Diphenylamine compounds, their preparation methods, applications, and pharmaceutical compositions

Abstract

This invention provides a diphenylamine compound, its preparation method, and its applications, as well as a pharmaceutical composition containing the diphenylamine compound. This diphenylamine compound can effectively and selectively activate the key protein BAX in the apoptosis process at the molecular level. Simultaneously, it exhibits significant killing effects and high selectivity against cancer cells, especially human histiocytic lymphoma cells U937 and human monocytic leukemia cells THP-1, showing potential for development into novel anti-acute myeloid leukemia drugs and possessing good market prospects. Furthermore, this invention also verifies that the combined use of diphenylamine compounds and Navitoclax can enhance the killing effect against hematologic malignancies and drug-resistant solid tumors.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, and more particularly to a diphenylamine compound or a pharmaceutically acceptable salt thereof, as well as methods for preparing the diphenylamine compound, its application, and pharmaceutical compositions containing the compound. Background Technology

[0002] Apoptosis refers to the autonomous and orderly death of cells controlled by genes in order to maintain homeostasis. BCL-2 family proteins are key checkpoints for apoptosis; therefore, targeting BCL-2 family protein members is one of the most promising therapeutic strategies for dysfunctional apoptosis-related diseases. The successful launch of venetoclax validates this target and its enormous market potential.

[0003] BAX protein is a pro-apoptotic protein of the BCL-2 family and an effector protein essential for mitochondrial outer membrane permeability and apoptosis. Tumor cells resist apoptosis in various ways. Traditional anti-apoptotic inhibitors are ineffective in cell lines with downregulated or inhibited BH3-only protein expression or those resistant to inhibitors. Designing small-molecule agonists targeting BAX protein can directly activate its pro-apoptotic function and induce apoptosis, potentially becoming a next-generation cancer treatment. Related studies have shown that the combination of BAX agonists and the BCL-2 family anti-apoptotic inhibitor Navitoclax can treat drug-resistant hematologic malignancies and biomarker-driven refractory solid tumors, demonstrating good selectivity and application prospects. Summary of the Invention

[0004] The technical problem this invention aims to solve is the lack of BAX small molecule activators with high selectivity and high affinity. This invention provides a diphenylamine compound, its preparation method, and its applications, as well as a pharmaceutical composition containing this diphenylamine compound. This diphenylamine compound can effectively and selectively activate the key protein BAX in the apoptosis process at the molecular level. Simultaneously, it exhibits significant killing effects and high selectivity against cancer cells, especially human histiocytic lymphoma cells U937 and human monocytic leukemia cells THP-1, showing potential for development into novel anti-acute myeloid leukemia drugs and possessing good market prospects. Furthermore, this invention also verifies that the combined use of diphenylamine compounds and Navitoclax can enhance the killing effect against hematologic malignancies and drug-resistant solid tumors.

[0005] Specifically, the present invention provides a diphenylamine compound or a pharmaceutically acceptable salt thereof, characterized in that the diphenylamine compound has a structural formula of any one of the following formulas IV:

[0006]

[0007] In Equation I, R 1 Selected from any of the following substituents: ortho-, meta-, para-substituted halogen or carboxylic acid substituents, aromatic ring groups having para-substituted substituents, or para-substituted unsaturated rings; R 2 The substituted nitro, sulfone, or cyclopentanone group is located at the ortho position; R 3 Selected from any of the following substituents:

[0008]

[0009] In Equation II, R 4 For ortho-substituted nitro, sulfone, or cyclopentanone groups, R 5 A pyrrole ring substituted with an ortho- or meta-carboxylic acid;

[0010] In Equation III, R 6 Selected from any of the following substituents: ortho-nitro-substituted benzene ring, and 2,3-ethylenedioxyphenyl group; R 7 A furan ring or pyridine ring substituted with an ortho-carboxylic acid; R 8 It is a halogen substituent that is substituted at the ortho, meta, or para position, or an isopropyl group that is substituted at the meta position;

[0011] In equation IV, R 9 Selected from any of the following substituents: halogen substitutions at the ortho, meta, and para positions; aromatic ring groups with different substitutions at the para position; and para-substituted unsaturated rings; R 10 For nitro, sulfone, and cyclopentanone groups, R 11 It is a carboxyl or carboxylic acid ester;

[0012] In formula V, R 12 Selected from any of the following substituents: aromatic ring groups with different para-substitutions, and para-substituted unsaturated rings; R 13 It is benzoyl or sulfone.

[0013] Preferably, R 1 The substituents are selected from any of the following: benzene ring, thiophene, thiazole ring, piperidine, and morpholine; and / or, preferably, R 9 The substituent is selected from any of the following: benzene ring, thiophene, thiazole ring, piperidine, and morpholine.

[0014] Specifically, the diphenylamine compounds are any one of the following compounds:

[0015]

[0016]

[0017]

[0018]

[0019] The present invention also provides a method for preparing the above-mentioned diphenylamine compounds.

[0020] Compounds I-1, I-2, and I-3 were prepared using the following reaction route:

[0021]

[0022] The reaction conditions for each step in the above reaction route are: (a) Pd(OAc)2, dppf, CuCl, Cs2CO3, DMF; (b) Cs2CO3, DMF; (c) Fe, NH4Cl, EtOH, THF, H2O; (d) Pd2(dba)3, Xantphos, Cs2CO3, 1,4-dioxane; (e) 2M NaOH, MeOH, THF.

[0023] Compound I-4 was prepared using the following reaction route:

[0024]

[0025] The reaction conditions for each step in the above reaction route are: (a) Pd(OAc)2, dppf, CuCl, Cs2CO3, DMF; (b) Cs2CO3, DMF; (c) Fe, NH4Cl, EtOH, THF, H2O; (d) Pd2(dba)3, Xantphos, Cs2CO3, 1,4-dioxane; (e) 2M NaOH, MeOH, THF.

[0026] Compounds I-5 to I-21 were prepared using reaction routes 1 and 2 as follows:

[0027] Reaction route 1:

[0028]

[0029] The reaction conditions for each step in the above reaction route are: (a) Pd(OAc)2, dppf, CuCl, Cs2CO3, DMF; (b) Cs2CO3, DMF; (c) Fe, NH4Cl, EtOH, THF, H2O; (d) Pd2(dba)3, Xantphos, Cs2CO3, 1,4-dioxane; (e) 2M NaOH, MeOH, THF.

[0030] Reaction route 2:

[0031]

[0032] The reaction conditions for each step in the above reaction route are: (a) Pd(OAc)2, dppf, CuCl, Cs2CO3, DMF; (b) Cs2CO3, DMF; (c) Fe, NH4Cl, EtOH, THF, H2O; (d) Pd2(dba)3, Xantphos, Cs2CO3, 1,4-dioxane; (e) 2M NaOH, MeOH, THF.

[0033] Compound I-22 was prepared using the following reaction route:

[0034]

[0035] The reaction conditions for each step in the above reaction route are as follows: (a) Pd(OAc)2, dppf, CuCl, Cs2CO3, DMF; (b) Cs2CO3, THF; (c) Fe, NH4Cl, EtOH, THF, H2O; (d) Pd2(dba)3, Xantphos, Cs2CO3, 1,4-dioxane; (e) TABF, THF, H2O, 60℃; (f) TEMPO, PhI(OAc)2, DCM, rt; (g) K2CO3, MeOH, 90965-06-3, THF; (h) 2M NaOH, MeOH, THF;

[0036] Compound II-1 was prepared using the following reaction route:

[0037]

[0038] The reaction conditions for each step in the above reaction route are: (a) Cs2CO3, Dry THF, reflux; (b) Pd(OAc)2, dppf, CuCl, Cs2CO3, Dry DMF, 80℃; (c) 1,3-Dibromobenzene, Pd2(dba)3, Xantphos, Cs2CO3, 1,4-Dioxane, reflux; (d) 2M NaOH, MeOH, THF; (e) Fe, NH4Cl, EtOH, THF, H2O.

[0039] Compounds II-2 and II-3 were prepared using the following reaction route:

[0040]

[0041] The reaction conditions for each step in the above reaction route are: (a) Cs2CO3, Dry THF, reflux; (b) Pd(OAc)2, dppf, CuCl, Cs2CO3, Dry DMF, 80℃; (c) 1,3-Dibromobenzene, Pd2(dba)3, Xantphos, Cs2CO3, 1,4-Dioxane, reflux; (d) 2M NaOH, MeOH, THF; (e) Fe, NH4Cl, EtOH, THF, H2O.

[0042] Compound II-4 was prepared using the following reaction route:

[0043]

[0044] The reaction conditions for each step in the above reaction route are as follows: (a) 1-Fluoro-2-nitrobenzene, Cs2CO3, DryTHF, reflux; (b) Pd(OAc)2, dppf, CuCl, Cs2CO3, Dry DMF, 80℃; (c) 1,3-Dibromobenzene, Pd2(dba)3, Xantphos, Cs2CO3, 1,4-Dioxane, reflux; (d) 30% aq NaOH, MeOH, THF, 100℃, 3M HCl;

[0045] Compounds III-1 to III-4 were prepared using the following reaction route:

[0046]

[0047] Compound IV-1 was prepared using the following reaction route:

[0048]

[0049] The reaction conditions for each step in the above reaction route are as follows: (a) Pd(OAc)2, dppf, CuCl, Cs2CO3, DMF; (b) Chloroacetyl Chloride, AcONa, AcOH, THF; (c) NaH, Dry DMF; (d) BH3-THF, THF; (e) Pd2(dba)3, Ruphos, Cs2CO3, 1,4-dioxane, 110℃; (f) Fe, NH4Cl, EtOH, THF, H2O; (g) Pd2(dba)3, Xantphos, Cs2CO3, 1,4-dioxane; (h) 2M NaOH, MeOH, THF, 67℃.

[0050] Compounds V-1 to V-3 were prepared using the following reaction route:

[0051]

[0052] The reaction conditions for each step in the above reaction route are: (a) PdCl2(MeCN)2, CuCl2, 1,2-ClCH2CH2Cl; (b) Pd(OAc)2, dppf, CuCl, Cs2CO3, DMF; (c) Cs2CO3, THF; (d) Fe, NH4Cl, MeOH, THF, H2O; (e) Pd2(dba)3, Xantphos, Cs2CO3, 1,4-dioxane; (f) 2M NaOH, MeOH, THF.

[0053] Compounds VI-1 and VI-2 were prepared using the following reaction route:

[0054]

[0055] The reaction conditions for each step in the above reaction route are: (a) Cs2CO3, THF, reflux; (b) Fe, NH4Cl, EtOH, THF, H2O; (c) Pd2(dba)3, Xantphos, Cs2CO3, 1,4-dioxane; (d) 2M NaOH, MeOH, THF, 67℃; (e) HATU, DIEA, Dry DMF; (f) 2M NaOH, MeOH, THF, 67℃.

[0056] The present invention also provides the use of any of the preceding diphenylamine compounds or pharmaceutically acceptable salts thereof in the preparation of pharmaceutical compositions for the treatment or prevention of cancer. Preferably, the cancer is acute myeloid leukemia, a hematologic malignancy, or a solid tumor.

[0057] The present invention also provides a pharmaceutical composition for treating or preventing cancer, characterized in that it comprises a diphenylamine compound as described in any of the preceding claims or a pharmaceutically acceptable salt thereof. Preferably, the cancer is a hematologic malignancy or a drug-resistant solid tumor, and the pharmaceutical composition further comprises a BCL-xL inhibitor; more preferably, the BCL-xL inhibitor is Navitoclax. Attached Figure Description

[0058] Figure 1 This is a schematic diagram of the thermal stability migration experiment in Test Example 3 of the present invention.

[0059] Figure 2 This is a Western blot image of the thermostable migration of cells in Test Example 3 of this invention.

[0060] Figure 3 This is the result of Western blotting grayscale quantitative analysis of cell thermostable migration in Test Example 3 of this invention.

[0061] Figure 4 This is a 6A7 immunoprecipitation (IP) western blot image of BAX after I-2 and BTSA1 treatment in Test Example 4 of this invention.

[0062] Figure 5 This is the molecular sieve chromatogram of Test Example 5 of the present invention.

[0063] Figure 6 This is a graph showing the oligomerization test results of BAX after treatment with compound I-2 and BTSA1 in Test Example 5 of the present invention.

