Alkynylphenylbenzamide compounds and their applications

Abstract

The present invention relates to alkynylphenylbenzamide compounds and their applications. The alkynylphenylbenzamide compounds of the present invention have a structure represented by formula (I), and such compounds can be used as protein kinase inhibitors, effectively inhibit the activity of TRK protein kinase, and inhibit the proliferation, metastasis, and invasion of various tumor cells, while simultaneously possessing relatively excellent pharmacokinetics and low toxicity.

Description

Technology Field

[0001] The present invention relates to the field of pharmaceutical chemistry, and in particular to alkynylphenylbenzamide compounds and their applications. Background Technology

[0002] Tropomyosin receptor kinases (TRKs) belong to the receptor tyrosine kinase (RTK) family and include three subtypes: TRKA, TRKB, and TRKC, which are encoded by the NTRK1, NTRK2, and NTRK3 genes, respectively. TRKs are a type of transmembrane protein composed of an extracellular ligand binding domain, a transmembrane domain (TM), and an intracellular domain; TRKs primarily act by binding to neurotrophic factors (NTs). Neurotrophic factors are a type of protein molecule produced by neuronal tissues (such as muscle) and astrocytes, and are necessary for the growth and survival of neurons. Currently, the four main neurotrophic factors identified are NGF (nerve growth factor), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), and neurotrophic factor 4 (NT-4). NGF binds to TRKA, while BDNF and NT-4 bind to TRKA. Although NT-3 can bind to three TRK proteins, it has a stronger binding ability to TRKC. When activated by signal induction, TRK sequentially activates downstream signaling pathways through self-dimerization and phosphorylation to implement various physiological functions of the cell. The downstream signaling pathways of TRK include the MAPK, PI3K / AKT, and PLCγ / PKC pathways. These signaling pathways regulate various physiological activities related to neurons, such as physiological processes like cell proliferation, differentiation, metastasis, and apoptosis; the elasticity of neural synapses; the growth and repair of neural dendrites; the prevention and repair of neuronal degradation; and the maintenance of sensory neurons.

[0003] Numerous studies have shown that TRK overexpression, gene fusions, and mononucleotide alterations are closely associated with the development of various types of tumors, such as non-small cell lung cancer, breast cancer, colon cancer, prostate cancer, thyroid cancer, malignant melanoma, neuroblastoma, and mammary gland-like secretory carcinoma. The most common mechanism of abnormal TRK activation is gene fusion. The first NTRK fusion gene discovered in medical research was the TPM3-NTRK1 fusion gene found in colon cancer samples; as research deepened, researchers successively discovered various types of fusion genes, including CD74-NTRK1, ETV6-NTRK2, QKI-NTRK2, and ETV6-NTRK3. TRK fusion proteins expressed by NTRK fusion genes can induce abnormal cell proliferation and promote tumor development by continuously activating downstream signaling pathways without binding to ligands. Therefore, TRK is considered an effective target for anticancer therapy.

[0004] Currently, Larotrectinib, a TRK selective inhibitor developed by LOXO in the US, was approved by the FDA in 2018 and launched, Entrectinib, a TRK inhibitor developed by Roche Pharmaceuticals, was launched in Japan in June 2019, Belizatinib, developed by TESARO, is undergoing clinical trials, and other multi-target inhibitors such as Cabozanitinib, Sitravatinib, and Altiratinib also have excellent TRK inhibitory activity.

[0005] NTRK gene point mutations resulting from the continuous use of TRK inhibitors are a major cause of drug resistance in tumors. Clinical studies have already identified G595R, G667C, F589L, G667S mutations in NTRK1 and G623R, G696A mutations in NTRK3 in succession, but there are currently no inhibitors on the market that target these mutations, and second-generation TRK inhibitors LOXO-195, TPX-0005, and ONO-5390556 are currently in clinical trials. The problem to be solved

[0006] Based on this, the present invention provides an alkynylphenylbenzamide-based compound, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof that can be used as a protein kinase inhibitor to effectively inhibit the activity of TRK protein kinase and inhibit the proliferation, metastasis, and invasion of various tumor cells, and in particular has excellent pharmacokinetics and anti-drug resistance. means of solving the problem

[0007] The specific technical solutions are as follows.

[0008] As an alkynylphenylbenzamide compound having a structure represented by formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a prodrug molecule thereof;

[0009]

[0010] (I)

[0011] In the above equation (I), R1 is arbitrarily C1~C 20 Selected from alkyl;

[0012] R2 is optionally H, halogen, C1~C 20 Alkyl, C1~C 20 Alkoxy or halogen-substituted C1~C 20 Selected from alkyls;

[0013] R3 is optionally selected from C1-C4 alkyl groups substituted with H or fluorine, and 5-6 member heterocyclyl groups comprising 1-3 substituted or unsubstituted N ring atoms;

[0014] R4 is optionally H, halogen, nitro, or substituted or unsubstituted C1~C 20 Alkyl, substituted or unsubstituted C1~C 20 Selected from an alkoxy, a 5-10-membered heterocyclic group comprising 1-3 substituted or unsubstituted N ring atoms, and a 5-10-membered heteroaryl comprising 1-3 substituted or unsubstituted N ring atoms;

[0015] R5 is -NR6R7 and;

[0016] R6 and R7 are each independently -(CH2) m NR8R9, -(CH2) n CR 10 R 11 R 12 , -(CH2) p OR 12 Selected from, or R6, R7 form a single ring, condensation ring, spiro ring, or cross-linked ring comprising a substituted or unsubstituted heteroatom together with a nitrogen atom connected thereto;

[0017] R8 and R9 are independently H and C1~C 20 Selected from alkyls; or R8, R9 form a single ring, condensation ring, spiro ring, or cross-linked ring comprising 1-3 heteroatoms substituted or unsubstituted together with nitrogen atoms connected thereto;

[0018] R 10 , R 11 It forms a single ring, condensation ring, spiro ring, or cross-linked ring containing 1 to 3 substituted or unsubstituted heteroatoms together with carbon atoms connected to them;

[0019] R 12 is H, C1~C20 Selected from alkyls;

[0020] m, n, and p are each independently selected from integers 0 to 10.

[0021] In some of the embodiments, R4 is optionally H, halogen, nitro, C1~C 10 Alkyl, halogen-substituted C1~C 10 Alkyl, C1~C 10 Alkoxy, halogen-substituted C1~C 10 Alkoxy, -(CH2) x NR 17 R 18 , 1-5 R 19 A 5-10-membered heterocyclic group containing 1-3 N ring atoms substituted or unsubstituted with R, and 1-5 R 19 Selected from 5-10 member heteroaryls containing 1-3 N ring atoms substituted or unsubstituted; x is an integer from 1 to 5;

[0022] R 17 , R 18 1-5 R atoms along with nitrogen atoms connected to them 19 Forming a morpholinyl, pyrrolidinyl, piperidinyl, or piperazinyl substituted or unsubstituted;

[0023] Each R 19 Each is independently selected from C1-C5 alkyls.

[0024] In some of the embodiments, R4 is optionally H, halogen, nitro, C1-C8 alkyl, halogen-substituted C1-C8 alkyl, C1-C8 alkoxy, halogen-substituted C1-C8 alkoxy, -(CH2) x NR 17 R 18 , 1-5 R 19 A 5-6-membered heterocyclic group containing 1-3 N ring atoms substituted or unsubstituted with R, and 1-5 R 19Selected from 5-6-membered heteroaryls containing 1-3 N ring atoms substituted or unsubstituted; x is an integer from 1 to 5.

[0025] In some of the embodiments, R4 is optionally H, halogen, nitro, C1-C4 alkyl, halogen-substituted C1-C4 alkyl, C1-C4 alkoxy, halogen-substituted C1-C4 alkoxy, -(CH2) x NR 17 R 18 , 1-3 R 19 A 5-6-membered heterocyclic group containing 1-3 N ring atoms substituted or unsubstituted with R, 1-3 R 19 Selected from 5-6-membered heteroaryls containing 1-3 N ring atoms substituted or unsubstituted; x is an integer from 1 to 5.

[0026] In some of the embodiments, R4 is optionally H, halogen, nitro, C1-C4 alkyl, halogen-substituted C1-C4 alkyl, C1-C4 alkoxy, halogen-substituted C1-C4 alkoxy, -(CH2) x NR 17 R 18 , 1-3 R 19 Selected from imidazolyls substituted or unsubstituted; x is 1, 2, or 3;

[0027] R 17 , R 18 1-3 R atoms along with nitrogen atoms connected to them 19 Forming a piperazinyl that is substituted or unsubstituted;

[0028] Each R 19 Each is independently selected from C1-C5 alkyls.

[0029] In some of the embodiments, R4 is optionally H, halogen, nitro, methyl, ethyl, propyl, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoroethyl, , or Selected from;

[0030] Each R 19 Each is independently selected from methyl, ethyl, and propyl.

[0031] In some of the embodiments, R6 and R7 are each independently -(CH2) m NR8R9, -(CH2) n CR 10 R 11 R 12 , -(CH2) p OR 12 Selected from, or R6, R7 with 1-5 R atoms along with nitrogen atoms connected to them. 13 Forming a 3-15 member single ring, condensation ring, spiro ring, or cross-linked ring comprising 1-3 heteroatoms substituted or unsubstituted with, wherein the heteroatoms are selected from O, N, and S;

[0032] R8 and R9 are each independently selected from H, C1–C5 alkyl; or R8 and R9 have 1–5 R atoms together with nitrogen atoms connected thereto. 13 Forming a 3-10 member single ring, condensation ring, spiro ring, or cross-linked ring comprising 1-3 heteroatoms substituted or unsubstituted with, wherein the heteroatoms are selected from O and N;

[0033] R 10 , R 11 It has 1-5 R atoms along with carbon atoms connected to them. 13 Forming a 3-10 member single ring, condensation ring, spiro ring, or cross-linked ring comprising 1-3 heteroatoms substituted or unsubstituted with, wherein the heteroatoms are selected from O and N;

[0034] R 12 is selected from H, C1-C5 alkyl;

[0035] Each R 13Each is independently H, C1-C5 alkyl, C1-C5 alkanoyl, hydroxyl, hydroxyl-substituted C1-C5 alkyl, amino-substituted C1-C5 alkyl, amino-substituted C1-C5 alkoxy, C3-C7 cycloalkyl-substituted C1-C3 alkyl, -NR 15 R 16 , 1-5 R 14 Selected from a 3-10 member single ring, condensation ring, spiro ring, or bridging ring containing 1-3 heteroatoms substituted or unsubstituted, and the heteroatoms are selected from O and N;

[0036] R 14 , R 15 , R 16 Each is independently selected from H, C1-C5 alkyl;

[0037] m, n, and p are each independently selected from integers 0-5.

[0038] In some of the embodiments, R6 and R7 are each independently -(CH2) m NR8R9, -(CH2) p OR 12 Selected from, or R6, R7 with 1-3 R atoms along with nitrogen atoms connected to them. 13 Forming a morpholineyl, pyrrolidineyl, piperidineyl, or piperazinyl substituted or unsubstituted with;

[0039] R8 and R9 are each independently selected from H, C1–C5 alkyl, or R8 and R9 have 1–5 R atoms together with the nitrogen atoms connected thereto. 13 Forming a morpholineyl, pyrrolidineyl, piperidineyl, or piperazinyl substituted or unsubstituted with;

[0040] R 12 is selected from H, C1-C5 alkyl;

[0041] Each R 13Each independently consists of H, C1-C5 alkyl, C1-C5 alkanoyl, hydroxyl, -NR 15 R 16 , 1-2 R 14 Oxetanyl substituted or unsubstituted with 1-4 R groups 14 Selected from morpholine that is substituted or unsubstituted;

[0042] R 14 , R 15 , R 16 Each is independently selected from H, C1-C3 alkyl;

[0043] m and p are each independently selected from 1, 2, 3, 4 or 5.

[0044] In some of the embodiments, R5 is selected from any one of the following groups:

[0045] , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , .

[0046] In some of the embodiments, R4 is a halogen; R5 is -NR6R7;

[0047] R6 and R7 are each independently -(CH2) m NR8R9, -(CH2) p OR 12 Selected from, or R6, R7 with 1-3 R atoms connected to them. 13 Forming a morpholineyl, pyrrolidineyl, piperidineyl, or piperazinyl substituted or unsubstituted with;

[0048] R8 and R9 are each independently selected from H, C1-C3 alkyl, or R8 and R9 have 1-2 R atoms together with the nitrogen atoms connected thereto. 13 Forming a morpholineyl, pyrrolidineyl, piperidineyl, or piperazinyl substituted or unsubstituted with;

[0049] R 12 is selected from H, C1-C3 alkyl;

[0050] Each R 13 Each independently consists of H, C1-C3 alkyl, acetyl, hydroxyl, -NR 15R 16 , selected from oxetanyl and morpholinyl;

[0051] R 15 , R 16 Each is independently selected from H, C1-C3 alkyl;

[0052] m and p are each independently selected from 2, 3, or 4.

[0053] In some of the embodiments, R4 is Cl, and

[0054] R5 is , , , , , , , , , , , , , , , , Selected from.

[0055] In some of the embodiments, R4 is H, halogen, methyl, methoxy, trifluoromethyl, nitro, Selected from, and R5 is am.

[0056] In some of the embodiments, R4 is H, Selected from, and R5 is , , Selected from.

[0057] In some of the embodiments, R4 is And, R5 is , Selected from.

[0058] In some of the embodiments, R1 is optionally C1 to C 10 Selected from alkyls.

[0059] In some of the embodiments, R1 is optionally selected from C1 to C4 alkyls.

[0060] In some of the embodiments, R1 is optionally selected from methyl, ethyl, isopropyl, and tert-butyl.

[0061] In some of the embodiments, R2 is optionally H, halogen, C1~C 10 Alkyl, halogen-substituted C1~C 10 Selected from alkyls.

[0062] In some of the embodiments, R2 is optionally selected from H, halogen, C1-C4 alkyl, halogen-substituted C1-C4 alkyl, and C1-C4 alkoxy.

