3-(2-aminobenzo [d] thiazole-5-yl) benzamide derivative as well as preparation method and application thereof

Abstract

The invention discloses a series of 3-(2-aminobenzo [d] thiazole-5-yl) benzamide derivatives as well as a preparation method and application thereof, and relates to the technical field of organic medicine synthesis. According to the 3-(2-aminobenzo [d] thiazole-5-yl) benzamide derivatives, a benzimidazole part of an existing benzimidazole substituted quinazolinone derivative is replaced with benzothiazole, quinazolinone is replaced with benzamide, and a series of 3-(2-aminobenzo [d] thiazole-5-yl) benzamide derivatives are prepared. The derivatives not only can effectively inhibit ROR1 and downstream pathways thereof so as to induce G1 phase retardation and apoptosis of tumor cells, but also can significantly inhibit migration and invasion of the tumor cells. In addition, the selectivity of the derivatives to tumor cells is also greatly improved, and better safety is shown; the sensitivity of chemotherapeutic drugs such as paclitaxel (tylosin) and doxorubicin (doxorubicin) and EGFR (epidermal growth factor receptor) targeted drug gefitinib to drug-resistant cells can also be enhanced, and the derivative disclosed by the invention shows a relatively strong synergistic curative effect when being combined with the anti-tumor drug.

Description

Technical Field

[0001] The present invention relates to the technical field of organic drug synthesis, and in particular to a series of 3-(2-aminobenzo[d]thiazol-5-yl)benzamide derivatives and preparation methods and applications thereof. Background Art

[0002] Lung cancer is the leading cause of cancer mortality worldwide, with a five-year survival rate of only 15%. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases and is usually diagnosed at an advanced stage with a poor prognosis. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), including gefitinib, afatinib, erlotinib, and osimertinib, have been approved by the FDA as first-line treatments for advanced non-small cell lung cancer. Nevertheless, resistance to EGFR-TKIs has highlighted the need for new targets for NSCLC treatment.

[0003] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a member of the receptor tyrosine kinase family, characterized by an extracellular immunoglobulin-like domain, a frizzled domain, a kringle domain, and an intracellular tyrosine kinase domain. In addition, recent studies have shown that ROR1 plays a key role in maintaining the balance between the Src survival pathway and the p38 apoptosis pathway. Reduced expression of ROR1 leads to decreased phosphorylation levels of RTKs, including MET, ERBB3, and IGFIR. This leads to inhibition of the Src / PI3K pathway and dysregulation of the p38 apoptosis pathway, which in turn helps to inhibit cancer development. In view of these findings, ROR1 is expected to become a potential new target for the treatment of NSCLC.

[0004] Therapies using monoclonal antibodies or chimeric antigen receptor T cells (CAR-T) have demonstrated the therapeutic potential of ROR1 in human cancers. The use of ROR1-specific monoclonal antibodies has been shown to be an effective method to inhibit the growth of breast cancer and leukemia cells. In addition, immunotherapy with ROR1-specific CAR-T cells has shown considerable antitumor efficacy in ROR1-positive tumors. However, to our knowledge, only a few small molecule-based ROR1 inhibitors have been documented to date. Therefore, there is still a need to develop new small molecule ROR1 inhibitors. Based on this, we synthesized a series of benzimidazole-substituted quinazolinone derivatives based on the previous theory of small molecule ROR1 inhibitors, and identified a lead compound 9a (as shown in Formula 6) that showed promising antitumor activity. However, although the compound showed excellent in vitro antiproliferative activity, its selectivity for tumor cells was not very ideal. Compared with human normal embryonic lung fibroblasts MRC-5 cells, the selectivity index (SI) of the compound for H1975 cells was only 1.83. Therefore, further research is needed on new small molecule ROR1 inhibitors.

[0005]

[0006] Summary of the invention

[0007] The present invention structurally transforms the existing benzimidazole-substituted quinazolinone derivatives, replaces the benzimidazole part with benzothiazole, and replaces quinazolinone with benzamide, thereby disclosing a series of 3-(2-aminobenzo[d]thiazol-5-yl)benzamide derivatives, which greatly improve the selectivity index (SI) of existing drugs for lung cancer H1975 cells relative to human normal cells MRC-5 cells.

[0008] The technical solution adopted by the present invention is as follows:

[0009] 3-(2-aminobenzo[d]thiazol-5-yl)benzamide derivatives, the general structural formula of the 3-(2-aminobenzo[d]thiazol-5-yl)benzamide derivatives is shown in Formula 1:

[0010]

[0011] In Formula 1, R1 is selected from one of alkanes, cycloalkanes, alkanes containing heteroatoms or alkanes substituted with aromatic groups; and R2 is selected from one of alkanes, cycloalkanes or alkanes substituted with aromatic groups.

[0012] Preferably, the 3-(2-aminobenzo[d]thiazol-5-yl)benzamide derivative is one of the compounds shown in the following structure:

[0013]

[0014] The preparation method of the 3-(2-aminobenzo[d]thiazol-5-yl)benzamide derivative comprises the following steps:

[0015] (1) Synthesizing compound intermediate 3, the synthesis reaction formula of which is shown in Formula 2, the reaction conditions are: adding compound 1 dropwise to a dichloromethane solution containing compound 2, triethylamine and DMAP at 0°C to carry out an acylation reaction, after the reaction is completed, removing the solvent in vacuo, and the product is chromatographed on a silica gel column to obtain compound intermediate 3;

[0016]

[0017] (2) Synthesizing compound intermediate 5, the synthesis reaction formula of which is shown in Formula 3, the reaction conditions are: adding [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride to a solution of compound 4, biboric acid pinacol ester 8 and potassium acetate in 1,4-dioxane to react for 2 to 3 hours, so that a Suzuki coupling reaction of the borate ester occurs. After the reaction is completed, the mixture is cooled to room temperature, the solvent is removed in vacuo, dichloromethane and methanol are added to dissolve the crude product, potassium acetate is removed by suction filtration, and the concentrated filtrate is subjected to column chromatography to obtain compound 5;

[0018]

[0019] (3) Synthesizing compound intermediate 6, the synthesis reaction formula of which is shown in Formula 4, and the reaction conditions are: adding [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride to a mixed solution of intermediate compound 5, intermediate compound 3 and potassium carbonate in 1,4-dioxane and water to react for 0.5 to 3 hours, so that a Suzuki coupling reaction of boric acid ester occurs. After the reaction is completed, the mixture is cooled to room temperature, the solvent is removed in vacuo, dichloromethane and methanol are added to dissolve the crude product, potassium carbonate is removed by suction filtration, and the concentrated filtrate is subjected to column chromatography to obtain compound intermediate 6;

[0020]

[0021] (4) Synthesizing the target compound 7, the synthesis reaction formula of which is shown in Formula 5, and the reaction conditions are: at 0°C, a dichloromethane solution of differently substituted acyl chloride 9 is added dropwise to a dichloromethane solution containing compound 6, DMAP and triethylamine to cause an acylation reaction. After the reaction is completed, the solvent is removed in vacuo, dichloromethane and methanol are added to dissolve the crude product, and the product is purified by silica gel column chromatography to obtain the target compound 7;

[0022]

[0023] The application of the 3-(2-aminobenzo[d]thiazol-5-yl)benzamide derivative in the preparation of anti-tumor drugs.

