A cholate choline conjugate, its preparation method and application

Abstract

The present invention discloses a cholate choline conjugate, a preparation method thereof and an application thereof. The cholate choline conjugate has a structure shown in formula (I). The present invention provides a series of cholate choline conjugates with novel structures, which have the effect of reducing blood lipid and can be used for preparing drugs for treating hyperlipidemia, non-alcoholic fatty liver, hypercholesterolemia, diabetes, etc.; moreover, the preparation raw materials are simple and easy to obtain, the reaction conditions are mild, the operation is simple, the reaction yield is high, and it is environmentally friendly, being suitable for industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug synthesis, and particularly relates to a cholate choline conjugate, a preparation method thereof, and an application thereof. Background Art

[0002] The root cause of obesity is the imbalance between energy intake and consumption. Therefore, current treatment methods mainly focus on reducing calorie intake and increasing energy consumption. Given that long-term adherence to a weight-loss diet and taking medicine are generally unpopular among patients, weight-loss surgery seems to be the only reliable treatment option for obese people, despite the high cost of invasive surgery. As people age, the body's metabolic efficiency decreases, and food energy will be stored in the body in the form of more fat. Obesity not only causes joint pain, muscle soreness, cardiovascular diseases, and diabetes, but also makes people feel inferior, anxious, and depressed.

[0003] Recently, the laboratory of Kristina Schoonjans at the École Polytechnique Fédérale de Lausanne (EPFL) published the latest research results in *Nature Communications*, demonstrating that bile acids can cause our adipose tissue to burn, effectively consume fat, and combat obesity.

[0004] Bile acids, the yellow-green liquid secreted by the liver and usually stored in the gallbladder, are an important component of bile. During a meal, bile is released into the intestine to participate in the emulsification of dietary fat, enabling them to be broken down and absorbed by the digestive enzymes in the small intestine. This article demonstrates that bile acids can convert fat storage cells into fat-burning cells, a process known as thermogenesis, which helps the body maintain its body temperature in a cold environment. Research shows that after the bile acid receptor TGR5 is activated by "bile acid mimetics", it can induce white adipocytes to transform into beige adipocytes. In the blood, the accumulation of bile acids and their interaction with the TGR5 receptor can change the metabolic function of white adipocytes. Affected not only by the color of the cells, the researchers also found that bile acids increased the number of mitochondria in new adipocytes, indicating that these newly transformed beige adipocytes have a higher energy metabolism level. Bile acid mimetics also trigger lipolysis, the first step in the degradation of body fat by allowing adipocytes to use fatty acids as the main fuel source.

[0005] Choline, the hydroxide of β-hydroxyethyltrimethylamine, is a nitrogen-containing organic basic compound that can be completely ionized in aqueous solution, and its basic strength is similar to that of NaOH. Choline has an affinity for fats and can promote the transport of fats in the form of phospholipids from the liver through the blood or improve the utilization of fatty acids themselves in the liver, and prevent the abnormal accumulation of fat in the liver. Without choline, fat accumulates in the liver, resulting in fatty liver and being in a pathological state. Clinically, choline is used to treat cirrhosis, hepatitis and other liver diseases with good effects. On the other hand, choline and phospholipids have good emulsifying properties, can prevent cholesterol from depositing on the inner wall of blood vessels and remove some deposits, while improving the absorption and utilization of fats, and have the effect of preventing cardiovascular diseases induced by atherosclerosis caused by cholesterol deposition in blood vessels. In addition, choline can emulsify and decompose oils and fats, reduce the cholesterol and fat content in the blood, thereby softening blood vessels, reducing blood viscosity, enhancing blood circulation, and further preventing and treating diseases such as coronary heart disease, hypertension, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, and arteriosclerosis.

[0006] At present, there have been reports on the use of conjugates formed by bile acids and other substances for obesity, but there is no report on the conjugate of bile acid and choline, and the role of related substances in reducing fat still needs to be improved, and there are problems such as complex preparation processes and low yields. Clinically, there is an urgent need for new bile acid conjugate-related salts with better curative effects and easy to realize industrial preparation. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the present invention provides a cholate choline conjugate, its preparation method and application.

[0008] The cholate choline conjugate of the present invention is a tertiary amine containing an ester bond obtained by reacting a bile acid compound with a hydrohalide of dimethylaminoalkyl halide, or a tertiary amine containing an amide bond obtained by reacting a bile acid compound with N,N-dimethylalkanediamine and then reacting with an alkyl halide. This compound contains the structures of bile acid compounds and choline compounds. Among them, bile acid compounds are cholesterol derivatives synthesized in the liver, participate in many important physiological and metabolic reactions in the body, and also promote the intestinal absorption of nutrients; while choline compounds can emulsify and decompose oils and fats, reduce the cholesterol and fat content in the blood, thereby reducing blood viscosity, enhancing blood circulation, and further preventing and treating diseases such as hypertension, cerebral thrombosis, cerebral hemorrhage, and arteriosclerosis. The present invention discovers that this cholate choline conjugate can produce a synergistically enhanced therapeutic effect on hyperlipidemia, can effectively treat hyperlipidemia, and the animal experiment results are extremely significant, and the effect is better than that of a single bile acid compound or choline compound. The preparation method of this compound is simple, the yield is high, and it is easy to realize industrialization, providing a new solution idea for the preparation of drugs for treating hyperlipidemia or diseases related to hyperlipidemia.

