Patent Description:
Farnesoid X Receptor (FXR) is a member of the nuclear receptor family. It is mainly expressed in the liver, small intestine and other intestinal systems, and is involved in bile acid metabolism and cholesterol metabolism. Bile acids have a variety of physiological functions and play an important role in processes such as fat absorption, transport, distribution and cholesterol homeostasis. The farnesoid X receptor acts as a receptor for bile acids such as chenodeoxycholic acid, maintains the balance of bile acids in the body by regulating the expression of genes involved in bile acid metabolism. In addition, the farnesoid X receptor also plays an important role in the dynamic balance of glucose in the body and insulin resistance. Therefore, farnesoid X receptor agonists are expected to be developed into drugs for treatment of non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, gallstones, primary biliary cirrhosis, liver cirrhosis, liver fibrosis, diabetes, hypercholesterolemia, atherosclerosis, obesity, hypertriglyceridemia, etc.. Among them, the compound obeticholic acid is a selective farnesoid X receptor agonist, of which the chemical name is 3α,7α-dihydroxy-6α-ethyl-5β-cholan-<NUM>-oic acid. It is useful in treating primary biliary cirrhosis (PBC), non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver related diseases. At present, obeticholic acid has been approved to market in primary biliary cirrhosis, and it is in phase TTT clinical research for NASH.

Patent application <CIT> discloses a method for synthesizing chenodeoxycholic acid derivatives by using 3α-dihydroxyl-<NUM>-keto-5β-cholan-<NUM>-oic acid as a starting material through steps such as <NUM>-position alkylation. However, there are many shortcomings in this synthetic method, for example, all intermediates and products need to be purified by chromatographic column, the total yield of the reaction is very low (only <NUM>%), and carcinogenic reagents should be used in the reaction step.

In addition, the patent <CIT> discloses a method for synthesizing obeticholic acid using 3α-dihydroxyl-<NUM>-keto-5β-cholan-<NUM>-oic acid as a starting material through Aldol condensation and other steps. However, the synthesis method have many shortcomings such as the intermediates are not easy to separate, long reaction steps, low yield of the configuration conversion step, and the impurities in the final product are not easy to remove.

<CIT> discloses a method for the preparation of obeticholic acid via an N-heteroaryl-amide intermediate or an amide intermediate, wherein the N atom thereof is part of a heterocyclic group.

Therefore, there is still need in developing better method for synthesizing chenodeoxycholate.

In the first aspect of the present invention, a method for synthesizing the compound represented by Formula (A) is provided:
<CHM>.

In another preferred embodiment, during the process of "one-pot method" of catalytic hydrogenation and metal hydride reducing agent reduction, the intermediate product is not separated or purified.

In another preferred embodiment, the step (a) includes: in anhydrous methanol, the compound of Formula (VI) is used for catalytic hydrogenation, and then water and metal hydride are added to react to obtain a compound of Formula (V).

In another preferred embodiment, the compound represented by Formula (VI) is the following compound:
<CHM>.

In another preferred embodiment, the compound of Formula (A) is selected from:
<CHM>
<CHM>.

In another preferred embodiment, the obtained compounds of Formula I to IV have high purity, preferably have purity greater than <NUM>%, further preferably have purity greater than <NUM>%, and particularly preferably have purity greater than <NUM>%.

In another preferred embodiment, the obtained compound of Formula I to IV contains less than <NUM>% of related dimer substance Formula (B), particularly preferably less than <NUM>%.

In another preferred embodiment, the obtained compound of Formula I to IV contains less than <NUM>% of related isomer substance Formula (C), more preferably less than <NUM>%, and particularly preferably not detected.

In another preferred embodiment, in the step (a), the inert solvent is selected from the group consisting of C1-C4 alcohols, water, or the combinations thereof.

In another preferred embodiment, in the step (a), the alcohol is selected from the group consisting of methanol, ethanol, isopropanol, tert-butanol, or the combinations thereof.

In another preferred embodiment, in the step (a), the inert solvent is selected from the group consisting of anhydrous methanol, or the mixed solvent of methanol:water (v/v)=<NUM>:<NUM>-<NUM>:<NUM>, preferably methanol:water (v/v)=<NUM>:<NUM>-<NUM>:<NUM>.

In another preferred embodiment, in the step (a), the catalytic hydrogenation is carried out in the presence of a palladium/carbon catalyst under hydrogen atomsphere.

In another preferred embodiment, the reaction temperature in step (a) is <NUM> to <NUM>, preferably <NUM> to <NUM>, particularly preferably <NUM> to <NUM>.

In another preferred embodiment, the pressure of catalytic hydrogenation in step (a) is <NUM>,<NUM>-<NUM>,<NUM>,<NUM> Pa (<NUM>-<NUM> atm), preferably <NUM>,<NUM>-<NUM>,<NUM>,<NUM> Pa (<NUM>-<NUM> atm).

