A cucurbitacin b derivative a2, and a preparation method and application thereof

Abstract

The application discloses a cucurbitacin B derivative A2, a preparation method and application thereof, and the structural formula of the cucurbitacin B derivative A2 is provided. 50 The cucurbitacin B derivative A2 provided by the application has a strong inhibitory effect on the proliferation activity of a human non-small cell lung cancer cell line A549, and the IC value is only 0.009 muM, which is increased by nearly 10 times than the inhibitory activity of cucurbitacin B on the proliferation of A549, and the toxicity to normal cells L02 is reduced by nearly 10 times, and the selectivity coefficient is increased by nearly 100 times than cucurbitacin. The xenograft mouse experiment result is consistent with the cell result, and can be used as a candidate drug for treating human non-small cell lung cancer; the preparation method has the advantages of rich raw material source, mild reaction condition, convenient operation in reaction process, cheap and easily obtained reagent, low toxicity or non-toxicity, low cost and suitability for industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of anti-cancer drugs, in particular to a cucurbitacin B derivative A2 and a preparation method and application thereof. BACKGROUND

[0002] In 2018, the International Agency for Research on Cancer under the World Health Organization estimated the incidence and mortality of 36 types of cancer in 185 countries around the world. The “2018 Global Cancer Statistics” released shows that lung cancer, breast cancer in women and colorectal cancer are the three highest incidence cancers in the world, and the mortality rates are ranked first, fifth and second respectively. Although the statistical data of the International Agency for Research on Cancer in 2020 shows that the incidence of breast cancer has exceeded lung cancer for the first time to become the world's first cancer, the incidence of breast cancer and lung cancer accounts for 11.7% and 11.4% of the total number of global cancer cases respectively, but lung cancer is still the cancer with the highest mortality, accounting for 18.0% of the total number of cancer deaths.

[0003] About 85% of lung cancer pathological types are non-small cell lung cancer (NSCLC), and the early clinical manifestations of this type of lung cancer are not specific. Most NSCLC patients are in the advanced stage when clinically diagnosed, and cannot be treated by radical surgery. Radiotherapy and chemotherapy are the main means for treating advanced NSCLC. Although the introduction of specific tyrosine kinase inhibitors and immunotherapy has improved the overall treatment effect of NSCLC, these drugs are only used for the treatment of patients with certain genetic mutations, and with the prolongation of treatment time, treatment resistance and other conditions inevitably occur.

[0004] Therefore, developing drugs with strong inhibitory effect on the proliferation activity of NSCLC cells, wider application population and lower toxicity and side effects to improve the survival rate and even cure of NSCLC patients is the eternal goal of scientific researchers.

[0005] Cucurbitacin B is a class of tetracyclic triterpenoids isolated from plants such as Cucurbitaceae, and is the most abundant member of the cucurbitacin family. It has a wide range of pharmacological activities. Previous studies have shown that cucurbitacin B has multiple biological activities such as liver protection, anti-inflammatory and anti-tumor. Studies have shown that cucurbitacin B has inhibitory effect on liver cancer, breast cancer, laryngeal cancer, pancreatic cancer and other malignant tumor cell lines. Studies have shown that lung cancer treated with cucurbitacin B inhibits the phosphorylation of STAT3 in a dose- and time-dependent manner, thereby causing growth arrest and apoptosis. Cucurbitacin B has become a hot spot in cancer research due to its anti-tumor effect in various tumor cells. SUMMARY

[0006] The present application aims to provide a cucurbitacin B derivative A2 and a preparation method and application thereof, so as to provide a new compound with stronger inhibition on NSCLC cell proliferation, wider action on people and lower side effects, so as to improve the survival rate of NSCLC patients and contribute to the cure of NSCLC patients.

