Oleanolic acid triazole derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to an oleanolic acid triazole derivative as well as a preparation method and application thereof. The oleanolic acid derivative with a brand new structure provided by the invention has good cell proliferation inhibition activity on tumor cell lines, the inhibition effect is greatly improved compared with that of oleanolic acid, and the oleanolic acid derivative has the potential to be developed into a new anti-cancer drug.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to an oleanolic acid triazole derivative, a preparation method and an application thereof. Background Art

[0002] Cancer seriously endangers human health. It has numerous pathogenic factors, complex pathogenic mechanisms and great prevention and control difficulties, and is one of the main causes of global population death. The technologies for treating tumors include surgical treatment, chemotherapy, immunotherapy, radiotherapy, etc. Among them, small molecule anticancer drug chemotherapy still plays an important role. Existing chemotherapy drugs often cause serious adverse reactions due to problems such as selectivity and drug resistance. Therefore, it is still extremely urgent to develop new, highly efficient and low-toxic anticancer drugs to meet clinical needs.

[0003] Oleanolic acid (OA), belonging to pentacyclic triterpenoid natural products, has the chemical name of 3β-hydroxyolean-12-ene-28-oic acid, and is widely distributed in the plant kingdom in the form of the aglycone or free acid of triterpenoid saponins. Research shows that oleanolic acid and its derivatives have various pharmacological activities such as liver protection, anti-cancer, anti-atherosclerosis, anti-inflammatory, antibacterial, anti-HIV and anti-diabetes. Due to its low toxicity, significant biological activity and high natural abundance, oleanolic acid has become an important precursor compound for developing new bioactive molecules.

[0004] Certain breakthroughs have also been made in the anti-cancer aspect of oleanolic acid derivatives. Patent CN 103946231A discloses an oleanolic acid amide derivative, which has obvious inhibitory effects on a variety of tumor cell lines; CN106749486A discloses an oleanolic acid derivative with ethylenediamine as the linker arm, which has obvious inhibitory effects on a variety of tumor cell lines. However, these known derivatives with anti-cancer activity still have defects such as low biological activity, poor physicochemical properties or insufficient drug-likeness. Developing new oleanolic acid derivatives for inhibiting tumors is of great significance. Moreover, providing more derivatives based on oleanolic acid is also of great significance for enriching the access routes and drug selection of anti-tumor drugs. Summary of the Invention

[0005] In order to solve the above technical problems, one of the purposes of the present invention is to provide an oleanolic acid triazole derivative.

[0006] The technical solution adopted by the present invention is as follows:

[0007] 1. An oleanolic acid triazole derivative, characterized in that the general structural formula of the derivative is shown in Formula I:

[0008]

[0009] Among them, the R group is selected from any one of A1 - A7:

[0010]

[0011] The second object of the present invention is to provide a preparation method of the oleanolic acid triazole derivative as described above, and the synthetic route can be expressed as follows:

[0012]

[0013] Step a. Dissolve N-Boc-bromoethylamine (Compound 1) and sodium azide in N,N-dimethylformamide, heat, after the reaction is completed, dissolve the reaction solution in ethyl acetate, wash, dry, collect the organic phase and concentrate to obtain N-Boc-2-azido-ethylamine (Compound 2);

[0014] Step b. Remove the Boc protecting group of N-Boc-2-azido-ethylamine with 2 mol / L hydrochloric acid 1,4-dioxane solution at room temperature to obtain 2-azidoethylamine hydrochloride (Compound 3) for standby;

[0015] Step c. Dissolve oleanolic acid and 2-azidoethylamine hydrochloride in N,N-dimethylformamide, add HATU condensing reagent and triethylamine, stir at room temperature, after the reaction is completed, pour the reaction solution into water, extract with ethyl acetate, dry the organic phase with anhydrous sodium sulfate, and purify by silica gel column chromatography to obtain 28-(2-azidoethyl)amide oleanolic acid (Compound 4);

[0016] Step d. Dissolve 28-(2-azidoethyl)amide oleanolic acid in a mixed solution of dichloromethane and water, add an alkyne containing the corresponding substituent R (A1 - A7), then successively add copper sulfate pentahydrate and sodium ascorbate, react at room temperature, after the reaction is completed, pour the reaction solution into water, extract with ethyl acetate, dry the organic phase with anhydrous sodium sulfate and then spin-dry, and purify by silica gel column chromatography to obtain the required oleanolic acid triazole derivative.

