Structure of a class of egfr mutant covalent inhibitors containing indolinone structure and application thereof

Abstract

The application relates to a structure of an EGFR mutant covalent inhibitor containing an indole ketone structure and application thereof. The compound has a structure as shown in a general formula (I): the compound has simple synthesis process, high efficiency, and good in-vitro antitumor activity on a third-generation EGFR mutant H1975 cell.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, and in particular to the structure and application of a class of covalent inhibitors of EGFR mutants containing indole-3-methyl ... Background Technology

[0002] According to statistics from the World Health Organization, cancer has become the second leading cause of death worldwide. In 2018, 18.08 million new cancer cases were diagnosed, with lung cancer (2.09 million), breast cancer (2.09 million), colorectal cancer (1.8 million), prostate cancer (1.28 million), and skin cancer (non-melanoma, 1.04 million) being the five most common. The top five cancers causing death were lung cancer (1.76 million), colorectal cancer (862,000), stomach cancer (783,000), liver cancer (782,000), and breast cancer (627,000). The World Health Organization estimates that in the next 40 years, cancer deaths are projected to surpass those from ischemic heart disease.

[0003] Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein belonging to the ErbB receptor tyrosine kinase (RTK) family. The ErbB family of receptor tyrosine kinases consists of four distinct receptors: EGFR (also known as ErbB-1 / HER1), ErbB-2 (neu, HER2), ErbB-3 (HER3), and ErbB-4 (HER4). All proteins in this family possess an extracellular ligand-binding domain, a hydrophobic transmembrane domain, and a cytoplasmic tyrosine kinase domain. The intracellular tyrosine kinase domain of the ErbB receptor is highly conserved and lacks kinase activity. In contrast, the extracellular regions of the four receptors are less conserved, indicating different specificities in ligand binding.

[0004] ErbB receptors are activated by binding to growth factors of the EGF family. EGFR activation begins with ligand binding to the extracellular domain of the ErbB receptor, inducing EGFR dimerization, which stimulates its intracellular kinase domain. Receptor activation leads to autophosphorylation of specific tyrosine residues in the cytoplasmic tail. These phosphorylated residues are docking sites for proteins containing Src homology 2 (SH2) and phosphorylated tyrosine-binding (PTB) domains. Recruitment of these domains activates intracellular signaling pathways. This activation further recruits downstream signaling molecules that regulate EGFR function. It activates downstream signaling pathways, including the Ras / Raf / mitogen-activated protein kinase (MAPK) pathway and the PI3K-Akt pathway. Ras activation triggers a multi-step phosphorylation event, leading to MAPK activation, which in turn regulates gene transcription. EGFR is normally expressed in epithelial, mesenchymal, and neuronal tissues and is associated with cell proliferation, differentiation, and development.

[0005] Previous studies have shown that epidermal growth factor receptor (EGFR) is closely related to tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and apoptosis, making it an important target for the treatment of non-small cell lung cancer (NSCLC). Activating mutations in the EGFR tyrosine kinase domain have been identified as oncogenic drivers of NSCLC. EGFR mutations occur in exons 18-21, encoding its tyrosine kinase domain. In-frame deletions in exon 19 account for 44% of EGFR tyrosine kinase (TK) activating mutations, including amino acid residues Leu747 to G1u749. The major single-point mutation L858R in exon 21 accounts for 41% of EGFR TK activating mutations; G719S, G719A, and G719C activating mutations account for 10%; and duplication and / or insertion in exon 20 account for the remaining 5% of EGFR TK activating mutations.

[0006] In summary, lung cancer ranks second in both incidence and mortality among cancers worldwide, with non-small cell lung cancer (NSCLC) accounting for the largest proportion of all lung cancer patients. NSCLC accounts for approximately [percentage missing] of all lung cancer cases. [1-3] 80%-85% of NSCLC patients have epidermal growth factor receptor (EGFR) activating mutations, such as basal sequence deletion in exon 19 (Ex19del) or exon 21 (L858R). [4] Missense mutations in EGFR are a significant risk factor for NSCLC. Therefore, epidermal growth factor receptor tyrosine kinase (EGFR-TK) has become an important target for NSCLC treatment. To overcome the problems of abnormal pathway activation and drug resistance caused by EGFR protein mutations in vivo, four generations of EGFR tyrosine kinase inhibitors have been developed for clinical treatment.

