CN110981933A

CN110981933A - Method for efficiently synthesizing Aramchol

Info

Publication number: CN110981933A
Application number: CN201911354582.2A
Authority: CN
Inventors: 冯乙巳; 浦同俊; 朱天彩; 程俊; 张佳媛
Original assignee: Hefei University of Technology
Current assignee: Hefei University of Technology
Priority date: 2019-12-25
Filing date: 2019-12-25
Publication date: 2020-04-10

Abstract

The invention discloses a method for efficiently synthesizing Aramchol, which comprises the steps of firstly generating intermediate b-cholic acid methyl ester through esterification reaction of methanol and cholic acid molecules, then adopting alkylbenzene sulfonyl chloride with a specific structure to react with the cholic acid methyl ester in the presence of an acid-binding agent, selectively reacting with 3-hydroxyl of the cholic acid molecules to synthesize key intermediate c-3 α -O-alkylbenzene sulfonyl cholic acid methyl ester, then protecting free hydroxyl in the intermediate c, and removing after the amidation reaction is finished, so as to effectively avoid side reaction of acyl chloride and free hydroxyl, and the problems of serious emulsification phenomenon and difficult separation in the reaction process.

Description

Method for efficiently synthesizing Aramchol

Technical Field

The invention relates to a synthesis method of a known compound, in particular to a method for efficiently synthesizing Aramchol, belonging to the technical field of organic synthesis.

Background

Non-alcoholic fatty liver disease (NAFLD) is a metabolic stress liver injury closely related to insulin resistance and genetic susceptibility, which is a type of second-largest liver disease in America and a chronic liver disease which is more and more emphasized in China, and the NAFLD is always harmful to human health in recent years. Because the market lacks drugs capable of effectively treating the non-alcoholic fatty hepatitis, only weight-losing drugs (such as orlistat, sibutramine and the like), drugs for regulating lipid metabolism disorder (such as insulin sensitizer metformin and thiazolidinedione drugs) and liver-protecting anti-inflammatory drugs (such as vitamin E, ursodeoxycholic acid and the like) are clinically used for relieving the non-alcoholic fatty liver disease. Therefore, the development of novel and more effective drugs for the treatment of non-alcoholic fatty liver diseases is becoming more urgent.

Fatty Acid Bile Acid Conjugates (FABACs) formed by coupling saturated Fatty acids with cholic acid derivatives are a class of compounds developed in recent years for treating nonalcoholic steatohepatitis, Israel Galled developed a FABAC Fatty liver treatment drug Aramchol, which is a compound in which cholic acid is coupled to arachidic acid through an amide bond, and has a chemical name of 3 β -arachidoyl-7 α,12 α -di-hydroxy-5 β -cholestan-24-oic acid (formula I below), and has been currently subjected to phase II clinical studies, and has been shown to have the potential for treating cholesterol gallstones and atherosclerosis.

For the synthesis of Aramchol, the following main routes have been reported:

route 1: the preparation method comprises the steps of firstly reacting cholic acid with p-toluenesulfonyl chloride, generating corresponding p-benzene sulfonate from 3-hydroxyl of the cholic acid, generating corresponding 3-azido cholic acid from an obtained intermediate through nucleophilic substitution reaction with sodium azide, reducing azido to amino through catalytic reduction, and finally reacting with eicosanoyl chloride to generate a target compound Aramchol. The synthetic route has the advantages of few reaction steps, short process route and simpler operation. However, carboxyl and other hydroxyl in cholic acid can also react with p-toluenesulfonyl chloride, so that the reaction selectivity is poor, side reactions are more, an intermediate is not easy to separate and purify, the yield of the final product Aramchol is low, and the impurity content is high. In order to facilitate the separation of intermediates and products and reduce side reactions, there are reports in the literature that the carboxyl group of cholic acid is protected by esterification and then deprotected. The strategy can reduce side reactions generated by the reaction with carboxyl, and can also make the separation of reaction intermediates relatively easy; however, three hydroxyl groups of cholic acid are still exposed, three active sites are generated when the cholic acid reacts with tosyl chloride, the selectivity of the cholic acid to the three hydroxyl groups is not high, and more side reactions are still generated.

Route 2: in order to improve the yield of the target product, a 9-step reaction synthesis route is researched and developed. Performing methyl esterification protection on carboxyl of cholic acid, performing acetylation protection on three hydroxyls of the cholic acid by using acetic anhydride, and selectively removing acetyl protecting groups of 3-hydroxyl under specific conditions; and then reacting tosyl chloride with 3-hydroxyl of cholic acid, carrying out nucleophilic substitution on the activated 3-hydroxyl and sodium azide, carrying out hydrogenation reduction and ester hydrolysis to remove acetyl protection on 7-hydroxyl and 12-hydroxyl to obtain a key intermediate 3-aminocholic acid methyl ester synthesized by Aramchol, and carrying out amidation and ester hydrolysis reactions to obtain the target drug molecule Aramchol.

The reaction conditions of the route 2 are overall mild and controllable, safe and reliable, and have good reproducibility and fewer side reactions. However, the process is too long, the cost is high, and the yield is low (the total yield of Aramchol based on cholic acid is only 12.7%). In the reaction process, materials are difficult to circulate, a large amount of intermediate raw materials are consumed, the atom utilization rate is too low, and the environmental pollution is large. The route is only suitable for small-batch synthesis in a laboratory and is limited for industrialized mass preparation.