[0017] Preferably, in the step S1, based on 1 equivalent of oleanolic acid, 2-azidoethylamine hydrochloride is used in an amount of 0.5 - 5 equivalents, the HATU condensing reagent is used in an amount of 0.5 - 5 equivalents, and triethylamine is used in an amount of 0.5 - 10 equivalents. The amount of N,N-dimethylformamide used is 20 mL / g of oleanolic acid; more preferably, based on 1 equivalent of oleanolic acid, 2-azidoethylamine hydrochloride is used in an amount of 1.2 equivalents, the HATU condensing reagent is used in an amount of 1.2 equivalents, and triethylamine is used in an amount of 3 equivalents.

[0018] Preferably, in step S2, based on 1 equivalent of 28-(2-azidoethyl)amidooleanolic acid, the alkyne containing the corresponding substituent R is used in an amount of 0.5 - 5 equivalents, copper sulfate pentahydrate in an amount of 0.01 - 2 equivalents, and sodium ascorbate in an amount of 0.1 - 10 equivalents. The dichloromethane and water are mixed at a volume ratio of 2:1, and the amount used is 6 mL / 100 mg of 28-(2-azidoethyl)amidooleanolic acid. More preferably, based on 1 equivalent of 28-(2-azidoethyl)amidooleanolic acid, the alkyne containing the corresponding substituent R is used in an amount of 1 equivalent, copper sulfate pentahydrate in an amount of 0.15 equivalent, and sodium ascorbate in an amount of 2 equivalents.

[0019] Preferably, in step a, based on 1 equivalent of N-Boc-bromoethylamine, the amount of sodium azide used is 0.5 - 10 equivalents, preferably 2 equivalents, and the amount of N,N-dimethylformamide used is 1.5 mL / mmol of N-Boc-bromoethylamine. In step b, the concentration of the hydrogen chloride 1,4-dioxane solution is 0.5 - 4 mol / L, preferably 2 mol / L, and the amount used is 5 mL / mmol of N-Boc-2-azido-ethylamine.

[0020] Preferably, in step a, the heating temperature is 50 - 150 °C, preferably 110 °C, and the reaction time is 12 h.

[0021] A third object of the present invention is to provide an application of the oleanolic acid triazole derivative and / or its pharmaceutically acceptable salt as described above in the preparation of an anti-tumor drug.

[0022] Preferably, the pharmaceutically acceptable salts of the oleanolic acid triazole derivative include salts formed by the oleanolic acid triazole derivative with any one of inorganic acids, organic acids, alkali metals or alkaline earth metals.

[0023] Preferably, the inorganic acid is any one of hydrochloric acid, sulfuric acid, and phosphoric acid; the organic acid is any one of acetic acid, maleic acid, benzenesulfonic acid, p-toluenesulfonic acid, fumaric acid, tartaric acid, lactic acid, and citric acid; the alkali metal is any one of lithium, sodium, and potassium; and the alkaline earth metal is any one of calcium and magnesium.

[0024] A fourth object of the present invention is to provide an anti-cancer drug or pharmaceutical composition comprising a pharmaceutically effective dose of the oleanolic acid triazole derivative as described above, or comprising a pharmaceutically effective dose of a pharmaceutically acceptable salt of the oleanolic acid triazole derivative as described above.

