AKR1C3 / nrf2 dual-target inhibitor and pharmaceutical composition and application thereof

Abstract

The application discloses a kind of AKR1C3 / NRF2 dual-target inhibitor and its pharmaceutical composition and application, the compound has as shown in structure Formula I.The present application is aimed at the problem of tumor chemotherapy resistance, first design and synthesis can be targeted to AKR1C3 and NRF2 dual-target inhibitor, effectively cut off the positive feedback loop mediated by both chemotherapy resistance.Experiments prove that the compound of the present application is significantly superior to single-target inhibitor and their physical mixture in reversing drug resistance activity, synergistic effect is excellent, can significantly reverse antitumor drug resistance at cell level, has broad application prospect in preparation of drug-resistant tumor drugs.Ⅰ

Description

Technical Field

[0001] This invention relates to a compound, pharmaceutical compositions thereof, and applications, and more particularly to a class of AKR1C3 / NRF2 dual-target inhibitors, pharmaceutical compositions thereof, and applications. Background Technology

[0002] Chemotherapy resistance in tumors is one of the core causes of clinical cancer treatment failure, patient relapse, and death. The mechanisms of multidrug resistance in tumors are complex and diverse, involving multiple levels such as drug metabolism inactivation, enhanced drug efflux, abnormal DNA damage repair, and inhibition of apoptosis. Among these, the abnormal activation of aldehyde-ketone reductase 1C3 (AKR1C3) and nuclear factor erythroid 2-related factor 2 (NRF2) are two core pathways mediating chemotherapy resistance in tumors, and there is a positive feedback synergistic effect of cross-regulation between the two, jointly forming a resistance barrier that is difficult to overcome.

[0003] AKR1C3 is an important member of the aldehyde-ketone reductase superfamily and is highly expressed in various malignant tumors. Its mechanisms mediating tumor drug resistance mainly include two aspects: first, it directly inactivates many commonly used chemotherapeutic drugs such as anthracyclines (e.g., doxorubicin, daunorubicin) and platinum-based drugs (e.g., cisplatin, carboplatin) through reductive metabolism, significantly reducing their cytotoxicity; second, it promotes tumor cell proliferation, invasion, and anti-apoptosis by regulating signaling pathways such as prostaglandin metabolism and steroid hormone synthesis. Currently, S19-1035 is a reported highly selective AKR1C3 inhibitor, but it only inhibits AKR1C3 on a single target and cannot effectively block the NRF2-mediated compensatory resistance pathway, resulting in limited in vivo reversal of drug resistance. Currently, there are no marketed drugs specifically targeting AKR1C3.

[0004] NRF2 is a core transcription factor regulating cellular redox homeostasis. Under physiological conditions, NRF2 maintains intracellular redox balance and exerts a cytoprotective role by binding to the antioxidant response element (ARE) and upregulating the expression of downstream antioxidant target genes such as heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO1). However, in tumor cells, NRF2 is often abnormally and persistently activated. In addition to upregulating antioxidant genes, it can also significantly induce the expression of multidrug resistance-related proteins (such as MRP1 and P-gp), reducing oxidative damage and effective accumulation of chemotherapeutic drugs in tumor cells, which is one of the core mechanisms of acquired drug resistance in tumor cells. ML385 is currently a recognized NRF2 inhibitor and is widely used as a tool compound for mechanistic studies. However, it can only inhibit NRF2 as a single target and cannot block the direct metabolic inactivation of chemotherapeutic drugs mediated by AKR1C3, which severely limits its clinical translational potential.

[0005] More importantly, existing research has confirmed the existence of a synergistic positive feedback loop of drug resistance between the AKR1C3 and NRF2 pathways: reactive oxygen species (ROS) generated by AKR1C3 metabolizing chemotherapeutic drugs can further activate NRF2; and the abnormally activated NRF2 can then transcribe and upregulate AKR1C3 expression, leading to the metabolic inactivation of more drugs and the generation of more ROS, forming a vicious cycle. The existence of this positive feedback loop means that inhibiting one pathway will cause a compensatory enhancement of the other pathway, making it difficult for single-target inhibition strategies to completely block the drug resistance process.

[0006] As of the date of this application, the applicant has not found any prior art that discloses a covalently linked dual-target small molecule inhibitor capable of simultaneously targeting AKR1C3 and NRF2, nor has it found any technical inspiration for achieving intramolecular synergy by covalently fusing the core pharmacophores of S19-1035 and ML385 through a specific linker unit. Furthermore, the physical mixed-administration method of single-target inhibitors, due to pharmacokinetic mismatch and differences in tissue distribution, cannot achieve molecular-level synergy, making it difficult to completely block the aforementioned positive feedback resistance loop, resulting in less than ideal resistance reversal effects.

[0007] Therefore, developing a dual-target covalently linked inhibitor that can simultaneously target AKR1C3 and NRF2, and blocking two synergistic resistance pathways through backbone fusion and pharmacophore splicing strategies to achieve intramolecular synergistic effects, is of great scientific significance and clinical application value for solving the clinical problem of chemotherapy resistance in tumors. Summary of the Invention

[0008] Objectives of the invention: The first objective of this invention is to provide a class of AKR1C3 / NRF2 dual-target inhibitors; the second objective is to provide a pharmaceutical composition comprising the compound; and the third objective is to provide an application of the compound or a pharmaceutical composition thereof.

[0009] Technical solution: The present invention provides an AKR1C3 / NRF2 dual-target inhibitor, such as the compound shown in formula (I) or a pharmaceutically acceptable salt thereof:

[0010] Ⅰ

[0011] in:

[0012] R1 is selected from hydrogen, halogen, C1-C4 alkoxy, C1-C4 alkyl, C1-C4 haloalkyl, nitro, cyano, hydroxy, and amino.

[0013] R2 is selected from

[0014]

[0015] L1, L2, and L3 are each independently selected from one or more combinations of substituted or unsubstituted C1-C7 alkylene groups, substituted or unsubstituted phenylenediol groups, substituted or unsubstituted piperidinyl groups, substituted or unsubstituted piperazineyl groups, C1-C7 alkylamide groups, and C1-C7 alkoxy groups, where n = 0 or 1.

[0016] R3 is selected from

[0017]

[0018] X is selected from hydrogen, halogen, C1-C4 alkoxy, C1-C4 haloalkyl, nitro, cyano, hydroxyl, and amino.

[0019] Furthermore,

[0020] R1 is selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, trifluoromethyl, nitro, and cyano.

[0021] L1, L2, and L3 are each independently selected from one or more combinations of substituted or unsubstituted C1-C6 alkylene groups, substituted or unsubstituted 1,3-phenylenediol groups, substituted or unsubstituted 1,4-phenylenediol groups, substituted or unsubstituted piperidinidyl groups, substituted or unsubstituted piperazineidyl groups, C1-C6 alkylamide groups, and C1-C6 alkoxy groups; X is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, trifluoromethyl, nitro, and cyano.

[0022] Further, L1 is selected from substituted or unsubstituted C1-C6 alkylene groups, substituted or unsubstituted piperidinium groups; L2 is selected from substituted or unsubstituted 1,3-phenylenediol groups, substituted or unsubstituted 1,4-phenylenediol groups; L3 is selected from substituted or unsubstituted 1,3-phenylenediol groups, substituted or unsubstituted 1,4-phenylenediol groups; X is selected from hydrogen, fluorine, chlorine, and bromine.

[0023] Furthermore, R2 is selected from

[0024] Furthermore, the compound is selected from any of the following compounds:

[0025]

[0026]

[0027] Furthermore, the pharmaceutically acceptable salt is a salt formed by the compound and an acid selected from any of the following:

[0028] Hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, malic acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, ferulic acid.

[0029] "Pharmaceutically acceptable salts" refer to salts of compounds prepared by reacting a compound with a relatively non-toxic acid or base, containing specific substituents. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts, or similar salts. When a compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogen phosphates, dihydrogen phosphates), sulfuric acid (forming sulfates or bisulfates), hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; organic acid salts also include salts of organic acids such as amino acids (e.g., arginine), glucuronic acid, etc. Certain compounds contain both basic and acidic functional groups, thus allowing them to be converted into either a base or an acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner, followed by separation of the parent compound, thereby regenerating the free form of the compound. The free form of a compound differs from its various salt forms in certain physical properties, such as its solubility in polar solvents.

[0030] Pharmaceutically acceptable salts can be synthesized from parent compounds containing acid radicals or bases using conventional chemical methods. Generally, such salts are prepared by reacting these compounds, in their free acid or base form, with a stoichiometric amount of base or acid in water, an organic solvent, or a mixture of both. Non-aqueous media such as ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile are generally preferred.

[0031] The dosage form of the drug is selected from capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalers, ointments, suppositories, and patches. The carriers can be mixed arbitrarily and can be changed depending on the dosage form and administration method. Pharmaceutically acceptable carriers include, for example, excipients, binders, disintegrants, lubricants, flavoring agents, fragrances, colorings, or sweeteners.

[0032] "Pharmaceutically acceptable carriers" are excipients widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods to facilitate the dissolution of the active ingredient at a desired rate after administration to a subject, or to promote the effective absorption of the active ingredient after administration to a subject. The pharmaceutical excipients may be inert fillers or provide a function, such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient. The pharmaceutical excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0033] The pharmaceutical compositions described in this invention can be prepared using any method known to those skilled in the art, based on the disclosure. For example, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0034] The pharmaceutical compositions of this invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical compositions of this invention can also be controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0035] This invention provides the use of the above-mentioned AKR1C3 / NRF2 dual-target inhibitor or pharmaceutical composition thereof in the preparation of medicaments for the prevention and / or treatment of cancer and the reversal of tumor drug resistance.

[0036] Preferably, the drug is used in combination with other antitumor drugs that induce drug resistance, such as doxorubicin, sorafenib, osimertinib, and gefitinib.

[0037] Beneficial Effects: Compared with existing technologies, the beneficial effects of this invention are as follows: This invention provides for the first time a novel dual-target inhibitor formed by covalently fusing the core pharmacophores of an AKR1C3 inhibitor and an NRF2 inhibitor through a specific linker unit. This series of compounds can simultaneously block the AKR1C3-mediated chemotherapeutic drug metabolism pathway and the NRF2-mediated antioxidant resistance pathway, effectively breaking the positive feedback synergistic resistance loop between the two. Experiments show that its tumor resistance reversal activity is significantly better than that of the corresponding single-target inhibitor monotherapy, and even better than the physical combination of the two, exhibiting a significant intramolecular synergistic effect. The preferred compounds of this invention exhibit inhibitory activity against the AKR1C3 enzyme at the micromolar to nanomolar level, and inhibitory activity against NRF2 is significantly better than or equivalent to the positive control ML385. At the cellular level, they can effectively reverse the resistance of tumor cells to doxorubicin, providing new compound entities and therapeutic strategies for the treatment of drug-resistant tumors, and have broad prospects for clinical translation. Attached Figure Description

[0038] Figure 1 The antiproliferative activity of NRF2 inhibitor ML385 and AKR1C3 inhibitor S19-1035 in combination with doxorubicin against drug-resistant breast cancer cells;

[0039] Figure 2 The antiproliferative activity of NRF2 inhibitor ML385 and AKR1C3 inhibitor S19-1035 in combination with doxorubicin against drug-resistant breast cancer cells;

[0040] Figure 3 Combination index (CI) and doxorubicin dose reduction index (DOX DRI) for NRF2 inhibitor ML385 and AKR1C3 inhibitor S19-1035, respectively or in combination with doxorubicin.

[0041] Figure 4 The compound of this invention exhibits antiproliferative activity against doxorubicin-resistant breast cancer cells at 25 μM.

[0042] Figure 5 The antiproliferative activity of the compound of the present invention (25 μM) in combination with 25 μM doxorubicin against doxorubicin-resistant breast cancer cells;

[0043] Figure 6 Comparison of the reversal of doxorubicin resistance activity of preferred compounds with single-target molecules administered alone or in combination (****p < 0.0001). Detailed Implementation

[0044] The technical solution of the present invention will be further described below with reference to the embodiments.

[0045]

[0046] Reagents and reaction conditions: (i) 2-methylbenzoyl chloride, potassium carbonate, tetrahydrofuran / water, room temperature, 1 h; (ii) 2-bromopropionyl bromide, aluminum trichloride, dichloromethane, 0 °C, 2 h, 50 °C, 24 h; (iii) thiourea, ethanol, 70 °C, 5 h; (iv) 4-(chloromethyl)-3,5-dimethylisoxazole, potassium carbonate, N,N-dimethylformamide, 50 °C, 3 h; (v) lithium hydroxide, tetrahydrofuran / methanol / water, room temperature, 1 h; (vi) acid or base, N,N,N',N'-tetramethylchloroformamidine tetrafluoroborate, N-methylimidazolium, acetonitrile, room temperature, 2 h.

[0047]

[0048]

[0049] Reagents and reaction conditions: (i) 4-(chloromethyl)-3,5-dimethylisoxazole, potassium carbonate, N,N-dimethylformamide, 50°C, reaction time 3 h; (ii) lithium hydroxide, tetrahydrofuran / methanol / water mixed solvent, room temperature, reaction time 1 h; (iii) benzotriazol-1-yl-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature, reaction time 2 h; (iv) lithium hydroxide, tetrahydrofuran / methanol / water mixed solvent, room temperature, reaction time 1 h; (v) acid or base, N,N,N',N'-tetramethylchloroformamidinium tetrafluoroborate, N-methylimidazolium, acetonitrile, room temperature, reaction time 2 h.

[0050]

[0051] Reagents and reaction conditions: (i) 4-(chloromethyl)-3,5-dimethylisoxazole, potassium carbonate, N,N-dimethylformamide, 50°C, 3 h; (ii) trifluoroacetic acid, dichloromethane, room temperature, 12 h; (iii) acid, tetrafluoroboronic acid, N,N,N',N'-tetramethylchloroformamidinium, N-methylimidazole, acetonitrile, room temperature, 2 h; (iv) lithium hydroxide, tetrahydrofuran / methanol / water, room temperature, 1 h; (v) benzotriazol-1-yl-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature, 2 h.

[0052]

[0053] Reagents and reaction conditions: (i) methyl 5-bromopentanoate, cesium carbonate, acetonitrile, 70°C, 2 h; (ii) trifluoroacetic acid, dichloromethane, room temperature, 12 h; (iii) N,N,N',N'-tetramethylchloromethanedinium tetrafluoroborate, N-methylimidazole, acetonitrile, room temperature, 2 h; (iv) 1,4-dibromobutane, cesium carbonate, acetonitrile, room temperature, 2 h; (v) cesium carbonate, acetonitrile, 70°C, 24 h; (vi) lithium hydroxide, tetrahydrofuran / methanol / water, room temperature, 1 h; (vii) N,N,N',N'-tetramethylchloromethanedinium tetrafluoroborate, N-methylimidazole, acetonitrile, room temperature, 2 h; (viii) cesium carbonate, acetonitrile, 70°C, 24 h.

