Combretastatin microtubule targeting agent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly discloses a combretastatin microtubule targeting agent and a preparation method and application thereof.The synthesized target compounds 9a-9l and 9m-9q have good anti-proliferative activity on tested human cancer cell lines, the representative compound 9n has the optimal anti-tumor activity, the IC50 value range is 0.09-0.51 mu M, and in addition, the target compounds 9a-9l and 9m-9q have the good anti-proliferative activity on the tested human cancer cell lines. The anti-proliferative activity of the compound 9n to cis-platinum drug-resistant cells A549 / CDDP is 121.45 times that of cis-platinum, the anti-proliferative activity of the compound 9n to adriamycin drug-resistant cells MCF-7 / DOX is 74.88 times that of adriamycin, and the toxicity of the compound 9n to human normal hepatocytes (HL-7702) is low. In a HepG-2 xenotransplantation tumor model, the in-vivo anti-tumor activity of the compound 9n is superior to that of a positive drug CA-4, and the compound 9n has no obvious systemic toxicity. Therefore, the compound has the potential purpose of targeted treatment of liver cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry and relates to the design and synthesis of a microtubule targeting agent, and specifically to a combretin derivative, the synthesis of the combretin derivative and the use of the combretin derivative as a microtubule targeting agent in the preparation of anticancer drugs. Background Art

[0002] Today, cancer seriously affects people's quality of life and health. Although current tumor diagnostic methods and treatment strategies are rapidly developing, the recurrence rate and cancer-related mortality of patients are still very high. Currently, chemotherapy is still one of the main methods for treating malignant tumors, which mainly induces cell cycle arrest or cell death in cancer cells. However, drug resistance and toxic side effects are easily generated during chemotherapy, which in turn affects the treatment effect and ultimately becomes a major obstacle to cancer treatment. Therefore, it is urgent to develop a new microtubule-targeted anticancer drug with strong targeting, high efficacy and low toxicity.

[0003] Recently, microtubule-targeting drugs, such as paclitaxel and docetaxel, derived from natural products, have been recognized as first-line chemotherapy agents in clinical practice. Furthermore, natural products and their derivatives play a key role in drug development, particularly in the discovery of anticancer drugs, providing a variety of bioactive lead compounds for drug research. However, despite the tremendous success of these marketed compounds in cancer treatment, most suffer from significant drawbacks, such as high toxicity, limited selectivity, poor oral bioavailability, poor solubility, drug resistance, or complex synthesis. Therefore, the design and synthesis of a class of novel compretin derivatives with strong targeting, high efficacy, and low toxicity as microtubule-targeting antitumor drugs will play a significant role in promoting the development of anticancer drugs. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a microtubule targeting agent of the Kangpuruitin type. The present invention provides a new Kangpuruitin derivative as a microtubule targeting anti-tumor drug. While treating liver cancer, the targeted drug can overcome the shortcomings of existing cisplatin anti-tumor drugs such as easy drug resistance and large toxic side effects. The present invention further provides a method for synthesizing the microtubule targeting agent.

[0005] The present invention is achieved through the following technical solutions:

[0006] A Compretin-type microtubule targeting agent, the structural formula of which is shown below:

[0007]

[0008] Wherein, R1 is selected from H, OCH3, CH3, Cl, CH2Cl, F; R2 is selected from H, C(CH3)3, OC2H5, C3H7, OCH3, NO2, Br, Cl; R3 is selected from H, OCH3; R4 is selected from H, OCH3; R5 is selected from H, OCH3, CH3; R6 is selected from H, OCH3.

[0009] A further improvement of the present invention is:

[0010] A method for preparing a microtubule targeting agent of the Kangpuruiting type comprises the following steps:

[0011] (1) reacting compound 1 with sodium borohydride to synthesize compound 2;

[0012] (2) reacting compound 2 with phosphorus tribromide under organic base conditions to synthesize compound 3;

[0013] (3) reacting compound 3 with triphenylphosphine to synthesize compound 4;

[0014] (4) reacting compound 4 with compound 5 to synthesize compound 6;

[0015] (5) Compound 6 was reacted with a 1N methanol solution of hydrochloric acid to synthesize compound 7;

[0016] (6) reacting compound 7 with ethylene carbonate in the presence of an inorganic base to synthesize compound 8;

[0017] (7) Compound 8 is reacted with a substituted benzoic acid or a substituted cinnamic acid in the presence of a condensing agent to synthesize target compounds 9a-9l and 9m-9q;

[0018] The synthetic route is as follows:

[0019]

[0020] Wherein, R1 is selected from H, OCH3, CH3, Cl, CH2Cl, F; R2 is selected from H, C(CH3)3, OC2H5, C3H7, OCH3, NO2, Br, Cl; R3 is selected from H, OCH3; R4 is selected from H, OCH3; R5 is selected from H, OCH3, CH3; R6 is selected from H, OCH3.

[0021] A further improvement of the present invention is:

[0022] The organic base in step (2) is triethylamine; in step (6), the inorganic base is preferably potassium carbonate; and in step (7), the condensing agent is 1-ethyl-3(3-dimethylpropylamine)carbodiimide (EDCI) and 1-hydroxybenzotriazole (HOTB).

[0023] Furthermore, on a molar basis, in step (1), compound 1: sodium borohydride = 1:1.5, in step (2), compound 2: phosphorus tribromide: organic base = 1:0.5:1.5, in step (3), compound 3: triphenylphosphine = 1:1.2, in step (4), compound 4: compound 5: sodium hydride = 1:0.9:2.7, in step (5), compound 6: 1N hydrochloric acid / methanol solution = 1:0.5, in step (6), compound 7: ethylene carbonate: inorganic base = 1:3:3, and in step (7), compound 8: substituted benzoic acid (or substituted cinnamic acid): EDCI: HOBT = 1:1.2:1.5:1.5.

