Halogenated indole ketones and their preparation methods and applications, halogenated indole ketone derivatives and their applications, pharmaceutical compositions and their applications

Abstract

This invention provides halogenated indole ketone compounds, their preparation methods and applications, halogenated indole ketone derivatives and their applications, and pharmaceutical compositions and their applications, relating to the field of biomedical technology. The halogenated indole ketone compounds provided by this invention, through kinase spectrum analysis, show that in addition to possessing VEGFR, FGFR, and PDGFR kinase inhibitory activities, they also exhibit strong AXL kinase inhibitory activity, with an IC50 value of [missing value]. 50 The value reached a low nanomolar level, which is 45 times that of nintedanib in inhibiting AXL kinase activity. The halogenated indole ketone compounds provided by this invention exhibit better antitumor activity than nintedanib at both the cellular and in vivo levels, and have a better effect on inhibiting tumor invasion and metastasis than nintedanib, thus effectively treating malignant tumors and their invasion and metastasis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to halogenated indole ketone compounds and their preparation methods and applications, halogenated indole ketone derivatives and their applications, and pharmaceutical compositions and their applications. Background Technology

[0002] Indoleone derivatives possess a wide range of biological activities. Nintedanib (NDN), approved by the US FDA in 2014 as a treatment for idiopathic pulmonary fibrosis (IPF), is a derivative with an ester group (methoxyacyl group) substituted at the 6th carbon of an indoleone. IPF is a chronic, progressive fibrotic lung disease of unknown etiology, with a complex pathogenesis that remains unclear. Nintedanib is a triple-receptor tyrosine kinase inhibitor that acts on vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), and platelet-derived growth factor receptor (PDGFR). In addition to FDA approval for clinical use in the treatment of idiopathic pulmonary fibrosis, the European Medicines Agency has also approved nintedanib in combination with docetaxel for the treatment of advanced locally recurrent non-small cell lung cancer after first-line chemotherapy. Literature reports that nintedanib has very low bioavailability in vivo, only 5-10%. Therefore, there is still room for structural optimization of nintedanib.

[0003] Tumor metastasis is the leading cause of death in most cancer patients. Tumor metastasis is a multi-step process, mainly including three stages: 1) Invasion: In situ tumor cells increase their invasiveness through epithelial-mesenchymal transition (EMT), invading surrounding tissues and migrating to areas near blood vessels or lymphatic vessels. They then exit the bloodstream and enter the circulatory system, becoming circulating tumor cells (CTCs); 2) Circulation: Platelets directly adhere to the surface of CTCs, forming "microthrombi" structures that reduce the recognition and clearance by the immune system; 3) Colonization: CTCs colonize in "pre-metastatic niches" in distant organs, which are inflammatory environments with immunosuppressive characteristics formed under the influence of cytokines or exosomes secreted by the in situ tumor tissue, conducive to tumor cell colonization. Inhibiting tumor metastasis is one of the important strategies in cancer treatment.

[0004] AXL kinase belongs to the TAM family of receptor tyrosine kinases and is highly expressed in various cancers, including non-small cell lung cancer and breast cancer. Aberrant expression of AXL can activate and antagonize tumor cell apoptosis, promote tumor cell invasion and metastasis, and promote tumor angiogenesis, thereby driving tumor development and progression. Furthermore, high AXL expression is also associated with drug resistance to antitumor drugs. AXL has also become a popular target in antitumor drug development in recent years.

[0005] The pathogenesis of malignant tumors is highly complex, often involving multiple mechanisms, pathological processes, and genes, resulting from the combined effects of various factors. Multi-target drugs can simultaneously act on multiple pathological processes and mechanisms of the same disease, potentially leading to better therapeutic outcomes. However, existing nintedanib drugs have low AXL kinase activity, resulting in insufficient anti-tumor efficacy. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide halogenated indole ketone compounds, their preparation methods and applications, halogenated indole ketone derivatives and their applications, and pharmaceutical compositions and their applications. The halogenated indole ketone compounds and their derivatives provided by this invention possess strong AXL kinase inhibitory activity and high antitumor activity.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides halogenated indolone compounds, including racemic compounds having the structure of Formula I or shown, or chiral compounds having the structure of Formula II:

[0009]

[0010] Wherein, R1 is a halogen;

[0011] R2 includes hydrogen or C1-C4 alkyl groups;

[0012] R4 includes C1-C4 alkyl groups;

[0013] R3 includes C1-C4 alkyl, cycloalkyl, hydrocarbon group containing unsaturated bonds, heterocyclic group or alkylamine group;

[0014] In Equation II, the wavy line represents the R configuration or the S configuration.

[0015] Preferably, the cycloalkyl group comprises cyclopropyl or cyclobutyl;

[0016] The hydrocarbon group containing unsaturated bonds includes an alkenyl group;

[0017] The heterocyclic group includes any one of the following structures:

[0018]

[0019] The alkylamine group includes N,N,N-trimethylethylenediamine, 2-(N,N-dimethylamino)ethoxy, or 2-(N,N-dimethylamino)eththio.

[0020] Preferably, it has any one of the following structures:

[0021]

[0022]

[0023] This invention also provides a method for preparing the haloindolone compounds described in the above technical solution, comprising the following steps:

[0024] Intermediate 3 and intermediate 4 were subjected to a substitution reaction to obtain the haloindolone compound;

[0025] The intermediate 3 includes a racemic intermediate 3 or a chiral intermediate 3;

[0026]

[0027] Preferably, the preparation method of the racemic intermediate 3 includes the following steps:

[0028] The acyl chloride was substituted with N-methyl-4-nitroaniline to give intermediate 1;

[0029] The intermediate 1 was subjected to a nucleophilic substitution reaction with HR3 to obtain racemic intermediate 2;

[0030] The racemic intermediate 2 was subjected to a hydrogenation reduction reaction to obtain the racemic intermediate 3;

[0031]

[0032] Where X is a halogen.

[0033] Preferably, the method for preparing the chiral intermediate 3 includes the following steps:

[0034] The p-toluenesulfonyl chloride was substituted with a 2-hydroxycarboxylic acid ester to give compound 1;

[0035] The compound 1 was subjected to a hydrolysis reaction to obtain compound 2;

[0036] Compound 2 was subjected to a substitution reaction with thionyl chloride to obtain compound 3;

[0037] Compound 3 was subjected to a substitution reaction with N-methyl-4-nitroaniline to obtain compound 4;

[0038] Compound 4 was subjected to a nucleophilic substitution-transconfiguration reaction with HR3 to obtain chiral intermediate 2;

[0039] The chiral intermediate 2 was subjected to a hydrogenation reduction reaction to obtain the chiral intermediate 3;

[0040]

[0041] The configuration of chiral intermediate 2 is different from that of 2-hydroxycarboxylic acid ester, compound 1, compound 2, compound 3 and compound 4.

[0042] Preferably, the preparation method of the intermediate 4 includes the following steps:

[0043] N-acetylindolone and triethyl orthobenzoate were condensed to give intermediate 4;

[0044]

[0045] The present invention also provides halogenated indole ketone derivatives, including pharmaceutically acceptable salts and / or solvates of the halogenated indole ketone compounds described in the above technical solutions.

[0046] The present invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients; said active ingredient comprising one or more of the halogenated indolone compounds and halogenated indolone derivatives described in the above-described technical solutions.

[0047] The present invention also provides the application of the haloindolone compounds or haloindolone derivatives described in the above technical solutions in the preparation of drugs for kinase-mediated diseases;

[0048] The kinases include one or more of the following: tyrosine protein kinase receptor, serine / threonine protein kinase, tyrosine protein kinase, vascular endothelial growth factor receptor 2, platelet-derived growth factor receptor-α, fibroblast growth factor receptor 1, neurotrophic factor receptor 2, non-receptor tyrosine kinase, tyrosine protein kinase, 5'-AMP-activated protein kinase subunit α1 / β1 / γ1, glycogen synthase kinase-3β, ribosomal protein S6 kinase α-1, tyrosine protein kinase, bispecific mitogen-activated protein kinase kinase 1, aurora kinase B, bispecific protein kinase, epidermal growth factor type A receptor 2, insulin-like growth factor 1 receptor, epidermal growth factor type B receptor 4, mitogen-activated protein kinase kinase 1, serine / threonine protein kinase, nonspecific serine / threonine protein kinase, nonspecific serine / threonine protein kinase, and glucokinase.

[0049] The halogenated indole ketone compounds provided by this invention, through kinase spectrum analysis, show that in addition to the VEGFR, FGFR, and PDGFR kinase inhibitory activities possessed by nintedanib, they also exhibit strong AXL kinase inhibitory activity, IC50. 50 The value reached a low nanomolar level, which is 45 times more effective than nintedanib in inhibiting AXL kinase activity. The halogenated indole ketone compounds provided by this invention exhibit better antitumor activity than nintedanib at both the cellular and in vivo levels, and show better inhibition of tumor invasion and metastasis. Furthermore, the halogenated indole ketone compounds provided by this invention show inhibitory activities exceeding 90% against serine / threonine protein kinase (QIK), tyrosine protein kinase (LCK), platelet-derived growth factor receptor-α (PDGFRα), fibroblast growth factor receptor 1 (FGFR1), neurotrophic factor receptor 2 (TRKB), non-receptor tyrosine kinase (SRC), and tyrosine protein kinase (ABL1), demonstrating high kinase inhibitory activity and the ability to effectively treat malignant tumors and their invasion and metastasis.

[0050] The method for preparing halogenated indole ketone compounds provided by this invention is simple in process, easy to operate, low in production cost, and suitable for industrial production. Attached Figure Description

[0051] Figure 1 The graph shows the inhibitory activity of target compound 1 (X529) prepared in Example 1 against 80 kinases.

[0052] Figure 2 The graph shows the changes in tumor volume in mice under the action of the blank group, the nintedanib group, and the target compound (11b) group prepared in Example 1 during the process of anti-pancreatic cancer activity.

[0053] Figure 3 The graph shows the changes in tumor weight in mice under the action of the blank group, the nintedanib group, and the target compound (11b) group prepared in Example 1 during the process of anti-pancreatic cancer activity.

[0054] Figure 4 H&E staining (a) and Western Blot analysis (b) of mouse tumor tissues containing target compound 1 (11b) prepared for the control group, nintedanib group and Example 1 are shown. In Figure a, 1, 2, 3 and 4 represent sites randomly selected from the full-view lung sections of mice in each group, and their magnified microscopic images are provided. Detailed Implementation

[0055] This invention provides halogenated indole ketone compounds, characterized in that they comprise racemic compounds having the structure shown in Formula I or a chiral compound having the structure shown in Formula II:

[0056]

[0057] Wherein, R1 is a halogen; R2 includes hydrogen or C1-C4 alkyl; R4 includes C1-C4 alkyl; R3 includes C1-C4 alkyl, cycloalkyl, hydrocarbon group containing unsaturated bond, heterocyclic group or alkylamine group; the wavy line in Formula II represents the R configuration or S configuration.

[0058] In this invention, the halogen preferably includes fluorine, chlorine or bromine.

[0059] In this invention, the C1-C4 alkyl group preferably includes methyl, ethyl, propyl or butyl.