[0064] Figure 7 These are the cytotoxicity test results of compound I-2 in different cells of Test Example 6 of this invention.

[0065] Figure 8 The results of the cytotoxicity experiments of different doses of I-2 and Navitoclax on A549 and HCT116 cells in Test Example 7 of this invention are as follows.

[0066] Figure 9 The results are flow cytometry measurements of the effects of the positive control, BTSA1, and I-2 on U937 cells in Test Example 8 of this invention.

[0067] Figure 10 This is a Western blot image of the immunoprecipitation assay of THP-1 cells in Test Example 9 of this invention, which were incubated for 6 hours with different doses of I-2 and BTSA1.

[0068] Figure 11 This is the TMRE mitochondrial potential measurement result of THP-1 cells treated with different doses of I-2 for 6 h in Test Example 10 of the present invention.

[0069] Figure 12 These are Western blot images of BAX translocation and cytochrome c release in THP-1 cells after 6 hours of treatment with different doses of I-2 and BTSA1 in Test Example 11 of this invention.

[0070] Figure 13 These are Western blot images of apoptosis biomarkers in THP-1 cells after 4 hours of treatment with different doses of I-2 and BTSA1 in Test Example 12 of this invention. Detailed Implementation

[0071] In the following embodiments, reagents and materials not mentioned are all commercially available, and operating methods not mentioned are in accordance with conventional operating methods in the art. Unless otherwise specified, all descriptions in the following embodiments are in units of mass (mg), and room temperature refers to 20°C to 30°C.

[0072] Example 1: Preparation of methyl 5-(2-fluoro-5-nitrophenyl)-1H-pyrrole-2-carboxylate (compound 1)

[0073] The methyl 5-(2-fluoro-5-nitrophenyl)-1H-pyrrole-2-carboxylate (compound 1) of this embodiment was prepared by the following reaction:

[0074]

[0075] Methyl 5-bromo-1H-pyrrole-2-carboxylate (1 g, 4.9 mmol) and 2-fluoro-5-nitrophenylboronic acid (2.266 g, 12.25 mmol) were added to a 50 mL double-necked flask. Then, Cs₂CO₃ (3.993 g, 2.60 mmol), palladium acetate (0.055 g, 0.245 mmol), dppf (0.267 g, 0.49 mmol), and cuprous chloride (0.485 g, 4.9 mmol) were added sequentially. The flask was evacuated, replaced with nitrogen, and 20 mL of N₂ was added. Anhydrous DMF was used, and the reaction was evacuated again to replace N2. The reaction temperature was heated to 80℃ and reacted for 5.0 h. The reaction was then stopped and cooled to room temperature. The insoluble matter was removed by filtration, and the filtrate was collected and DMF was removed under reduced pressure. Then, 50 mL of ethyl acetate was added to redissolve the residue. The organic phase was washed with saturated NH4Cl (3 × 20 mL), water (3 × 20 mL), and saturated brine (3 × 20 mL), respectively. The solution was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 14:1) to give a white solid in 65% yield. The product is compound 1, and its NMR 1H spectrum results are as follows:

[0076] 1 H NMR (400MHz, d6-DMSO) δ: 12.65 (s, 1H), 9.00 (d, J = 2.2Hz, 1H), 8.19 (d, J = 5.3Hz, 1H), 7.58 (t, J = 9.9Hz, 1H), 6.93 (s, 1H), 6.73 (s, 1H), 3.82 (s, 3H).

[0077] Example 2: Preparation of 3-methyl-4-phenylphenol (compound 2)

[0078] 3-Methyl-4-phenylphenol (compound 2) in this embodiment was prepared by the following reaction:

[0079]

[0080] Add 4-bromo-3-methylphenol (2.5 g, 13.37 mmol) and phenylboronic acid (3.26 g, 26.74 mmol) to a 100 mL double-necked flask, followed by Cs₂CO₃ (8.72 g, 26.74 mmol), palladium acetate (0.15 g, 0.668 mmol), dppf (0.73 g, 1.34 mmol), and cuprous chloride (1.3 g, 13.37 mmol). Evacuate the flask to displace nitrogen, and add 40 mL of anhydrous DMF. Vacuum was applied again to replace N2, and the reaction temperature was heated to 80℃. After reacting for 5.0 h, the reaction was stopped and cooled to room temperature. The insoluble matter was removed by filtration, and the filtrate was collected to remove DMF under reduced pressure. Then, 50 mL of ethyl acetate was added to redissolve the residue. The organic phase was washed with saturated NH4Cl (3 × 20 mL), water (3 × 20 mL), and saturated brine (3 × 20 mL), respectively. The solution was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 14:1) to obtain a yellow oil with a yield of 65%. The product is compound 2, and its NMR 1H and mass spectrometry results are as follows:

[0081] 1 H NMR(400MHz,d6-DMSO)δ:9.35(s,1H),7.38(t,J=7.5Hz,2H),7.28(dd,J=13.9,7.0Hz ,3H),6.99(d,J=8.2Hz,1H),6.68(s,1H),6.66–6.57(m,1H),2.14(s,3H); ESI-MS:m / z 183.0[MH] - .

[0082] Example 3: Preparation of methyl 5-{2-[(3-methyl-4-phenylphenyl)oxy]-5-nitrophenyl}-1H-pyrrole-2-carboxylate (compound 3)

[0083] The methyl 5-{2-[(3-methyl-4-phenylphenyl)oxy]-5-nitrophenyl}-1H-pyrrole-2-carboxylate (compound 3) of this embodiment was prepared from compounds 1 and 2 via the following reaction:

[0084]

[0085] 3-Methyl-4-phenylphenol (compound 2, 0.21 g, 1.14 mmol) was added to a 25 mL two-necked flask. At room temperature, cesium carbonate (0.4 g, 1.23 mmol) and methyl 5-(2-fluoro-5-nitrophenyl)-1H-pyrrole-2-carboxylate (compound 1, 0.20 g, 0.76 mmol) were added sequentially. The mixture was evacuated to nitrogen, and 5 mL of anhydrous THF was added. The reaction was heated to reflux and reacted for 3.0 h. The reaction was then stopped, cooled to room temperature, and the THF was removed under reduced pressure. The residue was then redissolved in 25 mL of dichloromethane. The organic phase was washed with saturated NH4Cl (3 × 10 mL), water (3 × 10 mL), and saturated brine (3 × 10 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 6:1) to give a white solid in 75% yield. The product was compound 3, and its NMR spectrum was as follows:

[0086] 1 H NMR(400MHz,d6-DMSO)δ:12.47(s,1H),8.89(d,J=2.4Hz,1H),8.14(dd,J=9.1,2.5Hz,1H),7.45(t,J=7.2Hz,2H),7.37(t,J =7.9Hz, 3H), 7.29 (d, J = 8.3Hz, 1H), 7.16 (s, 1H), 7.08 (t, J = 7.9Hz, 2H), 6.90 (s, 1H), 6.85 (s, 1H), 3.81 (s, 3H), 2.23 (s, 3H).

[0087] Example 4: Preparation of methyl 5-{5-amino-2-[(3-methyl-4-phenylphenyl)oxy]phenyl}-1H-pyrrole-2-carboxylate (compound 4)

[0088] Methyl 5-{5-amino-2-[(3-methyl-4-phenylphenyl)oxy]phenyl}-1H-pyrrole-2-carboxylate (compound 4) of this embodiment was prepared from compound 3 via the following reaction:

[0089]

[0090] Methyl 5-{2-[(3-methyl-4-phenylphenyl)oxy]-5-nitrophenyl}-1H-pyrrole-2-carboxylate (compound 3, 0.36 g, 0.84 mmol) was added to a 25 mL two-necked flask, and an appropriate amount of THF was added to dissolve it. Then, 6 mL of LEtOH and 1.5 mL of [unspecified substance] were added. H2O was added, and then heated to 50℃ to completely dissolve methyl 5-{2-[(3-methyl-4-phenylphenyl)oxy]-5-nitrophenyl}-1H-pyrrole-2-carboxylate. After slight cooling, NH4Cl (0.135 g, 2.52 mmol) was added, followed by fractional addition of reduced iron powder (0.235 g, 4.21 mmol). The reaction was heated to 70℃ and allowed to proceed for 3.0 h. The reaction was then stopped, and the mixture was filtered through hot diatomaceous earth. The solution was concentrated under reduced pressure, and then 60 mL of ethyl acetate was added to redissolve the residue. The organic phase was washed with water (3 × 20 mL) and saturated brine (3 × 20 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 3:1) to give a yellow solid with a yield of 76%. The product is compound 4, and its NMR spectrum is as follows:

[0091] 1 H NMR(400MHz,d6-DMSO)δ:11.64(s,1H),7.39(t,J=7.4Hz,2H),7.30(dd,J=14.7,7.3Hz,3H),7.09(d,J=8.4Hz,1H),7.05(s,1H),6 .83(d,J=8.6Hz,1H),6.77(s,2H),6.68(d,J=8.2Hz,1H),6.64–6.57(m,1H),6.44(s,1H),5.17(s,1H),3.74(s,3H),2.14(s,3H).

[0092] Example 5: Preparation of methyl 5-{2-[(3-methyl-4-phenylphenyl)oxy]-5-[(2-nitrophenyl)amino]phenyl}-1H-pyrrole-2-carboxylate (compound I-1)

[0093] In this embodiment, methyl 5-{2-[(3-methyl-4-phenylphenyl)oxy]-5-[(2-nitrophenyl)amino]phenyl}-1H-pyrrole-2-carboxylate (compound I-1) was prepared by reacting compound 4 with 1-bromo-2-nitrobenzene via the following reaction:

[0094]

[0095] Methyl 5-{5-amino-2-[(3-methyl-4-phenylphenyl)oxy]phenyl}-1H-pyrrole-2-carboxylate (compound 4, 0.15 g, 0.377 mmol) was added to a 25 mL two-necked flask, followed by Cs₂CO₃ (0.31 g, 0.94 mmol), pd₂(dba)₃ (0.0345 g, 0.038 mmol), Xantphos (0.022 g, 0.038 mmol), and 1-bromo-2-nitrobenzene (0.152 g, 0.754 mmol). The flask was evacuated to nitrogen, and 5 mL of anhydrous 1,4- Dioxane was reacted at 105°C for 7.0 h, then the reaction was stopped and cooled to room temperature. The insoluble matter was removed by filtration, and the filtrate was collected to remove 1,4-dioxane under reduced pressure. The residue was then redissolved in 25 mL of ethyl acetate. The organic phase was washed with saturated NH4Cl (3 × 10 mL), water (3 × 10 mL), and saturated brine (3 × 10 mL), respectively. The solution was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 10:1) to obtain a red powder with a yield of 65%. The product was compound I-1, and its NMR (1H and 1C) spectra and mass spectrometry results were as follows:

[0096] 1 H NMR (400MHz, d6-DMSO) δ: 11.96 (s, 1H), 9.45 (s, 1H), 8.14 (d, J = 8.6Hz, 1H), 8.06 (d, J = 2.1Hz, 1H), 7.54 (t, J = 7.7Hz, 1H), 7.42 (t, J = 7.3Hz, 2H), 7 .38–7.24(m,5H),7.19(d,J=8.3Hz,1H),7.11(d,J=8.6Hz,1H),6.97(s,1 H),6.88(dd,J=17.4,9.3Hz,3H),6.69(s,1H),3.76(s,3H),2.20(s,3H); 13 C NMR(151MHz,d6-DMSO)δ161.1,156.5,149.9,142.9,141.0,137.4,136.8,135.8,133.6,132.5,131.6,129.5(2),128.7(2),1 27.3,126.7,125.2,124.8,124.6,123.3,122.4,119.5,118.4,117.3,116.9,115.2,112.1,51.7,20.8; HRMS(ESI)m / z:[M+Na] + calcd for C 31 H 25N3O5Na 542.1686, found 542.1686.

