Isosteviol derivative and application thereof in preparation of anti-cancer drugs

Abstract

The invention discloses an isosteviol derivative and application thereof in preparation of anticancer drugs, the isosteviol derivative has a general formula structure as shown in a formula 1 and can be prepared through a synthetic route as shown in a formula 2, and the obtained isosteviol derivative is used for preparing drugs for resisting colon cancer, ovarian cancer and liver cancer, especially colon cancer HCT116, ovarian cancer SKOV3 and liver cancer HepG2. The medicine is selected from at least one preparation form of tablets, capsules, granules, dripping pills, suspensions, syrups, enteric-coated preparations, emulsion suspensions and injections. The isosteviol derivative disclosed by the invention is small in toxic and side effects and high in bioavailability and clinical use value, provides a candidate lead compound for research and development of a novel inhibitor of an anti-cancer drug, and has potential application value in the field of targeted drugs for ovarian cancer.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to isosteviol derivatives and applications thereof in the preparation of anticancer drugs. Background Art

[0002] Malignant tumors are a type of disease that threatens human health and life. Currently, the treatment of malignant tumors often adopts a combination of surgery, radiotherapy, and chemotherapy. Chemotherapy is a systemic treatment that can eliminate cancer cells that have metastasized to distant sites. It plays an important role in comprehensive treatment. However, clinically, chemotherapy drugs have problems with efficacy, side effects, and drug resistance of tumor cells. Therefore, it is still urgent to find highly effective and low-toxic anti-tumor drugs from different angles.

[0003] Natural products are an important source of drug lead compounds and functional organic molecules. Screening drug lead compounds from natural products has always been an effective way to develop new drugs. Natural products have always been an important source of drug research and development due to their wide range of biological activities and low toxic side effects. The active ingredients in traditional Chinese medicine have become a current research hotspot. Isosteviol is isolated from the roots of Tripterygium wilfordii and has multiple biological activities, including anti-tumor, anti-inflammatory, and immunosuppressive effects. Its anti-tumor activity has been widely studied. Isosteviol exhibits cytotoxicity against a variety of human cancer cell lines by inducing apoptosis, but the anti-tumor efficacy of isosteviol (HCT116: IC 50 =24.8±2.43μM, SKOV3:IC 50 >100μM, HepG2:IC 50 >100μM) and physical and chemical properties need to be improved. By modifying the C-16 and C-19 of isosteviol, designing different substituents at different positions, and using the CCK8 method to measure the inhibitory effects of its compounds on three cancer cells, HCT116, SKOV3 and HepG2, it can provide a good starting point for the development of new anti-tumor drugs. Summary of the invention

[0004] In view of this, the main object of the present invention is to provide an isosteviol derivative.

[0005] Another object of the present invention is to provide the use of the above-mentioned isosteviol derivatives in the preparation of anticancer drugs, and the isosteviol derivatives have obvious anti-tumor cell activity.

[0006] The above-mentioned object of the present invention is achieved by the following technical solutions:

[0007] The present invention provides an isosteviol derivative having a general structure as shown in Formula 1:

[0008]

[0009] In formula 1, R1 and R2 are respectively shown in the following table:

[0010]

[0011]

[0012]

[0013] The present invention also provides a method for preparing the above-mentioned isosteviol derivative, and its synthetic route is selected from one of Formula 2:

[0014]

[0015] The present invention also provides the use of the isosteviol derivative or its pharmaceutically acceptable salt in the preparation of anticancer drugs.

[0016] Preferably, the cancer includes colon cancer, ovarian cancer and liver cancer.

[0017] More preferably, the cancer includes colon cancer HCT116, ovarian cancer SKOV3, and liver cancer HepG2.

[0018] The present invention provides an anti-cancer drug, which comprises an isosteviol derivative or a pharmaceutically acceptable solvate thereof. The structural formula of the isosteviol derivative is as described above, and the isosteviol derivative has a significant inhibitory effect on cancer cells, and is a new type of anti-cancer drug.

[0019] Preferably, the weight percentage of the isosteviol derivative or its pharmaceutically acceptable solvate in the drug is 5% to 100%.

[0020] Preferably, the drug further comprises a pharmaceutically acceptable carrier or diluent.

[0021] "Pharmaceutically acceptable carrier" refers to inactive ingredients in drugs, including but not limited to calcium carbonate, calcium phosphate, various sugars such as lactose, mannitol, starch, cyclodextrin, magnesium stearate, cellulose, magnesium carbonate, acrylic acid polymers or methacrylic acid polymers, gelatin, water, polyethylene glycol, propylene glycol, ethylene glycol, castor oil, hydrogenated castor oil, polyethoxylated hydrogenated castor oil, sesame oil, corn oil, and peanut oil.

[0022] "Pharmaceutically acceptable diluents" include, but are not limited to, starch (such as corn starch, wheat starch, potato starch, etc.), lactose, dextrin, sucrose, pregelatinized starch, microcrystalline cellulose, inorganic salts (such as calcium hydrogen phosphate, calcium sulfate, residual calcium acid, etc.) and mannitol.

[0023] Preferably, the dosage form of the drug is selected from at least one of tablets, capsules, granules, pellets, suspensions, syrups, enteric-coated preparations, emulsion suspensions and injections.

[0024] The present invention also provides the use of isosteviol derivatives or pharmaceutically acceptable salts thereof in the preparation of HCT116, SKOV3 and HepG2 cell proliferation inhibitors.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention confirms through in vitro experiments that isosteviol derivatives have strong anti-colon cancer HCT116, ovarian cancer SKOV3 and liver cancer HepG2 activities, and have obvious inhibitory effects on the growth and cell activity of HCT116, SKOV3 and HepG2 cells, and can be used as lead compounds for the treatment of cancer for the preparation of cancer drugs.

[0027] (2) The isosteviol derivatives of the present invention not only have the potential to be developed into a new generation of anticancer drugs, but also have potential application value in the field of targeted cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is to detect the inhibitory effect of compound 7 at different concentrations on the SKOV3 cell cycle in Example 9.

[0029] Figure 2 This is to detect the inhibitory effect of compound 18 at different concentrations on the HCT116 cell cycle in Example 9.

[0030] Figure 3 This is to detect the inhibitory effect of compound 7 at different concentrations on the SKOV3 cell cycle in Example 10.

[0031] Figure 4 This is to detect the inhibitory effect of compound 18 at different concentrations on HCT116 cell apoptosis in Example 10.

[0032] Figure 5 It is the general structure of the isosteviol derivatives in the present invention. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention is clearly and completely described below in conjunction with the accompanying drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0034] Unless otherwise specified in the following examples, all reagents and materials used were commercially available.

[0035] The isosteviol derivatives in the following examples are an isosteviol derivative having a general structure as shown in Formula 1:

[0036]

[0037] In formula 1, R1 and R2 are respectively shown in the following table:

[0038]

[0039]

[0040]

[0041] The following examples also provide a method for preparing the above-mentioned isosteviol derivatives, using a synthetic route as shown in Formula 2:

[0042]

[0043] Example 1

[0044] (1) Preparation of compound a: Dissolve isosteviol (200 mg, 0.636 mmol) in N,N-dimethylformamide (DMF), then add 87.0 μL of bromopropane (0.954 mmol) and 80.1 mg of sodium bicarbonate (0.954 mmol), and stir the mixture at room temperature for 5 hours.