[0060] In this invention, the cycloalkyl group includes cyclopropyl or cyclobutyl.

[0061] In this invention, the hydrocarbon group containing unsaturated bonds preferably includes an alkenyl group, and in specific embodiments it can be a vinyl or propene group.

[0062] In this invention, the heterocyclic group preferably comprises any one of the following structures:

[0063]

[0064] In this invention, the alkylamine group preferably includes N,N,N-trimethylethylenediamine, 2-(N,N-dimethylamino)ethoxy, or 2-(N,N-dimethylamino)eththio.

[0065] In this invention, the haloindolone compound preferably has any one of the following structures:

[0066]

[0067]

[0068] The present invention also provides a method for preparing the haloindolone compound described in the above technical solution, comprising the following steps: subjecting intermediate 3 and intermediate 4 to a substitution reaction to obtain the haloindolone compound; wherein intermediate 3 includes racemic intermediate 3 or chiral intermediate 3;

[0069]

[0070] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0071] In this invention, the substitution reaction preferably includes: mixing intermediate 3, intermediate 4, and an organic solvent (denoted as the first organic solvent), carrying out a first substitution reaction under a protective atmosphere, and then adding an organic base (denoted as the first organic base) to carry out a second substitution reaction. In this invention, the molar ratio of intermediate 3 to intermediate 4 is preferably 1:1 to 1.2, and in specific embodiments it can be 1:1, 1:1.05, 1:1.1, 1:1.15, or 1:1.2. In this invention, the first organic solvent preferably includes one or more of dimethylformamide, acetonitrile, and dimethyl sulfoxide. This invention does not have a special limitation on the amount of the first organic solvent, as long as it ensures the substitution reaction proceeds smoothly. In this invention, the first organic base is preferably piperidine and / or dimethylamine. In this invention, the molar ratio of intermediate 3 to the first organic base is preferably 1:1.5 to 3, and in specific embodiments it can be 1:1.5, 1:2, 1:2.5, or 1:3. In this invention, the protective atmosphere preferably includes nitrogen, argon, or helium.

[0072] In this invention, the temperature of the substitution reaction is preferably 70-90°C, and in specific embodiments it can be 70°C, 75°C, 80°C, 85°C or 90°C; the time of the first substitution reaction is preferably 1-2 hours, and in specific embodiments it can be 1 hour, 1.5 hours or 2 hours; the time of the second substitution reaction is preferably 2-3 hours, and in specific embodiments it can be 2 hours, 2.5 hours or 3 hours.

[0073] In this invention, when racemic intermediate 3 is used, a haloindolone compound having the structure shown in Formula I is obtained; when chiral intermediate 3 is used as a raw material, a haloindolone compound having the structure shown in Formula II is obtained.

[0074] After the substitution reaction is completed, the present invention preferably further includes: adding water to the reaction system obtained by the substitution reaction to precipitate a solid, filtering, grinding the obtained solid with methanol, filtering, washing the obtained filter cake with methanol and ethyl acetate, and drying to constant weight to obtain the haloindolone compound.

[0075] In this invention, the preparation method of the racemic intermediate 3 preferably includes the following steps:

[0076] The acyl chloride was substituted with N-methyl-4-nitroaniline to give intermediate 1;

[0077] The intermediate 1 was subjected to a nucleophilic substitution reaction with HR3 to obtain racemic intermediate 2;

[0078] The racemic intermediate 2 was subjected to a hydrogenation reduction reaction to obtain the racemic intermediate 3;

[0079]

[0080] Wherein, X is a halogen, and the halogen preferably includes fluorine, chlorine, bromine or iodine.

[0081] In this invention, the preferred method for preparing the acyl chloride includes the following steps: subjecting a halocarboxylic acid and thionyl chloride to a substitution reaction to obtain the acyl chloride;

[0082]

[0083] In this invention, the preparation route of the racemic intermediate 3 is as follows:

[0084]

[0085] This invention involves a substitution reaction (referred to as the third substitution reaction) between a halocarboxylic acid and thionyl chloride to obtain an acyl chloride.

[0086] In this invention, the molar ratio of the halocarboxylic acid to thionyl chloride is preferably 1:1 to 5, and in specific embodiments it can be 1:1, 1:2, 1:3, 1:4 or 1:5.

[0087] In this invention, the third substitution reaction is preferably carried out in the absence of a solvent or in the presence of an organic solvent (denoted as the second organic solvent). In this invention, the second organic solvent preferably includes chloroform and / or acetonitrile. This invention does not have a particular limitation on the amount of the second organic solvent used, as long as it is sufficient to ensure the smooth progress of the third substitution reaction.

[0088] In this invention, the temperature of the third substitution reaction is preferably 65-85°C, and in specific embodiments it can be 65°C, 70°C, 75°C, 80°C or 85°C; the time of the third substitution reaction is preferably 2-8h, and in specific embodiments it can be 2h, 3h, 4h, 5h, 6h, 7h or 8h.

[0089] After completing the third substitution reaction, the present invention preferably further includes: removing unreacted raw materials (e.g., thionyl chloride) from the reaction system obtained by the third substitution reaction under reduced pressure to obtain acyl chloride, which is then directly carried out in the next reaction without purification.

[0090] After obtaining the acyl chloride, the present invention performs a substitution reaction (referred to as the fourth substitution reaction) with N-methyl-4-nitroaniline to obtain intermediate 1.

[0091] In this invention, the molar ratio of the acyl chloride to N-methyl-4-nitroaniline is preferably 1 to 1.2:1, and in specific embodiments it can be 1:1, 1.05:1, 1.1:1, 1.15:1 or 1.2:1.

[0092] In this invention, the fourth substitution reaction is preferably carried out in the presence of a catalyst (denoted as the first catalyst), an acid remover (denoted as the first acid remover), and an organic solvent (denoted as the third organic solvent). In this invention, the first catalyst preferably comprises N,N-dimethylaminopyridine (DMAP) and / or pyridine; the molar ratio of N-methyl-4-nitroaniline to the first catalyst is preferably 1:0.05 to 0.2, and in specific embodiments it can be 1:0.05, 1:0.1, 1:0.13, 1:0.15, or 1:2. In this invention, the first acid-removing agent preferably comprises an organic amine, and in specific embodiments may include triethylamine and / or N-ethyl-N,N-diisopropylamine; the molar ratio of N-methyl-4-nitroaniline to the first acid-removing agent is preferably 1:1 to 1.5, and in specific embodiments may be 1:1, 1:1.05, 1:1.1, 1:1.15, 1:1.2, 1:1.25, 1:1.3, 1:1.35, 1:1.4, 1:1.45, or 1:1.5. In this invention, the third organic solvent preferably comprises one or more of anhydrous acetonitrile, tetrahydrofuran, and dichloromethane; this invention does not have a special limitation on the amount of the third organic solvent, as long as it is sufficient to ensure the smooth progress of the fourth substitution reaction.

[0093] In this invention, the temperature of the fourth substitution reaction is preferably 15-45°C, and in specific embodiments it can be 15°C, 20°C, 25°C, 30°C, 35°C, 40°C or 45°C; the time of the fourth substitution reaction is preferably 2-3 hours, and in specific embodiments it can be 2 hours, 2.5 hours or 3 hours.

[0094] After completing the fourth substitution reaction, the present invention preferably further includes: removing the solvent under reduced pressure, adding water to separate the layers, obtaining an organic phase and an aqueous phase respectively; extracting the aqueous phase with dichloromethane to obtain a dichloromethane phase; combining the organic phase and the dichloromethane phase, washing with saturated brine, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, and purifying by silica gel column chromatography to obtain intermediate 1. In the present invention, the eluent used for the silica gel column chromatography purification preferably includes petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is preferably 4:1 to 1:1.

[0095] After obtaining intermediate 1, the present invention performs a nucleophilic substitution reaction between intermediate 1 and HR3 to obtain racemic intermediate 2.

[0096] In this invention, the molar ratio of intermediate 1 to HR3 is preferably 1:1 to 3, and in specific embodiments it can be 1:1, 1:1.5, 1:2, 1:2.5, or 1:3. In this invention, HR3 preferably comprises any one of the following structural formulas:

[0097]

[0098] In this invention, the nucleophilic substitution reaction is preferably carried out in the presence of an acid-scavenging agent (denoted as the second acid-scavenging agent) and an organic solvent (denoted as the fourth organic solvent). In this invention, the second acid-scavenging agent preferably comprises an organic amine, more preferably triethylamine and / or N-ethyl-N,N-diisopropylamine; the molar ratio of intermediate 1 to the second acid-scavenging agent is preferably 1:1 to 3, and in specific embodiments it can be 1:1, 1:1.5, 1:2, 1:2.5, or 1:3. In this invention, the fourth organic solvent preferably comprises acetonitrile and / or dioxane; this invention does not have a special limitation on the amount of the fourth organic solvent, as long as it is sufficient to ensure the smooth progress of the nucleophilic substitution reaction.

[0099] In this invention, the temperature of the nucleophilic substitution reaction is preferably 80-100°C, and in specific embodiments it can be 80°C, 85°C, 90°C, 95°C or 100°C; the time of the nucleophilic substitution reaction is preferably 10-20h, and in specific embodiments it can be 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h or 20h.

[0100] After completing the nucleophilic substitution reaction, the present invention preferably further includes: removing the solvent from the reaction system obtained by the nucleophilic substitution reaction under reduced pressure, and purifying it by silica gel column chromatography to obtain racemic intermediate 2. In the present invention, the eluent used for the silica gel column chromatography purification preferably includes ethyl acetate and a dichloromethane-methanol mixed solvent in sequence, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solvent is preferably 4 to 10:1, and in specific embodiments it can be 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0101] After obtaining racemic intermediate 2, the present invention performs a hydrogenation reduction reaction on racemic intermediate 2 (denoted as the first hydrogenation reduction reaction) to obtain racemic intermediate 3.

[0102] In this invention, the first hydrogenation reduction reaction preferably includes: mixing racemic intermediate 2, a hydrogenation catalyst, and an organic solvent (denoted as the fifth organic solvent), and carrying out the first hydrogenation reduction reaction under a hydrogen atmosphere. In this invention, the hydrogenation catalyst preferably includes palladium on carbon or stannous chloride. In this invention, the mass of the hydrogenation catalyst is preferably 5-20% of the mass of racemic intermediate 2, and in specific embodiments, it can be 5%, 10%, 15%, or 20%. In this invention, the fifth organic solvent preferably includes anhydrous ethanol, methanol, etc.; this invention does not have a special limitation on the amount of the fifth organic solvent, as long as it is sufficient to ensure the smooth progress of the first hydrogenation reduction reaction.

[0103] In this invention, the temperature of the first hydrogenation reduction reaction is preferably room temperature; the time of the first hydrogenation reduction reaction is preferably 12-48 hours, and in specific embodiments it can be 12 hours, 15 hours, 20 hours, 24 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours or 48 hours; the pressure of the hydrogen gas is preferably 0.1-0.505 MPa, and in specific embodiments it can be 0.101 MPa, 0.2 MPa, 0.3 MPa, 0.4 MPa, 0.5 MPa or 0.505 MPa; as the hydrogen gas pressure increases, the time of the first hydrogenation reduction reaction is shortened.

