Cucurbitacine B ester derivatives and application thereof in drugs for resisting various cancer cells

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to cucurbitacine B ester derivatives and application thereof in drugs for resisting various cancer cells. A series of cucurbitacine B ester derivatives are synthesized by carrying out structural modification on the original drug cucurbitacine B, and the proliferation inhibition effects of the derivatives on liver cancer, prostatic cancer and breast cancer cells are verified through cell proliferation experiments. Test results show that the effect of most cucurbitacine B ester derivatives is better than that of cucurbitacine B, and particularly, the anti-cancer effects of the derivatives Z1, Z3 and Z7 are better.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a class of cucurbitacin B ester derivatives and applications thereof in drugs against various cancer cells. Background Art

[0002] Natural products are an important source of clinical drugs. According to statistics, nearly half of the chemical drugs currently in use are directly derived from natural products, or are derivatives derived from natural products as lead compounds through structural modification.

[0003] Melon pedicles are traditional medicinal plants in my country. Modern research shows that the α,β-unsaturated carbonyl structure cucurbitacin compounds contained in melon pedicles have a wide range of biological and pharmacological activities. Cucurbitacin compounds have multiple biological activities such as anti-tumor, liver protection, anti-inflammatory, improving body immunity, gastrointestinal effects, etc. The α,β-unsaturated group contained in this type of compound is the key functional group for its activity.

[0004] Cucurbitacin B has the chemical structure:

[0005]

[0006] It is a tetracyclic triterpenoid compound with high cytotoxic activity isolated from plants such as the Cucurbitaceae family. It is relatively abundant in Cucurbitaceae plants and has a wide range of pharmacological activities.

[0007] Currently, the derivatization of cucurbitacin B is relatively limited, and mainly focuses on the modification of the 2-hydroxyl group and the side chain. At the same time, due to the low reactivity of the 2-hydroxyl group, the derivatives obtained are mostly simple organic acid derivatives such as methyl ester and ethyl ester, and the pharmacological activity is limited. Summary of the invention

[0008] The purpose of the present invention is to provide a class of cucurbitacin B ester derivatives and preparation methods and uses. The present invention synthesizes a class of novel cucurbitacin B ester derivatives, which have better anticancer activity than cucurbitacin B. The structure of the cucurbitacin B ester derivatives of the present invention is as follows:

[0009]

[0010] Where R is:

[0011]

[0012] The preferred chemical names and chemical structural formulas of the derivatives are as follows:

[0013] Z1: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-16-hydroxy-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2-yl-2-chloroacetate

[0014]

[0015] Z2: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,10,11,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2,16-diylbis(2-chloroacetate)

[0016]

[0017] Z3: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-16-hydroxy-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2-yl-2-methylbenzoate

[0018]

[0019] Z4: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,10,11,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2,16-diylbis(2-methylbenzoate)

[0020]

[0021] Z5: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-16-hydroxy-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2-yl-2-nitrobenzoate

[0022]

[0023] Z6: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,10,11,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2,16-diylbis(2-nitrobenzoate)

[0024]

[0025] Z7: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-16-hydroxy-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2-yl-4-cyanobenzoate

[0026]

[0027] Z8: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,10,11,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2,16-diylbis(4-cyanobenzoate)

[0028]

[0029] The reaction equation and preparation method steps of the cucurbitacin B derivative of the present invention are as follows:

[0030]

[0031] 1) Add an appropriate amount of organic solvent to dissolve cucurbitacin B, then add 4-dimethylaminopyridine (DMAP) and an acyl chloride reagent, and react at 30-60° C. for 12-24 hours;

[0032] Wherein, the organic solvent is anhydrous dichloromethane or 1,2-dichloroethane.

[0033] The molar ratio of cucurbitacin B, DMAP and acyl chloride reagent is 1:1:0.7-3.

[0034] The acyl chloride reagent is chloroacetyl chloride, o-toluoyl chloride, 2-nitrobenzoyl chloride or 4-cyanobenzoyl chloride.

[0035] 2) After the reaction is complete, stop the reaction; concentrate the reaction solution under reduced pressure, and separate the target products (Z1-Z8) by silica gel column chromatography.

[0036] When eluting through silica gel column chromatography, first use petroleum ether for elution, and then use petroleum ether:ethyl acetate = 6:1-1:1 for gradient elution.

