Diosgenin derivative containing 1, 2, 3-triazole structure fragment as well as preparation method and application of diosgenin derivative

Abstract

The invention discloses a diosgenin derivative containing a 1, 2, 3-triazole structure fragment as well as a preparation method and application of the diosgenin derivative, and belongs to the field of natural medicinal chemistry and medicinal chemistry. Based on a molecular heterozygosis principle, diosgenin is used as a lead compound and is heterozygous with a 1, 2, 3-triazole structure fragment with different substituent groups to obtain a derivative shown as a general formula (I) or (I). The diosgenin derivative containing the 1, 2, 3-triazole fragment, disclosed by the invention, has good anti-breast cancer activity. Pharmacological experiment results show that the anti-tumor activity of part of the derivatives on human breast cancer cells is obviously superior to that of diosgenin, and the toxicity on human normal gastric mucosa epithelial cells is relatively small, so that the derivatives are expected to be further developed into anti-tumor drugs. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of natural drug chemistry and medicinal chemistry, and specifically relates to a diosgenin derivative containing a 1,2,3-triazole fragment, a preparation method thereof and a medical use thereof. Background Art

[0002] Breast cancer is the leading type of cancer in women and one of the leading causes of cancer death worldwide. Due to the mutation and drug resistance of different breast cancer cell lines to traditional therapies, the treatment effect is becoming increasingly poor. Therefore, it is crucial to develop safe and effective anti-breast cancer drugs.

[0003] In clinical drug use, more than one-third of drugs are produced by natural products and structural modification of them as lead compounds. Diosgenin is a natural steroid compound obtained mainly from the tubers of plants such as Dioscorea zingiberensis and Dioscorea zingiberensis through hydrolysis, fermentation, extraction and other methods. Diosgenin is abundant in natural resources and is one of the important raw materials for the production of steroid hormone drugs and contraceptives. Diosgenin shows good potential in the treatment of various cancers, such as breast cancer, laryngeal cancer, esophageal cancer, liver cancer, gastric cancer, lung cancer, cervical cancer, prostate cancer, glioma and leukemia. Diosgenin has moderate broad-spectrum anti-cancer activity, and diosgenin has no obvious biological toxicity. However, diosgenin has defects such as poor solubility in water and low bioavailability, and its cytotoxicity to breast cancer needs to be improved. Therefore, it is necessary to further modify the structure of diosgenin in order to obtain diosgenin anti-breast cancer active lead compounds with good efficacy and high bioavailability.

[0004] 1,2,3-Triazole is an important class of nitrogen-containing heterocyclic compounds with wide application value in the field of medicinal chemistry. In addition, heterocomplexes containing 1,2,3-triazole can exert dual or multiple anticancer mechanisms, constituting an important source for the development of new anticancer candidates. Summary of the invention

[0005] In order to solve the defects and shortcomings in the prior art, based on the principle of molecular hybridization, the present invention uses diosgenin as a lead compound and hybridizes it with 1,2,3-triazole structural fragments with different substituents to obtain a derivative represented by formula (I) or (II).

[0006] The primary purpose of the present invention is to obtain a diosgenin intermediate with a new modifiable site by structurally modifying diosgenin, and then hybridize the diosgenin intermediate with a triazole structural fragment to provide a series of diosgenin derivatives with significant anti-tumor activity.

[0007] Another object of the present invention is to provide a method for preparing the diosgenin derivative containing the 1,2,3-triazole structural fragment.

[0008] Another object of the present invention is to provide the use of the diosgenin derivative containing the 1,2,3-triazole structural fragment in the preparation of anti-tumor drugs.

[0009] The present invention provides the following detailed technical solutions:

[0010] The diosgenin derivative containing 1,2,3-triazole provided by the present invention has a structure as shown in formula (I) or (II), or a pharmaceutically acceptable salt of the derivative;

[0011]

[0012] Wherein, R is a substituted or unsubstituted alkyl, benzyl or heterocyclic structure; the substituent is selected from hydroxyl, C1-C6 alkyl, cycloalkyl, fluorine, chlorine, bromine, cyano or amino.

[0013] Wherein, when R is a substituted or unsubstituted alkyl structure, R in formula (I) or (II) is selected from the following groups;

[0014]

[0015] Wherein, when R is a substituted or unsubstituted benzyl structure, R in formula (I) or (II) is selected from the following groups;

[0016]

[0017] Wherein, when R is a substituted or unsubstituted heterocyclic structure, R in formula (I) or (II) is selected from the following groups;

[0018]

[0019] Preferably, when R is a substituted or unsubstituted alkyl structure, R in formula (I) or (II) is selected from the following groups;

[0020]

[0021] Preferably, when R is a substituted or unsubstituted benzyl structure, R in formula (I) or (II) is selected from the following groups;

[0022]

[0023] The further preferred diosgenin derivatives containing 1,2,3-triazole structural fragments in the present invention are selected from the following structures:

[0024]

[0025] The diosgenin derivative containing a 1,2,3-triazole structural fragment represented by formula (I) or (II) of the present invention is prepared by the following method:

[0026]

[0027] a: pyridine, acetic anhydride;

[0028] b: glacial acetic acid, sodium cyanoborohydride;

[0029] c: Jones reagent;

[0030] d: propargylamine, TBTU, DIPEA;

[0031] e: azide, sodium ascorbate, CuSO4·5H2O;

[0032] f: alkali, preferably potassium hydroxide or sodium hydroxide;

[0033] The preparation method of the diosgenin derivative containing a 1,2,3-triazole structural fragment represented by formula (I) or (II) of the present invention comprises the following steps:

[0034] (1) dissolving diosgenin in a solvent, adding Ac2O and pyridine, stirring at room temperature, and refluxing the crude product with anhydrous ethanol to purify the intermediate compound 2;

