Novel tubulin and metalloproteinase double-target inhibitor as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly discloses a novel tubulin and metalloproteinase double-target inhibitor as well as a preparation method and application of the novel tubulin and metalloproteinase double-target inhibitor. A novel tubulin and metalloproteinase double-target inhibitor is obtained as a compound with a targeting function. A verification result shows that the synthesized target compound shows good anti-proliferative activity on tested cancer cells, 15j has the optimal anti-tumor activity, the IC50 value range of the target compound is 0.154-0.296 mu M, and the tumor inhibition rate of the target compound on human lung cancer transplantation tumors is 74.7% (20 mg / kg). Besides, the anti-proliferative activity of the 15j to cis-platinum drug-resistant cells A549 / CDDP is 136.70 times that of the cis-platinum, the anti-proliferative activity of the 15j to adriamycin drug-resistant cells MCF-7 / DOX is 71.14 times that of the adriamycin, and the toxicity of the 15j to human normal cells (LO2 and MCF-10A) is lower than that of positive drugs such as the cis-platinum, the adriamycin and the paclitaxel. Therefore, the compound has the potential purpose of targeted treatment of lung cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and relates to the design and synthesis of a microtubule targeting agent, and in particular to a novel microtubule protein and metalloproteinase dual-point inhibitor, a synthesis method thereof, and application in the anti-tumor field. Background Art

[0002] Today, lung cancer has become one of the most common and deadly malignant tumors in the world, among which non-small cell lung cancer (NSCLC) accounts for about 85% of all newly diagnosed cases, which has seriously affected people's quality of life and health. Although current tumor diagnostic methods and treatment strategies are developing rapidly, targeted drugs for the treatment of non-small cell lung cancer have also made undeniable progress, but the chemotherapy response rate and prognosis of patients still need to be improved. At present, chemotherapy is still one of the main methods for the treatment of malignant tumors. However, drug resistance and toxic side effects are prone to occur during chemotherapy, which in turn affects the treatment effect and ultimately constitutes a major obstacle to the widespread use of drugs. Therefore, it is urgent to develop a new type of microtubule-targeted anticancer drug with strong targeting, high efficacy and low toxicity.

[0003] In recent years, microtubule-targeted drugs have been widely used in the treatment of various solid tumors, including non-small cell lung cancer. These drugs are mainly derived from taxanes (such as paclitaxel and docetaxel) and vinca alkaloids (such as vincristine and vinblastine). Natural products and their derivatives play a key role in drug development, especially in anticancer drug research. They provide a variety of biologically active lead compounds for drug research. Although these compounds that have been listed have achieved great success in cancer treatment, they generally have obvious defects, such as high toxicity, insufficient selectivity, low oral bioavailability, poor solubility, drug resistance or complex synthesis process. Therefore, the present invention designs and synthesizes a new type of microtubule protein and matrix metalloproteinase inhibitors as microtubule-targeted antitumor drugs, which have strong targeting, high efficacy and low toxicity. Summary of the invention

[0004] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a novel microtubule protein and metalloproteinase dual-target inhibitor compound, which can overcome the shortcomings of existing anti-tumor drugs such as large toxic side effects and poor oral availability while treating lung cancer. Another purpose of the present invention is to provide a method for synthesizing the microtubule protein and metalloproteinase dual-target inhibitor.

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

[0006] A novel dual-target inhibitor of tubulin and metalloproteinase, the general structural formula of which is shown below

[0007]

[0008] Wherein, R1 is selected from H, F, Cl, NO2, OCH3; R2 is selected from H, CH3, F, Cl, Br, NO2, OCH3; R3 is selected from H, OCH3;

[0009] Furthermore, the novel dual-target inhibitor of tubulin and metalloproteinase is selected from the compounds with the following structures:

[0010]

[0011]

[0012] A further improvement of the present invention is:

[0013] A method for preparing a novel dual-target inhibitor of tubulin and metalloproteinase comprises the following steps:

[0014] (1) reacting compound 1 with compound 2 under organic base conditions to synthesize compound 3;

[0015] (2) reacting compound 3 with pyridine to synthesize compound 4;

[0016] (3) reacting compound 4 with compound 5 under the action of an inorganic base to synthesize compound 6;

[0017] (4) reacting compound 6 with compound 10 in the presence of a condensing agent to synthesize compound 11;

[0018] The synthetic route is as follows:

[0019]

[0020] Among them, R1 is selected from H, F, Cl, NO2, OCH3; R2 is selected from H, CH3, F, Cl, Br, NO2, OCH3; R3 is selected from H, OCH3.

[0021] Furthermore, the organic base in step (1) is a bicyclic amidine; in terms of molar amount, compound 1: compound 2 = 1: 1-2;

[0022] And / or, in step (2), in terms of molar amount, compound 3:pyridine=1:60-65;

[0023] And / or, in step (3), the inorganic base is potassium hydroxide; in terms of molar amount, compound 4: compound 5 = 1: 0.8 to 1.2;

[0024] And / or, in step (4), the condensing agent is 1-ethyl-3(3-dimethylpropylamine)carbodiimide (EDCI) and 4-dimethylaminopyridine (DMAP); in terms of molar amount, compound 6﹕compound 10﹕EDCI﹕DMAP=1﹕1~1.5﹕1.5~2﹕1.5~2.

[0025] Furthermore, the reaction temperature of step (1) is -5 to 5°C and the reaction time is 10 to 14 hours;

[0026] And / or, the reaction temperature of step (2) is 110-130° C. and the reaction time is 8-12 h;

[0027] And / or, the reaction temperature of step (3) is -5 to 5°C and the reaction time is 10 to 14 hours;

[0028] And / or, the reaction temperature of step (4) is room temperature and the reaction time is 8 to 12 hours;

[0029] And / or, steps (1) to (4) further include a purification process.

[0030] A further improvement of the present invention is:

[0031] A method for preparing a novel dual-target inhibitor of tubulin and metalloproteinase comprises the following steps:

[0032] (1) reacting compound 12 with compound 13 under an inorganic base to synthesize compound 14;

[0033] (2) reacting compound 14 with compound 10 in the presence of a condensing agent to synthesize compound 15;

[0034] The synthetic route is as follows:

[0035]

[0036] Among them, R1 is selected from H, F, Cl, NO2, OCH3; R2 is selected from H, CH3, F, Cl, Br, NO2, OCH3; R3 is selected from H, OCH3.

[0037] Furthermore, in step (1), the inorganic base is potassium hydroxide, and the molar ratio of compound 12:compound 13 is 1:0.8-1.2;

[0038] And / or, in step (2), the condensing agent is 1-ethyl-3 (3-dimethylpropylamine) carbodiimide and 4-dimethylaminopyridine; in terms of molar amount, compound 14: compound 10: EDCI: DMAP = 1: 1-1.5: 1.5-2.5: 1.5-2.5.

[0039] Furthermore, the reaction temperature of step (1) is -5 to 5°C and the reaction time is 10 to 14 hours;

[0040] And / or, the reaction temperature of step (2) is room temperature and the reaction time is 8 to 12 hours.

[0041] And / or, steps (1) and (2) further include a purification process.

[0042] A further improvement of the present invention is:

[0043] Application of a novel dual-target inhibitor of tubulin and metalloproteinase in the preparation of anti-lung cancer drugs.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The present invention introduces a new chalcone derivative into an aminophosphoric acid ester derivative to obtain a new dual-target inhibitor of tubulin and metalloproteinase as a compound with targeting function. Studies have shown that this type of dual-target inhibitor of tubulin and metalloproteinase has good anti-lung cancer activity, represented by compound 15j, which has stronger anti-proliferation ability against A549 cells than CDDP and DOX, and has lower toxicity against normal human liver cells LO2 cells and breast epithelial cells MCF-10A. In addition, the drug showed good inhibitory effects on cisplatin-resistant cells A549 / CDDP, doxorubicin-resistant cells MCF-7 / DOX and paclitaxel-resistant cells A549 / Paclitaxel. In vitro research results show that this type of dual-target inhibitor of tubulin and metalloproteinase has potential use in targeted treatment of lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the in vivo anti-tumor effect of compound 15j in the present invention; (A) images of excised tumors in each group, (B) tumor volume in each dosing group, (C) tumor mass in each dosing group, and (D) weight changes of mice in each group over 21 days. **p<0.01

[0047] Figure 2 This is the H&E staining of the main organs such as the heart, liver, spleen, lung, and kidney of mice after treatment with compound 15j in the example of the present invention for 21 days. DETAILED DESCRIPTION

[0048] The present invention is described in detail below in conjunction with specific embodiments.