Line 3: in CN109503693A, the subject group reports that cholic acid and arachidic acid are used as raw materials, and through the difference of spatial positions and activities of three hydroxyl groups of cholic acid molecules, alkylbenzenesulfonyl chloride with a specific structure is selectively reacted with 3-hydroxyl group of cholic acid molecules in the presence of tertiary amine organic alkali, and then NaN is used for reaction₃Nucleophilic substitution reaction with the said compound to generate 3 β -azido-7 α,12 α -dihydroxyl methyl cholate, catalytic hydrogenation reduction by Pt/C), amidation reaction with arachidylchlorideTo 3 β -arachidic amide group-7 α,12 α -dihydroxy cholic acid methyl ester, finally hydrolyzing ester group under proper condition, removing protection of methyl ester to obtain the target product Aramchol.

The reaction steps for synthesizing the Aramchol by the route are less, the process route is short, the selectivity is high, and the final product quality is high. However, in the subsequent studies, it was found that when 3C-hydroxyl group of methyl cholate is substituted by alkyl benzene sulfonyl chloride, the conversion rate is relatively low, and the emulsification phenomenon is severe during the separation treatment. In the amidation reaction process of arachidic acid chloride and amino, the acid chloride can partially react with hydroxyl at 7C-position and 12C-position of cholic acid molecule, causing unnecessary side reaction and influencing the yield and quality of final product. Therefore, in order to improve the reaction yield and ensure the product quality, the industrial route more meets the requirement of industrial production, and the protection and removal of free hydroxyl are necessary.

The protection method of the hydroxyl group mainly comprises ester formation and ether formation, and the ester is relatively stable under neutral over-acidic conditions, so that the hydroxyl group sensitive to acid can be protected by the method of ester formation. Commonly used such protecting groups are: acetyl-COC₂H₅formyl-COCH₃Tribromoethoxycarbonyl, trichloroethoxycarbonyl, and the like. Another way commonly used for protecting alcohols ROH is to prepare ethers (ROR') which have a considerable stability to oxidizing or reducing agents. ROCH of the methylether type commonly used₃Benzyl ethers, trityl ethers, alkylsilyl ethers, tetrahydropyranyl ethers, allyl ethers, and the like.

Disclosure of Invention

The process for synthesizing Aramchol by using cholic acid and arachidic acid as raw materials reported in CN109503693A has several problems, although the overall yield is improved, and the mass production of Arachol is limited. The method mainly comprises the following steps:

1. because the hydroxyl groups at the 7 th position and the 12 th position in the cholic acid molecule are exposed, the emulsification phenomenon is serious during the post-treatment of the intermediate, the separation is difficult, and the mass production operation is not facilitated.

2. In the amidation reaction process of arachidylic chloride and amino, acyl chloride can react with hydroxyl, so that side reactions are more, and the yield and quality of the product are reduced.

The invention adopts proper hydroxyl protecting group and reaction condition on the basis of the route reported in CN109503693A, protects free hydroxyl after obtaining a 3 α -O-alkyl benzene sulfonyl methyl cholate intermediate, and then removes the free hydroxyl after finishing amidation reaction, thereby effectively avoiding the side reaction of acyl chloride and free hydroxyl, and the problems of serious emulsification phenomenon and difficult separation in the reaction process.

We comprehensively consider the Aramchol synthesis condition, the nature and chemical environment of the needed protected hydroxyl, each intermediate structure and the deprotection condition, we protect the free hydroxyl (formula 2 below) after selectively obtaining the 3 α -O-alkyl benzene acyl methyl cholate intermediate, and select hydroxyl protection in this step, which requires that the protection group has certain stability to acid and alkali, and needs heat resistance and difficult hydrogenolysis, and because the steric hindrance of the hydroxyl at the 7-position and 12-position in the 3 α -O-alkyl benzene acyl cholate molecule is larger, it is not suitable to select the protection group sensitive to the steric hindrance, and needs to add a proper amount of catalyst to catalyze the reaction in order to improve the hydroxyl reaction activity and the conversion rate.

The relevant synthetic route is as follows:

wherein R is alkyl of 1-5 carbons (such as methyl, ethyl, propyl, isopropyl, etc.) or acyl of 1-5 carbons (such as formyl, acetyl, etc.).

The method for efficiently synthesizing Aramchol comprises the following steps:

step 1: synthesis of intermediate b

The method comprises the steps of adopting mature esterification reaction to protect hydroxyl of cholic acid, taking methanol as a solvent at room temperature, reacting the cholic acid with a condensing agent acetyl chloride at a ratio of 1:2 for 6 hours, and slowly pouring a large amount of saturated NaHCO into a reaction liquid after the reaction is finished₃Filtering the solution, washing the solution for 3 times by using deionized water and saturated saline solution respectively, and drying the obtained white to obtain an intermediate b, namely methyl cholate;

the detailed preparation process of the step is shown in CN 109503693A.

Step 2: synthesis of intermediate c

In the step, alkylbenzene sulfonyl chloride (such as p-tert-butylbenzene sulfonyl chloride) with a specific structure and an intermediate b are used as raw materials, the alkylbenzene sulfonyl chloride and the 3-hydroxyl of the methyl cholate are selectively subjected to sulfonyl esterification reaction in the presence of a catalyst DMAP and an acid-binding agent quaternary ammonium base, and after the reaction is finished, the alkylbenzene sulfonyl chloride and the intermediate b are separated by column chromatography to obtain a pure intermediate c.