[0007] This invention utilizes the structure of osimertinib compound and EGFRT790M By studying the characteristics of mutant protein binding and using molecular docking as an aid, a new class of structural compounds was designed. Subsequently, through further chemical synthesis and biological activity evaluation, an efficient and highly selective inhibitor against EGFR mutants was sought. Summary of the Invention

[0008] One of the objectives of this invention is to provide the structure of a class of covalent inhibitors of EGFR mutants containing indole-3-one structures.

[0009] Another object of the present invention is to provide isomers or any mixtures thereof in these forms or pharmaceutical salts thereof, as well as adjuvants or oncological agents in medicine.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned compound.

[0011] Another object of the present invention is to provide the antitumor use of the above-described compound and the compound.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] A derivative of a covalent inhibitor of EGFR mutants containing an indole ketone structure, the compound having a structure as shown in general formula (I):

[0014]

[0015] in,

[0016] R1 is a halogen or methyl group;

[0017] R2 is hydrogen, an alkane with or without substitution, a halogen, or an aryl group with or without substitution.

[0018] Furthermore, R2 is selected from: hydrogen, alkyl, cyano, alkoxy, fluorine, chlorine, bromine, iodine, trifluoromethyl, substituted or unsubstituted aryl; preferably, R2 is selected from halogen atoms.

[0019] The present invention also provides a method for preparing the aforementioned derivative or its stereoisomer, its salt or its solvate, comprising the following steps:

[0020]

[0021] The present invention also provides a composition comprising the compound or its stereoisomer, or any mixture thereof or its pharmaceutical salt, and a pharmaceutical adjuvant or oncology preparation; preferably, the composition is selected from granules, pills, tablets, oral liquids, injections, powders, etc.

[0022] The compound or its isomers or any mixtures thereof or their pharmaceutical salts are used in the preparation of antitumor drugs. Preferably, the tumor is a human tumor cell line including human lung cancer cell A549 (human nonsmall cell lung cancer cell) and human lung cancer cell H1975 (human nonsmall cell lung cancer cell).

[0023] The present invention also provides a method for preparing an antitumor drug. The method involves applying the compound or its isomers or any mixtures thereof, or pharmaceutical salts thereof, along with pharmaceutical adjuvants or tumor preparations, to tumor cells or their environment; preferably, the tumor cells are lung cancer cells A549 and H1975.

[0024] As used herein, the term "substituted" means that any one or more hydrogen atoms on a specified atom or group are substituted with a selected specified group, provided that the substitution does not exceed the general valence of the specified atom. Unless otherwise specified, substituents are named to the central structure. For example, it can be understood that when (cycloalkyl)alkyl is a possible substituent, the point of connection of the substituent to the central structure is in the alkyl moiety.

[0025] Combinations of substituents and variables are permitted only when these combinations yield stable compounds or useful synthetic intermediates. A stable compound or stable structure implies that the compound is sufficiently stable to be isolated from the reaction mixture with useful purity, subsequently formulated to form an effective therapeutic agent.

[0026] The term "halogen" or "halogen atom" refers to chlorine, bromine, fluorine, and iodine.

[0027] When referring to substituents, such as alkenyl, alkynyl, alkyl, halogen, aryl, heteroaryl, alkoxy, cycloalkyl, hydroxyl, amino, mercapto, or phosphinyl, or when these substituents specifically refer to a particular alkenyl, alkynyl, alkyl, halogen, aryl, heteroaryl, alkoxy, cycloalkyl, hydroxyl, amino, mercapto, or phosphinyl group, it refers to one to three of the aforementioned substituents. For example, methylphenyl refers to a phenyl group with one to three methyl-substituted groups.

