WO2017101789A1 - Compound having anti-cancer effect, and method for preparation thereof and application thereof - Google Patents

Abstract

The present invention provides a compound having the structure (I), and pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, or prodrug thereof, and a method for preparing said compound; also provided is an application of a pharmaceutical composition, containing the described substances, in the preparation of an anti-cancer drug.

Description

Compound with anticancer effect and preparation method and application thereof Technical field

The invention relates to a compound, a preparation method and application thereof, and particularly to a compound with selective anticancer activity, a preparation method and application thereof.

Background technique

At present, the treatment of cancer in the clinic often has the tragedy of "cancer death", especially the liver cancer, colon cancer, cervical cancer and other malignant cancers. There is no specific drug at present. The reason is that chemotherapy is one of the main ways of cancer. Chemotherapy drugs often cause serious side effects on normal organisms while killing cancer cells, such as nephrotoxicity, hepatotoxicity, neurotoxicity and the like. It is the goal of pharmacy workers to obtain anti-cancer drugs with high efficiency, low toxicity and strong selectivity. It is gratifying that the latest researches at home and abroad have found that compounds with strong cytotoxicity have good drug-forming properties, which is in line with the development trend of contemporary precision medicine. (Cancer Cell, 2015, 28, 240-252; Int. J. Mol. Sci. 2015, 16(7), 16401-16413).

The invention has a bile acid component having antitumor and anti-hepatic biological activities, mainly comprising cholic acid (CA), deoxycholic acid (DCA), ursodeoxycholic acid (UCA), hyodeoxycholic acid (HCA), Goose deoxycholic acid (CDCA), lithocholic acid (LCA) and the natural drug tetramethylpyrazine (TMP) were used as starting materials to chemically combine to synthesize the compounds of the present invention. The activity evaluation of the compounds mainly focused on anti-tumor (especially liver cancer, intestinal cancer, etc.), and the analogs were tested on four cancer cell lines (HepG-2, HT-29, Hela, MCF-7), respectively. Primary hepatoma cell lines (HCC3787C1, HCC4172A2, HCC4477B2, HCC4484B1, HCC5850A1, HCC5864D1, HCC5953B1, HCC633A5, and HCC061A2) and non-cancer cell lines (NGF-induced PC12 cells, renal epithelial cells MDCK, mouse embryonic fibroblasts 3T3) Cytotoxic activity.

Summary of the invention

A first object of the present invention is to provide a compound having the structure of Formula 1 and a process for the preparation thereof.

A second object of the present invention is to provide the use of a compound of formula 1 for the preparation of an anticancer drug.

A third object of the present invention is to provide a kit having an anticancer effect.

The object of the present invention is achieved by the following technical solutions:

A compound having the structure of Formula 1, a pharmaceutically acceptable salt, stereoisomer, isotope label, solvate, polymorph or prodrug thereof:

取代基中的任意一种；Wherein R _{1 -} R ₄ are -OH, -H or

Any of the substituents;

取代基中的任意一种；R5 is -COOH,

Any of the substituents;

取代基。And at least one of R ₁ -R ₅

Substituent.

The present application further provides a pharmaceutical composition comprising the above-described compound, pharmaceutically acceptable salt, stereoisomer, isotope label, solvate, polymorph or prodrug thereof, and pharmaceutically acceptable a. The pharmaceutical compositions include, but are not limited to, oral dosage forms, parenteral dosage forms, topical dosage forms, and rectal administration dosage forms. In some embodiments, the pharmaceutical composition may be an oral tablet, capsule, pill, powder, sustained release preparation, solution or suspension, sterile solution, suspension or emulsion for parenteral injection, A topical ointment or cream, or a suppository for rectal administration.

Preparation method of the compound of the invention:

Reaction Scheme 1: The cholic acid component is dissolved in an organic solvent and reacted with 2-bromomethyl-3,5,6-trimethylpyrazine (intermediate compound 1) under basic conditions to obtain the present invention. Compound I;

Compound I is further reacted with ligustrazine acid (intermediate compound 2) under the action of a catalyst and a condensing agent to give a compound II of the present invention.

Reaction route map:

Reaction conditions and reagents: (a) Benzene, reflux, 10h; (b) CCl ₄ , NBS, hv, reflux, 12h; (c) DMF, K ₂ CO ₃ , N ₂ , 85 ° C, 1.5 h; H ₂ O, KMnO ₄ , 37 ° C, 6 h; (e) dry CH ₂ Cl ₂ , EDCI, DMAP, 12h.

Wherein, in the compound I, R _{1 -} R ₄ are each -OH or -H, and at least one of R ₁ , R ₂ and R ₄ is -OH;

且R₁、R₂、R₄中至少有一个

In compound II, R ₃ is -OH or -H, and R ₁ , R ₂ and R ₄ are -OH, -H or

And at least one of R ₁ , R ₂ , and R ₄

The specific structure of each compound is shown in Table 1.

Table 1 Compound I and II structural formula

Reaction Scheme 2: The cholic acid component is dissolved in an organic solvent, and reacted with benzyl bromide under basic conditions to obtain an intermediate compound 3; the intermediate compound 3 is further reacted with ligustrazine by a catalyst and a condensing agent (middle) The compound 2) is reacted to obtain the compound III of the present invention; the compound III is further reduced by hydrogen to obtain the compound IV of the present invention.

Reaction route map:

a, THF, Reflux, K ₂ CO ₃ ; b, CH ₂ Cl ₂ , EDCI/DMAP, c, THF, Pd/C, Pd(OH) ₂ /C

且R₁、R₂、R₄中至少有一个

Wherein, in compounds III and IV, R ₃ is -OH or -H, and R ₁ , R ₂ and R ₄ are -OH, -H or

And at least one of R ₁ , R ₂ , and R ₄

.

The specific structure of each compound is shown in Table 2.

Table 2 Compound III and IV structural formula

Further, the above reaction is carried out at -20 ° C to 250 ° C; the organic solvent is an ether, an alcohol, an alkane, an aromatic hydrocarbon, a ketone, an alkyl halide, an amide, a nitrile, an ester having 1 to 20 carbon atoms or various ratios thereof a mixture; the base used is triethylamine or potassium carbonate; the catalyst is 1-hydroxybenzotriazole (HOBT); the condensing agent is 1-ethyl-3-(3-dimethylaminopropyl) carbon Any one or more of diimine hydrochloride (EDCI), 1,3-dicyclohexylcarbodiimide (DCC) or 4-dimethylaminopyridine (DMAP).

Further, in the above preparation method, the molar ratio of the cholic acid component to the 2-bromomethyl-3,5,6-trimethylpyrazine (intermediate compound 1) is 1:0.1 to 1:10; The molar ratio of the component to the base is 1:0.1 to 1:10; the molar ratio of the compound II to the ligustrazine acid (intermediate compound 2) is 1:0.1 to 1:10; the molar ratio of the compound II to the condensing agent is 1:0.1 to 1:10; and the molar ratio to the catalyst is 1:0.1 to 1:10.

The invention further provides the use of a compound of formula 1, a pharmaceutically acceptable salt, stereoisomer, isotopic label, solvate, polymorph or prodrug thereof for the preparation of an anticancer drug.

Further, the cancer is liver cancer, colon cancer, cervical cancer, breast cancer.

The invention also provides a method of treating cancer, the method comprising administering an effective amount of a compound of Formula 1, or a pharmaceutically acceptable salt, stereoisomer, isotopic label, solvate, polymorph or prodrug thereof For individuals with this need. The individual can be a mammal, such as a human.

Further, the effective amount of the compound of Formula 1 or a pharmaceutically acceptable salt, stereoisomer, isotope label, solvate, polymorph or prodrug thereof is from 1 to 10 mg/kg·day.

Further, the cancer is liver cancer, colon cancer, cervical cancer, breast cancer.

In some embodiments, the pharmaceutical composition and the at least one therapeutic agent are each combined in a separate dosage form into a combined product, such as a kit. The effective amount of the combination product of a therapeutically effective amount of a compound of Formula 1, or a pharmaceutically acceptable salt, stereoisomer, isotopic label, solvate, polymorph or prodrug thereof, is from 1 to 10 mg/kg day. .

Further, the anticancer drug is any one or more of cyclophosphamide, 5-fluorouracil, paclitaxel, doxorubicin, etoposide, irinotecan, oxaliplatin, cisplatin or japonica.

"Pharmaceutically acceptable" as used herein refers to molecular entities and compositions that are physiologically tolerable in humans and that generally do not produce an allergic or similar untoward reaction. By "therapeutically effective amount" is meant an amount sufficient to cause an improvement in a clinically significant condition/symptom in a host. The "solvate" refers to a combination of a compound of the present application and a solvent molecule formed by solvation. By "polymorph" is meant a compound of the present application that exists in a different lattice form. By "isotopic label" is meant a compound of the present application that is labeled by an isotope. The "pharmaceutically acceptable prodrug" refers to any pharmaceutically acceptable salt, ester, ester salt or other derivative of a compound of the present application which can be provided directly or indirectly after use to a recipient. a compound or a pharmaceutically active metabolite or residue thereof. The "stereoisomer" refers to an isomer produced by the arrangement of atoms in a molecule in a spatially different manner.

The compound of the invention has an inhibitory effect on various cancer cells. The inhibition rate of intraperitoneal injection of 30 mg/kg on H22 tumor-bearing mice is 60.16%, which is equivalent to the positive drug cyclophosphamide; and for such as nerve cell kidney cells, mouse embryos Normal cytotoxicity such as fibroblasts is very small. Normal mice have an intraperitoneal injection of LD ₅₀ >900 mg/kg, indicating that the drug is safe and can be used for the prevention and treatment of cancers such as liver cancer, colon cancer and cervical cancer.

Experimental Example 1 The effect of the composition of the present invention on the proliferation of cancer cells and non-cancer cell lines was observed by the AlamarBlue method.

1. Instruments and materials

Thermo 311 CO ₂ incubator; Haier biosafety cabinet; Molecular Devices; Xiangyi L530 desktop low speed centrifuge; Olympus IX51 inverted fluorescence microscope, RPMI-1640 medium, fetal bovine serum, 0.25% trypsin solution , phosphate buffer (Thermo Fisher Shanghai Co., Ltd.); sigma dimethyl sulfoxide (DMSO), resazurin;

Human breast cancer cell line MCF-7; human hepatoma cell line HepG-2; mouse embryonic fibroblast 3T3; human colon cancer cell line HT-29; human cervical cancer cell line Hela; canine renal epithelial cell MDCK; mouse adrenal gland Chromosome cell PC12;

Experimental drug: Compound (1-30) of the present invention (prepared as in Example 3-32); ligustrazine (TMP), cholic acid (CA), deoxycholic acid (DCA), ursodeoxycholic acid (UCA), Hyodeoxycholic acid (HCA), chenodeoxycholic acid (CDCA), lithocholic acid (LCA); positive drug doxorubicin (HY-15142; Shanghai Qianyuan Biomedical Technology Co., Ltd.).

2. Method

2.1 Cultivation of different cell lines

MCF-7, HepG2, 3T3, HT-29, Hela, MDCK, and NGF-PC12 cells were cultured in 1640 medium containing 10% fetal bovine serum, and incubated at 37 ° C in a 5% CO ₂ incubator. The cells were all grown in an adherent state, and the growth was observed under an inverted microscope, and subcultured when the number of cells was appropriate.

2.2 inhibition of proliferation of tumor cells and normal cells

Cell test: MCF-7, HepG2, HT-29, Hela, 3T3, MDCK, NGF-PC12 cells in logarithmic growth phase were seeded in 96-well culture plates at 3×103/well in 5%. After incubating at 37 ° C for 4 h in a CO2 humidification incubator, add 10 μL to each well to be tested. Compounds or positive drugs, 9 drug concentrations were tested such that the final drug concentrations were 100, 31.6, 10, 3.16, 1, 0.31, 0.1, 0.03, and 0.01 μM, respectively. At the same time, a positive control group (100% inhibition) and a negative control group (0% inhibition) were set. The drug group was repeated 2 wells per concentration, and the positive control group and the negative control group were repeated 6 wells. After 6 days of incubation in the incubator, the subsequent AlamarBlue was followed. Test operation

AlamarBlue test operation: Incubate with 10 μL of AlamarBlue reagent per well for 1-4 h, shake for 1-2 min, MD microplate reader EX: 560 nm, EM: 590 nm wavelength to measure the fluorescence value, record the results, calculate the cell inhibition rate (%) = (A0% inhibition - A administration) / (A0% inhibition - A100% inhibition) × 100%, and then using GraphPad Prism 5.0 or MATILAB software using nonlinear regression method (often using four-parameter fitting curve equation) to obtain drug dose response curves to obtain IC ₅₀ value of the compound and other relevant parameters.

3. Results

3.1 The IC ₅₀ values of the compounds of the present invention against five tumor cell lines (MCF-7, HepG2, HT-29, Hela) and normal cells (3T3, MDCK, NGF-induced PC12) are shown in Table 3.

Table 3 IC ₅₀ values of different drugs against tumor cell lines and normal cells

Note: “—” means: According to the results of the cytotoxic test, when the effect on cancer cells is not obvious, it is considered to have low activity, and no toxicity test for non-cancer cells is performed.

As can be seen from Table 3, the compound of the present invention exhibits proliferation-inhibiting activity against various cancer cells, especially Compound 10, and its antitumor activity is not only superior to the raw material, but also to human colon cancer cell line HT-29 and human cervical cancer cells. The anti-proliferative activity of Hela was better than that of the positive drug doxorubicin; the cytotoxicity of NGF-induced PC12 cells, canine renal epithelial cells MDCK and mouse embryonic fibroblasts 3T3 was significantly lower than that of the positive drug doxorubicin.