[0054] Example 1: Synthesis of the main intermediate

[0055] Synthesis of intermediate 2a: Indoline 1a (600 μL, 5.04 mmol) was dissolved in THF / H2O (6 / 2 mL) and kept below 0 °C. Potassium carbonate (1.741 g, 12.61 mmol) was slowly added, and the mixture was stirred for 5 min. Then, o-methylbenzoyl chloride (684 μL, 5.28 mmol) was added dropwise, and the reaction was continued at room temperature. After 1 h, the reaction was completed. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was distilled under reduced pressure to give a pale yellow solid 2a (1.1 g, yield 92%).

[0056] Synthesis of intermediate 3a: Intermediate 2a (400 mg, 1.69 mmol) was dissolved in DCM (8 mL) and placed below 0 °C. Aluminum chloride (675 mg, 5.06 mmol) was slowly added, and the mixture was stirred at low temperature for 1 h. Then, 2-bromopropionyl bromide (529.6 μL, 5.06 mmol) was added dropwise, and the mixture was stirred at low temperature for 1 h. The reaction was then continued at 50 °C. After 24 h, the reaction was complete. Water was added to the reaction system, and the mixture was extracted with DCM. The DCM layer was distilled under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain a white solid 3 (200 mg, yield 32%).

[0057] Synthesis of intermediate 4a: Ethanol (8 mL) was added to intermediate 3a (400 mg, 1.075 mmol), the mixture was heated to 70 °C and stirred under reflux until partially dissolved. Thiourea (122.8 mg, 1.613 mmol) was then added, and the mixture was stirred for several minutes until the reaction solution gradually became clear. The reaction was completed after 5 h, at which point a white solid precipitated in the reaction system. The reaction system was filtered, washed with ethanol, and dried to obtain white solid 1a (270 mg, yield 75%).

[0058] Synthesis of intermediate 4b: Following the synthesis method of intermediate 4a, 2-bromo-1-(3-chlorophenyl)-1-propanone was used as the starting material to obtain a white solid with a yield of 60%.

[0059] Synthesis of intermediate 4c: Following the synthesis method of intermediate 4a, 2-bromo-1-(4-bromophenyl)-1-propanone was used as the starting material to obtain a white solid with a yield of 74%.

[0060] Synthesis of intermediate 6l: 5l (170 μL, 1.31 mmol) was dissolved in DMF (6 mL), potassium carbonate (272.5 mg, 1.97 mmol) was added, and the mixture was stirred at 50 °C for 15 min. Then, 4-chloromethyl-3,5-dimethylisoxazole (179 μL, 1.45 mmol) was added, and the reaction was continued at 50 °C. After 3 h, the reaction was completed, water was added to the reaction solution, and the mixture was extracted with ethyl acetate solution and washed with saturated brine. The ethyl acetate layer was distilled under reduced pressure and dried to give a pale yellow solid 6a-l (243 mg, yield 71%).

[0061] Synthesis of intermediate 6b-k: Following the synthesis method of intermediate 6l, 5b-k was used as the starting material to obtain a white solid with a yield of 76%.

[0062] Synthesis of intermediate 7a: Intermediate 6a (381 mg, 1.31 mmol) was dissolved separately in THF / MeOH (6 / 2 mL), and 2N lithium hydroxide solution (5 mL) was added. The reaction was carried out at room temperature. After 1 h, the reaction was completed. The organic solvent in the reaction system was removed by vacuum distillation. The pH was adjusted by adding dilute hydrochloric acid, and a white solid precipitated. The solid was filtered, the filter cake was collected, and dried to obtain a white solid 7a-l (263 mg, yield 81%).

[0063] Synthesis of intermediate 7b-l: Following the synthesis method of intermediate 7a, intermediate 6b-l was used as the raw material to obtain a white solid with a yield of 90%.

[0064] Synthesis of intermediate 9a: Intermediate 8a (350 mg, 2.12 mmol) was dissolved in DMF (6 mL), and 7a (534 mg, 1.93 mmol) was added. DIEA (624 μL, 3.86 mmol) was added to the reaction system, followed by HATU (1.1 g, 2.9 mmol). The reaction was carried out at room temperature. After 2 h, the reaction solution was extracted with water and ethyl acetate solution, and then washed with saturated brine. The ethyl acetate layer was distilled under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain oily liquid 9a (630 mg, yield 77%).

[0065] Synthesis of intermediate 9b: Following the synthesis method of intermediate 9a, using intermediates 7b and 8a as raw materials, an oily liquid was obtained with a yield of 75%.

[0066] Synthesis of intermediate 9c: Following the synthesis method of intermediate 9a, using intermediates 7c and 8a as raw materials, an oily liquid was obtained with a yield of 72%.

[0067] Synthesis of intermediate 9d: Following the synthesis method of intermediate 9a, using intermediates 7d and 8a as raw materials, an oily liquid was obtained with a yield of 76%.

[0068] Synthesis of intermediate 9e: Following the synthesis method of intermediate 9a, using intermediates 7l and 8b as raw materials, an oily liquid was obtained with a yield of 74%.

[0069] Synthesis of intermediate 9f: Following the synthesis method of intermediate 9a, using intermediates 7f and 8a as raw materials, an oily liquid was obtained with a yield of 72%.

[0070] Synthesis of intermediate 9g: Following the synthesis method of intermediate 9a, using intermediates 7g and 8a as raw materials, an oily liquid was obtained with a yield of 73%.

[0071] Synthesis of intermediate 9h: Following the synthesis method of intermediate 9a, using intermediates 7h and 8a as raw materials, an oily liquid was obtained with a yield of 76%.

[0072] Synthesis of intermediate 9i: Following the synthesis method of intermediate 9a, using intermediates 7i and 8a as raw materials, an oily liquid was obtained with a yield of 77%.

[0073] Synthesis of intermediate 9l: Following the synthesis method of intermediate 9a, using intermediates 7l and 8a as raw materials, an oily liquid was obtained with a yield of 74%.

[0074] Synthesis of intermediate 9j-k,ms: Following the synthesis method of intermediate 9a, using intermediates 7l and 8c-k as raw materials, an oily liquid was obtained with a yield of 75%.

[0075] Synthesis of intermediate 10a-s: Following the synthesis method of intermediate 7a, intermediate 9a-s was used as the raw material to obtain a white solid with a yield of 84%.

[0076] Synthesis of intermediate 12a: Following the synthesis method of intermediate 6a, intermediate 11a was used as the raw material to obtain a white solid with a yield of 91%.

[0077] Synthesis of intermediate 13a: Intermediate 12a (655 mg, 1.15 mmol) was dissolved in DCM (10 mL), and TFA (2 mL) was added. The reaction was carried out at room temperature. After 12 h, saturated sodium bicarbonate solution was added to the reaction system to adjust the pH until no more bubbles were produced when sodium bicarbonate solution was added dropwise. The mixture was extracted with DCM, and the DCM layer was distilled under reduced pressure and dried to give a pale yellow solid 2a (520 mg, yield 91%).

[0078] Synthesis of intermediate 15a-c: Following the synthesis method of intermediate 9a, using intermediates 13a and 14a-c as raw materials, an oily liquid was obtained with a yield of 74%.

[0079] Synthesis of intermediate 16a-c: Following the synthesis method of intermediate 9a, intermediate 15a-c was used as the raw material to obtain a white solid with a yield of 85%.

[0080] Synthesis of intermediate 18a: Intermediate 17a (100 mg, 0.53 mmol) was dissolved in acetonitrile (8 mL). Cesium carbonate (262 mg, 0.81 mmol) was added to the reaction system, and the mixture was stirred at 70 °C for 10 min. Then, methyl 5-bromopentanoate (76 μL, 0.53 mmol) was added, and the reaction was continued at 70 °C. The reaction was completed after 2 h. The reaction system was filtered to remove insoluble cesium carbonate. The filtrate was prepared into sand and purified by column chromatography to obtain a pale yellow solid 18a (120 mg, yield 75%).

[0081] Synthesis of intermediate 19a: Following the synthesis method of intermediate 13a, using intermediate 18a as raw material, a pale yellow solid was obtained with a yield of 85%.

[0082] Synthesis of intermediate 21a: Following the synthesis method of intermediate 6a, using 3-aminophenol and 7l as raw materials, a pale yellow solid was obtained with a yield of 90%.

[0083] Synthesis of intermediate 22a: Intermediate 21a (300 mg, 0.89 mmol) was dissolved in a large amount of acetonitrile (30 mL). Cesium carbonate (433 mg, 1.33 mmol) was added to the reaction system, and the mixture was stirred at room temperature for 10 min. Then, 1,4-dibromobutane (1.072 mL, 8.97 mmol) was added, and the reaction was continued at room temperature. The reaction was completed after 2 h. The reaction system was filtered to remove insoluble cesium carbonate. The filtrate was prepared into sand and purified by column chromatography to obtain a pale yellow solid 22a (300 mg, yield 72%).

[0084] Synthesis of intermediate 23a: Following the synthesis method of intermediate 18a, using intermediates 19a and 22a as raw materials, a pale yellow solid was obtained with a yield of 76%.

[0085] Synthesis of intermediate 24a: Following the synthesis method of intermediate 7a, intermediate 23a was used as the raw material to obtain a white solid with a yield of 95%.

[0086] Synthesis of intermediate 26a: Intermediate 4a (213 mg, 0.61 mmol) and monomethyl isophthalate (121 mg, 0.67 mmol) were dissolved in acetonitrile (8 mL). N-methylimidazole (171 μL, 2.13 mmol) was added to the reaction system, and the mixture was stirred at room temperature for 10 min. Then, TCFH (205 mg, 0.73 mmol) was added, and the reaction was continued at room temperature. The reaction was completed after 2 h, at which point a solid precipitated from the reaction system. The reaction system was filtered, washed with acetonitrile, and dried to obtain a pale yellow solid 26a (444 mg, yield 84%).

[0087] Synthesis of intermediate 27a: Following the synthesis method of intermediate 7a, intermediate 26a was used as the raw material to obtain a white solid with a yield of 94%.

[0088] Example 2: Synthesis of 2-[(3,5-dimethylisoxazo-4-yl)methoxy]-N-[3-[4-[4-[[5-methyl-4-(1-(2-methylbenzoyl)indoline-5-yl)thiazo-2-yl]carbamoyl]phenoxy]butoxy]phenyl]benzamide (compound 4)

[0089] Following the synthesis method of intermediate 26a, intermediates 21a and 27a were used as raw materials to obtain a pale yellow solid compound 4. 1H NMR (500 MHz, DMSO-d6) δ 12.37 (d, J = 54.4 Hz, 1H), 10.07 (d, J = 18.5Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 8.11 (d, J = 8.6 Hz, 2H), 7.66 – 7.55 (m,3H), 7.52 (t, J = 7.9 Hz, 1H), 7.41 – 7.23 (m, 6H), 7.23 – 7.04 (m, 5H), 6.69– 6.62 (m, 1H), 5.05 (s, 2H), 4.15 (d, J = 6.2 Hz, 2H), 4.07 – 3.94 (m, 2H),3.75 (s, 2H), 2.39 (s, 3H), 2.29 (d, J = 10.6 Hz, 3H), 2.19 (s, 3H), 1.90 (s,4H). 13 C NMR (126 MHz, DMSO) δ 168.68, 168.27, 164.94, 164.75, 162.53, 160.06,159.37, 155.67, 141.99, 140.59, 137.90, 133.87, 133.27, 132.27, 130.88,130.66, 130.01, 129.53, 127.62, 126.42, 126.35, 125.92, 125.07, 124.48,121.59, 120.91, 116.61, 114.76, 114.22, 111.97, 110.50, 110.12, 105.97,68.06, 67.49, 60.35, 55.36, 50.20, 25.86, 25.79, 19.06, 14.54, 14.40, 12.32,11.11, 11.07, 10.06, 10.02. HPLC (90% acetonitrile in water with 1‰ HCOOH):t R = 8.877 min, 96.26%. ESI-HRMS (m / z): [M + H] + , calcd. for C 50 H 47 N5O7S,862.3230, found, 862.3324.

[0090] Example 3: Synthesis of 2-[(3,5-dimethylisoxazo-4-yl)methoxy]-N-[3-[[5-methyl-4-(1-(2-methylbenzoyl)indoline-5-yl)thiazo-2-yl]carbamoyl]phenyl]benzamide (compound 2)

[0091] Following the synthesis method of intermediate 26a, using intermediates 10e and 4a as raw materials, a pale yellow solid compound 2 was obtained. 1 H NMR (500 MHz, DMSO-d6) δ 12.61 (s, 1H), 10.34 (s, 1H), 8.42 (s, 1H), 8.26 (d, J = 8.4 Hz, 1H), 7.83 (t, J = 10.0 Hz, 1H), 7.73 (d, J = 8.1 Hz,1H), 7.61 – 7.54 (m, 3H), 7.51 (m, J = 7.9, 1.8 Hz, 1H), 7.45 (t, J = 7.9 Hz,1H), 7.32 (m, J = 11.1, 6.8 Hz, 5H), 7.10 (t, J = 7.5 Hz, 1H), 5.04 (s, 2H), 3.73 (t, J = 8.2 Hz, 2H), 3.13 (t, J = 8.7 Hz, 2H), 2.67 (s, 3H), 2.38 (s, 3H), 2.28 (s, 3H), 2.17 (s, 3H). 13 C NMR (126 MHz, DMSO) δ 168.69, 168.11,165.52, 160.04, 155.58, 142.05, 139.81, 137.91, 133.88, 133.32, 132.16,130.88, 129.75, 129.52, 129.39, 127.66, 126.88, 126.43, 125.93, 125.10,123.54, 123.29, 121.57, 121.21, 119.84, 116.63, 114.36, 110.55, 60.37, 55.37,50.21, 19.06, 14.55, 12.35, 11.13, 10.09. HPLC (90% acetonitrile in water with 1‰ HCOOH): t R = 8.877 min, 97.78%. ESI-HRMS (m / z): [M + H]+ , calcd. forC 40 H 35 N5O5S, 698.2392, found, 698.2480.