[0028] Unless otherwise stated, the compounds of this invention are understood to include both their free state and their salts. The term "salt" means an acidic and / or basic salt formed from inorganic and / or organic acids and bases.

[0029] By adopting the above technical solution, this invention synthesizes a series of derivatives of EGFR mutant covalent inhibitors containing indole ketone structures based on the 2-hydroxy-5-aminoindole ketone structure, and finds that the compounds have good inhibitory effects on tumor cells, such as human lung cancer cells A549 and H1795, providing a scientific basis and research direction for the development of new EGFR-TKI drugs based on overcoming the L858R / T790M mutation. Detailed Implementation

[0030] The present invention will be further illustrated below through examples. It should be understood that the methods described in the examples are merely illustrative and not intended to limit the invention. Simple modifications to the preparation methods of the present invention within the framework of the present invention's concept are all within the scope of the invention. All raw materials and solvents used in the examples are commercially available products.

[0031] Example 1: Synthesis of (Z)N-(2-((5-chloro-2-((2-oxo-3-(3,4,5-trimethoxybenzyl)indololin-5-yl)amino)pyrimidin-4-yl)amino)phenyl)acrylamide (1)

[0032]

[0033] (1) Preparation of intermediate 1

[0034] The synthesis steps of intermediate 1 are as follows.

[0035] o-Phenylated diamine (0.1 g, 0.545 mmol) was added together with 2,4,5-trichloropyrimidine (0.058 g, 0.545 mmol) and DIPEA (0.141 g, 1.09 mmol) into isopropanol (10 mL). The reaction was refluxed at 80 °C. The completion of the reaction was monitored by TLC. A white solid was collected to give intermediate 1.

[0036] (2) Preparation of intermediate a1

[0037] Intermediate 1 (0.1 g, 0.39 mmol) was dissolved in 5 mL of acetonitrile containing sodium bicarbonate (0.04 g, 0.47 mmol). The mixture was stirred at room temperature for 30 min. Acryloyl chloride (0.035 g, 0.39 mmol) was added dropwise under ice bath conditions. After the addition was complete, the reaction was monitored by TLC. The reaction mixture was placed in ice water, the filter cake was collected, and dried under vacuum to obtain intermediate a1.

[0038] (3) Preparation of intermediate b1

[0039] Commercially available 2-hydroxy-5-aminoindolone (0.1 g, 0.674 mmol) was added to a flask containing 15 mL of dichloromethane. After the addition of triethylamine (0.204 g, 2.02 mmol), a dichloromethane solution of Boc anhydride (0.44 g, 2.02 mmol) was added dropwise under ice bath conditions. The reaction was monitored by TLC. After the reaction was complete, intermediate b1 was obtained by separation using an EA:PE = 1:2 column.

[0040] (4) Preparation of intermediate b2

[0041] Intermediate b1 (0.1 g, 0.402 mmol) was dissolved in 10 mL of ethanol, and a catalytic amount of hexahydropyridine was added. Under stirring, 3,4,5-benzaldehyde (0.087 g, 0.443 mmol) was added dropwise, and the mixture was refluxed at 80 °C. The reaction was monitored by TLC. After the reaction was complete, intermediate b2 was obtained by separation using an EA:PE = 1:3 column.

[0042] (5) Preparation of intermediate b3

[0043] Intermediate b2 (0.1 g, 0.234 mmol) was dissolved in 10 mL of 1,4-dioxane, and concentrated hydrochloric acid (0.043 g, 1.17 mmol) was added. The reaction was monitored by TLC under stirring at room temperature. After the reaction was complete, the reaction mixture was placed in an aqueous solution of NaOH, the filter cake was collected, and dried under vacuum to obtain intermediate b3.

