A benzopyridine compound and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a benzopyridine compound and a preparation method and application thereof. The benzopyridine compound has high STAT3 selectivity, can target STAT3 and inhibit STAT3 phosphorylation, significantly inhibit the activation of a STAT3 signal path, inhibit the growth and proliferation of cells with high STAT3 expression, and achieve the effects of inhibiting the growth and proliferation of tumor cells and inducing tumor cell apoptosis. In particular, the benzopyridine compound also has a significant inhibitory effect on drug-resistant tumor cells. Further, the benzopyridine compound has a synergistic advantage when used in combination with a chemotherapeutic drug, has good drug properties, and is very suitable for the preparation of anticancer drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a benzopyridine compound, its preparation method, and its application. Background Technology

[0002] STAT3 (signal transducer and activator of transcription 3) is a transcription factor closely related to cell proliferation and survival, and it is overactivated in various solid tumors and hematological malignancies. Upon stimulation by upstream cytokines or growth factors, STAT3 is phosphorylated by receptor-associated kinases, then forms a dimer and enters the cell nucleus, thereby exerting a transcriptional regulatory role. It regulates the expression of downstream target genes, including Cyclin D1, c-Myc, Bcl-xL, and Mcl-1, playing a crucial role in regulating cellular physiological processes. Currently, many publicly available small-molecule drugs targeting STAT3 have limited further development and clinical application due to their low activity and selectivity. Therefore, there is an urgent need to develop small-molecule inhibitors of STAT3 with high activity and selectivity.

[0003] Leukemia is a common malignant clonal disease of hematopoietic cells, characterized by the massive proliferation and accumulation of leukemic cells in the bone marrow and other hematopoietic tissues, as well as infiltration into other non-hematopoietic organs. Clinical manifestations include varying degrees of anemia, bleeding, infection, hepatomegaly, splenomegaly, lymphadenopathy, and bone pain. In my country, the incidence of leukemia is approximately 3-4 cases per 100,000 people. Among children and adults under 35 years of age, leukemia ranks first in mortality from malignant tumors. Furthermore, leukemia seriously endangers the lives and health of patients and increases the medical burden on patients' families and society. For decades, chemotherapy has remained the first-line treatment for leukemia, but the existence of drug resistance has greatly limited its clinical use.

[0004] Currently, with the development of various new targeted drugs, leukemia treatment is no longer limited to single chemotherapy regimens, but is gradually shifting from high-intensity chemotherapy to low-intensity chemotherapy combined with other drug therapies. Therefore, it is essential to develop new targeted drugs and explore effective combination therapy regimens. STAT3 overactivation can be observed in peripheral blood and bone marrow samples from most leukemia patients, and studies have found that STAT3 can promote the proliferation and survival of leukemia stem cells, thereby causing leukemia relapse and drug resistance ([1] L. Chen, Z. Guo, Y. Zhou, et al. microRNA-1246-containing extracellular vesicles from acute myeloid leukemia cells promote the survival of leukemia stem cells via the LRIG1-meditated STAT3 pathway[J]. Aging(AlbanyNY), 2021, 13(10): 13644-13662. [2] Maria L. Amaya, Anagha Inguva, Courtney L. Jones, et al. STAT3 Plays a Critical Role in Mitochondrial Function and Survival of Primary AML Cells[J].Blood,2019,134(Supplement_1):1275-1275.[3]MLAmaya,A.Inguva,S.Pei,et al.The STAT3-MYC axis promotes survival of leukemia stem cells by regulating SLC1A5 and oxidative phosphorylation[J].Blood,2022,139(4):584-596.). Therefore, developing inhibitors targeting STAT3 for the treatment of tumors including leukemia or overcoming chemotherapy resistance in leukemia may have great potential. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing commonly used anti-leukemia drugs, such as drug resistance, and the low activity and low selectivity of small molecule drugs targeting STAT3, and to provide a benzopyridine compound.

[0006] The purpose of this invention is to provide a method for preparing the benzopyridine compounds.

[0007] Another object of the present invention is to provide the use of the benzopyridine compounds in the preparation of STAT3 inhibitors.

[0008] Another object of the present invention is to provide the use of the benzopyridine compounds in the preparation of anticancer drugs.

[0009] Based on this, the present invention also provides a composition for treating leukemia.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] A benzopyridine compound having the structure of Formula I:

[0012]

[0013] Wherein, R1 is hydrogen or phenyl;

[0014] R2 is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted C3-C8 cycloalkyl, unsubstituted or substituted C3-C8 heterocycloalkyl, unsubstituted or substituted phenyl, unsubstituted or substituted heteroaryl, unsubstituted or substituted C1-C6 alkylamino, and unsubstituted or substituted C4-C8 oxo-nitroheterocycle; wherein the substitution means that at least one site is substituted by the following substituents: halogen, cyano, amino, nitro, hydroxyl, trifluoromethyl, methylthio, morpholino, C1-C6 alkyl, C1-C6 alkoxy, C3-C8 cycloalkyl, and C1-C6 alkylamino;

[0015] R3 is a non-substituted or substituted 3- to 8-membered heteroaryl group containing 1 to 3 heteroatoms of N, O or S, wherein the substitution means that at least one site is substituted by the following substituents: halogen, cyano, amino, nitro, hydroxyl, trifluoromethyl, methylthio, C1-C6 alkyl, C1-C6 alkoxy, C3-C8 cycloalkyl, C1-C6 alkylamino.

[0016] Preferably, R1 is hydrogen or phenyl;

[0017] R2 is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, and unsubstituted or substituted C1-C6 alkoxy; the substitution means that at least one site is substituted by the following substituents: morpholino, C1-C6 alkoxy;

[0018] R3 is a non-substituted or substituted 3- to 6-membered heteroaryl group containing 1-2 heteroatoms of N, O or S, wherein the substitution means that at least one site is substituted by one of the following substituents: nitro, C1-C6 alkoxy, C1-C6 alkyl, or trifluoromethyl.