[0035] (2) Dissolving the intermediate compound 2 in a solvent, adding glacial acetic acid and NaBH3CN, stirring at room temperature, and separating and purifying the crude product to obtain the intermediate compound 3;

[0036] (3) Dissolve the intermediate compound 3 in a solvent, add Jones reagent dropwise at room temperature until the solution turns orange, stir at room temperature, and separate and purify the crude product to obtain the intermediate compound 4;

[0037] (4) dissolving the intermediate compound 4 in a solvent, adding propargylamine, benzotriazoletetramethyltetrafluoroboric acid (TBTU) and N,N-diisopropylethylamine (DIPEA), stirring at room temperature, and separating and purifying the crude product to obtain the intermediate compound 5;

[0038] (5) dissolving the intermediate compound 5 and the azide in a solvent, adding CuSO4·5H2O and sodium ascorbate, stirring at room temperature, and separating and purifying the crude product to obtain the diosgenin derivative represented by formula (I);

[0039] (6) The diosgenin derivative represented by formula (I) is dissolved in a solvent, potassium hydroxide or sodium hydroxide is added, and the mixture is stirred at room temperature. The crude product is separated and purified to obtain the diosgenin derivative represented by formula (II).

[0040] The azide synthesis method of the present invention is:

[0041] Dissolve the halide in a solvent, add NaN3, and stir at room temperature to obtain the corresponding azide. The reaction solution can be used directly in the next step without treatment.

[0042] Wherein, the halogenated compound includes iodomethane, ethyl bromide, 2-bromopropane, isobutyl bromide, (bromomethyl)cyclopropane, (bromomethyl)cyclohexane, 2-bromoethanol, benzyl bromide, 4-methylbenzyl bromide, 4-fluorobenzyl bromide, 4-chlorobenzyl chloride, 4-bromobenzyl bromide, 4-cyanobenzyl bromide, 4-chloromethylpyridine hydrochloride, 3-(2-bromoethyl)indole, and 2-(chloromethyl)benzimidazole.

[0043] The solvent is selected from chloroform, dichloromethane, methanol, acetone, tetrahydrofuran, a mixed solution of dichloromethane and water, a mixed solution of dichloromethane and methanol, a mixed solution of tetrahydrofuran and acetone, petroleum ether, ethyl acetate, N,N-dimethylacetamide or N,N-dimethylformamide.

[0044] The crude product of the present invention is separated and purified, which means that the obtained crude product is separated and purified to obtain a fine product, and the separation and purification method is a combination of one or more methods of extraction, silica gel column chromatography, and thin layer chromatography. Among them, the eluent used for silica gel column chromatography purification is a petroleum ether / ethyl acetate mixed solution, or a dichloromethane / methanol mixed solution.

[0045] The present invention also provides a pharmaceutical composition, which comprises a diosgenin derivative containing a 1,2,3-triazole fragment or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0046] The present invention also provides use of the diosgenin derivative containing the 1,2,3-triazole fragment or a pharmaceutically acceptable salt thereof in preparing a drug for treating tumor diseases.

[0047] The present invention also provides the use of the pharmaceutical composition in preparing medicines for treating tumor diseases.

[0048] The tumor disease described in the present invention is preferably breast cancer, and the breast cancer includes estrogen receptor positive breast cancer and triple negative breast cancer.

[0049] Beneficial effects of the present invention:

[0050] The synthesis method of the present invention has simple and efficient conditions, no need for heating, low equipment requirements and high yield. The method of hybridizing natural products and 1,2,3-triazole structural fragments proposed in the present invention is used to develop new anticancer drugs, and realize the development of multi-target and multifunctional anti-breast cancer drugs. The diosgenin derivatives containing 1,2,3-triazole fragments provided by the present invention have good anti-breast cancer activity. The results of pharmacological experiments show that the anti-tumor activity of some derivatives on human breast cancer cells (MDA-MB-231, MDA-MB-468 and MCF-7) is significantly better than that of diosgenin, and the toxicity to normal human gastric mucosal epithelial cells is relatively small, so it is expected to be further developed into anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The effect of compound 61 on apoptosis of MCF-7 cells. (A) Flow cytometry analysis of apoptosis of MCF-7 cells treated with 61 at different concentrations for 48 hours; (B) Relative proportions of MCF-7 cells at different apoptotic stages treated with 61 at different concentrations. *p<0.05, **p<0.01, ***p<0.001, compared with cells at the corresponding apoptotic stages of the control group.

[0052] Figure 2 The effect of compound 6l on the apoptotic proteins Bax / Bcl-2 / Caspase8 / Cytochrome C / Caspase3 / Parp in MCF-7 cells. (A) The expression of apoptotic proteins in MCF-7 cells treated with 6l at different concentrations for 48 hours; (B) The effect of 6l at different concentrations on the relative content (%) of apoptotic proteins in MCF-7 cells. *p<0.05, **p<0.01 and ***p<0.001, compared with the corresponding proteins in the control group.

