Method for preparing intermediate of isoxazoline type drug

Abstract

Provided in the present application is a method for preparing an acid salt of a compound of formula (I), which is prepared by means of Boc anhydride protection, reaction with a carbene reagent, and successive deprotection reaction with an alkaline reagent and an acidic reagent. The method has a high yield and involves mild reaction conditions, and is thus suitable for industrial production. Further provided in the present application is the use of the compound of formula (I) as an intermediate in the synthesis of an isoxazoline type drug.

Description

Process for preparing isoxazoline drug intermediates

[0001] This application claims priority to the Chinese patent application No. 202411075234.2, filed on August 7, 2024, entitled “Process for preparing isoxazoline drug intermediates”, to the Chinese patent application No. 202511044343.2, filed on July 28, 2025, entitled “Process for preparing isoxazoline drug intermediates”, both of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0002] The present application belongs to the field of medicinal chemistry, and specifically relates to a process for preparing an isoxazoline drug intermediate and applications thereof. BACKGROUND

[0003] Isoxazoline compounds are a class of effective insecticides and acaricides, which can interfere with the transmembrane signal transmission of the nervous system by antagonizing the gamma-aminobutyric acid receptor and glutamate receptor-gated chloride ion channels, so that chloride ions cannot penetrate into the postsynaptic membrane, leading to disorder of the insect nervous system and then death. As an important class of antiparasitic drugs, isoxazoline compounds have attracted attention due to their high insecticidal activity, wide spectrum of antiparasitic activity, and good safety. In agriculture, non-agriculture, livestock and pet care, isoxazoline drugs are widely used for the prevention and treatment of various parasitic infections or infestations.

[0004] The present applicant has described a kind of isoxazoline compound in CN202410482503.0, which has good insecticidal and acaricidal activity, and is expected to be developed as a clinical drug, and its structure is shown as formula (LN-4):

[0005] Currently, the acid salt of formula (I) compound is generally used as an intermediate in the synthesis of the compound of formula (LN-4) and isoxazoline drugs with similar structures, and its structure is shown as follows:

[0006] The prior art has reported the synthesis method of the compound of formula (I), including the method for preparing the compound of formula (c) from the compound of formula (a) reported in the literature Large-Scale Cyclopropanation of Butyl Acrylate with Difluorocarbene and Classical Resolution of a Key Fluorinated Building Block (Org. Process Res. Dev., 2022, 26, 683-697); and the method for preparing the compound of formula I from the compound of formula (c) reported in the patent literature CN107889487 A. The synthetic route is as follows:

[0007] However, in the reaction for preparing the compound of formula (II) from the compound (c), the Curtius rearrangement reaction is used, which requires the use of the explosive azide compound-diphenyl phosphorazide (DPPA), and it is difficult to carry out safe large-scale production, and the yield of the synthetic route is low. Therefore, it is urgent to develop an efficient, economical and more environmentally safe preparation method of the intermediate compound of formula (I), and apply it to the synthesis of isoxazoline drugs. SUMMARY

[0008] The present application relates to a preparation method of an isoxazoline drug intermediate and application thereof.

[0009] The first aspect of the present application provides a preparation method of a compound of formula (I) or an acid salt of a stereoisomer thereof as an intermediate, comprising the following steps:

[0010] Step 1: removing the formyl group of the compound of formula (III) under a basic reagent to obtain the compound of formula (II);

[0011] Step 2: removing the Boc protecting group of the compound of formula (II) with an acidic reagent to obtain the compound of formula (I) or an acid salt of a stereoisomer thereof;

[0012] The acid in the acid salt is an inorganic acid or an organic acid.

[0013] In some embodiments, the acid in the acid salt is selected from hydrogen chloride, hydrogen bromide, sulfuric acid, trifluoroacetic acid, methanesulfonic acid, formic acid, p-toluenesulfonic acid; preferably, the acid in the acid salt is selected from hydrogen chloride.

[0014] In some embodiments, the basic reagent in step 1 is selected from one or more of any combination of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, triethylamine, diisopropylethylamine, N-methylmorpholine (NMM), pyridine, ethylenediamine (EDA), monoethanolamine (MEA), 4-N,N-dimethylaminopyridine (DMAP), 1,8-diazabicycloundec-7-ene (DBU), potassium tert-butoxide, sodium tert-butoxide; preferably, the basic reagent is selected from sodium hydroxide.

[0015] In some embodiments, the molar ratio of the compound of formula (III) to the basic reagent in step 1 is 1:1-1:10, preferably, the molar ratio of the compound of formula (III) to the basic reagent is 1:1.2-1:4.

[0016] In some embodiments, the acidic reagent in step 2 is selected from hydrogen chloride, hydrogen bromide, sulfuric acid, trifluoroacetic acid, methanesulfonic acid, formic acid, p-toluenesulfonic acid; preferably, the acidic reagent is selected from hydrogen chloride.

[0017] In some embodiments, the molar ratio of the compound of formula (II) to the acidic reagent in step 2 is 1:1-1:15, preferably, the molar ratio of the compound of formula (II) to the acidic reagent is 1:1.5-1:6.

[0018] In some embodiments, the method further comprises the following steps:

[0019] Step 01: the compound of formula (V) is subjected to a protection reaction by Boc anhydride to obtain a compound of formula (IV);

[0020] Step 02: the compound of formula (IV) is reacted with a carbene reagent to obtain a compound of formula (III), the carbene reagent is selected from one or more of any combination of (triphenylphosphoranylidene) difluoroacetate, ethyl difluorobromoacetate, fluorosulfonyl methyl difluoroacetate, trimethylsilyl 2-(fluorosulfonyl) difluoroacetate, bromodifluoromethyltrimethylsilane; preferably, the carbene reagent is selected from ethyl difluorobromoacetate.

[0021] The second aspect of the present application provides a method for preparing a compound of formula (III) or a stereoisomer thereof:

[0022] which is prepared from a compound of formula (V):

[0023] In some embodiments, the method for preparing a compound of formula (III) or a stereoisomer thereof from a compound of formula (V) comprises the following steps:

[0024] Step 01: protecting reaction of compound of formula (V) with Boc anhydride to obtain compound of formula (IV);

[0025] Step 02: reacting compound of formula (IV) with a carbene reagent to obtain compound of formula (III) or stereoisomer thereof.

[0026] In some embodiments, the carbene reagent of step 02 described above is selected from one or more of any combination of (triphenylphosphoranylidene) difluoroacetic acid inner salt, ethyl difluorobromoacetate, fluorosulfonyl methyl difluoroacetate, trimethylsilyl 2-(fluorosulfonyl) difluoroacetate, bromodifluoromethyltrimethylsilane; preferably, the carbene reagent is selected from ethyl difluorobromoacetate.