[0049] Compound 10 used in the present invention was prepared according to the method reported in the literature (Eur. J. Med. Chem. 2020, 189, 112067).

[0050]

[0051] Among them, R1 is selected from H, F, Cl, NO2, OCH3; R2 is selected from H, CH3, F, Cl, Br, NO2, OCH3; R3 is selected from H, OCH3.

[0052] Example 1: Preparation of Compound 3

[0053]

[0054] 1-(4-methoxy-2-(2-methyl-3-alkyn-2-yl)oxy)phenyl)ethanone (3). Compound 1 (10 mmol) was dissolved in dry CH3CN (100 mL), and then DBU (10 mmol), CuCl2·2H2O (0.3 mol%) and compound 2 (15 mmol) were added at 0°C, stirred at the same temperature for 5 h, and monitored by thin layer chromatography. Then the pH was adjusted to pH=2 with 1N HCl solution, the solvent was removed under reduced pressure, and then a large amount of ice water was added to collect the corresponding precipitated solid. The obtained crude product was washed with petroleum ether and dried in vacuo to obtain compound 3. Yellow solid, 1.75 g, yield 78.8%. 1 HNMR (400MHz, CDCl3) δ7.75(d,J=8.8Hz,1H),7.18(d,J=2.4Hz,1H),6.58(dd,J=8.8,2.4Hz,1H),3.82(s,3H),2.67(s,1H),2.56(s,3H),1.74(s,6H).

[0055] Example 2 Preparation of Compound 4

[0056]

[0057] 1-(5-Methoxy-2,2-dimethyl-2H-propoxy-8-yl)ethanone (4). Compound 3 (10 mmol) was dissolved in 50 mL of dry pyridine, stirred at 120 ° C overnight, and the reaction was monitored by TLC. After the reaction was completed, the solvent was removed under reduced pressure, and then 1N HCl solution was added to adjust the pH to 2-3, and then dichloromethane (100 ml) was added, washed with saturated brine, and the organic phase was collected and dried over anhydrous Na2SO4. The solvent was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain compound 4. Yellow oil, 1.55 g, yield 69.8%. 1HNMR(400MHz, CDCl3)δ7.72(d,J=8.9Hz,1H),6.64(d,J=10.0Hz,1H),6.45(d, J=8.9Hz,1H),5.59(d,J=10.0Hz,1H),3.83(s,3H),2.59(s,3H),1.47(s,6H).

[0058] Example 3 Preparation of Compound 6

[0059]

[0060] (E)-3-(3-hydroxy-4-methoxyphenyl)-1-(5-methoxy-2,2-dimethyl-2H-propoxy-8-yl)prop-2-en-1-one (6). Compound 4 (6.46 mmol) and compound 5 (6.46 mmol) were dissolved in methanol (30 mL), and then a 50% aqueous solution of KOH (15 mL) was added dropwise to the reaction at 0°C, stirred overnight at the same temperature, and the reaction was monitored by thin layer chromatography. After the reaction was completed, the mixture was poured into a large amount of ice water, the pH was adjusted to 2-3 with 4N HCl solution, and the yellow precipitate was collected by filtration to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 6. Yellow solid, 1.69 g, yield 71.6%. 1 HNMR(400MHz,DMSO_d6)δ9.32(s,1H),7.55(d,J=8.8Hz,1H),7.45(s,2H),7.12–7.10(m,2H),6.99(d,J=8.9Hz,1 H),6.70(d,J=8.9Hz,1H),6.62(d,J=10.0Hz,1H),5.79(d,J=10.0Hz,1H),3.86(s,3H),3.81(s,3H),1.47(s,6H). 13 CNMR(125MHz,DMSO_d6)δ189.13,158.30,153.10,150.56,147.30,142.16,131.62,129.86,128.19,124.85,122 .02,121.66,116.51,114.05,112.58,110.31,104.67,77.26,56.43,56.07,27.99.HR-MS(m / z)(ESI):calcdfor C 22 H 22 O5[M+H] + :367.1545; found:367.1542.Purity:98.52%(byHPLC).

[0061] Example 4 Preparation of Compounds 11a-111

[0062]

[0063] Compound 10 (0.229 mmol) was dissolved in anhydrous dichloromethane (5 mL) solution, EDCI (0.382 mmol), DAMP (0.382 mmol) and compound 6 (0.191 mmol) were added, stirred at room temperature overnight, and monitored by TLC. After the reaction was completed, the reaction mixture was diluted with dichloromethane, then washed with brine, the organic phase was dried over anhydrous Na2SO4, and concentrated under reduced pressure. After the solvent was removed, the obtained crude product was purified by silica gel column chromatography to obtain the desired target products 11a-11l.

[0064] (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-propoxy-8-yl)-3-oxy-1-en-1-yl)phenyl 2-(4-((dioxyphosphino)(phenyl)methyl)amino)phenyl)acetate (11a). Yellow solid, 100 mg, yield 72.5%. 1 HNMR(500MHz,CDCl3)δ7.73(d,J=8.8Hz,1H),7.63–7.54(m,2H),7.52–7.47(m,2H),7.43(dd,J=8.5,2.0Hz,1H),7.36(t,J=7 .6Hz,2H),7.30(d,J=4.7Hz,1H),7.26(d,J=2.0Hz,1H),7.16(d,J=8.5Hz,2H),6.95(d,J=8.5Hz,1H),6.72(d,J=10.0Hz,1H) ,6.62(d,J=8.5Hz,2H),6.54(d,J=8.9Hz,1H),5.64(d,J=10.0Hz,1H),4.79(d,J=24.3Hz,1H),4.20–4.10(m,2H),4.00–3.94 (m,1H),3.91(s,3H),3.76(s,3H),3.74(s,2H),3.73–3.66(m,1H),1.47(s,6H),1.32(t,J=7.1Hz,3H),1.14(t,J=7.1Hz,3H). 13CNMR (125MHz, CDCl3) δ189.81,169.89,158.38,153.66,152.58,145.56,145.44,140.36,140 .16,135.84,131.67,130.14,128.79,128.74,128.66,128.64,128.03,128.00,127.89,127.8 5,127.52,126.22,123.20,122.01,121.70,116.75,114.02,112.40,110.46,103.47,63.41, 64.34,56.72,55.90,55.81,55.53,40.10,27.98,16.49,16.24.HR-MS(m / z)(ESI):calcdforC 41 H 44 NO9P[M+H] + :726.2832; found:726.2827.Purity:96.05%(byHPLC).

[0065] (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-propoxy-8-yl)-3-oxy-1-en-1-yl)phenyl 2-(4-((dioxyphosphino)(p-methyl)amino)phenyl)acetate (11b). Yellow solid, 80 mg, yield 56.7%. 1 HNMR (500MHz, CDCl3) δ7.69(d,J=8.8Hz,1H),7.59–7.51(m,2H),7.39(dd,J=8.5,1.9Hz,1H),7.35(d,J=1.5Hz,1H),7.33(d,J=1 .5Hz,1H),7.22(d,J=1.9Hz,1H),7.13(d,J=3.1Hz,2H),7.11(d,J=3.8Hz,2H),6.92(d,J=8.5Hz,1H),6.68(d,J=10.0Hz,1H),6.5 8(d,J=8.4Hz,2H),6.50(d,J=8.9Hz,1H),5.61(d,J=10.0Hz,1H),4.72(d,J=24.1Hz,1H),4.16–4.06(m,2H),3.96–3.91(m,1H),3 .88(s,3H),3.73(s,3H),3.72(s,2H),3.69–3.64(m,1H),2.30(s,3H),1.43(s,6H),1.29(t,J=7.1Hz,3H),1.13(t,J=7.1Hz,3H). 13CNMR (125MHz, CDCl3) δ189.81,169.91,158.38,153.66,152.59,145.63,145.51,140.36,140.1 7,137.72,137.69,132.67,131.67,130.12,129.38,129.37,128.79,128.73,127.76,127.72,1 27.54,126.21,123.11,122.01,121.70,116.74,114.03,112.39,110.46,103.47,63.36,63.29 ,56.41,55.90,55.81,55.21,40.10,27.98,21.16,16.50,16.28.HR-MS(m / z)(ESI):calcdforC 42 H 46 NO9P[M+H] + :740.2988; found:740.2992.Purity:98.03%(byHPLC).

