CN114133421A - Preparation method of beta-mouse cholic acid - Google Patents
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Abstract
The invention discloses a preparation method of beta-murine cholic acid, which takes hyocholic acid (3 alpha, 6 alpha, 7 alpha trihydroxy-5 beta-cholanic acid) as a starting material to prepare the beta-murine cholic acid (3 alpha, 6 beta, 7 beta trihydroxy-5 beta-cholanic acid). The mouse deoxycholic acid synthesized and prepared by the method has the advantages of easily available raw materials, simple reaction, less side reaction and high conversion rate, and is suitable for mass production.
Description
Technical Field
The invention belongs to the field of organic chemical synthesis, and particularly relates to a preparation method of beta-murine cholic acid.
Background
Bile acid is an important component of bile, is mainly present in the enterohepatic circulatory system and plays a certain role in protection through recirculation; bile acid is an important endogenous molecule associated with numerous biological functions, including the absorption and excretion of cholesterol, and plays a role in promoting lipid emulsification and enhancing pancreatic lipolysis in metabolism; in clinical and disease diagnosis, bile acid plays an important role in the research of liver, gallbladder, gastrointestinal diseases.
Murine Cholic Acid (MCA) is an important bile acid, and three different configurations exist, namely alpha-murine cholic acid (alpha-MCA), beta-murine cholic acid (beta-MCA) and omega-murine cholic acid (omega-MCA), and the structures of the three configurations are shown in the following formula.
Murine cholic acid is a hepatoprotective agent, is beneficial to chronic cholestatic liver diseases, and taurine-beta-murine cholic acid in urine is identified as a promising biomarker to promote clinical monitoring of traditional Chinese medicine Polygonum Multiflorum (PMR) -induced hepatotoxicity and can be used as a potential therapeutic target. However, since most CYPs produce a variety of hydroxylated products, it is difficult to characterize these metabolites without knowing their chemical composition and composition, and thus the synthesis of these bile acids is required to gain further insight into the biokinetics of bile acids, and understanding of this conversion can facilitate the development of cholestasis drugs, providing a standard for further research in the future.
At present, the preparation of the mouse cholic acid is mainly obtained by two methods of animal bile extraction and artificial synthesis. The animal bile is mostly extracted from the bile of the rat, the quantity of the bile of the rat is limited, and the extraction is troublesome; the artificial synthesis is mainly obtained by a series of chemical reactions of chenodeoxycholic acid and hyodeoxycholic acid, and the murine cholic acid obtained by the two methods needs to introduce hydroxyl into 6-position or 7-position carbon atoms respectively, so that the process difficulty is high, side reactions are multiple, the conversion rate is low, and the method is not suitable for mass production, so that the process cost of the murine cholic acid is high and the price is high. Therefore, it is necessary to develop a method for synthesizing beta-mucic acid with simple process and high conversion rate.
Disclosure of Invention
The invention aims to: the preparation method of the beta-mouse cholic acid has the advantages of easily available raw materials, simple reaction, less side reaction, high conversion rate and suitability for mass production.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of beta-murine cholic acid (3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanic acid) comprises the following steps of taking hyocholic acid (3 alpha, 6 alpha, 7 alpha trihydroxy-5 beta-cholanic acid) as a raw material:
wherein:
r1 is C1-C4 straight-chain alkyl or branched-chain alkyl;
r2 is C1-C4 straight-chain alkyl or branched-chain alkyl;
r3 is C1-C4 straight-chain alkyl, branched-chain alkyl, methylsulfonyl or p-nitrobenzyl.
In the invention, the reaction process of the step a is as follows: dissolving the compound A in alcohol, adding a catalyst, carrying out reflux reaction for 2 hours, adding a sodium bicarbonate solution with the concentration of 1% to terminate the reaction after the reaction is finished, distilling to remove the solvent, filtering and drying to obtain a compound B;
the catalyst is organic acid or inorganic acid, and further preferably hydrochloric acid, sulfuric acid or p-toluenesulfonic acid; the mass ratio of the catalyst to the compound A is 0.1-0.5: 100.
The alcohol is straight chain alcohol or branched chain alcohol containing 1-4 carbon atoms, and the volume weight ratio of the alcohol to the compound A is 4-8 ml:1 g.
