An N-aryl-N-arylalkynyl-arylacetamide compound and pharmaceutical use thereof

Abstract

This invention provides an N-aryl-N-arylpropynyl-arylacetamide compound and its pharmaceutical uses. The provided compounds include their prodrugs, tautomers, stereoisomers, solvates, isotopic derivatives, or pharmaceutically acceptable salts thereof. The compounds of this invention can serve as the first covalent inhibitor of POLQ. In vitro enzyme activity inhibition studies show that the compounds of this invention have strong inhibitory effects on POLQ enzymes and can serve as promising compounds for the treatment of POLQ-mediated diseases. Further studies have shown that typical compounds I-1, I-13, and I-25 have considerably long-lasting inhibitory activity against POLQ enzymes and good cellular activity. The synthetic method of this invention is simple and convenient, facilitating large-scale industrial production and application. The general structural formula of this invention is shown in Formula I:

Description

Technical Field

[0001] This invention relates to the field of chemical pharmaceuticals, specifically to an N-aryl-N-arylpropynyl-arylacetamide compound and its pharmaceutical uses, which is an arylacetamide compound used as a covalent inhibitor of POLQ, as well as the preparation method and uses of the compound. Background Technology

[0002] DNA double-strand damage repair (DSBs) is crucial for genomic stability and cell survival. There are three main DSB repair pathways: homologous recombination (HR), non-homologous end joining (NHEJ), and microhomologous end joining (MMEJ). HR repair is a high-fidelity, error-free DSB repair pathway; HR dysfunction can lead to genomic instability and tumorigenesis. HR loss is particularly common in gynecological tumors, accounting for approximately 10% of all gynecological tumors. In the context of HR loss, the DNA polymerase theta (POLQ / Pol theta / POLθ)-mediated microhomologous end joining (MMEJ) repair pathway is an important backup repair pathway. Multiple studies have demonstrated that inhibiting Pol theta has a synthetic lethal effect on HR loss. Therefore, POLQ is a potential DNA damage repair target for the treatment of HR-deficient tumors.

[0003] Currently, several small molecule inhibitors targeting the POLQ polymerase domain have been reported. Among them, ART4215 is the only orally administered, specific small molecule POLQ inhibitor that has entered the clinical development stage, but its structure has not yet been disclosed. To date, all reported Pol theta inhibitors are non-covalent reversible inhibitors (Maria Chiara Pismataro, Andrea Astolfi, et al. Small Molecules Targeting DNA Polymerase Theta (POLθ) as Promising Synthetic Lethal Agents for Precision Cancer Therapy. J. Med. Chem. 2023). No covalent inhibitors have been reported. Covalent inhibitors, while binding to the target protein, can form covalent bonds with electrophilic amino acid residues on the target protein near the binding site. Compared with non-covalent inhibitors, covalent inhibitors have a series of advantages such as strong efficacy, long duration of action, and high specificity. The POLQ polymerase domain allosteric pocket contains electrophilic cysteine ​​residues (Cys2411), which meets the conditions for developing covalent inhibitors.

[0004] Therefore, designing and developing a POLQ covalent inhibitor with strong efficacy and excellent drug-like properties has significant research and application value. Summary of the Invention

[0005] The purpose of this invention is to overcome the lack of POLQ covalent inhibitors and provide an N-aryl-N-arylyynylpropylated-arylacetamide compound, which is a compound with an arylacetamide structure that serves as a POLQ covalent inhibitor.

[0006] The N-aryl-N-arylpropynyl-arylacetamide compounds, or their prodrugs, tautomers, stereoisomers, solvates, isotope derivatives, or pharmaceutically acceptable salts thereof provided by this invention, have the general structural formula shown in Formula I:

[0007]

[0008] Where X is selected from CH or N;

[0009] R1 is selected from hydrogen, halogen, amino, cyano, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, and halogenated C1-C6 alkyl;

[0010] R2 is selected from hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 fluorinated alkyl, C1-C6 fluorinated alkoxy, and C3-C6 cycloalkyl.

[0011] Ar is a benzene ring or an aromatic heterocycle, including thiazole ring, pyrazole ring, pyridine ring, pyrimidine ring, pyridazine ring, and pyrazine ring;

[0012] R3 is selected from hydrogen, C 1- C3 alkyl;

[0013] n is selected from 0, 1, 2, 3;

[0014] R4 is selected from hydrogen, C 1- C6 alkyl, C3-C6 cycloalkyl and their deuterated derivatives;

[0015] L is selected from, but not limited to, [the following].

[0016] Furthermore, the compound of general formula I, or its prodrug, tautomer, stereoisomer, solvate, isotope derivative, or pharmaceutically acceptable salt thereof;

[0017] Wherein: X is preferably selected from CH or N;

[0018] R1 is preferably selected from hydrogen or halogen;

[0019] R2 is preferably derived from hydrogen and has a C1-C3 fluorinated alkyl group;

[0020] Ar is preferably selected from benzene ring, pyridine ring, pyrimidine ring, pyridazine ring, and pyrazine ring;

[0021] n is preferably selected from 0 and 1;

[0022] R3 and R4 are independently selected from hydrogen and C. 1- C3 alkyl;

[0023] L is preferred from

[0024] Furthermore, the compound is selected from the following structures:

[0025]

[0026]

[0027]

[0028]

[0029] The preparation method of the compound of formula I shown in this invention is achieved through the following steps, wherein the compound includes the preparation method of its prodrug, tautomer, stereoisomer, solvate, isotope derivative or pharmaceutically acceptable salt thereof.

[0030] The compound is prepared by the following steps:

[0031]

[0032] Step 1: Compound A undergoes a nucleophilic substitution reaction with bromopropyne under the action of a strong base to give compound B;

[0033] Step 2: Compound B reacts with compound C in an acid-amine condensation reaction to yield compound D;

[0034] Step 3: Compound D and compound E undergo a Sonogashira coupling reaction to give compound F;

[0035] Step 4: Compound F undergoes a condensation reaction with an acid under the conditions of a condensing agent and an organic base to obtain the compound with the structure shown in Formula I.

[0036] R1-R4, n, and X are as described above.

[0037] The present invention also provides a pharmaceutical composition comprising a compound of Formula I shown in the present invention, or a prodrug, tautomer, solvate, isotope derivative, or a pharmaceutically acceptable salt thereof.

[0038] The present invention also provides a pharmaceutical composition comprising a compound of Formula I shown herein, or a prodrug, tautomer, solvate, isotope derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0039] A second object of the present invention is to provide the use of the compound of Formula I shown herein, or its prodrug, tautomer, stereoisomer, solvate, isotope derivative or pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating diseases mediated by POLQ.

[0040] In vitro enzyme activity inhibition studies showed that the compounds of this invention have a strong inhibitory effect on POLQ enzymes and can be considered as prospective compounds for the treatment of POLQ-mediated diseases. Further studies have shown that typical compounds I-1, I-13, and I-25 have fairly long-lasting inhibitory activity against POLQ enzymes and good cellular activity.

[0041] The administration of the compounds of the present invention or pharmaceutically acceptable salts thereof can be carried out in pure form or as a suitable pharmaceutical composition by any acceptable route of administration of a medicament for similar use. The pharmaceutical compositions of the present invention can be prepared by combining the compounds of the present invention with suitable pharmaceutically acceptable excipients. The pharmaceutical compositions of the present invention can be formulated into solid, semi-solid, liquid, or gaseous formulations.

[0042] In some embodiments, the POLQ-mediated disease is cancer or a tumor-related disease.

[0043] Furthermore, in the uses provided by the present invention, the cancer or tumor includes solid tumors and hematologic tumors: the solid tumors include breast cancer, colorectal cancer, cervical cancer, ovarian cancer, prostate cancer, gastric cancer (including gastrointestinal junction cancer), esophageal cancer, head and neck cancer, and lung cancer; the hematologic tumors include lymphoma and leukemia.

[0044] The object of the present invention also includes providing a method for preventing and / or treating POLQ-mediated diseases, comprising administering to a patient a therapeutically effective dose of a compound of general formula I, or a prodrug, tautomer, stereoisomer, solvate, isotope-based organism, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention: further, the POLQ-mediated disease is cancer or a tumor-related disease; further, the use provided by the present invention includes the cancer or tumor comprising solid tumors and hematologic malignancies: the solid tumors include breast cancer, colorectal cancer, cervical cancer, ovarian cancer, prostate cancer, gastric cancer (including gastrointestinal junction cancer), esophageal cancer, head and neck cancer, and lung cancer; the hematologic malignancies include lymphoma and leukemia.