[0024] The use of the 3-(2-aminobenzo[d]thiazol-5-yl)benzamide derivative in the preparation of ROR1 inhibitors.

[0025] In summary, compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] The 3-(2-aminobenzo[d]thiazol-5-yl)benzamide derivatives prepared by the present invention can not only effectively inhibit ROR1, but also show the best anti-cancer properties and good safety; they can also greatly improve the selectivity index (SI) of H1975 cells by blocking the Src survival pathway and activating the p38 apoptosis pathway, significantly inhibit the migration and invasion of H1975 cells in vitro, and induce H1975 cell cycle arrest in the G1 phase. In addition, the 3-(2-aminobenzo[d]thiazol-5-yl)benzamide derivatives prepared by the present invention can also enhance the sensitivity of chemotherapy drugs paclitaxel (Taxol) and doxorubicin (doxorubicin) and EGFR targeted drug gefitinib to their drug-resistant cells, and the derivatives of the present invention combined with the above anti-tumor drugs show a strong synergistic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The effect of compound 7h on cell proliferation, wherein A is the MTT assay to detect the inhibitory effect of different concentrations of 7h on the proliferation of lung cancer A549, PC9 and H1975 cells; B and C are the colony formation and statistical analysis of different concentrations of compound 7h inhibiting H1975 cells, respectively; the data in the figures are expressed as the mean ± SD of three independent experiments;

[0028] Figure 2 It is the docking model diagram of compound 7h in ROR1 (PDB entry code: 6TU9), where A is the binding conformation diagram of ROR1 compound 7h in the active center; B is the 2D binding mode diagram of 7h and ROR1;

[0029] Figure 3 The CETSA test result diagram of compound 7h, wherein A and B are the thermal stability diagram and statistical diagram of compound 7h and DMSO, respectively; C and D are the degradation diagram and statistical diagram of ROR1, respectively;

[0030] Figure 4 The effect of compound 7h on the ROR1 signaling pathway, wherein A is a graph showing the changes in ROR1 after H1975 cells were treated with different concentrations of 7h for 48 hours, and B is a graph showing the changes in Src after H1975 cells were treated with different concentrations of 7h for 48 hours; B is a graph showing the changes in p38 downstream of ROR1 after H1975 cells were treated with different concentrations of 7h for 48 hours; Image J was used to quantify the changes in the corresponding proteins, and the statistical graph represents the mean ± SD of three data (n = 3);

[0031] Figure 5The effect of compound 7h on the migration and invasion of H1975 cells, where A and B are the results and statistical graphs of the migration ability of H1975 cells after 24 and 48 hours of treatment with compound 7h (0, 1.75, 3.5 and 7 μM) by Transwell assay; C and D are the results and statistical graphs of the invasion ability of H1975 cells after 24 hours of incubation with compound 7h and the test compound; the scratch area and the number of invading cells in this figure were obtained by Image J. The data are expressed as mean ± SD, calculated from three random fields of three replicates;

[0032] Figure 6 The effect of compound 7h on the expression of migration and invasion-related proteins, wherein A to F respectively evaluate the changes of migration and invasion-related proteins in H1975 cells treated with compound 7h (0, 1.75, 3.5 and 7 μM) for 48 hours using Western blot analysis and related statistical graphs; the graph uses Image J to quantify the changes of the corresponding proteins, and each bar represents the mean ± standard deviation (n = 3);

[0033] Figure 7 The experimental results of compound 7h inducing cell cycle arrest are shown in Figure 1, where A is the result of PI staining to detect the effect of compound 7h on the cell cycle distribution of H1975 cells; B is a quantitative histogram of different cell cycle phases of H1975 cells after 7h treatment; C to F are bar graphs of protein expression related to the G1 phase analyzed by western blot after cells were treated with 7h for 24 hours, and the changes of corresponding proteins were quantified by Image J. Each bar represents the mean ± standard deviation (n = 3). DETAILED DESCRIPTION

[0034] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0035] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0036] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0037] The present application will be described in detail below with reference to embodiments and experimental data.

[0038] Example 1

[0039] In this example, target compounds 7a to 7r were prepared according to the following four steps.

[0040] (1) Synthesize compound intermediate 3a. The synthesis reaction formula is shown in Formula 7. The reaction conditions are: at 0°C, a dichloromethane solution of 3-bromobenzoyl chloride (compound 1) (2.18 g, 10 mmol) was added dropwise to a dichloromethane solution containing 4-fluorobenzylamine (compound 2a) (1.2 g, 10 mmol), DMAP (122.17 mg, 1 mmol) and triethylamine (1.52 g, 15 mmol), and the reaction was carried out for 10 minutes; then the temperature was raised to 30°C and the reaction was carried out for 1 hour (TLC monitoring of the completion status). After the reaction, the solvent was removed in vacuo, dichloromethane and methanol were added to dissolve the crude product, and the product was purified by silica gel column chromatography using dichloromethane:methanol=100:1 as eluent to obtain 3-bromo-N-(4-fluorobenzyl)benzamide (compound 3a), (2.94 g, 9.56 mmol) as a white solid, with a yield of 95.6%, ESI-MS: m / z 309.0 [M+H] + .

[0041]

[0042] In a similar manner, compound 1 and different primary amine compounds 2b-g were used to prepare the corresponding compounds 3b-g:

[0043] Compound 3b, 3-bromo-N-(cyclopropylmethyl)benzamide, was obtained as a white solid with a yield of 92.7%. ESI-MS: m / z 255.0 [M+H] + .

[0044] Compound 3c, 3-bromo-N-isopentylbenzamide, was obtained as a white solid with a yield of 90.3%. ESI-MS: m / z 271.0 [M+H] + .

[0045] Compound 3d, 3-bromo-N-(4-methylbenzyl)benzamide, was obtained as a white solid with a yield of 94.2%. ESI-MS: m / z 305.0 [M+H] + .

[0046] Compound 3e, 3-bromo-N-(2-methoxyethyl)benzamide, was obtained as a white solid with a yield of 96.1%. ESI-MS: m / z 259.0 [M+H] + .

[0047] Compound 3f, 3-bromo-N-(4-methoxybenzyl)benzamide, was obtained as a white solid with a yield of 93.6%. ESI-MS: m / z 321.0 [M+H] + .

[0048] The obtained compound 3g, namely 3-bromo-N-propylbenzamide, was a white solid with a yield of 90.3%. ESI-MS: m / z 243.0 [M+H] + The structural formulas of compounds 3b-g are shown below:

[0049]

[0050] (2) Synthesize compound intermediate 5, the synthesis reaction formula of which is shown in Formula 3, and the reaction conditions are: 5-bromobenzo[d]thiazol-2-amine (1.72 g, 7.5 mmol) (compound 4), KOAc (2.21 g, 22.5 mmol), bis(pinacolato)diborane (3.81 g, 15 mmol) (compound 8) and Pd(dppf)Cl2·CH2Cl2 (0.29 g, 0.35 mmol) were added to 30 mL of anhydrous 1,4-dioxane solution and stirred at 100°C for 2 hours under argon. After the reaction, the mixture was cooled to room temperature, 1,4-dioxane was removed under reduced pressure, 100 mL of saturated brine was added, and the mixture was extracted with ethyl acetate (3×80 mL), then dried over anhydrous magnesium sulfate, and purified by column chromatography to obtain 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]thiazole-2-amine (Compound 5) (yield>90%).