4 Conclusion

The compound of the present invention has good inhibitory activity against various cancer cells (IC ₅₀ <5.7 μM), but does not have any killing effect on various normal cells at a higher dose (IC ₅₀ >80 μM), and has good cytotoxicity selection. Sexual effect.

Experimental Example 2 AlamarBlue Method to Observe the Effect of the Invention on the Proliferation of Hepatoma Cells and the Response Rate in Liver Cancer

1. Instruments and materials

Thermo 311 CO ₂ incubator; Haier biosafety cabinet; Molecular Devices microplate reader; Xiangyi brand L530 desktop low speed centrifuge; Olympus IX51 inverted fluorescence microscope, DMEM/F12 medium, fetal bovine serum, 0.25% trypsin solution , phosphate buffer (Thermo Fisher Shanghai Co., Ltd.); sigma dimethyl sulfoxide (DMSO), resazurin;

The primary liver cancer cell lines (human hepatoma cell lines HCC3787C1, HCC4172A2, HCC4477B2, HCC4484B1, HCC5850A1, HCC5864D1, HCC5953B1, HCC633A5 and HCC061A2) were prepared as follows from the liver cancer tissues of 9 independent liver cancer patients according to the following method. The fresh liver cancer tissue after surgical resection was washed in PBS (GIBCO, 10010-23), and cut into 0.5-1 mm3 pieces in a 100 mm culture dish using an ophthalmic forceps, scissors, etc., and laid flat on the bottom of the dish. And add 10 ml of DMEM/F12 cell culture medium (GIBCO, 11320-033) containing 10% FBS (GIBCO, 10099-141), 1% Pen-Strep antibiotic (GIBCO, 15140-122) to the culture dish, and put into 37 The culture was carried out in an incubator of °C 5% CO2. The next day, under the inverted microscope, the cells were observed to swim around the tissue block, and then gradually extended to grow into a halo that could be observed by the naked eye. After 5-7 days, the tissue cells in the center of the tissue block gradually necrotic and floating, and the floating small pieces can be discarded with the change of the liquid, and the adherent cells extending around the tissue block gradually form a layer. Using fibroblasts and tumor cells to digest, the cells were digested with 0.25% trypsin solution (GIBCO, 25200056) to continuously remove fibroblasts. After multiple passages, fibroblasts were not visible to the naked eye in the culture dish and could be used for subsequent experiments when they were continuously grown for passage.

Experimental drug: compound of the present invention (prepared according to Example 10); chemotherapeutic drug Doxorubicin (HY-15142; Shanghai Qianyuan Biomedical Technology Co., Ltd.).

2. Method

2.1 Cultivation of different cell lines

9 liver cancer cell lines HCC3787C1, HCC4172A2, HCC4477B2, HCC4484B1, HCC5850A1, HCC5864D1, HCC5953B1, HCC633A5 and HCC061A2 were cultured in DMEM/F12 medium containing 10% fetal bovine serum and placed in a 5% CO ₂ incubator at 37 °C. Medium temperature education. The cells were all grown in an adherent state, and the growth was observed under an inverted microscope, and subculture was performed when the cell confluence rate reached 80%-90%. The proportion and quantity of passages are subject to the experimental needs. The ratio of subculture of this liver cancer cell line is generally 1:2～1:3.

2.2 Proliferation inhibition of different liver cancer tumor cell lines

Cell test: 9 liver cancer cell lines HCC3787C1, HCC4172A2, HCC4477B2, HCC4484B1, HCC5850A1, HCC5864D1, HCC5953B1, HCC633A5 and HCC061A2 in logarithmic growth phase, 1~3×10 ³ /well (predetermined for each liver cancer cell in the experiment) The optimal seeding density of the strain was inoculated in a 96-well culture plate, and after incubation at 37 ° C for 4 hours in a humidified incubator containing 5% CO ₂ , 10 μL of the compound of the present invention was added to each well to test 9 drug concentrations. The final drug concentration was 100, 31.6, 10, 3.1, 1, 0.3, 0.1, 0.03 and 0.01 μM, respectively, and the QC reference compound Doxorubicin was added simultaneously in the test of each liver cancer cell line, and the final drug concentration was 10 in turn. , 3.16, 1, 0.31, 0.1, 0.03, 0.01, 0.003, and 0.001 μM. In addition, a positive control group (100% inhibition) and a negative control group (0% inhibition) were set at the same time. The drug group was repeated for 2 wells per concentration, and the positive control group and the negative control group were repeated for 6 wells. After the culture was continued for 6 days in the incubator, follow-up AlamarBlue test operation;

AlamarBlue test procedure: Incubate with 10 μL of AlamarBlue reagent per well for 1-4 h, shake for 1-2 min, MD microplate reader EX: 560 nm, EM: 590 nm wavelength to measure the fluorescence value, record the results, calculate the cell inhibition rate of the compound of the invention ( %)=(A _{0% inhibition-} A _{administration} )/(A _{0% inhibition-} A _{100% inhibition} )×100%, and then using the method of nonlinear regression using GraphPad Prism 5.0 or MATILAB software (usually using four-parameter fitting The curve equation was plotted to obtain a drug dose response curve, thereby obtaining an IC ₅₀ value of the compound of the present invention acting on 9 liver cancer cell lines.

3. Results

3.1 The IC ₅₀ values of the compound 10 of the present invention against 9 liver cancer cell lines (HCC3787C1, HCC4172A2, HCC4477B2, HCC4484B1, HCC5850A1, HCC5864D1, HCC5953B1, HCC633A5 and HCC061A2) are shown in Table 4.

Table 4 IC ₅₀ values of compound 10 of the present invention against 9 liver cancer cell lines

cell IC ₅₀ (μM) HCC3787C1 10.31 HCC4172A2 10.54 HCC4477B2 3.25 HCC4484B1 5.27

HCC5850A1 4.51 HCC5864D1 9.46 HCC5953B1 6.46 HCC633A5 4.44 HCC061A2 6.85

As can be seen from Table 4, the compound 10 of the present invention has a good inhibitory activity against 7 liver cancer cell lines (HCC4477B2, HCC4484B1, HCC5850A1, HCC5864D1, HCC5953B1, HCC633A5 and HCC061A2) (IC ₅₀ <10 μM), and 2 liver cancer cells. The inhibitory activity of HCC4172A2 and HCC3787C1 is about ₅₀ μM. Therefore, the compound of the present invention has a good inhibitory effect on proliferation of liver cancer cells and has a good response rate in liver cancer cells.

Experimental Example 3 Antitumor effect of Compound 10 of the present invention on H22 sarcoma mice

Material

1.1 Experimental animals and tumor strains:

Healthy male ICR mice, weighing 18-22 g, were purchased from the Experimental Animal Center of Weitonglihua Experimental Animal Technology Co., Ltd. (Qualification No. SCXK (Beijing) 2011-0004); Mouse sarcoma H22 was donated by Xi'an Jiaotong University. Tumor-bearing H22 mice were passaged every 5 days.

1.2 experimental drugs:

The compound 10 of the present invention was subjected to liquid chromatography (HPLC) analysis for purity identification, and the purity was ≥98%, which met the experimental requirements. The powder was sealed and stored at 4 °C.

Cyclophosphamide for injection: Shanxi Pude Pharmaceutical Co., Ltd. (production batch number: 04140202)

2. Method

2.1 Establishment of model animals

The H22 mice that had been inoculated for 7 days were sacrificed by cervical dislocation. The ascites was taken under aseptic conditions, washed twice with RPMI1640 medium, and then made into 2×10 ⁷ /ml cell suspension with sterile physiological saline. H22 cell suspension The mice were inoculated subcutaneously in the right flank of the mice, and each of them was 0.1 ml, and 72 cells were inoculated to prepare a solid tumor model.

2.2 Experimental grouping

Seventy-two mice were randomly divided into 6 groups according to body weight, namely normal control group, positive drug (cyclophosphamide) group, model group, low-dose group, medium-dose group and high-dose group of the present invention; 12 rats in each group.

2.3 method of administration

The compound 10 of the present invention is prepared by using soybean oil for injection, the low dose group is 10 mg/kg, the middle dose group is 30 mg/kg, the high dose group is 90 mg/kg, and the negative control group is injected with the same volume of injection soybean oil. The positive control group was injected with cyclophosphamide injection 30 mg/kg, and intraperitoneally injected every other day for 12 days. During the period, the general activity, fur, feces, body weight, etc. of the mice were observed daily. 24 hours after the last administration, mice subcutaneously inoculated with H22 were sacrificed by cervical dislocation, and tumors, liver, spleen and kidney were taken.

2.4 Calculation of organ index

After the end of all the solid tumor groups, the body weight of the mice was weighed and then sacrificed, and the weights of the spleen, kidney and liver were weighed using an electronic balance. The spleen index is the spleen weight (mg) of each group of mice/body weight (g), liver index is liver weight (100 g) / mouse body weight (g), kidney index is kidney weight (mg) / mouse Weight (g).

2.5 Calculation of tumor inhibition rate

The solid tumor-negative control group was weighed the next day, and the mice were sacrificed (the blood was collected from the eyeball, and the serum was collected for subsequent experiments). The mice were sacrificed by cervical dislocation, the tumor tissues were dissected, and the electronic balance was weighed to calculate the tumor inhibition rate.

2.6 statistical processing

All data were statistically analyzed using the SPSS 11.0 software package, and the comparison of the count data was performed using the х ² test. P < 0.05 was statistically significant.

3. Results

3.1 In vivo anticancer activity of the compound 10 of the invention

The results are shown in Table 5.

Table 5 Inhibition of Compound 22 of the present invention on H22 tumor-bearing mice (x±s)

Group Weight change (g) Tumor weight (g) Tumor inhibition rate (%) Normal control group 4.27±1.19 —— —— Model group 6.48±0.84* 1.28±0.42 ——

Cyclophosphamide group 4.37±1.26 0.49±0.29* 61.72% Low dose group of the composition of the invention 4.21±1.08 0.69±0.30 46.09% Dosage group in the composition of the invention 4.52±1.78 0.51±0.26* 60.16% High dose group of the composition of the invention 5.49±0.97 0.63±0.35* 50.78%

Note: *Compared with normal control group: *P<0.05, **P<0.01

As shown in Table 5, the compound 10 of the present invention had no effect on the weight gain of H22 sarcoma mice, and the tumor inhibition effect was obvious. The tumor growth inhibition rates of the low, medium and high dose groups on H22 sarcoma mice were 46.09% and 60.16%, respectively. And 50.78%, and the high-dose group has an anti-tumor effect comparable to the positive drug cyclophosphamide.

3.2 Changes of liver, spleen and kidney index of H22 mice in each group

The results are shown in Table 6.

Table 6 Effect of Compound 10 of the present invention on liver, spleen and kidney index of H22 mice (x±s)

Note: *Compared with the blank control group: *P<0.05, **P<0.01

As shown in Table 6, compared with the normal control group, the liver and spleen index of the model group increased, the spleen index changed significantly (P<0.05), and the kidney index decreased. After treatment, cyclophosphamide and the compound of the present invention can reduce the liver and spleen index of H22 sarcoma mice to near normal levels.

4 Conclusion

4.1 Compound 10 of the present invention significantly inhibited the growth of H22 mouse sarcoma.

4.2 The compound 10 of the present invention can modulate the liver and spleen index of H22 sarcoma mice.

Experimental Example 4 Acute Toxicity Test of Compound 10 of the Present Invention

Material

1.1 experimental animals

Healthy ICR mice, 12 males and 12 females, weighing 18-22 g, purchased from the Laboratory Animal Center of Vitallihua Laboratory Animal Technology Co., Ltd. (Qualification No. SCXK (Beijing) 2006-0009)

1.2 experimental drugs

The compound 10 of the present invention was subjected to liquid chromatography (HPLC) analysis for purity identification, and the purity was ≥98%, which met the experimental requirements. The powder was sealed and stored at 4 °C. 450 mg of this compound was dissolved in an appropriate amount of ethyl acetate, and then ultrasonically suspended with 5.0 ml of soybean oil for injection, and the solvent ethyl acetate was recovered to be tasteless. Now available.

2. Method

Forty mice were randomly divided into 4 groups according to their body weight, with 10 rats in each group. The experimental group was administered intraperitoneally with the mice, and the control group was given the same amount of soybean oil for injection. Before the administration, the mice were fasted to avoid overnight water (12 hours in a row), and the administration was started at 9:00 am on the day of administration (900 mg/kg), and the rats were fed for 6 hours after continuous observation for 14 days.

3. Results

After intraperitoneal administration (900 mg/kg), individual mice showed mild writhing, and the symptoms disappeared within 1 hour. No death occurred within 12 hours after administration, and no obvious side effects were observed in the blank group. After 14 days, the mice in each group were in good condition and no death was observed. After the cervical spine was dislocated, the organs such as heart, liver, spleen, lung, kidney, adrenal gland and thyroid were observed. No abnormal changes were observed. Acute toxicity test of compound 26 mice by intraperitoneal injection showed that the drug had an LD ₅₀ >900 mg/kg.

