Method for preparing diarylisoxazoline derivatives

Abstract

The present disclosure provides methods for making enantiomerically pure compounds of formula (1). [Formula 1] TIFF2024531064000028.tif42155

Description

Technical Field

[0001] Cross-Reference to Related Patent Applications This patent application claims priority from U.S. Provisional Patent Application No. 63 / 231,858, filed on August 11, 2021, and U.S. Provisional Patent Application No. 63 / 306,240, filed on February 3, 2022, the entire disclosures of each of which are incorporated herein by reference.

Background Art

[0002] The compound of formula (1) shown below: 5-[(5S)-4,5-dihydro-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-3-isoxazolyl]-3-methyl-N-[2-oxo-2-[(2-propyn-1-yl)amino]ethyl]-2-thiophenecarboxamide

Chemical Formula

[0003] The compound of formula (1) is further described in WO2016 / 077158, which is incorporated herein by reference, and belongs to a well-known class of isoxazoline derivatives having insecticidal and acaricidal activity, which can be used in the fields of agriculture, forestry, lawn, home, wood products, seedling crop protection, and veterinary medicine. For example, such isoxazolines are disclosed in WO2010 / 070068 and WO2013 / 079407, which are incorporated herein by reference.

[0004] The production of pure enantiomers is costly and time-consuming. A method for preparing rotilaner, another isoxazoline derivative, is described in WO2014 / 090918 (incorporated herein by reference), in which the (S)-enantiomer is the following carboxylic acid: [ka] It is prepared by splitting the diastereomer salt by crystallization, then repeating the racemization cycle, and then further splitting the diastereomer salt formation. The method of splitting and performing the racemization and splitting cycle is laborious and costly. It is advantageous to directly form the desired (S)-enantiomer. Direct formation of certain 5-aryl-5-trifluoromethyl-4,5-dihydro-isoxazole enantiomers is known in the art and includes those described in US2014 / 0206633;US2014 / 0350261;WO2013 / 116236;WO2014 / 081800;Angew, Chem.Int.Ed.2010,49,5762-7566;and WO2017 / 176948 (each of which is incorporated herein by reference).

[0005] The present invention provides a method for producing the compound of formula (1) using a quinalkaloid-directed asymmetric hydroxylamine / enone cascade reaction, thereby avoiding costly and laborious cycles of reconciliation, racemization, and further reconciliation. [Brief explanation of the drawing]

[0006] [Figure 1] The image shows superimposed chiral chromatograms of 3-methyl-N-[2-oxo-2-[(2-propyne-1-yl)amino]ethyl]-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxamide (bottom line), 5S-enantiomer reference sample (middle line), and 5R-enantiomer reference sample (top line). [Figure 2] This shows the HPLC purity of 3-methyl-N-[2-oxo-2-[(2-propyne-1-yl)amino]ethyl]-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxamide (top line) compared to the blank (bottom line). [Figure 3] This shows a comparison of 1H NMR between 3-methyl-N-[2-oxo-2-[(2-propyne-1-yl)amino]ethyl]-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxamide (lower line) and the reference sample (upper line). [Figure 4] The 1H NMR data for 3-methyl-N-[2-oxo-2-[(2-propyne-1-yl)amino]ethyl]-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxamide is shown. [Overview of the project]

[0007] In one embodiment, the present invention relates to an enantiomerically pure compound of formula (1). [ka] A method for preparing, (i) The compound of formula (2) is hydroxylamine [ka] (wherein X is selected from the group consisting of halogens and -C(O)OR4 (wherein R4 is C1-C4 alkyl), a suitable base, and the compound of formula (3) [ka] (In the formula, Y - is an anion, R1 is selected from the group consisting of hydrogen and methoxy. R2 is selected from the group consisting of ethyl and vinyl. R3 is selected from the group consisting of aryls substituted with 1 to 5 substituents optionally independently selected from the group consisting of nitro, halogen, amino, trifluoromethyl, C1-C4 alkyl, C1-C4 alkoxy, and benzyloxy, and heteroaryls substituted with 1 to 3 substituents optionally independently selected from the group consisting of halogen, trifluoromethyl, C1-C4 alkyl, and C1-C4 alkoxy. The step of reacting with to obtain the compound of formula (4), [ka] (ii) A step of converting X of the compound of formula (4) to a carboxylic acid to obtain the compound of formula (5), [ka] (iii) Optionally, the compound of formula (5) is C 1~5 Alcohol, C 2~5 Alkylcyanide, C 3~9 Alkyl ketone, C 2~8 Alkyl ethers, and C 2~8 A solvent selected from the group consisting of alkyl acetates, and optionally water and C 5~8 A step of crystallization using a poor solvent selected from the group consisting of hydrocarbons, Furthermore (iv) The step of coupling the compound of formula 5 with a suitable amine, The present invention relates to a method wherein the appropriate amine is either 2-amino-propargyl-acetamide, or an amine resulting from a sequential reaction in which carboxyl-protected glycine is subsequently deprotected, if necessary, and coupled with propargylamine.

[0008] The present invention is further described by Scheme 1. In Scheme 1, all products can be isolated and purified by techniques well known in the art (e.g., extraction, evaporation, trituration, chromatography, and recrystallization). [ka]

[0009] Step 1 of Scheme 1 illustrates a quinalkaloid-directed asymmetric hydroxylamine / enone cascade reaction in which the compound of formula (2) (wherein X is selected from the group consisting of halogens and -C(O)OR4 (wherein R4 is C1-C4 alkyl)) is used with hydroxylamine and a suitable base in the presence of the compound of formula (3) to obtain the enantiomerically pure compound of formula (4).

[0010] Those skilled in the art will understand that compounds of formula (2) exist as geometric isomers. In compounds of formula (2), the bond from the double bond to the CF3 group results in such geometric isomers (including E-isomers, Z-isomers, and mixtures thereof), and the present invention encompasses the use of E-isomers, Z-isomers, and mixtures thereof in any ratio. Particularly preferred compounds of formula (2) are those in which X is chloro or bromo, more preferably bromo. Other particularly preferred compounds of formula (2) are those in which X is -C(O)OR4, and R4 is selected from the group of methyl and ethyl, more preferably methyl. Particularly preferred compounds of formula (3) are those in which R1 is methoxy.

[0011] The compound of formula (3) is typically used in molar ratios of typically 0.001 to 10, more typically 0.01 to 1, and even more typically 0.05 to 0.5, relative to the compound of formula (2).

[0012] Examples of suitable bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, barium hydroxide, cesium hydroxide, sodium phosphate, potassium phosphate, sodium methoxide, potassium methoxide, and potassium t-butoxide. In one embodiment, a suitable base is selected from the group consisting of lithium hydroxide, sodium hydroxide, potassium hydroxide, barium hydroxide, cesium hydroxide, sodium phosphate, potassium phosphate, sodium methoxide, potassium methoxide, potassium t-butoxide, and mixtures thereof. Typically, with respect to the compound of formula (2), the base is used in a molar ratio of 1 to 10, more typically 1 to 5, and even more typically 2 to 4. Naturally, those skilled in the art will understand that when hydroxylamine is used as a salt, additional bases may be used.

[0013] The reaction illustrated in step 1 of Scheme 1 is carried out in a solvent (e.g., lower alcohols (e.g., methanol, ethanol, and isopropanol), chlorinating solvents (e.g., methylene chloride and chloroform), ether solvents (e.g., tetrahydrofuran, 2-methyltetrahydrofuran, diisopropyl ether, and methyl-t-butyl ether, t-amyl methyl ether, ethyl-t-butyl ether), aromatic solvents (e.g., toluene, chlorobenzene, and benzotrifluoride), or alkane solvents (e.g., hexane, heptane, methylcyclohexane, and cyclohexane), as well as mixtures of such solvents). Water may be added to the reactants. The reaction is carried out typically at temperatures of -50°C to 50°C, more typically -40°C to 0°C, more typically -40°C to -10°C, and even more typically -30°C to -20°C, and generally takes 1 to 48 hours.