[0063] In some of the embodiments, R2 is optionally selected from hydrogen, fluorine, methyl, ethyl, isopropyl, tert-butyl, difluoromethyl, difluoroethyl, trifluoromethyl, or trifluoroethyl.

[0064] In some of the embodiments, R3 is selected from H, difluoromethyl, difluoroethyl, trifluoromethyl, or trifluoroethyl.

[0065] In some of the embodiments, the alkynylphenylbenzamide compound has a structure represented by formula (II).

[0066]

[0067] (II)

[0068] Another object of the present invention is to provide the application of the alkynylphenylbenzamide compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a prodrug molecule thereof, in the manufacture of TRK inhibitors.

[0069] Another object of the present invention is to provide the application of the alkynylphenylbenzamide compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a prodrug molecule thereof, in the manufacture of a drug for preventing and / or treating diseases mediated by TRK tyrosine kinase.

[0070] In some of the embodiments, the disease mediated by the TRK tyrosine kinase is a tumor, preferably non-small cell lung cancer, breast cancer, colon cancer, prostate cancer, thyroid cancer, malignant melanoma, neuroblastoma, or mammary gland-like secretory carcinoma.

[0071] Another object of the present invention is to provide a pharmaceutical composition for preventing and / or treating tumors, said pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable adjuvant, said active ingredient comprising the alkynylphenylbenzamide-based compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof or a prodrug molecule thereof. Effects of the invention

[0072] The alkynylphenylbenzamide compound provided in the present invention possesses potent inhibitory activity against TRK kinases and potent inhibitory activity against the proliferation of wild-type and drug-resistant cells of Ba / F3-TRKs stable strains. For example, it can be used in the manufacture of drugs for the prevention or treatment of diseases mediated by TRK tyrosine kinases, such as non-small cell lung cancer, breast cancer, colon cancer, prostate cancer, thyroid cancer, malignant melanoma, neuroblastoma, and mammary gland-like secretory carcinoma, while simultaneously possessing excellent pharmacokinetics and low toxicity. Brief explanation of the drawing

[0073] Figure 1 shows the in vivo antitumor activity of compound XS3-55. Figure 2 shows the effect of compound XS3-55 on the body weight of mice. Specific details for implementing the invention

[0074] In the following examples of the present invention, experimental methods for which specific conditions are not specified generally follow conventional conditions or conditions provided by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0075] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by a person skilled in the art of this invention. The terms used in the specification of this invention are merely for describing specific embodiments and are not intended to limit the invention.

[0076] The terms “include” and “comprising” and any variations thereof in the present invention are intended to include non-exclusive inclusions, such that, for example, a process, method, apparatus, product, or device comprising a series of steps is not necessarily limited to the listed steps or modules, optionally includes additional steps not listed, or optionally includes additional steps inherent to such process, method, product, or device.

[0077] The term "plural" as used in the present invention means two or more. "And / or" is used to describe the association relationship of associated objects and implies that three types of relationships may exist; for example, A and / or B may mean three cases: only A exists, A and B exist simultaneously, or only B exists. The symbol " / " generally indicates that the preceding and succeeding associated objects are in an "or" relationship.

[0078] In the compound according to the present invention, any variable (e.g., R 10 , R 11Where (etc.) appears one or more times in any component, each definition that appears is independent of the other definitions that appear each time. Likewise, such combinations are permitted insofar as the combination of substituents and variables stabilizes the compound. A line drawn from a substituent to a ring system indicates that the indicated bond can be connected to any substitutable ring atom. If the ring system is a polyhedron, it means that such a bond is connected only to any suitable carbon atom of an adjacent ring. Those skilled in the art should understand that by selecting the substituents and substitution patterns of the compounds of the present invention, it is possible to provide compounds that are chemically stable and easily synthesized from raw materials readily obtainable through the art of the art and the methods presented below. It should be understood that if a substituent itself is substituted with one or more groups, these groups may be on the same carbon atom or different carbon atoms insofar as they stabilize the structure.

[0079] As used in this text, the term "alkyl" refers to saturated aliphatic hydrocarbon groups of branched and straight chains having a specific number of carbon atoms. For example, "C1-C 20 In "alkyl", "C1-C 20 The definition of “includes groups having 1, 2, 3, 4, 5, 6, or 20 carbon atoms arranged in a straight or branched chain. The term “cycloalkyl” means a monocyclic saturated aliphatic hydrocarbon group having a specific number of carbon atoms. For example, “C3–C7 cycloalkyl” includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc.

[0080] The term "alkoxy" used in the text refers to a group in which an alkyl group is directly connected to oxygen, that is, a group having an -O-alkyl structure such as -OCH3, -OCH2CH3, -OCH2CH2CH3, -O-CH2CH(CH3)2, -OCH2CH2CH2CH3, -O-CH(CH3)2, etc.

[0081] The term "heterocyclic group" as used in the text refers to a non-aromatic heterocyclic group comprising one or more heteroatoms selected from O, N, and S, such as piperidineyl, tetrahydropyrrolyl (pyrrolidineyl), morpholineyl, piperazinyl, etc. The connection of heterocyclic substituents can be made through carbon atoms or heteroatoms.

[0082] The term "heteroaryl" as used in the text refers to an aromatic ring comprising one or more heteroatoms selected from O, N, or S, and heteroaryls within the scope of the present invention include, but are not limited to, quinolinyl, pyrazolyl, pyrrolyl, thienyl, furyl, pyridyl, pyrimidinyl, pyrazinyl, triazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridazinyl, benzofuryl, benzothienyl, benzoxazolyl, indolyl, etc.; Additionally, "heteroaryl" is understood to include N-oxide derivatives of any nitrogen-containing heteroaryl. The linkage of heteroaryls can be formed through carbon atoms or heteroatoms.

[0083] The term "substituted" used in the text means substituting a hydrogen group in a specific structure with a specified substituent group.

[0084] As understood by those skilled in the art, the term "halogen" as used in this text refers to substances including chlorine, fluorine, bromine, and iodine.

[0085] The present invention comprises the free form of the compound of Formula (I) or Formula (II), as well as its pharmaceutically acceptable salts, its stereoisomers, and its prodrug molecules. The term “free form” means a compound in a form other than a salt. The pharmaceutically acceptable salts included in the present invention include all typical pharmaceutically acceptable salts of the free form of the compound of Formula (I) or Formula (II), as well as exemplary salts of the specific compound according to the text. The free form of the specific salt of said compound can be separated using techniques known in the art. For example, the corresponding salt can be regenerated into a free form by treating it with a suitable aqueous solution of a base, such as a dilute aqueous solution of NaOH, a dilute aqueous solution of potassium carbonate, a dilute aqueous solution of ammonia, and a dilute aqueous solution of sodium bicarbonate. Although the free form differs to some extent from the respective salt form in some physical properties, such as solubility in polar solvents, for the purposes of the present invention, these salt and base salts are equivalent to their respective free forms in other pharmaceutical aspects.

[0086] Pharmaceutically acceptable salts of the present invention can be synthesized from compounds of the present invention comprising a basic portion or an acidic portion through conventional chemical methods. Generally, salts of basic compounds are prepared by ion exchange chromatography or by reacting a free base with a stoichiometric amount or excess of an inorganic or organic acid in the form of a desired salt in a suitable solvent or a combination of various solvents. Similarly, salts of acidic compounds are formed by reacting with a suitable inorganic or organic salt.

[0087] Accordingly, a pharmaceutically acceptable salt of the compound of the present invention comprises a conventional non-toxic salt of the compound of the present invention formed by reacting the basic compound of the present invention with an inorganic acid or an organic acid. For example, conventional non-toxic salts include salts obtained by preparing from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc., and acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, It also includes salts obtained by preparing organic acids such as 2-acetoxybenzoic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and trifluoroacetic acid.

[0088] When the compound of the present invention is acidic, a suitable "pharmaceuticalally acceptable salt" means a salt prepared from a pharmaceutically acceptable, non-toxic base including an inorganic base and an organic base. Salts obtained from inorganic bases include aluminum salt, ammonium salt, calcium salt, cuprous salt, iron salt, ferrous salt, lithium salt, magnesium salt, manganese salt, manganese salt, potassium salt, sodium salt, zinc salt, etc. Particularly preferably, ammonium salt, calcium salt, magnesium salt, potassium salt, and sodium salt are included.In a salt obtained from a pharmaceutically acceptable organic non-toxic base, said base comprises a salt of a primary amine, a secondary amine, and a tertiary amine, and the substituted amine is a naturally occurring substituted amine, a cyclic amine, and arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, aminoglucose, histidine, It includes basic ion exchange resins such as hydroxocobalamin, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc.

[0089] Berg et al., “Pharmaceutical Salts” J. Pharm. Sci.'1977: 66: 1-19 describes in more detail the preparation of the pharmaceutically acceptable salts described above and other typical pharmaceutically acceptable salts.

[0090] The present invention will be described in more detail in conjunction with the specific embodiments below.

[0091] Example 1: Preparation of 3-(imidazo[1,2-b]pyridazine-3-ylethinyl)-2-methyl-N-(3-((4-methylpiperazine-1-yl)methyl)-5-(trifluoromethyl)phenyl)benzamide (named XS116)

[0092]

[0093] Step 1: Preparation of methyl 2-methyl-3-((trimethylsilyl)ethynyl)benzoate (Compound 2)

[0094]

[0095] 10 g (36 mmol) of compound, 1,689 mg (3.6 mmol) of cuprous iodide, 1.27 g (1.8 mmol) of bis(triphenylphosphine)palladium dichloride, 150 mL of anhydrous acetonitrile, and 9.3 g (72 mmol) of N,N-diisopropylethylamine were added to a 500 mL three-necked flask, the mixture was replaced with argon gas, the reaction system was sealed, and then 10.6 g (108 mmol) of trimethylsilylacetylene was injected using a syringe and stirred at 60 °C for 6 hours. The reaction solution was filtered through diatomaceous earth and the solvent was spin-dried to obtain a black mixture, which was then used in the next step of the reaction.

[0096] Step 2: Preparation of methyl 2-ethynyl-2-methylbenzoate (Compound 3)

[0097]

[0098] The crude product from the previous step was dissolved in methanol, and approximately 20 mL of a 1 mol / L tetrahydrofuran solution of tetrabutylammonium fluoride was added and stirred at room temperature for 2 hours. After spin-drying the reaction system, 4 g of a yellowish-brown oily substance was obtained by column chromatography (total yield of 63% from the two steps).

[0099] 1 H NMR (400 MHz, DMSO- d 6) δ 7.80 (s, 1H), 7.78 (s, 1H), 7.58 (s, 1H), 4.27 (s, 1H), 3.85 (s, 3H), 2.37 (s, 3H). LC-MS (ESI) m / z 175.5[M+H] + .

[0100] Step 3: Preparation of 3-ethynyl-2-methylbenzoic acid (Compound 4)

[0101]

[0102] 1.5 g (9 mmol) of Compound 3 was dissolved in a mixed solvent of tetrahydrofuran, methanol, and water in a volume ratio of 10:1:5, and then 1.8 g (40 mmol) of lithium hydroxide hydrate was added and stirred at 60°C for 1 hour. After filtration, the reaction system was spin-dried, and then a 4 M hydrochloric acid solution was added until the reaction system became acidic, at which point a white solid precipitated; the white solid was collected by filtration and dried to obtain 900 mg (yield 65%) of white solid.

[0103] 1 H NMR (400 MHz, DMSO- d 6) δ 13.09 (s, 1H), 7.77 (dd, J =7.8, 0.9Hz, 1H), 7.62 (d, J =7.6Hz, 1H), 7.29 (t, J=7.7Hz, 1H), 4.47 (s, 1H), 2.61 (s, 3H). LC-MS (ESI) m / z 158.9[MH] - .

[0104] Step 4: Preparation of 3-ethynyl-2-methyl-N-(3-((4-methylpiperazine-1-yl)methyl)-5-(trifluoromethyl)phenyl)benzamide (Compound 6)

[0105]

[0106] 2.3 g of compound 4 and 3.4 g of compound 5 were dissolved in 40 mL of N,N-dimethylformamide (DMF), and 9.12 g (24 mmol) of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and 2.3 g (18 mmol) of N,N-diisopropylethylamine were added. The mixture was heated and stirred at 70°C for 2 hours. After spin-drying the reaction system, water was added and extracted with ethyl acetate, washed with water, dried with anhydrous sodium sulfate, and spin-dried the solvent, 3.4 g (yield 70%) of yellow oily substance was obtained by column chromatography.

[0107] 1 H NMR (400 MHz, DMSO- d 6) δ 10.52 (s, 1H), 8.17 (s, 1H), 8.10 (s, 1H), 8.00(d, J =8.0Hz, 1H), 7.91 (m, 1H), 7.47 (d, J=8.0Hz, 1H), 7.34 (s, 1H), 4.51 (s, 1H),3.53 (s, 2H), 2.46 (s, 3H), 2.39 (s, 4H), 2.33 (s, 4H), 2.15 (s, 3H). LC-MS (ESI) m / z 416.3 [M+H] + .

[0108] Step 5: Preparation of 3-(imidazo[1,2-b]pyridazine-3-ylethinyl)-2-methyl-N-(3-((4-methylpiperazine-1-yl)methyl)-5-(trifluoromethyl)phenyl)benzamide (XS116)

[0109]

[0110] 210 mg (0.51 mmol) of Compound 6 and 120 mg (0.61 mmol) of Compound 7 were dissolved in 10 mL of anhydrous N,N-dimethylformamide (DMF), after which 19 mg (0.1 mmol) of cuprous iodide, 35 mg (0.05 mmol) of bis(triphenylphosphine)palladium dichloride, and 131 mg (1.02 mmol) of N,N-diisopropylethylamine were added, the system was replaced with argon gas, and the reaction system was sealed. The mixture was heated and stirred at 80°C and reacted overnight. The solution was filtered through diatomite, and the filtrate was spin-dried. Subsequently, 70 mg (yield 26%) of a yellowish-white solid was obtained by column chromatography.