The volume weight ratio of the sodium bicarbonate solution to the compound A is 1ml:1 g.
In the invention, the reaction process of the step b is as follows: suspending the compound B in isopropyl ether, adding Novoxin-435 lipase, heating to 40 ℃, adding an ester substance, maintaining the temperature at 35-40 ℃ for reaction for 6 hours, filtering after the reaction is finished, and removing the solvent from the filtrate through vacuum distillation to obtain a compound C;
the esters are straight-chain and branched-chain acetates containing 1-4 carbon atoms, and the mass ratio of the esters to the compound B is 1.5-3: 1;
the volume-mass ratio of the isopropyl ether to the compound B is 5-10 ml:1 g;
the mass ratio of the novacin-435 lipase to the compound B is 2-5: 1;
the volume-mass ratio of the sodium hydroxide solution to the compound B is 10-11 ml:1 g.
In the invention, the reaction process of the step c is as follows: dissolving the compound C in tetrahydrofuran, adding triphenylphosphine, acid and diethyl azodicarboxylate, stirring at room temperature for 5-6 h, adding water to stop the reaction after the reaction is finished, removing tetrahydrofuran by vacuum distillation to obtain an oily substance, dissolving the oily substance in ethyl acetate with the volume of 4 times, washing with 1% sodium bicarbonate solution to remove unreacted acid, concentrating the solution, purifying by column chromatography, collecting eluent containing the compound D, and concentrating to obtain a purified compound D;
the acid is C1-C4 straight chain or branched chain acid, one of methanesulfonic acid and p-nitrobenzoic acid, preferably formic acid, methanesulfonic acid and p-nitrobenzoic acid, and preferably formic acid.
The column chromatography purification is a silica gel column, the particle size of the silica gel is 300-400 meshes, and an eluant for the column chromatography purification is a solvent formed by mixing ethyl acetate and petroleum ether according to a volume ratio of 1: 2.
The volume-mass ratio of the tetrahydrofuran to the compound C is 3-8 ml:1 g;
the mass ratio of the triphenylphosphine to the compound C is 2.5-4: 1;
the mass ratio of the acid to the compound C is 2.5-5: 1;
the amount ratio of the diethyl azodicarboxylate to the compound C is 3-5: 1.
In the invention, the reaction process of the step d is as follows: suspending the compound D prepared in the step c in water, adding strong base, heating to hydrolyze for 2-8 h, adding acid to crystallize after complete hydrolysis, filtering and washing to obtain a compound E, namely a target product 3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanic acid;
the hydrolysis temperature in the step d is 50-100 ℃, and further preferably 60-80 ℃.
The strong base is an inorganic base, further preferably sodium hydroxide and potassium hydroxide, and further preferably sodium hydroxide; the mass ratio of the sodium hydroxide to the compound D is 30-50: 100.
The acid used for the crystallization is an inorganic acid or an organic acid, preferably an inorganic acid, and further preferably hydrochloric acid having a concentration of 6M.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention takes the hyocholic acid (3 alpha, 6 alpha, 7 alpha trihydroxy-5 beta-cholanic acid) as the initial raw material to synthesize the beta-mouse cholic acid (3 alpha, 6 beta, 7 beta trihydroxy-5 beta-cholanic acid), and the raw material has wide source and low price;
(2) the synthesis route of the invention has simple process route, less side reaction and high conversion rate, and is suitable for mass production;
(3) the beta-muricacid obtained by the method has high purity and few impurities, and can provide data and standard samples for subsequent research.
Drawings
FIG. 1 is a flow chart of the synthesis of beta-mucic acid.
Detailed Description
The present invention will be further described with reference to the following examples, but is not limited thereto.
