Process for preparation of lotilaner

Abstract

The present disclosure encompasses a process for preparing Lotilaner in high purity and quality. The process provides Lotilaner in high pharmaceutical grade and high yield. In some embodiments, there is provided a process for purifying benzyl (S)-3-methyl-5-(5-(3,4,5- trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl) thiophene-2-carboxylate (Compound A) comprising crystallizing a racemic form of Compound A while obtaining a mother-liquor ("ML") enriched with the S-enantiomer of Compound A, and isolating the S- enantiomer of Compound A from the ML. In some embodiments, there is provided a process for preparing Lotilaner comprising purifying benzyl (S)-3-methyl-5-(5-(3,4,5- trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydro isoxazol-3-yl) thiophene-2-carboxylate and converting the purified compound to Lotilaner.

Description

Attorney Docket: API092-W001 (2222-238 PCT)PROCESS FOR PREPARATION OF LOTILANERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, Indian Provisional Application No. 202511003536 filed January 15, 2025. The entire contents of the foregoing application are incorporated by reference herein.FIELD OF THE DISCLOSURE

[0002] The present disclosure encompasses a process for preparing Lotilaner in high purity and quality. The process provides Lotilaner in high pharmaceutical grade and high yield.BACKGROUND OF THE DISCLOSURE

[0003] Lotilaner, chemical name 3-Methyl-2V-[2-oxo-2-(2,2,2-trifluoroethylamino)ethyl]-5-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4 / f-l,2-oxazol-3-yl] thiophene-2-carboxamide, has the following chemical structure:

[0004] Lotilaner is a veterinary drug administered orally for the treatment and prevention of flea and tick infestations in dogs. Lotilaner has also been investigated for use in the killing of ticks attached to human skin. It is currently developed for the treatment of eye infection and blepharitis.

[0005] The compound is described in International Publication No. WO 2010 / 070068. Processes for preparing Lotilaner are disclosed in WO 2014090918, WO 2022016490 and WO 2022020585 and IPCOM000274548D. The entire contents of each of the foregoing publications is incorporated by reference herein.

[0006] The compound patent publication, WO 2010 / 070068 discloses general preparation of isooxazoline compounds, however the stereochemistry is not indicated, and the final compounds are only reported to be chemically purified by preparative HPLC. W02014090918 discloses a synthetic process for preparing Lotilaner, which includes kinetic resolution of the key intermediate 3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylic acid (referred to herein as Compound 5-Rac) using (R)-l-(p-tolyl)ethan-l -amine for the salt formation. WO 2022016490 and WOAttorney Docket: API092-W001 (2222-238 PCT)2022020585 disclose another synthetic process and describe improving the enantiomeric purity of the key intermediate 3-methyl-5-[(5S)-5-(3,4,5- trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazol-3-yl]thiophene-2-carboxylic acid (Referred to herein as Compound 5) by crystallization from specified solvents.

[0007] The present disclosure provides a process for obtaining Lotilaner at high chemical and enantiomeric purity, which is of extreme importance for preparing high-quality pharmaceutical composition, particularly sterile pharmaceutical composition for treatment of eye-diseases.SUMMARY OF THE INVENTION

[0008] The present disclosure encompasses a process for preparing Lotilaner in high purity and quality. The process provides Lotilaner in high pharmaceutical grade and high yield.

[0009] In one embodiment, the present disclosure encompasses a process for purifying benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl) thiophene-2-carboxylate (referred to herein as Compound A) comprising crystallizing a racemic form of Compound A while obtaining a mother-liquor ("ML") enriched with the S-enantiomer of Compound A, and isolating the S -enantiomer of Compound A from the ML.

[0010] Particularly, the present disclosure provides a process for purifying benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (S-enantiomer of Compound A) comprising crystallizing a racemic form of Compound A while obtaining a mother-liquor enriched with the S-enantiomer of Compound A, and isolating the S-enantiomer of Compound A from the mother liquor.

[0011] The present disclosure further provides a process for purifying benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (S-enantiomer of Compound A) from a solution comprising S-enantiomer and R-enantiomer of Compound A and which is enriched in S-enantiomer, preferably in acetonitrile; comprising crystallizing racemic form of Compound A from the solution, to obtain a motherliquor which is further enriched with the S-enantiomer of Compound A, and isolating the S-enantiomer of Compound A from the mother liquor.