[0097] Example 6: Preparation of 5-{2-[(3-methyl-4-phenylphenyl)oxy]-5-[(2-nitrophenyl)amino]phenyl}-1H-pyrrole-2-carboxylic acid (compound I-2)

[0098] In this embodiment, 5-{2-[(3-methyl-4-phenylphenyl)oxy]-5-[(2-nitrophenyl)amino]phenyl}-1H-pyrrole-2-carboxylic acid (compound I-2) was prepared from compound I-1 via the following reaction:

[0099]

[0100] Methyl 5-{2-[(3-methyl-4-phenylphenyl)oxy]-5-[(2-nitrophenyl)amino]phenyl}-1H-pyrrole-2-carboxylate (compound I-1, 0.10 g, 0.193 mmol) was added to a 10 mL single-necked flask, dissolved in an appropriate amount of THF, followed by 0.6 mL of MeOH, and then 0.6 mL of 2M NaOH was added dropwise. The reaction was allowed to proceed at room temperature for 4.0 h, after which the reaction was stopped. Then, 0.40 mL of 3M HCl was added dropwise to adjust the pH to 5-6, and 30 mL of ethyl acetate was added. The organic phase was washed with water (3 × 10 mL) and saturated brine (3 × 10 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography (DCM:MeOH = 1:1) to obtain a red powder with a yield of 91%. The product was compound I-2, and its NMR 1H and 1C spectra and mass spectrometry results were as follows:

[0101] 1 H NMR(400MHz,d6-DMSO)δ:11.76(s,1H),9.44(s,1H),8.14(d,J=8.5Hz,1H),8.06(s,1H),7.54(t,J=7.6Hz,1H),7.42(t,J=7.2Hz,2H),7.38– 7.24(m,5H),7.19(d,J=8.3Hz,1H),7.10(d,J=8.6Hz,1H),6.98(s,1H),6.88(dd,J=13.5,7.6Hz,2H),6.78(s,1H),6.69(s,1H),2.21(s,3H); 13C NMR(151MHz,d6-DMSO)δ162.2,156.5,149.8,143.0,141.0,137.4,136.8,135.8,133.5,131.9,131.6,129.5(2),12 8.7(2),127.3,126.7,125.0,124.4,122.4,119.5,118.4,117.3,116.5,115.3,111.8,20.8; HRMS(ESI)m / z:[M+Na] + calcd for C 30 H 23 N3O5Na 528.1530, found 528.1534.

[0102] Example 7: Preparation of 5-{2-[(3-methyl-4-phenylphenyl)oxy]-5-[(2-nitrophenyl)amino]phenyl}-1H-pyrrole-3-carboxylic acid (compound I-3)

[0103] The methyl 5-bromo-1H-pyrrole-2-carboxylate in Example 1 was replaced with methyl 5-bromo-1H-pyrrole-3-carboxylate. The remaining reaction process, required raw materials and reagents, and preparation method were the same as in Examples 1-6, yielding the product 5-{2-[(3-methyl-4-phenylphenyl)oxy]-5-[(2-nitrophenyl)amino]phenyl}-1H-pyrrole-3-carboxylic acid (compound I-3), with the following structural formula:

[0104]

[0105] The product is a red powder, and its NMR 1H and 1C spectra and mass spectra are as follows:

[0106] 1 H NMR(400MHz,d6-DMSO)δ:11.76(s,1H),9.44(s,1H),8.13(d,J=8.5Hz,1H),7.79(s,1H),7.53(t,J=7.7Hz,1H),7.41(d,J=7.5Hz,3H ),7.34(d,J=7.4Hz,3H),7.22(t,J=7.8Hz,3H),7.07(d,J=8.6Hz,1H),7.00(s,1H),6.95(s,1H),6.88(t,J=8.2Hz,2H),2.21(s,3H); 13C NMR(151MHz,d6-DMSO)δ165.6,156.5,149.5,143.0,141.1,137.4,136.9,136.7,135.8,133.5,131.6,129.5(2),128.7(2),12 7.9,127.3,126.7,125.5,125.2,124.4,123.2,122.2,119.8,118.3,117.5,117.2,115.6,111.0,20.8; HRMS(ESI)m / z:[M+Na] + calcd for C 30 H 23 N3O5Na 528.1530, found 528.1530.

[0107] Example 8: Preparation of 5-{2-[(3-bromophenyl)oxy]-5-[(2-nitrophenyl)amino]phenyl}-1H-pyrrole-2-carboxylic acid (compound I-4)

[0108] The 3-methyl-4-phenylphenol (compound 2) in Example 3 was replaced with 3-bromophenol, and the other required raw materials, reagents and preparation methods were the same as in Examples 3-6, to prepare the product 5-{2-[(3-bromophenyl)oxy]-5-[(2-nitrophenyl)amino]phenyl}-1H-pyrrole-2-carboxylic acid (compound I-4), with the following structural formula:

[0109]

[0110] The product is a red powder, and its NMR 1H and 1C spectra and mass spectra are as follows:

[0111] 1 H NMR(400MHz,d6-DMSO)δ:11.53(s,1H),9.43(s,1H),8.13(d,J=8.1Hz,1H),8.02(s,1H),7.53(s,1H),7.28(t,J =9.7Hz,4H),7.16(s,1H),7.10(d,J=8.4Hz,1H),6.98(d,J=7.1Hz,1H),6.88(s,1H),6.61(s,1H),6.53(s,1H); 13C NMR(151MHz,d6-DMSO)δ158.6,148.5,142.8,136.6,133.7,132.2,129.6,126.6,126.2 ,125.8,124.1,122.8,120.2,118.4,117.4,116.6,114.7,111.4; HRMS(ESI)m / z:[M+Na] + calcd forC 23 H 16 BrN3O5Na 516.0166, found 516.0170.

[0112] Example 9: Preparation of 5-(2-{[4-(hexahydropyridin-1-yl)phenyl]oxy}-5-[(2-nitrophenyl)amino]phenyl)-1H-pyrrole-2-carboxylic acid (compound I-5)

[0113] The 3-methyl-4-phenylphenol (compound 2) in Example 3 was replaced with 4-(hexahydropyridin-1-yl)phenol. The other raw materials, reagents and preparation methods were the same as in Examples 3-6. The product 5-(2-{[4-(hexahydropyridin-1-yl)phenyl]oxy}-5-[(2-nitrophenyl)amino]phenyl)-1H-pyrrole-2-carboxylic acid (compound I-5) was prepared, with the following structural formula:

[0114]

[0115] The product is a red solid, and its NMR 1H spectrum and mass spectrometry results are as follows:

[0116] 1 H NMR(600MHz,d6-DMSO)δ12.33(s,1H),11.63(s,1H),9.41(s,1H),8.14(dd,J=8.6, 1.5Hz,1H),7.98(d,J=2.6Hz,1H),7.52(ddd,J=8.5,7.0,1.5Hz,1H),7.21(dd,J=8. 7,2.5Hz,1H),7.19(d,J=8.6Hz,1H),6.99–6.91(m,4H),6.89(d,J=8.7Hz),6.86(m ,1H),6.80–6.76(m,1H),6.70(m,1H),3.12–3.01(m,4H),1.62(s,4H),1.51(m,2H); 13C NMR(151MHz,d6-DMSO)δ162.1,151.8,149.0,143.3,136.7,134.6,133.3,132.2,126.6,125.1,124 .8,124.2,123.7,120.1,119.8,118.1,117.1,116.3,111.7,50.8,25.8,24.2; HRMS(ESI)m / z:[M+H] + calcd for C 28 H 27 N4O5 499.1976, found 499.1979.

[0117] Example 10: Preparation of 5-(2-{[4-(hexahydropyridin-1-yl)phenyl]oxy}-5-(2-methylsulfonylphenylamino)phenyl)-1H-pyrrole-2-carboxylic acid (compound I-6)

[0118] Replacing 1-bromo-2-nitrobenzene in Example 5 with 2-methylsulfonylbromobenzene, and using the same raw materials, reagents, and preparation methods as in Examples 5-6, the product 5-(2-{[4-(hexahydropyridin-1-yl)phenyl]oxy}-5-(2-methylsulfonylphenylamino)phenyl)-1H-pyrrole-2-carboxylic acid (compound I-6) was prepared, with the following structural formula:

[0119]

[0120] The product is a pale yellow solid. Its NMR (1H and 1C) spectrum and mass spectrometry results are as follows:

[0121] 1 H NMR(600MHz,d6-DMSO)δ12.29(s,1H),11.64(s,1H),7.86(d,J=2.6Hz,1H),7.78(s,1H),7 .77(d,J=8.2,1.4Hz,1H)7.52(t,J=7.8Hz,1H),7.23(d,J=8.4Hz,1H),7.15(dd,J=8.7,2.5 Hz,1H),7.00(t,J=7.5Hz,1H),6.95-6.89(m,4H),6.87(d,J=8.7Hz,1H),6.80–6.74(m,1H) ,6.71–6.64(m,1H),3.27(s,3H),3.09–2.99(m,4H),1.66–1.56(m,4H),1.56–1.46(m,2H); 13C NMR(151MHz,d6-DMSO)δ162.3,150.4,149.5,148.7,144.4,136.2,135.7,132.2,129.8,124.9,124.4,124 .0,122.6,122.3,120.6,119.4,118.1,116.6,116.1,111.6,50.9,42.9,25.9,24.3; HRMS(ESI)m / z:[M+Na] + calcd forC 29 H 29 N3O5SNa 554.1720, found 554.1720.

[0122] Example 11: Preparation of 5-(2-{[4-(hexahydropyridin-1-yl)phenyl]oxy}-5-(1-indanone)-1H-pyrrole-2-carboxylic acid (compound I-7)

[0123] Replacing 1-bromo-2-nitrobenzene with 7-bromo-1-indanone in Example 5, and using the same raw materials, reagents, and preparation methods as in Examples 5-6, yielded the product 5-(2-{[4-(hexahydropyridin-1-yl)phenyl]oxy}-5-(1-indanone)-1H-pyrrole-2-carboxylic acid (compound I-7), with the following structural formula:

[0124]

[0125] The product is a creamy white solid. Its NMR (1H and 1C) spectrum and mass spectrometry results are as follows:

[0126] 1 H NMR(600MHz,d6-DMSO)δ12.39(br,1H),11.65(s,1H),9.09(s,1H),7.87(s, 1H),7.43(t,J=7.8Hz,1H),7.22(dd,J=8.7,2.6Hz,1H),7.04(t,J=7.3Hz,1H ),6.94-6.88(m,4H),6.87(d,J=8.7Hz,1H),6.82(d,J=7.3Hz,1H),6.75(m, 1H),6.71–6.64(m,1H),3.04(m,6H),2.68(m,2H),1.62(m,4H),1.50(m,2H); 13C NMR(151MHz,d6-DMSO)δ208.2,162.4,157.4,150.2,149.5,148.7,144.9,137.2,135.6,132.1,124.0,121.9 ,120.9,120.5,119.3,118.1,116.0,115.4,111.6,109.4,50.9,36.4,25.9,25.5,24.3; HRMS(ESI)m / z:[M+H] + calcd for C 31 H 30 N3O4 508.2231, found 508.2235.

[0127] Example 12: Preparation of 2-bromo-4-[(2-nitrophenyl)oxy]aniline (compound 5)

[0128] The 2-bromo-4-[(2-nitrophenyl)oxy]aniline (compound 5) of this embodiment was prepared by reacting 4-amino-3-bromophenol with 1-fluoro-2-nitrobenzene via the following reaction:

[0129]

[0130] 4-Amino-3-bromophenol (0.90 g, 4.79 mmol) was added to a 50 mL two-necked flask. At room temperature, cesium carbonate (1.87 g, 5.74 mmol) and 1-fluoro-2-nitrobenzene (0.74 g, 5.27 mmol) were added sequentially. The mixture was evacuated to remove nitrogen, and 25 mL of anhydrous THF was added. The reaction was heated to reflux and reacted for 3.0 h. The reaction was then stopped and cooled to room temperature. THF was removed under reduced pressure, and the residue was redissolved in 50 mL of dichloromethane. The organic phase was washed with saturated NH4Cl (3 × 10 mL), water (3 × 10 mL), and saturated brine (3 × 10 mL), respectively. The mixture was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 9:1) to give a yellow solid (1.25 g, 4.07 mmol, yield 85%).

[0131] The NMR 1H spectrum results of the product are as follows:

[0132] 1H NMR(400MHz,d6-DMSO)δ:7.99(d,J=8.1Hz,1H),7.61(t,J=7.9Hz,1H),7.25(t,J=7.8Hz,1H),7.21(d, J=2.1Hz, 1H), 6.98 (d, J=8.4Hz, 1H), 6.93 (dd, J=8.8, 2.0Hz, 1H), 6.85 (d, J=8.7Hz, 1H), 5.33 (s, 2H).