[0045] The compound of general formula I of the present invention, or its prodrug, tautomer, stereoisomer, solvate, isotope derivative or pharmaceutically acceptable salt thereof, may be administered in combination with one, two or more other anticancer agents or immune checkpoint modulators used to treat cancer or tumors.

[0046] Furthermore, the additional anticancer agents or immune checkpoint modulators used to treat cancer or tumors include PARP inhibitors, ATR inhibitors, ATM inhibitors, WEEI inhibitors, topoisomerase inhibitors, and DNA-damaging chemotherapeutic agents.

[0047] Furthermore, the DNA-damaging chemotherapy drugs include cisplatin, bleomycin, gemcitabine, and docetaxel; the topoisomerase inhibitors include etoposide and irinotecan.

[0048] The compound of general formula I of the present invention, or its prodrug, tautomer, stereoisomer, solvate, isotope derivative or pharmaceutically acceptable salt thereof, may be used in combination with one, two or more other treatments for treating cancer or tumors (such as radiotherapy).

[0049] When the compounds of the present invention, or their prodrugs, tautomers, stereoisomers, solvates, isotope derivatives, or pharmaceutically acceptable salts thereof, are administered in combination with other anticancer agents or immune checkpoint inhibitors for the treatment of cancer or tumors, the compounds of the present invention or their pharmaceutically acceptable salts may provide enhanced anticancer effects.

[0050] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0051] The compounds and derivatives provided by this invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

[0052] The term "alkyl" refers to a group consisting of a straight-chain or branched saturated hydrocarbon group. The minimum and maximum carbon atom content in a hydrocarbon group are indicated by a prefix, for example, the prefix C. a-b Alkyl groups refer to any alkyl group containing one to two carbon atoms ("a" to "b"). For example, C 1-6 Alkyl groups are straight-chain or branched alkyl groups containing 1-6 carbon atoms. C 1-6 Examples of alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6).

[0053] The term "cycloalkyl" refers to a saturated or unsaturated cyclic compound with substituents. For example, "3-8 saturated cycloalkyl" refers to a saturated cycloalkyl compound with 3-8 carbon atoms in the ring.

[0054] The term "heterocyclic group" refers to a substituent in a saturated or unsaturated cyclic hydrocarbon, wherein the cyclic hydrocarbon carries at least one cyclic heteroatom (including but not limited to O, S, or N). For example, "3-8 membered saturated heterocyclic group" refers to a saturated heterocyclic group with 3-8 ring atoms.

[0055] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0056] The term "pharmaceutically acceptable" means that a carrier, delivery substance, diluent, excipient, and / or the salt formed therefrom is generally chemically or physically compatible with other components constituting a drug dosage form and physiologically compatible with the receptor.

[0057] The term "pharmaceutically acceptable salt" refers to salts prepared by conventional methods, including but not limited to organic acid salts, inorganic acid salts, organic base salts, and inorganic base salts. Organic acid salts include, but are not limited to, oxalates, lactates, p-toluenesulfonates, maates, citrates, fumarates, camphorsulfonates, and methanesulfonates. Inorganic acid salts include, but are not limited to, nitrates, sulfates, hydrohalates, and phosphates. Organic base salts include, but are not limited to, sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, triethylamine, and tert-butylamine. Inorganic base salts include, but are not limited to, sodium hydroxide, potassium hydroxide, and lithium hydroxide.

[0058] The term "isotope-labeled compound" refers to a compound obtained by replacing one or more atoms with their corresponding isotopes. For example, a compound obtained by replacing one or more hydrogen atoms (H) with deuterium (D) or tritium (T); or a compound obtained by replacing one or more carbon atoms with their corresponding isotopes. 12 Carbon 11 or carbon 13 The compound obtained after substitution.

[0059] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include, but are not limited to, oral, parenteral (intravenous, intramuscular or subcutaneous) and topical administration.

[0060] The beneficial effects of this invention are:

[0061] This invention designs a class of N-aryl-N-arylpropynyl-arylacetamide compounds, which can serve as the first covalent inhibitor of POLQ. In vitro enzyme activity studies show that the compounds of this invention have strong inhibitory effects on POLQ enzymes and can be considered as promising compounds for the treatment of POLQ-mediated diseases. Further studies have shown that typical compounds I-1, I-13, and I-25 have fairly long-lasting inhibitory activity against POLQ enzymes and good cellular activity. In addition, this invention studies a specific synthetic method, which is simple, convenient, and conducive to large-scale industrial production and application.

[0062] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention. Attached Figure Description

[0063] Figure 1 IC50 of compound I-1 against POLQ 50 Changes with incubation time. Detailed Implementation

[0064] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The specific embodiments described below are for illustrative purposes only and should not be construed as limiting the scope of the invention to the following examples. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0065] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0066] Preparation of intermediates:

[0067] Preparation of Intermediate A: 5-Bromopyridine-3-methylamine (Intermediate A)

[0068]

[0069] Step 1: Preparation of 3-bromo-5-chloromethylpyridine (compound A1)

[0070] 5-Bromopyridine-3-methanol (I-1, 9 g, 48.0 mmol) was dissolved in 60 mL of DCM, cooled to 0 °C, and thionyl chloride (22.8 g, 191.4 mmol) was added dropwise. The mixture was stirred overnight at room temperature. The reaction solution was poured into 20 mL of ice water, alkalized with 1 N NaOH aqueous solution, extracted with ethyl acetate, and the organic layers were combined. The mixture was washed with saturated NaCl solution, dried over anhydrous Na₂SO₄, concentrated, and purified by silica gel column chromatography to give a white solid. Yield 70%; ESI-MS: m / z = 206.0, 207.9 [M+H] + .

[0071] Step 2: Preparation of 5-bromopyridine-3-methylamine (intermediate A)

[0072] 3-Bromo-5-chloromethylpyridine (I-2, 7 g, 34.0 mmol) was dissolved in 150 mL of 7N ammonia-methanol solution, heated to 60 °C and stirred overnight. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography to obtain a pale yellow solid. Yield 31%; ESI-MS: m / z = 187.1, 189.1 [M+H] + .

[0073] Preparation of Intermediate B: N-(3-(5-aminomethyl)pyridin-3-yl)propyl-2-yn-1-yl)-2-(2,4-bis(trifluoromethylphenyl)-N-(4-fluorophenyl)acetamide (Intermediate B)

[0074]

[0075] Step 1: Preparation of 4-fluoro-N-propyl-2-ynylaniline (compound B1)

[0076] 10 g (107.3 mmol, 9.80 mL) of p-fluoroaniline was dissolved in 50 mL of DMF, and sodium hydride (4.89 g, 112.7 mmol, 60% purity) was added. The mixture was stirred at room temperature for 30 min, and then 3-bromo-1-propyne (16.77 g, 112.7 mmol, 19.2 mL) was slowly added dropwise. The mixture was stirred for another 2 h. The reaction solution was diluted with saturated NaHCO3 solution, extracted with ethyl acetate, and the organic layers were combined. The mixture was washed with saturated NH4Cl solution, dried over anhydrous Na2SO4, concentrated, and purified by silica gel column chromatography to obtain a reddish-brown oily liquid. Yield: 47%; ESI-MS: m / z = 150.0 [M+H] + .

[0077] Step 2: Preparation of 2-(2,4-bis(trifluoromethyl)phenyl-N-(4-fluorophenyl)-N-prop-2-yn-1-ylacetamide (compound B2)

[0078] Compound B1 (500.0 mg, 3.33 mmol) and 2,4-bis(trifluoromethyl)phenylacetic acid (905.7 mg, 3.33 mmol) were dissolved in 25 mL of THF. N,N-diisopropylethylamine (865.8 mg, 6.66 mmol) and T3P (4.23 g, 6.66 mmol, 50% purity) were added, and the mixture was reacted at 80 °C for 2 h. After cooling to room temperature, water was added, and the mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated NaCl solution, dried over anhydrous Na₂SO₄, concentrated, and purified by silica gel column chromatography to give a white solid. Yield: 51%; ESI-MS: m / z = 404.1 [M+H] + . 1 H NMR (400MHz, CDCl3) δ7.87(s,1H),7.79(d,J=8.0Hz,1H),7.55(d,J=8.0Hz,1H),7.39–7.30 (m,2H),7.19(dd,J1=8.9,J2=8.0Hz,2H),4.50(d,J=2.5Hz,2H),3.63(s,2H),2.26(t,1H).