[0044] (6) Synthesis of (Z)N-(2-((5-chloro-2-((2-oxo-3-(3,4,5-trimethoxybenzyl)indololin-5-yl)amino)pyrimidin-4-yl)amino)phenyl)acrylamide

[0045] Intermediate a1 (0.1 g, 0.234 mmol), intermediate b3 (0.1 g, 0.234 mmol), and p-toluenesulfonic acid monohydrate (0.1 g, 0.234 mmol) were added sequentially to 50 mL of sintered cake, along with 15 mL of isopropanol as solvent. The mixture was refluxed at 80 °C, and the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was placed in a saturated aqueous solution of sodium bicarbonate, and the target compound T1 was obtained by separation using a MeOH:DCM column chromatography at a ratio of 1:30.

[0046] Example 2: Synthesis of (Z)-N-(2-((5-chloro-2-((3-(2-cyanobenzylidene)-2-oxoindoline-5-yl)amino)pyrimidin-4-yl)amino)phenyl)acrylamide

[0047] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the raw material for preparing intermediate b2 was replaced with 2-cyanobenzaldehyde, while the reaction conditions and molar amounts of the materials remained unchanged. A yellow solid compound T2 was obtained.

[0048] Example 3: Synthesis of (Z)-N-(2-((2-((3-(4-bromobenzyl)-2-oxoindoline-5-yl)amino)-5-chloropyrimidin-4-yl)amino)phenyl)acrylamide

[0049] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the starting material for preparing intermediate b2 was replaced with 4-bromobenzaldehyde, while the reaction conditions and molar amounts of the materials remained unchanged. A yellow solid compound T3 was obtained.

[0050] Example 4: Synthesis of (Z)-N-(2-((2-((3-(2-bromobenzyl)-2-oxoindoline-5-yl)amino)-5-chloropyrimidin-4-yl)amino)phenyl)acrylamide

[0051] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the starting material for preparing intermediate b2 was replaced with 2-bromobenzaldehyde, while the reaction conditions and molar amounts of the materials remained unchanged. A yellow solid compound T4 was obtained.

[0052] Example 5: Synthesis of (Z)-N-(2-((2-((3-benzylidene-2-oxoindoline-5-yl)amino)-5-chloropyrimidin-4-yl)amino)phenyl)acrylamide

[0053] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the raw material for preparing intermediate b2 was replaced with benzaldehyde, while the reaction conditions and molar amounts of the materials remained unchanged. A yellow solid compound T5 was obtained.

[0054] Example 6: Synthesis of (Z)-N-(2-((2-((3-(3-fluorobenzyl)-2-oxoindoline-5-yl)amino)-5-chloropyrimidin-4-yl)amino)phenyl)acrylamide

[0055] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the starting material for preparing intermediate b2 was replaced with 3-fluorobenzaldehyde, while the reaction conditions and molar amounts of the materials remained unchanged. A yellow solid compound T6 was obtained.

[0056] Example 7: Synthesis of (Z)-N-(2-((2-((3-(4-methylbenzyl)-2-oxoindoline-5-yl)amino)-5-chloropyrimidin-4-yl)amino)phenyl)acrylamide

[0057] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the starting material for preparing intermediate b2 was replaced with 4-methylbenzaldehyde, while the reaction conditions and molar amounts of the materials remained unchanged. A yellow solid compound T7 was obtained.

[0058] Example 8: Synthesis of (Z)-N-(2-((2-((3-(2-methylbenzyl)-2-oxoindoline-5-yl)amino)-5-chloropyrimidin-4-yl)amino)phenyl)acrylamide

[0059] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the starting material for preparing intermediate b2 was replaced with 2-methylbenzaldehyde, while the reaction conditions and molar amounts of the materials remained unchanged. A yellow solid compound T8 was obtained.

[0060] Example 9: Synthesis of (Z)-N-(2-((2-((3-(3-chlorobenzyl)-2-oxoindoline-5-yl)amino)-5-chloropyrimidin-4-yl)amino)phenyl)acrylamide

[0061] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the starting material for preparing intermediate b2 was replaced with 3-chlorobenzaldehyde, while the reaction conditions and molar amounts of the materials remained unchanged. A yellow solid compound T9 was obtained.