As shown in figure 1, the synthesis method of beta-murine cholic acid adopts the starting raw material of hyocholic acid, and comprises the following specific steps:
a. preparation of Compound B
Dissolving 3 alpha, 6 alpha, 7 alpha trihydroxy-5 beta-cholanic acid (hyocholic acid) in alcohol with the volume 4-8 times of the weight of the hyocholic acid, adding a catalyst with the mass ratio of the substance to the hyocholic acid being 0.1-0.5: 100, carrying out reflux reaction for 2 hours, adding a 1% sodium bicarbonate solution with the same volume as the weight of the hyocholic acid after the reaction is finished, terminating the reaction, distilling to remove the solvent, filtering and drying to obtain a compound B.
b. Preparation of Compound C
Suspending the compound B prepared in the previous step in isopropyl ether with the volume mass ratio of the compound B to the isopropyl ether of 5-10 ml:1g, adding 20-50 wt% of Novoxin-435 lipase, heating to 40 ℃, adding ester with the amount of 1.5-3.0 times of the substance, maintaining the temperature at 35-40 ℃ for reaction for 6 hours, filtering after the reaction is finished, and removing the solvent from the filtrate through vacuum distillation to obtain the compound C.
c. Preparation of Compound D
Dissolving the compound C in the previous step in tetrahydrofuran with the volume being 3-8 times that of the compound C, adding triphenylphosphine with the amount being 2.5-4.0 times that of the substance, acid with the amount being 2.5-5.0 times that of the substance and diethyl azodicarboxylate with the amount being 3.0-5.0 times that of the substance, and stirring for reaction at room temperature for 5-6 hours. After the reaction is finished, water is added to terminate the reaction, tetrahydrofuran is removed by vacuum distillation, the obtained oil is dissolved in ethyl acetate with 4 times of volume, unreacted acid is removed by washing with 1% sodium bicarbonate solution, the solution is concentrated to the original volume of 1/5, and the mixture is applied to a silica gel column and eluted by using a mixed solvent of ethyl acetate and petroleum ether with the volume ratio of 1: 2. Collecting eluate containing compound D, concentrating the eluate to original volume 1/5, precipitating a large amount of crystals, and filtering to obtain compound D.
d. Preparation of Compound E
Suspending the compound D in 10 times of water by weight volume, adding 30-50% of sodium hydroxide, heating to 60-80 ℃ for hydrolysis, cooling to room temperature after complete hydrolysis, adjusting the solution to be acidic and crystallized by 6M hydrochloric acid, filtering and washing to obtain a compound E.
Example 1
The preparation method of the beta-murine cholic acid comprises the following steps:
a. preparation of 3 alpha, 6 alpha, 7 alpha trihydroxy-5 beta-cholanic acid methyl ester
50g of hyocholic acid is added into a 500ml three-neck flask, 200ml of methanol is used for stirring and dissolving, 0.25ml of concentrated sulfuric acid is added, the temperature is increased, reflux reaction is carried out for 2 hours, and sampling is carried out to detect that the residue of the hyocholic acid is 0.732%. Adding 50ml of 1% sodium bicarbonate solution, stirring uniformly, distilling under vacuum, removing methanol to obtain a large amount of white crystals, filtering, washing a filter cake with 200ml of water, drying in a hot air circulation oven at 75 ℃ for 8h to obtain 49.8g of 3 alpha, 6 alpha, 7 alpha trihydroxy-5 beta-methyl cholanate, and detecting the purity by HPLC (high performance liquid chromatography) of 99.347%.
b. Preparation of 3 alpha-acetoxyl-6 alpha, 7 alpha dihydroxy-5 beta-cholanic acid methyl ester
49.8g of 3 alpha, 6 alpha, 7 alpha trihydroxy-5 beta-methyl cholanate, 250ml of isopropyl ether and 10g of novacin-435 lipase are added into a 500ml three-neck flask, the temperature is raised to 40 ℃, 26ml of ethyl acetate is added, and the temperature is maintained at 35-40 ℃ for reaction for 6 hours. Sampling to detect that the residue of 3 alpha, 6 alpha, 7 alpha trihydroxy-5 beta-methyl cholanate is 0.323%, and filtering. The filtrate was vacuum distilled to remove the solvent to give 51.3g of methyl 3 α -acetoxy-6 α, 7 α dihydroxy-5 β -cholanate with a purity of 98.932% by HPLC.