[0079] Step 3: Preparation of N-(3-(5-aminomethyl)pyridin-3-yl)propyl-2-yn-1-yl)-2-(2,4-bis(trifluoromethylphenyl)-N-(4-fluorophenyl)acetamide (intermediate B)

[0080] Compound B2 (100.0 mg, 0.25 mmol) and compound A (56.1 mg, 0.30 mmol) were dissolved in 5 mL of DMF. Triethylamine (150 mg, 1.50 mmol), PdCl2(PPh3)2 (0.03 g, 0.05 mmol), and CuI (0.01 g, 0.05 mmol) were then added. The mixture was reacted at 80 °C for 2 h under a N2 atmosphere. After cooling to room temperature, water was added, and the mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated NaCl solution, dried over anhydrous Na2SO4, concentrated, and purified by silica gel column chromatography to obtain a grayish-brown oily liquid. Yield: 38%; ESI-MS: m / z = 510.1 [M+H] + .

[0081] Preparation of Intermediate C: 2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)-N-(3-(5-((methylamino)methyl)pyridin-3-yl)prop-2-yn-1-yl)acetamide (Intermediate C)

[0082]

[0083] Step 1: Preparation of 1-(5-bromopyridin-3-yl)-N-methylmethylamine (compound C1)

[0084] 5-Bromopyridine-3-carboxaldehyde (930 mg, 5.0 mmol) was added to a reaction flask, dissolved in MeOH solvent, followed by methylamine solution (155 mg, 5.0 mmol). A few drops of acetic acid were added to adjust the pH of the reaction solution to 3-4, and the mixture was stirred at room temperature for 30 min. Then, sodium cyanoborohydride (465 mg, 7.5 mmol) was added, and the reaction was stirred at room temperature for 3 h until completion. The reaction progress was monitored by TLC. The mixture was diluted with water, extracted with ethyl acetate, and the organic layer was washed with brine. The organic phases were combined, dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated. The crude product was purified by column chromatography to obtain a white solid. The yield was 53%; ESI-MS: m / z = 200.2 [M+H]. + .

[0085] Step 2: Preparation of 2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)-N-(3-(5-((methylamino)methyl)pyridin-3-yl)prop-2-yn-1-yl)acetamide (intermediate C)

[0086] The preparation of intermediate C followed the synthesis of intermediate B. Intermediate A was replaced with an equimolar amount of 1-(5-bromopyridin-3-yl)-N-methylmethylamine (C1), while the remaining starting materials, reagents, and preparation method were the same as for intermediate B, yielding a yellow oily liquid. The yield was 42%; ESI-MS: m / z = 524.0 [M+H] + .

[0087] Preparation of Intermediate D: N-(3-(3-(aminomethyl)phenyl)prop-2-yn-1-yl)-2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamide (Intermediate D)

[0088]

[0089] The preparation of intermediate D was performed following the synthesis of compound B. Intermediate A was replaced with an equimolar amount of 3-iodobenzylamine, and the remaining starting materials, reagents, and preparation method were the same as for intermediate B, yielding a yellow oily liquid. The yield was 53%; ESI-MS: m / z = 509.0 [M+H] + .

[0090] Preparation of Intermediate E: N-(3-(4-(aminomethyl)phenyl)prop-2-yn-1-yl)-2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamide (Intermediate E)

[0091] The preparation of intermediate E was performed following the synthesis of compound B. Intermediate A was replaced with an equimolar amount of p-iodobenzylamine, and the remaining starting materials, reagents, and preparation method were the same as for intermediate B, yielding a yellow oily liquid. The yield was 56%; ESI-MS: m / z = 509.0 [M+H]+ .

[0092] Preparation of Intermediate F: N-(3-(3-(2-aminoethyl)phenyl)prop-2-yn-1-yl)-2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamide (compound F)

[0093] The preparation of intermediate F was performed following the synthesis of compound B. Intermediate A was replaced with an equimolar amount of 2-(3-iodophenyl)ethyl-1-amine, and the remaining starting materials, reagents, and preparation method were the same as for intermediate B, yielding a yellow oily liquid. The yield was 51%; ESI-MS: m / z = 523.1 [M+H] + .

[0094] Preparation of intermediate G: N-(3-(3-aminophenyl)prop-2-yn-1-yl)-2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamide (compound G)

[0095] The preparation of intermediate G was performed following the synthesis of compound B. Intermediate A was replaced with an equimolar amount of m-iodoaniline, and the remaining starting materials, reagents, and preparation method were the same as for intermediate B, yielding a brown oily liquid. The yield was 41%; ESI-MS: m / z = 495.1 [M+H] + .

[0096] Preparation of intermediate H: N-(3-(5-aminopyridin-3-yl)prop-2-yn-1-yl)-2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamide (compound H)

[0097] The preparation of intermediate H was performed following the synthesis of compound B. Intermediate A was replaced with an equimolar amount of 5-bromopyridin-3-amine, and the remaining starting materials, reagents, and preparation method were the same as for intermediate B, yielding a brown oily liquid. The yield was 64%; ESI-MS: m / z = 496.1 [M+H] + .

[0098] Preparation of Intermediate J: N-(3-(6-aminopyridin-3-yl)prop-2-yn-1-yl)-2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamide (Compound J)

[0099] The preparation of intermediate J was performed following the synthesis of compound B. Intermediate A was replaced with an equimolar amount of 5-bromopyridin-2-amine, and the remaining starting materials, reagents, and preparation method were the same as for intermediate B, yielding a brown oily liquid. The yield was 47%; ESI-MS: m / z = 496.1 [M+H] + .

[0100] Preparation of intermediate K: N-(3-(6-aminopyridazin-3-yl)prop-2-yn-1-yl)-2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamide (compound K)

[0101] The preparation of intermediate K was performed following the synthesis of compound B. Intermediate A was replaced with an equimolar amount of 6-bromopyridazine-3-amine, and the remaining starting materials, reagents, and preparation method were the same as for intermediate B, yielding a brown oily liquid. The yield was 38%; ESI-MS: m / z = 497.1 [M+H] + .

[0102] Preparation of intermediate L: N-(3-(5-(aminomethyl)pyridin-3-yl)prop-2-yn-1-yl)-2-(3,5-bis(trifluoromethyl)pyridin-2-yl)-N-(4-fluorophenyl)acetamide (compound L)

[0103]

[0104] Step 1: Preparation of 2-(3,5-bis(trifluoromethyl)pyridin-2-yl)-N-(4-fluorophenyl)-N-(prop-2-yn-1-yl)acetamide (compound L2)

[0105] Compound B1 (500.0 mg, 3.33 mmol) and 2-(3,5-bis(trifluoromethylpyridin-2-yl)acetic acid (909.1 mg, 3.33 mmol) were dissolved in 25 mL of THF. N,N-diisopropylethylamine (865.8 mg, 6.66 mmol) and T3P (4.25 g, 6.66 mmol, 50% purity) were added, and the mixture was reacted at 80 °C for 2 h. After cooling to room temperature, water was added, and the mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated NaCl solution, dried over anhydrous Na₂SO₄, concentrated, and purified by silica gel column chromatography to give a white solid. Yield: 58%; ESI-MS: m / z = 405.1 [M+H] + .

[0106] Step 2: Preparation of N-(3-(5-(aminomethyl)pyridin-3-yl)prop-2-yn-1-yl)-2-(3,5-bis(trifluoromethyl)pyridin-2-yl)-N-(4-fluorophenyl)acetamide (compound L)

[0107] Compound L2 (100.0 mg, 0.25 mmol) and compound A (56.1 mg, 0.30 mmol) were dissolved in 5 mL of DMF. Triethylamine (150 mg, 1.50 mmol), PdCl2(PPh3)2 (0.03 g, 0.05 mmol), and CuI (0.01 g, 0.05 mmol) were then added. The mixture was reacted at 80 °C for 2 h under a N2 atmosphere. After cooling to room temperature, water was added, and the mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated NaCl solution, dried over anhydrous Na2SO4, concentrated, and purified by silica gel column chromatography to obtain a grayish-brown oily liquid. Yield: 52%; ESI-MS: m / z = 511.1 [M+H] + .