[0025] The beneficial effects of the present invention are as follows:

[0026] This application constructs an oleanolic acid triazole derivative library based on click chemistry technology and discovers potential anti-cancer drugs through in-situ screening. A new class of oleanolic acid derivatives with a completely novel structure provided by this application has good cell proliferation inhibitory activity against tumor cell lines, and the inhibitory effect is significantly improved compared with oleanolic acid (OA), overcoming the problems of fewer structural types, poor activity, poor drug-likeness, and strong toxic and side effects of existing oleanolic acid anti-tumor derivatives, and has the potential to develop into new anti-cancer drugs. Detailed implementation mode

[0027] Unless otherwise specified, the terms used herein have the meanings commonly understood by those skilled in the art. Without special instructions, various raw materials, reagents, instruments, and equipment used herein can be obtained through market purchases or can be prepared by existing methods.

[0028] The technical solutions of the present invention will be described in more detail below with reference to the examples:

[0029] 1. Synthesis of N-Boc-2-azido-ethylamine (Compound 2)

[0030]

[0031] Add N-Boc-bromoethylamine (448 mg, 2 mmol) and sodium azide (143 mg, 2.2 mmol) to a round-bottom flask, add the solvent N,N-dimethylformamide (3 mL), heat to 110 °C and stir for 6 hours. Monitor the reaction by TLC. After the reaction is completed, cool the reaction solution to room temperature, add ethyl acetate to the mixture, and wash with water 3 times. Add anhydrous sodium sulfate to remove the water in the organic phase, collect the organic phase, and distill off the solvent from the organic phase under reduced pressure to obtain the final product, namely N-Boc-2-azido-ethylamine, as a colorless oily liquid of 370 mg, with a yield of 99%. 1 H NMR (400 MHz, DMSO-d6): δ 7.07 (t, J = 12.0 Hz, 1H), 3.29 (t, J = 12.0 Hz, 2H), 3.13 - 3.09 (m, 2H), 1.38 (s, 9H). 13 C NMR (100 MHz, DMSO-d6): δ 155.63, 77.91, 50.04, 28.23.

[0032] 2. Synthesis of 2-azidoethylamine hydrochloride (Compound 3)

[0033]

[0034] N-Boc-2-azido-ethylamine (372 mg, 2 mmol) was placed in a round-bottom flask, and 2 M hydrogen chloride 1,4-dioxane solution (10 mL) was added. The mixture was stirred at room temperature for 5 hours. The reaction was monitored by TLC. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain the final product, 2-azidoethylamine hydrochloride, as a white solid.

[0035] 3. Synthesis of 28-(2-azidoethyl)amido oleanolic acid (Compound 4)

[0036]

[0037] Oleanolic acid (1005 mg, 2.2 mmol) was placed in a round-bottom flask, and HATU condensation reagent (912 mg, 2.4 mmol) was added. The mixture was dissolved in solvent N,N-dimethylformamide (20 mL) and stirred at room temperature for 10 minutes. Then, 2-azidoethylamine hydrochloride and triethylamine (606 mg, 6 mmol) were added successively. The reaction was continued to stir at room temperature for 8 hours. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was poured into 200 mL of water for dilution, extracted with ethyl acetate (200 mL × 3), and the organic phases were combined. The obtained organic phase was dried over anhydrous sodium sulfate and purified by silica gel column chromatography to obtain Compound 4, 28-(2-azidoethyl)amido oleanolic acid, as a white solid, 745 mg, with a yield of 71%. 1 H NMR (400 MHz, DMSO-d6): δ 7.53 (s, 1H), 5.20 (s, 1H), 4.30 (d, J = 4.0 Hz, 1H), 3.30 - 3.29 (m, 2H), 3.27 - 3.23 (m, 1H), 3.13 - 3.07 (m, 1H), 3.01 - 2.96 (m, 1H), 2.80 - 2.76 (m, 1H), 1.96 - 1.88 (m, 1H), 1.82 - 1.79 (m, 2H), 1.68 - 1.61 (m, 1H), 1.59 - 1.54 (m, 2H), 1.50 - 1.38 (m, 7H), 1.36 - 1.28 (m, 2H), 1.23 - 1.18 (m, 1H), 1.11 - 1.04 (m, 5H), 0.94 - 0.84 (m, 14H), 0.67 - 0.65 (m, 7H). 1313C NMR(100MHz, DMSO-d6): δ 176.77, 143.96, 121.53, 76.84, 54.85, 50.01, 47.15, 46.01, 45.30, 41.22, 38.40, 38.09, 36.60, 33.63, 32.95, 32.49, 32.42, 30.44, 28.24, 26.96, 25.70, 23.52, 22.95, 22.28, 18.03, 16.66, 16.04, 15.11.

