Halicogenin derivative with anti-tumor activity as well as preparation method and application thereof

Abstract

The invention relates to the fields of natural medicines and medicinal chemistry, and relates to a heicogenin derivative as well as a preparation method and application thereof. The invention particularly relates to a preparation method for introducing amino acid, chlorambucil benzoate and chlorambucil butyrate derivatives at C3-OH sites of a heicogenin mother nucleus structure and application of the derivatives in preparation of anti-tumor drugs. The structure of the heicogenin spliced amino acid, chlorambucil benzoate and chlorambucil benzoate derivative and the pharmaceutically acceptable salt thereof is shown as a general formula I, II or III. Pharmacological experiments prove that the heicogenin derivative disclosed by the invention has a relatively good anti-tumor effect on human breast cancer cell lines and relatively low toxicity on human normal gastric mucosa epithelial cells GES-1, so that the heicogenin derivative has the potential of being developed into clinical anti-cancer drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of natural medicine and medicinal chemistry, and to a hecosapogenin derivative with anti-tumor activity, a preparation method and an application thereof, and in particular to a hecosapogenin derivative with amino acids, benzoic acid mustard and chlorambucil introduced into the C3-OH site of the hecosapogenin parent nucleus structure, a preparation method thereof and an application thereof in the preparation of anti-tumor drugs. Background Art

[0002] Cancer is currently treated with a combination of surgery, chemotherapy, and radiotherapy. If necessary, targeted therapy, biological therapy, and other methods can be combined to improve the prognosis and treatment effect. Current clinical practice shows that chemotherapy is still one of the most effective methods for treating cancer, but its adverse side effects seriously limit its clinical application. Therefore, there is an urgent need to develop safe and effective new anticancer drugs. Natural products are one of the important sources of drug discovery, and the development of safe and effective anticancer drugs based on the structural modification of natural products has become a research hotspot for many scientific researchers.

[0003] Hecogenin (HG) is naturally present in the leaves of Agave plants and is one of the raw materials for synthesizing steroid hormones. In recent years, pharmacological studies have found that hecogenin has pharmacological activities such as anti-tumor, lowering blood cholesterol content, anti-diabetes, antioxidant, and anti-inflammatory. However, it only has weak to moderate activity against tumors, which seriously hinders its further clinical application. Therefore, it is necessary to modify its structure to improve its water solubility and anti-tumor activity.

[0004] Nitrogen mustards belong to biological alkylating agents, which are cytotoxic substances. From a structural point of view, this type of anti-tumor compound consists of two parts: an alkylation part and a carrier part. The alkylation part is the main part and also the functional group of anti-tumor activity. Chemical modification of the carrier part can improve the various pharmacokinetic properties of this type of drug in the body, thus affecting the selectivity, toxicity and anti-tumor activity of the drug. At present, there are several nitrogen mustard derivatives in clinical practice, such as melphalan, chlorambucil, cyclophosphamide, etc. However, they show certain drug resistance during treatment. Therefore, it is of great value to chemically modify nitrogen mustard drugs to improve their efficacy and reduce toxicity. It is a common strategy to combine natural products with nitrogen mustard drugs, which can not only improve activity, reduce toxicity, and improve drug properties, but also improve multidrug resistance.

[0005] The present invention uses hecosapogenin as a lead compound, utilizes the splicing principle, introduces amino acids, benzoic acid mustard and chlorambucil into C3-OH of its molecular structure, and designs and synthesizes hecosapogenin derivatives shown in general formulas I, II and III. Summary of the invention

[0006] The technical problem to be solved by the present invention is to design and synthesize hecosapogenin derivatives with good anti-tumor activity, and further provide a pharmaceutical composition containing the derivatives and the use of the derivatives and the pharmaceutical composition in the preparation of anti-tumor drugs.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] In the first aspect, the present invention provides a hecosapogenin derivative or a pharmaceutically acceptable salt thereof represented by general formula I, II or III:

[0009]

[0010] Among them, R 1 and R 2 Selected from one of the following groups:

[0011]

[0012] As an optional mode, in the above-mentioned hecosapogenin derivative or its pharmaceutically acceptable salt, the hecosapogenin derivative is selected from:

[0013]

[0014] In a second aspect, the present invention provides a pharmaceutical composition comprising the hecosapogenin derivative or pharmaceutically acceptable salt described in the first aspect and a pharmaceutically acceptable carrier.

[0015] As an optional mode, in the above-mentioned pharmaceutical composition, the pharmaceutical composition can be prepared into tablets, capsules or granules.