[0019] More preferably, R1 is hydrogen or phenyl;

[0020] R2 is an unsubstituted or substituted C1-C3 alkoxy group; the substitution refers to the substitution of one site by the following substituents: morpholino, methoxy;

[0021] R3 is a non-substituted or substituted 3- to 6-membered heteroaryl group containing 1-2 heteroatoms of N, O or S, wherein the substitution refers to the substitution of one site by the following substituents: nitro, C1-C3 alkoxy, C1-C3 alkyl, trifluoromethyl.

[0022] Specifically, the benzopyridine compounds have any of the following structures:

[0023]

[0024] Furthermore, the benzopyridine compounds also include their pharmaceutically acceptable salts or solvates.

[0025] Furthermore, this invention also claims protection for a method for preparing the benzopyridine compound, the synthetic route of which is as follows:

[0026]

[0027] Specifically, the following steps are included:

[0028] Compound A, compound B, condensing agent, and basic reagent are dissolved in an organic solvent, stirred at room temperature until the reaction is complete, extracted, and filtered through a column to obtain the final product.

[0029] Furthermore, the room temperature for the room temperature stirring reaction is 15–35°C, and the reaction time is 5–8 hours.

[0030] Furthermore, the condensing agent is HATU or other commonly used condensing agents; the basic reagent is DIPEA or other commonly used basic reagents; and the organic solvent is DMF or other commonly used organic solvents in organic synthesis.

[0031] Furthermore, the extraction is performed using ethyl acetate and water.

[0032] Furthermore, after extraction, the organic phase is collected, concentrated, and purified by column chromatography.

[0033] The benzopyridine small molecule compounds constructed in this invention have good drug-like properties, high selectivity and strong affinity for STAT3 protein, and can specifically inhibit the activation of the STAT3 signaling pathway, thus serving as STAT3 specific inhibitors.

[0034] Therefore, this invention claims protection for the use of the benzopyridine compounds or their pharmaceutically acceptable salts or solvates in the preparation of STAT3 inhibitors.

[0035] In addition, this benzopyridine compound also has significant anticancer effects, with clear targets and mechanisms of action, and can significantly inhibit the growth and proliferation of various tumor cells.

[0036] Furthermore, the present invention also claims the use of the benzopyridine compounds or their pharmaceutically acceptable salts or solvates in the preparation of anticancer drugs.

[0037] Preferably, the cancers include, but are not limited to: acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, extrahepatic duct cancer, bladder cancer, synovial sarcoma, skin cancer, brainstem glioma, brain tumor, bronchial adenoma, Burkitt's lymphoma, carcinoid tumor, unknown primary cancer, central nervous system lymphoma, cervical cancer, gastric cancer, kidney cancer, laryngeal cancer, leukemia, liver cancer, non-small cell lung cancer, melanoma, rectal cancer, salivary gland cancer, sarcoma, small bowel cancer, soft tissue sarcoma, uterine sarcoma, testicular cancer, breast cancer, ovarian cancer, rhabdoid tumor, oral cancer, fallopian tube tumor, peritoneal tumor, bone cancer, prostate tumor, glioma, glioblastoma, and myeloma.

[0038] More preferably, the anticancer effect is against leukemia, including but not limited to one or more of acute leukemia, chronic leukemia, and special types of leukemia. This benzopyridine compound can reverse chemotherapy resistance in leukemia cells, overcoming the limitations of drug use in leukemia treatment. It has broad applications in the subsequent development of novel drugs targeting STAT3 and in the preparation of drugs for treating tumors and delaying chemotherapy resistance in leukemia.

[0039] As a preferred embodiment, the dosage form of the drug includes injections, capsules, tablets, pills, or granules.

[0040] As a preferred embodiment, the drug comprises a pharmaceutically acceptable carrier.

[0041] The term "pharmaceutically acceptable" means that a carrier, delivery substance, diluent, excipient, and / or the salt formed therefrom is generally chemically or physically compatible with other components constituting a drug dosage form and physiologically compatible with receptors.

[0042] The terms "salt," "acceptable salt," and "pharmaceutical salt" refer to acidic and / or basic salts formed by the above-described compounds or their stereoisomers with inorganic and / or organic acids and bases, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly during the final separation and purification of the compounds. Alternatively, they can be obtained by mixing the above-described compounds or their stereoisomers with an appropriate (e.g., equimolar) amount of acid or base. These salts may be obtained by precipitating in solution and collecting by filtration, or by recovery after solvent evaporation, or by freeze-drying after reaction in an aqueous medium.

[0043] Specifically, pharmaceutically acceptable salts include, but are not limited to: sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, hydrogen tartrates, ascorbic acid salts, succinates, maleates, gentianates, fumarates, gluconates, glucurons, glycosides, formates, benzoates, glutamates, methanesulfonates (methanesulfonates), ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and bis(hydroxynaphthyl) salts; or ammonium salts (e.g., primary amine salts, secondary amine salts, tertiary amine salts, quaternary ammonium salts), metal salts (e.g., sodium salts, potassium salts, calcium salts, magnesium salts, manganese salts, iron salts, zinc salts, copper salts, lithium salts, aluminum salts).

[0044] Furthermore, the present invention also provides an anti-leukemia composition comprising the benzopyridine compound or a pharmaceutically acceptable salt thereof, a solvate, and a leukemia chemotherapy drug.

[0045] Furthermore, the leukemia chemotherapy drugs include doxorubicin or cytarabine.