[0053] Figure 3 The effect of compound 6l on the cell cycle of MCF-7 cells. (A) Flow cytometry analysis of the cell cycle of MCF-7 cells treated with 6l at different concentrations for 48 h; (B) The relative proportion of DNA in MCF-7 cells at different stages under 6l treatment. *p<0.05, **p<0.01, ***p<0.001, compared with the control group. DETAILED DESCRIPTION

[0054] The preferred embodiments of the present invention are described below. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention. If specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this area or the product instructions are used. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0055] Example 1

[0056]

[0057] Dissolve diosgenin (30.0 g, 72.4 mmol) in CH2Cl2 (210 mL) solution, add Ac2O (14.8 g, 144.9 mmol) and pyridine (60 mL), stir at room temperature for 6 h, and monitor the reaction by thin layer chromatography (petroleum ether: ethyl acetate = 3:1). The reaction solution is extracted with distilled water (300 mL × 3), washed with saturated NaHCO3 solution (300 mL × 3), washed with saturated brine (300 mL × 3), dried with anhydrous Na2SO4, filtered to remove solids, concentrated under reduced pressure, and the crude product is purified by refluxing with anhydrous ethanol to obtain a white solid powder with a yield of 97.1% and mp of 194.8-195.6 ° C. 1 HNMR (600MHz, CDCl3, δ): 5.37 (d, J=4.9Hz, 1H, H-6), 4.60 (m, 1H, H-3), 4.41 (dd, J=14.8, 7.2Hz, 1H, H-16), 3.47 (dd, J=10.1, 4.1Hz, 1H, H-26), 3.37 (t ,J=11.0Hz,1H,H-26),2.03(s,3H,Ac-CH3),1.03(s,3H,19-CH3),0.97(d, J=7.0Hz,3H,21-CH3),0.78(s,3H,18-CH3),0.78(d,J=5.6Hz,3H,27-CH3). 13 C NMR (150MHz, CDCl3, δ): 170.66 (Ac-CO), 139.84 (C-5), 122.51 (C-6), 109.41 (C-22), 80.95 (C-16), 74.03 (C-3), 66.99(C-26),62.23(C-17),56.57(C-14),50.08(C-9),41.76(C-20),40.40(C-12),39.87(C-13),38.24(C-4),3 7.10(C-1),36.88(C-10),32.18(C-7),31.99(C-15),31.55(C-8),30.45(C-23),29.85(C-25),28.95(C-2),27. 88(C-24),21.57(Ac-CH3),20.95(C-11),19.47(C-19),17.28(C-27),16.42(C-18),14.67(C-21).ESI-HRMS:m / z 457.3330[M+H]+ (Calcd for C 29 H 45 O4,457.3312).

[0058] Example 2

[0059]

[0060] The intermediate compound 2 (20.0 g, 43.9 mmol) was dissolved in CH2Cl2 (80 mL) solution, glacial acetic acid (20 mL) and NaBH3CN (5.53 g, 87.8 mmol) were added, and the mixture was stirred at room temperature for 8 h. The reaction was monitored by thin layer chromatography (petroleum ether: ethyl acetate = 15:1) to complete the reaction. The reaction solution was alkalized with saturated Na2CO3 solution, washed with distilled water (100 mL × 3), washed with saturated brine (100 mL × 3), dried with anhydrous Na2SO4, filtered to remove the solid, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 20:1-15:1) to obtain a white solid powder with a yield of 81.0%. mp: 107.2-108.6 ° C. 1 H NMR (600MHz, CDCl3, δ): 5.36 (d, J=5.0Hz, 1H, H-6), 4.59 (m, 1H, H-3), 4.30 (m, 1H ,H-16),3.49(dd,J=10.6,6.0Hz,1H,H-26),3.43(dd,J=10.6,6.0Hz,1H,H-26), 3.32(td,J=8.3,3.7Hz,1H,H-22),2.02(s,3H,Ac-CH3),1.03(s,3H,19-CH3),1. 00(d,J=6.7Hz,3H,21-CH3),0.91(d,J=6.8Hz,3H,27-CH3),0.80(s,3H,18-CH3). 13C NMR (150MHz, CDCl3, δ): 170.68 (Ac-CO), 139.81 (C-5), 122.52 (C-6), 90.50 (C-22), 83.35 (C-16), 74.02 (C-3), 6 8.22(C-26),65.22(C-17),57.03(C-14),50.13(C-9),40.83(C-12),39.54(C-13),38.22(C-4),38.07(C-20),3 7.13(C-1),36.84(C-10),35.89(C-25),32.36(C-7),32.12(C-15),31.69(C-8),30.61(C-24),30.30(C-23),27 .88(C-2),21.57(Ac-CH3),20.78(C-11),19.46(C-19),19.06(C-21),16.77(C-27),16.58(C-18).ESI-HRMS:m / z 459.3472[M+H] + (Calcd for C 29 H 47 O4,459.3469).

[0061] Example 3

[0062]

[0063] Preparation of Jones reagent: Dissolve CrO3 (13.4 g) in a small amount of water, slowly add concentrated sulfuric acid (12 mL), stir until dissolved, and dilute to 50 mL with distilled water.

[0064] The intermediate compound 3 (10.0 g, 21.8 mmol) was dissolved in a mixed solution of THF (50 mL) and acetone (50 mL), and Jones reagent was added dropwise at room temperature until the solution turned orange, and stirred at room temperature for 2 h. The reaction was monitored to be complete by thin layer chromatography (petroleum ether: ethyl acetate = 5:1). The reaction solution was filtered to remove the precipitate, concentrated under reduced pressure, washed with saturated brine (100 mL × 3), dried with anhydrous Na2SO4, and concentrated under reduced pressure. Purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1-4:1) to obtain a white solid powder with a yield of 76.0%. mp: 124.5-125.2°C. 11H NMR (600 MHz, CDCl3, δ): 5.37 (d, J = 4.8 Hz, 1H, H-6), 4.60 (m, 1H, H-3), 4.33 (m, 1H, H-16), 3.35 (td, J = 8.6, 2.4 Hz, 1H, H-22), 2.52 (m, 1H, H-25), 2.03 (s, 3H, Ac-CH3), 1.18 (d, J = 6.9 Hz, 3H, 27-CH3), 1.03 (s, 3H, 19-CH3), 1.00 (d, J = 6.7 Hz, 3H, 21-CH3), 0.79 (s, 3H, 18-CH3). 13 13C NMR (150 MHz, CDCl3, δ): 181.16 (C-26), 170.74 (Ac-CO), 139.83 (C-5), 122.47 (C-6), 90.43 (C-22), 83.66 (C-16), 74.03 (C-3), 64.98 (C-17), 57.04 (C-14), 50.11 (C-9), 40.85 (C-12), 39.53 (C-13), 39.50 (C-25), 38.22 (C-4), 38.16 (C-20), 37.13 (C-1), 36.84 (C-10), 32.30 (C-7), 32.10 (C-15), 31.67 (C-8), 31.56 (C-24), 31.07 (C-23), 27.88 (C-2), 21.57 (Ac-CH3), 20.76 (C-11), 19.46 (C-19), 18.97 (C-21), 17.39 (C-27), 16.52 (C-18). ESI-HRMS: m / z 473.3220 [M+H] + (Calcd for C 29 H 45 O5, 473.3262).