[0111] 1 H NMR (400 MHz, Chloroform- d ) δ 8.50 (dd, J =4.4, 1.6Hz, 1H), 8.07 (s, 1H), 8.02 (dd, J =9.2, 1.7Hz, 1H), 7.94 (s, 1H), 7.81 (d, J =4.5Hz, 2H), 7.73 (dd, J =7.7, 1.4Hz, 1H), 7.50 (d, J =7.8Hz, 1H), 7.41 (s, 1H), 7.32 (t, J =7.7Hz, 1H), 7.16 (dd,J =9.2, 4.4Hz, 1H), 3.62 (s, 2H), 2.76 (s, 3H), 2.64 (s, 8H), 2.41 (s, 3H). LC-MS (ESI) m / z 533.3[M+H] + .

[0112] Example 2: Preparation of 3-(imidazo[1,2-a]pyrimidine-3-ylethinyl)-2-methyl-N-(3-((4-methylpiperazine-1-yl)methyl)-5-(trifluoromethyl)phenyl)benzamide (named XS2-161)

[0113]

[0114] The synthesis method is the same as in Example 1.

[0115] 1 H NMR (400 MHz, Chloroform- d ) δ 9.06 (s, 1H), 8.64 (dd, J =4.1, 2.0Hz, 1H), 8.60 (dd, J =6.8, 2.0Hz, 1H), 8.12 (s, 1H), 8.03 (s, 1H), 7.90 (s, 1H), 7.58 (dd, J =7.8, 1.3Hz, 1H), 7.50 (dd, J =7.8, 1.3Hz, 1H), 7.40 (s, 1H), 7.25 (t, J =7.7Hz, 1H), 7.09 (dd, J =6.8, 4.1Hz, 1H), 3.70 (s, 2H), 3.11 (q, J =7.3Hz, 4H), 2.91 (d, J =5.0Hz, 4H), 2.73 (s, 3H), 2.61 (s, 3H). LC-MS (ESI) m / z 533.2[M+H] + .

[0116] Example 3: Preparation of 3-(imidazo[1,2-b]pyridazine-3-ylethinyl)-2-methyl-N-(3-(trifluoromethyl)phenyl)benzamide (named XS2-106)

[0117]

[0118] Step 1: Preparation of methyl 3-(imidazo[1,2-b]pyridazine-3-ylethinyl)-2-methylbenzoate (Compound 3)

[0119]

[0120] 2.5 g (14 mmol) of Compound 1 and 3.4 g (17 mmol) of Compound 2 were dissolved in 40 mL of anhydrous N,N-dimethylformamide (DMF), after which 533 mg (2.8 mmol) of cuprous iodide, 982 mg (1.4 mmol) of bis(triphenylphosphine)palladium dichloride, and 3.6 g (28 mmol) of N,N-diisopropylethylamine were added, the mixture was switched to argon gas, and the reaction system was sealed. The mixture was heated and stirred at 80°C and reacted overnight. The solution was filtered through diatomite, and the filtrate was spin-dried to obtain 1.38 g (yield 34%) of a yellow powdery solid by column chromatography.

[0121] 1 H NMR (400 MHz, DMSO- d 6) δ 8.74 (d, J =4.3Hz, 1H), 8.27 (d, J =8.9Hz, 2H), 7.83 (d, J =7.7Hz, 1H), 7.75 (d, J =7.5Hz, 1H), 7.44-7.41 (m, 1H), 7.39 (d, J =7.6Hz, 1H), 3.85(s, 3H), 2.76 (s, 3H). LC-MS (ESI) m / z 292.3[M+H] + .

[0122] Step 2: 3-(imidazo[1,2-b]pyridazine-3-ylethynyl)-2-methylbenzoic acid (compound 4)

[0123]

[0124] 1.38 g (4.7 mmol) of Compound 3 was dissolved in a mixed solvent of tetrahydrofuran, methanol, and water in a volume ratio of 10:1:5, and then 995 mg (24 mmol) of hydrated lithium hydroxide was added and stirred at 60°C for 1 hour. After filtration, the reaction system was spin-dried, and then a 4 M hydrochloric acid solution was added until the reaction system became acidic, at which point a solid precipitated; the solid was collected by filtration and dried to obtain 1.05 g (yield 81%) of yellow solid.

[0125] 1 H NMR (400 MHz, DMSO- d 6) δ 13.14 (s, 1H), 8.72 (d, J =3.9Hz, 1H), 8.32-8.19 (m, 2H), 7.83 (d, J =7.6Hz, 1H), 7.75 (d, J =7.3Hz, 1H), 7.44-7.34 (m, 2H), 2.76 (s, 3H). LC-MS (ESI) m / z 276.8[MH] - .

[0126] Step 3: 3-(imidazo[1,2-b]pyridazine-3-ylethinyl)-2-methyl-N-(3-(trifluoromethyl)phenyl)benzamide(XS2-106)

[0127]

[0128] 100 mg (0.36 mmol) of Compound 4 and 48 mg (0.3 mmol) of Compound 5 were dissolved in 10 mL of N,N-dimethylformamide (DMF), and then 137 mg (0.36 mmol) of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and 77 mg (0.6 mmol) of N,N-diisopropylethylamine were added. The mixture was heated and stirred at 80°C and reacted overnight. After spin-drying the reaction system, 51 mg (yield 40%) of a yellowish-white solid was obtained by column chromatography.

[0129] 1H NMR (400MHz, Chloroform- d ) δ 8.51 (d, J =4.1Hz, 1H), 8.03 (dd, J =23.3, 14.3Hz, 3H), 7.89 (d, J =7.4Hz, 1H), 7.73 (d, J =6.6Hz, 2H), 7.50 (ddd, J =24.8, 16.6, 7.8Hz, 3H), 7.32 (t, J =7.7Hz, 1H), 7.17 (dd, J =9.1, 4.4Hz, 1H), 2.76 (s, 3H). LC-MS (ESI) m / z 419.2[M-H] - .

[0130] Example 4: Preparation of N-(3-fluoro-5-(trifluoromethyl)phenyl)-3-(imidazo[1,2-b]pyridazine-3-ylethinyl)-2-methylbenzamide (named XS2-109)

[0131]

[0132] 합성 방법은 실시예 3과 같다.

[0133] 1 H NMR (400MHz, Chloroform- d ) δ 8.53 (d, J =4.2Hz, 1H), 8.08 (s, 2H), 7.91 (d, J =10.1Hz, 1H), 7.81 (s, 1H), 7.74 (d, J =7.8Hz, 1H), 7.59 (s, 1H), 7.50 (d, J =7.5Hz, 1H), 7.33 (t, J =7.7Hz, 1H), 7.19 (s, 1H), 7.16 (d, J =8.2Hz, 1H), 2.75 (s, 3H). LC-MS (ESI) m / z 437.6[M-H] - .

[0134] Example 5: Preparation of N-(3-chloro-5-(trifluoromethyl)phenyl)-3-(imidazo[1,2-b]pyridazine-3-ylethinyl)-2-methylbenzamide (named XS2-112)

[0135]

[0136] The synthesis method is the same as in Example 3.

[0137] 1 H NMR (400 MHz, Chloroform- d ) δ 8.50 (dd, J =4.4, 1.5Hz, 1H), 8.06 (d, J =4.2Hz, 2H), 8.01 (dd, J =9.2, 1.6Hz, 1H), 7.85 (s, 1H), 7.80 (s, 1H), 7.74 (d, J =6.7Hz, 1H), 7.48 (d, J =6.9Hz, 1H), 7.44 (s, 1H), 7.32 (t, J =7.7Hz, 1H), 7.16 (dd, J =9.2, 4.4Hz, 1H), 2.75 (s, 3H). LC-MS (ESI) m / z 455.5[M+H] + .

[0138] Example 6: Preparation of 2-methyl-3-((6-morpholinimidazo[1,2-b]pyridazine-3-yl)ethynyl)-N-(3-(trifluoromethyl)phenyl)benzamide (named XS3-23)

[0139]

[0140] Step 1: Preparation of 3-ethynyl-2-methyl-N-(3-(trifluoromethyl)phenyl)benzamide (Compound 3)

[0141]

[0142] 120 mg (0.74 mmol) of Compound 1 and 101 mg (0.62 mmol) of Compound 2 were dissolved in 15 mL of N,N-dimethylformamide (DMF), and then 353 mg (0.93 mmol) of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and 160 mg (1.24 mmol) of N,N-diisopropylethylamine were added. The mixture was heated and stirred at 80°C for 2 hours. After spin-drying the reaction system, water was added and extracted with ethyl acetate, washed with water, dried with anhydrous sodium sulfate, and the solvent was spin-dried. Subsequently, 150 mg (yield 80%) of a yellow oily substance was obtained by column chromatography.

[0143] 1 H NMR (400 MHz, DMSO- d 6) δ 10.75 (s, 1H), 8.26 (d, J =2.0Hz, 1H), 7.95-7.90 (m, 1H), 7.63-7.58 (m, 2H), 7.53 (dd, J =7.7, 1.4Hz, 1H), 7.49-7.45 (m, 1H), 7.35 (t, J =7.7Hz, 1H), 4.52 (s, 1H), 2.47 (s, 3H). LC-MS (ESI) m / z 304.1 [M+H] + .

[0144] Step 2: Preparation of 4-(3-iodoimidazo[1,2-b]pyridazine-6-yl)morpholine (Compound 5)

[0145]

[0146] 300 mg (1.07 mmol) of Compound 4 was dissolved in 15 mL of dimethyl sulfoxide (DMSO), and then 280 mg (3.22 mmol) of morpholine and 744 mg (12.84 mg) of potassium fluoride were added. The mixture was heated and stirred at 120°C for 3 hours. After filtration, the reaction system was spin-dried, and 260 mg (yield 74%) of yellow powdery solid was obtained by column chromatography.

[0147] 1 H NMR (400 MHz, Chloroform- d ) δ 7.70 (d, J =9.8Hz, 1H), 7.64 (s, 1H), 6.82 (d, J =9.9Hz, 1H), 3.89 (t, J =4.8Hz, 4H), 3.57 (t, J =4.9Hz, 4H). LC-MS (ESI) m / z 331.0 [M+H] + .

[0148] Step 3: Preparation of 2-methyl-3-((6-morpholinimidazo[1,2-b]pyridazine-3-yl)ethynyl)-N-(3-(trifluoromethyl)phenyl)benzamide (XS3-23)

[0149]

[0150] 138 mg (0.45 mmol) of Compound 3 and 300 mg (0.91 mmol) of Compound 5 were dissolved in 10 mL of anhydrous N,N-dimethylformamide (DMF), after which 7 mg (0.036 mmol) of cuprous iodide, 21 mg (0.023 mmol) of trisdibenzylideneacetone-dipalladium, 9 mg (0.045 mmol) of tritert-butylphosphine, and 124 mg (0.9 mmol) of potassium carbonate were added, the system was switched to argon gas, and the reaction system was sealed. The mixture was heated and stirred at 80°C and reacted overnight. The solution was filtered through diatomite, and the filtrate was spin-dried to obtain 120 mg (yield 52%) of a yellowish-white solid by column chromatography.

[0151] 1 H NMR (400 MHz, Chloroform- d ) δ 8.06-7.98 (m, 2H), 7.91 (d, J =8.1Hz, 1H), 7.87-7.74 (m, 2H), 7.64 (d, J =7.7Hz, 1H), 7.54 (t, J =8.0Hz, 1H), 7.47 (d,J =6.5Hz, 2H), 7.30 (d, J =7.6Hz, 1H), 6.90 (d, J =9.5Hz, 1H), 3.87 (t, J =4.7Hz, 4H), 3.57 (t, J =4.8Hz, 4H), 2.73 (s, 3H). LC-MS (ESI) m / z 504.2 [MH] - .

[0152] Example 7: Preparation of N-(3-chloro-5-(trifluoromethyl)phenyl)-2-methyl-3-((6-mopolinoimidazo[1,2-b]pyridazine-3-yl)ethynyl)benzamide (named XS3-61)

[0153]

[0154] The synthesis method is the same as in Example 6.

[0155] 1 H NMR (400 MHz, DMSO- d 6) δ 10.92 (s, 1H), 8.13 (d, J =2.5Hz, 2H), 7.98 (d, J =10.0Hz, 2H), 7.70 (dd, J =7.7, 1.4Hz, 1H), 7.61 (s, 1H), 7.56 (dd, J =7.7, 1.4Hz, 1H), 7.42 (t, J =7.7Hz, 1H), 7.32 (d, J =9.8Hz, 1H), 3.74 (t, J =4.8Hz, 4H), 3.53 (t, J =4.8Hz, 4H), 2.63 (s, 3H). LC-MS (ESI) m / z 539.8 [M+H] + .

[0156] Example 8: Preparation of N-(3-fluoro-5-(trifluoromethyl)phenyl)-2-methyl-3-((6-methylimidazopyrazine-3-yl-3-yl)benzamide (designated XS4-80)

[0157]

[0158] The synthesis method is the same as in Example 6.

[0159] 1 H NMR (400 MHz, DMSO- d6) δ 10.94 (s, 1H), 8.02-7.96 (m, 2H), 7.96-7.89 (m, 2H), 7.70 (dd, J =7.8, 1.4Hz, 1H), 7.55 (dd, J =7.8, 1.4Hz, 1H), 7.42 (dd, J =8.9, 6.3Hz, 2H), 7.31 (d, J =10.0Hz, 1H), 3.74 (t, J =4.8Hz, 4H), 3.53 (t, J =4.8Hz, 4H), 2.63 (s, 3H). HRMS (ESI) for C 27 H21F4N5O2[M+H] + : Calculated value (calcd) 524.1704, Measured value (found) 524.1686.

[0160] Example 9: Preparation of 2-methyl-N-(3-methyl-5-(trifluoromethyl)phenyl)-3-((6-methyl-1,2-B]pyridazine-3-yl)benzamide (designated XS4-81)

[0161]

[0162] The synthesis method is the same as in Example 6.