[0046] The present invention has the following beneficial effects:

[0047] The benzopyridine compounds provided by this invention have high STAT3 selectivity, can target STAT3 and inhibit STAT3 phosphorylation, significantly inhibit the activation of the STAT3 signaling pathway, inhibit the growth and proliferation of cells with high STAT3 expression, and achieve the effects of inhibiting tumor cell growth and proliferation and inducing tumor cell apoptosis. In particular, they have a significant inhibitory effect on drug-resistant tumor cells. Furthermore, when used in combination with chemotherapy drugs, they have the advantage of synergistic effect. They have good drug properties and are very suitable for the preparation of anticancer drugs. Attached Figure Description

[0048] Figure 1The following graphs illustrate the cell viability of MOLM-13 leukemia cells treated with benzopyridine compounds at a concentration of 5 μM for 72 h (A); the cell viability of Mv4-11 leukemia cells treated with benzopyridine compounds at a concentration of 5 μM for 72 h (B); the cell viability of MOLM-13 leukemia cell line treated with compounds W1310 and W1307 at different concentrations for 72 h (C); and the cell viability of Mv4-11 leukemia cell line treated with compounds W1310 and W1307 at different concentrations for 72 h (D).

[0049] Figure 2 The following graphs show the statistical data of cell viability after treating various tumor cells with different concentrations of compound W1307 in Example 2 for 72 hours (A); flow cytometry results of the effect of EdU on the proliferation of leukemia cell lines (B); and statistical data of the effect of EdU on the proliferation of leukemia cell lines (C).

[0050] Figure 3 Figure (A) shows the results of flow cytometry analysis of the effect of compound W1307 on apoptosis in leukemia cells in Example 3; Figure (B) shows the statistical data of flow cytometry analysis of the effect of compound W1307 on apoptosis in leukemia cells; and Figure (C) shows the expression of apoptosis proteins in leukemia cells after treatment with compound W1307.

[0051] Figure 4 This is a Western blot diagram showing the effect of compound W1307 from Example 4 on proteins related to the STAT3 signaling pathway in leukemia cells.

[0052] Figure 5 Figure (A) shows the results of the affinity assay between compound W1307 and STAT3 protein in Example 5; Western blot diagram of compound W1307 binding to STAT3 protein in cells (B); Western blot diagram of immunoprecipitation protein blotting of compound W1307 inhibiting STAT3 dimerization in cells (C); and statistical graph of reporter gene experimental data on the inhibition of STAT3 transcriptional activity by compound W1307 (D).

[0053] Figure 6 The following are statistical diagrams of the fluorescence distribution in mice during in vivo imaging during the drug administration period in Example 6 (A); the percentage of residual MOLM-13-EGFP / Luc cells in the bone marrow of mice after drug administration (B); and the percentage of residual MOLM-13-EGFP / Luc cells in the spleen of mice after drug administration (C).

[0054] Figure 7The following are statistical graphs showing the results of sensitivity assays of MOLM-13 and MOLM-13 / AR to Ara-C in Example 7 (A); the following are statistical graphs showing the cell viability of Ara-C resistant leukemia strains treated with different concentrations of compound W1307 (B); the following are statistical graphs showing the effect of combined administration of compound W1307 and Ara-C on cell viability (C); the following are statistical graphs showing the EdU assay results of combined administration of compound W1307 and Ara-C (D); and the following are statistical graphs showing the EdU assay results of combined administration of compound W1307 and Ara-C (E).

[0055] Figure 8 Figure (A) shows the results of flow cytometry analysis of the effect of compound W11307 combined with Ara-C on apoptosis of leukemia cells in Example 8; Figure (B) shows the statistical data of flow cytometry analysis of the effect of compound W11307 combined with Ara-C on apoptosis of leukemia cells; and Figure (C) shows the Western blot of apoptosis protein expression in leukemia cells after treatment with compound W11307 combined with Ara-C. Detailed Implementation

[0056] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0057] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0058] Example 1: Preparation of a benzopyridine compound W1300 (7-methoxy-N-(5-nitrothiazolyl)-2-phenylquinoline-4-carboxamide)

[0059] The specific synthetic routes for benzopyridine compounds are as follows:

[0060]

[0061] Specifically, the following steps are included:

[0062] 7-Methoxy-2-phenylquinoline-4-carboxylic acid (600 mg, 2.15 mmol), 5-nitrothiazol-2-amine (374 mg, 2.58 mmol), HATU (980.9 mg, 2.58 mmol), and DIPEA (1.14 mL, 6.45 mmol) were dissolved in 10 mL of DMF and reacted with the solution at room temperature for 5 hours. After the reaction was complete, the solution was diluted with ethyl acetate, extracted with water, and the organic phases were combined. The solutions were then evaporated to dryness and passed through a column (PE / EA = 1 / 1) to give 406 mg of a white solid, which was compound W1300, with a yield of 54.7%.

[0063] 1 H NMR (500MHz, DMSO-d6) δ12.98 (s, 1H), 8.76 (m, 2H), 8.56 (d, J = 7.4Hz, 2H), 8.20 (d d,J=9.2,3.6Hz,1H),7.56(ddd,J=13.7,11.0,5.3Hz,4H),,3.98(d,J=3.4Hz,3H).

[0064] Example 2: Preparation of a benzopyridine compound W1301 (7-(2-methoxyethoxy)-N-(5-nitrothiazolyl-2-yl)-2-phenylquinoline-4-carboxamide)

[0065]

[0066] Referring to Example 1, the difference is that in this example, 7-methoxy-2-phenylquinoline-4-carboxylic acid is replaced with an equimolar amount of 7-(2-methoxyethoxy)-2-phenylquinoline-4-carboxylic acid. The other reagents, parameters, and procedures are the same as in Example 1, yielding 408 mg of a white solid, which is compound W1301, with a yield of 42.17%.