In another preferred embodiment, in the step (a), the metal hydride reducing agent includes borohydrides, lithium tri-tert-butoxyaluminum hydride, preferably borohydrides, more preferably selected from sodium borohydride and potassium borohydride, particularly preferably sodium borohydride.

In another preferred embodiment, in the step (a), the molar ratio of the metal hydride to the compound of Formula (VI) is <NUM>:<NUM> to <NUM>:<NUM>, more preferably <NUM>:<NUM> to <NUM>:<NUM>, particularly preferably <NUM>:<NUM>.

In another preferred embodiment, in the step (a), the reduction of the metal hydride is carried out under alkaline conditions. Preferably, the alkaline condition is in the presence of sodium hydroxide or potassium hydroxide.

In another preferred embodiment, the molar ratio of the added amount of sodium hydroxide or potassium hydroxide to the compound of Formula (VI) is <NUM>:<NUM> to <NUM>:<NUM>, more preferably <NUM>:<NUM> to <NUM>:<NUM>.

In another preferred embodiment, the method further comprises: preparing the compound of Formula (VI) by the following method:.

In another preferred embodiment, in the step a), the molar ratio of the amount of the compound
<CHM>
added to the compound of Formula (VII) is <NUM>:<NUM> to <NUM>:<NUM>, more preferably <NUM>:<NUM> to <NUM>:<NUM>, particularly preferably <NUM>:<NUM>~<NUM>:<NUM>.

In another preferred embodiment, the condensing agent in the step a) is selected from the group consisting of N,N'-carbonyldiimidazole (CDI), EDCl, DIC, DCC, HATU, HBTU, TBTU and PyBOP; preferably HATU, HBTU and PyBOP; particularly preferably PyBOP.

In another preferred embodiment, in the step a), the molar ratio of the added amount of the condensing agent to the compound of Formula (VII) is <NUM>:<NUM> to <NUM>:<NUM>, more preferably <NUM>:<NUM> to <NUM>:<NUM>, particularly preferably <NUM>:<NUM>.

In another preferred embodiment, the step a) also includes the step of adding activator, and the activator is selected from the group consisting of DMAP, HOBt, <NUM>-PPY, DIPEA and Et<NUM>N; preferably DIPEA.

In another preferred embodiment, the step a) is carried out in an aprotic solvent; preferably, the aprotic solvent is selected from the group consisting of dichloromethane, acetonitrile, N,N-dimethylmethyl amide, dimethyl sulfoxide, or the combinations thereof; preferably N,N-dimethylformamide.

In another preferred embodiment, the step b) is carried out in a solvent, and the solvent is selected from the group consisting of dichloromethane, acetonitrile, dimethyl sulfoxide, tetrahydrofuran, <NUM>,<NUM>-dioxane, or the combinations thereof; preferably dichloromethane.

In another preferred embodiment, the Lewis acid in the step b) is selected from the group consisting of hydrochloric acid, acetic acid, p-toluenesulfonic acid, boron trifluoride ether solution, boron trifluoride acetonitrile solution, or the combinations thereof; preferably boron trifluoride ether solution.

In another preferred embodiment, in the step b), the molar ratio of the added amount of the Lewis acid to the compound of Formula (VIII) is <NUM>:<NUM> to <NUM>:<NUM>, more preferably <NUM>:<NUM> to <NUM>:<NUM>.

In another preferred embodiment, the method further comprises the step: preparing the compound of Formula (VIII) by the following method:.

wherein, the definition of each group is as described in the first aspect of the invention.

In another preferred embodiment, in the step c), the condensing agent is selected from the group consisting of N,N'-carbonyldiimidazole (CDI), EDCl, DIC, DCC, HATU, HBTU, TBTU and PyBOP, or the combinations thereof; preferably HATU, HBTU and PyBOP, or the combinations thereof; particularly preferably PyBOP.

In another preferred embodiment, in the step c), the molar ratio of the added amount of the compound
<CHM>
to the compound of Formula (IX) is <NUM>:<NUM> to <NUM>:<NUM>, more preferably <NUM>:<NUM> to <NUM>:<NUM>, particularly preferably <NUM>:<NUM> to <NUM>:<NUM>.

In another preferred embodiment, in the step c), the molar ratio of the added amount of the condensing agent to the compound of Formula (IX) is <NUM>:<NUM> to <NUM>:<NUM>, more preferably <NUM>:<NUM> to <NUM>:<NUM>, particularly preferably <NUM>:<NUM>.

In another preferred embodiment, the step c) also includes the step of adding activator, and the activator is selected from the group consisting of DMAP, HOBt, <NUM>-PPY, DIPEA and Et<NUM>N; preferably DIPEA.