[0037] Beneficial effects:

[0038] Cucurbitacin B and its derivatives have inhibitory effects on HePG2 cells, 22RV1 cells and MCF-7 cells, and the inhibitory effects of most of the cucurbitacin B ester derivatives of the present invention are greater than that of cucurbitacin B. The cucurbitacin B ester derivatives Z1, Z2, Z3, Z5 and Z7 of the present invention all have anticancer activity, among which the effects of Z1, Z3 and Z7 are more significant. Therefore, the cucurbitacin B ester derivatives of the present invention can be developed as potential anticancer lead derivatives. DETAILED DESCRIPTION

[0039] In order to more clearly illustrate the purpose, technical solutions and advantages of the present invention, the present invention is further described in detail below in conjunction with tables and specific examples.

[0040] Example 1

[0041] Dissolve cucurbitacin B (100 mg, 0.18 mmol) in anhydrous dichloromethane (50 ml), then add 4-dimethylaminopyridine (22 mg, 0.18 mmol) and chloroacetyl chloride (0.13 mmol), stir and heat at 40°C for 12 h, concentrate under reduced pressure, and obtain the target derivative Z1 by silica gel column chromatography in a yield of 39%.

[0042] Z1: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-16-hydroxy-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2-yl-2-chloroacetate

[0043] 1H NMR(400MHz,Chloroform-d)δ7.19(d,J=15.6Hz,1H),6.37(d,J=15.6Hz,1H),5.77(d,J=5.5Hz,1H),5.33-5.20(m,1 H),4.40(dt,J=12.8,4.5Hz,1H),3.90(d,J=3.1Hz,2H),3.26(d,J=14.6Hz,1H),2.81-2.65(m,3H),2.42(dd,J=19.6 ,7.8Hz,1H),2.35-2.26(m,1H),2.03(s,3H),2.00(s,1H),1.95(d,J=6.0Hz,1H),1.90(d,J=5.7Hz,1H),1.68(s,2H) ,1.60(s,3H),1.42(s,3H),1.39(s,1H),1.33(s,3H),1.28(d,J=9.7Hz,7H),1.24(s,1H),1.08(s,3H),1.02(s,3H).

[0044] 13 C NMR(101MHz,Chloroform-d)δ213.00,211.55,200.94,169.98,166.84,153.60,140.51,120.41,118.95,79.17,77.69,75.70,71.70,54.23,5 0.36,50.20,48.59,48.46,48.17,43.04,42.14,40.71,36.05,33.81, 29.45,27.23,26.02,23.88,23.61,21.96,21.36,20.20,19.85,18.79.

[0045] Example 2

[0046] Cucurbitacin B (100 mg, 0.18 mmol) was dissolved in anhydrous 1,2-dichloroethane (50 ml), and then 4-dimethylaminopyridine (22 mg, 0.18 mmol) and chloroacetyl chloride (0.54 mmol) were added. The mixture was stirred and heated at 65°C for 12 h, concentrated under reduced pressure, and the target derivative Z2 was obtained by silica gel column chromatography in a yield of 65%.

[0047] Z2: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,10,11,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2,16-diylbis(2-chloroacetate)

[0048] 1 H NMR(400MHz,Chloroform-d)δ7.18(d,J=15.6Hz,1H),6.37(d,J=15.6Hz,1H),5.78(d,J=5.6Hz,1H),5.52(dd,J=13.6,5.5Hz, 1H),5.26(t,J=8.0Hz,1H),4.17(d,J=8.4Hz,2H),3.90(d,J=2.9Hz,2H),3.25(d,J=14.7Hz,1H),2.81(d,J=12.7Hz,1H),2.77 -2.68(m,2H),2.42(dd,J=19.3,7.4Hz,1H),2.17(ddd,J=12.5,5.7,3.5Hz,1H),2.02(s,4H),1.93(dd,J=19.7,5.7Hz,1H),1. 59(s,4H),1.53(s,3H),1.41(s,3H),1.39(s,1H),1.31(s,3H),1.28(d,J=5.1Hz,6H),1.23(s,1H),1.09(s,3H),1.02(s,3H).

[0049] 13 C NMR(101MHz,Chloroform-d)δ211.79,204.80,200.89,169.95,166.83,166.70,153.62,139.41,120.83,118.91,79.16,77.64,75.60,74.98,54.18, 51.37,50.14,48.61,48.41,48.10,42.99,42.09,40.71,40.68,34.23,31. 86,28.73,27.15,26.05,23.82,23.59,21.94,21.31,20.10,19.90,18.70.

