CN110804082A - Cholic acid derivative and preparation method and application thereof - Google Patents

Cholic acid derivative and preparation method and application thereof Download PDF

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CN110804082A
CN110804082A CN201911166718.7A CN201911166718A CN110804082A CN 110804082 A CN110804082 A CN 110804082A CN 201911166718 A CN201911166718 A CN 201911166718A CN 110804082 A CN110804082 A CN 110804082A
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王玉成
王菊仙
何红伟
牛伟萍
牛伟晓
张国宁
朱梅
王明华
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Institute of Medicinal Biotechnology of CAMS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton

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Abstract

The invention relates to the technical field of medicine synthesis, and provides a cholic acid derivative and a preparation method and application thereof. The cholic acid derivative provided by the invention has the obvious effect of inhibiting hepatocyte apoptosis, can effectively inhibit hepatocyte damage induced by glycochenodeoxycholic acid, and has a partial derivative inhibition rate superior to that of positive control tauroursodeoxycholic acid; the cholic acid derivative provided by the invention provides reference for research and development of liver protection medicaments, and has a wide application prospect in preparation of liver protection medicaments.

Description

Cholic acid derivative and preparation method and application thereof
Technical Field
The invention relates to the technical field of medicine synthesis, and particularly relates to a cholic acid derivative and a preparation method and application thereof.
Background
Liver injury is a common pathological state in clinic, and causes of liver injury include hepatitis virus, alcohol, drug abuse, free fatty acid metabolic syndrome, inflammation process and genetic diseases which affect liver immune mediation, and the like. Apoptosis of hepatocytes is a characteristic of liver damage, and hepatocytes have a strong regenerative function through cell proliferation. For example, 70% of the liver regenerates to its original mass within about 1 week after hepatectomy. However, when cell loss exceeds a certain threshold, liver regeneration is insufficient to counteract hepatocyte apoptosis, functional hepatocytes are replaced by fibrotic scars, liver function is impaired, and chronic liver disease ultimately results. Therefore, designing and synthesizing compounds capable of inhibiting hepatocyte apoptosis has important clinical therapeutic value.
Bile acids (bile acids) are a class of steroid molecules with unique physicochemical and biological properties. On the one hand, certain bile acids can cause hepatocyte apoptosis, which is a common pathological feature of cholestatic liver diseases. For example, glycochenodeoxycholic acid (GCDCA), which is the binding form of glycine to chenodeoxycholic acid (CDCA), has been shown to induce apoptosis of hepatocytes by promoting ligand-independent oligomerization of the death receptor Fas/CD 95. On the other hand, certain cholic acids and derivatives thereof have been also shown to inhibit apoptosis of hepatocytes and to protect liver. Studies have shown that ursodeoxycholic acid (UDCA) exerts a cytoprotective effect by modulating the classical mitochondrial pathway, thereby lowering the apoptosis threshold of several cell types. UDCA can also inhibit apoptosis by modulating the E2F-1/p53/Bax pathway in animal models. Tauroursodeoxycholic acid (TUDCA), a form of taurine bound to UDCA, can also inhibit rat hepatocyte apoptosis by activating various pathways. In addition, UDCA, TUDCA and obeticholic acid (OCA, 6-ethyl derivative of chenodeoxycholic acid) show therapeutic effects on primary cholangitis. However, there are still few organic substances that can inhibit apoptosis of hepatocytes.
Disclosure of Invention
In view of the above, the present invention aims to provide cholic acid derivatives, and a preparation method and applications thereof. The cholic acid derivative provided by the invention can effectively inhibit hepatocyte damage induced by glycochenodeoxycholic acid, and the activity of inhibiting hepatocyte apoptosis of part of the derivative is better than that of positive control tauroursodeoxycholic acid, so that the derivative is expected to be developed into a novel liver protection medicament.
In order to achieve the above object, the present invention provides the following technical solutions:
a cholic acid derivative has a structure shown in formula I:
Figure BDA0002287654050000021
in formula I: r1Is any one of the following groups:
Figure BDA0002287654050000022
H；
R2is any one of the following groups:
Figure BDA0002287654050000023
H、-CH2CH3；
R3is any one of the following groups:
Figure BDA0002287654050000024
H；
R1' is any one of the following groups:
Figure BDA0002287654050000025
H；
R2' is any one of the following groups:H、-CH2CH3；
R3' is any one of the following groups:H；
y is any one of the following groups:
Figure BDA0002287654050000028
the invention provides a preparation method of the cholic acid derivative in the scheme, which comprises the following steps:
when R is1And R1' same, R2And R2' same, R3And R3' same, when Y is the following group,
Figure BDA0002287654050000031
the preparation method of the cholic acid derivative comprises the following steps:
(1) reacting a compound with a structure shown in a formula A with N-hydroxysuccinimide under the action of a condensing agent to obtain an intermediate with a structure shown in a formula 1-1;
Figure BDA0002287654050000032
(2) mixing an intermediate with a structure shown in a formula 1-1, a compound Y1 and a solvent for reaction to obtain a cholic acid derivative; the compound Y1 has one of the following structures:
Figure BDA0002287654050000033
when R is1And R1' same, R2And R2' same, R3And R3' same, when Y is the following group:
Figure BDA0002287654050000034
the preparation method of the cholic acid derivative comprises the following steps:
(i) reacting a compound with a structure shown in a formula A with 3, 4-dihydro-2H-pyran under the action of a catalyst to obtain an intermediate with a structure shown in a formula 2-1;
Figure BDA0002287654050000041
(ii) under the action of HOBT, EDCI and DBU, carrying out condensation reaction on an intermediate with a structure shown in a formula 2-1 and a compound Y2 to obtain an intermediate with a structure shown in a formula 2-2;
Figure BDA0002287654050000042
the compound Y2 has one of the following structures:
Figure BDA0002287654050000043
(iii) reacting the intermediate with the structure shown as the formula 2-2 with pyridinium p-toluenesulfonate in a solvent to obtain a cholic acid derivative;
when R is1And R1' not identical and/or R2And R2' not identical and/or R3And R3' when not identical, the preparation method of the cholic acid derivative comprises the following steps:
(a) under the action of an acid binding agent, reacting a compound with a structure shown in a formula A or a compound with a structure shown in a formula B with tert-butyldimethylsilyl trifluoromethanesulfonate to respectively obtain an intermediate with a structure shown in a formula 3-1 and an intermediate with a structure shown in a formula 4-1;
Figure BDA0002287654050000044
Figure BDA0002287654050000051
(b) under the action of HOBT, EDCI and DBU, carrying out condensation reaction on an intermediate with a structure shown in a formula 3-1 and a compound with a structure shown in a formula C to obtain an intermediate with a structure shown in a formula 3-2;
Figure BDA0002287654050000053
wherein the Y group is the same as in formula I;
(c) carrying out hydrogenation reaction on the intermediate with the structure shown in the formula 3-2 in ethanol under the action of a hydrogen environment and a Pd/C catalyst to obtain the intermediate with the structure shown in the formula 3-3;
Figure BDA0002287654050000054
(d) under the action of HOBT, EDCI and DBU, reacting an intermediate with a structure shown in a formula 4-1 with an intermediate with a structure shown in a formula 3-3 to obtain an intermediate with a structure shown in a formula 3-4;
Figure BDA0002287654050000055
(e) reacting the intermediate with the structure shown in the formula 3-4 with hydrogen fluoride pyridine in a solvent to obtain the cholic acid derivative.