[0067] 1 H NMR(500MHz,DMSO-d6)δ8.73(s,1H),8.36-8.31(m,3H),8.19(d,J=9.3Hz,1H),7.59(t,J=7.3Hz,2H) ,7.56-7.52(m,2H),7.34(dd,J=9.2,2.6Hz,1H),4.35-4.30(m,2H),3.77-3.74(m,2H),3.35(s,3H).

[0068] Example 3: Preparation of a benzopyridine compound W1302 (7-(3-morpholinopropoxy)-N-(5-nitrothiazolyl-2-yl)-2-phenylquinoline-4-carboxamide)

[0069]

[0070] Referring to Example 1, the difference is that in this example, 7-(2-methoxyethoxy)-2-phenylquinoline-4-carboxylic acid is replaced with an equimolar amount of 7-(3-morpholinopropoxy)-2-phenylquinoline-4-carboxylic acid. The other reagents, parameters and operations are the same as in Example 1, yielding 450 mg of white solid, which is compound W1302, with a yield of 40.32%.

[0071] 1H NMR (500MHz, DMSO-d6) δ8.64(s,1H),8.58(d,J=9.3Hz,1H),8.38(s,1H),8.29(d,J=7.7Hz,2H),7.57(t,J=7.4Hz,2H),7.53-7.50( m,2H),7.27(dd,J=9.3,2.4Hz,1H),4.25(t,J=6.0Hz,2H),3.73(s,4H),3.03-2.96(m,2H),2.93(s,4H),2.11(q,J=6.7,6.1Hz,2H).

[0072] Example 4: Preparation of a benzopyridine compound W1303 (7-methoxy-N-(3-methoxy-1,2,4-thiadiazol-5-yl)-2-phenylquinoline-4-carboxamide)

[0073]

[0074] Referring to Example 1, the difference is that 5-nitrothiazol-2-amine is replaced with an equimolar amount of 3-methoxy-1,2,4-thiadiazole-5-amine. The other reagents, parameters and operations are the same as in Example 1, yielding 409 mg of white solid, which is compound W1303, with a yield of 48.52%.

[0075] 1 H NMR (500MHz, DMSO-d6) δ13.86 (s, 1H), 8.38 (s, 1H), 8.37-8.34 (m, 2H), 8.23 ​​(d, J = 9. 3Hz,1H),7.61-7.53(m,4H),7.35(dd,J=9.3,2.6Hz,1H),4.01(s,3H),3.98(s,3H).

[0076] Example 5: Preparation of a benzopyridine compound W1304 (7-methoxy-N-(4-methylthiazolyl)-2-phenylquinoline-4-carboxamide)

[0077]

[0078] Referring to Example 1, the difference is that 5-nitrothiazol-2-amine was replaced with an equimolar amount of 4-methylthiazol-2-amine. The other reagents, parameters and operations were the same as in Example 1, yielding 406 mg of white solid, which was compound W1304, with a yield of 50.35%.

[0079] 1H NMR (500MHz, DMSO-d6) δ12.98(s,1H),8.36(d,J=7.4Hz,2H),8.29(d,J=3.7Hz,1H),8.20(dd,J=9.2,3.6Hz,1H),7.56( ddd,J=13.7,11.0,5.3Hz,4H),7.34(dt,J=9.2,2.9Hz,1H),6.92(s,1H),3.98(d,J=3.4Hz,3H),2.34(d,J=3.0Hz,3H).

[0080] Example 6 Preparation of a benzopyridine compound W1305 (7-methoxy-2-phenyl-N-(4-(trifluoromethyl)thiazolyl)quinoline-4-carboxamide)

[0081]

[0082] Referring to Example 1, the difference is that 5-nitrothiazol-2-amine was replaced with an equimolar amount of 4-(tert-butyl)thiazol-2-amine. The other reagents, parameters and operations were the same as in Example 1, and 438 mg of white solid was obtained, which was compound W1305, with a yield of 47.48%.

[0083] 1 H NMR(500MHz,DMSO-d6)δ13.50(s,1H),8.37(d,J=7.6Hz,3H),8.23(d,J=9.2Hz, 1H),8.13(s,1H),7.61-7.53(m,4H),7.35(dd,J=9.2,2.6Hz,1H),3.99(s,3H).

[0084] Example 7 Preparation of a benzopyridine compound W1306 (7-methoxy-2-phenyl-N-(thiazolyl-2-yl)quinoline-4-methamide)

[0085]

[0086] Referring to Example 1, the difference is that 5-nitrothiazol-2-amine was replaced with an equimolar amount of thiazol-2-amine. The other reagents, parameters and operations were the same as in Example 1, yielding 414 mg of white solid, which was compound W1306, with a yield of 53.34%.

[0087] 1H NMR(500MHz,DMSO-d6)δ13.05(s,1H),8.36(d,J=7.3Hz,2H),8.29(s,1H),8.16(d,J=9.2H z,1H),7.62-7.52(m,5H),7.39(d,J=3.5Hz,1H),7.34(dd,J=9.2,2.5Hz,1H),3.98(s,3H).

[0088] Example 8: Preparation of a benzopyridine compound W1307 (7-methoxy-N-(acetazol-2-yl)-2-phenylquinoline-4-methamide)

[0089]

[0090] Referring to Example 1, the difference is that 5-nitrothiazol-2-amine is replaced with an equimolar amount of oxazol-2-amine. The other reagents, parameters and operations are the same as in Example 1, yielding 399 mg of white solid, which is compound W1307, with a yield of 53.79%.

[0091] 1 H NMR (500MHz, DMSO-d6) δ12.12(s,1H),8.35(d,J=7.4Hz,2H),8.24(s,1H),8.16(d,J=8.5Hz, 1H),8.00(s,1H),7.60-7.52(m,4H),7.35(dd,J=9.2,2.4Hz,1H),7.24(s,1H),3.98(s,3H).

[0092] Example 9: Preparation of a benzopyridine compound W1308 (7-methoxy-2-phenyl-N-(1,3,4-thiadiazol-2-yl)quinoline-4-methamide).