[0104] After the first hydrogenation reduction reaction is completed, the present invention preferably further includes: filtering the reaction system obtained from the first hydrogenation reduction reaction, removing the solvent from the filtrate under reduced pressure, and obtaining racemic intermediate 3.

[0105] In this invention, the method for preparing the chiral intermediate 3 preferably includes the following steps:

[0106] The p-toluenesulfonyl chloride was substituted with a 2-hydroxycarboxylic acid ester to give compound 1;

[0107] The compound 1 was subjected to a hydrolysis reaction to obtain compound 2;

[0108] Compound 2 was subjected to a substitution reaction with thionyl chloride to obtain compound 3;

[0109] Compound 3 was subjected to a substitution reaction with N-methyl-4-nitroaniline to obtain compound 4;

[0110] Compound 4 was subjected to a nucleophilic substitution-transconfiguration reaction with HR3 to obtain chiral intermediate 2;

[0111] The chiral intermediate 2 was subjected to a hydrogenation reduction reaction to obtain the chiral intermediate 3;

[0112]

[0113] The configuration of the chiral intermediate is different from that of the 2-hydroxycarboxylic acid ester, compound 1, compound 2, compound 3 and compound 4.

[0114] In this invention, the preparation route of the chiral intermediate 3 is as follows:

[0115]

[0116] In this invention, a substitution reaction (referred to as the fifth substitution reaction) is carried out between toluenesulfonyl chloride and 2-hydroxycarboxylic acid ester to obtain compound 1.

[0117] In this invention, the molar ratio of the 2-hydroxycarboxylic acid ester to toluenesulfonyl chloride is preferably 1:1 to 1.3, and in specific embodiments it can be 1:1, 1:1.05, 1:1.1, 1:1.15, 1:1.2, 1:1.25 or 1:1.3. In this invention, the toluenesulfonyl chloride is preferably added dropwise under ice bath conditions.

[0118] In this invention, the fifth substitution reaction is preferably carried out in the presence of an organic solvent (denoted as the sixth organic solvent) and an organic base (denoted as the second organic base).

[0119] In this invention, the sixth organic solvent preferably includes dichloromethane and / or chloroform. There is no particular limitation on the amount of the sixth organic solvent used, as long as it is sufficient to ensure the smooth progress of the fifth substitution reaction.

[0120] In this invention, the second organic base preferably comprises an organic amine, more preferably triethylamine or N-ethyl-NN-diisopropylamine. In this invention, the molar ratio of the 2-hydroxycarboxylic acid ester to the second organic base is preferably 1:1 to 2, and in specific embodiments it can be 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, or 1:2.

[0121] In this invention, the temperature of the fifth substitution reaction is preferably room temperature; the time of the fifth substitution reaction is preferably 10 to 30 hours, and in specific embodiments it can be 10 hours, 15 hours, 20 hours, 25 hours or 30 hours.

[0122] After completing the fifth substitution reaction, the present invention preferably further includes: adding water to the reaction system obtained by the fifth substitution reaction, separating the phases to obtain an organic phase and an aqueous phase, extracting the aqueous phase with dichloromethane to obtain a dichloromethane phase; combining the organic phase and the dichloromethane phase, washing with saturated brine, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure to constant weight to obtain compound 1, which is then directly proceeded to the next reaction without purification.

[0123] After obtaining compound 1, the present invention performs a hydrolysis reaction on compound 1 to obtain compound 2.

[0124] In this invention, the hydrolysis reaction is preferably carried out under alkaline conditions. The alkaline condition preferably includes an alkali metal hydroxide, more preferably NaOH and / or KOH. The alkaline is preferably used in the form of an alkaline aqueous solution, and the concentration of the alkaline aqueous solution is preferably 2-3 mol / L. In specific embodiments, it can be 2.1 mol / L, 2.2 mol / L, 2.3 mol / L, 2.4 mol / L, 2.5 mol / L, 2.6 mol / L, 2.7 mol / L, 2.8 mol / L, 2.9 mol / L, or 3 mol / L. The alkaline aqueous solution is preferably added dropwise under ice bath conditions.

[0125] In this invention, the molar ratio of the 2-hydroxycarboxylic acid ester to the base is preferably 1:1.5 to 2, and in specific embodiments it can be 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2.

[0126] In this invention, the hydrolysis reaction temperature is preferably room temperature; the hydrolysis reaction time is preferably 2 to 3 hours, and in specific embodiments it can be 2 hours, 2.5 hours or 3 hours.

[0127] After the hydrolysis reaction is completed, the present invention preferably further includes: under ice bath conditions, adding concentrated hydrochloric acid to the reaction system obtained by the hydrolysis reaction to adjust the pH value to 2-3, extracting three times with ethyl acetate, combining the organic phases, washing with saturated brine, drying with anhydrous sodium sulfate, filtering, and concentrating the filtrate under reduced pressure to obtain compound 2.

[0128] After obtaining compound 2, the present invention performs a substitution reaction (referred to as the sixth substitution reaction) on compound 2 with thionyl chloride to obtain compound 3.

[0129] In this invention, the molar ratio of compound 2 to thionyl chloride is preferably 1:1 to 5, and in specific embodiments it can be 1:1, 1:2, 1:3, 1:4 or 1:5; the thionyl chloride is preferably added dropwise.

[0130] In this invention, the sixth substitution reaction is preferably carried out in the presence of an organic solvent (denoted as the seventh organic solvent). In this invention, the seventh organic solvent preferably includes dichloromethane and / or chloroform. This invention does not have a particular limitation on the amount of the seventh organic solvent, as long as it is sufficient to ensure the smooth progress of the sixth substitution reaction.

[0131] In this invention, the temperature of the sixth substitution reaction is preferably 65-85°C, and in specific embodiments it can be 65°C, 70°C, 75°C, 79°C, 80°C or 85°C; the time of the sixth substitution reaction is preferably 2-8h, and in specific embodiments it can be 2h, 3h, 4h, 5h, 6h, 7h or 8h.

[0132] After completing the sixth substitution reaction, the present invention preferably further includes: removing unreacted raw materials (e.g., thionyl chloride) from the reaction system obtained by the sixth substitution reaction under reduced pressure to obtain compound 3, which is then directly carried out in the next reaction without purification.

[0133] After obtaining compound 3, the present invention performs a substitution reaction (referred to as the seventh substitution reaction) on compound 3 with N-methyl-4-nitroaniline to obtain compound 4.

[0134] In this invention, the molar ratio of compound 3 to N-methyl-4-nitroaniline is preferably 1 to 1.2:1, and in specific embodiments it can be 1:1, 1.05:1, 1.1:1, 1.15:1 or 1.2:1.

[0135] In this invention, the substitution-transconfiguration reaction is preferably carried out in the presence of a catalyst (denoted as the second catalyst), an acid remover (denoted as the third acid remover), and an organic solvent (denoted as the eighth organic solvent). In this invention, the second catalyst preferably comprises N,N-dimethylaminopyridine (DMAP) and / or pyridine; the molar ratio of N-methyl-4-nitroaniline to the second catalyst is preferably 1:0.05 to 0.2, and in specific embodiments it can be 1:0.05, 1:0.1, 1:0.13, 1:0.15, or 1:2. In this invention, the third acid-removing agent preferably comprises an organic amine, and in specific embodiments may include triethylamine and / or N-ethyl-N,N-diisopropylamine; the molar ratio of N-methyl-4-nitroaniline to the third acid-removing agent is preferably 1:1 to 2, and in specific embodiments may be 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, or 1:2. In this invention, the eighth organic solvent preferably comprises one or more of anhydrous acetonitrile, tetrahydrofuran, and dichloromethane; this invention does not have a special limitation on the amount of the eighth organic solvent, as long as it is sufficient to ensure the smooth progress of the substitution-transconfiguration reaction.

[0136] In this invention, the temperature of the substitution-transconfiguration reaction is preferably 15-45°C, and in specific embodiments it can be 15°C, 20°C, 25°C, 30°C, 35°C, 40°C or 45°C; the time of the substitution-transconfiguration reaction is preferably 2-3 hours, and in specific embodiments it can be 2 hours, 2.5 hours or 3 hours.

[0137] After completing the substitution-transconfiguration reaction, the present invention preferably further includes: removing the solvent under reduced pressure, adding water to separate the layers, obtaining an organic phase and an aqueous phase respectively; extracting the aqueous phase with dichloromethane to obtain a dichloromethane phase; combining the organic phase and the dichloromethane phase, washing with saturated brine, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, and purifying by silica gel column chromatography to obtain compound 4. In the present invention, the eluent used for the silica gel column chromatography purification preferably includes petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is preferably 4:1 to 1:1. In specific embodiments, the volume ratios of petroleum ether to ethyl acetate are 4:1 and 1:1, respectively.

[0138] After obtaining compound 4, the present invention performs a nucleophilic substitution-transconfiguration reaction on compound 4 with HR3 to obtain chiral intermediate 2.

[0139] In this invention, the molar ratio of compound 4 to HR3 is preferably 1:1 to 3, and in specific embodiments it can be 1:1, 1:1.5, 1:2, 1:2.5 or 1:3.

[0140] In this invention, the nucleophilic substitution-transconfiguration reaction is preferably carried out in the presence of an acid-scavenging agent (denoted as the fourth acid-scavenging agent) and an organic solvent (denoted as the ninth organic solvent). In this invention, the fourth acid-scavenging agent preferably comprises an organic amine, more preferably triethylamine and / or N-ethyl-N,N-diisopropylamine; the molar ratio of compound 4 to the fourth acid-scavenging agent is preferably 1:1 to 3.5, and in specific embodiments can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, or 1:3.5. In this invention, the ninth organic solvent preferably comprises acetonitrile and / or dioxane; this invention does not have a special limitation on the amount of the ninth organic solvent, as long as it ensures the smooth progress of the nucleophilic substitution-transconfiguration reaction.

[0141] In this invention, the temperature of the nucleophilic substitution-transconfiguration reaction is preferably 80-100°C, and in specific embodiments it can be 80°C, 85°C, 90°C, 95°C or 100°C; the time of the nucleophilic substitution-transconfiguration reaction is preferably 10-24h, and in specific embodiments it can be 10h, 12h, 14h, 16h, 18h, 20h, 22h or 24h.

[0142] After completing the nucleophilic substitution-transconfiguration reaction, the present invention preferably further includes: removing the solvent from the reaction system obtained by the nucleophilic substitution-transconfiguration reaction under reduced pressure, and purifying it by silica gel column chromatography to obtain racemic intermediate 2. In the present invention, the eluent used for the silica gel column chromatography purification preferably includes ethyl acetate and a dichloromethane-methanol mixed solvent in sequence, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solvent is preferably 4 to 10:1, and in specific embodiments it can be 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0143] After obtaining chiral intermediate 2, the present invention performs a hydrogenation reduction reaction on chiral intermediate 2 (denoted as the second hydrogenation reduction reaction) to obtain chiral intermediate 3. In the present invention, the conditions of the second hydrogenation reduction reaction are the same as those of the first hydrogenation reduction reaction, and will not be described in detail here.