[0051]

[0052] (3) Synthesize compound intermediate 6a, whose synthesis reaction formula is shown in Formula 8, and the reaction conditions are: add [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride to a mixed solution of 1,4-dioxane (30 mL) and water (10 mL) containing intermediate compound 5 (1.38 g, 5 mmol), intermediate compound 3a (1.54 g, 5 mmol) and potassium carbonate (2.07 g, 15 mmol), heat the mixture to 100°C, and stir under argon for 5 hours. Suzuki coupling reaction of boric acid ester occurs. After the reaction is completed, the mixture is cooled to room temperature, the solvent is removed in vacuo, dichloromethane and methanol are added to dissolve the crude product, potassium carbonate is removed by suction, and the product is purified by silica gel column chromatography using dichloromethane / methanol to obtain a yellow solid 3-(2-aminobenzo[d]thiazol-5-yl)-N-(4-fluorobenzyl)benzamide (compound intermediate 6a) (1.71 g, 4.52 mmol, yield: 90.4%).

[0053]

[0054]

[0055] In a similar manner, compound 5 and compound intermediate 3b-g were used to prepare the corresponding compound 6b-g:

[0056] Compound 6b, 3-(2-aminobenzo[d]thiazol-5-yl)-N-(cyclopropylmethyl)benzamide, was obtained as a yellow solid with a yield of 85.6%. ESI-MS: m / z 324.4 [M+H] + .

[0057] Compound 6c, 3-(2-aminobenzo[d]thiazol-5-yl)-N-isopentylbenzamide, was obtained as a yellow solid with a yield of 80.4%. ESI-MS: m / z 340.4 [M+H] + .

[0058] Compound 6d, 3-(2-aminobenzo[d]thiazol-5-yl)-N-(4-methylbenzyl)benzamide, was obtained as a yellow solid with a yield of 86.5%. ESI-MS: m / z 374.4 [M+H] + .

[0059] Compound 6e, 3-(2-aminobenzo[d]thiazol-5-yl)-N-(2-methoxyethyl)benzamide, was obtained as a yellow solid with a yield of 90.8%. ESI-MS: m / z 328.4 [M+H] + .

[0060] Compound 6f, ie, 3-(2-aminobenzo[d]thiazol-5-yl)-N-(4-methoxybenzyl)benzamide, was obtained as a yellow solid with a yield of 88.1%. ESI-MS: m / z 390.4 [M+H] + .

[0061] Compound 6g, namely 3-(2-aminobenzo[d]thiazol-5-yl)-N-propylbenzamide, was obtained as a yellow solid with a yield of 78.9%. ESI-MS: m / z 312.4 [M+H] + The structural formulas of compounds 6b-g are shown below:

[0062]

[0063] (4) Synthesis of target compound 7a, the synthesis reaction formula of which is shown in Formula 9, the reaction conditions are: a solution of compound intermediate 6a (0.377 g, 1 mmol) and triethylamine (0.152 g, 1.5 mmol) in anhydrous dichloromethane (CH2Cl2, 25 mL) was stirred for 5 minutes. Subsequently, a solution of acetyl chloride (0.0785 g, 1 mmol) (compound 9a) in CH2Cl2 (5 mL) was added dropwise to the reaction mixture under ice-cold conditions using a dropping funnel, and the contents were further stirred at ambient temperature for 2 hours. After the reaction was completed, the mixed solution was poured into 30 mL of saturated brine, extracted with dichloromethane (30 mL×3), the combined organic fractions were washed three times with saturated NaHCO3 aqueous solution, dried over anhydrous MgSO4, and the solvent was removed under reduced pressure to obtain a solid residue, which was purified by silica gel column chromatography with dichloromethane / methanol to obtain the target compound 3-(2-acetylaminobenzo[d]thiazol-5-yl)-N-(4-fluorobenzyl)benzamide (target compound 7a) (0.315 g, 0.752 mmol) as a white solid with a yield of 75.2%. mp230.7–233.7°C.

[0064] Related spectral information of target compound 7a:

[0065] 1 H NMR (600MHz, DMSO-d6) δ12.41(s,1H),9.24(t,J=6.0Hz,1H),8.27(s,1H),8.09(d,J=1.8Hz,1H),8.08(d,J=8.2Hz,1H),7.91(dd,J=12.5, 7.7Hz,2H),7.68(dd,J=8.2,1.9Hz,1H),7.59(t,J=7.7Hz,1H),7.42–7.37(m,2H),7.19–7.14(m,2H),4.51(d,J=6.1Hz,2H),2.23(s,3H).

[0066] 13C NMR(150MHz,DMSO)δ170.01,166.52,161.64(162.44,160.84,d,J=242.1),15 9.18,149.86,140.63,138.36,136.30(136.31,136.29,d,J=205.8),135.32,1 31.52,130.26,129.71(129.74,129.68,d,J=195.9),129.61,127.01,126.01 ,122.98,122.67,119.01,115.51(115.58,115.44,d,J=174.4),42.47,23.28.

[0067] 19 F NMR(565MHz, DMSO)δ-115.99–-116.19(m),-116.21,-116.22–-116.41(m).

[0068] HRMS(ESI):C 23 H 19 FN3O2S.[M+H] + m / z:calcd.for 420.1104, found 420.1163;

[0069]

[0070] In a similar manner, intermediate compound 6b-g and different substituted acid chlorides (compound 9) were used to prepare the corresponding compounds 7b-r:

[0071] 3-(2-Acetylaminobenzo[d]thiazol-5-yl)-N-(cyclopropylmethyl)benzamide (7b), white solid, yield 76.2%. mp 269.2-272.7℃. Related spectrum information:

[0072] 1H NMR (600MHz, DMSO-d6) δ12.41 (s, 1H), 8.75 (t, J = 5.7Hz, 1H), 8.20 (d, J = 2.0Hz, 1H),8.09–8.04(m,2H),7.89(d,J=7.7Hz,1H),7.85(d,J=7.7Hz,1H),7.67(dd,J =8.2,1.8Hz,1H),7.57(t,J=7.7Hz,1H),3.18(t,J=6.2Hz,2H),2.22(s,3H),1.0 6(ddt,J=15.0,8.2,3.9Hz,1H),0.44(dt,J=8.1,3.0Hz,2H),0.27–0.21(m,2H).

[0073] 13 C NMR(150MHz,DMSO-d6)δ170.08,166.54,159.17,149.82,140.54,138.43,135.67,131.4 7,130.06,129.55,126.91,125.95,123.01,122.67,118.97,44.09,23.26,11.48,3.83.

[0074] HRMS(ESI):C 20 H 19 N3O2S[M+Na] + m / z:calcd.for 365.1198, found 388.1076.

