Isosteviol derivatives and their use in the preparation of anticancer drugs

Abstract

The application discloses isosiphanol derivatives and application thereof in preparation of anticancer drugs, and relates to isosiphanol derivatives with a general structure as shown in formula 1, which can be prepared through a synthetic route as shown in formula 2, and the obtained isosiphanol derivatives are used for preparing anticancer drugs for colon cancer, ovarian cancer and liver cancer, in particular, colon cancer HCT116, ovarian cancer SKOV3 and liver cancer HepG2, and the drugs are at least one preparation form selected from tablet, capsule, granule, drop pill, suspension, syrup, enteric preparation, emulsion suspension and injection. The isosiphanol derivatives have small toxic and side effects, high bioavailability and high clinical use value, provide a candidate lead compound for new inhibitor research and development of anticancer drugs, and have potential application value in the field of targeted drugs for ovarian cancer.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to isosteviol derivatives and their application in the preparation of anticancer drugs. Background Technology

[0002] Malignant tumors are a class of diseases that threaten human health and life. Currently, the treatment of malignant tumors often employs a comprehensive approach combining surgery, radiotherapy, and chemotherapy. Chemotherapy is a systemic treatment that can eliminate cancer cells that have metastasized to distant sites, playing an important role in comprehensive treatment. However, in clinical practice, chemotherapy drugs have issues with efficacy, side effects, and drug resistance in tumor cells. Therefore, finding highly effective and low-toxicity anti-tumor drugs from different perspectives remains an urgent task.

[0003] Natural products are an important source of lead compounds and functional organic molecules for drugs, and screening for drug lead compounds from natural products has always been an effective approach to new drug development. Due to their broad biological activities and low toxicity, natural products have always been an important source for drug research and development, and the active ingredients in traditional Chinese medicine have become a current research hotspot. Isostevirol, isolated from the roots of Tripterygium wilfordii, possesses various biological activities, including antitumor, anti-inflammatory, and immunosuppressive effects, and its antitumor activity has been extensively studied. Isostevirol exhibits cytotoxicity against various human cancer cell lines by inducing apoptosis, but its antitumor efficacy (HCT116: IC50) remains limited. 50 =24.8±2.43μM, SKOV3:IC 50 >100μM, HepG2: IC 50 The physicochemical properties (>100 μM) need improvement. By modifying isosteviol C-16 and C-19, and designing compounds with different substituents at different positions, the inhibitory effects of these compounds on three cancer cells (HCT116, SKOV3, and HepG2) were measured using the CCK8 assay. This can provide a good starting point for the development of novel antitumor drugs. Summary of the Invention

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

[0005] Another objective of this invention is to provide the application of the above-mentioned isosteviol derivatives in the preparation of anticancer drugs, wherein the isosteviol derivatives have significant antitumor cell activity.

[0006] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0007] This invention provides an isosteviol derivative having the general formula structure shown in Formula 1:

[0008]

[0009] In Equation 1, R1 and R2 are shown in the table below:

[0010]

[0011]

[0012]

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

[0014]

[0015] The present invention also provides the use of the above-mentioned isosteviol derivatives or their pharmaceutically acceptable salts in the preparation of anticancer drugs.

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

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

[0018] This invention provides an anticancer drug comprising isosteviol derivatives or pharmaceutically acceptable solvates thereof, wherein the isosteviol derivatives have the structural formula described above and exhibit significant inhibitory effects on cancer cells, thus representing a novel anticancer 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 also includes a pharmaceutically acceptable carrier or diluent.

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

[0022] "Pharmaceuticalally acceptable diluents" include, but are not limited to, starches (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 the following formulations: 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) This invention demonstrates through in vitro experiments that isosteviol derivatives have strong anti-colon cancer HCT116, ovarian cancer SKOV3 and liver cancer HepG2 activities, and have significant inhibitory effects on the growth and cell activity of HCT116, SKOV3 and HepG2 cells. They can be used as lead compounds for cancer treatment in the preparation of cancer drugs.