[0007] To achieve the above-mentioned purpose, the present application provides a cucurbitacin B derivative A2 and a preparation method and application thereof, wherein the cucurbitacin B derivative A2 has the following structural formula:

[0008]

[0009] A preparation method of the cucurbitacin B derivative A2, and the steps are as follows:

[0010] 1) Put 4-fluoroaniline, triethyl orthoformate and methyl hydrazine formate into anhydrous methanol and reflux;

[0011] 2) After the reaction is completed, sodium methoxide is added to obtain a magenta precipitate, then distilled water is added to the reaction solution to precipitate the solid, and the solid is repeatedly washed with distilled water until the product becomes white, then the obtained product is subjected to a substitution reaction with chloroacetic acid to obtain an intermediate 1a;

[0012] 3) The intermediate 1a and cucurbitacin B are added to a 1,2-dichloroethane solution, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are used as catalysts, and the solution is continuously stirred at a speed of 200-400 rpm at 60°C until the reaction is completed by TLC monitoring;

[0013] 4) After the reaction is completed, the reaction solution is poured into distilled water with a volume of 1.5 times the volume of the 1,2-dichloroethane solution, and the solution is extracted with ethyl acetate three times with a volume of 1.5 times the volume of the 1,2-dichloroethane solution, the ethyl acetate layer is collected and washed with saturated brine three times, the organic phase is dried with anhydrous sodium sulfate, the ethyl acetate is removed by rotary evaporation under reduced pressure, and the product is purified to obtain the cucurbitacin B derivative A2.

[0014] The reaction formula of the cucurbitacin B derivative A2 is as follows:

[0015]

[0016] Preferably, in step 1), the molar ratio of 4-fluoroaniline, triethyl orthoformate and methyl hydrazine formate is 1:1.5:1.5, and the reflux temperature is 70°C.

[0017] Preferably, in step 2), the amount of sodium methoxide added is 1.5 times the amount of 4-fluoroaniline, and the molar ratio of the white product to chloroacetic acid is 1:1.5, and anhydrous potassium carbonate and acetonitrile are used as catalysts, and the reaction is completed by TLC monitoring.

[0018] Preferably, the substance amount ratio of 4-fluorophenyl triazolone: Cucurbitacin B: 1,2-dichloroethane solution in step 3) is 1.2:1:100.

[0019] Preferably, the parameter setting of the reduced pressure rotary evaporation in step 4) is 1.3-2.0 kPa of pressure and 30-40℃ of water bath temperature.

[0020] Preferably, the purification method in step 4) is silica gel chromatography; and the silica gel chromatography is used to elute with dichloromethane methanol solution as developing agent in a volume ratio of 100:1-30:1.

[0021] A use of the Cucurbitacin B derivative A2 in an antitumor drug.

[0022] Therefore, the Cucurbitacin B derivative A2, the preparation method and the application thereof provided by the present application have the following specific technical effects:

[0023] (1) The Cucurbitacin B derivative A2 provided by the present application has a strong inhibitory effect on the proliferation activity of human non-small cell lung cancer cell line A549, and the IC 50 value is only 0.009 μM, which is nearly 10 times higher than the inhibitory activity of Cucurbitacin B on A549 proliferation, and the toxicity to normal cells L02 is reduced by nearly 10 times, and the selectivity coefficient is increased by nearly 100 times than Cucurbitacin; the xenotransplantation mouse experiment results are consistent with the cell results, and can be used as a candidate drug for treating human non-small cell lung cancer;

[0024] (2) The preparation method provided by the present application has the advantages of rich raw material sources, mild reaction conditions, convenient operation, cheap and easily available reagents, low toxicity or no toxicity, low cost, and suitability for industrial production.

[0025] The technical solutions of the present application will be further described in detail below with the aid of the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the description of the embodiments of the present application will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0027] Figure 1 is the anti-proliferation activity of Cucurbitacin and its derivative A2 on human lung cancer A549 cells and the toxicity results on L02 normal cells;

[0028] Figure 2The results of the influence of cucurbitacin B and derivative A2 on tumors in xenotransplant mice. DETAILED DESCRIPTION

[0029] The technical solutions of the present application are further described below by means of the drawings and examples.

[0030] In order to make the purpose, technical solutions and advantages of the present application more clear, thorough and complete, the technical solutions of the present application are clearly and completely described below by means of the drawings and examples. The following detailed description is the description of examples, which aims to provide further detailed description of the present application. Unless otherwise specified, all technical terms used in the present application have the same meaning as generally understood by the general technical personnel in the field to which the present application belongs.