[0062] Example 10: Synthesis of (Z)-N-(2-((2-((3-(2,4-dichlorobenzyl)-2-oxoindoline-5-yl)amino)-5-chloropyrimidin-4-yl)amino)phenyl)acrylamide

[0063] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the starting material for preparing intermediate b2 was replaced with 2,4-dichlorobenzaldehyde, while the reaction conditions and molar amounts of the materials remained unchanged. A yellow solid compound T10 was obtained.

[0064] Example 11: Synthesis of (Z)-N-(2-((2-((3-(2-trifluoromethylbenzyl)-2-oxoindoline-5-yl)amino)-5-chloropyrimidin-4-yl)amino)phenyl)acrylamide

[0065] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the starting material for preparing intermediate b2 was replaced with 2-trifluoromethylbenzaldehyde, while the reaction conditions and molar amounts of the materials remained unchanged. A yellow solid compound T11 was obtained.

[0066] Example 12: Synthesis of (Z)-N-(2-((2-((3-(2-iodobenzylidene)-2-oxoindoline-5-yl)amino)-5-chloropyrimidin-4-yl)amino)phenyl)acrylamide

[0067] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the raw material for preparing intermediate b2 was replaced with 2-iodobenzaldehyde, while the reaction conditions and molar amounts of the materials remained unchanged. A yellow solid compound T12 was obtained.

[0068] Example 13: Synthesis of (Z)-N-(2-((2-((3-(4-trifluoromethylbenzyl)-2-oxoindoline-5-yl)amino)-5-chloropyrimidin-4-yl)amino)phenyl)acrylamide

[0069] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the starting material for preparing intermediate b2 was replaced with 4-trifluoromethylbenzaldehyde, while the reaction conditions and molar amounts of the materials remained unchanged. A yellow solid compound T13 was obtained.

[0070] Example 14: Synthesis of (Z)-N-(2-((2-((3-(4-isopropylbenzyl)-2-oxoindoline-5-yl)amino)-5-chloropyrimidin-4-yl)amino)phenyl)acrylamide

[0071] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the raw material for preparing intermediate b2 was replaced with 4-isopropylbenzaldehyde, while the reaction conditions and molar amounts of the materials remained unchanged. A yellow solid compound T14 was obtained.

[0072] Example 15: Synthesis of (Z)-N-(2-((2-((3-(3-cyanobenzylidene)-2-oxoindoline-5-yl)amino)-5-chloropyrimidin-4-yl)amino)phenyl)acrylamide

[0073] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the raw material for preparing intermediate b2 was replaced with 3-cyanobenzaldehyde, while the reaction conditions and molar amounts of the materials remained unchanged. A yellow solid compound T15 was obtained.

[0074] Example 16: Synthesis of (Z)-N-(2-((2-((3-(3-phenoxybenzyl)-2-oxoindoline-5-yl)amino)-5-chloropyrimidin-4-yl)amino)phenyl)acrylamide

[0075] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the starting material for preparing intermediate b2 was replaced with 3-phenoxybenzaldehyde, while the reaction conditions and molar amounts of the materials remained unchanged. A yellow solid compound T16 was obtained.

[0076] Example 17: Synthesis of (Z)-N-(2-((2-((3-(4-trifluoromethylbenzyl)-2-oxoindoline-5-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)acrylamide

[0077] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the initial starting material 2,4,5-trichloropyrimidine is replaced with 2,4-dichloro-5-fluoropyrimidine, and the starting material for preparing intermediate b2 is replaced with 4-trifluoromethylbenzaldehyde. The reaction conditions and molar amounts of the materials remain unchanged. A yellow solid compound T17 is obtained.