[0144] In this invention, the preparation method of intermediate 4 preferably includes the following steps: condensing N-acetylindolone and triethyl orthobenzoate to obtain intermediate 4.

[0145] In this invention, the preparation route of intermediate 4 is as follows:

[0146]

[0147] In this invention, the molar ratio of N-acetylindolone to triethyl orthobenzoate is preferably 1:1 to 3.5, and in specific embodiments it can be 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4 or 1:3.5.

[0148] In this invention, the condensation reaction is preferably carried out in the presence of acetic anhydride and under a protective atmosphere. In this invention, the molar ratio of N-acetylindolone to acetic anhydride is preferably 1:15 to 25, and in specific embodiments it can be 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, or 1:25. In this invention, the protective atmosphere preferably includes nitrogen, argon, or helium.

[0149] In this invention, the temperature of the condensation reaction is preferably 100-135°C, and in specific embodiments it can be 100°C, 105°C, 110°C, 115°C, 120°C, 125°C, 130°C or 135°C; the time of the condensation reaction is preferably 1-4 hours, and in specific embodiments it can be 1 hour, 2 hours, 3 hours or 4 hours.

[0150] After the condensation reaction is completed, the present invention preferably further includes: cooling the reaction system obtained by the condensation reaction to room temperature, precipitating a solid, adding petroleum ether, stirring in an ice bath, filtering under reduced pressure, washing with petroleum ether, rinsing the filter cake with petroleum ether, and drying to constant weight to obtain intermediate 4.

[0151] The present invention also provides halogenated indole ketone derivatives, including pharmaceutically acceptable salts and / or solvates of the halogenated indole ketone compounds described in the above technical solutions.

[0152] In this invention, the pharmaceutically acceptable salt preferably includes salts of organic acids or salts of inorganic acids; the organic acids preferably include methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, 1-naphthalenesulfonic acid, 2-naphthalenesulfonic acid, acetic acid, trifluoroacetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid, or mandelic acid; the inorganic acid salt preferably includes hydrochloric acid, hydrobromic acid, sulfuric acid, or phosphoric acid.

[0153] The present invention does not impose any particular limitation on the preparation method of pharmaceutically acceptable salts of the haloindolone compounds, and any salt-forming method well known to those skilled in the art can be used.

[0154] In this invention, the solvate preferably comprises a hydrate.

[0155] The present invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients; said active ingredient comprising one or more of the halogenated indolone compounds and halogenated indolone derivatives described in the above-described technical solutions.

[0156] In this invention, the dosage form of the pharmaceutical composition preferably includes injection, tablet, capsule, pill, suspension or emulsion; the administration method of the pharmaceutical composition preferably includes oral, spray or injection, and the injection preferably includes subcutaneous injection, intravenous injection or intramuscular injection.

[0157] This invention does not specifically limit the pharmaceutically acceptable excipients used; any pharmaceutically acceptable excipient well known to those skilled in the art can be used, specifically one or more of the following: solubilizers, antioxidants, osmotic pressure regulators, suspending agents, fillers, disintegrants, wetting agents, and lubricants. In this invention, the solubilizer preferably includes one or more of polysorbate 80, lecithin, and polyethylene glycol; the antioxidant preferably includes vitamin C; the osmotic pressure regulator preferably includes sodium chloride and / or glucose; the suspending agent preferably includes glycerin; the filler preferably includes one or more of microcrystalline cellulose, starch, pregelatinized starch, calcium hydrogen phosphate, and calcium sulfate; the disintegrant preferably includes one or more of sodium carboxymethyl starch, low-substituted hydroxypropyl methylcellulose, croscarmellose sodium, sodium carboxymethyl cellulose, and croscarmellose; the wetting agent preferably includes a 50-75 v / v% aqueous ethanol solution; and the lubricant preferably includes one or more of sodium stearate fumarate, magnesium stearate, calcium stearate, or silica.

[0158] In this invention, the mass content of the active component in the pharmaceutical composition is preferably 1-90%, and in specific embodiments it can be 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%.

[0159] This invention also provides the application of the halogenated indole ketone compounds or the halogenated indole ketone derivatives described in the above-mentioned technical solutions in the preparation of drugs for kinase-mediated diseases; the kinases include one or more of the following: tyrosine protein kinase receptor, serine / threonine protein kinase, tyrosine protein kinase, vascular endothelial growth factor receptor 2, platelet-derived growth factor receptor-α, fibroblast growth factor receptor 1, neurotrophic factor receptor 2, non-receptor tyrosine kinase, tyrosine protein kinase, 5'-AMP-activated protein kinase subunit α1 / β1 / γ1, glycogen synthase kinase-3β, ribosomal protein S6 kinase α-1, tyrosine protein kinase, bispecific mitogen-activated protein kinase kinase 1, aurora kinase B, bispecific protein kinase, epidermal growth factor type A receptor 2, insulin-like growth factor 1 receptor, epidermal growth factor type B receptor 4, mitogen-activated protein kinase kinase 1, serine / threonine protein kinase, nonspecific serine / threonine protein kinase, nonspecific serine / threonine protein kinase, and glucokinase.

[0160] In this invention, the drug preferably includes one or more of the following: antitumor drugs, anti-inflammatory drugs, antihypertensive drugs, drugs for treating cardiovascular diseases, drugs for treating diabetes, and drugs for treating autoimmune diseases. In this invention, the tumor preferably includes one or more of the following: pancreatic cancer, colorectal cancer, lung cancer, liver cancer, and stomach cancer.

[0161] The halogenated indole ketone compounds provided by this invention, through kinase spectrum analysis, show that in addition to the VEGFR, FGFR, and PDGFR kinase inhibitory activities possessed by the lead compound nintedanib, they also exhibit strong AXL kinase inhibitory activity, IC50. 50 The value reached a low nanomolar level, which is 45 times more potent than nintedanib in inhibiting AXL kinase activity. The halogenated indole ketones provided by this invention exhibit better antitumor activity than nintedanib at both the cellular and in vivo levels, and demonstrate better inhibition of tumor invasion and metastasis. Furthermore, the halogenated indole ketones provided by this invention show inhibitory activities exceeding 90% against serine / threonine protein kinase (QIK), tyrosine protein kinase (LCK), platelet-derived growth factor receptor-α (PDGFRα), fibroblast growth factor receptor 1 (FGFR1), neurotrophic factor receptor 2 (TRKB), non-receptor tyrosine kinase (SRC), and tyrosine protein kinase (ABL1). These high kinase inhibitory activities effectively treat malignant tumors and their invasion and metastasis, demonstrating significant potential for application in the preparation of drugs for kinase-mediated diseases (especially tumors).

[0162] To further illustrate the present invention, the following detailed descriptions, in conjunction with embodiments, illustrate the halogenated indole ketone compounds and their preparation methods and applications, halogenated indole ketone derivatives and their applications, and pharmaceutical compositions and their applications. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0163] Example 1

[0164]

[0165] (1) Synthesis of intermediate 1: 2-Bromopropionic acid (1.51 g, 10 mmol) was dissolved in 3 mL of thionyl chloride and refluxed at 80 °C for 5 h. The remaining thionyl chloride was removed under reduced pressure, and the crude product was directly dissolved in 25 mL of acetonitrile to obtain a 0.4 M (mol / L) 2-bromopropionyl chloride solution. N-methyl-4-nitroaniline (10 mmol, 1.52 g), DMAP (1 mmol, 0.122 g), anhydrous acetonitrile (10 mL), and triethylamine (1.5 mL) were added. The 2-bromopropionyl chloride solution was added using a syringe under ice bath conditions, and the reaction was carried out at room temperature for 2–3 h. The reaction was confirmed to be complete by TLC. The solvent was removed under reduced pressure, 5 mL of water was added, and the phases were separated to obtain an organic phase and an aqueous phase. The aqueous phase was extracted twice with dichloromethane to obtain a dichloromethane phase. The organic phase and the dichloromethane phase were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate volume ratio of 4:1 and 1:1, respectively) to obtain intermediate 1 with a yield of 75%.

[0166] (2) Synthesis of intermediate 2: Intermediate 1 (5 mmol, 1.43 g) was dissolved in 15 mL of 1,4-dioxane, and N-ethylpiperazine (7.5 mmol, 0.86 g) and diisopropylethylamine (10 mmol) were added. The reaction was heated to 100 °C for 16 h, and the reaction was confirmed to be complete by TLC. The solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (eluting successively with ethyl acetate and dichloromethane:methanol in a volume ratio of 5:1) to obtain intermediate 2 in 46% yield. 1 H NMR(400MHz,Chloroform-d)δ8.28(d,J=9.0Hz,2H),7.48(d,J=9.0Hz,2H),3.72(q,J=6.7Hz,1H),3.36 (s, 3H), 2.61 (d, J = 6.7Hz, 2H), 2.42 (p, J = 6.7, 6.3Hz, 8H), 1.17 (d, J = 6.7Hz, 3H), 1.09 (t, J = 7.2Hz, 3H).

[0167] (3) Synthesis of intermediate 3: Intermediate 2 (5 mmol, 1.6 g) was dissolved in anhydrous ethanol (20 mL), 10% palladium on carbon (215 mg) was added, hydrogen gas (0.101 MPa) was passed through, and the reaction was carried out at room temperature for 24 h. The reaction was detected by TLC to be complete. The mixture was filtered, and the solvent was removed from the filtrate under reduced pressure to obtain 1.3 g of brown oily product. 1H NMR(400MHz,Chloroform-d)δ8.28(d,J=9.0Hz,2H),7.48(d,J=9.0Hz,2H),3.36(s,3H ),3.31(m,1H),2.61(m,2H),2.42(m,8H),1.17(d,J=6.7Hz,3H),1.09(t,J=7.2Hz,3H).

[0168] (4) Synthesis of intermediate 4: 9 mL of acetic anhydride and 6-chloroacetylindolone (1.3 g, 5.6 mmol) were added sequentially, followed by the slow addition of triethyl orthobenzoate (3.8 g, 16.8 mmol). The reaction was carried out under nitrogen protection at 125 °C for 2 h (TLC monitoring, developing solvent: ethyl acetate: petroleum ether volume ratio = 1:4). After the reaction was complete, the mixture was cooled to room temperature, and a solid precipitated. Petroleum ether (40 mL) was added, and the mixture was stirred in an ice bath for 1 h. The mixture was then filtered under reduced pressure, washed with petroleum ether, and the filter cake was rinsed with petroleum ether (20 mL). The mixture was dried under vacuum at 45 °C to constant weight to obtain intermediate 4, a light yellow solid of 1.7 g, with a yield of 81%.