c. Preparation of 3 alpha-acetoxyl-6 beta, 7 beta-diformyloxy-5 beta-cholanic acid methyl ester
51.3g of 3 alpha-acetoxyl-6 alpha, 7 alpha dihydroxy-5 beta-cholanic acid methyl ester is added into a 500ml three-neck flask, stirred and dissolved by 160ml of tetrahydrofuran, 73g of triphenylphosphine, 12ml of anhydrous formic acid and 52ml of diethyl azodicarboxylate are added, stirred and reacted for 6 hours at room temperature, and the residue of the 3 alpha-acetoxyl-6 alpha, 7 alpha dihydroxy-5 beta-cholanic acid methyl ester is measured to be 0.047% by sampling. 50ml of water was added and the tetrahydrofuran was distilled off in vacuo to give an oil. Adding 200ml of ethyl acetate, stirring for dissolving, standing for layering, washing the ethyl acetate solution by using 2 x 200ml of 1% sodium bicarbonate solution, removing unreacted formic acid, carrying out vacuum distillation on the solution to collect about 150ml of distillate, pouring the concentrated solution into a silica gel column for chromatography, eluting by using a mixed solvent of ethyl acetate and petroleum ether in a volume ratio of 1:2, collecting about 400ml of eluent containing 3 alpha-acetoxyl-6 beta, 7 beta-diformyloxy-5 beta-methyl cholate, carrying out vacuum distillation on the eluent to collect about 300ml of distillate, precipitating a large amount of crystals in a distillation bottle, filtering to obtain 43.7g of 3 alpha-acetoxyl-6 beta, 7 beta-diformyloxy-5 beta-methyl cholate, and carrying out sampling HPLC (high performance liquid chromatography) to detect that the purity is 98.668%.
d. Preparation of 3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanic acid
Adding 43.7g of 3 alpha-acetoxyl-6 beta, 7 beta-diformyloxy-5 beta-cholanic acid methyl ester, 437ml of water and 13.2g of sodium hydroxide into a 1000ml three-neck flask, heating to 60 ℃, maintaining the temperature at 60 ℃, stirring for 6h, sampling to detect that the 3 alpha-acetoxyl-6 beta, 7 beta-diformyloxy-5 beta-cholanic acid methyl ester is 0.273%, cooling to room temperature, adding 30ml of concentrated hydrochloric acid, adjusting the solution to be acidic and crystallized, filtering and washing, drying a filter cake to obtain 34.6g of 3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanic acid, and the HPLC purity is 98.521%.
Example 2
The preparation method of the beta-murine cholic acid comprises the following steps:
a. preparation of 3 alpha, 6 alpha, 7 alpha trihydroxy-5 beta-cholanic acid ethyl ester
50g of hyocholic acid is added into a 500ml three-neck flask, 300ml of absolute ethyl alcohol is used for stirring and dissolving, 0.5ml of concentrated hydrochloric acid is added, the temperature is increased, reflux reaction is carried out for 2 hours, and the residue of the hyocholic acid is measured to be 0.679% by sampling. Adding 50ml of 1% sodium bicarbonate solution, stirring uniformly, distilling under vacuum, removing ethanol to obtain a large amount of white crystals, filtering, washing the filter cake with 200ml of water, drying in a hot air circulation oven at 75 ℃ for 8h to obtain 49.2g of 3 alpha, 6 alpha, 7 alpha trihydroxy-5 beta-cholanic acid ethyl ester, wherein the purity is 99.236% by HPLC detection.
b. Preparation of 3 alpha-acetoxyl-6 alpha, 7 alpha dihydroxy-5 beta-cholanic acid ethyl ester
Adding 49.2g of 3 alpha, 6 alpha, 7 alpha trihydroxy-5 beta-cholanic acid ethyl ester, 350ml of isopropyl ether and 15g of novacin-435 lipase into a 500ml three-neck flask, heating to 40 ℃, adding 30ml of isopropenyl acetate, and reacting for 6 hours at the temperature of 40 ℃. Sampling to detect the residue of 3 alpha, 6 alpha, 7 alpha trihydroxy-5 beta-cholanic acid ethyl ester as 0.257%, and filtering. The filtrate was vacuum distilled to remove the solvent to give 50.9g of ethyl 3 α -acetoxy-6 α, 7 α dihydroxy-5 β -cholanate with a purity of 98.855% by HPLC.