[0012] In another embodiment, the present disclosure encompasses Compound A having an enantiomeric purity of at least 99%, preferably at least 99.5%, more preferably at least 99.9%.Attorney Docket: API092-W001 (2222-238 PCT)

[0013] In one embodiment, the present disclosure encompasses the use of Compound A in preparing Lotilaner. In specific embodiment, the present disclosure encompasses the use of Compound A in preparing enantiomerically pure Lotilaner.

[0014] In another embodiment, the present disclosure encompasses a process for preparing Lotilaner comprising purifying benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate and converting the high-pure compound to Lotilaner.

[0015] In another embodiment, the present disclosure encompasses Lotilaner having an enantiomeric purity of at least 99%, preferably at least 99.5%, more preferably at least 99.9%, more preferably at least 99.95% prepared by the process of the present disclosure.DETAILED DESCRIPTION OF THE INVENTION

[0016] The present disclosure encompasses a process for preparing Lotilaner in high purity and quality. The process provides Lotilaner in high pharmaceutical grade and high yield.

[0017] The applicant of the present disclosure developed a process for preparing Lotilaner, and evaluated several means of enantiomeric purification, including those described in the earlier publications describing Lotilaner.

[0018] Enantiomeric purification of Compound 5 using chiral amines, for example, had inconsistent results, and resulted in Compound 5 having enantiomeric purity of lower than about 99%.

[0019] To further evaluate, the experimental procedures of WO 2022020585 were reviewed, and it was noted that the chiral purity of Compound 5 obtained in this publication is either low, or it can be achieved after several purification cycles, requiring careful control of specific process parameters and conditions and with expected lower yield and possible formation of Des-bromo impurity.

[0020] The present disclosure provides a process for obtaining a high yield of Lotilaner, at high chemical and enantiomeric purity, which is of extreme importance for preparing high-quality pharmaceutical composition, particularly sterile pharmaceutical composition for treatment of eye-diseases.

[0021] In particular, the process of the present disclosure provides a convenient and highly efficient preparation of enantiomerically pure Compound A which can be used to prepare Lotilaner having very high enantiomeric purity. The process does not require the use of chiral column chromatography, and can achieve a high enantiomeric purification of Compound A, typically in a single step. The process therefore provides a highly efficient production of anAttorney Docket: API092-W001 (2222-238 PCT)enantiomerically pure starting material for the preparation of Lotilaner. Moreover, the process of the present disclosure enables the consistent production of enantiomerically pure Compound A, thereby facilitating the production of enantiomerically pure Lotilaner.

[0022] As used herein, the term Compound 5 refers to (S)-3-methyl-5-(5-(3, 4, 5-trichlorophenyl)-5-(trifluoromethyl)-4, 5-dihydroisoxazol-3-yl) thiophene-2-carboxylic acid, of the following structure:Compound 5

[0023] As used herein, the term Compound 5-Rac refers to 3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylic acid- of the following structure:Compound 5-Rac

[0024] As used herein, the term Compound A or S-enantiomer of Compound A refers to benzyl (S)-3-methyl-5-(5-(3, 4, 5-trichlorophenyl)-5-(trifluoromethyl)-4, 5-dihydroisoxazol-3-yl) thiophene-2-carboxylate, of the following structure:Compound A or S-enantiomer of Compound A

[0025] As used herein, the term R- enantiomer of Compound A refers to benzyl (R)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate, of the following structure:Attorney Docket: API092-W001 (2222-238 PCT)R-enantiomer of Compound A

[0026] As used herein, the term racemic form of Compound A or Compound A-Rac refers to benzyl 3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate of the following structure:Compound A-Rac