[0009] Specifically, in the first aspect of the present invention, there is provided a cholate choline conjugate having a structure as shown in formula (I):

[0010]

[0011] Wherein, R1 is hydrogen or an alkyl group; R2 and R3 are each independently hydrogen or a hydroxyl group; Z is NH or an oxygen atom; X is a halogen atom; n is an integer from 1 to 5; the wavy line represents that the stereochemical position is optionally one of endo and exo.

[0012] According to a specific embodiment of the present invention, R1 is hydrogen or a C1-C6 alkyl group.

[0013] According to a specific embodiment of the present invention, R1 is hydrogen, methyl or ethyl.

[0014] According to a specific embodiment of the present invention, X is a chlorine atom, a bromine atom or an iodine atom.

[0015] According to a specific embodiment of the present invention, the cholate choline conjugate is selected from any one of the following compounds:

[0016]

[0017] In the second aspect of the present invention, there is provided a method for preparing the aforementioned cholate choline conjugate, comprising: reacting a compound shown in formula (1) with a compound shown in formula (2) or formula (3) to form a tertiary amine, and then reacting the tertiary amine with a haloalkane to form a quaternary ammonium salt, which is the cholate choline conjugate;

[0018]

[0019] Wherein, the definitions of R1, R2, R3, X and n are as described above.

[0020] According to a specific embodiment of the present invention, in the reaction of the compound shown in formula (1) with the compound shown in formula (2): the molar ratio of the compound shown in formula (1) to the compound shown in formula (2) is 1:1 to 2; and / or, the reaction conditions include the presence of a base and a solvent; preferably, the base is a carbonate, more preferably potassium carbonate; preferably, the solvent is one of dichloromethane, chloroform, isopropanol, acetone, butanone, dimethyl sulfoxide, N,N-dimethylformamide, and more preferably N,N-dimethylformamide.

[0021] According to a specific embodiment of the present invention, in the reaction of the compound shown in formula (1) with the compound shown in formula (3): the molar ratio of the compound shown in formula (1) to the compound shown in formula (3) is 1:1 to 2; and / or, the reaction conditions include the presence of an amide condensation reagent and a solvent; preferably, the amide condensation reagent is one of O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-propylphosphonic anhydride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1,3-dicyclohexylcarbodiimide, N,N-carbonyldiimidazole, more preferably N,N-carbonyldiimidazole; preferably, the solvent is a C1-C4 halogenated hydrocarbon, more preferably chloroform.

[0022] In a second aspect of the present invention, there is provided the use of the aforementioned cholate choline conjugate in the preparation of a medicament for treating hyperlipidemia. Preferably, the hyperlipidemia is hyperlipidemia induced by a high-fat diet.

[0023] The beneficial effects of the present invention are as follows:

[0024] The present invention provides a series of cholate choline conjugates with novel structures, which have the effect of reducing blood lipids and can be used in the preparation of medicaments for treating hyperlipidemia, non-alcoholic fatty liver, hypercholesterolemia, diabetes, etc. Moreover, the preparation raw materials are simple and easily available, the reaction conditions are mild, the operation is simple, the reaction yield is high, and it is environmentally friendly, being suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings used in the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0026] Figure 1 It is the synthetic route of obeticholic acid-C2-ester derivatives in Example 1;

[0027] Figure 2 It is the synthetic route of deoxycholic acid-C2-ester derivatives in Example 3;

[0028] Figure 3 It is the synthetic route of ursodeoxycholic acid-C2-ester derivatives in Example 5;

[0029] Figure 4 It is the synthetic route of chenodeoxycholic acid-C2-ester derivatives in Example 6;

[0030] Figure 5 It is the synthetic route of obeticholic acid-C2-amide derivatives in Example 7;

[0031] Figure 6 Synthetic route of deoxycholic acid-C2-amide derivatives for Example 8;

[0032] Figure 7 Synthetic route of ursodeoxycholic acid-C2-amide derivatives for Example 9;

[0033] Figure 8 Synthetic route of chenodeoxycholic acid-C2-amide derivatives for Example 10;

[0034] Figure 9 Mass spectrum of obeticholic acid-C2-ester intermediate for Example 1;

[0035] Figure 10 Mass spectrum of obeticholic acid-C2-ester derivatives for Example 1;

[0036] Figure 11 Mass spectrum of obeticholic acid-C3-ester intermediate for Example 2;