Preferably, the condensing agent in step (1) comprises one or more of N- (3-dimethylamidopropyl) -N' -ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide and diisopropylcarbodiimide.
Preferably, the reaction temperature in the step (1) is 0-60 ℃, and the reaction time is 1-24 hours; the reaction temperature in the step (2) is 0-60 ℃, and the reaction time is 1-10 h.
Preferably, the catalyst in step (i) comprises one or more of p-toluenesulfonic acid monohydrate, 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone and bis (trimethylsilyl) sulfate, perchloric acid and tetrachlorosilane.
Preferably, the reaction in the step (i) is carried out at room temperature for 1-12 h; the reaction temperature in the step (ii) is 0-60 ℃, and the reaction time is 2-24 hours; the reaction temperature in the step (iii) is 0-100 ℃, and the reaction time is 2-24 h.
Preferably, the acid-binding agent in step (a) and step (d) independently comprises one or more of N, N-diisopropylethylamine, 2, 6-dimethylpyridine, pyridine and triethylamine.
Preferably, the reaction temperature in the step (a) is 0-60 ℃, and the reaction time is 1-24 hours; the reaction temperature in the step (b) is 0-60 ℃, and the reaction time is 1-24 hours; the pressure of the reaction in the step (c) is 30-60 psi, and the time is 8-48 h; the reaction temperature in the step (d) is 0-60 ℃, and the reaction time is 1-24 hours; the reaction temperature in the step (e) is 0-80 ℃, and the reaction time is 1-24 hours.
The invention provides application of the cholic acid derivative in the scheme in preparation of liver protection medicaments.
The invention provides a cholic acid derivative which has a structure shown in a formula I. The cholic acid derivative provided by the invention has the obvious effect of inhibiting hepatocyte apoptosis, can effectively inhibit hepatocyte damage induced by glycochenodeoxycholic acid, has a partial derivative inhibition rate superior to that of positive control tauroursodeoxycholic acid, provides a reference for research and development of liver protection medicaments, and has a wide application prospect in preparation of the liver protection medicaments. The results of the examples show that the inhibition rate of the cholic acid derivative for inhibiting the hepatocyte apoptosis can reach 105.24%, and the inhibition rate of the positive control tauroursodeoxycholic acid is 41.03%.
Detailed Description
The invention provides a cholic acid derivative which has a structure shown in a formula I:
Figure BDA0002287654050000071
in formula I: r1Is any one of the following groups:
Figure BDA0002287654050000072
H；
R2is any one of the following groups:
Figure BDA0002287654050000073
H、-CH2CH3；
R3is any one of the following groups:
Figure BDA0002287654050000074
H；
R1' is any one of the following groups:
Figure BDA0002287654050000075
H；
R2' is any one of the following groups:
Figure BDA0002287654050000076
H、-CH2CH3；
R3' is any one of the following groups:
Figure BDA0002287654050000077
H；
y is any one of the following groups:
Figure BDA0002287654050000078
the invention provides a preparation method of the cholic acid derivative in the scheme, which comprises the following steps:
when R is1And R1' same, R2And R2' same, R3And R3' same, when Y is the following group,
Figure BDA0002287654050000079
Figure BDA0002287654050000081
the preparation method of the cholic acid derivative comprises the following steps:
(1) reacting a compound with a structure shown in a formula A with N-hydroxysuccinimide under the action of a condensing agent to obtain an intermediate with a structure shown in a formula 1-1;
Figure BDA0002287654050000082
(2) mixing an intermediate with a structure shown in a formula 1-1, a compound Y1 and a solvent for reaction to obtain a cholic acid derivative; the compound Y1 has one of the following structures:
Figure BDA0002287654050000083
the specific synthetic route is shown as formula I:
Figure BDA0002287654050000084
the method comprises the following steps of reacting a compound with a structure shown in a formula A with N-hydroxysuccinimide under the action of a condensing agent to obtain an intermediate with a structure shown in a formula 1-1; in the present invention, the condensing agent in the step (1) preferably includes one or more of N- (3-dimethylamidopropyl) -N' -ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide and diisopropylcarbodiimide; the solvent for reaction in the step (1) is preferably one or more of super-dry DMF (N, N-dimethylformamide), super-dry DMSO (dimethyl sulfoxide), super-dry DCM (dichloromethane) and super-dry THF (tetrahydrofuran); the reaction temperature in the step (1) is preferably 0-60 ℃, more preferably 25-45 ℃, and the time is preferably 1-24 hours, more preferably 8-12 hours; the reaction is preferably carried out under the protection of argon; in the present invention, the molar ratio of the compound having the structure represented by formula a, the condensing agent, and N-hydroxysuccinimide is preferably 1:1.5: 1.5.
After the reaction in the step (1) is finished, the product liquid and water are preferably mixed and then extracted by ethyl acetate, and the obtained organic phase is sequentially washed by saturated sodium carbonate, dried by anhydrous sodium sulfate and concentrated under reduced pressure to obtain a white powdery product, namely an intermediate with a structure shown in a formula 1-1.