[0092] Example 4: Synthesis of 2-[(3,5-dimethylisoxazo-4-yl)methoxy]-N-[3-[[5-methyl-4-(1-(2-methylbenzoyl)indoline-5-yl)thiazo-2-yl]carbamoyl]benzyl]benzamide (compound 5)

[0093] Following the synthesis method of intermediate 26a, using intermediates 10j and 4a as raw materials, a pale yellow solid compound 5 was obtained. 1 H NMR (500 MHz, DMSO-d6) δ 12.61 (s, 1H), 8.61 (t, J = 6.0 Hz, 1H), 8.27(d, J = 8.4 Hz, 1H), 8.08 – 7.95 (m, 2H), 7.67 – 7.63 (m, 1H), 7.59 (s, 2H),7.50 – 7.43 (m, 2H), 7.41 – 7.29 (m, 5H), 7.26 (d, J = 8.3 Hz, 1H), 7.08 (t,J = 7.5 Hz, 1H), 5.04 (s, 2H), 4.52 (d, J = 5.9 Hz, 2H), 3.75 (t, J = 8.4 Hz,2H), 3.15 (t, J = 8.6 Hz, 2H), 2.35 (s, 3H), 2.31 (s, 3H), 2.12 (s, 3H). 13CNMR (126 MHz, DMSO) δ 168.68, 168.17, 166.14, 165.31, 159.95, 155.86, 154.88,142.05, 140.23, 137.91, 133.88, 133.31, 132.53, 132.10, 131.45, 131.30,130.88, 130.30, 129.53, 128.88, 127.64, 126.94, 126.43, 125.93, 125.47,125.09, 121.47, 121.15, 116.62, 114.14, 110.37, 60.22, 55.38, 50.21, 42.84,28.00, 26.82, 21.52, 19.07, 12.35, 11.09, 10.04. HPLC (90% acetonitrile inwater with 1‰ HCOOH): t R = 5.133 min, 98.68%. ESI-HRMS (m / z): [M + H] + ,calcd. for C 41 H 37 N5O5S, 712.2549, found, 712.2634.

[0094] Example 5: Synthesis of 2-[(3,5-dimethylisoxazo-4-yl)methoxy]-N-[4-[[5-methyl-4-(1-(2-methylbenzoyl)indoline-5-yl)thiazo-2-yl]carbamoyl]phenyl]benzamide (compound 6)

[0095] Following the synthesis method of intermediate 26a, intermediates 10k and 4a were used as raw materials to obtain a pale yellow solid compound 6. 1H NMR (500 MHz, DMSO-d6) δ 12.48 (s, 1H), 10.48 (s, 1H), 8.27 (d, J = 8.3Hz, 1H), 8.11 (d, J = 8.5 Hz, 2H), 7.76 (d, J = 8.3 Hz, 2H), 7.62 – 7.49 (m,4H), 7.33 (m, J = 20.0, 15.1, 7.2 Hz, 5H), 7.11 (t, J = 7.5 Hz, 1H), 5.05 (s,2H), 3.74 (t, J = 8.4 Hz, 2H), 3.15 (q, J = 11.5 Hz, 2H), 2.39 (s, 3H), 2.30 (s, 3H), 2.18 (s, 3H). 13 C NMR (126 MHz, DMSO) δ 168.68, 168.16, 165.72,164.70, 160.05, 155.60, 155.01, 143.24, 142.02, 137.91, 133.88, 133.30,132.33, 131.36, 130.88, 129.80, 129.70, 129.53, 127.63, 127.04, 126.65,126.43, 125.93, 125.09, 121.59, 121.03, 118.95, 116.62, 114.29, 110.53,60.35, 60.23, 50.21, 28.00, 21.22, 19.07, 14.55, 12.35, 11.13, 10.07. HPLC(90% acetonitrile in water with 1‰ HCOOH): t R = 6.533 min, 98.15%. ESI-HRMS(m / z): [M + H] + , calcd. for C 40 H 35 N5O5S, 698.2392, found, 698.2480.

[0096] Example 6: Synthesis of 2-[(3,5-dimethylisoxazo-4-yl)methoxy]-N-[5-methyl-4-(1-(2-methylbenzoyl)indoline-5-yl)thiazo-2-yl]benzamide (compound 7)

[0097] Following the synthesis method of intermediate 26a, using intermediates 7l and 4a as raw materials, a pale yellow solid compound 7 was obtained. 1 H NMR (500 MHz, DMSO-d6) δ 11.71 (s, 1H), 8.26 (d, J = 8.3 Hz, 1H), 7.79(d, J = 7.8 Hz, 1H), 7.68 – 7.58 (m, 1H), 7.55 (d, J = 9.1 Hz, 2H), 7.36 (m,J = 34.5, 17.7, 7.7 Hz, 5H), 7.15 (t, J = 7.5 Hz, 1H), 5.13 (s, 2H), 3.75 (t,J = 8.4 Hz, 2H), 3.24 – 3.10 (m, 2H), 2.43 (s, 3H), 2.30 (s, 2H), 2.26 (d, J = 16.1 Hz, 3H). 13 C NMR (126 MHz, DMSO) δ 168.68, 168.62, 163.84, 160.02,156.50, 153.48, 144.58, 142.04, 137.92, 133.88, 133.77, 133.32, 131.22,130.88, 130.72, 129.52, 127.39, 126.42, 125.93, 124.98, 122.58, 121.84,121.19, 116.56, 114.59, 110.32, 60.72, 50.20, 28.02, 26.82, 19.05, 12.47,11.18, 10.12. HPLC (90% acetonitrile in water with 1‰ HCOOH): t R = 5.323 min,98.85%. ESI-HRMS (m / z): [M + H] + , calcd. for C 33 H 30 N4O4S, 579.2021, found,579.2095.

[0098] Example 7: Synthesis of 2-[(3,5-dimethylisoxazol-4-yl)methoxy]-N-[3-[4-[4-[5-[[4-[[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazolyl-2-yl]carbamoyl]benzyl]amino]-5-oxopentyl]piperazin-1-yl]butoxy]phenyl]benzamide (compound 3)

[0099] Following the synthesis method of intermediate 26a, intermediates 24a and 4a were used as raw materials to obtain a pale yellow solid compound 3. 1 H NMR (500 MHz, DMSO-d6) δ 12.62 (s, 1H), 9.42 (s, 1H), 8.42 (s, 1H), 8.27(d, J = 8.3 Hz, 1H), 8.00 (d, J = 10.0 Hz, 2H), 7.65 (d, J = 7.8 Hz, 1H), 7.59 (d, J = 6.9 Hz, 2H), 7.52 – 7.44 (m, 3H), 7.37 (m, J = 28.2, 14.7, 7.3Hz, 6H), 7.25 (d, J = 7.2 Hz, 2H), 7.12 (m, J = 8.1, 2.5 Hz, 1H), 7.04 – 6.99(m, 1H), 4.97 (s, 2H), 4.36 (d, J = 5.9 Hz, 2H), 4.02 (d, J = 6.0 Hz, 2H), 3.75 (t, J = 8.4 Hz, 2H), 3.15 (t, J = 8.5 Hz, 2H), 2.95 (s, 6H), 2.36 (s,4H), 2.30 (s, 3H), 2.19 (d, J = 22.1 Hz, 7H), 1.65 (d, J = 92.2 Hz, 9H), 1.31– 1.21 (m, 1H). 13C NMR (126 MHz, DMSO) δ 167.92, 165.42, 165.26, 160.05,159.02, 151.57, 140.77, 136.38, 132.58, 131.88, 130.89, 130.14, 129.00,127.83, 127.64, 126.88, 126.67, 126.44, 125.88, 125.06, 121.47, 121.17,119.92, 118.49, 116.64, 114.18, 113.42, 110.81, 60.34, 55.37, 50.21, 49.08,42.30, 27.99, 26.56, 19.05, 12.34, 11.08, 10.07. HPLC (90% acetonitrile inwater with 1‰ HCOOH): t R = 2.863 min, 96.91%. ESI-HRMS (m / z): [M + H] + ,calcd. for C 60 H 66 N8O7S, 1043.4809, found, 1043.4866.

[0100] Example 8: Synthesis of N1-[2-[(3,5-dimethylisoxazo-4-yl)methoxy]phenyl]-N3-[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazo-2-yl]isophthalamide (compound 16)

[0101] Following the synthesis method of intermediate 26a, intermediates 16a and 4a were used as raw materials to obtain a pale yellow solid compound 16. 1H NMR (500 MHz, DMSO-d6) δ 12.64 (s, 1H), 9.64 (s, 1H), 8.61 (d, J = 13.5Hz, 1H), 8.25 (t, J = 8.3 Hz, 2H), 8.09 (d, J = 7.7 Hz, 1H), 7.72 – 7.51 (m,3H), 7.41 – 7.17 (m, 5H), 7.07 – 6.99 (m, 1H), 4.96 (s, 2H), 3.72 (t, J = 8.5Hz, 1H), 3.13 (dd, J = 13.6, 5.7 Hz, 2H), 2.46 (s, 2H), 2.33 (s, 3H), 2.27 (s, 2H), 2.14 (s, 3H). 13 C NMR (126 MHz, DMSO) δ 168.70, 167.89, 165.00,160.00, 151.80, 142.08, 137.89, 135.24, 133.88, 133.34, 131.76, 131.46,130.89, 129.53, 129.41, 127.83, 127.75, 127.66, 126.96, 126.42, 125.93,125.09, 121.62, 121.37, 116.63, 114.69, 110.78, 60.58, 55.37, 50.20, 27.99,21.22, 19.06, 12.37, 11.09, 10.06. HPLC (90% acetonitrile in water with 1‰HCOOH): t R = 4.693 min, 96.97%. ESI-HRMS (m / z): [M + H] + , calcd. forC 40 H 35 N5O5S, 698.2392, found, 698.2480.

[0102] Example 9: Synthesis of N-[2-[(3,5-dimethylisoxazol-4-yl)methoxy]phenyl]-3-[2-[[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazo-2-yl]amino]-2-oxoethyl]benzamide (compound 17)

[0103] Following the synthesis method of intermediate 9a, using intermediates 16b and 4a as raw materials, a pale yellow solid compound 17 was obtained. 1 H NMR (500 MHz, DMSO-d6) δ 12.41 (s, 1H), 9.47 (s, 1H), 8.24 (d, J = 8.5Hz, 1H), 7.89 (s, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.65 (d, J = 7.9 Hz, 1H),7.59 – 7.50 (m, 3H), 7.47 (t, J = 7.7 Hz, 1H), 7.33 (dt, J = 21.3, 7.1 Hz,4H), 7.23 (m, J = 5.5, 1.9 Hz, 2H), 7.02 (m, J = 8.4, 6.1, 2.6 Hz, 1H), 4.97(s, 2H), 3.84 (s, 2H), 3.78 – 3.64 (m, 2H), 3.13 (t, J = 8.3 Hz, 2H), 2.46(s, 2H), 2.35 (s, 3H), 2.29 (s, 3H), 2.16 (s, 3H). 13 C NMR (126 MHz, DMSO) δ170.80, 169.26, 167.88, 165.49, 160.00, 151.57, 135.94, 135.11, 132.98,130.88, 129.01, 128.01, 126.66, 126.43, 126.10, 126.03, 121.54, 120.79,116.62, 114.42, 110.77, 60.45, 60.22, 50.19, 41.96, 40.52, 40.44, 40.35,40.27, 40.19, 40.10, 40.02, 39.85, 39.68, 39.52, 28.01, 21.23, 19.04, 14.56,12.35, 11.10, 10.07. HPLC (90% acetonitrile in water with 1‰ HCOOH): t R =4.790 min, 96.40%. ESI-HRMS (m / z): [M + H] + , calcd. for C 41 H 37N5O5S, 712.2549, found, 712.2634.

[0104] Example 10: Synthesis of 2-[2-[(3,5-dimethylisoxazo-4-yl)methoxy]phenyl]-N-[5-methyl-4-(1-(2-methylbenzoyl)indoline-5-yl)thiazo-2-yl]acetamide (compound 1)

[0105] Following the synthesis method of intermediate 26a, using intermediates 7k and 4a as raw materials, a pale yellow solid compound 1 was obtained. 1 H NMR (500 MHz, DMSO-d6) δ 12.08 (d, J = 58.3 Hz, 1H), 8.26 (d, J = 8.3 Hz, 1H), 7.55 (s, 2H), 7.31 (m, 6H), 7.12 (d, J = 8.2 Hz, 1H), 6.95 (t, J = 7.4Hz, 1H), 4.87 (s, 2H), 3.75 (t, J = 8.3 Hz, 2H), 3.71 (s, 2H), 3.15 (t, J =8.3 Hz, 2H), 2.46 (s, 3H), 2.34 (s, 3H), 2.31 (s, 3H), 2.14 (s, 3H). 13 C NMR(126 MHz, DMSO-d6) δ 169.63, 168.67, 167.83, 160.03, 156.68, 154.28, 144.42,133.87, 133.28, 131.84, 131.41, 130.88, 129.52, 128.75, 127.53, 126.43,125.93, 125.00, 124.15, 121.06, 120.40, 116.59, 112.43, 110.79, 59.49, 50.20,36.90, 34.14, 29.48, 28.01, 19.04, 14.41, 12.34, 11.00, 9.97. HPLC (90%acetonitrile in water with 1‰ HCOOH): t R = 8.308 min, 96.95%. ESI-HRMS (m / z):[M + H] + , calcd. for C 34 H 32N4O4S, 593.2144, found, 593.2250.

[0106] Example 11: Synthesis of 2-[(3,5-dimethylisoxazol-4-yl)methoxy]-N-[3-[2-[[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazolyl-2-yl]amino]-2-oxoethyl]phenyl]benzamide (compound 8)

[0107] Following the synthesis method of intermediate 26a, using intermediates 10l and 4a as raw materials, a pale yellow solid compound 8 was obtained. 1 H NMR (500 MHz, DMSO-d6) δ 12.39 (s, 1H), 10.17 (s, 1H), 8.25 (d, J = 8.7Hz, 1H), 7.70 (s, 1H), 7.63 – 7.47 (m, 4H), 7.43 – 7.25 (m, 7H), 7.12 – 7.03(m, 2H), 5.05 (s, 2H), 3.75 (d, J = 8.3 Hz, 2H), 3.73 (s, 2H), 3.15 (q, J =10.0, 8.4 Hz, 2H), 2.47 (s, 3H), 2.39 (s, 3H), 2.30 (s, 3H), 2.19 (s, 3H). 13 CNMR (101 MHz, DMSO-d6) δ 169.42, 168.20, 165.18, 160.10, 155.53, 154.22,144.49, 141.99, 139.61, 137.88, 136.14, 133.85, 133.34, 132.05, 131.31,130.87, 129.78, 129.53, 129.23, 127.52, 126.81, 126.43, 125.91, 124.91,121.51, 120.75, 120.41, 118.17, 116.62, 114.17, 110.54, 60.24, 50.19, 49.08,42.25, 27.98, 19.05, 12.36, 11.12, 10.09. HPLC (95% acetonitrile in waterwith 1‰ HCOOH): t R = 3.824 min, 98.39%. ESI-HRMS (m / z): [M + H] +, calcd. forC 41 H 37 N5O5S, 712.2515, found, 712.2634.

