A method for preparing cucurbitacin B derivatives by strong acid dehydration and peroxyacid oxidation and its application

Abstract

The present invention belongs to the field of biopharmaceutical technology, and specifically relates to a method and application for preparing cucurbitacin B derivatives by strong acid dehydration and peroxyacid oxidation. The present invention utilizes strong acid to catalyze the dehydration of cucurbitacin B and peroxyacid to oxidize the carbon-carbon double bond of cucurbitacin B, provides a variety of structural modification methods for cucurbitacin B, and synthesizes a series of novel cucurbitacin B derivatives. Subsequently, by using human breast cancer MCF-7 cells and human kidney epithelial HEK-293 cells to verify its proliferation inhibition effect and specificity, it was found that the anti-tumor activity of the compound was retained, and the selectivity index was improved compared to cucurbitacin B, which is worthy of further study.

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 structural formula:

[0003]

[0004] Cucurbitacin B is a highly cytotoxic tetracyclic triterpenoid isolated from plants in the Cucurbitaceae family, where it is abundant. Modern research indicates that cucurbitacin B possesses multiple benefits, including anti-inflammatory, anti-tumor, lipid-lowering, hepatoprotective, and immunomodulatory effects. Its anti-tumor activity can effectively slow cancer cell proliferation by inducing cell cycle arrest. It can inhibit tumor cell invasion and migration, and reduce tumor angiogenesis, thereby limiting nutrient supply and slowing tumor growth.

[0005] Due to the excellent activity of cucurbitacin B and its high abundance in plants, attempts to modify its structure to obtain derivatives with improved activity, enhanced selectivity, and reduced toxicity have been ongoing. However, due to its chemical structure, which includes multiple rings and functional groups such as hydroxyl groups, ester groups, and olefins, the methods for modifying cucurbitacin B have been limited to esterification or etherification. This has greatly limited the structural diversity of cucurbitacin B derivatives.

[0006] Current literature and patent reports mostly focus on modifications at the 2-OH, 16-OH, or terminal acetyl groups. Furthermore, current methods for preparing cucurbitacin B derivatives are limited and yield low yields. Furthermore, excessively large molecular weights are also detrimental to drugability studies 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-tetrahydro-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 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] The molar ratio of cucurbitacin B to p-toluenesulfonic acid is 1:1.

[0023] 2) After the reaction is complete, the reaction is stopped, the mixture is concentrated under reduced pressure, and the target product S1 is obtained by preparative liquid phase separation. The preparative liquid phase conditions are as follows: isocratic elution and collection using a mobile phase ratio of MeOH:H2O = 60:40, and an ultraviolet absorption wavelength of 220 nm.

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

[0025]

[0026] 1) Dissolve cucurbitacin B in anhydrous dichloromethane, then add methanesulfonic acid as an acid reagent, and react at 40°C for 12 hours to obtain S2, and react for 18 hours to obtain S3;

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

[0028] 2) After the reaction is complete, the reaction is stopped; the product is concentrated under reduced pressure, and the target products S2 and S3 are obtained by preparative liquid separation.

[0029] The conditions for preparing the liquid phase are as follows: 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 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 2 hours;

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

[0034] 2) After the reaction is complete, the reaction is stopped, the mixture is concentrated under reduced pressure, and the target product S4 is obtained by preparative liquid phase separation. The preparative liquid phase conditions are as follows: isocratic elution and collection using a mobile phase ratio of MeOH:H2O = 60:40, and an ultraviolet absorption wavelength of 220 nm.

[0035] IV: The reaction equation and preparation 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 the silica gel column chromatography, use petroleum ether:ethyl acetate = 5:1-1:2 for gradient elution and collect.

[0040] Beneficial effects:

[0041] By utilizing strong acid-catalyzed dehydration of cucurbitacin B and peroxyacid-catalyzed oxidation of the carbon-carbon double bond of cucurbitacin B, novel cucurbitacin B derivatives S1-S4 and H were synthesized. These derivatives, characterized by structural variations in the cucurbitacin B skeleton, are relatively novel and enrich the structural repertoire of cucurbitacin B derivatives. Furthermore, while cucurbitacin B possesses up to two secondary hydroxyl groups and one tertiary hydroxyl group, a series of structurally diverse cucurbitacin B dehydrated derivatives can be obtained in high yields by varying the type, amount, and reaction time of the acid. Compounds S4 and H, in particular, exhibit excellent reaction specificity, achieving yields of 59% and 83% respectively. The results demonstrate that the reactivity of methanesulfonic acid and p-toluenesulfonic acid with different hydroxyl groups is influenced by a range of factors, including steric hindrance, temperature, and reaction time. This study is of considerable research significance and provides a methodological reference for the structural modification of similar natural products.

