An isoxazoline thien compound and use thereof

Abstract

The present application provides a kind of isoxazoline thiofene compound and its application, the isoxazoline compound has the structure as shown in formula I, the compound of the present application can be used to prevent and treat parasitic infection, has good prevention and treatment effect, shows good pharmacokinetic properties in mouse in vivo.

Description

[0001] This application claims priority to patent application number 202411723056.X, which was filed on November 28, 2024, and is entitled "An Isoxazoline Thiophene Compound and Its Application Thereof"—applied to the priority claim. Technical Field

[0002] This invention belongs to the field of pharmaceutical compound technology, specifically relating to an isoxazoline thiophene compound and its applications. Background Technology

[0003] Animal ectoparasites pose a significant threat to the health of companion animals and the production performance of livestock and poultry. Effective control of animal ectoparasite infections has always been a goal of the agricultural and veterinary sectors (WOODS DJ, KNAUER C S. Discovery of veterinary antiparasitic agents in the 21st century: a view from industry[J]. Int J Parasitol, 2010, 40(10): 1177-81.). Isoxazolines are a new class of insecticides for pest control. These drugs mainly act on the γ-aminobutyric acid (GABA)-gated chloride channels in invertebrates (MITA T, KIKUCHI T, MIZUKOSHI T, et al. Isoxazoline-substituted benzamide compound and noxious organism control agent [J]. WO Patent, 2005,2005085216: A1. & OZOE Y, ASAHI M, OZOE F, et al. The antiparasiticisoxazoline A1443 is a potent blocker of insect ligand-gated chloride channels [J]. Biochemical and biophysical research communications, 2010, 391(1): 744-9.). Its site of action differs from other insecticides that act on this ion channel (such as Dieldrin and Fipronil), allowing it to overcome resistance issues that have arisen with existing insecticides. The International Resistant Pesticides Committee (IRAC) classifies it as Group 30.Following the development of the insecticidal active ingredient fluranal by researchers from Nissan Chemical Industries of Japan and DuPont of the United States (CASIDA J E. Golden age of RyR and GABA-Rdiamide and isoxazoline insecticides: common genesis, serendipity, surprises, selectivity, and safety [J]. Chemical research in toxicology, 2015, 28(4):560-6.), aforanal, saloranal, and loteranal were subsequently developed. Currently, these four isoxazoline drugs are used for pet deworming. The global sales of this class of dewormers (including compound preparations) exceed US$2 billion annually, making them the mainstay of daily deworming medications for companion animals. Three of the currently marketed fluranal drugs contain the same N-trifluoroethylglycine tail chain. Insects' ability to metabolize this fragment is enhanced, potentially leading to multidrug resistance to fluranal dewormers. Therefore, developing a new generation of safer, more convenient, more effective, and more environmentally friendly lanatin derivatives can overcome the common problem of drug resistance in anthelmintics and provide pet owners with new medication options. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide an isoxazoline thiophene compound and its application.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] On one hand, the present invention provides an isoxazoline compound having the structure shown in Formula I:

[0007]

[0008] in,

[0009] Ring A is selected from 5-10 heteroaryl groups containing 1-3 heteroatoms;

[0010] R1 is selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo-C1-C6 alkyl, deuterated C1-C6 alkyl, halo-C2-C6 alkenyl, halo-C2-C6 alkynyl, halogen, cyano, nitro, -C(O)NR a R b -C(O)R a -C(O)OR a -OR a -R a OR b -OC(O)Ra -OC(O)OR a -OC(O)NR a R b -NR a R b -SR a -S(O)R a -S(O)2R a Or a 3-10 saturated or unsaturated ring containing 0-3 heteroatoms, wherein the 3-10 saturated or unsaturated ring containing 0-3 heteroatoms may optionally be surrounded by 1-3 R... a replace;

[0011] R2 is selected from hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo-C1-C6 alkyl, deuterated C1-C6 alkyl, halo-C2-C6 alkenyl, and halo-C2-C6 alkynyl.

[0012] R3 is selected from hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo-C1-C6 alkyl, deuterated C1-C6 alkyl, halo-C2-C6 alkenyl, and halo-C2-C6 alkynyl.

[0013] R4 and R5 are each independently selected from hydrogen, any C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo-C1-C6 alkyl, deuterated C1-C6 alkyl, halo-C2-C6 alkenyl, halo-C2-C6 alkynyl, halogen, cyano, nitro, -C(O)NHR6, -C(O)R6, -C(O)OR6, -OR6, -OC(O)R6, -OC(O)OR6, -NHR6, -SR6, -S(O)R6, -S(O)2R6, or a 3-10 member saturated or unsaturated ring containing 0-3 heteroatoms, wherein the 3-10 member saturated or unsaturated ring containing 0-3 heteroatoms may optionally be substituted with 1-3 R6s;

[0014] R6 is selected from hydrogen, C1-C6 alkyl groups, and compounds with 1-3 R groups. a Substituted C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo-C1-C6 alkyl, deuterated C1-C6 alkyl, halo-C2-C6 alkenyl, halo-C2-C6 alkynyl, halogen, cyano, nitro, -C(O)NHR a -C(O)NR a R b -C(O)R a -C(O)OR a -OR a -R a OR b -OC(O)R a -OC(O)ORa -OC(O)NR a R b -NR a R b -SR a -S(O)R a -S(O)2R a Or a 3-10 saturated or unsaturated ring containing 0-3 heteroatoms, wherein the 3-10 saturated or unsaturated ring containing 0-3 heteroatoms may optionally be surrounded by 1-3 R... a replace;

[0015] Each R a R b Each is independently selected from hydrogen, deuterium, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo-C1-C6 alkyl, deuterated C1-C6 alkyl, halo-C2-C6 alkenyl, halo-C2-C6 alkynyl, halogen, cyano, nitro, amino, carboxyl, carbonyl, hydroxyl, hydroxyalkyl, alkoxy, haloalkoxy, deuteratedalkoxy, C3-C6 cycloalkyl, halo-C3-C6 cycloalkyl, alkoxy-substituted C3-C6 cycloalkyl, and alkyl groups substituted with 1-3 R groups. c Substituted C3-C6 cycloalkyl, C3-C6 heterocyclic, halogenated C3-C6 heterocyclic, alkyl-substituted C3-C6 heteroaryl, -OR c -NHR c -S(O)2R c -OR c OR d -R c OR d or -OC(O)R c Each R c R d Each is independently selected from halogen, cyano, hydroxyl, C1-C6 alkyl, and halo-C1-C6 alkyl; and

[0016] m is an integer from 0 to 5, such as 0, 1, 2, 3, 4 or 5;

[0017] n is an integer between 0 and 2, such as 0, 1, or 2.

[0018] In some embodiments, the isoxazoline compound has the structure shown in Formula Ia:

[0019]

[0020] R1, R2, R3, R4, R5, R6, R a R b R c R d m and n are as defined above.

[0021] In other embodiments, the isoxazoline compound has the structure shown in Formula Ib:

[0022]

[0023] R1, R2, R3, R4, R5, R6, R a R b R c R d m and n are as defined above.

[0024] In some embodiments, R1 is selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkyl, or halo-C1-C6 alkoxy.

[0025] In other implementations, R1R1 is chlorine.

[0026] In other embodiments, R2 is selected from hydrogen, halogen, C1-C6 alkyl, and halogenated C1-C6 alkyl;

[0027] In other embodiments, R2 is trifluoromethyl.

[0028] In other embodiments, R3 is selected from hydrogen, halogen, C1-C6 alkyl, and halogenated C1-C6 alkyl;

[0029] In other embodiments, R3 is methyl.

[0030] In other embodiments, R4 is hydrogen or a C1-C6 alkyl group.

[0031] In other embodiments, R5 is selected from any C1-C6 alkyl group substituted with R6 or a 3- to 6-membered saturated or unsaturated ring containing 0-3 heteroatoms, wherein the 3- to 6-membered saturated or unsaturated ring containing 0-3 heteroatoms may optionally be substituted with 1-3 R6 atoms.

[0032] In other implementations, R5 is selected from... , , , , , , , , , , , , , , , , , , , , , , , or The wavy line represents the connection site of the functional group.

[0033] In other embodiments, the isoxazoline compound is any one of the following compounds:

[0034] , , , , , , , , , , , , , , , , , , , , , , , , or .

[0035] On the other hand, the present invention also provides tautomers, enantiomers, diastereomers, meso compounds, racemates, or pharmaceutically acceptable salts of the isoxazoline compounds as described above.

[0036] On the other hand, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the isoxazoline compound as described above, its tautomers, enantiomers, diastereomers, mesosomes, racemates, or pharmaceutically acceptable salts; and a pharmaceutically acceptable carrier or excipient.

[0037] On the other hand, the present invention provides the use of isoxazoline compounds or their tautomers, enantiomers, diastereomers, meso compounds, racemates, or pharmaceutically acceptable salts, or pharmaceutical compositions as described above, in the control of ectoparasitic infections in animals.

[0038] the term:

[0039] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0040] The term "isomer" includes enantiomers, diastereomers, and geometric (or conformational) isomers of a given structure. For example, the present invention includes R and S configurations for each asymmetry center, Z and E double bond isomers, Z and E conformational isomers, single stereochemical isomers and mixtures of enantiomers, diastereomers, and geometric (or conformational) isomers.

[0041] The term "pharmaceutically acceptable salt" refers to salts such as their acid addition salts and / or base salts. Suitable acid addition salts are formed from acids, which form non-toxic salts, such as hydrochlorides / chlorides. Suitable base salts are formed from bases, which form non-toxic salts, such as calcium and sodium salts. Hemisalts of acids and bases can also be formed, such as hemisulfates and hemicalcium salts.

[0042] The term “therapeutic effective amount” means the amount of the compound of the present invention that (i) treats a specific disease, symptom or disorder; (ii) reduces, alleviates or eliminates one or more symptoms of a specific disease, symptom or disorder; or (iii) prevents or delays the onset of one or more symptoms of a specific disease, symptom or disorder described in this application.

[0043] The term "pharmaceutically acceptable carrier or excipient" refers to a non-toxic carrier, excipient, or medium that does not impair the pharmacological activity of the compound formulated with it.

[0044] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, and more preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples of lower alkyl groups containing 1 to 6 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc.

[0045] The term "alkenyl" refers to an aliphatic hydrocarbon having at least one carbon-carbon double bond, including straight-chain and branched chains having at least one carbon-carbon double bond. In some embodiments, the alkenyl group has 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, or 2 to 4 carbon atoms. For example, the term "C 2-6"Alkenyl" includes straight-chain or branched unsaturated groups with 2 to 6 carbon atoms (having at least one carbon-carbon double bond), including but not limited to vinyl, 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, etc.

[0046] The term "alkynyl" refers to an aliphatic hydrocarbon having at least one carbon-carbon triple bond, including straight-chain and branched chains having at least one carbon-carbon triple bond. In some embodiments, the alkynyl group has 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, or 3 to 6 carbon atoms. For example, "C 2-6 "Alkyne" includes straight-chain or branched unsaturated groups with 2 to 6 carbon atoms (having at least one carbon-carbon triple bond).

[0047] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), wherein the alkyl group is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy.

[0048] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 6 carbon atoms (e.g., 3, 4, 5, or 6 carbon atoms), and most preferably 5 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

[0049] The term "spirocycloalkyl" refers to a polycyclic group consisting of 5 to 20 quintile rings sharing a single carbon atom (called a spiro atom). It may contain one or more double bonds, but each ring does not have a fully conjugated π-electron system. Preferably, it is 6 to 14 quintiles, more preferably 7 to 10 quintiles (e.g., 7, 8, 9, or 10 quintiles). Spirocycloalkyl groups are classified as monospirocycloalkyl, bispirocycloalkyl, or polyspirocycloalkyl groups based on the number of shared spiro atoms between rings. Monospirocycloalkyl and bispirocycloalkyl groups are preferred, more preferably 4 / 4, 4 / 5, 4 / 6, 5 / 5, or 5 / 6 quintile monospirocycloalkyl groups.

[0050] The term "fused cycloalkyl" refers to a 5- to 20-membered polycyclic aromatic hydrocarbon group in which each ring in the system shares an adjacent pair of carbon atoms with other rings in the system. One or more rings may contain one or more double bonds, but each ring does not have a fully conjugated π-electron system. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered. Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused cycloalkyl, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic alkyl.