In another preferred embodiment, the step c) is carried out in an aprotic solvent; preferably, the aprotic solvent is selected from the group consisting of dichloromethane, acetonitrile, N,N-dimethylmethyl amide, dimethyl sulfoxide, or the combinations thereof; preferably N,N-dimethylformamide.

In another preferred embodiment, the base in step d) is selected from the group consisting of sodium hydroxide, potassium hydroxide, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium hydride, lithium diisopropylamide (LDA), or the combinations thereof; preferably lithium diisopropylamide.

In another preferred embodiment, in the step d), the molar ratio of the added amount of trimethylchlorosilane to the compound of Formula (X) is <NUM>:<NUM> to <NUM>:<NUM>, more preferably <NUM>:<NUM> to <NUM>:<NUM>, particularly preferably is <NUM>:<NUM>.

In another preferred embodiment, in the step d), the molar ratio of the added amount of the base to the compound of Formula (X) is <NUM>:<NUM> to <NUM>:<NUM>, more preferably <NUM>:<NUM> to <NUM>:<NUM>, particularly preferably <NUM>:<NUM>.

In another preferred embodiment, the step d) is carried out in a solvent selected from the group consisting of dichloromethane, tetrahydrofuran, ether, toluene, <NUM>,<NUM>-dioxane, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide or the mixed solvents thereof, more preferably tetrahydrofuran.

In another preferred embodiment, the salt-forming and crystallization comprises:.

In another preferred embodiment, the weight ratio of the 3α,7α-dihydroxyl-6α-ethyl-5β-cholan-<NUM>-oic acid to pure water is <NUM>:<NUM> to <NUM>:<NUM>, more preferably <NUM>:<NUM> to <NUM>:<NUM>, particularly preferably <NUM>:<NUM>.

In another preferred embodiment, the weight ratio of the 3α,7α-dihydroxyl-6α-ethyl-5β-cholan-<NUM>-oic acid to the sodium hydroxide aqueous solution is <NUM>:<NUM> to <NUM>:<NUM>, more preferably <NUM>:<NUM> to <NUM>:<NUM>, particularly preferably <NUM>:<NUM> to <NUM>:<NUM>.

In another preferred embodiment, the concentration of the sodium hydroxide aqueous solution is <NUM>-<NUM> mol. L-<NUM>, more preferably <NUM>-<NUM> mol. L-<NUM>, particularly preferably <NUM> mol.

In another preferred embodiment, the concentration of the aqueous solution containing metal ions Mg<NUM>+ is <NUM>-<NUM>, more preferably <NUM>-<NUM>, particularly preferably <NUM>-<NUM> mol·L-<NUM>.

In another preferred embodiment, the concentration of the aqueous solution containing metal ions Ca<NUM>+ is <NUM>-<NUM>, more preferably <NUM>-<NUM>, particularly preferably <NUM>-<NUM> mol·L-<NUM>.

After long time and in-depth research, the inventors have obtained a method for preparing 3α,7α-dihydroxyl-6α-ethyl-5β-cholan-<NUM>-oic acid or salts thereof. The method has the characteristics such as short synthetic route, easy purification of intermediates and mild reaction conditions, and the obtained corresponding cholic acid or cholate has higher purity and better product quality, thus being suitable for pharmaceutical production. Based on the above findings, the inventor completed the present invention.

The compounds of the Formula (A) structure of the present invention can be prepared by the following general scheme I and II:
<CHM>.

The reaction is carried out in the presence of a condensing agent and/or an activator in an aprotic solvent at -<NUM> to <NUM>. The condensing agent is selected from N,N'-carbonyldiimidazole (CDI), EDCl, DIC, DCC, HATU, HBTU, TBTU and PyBOP, more preferably is PyBOP. The activator is selected from DMAP, HOBt, <NUM>-PPY, DIPEA and Et<NUM>N, more preferably is DIPEA; the aprotic solvent is dichloromethane, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, or the mixed solvent thereof, more preferably is N,N-dimethylformamide; the reaction temperature is preferably -<NUM>~<NUM>, more preferably is <NUM>-<NUM>.

(<NUM>) Obtaining the compound of Formula (V) by hydrogenating the compound of Formula (VI) and reducing with metal hydride reducing agent through the "one-pot method" to
The reaction in this step is carried out in a protic solvent, and existance of an alkaline aqueous solution at <NUM> to <NUM>. The hydrogenation reaction uses palladium/carbon as a catalyst under <NUM>,<NUM>-<NUM>,<NUM>,<NUM> Pa (<NUM>-<NUM> atm) of hydrogen; the metal hydride reducing agent can be sodium borohydride or potassium borohydride, more preferably is sodium borohydride; the protic solvent is selected from methanol, ethanol, isopropanol, tert-butanol, water or the mixtures thereof, preferably is the mixture of methanol and water; the alkaline aqueous solution is selected from sodium hydroxide aqueous solution and potassium hydroxide aqueous solution. The reaction temperature is <NUM> to <NUM>, more preferably <NUM> to <NUM>.