c. Preparation of 3 alpha-acetoxy-6 beta, 7 beta-dimethyl sulfonate-5 beta-cholanic acid ethyl ester
50.9g of ethyl 3 alpha-acetoxyl-6 alpha, 7 alpha dihydroxy-5 beta-cholanate is added into a 500ml three-neck flask, the mixture is stirred and dissolved by 250ml of tetrahydrofuran, 80g of triphenylphosphine, 20ml of methanesulfonic acid and 60ml of diethyl azodicarboxylate are added, the mixture is stirred and reacted for 6 hours at room temperature, and the residue of the ethyl 3 alpha-acetoxyl-6 alpha, 7 alpha dihydroxy-5 beta-cholanate is 0.126 percent by sampling. 50ml of water was added and the tetrahydrofuran was distilled off in vacuo to give an oil. Adding 200ml ethyl acetate, stirring for dissolving, standing for layering, washing ethyl acetate solution with 2 x 200ml sodium bicarbonate solution with concentration of 1%, removing unreacted methanesulfonic acid, vacuum distilling the solution to collect about 150ml distillate, pouring the concentrated solution into silica gel column for column chromatography, and eluting with mixed solvent of ethyl acetate and petroleum ether at volume ratio of 1: 2. The collected part is about 400ml of eluent of 3 alpha-acetoxyl group-6 beta, 7 beta dimethyl sulfonate-5 beta-ethyl cholanate, the eluent is subjected to vacuum distillation to collect about 300ml of distillate, a large amount of crystals are separated out from a distillation flask at the moment, the crystals are filtered to obtain 43.5g of 3 alpha-acetoxyl group-6 beta, 7 beta dimethyl sulfonate-5 beta-ethyl cholanate, and the purity of the ethyl cholanate is 98.854% by sampling HPLC detection.
d. Preparation of 3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanic acid
Adding 43.5g of 3 alpha-acetoxyl-6 beta, 7 beta-dimethyl sulfonate-5 beta-cholanic acid ethyl ester, 435ml of water and 17.5g of sodium hydroxide into a 1000ml three-neck flask, heating to 70 ℃, maintaining the temperature at 60 ℃, stirring for 4 hours, sampling and detecting that the 3 alpha-acetoxyl-6 beta, 7 beta-dimethyl sulfonate-5 beta-cholanic acid ethyl ester is 0.124%, cooling to room temperature, adding 40ml of concentrated hydrochloric acid, adjusting the solution to be acidic and crystallized, filtering and washing, drying a filter cake to obtain 34.4g of 3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanic acid, wherein the HPLC purity is 98.723%.
Example 3
The preparation method of the beta-murine cholic acid comprises the following steps:
a. preparation of 3 alpha, 6 alpha, 7 alpha trihydroxy-5 beta-cholanic isopropyl ester
50g of hyocholic acid is added into a 500ml three-neck flask, 400ml of isopropanol is used for stirring and dissolving, 1g of p-toluenesulfonic acid is added, the temperature is increased, reflux reaction is carried out for 2 hours, and the residue of the hyocholic acid is measured to be 0.524% by sampling. Adding 50ml of 1% sodium bicarbonate solution, stirring uniformly, distilling in vacuum, removing isopropanol to obtain a large amount of white crystals, filtering, washing a filter cake with 200ml of water, drying in a hot air circulation oven at 75 ℃ for 8 hours to obtain 50.3g of 3 alpha, 6 alpha, 7 alpha trihydroxy-5 beta-isopropyl cholate, wherein the purity is 99.236% by HPLC (high performance liquid chromatography).
b. Preparation of 3 alpha-acetoxyl-6 alpha, 7 alpha dihydroxy-5 beta-cholanic acid isopropyl ester
50.3g of isopropyl 3 alpha, 6 alpha, 7 alpha trihydroxy-5 beta-cholate, 500ml of isopropyl ether and 20g of novacin-435 lipase are added into a 500ml three-neck flask, the temperature is raised to 40 ℃, 30ml of butyl acetate is added, and the temperature is maintained at 40 ℃ for reaction for 4 hours. Sampling to detect that the residue of isopropyl 3 alpha, 6 alpha, 7 alpha trihydroxy-5 beta-cholate is 0.221%, and filtering. The filtrate was vacuum distilled to remove the solvent to give 51.5g of isopropyl 3 α -acetoxy-6 α, 7 α dihydroxy-5 β -cholate having a purity of 99.043% by HPLC.
