CN113173963A - Anti-tumor betulin derivative, preparation method and application thereof - Google Patents

Abstract

The invention discloses an anti-tumor betulin derivative, a preparation method and an application thereof, wherein the preparation method comprises the following steps: adding betulin into a reactor, sulfonating by acyl chloride, reacting with indoleacetic acid to obtain a betulin derivative crude product, and purifying the crude product by silica gel column purification and high performance liquid chromatography separation purification to obtain a betulin derivative; the betulin derivative has significant inhibitory activity on human ovarian cancer cell (SKOV3), human leukemia cell (K562) and human melanoma cell (A875), wherein the betulin derivative has half Inhibitory Concentration (IC) of human melanoma cell (A875)₅₀) 12.39 mug/mL, the effect is similar to that of positive medicine cisplatin (IC)₅₀10.67 μ g/mL) indicating the potential of the derivatives of the invention for use in the preparation of anti-human melanoma drugs.

Description

Anti-tumor betulin derivative, preparation method and application thereof

Technical Field

The invention belongs to the technical field of chemical drug synthesis, and relates to an anti-tumor betulin derivative, a preparation method and application thereof.

Background

Betulin, also called betulin ester and betulin, belongs to lupane pentacyclic triterpenes, and is a natural product widely existing in various plants. Has wide biological activity, especially has excellent inhibitory activity on the proliferation of tumor cells, so that the betulin is more favored by people in the related technical field, but the betulin has low selectivity, low bioavailability and poor solubility which does not meet the requirement of clinical application. In order to make natural products perform their desired functions, attempts have been made to modify their structure in order to improve their properties. Therefore, a great deal of research on this aspect is carried out, for example, Chrobak et al (Molecules, 2016, 21(9): 1123-1135) introduce phosphate into the structure of betulin to obtain a series of betulin with phosphate structure, although these compounds have good anti-tumor activity, the compounds contain too many ester groups in the molecule, so that the solubility of the compounds in water is reduced, and the main purpose of the betulin structure modification is not achieved; bebenek et al (Medicinal Chemistry Research, 2017, 26(1): 1-8) and the like connect a series of hydrocarbyl chains with different lengths and saturation degrees on the 28-position hydroxyl of the betulin, but the compounds kill tumor cells and simultaneously damage normal colon cells to a certain extent, so that the betulin derivative obtained by the method has defects.

Disclosure of Invention

In order to solve the technical problems, the invention provides an anti-tumor betulin derivative, which has a molecular structure shown in a formula (I):

an object of the present invention is to provide a method for preparing an anti-tumor betulin derivative, comprising the steps of:

(1) taking betulin as a raw material, adding an organic solvent A at room temperature for full dissolution, slowly adding oxalyl chloride at the temperature of-10-0 ℃, adding 2-3 drops of DMF (N, N-dimethylformamide), heating to room temperature for reaction for 2-5 hours, and removing redundant thionyl chloride after the reaction is finished to obtain a compound 1;

(2) dissolving the compound 1 in an organic solvent B, adding indoleacetic acid, stirring and reacting for 4-8 h at room temperature, and displaying no progress of the reaction by thin-layer chromatography. Adding sodium bicarbonate solution into the reaction solution, extracting with dichloromethane or ethyl acetate, washing the organic phase with saturated saline solution and 5% hydrochloric acid solution in sequence, drying, concentrating, separating and purifying the crude product with silica gel column to obtain crude product, and separating and purifying the crude product with high performance liquid chromatography to obtain the target product;

in the preparation method, in the step (1), the organic solvent A is dichloromethane, acetone, acetonitrile, tetrahydrofuran, N-dimethylformamide or diethyl ether;

the feeding molar ratio of the betulin to the oxalyl chloride is 1: 2.0-4.0;

in the preparation method, in the step (2), the organic solvent B is dichloromethane, ethyl acetate, chloroform, dimethyl sulfoxide or acetone;

the feeding molar ratio of the indoleacetic acid to the compound 2 is 1.2: 1.8-2.5: 3.0;

the silica gel column eluent adopts a mixed liquid system of petroleum ether and acetone, and the volume ratio of the petroleum ether to the acetone is 10-20: 1;

the high performance liquid chromatography separation and purification conditions are as follows:

a chromatographic column: a C18 column or a C8 column; mobile phase: a methanol-water solution, wherein the volume ratio of methanol to water is 68: 36; the detection wavelength is 278-320 nm; column temperature: 25-40 ℃; the sampling amount is 0.5-1.2 mL each time; and (3) collecting chromatographic peaks at the flow rate of 10-15 mL/min for 40-55 min, accumulating for multiple times, and drying to obtain the target product.