[0065] Example 4

[0066]

[0067] The intermediate compound 4 (5.0 g, 10.6 mmol) was dissolved in CH2Cl2 (80 mL) solution, and propargylamine (1.17 g, 21.2 mmol), TBTU (4.08 g, 12.72 mmol) and DIPEA (2.74 g, 21.2 mmol) were added, and stirred at room temperature for 2 h. The reaction was monitored by thin layer chromatography (petroleum ether: ethyl acetate = 1:1). The reaction solution was washed with distilled water (100 mL × 3), washed with saturated brine (100 mL × 3), dried with anhydrous Na2SO4, and filtered to remove the solid. The mixture was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 9:1-5:1) to obtain the intermediate compound 5 with a yield of 81.0%. mp: 139.8-140.6 °C. 1 HNMR (600MHz, CDCl3, δ): 6.51 (brs, 1H, NH), 5.37 (d, J = 5.1Hz, 1H, H-6), 4.59 (m,1H,H-3),4.33(m,H,H-16),4.02(m,2H,28-CH2),3.31(td,J=2.6,8.6Hz,1 H,H-22),2.45(s,1H,H-30),2.02(s,3H,Ac-CH3),1.11(d,J=6.8Hz,3H,27-CH 3),1.03(s,3H,19-CH3),0.98(d,J=6.8Hz,3H,21-CH3),0.80(s,3H,18-CH3). 13 C NMR (150MHz, CDCl3, δ): 176.40 (C-26), 170.71 (Ac-CO), 139.88 (C-5), 122.41 (C-6), 91.45 (C-22), 83.77 (C-16), 80.09 (C- 29),73.99(C-3),71.51(C-30),64.51(C-17),57.03(C-14),50.08(C-9),40.83(C-12),39.94(C-13),39.50(C-25),38.58( C-4),38.21(C-20),37.10(C-1),36.82(C-10),33.56(C-28),32.42(C-7),32.09(C-15),31.69(C-8),30.62(C-24),28.96 (C-23),27.85(C-2),21.57(Ac-CH3),20.75(C-11),19.48(C-29),18.73(C-21),17.93(C-27),16.71(C-18).ESI-HRMS:m / z 510.3548[M+H] + (Calcd for C32 H 48 NO4,510.3578).

[0068] Example 5

[0069]

[0070] Preparation of (azidomethyl)cyclohexane: (bromomethyl)cyclohexane (400 mg, 2.26 mmol) was dissolved in DMF (50 mL), and NaN3 (293.6 mg, 4.52 mmol) was added. The mixture was stirred at room temperature for 4 h, and the reaction was complete after monitoring by thin layer chromatography (petroleum ether: ethyl acetate = 5:1). The reaction solution was used directly in the next step without further treatment.

[0071] The intermediate compound 5 (150 mg, 0.295 mmol) was added to the DMF solution of the azide prepared in the previous step, and 5 mL CH2Cl2 and 5 mL H2O were added. Sodium ascorbate (233.6 mg, 1.18 mmol) and CuSO4·5H2O (73.8 mg, 0.295 mmol) were added under stirring. The reaction was stirred at room temperature for 4 h. The reaction was complete by TLC (petroleum ether: ethyl acetate = 1:1). The reaction solution was washed with distilled water (100 mL × 3), washed with saturated brine (100 mL × 3), dried over anhydrous Na2SO4, filtered to remove the solid, and concentrated under reduced pressure to obtain a dark green solid. Purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1-1:1), 6 g of white solid powder was obtained, with a yield of 83.6%. mp: 117.8-119.5 ° C. 1 HNMR(600MHz,CDCl3,δ):7.49(s,1H,H-30),6.66(br s,1H,NH),5.36(d,J=5.0Hz,1H,H-6),4.59(m,1H,H-3),4.48(m,2H,28-CH2),4.28(m,1H,H-16),4.12(d,J=7.2Hz,2H,31-CH2),3.26(t,J=7. 2Hz,1H,H-22),2.02(s,3H,Ac-CH3),1.10(d,J=6.8Hz,3H,27-CH3),1.03(s,3H,19-CH3),0.94(d,J=6.8Hz,3H,21-CH3),0.73(s,3H,18-CH3). 13C NMR (150MHz, CDCl3, δ): 176.82 (C-26), 170.70 (Ac-CO), 144.79 (C-29), 139.80 (C-5), 122.92 (C-30), 122.47 (C-6), 90.87 (C-22), 83.55 ( C-16),74.01(C-3),64.72(C-17),57.01(C-14),56.73(C-31),50.09(C-9),40.76(C-12),40.59(C-25),39.51(C-13),38.84(2C,C-33and C-37),38.36(C-20),38.21(C-4),37.11(C-1),36.83(C-10),34.87(C-28),32.84(C-24),32.38(C-7) ,32.09(C-15),31.64(C-8),30.94(C-23),30.59(C-32),27.86(C-2),26.16(C-35),25.61(2C,C-34and C-36),21.58(Ac-CH3),20.76(C-11),19.49(C-19),18.80(C-21),18.00(C-17),16.54(C-18).ESI-HRMS:m / z649.4688[M+H] + (Calcd for C 39 H 61 N4O4,649.4687).