[0045] (2) The reaction mixture was filtered and the residue was washed with ethyl acetate (3×30 mL). Then, the ethyl acetate solution was washed with hydrochloric acid (3×30 mL), saturated sodium bicarbonate (3×30 mL) and saturated brine (3×30 mL), respectively. Subsequently, the organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure. Finally, it was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 20:1) to obtain pure target compound a with a yield of 73.7%.

[0046] (3) 1H NMR (500 MHz, CDCl3) δ 4.05-3.89 (m, 2H), 2.61 (dd, J = 18.6,3.8 Hz, 1H), 2.17 (d,J=13.4Hz,1H),1.92-1.83(m,1H),1.83-1.73(m,2H),1.73-1.63(m,4H),1.63-1.56(m,3H),1.55-1.44(m,3H),1.43-1.29(m,2H ),1.27-1.19(m,1H),1.17(s,4H),1.15-1.07(m,1H),1.04-0.97(m,1H),0.96(s,4H),0.94(s,1H),0.93-0.83(m,2H),0.69(s,3H); 13 C NMR (125MHz, CDCl3) δ222.7,177.5,66.0,57.2,54.8,54.4,48.8,48.5,43.9,41.6,40.0,39. 6,38.1,38.0,37.4,29.1,22.0,21.8,20.4,19.9,19.1,13.5,10.9; HRMS(ESI,m / z)Calcd.for C 23 H 36 O3[M+H]+:361.2737,Found:361.2719.

[0047] Example 2

[0048] (1) Preparation of compound b: Compound a (50 mg, 0.14 mmol) and NH2OH·HCl (15.0 mg, 0.21 mmol) were dissolved in ethanol, and the mixture was stirred at 60°C in the presence of NaHCO3 for 4 hours.

[0049] (2) The reaction mixture was filtered and the residue was washed with ethyl acetate (3×30 mL). Then, the ethyl acetate solution was washed with saturated sodium bicarbonate (3×30 mL) and saturated brine (3×30 mL), respectively. Subsequently, the organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure. Finally, it was purified by silica gel column chromatography to obtain pure target compound b (yield 49.5%).

[0050] (3) Compound b: 1H NMR (500MHz, CDCl3) δ4.01-3.87(m,2H),2.95(dd,J=18.6,3.2Hz,1H),2.18(d,J=12.9Hz,1 H),1.99(d,J=18.7Hz,1H),1.91-1.80(m,2H),1.76-1.69(m,2H),1.69-1.65(m,2H),1.64-1 .56(m,2H),1.49-1.42(m,2H),1.41-1.36(m,2H),1.33-1.21(m,3H),1.18(s,3H),1.10(s, 4H),1.07(s,1H),1.03-0.99(m,1H),0.96(t,J=7.4Hz,3H),0.92-0.84(m,1H),0.77(s,3H); 13 C NMR (125MHz, CDCl3) δ177.6,170.1,65.8,57.1,56.3,54.9,53.4,43.8,43.7,40.9,40.6,40. 0,39.4,38.0,36.7,28.9,22.1,21.9,21.7,20.4,18.9,13.3,10.7; HRMS(ESI,m / z)Calcd.for C 23 H 37 NO3[M+H] + :376.2802,Found:376.2821.

[0051] Example 3

[0052] (1) Preparation of compounds 1, 14-21: Compound b (0.13 mmol) was dissolved in dichloromethane (2 mL), compound (0.26 mmol), EDCI and DMAP were added, and the mixture was heated at 60° C. for 12 h.

[0053] (2) The reaction mixture was filtered and the residue was washed with ethyl acetate (3×30 mL). Then, the ethyl acetate solution was washed with hydrochloric acid (3×30 mL), saturated sodium bicarbonate (3×30 mL) and saturated brine (3×30 mL), respectively. Subsequently, the organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure. Finally, it was purified by silica gel column chromatography to obtain pure target compound 1

[0054] (yield 70.8%), 14-21 (yield 70.8%, 50.2%, 60.4%, 67.3%, 65.3%, 61.6%, 65.2%, 60.2%).

[0055] (3) Compound 1: 11H NMR (500 MHz, CDCl3) δ 7.77 (d, J = 13.5 Hz, 1H), 7.65 (q, J = 7.0 Hz, 4H), 6.62 (d, J = 13.5 Hz, 1H), 4.05 - 3.92 (m, 2H), 3.07 (d, J = 18.0 Hz, 1H), 2.18 (d, J = 11.0 Hz, 1H), 2.09 (d, J = 15.5 Hz, 1H), 1.89 (d, J = 11.0 Hz, 1H), 1.84 - 1.75 (m, 3H), 1.74 - 1.63 (m, 5H), 1.59 (d, J = 11.0 Hz, 1H), 1.54 - 1.38 (m, 4H), 1.29 (s, 1H), 1.23 (s, 3H), 1.18 (s, 3H), 1.13 - 1.07 (m, 2H), 1.04 - 0.99 (m, 1H), 0.96 (t, J = 6.0 Hz, 3H), 0.92 - 0.85 (m, 1H), 0.76 (s, 3H). 13 13C NMR (125 MHz, CDCl3) δ 176.58, 175.08, 162.04, 141.27, 135.51, 126.03, 125.90, 123.61, 123.59, 123.55, 123.53, 116.60, 63.55, 54.80, 53.89, 52.23, 51.16, 42.70, 41.56, 38.49, 37.55, 37.05, 36.45, 35.77, 35.53, 26.80, 19.62, 19.30, 18.04, 16.74, 11.24, 8.42. HRMS (ESI, m / z) Calcd. for C 32 1 41 19H3F3NO4 [M + H] + : 573.6972, Found: 573.7012.

[0056] (4) Compound 14: 11H NMR (500 MHz, CDCl3) δ: 7.64 - 7.61 (m, 1H), 7.21 (dd, J = 3.5, 0.6 Hz, 1H), 6.54 (dd, J = 3.5, 1.7 Hz, 1H), 4.05 - 3.95 (m, 2H), 2.21 - 2.16 (m, 2H), 1.93 - 1.88 (m, 1H), 1.83 - 1.73 (m, 3H), 1.71 - 1.62 (m, 6H), 1.57 - 1.41 (m, 4H), 1.33 - 1.27 (m, 3H), 1.25 (s, 3H), 1.20 (s, 3H), 1.15 - 1.07 (m, 2H), 0.98 (dd, J = 9.0, 5.9 Hz, 3H), 0.92 - 0.85 (m, 1H), 0.76 (s, 3H); 13 13C NMR (125 MHz, CDCl3) δ: 179.51, 177.55, 156.62, 146.69, 143.64, 118.34, 111.95, 66.03, 57.26, 56.39, 54.71, 45.24, 44.01, 40.93, 40.77, 40.00, 39.45, 38.83, 38.20, 37.97, 29.25, 22.14, 22.02, 21.72, 20.46, 19.17, 13.62, 10.88. HRMS (ESI, m / z) Calcd. for C 28 H 39 NO5[M + H] + : 470.2908, Found: 470.1991.