The structure of the alkylbenzene sulfonyl chloride is as follows:

in the above alkylbenzenesulfonylchlorides of specific structure, R₁、R₂、R₃、R₄Is H or a small volume alkyl substituent, wherein a small volume alkyl substituent refers to a straight chain alkyl group of less than 3C, such as-CH₃、-CH₂CH₃And the like. R₁、R₂、R₃、R₄In mono-or disubstituted substitution form, i.e. R₁、R₂、R₃、R₄One or two of which are small volume alkyl substituents, the others being all H, the substituents preferably being in the ortho or meta position to the sulfonyl chloride. Such as o-methylbenzenesulfonyl chloride, m-methylbenzenesulfonyl chloride, 2, 4-dimethylbenzenesulfonyl chloride or the like; o-methylbenzenesulfonyl chloride is preferred.

In the above alkylbenzenesulfonylchlorides of specific structure, R is in the para-position₅Is H or a bulky substituent, wherein bulky substituent means a non-linear alkyl group of greater than 3C, e.g. R₅T-butyl, t-amyl, isobutyl, neopentyl, or the like; tert-butyl or tert-amyl is preferred.

When R is₅When it is a bulky substituent, R₁、R₂、R₃、R₄Are all H.

The detailed preparation process of the step is shown in CN 109503693A.

And step 3: synthesis of intermediate d

Hydroxyl protection is selected in the step, the protective group is required to have certain stability to acid and alkali, heat resistance is required, hydrogenolysis is difficult, most of silyl ether protective groups are sensitive to acid, the volume of the silyl ether stable to acid is large, the steric hindrance effect is large, and the silyl ether protective group is not suitable for selection, so the hydroxyl is protected by a mode of generating ester or alkyl ether in the step.

Protecting by ester formation:

in this step, the R group is an acyl group having 1 to 5 carbons (e.g., formyl, acetyl, etc.).

And (3) mixing the intermediate c and an acylating reagent according to a molar ratio of 1: dissolving 1-1:2 in a solvent, adding 1-10 mol% of a catalyst for catalytic reaction, adding alkali which is equal to the intermediate c in mole as an acid-binding agent, stirring for reaction, adding a proper amount of dichloromethane after the reaction is finished, adjusting the pH value with hydrochloric acid under an ice bath condition, separating liquid, washing an organic phase with saturated salt water, collecting the organic phase, drying the organic phase with anhydrous magnesium sulfate, and performing rotary evaporation to remove the solvent to obtain a crude product of the intermediate d.

The acylating agent can be selected from acyl chloride, acid anhydride, acid and the like with 1-5C, such as formic acid, acetyl chloride, trichloroethoxycarbonyl chloride, tribromoethoxycarbonyl chloride, acetic anhydride or formic acetic anhydride, and the like, and acetic anhydride or formic acid is preferred.

The solvent is selected from one or more of dichloromethane, trichloromethane, petroleum ether, methyl tert-butyl ether, ethyl acetate, pyridine, furan and the like, and preferably one or more of dichloromethane, methyl tert-butyl ether and pyridine.

The acid-binding agent is quaternary ammonium base such as triethylamine, pyridine, pyrazine, pyrimidine, aryl, alkyl tertiary amine such as tri-tert-butyl amine, diethylphenyl amine, di-tert-butyl benzyl amine, triphenylamine, etc., preferably pyridine or triethylamine.

The catalyst is selected from one or a combination of more of DMAP (4-dimethylaminopyridine), DCC (dicyclohexylcarbodiimide), DIC (N, N' -diisopropylcarbodiimide), EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and the like, and preferably DMAP.

In the above-mentioned ester formation protection reaction, the reaction temperature is controlled to 0 to 60 ℃ and preferably 20 to 30 ℃.

In the above-mentioned ester-forming protection reaction, the reaction system has a pH of >7, preferably 11 to 14.

In the above-mentioned ester formation protection reaction, the amount of the catalyst to be used is controlled to 0.1 to 2 times, preferably 0.5 to 1eq, the molar number of the hydroxyl group.

Ether formation protection:

in this step, the R group is an alkyl group having 1 to 5 carbons (e.g., methyl, ethyl, propyl, isopropyl, etc.).

And (3) mixing the intermediate c and the halogenated alkane according to a molar ratio of 1: dissolving 1-1:2 in a solvent, adding 1-10 mol% of a catalyst for catalytic reaction, adding alkali which is equal to the intermediate c in mol and is used as an acid-binding agent for stirring reaction, adding a proper amount of dichloromethane after the reaction is finished, adjusting the pH value with hydrochloric acid under an ice bath condition, separating liquid, washing an organic phase with saturated saline solution, collecting the organic phase, drying the organic phase with anhydrous magnesium sulfate, performing rotary evaporation to remove the solvent, and performing column chromatography separation to obtain an intermediate d.

The halogenated alkane is selected from halogenated hydrocarbon with small space volume, such as methyl iodide, ethyl bromide or propyl bromide, etc., preferably methyl iodide.

The solvent is selected from one or more of dichloromethane, trichloromethane, petroleum ether, methyl tert-butyl ether, DMF, etc., preferably trichloromethane or DMF.

The acid-binding agent is selected from quaternary ammonium base such as triethylamine, pyridine, DMF, pyrazine, pyrimidine, aryl, alkyl tertiary amine such as tri-tert-butyl amine, diethylphenyl amine, di-tert-butyl benzyl amine, triphenylamine, etc., preferably DMF or triethylamine.

The catalyst is selected from silver oxide, silver chloride or silver iodide, etc., preferably silver oxide.