[0027] The third aspect of the present application provides a method for preparing an acid salt of compound of formula (I), comprising the following steps:

[0028] (a) taking compound of formula (V) as starting material, protecting reaction with Boc anhydride to obtain compound of formula (IV);

[0029] (b) reacting compound of formula (IV) with a carbene reagent to obtain compound of formula (III);

[0030] (c) removing the formyl group of compound of formula (III) under basic reagent to obtain compound of formula (II);

[0031] (d) removing the Boc protecting group of compound of formula (II) with an acidic reagent to obtain an acid salt of compound of formula (I).

[0032] The fourth aspect of the present application provides the use of an acid salt of compound of formula (I) or stereoisomer thereof as an intermediate for preparing an isoxazoline compound or a pharmaceutically acceptable salt thereof.

[0033] In some embodiments, the isoxazoline compound or a pharmaceutically acceptable salt thereof has a structure as shown in formula (LN):

[0034] wherein R1, R2, R3are independently selected from H, halogen or C1-C6 haloalkyl.

[0035] In some preferred embodiments, the R1, R2, R3are independently selected from H, F, Cl, CF3.

[0036] In some embodiments, the preparation of the isoxazoline compound or a pharmaceutically acceptable salt thereof comprises the following steps:

[0037] Step (a): reacting a compound of formula (I) or an acid salt of a stereoisomer thereof with compound 2 in the presence of an optional acid binding agent to obtain compound 3;

[0038] Step (b): reacting compound 3 in a basic solution to obtain compound 4;

[0039] Step (c): reacting compound 4 with compound 5 in the catalysis of a condensing reagent to obtain an isoxazoline compound of formula (LN) or a pharmaceutically acceptable salt thereof.

[0040] In some embodiments, in step (a), the compound of formula (I) or an acid salt of a stereoisomer thereof is a hydrochloride salt.

[0041] In some embodiments, in step (a), the compound of formula (I) or an acid salt of a stereoisomer thereof is an acid salt of a compound of formula (I), or an acid salt of a stereoisomer of a compound of formula (I); preferably a hydrochloride salt of a compound of formula (I), or a hydrochloride salt of a stereoisomer of a compound of formula (I).

[0042] In some embodiments, in step (a),

[0043] The acid binding agent is selected from one or more of any combination of sodium carbonate, potassium carbonate, cesium carbonate, triethylamine, diisopropylethylamine, or no acid binding agent is used; preferably, the acid binding agent is selected from triethylamine.

[0044] The molar ratio of the compound of formula (I) or an acid salt of a stereoisomer thereof to the compound 2 is 1:0.6 to 1:2, preferably the molar ratio of the compound of formula (I) or an acid salt of a stereoisomer thereof to the compound 2 is 1:0.9 to 1:1.2.

[0045] The solvent of the reaction is selected from one or more of any combination of N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dioxane, toluene, xylene, anisole, preferably the solvent is selected from toluene.

[0046] In some embodiments, in step (b),

[0047] The basic solution is selected from one or more of any combination of aqueous ammonia, hydrazine hydrate, methylhydrazine solution, methylamine solution; preferably the basic solution is selected from methylamine solution.

[0048] The solvent of the reaction further comprises an organic solvent selected from one or more of any combination of toluene, tetrahydrofuran, ethyl acetate, dichloromethane, methanol, ethanol, isopropanol, methyl tert-butyl ether, acetonitrile, preferably the organic solvent is selected from ethanol.

[0049] The molar ratio of the compound 3 to the basic reagent in the basic solution is 1:1.5-1:20; preferably, the molar ratio of the compound 3 to the basic reagent in the basic solution is 1:3-1:10.

[0050] In some embodiments, in step (c),

[0051] The condensing reagent is selected from one or more of any combination of 2-(7-azabenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-benzotriazolyl-tetramethyluronium hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexyl carbodiimide, diisopropyl carbodiimide, benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, benzotriazol-1-yl oxy-tris(dimethylamino) phosphonium hexafluorophosphate, carbonyldiimidazole; preferably, the condensing reagent is selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0052] The catalyst used in the reaction is selected from at least one of 4-dimethylaminopyridine, 1-hydroxybenzotriazole;

[0053] The solvent of the reaction is selected from one or more of any combination of toluene, tetrahydrofuran, ethyl acetate, dichloromethane, methyl tert-butyl ether, acetonitrile, preferably dichloromethane;

[0054] The molar ratio of the compound 4 to the compound 5 is 1:0.8-1:1.5; preferably, the molar ratio of the compound 4 to the compound 5 is 1:0.9-1:1.2.

[0055] In more preferred embodiments, the isoxazoline compound is selected from the following specific structures:

[0056] The fifth aspect of the present application provides a method for preparing an isoxazoline compound represented by formula (LN) or a pharmaceutically acceptable salt thereof from an acid salt of a compound of formula (I) or a stereoisomer thereof, comprising the following steps:

[0057] Step (a): reacting the compound of formula (I) or the acid salt of a stereoisomer thereof with compound 2 in the presence of an optional acid binding agent to obtain compound 3;

[0058] Step (b): reacting compound 3 in a basic solution to obtain compound 4;

[0059] Step (c): reacting compound 4 with compound 5 in the presence of a condensing reagent to obtain the isoxazoline compound represented by formula (LN) or a pharmaceutically acceptable salt thereof.

[0060] In some embodiments, the acid salt of the compound of formula (I) or its stereoisomer in step (a) is preferably a hydrochloride salt.

[0061] In some embodiments, the acid binding agent in step (a) is selected from one or more of any combination of sodium carbonate, potassium carbonate, cesium carbonate, triethylamine, diisopropylethylamine, or no acid binding agent is used; preferably, the acid binding agent is selected from triethylamine.

[0062] In some embodiments, the molar ratio of the acid salt of the compound of formula (I) or its stereoisomer to compound 2 in step (a) is 1 :0.6 to 1 :2; preferably, the molar ratio of the acid salt of the compound of formula (I) or its stereoisomer to compound 2 is 1 :0.9 to 1 :1.2.

[0063] In some embodiments, the reaction solvent in step (a) is selected from one or more of any combination of N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dioxane, toluene, xylene, anisole, preferably, the solvent is selected from toluene.

[0064] In some embodiments, the basic solution in step (b) is selected from one or more of any combination of aqueous ammonia, hydrazine hydrate, methylhydrazine solution, methylamine solution; preferably, the basic solution is selected from methylamine solution.