[0051] The term "bridged cycloalkyl" refers to a 5- to 20-membered polycyclic carbon group in which any two rings share two non-directly bonded carbon atoms. It may contain one or more double bonds, but each ring does not have a fully conjugated π-electron system. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered. Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged cycloalkyl, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic.

[0052] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent comprising 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), but excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, it comprises 3 to 12 ring atoms, wherein 1 to 4 are heteroatoms; more preferably, it comprises 3 to 8 ring atoms, wherein 1 to 3 are heteroatoms; most preferably, it comprises 5 to 6 ring atoms, wherein 1 to 2 or 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, imidazoalkyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, dihydroimidazoyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc., preferably tetrahydropyranyl, piperidinyl, or pyrrolidinyl. Polycyclic heterocyclic groups include spirocyclic heterocyclic groups, fused heterocyclic groups, and bridged heterocyclic groups.

[0053] The term "spiroheterocyclic group" refers to a polycyclic heterocyclic group consisting of 5 to 20 cyclic rings sharing a single atom (called a spiro atom), wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. It may contain one or more double bonds, but each ring does not have a fully conjugated π-electron system. Preferably, it is 6 to 14 cyclic, more preferably 7 to 10 cyclic. Spiroheterocyclic groups are classified into monospirocyclic, bispirocyclic, or polyspirocyclic groups based on the number of shared spiro atoms between rings, with monospirocyclic and bispirocyclic groups being preferred, and 4 / 4, 4 / 5, 4 / 6, 5 / 5, or 5 / 6 monospirocyclic groups being more preferred.

[0054] The term "fused heterocyclic group" refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system. One or more rings may contain one or more double bonds, but each ring does not have a fully conjugated π-electron system. One or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered. Depending on the number of constituent rings, it can be classified as a bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclic group, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic group.

[0055] The term "bridged heterocyclic group" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two non-directly connected atoms. It may contain one or more double bonds, but each ring does not have a fully conjugated π-electron system. One or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered. Depending on the number of constituent rings, it can be classified as a bicyclic, tricyclic, tetracyclic, or polycyclic bridged heterocyclic group, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic.

[0056] The heterocyclic group includes heterocyclic groups (including monocyclic, spirocyclic, fused heterocyclic, and bridged heterocyclic groups) fused to an aryl, heteroaryl, or cycloalkyl ring as described above, wherein the ring connected to the parent structure is a heterocyclic group, and non-limiting examples include:

[0057] , , The wavy line represents the connection site of the functional group.

[0058] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl.

[0059] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 10-membered, containing 1 to 3 heteroatoms; more preferably 5- or 6-membered, containing 1 to 2 heteroatoms; preferred examples include imidazolyl, furanyl, thiophenel, thiazolyl, pyrazolyl, oxazolyl, pyrrolel, tetrazolyl, pyridinyl, pyrimidinyl, thiadiazole, pyrazinyl, and pyridazinyl.

[0060] The heteroaryl group includes, as described above, a heteroaryl group fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, and non-limiting examples include:

[0061] , , , , and The wavy line represents the connection site of the functional group.

[0062] The term “saturated or unsaturated ring” includes the aforementioned aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups.

[0063] The term "hydroxyalkyl" refers to an alkyl group that has been substituted with a hydroxyl group, wherein the alkyl group is as defined above.

[0064] The term "halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above.

[0065] The term "haloalkoxy" refers to an alkoxy group that is substituted by one or more halogens, wherein the alkoxy group is as defined above.

[0066] The term “deuterated alkyl” refers to an alkyl group that is replaced by one or more deuterium atoms, wherein the alkyl group is as defined above.

[0067] The term “deuterated alkoxy” refers to an alkoxy group that is replaced by one or more deuterium atoms, where the alkoxy group is as defined above.

[0068] The term "cycloalkylalkyl" refers to an alkyl group that is substituted by one or more cycloalkyl groups, wherein the cycloalkyl and alkyl groups are as defined above.

[0069] The term "cycloalkyloxy" refers to -O-cycloalkyl, where the cycloalkyl is as defined above.

[0070] The term "heterocyclic alkyl" refers to an alkyl group that is substituted with one or more heterocyclic groups, wherein the heterocyclic group and the alkyl group are as defined above.

[0071] The term "arylalkyl" refers to an alkyl group that is substituted with one or more aryl groups, wherein the aryl and alkyl groups are as defined above.

[0072] The term "hydroxyl group" refers to the -OH group.

[0073] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0074] The term "amino" refers to -NH2.

[0075] The term "cyano" refers to -CN.

[0076] The term "nitro" refers to -NO2.

[0077] The term "carboxyl group" refers to -C(O)OH.

[0078] In this invention, the defined carbon number range of a group refers to any integer number of carbon atoms included within the defined range. For example, C1-C6 means that the number of carbon atoms in the group can be 1, 2, 3, 4, 5 or 6, C2-C6 means that the number of carbon atoms in the group can be 2, 3, 4, 5 or 6, and so on for other groups.

[0079] Compared with the prior art, the present invention has the following beneficial effects:

[0080] The compounds of this invention can be used to prevent and treat parasitic infections, exhibiting good preventive and therapeutic effects and demonstrating good pharmacokinetic properties in mice. Detailed Implementation

[0081] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0082] The abbreviations of some of the reaction reagents involved in this invention are shown below:

[0083] DMF: N,N-Dimethylformamide

[0084] HATU: 2-(7-Azobenzotriazole)-N,N,N',N'-Tetramethylurea hexafluorophosphate

[0085] DIPEA: N,N-Diisopropylethylamine

[0086] DCM: Dichloromethane

[0087] T4P: 1-Butylphosphine

[0088] TFA: Trifluoroacetic acid

[0089] Pd(dppf)Cl2: [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride

[0090] THF: Tetrahydrofuran

[0091] NCS: N-chlorosuccinimide.

[0092] Example 001 Preparation of 3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-N-(1-(3,3,3-trifluoropropionyl)pyrrolidine-3-yl)thiophene-2-carboxamide (1)

[0093]

[0094] Step 1: Compound 1-1 (15.0 g, 84 mmol, 1 eq.) was dissolved in DCE (150 mL). Acetyl chloride (13.3 g, 169 mmol, 2 eq.) and AlCl3 (16.9 g, 127 mmol, 1.50 eq.) were added at 0 °C, and the mixture was stirred at 25 °C for 3 hours. The reaction was quenched with NaHCO3 (100 mL), and the mixture was extracted with ethyl acetate (100 mL x 3). The organic phase was washed with saturated brine (100 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (SiO2, ethyl acetate / n-heptane, 0–5%) to obtain compound 1-2 (13.2 g, 71% yield). MS-ESI calculated value [M+H] + 219, measured value 219.

[0095] Step 2: Compounds 1-2 (1.20 g, 6.05 mmol, 1 eq.) were dissolved in DMF (100 mL) and MeOH (100 mL), and TEA (18.2 g, 180 mmol, 3 eq.) and Pd(dppf)Cl2 (4.40 g, 6.02 mmol, 0.1 eq.) were added. The mixture was stirred at 90 °C under a carbon monoxide atmosphere for 12 hours. H2O (100 mL) was added to the reaction solution, and ethyl acetate (50 mL x 3) was added for extraction. The organic phase was washed with saturated brine (50 mL x 3), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (SiO2, ethyl acetate / n-heptane, 5–10%) to obtain compounds 1-3 (7.80 g, 78% yield). MS-ESI calculated value [M+H] + 199, measured value 199.

[0096] Step 3: Compounds 1-3 (13.2 g, 60.2 mmol, 1 eq.) and 1-4 (2.51 g, 9.08 mmol, 1.5 eq.) were dissolved in trifluorotoluene (10 mL) and toluene (10 mL), and Cs₂CO₃ (394 mg, 1.21 mmol, 0.2 eq.) was added. The mixture was stirred at 110 °C for 12 hours. H₂O (20 mL) was added to the reaction solution, and the mixture was extracted with DCM (20 mL x 3). The organic phase was washed with saturated brine (120 mL x 2), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in toluene (10 mL), and SOCl₂ (1.44 g, 12.1 mmol, 2 eq.) was added. The mixture was stirred at 70 °C under a N₂ atmosphere for 12 hours, and then concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (SiO2, ethyl acetate / n-heptane, 0-5%) to obtain compounds 1-5 (1.70 g, yield 61%). 1 H NMR (400 MHz, CDCl3) δ 7.51 (s, 1H), 7.37 (d, J = 1.2 Hz,1H), 7.35 (s, 2H), 3.92 (s, 3H), 2.59 (s, 3H).

[0097] Step 4: Add NaOH (594 mg, 14.8 mmol, 4 eq.) to a solution of NH₂OH·HCl (774 mg, 11.1 mmol, 3 eq.) and tetrabutylammonium bromide (119 mg, 371 μmol, 0.1 eq.) in H₂O (4 mL). Stir at 25 °C for 0.5 h. Then add 1–5 (1.70 g, 3.71 mmol, 1 eq.) of THF (20 mL) solution and stir at 25 °C for 0.5 h. After the reaction is complete, quench with NH₄Cl, then add H₂O (50 mL). Extract with ethyl acetate (50 mL x 3). Wash the organic phase with saturated brine (50 mL), dry to anhydrous magnesium sulfate, and concentrate by filtration under reduced pressure. The crude product was purified by column chromatography (SiO2, ethyl acetate / n-heptane, 0-20%) to give compounds 1-6 (1.36 g, 77% yield). 1 H NMR (400 MHz, CDCl3) δ 7.64 (s, 2H), 7.11 (s, 1H), 4.08 (d, J = 17.2 Hz, 1H), 3.91 (s, 3H), 3.68 (d, J = 17.2 Hz, 1H), 2.56 (s, 3H).

[0098] Step 5: Compounds 1-6 (1.36 g, 2.87 mmol, 1 eq.) were dissolved in THF (20 mL) and H2O (5 mL), and LiOH·H2O (362 mg, 8.63 mmol, 3 eq.) was added. The mixture was stirred at 25 °C for 2 hours. The pH of the reaction solution was adjusted to 5-7 with 1M HCl, and the solution was extracted with ethyl acetate (50 mL x 2). The organic phase was washed with saturated brine (50 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain crude compound 1-7 (1.20 g, 90% yield). 1 H NMR (400 MHz, CDCl3) δ7.64 (s, 2H), 7.15 (s, 1H), 4.09 (d, J = 17.2 Hz, 1H), 3.69 (d, J = 17.2 Hz, 1H), 2.58 (s, 3H).

[0099] Step 6: HATU (65 mg, 172 μmol, 1.2 eq.) and DIPEA (55 mg, 430 μmol, 3 eq.) were added to a DMF (1 mL) solution of compounds 1-7 (60 mg, 143 μmol, 1 eq.), and the mixture was stirred at 25 °C for 10 min. Compounds 1-8 (60 mg, 322 μmol, 1.23 eq.) were added to the mixture, and the mixture was stirred at 25 °C for 30 min. H₂O (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL x 3). The organic phase was washed with saturated brine (20 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO₂, ethyl acetate / n-heptane, 0–50%) to give compounds 1-9 (80 mg, 48% yield). 1 H NMR (400 MHz, CDCl3) δ 7.64 (s, 2H), 7.07 (s, 1H), 5.96 - 5.80 (m,1H), 4.67 - 4.51 (m, 1H), 4.07 (d, J = 17.2 Hz, 1H), 3.78 - 3.71 (m, 1H),3.68(d, J = 17.2 Hz, 1H), 3.57 - 3.42 (m, 2H), 3.40 - 3.22 (m, 1H), 2.51 (s, 3H),2.34 - 2.18 (m, 1H), 2.02 - 1.87 (m, 1H), 1.49 (s, 9H).

[0100] Step 7: Dissolve compounds 1-9 (80 mg, 127 μmol, 1 eq.) in DCM (2 mL), add a dioxane solution of hydrogen chloride (4 M, 1 mL), and stir at 25 °C for 12 hours. After the reaction is complete, concentrate under reduced pressure to obtain the hydrochloride salt of compounds 1-10 (70 mg, 97% yield).