According to a preferred embodiment of the above preparation method, the method comprises the following steps:

(1) taking 15g of betulin as a raw material, adding acetone at room temperature, fully dissolving, slowly adding 4.5g of oxalyl chloride at the temperature of-5-0 ℃, adding 2-3 drops of DMF (N, N-dimethylformamide), raising the temperature, reacting for 5 hours, and removing redundant thionyl chloride after the reaction is finished to obtain a compound 1;

(2) 7.5g of Compound 1 was dissolved in methylene chloride, and 6.4g of indoleacetic acid was added thereto, and the reaction was stirred at room temperature for 6 hours, and the reaction was not progressed by thin layer chromatography. Adding a sodium bicarbonate solution into the reaction solution, extracting with dichloromethane or ethyl acetate, washing an organic phase with saturated saline solution and 5% hydrochloric acid solution in sequence, drying, concentrating into an extract, eluting the extract by silica gel column chromatography, performing gradient elution by using a mixed solution of petroleum ether and acetone with the volume of 15:1, collecting eluent, and concentrating to obtain a crude product of the target product; separating the crude target product by using high performance liquid chromatography, wherein the chromatographic conditions are as follows: and (2) taking a methanol-water solution as a mobile phase, taking a C18 column as a stationary phase, setting the flow rate of the mobile phase at 15mL/min, setting the ultraviolet detection wavelength at 285-290 nm, setting the column temperature at 25 ℃, setting the sample amount at 1.0-1.2 mL each time, collecting chromatographic peaks with the retention time of 44.5min, accumulating for multiple times, and drying with anhydrous sodium sulfate to obtain the target product.

The derivatives of the invention are useful for the treatment of human ovarian cancer, human leukemia and human melanoma.

The derivative of the invention is used for preparing anti-human melanoma drugs.

Compared with the prior art, the invention has the beneficial effects that:

the betulin derivative has obvious inhibitory activity on human ovarian cancer cells (SKOV3), human leukemia cells (K562) and human melanoma cells (A875), wherein the betulin derivative has half Inhibitory Concentration (IC) of the human melanoma cells (A875)₅₀) 12.39 mu g/mL, and the effect is slightly similar to that of positive medicament cisplatin (IC)₅₀10.67 μ g/mL), indicating that the compounds of the present invention are useful in the preparation of medicaments against human melanoma.

Drawings

FIG. 1: example 1 is nuclear magnetic resonance hydrogen spectrum of anti-tumor betulin derivatives.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments for the purpose of making the objects, technical solutions and advantages of the present invention more apparent, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples.

Example 1

(1) Taking 15g of betulin as a raw material, adding acetone at room temperature, fully dissolving, slowly adding 4.5g of oxalyl chloride at the temperature of-5-0 ℃, adding 2-3 drops of DMF (N, N-dimethylformamide), raising the temperature, reacting for 5 hours, and removing redundant thionyl chloride after the reaction is finished to obtain a compound 1;

(2) 7.5g of Compound 1 was dissolved in methylene chloride, and 6.4g of indoleacetic acid was added thereto, and the reaction was stirred at room temperature for 6 hours, and the reaction was not progressed by thin layer chromatography. Adding a sodium bicarbonate solution into the reaction solution, extracting with dichloromethane or ethyl acetate, washing an organic phase with saturated saline solution and 5% hydrochloric acid solution in sequence, drying, concentrating into an extract, eluting the extract by silica gel column chromatography, performing gradient elution by using a mixed solution of petroleum ether and acetone with the volume of 15:1, collecting eluent, and concentrating to obtain a crude product of the target product; separating the crude target product by using high performance liquid chromatography, wherein the chromatographic conditions are as follows: and (2) taking a methanol-water solution as a mobile phase, taking a C18 column as a stationary phase, setting the flow rate of the mobile phase at 15mL/min, setting the ultraviolet detection wavelength at 285-290 nm, setting the column temperature at 25 ℃, setting the sample amount at 1.0-1.2 mL each time, collecting chromatographic peaks with the retention time of 44.5min, accumulating for multiple times, and drying with anhydrous sodium sulfate to obtain the target product. The yield was 79.09%.

And (3) nuclear magnetic resonance hydrogen spectrum detection:

the sample was placed in a sample tube, and 0.5mL of CDCL3 (deuterated chloroform) was injected into the sample tube with a syringe to dissolve the sample sufficiently. The sample and the reagent are required to be fully mixed, the solution is clear and transparent, and has no suspended matters or other impurities, and a nuclear magnetic resonance hydrogen spectrogram is obtained through nuclear magnetic resonance identification, and the result is shown in figure 1.