[0065] In some embodiments, the solvent for the reaction in step (b) further comprises an organic solvent selected from one or more of any combination of toluene, tetrahydrofuran, ethyl acetate, dichloromethane, methanol, ethanol, isopropanol, methyl tert-butyl ether, acetonitrile, preferably, the organic solvent is selected from ethanol.

[0066] In some embodiments, the molar ratio of compound 3 to the basic reagent in the basic solution in step (b) is 1 :1.5 to 1 :20; preferably, the molar ratio of compound 3 to the basic reagent in the basic solution is 1 :3 to 1 :10.

[0067] In some embodiments, in step (c), the condensing reagent is selected from one or more of any combination of 2-(7-azabenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazolyl-tetramethyluronium hexafluorophosphate (HBTU), l-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), benzotriazol-1-yl oxy-tris(dimethylamino) phosphonium hexafluorophosphate (BOP), carbonyldiimidazole (CDI); preferably, the condensing reagent is selected from l-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI); and the catalyst used in the reaction is selected from at least one of 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBt), and the like.

[0068] In some embodiments, in step (c), the solvent of the reaction is selected from one or more of any combination of toluene, tetrahydrofuran, ethyl acetate, dichloromethane, methyl tert-butyl ether, acetonitrile; preferably, the solvent is selected from dichloromethane.

[0069] In some embodiments, in step (c), the molar ratio of compound 4 to compound 5 is 1:0.8 to 1:1.5; preferably, the molar ratio of compound 4 to compound 5 is 1:0.9 to 1:1.2.

[0070] In some embodiments, the isoxazoline compound of formula (LN) is selected from the following specific structures:

[0071] The sixth aspect of the present application provides a compound of formula (III) or a stereoisomer thereof:

[0072] Explanation and Definitions

[0073] The following terms and phrases, as used herein, are intended to have the following meanings unless otherwise indicated. A particular term or phrase should not be construed as being indefinite or unclear in the absence of a specific definition, but should be understood according to its ordinary meaning.

[0074] The term "pharmaceutically acceptable" means those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0075] The term "pharmaceutically acceptable salt" refers to derivatives obtained from the compounds of this application prepared with relatively non-toxic acids or bases. These salts can be prepared during the synthesis, isolation, and purification of the compounds, or by reacting the purified free form of the compounds with suitable acids or bases. When the compounds contain relatively acidic functional groups, they react with alkali metal, alkaline earth metal hydroxides, or organic amines to yield base addition salts, including alkali metal and alkaline earth metal-based cations, as well as non-toxic ammonium, quaternary ammonium, and amine cations, and also encompassing amino acid salts. When the compounds contain relatively basic functional groups, they react with organic or inorganic acids to yield acid addition salts.

[0076] The term "isomer" as used in this application includes geometric isomers and stereoisomers, such as blocked trans isomers, cis-trans isomers, enantiomers, diastereomers, tautomers, and their racemic mixtures and other mixtures, all of which are within the scope of this application. The term "enantiomer" refers to stereoisomers that are mirror images of each other. The term "tautomer" refers to a functional group isomer that has different hydrogen bonding sites through one or more double bond shifts; for example, a ketone and its enol form are keto-enol tautomers. The term "diastereomer" refers to stereoisomers of a molecule having two or more chiral centers and being non-mirror images of each other. The term "cis-trans isomer" refers to different spatial configurations of a molecule where double bonds or single bonds of cyclic carbon atoms cannot rotate freely. The term "blocked trans isomer" refers to a stereoisomer that can be separated due to impeded or very slow single bond rotation. The stereoisomers of the compounds in this application can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. For example, an enantiomer of a compound in this application can be prepared by asymmetric catalysis or chiral derivative derivatization. Alternatively, a single stereoisomer can be obtained from a mixture using chiral resolution techniques. Alternatively, it can be prepared directly from chiral starting materials. The separation of optically pure compounds in this application is typically accomplished using preparative chromatography, employing a chiral column to achieve the separation of chiral compounds.

[0077] The absolute stereoconfiguration of a compound can be confirmed using conventional techniques in the art. For example, single-crystal X-ray diffraction can be used, or the absolute configuration of the compound can be confirmed by examining the chiral structure of the starting material and the reaction mechanism of asymmetric synthesis. Alternatively, after resolution, the stereoconfiguration can be determined by comparing it with a product whose absolute configuration is known. Compounds marked "Absolute configuration unknown / undetermined" in this article are typically racemic compounds resolved into single isomers by chiral preparative supercritical fluid chromatography (SFC), followed by characterization and testing.

[0078] When any variable (e.g., Rd) appears more than once in the composition or structure of a compound, its definition is independent in each case. For example, Rdrepresents cyclopentyl substituted with 3 Rd, and each Rdhas independent options.

[0079] The term "optionally present" means both "present" or "absent". For example, the term "reacting in the presence of an optional acid binding agent" means that the reaction can be carried out in the presence of an acid binding agent or without using an acid binding agent.

[0080] The term "alkyl" means straight chain or branched chain saturated hydrocarbon groups. Preferred alkyl groups are C 1-6 alkyl groups, more preferred C 1-4 alkyl groups. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, and the like. The term "haloalkyl" means an alkyl group in which one or more hydrogens are replaced by halogen atoms.

[0081] The term "halogen" means a fluorine, chlorine, bromine, or iodine atom.

[0082] The term "haloalkyl" means an alkyl group in which one or more hydrogens are replaced by halogen atoms. Preferred haloalkyl groups are haloC 1-6 alkyl groups, more preferred haloC 1-4 alkyl groups. Examples of haloalkyl groups include, but are not limited to, monofluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, tribromomethyl, 2,2,2-trifluoroethyl, 2,2,2-trichloroethyl, and the like. Alkyl groups are as defined above.

[0083] In the present application, the term "amide reaction" refers to the use of a condensing agent and an organic base. The condensing agent includes, but is not limited to, carbodiimide-based condensing agents, carbonium salt-based condensing agents, phosphonium salt-based condensing agents, imidazole derivative-based condensing agents, and the like.

[0084] The carbodiimide-based condensing agent is selected from the group consisting of dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and the like.

[0085] The carbonium salt-based condensing agent includes, but is not limited to, 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazol-tetramethyluronium hexafluorophosphate (HBTU), and the like.

[0086] The phosphonium salt-based condensing agent includes, but is not limited to, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP).

[0087] The imidazole derivative-based condensing agent includes, but is not limited to, carbonyldiimidazole, and the like.

[0088] The amide condensation generally requires the addition of a catalyst or activator selected from 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBt), and the like.