[0057] (5) Compound 15: 1 1H NMR (500 MHz, CDCl3) δ: 7.09 (d, J = 3.3 Hz, 1H), 6.12 (d, J = 3.2 Hz, 1H), 4.02 - 3.95 (m, 2H), 2.38 (s, 3H), 2.19 - 2.09 (m, 2H), 1.96 - 1.86 (m, 2H), 1.81 - 1.75 (m, 2H), 1.75 - 1.71 (m, 1H), 1.67 - 1.61 (m, 4H), 1.61 - 1.56 (m, 2H), 1.54 - 1.50 (m, 1H), 1.47 - 1.41 (m, 3H), 1.28 - 1.24 (m, 2H), 1.22 (s, 3H), 1.17 (s, 3H), 1.02 - 0.98 (m, 1H), 0.95 (t, J = 7.6 Hz, 4H), 0.90 - 0.84 (m, 1H), 0.73 (s, 3H); 13C NMR(125MHz, CDCl3)δ:179.10,177.52,157.74,156.70,141.78,119.79,108.50,65.96,57.16,56.32,54.63,45.11,43.92,40.83,4 0.70,39.92,39.35,38.70,38.12,37.90,29.16,22.09,21.94,21.66,20.38,19.09,14.14,13.53,10.83.HRMS(ESI,m / z)Calcd.for C 28 H 38 BrNO5[M+H] + :548.2013,Found:548.1859.

[0058] (6) Compound 16: 1 H NMR (500MHz, CDCl3) δ: 7.18 (dd, J=3.5, 1.2Hz, 1H), 6.33 (d, J=3.5Hz, 1H), 4.03-3 .93(m,2H),2.20-2.10(m,2H),1.91-1.86(m,1H),1.80-1.74(m,3H),1.72-1.57(m ,6H),1.54-1.39(m,4H),1.31-1.25(m,2H),1.22(s,3H),1.17(s,3H),1.13-1.06 (m,2H),1.03-0.99(m,1H),0.96(t,J=6.8Hz,3H),0.91-0.84(m,1H),0.73(s,3H); 13 CNMR(125MHz, CDCl3)δ:179.72,177.52,155.53,142.73,141.53,120.47,109.05,65.99,57.10,56.25,54.54,45.24,43.91,40.8 6,40.64,39.90,39.34,38.73,38.11,37.89,29.13,22.06,21.93,21.65,20.36,19.06,13.52,10.86.HRMS(ESI,m / z)Calcd.forC 28 H 38 ClNO5[M+H] + :504.2519,Found:504.0534.

[0059] (7) Compound 17: 11H NMR (500 MHz, CDCl3) δ: 7.13 (d, J = 3.5 Hz, 1H), 6.46 (d, J = 3.5 Hz, 1H), 4.01 - 3.91 (m, 2H), 2.17 - 2.07 (m, 2H), 1.89 - 1.83 (m, 1H), 1.78 - 1.68 (m, 4H), 1.67 - 1.61 (m, 4H), 1.60 - 1.54 (m, 1H), 1.51 - 1.48 (m, 1H), 1.46 - 1.36 (m, 3H), 1.28 - 1.24 (m, 2H), 1.20 (s, 3H), 1.15 (s, 3H), 1.11 - 1.03 (m, 2H), 1.00 - 0.96 (m, 1H), 0.93 (t, J = 7.4 Hz, 3H), 0.89 - 0.81 (m, 1H), 0.71 (s, 3H); 13 13C NMR (125 MHz, CDCl3) δ: 179.62, 177.40, 155.36, 145.06, 127.83, 120.48, 113.99, 65.89, 57.00, 56.16, 54.45, 45.15, 43.81, 40.78, 40.55, 39.81, 39.26, 38.64, 38.02, 37.81, 29.05, 22.00, 21.86, 21.57, 20.28, 18.98, 13.44, 10.82. HRMS (ESI, m / z) Calcd. for C 29 H 41 NO5[M + H] + : 484.3065, Found: 484.0853.

[0060] (8) Compound 18: 1 1H NMR (500 MHz, CDCl3) δ: 7.28 (d, J = 3.8 Hz, 1H), 7.13 (d, J = 3.8 Hz, 1H), 3.93 (d, J = 7.9 Hz, 2H), 2.17 (s, 3H), 1.88 (d, J = 4.6 Hz, 2H), 1.85 (s, 3H), 1.77 (s, 4H), 1.75 (s, 2H), 1.71 (s, 2H), 1.34 (s, 3H), 1.24 (s, 3H), 1.18 (s, 6H), 0.95 (s, 3H), 0.75 (s, 3H); 1313C NMR (125 MHz, CDCl3) δ: 177.25, 175.53, 131.27, 130.41, 119.94, 117.17, 112.09, 66.03, 57.22, 56.87, 56.69, 45.85, 40.23, 40.06, 40.02, 39.95, 39.19, 37.98, 37.45, 35.30, 28.88, 23.39, 21.99, 20.10, 19.98, 19.01, 17.75, 10.82. HRMS (ESI, m / z) Calcd. for C 28 H 38 N2O7 [M + H] + : 515.2759, Found: 515.2929.

[0061] (9) Compound 19: 1 1H NMR (500 MHz, CDCl3) δ: 7.83 (dd, J = 3.7, 1.2 Hz, 1H), 7.57 (dd, J = 5.0, 1.2 Hz, 1H), 7.11 (dd, J = 4.9, 3.8 Hz, 1H), 4.02 - 3.93 (m, 2H), 2.19 - 2.10 (m, 2H), 1.91 - 1.85 (m, 1H), 1.81 - 1.73 (m, 3H), 1.72 - 1.57 (m, 6H), 1.54 - 1.50 (m, 1H), 1.48 - 1.43 (m, 2H), 1.42 - 1.37 (m, 1H), 1.30 - 1.25 (m, 2H), 1.23 (s, 3H), 1.17 (s, 3H), 1.13 - 1.05 (m, 2H), 1.02 - 0.98 (m, 1H), 0.96 (t, J = 7.4 Hz, 3H), 0.90 - 0.83 (m, 1H), 0.73 (s, 3H); 13 13C NMR (125 MHz, CDCl3) δ: 179.13, 177.44, 159.79, 133.79, 132.60, 132.15, 127.82, 65.93, 57.05, 56.19, 54.52, 45.02, 43.85, 40.80, 40.61, 39.85, 39.35, 38.66, 38.07, 37.85, 29.09, 22.03, 21.89, 21.60, 20.32, 19.02, 13.42, 10.88. HRMS (ESI, m / z) Calcd. for C 28 H 39 NO4S [M + H] + : 486.2680, Found: 486.3421.

[0062] (10) Compound 20: 1 H NMR (500 MHz, CDCl3) δ: 7.66 (d, J = 4.0 Hz, 1H), 6.96 (d, J = 4.0 Hz, 1H), 4.00 (td, J = 6.5, 1.0 Hz, 2H), 2.22 - 2.08 (m, 2H), 1.93 - 1.86 (m, 1H), 1.82 - 1.65 (m, 8H), 1.63 - 1.58 (m, 1H), 1.55 - 1.52 (m, 1H), 1.50 - 1.45 (m, 2H), 1.44 - 1.40 (m, 1H), 1.31 - 1.27 (m, 2H), 1.24 (s, 3H), 1.19 (s, 3H), 1.14 - 1.07 (m, 2H), 0.99 (dd, J = 9.0, 5.8 Hz, 3H), 0.92 - 0.83 (m, 2H), 0.75 (s, 3H); 13 C NMR (125 MHz, CDCl3) δ: 179.42, 177.54, 159.00, 137.81, 133.68, 130.17, 127.36, 66.08, 57.14, 56.24, 54.57, 45.20, 43.95, 40.92, 40.69, 39.95, 39.43, 38.73, 38.17, 37.96, 29.15, 22.09, 21.99, 21.68, 20.40, 19.09, 13.53, 10.98. HRMS (ESI, m / z) Calcd. for C 28 H 38 ClNO4S [M + H] + : 520.2290, Found: 520.0744.

