N-ortho-aminobenzoyltryptamine derivative, preparation method, composition, and use

Abstract

The present invention relates to the technical field of medicine, and specifically relates to an N-ortho-aminobenzoyltryptamine derivative, a preparation method, a composition, and a use. The structural formula is as shown, wherein any substituent in R1-4 is methyl, methoxy, halogen, nitro or trifluoromethyl, and the other substituents are hydrogen atoms; or R1-4 are combination substituents of a plurality of groups of methyl, methoxy, halogen or hydrogen atoms; and R5-8 are methyl, methoxy, halogen, trifluoromethyl or hydrogen atoms. The compound of the present invention exhibits excellent anti-tumor activity for various human tumors, especially for breast cancer, in particular triple-negative breast cancer, has potential for anti-tumor drug development, and can be used for preparing anti-tumor drugs.

Description

N-o-aminobenzoyltryptamine derivatives, preparation methods, compositions and applications Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to an N-o-aminobenzoyltryptamine derivative, a preparation method, a composition and an application thereof. Background Art

[0002] Breast cancer is a malignant tumor characterized by uncontrolled proliferation of mammary epithelial cells, leading to malignant transformation. Breast cancer ranks first among female malignancies worldwide in terms of morbidity and mortality, posing a serious threat to women's health. Comprehensive treatment for breast cancer primarily relies on surgery, supplemented by chemotherapy, endocrine therapy, immunotherapy, and radiation therapy. While cure rates for early-stage breast cancer are high with standard treatment, cure rates for late-stage breast cancer are lower, particularly for triple-negative breast cancer, which has a worse prognosis than other types. Triple-negative breast cancer is defined as breast cancer that is negative for estrogen receptors (ER), progesterone receptors (PR), and the proto-oncogene Her-2 oncogene based on immunohistochemistry. This lacks endocrine and anti-HER-2 therapeutic targets, resulting in minimal benefit from endocrine and immunotherapy. Treatment options are limited, and recurrence and metastasis are highly likely. Compared to other types of breast cancer, triple-negative breast cancer is more sensitive to chemotherapy. Current first-line clinical treatment regimens primarily consist of a combination of taxanes and anthracyclines. Once drug resistance develops during treatment, there are fewer effective alternatives.

[0003] Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an N-o-aminobenzoyltryptamine derivative, a preparation method, a composition and an application thereof, so as to improve the anti-breast cancer effect.

[0005] The present invention provides an N-o-aminobenzoyltryptamine derivative, the structural formula of which is as follows:

[0006] Among them, R 1-4 Any one of the substituents is methyl, methoxy, halogen, nitro or trifluoromethyl, and the other substituents are hydrogen atoms; or R 1-4 is substituted by a combination of multiple groups of methyl, methoxy, halogen or hydrogen atoms;

[0007] R 5-8 is a methyl group, a methoxy group, a halogen group, a trifluoromethyl group or a hydrogen atom.

[0008] R 1-4 Any one of the substituents is methyl, methoxy, halogen, nitro or trifluoromethyl, and the other substituents are hydrogen atoms, which means R 1-4Only one hydrogen atom in the group is replaced by a methyl group, a methoxy group, a halogen group, a nitro group, or a trifluoromethyl group, and the other hydrogen atoms remain unchanged. 1-4 The combination substitution of multiple groups of methyl, methoxy, halogen or hydrogen atoms means that one or more of R1, R2, R3 or R4 is methyl, methoxy, halogen or hydrogen atom.

[0009] R 5-8 is a methyl group, a methoxy group, a halogen group or a hydrogen atom, which means that one or more of R5, R6, R7 or R8 is a methyl group, a methoxy group, a halogen group or a hydrogen atom.

[0010] Preferably, the R 1-8 Not all hydrogen atoms.

[0011] Preferably, R1 and R3 are hydrogen atoms, R2 is halogen, and R4 is halogen or hydrogen atoms. More preferably, R1 and R3 are hydrogen atoms, R2 is fluorine or chlorine, and R4 is fluorine or hydrogen atoms. More preferably, R1 and R3 are hydrogen atoms, R2 is fluorine, and R4 is hydrogen atoms. In this case, R 5-8 Preferred is a hydrogen atom.

[0012] Preferably, R6 is a halogen, and R5, R7, and R8 are hydrogen atoms; or R7 is a halogen, and R5, R6, and R8 are hydrogen atoms; or R5 and R6 are halogen, and R7 and R8 are hydrogen atoms; or R5 is a halogen or methyl, and R 6-8 is a hydrogen atom; or, R8 is a halogen, a methyl group or a methoxy group, R 5-7 is a hydrogen atom; or R5 and R8 are halogen, and R6 and R7 are hydrogen atoms. In this case, preferably R1 and R3 are hydrogen atoms, R2 is fluorine, and R4 is a hydrogen atom.

[0013] Preferably, R6 is fluorine, R5, R7, and R8 are hydrogen atoms; or R7 is bromine, R5, R6, and R8 are hydrogen atoms; or R5 and R6 are fluorine, R7, and R8 are hydrogen atoms; or R5 is fluorine, chlorine, bromine, or methyl, and R 6-8 is a hydrogen atom; or, R8 is fluorine, chlorine, bromine, methyl or methoxy, R 5-7 is a hydrogen atom; or R5 and R8 are fluorine, and R6 and R7 are hydrogen atoms. In this case, preferably R1 and R3 are hydrogen atoms, R2 is fluorine, and R4 is a hydrogen atom.

[0014] Preferably, R5 is fluorine, R 6-8 is a hydrogen atom; or R6 is fluorine, R5, R 7-8 is a hydrogen atom; or R8 is fluorine, R 5-7 is a hydrogen atom; or R5, R8 are fluorine, R 6-7 is a hydrogen atom; or R 5-6 is fluorine, R 7-8In this case, it is preferred that R1 and R3 are hydrogen atoms, R2 is fluorine, and R4 is a hydrogen atom.

[0015] Some of the specific compounds are as follows:

[0016] The present invention provides a method for preparing an N-o-aminobenzoyltryptamine derivative, comprising the following steps:

[0017] The substituted phenylaminobenzoic acid and the tryptamine derivative are mixed and reacted to obtain the N-o-aminobenzoyltryptamine derivative; the reaction scheme is as follows:

[0018] where R a represents any one or more substituents R on the benzene ring 1-4 ; R b represents any one or more substituents R 5-8 .

[0019] The reaction solvent was dichloromethane.

[0020] Preferably, the preparation method of the substituted anilinobenzoic acid is to react substituted 2-bromobenzoic acid with 3,4,5-trimethoxyaniline to obtain the substituted anilinobenzoic acid. The reaction scheme is as follows:

[0021] The catalyst for the above reaction is metallic copper, the reaction solvent is N,N-dimethylformamide, the reaction temperature is 60-80° C., and the reaction time is 4-8 hours.

[0022] The preparation method of the tryptamine derivative IV is as follows: a substituted indole (compound I) is reacted with 1-(dimethylamino)-2-nitroethylene to obtain compound II; compound II is reduced with sodium borohydride to obtain compound III; compound III is reduced under the action of zinc to obtain tryptamine derivative IV; the reaction scheme is as follows:

[0023] In the reaction to obtain compound II, the solvent is preferably dichloromethane, and trifluoroacetic acid is added to the reaction system.

[0024] In the reaction to obtain compound III, the solvent is preferably a mixture of N,N-dimethylformamide and methanol.

[0025] In the reaction to obtain compound IV, the solvent was methanol, dilute hydrochloric acid was added to the reaction system, and the reaction temperature was 70°C.

[0026] The present invention provides a composition comprising the N-ophthalamide derivative and paclitaxel, wherein the molar ratio of the N-ophthalamide derivative to paclitaxel is 1-2:1.

[0027] The present invention provides an application of an N-ophthalmidine derivative, which is used to prepare a drug for treating breast cancer, liver cancer, prostate cancer, gastric cancer, or colorectal cancer. Preferably, the drug is an anti-triple-negative breast cancer drug; the drug is an anti-resistant triple-negative breast cancer drug, where the resistance is paclitaxel-resistant; or the drug is a drug that enhances the sensitivity of drug-resistant breast cancer to paclitaxel. The combined use of these drugs can enhance the sensitivity to paclitaxel and reduce the dosage of paclitaxel used in drug-resistant breast cancer.

[0028] The beneficial effects of the present invention are that the present invention discloses a novel class of N-ophthalmidine derivatives, the synthesis route of the compounds is green and environmentally friendly, simple and efficient, and the raw materials are easily available. The in vitro anti-tumor activity verified by in vivo and in vitro experiments shows that the compounds exhibit good anti-various human tumor activity, especially against breast cancer, especially triple-negative breast cancer, and have the potential for development of anti-tumor drugs. They can be used to prepare anti-tumor drugs, provide new ideas and new targets for the treatment of triple-negative breast cancer, and contribute to the development of anti-triple-negative breast cancer drugs with new targets, high efficiency and low toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 is a graph showing the anti-proliferative effects of N-o-aminobenzoyltryptamine derivatives on various human triple-negative breast cancer cell lines.

[0030] FIG2 is a graph showing the anti-proliferative effects of N-o-aminobenzoyltryptamine derivatives on various human triple-negative breast cancer cell lines.

[0031] FIG3 is a graph showing the anti-proliferative effects of N-o-aminobenzoyltryptamine derivatives on various human triple-negative breast cancer cell lines.

[0032] Figure 4 shows that NHWL09038 significantly reduced the proportion of proliferating cells in MDA-MB-231 and HCC-1806 cells compared with the Control group.

[0033] Among them, Figure 4(A) is a diagram showing the effect of different concentrations of the compound NHWL09038 on the proliferation of MDA-MB-231 cells, Figure 4(B) is a specific comparison diagram of different concentrations of the compound NHWL09038 on the proliferation of MDA-MB-231 cells, Figure 4(C) is a diagram showing the effect of different concentrations of the compound NHWL09038 on the proliferation of HCC-1806 cells, and Figure 4(D) is a specific comparison diagram of different concentrations of the compound NHWL09038 on the proliferation of HCC-1806 cells.

[0034] Figure 5 is a graph showing the effects of different concentrations of compound NHWL09038 on apoptosis of MDA-MB-231 and HCC-1806 cells compared with the control group.

[0035] 5(A) shows the effect of different concentrations of NHWL09038 on apoptosis of MDA-MB-231 cells compared with the control group; FIG5(B) shows the effect of different concentrations of NHWL09038 on apoptosis of HCC-1806 cells compared with the control group.

[0036] Figure 6 shows the effects of different concentrations of compound NHWL09038 on the invasion and migration of MDA-MB-231 and HCC-1806 cells.

[0037] Among them, Figure 6(A) is a statistical graph showing the effect of different concentrations of compound NHWL09038 on the invasion of MDA-MB-231 cells; Figure 6(B) is a statistical graph showing the effect of different concentrations of compound NHWL09038 on the migration of MDA-MB-231 cells; Figure 6(C) is a statistical graph showing the effect of different concentrations of compound NHWL09038 on the invasion of HCC-1806 cells; Figure 6(D) is a statistical graph showing the effect of different concentrations of compound NHWL09038 on the migration of HCC-1806 cells.

[0038] FIG7 shows that the compound NHWL09038 significantly inhibits the growth rate of human triple-negative breast cancer HCC-1806 cells.

[0039] Among them, Figure 7(A) is a photograph of the compound NHWL09038 inhibiting the growth of human triple-negative breast cancer HCC-1806 cells; Figure 7(B) is a statistical graph of the tumor volume of each group; Figure 7(C) is a statistical graph of the tumor volume of each group; Figure 7(D) is a statistical graph of the tumor weight of each group; and Figure 7(E) is a statistical graph of the weight of nude mice in each group.

[0040] Figure 8 shows the changes in the resistance index of compound NHWL09038 and paclitaxel in wild-type triple-negative breast cancer cells and paclitaxel-resistant triple-negative breast cancer cells.

[0041] FIG9 is a graph showing cell viability of the compound NHWL09038 in combination with paclitaxel at different concentrations in MDA-MB-231 paclitaxel-resistant cells and HCC-1806 paclitaxel-resistant cells.

[0042] Among them, Figure 9(A) shows the cell survival rate of compound NHWL09038 and paclitaxel at different concentrations in combination with MDA-MB-231 paclitaxel-resistant cells; Figure 9(B) is a statistical graph of the CI index at different concentrations of Figure 9(A); Figure 9(C) shows the cell survival rate of compound NHWL09038 and paclitaxel at different concentrations in combination with HCC-1806 paclitaxel-resistant cells; Figure 9(D) is a statistical graph of the CI index at different concentrations of Figure 9(C).

[0043] FIG10 shows the ability of the compound NHWL09038 combined with paclitaxel to significantly inhibit cell colony formation in MDA-MB-231 paclitaxel-resistant cells and HCC-1806 paclitaxel-resistant cells.

[0044] Among them, Figure 10 (A) is a graph showing that the combination of compound NHWL09038 and paclitaxel can significantly inhibit the colony formation ability of MDA-MB-231 paclitaxel-resistant cells; Figure 10 (B) is a statistical graph showing the effect of the combination of compound NHWL09038 and paclitaxel on the colony formation ability of MDA-MB-231 paclitaxel-resistant cells in Figure 10 (A); Figure 10 (C) is a graph showing that the combination of compound NHWL09038 and paclitaxel can significantly inhibit the colony formation ability of HCC-1806 paclitaxel-resistant cells;

[0045] FIG10(D) is a statistical graph showing the effect of the combination of compound NHWL09038 and paclitaxel on the colony-forming ability of paclitaxel-resistant HCC-1806 cells ( FIG10(C) ).