[0050] Example 3

[0051] Z3: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-16-hydroxy-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2-yl-2-methylbenzoate

[0052] The preparation method was the same as that in Example 1, and 0.13 mmol of o-toluoyl chloride was added to carry out the reaction to obtain the product Z3 with a yield of 41%. 1 H NMR(400MHz,Chloroform-d)δ7.98(d,J=7.9Hz,1H),7.40(t,J=7.5Hz,1H),7.25(s,2H),7.07(d,J=15.6Hz,1H),6.48(d,J=15.6Hz,1H),5. 82(d,J=5.4Hz,1H),5.72(dd,J=13.7,5.4Hz,1H),4.36(t,J=8.0Hz,1H),3.27(d,J=14.6Hz,1H),2.91(d,J=12.9Hz,1H),2.70(d,J=14.6Hz ,1H),2.60(s,3H),2.51(d,J=7.0Hz,1H),2.47-2.37(m,1H),2.25(d,J=11.3Hz,1H),2.02(s,3H),1.99(s,1H),1.88(dd,J=13.4,9.2Hz,1H ),1.64(d,J=6.1Hz,2H),1.55(s,3H),1.45(s,3H),1.39(d,J=3.7Hz,5H),1.33(s,4H),1.28(s,1H),1.25(s,3H),1.12(s,3H),0.99(s,3H).

[0053] 13C NMR(101MHz,Chloroform-d)δ212.57,205.86,202.56,170.41,166.77,152.21,140 .49,139.92,132.31,131.70,131.00,129.17,125.85,120.74,120.41,79.47,78.3 3,73.80,71.45,58.29,51.50,50.82,48.84,48.68,48.23,45.47,42.53,34.56,32.30,29.83,28.97,26.56,26.10,24.06,22.10,21.78,21.48,20.14,20.03,19.01.

[0054] Example 4

[0055] Z4: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,10,11,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2,16-diylbis(2-methylbenzoate)

[0056] The preparation method was the same as that of Example 2, and 0.54 mmol of o-toluoyl chloride was added to carry out the reaction to obtain the product Z4 with a yield of 71%. 11H NMR (400 MHz, Chloroform-d) δ 8.00 (dd, J = 8.1, 1.5 Hz, 1H), 7.63 (dd, J = 7.8, 1.5 Hz, 1H), 7.39 (dtd, J = 20.5, 7.5, 1.6 Hz, 2H), 7.30 - 7.14 (m, 4H), 6.80 (d, J = 15.5 Hz, 1H), 6.44 (d, J = 15.5 Hz, 1H), 5.82 (d, J = 5.4 Hz, 1H), 5.75 (dd, J = 13.6, 5.5 Hz, 1H), 5.58 (t, J = 8.0 Hz, 1H), 3.33 (d, J = 14.7 Hz, 1H), 2.93 (d, J = 12.9 Hz, 1H), 2.86 (d, J = 7.3 Hz, 1H), 2.81 (d, J = 14.6 Hz, 1H), 2.61 (s, 3H), 2.57 (s, 3H), 2.47 (dd, J = 20.3, 8.0 Hz, 1H), 2.34 - 2.24 (m, 1H), 2.19 - 2.12 (m, 1H), 2.09 (d, J = 8.0 Hz, 1H), 2.01 (s, 3H), 1.96 (d, J = 5.6 Hz, 1H), 1.70 (q, J = 13.0 Hz, 1H), 1.52 (d, J = 14.0 Hz, 1H), 1.47 (s, 3H), 1.42 (s, 3H), 1.41 (s, 3H), 1.35 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H), 1.16 (s, 3H), 1.10 (s, 3H).

[0057] 13 13C NMR (101 MHz, CDCl3) δ 212.07, 205.72, 201.22, 169.71, 166.72, 166.70, 152.61, 140.46, 140.15, 139.87, 132.33, 131.91, 131.69, 130.96, 129.94, 129.67, 129.04, 125.83, 125.68, 120.58, 119.26, 79.28, 77.95, 73.70, 73.67, 54.57, 51.45, 50.35, 48.72, 48.53, 48.09, 43.61, 42.25, 34.47, 32.22, 28.92, 26.45, 25.41, 23.94, 23.87, 22.09, 21.77, 21.65, 21.40, 20.13, 19.93, 18.78.