[0072] Example 6

[0073]

[0074] The target compound 6i was prepared by referring to the synthesis method of Example 5. White solid powder, yield: 86.0%, mp 94.1-95.6℃. 11H NMR (600 MHz, CDCl3, δ): 7.42 (s, 1H, H-30), 7.14 (m, 4H, Ar-H), 6.68 (br s, 1H, N-H), 5.41 (s, 2H, 31-CH2), 5.36 (d, J = 4.6 Hz, 1H, H-6), 4.58 (m, 1H, H-3), 4.43 (m, 2H, 28-CH2), 4.26 (m, 1H, H-16), 3.24 (td, J = 1.9 Hz, 8.9 Hz, H-22), 2.32 (s, 3H, 38-CH3), 2.02 (s, 3H, Ac-CH3), 1.07 (d, J = 6.7 Hz, 3H, 27-CH3), 1.03 (s, 3H, 19-CH3), 0.92 (d, J = 6.7 Hz, 3H, 21-CH3), 0.70 (s, 3H, 18-CH3). 13 13C NMR (150 MHz, CDCl3, δ): 176.73 (C-26), 170.68 (Ac-CO), 145.47 (C-35), 139.77 (C-5), 138.77 (C-32), 131.65 (C-29), 129.85 (2C, C-34 and C-36), 128.19 (2C, C-33 and C-37), 122.44 (C-6), 122.12 (C-30), 90.81 (C-22), 83.50 (C-16), 73.99 (C-3), 64.69 (C-17), 56.98 (C-14), 54.05 (C-31), 50.07 (C-9), 40.72 (C-12), 40.54 (C-25), 39.47 (C-13), 38.29 (C-20), 38.18 (C-4), 37.08 (C-1), 36.79 (C-10), 34.89 (C-28), 32.76 (C-24), 32.34 (C-7), 32.06 (C-15), 31.60 (C-8), 30.89 (C-23), 27.83 (C-2), 21.54 (Ac-CH3), 21.26 (C-38), 20.72 (C-11), 19.46 (C-19), 18.77 (C-21), 17.94 (C-27), 16.48 (C-18). ESI-HRMS: m / z 657.4371 [M+H]+ (Calcd for C 40 H 57 N4O4, 657.4374).

[0075] Example 7

[0076]

[0077] The target compound 6k was prepared by referring to the synthesis method of Example 5. White solid powder, yield: 91.6%, mp: 136.1-137.2℃. 1 HNMR (600MHz, CDCl3, δ): 7.49 (s, 1H, H-30), 7.33 (d, J = 8.4Hz, 2H, Ar-H), 7.18 (d, J = 8.0Hz, 2H, Ar-H), 6.67 (br s,1H,NH),5.44(s,2H,31-CH2),5.37(d,J=4.4Hz,1H,H-6),4.60(m,1H,H-3),4.46(m,2H,28-CH2),4.27(m,1H,H-16),3.25(t,J=8.6Hz ,H-22),2.02(s,3H,Ac-CH3),1.08(d,J=6.7Hz,3H,27-CH3),1.03(s,3H,19-CH3),0.93(d,J=6.7Hz,3H,21-CH3),0.71(s,3H,18-CH3). 13 C NMR (150MHz, CDCl3, δ): 176.85 (C-26), 170.70 (Ac-CO), 145.74 (C-32), 139.82 (C- 5),134.98(C-35),133.14(C-29),129.50(2C,C-34andC-36),129.46(2C,C-33and C-37),122.45(2C,C-6and C-30),90.89(C-22),83.57(C-16),74.01(C-3),64.68(C-17),57.02(C-14),53.58(C-31),50.10(C -9),40.76(C-12),40.49(C-25),39.50(C-13),38.37(C-20),38.22(C-4),37.11(C-1),36.83(C-10 ),34.85(C-28),32.88(C-24),32.40(C-7),32.10(C-15),31.64(C-8),30.87(C-23),27.87(C-2),2 1.58(Ac-CH3),20.76(C-11),19.50(C-19),18.78(C-21),17.96(C-27),16.53(C-18).ESI-HRMS:m / z 677.3858[M+H] + (Calcd for C 39 H 54 ClN4O4,677.3828).

[0078] Example 8

[0079]

[0080] The target compound 61 was prepared by referring to the synthesis method of Example 5. White solid powder, yield: 82.0%, mp: 135.5-137.1°C. 1 HNMR(600MHz,CDCl3,δ):7.48(br s,2H,Ar-H),7.47(s,1H,H-30),7.12(d,J=7.8Hz,2H,Ar-H),6.67(m,1H,NH),5.42(s,2H,31-CH2),5.37(d,J=4. 4Hz,1H,H-6),4.59(m,1H,H-3),4.45(m,2H,28-CH2),4.27(m,1H,31-H-16),3.25(t,J=8.7Hz,1H,H-22),2.02(br s,3H,Ac-CH3),1.08(d,J=6.7Hz,3H,27-CH3),1.03(s,3H,19-CH3),0.93(d,J=6.9Hz,3H,21-CH3),0.71(s,3H,18-CH3). 13 C NMR (150MHz, CDCl3, δ): 176.84 (C-26), 170.70 (Ac-CO), 145.80 (C-32), 13 9.82(C-5),133.71(C-29),132.41(C-34,C-36),129.76(C-33,C-37),123. 07(C-35),122.45(C-6),122.41(C-30),90.88(C-22),83.56(C-16),74.00 (C-3),64.67(C-17),57.01(C-14),53.59(C-31),50.09(C-9),40.75(C-12 ),40.47(C-25),39.49(C-13),38.36(C-20),38.21(C-4),37.10(C-1),36. 82(C-10),34.88(C-28),32.86(C-24),32.39(C-7),32.09(C-15),31.63(C -8),30.85(C-23),27.86(C-2),21.57(Ac-CH3),20.75(C-11),19.49(C-19 ),18.78(C-21),17.95(C-27),16.52(C-18).ESI-HRMS:m / z721.3303[M+H]+ (Calcd for C 39 H 54 BrN4O4,721.3323).

