Polysubstituted aromatic nitrogen heterocyclic compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a polysubstituted aromatic nitrogen heterocyclic compound as well as a preparation method and application thereof. The polysubstituted aromatic nitrogen heterocyclic compound and the pharmaceutical salt thereof have excellent microtubule aggregation inhibition effect, various tumor cell proliferation inhibition activity and in-vivo anti-tumor activity, and can be applied to preparation of drugs for preventing or treating tumor-related diseases; the tumor-related diseases comprise benign and malignant tumors and other diseases caused by tumors.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to an anti-tumor drug - CA-4 type tubulin aggregation inhibitor and its application. Background Art

[0002] Cancer is currently one of the most significant threats to human health. Current anti-tumor drugs can be categorized by their targets, including cytotoxic drugs, hormonal drugs (such as tamoxifen and letrozole), biological response modifiers (such as interleukin-2 and interferon), and molecularly targeted drugs (such as trastuzumab and gefitinib). Cytotoxic drugs, the largest category, can be further subdivided into DNA-targeting drugs (such as alkylating agents, platinums, and anthracyclines), microtubule-targeting drugs (such as vincristine and paclitaxel), nucleic acid transcription-targeting drugs (such as aclarubicin), metabolic inhibitors (such as methotrexate, 5-fluorouracil, and cytarabine), and topoisomerase inhibitors (such as irinotecan and etoposide), based on their mode of action and target. Microtubule-targeting drugs are an important component of chemotherapy, exerting inhibitory effects on a wide range of tumor cell types. Microtubule targeting agents can be divided into two major categories based on their effects: microtubule aggregation inhibitors (i.e., microtubule destabilizers, represented by vinca alkaloids, colchicine, podophyllotoxin, and combretastatins) and microtubule aggregation promoters (i.e., microtubule stabilizers, represented by paclitaxel and epothilones). Among them, colchicine-type microtubule aggregation inhibitors not only have the ability to inhibit tubulin polymerization, but also have the effect of specifically targeting tumor blood vessels and destroying existing tumor blood vessels, thereby "starving" the tumor. Most of these drugs are not substrates for drug efflux transporters and do not have multidrug resistance. They have shown activity in inhibiting tumor growth in a variety of tumors, including breast cancer, non-small cell lung cancer, ovarian cancer, gastric cancer, and liver cancer. Therefore, they have become a class of anti-tumor drugs that have attracted attention in the industry.

[0003] Studies have revealed that Combretastatin A-4 (CA-4), a microtubule aggregation inhibitor targeting the colchicine binding site, is a series of cis-stilbene natural products isolated in 1982 from the bark or stem of the African shrub willow (Combretum caffrum). It has strong activity in inhibiting microtubule aggregation and selectively inhibiting tumor angiogenesis, and can overcome P-glycoprotein (P-gp)-mediated multidrug resistance, showing significant inhibitory effects on various types of cancer cell lines. However, due to its poor water solubility and chemical instability (easy conversion to an inactive trans configuration), its in vivo effect is not ideal. To date, no CA-4-type microtubule aggregation inhibitor has been approved for marketing. Therefore, the search for new CA-4-type microtubule aggregation inhibitors with stronger activity and fewer side effects is of great clinical value. Summary of the Invention

[0004] The purpose of the present invention is to provide an anti-tumor drug, CA-4 type microtubule aggregation inhibitor, which has novel structure, significant activity, good resistance to drug resistance and few toxic and side effects.

[0005] The anti-tumor drug provided by the present invention, the CA-4 type tubulin aggregation inhibitor, is a polysubstituted aromatic nitrogen heterocyclic compound, and its structural formula is shown in the following formula I or II:

[0006]

[0007] The heterocyclic scaffold is selected from imidazo[5,4-c]pyridin-2-one, quinazoline, pyrido[2,3-d]pyrimidine, 5,6,7,8-tetrahydroquinazoline, 6,7-dihydrothieno[3,2-d]pyrimidine, 6,7-dihydro-5H-cyclopenta[2,1-d]pyrimidine, 9-methylpurine, benzo[2,1-d]imidazole, 1H-indole, and 2,3-dihydro-1H-benzo[2,1-d]imidazol-2-one; R 1 Derived from methoxy; R 2 It is selected from a substituted aromatic ring (the substituent is an alkoxy group, a halogen atom, an alkyl group, a substituted alkyl group, a hydroxyl group, or an amino group), a methyl group, a chlorine atom, a bromine atom, or a hydrogen atom; X is selected from an oxygen atom or a nitrogen atom; and n is 0 or 1.

[0008] Preferred compounds in the present invention include but are not limited to compounds having the following structures:

[0009]

[0010]

[0011] The present invention also includes pharmaceutically acceptable salts of the polysubstituted aromatic nitrogen heterocyclic compounds, such as salts formed with inorganic acids such as hydrohalic acid, sulfuric acid, phosphoric acid, and nitric acid, and organic acids such as citric acid, fumaric acid, maleic acid, oxalic acid, malic acid, lactic acid, and camphorsulfonic acid.

[0012] The present invention uses commercially available compounds as starting materials and successfully obtains target compounds, including compounds 1-39, through a series of nucleophilic substitution reactions, reduction reactions, condensation reactions, and metal coupling reactions.

[0013] The present invention also provides the use of the above-mentioned compound and its pharmaceutically acceptable salt and a composition containing the compound or its salt in the preparation of a drug for preventing or treating tumor-related diseases.

[0014] The tumor-related diseases can specifically be thyroid cancer, head and neck squamous cell carcinoma, cervical cancer, uterine cancer, ovarian cancer, breast cancer, colorectal cancer, pancreatic cancer, esophageal cancer, osteosarcoma, kidney cancer, gastric cancer, lung cancer, liver cancer, melanoma, leukemia, lymphoma, prostate cancer, bladder cancer, brain glioma, malignant glioblastoma, nasopharyngeal carcinoma, neuroendocrine cancer, undifferentiated cancer, stromal sarcoma, choriocarcinoma, malignant hydatidiform mole, malignant teratoma and other malignant tumors and benign tumors, but are not limited thereto.

[0015] The present invention conducted an in vitro human tumor cell proliferation inhibition activity test experiment on the compound, and the results showed that the compound significantly inhibited the proliferation activity of tumor cells; the results of the in vitro microtubule self-assembly experiment showed that the compound could significantly inhibit microtubule aggregation; the results of the immunofluorescence detection microtubule morphology experiment showed that the compound could significantly inhibit microtubule aggregation; the results of the verapamil co-incubation experiment and the rhodamine efflux experiment showed that the compound was not a P-gp substrate and was not affected by P-gp efflux; the results of the in vitro cell P-gp expression detection experiment showed that the compound could significantly inhibit cell P-gp expression; the results of the colony inhibition experiment showed that the compound could significantly inhibit the formation of tumor cell colonies; the results of the in vitro cell cycle experiment showed that the compound It can significantly arrest cells in the G2 / M phase; the results of in vitro cell apoptosis experiments showed that the compound can significantly promote cell apoptosis; the results of in vitro liver microsome metabolic stability experiments showed that the compound has good metabolic stability; the results of physicochemical property determination experiments showed that the compound has good water solubility, and other physicochemical properties are within a reasonable range of drug-like characteristics and comply with the five principles of drug-like properties; further animal-level tumor treatment effect experiments were carried out, and the results showed that the compound can significantly inhibit tumor growth in vivo and has no significant effect on the weight of mice; the results of hematoxylin-eosin staining experiments showed that the compound has no significant effect on major organs of mice such as the heart, liver, spleen, lungs, and kidneys; the results of routine blood tests showed that the compound has no significant effect on blood biochemical indicators.

[0016] The present invention provides and demonstrates that polysubstituted aromatic nitrogen heterocyclic compounds or pharmaceutically acceptable salts thereof have significant anti-tumor effects, which have a good inhibitory effect on tumor growth in in vitro and in vivo anti-tumor experiments through the regulatory mechanism of inhibiting microtubule aggregation, escaping P-gp efflux, and inhibiting tumor cell growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of compound 32 inhibiting tubulin in vitro show that compound 32 can significantly inhibit tubulin aggregation.

[0018] Figure 2 The morphology of tubulin observed under a confocal microscope shows that compound 32 significantly inhibits tubulin aggregation.

[0019] Figure 3 The results of the colony inhibition experiment and the normal cell proliferation inhibition experiment showed that compound 32 significantly inhibited the formation of tumor cell colonies and had low toxicity to normal cells.

[0020] Figure 4 The results of in vitro cell cycle experiments showed that compound 32 significantly arrested cells in the G2 / M phase.

[0021] Figure 5 The results of in vitro cell apoptosis experiments show that compound 32 can significantly promote cell apoptosis.

[0022] Figure 6 The results of the verapamil co-incubation experiment show that compound 32 is not a P-gp substrate and is not affected by P-gp.

[0023] Figure 7 The results of the rhodamine efflux experiment show that compound 32 is not a P-gp substrate and does not affect the P-gp efflux function.