[0016] In a third aspect, the present invention provides a method for preparing the hecosapogenin derivative or a pharmaceutically acceptable salt thereof according to the first aspect, the method comprising the following steps:

[0017] (1) Dissolve hecosapogenin 1 in a solvent, then add a Boc-protected amino acid, react at room temperature under DCC and DMAP conditions, monitor the completion of the reaction by TLC, load the resulting product onto a silica gel column, and use a petroleum ether / ethyl acetate mixed solution for gradient elution to obtain intermediates 2a-2f;

[0018] (2) Dissolve the intermediate 2a-2f in a solvent, then add trifluoroacetic acid (TFA) to remove the Boc protection, react at room temperature, monitor the completion of the reaction by TLC, extract the reaction solution, load the obtained product onto a silica gel column chromatography, and use a dichloromethane / methanol mixed solution for gradient elution to obtain intermediates 3a-3f;

[0019] (3) The intermediates 3a-3f were dissolved in a solvent and reacted with benzoic acid mustard or chlorambucil under EDCI and DMAP conditions at room temperature. The reaction was monitored by TLC. The obtained product was loaded onto a silica gel column chromatography and gradient eluted with a petroleum ether / ethyl acetate mixed solution to obtain the target compounds 4a-4f or 5a-5f.

[0020]

[0021] a: dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP);

[0022] b: trifluoroacetic acid (TFA);

[0023] c: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), DMAP;

[0024] d: EDCI, DMAP.

[0025] In a fourth aspect, the present invention provides use of the hecosapogenin derivative or a pharmaceutically acceptable salt thereof as described in the first aspect in the preparation of a drug for treating tumor diseases.

[0026] In a fifth aspect, the present invention provides use of the pharmaceutical composition described in the second aspect in the preparation of a drug for treating tumor diseases.

[0027] As an optional mode, in the above application, the tumor is breast cancer.

[0028] Beneficial effects of the present invention:

[0029] (1) The hecosapogenin derivatives or pharmaceutically acceptable salts thereof of the present invention are based on hecosapogenin as a lead compound, and amino acids, benzoic acid mustard and chlorambucil are introduced into C3-OH of the molecular structure by the principle of splicing to design and synthesize hecosapogenin derivatives of general formula I, II or III. The spliced ​​compounds and their pharmaceutical compositions and preparations have good anti-tumor activity.

[0030] (2) Pharmacological experiments have shown that the hecosapogenin derivatives of the present invention have good anti-tumor effects on human breast cancer cell lines and relatively low toxicity to human normal gastric mucosal epithelial cells GES-1, and therefore have the potential to be developed into clinical anti-cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Effects of compound 5d on the morphology of MDA-MB-231 cells.

[0032] Figure 2: Effect of compound 5d on apoptosis of MDA-MB-231 cells under Hoechst 33342 / PI double staining.

[0033] Figure 3 :Effects of compound 5d on apoptosis of MDA-MB-231 cells. (A) Flow cytometry analysis of apoptosis of MDA-MB-231 cells treated with 5d at different concentrations for 48 h; (B) Relative proportions of different apoptotic stages of MDA-MB-231 cells treated with 5d at different concentrations. * p<0.05, ** p<0.01, *** p<0.001, compared with the cells in the corresponding apoptotic stage of the control group. DETAILED DESCRIPTION

[0034] Preparation Examples Section

[0035] Example 1

[0036] In a 50 mL dry eggplant-shaped bottle, add hecosapogenin (300 mg, 0.69 mmol) and 10 mL CH 2 Cl 2 After stirring and dissolving, DMAP (170.0 mg, 1.39 mmol), DCC (287.1 mg, 1.39 mmol) and 4-(Boc-amino)-butyric acid (282.9 mg, 1.39 mmol) were added and stirred at room temperature for 8 h. The reaction was monitored by thin layer chromatography (petroleum ether: ethyl acetate volume ratio = 3:1). The raw materials were basically reacted. The target compound 2b was separated and purified by silica gel column chromatography as a white powder with a yield of 81.2%. 1 HNMR (600MHz, CDCl 3 ,δ):4.67(m,1H,H-3),4.60(s,1H,NH),4.33(dd,J=14.5,6.8Hz,1H,H-16),3.48(d, J=10.3Hz,1H,H-26),3.34(t,J=11.0Hz,1H,H-26),3.14(d,J=5.1Hz,2H,GABA-α-CH 2 ),2.30(t,J=7.3Hz,2H,GABA-γ-CH 2 ),1.43(s,9H,Boc-CH 3 ,×3),1.05(d,J=6.9Hz,3H,21-CH 3 ),1.03(s,3H,19-CH 3 ),0.91(s,3H,18-CH 3),0.78(d,J=6.3Hz,3H,27-CH 3 ). 13 CNMR (150MHz, CDCl 3 ,δ):213.52(C-12),172.89(GABA-COO),156.05(Boc-COO),109.38(C-22),79.31(C-16),79.31(Boc-quart.-C),73.39( C-3),67.02(C-26),55.80(C-17),55.45(C-14),55.23(C-9),53.65(C-13),44.57(C-5),42.32(C-20),40.13(GABA-α-CH 2 ),37.87(C-11),36.37(C-4),36.21(C-1),34.44(C-10),33.92(C-7),32.10(GABA-γ-CH 2 ),31.59(C-15),31.54(C-8),31.26(C-23),30.32(C-25),28.91(C-24),28.54(Boc-CH 3 ,×3),28.29(C-2),27.34(C-6),25.46(GABA-β-CH 2 ),17.26(C-19),16.15(C-27),13.40(C-18),12.01(C-21).ESI-HRMS:m / z616.4187[M+H] + (Calcd for C 36 H 58 NO 7 ,616.4208).