[0089] In the present application, the Boc protecting group is the abbreviation of t-Butyloxycarbonyl, which is a commonly used amino protecting group in organic synthesis.

[0090] In the present application, the amino protecting group includes but is not limited to benzyloxycarbonyl (Cbz), t-butyloxycarbonyl (Boc), formyloxycarbonyl (Fmoc), p-methoxybenzyl (PMB), benzyl (Bn), trityl (Trt), p-toluenesulfonyl (Tos), phthaloyl (Pht), and the like.

[0091] In the present application, deprotection refers to the removal of the amino protecting group, and the deprotection reagent includes but is not limited to Pd-C / H2, PdCl2, Na / ammonia, trifluoroacetic acid (TFA), trifluoroacetic acid / dichloromethane, HCl / 1,4-dioxane, HCl / methanol, HCl / ethanol, HCl / ethyl acetate, concentrated hydrochloric acid, sulfuric acid, hydrobromic acid, hydrazine hydrate, ammonia, methylhydrazine solution, aqueous methylamine, methylamine organic reagent solution, and the like.

[0092] In the present application, the acid-binding agent includes an organic base or an inorganic base, wherein the inorganic base includes but is not limited to potassium carbonate, sodium carbonate, potassium hydroxide, sodium hydroxide, lithium hydroxide, and the like.

[0093] In the present application, the organic base includes but is not limited to triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), N-methylmorpholine (NMM), pyridine (Py), ethylenediamine (EDA), monoethanolamine (MEA), 4-N,N-dimethylaminopyridine (DMAP), 1,8-diazabicycloundec-7-ene (DBU), potassium tert-butoxide, sodium tert-butoxide, and the like.

[0094] In the present application, the "compound (LN-4)", "compound of formula (LN-4)", or "drug of formula (LN-4)" all refer to the compound of the following structure.

[0095] The present application provides a preparation method and application of an isoxazoline drug intermediate. The preparation method of the compound of formula (I) or the acid salt of the stereoisomer thereof as an intermediate has a higher yield, mild reaction conditions, and avoids the use of explosive azide compounds, and is a high-efficiency, economical and more environmentally friendly method, which is suitable for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0096] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

[0097] Figure 1 is the mean plasma concentration-time profile of compound (LN-4). DETAILED DESCRIPTION

[0098] To make the objects, technical solutions, and advantages of the present application clearer, the following further describes the present application with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the present application are within the scope of protection of the present application.

[0099] The present application is described in detail below through embodiments, but does not mean any unfavorable limitation on the present application. The present application has been described in detail herein, and the specific implementation manners thereof are also disclosed. It will be obvious to those skilled in the art to make various changes and improvements to the specific implementation manners of the present application without departing from the spirit and scope of the present application.

[0100] Summary of experimental instruments:

[0101] The structure of the compound of the present application is determined by nuclear magnetic resonance (NMR) or / and liquid chromatography-mass spectrometry (LC-MS). The NMR chemical shift (δ) is given in units of parts per million (ppm). The NMR determination is performed using a Bruker Neo 400M or a Bruker Ascend 400 nuclear magnetic instrument, and the determination solvent is deuterated dimethyl sulfoxide (DMSO-d6), heavy water (D2O), and the internal standard is tetramethylsilane (TMS).

[0102] The determination of liquid chromatography-mass spectrometry (LC-MS) is performed using an Agilent 1260-6125B single quadrupole mass spectrometer, a Welch Biomate column (C18, 2.7 μm, 4.6 x 50 mm), or a waters H-Class SQD2, a Welch Ultimate column (XB-C18, 1.8 μm, 2.1 x 50 mm), and the ion source of the mass spectrometer is electrospray ionization.

[0103] The determination of high performance liquid chromatography (HPLC) is performed using a Waters e2695-2998 or a Waters ARC and an Agilent 1260 or an Agilent Poroshell HPH high performance liquid chromatograph.

[0104] The starting materials in the embodiments of the present application are known and commercially available, or can be synthesized by using or according to the methods known in the art.

[0105] Unless otherwise specified, all reactions in the present application are carried out under continuous magnetic stirring, the solvent is dry solvent, and the reaction temperature unit is Celsius or °C. Unless otherwise specified, room temperature refers to 25±5°C.

[0106] Example 1 Preparation of hydrochloride salt of compound of formula (I)

[0107] Reaction scheme:

[0108] Operation steps:

[0109] Step 1: Compound V 100 g (1.407 mol), tetrahydrofuran (THF) 1000 mL, DMAP 1.72 g (0.014 mol) were sequentially added to a reaction bottle under 20-35°C, and stirred and dissolved; di-tert-butyl dicarbonate (Boc2O) 368.5 g (1.688 mol) was slowly added dropwise to the reaction system, and incubated at 20-35°C for 12 h with stirring; the reaction liquid was evaporated to dryness, 1000 mL of methyl tert-butyl ether and 1000 mL of 5 wt% aqueous citric acid solution were added, stirred for 15-25 min, and separated; the organic phase was washed twice with 600 mL of 5 wt% aqueous citric acid solution (×2) for 2 times, and washed once with 600 mL of saturated NaHCO3 aqueous solution, and the organic phase was dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure to obtain compound IV 237.2 g with a purity of 96.7% (determined by gas chromatography (GC)) and a yield of 98.5%.

[0110] 1 H NMR (400 MHz, DMSO-d6): δ 9.16 (s, 1H), 6.60-6.53 (m, 1H), 5.59-5.55 (d, 1H), 5.05-5.03 (m, 1H), 1.52-1.42 (m, 9H).

[0111] Mass spectrum (GC-MS) m / z: 71.1 [M-Boc].

[0112] Step 2: 2000 mL of toluene was measured in a reaction bottle at 20-35 °C and heated to 110 °C; 200 g of compound IV (1.168 mol), 830.0 g of ethyl difluorobromoacetate (4.089 mol) and 3.77 g of tetrabutylammonium bromide (0.012 mol) were weighed in another reaction bottle and mixed well; the above mixture was slowly added to the toluene solution at 110 °C, and the dropwise time was 1.5-2.5 h; after dropping, the reaction was continued for 2 h; 237.1 g of ethyl difluorobromoacetate (1.168 mol) and 3.77 g of tetrabutylammonium bromide (0.012 mol) were added, and the dropwise time was 0.5-1 h; after dropping, the reaction was continued for 2-4 h; the system was washed with 2000 mL of saturated NaCl aqueous solution for 3 times, and dried with anhydrous Na2SO4; filtered and evaporated to obtain 241.4 g of compound III with a purity of 94.5% (GC) and a yield of 93.4%.