[0075] N-(Cyclopropylmethyl)-3-(2-propionamidobenzo[d]thiazol-5-yl)benzamide (7c), white solid, yield 73.2%. mp 238.8-241.9℃. Related spectrum information:

[0076] 1H NMR(600MHz,DMSO-d6)δ12.37(s,1H),8.75(t,J=5.7Hz,1H),8.20(d,J=2.0Hz,1H), 8.09–8.05(m,2H),7.89(d,J=8.0Hz,1H),7.86(d,J=7.7Hz,1H),7.67(dd,J=8.2,1. 8Hz,1H),7.57(t,J=7.7Hz,1H),3.18(t,J=6.2Hz,2H),2.52(q,J=7.4Hz,2H),1.12( t,J=7.5Hz,3H),1.05(pd,J=7.3,3.6Hz,1H),0.46–0.42(m,2H),0.26–0.23(m,2H).

[0077] 13 C NMR(150MHz,DMSO-d6)δ173.63,166.50,159.21,149.88,140.55,138.42,135.69,131.49, 130.05,129.53,126.91,125.96,122.95,122.66,118.92,44.08,28.93,11.49,9.40,3.83.

[0078] HRMS(ESI):C 21 H 21 N3O2S[M+Na] + m / z:calcd.for 379.1354, found 402.1234.

[0079] N-(Cyclopropylmethyl)-3-(2-pivalamidobenzo[d]thiazol-5-yl)benzamide (7d), white solid, yield 68.2%. mp 211.8-214.9℃. Related spectrum information:

[0080] 1 H NMR (600MHz, DMSO-d6) δ12.10(s,1H),8.73(t,J=5.7Hz,1H),8.18(t,J=1.9Hz,1H),8.08(d,J=8.2Hz,1H),8.02(d,J=1.8Hz,1H),7.90–7.83(m,2H ),7.67(dd,J=8.2,1.7Hz,1H),7.57(t,J=7.7Hz,1H),3.18(t,J=6.2Hz,2 H),1.28(s,9H),1.09–1.02(m,1H),0.47–0.40(m,2H),0.27–0.22(m,2H).

[0081] 13 C NMR(150MHz,DMSO-d6)δ178.09,166.44,160.00,149.84,140.63,138.52,135.77,131.5 6,130.09,129.49,126.87,126.11,123.05,122.63,118.74,44.07,26.96,11.50,3.83.

[0082] HRMS(ESI):C 23 H 25 N3O2S[M+Na] + m / z:calcd.for 407.1667,found 430.1540.

[0083] N-(Cyclopropylmethyl)-3-(2-(4-fluorobenzamido)benzo[d]thiazol-5-yl)benzamide (7e), white solid, yield 71.2%. mp 208.4.8-211.5℃. Related spectrum information:

[0084] 1 H NMR (600MHz, DMSO-d6) δ13.00(s,1H),8.75(t,J=5.7Hz,1H),8.26–8.21(m,3H),8.14(d,J=8.2Hz,1H),8.10(s,1H),7.89(dd,J=19.5,7.9Hz,2H),7.7 2(dd,J=8.2,1.8Hz,1H),7.59(t,J=7.7Hz,1H),7.42(t,J=8.8Hz,2H),3.19 (t,J=6.2Hz,2H),1.10–1.03(m,1H),0.47–0.43(m,2H),0.27–0.24(m,2H).

[0085] 13 C NMR (150MHz, DMSO-d6) δ166.43,165.32(166.16,164.49,d,J=251.6),140.55,138.64,135.76,131.79(131.82,131.76,d,J=9.8 ),129.8(130.10,129.51,d,J=87.8),126.95,126.10,123.35,122.77,116.21(116.28,116.14,d,J=22.0),44.08,11.51,3.84.

[0086] 19 F NMR (565 MHz, DMSO) δ-106.39.

[0087] HRMS(ESI):C 25 H 20 N3O2S[M+H] + m / z:calcd.for 445.1260, found 446.1316.

[0088] 3-(2-Acetylaminobenzo[d]thiazol-5-yl)-N-isopentylbenzamide (7f), white solid, yield 68.2%. mp 206.6-209.9℃. Related spectrum information:

[0089] 1 H NMR (600MHz, DMSO-d6) δ12.41(s,1H),8.60(s,1H),8.17(s,1H),8.06(d,J=8.0Hz,2H),7.88(d,J=7.7Hz,1H),7.83(d,J=7.7Hz,1H),7.66 (d,J=8.2Hz,1H),7.56(d,J=8.0Hz,1H),3.33–3.29(m,2H),2.22(s,3H),1.62(dt,J=13.9,7.1Hz,1H),1.44(d,J=8.2Hz,2H),0.90(s,6H).

[0090] 13 C NMR(150MHz,DMSO-d6)δ170.00,166.33,159.17,149.86,140.53,138.44,135.81,131.47,1 29.96,129.49,126.87,125.90,122.98,122.66,118.99,38.64,38.00,25.84,23.28,22.94.

[0091] HRMS(ESI):C 21 H 23 N3O2S[M+Na] + m / z:calcd.for 381.1511,found 404.1385.

[0092] N-isopentyl-3-(2-pivalamidobenzo[d]thiazol-5-yl)benzamide (7 g), white solid, yield 66.2%. mp 206.7.8-210.1℃. Related spectrum information:

[0093] 1H NMR(600MHz,DMSO-d6)δ12.13(s,1H),8.60(s,1H),8.16(s,1H),8.09(d,J= 8.1Hz,1H),8.01(s,1H),7.88(d,J=7.7Hz,1H),7.84(d,J=7.7Hz,1H),7.66 (d,J=8.3Hz,1H),7.57(t,J=7.8Hz,1H),3.32(q,J=6.8Hz,2H),1.63(dt,J= 13.8,7.1Hz,1H),1.45(q,J=7.3Hz,2H),1.29(s,9H),0.91(d,J=6.7Hz,6H).

[0094] 13 C NMR (150MHz, DMSO-d6) δ178.09,166.37,160.00,149.83,140.62,138.52,135.85,131.56,130. 03,129.48,126.80,126.04,123.04,122.63,118.73,38.62,37.99,27.47,26.96,25.83,22.94.

[0095] HRMS(ESI):C 24 H 29 N3O2S[M+H] + m / z:calcd.for 423.1980, found 424.2046.

[0096] 3-(2-(4-Fluorobenzamido)benzo[d]thiazol-5-yl)-N-isopentylbenzamide (7h), white solid, yield 74.3%. mp 208.4-210.9℃. Related spectrum information:

[0097] 1H NMR(600MHz,DMSO-d6)δ12.98(s,1H),8.61(t,J=5.6Hz,1H),8.26–8.22(m,2H),8.19( t,J=1.9Hz,1H),8.13(d,J=8.3Hz,1H),8.09(s,1H),7.90(d,J=7.9Hz,1H),7.85(d,J= 7.7Hz,1H),7.71(dd,J=8.2,1.8Hz,1H),7.58(t,J=7.7Hz,1H),7.45–7.38(m,2H),3.3 4–3.30(m,2H),1.64(hept,J=6.7Hz,1H),1.45(q,J=7.0Hz,2H),0.92(d,J=6.6Hz,6H).