[0066] (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-propoxy-8-yl)-3-oxy-1-en-1-yl)phenyl 2-(4-((dioxyphosphino)(4-methoxyphenyl)methyl)amino)phenyl)acetate (11c). Yellow solid, 120 mg, yield 83.3%. 1 HNMR(500MHz, CDCl3)δ7.69(d,J=8.8Hz,1H),7.59–7.51(m,2H),7.42–7.38(m,2H),7.36(d,J=1.5Hz,1H),7.22(d,J=1.9 Hz,1H),7.12(d,J=8.4Hz,2H),6.92(d,J=8.5Hz,1H),6.86(d,J=8.5Hz,2H),6.68(d,J=10.0Hz,1H),6.58(d,J=8.4Hz,2H ),6.50(d,J=8.9Hz,1H),5.61(d,J=10.0Hz,1H),4.70(d,J=23.8Hz,1H),4.13–4.06(m,2H),3.96–3.91(m,1H),3.87(s,3 H),3.76(s,3H),3.73(s,3H),3.72(s,2H),3.70–3.65(m,1H),1.43(s,6H),1.28(t,J=7.1Hz,3H),1.13(t,J=7.1Hz,3H). 13CNMR (125MHz, CDCl3) δ189.81,169.93,159.37,158.39,153.67,152.59,145.61,145.49 ,140.38,140.16,131.68,130.13,129.01,128.97,128.77,128.74,127.56,126.21,123. 13,122.01,121.68,116.75,114.10,114.06,112.41,110.46,103.49,63.38,63.30,56. 02,55.92,55.82,55.27,54.80,40.10,27.98,16.52,16.33.HR-MS(m / z)(ESI):calcdfor C 42 H 46 NO 10 P[M+H] + :756.2938; found:756.2943.Purity:96.34%(byHPLC).

[0067] (E)-2-methoxy-5-(3-(5-methoxy-2,2-dimethyl-2h-propoxy-8-yl)-3-oxy-1-en-1-yl)phenyl 2-(4-(dioxyphosphino)(4-fluorophenyl)methyl)amino)phenyl)acetate (11d). Yellow solid, 100 mg, yield 70.4%. 1 HNMR (400MHz, CDCl3) δ7.69(d,J=8.8Hz,1H),7.60–7.50(m,2H),7.42–7.39(m,3H),7.30(d,J=8.4Hz,2H),7.22(d,J= 2.0Hz,1H),7.13(d,J=8.4Hz,2H),6.92(d,J=8.5Hz,1H),6.68(d,J=10.0Hz,1H),6.55(d,J=8.4Hz,2H),6.50(d,J=8.9 Hz,1H),5.61(d,J=10.0Hz,1H),4.86–4.82(m,1H),4.73(dd,J=24.1,7.4Hz,1H),4.16–4.08(m,2H),4.01–3.95(m,1H) ,3.86(s,3H),3.82–3.76(m,1H),3.73(s,3H),3.70(s,2H),1.43(s,6H),1.29(d,J=7.1Hz,3H),1.15(t,J=7.1Hz,3H). 13CNMR (100MHz, CDCl3) δ189.79,169.84,158.38,153.63,152.56,145.30,145.15,140.33,140.1 2,134.58,134.55,133.80,133.76,131.67,130.21,129.20,129.15,128.85,128.83,128.77,1 28.70,127.49,126.21,123.48,122.04,121.67,116.76,114.00,112.41,110.44,103.48,63.5 1,63.38,56.33,55.90,55.80,54.83,40.05,27.97,16.50,16.31.HR-MS(m / z)(ESI):calcdforC 41 H 43 NFO9P[M+H] + :744.2737; found:744.2738.Purity:97.87%(byHPLC).

[0068] (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-propoxy-8-yl)-3-oxy-1-en-1-yl)phenyl 2-(4-((4-chlorophenyl)(dioxyphosphino)methyl)amino)phenyl)acetate (11e). Yellow solid, 85 mg, yield 70.4%. 1 HNMR(500MHz, CDCl3)δ7.69(d,J=8.8Hz,1H),7.59–7.51(m,2H),7.46–7.43(m,2H),7.40(dd,J=8.5,2.0Hz,1H),7.22( d,J=2.0Hz,1H),7.13(d,J=8.4Hz,2H),7.02(t,J=8.5Hz,2H),6.92(d,J=8.5Hz,1H),6.69(d,J=10.0Hz,1H),6.56(d,J= 8.5Hz,2H),6.50(d,J=8.9Hz,1H),5.61(d,J=10.0Hz,1H),4.73(d,J=24.1Hz,1H),4.16–4.07(m,2H),3.99–3.94(m,1H ),3.87(s,3H),3.78–3.75(m,1H),3.74(s,3H),3.73(s,2H),1.44(s,6H),1.28(t,J=7.1Hz,3H),1.14(t,J=7.1Hz,3H). 13CNMR (125MHz, CDCl3) δ189.81,169.87,163.47,161.51,158.39,153.64,152.57,145.36,145 .24,140.35,140.14,131.67,130.20,129.51,129.47,129.41,128.79,128.71,127.49,126. 23,123.42,122.05,121.68,116.77,115.73,115.56,114.01,112.43,110.46,103.49,63.44 ,63.34,56.05,55.91,55.81,54.84,40.06,27.98,16.49,16.29.HR-MS(m / z)(ESI):calcdfor C 41 H 43 NClO9P[M+H] + :760.2442; found:760.2444.Purity:97.61%(byHPLC).

[0069] (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-propoxy-8-yl)-3-oxy-1-en-1-yl)phenyl 2-(4-((4-bromophenyl)(dioxyphosphino)methyl)amino)phenyl)acetate (11f). Yellow solid, 140 mg, yield 91.5%. 1 HNMR (500MHz, CDCl3) δ7.69(d,J=8.8Hz,1H),7.59–7.51(m,2H),7.46(d,J=8.3Hz,2H),7.41(dd,J=8.5,2.0Hz,1H),7.36(d,J =2.1Hz,1H),7.34(d,J=2.1Hz,1H),7.23(d,J=2.0Hz,1H),7.13(d,J=8.4Hz,2H),6.93(d,J=8.5Hz,1H),6.69(d,J=10.0Hz,1H ),6.55(d,J=8.5Hz,2H),6.51(d,J=8.9Hz,1H),5.62(d,J=10.0Hz,1H),4.71(d,J=24.4Hz,1H),4.16–4.07(m,2H),4.01–3.95 (m,1H),3.88(s,3H),3.81–3.75(m,1H),3.74(s,3H),3.73(s,2H),1.44(s,6H),1.29(t,J=7.1Hz,3H),1.16(t,J=7.1Hz,3H). 13CNMR (125MHz, CDCl3) δ189.86,169.87,158.39,153.65,152.56,145.25,145.13,140.37,14 0.12,135.08,131.81,131.79,131.68,130.23,129.54,129.50,128.79,128.73,127.50,126 .22,123.53(s),122.08,121.97,121.94,121.69,116.78,114.02,112.42,110.46,63.55,6 3.43,56.28,55.92,55.83,55.08,40.07,27.99,16.51,16.32.HR-MS(m / z)(ESI):calcdforC 41 H 43 NBrO9P[M+H] + :804.1937; found:804.1939.Purity:96.84%(byHPLC).

[0070] (E)-2-methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-propoxy-8-yl)-3-oxy-1-en-1-yl)phenyl 2-(4-(dioxyphosphino)(4-nitrophenyl)methyl)amino)phenyl)acetate (11 g). Yellow solid, 90 mg, yield 61.2%. 1 HNMR(500MHz, CDCl3)δ8.20(d,J=8.6Hz,2H),7.68(t,J=5.7Hz,2H),7.65(d,J=2.1Hz,1H),7.58–7.50(m,2H),7.41( dd,J=8.5,1.8Hz,1H),7.21(d,J=1.9Hz,1H),7.14(d,J=8.4Hz,2H),6.93(d,J=8.5Hz,1H),6.69(d,J=10.0Hz,1H),6. 53(d,J=8.4Hz,2H),6.51(d,J=8.9Hz,1H),5.61(d,J=10.0Hz,1H),4.85(d,J=25.1Hz,1H),4.18–4.09(m,2H),4.06– 4.00(m,1H),3.91–3.85(m,4H),3.76(s,3H),3.73(s,2H),1.44(s,6H),1.30(t,J=7.1Hz,3H),1.18(t,J=7.1Hz,3H). 13CNMR (125MHz, CDCl3) δ189.82,169.78,158.40,153.62,152.53,147.67,144.94,144.8 3,144.06,140.32,140.09,131.67,130.35,128.80,128.72,128.69,127.38,126.26,1 23.97,123.82,122.15,121.67,116.79,113.98,112.47,110.45,103.51,63.84,63.59 ,56.67,55.94,55.83,55.49,39.98,27.99,16.49,16.32.HR-MS(m / z)(ESI):calcdforC 41 H 43 N2O 11 P[M+H] + :771.2682; found:771.2684.Purity:98.22%(byHPLC).