[0108] Preparation of intermediate M: N-(3-(5-aminopyridin-3-yl)prop-2-yn-1-yl)-2-(3,5-bis(trifluoromethyl)pyridin-2-yl)-N-(4-fluorophenyl)acetamide (compound M)

[0109]

[0110] The preparation of intermediate M was performed following the synthesis of compound L. Intermediate A was replaced with an equimolar amount of 3-iodobenzylamine; the remaining starting materials, reagents, and preparation method were the same as in L, yielding a brown oily liquid. The yield was 51%; ESI-MS: m / z = 497.1 [M+H] + .

[0111] Preparation of intermediate N: N-(3-(5-(aminomethyl)pyridin-3-yl)prop-2-yn-1-yl)-2-(3,5-bis(trifluoromethyl)pyridin-2-yl)-N-(4-fluorophenyl)acetamide (compound N)

[0112] The preparation of intermediate N was performed following the synthesis of compound L. Intermediate A was replaced with an equimolar amount of 5-bromopyridin-3-amine; the remaining starting materials, reagents, and preparation method were the same as in L, yielding a brown oily liquid. The yield was 41%; ESI-MS: m / z = 511.0 [M+H] + .

[0113] Preparation of intermediate O: 2-(3,5-bis(trifluoromethylpyridin-2-yl)-N-(4-fluorophenyl)-N-(3-(5-(((methylamino)methyl)pyridin-3-yl)prop-2-yn-1-yl)acetamide (compound O)

[0114] The preparation of intermediate O was performed following the synthesis of compound L. Intermediate A was replaced with an equimolar amount of 1-(5-bromopyridin-3-yl)-N-methylmethylamine (C1), with the remaining starting materials, reagents, and preparation method remaining the same as in L, yielding a pale yellow oily liquid. The yield was 52%; ESI-MS: m / z = 525.1 [M+H]+ .

[0115] The structural formulas of this series of intermediates are shown in Table 1 below.

[0116] Table 1. Structural Formulas of the Intermediates in this Series

[0117]

[0118]

[0119] Example 1: Preparation of N-(5-(3-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamido)prop-1-ynyl)pyridine-3-methyl)acrylamide (compound I-1)

[0120]

[0121] Intermediate B (100.0 mg, 0.20 mmol) was dissolved in 5 mL of DCM, then triethylamine (60 mg, 0.60 mmol) was added. The mixture was cooled to 0 °C, and acryloyl chloride (20.0 mg, 0.20 mmol) was added dropwise. The reaction was carried out at room temperature for 1 h. Saturated NaHCO3 solution was added, and the mixture was extracted with dichloromethane. The organic layers were combined, washed with saturated NaCl solution, dried over anhydrous Na2SO4, concentrated, and purified by silica gel column chromatography to give a grayish-white solid. Yield 24%; ESI-MS: m / z = 564.1 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.51 (s, 2H), 7.88 (s, 1H), 7.80 (d, J = 7.6Hz, 1H), 7.65 (s, 1H),7.55(d,J=8.0Hz,1H),7.43-7.33(m,2H),7.22(t,J=8.4Hz,2H),6.38(dd, J1=16.0,J2=1.2Hz,1H),6.15(dd,J1=16.0,J2=10.4Hz,1H),6.07(s,1H),5.75 (dd,J1=10.0,J2=1.2Hz,1H),4.73(s,2H),4.54(d,J=6.4Hz,2H),3.65(s,2H).

[0122] Example 2: Preparation of 2-(2,4-bis(trifluoromethyl)phenyl-N-(3-(5-(2-chloroacetamido)methylpyridin-3-yl)prop-2-ynyl-1-yl)-N-(4-fluorophenyl)acetamide (compound I-2)

[0123] The preparation of target compound I-2 was performed following the synthesis of compound I-1. Acryloyl chloride was replaced with an equimolar amount of chloroacetyl chloride, and the remaining starting materials, reagents, and preparation method were the same as in Example 1, yielding a white solid. The yield was 27%; ESI-MS: m / z = 586.1 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.52(d,J=10.4Hz,1H),8.36(d,J=20.8Hz,1H),7.87(s,1H),7.79(s,1H),7.69(d,J=19.2Hz,1H),7.56(d,J=8 .0Hz,1H),7.39(s,2H),7.22(d,J=10.4Hz,2H),4.82-4.64(m,2H),4.52(d,J=6.4Hz,2H),4.22-4.12(m,1H),3.68(d,J=10.0Hz,2H).

[0124] Example 3: Preparation of 2-(2,4-bis(trifluoromethyl)phenyl-N-(4-fluorophenyl)-N-(3-(5-vinylsulfonamidemethyl)pyridin-3-yl)propyl-2-yn-1-yl)acetamide (compound I-3)

[0125] The preparation of target compound I-3 was performed following the synthesis of compound I-1. Acryloyl chloride was replaced with an equimolar amount of vinylsulfonyl chloride; the remaining starting materials, reagents, and preparation method were the same as in Example 1, yielding a white solid. The yield was 35%; ESI-MS: m / z = 600.1 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.54(d,J=2.0Hz,1H),8.48(d,J=2.4Hz,1H),7.88(s,1H ),7.80(d,J=8.0Hz,1H),7.72(t,1H),7.56(d,J=8.0Hz,1H),7.46-7.34(m,2H ),7.24(t,J=8.4Hz,2H),6.54(dd,J1=16.4,J2=10.0Hz,1H),6.32(d,J=16.4H z,1H),6.01(d,J=9.6Hz,1H),4.75(s,2H),4.24(d,J=6.4Hz,2H),3.66(s,2H).

[0126] Example 4: Preparation of 5-(3-(2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)pyridin-3-yl)methyl)aminosulfonyl fluoride (compound I-4)

[0127] The preparation of target compound I-4 was performed following the synthesis of compound I-1. Acryloyl chloride was replaced with an equimolar amount of fluorosulfonyl chloride; the remaining raw materials, reagents, and preparation method were the same as in Example 1, yielding a yellow solid. The yield was 42%; ESI-MS: m / z = 592.0 [M+H]. + . 1 H NMR (400MHz, CDCl3) δ8.61-8.55(m,1H),8.52-8.48(m,1H),7.87(s,1H),7.84-7.69(m,2H),7.54(d,J=8.0Hz,1H),7.43-7.34(m,2H),7.26-7.19 (m,1H),7.14(t,J=8.2Hz,1H),4.73(d,J=7.2Hz,1H),4.65(s,1H),4.55 (d,J=6.4Hz,1H),4.46(s,1H),4.37-4.23(m,1H),3.65(d,J=9.2Hz,2H).

[0128] Example 5: Preparation of N-(5-(3-(2-(2,4-bis(trifluoromethylphenyl)-N-(4-fluorophenyl)acetamido)propyl-1-ynylpyridin-3-yl)methyl)-3-chloropropionamide (compound I-5)

[0129] The preparation of target compound I-5 was performed following the synthesis of compound I-1. Acryloyl chloride was replaced with an equimolar amount of 3-chloroacetyl chloride; the remaining starting materials, reagents, and preparation method were the same as in Example 1, yielding a white solid. The yield was 42%; ESI-MS: m / z = 600.1 [M+H]. + . 1 H NMR (400MHz, CDCl3) δ8.54-8.36(m,2H),7.88(s,1H),7.80(d,J=8.0Hz,1H),7.64(s,1H),7.55(d,J=8.0Hz,1H),7.43-7.34(m,2H),7.2 7-7.16(m,2H),4.74(s,2H),4.52(dd,J1=16.0,J2=6.0Hz,2H),4.24(s,1H),3.87(t,J=6.4Hz,1H),3.65(s,2H),2.71(t,J=6.4Hz,1H).

[0130] Example 6: Preparation of N-(5-(3-(2-(2,4-bis(trifluoromethylphenyl)-N-(4-fluorophenyl)acetamido)propyl-1-ynyl)pyridin-3-yl)methyl)but-2-yne (compound I-6)

[0131]

[0132] Intermediate B (100.0 mg, 0.20 mmol) was dissolved in 5 mL of ultra-dry DCM. 2-Butynedic acid (16.8 mg, 0.20 mmol), DCC (41.0 mg, 0.20 mmol), and DMAP (41.0 mg, 0.02 mmol) were added at room temperature. The mixture was stirred at room temperature, and the reaction was monitored by TLC. After 2 h, the reaction was completed. The mixture was extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. The mixture was then filtered, and the solvent was evaporated. The crude product was purified by column chromatography to give the target compound I-6 as a white solid. The yield was 31%; ESI-MS: m / z = 576.1 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.52(d,J=2.0Hz,1H),8.47(d,J=2.4Hz,1H),7.88(s,1H),7.80(d,J=8.0Hz,1H),7.63(s,1H),7.56 (d,J=8.4Hz,1H),7.46-7.31(m,2H),7.23(t,J=8.4Hz,2H),4.75(s,2H),4.49(d,J=6.4Hz,2H),3.66(s,2H),1.99(s,3H).