[0098] 13 C NMR (150 MHz, DMSO)δ 13 C NMR (151MHz, DMSO) δ166.45,165.31(166.14,164.48,d,J=251.1),140.53,138.63,135.80,132.61,132.55,131.78(131.81,131.74,d,J=9.4) ,129.80(130.06,129.55,d,J=77.4),126.86,126.01,123.33,122.79, 116.21(116.28,116.14,d,J=22.1)116.02,38.59,38.01,25.82,22.92.

[0099] 19 F NMR (565 MHz, DMSO) δ -106.42.

[0100] HRMS(ESI):C 26 H 24 FN3O2S[M+Na] + m / z:calcd.for 461.1573,found 484.1443.

[0101] N-(4-methylbenzyl)-3-(2-pivalamidobenzo[d]thiazol-5-yl)benzamide (7i), white solid, yield 66.8%. mp101.9-104.9℃. Related spectrum information:

[0102] 1H NMR (600MHz, DMSO-d6) δ12.11(s,1H),9.18(t,J=6.0Hz,1H),8.23(d,J=1.9 Hz,1H),8.08(d,J=8.3Hz,1H),8.02(d,J=1.8Hz,1H),7.89(dt,J=7.9,2.1Hz ,2H),7.67(dd,J=8.2,1.8Hz,1H),7.59(t,J=7.7Hz,1H),7.23(d,J=7.7Hz, 2H),7.13(d,J=7.7Hz,2H),4.47(d,J=5.9Hz,2H),2.26(s,3H),1.28(s,9H).

[0103] 13 C NMR (150MHz, DMSO) δ178.14,166.54,159.99,149.82,140.68,138.45,137.03,136.30,135.46,131. 58,130.27,129.61,129.32,127.73,126.92,126.12,123.05,122.64,118.73,42.90,26.95,21.13.

[0104] HRMS(ESI):C 27 H 27 N3O2S[M+Na] + m / z:calcd.for 457.1824, found 480.1694.

[0105] 3-(2-(Cyclohexanecarboxamido)benzo[d]thiazol-5-yl)-N-(4-methylbenzyl)benzamide (7j), white solid, yield 66.8%. mp 233.7-236.7℃. Related spectrum information:

[0106] 1H NMR (600MHz, DMSO-d6) δ12.27(s,1H),9.13(t,J=6.4Hz,1H),8.19(s,1H),8.01(d,J=8.3Hz,2H),7. 84(t,J=9.4Hz,2H),7.61(d,J=8.3Hz,1H),7.53(t,J=7.8Hz,1H),7.17(d,J=7.7Hz,2H),7.08(d,J= 7.7Hz,2H),4.42(d,J=5.9Hz,2H),2.45(s,1H),2.21(s,3H),1.80(d,J=12.5Hz,2H),1.70(d,J=12. 5Hz,2H),1.59(d,J=12.1Hz,1H),1.39(d,J=12.4Hz,1H),1.35(d,J=12.8Hz,1H),1.24–1.13(m,3H).

[0107] 13 C NMR (150MHz, DMSO-d6) δ175.71,166.45,159.26,149.88,140.61,138.37,137.08,136.26,135.46,131.54,130. 18,129.58,129.32,127.73,127.00,126.01,122.95,122.63,118.92,44.02,42.90,29.18,25.73,25.51,21.15.

[0108] HRMS(ESI):C 29 H 29 N3O2S[M+Na] + m / z:calcd.for 483.1980, found 506.1853.

[0109] 3-(2-Acetylaminobenzo[d]thiazol-5-yl)-N-(2-methoxyethyl)benzamide (7k), white solid, yield 71.7%. mp210.9-213.9℃. Related spectrum information:

[0110] 1H NMR (600MHz, DMSO-d6) δ12.41(s,1H),8.73(t,J=5.3Hz,1H),8.21(d,J=2.0Hz,1H),8.09–8.04(m,2H),7.92–7.87(m,1H) ,7.85(d,J=7.7Hz,1H),7.67(dd,J=8.2,1.8Hz,1H),7.57(t,J=7.7Hz,1H),3.49–3.49(m,4H),3.27(s,3H),2.22(s,3H).

[0111] 13 C NMR(150MHz,DMSO-d6)δ170.07,166.59,159.28,149.88,140.54,138.37,135.49,13 1.50,130.10,129.52,126.94,125.96,122.93,122.64,118.97,71.00,58.43,23.32.

[0112] HRMS(ESI):C 19 H 19 N3O3S[M+Na] + m / z:calcd.for 369.1147,found 392.1031.

[0113] N-(2-methoxyethyl)-3-(2-propionamidobenzo[d]thiazol-5-yl)benzamide (7l), white solid, yield 65.9%. mp 207.4-210.7℃. Related spectrum information:

[0114] 1 H NMR (600MHz, DMSO-d6) δ12.35(s,1H),8.71(t,J=5.3Hz,1H),8.20(s,1H),8.06(d,J=6.3Hz,2H),7.89(d,J=7.7Hz,1H),7.85(d,J=7. 7Hz,1H),7.66(d,J=8.2Hz,1H),7.56(t,J=7.7Hz,1H),3.54–3.48(m,4H),3.27(s,3H),2.53(t,J=7.5Hz,2H),1.12(t,J=7.5Hz,3H).

[0115] 13C NMR(150MHz,DMSO-d6)δ173.58,166.59,159.20,149.91,140.54,138.38,135.49,131.5 0,130.10,129.52,126.94,125.97,122.93,122.65,118.94,71.00,58.43,28.93,9.40.

[0116] HRMS(ESI):C 20 H 21 N3O3S[M+Na] + m / z:calcd.for 383.1304,found 406.1181.

[0117] 3-(2-Butyrylaminobenzo[d]thiazol-5-yl)-N-(2-methoxyethyl)benzamide (7m), white solid, yield 64.3%. mp 165.7-169.2℃. Related spectrum information:

[0118] 1 H NMR (600MHz, DMSO-d6) δ12.38(s,1H),8.73(t,J=5.1Hz,1H),8.21(d,J=1.9H z,1H),8.07(dd,J=5.0,3.2Hz,2H),7.90(d,J=8.0Hz,1H),7.85(d,J=7.7Hz,1 H),7.67(dd,J=8.3,1.7Hz,1H),7.57(t,J=7.7Hz,1H),3.49–3.46(m,4H),3.2 7(s,3H),2.48(d,J=7.3Hz,2H),1.65(h,J=7.4Hz,2H),0.92(t,J=7.4Hz,3H).

[0119] 13 C NMR(150MHz,DMSO-d6)δ172.76,166.59,159.13,149.88,140.54,138.39,135.49,131.49,1 30.10,129.52,126.93,125.98,122.96,122.65,118.95,71.00,58.43,37.47,18.49,14.02.

[0120] HRMS(ESI):C 21 H 23 N3O3S[M+Na] +m / z:calcd.for 397.1460, found 420.1337.

[0121] N-(2-methoxyethyl)-3-(2-pivalamidobenzo[d]thiazol-5-yl)benzamide (7n), white solid, yield 73.1%. 173.2-176.9℃. Related spectrum information:

[0122] 1 H NMR (600MHz, DMSO-d6) δ12.12(s,1H),8.74–8.70(m,1H),8.19(s,1H),8.08(d,J=8.1Hz,1H),8.02(s,1H),7.89(d,J=7.8 Hz,1H),7.85(d,J=7.7Hz,1H),7.67(d,J=8.2Hz,1H),7.57(t,J=7.6Hz,1H),3.48–3.47(m,4H),3.27(s,3H),1.28(s,9H).