[0101] Step 8: Add DIPEA (41.3 mg, 319 μmol, 3 eq.) and HATU (48.6 mg, 127 μmol, 1.2 eq.) to a DMF (1 mL) solution of 1-11 (16.3 mg, 127 μmol, 1.2 eq.), and then add 1-10 (60 mg, 106 μmol, 1 eq.) of hydrochloride to the mixture. Stir at 25 °C for 2 h. Add H2O (10 mL) and extract with ethyl acetate (10 mL x 3). Wash the organic phase with saturated brine (10 mL), dry to anhydrous magnesium sulfate, filter, and concentrate under reduced pressure. The crude product was purified by preparative high performance liquid chromatography (HPLC) (column: Xtimate C18, 30×150mm ID, 5µm; mobile phase: [0.1% formic acid aqueous solution-acetonitrile], gradient: (70%-90%, 10 min, 95%, 2 min, 70%, 3 min)) to obtain compound 1 (14.0 mg, yield 20%). 1 ¹H NMR (400 MHz, CDCl₃) δ 7.63 (s, 2H), 7.08 (d, J = 1.2 Hz, 1H), 5.98 (d, J = 6.4 Hz, 1H), 4.82 - 4.56 (m, 1H), 4.12 - 3.55 (m, 6H), 3.17 - 3.22 (m, 2H), 2.51 (s, 3H), 2.44 (s, 1H), 2.21 - 2.00 (m, 1H). MS-ESI calculated values ​​[M+H] + 636, measured value 636.

[0102] Example 002 Preparation of 3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-N-(1-(2,2,2-trifluoroacetyl)pyrrolidine-3-yl)thiophene-2-carboxamide (2)

[0103]

[0104] Step 1: DIPEA (103 mg, 798 μmol, 3 eq.) and HATU (121 mg, 319 μmol, 1.2 eq.) were added to a 1-10 (150 mg, 266 μmol, 1 eq.) DMF (2 mL). Trifluoroacetic acid (45 mg, 399 μmol, 1.5 eq.) and trifluoroacetic anhydride (55 mg, 266 μmol, 1 eq.) were added to the mixture, and the mixture was stirred at 25 °C for 2 hours. H₂O (10 mL) was added, and the mixture was extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated brine (10 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by preparative thin-layer chromatography (SiO₂, ethyl acetate) to give compound 2 (51.7 mg, 31% yield). 1 ¹H NMR (400 MHz, CDCl₃) δ 7.63 (s, 2H), 7.08 (d, J = 1.2 Hz, 1H), 5.88 (d, J = 6.4 Hz, 1H), 4.82–4.56 (m, 1H), 4.12–3.55 (m, 6H), 2.51 (s, 3H), 2.44 (s, 1H), 2.21–2.00 (m, 1H). MS-ESI calculated values ​​[M+H] + 622, measured value 622.

[0105] Example 003 Preparation of 3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-N-(2-((trifluoromethyl)sulfonamido)ethyl)thiophene-2-carboxamide (3)

[0106]

[0107] Step 1: DIPEA (169 mg, 1.30 mmol, 3 eq.) and HATU (198 mg, 523 μmol, 1.2 eq.) were added to a DMF (0.5 mL) solution of 1-7 (200 mg, 436 μmol, 1 eq.), and 3-1 (83 mg, 523 μmol, 1.2 eq.) was added to the mixture. The mixture was stirred at 25 °C for 1 hour. H2O (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL x 3). The organic phase was washed with saturated brine (20 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO2, ethyl acetate / n-heptane, 0–60%) to give compound 3-2 (200 mg, 76% yield).1 HNMR (400 MHz, CDCl3) δ 7.64 (s, 2H), 7.07 (s, 1H), 6.93 - 6.74 (m, 1H), 4.94( s, 1H), 4.07 (d, J = 17.2 Hz, 1H), 3.67 (d, J = 17.2 Hz, 1H), 3.59 - 3.50 (m, 2H), 3.40 (q, J = 5.6 Hz, 2H), 2.52 (s, 3H), 1.46 (s, 9H).

[0108] Step 2: Compound 3-2 (200 mg, 332 μmol, 1 eq.) was dissolved in DCM (2 mL), and a dioxane solution of hydrogen chloride (4 M, 2 mL) was added. The mixture was stirred at 25 °C for 12 hours. After the reaction was complete, the solution was concentrated under reduced pressure to give the hydrochloride salt of compound 3-3 (175 mg, 97% yield). MS-ESI calculated value [M+H] + 500, measured value 500.

[0109] Step 3: The hydrochloride of compound 3-3 (50 mg, 99.8 μmol, 1 eq.) was dissolved in DCM (1 mL), and triethylamine (30 mg, 299 μmol, 3 eq.) was added. Then, 3-4 (18 mg, 109 μmol, 1.1 eq.) was added at 0 °C, and the mixture was stirred at 25 °C for 1 hour. H₂O (10 mL) was added, and the mixture was extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated brine (10 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by preparative high performance liquid chromatography (HPLC) (column: Ultimate C18, 30*150mm ID, 5um; mobile phase: [0.1% formic acid aqueous solution-acetonitrile], gradient: (73%-93%, 10 min, 95%, 2 min, 73%, 3 min)) to obtain compound 3 (3.46 mg, yield 5%). 1¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 2H), 7.09 (s, 1H), 6.52–6.37 (m, 1H), 6.29 (t, J = 5.0 Hz, 1H), 4.08 (d, J = 17.2 Hz, 1H), 3.72–3.69 (m, 1H), 3.67 (t, J = 5.6 Hz, 2H), 3.60–3.52 (m, 2H), 2.52 (s, 3H). MS-ESI calculated values ​​[M+H] + 632, measured value 632.

[0110] Example 004 Preparation of 3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-N-((1-(2,2,2-trifluoroethyl)amino)cyclopropyl)methyl)thiophene-2-carboxamide (4)

[0111]

[0112] Step 1: Trifluoroethyl trifluoromethanesulfonate (934 mg, 4.02 mmol, 1.5 eq.) and Et3N (543 mg, 5.37 mmol, 746 μL, 2 eq.) were added to a 10 mL solution of THF (500 mg, 2.68 mmol, 1 eq.) at 70°C. o The mixture was stirred at C for 12 hours. The reaction solution was concentrated under reduced pressure to give crude compound 4-2 (700 mg, 97% yield). MS-ESI calculated value [M+H] + 269, measured value 269.

[0113] Step 2: Add a 4 M dioxane solution (5 mL) to a 4-2 (250 mg, 931 μmol, 1 eq.) dioxane solution (5 mL), 25 o The mixture was stirred at C for 16 hours. The reaction solution was concentrated under reduced pressure, and the crude product was slurried with ethyl acetate (15 mL) to give the hydrochloride of compound 4-3 (176 mg, yield 92%). 1 H NMR (400 MHz, DMSO-d6)δ 8.13 (s, 3H), 3.43 (d, J = 10.4 Hz, 2H), 2.86 (d, J = 5.6 Hz, 2H), 0.70 (s, 4H).

[0114] Step 3: HATU (49 mg, 130 μmol, 1.2 eq.) and DIPEA (42 mg, 327 μmol, 3 eq.) were added to a DMF (1 mL) solution of 1-7 (50 mg, 109 μmol, 1 eq.), and the mixture was stirred at 25 °C for 10 min. 4-3 (26 mg, 130 μmol, 1.2 eq.) of hydrochloride was added to the mixture, and the mixture was stirred at 25 °C for 10 min. H₂O (10 mL) was added, and the mixture was extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated brine (10 mL x 2), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by preparative thin-layer chromatography (SiO₂, n-heptane:ethyl acetate = 1:1) to give compound 4 (13.8 mg, 20% yield). 1 ¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 2H), 7.09 (s, 1H), 6.49–6.11 (m, 1H), 4.08 (d, J = 17.2 Hz, 1H), 3.69 (d, J = 16.8 Hz, 1H), 3.42 (d, J = 6.0 Hz, 2H), 3.32 (d, J = 9.6 Hz, 2H), 2.52 (s, 3H), 1.61 (d, J = 2.8 Hz, 1H), 0.79–0.73 (m, 2H), 0.72–0.63 (m, 2H). MS-ESI calculated values ​​[M+H] + 608, measured value 608.

[0115] Example 005 Preparation of 3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-N-((1-(2,2,2-trifluoroethyl)amino)cyclobutyl)methyl)thiophene-2-carboxamide (5)

[0116]

[0117] Step 1: Trifluoroethyl trifluoromethanesulfonate (869 mg, 3.74 mmol, 1.5 eq.) and triethylamine (505 mg, 4.99 mmol, 2 eq.) were added to a 5-1 (0.50 g, 2.49 mmol, 1 eq.) solution of THF (40 mL), and the solution was heated to 70 mL. oStirred at C for 12 hours. Add H2O (10 mL) and extract with ethyl acetate (20 mL x 3). Wash the organic phase with saturated brine (20 mL x 2), dry to anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to give compound 5-2 (700 mg, 99% yield).

[0118] Step 2: Add TFA (707 mg, 6.20 mmol, 2 mL, 1.77 eq.) to a 5-2 (700 mg, 3.49 mmol, 1 eq.) DCM (10 mL) solution. o The mixture was stirred at C for 4 hours. The reaction solution was concentrated under reduced pressure, and the crude product was slurried with DCM (50 mL) to give trifluoroacetate of compound 5-3 (425 mg, yield 41%). 1 H NMR (400 MHz, DMSO-d6) δ 7.78 - 7.32 (m, 3H), 3.20 (q, J = 10.0 Hz, 2H), 2.96 (s, 2H), 1.97 -1.83 (m, 4H), 1.76 - 1.60 (m, 2H).

[0119] Step 3: HATU (49.7 mg, 130 μmol, 1.2 eq.) and DIPEA (42.2 mg, 327 μmol, 3 eq.) were added to a DMF (1 mL) solution of 1-7 (48.4 mg, 163 μmol, 1.5 eq.), and the mixture was stirred at 25 °C for 10 min. Trifluoroacetate (5-3) was added to the mixture, and the mixture was stirred at 25 °C for 10 min. H₂O (10 mL) was added, and the mixture was extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated brine (10 mL x 2), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by preparative thin-layer chromatography (SiO₂, n-heptane:ethyl acetate = 1:1) to give compound 5 (17.7 mg, yield 26%). 1¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 2H), 7.09 (s, 1H), 6.44 (t, J = 4.4 Hz, 1H), 4.07 (d, J = 17.2 Hz, 1H), 3.67 (d, J = 17.2 Hz, 1H), 3.56 (d, J = 4.8 Hz, 2H), 3.24 - 3.04 (m, 2H), 2.49 (s, 3H), 2.06 -1.84 (m, 6H), 1.55 - 1.42 (m, 1H). MS-ESI calculated values ​​[M+H] + 622, measured value 622.

[0120] Example 006 Preparation of 3-methyl-N-(2-oxo-2-(2-(trifluoromethyl)azacyclobutane-1-yl)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethane)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (6)

[0121]

[0122] Step 1: Add DIPEA (463 mg, 3.58 mmol, 624 μL, 3 eq.) and HATU (545 mg, 1.43 mmol, 1.2 eq.) to a DMF (10 mL) solution of 6-1 (0.25 g, 1.19 mmol, 1 eq.) and 6-2 (193 mg, 1.19 mmol, 1 eq.), and incubate for 20 minutes. o Stirred at C for 12 hours. Add H2O (10 mL) and extract with ethyl acetate (10 mL x 2). Wash the organic phase with saturated brine (15 mL x 2), dry to anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify the crude product by column chromatography (SiO2, ethyl acetate / n-heptane, 10% ~ 60%) to give compound 6-3 (320 mg, yield 84%). 1 H NMR (400 MHz, CDCl3) δ 7.43 - 7.30 (m, 5H), 5.54 (s, 1H), 5.14 (s, 2H), 4.80 (s, 1H), 4.33 - 4.15 (m, 1H), 4.03 - 3.91 (m, 1H), 3.85 - 3.61 (m, 1H), 2.65 (s, 1H), 2.42 (s, 1H).