[0093]

[0094] Referring to Example 1, the difference is that 5-nitrothiazol-2-amine was replaced with an equimolar amount of 1,3,4-thiadiazole-2-amine. The other reagents, parameters and operations were the same as in Example 1, yielding 387 mg of white solid, which was compound W1308, with a yield of 49.72%.

[0095] 1 H NMR (500MHz, DMSO-d6) δ9.33(s,1H),8.36(s,1H),8.35(s,2H),8.15(d,J=9.2Hz,1H),7.60-7.53(m,4H),7.33(dd,J=9.2,2.4Hz,1H),3.98(s,3H).

[0096] Example 10 Preparation of a benzopyridine compound W1309 (7-hydroxy-N-(5-nitrothiazo-2-yl)quinoline-4-carboxamide)

[0097]

[0098] Referring to Example 1, the difference is that 7-methoxy-2-phenylquinoline-4-carboxylic acid was replaced with an equimolar amount of quinoline-4-carboxylic acid. The other reagents, parameters and operations were the same as in Example 1, yielding 366 mg of white solid, which was compound W1309, with a yield of 53.87%.

[0099] 1 H NMR (500MHz, DMSO-d6) δ9.10(d,J=4.3Hz,1H),8.74(s,1H),8.21(d,J=8.3Hz,1H),8.15(d,J=8.4Hz,1H),7.89-7.86(m,2H),7.75-7.72(m,1H).

[0100] Example 11 Preparation of a benzopyridine compound W1310 (7-hydroxy-N-(5-nitrothiazolyl)-2-phenylquinoline-4-carboxamide)

[0101]

[0102] Referring to Example 1, the difference is that 7-methoxy-2-phenylquinoline-4-carboxylic acid was replaced with an equimolar amount of 2-phenylquinoline-4-carboxylic acid. The other reagents, parameters and operations were the same as in Example 1, yielding 408 mg of white solid, which was compound W1310, with a yield of 48.41%.

[0103] 1 H NMR (500MHz, DMSO-d6) δ8.77(s,1H),8.55(s,1H),8.37(d,J=7.5Hz,2H),8.28(d,J=8.4Hz,1H),8.20(d ,J=8.4Hz,1H),7.89(t,J=7.6Hz,1H),7.72(t,J=7.6Hz,1H),7.61(t,J=7.3Hz,2H),7.57-7.53(m,1H).

[0104] The human leukemia cell lines MOLM-13, K562, and Mv4-11, lung cancer cells PC9 and A549, and gastric cancer cells AGS and MGC803 used in this invention were all cultured in RPIM-1640 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. Breast cancer cell lines MDA-MB231-4175, MDA-MB-231, and MDA-MB-468, human embryonic kidney cells 293T, and liver cancer cells MHCC-97H were all cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. MOLM-13-EGFP / Luc cells were created by infecting MOLM-13 cells with a lentivirus carrying the EGFP / Luc tag, enabling them to express EGFP and Luciferase. MOLM-13 / AR cells were induced in leukemia cells to develop resistance to Ara-C through an in vitro low-concentration gradient escalation method, resulting in an Ara-C-resistant MOLM-13 cell line. This line was then maintained in complete medium containing Ara-C. All cells were cultured in a 37°C, 5% CO2 incubator.

[0105] Application Example 1: Benzopyridine compounds inhibit the cell viability of leukemia cells

[0106] 1. Experimental Methods

[0107] CCK8 assay for cell proliferation: Tumor cells in logarithmic growth phase (leukemia cell lines MOLM-13 and Mv4-11) were used at a concentration of (3–10) × 10⁻⁶. 3 Cells were seeded at a density of 100 μL / well in 96-well plates and cultured for 12–24 h. Three groups were then established: a control group (no drug administration), a blank control group, and a drug-administered group. Cells in the control group received an equal volume of culture medium; cells in the blank control group received only culture medium; cells in the drug-administered group received a specified concentration of benzopyridine compound and were cultured for another 72 h. Then, 10 μL / well of CCK8 assay reagent was added to each well of the 96-well plate in the dark, and the plates were incubated at 37°C in the dark for 1–4 h. The absorbance (OD) at 450 nm was measured using a microplate reader. Each experiment was independently repeated three times.

[0108] Cell viability (%) = [OD(drug-treated) - OD(blank)] / [OD(control) - OD(blank)] × 100%. Finally, nonlinear regression was performed using GraphPad Prism 8 software to obtain the corresponding half-maximal inhibitory concentration (IC50). 50 ).

[0109] 2. Experimental Results

[0110] Experimental results are as follows Figure 1 As shown, Figure 1Results A showed that multiple benzopyridine small molecule inhibitors could inhibit the cell viability of MOLM-13 leukemia cells after treatment at a concentration of 5 μM for 72 h, among which compounds W1300, W1301, W1302, W1307, W1309, and W1310 showed better inhibitory effects. Figure 1 Results B showed that several benzopyridine small molecule inhibitors could inhibit the cell viability of leukemia cells Mv4-11 at a concentration of 5 μM, among which compounds W1300, W1302, W1307 and W1310 showed better effects.

[0111] Figure 1 The C-results showed that compounds W1310 and W1307, when treated with the leukemia cell line MOLM-137 at different concentrations for 2 h, were able to inhibit the growth of leukemia cells MOLM-13 in a dose-dependent manner, with IC50 values ​​of 0.9897 μM and 1.876 μM, respectively. Figure 1 The results showed that W1310 and W1307, at different concentrations, could dose-dependently inhibit the growth of leukemia cell line Mv4-11 after treatment for 72 hours, with IC50 values ​​of 1.313 μM and 0.2095 μM, respectively, both exhibiting good inhibitory activity against leukemia cells.