[0133] Example 7: Preparation of N-(5-(3-(2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamido)propyl-1-ynylpyridin-3-yl)methyl)-2-oxopropionamide (compound I-7)

[0134] The preparation of target compound I-7 was performed following the synthesis of compound I-6. 2-Butynic acid was replaced with an equimolar amount of pyruvic acid; the remaining starting materials, reagents, and preparation method were the same as in Example 6, yielding a white solid. The yield was 53%; ESI-MS: m / z = 580.1 [M+H]. + . 1 H NMR (400MHz, CDCl3) δ8.53(d,J=2.0Hz,1H),8.49(d,J=2.0Hz,1H),7.88(s,1H),7.80(d,J=8.0Hz,1H),7.60(s,1H),7.55 (d,J=8.4Hz,1H),7.43-7.34(m,2H),7.23(t,J=8.4Hz,2H),4.74(s,2H),4.49(d,J=6.4Hz,2H),3.65(s,2H),2.54(s,3H).

[0135] Example 8: Preparation of 2-(2,4-bis(trifluoromethyl)phenyl-N-(3-(5-((2,2-dichloroacetamido)methyl)pyridin-3-yl)propyl-2-ynyl)-N-(4-fluorophenyl)acetamide (compound I-8)

[0136] The preparation of target compound I-8 was performed following the synthesis of compound I-6. 2-Butynic acid was replaced with an equimolar amount of dichloroacetic acid; the remaining starting materials, reagents, and preparation method were the same as in Example 6, yielding a white solid. The yield was 37%; ESI-MS: m / z = 620.0 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.55(s,1H),8.51(s,1H),7.88(s,1H),7.80(d,J=8.4Hz,1H),7.62(s,1H),7.55( d,J=8.4Hz,2H),7.42-7.34(m,2H),7.27-7.19(m,2H),4.75(s,2H),4.54(d,J=6.0Hz,2H),3.66(s,2H).

[0137] Example 9: Preparation of 5-(3-(2,4-bis(trifluoromethylphenyl)-N-(4-fluorophenyl)acetamido)prop-1-ynyl-1-yl)pyridin-3-yl)methyl)-4-dimethylaminobut-2-enamide (compound I-9)

[0138] The preparation of target compound I-9 was performed following the synthesis of compound I-6. 2-Butynic acid was replaced with an equimolar amount of (E)-4-(dimethylamino)but-2-enoic acid; the remaining starting materials, reagents, and preparation method were the same as in Example 6, yielding a white solid. The yield was 49%; ESI-MS: m / z = 621.2 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.56(s,1H),8.47(s,1H),7.96(s,1H),7.83(t,J=8.0Hz,1H),7.68(s,1H),7.45(d,J=8.0Hz,2H),7.32(d,J =7.8Hz,1H),7.34-7.04(m,2H),6.66(m,1H),6.01(d,J=7.8Hz,1H),4.56(s,2H),3.78(s,2H),3.39(d,J=7.6Hz,2H),2.33(s,6H).

[0139] Example 10: Preparation of 2-(2,4-bis(trifluoromethyl)phenyl-N-(3-(5-(((2-cyanoacetamido)methyl)pyridin-3-yl)prop-2-yn-1-yl)-N-(4-fluorophenyl)acetamide (compound I-10)

[0140] The preparation of target compound I-10 was performed following the synthesis of compound I-6. 2-Butynic acid was replaced with an equimolar amount of cyanoacetic acid; the remaining starting materials, reagents, and preparation method were the same as in Example 6, yielding a white solid. The yield was 32%; ESI-MS: m / z = 577.1 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.55(s,1H),8.50(s,1H),7.88(s,1H),7.80(d,J=8.0Hz,1H),7.63(s,1H),7.55(d,J=8.4 Hz,1H),7.41-7.35(m,2H),7.23(t,J=8.4Hz,2H),6.56(s,1H),4.75(s,2H),4.50(d,J=6.0Hz,2H),3.66(s,2H).

[0141] Example 11: Preparation of N-((5-(3-(2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)pyridin-3-yl)methyl)propionamide (compound I-11)

[0142] The preparation of target compound I-11 was performed following the synthesis of compound I-6. 2-Butynic acid was replaced with an equimolar amount of propynic acid; the remaining starting materials, reagents, and preparation method were the same as in Example 6, yielding a pale white solid. The yield was 53%; ESI-MS: m / z = 562.1 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.75(d,J=2.4Hz,1H),8.57(d,J=2.0Hz,1H),8.04(s,1H),7.79(d,J=8.0Hz,1H),7.63(s,1H),7.56 (d,J=8.4Hz,1H),7.46-7.28(m,2H),7.23(t,J=8.4Hz,2H),4.75(s,2H),4.49(d,J=6.4Hz,2H),3.66(s,2H),2.85(s,1H).

[0143] Example 12: Preparation of N-((5-(3-(2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)pyridin-3-ylmethyl)-N-methylacrylamide (compound I-12)

[0144]

[0145] Intermediate C (105.0 mg, 0.20 mmol) was dissolved in 5 mL of DCM, then triethylamine (60 mg, 0.60 mmol) was added. The mixture was cooled to 0 °C, and acryloyl chloride (20.0 mg, 0.20 mmol) was added dropwise. The reaction was carried out at room temperature for 1 h. Saturated NaHCO3 solution was added, and the mixture was extracted with dichloromethane. The organic layers were combined, washed with saturated NaCl solution, dried over anhydrous Na2SO4, concentrated, and purified by silica gel column chromatography to give a white solid. Yield: 41%; ESI-MS: m / z = 578.1 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.53(d,J=12.0Hz,1H),8.45(d,J=16.0Hz,1H),7.88(s,1 H),7.80(d,J=8.0Hz,1H),7.62(s,1H),7.56(d,J=8.0Hz,1H),7.42-7.34(m,2H) ,7.22(t,J=8.4Hz,2H),6.66(dd,J1=16.8,J2=10.4Hz,1H),6.45(m,1H),5.81(m ,1H),4.74(s,2H),4.64(d,J=14.0Hz,2H),3.66(s,2H),3.05(d,J=10.0Hz,3H).

[0146] Example 13: Preparation of N-(3-(3-(2-(2,4-bis(trifluoromethyl)phenyl))-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)benzyl)acrylamide (compound I-13)

[0147]

[0148] Intermediate D (101.0 mg, 0.20 mmol) was dissolved in 5 mL of DCM, then triethylamine (60 mg, 0.60 mmol) was added. The mixture was cooled to 0 °C, and acryloyl chloride (20.0 mg, 0.20 mmol) was added dropwise. The reaction was carried out at room temperature for 1 h. Saturated NaHCO3 solution was added, and the mixture was extracted with dichloromethane. The organic layers were combined, washed with saturated NaCl solution, dried over anhydrous Na2SO4, concentrated, and purified by silica gel column chromatography to give a white solid. The yield was 27%; ESI-MS: m / z = 563.1 [M+H] + . 1H NMR (400MHz, CDCl3) δ7.88(s,1H),7.79(d,J=8.0Hz,1H),7.56(d,J=8.0Hz,1H),7.43-7.35(m,2H),7.31(s,1H),7.29(s,3H),7.21(t ,J=8.4Hz,2H),6.42-6.34(m,1H),6.15(dd,J1=16.8,J2=10.4Hz,1H),5.73(m,1H),4.72(s,2H),4.52(d,J1=6.0Hz,2H),3.65(s,2H).

[0149] Example 14: Preparation of N-(3-(6-(2,4-bis(trifluoromethyl)phenyl)-4-(4-fluorophenyl)-5-acetamido-1-yn-1-yl)benzyl)-2-chloroacetamide (compound I-14)

[0150] The preparation of target compound I-14 was performed following the synthesis of compound I-13. Acryloyl chloride was replaced with an equimolar amount of chloroacetyl chloride; the remaining starting materials, reagents, and preparation method were the same as in Example 13, yielding a white solid. The yield was 37%; ESI-MS: m / z = 584.1 [M+H]. + . 1 H NMR (400MHz, CDCl3) δ7.69 (s, 1H), 7.51 (d, J = 8.0Hz, 1H), 7.42-7.36 (m, 2H), 7.27-7.16 (m, 2H), 7.01 (d, J = 7. 8Hz,1H),7.00-6.95(m,2H),6.90-6.84(m,2H),4.74(s,2H),4.52(dd,J1=16.0,J2=6.0Hz,2H),4.21(s,1H).