[0037] Figure 12 Mass spectrum of obeticholic acid-C3-ester derivatives for Example 2;

[0038] Figure 13 Mass spectrum of deoxycholic acid-C2-ester intermediate for Example 3;

[0039] Figure 14 Mass spectrum of deoxycholic acid-C2-ester derivatives for Example 3;

[0040] Figure 15 1H NMR spectrum of deoxycholic acid-C2-ester derivatives for Example 3;

[0041] Figure 16 Mass spectrum of deoxycholic acid-C3-ester intermediate for Example 4;

[0042] Figure 17 Mass spectrum of deoxycholic acid-C3-ester derivatives for Example 4;

[0043] Figure 18 Mass spectrum of obeticholic acid-C2-amide intermediate for Example 7;

[0044] Figure 19 Mass spectrum of obeticholic acid-C2-amide derivatives for Example 7;

[0045] Figure 20 Mass spectrum of deoxycholic acid-C2-amide intermediate for Example 8;

[0046] Figure 21 Mass spectrum of deoxycholic acid-C2-amide derivatives for Example 8;

[0047] Figure 22 1H NMR spectrum of deoxycholic acid-C2-amide derivative in Example 8;

[0048] Figure 23 Effect of obeticholic acid and its derivatives on cell viability;

[0049] Figure 24 Effect of combined induction concentration of oleic acid and palmitic acid on cell viability;

[0050] Figure 25 Effect of obeticholic acid and its derivatives on FFA-induced steatosis in HepG2 cells;

[0051] Figure 26 Effect of deoxycholic acid and its derivatives on FFA-induced steatosis in HepG2 cells;

[0052] Figure 27 Effect of obeticholic acid and its derivatives on body weight gain in high-fat diet-fed mice;

[0053] Figure 28 Effect of obeticholic acid and its derivatives on liver weight and liver index in high-fat diet-induced mice;

[0054] Figure 29 Effect of obeticholic acid and its derivatives on plasma biochemical analysis in high-fat diet-induced mice. Detailed implementation manners

[0055] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0056] Example 1 Obeticholic acid-C2-ester derivatives

[0057] The synthesis route is as Figure 1 shown as follows:

[0058] 1. Synthesis of obeticholic acid-C2-ester intermediate

[0059] Add 0.84 g of obeticholic acid, 5 ml of DMF, and 3.0 g of potassium carbonate to a reaction flask, stir at room temperature for 10 min, add 0.46 g of 2-(dimethylamino)ethyl bromide hydrobromide, stir and react overnight, add 10 ml of water and 20 ml of DCM for extraction, separate the aqueous layer, and concentrate the organic layer to dryness. Purify by column chromatography (DCM / MeOH = 10:1) to obtain 0.6 g of an oily substance with a yield of 64.2%. Mass spectrometry analysis is as Figure 9As shown, MS (m / z): 492.4 [M+H] + 。

[0060] 2. Synthesis of quaternary ammonium salt

[0061] Add the intermediate of obeticholic acid-C2-ester obtained in the previous step and 10 ml of DCM to the reaction flask, dissolve completely at room temperature, add 0.5 g of methyl bromide, stir overnight at room temperature, filter to obtain 0.5 g of white solid, which is the obeticholic acid-C2-ester derivative, with a yield of 83.3%. Mass spectrometry analysis is as Figure 10 shown, MS (m / z): 506.4 [M] + 。

[0062] Example 2 Obeticholic acid-C3-ester derivative

[0063] Refer to the method of Example 1 above, replace 2-(dimethylamino)ethyl bromide hydrobromide in Example 1 with 3-(dimethylamino)propyl chloride hydrochloride, and operate according to law to obtain. Mass spectrometry analysis of the obeticholic acid-C3-ester intermediate is as Figure 11 shown, MS (m / z): 506.3 [M+H] + , and mass spectrometry analysis of the obeticholic acid-C3-ester derivative is as Figure 12 shown, MS (m / z): 520.4 [M+H] + 。

[0064] Example 3 Deoxycholic acid-C2-ester derivative

[0065] The synthesis route is as Figure 2 shown, specifically as follows:

[0066] 1. Synthesis of deoxycholic acid-C2-ester intermediate

[0067] Add 0.78 g of deoxycholic acid, 5 ml of DMF, and 3.0 g of potassium carbonate to the reaction flask, stir at room temperature for 10 min, add 0.46 g of 2-(dimethylamino)ethyl bromide hydrobromide, stir and react overnight, add 10 ml of water and 20 ml of DCM for extraction, separate the aqueous layer, and concentrate the organic layer to dryness. Purify by column chromatography (DCM / MeOH = 10:1) to obtain 0.5 g of oily substance, with a yield of 54.3%. Mass spectrometry analysis is as Figure 13 shown, MS (m / z): 464.4 [M+H] + 。