[0163] 1 H NMR (400 MHz, DMSO- d 6) δ 10.68 (s, 1H), 8.05-7.90 (m, 3H), 7.80 (s, 1H), 7.68 (dd, J =7.7, 1.4Hz, 1H), 7.52 (dd, J =7.7, 1.4Hz, 1H), 7.40 (t, J =7.7Hz, 1H), 7.34-7.28 (m, 2H), 3.74 (dd, J =5.8, 3.9Hz, 4H), 3.53 (t, J =4.8Hz, 4H), 2.62 (s, 3H), 2.40 (s, 3H). HRMS (ESI) for C 28 H 24 F3N5O2[M+H] + : Calculated value (calcd) 520.1955, Measured value (found) 520.1939.

[0164] Example 10: Preparation of N-(3-methoxy-5-(trifluoromethyl)phenyl)-2-methyl-3-((6-methylimidazopyrazine-3-yl-3-yl)benzamide (named XS4-72)

[0165]

[0166] The synthesis method is the same as in Example 6.

[0167] 1 H NMR (400 MHz, DMSO- d 6) δ 10.72 (s, 1H), 7.97 (d, J =9.9Hz, 1H), 7.93 (s, 1H), 7.81 (s, 1H), 7.68 (dd, J =7.8, 1.4Hz, 1H), 7.62 (t, J =2.2Hz, 1H), 7.53 (dd, J =7.8, 1.4Hz, 1H), 7.41 (t, J =7.7Hz, 1H), 7.31 (d, J =9.9Hz, 1H), 7.01 (t, J =2.0Hz, 1H), 3.84 (s, 3H), 3.74 (dd, J =5.8, 3.8Hz, 4H), 3.53 (t, J =4.8Hz, 4H), 2.62 (s, 3H). HRMS (ESI) for C 27 H21ClF3N5O2[M+H] + : Calculated value (calcd) 536.1904, Measured value (found) 536.1919.

[0168] Example 11: Preparation of N-(3,5-bis(trifluoromethyl)phenyl)-2-methyl-3-((6-methylimidazopyrazine-3-yl-3-yl)benzamide (named XS4-76)

[0169]

[0170] The synthesis method is the same as in Example 6.

[0171] 1 H NMR (400 MHz, DMSO- d 6) δ 11.07 (s, 1H), 8.44 (s, 2H), 7.98 (d, J =10.0Hz, 2H), 7.85 (s, 1H), 7.71 (dd, J=7.8, 1.4Hz, 1H), 7.59 (dd, J =7.8, 1.4Hz, 1H), 7.43 (t, J =7.7Hz, 1H), 7.32 (d, J =9.5Hz, 1H), 3.74 (t, J =4.8Hz, 4H), 3.53 (t, J =4.8Hz, 4H), 2.64 (s, 3H). HRMS (ESI) for C 28 H21F6N5O2[M+H] + : Calculated value (calcd) 574.1672, Measured value (found) 574.1676.

[0172] Example 12: Preparation of 2-methyl-3-((6-methylimidazopyrazine-3-yl)ethynyl)-N-(3-nitro-5-(trifluoromethyl)phenyl)benzamide (designated XS4-77)

[0173]

[0174] The synthesis method is the same as in Example 6.

[0175] 1 H NMR (400 MHz, DMSO- d 6) δ 11.20 (s, 1H), 8.95 (t, J =2.1Hz, 1H), 8.53 (s, 1H), 8.23 ​​(d, J =2.1Hz, 1H), 7.97 (d, J =9.9Hz, 1H), 7.93 (s, 1H), 7.72 (dd, J =7.8, 1.4Hz, 1H), 7.60 (dd, J =7.7, 1.4Hz, 1H), 7.44 (t, J =7.7Hz, 1H), 7.31 (dd, J =10.1, 1.6Hz, 1H), 3.74 (t, J =4.8Hz, 4H), 3.53 (t, J =4.9Hz, 4H), 2.65 (s, 3H). HRMS (ESI) for C 27 H21F3N6O4[M+H] + : Calculated value (calcd) 551.1649, Measured value (found) 551.1667.

[0176] Example 13: Preparation of 2-methyl-N-(3-((4-methylpiperazine-1-yl)methyl)-5-(trifluoromethyl)phenyl)-3-((6-morpholinimidazo[1,2-b]pyridazine-3-yl)ethynyl)benzamide (named XS3-68)

[0177]

[0178] The synthesis method is the same as in Example 6.

[0179] 1 H NMR (400 MHz, Chloroform- d ) δ 8.22 (s, 1H), 7.98 (s, 1H), 7.82 (s, 1H), 7.77 (s, 1H), 7.70 (d, J =9.8Hz, 1H), 7.62 (dd, J =7.7, 1.4Hz, 1H), 7.45-7.40 (m, 2H), 7.26 (d, J =7.7Hz, 1H), 6.86 (d, J =9.9Hz, 1H), 3.88-3.84 (m, 4H), 3.59 (s, 2H), 3.57-3.53 (m, 4H), 2.71 (s, 3H), 2.52 (s, 8H), 2.32 (s, 3H). LC-MS (ESI) m / z 618.3 [M+H] + .

[0180] Example 14: Preparation of (S)-3-((6-(3-hydroxypyrrolidin-1-yl)imidazo[1,2-b]pyridazine-3-yl)ethynyl)-2-methyl-N-(3-(trifluoromethyl)phenyl)benzamide (named XS3-35)

[0181]

[0182] The synthesis method is the same as in Example 6.

[0183] 1 H NMR (400 MHz, Chloroform- d ) δ 8.16 (s, 1H), 8.05 (s, 1H), 7.91 (d, J =8.1Hz, 1H), 7.74 (s, 1H), 7.65-7.57 (m, 2H), 7.54 (t, J =7.9Hz, 1H), 7.46 (d, J =7.8Hz, 1H), 7.41 (d, J =7.9Hz, 1H), 7.24 (t, J =7.6Hz, 1H), 6.58 (d, J=8.8Hz, 1H), 5.03 (d, J =3.7Hz, 1H), 4.42 (s, 1H), 3.62-3.50 (m, 3H), 3.40 (d, J =11.3Hz, 1H), 2.67 (s, 3H), 2.10-1.98 (m, 1H), 1.92 (d, J =12.9Hz, 1H).

[0184] .LC-MS (ESI) m / z 504.2 [MH] - .

[0185] Example 15: Preparation of (S)-N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-(3-hydroxypyrrolidin-1-yl)imidazo[1,2-b]pyridazine-3-yl)ethynyl)-2-methylbenzamide (named XS3-58)

[0186]

[0187] The synthesis method is the same as in Example 6.

[0188] 1 H NMR (400 MHz, Chloroform- d ) δ 8.27 (s, 1H), 8.08 (s, 1H), 7.86 (s, 1H), 7.72 (s, 1H), 7.64-7.59 (m, 2H), 7.44 (d, J =1.8Hz, 1H), 7.39 (dd, J =7.7, 1.4Hz, 1H), 7.25 (t, J =7.7Hz, 1H), 6.60 (d, J =9.6Hz, 1H), 4.66 (s, 1H), 3.70-3.54 (m, 5H), 2.71 (s, 3H), 2.16 (dd, J =7.9, 3.9Hz, 2H). LC-MS (ESI) m / z 540.0 [M+H] + .

[0189] Example 16: Preparation of (S)-3-((6-(3-hydroxypyrrolidin-1-yl)imidazo[1,2-b]pyridazine-3-yl)ethinyl)-2-methyl-N-(3-((4-methylpiperazine-1-yl)methyl)-5-(trifluoromethyl)phenyl)benzamide (named XS3-36)

[0190]

[0191] The synthesis method is the same as in Example 6.

[0192] 1 H NMR (400 MHz, Chloroform- d) δ 8.18 (s, 1H), 8.02 (s, 1H), 7.79 (s, 1H), 7.72 (s, 1H), 7.63-7.58 (m, 2H), 7.41 (d, J =5.1Hz, 2H), 7.25 (t, J =7.7Hz, 1H), 6.58 (d, J =9.7Hz, 1H), 4.64 (dd, J =4.3, 2.2Hz, 1H), 3.68-3.56 (m, 7H), 2.74 (s, 3H), 2.51 (s, 8H), 2.31 (s, 3H), 2.17-2.09 (m, 2H). LC-MS (ESI) m / z 618.3 [M+H] + .

[0193] Example 17: Preparation of 3-((6-(4-(dimethylamino)piperidine-1-yl)imidazo[1,2-b]pyridazine-3-yl)ethynyl)-2-methyl-N-(3-(trifluoromethyl)phenyl)benzamide (named XS3-57)

[0194]

[0195] 합성 방법은 실시예 6과 같다.

[0196] 1 H NMR (400MHz, DMSO- d 6) δ 10.78 (s, 1H), 8.27 (d, J =2.3Hz, 1H), 7.95 (d, J =2.6Hz, 1H), 7.93 (d, J =3.2Hz, 1H), 7.90 (s, 1H), 7.68 (dd, J =7.7, 1.4Hz, 1H), 7.61 (t, J =8.1Hz, 1H), 7.55 (dd, J =7.7, 1.4Hz, 1H), 7.48 (d, J =7.8Hz, 1H), 7.42 (t, J =7.7Hz, 1H), 7.34 (d, J =10.0Hz, 1H), 4.30 (d, J =13.4Hz, 2H), 2.97 (t, J =12.0Hz, 2H), 2.65 (s, 3H), 2.34 (s, 6H), 1.90 (d,J =12.1Hz, 2H), 1.57-1.41 (m, 3H). LC-MS (ESI) m / z 547.2 [M+H] + .

[0197] Example 18: Preparation of N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-(4-(dimethylamino)piperidine-1-yl)imidazo[1,2-b]pyridazine-3-yl)ethynyl)-2-methylbenzamide (named XS3-56)

[0198]

[0199] The synthesis method is the same as in Example 6.

[0200] 1 H NMR (400 MHz, Chloroform- d ) δ 8.30 (s, 1H), 8.09 (d, J =2.3Hz, 1H), 7.86 (s, 1H), 7.74 (s, 1H), 7.66-7.60 (m, 2H), 7.44 (s, 1H), 7.40 (dd, J =7.7, 1.4Hz, 1H), 7.28-7.23 (m, 1H), 6.90 (d, J =9.9Hz, 1H), 4.26 (d, J =12.9Hz, 2H), 2.98 (t, J =12.8Hz, 2H), 2.72 (s, 3H), 2.41 (t, J =11.2Hz, 1H), 2.33 (s, 6H), 2.01-1.93 (m, 2H), 1.66-1.54 (m, 2H). LC-MS (ESI) m / z 579.3 [MH] - .

[0201] Example 19: Preparation of 3-((6-(4-(dimethylamino)piperidine-1-yl)imidazo[1,2-b]pyridazine-3-yl)ethynyl)-2-methyl-N-(3-((4-methylpiperazine-1-yl)methyl)-5-(trifluoromethyl)phenyl)benzamide (named XS3-67)

[0202]

[0203] The synthesis method is the same as in Example 6.

[0204] 1 H NMR (400 MHz, DMSO- d 6) δ 10.75 (s, 1H), 8.15 (s, 1H), 7.92 (d, J =9.8Hz, 2H), 7.89 (s, 1H), 7.67 (d, J=6.4Hz, 1H), 7.54 (d, J =6.9Hz, 1H), 7.40 (t, J =7.7Hz, 1H), 7.37 (s, 1H), 7.32 (d, J =10.0Hz, 1H), 4.26 (d, J =13.0Hz, 2H), 3.54 (s, 2H), 2.96 (t, J =12.1Hz, 2H), 2.64 (s, 3H), 2.45-2.29 (m, 8H), 2.18 (s, 6H), 2.16 (s, 3H), 2.00 (q, J =7.5Hz, 1H), 1.83 (d, J =11.5Hz, 2H), 1.44 (q, J =10.5, 9.3Hz, 2H). LC-MS (ESI) m / z 658.3 [M+H] + .

[0205] Example 20: Preparation of N-(3-chloro-5-(trifluoromethyl)phenyl)-2-methyl-3-((6-(4-methylpiperazine-1-yl)imidazo[1,2-b]pyridazine-3-yl)ethynyl)benzamide (named XS3-51)

[0206]

[0207] 합성 방법은 실시예 6과 같다.

[0208] 1 H NMR (400MHz, Chloroform- d ) δ 8.08 (s, 2H), 7.86 (s, 1H), 7.83 (d, J =3.4Hz, 1H), 7.78-7.72 (m, 1H), 7.66 (d, J =7.6Hz, 1H), 7.46 (d, J =7.7Hz, 1H), 7.43 (s, 1H), 7.32 (d, J =7.7Hz, 1H), 6.89 (d, J =9.9Hz, 1H), 3.80 (s, 4H), 3.13 (s, 3H), 2.74 (s, 3H), 2.53 (d, J =21.6Hz, 4H). LC-MS (ESI) m / z 553.2 [M+H] + .

[0209] Example 21: Preparation of (R)-N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-(3,4-dimethylpiperazine-1-yl)imidazo[1,2-b]pyridazine-3-yl)ethinyl)-2-methylbenzamide (named XS3-52)

[0210]

[0211] The synthesis method is the same as in Example 6.

[0212] 1 H NMR (400 MHz, DMSO- d 6) δ 10.93 (s, 1H), 8.13 (s, 2H), 8.06 (s, 1H), 7.97 (s, 1H), 7.71 (d, J =7.2Hz, 1H), 7.62 (s, 1H), 7.58 (d, J =7.3Hz, 1H), 7.44 (t, J =7.6Hz, 1H), 7.39 (d, J =9.5Hz, 1H), 3.13―3.06 (m, 4H), 2.91 (s, 2H), 2.75 (m, 1H), 2.65 (s, 3H), 2.46 (s, 3H), 1.24 (s, 3H). LC-MS (ESI) m / z 567.2 [M+H] + .

[0213] Example 22: Preparation of N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-(4-hydroxypiperidin-1-yl)imidazo[1,2-b]pyridazine-3-yl)ethinyl)-2-methylbenzamide (named XS3-55)

[0214]

[0215] The synthesis method is the same as in Example 6.