[0151] Example 15: Preparation of N-(3-(3-(2-(2,4-bis(trifluoromethyl)phenyl))-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)benzyl)propanolamide (compound I-15)

[0152]

[0153] Intermediate D (110.0 mg, 0.20 mmol) was dissolved in 5 mL of ultra-dry DCM. Propynoic acid (15.8 mg, 0.20 mmol), DCC (41.0 mg, 0.20 mmol), and DMAP (41.0 mg, 0.02 mmol) were added at room temperature. The mixture was stirred at room temperature, and the reaction was monitored by TLC. After 2 h, the reaction was completed. The mixture was extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. The mixture was then filtered, and the solvent was evaporated. The crude product was purified by column chromatography to give the target compound I-15 as a white solid. The yield was 46%; ESI-MS: m / z = 561.1 [M+H] + . 1 H NMR (400MHz, CDCl3) δ7.88(s,1H),7.79(d,J=8.0Hz,1H),7.56(d,J=8.0Hz,1H),7.38(dd,J1=8.8,J2=5.0Hz,2H) ,7.30(s,3H),7.28(s,1H),7.22(t,J=8.4Hz,2H),4.73(s,2H),4.48(d,J=6.0Hz,2H),3.65(s,2H),2.86(s,1H).

[0154] Example 16: Preparation of N-(3-(3-(2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)benzyl)but-2-ynylamide (compound I-16)

[0155] The preparation of target compound I-16 was performed following the synthesis of compound I-15. Propynoic acid was replaced with an equimolar amount of 2-butynic acid; the remaining starting materials, reagents, and preparation method were the same as in Example 15, yielding a white solid. The yield was 34%; ESI-MS: m / z = 575.1 [M+H] + . 1 H NMR (400MHz, CDCl3) δ7.88(s,1H),7.79(d,J=8.0Hz,1H),7.57(d,J=8.0Hz,1H),7.38(dd,J1=8.8,J2=4.8Hz,2H),7.30(d,J =2.0Hz,2H),7.28(s,1H),7.27(s,1H),7.22(t,J=8.4Hz,2H),4.73(s,2H),4.46(d,J=6.0Hz,2H),3.65(s,2H),1.98(s,3H).

[0156] Example 17: Preparation of 2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)-N-(3-(3-(vinylsulfonylaminomethyl)phenyl)prop-2-yn-1-yl)acetamide (compound I-17)

[0157] The preparation of target compound I-17 was performed following the synthesis of compound I-13. Acryloyl chloride was replaced with an equimolar amount of vinylsulfonyl chloride; the remaining starting materials, reagents, and preparation method were the same as in Example 13, yielding a white solid. The yield was 44%; ESI-MS: m / z = 599.1 [M+H]. + . 1 H NMR (400MHz, CDCl3) δ7.88(s,1H),7.79(d,J=8.0Hz,1H),7.56(d,J=8.0Hz,1H),7.42-7.36(m,2H),7.35(s,1H),7.31(d,J=1.4Hz,3H),7.22(t,J =8.4Hz,2H),6.52(dd,J1=16.4,J2=10.0Hz,1H),6.30(d,J=16.6Hz,1H), 5.97(d,J=10.0Hz,1H),4.73(s,2H),4.20(d,J=6.4Hz,2H),3.65(s,2H).

[0158] Example 18: Preparation of 3-(3-(2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)benzyl)aminosulfonyl fluoride (compound I-18)

[0159] The preparation of target compound I-18 was performed following the synthesis of compound I-13. Acryloyl chloride was replaced with an equimolar amount of fluorosulfonyl chloride; the remaining starting materials, reagents, and preparation method were the same as in Example 13, yielding a yellow solid. The yield was 53%; ESI-MS: m / z = 591.1 [M+H] + . 1 H NMR (400MHz, CDCl3) δ7.87(s,1H),7.78(d,J=7.8Hz,1H),7.54(d,J=8.0Hz,1H),7.42-7.35(m,2H),7.35(d,J=4.0Hz,2H),7 .34(d,J=1.4Hz,2H),7.34-7.27(m,1H),7.21(t,J=8.4Hz,1H),4.69(s,2H),4.41(dd,J1=6.0,J2=1.8Hz,2H),3.65(s,2H).

[0160] Example 19: Preparation of N-(4-(3-(2-(2,4-bis(trifluoromethyl)phenyl))-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)benzyl)acrylamide (compound I-19)

[0161]

[0162] The preparation of target compound I-19 was performed following the synthesis of compound I-13. Intermediate D was replaced with an equimolar amount of intermediate E, and the remaining starting materials, reagents, and preparation method were the same as in Example 13, yielding a brown oily liquid. The yield was 85%; ESI-MS: m / z = 563.1 [M+H] + .

[0163] Example 20: Preparation of N-(3-(3-(2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)phenethyl)acrylamide (compound I-20)

[0164] The preparation of target compound I-20 was performed following the synthesis of compound I-13. Intermediate D was replaced with an equimolar amount of intermediate F, and the remaining starting materials, reagents, and preparation method were the same as in Example 13, yielding a brown oily liquid. The yield was 69%; ESI-MS: m / z = 577.1 [M+H] + .

[0165] Example 21: Preparation of N-(3-(3-(2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)phenyl)acrylamide (compound I-21)

[0166] The preparation of target compound I-21 was performed following the synthesis of compound I-13. Intermediate D was replaced with an equimolar amount of intermediate G, and the remaining starting materials, reagents, and preparation method were the same as in Example 13, yielding a brown oily liquid. The yield was 63%; ESI-MS: m / z = 549.1 [M+H] + . 1 H NMR (400MHz, CDCl3) δ7.87(s,1H),7.81(d,J=8.0Hz,1H),7.65(s,1H),7.57(d,J=8.4Hz,2H),7.38(dd,J1=8.8,J2=5.0Hz,2H),7.25-7.16( m,3H),7.13(d,J=7.8Hz,1H),6.47(d,J=16.8Hz,1H),6.26(dd,J1=16.8,J2=10.4Hz,1H),5.83(d,J=10.4Hz,1H),4.73(s,2H),3.65(s,2H).

[0167] Example 22: Preparation of N-(5-(3-(2-(2,4-bis(trifluoromethyl)phenyl))-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)pyridin-3-yl)acrylamide (compound I-22)

[0168] The preparation of target compound I-21 was performed following the synthesis of compound I-13. Intermediate D was replaced with an equimolar amount of intermediate H, and the remaining starting materials, reagents, and preparation method were the same as in Example 13, yielding a yellow solid. The yield was 54%; ESI-MS: m / z = 550.1 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.54(d,J=2.4Hz,1H),8.35(s,1H),8.28(s,1H),7.88(s,1H),7.81(d,J=8.0Hz,1H),7.57(d,J=8.0Hz,1H),7.38(dd,J1=8.8, J2=4.8Hz,2H),7.23(t,J=8.4Hz,2H),6.51(d,J=16.8Hz,1H),6.28(dd,J1 =16.8, J2=10.4Hz,1H),5.89(d,J=10.4Hz,1H),4.75(s,2H),3.66(s,2H).

[0169] Example 23: Preparation of N-(5-(3-(2-(2,4-bis(trifluoromethyl)phenyl))-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)pyridin-2-yl)acrylamide (compound I-23)

[0170] The preparation of target compound I-23 was performed following the synthesis of compound I-13. Intermediate D was replaced with an equimolar amount of intermediate J, and the remaining starting materials, reagents, and preparation method were the same as in Example 13, yielding a white solid. The yield was 75%; ESI-MS: m / z = 550.1 [M+H]. + . 1 H NMR (400MHz, CDCl3) δ8.28(d,J=7.8Hz,1H),7.98(s,1H),7.88(s,1H),7.80(d,J=8.0Hz,1H),7.69(d,J=8.8Hz,1H),7.56(d,J=8.0Hz,1H), 7.38(dd,J1=8.8,J2=4.8Hz,2H),7.22(t,J=8.4Hz,1H),6.50(d,J=16.8Hz,1H),6.28(dd,J1=17.2,J2=10.4Hz,1H),5.87(d,J=10.4Hz,1H).