[0027] A compound may be referred to herein as chemically pure or purified compound or as substantially free of any other compounds. As used herein, the terms "chemically pure" or "purified" or "substantially free of any other compounds" refer to a compound that is substantially free of any impurities including enantiomers of the subject compound, diastereomers or other isomers. A chemically pure or purified compound or a compound that is substantially free of any other compound will be understood to mean that it contains will be understood to mean that the pure compound contains about 20% (w / w) or less, about 10% (w / w) or less, about 5% (w / w) or less, about 2% (w / w) or less, about 1% (w / w) or less, or about 0% of any other compound as measured, for example, by HPLC. Thus, pure or purified Lotilaner or Lotilaner intermediate described herein as substantially free of any compounds would be understood to contain greater than about 80% (w / w), greater than about 90% (w / w), greater than about 95% (w / w), greater than about 98% (w / w), greater than about 99% (w / w), or about 100% of the subject Lotilaner or Lotilaner intermediate. In some embodiments of the disclosure, the described pure or purified Lotilaner or Lotilaner intermediate may contain from about 1% to about 20% (w / w), from about 5% to about 20% (w / w), or from about 5% to about 10% (w / w) of one or more other compounds. Particularly, according to any aspect or embodiment, Lotilaner or Lotilaner intermediate, which is substantially free of any otherAttorney Docket: API092-W001 (2222-238 PCT)compounds, may contain no more than about 5%, no more than about 3%, no more than about 2%, no more than about 1%, no more than about 0.5%, no more than about 0.2%, no more than about 0.1% or no more than about 0.05%, of any other compounds.

[0028] A thing, e.g., a reaction mixture, may be characterized herein as being at, or allowed to come to “room temperature” or “ambient temperature,” often abbreviated as “RT.” This means that the temperature of the thing is close to, or the same as, that of the space, e.g., the room or fume hood, in which the thing is located. Typically, room temperature is from about 20°C to about 30°C, or about 22 °C to about 27°C, or about 25°C.

[0029] The amount of solvent employed in a chemical process, e.g., a reaction or crystallization, may be referred to herein as a number of “volumes” or “vol” or “V.” For example, a material may be referred to as being suspended in 10 volumes (or 10 vol or 10V) of a solvent. In this context, this expression would be understood to mean milliliters of the solvent per gram of the material being suspended, such that suspending 5 grams of a material in 10 volumes of a solvent means that the solvent is used in an amount of 10 milliliters of the solvent per gram of the material that is being suspended or, in this example, 50 mL of the solvent. In another context, the term "v / v" may be used to indicate the number of volumes of a solvent that are added to a liquid mixture based on the volume of that mixture. For example, adding solvent X (1.5 v / v) to a 100 ml reaction mixture would indicate that 150 mL of solvent X was added.

[0030] A process or step may be referred to herein as being carried out "overnight. " This refers to a time interval, e.g., for the process or step, that spans the time during the night, when that process or step may not be actively observed. This time interval is from about 8 to about 20 hours, or about 10-18 hours, in some cases about 16 hours.

[0031] As used herein, the term “reduced pressure” refers to a pressure that is less than atmospheric pressure. For example, reduced pressure is about 10 mbar to about 50 mbar.

[0032] As used herein and unless indicated otherwise, the term "ambient conditions" refer to atmospheric pressure and a temperature of from about 22°C to about 28°C or from about 22°C to about 24°C.

[0033] As used herein, the term “crude Compound A” preferably refers to Compound A which contains, consists essentially of, or consists of, a mixture of S- and R-enantiomers of Compound A, and which is enriched in S-enantiomer. As used herein, the term “a solution containing a mixture of S-enantiomer and R-enantiomer of Compound A which is enriched in S-enantiomer” refers to a solution containing, consisting essentially of, or consisting of, a mixture of S- and R-enantiomers of Compound A which is enriched in S-enantiomer ofAttorney Docket: API092-W001 (2222-238 PCT)Compound A, in a solvent (preferably in any aspect or embodiment of the disclosure, the solvent is acetonitrile). Particularly, according to any aspect or embodiment the ratio of S-enantiomer of Compound A to R-enantiomer of Compound A is: about 60:40 to about 95:5, or about 65:35 to about 90:10; or about 70:30 to about 92:8, or about 75:25 to about 91:9. More particularly, the ratio of S -enantiomer of Compound A to R-enantiomer of Compound A is about 80:20 to about 90:10; or about 85:15 to about 89:11; or about 82:18 to about 91:9; or about 88:12 to about 90:9; or about 84:16 to about 90:10, or about 84 :16 to about 89:11; or about 85:15 to about 89:11. Preferably, the crude Compound A which is enriched in S-enantiomer may be isolated from a reaction mixture, for example by following reaction-work up. Particularly, “crude Compound A” refers to Compound A which can be obtained by extraction after reaction work up. Preferably, “crude Compound A” that is especially suitable for the process of the present disclosure may be obtained by reaction of benzyl 3-methyl-5-(4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)but-2-enoyl)thiophene-2-carboxylate, (2S)-1-(anthracen-9-ylmethyl)-2-((R)-hydroxy (6-methoxyquinolin-4-yl) methyl)-5-vinylquinuclidin-l-ium chloride and N-hydroxylamine or a salt thereof, and a base, in a solvent (preferably wherein the solvent is a mixture of dichloromethane and methyl tert-butylether).