[0031] The device for reflux in the examples is a heat collecting constant temperature heating magnetic stirrer (Shanghai Yukang Science and Technology Instrument and Equipment Co., Ltd.); the instrument for TLC monitoring is a Goodsee-10 type thin layer chromatography imaging instrument produced by Shanghai Kezhe Biochemical Technology Co., Ltd.; the silica gel chromatographic column is self-packed, and the silica gel used is produced by Qingdao Haoyang Chemical Co., Ltd.; the rotary evaporation under reduced pressure is completed by a rotary evaporator (Shenxiang Science and Technology Co., Ltd.); the enzyme label instrument is produced by the company; the electronic scale is produced by Shanghai Jingtian Electronic Instrument Co., Ltd.; the vernier caliper is produced by Zhejiang Wenzhou Sanhe Measuring Instrument Co., Ltd.; and the nuclear magnetic resonance spectrum data is measured by a nuclear magnetic resonance spectrometer (BRUKER AV-300, Switzerland).

[0032] 4-fluoroaniline, triethyl orthoformate, methyl hydrazine carbonate, anhydrous methanol, sodium methoxide, chloroacetic acid, cucurbitacin B, 1,2-dichloroethane, 1-hydroxybenzotriazole, ethyl acetate, dichloromethane, anhydrous sodium sulfate are purchased from Macklin Reagent Co., Ltd., and the purity is analytical pure.

[0033] Human lung cancer A549 cell strain and L02 cell strain are purchased from Pnnsay Company; DMEM medium, fetal bovine serum (FBS), 0.25% trypsin are purchased from American Gibco BRL Company; penicillin and streptomycin are purchased from Solabio Company, and Balbc / nude mice are purchased from Shanghai Lingchang Company.

[0034] Example 1

[0035] A cucurbitacin B derivative A2 is prepared, and the steps are as follows:

[0036] 1) 4-fluoroaniline 1.11 g, triethyl orthoformate 2.22 g, methyl hydrazine carbonate 1.35 g are accurately weighed and placed in a 100 mL round-bottom flask, 50 mL of anhydrous methanol is added, and reflux is carried out at 70°C for about 8 hours under magnetic stirring. After TLC monitoring, 1.665 g of sodium methoxide is added, and the reflux reaction is continued for 4 hours to obtain a magenta solution.

[0037] 2) Add 100 mL distilled water to the reaction solution, and precipitate the rose solid. After suction filtration, repeatedly rinse with distilled water until the product becomes white solid, and dry at 60°C overnight.

[0038] 3) Take 0.91 g of the product obtained above, 1.40 g of anhydrous potassium carbonate, and 0.71 g of chloroacetic acid, and add them to a round-bottom flask. Add 30 mL of acetonitrile, and reflux the reaction under magnetic stirring. After the reaction is completed as monitored by TLC, remove the acetonitrile under reduced pressure to obtain the crude product 1a. Purify the product 1a by column chromatography using dichloromethane and methanol (10:1 by volume) as the mobile phase.

[0039] 4) Take 0.284 g of the intermediate 1a and 0.56 g of cucurbitacin B, and add them to 10 mL of 1,2-dichloroethane. Use 1-hydroxybenzotriazole (HOBT) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) as catalysts, and continuously stir at 200 rpm at 60°C until the reaction is completed as monitored by TLC.

[0040] 5) After the reaction is completed, pour the reaction solution into 15 mL of water, add 15 mL of ethyl acetate, extract three times, collect the ethyl acetate layer, wash with saturated brine three times, remove water with anhydrous sodium sulfate, and remove the ethyl acetate under reduced pressure with a water bath temperature of 30-40°C at 1.3-2.0 kPa to obtain a white oil.

[0041] 6) Purify the product by silica gel chromatography using dichloromethane and methanol (100:1-30:1) as the developing agent to elute the target product, cucurbitacin B derivative A2.

[0042] Example Two

[0043] Determine the nuclear magnetic resonance hydrogen spectrum and carbon spectrum of the obtained cucurbitacin B derivative A2 as follows: take 20 mg of the cucurbitacin B derivative A2 prepared in Example One, load it into a nuclear magnetic tube, add 0.7 mL of deuterated chloroform to dissolve it, and then determine the nuclear magnetic resonance hydrogen spectrum and carbon spectrum by using a BRUKER AV-300 nuclear magnetic resonance instrument.