[0112] The following application examples use compound W1307 as an example; the effects of other compounds are similar.

[0113] Application Example 2: Compound W1307 inhibits tumor cell growth and proliferation

[0114] 1. Experimental Methods

[0115] EdU assay for cell proliferation: Tumor cells in logarithmic growth phase (breast cancer cell lines MDA-MB231-4175, MDA-MB-231, MDA-MB-468, liver cancer cells MHCC-97H, lung cancer cells PC9, A549, gastric cancer cells AGS, MGC803, leukemia cell lines MOLM-13, K562) were analyzed using (20-30)×10⁻⁶ cells / cells. 4Cells were seeded in six-well plates and treated with different concentrations of W1307. After culturing for 48 hours, EdU working solution of appropriate concentration was added, and the cells were incubated for 2 hours. The culture medium was discarded by centrifugation, and 1 mL of 4% paraformaldehyde fixative was added to resuspend the cells. The cells were fixed at room temperature for 15 minutes. The fixative was removed by centrifugation, and the cells were washed three times with 3% BSA wash buffer. The wash buffer was removed, and 1 mL of 0.3% Triton X-100 permeabilization buffer was added. The cells were incubated at room temperature for 15 minutes. The permeabilization buffer was removed by centrifugation, and the cells were washed three times with wash buffer. Click reaction buffer was prepared (for one sample: Click Reaction Buffer: 430 μL, CuSO4). 4: 20 μL of Azide 555:1 μL, Click Additive Solution:50 μL were added to each sample, and the cells were centrifuged to remove the washing buffer. 500 μL of Click reaction solution was added to each sample to resuspend the cells, and the cells were incubated at room temperature in the dark for 30 min. The Click reaction solution was removed by centrifugation, and the cells were washed three times with washing buffer. Cell proliferation was then detected using the PE channel of a flow cytometer.

[0116] 2. Experimental Results

[0117] Experimental results are as follows Figure 2 As shown, Figure 2 Results A showed that compound W1307, at different concentrations, significantly inhibited the growth of various tumor cells in a dose-dependent manner after 72 hours of treatment. Figure 2 Figures B and C show the effect of EdU assay on the proliferation of W1307 on leukemia cell lines. The results show that W1307 treatment for 48 hours can dose-dependently inhibit the proliferation of leukemia cells.

[0118] Application Example 3: Compound W1307 induces apoptosis in leukemia cells

[0119] 1. Experimental Methods

[0120] (1) Flow cytometry detection of cell apoptosis

[0121] Cells in the logarithmic growth phase (leukemia cell lines MOLM-13 and K562) were harvested at a concentration of (20–30) × 10⁻⁶. 4 Cells were seeded in six-well plates and treated with different concentrations of compound W1307. After culturing for 48 h, the culture medium was removed by centrifugation, and the cells were washed twice with pre-cooled PBS. The cells were resuspended in 400 μL of 1×Annexin V binding solution, and 2.5 μL of Annexin V-FITC staining solution was added to the sample. The mixture was then incubated on ice in the dark for 15 min. Then, 5 μL of PI staining solution was added, and the mixture was incubated on ice in the dark for 5 min. The cells were then detected by flow cytometry.

[0122] (2) Western blotting detection of apoptosis-related protein expression

[0123] Cells were collected after treatment with compound W1307, centrifuged to remove the culture medium, and then lysed with an appropriate amount of RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. Cells were lysed on ice, vortexed every 5 minutes, and centrifuged at 15,000 rpm for 15 minutes at 4°C after 20 minutes. Protein supernatant was separated, and 5× loading buffer was added for boiling denaturation. Protein samples were then separated by polyacrylamide gel SDS-PAGE electrophoresis, transferred to nitrocellulose membranes (PVDF membranes), blocked with 5% BSA for 1 hour, and incubated overnight at 4°C with the corresponding primary antibody. The next day, the cells were incubated with secondary antibody at room temperature for 1 hour, and protein expression levels were detected using a Bio-RAD imaging system.

[0124] 2. Experimental Results

[0125] Experimental results are as follows Figure 3 As shown, Figure 3 A and B in the figure show that after treatment with compound W1307 for 48 h, apoptosis of leukemia cells was induced in a dose-dependent manner. Figure 3 The C in the figure shows that treatment with compound W1307 can induce the expression of apoptosis proteins in leukemia cells.

[0126] Application Example 4: Compound W1307 affects the STAT3 signaling pathway in leukemia cells.

[0127] 1. Experimental Methods

[0128] After compound W1307 was treated using the method in Example 3, the expression levels of STAT3 signaling pathway-related proteins (pY705-STAT3, T-STAT3, C-Myc, Mcl-1, Bcl-2) in leukemia cells were determined using the protein immunoblotting method in Example 3.

[0129] 2. Experimental Results

[0130] Experimental results are as follows Figure 4 As shown, compound W1307 significantly inhibited the phosphorylation level of STAT3-Y705 with increasing concentration, and also significantly inhibited the expression of downstream proteins of the STAT3 signaling pathway, including c-Myc, Mcl-1, and Bcl-2, which are related to proliferation, cell cycle, and anti-apoptosis.

[0131] Application Example 5: Compound W1307 binds to STAT3 protein and inhibits STAT3 dimerization and transcriptional activity.

[0132] 1. Experimental Methods

[0133] (1) Surface plasmon resonance (SPR)

[0134] After STAT3 protein purification, the concentration was adjusted to 0.1 mg / mL with PBS and immobilized on a CM5 chip. Compound W1307 was dissolved in PBS buffer containing 0.05% p20 and 2% DMSO, then diluted 1 / 2 times to a specific concentration before injection. The corresponding response values ​​were analyzed, and K was fitted using software. d Value. See results. Figure 5 A in the middle.