4 Conclusion

The compound of the present invention 10 has a high safety by intraperitoneal injection of LD ₅₀ >900 mg/kg into normal mice.

detailed description

Example 1 Preparation of 2-bromomethyl-3,5,6-trimethylpyrazine (Intermediate Compound 1)

According to "Optimization of the Synthesis Process of Intermediate 2-Bromomethyl-3,5,6-Trimethylpyrazine". Xu Xuan, Wang Penglong, Han Qiujun, et al. Anhui Medicine, 2013, 17(9): 1467– Method prepared by 1470". Weigh 20.00g (0.15mol) of anhydrous ligustrazine, 23.54g NBS N- bromosuccinimide (0.15 mol, finely ground before use) was placed in 250mL 3-necked flask, 100mL CCl ₄ as the reaction solvent, 4 Irradiation with an 85W incandescent lamp and reflux reaction at 95 ° C for 1 h. TLC [V (petroleum ether): V (acetone) = 3:1] The reaction was almost complete; after cooling, it was filtered, and the filtrate was collected, and the solvent was evaporated under reduced pressure to give a purple-yellow viscous liquid (content: 60%). HRMS (ESI) m/z: 216.00 135 [M+H] ⁺ , calcd. for C ₈ H ₁₁ BrN ₂ 216.00851.

Example 2 Preparation of 3,5,6-trimethylpyrazine-2-carboxylic acid (Intermediate Compound 2)

According to "Synthesis of a Novel Ligustrazine Derivative and Its Anticancer Activity", Northwest Pharmaceutical Journal, 29(1): 58-64". 10.0 g (73.53 mmol) of ligustrazine was suspended in 100.0 mL of distilled water, and 11.62 g (73.53 mmol) of potassium permanganate was weighed and added in 3 portions, and stirred at 37 ° C for 24 hours with magnetic stirring. The mixture was cooled, filtered, and the filtrate was evaporated to EtOAc EtOAc EtOAc EtOAc EtOAc Dry, acetone was recrystallized to give a white solid, yield: 47.8%, mp: 162 - 163.

Preparation of Compound 1 of Example 3

Weigh 2.19 mmol of 2-bromomethyl-3,5,6-trimethylpyrazine and 2.19 mmol of deoxycholic acid in a 50 ml round bottom flask, add 25 ml of DMF, and add 3 mmol of potassium carbonate after the mixture is dissolved. , 85 deg.] C was stirred for 2h, TLC the reaction was monitored starting material disappeared, the reaction was stopped, the reaction solution was added to the dispersion amounts of saturated NaCl, extracted twice with 300ml portions of ethyl acetate, ethyl acetate layer was dried over anhydrous Na ₂ SO _4, and concentrated under reduced pressure The residue was dissolved in 3 ml of chloroform, and 2.8 g of silica gel was added to dryness under reduced pressure. The eluent was eluted with petroleum ether: acetone = 8:1 to give a white solid. Mp: 83.6 - 84.3 ° C, yield 58.4%. HRMS (ESI) m/z: 527.5201 [M+H] ⁺ , calcd. for C ₃₂ H ₅₁ N ₂ O ₄ 527.3849.

Preparation of Compound 2 of Example 4

Weighed 1.2 mmol of compound 1 and 1.8 mmol of ligustrazine acid were placed in a 50 ml round bottom flask, and 25 ml of CH ₂ Cl ₂ was added. After the mixture was dissolved, EDCI 1.8 mmol and DMAP 0.18 mmol were added, respectively, and stirred at room temperature for 12 h. The reaction materials were monitored by TLC. The reaction disappeared, the reaction was stopped, and the mixture was concentrated under reduced pressure. EtOAc (EtOAc) White amorphous solid, mp: 60.5 - 61.2 ° C, yield 52.7%. ¹ H-NMR (CDCl ₃ ) (ppm): 0.67 (s, 3H, H-18), 0.96 (s, 3H, H-19), 0.97 (d, J = 7.0 Hz, 3H, H-21), 3.99 (m, 1H, H-12), 5.04 (m, 1H, H-3), 5.19 (t, J = 12.5 Hz, 2H, CH ₂ -2'), 2.72 (s, 3H, CH ₃ -6"), 2.57 (s, 3H, CH ₃ -5"), 2.56 (s, 3H, CH ₃ -3"), 2.54 (s, 3H, CH ₃ -6 '), 2.52 (s, 3H, CH ₃ -5'), 2.51 (s, 3H, CH ₃ -3'), 1.00 - 2.50 (27H, methyl-and methylene-of steroid structure). ¹³ C-NMR (CDCl3) (ppm): 35.0 (C1), 26.5 (C2), 76.2 (C3), 32.1 (C4), 42.0 (C5), 27.0 (C6), 26.0 (C7), 36.0 (C8), 33.7 (C9), 34.2 (C10), 28.7 (C11), 73.1 (C12), 46.5 (C13), 48.2 (C14), 23.6 (C15), 27.4 (C16), 47.3 (C17), 12.7 (C18), 23.1 (C19), 35.1 (C20), 17.3 (C21), 31.1 (C22), 30.9 (C23), 173.8 (C24); pyrazine ring: 64.9 (2'-CH ₂ ), 151.2 (C2'), 144.9 ( C3'), 148.9 (C5'), 149.1 (C6'), 166.8 (2"-C=O), 154.5 (C2"), 140.4 (C3"), 149.3 (C5"), 150.5 (C6"), 21.6 (6'-CH ₃ ), 21.4 (5'-CH ₃ ), 20.4 (3'-CH ₃ ), 22.5 (6"-CH ₃ ), 22.1 (5"-CH ₃ ), 21.6 (3"- CH ₃ ). HRMS (ESI) m/z: 675.44635 [M+H] ⁺ , calcd. for C ₄₀ H ₅₈ N ₄ O ₅ 674.44072.

Preparation of intermediate benzyl deoxycholate: Weigh 5.0 mmol of benzyl bromide and 5.0 mmol of deoxycholic acid into a 50 ml round bottom flask, add 25 ml of DMF. After the mixture is dissolved, add 5 mmol of potassium carbonate and stir at 85 ° C for 2 h. , 85 deg.] C was stirred for 2h, TLC the reaction was monitored starting material disappeared, the reaction was stopped, the reaction solution was added a large amount of saturated NaCl solution dispersion, 400ml and extracted twice with ethyl acetate, ethyl acetate layer was dried over anhydrous Na ₂ SO _4, and concentrated under reduced pressure The residue was dissolved in 5 ml of chloroform, and 3.8 g of silica gel was added to dryness under reduced pressure. The eluent was eluted with petroleum ether: acetone = 5:1 to give white solid. White powder, mp: 136.5–137.3 ° C, yield 91.8%.

Preparation of Compound 3 of Example 5

Weighed 3.0 mmol of benzyl deoxycholate and 4.0 mmol of tetramethylpyrazine in a 50 ml round bottom flask, and added 30 ml of CH ₂ Cl ₂ . After the mixture was dissolved, EDCI 4.0 mmol and DMAP 0.4 mmol were added, respectively, and stirred at room temperature for 12 h, TLC The reaction raw materials were basically disappeared, the reaction was stopped, the reaction solution was washed twice with 60 ml of a saturated NaCl solution, the CH ₂ Cl ₂ layer was dried over anhydrous Na ₂ SO 4 and concentrated under reduced pressure, and 3.2 g of silica gel was added to dryness under reduced pressure. Petroleum ether: acetone = 7:1 eluted to give a white solid, 0.924 g. White amorphous solid, mp: 47.1 - 48.0 ° C, yield 48.9%. ¹ H-NMR (CDCl ₃ ) (ppm): 0.67 (s, 3H, H-18), 0.97 (s, 3H, H-19), 0.98 (d, J = 7.5 Hz, 3H, H-21), 3.99 (m, 1H, H-12), 5.05 (m, 1H, H-3), 5.14, 5.11 (ea, d, J = 12.5 Hz, 1H, CH ₂ -1'), 7.37 (m, 5H, H-2', 3', 4', 5', 6'), 2.72 (s, 3H, CH ₃ -6"), 2.58 (s, 3H, CH ₃ -5"), 2.57 (s, 3H, CH ₃ -3"), 1.00 - 2.50 (27H, methyl-and methylene-of steroid structure). ¹³ C-NMR (CDCl ₃ ) (ppm): 35.0 (C1), 26.5 (C2), 76.2 (C3), 32.3 (C4), 42.1 (C5), 27.0 (C6), 26.1 (C7), 36.0 (C8), 33.7 (C9), 34.2 (C10), 28.7 (C11), 73.1 (C12), 46.5 (C13), 48.2 (C14), 23.6 (C15), 27.4 (C16), 47.5 (C17), 12.7 (C18), 23.1 (C19), 35.0 (C20), 17.3 (C21), 31.3 (C22), 30.9 (C23), 174.0 (C24), 66.1 (1'-CH ₂ ), 136.1 (C1'), 128.2 (C2', 6'), 128.5 (C3', 5 '), 128.2 (C4'); pyrazine ring: 165.8 (2"-C=O), 153.9 (C2"), 140.5 (C3"), 149.2 (C5"), 150.4 (C6"), 22.5 (6" -CH ₃ ),22.1(5"-CH ₃ ),21.6(3"-CH ₃ ).HRMS(ESI) m/z:653.39187[M+Na] ⁺ ,calcd.for C ₃₉ H ₅₄ N ₂ O ₅ 630.40327.

Preparation of Compound 4 of Example 6

Weighed 1.2 mmol of compound 3 was placed in a 50 ml round bottom flask, and 30 ml of THF was added. After the mixture was dissolved, Pd/C 75 mg was added, and hydrogen was reduced. The mixture was stirred at room temperature for 8 hours. The reaction material was almost disappeared by TLC, the reaction was stopped, and Pd/C was removed by filtration. The reaction mixture was concentrated under reduced pressure. The residue was purified eluting with ₃ ml of CHCl ₃ and then evaporated to dryness. White amorphous solid, mp: 182.8 - 183.5 ° C, yield 58.4%. ¹ H-NMR (CDCl ₃ ) (ppm): 0.69 (s, 3H, H-18), 0.96 (s, 3H, H-19), 0.98 (d, J = 6.0 Hz, 3H, H-21), 4.01 (m, 1H, H-12), 5.04 (m, 1H, H-3), 2.73 (s, 3H, CH ₃ -6'), 2.57 (s, 3H, CH ₃ -5'), 2.57 (s, 3H, CH ₃ -3'), 1.00 - 2.50 (28H, methyl-and methylene-of steroid structure). ¹³ C-NMR (CDCl ₃ ) (ppm): 35.0 (C1), 26.5 (C2), 76.2 (C3), 32.1 (C4), 42.1 (C5), 27.0 (C6), 26.0 (C7), 36.0 (C8), 33.7 (C9), 34.2 (C10), 28.7 (C11), 73.2 (C12), 46.5 (C13), 48.2 (C14), 23.6 (C15), 27.5 (C16), 47.3 (C17), 12.7 (C18), 23.1 (C19), 35.1 (C20), 17.3 (C21), 31.0 (C22), 30.7 (C23), 179.0 (C24). pyrazine ring: 165.6 (2'-C=O), 154.1 (C2'), 140.3 (C3'), 149.3 (C5'), 150.6 (C6'), 22.4 (6'-CH ₃ ), 22.0 (5'-CH ₃ ), 21.4 (3'-CH ₃ ). HRMS (ESI) m/z: 541.36188 [M+ H] ⁺ ,calcd.for C ₃₂ H ₄₈ N ₂ O ₅ 540.35632.

Preparation of Compound 5 of Example 7

Weigh 2.19 mmol of 2-bromomethyl-3,5,6-trimethylpyrazine and 2.19 mmol of cholic acid in a 50 ml round bottom flask, add 25 ml of DMF, and add 3 mmol of potassium carbonate after the mixture is dissolved. After stirring at °C for 2 h, the reaction material was almost disappeared by TLC. The reaction was stopped. The reaction mixture was dissolved in a large amount of saturated NaCl solution. The mixture was extracted twice with 300 ml of ethyl acetate. The ethyl acetate layer was dried over anhydrous Na.sub.2SO. After dissolving, 2.8 g of silica gel was added to dryness under reduced pressure, and the eluent was eluted with petroleum ether: acetone = 8:1 to give a white solid. Mp: 95.4 - 96.3 ° C, yield 55.4%. HRMS (ESI) m/z: 543.6199 [M+H] ⁺ , calcd. for C ₃₂ H ₅₁ N ₂ O ₅ 543.3798.

Preparation of Compound 8 of Example 8

Weighed 1.2 mmol of compound 5 and 1.8 mmol of ligustrazine in a 50 ml round bottom flask, and added 25 ml of CH ₂ Cl ₂ . After the mixture was dissolved, EDCI 1.8 mmol and DMAP 0.18 mmol were added, respectively, and stirred at room temperature for 12 h. The reaction materials were monitored by TLC. After disappearing, the reaction was stopped, concentrated under reduced pressure, and the mixture was evaporated to dryness with diethyl ether (2.2 g), and the eluent was eluted with petroleum ether: acetone = 7:1 to give a white solid. White amorphous solid, mp: 85.7-86.5 ℃ , yield 47.8% 1 H-NMR (CDCl3) (ppm):. 0.68 (s, 3H, H-18), 0.93 (s, 3H, H-19), 0.96 ( d, J = 5.5 Hz, 3H, H-21), 4.90 (m, 1H, H-3), 3.87 (m, 1H, H-7), 3.98 (m, 1H, H-12), 5.19 (brs , 2H, CH ₂ -2'), 2.71 (s, 3H, CH ₃ -6"), 2.56 (s, 3H, CH ₃ -5"), 2.56 (s, 3H, CH ₃ -3"), 2.54 (s, 3H, CH ₃ -6'), 2.52 (s, 3H, CH ₃ -5'), 2.51 (s, 3H, CH ₃ -3'), 1.00 - 2.50 (26H, methyl-and methylene-of Steroid structure. ¹³ C-NMR (CDCl ₃ ) (ppm): 35.1 (C1), 26.7 (C2), 76.2 (C3), 34.9 (C4), 41.3 (C5), 34.8 (C6), 68.2 (C7) , 39.5 (C8), 26.6 (C9), 34.4 (C10), 28.4 (C11), 72.9 (C12), 46.5 (C13), 42.0 (C14), 23.1 (C15), 27.4 (C16), 47.1 (C17) , 12.5 (C18), 22.5 (C19), 35.2 (C20), 17.3 (C21), 31.0 (C22), 30.8 (C23), 173.9 (C24); pyrazine ring: 64.9 (2'-CH ₂ ), 151.2 ( C2'), 144.9 (C3'), 148.9 (C5'), 149.1 (C6'), 165.8 (2"-C=O), 153.9 (C2"), 140.5 (C3"), 149.2 (C5"), 150.4 (C6"), 21.5 (6'-CH ₃ ), 21.4 (5'-CH ₃ ), 20.4 (3'-CH ₃ ), 22.6 (6"-CH ₃ ), 22.1 (5"-CH ₃ ) , 21.6 (3"-CH ₃ ). HRMS (ESI) m/z: 691.44092 [M+H] ⁺ , calcd. for C ₄₀ H ₅₈ N ₄ O ₆ 690.43564.