[0037] The structure of compound 2b is shown below:

[0038]

[0039] Example 2

[0040] In a 50 mL dry eggplant-shaped bottle, intermediate 2b (250 mg, 0.40 mmol) and 5 mL CH 2 Cl 2 After stirring and dissolving, 1 mL of TFA was added and stirred at room temperature for 5 h. The reaction was monitored by thin layer chromatography (dichloromethane: methanol volume ratio = 15:1). The raw materials were basically reacted. 10 mL of water was added to the reaction solution, and the mixture was extracted three times with 5 mL of dichloromethane, washed with saturated brine, and anhydrous Na 2 SO4 After drying, filtering and concentration, the target compound 3b was obtained by separation and purification by silica gel column chromatography as a white powder with a yield of 81.2%. 1 HNMR (600MHz, CDCl 3 ,δ):4.65(m,1H,H-3),4.33(dd,J=14.2,7.0Hz,1H,H-16),3.48(d,J=10.0Hz,1H,H-26),3.34(t,J=11.0Hz,1H,H-26),3.06(m,2H,GABA-α-CH 2 ),2.40(m,2H,GABA-γ-CH 2 ),1.05(d,J=6.9Hz,3H,21-CH 3 ),1.03(s,3H,19-CH 3 ),0.90(s,3H,18-CH 3 ),0.78(d,J=6.3Hz,3H,27-CH 3 ). 13 C NMR (150 MHz, CDCl 3 ,δ):213.40(C-12),172.28(GABA-COO),109.36(C-22),79.29(C-16),73.94(C-3),67.01(C-26),5 5.83(C-17),55.44(C-14),55.23(C-9),53.67(C-13),44.55(C-5),42.32(C-20),39.51(GABA-α-CH 2 ),37.86(C-11),36.33(C-4),36.19(C-1),34.43(C-10),33.88(C-7),31.66(GABA-γ-CH 2 ),31.59(C-15),31.54(C-8),31.26(C-23),30.32(C-25),28.91(C-24),28.30(C-2),27.31(C-6),23.02(GABA-β-CH 2 ),17.26(C-19),16.15(C-27),13.40(C-18),12.02(C-21).ESI-HRMS:m / z516.3658[M+H] + (Calcd for C 31 H 50 NO 5 ,516.3684).

[0041] The structure of compound 3b is shown below:

[0042]

[0043] Example 3

[0044] The target compound 3c was prepared by referring to the synthesis method of Example 2. It was white powder with a yield of 84.7%. 1 HNMR (600MHz, CDCl 3 ,δ):4.73(m,1H,H-3),4.32(m,1H,H-16),3.47(m,1H,H-26),3.33(t,J=11. 0Hz,1H,H-26),3.25(d,J=4.5Hz,1H,Val-α-CH),1.05(d,J=6.9Hz,3H,21-CH 3 ),1.03(s,3H,19-CH 3 ),0.96(d,J=7.0Hz,3H,Val-CH 3 ),0.91(s,3H,18-CH 3 ),0.89(d,J=6.8Hz,3H,Val-CH 3 ),0.78(d,J=6.3Hz,3H,27-CH 3 ). 13 C NMR (150 MHz, CDCl 3 ,δ):213.46(C-12),174.96(Val-COO),109.37(C-22),79.30(C-16),73.66(C-3),67.01(C-26),60 .00(Val-α-CH),55.79(C-17),55.43(C-14),55.22(C-9),53.64(C-13),44.56(C-5),42.32(C-20) ,37.86(C-11),36.37(C-4),36.20(C-1),34.43(C-10),33.96(C-7),32.25(Val-β-CH),31.59(C-1 5),31.54(C-8),31.25(C-23),30.31(C-25),28.91(C-24),28.28(C-2),27.38(C-6),19.39(Val-CH 3 ),17.32(Val-CH 3 ),17.26(C-19),16.15(C-27),13.39(C-18),12.02(C-21).ESI-HRMS:m / z530.3803[M+H]+ (Calcd for C 32 H 52 NO 5 ,530.3840).