After an intermediate with a structure shown in a formula 1-1 is obtained, the intermediate with the structure shown in the formula 1-1, a compound Y1 and a solvent are mixed for reaction to obtain the cholic acid derivative. In the present invention, the kind of the solvent reacted in the step (2) is the same as that reacted in the step (1), and is not described herein again; the reaction temperature in the step (2) is preferably 0-60 ℃, more preferably 25-45 ℃, and the time is preferably 1-10 hours, more preferably 2-4 hours; the reaction is preferably carried out under the protection of argon; in the invention, the intermediate with the structure shown in the formula 1-1 is preferably mixed with a solvent, and then the compound Y1 is dripped, wherein the reaction time is calculated from the completion of the dripping of the compound Y1; in the present invention, the molar ratio of the intermediate having the structure represented by formula 1-1 to compound Y1 is preferably 2:1.
After the reaction in the step (2) is completed, the product liquid is preferably mixed with water, then the mixture is extracted by ethyl acetate, the obtained organic phase is dried by anhydrous sodium sulfate and then is concentrated under reduced pressure, and then the concentrated product is purified by silica gel column chromatography to obtain the cholic acid derivative.
When R is1And R1' same, R2And R2' same, R3And R3' same, when Y is the following group:
Figure BDA0002287654050000091
the preparation method of the cholic acid derivative comprises the following steps:
(i) reacting a compound with a structure shown in a formula A with 3, 4-dihydro-2H-pyran under the action of a catalyst to obtain an intermediate with a structure shown in a formula 2-1;
(ii) under the action of HOBT, EDCI and DBU, carrying out condensation reaction on an intermediate with a structure shown in a formula 2-1 and a compound Y2 to obtain an intermediate with a structure shown in a formula 2-2;
Figure BDA0002287654050000102
the compound Y2 has one of the following structures:
Figure BDA0002287654050000103
(iii) reacting the intermediate with the structure shown as the formula 2-2 with pyridinium p-toluenesulfonate in a solvent to obtain a cholic acid derivative;
the specific reaction route is shown as the formula II:
Figure BDA0002287654050000104
the compound with the structure shown in the formula A and 3, 4-dihydro-2H-pyran react under the action of a catalyst to obtain an intermediate with the structure shown in the formula 2-1. In the present invention, the catalyst preferably comprises one or more of p-toluenesulfonic acid monohydrate, 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone and bis (trimethylsilyl) sulfate, perchloric acid and tetrachlorosilane; the solvent for reaction in the step (i) is preferably one or more of dichloromethane, DMF, DMSO, THF and 1, 2-dichloroethane; the reaction temperature in the step (i) is preferably room temperature, and the time is preferably 1-12 h, and more preferably 1-3 h; the invention protects the hydroxyl group in the compound with the structure shown in the formula A through the step (i).
In the present invention, when R is in the structural compound represented by formula A1、R2Or R3When the intermediate is hydrogen or ethyl, R in the structural formula of the intermediate with the structure shown in the formula 2-1 and the intermediate with the structure shown in the formula 2-21、R2And R3Also hydrogen or ethyl; when R in the compound of the formula A1、R2Or R3In the case of a hydroxyl group, the hydroxyl group is also reacted with 3, 4-dihydro-2H-pyran, i.e., in this case, an intermediate having a structure represented by formula 2-1, or a compound having a structure represented by formula 2-2Structural formula of intermediate R1、R2Or R3Is composed of
Figure BDA0002287654050000111
Namely, hydroxyl groups in the structure of the compound shown in the formula A need to be protected; in a specific embodiment of the present invention, the molar amount of 3, 4-dihydro-2H-pyran is preferably determined according to the molar amount of hydroxyl groups in the compound of the structure of formula A, the ratio of the molar amount of hydroxyl groups to the molar amount of 3, 4-dihydro-2H-pyran in the compound of the structure of formula A preferably being 1:1 to 1.5.
After the reaction in step (i) is completed, the invention preferably mixes the product liquid with water, then extracts with ethyl acetate, dries the obtained organic phase with anhydrous sodium sulfate, then concentrates under reduced pressure, and then purifies the concentrated product by silica gel column chromatography to obtain the intermediate with the structure shown in formula 2-1.
After an intermediate with a structure shown in a formula 2-1 is obtained, the intermediate with the structure shown in the formula 2-1 and a compound Y2 are subjected to condensation reaction under the action of HOBT (1-hydroxybenzotriazole), EDCI (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and DBU (1, 8-diazabicycloundecene-7-ene) to obtain the intermediate with the structure shown in the formula 2-2. In the present invention, the solvent for the reaction in the step (ii) is preferably anhydrous DMF; the reaction temperature in the step (ii) is preferably 0-60 ℃, more preferably 25-45 ℃, and the time is preferably 2-24 hours, more preferably 8-12 hours; the reaction is preferably carried out under the protection of argon; the preparation method preferably comprises the steps of stirring and mixing the intermediate with the structure shown in the formula 2-1, HOBT, EDCI, DBU and a solvent for 0.5-1 h at the temperature of 0-60 ℃, and then adding a compound Y2 for reaction; in the present invention, the molar ratio of the intermediate having the structure represented by formula 2-1, compound Y2, HOBT, EDCI, and DBU is preferably 1:0.5:1.2:2: 2.
After the reaction in the step (ii), the product liquid is preferably mixed with water, then the mixture is extracted by ethyl acetate, the obtained organic phase is dried by anhydrous sodium sulfate and then is concentrated under reduced pressure, and the concentrated crude product can be directly subjected to the next reaction without purification.
After an intermediate with a structure shown in a formula 2-2 is obtained, the intermediate with the structure shown in the formula 2-2 and pyridinium p-toluenesulfonate (PPTS) are reacted in a solvent to obtain the cholic acid derivative. In the present invention, the solvent for the reaction in step (iii) is preferably one or more of methanol, DCM, THF, DMSO and dichloroethane; the reaction temperature in the step (iii) is preferably 0-100 ℃, more preferably 50-80 ℃, and the time is preferably 2-24 hours, more preferably 8-12 hours; in the present invention, the molar ratio of the intermediate having the structure represented by formula 2-2 to pyridinium p-toluenesulfonate is preferably 1: 1; according to the invention, the hydroxyl protecting group in the intermediate with the structure shown in the formula 2-2 is removed through the reaction in the step (iii), so that the cholic acid derivative is obtained.
After the reaction in step (iii) is completed, the present invention preferably performs reduced pressure distillation on the product liquid to remove the solvent, and performs silica gel column chromatography purification on the residue to obtain the cholic acid derivative.