[0081] Example 9

[0082]

[0083] Compound 6k (135.3 mg, 0.2 mmol) was dissolved in MeOH (6 mL), and KOH (33.6 mg, 0.6 mmol) was added. The mixture was stirred at room temperature for 2 h. The reaction was complete by TLC (dichloromethane: methanol = 15: 1). After concentration under reduced pressure, the mixture was washed with distilled water (100 mL × 3), washed with saturated brine (100 mL × 3), dried over anhydrous Na2SO4, filtered to remove the solid, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane: methanol = 70: 1-15: 1) to obtain white solid powder 7k with a yield of 89.5%. mp: 132.0-133.4 ° C. 1 HNMR (600MHz, CDCl3, δ): 7.49 (s, 1H, H-30), 7.33 (d, J=7.0Hz, 2H, Ar-H), 7.18 (d, J=7.0Hz, 2H, Ar-H), 6.69 (br s, 1H, NH), 5.44 (s, 2H, 31-CH2), 5.34 (br s,1H,H-6),4.46(m,2H,28-CH2),4.27(m,1H,H-16),3.51(m,1H,H-3),3.25(t,J=8.8Hz,H-22),1.09 (d,J=6.7Hz,3H,27-CH3),1.02(s,3H,19-CH3),0.93(d,J=6.8Hz,3H,21-CH3),0.72(s,3H,18-CH3). 13C NMR (150MHz, CDCl3, δ): 176.88(C-26), 145.86(C-32), 140.94(C-5), 135.00(C-35), 133.12(C-29), 129.50(2C, C-34and C-36), 129.47(2C, C-33and C-37),122.48(C-6),121.53(C-30),90.82(C-22),83.57(C-16),71.86(C-3),64.73(C-17),57.11(C-1 4),53.59(C-31),50.21(C-9),42.41(C-4),40.78(C-12),40.55(C-25),39.56(C-13),38.33(C-20),37 .38(C-1),36.75(C-10),34.81(C-28),32.76(C-24),32.41(C-7),32.12(C-15),31.75(C-8),31.69(C- 2),30.89(C-23),20.82(C-11),19.59(C-19),18.80(C-21),17.93(C-27),16.54(C-18).ESI-HRMS:m / z 635.3738[M+H] + (Calcd for C 37 H 52 ClN4O3,635.3722).

[0084] Example 10

[0085]

[0086] The target compound 71 was prepared by referring to the synthesis method of Example 9. White solid powder, yield: 79.0%, mp: 133.5-134.9°C. 1HNMR (600MHz, CDCl3, δ): 7.48 (br s, 2H, 2H, Ar-H), 7.47 (s, 1H, H-30), 7.11 (d, J = 8.3Hz, 2H, Ar-H), 6.71 (br s,1H,NH),5.42(s,2H,31-CH2),5.34(d,J=4.1Hz,H-6),4.44(m,2H,28-CH2),4.27(m,1H,H-16),3.51(m,1H,H-3),3.25(t,J =7.3Hz,1H,H-22),1.08(d,J=6.8Hz,3H,27-CH3),1.02(s,3H,19-CH3),0.93(d,J=6.8Hz,3H,21-CH3),0.71(s,3H,18-CH3). 13 C NMR (150MHz, CDCl3, δ): 176.87(C-26), 145.79(C-32), 140.95(C-5), 133.74(C-29), 132.39(2C, C-34and C-36), 129.76(2C, C-33and C-37),123.05(C-35),122.39(C-6),121.49(C-30),90.79(C-22),83.56(C-16),71.81(C-3),64.70(C-17) ,57.08(C-14),53.57(C-31),50.19(C-9),42.39(C-4),40.76(C-12),40.50(C-25),39.54(C-13),38.31(C -20),37.37(C-1),36.74(C-10),34.88(C-28),32.74(C-24),32.39(C-7),32.10(C-15),31.72(C-2),31.6 8(C-8),30.84(C-23),20.81(C-11),19.58(C-19),18.79(C-21),17.91(C-27),16.52(C-18).ESI-HRMS:m / z 679.3205[M+H] + (Calcd for C 37 H 52 BrN4O3,679.3217).

[0087] Example 11: In vitro cell antiproliferative activity test

[0088] Human breast cancer cells MDA-MB-231, MDA-MB-468, and MCF-7 in logarithmic growth phase and human gastric mucosal cells GES-1 were selected and cultured at 1×104 / cell / well, 100 μL / well was seeded into a 96-well plate. After overnight attachment, the culture medium was removed and a series of concentrations (1-100 μM) of compounds prepared in DMSO were added (the final concentration of DMSO was kept at one thousandth, that is, the corresponding reserve solution was added to the cell culture medium according to the corresponding reserve solution concentration (such as 1 μL was added to 999 μL of cell culture medium)). In addition, cisplatin (1.875-120 μM or 1-100 μM) was used as a positive control (dissolved in physiological saline). After 48 hours of treatment, 10 μL of CCK-8 solution (YEASEN, Shanghai, CN) was added to each 96-well incubator and incubated for 2 hours. The absorbance was measured at a wavelength of 450 nm by an enzyme reader. The relative inhibition rate of each well was calculated according to the formula "cell viability*(%) = [A(drug group)-A(blank group)] / [A(control group)-A(blank group)]×100" and the IC was calculated based on the inhibition rate. 50 value.