[0108] Example 12: Synthesis of 1-[2-[(3,5-dimethylisoxazol-4-yl)methoxy]benzoyl]-N-[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazolyl-2-yl]piperidine-3-carboxamide (compound 9)

[0109] Following the synthesis method of intermediate 26a, using intermediates 10m and 4a as raw materials, a pale yellow solid compound 9 was obtained. 1 H NMR (400 MHz, DMSO-d6) δ 12.12 (d, J = 45.5 Hz, 1H), 8.26 (m, 2H), 7.67(dd, J = 7.7, 1.9 Hz, 1H), 7.56 – 7.42 (m, 3H), 7.34 (m, 3H), 7.22 (d, J =8.3 Hz, 1H), 7.05 (t, J = 7.5 Hz, 1H), 5.06 (s, 2H), 3.74 (t, J = 8.3 Hz,2H), 3.53 (q, J = 6.4 Hz, 2H), 3.13 (t, J = 8.5 Hz, 2H), 2.65 (t, J = 6.7 Hz,2H), 2.47 (s, 3H), 2.42 (s, 3H), 2.29 (s, 3H), 2.22 (s, 3H), 1.99 (m, 1H), 1.37 – 1.24 (m, 2H), 1.18 (t, J = 7.1 Hz, 2H). 13C NMR (101 MHz, DMSO) δ168.68, 167.87, 167.75, 167.68, 166.93, 160.05, 160.00, 154.04, 141.99,137.87, 133.85, 133.34, 130.88, 129.54, 128.16, 127.52, 127.01, 126.70,126.43, 125.91, 124.98, 121.80, 120.82, 116.62, 113.98, 113.77, 110.70,60.14, 50.19, 48.48, 43.22, 41.50, 27.97, 19.04, 12.33, 11.17, 11.11, 10.25,10.08. HPLC (90% acetonitrile in water with 1‰ HCOOH): t R = 7.364 min,99.39%. ESI-HRMS (m / z): [M + H] + , calcd. for C 39 H 39 N5O5S, 690.2672, found, 690.2808.

[0110] Example 13: Synthesis of 1-[2-[(3,5-dimethylisoxazo-4-yl)methoxy]benzoyl]-N-[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazo-2-yl]piperidine-4-carboxamide (compound 10)

[0111] Following the synthesis method of intermediate 26a, intermediates 10n and 4a were used as raw materials to obtain a pale yellow solid compound 10. 1H NMR (500 MHz, DMSO-d6) δ 12.11 (s, 1H), 8.24 (d, J = 8.1 Hz, 1H), 7.53(s, 2H), 7.37 (m, 4H), 7.19 (d, J = 42.9 Hz, 3H), 7.04 (q, J = 8.1 Hz, 1H),4.97 (q, J = 9.8, 8.4 Hz, 2H), 4.48 (t, J = 17.2 Hz, 1H), 3.74 (t, J = 8.4Hz, 2H), 3.27 – 3.05 (m, 2H), 3.04 – 2.81 (m, 2H), 2.72 (d, J = 13.5 Hz, 2H),2.44 (d, J = 39.1 Hz, 6H), 2.30 (s, 3H), 2.19 (s, J = 8.5 Hz, 3H), 2.05 –1.72 (m, 2H), 1.71 – 1.47 (m, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 173.10,168.67, 167.76, 167.75, 160.05, 159.91, 154.23, 144.55, 140.72, 137.95,133.82, 133.31, 130.88, 130.57, 130.43, 127.82, 126.41, 125.92, 124.95,121.79, 121.66, 120.63, 116.62, 113.91, 111.81, 110.75, 110.55, 60.16, 50.18,45.99, 41.36, 28.67, 28.18, 19.02, 12.34, 11.11, 10.09. HPLC (85%acetonitrile in water with 1‰ HCOOH): t R = 6.649 min, 95.97%. ESI-HRMS (m / z):[M + H] + , calcd. for C 39 H 39 N5O5S, 690.2672, found, 690.2808.

[0112] Example 14: Synthesis of 2-[(3,5-dimethylisoxazol-4-yl)methoxy]-N-[2-[[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazo-2-yl]amino]-2-oxoethyl]benzamide (compound 11)

[0113] Following the synthesis method of intermediate 26a, using intermediates 10o and 4a as raw materials, a pale yellow solid compound 11 was obtained. 1 H NMR (300 MHz, DMSO-d6) δ 12.21 (d, J = 36.0 Hz, 1H), 8.53 (t, J = 5.5Hz, 1H), 8.27 (d, J = 8.5 Hz, 1H), 7.82 (d, J = 7.4 Hz, 1H), 7.68 – 7.50 (m,3H), 7.41 – 7.28 (m, 5H), 7.12 (t, J = 7.5 Hz, 1H), 5.14 (s, 2H), 4.20 (d, J= 5.5 Hz, 2H), 3.78 (d, J = 8.8 Hz, 1H), 3.17 (d, J = 8.4 Hz, 2H), 2.50 (s, 3H), 2.46 (s, 3H), 2.32 (s, 3H), 2.27 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ168.68, 168.46, 168.11, 165.72, 160.05, 156.31, 154.03, 144.51, 142.00,137.88, 133.85, 133.35, 132.91, 131.26, 131.09, 130.87, 129.54, 127.53,126.43, 125.91, 124.99, 123.56, 121.65, 120.80, 116.61, 114.62, 110.27,60.53, 55.38, 50.19, 43.02, 27.98, 19.05, 12.41, 11.24, 10.19. HPLC (95%acetonitrile in water with 1‰ HCOOH): t R = 3.910 min, 99.21%. ESI-HRMS (m / z):[M + H] + , calcd. for C 35 H 33N5O5S, 636.2202, found, 636.2318.

[0114] Example 15: Synthesis of 2-[(3,5-dimethylisoxazol-4-yl)methoxy]-N-[3-[[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazo-2-yl]amino]-3-oxopropyl]benzamide (compound 12)

[0115] Following the synthesis method of intermediate 26a, intermediates 10p and 4a were used as raw materials to obtain a pale yellow solid compound 12. 1 H NMR (400 MHz, DMSO-d6) δ 12.12 (d, J = 45.6 Hz, 1H), 8.41 – 8.11 (m,2H), 7.68 (dd, J = 7.7, 1.8 Hz, 1H), 7.57 – 7.44 (m, 3H), 7.41 – 7.25 (m,4H), 7.22 (d, J = 8.3 Hz, 1H), 7.05 (t, J = 7.5 Hz, 1H), 5.06 (s, 2H), 3.87 –3.63 (m, 2H), 3.54 (q, J = 6.5 Hz, 2H), 3.19 – 3.10 (m, 2H), 2.65 (t, J = 6.7Hz, 2H), 2.50 (s, 3H), 2.42 (s, 3H), 2.29 (s, 3H), 2.23 (s, 3H). 13 C NMR (101MHz, DMSO-d6) δ 170.00, 168.67, 168.16, 165.68, 159.95, 155.87, 154.10,144.36, 141.96, 137.88, 133.85, 133.33, 132.30, 131.34, 130.87, 130.63,129.53, 127.50, 126.43, 125.91, 124.96, 124.80, 121.53, 120.57, 116.60,114.44, 110.34, 60.41, 50.18, 35.57, 35.26, 27.98, 19.04, 12.37, 11.18,10.17. HPLC (90% acetonitrile in water with 1‰ HCOOH): t R= 6.026 min,97.58%. ESI-HRMS (m / z): [M + H] + , calcd. for C 36 H 35 N5O5S, 650.2359, found, 650.2472.

[0116] Example 16: Synthesis of 2-[(3,5-dimethylisoxazol-4-yl)methoxy]-N-[4-[[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazo-2-yl]amino]-4-oxobutyl]benzamide (compound 13)

[0117] Following the synthesis method of intermediate 26a, intermediates 10q and 4a were used as raw materials to obtain a pale yellow solid compound 13. 1 H NMR (400 MHz, DMSO-d6) δ 12.08 (s, 1H), 8.25 (d, J = 8.3 Hz, 1H), 8.09(d, J = 5.7 Hz, 1H), 7.61 – 7.20 (m, 9H), 7.04 (t, J = 7.5 Hz, 1H), 5.04 (s,2H), 3.74 (t, J = 8.2 Hz, 2H), 3.24 (q, J = 6.5 Hz, 2H), 3.18 – 3.02 (m, 2H),2.47 (s, 3H), 2.37 (m, 2H), 2.29 (s, 3H), 2.23 (s, 3H), 1.83 – 1.68 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 171.23, 168.67, 168.14, 166.05, 160.03, 155.69,154.24, 144.32, 141.95, 137.88, 133.86, 133.32, 131.87, 131.36, 130.87,130.21, 129.53, 127.50, 126.43, 125.85, 124.96, 121.40, 120.48, 116.60,114.20, 110.48, 60.24, 50.19, 38.85, 32.78, 27.99, 25.23, 19.05, 12.36,11.14, 10.13. HPLC (90% acetonitrile in water with 1‰ HCOOH): tR = 6.120 min,97.66%. ESI-HRMS (m / z): [M + H] + , calcd. for C 37 H 37 N5O5S, 664.2515, found, 664.2617.

[0118] Example 17: Synthesis of 2-[(3,5-dimethylisoxazo-4-yl)methoxy]-N-[5-[[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazo-2-yl]amino]-5-oxopentyl]benzamide-methane (1 / 1) (Compound 14)

[0119] Following the synthesis method of intermediate 26a, using intermediates 10r and 4a as raw materials, a pale yellow solid compound 14 was obtained. 1 H NMR (500 MHz, DMSO-d6) δ 12.06 (s, 1H), 8.25 (d, J = 8.3 Hz, 1H), 8.03 (t, J = 5.7 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.53 (m, 2H), 7.46 (t, J = 7.9Hz, 1H), 7.41 – 7.25 (m, 4H), 7.23 (d, J = 8.4 Hz, 1H), 7.05 (t, J = 7.5 Hz,1H), 5.03 (s, 2H), 3.74 (t, J = 8.5 Hz, 2H), 3.22 (q, J = 6.4 Hz, 2H), 3.14(t, J = 8.5 Hz, 2H), 2.47 (s, 3H), 2.41 (s, 3H), 2.38 (d, J = 7.9 Hz, 2H), 2.30 (s, 3H), 2.22 (s, 3H), 1.55 (m, 2H), 1.42 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 171.46, 168.22, 165.79, 160.00, 155.81, 154.26, 137.91, 133.87,133.30, 131.92, 130.87, 130.27, 129.53, 127.50, 126.42, 125.92, 125.65,124.96, 121.44, 120.42, 116.60, 114.17, 110.46, 60.25, 50.19, 38.99, 34.87,28.91, 28.00, 22.52, 19.03, 12.34, 11.10, 10.09. HPLC (90% acetonitrile inwater with 1‰ HCOOH): t R = 6.586 min, 99.06%. ESI-HRMS (m / z): [M + H] + ,calcd. for C 39 H 43 N5O5S, 678.2619, found, 678.2743.

[0120] Example 18: Synthesis of 2-[(3,5-dimethylisoxazol-4-yl)methoxy]-N-[6-[[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazo-2-yl]amino]-6-oxohexyl]benzamide (compound 15)

[0121] Following the synthesis method of intermediate 26a, using intermediates 10s and 4a as raw materials, a pale yellow solid compound 15 was obtained. 1H NMR (300 MHz, CDCl3-d) δ 10.59 (d, J = 88.4 Hz, 1H), 8.33 – 7.99 (m,2H), 7.59 – 7.33 (m, 2H), 7.31 – 6.75 (m, 8H), 4.77 (s, 2H), 4.20 (d, J = 9.8Hz, 1H), 3.62 (t, J = 8.3 Hz, 1H), 3.18 (q, J = 6.6 Hz, 2H), 3.00 (d, J = 9.0Hz, 2H), 2.35 (s, 3H), 2.27 (s, 3H), 2.18 (m, 6H), 1.93 (d, J = 7.8 Hz, 2H),1.38 (t, J = 7.7 Hz, 2H), 1.19 (m, 2H), 0.95 (m, 2H). 13 C NMR (151 MHz, CDCl3-d) δ 170.86, 169.18, 168.41, 164.94, 159.56, 156.23, 154.89, 144.26, 141.89,137.44, 133.86, 132.61, 132.50, 131.30, 130.68, 129.83, 129.24, 127.98,126.52, 126.08, 125.49, 124.68, 122.15, 121.30, 117.17, 112.66, 109.71,60.42, 50.13, 39.49, 35.41, 29.10, 28.12, 26.36, 24.70, 19.01, 12.19, 11.15,10.06. HPLC (90% acetonitrile in water with 1‰ HCOOH): t R = 7.135 min,97.91%. ESI-HRMS (m / z): [M + H] + , calcd. for C 39 H 41 N5O5S, 692.2828, found,692.2950.

[0122] Example 19: Synthesis of N-[2-[(3,5-dimethylisoxazol-4-yl)methoxy]phenyl]-4-[2-[[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazo-2-yl]amino]-2-oxoethyl]benzamide (compound 18)

[0123] Following the synthesis method of intermediate 9a, intermediates 16c and 4a were used as raw materials to obtain a pale yellow solid compound 18. 1 H NMR (500 MHz, DMSO-d6) δ 12.41 (s, 1H), 9.43 (s, 1H), 8.26 (d, J = 8.2Hz, 1H), 7.87 (d, J = 7.8 Hz, 2H), 7.68 (d, J = 7.8 Hz, 1H), 7.61 – 7.19 (m,10H), 7.02 (dd, J = 8.4, 2.5 Hz, 1H), 4.98 (s, 2H), 3.86 (s, 2H), 3.75 (t, J= 8.5 Hz, 2H), 3.22 – 3.10 (m, 2H), 2.47 (s, 3H), 2.37 (s, 3H), 2.30 (s, 3H), 2.18 (s, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 169.10, 168.68, 167.89, 165.34,160.02, 154.15, 151.45, 144.54, 139.19, 137.90, 133.87, 133.56, 133.33,131.29, 130.88, 129.84, 129.52, 128.00, 127.94, 127.53, 126.54, 126.42,125.93, 125.82, 124.97, 121.48, 120.81, 116.64, 114.26, 110.79, 60.39, 50.20,41.95, 28.01, 19.04, 12.35, 11.09, 10.08. HPLC (80% acetonitrile in water with 1‰ HCOOH): t R = 8.006 min, 95.90%. ESI-HRMS (m / z): [M + H] + , calcd. forC 41 H 37N5O5S, 712.2515, found, 712.2633.