[0024] Furthermore, the reaction temperature of step (1) is 0-5°C and the time is 6 hours; the reaction temperature of step (2) is 0°C and the time is 30 minutes; the reaction temperature of step (3) is 110°C, reflux, and the time is 12 hours; the reaction temperature of step (4) is 0-5°C for 30 minutes, and then the reaction time is 8 hours at room temperature; the reaction temperature of step (5) is room temperature and the time is 6 hours; the reaction temperature of step (6) is 100°C and the time is 12 hours; the reaction temperature of step (7) is room temperature and the time is 10 to 12 hours.

[0025] Furthermore, steps (1) to (7) also include a purification process.

[0026] A further improvement of the present invention is:

[0027] Application of the above-mentioned Compretin-type microtubule targeting agent in the preparation of anticancer drugs.

[0028] The beneficial effects of the present invention are:

[0029] The present invention introduces substituted benzoic acids or substituted cinnamic acids into the CA-4 backbone to produce a novel Compretin derivative as a microtubule-targeting drug. Studies have shown that this class of microtubule-targeting agents exhibits excellent anti-liver cancer activity. Compound 9n, a representative example, exhibits stronger anti-proliferative activity against HepG-2 cells than CA-4 and lower toxicity against normal human liver cells, HL-7702. Furthermore, this drug exhibits significant inhibitory effects against both cisplatin-resistant A549 / CDDP and doxorubicin-resistant MCF-7 / DOX cells. In vitro studies suggest that this class of microtubule-targeting agents has potential as a targeted treatment for liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the in vivo anti-tumor effect of compound 9n in the examples of the present invention;

[0031] Among them, (A) images of resected tumors in each group, (B) tumor volumes in each drug-treated group, (C) tumor weights in each drug-treated group, and (D) changes in body weight of mice in each group over 21 days. *p<0.05, **p<0.01;

[0032] Figure 2 This is H&E staining of the heart, liver, spleen, lung, kidney and other major organs of mice after 21 days of treatment with compound 9n in the examples of the present invention. DETAILED DESCRIPTION

[0033] The present invention is described in detail below with reference to specific embodiments.

[0034] The present invention provides a Compretin-type microtubule targeting agent, the structural formula of which is shown below:

[0035]

[0036] Wherein, R1 is selected from H, OCH3, CH3, Cl, CH2Cl, F; R2 is selected from H, C(CH3)3, OC2H5, C3H7, OCH3, NO2, Br, Cl; R3 is selected from H, OCH3; R4 is selected from H, OCH3; R5 is selected from H, OCH3, CH3; R6 is selected from H, OCH3.

[0037] The synthetic route is as follows:

[0038]

[0039] Example 1: Preparation of Compound 2

[0040] Synthesis of (3,4,5-trimethoxyphenylmethanol) (2). Compound 1 (10.0 g, 51 mmol) and dry methanol (MeOH, 30 mL) were added to a 50 mL round-bottom flask, and the mixture was stirred at 0-5 ° C. NaBH4 was then added to the reaction, and the mixture was stirred at the same temperature for 6 hours. The reaction was monitored by thin layer chromatography (TLC). After the reaction was completed, the reaction was quenched with ice water, and water (200 mL), saturated NaCl solution (100 mL) and DCM (200 mL) were added for washing and extraction. The organic phase was dried over anhydrous sodium sulfate (Na2SO4) and concentrated under reduced pressure to obtain compound 2. It was a transparent oil (yield 95.1%). 1 H NMR (400MHz, CDCl3) δ6.51 (s, 2H), 4.52 (s, 2H), 3.76 (d, J = 3.5Hz, 9H), 2.83 (s, 1H). HR-MS (m / z) (ESI): calcd for C 10 H 15 O4[M+H] +:199.0970; found:199.0979.

[0041] Example 2: Preparation of Compound 3

[0042] Synthesis of 5-bromoethyl-1,2,3-trimethoxybenzene (3). Compound 2 was dissolved in dry DCM (20 mL), PBr3 was added dropwise to the reaction flask at 0°C, and the mixture was stirred at the same temperature for 30 minutes. The reaction was monitored by TLC. After completion of the reaction, the reaction was quenched with ice water, and water (200 mL), saturated NaCl solution (100 mL) and DCM (100 mL) were added for washing and extraction. The organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel chromatography eluted with PE / EA to obtain compound 3. White solid (yield 83.69%). 1 H NMR(400MHz, CDCl3)δ6.62(s,2H),4.46(s,2H),3.87(s,6H),3.84(s,3H).HR-MS(m / z)(ESI):calcd for C 10 H 14 BrO3[M+H] + :261.0126;found:261.0122.

[0043] Example 3: Preparation of Compound 4

[0044] Synthesis of 3,4,5-trimethoxybenzyltriphenylphosphonium bromide (4). PPh3 was added to a toluene solution of compound 3, and the resulting mixture was stirred under reflux for 12 h. The reaction was monitored by TLC. After the reaction was complete, the reaction mixture was cooled at room temperature, and the solvent was removed under reduced pressure. The white solid was washed with toluene and dried in vacuo to obtain compound 4 as a white solid (yield 91.8%). 1 H NMR (400MHz, CDCl3) δ7.75–7.70(m,9H),7.60–7.56(m,6H),6.46(d,J=2.6Hz,2H ),5.36(d,J=14.1Hz,2H),3.72(s,3H),3.47(s,6H).HR-MS(m / z)(ESI):calcdfor C 28 H 29 O3P[M+H] + :444.1854; found:444.1824.