[0063] (11) Compound 21: 1 H NMR (500 MHz, CDCl3) δ: 7.62 (d, J = 4.0 Hz, 1H), 7.10 (d, J = 4.0 Hz, 1H), 4.01 (t, J = 6.4 Hz, 2H), 2.22 - 2.09 (m, 2H), 1.94 - 1.87 (m, 1H), 1.83 - 1.66 (m, 8H), 1.64 - 1.58 (m, 1H), 1.56 - 1.40 (m, 4H), 1.32 - 1.27 (m, 2H), 1.24 (s, 3H), 1.19 (s, 3H), 1.15 - 1.08 (m, 2H), 1.00 (dd, J = 9.3, 5.5 Hz, 4H), 0.92 - 0.85 (m, 1H), 0.75 (s, 3H); 13C NMR(125MHz, CDCl3)δ:179.44,177.57,158.91,134.37,133.13,131.02,120.77,66.10,57.15,56.26,54.59,45.22,43.96,40.9 4,40.71,39.97,39.44,38.75,38.18,37.98,29.17,22.11,22.01,21.70,20.42,19.11,13.55,11.02.HRMS(ESI,m / z)Calcd.for C 29 H 41 NO4S[M+H] + :500.2836,Found:500.2084.

[0064] Example 4

[0065] (1) Preparation of compounds 2 and 3: Dissolve isosteviol (60 mg, 0.2 mmol) in acetonitrile, add chloroacetylchloroaniline (0.21 mmol), and heat the mixture at 80° C. for 24 h.

[0066] (2) After the reaction was completed, the reaction mixture was filtered and the residue was washed with ethyl acetate (3×30 mL). Then, the ethyl acetate solution was washed with water (3×30 mL). Subsequently, the organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure. Finally, it was purified by silica gel column chromatography to obtain pure target compounds 2 and 3 (2: yield 81.2%; 3: yield 82.2%).

[0067] (3) Compound 2: 1 H NMR (500MHz, CDCl3) δ8.28(d,J=10.0Hz,1H),7.90(d,J=10.0Hz,2H),7.58(d,J=10.5Hz,2H),4.78-4.50(m,2H),2.53(s,3H),2.20(s ,1H),1.93(s,2H),1.81-1.61(m,7H),1.50(d,J=10.5Hz,3H),1.36(s,2H),1.28(s,3H),1.20-1.01(m,5H),0.92(s,3H),0.66(s,3H). 1313C NMR(125 MHz, CDCl3) δ 222.46, 197.05, 176.13, 165.53, 141.41, 133.26, 129.84, 119.00, 63.15, 56.95, 54.59, 54.11, 48.71, 48.41, 44.01, 41.32, 39.56, 39.44, 38.04, 37.91, 37.20, 28.95, 26.48, 21.89, 20.30, 19.81, 18.90, 13.48. HRMS(ESI, m / z) Calcd. for C 30 H 39 NO5[M + H] + : 494.2862, Found: 494.2853.

[0068] (4) Compound 3: 1 1H NMR(500 MHz, CDCl3) δ 7.95(s, 1H), 7.60 - 7.56(m, 2H), 7.53(d, J = 10.5 Hz, 2H), 7.28 - 7.27(m, J = 2.5, 0.5 Hz, 1H), 7.13 - 7.04(m, 1H), 4.75(d, J = 19.0 Hz, 1H), 4.59(d, J = 19.0 Hz, 1H), 2.62(dd, J = 23.0, 2.5 Hz, 1H), 2.28(d, J = 17.0 Hz, 1H), 2.06 - 1.99(m, 1H), 1.88 - 1.69(m, 6H), 1.65 - 1.51(m, 4H), 1.47 - 1.38(m, 2H), 1.35(s, 3H), 1.29 - 1.22(m, 4H), 1.20 - 1.12(m, 1H), 1.02 - 0.97(m, 3H), 0.74(s, 3H). 13 13C NMR(125 MHz, CDCl3) δ 222.12, 175.88, 165.07, 143.63, 136.15, 131.17, 128.08, 126.66, 124.71, 122.96, 120.08, 63.33, 56.99, 54.68, 54.17, 48.70, 48.41, 44.03, 41.37, 39.60, 39.44, 38.06, 37.97, 37.20, 29.71, 29.00, 21.99, 20.33, 19.83, 18.94, 13.52. HRMS(ESI, m / z) Calcd. for C 32 H 39 NO5S[M + H] + : 533.2620, Found: 533.1046.

[0069] Example 5

[0070] (1) Preparation of compound 4-13: Dissolve isosteviol (50 mg, 0.16 mmol) in methanol (MeOH) (2 mL), and add benzenesulfonyl hydrazide compound and hydrazide compound (0.24 mmol). Then, the mixture was heated at 80° C. for 12 h in a heating device, and the reaction was monitored by TLC. The reaction was stopped after the raw materials reacted. The reaction solution was extracted with ethyl acetate and water for 3 times, the organic phase was collected, washed with saturated brine for 3 times, and then dehydrated with anhydrous Na2SO4. The solvent was removed in vacuo and purified by silica gel column chromatography (petroleum ether: acetone = 4:1) to obtain the target compound 4-13 (4: yield 69.2%; 5: yield 69.5%; 6: yield 69.4%; 7: yield 70.4%; 8: yield 80.4%; 9: yield 65.3%; 10: yield 62.5%; 11: yield 60.8%; 12: yield 72.4%; 13: yield 78.6%;).

[0071] (2) Compound 4: 1 H NMR (500MHz, CDCl3) δ7.94(d,J=7.0Hz,2H),7.59(t,J=7.5Hz,1H),7.50(t,J=8.0 Hz,2H),2.67(d,J=18.0Hz,1H),2.19(t,J=4.5Hz,1H),1.74-1.69(m,2H),1.53(d, J=13.0Hz,3H),1.47-1.44(m,1H),1.40(s,1H),1.36(s,1H),1.33(s,1H),1.30(s, 2H),1.23(s,3H),1.13-1.02(m,3H),1.00(s,3H),0.90-0.87(m,4H),0.81(s,3H). 13 C NMR (125MHz, CDCl3) δ183.48,171.35,138.39,132.96,128.70,127.91,56.95,55.80,54.75,44.85,43.63,41.08,39.75,39 .10,38.08,37.72,31.94,29.71,29.21,22.70,22.68,22.00,21.52,20.24,18.90,14.14,13.49.HRMS(ESI,m / z)Calcd.for C 26 H 36 N2O4S[M+H] + :472.2396,Found:472.9126.

