaction selectivity is not good and causes a wide side reaction. Therefore, the intermediate purification is not easy and the final product Aramchol yield is low.

CN109503693 describes another synthetic route as shown in Scheme 5.

However, also in this case, the yield of the procedure is very low (based on the total yield is 12.7% Aramchol to cholic acid) and it is suitable only for synthesis of small quantity of Aramchol in a laboratory.

Further disadvantages are that the procedure involves a great number of steps and high costs.

In view of the above, there is a need for a new, easy and industrially scalable procedure for Aramchol synthesis.

Therefore, the aim of the present invention is to provide a new method for the synthesis of Aramchol with high yield of pure product that is easy and industrially scalable. DISCLOSURE OF INVENTION

The aforementioned objective has been met according to the method of claim 1 and intermediate of claim 11. Preferred embodiments are set out within the dependent claims.

In particular, the present inventors had developed an improved process for the preparation of pure Aramchol that is industrially feasible and viable, with the use of industrially friendly solvents and does not require cumbersome work up, repeated purifications and time delaying steps. According to a first aspect of the invention, it is provided a method for the preparation of 3β-arachidylamido- 7α,12α-dihydroxy-5β-cholan-24-carboxylic acid comprising the step of: d) reacting a compound (IV) with methane sulphonyl chloride to obtain methyl 7α,12α-diacetyloxy-3α-mesyloxy-5β- cholan-24-oate (V).

Advantageously, the present invention provides an easy, high yield, cost effective and industrially scalable process for Aramchol preparation. A further advantage of the present invention is the selectivity of the acylation of 3-hydroxy group of methyl cholate using mesyl chloride.

In a further embodiment, the method of the present invention comprises, after step d), the steps of: e) reacting compound (V) with sodium azide to provide methyl 3β-azido-7α,12α-diacetyloxy-5β-cholan-24-oate (VI);

In one embodiment, the method of the present invention further comprises, after step e), the following steps: f) reduction of compound (VI) using a reducing agent followed by salt formation with an acid and obtaining the free amine in the presence of a base to yield methyl 3β- azido-7α,12α-diacetyloxy-5β-cholan-24-oate (VII); g) treating compound (VII) with arachidic acid in the presence of a coupling agent and HOBt/HOAt to form methyl 3β-arachidylamido-7α,12α-diacetyloxy-5β-cholan-24-oate h) hydrolysing compound (VIII) using a base to get 3β- arachidylamido-7α,12α-dihydroxy-5β-cholan-24-carboxylic acid (IX).

Preferably, in a further embodiment, the method of the present invention comprises, before step d), the steps of: a) treating cholic acid (I) with an esterification reagent to provide compound (II);

b) reacting compound (II) with an acylation reagent, an organic base and DMAP to yield methyl 3α,7α,12α- triacetyloxy-5β-cholan-24-oate (III); c) reacting compound (III) with a base in a polar protic solvent to get methyl 7α,12α-diacetyloxy-3α-hydroxy-5β- cholan-24-oate (IV). The present invention involves the acetylation of all three hydroxyl (3, 7 and 12) of methyl cholate and then selective hydrolysis of 3-hydroxy group. The hydrolysis of 3-acetyl is a very selective reaction when performed using suitable reactants which avoid the formation of other impurities . Step a): methylation of cholic acid

The preparation methyl cholate (compound (II)) is performed starting from cholic acid. The reaction can be performed in methanol solvent using an esterification reagent selected from the group consisting of methane sulfonic acid, para-toluene sulfonic acid, acetyl chloride, sulfuric acid, thionyl chloride. The preferred and cost effective esterification reagents are acetyl chloride and methane sulphonic acid. Step a) is performed within a wide range of temperature from 30-80°C, preferably 50-70°C. After complete conversion of the starting material, the product is isolated by filtration at acidic pH.