[0216] 1 H NMR (400 MHz, DMSO- d 6) δ 10.93 (s, 1H), 8.14 (d, J =1.8Hz, 2H), 7.94-7.88 (m, 2H), 7.69 (dd, J =7.7, 1.4Hz, 1H), 7.61 (s, 1H), 7.56 (dd, J =7.7, 1.4Hz, 1H), 7.42 (t, J =7.7Hz, 1H), 7.32 (d, J =10.0Hz, 1H), 4.71 (d, J=4.2Hz, 1H), 4.00-3.93 (m, 2H), 3.74 (m, 1H), 3.25 (t, J =13.1Hz, 2H), 2.64 (s, 3H), 1.82 (d, J =12.8Hz, 2H), 1.49-1.40 (m, 2H). LC-MS (ESI) m / z 552.1[MH] - .

[0217] Example 23: Preparation of 3-((6-(4-acetylpiperazine-1-yl)imidazo[1,2-b]pyridazine-3-yl)ethinyl)-N-(3-chloro-5-(trifluoromethyl)phenyl)-2-methylbenzamide (named XS3-54)

[0218]

[0219] The synthesis method is the same as in Example 6.

[0220] 1 H NMR (400 MHz, DMSO- d 6) δ 10.93 (s, 1H), 8.14 (d, J =1.7Hz, 2H), 7.98 (d, J =9.9Hz, 1H), 7.93 (s, 1H), 7.71 (dd, J =7.7, 1.3Hz, 1H), 7.61 (d, J =2.0Hz, 1H), 7.57 (dd, J =7.7, 1.4Hz, 1H), 7.43 (t, J =7.7Hz, 1H), 7.34 (d, J =10.0Hz, 1H), 3.66-3.53 (m, 8H), 2.65 (s, 3H), 2.04 (s, 3H). LC-MS (ESI) m / z581.5 [M+H] + .

[0221] Example 24: Preparation of N-(3-chloro-5-(trifluoromethyl)phenyl)-2-methyl-3-((6-((2-morpholinoethyl)amino)imidazo[1,2-b]pyridazine-3-yl)ethynyl)benzamide (named XS3-53)

[0222]

[0223] The synthesis method is the same as in Example 6.

[0224] 1 H NMR (400 MHz, Chloroform- d) δ 8.28 (s, 1H), 8.08 (s, 1H), 7.87 (s, 1H), 7.66 (d, J =7.7Hz, 2H), 7.47 (d, J =7.5Hz, 1H), 7.44 (s, 1H), 7.34 (d, J =7.9Hz, 1H), 7.31 (s, 1H), 6.73 (s, 2H), 4.01 (s, 4H), 3.78 (s, 2H), 3.19 (s, 2H), 3.05 (s, 4H), 2.71 (s, 3H). LC-MS (ESI) m / z583.2 [M+H] +

[0225] Example 25: Preparation of 3-((6-(4-hydroxypiperidin-1-yl)imidazo[1,2-b]pyridazine-3-yl)ethynyl)-2-methyl-N-(3-(((4-methylpiperazine-1-yl)methyl)-5-(trifluoromethyl)phenyl)benzamide (named XS3-81)

[0226]

[0227] 합성 방법은 실시예 6과 같다.

[0228] 1 H NMR (400MHz, Chloroform- d ) δ 8.36 (s, 1H), 8.01 (s, 1H), 7.80 (s, 1H), 7.73 (s, 1H), 7.62 (d, J =9.9Hz, 1H), 7.59 (dd, J =7.8, 1.4Hz, 1H), 7.43-7.38 (m, 2H), 7.24 (t, J =7.7Hz, 1H), 6.88 (d, J =9.9Hz, 1H), 4.0-3.91 (m, 3H), 3.59 (s, 2H), 3.28 (t, J =13.1Hz, 2H), 2.70 (s, 3H), 2.51 (s, 8H), 2.31 (s, 3H), 1.99-1.95 (m, 2H), 1.69 -1.61 (m, 2H).LC-MS (ESI) m / z632.3[M+H] + .

[0229] Example 26: Preparation of N-(3-chloro-5-(trifluoromethyl)phenyl)-2-methyl-3-((6-(4-morpholinepiperidin-1-yl)imidazo[1,2-b]pyridazine-3-yl)ethynyl)benzamide (named XS3-130)

[0230]

[0231] The synthesis method is the same as in Example 6.

[0232] 1 H NMR (400 MHz, DMSO- d 6) δ 10.93 (s, 1H), 8.13 (s, 2H), 7.92 (d, J =9.8Hz, 2H), 7.69 (d, J =7.3Hz, 1H), 7.61 (s, 1H), 7.56 (d, J =7.4Hz, 1H), 7.42 (t, J =7.7Hz, 1H), 7.33 (d, J =9.8Hz, 1H), 4.26 (d, J =13.0Hz, 2H), 3.54 (s, 4H), 2.97 (t, J =12.3Hz, 2H), 2.64 (s, 3H), 2.45 (s, 5H), 1.86 (d, J =12.8Hz, 2H), 1.45 (d, J =12.2Hz, 2H). HRMS (ESI) for C 32 H 30 ClF3N6O2[M+H] + : Calculated value (calcd) 623.2144, Measured value (found) 623.2126.

[0233] Example 27: Preparation of N-(3-chloro-5-(trifluoromethyl)phenyl)-2-methyl-3-((6-(4-(oxetanyl-3-yl)piperazine-1-yl)imidazo[1,2-b]pyridazine-3-yl)ethinyl)benzamide (named XS3-138)

[0234]

[0235] The synthesis method is the same as in Example 6.

[0236] 1 H NMR (400 MHz, DMSO- d 6) δ 10.92 (s, 1H), 8.13 (s, 2H), 7.98-7.87 (m, 2H), 7.69 (d, J =8.2Hz, 1H), 7.61 (s, 1H), 7.56 (d, J =6.9Hz, 1H), 7.42 (t, J =7.7Hz, 1H), 7.34 (d, J=9.9Hz, 1H), 4.55 (t, J =6.5Hz, 2H), 4.47 (t, J =6.1Hz, 2H), 3.60 (t, J =5.1Hz, 4H), 3.45 (m, 1H), 2.63 (s, 3H), 2.40 (t, J =5.0Hz, 4H). HRMS (ESI) for C 30 H 26 ClF3N6O2[M+H] + : Calculated value (calcd) 595.1831, Measured value (found) 595.1811.

[0237] Example 28: Preparation of N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-((2-(dimethylamino)ethyl)(methyl)amino)imidazo[1,2-b]pyridazine-3-yl)ethynyl)-2-methylbenzamide (named XS3-134)

[0238]

[0239] The synthesis method is the same as in Example 6.

[0240] 1 H NMR (400 MHz, DMSO- d 6) δ 10.92 (s, 1H), 8.13 (d, J =1.8Hz, 2H), 7.90-7.82 (m, 2H), 7.68 (dd, J =7.7, 1.4Hz, 1H), 7.60 (d, J =1.8Hz, 1H), 7.55 (dd, J =7.8, 1.3Hz, 1H), 7.41 (t, J =7.7Hz, 1H), 7.14 (d, J =10.0Hz, 1H), 3.64 (t, J =6.7Hz, 2H), 3.09 (s, 3H), 2.63 (s, 3H), 2.45 (t, J =6.7Hz, 2H), 2.16 (s, 6H). HRMS (ESI) for C 28 H 26 ClF3N6O [M+H] + : Calculated value (calcd) 555.1881, Measured value (found) 555.1862.

[0241] Example 29: Preparation of N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-((2-methoxyethyl)amino)imidazo[1,2-b]pyridazine-3-yl)ethynyl)-2-methylbenzamide (named XS3-135)

[0242]

[0243] The synthesis method is the same as in Example 6.

[0244] 1 H NMR (600 MHz, DMSO- d 6) δ 10.93 (s, 1H), 8.14-8.09 (m, 2H), 7.81-7.73 (m, 2H), 7.67 (dd, J =7.7, 1.3Hz, 1H), 7.59 (d, J =1.9Hz, 1H), 7.54 (dd, J =7.7, 1.3Hz, 1H), 7.41 (t, J =7.7Hz, 1H), 7.27 (t, J =5.5Hz, 1H), 6.82 (d, J =9.6Hz, 1H), 3.54 (t, J =5.5Hz, 2H), 3.47-3.45 (m, 2H), 3.26 (s, 3H), 2.63 (s, 3H). HRMS (ESI) for C 26 H21ClF3N5O2[M+H] + : Calculated value (calcd) 528.1409, Measured value (found) 528.1394.

[0245] Example 30: Preparation of N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-((4-hydroxybutyl)amino)imidazole[1,2-b]pyridazine-3-yl)ethynyl)-2-methylbenzamide (named XS3-139)

[0246]

[0247] The synthesis method is the same as in Example 6.

[0248] 1 H NMR (400 MHz, DMSO- d 6) δ 10.92 (s, 1H), 8.12 (s, 2H), 7.76 (d, J =9.6Hz, 2H), 7.68 (dd, J =7.7, 1.4Hz, 1H), 7.61 (s, 1H), 7.55 (dd, J =7.7, 1.4Hz, 1H), 7.41 (t, J=7.7Hz, 1H), 7.16 (t, J =5.3Hz, 1H), 6.77 (d, J =8.9Hz, 1H), 4.38 (t, J =5.1Hz, 1H), 3.43-3.37 (m, 2H), 3.30-3.25 (m, 2H), 2.65 (s, 3H), 1.70-1.59 (m, 2H), 1.53-1.46 (m, 2H). HRMS (ESI) for C 27 H23ClF3N5O2[M+H] + : Calculated value (calcd) 542.1565, Measured value (found) 542.1552.

[0249] Example 31: Preparation of 3-((6-(4-aminopiperidin-1-yl)imidazole[1,2-b]pyridazine-3-yl)ethinyl)-N-(3-chloro-5-(trifluoromethyl)phenyl)-2-methylbenzamide (named XS3-131)

[0250]

[0251] The synthesis method is the same as in Example 6.

[0252] 1 H NMR (400 MHz, DMSO- d 6) δ 10.96 (s, 1H), 8.15 (s, 2H), 7.96 (d, J =9.9Hz, 1H), 7.92 (s, 1H), 7.70 (dd, J =7.8, 1.3Hz, 1H), 7.61 (d, J =2.3Hz, 1H), 7.57 (dd, J =7.7, 1.3Hz, 1H), 7.43 (t, J =7.7Hz, 1H), 7.34 (d, J =10.0Hz, 1H), 4.23 (d, J =13.4Hz, 2H), 3.17 (s, 2H), 3.12-3.02 (m, 2H), 2.65 (s, 3H), 2.63 (m, 1H), 1.89 (d, J =11.2Hz, 2H), 1.48 (q, J =11.7, 10.8Hz, 2H). HRMS (ESI) for C 28 H 24 ClF3N6O [M+H]+ : Calculated value (calcd) 553.1725, Measured value (found) 553.1708.

[0253] Example 32: Preparation of (R)-3-((6-(3-aminopiperidin-1-yl)imidazole[1,2-b]pyridazine-3-yl)ethinyl)-N-(3-chloro-5-(trifluoromethyl)phenyl)-2-methylbenzamide (named XS3-137)

[0254]

[0255] The synthesis method is the same as in Example 6.

[0256] 1 H NMR (400 MHz, DMSO- d 6) δ 10.93 (s, 1H), 8.13 (s, 2H), 7.92-7.85 (m, 2H), 7.69 (d, J =7.6Hz, 1H), 7.60 (s, 1H), 7.55 (d, J =7.6Hz, 1H), 7.42 (t, J =7.7Hz, 1H), 7.28 (d, J =10.0Hz, 1H), 4.04 (dd, J =10.6, 6.5Hz, 2H), 2.98 (m, 1H), 2.78-2.69 (m, 2H), 2.65 (s, 3H), 1.86 (m, 1H), 1.74 (m, 1H), 1.61-1.43 (m, 2H). HRMS (ESI) for C 28 H 24 ClF3N6O [M+H] + : Calculated value (calcd) 553.1725, Measured value (found) 553.1706.

[0257] Example 33: Preparation of (S)-3-((6-(3-aminopiperidin-1-yl)imidazole[1,2-b]pyridazine-3-yl)ethinyl)-N-(3-chloro-5-(trifluoromethyl)phenyl)-2-methylbenzamide (named XS3-136)

[0258]

[0259] The synthesis method is the same as in Example 6.

[0260] 1 H NMR (400 MHz, DMSO- d 6) δ 10.93 (s, 1H), 8.13 (s, 2H), 7.95-7.84 (m, 2H), 7.69 (d, J=7.6Hz, 1H), 7.60 (s, 1H), 7.55 (d, J =7.6Hz, 1H), 7.42 (t, J =7.7Hz, 1H), 7.28 (d, J =10.0Hz, 1H), 4.04 (dd, J =11.5, 5.9Hz, 2H), 3.04-2.93 (m, 2H), 2.80-2.70 (m, 2H), 2.65 (s, 3H), 1.86 (d, J =12.0Hz, 1H), 1.74 (m, 1H), 1.53 (q, J =11.8Hz, 1H). HRMS (ESI) for C 28 H 24 ClF3N6O [M+H] + : Calculated value (calcd) 553.1725, Measured value (found) 553.1703.

[0261] Example 34: Preparation of N-(3-chloro-5-(trifluoromethyl)phenyl)-3-(6-(4-hydroxy-4-methylpiperidine-1-yl)imidazo[1,2-b]pyridazine-3-yl)ethynyl)-2-methylbenzamide (named XS3-153)

[0262]

[0263] The synthesis method is the same as in Example 6.