Preparation of intermediate benzyl cholate: Weigh 5.0 mmol of benzyl bromide and 5.0 mmol of cholic acid in a 50 ml round bottom flask, add 25 ml of DMF. After the mixture is dissolved, add 5 mmol of potassium carbonate, stir at 85 ° C for 2 h, stir at 85 ° C. 2h, TLC monitoring the reaction material disappeared, the reaction was stopped, the reaction solution was added to a large amount of saturated NaCl solution, and 400 ml of ethyl acetate was extracted twice. The ethyl acetate layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The chloroform was dissolved, and 3.8 g of silica gel was added to dryness under reduced pressure. The eluent was eluted with petroleum ether: acetone = 5:1 to give a white solid. White powder, mp: 177.8–178.6°C, yield 90.1%.

Preparation of Compound 7 of Example 9

The intermediate benzyl cholate 3.0 mmol and the tetramethylpyrazine 4.0 mmol were weighed into a 50 ml round bottom flask, and 30 ml of CH ₂ Cl ₂ was added. After the mixture was dissolved, EDCI 4.0 mmol and DMAP 0.4 mmol were added, respectively, and stirred at room temperature for 12 h, TLC The reaction raw materials were basically disappeared, the reaction was stopped, the reaction solution was washed twice with 60 ml of a saturated NaCl solution, and the CH ₂ Cl ₂ layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The de-agent was petroleum ether: acetone = 7:1 eluted to give a white solid, 0.928 g. White amorphous solid, mp: 169.0 - 169.6 ° C, yield 47.9%. ¹ H-NMR (CDCl ₃ ) (ppm): 0.69 (s, 3H, H-18), 0.95 (s, 3H, H-19), 0.98 (d, J = 6.0 Hz, 3H, H-21), 4.91 (m, 1H, H-3), 3.88 (m, 1H, H-7), 4.00 (m, 1H, H-12), 5.15, 5.11 (ea,d,J=12.5 Hz, 1H, CH ₂ -1'), 7.37 (m, 5H, H-2', 3', 4', 5', 6'), 2.72 (s, 3H, CH ₃ -6"), 2.57 (s, 3H, CH ₃ -5"), 2.57 (s, 3H, CH ₃ -3"), 1.00 - 2.50 (26H, methyl-and methylene-of steroid structure). ¹³ C-NMR (CDCl3) (ppm): 35.1 (C1), 26.8 (C2), 76.2 (C3), 34.9 (C4), 41.3 (C5), 34.8 (C6), 68.2 (C7), 39.6 (C8), 26.7 (C9), 34.4 (C10), 28.4 (C11), 72.9 (C12), 46.5 (C13), 42.0 (C14), 23.1 (C15), 27.4 (C16), 47.2 (C17), 12.5 (C18), 22.5 (C19), 35.1 (C20), 17.3 (C21), 31.3 (C22), 30.8 (C23), 174.0 (C24), 66.1 (1'-CH2), 136.1 (C1'), 128.2 (C2', 6 '), 128.5 (C3', 5'), 128.2 (C4'); pyrazine ring: 165.8 (2"-C=O), 153.9 (C2"), 140.6 (C3"), 149.3 (C5"), 150.4 (C6"), 22.6 (6"-CH ₃ ), 22.1 (5"-CH ₃ ), 21.6 (3"-CH ₃ ). HRMS (ESI) m/z: 647.40668 [M+H] ⁺ , calcd. For C ₃₉ H ₅₄ N ₂ O ₆ 646.39819.

Preparation of Compound 8 of Example 10

Weighed compound 7 1.2mmol was placed in a 50ml round bottom flask, 30ml THF was added, after the mixture was dissolved, Pd/C 78mg was added, hydrogen was reduced, and stirred at room temperature for 10h. The reaction material disappeared by TLC, the reaction was stopped, and Pd/C was removed by filtration. The reaction mixture was concentrated under reduced pressure. The residue was purified eluting with ₃ ml of CHCl ₃ and then evaporated to dryness. White amorphous solid, mp: 132.6 - 133.5 ° C, yield 88.4%. 1H-NMR (CDCl ₃ ) (ppm): 0.70 (s, 3H, H-18), 0.93 (s, 3H, H-19), 0.98 ( d, J = 5.5 Hz, 3H, H-21), 4.89 (m, 1H, H-3), 3.88 (m, 1H, H-7), 4.00 (m, 1H, H-12), 2.73 (s , 3H, CH ₃ -6'), 2.57 (s, 3H, CH ₃ -3 ', 5'), 1.00 - 2.50 (27H, methyl-and methylene-of steroid structure). 13C-NMR (CDCl ₃ ) ( Ppm): 35.1 (C1), 26.7 (C2), 76.3 (C3), 34.9 (C4), 41.3 (C5), 34.8 (C6), 68.3 (C7), 39.5 (C8), 26.6 (C9), 34.4 ( C10), 28.3 (C11), 73.0 (C12), 46.5 (C13), 41.9 (C14), 23.2 (C15), 27.5 (C16), 47.0 (C17), 12.5 (C18), 22.5 (C19), 35.2 ( C20), 17.3 (C21), 30.9 (C22), 30.7 (C23), 178.3 (C24); pyrazine ring: 165.6 (2'-C=O), 154.0 (C2'), 140.3 (C3'), 149.2 ( C5 '), 150.6 (C6' ), 22.5 (6'-CH 3), 22.0 (5'-CH 3), 21.3 (3'-CH 3) .HRMS (ESI) m / z: 555.34312 [MH] - ,calcd.for C ₃₂ H ₄₈ N ₂ O ₆ 556.35124.

Preparation of Compound 9 of Example 11

Weigh 2.19 mmol of 2-bromomethyl-3,5,6-trimethylpyrazine and 2.19 mmol of hyodeoxycholic acid in a 50 ml round bottom flask, add 25 ml of DMF, and add 3 mmol of potassium carbonate after the mixture is dissolved. , 85 deg.] C was stirred for 2h, TLC the reaction was monitored starting material disappeared, the reaction was stopped, the reaction solution was added to the dispersion amounts of saturated NaCl, extracted twice with 300ml portions of ethyl acetate, ethyl acetate layer was dried over anhydrous Na ₂ SO _4, and concentrated under reduced pressure The residue was dissolved in 3 ml of chloroform, and 2.8 g of silica gel was added to dryness under reduced pressure. The eluent was eluted with petroleum ether: acetone = 8:1 to give a white solid. Mp: 65.3 - 66.1 ° C, yield 51.6%. 1H-NMR (CDCl ₃ ) (ppm): 0.62 (s, 3H, H-18), 0.90 (s, 3H, H-19), 0.91 (d, J = 6.0 Hz, 3H, H-21), 3.61 (m, 1H, H-3), 4.05 (m, 1H, H-6), 5.19 (t, J = 12.5 Hz, 2H, CH ₂ -2'), 2.53 (s, 3H, CH ₃ -6'), 2.51 (s, 3H, CH ₃ -5'), 2.51 (s, 3H, CH ₃ -3'), 1.00 - 2.50 (28H, methyl-and methylene- 13C-NMR (CDCl ₃ ) (ppm): 35.6 (C1), 29.2 (C2), 71.5 (C3), 28.1 (C4), 48.5 (C5), 68.0 (C6), 35.0 (C7) , 34.8 (C8), 39.8 (C9), 35.9 (C10), 20.7 (C11), 39.9 (C12), 42.8 (C13), 56.1 (C14), 24.2 (C15), 30.2 (C16), 55.9 (C17) , 12.0 (C18), 23.5 (C19), 35.3 (C20), 18.2 (C21), 31.1 (C22), 30.9 (C23), 173.9 (C24); pyrazine ring: 64.9 (2'-CH ₂ ), 151.2 ( C2 '), 144.9 (C3' ), 149.0 (C5 '), 149.0 (C6'), 21.6 (6'-CH 3), 21.4 (5'-CH 3), 20.5 (3'-CH 3) .HRMS (ESI) m/z: 527.38214 [M+H] ⁺ , calcd. for C ₃₂ H ₅₀ N ₂ O ₄ 526.37706.

Preparation of Compound 10 of Example 12

Weigh about 1.2 mmol of compound 9 and 1.8 mmol of ligustrazine acid in a 50 ml round bottom flask, and add 25 ml of CH ₂ Cl ₂ . After the mixture was dissolved, add EDCI 1.8 mmol and DMAP 0.18 mmol, respectively, stir at room temperature for 12 h, and monitor the reaction materials by TLC. Basically disappeared, the reaction was stopped, concentrated under reduced pressure, and 2.2 g of silica gel was added to dryness under reduced pressure. The eluent was eluted with petroleum ether: acetone = 8:1, white semi-solid 0.181 gmp: 72.3 - 73.1 ° C, yield 18.4% .1H-NMR (CDCl ₃ ) (ppm): 0.67 (s, 3H, H-18), 1.01 (s, 3H, H-19), 0.92 (d, J = 6.5 Hz, 3H, H-21), 5.04 (m, 1H, H-3), 5.51 (m, 1H, H-6), 5.20 (t, J = 12.5 Hz, 2H, CH2-2'), 2.48 - 2.71 (s, each, 3H, 9 _{× CH 3 -pyrazine ring), 1.00-2.50} (25H, methyl-and methylene-of steroid structure) .13C-NMR (CDCl 3) (ppm): 35.3 (C1), 26.5 (C2), 75.5 (C3), 26.4 (C4), 45.6 (C5), 72.9 (C6), 31.3 (C7), 34.8 (C8), 39.9 (C9), 36.3 (C10), 20.7 (C11), 39.9 (C12), 42.9 (C13), 56.1 (C14), 24.1 (C15), 28.1 (C16), 55.9 (C17), 12.0 (C18), 23.3 (C19), 35.1 (C20), 18.2 (C21), 31.1 (C22), 30.9 (C23), 173.8(C24); pyrazine ring: 65.0 (2'-CH2), 151.2 (C2'), 144.9 (C3'), 149.0 (C5'), 149.0 (C6'), 165.5 (2"-C=O), 154.1(C2"), 140.4(C3"), 1 49.3(C5"), 150.2(C6"), 165.7(2"'-C=O), 154.0(C2"'), 140.3(C3"'), 149.2(C5"'), 150.5(C6"), 21.6(6'-CH ₃₎ , 21.5(5'-CH ₃ ), 20.5(3'-CH ₃ ), 22.6(6"-CH ₃ ), 22.1(5"-CH ₃ ), 21.7(3"- _{CH 3), 22.5 (6 "} '- CH 3), 22.1 (5"' - CH 3), 21.6 (3 "'- CH 3) .HRMS (ESI) m / z: 823.51044 [m + H] +, Calcd.for C ₄₈ H ₆₆ N ₆ O ₆ 822.50438.

Preparation of Compound 11 of Example 13

This structure was isolated in the preparation of compound 10 as a white semi-solid 0.149 g. Semisolid, yield 22.1% .1H-NMR (CDCl 3) (ppm): 0.66 (s, 3H, H-18), 1.04 (s, 3H, H-19), 0.92 (d, J = 6.5Hz, 3H, H-21), 3.61 (m, 1H, H-3), 5.47 (m, 1H, H-6), 5.20 (t, J = 12.5 Hz, 2H, CH ₂ -2'), 2.71 (s, 3H) , CH ₃ -6"), 2.56 (brs, 6H, CH ₃ -3", 5"), 2.54 (s, 3H, CH ₃ -6'), 2.52 (s, 3H, CH ₃ -5'), 2.52 (s, 3H, CH ₃ -3'), 1.00 - 2.50 (27H, methyl-and methylene-of steroid structure). 13C-NMR (CDCl ₃ ) (ppm): 35.4 (C1), 30.2 (C2), 71.2 (C3), 28.1 (C4), 45.5 (C5), 73.3 (C6), 31.3 (C7), 34.8 (C8), 39.9 (C9), 36.2 (C10), 20.7 (C11), 39.9 (C12), 42.9 (C13), 56.1 (C14), 24.1 (C15), 30.3 (C16), 55.9 (C17), 12.0 (C18), 23.3 (C19), 35.3 (C20), 18.2 (C21), 31.1 (C22), 30.9 (C23), 173.8 (C24); pyrazine ring: 64.9 (2'-CH2), 151.2 (C2'), 144.9 (C3'), 149.0 (C5'), 149.0 (C6'), 165.7 (2"- C = O), 154.0 (C2 "), 140.4 (C3"), 149.3 (C5 "), 150.2 (C6"), 21.6 (6'-CH 3), 21.4 (5'-CH 3), 20.5 (3 '-CH ₃ ), 22.5(6"-CH ₃ ), 22.1(5"-CH ₃ ), 21.6(3"-CH ₃ ).HRMS(ESI)m/z:675.44873[M+H] ⁺ ,calcd .for C ₄₀ H ₅₈ N ₄ O ₅ 674.44072.