[0027] (2) The isosteviol derivatives in this 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. Attached Figure Description

[0028] Figure 1 Example 9 was used to test the inhibitory effect of different concentrations of compound 7 on the SKOV3 cell cycle.

[0029] Figure 2 Example 9 was used to test the inhibitory effect of different concentrations of compound 18 on the HCT116 cell cycle.

[0030] Figure 3 Example 10 was used to test the inhibitory effect of different concentrations of compound 7 on the SKOV3 cell cycle.

[0031] Figure 4 Example 10 was used to detect the inhibitory effect of different concentrations of compound 18 on apoptosis in HCT116 cells.

[0032] Figure 5 This is the general formula structure of the isosteviol derivative in this invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

[0035] The following examples illustrate an isosteviol derivative having the general formula shown in Formula 1:

[0036]

[0037] In Equation 1, R1 and R2 are shown in the table below:

[0038]

[0039]

[0040]

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

[0042]

[0043] Example 1

[0044] (1) Preparation of compound a: Isostevioside (200 mg, 0.636 mmol) was dissolved in N,N-dimethylformamide (DMF), and then 87.0 μL of bromopropane (0.954 mmol) and 80.1 mg of sodium bicarbonate (0.954 mmol) were added. The mixture was stirred at room temperature for 5 hours.

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

[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 for 4 hours in the presence of NaHCO3.

[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, the mixture was purified by silica gel column chromatography to give 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). The ethyl acetate solution was then washed with hydrochloric acid (3 × 30 mL), saturated sodium bicarbonate (3 × 30 mL), and saturated saline solution (3 × 30 mL), respectively. The organic layer was then dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure. Finally, the mixture 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: Isostevioside (60 mg, 0.2 mmol) was dissolved in acetonitrile, and chloroacetylchloroaniline compound (0.21 mmol) was added. The mixture was heated at 80 °C for 24 h.

[0066] (2) After the reaction was complete, 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, the mixture was purified by silica gel column chromatography to give 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: Isostevioside (50 mg, 0.16 mmol) was dissolved in methanol (MeOH) (2 mL), and benzenesulfonyl hydrazide compound and hydrazide compound (0.24 mmol) were added. The mixture was then heated at 80°C for 12 hours in a heating device. The reaction was monitored by TLC. After the reactants had reacted completely, the reaction was stopped. The reaction solution was extracted three times with ethyl acetate and water. The organic phase was collected, washed three times with saturated brine, and then dehydrated with anhydrous Na2SO4. The solvent was removed under vacuum, and the solution was 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 1 36 2N2O4 [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 14N4O3 [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, isosteviol (200 mg, 0.628 mmol) was added to a solution of N,N-dimethylformamide (DMF) (1.5 mL) with 1,2-dibromoethane (81 μL, 0.942 mmol) and K₂CO₃ (130.2 mg, 0.942 mmol). 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 with petroleum ether / ethyl acetate (5:1) as eluent to give pure compound III as a white amorphous powder, yield: 56.4%.

[0083] To a solution of compound III (50 mg, 0.118 mmol) in DMF (1.5 mL), K₂CO₃ (24.46 mg, 0.177 mmol), piperazine (81 mg, 0.94 mmol), and N-butylpiperazine (33.57 mg, 0.236 mmol) were added. 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 give pure target compound IV. Yields: 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 reacted in a solution of DMF (1.5 mL) with 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), and 1-bromo-4-nitrobenzene (55.1 μL, 0.464 mmol) 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 give pure target compounds 23-26 in yields of 57.8%, 60.3%, 54.9%, and 50.3%, respectively.

[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 seeding: EGFR-sensitive mutants HCT116, SKOV3, and HepG2 cells in the exponential growth phase were digested and suspended in DMEM / McCoy's 5A medium containing 10% fetal bovine serum. Tumor cells were seeded at a rate of 0.5 × 10⁶ cells / year. 4Inoculate 100 μL of each sample per mL into a 96-well plate and incubate at 37°C in a CO2 incubator for 12 h.