[0124] Example 20: Synthesis of 2-[(3,5-dimethylisoxazo-4-yl)methoxy]-3-methoxy-N-[5-methyl-4-(1-(2-methylbenzoyl)indoline-5-yl)thiazo-2-yl]benzamide (compound 19)

[0125] Following the synthesis method of intermediate 26a, using intermediates 7a and 4a as raw materials, a pale yellow solid compound 19 was obtained. 1 H NMR (400 MHz, DMSO-d6) δ 11.99 (d, J = 49.0 Hz, 1H), 8.26 (d, J = 8.3Hz, 1H), 7.55 (d, J = 6.4 Hz, 2H), 7.40 – 7.07 (m, 7H), 4.89 (s, 2H), 3.91(s, 3H), 3.75 (t, J = 8.3 Hz, 2H), 3.15 (t, J = 8.4 Hz, 2H), 2.46 (s, 3H), 2.30 (s, 3H), 2.22 (s, 3H), 2.14 (s, 3H). 13 C NMR (151 MHz, DMSO) δ 169.03,168.68, 164.37, 160.04, 153.77, 153.23, 144.87, 144.70, 142.05, 137.91,133.88, 133.33, 131.27, 130.87, 130.23, 129.52, 129.25, 127.50, 126.42,125.93, 125.32, 125.02, 121.16, 116.58, 116.32, 110.98, 63.80, 56.55, 50.20,28.00, 19.04, 12.42, 10.75, 9.69. HPLC (90% acetonitrile in water with 1‰HCOOH): t R = 5.394 min, 98.01%. ESI-HRMS (m / z): [M + H] + , calcd. forC 34 H 32 N4O5S, 609.2093, found, 609.2175.

[0126] Example 21: Synthesis of 2-[(3,5-dimethylisoxazo-4-yl)methoxy]-N-[5-methyl-4-(1-(2-methylbenzoyl)indoline-5-yl)thiazo-2-yl]-4-(trifluoromethyl)benzamide (compound 20)

[0127] Following the synthesis method of intermediate 26a, using intermediates 7b and 4a as raw materials, a pale yellow solid compound 20 was obtained. 1 H NMR (500 MHz, CDCl3-d) δ 10.89 (d, J = 29.0 Hz, 1H), 8.67 – 8.25 (m,2H), 7.73 – 7.30 (m, 7H), 7.05 (s, 1H), 5.18 (d, J = 8.5 Hz, 2H), 4.57 – 4.24(m, 1H), 3.96 – 3.70 (m, 1H), 3.28 (dd, J = 30.8, 11.8 Hz, 2H), 2.69 – 2.54(m, 3H), 2.50 (s, 3H), 2.42 (s, 3H), 2.39 – 2.11 (m, 3H). 13 C NMR (151 MHz, CDCl3-d) δ 168.03, 159.56, 158.16, 155.23, 151.68, 140.86, 136.57, 134.67,134.45, 132.65, 131.46, 129.67, 128.78, 128.18, 126.41, 125.52, 125.05,124.52, 123.87, 122.39, 118.20, 118.17, 118.15, 115.98, 112.15, 109.34,107.49, 60.67, 49.17, 27.12, 18.03, 11.47, 10.36, 9.14. HPLC (90%acetonitrile in water with 1‰ HCOOH): t R = 6.380 min, 97.49%. ESI-HRMS (m / z):[M + H] + , calcd. for C 34 H 29 F3N4O4S, 647.1862, found, 647.1936.

[0128] Example 22: Synthesis of 2-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(5-methyl-4-(1-(2-methylbenzoyl)indoline-5-yl)thiazolyl)-4-nitrobenzamide (compound 21)

[0129] Following the synthesis method of intermediate 26a, using intermediates 7c and 4a as raw materials, a pale yellow solid compound 21 was obtained. 1 H NMR (500 MHz, DMSO-d6) δ 12.36 (s, 1H), 8.26 (d, J = 8.6 Hz, 1H), 8.15 (d, J = 2.1 Hz, 1H), 7.96 (dd, J = 8.4, 2.0 Hz, 1H), 7.86 (d, J = 8.4 Hz,1H), 7.60 – 7.46 (m, 2H), 7.35 (ddd, J = 21.2, 11.1, 4.2 Hz, 4H), 5.25 (s,2H), 3.75 (t, J = 8.3 Hz, 2H), 3.16 (s, 2H), 2.46 (m, 6H), 2.30 (s, 3H), 2.25 (s, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 168.69, 168.57, 163.44, 160.15, 156.39,153.48, 150.13, 142.09, 137.88, 133.86, 133.36, 130.94, 130.87, 130.40,129.54, 127.52, 126.42, 125.92, 125.00, 121.51, 116.60, 116.36, 109.99,109.03, 61.14, 55.39, 50.20, 27.99, 19.05, 12.42, 11.16, 10.12. HPLC (90%acetonitrile in water with 1‰ HCOOH): t R = 5.268 min, 98.06%. ESI-HRMS (m / z):[M + H] + , calcd. for C 33 H 29 N5O6S, 624.1839, found, 624.1942.

[0130] Example 23: Synthesis of 4-chloro-2-[(3,5-dimethylisoxazol-4-yl)methoxy]-N-[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazolyl-2-yl]benzamide (compound 22)

[0131] Following the synthesis method of intermediate 26a, using intermediates 7d and 4a as raw materials, a pale yellow solid compound 22 was obtained. 1 H NMR (500 MHz, CDCl3-d) δ 10.80 (d, J = 26.5 Hz, 1H), 8.42 (d, J = 8.4Hz, 1H), 8.26 (t, J = 9.2 Hz, 1H), 7.61 – 7.41 (m, 2H), 7.38 – 7.30 (m, 3H),7.28 – 7.01 (m, 3H), 5.10 (s, 2H), 4.39 (t, J = 8.5 Hz, 1H), 3.82 (t, J = 8.4Hz, 1H), 3.31 (t, J = 8.5 Hz, 1H), 3.24 (t, J = 8.5 Hz, 1H), 2.58 – 2.29 (m, 12H). 13 C NMR (151 MHz, CDCl3-d) δ 167.98, 159.94, 158.16, 155.62, 151.84,140.77, 138.98, 136.59, 132.83, 131.43, 129.66, 128.78, 128.17, 126.35,125.52, 125.05, 124.53, 123.87, 121.95, 120.86, 117.81, 115.94, 112.97,112.12, 107.60, 60.61, 49.16, 27.12, 18.03, 11.47, 10.38, 9.14, 9.03. HPLC(90% acetonitrile in water with 1‰ HCOOH): t R = 6.829 min, 98.30%. ESI-HRMS(m / z): [M + H] + , calcd. for C 33 H 29 ClN4O4S, 612.1598, found, 613.1677.

[0132] Example 24: Synthesis of 2-[(3,5-dimethylisoxazo-4-yl)methoxy]-4-methyl-N-[5-methyl-4-(1-(2-methylbenzoyl)indoline-5-yl)thiazo-2-yl]benzamide (compound 23)

[0133] Following the synthesis method of intermediate 26a, using intermediates 7e and 4a as raw materials, a pale yellow solid compound 23 was obtained. 1 H NMR (500 MHz, CDCl3-d) δ 10.93 (d, J = 26.2 Hz, 1H), 8.41 (d, J = 8.3Hz, 1H), 8.19 (dd, J = 10.6, 7.7 Hz, 1H), 7.66 – 7.30 (m, 5H), 7.28 – 6.91(m, 3H), 5.08 (s, 2H), 4.39 (t, J = 8.4 Hz, 1H), 3.81 (t, J = 8.3 Hz, 1H), 3.31 (t, J = 8.5 Hz, 1H), 3.24 (t, J = 8.5 Hz, 1H), 2.56 (s, 2H), 2.50 (s, 3H), 2.46 (s, 3H), 2.42 (s, 3H), 2.37 (s, 3H), 2.30 (s, 1H). 13 C NMR (151 MHz, CDCl3-d) δ 167.72, 160.94, 158.25, 155.38, 152.17, 144.41, 140.67, 136.62,132.91, 131.60, 129.64, 128.76, 128.14, 126.30, 125.51, 125.03, 124.52,123.90, 122.50, 120.50, 116.53, 115.90, 113.14, 112.09, 108.10, 60.15, 49.17,47.06, 27.12, 20.86, 18.03, 11.46, 10.33, 9.12. HPLC (90% acetonitrile inwater with 1‰ HCOOH): t R = 6.437 min, 99.12%. ESI-HRMS (m / z): [M + H] + ,calcd. for C 34 H 32N4O4S, 593.2144, found, 593.2225.

[0134] Example 25: Synthesis of 2-[(3,5-dimethylisoxazo-4-yl)methoxy]-4-fluoro-N-[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazo-2-yl]benzamide (compound 24)

[0135] Following the synthesis method of intermediate 26a, using intermediates 7f and 4a as raw materials, a pale yellow solid compound 24 was obtained. 1 H NMR (500 MHz, CDCl3-d) δ 10.78 (d, J = 25.3 Hz, 1H), 8.63 – 8.18 (m,2H), 7.71 – 7.46 (m, 2H), 7.32 (m, 4H), 6.91 (m, 2H), 5.08 (s, 2H), 4.39 (t,J = 8.4 Hz, 1H), 3.81 (t, J = 8.3 Hz, 1H), 3.31 (t, J = 8.5 Hz, 1H), 3.24 (t,J = 8.4 Hz, 1H), 2.56 (s, 2H), 2.53 (s, 2H), 2.51 (s, 1H), 2.46 (s, 1H), 2.42(s, 2H), 2.40 (s, 2H), 2.38 (s, 1H), 2.30 (s, 1H). 13 C NMR (151 MHz, CDCl3-d)δ 168.96, 166.92, 165.22, 160.97, 159.16, 157.76, 152.95, 141.76, 137.62,134.92, 134.85, 133.93, 132.44, 130.67, 129.18, 127.35, 126.54, 126.06,125.54, 124.88, 116.95, 113.13, 109.83, 108.59, 101.48, 61.63, 50.18, 28.14,19.05, 12.48, 11.40, 10.16, 1.02. HPLC (90% acetonitrile in water with 1‰HCOOH): t R = 5.828 min, 98.56%. ESI-HRMS (m / z): [M + H] + , calcd. forC33 H 29 FN4O4S, 597.1894, found, 597.1985.

[0136] Example 26: Synthesis of 4-bromo-2-[(3,5-dimethylisoxazol-4-yl)methoxy]-N-[3-[2-[[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazolyl]amino]-2-oxoethyl]phenyl]benzamide (compound 25)

[0137] Following the synthesis method of intermediate 26a, using intermediate 7g and 4a as raw materials, a pale yellow solid compound 25 was obtained. 1 H NMR (500 MHz, DMSO-d6) δ 11.76 (d, J = 36.4 Hz, 1H), 8.25 (d, J = 8.3Hz, 1H), 7.71 – 7.60 (m, 2H), 7.54 (d, J = 1.6 Hz, 2H), 7.47 – 7.23 (m, 5H), 5.16 (s, 2H), 3.75 (t, J = 8.5 Hz, 2H), 3.15 (t, J = 8.5 Hz, 2H), 2.50 – 2.32 (m, 6H), 2.32 – 2.07 (m, 6H). 13 C NMR (151 MHz, DMSO) δ 168.72, 163.36,160.05, 157.01, 153.43, 144.64, 142.05, 137.90, 133.87, 133.34, 132.02,131.17, 130.88, 129.53, 127.41, 126.48, 126.43, 125.92, 124.97, 124.69,122.32, 121.33, 117.71, 116.57, 110.09, 61.14, 50.20, 28.00, 19.03, 12.44,11.15, 10.09. HPLC (90% acetonitrile in water with 1‰ HCOOH): t R = 7.150 min,97.87%. ESI-HRMS (m / z): [M + H] + , calcd. for C 33 H 29BrN4O4S, 657.1093, found, 657.1159.

[0138] Example 27: Synthesis of 2-[(3,5-dimethylisoxazo-4-yl)methoxy]-5-methyl-N-[5-methyl-4-(1-(2-methylbenzoyl)indoline-5-yl)thiazo-2-yl]benzamide (compound 26)

[0139] Following the synthesis method of intermediate 26a, using intermediates 7h and 4a as raw materials, a pale yellow solid compound 26 was obtained. 1 H NMR (500 MHz, CDCl3-d) δ 10.98 (d, J = 27.5 Hz, 1H), 8.42 (d, J = 8.4Hz, 1H), 8.11 (d, J = 9.8 Hz, 1H), 7.66 – 7.42 (m, 2H), 7.33 (m, 5H), 7.05(d, J = 8.0 Hz, 1H), 5.05 (s, 2H), 4.39 (t, J = 8.4 Hz, 1H), 3.81 (t, J = 8.3Hz, 1H), 3.31 (t, J = 8.6 Hz, 1H), 3.24 (t, J = 8.4 Hz, 1H), 2.50 – 2.41 (m,6H), 2.39 (s, 3H), 2.37 (s, 2H), 2.35 (s, 1H), 2.30 (s, 1H). 13 C NMR (126 MHz, CDCl3-d) δ 168.74, 162.07, 159.30, 154.41, 153.15, 144.90, 141.70, 137.61,134.71, 132.87, 132.32, 130.68, 129.18, 127.36, 126.54, 126.07, 125.54,124.91, 121.67, 119.91, 116.93, 113.76, 113.13, 109.19, 61.46, 50.19, 28.14,20.45, 19.08, 12.52, 11.36, 10.17. HPLC (90% acetonitrile in water with 1‰HCOOH): t R = 6.507 min, 97.42%. ESI-HRMS (m / z): [M + H] +, calcd. forC 34 H 32 N4O4S, 593.2144, found, 593.2228.