[0014] Typical compounds of formula (3) include (R)-[(2S)-1-[(3,5-bis-trifluoromethylphenyl)methyl]-5-vinyl-quinuclidin-1-ium-2-yl]-(6-methoxy-4-quinolyl)methanol bromide, (R)-[(2S)-1-[(3,5-bis-trifluoromethylphenyl)methyl]-5-vinyl-quinuclidin-1-ium-2-yl]-(6-methoxy-4-quinolyl)methanol chloride, and (R)-[(2S)-1-[(3,5-bis-trifluoromethylphenyl)methyl]-5-vinyl-quinuclidin-1-ium-2-yl]-(4-quinolyl) Examples include methanol bromide, (R)-[(2S)-1-[(2,3,5-trifluorophenyl)methyl]-5-vinyl-quinuclidin-1-ium-2-yl]-(6-methoxy-4-quinolyl)methanol bromide, (R)-[(2S)-1-[(3,5-di-t-butylphenyl)methyl]-5-vinyl-quinuclidin-1-ium-2-yl]-(6-methoxy-4-quinolyl)methanol bromide, and (R)-[(2S)-1-[(anthracene-9-yl)methyl]-5-vinyl-quinuclidin-1-ium-2-yl]-(6-methoxy-4-quinolyl)methanol bromide.

[0015] Step 2 of Scheme 1 illustrates the step of converting X in the compound of formula (4) to the carboxylic acid of the compound of formula (5). The compound of formula (4) in which X is a halogen can be converted to the compound of formula (5) by metallation of the X position by halogen metal exchange with a Grignard reagent or alkyllithium, and then reacting the metallated species with carbon dioxide or a reagent that can produce a carboxylic acid. Such reactions are readily performed and well known. See WO2014 / 090918. The compound of formula (4) in which X is -C(O)OR4 can be readily converted to the compound of formula (5) by hydrolysis. Such reactions are readily performed and well known.

[0016] Step 3 of Scheme 1 involves converting the compound of formula (5) to a suitable amine (the compound of formula (6), which is 2-amino-propargyl-acetamide). [ka] Alternatively, the procedure shows a step of coupling a carboxyl-protected glycine (which is then deprotected as needed and coupled with propargylamine) with an amine obtained from a series of reactions to obtain the compound of formula (1).

[0017] The step of coupling a carboxylic acid or activated carboxylic acid derivative (e.g., an acid halide) with an amine in this manner to produce an amide is well known in the art. The use, deprotection, and amide coupling with carboxyl-protected glycine are also readily achieved. See WO2010 / 070068 and WO2014 / 090918.

[0018] As used herein, the term “enantiomerically pure” refers to an (S)-enantiomer present in greater than 90% (i.e., an enantiomer excess of 80% or more (i.e., ee)). In one embodiment, the term “enantiomerically pure” refers to an (S)-enantiomer present in greater than 92% (i.e., an ee of 84% or more). In one embodiment, the term “enantiomerically pure” refers to an (S)-enantiomer present in greater than 94% (i.e., an ee of 88% or more). In one embodiment, the term “enantiomerically pure” refers to an (S)-enantiomer present in greater than 95% (i.e., an ee of 90% or more). In one embodiment, the term “enantiomerically pure” refers to an (S)-enantiomer present in greater than 96% (i.e., an ee of 92% or more). In one embodiment, the term “enantiomerically pure” refers to an (S)-enantiomer present in greater than 97% (i.e., an ee of 94% or more). In one embodiment, the term “enantiomerically pure” refers to the (S)-enantiomer present in greater than 98% (i.e., 96% or more ee). In one embodiment, the term “enantiomerically pure” refers to the (S)-enantiomer present in greater than 99% (i.e., 98% or more ee). In one embodiment, the term “enantiomerically pure” refers to the (S)-enantiomer present in greater than 99.8% (i.e., 99.6% or more ee).

[0019] The use of a poor solvent may be advantageous. In this context, "poor solvent" refers to a solvent in which the solubility of the compound of formula (5) is significantly lower than that of the selected solvent. When a poor solvent is used, it is preferable that the poor solvent is miscible with the selected solvent.

[0020] Furthermore, the present invention is a method for producing an enantiomerically pure isoxazoline compound of formula (1), characterized by improving the enantiomer purity of the compound of formula (5), C 1~5 Methods including crystallization from alcohol / water are also provided. In a preferred embodiment, C1~5 The alcohol:water ratio is about 9:1 (v / v). In a preferred embodiment, C 1~5 the alcohol is isopropanol. In a further preferred embodiment, C 1~5 the alcohol is isopropanol, and the isopropanol:water ratio is 9:1 (v / v).

[0021] The present invention also provides a method for producing an enantiomerically pure compound of formula (1), characterized by improving the enantiomeric purity of a compound of formula (5), C 3~9 the method comprising crystallization from alkyl ketone / water. In a preferred embodiment, C 3~9 the alkyl ketone:water ratio is about 9:1 (v / v). In a preferred embodiment, C 3~9 the alkyl ketone is acetone. In a further preferred embodiment, C 3~9 the alkyl ketone is acetone, and the acetone:water ratio is 9:1 (v / v).

[0022] Preferred poor solvents are C 5~8 hydrocarbons and water. Specifically, preferred poor solvents are selected from the group consisting of water, pentane, hexane, heptane, cyclohexane, and methylcyclohexane. A particularly preferred poor solvent is methylcyclohexane. The ratio of good solvent:poor solvent is not critical, and is typically in the range of 2:1 to 1:6 (v / v).

[0023] The present invention also provides a method for producing an enantiomerically pure isoxazoline compound of formula (1), characterized by improving the enantiomeric purity of a compound of formula (5), C 1~5 the method comprising crystallization from an alcohol and C 5~8 a hydrocarbon. In a preferred embodiment, C 1~5 the alcohol is selected from the group consisting of ethanol and isopropanol.

[0024] Furthermore, the present invention is a method for producing an enantiomerically pure isoxazoline compound of formula (1), characterized by improving the enantiomer purity of the compound of formula (5), C 2~8 Alkyl ethers and C 5~8 Methods including crystallization from hydrocarbons are also provided. In a preferred embodiment, C 2~8 The alkyl ether is selected from the group consisting of tetrahydrofuran and 2-methyltetrahydrofuran.

[0025] Furthermore, the present invention is a method for producing an enantiomerically pure isoxazoline compound of formula (1), characterized by improving the enantiomer purity of the compound of formula (5), C 2~8 Alkyl acetate and C 5~8 Methods including crystallization from hydrocarbons are also provided. In a preferred embodiment, C 2~8 Alkyl acetates are selected from the group consisting of ethyl acetate and isopropyl acetate.

[0026] Furthermore, the present invention is a method for producing an enantiomerically pure isoxazoline compound of formula (1), characterized by improving the enantiomer purity of the compound of formula (5), C 3~9 Alkyl ketones and C 5~8 Methods including crystallization from hydrocarbons are also provided. In a preferred embodiment, C 3~9 Alkyl ketones are selected from the group consisting of acetone and methyl ethyl ketones.

[0027] As used herein, the term "halogen" refers to fluorine, chlorine, bromine, and iodine atoms. More specifically, the term "halogen" refers to fluorine, chlorine, and bromine atoms. More specifically, the term "halogen" refers to chlorine and bromine atoms.

[0028] Y - The related term "anion" refers to a negatively charged organic or inorganic group. For example, Y -This may be a tosylate, brosylate, mesylate, nosylate, triflate, acetate, or a halide, sulfuric acid, phosphoric acid, hydroxide, boron tetrafluoride, etc. In one embodiment, Y - is a halide. In one embodiment, Y - It is a chloride or bromide.