[0042] Subsequent cytotoxicity testing of the synthesized derivatives demonstrated that the derivatives exhibited inhibitory effects against both MCF-7 and 293T cells, with the epoxy derivative H exhibiting superior activity compared to cucurbitacin B. Furthermore, derivatives S1-S3 exhibited higher specificity against tumor cells. Furthermore, the synthesized compounds possessed a smaller molecular weight, suggesting improved drugability and, therefore, potential development. 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 with reference to specific embodiments.

[0044] Example 1

[0045] Cucurbitacin B (100 mg, 0.18 mmol) was dissolved in anhydrous dichloromethane (30 ml), and p-toluenesulfonic acid (28.84 μl, 0.18 mmol) was added. 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 set at 220 nm) to obtain the target derivative S1 in 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. 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 at 220 nm) to obtain the target derivative S2 in 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. 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 set at 220 nm) to obtain the target derivative S3 in 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. 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 set at 220 nm) to obtain the target derivative S4 in 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] Cucurbitacin B (100 mg, 0.18 mmol) was dissolved in anhydrous dichloromethane (30 ml), and then m-chloroperbenzoic acid (92.8 mg, 0.54 mmol) was added. The mixture was stirred and heated at 40°C for 12 h. The mixture was concentrated under reduced pressure and separated 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.

[0073] Example 6

[0074] Tumor cell inhibitory activity

[0075] 1. Experimental drugs

[0076] Different concentrations of cucurbitacin B and cucurbitacin B derivatives (S1~S4 and H).

[0077] 2. Cell Lines

[0078] 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. Both cells were routinely cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator. The medium was changed every 24 hours. When cells reached saturation, they were digested and passaged with 0.25% trypsin, with passages every 2-3 days. Cells in the logarithmic growth phase were used for these experiments.

[0079] 3. Preparation of DMEM culture medium

[0080] Add 5 mL of fetal bovine serum, 0.5 mL of penicillin-streptomycin solution (200,000 U / mL), and DMEM culture medium to finally make up the volume to 50 mL.

[0081] 4. Cell Viability Detection

[0082] The MTT [3-(4,5)-bismethyl-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.

[0083] Take the above cell lines in the logarithmic growth phase and inoculate them into 96-well plates at a density of 5,000 cells per well, and culture them in an incubator. 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 to different concentrations and add them to the cell wells (DMSO concentration ≤ 0.1%). At the same time, set up a control group (only culture medium and cells, no test drug) and a blank group (only culture medium, no cells and test drug). After incubation for 48 hours, add 20ul of MTT (prepared to 5mg / mL with PBS buffer) to each well of the 96-well plate. After placing at 37°C for 4 hours, remove the supernatant. Add 200ul of DMSO to each well and shake for 30 minutes. 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.

[0084] Growth inhibition rate = (1-survival rate) × 100% = [1-(OD experimental - OD blank) / (OD control - OD blank)] × 100% (where OD experimental 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)

[0085] 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 cucurbitacin B derivatives of the present invention on tumor cells. 50 Value (uM) and selection index SI.

[0086] Table 1

[0087]

[0088] a Selection index SI = IC 50 (293T) / IC 50 (MCF-7)

[0089] The results showed that cucurbitacin B derivatives S1~S4 and H had an inhibitory effect on the proliferation of MCF-7 cells and 293T cells. In addition, the derivatives were further evaluated by IC using the selectivity index.50 Analysis revealed that S1–S3 exhibited significantly weaker inhibitory effects on the growth of human renal epithelial 293T cells, suggesting a higher safety profile than cucurbitacin B. Among these, S3 exhibited the best antiproliferative selectivity index, with an SI value of 3.72. Derivative H exhibited even higher activity. In summary, these new cucurbitacin B derivatives demonstrated high tumor specificity and bioactivity in MCF-7 cell proliferation assays, worthy of further investigation.

Claims

1. A cucurbitacin B derivative, characterized in that: The structure of the cucurbitacin B derivative is shown below: 。 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 a strong 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; The strong acid reagent is p-toluenesulfonic acid or methanesulfonic acid; The oxidant is m-chloroperbenzoic acid m-CPBA; The molar ratio of cucurbitacin B to the acid reagent is 1:0.25-1; The molar ratio of cucurbitacin B to the oxidant is 1:3; The reaction temperature is 30-40°C and the reaction time is 2-24h; The separation H was performed by silica gel column chromatography with a gradient elution of petroleum ether: ethyl acetate = 5:1-1:2 and the separation was carried out. The separation S1-S4 was performed by preparative liquid phase separation with a mobile phase of MeOH: H2O = 60:40 and the separation was carried out isocratically.

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

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