[0123] Step 2: Add Pd / C (64 mg, 10% purity, 20% w / w) to a 6-3 (320 mg, 1.01 mmol, 1 eq.) MeOH (10 mL) solution and incubate at 15 psi under a hydrogen atmosphere for 20 minutes. o The mixture was stirred at C for 3 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give compound 6-4 (170 mg, 92% yield). 1 H NMR (400 MHz, CDCl3) δ 4.75 (s, 1H), 4.28 - 3.98 (m, 2H), 3.44 - 3.21 (m, 2H), 2.70 - 2.56 (m, 1H), 2.50 - 2.30 (m, 1H).

[0124] Step 3: Add HATU (49 mg, 130 μmol, 1.2 eq.) and DIPEA (42 mg, 327 μmol, 3 eq.) to DMF (1 mL) solutions of 1-7 (50 mg, 109 μmol, 1 eq.) and 6-4 (23 mg, 130 μmol, 1.2 eq.), and incubate at 25°C. o Stir at C for 1 hour. Add H2O (10 mL) and extract with ethyl acetate (10 mL x 3). Wash the organic phase with saturated brine (20 mL x 2), dry to anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify the crude product by preparative thin-layer chromatography (SiO2, n-heptane:ethyl acetate = 1:1) to give compound 6 (25.6 mg, yield 37%). 1 ¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 2H), 7.09 (s, 1H), 6.75 (s, 1H), 4.98–4.70 (m, 1H), 4.38–4.12 (m, 3H), 4.07 (d, J = 16.9 Hz, 1H), 4.04–3.84 (m, 1H), 3.68 (d, J = 17.1 Hz, 1H), 2.79–2.64 (m, 1H), 2.54 (s, 3H), 2.50–2.39 (m, 1H). MS-ESI calculated values ​​[M+H] + 622, measured value 622.

[0125] Example 007 Preparation of 3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-N-((1-(2,2,2-trifluoroethoxy)cyclopropyl)methyl)thiophene-2-carboxamide (7)

[0126]

[0127] Step 1: Add NaH (133 mg, 3.33 mmol, 60% purity, 2.5 eq.) to a 7-1 solution (0.25 g, 1.33 mmol, 1 eq.) of THF (10 mL). o Stirring at C for 0.5 hours. Then add trifluoroethyl trifluoromethanesulfonate (464 mg, 2.00 mmol, 1.5 eq.), 20 o The mixture was stirred at C for 12 hours. The reaction was quenched with NH4Cl (5 mL), and extracted with ethyl acetate (10 mL x 2). The organic phase was washed with saturated brine (15 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to give compound 7-2 (0.35 g, 97% yield). MS-ESI calculated value [M+H] + 270, measured value 270.

[0128] Step 2: Add TFA (1 mL) to a 7-2 (250 mg, 928 μmol, 1 eq.) solution of DCM (10 mL), 20 o The mixture was stirred at C for 6 hours. The reaction solution was concentrated under reduced pressure to give trifluoroacetate of compound 7-3 (250 mg, 95% yield).

[0129] Step 3: Add HATU (67 mg, 176 μmol, 1.5 eq.) and DIPEA (45 mg, 353 μmol, 61 μL, 3 eq.) to a 1-7 (54 mg, 117 μmol, 1 eq.) solution of DMF (2 mL), and incubate at 25°C. o Stir at C for 10 minutes. Then add 7-3 (33 mg, 117 μmol, 1 eq.) of trifluoroacetate and stir at 25°C. o Stir at C for 10 min. Add H2O (3 mL) and extract with ethyl acetate (5 mL x 2). Wash the organic phase with saturated brine (10 mL), dry to anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify the crude product by preparative thin-layer chromatography (SiO2, n-heptane:ethyl acetate = 1:1) to give compound 7 (17 mg, yield 23%). 1¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 2H), 7.09 (s, 1H), 6.20 (s, 1H), 4.08 (d, J = 17.2 Hz, 1H), 3.95 (q, J = 8.8 Hz, 2H), 3.68 (d, J = 17.2 Hz, 1H), 3.61 (d, J = 5.6 Hz, 2H), 2.52 (s, 3H), 1.08–0.97 (m, 2H), 0.79–0.71 (m, 2H). MS-ESI calculated values ​​[MH] - 607, measured value 607.

[0130] Example 008 Preparation of 3-methyl-N-(2-oxo-2-(2-(trifluoromethyl)pyrrolidone-1-yl)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethane)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (8)

[0131]

[0132] Step 1: Add DIPEA (885 mg, 6.85 mmol, 1.19 mL, 3 eq.) and HATU (1.04 g, 2.74 mmol, 1.2 eq.) to a DMF (10 mL) solution of 8-1 (0.4 g, 2.28 mmol, 1 eq.) and 8-2 (400 mg, 2.28 mmol, 1 eq.), 20 o Stirred at C for 12 hours. Add H2O (10 mL) and extract with ethyl acetate (10 mL x 2). Wash the organic phase with saturated brine (15 mL x 2), dry to anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to give compound 8-3 (670 mg, 99% yield).

[0133] Step 2: Add a 4 M, 5 mL solution of dioxane (hydrogen chloride) to a 5 mL solution of 8-3 (670 mg, 2.26 mmol, 1 eq.) of dioxane, and incubate at 20°C. o The mixture was stirred at C for 12 hours. The reaction solution was concentrated under reduced pressure, and the crude product was slurried with ethyl acetate (15 mL) to give the hydrochloride of compound 8-4 (500 mg, yield 95%). 1HNMR (400 MHz, DMSO-d6) δ8.48 - 8.01 (m, 3H), 4.85 - 4.69 (m, 1H), 3.89 (s, 2H), 3.57 (dd, J = 5.3,8.7 Hz, 2H), 2.17 - 1.91 (m, 4H).

[0134] Step 3: Add DIPEA (42 mg, 327 μmol, 3 eq.) and HATU (49 mg, 130 μmol, 1.2 eq.) to a 1-7 (50 mg, 109 μmol, 1 eq.) solution of DMF (2 mL), and incubate at 25°C. o Stir at C for 10 minutes. Add 8-4% hydrochloride (30 mg, 130 μmol, 1.2 eq.) and stir at 25°C. o Stir at C for 10 minutes. Add H2O (10 mL) and extract with ethyl acetate (10 mL x 3). Wash the organic phase with saturated brine (10 mL x 2), dry to anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify the crude product by preparative thin-layer chromatography (SiO2, n-heptane:ethyl acetate = 1:1) to give compound 8 (21.7 mg, yield 31%). 1 ¹H NMR (400 MHz, CDCl₃) δ 7.66 (s, 2H), 7.11 (s, 1H), 7.00 (s, 1H), 4.85 (t, J = 7.6 Hz, 1H), 4.48–4.30 (m, 1H), 4.26–4.16 (m, 1H), 4.09 (d, J = 17.2 Hz, 1H), 3.76–3.49 (m, 3H), 2.57 (s, 3H), 2.32–2.04 (m, 4H). MS-ESI calculated values ​​[M+H] + 636, measured value 636.

[0135] Example 009 Preparation of N-(2-((1-cyanocyclopropyl)amino)-2-oxoethyl)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (9)

[0136]

[0137] Step 1: Add DIPEA (741 mg, 5.73 mmol, 999 μL, 3 eq.) and HATU (872 mg, 2.29 mmol, 1.2 eq.) to a 10 mL solution of DMF containing 6-1 (400 mg, 1.91 mmol, 1 eq.) and 9-1 (226 mg, 1.91 mmol, 1 eq.). o Stir at C for 12 hours. At 25°C... o Stirred at C for 12 hours. Add H2O (10 mL) and extract with ethyl acetate (10 mL x 2). Wash the organic phase with saturated brine (15 mL x 2), dry to anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify the crude product by column chromatography (SiO2, ethyl acetate / n-heptane, 30% ~ 60%) to give compound 9-2 (500 mg, 95% yield).

[0138] Step 2: Add Pd / C (0.1 g, 10% purity, 20% w / w) to a 9-2 (0.5 g, 1.83 mmol, 1 eq.) MeOH (10 mL) solution and incubate at 20°C under a hydrogen atmosphere of 15 Psi. o The mixture was stirred at C for 3 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give compound 9-3 (230 mg, 90% yield).

[0139] Step 3: Add HATU (49 mg, 130 μmol, 1.2 eq.) and DIPEA (42 mg, 327 μmol, 3 eq.) to a 1-7 (50 mg, 109 μmol, 1 eq.) solution of DMF (1 mL), 20 o Stir at C for 10 minutes. Add 9-3 (22 mg, 130 μmol, 1.2 eq.) and stir at 25°C. o Stir at C for 10 min. Add H2O (5 mL) and extract with ethyl acetate (10 mL x 2). Wash the organic phase with saturated brine (10 mL), dry to anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify the crude product by preparative thin-layer chromatography (SiO2, n-heptane:ethyl acetate = 1:1) to give compound 9 (7.9 mg, yield 12%). 1¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 2H), 7.17–7.04 (m, 2H), 6.79 (s, 1H), 4.17–4.12 (m, 2H), 4.08 (d, J = 17.2 Hz, 1H), 3.69 (d, J = 17.2 Hz, 1H), 2.56 (s, 3H), 1.63–1.60 (m, 2H), 1.34 (s, 2H). MS-ESI calculated values ​​[M+H] + 579, measured value 579.

[0140] Example 010 Preparation of N-(2-oxo-2-((2,2,2-trifluoroethyl)amino)ethyl)-3-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)benzo[c]thiophene-1-carboxamide (10)

[0141]

[0142] Step 1: n-BuLi (2.43 g, 38.0 mmol, 2.5 M, 15.2 mL, 1.3 eq.) was added to a 10⁻¹ (5.00 g, 29.2 mmol, 1 eq.) THF solution (100 mL) at -70 °C. After stirring at -70 °C for 0.5 h, a 20 mL THF solution of dimethyl oxalate (5.17 g, 43.8 mmol, 1.5 eq.) was added. At 25 °C... o Stirred at C for 12 hours. Quenched with NH4Cl (30 mL), extracted with ethyl acetate (50 mL x 2). The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by column chromatography (SiO2, ethyl acetate / n-heptane, 5% ~ 20%) to give compound 10⁻² (1.66 g, 31% yield). 1 H NMR (400 MHz, CDCl3) δ 7.70 (d, J = 7.6 Hz, 1H), 7.52 (td,J = 7.6, 1.2 Hz, 1H), 7.39 - 7.29 (m, 2H), 3.98 (s, 3H), 2.62 (s, 3H).

[0143] Step 2: AIBN (213 mg, 1.30 mmol, 0.2 eq.) and NBS (1.50 g, 8.46 mmol, 1.3 eq.) were added to a 10⁻² (1.16 g, 6.51 mmol, 1 eq.) solution of CCl₄ (20 mL). The mixture was stirred at 70 °C under N₂ atmosphere for 12 hours and then concentrated under reduced pressure. The crude product was purified by column chromatography (SiO₂, ethyl acetate / n-heptane, 5% ~ 10%) to obtain compound 10⁻³ (1.58 g, 94% yield).

[0144] Step 3: NaSH (554 mg, 6.92 mmol, 70% purity, 1 eq.) was added to a 10⁻³ (1.78 g, 6.92 mmol, 1 eq.) solution of DMF (20 mL) at 0 °C, and stirred at 0 °C for 3 h. The mixture was quenched with H₂O (10 mL), extracted with ethyl acetate (10 mL x 2) and concentrated under reduced pressure. The organic phase was washed with saturated brine (10 mL x 2), dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by column chromatography (SiO₂, ethyl acetate / n-heptane, 5% ~ 20%) to obtain compound 10⁻⁴ (620 mg, yield 46%).

[0145] Step 4: A solution of Br2 (91 mg, 572 μmol, 1.1 eq.) in ACN (0.5 mL) was added at 0°C to a solution of 10⁻⁴ (100 mg, 520 μmol, 1 eq.) in THF (0.5 mL), H₂O (0.7 mL), and AcOH (0.9 mL). The mixture was stirred at 25°C for 12 hours. The mixture was quenched with Na₂S₂O₃ (5 mL), extracted with ethyl acetate (10 mL x 2), washed with saturated brine (10 mL), dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by column chromatography (SiO₂, ethyl acetate / n-heptane, 0–5%) to obtain compound 10⁻⁵ (50 mg, yield: 35%). MS-ESI calculated value [M+H] + 271, measured value 271.