[0133] Example 13: Preparation of ethyl 5-{2-amino-5-[(2-nitrophenyl)oxy]phenyl}furan-2-carboxylate (compound 6)

[0134] In Example 2, 4-bromo-3-methylphenol was replaced with 2-bromo-4-[(2-nitrophenyl)oxy]aniline, and phenylboronic acid was replaced with pinacol 2-(ethoxycarbonyl)furan-5-boronate. The other required raw materials, reagents, and preparation methods were the same as in Example 2, yielding ethyl 5-{2-amino-5-[(2-nitrophenyl)oxy]phenyl}furan-2-carboxylate (compound 6), with the following structural formula:

[0135]

[0136] The product was a white solid, and the NMR 1H spectrum results were as follows:

[0137] 1 H NMR(400MHz,d6-DMSO)δ:8.01(d,J=7.8Hz,1H),7.61(t,J=7.8Hz,1H),7.41(d,J=3.3Hz,1H),7.33(s,1H),7.24(t,J=7.7 Hz,1H),7.06(d,J=3.3Hz,1H),7.02–6.88(m,3H),5.76(s,1H),5.64(s,2H),4.29(q,J=7.0Hz,3H),1.29(t,J=7.1Hz,4H).

[0138] Example 14: Preparation of 5-{2-[(3-bromophenyl)amino]-5-[(2-nitrophenyl)oxy]phenyl}furan-2-carboxylic acid (compound II-1)

[0139] Methyl 5-{5-amino-2-[(3-methyl-4-phenylphenyl)oxy]phenyl}-1H-pyrrole-2-carboxylate (compound 4) in Example 5 was replaced with ethyl 5-{2-amino-5-[(2-nitrophenyl)oxy]phenyl}furan-2-carboxylate, and 1-bromo-2-nitrobenzene was replaced with 1,3-dibromobenzene. The remaining raw materials, reagents, and preparation methods were the same as in Example 5. The product ethyl 5-{2-[(3-bromophenyl)amino]-5-[(2-nitrophenyl)oxy]phenyl}furan-2-carboxylate was then hydrolyzed according to Example 6 to prepare 5-{2-[(3-bromophenyl)amino]-5-[(2-nitrophenyl)oxy]phenyl}furan-2-carboxylic acid (compound II-1), with the following structural formula:

[0140]

[0141] The product is a yellow solid. Its NMR (1H and 1C) spectrum and mass spectrometry results are as follows:

[0142] 1 H NMR(400MHz,d6-DMSO)δ:8.06(d,J=8.2Hz,1H),8.00(s,1H),7.70(t,J=7.9Hz,1H),7.49(s,1H),7.36(t,J=7.7Hz,2H) ,7.25(d,J=8.3Hz,1H),7.06(t,J=7.8Hz,2H),6.90(s,1H),6.83(d,J=4.5Hz,2H),6.79(s,1H),6.72(d,J=8.2Hz,1H); 13 C NMR(151MHz,d6-DMSO)δ158.8,152.7,150.3,149.9,148.1,141.4,135.6,134.6,131.5,128.4,12 7.6,126.1,124.6,122.8,121.2,121.1,119.8,117.2,116.0,113.7,111.9; HRMS(ESI)m / z:[M+Na] + calcd for C 23 H 15 BrN2O6Na 517.0006, found 517.0002.

[0143] Example 15: Preparation of 2-bromo-4-(2,3-dihydrobenzo[2,1-b][1,4]dioxane-5-yloxy)aniline (compound 7)

[0144] Replace 4-amino-3-bromophenol with 2,3-ethadioxyphenol in Example 12, and replace 1-fluoro-2-nitrobenzene with 2-bromo-4-fluoro-1-nitrobenzene. The other required raw materials, reagents and preparation methods are the same as in Example 12, yielding the product 5-[(3-bromo-4-nitrophenyl)oxy]-2,3-dihydrobenzo[2,1-b][1,4]dioxane. Then, reduce the nitro group to an amino group according to Example 4 to obtain 2-bromo-4-(2,3-dihydrobenzo[2,1-b][1,4]dioxane-5-yloxy)aniline (compound 7), with the following structural formula:

[0145]

[0146] The product is a yellow solid with a yield of 89%. Its NMR 1H spectrum results are as follows:

[0147] 1 H NMR (400MHz, CDCl3) δ: 7.12 (s, 1H), 6.90–6.82 (m, 1H), 6.72 (t, J = 8.3Hz, 2H), 6 .64(d,J=8.3Hz,1H), 6.42(d,J=8.1Hz,1H), 4.29(d,J=3.0Hz,4H), 3.93(s,2H).

[0148] Example 16: Preparation of 5-[5-(2,3-dihydrobenzo[2,1-b][1,4]dioxane-5-yloxy)-2-{[3-(propyl-2-yl)phenyl]amino}phenyl]furan-2-carboxylic acid (compound II-2)

[0149] In Example 15, 4-bromo-3-methylphenol was replaced with 2-bromo-4-(2,3-dihydrobenzo[2,1-b][1,4]dioxane-5-yloxy)aniline, and phenylboronic acid was replaced with pinacol 2-(ethoxycarbonyl)furan-5-boronate. The other raw materials, reagents, and preparation methods were the same as in Example 2, and the product ethyl 5-[2-amino-5-(2,3-dihydrobenzo[2,1-b][1,4]dioxane-5-yloxy)phenyl]furan-2-carboxylate was prepared.

[0150] Then, methyl 5-{5-amino-2-[(3-methyl-4-phenylphenyl)oxy]phenyl}-1H-pyrrole-2-carboxylate (compound 4) of Example 5 was replaced with the aforementioned product ethyl 5-[2-amino-5-(2,3-dihydrobenzo[2,1-b][1,4]dioxane-5-yloxy)phenyl]furan-2-carboxylate, and 1-bromo-2-nitrobenzene was replaced with 1-bromo-3-isopropylbenzene. The other required raw materials, reagents and preparation methods were the same as in Example 5, to obtain the product ethyl 5-[5-(2,3-dihydrobenzo[2,1-b][1,4]dioxane-5-yloxy)-2-{[3-(propyl-2-yl)phenyl]amino}phenyl]furan-2-carboxylate. Then, hydrolysis was performed according to Example 6 to obtain 5-[5-(2,3-dihydrobenzo[2,1-b][1,4]dioxane-5-yloxy)-2-{[3-(prop-2-yl)phenyl]amino}phenyl]furan-2-carboxylic acid (compound II-2), with the following structural formula:

[0151]

[0152] The product is a pale yellow solid. Its NMR (1H and 1C) spectrum and mass spectrometry results are as follows:

[0153] 1 H NMR(400MHz,d6-DMSO)δ:7.58(s,1H),7.40–7.24(m,3H),7.04(d,J=7.1Hz,1H),6.94(dd,J=12.5,7.9Hz,2H),6.88–6.79(m,1H) ,6.75(d,J=7.1Hz,1H),6.63(d,J=20.3Hz,3H),6.54(s,1H),4.26(d,J=6.8Hz,4H),2.73(d,J=6.6Hz,1H),1.12(d,J=6.6Hz,6H); 13 C NMR(151MHz,d6-DMSO)δ159.9,153.8,153.4,149.8,146.0,145.5,144.7,136.4,135.1,129.5,126.8,125 .4,120.9,119.6,118.7,117.2,115.2,113.6,113.4112.9,111.8,64.5,33.9,24.3; HRMS(ESI)m / z:[M+Na] + calcd for C 30 H 29 NO6Na 494.1574,found494.1570.

[0154] Example 17: Preparation of 5-{5-(benzenesulfonamide)-2-{[4-(thiophen-2-yl)phenyl]oxy}phenyl}-1H-pyrrole-2-carboxylic acid (compound III-1)

[0155] The 5-{5-(benzenesulfonamide)-2-{[4-(thiophen-2-yl)phenyl]oxy}phenyl}-1H-pyrrole-2-carboxylic acid (compound III-1) in this embodiment was prepared using the following reaction route:

[0156]

[0157] Methyl 5-(5-amino-2-{[4-(thiophen-2-yl)phenyl]oxy}phenyl)-1H-pyrrole-2-carboxylate (0.1 g, 0.26 mmol) was added to a 10 mL two-necked flask. At room temperature, 5 mL of anhydrous dichloromethane was added, and the mixture was in an ice bath for 2 min. Then, 89 μL (0.64 mmol) of triethylamine was slowly added, followed by slow dropwise addition of benzenesulfonyl chloride (0.091 g, 0.51 mmol) in an ice bath. The reaction was allowed to proceed for 3.0 h at room temperature, at which point the reaction was stopped, and 20 μL of triethylamine was added. The residue was redissolved in dichloromethane, and the organic phase was washed with saturated NH4Cl (3 × 10 mL), water (3 × 10 mL), and saturated brine (3 × 10 mL), respectively. After drying with anhydrous Na2SO4, the solution was concentrated under reduced pressure and purified by column chromatography (PE:EA = 4:1) to give a yellow solid methyl 5-{5-(benzenesulfonylamino)-2-{[4-(thiophene-2-yl)phenyl]oxy}phenyl}-1H-pyrrole-2-carboxylate (0.067 g, 0.13 mmol, yield 51%). Subsequently, hydrolysis was performed according to the hydrolysis conditions of Example 6 to give a pale yellow solid. The NMR spectra (1H and 1C) and mass spectra of the product are as follows:

[0158] 1 H NMR(600MHz,d6-DMSO)δ12.32(br,1H),11.61(s,1H),10.33(s,1H),8.25(t,J=2.6Hz,1H), 7.99(dd,J=7.2,1.3Hz,2H),7.70(dd,J=8.8,2.5Hz,1H),7.66–7.58(m,3H),7.55(t,J=7.2H z,2H),7.49(dd,J=5.0,1.0Hz,1H),7.40(dd,J=3.5,1.0Hz,1H),7.10(dd,J=5.0,3.7Hz,2H ),7.07(d,J=8.8Hz,1H),6.99(dt,J=8.7,2.0Hz,2H),6.75–6.73(m,1H),6.49–6.47(m,1H); 13C NMR(151MHz,d6-DMSO)δ162.1,156.8,149.4,143.2,139.9,134.3,133.4,131.9,129.7,129.4,128.9,12 7.6,127.2,125.7,124.8,124.5,123.7,122.6,122.0,121.6,118.5,116.2,111.2; HRMS(ESI)m / z:[M+Na] + calcd forC 27 H 20 N2O5S2Na 539.0706,found 539.0703.

[0159] Example 18: Preparation of 5-[5-(phenylcarbonylamino)-2-{[4-(thiophen-2-yl)phenyl]oxy}phenyl]-1H-pyrrole-2-carboxylic acid (compound III-2)

[0160] Replacing benzenesulfonyl chloride with benzoyl chloride in Example 17, and using the same raw materials, reagents, and preparation method as in Example 17, yielded 5-[5-(phenylcarbonylamino)-2-{[4-(thiophen-2-yl)phenyl]oxy}phenyl]-1H-pyrrole-2-carboxylic acid (compound III-2), with the following structural formula:

[0161]

[0162] The product is a white solid. Its NMR (1H and 1C) spectrum and mass spectrometry results are as follows:

[0163] 1 H NMR(600MHz,d6-DMSO)δ12.31(br,1H),11.69(s,1H),10.25(s,1H),7.78(dt,J=7.2 ,1.3Hz,2H),7.63(tt,J=7.3,2.0Hz,2H),7.61–7.55(m,5H),7.49(dd,J=5.0,1.1Hz ,1H),7.40(dd,J=3.5,1.0Hz,1H),7.10(dd,J=5.1,3.7Hz,1H),6.95(dd,J=8.8,2.5 Hz,1H),6.93–6.88(m,3H),6.73(dd,J=3.8,2.3Hz,1H),6.40(dd,J=3.8,2.4Hz,1H); 13C NMR(151MHz,d6-DMSO)δ166.0,162.2,157.3,148.4,143.3,136.1,135.2,132.3,132.2,129.2,12 8.9,128.1,127.6,125.6,124.4,123.7,121.8,121.5,118.2,116.1,111.0; HRMS(ESI)m / z:[M+Na] + calcd for C 28 H 20 N2O4SNa 503.1036, found 503.1040.