[0044] Effect Example One

[0045] Detect the effect of the cucurbitacin B derivative A2 prepared in Example One on the proliferation of human non-small cell lung cancer cell line A549, and use cucurbitacin B as a control by using the sulforhodamine B (SRB) colorimetric method as follows:

[0046] S1-1, A549 and L02 cell lines in logarithmic growth phase were inoculated in 96-well plates containing 10% FBS DMEM medium at 5000-8000 CFU per well (150 μL of medium per well), and cultured in a cell incubator overnight.

[0047] S1-2, 10, 2, 0.4, 0.8, 0.016, 0.008, 0.004, 0.002, 0.0004, and 0.00008 μM of cucurbitacin B derivative A2 and cucurbitacin B were added to the medium, respectively, and after 72 h of incubation, the 96-well plates were discarded, 200 μL of 10% trichloroacetic acid was gently added, and the plates were fixed in a 4°C refrigerator for 30 min, the liquid was discarded, and 5 biological replicates were set for each concentration.

[0048] S1-3, 300 μL of double distilled water was added and washed three times, and dried at room temperature for 1 h, 80 μL of 4% SRB was added, and after 20 min of staining, the staining solution was discarded, and washed with 1% acetic acid three times, and dried at room temperature.

[0049] S1-4, dissolved with 200 μL of unbuffered Trisbase (pH = 10.5) at a concentration of 10 mM, and plate was shaken for 5 min.

[0050] S1-5, the absorbance value was measured at 545 nm using a microplate reader, and the inhibition rate was calculated according to the following formula

[0051] Inhibition rate (%) = (1 - OD value of the dosing well / OD value of the control well)

[0052] The half maximal inhibitory concentration was obtained according to the inhibition rate and the dosing concentration, and the results are shown in Figure 1 and Table 1, wherein Figure 1 the left graph in Part A of Table 1 shows the results of the effects of different concentrations of cucurbitacin B (CuB) and derivative A2 (A2) on L02 cell lines, Figure 1 the right graph in Part A of Table 1 shows the inhibition results of different concentrations of CuB and A2 on A549 cell lines; Figure 1 Part B of Table 1 is the quantitative analysis results of Part A.

[0053] Table 1

[0054]

[0055] Example 2

[0056] The in vivo anti-tumor activity of cucurbitacin B derivative A2 was detected by xenotransplantation, and the method was as follows:

[0057] S2-1, Male athymic BALB / C nude mice with a body weight of 16-18 g were purchased and raised under SPF level for 7 days. A549 cells (1 x 10 7 ) suspended in 125 μL DMEM medium were injected into the armpit of the mice.

[0058] S2-2, When the tumor volume of each group increased to about 100 mm 3 , the mice were randomly divided into 3 groups, 6 mice in each group. The dosing group was administered with a dose of 0.5 mg / kg, and 5% DMSO / 30% PEG400 / 65% double distilled water was used as a vehicle for intraperitoneal injection. The blank control group was only injected with the same dose of vehicle.

[0059] S2-3, The drug was administered once a day for the first 6 days, and then considering the tolerance of the mice, the administration was changed to once every 2 days, for a total of 14 times.

[0060] S2-4, The body weight and tumor volume of the mice were measured every 2 days, and after the administration was completed, the mice were sacrificed, the tumor was removed and weighed on an electronic scale. The size of the tumor was determined using a vernier caliper, and the tumor volume (mm 3 ) and tumor inhibition rate (%) were calculated using the following standard formula:

[0061] Tumor volume = (L x W 2 ) / 2, (L is length, W is width);

[0062] Tumor inhibition rate (%) = (1 - average tumor weight of treatment group / average tumor weight of control group) x 100%.

[0063] The results are shown in Figure 2 , wherein Figure 2 Part A is the in vivo anti-tumor experiment process, Part B is the survival rate of nude mice in each group, Part C is the average weight of the tumor obtained after dissection of each group after administration, Part D is the change trend of the tumor volume of each group, Part E is the change trend of the body weight of nude mice in each group, and Part F is the tumor obtained after dissection after administration.