[0024] Figure 8 The results of the in vitro cell P-gp expression detection experiment show that compound 32 can significantly inhibit cell P-gp expression.

[0025] Figure 9 The results of the in vitro liver microsome metabolic stability experiment show that compound 32 has good metabolic stability.

[0026] Figure 10 The experimental results showed that compound 32 significantly inhibited tumor growth in the animal model of paclitaxel-resistant subcutaneous transplanted lung cancer cells, had no significant effect on the body weight of mice, had no significant effect on the main organs of mice such as the heart, liver, spleen, lungs and kidneys, and had no significant effect on the blood biochemical indicators of mice.

[0027] Figure 11The molecular docking simulation results show that compound 32 and CA-4 exhibit highly similar binding modes at the colchicine binding site.

[0028] Figure 12 Schematic diagram of the antitumor effects of polysubstituted aromatic nitrogen heterocycles. This class of compounds can inhibit tubulin aggregation, disrupt cell cycle activity, induce apoptosis, and overcome P-gp-mediated multidrug resistance. They also possess excellent safety and drugability, demonstrating significant antitumor effects in vitro and in vivo, overcoming multidrug resistance. DETAILED DESCRIPTION

[0029] The present invention is further described below through embodiments with reference to the accompanying drawings.

[0030] Example 1, 6-(3-fluoro-4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)oxy]-2,3-dihydro-1H-imidazo[5,4-c]pyridin-2-one (1)

[0031]

[0032] 2,6-Dichloro-3-nitropyridin-4-amine (2.08 g, 10 mmol), 3,4,5-trimethoxyaniline (1.84 g, 10 mmol), and DIPEA (2.58 g, 20 mmol) were added to a 100 mL eggplant-shaped flask, dissolved in 30 mL of DMF, and stirred at 50°C for 5 hours. The reaction was monitored for completion using a TLC plate. The hot reaction solution was poured into 500 mL of ice water and filtered. The filter cake was dissolved in ethyl acetate and washed twice with saturated NaCl solution. The organic phase was dried over anhydrous sodium sulfate and spin-dried. The dry-phase column was then purified by column chromatography (EA / PE = 1:4, v / v) to obtain a red solid (1a) with a yield of 85%. 1 H NMR (400MHz, CDCl3) δ6.09(s,2H),4.97(s,1H),3.81(s,6H),3.79(s,3H). 13 C NMR(151MHz, CDCl3)δ153.80,152.25,131.97,99.20,92.95,61.07,56.02.ESI-MS(m / z):355.8(M+H + ).ESI-HRMS(m / z):calcd forC 14 H 14 ClN3O6[M+H + ],356.0644; found,356.0646.

[0033] Compound 1a (0.36 g, 1.0 mmol), 3-fluoro-4-methoxyphenylboronic acid (0.34 g, 2.0 mmol), potassium carbonate (0.28 g, 2.0 mmol), bistriphenylphosphine palladium dichloride (0.06 g, 0.1 mmol), 2 mL of water, and 10 mL of dioxane were added to a 50 mL Shrek tube. The atmosphere was replaced with nitrogen three times and stirred at 100°C overnight. The reaction was monitored for completion by TLC. The reaction was quenched with water and extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, and the organic phases were combined and dried by spin drying. The residue was then purified by column chromatography (EA / PE = 1:2, v / v) to obtain a red solid (1b) in an 89% yield. 1 HNMR (400MHz, CDCl3) δ7.57-7.49(m,2H),6.94(t,J=8.6Hz,1H),6.75(s,1H),6.53(s,2H),6.24(s,2H),3.91(s,3H),3.89(s,3H),3.85(s,6H). 13 C NMR (151MHz, CDCl3) δ157.68,154.32,154.30,153.39,153.08,151.69,151.45,149.63,149.56,149.07,135 .38,129.63,129.59,123.29,123.27,119.46,114.66,114.53,113.09,102.28,99.68,61.07,56.27,56.24. 13 C NMR(151MHz, CDCl3)δ153.80,152.25,131.97,99.20,92.95,61.07,56.02.ESI-MS(m / z):445.9(M+H + ).ESI-HRMS(m / z):calcd for C 21 H 20 FN3O7[M+H + ],446.1358; found,446.1368.

[0034] Compound 1b (0.46 g, 1.0 mmol), iron powder (0.28 g, 5.0 mmol), concentrated hydrochloric acid (0.5 mL), and isopropanol (5 mL) were added to a 25 mL eggplant-shaped flask and stirred at 70°C for 4 hours. The reaction was monitored for completion by TLC. The reaction was quenched with saturated sodium bicarbonate solution and extracted three times with ethyl acetate. The organic phases were combined and dried, then purified by column chromatography (EA / PE = 1:1, v / v) to obtain 0.34 g of a red solid (1c) in an 82% yield. 1H NMR (400MHz, CDCl3) δ7.57-7.49(m,2H),6.92(t,J=8.7Hz,1H),6.82(s,1H),6.51(s,2H),3.89(s,3H),3.87(s,3H),3.83(s,6H). 13 C NMR (151MHz, CDCl3) δ153.38,153.25,152.13,151.63,150.85,147.54,147.46,144.66,134.35,132.59,132.54 ,121.55,121.53,116.12,113.78,113.65,113.18,103.35,98.04,61.05,56.31,56.17.ESI-MS(m / z):415.9(M+H + ).ESI-HRMS(m / z):calcd for C 21 H 22 FN3O5[M+H + ],416.1616; found,416.1624. White solid (1), yield 79%. 1 HNMR(400MHz,DMSO-d6)δ11.32(s,1H),11.29(s,1H),7.67(d,J=10.7Hz,2H),7.31 (s,1H),7.19(t,J=8.3Hz,1H),6.64(s,2H),3.85(s,3H),3.76(s,6H),3.69(s,3H). 13 C NMR(151MHz,DMSO-d6)δ155.49,153.53,152.79,151.18,150.19,147.68,147.60,145.63,145.06,140.04,134.68,132.3 3,132.29,122.49,122.47,114.28,113.78,113.65,113.55,99.42,97.70,60.65,56.51,56.49.ESI-MS(m / z):441.9(M+H + ).ESI-HRMS(m / z):calcd forC 22 H 20 FN3O6[M+H + ],442.1409; found,442.1408.

[0035] Example 2, 2-[(3-fluoro-4-methoxyphenyl)amino]-4-[(3,4,5-trimethoxyphenyl)amino]quinazoline (2)

[0036]

[0037] The preparation method is as described in Example 1. The product is a white solid (2) with a yield of 65%. 1 H NMR (400MHz, DMSO-d6) δ9.60(s,1H),9.19(s,1H),8.35(d,J=7.8Hz,1H),8.02-7.93(m,1H),7.69(t,J=7.6Hz,1 H),7.50(d,J=8.7Hz,2H),7.30(t,J=7.7Hz,1H),7.14(s,2H),7.05(t,J=9.3Hz,1H),3.80(s,9H),3.71(s,3H). 13 C NMR(151MHz,DMSO-d6)δ158.97,156.73,153.06,152.36,150.77,141.61,141.54,135.42,134.49,133.53,125.92,12 3.49,122.35,115.01,114.54,114.52,112.19,107.83,107.67,101.41,60.53,56.79,56.21.ESI-MS(m / z):451.0(M+H + ).ESI-HRMS(m / z):calcd for C 24 H 23 FN4O4[M+H + ],451.1776; found,451.1780.

[0038] Example 3, 2-(3-fluoro-4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)amino]quinazoline (3)

[0039]

[0040] The preparation method is as described in Example 1. The product is a white solid (3) with a yield of 63%. 1H NMR (400MHz, DMSO-d6) δ9.60(s,1H),9.19(s,1H),8.35(d,J=7.8Hz,1H),8.02-7.93(m,1H),7.69(t,J=7.6Hz,1 H),7.50(d,J=8.7Hz,2H),7.30(t,J=7.7Hz,1H),7.14(s,2H),7.05(t,J=9.3Hz,1H),3.80(s,9H),3.71(s,3H). 13 C NMR(151MHz,DMSO-d6)δ158.97,156.73,153.06,152.36,150.77,141.61,141.54,135.42,134.49,133.53 ,125.92,123.49,122.35,115.01,114.54,114.52,112.19,107.83,107.67,101.41,60.53,56.79,56.21. 1 HNMR (400MHz, CDCl3) δ8.35(s,1H),8.34-8.31(m,1H),7.98(d,J=8.4Hz,1H),7.91(d,J=8.2Hz,1H),7.79(t,J =7.7Hz,1H),7.52(t,J=7.3Hz,1H),7.25(s,2H),7.04(t,J=8.5Hz,1H),3.98(s,3H),3.97(s,6H),3.91(s,3H). 13 C NMR (151MHz, CDCl3) δ154.08,153.38,145.38,145.19,137.29,134.64,134.07,130.99,129.01,126.07, 120.50,117.96,112.68,110.32,109.18,106.76,103.72,60.31,55.71,55.45.ESI-MS(m / z):436.0(M+H + ).ESI-HRMS(m / z):calcd for C 24 H 22 FN3O4[M+H + ],436.1667; found,436.1671.