[0068] 2. Synthesis of quaternary ammonium salt

[0069] Add the deoxycholic acid-C2-ester intermediate obtained in the previous step and 10 ml of DCM to the reaction flask. After complete dissolution at room temperature, add 0.5 g of methyl bromide and stir at room temperature overnight. Filter to obtain 0.4 g of white solid, which is the deoxycholic acid-C2-ester derivative with a yield of 66.7%. Mass spectrometry analysis is as Figure 14 shown, MS(m / z): 478.4[M] + . The hydrogen spectrum is as Figure 15 shown, 1 H NMR(600MHz,dmso)δ4.45(d,J=3.7Hz,1H,-OCH-),4.41(s,2H,-OCH2-),4.20(d,J=3.9Hz,1H,-OCH-),3.76(d,J=2.2Hz,1H),3.6

[0070] 7-3.60(m,2H,-NCH2-),3.35(m,1H),3.11(s,9H,-NCH3*3),2.39-2.31(m,1H),2.26-2.18(m,1H),1.82-1.39(m,11H),1.39-1.20(m,10H),1.15(d,J=11.8Hz,2H),1.09-0.93(m,2H),0.90(d,J=6.3Hz,3H,-CH3),0.85(dd,J=11.3,8.7Hz,1H),0.82(s,3H,-CH3),0.57(s,3H,-CH3).

[0071] Example 4 Deoxycholic acid-C3-ester derivative

[0072] Referring to the method of Example 3 above, replace 2-(dimethylamino)ethyl bromide hydrobromide in Example 3 with 3-(dimethylamino)propyl chloride hydrochloride and operate according to the law to obtain. The mass spectrometry analysis of the deoxycholic acid-C3-ester intermediate is as Figure 16 shown, MS(m / z): 478.6[M+H] + , and the mass spectrometry analysis of the deoxycholic acid-C3-ester derivative is as Figure 17 shown, MS(m / z): 492.3[M+H] + .

[0073] Example 5 Ursodeoxycholic acid-C2-ester derivative

[0074] The synthesis route is as Figure 3 shown, specifically as follows:

[0075] 1. Synthesis of ursodeoxycholic acid-C2-ester intermediate

[0076] Add 0.78 g of ursodeoxycholic acid, 5 ml of DMF, and 3.0 g of potassium carbonate to a reaction flask. Stir at room temperature for 10 min, add 0.46 g of 2-(dimethylamino)ethyl bromide hydrobromide, stir the reaction overnight, add 10 ml of water and 20 ml of DCM for extraction. Separate the aqueous layer, and concentrate the organic layer to dryness. Purify by column chromatography (DCM / MeOH = 10:1) to obtain 0.53 g of an oily substance with a yield of 57.6%. 464.4 [M+H] + 。

[0077] 2. Synthesis of quaternary ammonium salt

[0078] Add the intermediate of ursodeoxycholic acid-C2-ester from the previous step and 10 ml of DCM to a reaction flask. Dissolve completely at room temperature, add 0.5 g of methyl bromide, stir at room temperature overnight, filter to obtain 0.48 g of a white solid, which is the derivative of ursodeoxycholic acid-C2-ester with a yield of 75%. 478.4 [M] + 。

[0079] Example 6 Chenodeoxycholic acid-C2-ester derivatives

[0080] The synthesis route is as Figure 4 shown as follows:

[0081] 1. Synthesis of chenodeoxycholic acid-C2-ester intermediate

[0082] Add 0.78 g of chenodeoxycholic acid, 5 ml of DMF, and 3.0 g of potassium carbonate to a reaction flask. Stir at room temperature for 10 min, add 0.46 g of 2-(dimethylamino)ethyl bromide hydrobromide, stir the reaction overnight, add 10 ml of water and 20 ml of DCM for extraction. Separate the aqueous layer, and concentrate the organic layer to dryness. Purify by column chromatography (DCM / MeOH = 10:1) to obtain 0.55 g of an oily substance, which is the derivative of chenodeoxycholic acid-C2-ester with a yield of 59.8%. 464.4 [M+H] + 。

[0083] 2. Synthesis of quaternary ammonium salt

[0084] Add the intermediate of chenodeoxycholic acid-C2-ester from the previous step and 10 ml of DCM to a reaction flask. Dissolve completely at room temperature, add 0.5 g of methyl bromide, stir at room temperature overnight, filter to obtain 0.5 g of a white solid with a yield of 75.8%. 478.4 [M] + 。

[0085] Example 7 Obeticholic acid-C2-amide derivatives

[0086] The synthesis route is as Figure 5 shown as follows:

[0087] 1. Synthesis of obeticholic acid-C2-amide intermediate

[0088] Add 0.84 g of obeticholic acid, 0.20 g of N,N-dimethylethylenediamine, 20 ml of DCM, and 0.35 g of CDI to a reaction flask. Stir the reaction at room temperature overnight. Add 10 ml of water for extraction. Separate the aqueous layer. Concentrate the organic layer to dryness. Purify by column chromatography (DCM / MeOH = 10:1). Concentrate under reduced pressure to obtain 0.65 g of a foamy solid with a yield of 69.5%. Mass spectrometry analysis is as follows Figure 18 shown, MS(m / z): 491.6[M+H] + .