[0029] The term "aryl" refers to phenyl, naphthyl, anthracenyl, etc. In one embodiment, "aryl" is phenyl. In one embodiment, "aryl" is anthracene-9-yl.

[0030] The term "heteroaryl" refers to a fully unsaturated ring containing at least one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur, and includes pyridyl, pyrimidyl, pyrazinyl, indolyl, quinolinyl, acridinyl, and others.

[0031] "C1-C4 alkyl" refers to a linear or branched alkyl group having 1 to 4 carbon atoms.

[0032] The term "C1-C4 alkoxy" refers to C1-C4 alkyl groups bonded via an oxygen atom.

[0033] When used in reference to measurable numerical variables, the term "approximately" refers to the indicated value of the variable and all values ​​of the variable that are either within the experimental error of the indicated value or within ±10% of the indicated value, whichever is greater.

[0034] "C 1~5 The term "alcohol" refers to linear or branched alkanols having 1 to 5 carbon atoms, such as methanol, ethanol, n-propanol, iso-propanol, 1-butanol, and 1,3-propanediol.

[0035] "C 2~5The term "alkylcyanide" refers to linear or branched alkylcyanides having a total of 2 to 5 carbon atoms, such as acetonitrile, propionitrile, and butyronitrile.

[0036] "C 3~9 The term "alkyl ketone" refers to a linear, branched, or cyclic alkyl group having an oxo group and a total of 3 to 9 carbon atoms, such as acetone, methyl ethyl ketone, and cyclohexanone.

[0037] "C 2~8 The term "alkyl ether" refers to linear, branched, or cyclic alkyl ethers having a total of 2 to 8 carbon atoms, such as diethyl ether, methyl t-butyl ether, t-amyl methyl ether, ethyl t-butyl ether, tetrahydrofuran (THF), 2-methyl THF, and dioxane.

[0038] "C 3~8 The term "alkyl acetate" refers to linear or branched alkyl esters of acetic acid that have a total of 3 to 8 carbon atoms, such as methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, and isobutyl acetate.

[0039] "C 5~8 The term "hydrocarbon" refers to linear, branched, or cyclic saturated alkyl hydrocarbons, such as pentane, hexane, heptane, octane, cyclopentane, cyclohexane, and methylcyclohexane.

[0040] The terms "crystallize," "the act of crystallization," and "crystallization" should be understood to refer to precipitation and slurry processes that do not involve complete dissolution following complete dissolution. Slurry processes include processes that involve continued stirring following precipitation after complete dissolution.

[0041] The compound of formula (1) is known in the art as a useful active ingredient for use in controlling pests (WO2016 / 077158). The term “pests” includes internal parasites, preferably ectoparasites, in the context of animal bodies, both external and internal, as well as in the field of hygiene. Exoparasites should be understood to specifically include insects, mites (ticks and mites), and fish-parasitic crustaceans (fish lice). Specific pests include fleas, ticks, mites, flies, worms, lice, and crustaceans. More specific pests include fleas, mites, lice, and fish lice.

[0042] In this specification, animals should be understood to include vertebrates. In this context, the term vertebrate should be understood to refer to mammals, including, for example, fish, amphibians, reptiles, birds, and humans. A preferred group of vertebrates according to the present invention includes warm-blooded animals, which include domestic animals such as cattle, horses, pigs, sheep, and goats; poultry such as chickens, turkeys, guinea fowl, and geese; fur-bearing animals such as minks, foxes, chinchillas, and rabbits; as well as companion animals such as ferrets, guinea pigs, rats, hamsters, cats, and dogs, and humans. A further preferred group of vertebrates according to the present invention includes fish, which include salmonids, such as salmon, trout, or whitefish.

[0043] The compound of formula (1) can be administered alone or in the form of a composition. In practice, the compound is usually administered in the form of a composition, i.e., mixed with at least one acceptable excipient. The proportion and nature of any acceptable excipient is determined by the disorder or condition being treated, other relevant circumstances, the chosen route of administration, and standard practices such as those in the fields of veterinary medicine and pharmaceuticals.

[0044] The present invention is further illustrated by the following embodiments. These embodiments are for illustrative purposes only and are not intended to limit the present invention in any way.

[0045] Example 1 (5S)-3-(5-bromo-4-methyl-2-thienyl)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole [ka] (Z / E)-1-(5-bromo-4-methyl-2-thienyl)-4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)buta-2-en-1-one (1.0 g, 2.1 mmol) and (R)-[(2S)-1-[[3,5-bis(trifluoromethyl)phenyl]methyl]-5-vinyl-quinuclidine-1-ium-2-yl]-(6-methoxy-4-quinol)methanol bromide (135 mg, 0.2138 mmol, 0.1 equivalents) were combined in 100 mL of dichloromethane under nitrogen. The solution was cooled to a temperature of -15°C to -10°C, and hydroxylamine aqueous solution (386 μL, 6.25 mmol, 16.2 mol / L, 3.0 equivalents) and sodium hydroxide (0.70 mL, 7.0 mmol, 10 M, 3.3 equivalents) were slowly added to the reaction mixture while maintaining an internal temperature of -10°C. After stirring at -10°C for 7 hours, the condenser was stopped, and the reaction was allowed to complete by stirring the mixture overnight at room temperature. Chiral HPLC showed 90.3% S-isomers and 9.7% R-isomers. The reaction mixture was transferred to a round-bottom flask and concentrated under reduced pressure at room temperature to obtain a solid. The solid was dissolved in ethyl acetate (3 mL) and purified by automated flash chromatography using silica gel, eluting with Depositphotos:hexane (1:1). The solvent was removed from the fraction containing the product under reduced pressure at 40°C to obtain a pale yellow solid (0.833 g, 81%).

[0046] Example 2 (5S)-3-(5-bromo-4-methyl-2-thienyl)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole [ka] (Z / E)1-(5-bromo-4-methyl-2-thienyl)-4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)buta-2-en-1-one (10.0 g, 20.9 mmol) and (R)-[(2S)-1-[[3,5-bis(trifluoromethyl)phenyl]methyl]-5-vinyl-quinuclidine-1-ium-2-yl]-(6-methoxy-4-quinolyl)methanol bromide (2.14 mmol, 0.1 equivalents) were combined in dichloromethane (250 mL) under nitrogen. The solution was cooled to a temperature of -10°C to -15°C, and a solution of hydroxylamine in water (3.9 mL, 63.2 mmol, 16.2 mol / L, 3.0 equivalents) and sodium hydroxide (7.0 mL, 70 mmol, 10 M, 3.3 equivalents) were slowly added while maintaining the internal temperature at -10°C to -15°C. After stirring at -15°C to -10°C for 18 hours, the reaction mixture was transferred to a round-bottom flask and concentrated under reduced pressure at room temperature to obtain a solid. The solid was then dissolved in ethanol (90 mL) at 50°C and stirred at 50°C (water bath) for 30 minutes, and then water (300 mL) was slowly added dropwise while stirring to obtain a suspension. The suspension was filtered, and recrystallization was repeated once to obtain a free-flowing solid. The solid was dried in a vacuum oven at 25-30°C to obtain 10.34 g of the product. The solid was evaluated by chiral HPLC, revealing 91.0% S-isomers and 9.0% R-isomers.