[0169] (5) Synthesis of target compound 1 (R1 is chlorine, R2 is methyl, R3 is N-ethylpiperazinyl): Intermediate 4 (1.27 mmol, 0.35 g) was dissolved in DMF (5 mL), and intermediate 3 (1.2 mmol, 0.438 g) was added. Under nitrogen protection, the mixture was heated to 80 °C and stirred for 1 h. Piperidine (0.22 mL) was added, and stirring was continued for 2 h. 10 mL of water was added to the reaction system to precipitate a solid. The solid was filtered, ground with 2 mL of methanol, filtered, and the filter cake was washed with 2 mL of methanol and 2 mL of ethyl acetate. The mixture was dried to constant weight to obtain target compound 1 (compound number X529 or 11b), a yellow solid with a yield of 38%. 1 H NMR (400MHz, DMSO-d6) δ11.97(s,1H),10.96(s,1H),7.67-7.54(m,3H),7.52(d,J=7.0H z,1H),7.47(d,J=7.1Hz,1H),7.16(d,J=8.5Hz,2H),6.89(d,J=2.0Hz,2H),6.87(s,1H) ,6.61(dd,J=8.4,2.0Hz,1H),5.74(d,J=8.3Hz,1H),4.11-3.67(m,1H),3.06(s,3H),2. 96(m,4H),2.78-2.53(m,4H),2.35(m,2H),1.20(t,J=7.2Hz,3H),0.95(d,J=6.5Hz,3H). 13C NMR (101MHz, DMSO) δ171.21,170.52,156.87,140.00,138.24,132.75,130.84,129.98,129.95,129.09,128.89,128.4 4,128.25,124.12,123.29,120.19,119.40,109.62,97.97,57.64,51.22,51.11,45.94,37.22,11.91,9.55.HR-ESI-MS for C 31 H 35 ClN5O2[M+H] + ,calculatedfor 544.2479,found,544.2474.

[0170] Example 2

[0171] Synthesis of target compound 2 (R1 is chlorine, R2 is methyl, R3 is 4-(N,N-dimethyl)aminopiperidinyl): Target compound 2 was prepared according to the method of Example 1, except that N-ethylpiperazine was replaced with 4-(N,N-dimethyl)aminopiperidinyl.

[0172]

[0173] Target compound 2: 1 H NMR (400MHz, DMSO-d6) δ7.54 (ddd, J=38.8, 16.0, 7.2Hz, 5H), 7.13 (d, J=8.1Hz, 2H) ,6.88(d,J=8.8Hz,3H),6.61(d,J=8.2Hz,1H),5.74(d,J=8.2Hz,1H),3.05(s,3H), 3.03-2.95(m,1H),2.80(m,1H),2.68(m,1H),2.60(s,6H),2.25(m,1H),2.20-2.08 (m,1H),2.06-1.93(m,1H),1.83(m,2H),1.54-1.34(m,2H),0.93(d,J=6.2Hz,3H). 13C NMR (101MHz, DMSO) δ171.60,170.38,156.76,140.35,138.09,132.70,130.86,129.97,129.91,129.05,128.90,128.38,12 8.24,124.13,123.97,123.27,120.19,119.39,109.57,97.88,62.70,57.88,48.14,46.56,37.21,26.51,11.86.HR-ESI-MS forC 32 H 37 ClN5O2[M+H] + ,calculated for 558.2636,found,558.2630.

[0174] Example 3

[0175] Synthesis of target compound 3 (R1 is chlorine, R2 is methyl, R3 is morpholino): Target compound 3 was prepared according to the method of Example 1, except that N-ethylpiperazine was replaced with morpholino.

[0176]

[0177] Target compound 3: 1 H NMR(500MHz,Chloroform-d)δ11.84(s,1H),8.87(s,1H),7.47(dq,J=16.0,7.4Hz,3 H),7.38(d,J=7.2Hz,1H),7.32(d,J=7.3Hz,1H),6.92(d,J=8.6Hz,2H),6.88(s,1H), 6.70(d,J=8.5Hz,2H),6.55(d,J=8.3Hz,1H),5.80(d,J=8.3Hz,1H),3.52(m,4H),3. 11(s,3H),3.02-2.94(m,1H),2.41(m,2H),2.23-2.10(m,2H),1.02(d,J=6.6Hz,3H). 13CNMR (126MHz, CDCl3) δ172.36,171.22,167.37,158.33,140.24,137.88,135.72,132.36,130.53,129.59,129.57,129.07,12 8.70,128.49,128.00,125.16,123.99,122.72,118.21,110.45,98.51,67.16,59.05,51.93,49.40,37.42,11.91.HR-ESI-MS forC 29 H 30 ClN4O3[M+H] + ,calculated for517.2006,found,517.2001.

[0178] Example 4

[0179] Synthesis of target compound 4 (R1 is chlorine, R2 is methyl, R3 is N-methylperiperazinyl): Target compound 2 was prepared according to the method of Example 1, except that N-ethylperiperazine was replaced with N-methylperiperazine.

[0180]

[0181] Target compound 4: 1 H NMR (500MHz, Methanol-d4) δ7.61(dq,J=10.5,5.8,4.2Hz,3H),7.49(t,J=9.2Hz,2H),7.15(d,J=8.5Hz,2H),6.94(dd,J=5.1,3.2Hz,3H),6.58(dd,J=8.4, 1.8Hz,1H),5.87(d,J=8.4Hz,1H),3.48-3.38(m,1H),3.33(s,3H),3.30-3.1 7(m,6H),3.06-2.96(m,2H),2.90(s,3H),1.95(m,2H),1.10(d,J=6.0Hz,3H). 13C NMR(126MHz,MeOD)δ170.90,156.90,137.53,132.69,130.18,129.67,129.35,129.12,128.67,128.57,127.68,127.22,124.8 5,123.81,122.74,120.05,119.38,109.31,98.10,58.29,57.97,54.76,50.68,44.18,43.56,42.52,36.36,24.74.HR-ESI-MS for C 31 H 35 ClN5O2[M+H] + ,calculated for 544.2479, found ,544.2474.

[0182] Example 5

[0183] Synthesis of target compound 5 (R1 is chlorine, R2 is hydrogen, R3 is N-methylpiperazinyl): Target compound 2 was prepared according to the method of Example 1, except that bromoacetic acid was used instead of 2-bromopropionic acid and N-methylpiperazine was used instead of N-ethylpiperazine.

[0184]

[0185] Target compound 5: 1 H NMR (400MHz, DMSO-d6) δ11.97(s,1H),10.87(s,1H),7.57(q,J=6.7,6.3Hz,3H),7.49(d,J=7.1Hz,2H),7.11(d,J=8.2Hz,2H),6 .88-6.82(m,3H),6.61(dd,J=8.4,2.0Hz,1H),5.72(d,J=8.3Hz,1H),3.06(s,3H),2.79-2.64(m,2H),2.28(m,8H),2.16(s,3H). 13 C NMR (126MHz, DMSO) δ170.53,168.99,156.96,140.19,138.16,132.76,130.82,129.95,128.95,128 .19,123.96,123.33,120.19,119.38,109.55,97.86,59.46,54.88,52.51,45.91,37.13.HR-ESI-MS for C 29 H 30 ClN5O2[M+H] +,calculated for516.2144,found,516.2161.

[0186] Example 6

[0187] Synthesis of target compound 6 (R1 is chlorine, R2 is hydrogen, R3 is N-ethylpiperazinyl): Target compound 2 was prepared according to the method of Example 1, except that bromoacetic acid was used instead of 2-bromopropionic acid.

[0188]

[0189] 1 H NMR (400MHz, DMSO-d6) δ11.99(s,1H),10.89(s,1H),7.58(q,J=6.9,6.4Hz,3H),7.49(d,J=6.9Hz,2H),7.12(d,J=8.2Hz,2H),6.9 0-6.79(m,3H),6.61(d,J=8.4Hz,1H),5.73(d,J=8.4Hz,1H),3.06(m,5H),2.81-2.33(m,8H),2.45(m,2H),1.09(t,J=7.1Hz,3H). 13 CNMR(101MHz,DMSO)δ170.54,168.69,156.84,138.20,132.78,130.86,129.98,128.94,128.24,123.8 6,123.31,120.20,119.39,109.59,97.97,51.74,51.54,50.95,46.16,37.16,29.47,10.80.HR-ESI-MS for C 30 H 32 ClN5O2[M+H] + ,calculated for530.2301,found,530.2317.

[0190] Example 7

[0191] Synthesis of target compound 7 (R1 is chlorine, R2 is hydrogen, R3 is N,N,N-trimethylethylenediamine): Target compound 2 was prepared according to the method of Example 1, except that bromoacetic acid was used instead of 2-bromopropionic acid and N,N,N-trimethylethylenediamine was used instead of N-ethylpiperazine.

[0192]

[0193] 1H NMR (400MHz, DMSO-d6) δ11.97(s,1H),10.87(s,1H),7.57(q,J=6.9,6.4Hz,3H),7.49(d,J=6.9Hz,2H),7.12(d,J=8.3Hz,2H),6.8 5(d,J=8.2Hz,3H),6.61(d,J=9.6Hz,1H),5.72(d,J=8.3Hz,1H),3.43(s,3H),3.01(m,4H),2.70(m,2H),2.50(s,6H),2.20(s,3H). 13 CNMR(101MHz,DMSO)δ170.54,168.83,156.96,140.22,138.19,137.57,132.78,130.79,129.92,128.96,12 8.20,123.93,123.34,120.18,119.38,109.57,97.90,70.26,66.54,59.89,53.28,46.11,37.22.HR-ESI-MS for C 29 H 32 ClN5O2[M+2H] + ,calculated for 519.2215, found ,519.2185.

[0194] Example 8

[0195] Synthesis of target compound 8 (R1 is chlorine, R2 is methyl, R3 is 4-hydroxyethylpiperazine): Target compound 2 was prepared according to the method of Example 1, replacing N-ethylpiperazine with 4-hydroxyethylpiperazine.

[0196]

[0197] Target compound 8: ¹H NMR (400MHz, DMSO-d6) δ 11.96 (s, 1H), 10.88 (s, 1H), 7.65–7.49 (m, 4H), 7.44 (d, J = 7.6 Hz, 1H), 7.12 (d, J = 8.3 Hz, 2H), 6.92–6.82 (m, 3H), 6.61 (dd, J = 8.4, 2.0 Hz, 1H), 5.74 (d, J = 8.3 Hz, 1H), 3.48 (m, 2H), 3.05 (s, 3H), 3.00 (d, J = 6.4 Hz, 1H), 2.46–2.25 (m, 8H), 2.10 (m, 2H), 0.92 (d, J = 6.4 Hz, 3H). 13C NMR (101MHz, DMSO) δ171.55,170.55,157.00,140.35,138.19,137.90,132.74,130.77,129.94,129.13,128.81,128.38 ,128.20,124.07,123.33,120.18,119.38,109.56,97.87,60.42,58.57,57.95,53.72,48.09,37.15,11.70.HR-ESI-MS for C 31 H 34 ClN5O3[M+H] + ,calculated for 560.2423,found,560.2421.

[0198] Example 9

[0199] Synthesis of target compound 9 (R1 is bromine, R2 is methyl, R3 is N-methylpiperazinyl): Target compound 2 was prepared according to the method of Example 1, except that N-methylpiperazine was used instead of N-ethylpiperazine and 6-bromo-N-acetylindolone was used instead of 6-chloro-N-acetylindolone.