When R is1And R1' not identical and/or R2And R2' not identical and/or R3And R3' when not identical, the preparation method of the cholic acid derivative comprises the following steps:
(a) under the action of an acid binding agent, reacting a compound with a structure shown in a formula A or a compound with a structure shown in a formula B with tert-butyldimethylsilyl trifluoromethanesulfonate to respectively obtain an intermediate with a structure shown in a formula 3-1 and an intermediate with a structure shown in a formula 4-1;
Figure BDA0002287654050000121
(b) under the action of HOBT, EDCI and DBU, carrying out condensation reaction on an intermediate with a structure shown in a formula 3-1 and a compound with a structure shown in a formula C to obtain an intermediate with a structure shown in a formula 3-2;
wherein the Y group is the same as in formula I;
(c) carrying out hydrogenation reaction on the intermediate with the structure shown in the formula 3-2 in ethanol under the action of a hydrogen environment and a Pd/C catalyst to obtain the intermediate with the structure shown in the formula 3-3;
Figure BDA0002287654050000132
(d) under the action of HOBT, EDCI and DBU, reacting an intermediate with a structure shown in a formula 4-1 with an intermediate with a structure shown in a formula 3-3 to obtain an intermediate with a structure shown in a formula 3-4;
Figure BDA0002287654050000133
(e) reacting the intermediate with the structure shown in the formula 3-4 with hydrogen fluoride pyridine in a solvent to obtain the cholic acid derivative.
The specific reaction route is shown as the formula III:
Figure BDA0002287654050000141
the compound with the structure shown in the formula A and tert-butyldimethylsilyl trifluoromethanesulfonate react under the action of an acid-binding agent to obtain an intermediate with the structure shown in the formula 3-1. In the invention, the acid-binding agent preferably comprises one or more of N, N-diisopropylethylamine, 2, 6-dimethylpyridine, pyridine and triethylamine; the solvent for the reaction in the step (a) is preferably one or more of anhydrous DCM, anhydrous DMF and anhydrous THF; the reaction temperature in the step (a) is preferably 0-60 ℃, more preferably 0-35 ℃, and the time is preferably 1-24 hours, more preferably 2-4 hours; the reaction is preferably carried out under the protection of argon; the compound with the structure shown in the formula A, the acid binding agent and the solvent are preferably mixed, then the tert-butyl dimethyl silyl trifluoromethanesulfonate is dropwise added under the ice bath condition, the ice bath is removed after the dropwise addition is finished, and the reaction is carried out under the reaction temperature condition.
In the present invention, when R is in the structural compound represented by formula A1、R2Or R3When the intermediate is hydrogen or ethyl, R in the structural formula of the intermediate with the structure shown in the formula 3-1, the intermediate with the structure shown in the formula 3-2 and the intermediate with the structure shown in the formula 3-41、R2And R3Also hydrogen or ethyl; when R in the compound of the formula A1、R2Or R3In the case of a hydroxyl group, the hydroxyl group is also reacted with t-butyldimethylsilyl trifluoromethanesulfonate, i.e., in this case, R in the structural formula of the intermediate having the structure represented by formula 3-1, the intermediate having the structure represented by formula 3-2, and the intermediate having the structure represented by formula 3-41、R2Or R3Is composed of
Figure BDA0002287654050000142
Namely, hydroxyl groups in the structure of the compound shown in the formula A need to be protected; in a specific embodiment of the present invention, the molar amount of tert-butyldimethylsilyl trifluoromethanesulfonate is preferably determined on the basis of the molar amount of hydroxyl groups in the compound of the formula A, which is preferably in a ratio of 1:2 to 3.
Under the action of an acid binding agent, the compound with the structure shown in the formula B reacts with tert-butyldimethylsilyl trifluoromethanesulfonate to obtain an intermediate with the structure shown in the formula 4-1. In the present invention, the reaction conditions, raw material molar ratio and post-treatment method for the reaction of the compound having the structure shown in formula B and tert-butyldimethylsilyl trifluoromethanesulfonate are the same as those for the reaction of the compound having the structure shown in formula A and tert-butyldimethylsilyl trifluoromethanesulfonate, and thus are not described herein again.
In the present invention, the hydroxyl groups in the compound of formula B need to be protected, and the specific situation is the same as that of the compound of formula a, and will not be described herein again.
After the reaction in the step (a) is finished, the invention preferably mixes the obtained product liquid with water, then extracts the mixture by ethyl acetate, dries the obtained organic phase by anhydrous sodium sulfate, then carries out decompression concentration, and then carries out silica gel column chromatography purification on the concentrated product to obtain the intermediate with the structure shown in the formula 3-1.
After the intermediate with the structure shown in the formula 3-1 is obtained, the intermediate with the structure shown in the formula 3-1 and the compound with the structure shown in the formula C are subjected to condensation reaction under the action of HOBT, EDCI and DBU, so that the intermediate with the structure shown in the formula 3-2 is obtained. In the invention, the solvent for reaction in the step (b) is preferably one or more of super-dry DMF, super-dry THF, super-dry DCM and super-dry DMSO; the reaction temperature in the step (b) is preferably 0-60 ℃, more preferably 0-35 ℃, and the time is preferably 1-24 hours, more preferably 8-12 hours; the reaction is preferably carried out under the protection of argon; in the present invention, the molar ratio of the intermediate having the structure represented by formula 3-1, the intermediate having the structure represented by formula C, HOBT, EDCI and DBU is preferably 1:1.5:1.5:2.5: 2.5.
After the reaction in the step (b) is completed, the invention preferably mixes the product liquid with water, then extracts with ethyl acetate, dries the obtained organic phase with anhydrous sodium sulfate, then concentrates under reduced pressure, and then purifies the concentrated product by silica gel column chromatography to obtain the intermediate with the structure shown in the formula 3-2.
After the intermediate with the structure shown in the formula 3-2 is obtained, the intermediate with the structure shown in the formula 3-2 is subjected to hydrogenation reaction in ethanol under the hydrogen environment and the action of a Pd/C catalyst to obtain the intermediate with the structure shown in the formula 3-3. In the invention, the pressure of the reaction in the step (c) is preferably 30-60 psi, more preferably 40-60 psi, and the time is preferably 8-48 h, more preferably 8-24 h; the temperature of the reaction is preferably room temperature; the reaction is preferably carried out in a medium-pressure reaction flask; the present invention hydrogenates an intermediate having a structure represented by formula 3-2 through step (c).