[0164] Example 19: Preparation of methyl 5-(2-amino-5-nitrophenyl)-1H-pyrrole-2-carboxylate (compound 8)

[0165] Replacing 4-bromo-3-methylphenol with 2-bromo-4-nitroaniline and phenylboronic acid with 5-(methoxycarbonyl)pyrrole-2-boronic acid pinacol ester, the remaining raw materials, reagents, and preparation methods are the same as in Example 2, yielding the product methyl 5-(2-amino-5-nitrophenyl)-1H-pyrrole-2-carboxylate (compound 8), with the following structural formula:

[0166]

[0167] The product is a yellow solid, and the NMR 1H spectrum results are as follows:

[0168] 1 H NMR(400MHz,d6-DMSO)δ12.26(s,1H),8.13(d,J=2.7Hz,1H),7.94(dd,J=9.1,2.7Hz,1H),6.9 2(dd,J=3.7,2.3Hz,1H),6.81(d,J=9.1Hz,1H),6.59(s,2H),6.52–6.46(m,1H),3.79(s,3H).

[0169] Example 20: Preparation of methyl 10-nitro-6,7-dihydro-5H-benzo[e]pyrrolo[2,1-g][1,4]diazacycloheptane-3-carboxylate (compound 9)

[0170] The methyl 10-nitro-6,7-dihydro-5H-benzo[e]pyrrolo[2,1-g][1,4]diazacycloheptane-3-carboxylate (compound 9) of this embodiment was prepared from compound 8 via the following reaction:

[0171]

[0172] Methyl 5-(2-amino-5-nitrophenyl)-1H-pyrrole-2-carboxylate (compound 8, 0.206 g, 0.79 mmol) was added to a 10 mL single-necked flask, and 4 mL of anhydrous THF was added to dissolve it. Then, 50 μL of glacial acetic acid and sodium acetate (0.071 g, 0.87 mmol) were added sequentially, followed by the slow addition of chloroacetyl chloride (70 μL, 0.87 mmol). The reaction was carried out at room temperature for 3 h, and the reaction was stopped. Saturated NaHCO3 solution was slowly added to quench the glacial acetic acid, and then 50 mL of ethyl acetate was added. The organic phase was washed with saturated water (3 × 10 mL) and saturated brine (3 × 10 mL), respectively, dried over anhydrous Na2SO4, and concentrated under reduced pressure.

[0173] Then, the crude product obtained in the previous step was redissolved in 7 mL of anhydrous DMF, and 60% NaH (0.068 g, 1.7 mmol) was added under ice bath. After the addition of sodium hydride, the reaction solution turned red. The reaction was carried out at room temperature for 2 h, and then an appropriate amount of 0.1 M HCl was added to neutralize the excess sodium hydride. The mixture was then filtered, washed with a small amount of EA, and dried under vacuum to obtain a yellow solid.

[0174] The yellow solid obtained in the previous step was redissolved in 2 mL of anhydrous THF, cooled in an ice bath for 2 min, and BH3-THF solution (1 M, 2.72 mL, 4 eq) was slowly added. The mixture was stirred at room temperature for 6 h to stop the reaction. Then, 1.6 mL of 2 M HCl solution was added dropwise very slowly. A large number of bubbles were produced with the first drop. After the addition was complete, the mixture was heated at 70 °C for 1.5 h to stop the reaction. The mixture was then allowed to return to room temperature. Then, 1.6 mL of 2 M NaOH was added dropwise in an ice bath to adjust the pH to 7.0. Then, 50 mL of ethyl acetate was added. The organic phase was washed with saturated water (3 × 10 mL) and saturated brine (3 × 10 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. A red powder was obtained and used directly in the next step without purification. The NMR spectrum of the product was as follows:

[0175] 1 H NMR(400MHz,dmso)δ8.26(d,J=2.7Hz,1H),7.98(t,J=4.1Hz,1H),7.89(dd,J=9.3,2.6Hz,1H),6.94(d,J=4 .2Hz,1H),6.80(d,J=9.2Hz,1H),6.55(d,J=4.2Hz,1H),4.91–4.80(m,2H),3.73(s,3H),3.61–3.53(m,2H).

[0176] Example 21: Preparation of methyl 7-(2-methyl-1-phenylphenyl-4-yl)-10-nitro-6,7-dihydro-5H-benzo[e]pyrrolo[2,1-g][1,4]diazacycloheptane-3-carboxylate (compound 10)

[0177] In this embodiment, methyl 7-(2-methyl-1-phenylphenyl-4-yl)-10-nitro-6,7-dihydro-5H-benzo[e]pyrrolo[2,1-g][1,4]diazacycloheptane-3-carboxylate (compound 10) was prepared by reacting compound 9 with 4-bromo-2-methylbiphenyl as follows:

[0178]

[0179] 10-Nitro-6,7-dihydro-5H-benzo[e]pyrrolo[2,1-g][1,4]diazacycloheptan-3-carboxylate (compound 9, 0.33 g, 1.15 mmol) was added to a 50 mL two-necked flask, followed by Cs₂CO₃ (0.94 g, 2.89 mmol), pd₂(dba)₃ (0.13 g, 12.5% ​​mol), Ruphos (0.065 g, 12.5% ​​mmol), and 4-bromo-2-methylbiphenyl (0.51 mL, 1.73 mmol). The flask was evacuated, N₂ was replaced, and 22.5 mL of N₂ was added. The reaction was carried out with mL of anhydrous 1,4-dioxane, heated to 115℃, and reacted for 16.0 h. The reaction was then stopped and cooled to room temperature. The insoluble matter was removed by filtration, and the filtrate was collected to remove 1,4-dioxane under reduced pressure. The residue was then redissolved in 50 mL of ethyl acetate. The organic phase was washed with saturated NH4Cl (3 × 10 mL), water (3 × 10 mL), and saturated brine (3 × 10 mL), respectively. The solution was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 91:9) to give a red solid (0.33 g, 0.73 mmol, yield 64%). The NMR spectrum of the product was as follows:

[0180] 1H NMR(600MHz,d6-DMSO)δ8.38(d,J=2.8Hz,1H),8.11(dd,J=9.1,2.8Hz,1H),7.42( dd,J=10.5,4.6Hz,2H),7.36–7.28(m,3H),7.24(d,J=9.1Hz,1H),7.14(d,J=8.3Hz ,1H),7.04–7.00(m,1H),6.98(t,J=3.5Hz,1H),6.91(dd,J=8.3,2.4Hz,1H),6.68 (t,J=4.7Hz,1H),4.82–4.77(m,2H),4.17–4.11(m,2H),3.78(s,3H),2.18(s,3H).

[0181] Example 22: Preparation of 7-(2-methyl-1-phenylphenyl-4-yl)-10-[(2-nitrophenyl)amino]-6,7-dihydro-5H-benzo[e]pyrrolo[2,1-g][1,4]diazacycloheptane-3-carboxylic acid (compound IV-1)

[0182] Methyl 5-{2-[(3-methyl-4-phenylphenyl)oxy]-5-nitrophenyl}-1H-pyrrolo-2-carboxylate (compound 3) in Example 3 was replaced with methyl 7-(2-methyl-1-phenylphenyl-4-yl)-10-nitro-6,7-dihydro-5H-benzo[e]pyrrolo[2,1-g][1,4]diazacycloheptane-3-carboxylate (compound 10). The other required raw materials, reagents, and preparation methods were the same as in Examples 4-6, yielding the product 7-(2-methyl-1-phenylphenyl-4-yl)-10-[(2-nitrophenyl)amino]-6,7-dihydro-5H-benzo[e]pyrrolo[2,1-g][1,4]diazacycloheptane-3-carboxylic acid (compound IV-1), with the following structural formula:

[0183]

[0184] The product is a red solid, and its NMR 1H and 1C spectra and mass spectra are as follows:

[0185] 1H NMR(600MHz,d6-DMSO)δ12.40(br,1H),9.43(s,1H),8.13(dd,J=8.5,1.4Hz,2H),7.55(t,J=8.52Hz,1H), 7.53(d,J=2.4Hz,1H),7.42–7.34(m,4H),7.30(d,J=8.6Hz,1H),7.28(t,J=7.4Hz,1H),7.25(dd,J=8.3,1 .3Hz,1H),6.97(d,J=8.5Hz,1H),6.91(t,J=8.3Hz,1H),6.90(d,J=4.0Hz,1H),6.67(d,J=2.3Hz,1H),6.5 8(dd,J=8.5,2.4Hz,1H),6.44(d,J=4.0Hz,1H),4.67(t,J=5.7Hz,2H),4.02(t,J=6.0Hz,1H),2.13(s,1H). 13 C NMR (150MHz, d6-DMSO) δ162.5,148.5,142.1,141.7,139.5,139.3,137.3,136.5,135.7,134.4,132.6,131.4,130.6,130.1,129.5(2 ),128.6(2),126.8,126.7,125.0,124.5,123.2,118.8,117.9,117.8,117.7,114.3,107.7,55.4,43.4,21.0; HRMS(ESI)m / z:[M+Na] + calcdfor C 32 H 26 N4O4Na 553.1846, found 553.1850.

[0186] Example 23: Preparation of 2-bromo-4-phenylthiophene (compound 11)

[0187] The 2-bromo-4-phenylthiophene (compound 11) in this embodiment was prepared by reacting trimethylphenylsilane and 2-bromothiophene as follows:

[0188]

[0189] PdCl2(MeCN)2 (0.04 g, 0.153 mmol) and CuCl2 (0.824 g, 6.2 mmol) were added to a 25 mL double-necked flask. The mixture was evacuated and N2 was replaced. Then, 2-bromothiophene (0.5 g, 3.1 mmol) and trimethylphenylsilane (0.922 g, 6.2 mmol) were added to another 10 mL double-necked flask, along with 4 mL of 1,2-dichloroethane, to completely dissolve the starting materials. The mixture was evacuated and N2 was replaced. The mixed solution was then added to the 25 mL double-necked flask. The reaction temperature was raised to 80 °C and the reaction was carried out for 12.0 h. The reaction was then stopped and cooled to room temperature. The insoluble matter was removed by filtration. The filtrate was collected and subjected to direct column chromatography with silica gel. The solution was eluted with pure petroleum ether to obtain a white solid (i.e., compound 11), which was used directly in the next step.

[0190] Example 24: Preparation of 2-(2-fluoro-5-nitrophenyl)-4-phenylthiophene (compound 12)

[0191] Methyl 5-bromo-1H-pyrrole-2-carboxylate (compound 1) was replaced with 2-bromo-4-phenylthiophene, and the remaining raw materials, reagents, and preparation methods were the same as in Example 1, to prepare the product 2-(2-fluoro-5-nitrophenyl)-4-phenylthiophene (compound 12), with the following structural formula:

[0192]

[0193] The product was a white solid, and the NMR 1H spectrum results were as follows:

[0194] 1 H NMR(600MHz,d6-DMSO)δ8.82(dd,J=6.1,1.7Hz,1H),8.36(s,1H),8.27-8.23(m,1H),8.14(s,1 H), 7.85 (d, J = 7.8Hz, 2H), 7.67 (t, J = 9.5Hz, 1H), 7.45 (t, J = 7.5Hz, 1H), 7.34 (t, J = 7.3Hz, 1H).

[0195] Example 25: Preparation of 3-({4-[(2-nitrophenyl)amino]-2-(4-phenylthiophen-2-yl)phenyl}oxy)benzoic acid (compound V-1)

[0196] The 3-methyl-4-phenylphenol (compound 2) in Example 3 was replaced with methyl 3-hydroxybenzoate, and the methyl 5-(2-fluoro-5-nitrophenyl)-1H-pyrrole-2-carboxylate (compound 1) was replaced with 2-(2-fluoro-5-nitrophenyl)-4-phenylthiophene. The other required raw materials, reagents, and preparation methods were the same as in Examples 3-6, yielding the product 3-({4-[(2-nitrophenyl)amino]-2-(4-phenylthiophene-2-yl)phenyl}oxy)benzoic acid (compound V-1), with the following structural formula:

[0197]

[0198] The product is a red powder, and its NMR 1H and 1C spectra and mass spectra are as follows:

[0199] 1 H NMR(600MHz,d6-DMSO)δ13.06(br,1H),9.48(s,1H),8.15(dd,J=8.5,1.2Hz,1H),8 .12(d,J=1.2Hz,1H),8.10(d,J=2.5Hz,1H),7.89(s,1H),7.73(d,J=7.9Hz,2H),7.7 0(d,J=7.7Hz,1H),7.55(t,J=8.0Hz,2H),7.50–7.46(m,1H),7.40(t,J=7.7Hz,2H), 7.38–7.34(m,2H),7.31–7.26(m,2H),7.20(d,J=8.6Hz,1H),6.91(t,J=7.7Hz,1H); 13 CNMR(151MHz,d6-DMSO)δ167.2,157.6,148.3,142.8,141.5,137.8,136.9,136.7,135.3,133.9,133.6,130.8,12 9.3,127.7,126.7,126.4,125.6,125.4,124.8,124.4,123.2,123.1,122.0,118.5,117.4; HRMS(ESI)m / z:[M+Na] + calcd for C 29 H 20 N2O5SNa 531.0985, found 531.0984.