[0071] (E)-2-methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-propoxy-8-yl)-3-oxy-1-en-1-yl)phenyl 2-(4-((dioxyphosphino)(3-fluorophenyl)methyl)amino)phenyl)acetate (11h). Yellow solid, 110 mg, yield 77.5%. 1 HNMR (500MHz, CDCl3) δ7.69(d,J=8.8Hz,1H),7.59–7.51(m,2H),7.40(dd,J=8.5,2.0Hz,1H),7.32–7.25(m,3H),7.23(d,J=2. 0Hz,1H),7.19(dd,J=9.5,1.2Hz,1H),7.14(d,J=8.4Hz,2H),6.96(t,J=8.1Hz,1H),6.92(d,J=8.5Hz,1H),6.69(d,J=10.0Hz, 1H),6.57(d,J=8.4Hz,2H),6.51(d,J=8.9Hz,1H),5.61(d,J=5.7Hz,1H),4.74(d,J=24.5Hz,1H),4.17–4.08(m,2H),4.01–3.9 3(m,1H),3.88(s,3H),3.81–3.76(m,1H),3.74(s,3H),3.73(s,2H),1.44(s,6H),1.29(t,J=7.1Hz,3H),1.15(t,J=7.1Hz,3H). 13CNMR(125MHz, CDCl3)δ189.81,169.84,158.38,153.65,152.57,145.30,145.18,140.35, 140.15,138.79,131.67,130.22,128.81,128.72,127.48,126.24,123.53,122.06,121.7 1,116.76,115.10,114.89,114.72,114.67,113.98,112.41,110.46,103.47,63.52,63.4 7,56.44,55.92,55.81,55.25,40.08,27.98,16.48,16.26.HR-MS(m / z)(ESI):calcdforC 41 H 43 FN2O9P[M+H] + :744.2737; found:744.2733.Purity:97.15%(byHPLC).

[0072] (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-propoxy-8-yl)-3-oxy-1-en-1-yl)phenyl 2-(4-((3-chlorophenyl)(dioxyphosphino)methyl)amino)phenyl)acetate (11i). Yellow solid, 102 mg, yield 70.3%. 1 HNMR (500MHz, CDCl3) δ7.67(d,J=8.8Hz,1H),7.56–7.49(m,2H),7.44(d,J=1.1Hz,1H),7.38(dd,J=8.5,1.9Hz,1H),7.34(dd,J=6.8, 1.2Hz,1H),7.24(d,J=3.2Hz,1H),7.23(d,J=2.0Hz,1H),7.20(d,J=2.0Hz,1H),7.12(d,J=8.4Hz,2H),6.90(d,J=8.5Hz,1H),6.66(d ,J=10.0Hz,1H),6.54(d,J=8.5Hz,2H),6.48(d,J=8.9Hz,1H),5.59(d,J=10.0Hz,1H),4.70(d,J=24.5Hz,1H),4.15–4.06(m,2H),4.0 0–3.92(m,1H),3.85(s,3H),3.79–3.74(m,1H),3.72(s,3H),3.71(s,2H),1.41(s,6H),1.27(t,J=7.1Hz,3H),1.13(t,J=7.1Hz,3H).13 CNMR(125MHz, CDCl3)δ189.83,169.87,158.39,153.65,152.58,145.26,145.14,140.37,140.15, 138.30,134.61,134.59,131.67,130.25,129.93,128.78,128.73,128.25,127.94,127.90,127.50 ,126.23(s),126.02,125.98,123.54,122.05,121.69,116.76,113.97,112.42,110.47,103.49,6 3.56,63.50,56.39,55.92,55.82,55.20,40.08,27.99,16.48,16.25.HR-MS(m / z)(ESI):calcdfor C 41 H 43 ClN2O9P[M+H] + :760.2442; found:760.2439.Purity:98.39%(byHPLC).

[0073] (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-propoxy-8-yl)-3-oxy-1-en-1-yl)phenyl 2-(4-((dioxyphosphino)(3-nitrophenyl)methyl)amino)phenyl)acetate (11j). Yellow solid, 120 mg, yield 81.6%. 1HNMR(500MHz,CDCl3)δ8.34(d,J=1.8Hz,1H),8.14(d,J=8.1Hz,1H),7.83(d,J=7.6Hz,1H),7.68(d,J=8.8Hz,1H),7.54(d,J=6.1Hz,2H),7.51(d,J=8.2Hz,1H),7.41(dd,J=8.5,2.0Hz,1H),7.21(d,J=2.0Hz,1H),7.14(d,J=8.5Hz,2H),6.92(d,J=8.5Hz,1H),6.68(d,J=10.0Hz,1H),6.56(d,J=8.5Hz,2H),6.50(d,J=8.9Hz,1H),5.61(d,J=10.0Hz,1H),4.86(d,J=24.7Hz,1H),4.19–4.11(m,2H),4.06–4.02(m,1H),3.94–3.89(m,1H),3.87(s,3H),3.75(s,3H),3.73(s,2H),1.44(s,6H),1.30(t,J=7.1Hz,3H),1.18(t,J=7.1Hz,3H). 13 CNMR(125MHz,CDCl3)δ189.82,169.78,158.39,153.63,152.54,148.51,144.90,144.79,140.33,140.11,138.82,133.81,133.77,131.67,130.36,129.67,128.80,128.70,127.39,126.25,123.96,123.06,122.80,122.76,122.13,121.69,116.78,113.98,112.45,110.46,103.49,63.82,63.54,56.34,55.94,55.82,55.15,40.00,27.99,16.49,16.29.HR-MS(m / z)(ESI):calcdforC 41 H 43 N2O 11 P[M+H] + :771.2682;found:771.2684.Purity:95.63%(byHPLC).

[0074] (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-propoxy-8-yl)-3-oxy-1-en-1-yl)phenyl 2-(4-((dioxyphosphino)(3,4-dimethoxyphenyl)methyl)amino)phenyl)acetate (11k). Yellow solid, 129 mg, yield 86%. 1 HNMR (500MHz, CDCl3) δ7.69(d,J=8.8Hz,1H),7.59–7.51(m,2H),7.40(dd,J=8.5,2.1Hz,1H),7.23(d,J=2.0Hz,1H),7.13(d,J =8.4Hz,2H),7.02–7.00(m,2H),6.92(d,J=8.6Hz,1H),6.82(d,J=8.7Hz,1H),6.68(d,J=10.0Hz,1H),6.60(d,J=8.3Hz,2H),6 .50(d,J=8.9Hz,1H),5.61(d,J=10.0Hz,1H),4.68(d,J=23.7Hz,1H),4.16–4.06(m,2H),3.98–3.93(m,1H),3.88(s,3H),3.87 (s,3H),3.84(s,3H),3.74(s,3H),3.73(s,2H),3.72–3.67(m,1H),1.44(s,6H),1.29(t,J=7.1Hz,3H),1.14(t,J=7.1Hz,3H). 13 CNMR (125MHz, CDCl3) δ189.79,169.92,158.38,153.64,152.58,149.12,148.76,145.69,145.57,1 40.36,140.14,131.67,130.14,128.76,128.72,128.12,127.49,126.20,123.22,122.05,121.66, 120.26,120.21,116.75,114.05,112.42,111.12,110.79,110.76,110.44,103.48,63.39,63.31,5 6.44,55.96,55.90,55.87,55.80,55.23,40.08,27.98,16.53,16.37.HR-MS(m / z)(ESI):calcdforC 43 H 48 NO 11 P[M+H] +:786.3043; found:786.3053.Purity:97.43%(byHPLC).

[0075] (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-propoxy-8-yl)-3-oxy-1-en-1-yl)phenyl 2-(4-((dioxyphosphino)(3,4,5-trimethoxyphenyl)methyl)amino)phenyl)acetate (111). Yellow solid, 135 mg, yield 86.5%. 1 HNMR(500MHz, CDCl3)δ7.68(d,J=8.8Hz,1H),7.59–7.51(m,2H),7.41(dd,J=8.5,2.0Hz,1H),7.23(d,J=2.0Hz,1H),7.15 (d,J=8.4Hz,2H),6.92(d,J=8.5Hz,1H),6.70(d,J=2.1Hz,2H),6.68(d,J=10.0Hz,1H),6.61(d,J=8.5Hz,2H),6.50(d,J= 8.9Hz,1H),5.61(d,J=10.0Hz,1H),4.66(d,J=23.9Hz,1H),4.16–4.07(m,2H),4.00–3.93(m,1H),3.87(s,3H),3.84(s,6 H),3.81(s,3H),3.75(s,2H),3.74(s,3H),3.72–3.68(m,1H),1.45(s,6H),1.28(t,J=7.1Hz,3H),1.15(t,J=7.1Hz,3H). 13 CNMR (100MHz, CDCl3) δ189.82,169.92,158.37,153.62,153.39,153.36,152.55,145.70,145.55,1 40.35,140.13,137.64,137.61,131.65,131.51,131.48,130.19,128.79,128.70,127.38,126.22, 123.34,122.14,121.67,116.75,113.99,112.43,110.44,104.79,104.75,103.47,63.44,63.35,6 0.86,57.20,56.18,55.90,55.79,55.70,40.04,27.98,16.52,16.34.HR-MS(m / z)(ESI):calcdforC 44 H 50 NO 12P[M+H] + :816.3148; found:816.3155.Purity:98.23%(byHPLC).

