Method for preparing cucurbitacine B derivative through strong acid dehydration and peroxy acid oxidation and application of cucurbitacine B derivative

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a method for preparing a cucurbitacine B derivative through strong acid dehydration and peroxy acid oxidation and application of the cucurbitacine B derivative. According to the invention, strong acid is utilized to catalyze dehydration of cucurbitacine B, and peroxy acid is utilized to oxidize carbon-carbon double bonds of cucurbitacine B, so that a plurality of structural modification methods of cucurbitacine B are provided, and a series of novel cucurbitacine B derivatives are synthesized. Subsequently, human breast cancer MCF-7 cells and human kidney epithelium HEK-293 cells are used for verifying the proliferation inhibition effect and specificity of the compound, it is found that the anti-tumor activity of the compound is reserved, the selection index is improved compared with cucurbitacine B, and the compound is worthy of further research.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and specifically relates to a method for preparing cucurbitacin B derivatives by strong acid dehydration and peroxyacid oxidation and its application. Background Art

[0002] Cucurbitacin B has the chemical structure:

[0003]

[0004] It is a tetracyclic triterpenoid compound with high cytotoxic activity isolated from plants such as the Cucurbitaceae family. It is abundant in Cucurbitaceae plants. Modern research shows that cucurbitacin B has multiple effects such as anti-inflammatory, anti-tumor, lipid-lowering, liver protection and immunomodulatory. In terms of anti-tumor activity, cucurbitacin B can effectively slow down the proliferation of cancer cells by inducing cancer cell cycle arrest. It can inhibit the invasion and migration of tumor cells. It can also reduce the formation of new blood vessels in tumors, thereby limiting the nutrient supply of tumors and slowing down tumor growth.

[0005] Due to the excellent activity of cucurbitacin B and its high content in plants, attempts to modify its structure to obtain derivatives with better activity, stronger selectivity and less toxicity have never stopped. However, since the chemical structure of cucurbitacin B includes multiple ring structures and functional groups such as hydroxyl, ester, and olefin, the modification methods of cucurbitacin B have been limited to esterification or etherification. This has greatly affected the diversity of the structure of cucurbitacin B derivatives.

[0006] Currently, most of the reports in the literature and patents focus on modification at the 2-OH, 16-OH or terminal acetyl groups, and the current preparation methods for cucurbitacin B derivatives are single and have low yields. At the same time, too large a molecular weight is not conducive to the drugability research of cucurbitacin B. Summary of the invention

[0007] The purpose of the present invention is to provide a novel cucurbitacin B derivative and a preparation method and application thereof by strong acid dehydration and peroxyacid oxidation, wherein the anticancer activity and specificity thereof are improved compared with cucurbitacin B.

[0008] The chemical names and chemical structural formulas of the novel cucurbitacin B derivatives of the present invention are as follows:

[0009] S1: (E)-6-((3R)-3,16-dihydroxy-4,4,9,13,14-pentamethyl-2,11-dioxo-2,3,4,7,8,9,10,11,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17-yl)-2-methyl-5-oxohept-3,6-dien-2-yl acetate

[0010]

[0011] S2: (3S)-3,16-dihydroxy-17-((E)-2-hydroxy-6-methyl-3-oxohept-4,6-dien-2-yl)-4,4,9,13,14-pentamethyl-3,4,7,8,9,10,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-2,11-dione

[0012]

[0013] S3: (3R)-3,16-dihydroxy-17-((E)-2-hydroxy-6-methyl-3-oxohept-4,6-dien-2-yl)-4,4,9,13,14-pentamethyl-3,4,7,8,9,10,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-2,11-dione

[0014]

[0015] S4: (2S)-2,16-dihydroxy-17-((E)-2-hydroxy-6-methyl-3-oxohept-4,6-dien-2-yl)-4,4,9,13,14-pentamethyl-1,4,7,8,9,10,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-3,11(2H)-dione

[0016]

[0017] H: (E)-6-(2,8-dihydroxy-4,4,6b,9a,11a-pentamethyl-3,11-dioxanedecahydrocyclopenta[1,2]phenanthro[8a,9-b]oxy-9-yl)-6-hydroxy-2-methyl-5-oxohept-3-en-2-yl acetate

[0018]

[0019] Wherein, Ⅰ: The reaction equation and preparation method steps of cucurbitacin B derivative S1 are as follows:

[0020]

[0021] 1) Dissolve cucurbitacin B in anhydrous dichloromethane, then add p-toluenesulfonic acid as an acid reagent, and react at 40°C for 24 hours;

[0022] Wherein, the molar ratio of cucurbitacin B to p-toluenesulfonic acid is 1:1.