After the reaction in the step (c) is completed, the invention preferably filters the obtained product liquid by using kieselguhr, then removes the solvent by reduced pressure evaporation, and performs silica gel column chromatography purification on the residue to obtain the intermediate with the structure shown in the formula 3-3.
After obtaining the intermediate with the structure shown in the formula 4-1 and the intermediate with the structure shown in the formula 3-3, the invention reacts the intermediate with the structure shown in the formula 4-1 and the intermediate with the structure shown in the formula 3-3 under the action of HOBT, EDCI and DBU to obtain the intermediate with the structure shown in the formula 3-4. In the invention, the solvent for reaction in the step (d) is preferably one or more of super-dry DMF, super-dry THF, super-dry DCM and super-dry DMSO; the reaction temperature in the step (d) is preferably 0-60 ℃, more preferably 20-35 ℃, and the time is preferably 1-24 hours, more preferably 8-12 hours; the reaction is preferably carried out under the protection of argon; in the present invention, the molar ratio of the intermediate having the structure represented by formula 4-1, the intermediate having the structure represented by formula 3-3, HOBT, EDCI and DBU is preferably 1:1.2:1.5:2.5: 2.5; according to the invention, preferably, the intermediate with the structure shown in the formula 4-1, HOBT, EDCI, DBU and a solvent are stirred and mixed for 0.5-1 h at the temperature of 0-60 ℃, and then the intermediate with the structure shown in the formula 3-3 is added for reaction.
After the reaction in the step (d) is finished, the product liquid is preferably mixed with water, then the mixture is extracted by ethyl acetate, the obtained organic phase is dried by anhydrous sodium sulfate and then is concentrated under reduced pressure, and then the concentrated product is purified by silica gel column chromatography to obtain an intermediate with a structure shown in a formula 3-4.
After an intermediate with a structure shown in a formula 3-4 is obtained, the intermediate with the structure shown in the formula 3-4 and hydrogen fluoride pyridine react in a solvent to obtain the cholic acid derivative. In the present invention, the solvent for the reaction in step (e) is preferably one or more of tetrahydrofuran, DMF and DCM; the reaction temperature in the step (e) is preferably 0-80 ℃, more preferably 20-35 ℃, and the time is preferably 1-24 hours, more preferably 2-3 hours; in the invention, the dosage ratio of the hydrogen fluoride pyridine to the intermediate with the structure shown in the formula 3-4 is preferably 1.7mL:1 mmol; the intermediate with the structure shown in the formula 3-4 is preferably dissolved in a solvent, and then the hydrogen fluoride pyridine is dripped for reaction, wherein the reaction time is calculated from the completion of the dripping of the hydrogen fluoride pyridine; the invention removes the hydroxyl protecting group in the intermediate with the structure shown in the formula 3-4 through the step (e).
After the reaction in the step (e) is completed, the invention preferably mixes the obtained product feed liquid with saturated sodium bicarbonate solution, then uses ethyl acetate for extraction, dries the obtained organic phase with anhydrous sodium sulfate, then carries out decompression concentration, and then carries out silica gel column chromatography purification on the concentrated product to obtain the cholic acid derivative.
The invention also provides application of the cholic acid derivative in the scheme in preparation of liver protection medicaments. The present invention is not particularly limited to the specific method of application, and may be applied according to a method known to those skilled in the art.
The embodiments of the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Synthesis of (4R,4'R) -N, N' - (ethane-1, 2-diyl) bis (4- ((3R,5S,7S,8R,9S,10S,13R,14S,17R) -3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthreneanthracen-17-yl) pentanamide); denoted compound 1a, the procedure is as follows:
ursodeoxycholic acid (10g, 25.47mmol), EDCI (4.40g, 38mmol), N-hydroxysuccinimide (3.5g, 30.5mmol) and anhydrous DMF (50mL) were added sequentially to the flask and the reaction was stirred at 35 ℃ under argon for 12 h. Water (120mL) was added to the reaction and extracted with ethyl acetate (3X 50 mL). The combined organic phases were washed with saturated NaHCO3The solution (3X 50mL) was washed and Na was added2SO4And (5) drying. The extract was concentrated under reduced pressure to give the product (1-1) (8.08g, 64%) as a white powder.1HNMR(600MHz,CD3OD-d4)δ3.55-3.46(m,2H),2.84(s,4H),2.72-2.64(m,1H),2.62-2.52(m,1H),2.07(dt,J＝12.8,3.4Hz,1H),1.98-1.79(m,4H),1.68-1.18(m,16H),1.13(q,J＝9.5Hz,1H),1.09-1.04(m,1H),1.03-0.98(m,6H),0.76–0.73(m,3H).
A solution of (1-1) (1.0g, 2.04mmol), ethylenediamine (0.061g, 1.02mmol) and anhydrous DMF (10mL) was stirred at 35 ℃ for 10 h. Water (40mL) was added to the reaction and extracted with ethyl acetate (3X 30 mL). The combined extracts were extracted with anhydrous Na2SO4Dried and concentrated under reduced pressure and purified by silica gel column chromatography. Eluting with 1:40 to 1:15 methanol-dichloromethane to obtain final productProduct as a white powder (0.5195 g, 61%); mp; 149-153 ℃;
1H NMR(600MHz,CD3OD-d4)δ3.53-3.44(m,4H),3.27(s,4H),2.28-2.20(m,2H),2.12-2.00(m,4H),1.95–1.87(m,4H),1.86-1.75(m,6H),1.66-1.00(m,38H),0.99-0.95(m,12H),0.72(s,6H).13C NMR(151MHz,CD3OD-d4)δ177.19,72.04(d,J＝26.2Hz),57.55,56.56,44.82,44.51,44.05,41.61,40.73,40.09,38.63,38.03,36.94,36.10,35.19,34.24,33.35,31.06,29.73,27.99,23.95,22.40,19.06,12.69.HRMS-ESI(m/z):Calcd.forC50H84N2O6(M+H)+:809.6408,(M+Na)+:831.6227；Found:831.6274.
example 2
Synthesis of ethane-1, 2-diyl (4R,4' R) -bis (4- (((3R,5S,7S,8R,9S,10S,13R,14S,17R) -3, 7-dihydroxy-10, 13-hexadecyldimethyl-1H-cyclopenta [ a ] phenanthryl-17-yl) valerate), denoted as compound 2a, as follows:
ursodeoxycholic acid (3g,7.64mmol),1, 4-dioxane (15mL), 3, 4-dihydro-2H-pyran (1.93g,22.9mmol) and p-toluenesulfonic acid hydrate (0.29g,1.53mmol) were sequentially added to the flask, and stirred at room temperature for 3H. Water (50mL) was added to the reaction and extracted with ethyl acetate (3X 30 mL). The combined extracts were extracted with anhydrous Na2SO4Dried, concentrated under reduced pressure, and purified by silica gel column chromatography. Elution with ethyl acetate-n-hexane from 1:10 to 1:5 gave (2-1) (2.8g, 65%) as a white powder.1H NMR(600MHz,Chloroform-d)δ3.94–3.82(m,3H),3.64–3.32(m,3H),2.43–2.34(m,1H),2.29–2.20(m,1H),2.01–0.96(m,22H),0.95–0.91(m,6H),0.69–0.63(m,3H).