[0076] Example 5: Preparation of Compound 14

[0077]

[0078] (E)-3-(3-Hydroxy-4-ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)acetone-2-ene-1-one (14). The synthesis process of compound 14 is the same as that of compound 6. Yellow solid, 2.4 g, yield 69.8%. 1 HNMR(500MHz, CDCl3)δ7.75(d,J=15.5Hz,1H),7.36(d,J=15.5Hz,1H),7.31(s,1H),7.28 (s,2H),7.14(d,J=8.3Hz,1H),6.88(d,J=8.3Hz,1H),3.95(d,J=0.9Hz,6H),3.94(s,6H). 13 CNMR (125MHz, CDCl3) δ189.18,153.14,148.95,145.94,144.77,142.29,133.77,128.52,1 23.06,119.80,112.82,110.63,105.94,61.02,56.39,56.06.HR-MS(m / z)(ESI):calcdforC 19 H 20 O6[M+H] + :345.1338; found:345.1336.Purity:99.73%(byHPLC).

[0079] Example 6: Preparation of Compounds 15a-15l

[0080]

[0081] EDCI (0.408 mmol), DAMP (0.408 mmol) and compound 14 (0.204 mmol) were added to a solution of compound 10 (0.245 mmol) in anhydrous dichloromethane (5 mL) and stirred overnight at room temperature. The reaction process was monitored by thin layer chromatography. After the reaction was completed, the reaction mixture was diluted with dichloromethane, then washed with saturated brine and dried over anhydrous sodium sulfate. After removing the solvent, the obtained crude product was purified by silica gel column chromatography to obtain the desired target products 15a-15l.

[0082] (E)-2-Methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenyl 2-(4-((dioxyphosphino)(phenyl)methyl)amino)phenyl)acetate (15a). Yellow solid, 120 mg, yield 83.9%. 1 HNMR(500MHz, CDCl3)δ7.71(d,J=15.6Hz,1H),7.48–7.46(m,2H),7.43(dd,J=8.5,2.0Hz,1H),7.35–7.3 3(m,2H),7.31(d,J=4.3Hz,1H),7.27(d,J=4.3Hz,2H),7.25(s,2H),7.14(d,J=8.5Hz,2H),6.93(d,J=8.5 Hz,1H),6.60(d,J=8.5Hz,2H),4.76(d,J=24.3Hz,1H),4.17–4.07(m,2H),3.95(s,6H),3.94(s,3H),3.93 –3.89(m,1H),3.74(s,2H),3.73(s,3H),3.70–3.64(m,1H),1.29(t,J=7.1Hz,3H),1.11(t,J=7.1Hz,3H). 13 CNMR (125MHz, CDCl3) δ189.13,170.07,153.16,145.61,145.49,143.79,142 .38,140.29,135.84,133.68,130.22,128.64,128.03,127.96,127.90,127.8 6,122.99,121.87,120.38,114.02,112.32,106.04,63.43,63.34,61.00,56 .69,56.45,55.93,55.49,40.02,16.49,16.23.HR-MS(m / z)(ESI):calcdforC 38 H 42 NO 10 P[M+H] + :704.2625; found:704.2624.Purity:97.02%(byHPLC).

[0083] (E)-2-Methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenyl 2-(4-((dioxyphosphino)(p-methyl)amino)phenyl)acetate (15b). Yellow solid, 110 mg, yield 75.3%. 1HNMR (500MHz, CDCl3) δ7.70(d,J=15.5Hz,1H),7.42(d,J=8.4Hz,1H),7.34(d,J=1.9Hz,1H),7.33(d,J= 1.6Hz,2H),7.28(d,J=15.6Hz,1H),7.24(s,2H),7.12(d,J=8.2Hz,4H),6.92(d,J=8.5Hz,1H),6.59(d, J=8.5Hz,2H),4.72(d,J=24.1Hz,1H),4.15–4.05(m,2H),3.98–3.95(m,1H),3.93(s,6H),3.92(s,3H), 3.73(s,2H),3.72(s,3H),3.70–3.64(m,1H),2.29(s,3H),1.28(t,J=7.1Hz,3H),1.12(t,J=7.1Hz,3H). 13 CNMR (125MHz, CDCl3) δ189.11,170.07,153.16,145.70,145.58,143.78,142.39 ,140.30,137.72,137.69,133.68,132.66,130.20,129.37,128.64,127.97,127. 78,122.90,121.87,120.37,114.03,112.32,106.05,63.37,63.28,60.99,56.45 ,56.39,55.92,55.19,40.02,21.15,16.49,16.27.HR-MS(m / z)(ESI):calcdforC 39 H 44 NO 10 P[M+H] + :718.2781; found:718.2784.Purity:96.07%(byHPLC).

[0084] (E)-2-Methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenyl 2-(4-(dioxyphosphino)(4-methoxyphenyl)methyl)amino)phenyl)acetate (15c). Yellow solid, 130 mg, yield 87.2%. 1HNMR(500MHz, CDCl3)δ7.72(d,J=15.6Hz,1H),7.43(dd,J=19.7,7.6Hz,3H),7.36(s,1H),7.32(d,J=15.8H z,1H),7.27(s,2H),7.16(d,J=8.1Hz,2H),6.94(dd,J=8.5,3.2Hz,1H),6.88(d,J=8.3Hz,2H),6.63(d,J=8. 3Hz,2H),4.88–4.85(m,1H),4.74(dd,J=23.8,5.5Hz,1H),4.16–4.10(m,2H),3.95(s,9H),3.91–3.85(m,1 H),3.78(s,3H),3.76(s,2H),3.74(s,3H),3.71–3.67(m,1H),1.31(t,J=7.1Hz,3H),1.16(t,J=7.1Hz,3H). 13 CNMR (125MHz, CDCl3) δ189.08,170.09,159.38,153.16,145.69,145.57,143.77 ,142.37,140.28,133.67,130.21,129.02,128.98,128.67,127.97,127.61,122. 92,121.88,120.34,114.09,114.05,112.33,106.04,63.37,63.27,61.00,56.44 ,56.00,55.93,55.26,54.79,40.01,16.52,16.33.HR-MS(m / z)(ESI):calcdforC 39 H 44 NO 11 P[M+H] + :734.2730; found:734.2736.Purity:97.28%(byHPLC).

[0085] (E)-2-Methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenyl 2-((dioxyphosphino)(4-nitrophenyl)methyl)amino)phenyl)acetate (15d). Yellow solid, 140 mg, yield 91.5%. 1HNMR(400MHz, CDCl3)δ8.20(d,J=8.2Hz,2H),7.72–7.66(m,3H),7.44(d,J=8.2Hz,1H ),7.30(d,J=17.8Hz,2H),7.25(s,2H),7.15(d,J=8.0Hz,2H),6.94(d,J=8.4Hz,1H), 6.55(d,J=8.0Hz,2H),4.97–4.83(m,2H),4.18–4.12(m,2H),4.07–4.01(m,1H),3.94 (s9H),3.90–3.85(m,1H),3.76(s,5H),1.30(t,J=7.1Hz,3H),1.19(t,J=7.1Hz,3H). 13 CNMR(100MHz, CDCl3)δ189.12(s),169.94(s),153.14,153.08,147.65,147.62,144. 97,144.83,144.01,143.72,142.38,140.18,133.64,130.41,128.70,128.65,128.5 2,127.98,123.81,123.78,121.95,120.42,113.96,112.36,106.04,63.83,63.59,6 1.00,56.78,56.44,55.96,55.30,39.87,16.48,16.30.HR-MS(m / z)(ESI):calcdfor C 38 H 41 N2O 12 P[M+H] + :749.2475; found:749.2476.Purity:97.40%(byHPLC).