[0046] FIG11 shows the ability of the compound NHWL09038 to significantly inhibit cell proliferation in combination with paclitaxel in MDA-MB-231 paclitaxel-resistant cells and HCC-1806 paclitaxel-resistant cells.

[0047] 11(A) shows that the combination of compound NHWL09038 and paclitaxel can significantly inhibit cell proliferation in paclitaxel-resistant MDA-MB-231 cells; FIG11(B) is a statistical analysis of FIG11(A) compared with the Control group;

[0048] FIG11(C) shows that the combination of compound NHWL09038 and paclitaxel can significantly inhibit cell proliferation in paclitaxel-resistant HCC-1806 cells; FIG11(D) is a statistical analysis diagram of FIG11(C) compared with the Control group.

[0049] FIG12 shows that the combination of compound NHWL09038 and paclitaxel can significantly increase the proportion of cell apoptosis in MDA-MB-231 paclitaxel-resistant cells and HCC-1806 paclitaxel-resistant cells.

[0050] 12(A) shows that the combination of compound NHWL09038 and paclitaxel can significantly increase the proportion of cell apoptosis in MDA-MB-231 paclitaxel-resistant cells; FIG12(B) shows that the combination of compound NHWL09038 and paclitaxel can significantly increase the proportion of cell apoptosis in HCC-1806 paclitaxel-resistant cells.

[0051] Among them, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0052] Example 1

[0053] A method for preparing an N-o-aminobenzoyltryptamine derivative, the synthetic route of which is as follows:

[0054] The specific steps are as follows:

[0055] (1) 1-(Dimethylamino)-2-nitroethylene (1.1 mmol, 1.1 eq) was dissolved in dichloromethane and stirred in an ice bath for 5 min. Trifluoroacetic acid (FTA) (10 mmol, 10 eq) was added and stirring continued for about half an hour. Substituted indole (Compound I, 1 mmol, 1 eq) was dissolved in DCM and added to the mixed solution. The mixture was reacted at room temperature for about 2 h and detected by TLC. After the reaction was complete, the mixture was neutralized with saturated sodium bicarbonate, extracted with EA, washed with water, and concentrated by rotary evaporation under reduced pressure to obtain Compound II without further purification.

[0056] (2) Compound II (1 mmol, 1 eq) was dissolved in N,N-dimethylformamide (10 mmol / L) and methanol (10 mmol / L), and sodium borohydride (11 mmol, 11 eq) was added at 0°C. After stabilization, the mixture was allowed to react at room temperature for about 8 h. The reaction was detected by TLC, and dilute hydrochloric acid was added to quench the mixture in an ice bath. The mixture was extracted with EA and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and the crude product of the reaction solution was purified by silica gel flash column chromatography to obtain compound III.

[0057] (3) An appropriate amount of compound III (1 mmol, 1 eq) was weighed and dissolved in methanol. The mixture was evacuated and protected with argon. Zinc powder (23 mmol, 23 eq) and 1 M dilute hydrochloric acid (9 mmol, 9 eq) were added with stirring. The mixture was heated under reflux at 70°C for 5-8 h. After the reaction was complete, the mixture was cooled and neutralized with 2 M sodium hydroxide. The mixture was extracted three times with dichloromethane and concentrated to obtain compound IV without further purification.

[0058] (4) Substituted o-bromobenzoic acid V (1 mmol, 1 eq), copper powder (0.1 mmol, 0.1 eq), and potassium carbonate (2 mmol, 2 eq) were placed in a dry 100 mL round-bottom flask. An appropriate amount of N,N-dimethylformamide was added to dissolve the mixture. Under argon protection, 3,4,5-trimethoxyaniline (1.2 mmol, 1.2 eq) was added and heated at 70°C for about 5 h. After the reaction was complete, the reaction solution was cooled to room temperature, the pH was adjusted to 3, and the mixture was extracted with ethyl acetate. The crude product was purified by silica gel flash column chromatography to obtain compound IV.

[0059] (5) The intermediate substituted benzoic acid (1 eq), 5-methoxytryptamine (1.2 eq), EDCI (1.2 eq), HOBt (1.2 eq), and triethylamine (2 eq) were weighed into a round-bottom flask and dissolved in dichloromethane. The mixture was stirred magnetically at room temperature. The reaction was complete after about 12 h. The reaction solution was quenched with water, extracted with ethyl acetate, washed three times with water, concentrated, and purified by column chromatography with petroleum ether and ethyl acetate to obtain the desired solid product, i.e., the N-anthranilamide tryptamine derivative.

[0060] By using the above method, specific compounds such as Examples 1-54 were obtained depending on the substituents of the raw materials.

[0061] Example 1

[0062] N-(2-(1H-indol-3-yl)ethyl)-5-methoxy-2-((3,4,5-trimethoxyphenyl)amino)benzamide: light yellow solid (76.4% yield). 1 H NMR (500MHz, CDCl3) δ8.12(s,2H),7.64(d,J=7.9Hz,1H),7.38(d,J=8.1Hz,1H),7.27(d,J=3.6Hz,1H),7.21(t,J=7.5Hz,1H),7.13(t,J=7 .5Hz,1H),7.01(s,1H),6.93-6.85(m,2H),6.49(s,1H),6.26(s,2H),3.82(s,3H),3.80-3.74(m,8H),3.71(s,3H),3.08(t,J=6.6Hz,2H). 13 C NMR (126MHz, CDCl3) δ168.6,153.8,152.8,139.6,137.9,136.4,132.7,127.3,122.3(t,J=11.8Hz),1 19.9,119.6,118.6,118.3,112.9,112.6,111.3,96.4,77.2,77.0,76.7,61.0,56.0,55.7,40.2,25.0.

[0063] Example 2

[0064] N-(2-(1H-indol-3-yl)ethyl)-5-chloro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: light yellow solid (75.9% yield). 11H NMR (500 MHz, CDCl3) δ 9.14 (s, 1H), 8.17 (s, 1H), 7.65 (d, J = 7.9 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.25 - 7.13 (m, 5H), 7.08 (s, 1H), 6.40 (s, 2H), 6.22 (s, 1H), 3.83 (s, 3H), 3.80 (s, 6H), 3.75 (dd, J = 12.6, 6.3 Hz, 2H), 3.10 (t, J = 6.6 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 168.3, 153.8, 144.5, 137.1, 136.4, 134.1, 131.9, 127.2 (d, J = 15.7 Hz), 122.4, 122.0 (d, J = 10.9 Hz), 119.7, 119.2, 118.6, 116.7, 112.8, 111.4, 99.2, 77.3, 77.1 - 76.9 (m), 76.8, 61.0, 56.1, 40.3, 25.1.

[0065] Example 3

[0066] N-(2-(1H-indol-3-yl)ethyl)-4,5-difluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: light yellow solid (68.6% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.21 (s, 1H), 7.64 (d, J = 7.4 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H), 7.25 - 7.19 (m, 1H), 7.15 (t, J = 6.8 Hz, 1H), 7.08 (s, 1H), 7.01 (dd, J = 19.4, 11.4 Hz, 2H), 6.38 (s, 2H), 6.11 (s, 1H), 3.79 (dd, J = 38.1, 9.4 Hz, 11H), 3.10 (s, 2H). 13 13C NMR (126 MHz, CDCl3) δ 167.9, 153.9, 143.9, 141.3, 136.8, 136.5, 134.5, 127.2, 122.5, 122.1, 119.7, 118.6, 115.9 (d, J = 18.6 Hz), 113.1, 112.8, 111.4, 103.6 (d, J = 21.6 Hz), 99.7, 77.3, 77.2 - 76.9 (m), 76.8, 61.0, 56.1, 40.2, 25.1.

[0067] Example 4

[0068] N-(2-(1H-indol-3-yl)ethyl)-4-methyl-2-((3,4,5-trimethoxyphenyl)amino)benzamide: yellow solid (60.5% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.37 (s, 1H), 8.18 (s, 1H), 7.65 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.22 (t, J = 7.5 Hz, 1H), 7.13 (dd, J = 12.3, 7.4 Hz, 3H), 7.06 (s, 1H), 6.49 (d, J = 8.0 Hz, 1H), 6.45 (s, 2H), 6.23 (s, 1H), 3.85 (s, 3H), 3.81 (s, 6H), 3.76 (dd, J = 12.6, 6.4 Hz, 2H), 3.10 (t, J = 6.6 Hz, 2H), 2.24 (s, 3H). 13 13C NMR (126 MHz, CDCl3) δ 169.5, 153.7, 146.0, 142.7, 137.6, 136.5, 133.8, 127.3 (d, J = 12.3 Hz), 122.3, 122.13, 119.53, 118.7 (d, J = 5.7 Hz), 115.6, 115.3, 113.0, 111.3, 99.2, 77.3, 77.1 - 76.9 (m), 76.8, 61.0, 56.1, 40.0, 25.2, 21.7.

[0069] Example 5

[0070] N-(2-(1H-indol-3-yl)ethyl)-4-fluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (75.3% yield). 11H NMR (500 MHz, CDCl3) δ 9.61 (s, 1H), 8.18 (s, 1H), 7.65 (d, J = 7.9 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.15 (dt, J = 14.7, 7.3 Hz, 2H), 7.08 (s, 1H), 6.89 (d, J = 11.9 Hz, 1H), 6.44 (s, 2H), 6.31 (t, J = 8.0 Hz, 1H), 6.16 (s, 1H), 3.85 (s, 3H), 3.82 (s, 6H), 3.77 (dd, J = 12.5, 6.2 Hz, 2H), 3.10 (t, J = 6.6 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 168.9, 166.4, 164.4, 153.8, 148.8 (d, J = 11.8 Hz), 136.5, 134.6, 129.4 (d, J = 11.0 Hz), 127.3, 122.4, 122.1, 119.6, 118.7, 113.5, 112.9, 111.3, 104.4, 104.2, 101.1, 100.9, 100.2, 77.3, 77.1 - 76.9 (m), 76.8, 61.0, 56.1, 40.1, 25.2.

[0071] Example 6

[0072] N-(2-(1H-indol-3-yl)ethyl)-3-fluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (68.8% yield). 1 1H NMR (500 MHz, CDCl3) δ 8.02 (s, 1H), 7.73 (s, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.19 (dd, J = 18.5, 10.5 Hz, 2H), 7.11 (t, J = 7.4 Hz, 1H), 6.97 (dt, J = 12.7, 6.5 Hz, 1H), 6.91 (s, 1H), 6.61 (s, 1H), 6.03 (s, 2H), 3.79 (s, 3H), 3.75 (s, 6H), 3.71 (dd, J = 12.6, 6.3 Hz, 2H), 2.99 (t, J = 6.6 Hz, 2H). 1313C NMR (126 MHz, CDCl3) δ 167.6, 156.8, 154.8, 153.6, 140.1, 136.4, 132.7, 130.5 (d, J = 12.4 Hz), 128.0, 127.2, 123.8 (d, J = 3.2 Hz), 122.5 (d, J = 7.9 Hz), 122.3, 122.1, 119.5, 118.9, 118.7 (d, J = 13.9 Hz), 112.7, 111.3, 94.8, 77.3, 76.8, 61.0, 56.0, 40.1, 24.9.

[0073] Example 7

[0074] N-(2-(1H-indol-3-yl)ethyl)-5-bromo-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (76.0% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.16 (s, 1H), 8.21 (s, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.33 - 7.27 (m, 2H), 7.23 (t, J = 7.5 Hz, 1H), 7.15 (t, J = 8.8 Hz, 2H), 7.08 (s, 1H), 6.40 (s, 2H), 6.23 (d, J = 10.5 Hz, 1H), 3.83 (s, 3H), 3.80 (s, 6H), 3.75 (dd, J = 12.5, 6.4 Hz, 2H), 3.10 (t, J = 6.6 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 168.2, 153.8, 145.0, 137.0, 136.4, 134.7, 134.1, 130.0, 127.2, 122.4, 122.1, 119.7, 119.7, 118.6, 117.0, 112.8, 111.4, 108.8, 99.3, 77.3, 77.0, 76.8, 61.0, 56.1, 40.3, 25.1.

[0075] Example 8

[0076] N-(2-(1H-indol-3-yl)ethyl)-2-methyl-6-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (70.5% yield). 11H NMR (500 MHz, CDCl3) δ 7.97 (s, 1H), 7.60 (d, J = 7.7 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.18 (t, J = 7.4 Hz, 1H), 7.10 (d, J = 7.3 Hz, 3H), 6.91 (s, 1H), 6.70 - 6.59 (m, 2H), 6.27 (s, 2H), 5.87 (s, 1H), 3.85 (d, J = 6.2 Hz, 2H), 3.82 (s, 3H), 3.78 (s, 6H), 3.08 (t, J = 6.1 Hz, 2H), 2.23 (s, 3H). 13 13C NMR (126 MHz, CDCl3) δ 169.3, 153.8, 141.6 138.9, 136.4, 135.4, 132.7, 129.7, 127.1, 126.5, 122.4, 122.3, 122.3, 119.6, 118.7, 114.6, 112.4, 111.3, 96.6, 61.1, 56.1, 39.6, 25.3, 20.0.