(2-1) (0.5g, 0.9mmol), HOBT (0.15g,1.08mmol), EDCI (0.35g,1.8 mmol), DBU (0.27g,1.8mmol) and anhydrous DMF were put into a reaction flask in this order, stirred at 30 ℃ for 0.5h, added with ethylene glycol (0.028g,0.45mmol) to the above system, and stirred under argon atmosphere at 35 ℃ for another 12 h. Water (40mL) was added to the reaction and extracted with ethyl acetate (3X 30 mL). The combined extracts were extracted with anhydrous Na2SO4Drying and concentrating under reduced pressure to obtain crude product (2-2) (0) as yellow oil577g) the next step was carried out without further purification.
(2-2) (0.577g, 0.503mmol), methanol (10mL) and pyridinium p-toluenesulfonate (PPTS, 0.135g, 0.503mmol) were put in this order in a reaction flask and stirred at 65 ℃ for 12 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography. Elution with 1: 100 methanol-dichloromethane afforded the final product as a white powder (0.240g, 61%, from 2-1). mp: 99-102 ℃;1H NMR(600MHz,Chloroform-d)δ4.27(s,4H),3.77–3.38(m,5H),2.42–2.33(m,2H),2.30–2.20(m,2H),2.06–1.95(m,3H),1.95–0.98(m,60H),0.94(d,J＝9.3Hz,13H),0.68(s,6H).13C NMR(151MHz,Chloroform-d)δ174.11,71.58,71.47,62.18,55.95,55.07,43.92,43.89,42.59,40.31,39.38,37.46,37.05,35.48,35.08,34.22,31.37,31.12,30.52,28.76,27.08,23.54,21.33,18.55,12.30.
HRMS-ESI(m/z):Calcd.for C50H82O8(M+H)+:811.6089,(M+Na)+:833.5908；Found:833.5912.
example 3
The synthesis of 2- (2- (((R) -4- ((3R,5S,7R,8R,9S,10S,13R,14S,17R) -3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) pentanoyl) oxy) ethoxy) ethyl (R) -4- ((3R,5S,7S,8R,9S,10S,13R,14S,17R) -3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) pentanoate, denoted compound 4a step as follows:
a solution of ursodeoxycholic acid (6.0g, 15.28mmol), DIEA (9.86g, 76.40mmol) and anhydrous DMF (60mL) was stirred at 0 ℃ for 0.5h, then tert-butyldimethylsilyl trifluoromethanesulfonate (14.14g, 53.49mmol) was added dropwise to the above system via syringe and stirred under argon atmosphere at room temperature for 2 h. Water was added to the above reaction solution, which was extracted with ethyl acetate (3X 30mL), followed by extraction with anhydrous Na2SO4Dried and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography. Elution with ethyl acetate-n-hexane from 1:30 to 1:20 gave (3-1) as a white powder (5.98 g; 63%);1H NMR(400MHz,Chloroform-d)δ3.74–3.60(m,1H),3.59–3.44(m,1H),2.46–2.33(m,1H),2.32–2.17(m,1H),2.00–1.90(m,1H),1.88–0.94(m,22H),0.94–0.80(m,24H),0.64(s,3H),0.13–0.01(m,12H).
(3-1) (3.2g, 5.16mmol), HOBT (1.045,7.74mmol), EDCI (2.473g,12.9m mol), DBU (1.96g,12.9mmol) and anhydrous DMF were put into a reaction flask in this order, stirred at 30 ℃ for 0.5h, and 2- [2- (benzyloxy) ethoxy ] ethyl acetate was added to the above system]Ethanol (1.519g,7.74mmol) and stirring at 35 ℃ under argon atmosphere for 12 h. Water (40mL) was added to the reaction and extracted with ethyl acetate (3X 30 mL). The combined extracts were extracted with anhydrous Na2SO4Drying, concentrating under reduced pressure, and purifying by silica gel column chromatography. Elution with ethyl acetate-n-hexane from 1:30 to 1:15 gave (3-2) as a white powder (2.64g, 64%);1H NMR(500MHz,Chloroform-d)δ7.37–7.27(m,5H),4.58(s,2H),4.23(t,J＝4.8Hz,2H),3.76–3.59(m,8H),3.58–3.43(m,1H),2.45–2.31(m,1H),2.29–2.15(m,1H),2.05–0.94(m,32H),0.94–0.90(m,6H),0.90–0.82(m,24H),0.63(s,3H),0.08–0.02(m,12H).
to a medium pressure reaction flask were added (3-2) (1.715g,2.15mmol), Pd/C (0.43g, w/w 0.1:1) and ethanol (15 mL). N for air of the mixture2(g) Replacement 3 times, then reuse of H2(g) The replacement was performed 3 times. Hydrogenation was carried out at room temperature under a pressure of 45psi for 12 h. With N2(g) Replacement of 3 times H2(g) The reaction mixture was filtered through celite, and the solvent was removed under reduced pressure. Purification by silica gel column chromatography (ethyl acetate/n-hexane, 1:15 to 1:5, v: v) afforded the product (3-3) as a white powder: (1.17g, 77%).1HNMR(500MHz,Chloroform-d)δ4.33–4.18(m,2H),3.81–3.44(m,8H),2.45–2.31(m,1H),2.31–2.16(m,2H),2.06–1.88(m,1H),1.85–0.94(m,28H),0.93–0.86(m,23H),0.64(s,3H),0.10–0.01(m,11H).