[0264] 1 H NMR (400 MHz, DMSO- d 6) δ 10.93 (s, 1H), 8.13 (t, J =2.1Hz, 2H), 7.90 (d, J =10.2Hz, 2H), 7.69 (dd, J =7.7, 1.4Hz, 1H), 7.61 (d, J =1.8Hz, 1H), 7.56 (dd, J =7.7, 1.4Hz, 1H), 7.42 (t, J =7.7Hz, 1H), 7.32 (d, J =10.0Hz, 1H), 4.39 (s, 1H), 3.92-3.83 (m, 2H), 3.44-3.37 (m, 2H), 2.64 (s, 3H), 1.55 (t, J=5.6Hz, 4H), 1.15 (s, 3H). HRMS (ESI) for C 29 H 25 ClF3N5O2[M+H] + : Calculated value (calcd) 568.1722, Measured value (found) 568.1699.

[0265] Example 35: Preparation of 2-methyl-N-(3-(4-methyl-1H-imidazole-1-yl)-5-(trifluoromethyl)phenyl)-3-((6-morpholinomidazo[1,2-b]pyrido-3-ylethinyl)benzamide (named XS3-91)

[0266]

[0267] Step 1: Preparation of 4-methyl-1-(3-nitro-5-(trifluoromethyl)phenyl)-1H-imidazole (Compound 3)

[0268]

[0269] 1 g (4.5 mmol) of Compound 1 and 800 mg (9.5 mmol) of Compound 2 were dissolved in 20 mL of dimethyl sulfoxide (DMSO), and then 850 mg (7 mmol) of potassium carbonate was added. The mixture was heated and stirred at 120°C and reacted overnight. After spin-drying the reaction system, 430 mg (yield 36%) of a yellowish-white solid was obtained by column chromatography.

[0270] 1 H NMR (400 MHz, Chloroform- d ) δ 8.46 (q, J =1.9Hz, 2H), 8.02-7.97 (m, 2H), 7.17 (s, 1H), 2.36 (d, J =1.0Hz, 3H). LC-MS (ESI) m / z 272.1[M+H] + .

[0271] Step 2: Preparation of 3-(4-methyl-1H-imidazole-1-yl)-5-(trifluoromethyl)aniline (Compound 4)

[0272]

[0273] 430 mg (1.59 mmol) of Compound 3 was dissolved in a mixed solvent with an ethanol:water ratio of 7:3, hydrochloric acid was added to make the reaction system weakly acidic, and then 444 mg (7.93 mmol) of iron was added. The mixture was heated and stirred at 70°C for 2 hours. After filtering with diatomaceous earth and spin-drying the reaction system, the crude product was used in the next step of the reaction.

[0274] Step 3: Preparation of 3-ethynyl-2-methyl-N-(3-(4-methyl-1H-imidazole-1-yl)-5-(trifluoromethyl)phenyl)benzamide (Compound 6)

[0275]

[0276] 270 mg (1.12 mmol) of Compound 4 and 215 mg (1.3 mmol) of Compound 5 were dissolved in 15 mL of N,N-dimethylformamide (DMF), and then 638 mg (1.68 mmol) of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and 216 mg (1.68 mmol) of N,N-diisopropylethylamine (DIPEA) were added. The mixture was heated and stirred at 80°C and reacted overnight. After spin-drying the reaction system, water was added, the mixture was extracted with ethyl acetate and washed with water, dried with anhydrous sodium sulfate, and the solvent was spin-dried. Subsequently, 220 mg (yield 51%) of a yellow oily substance was obtained by column chromatography.

[0277] 1 H NMR (400 MHz, Chloroform- d ) δ 9.41 (s, 1H), 8.22 (s, 1H), 7.91 (s, 1H), 7.73 (s, 1H), 7.55 (d, J =7.6Hz, 1H), 7.43 (d, J =7.2Hz, 1H), 7.34 (s, 1H), 7.19 (t, J=7.7Hz, 1H), 7.09 (s, 1H), 3.34 (s, 1H), 2.58 (s, 3H), 2.25 (s, 3H). LC-MS (ESI) m / z 384.1[M+H] + .

[0278] Step 4: Preparation of 2-methyl-N-(3-(4-methyl-1H-imidazole-1-yl)-5-(trifluoromethyl)phenyl)-3-((6-morpholinomidazo[1,2-b]pyrido-3-ylethinyl)benzamide (designated XS3-91)

[0279]

[0280] 150 mg (0.39 mmol) of Compound 6 and 130 mg (0.47 mmol) of Compound 7 were dissolved in 10 mL of anhydrous N,N-dimethylformamide (DMF), after which 6 mg (0.03 mmol) of cuprous iodide, 17 mg (0.019 mmol) of trisdibenzylideneacetone-dipalladium, 8 mg (0.039 mmol) of tritert-butylphosphine, and 107 mg (0.78 mmol) of potassium carbonate were added, the system was switched to argon gas, and the reaction system was sealed. The mixture was heated and stirred at 80°C and reacted overnight. The solution was filtered through diatomite, and the filtrate was spin-dried to obtain 40 mg (yield 18%) of a yellowish-white solid by column chromatography.

[0281] 1 H NMR (400 MHz, DMSO- d 6) δ 10.92 (s, 1H), 8.20 (d, J =1.6Hz, 2H), 8.10 (s, 1H), 7.98 (d, J =9.9Hz, 1H), 7.93 (s, 1H), 7.76 (s, 1H), 7.72-7.69 (m, 1H), 7.57 (d, J =7.4Hz, 1H), 7.48 (s, 1H), 7.44 (d, J =7.7Hz, 1H), 7.32 (d, J =10.0Hz, 1H), 3.77-3.72 (m, 4H), 3.54 (t, J=4.9Hz, 4H), 2.65 (s, 3H), 2.19 (d, J =1.0Hz, 3H). LC-MS (ESI) m / z 586.6[M+H] + .

[0282] Example 36: Preparation of 3-((6-(4-hydroxypiperidin-1-yl)imidazo[1,2-b]pyridazine-3-yl)ethynyl)-2-methyl-N-(3-(4-methyl-1H-imidazole-1-yl)-5-(trifluoromethyl)phenyl)benzamide (named XS3-87)

[0283]

[0284] The synthesis method is the same as in Example 35.

[0285] 1 H NMR (400 MHz, Chloroform- d ) δ 8.25 (s, 1H), 7.97 (s, 1H), 7.88 (s, 1H), 7.83 (s, 1H), 7.75-7.70 (m, 2H), 7.68 (d, J =7.7Hz, 1H), 7.47 (d, J =7.5Hz, 1H), 7.42 (s, 1H), 7.33 (t, J =7.7Hz, 1H), 7.13 (s, 1H), 6.93 (d, J =9.9Hz, 1H), 4.05-3.97 (m, 3H), 3.37-3.29 (m, 2H), 2.78 (s, 3H), 2.33 (d, J =1.0Hz, 3H), 2.03 (d, J =9.6Hz, 2H), 1.69 (d, J =9.5Hz, 3H). LC-MS (ESI) m / z 600.0[M+H] + .

[0286] Example 37: IC50 of a compound against TRKs kinase 50 test

[0287] Kinase activity detection: Z´-LYTE™ technology (fluorescence-based detection based on the difference in sensitivity between phosphorylated and non-phosphorylated polypeptides to protein hydrolytic cleavage, enzyme-linked form) was applied, the fluorescence resonance energy transfer (FRET) principle was adopted, Z´-LYTE™ FRET peptide substrates were used, and the inhibitory activity of the compound against TRKs (TRK1, TRK2, TRK3) kinases (Life Technologies, PV3144, PV3616, PV3617) was detected by a secondary reaction.

[0288] Enzyme reaction: 5 μL of enzyme-substrate system [50 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) pH 7.5, 0.01% BRIJ-35, 10 mM magnesium chloride (MgCl2), 1 mM ethylene glycolbis(2-aminoethyl ether)tetraacetic acid (EGTA), 2 μM Tyr O1 peptide substrate] was added to a 384-well plate, 5 nL of the compound (concentration gradient) was transferred using an echo520 ultramicro liquid pipetting system, and the mixture was shaken at room temperature for 10-20 minutes. Then, 200 nL, 12.5 nL, and 25 nL of ATP (final concentrations of 400 μM, 25 μM, and 50 μM, respectively) were transferred using an echo520 ultramicro liquid pipetting system and the mixture was shaken to mix uniformly. Afterward, centrifugation was performed, and the mixture was reacted in a dark place at 30°C for 1.5 hours.

[0289] Detection reaction: 2.5 μL of development solution (1:128 dilution) was added to each well and incubated in a dark place at 37°C for 1 hour, then 5 μL of stop reagent was added.

[0290] Plate Reading: Fluorescence signals (excitation wavelength 400 nm, emission wavelengths 460 nm and 535 nm) were detected using a multi-label microplate reader (Perkin Elmer EnVision Multimode Plate Reader).

[0291] Calculation: The inhibition rate of each well was calculated from the fully active well and the control signal well, and the data analysis method is as follows.

[0292] Phosphorylation ratio = 1 - {(Emission ratio × F100% - C100%) / [C0% - C100% + Emission ratio × (F100% - F0%)]} × 100;

[0293] Inhibition rate = 100X(1-compound phosphorylation rate / negative control phosphorylation rate).

[0294] IC 50 The value was calculated by medical GraphPad Prism (GraphPad Prism 5.0) software.

[0295] The results of the kinase activity test are as shown in Table 1.

[0296] Table 1: Compound kinase activity test results (IC 50 : nM) Compound number TRKA TRKB TRKC XS116 ** ** ** XS2-161 *** **** *** XS2-106 *** **** XS2-109 *** **** *** XS2-112 *** *** *** XS3-23 ** ** ** XS3-61 ** ** ** XS4-80 ** ** XS4-81 ** ** XS4-72 ** ** ** XS4-76 ** ** ** XS4-77 ** ** ** XS3-68 * * * XS3-35 * ** * XS3-58 ** ** ** XS3-36 ** ** * XS3-57 * * * XS3-56 ** ** ** XS3-67 * ** * XS3-51 ** ** * XS3-52 ** ** ** XS3-55 * ** ** XS3-54 * ** * XS3-53 * ** * XS3-81 * * * XS3-130 ** ** * XS3-138 ** * * XS3-134 ** ** ** XS3-135 ** ** ** XS3-139 ** ** ** XS3-131 ** ** ** XS3-137 ** ** ** XS3-136 ** ** ** XS3-153 ** ** ** XS3-91 * * * XS3-87 * * *

[0297] IC 50 : <10nM=*; 10-100nM=**; 100-1000nM=***; >1uM=****.

[0298] As can be seen from the data in Table 1, the alkynylphenylbenzamide compounds of the present invention have very strong inhibitory activity against TRKs kinase.

[0299] Example 38: Study on the cell proliferation inhibitory activity of Ba / F3-TRKs stable strains

[0300] The BaF3 cells (mouse pre-B cells) used in this experiment were purchased from a Japanese cell bank, and the BaF3-CD74-NTRK1, BaF3-ETV6-NTRK2, and BaF3-ETV6-NTRK3 monoclonal stable strains were all established in this laboratory and confirmed to be fully accurate through experiments such as positive drug activity, protein expression, and gene sequencing.

[0301] The brief steps for constructing stable strains are as follows: a pCDNA3.1(+) plasmid vector containing genes such as CD74-NTRK1, ETV6-NTRK2, and ETV6-NTRK3 was constructed; the plasmids were electro-transformed into Ba / F3 cells using the Amaxa® Cell Line Nucleofector® Kit V; 48 hours after electro-transformation, screening was continued for 2 weeks by adding Geneticin (G418) at a final concentration of 1000 μg / ml and removing Interleukin 3 (IL3) to obtain polyclonal stable strains; then monoclones were selected using the limiting dilution method; and the stable strains were confirmed using positive drugs, Western Blot (WB), and gene sequencing; and the monoclones confirmed to be completely accurate can be used to study the cell proliferation inhibitory activity of the inhibitor.

[0302] Study of cell proliferation inhibitory activity: Logarithmic growth phase cells were seeded into 96-well plates at a rate of 8,000–12,000 cells / well, and different concentrations of inhibitor (0–10 μM) were added the following day, followed by incubation for 72 hours; subsequently, 10 μL of Cell Counting Kit-8 (CCK-8 reagent) was added to each well and incubated for 1–3 hours; and then, absorbance was measured at 450 nm and 650 nm using a Supermicroplate reader. The half-inhibitory concentration (IC10) was determined using medical GraphPad Prism software (GraphPad Prism 8.0.0). 50 ) calculated.

[0303] The test results are as shown in Table 2.

[0304] Table 2: Compound cell activity test results (IC 50 : nM) Compound number CD74-NTRK1 ETV6-NTRK2 ETV6-NTRK3 XS116 *** *** *** XS2-161 **** **** **** XS2-106 *** *** XS2-112 *** *** *** XS3-23 ** ** ** XS3-61 ** ** XS4-80 ** ** ** XS4-81 ** ** * XS4-72 ** ** * XS4-76 ** ** ** XS4-77 ** ** * XS3-68 * ** * XS3-35 ** *** ** XS3-58 *** *** ** XS3-36 ** ** ** XS3-57 ** *** ** XS3-56 *** *** ** XS3-67 *** *** ** XS3-51 *** *** ** XS3-52 ** *** ** XS3-55 ** ** ** XS3-54 ** ** ** XS3-53 ** *** ** XS3-81 ** * * XS3-130 ** ** ** XS3-138 ** ** * XS3-134 *** *** ** XS3-135 *** *** ** XS3-139 ** ** ** XS3-131 *** *** ** XS3-137 *** *** ** XS3-136 *** *** ** XS3-153 ** ** ** XS3-87 * ** * XS3-91 ** ** *

[0305] IC 50: <10nM=*; 10-100nM=**; 100-1000nM=***; >1uM=****.

[0306] As can be seen from the data in Table 2, the alkynylphenylbenzamide compounds of the present invention have very strong inhibitory activity against cell proliferation of Ba / F3-TRKs stable strains.