[0047] Example 3a (5S)-3-(5-bromo-4-methyl-2-thienyl)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole [ka] (Z / E)1-(5-bromo-4-methyl-2-thienyl)-4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)buta-2-en-1-one (50.0 g, 104.5 mmol) and (R)-[(2S)-1-[[3,5-bis(tert-butyl)phenyl]methyl]-5-vinyl-quinuclidin-1-ium-2-yl]-(6-methoxy-4-quinolyl)methanol bromide (0.11 equivalents) were combined in dichloromethane (100 mL) and ethyl t-butyl ether (400 mL). The reaction mixture was stirred at 30°C for 30 minutes, then cooled to -20°C, and a 50% aqueous solution of hydroxylamine (40 mL, 313 mmol, 3.0 equivalents) and sodium hydroxide (34.5 mL, 345 mmol, 10 M, 3.3 equivalents) were slowly added while maintaining the internal temperature at -15°C to -20°C. After stirring at -15°C to -20°C for 18 hours, an aqueous hydrochloric acid solution (1 N, 500 mL) was added, and the reaction mixture was stirred at 15°C to 20°C, then the stirring was stopped, and the phases were separated after 30 minutes. The organic layer was extracted with an aqueous hydrochloric acid solution (1 N, 75 mL), the layer was separated, and the organic layer was extracted again with an aqueous hydrochloric acid solution (1 N, 100 mL). The organic layer was separated, extracted with an aqueous saturated sodium bicarbonate solution (75 mL), the layer was separated, and the organic layer was extracted again with an aqueous saturated sodium bicarbonate solution (100 mL). The layers were separated, and the organic layer was dried with sodium sulfate (10 g). The organic layer was filtered, the cake was washed with ethyl t-butyl ether (50 mL), then montmorillonite clay (50 g) was added, and the mixture was stirred at 10°C to 20°C. After 2 hours, the reaction mixture was filtered, the cake was rinsed with ethyl t-butyl ether (50 mL), the filtrate was concentrated to approximately 100 mL, THF was added twice, and the mixture was concentrated again to approximately 100 mL, then THF (150 mL) was added to obtain the title compound as a solution in THF. The solution was evaluated by chiral HPLC, which showed 96.5% S-isomers and 3.5% R-isomers.

[0048] Example 3b 3-Methyl-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxylic acid [ka] A 22% solution of (5S)-3-(5-bromo-4-methyl-2-thienyl)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole in THF (185.0 g, 374.8 mmol) was cooled to 0°C to 5°C. A solution of ethylmagnesium chloride in THF (2 M, 300 mL, 1.6 equivalents) was added dropwise while maintaining the internal temperature below 10°C. The reaction mixture was stirred at 15°C to 20°C for 2 to 4 hours. Then, concentrated sulfuric acid (50 mL) was passed through, followed by the introduction of carbon dioxide gas (58 g, 3.5 equivalents) below the surface at 0°C to 5°C. The reaction mixture was stirred at 0°C to 5°C for 2 hours, and an 8% aqueous sodium chloride solution (601 g) was added dropwise at below 10°C, followed by the addition of a 37% aqueous hydrochloric acid solution (92.5 g) at below 0°C to obtain the title compound.

[0049] Example 4a (5S)-3-(5-bromo-4-methyl-2-thienyl)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole [ka] A mixture of butenone bromothiophene (658 g), (R)-[(2S)-1-[(3,5-di-t-butylphenyl)methyl]-5-vinyl-quinuclidine-1-ium-2-yl]-(6-methoxy-4-quinolyl)methanol bromide (57 g), dichloromethane (1120 g), and methyl tert-butyl ether (MTBE) (2586 g) was added at -30°C to a solution of hydroxylamine hydrochloride (261 g) in water (333 g, pre-cooled to 0°C), followed by the addition of an aqueous sodium hydroxide solution (32%, 548 g) at the same temperature. The reaction mixture was stirred at -30°C for several hours until the conversion was complete. The reaction mixture was heated to 0-5°C and transferred to a quench solution consisting of hydrochloric acid (37%, 286 g), ethanol (468 g), and water (600 g). The mixture was heated to 40°C, and the pH was confirmed to be 5-6, after which the phase was separated. The organic layer was concentrated under reduced pressure, and the distillate was replaced with fresh methyl tert-butyl ether (2 cycles, 1777 g each). The mixture was then briefly heated under reflux and cooled to -10°C to induce catalyst precipitation. The resulting suspension was filtered and optionally extracted with a solution of hydrochloric acid (37%, 240 g), sodium chloride (240 g), and water (1080 g), and optionally filtered through a bleached clay filter bed. The filtrate was washed with saturated bicarbonate solution (1200 g), and the organic layer was stored as an MTBE solution containing the product (S)-isoxazole bromothiophene.

[0050] Example 4b 3-Methyl-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxylic acid [ka] The reaction mixture produced from Example 4a ((5S)-3-(5-bromo-4-methyl-2-thienyl)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole in MTBE) was placed in a reactor and concentrated. The distillate was replaced with fresh THF (2 cycles, 2136 g each). Ethyl magnesium chloride (approximately 25% in tetrahydrofuran, 933 g) was added after it had cooled to -10°C. After the completion of the reaction (HPLC), carbon dioxide gas (236 g) was added as quickly as possible below the surface at an internal temperature of -1°C. The reaction mixture was stirred at an internal temperature of 0°C. After the completion of the reaction (HPLC), the reaction mixture was quenched by slowly adding a mixture containing sodium chloride (110 g), water (2235 g), and 37% hydrochloric acid (283 g) at ambient temperature. After mixing and precipitation, the phases were separated. The organic layer was concentrated, and the distillate was replaced with fresh acetonitrile (2 cycles, 1915 g each). The reaction mixture was briefly heated to obtain a clear solution, which was then cooled to -10°C. The product was isolated by centrifugation and washed with pre-cooled acetonitrile (460 g). The moist (S)-isoxazolthiophenecarboxylic acid was dried in a vacuum dryer at 50°C and below 100 mbar. The dry yield was 82% of the theoretical yield. Purity: 100%, Chiral purity: 99.8a%.

[0051] Example 4c 3-Methyl-N-[2-oxo-2-[(2-propyne-1-yl)amino]ethyl]-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxamide [ka] Dried S-isoxazolthiophenecarboxylic acid (from Example 4b, 10 g) and toluene (125 g) were placed in a reactor, and the mixture was heated to 110°C. Thionyl chloride (7.0 g) was slowly added to the reaction mixture. After the reaction was complete, toluene was removed by distillation under vacuum at NMT 50°C, and the residue was diluted with fresh dichloromethane (82.5 g). In a separate reactor, 2-amino-propargyl-acetamide HCl (3.4 g) was suspended in dichloromethane (100 g), and triethylamine (6.9 g) was added at ambient temperature. The resulting mixture was cooled to 0°C, and the acyl chloride reaction mixture in the dichloromethane was added over 45 minutes at 0°C. The combined reaction mixture was stirred at 0°C for a further 1 to 8 hours, and the conversion was confirmed by IPC.

[0052] After sufficient conversion (IPC), the mixture was extracted with 1M hydrochloric acid (a mixture of 37% HCl (4.8g) and water (38.7g)), followed by extraction with saturated sodium bicarbonate solution (4.2g sodium bicarbonate, 48g water), and finally with water (52.5g). The majority of the organic layer was removed under vacuum at 40°C, and methyl tert-butyl ether (23.8 g) was added. The mixture was stirred at 25°C, and heptane (47.6 g) was slowly added to precipitate the product. The product (3-methyl-N-[2-oxo-2-[(2-propyne-1-yl)amino]ethyl]-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxamide) was isolated by filtration and washed with a mixture of methyl tert-butyl ether (4.3 g) and heptane (20.7 g). The product was dried under vacuum at 45°C. Crystallization of the product may be carried out as needed. Yield: 11.2 g. Purity: >98.7%, chiral purity >99.87%. 1 The 1H NMR spectrum matched that of the genuine sample.