[0113] 1 H NMR (400 MHz, DMSO-d6): δ 9.04 (s, 1H), 3.30-3.20 (m, 1H), 2.24-2.14 (m, 1H), 1.74-1.65 (m, 1H), 1.50-1.42 (m, 9H).

[0114] Mass (GC-MS) m / z: 121.0 [M-Boc].

[0115] Step 3: 221.2 g of compound III (1 mol) was dissolved in 2212 mL of tetrahydrofuran at 20-35 °C, and 22.1 g of solid NaCl was added, and the temperature was reduced to -10 °C to -5 °C by stirring; 6400 g of 1 wt% NaOH (64 g, 1.6 mol) saturated NaCl aqueous solution was prepared and slowly added to the above system, and the dropwise time was 1.5-2.5 h; after dropping, the reaction was continued at -10 °C to -5 °C for 2 h; the reaction system was separated, and the aqueous phase was back extracted with 1000 mL of tetrahydrofuran for 2 times; the combined organic phase was dried with anhydrous Na2SO4, filtered, and evaporated under reduced pressure to obtain 183.9 g of compound II with a purity of 95.2% (GC) and a yield of 95.2%.

[0116] 1 H NMR (400 MHz, DMSO-d6): δ 7.38 (s, 1H), 3.08 (s, 1H), 1.82-1.72 (m, 1H), 1.55-1.44 (m, 10H).

[0117] Mass (GC-MS) m / z: 93.1 [M-Boc].

[0118] Step 4: Compound II 100 g (0.518 mol) was dissolved in ethyl acetate 200 mL at 20-35 °C, and the solution was stirred while cooling to -5-0 °C. A solution of HCl / ethyl acetate 1554 mL (HCl 2 mol / L (M), 3.108 mol) was added dropwise to the reaction system. After the dropwise addition was completed, the temperature was raised to 20-35 °C and the reaction was allowed to proceed for 3-6 h. To the reaction system was added n-heptane 200 mL, and the stirring was continued for 0.5-1 h. The mixture was filtered, the filter cake was washed with n-heptane, and the product was dried under reduced pressure to give compound I hydrochloride 52.5 g in a yield of 78.2%.

[0119] 1 H NMR (400 MHz, D2O): δ 3.35-3.28 (m, 1H), 2.03-1.93 (m, 1H), 1.83-1.82 (d, 1H).

[0120] Mass (MS) m / z: 94.1 [M+H] + .

[0121] Example 2: Preparation of compound I hydrochloride

[0122] Reaction scheme: same as Example 1.

[0123] Operation steps:

[0124] Step 1: Compound V 50 g (0.703 mol), toluene 500 mL, DMAP 0.86 g (0.007 mol) were sequentially added to a reaction bottle, and the mixture was stirred to dissolve. The temperature was controlled at 0-10 °C, and Boc2O 161.1 g (0.738 mol) was slowly added dropwise to the reaction system. After the dropwise addition was completed, the temperature was maintained at 0-10 °C, and the stirring was continued for 6-8 h. The organic phase was washed twice with 5 wt% aqueous citric acid 250 mL, dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure to give compound IV 120.4 g with a purity of 95.5% (GC) and a yield of 100%.

[0125] Step 2: 1000 mL of toluene was measured in a reaction bottle at 20-35 °C and heated to 110 °C; 100 g of compound IV (0.584 mol), 237.1 g of ethyl difluorobromoacetate (1.168 mol) and 18.83 g of tetrabutylammonium bromide (0.058 mol) were weighed in another reaction bottle and mixed well; the above mixture was slowly added to the toluene solution at 110 °C, and the dropwise time was 1.5-2.5 h; after dropping, the reaction was continued for 4 h; 59.3 g of ethyl difluorobromoacetate (0.292 mol) and 1.88 g of tetrabutylammonium bromide (0.006 mol) were added, and the dropwise time was 0.5-1 h; after dropping, the reaction was continued for 2-4 h to obtain compound III reaction liquid, the purity of compound III was 93.5% (GC) and the yield was 100%.

[0126] Step 3: The compound III reaction liquid obtained in step 2 was cooled to 0-10 °C; 1168 g of 4 wt% NaOH (46.72 g, 1.168 mol) aqueous solution was slowly added to the above system, and the temperature was kept at 0-5 °C; after dropping, the temperature was kept at 0-10 °C for 1.5-3 h; the reaction system was separated, and the organic phase was dried with anhydrous sodium sulfate; filtration was performed, and the filter cake was washed with a small amount of toluene to obtain compound II reaction liquid, the purity of compound II was 93.6% (GC) and the yield was 100%.

[0127] Step 4: The compound II reaction liquid obtained in step 3 was cooled to -5 °C-0 °C and stirred; 1752 mL of HCl / ethyl acetate (HCl 3.504 mol) solution was added dropwise to the reaction system; after dropping, the temperature was raised to 20-35 °C and reacted for 6-7 h; 226 mL of n-heptane was added to the reaction system, and the stirring was continued for 0.5-1 h to precipitate a large amount of solid; filtration was performed, and the filter cake was washed with n-heptane and dried under reduced pressure to obtain 58.6 g of hydrochloride of compound I, with a yield of 77.5%.

[0128] Comparative Example 1:

[0129] According to the method reported in document CN107889487A, the hydrochloride of compound I was prepared:

[0130] Step 1: Synthesis of tert-butyl N-(2,2-difluorocyclopropyl)carbamate

[0131] To a stirred solution of 2,2-difluorocyclopropane-l-carboxylic acid 8.56 g (70.12 mmol) in t-butyl alcohol (t-BuOH, 100 mL) was added TEA 11.83 mL (84.15 mmol) followed by diphenyl phosphorazide 18.09 mL (84.15 mmol) at 20-30 °C. The reaction was then heated at 90 °C for 18 h. The reaction was monitored by thin layer chromatography (eluent: n-heptane, 10% ethyl acetate (EtOAc), staining: KMnO4). The solvent was removed under reduced pressure and the obtained crude residue was purified by column chromatography (100 g silica gel, eluent: n-heptane, 0-10% EtOAc) to give the target product as a white solid 7.5 g, 97.3% purity (GC), 55.6% yield.