[0045] Example 4: Preparation of Compound 5

[0046] Synthesis of 3-((tert-butyldimethylsilyl)oxy)-4-methoxybenzaldehyde (5). 3-Hydroxy-4-methoxybenzaldehyde and triethylamine (Et3N) were added to a round-bottom flask and dissolved in DCM (100 mL). TBDMSCl was added in batches, and the resulting mixture was stirred at room temperature for 6 h. The reaction was monitored by TLC. After completion of the reaction, the reaction solution was washed and extracted with water (200 mL), saturated NaCl solution (100 mL), and DCM (200 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain compound 5. It was a yellow oil (yield 93.6%). 1 H NMR spectrum. 1 H NMR (400MHz, CDCl3) δ9.84 (s, 1H), 7.39 (d, J = 1.8Hz, 1H), 7.37–7.35 (m, 1H), 6.95 (d,J=8.0Hz,1H),3.86(s,3H),0.99(s,9H),0.18(s,6H).HR-MS(m / z)(ESI):calcd for C 14 H 23 O3Si[M+H] + :267.1416; found:267.1447.

[0047] Example 5: Preparation of Compound 6

[0048] Synthesis of (Z)-tert-butyl(2-methoxy-5-(3,4,5-trimethoxyphenyl)phenoxy)dimethylsilane (6). Compound 4 was dissolved in anhydrous DCM (50 mL), NaH was added at 0-5°C, and the resulting mixture was stirred at 0-5°C for 0.5 h. Compound 5 was then dissolved in anhydrous DCM (20 mL) and added dropwise to the reaction, which was stirred at room temperature overnight. The reaction was monitored by TLC. After completion of the reaction, the reaction was quenched with ice water and the extract was washed with DCM (200 mL) and saturated sodium chloride solution (200 mL). The organic layer was dried over anhydrous Na2SO4 and the organic phase was concentrated under reduced pressure. The crude product was purified by silica gel chromatography eluted with PE / EA to obtain compound 6. It was a yellow oil (yield 44%). 1HNMR (600MHz, CDCl3) δ6.85(dd,J=8.3,2.0Hz,1H),6.79(d,J=2.0Hz,1H),6.73(d,J=8.3Hz,1H),6.50(s,2H),6.47(d,J=12. 1Hz,1H),6.42(d,J=12.1Hz,1H),3.83(s,3H),3.78(s,3H),3.70(s,6H),0.93(s,9H),0.06(s,6H).HR-MS(m / z)(ESI):calcd for C 24 H 35 O5Si[M+H] + :431.2254; found:431.2246.

[0049] Example 6: Preparation of Compound 7

[0050] Synthesis of (Z)-2-methoxy-5-(3,4,5-trimethoxyphenyl)phenol (7). A solution of compound 6 in MeOH (15 mL) was added to PTSA, and the mixture was stirred at room temperature for 6 hours. The reaction was monitored by TLC. After the reaction was completed, the resulting reaction solution was washed and extracted with DCM (2×100 mL), water (100 mL) and saturated NaCl solution (100 mL). The organic layer was dried over anhydrous Na2SO4 and the organic phase was concentrated under reduced pressure. The crude product was purified by silica gel chromatography eluted with PE / EA to obtain the target compound 7 as a white solid (yield 94.7%). 1 HNMR (600MHz, CDCl3) δ6.92(d,J=1.9Hz,1H),6.80(dd,J=8.3,1.9Hz,1H),6.73(d,J=8.3Hz,1H),6.53(s,2H),6.47(d,J =12.2Hz,1H),6.41(d,J=12.2Hz,1H),5.55(s,1H),3.86(s,3H),3.84(s,3H),3.70(s,6H).HR-MS(m / z)(ESI):calcdfor C 18 H 21 O5[M+H] + :317.1389; found:317.1393.

[0051] Example 7: Preparation of Compound 8

[0052] Synthesis of (Z)-2-(2-methoxy-5-(3,4,5-trimethoxyphenyl)phenoxy)ethylene glycol (8). To a DMF (10 mL) solution of compound 7 was added ethylene carbonate and K2CO3, and the resulting mixture was stirred at 100°C under the protection of N2 for 12 hours. The reaction was monitored by TLC. After completion of the reaction, the resulting reaction solution was washed and extracted with water (200 mL), saturated NaCl solution (100 mL) and DCM (2×100 mL). The organic layer was dried over anhydrous Na2SO4 and the organic phase was concentrated under reduced pressure. The crude product was purified by silica gel chromatography eluted with PE / EA to obtain the target compound 8 as a white solid (yield 92.6%). 1 HNMR (600MHz, CDCl3) δ6.91(dd,J=8.3,1.9Hz,1H),6.86(d,J=1.9Hz,1H),6.78(d,J=8.3Hz,1H),6.51(s,2H),6.48(d ,J=12.1Hz,1H),6.45(d,J=12.1Hz,1H),3.93–3.92(m,2H),3.84–3.82(m,8H),3.70(s,6H).HR-MS(m / z)(ESI):calcd for C 27 H 29 O7[M+H] + :361.1651; found:361.1673

[0053] Example 8: Preparation of Compounds 9a-9l and 9m-9q

[0054] The structural formulas of compounds 9a-9l and 9m-9q are shown in Table 1:

[0055]

[0056]

[0057] Table 1. Structures of Compretin derivatives

[0058] The specific preparation process is:

[0059] A substituted benzoic acid or substituted cinnamic acid was dissolved in anhydrous DCM (5 mL), followed by the addition of EDCI, HBOT, and compound 8, and stirred at room temperature overnight. The reaction was monitored by TLC. After completion, DCM (200 mL) and saturated NaCl solution were added to the reaction mixture for extraction and washing. The organic phase was dried over anhydrous MgSO4 and concentrated under reduced pressure. The resulting crude product was purified by silica gel column chromatography to afford the final products 9a-9l and 9m-9q.