Step b): acetylation in positions 3, 7 and 12 Compound (II) is treated with an acylation reagent, such as acetic anhydride, an organic base and DMAP to yield methyl 3α,7α,12α-triacetyloxy-5β-cholan-24-oate (III). The organic base can be selected from the group consisting of triethylamine, pyridine, N-methyl morphine, and diisoproylethyl amine; preferably trimethylamine or N-methyl morpholine. Moreover, it can be used a solvent selected from the group consisting of methyl isobutyl ketone, ethyl acetate, methylene dichloride, THF, 1,4-dioxane, acetonitrile, DMF and DMSO. Step b) can be performed in wide range of temperature from 40-120°C; preferably 80-120°C. After aqueous work up and distillation of the organic phase, a semisolid compound III is obtained which can be isolated as a solid through the treatment with suitable solvents selected from the group consisting of diisopropyl ether, diethyl ether, heptane, and pentane. To make procedure short, semisolid compound (III) can be used directly in the next step of hydrolysis.

Step c): hydrolysis in position 3

The semisolid compound (III) is selectively hydrolyzed using a base selected from the group consisting of sodium carbonate, potassium carbonate, sodium methoxide, sodium ethoxide, potassium tertiary butoxide and sodium tertiary butoxide; preferably sodium carbonate. This reaction can be performed in polar protic solvents selected from the group consisting of methanol, ethanol, propanol, water and butanol at temperature range of 0-50°C. After aqueous work up and distillation of the organic phase, semisolid compound (IV) is obtained, which can be isolated as solid by treating it with suitable solvents selected from the group consisting of diisopropyl ether, diethyl ether, heptane, and pentane. To make the procedure easy, short and convenient, semisolid compound (IV) can be used as such in the next step.

Step d): mesylation in position 3

The semisolid compound (IV) is reacted with methane sulphonyl chloride in the presence of a suitable base to provide methyl 7α,12α-diacetyloxy-3α-mesyloxy-5β-cholan-24- oate (V). The reaction can be performed in a solvent selected from the group consisting of ethyl acetate, dichloromethane, toluene, chloroform, tetrahydrofuron, methylisobutyl ketone and mixture therof; preferably ethyl acetate and dichloromethane. The base can be selected from the group consisting of triethyl amine, pyridine, diisopropyl ethylamine, N-methyl morpholine, sodium carbonate, potassium carbonate, sodium bicarbonate and sodium acetate; preferably triethyl amine.

After completion of the reaction, it is quenched with water and the organic layer is evaporated to get semisolid methyl 7α,12α-diacetyloxy-3α-mesyloxy-5β-cholan-24-oate (V) which can be purified and isolated by crystallization using suitable solvent like cychlohexane, hexane, heptane and diisopropyl ether.

Step e): Azidation

Compound (V) is reacted with sodium azide to get methyl 3β-azido-7α,12α-diacetyloxy-5β-cholan-24-oate (VI). The solvent can be selected from the group consisting of DMF, DMSO, DMAc, acetonitrile, ethanol, and methanol. During this reaction, azide nucleophile reacts from opposite side of the mesylate group, which provides the inversion of the stereochemistry of position 3. Mole ratio of sodium azide in respect to compound V ranges from 1.0 to 4.5.

Step f): reduction in position 3

Reduction of compound (VI) can be performed using a reducing reagent and then performing the purification/isolation through the formation of an acid salt followed by obtaining the free amine to yield pure 3β-amino- 7α,12α-diacetyloxy-5β-cholan-24-oate (VII). The present reaction can be performed in solvents selected from the group consisting of methanol, tetrahydrofuron, isopropyl alcohol, n-propanol, 2-butanol and ethanol; preferably in methanol or isopropyl alcohol. For the reduction of the azide to amine, different reducing reagents can be used under hydrogen atmosphere selected from the group consisting of Pd/C, Pt/C and Raney Nickel. Triphenyl phosphine may also be used for said reaction. The most suitable reducing agent found to be 10% Pd/C under hydrogen pressure in the range of 1 - 5kg. After reaction completion, the catalyst is separated by filtration and the collective filtrate is evaporated to get crude amine intermediate (VII), which contains isomer impurity (X) in range of 0.5% to 3%.