Experimental example 2 antitumor pharmacological study of the Compound of the present invention

(1) In vitro testing: dissolving betulin derivative and betulin with dimethyl sulfoxide (DMSO), respectively, preparing into stock solution with concentration of 2mg/mL with culture solution (RPMI 1640 culture solution containing 10% fetal calf serum), and storing at-20 deg.C. Before use, the final concentrations of the betulin derivative and the betulin are respectively 2.5. mu.g/mL, 5.0. mu.g/mL, 10. mu.g/mL, 25.0. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL and 200. mu.g/mL by diluting with the culture solution, and the final concentration of DMSO in any diluted test solution is controlled to be below 0.01%. Human gastric adenocarcinoma cell line (BGC-823), human ovarian cancer cell line (SKOV3), human leukemia cell line (K562), human esophageal cancer cell line (Ec9706), and human melanoma cell line (A875) were digested with 0.25% trypsin, suspended in RPMI1640 culture medium containing 10% fetal bovine serum, gently blown with a glass dropper to form a single cell suspension, and then diluted with the culture medium to a concentration of 20000 cells/mL. Cisplatin was dissolved in DMSO, and the resulting solution was prepared into a stock solution containing 1mg/mL of cisplatin (10% fetal bovine serum in RPMI1640 culture medium) and stored at-20 ℃. Before use, the cells were diluted with the culture medium to final concentrations of 50. mu.g/mL and 25. mu.g/mL, respectively12.5. mu.g/mL, 6.25. mu.g/mL, 3.12. mu.g/mL, 1.56. mu.g/mL, 0.78. mu.g/mL and 0.39. mu.g/mL, and wherein the final concentration of DMSO in any of the diluted test samples is controlled to be 0.01% or less, as a positive control. 100 mul of well-grown tumor cells (about 2000 cells/well) are added into each well of a 96-well culture plate, after the cells are cultured for 24 hours and completely attached, 100 mul of test solution (betulin derivatives, betulin and cisplatin) is respectively added into each well, six duplicate wells are arranged in parallel in each group, and a negative control (culture medium and cancer cells are added, no medicine is added) and a blank control (culture medium is added only, no cancer cells are added) are arranged. Placing at 37 ℃ in CO₂Culturing at 5% concentration for 72 h. Then, 20. mu.L (5mg/L) of MTT solution was added to each well, the culture was continued for 4 hours, the culture solution was discarded, 100. mu.L of DMSO was added to each well, and after the crystals were completely dissolved, the absorbance value at 490nm, that is, the OD value, was measured using a microplate reader. And calculating the cell proliferation inhibition rate. The experiment was repeated 3 times for each group of samples and the average value was taken.

And (3) testing results: the negative control group has good cell growth and no growth inhibition phenomenon, which indicates that the test method is feasible. IC measured by test solution group₅₀The values are shown in Table 1.

TABLE 1 median Inhibitory Concentration (IC) of the compounds of the invention on cancer cells₅₀)

The test results in table 1 show that the betulin derivative of the present invention has significant inhibitory activity on human ovarian cancer cells (SKOV3), human leukemia cells (K562) and human melanoma cells (a875), wherein the betulin derivative has half inhibitory concentration (IC 875) against human melanoma cells (a875)₅₀) 12.39 mu g/mL, and the effect is slightly similar to that of positive medicament cisplatin (IC)₅₀10.67 μ g/mL) indicating that the betulin derivatives of the present invention show great potential in the treatment of cancer.

(2) In vivo test: effect of the derivatives of the present invention on human melanoma cells in nude mice

Selecting tumor tissue in vigorous growth stage, and shearing into 2mm in sterile physiological saline solution³The left and right small blocks are inoculated under the aseptic environment to the right axilla subcutaneous part of the nude mouse. Measuring the diameter of the transplanted tumor by using a vernier caliper for the subcutaneous transplanted tumor of the nude mouse until the tumor grows to 100-200 mm³Then, the animals were randomly divided into a blank control group (control group 1), a positive drug control group (control group 2), a betulin control group (control group 3), and a betulin derivative (experimental group), except for the blank group, the administration dose of each group of mice was 20mg/kg, 1 time per day, and the mice were continuously cultured for 21 days with cisplatin as the positive drug, and distilled water of the same amount was administered to the blank group. During the experiment, the diameter of the transplanted tumor was measured 2 times per week, and the tumor volume, the relative tumor volume and the relative tumor proliferation rate were calculated while weighing the mouse body weight. Tumor Volume (TV) 1/2 × a × b²Wherein a and b are respectively length and width; relative Tumor Volume (RTV) ═ V_t/V_oIn which V is_tFor each measurement, tumor volume, V_oTumor volume was measured at the time of dosing. Relative tumor proliferation rate T/C (%) ═ T_RTV/C_RTV)×100％，T_RTVIs RTV, C of the treatment group_RTVIs the RTV of the negative control group. The results are shown in Table 2.