[0086] (E)-2-Methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenyl 2-(4-((4-bromophenyl)(dioxyphosphino)methyl)amino)phenyl)acetate (15e). Yellow solid, 138 mg, yield 86.8%. 1HNMR(400MHz, CDCl3)δ7.70(d,J=15.5Hz,1H),7.45–7.42(m,3H),7.37–7.34(m,3H),7.30(d,J=1 5.4Hz,1H),7.25(s,2H),7.14(d,J=8.1Hz,2H),6.92(dd,J=8.5,2.5Hz,1H),6.57(d,J=8.5Hz,2H ),4.90–4.86(m,1H),4.72(dd,J=24.3,7.1Hz,1H),4.17–4.08(m,2H),4.02–3.96(m,1H),3.93(s ,9H),3.83–3.77(m,1H),3.75(s,2H),3.72(s,3H),1.29(t,J=7.1Hz,3H),1.16(t,J=7.1Hz,3H). 13 CNMR(100MHz,CDCl3)δ189.08,170.03,153.16,145.33,145.22,143.75,142.3 8,140.25,135.14,133.67,131.78,130.30,129.57,129.53,128.65,127.98,1 23.30,121.94,121.90,120.36,114.02,112.34,106.05,63.53,63.47,61.01, 56.45,56.25,55.93,55.05,39.97,16.50,16.32.HR-MS(m / z)(ESI):calcdforC 38 H 41 BrNO 10 P[M+H] + :782.1730; found:782.1739.Purity:97.80%(byHPLC).

[0087] (E)-2-Methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenyl 2-(4-((dioxyphosphino)(m-methyl)methyl)amino)phenyl)acetate (15f). Yellow solid, 126 mg, yield 86.3%. 1HNMR(500MHz, CDCl3)δ7.74(d,J=15.5Hz,1H),7.46(dd,J=8.5,1.9Hz,1H),7.37(d,J=2.0Hz,1H),7.34–7.30 (m,3H),7.28(s,2H),7.24(t,J=7.5Hz,1H),7.17(d,J=8.3Hz,2H),7.10(d,J=7.3Hz,1H),6.96(d,J=8.5Hz,1H ),6.64(d,J=8.4Hz,2H),4.75(d,J=24.2Hz,1H),4.19–4.11(m,2H),3.98(s,6H),3.97(s,3H),3.965–3.91(m ,1H),3.78(s,2H),3.76(s,3H),3.72–3.66(m,1H),2.36(s,3H),1.32(t,J=7.1Hz,3H),1.14(t,J=7.1Hz,3H). 13 CNMR (125MHz, CDCl3) δ189.10,170.09,153.15,145.72,145.60,143.77,142.38,140.2 9,138.26,135.73,133.68,130.22,128.84,128.82,128.65,128.50,128.46,127.97,1 25.01,124.97,122.90,121.86,120.36,113.98,112.32,106.04,63.42,63.27,60.99, 56.66,56.44,55.91,55.46,40.02,21.49,16.48,16.21.HR-MS(m / z)(ESI):calcdforC 39 H 44 NO 10 P[M+H] + :718.2781; found:718.2792.Purity:96.83%(byHPLC).

[0088] (E)-2-Methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenyl 2-(4-((dioxyphosphino)(3-methoxyphenyl)methyl)amino)phenyl)acetate (15 g). Yellow solid, 140 mg, yield 93.3%. 1HNMR (500MHz, CDCl3) δ7.74(d,J=15.5Hz,1H),7.47(dd,J=8.5,2.0Hz,1H),7.37(d,J=2.0Hz,1H),7.33(d,J=15.6Hz,1 H),7.30(s,1H),7.28(s,2H),7.17(d,J=8.5Hz,2H),7.08(d,J=7.5Hz,1H),7.05(d,J=1.6Hz,1H),6.97(d,J=8.5Hz,1H) ,6.84(d,J=8.2Hz,1H),6.64(d,J=8.5Hz,2H),4.76(d,J=24.3Hz,1H),4.20–4.12(m,2H),4.02–3.99(m,1H),3.98(s,6H ),3.97(s,3H),3.81(s,3H),3.79(s,2H),3.78(s,3H),3.76–3.69(m,1H),1.33(t,J=7.1Hz,3H),1.17(t,J=7.1Hz,3H). 13 CNMR (125MHz, CDCl3) δ189.17,170.10,159.85,153.15,145.66,145.54,143.81,142 .38,140.29,137.49,133.68,130.22,129.63,128.64,127.96,123.01,121.87,120. 39,120.29,120.25,114.01,113.52,113.47,112.33,106.05,63.50,63.38,61.00,5 6.72,56.45,55.94,55.52,55.26,40.02,16.49,16.26.HR-MS(m / z)(ESI):calcdforC 39 H 44 NO 11 P[M+H] + :734.2730; found:734.2736.Purity:97.09%(byHPLC).

[0089] (E)-2-Methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenyl 2-(4-((dioxyphosphino)(3-fluorophenyl)methyl)amino)phenyl)acetate (15h). Yellow solid, 90 mg, yield 61.2%. 1HNMR(500MHz, CDCl3)δ7.75(d,J=15.5Hz,1H),7.47(dd,J=8.5,1.7Hz,1H),7.38(d,J=1.9Hz,1H),7.35–7.31(m ,3H),7.29(s,2H),7.23(d,J=9.5Hz,1H),7.19(d,J=8.4Hz,2H),7.01(d,J=8.1Hz,1H),6.97(d,J=8.6Hz,1H),6. 62(d,J=8.5Hz,2H),4.92–4.88(m,1H),4.79(dd,J=24.4,7.4Hz,1H),4.21–4.12(m,2H),4.05–3.99(m,1H),3.98 (s,6H),3.97(s,3H),3.88–3.82(m,1H),3.79(s,2H),3.78(s,3H),1.33(t,J=7.1Hz,3H),1.19(t,J=7.1Hz,3H). 13 CNMR(125MHz,CDCl3)δ189.13,170.03,164.02,162.05,153.16,145.36,145.24,1 43.78,142.40,140.28,138.81,133.67,130.29,128.62,127.98,123.55,123.31,1 21.88,120.40,115.09,114.89,114.67,113.98,112.34,106.05,63.52,63.46,61. 00,56.46,56.41,55.94,55.21,40.00,16.47,16.25.HR-MS(m / z)(ESI):calcdforC 38 H 41 FNO 10 P[M+H] + :722.2530; found:722.2531.Purity:96.03%(byHPLC).

[0090] (E)-2-Methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenyl 2-(4-((3-chlorophenyl)(dioxyphosphino)methyl)amino)phenyl)acetate (15i). Yellow solid, 95 mg, yield 64.2%. 1HNMR(500MHz, CDCl3)δ7.73(d,J=15.5Hz,1H),7.49–7.45(m,2H),7.39(d,J=7.1Hz,1H),7.37(d, J=1.5Hz,1H),7.33–7.29(m,3H),7.27(s,2H),7.18(d,J=8.3Hz,2H),6.96(d,J=8.5Hz,1H),6.61( d,J=8.4Hz,2H),4.75(d,J=24.5Hz,1H),4.21–4.11(m,2H),4.04–3.99(m,1H),3.97(s,6H),3.96( s,3H),3.92–3.81(m,1H),3.78(s,2H),3.77(s,3H),1.32(t,J=7.1Hz,3H),1.18(t,J=7.1Hz,3H). 13 CNMR(125MHz,CDCl3)δ189.13,170.03,153.16,145.31,145.19,143.78,142.39,1 40.28,138.30,134.62,133.68,130.32,129.92,128.63,128.24,127.98,127.94,1 27.69,126.04,123.34,121.89,120.40,113.97,112.34,106.05,63.55,63.49,61. 01,56.46,56.37,55.94,55.17,40.01,16.47,16.24.HR-MS(m / z)(ESI):calcdforC 38 H 41 ClNO 10 P[M+H] + :728.2235; found:728.2236.Purity:97.19%(byHPLC).

[0091] (E)-2-Methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenyl 2-(4-((dioxyphosphino)(3-nitrophenyl)methyl)amino)phenyl)acetate (15j). Yellow solid, 125 mg, yield 81.7%. 1HNMR (500MHz, CDCl3) δ8.33(d,J=1.6Hz,1H),8.13(d,J=8.0Hz,1H),7.83(d,J=7.6Hz,1H),7.69(d,J=15.5Hz,1H),7. 52(t,J=7.9Hz,1H),7.43(dd,J=8.5,1.8Hz,1H),7.32(d,J=1.9Hz,1H),7.28(d,J=15.6Hz,1H),7.23(s,2H),7.14(d, J=8.4Hz,2H),6.93(d,J=8.5Hz,1H),6.57(d,J=8.5Hz,2H),4.88–4.81(m,2H),4.19–4.10(m,2H),4.07–4.01(m,1H), 3.93(s,6H),3.92(s,3H),3.91–3.83(m,1H),3.75(s,3H),3.72(s,2H),1.29(t,J=7.1Hz,3H),1.17(t,J=7.1Hz,3H). 13 CNMR (125MHz, CDCl3) δ189.17,169.98,153.16,148.51,144.96,144.85,143.76, 142.41,140.22,138.80,133.83,133.66,130.43,129.66,128.56,127.99,123.7 5,123.06,122.79,121.94,120.44,113.98,112.38,106.08,63.85,63.56,61.01 ,56.46,56.30,55.97,55.11,39.92,16.48,16.28.HR-MS(m / z)(ESI):calcdforC 38 H 41 N2O 12 P[M+H] + :749.2475; found:749.2478.Purity:97.11%(byHPLC).