It is difficult to purify said compound with simple solvent purification without losing notable yield. If intermediate (VII) is used for next stages to make Aramchol, it results in the formation of 1% to 4% isomer impurity (XI) in final product, which is difficult to eliminate with simple solvent purifications. Thus, it is important to use pure amine intermediate

(VII) in next stages.

To isolate amine intermediate, semisolid compound (VII) is reacted with an acid selected from the group consisting of L-(D)- tartaric acid and L-mendelic acid and isolated in form of salt as shown in Scheme 6 (for L-D-tartaric acid).

The preferred acid for salt formation is tartaric acid which helps to eliminate maximum of isomer impurity up to 0.05%.

Then, the isolated tartaric acid salt of amine intermediate purified with suitable solvent leads to control isomer impurity up to 0.01%. Furthermore, the wet salt as such is processed to obtain the free base using a mild base and to isolate substantial pure amine intermediate (VII).

The salt formation can be performed using a suitable solvent selected from the group consisting of propanol, methanol, ethanol, ethyl acetate, methyl isobutyl ketone, acetone, isopropyl alcohol and mixture thereof; preferably ethyl acetate or propanol.

The impact of amine intermediate (VII) purification with a salt was studied on different starting materials and summarized in Table 1. Results clearly indicates the necessity and the importance of salt purification procedure to get substantial pure Aramchol.

Table 1

All experiments in Table 1 were performed using starting material intermediate (VII) containing 2.1% impurity X. Step g): conjugation with arachidic acid

The reaction of amine intermediate (VII) and arachidic acid was performed in the presence of a coupling reagent, and hydroxybenzotriazole/ 1-hydroxy-7-azabenzotriazole

(HOBt/HOAt) in suitable polar solvent. The coupling reagent is selected from the group consisting of dicyclohexyl carbodiimide (DCC), N- (3-Dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (EDC. HC1), Diethyl cyanophosphonate, 2- ( 1H-benzotriazole-l-yl)-1,1,3,3-tetra- methylaminium-tetra-fluroborate (TBTU), carbonyl di- imidazole (CDI). Preferably, N-(3-Dimethylaminopropyl)-N- ethylcarbodiimide hydrochloride (EDC.HC1) and TBTU can be used to provide better results. The more preferable and cost effective reagent suitable for this reaction was EDC.HC1.

Different inorganic and organic bases can be used for this reaction. Organic bases are selected from the group consisting of triethyl amine (TEA), diisopropyl ethyl amine, Diethyl amine, N-methyl morpholine, n-methyl pyrrolidine. Triethyl amine and n-Methyl morpholine can be used for said reaction in combination with EDC.HC1 to provide better results. To avoid the formation of the impurity (XI) during the reaction, HOBt and HOAt reagents can be used effectively. The reaction was performed in a solvent selected from the group consisting of methylene dichloride, ethyl acetate, chloroform, tetrahydrofuran, acetonitrile, N,N-dimethyl formamide, N,N-dimethyl acetamide, 1,4-dioxane or in a mixture thereof. The preferred solvents are ethyl acetate and methylene dichloride. The present reaction can be performed in wide range of temperature from -20°C to 80°C, preferably -10°C to 50°C. Reaction conversion is completed in 3-20 hrs.

After complete conversion, the starting material is quenched with a suitable diluted aqueous solution of a base like sodium bicarbonate or sodium carbonate. The excess of arachidic acid is removed by washing with an aqueous solution of inorganic base like but not limited to sodium bicarbonate, sodium carbonate, potassium carbonate etc. Preferably, a solution of sodium bicarbonate can be used to get better results. Further unreacted amine content is removed by washing with a diluted aqueous acid, preferably selected from the group consisting of hydrochloric acid, sulphuric acid, citric acid, acetic acid and phosphoric acid. The organic layer is evaporated to provide semisolid methyl 3β- arachidylamido-7α,12α-diacetyloxy-5β-cholan-24-oate (VIII). Compound (VIII) can be isolated as solid with suitable solvents preferably selected from the group consisting of cyclohexane, toluene, diisopropyl ether and diethyl ether.