[0072] (3) Compound 5: 1 H NMR (500 MHz, CDCl3) δ 7.94 (dd, J = 9.0, 5.0 Hz, 2H), 7.15 (t, J = 10.0 Hz, 2H), 2.65 (d, J = 15.0 Hz, 1H), 2.21 - 2.11 (m, 1H), 1.81 (t, J = 5.0 Hz, 2H), 1.73 - 1.62 (m, 3H), 1.56 (d, J = 10.0 Hz, 1H), 1.49 (d, J = 10.0 Hz, 2H), 1.44 - 1.39 (m, 2H), 1.36 - 1.31 (m, 3H), 1.19 (s, 3H), 1.09 - 1.03 (m, 2H), 0.98 (s, 3H), 0.87 - 0.84 (m, 3H), 0.79 (s, 3H). 13 C NMR (125 MHz, CDCl3) δ 183.75, 171.43, 166.33, 164.30, 130.76, 115.81, 56.93, 55.75, 54.72, 44.93, 43.62, 41.07, 39.70, 39.13, 38.05, 31.93, 29.71, 29.37, 29.23, 22.70, 21.99, 21.58, 20.28, 18.85, 14.13, 13.5. HRMS (ESI, m / z) Calcd. for C 26 H 35 FN2O4S [M + H] + : 490.2302, Found: 490.8087.

[0073] (4) Compound 6: 1 H NMR (500 MHz, CDCl3) δ 7.84 (d, J = 10.0 Hz, 2H), 6.94 (d, J = 10.0 Hz, 2H), 3.84 (s, 3H), 2.65 (d, J = 20.0 Hz, 1H), 2.18 - 2.12 (m, 1H), 1.84 - 1.78 (m, 2H), 1.73 - 1.66 (m, 3H), 1.55 - 1.49 (m, 3H), 1.45 - 1.37 (m, 3H), 1.36 - 1.30 (m, 3H), 1.20 (s, 3H), 1.09 - 1.02 (m, 3H), 0.99 (s, 3H), 0.88 - 0.84 (m, 3H), 0.78 (s, 3H). 13C NMR (125MHz, CDCl3) δ183.51,163.14,130.10,130.03,114.11,113.87,56.95,55.63,54.76,44.85,43.68,43.62,41.07,39 .75,39.09,37.69,37.65,31.93,29.70,29.19,22.70,22.01,21.52,20.27,18.89,14.13,13.50.HRMS(ESI,m / z)Calcd.for C 27 H 38 N2O5S[M+H] + :502.6700,Found:502.9133.

[0074] (5) Compound 7: 1 H NMR (500MHz, MeOD) δ7.78(d,J=10.0Hz,2H),7.63(d,J=10.0Hz,2H),2.67(d,J=15.0Hz,1H),2.11(d,J=15.0Hz,1H),1.86-1.79(m,2H),1.73-1.64(m ,3H),1.57-1.49(m,2H),1.42-1.31(m,4H),1.27(s,1H),1.24(s,1H),1.1 8(s,3H),1.09-1.03(m,2H),0.97(s,3H),0.94-0.81(m,3H),0.75(s,3H). 13 C NMR(125MHz,MeOD)δ180.61,172.60,137.66,131.87,129.38,127.74,56.84,55.69,54.59,53.43,44.82,43.44,40.93 ,40.82,39.81,39.10,38.03,37.97,37.83,29.58,28.85,21.80,21.53,20.17,18.84,13.06.HRMS(ESI,m / z)Calcd.for C 26 H 35 BrN2O4S[M+H] + :551.5400,Found:551.0521.

[0075] (6) Compound 8: 11H NMR (500 MHz, CDCl3) δ 7.64 (d, J = 10.0 Hz, 2H), 7.60 - 7.18 (m, 3H), 3.29 - 3.07 (m, 1H), 2.81 (s, 1H), 1.98 (d, J = 15.0 Hz, 1H), 1.78 - 1.44 (m, 6H), 1.35 (d, J = 15.0 Hz, 2H), 1.30 - 1.23 (m, 2H), 1.17 (s, 3H), 1.11 (d, J = 15.0 Hz, 3H), 1.03 (s, 3H), 1.03 (s, 1H), 0.97 (d, J = 11.9 Hz, 1H), 0.93 - 0.78 (m, 2H), 0.74 (s, 3H). 13 13C NMR (125 MHz, CDCl3) δ 184.40, 180.05, 135.59, 132.25, 131.38, 128.50, 127.82, 60.86, 59.80, 58.73, 49.04, 47.34, 44.77, 43.80, 43.05, 41.94, 41.76, 35.70, 34.83, 33.72, 33.41, 32.57, 26.40, 25.50, 24.28, 22.77, 17.45, 16.95. HRMS (ESI, m / z) Calcd. for C 27 H 36 N2O4 [M + H] + : 437.2759, Found: 437.2293.

[0076] (7) Compound 9: 1 1H NMR (500 MHz, CDCl3) δ 11.97 (s, 1H), 10.33 (s, 1H), 7.70 (dd, J = 15.0, 10.0 Hz, 4H), 2.01 (d, J = 15.5 Hz, 1H), 1.91 (d, J = 20.0 Hz, 1H), 1.83 - 1.61 (m, 5H), 1.61 - 1.54 (m, 2H), 1.48 (s, 1H), 1.45 - 1.35 (m, 3H), 1.34 - 1.22 (m, 3H), 1.11 (s, 3H), 1.07 (s, 2H), 1.06 (s, 3H), 1.00 - 0.82 (m, 3H), 0.78 (s, 3H). 13C NMR(125MHz,Pyr)δ179.75,135.85,134.25,131.38,129.96,123.92,122.49,56.75,55.85,54.86,45.24,43.58 ,41.07,39.93,39.37,38.95,38.33,38.06,29.21,22.67,22.18,20.56,19.39,13.27.HRMS(ESI,m / z)Calcd.for C 27 H 35 BrN2O3[M+H] + :514.1831,Found:514.4161.

[0077] (8) Compound 10: 1 H NMR (500MHz, CDCl3) δ11.97(s,1H),10.13(s,1H),7.78(d,J=10.0Hz,2H),7.01(d,J=10.0Hz,2H),3.81(s,3H),2.02(d,J=20 .0Hz,1H),1.92(d,J=20.0Hz,1H),1.57(s,7H),1.46-1.24(m,8H),1.13(s,3H),1.07(s,3H),1.01-0.83(m,4H),0.80(s,3H). 13 CNMR (125MHz, CDCl3) δ184.62,133.21,117.77,60.83,59.84,59.15,58.70,48.94,48.84,47.41,44. 85,43.80,43.05,42.01,41.81,32.82,25.71,25.54,24.34,22.81,17.20.HRMS(ESI,m / z)Calcd.for C 28 H 38 N2O4[M+H] + :467.2865,Found:467.2655.