[0200] Example 26: Preparation of 2-[(3-methyl-4-phenylphenyl)oxy]-5-[(2-nitrophenyl)amino]benzoic acid (compound 13)

[0201] Methyl 5-(2-fluoro-5-nitrophenyl)-1H-pyrrole-2-carboxylate (compound 1) in Example 3 was replaced with methyl 2-fluoro-5-nitrobenzoate. The other required raw materials, reagents, and preparation methods were the same as in Examples 3-6, yielding the product 2-[(3-methyl-4-phenylphenyl)oxy]-5-[(2-nitrophenyl)amino]benzoic acid (compound 13), with the following structural formula:

[0202]

[0203] The product is a red powder, and the NMR 1H spectrum results are as follows:

[0204] 1 H NMR(600MHz,d6-DMSO)δ13.02(br,1H),9.39(s,1H),8.13(dd,J=8.5,1.5Hz,1H),7.77(d,J=2.8Hz,1H),7.38–7.30(m,3H ),7.24(dd,J=8.6,3.9Hz,1H),7.16(dd,J=12.5,8.4Hz,2H),6.94–6.88(m,2H),6.79(dd,J=8.3,2.6Hz,1H),2.21(s,1H).

[0205] Example 27: Preparation of 1-[({2-[(3-methyl-4-phenylphenyl)oxy]-5-[(2-nitrophenyl)amino]phenyl}carbonyl)amino]cyclopropane-1-carboxylic acid (compound VI-1)

[0206] In this embodiment, 1-[({2-[(3-methyl-4-phenylphenyl)oxy]-5-[(2-nitrophenyl)amino]phenyl}carbonyl)amino]cyclopropane-1-carboxylic acid (compound VI-1) was prepared from compound 13 and methyl 1-aminocyclopropane-1-carboxylic acid via the following reaction:

[0207]

[0208] 2-[(3-methyl-4-phenylphenyl)oxy]-5-[(2-nitrophenyl)amino]benzoic acid (compound 13, 0.12 g, 0.273 mmol) was added to a 10 mL single-necked flask, followed by HATU (0.16 g, 0.41 mmol), 3 mL of anhydrous DMF, and DIEA (0.088 g, 0.68 mmol). The reaction was carried out at room temperature for 20 min, followed by the addition of methyl 1-aminocyclopropane-1-carboxylate (0.038 g, 0.33 mmol). The reaction was then stopped after 3.0 h at room temperature. The residue was then redissolved with 25 mL of ethyl acetate. The organic phase was washed with saturated NH4Cl (3 × 10 mL), water (3 × 10 mL), and saturated brine (3 × 10 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 4:1) to obtain a red solid. This solid was then hydrolyzed under the conditions of Example 6 to obtain 1-[({2-[(3-methyl-4-phenylphenyl)oxy]-5-[(2-nitrophenyl)amino]phenyl}carbonyl)amino]cyclopropane-1-carboxylic acid (compound VI-1). The NMR spectra (1H and 1C) and mass spectrometry results of the product are as follows:

[0209] 1 H NMR(600MHz,d6-DMSO)δ13.11(br,1H),9.39(s,1H),8.88(s,1H),8.13(dd,J=8.5,1.5Hz,1H) ,7.60–7.53(m,2H),7.50–7.40(m,3H),7.36(d,J=7.2Hz,1H),7.31(dd,J=7.0,1.4Hz,2H),7.2 4(dd,J=8.6,3.9Hz,1H),7.18(d,J=8.4Hz,1H),7.12(d,J=8.4Hz,1H),6.97–6.88(m,2H),6.82 (dd,J=8.4,2.6Hz,1H),2.20(s,1H),1.34(dd,J=7.9,4.6Hz,1H),0.88(dd,J=7.9,4.6Hz,1H); 13 C NMR(151MHz,d6-DMSO)δ174.1,165.9,156.7,150.5,142.4,141.2,137.1,136.9,136.5,135.7,134.3,131.3,129.7, 129.5,128.7,127.7,127.3,126.7,125.6,121.9,1199,118.7,117.3,115.8,33.5,20.8,16.9; HRMS(ESI)m / z:[M+Na] + calcd for C30 H 25 N3O6Na546.1636, found 546.1637.

[0210] Test Example 1: Affinity test of some compounds with BAX, Bcl-xL, Bcl-2 and Mcl-1

[0211] This test example is used to test the affinity of some of the compounds prepared in the examples with BAX, Bcl-xL, Bcl-2 and Mcl-1.

[0212] In this test example, a Bim-SAHB peptide, FITC-betaAla-EIWIAQELR-S5-IGD-S5-FNAYYA (S5 and S5 need to be stapled), was synthesized at the N-terminus to test the affinity of the compound with BAX. The specific procedures are as follows:

[0213] The fluorescent peptide and the test compound were dissolved in HEPES buffer solution, mixed well, and incubated at room temperature in the dark for 20 min. Then, the target protein BAX was added to make the total volume of each solution 200 μL, mixed well, and incubated at room temperature in the dark for 20 min. The final concentrations of BAX protein and FITC-Bim-SAHB were 2.2 μM and 50 nM, respectively. The final concentrations of the test compound in the system were 24 nM, 98 nM, 391 nM, 1.6 μM, 6.3 μM, 25 μM, and 100 μM, respectively.

[0214] Transfer 60 μL each of the above solution and the calibration solution to black 384-well plates (three parallel groups). Immediately detect fluorescence polarization on a microplate reader using 485 nm excitation wavelength and 535 nm emission wavelength. Set the fluorescence polarization value of the calibration solution to 20 mP. Two control groups were also established: one control group contained only BAX and FITC-Bim-SAHB in the reaction system (equivalent to 0% inhibition rate), and the other control group contained only FITC-Bim-SAHB peptide in the reaction system.

[0215] The protein inhibition rate was calculated based on the polarization values ​​of the control group and the tested compounds. The IC50 was determined by plotting the protein inhibition rate against the logarithm of the compound concentration. 50 Value. According to the formula K i =[I] 50 / ([L] 50 / K d +[P]0 / K d+1 The competitive inhibition constant K between the compound and the protein was derived and calculated. i Value, in the formula [I] 50[L] represents the compound concentration required for a 50% protein inhibition rate. 50 K represents the concentration of free FAM-Bid when the protein inhibition rate is 50%. d [P]0 represents the dissociation constant between the target protein and the FITC-Bim-SAHB polypeptide, and [P]0 represents the concentration of free protein when the protein inhibition rate is 0%. Specific results are shown in Table 1.

[0216] Then, a 6-carboxyfluorescein succinimide (FAM) ester labeled with its N-terminus was synthesized as a fluorescent tag for FAM affinity testing. His-Bcl-xL protein, His-Bcl-2 protein, or His-Mcl-1 protein, along with the test small molecule compound, were dissolved in phosphate buffer solution and incubated at 37°C in the dark for 30 min. Then, the FAM-Bid peptide was added, mixed, and incubated at 37°C in the dark for 20 min. The final concentrations of His-Bcl-xL protein, His-Bcl-2 protein, His-Mcl-1 protein, and FAM-Bid peptide were 145 nM, 420 nM, 285 nM, and 10 nM, respectively. The final concentrations of the test compounds in the system were 24 nM, 98 nM, 391 nM, 1.6 μM, 6.3 μM, 25 μM, and 100 μM, respectively.

[0217] Add 60 μL of each of the above reaction solutions to a 384-well plate (three parallel groups), and immediately detect fluorescence polarization using a microplate reader. The fluorescence polarization value (mP) is measured at an emission wavelength of 535 nm generated by excitation at 485 nm. Simultaneously, two control groups were established: one control group contained only Bcl-xL, Bcl-2, or Mcl-1 and FAM-Bid (equivalent to 0% inhibition rate), and the other control group contained only FAM-Bid peptide.

[0218] The protein inhibition rate was calculated based on the polarization values ​​of the control group and the tested compounds. The IC50 was determined by plotting the protein inhibition rate against the logarithm of the compound concentration. 50 Value. According to the formula Ki = [I] 50 / ([L] 50 / K d +[P]0 / K d+1 The competitive inhibition constant K between the compound and the protein was derived and calculated. i Value, in the formula [I] 50 [L] represents the compound concentration required for a 50% protein inhibition rate. 50 K represents the concentration of free FAM-Bid when the protein inhibition rate is 50%. d ρ is the dissociation constant between the target protein and the FAM-Bid peptide, and [P]0 is the concentration of free protein when the protein inhibition rate is 0%. Specific results are shown in Table 1 below:

[0219] Table 1 shows the effects of the compounds in the examples on BAX and Bcl-X. L IC50 of Bcl-2 and Mcl-1 proteins 50 and K i value

[0220]

[0221]

[0222] Note a: All values ​​in Table 1 are averages of three parallel groups; in Table 1, NA indicates no obvious activity and therefore was not tested.

[0223] As shown in Table 1, most formic acid compounds exhibit the same order of magnitude affinity for the positive BTSA1 protein on the BAX protein. Among them, phenylpyrrole compound I-2 shows a slightly stronger affinity than BTSA1. Simultaneously, phenylpyrrole compounds are slightly more active than phenylfuran compounds and other related series. The activity is comparable when the carboxyl group on the pyrrole is located at the ortho or meta position (component I-2 vs. I-3). When the carboxyl group in the molecule is esterified, the activity decreases, indicating that the carboxyl group is an essential group for activity (component I-1 vs. I-2). Nitro compounds show slightly stronger activity than sulfones and indanones (component I-5 vs. I-6 or I-7).

[0224] Meanwhile, most compounds exhibited poor binding affinity to BCL-2 anti-apoptotic proteins, demonstrating good selectivity for the BCL-2 family of proteins. Test Example 2: Inhibitory activity of some compounds on several tumor cell lines.

[0225] This test case demonstrates the inhibitory activity of the compound prepared in the previous example on several tumor cell lines. The specific procedures are as follows:

[0226] Human histiocytic lymphoma cells U937, human monocytic leukemia cells THP-1, and human acute promyelocytic leukemia cells NB4 were cultured in RPMI 1640 medium containing 10% fetal bovine serum, while human myeloid monocytic leukemia cells MV-4-11 were cultured in IMDM medium containing 10% fetal bovine serum. Cells were seeded in 96-well opaque white culture plates at a concentration of 15,000 cells / 100 μL for U937, NB4, and MV-4-11, and 10,000 cells / 100 μL for THP-1. The zero-conditioner group received only culture medium.

[0227] Compounds were added to 96-well plates at eight different concentrations: 40 μM, 20 μM, 10 μM, 7.5 μM, 5 μM, 2.5 μM, 1.25 μM, and 0.625 μM, with three replicates for each concentration. The compounds and cells were first incubated in serum-free culture media at 37°C in a CO2 incubator for 2.5 hours. Serum was then added to bring the final concentration to 10%. After incubation for 48 hours, the 96-well plates were removed from the incubator and allowed to equilibrate at room temperature for 30 minutes. An equal volume (100 μL) of CellTiter-Glo reagent was added to each well, and the plates were mixed on a shaker for 2 minutes to induce cell lysis. The plates were then incubated at room temperature in the dark for 10 minutes to stabilize the chemiluminescence signal and the chemiluminescence values ​​were recorded. Calculate cell viability = (experimental group absorbance - zero-adjustment group absorbance) / (control group absorbance - zero-adjustment group absorbance). Plot cell viability against the logarithm of compound concentration to determine the IC50. 50 Values. See Table-2 for specific results.

[0228] Table 2. Proliferation inhibition results of the compounds in the examples on NB4, U937, THP-1, and MV-4-11 cell lines.

[0229]

[0230]

[0231] Note a: All values ​​in Table 2 are the average of three parallel sets; in Table 2, NT indicates untested.