[0077] Example 9

[0078] N-(2-(1H-indol-3-yl)ethyl)-3-methyl-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (62.1% yield). 1 1H NMR (500 MHz, CDCl3) δ 8.05 (s, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 7.9 Hz, 2H), 7.28 (d, J = 7.4 Hz, 1H), 7.18 (t, J = 7.5 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 7.02 (t, J = 7.6 Hz, 1H), 6.73 (s, 1H), 6.51 (s, 1H), 5.83 (s, 2H), 3.79 (s, 3H), 3.70 (s, 8H), 2.98 (t, J = 6.3 Hz, 2H), 2.17 (s, 3H). 13 13C NMR (126 MHz, CDCl 3) δ 16 8.9, 153.7, 141.9, 139.9, 136.4, 134.6, 133.8, 131.7, 129.4, 127.2, 125.9, 123.7, 122.3, 122.1, 1 19.3, % 118.6, 112.5, 111.2, 93.9, 77.3, 77.0, 76.8, 61.1, 56.0, 39.9, 24.9, 18.9.

[0079] Example 10

[0080] N-(2-(1H-indol-3-yl)ethyl)-2-fluoro-6-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (66.4% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.78 (s, 1H), 8.25 (s, 1H), 7.66 (d, J = 7.9 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 7.17 - 7.10 (m, 2H), 7.09 (s, 1H), 7.01 (d, J = 8.5 Hz, 1H), 6.85 - 6.75 (m, 1H), 6.45 (s, 2H), 6.40 (dd, J = 12.3, 8.1 Hz, 1H), 3.85 (s, 3H), 3.84 - 3.77 (m, 8H), 3.11 (t, J = 6.8 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 165.9 (d, J = 1.8 Hz), 162.8, 160.9, 153.7, 148.6 (d, J = 5.6 Hz), 137.0, 136.4, 134.3, 132.1 (d, J = 12.7 Hz), 127.2, 122.1 (d, J = 5.3 Hz), 119.4, 118.6, 112.7, 111.2, 110.6 (d, J = 2.2 Hz), 105.6 (d, J = 14.5 Hz), 104.2, 104.0, 100.1, 77.3, 76.8, 60.9, 56.1, 40.0, 25.1.

[0081] Example 11

[0082] N-(2-(1H-indol-3-yl)ethyl)-2,4-difluoro-6-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (68.5% yield). 11H NMR (500 MHz, CDCl3) δ 10.17 (s, 1H), 8.17 (s, 1H), 7.65 (d, J = 7.7 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.22 (t, J = 7.3 Hz, 1H), 7.14 (t, J = 7.2 Hz, 1H), 7.09 (s, 1H), 6.79 - 6.69 (m, 1H), 6.63 (d, J = 11.4 Hz, 1H), 6.44 (s, 2H), 6.18 - 6.07 (m, 1H), 3.82 (dd, J = 21.7, 11.4 Hz, 11H), 3.11 (t, J = 6.4 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 165.6, 153.8, 136.5, 135.9, 135.2, 127.2, 122.2 (d, J = 17.1 Hz), 119.5, 118.7, 112.8, 111.2, 101.2, 96.8, 96.6, 92.9, 92.7, 92.5, 77.3, 76.8, 61.0, 56.1, 40.0, 25.1.

[0083] Example 12

[0084] N-(2-(1H-indol-3-yl)ethyl)-4-(trifluoromethyl)-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (70.5% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.38 (s, 1H), 8.18 (s, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.52 (s, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.22 (dd, J = 14.4, 6.9 Hz, 2H), 7.14 (d, J = 7.5 Hz, 1H), 7.07 (s, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.42 (s, 2H), 6.27 (s, 1H), 3.84 (s, 3H), 3.82 - 3.73 (m, 8H), 3.11 (t, J = 6.5 Hz, 2H). 1313C NMR (126 MHz, CDCl3) δ 168.5, 153.9, 146.2, 136.4 (d, J = 12.8 Hz), 134.4, 133.9, 133.6, 128.0, 127.2, 124.7, 122.5 (d, J = 12.3 Hz), 122.1, 120.3, 119.7, 118.6, 113.4, 112.7, 111.7 (d, J = 3.8 Hz), 111.4, 99.3, 77.3, 76.8, 61.0, 56.1, 40.2, 25.1.

[0085] Example 13

[0086] N-(2-(1H-indol-3-yl)ethyl)-5-(trifluoromethyl)-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (78.5% yield).<000照原文格式保留 1 1H NMR (500 MHz, CDCl3) δ 9.63 (s, 1H), 8.16 (s, 1H), 7.66 (d, J = 7.9 Hz, 1H), 7.42 (dd, J = 15.7, 6.1 Hz, 3H), 7.25 - 7.20 (m, 2H), 7.15 (t, J = 7.5 Hz, 1H), 7.憨豆先生 10 (s, 1H), 6.45 (s, 2H), 6.28 (s, 1H), 3.85 (s, 3H), 3.82 (s, 6H), 3.78 (dd, J = 12.7, 6.4 Hz, 2H), 3.13 (t, J = 6.6 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 168.6, 153.9, 149.1, 136.5, 135.9, 135.0, 128.9, 127.2, 125.3, 124.8 (d, J = 3.7 Hz), 122.4, 122.1, 119.8, 118.6 (d, J = 10.2 Hz), 118.4, 116.4, 114.3, 112.8, 111.4, 100.6, 77.3, 76.8, 61.0, 56.1, 40.3, 25.2.

[0087] Example 14

[0088] N-(2-(1H-indol-3-yl)ethyl)-3,4-difluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (68.3% yield). 11H NMR (500 MHz, CDCl3) δ 8.25 (s, 1H), 8.13 (s, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 7.18 - 7.14 (m, 1H), 7.11 (t, J = 7.4 Hz, 1H), 6.96 (s, 1H), 6.73 (dd, J = 16.1, 8.6 Hz, 1H), 6.45 (s, 1H), 6.09 (s, 2H), 3.80 (s, 3H), 3.77 (s, 6H), 3.70 (dd, J = 12.4, 6.2 Hz, 2H), 3.01 (t, J = 6.6 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 167.3, 153.9, 153.5, 152.0, 144.5, 142.5, 139.0, 136.4, 133.5 (d, J = 8.7 Hz), 133.2, 127.2, 123.4 (d, J = 4.9 Hz), 122.3, 122.1, 121.9, 119.6, 118.6, 112.6, 111.3, 109.2 (d, J = 18.2 Hz), 95.7, 77.3, 76.8, 61.0, 56.1, 40.2, 24.9.

[0089] Example 15

[0090] N-(2-(1H-indol-3-yl)ethyl)-4-fluoro-5-methyl-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (65.3% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.35 (s, 1H), 8.24 (s, 1H), 7.66 (d, J = 7.9 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.23 (t, J = 7.5 Hz, 1H), 7.14 (t, J = 7.4 Hz, 1H), 7.08 (s, 1H), 7.00 (d, J = 8.2 Hz, 1H), 6.91 (d, J = 12.4 Hz, 1H), 6.41 (s, 2H), 6.19 (s, 1H), 3.84 (s, 3H), 3.81 (s, 6H), 3.75 (dd, J = 12.5, 6.3 Hz, 2H), 3.10 (t, J = 6.6 Hz, 2H), 2.08 (s, 3H). 1313C NMR (126 MHz, CDCl3) δ 168.9, 164.6, 162.6, 153.8, 146.2 (d, J = 11.3 Hz), 137.2, 136.4, 134.0, 130.5 (d, J = 8.0 Hz), 127.4, 122.3, 122.1, 119.6, 118.6, 114.0 - 113.6 (m), 112.9, 111.4, 101.7, 101.4, 99.2, 77.3, 76.8, 61.0, 56.1, 40.3, 25.1, 13.7 (d, J = 2.5 Hz).

[0091] Example 16

[0092] N-(2-(1H-indol-3-yl)ethyl)-4-fluoro-2-methyl-6-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (59.7% yield). 1 1H NMR (500 MHz, CDCl3) δ 8.21 (s, 1H), 7.60 (d, J = 7.9 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H), 7.18 (t, J = 7.5 Hz, 1H), 7.09 (t, J = 7.4 Hz, 1H), 6.97 (s, 1H), 6.93 (s, 1H), 6.79 - 6.67 (m, 1H), 6.34 - 6.26 (m, 3H), 5.93 (t, J = 5.4 Hz, 1H), 3.86 - 3.80 (m, 5H), 3.78 (s, 6H), 3.09 (t, J = 6.6 Hz, 2H), 2.19 (s, 3H). 13 13C NMR (126 MHz, CDCl3) δ 168.8, 164.4, 162.5, 153.8, 144.6 (d, J = 11.7 Hz), 137.7 (d, J = 10.1 Hz), 137.4, 136.5, 133.8, 127.1, 122.2, 120.9 (d, J = 2.1 Hz), 119.5, 118.5, 112.3, 111.3, 108.2, 108.1, 100.2, 100.0, 98.4, 77.3, 76.8, 6y.0, 56.1, 39.8, 25.2, 20.2.

[0093] Example 17

[0094] N-(2-(1H-indol-3-yl)ethyl)-4-bromo-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (67.8% yield). 1 H NMR (500 MHz, CDCl3) δ 9.43 (s, 1H), 8.20 (s, 1H), 7.64 (d, J = 7.4 Hz, 1H), 7.39 (d, J = 11.1 Hz, 2H), 7.25 - 7.20 (m, 1H), 7.18 - 7.11 (m, 1H), 7.08 (s, 1H), 7.02 (d, J = 8.1 Hz, 1H), 6.74 (d, J = 7.8 Hz, 1H), 6.43 (s, 2H), 6.21 (s, 1H), 3.83 (d, J = 16.9 Hz, 9H), 3.75 (d, J = 5.5 Hz, 2H), 3.10 (s, 2H). 13 C NMR (126 MHz, CDCl3) δ 168.9, 153.7, 147.4, 136.4 (d, J = 2.7 Hz), 134.3, 128.6, 127.2, 126.8, 122.4, 122.1, 120.1, 119.6, 118.6, 117.4, 116.0, 112.8, 111.4, 99.7, 77.3, 76.8, 61.0, 56.1, 40.1, 25.1.

[0095] Example 18

[0096] N-(2-(1H-indol-3-yl)ethyl)-2-(trifluoromethyl)-6-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (70.0% yield). 1 H NMR (500 MHz, CDCl3) δ 8.02 (s, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.40 (d, J = 8.3 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 7.10 (t, J = 7.2 Hz, 2H), 7.01 (s, 1H), 6.78 (s, 1H), 6.30 (s, 2H), 6.07 (t, J = 5.3 Hz, 1H), 3.89 - 3.84 (m, 2H), 3.83 (s, 3H), 3.79 (s, 6H), 3.09 (t, J = 6.7 Hz, 2H). 1313C NMR (126 MHz, CDCl3) δ 166.9, 153.8, 143.4, 137.3, 136.3, 133.8, 130.0, 128.0, 127.7, 127.0, 122.6, 122.3 (d, J = 14.1 Hz), 119.5, 119.1, 118.6, 116.8 (d, J = 4.9 Hz), 112.2, 111.2, 98.2, 77.3, 76.8, 61.0, 56.1, 40.0, 24.9.

[0097] Example 19

[0098] N-(2-(1H-indol-3-yl)ethyl)-4-chloro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (75.0% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.48 (s, 1H), 8.28 (s, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.22 (dd, J = 10.9, 4.5 Hz, 2H), 7.17 - 7.05 (m, 3H), 6.58 (dd, J = 8.4, 1.9 Hz, 1H), 6.43 (s, 2H), 6.24 (t, J = 5.2 Hz, 1H), 3.85 (s, 3H), 3.81 (s, 6H), 3.75 (dd, J = 12.5, 6.4 Hz, 2H), 3.10 (t, J = 6.6 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 168.8, 1, 147.4, 138.3, 136.4, 134.3, 128.6, 127.2, 122.2 (d, J = 19.8 Hz), 119.5, 118., 117.2, 115.5, 114.3, 112.7, 111.3, 99.8, 77.3, 76.8, 61.0, 56.0, 40.1, 25.1.

[0099] Example 20

[0100] N-(2-(1H-indol-3-yl)ethyl)-2-chloro-6-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (69.5% yield). 11H NMR (500 MHz, CDCl3) δ 8.13 (s, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.30 (s, 1H), 7.19 (t, J = 7.5 Hz, 1H), 7.11 (dt, J = 17.7, 8.3 Hz, 3H), 7.04 (s, 1H), 6.79 (d, J = 7.7 Hz, 1H), 6.32 (s, 2H), 6.23 (t, J = 5.4 Hz, 1H), 3.89 - 3.84 (m, 2H), 3.83 (s, 3H), 3.78 (s, 6H), 3.12 (t, J = 6.7 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 165.5, 152.7, 143.7, 136.4, 135.3, 132.5, 130.1, 129.7, 126.1, 121.6, 121.4, 121.2, 119.3, 118.4, 117.6, 113.0, 111.3, 110.2, 97.1, 76.3, 75.8, 60.0, 55.0, 38.9, 24.0.

[0101] Example 21

[0102] N-(2-(1H-indol-3-yl)ethyl)-3,5-difluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (64.4% yield). 1 1H NMR (500 MHz, CDCl3) δ 8.12 (s, 1H), 7.55 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.23 (dd, J = 8.2, 2.1 Hz, 1H), 7.20 (t, J = 7.6 Hz, 1H), 7.15 - 7.06 (m, 2H), 7.02 - 6.95 (m, 1H), 6.84 - 6.78 (m, 2H), 5.88 (s, 2H), 3.78 (s, 3H), 3.72 (s, 6H), 3.68 (dd, J = 12.6, 6.4 Hz, 2H), 2.94 (t, J = 6.6 Hz, 2H). 1313C NMR (126 MHz, CDCl3) δ 165.6, 159.0 (d, J = 12.0 Hz), 157.7 (d, J = 12.0 Hz), 157.1 (d, J = 11.9 Hz), 155.7 (d, J = 11.7 Hz), 153.6, 140.6, 136.3, 132.3, 130.3 (d, J = 5.6 Hz), 127.1, 130.0 (dd, J = 13.5, 3.6 Hz), 122.1 (d, J = 13.1 Hz), 119.4, 118.5, 112.4, 111.5 - 111.0 (m), 107.5, 107.2, 107.0, 93.6, 77.3, 76.8, 61.0, 55.9, 40.2, 24.8.