[0053] The enantiomer purity of the product was quantified by HPLC using a chiral column (Daicel Chiralpak AS-3R, 150 × 4.6 mm, 3 μm). Retention times (see Table 1 below) are related to the use of a solvent system containing a water / acetonitrile 55:45 (v / v) mixture in each case. The eluent was used in homogeneous concentration mode at a flow rate of 1.5 ml / min. Figure 1 shows a superimposed chiral chromatogram of the 3-methyl-N-[2-oxo-2-[(2-propyne-1-yl)amino]ethyl]-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxamide product (lower line). The middle line shows the chromatogram of the reference sample (i.e., enantiomerically pure) of 3-methyl-N-[2-oxo-2-[(2-propyne-1-yl)amino]ethyl]-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxamide. The upper line shows the chromatogram of the reference sample (i.e., enantiomerically pure) of 3-methyl-N-[2-oxo-2-[(2-propyne-1-yl)amino]ethyl]-5-[(5R)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxamide. The peak results in Figure 1 are shown in Table 1 below. [Table 1]

[0054] Figure 2 shows the HPLC purity of the product (upper line) of Example 4c compared to the blank (lower line). The peak results of the product in Figure 2 are shown in Table 2 below. [Table 2]

[0055] Figure 3 shows the relationship between the product of Example 4c (underlined) and the API reference sample (toplined). 1This shows a comparison of 1H NMR.

[0056] Figure 4 shows the product of Example 4c. 1 This is 1H NMR data.

[0057] Example 5 3-Methyl-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxylic acid [ka] A 22% solution of (5S)-3-(5-bromo-4-methyl-2-thienyl)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole in THF (185.0 g, 374.8 mmol) was cooled to 0°C to 5°C. A 2M solution of ethylmagnesium chloride in THF (300 mL, 1.6 equivalents) was added dropwise while maintaining the internal temperature below 10°C. The reaction mixture was stirred at 15°C to 20°C for 2 to 4 hours. Then, concentrated sulfuric acid (50 mL) was passed through, followed by the introduction of carbon dioxide gas (58 g, 3.5 equivalents) below the surface at 0°C to 5°C. The reaction mixture was stirred at 0°C to 5°C for 2 hours, and 8% sodium chloride aqueous solution (601 g) was added dropwise at below 10°C, followed by 37% hydrochloric acid aqueous solution (92.5 g) at below 0°C.

[0058] The reaction mixture was stirred at 10°C–15°C for 30 minutes, then the stirring was stopped and the postphase was separated after 30 minutes. The organic layer was concentrated under vacuum to approximately 370 mL, followed by the addition of THF (1850 mL) and concentration under vacuum three times, resulting in a concentration of approximately 370 mL–555 mL. After confirming that the reaction mixture was dry, the cycle of adding acetonitrile (925 mL) followed by vacuum concentration to approximately 555 mL–740 mL was performed three times. The reaction mixture was heated to 75°C and gradually cooled to 50°C over 1 hour. Product seed (1.85 g) was added at 50°C, and the reaction mixture was stirred at 50°C for 30 minutes. The batch was gradually cooled to -10°C over 3 hours and maintained at -10°C for 2 hours. The batch was filtered, and the cake was washed with cold acetonitrile (93–185 mL). The wet cake was dried under vacuum at 50°C for 12 hours to obtain 110 g of the title compound. The product was evaluated by chiral HPLC, and over 99.9% of the S-isomers were observed.

[0059] The above product seeds were prepared as follows: A 300 mL solution of (5S)-3-(5-bromo-4-methyl-2-thienyl)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole in THF (48.93 g, 99.1 mmol) was cooled to 0°C to 5°C. A 2 M solution of ethyl magnesium chloride in THF (80 mL) was added dropwise while maintaining the internal temperature below 10°C. The reaction mixture was stirred at 15°C to 20°C for 2 to 4 hours. Then, concentrated sulfuric acid (50 mL) was passed through, followed by the introduction of carbon dioxide gas (25 g, 3.5 equivalents) below the surface at 0°C to 5°C. The reaction mixture was stirred at 0°C to 5°C for 6 hours, and a 5% sodium chloride aqueous solution (157 g) was added dropwise below 10°C, followed by a 37% hydrochloric acid aqueous solution (25 g) being added dropwise below 0°C. The reaction mixture was stirred at 10°C to 15°C for 30 minutes, then the stirring was stopped, and the phase was separated after 30 minutes. The organic layer was concentrated and the solvent was removed. 50 ml of heptane was added to the mixture, and the solvent was removed. The crude product was dissolved in 50 ml of EA and 100 ml of heptane at 40°C. A further 1000 ml of heptane was slowly added dropwise to the mixture. The mixture was then stirred at 40°C for 15 hours. The mixture was filtered to obtain a wet cake. The wet cake was slurryed with acetone at 20°C. The mixture was filtered, and the wet cake was dried under vacuum at 50°C for 3 hours to obtain 9.7 g of product. The product was evaluated by chiral HPLC, and more than 99.9% of the S-isomer was observed. Example 6 3-Methyl-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxylic acid [ka]

[0060] 2-Bromo-3-methyl-5-acetylthiophene (20 g), p-toluenesulfonic acid monohydrate (2.3 g), and ethylene glycol (11.3 g) were combined in toluene (120 mL) and heated with stirring at 115 °C for 12 hours, while water was collected using a Dean-Stark trap. The reaction mixture was then cooled and quenched with saturated sodium bicarbonate aqueous solution (40 mL). The organic layer was separated, washed twice with water (40 mL), and concentrated under vacuum at 60 °C to obtain 2-(5-bromo-4-methyl-2-thienyl)-2-methyl-1,3-dioxolane.

[0061] 2-(5-bromo-4-methyl-2-thienyl)-2-methyl-1,3-dioxane (25.2 g) and THF (50 mL) were combined and cooled in an ice / water bath. Ethyl magnesium chloride (2.0 M, 75 mL) in the THF was added while stirring, maintaining the temperature at 10°C to 30°C in an ice / water bath. The reaction mixture was then warmed to ambient temperature. After 90 minutes, the reaction mixture was cooled to 0°C to 5°C in an ice / water bath, and carbon dioxide gas was bubbling beneath the surface of the reactants at 5°C to 14°C for 30 minutes. The reaction mixture was warmed to ambient temperature and stirred overnight. The reaction mixture was cooled to 0°C to 10°C, and 75 mL of saturated brine aqueous solution was added at 10°C to 35°C. The pH was then adjusted to approximately 1 with 37% HCl aqueous solution. Ethyl acetate (50 mL) and water (25 mL) were added, and the reaction mixture was stirred. The aqueous layer was separated, and the organic layer was washed with saturated brine aqueous solution (3 × 50 mL). The washed organic layer was concentrated under vacuum at 40°C to obtain 3-methyl-5-(2-methyl-1,3-dioxolan-2-yl)thiophene-2-carboxylic acid (19.2 g) as a red oily product (coagulated during storage at ambient temperature). MS:ESI+:228.96;ESI-:226.98.

[0062] 19.2 g of 3-methyl-5-(2-methyl-1,3-dioxolan-2-yl)thiophene-2-carboxylic acid, 24.9 g of potassium carbonate, and 60 mL of dimethylformamide (DMF) were combined. The reaction mixture was cooled to 0–5°C in an ice / water bath, and then 13.1 mL of methyl iodide was added dropwise while maintaining the temperature at 0–5°C. The reaction mixture was stirred at ambient temperature for 1 hour, then cooled to 0–10°C and quenched with water (180 mL) and ethyl acetate (180 mL). The aqueous layer was separated, and the organic layer was washed with water (2 × 60 mL) and aqueous brine (60 mL). The organic layer was then evaporated under vacuum at 40°C to obtain methyl 3-methyl-5-(2-methyl-1,3-dioxolan-2-yl)thiophene-2-carboxylate (21.3 g) as a red oily product. MS:ESI+243.00.