[0171] Example 24: Preparation of N-(6-(3-(2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)pyridazin-3-yl)acrylamide (compound I-24)

[0172] The preparation of target compound I-24 was performed following the synthesis of compound I-13. Intermediate D was replaced with an equimolar amount of intermediate K, and the remaining starting materials, reagents, and preparation method were the same as in Example 13, yielding a white solid. The yield was 33%; ESI-MS: m / z = 551.1 [M+H] + .

[0173] Example 25: Preparation of N-((5-(3-(2-(3,5-bis(trifluoromethyl)pyridin-2-yl)-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)pyridin-3-yl)methyl)acrylamide (I-25)

[0174] The preparation of target compound I-25 was performed following the synthesis of compound I-1. Intermediate D was replaced with an equimolar amount of intermediate L, and the remaining starting materials, reagents, and preparation method were the same as in Example 13, yielding a white solid. The yield was 42%; ESI-MS: m / z = 565.1 [M+H] + .

[0175] Example 26: Preparation of N-(5-(3-(2-(3,5-bis(trifluoromethyl)pyridin-2-yl)-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)pyridin-3-yl)acrylamide (compound I-26)

[0176] The preparation of target compound I-26 was performed following the synthesis of compound I-13. Intermediate D was replaced with an equimolar amount of intermediate M, and the remaining starting materials, reagents, and preparation method were the same as in Example 13, yielding a white solid. The yield was 37%; ESI-MS: m / z = 551.1 [M+H] + .

[0177] Example 27: Preparation of N-(3-(3-(2-(3,5-bis(trifluoromethyl)pyridin-2-yl)-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)benzyl)acrylamide (I-27)

[0178] The preparation of target compound I-27 was performed following the synthesis of compound I-13. Intermediate D was replaced with an equimolar amount of intermediate N, and the remaining starting materials, reagents, and preparation method were the same as in Example 13, yielding a white solid. The yield was 85%; ESI-MS: m / z = 564.1 [M+H] + .

[0179] Example 28: Preparation of (E)-N-((5-(3-(2-(3,5-bis(trifluoromethyl)pyridin-2-yl)-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)pyridin-3-yl)methyl)but-2-enamide (I-28)

[0180]

[0181] Intermediate L (102.0 mg, 0.20 mmol) was dissolved in 5 mL of ultra-dry DCM. (E)-4-(dimethylamino)but-2-enoic acid (25.8 mg, 0.20 mmol), DCC (41.0 mg, 0.20 mmol), and DMAP (41.0 mg, 0.02 mmol) were added at room temperature. The mixture was stirred at room temperature, and the reaction was monitored by TLC. After 2 h, the reaction was completed. The mixture was extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. The mixture was then filtered, and the solvent was evaporated. The crude product was purified by column chromatography to give the target compound I-28 as a white solid. The yield was 31%; ESI-MS: m / z = 622.1 [M+H] + .

[0182] Example 29: Preparation of (E)-N-((5-(3-(2-(3,5-bis(trifluoromethyl)pyridin-2-yl)-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)pyridin-3-yl)methyl)-3-cyanoacrylamide (I-29)

[0183] The preparation of target compound I-29 was performed following the synthesis of compound I-28. (E)-4-(dimethylamino)but-2-enoic acid was replaced with an equimolar amount of (E)-3-cyanoacrylic acid; the remaining starting materials, reagents, and preparation method were the same as in Example 28, yielding a white solid. The yield was 47%; ESI-MS: m / z = 590.1 [M+H] + .

[0184] Example 30: Preparation of (E)-N-((5-(3-(2-(3,5-bis(trifluoromethyl)pyridin-2-yl)-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)pyridin-3-yl)methyl)-3-fluoroacrylamide (compound I-30)

[0185] The preparation of target compound I-30 was performed following the synthesis of compound I-28. (E)-4-(dimethylamino)but-2-enoic acid was replaced with an equimolar amount of (E)-3-fluoroacrylic acid; the remaining starting materials, reagents, and preparation method were the same as in Example 28, yielding a white solid. The yield was 43%; ESI-MS: m / z = 583.1 [M+H] + .

[0186] Example 31: Preparation of (E)-N-((5-(3-(2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)pyridin-3-yl)methyl)-2-cyanoacrylamide (I-31)

[0187]

[0188] The preparation of target compound I-31 was performed following the synthesis of compound I-15. Propynoic acid was replaced with an equimolar amount of (E)-3-cyanoacrylic acid; the remaining starting materials, reagents, and preparation method were the same as in Example 15, yielding a white solid. The yield was 46%; ESI-MS: m / z = 589.1 [M+H] + .

[0189] Example 32: Preparation of N-((5-(3-(2-(2,4-bis(trifluoromethyl)phenyl)-N-(4-fluorophenyl)acetamido)prop-1-yn-1-yl)pyridin-3-yl)methyl)-2-fluoroacrylamide (I-32)

[0190] The preparation of target compound I-32 was performed following the synthesis of compound I-15. Propynoic acid was replaced with an equimolar amount of (E)-3-fluoroacrylic acid; the remaining starting materials, reagents, and preparation method were the same as in Example 15, yielding a white solid. The yield was 53%; ESI-MS: m / z = 582.1 [M+H] + .

[0191] Example 33: Preparation of N-((5-(3-(2-(3,5-bis(trifluoromethylpyridin-2-yl)-N-(4-fluorophenyl)acetamyl)prop-1-yn-1-yl)pyridin-3-yl)methyl)propynamide (I-33)

[0192]

[0193] The preparation of target compound I-33 was performed following the synthesis of compound I-28. (E)-4-(dimethylamino)but-2-enoic acid was replaced with an equimolar amount of propynic acid; the remaining starting materials, reagents, and preparation method were the same as in Example 28, yielding a pale yellow solid. The yield was 41%; ESI-MS: m / z = 563.1 [M+H] + .

[0194] Example 34: Preparation of N-((5-(3-(2-(3,5-bistrifluoromethylpyridin-2-yl)-N-(4-fluorophenyl)acetamyl)prop-1-yn-1-yl)pyridin-3-yl)methyl)sulfonyl fluoride (I-34)

[0195]

[0196] Intermediate L (100.0 mg, 0.20 mmol) was dissolved in 5 mL of DCM, then triethylamine (60 mg, 0.60 mmol) was added, the mixture was cooled to 0 °C, and fluorosulfonyl chloride (22.0 mg, 0.20 mmol) was added dropwise. The reaction was carried out at room temperature for 1 h, followed by the addition of saturated NaHCO3 solution and extraction with dichloromethane. The organic layers were combined, washed with saturated NaCl solution, dried over anhydrous Na2SO4, concentrated, and purified by silica gel column chromatography to obtain a grayish-white solid. The yield was 37%; ESI-MS: m / z = 592.1 [M+H] + .

[0197] Example 35: Preparation of N-((5-(3-(2-(3,5-bis(trifluoromethylpyridin-2-yl)-N-(4-fluorophenyl)acetamyl)prop-1-yn-1-yl)pyridin-3-yl)methyl)-N-methylacrylamide (I-35)

[0198]

[0199] The preparation of target compound I-35 was performed following the synthesis of compound I-12. Intermediate B was replaced with an equimolar amount of intermediate O, and the remaining starting materials, reagents, and preparation method were the same as in Example 12, yielding a grayish-white solid. The yield was 33%; ESI-MS: m / z = 579.1 [M+H] + .

[0200] Example 36: Preparation of 2-(3,5-bis(trifluoromethylpyridin-2-yl)-N-(4-fluorophenyl)-N-(3-(5-(ethylenesulfonamide methyl)pyridin-3-yl)prop-2-yn-1-yl)acetamide (I-36)

[0201] The preparation of target compound I-36 was performed following the synthesis of compound I-34. Fluorosulfonyl chloride was replaced with an equimolar amount of vinylsulfonyl chloride; the remaining starting materials, reagents, and preparation method were the same as in Example 34, yielding a white solid. The yield was 61%; ESI-MS: m / z = 601.1 [M+H] + .