To make the procedure short, semisolid compound VIII as such can be used in the next step of hydrolysis to make Aramchol .

Step h): hydrolysis

The hydrolysis can be performed in the presence of a base preferably selected from the group consisting of sodium hydroxide, potassium hydroxide, sodium carbonate, ammonia, sodium methoxide and sodium ethoxide; more preferably sodium hydroxide or potassium hydroxide. The hydrolysis reaction can be performed in a solvent selected from the group consisting of water, methanol, ethanol, isopropyl alcohol, butanol, acetone, methyl etheyl ketone and mixtures thereof; preferably alcoholic solvent and water. It can be performed in wide range of temperature from -10°C to 110°C; preferably 0-100°C. After complete conversion of starting materials, aqueous work up can be performed to isolate Aramchol at acidic pH.

The isolated Aramchol can be further purified using ester solvents preferably selected from the group consisting of ethyl acetate, methyl acetate, tert-butyl acetate or ketone solvents preferably selected from the group consisting of acetone, methylisobutyl ketone, ethylmethyl ketone and mixtures thereof with water to provide pure Aramchol .

In another embodiment, purification is performed using mixtures of alcohol solvents like methanol, ethanol, propanol, isopryl alchohol along with water to provide pure Aramchol. The preferred solvent combination is a mixture of ethyl acetate and water in different ratio. The preferred ratio organic solvent/water varies from 0.5% to 99.5%. In another embodiment, for Aramchol purification, the ratio organic solvent/water varies from 50-50%. In another embodiment, the preferred ratio was 30-70% to get better results .

The method of the present invention comprises eight stages, wherein each and every stage is very simple, easy to perform, high yielding and provides good quality of intermediates and final active principle. To make the procedure short, intermediates II and III can be used as such in thenext respective steps without isolation. Also intermediate VIII can be used as such. Therefore, the procedure can be performed to have only five isolating steps and without affecting the quality of intermediates and final product. This makes the procedure of present invention short, cost effective, high yielding and industrially scalable.

Advantageously, one of the differences between the present invention and the synthetic method illustrated in CN109503693 is the use of 7α,12α-diacetyloxy-3α-mesyloxy-5β- cholan-24-oate (V) to prepare azide intermediate (VI) instead of methyl 7α,12α-diacetyloxy-3α-tosyloxy-5β-cholan- 24-oate.

In the present invention, methyl 7α,12α-diacetyloxy-3α- mesyloxy-5β-cholan-24-oate is used to make substantial pure amine intermediate (VII) and subsequently Aramchol with comparatively very high yield and better quality.

The overall yield of Aramchol as per the method of the present invention is about 65% based on cholic acid which is fivefold more than the procedure disclosed in CN109503693 (12.7% only).

Moreover, the procedure disclosed in CN109503693 is not capable of producing good quality of Aramchol, whereas the method of the present invention allows the production of pure Aramchol and control of impurities as per ICH Q3 guidelines. The procedure is capable of producing Aramchol having optical purity up to 99.9%.

The procedure disclosed in CN109503693 is suitable for the preparation of small quantities of Aramchol only and is not industrially scalable, whereas the procedure of present invention has been developed considering safety aspects and operation ease for each stage which make this procedure industrially scalable in production plants. The method is capable of producing bulk quantity of Aramchol up to hundreds kg scale which makes it suitable for commercial mass production.

According to a further aspect of the invention, it is provided the preparation of a pure amine intermediate (VII) having HPLC chromatography purity greater than 99.5 %. Yet another aspect of the present invention relates to the preparation of substantially pure amine intermediate (VII) having HPLC chromatography purity up to 99.9% and isomer impurity of formula (X) controlled up to 0.01%.

Yet another aspect of the present invention relates to preparation of pure Aramchol having HPLC chromatography purity greater than 99.5%.