In the above ether-forming protection reaction, the reaction temperature is controlled to 10 to 80 ℃ and preferably 30 to 40 ℃.

In the above ether-forming protection reaction, the reaction system has a pH of >7, preferably 11 to 14.

In the above ether-forming protection reaction, the amount of the catalyst is controlled to be 1-20%, preferably 10% of the mole number of the hydroxyl group.

And 4, step 4: synthesis of intermediate e

Dissolving the intermediate d, ammonium chloride with the same mole and 5-10eq (calculated by the molar weight of the intermediate d) of sodium azide in anhydrous DMF, heating to 120-130 ℃, stirring and refluxing for 5-8 hours, pouring the reaction liquid into ice water after the reaction is finished to quench the reaction, extracting an organic phase by using dichloromethane, separating the liquid, collecting the organic phase, evaporating the organic solvent to obtain wine red oily liquid, and separating and purifying by using a chromatographic column to obtain the intermediate e.

And 5: synthesis of intermediate f

Taking the intermediate e as a raw material, taking methanol as a solvent, taking 10% Pt/C as a catalyst, introducing sufficient hydrogen, and reacting for 60 hours at room temperature; after the reaction is finished, the catalyst is filtered, the methanol is evaporated to obtain white powder, and the intermediate f is obtained by chromatographic column separation and purification.

Reference documents: pore V S, Aher N G, Kumar M, et al design and synthesis of fluconazole/double acid coupling use reaction [ J ]. Tetrahedron,2006,62(48): 11178-.

Step 6: synthesis of intermediate g

Firstly, the arachidic acid is put in N₂Under protective conditions and SOCl₂Refluxing to obtain arachidylic chloride, and evaporating under reduced pressure to remove SOCl₂Then, dissolving the obtained product in dichloromethane, slowly dropwise adding the obtained product into anhydrous DMF solution in which the intermediate f and 2eq (calculated by the intermediate f) of triethylamine are dissolved, and reacting for 12 hours at room temperature; after the reaction is finished, dichloromethane is added for dissolution, then 5% dilute hydrochloric acid, saturated sodium bicarbonate solution and saturated saline are used for washing in sequence, and the intermediate g is obtained through chromatographic column separation and purification.

Reference documents: CN109503693A, a new process for synthesizing Aramchol from arachidic acid and cholic acid.

And 7: synthesis of target product

In the step, the protection of hydroxyl is removed firstly, and then the protection of methyl ester is removed.

When R group is acyl with 1-5 carbons, removing hydroxyl protecting group can be selected from sodium methoxide or sodium ethoxide and intermediate g to be dissolved in methanol or ethanol, heating and refluxing for 12h, removing solvent after reaction, washing solid residue with clear water, and drying. Dissolving the dried solid into a methanol solution, adding 1.5eq of NaOH in the molar amount of the intermediate g for three times, stirring and reacting for 12 hours at the temperature of 30 ℃, removing the methanol after the reaction is finished, adding distilled water for dissolving, stirring uniformly, adjusting the pH to 1-2, separating out a white substance, extracting with ethyl acetate, evaporating the solvent, and separating and purifying by a chromatographic column to obtain the target product.

When the R group is an alkyl group of 1 to 5 carbons, a Lewis acid such as BBr is generally used₃And Me₃Heating SiI and the like in an aqueous solution, stirring and removing, washing and drying the obtained solid, dissolving the dried solid into a methanol solution, adding 1.5eq of NaOH in the molar amount of an intermediate g for three times, stirring and reacting at 30 ℃ for 12 hours, removing methanol after the reaction is finished, adding distilled water for dissolving, stirring uniformly, adjusting the pH to 1-2, separating out a white substance, extracting with ethyl acetate, evaporating to remove the solvent, and separating and purifying by a chromatographic column to obtain the target product.

Reference documents: the synthesis of Aramchol, a lipid metabolism regulating drug, Aramchol, is carried out by Lanzhu, Van der Ware, Shiwei et al (J.). J. China J. Pharmaol, 2013,48(11): 920-containing 923).

The invention has the beneficial effects that:

1. the problems of serious emulsification and difficult separation in the reaction process are successfully solved.

Because each intermediate molecule contains hydrophilic hydroxyl, the emulsification phenomenon is very serious in the cleaning treatment process, and the separation efficiency is greatly reduced. Free hydroxyl is protected, the hydrophilicity of molecules is greatly improved, and cheap clean water can be selected to remove impurities in the separation process, so that the method is convenient and economical. Solves the emulsification problem, has high efficiency and simple separation, and provides guarantee for mass production of products.

2. Can effectively avoid side reaction and improve the overall yield of the product.

In the process of generating acylation reaction by reacting arachidic acyl chloride with amino, due to the existence of exposed hydroxyl, acyl chloride can react with amino and hydroxyl, so that a large amount of unnecessary byproducts are generated, the separation difficulty is increased, and the waste of reactants is also caused. The hydroxyl is protected, so that the normal reaction of amidation is not influenced, the generation of unnecessary side reaction can be greatly reduced, the separation means is simplified, the yield of the product is improved, and the whole yield can reach 56.7 percent or more.

3. The method provided by the invention is simple to operate, mild in condition, good in stability and high in yield, and can be stably operated in a kilogram-level reaction through gradual amplification research, so that the method provides technical support for industrial mass production.