[0023] 2) After the reaction is complete, stop the reaction, concentrate under reduced pressure, and separate the target product S1 by preparative liquid phase separation. The conditions for the preparative liquid phase are that the mobile phase ratio is MeOH:H2O=60:40, isocratic elution and collection, and the ultraviolet absorption wavelength is set to 220nm.

[0024] Ⅱ: The reaction equations and preparation steps of cucurbitacin B derivatives S2 and S3 are as follows:

[0025]

[0026] 1) using anhydrous dichloromethane to dissolve cucurbitacin B, then adding an acid reagent methanesulfonic acid, reacting at 40°C for 12h to obtain S2, and reacting for 18h to obtain S3;

[0027] Wherein, the molar ratio of cucurbitacin B to methanesulfonic acid is 1:0.5.

[0028] 2) After the reaction is complete, stop the reaction; concentrate under reduced pressure, and separate the target products S2 and S3 by preparative liquid phase separation.

[0029] The conditions for preparing the liquid phase are that the mobile phase ratio is MeOH:H2O=60:40 for isocratic elution and collection, and the ultraviolet absorption wavelength is set to 220 nm.

[0030] III: The reaction equation and preparation method steps of cucurbitacin B derivative S4 are as follows:

[0031]

[0032] 1) Dissolve cucurbitacin B in anhydrous dichloromethane, then add methanesulfonic acid as an acid reagent and react at 40°C for 2h;

[0033] Wherein, the molar ratio of cucurbitacin B to methanesulfonic acid is 1:0.25.

[0034] 2) After the reaction is complete, stop the reaction, concentrate under reduced pressure, and separate the target product S4 by preparative liquid phase separation. The conditions for the preparative liquid phase are that the mobile phase ratio is MeOH:H2O=60:40, isocratic elution and collection, and the ultraviolet absorption wavelength is set to 220nm.

[0035] IV: The reaction equation and preparation method steps of cucurbitacin B derivative H are as follows:

[0036]

[0037] 1) Dissolve cucurbitacin B in anhydrous dichloromethane, then add an oxidant and react at 40°C for 12 hours;

[0038] The oxidant is m-chloroperbenzoic acid (m-CPBA); the molar ratio of cucurbitacin B to m-CPBA is 1:3.

[0039] 2) After the reaction is complete, stop the reaction, concentrate under reduced pressure, and separate by silica gel column chromatography to obtain the target product H. When eluting by silica gel column chromatography, use petroleum ether: ethyl acetate = 5:1-1:2 for gradient elution and collect.

[0040] Beneficial effects:

[0041] The present invention synthesizes novel cucurbitacin B derivatives S1-S4 and H by using strong acid to catalyze the dehydration of cucurbitacin B and peroxyacid to oxidize the carbon-carbon double bond of cucurbitacin B. The derivatives synthesized by the present invention are derivatives with changes in the skeleton structure of cucurbitacin B, which are relatively novel and enrich the structural types of cucurbitacin B derivatives. At the same time, although cucurbitacin B has up to two secondary hydroxyl groups and one tertiary hydroxyl group, a total of three hydroxyl groups, a series of cucurbitacin B dehydrate derivatives with different structures can be obtained by changing the type, amount and reaction time of the acid. In particular, the synthesized compounds S4 and H have yields of up to 59% and 83%, respectively, and have good reaction specificity. It shows that the reaction activity of methanesulfonic acid and p-toluenesulfonic acid with different hydroxyl groups is affected by a series of factors such as compound steric hindrance, temperature, reaction time, etc., which has certain research significance and provides a method reference for the related structural modification of natural products with similar structures.

[0042] The cytotoxic activity of the synthesized derivatives was subsequently tested, and the experimental results showed that the derivatives had an inhibitory effect on both MCF-7 cells and 293T cells, and the activity of the epoxy derivative H was better than that of cucurbitacin B. At the same time, derivatives S1 to S3 showed higher specificity for tumor cells. In addition, the synthesized compounds of the present invention have a smaller molecular weight, which means better drugability, and thus have certain development value. DETAILED DESCRIPTION

[0043] 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 specific embodiments.

[0044] Example 1

[0045] Cucurbitacin B (100 mg, 0.18 mmol) was dissolved in anhydrous dichloromethane (30 ml), and then p-toluenesulfonic acid (28.84 ul, 0.18 mmol) was added, and the mixture was stirred and heated at 40°C for 24 h, concentrated under reduced pressure, and subjected to preparative liquid separation (mobile phase ratio of MeOH:H2O=60:40, isocratic elution and collection, UV absorption wavelength was set to 220 nm) to obtain the target derivative S1 with a yield of 20.3%.