[0132] Step 2: Synthesis of 2,2-difluorocyclopropan-l -amine hydrochloride

[0133] To a stirred solution of tert-butyl N-(2,2-difluorocyclopropyl)carbamate 7.2 g (37.27 mmol) in 1,4-dioxane (5 mL) was added 4 M HCI in 1,4-dioxane 37.27 mL. The reaction was stirred at room temperature for 3 h. The reaction progress was monitored by thin layer chromatography (eluent: n-heptane, 10% EtOAc, staining: KMnO4). The reaction was filtered and the filter cake was washed with 1,4-dioxane and dried under reduced pressure to give the target product as a white solid 2.8 g, 57.9% yield.

[0134] Example 3: Preparation of compound of formula (I) trifluoroacetate salt

[0135] Compound II 10 g (0.052 mol) was dissolved in dichloromethane 60 mL, and the solution was stirred at 20-35 °C and cooled to -5-0 °C; trifluoroacetic acid 35.6 g (0.312 mol) was added dropwise to the reaction system; after the dropwise addition was completed, the temperature was raised to 20-35 °C and the reaction was carried out for 4-5 h; the reaction mixture was filtered, the filter cake was washed with dichloromethane, and the product was dried under reduced pressure to give compound I trifluoroacetate salt 7.82 g, with a yield of 72.6%.

[0136] Example 4: Synthesis of 4-[5-(3,5-dichloro-4-fluorophenyl)-4,5-dihydro-5- (trifluoromethyl)-3-isoxazolyl]-N-(2-((2,2-difluorocyclopropyl)amino)2-oxoethyl) methylbenzamide (Formula LN-4)

[0137] Reaction Scheme:

[0138] Procedure:

[0139] Step 1: 2,2-difluorocyclopropyl-1-amine hydrochloride 10 g (0.077 mol), compound 2: phthaloylglycine chloride 18.1 g (0.081 mol) were added into toluene (200 mL) at 20-35 °C; the temperature was raised to 108-115 °C and stirred for 2-4 h; the temperature was lowered to room temperature, a large amount of white solid was precipitated; the filter cake was slurried with 5 wt% saturated NaHC03 aqueous solution 100 mL for 0.5-1 h; the filter cake was filtered and dried under reduced pressure to give compound 3: 20.1 g, purity 98.0%, yield 93.4%.

[0140] 1 H NMR (400 MHz, DMSO-d6): δ 8.70 (s, 1H), 7.94-7.86 (m, 4H), 4.28-4.20 (m, 2H), 3.32-3.28 (d, 1H), 1.95-1.85 (m, 1H), 1.52-1.45 (m, 1H).

[0141] Mass (MS) m / z: 281.1 [M+H] + .

[0142] Step 2: 20-35 °C, compound 3: 20 g (0.071 mol) was dissolved in 30 wt% methylamine ethanol solution (73.9 g, 0.713 mol); the reaction was stirred at room temperature for 2-4 h; then, the residual methylamine was distilled off under reduced pressure; THF 200 mL was added, and the mixture was slurried for 1 h, filtered, and the filter cake was washed with a small amount of THF; hydrogen chloride ethyl acetate solution (HCl, 0.143 mol) was added to salt out the product for 1-3 h, filtered, and dried under reduced pressure to give compound 4: 11.3 g, purity 97.08%, yield 85.2%.

[0143] 1 H NMR (400 MHz, DMSO-d6): δ 8.30 (s, 3H), 3.62-3.54 (m, 2H), 3.39-3.32 (m, 1H), 1.98-1.88 (m, 1H), 1.57 (s, 1H).

[0144] Mass (MS) m / z: 151.1 [M+H] + .

[0145] Step 3: Compound 5: 23.2 g (0.053 mol), compound 4: 10 g (0.053 mol), 1-hydroxybenzotriazole (HOBT) 2.9 g (0.021 mol), ethyl acetate 200 mL, triethylamine 2.2 g (0.022 mol) were added into the reaction bottle in turn at 20-30 °C, stirred uniformly, then EDCI was added in five batches (2.44 g each time, 0.013 mol), added once every 5 min, and the reaction was carried out at 20-30 °C for 3 h. The sample was detected until the reaction was completed. 100 mL of purified water was added, and the pH was adjusted to 4.0-4.5 with 3M hydrochloric acid solution. After standing and separating, 100 mL of purified water was added to the organic phase, and the pH was adjusted to 8.0-9.0 with 30 wt% sodium hydroxide solution. After standing and separating, the organic phase was washed with 100 mL of purified water for 15 min, and then separated. The organic phase was concentrated to 1 / 5 to 1 / 4 of the original volume under vacuum, filtered at 55-60 °C, then 90 mL of n-heptane was slowly added dropwise, and the crystals were aged at room temperature for 1 h, filtered, and dried under reduced pressure to obtain the product compound 27.2 g of formula (LN-4), with a purity of 99.8% and a yield of 90.2%.

[0146] Mass (MS) m / z: 568.0 [M+H] + .

[0147] 1 H NMR (DMSO-d6, 400 MHz) δ 8.57 (t, J = 6.0 Hz, 1H), 8.43 (s, 1H), 7.81 (d, J = 6.4 Hz, 2H), 7.60 (d, J = 6.0 Hz, 2H), 7.49 (d, J = 8.4 Hz, 1H), 4.41-4.29 (m, 2H), 3.94-3.82 (m, 2H), 3.34-3.29 (s, 1H), 2.41 (s, 3H), 1.95-1.85 (m, 1H), 1.53-1.44 (m, 1H).

[0148] II. Biological activity experiment

[0149] Test Example 1: Inhibition test of in vitro mite contact killing activity at different concentrations

[0150] 1. Purpose of the test:

[0151] The contact killing effect and effective concentration of the isoxazoline drug (compound of formula (LN-4)) prepared from the compound of formula I on chicken skin mites were evaluated by the filter paper drug film method.

[0152] 2. Test materials:

[0153] 2.1 Test drug: First, mix propylene glycol and Tween 80 at 1:3 (w / w) at 40°C until uniform, then add a certain amount of the compound of formula (LN-4) to prepare a 1% (w / v) test drug solution. Before use, dilute with water to the target concentration.

[0154] 2.2 Test material: Neutral filter paper (size: 7 x 10 cm). Chicken mite is derived from clinical and cultured to physiological state.

[0155] 2.3 Instrumentation: PRX700 series intelligent artificial climate chamber.

[0156] 3. Test method:

[0157] Dilute the test drug solution with purified water to 5 μg / mL, 10 μg / mL and 20 μg / mL, respectively, soak the neutral filter paper, naturally air dry, fold and bind to prepare filter paper bags, and prepare three for each concentration of drug. Select 20 chicken mites with a hook pen into each filter paper bag, seal, and place in an artificial climate chamber for 48 h. Observe the death of chicken mites in filter paper bags at different concentrations and count the mortality rate.