[0202] Example 37: Inhibitory effect of some compounds on in vitro POLQ activity

[0203] 1. Experimental Methods

[0204] The ability of compounds to bind to and inhibit POLQ activity in vitro was tested using the Picogreen assay. POLQ enzyme, primer-template double-stranded (PTD) solution, and dNTP working solution were prepared in 1X test buffer. 5 μL of Polθ enzyme, primer-template double-stranded (PTD) solution, and 1X reaction buffer were added to each well of a 384-well plate. After centrifugation, the plate was incubated at room temperature for 30 min. Then, 5 μL of 2X dNTP working solution was added to each well, centrifuged, and incubated at room temperature for another 1 hour. Finally, Picogreen diluted 1:80 was added to terminate the reaction. After 1 hour, the luminescence intensity was measured using a microplate reader (excitation wavelength 485 nm, emission wavelength 520 nm). The inhibition rate (%) was calculated as (Max - Signal) / (Max - Min) * 100%, where Max represents the luminescence signal intensity of the positive control well without the compound, Min represents the luminescence signal intensity of the negative control well without the enzyme, and Signal represents the luminescence signal intensity of the test compound. The final results were plotted in Graphpad Prism to obtain curves and IC50 values. 50 .

[0205] 2. Experimental results: See Table 2 for details.

[0206] Table 2 shows the inhibitory effects of some compounds on POLQ activity.

[0207]

[0208]

[0209] Where "++++" represents IC 50 Value ≤ 5nM; "+++" indicates 5nM ≤ IC 50 Value ≤ 10nM; "++" indicates 10nM ≤ IC 50 Value ≤ 50 nM; "+" indicates 50 nM ≤ IC 50 Value ≤100nM; "-" indicates not measured.

[0210] As can be seen from Table 1, most of the compounds of the present invention can effectively inhibit the in vitro enzyme activity of POLQ at low nanomolar concentrations, and the half-maximal inhibitory concentration of most compounds is less than 5 nM, which indicates that the compounds of the present invention have strong inhibitory activity against POLQ.

[0211] Example 38: Verification of the covalent mechanism of compound I-1

[0212] 1. Experimental Methods

[0213] When proteins and covalent inhibitors are incubated for different durations, compared to non-covalent inhibitors, as the incubation time increases, more and more binding sites are occupied by the covalent inhibitor, increasing the half-inhibitory concentration (IC50). 50The trend will be downward. The test method is the same as in Example 37.

[0214] 2. Experimental Results: Specific results are shown in Table 3. Figure 1 .

[0215] Table 3. Verification results of the covalent mechanism

[0216]

[0217] Enzymatic verification of compound I-1 showed that the IC50 of compound I-1 increased with prolonged incubation time. 50 The value showed a decreasing trend, indicating enhanced inhibitory activity. This suggests that compound I-1 can persistently inhibit the in vitro enzyme activity of POLQ, which is consistent with the typical characteristics of covalent inhibitors.

[0218] Example 39: IC50 of compounds I-13 and I-25 against POLQ 50 Changes with incubation time

[0219] 1. Experimental Methods

[0220] Given that compound I-1 exhibits typical characteristics of a covalent inhibitor, the inhibitory activity (IC50) of compounds I-13 and I-25 against POLQ was further evaluated. 50 Changes with incubation time. The experimental method is the same as in Example 37.

[0221] 2. Experimental results: See Table 4 for details.

[0222] Table 4. IC50 values ​​of compounds I-13 and I-25 against POLQ 50 Changes with incubation time

[0223]

[0224] IC50 of compounds I-13 and I-25 against POLQ 50 The results of changes with incubation time showed that, with the extension of incubation time, I-13 and I-25 reduced the inhibitory effect of POLQ on IC50. 50 It significantly reduces and enhances inhibitory activity, providing sustained inhibition of POLQ enzyme activity in vitro, and is a covalent inhibitor of POLQ.

[0225] Example 40: Inhibitory effect of some compounds on clonogenicity of MDA-MB-436 and DLD-1BRCA2(- / -) cells

[0226] 1. Experimental Methods

[0227] To demonstrate the crucial role of covalent bonding in the molecular activity of compounds, the acrylamide groups in molecules I-1, I-13, and I-25 were replaced with propionamide or the acrylamide groups were removed. Changes in molecular biological activity were observed, and II-1, II-2, and II-3 were synthesized respectively. The specific structures are shown in Table 4 below.

[0228] Table 4 Chemical structures of II-1, II-2 and II-3

[0229]

[0230] The preparation of molecule II-1 was performed following the synthesis of compound I-1, using propionic acid as the starting material. All other synthetic methods were the same as for I-1, yielding a pale yellow solid. The yield was 30%; ESI-MS: m / z = 566.1 [M+H]. + ;

[0231] The preparation of molecule II-2 was performed following the synthesis of compound I-23, using propionic acid as the starting material. Other synthetic methods were the same as for I-23, yielding a pale yellow powder. The yield was 43%; ESI-MS: m / z = 567.1 [M+H]. + ;

[0232] The preparation of molecule II-3 was performed following the synthesis of intermediate B, using pyridazinamine as the starting material instead of 5-bromopyridin-3-methylamine. Other synthetic methods were the same as for intermediate B, yielding a white solid. Yield: 35%; ESI-MS: m / z = 497.1 [M+H] + .

[0233] The purpose of this experiment was to detect the inhibitory effect of the invented compound on colony formation of MDA-MB-436 and DLD-1BRCA2(- / -) cells. MDA-MB-436 and DLD-1BRCA2(- / -) cells were cultured and seeded at a density of 1000 cells / well. The culture plates were incubated overnight at 37°C, 5% CO2, and 100% relative humidity. Then, MDA-MB-436 and DLD-1BRCA2(- / -) cells were treated with different concentrations of the compound and incubated for 144 hours at 37°C, 5% CO2, and 100% relative humidity. After equilibration to room temperature, 50 μL / well of CellTiter-Glo working solution was added, and the cells were incubated at room temperature for 10 minutes in the dark. Fluorescence was then measured using a chemiluminescence module on a TECAN microplate reader, and the data were read. The IC50 was obtained by four-parameter fitting of the data. 50 The value is used to calculate the biological activity of the compound.

[0234] 2. Experimental results: See Table 5 for details.

[0235] Table 5 shows the inhibitory activity of some compounds on the colony formation of MDA-MB-436 and DLD-1BRCA2(- / -) cells.

[0236]

[0237]

[0238] Colony formation inhibition results in MDA-MB-436 and DLD-1BRCA2(- / -) cell lines showed that the reduction of the acrylamide double bond in I-1 and I-25 cells resulted in a 2-3 fold decrease in molecular activity; removal of the acrylamide group (II-3) led to almost complete loss of molecular activity. The significant difference in cell activity before and after double bond reduction fully demonstrates the crucial role of covalent bonding in cell viability.

Claims

1. An N-aryl-N-arylpropynyl-arylacetamide compound, characterized in that, Its general structural formula is shown in Formula I: ； Where X is selected from CH or N; R1 is selected from hydrogen, halogen, amino, cyano, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, and halogenated C1-C6 alkyl; R2 is selected from hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 fluoroalkyl, C1-C6 fluoroalkoxy, and C3-C6 cycloalkyl. Ar can be a benzene ring or a pyridine ring, pyrimidine ring, pyridazine ring, or pyrazine ring; R3 is selected from hydrogen or C1-C3 alkyl groups; n is selected from 0, 1, 2, 3; R4 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl; L is selected from 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 ； The compounds include their tautomers, stereoisomers, isotopic derivatives, or pharmaceutically acceptable salts thereof.

2. The compound according to claim 1, characterized in that, In Formula I X is selected from CH or N; R1 is selected from hydrogen or halogens; R2 is selected from hydrogen, and C1-C3 fluorinated alkyl groups; Ar is selected from benzene ring, pyridine ring, pyrimidine ring, pyridazine ring, and pyrazine ring; n is selected from 0, 1; R3 and R4 are independently selected from hydrogen and C1-C3 alkyl groups; L is selected from 、 、 、 、 、 、 、 、 、 、 、 。 3. The compound according to claim 1, characterized in that, Selected from the following compounds: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 4. The use of the compound of claim 1 in the preparation of a medicament for treating POLQ-mediated diseases, characterized in that, The drug is made from the compound and pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that, The POLQ-mediated disease is a cancer or tumor-related disease, wherein the cancer or tumor is selected from solid tumors and hematologic malignancies, wherein the solid tumor is selected from breast cancer, colorectal cancer, cervical cancer, ovarian cancer, prostate cancer, gastric cancer, esophageal cancer, head and neck cancer or lung cancer; and the hematologic malignancies are selected from lymphoma or leukemia.

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