[0140] Example 28: Synthesis of 2-[(3,5-dimethylisoxazo-4-yl)methoxy]-5-methoxy-N-[5-methyl-4-(1-(2-methylbenzoyl)indoline-5-yl)thiazo-2-yl]benzamide (compound 27)

[0141] Following the synthesis method of intermediate 26a, using intermediates 7i and 4a as raw materials, a pale yellow solid compound 27 was obtained. 1 H NMR (500 MHz, CDCl3-d) δ 11.07 (d, J = 28.9 Hz, 1H), 8.42 (d, J = 8.4Hz, 1H), 7.90 – 7.77 (m, 1H), 7.57 (d, J = 25.5 Hz, 2H), 7.42 – 7.30 (m, 3H),7.21 – 7.00 (m, 3H), 5.03 (s, 2H), 4.39 (t, J = 8.4 Hz, 1H), 3.87 (s, 3H),3.81 (t, J = 8.2 Hz, 1H), 3.31 (t, J = 8.6 Hz, 1H), 3.24 (t, J = 8.4 Hz, 1H), 2.57 (s, 2H), 2.47 (s, 1H), 2.46 (s, 2H), 2.43 (s, 1H), 2.42 (s, 2H), 2.36 (s, 2H), 2.34 (s, 1H), 2.30 (s, 1H). 13C NMR (151 MHz, CDCl3-d) δ 167.80,160.74, 158.29, 154.00, 152.04, 149.54, 144.02, 140.75, 136.60, 132.92,130.38, 129.66, 128.77, 128.16, 126.39, 125.52, 125.04, 124.53, 123.89,120.28, 120.12, 115.93, 115.19, 114.31, 112.13, 108.20, 61.33, 54.87, 49.17,27.12, 18.04, 11.46, 10.28, 9.10. HPLC (90% acetonitrile in water with 1‰HCOOH): t R = 5.748 min, 99.00%. ESI-HRMS (m / z): [M + H] + , calcd. forC 34 H 32 N4O5S, 609.2093, found, 609.2178.

[0142] Example 29: Synthesis of 5-chloro-2-[(3,5-dimethylisoxazol-4-yl)methoxy]-N-[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazolyl-2-yl]benzamide (compound 28)

[0143] Following the synthesis method of intermediate 26a, using intermediates 7j and 4a as raw materials, a pale yellow solid compound 28 was obtained. 1 H NMR (500 MHz, CDCl3-d) δ 10.88 (d, J = 27.6 Hz, 1H), 8.42 (d, J = 8.3Hz, 1H), 8.34 – 8.22 (m, 1H), 7.59 – 7.40 (m, 3H), 7.35 – 7.30 (m, 3H), 7.08(m, 2H), 5.09 (s, 2H), 4.40 (t, J = 8.0 Hz, 1H), 3.82 (t, J = 8.4 Hz, 1H), 3.31 (t, J = 8.7 Hz, 1H), 3.25 (t, J = 8.5 Hz, 1H), 2.59 – 2.30 (m, 12H). 13CNMR (126 MHz, CDCl3-d) δ 160.60, 159.18, 154.86, 152.78, 133.77, 132.42,130.71, 130.68, 129.20, 128.27, 127.41, 126.74, 126.59, 126.51, 126.07,125.76, 125.54, 124.89, 121.78, 116.98, 115.03, 108.75, 61.82, 50.19, 28.13,19.09, 12.53, 11.42, 10.19. HPLC (90% acetonitrile in water with 1‰ HCOOH):t R = 7.009 min, 98.11%. ESI-HRMS (m / z): [M + H] + , calcd. for C 33 H 29 ClN4O4S,613.1598, found, 613.1677.

[0144] Example 30: Synthesis of 2-[(3,5-dimethylisoxazo-4-yl)methoxy]-3-methoxy-N-[3-[2-[[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazo-2-yl]amino]-2-oxoethyl]phenyl]benzamide (compound 29)

[0145] Following the synthesis method of intermediate 26a, using intermediates 10a and 4a as raw materials, a pale yellow solid compound 29 was obtained. 1 H NMR (600 MHz, DMSO-d6) δ 12.37 (s, 1H), 10.21 (s, 1H), 8.25 (d, J = 8.3Hz, 1H), 7.75 (s, 1H), 7.63 – 7.45 (m, 3H), 7.44 – 7.25 (m, 5H), 7.23 – 7.18(m, 2H), 7.11 – 7.05 (m, 2H), 4.83 (s, 2H), 3.88 (s, 3H), 3.75 (s, 2H), 3.73(d, J = 8.2 Hz, 2H), 3.20 – 3.09 (m, 2H), 2.47 (s, 3H), 2.30 (s, 3H), 2.17(s, 3H), 2.10 (s, 3H). 13C NMR (151 MHz, DMSO) δ 169.43, 168.71, 168.67,165.30, 160.06, 154.22, 153.27, 144.52, 144.07, 142.01, 139.65, 137.90,136.04, 133.86, 133.31, 133.03, 131.33, 130.87, 129.52, 129.14, 127.52,126.42, 125.92, 125.20, 124.98, 120.74, 120.56, 118.45, 116.61, 114.91,111.17, 63.72, 56.45, 50.19, 42.28, 40.55, 28.00, 19.03, 12.34, 10.68, 9.68.HPLC (90% acetonitrile in water with 1‰ HCOOH): t R = 4.266 min, 99.26%. ESI-HRMS (m / z): [M + H] + , calcd. for C 42 H 39 N5O6S, 742.2621, found, 742.2699.

[0146] Example 31: Synthesis of 2-[(3,5-dimethylisoxazol-4-yl)methoxy]-N-[3-[2-[[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazo-2-yl]amino]-2-oxoethyl]phenyl]-4-(trifluoromethyl)benzamide (compound 30)

[0147] Referring to the synthesis method of intermediate 26a, using intermediates 10b and 4a as raw materials, a pale yellow solid compound 30 was obtained. 1H NMR (600 MHz, CDCl3-d) δ 9.72 (s, 2H), 8.38 (dd, J = 22.2, 8.2 Hz, 2H), 7.62 – 7.33 (m, 5H), 7.25 (m, 4H), 7.10 (s, 1H), 6.98 (t, J = 10.1 Hz, 1H),5.09 (s, 2H), 4.31 (s, 1H), 3.74 (t, J = 8.4 Hz, 1H), 3.55 (s, 2H), 3.24 –3.03 (m, 2H), 2.48 (s, 6H), 2.38 – 2.23 (m, 6H). 13 C NMR (151 MHz, CDCl3-d) δ169.27, 168.35, 161.28, 159.76, 155.93, 138.50, 134.96, 134.74, 134.65,133.62, 132.32, 131.14, 130.69, 130.31, 130.02, 129.79, 129.23, 127.98,127.43, 126.50, 126.06, 125.49, 125.28, 124.65, 121.70, 120.45, 119.19,119.16, 118.61, 117.19, 113.38, 109.88, 109.86, 109.14, 61.28, 50.12, 42.93,28.09, 19.02, 12.17, 11.27, 10.07. HPLC (90% acetonitrile in water with 1‰HCOOH): t R = 4.597 min, 99.15%. ESI-HRMS (m / z): [M + H] + , calcd. forC 42 H 36 F3N5O5S, 780.2389, found, 780.2447.

[0148] Example 32: Synthesis of 2-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(3-(2-((5-methyl-4-(1-(2-methylbenzoyl)indoline-5-yl)thiazolyl)amino)-2-oxoethyl)phenyl)-4-nitrobenzamide (compound 31)

[0149] Following the synthesis method of intermediate 26a, using intermediates 10c and 4a as raw materials, a pale yellow solid compound 31 was obtained. 1 H NMR (500 MHz, CDCl3-d) δ 9.71 (s, 2H), 8.46 (dd, J = 8.6, 1.3 Hz, 1H), 8.37 (d, J = 8.4 Hz, 1H), 8.05 – 7.97 (m, 2H), 7.55 – 7.31 (m, 4H), 7.28 –7.23 (m, 3H), 7.14 (d, J = 5.7 Hz, 1H), 7.02 – 6.97 (m, 1H), 5.18 (s, 2H), 4.34 (t, J = 8.4 Hz, 1H), 3.76 (t, J = 8.1 Hz, 1H), 3.55 (d, J = 5.5 Hz, 2H),3.17 (t, J = 8.4 Hz, 1H), 3.11 (t, J = 8.4 Hz, 1H), 2.54 (s, 3H), 2.50 (s,2H), 2.42 (s, 6H), 2.25 (s, 1H). 13 C NMR (151 MHz, CDCl3-d) δ 168.38, 167.32,159.60, 158.64, 154.91, 153.59, 149.35, 143.38, 140.96, 137.20, 133.72,132.90, 131.37, 130.08, 129.69, 128.99, 128.26, 126.98, 126.53, 125.49,125.06, 124.77, 124.45, 123.64, 120.71, 119.55, 117.66, 116.20, 115.97,112.39, 107.83, 106.98, 60.61, 49.10, 41.80, 27.07, 18.00, 11.15, 10.33,9.10. HPLC (90% acetonitrile in water with 1‰ HCOOH): t R = 4.074 min, 99.16%.ESI-HRMS (m / z): [M + H] + , calcd. for C 41 H 36 N6O7S, 757.2366, found, 757.2433.

[0150] Example 33: Synthesis of 4-chloro-2-[(3,5-dimethylisoxazol-4-yl)methoxy]-N-[3-[2-[[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazolyl]amino]-2-oxoethyl]phenyl]benzamide (compound 32)

[0151] Following the synthesis method of intermediate 26a, using intermediates 10d and 4a as raw materials, a pale yellow solid compound 32 was obtained. 1 H NMR (600 MHz, DMSO-d6) δ 12.37 (s, 1H), 10.15 (s, 1H), 8.25 (d, J = 8.3Hz, 1H), 7.67 (s, 1H), 7.63 – 7.47 (m, 3H), 7.47 – 7.26 (m, 7H), 7.17 (dd, J= 8.1, 1.8 Hz, 1H), 7.06 (d, J = 7.7 Hz, 1H), 5.10 (s, 2H), 3.76 (s, 2H),3.74 (s, 2H), 3.14 (t, J = 8.3 Hz, 2H), 2.47 (s, 3H), 2.40 (s, 3H), 2.30 (s,3H), 2.19 (s, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 169.38, 168.68, 168.40,164.26, 160.07, 156.40, 154.21, 144.52, 142.01, 139.43, 137.90, 136.17,136.15, 133.87, 133.32, 131.33, 131.09, 130.87, 129.52, 129.24, 127.52,126.42, 125.92, 125.78, 125.06, 124.97, 121.38, 120.75, HPLC (90% acetonitrile in water with 1‰ HCOOH): t R = 4.793 min,98.77%. ESI-HRMS (m / z): [M + H] +, calcd. for C 41 H 36 ClN5O5S, 746.2126, found,746.2196.

[0152] Example 34: Synthesis of 2-[(3,5-dimethylisoxazol-4-yl)methoxy]-4-fluoro-N-[3-[2-[[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazolyl]amino]-2-oxoethyl]phenyl]benzamide (compound 33)

[0153] Following the synthesis method of intermediate 26a, using intermediates 10f and 4a as raw materials, a pale yellow solid compound 33 was obtained. 1 H NMR (600 MHz, CDCl3-d) δ 9.67 (s, 1H), 8.62 – 8.05 (m, 2H), 7.66 – 7.27(m, 6H), 7.26 – 6.91 (m, 5H), 6.91 – 6.72 (m, 2H), 5.02 (s, 2H), 4.32 (s,1H), 3.75 (s, 1H), 3.70 (s, 2H), 3.13 (dd, J = 34.1, 11.3 Hz, 2H), 2.56 –2.14 (m, 12H). 13 C NMR (151 MHz, CDCl3-d) δ 169.15, 168.48, 166.36, 164.67,161.79, 159.73, 157.25, 157.18, 138.92, 134.89, 134.82, 134.08, 130.72,130.12, 129.29, 127.95, 126.49, 126.08, 125.50, 125.18, 124.53, 121.39,120.44, 118.74, 118.35, 117.24, 113.46, 109.59, 109.45, 109.19, 101.01,100.83, 61.17, 50.13, 43.25, 34.67, 28.05, 20.69, 12.16, 11.30, 10.08. HPLC(90% acetonitrile in water with 1‰ HCOOH): t R = 4.344 min, 98.90%. ESI-HRMS(m / z): [M + H] +, calcd. for C 41 H 36 FN5O5S, 730.2421, found, 730.2494.

[0154] Example 35: Synthesis of 4-bromo-2-[(3,5-dimethylisoxazol-4-yl)methoxy]-N-[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazolyl-2-yl]benzamide (compound 34)

[0155] Following the synthesis method of intermediate 26a, using intermediate 10g and 4a as raw materials, a pale yellow solid compound 34 was obtained. 1 H NMR (500 MHz, CDCl3-d) δ 9.73 (s, 1H), 8.38 (d, J = 8.4 Hz, 1H), 8.19(d, J = 8.4 Hz, 1H), 7.57 – 7.29 (m, 8H), 7.27 (s, 2H), 7.18 – 6.94 (m, 3H), 5.06 (s, 2H), 4.34 (t, J = 8.3 Hz, 1H), 3.77 (t, J = 8.3 Hz, 1H), 3.63 (s,2H), 3.17 (d, J = 8.6 Hz, 1H), 3.11 (t, J = 8.3 Hz, 1H), 2.51 (s, 3H), 2.50(s, 2H), 2.43 – 2.34 (m, 6H), 2.25 (t, J = 3.6 Hz, 1H). 13C NMR (151 MHz, CDCl3-d) δ 168.24, 167.25, 160.75, 158.80, 155.21, 153.05, 137.78, 136.45,133.51, 133.03, 129.66, 129.11, 128.76, 128.20, 126.92, 126.37, 126.26,125.49, 125.04, 124.88, 124.49, 124.35, 124.25, 123.60, 120.02, 119.30,117.55, 116.13, 115.37, 108.27, 60.18, 49.10, 42.16, 28.68, 27.09, 18.02,11.18, 10.27, 9.04. HPLC (90% acetonitrile in water with 1‰ HCOOH): t R =4.925 min, 95.99%. ESI-HRMS (m / z): [M + H] + , calcd. for C 41 H 36 BrN5O5S,790.1621, found, 790.1691.