Yet another aspect the present invention relates to preparation of substantially pure Aramchol having optical purity up to 99.9% and isomer impurity of formula (XI) controlled up to 0.01%.

In a further aspect of the invention, it is provided the intermediate (V).

BEST MODE FOR CARRYING OUT THE INVENTION

In the following, the present invention shall be illustrated by means of some examples, which are not construed to be viewed as limiting the scope of the invention.

Examples

Example 1: Preparation of methyl cholate (II) Cholic acid (100gm, 0.224mol) is charged in clean and dry round bottom flask containing methanol (300ml) and methane sulphonic acid (1.6 g, 0.016 mol). Reaction mass is heated to reflux temperature under stirring and maintained for 3hrs. After complete conversion, methanol from reaction mass is distilled out about two volumes. Reaction mass is cooled to 20-25°C and pH adjusted to 4.5 using methanolic sodium hydroxide solution. Mass is cooled to 0-5°C and the precipitated product is isolated by filtration on buchner funnel. The wet material is washed with chilled methanol, suck dried, unloaded and dried below 60°C under vacuum to get methyl cholate. Chromatographic purity by HPLC: 99.5%, Yield = 102 g

Example 2: Preparation of methyl 7α,12α -diacetyloxy- 3α-mesyloxy -5β-cholan-24-oate (IV)

Methyl cholate (100gm, 0.237mol) is taken in dry and clean RBF containing methyl isobutyl ketone (500 ml). To the reaction mass, acetic anhydride (96.6 gm, 0.946 mol), trimethylamine (95.76 g, 0.946 mol) and DMAP (1.44 g, 0.0118 mol) are added and heated to reflux temperature. Reaction mass is maintained at reflux until complete conversion. The reaction is quenched with water (950 ml) and top organic layer is separated at about 65°C. The organic layer is distilled completely under reduced pressure below 60°C to get semisolid residue. To the residue in RBF, methanol (650 ml) and sodium carbonate (12.54 g, 0.118 mol) are added at 25-30°C. Reaction mass is warmed to 35-40°C and maintained under stirring for 6 hours to get complete reaction conversion. Organic volatiles are distilled completely under vacuum below 60°C. Ethyl acetate (650 ml) and water (390 ml) are added to the residue and stirred to get clear solution. The organic layer is separated and washed with water (260 ml). The organic layer is distilled out completely under reduced pressure below 55°C to get semisolid residue.

To the residue in RBF, triethylamine (47.53 g, 0.469 mol) and ethyl acetate (595 ml) are added and cooled to 0- 5°C. Methane sulfonyl chloride (40.36 gm, 0.352 mol) is added slowly maintaining the mass temperature below 5°C. The mass is stirred for 2 hours at the same temperature to get complete conversion. Water is added to the reaction mass and the organic layer is separated at 25-30°C. The organic layer is distilled out completely under reduced pressure below 50°C to get oily residue. Cyclohexane is added to the residue and the mass is heated to reflux temperature and maintained for 2 hours. Then, the mass is cooled to 25-30°C and precipitated solid is filtered on buchner funnel. The wet cake is washed with cyclohexane and dried under vacuum below 60°C to get methyl 7α,12α-diacetyloxy-3α-mesyloxy-5β-cholan- 24-oate.

Chromatographic purity by HPLC: 99.2%, Yield = 132gm. Example 3: Preparation of methyl 3β-azido-7α,12α diacetyloxy-5β-cholan-24-oate (V) Methyl 7α,12α-diacetyloxy-3α-mesyloxy-5β-cholan-24- oate (124 g, 0.212 mol) is taken in clean and dry RBF containing DMF (496 ml). Sodium azide (40.95gm, 0.630mol) and ammonium chloride (22.46gm, 0.419mol) are added to the mass and heated to 90-100°C for 3 hours. After complete conversion, the mass is cooled to 25-30°C and the reaction is quenched with water (250 ml). Ethyl acetate (868 ml) is added to the mass and stirred for 30 min. The organic layer is separated and washed with water (250ml) once again. The organic layer is distilled under reduced pressure below 60°C to get a residue. Methanol (186ml) is added to the residue and the mass is heated to reflux to get a clear solution.