[0063] p-toluenesulfonic acid monohydrate (1.7 g), methyl 3-methyl-5-(2-methyl-1,3-dioxolan-2-yl)thiophene-2-carboxylate (21.3 g), acetone (140 mL), and water (14 mL) were combined and stirred at 35°C for 2 hours, then cooled to 20°C. Sodium bicarbonate (1.5 g) was then added, and the reaction mixture was stirred at 20°C for 10 minutes. The mixture was then concentrated under vacuum at 40°C to obtain a residue. The residue was dissolved in 200 mL of ethyl acetate and washed with water (50 mL). The layers were separated, and the organic layer was washed with water (2 × 50 mL). The organic layer was concentrated under vacuum at 40°C to obtain a residue, which was purified by flash chromatography using a mixture of MTBE (0-15% v / v) in n-heptane to obtain methyl 5-acetyl-3-methylthiophene-2-carboxylate (4.9 g). 1 H NMR(500MHz,CDCl3)δ ppm 2.51(d,J=5.87Hz,6H)3.85(s,3H)7.43(s,1H). 13C NMR(126MHz,CDCl3)δ ppm 15.85(s,1C)26.80(s,1C)52.06(s,1C)76.74(s,1C)77.00(s,1C)77.26(s,1C)132 .65(s,1C)135.25(s,1C)145.37(s,1C)146.02(s,1C)162.57(s,1C)190.78(s,1C).

[0064] Methyl 5-acetyl-3-methylthiophene-2-carboxylate (4.1 g), 2,2,2-trifluoro-1-(3,4,5-trichlorophenyl)ethanone (5.74 g), triethylamine (8.4 mL), and MTBE (41 mL) were combined, and the reaction mixture was heated to approximately 57°C. After 3 hours, the reaction mixture was cooled to ambient temperature and stirred for 12 hours. The reaction mixture was then cooled to 0-5°C, and thionyl chloride (2.3 mL) was added dropwise while maintaining the temperature at 0-10°C. The reaction mixture was then warmed to ambient temperature and stirred overnight. The mixture was then diluted with MTBE (45 mL) and cooled to 0-5°C. A mixture of saturated sodium bicarbonate aqueous solution (45 mL) and water (45 mL) was added dropwise. The reaction mixture was then combined with ethyl acetate (60 mL), and the layers were separated. The aqueous layer was extracted with ethyl acetate (41 mL), the organic layers were combined, and washed with brine aqueous solution (2 × 40 mL). Next, the organic layer was evaporated under vacuum at 30°C to 40°C to obtain a residue. The residue was suspended in ethanol (50 mL), stirred for 1 hour, and then cooled to 0°C to 5°C. Water (50 mL) was added dropwise while stirring at 0°C to 5°C, and the mixture was stirred for 3 hours to obtain a solid. The solid was collected by filtration, washed with a pre-cooled 1:3 ethanol / water mixture (2 × 10 mL), and dried under vacuum at 35°C to 40°C to obtain methyl 3-methyl-5-[(E / Z)-4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)buta-2-enoyl]thiophene-2-carboxylate (8.43 g) as a brown solid. E / Z ratio: 77:23 1 (By 1H NMR).

[0065] Methyl 3-methyl-5-[(E / Z)-4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)buta-2-enoyl]thiophene-2-carboxylate (500 mg), (R)-[(2S)-1-[[3,5-bis(trifluoromethyl)phenyl]methyl]-5-vinyl-quinuclidin-1-ium-2-yl]-(6-methoxy-4-quinolyl)methanol bromide (69 mg), and DCM (50 mL) were combined and cooled to -10 to -15°C. A mixture of pre-cooled sodium hydroxide aqueous solution (10 N, 0.33 mL) and hydroxylamine aqueous solution (50%, 0.223 mL) was added dropwise through a syringe while maintaining the temperature at -10 to -15°C. After 5 hours, aqueous hydrochloric acid (2N, 25 mL) was slowly added, and the reaction mixture was then heated to 10°C-15°C. The layers were then separated, the organic layer was washed with water (twice, 25 mL), and evaporated under vacuum at 50°C to obtain methyl 3-methyl-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxylate (640 mg), which was used in the next step without further purification.

[0066] Methyl 3-methyl-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxylate (640 mg) was combined with THF (5 mL) twice in succession and evaporated to obtain a residue, which was then combined with THF (4.2 mL), water (1.6 mL), and aqueous sodium hydroxide solution (10 N, 0.22 mL). The reaction mixture was then heated to 60 °C with stirring. After 4 hours, the reaction mixture was evaporated until almost dry to obtain a residue, which was then divided into ethyl acetate (50 mL) and aqueous hydrochloric acid solution (0.5 N HCl, 25 mL). The layers were separated, the organic layer was washed with water (2 × 25 mL), and evaporated under vacuum at 50 °C to obtain a residue. The residue was combined with toluene (5 mL) and then evaporated under vacuum at 60 °C to obtain the title compound as a foamy solid (450 mg). S / R ratio: 89:11. 1H NMR(500MHz,CDCl3)δ ppm 2.53-2.60(m,3H)3.63-3.73(m,1H)4.03-4.12(m,1H)7.12-7.14(m,1H)7.60-7.65(m,2H).

[0067] Example 7 Methyl 3-methyl-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxylate [ka] 3-methyl-2-thiophenecarboxylic acid (2.5 g) and THF (5 mL) were combined at ambient temperature, and then 2,2,6,6-tetramethylpiperidinyl magnesium chloride-lithium chloride complex (50 mL, 0.94 M in THF) was added via syringe over 15 minutes while controlling the temperature to below 45°C. The reaction mixture was stirred at 25°C for 1 hour, and then N-methoxy-N-methylacetamide (5.0 mL) was added via syringe while controlling the temperature to below 40°C. After stirring at ambient temperature for approximately 90 minutes, the reaction mixture was cooled to 0-5°C, and aqueous hydrochloric acid solution (2 M, 100 mL) was added while controlling the temperature to below 45°C. MTBE (100 mL) was added, the layers were separated, and the aqueous layer was extracted with MTBE (50 mL). The combined organic layers were washed with brine aqueous solution (2 × 25 mL) and evaporated under vacuum at 45°C to obtain 5-acetyl-3-methyl-thiophene-2-carboxylic acid (4.8 g) as a yellow solid.

[0068] 5-acetyl-3-methylthiophene-2-carboxylic acid (4.8 g) was combined with potassium carbonate (3.0 equivalents) and DMF (30 mL), and then methyl iodide (2.5 equivalents) was added dropwise. After 45 minutes, water (90 mL) and MTBE (120 mL) were added with stirring, and the layers were separated. The aqueous layer was extracted with MTBE (60 mL). The combined organic layers were washed with water (2 × 30 mL) and then evaporated under vacuum at 55°C to obtain methyl 5-acetyl-3-methylthiophene-2-carboxylate (4.5 g).

[0069] Methyl 5-acetyl-3-methylthiophene-2-carboxylate (4.5 g), 2,2,2-trifluoro-1-(3,4,5-trichlorophenyl)ethanone (3.66 g), triethylamine (2.9 mL), and MTBE (30 mL) were combined, and the reaction mixture was heated to approximately 60°C. After 6.5 hours, an additional 2.0 mL of triethylamine was added, and heating was continued at 60°C for 3 hours. The reaction mixture was cooled to 0°C-5°C, and thionyl chloride (1.7 mL) was added dropwise while maintaining the temperature below 12°C. The reaction mixture was then warmed to ambient temperature, stirred for 1 hour, diluted with MTBE (30 mL), then cooled to 10°C, and subsequently a mixture of saturated sodium bicarbonate aqueous solution (30 mL) and water (30 mL) was slowly added. The layers were then separated, and the aqueous layer was extracted with MTBE (30 mL). The combined organic layers were washed with brine aqueous solution (2 × 30 mL) and evaporated under vacuum at 30°C to 40°C to obtain a residue. The residue was suspended twice in ethanol (30 mL) and evaporated until almost dry. The residue was then suspended in ethanol (30 mL) and stirred at 0°C to 5°C for 1 hour to obtain a solid. The solid was collected by filtration, washed with a pre-cooled 1:3 ethanol / water mixture (2 × 10 mL), and dried under vacuum at 40°C to obtain methyl 3-methyl-5-[(E / Z)-4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)buta-2-enoyl]thiophene-2-carboxylate (2.54 g) (almost pure E isomer). 1 The result was obtained by 1H NMR.