[0089] The experimental results of the compounds are shown in Table 1 below.

[0090] Table 1: Inhibitory activities of diosgenin derivatives containing 1,2,3-triazole structure fragments on tumor cells and normal cells (IC 50 , μM)

[0091]

[0092]

[0093] The results of in vitro anti-tumor experiments show that compared with the parent compound diosgenin, the diosgenin derivatives containing 1,2,3-triazole structural fragments of the present invention have better anti-proliferative activity against three breast cancer cell lines, and are relatively less toxic to human normal gastric mucosal epithelial cells GES-1, showing good selectivity and the potential for further preparation as drugs for the treatment of breast cancer. Among them, derivatives 6k, 6l and 7l showed significant anti-proliferative activity against MDA-MB-231 cells and MCF-7 cells, IC 50 The value was less than 10 μM. Derivative 6l showed the strongest cytotoxicity against MCF-7 cells, IC 50 The value was 6.2 μM, which was significantly stronger than diosgenin.

[0094] Example 12: Effect of Compound 61 on Apoptosis of MCF-7 Cells

[0095] (1) Experimental methods:

[0096] MCF-7 cells were seeded in 24-well plates (2×10 5 / well), 1, 3 and 9 μmol / L doses of compound 61 were prepared with DMSO and added to 24-well plates respectively, placed at 37°C for 48h, trypsin digestion and collection of cells, then the excess trypsin was washed with cold PBS, centrifuged to remove the supernatant, and the culture medium containing 1‰ DMSO was used as the control group. The cells were resuspended with 1× binding buffer in the Annexin / PI double staining kit (Solarbio, Beijing, CN), and then a certain amount of Annexin was added for incubation for 15min, and then a certain amount of PI and a certain amount of PBS were added to prepare for the flow cytometer (CytoFlex, Beckmancoulter, CA, USA) to detect the apoptosis of each group of cells, and the corresponding control tubes with single staining of Annexin or single staining of PI or no staining were prepared at the same time before the sample loading test to adjust the instrument voltage gain and fluorescence channel compensation.

[0097] (2) Experimental results:

[0098] After treatment with compound 61 (1, 3, 9 μmol / L) for 48 h, the effects of each drug-treated group on cell apoptosis were as follows: Figure 1 A and Figure 1 As shown in Figure B, it can be seen that with the increase in the dosage, the proportion of apoptotic cells increased significantly. The results showed that compound 6l could significantly reduce the proportion of normal cells at concentrations of 1, 3, and 9 μmol / L (p<0.05), and it was dose-dependent. At all doses, the proportion of early apoptosis and late apoptosis of cells was significantly increased compared with the control group. The above results suggest that 6l can cause apoptosis of MCF-7 cells in a dose-dependent manner.

[0099] Example 13: Effect of Compound 61 on apoptotic proteins in MCF-7 cells

[0100] (1) Experimental methods:

[0101] Antibodies Bax (50599-2-Ig, Proteintech, Wuhan, CN), Bcl-2 (15071, CST, Boston, MA, USA), Cleaved caspase 8 (sc-166320, Santa Cruz, Shanghai, CN), Caspase 3 (66470-2-Ig, Proteintech, Wuhan, CN), Parp (9532, CST, Boston, MA, USA). MCF-7 cells were cultured at 1 × 10 6Cells were seeded into 6-well plates at 100 μg / well. The medium containing 1‰ DMSO was used as the control group. Different concentrations of 6l were used for 48 h. Cells were lysed and proteins were extracted using 1× cell lysis buffer (9803, CST, CA, USA). Protein quantification was then performed using a BCA kit (23225, Thermo, CA, USA). Mixed proteins were separated by SDS-PAGE, and the concentration of the separation gel used was 12%. The gel preparation process was performed according to the instructions in the SDS PAGE gel preparation kit. The total cell protein loading was controlled to be 20 μg. After SDS-PAGE gel separation, it was transferred to a PVDF membrane (Merck millipore, CA, USA). After blocking with 5% skim milk, the first and second antibodies were incubated at 4°C, and HRP development detection (Merck millipore, WBULS0500) and chemiluminescence development instrument (LAS4000, GE healthcare) were performed to detect chemiluminescence. Photos were taken and grayscale analysis was performed using Image J.

[0102] (2) Experimental results:

[0103] After treatment with compound 61 at 1, 3, and 9 μmol / L for 48 h, the expression levels of apoptotic protein Bax and cleaved Parp were significantly upregulated (Parp: a substrate of apoptosis), while the expression levels of anti-apoptotic protein Bcl-2 and anti-apoptotic protein Bcl-xl were significantly downregulated ( Figure 2 A). The ratio of Bcl-2 / Bax represents the cell's ability to resist apoptosis. It can be seen that with the increase in dose, the cell's ability to resist apoptosis significantly decreased. In addition, for the exogenous apoptosis pathway protein caspase8, compound 6l seems to favor the exogenous apoptosis pathway at low doses, and the intrinsic apoptosis pathway at high doses. However, for the cleavage of caspase3 and Parp, only 3 and 9 μmol / L showed a significant increase compared with the control group, which means that the cells feel the apoptotic pressure and progress towards mitochondrial-mediated apoptosis, suggesting that with the increase in concentration, compound 6l induces apoptosis in MCF-7 cells in a dose-dependent manner ( Figure 2 B).