Detailed Description

Example 1:

1. synthesis of intermediate b (cholic acid methyl ester)

Slowly dripping 10mL of acetyl chloride into 100mL of 0 ℃ methanol solution, continuously and uniformly stirring under the condition of ice salt water bath, adding 10g of cholic acid after the temperature is reduced back to 0-5 ℃, continuously stirring for 30min, removing the ice bath, naturally returning to the room temperature, and continuously reacting for 12 h; after the reaction is finished, saturated NaHCO is used₃Washing the solution, filtering to obtain white solid product b, and detecting purity by liquid chromatography>99%, yield 99%, structure determined by NMR.

¹H NMR(600MHz,cdcl₃)δ3.94(s,1H),3.82(s,1H),3.64(s,3H),3.42(t,J＝10.8Hz,1H),2.35(ddd,J＝14.6,9.8,4.5Hz,1H),2.26–2.12(m,3H),2.03(d,J＝8.5Hz,1H),1.94–1.21(m,18H),1.09(ddd,J＝24.0,14.9,8.9Hz,1H),0.96(d,J＝6.2Hz,3H),0.88(s,3H),0.66(s,3H).

2. Synthesis of intermediate c (3 α -O-p-tert-butylbenzenesulfonyl-7 α,12 α -dihydroxycholic acid methyl ester)

Dissolving 1g of the intermediate b into 10mL of pyridine solvent under the ice bath condition, adding 0.2g of diethyl benzylamine, stirring and mixing uniformly, adding 2g of p-tert-butylbenzene sulfonyl chloride, continuously stirring for 1h, removing the ice bath, and reacting for 3h at 30 ℃; after the reaction is finished, adding 20mL of dichloromethane, uniformly mixing, adding diluted hydrochloric acid to adjust the pH to be less than 7, washing with saturated saline solution for three times, separating liquid, drying, evaporating dichloromethane to obtain a white solid, and adding cyclohexane to a crude product: column chromatography with ethyl acetate 5:1(v/v) gave a yield of 85%.

¹H NMR(600MHz,cdcl₃)δ7.81(d,J＝8.6Hz,2H),7.52(d,J＝8.6Hz,2H),3.96(s,1H),3.83(d,J＝2.6Hz,1H),3.66(s,3H),2.40–2.32(m,2H),2.28–2.01(m,2H),1.95–1.38(m,16H),1.35(s,9H),1.30–1.23(m,2H),1.12(ddd,J＝24.5,12.1,6.1Hz,2H),0.97(d,J＝6.3Hz,3H),0.86(s,3H),0.67(s,3H).

3. Synthesis of intermediate d (3 α -O-p-tert-butylbenzenesulfonyl-7 α,12 α -diacetoxycholic acid methyl ester)

Dissolving 1g of the intermediate c in 10mL of acetic anhydride, adding 20mL of pyridine, stirring and stirring for reaction for 5 hours, adding a proper amount of dichloromethane after the reaction is finished, regulating the pH value to be 1-2 by using hydrochloric acid under an ice bath condition, separating liquid, washing an organic phase by using saturated saline solution, collecting the organic phase, drying the organic phase by using anhydrous magnesium sulfate, carrying out rotary evaporation to remove the solvent, and carrying out column chromatography separation (ethyl acetate: petroleum ether is 3:5, v/v), thus obtaining an intermediate d. After the hydroxyl is protected, the hydrophilic group is changed into the hydrophobic group, so that emulsification is not needed during washing, and the separation is simple.

1H NMR(600MHz,cdcl3)δ7.81(d,J＝8.6Hz,2H),7.52(d,J＝8.6Hz,2H),3.96(s,1H),3.83(d,J＝2.6Hz,1H),3.66(s,3H),2.40–2.32(m,2H),2.28–2.01(m,2H),2.12(d,J＝2.6Hz,3H),2.07–2.01(m,3H),1.95–1.38(m,16H),1.39–1.32(m,9H),0.89–0.82(m,3H),0.79(t,J＝7.5Hz,3H),0.72–0.66(m,3H).

4. Synthesis of intermediate e (3 β -azido-7 α,12 α -diacetoxycholic acid methyl ester)

1g of intermediate c, 0.14g of ammonium chloride and 0.45g of NaN are weighed out₃Dissolving in 15mL of DMF, adding 0.1g of triethylamine, heating and stirring for reaction for 6h, wherein the reaction temperature is 70 ℃. After the reaction is finished, cooling the reaction solutionAfter the mixture is cooled to room temperature, 30mL of dichloromethane is added and mixed uniformly, the mixture is washed twice with saturated brine, dried and evaporated to remove dichloromethane, petroleum ether, ethyl acetate 10: 1(v/v) column chromatography.

¹H NMR(600MHz,cdcl₃)δ3.90(s,1H),3.82(s,1H),3.62(s,3H),3.40(t,J＝10.8Hz,1H),2.34(ddd,J＝14.6,9.8,4.5Hz,1H),2.12(d,J＝2.6Hz,3H),2.07–2.01(m,3H)2.00(d,J＝8.5Hz,1H),1.94–1.21(m,18H),1.09(ddd,J＝24.0,14.9,8.9Hz,1H),0.95(d,J＝6.2Hz,3H),0.87(s,3H),0.67(s,3H).

5. Synthesis of intermediate f (3 β -amino-7 α,12 α -diacetoxycholic acid methyl ester)

Dissolving 1g of the purified intermediate d in 20mL of methanol, adding 0.1g of 10% palladium/carbon catalyst, continuously introducing hydrogen, and reacting for 60 hours at room temperature; after the reaction, the catalyst was filtered off, the methanol was evaporated to give a white powder, which was purified by column chromatography (ethyl acetate: petroleum ether: 20:1, v/v) to obtain intermediate f.