[0038] Example 1

[0039] Synthesis of Compound 5

[0040]

[0041] 28-(2-Azidoethyl)amidooleanolic acid (100 mg, 0.2 mmol) was placed in a round-bottom flask, and added to a mixed solution of dichloromethane (4 mL) and water (2 mL). 3,5-Dichloro-N-(2-methylbut-3-yn-2-yl)benzamide (51 mg, 0.2 mmol) was added, followed by copper(II) sulfate pentahydrate (10 mg, 0.04 mmol) and sodium ascorbate (79 mg, 0.4 mmol) successively. The reaction was carried out overnight at room temperature. The reaction was monitored by thin-layer chromatography. After the reaction was completed, water (100 mL) was added, and the mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain white solid Compound 5 (105 mg, 69.5%). 1 1H NMR(400MHz, DMSO-d6): δ 8.56(s, 1H), 7.85 - 7.82(m, 3H), 7.78(s, 1H), 7.36(s, 1H), 5.17(s, 1H), 4.36 - 4.32(m, 2H), 4.28(d, J = 8.0 Hz, 1H), 3.55 - 3.48(m, 1H), 3.42 - 3.37(m, 1H), 3.00 - 2.95(m, 1H), 2.72 - 2.67(m, 1H), 1.91 - 1.84(m, 1H), 1.78 - 1.75(m, 2H), 1.69(s, 6H), 1.65 - 1.58(m, 1H), 1.49 - 1.24(m, 11H), 1.17 - 1.14(m, 1H), 1.04 - 1.00(m, 5H), 0.88 - 0.81(m, 14H), 0.65 - 0.62(m, 7H). 1313C NMR(100MHz, DMSO-d6): δ 176.87, 163.03, 152.28, 143.91, 138.34, 134.07, 130.40, 126.39, 121.65, 121.57, 76.83, 54.79, 51.41, 48.40, 47.10, 45.95, 45.28, 41.17, 40.43, 38.38, 38.06, 36.55, 32.90, 32.32, 30.08, 28.23, 28.16, 26.96, 25.64, 23.50, 22.92, 22.28, 17.99, 16.71, 16.03, 15.08.

[0042] Example 2

[0043] Synthesis of Compound 6

[0044]

[0045] The synthesis method was the same as that in Example 1, except that 1-chloropent-1-yne was used instead of 3,5-dichloro-N-(2-methylbut-3-yn-2-yl)benzamide to obtain the target compound 6, a white solid (91 mg, 72.6%). 1 1H NMR(400MHz, DMSO-d6): δ 7.81(s, 1H), 7.39(s, 1H), 5.17(s, 1H), 4.35(t, J = 16.0Hz, 2H), 4.30(d, J = 8.0Hz, 1H), 3.68(t, J = 12.0Hz, 1H), 3.53 - 3.47(m, 1H), 3.41 - 3.37(m, 1H), 3.01 - 2.95(m, 1H), 2.75 - 2.68(m, 3H), 2.06 - 1.99(m, 2H), 1.92 - 1.86(m, 1H), 1.79 - 1.75(m, 2H), 1.66 - 1.59(m, 2H), 1.47 - 1.23(m, 11H), 1.18 - 1.15(m, 1H), 1.09 - 1.01(m, 5H), 0.91 - 0.82(m, 14H), 0.66 - 0.64(m, 4H), 0.57(s, 3H). 1313C NMR(100MHz, DMSO-d6): δ 176.86, 145.22, 143.90, 122.29, 121.59, 76.83, 54.79, 48.40, 47.10, 45.97, 45.31, 44.67, 41.17, 40.39, 38.85, 38.39, 38.06, 36.57, 33.57, 32.91, 32.56, 32.30, 31.86, 30.43, 28.24, 26.97, 26.89, 25.69, 23.53, 22.90, 22.35, 22.26, 18.01, 16.62, 16.04, 15.10.