A solution of chenodeoxycholic acid (5.0g, 12.74mmol), DIEA (8.22g, 63.68mmol) and anhydrous DMF (60mL) was stirred at 0 ℃ for 0.5h, then tert-butyldimethylsilyl trifluoromethanesulfonate (11.78g,44.58mmol) was added dropwise to the above system via syringe and stirred at room temperature under argon atmosphere for 2 h. Water was added to the reaction, and extracted with ethyl acetate (3X 30 mL). The combined substance is treated with anhydrous Na2SO4Drying and concentrating under reduced pressure, and purifying by silica gel column chromatographyAnd (5) purifying. Elution with ethyl acetate-n-hexane from 1:30 to 1:20 gave (4-1) as a white powder (4.83g, 63%);
(4-1) (0.10g,0.161mmol), HOBT (0.027g,0.201mmol), EDCI (0.0642g,0.335mmol), DBU (0.051g,0.335mmol), anhydrous DMF were sequentially charged into a reaction flask, stirred at 30 ℃ for 0.5h, (3-3) (0.095g,0.134mmol) was added to the above system, and stirred at 35 ℃ for 12h under argon shield. Water (40mL) was added to the reaction and extracted with ethyl acetate (3X 30 mL). The combined extracts were extracted with anhydrous Na2SO4Drying, concentration under reduced pressure, and purification by silica gel column chromatography (ethyl acetate/n-hexane 1:20) gave the product (3-4) (0.16g, 93%).1H NMR(500MHz,Chlor oform-d)δ4.22(t,J＝4.7Hz,4H),3.84–3.32(m,8H),2.45–2.30(m,2H),2.30–2.18(m,4H),2.07–0.92(m,67H),0.92–0.79(m,46H),0.63(d,J＝9.5Hz,6H),0.12–0.01(m,25H).
(3-4) (0.16g,0.123mmol) and tetrahydrofuran (THF,4mL) were added to a plastic reaction flask at room temperature, then hydrogenated fluoropyridine (0.21mL) was added dropwise with stirring and the reaction was stirred at room temperature for 2 h. The reaction mixture was added dropwise to saturated NaHCO3To the solution, and extracted with ethyl acetate (3X 30 mL). The combined extracts were extracted with anhydrous Na2SO4Drying, concentrating under reduced pressure, and purifying by silica gel column chromatography. Elution with ethyl acetate-n-hexane at 1:15 gave the final product as a white oil (0.070g, 61%); .1H NMR(600MHz,Chloroform-d)δ4.26–4.19(m,4H),3.87–3.82(m,1H),3.69(t,J＝4.8Hz,4H),3.63–3.54(m,2H),3.50–3.42(m,1H),2.43–2.34(m,2H),2.30–2.16(m,3H),2.03–0.96(m,29H),0.95–0.90(m,12H),0.67(d,J＝10.7Hz,6H).13C NMR(101MHz,Chloroform-d)δ174.25,174.22,72.03,71.45,71.37,69.15,68.51,65.60,63.34,55.81,55.75,54.90,50.48,43.77,42.71,42.46,41.50,40.16,39.88,39.67,39.44,39.21,37.31,36.89,35.38,35.35,35.29,35.07,34.96,34.63,34.10,32.86,31.15,31.14,30.96,30.94,30.67,30.59,30.34,28.66,28.20,26.92,23.74,23.42,22.80,21.20,20.60,19.21,18.42,18.32,13.76,12.17,11.81.
HRMS-ESI(m/z):Calcd.For C52H86O9(M+H)+:855.6351,(M+Na)+:877.6170,(M-H)+:853.6193；Found:853.6152.
Examples 4 to 19
Other conditions were the same as in example 1 except that ethylenediamine (i.e., compound Y1) was replaced with only compound Y1 selected in accordance with the structure of the Y group in table 1, and the obtained products were designated as compounds 1b to 1 f.
Other conditions were the same as in example 2 except that ethylene glycol (i.e., compound Y2) was replaced and compound Y2 was selected in accordance with the structure of the Y group in Table 1, and the obtained products were designated as compounds 2b to 2 g.
Other conditions were the same as in example 2 except that the starting material was replaced with chenodeoxycholic acid; and replacing ethylene glycol (i.e., compound Y2), selecting compound Y2 according to the structure of the Y group in Table 1, and marking the obtained products as compounds 3 a-3 e.
Example 20
The activity of cholic acid derivatives prepared in examples 1 to 19 in inhibiting hepatocyte apoptosis was measured, and GCDCA (glycochenodeoxycholic acid) was used to induce hepatocyte apoptosis in primary mice, and hepatocytes induced by GCDCA were co-treated with the target compound. The activity of inhibiting hepatocyte apoptosis in vitro was quantified by measuring the level of caspase3/7 (caspase3/7), UDCA (ursodeoxycholic acid), CDCA (chenodeoxycholic acid), TUDCA (tauroursodeoxycholic acid) and OCA (obeticholic acid) as positive control compounds.
(1) Isolation and culture of hepatocytes
Male BALB/C mice weighing 20-30g (age: 10 weeks) were maintained at a circadian rhythm of 12h, and were freely fed standard diet and water until the day of the experiment.
Hepatocytes were isolated by collagenase perfusion technique. Freshly isolated hepatocytes at 1X 106The cells/mL were suspended in a mixture of William E medium at a density. The culture medium was Williama E medium supplemented with 10% FBS, 100 units/ml penicillin G sodium and 100 mg/ml streptomycin sulfate. The hepatocytes were maintained at 37.0 ℃ with 5% CO2And an incubator in a humid atmosphere. The medium was changed 4h after cell inoculation to minimize contamination of dead cells. Separating the liver cellsIncubations were carried out in different William E media for 4h, which were divided into a group with/without any compound added (used as a blank control)/a group of mixtures with the target compounds (1 a-4a) (20. mu.M) and GCDCA (200. mu.M), respectively;
(2) caspase3/7 activity
Caspase3/7 activity was measured using the Caspase3/7 assay kit (Promage, G8091) according to the manufacturer's instructions.
Inhibition was 1- (compound caspase-blank group caspase)/(GCDCA group caspase-blank group caspase), and the results are shown in table 1.
Compounds 1a-4a have the formula:
Figure BDA0002287654050000221
in the general formula R2、R2', Y and the chirality of the hydroxyl group at position 7 are specified in Table 1.