¹H NMR(600MHz,cdcl₃)δ3.94(s,1H),3.82(s,1H),3.63(s,3H),3.40(t,J＝10.8Hz,1H),2.34(ddd,J＝14.6,9.8,4.5Hz,1H),2.26–2.14(m,3H),2.12(d,J＝2.6Hz,3H),2.07–2.01(m,3H)，2.00(d,J＝8.5Hz,1H),1.94–1.21(m,18H),1.09(ddd,J＝24.0,14.9,8.9Hz,1H),0.95(d,J＝6.2Hz,3H),0.87(s,3H),0.68(s,3H).

6. Synthesis of intermediate g (3 β -arachidonamide-7 α,12 α -diacetoxycholic acid methyl ester)

Weigh 0.5g of arachidic acid and add to 5mL of SOCl₂Heating and refluxing for 3h, and evaporating to remove SOCl under reduced pressure₂Dissolving the obtained product in 5mL of dichloromethane, slowly and dropwise adding the obtained product into 15mL of anhydrous DMF solution dissolved with 1g of intermediate f and 0.1g of triethylamine, and reacting for 12h at room temperature; after the reaction, 20mL of dichloromethane was added to dissolve the product, and the product was washed with 5% dilute hydrochloric acid, saturated sodium bicarbonate solution and saturated brine in this order, and purified by column chromatography (ethyl acetate: petroleum ether: 3:5, v/v) to obtain intermediate g. When the hydroxyl group is not protected, arachidylchloride is easy to react with the hydroxyl group, and has many side reactions and difficult separation. When the free hydroxyl group of cholic acid molecule is protected, arachidylchloride is only reacted with amino groupThe reaction is carried out, almost no by-product is generated, and the product yield is obviously improved compared with that when the hydroxyl is not protected. After the hydroxyl is protected, the hydrophilic group is changed into the hydrophobic group, so that emulsification is not needed during washing, and the separation is simple.

¹H NMR(600MHz,cdcl₃)δ3.92(s,1H),3.83(s,1H),3.64(s,3H),3.40(t,J＝10.8Hz,1H),2.34(ddd,J＝14.6,9.8,4.5Hz,1H),2.32–2.14(m，30H),2.12(d,J＝2.6Hz,3H),2.07–2.01(m,3H)，2.00-0.98(m，34H),0.93(d,J＝6.3Hz,3H),0.88(s,3H),0.66(s,3H).

7. Synthesis of target product Aramchol (3 β -arachidamido-7 α,12 α -dihydroxy-5-cholestane-24-acid)

Weighing 1g of intermediate f and 0.8g of sodium ethoxide, dissolving the intermediate f and the sodium ethoxide in 20mL of methanol solution, heating and refluxing for 12h, removing the solvent after the reaction is finished, washing the solid residue with clear water, and drying. Dissolving the dried solid into 20ml of methanol solution, adding 1.5eq of NaOH into the methanol solution for three times, stirring the mixture at 30 ℃ for reaction for 12 hours, removing the methanol after the reaction is finished, adding distilled water for dissolving, stirring the mixture evenly, adjusting the pH value to be 1-2, separating out white substances, extracting the white substances by using ethyl acetate, evaporating the solvent, and separating and purifying the white substances by using a chromatographic column (the ethyl acetate: petroleum ether is 1:2, v/v), wherein the yield is 97.8 percent.

¹H NMR(600MHz,cdcl₃)δ3.90(s,1H),3.82(s,1H),,3.40(t,J＝10.8Hz,1H),2.34(ddd,J＝14.6,9.8,4.5Hz,1H),2.26–0.98(m，65H),0.95(d,J＝6.2Hz,3H),0.87(s,3H),0.67(s,3H).

Example 2:

intermediate b was synthesized as in example 1.

Intermediate c was synthesized as in example 1.

Synthesis of intermediate d

Dissolving 1g of intermediate c in 20mL of methyl tert-butyl ether, adding 5mL of pyridine, slowly dropwise adding 3mL of acetyl chloride, stirring and stirring for reaction for 3 hours, adding 10mL of methyl tert-butyl ether after the reaction is finished, adjusting the pH to 1-2 with hydrochloric acid under the ice bath condition, separating liquid, washing an organic phase with saturated saline solution, collecting the organic phase, drying the organic phase with anhydrous magnesium sulfate, performing rotary evaporation to remove the solvent, and performing column chromatography separation (ethyl acetate: petroleum ether is 3:5, v/v) to obtain an intermediate d, wherein the yield is 98.8%

1H NMR(600MHz,cdcl3)δ7.81(d,J＝8.6Hz,2H),7.52(d,J＝8.6Hz,2H),3.96(s,1H),3.83(d,J＝2.6Hz,1H),3.66(s,3H),2.40–2.32(m,2H),2.28–2.01(m,2H),2.10(d,J＝2.6Hz,3H),2.01(m,3H),1.95–1.38(m,16H),1.39–1.32(m,9H),0.89–0.82(m,3H),0.79(t,J＝7.5Hz,3H),0.72–0.66(m,3H).

The synthesis of intermediates e-g and of the final product h are the same as in example 1.

Example 3:

intermediate b was synthesized as in example 1.

Intermediate c was synthesized as in example 1.