[0060] (Z)-2-(2-methoxy-5-(3,4,5-trimethoxybenzoic acid)phenoxy)benzoic acid ethyl ester (9a). White solid, yield 81.9%. 1 H NMR (400MHz, CDCl3) δ8.04–8.01(m,2H),7.55(t,J=7.4Hz,1H),7.42(t,J=7.7Hz,2H),6.93–6.88(m,2H),6.77(d,J=8.0Hz,1H) ,6.51(s,2H),6.48(d,J=12.2Hz,1H),6.44(d,J=12.1Hz,1H),4.61–4.58(m,2H),4.21–4.18(m,2H),3.81(s,6H),3.68(s,6H). 13 C NMR (100MHz, CDCl3) δ166.47,152.97,149.05,147.53,137.10,133.08,132.90,129.95,129.90,129.76,129.47 ,129.04,128.35,122.97,115.17,111.72,105.93,67.32,63.37,60.92,55.99,55.96.HR-MS(m / z)(ESI):calcd for C 27 H 29 O7[M+H] + :465.1913; found:465.1951.Purity 97.80% (by HPLC).

[0061] (Z)-2-(2-Methoxy-5-(3,4,5-trimethoxyphenyl)phenoxy)ethyl 4-(tert-butyl)benzoate (9b). White solid. Yield: 83.8%. 1 H NMR (400MHz, CDCl3) δ7.96(d,J=8.2Hz,2H),7.43(d,J=8.3Hz,2H),6.91(d,J=8.0Hz,2H),6.77(d,J=8.0Hz,1H),6.51(s,2H), 6.47(d,J=12.2Hz,1H),6.44(d,J=12.1Hz,1H),4.60–4.58(m,2H),4.22–4.16(m,2H),3.82(s,6H),3.68(s,6H),1.32(s,9H). 13C NMR (100MHz, CDCl3) δ166.49,156.74,152.96,149.03,147.59,137.11,132.88,129.96,129.63,129.50,129.03,127.10 ,125.32,122.88,115.10,111.74,105.95,67.32,63.17,60.91,56.01,55.95,35.09,31.12.HR-MS(m / z)(ESI):calcdfor C 31 H 37 O7[M+H] + :521.2539; found:521.2572.Purity97.73% (by HPLC).

[0062] (Z)-2-(2-Methoxy-5-(3,4,5-trimethoxyphenyl)phenoxy-4-ethoxybenzoate (9c). White solid. Yield 82.7%. 1 H NMR (400MHz, CDCl3) δ7.95(d,J=8.0Hz,2H),6.91(d,J=8.0Hz,2H),6.88–6.86(m,3H),6.76(d,J=8.1Hz,1H),6.50(s,2H),6.46(d,J=12.2 Hz,1H),6.43(d,J=12.1Hz,1H),4.60–4.57(m,2H),4.22–4.18(s,2H),4.08–4.03(m,2H),3.80(s,6H),3.68(s,6H),1.41(t,J=7.0Hz,3H). 13 C NMR (100MHz, CDCl3) δ166.22,162.87,152.96,149.04,147.60,137.11,132.90,131.79,129.96,129.49,129.02,122.8 9,122.08,115.15,114.01,111.73,105.94,67.41,63.69,63.07,60.91,56.00,55.95,14.68.HR-MS(m / z)(ESI):calcd for C 29 H 32 O8Na[M+Na] + :531.1995; found:531.1995.Purity 97.44% (by HPLC).

[0063] (Z)-2-(2-methoxy-5-(3,4,5-trimethoxyphenyl)phenoxy)4-propylbenzoic acid ethyl ester (9d). White solid, yield 83.3%. 1 H NMR (400MHz, CDCl3) δ7.93(d,J=8.2Hz,2H),7.21(d,J=8.1Hz,2H),6.91–6.89(m,2H),6.77(d,J=8.1Hz,1H),6.51(s,2H),6.48(d,J=12.3Hz,1H),6 .44(d,J=12.1Hz,1H),4.59–4.56(m,2H),4.20–4.17(m,2H),3.81(s,6H), 3.68(s,6H),2.64–2.60(m,2H),1.67–1.59(m,2H),0.92(t,J=7.3Hz,3H). 13 C NMR (100MHz, CDCl3) δ166.54,152.97,149.04,148.43,147.57,137.10,132.90,129.55,129.80,129.49(s),129.03,128.47 ,127.39,122.90,115.12,111.72,105.93,67.34,63.18,60.91,55.99,55.95,38.05,24.26,13.76.HR-MS(m / z)(ESI):calcd for C 30 H 34 O7Na[M+Na] + :529.2202; found:529.2229.Purity 97.24% (by HPLC).

[0064] (Z)-2-(2-methoxy-5-(3,4,5-trimethoxyphenyl)phenoxy 3,4,5-trimethoxybenzoic acid ethyl ester (9e). White solid. Yield 74.6%. 1 H NMR(400MHz, CDCl3)δ7.28(s,2H),6.90–6.89(m,2H),6.77(d,J=8.8Hz,1H),6.49(s,2H),6.46(d,J=12.4Hz,1H), 6.42(d,J=12.3Hz,1H),4.60–4.57(m,2H),4.19–4.17(m,2H),3.89(s,3H),3.87(s,6H),3.81(s,6H),3.68(s,6H). 13C NMR (100MHz, CDCl3) δ166.08,152.98,152.90,149.02,147.54,142.30,137.13,132.86,129.99,129.40,129.09,124.9 2,122.93,115.03,111.76,107.00,105.92,67.29,63.41,60.93,60.89,56.26,56.01,55.95.HR-MS(m / z)(ESI):calcd for C 30 H 34 O 10 Na[M+Na] + :577.2050; found:577.2051.Purity 97.46% (by HPLC).

[0065] (Z)-2-(2-methoxy-5-(3,4,5-trimethoxyphenyl)phenoxy 3-methylbenzoic acid ethyl ester (9f). White solid. Yield 74.8%. 1 HNMR (400MHz, CDCl3) δ7.85–7.81(m,2H),7.38–7.27(m,2H),6.92–6.89(m,2H),6.77(d,J=8.1Hz,1H),6.50(s,2H),6.48 (d,J=12.3Hz,1H),6.44(d,J=12.1Hz,1H),4.59–4.57(m,2H),4.20–4.18(m,2H),3.81(s,6H),3.68(s,6H),2.38(s,3H). 13 C NMR (100MHz, CDCl3) δ166.63,152.98,149.08,147.57,138.10,137.12,133.84,132.91,130.26,129.96,129.92,129.48,12 9.04,128.24,126.91,122.96,115.21,111.74,105.93,67.36,63.32,60.91,55.99,55.95,21.26.HR-MS(m / z)(ESI):calcd for C 28 H 32 O7[M+H] + :479.2070; found:479.2079.Purity97.64% (by HPLC).