In a preferred embodiment, the compound of Formula (A) is prepared by the following method:
<CHM>.

The reaction is carried out in the presence of a condensing agent and/or an activator, in an aprotic solvent at -<NUM> to <NUM>. The condensing agent is N,N'-carbonyl diimidazole (CDI), EDC1, DIC, DCC, HATU, HBTU, TBTU and PyBOP, more preferably is PyBOP; the activating agent is DMAP, HOBt, <NUM>-PPY, DIPEA and Et<NUM>N, more preferably is DIPEA; the aprotic solvent is dichloromethane, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, or the mixture, more preferably is N,N-dimethyl formamide. The reaction temperature is preferably -<NUM>~<NUM>, more preferably is <NUM>-<NUM>.

(<NUM>) Obtaining the compound of Formula (VIII) by reacting the compound of Formula (X) with trimethylchlorosilane with the presence of base to.

The reaction can be carried out in an aprotic solvent, and the base is selected from sodium hydroxide, potassium hydroxide, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium hydride or lithium diisopropylamide (LDA), more preferably is lithium diisopropylamide (LDA); the aprotic solvent is selected from dichloromethane, tetrahydrofuran, ether, toluene, <NUM>,<NUM>-dioxane, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide or the mixtures thereof, more preferably is tetrahydrofuran. The reaction temperature is preferably -<NUM> to <NUM>, more preferably -<NUM> to <NUM>. The compound of Formula (VIII) can be directly used in the next reaction without further purification.

(<NUM>) Obtaining the compound of Formula (VI) by reacting the compound of Formula (VIII) with acetaldehyde with the presence of Lewis acid.

In this step, the Lewis acid is preferably boron trifluoride etherate; the solvent is selected from dichloromethane, acetonitrile, dimethyl sulfoxide, tetrahydrofuran, <NUM>,<NUM>-dioxane, or the mixtures thereof; more preferably is dichloromethane. The reaction temperature is preferably -<NUM> to <NUM>, more preferably is -<NUM> to <NUM>.

(<NUM>) Obtaining the compound of Formula (V) by hydrogenating the compound of Formula (VI) and reducing with metal hydride reducing agent in the "one-pot method" The reaction in this step is carried out in a protic solvent, an alkaline aqueous solution at <NUM> to <NUM>. The hydrogenation reaction use palladium/carbon as a catalyst under <NUM>,<NUM>-<NUM>,<NUM>,<NUM> Pa (<NUM>-<NUM> atm) of hydrogen; the metal hydride reducing agent can be sodium borohydride or potassium borohydride, more preferably is sodium borohydride. The protic solvent is selected from methanol, ethanol, isopropanol, tert-butanol, water or the mixtures thereof, preferably is the mixture of methanol and water. The alkaline aqueous solution is selected from sodium hydroxide aqueous solution and potassium hydroxide aqueous solution. The reaction temperature is <NUM> to <NUM>, more preferably <NUM> to <NUM>.

The present invention will be further illustrated below with reference to the specific examples. It should be understood that these examples are only to illustrate the invention but not to limit the scope of the invention. The experimental methods with no specific conditions described in the following examples are generally performed under the conventional conditions, or according to the manufacturer's instructions. Unless indicated otherwise, parts and percentage are calculated by weight.

Of the following examples, examples <NUM>, <NUM>, <NUM> and <NUM>-<NUM> are examples of the present invention and examples <NUM> and <NUM> are for comparison only.

Into the flask were added 3α-hydroxyl-<NUM>-ethidene-<NUM>-keto-5β-cholan-<NUM>-oic acid (Formula (VII), <NUM>, <NUM>. 144mol), PyBOP (<NUM>, <NUM>. 173mol) and N, N-dimethylformamide (<NUM>) were added successivly; the mixture was stirred under ice bath and cooled to <NUM>. DIPEA (<NUM>, <NUM>. 576mol) was added, and stirred for <NUM> at <NUM>. Under the protection of nitrogen, ammonium chloride (<NUM>, <NUM>. 230mol) and N,N-dimethylformamide (<NUM>) were added. The mixture was warmed to room temperature and stirred overnight. Under stirring, the reaction mixture was slowly poured into <NUM> of <NUM>% sodium bicarbonate aqueous solution, and solids were precipitated, stirred for <NUM> and filtered, washed with pure water (<NUM>), and dried under vacuum to obtain a crude product. Ethyl acetate (<NUM>) was added to the crude product, refluxed and pulped for <NUM>, then slowly cooled to about <NUM>, filtered, and the filter cake was washed with ethyl acetate (<NUM>), and dried under vacuum to obtain the title compound (<NUM>, yield <NUM>%) , HPLC purity <NUM>%. <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ: <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (q, J=<NUM>, <NUM>, <NUM>), <NUM>(d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>). LC-MS: <NUM> (M+H)+.