[0064] Result analysis

[0065] The nuclear magnetic resonance hydrogen spectrum and carbon spectrum data of the cucurbitacin B derivative A2 prepared in Example 1 are as follows:

[0066] White powder; yield 28%; m.p. 1H NMR (300 MHz, CDC13) δ 7.73 (s, 1H), 7.60-7.50 (m, 2H), 7.24-7.15 (m, 2H), 7.08 (d, J = 15.6 Hz, 1H), 6.49 (d, J = 15.6 Hz, 1H), 5.82 (d, J = 5.2 Hz, 1H), 5.57 (dd, J = 13.6, 5.5 Hz, 1H), 4.85-4.69 (m, 2H), 4.37 (t, J = 7.8 Hz, 1H), 4.30 (s, 1H), 3.25 (d, J = 14.8 Hz, 1H), 2.85 (d, J = 12.4 Hz, 1H), 2.71 (d, J = 14.7 Hz, 1H), 2.51 (d, J = 7.1 Hz, 1H), 2.48-2.36 (m, 1H), 2.26-2.15 (m, 1H), 2.02 (s, 3H), 2.00-1.79 (m, 4H), 1.64 (d, J = 4.2 Hz, 1H), 1.58 (s, 3H), 1.56 (s, 3H), 1.46 (s, 3H), 1.37 (s, 3H), 1.32 (s, 3H), 1.27 (s, 3H), 1.10 (s, 3H), 0.99 (s, 3H). 13 C NMR (126 MHz, CDC13) δ 211.18, 203.50, 201.41, 169.22, 165.72, 161.65, 159.68, 151.08 (d, J = 11.25 Hz), 138.42, 133.54, 128.78, 123.16 (d, J = 8.75 Hz, 2C), 119.84, 119.29, 115.60 (d, J = 23.75 Hz, 2C), 78.31, 77.20, 73.79, 70.28, 57.17, 50.25, 49.65, 47.65, 47.44, 47.06, 45.53, 44.31, 41.35, 33.25, 30.88, 28.68, 27.59, 25.42, 24.96, 22.92, 22.84, 20.93, 20.25, 18.98, 18.88. ESI-HRMS (m / z) calcd for C 42 H 52 FN3O 10 [M+Na] + : 800.3534, found: 800.3532.

[0067] From the nuclear magnetic resonance results, it can be seen that the structure of cucurbitacin B derivative A2 prepared in Example One is shown in the following figure, and the purity is 98%. The reaction formula for preparing cucurbitacin B derivative A2 is as follows:

[0068] From the nuclear magnetic resonance results, it can be seen that the structure of cucurbitacin B derivative A2 prepared in Example One is shown in the following figure, and the purity is 98%. The reaction formula for preparing cucurbitacin B derivative A2 is as follows:

[0069] By Figure 1 As can be seen from Table 1, the cucurbitacin B derivative A2 prepared in Example 1 showed very strong anti-proliferative activity in A549 cells, with an IC 50 value of 0.009 ± 0.003 μM, which was nearly 10 times higher than the anti-proliferative activity of cucurbitacin B (the IC 50 value of cucurbitacin B was 0.019 ± 0.022 μM), and the toxicity to normal cells was nearly 10 times lower than that of the lead cucurbitacin B (the IC 50 value of the toxicity of the cucurbitacin B derivative A2 prepared in Example 1 to L02 cells was 0.1 ± 0.008 μM, and the IC 50 value of cucurbitacin B was 0.011 ± 0.003 μM), and the selectivity coefficient was nearly 100 times higher than that of cucurbitacin B (the selectivity coefficient of the cucurbitacin B derivative A2 was 11.11, and the selectivity coefficient of cucurbitacin B was 0.58).