[0066] (Z)-2-(2-methoxy-5-(3,4,5-trimethoxyphenyl)phenoxy)4-nitrobenzoic acid ethyl ester (9 g). White solid, yield 74.0%. 1 HNMR (400MHz, CDCl3) δ8.27–8.25(m,2H),8.19–8.17(m,2H),6.92(d,J=8.3Hz,2H),6.78(d,J=8.0Hz,1H),6.50(s,2 H),6.46(d,J=12.2Hz,1H),6.43(d,J=12.1Hz,1H),4.65–4.61(m,2H),4.20–4.18(m,2H),3.81(s,6H),3.69(s,6H). 13 C NMR (100MHz, CDCl3) δ164.55,153.01,150.58,149.10,147.29,137.14,135.30,132.88,130.90,129.95,129.35 ,129.11,123.51,123.29,115.27,111.71,105.91,67.13,64.22,60.90,55.97,55.94.HR-MS(m / z)(ESI):calcd for C 27 H 29 NO9[M+H] + :510.1764; found:510.1755.Purity 98.41% (by HPLC).

[0067] (Z)-2-(2-methoxy-5-(3,4,5-trimethoxyphenyl)phenoxy 3-chlorobenzoic acid ethyl ester (9h). White solid, yield 85.5%. 1 H NMR (400MHz, CDCl3) δ7.99–7.97(m,1H),7.92–7.89(m,1H),7.52–7.51(m,1H),7.36(t,J=7.9Hz,1H),6.93–6.90(m,2H),6.78(d,J=8. 9Hz,1H),6.51(s,2H),6.48(d,J=12.2Hz,1H),6.44(d,J=12.1Hz,1H),4.60–4.58(m,2H),4.19–4.17(m,2H),3.81(s,6H),3.69(s,6H). 13C NMR (100MHz, CDCl3) δ165.23,152.99,149.11,147.44,134.48,133.10,132.91,131.67,129.95,129.79,129.69,12 9.44,129.06,127.91,123.13,115.30,111.71,105.93,67.27,63.74,60.91,56.04,55.95.HR-MS(m / z)(ESI):calcd forC 27 H 28 ClO7[M+H] + :499.1524; found:499.1526.Purity 97.41% (by HPLC).

[0068] (Z)-2-(2-methoxy-5-(3,4,5-trimethoxyphenyl)phenoxy)ethyl 3-(chloromethyl)benzoate (9i) was obtained as a white solid in a yield of 76.6%. 1 H NMR (400MHz, CDCl3) δ8.01–7.97(m,2H),7.58(d,J=7.7Hz,1H),7.42(t,J=7.7Hz,1H),6.92–6.90(m,2H),6.77(d,J=8.9Hz,1H),6.5 1(s,2H),6.47(d,J=12.2Hz,1H),6.44(d,J=12.1Hz,1H),4.60–4.57(m,4H),4.20–4.18(m,2H),3.81(d,J=0.8Hz,6H),3.68(s,6H). 13 C NMR (100MHz, CDCl3) δ165.92,152.99,149.08,147.50,137.87,137.11,133.23,132.92,130.44,129.94,129.81,129.77,12 9.45,129.06,128.91,123.05,115.22,111.73,105.93,67.28,63.59,60.91,55.99,55.97,45.51.HR-MS(m / z)(ESI):calcd for C 28 H 30 ClO7[M+H] + :513.1680; found:513.1674.Purity 97.64%(byHPLC).

[0069] (Z)-2-(2-methoxy-5-(3,4,5-trimethoxyphenyl)phenoxy-3-fluorobenzoic acid ethyl ester (9j). White solid. Yield 75.8%. 1 H NMR (400MHz, CDCl3) δ7.82(d,J=8.3Hz,1H),7.71(d,J=9.3Hz,1H),7.43–7.38(m,1H),7.28–7.23(m,1H),6.92(d,J=6.5Hz,2H),6.78(d,J =8.8Hz,1H),6.51(s,2H),6.49(d,J=12.3Hz,1H),6.45(d,J=12.1Hz,1H),4.61–4.59(m,2H),4.20–4.18(m,2H),3.82(s,6H),3.69(s,6H). 13 C NMR (100MHz, CDCl3) δ165.33,163.72,161.24,152.98,149.08,147.43,137.11,132.91,132.08,129.95,129.43,129.06,12 5.52,123.10,120.25,120.04,116.75,116.52,115.23,111.71,67.24,63.68,60.90,55.95,55.88.HR-MS(m / z)(ESI):calcd for C 27 H 29 FO7[M+H] + :483.1819; found:483.1793.Purity 98.23% (by HPLC).

[0070] (Z)-2-(2-methoxy-5-(3,4,5-trimethoxyphenyl)phenoxy)4-bromobenzoic acid ethyl ester (9k). White solid, yield 82.6%. 1 H NMR (400MHz, CDCl3) δ7.91–7.86(m,2H),7.56–7.54(m,2H),6.92–6.88(m,2H),6.77(d,J=8.8Hz,1H),6.50(s,2H),6.4 8(d,J=12.2Hz,1H),6.44(d,J=12.1Hz,1H),4.59–4.56(m,2H),4.19–4.16(m,2H),3.81(d,J=3.3Hz,6H),3.68(s,6H). 13CNMR(100MHz, CDCl3)δ165.70,153.41,152.99,149.08,147.45,137.13,132.88,131.69,131.29,129.95,129.41,129.0 7,128.82,128.20,123.09,115.23,111.71,105.93,103.32,67.26,63.62,60.91,56.12,55.96.HR-MS(m / z)(ESI):calcd for C 27 H 28 BrO7[M+H] + :543.1018; found:543.1007.Purity97.65% (by HPLC).