[0070] Methyl 3-methyl-5-[(E / Z)-4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)buta-2-enoyl]thiophene-2-carboxylate (500 mg), (R)-[(2S)-1-[[3,5-bis(trifluoromethyl)phenyl]methyl]-5-vinyl-quinuclidin-1-ium-2-yl]-(6-methoxy-4-quinolyl)methanol bromide (69 mg), and DCM (50 mL) were combined and cooled to -10 to -15°C. A mixture of pre-cooled sodium hydroxide aqueous solution (10N, 0.33 mL) and hydroxylamine aqueous solution (50%, 0.223 mL) was added dropwise through a syringe with stirring while maintaining the temperature at -10 to -15°C. After 5 hours at -10 to -15°C, the mixture was analyzed. S / R ratio: 89:11.

[0071] Example 8 Methyl 3-methyl-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxylate Methyl 3-methyl-5-[(E / Z)-4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)buta-2-enoyl]thiophene-2-carboxylate (500 mg), (R)-[(2S)-1-[[3,5-bis(trifluoromethyl)phenyl]methyl]-5-vinyl-quinuclidin-1-ium-2-yl]-(6-methoxy-4-quinolyl)methanol bromide (69 mg), and toluene / methylcyclohexane (1:1 (v / v) 50 mL) were added and cooled to -10°C to -15°C. A mixture of pre-cooled sodium hydroxide aqueous solution (10N, 0.33 mL) and hydroxylamine aqueous solution (50%, 0.223 mL) was added dropwise through a syringe with stirring while maintaining the temperature at -10°C to -15°C. After 46 hours at -10°C to -15°C, the mixture was analyzed. S / R ratio: 92:8.

[0072] Example 9 Methyl 3-methyl-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxylate Methyl 3-methyl-5-[(E / Z)-4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)buta-2-enoyl]thiophene-2-carboxylate (500 mg), (R)-[(2S)-1-[[3,5-bis(t-butyl)phenyl]methyl]-5-vinyl-quinuclidin-1-ium-2-yl]-(6-methoxy-4-quinolyl)methanol bromide (69 mg), and DCM (50 mL) were added and cooled to -10 to -15°C. A mixture of pre-cooled sodium hydroxide aqueous solution (10N, 0.33 mL) and hydroxylamine aqueous solution (50%, 0.223 mL) was added dropwise through a syringe with stirring while maintaining the temperature at -10 to -15°C. After 18 hours at -10 to -15°C, the mixture was analyzed. S / R ratio: 81:19.

[0073] Example 10 Methyl 3-methyl-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxylate Methyl 3-methyl-5-[(E / Z)-4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)buta-2-enoyl]thiophene-2-carboxylate (500 mg), (R)-[(2S)-1-[[3,5-bis(t-butyl)phenyl]methyl]-5-vinyl-quinuclidin-1-ium-2-yl]-(6-methoxy-4-quinolyl)methanol bromide (69 mg), and DIPE (50 mL) were added and cooled to -10 to -15°C. A mixture of pre-cooled sodium hydroxide aqueous solution (10N, 0.33 mL) and hydroxylamine aqueous solution (50%, 0.223 mL) was added dropwise through a syringe with stirring while maintaining the temperature at -10 to -15°C. After 18 hours at -10 to -15°C, the mixture was analyzed. S / R ratio: 88:12.

[0074] Example 11 Methyl 3-methyl-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazole-3-yl]thiophene-2-carboxylate Methyl 3-methyl-5-[(E / Z)-4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)buta-2-enoyl]thiophene-2-carboxylate (500 mg), (R)-[(2S)-1-[[3,5-bis(t-butyl)phenyl]methyl]-5-vinyl-quinuclidin-1-ium-2-yl]-(6-methoxy-4-quinolyl)methanol bromide (69 mg), diisopropyl ether (40 mL), and DCM (10 mL) were combined and cooled to -10 to -15°C. A mixture of pre-cooled sodium hydroxide aqueous solution (10N, 0.33 mL) and hydroxylamine aqueous solution (50%, 0.223 mL) was added dropwise through a syringe with stirring while maintaining the temperature at -10 to -15°C. After 18 hours at -10 to -15°C, the mixture was analyzed. S / R ratio: 91:9.

[0075] For completeness reasons, various aspects of this disclosure are set forth in the following numbered clauses.