[0045] The structure of compound 3c is shown below:

[0046]

[0047] Example 4

[0048] The target compound 3d was prepared by referring to the synthesis method of Example 2. It was white powder with a yield of 84.7%. 1 HNMR (600MHz, CDCl 3 ,δ):4.71(m,1H,H-3),4.33(dd,J=13.8,6.9Hz,1H,H-16),3.77(brs,1H,Pro- α-CH),3.47(m,1H,H-26),3.33(t,J=11.2Hz,1H,H-26),3.10(m,1H,Pro-δ-CH 2 ),2.95(m,1H,Pro-δ-CH 2 ),1.05(d,J=6.9Hz,3H,21-CH 3 ),1.03(s,3H,19-CH 3 ),0.91(s,3H,18-CH 3 ),0.77(d,J=6.3Hz,3H,27-CH 3 ). 13 C NMR (150 MHz, CDCl 3 ,δ):213.44(C-12),174.49(Pro-COO),109.37(C-22),79.29(C-16),74.09(C-3),67.00(C-26 ),59.89(Pro-α-CH),55.78(C-17),55.41(C-14),55.21(C-9),53.63(C-13),47.06(Pro-δ-CH 2 ),44.53(C-5),42.31(C-20),37.85(C-11),36.33(C-4),36.19(C-1),34.42(C-10), 33.82(C-7),31.57(C-15),31.53(C-8),31.25(C-23),30.30(C-25),29.82(Pro-β-CH 2),28.90(C-24),28.26(C-2),27.25(C-6),25.44(Pro-γ-CH 2 ),17.25(C-19),16.13(C-27),13.39(C-18),12.01(C-21).ESI-HRMS:m / z528.3678[M+H] + (Calcd for C 32 H 50 NO 5 ,528.3684).

[0049] The structure of compound 3d is shown below:

[0050]

[0051] Example 5

[0052] The target compound 3f was prepared by referring to the synthesis method of Example 2. It was white powder with a yield of 79.3%. 1 HNMR (600MHz, CDCl 3 ,δ):8.19(brs,1H,Trp-NH),7.62(d,J=7.8Hz,1H,Trp-Ph),7.35(d,J=8.2Hz,1H,Trp-P h),7.18(t,J=7.2Hz,1H,Trp-Ph),7.11(t,J=7.8Hz,1H,Trp-Ph),7.06(s,1H,Trp-δ-CH) ,4.67(m,1H,H-3),4.34(dd,J=14.1,7.0Hz,1H,H-16),3.77(t,J=6.7Hz,1H,Trp-α-CH) ,3.48(m,1H,H-26),3.34(t,J=11.0Hz,1H,H-26),3.25(dd,J=14.3,7.9Hz,1H,Trp-β-CH 2 ),3.01(dd,J=14.3,7.9Hz,1H,Trp-β-CH 2 ),1.06(d,J=6.9Hz,3H,21-CH 3 ),1.04(s,3H,19-CH 3 ),0.88(s,3H,18-CH 3 ),0.78(d,J=6.3Hz,3H,27-CH 3 ). 13 C NMR (150 MHz, CDCl 3,δ):213.52(C-12),174.88(Trp-COO),136.37(Trp-Ph),127.69(Trp-Ph),123.05(Trp-δ-C),122.28(Trp-Ph),119. 59(Trp-Ph),119.01(Trp-Ph),111.42(Trp-Ph),111.30(Trp-γ-C),109.38(C-22),79.30(C-16),73.95(C-3),67.01( C-26),55.79(C-17),55.41(C-14),55.30(C-9),55.22(Trp-α-CH),53.69(C-13),44.51(C-5),42.32(C-20),37.85(C -11),36.32(C-4),36.17(C-1),34.41(C-10),33.71(C-7),31.56(C-15),31.53(C-8),31.25(C-23),30.93(Trp-β-CH 2 ),30.31(C-25),28.90(C-24),28.25(C-2),27.24(C-6),17.26(C-19),16.14(C-27),13.40(C-18),11.99(C-21).ESI-HRMS:m / z617.3906[M+H] + (Calcd for C 38 H 53 N 2 O 5 ,617.3949).