[0089] 2. Synthesis of quaternary ammonium salt

[0090] Add the obeticholic acid-C2-amide intermediate from the previous step and 10 ml of DCM to a reaction flask. Dissolve completely at room temperature. Add 0.5 g of methyl bromide and stir at room temperature overnight to obtain 0.5 g of a white solid, which is the obeticholic acid-C2-amide derivative with a yield of 83.3%. Mass spectrometry analysis is as follows Figure 19 shown, MS(m / z): 505.3[M] + .

[0091] Example 8 Deoxycholic acid-C2-amide derivative

[0092] The synthesis route is as follows Figure 6 shown, specifically as follows

[0093] 1. Synthesis of deoxycholic acid-C2-amide intermediate

[0094] Add 0.78 g of deoxycholic acid, 0.18 g of N,N-dimethylethylenediamine, 20 ml of DCM, and 0.35 g of CDI to a reaction flask. Stir the reaction at room temperature overnight. Add 10 ml of water for extraction. Separate the aqueous layer. Concentrate the organic layer to dryness. Purify by column chromatography (DCM / MeOH = 10:1) to obtain 0.55 g of a white solid with a yield of 59.8%. Mass spectrometry analysis is as follows Figure 20 shown, MS(m / z): MS(m / z): 463.4[M+H] +

[0095] 2. Synthesis of quaternary ammonium salt

[0096] Add the deoxycholic acid-C2-amide intermediate from the previous step and 10 ml of DCM to a reaction flask. Dissolve completely at room temperature. Add 0.5 g of methyl bromide and stir at room temperature overnight. Filter to obtain 0.45 g of a white solid, which is the deoxycholic acid-C2-amide derivative with a yield of 68.2%. Mass spectrometry analysis is as follows Figure 21 shown, MS(m / z): 479.1[M] + . The hydrogen spectrum is as follows Figure 22 shown 11H NMR (600 MHz, dmso) δ 8.18 (t, J = 5.7 Hz, 1H, -NH-), 4.46 (d, J = 4.1 Hz, 1H, -OCH-), 4.18 (d, J = 4.0 Hz, 1H, -OCH-), 3.76 (d, J = 3.3 Hz, 1H), 3.48 - 3.40 (m, 2H, -NCH2-), 3.33 (m, 3H, -NCH2-, -CH-), 3.08 (s, 9H, -NCH3*3), 2.17 - 2.06 (m, 1H), 2.03 - 1.94 (m, 1H), 1.82 - 1.67 (m, 4H), 1.66 - 1.38 (m, 7H), 1.38 - 1.20 (m, 10H), 1.19 - 1.09 (m, 3H), 1.06 - 0.93 (m, 2H), 0.9

[0097] 0 (d, J = 6.5 Hz, 3H, -CH3), 0.86 (dd, J = 14.1, 3.0 Hz, 1H), 0.82 (s, 3H, -CH3), 0.5

[0098] 6 (s, 3H, -CH3)

[0099] Example 9 Ursodeoxycholic acid - C2 - amide derivatives

[0100] The synthetic route is as Figure 7 shown below:

[0101] 1. Synthesis of Ursodeoxycholic acid - C2 - amide intermediate

[0102] Add 0.78 g of ursodeoxycholic acid, 0.21 g of N,N - dimethylethylenediamine, 20 ml of DCM, and 0.35 g of CDI to the reaction flask. Stir the reaction at room temperature overnight. Add 10 ml of water for extraction. Separate the aqueous layer, and concentrate the organic layer to dryness. Purify by column chromatography (DCM / MeOH = 10:1) to obtain 0.57 g of a foamy solid with a yield of 62%. 463.5 [M + H] + .

[0103] 2. Synthesis of quaternary ammonium salt

[0104] Add the ursodeoxycholic acid - C2 - amide intermediate from the previous step to a reaction flask, dissolve it in 10 ml of DCM at room temperature. Add 0.5 g of methyl bromide and stir at room temperature overnight. Filter to obtain 0.51 g of a foamy solid, which is the ursodeoxycholic acid - C2 - amide derivative with a yield of 73.9%. 477.4 [M] + .