[0103] Example 22

[0104] N-(2-(1H-indol-3-yl)ethyl)-2-methoxy-6-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (78.8% yield). 1 1H NMR (500 MHz, CDCl3) δ 10.26 (s, 1H), 8.31 (s, 1H), 7.81 (s, 1H), 7.66 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.15 - 7.09 (m, 2H), 7.08 (d, J = 1.4 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.45 (s, 2H), 6.23 (d, J = 8.2 Hz, 1H), 3.83 (s, 3H), 3.82 - 3.78 (m, 8H), 3.51 (s, 3H), 3.10 (t, J = 6.6 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 168.2, 158.8, 153.5, 148.6, 137.8, 136.3, 133.5, 131.5, 127.4, 122.1 (d, J = 16.8 Hz), 119.4, 118.8, 113.2, 111.2, 108.5, 106.2, 100.1, 99.5, 92.5, 77.3, 76.8, 61.0, 56.0, 55.5, 39.9, 24.9.

[0105] Example 23

[0106] N-(2-(1H-indol-3-yl)ethyl)-5-fluoro-4-methyl-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (74.5% yield). 1 1H NMR (500 MHz, CDCl3) δ 8.83 (s, 1H), 8.29 (s, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.22 (t, J = 7.5 Hz, 1H), 7.16 - 7.08 (m, 2H), 7.04 (d, J = 1.6 Hz, 1H), 6.93 (d, J = 9.9 Hz, 1H), 6.42 - 6.26 (m, 3H), 3.83 (s, 3H), 3.79 (s, 6H), 3.73 (dd, J = 12.5, 6.4 Hz, 2H), 3.07 (t, J = 6.7 Hz, 2H), 2.19 (s, 3H). 13 13C NMR (126 MHz, CDCl3) δ 168.3, 154.9, 153.8, 153.0, 141.6, 138.3, 136.5, 133.4, 129.6 (d, J = 18.3 Hz), 127.2, 122.2 (d, J = 19.4 Hz), 119.6, 118.9 (d, J = 3.8 Hz), 118.7, 117.3 (d, J = 5.5 Hz), 113.4, 113.3, 112.8, 111.4, 98.2, 61.1, 56.1, 40.1, 25.2, 15.0 (d, J = 2.7 Hz).

[0107] Example 24

[0108] N-(2-(1H-indol-3-yl)ethyl)-5-fluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (70.6% yield). 11H NMR (500 MHz, CDCl3) δ 8.78 (s, 1H), 8.33 (s, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.27 - 7.18 (m, 2H), 7.13 (t, J = 7.4 Hz, 1H), 7.04 (s, 1H), 7.01 - 6.94 (m, 2H), 6.37 (t, J = 5.4 Hz, 1H), 6.34 (s, 2H), 3.83 (s, 3H), 3.78 (s, 6H), 3.74 (dd, J = 12.6, 6.4 Hz, 2H), 3.08 (t, J = 6.6 Hz, 2H), 3.08 (t, J = 6.6 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 168.2 (d, J = 1.6 Hz), 156.0, 154.1, 153.7, 141.5, 138.1, 136.4, 133.2, 127.1, 122.2 (d, J = 7.7 Hz), 119.8 (d, J = 5.5 Hz), 119.5, 119.1, 119.0, 118.5, 117.7 (d, J = 7.0 Hz), 113.8, 113.7, 112.5, 111.3, 97.8, 77.3, 76.8, 61.0, 55.9, 40.1, 25.0.

[0109] Example 25

[0110] N-(2-(1H-indol-3-yl)ethyl)-3-nitro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: red solid (67.5% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.22 (s, 1H), 8.22 (s, 1H), 8.18 - 8.09 (m, 1H), 7.80 (d, J = 7.5 Hz, 1H), 7.53 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H), 7.02 (t, J = 7.9 Hz, 1H), 6.93 (d, J = 1.4 Hz, 1H), 6.42 (t, J = 5.5 Hz, 1H), 6.12 (s, 2H), 3.75 (s, 3H), 3.74 (s, 6H), 3.44 (dd, J = 12.9, 6.7 Hz, 2H), 2.79 (t, J = 7.0 Hz, 2H). 1313C NMR (126 MHz, CDCl3) δ 166.3, 153.7, 139.8, 137.8, 136.9, 136.3, 135.9, 134.1, 128.8, 128.4, 127.1, 122.2, 121.8, 120.2, 119.5, 118.5, 112.5, 111.3, 95.8, 77.3, 76.8, 61.0, 56.0, 40.2, 24.7.

[0111] Example 26

[0112] 4-fluoro-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (76.2% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.64 (s, 1H), 8.25 (s, 1H), 7.30 - 7.24 (m, 1H), 7.18 (dd, J = 8.6, 6.4 Hz, 1H), 7.05 (s, 2H), 6.93 - 6.84 (m, 2H), 6.43 (s, 2H), 6.31 (td, J = 8.5, 2.3 Hz, lH), 6.25 (t, J = 5.4 Hz, 1H), 3.84 (s, 3H), 3.81 (d, J = 2.5 Hz, 9H), 3.74 (dd, J = 12.5, 6.3 Hz, 2H), 3.06 (t, J = 6.6 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 168.9, 166.3, 164.4, 154.0, 153.7, 148.7 (d, J = 11.7 Hz), 136.4, 134.3, 131.5, 129.5 (d, J = 11.0 Hz), 127.6, 122.9, 113.3, 112.5 (d, J = 3.5 Hz), 112.1, 104.4, 104.2, 101.0, 100.8, 100.3, 99.8, 77.3, 77.1 - 76.9 (m), 76.8, 61.0, 56.0, 55.8, 40.1, 25.1.

[0113] Example 27

[0114] N-(2-(5-chloro-1H-indol-3-yl)ethyl)-4-fluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (73.3% yield).1 1H NMR (500 MHz, CDCl3) δ 9.59 (s, 1H), 8.52 (s, 1H), 7.57 (s, 1H), 7.28 (d, J = 8.6 Hz, 1H), 7.23 (dd, J = 8.4, 6.5 Hz, 1H), 7.13 (d, J = 8.6 Hz, 1H), 7.08 (s, 1H), 6.89 (dd, J = 11.9, 1.9 Hz, 1H), 6.43 (s, 2H), 6.37 - 6.31 (m, 1H), 6.28 (t, J = 5.4 Hz, 1H), 3.84 (s, 3H), 3.79 (s, 6H), 3.71 (dd, J = 12.6, 6.4 Hz, 2H), 3.03 (t, J = 6.7 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 169.0, 166.4, 164.4, 153.7, 148.6 (d, J = 11.8 Hz), 136.4, 134.7, 134.2, 129.5 (d, J = 11.0 Hz), 128.3, 125.2, 123.6, 122.4, 118.1, 113.3 (d, J = 1.6 Hz), 1,12.6, 112.5 (d, J = 21.4 Hz), 104.5, 104.3, 101.0, 100.8, 99.9, 77.3, 76.8, 61.0, 56.0, 40.2, 25.1.

[0115] Example 28

[0116] 4 - fluoro - N - (2 - (6 - methoxy - 1H - indol - 3 - yl)ethyl)-2 - ((3,4,5 - trimethoxyphenyl)amino)benzamide: white solid (76.1% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.62 (s, 1H), 8.18 (s, 1H), 7.49 (d, J = 8.6 Hz, 1H), 7.19 (dd, J = 8.3, 6.6 Hz, 1H), 6.95 (s, 1H), 6.90 (dd, J = 11.9, 1.9 Hz, 1H), 6.86 (s, 1H), 6.79 (dd, J = 8.6, 1.6 Hz, 1H), 6.43 (s, 2H), 6.35 - 6.28 (m, 1H), 6.23 (t, J = .2 Hz, 1H), 3.84 (s, 3H), 3.83 (s, 3H), 3.80 (s, 6H), 3.73 (dd, J = 12.5, 6.3 Hz, 2H), 3.05 (t, J = 6.6 Hz, 2H). 1313C NMR (126 MHz, CDCl3) δ 168.9, 166.3, 164.3, 156.6, 153.7, 148.6 (d, J = 11.6 Hz), 137.2, 136.4, 134.3, 129.4 (d, J = 11.0 Hz), 121.6, 120.8, 119.2, 113.4, 112.7, 109.6, 104.4, 104.2, 101.0, 100.8, 99.9, 94.7, 77.3, 76.8, 61.0, 56.0, 55.6, 40.1, 25.2.

[0117] Example 29

[0118] 4-fluoro-N-(2-(6-fluoro-1H-indol-3-yl)ethyl)-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (62.0% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.60 (s, 1H), 8.30 (s, 1H), 7.53 (dd, J = 8.5, 5.3 Hz, 1H), 7.20 (dd, J = 8.4, 6.5 Hz, 1H), 7.09 - 7.00 (m, 2H), 6.95 - 6.83 (m, 2H), 6.43 (s, 2H), 6.36 - 6.28 (m, 1H), 6.20 (t, J = 5.0 Hz, 1H), 3.84 (s, 3H), 3.81 (s, 6H), 3.73 (dd, J = 12.6, 6.4 Hz, 2H), 3.06 (t, J = 6.7 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 168.9, 166.4, 164.4, 161.0, 159.1, 153.71, 148.7 (d, J = 11.7 Hz), 136.5 - 136.2 (m), 134.3, 129.4 (d, J = 11.0 Hz), 123.8, 122.3 (d, J = 3.4 Hz), 119.4 (d, J = 10.2 Hz), 113.3 (d, J = 1.7 Hz), 112.9, 108.4, 108.2, 104.5, 104.3, 101.1, 100.8, 99.8, 97.7, 97.5, 77.3, 76.8, 61.0, 56.0, 40.0, 25.2.

[0119] Example 30

[0120] 4-fluoro-N-(2-(5-fluoro-1H-indol-3-yl)ethyl)-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (60.8% yield). 1 H NMR (500 MHz, CDCl3) δ 9.60 (s, 1H), 8.30 (s, 1H), 7.33 - 7.24 (m, 2H), 7.24 - 7.16 (m, 1H), 7.11 (s, 1H), 6.95 (t, J = 8.3 Hz, 1H), 6.90 (d, J = 11.2 Hz, 1H), 6.44 (s, 2H), 6.33 (t, J = 7.3 Hz, 1H), 6.21 (s, 1H), 3.84 (s, 3H), 3.81 (s, 6H), 3.72 (dd, J = 12.4, 6.2 Hz, 2H), 3.04 (t, J = 6.5 Hz, 2H). 13 C NMR (126 MHz, CDCl3) δ 168.96 (s), 166.37 (s), 164.39 (s), 158.68 (s), 156.81 (s), 153.70 (s), 148.67 (d, J = 11.7 Hz), 136.4, 134.3, 132.8, 129.4 (d, J = 11.0 Hz), 127.6 (d, J = 9.4 Hz), 123.9, 113.3, 113.0 (d, J = 4.7 Hz), 112.0 (d, J = 9.8 Hz), 110.7, 110.5, 104.5, 104.3, 103.6, 103.5, 101.0, 100.8, 99.9, 61.0, 56.0, 40.0, 25.2.

[0121] Example 31

[0122] 4-fluoro-N-(2-(5-methyl-1H-indol-3-yl)ethyl)-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (65.5% yield). 11H NMR (500 MHz, CDCl3) δ 9.65 (s, 1H), 8.20 (s, 1H), 7.41 (s, 1H), 7.28 (d, J = 8.3 Hz, 1H), 7.21 - 7.13 (m, 1H), 7.05 (d, J = 9.2 Hz, 2H), 6.95 - 6.86 (m, 1H), 6.44 (s, 2H), 6.31 (t, J = 7.2 Hz, 1H), 6.22 (s, 1H), 3.85 (s, 3H), 3.81 (s, 6H), 3.74 (dd, J = 12.3, 6.2 Hz, 2H), 3.07 (t, J = 6.5 Hz, 2H), 2.44 (s, 3H). 13 13C NMR (126 MHz, CDCl3) δ 168.9, 166.3, 164.3, 153.7, 148.6 (d, J = 11.7 Hz), 136.4, 134.7, 134.2, 129.4 (d, J = 11.0 Hz), 128.8, 127.4, 123.9, 122.3, 118.3, 113.4, 112.2, 111.0, 104.4, 104.2, 101.0, 100.7, 99.8, 61.0, 56.0, 40.2, 25.1, 21.4.