Synthesis of intermediate d

Dissolving 1g of intermediate c in 20mL of dichloromethane, adding 5mL of triethylamine, 1.1g of methyl iodide and 0.1g of silver chloride, stirring and reacting at 30 ℃ for 3 hours, after the reaction is finished, adjusting the pH to 3-4 with hydrochloric acid under an ice bath condition, separating liquid, washing an organic phase with saturated saline solution, collecting the organic phase, drying the organic phase with anhydrous magnesium sulfate, performing rotary evaporation to remove the solvent, and performing column chromatography separation (ethyl acetate: petroleum ether: 3:5, v/v) to obtain intermediate d, wherein the yield is 95.6%.

1H NMR(600MHz,cdcl3)δ7.81(d,J＝8.6Hz,2H),7.52(d,J＝8.6Hz,2H),3.96(s,1H),3.83(d,J＝2.6Hz,1H),3.66(s,3H),3.14((s,6H),1.95–1.38(m,16H),1.39–1.32(m,9H),0.89–0.82(m,3H),0.79(t,J＝7.5Hz,3H),0.72–0.66(m,3H).

Example 4:

intermediate b was synthesized as in example 1.

Intermediate c was synthesized as in example 1.

Synthesis of intermediate d

Dissolving 1g of intermediate c in 20mL of dichloromethane, adding 5mL of pyridine, 1.1g of methyl iodide and 0.1g of silver iodide, stirring and reacting at 50 ℃ for 3 hours, after the reaction is finished, adjusting the pH to 1-2 with hydrochloric acid under an ice bath condition, separating liquid, washing an organic phase with saturated saline solution, collecting the organic phase, drying the organic phase with anhydrous magnesium sulfate, performing rotary evaporation to remove the solvent, and performing column chromatography separation (ethyl acetate: petroleum ether: 3:5, v/v) to obtain intermediate d, wherein the yield is 94.5%.

1H NMR(600MHz,cdcl3)δ7.81(d,J＝8.6Hz,2H),7.52(d,J＝8.6Hz,2H),3.96(s,1H),3.83(d,J＝2.6Hz,1H),3.66(s,3H),3.12(s,6H),1.95–1.38(m,16H),1.39–1.32(m,9H),0.88–0.82(m,3H),0.78(t,J＝7.5Hz,3H),0.72–0.65(m,3H).

Intermediates e-g were synthesized as in example 1

Final product Aramchol synthesis

1g of intermediate g and 0.6g of Me were weighed out₃SiI, dissolved in an aqueous solution, was heated to 60 ℃ and stirred for 12 hours. After the obtained solid was washed and dried, the dried solid was dissolved in 20mL of water: adding 1.5eq of sodium ethoxide with the molar amount of the intermediate g into the solution 1:4 for three times, stirring and reacting for 12 hours at the temperature of 30 ℃, removing the methanol after the reaction is finished, adding distilled water for dissolving, stirring uniformly, adjusting the pH to 1-2, separating out a white substance, extracting with ethyl acetate, evaporating the solvent, and separating and purifying by a chromatographic column to obtain the target product with the yield of 94.2%.

Example 5:

intermediate b was synthesized as in example 1.

Intermediate c was synthesized as in example 1.

Synthesis of intermediate d

Dissolving 1g of intermediate c in 20mL of DMF, reacting 1g of methyl iodide and 0.12g of silver oxide under stirring at 20 ℃ for 3 hours, adjusting the pH to 1-2 with hydrochloric acid under an ice bath condition after the reaction is finished, separating liquid, washing an organic phase with saturated saline solution, collecting the organic phase, drying the organic phase with anhydrous magnesium sulfate, performing rotary evaporation to remove the solvent, and performing column chromatography separation (ethyl acetate: petroleum ether is 3:5, v/v), thus obtaining an intermediate d with the yield of 93.3%.

1H NMR(600MHz,cdcl3)δ7.81(d,J＝8.6Hz,2H),7.52(d,J＝8.6Hz,2H),3.96(s,1H),3.83(d,J＝2.6Hz,1H),3.66(s,3H),3.12((s,6H),1.95–1.38(m,16H),1.39–1.32(m,9H),0.88–0.82(m,3H),0.78(t,J＝7.5Hz,3H),0.72–0.65(m,3H).

The synthesis of intermediates e-g and of the final product h are as in example 4.

Claims

1. A method for efficiently synthesizing Aramchol is characterized by comprising the following steps:

the method comprises the steps of firstly, using cholic acid as a raw material, generating an intermediate b-cholic acid methyl ester through esterification reaction of methanol and cholic acid molecules, then using alkylbenzene sulfonyl chloride with a specific structure to react with the cholic acid methyl ester in the presence of an acid-binding agent, selectively reacting with 3-hydroxyl of the cholic acid molecules, and synthesizing a key intermediate c-3 α -O-alkylbenzene sulfonyl cholic acid methyl ester, then protecting free hydroxyl in the intermediate c, and removing after the amidation reaction is finished, so as to effectively avoid side reaction of acyl chloride and free hydroxyl, and the problems of serious emulsification phenomenon and difficult separation in the reaction process;

the reaction route is as follows:

wherein R is alkyl of 1-5 carbons or acyl of 1-5 carbons.