[0146] Step 5: Pd(dppf)Cl2 (80 mg, 110 μmol, 0.1 eq.) and Cs2CO3 (1.08 g, 3.31 mmol, 3 eq.) were added to a solution of 10⁻⁵ (300 mg, 1.10 mmol, 1 eq.) and potassium isopropenyltrifluoroborate (245 mg, 1.66 mmol, 1.5 eq.) in dioxane (5 mL) and H₂O (1 mL). The mixture was stirred at 90°C for 12 hours. The mixture was quenched with H₂O (10 mL), extracted with ethyl acetate (10 mL x 3), washed with saturated brine (10 mL x 2), dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by column chromatography (SiO₂, ethyl acetate / n-heptane, 0–5%) to obtain compound 10⁻⁶ (100 mg, 38% yield). 1 H NMR (400 MHz, CDCl3) δ 8.47 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.35 (ddd, J = 8.8, 6.4, 1.2 Hz, 1H), 7.20 (ddd, J = 8.8, 6.4, 1.2 Hz, 1H), 5.62 (s, 1H), 5.55 - 5.47 (m, 1H), 3.99 (s, 3H), 2.37 (s, 3H).

[0147] Step 6: Potassium osmium tetroxide (13.4 mg, 43.0 μmol, 0.1 eq.) and sodium periodate (211 mg, 990 μmol, 2.3 eq.) were added to a 10⁻⁶ solution of THF (2 mL) and H₂O (1 mL) and stirred at 25°C for 12 h. The mixture was quenched with Na₂S₂O₃ (5 mL), extracted with ethyl acetate (10 mL x 2) and concentrated under reduced pressure. The organic phase was washed with saturated brine (10 mL), dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by preparative thin-layer chromatography (SiO₂, n-heptane:ethyl acetate = 5:1) to give compound 10⁻⁷ (30 mg, yield: 29%). 1 H NMR (400 MHz, CDCl3) δ 8.78 - 8.59 (m, 1H), 8.55 - 8.40 (m, 1H), 7.63- 7.34 (m, 2H), 4.05 (s, 3H), 2.77 (s, 3H).

[0148] Step 7: Cs₂CO₃ (8 mg, 25.6 μmol, 0.2 eq.) was added to a solution of 10⁻⁷ (30 mg, 128 μmol, 1 eq.) and 1⁻⁴ (46 mg, 166 μmol, 1.3 eq.) in toluene (2 mL) and trifluorotoluene (2 mL), and stirred at 110°C for 12 hours. The mixture was quenched with H₂O (10 mL), extracted with ethyl acetate (10 mL x 3), washed with saturated brine (10 mL x 2), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to give compound 10⁻⁸ (49 mg, crude). 1 H NMR(400 MHz, CDCl3) δ 8.53 (dt, J = 7.2, 2.8 Hz, 2H), 8.08 (s, 1H), 7.51 7.48(m, 2H), 7.41 (s, 2H), 4.07 (s, 3H).

[0149] Step 8: NaOH (12 mg, 324 μmol, 4 eq.), NH₂OH·HCl (17 mg, 243 μmol, 3 eq.), and tetrabutylammonium bromide (3 mg, 8.10 μmol, 0.1 eq.) were dissolved in H₂O (1 mL) and stirred at 25°C for 1 hour. Then, a solution of 10⁻⁸ (40 mg, 81.0 μmol, 1 eq.) in THF (2 mL) was added. The mixture was stirred at 25°C for 11 hours. The mixture was quenched with H₂O (10 mL), extracted with ethyl acetate (10 mL x 3), and concentrated under reduced pressure. The organic phase was washed with saturated brine (10 mL x 2), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain compound 10⁻⁹ (40 mg, crude). 1 HNMR (400 MHz, CDCl3)δ 8.50 (d, J = 8.8 Hz, 1H), 8.39 (d, J = 8.8 Hz, 1H), 8.08 (s, 2H), 7.70 (s,2H), 4.27 (d, J =16.8 Hz, 1H), 4.03 (s, 3H), 3.88 (d, J = 16.8 Hz, 1H).

[0150] Step 9: LiOH·H2O (10 mg, 235 μmol, 3 eq.) was added to a solution of 10⁻⁹ (40 mg, 78.6 μmol, 1 eq.) in THF (1 mL) and H2O (0.1 mL). The mixture was stirred at 25°C for 12 hours. The pH was adjusted to 5–7 with 1 N HCl, and the mixture was extracted with ethyl acetate (10 mL x 2). The organic phase was washed with saturated brine (10 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain compound 10⁻¹⁰ (40 mg, crude product). 1 H NMR (400 MHz, CDCl3) δ 8.51 (d, J =8.8 Hz, 1H), 8.43 (d, J = 8.8 Hz, 1H), 8.11 (s, 2H), 7.71 (s, 2H), 4.29 (d, J= 16.8 Hz,1H), 3.90 (d, J = 16.8 Hz, 1H).

[0151] Step 10: Add HATU (36 mg, 97.0 μmol, 1.2 eq.) and DIPEA (31 mg, 242 μmol, 3 eq.) to a 10⁻¹⁰ (40 mg, 80.8 μmol, 1 eq.) solution of DMF (1 mL), 20 o Stir at C for 10 minutes. Then add 10⁻¹¹ (15 mg, 97.0 μmol, 1.2 eq.) and stir at 25°C. o Stir at C for 10 minutes. Add H2O (10 mL) and extract with ethyl acetate (10 mL x 3). Wash the organic phase with saturated brine (10 mL x 2), dry to anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify the crude product by preparative thin-layer chromatography (SiO2, n-heptane:ethyl acetate = 1:2) to give compound 10 (2.9 mg, yield 5%). 1 ¹H NMR (400 MHz, CDCl₃) δ 8.40 – 8.32 (m, 1H), 8.30 – 8.20 (m, 1H), 7.70 (s, 2H), 7.49 – 7.36 (m, 2H), 6.94 (t, J = 4.8 Hz, 1H), 6.59 (s, 1H), 4.36 – 4.20 (m, 3H), 4.08 – 3.95 (m, 2H), 3.88 (d, J = 16.9 Hz, 1H). MS-ESI calculated values ​​[M+H] + 632, measured value 632.

[0152] Example 011 Preparation of 3-methyl-N-(2-oxo-2-(3-(trifluoromethyl)azacyclobutane-1-yl)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethane)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (11)

[0153]

[0154] Step 1: Add DIPEA (442 mg, 3.42 mmol, 596 μL, 3 eq.) and HATU (520 mg, 1.37 mmol, 1.2 eq.) to a 10 mL solution of DMF containing 8-1 (0.2 g, 1.142 mmol, 1 eq.) and 11-1 (184 mg, 1.14 mmol, 1 eq.). o Stirred at C for 12 hours. Add H2O (10 mL) and extract with ethyl acetate (10 mL x 2). Wash the organic phase with saturated brine (15 mL x 2), dry to anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to give compound 11-2 (320 mg, 99% yield). 1 H NMR (400 MHz, CDCl3) δ 5.23 (s, 1H), 4.42 - 4.28 (m, 1H), 4.27 - 4.18 (m, 2H), 4.18 - 4.07 (m, 1H), 3.77 (dd, J = 4.8, 10.0 Hz, 2H), 3.43 - 3.27 (m, 1H), 1.47 (s, 9H).

[0155] Step 2: Add a 4 M, 5 mL solution of dioxane-1,4-dioxane (11-2, 320 mg, 1.13 mmol, 1 eq.) to a 5 mL solution of 1,4-dioxane, and incubate at 20°C. o The mixture was stirred at C for 12 hours. The reaction solution was concentrated under reduced pressure to give the hydrochloride of compound 11-3 (230 mg, yield: 92%). 1 H NMR (400 MHz, DMSO-d6) δ 8.31 (s, 3H), 4.48- 4.41 (m, 1H), 4.26 (dd, J = 5.6, 9.6 Hz, 1H), 4.22 - 4.13 (m, 1H), 3.78 -3.68 (m, 1H), 3.68 - 3.58 (m, 3H).

[0156] Step 3: Add HATU (69 mg, 183 μmol, 1.2 eq.) and DIPEA (59 mg, 457 μmol, 3 eq.) to a 1-7 (70 mg, 152 μmol, 1 eq.) solution of DMF (1 mL), and incubate at 25°C. o Stir at C for 10 minutes. Then add 11-3% hydrochloride (40 mg, 183 μmol, 1.2 eq.) and stir at 25°C. o Stir at C for 10 min. Add H2O (10 mL) and extract with ethyl acetate (10 mL x 3). Wash the organic phase with saturated brine (10 mL x 2), dry to anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify the crude product by preparative thin-layer chromatography (SiO2, n-heptane:ethyl acetate = 0:1) to give compound 11 (33.3 mg, yield 35%). 1 ¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 2H), 7.09 (s, 1H), 6.75 (t, J = 4.0 Hz, 1H), 4.46–4.37 (m, 1H), 4.33–4.24 (m, 2H), 4.22–4.12 (m, 1H), 4.11–3.94 (m, 3H), 3.68 (d, J = 17.2 Hz, 1H), 3.49–3.31 (m, 1H), 2.54 (s, 3H). MS-ESI calculated values ​​[M+H] + 622, measured value 622.

[0157] Example 012 Preparation of N-(2-(3-fluorozacricyclobutane-1-yl)-2-oxoethyl)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (12)

[0158]

[0159] Step 1: Add T4P (2.01 g, 2.80 mmol, 50% ethyl acetate solution, 1.5 eq.) and DIPEA (724 mg, 5.60 mmol, 3 eq.) to a DMF (5 mL) solution of 8-1 (327 mg, 1.86 mmol, 1 eq.) and 12-1 (250 mg, 2.24 mmol, 1.2 eq.), 20 oStirred at C for 0.5 hours. Add H2O (10 mL) and extract with ethyl acetate (10 mL x 2). Wash the organic phase with saturated brine (15 mL x 2), dry to anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to give compound 12-2 (330 mg, yield 76%). 1 H NMR (400 MHz, CDCl3) δ 4.53 - 4.38 (m, 1H), 3.77 (s, 2H), 1.96 - 1.74 (m, 2H), 1.69 - 1.58 (m, 2H), 1.46 (s, 9H).

[0160] Step 2: Add TFA (98 mg, 861 μmol, 1 mL, 1 eq.) to a 12-2 (200 mg, 861 μmol, 1 eq.) solution of DCM (5 mL), and incubate at 25°C. o The mixture was stirred at C for 2 hours. The reaction solution was concentrated under reduced pressure to give the TFA salt of compound 12-3 (240 mg, crude product).

[0161] Step 3: Add HATU (49 mg, 130 μmol, 1.2 eq.) and DIPEA (42 mg, 327 μmol, 3 eq.) to a 1-7 (50 mg, 109 μmol, 1 eq.) solution of DMF (1 mL), and incubate at 25°C. o Stir at C for 10 minutes. Then add 12-3g of TFA salt (67 mg, 272 μmol, 2.5 eq.) and stir at 25°C. o Stir at C for 10 minutes. Add H2O (10 mL) and extract with ethyl acetate (10 mL x 3). Wash the organic phase with saturated brine (10 mL x 2), dry to anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify the crude product by preparative thin-layer chromatography (SiO2, n-heptane:ethyl acetate = 0:1) to give compound 12 (49.4 mg, yield 79%). 1 ¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 2H), 7.09 (s, 1H), 6.79 (s, 1H), 5.58–5.19 (m, 1H), 4.55–4.15 (m, 4H), 4.11–3.96 (m, 3H), 3.68 (d, J = 17.2 Hz, 1H), 2.54 (s, 3H). MS-ESI calculated values ​​[M+H] + 572, measured value 572.

[0162] Example 013 Preparation of N-(2-(3-hydroxy-3-(trifluoromethyl)azacyclobutane-1-yl)-2-oxoethyl)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethane)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (13)

[0163]

[0164] Step 1: Add DIPEA (1.09 g, 8.44 mmol, 1 eq.) and T4P (3.04 g, 4.22 mmol, 50% ethyl acetate solution, 1.5 eq.) to a solution of 8-1 hydrochloride (500 mg, 2.81 mmol, 1 eq.) and 13-1 (493 mg, 2.81 mmol, 1 eq.) in DCM (3 mL). In a 25°C solution... o Stirred at C for 2 hours. Dilute with H2O (20 mL), extract with ethyl acetate (20 mL x 3), wash the combined organic phases with NaHCO3 (20 mL x 2), dry with MgSO4, filter, concentrate under reduced pressure to give crude compound 13-2 (600 mg, yield 71%). 1 H NMR (400 MHz, CDCl3) δ 5.40 - 5.11 (m,1H), 4.51 - 4.30 (m, 2H), 4.28 - 4.03 (m, 2H), 3.94 - 3.67 (m, 2H), 1.47 (s,9H).