[0041] Example 4, 2-(3-fluoro-4-methoxyphenyl)-6-methoxy-4-[(3,4,5-trimethoxyphenyl)amino]quinazoline (4)

[0042]

[0043] The preparation method is as described in Example 1. The product is a white solid (4) with a yield of 67%. 1 H NMR (400MHz, DMSO-d6) δ9.59 (s, 1H), 8.24 (d, J = 8.7Hz, 1H), 8.18 (d, J = 12.3Hz, 1H), 7.94 (s, 1H), 7.78 (d, J = 8.9Hz, 1H),7.51(d,J=9.8Hz,1H),7.37(s,2H),7.30(t,J=8.6Hz,1H),3.97(s,3H),3.91(s,3H),3.88(s,6H),3.71(s,3H). 13 C NMR(151MHz,DMSO-d6)δ157.71,157.34,156.31,156.29,153.02,152.63,151.02,149.22,149.15,146.15,135.86,134.17,132.15,132.1 1,130.08,124.59,124.51,124.49,115.12,115.00,114.82,113.91,103.13,100.27,60.69,56.60,56.55,56.33.ESI-MS(m / z):466.0(M+H + ).ESI-HRMS(m / z):calcd for C 25 H 24 FN3O5[M+H + ],466.1773; found,466.1780.

[0044] Example 5, 2-(3-fluoro-4-methoxyphenyl)-6,7-dimethoxy-4-[(3,4,5-trimethoxyphenyl)amino]quinazoline (5)

[0045]

[0046] The preparation method is as described in Example 1. The product is a white solid (5) with a yield of 60%. 1 H NMR (400MHz, DMSO-d6) δ9.46(s,1H),8.24(d,J=8.6Hz,1H),8.18(d,J=13.0Hz,1H),7.86(s ,1H),7.35-7.23(m,4H),3.99(s,3H),3.97(s,3H),3.92(s,3H),3.88(s,6H),3.71(s,3H). 13C NMR(151MHz,DMSO-d6)δ156.79,156.75,156.74,154.90,153.02,152.62,151.01,149.20,149.15,149.08,148.02,136.08,133.99,132.36,1 32.32,124.42,124.40,115.07,114.94,113.90,107.99,107.96,102.6 0,100.16,60.69,56.79,56.60,56.32,56.30.ESI-MS(m / z):495.9(M+H + ).ESI-HRMS(m / z):calcd for C 26 H 26 FN3O6[M+H + ],496.1878; found,496.1884.

[0047] Example 6, 2-(3-fluoro-4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)amino]pyrido[2,3-d]pyrimidine (6)

[0048]

[0049] The preparation method is as described in Example 1. The product is a white solid (6) with a yield of 72%. 1 H NMR (400MHz, DMSO-d6) δ9.99(s,1H),9.06(s,1H),8.98(d,J=8.3Hz,1H),8.31(d,J=8.5Hz,1H),8.22(d,J= 12.9Hz,1H),7.64-7.59(m,1H),7.41(s,2H),7.36(t,J=8.6Hz,1H),3.94(s,3H),3.88(s,6H),3.71(s,3H). 13 CNMR(151MHz,DMSO-d6)δ161.54,161.52,159.61,159.22,156.75,153.04,152.59,150.98,150.05,149.98,135.44,134.46,133.0 6,131.54,131.50,125.43,125.41,121.69,115.71,115.58,114.03,109.30,100.20,60.69,56.68,56.32.ESI-MS(m / z):437.0(M+H + ).ESI-HRMS(m / z):calcd for C23 H 21 FN4O4[M+H + ],437.1602; found,437.1625.

[0050] Example 7, 2-(3-fluoro-4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)amino]-5,6,7,8-tetrahydroquinazoline (7)

[0051]

[0052] The preparation method is as described in Example 1. White solid (7), yield 61%. 1 H NMR (400MHz, DMSO-d6) δ8.23(s,1H),8.11(d,J=8.4Hz,1H),8.04(d,J=13.1Hz,1H),7.29(s,2H),7. 25(d,J=8.8Hz,1H),3.90(s,3H),3.82(s,6H),3.67(s,3H),2.72(s,2H),2.58(s,2H),1.83(s,4H). 13 C NMR(151MHz,DMSO-d6)δ163.12,158.76,158.24,158.22,152.88,152.57,150.97,149.15,149.08,136.56,133.40,131.91,131.87, 124.24,124.22,114.86,114.73,113.87,112.19,99.43,60.63,56.57,56.18,32.47,22.89,22.27,22.24.ESI-MS(m / z):440.0(M+H + ).ESI-HRMS(m / z):calcd for C 24 H 26 FN3O4[M+H + ],440.1980; found,440.1987.

[0053] Example 8, 2-(3-fluoro-4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)amino]-6,7-dihydrothieno[3,2-d]pyrimidine (8)

[0054]

[0055] The preparation method is as described in Example 1. The product is a white solid (8) with a yield of 73%. 1H NMR (400MHz, DMSO-d6) δ8.70 (s, 1H), 8.10 (d, J = 8.5Hz, 1H), 8.03 (d, J = 13.0Hz, 1H), 7.27 (t, J = 8.4Hz, 1H),7.24(s,2H),3.90(s,3H),3.81(s,6H),3.66(s,3H),3.42(d,J=8.2Hz,2H),3.30(d,J=9.0Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ168.84,158.56,158.54,154.36,152.93,152.60,150.99,149.21,149.14,136.11,133.55,131.34,1 31.30,124.23,124.21,114.93,114.81,114.69,114.01,99.19,60.62,56.60,56.17,37.08,29.86.ESI-MS(m / z):443.9(M+H + ).ESI-HRMS(m / z):calcd for C 22 H 22 FN3O4S[M+H + ],444.1388; found,444.1394.

[0056] Example 9, 2-(3-fluoro-4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)amino]-6,7-dihydro-5H-cyclopenta[2,1-d]pyrimidine (9)

[0057]

[0058] The preparation method is as described in Example 1. White solid (9) with a yield of 70%. 1 H NMR (400MHz, DMSO-d6) δ8.68(s,1H),8.15(d,J=8.8Hz,1H),8.08(d,J=14.6Hz,1H),7.31(s,2H),7.27(t ,J=8.4Hz,1H),3.90(s,3H),3.83(s,6H),3.66(s,3H),2.88(q,J=8.4,7.4Hz,4H),2.09(t,J=7.8Hz,2H). 13CNMR(151MHz,DMSO-d6)δ172.62,160.81,160.79,156.89,153.00,152.58,150.97,149.22,149.15,136.63,133.21,131.96,131.92 ,124.42,124.40,116.16,115.04,114.91,113.90,98.54,98.45,60.63,56.59,56.17,34.37,27.75,21.70.ESI-MS(m / z):425.9(M+H + ).ESI-HRMS(m / z):calcd for C 23 H 24 FN3O4[M+H + ],426.1824; found,426.1833.

[0059] Example 10, 2-(3-fluoro-4-methoxyphenyl)-9-methyl-6-[(3,4,5-trimethoxyphenyl)amino]purine (10)

[0060]

[0061] The preparation method is as described in Example 1. White solid (10) with a yield of 78%. 1 H NMR (400MHz, CDCl3) δ8.38-8.28(m,2H),7.89(s,1H),7.74(s,1H),7.32(s,1H),7.05(s,1H),4.01(s,9H),3.95(s,3H),3.91(s,3H). 13 C NMR (151MHz, CDCl3) δ158.04,153.37,153.09,151.55,151.47,150.91,149.30,149.23,141.17,135.25,133.86,13 2.16,132.12,124.16,118.83,116.00,115.87,112.62,97.34,61.08,56.31,56.16,29.77.ESI-MS(m / z):439.9(M+H + ).ESI-HRMS(m / z):calcd for C 22 H 22 FN5O4[M+H + ],440.1729; found,440.1733.

[0062] Example 11, 2-(3-fluoro-4-methoxyphenyl)-9-methyl-6-[methyl(3,4,5-trimethoxyphenyl)amino]purine (11)

[0063]

[0064] Compound 10 (0.44 g, 1.0 mmol), iodomethane (0.28 g, 2.0 mmol), potassium carbonate (0.28 g, 2.0 mmol) and DMF (5 mL) were added to a 25 mL two-necked flask and stirred at room temperature for 5 hours. The reaction was monitored by TLC plate for completion. The reaction was quenched by adding water, extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, and the organic phases were combined and spin-dried. The dry method was applied to a column and purified by column chromatography (EA / PE = 1:2, v / v) to obtain a white solid (11) with a yield of 94%. 1 HNMR (400MHz, CDCl3) δ8.38-8.28(m,2H),7.89(s,1H),7.74(s,1H),7.32(s,1H),7.05(s,1H),4.01(s,9H),3.95(s,3H),3.91(s,3H). 13 CNMR(151MHz, CDCl3)δ158.04,153.37,153.09,151.55,151.47,150.91,149.30,149.23,141.17,135.25,133.86,13 2.16,132.12,124.16,118.83,116.00,115.87,112.62,97.34,61.08,56.31,56.16,29.77.ESI-MS(m / z):454.0(M+H + ).ESI-HRMS(m / z):calcd forC 23 H 24 FN5O4[M+H + ],454.1885; found,454.1893.