[0123] 13 C NMR(150MHz,DMSO-d6)δ179.86,178.10,166.62,160.01,149.83,140.63,138.47,135.52,13 1.57,130.17,129.51,126.87,126.11,123.04,122.63,118.73,70.98,58.42,27.47,26.96.

[0124] HRMS(ESI):C 22 H 25 N3O3S[M+Na] + m / z:calcd.for 411.1617, found 434.1494.

[0125] 3-(2-Benzamidobenzo[d]thiazol-5-yl)-N-(2-methoxyethyl)benzamide (7o), white solid, yield 72.2%. mp121.0-123.5℃. Related spectrum information:

[0126] 1H NMR (600MHz, DMSO-d6) δ12.94(s,1H),8.75(d,J=5.8Hz,1H),8.22(s,1H),8.14(t,J=7.4Hz,3H),8.10(s,1H),7.91(d,J=7.6Hz,1H ),7.87(d,J=7.7Hz,1H),7.72(d,J=8.3Hz,1H),7.68(t,J=7.5Hz,1H),7.59(q,J=7.5,6.4Hz,3H),3.61–3.53(m,4H),3.28(s,3H).

[0127] 13 C NMR(150MHz,DMSO-d6)δ166.73,140.56,138.56,135.48,133.44,132.35,130.21 ,129.59,129.16,128.83,126.94,126.07,123.31,122.76,118.86,70.94,58.41.

[0128] HRMS(ESI):C 24 H 21 N3O3S[M+Na] + m / z:calcd.for 431.1304, found 454.1187.

[0129] N-(4-methoxybenzyl)-3-(2-propionamidobenzo[d]thiazol-5-yl)benzamide (7p), white solid, yield 72.2%. mp 233.6-236.4℃. Related spectrum information:

[0130] 1 H NMR (600MHz, DMSO-d6) δ12.36(s,1H),9.16(s,1H),8.24(s,1H),8.07(s,2H),7.94–7.85(m,2H),7.67(d,J=8.4Hz,1H),7.58(t,J=7.9 Hz,1H),7.27(d,J=8.3Hz,2H),6.89(d,J=8.2Hz,2H),4.45(d,J=5.9Hz,2H),3.71(s,3H),2.52(d,J=7.6Hz,2H),1.12(t,J=7.5Hz,3H).

[0131] 13C NMR(150MHz,DMSO-d6)δ173.77,166.60,159.34,158.77,149.98,140.70,138.47,135.56,132.12,131.6 1,130.30,129.73,129.21,127.08,126.08,123.05,122.76,119.02,114.28,55.62,42.71,29.04,9.51.

[0132] HRMS(ESI):C 25 H 23 N3O3S[M+Na] + m / z:calcd.for 445.1460, found 468.1336.

[0133] N-(4-methoxybenzyl)-3-(2-pivalamidobenzo[d]thiazol-5-yl)benzamide (7q), white solid, yield 68.6%. mp 194.6-197.5℃. Related spectrum information:

[0134] 1 H NMR (600MHz, DMSO-d6) δ12.11(s,1H),9.16(s,1H),8.22(s,1H),8.07(d,J=8.3Hz,1H),8.02(s,1H),7.89(s,2H),7.66(d, J=8.4Hz,1H),7.58(t,J=7.6Hz,1H),7.27(d,J=8.2Hz,2H),6.89(d,J=8.2Hz,2H),4.45(d,2H),3.71(s,3H),1.28(s,9H).

[0135] 13 C NMR(150MHz,DMSO-d6)δ178.16,166.52,160.00,158.67,149.81,140.67,138.45,135.48,132.00,131.5 7,130.26,129.62,129.12,126.91,126.10,123.05,122.64,118.73,114.17,55.51,42.61,26.95,26.79.

[0136] HRMS(ESI):C 27 H 27 N3O3S[M+Na] + m / z:calcd.for 473.1773, found 496.1642.

[0137] 3-(2-Pivalamidobenzo[d]thiazol-5-yl)-N-propylbenzamide (7r), white solid, yield 73.1%. mp 215.4-218.4℃. Related spectrum information:

[0138] 1 H NMR (600MHz, DMSO-d6) δ12.12(s,1H),8.64(t,J=5.7Hz,1H),8.16(s,1H),8.08(d,J=8.2Hz,1H),8.01(s,1H),7.87(d,J=7.7Hz,1H),7.84(d, J=7.7Hz,1H),7.66(d,J=8.2Hz,1H),7.57(t,J=7.7Hz,1H),3.25(q,J=6.6Hz,2H),1.55(h,J=7.3Hz,2H),1.28(s,9H),0.90(t,J=7.4Hz,3H).

[0139] 13 C NMR(150MHz,DMSO-d6)δ178.10,166.47,160.01,149.84,140.62,138.51,135.86,131.5 6,130.04,129.48,126.81,126.05,123.04,122.64,118.73,41.52,26.97,22.89,11.98.

[0140] HRMS(ESI):C 22 H 25 N3O2S[M+Na] + m / z:calcd.for 395.1667, found 418.1541.

[0141] The structures of target compounds 7a~7r are as follows:

[0142]

[0143] Example 2

[0144] In this example, the anti-tumor activity and other related properties of the target compounds 7a to 7r prepared in Example 1 were determined:

[0145] (1) Cytotoxicity was determined by MTT assay. Cells were seeded in 96-well plates (3 × 10 3Cells were cultured with different concentrations of target compounds 7a to 7r for 72 h. Cell viability was then measured using a multiwell spectrophotometer (Thermo Scientific, VARIOSKAN LUX, USA). SPSS was used to analyze the data and calculate the half inhibitory concentration (IC) of each drug. 50 As shown in Table 1, all compounds showed a series of antiproliferative activities against the tested cells. Among them, compound 7h showed the most obvious antiproliferative activity, with IC 50 The values ​​were 18.16, 8.11 and 3.5 μM, respectively. In addition, the proliferation of cells treated with 7h for 72 hours was inhibited in a dose-dependent manner. Compared with compounds 7d, 7i, 7n, 7q and 7r, compound 7g showed excellent antiproliferative activity against H1975 cells. In addition, when R2 was replaced by a straight-chain alkane, the antiproliferative activity of these compounds against the three NSCLC cell lines was reduced. Subsequently, the cytotoxicity of the synthesized compounds on human normal embryonic lung fibroblasts MRC-5 cells was evaluated in vitro. The antiproliferation results showed that the inhibitory activity of 7h on MRC-5 cells was significantly weakened, and the IC 50 The value exceeded 80 μM, which was at least 22 times lower than the inhibitory activity of H1975 cells. It is worth noting that the SI of compound 7h against H1975 cells was 22.86, while the SI of the lead compound screened by existing benzimidazole-substituted quinazolinone derivatives was 1.83. Compound 7h was at least 12 times that of the lead compound, indicating that compound 7h had better safety.