[0200]

[0201] Target compound 9: 1 H NMR (400MHz, Methanol-d4) δ7.59 (dd, J=14.8, 6.7Hz, 3H), 7.45 (dd, J=15.4, 7.0 Hz,2H),7.19-7.03(m,3H),6.91(d,J=8.2Hz,2H),6.71(d,J=8.1Hz,1H),5.81(d ,J=8.3Hz,1H),3.16(s,3H),3.15-3.12(m,1H),2.84-2.76(m,2H),2.71-2.60(m ,2H),2.57(s,3H),2.49-2.38(m,2H),1.87-1.76(m,2H),1.09(d,J=6.5Hz,3H). 13C NMR(101MHz,MeOD)δ172.69,170.71,157.14,139.53,138.45,137.73,132.66,130.18,129.33,128.68,128.52,127.80,12 3.87,123.20,122.93,119.72,116.54,112.16,98.00,58.35,54.09,44.32,43.31,36.37,22.37,21.68,11.48.HR-ESI-MS for C 30 H 33 BrN5O2[M+H] + ,calculated for 574.1818, found ,574.1812.

[0202] Example 10

[0203] Synthesis of target compound 10 (R1 is bromine, R2 is hydrogen, R3 is N-ethylpiperazinyl): Target compound 2 was prepared according to the method of Example 1, except that 2-bromopropionic acid was replaced with 2-bromoacetic acid and 6-chloro-N-acetylindolone was replaced with 6-bromo-N-acetylindolone.

[0204]

[0205]

[0206] Target compound 10: 1 H NMR(400MHz,DMSO-d6)δ11.99(s,1H),10.88(s,1H),7.67-7.53(m,3H),7.4 9(dd,J=7.9,1.6Hz,2H),7.12(d,J=8.2Hz,2H),6.99(d,J=1.9Hz,1H),6.85 (d,J=8.5Hz,2H),6.74(dd,J=8.3,1.9Hz,1H),5.67(d,J=8.3Hz,1H),3.05( s,3H),2.75(s,2H),2.68-2.53(m,2H),2.39(m,8H),1.02(t,J=7.1Hz,3H). 13C NMR (126MHz, DMSO) δ170.37,168.88,157.11,138.37,132.71,130.88,129.98,128.89,128.22,123.97 ,123.66,123.01,119.81,116.26,112.36,97.91,70.22,58.89,52.01,51.69,37.18,11.26.HR-ESI-MS for C 30 H 32 BrN5O2[M+2H] + ,calculated for 573.1739, found ,573.1748.

[0207] Example 11

[0208] Synthesis of target compound 11 (R1 is bromine, R2 is hydrogen, R3 is 4-(N,N-dimethyl)aminopiperidinyl): Target compound 2 was prepared according to the method of Example 1, except that 2-bromopropionic acid was replaced with 2-bromoacetic acid, N-ethylpiperazine was replaced with 4-(N,N-dimethyl)aminopiperidinyl, and 6-chloro-N-acetylindolone was replaced with 6-bromo-N-acetylindolone.

[0209]

[0210] Target compound 11: 1 H NMR (400MHz, DMSO-d6) δ11.99 (s, 1H), 10.87 (s, 1H), 7.57 (q, J = 6.5Hz, 3H), 7.49(d,J=6.5Hz,2H),7.11(d,J=8.3Hz,2H),6.99(d,J=1.8Hz,1H),6.84(d ,J=8.4Hz,2H),6.73(dd,J=8.3,1.9Hz,1H),5.68(d,J=8.3Hz,1H),3.05(s, 3H),2.70(s,2H),2.59(m,2H),2.26(s,6H),2.17(m,1H),1.89-1.65(m,6H). 13C NMR (101MHz, DMSO) δ170.40,169.19,159.67,157.05,140.30,138.43,132.76,130.83,129.94,128.91,128 .01,123.70,122.98,119.79,116.21,112.29,97.91,61.95,59.43,52.32,41.36,37.18,27.86.HR-ESI-MS for C 31 H 34 BrN5O2[M+2H] + ,calculated for 589.1933, found ,589.2031.

[0211] Example 12

[0212] Synthesis of target compound 12 (R1 is bromine, R2 is hydrogen, R3 is morpholino): Target compound 2 was prepared according to the method of Example 1, except that 2-bromopropionic acid was replaced with 2-bromoacetic acid, N-ethylpiperazine was replaced with morpholino, and 6-chloro-N-acetylindolone was replaced with 6-bromo-N-acetylindolone.

[0213]

[0214] Target compound 12: 1 H NMR (400MHz, DMSO-d6) δ12.01(s,1H),10.87(s,1H),7.58(q,J=6.3Hz,3H),7.50(dd,J=7.6,1.9Hz,2H),7.11(d,J=8.3Hz,2H),7.00(d,J=1.9 Hz,1H),6.85(d,J=8.3Hz,2H),6.74(dd,J=8.4,1.9Hz,1H),5.67(d,J=8.4Hz,1H),3.51(s,3H),3.07(m,4H),2.90(s,2H),2.48-2.34(m,4H). 13 C NMR (101MHz, DMSO) δ170.39,169.85,156.95,138.44,132.76,130.87,130.00,128.88,128.11,123.77 ,123.67,122.99,119.80,116.27,112.35,98.02,70.25,56.44,53.48,44.94,42.63,37.23.HR-ESI-MS forC 28 H 27BrN4O3[M+2H] + ,calculated for548.1246,found,548.1256.

[0215] Example 13

[0216] Synthesis of target compound 13 (R1 is bromine, R2 is hydrogen, R3 is N-methylperiperazinyl): Target compound 2 was prepared according to the method of Example 1, except that 2-bromopropionic acid was replaced with 2-bromoacetic acid, N-ethylpiperazine was replaced with N-methylperiperazine, and 6-chloro-N-acetylindolone was replaced with 6-bromo-N-acetylindolone.

[0217]

[0218]

[0219] Target compound 13: 1 H NMR (400MHz, DMSO-d6) δ12.01(s,1H),10.87(s,1H),7.58(d,J=7.3Hz,3H),7.49(d,J=7.0Hz,2H),7.11(d,J=8.1Hz,2H),7.00(s,1H),6.84( d,J=8.3Hz,2H),6.74(d,J=8.2Hz,1H),5.67(d,J=8.3Hz,1H),3.06(s,3H),2.94(s,2H),2.47(m,4H),2.39(m,4H),2.23(s,3H),1.59(m,2H). 13 C NMR (101MHz, DMSO) δ170.38,157.05,138.41,132.77,130.85,129.97,128.90,128.08,123.71,12 2.99,119.80,116.20,112.29,97.91,58.16,56.41,53.67,53.58,46.68,37.23,27.28.HR-ESI-MS for C 30 H 32 BrN5O2[M+2H] + ,calculated for 575.1875, found ,575.1921.

[0220] Example 14

[0221] Synthesis of target compound 14 (R1 is bromine, R2 is hydrogen, R3 is N,N,N-trimethylethylenediamine): Target compound 2 was prepared according to the method of Example 1, except that 2-bromopropionic acid was replaced with 2-bromoacetic acid, N-ethylpiperazine was replaced with N,N,N-trimethylethylenediamine, and 6-chloro-N-acetylindolone was replaced with 6-bromo-N-acetylindolone.

[0222]

[0223] Target compound 14: 1 H NMR (400MHz, DMSO-d6) δ11.98(s,1H),10.86(s,1H),7.56(q,J=7.2,6.5Hz,3H),7.48(d,J=6.7Hz,2H),7.12(d,J=8.3Hz,2H),6.99(d,J=1.8Hz,1H) ,6.85(d,J=8.3Hz,2H),6.73(dd,J=8.4,1.9Hz,1H),5.67(d,J=8.3Hz,1H ),3.43(s,3H),3.06(m,3H),2.82-2.64(m,2H),2.50(s,6H),2.20(m,4H). 13 C NMR (101MHz, DMSO) δ170.39,168.84,157.13,140.26,138.41,132.75,130.81,129.93,128.92, 128.10,123.71,122.99,119.84,116.21,112.33,97.89,70.25,66.52,53.27,46.21.HR-ESI-MS for C 29 H 32 BrN5O2[M+H] + ,calculated for562.1812,found,562.1803.

[0224] Example 15

[0225] Synthesis of target compound 15 (R1 is bromine, R2 is hydrogen, R3 is 4-hydroxyethylpiperazine): Target compound 2 was prepared according to the method of Example 1, except that 2-bromoacetic acid was used instead of 2-bromopropionic acid; 4-hydroxyethylpiperazine was used instead of N-ethylpiperazine; and 6-bromo-N-acetylindolone was used instead of 6-chloro-N-acetylindolone.

[0226]

[0227] Target compound 15:1 H NMR (400MHz, DMSO-d6) δ11.98(s,1H),10.86(s,1H),7.57(t,J=7.4Hz,3H),7.48(d,J=6.5Hz,2H),7.11(d,J=8.4Hz,2H),6.99(s,1H),6.84( d,J=8.5Hz,2H),6.73(d,J=10.1Hz,1H),5.68(d,J=8.3Hz,1H),3.46(m,2H),3.45(s,3H),3.10(s,2H),2.79-2.64(m,2H),2.36-2.11(m,8H). 13 C NMR (126MHz, DMSO) δ170.37,157.15,140.20,138.38,132.72,130.86,129.96,128.91,128.16,123.98 ,123.69,123.00,119.80,116.21,112.32,97.87,60.43,59.50,58.66,53.38,52.56,37.18.HR-ESI-MS for C 30 H 32 BrN5O3[M+2H] + ,calculated for591.1824,found,591.1768.

[0228] Example 16

[0229] Synthesis of target compound 16 (R1 is bromine, R2 is methyl, R3 is 4-hydroxyethylpiperazinyl): Target compound 2 was prepared according to the method of Example 1, except that N-ethylpiperazine was replaced with 4-hydroxyethylpiperazinepiperazine and 6-chloro-N-acetylindolone was replaced with 6-bromo-N-acetylindolone.

[0230]

[0231] Target compound 16: 1H NMR (400MHz, DMSO-d6) δ11.98(s,1H),10.98(s,1H),7.59(p,J=7.8Hz,3H),7.4 9(dd,J=21.4,7.2Hz,2H),7.16(d,J=8.2Hz,2H),7.03(s,1H),6.88(d,J=8.3Hz ,2H),6.73(d,J=9.8Hz,1H),5.69(d,J=8.3Hz,1H),5.28(s,1H),3.74(m,2H),3 .23(t,J=7.4Hz,1H),2.95-2.79(m,2H),2.64(s,3H),2.51(m,8H),0.95(s,3H). 13 C NMR (126MHz, DMSO) δ171.19,170.34,157.04,140.10,138.51,138.10,132.73,130.86,129.97,129.08,128.86,128.44 ,124.19,123.66,122.96,119.80,116.24,112.37,97.95,58.42,57.66,52.50,52.31,45.84,37.22,11.74.HR-ESI-MS for C 31 H 34 BrN5O3[MH] - ,calculated for 602.1772,found,602.1750.

[0232] Example 17

[0233] Synthesis of target compound 17 (R1 is chlorine, R2 is methyl, R3 is N-ethylpiperazinyl): Target compound 2 was prepared according to the method of Example 1, the only difference from Example 1 being that the synthesis of intermediate 2 was as follows.