[0104] Example 14: Effect of Compound 61 on MCF-7 Cell Cycle

[0105] (1) Experimental methods:

[0106] MCF-7 cells were grown at 2×10 51×10 cells / well were seeded into a 24-well plate, and 1, 3, and 9 μmol / L of compound 61 were prepared with DMSO and added to the 24-well plate. The plates were placed at 37°C for 48 h, and the cells were collected by trypsin digestion. The excess trypsin was then washed with cold PBS, and the supernatant was removed by centrifugation. The cells were washed with PBS and the cell concentration was adjusted to about 1×10 6 / mL, and the cells were obtained by centrifugation again, and fixed with 70% ethanol at 4°C overnight, and the excess fixative was washed off with PBS before staining; then the cells were resuspended with RNase A solution in the cell cycle kit (Solarbio, Beijing, CN), and the cells were bathed in 37°C water for 30 minutes, and then PI staining solution was added and mixed, and incubated at 4°C in the dark for 30 minutes. After staining, all samples were prepared for detection on the PI channel of the flow cytometer (CytoFlex, Beckman coulter, CA, USA).

[0107] (2) Experimental results:

[0108] After treatment with compound 61 (1, 3, 9 μmol / L) for 48 h, the effects of each drug-treated group on the cell cycle were as follows: Figure 3 As shown in A and 3B, it can be seen that with the increase in the dosage, the number of cells in the G2 / M phase increased significantly. The results showed that compound 6l could significantly reduce the G0 / G1 ratio (p<0.05) at concentrations of 1, 3, and 9 μmol / L, and increase the G2 / M ratio in a dose-dependent manner. This indicates that compound 6l causes cell damage as the dose increases. In the case of damage, the cells start the self-repair mechanism and stagnate at the G2 / M checkpoint. Therefore, compound 6l can cause G2 / M phase arrest of cells.

[0109] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A diosgenin derivative containing a 1,2,3-triazole structural fragment or a pharmaceutically acceptable salt thereof, characterized in that: Its structure is shown in formula (Ⅰ) or (ⅠⅠ); Wherein, R is a substituted or unsubstituted alkyl, benzyl or heterocyclic structure; the substituent is selected from hydroxyl, C1-C6 alkyl, cycloalkyl, fluorine, chlorine, bromine, cyano or amino.

2. The diosgenin derivative containing a 1,2,3-triazole structural fragment or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: When R is a substituted or unsubstituted alkyl structure, R in formula (I) or (II) is selected from the following groups; When R is a substituted or unsubstituted benzyl structure, R in formula (I) or (II) is selected from the following groups; When R is a substituted or unsubstituted heterocyclic structure, R in formula (I) or (II) is selected from the following groups; 3. The diosgenin derivative containing a 1,2,3-triazole structural fragment or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: When R is a substituted or unsubstituted alkyl structure, R in formula (I) or (II) is selected from the following groups; When R is a substituted or unsubstituted benzyl structure, R in formula (I) or (II) is selected from the following groups; 4. The diosgenin derivative containing a 1,2,3-triazole structural fragment or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: The diosgenin derivative containing a 1,2,3-triazole structural fragment is selected from the following structures 5. The method for preparing the diosgenin derivative containing a 1,2,3-triazole structural fragment or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: a: pyridine, acetic anhydride; b: glacial acetic acid, sodium cyanoborohydride; c: Jones reagent; d: propargylamine, TBTU, DIPEA; e: azide, sodium ascorbate, CuSO4·5H2O; f: potassium hydroxide or sodium hydroxide; (1) dissolving diosgenin in a solvent, adding acetic anhydride and pyridine, stirring at room temperature, and refluxing the crude product with anhydrous ethanol to purify the intermediate compound 2; (2) Dissolving the intermediate compound 2 in a solvent, adding glacial acetic acid and NaBH3CN, stirring at room temperature, and separating and purifying the crude product to obtain the intermediate compound 3; (3) Dissolve the intermediate compound 3 in a solvent, add Jones reagent dropwise at room temperature until the solution turns orange, stir at room temperature, and separate and purify the crude product to obtain the intermediate compound 4; (4) Dissolving the intermediate compound 4 in a solvent, adding propargylamine, TBTU and DIPEA, stirring at room temperature, and separating and purifying the crude product to obtain the intermediate compound 5; (5) dissolving the intermediate compound 5 and the azide in a solvent, adding CuSO4·5H2O and sodium ascorbate, stirring at room temperature, and separating and purifying the crude product to obtain the diosgenin derivative represented by formula (I); (6) dissolving the diosgenin derivative represented by formula (I) in a solvent, adding potassium hydroxide or sodium hydroxide, stirring at room temperature, and separating and purifying the crude product to obtain the diosgenin derivative represented by formula (II); The solvent is selected from chloroform, dichloromethane, methanol, acetone, tetrahydrofuran, a mixed solution of dichloromethane and water, a mixed solution of dichloromethane and methanol, a mixed solution of tetrahydrofuran and acetone, petroleum ether, ethyl acetate, N,N-dimethylacetamide or N,N-dimethylformamide.

6. An intermediate compound, characterized in that: The intermediate compound is used to prepare the diosgenin derivative containing a 1,2,3-triazole fragment or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, and the structural formula of the intermediate compound is:

7. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the diosgenin derivative containing a 1,2,3-triazole fragment or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

8. Use of the diosgenin derivative containing a 1,2,3-triazole fragment or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 in the preparation of a drug for treating tumor diseases.

9. Use of the pharmaceutical composition according to claim 7 in the preparation of drugs for treating tumor diseases.

10. The use according to claim 8 or claim 9, characterized in that: The tumor disease is breast cancer, which includes estrogen receptor positive breast cancer and triple negative breast cancer.

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