Preparation of benzyl hyodeoxycholate: 5.0 mmol of benzyl bromide and 5.0 mmol of hyodeoxycholic acid were placed in a 50 ml round bottom flask, and 25 ml of DMF was added. After the mixture was dissolved, 5 mmol of potassium carbonate was added, and the mixture was stirred at 85 ° C for 2 h, stirring at 85 ° C. 2h, TLC monitoring the reaction material disappeared, the reaction was stopped, the reaction solution was added to a large amount of saturated NaCl solution, and 400 ml of ethyl acetate was extracted twice. The ethyl acetate layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The chloroform was dissolved, and 3.8 g of silica gel was added to dryness under reduced pressure. The eluent was eluted with petroleum ether: acetone = 5:1 to give a white solid. White powder, mp: 132.4–133.3°C, yield 92.7%.

Preparation of Compound 12 of Example 14

Weighed 3.0 mmol of benzyl hyodeoxycholate and 4.0 mmol of tetramethylpyrazine in a 50 ml round bottom flask, and added 30 ml of CH ₂ Cl ₂ . After the mixture was dissolved, EDCI 4.0 mmol and DMAP 0.4 mmol were added, respectively, and stirred at room temperature for 12 h, TLC The reaction raw materials were basically disappeared, the reaction was stopped, the reaction solution was washed twice with 60 ml of a saturated NaCl solution, and the CH ₂ Cl ₂ layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The de-agent was petroleum ether: acetone = 7:1 eluted to give a white solid, 0.391 g. Mp: 55.3 - 56.1 ° C, yield 20.7%. 1H-NMR (CDCl ₃ ) (ppm): 0.66 (s, 3H, H-18), 1.04 (s, 3H, H-19), 0.92 (d, J = 6.5 Hz, 3H, H-21), 3.64 (m, 1H, H-3), 5.48 (m, 1H, H-6), 5.14, 5.11 (ea, d, J = 12.5 Hz, 1H, CH ₂ - 1'), 7.37 (m, 5H, H-2', 3', 4', 5', 6'), 2.71 (s, 3H, CH3-6"), 2.57 (brs, 6H, CH ₃ -3) ", 5"), 1.00 - 2.50 (26H, methyl-and methylene-of steroid structure). 13C-NMR (CDCl ₃ ) (ppm): 35.4 (C1), 30.2 (C2), 71.2 (C3), 28.1 ( C4), 45.5 (C5), 73.3 (C6), 31.3 (C7), 34.8 (C8), 39.9 (C9), 36.2 (C10), 20.7 (C11), 39.9 (C12), 42.9 (C13), 56.1 ( C14), 24.2 (C15), 30.2 (C16), 55.9 (C17), 12.0 (C18), 23.4 (C19), 35.3 (C20), 18.2 (C21), 31.0 (C22), 30.9 (C23), 174.1 ( C24), 66.1 (1'-CH ₂ ), 136.1 (C1'), 128.3 (C2', 6'), 128.6 (C3', 5'), 128.2 (C4'); pyrazine ring: 165.7 (2"- C = O), 154.0 (C2 "), 140.4 (C3"), 149.4 (C5 "), 150.2 (C6"), 22.6 (6 "-CH 3), 22.1 (5" -CH 3), 21.7 (3 "-CH ₃ ). HRMS (ESI) m/z: 631.40991 [M+H] ⁺ , calcd. for C ₃₉ H ₅₄ N ₂ O ₅ 630.40327.

Preparation of Compound 13 of Example 15

The structure obtained is isolated in the preparation of compound 12 as a white solid 0.448gmp: 61.3-62.1 ℃, yield 19.2% .1H-NMR (CDCl 3) (ppm): 0.66 (s, 3H, H-18), 1.09 ( s, 3H, H-19), 0.92 (d, J = 6.5 Hz, 3H, H-21), 5.04 (m, 1H, H-3), 5.50 (m, 1H, H-6), 5.14, 5.11 (ea,d,J=12.0Hz,2H,CH ₂ -1'), 7.37(m,5H,H-2',3',4',5',6'),2.55–2.72(s,each , 3H, 6 × CH ₃ -pyrazine ring), 1.00 - 2.50 (26H, methyl-and methylene-of steroid structure). 13C-NMR (CDCl3) (ppm): 35.3 (C1), 26.5 (C2), 75.6 ( C3), 26.4 (C4), 45.6 (C5), 72.9 (C6), 31.3 (C7), 34.8 (C8), 39.8 (C9), 36.3 (C10), 20.7 (C11), 39.9 (C12), 42.9 ( C13), 56.1 (C14), 24.2 (C15), 28.1 (C16), 55.9 (C17), 12.0 (C18), 23.3 (C19), 35.1 (C20), 18.3 (C21), 31.0 (C22), 30.9 ( C23), 174.0 (C24), 66.1 (1'-CH2), 136.1 (C1'), 128.3 (C2', 6'), 128.6 (C3', 5'), 128.2 (C4'); pyrazine ring: 165.7 (2"-C=O), 154.1 (C2"), 140.4 (C3"), 149.3 (C5"), 150.6 (C6"), 165.6 (2"'-C=O), 154.1 (C2"') , 140.3 (C3"'), 149.3 (C5"'), 150.2 (C6"), 22.6 (6"-CH ₃ ), 22.2 (5"-CH ₃ ), 21.7 (3"-CH ₃ ), 22.5 ( 6"'-CH ₃ ),22.1(5"'-CH ₃ ),21.6(3"'-CH ₃ ).HRMS(ESI)m/z:779.47357[M+H] ⁺ ,calcd .for C ₄₇ H ₆₂ N ₄ O ₆ 778.46694.

Preparation of Compound 14 of Example 16

Weigh about 0.3 mmol of compound 12 and place it in a 50 ml round bottom flask. Add 30 ml of THF. After the mixture is dissolved, add Pd/C 40 mg, hydrogen reduction, and stir at room temperature for 10 h. The reaction material is almost disappeared by TLC, the reaction is stopped, and Pd/ is removed by filtration. C, the reaction solution was concentrated under reduced pressure, the residue was re-dissolved with ₃ ml of CHCl ₃ , and then added with 2.2 g of silica gel and evaporated to dryness. The eluent was eluted with petroleum ether: acetone = 5:1 to give white solid 0.147 g mp:39.1– 39.9 ° C, yield 90.6%. 1H-NMR (CDCl ₃ ) (ppm): 0.69 (s, 3H, H-18), 1.05 (s, 3H, H-19), 0.95 (d, J = 6.5 Hz, 3H , H-21), 3.64 (m, 1H, H-3), 5.48 (m, 1H, H-6), 2.72 (s, 3H, CH ₃ -6"), 2.57 (brs, 6H, CH ₃ - 3", 5"), 1.00 - 2.50 (28H, methyl-and methylene-of steroid structure). 13C-NMR (CDCl ₃ ) (ppm): 35.4 (C1), 30.1 (C2), 71.3 (C3), 28.1 (C4), 45.5 (C5), 73.4 (C6), 31.3 (C7), 34.8 (C8), 39.9 (C9), 36.2 (C10), 20.7 (C11), 39.9 (C12), 43.0 (C13), 56.0 (C14), 24.2 (C15), 30.2 (C16), 56.0 (C17), 12.1 (C18), 23.3 (C19), 35.4 (C20), 18.3 (C21), 31.0 (C22), 30.8 (C23), 178.8 (C24); pyrazine ring: 165.6 (2'-C = O), 154.1 (C2 '), 140.3 (C3'), 149.3 (C5 '), 150.4 (C6'), 22.5 (6'-CH 3), 22.1(5'-CH ₃ ), 21.5(3'-CH ₃ ).H RMS (ESI) m / z: 541.36359 [M + H] ⁺ , calcd. for C ₃₂ H ₄₈ N ₂ O ₅ 540.35632.

Preparation of Compound 15 of Example 17

Weigh about 0.3 mmol of compound 13 and place it in a 50 ml round bottom flask. Add 30 ml of THF. After the mixture is dissolved, add Pd/C 40 mg, hydrogen reduction, and stir at room temperature for 10 h. The reaction material is almost disappeared by TLC, the reaction is stopped, and Pd/ is removed by filtration. C, the reaction mixture was concentrated under reduced pressure, the residue was reconstituted with 3mlCHCl _3, was added 2.2g of silica gel and evaporated to dryness under reduced pressure mixed sample, eluent petroleum ether: acetone = 5: 1 as eluent to give a white solid 0.147g. Mp: 88.3 - 89.0 ° C, yield 91.5%. 1H-NMR (CDCl ₃ ) (ppm): 0.70 (s, 3H, H-18), 1.09 (s, 3H, H-19), 0.95 (d, J = 6.5 Hz, 3H, H-21), 5.04 (m, 1H, H-3), 5.51 (m, 1H, H-6), 2.55–2.73 (s, each, 3H, 6×CH3-pyrazine ring), 13C-NMR (CDCl ₃ ) (ppm): 35.3 (C1), 26.4 (C2), 75.6 (C3), 26.4 (C4), 45.6 (C5) ), 73.0 (C6), 31.3 (C7), 34.8 (C8), 39.9 (C9), 36.3 (C10), 20.7 (C11), 40.0 (C12), 43.0 (C13), 56.2 (C14), 24.2 (C15) ), 28.1 (C16), 56.0 (C17), 12.0 (C18), 23.3 (C19), 35.2 (C20), 18.3 (C21), 31.0 (C22), 30.8 (C23), 178.7 (C24); pyrazine ring: 165.6 (2'-C=O), 154.1 (C2'), 140.3 (C3'), 149.3 (C5'), 150.6 (C6'), 165.5 (2"-C=O), 154.1 (C2"), 140.3(C3"), 149.3(C5"), 150.3(C6'), 22.5(6'-CH ₃ ), 22.1(5'-CH ₃ ), 21.5(3'-CH ₃ ), 22.4(6"- CH ₃ ), 22.0 (5"-CH ₃ ), 21.5 (3"-CH ₃ ). HRMS (ESI) m/z:689.42712 [M+H] ⁺ ,calcd.for C ₄₀ H ₅₆ N ₄ O ₆ 688.41999 .

Preparation of Compound 16 of Example 18

Weigh 2.19 mmol of 2-bromomethyl-3,5,6-trimethylpyrazine and 2.19 mmol of ursodeoxycholic acid in a 50 ml round bottom flask, add 25 ml of DMF, and add 3 mmol of carbonic acid after the mixture is dissolved. Potassium, stirring at 85 ° C for 2 h, TLC monitoring the reaction material disappeared, the reaction was stopped, the reaction solution was added to a large amount of saturated NaCl solution dispersion, 300 ml of ethyl acetate was extracted twice, and the ethyl acetate layer was dried over anhydrous Na ₂ SO ₄ After concentration, the residue was dissolved in 3 ml of chloroform, and 2.8 g of silica gel was added and evaporated to dryness. The eluent was eluted with petroleum ether: acetone = 8:1 to give a white solid. Mp: 46.2 - 46.9 ° C, yield 51.7%. 1H-NMR (CDCl ₃ ) (ppm): 0.65 (s, 3H, H-18), 0.94 (s, 3H, H-19), 0.91 (d, J = 6.0Hz, 3H, H-21), 3.58 (brs, 2H, H-3, 7), 5.19 (t, J = 12.5 Hz, 2H, CH ₂ -2'), 2.52 (s, 3H, CH ₃ - 6'), 2.51 (s, 3H, CH ₃ -5'), 2.50 (s, 3H, CH ₃ -3'), 1.00 - 2.50 (28H, methyl-and methylene-of steroid structure). 13C-NMR ( CDCl ₃ ) (ppm): 35.0 (C1), 30.3 (C2), 71.33 (C3), 37.3 (C4), 42.5 (C5), 36.9 (C6), 71.29 (C7), 43.7 (C8), 39.2 (C9) ), 34.1 (C10), 21.2 (C11), 40.1 (C12), 43.8 (C13), 55.8 (C14), 26.9 (C15), 28.6 (C16), 54.9 (C17), 12.1 (C18), 23.4 (C19) ), 35.2 (C20), 18.4 (C21), 31.1 (C22), 31.0 (C23), 173.8 (C24); pyrazine ring: 64.9 (2'-CH ₂ ), 151.2 (C2'), 144.9 (C3') , 149.0 (C5'), 149.0 (C6'), 21.6 (6'-CH ₃ ), 21.4 (5'-CH ₃ ), 20.4 (3'-CH ₃ ). HRMS (ESI) m/z: 527.38373 [ M+H] ⁺ ,calcd.for C ₃₂ H ₅₀ N ₂ O ₄ 526.37706.