TABLE 1 Structure, physical data and anti-apoptotic Activity of Compounds 1a-5b
Figure BDA0002287654050000222
Figure BDA0002287654050000231
In table 1: a represents a structural general formula of which the structure is not applicable to the formula I, b represents that the melting point is not corrected, and c represents that the compound is oil;
according to the results in table 1, the cholic acid derivative provided by the invention has significant activity of inhibiting hepatocyte apoptosis, wherein the inhibition rate of hepatocyte apoptosis of a part of derivatives is even higher than that of positive control, and the inhibition rate of compound 4a for inhibiting hepatocyte apoptosis can reach 105.24%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (9)

1. A cholic acid derivative, which has a structure shown in formula I:
in formula I: r1Is any one of the following groups:
Figure FDA0002287654040000012
H；
R2is any one of the following groups:
Figure FDA0002287654040000013
H、-CH2CH3；
R3is any one of the following groups:
Figure FDA0002287654040000014
H；
R1' is any one of the following groups:
Figure FDA0002287654040000015
H；
R2' is any one of the following groups:
Figure FDA0002287654040000016
H、-CH2CH3；
R3' is any one of the following groups:H；
y is any one of the following groups:
Figure FDA0002287654040000018
n1＝1～10，Ra＝H，CH3、
Figure FDA0002287654040000019
n2＝1～8，Rb＝H，CH3、
Figure FDA00022876540400000110
Figure FDA00022876540400000111
n＝1～8。
2. the method for preparing the cholic acid derivative according to claim 1, comprising the steps of:
when R is1And R1' same, R2And R2' same, R3And R3' same, when Y is the following group,
Figure FDA0002287654040000021
n1＝1～10，Ra＝H，CH3、
Figure FDA0002287654040000022
n2＝1～8，Rb＝H，CH3；
the preparation method of the cholic acid derivative comprises the following steps:
(1) reacting a compound with a structure shown in a formula A with N-hydroxysuccinimide under the action of a condensing agent to obtain an intermediate with a structure shown in a formula 1-1;
Figure FDA0002287654040000023
(2) mixing an intermediate with a structure shown in a formula 1-1, a compound Y1 and a solvent for reaction to obtain a cholic acid derivative; the compound Y1 has one of the following structures:
Figure FDA0002287654040000024
n1＝1～10，Ra＝H，CH3、
n2＝1～8，Rb＝H，CH3；
when R is1And R1' same, R2And R2' same, R3And R3' same, when Y is the following group:
Figure FDA0002287654040000026
Figure FDA0002287654040000027
n＝1～8；
the preparation method of the cholic acid derivative comprises the following steps:
(i) reacting a compound with a structure shown in a formula A with 3, 4-dihydro-2H-pyran under the action of a catalyst to obtain an intermediate with a structure shown in a formula 2-1;
Figure FDA0002287654040000031
(ii) under the action of HOBT, EDCI and DBU, carrying out condensation reaction on an intermediate with a structure shown in a formula 2-1 and a compound Y2 to obtain an intermediate with a structure shown in a formula 2-2;
the compound Y2 has one of the following structures:
Figure FDA0002287654040000034
n＝1～8；
(iii) reacting the intermediate with the structure shown as the formula 2-2 with pyridinium p-toluenesulfonate in a solvent to obtain a cholic acid derivative;
when R is1And R1' not identical and/or R2And R2' not identical and/or R3And R3' when not identical, the preparation method of the cholic acid derivative comprises the following steps:
(a) under the action of an acid binding agent, reacting a compound with a structure shown in a formula A or a compound with a structure shown in a formula B with tert-butyldimethylsilyl trifluoromethanesulfonate to respectively obtain an intermediate with a structure shown in a formula 3-1 and an intermediate with a structure shown in a formula 4-1;
Figure FDA0002287654040000035
(b) under the action of HOBT, EDCI and DBU, carrying out condensation reaction on an intermediate with a structure shown in a formula 3-1 and a compound with a structure shown in a formula C to obtain an intermediate with a structure shown in a formula 3-2;
Figure FDA0002287654040000042
Figure FDA0002287654040000043
wherein the Y group is the same as in formula I;
(c) carrying out hydrogenation reaction on the intermediate with the structure shown in the formula 3-2 in ethanol under the action of a hydrogen environment and a Pd/C catalyst to obtain the intermediate with the structure shown in the formula 3-3;
Figure FDA0002287654040000044
(d) under the action of HOBT, EDCI and DBU, reacting an intermediate with a structure shown in a formula 4-1 with an intermediate with a structure shown in a formula 3-3 to obtain an intermediate with a structure shown in a formula 3-4;
Figure FDA0002287654040000045
(e) reacting the intermediate with the structure shown in the formula 3-4 with hydrogen fluoride pyridine in a solvent to obtain the cholic acid derivative.
3. The method according to claim 2, wherein the condensing agent in the step (1) comprises one or more of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide and diisopropylcarbodiimide.
4. The preparation method according to claim 2, wherein the reaction temperature in the step (1) is 0-60 ℃ and the reaction time is 1-24 h; the reaction temperature in the step (2) is 0-60 ℃, and the reaction time is 1-10 h.
5. The method of claim 2, wherein the catalyst in step (i) comprises one or more of p-toluenesulfonic acid monohydrate, 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone and bis (trimethylsilyl) sulfate, perchloric acid and tetrachlorosilane.
6. The preparation method according to claim 2, wherein the reaction in the step (i) is carried out at room temperature for 1-12 h; the reaction temperature in the step (ii) is 0-60 ℃, and the reaction time is 2-24 hours; the reaction temperature in the step (iii) is 0-100 ℃, and the reaction time is 2-24 h.
7. The preparation method according to claim 2, wherein the acid-binding agent in step (a) and step (d) independently comprises one or more of N, N-diisopropylethylamine, 2, 6-dimethylpyridine, pyridine and triethylamine.
8. The preparation method according to claim 2, wherein the reaction temperature in the step (a) is 0-60 ℃ and the reaction time is 1-24 h; the reaction temperature in the step (b) is 0-60 ℃, and the reaction time is 1-24 hours; the pressure of the reaction in the step (c) is 30-60 psi, and the time is 8-48 h; the reaction temperature in the step (d) is 0-60 ℃, and the reaction time is 1-24 hours; the reaction temperature in the step (e) is 0-80 ℃, and the reaction time is 1-24 hours.
9. The use of a cholic acid derivative according to claim 1 for the preparation of a hepatoprotective agent.
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