[0078] (9) Compound 11: 11H NMR (500 MHz, CDCl3) δ 10.08 (s, 1H), 9.47 (s, 1H), 8.79 (d, J = 5.0 Hz, 1H), 8.59 (d, J = 5.0 Hz, 1H), 2.85 (d, J = 20.0 Hz, 1H), 2.19 (d, J = 15.0 Hz, 1H), 1.98 (d, J = 20.0 Hz, 1H), 1.91 (s, 1H), 1.87 - 1.74 (m, 3H), 1.72 (s, 1H), 1.70 - 1.63 (m, 2H), 1.57 - 1.41 (m, 4H), 1.36 - 1.31 (m, 2H), 1.28 (s, 3H), 1.24 (s, 3H), 1.23 (s, 1H), 1.16 (s, 2H), 1.10 - 1.01 (m, 1H), 0.98 - 0.92 (m, 1H), 0.86 (s, 3H). 13 13C NMR (125 MHz, CDCl3) δ 158.37, 147.90, 144.40, 142.69, 57.17, 56.05, 54.85, 45.07, 43.94, 41.30, 41.00, 39.72, 39.01, 38.48, 37.73, 37.57, 29.71, 29.15, 22.30, 21.70, 20.50, 18.90, 13.89, 0.86. HRMS (ESI, m / z) Calcd. for C 25 H 34 N4O3 [M + H] + : 439.2664, Found: 439.1984.

[0079] (10) Compound 12: 1 1H NMR (500 MHz, MeOD) δ 9.06 (s, 1H), 8.96 (s, 1H), 8.65 (d, 1H), 8.33 (d, J = 5.0 Hz, 1H), 8.22 (d, J = 5.0 Hz, 1H), 7.47 (t, J = 10.0 Hz, 1H), 2.14 (d, J = 15.0 Hz, 1H), 1.90 - 1.76 (m, 5H), 1.74 - 1.66 (m, 4H), 1.53 - 1.46 (m, 4H), 1.31 (d, J = 15.0 Hz, 2H), 1.18 (s, 3H), 1.11 (d, J = 15.0 Hz, 2H), 1.03 (s, 3H), 0.88 (s, 2H), 0.82 (s, 3H). 13C NMR(125MHz,MeOD)δ218.66,180.61,176.04,163.24,151.35,150.58,148.05,138.89,136.52,130.09,123.76,122.99,122.89 ,56.88,55.85,54.69,45.28,43.48,40.93,39.88,39.16,37.86,28.89,21.82,20.42,18.87,13.21.HRMS(ESI,m / z)Calcd.for C 26 H 35 N3O3[M+H] + :437.2628,Found:437.1204.

[0080] (11) Compound 13: 1 H NMR (500MHz, CDCl3) δ10.64(d,J=10.0Hz,1H),8.57(d,J=10.0Hz,1H),2.77(d,J=20.0 Hz,1H),2.22(d,J=15.0Hz,1H),2.09-2.02(m,1H),1.97(d,J=25.0Hz,1H),1.90-1.83( m,3H),1.74(d,J=15.0Hz,2H),1.69-1.65(m,1H),1.65-1.59(m,3H),1.52-1.37(m,7H ),1.22(s,3H),1.13(s,3H),1.09(s,3H),1.04-0.96(m,2H),0.84(s,3H),0.68(s,1H). 13 C NMR (125MHz, CDCl3) δ182.23,170.70,167.06,57.25,56.20,55.16,44.50,44.28,43.66,41.40,41.04, 40.14,39.23,38.74,38.09,37.91,29.64,22.25,21.14,20.48,19.18,13.91.HRMS(ESI,m / z)Calcd.for C 22 H 34 N2O3[M+H] + :374.2569,Found:374.5970.

[0081] Example 6

[0082] At room temperature, 1,2-dibromoethane (81 μL, 0.942 mmol) and K2CO3 (130.2 mg, 0.942 mmol) were added to a solution of isosteviol (200 mg, 0.628 mmol) in N,N-dimethylformamide (DMF) (1.5 mL). After stirring for 9 hours, the reaction mixture was diluted with water and extracted with ethyl acetate (3×30 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was evaporated under reduced pressure. The crude material was subjected to column chromatography using petroleum ether / ethyl acetate (5:1) as the eluent to obtain pure compound III as a white amorphous powder with a yield of 56.4%.

[0083] To a solution of compound III (50 mg, 0.118 mmol) in DMF (1.5 mL) were added K2CO3 (24.46 mg, 0.177 mmol), piperazine (81 mg, 0.94 mmol) and N-butylpiperazine (33.57 mg, 0.236 mmol). After stirring for 12 hours, the reaction mixture was diluted with water and extracted with ethyl acetate (3×30 mL). The organic layer was collected, dried over anhydrous Na2SO4, filtered, and the filtrate was evaporated under reduced pressure. Finally, the residue was purified by silica gel column chromatography to obtain pure target compound IV. Yield: 57.6%, 70.1%.

[0084] Compound 22: 1 H NMR(500MHz, CDCl3)δ:4.19-4.08(m,2H),2.65-2.43(m,10H),2.33-2.28(m, 2H),2.18-2.12(m,1H),1.88-1.63(m,7H),1.63-1.56(m,2H),1.55-1.44(m,4 H),1.41-1.37(m,2H),1.37-1.32(m,1H),1.23(s,2H),1.17(s,3H),1.12-1.0 8(m,1H),1.03-0.98(m,1H),0.96(s,3H),0.88(t,J=7.4Hz,4H),0.70(s,3H); 13 C NMR(125MHz, CDCl3)δ:222.69,177.32,61.58,60.71,57.18,56.56,54.78,54.36,53.25(2C),53.20(2C),48.82,48.71,43 .87,41.64,39.92,39.56,38.14,38.05,37.41,29.06,21.80,20.42,19.97,19.01,13.53,12.05.HRMS(ESI,m / z)Calcd.for C 29H 48 N2O3[M+H] + :473.3745,Found:472.7200.

[0085] Example 7

[0086] Compound IV (100 mg, 0.232 mmol) was dissolved in DMF (1.5 mL) and K2CO3 (48.1 mg, 0.348 mmol), 2-chlorobenzyl bromide (59.6 μL, 0.464 mmol), 2-bromobenzyl bromide (61.0 μL, 0.464 mmol), 2-iodobenzyl bromide (65.6 μL, 0.464 mmol), 1-bromo-4-nitrobenzene (55.1 μL, 0.464 mmol) were added to a solution of compound IV (100 mg, 0.232 mmol) in DMF (1.5 mL) and reacted at room temperature. After stirring for 6 hours, the reaction mixture was diluted with water and extracted with ethyl acetate (3×30 mL). The organic layer was collected, dried over anhydrous Na2SO4, filtered, and the filtrate was evaporated under reduced pressure. Finally, the residue was purified by silica gel column chromatography to obtain pure target compounds 23-26 with yields of 57.8%, 60.3%, 54.9%, and 50.3%.

[0087] Compound 23: 1 H NMR(500MHz, CDCl3)δ:7.77-7.68(m,1H),7.34-7.27(m,1H),7.24-7.21(m,1H),6.89-6.80(m,1H ),4.13-3.99(m,2H),3.43(s,2H),2.58-2.54(m,2H),2.53-2.39(m,8H),2.11-2.04(m,1H),1.83 -1.57(m,7H),1.57-1.49(m,2H),1.49-1.36(m,4H),1.31-1.25(m,1H),1.20-1.15(m,2H),1.11- 1.10(m,3H),1.06-1.04(m,1H),0.96-0.92(m,1H),0.89(s,3H),0.85-0.79(m,1H),0.63(s,3H); 1313C NMR (125 MHz, CDCl3) δ: 222.54, 177.16, 140.42, 139.39, 130.30, 128.61, 127.90, 100.65, 66.32, 61.48, 56.98, 56.38, 54.58, 54.16, 53.28 (2C), 52.88 (2C), 48.63, 48.53, 43.69, 41.44, 39.72, 39.39, 37.96, 37.87, 37.22, 28.90, 21.62, 20.24, 19.81, 18.84, 13.38. HRMS (ESI, m / z) Calcd. for C 33 H 47 ClN2O3 [M + H] + : 555.3355, Found: 555.3415.