[0046] Example 3

[0047] Synthesis of Compound 7

[0048]

[0049] The synthesis method was the same as that in Example 1, except that 3-ethynylthiophene was used instead of 3,5-dichloro-N-(2-methylbut-3-yn-2-yl)benzamide to obtain the target compound 7, a white solid (90 mg, 71.2%). 1 1H NMR(400MHz, DMSO-d6): δ 8.35(s, 1H), 7.79(s, 1H), 7.64 - 7.62(m, 1H), 7.47(d, J = 8.0 Hz, 1H), 7.40(s, 1H), 5.14(s, 1H), 4.45(t, J = 12.0 Hz, 2H), 4.27(d, J = 8.0 Hz, 1H), 3.65 - 3.57(m, 1H), 3.44 - 3.38(m, 1H), 2.98 - 2.93(m, 1H), 2.73 - 2.69(m, 1H), 1.92 - 1.84(m, 1H), 1.73 - 1.69(m, 2H), 1.65 - 1.58(m, 1H), 1.51 - 1.24(m, 11H), 1.19 - 1.14(m, 1H), 1.10 - 1.00(m, 5H), 0.86 - 0.82(m, 11H), 0.74(s, 3H), 0.64 - 0.59(m, 4H), 0.49(s, 3H). 1313C NMR (100 MHz, DMSO-d6): δ 176.91, 143.91, 142.83, 132.26, 127.01, 125.72, 121.52, 121.27, 150.52, 76.83, 54.75, 48.58, 47.09, 45.98, 45.31, 41.12, 40.34, 38.76, 38.37, 36.52, 32.90, 32.54, 32.19, 30.41, 28.24, 26.95, 26.83, 25.66, 23.51, 22.85, 22.28, 17.91, 16.48, 16.02, 14.99.

[0050] Example 4

[0051] Synthesis of Compound 8

[0052]

[0053] The synthesis method was the same as that in Example 1, except that (8R,9S,13S,14S,17R)-17-ethynyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol was used instead of 3,5-dichloro-N-(2-methylbut-3-yn-2-yl)benzamide to obtain the target compound 8, a white solid (35 mg, 21.9%). 1 1H NMR (400 MHz, DMSO-d6): δ 8.76 (s, 1H), 7.76 (s, 1H), 7.40 (s, 1H), 6.93 (d, J = 8.0 Hz, 1H), 6.47 - 6.40 (m, 2H), 5.17 (t, J = 8.0 Hz, 1H), 4.99 (s, 1H), 4.38 - 4.35 (m, 2H), 4.29 (d, J = 4.0 Hz, 1H), 3.56 - 3.51 (m, 1H), 3.45 - 3.39 (m, 1H), 3.00 - 2.95 (m, 1H), 2.73 - 2.67 (m, 3H), 2.35 - 2.28 (m, 1H), 2.07 - 1.77 (m, 8H), 1.65 - 1.20 (m, 21H), 1.06 - 1.02 (m, 5H), 0.91 - 0.83 (m, 17H), 0.65 - 0.60 (m, 7H). 1313C NMR (100 MHz, DMSO-d6): δ 176.90, 154.89, 153.94, 143.88, 137.17, 130.41, 125.90, 122.70, 121.65, 114.93, 112.67, 81.16, 76.84, 54.79, 48.47, 47.51, 47.12, 46.70, 45.97, 45.31, 43.08, 41.19, 40.52, 38.39, 38.06, 37.25, 36.57, 33.56, 32.09, 32.57, 32.34, 30.41, 29.29, 28.23, 27.25, 26.98, 26.09, 25.72, 23.54, 22.91, 22.28, 18.02, 16.75, 16.04, 15.12, 14.43.