[0053] The structure of compound 3f is shown below:

[0054]

[0055] Example 6

[0056] In a 50 mL dry eggplant-shaped bottle, intermediate 3d (60 mg, 0.11 mmol) and 5 mL CH 2 Cl 2 After stirring and dissolving, DMAP (20.8 mg, 0.17 mmol), EDCI (32.6 mg, 0.17 mmol) and benzoic acid nitrogen mustard (35.5 mg, 0.14 mmol) were added and stirred at room temperature for 8 h. The reaction was monitored by thin layer chromatography (volume ratio of petroleum ether: ethyl acetate = 1.5:1). The raw materials were basically reacted. The target compound 4d was separated and purified by silica gel column chromatography as a white powder with a yield of 81.2%. 1HNMR(600MHz,CDCl 3 ,δ):7.54(brd,J=7.3Hz,2H,Ph-H),6.65(d,J=7.3Hz,2H,Ph-H),4.73(s,1H,H-3),4.58(s,1H,Pro-α-CH),4.32(dd,J=12.9,6.0Hz,1H,H-16),3.76(t,J=6.1Hz,4H,NCH 2 CH 2 Cl,×2),3.69(m,2H,Pro-δ-CH 2 ),3.63(t,J=6.1Hz,4H,NCH 2 CH 2 Cl,×2),3.47(m,1H,H-26),3.33(t,J=11.0Hz,1H,H-26),1.05(d,J=7.0Hz,3H,21-CH 3 ),1.03(s,3H,19-CH 3 ),0.90(s,3H,18-CH 3 ),0.77(d,J=6.3Hz,3H,27-CH 3 ). 13 CNMR(150MHz,CDCl 3 ,δ):213.51(C-12),172.04(Pro-COO),169.26(-CONH),147.78(Ph-C),130.08(Ph-C,×3),111.02(Ph-C,×2),109.36(C-22),79.29(C-16),74.00(C-3),66.99(C-26),59.98(Pro-α-CH),55.77(C-17),55.40(C-14),55.20(C-9),53.61(C-13),53.47(NCH 2 CH 2 Cl,×2),50.38(Pro-δ-CH 2 ),44.54(C-5),42.30(C-20),40.34(NCH 2 CH 2 Cl,×2),37.85(C-11),36.32(C-4),36.20(C-1),34.41(C-10),33.72(C-7),31.57(C-15),31.52(C-8),31.23(C-23),30.29(C-25),29.37(Pro-β-CH 2),28.89(C-24),28.27(C-2),27.22(C-6),25.70(Pro-γ-CH 2 ),17.24(C-19),16.13(C-27),13.38(C-18),12.02(C-21).ESI-HRMS:m / z771.3886[M+H] + (Calcd for C 43 H 61 Cl 2 N 2 O 6 ,771.3901).

[0057] The structure of compound 4d is shown below:

[0058]

[0059] Example 7

[0060] The target compound 3a was prepared by referring to the synthesis method of Example 2, and then the target compound 5a was prepared by referring to the synthesis method of Example 6. White powder, yield 84.4%. 1 HNMR (600MHz, CDCl 3 ,δ):7.07(d,J=8.1Hz,2H,Ph-H),6.63(d,J=7.8Hz,2H,Ph-H),5.91(s,1H,NH ),4.74(m,1H,H-3),4.33(dd,J=13.7,6.7Hz,1H,H-16),3.98(m,2H,Gly-α-CH 2 ),3.69(t,J=7.2Hz,4H,NCH 2 CH 2 Cl,×2),3.61(t,J=7.2Hz,4H,NCH 2 CH 2 Cl,×2),3.47(m,1H,H-26),3.33(t,J=11.0Hz,1H,H-26),1.05(d,J=6.9Hz,3H,21-CH 3 ),1.03(s,3H,19-CH 3 ),0.90(s,3H,18-CH 3 ),0.78(d,J=6.5Hz,3H,27-CH 3 ). 13 C NMR (150 MHz, CDCl 3,δ):213.43(C-12),172.98(-CONH),169.70(Gly-COO),144.24(Ph-C),129.86(Ph-C,×3),112.46(Ph-C,×2), 109.36(C-22),79.27(C-16),74.74(C-3),67.00(C-26),55.78(C-17),55.40(C-14),55.21(C-9),53.81(NCH 2 CH 2 Cl,×2),53.63(C-13),44.52(C-5),42.30(C-20),41.63(Gly-α-CH 2 ),40.54(NCH 2 CH 2 Cl,×2),37.84(C-11),36.28(C-4),36.15(C-1),35.61(Ph-CH 2 -CH 2 -CH 2 ),34.40(C-10),34.06(Ph-CH 2 ),33.79(C-7),31.55(C-15),31.52(C-8),31.24(C-23),30.30(C-25),28.89(C-24),28.24(C-2),27.30(Ph-CH 2 -CH 2 ),27.24(C-6),17.25(C-19),16.14(C-27),13.39(C-18),11.98(C-21).ESI-HRMS:m / z773.4010[M+H] + (Calcd for C 43 H 63 Cl 2 N 2 O 6 ,773.4058).