Then, the mass is cooled to 0-5°C and stirred for 2 hours to obtain product precipitation. The mass is filtered on buchner funnel, washed with chilled methanol and dried under vacuum below 50°C to provide the desired product.

Chromatographic purity by HPLC: 98.5%; Yield = 93gm Example 4: Preparation of methyl 3β-amino-7α,12α diacetyloxy-5β-cholan-24-oate (VI)

Methyl 3β-azido-7α, 12α-diacetyloxy-5β-cholan-24-oate (93 g, 0.175 mol), 10% Pd/C (9.3 g) and methanol (900 ml) are charged into a hydrogen pressure reactor. The reaction mass is flushed with nitrogen and hydrogen twice and then is maintained under 3 kg hydrogen pressure under stirring at 25-30°C for 10 hours. After complete conversion, the mass is filtered through a celite bed at and washed with methanol.

Collectively filtrate ml are distilled out completely under vacuum below 50°C to get residue. To the residue, n- propanol (900 ml) and L-(D)-tartaric acid (28.89 g, 0.19 mol) are added at 25-35°C. The mass is heated to 60°C and maintained for 1 hour. Then, the mass is cooled to 25-35°C, stirred for 3 hours and filtered on buchner funnel. Wet cake is washed with n-propanol and suck dried. The wet product is charged to a clean RBF containing ethyl acetate (900 ml) and sodium bicarbonate solution (5%) is added to adjust pH 7-7.2 at 25-35°C to get clear solutions of organic and aqueous layer. The organic layer is separated and washed with water (400 ml). The organic layer is distilled out completely under vacuum to get the desired product. Chromatographic purity by HPLC: 99.8%, Isomer impurity

X = 0.01%; Yield = 65gm

Example 5: Preparation of Aramchol (IX)

Arachidic Acid (33.35 g, 0.106 mol) and ethyl acetate (13.5vol) are taken in clean and dry RBF and stirred at 25- 30°C to get complete dissolution. The mass is cooled to 10°C- 15°C and HOBt (14.4gm, 0.106mol) and triethylamine (21.5gm, 0.21mol) are added to the reaction mass. Methyl 3β-amino- 7α,12α-diacetyloxy-5β-cholan-24-oate (65 g, 0.128 mol) and EDC.HC1 (24.55 g, 0.128 mol) are added to the reaction mass and stirred for 30 min. at 10-15°C. The mass is allowed to warm slowly to 25-30°C and stirred for 8 hours at the same temperature to obtain the complete conversion. The reaction is quenched with 5% sodium bicarbonate solution (300 ml) and stirred for 45 min. Top organic layer is separated and washed with 5% sodium bicarbonate solution (200 ml), 5% citric acid solution (300 ml) and followed by washing with water (250 ml). The organic layer is distilled completely under reduced pressure below 50°C to obtain a residue. To the residue, water (1000 ml) and sodium hydroxide

(153 g) are added and heated to reflux temperature for 5 hours. The reaction progress is monitored by HPLC analysis. After complete conversion of the starting material, the mass is cooled to 25-30°C, ethyl acetate (850ml) is added and the reaction mass pH is adjusted to 3.2 using hydrochloric acid.

The organic layer is separated, washed with water (510 ml) twice and distilled under reduced pressure below 50°C.

Ethyl acetate (510ml) and water (510ml) are added to the mass at 20-25°C, heated to 80-85°C and maintained 1 hour to obtain a clear solution. Then, the mass is cooled to 25- 30°C and maintained 4 hour at the same temperature to precipitate the product. The precipitated product filtered on buchner funnel, is washed with a mixture of ethyl acetate + water (1:1 ratio) and the wet cake is dried under vacuum below 60°C to provide Aramchol.

Chromatographic purity by HPLC: 99.78%, Isomer impurity = 0.01%, Yield = 62gm