[0094] (2) Drug addition: The test compound was prepared into a stock solution of a certain concentration by adding DMSO. Then, solutions of 30.0 μM, 20.0 μM, 10.0 μM, 5.0 μM and 1.0 μM were prepared in DMEM / McCoy's 5A medium containing 10% fetal bovine serum. The solutions were seeded into 96-well cell culture plates with three parallel replicates for each concentration. The cells were cultured in a 37℃, 5% CO2 incubator for 24 h. Cell morphology and growth changes were observed under an inverted microscope.

[0095] (3) Termination of culture: First, prepare a mixture containing 10% CCK-8. Take 450 μL of CCK-8 and dilute it with serum-free medium to 4500 μL. Add 100 μL of the CCK-8 and medium mixture to each well. Incubate at 37℃ and 5% CO2 for 1.5 h. Then, measure the absorbance (A value) at 450 nm using a microplate reader. Calculate the inhibition rate using the following formula: Inhibition rate (%) = (Average absorbance (A value) of the control group - Average absorbance (A value) of the drug-treated 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 0% isosteviol derivative and DMSO added. The blank group is the culture well with medium and CCK-8 solution but no cells. Statistical analysis of the data was performed using GraphPad Prism6, and the IC50 was calculated. 50 .

[0096] The results of the CCK-8 experiment are shown in Table 1. The results show that isosteviol derivatives have a significant inhibitory effect on the proliferation of HCT116, SKOV3 and HepG2 cells, indicating that isosteviol derivatives have the largest inhibitory gradient on ovarian cancer SKOV3 and have a relatively strong inhibitory effect.

[0097] Table 1

[0098]

[0099] Example 9

[0100] Flow cytometry for cell cycle detection

[0101] (1) Cell seeding: SKOV3 and HCT116 cells in the exponential growth phase were digested and suspended in McCoy's 5A medium containing 10% fetal bovine serum. Tumor cells were seeded at a rate of 1 × 10⁶ cells / year. 6 Inoculate 2 mL of each sample per well into a 6-well plate and incubate at 37°C in a CO2 incubator for 12 h.

[0102] (2) Drug addition: Compounds 7 and 18 were prepared into stock solutions of a certain concentration by adding DMSO. Then, solutions of 3.0 μM, 13.0 μM, 52.0 μM and 3.0 μM, 6.0 μM and 12.0 μM were prepared in DMEM medium containing 10% fetal bovine serum. These solutions were seeded into 6-well cell culture plates with three parallel replicates for each concentration. The plates were then cultured for 24 h at 37°C in a 5% CO2 incubator.

[0103] (3) Cell collection: Wash cells once with PBS, centrifuge at 1500 rpm for 5 min to collect cells, and adjust the cell concentration to 1×10⁻⁶. 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) Detection on the instrument: Detection was performed using a BD Accuri C6 flow cytometer.

[0106] Flow cytometry results 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 in a dose-dependent manner with increasing concentration. Furthermore, compared with the control, the number of S-phase tumor cells treated with 52.0 μM compound 7 was 1.23 times that of the control. Moreover, when treated with 3.0, 13.0, and 52.0 μM compound 7, the number of cells in G1 phase decreased from 41.7% in the control group to 33.0%, 26.0%, and 27.6%, respectively. These results indicate that compound 7 can arrest the cell cycle at the G2 / M phase.

[0107] Flow cytometry results as follows Figure 2 As shown, treatment with compound 18 resulted in a dose-dependent increase in the proportion of G1 phase tumor cells. Furthermore, compared to the control group, compound 18 led to a significant reduction in the number of S phase cells. The proportion of G1 phase cells increased from 46.1% (NC) to 45.0% (3.0 μM), 49.2% (6.0 μM), and 49.3% (12.0 μM). Simultaneously, the proportion of S phase cells decreased from 36.9% (NC) to 34.2% (3.0 μM), 33.1% (6.0 μM), and 32.5% (12.0 μM). These results indicate that compound 18 has an effect on the cell cycle during G1 phase arrest.