2. The method according to claim 1, characterized by comprising the steps of:

step 1: synthesis of intermediate b

step 2: synthesis of intermediate c

The method comprises the following steps of using alkylbenzene sulfonyl chloride with a specific structure and an intermediate b as raw materials, selectively carrying out sulfonyl esterification reaction with 3-hydroxyl of methyl cholate in the presence of a catalyst DMAP and an acid-binding agent quaternary ammonium base, and separating by column chromatography after the reaction is finished to obtain a pure intermediate c;

and step 3: synthesis of intermediate d

Protecting hydroxyl by generating ester or alkyl ether to obtain an intermediate d;

and 4, step 4: synthesis of intermediate e

Dissolving the intermediate d, equimolar ammonium chloride and 5-10 equivalent of sodium azide in anhydrous DMF, heating to 120-130 ℃, stirring and refluxing for 5-8 hours, pouring reaction liquid into ice water to quench the reaction after the reaction is finished, extracting an organic phase by using dichloromethane, separating liquid, collecting the organic phase, evaporating the organic solvent to obtain wine red oily liquid, and separating and purifying by using a chromatographic column to obtain an intermediate e;

and 5: synthesis of intermediate f

Taking the intermediate e as a raw material, taking methanol as a solvent, taking 10% Pt/C as a catalyst, introducing sufficient hydrogen, and reacting for 60 hours at room temperature; after the reaction is finished, filtering the catalyst, evaporating methanol to obtain white powder, and separating and purifying by a chromatographic column to obtain an intermediate f;

step 6: synthesis of intermediate g

Firstly, the arachidic acid is put in N₂Under protective conditions and SOCl₂Refluxing to obtain arachidylic chloride, and evaporating under reduced pressure to remove SOCl₂Then, dissolving the obtained product in dichloromethane, slowly dropwise adding the obtained product into an anhydrous DMF solution in which the intermediate f and 2eq of triethylamine are dissolved, and reacting for 12 hours at room temperature; after the reaction is finished, adding dichloromethane for dissolution, washing with 5% dilute hydrochloric acid, saturated sodium bicarbonate solution and saturated saline solution in sequence, and separating and purifying by a chromatographic column to obtain an intermediate g;

and 7: synthesis of target product

In the step, the protection of hydroxyl is removed firstly, and then the protection of methyl ester is removed, so that the target product is obtained.

3. The method of claim 2, wherein:

in step 3, protecting hydroxyl group by ester formation to obtain intermediate d, comprising the following steps:

dissolving the intermediate c and an acylation reagent in a solvent, adding a catalyst for catalytic reaction, adding alkali serving as an acid-binding agent, stirring for reaction, adding a proper amount of dichloromethane after the reaction is finished, regulating the pH value by using hydrochloric acid under an ice bath condition, separating liquid, washing an organic phase by using saturated salt water, collecting the organic phase, drying the organic phase by using anhydrous magnesium sulfate, and removing the solvent by rotary evaporation to obtain a crude product of the intermediate d; in this step, the R group is an acyl group having 1 to 5 carbons.

4. The production method according to claim 3, characterized in that:

the acylating reagent is acyl chloride, acid anhydride or acid with 1-5C;

the acid-binding agent is quaternary ammonium base;

the catalyst is selected from one or a combination of more of DMAP, DCC, DIC and EDC.

5. The production method according to claim 3, characterized in that:

in the ester formation protection reaction, the reaction temperature is controlled to be 0-60 ℃; the reaction system had a pH > 7.

6. The method of claim 2, wherein:

in step 3, protecting hydroxyl group by way of generating alkyl ether to obtain intermediate d, comprising the following steps:

dissolving the intermediate c and halogenated alkane in a solvent, adding a catalyst for catalytic reaction, adding alkali serving as an acid-binding agent, stirring for reaction, adding a proper amount of dichloromethane after the reaction is finished, regulating the pH value by using hydrochloric acid under an ice bath condition, separating liquid, washing an organic phase by using saturated saline solution, collecting the organic phase, drying the organic phase by using anhydrous magnesium sulfate, rotationally evaporating to remove the solvent, and carrying out column chromatography separation to obtain an intermediate d; in this step, the R group is an alkyl group having 1 to 5 carbons.

7. The method of claim 6, wherein:

the halogenated alkane is selected from methyl iodide, ethyl bromide or propyl bromide;

the acid-binding agent is quaternary ammonium base;

the catalyst is silver oxide, silver chloride or silver iodide.

8. The method of claim 6, wherein:

in the ether formation protection reaction, the reaction temperature is controlled at 10-80 ℃; the reaction system had a pH > 7.

9. The method of claim 2, wherein:

in step 7, when the R group is acyl with 1-5 carbons, removing the hydroxyl protecting group by dissolving sodium methoxide or sodium ethoxide and intermediate g in methanol or ethanol, heating and refluxing for 12h, removing the solvent after the reaction is finished, washing the solid residue with clear water, and drying; dissolving the dried solid into a methanol solution, adding NaOH, stirring and reacting for 12h at 30 ℃, removing the methanol after the reaction is finished, adding distilled water for dissolving, stirring uniformly, adjusting the pH to 1-2, separating out a white substance, extracting with ethyl acetate, evaporating the solvent, and separating and purifying by a chromatographic column to obtain the target product.

10. The method of claim 2, wherein:

in step 7, when the R group is alkyl with 1-5 carbons, removing the hydroxyl protecting group is to heat and stir Lewis acid and intermediate g in water solution to remove the hydroxyl protecting group, after washing and drying the obtained solid, dissolving the dried solid in methanol solution, adding NaOH, stirring and reacting for 12h at 30 ℃, removing methanol after the reaction is finished, adding distilled water to dissolve, stirring uniformly, adjusting pH to 1-2, separating out white substance, extracting with ethyl acetate, evaporating to remove the solvent, separating and purifying with a chromatographic column to obtain the target product.