[0065] Example 12, 6-(3-fluoro-4-methoxyphenyl)-1-methyl-4-[(3,4,5-trimethoxyphenyl)oxy]benzo[d]imidazole (12)

[0066]

[0067] The preparation method is as described in Example 1. The product is a white solid (12b), and the yield over two steps is 72%. 1H NMR (600MHz, CDCl3) δ7.19-7.15 (m, 2H), 7.08 (dd, J = 10.3, 1.7Hz, 1H), 7.01 (t, J = 8. 7Hz,1H),6.84(t,J=1.5Hz,1H),6.38(s,2H),3.92(s,3H),3.86(s,3H),3.83(s,6H). 13 C NMR (151MHz, CDCl3) δ155.78,154.23,154.07,153.35,151.71,151.56,151.55 ,150.72,148.77,148.70,144.28,144.26,144.21,144.20,135.68,130.61,130 .60,130.57,130.55,123.09,123.07,114.88,114.75,113.80,113.79,111.19 ,111.17,108.61,108.48,97.70,61.05,56.36,56.33.ESI-MS(m / z):447.9(M+H + ).ESI-HRMS(m / z):calcd for C 22 H 19 F2NO7[M+H + ],448.1202; found,448.1212. White solid (12) 0.34 g, two-step yield 76%. 1 H NMR (400MHz, CDCl3) δ7.97 (s, 1H), 7.32 (d, J = 28.4Hz, 3H), 7.07-6.96 (m, 2H), 6.43 (s, 2H), 3.94 (s, 6H), 3.86 (s, 3H), 3.79 (s, 6H). 13 C NMR (151MHz, CDCl3) δ153.83,153.36,153.04,151.73,148.73,147.17,147.10,143.64,137.22,136.43,134.42,134.38,134.28,13 4.25,122.97,122.95,115.15,115.03,113.75,113.73,109.65,102.80,96.82,61.02,56.42,56.15,31.46.ESI-MS(m / z):438.9(M+H + ).ESI-HRMS(m / z):calcd for C 24 H 23 FN2O5[M+H+ ],439.1664; found,439.1667.

[0068] Example 13, Synthesis of 6-(3-fluoro-4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)oxy]-1H-benzo[d]imidazole (13)

[0069]

[0070] The preparation method is as described in Example 1. The product is a white solid (13), and the yield over two steps is 81%. 1 H NMR (400MHz, CDCl3) δ8.09(s,1H),7.54(s,1H),7.27-7.17(m,2H),6.98(d,J=8.5Hz,2H),6.32(s,2H),3.90(s,3H),3.81(s,3H),3.69(s,6H). 13 C NMR (151MHz, CDCl3) δ153.89,153.29,152.81,151.66,146.99,146.91,141.44,135.95,134.43,134.39,134 .20,122.87,122.84,115.01,114.89,113.71,109.89,96.50,60.99,56.36,56.09.ESI-MS(m / z):424.9(M+H + ).ESI-HRMS(m / z):calcd for C 23 H 21 FN2O5[M+H + ],425.1507; found,425.1511.

[0071] Example 14, Synthesis of 6-(3-fluoro-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-indole (14)

[0072]

[0073] 4-Bromo-6-chloro-1H-indole (1.0 mmol), 3,4,5-trimethoxyphenylboronic acid (0.25 g, 1.2 mmol), PdCl2(dppf)2·CH2Cl2 (0.07 g, 0.1 mmol), potassium carbonate (0.28 g, 2.0 mmol), 2 mL of water, and 10 mL of dioxane were added to a 50 mL Shrek tube, replaced with nitrogen three times, and stirred at 80°C for 6 hours. Then, under nitrogen protection, the corresponding phenylboronic acid (2.0 mmol), bistriphenylphosphine palladium dichloride (0.06 g, 0.1 mmol), and potassium carbonate (0.28 g, 2.0 mmol) were added to the reaction solution, and stirred at 100°C overnight. After the reaction was completed as monitored by TLC plate, water was added to quench the reaction, and the product was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, and the organic phases were combined and dried by spin drying. The dry column was purified by column chromatography to obtain a white solid (14) with a yield of 80%. 1 HNMR (600MHz, DMSO-d6) δ11.32(s,1H),7.63(dd,J=13.0,2.3Hz,1H),7.59(s,1H),7.53(d,J=7.2Hz,1H),7.44(t,J=2.8 Hz,1H),7.36(d,J=1.6Hz,1H),7.25(t,J=8.9Hz,1H),6.99(s,2H),6.63(s,1H),3.89(s,3H),3.87(s,6H),3.74(s,3H). 13 C NMR(151MHz,DMSO-d6)δ153.46,153.10,151.49,146.54,146.46,137.51,137.23,136.97,135.25,135.21,133.98,132.90,127.18 ,125.45,123.29,123.26,118.12,114.81,114.72,114.69,108.87,106.19,100.88,60.56,56.57,56.41.ESI-MS(m / z):407.9(M+H + ).ESI-HRMS(m / z):calcd for C 24 H 22 FNO4[M+H + ],408.1606; found,408.1603.

[0074] Example 15, 6-(3-fluoro-4-methoxyphenyl)-2-methyl-4-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole (15)

[0075]

[0076] The preparation method is as described in Example 14. White solid (15) with a yield of 56%. 1 H NMR (600MHz, DMSO-d6) δ7.68-7.64(m,2H),7.55(d,J=9.9Hz,1H),7.53(s,1H),7.27-7.17(m,3H),3.89(s,9H),3.74(s,3H),2.55(s,3H). 13 C NMR(151MHz,DMSO-d6)δ153.36,153.08,151.47,146.67,146.59,137.57,134.79,134.75,134.17,133.41,123.4 7,123.45,120.07,114.99,114.87,114.67,114.66,106.72,60.51,56.56,56.45,15.15.ESI-MS(m / z):423.0(M+H + ).ESI-HRMS(m / z):calcd for C 24 H 23 FN2O4[M+H + ],423.1715; found,423.1719.

[0077] Example 16, 2-methoxy-5-[2-methyl-7-(3,4,5-trimethoxyphenyl)-3H-benzo[d]imidazol-5-yl]phenol (16)

[0078]

[0079] The preparation method is as described in Example 14. White solid (16) with a yield of 22%. 1 H NMR (600MHz, CDCl3) δ7.70 (s, 1H), 7.47 (s, 1H), 7.28 (s, 1H), 7.16 (d, J = 8.3Hz ,1H),6.98-6.95(m,3H),3.96(s,3H),3.93(s,6H),3.92(s,3H),2.66(s,3H). 13 C NMR (151MHz, CDCl3) δ152.93,150.97,145.42,145.26,137.07,135.34,134.67,133.71,120 .79,118.35,113.13,110.43,105.22,60.31,55.69,55.48,14.52.ESI-MS(m / z):421.0(M+H+ ).ESI-HRMS(m / z):calcd for C 24 H 24 N2O5[M+H + ],421.1758; found,421.1763.

[0080] Example 17, Synthesis of 6-(3-fluoro-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihydro-1H-benzo[d]imidazol-2-one (17)

[0081]

[0082] The preparation method is as described in Example 14. White solid (17a) with a yield of 55%. 1 H NMR (400MHz, DMSO-d6) δ10.92(s,1H),10.88(s,1H),7.18(s,1H),7.04(s,1H),6.75(s,2H),3.86(s,6H),3.70(s,3H). 13 CNMR(151MHz,DMSO-d6)δ155.89,153.51,137.62,132.12,131.98,126.75,12 5.14,123.27,113.07,110.39,105.92,60.42,56.24.ESI-MS(m / z):378.9(M+H + ). White solid (17), yield 76%. 1 H NMR (400MHz, DMSO-d6) δ11.03(s,1H),10.93(s,1H),7.74(d,J=15.1Hz,1H),7.62(d, J=8.3Hz,1H),7.42-7.35(m,2H),7.28(s,1H),7.00(s,2H),4.04(s,9H),3.87(s,3H). 13 C NMR(151MHz,DMSO-d6)δ156.29,153.50,153.05,151.43,146.65,146.58,137.40,134.36,134.32,133.31,132.58,131.42 ,126.92,123.83,123.08,123.06,119.85,114.65,114.51,106.05,105.91,60.43,56.55,56.26.ESI-MS(m / z):424.9(M+H +).ESI-HRMS(m / z):calcd for C 23 H 21 FN2O5[M+H + ],425.1507; found,425.1504.