[0076] Clause 1. A method for preparing an enantiomerically pure isoxazoline compound of formula (1), (i) A compound of formula (2) (wherein X is selected from the group consisting of halogens and -C(O)OR4 (wherein R4 is C1-C4 alkyl)), hydroxylamine and a suitable base and a compound of formula (3) (wherein Y -(where R1 is an anion, R2 is selected from the group consisting of hydrogen and methoxy, R3 is selected from the group consisting of ethyl and vinyl, and R3 is an aryl which is optionally substituted with 1 to 5 substituents independently selected from the group consisting of nitro, halogen, amino, trifluoromethyl, C1-C4 alkyl, C1-C4 alkoxy, and benzyloxy, and a heteroaryl which is optionally substituted with 1 to 3 substituents independently selected from the group consisting of halogen, trifluoromethyl, C1-C4 alkyl, and C1-C4 alkoxy) The step of reacting with to obtain the compound of formula (4), (ii) A step of converting X of the compound of formula (4) to the carboxylic acid of the compound of formula (5), (iii) Optionally, the compound of formula (5) is C 1~5 Alcohol, C 2~5 Alkylcyanide, C 3~9 Alkyl ketone, C 2~8 Alkyl ether, C 2~8 A solvent selected from the group consisting of alkyl acetates, and optionally water and C 5~8 A step of crystallization using a poor solvent selected from the group consisting of hydrocarbons, Furthermore (iv) The step of coupling the compound of formula 5 with a suitable amine, A method wherein the suitable amine is either 2-amino-propargyl-acetamide, or an amine resulting from a series of reactions in which carboxyl-protected glycine is then, if necessary, deprotected and subsequently coupled with propargylamine. Clause 2. The method according to Clause 1, wherein the suitable amine is 2-amino-propargyl-acetamide. Clause 3. The method according to Clause 1, wherein the suitable amine is an amine obtained from a series of reactions in which carboxyl-protected glycine is then, if necessary, deprotected and then coupled with propargylamine. The method described in any one of the clauses 1 to 3, wherein clause 4.X is a halogen. The method described in Clause 4, wherein Clause 5.X is a bromo. The method according to Clause 6.X is chloro. The method according to any one of the provisions 1 to 3, wherein provision 7.X is -C(O)OR4 (wherein R4 is a C1-C4 alkyl group). The method according to Clause 8, wherein R4 is methyl. The method according to Clause 9, wherein R4 is ethyl. Clause 10. The method described in any one of Clauses 1 to 9, wherein R1 is methoxy. Clause 11. The method according to any one of Clauses 1 to 10, wherein step (i) is performed at a temperature of -40°C to -10°C. Clause 12. The method according to any one of Clauses 1 to 10, wherein step (i) is performed at a temperature of -30°C to -20°C. Clause 13. The method according to any one of Clauses 1 to 10, wherein step (i) is performed at a temperature of approximately -30°C. Clause 14. The method according to any one of Clauses 1 to 13, wherein the reaction of the compound of formula (2) with hydroxylamine, the appropriate base, and the compound of formula (3) is carried out in the presence of a solvent system comprising dichloromethane and ether. Clause 15. The method according to Clause 14, wherein the ether is methyl t-butyl ether, ethyl t-butyl ether, diisopropyl ether, or t-amyl methyl ether. Clause 16. The method according to Clause 14, wherein the ether is methyl t-butyl ether or ethyl t-butyl ether. Clause 17. The method according to any one of Clauses 1 to 16, wherein the enantiomer excess of the compound of formula (4) is 80% or more. Clause 18. The method according to any one of Clauses 1 to 16, wherein the enantiomer excess of the compound of formula (4) is 93% or more. Clause 19. The method described in any one of Clauses 1 to 18, wherein step (iii) is performed. The method according to Clause 19, wherein the poor solvent is present in Clause 20(iii). The solvent in Clause 21(iii) is C 1~5The method described in Clause 19 or 20, which is an alcohol. The solvent in Clause 22.(iii) is C 2~5 The method according to clause 19 or 20, wherein the material is an alkylcyanide. The solvent in Clause 23(iii) is C 3~9 The method according to clause 19 or 20, wherein the alkyl ketone is used. The solvent in Clause 24(iii) is C 2~8 The method according to clause 19 or 20, wherein the alkyl ether is used. The solvent in Clause 25(iii) is C 2~8 The method according to clause 19 or 20, wherein the alkyl acetate is used. The C in Article 26(iii) 1~5 The method according to Clause 21, wherein the alcohol is isopropanol. The C in Article 27(iii) 1~5 The method according to Clause 21, wherein the alcohol is ethanol. The C in Article 28(iii) 2~5 The method according to Clause 22, wherein the alkylcyanide is acetonitrile. The C in Article 29(iii) 3~9 The method according to Clause 23, wherein the alkyl ketone is acetone. The C in Article 30(iii) 3~9 The method according to Clause 23, wherein the alkyl ketone is a methyl ethyl ketone. The C in Article 31(iii) 2~8 The method according to clause 24, wherein the alkyl ether is tetrahydrofuran. The C in Article 32(iii) 2~8 The method according to clause 24, wherein the alkyl ether is 2-methyltetrahydrofuran. The C in Article 33(iii) 2~8 The method according to Clause 25, wherein the alkyl acetate is ethyl acetate. The C in Article 34(iii) 2~8 The method according to Clause 25, wherein the alkyl acetate is isopropyl acetate. The method according to any one of the paragraphs 19 to 34, wherein the poor solvent in paragraph 35(iii) is water. The poor solvent in Article 36(iii) is C 5~8 A hydrocarbon as described in any one of the clauses 19 to 34. Article 37. The C 5~8 The method according to clause 36, wherein the hydrocarbon is pentane. Article 38. The relevant C 5~8 The method according to clause 36, wherein the hydrocarbon is hexane. Article 39. The C 5~8 The method according to clause 36, wherein the hydrocarbon is heptane. Article 40. The C 5~8 The method according to clause 36, wherein the hydrocarbon is cyclohexane. Article 41. The relevant C 5~8 The method according to clause 36, wherein the hydrocarbon is methylcyclohexane. Clause 42. The method according to any one of Clauses 1 to 41, wherein the enantiomer excess of the compound of formula (5) is 90% or more. Clause 43. The method according to any one of Clauses 1 to 41, wherein the enantiomer excess of the compound of formula (5) is 96% or more. Clause 44. The method according to any one of Clauses 1 to 41, wherein the enantiomer excess of the compound of formula (5) is 98% or more. Clause 45. The method according to any one of Clauses 1 to 41, wherein the enantiomer excess of the compound of formula (5) is 99% or more. Clause 46. The method according to any one of Clauses 1 to 41, wherein the enantiomer excess of the compound of formula (5) is 99.6% or more. Clause 47. The method according to any one of Clauses 1 to 46, wherein the appropriate base is selected from the group consisting of lithium hydroxide, sodium hydroxide, potassium hydroxide, barium hydroxide, cesium hydroxide, sodium phosphate, potassium phosphate, sodium methoxide, potassium methoxide, potassium t-butoxide, and mixtures thereof. Clause 48.Y -The method according to any one of the claims 1 to 47, wherein the material is selected from the group consisting of tosylate, brosylate, mesylate, nosylate, triflate, acetate, halide, sulfuric acid, phosphoric acid, hydroxide, and boron tetrafluoride. Article 49.Y - The method according to Article 48, wherein is a halogenated compound. Clause 50.Y - The method according to Article 49, wherein the substance is a chloride. Clause 51.Y - The method according to Article 49, wherein the bromide is used. Clause 52. A composition comprising the compound of formula (1) with an enantiomer purity of 98% or more. Clause 53. A composition comprising the compound of formula (1) with an enantiomer purity of 99% or more. Clause 54. A composition comprising the compound of formula (1) with an enantiomer purity of 99.8% or higher.

Claims

1. Enantiomerically pure isoxazoline compound of formula (1) 【Chemistry 1】 A method for producing, (i) Compound of formula (2) 【Chemistry 2】 (In the formula, X is halogen and -C(O)OR ４ (In the formula, R ４ is C １ ~C ４ (Selected from the group consisting of alkyl groups) The compound of hydroxylamine, a suitable base, and the compound of formula (3) 【Transformation 3】 (In the formula, Y － is an anion, R １ It is selected from the group consisting of hydrogen and methoxy, R ２ It is selected from the group consisting of ethyl and vinyl, R ３ is aryl, aryl substituted with 1 to 5 substituents independently selected from the group consisting of optionally nitro, halogen, amino, trifluoromethyl, C １ -C ４ alkyl, C １ -C ４ alkoxy, and benzyloxy, and heteroaryl, heteroaryl substituted with 1 to 3 substituents independently selected from the group consisting of optionally halogen, trifluoromethyl, C １ -C ４ alkyl, and C １ -C ４ alkoxy; and R is selected from the group consisting of the above) The step of reacting with to obtain the compound of formula (4), 【Chemistry 4】 (ii) X of the compound of formula (4) 【Transformation 5】 The step of converting to a carboxylic acid, (iii) Optionally, the compound of formula (5) above is C １～５ Alcohol, C ２～５ Alkylcyanide, C ３～９ Alkyl ketone, C ２～８ Alkyl ether, C ２～８ A solvent selected from the group consisting of alkyl acetates, and optionally water and C ５～８ A step of crystallization using a poor solvent selected from the group consisting of hydrocarbons, Furthermore (iv) The step of coupling the compound of formula 5 with a suitable amine, A method wherein the appropriate amine is 2-amino-propargyl-acetamide, or an amine resulting from a series of reactions in which carboxyl-protected glycine is subsequently deprotected if necessary and then coupled with propargylamine.

2. The method according to claim 1, wherein X is bromo.

3. X is -C(O)OR ４ (In the formula, R ４ The method according to claim 1, wherein ( is methyl).

4. R １ The method according to any one of claims 1 to 3, wherein is methoxy.

5. The method according to claim 1, wherein the appropriate amine is 2-amino-propargyl-acetamide.

6. The method according to claim 1, wherein the appropriate amine is an amine obtained from a series of reactions in which carboxyl-protected glycine is then, if necessary, deprotected and subsequently coupled with propargylamine.

7. The method according to claim 1, wherein the reaction between the compound of formula (2) and hydroxylamine, the appropriate base, and the compound of formula (3) is carried out in the presence of a solvent system comprising dichloromethane and an ether selected from the group consisting of tetrahydrofuran, 2-methyltetrahydrofuran, methyl-t-butyl ether, ethyl-t-butyl ether, diisopropyl ether, and t-amyl methyl ether.

8. The method according to claim 1, wherein the enantiomer excess of the compound of formula (5) is 90% or more.

9. The method according to claim 1, wherein crystallization of (iii) is performed.

10. The method according to claim 1, wherein crystallization of (iii) is performed and the enantiomer purity of the compound of formula (5) is 98% or higher.

11. The method according to claim 1, wherein crystallization of (iii) is performed, the solvent is acetonitrile, and the enantiomer purity of the compound of formula (5) is 98% or higher.

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