[0071] (Z)-2-(2-methoxy-5-(3,4,5-trimethoxyphenyl)phenoxy)4-chlorobenzoic acid ethyl ester (91). White solid, yield 78.2%. 1 H NMR (400MHz, CDCl3) δ7.94(d,J=10.6Hz,2H),7.38(d,J=8.5Hz,2H),6.92–6.90(m,2H),6.77(d,J=8.8Hz,1H),6.50(s,2H) ,6.48(d,J=12.6Hz,1H),6.44(d,J=12.2Hz,1H),4.59–4.57(m,2H),4.19–4.16(m,2H),3.81(d,J=2.1Hz,6H),3.68(s,6H). 13 C NMR (100MHz, CDCl3) δ165.59,152.99,149.07,147.44,139.52,137.12,132.89,131.16,129.95,129.42,129.07 ,128.70,128.36,123.08,115.20,111.71,105.92,67.26,63.60,60.91,55.96,55.84.HR-MS(m / z)(ESI):calcd for C 27 H 28 ClO7[M+H] + :499.1524; found:499.1525.Purity 97.88% (by HPLC).

[0072] Ethyl 2-(2-methoxy-5-((Z)-3,4,5-trimethoxyphenyl)phenoxycinnamate (9m). White solid. Yield 83.6%.1 H NMR (400MHz, CDCl3) δ7.69(d,J=16.0Hz,1H),7.52–7.50(m,2H),7.39–7.37(m,3H),6.92–6.89(m,2H),6.78(d,J=8.1 Hz,1H),6.51(s,2H),6.50–6.42(m,3H),4.50–4.47(m,2H),4.14–4.11(m,2H),3.84(s,3H),3.82(s,3H),3.70(s,6H). 13 C NMR (100MHz, CDCl3) δ166.84,152.98,148.98,147.44,145.28,137.10,134.33,132.92,130.40,129.92,129.51,129.04 ,128.92,128.15,122.93,117.69,114.87,111.60,105.91,67.22,62.87,60.93,55.99,55.97.HR-MS(m / z)(ESI):calcd for C 29 H 31 O7[M+H] + :491.2070; found:491.2063.Purity 98.15% (by HPLC).

[0073] 2-(2-Methoxy-5-((Z)-3,4,5-trimethoxyphenyl)phenoxy)ethyl (E)-3-(4-methoxyphenyl)acrylate (9n). White solid, yield 81.5%. 1 H NMR (400MHz, CDCl3) δ7.64(d,J=16.0Hz,1H),7.46(d,J=8.8Hz,2H),6.91–6.88(m,4H),6.77(d,J=8.0Hz,1H),6.51(s,2H),6.47(s,1H) ,6.44(d,J=12.4Hz,1H),6.33(d,J=16.0Hz,1H),4.48–4.46(m,2H),4.13–4.10(m,2H),3.83(d,J=2.1Hz,6H),3.82(s,3H),3.69(s,6H). 13C NMR (100MHz, CDCl3) δ167.17,161.45,152.98,148.97,147.48,144.95,137.09,132.92,129.92,129.82,129.52,129.02,12 7.08,122.88,115.13,114.86,114.34,111.60,105.91,67.27,62.71,60.93,55.99,55.97,55.40.HR-MS(m / z)(ESI):calcd for C 30 H 32 O8Na[M+Na] + :543.1995; found:543.1992.Purity 98.00% (by HPLC).

[0074] 2-(2-Methoxy-5-((Z)-3,4,5-trimethoxyphenyl)phenoxy)ethyl (E)-3-(p-tolyl)acrylate (9o). White solid. Yield 73.8%. 1 H NMR (400MHz, CDCl3) δ7.66(d,J=16.0Hz,1H),7.41(d,J=8.1Hz,2H),7.18(d ,J=8.0Hz,2H),6.92–6.89(m,2H),6.77(d,J=8.0Hz,1H),6.51(s,2H),6.49( d,J=12.3Hz,1H),6.46(d,J=12.1Hz,1H),6.41(d,J=13.2Hz,1H),4.49–4.47 (m,2H),4.13–4.11(m,2H),3.83(d,J=4.1Hz,6H),3.69(s,6H),2.36(s,3H). 13 C NMR (100MHz, CDCl3) δ167.01,152.99,149.00,147.47,145.27,140.83,137.12,132.92,131.62,129.93,129.93,129.65,129.5 1,129.03,128.14,122.91,116.59,114.90,111.62,105.93,67.26,62.78,60.92,55.99,55.97,21.50.HR-MS(m / z)(ESI):calcd for C 30 H 32 O7Na[M+Na] +:527.2046; found:527.2056.Purity98.18%(by HPLC).

[0075] 2-(2-Methoxy-5-((Z)-3,4,5-trimethoxyphenyl)phenoxy)ethyl (E)-3-(3,4-dimethoxyphenyl)acrylate (9p). White solid. Yield 83.9%. 1 H NMR (400MHz, CDCl3) δ7.62(d,J=15.9Hz,1H),7.08(d,J=8.3Hz,1H),7.04(d,J=1.2Hz ,1H),6.89(d,J=8.2Hz,2H),6.85(d,J=8.3Hz,1H),6.77(d,J=8.0Hz,1H),6.50(s,2H ),6.48(d,J=12.4Hz,1H),6.43(d,J=12.1Hz,1H),6.34(d,J=15.9Hz,1H),4.48–4.46 (m,2H),4.12–4.10(m,J=4.8Hz,2H),3.89(s,6H),3.82(d,J=5.2Hz,6H),3.69(s,6H). 13 C NMR (100MHz, CDCl3) δ167.04,152.98,151.18,149.19,148.97,147.45,145.15,137.11,132.90,129.91,129.49,129.02,127.33,12 2.90,122.74,115.38,114.84,111.58,111.00,109.57,105.91,67.26,62.73,60.91,55.97,55.96,55.88.HR-MS(m / z)(ESI):calcd for C 31 H 34 O9Na[M+Na] + :573.2101; found:573.2113.Purity 97.12% (by HPLC).