[0146] Table 1 Cytotoxic activity (IC 50 ,μM))

[0147]

[0148]

[0149] (2) In order to determine the potential therapeutic sensitization or synergistic effect of compounds 7a-7r on taxol-resistant A549 / T cells, doxorubicin-resistant MCF-7 / ADR cells, and gefitinib-resistant H1975 cells, we investigated the proliferation inhibitory activity of 5 μM 7a-7r combined with the above therapeutic drugs. The above cells in the logarithmic growth phase were seeded in 96-well plates, and after adherence, they were treated with 5 μM 7a-7r and different concentrations of taxol, doxorubicin or gefitinib to determine the IC values ​​of compounds 7a-7r against the above anti-tumor drugs. 50 As shown in Table 2, compounds 7a to 7r can reduce the resistance of A549 / T cells to Taxol, MCF-7 / ADR cells to Adriamycin, and H1975 cells to Gefitinib to varying degrees. The IC50 The values ​​were significantly reduced after combined use with 5μM compounds 7a~7r, indicating that the sensitivity of the above-mentioned drug-resistant cells to their corresponding therapeutic drugs was greatly improved. Among them, compounds 7g and 7h had the best activity in overcoming drug resistance and could significantly reduce the IC of taxol, doxorubicin and gefitinib on the corresponding drug-resistant cells. 50 The value of 7h was increased to enhance its proliferation inhibition activity. Since compound 7h also has certain anti-tumor activity, the subsequent experiments took compound 7h as an example.

[0150] Table 2 Effects of 5 μM of compounds 7a-7r on the sensitivity of various tumor cells to chemotherapy drugs

[0151]

[0152]

[0153] NT a , NT means not detected, and the above results are the average of three independent experiments.

[0154] (3) An inhibition experiment was performed on H1975 cell colony formation. H1975 cells were seeded in a 6-well plate at a density of 400 cells per well and incubated overnight. The cells were then treated with different concentrations of compound 7h (0, 1.75, 3.5, 7 μM) for 72 hours. The culture medium was replaced with fresh RPMI-1640 medium containing compound 7h every three days until colonies were clearly observed. The plates were then washed with PBS and fixed with 4% paraformaldehyde (Biosharp) for 30 minutes. After staining with 0.1% crystal violet (Solarbio), the cells were washed with PBS. Finally, the colonies were manually photographed and the number of colonies was analyzed using Image J. The results are shown in Figure 2. Figure 1 As shown in the figure, with the increase of concentration, the number of colonies in the treatment group gradually decreased. Especially when the concentration was 7 μM, the growth of cancer cells was completely inhibited. This shows that 7h can effectively inhibit the proliferation of H1975 cells in a concentration-dependent manner.

[0155] (4) Molecular docking experiment of compound 7h. In order to explore the potential binding target protein of compound 7h, we performed molecular docking simulation. Docking simulation was performed using MOE. The data were analyzed using the computational software PyMOL and MOE. The crystal structure was obtained from the PDB database for docking studies. The docking site was defined by the site finder application.

[0156] The results are as follows Figure 2As shown, 7h was observed to fit into the hydrophobic pocket and form three hydrogen bonds with residues Tyr554, Ile555, and Asp633 of ROR1, respectively. In addition, the benzene ring of isopentylbenzamide formed a π-π stacking with the Phe552 residue of ROR1.

[0157] (5) Cellular thermal shift assay: To further confirm the binding between ROR1 and compound 7h, the binding between 7h and ROR1 in H1975 cells was analyzed by cellular thermal shift assay (CETSA). 2 3×10 7 cells. After 24 h of incubation, the cells were treated with 10 μM 7h or DMSO for 4 h as drug-exposed cells and control cells. After harvesting and washing twice with PBS, the treated cells were resuspended with 900 μL PBS containing a cocktail of protease and phosphatase inhibitors and evenly divided into eight portions, each containing 100 μL of cells. Then, the experimental and control groups were heated at 37, 42, 47, 52, 57, and 62 ° C for 3 minutes, respectively. The samples underwent five freeze-thaw cycles and were then centrifuged at 14000g for 20 minutes at 4 ° C. The samples (80 μL) were transferred to Eppendorf tubes, 20 μL 5× loading buffer was added, and heated at 100 ° C for 10 minutes. Finally, the samples were placed on ice and analyzed by western blotting.

[0158] The DARTS assay was then performed. Cells were harvested and total proteins were isolated using RIPA lysis buffer containing 25, 50, or 100 μM compound 7h. After incubation at room temperature for 1 hour, pronase E was added to the lysate, and after incubation at room temperature for 10 minutes, protease inhibitors were added. The sample buffer was boiled for 10 minutes to stop the reaction and then analyzed by western blotting.

[0159] like Figure 3 As shown, the heat resistance of ROR1 was enhanced after treatment with compound 7h compared to GAPDH protein. It was observed that the compound increased the thermal stability tolerance of ROR1 from 57°C to 62°C. In addition, most of ROR1 was degraded after protease treatment of H1975 cells. However, in samples treated with the compound, the degradation of ROR1 was reduced in a dose-dependent manner. This indicates that compound 7h may directly bind to ROR1.

[0160] (6) Effect of compound 7h on ROR1 signaling pathway. H1975 cells were treated with vehicle (0.5% DMSO) or different concentrations of 7h (1.75, 3.5, 7 μM) for 48 hours and then lysed in RIPA lysis buffer to prepare protein samples. Denatured proteins were separated by electrophoresis in 10% SDS-PAGE and transferred to PVDF (polyvinylidene fluoride) (microporous) membranes. After blocking with 5% BSA for 1 hour, the membranes were incubated with primary antibodies at 4°C overnight. Subsequently, the membranes were washed three times with TBST and incubated with secondary antibodies at room temperature for 2 hours. The blots were imaged by ChemiDoc TM MP imaging system (Bio-Rad, USA).

[0161] ROR1 plays a key role in maintaining the balance between the Src survival pathway and the p38 apoptosis pathway. Western blot was used to detect the effects of compounds on ROR1, Src and p38 after 7 h. Figure 4 As shown, phosphorylation of ROR1 and its downstream survival pathway component p-Src was also found to be reduced by 7h. In addition, the p38 pro-apoptotic pathway was activated by 7h treatment, as evidenced by increased phosphorylation of p38 protein. Based on the above results, 7h may be a potential targeted ROR1 inhibitor.

[0162] (7) Effect of compound 7h on H1975 cell migration and invasion. Cancer cell migration is a hallmark of tumor invasion and metastasis and plays an important role in the progression of malignant tumors. Studies have shown that ROR1 plays an important role in the migration and invasion of tumor cells. Therefore, a wound healing assay was used to detect the effect of 7h on the migration ability of H1975 cells.

[0163] H1975 cells were seeded in 6-well plates at 5 × 10 5 cells. When the density reached 90%, the cells were scraped with a 200 μL pipette tip and gently washed with PBS to remove floating or detached cells. Subsequently, the cells were treated with different concentrations of 7h (0, 1.75, 3.5, 7 μM) in serum-free medium for 0, 24 and 48 hours. The scratch width was recorded using an inverted microscope and the results were analyzed by Image J.