[0046] S1: (E)-6-((3R)-3,16-dihydroxy-4,4,9,13,14-pentamethyl-2,11-dioxo-2,3,4,7,8,9,10,11,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17-yl)-2-methyl-5-oxohept-3,6-dien-2-yl acetate.

[0047] 1 H NMR (400MHz, Chloroform-d) δ7.47 (d, J=15.3Hz, 1H), 6.38 (d, J=

[0048] 15.3Hz,1H),5.97-5.87(m,1H),5.48(s,2H),5.14(t,J=8.0Hz,1H),4.39(s,1H),3.90(d,J=4.0H z,1H),3.14(d,J=14.7Hz,1H),2.74-2.67(m,2H),2.51(dd,J=5.5,3.2Hz,1H),2.41(dd,J=13.2, 5.1Hz,1H),2.24(d,J=13.2Hz,1H),2.06(d,J=15.6Hz,3H),2.01-1.98(m,1H),1.95(s,3H),1.75 (s,2H),1.41(s,3H),1.31(s,3H),1.25(s,3H),1.19(d,J=3.4Hz,5H),1.02(s,3H),0.79(s,3H).

[0049] 13 C NMR(101MHz,Chloroform-d)δ211.51,210.65,201.17,170.21,147.65,140.58,138.35,127.23,121.93,119.27,80.30,77.71,73.78, 54.47,50.10,48.68,48.42,48.02,46.86,43.17,42.56,38.91,36.42,24.24,23.88,23.83,21.13,20.72,20.20,19.95,18.86,18.32.

[0050] Example 2

[0051] Cucurbitacin B (100 mg, 0.18 mmol) was dissolved in anhydrous dichloromethane (30 ml), and then methanesulfonic acid (5.84 ul, 0.09 mmol) was added, and the mixture was stirred and heated at 40°C for 12 h, concentrated under reduced pressure, and subjected to preparative liquid separation (mobile phase ratio of MeOH:H2O=60:40, isocratic elution and collection, UV absorption wavelength set to 220 nm) to obtain the target derivative S2 with a yield of 33%.

[0052] S2: (3S)-3,16-dihydroxy-17-((E)-2-hydroxy-6-methyl-3-oxohept-4,6-dien-2-yl)-4,4,9,13,14-pentamethyl-3,4,7,8,9,10,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-2,11-dione

[0053] 1 H NMR (400MHz, Chloroform-d) δ7.47 (d, J=15.3Hz, 1H), 6.40 (d, J=

[0054] 15.4Hz,1H),5.94(d,J=5.7Hz,1H),5.47(d,J=5.4Hz,2H),5.30(d,J=1.1Hz,1H),4.39(d,J=9.6Hz,1H),3.90(d,J=4.4H z,1H),3.14(d,J=14.6Hz,1H),2.75(d,J=13.0Hz,1H),2.66(d,J=14.6Hz,1H),2.48(d,J=7.0Hz,1H),2.40(dd,J=13.3, 5.3Hz,1H),2.22(t,J=13.2Hz,1H),2.04(d,J=7.7Hz,2H),1.99(d,J=6.7Hz,1H),1.93(s,3H),1.85(dd,J=13.4,8.9Hz, 1H),1.63(s,3H),1.42(s,3H),1.38(d,J=4.5Hz,1H),1.32(s,3H),1.25(s,3H),1.18(s,2H),0.98(s,3H),0.80(s,3H).

[0055] 13C NMR(101MHz,Chloroform-d)δ212.09,210.79,203.00,147.95,140.71,138.24,127.31,122.02,119.48,80.34,78.11,71.26,5 8.05,50.94,48.72,48.49,48.14,46.88,45.39,42.78,38.94,36.39,24.23,24.10,23.92,21.11,20.17,20.03,18.85,18.31.

[0056] Example 3

[0057] S3: (3R)-3,16-dihydroxy-17-((E)-2-hydroxy-6-methyl-3-oxohept-4,6-dien-2-yl)-4,4,9,13,14-pentamethyl-3,4,7,8,9,10,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-2,11-dione