[0158] 4. Test results:

[0159] Table 1 Statistics of chicken mite death at different concentrations

[0160] 5. Test conclusion:

[0161] As shown in Table 1, the compound of formula (LN-4) prepared from the compound of formula I has good contact killing effect on chicken mites at different concentrations, and shows dose-dependent effect, with the highest killing activity at 20 μg / mL.

[0162] Test Example 2: Chicken mite killing test

[0163] 1. Purpose of the test:

[0164] To evaluate the treatment effect of the isoxazoline drug (compound of formula (LN-4)) prepared from the compound of formula I on chicken mites of laying hens at different doses.

[0165] 2. Test materials:

[0166] 2.1 Test drug: Compound of formula (LN-4), dosage: 0.5 mg / kg.bw or 0.3 mg / kg.bw.

[0167] 2.2 Test animals: Laying hens. Each group of 200 laying hens. (Laying hens and eggs are managed and recovered uniformly)

[0168] 3. Test method:

[0169] a) Preparation before test: One week before the test, the mite collectors were evenly arranged in the chicken house, and the number of mite collectors in each group of drugs was greater than or equal to five.

[0170] b) Start the test: Prepare the drug solution before the test, (1) Blank solvent: First, stir the propylene glycol and polysorbate 80 at a ratio of 1:3 (V / V) at 40°C to make a blank solvent for standby. (2) Drug solution: Weigh the raw material compound of formula (LN-4), add the prescribed amount of the above-mentioned blank solvent to make a 1% (m / v) solution, and stir it evenly at 40°C.

[0171] c) According to the number of test animals, calculate the volume of each test drug, mix it evenly with a small amount of drinking water, and then add it to an appropriate amount of drinking water, so that it is consumed within 3-5 hours. The same method is used to administer it once a week.

[0172] d) Data statistics: Count the number of chicken mites in each group of mite collectors before the first administration and before the second administration. Observe the number of chicken mites in each group of mite collectors once a week after the second administration.

[0173] 4. Test results:

[0174] The number of chicken mites in each group of mite collectors is shown in Table 2.

[0175] Table 2: Chicken mite statistics in the farm

[0176] 5. Test conclusion:

[0177] As shown in Table 2, the compound of formula (LN-4) prepared from the compound of formula I of the present application has excellent killing effect on chicken mites.

[0178] Test Example 3: Killing test of dog body surface fleas

[0179] 1. Purpose of the test:

[0180] To evaluate the effect of the isoxazoline drug (compound of formula (LN-4)) prepared from the compound of formula I of the present application on treating dog fleas.

[0181] 2. Test materials:

[0182] 2.1 Test drug: Compound of formula (LN-4), test solution preparation: First, stir the propylene glycol and polysorbate 80 at a ratio of 1:3 (V / V) at 40°C to make a blank solvent for standby. Weigh the compound of formula (LN-4) and add the prescribed amount of the above-mentioned blank solvent to make a 1% (m / v) test solution, and stir it evenly at 40°C.

[0183] 2.2 Dose: 2 mg / kg.

[0184] 2.3 Test animals: 6 dogs naturally infected with fleas, each dog using a comb cleaner to count fleas, all more than 10.

[0185] 3. Test method:

[0186] The test dogs were weighed, and according to the body weight, the appropriate amount of test product solution was orally fed to each dog according to the dosage, and the number of fleas on the surface of each test dog was counted using a comb cleaner at 3 days and 10 days after administration.

[0187] 4. Test results:

[0188] No fleas were found in each test dog at 3 days and 10 days after administration.

[0189] 5. Test conclusion:

[0190] The compound of formula (LN-4) prepared from the compound of formula I of the present application has excellent killing effect on fleas.

[0191] Test Example 4: Pharmacokinetic test on chickens

[0192] 1. Purpose of the test

[0193] The isoxazoline drug (compound of formula (LN-4)) prepared from the compound of formula I of the present application was determined to determine the change of blood concentration with time in the chicken body.

[0194] 2. Test materials

[0195] 2.1 Test animals: 3 healthy laying hens.

[0196] 2.2 Test drug: compound of formula (LN-4), preparation of test drug solution: first stir propylene glycol and polysorbate 80 at 1:3 (V / V) at 40℃ to make a blank solvent for standby, weigh the compound of formula (LN-4), add the prescribed amount of the above blank solvent to prepare a 1% (m / v) test drug solution, and stir evenly at 40℃.

[0197] 2.3 Instruments and equipment: Shimadzu high performance liquid chromatograph, AB SCIEX QTRAP 4500 mass spectrometer.

[0198] 3. Test method

[0199] The test drug solution was orally administered at a dose of 0.5 mg / kg. The chickens were laterally restrained and blood was collected from the wing vein. Blood samples of 1.5 mL were collected before administration and at 0.5, 1, 2, 4, 8, 24, 48, 72, 96, 120, 144, 168, 240, 336, 408 and 504 h after administration, and were placed in centrifuge tubes containing sodium heparin. The upper plasma was obtained by centrifugation at 4000 r / min for 10 min, and was stored at -20°C. After the plasma sample was naturally thawed, 0.2 mL of the plasma was accurately taken and placed in a 10 mL centrifuge tube, 0.8 mL of acetonitrile was added, and the mixture was vortexed for 5 min, centrifuged at 10000 r / min for 10 min, and the supernatant was passed through a 0.22 μm organic filter, and was detected by HPLC-MS / MS.

[0200] After oral administration, the blood concentration of the isoxazoline drug prepared in the present application in the chicken body changed with time, as shown in Table 3 and Figure 1.

[0201] 4. Test results

[0202] The blood concentration detection results of the compound (LN-4) are shown in Table 3 and Figure 1.

[0203] Table 3 Blood concentration change of compound (LN-4) in chicken body (ng / mL)

[0204] Note: "ND" means that the drug was not detected.

[0205] 5. Test conclusion:

[0206] The compound (LN-4) prepared from the compound of formula I has the advantages of rapid oral absorption, fast onset, long half-life, small dosage and longer duration.

[0207] The above only describes the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. Process for the preparation of a compound of formula (I) or of an acid salt of a stereoisomer thereof, characterized in that, comprising the steps of: Step 1: removal of the formyl group of the compound of formula (III) under a basic reagent to give the compound of formula (II); Step 2: deprotection of the Boc group of the compound of formula (II) with an acidic reagent to give the compound of formula (I) or an acid salt of a stereoisomer thereof; the acid in the acid salt is an inorganic acid or an organic acid.