[0135] (2) Cell heat transfer assay (CETSA)

[0136] Logarithmic growth phase cells (K562 leukemia cell line) were seeded in 15cm culture dishes. When the cell confluence density reached approximately 80%, DMSO and compound W1307 were added to a final concentration of 1μM, and the cells were treated for 1 hour. Cells were then collected, washed twice with PBS, centrifuged to remove the supernatant, and resuspended in 1mL of PBS containing PMSF and a phosphatase inhibitor. The cell suspension was aliquoted into 100μL PCR tubes. The PCR tubes were placed in a gradient PCR instrument and heated at different specific temperatures for 2 minutes, followed by incubation at room temperature for 3 minutes. Cell lysis and protein release were then performed by rapidly freezing cells in liquid nitrogen for 3 minutes, followed by thawing at room temperature for 3 minutes. After thawing, the cells were vortexed, and after three freeze-thaw cycles, the cells were transferred from the PCR tubes to 1.5mL EP tubes, centrifuged at 20000g for 20 minutes at 4℃, and the supernatant was collected. 5× loading buffer was added, and the cells were incubated at 100℃ for 5 minutes, followed by Western blotting analysis. Results are shown below. Figure 5 B in the middle.

[0137] (3) Co-immunoprecipitation (Co-IP)

[0138] Human embryonic kidney cells 293T were seeded at an appropriate density in 6-well plates. When the cells reached approximately 70%–80% confluence, transfection was performed. Two mixed transfection solutions, A and B, were prepared in 1.5 mL ep ep tracts (volume per well as follows: A: 5 μL Lipo2000 + Opti-MEM medium to a total volume of 100 μL; B: 2500 ng HA-STAT3 plasmid + 2500 ng Flag-STAT3 plasmid + Opti-MEM to a total volume of 100 μL). After standing for 5 min, the two solutions were gently mixed and incubated at 4°C for 15 min. The 6-well plates were then removed, the old medium was discarded, and 800 μL of antibiotic-free DMEM medium was added. The prepared transfection reagent was then added dropwise to each well (200 μL), gently mixed, and incubated for 6 h. The medium was then replaced with complete medium. 24 h after transfection, different concentrations of compounds were added for 24 h of treatment. Before protein collection, cells were stimulated with 100 ng / mL IL-6 for 30 min. Cells were then scraped off and collected along with the culture medium. After centrifugation and discarding the supernatant, IP lysis buffer was added, and lysis was performed on ice for 30 min. Following this, the cells were centrifuged at 15000 rpm for 15 min at 4°C. The supernatant protein was collected for BCA quantification, with four 30 μg protein aliquots from each group used as input. Simultaneously, magnetic bead pretreatment was performed: Anti-Flag Affinity Gel was gently mixed, and 15 μL of magnetic beads were aspirated into each tube. The tubes were washed twice with PBST, and then the same amount of protein was added to each tube. The tubes were incubated overnight on a rotary shaker at 4°C. The next day, samples were removed, centrifuged at 5000 rpm for 30 s, the supernatant was discarded, and the samples were washed twice with PBST. The PBST was discarded, and an equal volume of 1× loading buffer was added. The samples were boiled in a metal bath at 100°C for 5 min, followed by Western blotting analysis. Results are shown below. Figure 5 C in the middle.

[0139] (4) Dual-luciferase reporter gene assay

[0140] Human embryonic kidney 293T cells were seeded at an appropriate density in 96-well plates. When the cells adhered and grew to approximately 70%–80% confluence, transfection was performed. Two mixed transfection solutions, A and B, were prepared in 1.5 mL ep endothelial tubes (volume per well as follows: A: 0.25 μL Lipo2000 + Opti-MEM medium to a total volume of 5 μL; B: 50 ng pGL3-STAT3-promoter plasmid + 50 ng STAT3C plasmid + 40 ng TKRL plasmid + Opti-MEM to a total volume of 5 μL). After standing for 5 min, the two solutions were gently mixed and incubated at 4°C for 15 min. 10 μL of transfection reagent was added to each well, gently mixed, and incubated for 24 h. Then, different concentrations of the compound were added to each group for another 24 h. The Renilla luciferase detection substrate and buffer were prepared at a 1:100 ratio to prepare the Renilla luciferase working solution for later use. Remove the 96-well plate, discard the culture medium, add 50 μL of reporter gene cell lysis buffer to each well, vortex and mix for 10 min. Transfer 25 μL of lysis buffer to each well of the 96-well plate. Add 25 μL of firefly luciferase assay reagent to each well of the plate, vortex and mix for 5 min, and then analyze to obtain RLU1. Subsequently, add 25 μL of Renilla luciferase assay working solution to each well, vortex and mix for 5 min, and then analyze to obtain RLU2. Calculate the RLU1 / RLU2 ratio. See the results below. Figure 5 D in the middle.

[0141] 2. Experimental Results

[0142] The results are as follows Figure 5 As shown, Figure 5 ASPR experimental results showed that compound W1307 has a high affinity for STAT3 protein, K d The value is 1.74 × 10 6 mol / L; Figure 5 The results of the B CETSA experiment showed that compound W1307 can bind to and stabilize the STAT3 protein in cells; Figure 5 The C-results showed that W1307 was able to inhibit STAT3 dimerization in a dose-dependent manner; Figure 5 The results showed that W1307 was able to inhibit the transcriptional activity of STAT3 in a dose-dependent manner.

[0143] Application Example 6: Compound W1307 inhibits leukemia progression in an in vivo model.

[0144] 1. Experimental Methods

[0145] (1) Construction of MOLM-13-EGFP / Luc cells

[0146] Take MOLM-13 in the logarithmic growth phase at (30~35)×104 Inoculate 500 μL of the cells into small dishes. Add polybrene to a final concentration of 10 μg / mL, then add 1 mL of lentivirus expressing the plenti-EGFP-Luc plasmid. Mix well and incubate for 12–16 h. Afterward, replace with fresh medium and continue culturing. 48 h after virus infection, add puromycin to a final concentration of 2.5 μg / mL and screen for two consecutive weeks. Observe the degree of green fluorescence expression in the cells using a cell imaging system to verify successful cell construction.