[0123] Example 32

[0124] N-(2-(6-chloro-1H-indol-3-yl)ethyl)-4-fluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (67.8% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.58 (s, 1H), 8.31 (s, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.36 (s, 1H), 7.19 (dd, J = 8.6, 6.4 Hz, 1H), 7.11 - 7.02 (m, 2H), 6.89 (dd, J = 11.9, 2.2 Hz, 1H), 6.43 (s, 2H), 6.33 (td, J = 8.4, 2.2 Hz, 1H), 6.18 (t, J = 5.2 Hz, 1H), 3.84 (s, 3H), 3.81 (s, 6H), 3.73 (q, J = 6.5 Hz, 2H), 3.06 (t, J = 6.7 Hz, 2H). 1313C NMR (126 MHz, CDCl3) δ 169.0, 166.5, 164.5, 153.8, 148.8 (d, J = 11.6 Hz), 136.8, 136.5, 134.5, 129.5 (d, J = 11.1 Hz), 128.3, 126.0, 122.8, 120.3, 119.6, 113.4, 113.1, 111.3, 104.5, 104.4, 101.2, 101.0, 100.1, 61.1, 56.2, 40.2, 25.2.

[0125] Example 33

[0126] N-(2-(5-bromo-1H-indol-3-yl)ethyl)-4-fluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (66.0% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.58 (s, 1H), 8.47 (s, 1H), 7.72 (s, 1H), 7.27 - 7.18 (m, 3H), 7.05 (s, 1H), 6.88 (dd, J = 11.9, 1.6 Hz, 1H), 6.41 (s, 2H), 6.33 (dd, J = 11.5, 4.6 Hz, 1H), 6.24 (t, J = 5.1 Hz, 1H), 3.82 (s, 3H), 3.78 (s, 6H), 3.69 (dd, J = 12.5, 6.3 Hz, 2H), 3.01 (t, J = 6.6 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 169.0, 166.4, 164.4, 153.7, 148.6 (d, J = 11.8 Hz), 136.4, 134.9, 134.2, 129.5 (d, J = 11.1 Hz), 129.0, 125.0, 123.4, 121.2, 113.2, 112.8 (d, J = 13.6 Hz), 112.5, 104.6, 104.4, 101.0, 100.8, 99.8, 61.0, 56.0, 40.2, 25.​​​​​​​1H NMR (500 MHz, CDCl3) δ 9.58 (s, 1H), 8.30 (s, 1H), 7.51 (s, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.23 - 7.17 (m, 2H), 7.04 (s, 1H), 6.90 (dd, J = 11.9, 2.2 Hz, 1H), 6.43 (s, 2H), 6.36 - 6.30 (m, 1H), 6.17 (d, J = 4.9 Hz, 1H), 3.84 (s, 3H), 3.81 (s, 6H), 3.72 (dd, J = 12.7, 6.4 Hz, 2H), 3.06 (t, J = 6.7 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 169.1, 166.5, 164.5, 153.8, 148.7 (d, J = 11.6 Hz), 137.3, 136.6, 134.4, 129.6 (d, J = 10.9 Hz), 126.3, 122.8 (d, J = 13.6 Hz), 120.0, 115.8, 114.4, 113.4, 113.0, 104.6, 104.4, 101.2, 101.1 (d, J = 26.1 Hz), 100.0, 61.1, 56.1, 40.2, 25.2.

[0129] Example 35

[0130] N-(2-(7-chloro-1H-indol-3-yl)ethyl)-4-fluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (55.3% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.62 (s, 1H), 8.48 (s, 1H), 7.53 (d, J = 6.7 Hz, 1H), 7.20 (s, 2H), 7.12 (s, 1H), 7.05 (s, 1H), 6.90 (d, J = 11.1 Hz, 1H), 6.44 (s, 2H), 6.32 (s, 1H), 6.24 (s, 1H), 3.82 (d, J = 17.7 Hz, 9H), 3.74 (s, 2H), 3.07 (s, 2H). 1313C NMR (126 MHz, CDCl3) δ 168.9, 166.4, 164.4, 153.7, 148.7 (d, J = 11.7 Hz), 136.4, 134.3, 133.6, 129.4 (d, J = 11.0 Hz), 128.7, 122.7, 121.6, 120.3, 117.3, 116.8, 114.0, 113.2 (d, J = 1.6 Hz), 104.4, 104.2, 101.0, 100.8, 99.8, 61.0, 56.0, 40.1, 25.3.

[0131] Example 36

[0132] 4-fluoro-N-(2-(7-fluoro-1H-indol-3-yl)ethyl)-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (53.6% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.61 (s, 1H), 8.52 (s, 1H), 7.39 (d, J = 7.9 Hz, 1H), 7.20 (dd, J = 8.3, 6.6 Hz, 1H), 7.10 (s, 1H), 7.03 (td, J = 7.7, 4.9 Hz, 1H), 6.95 - 6.87 (m, 2H), 6.44 (s, 2H), 6.35 - 6.28 (m, 1H), 6.23 (t, J = 5.3 Hz, 1H), 3.84 (s, 3H), 3.81 (s, 6H), 3.74 (q, J = 6.4 Hz, 2H), 3.08 (t, J = 6.7 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 168.9, 166.4, 164.4, 153.7, 150.6, 148.7 (d, J = 12.0 Hz), 136.4, 134.3, 131.0 (d, J = 5.4 Hz), 129.4 (d, J = 11.1 Hz), 124.7 (d, J =​​​​​N-(2-(7-bromo-1H-indol-3-yl)ethyl)-4-fluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (72.7% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.60 (s, 1H), 8.34 (s, 1H), 7.65 - 7.64 (m, 1H), 7.64 - 7.63 (m, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.37 (d, J = 7.6 Hz, 1H), 7.19 (dd, J = 8.5, 6.5 Hz, 1H), 7.14 (s, 1H), 7.01 (t, J = 7.7 Hz, 1H), 6.90 (dd, J = 11.9, 2.0 Hz, 1H), 6.44 (s, 2H), 6.37 - 6.29 (m, 1H), 6.18 (t, J = 5.0 Hz, 1H), 3.84 (s, 3H), 3.82 (s, 6H), 3.74 (dd, J = 12.7, 6.4 Hz, 2H), 3.07 (t, J = 6.7 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 168.9, 166.4, 164.4, 153.7, 148.7 (d, J = 11.7 Hz), 136.4, 135.1, 134.4, 129.4 (d, J = 11.1 Hz), 128.4, 124.6, 122.7, 120.8, 117.9, 114.2, 113.2, 104.9, 104.4, 104.3, 101.0, 100.8, 99.9, 61.0, 56.1, 40.1, 25.4.

[0135] Example 38

[0136] 4-fluoro-N-(2-(7-methyl-1H-indol-3-yl)ethyl)-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (63.0% yield). 11H NMR (500 MHz, CDCl3) δ 9.63 (s, 1H), 8.17 (s, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.18 (dd, J = 8.6, 6.4 Hz, 1H), 7.11 - 7.01 (m, 3H), 6.90 (dd, J = 12.0, 2.2 Hz, 1H), 6.22 (t, J = 5.0 Hz, 1H), 3.85 (s, 3H), 3.81 (s, 6H), 3.76 (dd, J = 12.5, 6.4 Hz, 2H), 3.09 (t, J = 6.6 Hz, 2H), 2.50 (s, 3H). 13 13C NMR (126 MHz, CDCl3) δ 168.9, 166.31, 164.3, 153.7, 148.6 (d, J = 11.8 Hz), 136.4, 136.0, 134.3, 129.5 (d, J = 11.0 Hz), 126.7, 122.8, 121.8, 120.6, 119.8, 116.3, 113.3 (d, J = 17.7 Hz), 104.4, 104.2, 101.0, 100.8, 99.8, 61.0, 56.0, 40.1, 25.3, 16.5.

[0137] Example 39

[0138] 4-fluoro-N-(2-(6-methyl-1H-indol-3-yl)ethyl)-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (66.9% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.64 (s, 1H), 8.21 (s, 1H), 7.�2 (d, J = 8.1 Hz, 1H), 7.18 (dd, J = 8.鈥�, 5.8 Hz, 2H), 7.02 - 6.94 (m, 2H), 6.91 (dd, J = 11.9, 2.2 Hz, 1H), 6.44 (s, 2H), 6.31 (td, J = 8.4, 2.2 Hz, 1H), 6.24 (t, J = 5.2 Hz, 1H), 3.85 (s, 3H), 3.81 (s, 6H), 3.74 (dd, J = 12.5, 6.3 Hz, 2H), 3.07 (t, J = 6.6 Hz, 2H), 2.46 (s, 3H). 1313C NMR (126 MHz, CDCl3) δ 168.9, 166.3, 164.3, 153.7, 148.6 (d, J = 11.8 Hz), 136.9, 136.4, 134.2, 132.1, 129.5 (d, J = 11.0 Hz), 125.0, 121.5, 121.2, 118.2, 113.4 (d, J = 1.7 Hz), 112.5, 111.3, 104.4, 104.2, 101.0, 100.8, 99.8, 61.0, 56.0, 40.0, 25.2, 21.6.

[0139] Example 40

[0140] 4-fluoro-N-(2-(7-methoxy-1H-indol-3-yl)ethyl)-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (51.0% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.62 (s, 1H), 8.33 (s, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.16 (dd, J = 8.6, 6.4 Hz, 1H), 7.06 (t, J = 7.8 Hz, 2H), 6.89 (dd, J = 12.0, 2.3 Hz, 1H), 6.68 (d, J = 7.7 Hz, 1H), 6.44 (s, 2H), 6.31 (td, J = 8.3, 2.3 Hz, 1H), 6.15 (t, J = 5.2 Hz, 1H), 3.96 (s, 3H), 3.84 (s, 3H), 3.82 (s, 6H), 3.75 (q, J = 6.3 Hz, 2H), 3.08 (t, J = 6.6 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 168.9, 166.4, 164.4, 153.7, 148.7 (d, J = 11.8 Hz), 146.3, 136.5, 134.4, 129.4 (d, J = 11.0 Hz), 128.5, 127.0, 121.7, 120.1, 113.3 (d, J = 20.0 Hz), 111.4, 104.4, 104.2, 102.1, 101.0, 100.8, 99.9, 61.0, 56.1, 55.3, 40.1, 25.3.

[0141] Example 41

[0142] 4-fluoro-N-(2-(4-fluoro-1H-indol-3-yl)ethyl)-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (54.2% yield). 1 H NMR (500 MHz, CDCl3) δ 9.59 (s, 1H), 8.55 (s, 1H), 7.24 (dd, J = 8.5, 6.5 Hz, 1H), 7.14 (d, J = 8.1 Hz, 1H), 7.08 (td, J = 7.9, 5.2 Hz, 1H), 7.00 (s, 1H), 6.89 (dd, J = 11.9, 2.0 Hz, 1H), 6.76 (dd, J = 11.2, 7.7 Hz, 1H), 6.42 (s, 2H), 6.35 - 6.26 (m, 2H), 3.84 (s, 3H), 3.80 (s, 6H), 3.75 (dd, J = 12.6, 6.4 Hz, 2H), 3.16 (t, J = 6.7 Hz, 2H). 13 C NMR (126 MHz, CDCl3) δ 169.0, 166.3, 164.3, 158.0, 156.1, 153.7, 148.5 (d, J = 11.5 Hz), 139.2 (d, J = 11.6 Hz), 136.5, 134.2, 129.4 (d, J = 11.0 Hz), 122.6 (d, J = 7.8 Hz), 122.5, 116.0, 115.9, 113.5, 111.4 (d, J = 2.6 Hz), 107.5 (d, J = 3.5 Hz), 104.5 (d, J = 19.8 Hz), 104.2, 101.0, 100.8, 99.8, 61.0, 56.0, 40.8 (d, J = 1.5 Hz), 26.3.

[0143] Example 42

[0144] N-(2-(4-chloro-1H-indol-3-yl)ethyl)-4-fluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (62.5% yield). 11H NMR (500 MHz, CDCl3) δ 9.59 (s, 1H), 8.63 (s, 1H), 7.26 (td, J = 5.9, 3.0 Hz, 2H), 7.07 (dt, J = 4.8, 3.2 Hz, 3H), 6.90 (dd, J = 11.9, 2.1 Hz, 1H), 6.42 (s, 2H), 6.37 - 6.30 (m, 2H), 3.83 (s, 3H), 3.81 - 3.75 (m, 8H), 3.31 (t, J = 6.7 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 169.0, 166.3, 164.3, 153.7, 148.5 (d, J = 11.8 Hz), 137.9, 136.5, 134.2, 129.4 (d, J = 11.0 Hz), 126.0, 123.9, 122.6, 120.4, 113.5 (d, J = 1.6 Hz), 113.0, 110.2, 104.5, 104.3, 101.0, 100.8, 99.8, 61.0, 56.0, 41.3, 26.0.

[0145] Example 43

[0146] 4-fluoro-N-(2-(4-methoxy-1H-indol-3-yl)ethyl)-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (73.2% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.60 (s, 1H), 8.30 (s, 1H), 7.15 - 7.06 (m, 2H), 7.01 (d, J = 8.1 Hz, 1H), 6.94 (s, 1H), 6.88 (d, J = 11.9 Hz, 1H), 6.82 (s, 1H), 6.54 (d, J = 7.8 Hz, 1H), 6.42 (s, 2H), 6.29 (t, J = 8.1 Hz, 1H), 3.93 (s, 3H), 3.83 (s, 3H), 3.80 (s, 6H), 3.70 (dd, J = 11.3, 5.5 Hz, 2H), 3.21 (t, J = 6.1 Hz, 2H). 1313C NMR (126 MHz, CDCl3) δ 169.1, 166.2, 164.2, 153.9, 153.7, 148.4 (d, J = 11.6 Hz), 138.1, 136.5, 134.1, 129.4 (d, J = 10.9 Hz), 122.9, 121.5, 117.2, 113.8, 113.4, 105.1, 104.2, 104.0, 100.8, 100.6, 99.6 (d, J = 19.4 Hz), 61.0, 56.0, 55.2, 42.1, 25.6.