[0165] Step 2: Add TFA (1 mL) to a 13-2 (605 mg, 2.02 mmol, 1 eq.) solution of DCM (5 mL). At 25°C... o The mixture was stirred at C for 3 hours. After the reaction was complete, the solution was concentrated under reduced pressure to obtain the trifluoroacetate of crude compound 13-3 (600 mg, yield 94%).

[0166] Step 3: Add HATU (60 mg, 156 μmol, 1.2 eq.) and DIPEA (51 mg, 392 μmol, 3 eq.) to a 1-7 (60 mg, 130 μmol, 1 eq.) solution of DMF (1 mL), and incubate at 25°C. o Stir at C for 10 minutes. Add 13-3g of trifluoroacetate (60 mg, 192 μmol, 1.46 eq.) and stir at 25°C. oStir at C for 10 minutes. Add H2O (10 mL) and extract with ethyl acetate (10 mL x 3). Wash the organic phase with saturated brine (10 mL x 2), dry to anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify the crude product by preparative thin-layer chromatography (SiO2, n-heptane:ethyl acetate = 0:1) to give compound 13 (16.0 mg, yield 19%). 1 ¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 2H), 7.09 (s, 1H), 6.75 (t, J = 4.4 Hz, 1H), 4.48 (d, J = 10.0 Hz, 1H), 4.38 (d, J = 11.2 Hz, 1H), 4.26 (d, J = 9.6 Hz, 1H), 4.17 - 3.97 (m, 4H), 3.68 (d, J = 17.2 Hz, 1H), 3.47 (s, 1H), 2.53 (s, 3H). MS-ESI calculated values ​​[M+H] + 638, measured value 638.

[0167] Example 014 Preparation of N-(2-(3-fluoro-3-methylazacyclobutane-1-yl)-2-oxoethyl)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (14)

[0168]

[0169] Step 1: Compound 8-1 (500 mg, 2.85 mmol, 1.0 eq.), the hydrochloride salt of compound 14-1 (769 mg, 4.28 mmol, 1.5 eq.), HATU (1.63 g, 4.28 mmol, 1.5 eq.), and N,N-diisopropylethylamine (1.48 g, 11.4 mmol, 4.0 eq.) were dissolved in N,N-dimethylformamide (5 mL) for 20 minutes. o The reaction was carried out at C for 1 hour. The reaction was quenched with water (10 mL), extracted with ethyl acetate (15 mL x 2), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to give crude compound 14-2 (856 mg, 100% yield). 1H NMR (400 MHz, CDCl3) δ 4.59 - 4.23 (m, 4H), 3.85 - 3.78 (m, 2H), 1.80 - 1.64 (m, 3H), 1.47 (s, 9H).

[0170] Step 2: Dissolve compound 14-2 (856 mg, 2.85 mmol, 1.0 eq.) in DCM (1 mL) solution, and add trifluoroacetic acid (1.30 g, 11.4 mmol, 4.0 eq.) dropwise. o The reaction was carried out at C for 16 hours. The crude compound 14-3 was concentrated under reduced pressure to obtain the TFA salt (895 mg, 100% yield).

[0171] Step 3: Add HATU (60 mg, 156 μmol, 1.2 eq.) and DIPEA (50 mg, 392 μmol, 3 eq.) to a 1-7 DMF (1 mL) solution (60 mg, 130 μmol, 1 eq.) and incubate at 25°C. o Stir at C for 10 minutes. Then add 14-3 TFA salt (60 mg, 230 μmol, 1.76 eq.) and stir at 25°C. o Stir at C for 10 minutes. Add H2O (10 mL) and extract with ethyl acetate (10 mL x 3). Wash the organic phase with saturated brine (10 mL x 2), dry to anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify the crude product by preparative thin-layer chromatography (SiO2, n-heptane:ethyl acetate = 0:1) to give compound 14 (23.4 mg, yield 30%). 1 ¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 2H), 7.09 (s, 1H), 6.80 (s, 1H), 4.43–3.99 (m, 7H), 3.68 (d, J = 17.2 Hz, 1H), 2.54 (s, 3H), 1.75–1.65 (m, 3H). MS-ESI calculated values ​​[M+H] + 586, measured value 586.

[0172] Example 015 Preparation of N-(2-(cyclopropylamino)-2-oxoethyl)-3-(5-(3,5-dichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)benzo[c]thiophene-1-carboxamide (15)

[0173]

[0174] Step 1: Compounds 10-7 (290 mg, 1.23 mmol, 1 eq.) and 15-1 (451 mg, 1.85 mmol, 1.5 eq.) were dissolved in toluene (15 mL), and Cs₂CO₃ (80 mg, 247 μmol, 0.2 eq.) was added. The solution was heated at 110 °C. o The mixture was stirred at C for 16 hours. The reaction solution was poured into H₂O (10 mL) and extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated brine (10 mL x 2), dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO₂, n-heptane / ethyl acetate, 0–10%) to give 15⁻² (122 mg, 21% yield). MS-ESI calculated value [M+H] + 459, measured value 459.

[0175] Step 2: Dissolve NaOH (31 mg, 783 μmol, 4 eq.), tetrabutylammonium bromide (6 mg, 19 μmol, 0.1 eq.), and hydroxylamine hydrochloride (40 mg, 587 μmol, 3 eq.) in H2O (1 mL). After stirring at 0°C for 0.5 hours, add 15-2 (90 mg, 195 μmol, 1 eq.) of THF (2 mL) solution and heat at 25°C. o The mixture was stirred at C for 0.5 hours. The reaction was quenched with NH4Cl (10 mL), and the reaction solution was poured into H2O (20 mL). Extraction was performed with ethyl acetate (50 mL x 3). The organic phase was washed with saturated brine (10 mL x 2), dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure to give 15-3 (120 mg, crude product). MS-ESI calculated value [M+H] + 474, measured value 474.

[0176] Step 3: Dissolve compound 15-3 (120 mg, 253 μmol, 1 eq.) in THF (3 mL), H2O (1 mL), and MeOH (1 mL), then add LiOH·H2O (31 mg, 759 μmol, 3 eq.) and heat at 25°C. o The mixture was stirred at C for 5 hours. The pH of the reaction mixture was adjusted to 5-7 with 1N HCl, extracted with ethyl acetate (10 mL x 2), the organic phase was washed with brine (10 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to give 15-4 (100 mg, yield 85%). 1H NMR (400 MHz, CDCl3) δ8.52 (d, J = 8.8 Hz, 1H), 8.44 (d, J = 8.8 Hz, 1H), 7.95 (d, J = 0.8 Hz, 1H), 7.58 (d, J = 1.6 Hz, 2H), 7.46 - 7.37(m, 2H), 4.29 (d, J = 16.8 Hz, 1H), 3.92 (d, J = 16.8 Hz, 1H).

[0177] Step 4: Dissolve compound 15-4 (50 mg, 108 μmol, 1 eq.) in DMF (1 mL), add HATU (50 mg, 131 μmol, 1.21 eq.) and DIPEA (42 mg, 324 μmol, 3 eq.), and incubate at 25 °C. o Stirring at C for 0.5 hours, then adding the hydrochloride salt of compound 15-5 (20 mg, 132 μmol, 1.22 eq.), and stirring at 25°C. o The mixture was stirred at C for 0.5 hours. The reaction solution was poured into H2O (10 mL) and extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated brine (10 mL x 2), dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by preparative thin-layer chromatography (SiO2, ethyl acetate) to give 15 (23 mg, yield 38%). 1 H NMR (400 MHz, DMSO-d6) δ 8.68 (t, J = 6.0 Hz, 1H), 8.37 -8.29 (m, 1H), 8.25 - 8.16 (m, 1H), 8.08 (d, J = 4.4 Hz, 1H), 7.83 (t, J = 1.6Hz, 1H), 7.75 (d, J = 1.6 Hz, 2H), 7.45 - 7.36 (m, 2H), 4.54 (d, J = 8.4 Hz, 2H), 3.88 (d, J = 5.6 Hz, 2H), 2.70 - 2.63 (m, 1H), 0.70 - 0.57 (m, 2H), 0.48- 0.31 (m, 2H). MS-ESI calculated value [M+H] + 557, measured value 557.

[0178] Example 016 Preparation of 3-(5-(3,5-dichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-N-(2-oxo-2-((1-(trifluoromethane)cyclopropyl)amino)ethyl)benzo[c]thiophene-1-carboxamide (16)

[0179]

[0180] Step 1: Dissolve compound 15-4 (50 mg, 108 μmol, 1 eq.) in DMF (1 mL), add HATU (50 mg, 131 μmol, 1.21 eq.) and DIPEA (42 mg, 324 μmol, 3 eq.), and incubate at 25 °C. o Stirring at C for 0.5 hours, then adding the hydrochloride salt of compound 16-1 (29 mg, 132 μmol, 1.22 eq.), and stirring at 25°C. o The mixture was stirred at C for 0.5 hours. The reaction solution was poured into H2O (10 mL) and extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated brine (10 mL x 2), dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by preparative thin-layer chromatography (SiO2, n-heptane / ethyl acetate = 1:1) to give 16 (18 mg, yield 26%). 1 ¹H NMR (400 MHz, CDCl₃) δ 8.39 - 8.30 (m, 1H), 8.29 - 8.19 (m, 1H), 7.57 (d, J = 1.6 Hz, 2H), 7.47 (t, J = 1.6 Hz, 1H), 7.44 - 7.35 (m, 2H), 7.07 (t, J = 4.8 Hz, 1H), 6.77 (s, 1H), 4.31 - 4.16 (m, 3H), 3.87 (d, J = 16.4 Hz, 1H), 1.43 - 1.38 (m, 2H), 1.21 (s, 2H). MS-ESI calculated values ​​[M+H] + 624, measured value 624.

[0181] Example 017 Preparation of N-(2-(3-cyano-3-fluorozacricyclobutane-1-yl)-2-oxoethyl)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (17)

[0182]

[0183] Step 1: Dissolve compounds 1-7 (100 mg, 218 μmol, 1 eq.) and the hydrochloride salt of compound 17-1 (51 mg, 327 μmol, 1.5 eq.) in DMF (2 mL), add HATU (124 mg, 327 μmol, 1.5 eq.) and DIPEA (112 mg, 872 μmol, 4 eq.), 25 o The reaction was stirred at C for 2 hours. The reaction was quenched with H₂O (5 mL), and the mixture was extracted with ethyl acetate (5 mL x 2). The organic phases were combined, washed with saturated brine (5 mL x 3), dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure. The crude product obtained from the concentration was purified by prep-HPLC (Column: Ultimate C18, 30*150mm ID, 5µm; Mobilephase: A for H₂O (0.1% TFA) and B for CAN; Gradient: 65%-85%, 10 min, 100%, 2 min, 65%, 3 min; Flow rate: 30 mL / min) to give compound 17 (10 mg, yield 8%). 1 ¹H NMR (400 MHz, CDCl₃) δ 7.62 (s, 2H), 7.08 (s, 1H), 6.75 (t, J = 4.4 Hz, 1H), 4.90–4.45 (m, 4H), 4.10–4.03 (m, 3H), 3.71–3.63 (m, 1H), 2.51 (s, 3H). MS-ESI calculated value [M+H]+ 597, measured value 597.

[0184] Example 018 Preparation of 3-methyl-N-(2-oxo-2-((3-(trifluoromethyl)oxetane-3-yl)amino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethane)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (18)

[0185]

[0186] Step 1: Dissolve compound 8-1 (200 mg, 1.14 mmol, 1 eq.), compound 18-1 hydrochloride (203 mg, 1.14 mmol, 1 eq.), HATU (651 mg, 1.71 mmol, 1.5 eq.), and N,N-diisopropylethylamine (443 mg, 3.43 mmol, 3 eq.) in N,N-dimethylformamide (5 mL), 25o The reaction was carried out at C for 6 hours. The reaction was quenched with H₂O (10 mL), extracted with ethyl acetate (10 mL x 3), and the organic phases were combined. The organic phases were washed with saturated brine (10 mL x 2), dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by column chromatography (SiO₂, THF / n-heptane, 0–30%) to give compound 18-2 (200 mg, yield 59%).