[0088] Compound 24: 1 1H NMR (500 MHz, CDCl3) δ: 7.54 - 7.49 (m, 1H), 7.46 - 7.42 (m, 1H), 7.29 - 7.23 (m, 1H), 7.08 (t, J = 7.6 Hz, 1H), 4.20 - 4.07 (m, 2H), 3.59 (s, 2H), 2.65 - 2.61 (m, 2H), 2.60 - 2.42 (m, 8H), 2.18 - 2.13 (m, 1H), 1.90 - 1.64 (m, 7H), 1.64 - 1.56 (m, 2H), 1.56 - 1.44 (m, 2H), 1.42 - 1.37 (m, 2H), 1.37 - 1.31 (m, 1H), 1.26 - 1.18 (m, 2H), 1.17 (s, 3H), 1.13 - 1.08 (m, 1H), 1.03 - 0.98 (m, 1H), 0.96 (s, 3H), 0.92 - 0.85 (m, 1H), 0.70 (s, 3H); 13 13C NMR (125 MHz, CDCl3) δ: 222.62, 177.27, 137.53, 132.80, 130.87, 128.46, 127.24, 124.78, 61.79, 61.60, 57.14, 56.51, 54.74, 54.32, 53.40 (2C), 53.13 (2C), 48.77, 48.64, 43.83, 41.59, 39.87, 39.52, 38.10, 38.01, 37.36, 29.04, 21.76, 20.38, 19.94, 18.98, 13.50. HRMS (ESI, m / z) Calcd. for C 33 H 47BrN2O3[M+H] + :599.2850,Found:588.3996.

[0089] Compound 25: 1 H NMR(500MHz, CDCl3)δ:7.43(dd,J=7.5,1.4Hz,1H),7.34-7.29(m,1H),7.21(td,J=7.4,1.2Hz,1H),7.1 5(td,J=7.6,1.7Hz,1H),4.19-4.07(m,2H),3.60(s,2H),2.65-2.44(m,10H),2.17-2.12(m,1H),1.89- 1.63(m,7H),1.62-1.55(m,2H),1.55-1.43(m,2H),1.41-1.36(m,2H),1.36-1.30(m,1H),1.26-1.18(m ,2H),1.16(s,3H),1.12-1.07(m,1H),1.03-0.98(m,1H),0.96(s,3H),0.91-0.84(m,1H),0.69(s,3H); 13 C NMR(125MHz, CDCl3)δ:222.60,177.27,135.79,134.42,130.88,129.50,128.20,126.63,61.60,59.26,57.14,56.51,54.74,54.32,53.38(2C), 53.14(2C),48.76,48.65,43.83,41.59,39.87,39.52,38.10,38.01,37.37,29.04,21.77,20.38,19.95,18.98,13.50.HRMS(ESI,m / z)Calcd.for C 33 H 47 IN2O3[M+H] + :647.2711,Found:647.1413.

[0090] Compound 26: 1H NMR(500MHz, CDCl3)δ:8.09(d,J=6.3Hz,2H),6.81(d,J=9.4Hz,2H),4.25-4.12(m,2H),3.41-3.37(m,4H),2.66(t,J =5.8Hz,2H),2.65-2.61(m,4H),2.59(d,J=3.7Hz,1H),2.19-2.13(m,1H),1.91-1.85(m,1H),1.83-1.75(m,2H),1.7 2-1.65(m,3H),1.63-1.61(m,1H),1.61-1.56(m,1H),1.55-1.51(m,1H),1.48(dd,J=13.6,3.8Hz,1H),1.44-1.38(m ,3H),1.23(s,1H),1.19(s,4H),1.15-1.10(m,1H),1.05-0.98(m,1H),0.95(s,3H),0.94-0.86(m,1H),0.71(s,3H); 13 C NMR(125MHz, CDCl3)δ:222.63,177.30,154.91,138.55,126.05(2C),112.8 0(2C),61.09,57.12,56.52,54.70,54.30,52.81(2C),48.80,48.63,47.17 (2C),43.92,41.58,39.86,39.55,38.14,38.02,37.37,29.13,21.81,20.4 1,19.94,19.02,13.62.32,19.85,18.93,13.54.HRMS(ESI,m / z)Calcd.for C 32 H 45 N3O5[M+H] + :552.3439,Found:551.6603.

[0091] Example 8

[0092] Verification of the inhibitory effect of isosteviol derivatives on the proliferation of HCT116, SKOV3 and HepG2 cells

[0093] (1) Cell inoculation: HCT116, SKOV3 and HepG2 cells with EGFR sensitive mutations in the exponential growth phase were digested and made into suspension using DMEM / McCoy's 5A medium containing 10% calf serum. Tumor cells were seeded at a cell number of 0.5×10 4Each well of the plate was inoculated with 100 μL of the culture medium and cultured in a 37°C, CO2 incubator for 12 h.

[0094] (2) Drug addition: The test compound was added with DMSO to prepare a stock solution of a certain concentration, and then prepared into 30.0 μM, 20.0 μM, 10.0 μM, 5.0 μM, and 1.0 μM solutions respectively using DMEM / McCoy's 5A medium containing 10% calf serum. The solutions were inoculated in a 96-well cell culture plate, and three parallel wells were set for each concentration. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h, and the cell morphology and growth changes were observed under an inverted microscope.

[0095] (3) Termination of culture: First, prepare a mixed solution containing 10% CCK-8, take 450 μL of CCK-8 and dilute it to 4500 μL with serum-free culture medium, then add 100 μL of CCK-8 and culture medium mixed solution to each well, culture in a 37°C, 5% CO2 incubator for 1.5 hours, place on a microplate reader to measure the absorbance A value at a wavelength of 450 nm, and calculate the inhibition rate according to the following formula. Inhibition rate (%) = (average absorbance A value of the control group - average absorbance A value of the drug-added group) / (average absorbance A value of the control group - average absorbance A value of the blank group) × 100%; the control group is the culture well with a concentration of 0 isosteviol derivative and DMSO added, and the blank group is the culture well with culture medium and CCK-8 solution but no cells. GraphPad Prism6 was used for data statistical analysis and IC was calculated. 50 .

[0096] The results of the CCK-8 experiment are shown in Table 2, indicating that isosteviol derivatives have a significant inhibitory effect on the proliferation of HCT116, SKOV3 and HepG2 cells, indicating that isosteviol derivatives have the largest inhibition gradient on ovarian cancer SKOV3 and have a relatively strong inhibitory effect.

[0097] Table 2

[0098]

[0099] Example 9

[0100] Flow cytometry for cell cycle analysis

[0101] (1) Cell inoculation: SKOV3 and HCT116 cells in the exponential growth phase were digested and made into a suspension using McCoy's 5A medium containing 10% calf serum. Tumor cells were seeded at a cell number of 1×10 6 Each well of the plate was inoculated with 2 mL of culture medium and cultured in a 37°C, CO2 incubator for 12 h.