3α-Hydroxyl-<NUM>-ethidene-<NUM>-keto-5β-chole-<NUM>-alkamide (Compound <NUM>, <NUM>, <NUM>. 7mmol) and anhydrous methanol (<NUM>) were added into the flask, <NUM>% palladium on carbon catalyst (<NUM>) was added under stirring. The system was fully replaced by hydrogen, pressurized with hydrogen balloon, and reacted at <NUM> for <NUM>. The reaction solution was transferred to another flask, purified water (<NUM>) and sodium hydroxide (<NUM>) were added under stirring, and stirred at <NUM> for about <NUM>; filtered, and the mother liquor was transferred to another reaction flask. Sodium borohydride (<NUM>) was slowly added in batches, and the mixture was stirred in an oil bath at <NUM> for about <NUM> after that. The solution was concentrated under reduced pressure to remove the organic solvent, cooled in an ice bath, and 6N hydrochloric acid was slowly added dropwise with stirring to adjust the pH to about <NUM>, extracted with ethyl acetate (<NUM> × <NUM>), the organic phase was washed with water (<NUM>) and saturated brine (<NUM>) respectively, dried, and concentrated to obtain a crude solid. The title compound (<NUM>, yield <NUM>%) was obtained by recrystallization from mixture of butyl acetate/n-heptane (<NUM>:<NUM>) with HPLC purity of <NUM>%. <NUM>H NMR (<NUM>, DMSO-d6) δ: <NUM> (brs, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>). LC-MS: <NUM> (M-H)-, <NUM> (<NUM>-H)-.

Into the flask were added 3α-hydroxyl-<NUM>-ethidene-<NUM>-keto-5β-cholan-<NUM>-oic acid (Formula (VII), <NUM>, <NUM>. 144mol), PyBOP (<NUM>, <NUM>. 173mol) and N, N-dimethylformamide (<NUM>) successivly; the mixture was stirred under ice bath, cooled to <NUM>, and DIPEA (<NUM>, <NUM>. 576mol) was added, then stirred at <NUM> for <NUM>. Under the protection of nitrogen, N,O-dimethyl hydroxylamine hydrochloride (<NUM>, <NUM>. 288mol) and N,N-dimethylformamide (<NUM>) were added. The mixture was warmed to room temperature and stirred overnight. Under stirring, the reaction mixture was slowly poured into water (<NUM>), extracted with ethyl acetate (<NUM> × <NUM>), and then the organic phase was washed with saturated sodium bicarbonate solution (<NUM> × <NUM>), water (<NUM>) and brine (<NUM>), and dried over anhydrous sodium sulfate. The mixture was and concentrated to obtain a thick crude product. Ethyl acetate/petroleum ether (<NUM>:<NUM>) was added to the crude product to recrystallize, then refluxed and slowly cooled to about <NUM>. The solution was filtered, and the filter cake was washed with ethyl acetate/petroleum ether (<NUM> × <NUM>, EA/PE=<NUM>:<NUM>). After dried under vacuum, the title compound (<NUM>, yield <NUM>%) was obtained with HPLC purity of <NUM>%. <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ: <NUM> (q, J=<NUM>, <NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>); LC-MS: <NUM> (M+H)+.

3α-Hydroxyl-<NUM>-ethidene-<NUM>-keto-5β-chole-<NUM>-(N-methyl-N-methoxy)alkamide (Compound <NUM>, <NUM>, <NUM>. 88mmol) and anhydrous methanol (<NUM>) were added into the flask, <NUM>% palladium on carbon catalyst (<NUM>) were added under stirring, fully replaced by hydrogen, pressurized with the hydrogen balloon, and reacted at <NUM> for <NUM>. After filtration, the mother liquor was transferred to another flask, purified water (<NUM>) and sodium hydroxide (<NUM>) were added with stirring, and the mixture was stirred at <NUM> until the mixture was clear. Sodium borohydride (<NUM>) was slowly added in batches. After addition, the mixture was stirred in oil bath at <NUM> for about <NUM>, and concentrated under reduced pressure to remove the organic solvent. Then the residue was cooled in ice bath, 6N hydrochloric acid was slowly added dropwise under stirring to adjust the pH to about <NUM>, and extracted with ethyl acetate (<NUM> × <NUM>). The organic phase was washed with water (<NUM>) and saturated brine (<NUM>), dried, and concentrated to obtain crude solid. The title compound (<NUM>, yield <NUM>%) was obtained by crystallization from butyl acetate (<NUM>), of which the HPLC purity was greater than <NUM>%.