[0164] Figure 5 Typical images of scratches at 0 h, 24 h, and 48 h under different concentrations of 7h are shown. The results showed that untreated H1975 cells showed higher migration ability at 24h and 48h, and the scratch closure time was 24h and 48h, respectively. In addition, with the increase in concentration, the wound healing rate slowed down in the high concentration 7h group. The results showed that the migration ability of H1975 cells was significantly inhibited after 7h, and it was concentration- and time-dependent.

[0165] On the other hand, in order to more closely mimic the physiological biomechanics of the H1975 cell invasion process, mouse EHS tumor extract Matrigel, which consists of the main basement membrane components polymerized into a gel, was added to the higher chamber to determine the anti-invasive effects of the test compounds. Figure 5 As shown, at 7 μM, the number of invasive cells treated for 7 h was much lower than that of the control group, indicating that the test compound significantly inhibited the invasion ability of the H1975 cell line.

[0166] (8) Effect of compound 7h on the expression of migration and invasion-related proteins. To further explore the mechanism of action of 7h in cell migration and invasion, western blot was used to detect the effect of 7h on the expression levels of cell migration and invasion-related proteins. First, H1975 cells were starved in serum-free medium for 2 hours. At the same time, Matrigel (BD Biosciences) was quickly diluted 1:7 with the corresponding serum-free medium on ice, and 100 μL of diluted Matrigel was pre-coated on the polycarbonate membrane of the transwell upper chamber (Costar, USA). After 30 minutes, 100 μL of warm serum-free medium was added to the inner chamber to rehydrate the basement membrane layer for 1 hour. After removing the remaining medium, 200 μL of treated cells were incubated at 10 6 The cells were seeded into the upper chamber at a density of 10 cells / mL, and 600 μL of "complete medium" containing 20% ​​FBS was added to the lower chamber in parallel. At the same time, the test compound at the specified concentration was added to the upper chamber and incubated for 24 hours. After 24 hours, the non-invaded cells were carefully wiped with a cotton swab. The invaded cells on the lower side were washed, fixed and stained with crystal violet solution for 10 minutes. Images were acquired under an optical microscope (Motic, China). The invaded cells were quantified from three random fields of three replicates. All data are listed as mean ± SD, which are from three replicate views of three random fields of view. P < 0.05 was considered significant.

[0167] The results are as follows Figure 6 As shown in the figure, the expression levels of MMP2, ZEB1 and vimentin gradually decreased with the increase of 7h concentration. At the same time, 7h could inhibit the expression of N-cadherin in H1975 cells in a dose-dependent manner, while the expression level of E-cadherin increased, indicating that 7h could inhibit the migration and invasion ability of H1975 cells in a dose-dependent manner.

[0168] (9) Compound 7h induces cell cycle arrest. Abnormal proliferation of cancer cells caused by the loss of normal cell differentiation and apoptosis is usually associated with cell dysfunction cycle. To investigate the effect of compound 7h on tumor cell cycle progression, cell cycle analysis was performed by propidium iodide (PI) staining. H1975 cells were plated at 1×10 6The cells were seeded in 6-well plates at a density of 100 μg / mL overnight. The cells were then treated with 7h for 24 h. After treatment, the cells were collected and fixed with 70% ice-cold ethanol. All samples were processed according to the manufacturer's protocol of PI / RNase staining buffer and detected by flow cytometry.

[0169] The results are as follows Figure 7 As shown in the figure, with the increase of concentration, 7h can effectively arrest H1975 cells in the G1 phase, and the corresponding G2 / M phase cell cycle distribution gradually decreases. To further clarify the possible mechanism of 7h in cell cycle arrest, we detected regulatory proteins related to the G1 phase. As the concentration of 7h increased from 1.75μM to 7μM, the expression levels of cyclin D1, c-Myc and CDK2 decreased significantly in a dose-dependent manner. The results showed that 7h can induce H1975 cell cycle arrest in the G1 phase.

[0170] Finally, it should be noted that the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article, or device. Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0171] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. 3-(2-aminobenzo[d]thiazol-5-yl)benzamide derivatives, characterized in that The general structural formula of the 3-(2-aminobenzo[d]thiazol-5-yl)benzamide derivative is shown in Formula 1: In Formula 1, R1 is selected from one of alkanes, cycloalkanes, alkanes containing heteroatoms or alkanes substituted with aromatic groups; and R2 is selected from one of alkanes, cycloalkanes or alkanes substituted with aromatic groups.

2. The 3-(2-aminobenzo[d]thiazol-5-yl)benzamide derivative according to claim 1, characterized in that The 3-(2-aminobenzo[d]thiazol-5-yl)benzamide derivative is one of the compounds shown in the following structure:

3. The method for preparing the 3-(2-aminobenzo[d]thiazol-5-yl)benzamide derivative according to claim 1 or 2, characterized in that: The steps include: (1) Synthesizing compound intermediate 3, the synthesis reaction formula of which is shown in Formula 2, the reaction conditions are: adding compound 1 dropwise to a dichloromethane solution containing compound 2, triethylamine and DMAP at 0°C to carry out an acylation reaction, after the reaction is completed, removing the solvent in vacuo, and the product is chromatographed on a silica gel column to obtain compound intermediate 3; (2) Synthesizing compound intermediate 5, the synthesis reaction formula of which is shown in Formula 3, the reaction conditions are: adding [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride to a solution of compound 4, biboric acid pinacol ester 8 and potassium acetate in 1,4-dioxane to react for 2 to 3 hours, so that a Suzuki coupling reaction of the borate ester occurs. After the reaction is completed, the mixture is cooled to room temperature, the solvent is removed in vacuo, dichloromethane and methanol are added to dissolve the crude product, potassium acetate is removed by suction filtration, and the concentrated filtrate is subjected to column chromatography to obtain compound 5; (3) Synthesizing compound intermediate 6, the synthesis reaction formula of which is shown in Formula 4, and the reaction conditions are: adding [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride to a mixed solution of intermediate compound 5, intermediate compound 3 and potassium carbonate in 1,4-dioxane and water to react for 0.5 to 3 hours, so that a Suzuki coupling reaction of boric acid ester occurs. After the reaction is completed, the mixture is cooled to room temperature, the solvent is removed in vacuo, dichloromethane and methanol are added to dissolve the crude product, potassium carbonate is removed by suction filtration, and the concentrated filtrate is subjected to column chromatography to obtain compound intermediate 6; (4) Synthesizing the target compound 7, the synthesis reaction formula of which is shown in Formula 5, and the reaction conditions are: at 0°C, a dichloromethane solution of differently substituted acyl chloride 9 is added dropwise to a dichloromethane solution containing compound 6, DMAP and triethylamine to cause an acylation reaction. After the reaction is completed, the solvent is removed in vacuo, dichloromethane and methanol are added to dissolve the crude product, and the product is purified by silica gel column chromatography to obtain the target compound 7; 4. Use of the 3-(2-aminobenzo[d]thiazol-5-yl)benzamide derivative according to claim 1 or 2 in the preparation of antitumor drugs.

5. Use of the 3-(2-aminobenzo[d]thiazol-5-yl)benzamide derivative according to claim 1 or 2 in the preparation of ROR1 inhibitors.

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