[0078] Example 18: Synthesis of (Z)-N-(2-((2-((3-(2-trifluoromethylbenzyl)-2-oxoindoline-5-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)acrylamide

[0079] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the initial starting material 2,4,5-trichloropyrimidine is replaced with 2,4-dichloro-5-fluoropyrimidine, and the starting material for preparing intermediate b2 is replaced with 2-trifluoromethylbenzaldehyde. The reaction conditions and molar amounts of the materials remain unchanged. A yellow solid compound T18 is obtained.

[0080] Example 19: Synthesis of (Z)-N-(2-((2-((3-(4-bromobenzyl)-2-oxoindoline-5-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)acrylamide

[0081] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the initial starting material 2,4,5-trichloropyrimidine is replaced with 2,4-dichloro-5-fluoropyrimidine, and the starting material for preparing intermediate b2 is replaced with 4-bromobenzaldehyde. The reaction conditions and molar amounts of the materials remain unchanged. A yellow solid compound T19 is obtained.

[0082] Example 20: Synthesis of (Z)-N-(2-((2-((3-(2-methylbenzyl)-2-oxoindoline-5-yl)amino)-5-methylpyrimidin-4-yl)amino)phenyl)acrylamide

[0083] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the initial starting material 2,4,5-trichloropyrimidine is replaced with 2,4-dichloro-5-methylpyrimidine, and the starting material for preparing intermediate b2 is replaced with 2-methylbenzaldehyde. The reaction conditions and molar amounts of the materials remain unchanged. A yellow solid compound T20 is obtained.

[0084] Example 21: Synthesis of (Z)-N-(2-((2-((3-(2-cyanobenzylidene)-2-oxoindoline-5-yl)amino)-5-methylpyrimidin-4-yl)amino)phenyl)acrylamide

[0085] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the initial starting material 2,4,5-trichloropyrimidine is replaced with 2,4-dichloro-5-methylpyrimidine, and the starting material for preparing intermediate b2 is replaced with 2-cyanobenzaldehyde. The reaction conditions and molar amounts of the materials remain unchanged. A yellow solid compound T21 is obtained.

[0086] Example 22: Synthesis of (Z)-N-(2-((2-((3-(2-cyanobenzyl)-2-oxoindoline-5-yl)amino)-5-methylpyrimidin-4-yl)amino)phenyl)acrylamide

[0087] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the initial starting material 2,4,5-trichloropyrimidine is replaced with 2,4-dichloro-5-methylpyrimidine, and the starting material for preparing intermediate b2 is replaced with 2-cyanobenzaldehyde. The reaction conditions and molar amounts of the materials remain unchanged. A yellow solid compound T22 is obtained.

[0088] Example 23: Synthesis of (Z)-N-(2-((2-((3-(2-methoxybenzyl)-2-oxoindoline-5-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)acrylamide

[0089] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the initial raw material 2,4,5-trichloropyrimidine is replaced with 2,4-dichloro-5-fluoropyrimidine, and the raw material for preparing intermediate b2 is replaced with 2-methoxybenzaldehyde. The reaction conditions and molar amounts of the materials remain unchanged. A yellow solid compound T23 is obtained.

[0090] Example 24: Synthesis of (Z)-N-(2-((2-((3-(2-methylbenzyl)-2-oxoindoline-5-yl)amino)-5-fluoropyrimidin-4-yl)amino)phenyl)acrylamide

[0091] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the initial starting material 2,4,5-trichloropyrimidine is replaced with 2,4-dichloro-5-fluoropyrimidine, and the starting material for preparing intermediate b2 is replaced with 2-methylbenzaldehyde. The reaction conditions and molar amounts of the materials remain unchanged. A yellow solid compound T24 is obtained.

[0092] Example 25: Synthesis of (Z)-N-(2-((2-((3-benzylidene-2-oxoindoline-5-yl)amino)-5-methylpyrimidin-4-yl)amino)phenyl)acrylamide

[0093] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the initial starting material 2,4,5-trichloropyrimidine is replaced with 2,4-dichloro-5-methylpyrimidine, and the starting material for preparing intermediate b2 is replaced with benzaldehyde. The reaction conditions and molar amounts of the materials remain unchanged. A yellow solid compound T25 is obtained.