[0092] (E)-2-Methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenyl 2-(4-((dioxyphosphino)(3,4-dimethoxyphenyl)methyl)amino)phenyl)acetate (15k). Yellow solid, 126 mg, yield 80.8%. 1HNMR (500MHz, CDCl3) δ7.71(d,J=15.5Hz,1H),7.44(dd,J=8.5,1.9Hz,1H),7.34(d,J=2.0Hz,1H),7.29(d,J=15. 6Hz,1H),7.24(s,2H),7.14(d,J=8.4Hz,2H),7.03–6.99(m,2H),6.94(d,J=8.5Hz,1H),6.82(d,J=8.7Hz,1H),6. 61(d,J=8.4Hz,2H),4.68(d,J=23.7Hz,1H),4.16–4.06(m,2H),4.01–3.95(m,1H),3.94(s,6H),3.93(s,3H),3.8 6(s,3H),3.84(s,3H),3.75(s,3H),3.73(s,2H),3.71–3.66(m,1H),1.29(t,J=7.1Hz,3H),1.14(t,J=7.1Hz,3H). 13 CNMR (125MHz, CDCl3) δ189.10,170.09,153.14,149.12,148.77,145.74,145.62,143 .77,142.35,140.26,133.66,130.22,128.62,128.11,127.96,123.01,121.90,120. 35,120.27,120.22,114.04,112.33,111.11,110.80,106.02,63.40,63.30,60.99,5 6.43,55.96,55.92,55.87,55.21,39.99,16.52,16.36.HR-MS(m / z)(ESI):calcdforC 40 H 46 NO 12 P[M+Na] + :786.2655; found:786.2662.Purity:96.45%(byHPLC).

[0093] (E)-2-Methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenyl 2-(4-((dioxyphosphino)(3,4,5-trimethoxyphenyl)methyl)amino)phenyl)acetate (151). Yellow solid, 150 mg, yield 92.6%. 1HNMR (500MHz, CDCl3) δ7.71(d,J=15.5Hz,1H),7.44(dd,J=8.5,1.8Hz,1H),7.34(d,J=1.9Hz,1H),7.29(d,J =15.6Hz,1H),7.24(s,2H),7.16(d,J=8.2Hz,2H),6.94(d,J=8.5Hz,1H),6.70(d,J=1.2Hz,2H),6.62(d,J=8 .3Hz,2H),4.66(d,J=23.9Hz,1H),4.16–4.07(m,2H),4.01–3.96(m,1H),3.94(s,6H),3.93(s,3H),3.84(s, 6H),3.81(s,3H),3.77(s,2H),3.75(s,3H),3.73–3.67(m,1H),1.29(t,J=7.0Hz,3H),1.15(t,J=7.0Hz,3H). 13 CNMR(125MHz,CDCl3)δ189.13,170.08,153.40,153.15,145.75,145.63,143.77,1 42.40,140.27,137.67,133.66,131.50,130.27,128.55,127.99,123.16,121.94, 120.42,114.00,112.37,106.05,104.81,104.77,63.45,63.34,61.00,60.86,57. 04,56.45,56.19,55.94,55.84,39.88,16.52,16.34.HR-MS(m / z)(ESI):calcdfor C 41 H 48 NO 13 P[M+H] + :794.2941; found:794.2938.Purity:96.54%(byHPLC).

[0094] Example 7: In vitro antiproliferative activity assay

[0095] 1. Experimental Methods

[0096] The CCK-8 detection kit was used to detect the cytotoxicity of two tubulin / metalloproteinase inhibitors (11a-111 and 15a-151 obtained by chalcone derivatives or 1a coupled to aminophosphonate derivatives) against four human cancer cell lines (A549 (lung), NCI-H460 (lung), MDA-MB-231 (breast), MCF-7 (breast)). First, the antiproliferative activities of chalcone derivatives 11a-111 and 15a-151 containing aminophosphonic acid derivatives are shown in Table 2, with the lead compound chalcone, compound 6, cisplatin (CDDP) and doxorubicin (DOX) as positive controls. All cells were seeded in 96-well plates (3000 cells per well, 200 μL) and incubated overnight at 37°C in a humid environment containing 5% CO2. The cells were then treated with different concentrations of the test compounds under the same conditions for 48 hours. After 48 h of incubation, fresh CCK-8 solution (10 μL) was added and incubated at 37 °C for 2 h. The samples were recorded at a wavelength of 450 nm using a microplate reader, and the IC was calculated using SPSS software. 50 value.

[0097] Table 2 Antiproliferative activity of compounds 11a-111 and 15a-151 against different cancer cell lines

[0098]

[0099]

[0100] IC 50 ±SD(μM) a , half-inhibitory concentration, values ​​are expressed as the mean ± SD of three independent experiments. 1a / 10-2j b , an equimolar mixture of 1a and 10-2j. CDDP c , cisplatin. DOX d , doxorubicin.

[0101] Table 3 Antiproliferative activity of compound 15j on drug-resistant cells

[0102]

[0103] IC 50 ,mean±SD(μM) a , half inhibitory concentration, values ​​are expressed as the mean ± SD of three independent experiments. b , cisplatin. DOX c , doxorubicin. RF d (Drug resistance factor) = IC 50 (A549 / CDDP) / IC 50 (A549).RF e (Drug resistance factor) = IC50 (A549 / paclitaxel) / IC 50 (A549).RF f (Drug resistance factor) = IC 50 (MCF-7 / DOX) / IC 50 (MCF-7).

[0104] Table 4 Antiproliferative effect of compound 15j on normal human cells

[0105]

[0106] IC 50 ,mean±SD(μM) a , half inhibitory concentration, values ​​are expressed as the mean ± SD of three independent experiments. b , cisplatin. DOX c , doxorubicin. SI d (Selectivity Index) = IC 50 (LO2) / IC 50 (A549 / CDDP). SI e (Selectivity Index) = IC 50 (MCF-10A) / IC 50 (MCF-7).

[0107] 2. Experimental results

[0108] The results of in vitro experiments showed that the target compounds (11a-11l) had better antiproliferative activity than natural chalcone and compound 6. Among them, 11j had the strongest antiproliferative activity against four human cancer cells, with IC 50 The values ​​were 0.206 to 0.454 μM, respectively, which were significantly higher than those of natural chalcone (IC 50 =4.286-7.914 μM), compound 6 (IC 50 =1.036~1.221μM), CDDP (IC 50 =4.876-8.753 μM) and DOX (IC 50 =1.145-5.213 μM) were 9.44-28.84, 2.28-5.93, 15.73-26.36 and 3.45-17.43 times stronger. It is worth noting that, except for compound 11e (IC 50 =0.709~0.978μM)、11i(IC 50 =0.656~0.928μM)、11k(IC 50 =0.608~0.936μM) and 111(IC 50 =0.769~0.989μM) was slightly lower than 11a (IC 50=0.591~0.898μM), the anticancer activities of the remaining compounds were higher than those of 11a. In addition, the antiproliferative activities of 11h and 11j were compared with those of 11d and 11g, which had fluorine or nitro groups with strong electron absorption in the middle position of the benzene ring. In addition, the introduction of aminophosphonate fragments into chalcone derivatives can significantly enhance their antitumor activity.