[0070] By Figure 2 As can be seen from Part B of Table 1, after 8 doses of administration, the mice in the cucurbitacin B treatment group showed severe ascites, with a large amount of milk-white liquid accumulated in the abdomen of the mice, and the mice were in a state of emaciation. According to the requirements of animal ethics, the mice were sacrificed to alleviate their suffering. This phenomenon also occurred in the 12th and 14th doses of administration, while the A2 treatment group did not show this phenomenon. Moreover, at a dose of 0.5 mg / kg, the inhibition rates of the tumor growth of the cucurbitacin B treatment group and the A2 treatment group were 53% and 80%, respectively, which showed a significant difference compared with the blank control group.

[0071] Therefore, the cucurbitacin B derivative A2 provided in the present application has very strong inhibitory effect on the proliferation of human non-small cell lung cancer cell line A549, with an IC 50 value of only 0.009 μM, which is nearly 10 times higher than the inhibitory activity of cucurbitacin B on the proliferation of A549, and the toxicity to normal cells L02 is nearly 10 times lower, and the selectivity coefficient is nearly 100 times higher than that of cucurbitacin. The results of the xenotransplantation mouse experiment are consistent with the cell results, and the cucurbitacin B derivative A2 can be used as a candidate drug for treating human non-small cell lung cancer; the preparation method provided has the advantages of rich source of raw materials, mild reaction conditions, easy operation, cheap and easily available reagents, low toxicity or no toxicity, low cost, and suitability for industrial production.

[0072] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application but not to limit the same, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can still be modified or replaced equivalently, and these modifications or equivalent replacements should not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A cucurbitacin B derivative A2, characterized by, The structural formula is as follows:

2. A method for preparing cucurbitacin B derivative A2 as described in claim 1, characterized in that, The steps are as follows: 1) 4-fluoroaniline, triethyl orthoformate and methyl hydrazine formate are put into anhydrous methanol for reflux; 2) after the reaction is completed by TLC monitoring, sodium methoxide is added to obtain a magenta precipitate, then distilled water is added to the reaction solution to precipitate the solid, and the solid is repeatedly washed with distilled water until the product becomes white, then the obtained product is subjected to a substitution reaction with chloroacetic acid to obtain intermediate 1a; 3) intermediate 1a and cucurbitacin B are added to a 1,2-dichloroethane solution, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are used as catalysts, and the stirring is continuously carried out at 60 ℃ and a speed of 200-400 rpm until the reaction is completed by TLC monitoring; 4) after the reaction is completed, the reaction solution is poured into 1.5 times the volume of distilled water of the 1,2-dichloroethane solution, extracted with 1.5 times the volume of ethyl acetate of the 1,2-dichloroethane solution in three times, the ethyl acetate layer is collected and washed with saturated brine for three times, the organic phase is dried with anhydrous sodium sulfate, the ethyl acetate is removed by rotary evaporation under reduced pressure, and the product is purified to obtain cucurbitacin B derivative A2.

3. The preparation method of the withaferin B derivative A2 according to claim 2, characterized in that: In step 1), the molar ratio of 4-fluoroaniline: triethyl orthoformate: methyl hydrazine formate is 1:1.5:1.5; and the reflux temperature is 70 ℃.

4. The preparation method of the withaferin B derivative A2 according to claim 2, characterized in that: In step 2), the amount of sodium methoxide added is 1.5 times the amount of 4-fluoroaniline; the molar ratio of the white product to chloroacetic acid is 1:1.5, anhydrous potassium carbonate and acetonitrile are used as catalysts, and the reaction is completed by TLC monitoring.

5. The preparation method of the withaferin B derivative A2 according to claim 2, characterized in that: In step 3), the molar ratio of 4-fluorophenyl triazolone: cucurbitacin B: 1,2-dichloroethane solution is 1.2:1:

100.

6. The preparation method of the withaferin B derivative A2 according to claim 2, characterized in that: In step 4), the parameters for rotary evaporation under reduced pressure are set as follows: pressure 1.3-2.0 kPa, and water bath temperature 30-40 ℃.

7. The preparation method of the withaferin B derivative A2 according to claim 2, characterized in that: In step 4), the purification method is silica gel chromatography; and the silica gel chromatography is performed by using dichloromethane-methanol solution with a volume ratio of 100:1-30:1 as the developing agent for elution.

8. Use of the derivative A2 of cucurbitacin B according to claim 1 in the preparation of an antitumor medicament. The tumor is non-small cell lung cancer.

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