c. Preparation of 3 alpha-acetoxyl-6 beta, 7 beta-di-p-nitrobenzoate-5 beta-isopropyl cholate
51.5g of isopropyl 3 alpha-acetoxy-6 alpha, 7 alpha dihydroxy-5 beta-cholate is added into a 500ml three-neck flask, stirred and dissolved by 400ml of tetrahydrofuran, 100g of triphenylphosphine, 60g of p-nitrobenzoic acid and 80ml of diethyl azodicarboxylate are added, stirred and reacted for 6 hours at room temperature, and the residue of the isopropyl 3 alpha-acetoxy-6 alpha, 7 alpha dihydroxy-5 beta-cholate is 0.027 percent by sampling. 50ml of water was added and the tetrahydrofuran was distilled off in vacuo to give an oil. Adding 200ml ethyl acetate, stirring for dissolving, standing for layering, washing the ethyl acetate solution with 3 x 200ml sodium bicarbonate solution with concentration of 1%, removing unreacted p-nitrobenzoic acid, vacuum distilling the solution to collect about 150ml distillate, pouring the concentrated solution into a silica gel column for chromatography, and eluting with mixed solvent of ethyl acetate and petroleum ether with volume ratio of 1: 2. The collected eluate is about 400ml of 3 alpha-acetoxyl-6 beta, 7 beta-di-p-nitrobenzoate-5 beta-cholanate, the eluate is subjected to vacuum distillation to collect about 300ml of distillate, a large amount of crystals are separated out from a distillation flask at the moment, and the crystals are filtered to obtain 47.6g of 3 alpha-acetoxyl-6 beta, 7 beta-di-p-nitrobenzoate-5 beta-cholanate, wherein the purity of the 3 alpha-acetoxyl-6 beta, 7 beta-di-p-nitrobenzoate-5 beta-cholanate is 99.043% by sampling HPLC detection.
d. Preparation of 3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanic acid
Adding 47.6g of isopropyl 3 alpha-acetoxy-6 beta, 7 beta-di-p-nitrobenzoate-5 beta-cholanate, 476ml of water and 23.8g of sodium hydroxide into a 1000ml three-neck flask, heating to 80 ℃, maintaining the temperature at 60 ℃, stirring for 2 hours, sampling and detecting that ethyl 3 alpha-acetoxy-6 beta, 7 beta-di-p-nitrobenzoate-5 beta-cholanate is 0.056%, cooling to room temperature, adding 60ml of hydrochloric acid with the concentration of 6M, adjusting the solution to be acidic and crystallized, filtering and washing, drying a filter cake to obtain 36.7g of 3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanate with the HPLC purity of 99.014%.
The above examples show that the beta-murine cholic acid (3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanic acid) prepared by the method has the advantages of simple reaction, high conversion rate and high purity.
Claims (10)
1. The preparation method of the beta-murine cholic acid is characterized in that 3 alpha, 6 alpha, 7 alpha trihydroxy-5 beta-cholanic acid is used as a raw material to prepare the beta-murine cholic acid by the following steps:
wherein:
r1 is C1-C4 straight-chain alkyl or branched-chain alkyl;
r2 is C1-C4 straight-chain alkyl or branched-chain alkyl;
r3 is C1-C4 straight-chain alkyl, branched-chain alkyl, methylsulfonyl or p-nitrobenzyl.
2. The method for preparing beta-murine cholic acid according to claim 1, wherein the reaction process of the step a is as follows: dissolving the compound A in alcohol, adding a catalyst, carrying out reflux reaction for 2 hours, adding a sodium bicarbonate solution with the concentration of 1% to terminate the reaction after the reaction is finished, distilling to remove the solvent, filtering and drying to obtain a compound B;
the catalyst is organic acid or inorganic acid;
the alcohol is straight chain alcohol or branched chain alcohol containing 1-4 carbon atoms.
3. The method for producing β -murine cholic acid according to claim 2, wherein: the catalyst is hydrochloric acid, sulfuric acid or p-toluenesulfonic acid.
4. The method for producing β -murine cholic acid according to claim 2 or 3, wherein:
the mass ratio of the catalyst to the compound A is 0.1-0.5: 100;
the volume-mass ratio of the alcohol to the compound A is 4-8 ml:1 g;
the volume mass ratio of the sodium bicarbonate solution to the compound A is 1ml:1 g.