TABLE 2 inhibitory Effect of the Compounds of the present invention on human hepatoma cells

The test results in table 2 show that the betulin derivative of the present invention can significantly inhibit the growth of human melanoma cells, and has a Relative Tumor Volume (RTV) of 6.02 ± 3.15 and a relative tumor proliferation rate T/C of 29.40%, and the inhibitory effect is slightly reduced compared to cisplatin.

Claims (5)

1. An anti-tumor betulin derivative, which has a molecular structure represented by formula (I):

。

2. the preparation method of the derivative of the anti-tumor betulin is characterized by comprising the following steps:

(1) taking betulin as a raw material, adding an organic solvent A at room temperature for full dissolution, slowly adding oxalyl chloride at the temperature of-10-0 ℃, adding 2-3 drops of DMF (N, N-dimethylformamide), heating to room temperature for reaction for 2-5 hours, and removing redundant thionyl chloride after the reaction is finished to obtain a compound 1;

(2) dissolving the compound 1 in an organic solvent B, adding indoleacetic acid, stirring and reacting for 4-8 h at room temperature, and displaying no progress of the reaction by thin-layer chromatography. Adding sodium bicarbonate solution into the reaction solution, extracting with dichloromethane or ethyl acetate, washing the organic phase with saturated saline solution and 5% hydrochloric acid solution in sequence, drying, concentrating, separating and purifying the crude product with silica gel column to obtain crude product, and separating and purifying the crude product with high performance liquid chromatography to obtain the target product;

in the step (1), the organic solvent A is dichloromethane, acetone, acetonitrile, tetrahydrofuran, N-dimethylformamide or diethyl ether;

the feeding molar ratio of the betulin to the oxalyl chloride is 1: 2.0-4.0;

in the step (2), the organic solvent B is dichloromethane, ethyl acetate, chloroform, dimethyl sulfoxide or acetone;

the feeding molar ratio of the indoleacetic acid to the compound 2 is 1.2: 1.8-2.5: 3.0;

the silica gel column eluent adopts a mixed liquid system of petroleum ether and acetone, and the volume ratio of the petroleum ether to the acetone is 10-20: 1;

the high performance liquid chromatography separation and purification conditions are as follows:

a chromatographic column: a C18 column or a C8 column; mobile phase: a methanol-water solution, wherein the volume ratio of methanol to water is 68: 36; the detection wavelength is 278-320 nm; column temperature: 25-40 ℃; the sampling amount is 0.5-1.2 mL each time; and (3) collecting chromatographic peaks at the flow rate of 10-15 mL/min for 40-55 min, accumulating for multiple times, and drying to obtain the target product.

3. The method of claim 2, comprising the steps of:

(1) taking 15g of betulin as a raw material, adding acetone at room temperature, fully dissolving, slowly adding 4.5g of oxalyl chloride at the temperature of-5-0 ℃, adding 2-3 drops of DMF (N, N-dimethylformamide), heating to room temperature, reacting for 5 hours, and removing redundant thionyl chloride after the reaction is finished to obtain a compound 1;

(2) 7.5g of Compound 1 was dissolved in methylene chloride, and 6.4g of indoleacetic acid was added thereto, and the reaction was stirred at room temperature for 6 hours, and the reaction was not progressed by thin layer chromatography. Adding a sodium bicarbonate solution into the reaction solution, extracting with dichloromethane or ethyl acetate, washing an organic phase with saturated saline solution and 5% hydrochloric acid solution in sequence, drying, concentrating into an extract, eluting the extract by silica gel column chromatography, performing gradient elution by using a mixed solution of petroleum ether and acetone with the volume of 15:1, collecting eluent, and concentrating to obtain a crude product of the target product; separating the crude target product by using high performance liquid chromatography, wherein the chromatographic conditions are as follows: and (2) taking a methanol-water solution as a mobile phase, taking a C18 column as a stationary phase, setting the flow rate of the mobile phase at 15mL/min, setting the ultraviolet detection wavelength at 285-290 nm, setting the column temperature at 25 ℃, setting the sample amount at 1.0-1.2 mL each time, collecting chromatographic peaks with the retention time of 44.5min, accumulating for multiple times, and drying with anhydrous sodium sulfate to obtain the target product.

4. The derivative of claim 1 for use in the treatment of human ovarian cancer, human leukemia and human melanoma.

5. The use according to claim 4, wherein the derivative is used for preparing anti-human melanoma drugs.

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