[0058] Example 5

[0059] Z5: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-16-hydroxy-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2-yl-2-nitrobenzoate

[0060] The preparation method was the same as that of Example 1, and 0.13 mmol of 2-nitrobenzoyl chloride was added to carry out the reaction to obtain the product Z5 with a yield of 37%. 1 H NMR(400MHz,Chloroform-d)δ7.98-7.90(m,2H),7.73-7.59(m,2H),7.06(d,J=15.6Hz,1H),6.49(d,J=15.6Hz,1H),5.82(d,J=5.5H z,1H),5.74(dd,J=13.6,5.5Hz,1H),4.36(d,J=8.3Hz,1H),3.28(d,J=14.6Hz,1H),2.92(d,J=12.9Hz,1H),2.70(d,J=14.6Hz,1H), 2.51(d,J=7.1Hz,1H),2.41(dd,J=20.1,7.8Hz,1H),2.25(dt,J=9.4,4.0Hz,1H),2.02(s,3H),2.00(s,1H),1.97(d,J=2.4Hz,1H),1 .86(dd,J=9.7,3.7Hz,2H),1.58(s,3H),1.55(s,3H),1.43(s,1H),1.38(s,6H),1.32(s,3H),1.24(s,2H),1.08(s,3H),0.97(s,3H).

[0061] 13 C NMR (101MHz, CDCl3) δ212.31,205.39,202.56,170.46,164.63,152.23,147.7 8,139.57,133.21,131.95,130.41,127.45,124.06,121.02,120.38,79.49,7 8.31,75.16,71.41,58.21,51.48,50.78,48.78,48.60,48.18,45.40,42.47,34.27,31.89,28.79,26.56,26.06,23.99,22.09,21.37,20.05,20.00,18.95.

[0062] Example 6

[0063] Z6: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,10,11,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2,16-diylbis(2-nitrobenzoate)

[0064] The preparation method was the same as that of Example 2, and 0.54 mmol of 2-nitrobenzoyl chloride was added to carry out the reaction to obtain the product Z6 with a yield of 73%. 1 H NMR(400MHz,Chloroform-d)δ8.00-7.89(m,3H),7.76-7.56(m,5H),6.78(d,J=15.5Hz,1H),6.30(d,J=15.5Hz,1H),5.8 4(t,J=3.3Hz,1H),5.73(dd,J=13.7,5.5Hz,1H),5.53(t,J=8.2Hz,1H),3.28(d,J=14.7Hz,1H),2.90(d,J=13.0Hz,1H),2 .79-2.66(m,2H),2.50-2.38(m,1H),2.28-2.19(m,1H),2.06(d,J=8.1Hz,2H),1.96(s,3H),1.75(d,J=4.6Hz,2H),1.60 (d,J=13.0Hz,1H),1.43(s,3H),1.36(s,3H),1.33(s,3H),1.28(d,J=3.8Hz,6H),1.20(s,3H),1.10(s,3H),1.06(s,3H).

[0065] 13C NMR (101MHz, CDCl3) δ211.73,205.32,201.37,169.59,165.32,164.64,152.91,147.73,146.6 8,139.37,133.76,133.23,131.95,131.18,130.39,129.45,128.73,127.42,124.04,123.96, 121.05,119.18,78.98,77.68,75.94,75.09,53.76,51.48,50.20,48.58,48.54,48.03,42.25,41.54,34.17,31.83,28.73,26.52,25.60,23.81,23.61,22.00,21.28,20.02,19.86,18.59.

[0066] Example 7

[0067] Z7: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-16-hydroxy-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2-yl-4-cyanobenzoate

[0068] The preparation method was the same as that of Example 1, and 0.13 mmol of 4-cyanobenzoyl chloride was added to carry out the reaction to obtain the product Z5 with a yield of 35%. 1H NMR(400MHz,Chloroform-d)δ8.16(d,J=8.1Hz,2H),7.75(d,J=8.1Hz,2H),7.06(d,J=15.6Hz,1H),6.47(d,J=15.6Hz,1H),5.83 (d,J=5.4Hz,1H),5.71(dd,J=13.6,5.5Hz,1H),4.36(t,J=7.7Hz,1H),3.26(d,J=14.7Hz,1H),2.90(d,J=12.9Hz,1H),2.70(d,J= 14.7Hz,1H),2.51(d,J=7.0Hz,1H),2.43(dd,J=20.0,8.0Hz,1H),2.33-2.23(m,1H),2.01(s,3H),1.88(dd,J=13.6,9.1Hz,2H), 1.72(d,J=12.5Hz,2H),1.57(s,3H),1.54(s,3H),1.44(s,3H),1.38(s,6H),1.31(s,3H),1.24(s,1H),1.12(s,3H),0.99(s,3H).