[0061] The structure of compound 5a is shown below:

[0062]

[0063] Example 8

[0064] The target compound 5c was prepared by using compound 3c and referring to the synthesis method of Example 6. White powder, yield 84.4%. 1 HNMR (600MHz, CDCl 3,δ):7.07(d,J=8.6Hz,2H,Ph-H),6.64(d,J=8.6Hz,2H,Ph-H),5.89(d,J=8.8Hz,1H,NH),4.73(m,1H,H-3),4.52(dd,J=8.5,4.6Hz,1H,Val-α-CH),4.32(m,1H,H-16),3.69(t,J=7.2Hz,4H,NCH 2 CH 2 Cl,×2),3.62(t,J=6.8Hz,4H,NCH 2 CH 2 Cl,×2),3.48(m,1H,H-26),3.34(t,J=11.0Hz,1H,H-26),1.05(d,J=6.9Hz,3H,21-CH 3 ),1.04(s,3H,19-CH 3 ),0.93(d,J=6.9Hz,3H,Val-CH 3 ),0.91(s,3H,18-CH 3 ),0.89(d,J=6.9Hz,3H,Val-CH 3 ),0.78(d,J=6.3Hz,3H,27-CH 3 ). 13 C NMR(150MHz,CDCl 3 ,δ):213.42(C-12),172.76(-CONH),171.77(Val-COO),144.18(Ph-C),129.88(Ph-C,×3),112.54(Ph-C,×2),109.39(C-22),79.29(C-16),74.41(C-3),67.02(C-26),56.95(Val-α-CH),55.79(C-17),55.42(C-14),55.22(C-9),53.87(NCH 2 CH 2 Cl,×2),53.63(C-13),44.55(C-5),42.32(C-20),40.51(NCH 2 CH 2 Cl,×2),37.84(C-11),36.31(C-4),36.18(C-1),36.03(Ph-CH 2 -CH 2 -CH 2 ),34.42(C-10),34.14(Ph-CH 2),33.92(C-7),31.58(C-15),31.55(Val-β-CH),31.53(C-8),31.25(C-23),30.31(C-25),28.90(C-24),28.28(C-2),27.53(Ph-CH 2 -CH 2 ),27.32(C-6),19.06(Val-CH 3 ),17.95(Val-CH 3 ),17.26(C-19),16.14(C-27),13.40(C-18),12.00(C-21).ESI-HRMS:m / z 815.4511[M+H] + (Calcd for C 46 H 69 Cl 2 N 2 O 6 ,815.4527).

[0065] The structure of compound 5c is shown below:

[0066]

[0067] Example 9

[0068] The target compound 5d was prepared by using compound 3d according to the synthesis method of Example 6. White powder, yield 84.4%. 1 HNMR (600MHz, CDCl 3 ,δ):7.07(d,J=8.2Hz,2H,Ph-H),6.61(d,J=7.5Hz,2H,Ph-H),4.69(m,1H,H-3),4.43(dd, J=8.6,3.6Hz,1H,Pro-α-CH),4.32(dd,J=14.6,6.3Hz,1H,H-16),3.69(t,J=6.8Hz,4H,NCH 2 CH 2 Cl,×2),3.61(t,J=6.8Hz,4H,NCH 2 CH 2 Cl,×2),3.56(m,1H,Pro-δ-CH 2 ),3.47(m,1H,H-26),3.42(m,1H,Pro-δ-CH 2 ),3.33(t,J=11.0Hz,1H,H-26),1.05(d,J=6.9Hz,3H,21-CH 3),1.03(s,3H,19-CH 3 ),0.89(d,J=17.2Hz,3H,18-CH 3 ),0.78(d,J=6.3Hz,3H,27-CH 3 ). 13 C NMR(150MHz,CDCl 3 ,δ):213.50(C-12),172.17(-CONH),171.93(Pro-COO),144.33(Ph-C),129.85(Ph-C,×3),112.27(Ph-C,×2),109.37(C-22),79.30(C-16),73.99(C-3),67.00(C-26),59.09(Pro-α-CH),55.78(C-17),55.40(C-14),55.20(C-9),53.75(NCH 2 CH 2 Cl,×2),53.61(C-13),47.21(Pro-δ-CH 2 ),44.52(C-5),42.30(C-20),40.65(NCH 2 CH 2 Cl,×2),37.84(C-11),36.31(C-4),36.18(C-1),34.41(C-10),34.09(Ph-CH 2 -CH 2 -CH 2 ),33.72(C-7),33.68(Ph-CH 2 ),31.58(C-15),31.52(C-8),31.24(C-23),30.30(C-25),29.39(Pro-β-CH 2 ),28.89(C-24),28.26(C-2),27.20(C-6),26.48(Ph-CH 2 -CH 2 ),24.86(Pro-γ-CH 2 ),17.25(C-19),16.13(C-27),13.39(C-18),11.99(C-21).ESI-HRMS:m / z 813.4328[M+H] + (Calcd for C 46 H 67 Cl 2 N 2 O 6 ,813.4371)。