[0054] Example 5

[0055] Synthesis of Compound 9

[0056]

[0057] The synthesis method was the same as that in Example 1, except that ((1,1-dimethyl-2-propynyl)oxy)trimethylsilane was used instead of 3,5-dichloro-N-(2-methylbut-3-yn-2-yl)benzamide to obtain the target compound 9, a white solid (64 mg, 48.5%). 1 1H NMR (400 MHz, DMSO-d6): δ 7.86 (s, 1H), 7.40 (s, 1H), 5.18 (s, 1H), 3.37 (s, 2H), 4.29 (s, 1H), 3.49 - 3.41 (m, 2H), 2.98 (s, 1H), 2.72 (d, J = 12.0 Hz, 1H), 1.94 - 1.89 (m, 1H), 1.81 - 1.78 (m, 2H), 1.55 - 1.24 (m, 19H), 1.81 - 1.78 (m, 2H), 1.11 - 1.03 (m, 5H), 0.93 - 0.79 (m, 14H), 0.70 - 0.59 (m, 7H). 1313C NMR (100 MHz, DMSO-d6): δ 176.88, 154.10, 143.89, 121.60, 121.32, 76.82, 70.59, 54.78, 48.32, 47.10, 45.94, 45.30, 41.18, 40.42, 38.39, 38.07, 36.57, 34.50, 33.57, 32.90, 32.60, 32.32, 31.17, 31.00, 30.42, 29.83, 28.23, 26.95, 25.69, 23.56, 22.92, 22.24, 18.00, 16.68, 16.03, 15.11, 2.54, 2.25, 1.84.

[0058] Example 6

[0059] Synthesis of Compound 10

[0060]

[0061] The synthesis method was the same as that in Example 1, except that (R)-N-(prop-2-yn-1-yl)-2,3-dihydro-1H-inden-1-amine methanesulfonate was used instead of 3,5-dichloro-N-(2-methylbut-3-yn-2-yl)benzamide to obtain the target compound 10, a white solid (73 mg, 57.7%). 1 1H NMR (400 MHz, DMSO-d6): δ 7.90 (s, 1H), 7.41 - 7.34 (m, 1H), 7.22 - 7.13 (m, 3H), 5.16 (s, 1H), 4.37 (s, 2H), 4.30 (s, 1H), 4.15 (s, 1H), 3.82 (s, 2H), 3.54 - 3.49 (m, 1H), 3.41 - 3.39 (m, 1H), 3.00 - 2.89 (m, 2H), 2.76 - 2.68 (m, 2H), 2.38 - 2.26 (m, 2H), 1.91 - 1.85 (m, 1H), 1.79 - 1.74 (m, 2H), 1.65 - 1.59 (m, 1H), 1.51 - 1.23 (m, 11H), 1.17 - 1.14 (m, 1H), 1.05 - 1.01 (m, 5H), 0.88 - 0.80 (m, 14H), 0.65 - 0.56 (m, 7H). 1313C NMR (100 MHz, DMSO-d6): δ 176.89, 146.58, 145.35, 143.89, 143.27, 127.10, 125.88, 124.48, 124.26, 122.82, 121.60, 76.84, 61.93, 54.79, 48.44, 47.10, 45.31, 42.07, 41.16, 40.43, 38.84, 38.39, 36.56, 33.01, 32.90, 30.40, 29.94, 28.24, 26.90, 25.68, 23.49, 16.62, 16.03, 15.08.