[0187] Step 2: Compound 18-2 (100 mg, 335 μmol, 1 eq.) was added to dioxane (4 M, 2 mL) of hydrogen chloride, and heated for 25 minutes. o The reaction was carried out at C for 2 hours. The mixture was concentrated under reduced pressure to give the crude compound 18-3 hydrochloride (50 mg, yield 64%).

[0188] Step 3: Dissolve compounds 1-7 (47 mg, 103 μmol, 1 eq.) and the hydrochloride salt of compound 18-3 (48 mg, 205 μmol, 2 eq.) in DMF (2 mL), add HATU (58 mg, 154 μmol, 1.5 eq.) and DIPEA (53 mg, 410 μmol, 4 eq.), 25 mL o Stirred at C for 12 hours. Add water (10 mL) and extract with ethyl acetate (5 mL x 3). Wash the organic phase with saturated brine (5 mL x 2), dry to anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify the crude product by preparative thin-layer chromatography (SiO2, dichloromethane:methanol = 15:1) to give compound 18 (1.4 mg, yield: 2%). 1 ¹H NMR (400MHz, CDCl₃) δ 7.64 (s, 2H), 7.10 (s, 1H), 7.01 (br s, 1H), 6.70 – 6.59 (m, 1H), 4.27 – 4.03 (m, 5H), 3.87 – 3.65 (m, 3H), 2.53 (s, 3H). MS-ESI calculated values ​​[M+H] + 638, measured value 638.

[0189] Example 019 Preparation of N-(2-(3,3-difluoroazacyclobutane-1-yl)-2-oxoethyl)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (19)

[0190]

[0191] Step 1: Add DIPEA (556 mg, 4.30 mmol, 749 μL, 3 eq.) and HATU (654 mg, 1.72 mmol, 1.2 eq.) to a DMF (10 mL) solution of 6-1 (0.3 g, 1.43 mmol, 1 eq.) and compound 19-1 (186 mg, 1.43 mmol, 1 eq.) hydrochloride. o Stirred at C for 12 hours. Add H2O (10 mL) and extract with ethyl acetate (10 mL x 2). Wash the organic phase with saturated brine (15 mL x 2), dry to anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify the crude product by column chromatography (SiO2, ethyl acetate / n-heptane, 1:10 ~ 1:2) to give compound 19-2 (400 mg, 98% yield).

[0192] Step 2: Add Pd / C (80 mg, 10% purity, 20% w / w) to a 19-2 (400 mg, 1.407 mmol, 1 eq.) MeOH (10 mL) solution and incubate at 15 psi under a hydrogen atmosphere for 20 minutes. o The mixture was stirred at C for 3 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give compound 19-3 (200 mg, 95% yield).

[0193] Step 3: Add HATU (49.7 mg, 131 μmol, 1.5 eq.) and DIPEA (45.1 mg, 349 μmol, 4 eq.) to 2 mL solutions of DMF containing 1-7 (26.2 mg, 174 μmol, 2.0 eq.) and 19-3 (40 mg, 87.2 μmol, 1 eq.), and incubate at 25°C. o Stir at C for 1 hour. Add H2O (1 mL) and extract with ethyl acetate (1 mL x 3). Wash the organic phase with H2O (1 mL) and saturated brine (1 mL), dry to anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify the crude product by preparative thin-layer chromatography (SiO2, n-heptane:ethyl acetate = 1:2) to give compound 19 (25 mg, yield: 49%). 1 ¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 2H), 7.09 (s, 1H), 6.73 (s, 1H), 4.59–4.42 (m, 4H), 4.10–4.15 (m, 3H), 3.70–3.66 (m, 1H), 2.54 (s, 3H). MS-ESI calculated values ​​[M+H] +590, measured value 590.

[0194] Example 020 Preparation of 3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-N-(1-(2,2,2-trifluoroethyl)azacyclobutane-3-yl)methyl)thiophene-2-carboxamide (20)

[0195]

[0196] Step 1: The hydrochloride salt of compound 20-1 (0.5 g, 2.25 mmol, 1 eq.) was dissolved in THF (40 mL), and 2,2,2-trifluoroethyltrifluoromethanesulfonate (782 mg, 3.37 mmol, 1.5 eq.) and triethylamine (682 mg, 6.74 mmol, 936 μL, 3 eq.) were added. o The mixture was stirred at C for 12 hours. The reaction solution was concentrated under reduced pressure to give crude compound 20-2 (602 mg, 100% yield).

[0197] Step 2: Add TFA (1.02 g, 8.98 mmol, 4 eq.) to a 20-2 (602 mg, 2.24 mmol, 1 eq.) DCM (1 mL) solution. o The mixture was stirred at C for 1 hour. The reaction solution was concentrated under reduced pressure to give compound 20-3 (889 mg, 100% yield).

[0198] Step 3: Add HATU (62.2 mg, 164 μmol, 1.5 eq.) and DIPEA (56.4 mg, 436 μmol, 4 eq.) to 2 mL of DMF solutions of 1-7 (50 mg, 109 μmol, 1 eq.) and 20-3 (86.4 mg, 218 μmol, 2.0 eq.), 20 o Stirred at C for 0.5 hours. Add H2O (1 mL) and extract with ethyl acetate (1 mL x 3). Wash the organic phase with H2O (1 mL) and saturated brine (1 mL), dry to anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify the crude product by preparative thin-layer chromatography (SiO2, n-heptane:ethyl acetate = 1:2) to give compound 20 (21 mg, yield 32%). 1¹H NMR (400 MHz, CDCl₃) δ 7.65 (s, 2H), 7.09 (s, 1H), 6.74–6.72 (m, 1H), 4.11–4.07 (m, 1H), 3.74–3.56 (m, 4H), 3.17–3.04 (m, 4H), 2.55 (s, 3H), 2.13–2.03 (m, 2H). MS-ESI calculated values ​​[M+H] + 608, measured value 608.

[0199] Example 021 Preparation of 3-methyl-N-(2-oxo-2-((1-(trifluoromethyl)cyclopropyl)amino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethane)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (21)

[0200]

[0201] Step 1: Dissolve compounds 1-7 (40 mg, 87.2 μmol, 1 eq.) and the hydrochloride salt of compound 16-1 (38.1 mg, 174 μmol, 2.0 eq.) in DMF (2 mL), add HATU (50 mg, 131 μmol, 1.5 eq.) and DIPEA (45.1 mg, 349 μmol, 4 eq.), and heat at 20 °C. o Stir at C for 10 minutes. Add H2O (1 mL) and extract with ethyl acetate (1 mL x 3). Wash the organic phase with H2O (1 mL) and saturated brine (1 mL), dry to anhydrous magnesium sulfate, filter, and concentrate under reduced pressure. The crude product was purified by preparative thin-layer chromatography (SiO2, n-heptane / ethyl acetate = 1:1) to give compound 21 (20 mg, yield 37%). 1 ¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 2H), 7.10 (s, 1H), 6.72–6.36 (m, 2H), 4.14–4.12 (m, 2H), 4.10–4.05 (m, 1H), 3.70–3.66 (d, 1H), 2.54 (s, 3H), 1.41–1.18 (m, 4H). MS-ESI calculated values ​​[M+H] + 622, measured value 622.

[0202] Example 022 Preparation of 3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-N-(((S)-1-(2,2,2-trifluoroethyl)azacyclobutane-2-yl)methyl)thiophene-2-carboxamide (22)

[0203]

[0204] Step 1: The hydrochloride salt of compound 22-1 (0.5 g, 2.25 mmol, 1 eq.) was dissolved in THF (40 mL), and 2,2,2-trifluoroethyltrifluoromethanesulfonate (782 mg, 3.37 mmol, 1.5 eq.) and triethylamine (682 mg, 6.74 mmol, 936 μL, 3 eq.) were added. o The mixture was stirred at C for 12 hours. The reaction solution was concentrated under reduced pressure to give crude compound 22-2 (602 mg, 100% yield).

[0205] Step 2: Add TFA (1.02 g, 8.98 mmol, 4 eq.) to a 22-2 (602 mg, 2.24 mmol, 1 eq.) DCM (1 mL) solution. o The mixture was stirred at C for 1 hour. The reaction solution was concentrated under reduced pressure to give compound 22-3 (889 mg, 100% yield).

[0206] Step 3: Add HATU (62.2 mg, 164 μmol, 1.5 eq.) and DIPEA (56.4 mg, 436 μmol, 4 eq.) to 2 mL solutions of DMF containing 1-7 (50 mg, 109 μmol, 1 eq.) and 22-3 (86.4 mg, 218 μmol, 2.0 eq.), 20 o Stirred at C for 0.5 hours. Add H2O (1 mL) and extract with ethyl acetate (1 mL x 3). Wash the organic phase with H2O (1 mL) and saturated brine (1 mL), dry to anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify the crude product by preparative thin-layer chromatography (SiO2, n-heptane:ethyl acetate = 1:2) to give compound 22 (30 mg, yield 45%). 1¹H NMR (400MHz, CDCl₃) δ 7.65 (s, 2H), 7.11 (s, 1H), 6.74–6.72 (m, 1H), 4.11–4.07 (m, 1H), 3.74–3.56 (m, 4H), 3.17–3.04 (m, 4H), 2.55 (s, 3H), 2.13–2.03 (m, 2H). MS-ESI calculated values ​​[M+H] + 608, measured value 608.

[0207] Example 023 Preparation of 3-methyl-N-(2-oxo-2-(3,3,4,4-tetrafluoropyrrolidone-1-yl)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (23)

[0208]

[0209] Step 1: Add DIPEA (885 mg, 6.85 mmol, 1.19 mL, 3 eq.) and HATU (1.04 g, 2.74 mmol, 1.2 eq.) to a DMF (10 mL) solution of 8-1 (0.4 g, 2.28 mmol, 1 eq.) and 23-1 (410 mg, 2.28 mmol, 1 eq.). o Stirred at C for 12 hours. Add H2O (10 mL) and extract with ethyl acetate (10 mL x 2). Wash the organic phase with saturated brine (15 mL x 2), dry to anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to give compound 23-3 (680 mg, 99% yield).

[0210] Step 2: Add a 4 M, 5 mL solution of dioxane in hydrogen chloride to a 23-3 (680 mg, 2.27 mmol, 1 eq.) solution of dioxane (5 mL), and incubate at 20°C. o The mixture was stirred at C for 12 hours. The reaction solution was concentrated under reduced pressure, and the crude product was slurried with ethyl acetate (15 mL) to give the hydrochloride of compound 23-4 (520 mg, yield 97%).

[0211] Step 3: Add DIPEA (56.4 mg, 436 μmol, 4 eq.) and HATU (62.2 mg, 164 μmol, 1.5 eq.) to a 1-7 (50 mg, 109 μmol, 1 eq.) solution of DMF (2 mL), and incubate at 25°C. oStir at C for 10 minutes. Add 8-4% hydrochloride (30 mg, 130 μmol, 1.2 eq.) and stir at 25°C. o Stir at C for 10 minutes. Add H2O (1 mL) and extract with ethyl acetate (1 mL x 3). Wash the organic phase with H2O (1 mL) and saturated brine (1 mL), dry to anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify the crude product by preparative thin-layer chromatography (SiO2, n-heptane:ethyl acetate = 1:1) to give compound 8 (25 mg, yield 36%). 1 ¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 2H), 7.10 (s, 1H), 6.68–6.80 (m, 1H), 4.20–4.01 (m, 7H), 3.70–3.66 (m, 1H), 2.54 (s, 3H). MS-ESI calculated values ​​[M+H] + 640, measured value 640.