[0156] Example 36: Synthesis of 2-[(3,5-dimethylisoxazol-4-yl)methoxy]-5-methyl-N-[3-[2-[[5-methyl-4-(1-(2-methylbenzoyl)indololin-5-yl)thiazolyl]amino]-2-oxoethyl]phenyl]benzamide (compound 35)

[0157] Following the synthesis method of intermediate 26a, using intermediates 10h and 4a as raw materials, a pale yellow solid compound 35 was obtained. 1H NMR (500 MHz, CDCl3-d) δ 9.92 (s, 2H), 8.38 (d, J = 8.3 Hz, 1H), 8.11(d, J = 2.3 Hz, 1H), 7.56 – 7.29 (m, 6H), 7.27 (d, J = 5.3 Hz, 2H), 7.16 –6.83 (m, 4H), 5.01 (s, 2H), 4.33 (t, J = 8.5 Hz, 1H), 3.76 (t, J = 8.3 Hz,1H), 3.59 (s, 2H), 3.17 (t, J = 8.6 Hz, 1H), 3.11 (t, J = 8.4 Hz, 1H), 2.50(s, 2H), 2.39 (s, 6H), 2.35 (d, J = 5.6 Hz, 3H), 2.25 (s, 1H). 13 C NMR (126MHz, CDCl3-d) δ 168.94, 168.46, 162.83, 159.95, 154.30, 154.09, 144.50,141.87, 139.04, 137.47, 134.46, 133.84, 132.94, 132.19, 130.68, 130.01,129.21, 127.95, 126.48, 126.06, 125.51, 124.96, 124.65, 121.65, 120.29,118.51, 117.15, 113.36, 113.10, 109.85, 60.94, 50.12, 43.07, 28.09, 20.51,14.21, 12.21, 11.25, 10.07. HPLC (90% acetonitrile in water with 1‰ HCOOH):t R = 4.655 min, 99.29%. ESI-HRMS (m / z): [M + H] + , calcd. for C 42 H 39 N5O5S,726.2672, found, 726.2748.

[0158] Example 37: Synthesis of 2-[(3,5-dimethylisoxazo-4-yl)methoxy]-5-methoxy-N-[3-[2-[[5-methyl-4-(1-(2-methylbenzoyl)indoline-5-yl)thiazo-2-yl]amino]-2-oxoethyl]phenyl]benzamide (compound 36)

[0159] Following the synthesis method of intermediate 26a, using intermediates 10i and 4a as raw materials, a pale yellow solid compound 36 was obtained. 1 H NMR (500 MHz, CDCl3-d) δ 10.01 (s, 2H), 8.38 (d, J = 8.4 Hz, 1H), 7.84(d, J = 3.0 Hz, 1H), 7.56 – 7.28 (m, 6H), 7.28 – 6.92 (m, 6H), 4.99 (s, 2H), 4.34 (t, J = 8.3 Hz, 1H), 3.87 (s, 3H), 3.76 (t, J = 8.3 Hz, 1H), 3.62 (s,2H), 3.17 (t, J = 8.4 Hz, 1H), 3.11 (t, J = 8.4 Hz, 1H), 2.50 (s, 2H), 2.45(s, 3H), 2.42 (s, 1H), 2.39 (s, 2H), 2.35 (s, 3H), 2.25 (s, 1H). 13 C NMR (151MHz, CDCl3-d) δ 167.95, 167.32, 161.45, 158.91, 153.94, 153.07, 149.24,140.85, 137.96, 136.47, 133.45, 131.24, 129.66, 129.06, 128.19, 126.91,126.38, 125.49, 125.04, 124.50, 124.07, 123.61, 122.00, 120.67, 119.28,119.05, 117.54, 116.12, 114.72, 114.25, 112.33, 108.88, 60.71, 54.86, 49.10,42.16, 27.07, 18.02, 11.18, 10.19, 9.02. HPLC (90% acetonitrile in water with1‰ HCOOH): t R= 4.333 min, 98.99%. ESI-HRMS (m / z): [M + H] + , calcd. forC 42 H 39 N5O6S, 742.2621, found, 742.2697.

[0160] Example 38: Synthesis of N-[4-(3-chlorophenyl)-5-methylthiazol-2-yl]-1-[2-[(3,5-dimethylisoxazol-4-yl)methoxy]benzoyl]piperidine-4-carboxamide (compound 37)

[0161] Following the synthesis method of intermediate 26a, using intermediates 10n and 4b as raw materials, a pale yellow solid compound 37 was obtained. 1 H NMR (400 MHz, DMSO-d6) δ 12.14 (d, J = 11.3 Hz, 1H), 7.68 (t, J = 1.8Hz, 1H), 7.61 (dd, J = 7.8, 1.7 Hz, 1H), 7.53 – 7.33 (m, 3H), 7.31 – 7.11 (m,2H), 7.04 (q, J = 7.9 Hz, 1H), 5.05 – 4.87 (m, 2H), 4.58 – 4.38 (m, 1H), 3.35 (s, 1H), 3.03 – 2.86 (m, 1H), 2.84 – 2.61 (m, 2H), 2.48 (d, J = 7.2 Hz, 3H), 2.40 (s, 3H), 2.20 (d, J = 3.8 Hz, 3H), 1.88 (d, J = 12.8 Hz, 1H), 1.71 (dd,J = 12.9, 3.5 Hz, 1H), 1.59 – 1.24 (m, 2H). 13C NMR (151 MHz, DMSO-d6) δ173.24, 173.16, 167.76, 166.79, 160.02, 154.56, 154.31, 154.10, 142.82,137.41, 133.63, 130.76, 128.02, 127.82, 127.50, 126.80, 122.87, 121.81,113.93, 110.74, 60.18, 46.25, 46.03, 41.83, 28.65, 28.17, 12.26, 11.11,10.09. HPLC (90% acetonitrile in water with 1‰ HCOOH): t R = 7.451 min,99.18%. ESI-HRMS (m / z): [M + H] + , calcd. for C 29 H 29 ClN4O4S, 565.1598, found, 565.1687.

[0162] Example 39: Synthesis of N-[4-(4-bromophenyl)-5-methylthiazol-2-yl]-1-[2-[(3,5-dimethylisoxazol-4-yl)methoxy]benzoyl]piperidine-4-carboxamide (compound 38)

[0163] Following the synthesis method of intermediate 26a, using intermediates 10n and 4c as raw materials, a pale yellow solid compound 38 was obtained. 1H NMR (600 MHz, DMSO-d6) δ 12.09 (d, J = 16.1 Hz, 1H), 7.69 – 7.55 (m,4H), 7.45 – 7.35 (m, 1H), 7.27 – 7.13 (m, 2H), 7.04 (dt, J = 12.4, 7.4 Hz,1H), 5.01 – 4.91 (m, 2H), 4.47 (dd, J = 25.9, 12.9 Hz, 1H), 3.43 – 3.33 (m,1H), 3.03 – 2.77 (m, 2H), 2.77 – 2.63 (m, 2H), 2.46 (d, J = 9.8 Hz, 3H), 2.40(s, 3H), 2.19 (d, J = 4.9 Hz, 3H), 1.99 (s, 1H), 1.87 (d, J = 13.0 Hz, 1H), 1.75 – 1.66 (m, 1H), 1.63 – 1.39 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 173.20,167.77, 166.77, 160.06, 154.53, 154.28, 143.27, 134.54, 131.80, 130.39,127.81, 127.18, HPLC (90% acetonitrile inwater with 1‰ HCOOH): t R = 7.821 min, 99.47%. ESI-HRMS (m / z): [M + H] + ,calcd. for C 29 H 29 BrN4O4S, 609.1093, found, 609.1166.

[0164] Example 40: Synergistic Validation of the Reversal of Tumor Drug Resistance by Combined Administration of NRF2 Inhibitor and AKR1C3 Inhibitor

[0165] 1. Experimental Materials

[0166] The doxorubicin-resistant MCF-7 cells, namely MCF-7 / ADR, are a cell line constructed by our research group. Their resistance coefficient (RI) reaches 30.4 (RI < 5 indicates low resistance, 5-25 indicates moderate resistance, and > 25 indicates high resistance). Culture conditions were 37℃, 5% CO2. DMEM high-glucose medium was purchased from Jiangsu Kaiji Biotechnology Co., Ltd. (Nanjing, China). Fetal bovine serum (FBS) was purchased from Shanghai Xiaopeng Biotechnology. All culture media were supplemented with 10% FBS. 2 μM doxorubicin was added during cell culture to maintain resistance. The CCK-8 kit was purchased from Solarbio (Beijing, China).

[0167] 2. Experimental Methods

[0168] Cells were cultured in high-glucose DMEM medium containing 10% FBS, with doxorubicin added to maintain their resistance. Culture conditions were 37°C, 5% CO2, and saturated humidity. When cell density reached 80%–90%, cells were digested for 3 minutes at 37°C with 0.25% trypsin solution containing 0.01% EDTA. After observing the cells becoming rounded under a microscope, complete culture medium was added to terminate the digestion, and the cells were centrifuged at 1000 rpm for 5 minutes. The digestion solution was discarded, and fresh culture medium was added. Adherent cells were pipetted and mixed thoroughly, and the cells were transferred to new culture flasks according to the required cell volume. An appropriate amount of culture medium was added, and the cells were mixed and cultured further. A certain amount of cells was then taken and diluted to the required concentration using complete culture medium.

[0169] (1) Disperse the cells in a complete medium containing 10% FBS at a density of 3*10. 4 Cells were seeded at a density of 100 μL / mL in 96-well plates. The 96-well plates containing cells were placed in an incubator and incubated at 37°C and 5% CO2 for 18 h until the cells adhered.

[0170] (2) The test compound was serially diluted to the target concentration using complete culture medium. The original culture medium was discarded, and the cells were treated with blank complete culture medium or the test compound at the specified concentration at 37°C and 5% CO2 for 72 h. Three replicates were set up for each concentration.

[0171] (3) After 72 hours, discard the original culture medium and add incomplete culture medium containing 10% CCK-8 solution to each well under light-protected conditions. Incubate at 37°C for 2 hours and detect the OD value of each well at 450 nm wavelength using an ELISA reader.

[0172] (4) Data processing: Cell viability = (A 测试 -A 空白 ) / (A 阴性 -A 空白 )*100%. Where A 空白 For blank absorption of 96-well plate.

[0173] 3. Experimental Results

[0174] The results are as follows Figure 1 and Figure 2 As shown, both NRF2 inhibitor ML385 and AKR1C3 inhibitor S19-1035, when used alone in combination with doxorubicin, exhibited limited activity in reversing drug resistance. However, when both NRF2 inhibitors and AKR1C3 inhibitors were administered in combination with doxorubicin, the combination showed significantly greater activity in reversing doxorubicin resistance compared to the single-drug combination, indicating that NRF2 inhibitors and AKR1C3 inhibitors exert a synergistic activity in reversing drug resistance.

[0175] The Chou-Talalay method was used to calculate the combination index (CI) of the above-mentioned single-target inhibitors, alone or in combination with doxorubicin, and the dose reduction index (DOX DRI) of doxorubicin. Figure 3 The results are shown below. -Lg(CI) = 0~1 indicates a synergistic effect; -Lg(CI) > 1 indicates a very strong synergistic effect; the larger the -Lg(CI) value, the stronger the synergistic effect. DOX DRI: represents the multiple by which the dose of doxorubicin required for monotherapy is greater than the dose required for combination therapy to achieve the same efficacy (cell survival rate); the larger the DOX DRI value, the stronger the synergistic effect. Results are as follows... Figure 3 As shown, ML385 and S19-1035, when used alone in combination with doxorubicin, both exhibited a moderate to low-intensity synergistic effect, i.e., -Lg(CI) = 0~1; and both could reduce the dosage of doxorubicin while maintaining the same efficacy, i.e., DOX DRI > 0. The combination of NRF2 inhibitors, AKR1C3 inhibitors, and doxorubicin significantly increased both -Lg(CI) and DOX DRI values, indicating a very strong synergistic effect.

[0176] Example 41: Evaluation of the inhibitory activity of the compound AKR1C3 of the present invention

[0177] 1. Experimental Materials

[0178] The substrate (S)-(+)-1,2,3,4-tetrahydro-1-naphthol (Dairu Pharmaceutical Chiral Technology Co., Ltd.), oxidized coenzyme II free acid (Shanghai Yuanye Biotechnology Co., Ltd.), the compounds prepared in Examples 2-39, and the control compound indomethacin were all purchased from Anaiji. AKR1C protein (self-extracted from E. coli BL21(DE3), plasmid provided by Detai Biotechnology Co., Ltd.) and phosphate buffer were also used.

[0179] 2. Instruments

[0180] THERMO Varioskan Flash Full-Wavelength Multifunctional Microplate Reader.

[0181] 3. Experimental Methods

[0182] (1) Phosphate buffer (pH 7.0) Preparation of buffer: Dissolve 2.19 g of disodium hydrogen phosphate dodecahydrate and 6.84 g of sodium dihydrogen phosphate dihydrate in 470 mL of pure water and sonicate until all solids are dissolved. Adjust the pH to 7.0 precisely with dilute sodium hydroxide solution, then bring the volume to 500 mL and store at 4 °C.

[0183] (2) Preparation and dilution of 100 mM substrate (S)-(+)-1,2,3,4-tetrahydro-1-naphthol: Dissolve 22.23 mg of substrate in 1.5 mL of DMSO to prepare a 100 mM stock solution. Vortex and sonicate until completely dissolved. Store at -20°C for later use. Before each test, prepare the optimal substrate concentrations for different isoforms according to the Km values ​​of AKR1C1-AKR1C4, labeled as S1, S2, S3, and S4. Among them, AKR1C1 Km = 107.8 μM, AKR1C2 Km = 184.8 μM, AKR1C3 Km = 106.60 μM, and AKR1C4 Km = 230.45 μM.

[0184] (3) 2mM oxidized coenzyme II free acid NADP + Solution preparation: Weigh 12.1 mg of NADP + Dissolve in 138 mL of buffer. NADP + The solution should be prepared and used immediately, and should be kept away from light.

[0185] (4) Dilution of AKR1C1-AKR1C4: Select the protein concentrations that have the greatest response to the substrate and dilute them with buffer, labeling them E1, E2, E3, and E4. Dilute the extracted AKR1C1 80 times and label it E1; dilute AKR1C2 96 times and label it E2; dilute AKR1C3 84 times and label it E3; dilute AKR1C4 86 times and label it E4.

[0186] (5) Gradual dilution of compound solutions: The test compound should be prepared fresh for each use. Prepare a 100 mM stock solution of the test compound and the control compound using DMSO. Buffer is used for stepwise dilution of the compounds: 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 - 9 M.