[0232] As shown in Table 2, most of the compounds in the examples exhibited activities between 2 and 10 μM. The compounds with better activity were mainly phenylpyrrole carboxylic acids, almost all of which were stronger than the positive BTSA1. The conformationally inverted phenylthiophene compounds had the same activity as the phenylpyrrole compounds; among them, compound I-2 had the best activity.

[0233] Test Example 3: Thermostable migration of compound I-2 in BxPC-3 cells

[0234] When BAX proteins transition from an inert state to an activated state, they undergo complex conformational changes to localize on the outer mitochondrial membrane and undergo oligomerization to form pores.

[0235] Figure 1 This is a schematic diagram of the thermal stability migration experiment in Test Example 3 of the present invention.

[0236] like Figure 1As shown in the figure above, current research indicates that the activation process of BAX mainly includes the following stages: (1) Inactive BAX in the cytoplasm; (2) Under intracellular pressure, BH3-only protein binds to the trigger site; (3) loop α1-α2 changes from the closed state to the open state, and then the conformational change leads to the exposure of the 6A7 antigenic determinant cluster and the BAXBH3 domain (α2). α9 is released from the hydrophobic groove of BH3 and inserts into the mitochondrial membrane, completing the migration of BAX to the mitochondria; (4) BH3-only protein binds to the exposed hydrophobic groove of BH3, causing BAX core / latch separation, which promotes the formation of dimers and oligomers from BAX monomers; (5) Oligomers aggregate to form pores, allowing Cytochrome C to be released.

[0237] Based on the above principles, this test case investigates the binding of compound I-2 to BAX protein and its effect on the thermal stability of BAX protein. The specific operation procedure is as follows:

[0238] BxPC3 cells were seeded at 10 cm 2 In culture dishes, cells were grown to 85% confluence. The original culture medium was removed and replaced with serum-free medium, with compound I-2 added to a final concentration of 40 μM. BxPC3 cells were treated at 37°C for 60 min. Subsequently, the medium was removed, and cells were washed once with PBS. Cells were collected using a cell scraper and cultured at 1*10-1... 7 Cells were resuspended in PBS at a density of cells / mL and transferred to PCR tubes in 50 μL increments per sample. Samples were heated for 3 min for each min using a Biorad C1000 Touch Thermal Cycler at a concentration gradient (50, 52.1, 55.4, 59.4, 64.9, 69.2, 72.1, and 74 °C). All cells were lysed by three cycles of freeze-thaw in liquid nitrogen, and samples were centrifuged at 20,000 g for 15 min at 4 °C. The supernatant was collected, loaded with loading buffer, and subjected to Western blotting analysis.

[0239] Figure 2 These are Western blot images of the thermostable migration of cells in Test Example 3 of this invention. Figure 3 This is the result of Western blotting grayscale quantitative analysis of cell thermostable migration in Test Example 3 of this invention.

[0240] like Figure 2 , Figure 3 As shown, cell thermostability migration experiments demonstrate that compound I-2 can bind to BAX protein and increase its thermostability.

[0241] Test Example 4: The effect of compound I-2 on the BAX conformation

[0242] Upon activation (e.g., the BIM BH3 peptide binds to the BAX protein trigger site via its BH3 domain), the BAX protein gradually transitions from a closed to an open conformation as loop α1-α2 opens, fully exposing the 6A7 antigenic epitope on the α1 helix and the BH3 polypeptide domain. In healthy cells, soluble Bax protein cannot be bound by the monoclonal antibody 6A7 targeting amino acid positions 13-19 of the Bax N-terminal peptide. However, in apoptotic cells, if Bax undergoes a conformational change and inserts into the mitochondrial membrane, it can be recognized by 6A7. Therefore, the activated BAX with conformational changes can be detected using an IP (intracellular immunoassay) technique with a 6A7 antibody. Protein A / G, the BAX 6A7 antibody, and the activated BAX form a magnetic bead-antibody-antigen complex, which is then enriched by magnetic separation and elution.

[0243] Based on the above principles, this test example investigates the effect of compound I-2 on the BAX conformation. The specific procedures are as follows:

[0244] 1 μM BAX dissolved in 20 mM Hepes pH 7.2, 150 mM KCl buffer was treated with different concentrations of I-2 and BTSA1 and incubated at room temperature for 15 min (total volume 20 μL). Then, 280 μL of 3% BSA in PBS solution was added to the mixture, and after mixing, 30 μL was taken as input. 3 μL of 6A7 antibody (sc-23959, Santa Cruz) and pre-washed protein A / G beads (Santa Cruz) were added to the remaining solution, and the mixture was incubated at 4 °C for 2 h by rotation. The beads were collected by centrifugation and washed three times with 1 mL of 3% BSA buffer. Finally, the beads were dissolved in 25 μL of SDS / DTT loading buffer, and the samples were analyzed by SDS-PAGE electrophoresis and Western blot analysis using anti-BAX antibody (Cell Signaling Cat. 2772).

[0245] Figure 4 This is a 6A7 immunoprecipitation (IP) western blot image of BAX after I-2 and BTSA1 treatment in Test Example 4 of this invention.

[0246] like Figure 4 As shown, the BAX 6A7 immunoprecipitation assay indicates that compound I-2 can induce BAX protein activation in a concentration-dependent manner, thereby inducing the exposure of the BAX 6A7 antigenic determinant.

[0247] Test Example 5: Compound I-2 induces BAX to form oligomers.

[0248] BAX protein (final concentration 15 μM), small molecule compound or peptide (final concentration 100 μM) were added to HEPES buffer (20 mM HEPES, 150 mM NaCl, pH = 7.6, 0.5% CHAPS), mixed well and incubated at room temperature for 3 h. The sample was then loaded onto Superdex 75 10 / 300GL molecular sieves, and the oligomerization of BAX protein was observed.

[0249] Figure 5 This is the molecular sieve chromatogram of Test Example 5 of the present invention.

[0250] like Figure 5 As shown in the chromatogram, the peaks with retention volumes of 8–11 ml and 12–14 ml represent the oligomeric and monomeric forms of BAX protein, respectively, indicating that compound I-2 can induce BAX protein oligomerization in a time-dependent manner. In other words, compound I-2 can bind to BAX protein at the protein level and activate a conformational change in BAX, inducing BAX protein oligomerization.

[0251] Figure 6 This is a graph showing the oligomerization test results of BAX after treatment with compound I-2 and BTSA1 in Test Example 5 of the present invention.

[0252] like Figure 6 As shown, the BAX oligomerization experiment demonstrated that compound I-2 could induce BAX oligomerization in a time-dependent manner, indicating that compound I-2 can activate BAX protein and promote BAX protein oligomerization. Meanwhile, compounds BTSA1, I-3, I-4, II-4, IV-1, VI-4, IV-3, I-22, and VI-2, which showed good binding ability in the FP experiment, could all induce BAX oligomerization. However, the negative compound (i.e., compound T1742, structural formula shown below) did not induce BAX oligomerization.

[0253]

[0254] Test Example 6: Cytotoxicity Experiment of Compound I-2 on Different Cells

[0255] This test case examines the cytotoxicity of compound I-2. The experimental procedure is the same as that in test case 2. The cell lines used are NB4 (human acute promyelocytic leukemia cells), THP-1 (human monocytic leukemia cells), U937 (human histiocytic lymphoma cells), MV-4-11 (human myeloid monocytic leukemia cells), A549 (human non-small cell lung cancer cells), HCT116 (human colon cancer cells), SW480 (human colon adenocarcinoma cells), 293T (human embryonic kidney cells), OCI-AML3 (human acute myeloid leukemia cells), and BxPC-3 (human orthotopic pancreatic adenocarcinoma cells).

[0256] Figure 7 These are the cytotoxicity test results of compound I-2 in different cells of Test Example 6 of this invention.

[0257] like Figure 7 As shown, compound I-2 exhibits good cytotoxicity (i.e., cell proliferation inhibition activity) in hematologic malignancy cells while showing low cytotoxicity to normal human cells, but its inhibitory activity on solid tumor cells is poor.

[0258] Test Example 7: Combination Test of Compound I-2 with BCL-xL Inhibitor

[0259] Recently, there have been studies (Lopez A, Reyna DE, Gitego N, Kopp F, Zhou H, Miranda-Roman MA, (LU, Narayanagari SR, Chi P, Vilar E, Tsirigos A, Gavathiotis E. Co-targeting of BAX and BCL-XL proteins broadly overcomes resistance to apoptosis in cancer. Nat Commun., 2022, 13:1199.) reported that the main reason for the resistance of solid tumor cells to BAX agonists is the lack of BAX activation and BCL-xL dependence. Therefore, the inventors envisioned overcoming the resistance of solid tumor cells to BAX agonists through the combined use of a BAX agonist, compound I-2, and a BCL-xL inhibitor, Navitoclax.

[0260] This test case utilizes the combination of compound I-2 and Navitoclax in two cell lines, A549 and HCT116, to evaluate their synergistic effect. The specific procedure was as follows: Human non-small cell lung cancer cells (A549) were cultured in RPMI 1640 medium containing 10% fetal bovine serum, and human colon cancer cells (HCT116) were cultured in DMEM medium containing 10% fetal bovine serum. Cells were seeded in 96-well opaque white culture plates at a concentration of 10,000 cells / 100 μL; the zero-conditioner group received only culture medium.

[0261] Compound Navitoclax (eight different concentrations: 80 μM, 40 μM, 20 μM, 15 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM, with three replicates for each concentration) and a fixed concentration of compound I-2 were added to 96-well plates. Cells were seeded in the corresponding medium containing 10% serum and incubated at 37°C in a CO2 incubator for 12 hours, after which the old medium was removed. The corresponding compound and its solution dissolved in serum-free solution were added, and then serum was added to bring the concentration to 10% after 2.5 hours. The plates were incubated for another 48 hours, after which the 96-well plates were removed from the incubator and allowed to equilibrate at room temperature for 30 minutes. CellTiter-Glo reagent (100 μL) equal in volume to the cell culture medium was added to each well, and the plates were mixed on a shaker for 2 minutes to induce cell lysis. The plates were then incubated at room temperature in the dark for 10 minutes to stabilize the chemiluminescence signal and the chemiluminescence values ​​were recorded. Calculate cell viability = (experimental group absorbance - zero-adjustment group absorbance) / (control group absorbance - zero-adjustment group absorbance). Plot cell viability against the logarithm of compound concentration to determine the IC50. 50 value.

[0262] Figure 8 The results of the cytotoxicity experiments of different doses of I-2 and Navitoclax on A549 and HCT116 cells in Test Example 7 of this invention are as follows.

[0263] like Figure 8 As shown, the combination of Navitoclax and the sublethal BAX agonist I-2 can increase the killing effect on solid tumor cells. Test Example 8: Induction of apoptosis in leukemia cells by compound I-2.

[0264] Activation of the pro-apoptotic protein BAX leads to increased mitochondrial outer membrane permeability, loss of mitochondrial membrane potential, and release of apoptosis-related factors such as cytochrome C, thereby inducing apoptosis. The occurrence of apoptotic events can be detected by Annexin V / PI double staining flow cytometry.

[0265] This test case, based on the above principle, examines the effect of compound I-2 on inducing apoptosis in leukemia cells. The specific procedure is as follows:

[0266] First, cell counting is performed, and then an appropriate volume of cell suspension is selected for plating based on cell density. 1*102 cells are added to each well of a 6-well plate. 6 U937 cells were collected, and then different concentrations of compound I-2 or DMSO (DMSO final concentration was 0.4%) were added. The cells were cultured in serum-free medium for 2.5 h, and then 200 μL of FBS was added to the final concentration of 10%. After mixing, the cells were cultured for another 21.5 h. The cells were then collected, centrifuged at 200 g for 5 min at 25 °C, the supernatant was discarded, the pellet was collected, washed with 4 mL of PBS, and allowed to stand at room temperature for 5 min to allow the cells to recover from mechanical stress. FITC binding solution was added according to the cell density, and the cells were gently resuspended. Then, Annexin V-FITC and propidium iodide staining solution were added according to the instructions (Beyotime C1062L). The cells were incubated at room temperature in the dark for 15 min (the cells can be resuspended 2-3 times during incubation to improve the staining effect). The cells were then placed on ice in the dark. The blank tubes were first gated and the threshold was adjusted. Then, FITC and PI single staining tubes were added and the voltage was adjusted. Finally, the treated group samples were tested.