[0069] 13 C NMR (101MHz, CDCl3) δ212.66,205.29,202.53,170.45,164.17,152.21,139.4 6,133.47,132.37,130.47,121.05,120.35,118.12,116.73,79.45,78.31,74 .78,71.36,58.26,51.50,50.75,48.82,48.59,48.19,45.40,42.46,34.37,32.16,28.86,26.55,26.04,24.04,23.97,22.09,21.37,20.14,20.06,18.96.

[0070] Example 8

[0071] Z8: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,10,11,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2,16-diylbis(4-cyanobenzoate)

[0072] The preparation method was the same as that of Example 2, and 0.54 mmol of 4-cyanobenzoyl chloride was added to carry out the reaction to obtain the product Z8 with a yield of 66%.1 H NMR(400MHz,Chloroform-d)δ8.16(d,J=8.0Hz,2H),7.99(d,J=8.1Hz,2H),7.73(dd,J=17.7,8.0Hz,4H),6.91(d,J=15.5Hz,1H),6.46(d,J=1 5.6Hz,1H),5.79(d,J=5.4Hz,1H),5.73(dd,J=13.6,5.5Hz,1H),5.61(t,J=8.1Hz,1H),3.31(d,J=14.8Hz,1H),2.90(d,J=7.9Hz,2H),2.81(d ,J=14.8Hz,1H),2.46(dd,J=20.1,7.9Hz,1H),2.30(dt,J=12.4,4.3Hz,1H),2.17(dd,J=14.4,9.2Hz,1H),2.09(d,J=8.0Hz,1H),2.04(s,3H) ,1.96(dd,J=19.4,5.9Hz,1H),1.53(s,1H),1.46(d,J=6.9Hz,9H),1.37(d,J=6.0Hz,6H),1.29(s,3H),1.24(s,1H),1.15(s,3H),1.09(s,3H).

[0073] 13 C NMR (101MHz, CDCl3) δ211.87,204.99,201.22,169.79,164.40,164.17,153.19,139.59 ,133.78,133.42,132.37,130.47,130.23,120.83,119.18,118.08,118.05,116.78,116 .53,79.29,77.95,75.26,74.65,54.37,51.47,50.26,48.67,48.51,48.02,43.53,42.18,34.38,32.11,28.83,26.81,26.40,24.07,23.87,22.05,21.36,20.19,19.83,18.88.

[0074] Example 9

[0075] Tumor cell inhibitory activity

[0076] 1. Experimental drugs

[0077] Different concentrations of cucurbitacin B and cucurbitacin B derivatives (Z1~Z8).

[0078] 2. Cell lines

[0079] Human hepatoma cells (HePG2), human prostate cancer cells (22RV1) and human breast cancer cells (MCF-7) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. They were routinely cultured in a 37°C, 5% CO2 incubator using DMEM, 1640 and DMEM medium containing 10% fetal bovine serum, respectively. The medium was replaced every 24 hours. When the cells reached saturation, they were digested and passaged with 0.25% trypsin, and passaged once every 2-3 days. Cells in the logarithmic growth phase were selected for the experiment.

[0080] 3. Preparation of DMEM / 1640 culture medium

[0081] 5 mL of fetal bovine serum, 0.5 mL of penicillin-streptomycin solution (200,000 U / mL), add DMEM or 1640 culture medium and finally make up to 50 mL.

[0082] 4. Cell Viability Detection

[0083] The MTT [3-(4,5)-dimethyl-2-thiazol-(2,5)-phenyl tetrazolium blue bromide] method was used to determine the effects of cucurbitacin B ester derivatives on the proliferation activity of human hepatoma cell HePG2, human prostate cancer cell 22RV1 and human breast cancer cell MCF-7.

[0084] Take the above tumor cells in the logarithmic growth phase and inoculate them in a 96-well plate at a density of 5000 per well, and place them in an incubator for culture. After the cells adhere to the wall (usually 24 hours), discard the culture medium and dilute the DMSO stock solution of the test compound with the culture medium by half to different concentrations and add them to the cell wells (DMSO concentration ≤ 0.1%). At the same time, set up a control group (only add culture medium and cells, no test drug) and a blank group (only add culture medium, no cells and test drugs). After incubation for 48 and 72 hours, add 20ul of MTT (made into 5mg / mL with PBS buffer) to each well of the 96-well plate. After placing at 37°C for 4h, remove the supernatant. Add 200ul of DMSO to each well and shake for 15-30min. Finally, use an automatic microplate reader to detect the optical density (OD value) of each well at a wavelength of 490nm. Each group of experiments was repeated at least three times.