Into the flask were added 3α-hydroxyl-<NUM>-ethidene-<NUM>-keto-5β-cholan-<NUM>-oic acid (Formula (VII), <NUM>, <NUM>. 72mmol), PyBOP (<NUM>, <NUM>. 86mmol) and N, N- dimethylformamide (<NUM>) successivly. The mixture was stirred under ice bath, cooled to <NUM>, DIPEA (<NUM>, <NUM>. 88mmol) was added and stirring for <NUM> at <NUM>. Under nitrogen protection, hydroxylamine hydrochloride (<NUM>, <NUM>. 08mmol) was added. The mixture was warmed to room temperature and stirred for <NUM>. Under stirring, the reaction mixture was slowly poured into water (<NUM>) and stirred for <NUM> minutes. The solution was filtered, and the filter cake was washed with water (<NUM> × <NUM>), and dried under vacuum to obtain the title compound (<NUM>, yield <NUM>%), of which the HPLC purity was <NUM>%. <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ: <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (q, J=<NUM>, <NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>); LC-MS: <NUM> (M+H)+.

3α-Hydroxyl-<NUM>-ethidene-<NUM>-keto-5β-chole-<NUM>-hydroxyalkamide (Compound <NUM>, <NUM>, <NUM>. 46mmol) and anhydrous methanol (<NUM>) were added into the flask, <NUM>% palladium on carbon catalyst (<NUM>) were added under stirring. The system was fully replaced by hydrogen, pressurized with hydrogen balloon, and reacted at <NUM> for <NUM>. After filtration, the mother liquor was transferred to another flask, purified water (<NUM>) and sodium hydroxide (<NUM>) were added under stirring, and the mixture was stirred at <NUM> until the mixture was clear. Sodium borohydride (<NUM>) was slowly added in batches, then the mixture was stirred in an oil bath at <NUM> for about <NUM>. The organic solvent was removed by concentrating under reduced pressure. The residue was cooled in ice bath, 6N hydrochloric acid was slowly added dropwise with stirring to adjust the pH to about <NUM>, and extracted with ethyl acetate (<NUM> × <NUM>). The organic phase was washed with water (<NUM>) and saturated brine (<NUM>), dried and concentrated to obtain crude solid. The title compound (<NUM>, yield <NUM>%) was obtained by crystallizing with butyl acetate (<NUM>), of which the HPLC purity was greater than <NUM>%.

Into the flask were added 3α-hydroxyl-<NUM>-ethidene-<NUM>-keto-5β-cholan-<NUM>-oic acid (Formula (VII), <NUM>, <NUM>. 4mmol), PyBOP (<NUM>, <NUM>. 3mol) and N, N-dimethylformamide (<NUM>) successivly. The mixture was stirred under ice bath, cooled to <NUM>, DIPEA (<NUM>, <NUM>. 6mol) was added, then stirred at <NUM> for <NUM>. Under nitrogen protection, methylamine hydrochloride (<NUM>, <NUM>. 0mol) and N,N-dimethylformamide (<NUM>) were added. The mixture was warmed to room temperature and stirred for <NUM>. Under stirring, the reaction mixture was slowly poured into <NUM>% sodium bicarbonate aqueous solution (<NUM>), stirred and filtered. The filter cake was washed with water (<NUM> × <NUM>), and dried under vacuum to obtain the title compound (<NUM>, yield <NUM>%). HPLC purity was <NUM>%. <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ: <NUM> (d, J=<NUM>, <NUM>), <NUM> (q, J=<NUM>, <NUM>, <NUM>), <NUM>(d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>); LC-MS: <NUM> (M+H)+.

3α-Hydroxyl-<NUM>-ethidene-<NUM>-keto-5β-chole-<NUM>-carboxalkamide (Compound <NUM>, <NUM>, <NUM>. 32mmol) and anhydrous methanol (<NUM>) were added into the flask, <NUM>% palladium on carbon catalyst (<NUM>) were added under stirring. The system was fully replaced by hydrogen, pressurized with the hydrogen balloon, and reacted at <NUM> for <NUM>. After filtration, the mother liquor was transferred to another flask, purified water (<NUM>) and sodium hydroxide (<NUM>) were added with stirring, and the mixture was stirred at <NUM> until the mixture was clear. Sodium borohydride (<NUM>) was slowly added in batches. After addition, the mixture was stirred in oil bath at <NUM> for about <NUM>, and concentrated under reduced pressure to remove the organic solvent. The residue was cooled in an ice bath, 6N hydrochloric acid was slowly added dropwise under stirring to adjust the pH to about <NUM>, and extracted with ethyl acetate (<NUM> × <NUM>). The organic phase was washed with water (<NUM>) and saturated brine (<NUM>), dried, and concentrated to obtain a crude solid. The title compound (<NUM>, yield <NUM>%) was obtained by crystallizing with butyl acetate (<NUM>), of which the HPLC purity was greater than <NUM>%.