[0109] Based on the above results, we further designed and synthesized a series of new compounds 15a-15l, which are chalcone derivatives 1a conjugated with aminophosphonic acid derivatives. As shown in Table 2, 15d (IC 50 value is 0.328~0.570μM)、15f(IC 50 value is 0.309~0.574μM), 15h(IC 50 The antiproliferative activities of several 1a derivatives, including 1a (IC50 values ​​of 0.253-0.492 μM) and 15j (IC50 values ​​of 0.154-0.296 μM), against four human cancer cells were superior to those of the reference compound 1a (IC 50 The values ​​were 0.456-0.808 μM). Among them, 15j had the strongest antiproliferative activity against four types of human tumor cells, which were 2.73-2.96 times, 24.13-34.60 times, and 4.53-28.03 times that of 1a, CDDP, and DOX, respectively. It is worth noting that the antiproliferative effect of 15j on human cancer cells was 2.73-2.96 times higher than that of the mixed group of 1a and 10-2j, but its potency was slightly lower than that of paclitaxel. In addition, we found that the introduction of substituents such as methoxy, methyl, fluorine, and nitro groups at the middle position of the benzene ring can enhance the antiproliferative activity (15f vs 15b, 15g vs 15c, 15j vs 15d). The above results seem to indicate that the incorporation of substituents at the meta position of the benzene ring plays a crucial role in enhancing its cytotoxicity.

[0110] In addition, multidrug resistance is an important factor that hinders the progress of anti-tumor drugs. It often occurs in chemotherapy-induced cancer cell apoptosis and is a key challenge in drug development. Therefore, this study used three resistant cell lines, A549 / CDDP (cisplatin-resistant cells), A549 / TAX (paclitaxel-resistant cells), and MCF-7 / DOX (doxorubicin-resistant cells), as research objects to investigate the optimal antitumor activity of 15j against resistant cancer cells. As shown in Table 3, A549 / CDDP, A549 / TAX or MCF-7 / DOX cells showed no significant difference in their resistance to cisplatin (IC 50 value was 26.384 μM), paclitaxel (IC 50 value was 1.122 μM) or DOX (IC 50The drug resistance of 15j to A549 / CDDP, A549 / TAX and MCF-7 / DOX cells was consistent with the expected resistance factor, which was 5.27, 11.22 and 35.97, respectively. However, in contrast, 15j had a sustained efficacy against A549 / CDDP, A549 / TAX and MCF-7 / DOX cells, with an IC 50 The values ​​were 0.193-0.579 μM, and the resistance factors were only 1.25, 1.40, and 2.29. In addition, we also conducted experiments on two normal human cells, LO2 (liver) and MCF-10A (breast epithelial cells), and two cancer cells, A549 and MCF-7, to study the selectivity of 15j against normal human cells and cancer cells.

[0111] Unexpectedly, the results in Table 4 show that compound 15j has a higher selectivity index for LO2 and MCF-10A than CDDP (SI = 0.30 and 0.19), DOX (SI = 0.61 and 0.85) and paclitaxel (SI = 0.87 and 0.93), with SI values ​​of 14.34 and 10.70, respectively. In summary, compound 15j has excellent anticancer activity, overcomes drug resistance, and has high selectivity for both normal cells and cancer cells, and is a promising drug candidate for further study.

[0112] Example 8: In vivo anti-tumor activity experiment:

[0113] 1. Model building

[0114] Twenty-five 4-week-old nude mice were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. and raised under pathogen-free conditions. Logarithmically growing A549 cells were subcutaneously inoculated into the right side of BALB / c nude mice to establish an A549 xenograft model. When the tumor volume reached approximately 100 mm 3 At 14:00, mice were randomly divided into 5 experimental groups according to their body weight and tumor size: Group 1 was the control group, Group 2 was treated with cisplatin at a dose of 2 mg / kg, Group 3 was treated with 1a at a dose of 20 mg / kg, and Groups 4 and 5 were treated with 15j at a dose of 10 or 20 mg / kg, respectively. All experimental groups of mice were intraperitoneally injected once every 2 days for 3 consecutive weeks, and the changes in body weight and tumor volume were monitored at the same time. The tumor inhibition rate was calculated based on the treatment results of each compound.

[0115] 2. Experimental results

[0116] like Figure 1As shown in AC, compound 15j (10 or 20 mg / kg) exhibited significant antitumor activity in vivo in a dose-dependent manner, with tumor growth inhibition (TGI) of 53.3% and 74.7%, respectively. It is worth noting that when the dose of 15j was 20 mg / kg, the TGI reached 74.7%, which was even better than that of the cisplatin-treated group (TGI: 61.8%@2 mg / kg) or the 1a-treated group (TGI: 64.7%@20 mg / kg). In addition, it is worth noting that 15j did not cause significant weight loss at a dose of 10 or 20 mg / kg, while a significant weight loss was observed in the cisplatin-treated group ( Figure 1 D) On the contrary, Figure 2 The results of B further showed that the number of Ki67-positive tumor cells in the 15J-treated group (20 mg / kg) was significantly less than that in the other treatment groups, which was consistent with the antitumor activity results observed in vivo. More importantly, hematoxylin and eosin (H&E) staining of the main organs of mice (such as heart, liver, spleen, lung and kidney) further showed that 15J did not have any obvious toxic effects on the main organs of mice ( Figure 2 A).

[0117] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A novel dual-target inhibitor of tubulin and metalloproteinase, characterized in that: The general structural formula is shown below: Among them, R1 is selected from H, F, Cl, NO2, OCH3; R2 is selected from H, CH3, F, Cl, Br, NO2, OCH3; R3 is selected from H, OCH3.

2. A novel dual-target inhibitor of tubulin and metalloproteinase according to claim 1, characterized in that: A compound selected from the following structures:

3. The method for preparing a novel dual-target inhibitor of tubulin and metalloproteinase according to claim 1, characterized in that: The following steps are involved: (1) reacting compound 1 with compound 2 under organic base conditions to synthesize compound 3; (2) reacting compound 3 with pyridine to synthesize compound 4; (3) reacting compound 4 with compound 5 under the action of an inorganic base to synthesize compound 6; (4) reacting compound 6 with compound 10 in the presence of a condensing agent to synthesize compound 11; The synthetic route is as follows: Among them, R1 is selected from H, F, Cl, NO2, OCH3; R2 is selected from H, CH3, F, Cl, Br, NO2, OCH3; R3 is selected from H, OCH3.

4. The method for preparing a novel dual-target inhibitor of tubulin and metalloproteinase according to claim 3, characterized in that: The organic base in step (1) is a bicyclic amidine; in terms of molar amount, compound 1: compound 2 = 1: 1-2; And / or, in step (2), in terms of molar amount, compound 3:pyridine=1:60-65; And / or, in step (3), the inorganic base is potassium hydroxide; In terms of molar amount, compound 4﹕compound 5=1﹕0.8~1.2; and / or, in step (4), the condensing agent is 1-ethyl-3(3-dimethylpropylamine)carbodiimide and 4-dimethylaminopyridine; in terms of molar amount, compound 6﹕compound 10﹕EDCI﹕DMAP=1﹕1~1.5﹕1.5~2﹕1.5~2.

5. The method for preparing a novel dual-target inhibitor of tubulin and metalloproteinase according to claim 3, characterized in that: The reaction temperature of step (1) is -5 to 5°C and the reaction time is 10 to 14 hours; And / or, the reaction temperature of step (2) is 110-130° C. and the reaction time is 8-12 h; And / or, the reaction temperature of step (3) is -5 to 5°C and the reaction time is 10 to 14 hours; And / or, the reaction temperature of step (4) is room temperature and the reaction time is 8 to 12 hours; And / or, steps (1) to (4) further include a purification process.

6. The method for preparing a novel dual-target inhibitor of tubulin and metalloproteinase according to claim 1, characterized in that: The following steps are involved: (1) reacting compound 12 with compound 13 under an inorganic base to synthesize compound 14; (2) reacting compound 14 with compound 10 in the presence of a condensing agent to synthesize compound 15; The synthetic route is as follows: Among them, R1 is selected from H, F, Cl, NO2, OCH3; R2 is selected from H, CH3, F, Cl, Br, NO2, OCH3; R3 is selected from H, OCH3.

7. The method for preparing a novel dual-target inhibitor of tubulin and metalloproteinase according to claim 6, characterized in that: In step (1), the inorganic base is potassium hydroxide, and on a molar basis, the ratio of compound 12:compound 13 is 1:0.8-1.2; and / or, in step (2), the condensing agent is 1-ethyl-3(3-dimethylpropylamine)carbodiimide and 4-dimethylaminopyridine; on a molar basis, the ratio of compound 14:compound 10:EDCI:DMAP is 1:1-1.5:1.5-2.5:1.5-2.

5.

8. The method for preparing a novel dual-target inhibitor of tubulin and metalloproteinase according to claim 6, characterized in that: The reaction temperature of step (1) is -5 to 5°C and the reaction time is 10 to 14 hours; And / or, the reaction temperature of step (2) is room temperature and the reaction time is 8 to 12 hours; And / or, steps (1) and (2) further include a purification process.

9. Use of a novel dual-target inhibitor of tubulin and metalloproteinase as claimed in claim 1 or 2 in the preparation of anti-lung cancer drugs.

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