[0083] Example 18, 6-(3-hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihydro-1H-benzo[d]imidazol-2-one (18)

[0084]

[0085] The preparation method is as described in Example 14. White solid (18) with a yield of 73%. 1 H NMR(400MHz,DMSO-d6)δ10.81(s,1H),10.71(s,1H),9.09(s,1H),7.12(s,1H),7.02(d,J =11.4Hz,3H),6.94(d,J=8.2Hz,1H),6.78(s,2H),3.85(s,6H),3.76(s,3H),3.68(s,3H). 13 C NMR(151MHz,DMSO-d6)δ155.63,152.86,146.81,146.58,136.70,133.55,133.44,132.85,130.70,125.83 ,123.08,118.95,117.17,113.72,112.52,105.29,105.16,59.81,55.60,55.56.ESI-MS(m / z):422.9(M+H + ).ESI-HRMS(m / z):calcd for C 23 H 22 N2O6[M+H + ],423.1551; found,423.1554.

[0086] Example 19, 6-(3-fluoro-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole (19)

[0087]

[0088] The preparation method is as described in Example 14. White solid (19) with a yield of 48%. 1H NMR (600MHz, CDCl3) δ8.07 (s, 1H), 7.69 (s, 1H), 7.49 (d, J = 1.6Hz, 1H), 7.38-7.33 ( m,2H),7.02(d,J=8.6Hz,1H),7.00(s,2H),3.91(s,3H),3.85(s,3H),3.84(s,6H). 13 C NMR (151MHz, CDCl3) δ153.39,153.37,151.74,146.91,146.84,141.87,137.57,135.36,134.82,134.78,134 .26,123.01,122.99,121.78,115.15,115.02,113.75,105.93,60.89,56.38,56.12.ESI-MS(m / z):408.9(M+H + ).ESI-HRMS(m / z):calcd for C 23 H 21 FN2O4[M+H + ],409.1558; found,409.1561.

[0089] Example 20, 2-methoxy-5-[7-(3,4,5-trimethoxyphenyl)-3H-benzo[d]imidazol-5-yl]phenol (20)

[0090]

[0091] The preparation method is as described in Example 14. White solid (20) with a yield of 41%. 1 H NMR (400MHz, CDCl3) δ8.07(s,1H),7.69(s,1H),7.52(s,1H),7.25(s,1H),7.09(d,J=7 .5Hz,1H),6.98(s,2H),6.89(d,J=7.9Hz,1H),3.88(s,3H),3.82(s,3H),3.80(s,6H). 13 C NMR (151MHz, CDCl3) δ153.35,146.49,146.16,141.62,137.48,136.64,134.87,134.34, 122.06,118.89,114.01,111.30,105.90,60.89,56.10,56.03.ESI-MS(m / z):406.9(M+H + ).ESI-HRMS(m / z):calcd forC 23 H22 N2O5[M+H + ],407.1601; found,407.1591.

[0092] Example 21, 6-(6-methoxypyridin-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole (21)

[0093]

[0094] The preparation method is as described in Example 14. White solid (21) with a yield of 47%. 1 H NMR (400MHz, CDCl3) δ8.44(s,1H),8.08(s,1H),7.86(d,J=8.3Hz,1H),7.73(s,1H),7. 49(s,1H),7.01(s,2H),6.84(d,J=8.4Hz,1H),3.99(s,3H),3.88(s,3H),3.86(s,6H). 13 C NMR (151MHz, CDCl3) δ163.53,153.48,145.16,141.73,137.89,137.72,133.96,133 .54,130.47,121.80,110.79,105.86,60.92,56.15,53.63.ESI-MS(m / z):391.9(M+H + ).ESI-HRMS(m / z):calcd for C 22 H 21 N3O4[M+H + ],392.1605; found,392.1608.

[0095] Example 22, {2-methoxy-5-[7-(3,4,5-trimethoxyphenyl)-3H-benzo[d]imidazol-5-yl]phenyl}methanol (22)

[0096]

[0097] The preparation method is as described in Example 14. White solid (22) with a yield of 46%. 1 H NMR(400MHz, CDCl3)δ8.02(s,1H),7.59(s,1H),7.56(s,1H),7.48(s,2H), 6.97(s,2H),6.91(d,J=8.4Hz,1H),4.74(s,2H),3.85(s,6H),3.83(s,6H). 13C NMR (151MHz, CDCl3) δ156.73,153.36,141.54,137.60,136.44,134.25,133.97,129.55,127 .69,127.60,121.88,110.57,105.97,61.55,60.91,56.16,55.49.ESI-MS(m / z):421.0(M+H + ).ESI-HRMS(m / z):calcd for C 24 H 24 N2O5[M+H + ],421.1758; found,421.1762.

[0098] Example 23, 2-methoxy-5-[7-(3,4,5-trimethoxyphenyl)-3H-benzo[d]imidazol-5-yl]aniline (23)

[0099]

[0100] The preparation method is as described in Example 14. Pale yellow solid (23) with a yield of 53%. 1 H NMR (600MHz, CDCl3) δ8.18 (s, 1H), 7.72 (s, 1H), 7.53 (d, J = 1.6Hz, 1H), 7.02 -6.97(m,4H),6.86(d,J=8.0Hz,1H),3.89(s,3H),3.88(s,3H),3.87(s,6H). 13 C NMR (151MHz, CDCl3) δ153.51,146.97,141.02,137.70,137.36,136.43,134.49,134.11, 122.34,117.52,114.17,110.71,105.85,60.93,56.23,55.66.ESI-MS(m / z):405.9(M+H + ).ESI-HRMS(m / z):calcd for C 23 H 23 N3O4[M+H + ],406.1761; found,406.1765.

[0101] Example 24, Synthesis of 6-(4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole (24)

[0102]

[0103] The preparation method is as described in Example 14. White solid (24) with a yield of 44%. 1 H NMR (600MHz, CDCl3) δ8.07(s,1H),7.79(s,1H),7.60(d,J=8.6Hz,2H),7.54(s,1H),7.01(d,J=8.7Hz,4H),3.90(s,3H),3.88(s,6H),3.86(s,3H). 13 C NMR (151MHz, CDCl3) δ159.15,153.54,141.19,138.73,137.75,137.04,133.98,133.88,133.34, 129.19,128.54,122.42,114.34,113.00,105.83,60.94,56.21,55.40.ESI-MS(m / z):390.9(M+H + ).ESI-HRMS(m / z):calcd for C 23 H 22 N2O4[M+H + ],391.1652; found,391.1656.

[0104] Example 25, 6-(4-methylphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole (25)

[0105]

[0106] The preparation method is as described in Example 14. White solid (25) with a yield of 58%. 1 H NMR(600MHz, CDCl3)δ8.12(s,1H),7.81(s,1H),7.56(s,2H),7.55(s,1H), 7.27(d,J=7.2Hz,2H),6.98(s,2H),3.89(s,3H),3.88(s,6H),2.41(s,3H). 13 C NMR (151MHz, CDCl3) δ153.53,141.26,138.64,137.70,137.06,136.95,134.17, 129.58,127.40,122.33,105.83,60.95,56.22,21.10.ESI-MS(m / z):375.0(M+H + ).ESI-HRMS(m / z):calcd for C 23 H 22 N2O3[M+H+ ],375.1703; found,375.1703.

[0107] Example 26, 6-(4-ethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole (26)

[0108]

[0109] The preparation method is as described in Example 14. White solid (26) with a yield of 50%. 1 H NMR(400MHz, CDCl3)δ8.06(s,1H),7.75(s,1H),7.59-7.53(m,3H),7.01-6.96( m,4H),4.08(q,J=6.9Hz,2H),3.87(s,3H),3.85(s,6H),1.45(t,J=7.0Hz,3H). 13 C NMR (151MHz, CDCl3) δ158.44,153.44,141.39,137.60,136.79,134.25,133.83,128 .48,122.16,114.87,105.87,63.58,60.91,56.16,14.87.ESI-MS(m / z):405.0(M+H + ).ESI-HRMS(m / z):calcd for C 24 H 24 N2O4[M+H + ],405.1809; found,405.1811.

[0110] Example 27, 6-(4-cyclopropylphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole (27)

[0111]

[0112] The preparation method is as described in Example 14. White solid (27) with a yield of 44%. 1 H NMR (400MHz, CDCl3) δ8.16(s,1H),7.84(s,1H),7.57(d,J=6.7Hz,3H),7.19(d,J=7.9Hz,2H), 6.99(s,2H),3.92(s,9H),1.96(td,J=8.5,4.3Hz,1H),1.05-0.98(m,2H),0.79-0.73(m,2H). 13C NMR (151MHz, CDCl3) δ153.61,143.20,141.01,138.60,137.81,137.12,134.02,12 7.41,126.15,122.33,105.80,60.96,56.27,15.12,9.36.ESI-MS(m / z):401.0(M+H + ).ESI-HRMS(m / z):calcd forC 25 H 24 N2O3[M+H + ],401.1860; found,401.1860.