[0212] Example 024 Preparation of N-(2-(3-fluoro-3-(trifluoromethyl)azacyclobutane-1-yl)-2-oxoethyl)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (24)

[0213]

[0214] Step 1: Dissolve compounds 1-7 (50 mg, 109 μmol, 1 eq.) and the TFA salt of compound 24-1 (51.4 mg, 164 μmol, 1.5 eq.) in DMF (2 mL), add DIPEA (56.4 mg, 436 μmol, 4 eq.) and HATU (62.2 mg, 164 μmol, 1.5 eq.), and heat at 20 °C. o Stir at C for 1 hour. Add H2O (1 mL) and extract with ethyl acetate (1 mL x 3). Wash the organic phase with H2O (1 mL) and saturated brine (1 mL), dry to anhydrous magnesium sulfate, filter, and concentrate under reduced pressure. The crude product was purified by preparative thin-layer chromatography (SiO2, n-heptane / ethyl acetate = 1:3) to give compound 24 (25 mg, yield 36%). 1¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 2H), 7.09 (s, 1H), 6.69 (m, 1H), 4.59–4.05 (m, 7H), 3.70–3.66 (m, 1H), 2.54 (s, 3H). MS-ESI calculated values ​​[M+H] + 640, measured value 640.

[0215] Example 025 Preparation of N-(2-(3-fluoro-3-(fluoromethyl)azacyclobutane-1-yl)-2-oxoethyl)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (25)

[0216]

[0217] Step 1: Dissolve compounds 1-7 (50 mg, 109 μmol, 1 eq.) and the TFA salt of compound 24-1 (45.5 mg, 164 μmol, 1.5 eq.) in DMF (2 mL), add DIPEA (56.4 mg, 436 μmol, 4 eq.) and HATU (62.2 mg, 164 μmol, 1.5 eq.), and heat at 20 °C. o Stir at C for 1 hour. Add H2O (1 mL) and extract with ethyl acetate (1 mL x 3). Wash the organic phase with water (1 mL) and saturated brine (1 mL), dry to anhydrous magnesium sulfate, filter, and concentrate under reduced pressure. The crude product was purified by preparative thin-layer chromatography (SiO2, n-heptane / ethyl acetate = 1:3) to give compound 25 (25 mg, yield 38%). 1 ¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 2H), 7.09 (s, 1H), 6.79–6.77 (m, 1H), 4.75–4.59 (m, 2H), 4.42–4.24 (m, 4H), 4.09–4.05 (m, 3H), 3.70–3.66 (m, 1H), 2.54 (s, 3H). MS-ESI calculated values ​​[M+H] + 604, measured value 604.

[0218] Example 026 Preparation of 3-methyl-N-(2-oxo-2-((2,2,2-trifluoroethoxy)amino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (26)

[0219]

[0220] Step 1: Add DIPEA (443 mg, 3.43 mmol, 4.0 eq.) and HATU (488 mg, 1.28 mmol, 1.5 eq.) to a 5 mL solution of DMF containing 8-1 (150 mg, 856 μmol, 1 eq.) and 26-1 (195 mg, 1.28 mmol, 1.5 eq.). o Stir at C for 1 hour. Add H2O (5 mL) and extract with ethyl acetate (5 mL x 3). Wash the organic phase with water (5 mL x 3) and saturated brine (5 mL), dry to anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to give compound 26-2 (233 mg, 100% yield).

[0221] Step 2: Add 26-2 (233 mg, 856 μmol, 1 eq.) to a dioxane solution of hydrogen chloride (4 M, 2 mL), and incubate at 20°C. o The mixture was stirred at C for 16 hours. The reaction solution was concentrated under reduced pressure to give the crude compound 26-3 hydrochloride (178 mg, 100% yield).

[0222] Step 3: Dissolve compounds 1-7 (150 mg, 327 μmol, 1 eq.) and the hydrochloride salt of compound 26-3 (102 mg, 491 μmol, 1.5 eq.) in DMF (3 mL), add DIPEA (169 mg, 1.3 mmol, 4 eq.) and HATU (187 mg, 491 μmol, 1.5 eq.), and heat at 20 °C. o Stirred at C for 1 hour. Add water (1 mL) and extract with ethyl acetate (1 mL x 3). Wash the organic phase with H2O (1 mL) and saturated brine (1 mL), dry to anhydrous magnesium sulfate, filter, and concentrate under reduced pressure. The crude product was purified by preparative thin-layer chromatography (SiO2, n-heptane / ethyl acetate = 1:1) to give compound 26 (45 mg, yield 22%). 1 ¹H NMR (400 MHz, CDCl₃) δ 9.75 (br, 1H), 7.69 (s, 2H), 7.10 (s, 1H), 6.70 (m, 1H), 4.35–4.06 (m, 5H), 3.71–3.67 (m, 1H), 2.57 (s, 3H). MS-ESI calculated values ​​[M+H] + 612, measured value 612.

[0223] Biological test evaluation

[0224] Experimental Example 1: Test of the killing efficacy against mites in an in vitro model

[0225] Mite collection: Gently dab live mites from the cage using a cotton swab or small tweezers, with each group consisting of 17-26 live mites.

[0226] Drug treatment and detection methods: Using a pipette, 100 μL of different concentrations of drug (3% DMSO solution + 97% water to obtain a target final concentration of 30 μM) were added to each group of 24-well plates, ensuring full contact between the drug and the mites. After incubation at room temperature for 5 min, the mites were removed from each plate and transferred to another dry 24-well plate. The plate was sealed at the top and allowed to air dry for about 1 h. The activity of the mites was observed, and the number of dead mites / total number of mites was recorded. Then, the 24-well plates containing mites were incubated in an environment with high room temperature and high humidity. At 1 h, 2 h, 4 h, 6 h, 8 h, 22 h, and 24 h (time points after drug addition), the number of dead mites / total number of mites was counted, and the mite mortality rate was calculated. The endpoint of the test was 24 h after drug addition.

[0227] At the corresponding time points, a mite mortality rate greater than or equal to 90% was recorded as "+++"; a mite mortality rate less than 90% but greater than or equal to 50% was recorded as "++"; and a mite mortality rate less than 50% was recorded as "+". The experimental results are shown in Table 1 below.

[0228] Table 1

[0229]

[0230] The applicant declares that the above embodiments illustrate the isoxazoline compounds and their applications, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An isoxazoline compound, characterized in that, The isoxazoline compounds have the structure shown in Formula I: ； in, Ring A is selected from 5-10 heteroaryl groups containing 1-3 heteroatoms; R1 is selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo-C1-C6 alkyl, deuterated C1-C6 alkyl, halo-C2-C6 alkenyl, halo-C2-C6 alkynyl, halogen, cyano, nitro, -C(O)NR a R b -C(O)R a -C(O)OR a -OR a -R a OR b -OC(O)R a -OC(O)OR a -OC(O)NR a R b -NR a R b -SR a -S(O)R a -S(O)2R a Or a 3-10 saturated or unsaturated ring containing 0-3 heteroatoms, wherein the 3-10 saturated or unsaturated ring containing 0-3 heteroatoms may optionally be surrounded by 1-3 R... a replace; R2 is selected from hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo-C1-C6 alkyl, deuterated C1-C6 alkyl, halo-C2-C6 alkenyl, and halo-C2-C6 alkynyl. R3 is selected from hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo-C1-C6 alkyl, deuterated C1-C6 alkyl, halo-C2-C6 alkenyl, and halo-C2-C6 alkynyl. R4 and R5 are each independently selected from hydrogen, any C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo-C1-C6 alkyl, deuterated C1-C6 alkyl, halo-C2-C6 alkenyl, halo-C2-C6 alkynyl, halogen, cyano, nitro, -C(O)NHR6, -C(O)R6, -C(O)OR6, -OR6, -OC(O)R6, -OC(O)OR6, -NHR6, -SR6, -S(O)R6, -S(O)2R6, or a 3-10 member saturated or unsaturated ring containing 0-3 heteroatoms, wherein the 3-10 member saturated or unsaturated ring containing 0-3 heteroatoms may optionally be substituted with 1-3 R6s; R6 is selected from hydrogen, C1-C6 alkyl groups, and compounds with 1-3 R groups. a Substituted C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo-C1-C6 alkyl, deuterated C1-C6 alkyl, halo-C2-C6 alkenyl, halo-C2-C6 alkynyl, halogen, cyano, nitro, -C(O)NHR a -C(O)NR a R b -C(O)R a -C(O)OR a -OR a -R a OR b -OC(O)R a -OC(O)OR a -OC(O)NR a R b -NR a R b -SR a -S(O)R a -S(O)2R a Or a 3-10 saturated or unsaturated ring containing 0-3 heteroatoms, wherein the 3-10 saturated or unsaturated ring containing 0-3 heteroatoms may optionally be surrounded by 1-3 R... a replace; Each R a R b Each is independently selected from hydrogen, deuterium, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo-C1-C6 alkyl, deuterated C1-C6 alkyl, halo-C2-C6 alkenyl, halo-C2-C6 alkynyl, halogen, cyano, nitro, amino, carboxyl, carbonyl, hydroxyl, hydroxyalkyl, alkoxy, haloalkoxy, deuteratedalkoxy, C3-C6 cycloalkyl, halo-C3-C6 cycloalkyl, alkoxy-substituted C3-C6 cycloalkyl, and alkyl groups substituted with 1-3 R groups. c Substituted C3-C6 cycloalkyl, C3-C6 heterocyclic, halogenated C3-C6 heterocyclic, alkyl-substituted C3-C6 heteroaryl, -OR c -NHR c -S(O)2R c -OR c OR d -R c OR d or -OC(O)R c Each R c R d Each is independently selected from halogen, cyano, hydroxyl, C1-C6 alkyl, and halo-C1-C6 alkyl; and m is an integer between 0 and 5; n is an integer between 0 and 2.

2. The isoxazoline compound according to claim 1, characterized in that, The isoxazoline compounds have the structure shown in formula Ia: ； R1, R2, R3, R4, R5, R6, R a R b R c R d m and n are the same as those defined in claim 1.

3. The isoxazoline compound according to claim 1 or 2, characterized in that, The isoxazoline compounds have the structure shown in Formula Ib: ； R1, R2, R3, R4, R5, R6, R a R b R c R d m and n are the same as those defined in claim 1.

4. The compound of formula I according to claim 1, wherein R1 is selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, or halogenated C1-C6 alkoxy.

5. The isoxazoline compound according to claim 1, wherein R1 is chlorine.

6. The isoxazoline compound according to claim 1, wherein R2 is selected from hydrogen, halogen, C1-C6 alkyl, or halogenated C1-C6 alkyl.

7. The isoxazoline compound according to claim 1, wherein R2 is trifluoromethyl.

8. The isoxazoline compound according to claim 1, wherein R3 is selected from hydrogen, halogen, C1-C6 alkyl, or halogenated C1-C6 alkyl.

9. The isoxazoline compound according to claim 1, wherein R3 is methyl.

10. The isoxazoline compound according to claim 1, wherein R4 is selected from hydrogen or C1-C6 alkyl.

11. The isoxazoline compound according to claim 1, wherein R5 is selected from any C1-C6 alkyl group substituted with R6 or a 3- to 6-membered saturated or unsaturated ring containing 0-3 heteroatoms, wherein the 3- to 6-membered saturated or unsaturated ring containing 0-3 heteroatoms may optionally be substituted with 1-3 R6 atoms.

12. The isoxazoline compound according to claim 1, wherein R5 is selected from... , , , , , , , , , , , , , , , , , , , , , , , or The wavy line represents the connection site of the functional group.

13. The isoxazoline compound according to claim 1, characterized in that, The isoxazoline compound is any one of the following compounds: , , , , , , , , , , , , , , , , , , , , , , , , or .

14. A tautomer, enantiomer, diastereomer, meso compound, racemic compound, or pharmaceutically acceptable salt of any of the isoxazoline compounds according to any one of claims 1-13.

15. A pharmaceutical composition, characterized in that, It comprises a therapeutically effective amount of an isoxazoline compound as described in any one of claims 1-13 or its tautomer, enantiomer, diastereomer, meso compound, racemic compound, or pharmaceutically acceptable salt as described in claim 14; and a pharmaceutically acceptable carrier or excipient.

16. The use of an isoxazoline compound as described in any one of claims 1-13, or its tautomer, enantiomer, diastereomer, meso compound, racemic compound, or pharmaceutically acceptable salt as described in claim 14, or the pharmaceutical composition as described in claim 15, in the control of ectoparasitic infections in animals.

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