[0147] Example 44

[0148] N-(2-(4-bromo-1H-indol-3-yl)ethyl)-4-fluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (58.0% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.58 (s, 1H), 8.30 (s, 1H), 7.51 (s, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.23 - 7.17 (m, 2H), 7.04 (s, 1H), 6.90 (dd, J = 11.9, 2.2 Hz, 1H), 6.43 (s, 2H), 6.36 - 6.30 (m, 1H), 6.17 (d, J = 4.9 Hz, 1H), 3.84 (s, 3H), 3.81 (s, 6H), 3.72 (dd, J = 12.7, 6.4 Hz, 2H), 3.06 (t, J = 6.7 Hz, 2H). 13 13C NMR (126 MHz, None) δ 169.1, 166.4, 164.4, 153.8, 148.7 (d, J = 11.7 Hz), 137.9, 136.6, 134.3, 129.6 (d, J = 11.0 Hz), 125.3, 124.4, 124.0, 123.0, 114.1, 113.6, 113.5, 110.9, 104.6, 104.4, 101.1, 100.9, 99.9 (d, J = 6.7 Hz), 61.1, 56.1, 41.5, 25.9.

[0149] Example 45

[0150] 4-fluoro-N-(2-(4-methyl-1H-indol-3-yl)ethyl)-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (59.8% yield). 1 H NMR(500MHz,CDCl3)δ9.62(s,1H),8.19(s,1H),7.25 - 7.20(m,2H),7.09(t,J=7.6Hz,1H),7.03(d,J=2.1Hz,1H),6.94 - 6.84(m,2H),6.44(s, 2H),6.34(td,J=8.5,2.5Hz,1H),6.21(t,J=5.3Hz,1H),3.85(s,3H),3.82(s,6H),3.75(q,J=6.6Hz,2H),3.25(t,J=6.7Hz,2H),2.73(s,3H). 13 C NMR(126MHz,CDCl3)δ168.9,166.4,164.4,153.7,148.7(d,J=11.8Hz),136.9,136.4,134.3,130.6,129.4(d,J=11.1Hz),125.6,122.3(d,J=13.7Hz),121.3,113.4(d,J=13.0Hz),109.2,104.4,104.3,101.1,100.8,99.9,61.0,56.1,40.8,26.9,20.4.

[0151] Example 46

[0152] N-(2-(4,7-difluoro-1H-indol-3-yl)ethyl)-4-fluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (68.5% yield). 11H NMR (500 MHz, CDCl3) δ 9.57 (s, 1H), 8.69 (s, 1H), 7.29 - 7.24 (m, 1H), 7.04 (d, J = 1.4 Hz, 1H), 6.90 (dd, J = 12.0, 2.3 Hz, 1H), 6.78 (td, J = 10.0, 3.3 Hz, 1H), 6.69 - 6.61 (m, 1H), 6.43 (s, 2H), 6.35 (td, J = 8.4, 2.3 Hz, 1H), 6.30 (t, J = 5.2 Hz, 1H), 3.84 (s, 3H), 3.82 (s, 6H), 3.75 (dd, J = 12.7, 6.5 Hz, 2H), 3.14 (t, J = 6.7 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 171.3, 169.0, 166.4, 164.4, 153.7, 151.8, 148.6 (d, J = 11.8 Hz), 146.8, 144.9 (d, J = 2.9 Hz), 136.4, 134.3, 129.4 (d, J = 11.0 Hz), 126.97 - 126.47, 123.2, 118.7 (dd, J = 22.6, 5.3 Hz), 113.4 (d, J = 1.6 Hz), 112.6, 106.6 (dd, J = 19.1, 8.6 Hz), 104.4, 104.2, 103.9 (dd, J = 22.9, 6.8 Hz), 101.0, 100.8, 99.8, 61.0, 56.0, 40.7, 26.0.

[0153] Example 47

[0154] 4-fluoro-N-(2-(7-(trifluoromethyl)-1H-indol-3-yl)ethyl)-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (72.2% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.61 (s, 1H), 8.50 (s, 1H), 7.83 (d, J = 7.9 Hz, 1H), 7.48 (d, J = 7.4 Hz, 1H), 7.23 - 7.15 (m, 3H), 6.90 (dd, J = 12.0, 2.4 Hz, 1H), 6.45 (s, 2H), 6.34 (td, J = 8.3, 2.4 Hz, 1H), 6.15 (t, J = 5.3 Hz, 一H), 3.84 (s, 3H), 3.82 (s, 6H), 3.75 (q, J = 6.6 Hz, 2H), 3.12 (t, J = 6.8 Hz, 2H).13 13C NMR (126 MHz, CDCl3) δ 169.0, 166.5, 164.5, 153.8, 148.8 (d, J = 11.8 Hz), 136.4, 134.5, 131.9, 129.4 (d, J = 11.0 Hz), 128.9, 126.0, 123.2, 122.7, 119.9 (d, J = 4.5 Hz), 119.0, 113.7, 113.5, 113.2, 104.5, 104.3, 101.1, 100.9, 100.0, 61.0, 56.1, 40.1, 25.1.

[0155] Example 48

[0156] N-(2-(4,6-difluoro-1H-indol-3-yl)ethyl)-4-fluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (65.6% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.55 (s, 1H), 8.58 (s, 1H), 7.31 - 7.20 (m, 1H), 6.96 (s, 1H), 6.89 (dd, J = 11.9, 2.2 Hz, 1H), 6.85 (dd, J = 9.0, 1.5 Hz, 1H), 6.59 (dd, J = 15.2, 5.6 Hz, 1H), 6.42 (s, 2H), 6.35 (td, J = 8.3, 2.2 Hz, 1H), 6.28 (t, J = 5.4 Hz, 1H), 3.84 (s, 3H), 3.80 (s, 6H), 3.73 (q, J = 6.5 Hz, 2H), 3.12 (t, J = 6.7 Hz, 2H). 1313C NMR (126 MHz, CDCl3) δ 169.0, 166.4, 164.4, 160.2 (d, J = 12.1 Hz), 158.3 (d, J = 12.2 Hz), 157.3 (d, J = 14.8 Hz), 155.3 (d, J = 15.1 Hz), 153.7, 148.6 (d, J = 11.8 Hz), 138.1 (t, J = 14.3 Hz), 136.5, 134.2, 129.4 (d, J = 11.1 Hz), 122.6 (d, J = 2.7 Hz), 113.5 (d, J = 1.7 Hz), 112.6 (d, J = 19.2 Hz), 111.6 (d, J = 2.7 Hz), 104.5, 104.3, 101.0, 100.8, 99.8, 95.4, 95.2 (d, J = 4.7 Hz), 95.0, 94.0 (d, J = 4.4 Hz), 93.8 (d, J = 4.3 Hz), 61.0, 56.0, 40.6, 26.2.

[0157] Example 49

[0158] N-(2-(5,6-difluoro-1H-indol-3-yl)ethyl)-4-fluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (78.3% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.57 (s, 1H), 8.38 (s, 1H), 7.33 (dd, J = 10.4, 7.9 Hz, 1H), 7.23 (dd, J = 8.4, 6.5 Hz, 1H), 7.14 (dd, J = 10.4, 6.7 Hz, 1H), 7.08 (s, 1H), 6.89 (dd, J = 11.9, 1.8 Hz, 1H), 6.44 (s, 2H), 6.35 (m, J = 11.3, 4.9 Hz, 1H), 6.23 (t, J = 5.0 Hz, 1H), 3.84 (s, 3H), 3.81 (s, 6H), 3.71 (dd, J = 12.7, 6.5 Hz, 2H), 3.01 (t, J = 6.7 Hz, 2H). 1313C NMR (126 MHz, CDCl3) δ 169.0, 166.4, 164.4, 153.7, 149.0 (d, J = 15.8 Hz), 148.7 (d, J = 11.8 Hz), 147.2 (dd, J = 27.5, 15.5 Hz), 145.4 (d, J = 14.8 Hz), 136.4, 134.3, 131.3 (d, J = 10.5 Hz), 129.4 (d, J = 11.0 Hz), 123.4 (d, J = 3.6 Hz), 122.6 (d, J = 7.5 Hz), 113.3, 113.1 (d, J = 3.5 Hz), 105.2 (d, J = 19.0 Hz), 104.5, 104.3, 101.1, 100.9, 99.9, 99.3, 99.1, 61.0, 56.0, 40.0, 25.2.

[0159] Example 50

[0160] 4-fluoro-N-(2-(5-methoxy-7-methyl-1H-indol-3-yl)ethyl)-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (73.5% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.64 (s, 1H), 8.06 (s, 1H), 7.19 (dd, J = 8.7, 6.3 Hz, 1H), 7.05 (d, J = 2.1 Hz, 1H), 6.89 (dd, J = 11.8, 2.3 Hz, 2H), 6.71 (d, J = 1.0 Hz, 1H), 6.43 (s, 2H), 6.31 (td, J = 8.5, 2.4 Hz, 1H), 6.23 (t, J = 5.3 Hz, 1H), 3.84 (s, 3H), 3.81 (d, J = 4.5 Hz, 9H), 3.74 (dd, J = 12.4, 6.4 Hz, 2H), 3.05 (t, J = 6.5 Hz, 2H), 2.45 (s, 3H). 13 13C NMR (126 MHz, CDCl3) δ 168.9, 166.3, 164.4, 154.2, 153.7, 148.7 (d, J = 11.6 Hz), 136.4, 134.3, 131.2, 129.5 (d, J = 11.0 Hz), 127.0, 122.4, 121.7, 113.2 (dd, J = 27.6, 8.3 Hz), 104.4, 104.2, 101. (d, J = 11.0 Hz), 127.0, 122.4, 121.7, 113.2 (dd, J = 27.6, 8.3 Hz), 104.4, 104.2, 101.0, 100.8, 99.9, 97.8, 61.0, 56.0, 55.7, 40.1, 25.2, 16.6.

[0161] Example 51

[0162] N-(2-(4,5-difluoro-1H-indol-3-yl)ethyl)-4-fluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (80.2% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.56 (s, 1H), 8.56 (s, 1H), 7.28 (dd, J = 6.5, 4.2 Hz, 1H), 7.07 - 6.95 (m, 3H), 6.89 (dd, J = 11.9, 2.1 Hz, 1H), 6.42 (s, 2H), 6.34 (ddd, J = 11.3, 8.9, 3.8 Hz, 2H), 3.84 (s, 3H), 3.80 (s, 6H), 3.75 (dd, J = 12.8, 6.5 Hz, 2H), 3.15 (d, J = 6.8 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 171.3, 169.0, 166.3, 164.4, 153.7, 148.6 (d, J = 11.7 Hz), 145.1 (d, J = 11.6 Hz), 144.6 (d, J = 14.4 Hz), 143.2 (d, J = 11.4 Hz), 142.7 (d, J = 14.4 Hz), 136.5, 134.7 (d, J = 10.0 Hz), 134.2, 129.4 (d, J = 11.0 Hz), 124.2, 117.0 (d, J = 15.7 Hz), 113.5, 111.9, 111.7, 106.7 (dd, J = 7.5, 4.2 Hz), 104.5, 104.3, 101.0, 100.8, 99.8, 61.0, 56.0, 40.8, 26.2, 14.1.

[0163] Example 52

[0164] N-(2-(4-bromo-6-methoxy-1H-indol-3-yl)ethyl)-4-fluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (78.0% yield). 11H NMR (500 MHz, CDCl3) δ 9.59 (s, 1H), 8.16 (s, 1H), 7.27 (d, J = 9.3 Hz, 1H), 6.98 (d, J = 8.1 Hz, 2H), 6.90 (dd, J = 12.0, 2.0 Hz, 1H), 6.79 (s, 1H), 6.43 (s, 2H), 6.39 - 6.32 (m, 1H), 6.23 (t, J = 5.0 Hz, 1H), 3.84 (s, 3H), 3.81 (d, J = 3.9 Hz, 9H), 3.80 - 3.76 (dd, 2H), 3.28 (t, J = 6.7 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 169.0, 166.4, 164.4, 156.5, 153.8, 148.7 (d, J = 11.3 Hz), 138.2, 136.5, 134.5, 129.4 (d, J = 10.9 Hz), 122.8, 120.0, 114.2, 114.0, 113.6, 104.5, 104.3, 101.1, 100.9, 100.0, 94.4, 61.0, 56.1, 55.9, 41.2, 25.8.

[0165] Example 53

[0166] N-(2-(5,6-dimethoxy-1H-indol-3-yl)ethyl)-4-fluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (64.2% yield). 1 1H NMR (500 MHz, CDCl3) δ 9.65 (s, 1H), 8.09 (s, 1H), 7.18 (dd, J = 8.5, 6.5 Hz, 1H), 7.02 (s, 1H), 6.95 (s, 1H), 6.91 - 6.83 (m, 2H), 6.43 (s, 2H), 6.38 - 6.27 (m, 1H), 6.23 (t, J = 5.1 Hz, 1H), 3.89 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H), 3.81 (s, 6H), 3.74 (dd, J = 12.3, 6.2 Hz, 2H), 3.04 (t, J = 6.4 Hz, 2H). 13C NMR (126MHz, CDCl3) δ168.9,166.4,164.4,153.7,148.7(d,J=11.6Hz),147.2,145.0,136.4,134.3,130.6,129.4(d,J=1 1.0Hz),120.5,119.9,113.3,112.7,104.4,104.2,101.0,100.8,100.2,99.8,94.6,61.0,56.2,56.1,56.0,40.2,25.2.