[0234]

[0235] Synthesis of Compound 2: (R)-methyl lactate (60 mmol, 6.24 g) was dissolved in dichloromethane (36 mL), and triethylamine (11 mL) was added. A solution of p-toluenesulfonyl chloride (68.9 mmol, 13.14 g) in dichloromethane (45 mL) was added dropwise with stirring in an ice bath. The reaction was stirred at room temperature for 20 h, and TLC analysis showed complete reaction. Water (20 mL) was added to the reaction solution, the organic phase was separated, and the aqueous layer was extracted with dichloromethane (30 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product (Compound 1), which was directly used for the next reaction. A solution of NaOH (103 mmol, 4.124 g) dissolved in 37 mL of water was added dropwise with stirring in an ice bath. After the addition was complete, the ice bath was removed, and the reaction was stirred at room temperature for 2-3 h. TLC analysis showed complete reaction. The pH was adjusted to 2-3 by adding (10 mL) concentrated hydrochloric acid under ice bath conditions. The mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to constant weight to give compound 2. Compound 2: 1 H NMR (400MHz, Chloroform-d) δ10.77(s,1H),7.82(d,J=8.2Hz,2H),7.36(d,J=8.2Hz,2H),4.97(q,J=7.0Hz,1H),2.45(s,3H),1.54(d,J=7.0Hz,3H).

[0236] Synthesis of Compound 4: Compound 2 (49.3 mmol, 12.038 g) was dissolved in 1,2-dichloroethane (49 mL), and thionyl chloride (11 mL) was added dropwise under ice bath conditions. The mixture was refluxed at 79 °C for 6–7 h, and the solvent was removed under reduced pressure to obtain Compound 3. Acetonitrile (15 mL) was added to obtain a solution of Compound 3, which was directly used in the next reaction. In a reaction flask, N-methyl-4-nitroaniline (36.2 mmol, 5.5 g), DMAP (4.83 mmol, 0.59 g), anhydrous acetonitrile (49 mL), and triethylamine (10 mL) were added. The above solution of Compound 3 was added using a syringe under ice bath conditions. The reaction was carried out at room temperature for 2–3 h, and the reaction was monitored by TLC until complete. The solvent was removed under reduced pressure, 20 mL of water was added, and the phases were separated to obtain an organic phase and an aqueous phase. The aqueous phase was extracted twice with dichloromethane to obtain a dichloromethane phase. The organic and dichloromethane phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether:ethyl acetate volume ratios of 4:1 and 1:1, respectively) to obtain compound 4 in 50% yield. Compound 4: 1H NMR(400MHz,Chloroform-d)δ8.32(d,J=8.9Hz,2H),7.74(d,J=8.3Hz,2H),7.44(d,J=8.9Hz,2H ),7.32(d,J=8.1Hz,2H),5.02(q,J=6.6Hz,1H),3.33(s,3H),2.44(s,3H),1.38(d,J=6.6Hz,3H).

[0237] Synthesis of intermediate 2: Compound 4 (5.3 mmol, 2 g) was dissolved in 15 mL of 1,4-dioxane, and N-ethylpiperazine (10 mmol) and diisopropylethylamine (18 mmol, 2.29 g) were added. The reaction was heated to 100 °C for 16 h, and the reaction was confirmed to be complete by TLC. The solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (eluents were ethyl acetate, dichloromethane:methanol, v / v = 5:1) to give intermediate 2 in 46% yield. 1 H NMR(400MHz,Chloroform-d)δ8.32(d,J=8.9Hz,2H),7.46(d,J=9.0Hz,2H),3.72(q,J=6.7Hz,1H) ,3.35(s,3H),3.06-2.84(m,6H),2.77-2.67(m,4H),1.20(d,J=6.7Hz,3H), 0.9(t,J=7.1Hz,3H).

[0238] Target compound 17: 1 H NMR (400MHz, DMSO-d6) δ11.97(s,1H),10.96(s,1H),7.67-7.54(m,3H),7.52(d,J=7.0H z,1H),7.47(d,J=7.1Hz,1H),7.16(d,J=8.5Hz,2H),6.89(d,J=2.0Hz,2H),6.87(s,1H) ,6.61(dd,J=8.4,2.0Hz,1H),5.74(d,J=8.3Hz,1H),4.11-3.67(m,1H),3.06(s,3H),2. 96(m,4H),2.78-2.53(m,4H),2.35(m,2H),1.20(t,J=7.2Hz,3H),0.95(d,J=6.5Hz,3H).

[0239] Test Example 1

[0240] Using target compound 1 (denoted as X529) as a representative compound, the inhibitory activity against 80 kinases and the IC50 of inhibition against AXL and KDR (VEGFR2) kinases were analyzed. 50 Value test.

[0241] Nintedanib is a well-known oral small-molecule triple tyrosine kinase inhibitor. To explore whether the target compound provided in this invention differs from the lead compound nintedanib in terms of its target site, the inhibitory activity of target compound 1 against 80 kinases was measured. The experimental procedures are as follows:

[0242] Transfer 250 nmol of the 20 μM compound solution to each well of a 384 reaction plate for later use. Add 250 nmol of 100% DMSO to each of the negative control and blank wells. Prepare a 2.5-fold final concentration of kinase solution using 1×Kinase buffer. Add 10 μol of the 2.5-fold final concentration of kinase solution to each of the compound and positive control wells; add 10 μol of 1×Kinase buffer to each of the negative control wells. Centrifuge at 1000 rpm for 30 seconds, vortex to mix, and incubate at room temperature for 10 minutes. Prepare a 25 / 15-fold final concentration mixture of ATP and Kinase substrate 2 using 1×Kinase buffer. Add 15 μol of the 25 / 15-fold final concentration mixture of ATP and substrate to initiate the reaction. Centrifuge the 384-well plate at 1000 rpm for 30 seconds, vortex to mix, and incubate at room temperature for 60 minutes. Add 30 μL of stop assay solution to halt the kinase reaction, centrifuge at 1000 rpm for 30 seconds, and vortex to mix. Read the conversion rate using a Caliper EZ Reader II (microfluidic chip analyzer). Each kinase has duplicate wells, and the inhibition rate of each well is calculated using the following formula: Inhibition rate = (Compound well conversion rate - Blank conversion rate) / (Negative control conversion rate - Blank conversion rate) × 100%.

[0243] IC 50 The experimental design for the test is as follows:

[0244] Prepare 100-fold final concentration solutions of the compound in 384-well plates, starting at 1 μM and diluting 3-fold to create 10 concentration gradients. Using a pipette, transfer 250 nmol to each well of the 384-well plate according to the established protocol. Add 250 nmol of 100% DMSO to each negative control and blank control well. Prepare a 2.5-fold final concentration kinase solution using 1×Kinase buffer. Add 10 μL of the 2.5-fold final concentration kinase solution to each compound and positive control well; add 10 μL of 1×Kinase buffer to each negative control well. Centrifuge at 1000 rpm for 30 seconds, vortex to mix, and incubate at room temperature for 10 min. Prepare a 25 / 15-fold final concentration mixture of ATP and Kinase substrate 2 using 1×Kinase buffer. Add 15 μL of the 25 / 15-fold final concentration ATP and substrate mixture to initiate the reaction. Centrifuge the 384-well plate at 1000 rpm for 30 seconds, vortex to mix, and incubate at room temperature for 60 minutes. Add 30 μL of stop assay solution to stop the kinase reaction, centrifuge at 1000 rpm for 30 seconds, and vortex to mix. Read the conversion rate using Caliper EZ Reader II. After data processing, plot the logarithm of concentration on the X-axis and the percentage inhibition rate on the Y-axis. Use the log(inhibitor) vs. response-variable slope function of GraphPadPrism 8 software to fit dose-response curves, thereby obtaining the IC50 of each compound on the enzyme activity. 50 Value. Calculation formula: Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * Hillslope)). Test results are shown in Table 1 and... Figure 1 As shown.

[0245] Figure 1 The graph shows the inhibitory activity of target compound 1 (X529) prepared in Example 1 against 80 kinases.

[0246] Table 1 lists 25 kinases with inhibition rates greater than 50% among the 80 kinases measured by X529 (200 nM).

[0247]

[0248] Table 1 shows the data on the inhibition rate of target compound 1 against 80 kinases greater than 50%. Figure 1As shown in Table 1, compared with the kinase targets of nintedanib reported in the literature (J.Med.Chem.,2015,58,1053-1063.), target compound 1 not only showed good inhibitory activity against KDR, PDGFR and FGFR, but also showed good inhibitory activity against kinases AXL, QIK, TRKB, SRC and ABL1.

[0249] According to the data published by the original research company, nintedanib showed only weak inhibitory activity against AXL. Given that AXL kinase is overexpressed or activated in various cancers and is a significant factor contributing to resistance to chemotherapy and receptor tyrosine kinase inhibitors (TKIs), further quantitative analyses of AXL and KDR kinases by target compounds 1, 9, and 17 were performed, with nintedanib as a positive control. The results are shown in Table 2.

[0250] Table 2. Quantitative inhibitory activity tests of halogenated indole ketones and nintedanib against AXL and KDR kinases.

[0251] Test compound <![CDATA[IC of AXL kinase inhibitory activity 50 (nM)]]> <![CDATA[IC of KDR kinase inhibitory activity 50 (nM)]]> Target compound 1 3.75 1.525 Target compound 9 4.31 1.613 Target compound 17 2.91 1.455 Nidanip 169.7 1.046

[0252] Table 2 shows that the IC50 of target compound 1 against AXL kinase is... 50 The value is 3.75 nM, while the IC of Nydanib is 3.75 nM. 50 The concentration was 169.7 nM, with a difference of over 45-fold between the two. Furthermore, the inhibitory activity of target compound 1 and nintedanib against KDR was determined, showing similar results (IC50, IC50). 50 The values ​​were 1.525 nM and 1.046 nM, respectively. These results indicate that the KDR inhibitory activity of target compound 1 is similar to that of nintedanib, but it exhibits good inhibitory activity against AXL kinase, warranting further investigation.

[0253] Test Example 2

[0254] The halogenated indole ketone compounds prepared in the examples exhibited inhibitory activity (IC50) against tumor cells and tumor-associated fibroblasts. 50 Tests were conducted at μM.

[0255] The mouse fibroblast cell line (NIH3T3 cells, derived from Nanjing Kebai Biotechnology Co., Ltd.) was used. Its anti-fibroblast proliferation activity was tested using the CCK8 assay, with nintedanib as a positive control. The specific experimental steps are as follows: NIH3T3 cells were cultured in high-glucose DMEM containing 10 v / v% FBS, 1 v / v% penicillin, and streptomycin, and incubated at 37°C in a 5% CO2 saturated humidity incubator. The culture medium was replaced with fresh medium every other day. When the cells grew to cover 80-90% of the bottom wall of the culture flask, they were digested with 0.25% trypsin and passaged at a ratio of 1:3 to 1:5. Cells in the logarithmic growth phase were collected, and the cell suspension concentration was adjusted to 5 × 10⁶ cells / mL. 3 Cells were seeded per well into 96-well plates and incubated at 37°C with 5% CO2 for 12 hours. After cell attachment, the old culture medium was discarded, and FBS-free medium was added. Six concentration gradients were prepared for each compound, with three replicates for each concentration. After adding the medium, the plates were incubated for 48 hours. Then, 10 μL of CCK8 solution was added to each well in the dark, and the plates were incubated in a CO2 incubator for another 2–4 hours. The absorbance (OD value) at 450 nM was measured using a microplate reader. Finally, the data were analyzed, and the inhibition rate at each concentration was calculated using the following formula:

[0256]

[0257] The half-maximal inhibitory concentration (IC50) was calculated using GraphPadPrism 5.02 software. Each experiment was independently repeated three times, and the average value was taken to obtain the final data. The results are shown in Table 3.