[0058] Cucurbitacin B (100 mg, 0.18 mmol) was dissolved in anhydrous dichloromethane (30 ml), and then methanesulfonic acid (5.84 ul, 0.09 mmol) was added, and the mixture was stirred and heated at 40°C for 18 h, concentrated under reduced pressure, and subjected to preparative liquid separation (mobile phase ratio of MeOH:H2O=60:40, isocratic elution and collection, UV absorption wavelength was set to 220 nm) to obtain the target derivative S3 with a yield of 35%. 1 H NMR(400MHz,Chloroform-d)δ7.47(d,J=15.4Hz,1H),6.40(d,J=15.3Hz,1H),5.93-5.87(m,1H),5.47(d,J=5.3Hz,2 H),4.40(s,1H),4.12(d,J=2.8Hz,1H),3.14(d,J=14.6Hz,1H),2.97(d,J=13.3Hz,1H),2.67(d,J=14.7Hz,1H),2.50( d,J=7.3Hz,1H),2.46-2.34(m,2H),2.20(dd,J=19.8,13.2Hz,1H),2.10-2.03(m,1H),2.00(d,J=8.4Hz,2H),1.93(s ,3H),1.88-1.83(m,1H),1.42(s,3H),1.38(s,3H),1.30(s,3H),1.25(s,3H),1.07(s,3H),0.98(s,3H),0.84(s,3H).

[0059] 13 C NMR(101MHz,Chloroform-d)δ212.68,211.17,202.99,147.91,140.69,140.30,127.29,122.16,119.53,79.60,78.14,71.30,5 8.14,50.82,48.90,48.44,48.08,45.62,42.49,40.98,36.54,32.42,27.81,24.52,24.17,23.87,20.06,19.13,18.54,18.27.

[0060] Example 4

[0061] Cucurbitacin B (100 mg, 0.18 mmol) was dissolved in anhydrous dichloromethane (30 ml), and then methanesulfonic acid (2.92 ul, 0.045 mmol) was added, and the mixture was stirred and heated at 40°C for 2 h, concentrated under reduced pressure, and subjected to preparative liquid separation (mobile phase ratio of MeOH:H2O=60:40, isocratic elution and collection, UV absorption wavelength was set to 220 nm) to obtain the target derivative S4 with a yield of 59.1%.

[0062] S4: (2S)-2,16-dihydroxy-17-((E)-2-hydroxy-6-methyl-3-oxohept-4,6-dien-2-yl)-4,4,9,13,14-pentamethyl-1,4,7,8,9,10,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-3,11(2H)-dione

[0063] 1 H NMR (400MHz, Chloroform-d) δ7.48 (d, J=15.3Hz, 1H), 6.42 (d, J=

[0064] 15.3Hz,1H),5.81-5.75(m,1H),5.47(d,J=6.2Hz,2H),4.40(d,J=7.5Hz,2H),3 .27(d,J=14.6Hz,1H),2.72(t,J=12.6Hz,2H),2.52(d,J=6.8Hz,1H),2.40(dd, J=20.5,7.6Hz,1H),2.34-2.26(m,1H),1.94(s,3H),1.86(dd,J=13.2,9.0Hz,2 H),1.44(s,3H),1.39(s,1H),1.34(d,J=5.1Hz,6H),1.27(s,3H),1.24(s,2H),

[0065] 1.07(s,3H),0.99(s,3H).

[0066] 13 C NMR (101MHz, CDCl3) δ213.21,212.38,203.05,147.89,140.68,140.48,127.26,120.57,119.55,78.18,71.77,71.28,58.1 7,50.98,50.39,48.80,48.55,48.30,45.43,42.45,36.13,33.85,29.46,24.14,23.99,21.36,20.15,19.99,18.95,18.30.

[0067] Example 5

[0068] Dissolve cucurbitacin B (100 mg, 0.18 mmol) in anhydrous dichloromethane (30 ml), then add m-chloroperbenzoic acid (92.8 mg, 0.54 mmol), stir and heat at 40°C for 12 h, concentrate under reduced pressure, and separate by silica gel column chromatography to obtain the target derivative H in a yield of 83.2%.

[0069] H: (E)-6-(2,8-dihydroxy-4,4,6b,9a,11a-pentamethyl-3,11-dioxanedecahydrocyclopenta[1,2]phenanthro[8a,9-b]oxy-9-yl)-6-hydroxy-2-methyl-5-oxohept-3-en-2-yl acetate

[0070] 1 H NMR (400MHz, Chloroform-d) δ7.06 (d, J=15.6Hz, 1H), 6.48 (d, J=

[0071] 15.7Hz,1H),4.37(dd,J=8.0,4.6Hz,1H),4.34(d,J=7.6Hz,1H),3.32(d,J=5.3Hz,1H),3.21(d,J= 14.8Hz,1H),2.64(dd,J=13.4,7.1Hz,2H),2.45(d,J=7.0Hz,1H),2.33(dt,J=12.8,5.1Hz,1H),2.2 9-2.22(m,1H),2.00(d,J=2.3Hz,3H),1.89(t,J=7.1Hz,2H),1.84(d,J=4.9Hz,1H),1.54(d,J=7.2H z,6H),1.42(s,6H),1.35(s,3H),1.24(s,1H),1.18(s,1H),1.14(s,3H),0.96(s,3H),0.90(s,3H).