[0094] Example 26: Synthesis of (Z)-N-(2-((2-((3-(4-bromobenzyl)-2-oxoindoline-5-yl)amino)-5-methylpyrimidin-4-yl)amino)phenyl)acrylamide

[0095] The preparation of the intermediate is described in Example 1. The preparation of the target compound is also described in Example 1, except that the initial starting material 2,4,5-trichloropyrimidine is replaced with 2,4-dichloro-5-methylpyrimidine, and the starting material for preparing intermediate b2 is replaced with 4-bromobenzaldehyde. The reaction conditions and molar amounts of the materials remain unchanged. A yellow solid compound T26 is obtained.

[0096] The structures, physicochemical properties, and proton and carbon NMR spectra of the covalent inhibitors of EGFR mutants containing indole ketone structures are shown in Table 1.

[0097] Table 1. Physicochemical properties and NMR data of the target compounds

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105] Pharmacological Example 1:

[0106] Antitumor activity test.

[0107] The inhibitory activity of the compound against human lung cancer cells A549, human prostate cancer cells PC-3, human chronic myeloid leukemia cells K562, and human liver cancer cells SMMC-7721 was determined by the MTT assay.

[0108] Cell seeding steps (applicable to adherent cells and suspension cells).

[0109] Adherent cells:

[0110] 1: Collect cells in the logarithmic growth phase and adjust the cell suspension concentration to 5 × 10⁻⁶. 4 Add 100 μL to each well.

[0111] Incubate at 37°C with 2:5% CO2 until the cell monolayer covers the bottom of the well (96-well plate). Add drugs in a concentration gradient of (5μM, 2.5μM, 1.25μM, 0.625μM, 0.313μM). In principle, drugs can be added after the cells adhere to the plate.

[0112] Incubate at 37°C with 3:5% CO2 for 48 hours, then observe under an inverted microscope.

[0113] 4: Add 10 μL of MTT solution (5 mg / ml, i.e., 0.5% MTT) to each well and continue culturing for 4 h.

[0114] 5: Terminate the culture and carefully aspirate the culture medium from the well.

[0115] 6. Add 100 μL of dimethyl sulfoxide to each well and shake on a shaker at low speed for 10 min to fully dissolve the crystals. Measure the absorbance of each well at OD490 nm using an ELISA reader.

[0116] 7: Simultaneously set up zeroing wells (culture medium, MTT, dimethyl sulfoxide) and control wells (cells, drug dissolution medium of the same concentration, culture medium, MTT, dimethyl sulfoxide).

[0117] 1: Collect cells in the logarithmic growth phase and adjust the cell suspension concentration to 4.5 × 10⁻⁶. 4 Add 100 μL per well at a concentration of 1 ml.

[0118] 2:5% CO2, incubated at 37℃ for 24h, with concentration gradients of (5μM, 2.5μM, 1.25μM, 0.625μM, ...

[0119] In principle, the drug (0.313 μM) can be added after the cells have adhered to the cell wall.

[0120] 3: Incubate at 37℃ with 5% CO2 for 72 hours, then observe under an inverted microscope.

[0121] 4: Add 10 μL of MTT solution (5 mg / ml, i.e., 0.5% MTT) to each well and continue culturing for 4 h.

[0122] 5. Carefully aspirate the supernatant, add 100 μL of dimethyl sulfoxide to each well, and shake on a shaker at low speed for 10 min to fully dissolve the crystals. Measure the absorbance of each well at OD570 nm (630 nm calibration) using an ELISA reader.

[0123] 6: Simultaneously set up zeroing wells (culture medium, MTT, dimethyl sulfoxide) and control wells (cells, drug dissolution medium of the same concentration, culture medium, MTT, dimethyl sulfoxide), with 3 replicates for each group.