[0108] Example 10

[0109] Flow cytometry detection of cell apoptosis

[0110] (1) Cell seeding: SKOV3 and HCT116 cells in the exponential growth phase were digested. A suspension was prepared using McCoy's 5A medium containing 10% fetal bovine serum. Tumor cells were seeded at a rate of 1 × 10⁶ cells / year. 6 Inoculate 2 mL of each sample per well into a 6-well plate and incubate at 37°C in a CO2 incubator for 12 h.

[0111] (2) Drug addition: The test compounds 7 and 18 were prepared into stock solutions of a certain concentration by adding DMSO. Then, solutions of 3.0 μM, 13.0 μM, and 52.0 μM were prepared in McCoy's 5A medium containing 10% fetal bovine serum, and solutions of 3.0 μM, 6.0 μM, and 12.0 μM were prepared respectively. These solutions were seeded into 6-well cell culture plates with three parallel replicates for each concentration. The cells were then cultured in a 37°C, 5% CO2 incubator for 48 h.

[0112] (3) Cell collection: Wash cells once with PBS, centrifuge at 1500 rpm for 5 min to collect cells, and adjust the cell concentration to 1×10⁻⁶. 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] Apoptosis assays were performed on SKOV3 cells treated with different concentrations of compound 10 for 48 hours using V-APC and PI staining by flow cytometry. Figure 3 As shown, compound 10 significantly induced apoptosis in 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 was increased to 13.0 μM and 52.0 μM, the percentage of total apoptotic cells increased to 40.7% and 74.14%, respectively. Simultaneously, the proportions of necrotic SKOV3 cells were 2.35% (3.0 μM), 1.88% (13.0 μM), and 1.69% (30.0 μM), respectively. These results indicate that compound 13 can induce apoptosis, significantly increasing the number of apoptotic cells in a concentration-dependent manner.

[0116] Compound 18 significantly induced apoptosis in HCT116 cells in a dose-dependent manner. The apoptotic effect of compound 18 on the HCT116 cell line was observed using untreated cells as NC. 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 compound 8 at concentrations of 3.0, 6.0, and 12.0 μM, the late apoptosis rate changed from 19.4% and 19.7% to 41.2%. The apoptosis rates 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. These results indicate that compound 8 can induce late apoptosis, leading to a significant increase in apoptotic cells in a concentration-dependent manner.

[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 isosteviol derivatives have mild synthesis conditions, are not sensitive to water, oxygen, etc.; the reaction raw materials are readily 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.

[0118] The CCK8 assay showed that isosteviol derivatives inhibited the proliferation of tumor cell lines such as human colon cancer cell line HCT116, human ovarian cancer cell line SKOV3, and human hepatocellular carcinoma cell line HepG2, exhibiting significant anticancer activity (partial anticancer IC50 of the compound). 50 (Values ​​are shown in Table 1). Flow cytometry analysis showed that compound 7 had cell cycle arrest, causing SKOV3 cancer cells to arrest in the G2 / M phase, and compound 18 had cell cycle arrest, causing HCT116 cancer cells to arrest in the G1 phase, thus achieving anti-tumor effects and inducing apoptosis in cancer cells.

[0119] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. 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 invention, should be within the protection scope of the present invention.

Claims

1. An isostevirol derivative having the general formula shown in Formula 1: [Formula 1] In Equation 1, R1 and R2 are shown below.

2. The use of the isosteviol derivative of claim 1 in the preparation of anticancer drugs, wherein the cancer is selected from colon cancer, ovarian cancer and liver cancer.

3. The application according to claim 2, characterized in that, The drug uses the isosteviol derivative of claim 1 as the active ingredient, with a weight percentage of 5% to 100%.

4. The application according to claim 2, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

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

6. An anticancer drug, characterized in that, The drug includes the isosteviol derivative of claim 1.

7. The use of the isosteviol derivative of claim 1 in the preparation of HCT116, SKOV3 and HepG2 cell proliferation inhibitors.

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