[0085] Growth inhibition rate = (1-survival rate) × 100% = [1-(OD test-OD blank) / (OD control-OD blank)] × 100% (where OD test represents the average optical density of the test drug group, OD control represents the average optical density of the control group, and OD blank represents the average optical density of the control group)

[0086] The proliferation inhibitory activity of cucurbitacin B derivatives on HePG2 cells, 22RV1 cells and MCF-7 cells is shown in Table 1.

[0087] Table 1 Half inhibition rate IC of cucurbitacin B derivatives at different concentrations on tumor cells 50 Value(uM)

[0088]

[0089] Note: N / A means IC 50 >80uM or no inhibitory effect.

[0090] The results showed that cucurbitacin B ester derivatives Z1 to Z8 had strong proliferation inhibition effects on HePG2, 22RV1 and MCF-7 cells, and the inhibitory effects of most derivatives were stronger than cucurbitacin B. Among them, derivatives Z1, Z2, Z3, Z5 and Z7 all had strong anticancer activity, and derivatives Z1 and Z3 had more obvious effects on HePG2 liver cancer cells. Derivatives Z1, Z5 and Z7 had more obvious effects on MCF-7 breast cancer cells. At the same time, 2-OH monosubstituted derivatives had better inhibitory effects on cancer cell proliferation than 2-OH and 16-OH disubstituted derivatives, and the introduction of heteroatom-containing substituents made the derivatives have stronger inhibitory effects.

Claims

1. A cucurbitacin B ester derivative, characterized in that: The structure of the cucurbitacin B ester derivative is shown in the following formula: Where R is:

2. The cucurbitacin B ester derivative according to claim 1, characterized in that: The chemical names and chemical structural formulas of the cucurbitacin B ester derivatives are as follows: Z1: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-16-hydroxy-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2-yl-2-chloroacetate Z2: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,10,11,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2,16-diylbis(2-chloroacetate) Z3: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-16-hydroxy-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2-yl-2-methylbenzoate Z4: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,10,11,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2,16-diylbis(2-methylbenzoate) Z5: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-16-hydroxy-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2-yl-2-nitrobenzoate Z6: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,10,11,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2,16-diylbis(2-nitrobenzoate) Z7: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-16-hydroxy-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2-yl-4-cyanobenzoate Z8: (E)-17-(6-acetoxy-2-hydroxy-6-methyl-3-oxohept-4-en-2-yl)-4,4,9,13,14-pentamethyl-3,11-dioxo-2,3,4,7,8,9,10,11,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-2,16-diylbis(4-cyanobenzoate) 3. The cucurbitacin B ester derivative according to claim 1, characterized in that: The preparation method of the cucurbitacin B ester derivatives has the following steps: 1) Dissolve cucurbitacin B in an organic solvent, then add 4-dimethylaminopyridine and an acyl chloride reagent, and react at 30-60° C. for 12-24 hours; 2) After the reaction is complete, the organic solvent is removed by concentration under reduced pressure, and the target product, cucurbitacin B ester derivative, is obtained by separation by silica gel column chromatography.

4. The cucurbitacin B ester derivative according to claim 3, characterized in that: In step 1), the organic solvent is dichloromethane or 1,2-dichloroethane.

5. The cucurbitacin B ester derivative according to claim 3, characterized in that: In step 1), the acyl chloride reagent is chloroacetyl chloride, o-toluoyl chloride, 2-nitrobenzoyl chloride or 4-cyanobenzoyl chloride.

6. The cucurbitacin B ester derivative according to claim 3, characterized in that: In step 1), the molar ratio of cucurbitacin B, 4-dimethylaminopyridine and acyl chloride reagent is 1:1:0.7-3.

7. The cucurbitacin B ester derivative according to claim 3, characterized in that: In step 2), when eluting with silica gel column chromatography, petroleum ether is first used for elution, and then petroleum ether:ethyl acetate=6:1-1:1 is used for gradient elution.

8. A use of the cucurbitacin B ester derivatives as claimed in claim 1, characterized in that: The cucurbitacin B ester derivative is used for preparing a drug for inhibiting the proliferation of liver cancer cells, prostate cancer cells or breast cancer cells.

Images