[0113] Example 28, 2-methyl-5-[7-(3,4,5-trimethoxyphenyl)-3H-benzo[d]imidazol-5-yl]aniline (28)

[0114]

[0115] The preparation method is as described in Example 14. White solid (28), yield 48%. 1 H NMR (400MHz, CDCl3) δ8.05(s,1H),7.77(s,1H),7.55(s,1H),7.14(d,J=7.7H z,1H),7.00(dd,J=10.1,6.8Hz,4H),3.90(s,3H),3.88(s,6H),2.22(s,3H). 13 C NMR (151MHz, CDCl3) δ153.52,144.97,141.14,140.32,137.71,137.50,134.07,130.94, 122.53,121.50,117.83,113.98,105.85,60.94,56.22,17.06.ESI-MS(m / z):390.0(M+H + ).ESI-HRMS(m / z):calcd for C 23 H 23 N3O3[M+H + ],390.1812; found,390.1803.

[0116] Example 29, 2-chloro-5-[7-(3,4,5-trimethoxyphenyl)-3H-benzo[d]imidazol-5-yl]aniline (29)

[0117]

[0118] The preparation method is as described in Example 14. White solid (29), yield 49%. 1 H NMR (400MHz, CDCl3) δ8.05(s,1H),7.77(s,1H),7.55(s,1H),7.14(d,J=7.7H z,1H),7.00(dd,J=10.1,6.8Hz,4H),3.90(s,3H),3.88(s,6H),2.22(s,3H). 13 C NMR (151MHz, CDCl3) δ153.52,144.97,141.14,140.32,137.71,137.50,134.07,130.94, 122.53,121.50,117.83,113.98,105.85,60.94,56.22,17.06.ESI-MS(m / z):409.9(M+H + ).ESI-HRMS(m / z):calcd for C 22 H 20 ClN3O3[M+H + ],410.1266; found,410.1270.

[0119] Example 30, 2-methyl-5-[7-(3,4,5-trimethoxyphenyl)-3H-benzo[d]imidazol-5-yl]phenol (30)

[0120]

[0121] The preparation method is as described in Example 14. White solid (30), yield 76%. 1 H NMR (400MHz, CD3OD) δ8.20(s,1H),7.75(s,1H),7.54(s,1H),7.16-7.07(m,3H),7.05(s,2H),3.93(s,6H),3.84(s,3H),2.23(s,3H). 13 C NMR(151MHz,CD3OD)δ155.49,153.32,142.04,140.20,137.47,136.84,134.45,130.77, 123.15,121.50,118.02,113.04,105.87,59.79,55.26,14.51.ESI-MS(m / z):391.0(M+H + ).ESI-HRMS(m / z):calcd for C 23 H 22 N2O4[M+H +],391.1652; found,391.1653.

[0122] Example 31, 6-(3-fluoro-4-methylphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole (31)

[0123]

[0124] The preparation method is as described in Example 14. White solid (31) with a yield of 47%. 1 H NMR (600MHz, CD3OD) δ8.20(s,1H),7.78(s,1H),7.55(d,J=1.6Hz,1H),7.41(dd,J=7.8,1.8Hz,1H),7 .38(dd,J=11.2,1.9Hz,1H),7.32-7.28(m,1H),7.06(s,2H),3.93(s,6H),3.84(s,3H),2.30(s,3H). 13 CNMR(151MHz,CD3OD)δ162.39,160.78,153.34,142.35,141.32,141.27,137.54,135.37,134.23,131.54,131.50 ,123.05,122.94,122.47,122.45,121.34,113.24,113.09,105.93,59.78,55.27,12.75.ESI-MS(m / z):392.9(M+H + ).ESI-HRMS(m / z):calcd for C 23 H 21 FN2O3[M+H + ],393.1609; found,393.1612.

[0125] Example 32, 2-methoxy-5-[7-(2,3,4-trimethoxyphenyl)-3H-benzo[d]imidazol-5-yl]aniline (32)

[0126]

[0127] 6-Bromo-4-iodo-1H-benzo[d]imidazole (1.0 mmol), 2,3,4-trimethoxyphenylboronic acid (0.25 g, 1.2 mmol), PdCl2(dppf)2·CH2Cl2 (0.07 g, 0.1 mmol), potassium carbonate (0.28 g, 2.0 mmol), 2 mL of water, and 10 mL of dioxane were added to a 50 mL Shrek tube. The atmosphere was purged with nitrogen three times and stirred at 80°C for 6 hours. Then, under nitrogen protection, the corresponding phenylboronic acid (2.0 mmol), bistriphenylphosphine palladium dichloride (0.06 g, 0.1 mmol), and potassium carbonate (0.28 g, 2.0 mmol) were added to the reaction solution, and the mixture was stirred at 100°C overnight. After the reaction was complete as monitored by TLC plate, water was added to quench the reaction, and the product was extracted three times with ethyl acetate and dried over anhydrous sodium sulfate. The organic phases were combined and dried by spin-drying, and then purified by column chromatography to obtain a white solid (32) with a yield of 59%. 1 H NMR (400MHz, CDCl3) δ8.09(s,1H),7.93(s,1H),7.56(s,1H),7.25(d,J=9.6Hz,1H),7.07(d,J=7.7Hz, 2H),6.89(d,J=8.2Hz,1H),6.83(d,J=8.7Hz,1H),3.99(s,3H),3.93(s,3H),3.91(s,3H),3.58(s,3H). 13 C NMR (151MHz, CDCl3) δ153.65,150.96,146.84,142.55,140.94,136.68,136.38,134.73,131.19,125.93,124.90 ,123.61,123.33,117.51,115.84,114.22,110.75,108.47,61.49,61.33,56.15,55.67.ESI-MS(m / z):406.0(M+H + ).ESI-HRMS(m / z):calcd for C 23 H 23 N3O4[M+H + ],406.1761; found,406.1771.

[0128] Example 33, 2-methoxy-5-[7-(2-methoxyphenyl)-3H-benzo[d]imidazol-5-yl]aniline (33)

[0129]

[0130] The preparation method is as shown in Example 32. White solid (33) with a yield of 42%. 1H NMR(600MHz,CD3OD)δ8.10(s,1H),7.76(t,J=2.1Hz,1H),7.42(s,3H),7.14(d,J=10.0Hz,2H), 7.08(t,J=7.4Hz,1H),7.02(d,J=8.2Hz,1H),6.91(d,J=8.4Hz,1H),3.88(s,3H),3.79(s,3H). 13 C NMR (151MHz, CD3OD) δ156.96,147.26,141.42,136.41,136.40,134.62,130.91,129.08,127.09,125. 14,123.31,120.49,117.22,114.26,113.35,111.02,110.47,54.73,54.52.ESI-MS(m / z):346.0(M+H + ).ESI-HRMS(m / z):calcd for C 21 H 19 N3O2[M+H + ],346.1550; found,346.1553.

[0131] Example 34, 2-methoxy-5-[7-(4-methoxyphenyl)-3H-benzo[d]imidazol-5-yl]aniline (34)

[0132]

[0133] The preparation method is as shown in Example 32. White solid (34), yield 45%. 1 H NMR (400MHz, CDCl3) δ8.03 (s, 1H), 7.71 (d, J = 7.8Hz, 2H), 7.64 (s, 1H), 7.51 (s, 1H) ,7.02-6.94(m,4H),6.84(d,J=8.2Hz,1H),4.51(s,3H),3.88(s,3H),3.80(s,3H). 13 C NMR (151MHz, CDCl3) δ159.25,146.91,140.92,138.10,137.21,136.29,134.62,134.40,130.77,129. 69,129.06,121.92,117.57,114.28,114.22,111.82,110.70,55.64,55.33.ESI-MS(m / z):346.0(M+H +).ESI-HRMS(m / z):calcd for C 21 H 19 N3O2[M+H + ],346.1550; found,346.1554.

[0134] Example 35, 6-methyl-4-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole (35)

[0135]

[0136] The preparation method is as shown in Example 32. White solid (35) with a yield of 90%. 1 H NMR (600MHz, CDCl3) δ8.00(s,1H),7.40(s,1H),7.17(s,1H),6.95(s,2H),3.88(s,3H),3.87(s,6H),2.52(s,3H). 13 CNMR(151MHz,CDCl3)δ153.43,140.63,138.90,137.54,134.38,133.07,132.96 ,128.69,123.95,114.92,105.81,60.94,56.18,21.64.ESI-MS(m / z):299.0(M+H + ).ESI-HRMS(m / z):calcd for C 17 H 18 N2O3[M+H + ],299.1390; found,299.1391.