[0105] Example 10 Chenodeoxycholic acid - C2 - amide derivatives

[0106] The synthetic route is asFigure 8 As shown below:

[0107] 1. Synthesis of chenodeoxycholic acid-C2-amide intermediate

[0108] Add 0.78 g of chenodeoxycholic acid, 0.31 g of N,N-dimethylethylenediamine, 20 ml of DCM, and 0.35 g of CDI to the reaction flask. Stir the reaction at room temperature overnight. Add 10 ml of water for extraction. Separate the aqueous layer. Concentrate the organic layer to dryness. Purify by column chromatography (DCM / MeOH = 10:1) to obtain 0.56 g of a foamy solid with a yield of 60.9%. 463.4 [M+H] + .

[0109] 2. Synthesis of quaternary ammonium salt

[0110] Add the above-mentioned chenodeoxycholic acid-C2-amide intermediate and 10 ml of DCM to the reaction flask. Dissolve completely at room temperature. Add 0.5 g of methyl bromide. Stir at room temperature overnight. Filter to obtain 0.50 g of a foamy solid, which is the chenodeoxycholic acid-C2-amide derivative with a yield of 74.6%. 477.4 [M] + .

[0111] Example 11 Effect of the compound of the present invention on FFA-induced steatosis in HepG2 cells

[0112] 1. Culture of HepG2 cells

[0113] 1.1 Cell resuscitation

[0114] Wipe the laminar flow hood with 75% ethanol and disinfect it with ultraviolet light for 30 min. Take out the HepG2 cells cryopreserved in liquid nitrogen and quickly dissolve them in a water bath preheated to 37°C. Immediately centrifuge at 1200 prm for 5 min after complete dissolution. Place it in the laminar flow hood, wipe the cryotube with 75% alcohol, aspirate the cryoprotectant, add 1 mL of complete medium, transfer it to a new cell culture flask, add 5 mL of complete medium, and culture it in an incubator at 37°C and 5% CO2.

[0115] 1.2 Cell passage

[0116] When the cell morphology is normal and the cell confluence reaches about 70-80%, aspirate the culture medium, add PBS, 2-3 mL per flask, wash 2 times to wash away the cell metabolites. Add 1 mL of trypsin and let it stand for 1-2 minutes. Add 2 mL of complete medium to terminate the digestion. Transfer it to a 15 mL centrifuge tube, centrifuge at 1200 prm for 5 min, aspirate the culture medium, add 2 mL of PBS buffer, centrifuge at 1000 prm for 5 min, aspirate the PBS, add 1 mL of complete medium and pipette evenly, then transfer it to a culture flask, add 3 mL of complete medium, and culture it in an incubator at 37°C and 5% CO2.

[0117] 2. Establishment, grouping and administration of the HepG2 cell lipid deposition model

[0118] 2.1 Effects of obeticholic acid and its derivatives on cell viability

[0119] When the cells reached 80 - 90% confluence, a cell suspension of 5×10 4 cells / mL was prepared and inoculated into 96 - well plates at 100 μL / well. After 48 h of culture, the supernatant was discarded. Different concentrations of obeticholic acid and its derivatives (0, 25, 50, 100 μM) were added. Each group had 3 replicate wells. After 48 h of culture, 10 μL of CCK - 8 solution was added to each well. After 1.5 h of culture, the absorbance was measured at a wavelength of 450 nm using an enzyme - linked immunosorbent assay (ELISA) reader. The intervention concentration of obeticholic acid and its derivatives was selected according to the absorbance detection results.

[0120] 2.2 Determination of the concentration of oleic acid and palmitic acid combined induction (FFA)

[0121] Cells were inoculated into 96 - well plates at 5×10 4 cells / mL, 100 μL per well. After 24 h of culture, the supernatant was discarded. A mixed solution of oleic acid and palmitic acid (molar ratio 2:1) with different final concentrations (0.25, 0.5, 0.75, 1, 1.5, 2 mM) was added. Each group had 3 replicate wells. After 24 h of induction, the cell viability was detected by the CCK - 8 experiment according to "2.1". The non - toxic concentration range of the oleic acid and palmitic acid mixed solution was determined according to the absorbance value. The formation of lipid droplets in HepG2 cells was observed by Oil Red O staining: Cells were inoculated into 96 - well plates at 5×10 4 cells / mL, 100 μL per well. After attachment, a mixed solution of oleic acid and palmitic acid (molar ratio 2:1) with different final concentrations was added.

[0122] 2.3 Oil Red O staining

[0123] After the administration or model establishment in 96 - well plates, the medium in the culture wells was discarded, gently rinsed 2 times with PBS, fixed with 4% paraformaldehyde for about 15 min, and then gently rinsed 2 times with PBS again; 35 μL of Oil Red O staining solution was added to each well of the 96 - well plate for staining for 20 min. After rinsing 1 time with PBS, rinsed 1 time with 60% isopropanol, washed with PBS buffer to remove the excess Oil Red O staining solution, stained with hematoxylin for 1 min, rinsed with PBS two or three times, and observed the color picture under the microscope.