Preparation of Compound 17 of Example 19

Weigh about 1.2 mmol of compound 16 and 1.8 mmol of ligustrazine acid in a 50 ml round bottom flask, and add 25 ml of CH ₂ Cl ₂ . After the mixture was dissolved, add EDCI 1.8 mmol and DMAP 0.18 mmol, respectively, stir at room temperature for 12 h, and monitor the reaction materials by TLC. After the disappearance, the reaction was stopped and concentrated under reduced pressure. 2.2 g of silica gel was added and evaporated to dryness. The eluent was eluted with petroleum ether: acetone = 8:1 to give a white solid. White amorphous solid, mp: 59.1 - 59.9 ° C, yield 28.6%. 1H-NMR (CDCl ₃ ) (ppm): 0.68 (s, 3H, H-18), 1.00 (s, 3H, H-19), 0.92 ( d, J = 6.0 Hz, 3H, H-21), 5.00 (m, 1H, H-3), 3.60 (m, 1H, H-7), 5.20 (t, J = 12.5 Hz, 2H, CH ₂ - 2'), 2.72 (s, 3H, CH ₃ -6"), 2.58 (s, 3H, CH ₃ -5"), 2.57 (s, 3H, CH ₃ -3"), 2.54 (s, 3H, CH) ₃ -6 '), 2.52 (s, 3H, CH ₃ -5'), 2.51 (s, 3H, CH ₃ -3'), 1.00 - 2.50 (27H, methyl-and methylene-of steroid structure). 13C- NMR (CDCl ₃ ) (ppm): 34.7 (C1), 26.5 (C2), 75.7 (C3), 33.1 (C4), 42.4 (C5), 36.7 (C6), 71.2 (C7), 43.7 (C8), 39.1 (C9), 34.2 (C10), 21.5 (C11), 40.1 (C12), 43.7 (C13), 55.7 (C14), 26.9 (C15), 28.6 (C16), 54.9 (C17), 12.1 (C18), 23.3 (C19), 35.2 (C20), 18.4 (C21), 31.1 (C22), 31.0 (C23), 173.9 (C24); pyrazine ring: 65.0 (2'-CH2), 151.2 (C2'), 144.9 (C3' ), 149.0 (C5'), 149.0 (C6'), 165.7 (2"-C=O), 154.1 (C2"), 140.3 (C3"), 149.3 (C5"), 150.6 (C6"), 21.5 ( 6'-CH ₃ ), 21.2 (5'-CH ₃ ), 20.5 (3'-CH ₃ ), 22.6 (6"-CH ₃ ), 22.1 (5"-CH ₃ ), 21.7 (3"-CH ₃ HRMS (ESI) m / z: 675.44788 [M + H] ⁺ , calcd. for C ₄₀ H ₅₈ N ₄ O ₅ 674.44072.

Preparation of Compound 18 of Example 20

This structure was isolated in the preparation of compound 17 as a colorless oil of 0.155 g. Yield 15.7%.1H-NMR (CDCl ₃ ) (ppm): 0.68 (s, 3H, H-18), 1.01 (s, 3H, H-19), 0.91 (d, J = 6.5 Hz, 3H, H- 21), 5.02 (m, 1H, H-3), 5.11 (m, 1H, H-7), 5.17 (t, J = 12.5 Hz, 2H, CH ₂ -2'), 2.48 - 2.71 (s, each , 3H, 9 × CH ₃ -pyrazine ring), 1.00 - 2.50 (26H, methyl-and methylene-of steroid structure). 13C-NMR (CDCl ₃ ) (ppm): 34.6 (C1), 26.4 (C2), 75.5 (C3), 32.8 (C4), 40.0 (C5), 32.9 (C6), 75.4 (C7), 42.4 (C8), 39.6 (C9), 34.2 (C10), 21.6 (C11), 39.9 (C12), 43.6 (C13), 55.3 (C14), 25.9 (C15), 28.4 (C16), 55.0 (C17), 12.1 (C18), 23.3 (C19), 35.2 (C20), 18.3 (C21), 31.0 (C22), 30.9 (C23), 173.8 (C24); pyrazine ring: 64.9 (2'-CH ₂ ), 151.2 (C2'), 144.9 (C3'), 148.9 (C5'), 149.0 (C6'), 165.7 (2"- C=O), 154.1 (C2"), 140.2 (C3"), 149.3 (C5"), 150.6 (C6"), 165.7 (2"'-C=O), 153.9 (C2"'), 140.2 (C3) "'), 149.2 (C5"'), 150.7 (C6"), 21.4 (6'-CH ₃ ), 21.3 (5'-CH ₃ ), 20.5 (3'-CH ₃₎ , 22.6 (6"-CH ₃ ), 22.1(5"-CH ₃ ), 21.7(3"-CH ₃ ), 22.6(6"'-CH ₃ ), 22.2(5"'-CH ₃ ), 21.6(3"'-CH ₃ ) .HRMS (ESI) m/z: 823.51196 [M+H] ⁺ , calcd. for C ₄₈ H ₆₆ N ₆ O ₆ 822.50438.

Preparation of benzyl ursodeoxycholate: Weigh 5.0 mmol of benzyl bromide and 5.0 mmol of ursodeoxycholic acid into a 50 ml round bottom flask, add 25 ml of DMF. After the mixture is dissolved, add 5 mmol of potassium carbonate and stir at 85 ° C for 2 h. , 85 deg.] C was stirred for 2h, TLC the reaction was monitored starting material disappeared, the reaction was stopped, the reaction solution was added a large amount of saturated NaCl solution dispersion, 400ml and extracted twice with ethyl acetate, ethyl acetate layer was dried over anhydrous Na ₂ SO _4, and concentrated under reduced pressure The residue was dissolved in 5 ml of chloroform, and 3.8 g of silica gel was added to dryness under reduced pressure. The eluent was eluted with petroleum ether: acetone = 5:1 to give white solid. White powder, mp: 142.5–143.2 ° C, yield 90.5%.

Preparation of Compound 19 of Example 21

Weigh 3.0 mmol of ursodeoxycholic acid and 4.0 mmol of tetramethylpyrazine in a 50 ml round bottom flask, and add 30 ml of CH ₂ Cl ₂ . After the mixture was dissolved, add EDCI 4.0 mmol and DMAP 0.4 mmol, respectively, and stir at room temperature for 12 h. The reaction material was almost disappeared by TLC, the reaction was stopped, the reaction solution was washed twice with 60 ml of saturated NaCl solution, and the CH ₂ Cl ₂ layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The eluent was eluted with petroleum ether: acetone = 7:1 to give a white solid, 0.486 g. White amorphous solid, mp: 58.4 - 59.2 ° C, yield 25.7%. 1H-NMR (CDCl ₃ ) (ppm): 0.68 (s, 3H, H-18), 1.00 (s, 3H, H-19), 0.93 ( d, J = 6.5 Hz, 3H, H-21), 5.00 (m, 1H, H-3), 3.61 (m, 1H, H-7), 5.14, 5.11 (ea, d, J = 12.0 Hz, 1H , CH2-1'), 7.37 (m, 5H, H-2', 3', 4', 5', 6'), 2.73 (s, 3H, CH ₃ -6"), 2.58 (s, 3H, CH ₃ -5"), 2.57 (s, 3H, CH ₃ -3"), 1.00 - 2.50 (27H, methyl-and methylene-of steroid structure). 13C-NMR (CDCl ₃ ) (ppm): 34.7 (C1) ), 26.5 (C2), 75.7 (C3), 33.1 (C4), 42.5 (C5), 36.7 (C6), 71.2 (C7), 43.8 (C8), 39.2 (C9), 34.2 (C10), 21.2 (C11) ), 40.1 (C12), 43.8 (C13), 55.7 (C14), 26.9 (C15), 28.6 (C16), 55.0 (C17), 12.1 (C18), 23.3 (C19), 35.2 (C20), 18.4 (C21) ), 31.3 (C22), 31.0 (C23), 174.0 (C24), 66.1 (1'-CH2), 136.1 (C1'), 128.2 (C2', 6'), 128.5 (C3', 5'), 128.2 (C4'); pyrazine ring: 165.7 (2"-C=O), 154.1 (C2"), 140.3 (C3"), 149.2 (C5"), 150.5 (C6"), 22.5 (6"-CH ₃ ) , 22.1(5"-CH ₃ ), 21.6(3"-CH ₃ ).HRMS(ESI) m/z: 631.441046 [M+H] ⁺ ,calcd.for C ₃₉ H ₅₄ N ₂ O ₅ 630.40327.

Preparation of Compound 22 of Example 22

This structure was isolated in the preparation of compound 19, 0.334 g. White solid, mp: 147.2 - 148.0 ° C, yield 14.3%. 1H-NMR (CDCl ₃ ) (ppm): 0.68 (s, 3H, H-18), 1.07 (s, 3H, H-19), 0.91 (d) , J = 6.0 Hz, 3H, H-21), 5.02 (m, 2H, H-3, 7), 5.10 (brs, 2H, CH ₂ -1 '), 7.37 (m, 5H, H-2', 3',4',5',6'),2.56–2.75(s,each,3H,6×CH ₃ -pyrazine ring),1.00–2.50 (26H,methyl-and methylene-of steroid structure).13C- NMR (CDCl ₃ ) (ppm): 34.7 (C1), 26.5 (C2), 75.5 (C3), 32.9 (C4), 40.1 (C5), 32.9 (C6), 75.3 (C7), 42.4 (C8), 39.7 (C9), 34.2 (C10), 21.3 (C11), 40.0 (C12), 43.7 (C13), 55.4 (C14), 25.9 (C15), 28.3 (C16), 55.1 (C17), 12.1 (C18), 23.3 (C19), 35.1 (C20), 18.4 (C21), 31.3 (C22), 31.0 (C23), 173.9 (C24), 66.0 (1'-CH2), 136.2 (C1'), 128.1 (C2', 6' ), 128.5 (C3', 5'), 128.1 (C4'); pyrazine ring: 165.7 (2"-C=O), 153.9 (C2"), 140.3 (C3"), 149.1 (C5"), 150.5 ( C6 "), 165.7 (2"'- C = O), 153.7 (C2 "'), 140.3 (C3"'), 149.1 (C5 "'), 150.5 (C6"), 22.4 (6 "-CH 3) , 22.0(5"-CH ₃ ), 21.5(3"-CH ₃ ), 22.4(6"'-CH ₃ ), 22.0(5"'-CH ₃ ), 21.5(3"'-CH ₃ ).HRMS (ESI) m/z: 779.47418 [M+H] ⁺ , calcd. for C ₄₇ H ₆₂ N ₄ O ₆ 778.46694.

Preparation of Compound 21 of Example 23

Weigh about 0.6 mmol of compound 19 and place it in a 50 ml round bottom flask. Add 30 ml of THF. After the mixture is dissolved, add Pd(OH) ₂ /C 40 mg, hydrogen to reduce, stir at room temperature for 10 h, and the reaction material disappears by TLC to stop the reaction. Pd(OH) ₂ /C was removed by filtration, and the reaction mixture was concentrated under reduced pressure. The residue was re-dissolved with ₃ ml of CHCl ₃ , and then added to a mixture of 2.2 g of silica gel under reduced pressure. The eluent was eluted with petroleum ether: acetone = 5:1. A white solid was obtained as 0.301 g. White amorphous solid, mp: 101.9 - 102.7 ° C, yield 92.8%. 1H-NMR (CDCl ₃ ) (ppm): 0.71 (s, 3H, H-18), 1.01 (s, 3H, H-19), 0.96 ( d, J = 6.5 Hz, 3H, H-21), 5.01 (m, 1H, H-3), 3.62 (m, 1H, H-7), 2.73 (s, 3H, CH ₃ -6 '), 2.58 (s, 3H, CH ₃ -5'), 2.58 (s, 3H, CH ₃ -3'), 1.00 - 2.50 (28H, methyl-and methylene-of steroid structure). 13C-NMR (CDCl ₃ ) (ppm ): 34.7 (C1), 26.5 (C2), 75.7 (C3), 33.1 (C4), 42.4 (C5), 36.7 (C6), 71.3 (C7), 43.7 (C8), 39.2 (C9), 34.2 (C10) ), 21.2 (C11), 40.1 (C12), 43.8 (C13), 55.7 (C14), 26.9 (C15), 28.6 (C16), 55.0 (C17), 12.1 (C18), 23.3 (C19), 35.2 (C20) ), 18.4 (C21), 31.0 (C22), 30.8 (C23), 178.9 (C24); pyrazine ring: 165.6 (2'-C=O), 154.2 (C2'), 140.3 (C3'), 149.3 (C5) "), 150.6 (C6'), 22.5 (6'-CH ₃ ), 22.1 (5'-CH ₃ ), 21.6 (3'-CH ₃ ). HRMS (ESI) m/z: 541.3632 [M+H] ⁺ ,calcd.for C ₃₂ H ₄₈ N ₂ O ₅ 540.35632.