5. The method for preparing beta-murine cholic acid according to claim 1, wherein the reaction process of the step b is as follows: suspending the compound B in isopropyl ether, adding Novoxin-435 lipase, heating to 40 ℃, adding an ester substance, maintaining the temperature at 35-40 ℃ for reaction for 6 hours, filtering after the reaction is finished, and removing the solvent from the filtrate through vacuum distillation to obtain a compound C;
the ester substance is acetic ester of straight chain and branched chain alcohol containing 1-4 carbon atoms.
6. The method for producing β -murine cholic acid according to claim 5, wherein:
the volume-mass ratio of the isopropyl ether to the compound B is 5-10 ml:1 g;
the mass ratio of the novacin-435 lipase to the compound B is 2-5: 1;
the mass ratio of the esters to the compound B is 1.5-3: 1;
the volume-mass ratio of the sodium hydroxide solution to the compound B is 10-11 ml:1 g.
7. The method for preparing beta-murine cholic acid according to claim 1, wherein the reaction process of the step c is as follows: dissolving the compound C in tetrahydrofuran, adding triphenylphosphine, acid and diethyl azodicarboxylate, stirring at room temperature for 5-6 h for reaction, adding water to stop the reaction after the reaction is finished, removing tetrahydrofuran by vacuum distillation to obtain an oily substance, dissolving the oily substance in ethyl acetate with the volume of 4 times, washing with 1% sodium bicarbonate solution to remove unreacted acid, concentrating the solution, purifying by column chromatography, collecting an eluent containing the compound D, and concentrating to obtain a purified compound D;
the acid is one of C1-C4 straight chain or branched chain acid, methanesulfonic acid and p-nitrobenzoic acid;
the column chromatography purification is a silica gel column, and the particle size of the silica gel is 300-400 meshes;
the eluent for column chromatography purification is a solvent mixed by ethyl acetate and petroleum ether according to the volume ratio of 1: 2.
8. The method of preparing β -murine cholic acid according to claim 7, wherein:
the volume-mass ratio of the tetrahydrofuran to the compound C is 3-8 ml:1 g;
the mass ratio of the triphenylphosphine to the compound C is 2.5-4: 1;
the mass ratio of the acid to the compound C is 2.5-5: 1;
the mass ratio of the azodicarbonic acid diethyl ester to the compound C substance is 3-5: 1.
9. The method for preparing beta-murine cholic acid according to claim 1, wherein the reaction process of the step d is as follows: suspending the compound D prepared in the step c in water, adding strong base, heating to hydrolyze for 2-8 h, adding acid to crystallize after complete hydrolysis, filtering and washing to obtain a compound E, namely a target product 3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanic acid;
the strong base is an inorganic base;
the hydrolysis temperature is 50-100 ℃;
the acid used for crystallization is an inorganic acid or an organic acid.
10. The method of preparing β -murine cholic acid according to claim 9, wherein:
the strong base is sodium hydroxide, and the mass ratio of the sodium hydroxide to the compound D is 30-50: 100;
the hydrolysis temperature is 60-80 ℃;
the acid is hydrochloric acid with the concentration of 6M.
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| CN109369765A (en) * | 2018-12-10 | 2019-02-22 | 华南理工大学 | A kind of preparation method of cowardly acid |
| CN113336818A (en) * | 2021-05-31 | 2021-09-03 | 中山百灵生物技术股份有限公司 | Preparation method of alpha-murine cholic acid |
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| CN109369765A (en) * | 2018-12-10 | 2019-02-22 | 华南理工大学 | A kind of preparation method of cowardly acid |
| CN113336818A (en) * | 2021-05-31 | 2021-09-03 | 中山百灵生物技术股份有限公司 | Preparation method of alpha-murine cholic acid |
| CN113549123A (en) * | 2021-07-07 | 2021-10-26 | 中山百灵生物技术股份有限公司 | Preparation method of mouse deoxycholic acid |
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| 李阳 等: "一种合成α-鼠胆酸的新方法", 《精细化工》 * |
| 柴进: "齐墩果酸对阻塞性胆汁淤积大鼠胆酸转运蛋白OSTα/β 和BSEP的调控作用", 《局解手术学杂志》 * |
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