[0167] Example 54

[0168] N-(2-(4-chloro-6-methoxy-1H-indol-3-yl)ethyl)-4-fluoro-2-((3,4,5-trimethoxyphenyl)amino)benzamide: white solid (65.0% yield). 1 H NMR(500MHz, CDCl3)δ9.58(s,1H),8.39(s,1H),7.32-7.22(m,1H),6.93(s,1H),6.89(dd,J=11.9,1.6Hz,1H),6.77(s,1H),6 .72(s,1H),6.41(s,2H),6.32(m,J=13.4,9.5,3.4Hz,2H),3.83(s,3H),3.76(dd,J=16.7,7.0Hz,11H),3.25(t,J=6.6Hz,2H). 13 C NMR (126MHz, CDCl3) δ169.0,166.3,164.3,156.3,153.7,148.6(d,J=11.6Hz),138.3,136.5,134.2,129.4(d,J=11.0 Hz),126.2,122.5,118.6,113.5,112.9,110.5,104.4,104.3,101.0,100.8,99.8,93.7,61.0,56.0,55.7,41.1,26.0.

[0169] Example 55

[0170] Methodological study on MTT tumor drug sensitivity test

[0171] The target compound was accurately weighed and diluted to a concentration of 10 mmol / L as a mother solution, filtered for sterilization, and sealed and stored at 4 ° C for later use. Various tumor cells (MCF-7, SK-BR-3, MDA-MB-231, HS-578T, HCC-1806, HCT-116, HT-29, AGS, HGC-27, MKN28, HepG2, PC-3) were cultured in advance and grown to the logarithmic growth phase. The adherent cells in the logarithmic growth phase were then digested with trypsin (suspension cells do not need to be digested). 100 μl of cell suspension was added to each well, and 10 cells were added to each well. 10,000 to 10,000 cells were seeded in a 96-well plate, and 100 μl of PBS buffer was added to the surrounding wells. The cells were cultured in a 37°C cell culture incubator (5% CO2, 90% H2O) until the cells adhered. The culture medium containing the target compound was then added, and the experimental group was added according to the concentration gradient dilution (final concentrations of 100, 50, 25, 12.5, 6.25, 1.62, 0.54, 0.18, 0.06, and 0.02 μmol / L). 100 μl of the test compound was added to each well, with three parallel control wells set up for each concentration. The positive control group was treated with the same volume of cisplatin solution at the same concentration gradient, and the blank control group was treated with 100 μl of culture medium. The cultures were continued for 72 hours. 20 μl of MTT solution was added to each well and the cultures were continued for 4 hours. After that, the cultures were terminated and the culture medium was aspirated. 150 μl of DMSO was added to each well and shaken to fully dissolve the crystalline formazan. The absorbance was measured at 570 nm in a microplate reader. The inhibition rate of the target compound was calculated according to the formula, and the IC50 value was calculated using software. The results are shown in Figures 1, 2, and 3, indicating that N-phenyltryptamine salicylate compounds have significant anti-cancer cell proliferation effects.

[0172] Paclitaxel-resistant triple-negative breast cancer cells MDA-MB-231 / PTX and HCC-1806 / PTX (determined as resistant cells based on changes in resistance index and WB) were cultured to the logarithmic growth phase, and the adherent cells in the logarithmic growth phase were digested with trypsin digestion solution. 80 μl of cell suspension was added to each well, 3000-5000 cells per well, and plated in a 96-well plate. 100 μl of PBS buffer was added to the surrounding wells: the cells were cultured in a 37°C cell culture incubator (5% CO2, 90% H2O) until the cells adhered; then the culture medium containing the target compound to be tested (NHWL09038, paclitaxel) was added. The experimental group Add the test compound diluted according to a concentration gradient (the highest concentration is 3200nmol / L, 2-fold dilution, a total of 15 concentration gradients), add 20μl of the test sample to each well, and set up three parallel control wells for each concentration; add 20μl of fresh complete culture medium to the blank control group and continue to culture for 72h; add 20μl of MTT solution to each well and continue to culture for 4h; then terminate the culture and aspirate the culture medium in the well; add 150μl of DMSO to each well and shake to fully dissolve the crystalline formazan; place the plate in a microplate reader at 570nm to measure the absorbance value, calculate the inhibition rate of the target compound according to the formula, and then use the software to calculate the IC50 value.

[0173] The ratio of the IC50 of resistant triple-negative breast cancer cells to the IC50 of wild-type triple-negative breast cancer cells was used as the drug resistance index. The results, as shown in Figure 8, show that paclitaxel exhibited strong cytotoxicity in wild-type cells and significantly reduced activity in paclitaxel-resistant breast cancer cells. In contrast, NHWL09038 exhibited similar cytotoxicity in wild-type and paclitaxel-resistant breast cancer cells, indicating that NHWL09038 is still effective in paclitaxel-resistant breast cancer cells and has the potential to overcome paclitaxel resistance in breast cancer.

[0174] Combination drug analysis

[0175] Based on the Chou-Talalay method, the combination index (CI) was calculated to evaluate the interaction between NHWL09038 and paclitaxel in MDA-MB-231 / PTX and HCC-1806 / PTX cells. Drug-resistant cells were exposed to serially diluted NHWL09038 and PTX mixtures at varying concentrations for 72 hours. The combined cytotoxicity was assessed using the MTT assay. CompuSyn software was used to calculate the CI by comparing cell viability at varying concentrations of paclitaxel alone or in combination with the same concentration of NHWL09038. A CI < 1 indicates synergistic effect; a CI = 1 indicates additive effect; and a CI > 1 indicates antagonism between the two drugs. The results are shown in Figure 9. At low concentrations, NHWL09038 enhanced the sensitivity of paclitaxel-resistant breast cancer cells to paclitaxel.

[0176] Example 56

[0177] Tumor cell cycle detection experiment

[0178] The cell cycle refers to the entire process that continuously dividing cells go through from the end of one mitosis to the end of the next mitosis. During this process, the cell's genetic material replicates and doubles, and at the end of the division, it is evenly distributed between the two daughter cells. The cell cycle can be divided into interphase and mitosis. The interphase is often divided into the dormant period (G0), the early DNA synthesis period (G1), the DNA synthesis period (S), and the late DNA synthesis period (G2). The entire cycle can be expressed as G1-S-G2-M. DNA cycle detection can be used to reflect the status of each phase of the cell cycle, that is, the cell proliferation status. Utilizing the property of intracellular DNA that can bind to fluorescent dyes (such as propidium iodide (PI), the amount of DNA in each cell phase varies, and thus the amount of fluorescent dye bound varies, resulting in different fluorescence intensities detected by flow cytometry.

[0179] The specific method is:

[0180] 1. Breast cancer cells MDA-MB-231 and HCC-1806 were treated with NHWL09038 (0.1, 0.2, 0.4 μM), and a negative control group was set up. After 24 hours, the cells were digested and collected.

[0181] 2. Wash the cells once with PBS, collect them by centrifugation at 1500 rpm for 5 min, adjust the cell concentration to 1×106 / mL, and take 1 mL of single-cell suspension.

[0182] 3. After centrifugation of the prepared single-cell suspension, remove the supernatant and add 500uL of 70% pre-cooled ethanol to the cells for fixation for 2 hours to overnight. Store at 4°C and wash away the fixative with PBS before staining. If necessary, filter the cell suspension once with a 200-mesh cell sieve.

[0183] 4. Add 100 μl of RNase A solution to the cell pellet, resuspend the cells, and incubate in a 37°C water bath for 30 minutes.

[0184] 5. Add 400 μl of PI staining solution, mix well, and incubate at 4°C in the dark for 30 minutes.

[0185] 6. Test on the machine and record the red fluorescence at the excitation wavelength of 488nm.

[0186] The results are shown in FIG4 , which indicate that compound NHWL09038 has a strong G2 / M cycle arrest effect on breast cancer cells MDA-MB-231 and HCC-1806.

[0187] Example 57

[0188] Tumor cell apoptosis detection experiment

[0189] The Annexin V-FITC Apoptosis Detection Kit uses FITC-labeled recombinant Annexin V to detect phosphatidylserine (PS), which is expressed on the cell membrane during apoptosis. Detection can be performed using flow cytometry, fluorescence microscopy, or other fluorescence detection equipment. Annexins are a class of calcium-dependent phospholipid-binding proteins widely distributed in the cytoplasm of eukaryotic cells and involved in intracellular signal transduction. However, only Annexin V has been reported to regulate the activity of some PKCs. Annexin V selectively binds to phosphatidylserine (PS). PS is primarily distributed on the inner side of the cell membrane, adjacent to the cytoplasm. In the early stages of apoptosis, different cell types externalize phosphatidylserine to the cell surface, the outer side of the cell membrane. Exposed phosphatidylserine on the cell surface promotes coagulation and inflammation. Annexin V binds to externalized phosphatidylserine on the cell surface and blocks its procoagulant and proinflammatory activities. Annexin V labeled with the green fluorescent probe FITC, or Annexin V-FITC, allows for the direct and simple detection of phosphatidylserine externalization, a key hallmark of apoptosis, using flow cytometry or fluorescence microscopy. Furthermore, propidium iodide (PI) staining can stain necrotic cells or cells that have lost their membrane integrity in the late stages of apoptosis, producing red fluorescence. In necrotic cells, due to the loss of cell membrane integrity, Annexin V-FITC can enter the cytoplasm and bind to phosphatidylserine located on the inner side of the cell membrane, thereby also causing the necrotic cells to exhibit green fluorescence.

[0190] The specific steps are:

[0191] a. Treat MDA-MB-231 and HCC-1806 cells in a 6-well plate with NHWL09038 for 24 hours. Then, aspirate the cell culture medium into a suitable centrifuge tube, wash adherent cells once with PBS, and add an appropriate amount of trypsin (EDTA-free) to digest the cells. Incubate at room temperature until adherent cells can be dislodged by gentle pipetting, then aspirate the trypsin solution. Avoid over-digestion with trypsin. Note: The trypsinization step is critical for adherent cells. If the trypsinization time is too short, cells will require vigorous pipetting to dislodge, which can damage the cell membrane and lead to false positives for cell necrosis. If the trypsinization time is too long, this can also damage the cell membrane and result in false positives for cell necrosis. It can also affect the binding of phosphatidylserine to Annexin V-FITC on the cell membrane, interfering with apoptosis detection. Furthermore, the trypsin solution should be EDTA-free, as EDTA may interfere with the binding of Annexin V to phosphatidylserine.

[0192] b. Add the cell culture medium collected in step a. Gently pipette the cells to dislodge them, transfer them to a centrifuge tube, and centrifuge at 1000g for 5 minutes. Discard the supernatant, collect the cells, gently resuspend them in PBS, and count them. Note: Adding the cell culture medium from step a is very important. Firstly, it allows the collection of apoptotic or necrotic cells that have already been suspended. Second, the serum in the cell culture medium effectively inhibits or neutralizes residual trypsin. Residual trypsin will digest and degrade the subsequently added Annexin V-FITC, resulting in staining failure.

[0193] c. Take 50,000-100,000 resuspended cells, centrifuge at 1000g for 5 minutes, discard the supernatant, and add 200μl Annexin V-FITC binding solution to gently resuspend the cells.

[0194] d. Add 5 μl Annexin V-FITC and mix gently.

[0195] e. Add 10 μl of propidium iodide staining solution and mix gently.

[0196] f. Incubate at room temperature (20-25°C) in the dark for 10-20 minutes, then place in an ice bath. Use aluminum foil to protect from light. Resuspend the cells 2-3 times during the incubation period to improve staining.

[0197] g. Immediately after that, the detection was performed on the instrument. Annexin V-FITC showed green fluorescence and propidium iodide (PI) showed red fluorescence.

[0198] The results are shown in FIG5 , which show that NHWL09038 significantly induced apoptosis of MDA-MB-231 and HCC-1806.

[0199] The specific steps for drug-resistant cells are:

[0200] a. MDA-MB-231 / PTX cells were treated with NHWL09038 (0.2 μM), PTX (0.1 μM), and the combination group NHWL09038+PTX (0.2 μM+0.1 μM); HCC-1806 / PTX cells were treated with NHWL09038 (0.7 μM), PTX (0.4 μM), and the combination group NHWL09038+PTX (0.7 μM+0.4 μM). At the same time, a negative control group and a FITC and PI single-stained tube group without drug treatment were set up. After 72 hours, the cells were digested and collected.

[0201] b. Add the cell culture medium collected in step a, gently pipette off the cells, transfer them to a centrifuge tube, and centrifuge at 2000 rpm and 4°C for 5 minutes. Discard the supernatant, collect the cells, gently resuspend the cells in PBS, and count them.

[0202] c. Take 50,000-100,000 resuspended cells, centrifuge at 2000 rpm, 4°C for 5 minutes, discard the supernatant, and add 100 μl Annexin V-FITC binding solution to gently resuspend the cells.

[0203] d. Add 5 μl of Annexin V-FITC, mix gently, and incubate at room temperature in the dark for 5 minutes.

[0204] e. Add 5 μl of propidium iodide staining solution, mix gently, and then incubate in an ice bath in the dark for 5 minutes.

[0205] f. Immediately after that, the detection was performed on the instrument. Annexin V-FITC showed green fluorescence and propidium iodide (PI) showed red fluorescence.

[0206] The results are shown in Figure 12, which show that the apoptosis-inducing ability of the combination group was significantly better than that of the single-drug group. NHWL09038 significantly enhanced the apoptosis-inducing ability of paclitaxel in paclitaxel-resistant breast cancer cells MDA-MB-231 / PTX and HCC-1806 / PTX.