[0267] Figure 9 The results are flow cytometry measurements of the effects of the positive control, BTSA1, and I-2 on U937 cells in Test Example 8 of this invention.

[0268] like Figure 9 As shown, compound I-2 can induce apoptosis in U937 cells in a concentration-dependent manner.

[0269] Test Example 9: BAX 6A7 Immunoprecipitation Assay of Compound I-2 on THP-1 Cells

[0270] This test example demonstrates the BAX 6A7 immunoprecipitation assay of compound I-2 on THP-1 cells. The specific procedure is as follows:

[0271] Cells were counted, and an appropriate volume of cell suspension was selected for plating based on cell density. 2 x 10⁻⁶ cells were added to each well of a 6-well plate. 6THP-1 cells were cultured for 2.0 h in serum-free medium with different concentrations of the compound (DMSO final concentration was 0.4%). Then, 400 μL of FBS was added to a final concentration of 10%, and the cells were mixed and cultured for another 4 h. Cells were then collected, centrifuged at 200 g for 5 min at 25 °C, the supernatant was discarded, and the pellet was collected. The pellet was washed with 0.6 mL of ice-cold PBS and incubated on ice for 5 min to allow the cells to recover from mechanical stress. This washing was repeated twice. The pellet was then centrifuged again, the supernatant was discarded, and the pellet was collected. Each sample was lysed with 200 μL of 1% CHAPS lysis buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 1% CHAPS, with protease and phosphatase inhibitors added immediately before use), and then incubated on ice for 1 h.

[0272] After lysis, the sample was centrifuged (15000g, 10min, 4℃) and the supernatant was collected. 20μL of the supernatant was used as input. The diluted 6A7 antibody was then added to pre-washed protein A / G beads (Santa Cruz). The sample was incubated at 4℃ for 2h by inversion. The supernatant was removed by centrifugation, and the sample was washed twice with 400μL of PBS. The lysed protein solution was then added and incubated at 4℃ for 2h by inversion. The sample was washed twice again with 400μL of PBS. Finally, the beads were dissolved in 25μL of SDS / DTT loading buffer. The sample was analyzed by SDS-PAGE electrophoresis and Western blot analysis using anti-BAX antibody (Cell Signaling Cat.2772).

[0273] Figure 10 This is a Western blot image of the immunoprecipitation assay of THP-1 cells in Test Example 9 of this invention, which were incubated for 6 hours with different doses of I-2 and BTSA1.

[0274] like Figure 10 As shown, the results of the BAX 6A7 immunoprecipitation experiment at the cellular level indicate that I-2 can activate BAX protein, thereby causing a conformational change in BAX protein and completing the activation process of BAX protein.

[0275] Test Example 10: Detection Experiment of Mitochondrial Membrane Potential of Compound I-2

[0276] Maintaining mitochondrial membrane potential polarization is crucial for normal mitochondrial function. Mitochondrial membrane depolarization can alter mitochondrial membrane permeability and activate the mitochondrial apoptosis pathway. Therefore, we used TMRE staining to analyze the effects of compounds on mitochondrial membrane potential. Tetramethylrhodamine ethyl ester (TMRE) is a cell membrane-permeable orange-red cationic fluorescent probe that accumulates in intact mitochondria. Depolarization or a decrease in inactive mitochondrial membrane potential leads to reduced TMRE accumulation.

[0277] Based on the above principles, this test example performs an experiment to detect the mitochondrial membrane potential of compound I-2. The experimental procedure was performed according to the Beyotime Mitochondrial Membrane Potential Detection Kit (TMRE) (C2001S) instruction manual, as follows:

[0278] First, seed 1*10⁶ cells / well into 6-well plates, then add different concentrations of the compound (DMSO final concentration 0.4%) and incubate at 37°C for 2.5 h. Then, add 10% FBS to a total volume of 2 mL and continue incubation for 3.5 h. Collect cells, count them, and select the total cell count based on the cell density of the highest concentration treatment group. Centrifuge, discard the supernatant, add an appropriate volume of TMRE staining solution, and resuspend the cells to achieve a cell density of approximately 1*10⁶ cells / well. 6 Cells / mL, incubated at 37℃ for 30 min, centrifuged, supernatant discarded, cells resuspended in 1 mL of preheated 37℃ cell culture medium, centrifuged again, supernatant discarded, added another 1 mL of preheated 37℃ cell culture medium to resuspend the cells, centrifuged again, supernatant discarded. Add 400 μL of preheated cell culture medium to resuspend the cells, mix well, and add 100 μL to each well of a black or white opaque 96-well plate, with 3 replicates, for Fluoresense Intensity Assay (Ex: 540 nM, Em: 579 nM).

[0279] Figure 11 This is the TMRE mitochondrial potential measurement result of THP-1 cells treated with different doses of I-2 for 6 h in Test Example 10 of the present invention.

[0280] like Figure 11 As shown, the fluorescence intensity of the cell suspension gradually decreased with increasing concentration of compound I-2, indicating that the mitochondrial membrane potential was gradually lost with increasing concentration of compound I-2.

[0281] Test Example 11: Compound I-2-induced THP-1 cytochrome C release and BAX translocation experiment

[0282] Activation of the pro-apoptotic protein BAX leads to oligomerization and pore formation on the outer mitochondrial membrane, causing mitochondrial outer membrane permeability (MOMP). This results in the release of cytochrome C from the mitochondrial matrix into the cytoplasm. Therefore, the relative ratio of cytochrome C in the cytoplasm and mitochondria under different conditions reflects different degrees of apoptosis. Similarly, the relative ratio of BAX protein located in the outer mitochondrial membrane to that in the cytoplasm under different conditions indicates not only the degree of apoptosis but also the degree of BAX activation. To determine the effect of small molecule BAX agonists on the changes in the relative ratios of BAX protein and cytochrome C in the cytoplasm and mitochondria, it is generally necessary to first separate the cytoplasmic and mitochondrial proteins and then analyze the BAX and Cytochrome C content in each separately.

[0283] Based on the above principles, this test case demonstrates the induction of THP-1 cytochrome C release and BAX translocation by compound I-2. The specific procedures are as follows:

[0284] Cells were counted, and an appropriate volume of cell suspension was selected for plating based on cell density. 10 x 10⁻⁶ cells were added to each well of a 6-well plate. 6 THP-1 cells were cultured for 2.0 h in serum-free medium with different concentrations of the compound (DMSO final concentration was 0.4%). Then, 1 mL of FBS was added to a final concentration of 10%, and the cells were cultured for another 4 h. Cells were then collected, centrifuged at 200 g for 5 min at 25 °C, the supernatant was discarded, and the precipitate was collected. The precipitate was washed with 10 mL of ice-cold PBS and incubated on ice for 5 min to allow the cells to recover from mechanical stress. This washing process was repeated twice. The precipitate was then centrifuged again, the supernatant was discarded, and the precipitate was collected. Each sample was added with 500 μL of digitalis lysis buffer (20 mM Hepes, pH 7.2, 10 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 0.025% Digitonin (from 5% w / v stock, with protease inhibitor added immediately before use), and then incubated at 4 °C for 30 min.

[0285] After lysis, centrifugation (15000g, 10min, 4℃) was performed, and the supernatant was collected as the cytoplasmic fraction for BCA quantification. The precipitate was washed with 1mL of ice-cold PBS, incubated on ice for 10min, centrifuged to remove PBS, and 250μL of RIPA medium-strength lysis buffer was added. The mixture was incubated at 4℃ for 60min by inversion, centrifugation (15000g, 10min, 4℃) was performed, and the supernatant was collected as the mitochondrial protein fraction for BCA quantification. The cytoplasmic and mitochondrial fractions were analyzed by Western blot.

[0286] Figure 12These are Western blot images of BAX translocation and cytochrome C release in THP-1 cells after 6 hours of treatment with different doses of I-2 and BTSA1 in Test Example 11 of this invention.

[0287] like Figure 12 As shown, the content of cytoplasmic BAX protein gradually decreased with increasing compound I-2 concentration, while the content of cytochrome C gradually increased with increasing compound I-2 concentration; similarly, the content of mitochondrial BAX protein gradually increased with increasing compound I-2 concentration, while the content of cytochrome C gradually decreased with increasing compound I-2 concentration. These results indicate that compound I-2 can activate the translocation of cytoplasmic BAX protein to the outer mitochondrial membrane in THP-1 cells and activate the mitochondrial apoptosis pathway, prompting the release of cytochrome C from the mitochondria into the cytoplasm. Test Example 12: Effects of Compound I-2 on Key Proteins of the Apoptosis Pathway

[0288] Activation of pro-apoptotic proteins leads to increased permeability of the mitochondrial outer membrane, resulting in the release of apoptosis-related factors such as cytochrome C. Upon binding to Apaf-1 (apoptotic protease activating factor 1), these factors initiate a caspase cascade reaction. The cytochrome C / Apaf-1 complex activates caspase 9, which in turn activates caspase 3 and downstream caspases.

[0289] Based on the above principles, to better understand the pro-apoptotic mechanism of the compound, this test case examined the expression levels of PARP, caspase 3, and caspase 9, key apoptosis proteins in the apoptosis pathway of THP-1 cells, induced by compound I-2. The activated forms of PARP, caspase 3, and caspase 9 are key mediators of apoptosis. The specific procedures are as follows:

[0290] Add 1*10 to each well of the 6-well plate 6 THP-1 cells were cultured for 2 hours in serum-free medium, then different concentrations of compound I-2 or DMSO (DMSO final concentration 0.4%) were added. The cells were then supplemented with 200 μL of FBS to a final concentration of 10%, mixed, and cultured for another 4 hours. After centrifugation, the supernatant was discarded, and the precipitate was collected. The precipitate was washed twice with 2 mL of ice-cold PBS, centrifuged again, the supernatant was discarded, and the precipitate was collected. 100 μL of RIPA strong lysis buffer (Beyotime, containing EDTA, with 100x protease inhibitor added before use, but no phosphatase inhibitor) was added, and the cells were incubated in ice water for 30 minutes. The cells were then centrifuged at 15000g for 15 minutes at 4°C, and the supernatant was collected. BCA quantification was performed followed by Western blot analysis.

[0291] Figure 13 These are Western blot images of apoptosis biomarkers in THP-1 cells after 4 hours of treatment with different doses of I-2 and BTSA1 in Test Example 12 of this invention.

[0292] like Figure 13 As shown, compound I-2 degrades PARP into cleaved PARP, the main substrate for caspase activation, in a concentration-dependent manner. Furthermore, with increasing I-2 concentration, caspase 3 and caspase 9 levels significantly decreased, while the corresponding levels of cleaved caspase 3 and cleaved caspase 9 gradually increased. Therefore, compound I-2 can activate the apoptosis pathway and inhibit tumor cell proliferation.

Claims

1. A diphenylamine compound or a pharmaceutically acceptable salt thereof, characterized in that, The structural formula of the diphenylamine compound is any one of the following compounds: ， ， ， 。 2. The method for preparing diphenylamine compounds according to claim 1, characterized in that: in, Compounds I-1, I-2, and I-3 were prepared using the following reaction route: ； Compound I-4 was prepared using the following reaction route: ； Compound I-22 was prepared using the following reaction route: ； Compound II-1 was prepared using the following reaction route: ； Compounds II-2 and II-3 were prepared using the following reaction route: ； Compound II-4 was prepared using the following reaction route: ； Compound III-1 was prepared using the following reaction route: ； Compound IV-1 was prepared using the following reaction route: ； Compounds V-1 to V-3 were prepared using the following reaction route: ； Compound VI-1 and compound VI-2 were prepared using the following reaction route: 。 3. The use of the diphenylamine compound or a pharmaceutically acceptable salt thereof as described in claim 1 or 2 in the preparation of a pharmaceutical composition for the treatment or prevention of cancer, wherein the cancer is human histiocytic lymphoma, human monocytic leukemia, human acute promyelocytic leukemia, or human myeloid monocytic leukemia.

4. A pharmaceutical composition for treating or preventing cancer, characterized in that, It comprises a diphenylamine compound as described in claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the cancer is human histiocytic lymphoma, human monocytic leukemia, human acute promyelocytic leukemia, or human myeloid monocytic leukemia.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition further comprises a BCL-xL inhibitor.

6. The pharmaceutical composition according to claim 5, characterized in that: in, The BCL-xL inhibitor is Navitoclax.

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