[0069] The structure of compound 5d is shown below:

[0070]

[0071] Pharmacodynamics experiment part:

[0072] Effect Example 1: Cytotoxicity of the compounds of the present invention on various breast cancer cells

[0073] 1.1. Main experimental materials

[0074] Cell lines: human breast cancer cells MDA-MB-231, MDA-MB-468, MCF-7 and human normal gastric mucosal epithelial cells GES-1.

[0075] 1.2. Experimental methods: cell inhibitory activity assay

[0076] The above four types of cells were expressed at 1×10 4 Each well was inoculated with 100 μL / well into a 96-well plate. After overnight attachment, the culture medium was removed and a series of concentrations (1-100 μM) of the test compound 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). After 48 hours of treatment, 10% volume of CCK-8 solution was added to each 96-well plate and incubated in an incubator for 2 hours. The absorbance was measured at 450 nm, and the relative inhibition rate of each well was calculated according to the formula and the IC was calculated according to the inhibition rate. 50 value.

[0077] 1.3 Experimental results

[0078] Table 1: IC values ​​of the cytotoxic activities of the compounds in the examples against three human breast cancer cell lines and one human normal cell line 50 Value (μM)

[0079]

[0080] The pharmacological results are shown in Table 1. Compared with the parent compound hecosapogenin, the hecosapogenin derivatives of the present invention have stronger antiproliferative activity and tumor cell specificity against three breast cancer cell lines. The IC values ​​of most derivatives are 50 The values ​​were lower than 30 μM. Among them, derivatives 5a, 5c and 5d showed significant antiproliferative activity against MDA-MB-231 cells and MCF-7 cells, with IC 50The values ​​were all lower than 10 μM. In addition, compared with the positive control drug doxorubicin, the cytotoxicity of the hecosapogenin derivatives of the present invention to normal cells GES-1 was lower. In particular, derivative 5d had the strongest cytotoxic activity against MDA-MB-231 cells, with an IC 50 The value is 2.2μM.

[0081] Effect Example 2: Effect of Compound 5d on the Morphology of MDA-MB-231 Cells

[0082] Experimental methods

[0083] MDA-MB-231 cells were taken and 1×10 6 Each well of a 6-well plate was inoculated with 1×10 MDA-MB-231 cells and the corresponding volume of compound 5d prepared in DMSO (concentrations were 0.5, 1.5, and 4 μM, respectively) was added. In addition, the medium containing 1‰ DMSO was used as the control group and cultured for 24 h. The cell morphology was observed under a microscope. For staining, MDA-MB-231 cells were stained with 1×10 6 Cells were inoculated into 6-well plates. After overnight, the culture medium was removed and a series of concentrations (0.5, 1.5, 4 μM) of compound 5d in DMSO were added. After 48 h of cell treatment, the cells were stained with Hoechst 33342 / PI double staining kit. After staining with buffer, Hoechst 33342 and PI at room temperature for 30 min, the cells were photographed under a fluorescence microscope.