[0072] 13 C NMR (101MHz, CDCl3) δ212.46,212.02,202.53,170.47,152.24,120.32,79.46,78.41,72.29,70.99,67.06,58.64,54.69,49.77, 49.68,48.52,48.33,48.27,45.40,41.72,33.48,31.09,26.50,26.14,25.24,23.96,22.67,22.05,20.70,19.73,19.48,16.12.

[0074] Example 6

[0075] Tumor cell inhibitory activity

[0076] 1. Experimental drugs

[0077] Different concentrations of cucurbitacin B and cucurbitacin B derivatives (S1~S4 and H).

[0078] 2. Cell lines

[0079] Human breast cancer cells (MCF-7) and human renal epithelial cells (293T) 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 medium containing 10% fetal bovine serum. 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 culture medium

[0081] 5 mL of fetal bovine serum, 0.5 mL of penicillin-streptomycin solution (200,000 U / mL), add DMEM 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 derivatives on the proliferation activity of human breast cancer cells MCF-7 and human renal epithelial cells 293T cells.

[0084] Take the above cell lines in the logarithmic growth phase and inoculate them in 96-well plates at a density of 5000 per well, and place them in an incubator for culture. After the cells adhere to the wall, 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 drug). After incubation for 48 hours, add 20ul of MTT (made into 5mg / mL with PBS buffer) to each well of the 96-well plate. After placing at 37℃ for 4h, remove the supernatant. Add 200ul of DMSO to each well and shake for 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 inhibitory activity of cucurbitacin B derivatives on the proliferation of MCF-7 cells and 293T cells is shown in Table 1. Table 1 shows the half inhibitory concentration IC of the cucurbitacin B derivatives of the present invention on tumor cells 50 Value (uM) and selection index SI.

[0087] Table 1

[0088]

[0089] a Selection index SI = IC 50 (293T) / IC 50 (MCF-7)

[0090] The results showed that cucurbitacin B derivatives S1-S4 and H had proliferation inhibition effects on MCF-7 cells and 293T cells. In addition, the derivatives were further evaluated by IC50 Analysis showed that the inhibitory effects of S1 to S3 on the growth of human renal epithelial cells 293T were significantly weakened, indicating that they have higher safety than cucurbitacin B. Among them, S3 has the best antiproliferative selectivity index, with an SI value of 3.72. Derivative H showed higher activity. In summary, through the MCF-7 cell proliferation experiment, it was found that the new cucurbitacin B derivatives have high tumor specificity and biological activity, which deserves further study.

Claims

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

2. A method for preparing the cucurbitacin B derivative according to claim 1, characterized in that: The preparation method comprises: dissolving cucurbitacin B in dichloromethane, then adding an acid reagent or an oxidant to carry out strong acid dehydration and peroxyacid oxidation reaction, and after the reaction is complete, concentrating under reduced pressure to remove dichloromethane, and purifying by silica gel column chromatography or preparative liquid phase separation to obtain target products S1-S4 and compound H.

3. The method for preparing the cucurbitacin B derivative according to claim 2, characterized in that: The strong acid reagent is p-toluenesulfonic acid or methanesulfonic acid.

4. The method for preparing the cucurbitacin B derivative according to claim 2, characterized in that: The oxidant is m-chloroperbenzoic acid (m-CPBA).

5. The method for preparing the cucurbitacin B derivative according to claim 2, characterized in that: The molar ratio of cucurbitacin B to the acid reagent is 1:0.25-1.

6. The method for preparing the cucurbitacin B derivative according to claim 2, characterized in that: The molar ratio of cucurbitacin B to the oxidant is 1:

3.

7. The method for preparing the cucurbitacin B derivative according to claim 2, characterized in that: The reaction temperature is 30-40°C, and the reaction time is 2-24h.

8. The method for preparing the cucurbitacin B derivative according to claim 2, characterized in that: The separation H was performed by silica gel column chromatography with petroleum ether: ethyl acetate = 5:1-1:2 for gradient elution and collection, and the separation S1-S4 was performed by preparative liquid separation with a mobile phase of MeOH: H2O = 60:40 for isocratic elution and collection.

9. A use of the cucurbitacin B derivative according to claim 1, characterized in that: The cucurbitacin B derivative is used for preparing a medicine for inhibiting the proliferation of breast cancer or human kidney epithelial cells.

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