[0124] Cell growth inhibition rate = 1 - (OD value of drug-treated group - OD value of blank group) / (OD value of negative group - OD value of blank group) × 100%;

[0125] The embodiments of this invention are provided to illustrate the technical solutions of this invention, but the content of the embodiments is not limited thereto. The experimental results of some target compounds are shown in the table below.

[0126] The embodiments of this invention are provided to illustrate the technical solutions of this invention, but the content of the embodiments is not limited thereto. The experimental results of some target compounds are shown in Table 1.

[0127] Table 1. Inhibitory activity of covalent inhibitors of EGFR mutants containing indole-containing structures.

[0128]

[0129]

[0130] Data are presented as mean ± standard deviation.

[0131] a IC 50 (μM) indicates the concentration at which this compound inhibits cell growth by 50% compared to the control after 72 hours of cell culture.

[0132] b Reference drug: Osimertinib.

[0133] The in vitro antiproliferative activity of all target molecules against two human tumor cell lines, A549 and H1795, was tested using the MTT assay. The results showed that the half-maximal inhibitory concentration (IC50) was the highest among the target molecules. 50 The values ​​are expressed as follows, summarized in Table 1. The results show that, compared to the positive control drug osimertinib, the IC50 values ​​of most compounds are significantly higher. 50 The value is comparable to that of some compounds, IC 50 The value reaches the sub-micromolar level. According to IC... 50 The values ​​show that T16 has in vitro antitumor activity comparable to osimertinib, indicating that most of the compounds synthesized in this study exhibit extremely strong antitumor cell proliferation activity.

Claims

1. Compound of formula (Ⅰ) or its medicinal salt: ； (I) in, R1 is chlorine, fluorine, or methyl; R2 is hydrogen, cyano, halogen, trifluoromethyl, 4-isopropyl, or 3-phenoxy.

2. Compounds of formula (Ⅰ) or their medicinal salts: ； (I) T1: R1 = Chlorine; R2 = 3,4,5-Trimethoxy; T2: R1 = Chlorine; R2 = 2-Cyano; T3: R1 = chlorine; R2 = 4-bromine; T4: R1 = chlorine; R2 = 2-bromine; T5: R1 = chlorine; R2 = hydrogen; T6: R1 = chlorine; R2 = 3-fluorine; T7: R1 = Chlorine; R2 = 4-Methyl; T8: R1 = Chlorine; R2 = 2-Methyl; T9: R1 = chlorine; R2 = 3-chloro; T10: R1 = chlorine; R2 = 2,4-dichloro; T11: R1 = Chlorine; R2 = 2-Trifluoromethyl; T12: R1 = Chlorine; R2 = 2-Iodine; T13: R1 = Chlorine; R2 = 4-Trifluoromethyl; T14: R1 = Chlorine; R2 = 4-Isopropyl; T15: R1 = Chlorine; R2 = 3-Cyano; T16: R1 = Fluorine; R2 = 3-Phenoxy; T17: R1 = Fluorine; R2 = 4-Trifluoromethyl; T18: R1 = Fluorine; R2 = 2-Trifluoromethyl; T19: R1 = Fluorine; R2 = 4-Bromo; T20: R1 = Fluorine; R2 = 2-Methyl; T21: R1 = Fluorine; R2 = 4-Bromo; T22: R1 = Methyl; R2 = 2-Methyl; T23: R1 = methyl; R2 = 2-cyano; T24: R1 = methyl; R2 = 2-methoxy; T25: R1 = methyl; R2 = hydrogen; T26: R1 = methyl; R2 = 4-bromo.

3. The method for preparing the compound according to any one of claims 1-2, characterized in that: Includes the following steps: 。 4. A composition, characterized in that: It contains the compound according to any one of claims 1-2, and an adjuvant in medicine.

5. The use of the compound of any one of claims 1-2, or the composition of claim 4, in the preparation of an antitumor drug; wherein the antitumor drug is an anti-human lung cancer cell A549 or human lung cancer cell H1975 drug.

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