Into the reaction flask were added 3α-hydroxyl-<NUM>-keto-5β-cholin-<NUM>-alkanoic acid (Formula (IX), <NUM>, <NUM>. 26mol), PyBOP (<NUM>, <NUM>. 31mol) and N,N-dimethyl formamide (<NUM>); the mixture was stirred in an ice bath to about <NUM>, DIPEA (<NUM>, <NUM>. 91mol) was added, cooled to about <NUM>, and stirred for <NUM> minutes. Under the protection of nitrogen, ammonium chloride (<NUM>, <NUM>. 52mol) and N,N-dimethylformamide (<NUM>) were added, and the mixture was heated to room temperature and stirred for <NUM>. The mixture was stirred at <NUM> for <NUM> hours. Under stirring, the reaction mixture was slowly poured into <NUM>% sodium bicarbonate aqueous solution (<NUM>), the solid was precipitated, and stirred evenly for <NUM>. The mixture was filtered, washed with pure water (<NUM> × <NUM>), and dried under vacuum to obtain crude product. The crude product was transferred to a flask, added with tetrahydrofuran (<NUM>), refluxed and pulped. After cooled down and filtered, the residue was washed with tetrahydrofuran (<NUM>×<NUM>) and dried under vacuum to obtain the target compound (<NUM>, <NUM>%). <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ: <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>(d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>); ESI-MS (m/z): <NUM> (M+H)+.

Dry tetrahydrofuran (<NUM>) and lithium diisopropylamide (<NUM>, <NUM> heptane/tetrahydrofuran/ethylbenzene solution) were added to the flask successively, cooled to -<NUM> under nitrogen, and stirred for <NUM> minutes. Trimethyl chlorosilane (<NUM>, <NUM>. 385mol) was slowly dropped, and continued to stir for <NUM> at -<NUM>. The suspension of 3α-hydroxyl-<NUM>-keto-5β-chole-<NUM>-alkamide (Compound <NUM>) in tetrahydrofuran (<NUM> solids dispersed in <NUM> dry tetrahydrofuran uniformly) was dropped into the mixture in about <NUM> minutes, and continued to stir for <NUM> hour at about -<NUM>. After cooled in an ice bath, the reaction mixture was slowly added to saturated sodium bicarbonate solution to quench, and extracted with ethyl acetate (<NUM> × <NUM>). The organic phase was washed with saturated sodium bicarbonate solution (<NUM> × <NUM>) for several times, and finally washed with brine, dried and concentrated to obtain the crude product, which was directly used in the next reaction without purification. <NUM>H NMR (<NUM>, CD<NUM>OD) δ: <NUM> (q, J=<NUM>, <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>) , <NUM> (s, <NUM>) , <NUM> (s, <NUM>) , <NUM> (s, <NUM>); ESI-MS (m/z): <NUM> (M+H)+.

Into the flask were added 3α,<NUM>-bis(trimethylsiloxy)-5β-chole-<NUM>-en-<NUM>-alkamide (Compound <NUM>, <NUM>, <NUM>. 9mmol), dry dichloromethane (<NUM>) successivly, cooled to -<NUM> under nitrogen and stirred for <NUM>. Acetaldehyde (230ul, <NUM>. 8mmol) was quickly added to the mixed solution, and continued stirring for <NUM> minutes at about -<NUM>. The boron trifluoride ether in dichloromethane (<NUM> of <NUM>% boron trifluoride ether dissolved in <NUM> of dry dichloromethane) was added dropwise into the mixture, and continued stirring for <NUM> at - <NUM>, then heated to <NUM> and stirred for <NUM>. After cooled in an ice bath, the reaction mixture was slowly poured into saturated aqueous sodium bicarbonate solution under stirring to quench. After extracted with dichloromethane (<NUM> × <NUM>), the combined organic phases was washed with water and brine, dried and concentrated to give a crude product. The target product (<NUM>, yield <NUM>%) was obtained by crystallizing with mixed system of ethyl acetate and ethanol.

The method was similar to the preparation step <NUM> of Compound <NUM> in Example <NUM> to obtain Compound <NUM>, yield <NUM>%, HPLC purity was <NUM>%.