2. The method of claim 1, wherein, the acid in the acid salt is selected from hydrogen chloride, hydrogen bromide, sulfuric acid, trifluoroacetic acid, methanesulfonic acid, formic acid, p-toluenesulfonic acid; preferably, the acid in the acid salt is selected from hydrogen chloride.

3. The method according to claim 1 or 2, characterized in that, the basic reagent in step 1 is selected from one or more of any combination of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine, ethylenediamine, monoethanolamine, 4-N,N-dimethylaminopyridine, 1,8-diazabicycloundec-7-ene, potassium tert-butoxide, sodium tert-butoxide; preferably, the basic reagent is selected from sodium hydroxide.

4. The method according to claim 1 or 2, characterized in that, the acidic reagent in step 2 is selected from hydrogen chloride, hydrogen bromide, sulfuric acid, trifluoroacetic acid, methanesulfonic acid, formic acid, p-toluenesulfonic acid; preferably, the acidic reagent is selected from hydrogen chloride.

5. The method according to any one of claims 1 to 4, characterized in that, the molar ratio of the compound of formula (III) to the basic reagent in step 1 is 1:1 to 1:10; preferably, the molar ratio of the compound of formula (III) to the basic reagent is 1:1.2 to 1:

4.

6. The method according to any one of claims 1 to 5, characterized in that, the molar ratio of the compound of formula (II) to the acidic reagent in step 2 is 1:1 to 1:15; preferably, the molar ratio of the compound of formula (II) to the acidic reagent is 1:1.5 to 1:

6.

7. The method according to any one of claims 1 to 6, characterized in that, The method further comprises the steps of: Step 01: the compound of formula (V) is subjected to a Boc anhydride protection reaction to obtain a compound of formula (IV); Step 02: the compound of formula (IV) is reacted with a carbene reagent to obtain a compound of formula (III), the carbene reagent is selected from one or more of any combination of (triphenylphosphoranylidene) difluoroacetate, ethyl difluorobromoacetate, fluorosulfonyl methyl difluoroacetate, trimethylsilyl 2-(fluorosulfonyl) difluoroacetate, bromodifluoromethyltrimethylsilane; preferably, the carbene reagent is selected from ethyl difluorobromoacetate.

8. Process for the preparation of a compound of formula (III) or a stereoisomer thereof, characterized in that, The compound of formula (III) or a stereoisomer thereof is prepared from a compound of formula (V):

9. The method of claim 8, wherein, The method comprises the following steps: Step 01: the compound of formula (V) is subjected to a Boc anhydride protection reaction to obtain a compound of formula (IV); Step 02: the compound of formula (IV) is reacted with a carbene reagent to obtain a compound of formula (III) or a stereoisomer thereof, the carbene reagent is selected from one or more of any combination of (triphenylphosphoranylidene) difluoroacetate, ethyl difluorobromoacetate, fluorosulfonyl methyl difluoroacetate, trimethylsilyl 2-(fluorosulfonyl) difluoroacetate, bromodifluoromethyltrimethylsilane; preferably, the carbene reagent is selected from ethyl difluorobromoacetate.

10. Use of a compound of formula (I) or an acid salt of a stereoisomer thereof, prepared according to the process of any one of claims 1 to 7, as an intermediate in the preparation of an isoxazoline compound of formula (LN) or a pharmaceutically acceptable salt thereof: wherein R1, R2, R3are each independently selected from H, halogen or C1-C6haloalkyl; preferably, R1, R2, R3are each independently selected from H, F, Cl, CF3; The preparation of the isoxazoline compound or the pharmaceutically acceptable salt thereof comprises the following steps: Step (a): the acid salt of the compound of formula (I) or a stereoisomer thereof is reacted with compound 2 in the presence of an optional acid binding agent to obtain compound 3; preferably, the acid salt of the compound of formula (I) or a stereoisomer thereof is a hydrochloride salt; Step (b): compound 3 is reacted in a basic solution to obtain compound 4; Step (c): compound 4 is reacted with compound 5 in the catalysis of a condensation reagent to obtain an isoxazoline compound represented by formula (LN) or a pharmaceutically acceptable salt thereof.

11. Use according to claim 10, characterized in that, in step (a), The acid-binding agent is selected from one or more of any combination of sodium carbonate, potassium carbonate, cesium carbonate, triethylamine, diisopropylethylamine, or no acid-binding agent is used; preferably, the acid-binding agent is selected from triethylamine; The molar ratio of the compound of formula (I) or its stereoisomer of acid salt to the compound 2 is 1:0.6-1:2, preferably, the molar ratio of the compound of formula (I) or its stereoisomer of acid salt to the compound 2 is 1:0.9-1:1.2; The solvent of the reaction is selected from one or more of any combination of N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dioxane, toluene, xylene, anisole, preferably, the solvent is selected from toluene.

12. Use according to claim 10, characterized in that, In step (b), The basic solution is selected from one or more of any combination of aqueous ammonia, hydrazine hydrate, methylhydrazine solution, methylamine solution; preferably, the basic solution is selected from methylamine solution; The solvent of the reaction further comprises an organic solvent, the organic solvent is selected from one or more of any combination of toluene, tetrahydrofuran, ethyl acetate, dichloromethane, methanol, ethanol, isopropanol, methyl tert-butyl ether, acetonitrile, preferably, the organic solvent is selected from ethanol; The molar ratio of the compound 3 to the basic reagent in the basic solution is 1:1.5-1:20; preferably, the molar ratio of the compound 3 to the basic reagent in the basic solution is 1:3-1:

10.

13. Use according to claim 10, characterized in that, In step (c), The condensation reagent is selected from one or more of any combination of 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-benzotriazol-tetramethyluronium hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, dicyclohexyl carbodiimide, diisopropyl carbodiimide, benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, benzotriazol-1-yl oxy-tris(dimethylamine) phosphonium hexafluorophosphate, carbonyldiimidazole; preferably, the condensation reagent is selected from 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride; The catalyst used in the reaction is selected from at least one of 4-dimethylaminopyridine, 1-hydroxybenzotriazole; The solvent of the reaction is selected from one or more of any combination of toluene, tetrahydrofuran, ethyl acetate, dichloromethane, methyl tert-butyl ether, acetonitrile, preferably dichloromethane; The molar ratio of the compound 4 to the compound 5 is 1:0.8-1:1.5; preferably, the molar ratio of the compound 4 to the compound 5 is 1:0.9-1:1.

2.

14. Use according to any one of claims 10 to 13, characterized in that, The isoxazoline compounds of formula (LN) are selected from the following specific structures:

15. A compound of formula (III) or a stereoisomer thereof: ###00006### (III)

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