[0084] 2.2 Experimental Results

[0085] Effects of different concentrations (0.5, 1.5, 4 μM) on the morphology of MDA-MB-231 cells for 5 days Figure 1 As shown, it can be seen that with the increase in the dose of compound 5d, the morphology of MDA-MB-231 cells gradually changed from bright cells with clear edges to turbid cells with unclear edges and a small number of floating dead cells. After staining each group of cells with the Hoechst 33342 / PI double staining kit, it was found that with the increase in the dose of compound 5d, the number of early apoptotic cells (i.e., cells with collapsed nuclei that can be stained bright blue by Hoechst 33342) and the number of late apoptotic and necrotic cells (i.e., cells that can be stained by PI) showed an upward trend, indicating that compound 5d promoted the apoptosis of MDA-MB-231 cells in a dose-dependent manner ( Figure 2 ).

[0086] Effect Example 3: Effect of Compound 5d on Cell Apoptosis

[0087] 3.1 Experimental methods

[0088] MDA-MB-231 cells were taken and 2×10 5 Cells were inoculated into the corresponding 24-well plates, and the corresponding volume of compound 5d prepared in DMSO (concentrations were 0.5, 1.5, and 4 μM, respectively) was added. In addition, the medium containing 1‰ DMSO was used as the control group, and the culture was continued for 48 hours. After incubation, the medium was discarded, and the cells were washed twice with 2 mL PBS, digested with trypsin, and the cells were collected. Then, the excess trypsin was washed with cold PBS, and after centrifugation to remove the supernatant, the cells were resuspended with 1xbinding buffer in the Annexin / PI double staining kit (Solarbio, Beijing, CN), and then a certain amount of Annexin was added to incubate for 15 minutes, and then a certain amount of PI and a certain amount of PBS were added to prepare for flow cytometry (CytoFlex, Beckman coulter, CA, USA) detection, and the corresponding control tubes with single staining of Annexin or single staining of PI or no staining were prepared before loading the sample for detection to adjust the instrument voltage gain and fluorescence channel compensation.

[0089] 3.2 Experimental Results

[0090] After treatment with compound 5d (0.5, 1.5, 4 μM) for 48 h, the effects of each drug-treated group on cell apoptosis were as follows: Figure 3 A and Figure 3 As shown in B, it can be seen that with the increase in the dosage, the proportion of apoptotic cells increased significantly. The results showed that compound 5d could significantly reduce the proportion of normal cells at concentrations of 0.5, 1.5, and 4 μM (p<0.05). At all doses, the proportions of early apoptosis, late apoptosis, and necrosis of cells were significantly increased compared with the control group. The above results suggest that compound 5d can induce a strong apoptotic effect on MDA-MB-231 cells and is dose-dependent for late apoptosis and necrosis ( Figure 3 ).

[0091] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope of the present invention.

Claims

1. A hecosapogenin derivative or a pharmaceutically acceptable salt thereof represented by general formula Ⅰ, Ⅱ or Ⅲ, characterized in that: Wherein, R1 and R2 are independently selected from one of the following groups:

2. The hecosapogenin derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The hecosapogenin derivative is selected from:

3. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the hecosapogenin derivative or pharmaceutically acceptable salt according to claim 1 or claim 2 and a pharmaceutically acceptable carrier.

4. The method for preparing the hecosapogenin derivative or the pharmaceutically acceptable salt thereof according to claim 1 or claim 2, characterized in that: The preparation method comprises the following steps: (1) Dissolve hecosapogenin 1 in a solvent, then add a Boc-protected amino acid, react at room temperature under DCC and DMAP conditions, monitor the completion of the reaction by TLC, load the resulting product onto a silica gel column, and use a petroleum ether / ethyl acetate mixed solution for gradient elution to obtain intermediates 2a-2f; (2) Dissolve the intermediate 2a-2f in a solvent, then add trifluoroacetic acid (TFA) to remove the Boc protection, react at room temperature, monitor the completion of the reaction by TLC, extract the reaction solution, load the obtained product onto a silica gel column chromatography, and use a dichloromethane / methanol mixed solution for gradient elution to obtain intermediates 3a-3f; (3) The intermediates 3a-3f were dissolved in a solvent and reacted with benzoic acid mustard or chlorambucil under EDCI and DMAP conditions at room temperature. The reaction was monitored by TLC. The obtained product was loaded onto a silica gel column chromatography and gradient eluted with a petroleum ether / ethyl acetate mixed solution to obtain the target compounds 4a-4f or 5a-5f. a: dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP); b: trifluoroacetic acid (TFA); c: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), DMAP; d: EDCI, DMAP.

5. Use of the hecosapogenin derivative or a pharmaceutically acceptable salt thereof according to claim 1 or claim 2 in the preparation of a medicament for treating tumor diseases.

6. Use of the pharmaceutical composition according to claim 3 in the preparation of drugs for treating tumor diseases.

7. The use according to claim 5 or claim 6, characterized in that: The tumor is breast cancer.

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