[0137] Example 36, 6-chloro-4-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole (36)

[0138]

[0139] The preparation method is as shown in Example 32. White solid (36) with a yield of 79%. 1 H NMR (600MHz, CDCl3) δ8.06 (s, 1H), 7.62 (s, 1H), 7.32 (d, J = 1.6Hz, 1H), 6.91 (s, 2H), 3.89 (s, 3H), 3.87 (s, 6H). 13C NMR(151MHz, CDCl3)δ153.55,141.75,137.93,133.03,128.65,122.76,105.71,60.96,56.23.ESI-MS(m / z):318.9(M+H + ).ESI-HRMS(m / z):calcd for C 16 H 15 ClN2O3[M+H + ],319.0844; found,319.0847.

[0140] Example 37, 6-bromo-4-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole (37)

[0141]

[0142] The preparation method is as shown in Example 32. White solid (37), yield 18%. 1 H NMR (400MHz, CDCl3) δ8.05(s,1H),7.82(s,1H),7.47(s,1H),6.91(s,2H),3.92(s,3H),3.90(s,6H). 13 C NMR (151MHz, CDCl3) δ153.57,141.47,137.98,132.87,125.32,116.05,105.70,60.96,56.25.ESI-MS(m / z):362.9(M+H + ).ESI-HRMS(m / z):calcd for C 16 H 15 BrN2O3[M+H + ],363.0339; found,363.0342.

[0143] Example 38, 4-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole (38)

[0144]

[0145] The preparation method is as shown in Example 32. White solid (38), yield 93%. 1 H NMR (600MHz, CDCl3) δ7.95 (s, 1H), 7.57 (d, J = 4.6Hz, 1H), 7.33-7.29 (m, 2H), 6.99 (s, 2H), 3.85 (s, 3H), 3.82 (s, 6H). 13C NMR(151MHz, CDCl3)δ153.27,141.38,137.33,134.63,122.86,122.20,105.95,60.89,56.05.ESI-MS(m / z):285.0(M+H + ).ESI-HRMS(m / z):calcd for C 16 H 16 N2O3[M+H + ],285.1234; found,285.1237.

[0146] Example 39, 6-(Benzo[2,1-d][1,3]oxazepan-5-yl)-4-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole (39)

[0147]

[0148] The preparation method is as described in Example 32. White solid (39), yield 51%. 1 H NMR (600MHz, CDCl3) δ8.15(s,1H),8.11(s,1H),8.06(s,1H),7.87(s,1H),7.72–7.65(m,2H),7.60(s,1H),7.02(s,2H),3.91(s,3H),3.89(s,6H). 13 C NMR (151MHz, CDCl3) δ153.56,153.19,149.42,141.53,140.69,139.00,137.77,136.50, 134.10,125.70,122.52,119.33,111.09,105.85,60.97,56.24.ESI-MS(m / z):402.0(M+H + ).

[0149] Example 40, In vitro testing of the target compound's ability to inhibit human tumor cell proliferation

[0150] Tumor cells were inoculated in a 96-well plate and cultured for 24 hours. Samples of varying concentrations were then added. After 48 hours of culture, 20 μL of 5 mg / mL MTT solution was added. After another 4 hours of culture, the supernatant was discarded and 150 μL of DMSO was added to dissolve formazan. The OD value was measured at 540 nm on a microplate reader, and the inhibition rate and IC were calculated. 50 The results are shown in Table 1 and Table 2:

[0151] Table 1. Inhibitory activity of polysubstituted aromatic nitrogen heterocyclic compounds on tumor cell proliferation (IC50 ,μM).

[0152]

[0153]

[0154] The antitumor activity was determined by MTT assay, and the data were the average of three measurements. A549 is a human lung adenocarcinoma cell line, and A549 / T is a human paclitaxel-resistant lung adenocarcinoma cell line.

[0155] Table 2. Inhibitory activity of compound 32 on the proliferation of different tumor cell lines (IC 50 ,μM).

[0156]

[0157] The antitumor activity was determined by MTT assay, and the data were the average of three measurements. A549 / T is a human paclitaxel-resistant lung adenocarcinoma cell line.

[0158] Example 41, Tubulin Aggregation Inhibition Experiment: In Vitro Tubulin Self-Assembly Experiment

[0159] The inhibitory effect of test compound 32 on microtubule aggregation in vitro was determined by turbidimetry. The microtubule aggregation system reaction solution was pre-incubated on ice, and samples of different concentrations were added. A DMSO (4%, v / v) group was set as a negative control group, and a CA-4-treated group was set as a positive control. The absorbance was measured using a spectrophotometer for a total of 30 minutes, and a curve was drawn based on the absorbance ( Figure 1 ); The results showed that compound 32 could significantly inhibit tubulin aggregation, IC 50 was 1.8±0.6 μM (Table 3).

[0160] Table 3. Tubulin aggregation inhibitory activity of compound 32 (IC 50 , μM).

[0161]

[0162] The data are the average of three measurements.

[0163] Example 42, Tubulin Aggregation Inhibition Experiment: Immunofluorescence Detection of Tubulin Morphology Experiment

[0164] Tumor cells were seeded into confocal dishes (4000 cells / dish), cultured at 37°C and 5% carbon dioxide for 24 hours, and then treated with different concentrations of test compounds for 24 hours. A DMSO-treated group was set up as a negative control. The culture medium was discarded, washed twice with PBS, fixed with methanol for 15 minutes, washed three times with PBS, permeabilized with 0.1% Triton for 15 minutes, and washed three times with PBS. After blocking with 5% BSA at room temperature for 1 hour, the primary antibody was incubated at 4°C overnight, and rinsed three times with PBST for 5 minutes each time. Fluorescent secondary antibody was added, incubated at room temperature in the dark for 1 hour, and rinsed three times with PBST for 5 minutes each time. The morphology of tubulin was observed under a confocal microscope to detect the effect of the test compound on the microtubule structure, and relevant photos were taken ( Figure 2 ), the results showed that compound 32 could significantly inhibit the aggregation of microtubules, resulting in disordered microtubule structure and significantly reduced density. Even at low concentrations, it showed effective inhibition of tumor cell microtubule assembly.

[0165] Example 43, Colony Inhibition Assay and Normal Cell Proliferation Inhibition Assay

[0166] Tumor cells (1500 cells / well) were seeded into 6-well plates and incubated at 37°C for 24 hours. After the cells attached to the wall, different concentrations of compound 32 were added to treat the cells for 48 hours. A negative control group (DMSO) was set up. Fresh culture medium was replaced and cultured for 7-10 days. The culture medium was discarded, the cells were fixed with methanol, and stained with crystal violet dye. The results showed that compound 32 could significantly inhibit the formation of tumor cell colonies (such as Figure 3 MCF-10A cells were seeded in 96-well plates and cultured for 24 hours. Samples of different concentrations were then added. After 48 hours of culture, 20 μL of 5 mg / mL MTT solution was added. After another 4 hours of culture, the supernatant was discarded and 150 μL of DMSO was added to dissolve formazan. The OD value was measured at 540 nm on a microplate reader, and the inhibition rate was calculated ( Figure 3 ), the results showed that the area of ​​tumor cell colony formation decreased in a dose-dependent manner after treatment with compound 32, significantly inhibiting the formation of tumor cell colonies; compared with other positive controls, the toxicity of compound 32 to normal cells was significantly reduced.

[0167] Example 44, in vitro cell cycle assay

[0168] In a 6-well plate, 2 × 10 5 Cells were seeded at the number of cells / well and incubated at 37°C for 24 hours. After the cells attached to the wall, the cells were treated with different concentrations of compound 32 for 24 hours. A negative control group (2% DMSO) was set up at the same time. The cells were collected, washed twice with PBS, and then fixed with 75% ethanol at -20°C overnight. After staining with PI, the cells were tested by flow cytometry ( Figure 4), the results showed that compound 32 could arrest the tumor cell cycle at the G2 / M phase in a concentration-dependent manner.

[0169] Example 45, in vitro cell apoptosis experiment

[0170] In a 6-well plate, 2 × 10 5 Cells were seeded at a concentration of 100 μl / well and incubated at 37°C for 24 hours. After the cells adhered to the wall, they were treated with different concentrations of the test sample for 48 hours. A negative control group (2% DMSO) was also set up. The cells were collected, washed twice with PBS, and resuspended in 100 μl of 1× cell apoptosis binding solution. After double staining with PI and Annexin V, the cells were tested by flow cytometry ( Figure 5 ), the results showed that compound 32 could promote tumor cell apoptosis in a concentration-dependent manner.

[0171] Example 46, Verapamil Co-incubation Experiment

[0172] A549 / T tumor cells overexpressing P-gp were seeded in a 96-well plate and cultured at 37°C in a 5% CO2 environment for 24 hours. Five samples of different concentrations and 10 μM verapamil (P-gp inhibitor) were then added. After 48 hours of culture, 20 μL of 5 mg / mL MTT solution was added. After another 4 hours of culture, the supernatant was discarded and 150 μL of DMSO was added to dissolve the formazan. The OD value was measured at 540 nm on a microplate reader, and the inhibition rate was calculated ( Figure 6 ), the results showed that the antitumor effect of compound 32 was not affected by verapamil and it was not an efflux substrate of P-gp.