[0147] (2) Construction of mouse leukemia model

[0148] MOLM-13-EGFP / Luc cells in the logarithmic growth phase were washed twice with pre-cooled PBS and resuspended in PBS to obtain a density of 5 × 10⁶ cells / year. 6 A cell suspension of 100 μL was injected into mice via tail vein. Ten days after model establishment, mice were injected with potassium luciferin, and fluorescence distribution in the mice was observed using a small animal in vivo imaging system to confirm successful model establishment. Mice were randomly divided into three groups: a solvent control group, a 5 mg / kg W1307 drug group, and a 15 mg / kg W1307 drug group. The drugs were administered intraperitoneally daily for two weeks, and fluorescence intensity was monitored every seven days using an in vivo imaging system to observe leukemia progression. Two weeks after administration, mice were sacrificed and dissected. Bone marrow and spleen cells were isolated, and the proportion of MOLM-13-EGFP / Luc cells in the bone marrow and spleen was detected by flow cytometry.

[0149] (3) Experimental Results

[0150] The results are as follows Figure 6 As shown, Figure 6 A in the image is an in vivo imaging image taken during drug administration, showing that compound W1307 can inhibit the progression of leukemia in a mouse model. Figure 6 The compound W1307 in the B-group can reduce the proportion of residual MOLM-13-EGFP / Luc cells in mouse bone marrow; Figure 6 The C-value of compound W1307 can reduce the proportion of residual MOLM-13-EGFP / Luc cells in mouse spleen.

[0151] Application Example 7: Combination therapy of compound W1307 with Ara-C inhibited the proliferation of cytarabine (Ara-C)-resistant leukemia strains.

[0152] 1. Experimental Methods

[0153] After treatment with Ara-C, W1307, or Ara-C+W1307 drug combinations, the inhibitory effect of the compound combined with Ara-C on the proliferation of Ara-C resistant leukemia strains was determined according to the method in Application Example 2.

[0154] 2. Experimental Results

[0155] The results are as follows Figure 7 As shown, Figure 7 As can be seen from A, MOLM-13 and MOLM-13 / AR have different sensitivities to Ara-C. The IC50 of MOLM-13 is 0.1015 μM, while that of MOLM-13 / AR is 2.083 μM, with a resistance index of 20, indicating that the Ara-C resistant strain of leukemia was successfully constructed. Figure 7 Compound B, W1307, showed that after treatment of Ara-C resistant leukemia strains at different concentrations for 72 h, it significantly inhibited cell growth in a dose-dependent manner, with an IC50 of 3.564 μM. Figure 7 The combination of compound W1307 and Ara-C significantly inhibited the cell viability of Ara-C resistant leukemia strains. Figure 7 As shown in D and E, the combined use of compound W1307 and Ara-C significantly inhibited the proliferation of Ara-C resistant leukemia strains. Example 8: The combined use of compound W1307 and Ara-C induced apoptosis in Ara-C resistant leukemia strains.

[0156] 1. Experimental Methods

[0157] After treatment with Ara-C, W1307, or a combination of Ara-C and W1307, the effect of compound W1307 in inducing apoptosis in Ara-C resistant leukemia strains was determined, referring to the method in Application Example 3.

[0158] 2. Experimental Results

[0159] Experimental results are as follows Figure 8 As shown, Figure 8 As shown in A and B, flow cytometry analysis revealed that the combination of compound W1307 and Ara-C significantly induced apoptosis in Ara-C resistant leukemia strains. Figure 8 The C-value shows that the combined treatment of compound W1307 and Ara-C significantly induced the expression of apoptosis proteins in drug-resistant leukemia cell lines.

[0160] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A benzopyridine compound, characterized by, The benzopyridine compound has any one of the following structures: 。 2. The benzopyridine compound according to claim 1, wherein The benzopyridine compound also includes pharmaceutically acceptable salts thereof.

3. The method for preparing the benzopyridine compound according to claim 1, characterized in that, The synthetic route is as follows: Specifically comprising the following steps: Compound A, compound B, condensing agent and basic reagent are dissolved in an organic solvent, stirred at room temperature until the reaction is complete, extracted, and passed through a column to obtain the product. The definitions of R1, R2, and R3 are the same as the groups corresponding to any one of the structures of the benzopyridine compound in claim 1.

4. Use of a benzopyridine compound or a pharmaceutically acceptable salt thereof in the manufacture of a STAT3 inhibitor, characterized in that, The benzopyridine compound has any one of the following structures: 。 5. Use of a benzopyridine compound or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating cell MOLM-13 related leukemia, characterized in that, The benzopyridine compound has the following structure: 。 6. Use of a benzopyridine compound or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating Mv4-11 associated leukemia, characterized in that, The benzopyridine compound has the following structure: 。 7. Use of a benzopyridine compound or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating cell MOLM-13 and / or cell Mv4-11 related leukemia, characterized in that, The benzopyridine compound has any one of the following structures: 。 8. Use of a benzopyridine compound or a pharmaceutically acceptable salt thereof in the manufacture of an anticancer drug, characterized in that, The benzopyridine compound has any one of the following structures: ； The cancer is leukemia, breast cancer, liver cancer, non-small cell lung cancer, or gastric cancer.

9. A composition for treating leukemia, characterized in that, The benzopyridine compound or a pharmaceutically acceptable salt thereof in claim 1 and a leukemia chemotherapy drug are included.

10. The anti-leukemia composition of claim 9, wherein the compound is ###0002### The leukemia chemotherapy drug includes doxorubicin or cytarabine.

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