[0258] Table 3. Inhibitory activity of halogenated indole ketones against tumor cells and tumor-associated fibroblasts (IC50) 50 (μM)

[0259] Example NIH / 3T3 HUVEC Bxpc-3 MM231 Example NIH / 3T3 HUVEC Bxpc-3 MM231 Example 1 1.136 0.426 1.01 1.162 Example 10 3.198 1.571 3.144 3.972 Example 2 0.983 0.827 2.04 1.33 Example 11 >10 >10 >10 >10 Example 3 >10 >10 >10 >10 Example 12 2.314 1.935 3.544 4.271 Example 4 2.202 0.692 1.74 2.04 Example 13 >10 >10 >10 >10 Example 5 3.109 1.796 2.98 4.54 Example 14 5.983 3.192 3.626 3.862 Example 6 1.951 1.851 1.78 3.190 Example 15 5.891 4.713 5.912 6.413 Example 7 4.519 4.983 3.14 3.87 Example 16 2.109 1.984 2.144 2.256 Example 8 2.197 1.648 1.54 1.35 Example 17 1.681 0.435 1.071 1.135 Example 9 2.1405 0.952 1.89 1.44 Nidanip 4.343 1.686 5.34 >10

[0260] As shown in Table 3, compared with the lead compound nintedanib, the halogenated indole ketone compounds provided in this invention have better inhibitory activity against tumor cells such as pancreatic cancer cells Bxpc-3, triple-negative breast cancer cells MM231, and tumor-associated fibroblasts NIH / 3T3 due to changes in structure and target site.

[0261] Test Example 3

[0262] In vivo antitumor activity assay of target compound 1 (11b) in nude mice

[0263] The in vivo antitumor activity assay in nude mice was performed using BALB / c nude mice (5 weeks old, 18–20 g). Bxpc-3 pancreatic cancer cells (derived from Pronosei Biotechnology Co., Ltd.) cultured to passage 3–5 in the logarithmic growth phase were digested with trypsin and then prepared into a 1.0 × 10⁻⁶ medium. 7 Cell suspensions at a concentration of [number] cells / mL were injected subcutaneously into the right axilla of nude mice to establish a nude mouse Bxpc-3 pancreatic cancer xenograft model after tumor formation. When the tumor volume reached 100–150 mm², [further details needed]. 3 Mice were randomly divided into four groups (control group, nintedanib-50 mg / kg, 11b-25 mg / kg, and 11b-50 mg / kg group), with six mice in each group. Mice were orally administered the test compound once daily at the set dose for 14 consecutive days. Figure 2 The graph shows the changes in tumor volume in mice after treatment with the blank group, nintedanib group, and the target compound group prepared in Example 1 during the in vivo anti-pancreatic cancer activity. It can be seen that the tumor inhibition rate (TGI, calculated by volume) of target compound 1 at a dose of 25 mg / kg was 83.03%, and the TGI of nintedanib at a dose of 50 mg / kg was 68.10%. When the dose of target compound 1 was increased to 50 mg / kg, the TGI increased to 96.71%.

[0264] After euthanizing mice, fluid was found in tumor vesicles during tumor resection, especially in the control group and nintedanib group. Figure 3 The graph shows the changes in tumor weight in mice under the action of the blank group, nintedanib group and the target compound group prepared in Example 1 during the process of anti-pancreatic cancer activity. It can be seen that after removing the fluid from the tumor vesicles, the TGI (based on tumor weight) of target compound 1 was 56.46% and 71.00% at doses of 25 mg / kg and 50 mg / kg, respectively, while the TGI of nintedanib was 55.84% at a dose of 50 mg / kg.

[0265] The differences in weight and volume of TGI were thought to be related to the exudation of the interstitial fluid. Throughout the administration period, mice in the nintedanib group experienced weight loss, and one mouse died on day eleven. Mice in the control group and the target compound 1 group showed no significant changes in weight, and their behavior was normal.

[0266] Test Example 4

[0267] Target compound 1 (11b) inhibited lung metastasis of tumors in nude mice.

[0268] After the tumor treatment in mice was completed, lung tissue from one nude mouse was randomly selected from each experimental group and observed after hematoxylin-eosin (H&E) staining. Figure 4H&E staining images (a) and Western blotting analysis images (b) of mouse tumor tissues prepared with the target compound in Example 1 for the control group, nintedanib group, and mice were provided. In a, 1, 2, 3, and 4 represent sites randomly selected from full-view lung sections of mice in each group, and their magnified microscopic images are also provided. Lung tissues from mice in the control and nintedanib groups showed fibrotic-like pulmonary nodules, which is inconsistent with the normal physiological structure of the lungs. Under high magnification, the pulmonary nodule cells showed diffuse expansion, densely stained nuclei, and a large number of cells, similar to cancer cells, confirming lung metastasis. In contrast, the low-concentration target compound 1 treatment group showed only a few small nodules, while the high-concentration group showed no obvious nodules, a significant difference from the control and nintedanib groups. Under high magnification, the alveolar septa of nude mice treated with 50 mg / kg halogenated indolone compounds were thinner, and there was no abnormal enlargement of alveoli or cell nuclei.

[0269] Tumor metastasis is always accompanied by epithelial-mesenchymal transition (EMT), a crucial stage in the invasive growth of tumor cells. Through EMT, tumor cells infiltrate the surrounding stroma, creating a microenvironment that promotes tumor growth and metastasis. Studies have shown that most signaling pathways associated with EMT are related to the downregulation of E-cadherin expression. E-cadherin is a calcium-dependent transmembrane glycoprotein expressed in most epithelial tissues; its reduced expression can decrease cell adhesion, making tumor cells more susceptible to migration and metastasis. To investigate the effect of the target compound on the expression of EMT-related proteins, two tumor tissues were randomly selected from each group of mice for Western blot analysis. Figure 4 The results in section b showed that E-cadherin expression disappeared in the control group and the nintedanib group, while there was no change in the halogenated indole ketone group. The loss of E-cadherin expression in the control group and the nintedanib group is consistent with tumor metastasis. Figure 4 The results in group 11b also showed that halogenated indolone compounds effectively inhibited AXL kinase phosphorylation in a dose-dependent manner. Compared with the control group and the nintedanib group, the phosphorylation expression of AXL kinase was significantly reduced in the 25 mg / kg 11b group, while the phosphorylation expression of AXL kinase was completely eliminated in the 50 mg / kg 11b group. This indicates that halogenated indolone compounds can prevent the reduction of E-cadherin by inhibiting AXL activation, thereby inhibiting the EMT process.

[0270] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A haloindolone compound, characterized in that, This includes racemic compounds having the structure shown in Formula I or chiral compounds having the structure shown in Formula II: ； Among them, R1~R4 can have any of the following combinations: R1 is chlorine, R2 is methyl, and R3 is N-ethylpiperazinyl; R1 is chlorine, R2 is methyl, and R3 is 4-(N,N-dimethyl)aminopiperidinyl; R1 is chlorine, R2 is methyl, and R3 is N-methylperiperazinyl; R1 is chlorine, R2 is methyl, and R3 is 4-hydroxyethylpiperazinyl; R1 is bromine, R2 is methyl, and R3 is N-methylpiperazinyl; R1 is bromine, R2 is hydrogen, and R3 is N-ethylpiperazinyl; R1 is bromine, R2 is hydrogen, and R3 is morpholino. R1 is bromine, R2 is methyl, and R3 is 4-hydroxyethylpiperazinyl; R1 is chlorine, R4 is methyl, and R3 is N-ethylpiperazinyl; In Equation II, the wavy line represents the R configuration or the S configuration.

2. The method for preparing the haloindolone compound according to claim 1, characterized in that, Includes the following steps: Intermediate 3 and intermediate 4 were subjected to a substitution reaction to obtain the haloindolone compound; The intermediate 3 includes a racemic intermediate 3 or a chiral intermediate 3; 。 3. The preparation method according to claim 2, characterized in that, The preparation method of the racemic intermediate 3 includes the following steps: The acyl chloride was substituted with N-methyl-4-nitroaniline to give intermediate 1; The intermediate 1 was subjected to a nucleophilic substitution reaction with HR3 to obtain racemic intermediate 2; The racemic intermediate 2 was subjected to a hydrogenation reduction reaction to obtain the racemic intermediate 3; ； Where X is a halogen.

4. The preparation method according to claim 2, characterized in that, The preparation method of the chiral intermediate 3 includes the following steps: The p-toluenesulfonyl chloride was substituted with a 2-hydroxycarboxylic acid ester to give compound 1; The compound 1 was subjected to a hydrolysis reaction to obtain compound 2; Compound 2 was subjected to a substitution reaction with thionyl chloride to obtain compound 3; Compound 3 was subjected to a substitution reaction with N-methyl-4-nitroaniline to obtain compound 4; Compound 4 was subjected to a nucleophilic substitution-transconfiguration reaction with HR3 to obtain chiral intermediate 2; The chiral intermediate 2 was subjected to a hydrogenation reduction reaction to obtain the chiral intermediate 3; ； The configuration of chiral intermediate 2 is different from that of 2-hydroxycarboxylic acid ester, compound 1, compound 2, compound 3 and compound 4.

5. The preparation method according to claim 2, characterized in that, The preparation method of the intermediate 4 includes the following steps: N-acetylindolone and triethyl orthobenzoate were condensed to give intermediate 4; 。 6. A haloindolone derivative, characterized in that, It is a pharmaceutically acceptable salt and / or solvate of the halogenated indolone compound of claim 1.

7. A pharmaceutical composition, characterized in that, It includes an active ingredient and pharmaceutically acceptable excipients; the active ingredient includes one or more of the haloindolone compounds of claim 1 and the haloindolone derivatives of claim 6.

8. The use of the haloindolone compound of claim 1 or the haloindolone derivative of claim 6 in the preparation of a medicament for kinase-mediated diseases; The kinases include one or more of the following: serine / threonine protein kinase, tyrosine protein kinase, vascular endothelial growth factor receptor 2, platelet-derived growth factor receptor-α, fibroblast growth factor receptor 1, neurotrophic factor receptor 2, non-receptor tyrosine kinase, 5'-AMP-activated protein kinase subunit α1 / β1 / γ1, glycogen synthase kinase-3β, ribosomal protein S6 kinase α-1, bispecific mitogen-activated protein kinase kinase 1, aurora kinase B, bispecific protein kinase, epidermal growth factor type A receptor 2, insulin-like growth factor 1 receptor, epidermal growth factor type B receptor 4, mitogen-activated protein kinase kinase 1, and glucokinase.

9. The application according to claim 8, characterized in that, The tyrosine protein kinase includes a tyrosine protein kinase receptor; The serine / threonine protein kinases include nonspecific serine / threonine protein kinases.

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