[0187] (6) Sample loading: 200 μL per well. A total of 8 experimental groups were set up, with 3 replicates per group. 10 compounds were prepared.-4 M, 10 - 5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M consists of 6 test groups. Substrate diluent and NADP were added to each well. + Solutions, as well as compound diluents of different concentrations, buffers, and enzyme diluents. Each experimental group included a negative control group and a blank control group, with three replicates per group. The negative control group had substrate diluent and NADP added to each well. + Solution, buffer, enzyme dilution buffer; blank control group: substrate dilution buffer and NADP were added to each well. + Solution, Buffer. Incubate at 37℃ for 10 minutes. After incubation, measure the fluorescence absorbance of each group using a microplate reader under 340nm excitation and 460nm emission channels. Define A test as the fluorescence value of the test group, A negative as the fluorescence value of the negative group, and A blank as the fluorescence value of the blank group. Calculate the inhibition rate of each concentration of compound in the test group using the following formula (A... 测试 -A 空白 ) / (A 阴性 -A 空白 )×100%

[0188] The results were calculated using GraphPadPrism™ (GraphPad Software, San Diego, CA, USA) software in a non-linear regression analysis model, yielding the corresponding IC. 50 value.

[0189] 4. Experimental Results

[0190] Table 1. Inhibitory activity of the compounds of the present invention against AKR1C3 a

[0191]

[0192] a All data are the average of three parallel experiments. “++++” represents IC50. 50 Between 10-100nM, "+++" represents IC 50 Between 100-1000nM, "++" represents IC 50 Between 1000-10000nM, "+" represents IC. 50 Greater than 10000nM.

[0193] Table 2. Inhibitory activity and selectivity of preferred compounds against AKR1C1, AKR1C2, AKR1C3 and AKR1C4

[0194]

[0195] b Selective index (SI) n ) = IC 50 AKR1Cn / IC 50 AKR1C3 (n = 1, 2, 4). "++++" represents IC. 50 Between 10-100nM, "+++" represents IC 50 Between 100-1000nM, "++" represents IC 50 Between 1000-10000nM, "+" represents IC. 50 Greater than 10000nM.

[0196] Results Analysis: Overall, the activity test results showed that more than half of the 38 compounds exhibited single-digit micromolar inhibitory activity against AKR1C3. Among them, compound 10 showed the best activity with an IC50 value of [missing value]. 50 The value is less than 100 nM. AKR1C selectivity tests were performed on some preferred compounds, and it was found that these compounds all exhibited good selectivity for AKR1C3.

[0197] Example 42: Evaluation of the NRF2 inhibitory activity of the compound of the present invention

[0198] 1. Experimental Materials

[0199] ARE-Luc-EF1α-mCherry A549 cells (purchased from Beyotime Biotechnology, catalog number C8455) are stable A549 cell lines with multiple copies of the ARE binding site integrated into the genome. Line stability was maintained using 1 μg / mL puromycin. Culture conditions were 37℃, 5% CO2. DMEM high-glucose medium was purchased from Jiangsu Kaiji Biotechnology Co., Ltd. (Nanjing, China). Fetal bovine serum (FBS) was purchased from Shanghai Xiaopeng Biotechnology Co., Ltd., and 10% FBS was added to all culture media. A firefly luciferase assay kit was purchased from Beyotime Biotechnology (catalog number RG051S).

[0200] 2. Instruments

[0201] THERMO Varioskan Flash Full-Wavelength Multifunctional Microplate Reader.

[0202] 3. Experimental Methods

[0203] Cells were cultured in high-glucose DMEM medium containing 10% FBS, with puromycin added to maintain their resistance. Culture conditions were 37°C, 5% CO2, and saturated humidity. When cell density reached 80%–90%, cells were digested at 37°C for 3 minutes with a 0.25% trypsin solution containing 0.01% EDTA. After observing the cells becoming rounded under a microscope, complete culture medium was added to terminate the digestion, and the cells were centrifuged at 1000 rpm for 5 minutes. The digestion solution was discarded, and fresh culture medium was added. Adherent cells were pipetted and mixed thoroughly. Cells were then transferred to new culture flasks according to the required cell volume, and an appropriate amount of culture medium was added and mixed well before continuing culture. A certain amount of cells was taken and diluted to the required concentration using complete culture medium.

[0204] (1) Disperse the cells in a complete medium containing 10% FBS at a density of 3*10. 4 Cells were seeded at a density of 100 μL / mL in 96-well plates. The 96-well plates containing cells were placed in an incubator and incubated at 37°C and 5% CO2 for 18 h until the cells adhered.

[0205] (2) The test compound was serially diluted to the target concentration using complete culture medium. The original culture medium was discarded, and the cells were treated with complete culture medium or the specified concentration of the test compound at 37°C and 5% CO2 for 12 h. Three replicates were set up for each concentration.

[0206] (3) After 12 hours, add 100 μL of luciferase assay kit to each well under light-protected conditions, and use an ELISA reader to detect the chemiluminescence value of each well.

[0207] (4) Data processing: ARE luciferase activity (%): refers to the percentage of ARE-driven luciferase activity in C8455 cells treated with the drug relative to the solvent control group. Relative NRF2 inhibition titer (%, with ML385 as a reference) = (1 - ARE luciferase activity of the test compound) / (1 - ARE luciferase activity of ML385) × 100%. Nrf2 inhibition activity grading: based on the above relative NRF2 inhibition titer: potent (>90%); moderate (50%–90%); weak (<50%); no activity (<0%).

[0208] 4. Experimental Results

[0209] Table 3. Inhibitory activity of the compounds of the present invention against NRF2 c

[0210]

[0211] cAll data are the average of three parallel experiments. "++++" represents a potent NRF2 inhibitory potency (>90%), "+++" represents a moderate NRF2 inhibitory potency (50%–90%), "++" represents a weak NRF2 inhibitory potency (0–50%), and "+" represents an inactive NRF2 inhibitory potency (<0).

[0212] Results Analysis: Overall, the NRF2 inhibitory activity test results show that more than half of the 38 target compounds in this invention retained potent NRF2 inhibitory activity comparable to the positive control ML385. These results fully demonstrate that the AKR1C3 / NRF2 dual-target inhibitor designed in this invention can effectively retain NRF2 target inhibitory activity, successfully achieving synergistic retention of dual-target activity, and providing core target support for the drug resistance reversal efficacy of the compounds.

[0213] Example 43: Evaluation of the antiproliferative activity of the compounds of the present invention as sensitizers in combination with chemotherapeutic drugs

[0214] 1. Experimental reagents and materials

[0215] Same as Example 1

[0216] 2. Experimental Methods

[0217] Same as Example 1

[0218] 3. Experimental Results

[0219] This embodiment uses doxorubicin-resistant breast cancer MCF-7 / ADR cells as a model to systematically evaluate the cellular safety of the 38 AKR1C3 / NRF2 dual-target compounds of this invention when used alone, their chemosensitizing and resistance reversal activities when combined with doxorubicin, and to verify the synergistic effect of the dual-target molecules.

[0220] Safety evaluation results of compound used alone, such as Figure 4 As shown, after treatment at a final concentration of 25 μM for 72 h, the cell viability of most compound groups was higher than 80%, with no obvious non-specific cytotoxicity, demonstrating good in vitro safety and providing a sufficient safety window for subsequent combination therapy.

[0221] The results of the sensitizing activity of the compound in combination with doxorubicin are as follows: Figure 5 As shown, after combined administration of 25 μM compound and 25 μM doxorubicin, the cell survival rate of most compound groups decreased significantly compared with the doxorubicin monotherapy group. The survival rate of most combination groups dropped to below 50%, and some preferred compound groups even dropped to below 10%. This directly confirms that the compounds of the present invention can significantly enhance the killing effect of doxorubicin on drug-resistant tumor cells and have excellent potential for reversing chemotherapy resistance and enhancing sensitization.

[0222] The comparative experimental results of preferred compound 17 are as follows: Figure 6 As shown, at two drug concentrations of 1 μM and 50 μM, the cell survival rate of the compound 17 combined with doxorubicin group was significantly lower than that of the ML385 single drug combination group and the S19-1035 single drug combination group, and was significantly better than that of the physical combination group of the two (p<0.0001), which confirms that the covalently linked dual-target molecule of the present invention has an intramolecular synergistic effect that cannot be achieved by single-target formulations.

[0223] In summary, the dual-target compound of this invention possesses both good drug safety and excellent chemotherapy resistance reversal activity, and can effectively reverse the resistance of MCF-7 / ADR cells to doxorubicin. It is suitable for preparing pharmaceutical compositions for anti-tumor treatment, especially for overcoming chemotherapy resistance in tumors.

[0224] Example 44: Determination of the pure water solubility and apparent oil-water partition coefficient (LogP) of the compounds of the present invention

[0225] 1. Experimental reagents and materials

[0226] Pure water was purchased from Wahaha Group (Hangzhou, China), methanol for chromatography was purchased from Adamas (Shanghai, China), n-octanol was purchased from Bieder Pharmaceutical Co., Ltd. (Shanghai, China), the high performance liquid chromatograph was from Shimadzu Corporation (Japan), and the vacuum drying oven was from Shanghai Jinghong Experimental Equipment Co., Ltd. (Shanghai, China).

[0227] 2. Experimental Methods

[0228] Sample preparation:

[0229] (1) Place the solid compound to be tested in a vacuum drying oven at 45°C overnight to ensure that the sample is fully dried;

[0230] (2) Establishment of standard curve: Accurately weigh 1 mg of the analyte into a 1.5 mL EP tube and dissolve it in 1 mL of chromatographic methanol. Shake overnight at 37 °C to ensure complete dissolution of the compound, obtaining a 1000 μg / mL solution. At this time, the HPLC peak area should not change at different injection times. Dilute the completely dissolved compound solution with chromatographic methanol to obtain standard sample solutions of 500 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 20 μg / mL, 10 μg / mL, 5 μg / mL, 2 μg / mL and 1 μg / mL. Filter the prepared standard solutions through a 0.22 μM filter membrane. Take samples of each concentration of standard sample and analyze them 3 times by HPLC to obtain the peak area corresponding to different concentrations. Plot the standard curve with concentration as the x-axis and peak area as the y-axis.

[0231] (3) Pure water solubility test: Accurately weigh an excess of the compound into a 1.5 mL ep tube and dissolve it in a small amount of water. Shake overnight at 37°C to ensure the solution reaches saturation. Take the supernatant, filter it through a 0.22 μM filter membrane, and inject it for HPLC analysis. Repeat the test 3 times, and substitute the peak area into the standard curve to obtain the pure water solubility data of the tested compound;

[0232] (4) Oil-water partition coefficient test: In a 25 mL separatory funnel, add sufficient water to a small amount of n-octanol and sufficient n-octanol to a small amount of water, respectively. Mix thoroughly and allow to stand for separation. Obtain the organic layer of the former and the aqueous layer of the latter, i.e., a water-saturated n-octanol solution and a n-octanol-saturated aqueous solution. Add excess compound to the solution and shake overnight at 37°C to ensure saturation. Collect the supernatant using a 1 mL syringe preheated at 37°C, filter through a 0.22 μM filter membrane, and analyze by HPLC. Repeat the test three times. The logarithm of the peak area ratio between the n-octanol solution and the aqueous solution is the oil-water partition coefficient of the compound to be tested.

[0233] (5) Injection conditions: Gradient elution was used, with a flow rate of 1 mL / min. The mobile phase consisted of methanol and 1 / 1000 formic acid water in a ratio of methanol:water = 9:1. The injection volume was 10 μl.

[0234] 3. Experimental Results

[0235] Table 4. Test results of pure water solubility and apparent lipid-water partition coefficient (LogP) of the preferred compounds.

[0236]

[0237] "#" represents a solubility between 0 and 5 μg / mL, "##" represents a solubility between 5 and 10 μg / mL, and "###" represents a solubility greater than 10 μg / mL; "*" represents Log P < 0, "**" represents 0 < Log P < 2, "***" represents 2 < Log P < 4, and "****" represents Log P > 4.

[0238] Results Analysis: The LogP values ​​of the three preferred compounds were all in the range of 2–4. This range indicates suitable lipid-water partitioning, balancing cell membrane penetration and target binding affinity, which is beneficial for in vivo absorption and distribution. Regarding solubility, compound 8 showed a water solubility >10 μg / mL, superior to compounds 10 and 17. According to the solubility classification standards of the 2025 edition of the Pharmacopoeia of the People's Republic of China, all three compounds fall into the range of very slightly soluble to almost insoluble. This solubility level is relatively common among antitumor small molecule lead compounds and can be further improved through conventional methods such as solid dispersions, cyclodextrin inclusion complexes, nanocrystals, or liposome formulations, providing a basis for further drug development optimization.

Claims

1. An AKR1C3 / NRF2 dual-target inhibitor, characterized in that, Compounds of formula (I) or pharmaceutically acceptable salts thereof: Ⅰ in: R1 is selected from hydrogen, halogen, C1-C4 alkoxy, C1-C4 alkyl, C1-C4 haloalkyl, and nitro; R2 is selected from L1, L2, and L3 are each independently selected from one or more combinations of substituted or unsubstituted C1-C7 alkylene groups, substituted or unsubstituted phenyldiyl groups, substituted or unsubstituted piperidinyl groups, substituted or unsubstituted piperazinediyl groups, C1-C7 alkylamide groups, and C1-C7 alkoxy groups, where n = 0 or 1. R3 is selected from ， X is selected from hydrogen, halogen, C1-C4 alkoxy, C1-C4 haloalkyl, nitro, cyano, hydroxyl, and amino.

2. The AKR1C3 / NRF2 dual-target inhibitor according to claim 1, characterized in that, R1 is selected from hydrogen, fluorine, chlorine, bromine, methyl, methoxy, trifluoromethyl, nitro; L1 is selected from substituted or unsubstituted C1-C6 alkylene groups, substituted or unsubstituted piperidinium groups; L2 is selected from substituted or unsubstituted 1,3-phenylenediol groups, substituted or unsubstituted 1,4-phenylenediol groups; L3 is selected from substituted or unsubstituted 1,3-phenylenediol groups, substituted or unsubstituted 1,4-phenylenediol groups; X is selected from chlorine, bromine.

3. The AKR1C3 / NRF2 dual-target inhibitor according to claim 1, characterized in that, R2 is selected from 。 4. The AKR1C3 / NRF2 dual-target inhibitor according to claim 1, characterized in that, The compound is selected from any of the following compounds: 。 5. The AKR1C3 / NRF2 dual-target inhibitor according to claim 1, characterized in that, The pharmaceutically acceptable salt is a salt formed by a compound of formula (I) and an acid selected from any of the following: Hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, malic acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, ferulic acid.

6. A pharmaceutical composition, characterized in that, It comprises the AKR1C3 / NRF2 dual-target inhibitor of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, Its dosage forms include tablets, capsules, powders, pills, granules, injections, oral liquids, syrups, inhalers, ointments, patches, or suppositories.

8. The use of the AKR1C3 / NRF2 dual-target inhibitor of claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 6 in the preparation of a medicament for the prevention and / or treatment of cancer and the reversal of tumor drug resistance.

Images