3α,7α-Dihydroxyl-6α-ethyl-5β-cholan-<NUM>-oic acid (Compound <NUM>, <NUM>) and pure water (<NUM>) were added into a <NUM> reactor, and sodium hydroxide aqueous solution (<NUM> hydrogen sodium oxide dissolved in <NUM> water) was added with stirring, stirred at about <NUM> until it is almost clear. The mixture was filtered, washed with a small amount of water. The mother liquor was transferred to the reactor, and magnesium chloride aqueous solution (<NUM> magnesium chloride hexahydrate in <NUM> water) was slowly added dropwise, washed with <NUM> pure water and transferred together, then continued to stir for <NUM>. After filtered, the residue was washed with pure water to neutralize, and dried under vacuum at <NUM> to obtain the title compound (<NUM>, yield <NUM>%), purity <NUM>%, in which Formula (B) impurity was <NUM>%, and Formula (C) impurity was undetected.

<NUM>H NMR (<NUM>, DMSO-d6) δ: <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM> (s, <NUM>).

Infrared spectrum (IR) characteristic absorption peaks: <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM> and <NUM>±<NUM>-<NUM>.

Atomic absorption (Mg<NUM>+): <NUM>% (calculated value: <NUM>%).

<NUM> of Compound <NUM> was stirred with <NUM> of anhydrous methanol to dissolve to clear solution, then slowly dropped into sodium hydroxide methanol solution (<NUM>. 0eq NaOH dissolved in <NUM> methanol), and stirred at room temperature for <NUM>. The mixture was concentrated in water bath at <NUM>, then acetone (<NUM> × <NUM>) was added and concentrated to dryness. Finally, <NUM> of acetone was added to the residue to pulp for <NUM> hours, filtered, and dried under vacuum at room temperature for <NUM> hours to obtain the title compound <NUM>; yield: <NUM>%, purity <NUM>%, the Formula (B) impurity was <NUM>%, and the Formula (C) impurity hasn't been detected. <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ: <NUM>(s, <NUM>), <NUM>(s, <NUM>), <NUM>(s, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>(s, <NUM>).

Infrared spectrum (IR) characteristic absorption peaks: <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM> and <NUM>±<NUM>-<NUM>.

Ion chromatography IC (Na+): <NUM>% (calculated value: <NUM>%).

<NUM> of Compound <NUM> was stirred with <NUM> of methanol to dissolve to clear solution, then slowly dripped into potassium hydroxide methanol solution (<NUM> KOH dissolved in <NUM> methanol) and stirred at room temperature for <NUM>; After concentrated to dryness in <NUM> water bath, acetone (<NUM> × <NUM> ) was added to the residue to concentrate to dryness. <NUM> acetone was added to the residue and pulped for <NUM>, and filtered and dried under vacuum at room temperature for <NUM> to obtain the title Compound <NUM><NUM>; yield: <NUM>%, purity <NUM>%, Formula (B) impurity <NUM>%, Formula (C) impurity hasn't been detected.

<NUM>H NMR (<NUM>, DMSO-d<NUM>) δ: <NUM>(s, <NUM>), <NUM>(s, <NUM>), <NUM>(s, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>(s, <NUM>).

Ion chromatography IC (K+): <NUM>% (calculated value: <NUM>%).

<NUM> of Compound <NUM> was stirred with <NUM> water and sodium hydroxide aqueous solution (<NUM> solid dissolved in <NUM> water) to dissolve to clear; calcium chloride aqueous solution (<NUM> calcium chloride solid dissolved in <NUM> water) was added dropwise, stirred at room temperature for <NUM> hours, filtered, washed with pure water and dried. The residue was dried under vacuum at room temperature for <NUM> hours to obtain the title compound <NUM>; yield: <NUM>%, purity <NUM>%, Formula (B) impurity was <NUM>%, and Formula (C) impurity hasn't been detected.

<NUM>H NMR (<NUM>, DMSO-d<NUM>) δ: <NUM>(s, <NUM>), <NUM>(s, <NUM>), <NUM>(s, <NUM>), <NUM>(m, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>(s, <NUM>).

Infrared spectrum (IR) characteristic absorption peaks: <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>, <NUM>±<NUM>-<NUM>and <NUM>±<NUM>-<NUM>.

Claim 1:
A method for preparing a compound represented by General Formula (A):
<CHM>
wherein, n is <NUM> or <NUM>, and M is NH<NUM>+, alkali metal ion, alkaline earth metal ion or transition metal ion;
the method comprises the following steps:
(a) in an inert solvent, subjecting the compound of Formula (VI) to the catalytic hydrogenation and reducing with metal hydride reducing agent by "one-pot method"
<CHM>
to obtain the compound of Formula (V);
<CHM>
(b) salifying compound of Formula (V) and crystallizing in a solvent to obtain the compound of Formula (A);
wherein, R<NUM> and R<NUM> are both hydrogen, or R<NUM> is hydrogen, and R<NUM> is hydroxyl.