[0124] 2.4 Grouping and administration of HepG2 cells

[0125] Cells were at 5×10 4Inoculate at a density of

[0126] 3. Data Analysis

[0127] The experimental data were analyzed by variance using GraphPad Prism software and plotted. The experimental results were expressed as mean ± standard deviation (x ± s), and a P < 0.05 or P < 0.01 was considered statistically significant difference.

[0128] 4. Experimental Results

[0129] 4.1 Effects of Obeticholic Acid and Its Derivatives on Cell Viability

[0130] As Figure 23 The results of the effects of obeticholic acid and its derivatives on cell viability detected by CCK-8 showed that when the administration concentration of obeticholic acid and its derivatives was 50 μM, the cell survival rate could still reach more than 90%, and the cell state was observed to be good under the microscope. Therefore, we selected the concentration of obeticholic acid and its derivatives below 50 μM for subsequent experiments (compared with the blank control group, **** P < 0.0001).

[0131] 4.2 Determination of the Combined Induction Concentration of Oleic Acid and Palmitic Acid

[0132] The results of the effects of mixed solutions of oleic acid and palmitic acid at different concentrations (0.25 - 1.50 mM) on lipid droplet formation in cells within 24 h are as follows. Figure 24 As shown, after Oil Red O staining, no red lipid droplet aggregation was seen in the cytoplasm of the blank group. When the concentration of the mixed solution was 0.5 mM, a small amount of red lipid droplets could be seen in the cytoplasm, and as the induction concentration increased (0.25 - 1.50 mM), the number of red lipid droplets in the cells became more and more and was obvious. Considering the effects of mixed solutions at different concentrations on cell viability and lipid droplet formation, 1.0 mM was selected as the modeling concentration (compared with the blank control group, **** P < 0.0001).

[0133] 4.3 Effects of obeticholic acid and its derivatives on FFA-induced steatosis in HepG2 cells

[0134] The effects of obeticholic acid and its derivatives on FFA-induced steatosis in HepG2 cells are as follows. Figure 25 As shown by the Oil Red O staining results, after Oil Red O staining of normal HepG2 cells, the cells were closely bound to each other and no orange-red lipid droplets were seen. After 24 h in the model group, the lipid droplets increased significantly, showing a large number of orange-red lipid droplets. Compared with the model group, in the obeticholic acid, choline, and derivative groups (50 μM), after administering the drugs and intervening for 24 h first and then inducing the high-fat model for 24 h, only a small amount of orange-red lipid droplets were seen in the drug-administered groups. To quantify the lipid droplets in the cells, after treatment with 100% isopropanol, the absorbance of each group was measured at 490 nm, and there were significant differences between each drug-administered group and the FFA group. Among them, there were significant differences between obeticholic acid-C2-esters and obeticholic acid-C2-amides and the obeticholic acid + choline group. (Compared with the FFA group, **** P < 0.0001; compared with the obeticholic acid + choline group, #### P < 0.0001).

[0135] 4.4 Effects of deoxycholic acid and its derivatives on FFA-induced steatosis in HepG2 cells

[0136] The effects of deoxycholic acid and its derivatives on FFA-induced steatosis in HepG2 cells are as follows. Figure 26As shown by the Oil Red O staining results, after Oil Red O staining of HepG2 cells in the normal group, the cells were closely bound to each other, and no orange-red lipid droplets were seen. After 24 h in the model group, the lipid droplets increased significantly, showing a large number of orange-red lipid droplets. Compared with the model group, in the deoxycholic acid, choline, and derivative group (50 μM), after administration and intervention for 24 h and then induction of the high-fat model for 24 h, a small amount of orange-red lipid droplets were seen in each administration group. To quantify the lipid droplets in the cells, after treatment with 100% isopropanol, the absorbance of each group was measured at 490 nm, and there were significant differences between each administration group and the FFA group. Among them, there were significant differences between deoxycholic acid-C2-esters and deoxycholic acid-C3-esters and the deoxycholic acid + choline group. (Compared with the FFA group, ****P < 0.0001; compared with the obeticholic acid + choline group, #### P < 0.0001).

[0137] Example 12 Experimental study on the treatment of hyperlipidemia in animals with the compounds of the present invention

[0138] 1. Establishment of animal model and administration

[0139] Thirty C57 mice were randomly divided into 5 groups, namely the normal group (Control), the high-fat group (HFD), the obeticholic acid group (OCA), the obeticholic acid amide group (Amide), and the obeticholic acid ester group (Esters), with 6 C57 mice in each group. During the experiment, the normal group was fed with ordinary feed continuously for 8 weeks, and the remaining groups were all fed with HFD continuously for 8 weeks. Among them, the obeticholic acid group, the obeticholic acid amide group, and the obeticholic acid ester group were respectively intragastrically administered with 10 mg / kg / day of obeticholic acid, 14 mg / kg / day of the obeticholic acid-C2-amide derivative of Example 7, and 14 mg / kg / day of the obeticholic acid-C2-ester derivative of Example 1. The normal group and the high-fat group were intragastrically administered with the same volume of pure water. To study the effects of obeticholic acid and its derivatives on hyperlipidemia induced by high-fat feed.