[0076] 2-(2-Methoxy-5-((Z)-3,4,5-trimethoxyphenyl)phenoxy)ethyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (9q). White solid, yield 80.7%. 1HNMR(400MHz, CDCl3) δ7.59(d,J=15.9Hz,1H),6.91–6.88(m,2H),6.78–6.73(m,3H),6.50(s,2H),6.45(d,J=12.3Hz,1H),6.43(d,J=12.1Hz, 1H), 6.38 (dd, J=15.9, 1.1Hz, 1H), 4.48–4.46 (m, 2H), 4.48–4.46 (t, J=4.8Hz, 2H), 3.89–3.87 (m, 9H), 3.83 (s, 3H), 3.81 (s, 3H), 3.69 (s, 6H). 13 C NMR (100MHz, CDCl3) δ166.77,153.42,152.98,148.96,147.40,145.19,140.11,137.10,132.90,129.90,129.86,129.47,12 9.04,122.95,116.97,114.81,111.56,105.89,105.22,67.21,62.85,60.99,60.92,56.15,55.96.HR-MS(m / z)(ESI):calcd for C 32 H 37 O 10 [M+H] + :581.2387; found:581.2398.Purity 97.19% (by HPLC).

[0077] Example 9: In vitro anti-proliferative activity assay

[0078] (1) Experimental methods

[0079] The MTT assay was used to investigate the antiproliferative activity of compounds 9a-9q against four different types of human cancer cells, including liver cancer cells (HepG-2), lung cancer cells (NCI-H460), colon cancer cells (HCT-116), ovarian cancer cells (SK-OV-3), and normal human liver cells (HL-7702). CA-4 and paclitaxel, as well as two drug-resistant cell lines, cisplatin-resistant cells A549 / CDDP and doxorubicin-resistant cells MCF-7 / DOX, were used as controls. All cells were seeded in 96-well plates (5000 cells per well, 2000 μL) and incubated overnight at 37°C in a humidified atmosphere containing 5% CO2. The cells were then treated with different concentrations of the test compounds under the same conditions for 72 hours. After 72 hours of incubation, fresh MTT solution (10 μL) was added and incubated at 37°C for 4 hours. The samples were recorded at a wavelength of 570 nm using a microplate reader, and the IC values ​​were calculated using SPSS software. 50 value.

[0080] Table 2 Antiproliferative activity of compounds 9a-9l and 9m-9q against different cancer cell lines

[0081]

[0082]

[0083] IC 50 ±SD (μM) a , half-inhibitory concentration, values ​​are expressed as the mean ± SD of three independent experiments. SI b , selection index = IC 50 (HL-7702) / IC 50 (HepG-2).

[0084] Table 3 Antiproliferative activity of compound 9n against drug-resistant cells

[0085]

[0086] CDDP a , cisplatin; DOX b , doxorubicin; IC 50 (μM) c , half-inhibitory concentration, values ​​are expressed as the mean ± SD of three independent experiments. RF d , resistance factor = IC 50 (A549 / CDDP) / IC 50 (A549) or IC 50 (MCF-7 / DOX) / IC 50 (MCF-7).

[0087] (2) Experimental results

[0088] First, as shown in Table 2, in the HepG-2 cell experiment, target compounds 9a-9l (IC 50 =1.34~6.51μM) showed strong anticancer activity, but its antiproliferative activity was lower than that of CA-4 (IC 50 =0.14 μM) and paclitaxel (IC 50 =0.25 μM). Among them, 9b (IC 50 =2.33 μM), 9c (IC 50 =1.78μM), 9g(IC 50 =1.34μM)、9k(IC 50 =2.07 μM) and 91 (IC 50 =2.56 μM) had slightly stronger antiproliferative activity than 9a (IC 50=3.23μM), indicating that the introduction of electron-withdrawing or electron-donating substituents in the para-phenyl ring is beneficial to improving activity. This trend also applies to other cancer cell lines, such as NCI-H460, HCT-116, and SK-OV-3. We note that 9m-9p exhibited higher anticancer activity than 9a-9l against the tested human cancer cell lines, indicating that the introduction of cinnamic acid derivatives into CA-4 analogs has greater potential to enhance antiproliferative activity than benzoic acid derivatives. Interestingly, the results in Table 2 show that compound 9n exhibited the best antiproliferative activity among these CA-4 analogs against the tested cancer cells (HepG-2: 0.09μM, NCI-H460: 0.25μM, HCT-116: 0.20μM, SK-OV-3: 0.18μM), and was slightly better than AC-4 and paclitaxel. More importantly, compared with the control drug CA-4 (IC 50 =1.17 μM) and paclitaxel (IC 50 =1.03 μM), compared with compound 9n (IC 50 =3.16 μM) showed low cytotoxicity against normal human hepatocytes HL-7702, with a selectivity index (SI) of 35.11, lower than that of CA-4 (8.36) and paclitaxel (4.12), respectively. This indicates that compound 9n has high selectivity for both cancer cells and normal cells. Based on these findings, we selected compound 9n as a representative compound to further evaluate its anticancer mechanism.