[0207] Example 58

[0208] Breast cancer cell invasion and migration assay

[0209] Matrigel is a matrix component extracted from mouse EHS sarcoma, containing LN, type IV collagen, contact protein and heparin sulfate polysaccharide. It is spread on a polycarbonate filter membrane without polyvinylpyrrolidone and can reconstruct a membrane structure in DMEM culture medium. This membrane structure is very similar to the natural matrix membrane structure.

[0210] The pore size of the filter membrane is generally 8 μm, and the membrane pores are covered with Matrigel. Cells cannot pass through freely and must secrete hydrolases and undergo deformation movement to pass through the filter membrane covered with Matrigel1, which is similar to the situation in the body.

[0211] Treat MDA-MB-231 and HCC-1806 cells in a 6-well plate with NHWL09038 for 24 hours. Afterward, aspirate the cell culture medium into a suitable centrifuge tube, wash adherent cells once with PBS, and digest them with an appropriate amount of trypsin (EDTA-free). Collect and count the cells. Place a Martrigel-coated filter membrane between the upper and lower chambers of a Blind Well or MICS chamber, with the Martrigel-coated side facing the upper chamber. Add a chemoattractant, such as 20% serum-containing cell culture medium, to the lower chamber. Resuspended MDA-MB-231 and HCC-1806 cells are added to the upper chamber. Invasive cells, induced by the chemoattractant, 20% serum, initiate transmembrane migration. The time it takes for cells to transmembrane migration is related to the amount of Martrigel used, but observation after 36 hours is ideal. Most cells that passed through the filter adhered to the lower surface. Cells on the upper surface were removed with a cotton swab. The filter was then fixed with methanol and stained with 1% crystal violet. Matrigel was omitted from the migration assay, and the remaining steps were the same as for the invasion assay. Results are shown in Figure 6.

[0212] Example 59

[0213] Drug-resistant cell clone formation assay

[0214] Cells that survive by adhering to the surface do not necessarily proliferate and form colonies, but individual cells that can form clones must have proliferative activity. Therefore, the clone formation rate of individual cells can directly reflect the cell population dependency and its own proliferation capacity.

[0215] The specific operation steps are as follows: MDA-MB-231 / PTX and HCC-1806 / PTX cells in the logarithmic growth phase were obtained, digested, collected and counted, 500-1000 cells per well were seeded into six-well plates, and cultured overnight in a 37°C cell culture incubator (5% CO2, 90% H2O) until the cells adhered. MDA-MB-231 / PTX was treated with NHWL09038 (0.2μM), PTX (0.1μM), and the combination group NHWL09038 + PTX (0.2μM + 0.1μM); HCC-1806 / PTX was treated with NHWL09038 (0.35μM), PTX (0.2μM), and the combination group NHWL09038 + PTX (0.35μM + 0.2μM). After five days of drug treatment, the cells were replaced with fresh drug-free culture medium until visible cell colonies appeared, at which point the culture was terminated. Surviving cell colonies were washed three times with PBS, fixed with 1 ml of 4% paraformaldehyde per well for 15 minutes, washed once with PBS, stained with 1 ml of 0.1% crystal violet per well for 15 minutes, washed three to five times with PBS, and photographed and counted. As shown in Figure 10, the combination of NHWL09038 and PTX significantly inhibited colony formation in paclitaxel-resistant breast cancer cells, MDA-MB-231 / PTX and HCC-1806 / PTX.

[0216] Example 60

[0217] Breast cancer cell EdU proliferation assay

[0218] EdU (5-ethynyl-2'-deoxyuridine) is a thymidine analog that can be inserted into replicating DNA molecules during cell proliferation. Based on the conjugation reaction between EdU and the dye, efficient and rapid cell proliferation detection and analysis can be performed.

[0219] The specific procedure is as follows: MDA-MB-231 and HCC-1806 cells in a 96-well plate were treated with NHWL09038 for 24 hours. An equal volume of EdU working solution was added to the culture medium of the growing cells to a final EdU concentration of 10 μM. The cells were incubated in a 37°C CO2 incubator for 2 hours. After EdU labeling, all liquid was aspirated from the wells, and 100 μl of 4% paraformaldehyde was added to each well for fixation at room temperature for 15 minutes. After fixation, the fixative was removed, and the cells were washed three times (5 minutes each) with 100 μl of wash solution (3% BSA) per well. The wash solution was removed, and 100 μl of permeabilization solution (PBS containing 0.3% Triton X-100) was added to each well and incubated at room temperature for 15 minutes. The permeabilization solution was removed, and the cells were washed twice (5 minutes each) with 100 μl of wash solution per well. Add 50 μl of the prepared Azide 488 staining solution to each well and incubate on a shaker at room temperature in the dark for 30 minutes. Aspirate the staining solution and wash three times with wash buffer for 3-5 minutes each. After aspirating the wash buffer, add 100 μl of 1X Hoechst 33342 solution to each well and incubate at room temperature in the dark for 10 minutes. Aspirate the 1X Hoechst 33342 solution and wash three times with wash buffer for 3-5 minutes each. Observe and photograph under a fluorescence microscope and count the cells. Azide 488 emits green fluorescence with a maximum excitation wavelength of 495 nm and a maximum emission wavelength of 519 nm. Hoechst 33342 emits blue fluorescence with a maximum excitation wavelength of 346 nm and a maximum emission wavelength of 460 nm. The ratio of green fluorescence to blue fluorescence is calculated to represent the proportion of proliferating cells. As shown in Figure 4, NHWL09038 significantly inhibits the proliferation of MDA-MB-231 and HCC-1806 cells.

[0220] EdU proliferation assay of drug-resistant cells

[0221] MDA-MB-231 / PTX cells were treated with NHWL09038 (0.2μM), PTX (0.1μM), and the combination group NHWL09038 + PTX (0.2μM + 0.1μM) for 72 hours. HCC-1806 / PTX cells were treated with NHWL09038 (0.35μM), PTX (0.2μM), and the combination group NHWL09038 + PTX (0.35μM + 0.2μM) for 72 hours. The remaining steps were the same as the EdU proliferation assay described above for breast cancer cells. The results, shown in Figure 11, show that the combination group significantly inhibited the proliferation of paclitaxel-resistant breast cancer cells MDA-MB-231 / PTX and HCC-1806 / PTX compared to the single-drug group.

[0222] Example 61

[0223] Effects on the HCC-1806 triple-negative breast cancer model in vivo

[0224] This model, which involves subcutaneous implantation of triple-negative breast cancer cells (HCC-1806), boasts rapid tumor formation, a high tumorigenesis rate, a short experimental cycle, and excellent reproducibility, facilitating a variety of experimental studies. This study used immunohistochemistry, HE staining, and immunofluorescence to investigate the expression of the compound NHWL09038 during the proliferation of HCC-1806 triple-negative breast cancer cells in Balb / c nude mice, exploring its role in the proliferation of triple-negative breast cancer.

[0225] The study found that both the volume and number of cells injected subcutaneously into nude mice affected the success rate of this model. The optimal inoculation volume was 100 μL, with a cell count of 5,000,000 per mouse. This provided a reference for the cell inoculation volume and density used in this experiment. Tumor formation typically occurs around two weeks after tumor loading.

[0226] 1 Experimental animals

[0227] Healthy 4-week-old female Balb / c nude mice weighing 20-30 g were provided by Wenzheng Biological and met clean-grade standards. The relevant guidelines for the care and protection of laboratory animals were adhered to during the breeding and experiments.

[0228] Cell culture HCC-1806 cells were cultured and subcultured in 1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 50 U / mL streptomycin, 5% carbon dioxide, and 37°C. When the cells were approximately 80% of the logarithmic growth phase, they were digested with 0.25% trypsin. The digestion solution was collected and centrifuged, and the supernatant was removed. After washing twice with PBS, the cells were prepared into a cell suspension with 1640 basal medium for inoculation. The cell viability to be inoculated should be >90%, and the number of cells to be inoculated was 5×10 6 Prepare 100 μL of the solution for future use. Aliquot into 1.5 mL centrifuge tubes and store and transport in an insulated container using ice packs. (The ideal ice pack temperature is -20°C; -80°C will freeze the cells, reducing their survival rate.)

[0229] 2. Model making

[0230] Balb / c nude mice were injected subcutaneously with 100 μL of the cell suspension. After the experiment, HCC-1806 cell viability was monitored to reassure model success. When tumor volume reached 100 mm³, the mice were divided into groups and treated with NHWL09038 and the active agent doxorubicin via intraperitoneal injection for 14 consecutive days. Sterile syringes were used for administration to prevent infection in the nude mice. After model establishment, sterile bedding was used to prevent infection.

[0231] 3. Obtaining tumor tissue: After sacrificing the nude mice, the heart, liver, spleen, lung, kidney and tumor tissues were quickly removed and immersed in 4% paraformaldehyde to facilitate subsequent pathological examinations such as HE, immunohistochemistry, and immunofluorescence.

[0232] The results are shown in Figure 7. The above experiments confirmed that NHWL09038 has a significant inhibitory effect on the proliferation of the HCC-1806 triple-negative breast cancer model.

[0233] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of protection of the present application is limited to these examples. In line with the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.

[0234] The one or more embodiments of this application are intended to encompass all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of this application should be included in the scope of protection of this application.

Claims

1. An N-o-aminobenzoyltryptamine derivative, characterized in that: The structural formula is as follows: Among them, R 1-4 Any one of the substituents is methyl, methoxy, halogen, nitro or trifluoromethyl, and the other substituent is a hydrogen atom; or R 1-4 is substituted by a combination of methyl, methoxy, halogen or hydrogen atoms; R 5-8 It is a methyl group, a methoxy group, a halogen group, a trifluoromethyl group or a hydrogen atom.

2. The N-o-aminobenzoyltryptamine derivative according to claim 1, characterized in that: R1 and R3 are hydrogen atoms, R2 is halogen, and R4 is halogen or hydrogen atom.

3. The N-o-aminobenzoyltryptamine derivative according to claim 2, characterized in that: R1 and R3 are hydrogen atoms, R2 is fluorine or chlorine, and R4 is fluorine or a hydrogen atom.

4. The N-o-aminobenzoyltryptamine derivative according to claim 3, characterized in that: R1 and R3 are hydrogen atoms, R2 is fluorine, and R4 is a hydrogen atom.

5. The N-o-aminobenzoyltryptamine derivative according to any one of claims 1 to 4, characterized in that: R6 is a halogen, and R5, R7, and R8 are hydrogen atoms; or R7 is a halogen, and R5, R6, and R8 are hydrogen atoms; or R5 and R6 are halogen, and R7 and R8 are hydrogen atoms; or R5 is a halogen or a methyl group, and R 6-8 is a hydrogen atom; or, R8 is a halogen, a methyl group or a methoxy group, R 5-7 is a hydrogen atom; or R5 and R8 are halogen, and R6 and R7 are hydrogen atoms.

6. The N-o-aminobenzoyltryptamine derivative according to claim 5, characterized in that: R6 is fluorine, R5, R7, and R8 are hydrogen atoms; or R7 is bromine, R5, R6, and R8 are hydrogen atoms; or R5 and R6 are fluorine, R7, and R8 are hydrogen atoms; or R5 is fluorine, chlorine, bromine, or methyl, and R 6-8 is a hydrogen atom; or, R8 is fluorine, chlorine, bromine, methyl or methoxy, R 5-7 is a hydrogen atom; or R5 and R8 are fluorine, and R6 and R7 are hydrogen atoms.

7. The N-o-aminobenzoyltryptamine derivative according to claim 6, characterized in that: R5 is fluorine, R 6-8 is a hydrogen atom; or R6 is fluorine, R5, R 7-8 is a hydrogen atom; or R8 is fluorine, R 5-7 is a hydrogen atom; or R5 and R8 are fluorine, R 6-7 is a hydrogen atom; or R 5-6 is fluorine, R 7-8 A hydrogen atom.

8. A method for preparing the N-o-aminobenzoyltryptamine derivative according to any one of claims 1 to 7, characterized in that: The following steps are included: The substituted phenylaminobenzoic acid and the tryptamine derivative are mixed and reacted to obtain the N-o-aminobenzoyltryptamine derivative; the reaction route is as follows: Where R a represents any one or more substituents R on the benzene ring 1-4 ; R b represents any one or more substituents R 5-8 .

9. The preparation method according to claim 8, characterized in that: The preparation method of the substituted anilinobenzoic acid is to react substituted 2-bromobenzoic acid with 3,4,5-trimethoxyaniline to obtain the substituted anilinobenzoic acid. The reaction route is as follows: The preparation method of the tryptamine derivative IV is as follows: substituted indole reacts with 1-(dimethylamino)-2-nitroethylene to obtain compound II, compound II undergoes reduction reaction with sodium borohydride to obtain compound III, and compound III is reduced under the action of zinc to obtain tryptamine derivative IV; the reaction route is as follows:

10. A composition, characterized in that: The invention comprises the N-o-aminobenzoyltryptamine derivative as described in any one of claims 1 to 7, and paclitaxel.

11. A use of the N-o-aminobenzoyltryptamine derivative according to any one of claims 1 to 7, characterized in that: The N-o-aminobenzoyltryptamine derivative is used for preparing medicines for preventing breast cancer, liver cancer, prostate cancer, gastric cancer or colorectal cancer.

12. A use according to claim 11, characterized in that: The anti-breast cancer drug is an anti-drug-resistant breast cancer drug or a drug for enhancing the sensitivity of drug-resistant breast cancer to paclitaxel.

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