[0307] Example 39: Study on the inhibitory activity of Ba / F3-TRKs stabilized strains on drug-resistant cell proliferation

[0308] The BaF3 cells (mouse pre-B cells) used in this experiment were purchased from a Japanese cell bank, and BaF3-CD74-NTRK1-G667C, BaF3-CD74-NTRK1-F589L, BaF3-CD74-NTRK1-G595R, BaF3-CD74-NTRK1-G667A, BaF3-CD74-NTRK1-V573M, BaF3-ETV6-NTRK2-G639R, BaF3-ETV6-NTRK2-G709C, BaF3-ETV6-NTRK2-V617M, BaF3-ETV6-NTRK2-F633L, BaF3-ETV6-NTRK3-G696C, BaF3-ETV6-NTRK3-G696A, BaF3-ETV6-NTRK3-G623R, The monoclonal stable strains BaF3-ETV6-NTRK3-G623E, BaF3-ETV6-NTRK3-F617L, and BaF3-ETV6-NTRK3-V601M were all established in this laboratory and confirmed to be completely accurate through experiments such as positive drug activity, protein expression, and gene sequencing.

[0309] The brief steps for constructing a stable stock are as follows: BaF3-CD74-NTRK1-G667C, BaF3-CD74-NTRK1-F589L, BaF3-CD74-NTRK1-G595R, BaF3-CD74-NTRK1-G667A, BaF3-CD74-NTRK1-V573M, BaF3-ETV6-NTRK2-G639R, BaF3-ETV6-NTRK2-G709C, BaF3-ETV6-NTRK2-V617M, BaF3-ETV6-NTRK2-F633L, BaF3-ETV6-NTRK3-G696C, BaF3-ETV6-NTRK3-G696A, BaF3-ETV6-NTRK3-G623R, BaF3-ETV6-NTRK3-G623E, pCDNA3.1(+) plasmid vectors containing genes such as BaF3-ETV6-NTRK3-F617L and BaF3-ETV6-NTRK3-V601M were constructed; the plasmids were electro-transformed into Ba / F3 cells using the Amaxa® Cell Line Nucleofector® Kit V; 48 hours after electro-transformation, screening was continued for 2 weeks by adding geneticin (G418) at a final concentration of 1000 μg / ml and removing interleukin 3 (IL3) to obtain polyclonal stable strains; then monoclones were selected using the limiting dilution method; and stable strains were confirmed using positive drugs, Western Blot (WB), and gene sequencing; and monoclones confirmed to be completely accurate can be used to study the cell proliferation inhibitory activity of the inhibitor.

[0310] Study of cell proliferation inhibitory activity: Logarithmic growth phase cells were seeded into 96-well plates at a rate of 8,000–12,000 cells / well, and different concentrations of inhibitor (0–10 μM) were added the following day, followed by incubation for 72 hours; subsequently, 10 μL of Cell Counting Kit-8 (CCK-8 reagent) was added to each well and incubated for 1–3 hours; and then, absorbance was measured at 450 nm and 650 nm using a Supermicroplate reader. The half-inhibitory concentration (IC10) was determined using medical GraphPad Prism software (GraphPad Prism 8.0.0). 50 ) calculated.

[0311] The test results are as shown in Table 3.

[0312] Table 3: Results of compound drug resistance cell activity test (IC 50 : nM) Compound number XS3-55 XS3-68 XS3-81 XS3-87 BaF3-CD74-NTRK1-G667C * * * * BaF3-CD74-NTRK1-G667A * * * * BaF3-CD74-NTRK1-G595R **** *** *** *** BaF3-CD74-NTRK1-F589L *** *** *** ** BaF3-CD74-NTRK1-V573M ** ** * * BaF3-ETV6-NTRK2-G639R *** ** ** ** BaF3-ETV6-NTRK2-F633L *** *** *** ** BaF3-ETV6-NTRK2-V617M * * * * BaF3-ETV6-NTRK2-G709C * * ** * BaF3-ETV6-NTRK3-G696C * * * * BaF3-ETV6-NTRK3-G696A * * * * BaF3-ETV6-NTRK3-G623R *** ** ** ** BaF3-ETV6-NTRK3-G623E * * * * BaF3-ETV6-NTRK3-F617L ** ** ** * BaF3-ETV6-NTRK3-V601M * * * *

[0313] IC 50 : <10nM=*; 10-100nM=**; 100-1000nM=***; >1uM=****.

[0314] As can be seen from the data in Table 3, the alkynylphenylbenzamide compounds of the present invention have very strong inhibitory activity against drug-resistant cell proliferation of Ba / F3-TRKs stabilizers.

[0315] Example 40: IC of kinase selectivity of compound XS3-55 50 test

[0316] Kinase activity detection: Z´-LYTE™ technology (fluorescence-based detection based on the difference in sensitivity between phosphorylated and non-phosphorylated polypeptides to protein hydrolytic cleavage, enzyme-linked form) was applied, the fluorescence resonance energy transfer (FRET) principle was adopted, Z´-LYTE™ FRET peptide substrates were used, and the inhibitory activity of compound XS3-55, the control molecule XS4-128, and Ponatinib against Bcr-Abl, SRC, RET, PDGFRA, PDGFRB, VEGFR2, and Kit kinases was detected by secondary reaction.

[0317] Here, the structural formula of compound XS4-128 is as follows.

[0318]

[0319] The method of manufacturing this is as follows.

[0320]

[0321] Step 1: Preparation of methyl 4-methyl-3-((trimethylsilyl)ethynyl)benzoate (Compound 2)

[0322]

[0323] 1 g (3.6 mmol) of compound, 69 mg (0.36 mmol) of cuprous iodide, 127 mg (0.18 mmol) of bis(triphenylphosphine)palladium dichloride, 50 mL of anhydrous acetonitrile, and 934 mg (7.2 mmol) of N,N-diisopropylethylamine were added to a 100 mL three-necked flask, the mixture was replaced with argon gas, the reaction system was sealed, and 1.06 g (10.8 mmol) of trimethylsilylacetylene was injected using a syringe and stirred at 60 °C for 6 hours. The reaction mixture was filtered through diatomite and the solvent was spin-dried to obtain a black mixture, which was then used in the next step of the reaction.

[0324] Step 2: Preparation of methylmethyl 3-ethinyl-4-methylbenzoate (Compound 3)

[0325]

[0326] The crude product from the previous step was dissolved in methanol, and approximately 2 mL of a 1 mol / L tetrahydrofuran solution of tetrabutylammonium fluoride was added and stirred at room temperature for 2 hours. After spin-drying the reaction system, 470 mg of a yellowish-brown oily substance was obtained by column chromatography (75% yield of the two steps).

[0327] 1 H NMR (400 MHz, DMSO- d 6) δ 8.06 (d, J =1.5Hz, 1H), 7.76 (dd, J =7.5, 1.5Hz, 1H), 7.25 (dd, J =7.5, 1.0Hz, 1H), 4.25 (s, 1H), 3.86 (s, 3H), 2.39 (s, 3H). LC-MS (ESI) m / z 175.5[M+H] + .

[0328] Step 3: Preparation of 3-ethynyl-4-methylbenzoic acid (Compound 4)

[0329]

[0330] 400 mg (2.29 mmol) of Compound 3 was dissolved in a mixed solvent of tetrahydrofuran, methanol, and water in a volume ratio of 10:1:5, and then 482 mg (11.4 mmol) of lithium hydroxide hydrate was added and stirred at 60°C for 1 hour. After filtration, the reaction system was spin-dried, and then a 4 M hydrochloric acid solution was added until the reaction system became acidic, at which point a white solid precipitated; the solid was collected by filtration and dried to obtain 350 mg (yield 96%) of white solid.

[0331] 1 H NMR (400 MHz, DMSO- d 6) δ 13.02 (s, 1H), 7.93 (d, J =1.8Hz, 1H), 7.85 (dd, J =7.9, 1.9Hz, 1H), 7.43 (d,J =8.0Hz, 1H), 4.48 (s, 1H), 2.45 (s, 3H). LC-MS (ESI) m / z 160.9 [M+H] + .

[0332] Step 4: Preparation of N-(3-chloro-5-(trifluoromethyl)phenyl)-3-ethynyl-4-methylbenzamide (Compound 6)

[0333]

[0334] 300 mg (1.9 mmol) of Compound 4 and 305 mg (1.6 mmol) of Compound 5 were dissolved in 20 mL of N,N-dimethylformamide (DMF), and then 912 mg (2.4 mmol) of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and 413 mg (3.2 mmol) of N,N-diisopropylethylamine were added. The mixture was heated and stirred at 70°C for 2 hours. After spin-drying the reaction system, water was added and extracted with ethyl acetate, washed with water, dried with anhydrous sodium sulfate, and the solvent was spin-dried. Subsequently, 340 mg (yield 63%) of a yellow oily substance was obtained by column chromatography.

[0335] 1 H NMR (400 MHz, DMSO- d 6) δ 10.88 (s, 1H), 8.11 (d, J =1.8Hz, 2H), 7.64-7.57 (m, 2H), 7.54 (dd, J =7.7, 1.4Hz, 1H), 7.36 (t, J =7.7Hz, 1H), 4.51 (s, 1H), 2.47 (s, 3H). MS (ESI) m / z 335.8 [MH] - .

[0336] Step 5: Preparation of N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-(4-hydroxypiperidin-1-yl)imidazole[1,2-B]pyridazine-3-yl)ethinyl)-4-methylbenzamide (XS4-128)

[0337]

[0338] 70 mg (0.2 mmol) of Compound 6 and 83 mg (1.2 mmol) of Compound 7 were dissolved in 10 mL of anhydrous N,N-dimethylformamide (DMF), then 8 mg (0.016 mmol) of cuprous iodide, 19 mg (0.01 mmol) of bis(triphenylphosphine)palladium dichloride, and 145 mg (0.4 mmol) of N,N-diisopropylethylamine were added, the mixture was switched to argon gas, and the reaction system was sealed. The mixture was heated and stirred at 80°C and reacted overnight. The solution was filtered through diatomite, and the filtrate was spin-dried. Subsequently, 80 mg (yield 73%) of a yellowish-white solid was obtained by column chromatography.

[0339] 1 H NMR (400 MHz, DMSO- d 6) δ 10.66 (s, 1H), 8.25 (s, 2H), 8.16 (d, J =9.2Hz, 2H), 7.96 (s, 1H), 7.90 (dd, J =8.0, 1.9Hz, 1H), 7.55 (s, 1H), 7.52 (d, J =8.1Hz, 1H), 7.34 (s, 1H), 4.75 (d, J =4.2Hz, 1H), 3.98 (d, J =14.5Hz, 2H), 3.80-3.71(m, 1H), 3.26 (t, J =11.5Hz, 2H), 2.61 (s, 3H), 1.89-1.78(m, 2H), 1.54-1.40 (m, 2H). HRMS (ESI) for C 28 H23ClF3N5O2[M+H] + : Calculated value (calcd) 554.1565, Measured value (found) 554.1562.

[0340] Enzyme reaction: 5 μL of the enzyme-substrate system (50 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) pH 7.5, 0.01% BRIJ-35, 10 mM magnesium chloride (MgCl2), 1 mM ethylene glycolbis(2-aminoethyl ether)tetraacetic acid (EGTA), 2 μM Tyr O1 peptide substrate) was added to a 384-well plate, 5 nL of the compound (concentration gradient) was transferred using an echo520 ultramicro liquid pipetting system, and the mixture was shaken at room temperature for 10-20 minutes. Then, 200 nL, 12.5 nL, and 25 nL of ATP (final concentrations of 400 μM, 25 μM, and 50 μM, respectively) were transferred using an echo520 ultramicro liquid pipetting system and the mixture was shaken to mix uniformly. Afterward, centrifugation was performed, and the mixture was reacted in a dark place at 30°C for 1.5 hours.

[0341] Detection reaction: 2.5 μL of development solution (1:128 dilution) was added to each well and incubated in a dark place at 37°C for 1 hour, then 5 μL of stop reagent was added.

[0342] Plate Reading: Fluorescence signals (excitation wavelength 400 nm, emission wavelengths 460 nm and 535 nm) were detected using a multi-label microplate reader (Perkin Elmer EnVision Multimode Plate Reader).

[0343] Calculation: The inhibition rate of each well was calculated from the fully active well and the control signal well, and the data analysis method is as follows.

[0344] Phosphorylation ratio = 1 - {(Emission ratio × F100% - C100%) / [C0% - C100% + Emission ratio × (F100% - F0%)]} × 100;

[0345] Inhibition rate = 100X(1-compound phosphorylation rate / negative control phosphorylation rate).

[0346] IC 50The value was calculated by medical GraphPad Prism (GraphPad Prism 5.0) software.

[0347] The results of the kinase activity test are as shown in Table 4.

[0348] Table 4: Compound kinase selectivity activity test results (IC 50 : nM) Kinase XS3-55 XS4-128 Ponatinib Bcr-Abl >10000 128.8 3.9 SRC 526.3 20.3 2.0 RET >10000 10.7 3.3 VEGFR2 832.7 18.9 3.3 Kit 1669 93.5 25.1 PDGFRA 5887 19.9 3.9 PDGFRB >10000 118.9 18.0

[0349] As can be seen from the data in Table 4, XS3-55, a representative compound of the alkynylphenylbenzamide class of the present invention, has relatively weak inhibitory activity against various representative tyrosine kinases other than TRKA, TRKB, and TRKC and relatively excellent kinase selectivity. Since its kinase selectivity is much better than that of compounds XS4-128 and Ponatinib, the alkynylphenylbenzamide class of the present invention has excellent selectivity and low toxic side effects.

[0350] Example 41: Pharmacokinetic Evaluation

[0351] Pharmacokinetics and oral bioavailability were tested in SD rats. Depending on the drug solubility, a single dose was administered either orally or intravenously. Animal blood samples were collected at different time points (0, 0.5, 1, 2, 4, 6, 8, and 24 hours), and the supernatant was obtained by centrifugation after anticoagulation with the addition of heparin. Blood samples were analyzed by HPLC-MS, and DAS2.1 was used for data analysis to detect pharmacokinetic data such as half-life (T1 / 2), peak blood drug concentration (Cmax), peak time (Tmax), area under the drug-time curve (AUC), and bioavailability (BA). The pharmacokinetic data results for Compound XS3-55 and Compound 9o (European Journal of Medicinal Chemistry 179(2019) 470-482.) are shown in Table 5.