[0089] The results in Table 3 show that CDDP (IC 50 value 5.67μM) had a strong anticancer activity against CDDP-sensitive cells A549, but had a strong anticancer activity against CDDP-resistant cells A549 / CDDP (IC 50 The inhibitory effect of 9n on A549 / CDDP or MCF-7 / DOX cells was weaker (IC value 35.22μM), which was consistent with expectations. This trend was also observed in DOX. It is worth noting that 9n was more toxic to A549 / CDDP or MCF-7 / DOX cells, with an IC value of 50 The values ​​were 0.29 and 0.51 μM, respectively, and the inhibitory effects were nearly 121.45- and 74.88-fold that of CDDP or DOX. In addition, the resistance factors were lower (RF values ​​were 0.91 and 1.16, respectively) compared with CDDP (RF value 6.21) and DOX (RF value 6.24), indicating that 9n has great potential to overcome CDDP or DOX resistance.

[0090] Example 10: In vivo anti-tumor activity experiment:

[0091] (1) Model establishment

[0092] Twenty 6-week-old nude mice were purchased from the Center for Comparative Medicine of Yangzhou University and maintained under pathogen-free conditions. Logarithmically growing HepG-2 cells were subcutaneously inoculated into the right axilla of mice to establish a HepG-2 xenograft model. When the tumor volume in each group reached approximately 100 mm 3 Twenty nude mice were randomly divided into four groups (n=4): saline-treated, CA-4-treated (15 mg / kg), and 9n-treated (15 or 30 mg / kg). Dosing was performed every two days. Tumor volume and body weight were monitored every two days for 21 days. Three weeks after dosing, the mice were sacrificed, and tumors were excised. Tumor inhibition rates were calculated based on the treatment results.

[0093] (2) Experimental results

[0094] like Figure 1 The results shown in A to C show that compound 9n has a significant inhibitory effect on tumor growth compared with the control group, with tumor growth inhibition (TGI) rates reaching 58.3% and 70.7%, respectively, and in a dose-dependent manner. In addition, the TGI of 9n at a dose of 15 mg / kg was slightly lower than that of CA-4 (TGI rate of 61.2%), but at a high dose of 30 mg / kg, the TGI rate of 9n (TGI rate of 70.7%) was better than that of CA-4. In addition, there was no significant change in the body weight of mice after administration of compound 9n throughout the treatment period, indicating its high efficiency and low toxicity ( Figure 1 D). Importantly, the 9n-treated group showed no significant histopathological changes in major organs, including the liver, heart, lungs, spleen, and kidneys. Together, these results suggest that 9n may be a promising drug candidate due to its remarkable efficacy and low toxicity in the HepG-2 xenograft model.

[0095] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A Compretin-type microtubule targeting agent, characterized in that: The structural formula is as follows: Among them, R1 is selected from H, OCH3, CH3, Cl, CH2Cl, F; R2 is selected from H, C(CH3)3, OC2H5, C3H7, OCH3, NO2, Br, Cl; R3 is selected from H, OCH3; R4 is selected from H, OCH3; R5 is selected from H, OCH3, CH3; R6 is selected from H, OCH3.

2. The method for preparing a Compretin-type microtubule targeting agent according to claim 1, characterized in that: The following steps are involved: (1) reacting compound 1 with sodium borohydride to synthesize compound 2; (2) reacting compound 2 with phosphorus tribromide under organic base conditions to synthesize compound 3; (3) reacting compound 3 with triphenylphosphine to synthesize compound 4; (4) reacting compound 4 with compound 5 to synthesize compound 6; (5) reacting compound 6 with a 1N methanol solution of hydrochloric acid to synthesize compound 7; (6) reacting compound 7 with ethylene carbonate in the presence of an inorganic base to synthesize compound 8; (7) Compound 8 is reacted with a substituted benzoic acid or a substituted cinnamic acid in the presence of a condensing agent to synthesize target compounds 9a-9l and 9m-9q; The synthetic route is as follows: Among them, R1 is selected from H, OCH3, CH3, Cl, CH2Cl, F; R2 is selected from H, C(CH3)3, OC2H5, C3H7, OCH3, NO2, Br, Cl; R3 is selected from H, OCH3; R4 is selected from H, OCH3; R5 is selected from H, OCH3, CH3; R6 is selected from H, OCH3.

3. The method for preparing a Compretin-type microtubule targeting agent according to claim 2, characterized in that: The organic base in step (2) is triethylamine; in step (6), the inorganic base is preferably potassium carbonate; and in step (7), the condensing agent is 1-ethyl-3 (3-dimethylpropylamine) carbodiimide and 1-hydroxybenzotriazole.

4. A method for preparing a Compretin-type microtubule targeting agent according to claim 2 or 3, characterized in that: In terms of molar amount, in step (1), compound 1: sodium borohydride = 1:1.5; in step (2), compound 2: phosphorus tribromide: organic base = 1:0.5:1.5; in step (3), compound 3: triphenylphosphine = 1:1.2; in step (4), compound 4: compound 5: sodium hydride = 1:0.9:2.7; in step (5), compound 6: 1N hydrochloric acid / methanol solution = 1:0.5; in step (6), compound 7: ethylene carbonate: inorganic base = 1:3:3; in step (7), compound 8: substituted benzoic acid (or substituted cinnamic acid): EDCI: HOBT = 1:1.2:1.5:1.

5.

5. The method for preparing a Compretin-type microtubule targeting agent according to claim 2, characterized in that: The reaction temperature of step (1) is 0-5°C and the time is 6 hours; the reaction temperature of step (2) is 0°C and the time is 30 minutes; the reaction temperature of step (3) is 110°C, reflux, and the time is 12 hours; the reaction temperature of step (4) is 0-5°C for 30 minutes, and then the reaction time is 8 hours at room temperature; the reaction temperature of step (5) is room temperature and the time is 6 hours; the reaction temperature of step (6) is 100°C and the time is 12 hours; the reaction temperature of step (7) is room temperature and the time is 10 to 12 hours.

6. The method for preparing a Compretin-type microtubule targeting agent according to claim 2, characterized in that: Steps (1) to (7) also include a purification process.

7. Use of a Compretin-type microtubule targeting agent as claimed in claim 1 in the preparation of anticancer drugs.

Images