[0062] Example 7

[0063] Synthesis of Compound 11

[0064]

[0065] The synthesis method was the same as that in Example 1, except that N,N-diethylpropiolylamine was used instead of 3,5-dichloro-N-(2-methylbut-3-yn-2-yl)benzamide to obtain the target compound 11, a white solid (96 mg, 75.5%). 1 1H NMR (400 MHz, DMSO-d6): δ 8.25 (s, 1H), 7.49 (s, 1H), 5.17 (s, 1H), 4.43 (s, 2H), 4.31 - 4.24 (m, 3H), 3.53 - 3.40 (m, 2H), 3.61 - 2.92 (m, 5H), 2.73 - 2.68 (m, 1H), 1.92 - 1.85 (m, 1H), 1.80 - 1.76 (m, 2H), 1.66 - 1.59 (m, 1H), 1.50 - 1.21 (m, 18H), 1.08 - 1.01 (m, 5H), 0.85 - 0.83 (m, 14H), 0.66 - 0.61 (m, 7H). 13 13C NMR (100 MHz, DMSO-d6): δ 176.87, 143.91, 121.55, 76.82, 54.80, 48.58, 47.12, 46.07, 45.96, 45.28, 41.15, 40.40, 38.86, 38.38, 38.07, 36.57, 33.59, 32.91, 32.54, 32.31, 30.40, 29.07, 28.23, 26.96, 26.91, 25.70, 24.11, 23.54, 22.91, 22.25, 18.01, 16.64, 16.03, 15.10, 11.21.

[0066] Biological experiments

[0067] MTT assay for testing the cytotoxicity of compounds

[0068] Human cervical cancer cells (HeLa) and human ovarian cancer (SKOV3) cell lines were cultured in complete DMEM medium (90% DMEM, 10% fetal bovine serum, 100 units / mL penicillin, 100 mg / mL streptomycin). When the cell state was good, the cells were digested with trypsin, collected and adjusted to the appropriate concentration, and then seeded into 96-well microplates, with 100 μL of cell suspension (2000 cells) in each well. After culturing overnight at 37 °C in 5% CO2, different concentrations of the compound (final concentrations were 100, 33.3, 11.1, 3.7, 1.23, 0.411, 0.137 μM in sequence) were added to each well, 100 μL per well, and three replicates were set for each concentration. Incubation continued for 72 h. Then, 20 μL of MTT (5 mg / mL) was added to each well and incubation continued for 4 h. The supernatant was discarded, 150 μL of DMSO was added to each well and shaken for 20 minutes. The optical density value (OD) at 550 nm was read using a microplate reader.

[0069] Cell proliferation inhibition rate = (OD 阴性对照 - OD 试验 ) / (OD 阴性对照 - OD 空白 ) × 100%.

[0070] The cell proliferation inhibition rates under the action of compounds at different concentrations were analyzed using GraphPad Prism 9 software, and the half-maximal inhibitory concentration IC 50 value of the compound was calculated. The anti-tumor activity results are shown in Table 1.

[0071] Table 1. Inhibitory effects of oleanolic acid derivatives on cancer cell lines

[0072]

[0073]

[0074] It can be seen from the biological activity test results that the 7 triazole derivatives provided by the present invention all have good cell proliferation inhibitory activity against tumor cell lines. In particular, compounds 10 and 11 have a significantly improved inhibitory effect compared with oleanolic acid (OA), and are similar to the positive compound fluorouracil (5-Fu). This indicates that the oleanolic acid C28-position triazole derivatives provided by the present invention have the potential to be developed into novel anti-tumor drugs.

[0075] The above are only the preferred practical examples of the present invention and are not used to limit the present invention; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An oleanolic acid triazole derivative, characterized in that, The general structural formula of the derivative is shown in Formula (I): Among them, the R group is selected from any one of A1 - A7:

2. A preparation method of an oleanolic acid triazole derivative as described in claim 1, characterized in that, It includes the following steps: S1. Dissolve oleanolic acid and 2-azidoethylamine hydrochloride in N,N-dimethylformamide, add HATU condensation reagent and triethylamine, stir at room temperature. After the reaction is completed, pour the reaction solution into water, extract with ethyl acetate, dry the organic phase with anhydrous sodium sulfate, and purify by silica gel column chromatography to obtain 28-(2-azidoethyl) amide oleanolic acid; S2. Dissolve 28-(2-azidoethyl) amide oleanolic acid in a mixed solution of dichloromethane and water, add an alkyne containing the corresponding substituent R, then successively add copper sulfate pentahydrate and sodium ascorbate, react at room temperature. After the reaction is completed, pour the reaction solution into water, extract with ethyl acetate, dry the organic phase and then evaporate to dryness, and purify by silica gel column chromatography to obtain the desired oleanolic acid C28-triazole derivative.

3. A preparation method of an oleanolic acid triazole derivative as described in claim 2, characterized in that, The preparation method of the 2-azidoethylamine hydrochloride is as follows: Step a. Dissolve N-Boc-bromoethylamine and sodium azide in N,N-dimethylformamide, heat. After the reaction is completed, dissolve the reaction solution in ethyl acetate, wash, dry, collect the organic phase and concentrate to obtain N-Boc-2-azido-ethylamine; Step b. Remove the Boc protecting group of N-Boc-2-azido-ethylamine with a 1,4-dioxane solution of hydrogen chloride at room temperature to obtain 2-azidoethylamine hydrochloride.

4. A preparation method of an oleanolic acid triazole derivative as described in claim 2, characterized in that, In the step S1, based on 1 equivalent of oleanolic acid, 0.5 - 5 equivalents of 2-azidoethylamine hydrochloride, 0.5 - 5 equivalents of HATU condensation reagent, and 0.5 - 10 equivalents of triethylamine are used, and the amount of N,N-dimethylformamide used is 20 mL / g of oleanolic acid; in the step S2, based on 1 equivalent of 28-(2-azidoethyl) amide oleanolic acid, 0.5 - 5 equivalents of the alkyne containing the corresponding substituent R, 0.01 - 2 equivalents of copper sulfate pentahydrate, and 0.1 - 10 equivalents of sodium ascorbate are used, and the dichloromethane and water are mixed at a volume ratio of 2:1, and the amount used is 6 mL / 100 mg of 28-(2-azidoethyl) amide oleanolic acid.

5. A method for preparing an oleanolic acid triazole derivative as described in claim 3, characterized in that, In the step a, based on 1 equivalent of N-Boc-bromoethylamine, the amount of sodium azide used is 0.5 - 10 equivalents, and the amount of N,N-dimethylformamide used is 1.5 mL / mmol of N-Boc-bromoethylamine; in the step b, the concentration of the 1,4-dioxane solution of hydrogen chloride is 2 mol / L, and the amount used is 5 mL / mmol of N-Boc-2-azido-ethylamine.

6. A preparation method of an oleanolic acid triazole derivative as described in claim 3, characterized in that, In the step a, the heating temperature is 50 - 150 °C, and the reaction time is 12 h.

7. Use of an oleanolic acid triazole derivative as claimed in claim 1 and / or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug.

8. The application according to claim 7, characterized in that, The pharmaceutically acceptable salts of the oleanolic acid triazole derivatives include salts formed by the oleanolic acid triazole derivatives with any one of inorganic acids, organic acids, alkali metals or alkaline earth metals.

9. The application according to claim 8, characterized in that, The inorganic acid is any one of hydrochloric acid, sulfuric acid, and phosphoric acid; the organic acid is any one of acetic acid, maleic acid, benzenesulfonic acid, p-toluenesulfonic acid, fumaric acid, tartaric acid, lactic acid, and citric acid; the alkali metal is any one of lithium, sodium, and potassium; the alkaline earth metal is any one of calcium and magnesium.

10. An anticancer drug or pharmaceutical composition, characterized in that, It contains a pharmaceutically effective dose of the oleanolic acid triazole derivative as described in claim 1, or a pharmaceutically acceptable salt of the oleanolic acid triazole derivative as described in claim 1 containing a pharmaceutically effective dose.

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