[0102] (2) Dosing: Compounds 7 and 18 were added with DMSO to prepare stock solutions of certain concentrations, and then prepared into 3.0 μM, 13.0 μM, 52.0 μM and 3.0 μM, 6.0 μM, and 12.0 μM solutions respectively using DMEM culture medium containing 10% calf serum. The solutions were inoculated in a 6-well cell culture plate, and three parallel wells were set for each concentration. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h.

[0103] (3) Collect cells: Wash the cells once with PBS, collect them by centrifugation at 1500 rpm for 5 min, and adjust the cell concentration to 1×10 6 / mL, take 1mL of single cell suspension, centrifuge and remove the supernatant.

[0104] (4) Staining: Mix 1 mL of RNase A and 10 μL of PI solution evenly and react at room temperature for 30 min.

[0105] (5) On-machine detection: Detection was performed using a BD Accuri C6 flow cytometer.

[0106] The flow cytometry results are as follows Figure 1 As shown, compared with the control group, the proportion of S-phase tumor cells in cells treated with compound 7 increased with the increase of the administration concentration, in a dose-dependent manner. In addition, compared with the control, when the cells were treated with 52.0 μM of compound 7, the number of S-phase tumor cells was 1.23 times that of the control. Moreover, when treated with 3.0, 13.0 and 52.0 μM of compound 7, the number of cells in the G1 phase decreased from 41.7% in the control group to 33.0%, 26.0% and 27.6%. The results show that compound 7 can arrest the cell cycle in the G2 / M phase.

[0107] The flow cytometry results are as follows Figure 2 As shown, the proportion of tumor cells in the G1 phase increased in a dose-dependent manner after treatment with compound 18. In addition, compound 18 caused a significant decrease in the number of cells in the S phase compared with the control group. The proportion of cells in the G1 phase increased from 46.1% (NC) to 45.0% (3.0μM), 49.2% (6.0μM) and 49.3% (12.0μM). At the same time, the proportion of cells in the S phase decreased from 36.9% (NC) to 34.2% (3.0μM), 33.1% (6.0μM) and 32.5% (12.0μM). The results show that compound 8 has an effect on the cell cycle when it is arrested in the G1 phase.

[0108] Example 10

[0109] Flow cytometry detection of cell apoptosis

[0110] (1) Inoculation of cells: SKOV3 and HCT116 cells in the exponential growth phase were digested and made into a suspension using McCoy's 5A medium containing 10% calf serum. Tumor cells were seeded at a cell number of 1×10 6 Each well of the plate was inoculated with 2 mL of culture medium and cultured in a 37°C, CO2 incubator for 12 h.

[0111] (2) Drug addition: Test compounds 7 and 18 were added with DMSO to prepare a certain concentration of stock solution, and then prepared into 3.0 μM, 13.0 μM, 52.0 μM solutions and 3.0 μM, 6.0 μM, 12.0 μM solutions respectively using McCoy's 5A medium containing 10% calf serum. The solutions were inoculated into 6-well cell culture plates, and three parallel wells were set for each concentration. The cells were cultured in a 37°C, 5% CO2 incubator for 48 h.

[0112] (3) Cell collection: Wash the cells once with PBS, collect them by centrifugation at 1500 rpm for 5 min, and adjust the cell concentration to 1×10 6 / mL, take 1mL of single cell suspension, centrifuge and remove the supernatant.

[0113] (4) Staining: Resuspend in 500 μL Binding buffer, add 5 μL annexin V-APC and 10 μL propidium iodide (PI) solution, mix well and react in the dark for 5 min.

[0114] (5) On-machine testing

[0115] The apoptosis experiment of SKOV3 cells treated with different concentrations of compound 10 for 48 h was detected by flow cytometry using V-APC and PI staining. Figure 3 As shown. Compound 10 significantly induced apoptosis of SKOV3 cells in a dose-dependent manner. At 3.0 μM, the percentage of total apoptotic cells (right quadrant, UR+LR) increased from 36.22% (NC) to 40.00%. When the drug concentration increased to 13.0 μM and 52.0 μM, the percentage of total apoptotic cells increased to 40.7% and 74.14%, respectively. At the same time, the proportion of SKOV3 necrotic cells was 2.35% (3.0 μM), 1.88% (13.0 μM) and 1.69% (30.0 μM), respectively. The results showed that compound 13 could induce apoptosis and significantly increase apoptotic cells in a concentration-dependent manner.

[0116] Compound 18 can significantly induce apoptosis of HCT116 cells in a dose-dependent manner. Untreated cells were used as NC to observe the apoptotic effect of compound 18 on HCT116 cell lines. Figure 4As shown, the early apoptosis rate of compound 8 gradually decreased from 44.1% (NC) to 13.0%, 12.3% and 13.1%. After treatment with 3.0, 6.0 and 12.0 μM concentrations of compound 8, the late apoptosis rate changed from 19.4%, 19.7% to 41.2%. The apoptosis ratios of HCT116 cells treated with compound 8 were 32.4% (3.0 μM), 32.0% (6.0 μM) and 54.3% (12.0 μM), respectively. The results show that compound 8 can induce late apoptosis, resulting in a significant increase in apoptotic cells, and is concentration-dependent.

[0117] In summary, the examples disclose 26 isosteviol derivatives, among which compounds 7 and 18 have higher activity than isosteviol at the cellular level. The synthesis conditions of isosteviol derivatives are mild and they are insensitive to water, oxygen, etc. The reaction raw materials are easily available and inexpensive, the operation is simple, the products are easy to separate and purify, the yield is high, and they are easy to industrialize and produce.

[0118] The CCK8 method was used to determine that isosteviol derivatives had an inhibitory effect on the proliferation of tumor cell lines such as human colon cancer cell line HCT116, human ovarian cancer cell line SKOV3 and human liver cancer cell line HepG2, and had significant anticancer activity (partial anticancer IC 50 The values ​​are shown in Table 1). Flow cytometry showed that compound 7 had a cell cycle arrest effect, causing cancer cells SKOV3 to stay in the G2 / M phase, and compound 18 had a cell cycle arrest effect, causing cancer cells HCT116 to stay in the G1 phase, thereby achieving an anti-tumor effect and inducing cancer cell apoptosis.

[0119] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. An isosteviol derivative having a general structure as shown in Formula 1: In Formula 1, R1 and R2 are respectively as shown in the following table.

2. Use of the isosteviol derivative or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of anticancer drugs.

3. The use according to claim 2, characterized in that: Such cancers include colon cancer, ovarian cancer and liver cancer.

4. The use according to claim 2, characterized in that: The drug uses the isosteviol derivative or a pharmaceutically acceptable solvate thereof as claimed in claim 1 as an active ingredient, with a weight percentage of 5% to 100%.

5. The use according to claim 2, characterized in that: The medicament further includes a pharmaceutically acceptable carrier or diluent.

6. The use according to claim 2, characterized in that: The dosage form of the drug is selected from at least one of tablets, capsules, granules, pellets, suspensions, syrups, enteric-coated preparations, emulsion suspensions and injections.

7. An anti-cancer drug, characterized in that: The drug comprises the isosteviol derivative according to claim 1 or a pharmaceutically acceptable solvate thereof.

8. Use of the isosteviol derivative or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of HCT116, SKOV3 and HepG2 cell proliferation inhibitors.

Images