Preparation of Compound 22 of Example 24

Weigh about 0.3 mmol of compound 20 and place it in a 50 ml round bottom flask. Add 30 ml of THF. After the mixture is dissolved, add Pd(OH) ₂ /C 25 mg, hydrogen reduction, and stir at room temperature for 10 h. The reaction material disappears by TLC and the reaction is stopped. Pd(OH) ₂ /C was removed by filtration, and the reaction mixture was concentrated under reduced pressure. The residue was re-dissolved with ₃ ml of CHCl ₃ , and then added to a mixture of 2.2 g of silica gel under reduced pressure. The eluent was eluted with petroleum ether: acetone = 5:1. A white solid was obtained in an amount of 0.193 g. Mp: 147.2 - 148.0 ° C, yield 93.4%. 1H-NMR (CDCl ₃ ) (ppm): 0.71 (s, 3H, H-18), 1.07 (s, 3H, H-19), 0.93 (d, J = 6.0Hz, 3H, H-21), 5.02 (m, 1H, H-3), 5.12 (m, 1H, H-7), 2.57–2.80 (s, each, 3H, 6×CH ₃ -pyrazine ring) 13C-NMR (CDCl ₃ ) (ppm): 34.6 (C1), 26.4 (C2), 75.5 (C3), 32.8 (C4), 40.0 ( C5), 32.9 (C6), 75.4 (C7), 42.4 (C8), 39.6 (C9), 34.2 (C10), 21.3 (C11), 39.9 (C12), 43.7 (C13), 55.4 (C14), 25.9 ( C15), 28.4 (C16), 55.0 (C17), 12.1 (C18), 23.3 (C19), 35.2 (C20), 18.3 (C21), 30.8 (C22), 30.9 (C23), 178.6 (C24); pyrazine ring :165.6(2'-C=O),154.1(C2'),140.2(C3'),149.3(C5'),150.6(C6'),165.6(2"-C=O),153.9(C2") , 140.2 (C3"), 149.2 (C5"), 150.6 (C6'), 22.5 (6'-CH ₃ ), 22.0 (5'-CH ₃ ), 21.5 (3'-CH ₃ ), 22.4 (6" -CH3), 22.0 (5 "-CH 3), 21.5 (3" -CH 3) .HRMS (ESI) m / z: 689.42834 [m + H] +, calcd.for C 40 H 56 N 4 O 6 688.41999 .

Preparation of Compound 23 of Example 25

Weigh 2.19 mmol of 2-bromomethyl-3,5,6-trimethylpyrazine and 2.19 mmol of chenodeoxycholic acid in a 50 ml round bottom flask, add 25 ml of DMF, and add 3 mmol of carbonic acid after the mixture is dissolved. Potassium, stirring at 85 ° C for 2 h, TLC monitoring the reaction material disappeared, the reaction was stopped, the reaction solution was added to a large amount of saturated NaCl solution dispersion, 300 ml of ethyl acetate was extracted twice, and the ethyl acetate layer was dried over anhydrous Na ₂ SO ₄ After concentration, the residue was dissolved in 3 ml of chloroform, and 2.8 g of silica gel was added under reduced pressure to dryness. The eluent was eluted with petroleum ether: acetone = 8:1 to give white solid. Mp: 64.8 - 64.6 ° C, yield 55.4%. 1H-NMR (CDCl ₃ ) (ppm): 0.64 (s, 3H, H-18), 0.90 (s, 3H, H-19), 0.91 (d, J = 6.5 Hz, 3H, H-21), 3.44 (m, 1H, H-3), 3.84 (brs, 1H, H-7), 5.19 (t, J = 12.5 Hz, 2H, CH ₂ -2'), 2.53 (s, 3H, CH ₃ -6'), 2.51 (s, 3H, CH ₃ -5'), 2.50 (s, 3H, CH ₃ -3'), 1.00 - 2.50 (28H, methyl-and methylene- _{13 C} -NMR (CDCl ₃ ) (ppm): 35.3 (C1), 30.7 (C2), 71.9 (C3), 39.8 (C4), 41.5 (C5), 34.7 (C6), 68.3 (C7) , 39.6 (C8), 32.8 (C9), 35.0 (C10), 20.6 (C11), 39.4 (C12), 42.6 (C13), 50.4 (C14), 23.6 (C15), 28.1 (C16), 55.8 (C17) , 11.7 (C18), 22.8 (C19), 35.4 (C20), 18.2 (C21), 31.0 (C22), 31.0 (C23), 173.9 (C24); pyrazine ring: 64.9 (2'-CH ₂ ), 151.2 ( C2 '), 144.9 (C3' ), 148.9 (C5 '), 149.0 (C6'), 21.6 (6'-CH 3), 21.4 (5'-CH 3), 20.5 (3'-CH 3) .HRMS (ESI) m/z: 527.38440 [M+H]+, calcd. for C ₃₂ H ₅₀ N ₂ O ₄ 526.37706.

Preparation of Compound 24 of Example 26

Weigh about 1.2 mmol of compound 23 and 1.8 mmol of ligustrazine acid in a 50 ml round bottom flask, and add 25 ml of CH ₂ Cl ₂ . After the mixture was dissolved, add EDCI 1.8 mmol and DMAP 0.18 mmol, respectively, stir at room temperature for 12 h, and monitor the reaction materials by TLC. After the disappearance, the reaction was stopped, and the mixture was concentrated under reduced pressure. EtOAc (EtOAc): Oil, yield 46.1%.1H-NMR (CDCl ₃ ) (ppm): 0.66 (s, 3H, H-18), 0.95 (s, 3H, H-19), 0.92 (d, J = 6.0 Hz, 3H, H-21), 4.91 (m, 1H, H-3), 3.87 (brs, 1H, H-7), 5.19 (t, J = 12.5 Hz, 2H, CH ₂ -2'), 2.71 (s, 3H) , CH ₃ -6"), 2.56 (s, 3H, CH ₃ -5"), 2.55 (s, 3H, CH ₃ -3"), 2.53 (s, 3H, CH ₃ -6 '), 2.51 (s , 3H, CH3-5'), 2.51 (s, 3H, CH3-3'), 1.00 - 2.50 (27H, methyl-and methylene-of steroid structure). 13C-NMR (CDCl ₃ ) (ppm): 35.2 ( C1), 26.7 (C2), 76.3 (C3), 35.4 (C4), 41.4 (C5), 34.5 (C6), 68.4 (C7), 39.6 (C8), 32.8 (C9), 35.1 (C10), 20.6 ( C11), 39.4 (C12), 42.7 (C13), 50.4 (C14), 23.7 (C15), 28.1 (C16), 55.8 (C17), 11.8 (C18), 22.7 (C19), 35.3 (C20), 18.2 ( C21), 31.0 (C22), 31.0 (C23), 173.9 (C24); pyrazine ring: 64.9 (2'-CH ₂ ), 151.2 (C2'), 144.9 (C3'), 149.0 (C5'), 149.0 ( C6 '), 165.8 (2 " -C = O), 153.9 (C2"), 140.5 (C3 "), 149.2 (C5"), 150.4 (C6 "), 21.5 (6'-CH 3), 21.4 (5 '-CH ₃ ), 20.5 (3'-CH ₃ ), 22.6 (6"-CH ₃ ), 22.1 (5"-CH ₃ ), 21.6 (3"-CH ₃ ). HRMS (ESI) m/z: 675.44879[M+H] ⁺ ,calcd.for C ₄₀ H ₅₈ N ₄ O ₅ 674.44072.

Preparation of benzyl chenodeoxycholate: Weigh 5.0 mmol of benzyl bromide and 5.0 mmol of chenodeoxycholic acid into a 50 ml round bottom flask, add 25 ml of DMF. After the mixture is dissolved, add 5 mmol of potassium carbonate and stir at 85 ° C for 2 h. , 85 deg.] C was stirred for 2h, TLC the reaction was monitored starting material disappeared, the reaction was stopped, the reaction solution was added a large amount of saturated NaCl solution dispersion, 400ml and extracted twice with ethyl acetate, ethyl acetate layer was dried over anhydrous Na ₂ SO _4, and concentrated under reduced pressure The residue was dissolved in 5 ml of chloroform, and 3.8 g of silica gel was added to dryness under reduced pressure. The eluent was eluted with petroleum ether: acetone = 5:1 to give a white solid. White powder, mp: 146.9–147.7°C, yield 89.5%.

Preparation of Compound 25 of Example 27

Weigh 3.0 mmol of benzyl chenodeoxycholate and 4.0 mmol of tetramethylpyrazine in a 50 ml round bottom flask, and add 30 ml of CH ₂ Cl ₂ . After the mixture was dissolved, add EDCI 4.0 mmol and DMAP 0.4 mmol, respectively, and stir at room temperature for 12 h. The reaction material was almost disappeared by TLC, the reaction was stopped, the reaction solution was washed twice with 60 ml of saturated NaCl solution, and the CH ₂ Cl ₂ layer was dried over anhydrous Na ₂ SO 4 and concentrated under reduced pressure. Elution with petroleum ether: acetone = 7:1 gave a white solid. Mp: 64.8 - 64.6 ° C, yield 48.2%. 1H-NMR (CDCl ₃ ) (ppm): 0.65 (s, 3H, H-18), 0.96 (s, 3H, H-19), 0.92 (d, J = 6.0Hz, 3H, H-21), 4.92 (m, 1H, H-3), 3.87 (brs, 1H, H-7), 5.14, 5.10 (ea, d, J = 12.5 Hz, 1H, CH2-1 '), 7.36 (m, 5H, H-2', 3', 4', 5', 6'), 2.72 (s, 3H, CH ₃ -6"), 2.56 (s, 3H, CH ₃ -5) "), 2.56 (s, 3H, CH ₃ -3"), 1.00 - 2.50 (27H, methyl-and methylene-of steroid structure). 13C-NMR (CDCl ₃ ) (ppm): 35.2 (C1), 26.8 ( C2), 76.3 (C3), 35.2 (C4), 41.4 (C5), 34.5 (C6), 68.4 (C7), 39.6 (C8), 32.8 (C9), 35.1 (C10), 20.6 (C11), 39.4 ( C12), 42.7 (C13), 50.4 (C14), 23.7 (C15), 28.1 (C16), 55.8 (C17), 11.8 (C18), 22.7 (C19), 35.3 (C20), 18.3 (C21), 31.3 ( C22), 31.0 (C23), 174.0 (C24), 66.1 (1'-CH ₂ ), 136.1 (C1'), 128.2 (C2', 6'), 128.5 (C3', 5'), 128.1 (C4');pyrazine ring: 165.8 (2"-C=O), 153.8 (C2"), 140.6 (C3"), 149.2 (C5"), 150.3 (C6"), 22.5 (6"-CH ₃ ), 22.1 ( 5"-CH ₃ ), 21.5 (3"-CH ₃ ). HRMS (ESI) m/z: 631.40985 [M+H] ⁺ , calcd. for C ₃₉ H ₅₄ N ₂ O ₅ 630.40327.

Preparation of Compound 26 of Example 28

About 1.2 mmol of compound 25 was weighed into a 50 ml round bottom flask, and 30 ml of THF was added. After the mixture was dissolved, 78 mg of Pd/C was added, and hydrogen was reduced, and stirred at room temperature for 10 hours. The reaction material was almost disappeared by TLC, the reaction was stopped, and Pd/ was removed by filtration. C. The reaction mixture was concentrated under reduced pressure. The residue was purified eluting with ₃ ml of CHCl ₃ and then evaporated to dryness. Mp: 130.0 - 130.8 ° C, yield 90.7%. 1H-NMR (CDCl ₃ ) (ppm): 0.68 (s, 3H, H-18), 0.96 (s, 3H, H-19), 0.95 (d, J = 7.5 Hz, 3H, H-21), 4.92 (m, 1H, H-3), 3.87 (brs, 1H, H-7), 2.72 (s, 3H, CH ₃ -6'), 2.65 (s, 6H) , CH ₃ -3 ', 5'), 1.00 - 2.50 (28H, methyl-and methylene-of steroid structure). 13C-NMR (CDCl ₃ ) (ppm): 35.2 (C1), 26.7 (C2), 76.3 ( C3), 35.2 (C4), 41.4 (C5), 34.5 (C6), 68.5 (C7), 39.6 (C8), 32.8 (C9), 35.1 (C10), 20.6 (C11), 39.4 (C12), 42.7 ( C13), 50.4 (C14), 23.7 (C15), 28.1 (C16), 55.8 (C17), 11.8 (C18), 22.7 (C19), 35.4 (C20), 18.3 (C21), 31.0 (C22), 30.8 ( C23), 179.1 (C24); pyrazine ring: 165.7 (2'-C=O), 153.9 (C2'), 140.5 (C3'), 149.3 (C5'), 150.4 (C6'), 22.5 (6'- CH ₃ ), 22.0 (5'-CH ₃ ), 21.4 (3'-CH ₃ ). HRMS (ESI) m/z: 541.36346 [M+H] ⁺ ,calcd.for C ₃₂ H ₄₈ N ₂ O ₅ 540.35632 .

Preparation of Compound 27 of Example 29

2.19 mmol of 2-bromomethyl-3,5,6-trimethylpyrazine and 2.19 mmol of lithocholic acid were weighed and placed in a 50 ml round bottom flask, and 25 ml of DMF was added. After the mixture was dissolved, 3 mmol of potassium carbonate was added. 85 ℃ stirred for 2h, TLC the reaction was monitored starting material disappeared, the reaction was stopped, the reaction solution was added a large amount of saturated NaCl solution dispersion, 300ml and extracted twice with ethyl acetate, the ethyl acetate layer was dried over anhydrous Na ₂ SO _4, and concentrated under reduced pressure, The residue was dissolved in 3 ml of chloroform, and 2.8 g of silica gel was added to dryness under reduced pressure. The eluent was eluted with petroleum ether: acetone = 6:1 to give a white solid. Mp: 119.3 - 119.9 ° C, yield 52.7%. 1H-NMR (CDCl ₃ ) (ppm): 0.60 (s, 3H, H-18), 0.89 (s, 3H, H-19), 0.88 (d, J = 6.0Hz, 3H, H-21), 3.60 (m, H, H-3), 5.17 (t, J = 12.5 Hz, 2H, CH ₂ -2'), 2.51 (s, 3H, CH ₃ -6' ), 2.49 (s, 3H, CH ₃ -5'), 2.48 (s, 3H, CH ₃ -3'), 1.00 - 2.50 (29H, methyl-and methylene-of steroid structure). 13C-NMR (CDCl ₃ (ppm): 35.8 (C1), 30.5 (C2), 71.7 (C3), 36.4 (C4), 42.1 (C5), 27.2 (C6), 26.4 (C7), 40.2 (C8), 34.6 (C9), 35.3 (C10), 20.8 (C11), 40.4 (C12), 42.7 (C13), 56.5 (C14), 24.2 (C15), 28.1 (C16), 56.0 (C17), 12.0 (C18), 23.4 (C19), 35.4 (C20), 18.2 (C21), 31.1 (C22), 31.0 (C23), 173.8 (C24); pyrazine ring: 64.9 (2'-CH ₂ ), 151.2 (C2'), 144.9 (C3'), 148.9 (C5'), 149.0 (C6'), 21.6 (6'-CH ₃ ), 21.4 (5'-CH ₃ ), 20.4 (3'-CH ₃ ). HRMS (ESI) m/z: 511.38754 [M+ H] ⁺ , calcd.for C ₃₂ H ₅₀ N ₂ O ₃ 510.38214.