[0140] 2. Experimental results

[0141] 2.1 Effects of obeticholic acid and its derivatives on the weight gain of mice fed with high-fat feed

[0142] The body weight of the mice was recorded after the last intragastric administration every weekend. After 8 weeks of the experiment, as Figure 27 shown, compared with the Control group, the body weight of the HFD group increased significantly, the body weight of the OCA (obeticholic acid) group of mice increased slightly, and the Amide (obeticholic acid amide) group and the Esters (obeticholic acid ester) group significantly inhibited the weight gain of the mice.

[0143] 2.2 Effects of obeticholic acid and its derivatives on the liver weight and liver index of mice induced by high-fat feed

[0144] After the 8th week of the experiment, the mice were fasted but given water for 12 h. The next day, the mice were sacrificed by cervical dislocation. Blood was collected from the eyes of each mouse, and the liver and white adipose tissue were dissected out, rinsed with PBS, blotted dry with filter paper, weighed on an electronic balance, and the liver weight was recorded. The liver index was calculated as: liver weight / body weight × 100%. As Figure 28 shown, compared with the Control group, the liver weight of the HFD group increased significantly. The liver weight of the mice in the OCA (obeticholic acid) group increased. The Amide (obeticholic acid amide) group and the Esters (obeticholic acid ester) group significantly inhibited the increase in the liver weight of the mice. Also, compared with the Control group, the liver index of the HFD group increased significantly, while the Amide (obeticholic acid amide) group and the Esters (obeticholic acid ester) group significantly inhibited the increase in the liver index of the mice.

[0145] 2.3 Plasma biochemical analysis of obeticholic acid and its derivatives in high-fat diet-induced mice

[0146] The eyes of each mouse in each group were enucleated and blood was collected. The whole blood collected was placed in a centrifuge tube. After the blood sample was allowed to stand at 4 °C for 2 h, it was centrifuged at 3500 rpm for 20 min at 4 °C. The upper plasma was aspirated using a pipette and transferred to a new 1.5 mL sterile centrifuge tube and stored in a -80 °C refrigerator for later use. Detection was carried out using a relevant index chemical kit. As Figure 29 shown, compared with Control, HFD feeding led to a significant increase in TC, TG, LDL-C, and HDL-C in the plasma, indicating that the hyperlipidemia model was successfully established. All three drugs significantly reduced the levels of TC, TG, LDL-C, and HDL-C; however, there was a significant difference in the TC level between the OCA (obeticholic acid) group and the normal group.

[0147] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A cholate choline conjugate, characterized in that, The cholate choline conjugate is selected from any one of the following compounds: 。 2. A method for preparing the cholate-choline conjugate according to claim 1, characterized in that, The preparation method includes: reacting the compound shown in formula (1) with the compound shown in one of formula (2) or formula (3) to form a tertiary amine, and then reacting the tertiary amine with a haloalkane to form a quaternary ammonium salt, which is the cholate choline conjugate; ； X is a halogen atom.

3. The preparation method according to claim 2, characterized in that, In the reaction of the compound represented by formula (1) with the compound represented by formula (2): the molar ratio of the compound represented by formula (1) to the compound represented by formula (2) is 1:1 to 2; and / or, the reaction conditions include the presence of a base and a solvent, the base is a carbonate, and the solvent is one of dichloromethane, chloroform, isopropanol, acetone, methyl ethyl ketone, dimethyl sulfoxide, N,N -dimethylformamide.

4. The preparation method according to claim 3, wherein The base is potassium carbonate.

5. The preparation method according to claim 3, characterized in that, The solvent is N,N -dimethylformamide.

6. The preparation method according to claim 2, characterized in that, In the reaction of the compound shown in formula (1) with the compound shown in formula (3): the molar ratio of the compound shown in formula (1) to the compound shown in formula (3) is 1:1 to 2; and / or, the reaction conditions include the presence of an amide condensation reagent and a solvent, and the amide condensation reagent is O-benzotriazole- N,N,N',N' -tetramethylurea tetrafluoroborate, 2-(7-azabenzotriazole)- N,N,N',N' -tetramethylurea hexafluorophosphate, 1-propylphosphonic anhydride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1,3-dicyclohexylcarbodiimide, N,N -carbonyldiimidazole, and the solvent is a C1-C4 halogenated hydrocarbon.

7. The preparation method according to claim 6, wherein The amide condensation reagent is N,N -carbonyldiimidazole.

8. The preparation method according to claim 6, characterized in that, The solvent is chloroform.

9. Use of the cholate choline conjugate according to claim 1 in the preparation of a medicament for treating hyperlipidemia.

10. The use according to claim 9, wherein the hyperlipidemia is hyperlipidemia induced by a high-fat diet.

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