[0352]

[0353] compound Compound 9c Compound XS3-55 Administration method intravenous injection Oral administration intravenous injection Oral administration Dosage (mg / kg) 5 25 2.0 10.0 Half-life (h) 2.03 4.77 16.07 15.19 Peak time (h) 0.083 2 0.08 4.00 Peak blood drug concentration (ng / mL) 1968.37 63.3 46123.74 44066.54 Area under the drug-time curve (0-t)(h*ng / mL) 804.65 431.49 358128.95 560764.28 Area under the drug-time curve (0-∞) (h*ng / mL) 810.45 445.18 548798.57 878346.33 Cleaning rate (mL / h / kg) 1260.51 nd 3.65 nd Bioavailability (%) 10.72 31.32

[0354] Compound XS3-55 exhibits very strong inhibitory activity against TRKs kinases and against the proliferation of wild-type and drug-resistant cells of Ba / F3-TRKs stable strains. Additionally, since compound XS3-55 has excellent oral absorption characteristics, when orally administered to rats at a dose of 10 mg / kg, the half-life of compound XS3-55 is 15.19 hours, the peak blood drug concentration is high at 44066.54 ng / mL, the area under the drug-time curve is high at 878346.33 h*ng / mL, and the pharmacokinetic characteristics are significantly higher than those of the control compound 9o.

[0355] Example 42: In vivo tumor activity of compound XS3-55

[0356] Ba / F3-CD74-TRKA G667C The in vivo antitumor drug efficacy of compound XS3-55 by oral administration was evaluated in an allogeneic transplant mouse model. Cultured BaF3-CD74-TRKA G667C Collect the cells, centrifuge them, wash them twice with physiological saline, and then 1 x 10⁻⁶ 7 After adjusting to a density of 1 / mL, the mixture was placed on ice and immediately injected subcutaneously at a dose of 200μL per mouse into the right armpit of female CB17-SCID mice (purchased from Beijing Vital River, 6-8 weeks old). Nine days after modeling, the tumor volume was approximately 200mm³. 3When the animals had grown to a certain size, they were randomly divided into groups and administration was initiated. The control group consisted of 8 animals, while the compound XS3-55 was administered in 4 dose groups (50, 25, 12.5, and 6.25 mg / kg), with 6 animals per group. The administration method was as follows: Depending on the dosage, an appropriate amount of compound XS3-55 powder was taken and dissolved in a mixed solvent of 2% dimethyl sulfoxide (DMSO), 20% hydrogenated castor oil, 8% anhydrous ethanol, and 70% physiological saline to obtain a pale yellow to yellow transparent liquid, which was administered orally once daily; the control group was orally administered an equivalent volume of the mixed solvent. Body weight and tumor volume were recorded every 2 days.

[0357] The results are as shown in Figure 1. Compound XS3-55 administered once daily for 2 weeks CD74 TRKA G667C It inhibits growth in allogeneic mouse models containing mutations in a dose-dependent manner. A significant reduction in tumor size was observed after 2 days of treatment with the lowest dose of 6.25 mg / kg. After 12 days of treatment, compound XS3-55 demonstrated excellent in vivo antitumor efficacy at doses of 6.25 mg / kg / day, 12.5 mg / kg / day, 25 mg / kg / day, and 50 mg / kg / day, with TGI values ​​of 50.9%, 76.3%, 89.2%, and 91.6%, respectively, whereas control mice died on day 14. At the same time, in vivo studies indicated that all four different doses of compound XS3-55 did not have any apparent adverse effects on mouse body weight (Fig. 1A), demonstrating that compound XS3-55 possesses excellent safety.

[0358] Each technical feature of the aforementioned embodiments may be combined arbitrarily, and for the sake of brevity, not all possible combinations of each technical feature of the embodiments are described; however, as long as such combinations of technical features are not contradictory, they should be considered within the scope described herein.

[0359] The foregoing embodiments merely illustrate a few embodiments of the present invention, and while the description is relatively specific and detailed, the scope of the patent of the present invention should not be understood as being limited thereto. Those skilled in the art should note that various modifications and improvements can be made without departing from the concept of the present invention, and that all of these fall within the scope of protection of the present invention. Accordingly, the scope of protection of the patent of the present invention shall be in accordance with the appended claims.

Claims

Claim 1 As an alkynylphenylbenzamide compound having a structure represented by formula (I), or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, (I) In the above equation (I), R1 is C1~C 20 Selected from alkyl; R2 is H, halogen, C1~C 20 Alkyl, C1~C 20 Alkoxy or halogen-substituted C1~C 20 Selected from alkyls; R3 is selected from H, fluorine-substituted C1-C4 alkyls, and 5-6 member heterocyclyl groups containing 1-3 unsubstituted N ring atoms; R4 is selected from H, halogens, nitro, and C1-C 10 Alkyl, halogen-substituted C1~C 10 Alkyl, C1~C 10 Alkoxy, halogen-substituted C1~C 10 Alkoxy, -(CH2) x NR 17 R 18 , 1-5 R 19 A 5-10-membered heterocyclic group containing 1-3 N ring atoms, substituted or unsubstituted with R, and 1-5 R 19 Selected from 5-10 member heteroaryls containing 1-3 N ring atoms, substituted or unsubstituted with; x is an integer from 1 to 5; and R 17 , R 18 1-5 R atoms along with nitrogen atoms connected to them 19 Forming morpholinyl, pyrrolidinyl, piperidinyl, or piperazinyl substituted or unsubstituted with; and each R 19 Each is independently selected from C1-C5 alkyls; R5 is -NR6R7; and R6 and R7 are each independently -(CH2) m NR8R9, -(CH2) n CR 10 R 11 R 12 , and -(CH2) p OR 12 Selected from, or R6, R7 with 1-5 R atoms along with nitrogen atoms connected to them. 13 Forming a 3-15 member single ring, condensation ring, spiro ring, or bridging ring containing 1-3 heteroatoms substituted or unsubstituted with; the heteroatoms are selected from O, N, and S; R 8, R9 is each independently selected from H, C1–C5 alkyl; or R8, R9 are 1–5 R atoms together with nitrogen atoms connected thereto. 13 Forming a 3-10 member single ring, condensation ring, spiro ring, or cross-linked ring containing 1-3 heteroatoms substituted or unsubstituted with R, wherein the heteroatoms are selected from O and N; R 10 , R 11 It has 1-5 R atoms along with carbon atoms connected to them. 13 Forming a 3-10 member single ring, condensation ring, spiro ring, or cross-linked ring containing 1-3 heteroatoms substituted or unsubstituted with R, wherein the heteroatoms are selected from O and N; R 12 is selected from H and C1~C5 alkyl; each R 13 Each is independently H, C1-C5 alkyl, C1-C5 alkanoyl, hydroxyl, hydroxyl-substituted C1-C5 alkyl, amino-substituted C1-C5 alkyl, amino-substituted C1-C5 alkoxy, C3-C7 cycloalkyl-substituted C1-C3 alkyl, -NR 15 R 16 , 1-5 R 14 Selected from a 3-10 member single ring, condensation ring, spiro ring, or bridging ring containing 1-3 heteroatoms, substituted or unsubstituted with R, wherein the heteroatoms are selected from O and N; R 14 , R 15 and R 16 alkynylphenylbenzamide compounds or pharmaceutically acceptable salts thereof or stereoisomers thereof, wherein each is independently selected from H and C1-C5 alkyls; m, n, and p are each independently selected from integers 0 to 5. Claim 2 In claim 1, R4 is H, halogen, nitro, C1-C8 alkyl, halogen-substituted C1-C8 alkyl, C1-C8 alkoxy, halogen-substituted C1-C8 alkoxy, -(CH2) x NR 17 R 18 , 1-5 R 19 A 5-6-membered heterocyclic group containing 1-3 N ring atoms substituted or unsubstituted with R, and 1-5 R 19 Selected from 5-6-membered heteroaryls containing 1-3 N ring atoms substituted or unsubstituted; x is an integer from 1 to 5; and R 17 , R 18 1-5 R atoms along with nitrogen atoms connected to them 19 Forming morpholinyl, pyrrolidinyl, piperidinyl, or piperazinyl substituted or unsubstituted with; and each R 19 Alkynylphenylbenzamide compounds or pharmaceutically acceptable salts thereof or stereoisomers thereof, each characterized by being independently selected from C1-C5 alkyls. Claim 3 In paragraph 2, R4 is H, halogen, nitro, C1-C4 alkyl, halogen-substituted C1-C4 alkyl, C1-C4 alkoxy, halogen-substituted C1-C4 alkoxy, -(CH2) x NR 17 R 18 , 1-3 R 19 Selected from imidazolyls substituted or unsubstituted with; x is 1, 2 or 3, and R 17 , R 18 1-3 R atoms along with nitrogen atoms connected to them 19 Forming a piperazinyl substituted or unsubstituted with; each R 19 Alkynylphenylbenzamide compounds or pharmaceutically acceptable salts thereof or stereoisomers thereof, each characterized by being independently selected from C1-C5 alkyls. Claim 4 In paragraph 3, R4 is H, halogen, nitro, methyl, ethyl, propyl, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoroethyl, or Selected from; each R 19 Alkynylphenylbenzamide compounds or pharmaceutically acceptable salts thereof or stereoisomers thereof, each characterized by being independently selected from methyl, ethyl, and propyl. Claim 5 delete Claim 6 In paragraph 1, R6 and R7 are each independently -(CH2) m NR8R9, -(CH2) p OR 12 Selected from, or R6, R7 with 1-3 R atoms along with nitrogen atoms connected to them. 13 Forming a morpholineyl, pyrrolidineyl, piperidineyl, or piperazinyl substituted or unsubstituted with; R8 and R9 are each independently selected from H, C1–C5 alkyl, or R8 and R9 have 1–5 R atoms together with the nitrogen atoms connected thereto. 13 Forming a morpholineyl, pyrrolidineyl, piperidineyl, or piperazinyl substituted or unsubstituted with;R 12 is selected from H, C1-C5 alkyl; and each R 13 Each independently consists of H, C1-C5 alkyl, C1-C5 alkanoyl, hydroxyl, -NR 15 R 16 , 1-2 R 14 Oxetanyl substituted or unsubstituted with 1-4 R groups 14 Selected from morpholine that is substituted or unsubstituted;R 14 , R 15 , R 16 An alkynylphenylbenzamide compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that each is independently selected from H, C1-C3 alkyl; and m and p are each independently selected from 1, 2, 3, 4 or 5. Claim 7 In claim 1, R5 is an alkynylphenylbenzamide compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized by being selected from any one of the following groups: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , . Claim 8 In paragraph 1, R4 is a halogen; R5 is -NR6R7; and R6 and R7 are each independently -(CH2) m NR8R9, -(CH2) p OR 12 Selected from, or R6, R7 with 1-3 R atoms along with nitrogen atoms connected to them. 13 Forming a morpholineyl, pyrrolidineyl, piperidineyl, or piperazinyl substituted or unsubstituted with; R8 and R9 are each independently selected from H, C1-C3 alkyl, or R8 and R9 have 1-2 R atoms together with the nitrogen atoms connected thereto. 13 Forming a morpholineyl, pyrrolidineyl, piperidineyl, or piperazinyl substituted or unsubstituted with;R 12 is selected from H, C1-C3 alkyl; and each R 13 Each independently consists of H, C1-C3 alkyl, acetyl, hydroxyl, -NR 15 R 16 , selected from oxetanil and morpholinyl; R 15 , R 16 An alkynylphenylbenzamide compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that each is independently selected from H, C1-C3 alkyl; and m and p are each independently selected from 2, 3 or 4. Claim 9 In paragraph 8, R4 is Cl, and R5 is , , , , , , , , , , , , , , , , An alkynylphenylbenzamide-based compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized by being selected from. Claim 10 In paragraph 1, R4 is H, halogen, methyl, methoxy, trifluoromethyl, nitro, Selected from, and R5 is This or; or , R4 is H, Selected from, and R5 is , , Selected from; or, R4 is And, R5 is , An alkynylphenylbenzamide-based compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized by being selected from. Claim 11 An alkynylphenylbenzamide compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that, in claim 1, R1 is selected from C1-C4 alkyl; and / or, R2 is selected from H, halogen, C1-C4 alkyl, halogen-substituted C1-C4 alkyl, C1-C4 alkoxy. Claim 12 An alkynylphenylbenzamide compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that, in claim 11, R1 is selected from methyl, ethyl, isopropyl, tert-butyl; and / or, R2 is selected from hydrogen, fluorine, methyl, ethyl, isopropyl, tert-butyl, difluoromethyl, difluoroethyl, trifluoromethyl, or trifluoroethyl. Claim 13 In claim 1, an alkynylphenylbenzamide compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that R3 is selected from H, difluoromethyl, difluoroethyl, trifluoromethyl, or trifluoroethyl. Claim 14 In claim 1, an alkynylphenylbenzamide compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized by having a structure represented by formula (II). (II) Claim 15 In claim 1, the alkynylphenylbenzamide compound is characterized by being selected from the following compounds, an alkynylphenylbenzamide compound, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof. Claim 16 delete Claim 17 A pharmaceutical composition comprising an alkynylphenylbenzamide-based compound according to claim 1, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein the pharmaceutical composition is used for the prevention and / or treatment of a disease mediated by TRK tyrosine kinase, and the disease mediated by TRK tyrosine kinase is cancer. Claim 18 A pharmaceutical composition according to claim 17, characterized in that the cancer is non-small cell lung cancer, breast cancer, colon cancer, prostate cancer, thyroid cancer, malignant melanoma, neuroblastoma, or mammary gland-like secretory carcinoma. Claim 19 A pharmaceutical composition for preventing and / or treating tumors, comprising an active ingredient and a pharmaceutically acceptable adjuvant, wherein the active ingredient comprises an alkynylphenylbenzamide-based compound according to claim 1, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

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