[0173] Example 47, Rh123 efflux experiment

[0174] A549 / T cells were cultured at 1×10 5 Cells were seeded at a high density in 6-well plates. After 24 hours of culture, 15 μM Rh123 was added to each well and incubated with different concentrations of the test compound or DMSO (negative control) for 6 hours. After removing the unbound dye, the cells were resuspended and stored in the dark. The accumulation of Rh123 in the cells was detected by flow cytometry. Paclitaxel, a classic P-gp substrate, was used as a positive control ( Figure 7 ), the results showed that compound 32 was not an efflux substrate of P-gp and did not affect the efflux of Rh123.

[0175] Example 48, in vitro P-gp expression detection experiment

[0176] After treating tumor cells with different concentrations of compounds (DMSO was used as a negative control), the cells were collected and lysed. The obtained protein samples were denatured by heating, separated by electrophoresis, transferred to a membrane, blocked, and reacted with primary and secondary antibodies in sequence, and then exposed to light for color development ( Figure 8 ), the results showed that compound 32 could down-regulate the expression of P-gp in a concentration-dependent manner.

[0177] Example 49, In ​​vitro liver microsome metabolic stability experiment

[0178] The metabolism of the compounds in human or mouse liver microsomes was analyzed using LC-MS / MS, with ketanserin used as a control. The compounds were incubated with liver microsomes in potassium phosphate buffer, and samples were taken at 0, 5, 15, 30, and 45 minutes. After adding the stop solution, the samples were analyzed on the instrument. By applying the first-order kinetic equation, T was calculated. 1 / 2 and CLint(mic)(mL / min / kg)( Figure 9 , Table 4), the results showed that compound 32 was more stable in liver microsomes than CA-4, with a half-life of 63 minutes in mouse liver microsomes and 75 minutes in human liver microsomes.

[0179] Table 4. In vitro liver microsomal metabolic stability of preferred compounds 32 and CA-4.

[0180]

[0181] in, a T 1 / 2 :half life; b CL int(mic) : Hepatic microsomal clearance.

[0182] Example 50, Study on the physicochemical properties of compound 32

[0183] Methanol solutions of various compound concentrations were analyzed by HPLC at a wavelength of 254 nm to establish a standard curve comparing peak area and concentration. Subsequently, the test compounds were dissolved in phosphate buffer to prepare saturated solutions, analyzed by HPLC, and peak area values ​​were extracted. Solubility was calculated based on the standard curve (Table 5). The results showed that at pH 7.4, CA-4 had a water solubility of only 1.0 mg / mL, while compound 32 had a water solubility of 20.8 mg / mL, more than twenty times that of CA-4. Furthermore, the other physicochemical properties of compound 32 were within reasonable drug-like properties and met the five principles of drug-like properties.

[0184] Table 5. Physicochemical properties of compound 32.

[0185]

[0186] in, a MW: molecular weight; b HBA: hydrogen bond acceptor;c HBD: hydrogen bond donor; d cLogP: lipid-water partition coefficient; e tPSA: polar surface area.

[0187] Example 51, tumor therapeutic effect in a paclitaxel-resistant lung cancer cell subcutaneous transplanted tumor animal model.

[0188] The paclitaxel-resistant lung cancer cell line A549 / T was cultured and cells in the vigorous growth phase were selected for inoculation at a rate of 1×10 7 The cells were subcutaneously inoculated into 4-5 week old Balb / C female nude mice to establish a nude mouse tumor metastasis model. 3 Mice were randomly divided into four groups: group 1 (15 mg / kg compound 32), group 2 (30 mg / kg compound 32), group 3 (15 mg / kg paclitaxel), group 4 (15 mg / kg CA-4) and a blank control group (solvent). Intraperitoneal injection was performed every other day and the tumor diameter was measured with a vernier caliper. The tumor volume was calculated according to the following formula: tumor volume (mm 3 )=a×b 2 ×0.5 (where a represents the longest diameter, b represents the shortest diameter, and 0.5 is an empirical coefficient). When the tumor volume of the blank control group reaches 1000 mm 3 Afterwards, the nude mice were killed, the tumor tissues were removed, and the tumor weight was measured. The visceral tissues of the nude mice were further observed using H&E staining ( Figure 10 ), the results showed that compound 32 significantly inhibited tumor growth in vivo and had no significant effect on mouse body weight. Tissue staining also revealed no abnormal areas of interest in the heart, liver, kidney, lung, or spleen. Routine blood tests (Table 6) on mouse blood samples revealed no significant effect on blood biochemical parameters, demonstrating a high safety profile.

[0189] Table 6. Routine blood test report of nude mice after treatment with compound 32.

[0190]

[0191]

[0192] in, aWBC:white blood cell count.RBC:redblood cell count.RDWSD:redblood cell distribution width-cell volume.RDW-CV:red blood celldistribution width-cell size.PLT:platelet count.PCT:plateletocrit.MPV:meanplatelet volume.PDW:platelet distribution width.MCH:mean corpusularhemoglobin.MCHC:mean corpusularhemoglobin concentration.LY:lymphocyte.MON,monocytes.

[0193] Example 52, molecular docking experiment

[0194] use 13.5 Molecular docking studies were performed. The co-crystal structure of tubulin in complex with CA-4 was selected for docking simulation (PDB: 5LYJ). The 3D structure energy of the compound was minimized using the OPLS4 force field, and induced fit docking was used for docking analysis ( Figure 11 ), the results showed that compound 32 and CA-4 exhibited highly similar binding modes at the colchicine binding site. In addition, docking studies revealed hydrogen bonding interactions between the NH2 group of compound 32 and residue ASN-349, as well as cation-π interactions between the 1H-benzo[d]imidazole core of compound 32 and residue LYS-352. Based on this docking model, the binding driving force may mainly come from interactions resulting from the good fit between compound 32 and the β-tubulin binding site.

Claims

1. An anti-tumor drug---CA-4 type microtubule protein aggregation inhibitor, characterized in that: It is a polysubstituted aromatic nitrogen heterocyclic compound, and its structural formula is shown in the following formula I or II: The heterocyclic scaffold is selected from imidazo[5,4-c]pyridin-2-one, quinazoline, pyrido[2,3-d]pyrimidine, 5,6,7,8-tetrahydroquinazoline, 6,7-dihydrothieno[3,2-d]pyrimidine, 6,7-dihydro-5H-cyclopenta[2,1-d]pyrimidine, 9-methylpurine, benzo[2,1-d]imidazole, 1H-indole, 2,3-dihydro-1H-benzo[2,1-d]imidazol-2-one; R 1 From 1 to 3 alkoxy groups; R 2 To replace the aromatic ring, alkyl, halogen or hydrogen atom, the substituent on the aromatic ring is taken from alkoxy, halogen atom, alkyl, substituted alkyl, hydroxyl, amino, hydrogen atom; X is taken from oxygen atom, nitrogen atom or substituted nitrogen atom; n is 0 or 1.

2. The CA-4 class microtubule aggregation inhibitor according to claim 1, characterized in that The polysubstituted aromatic nitrogen heterocyclic compound is one of the following structural formulas:

3. The CA-4 class microtubule aggregation inhibitor according to claim 1 or 2, characterized in that: Also included are pharmaceutically acceptable salts of polysubstituted aromatic nitrogen heterocyclic compounds.

4. The CA-4 class microtubule aggregation inhibitor according to claim 3, characterized in that: The salt is specifically a salt formed by hydrohalic acid, sulfuric acid, phosphoric acid or nitric acid, or citric acid, fumaric acid, maleic acid, oxalic acid, malic acid, lactic acid or camphorsulfonic acid.

5. The method for preparing the CA-4 type tubulin aggregation inhibitor according to claim 1 or 2, characterized in that: Through a series of nucleophilic substitution reactions, reduction reactions, condensation reactions, and metal coupling reactions, the target compounds - polysubstituted aromatic nitrogen heterocyclic compounds - are obtained.

6. Use of the CA-4 class microtubule protein aggregation inhibitor according to any one of claims 1 to 4 in the preparation of drugs for preventing and treating tumor-related diseases.

7. The use according to claim 6, wherein the tumor-related disease is thyroid cancer, head and neck squamous cell carcinoma, cervical cancer, uterine cancer, ovarian cancer, breast cancer, colorectal cancer, pancreatic cancer, esophageal cancer, osteosarcoma, kidney cancer, gastric cancer, lung cancer, liver cancer, melanoma, leukemia, lymphoma, prostate cancer, bladder cancer, glioma, malignant glioblastoma, nasopharyngeal carcinoma, neuroendocrine cancer, undifferentiated cancer, stromal sarcoma, choriocarcinoma, malignant hydatidiform mole, malignant teratoma, malignant tumor and benign tumor.

8. A pharmaceutical composition for preventing and treating tumor-related diseases, comprising an effective amount of the CA-4 class microtubule protein aggregation inhibitor according to any one of claims 1 to 4.

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