Preparation of Compound 28 of Example 30

Weigh about 1.2 mmol of compound 27 and 1.2 mmol of ligustrazine acid in a 50 ml round bottom flask, and add 25 ml of CH ₂ Cl ₂ . After the mixture was dissolved, add EDCI 1.2 mmol and DMAP 0.12 mmol, respectively, and stir at room temperature for 12 h. After the disappearance, the reaction was stopped, concentrated under reduced pressure, and 2.2 g of silica gel was added to dryness under reduced pressure. The eluent was eluted with petroleum ether: acetone = 5:1 to yield 0.386 g of oil. Mp: 85.3 - 85.9 ° C, yield 48.9%. 1H-NMR (CDCl ₃ ) (ppm): 0.65 (s, 3H, H-18), 0.97 (s, 3H, H-19), 0.91 (d, J = 6.0 Hz, 3H, H-21), 5.01 (m, H, H-3), 5.20 (t, J = 12.5 Hz, 2H, CH ₂ -2'), 2.73 (s, 3H, CH ₃ -6) ), 2.58 (s, 3H, CH ₃ -5"), 2.57 (s, 3H, CH ₃ -3"), 2.54 (s, 3H, CH ₃ -6'), 2.53 (s, 3H, CH ₃ - 5'), 2.52 (s, 3H, CH ₃ -3'), 1.00 - 2.50 (28H, methyl-and methylene-of steroid structure). 13C-NMR (CDCl ₃ ) (ppm): 35.8 (C1), 27.1 (C2), 76.3 (C3), 32.2 (C4), 42.1 (C5), 26.6 (C6), 26.3 (C7), 40.1 (C8), 34.7 (C9), 35.1 (C10), 20.8 (C11), 40.4 (C12), 42.7 (C13), 56.5 (C14), 24.2 (C15), 28.2 (C16), 56.0 (C17), 12.0 (C18), 23.3 (C19), 35.3 (C20), 18.3 (C21), 31.1 (C22), 31.0 (C23), 173.9 (C24); pyrazine ring: 65.0 (2'-CH2), 151.2 (C2'), 144.9 (C3'), 149.0 (C5'), 149.0 (C6'), 165.5 (2 "-C = O), 154.0 (C2"), 140.5 (C3 "), 149.2 (C5"), 150.4 (C6 "), 21.7 (6'-CH 3), 21.5 (5'-CH 3) , 20.5 (3'-CH ₃ ), 22.6 (6"-CH ₃ ), 22.1 (5"-CH ₃ ), 21.7 (3"-CH _3). HRMS (ESI) m/z: 659.45245 [M+H ] ⁺ ,calcd.for C ₄₀ H ₅₈ N ₄ O ₄ 658.44581.

Preparation of benzyl choline: Weigh 5.0 mmol of benzyl bromide and 5.0 mmol of lithocholic acid in a 50 ml round bottom flask, add 25 ml of DMF. After the mixture is dissolved, add 2.0 mmol of potassium carbonate, add 5 mmol of potassium carbonate, 85 After stirring at °C for 2 h, stirring at 85 ° C for 2 h, the reaction material was almost disappeared by TLC, the reaction was stopped, the reaction mixture was added with a large amount of saturated NaCl solution, and then, 400 ml of ethyl acetate was extracted twice, and the ethyl acetate layer was dried over anhydrous Na ₂ SO ₄ . The organic layer was concentrated under reduced pressure, and the residue was evaporated, evaporated, evaporated. White powder, mp: 129.1–129.8°C, yield 91.2%.

Preparation of Compound 29 of Example 31

Weigh 3.0 mmol of benzyl bile cholate and 3.0 mmol of tetramethylpyrazine in a 50 ml round bottom flask, add 30 ml of CH ₂ Cl ₂ , and add EDCI 3.0 mmol and DMAP 0.3 mmol, respectively, and stir at room temperature for 12 h. TLC monitoring The reaction raw material disappeared, the reaction was stopped, the reaction solution was washed twice with 60 ml of saturated NaCl solution, and the CH ₂ Cl ₂ layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The solvent was petroleum ether: acetone = 7:1 eluting to give a white solid, 0.938 g. Mp: 102.6 - 103.3 ° C, yield 50.9%. 1H-NMR (CDCl ₃ ) (ppm): 0.65 (s, 3H, H-18), 0.98 (s, 3H, H-19), 0.92 (d, J = 6.5 Hz, 3H, H-21), 5.06 (m, H, H-3), 5.15, 5.12 (ea, d, J = 12.5 Hz, 1H, CH ₂ -1 '), 7.38 (m, 5H, H -2',3',4',5',6'), 2.74(s,3H,CH ₃ -6"), 2.59(s,3H,CH ₃ -5"), 2.58(s,3H,CH ₃ -3"), 1.00 - 2.50 (28H, methyl-and methylene-of steroid structure). 13C-NMR (CDCl ₃ ) (ppm): 35.8 (C1), 27.1 (C2), 76.3 (C3), 32.3 ( C4), 42.1 (C5), 26.6 (C6), 26.4 (C7), 40.1 (C8), 34.7 (C9), 35.1 (C10), 20.9 (C11), 40.4 (C12), 42.7 (C13), 56.5 ( C14), 24.2 (C15), 28.2 (C16), 56.0 (C17), 12.0 (C18), 23.3 (C19), 35.3 (C20), 18.3 (C21), 31.3 (C22), 31.0 (C23), 174.1 ( C24), 66.1 (1'-CH2), 136.1 (C1'), 128.2 (C2', 6'), 128.5 (C3', 5'), 128.2 (C4'); pyrazine ring: 165.8 (2"-C = O), 154.0 (C2 " ), 140.6 (C3"), 149.3 (C5 "), 150.4 (C6"), 22.6 (6 "-CH 3), 22.1 (5" -CH 3), 21.7 (3 " -CH ₃ ).HRMS (ESI) m/z: 637.39772 [M+Na] ⁺ , calcd. for C ₃₉ H ₅₄ N ₂ O ₄ 614.40836.

Preparation of Compound 32 of Example 32

Weighing compound 29 1.2mmol was placed in a 50ml round bottom flask, and 30ml of THF was added. After the mixture was dissolved, Pd/C 40mg was added, hydrogen was reduced, and stirred at room temperature for 10 hours. The reaction material was almost disappeared by TLC, the reaction was stopped, and Pd/C was removed by filtration. The reaction mixture was concentrated under reduced pressure. The residue was purified eluting with ₃ ml of CHCl ₃ and then evaporated to dryness. Mp: 184.5 - 185.2 ° C, yield 92.7%. ¹ H-NMR (CDCl ₃ ) (ppm): 0.68 (s, 3H, H-18), 0.99 (s, 3H, H-19), 0.95 (d, J) = 6.5 Hz, 3H, H-21), 5.07 (m, H, H-3), 2.74 (s, 3H, C H ₃ -6"), 2.59 (s, 3H, C H ₃ -5"), 2.59 (s, 3H, C H ₃ -3"), 1.00 - 2.50 (29H, methyl-and methylene-of steroid structure). ¹³ C-NMR (CDCl ₃ ) (ppm): 35.8 (C1), 27.1 (C2) ), 76.3 (C3), 32.3 (C4), 42.1 (C5), 26.6 (C6), 26.4 (C7), 40.1 (C8), 34.7 (C9), 35.1 (C10), 20.9 (C11), 40.4 (C12) ), 42.7 (C13), 56.5 (C14), 24.2 (C15), 28.2 (C16), 56.0 (C17), 12.0 (C18), 23.3 (C19), 35.3 (C20), 18.3 (C21), 31.3 (C22) ), 31.0 (C23), 174.1 (C24), 66.1 (1'- C H ₂ ), 136.1 (C1'), 128.2 (C2', 6'), 128.5 (C3', 5'), 128.2 (C4');pyrazinering:165.8(2" -C =O),154.0(C2"),140.6(C3"),149.3(C5"),150.4(C6"),22.6(6" -C H ₃ ),22.1 (5" -C H ₃ ), 21.7 (3" -C H ₃ ). HRMS (ESI) m/z: 525.36871 [M+H] ⁺ , calcd. for C ₃₂ H ₄₈ N ₂ O ₄ 524.36141.

Example 33

Take 10g of the compound prepared in any one of Examples 3 to 32, add an injection (including lyophilized powder injection and aseptic dry powder injection) appropriate excipients, and prepare according to the injection (including freeze-dried powder injection and aseptic dry powder injection) It is an anti-tumor drug injection.

Example 34

Take 10g of the compound prepared in any one of Examples 3 to 32, and add appropriate excipients to tablets (including slow-release tablets, matrix tablets, coated tablets, dispersible tablets, etc.), according to tablets (including controlled release tablets, matrix tablets, The coated tablets, dispersible tablets, and the like) are prepared into anti-tumor tablets.

Example 35

10 g of the compound prepared in any one of Examples 3 to 32 was added, and a suitable adjuvant was added to the capsule to prepare an antitumor drug capsule according to the capsule process.

Example 36

10 g of the compound prepared in any one of Examples 3 to 32 was added, and an appropriate adjuvant was added to the emulsion (including microemulsion, nanoemulsion, etc.) to prepare an antitumor drug emulsion according to the emulsion (including microemulsion, nanoemulsion, etc.).

Example 37

10 g of the compound prepared in any of Examples 3 to 32 was added to a granule suitable excipient, and an antitumor drug granule was prepared according to the granule process.

Example 38

10 g of the compound prepared in any one of Examples 3 to 32 was added to a sustained release controlled release agent as an appropriate adjuvant, and an antitumor drug sustained release controlled release agent was prepared according to the sustained release controlled release agent process.

Example 39

10 g of the compound prepared in any of Examples 3 to 32 was added, and an appropriate adjuvant was added to the oral solution, and an antitumor drug oral solution was prepared according to the oral liquid process.

Example 40

10 g of the compound prepared in any of Examples 3 to 32 was added to a suitable excipient of the liposome dosage form, and an antitumor drug liposome dosage form was prepared according to the liposome process.

Claims (10)

A compound having the structure of Formula 1, a pharmaceutically acceptable salt, stereoisomer, isotope label, solvate, polymorph or prodrug thereof:

among them, R _{1 -} R ₄ are -OH, -H or

Any of the substituents; R5 is -COOH,

Any of the substituents; And at least one of R ₁ -R ₅

Substituent. A compound, a pharmaceutically acceptable salt, a stereoisomer, an isotopic label, a solvate, a polymorph or a prodrug thereof, according to claim 1, wherein the compound is selected from the group consisting of:

A pharmaceutical composition comprising a compound according to any one of claims 1 or 2, a pharmaceutically acceptable salt, a stereoisomer, an isotope label, a solvate, a polymorph or a prodrug thereof, and A pharmaceutically acceptable carrier. A method of preparing a compound according to claim 1 or 2, wherein the method comprises: The cholic acid component is reacted with 2-bromomethyl-3,5,6-trimethylpyrazine under basic conditions; Or, reacting the cholic acid component with 2-bromomethyl-3,5,6-trimethylpyrazine under basic conditions, and further reacting the product obtained by the catalyst and the condensing agent with ligustrazine acid react; Alternatively, the cholic acid component is reacted with benzyl bromide under basic conditions, and the product obtained by the reaction is reacted with ligustrazine acid under the action of a catalyst and a condensing agent; Alternatively, the cholic acid component is reacted with benzyl bromide under basic conditions, and the product obtained by the reaction is reacted with ligustrazine acid under the action of a catalyst and a condensing agent, and further reduced by hydrogen. The method according to claim 4, wherein the cholic acid component is cholic acid, deoxycholic acid, ursodeoxycholic acid, hyodeoxycholic acid, chenodeoxycholic acid or lithocholic acid. Any of them. The use of a compound according to any one of claims 1 or 2, a pharmaceutically acceptable salt, stereoisomer, isotope label, solvate, polymorph or prodrug thereof for the preparation of an anticancer drug. The use according to claim 6, wherein the cancer is liver cancer, colon cancer, cervical cancer, breast cancer. A kit comprising a therapeutically effective amount of a compound of formula 1, a pharmaceutically acceptable salt, stereoisomer, isotope label, solvate, polymorph or prodrug thereof and an anticancer drug; wherein the compound of formula 1, Pharmaceutically acceptable salts, stereoisomers, isotopic labels, solvents thereof The therapeutically effective amount of the compound, polymorph or prodrug is from 1 to 10 mg/kg day. The kit according to claim 8, wherein the anticancer drug is cyclophosphamide, 5-fluorouracil, paclitaxel, doxorubicin, etoposide, irinotecan, oxaliplatin, cisplatin or Any one or several of the choices. The use of the kit according to claim 8 or 9 for the preparation of an anticancer drug.