[0034] The present disclosure encompasses a process for preparing Lotilaner in high purity and quality. The process provides Lotilaner in high pharmaceutical grade and high yield.

[0035] In one embodiment, the present disclosure encompasses a process for purifying benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (referred to herein as Compound A) comprising crystallizing a racemic form of Compound A while obtaining a mother-liquor ("ML") enriched with the S-enantiomer of Compound A, which is the desired enantiomeric form. The S -enantiomer of Compound A can then be isolated by crystallization, thus forming isolated solid form with improved enantiomeric purity. Typically, the enriched ML contain the S-enantiomer of Compound A in amount of at least from about 99.0% to about 99.3%.

[0036] The present disclosure also provides a process for purifying benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (S-enantiomer of Compound A) from a solution comprising S-enantiomer and R-enantiomer of Compound A and which is enriched in S-enantiomer, preferably in acetonitrile; comprising crystallizing racemic form of Compound A from the solution, to obtain a motherliquor which is further enriched with the S-enantiomer of Compound A, and isolating the S-enantiomer of Compound A from the mother liquor.Attorney Docket: API092-W001 (2222-238 PCT)

[0037] In specific embodiments, the above process comprises:a. Obtaining a solution of crude Compound A at a certain enantiomeric ratio in a solvent selected from acetonitrile, MTBE, methanol, ethyl acetate, iso propyl alcohol and heptane or mixture thereof;b. Crystallizing racemic form of Compound Ac. Isolating the crystallized racemic form of Compound A while the S-enantiomer of Compound A is maintained in the mother-liquord. Crystallizing the S-enantiomer of Compound A enantiomer from the mother- liquor e. Isolating the S-enantiomer of Compound A.

[0038] The present disclosure may comprise a process comprising:a. obtaining a solution containing a mixture of S-enantiomer and R-enantiomer of Compound A which is enriched in S-enantiomer, preferably at an enantiomeric ratio of S:R Compound A of: about 55:45 to about 98:2, in acetonitrile; b. crystallizing racemic form of Compound A from the solution;c. isolating the crystallized racemic form of Compound A while the S-enantiomer of Compound A is maintained in the mother-liquor;d. crystallizing the S-enantiomer of Compound A enantiomer from the mother-liquor;ande. isolating the S-enantiomer of Compound A.

[0039] The solution may contain Compound A as a mixture of S- and R-enantiomers in an enantiomeric ratio of: 60:40 to about 95:5, or about 65:35 to about 90:10; or about 70:30 to about 92:8, or about 75:25 to about 91:9, or about 80:20 to about 90:10; or about 85:15 to about 89:11. Alternatively, the solution may contain Compound A as a mixture of S- and R-enantiomers in an enantiomeric ratio of: about 80:20 to about 90:10; or about 82:18 to about 91:9; or about 88:12 to about 90:9; or about 84:16 to about 90:10, or about 84 :16 to about 89:11; or about 85:15 to about 89:11.

[0040] Typically, the mixture in step a) contains Compound A in enantiomeric ratio of S-enantiomer of Compound A to R-enantiomer of Compound A of about 80 to about 20, for example 85:15. Preferred solvents for the above described process are dichloromethane and methyl tert-butyl ether mixture (ratio about 1:1) for the reaction step, acetonitrile for the crystallization of racemic Compound A and acetonitrile and methanol mixture (ratio about 1 :2) for the crystallization of the S-enantiomer of Compound A. The solution is formed at temperature of from about 40°C to about 60°C.To achieve the crystallization in step b, the solution can be cooled down, to a temperature in which a solid forms, typically to a temperatureAttorney Docket: API092-W001 (2222-238 PCT)of from about 20°C to about 30°C to 0 to 10°C. The cooling can be achieved by maintain the solution at a temperature of about room temperature, or alternatively by actively cooling the solution, for example by applying cold water bath. After the solid is isolated, the mother-liquor is taken for additional crystallization, by adding an antisolvent selected from acetonitrile, MTBE, methanol, ethyl acetate, iso propyl alcohol and heptane or mixture thereof. Preferred solvents in this step are methanol and acetonitrile mixtures (preferred ratio about 1:2), particularly methanol. To aid crystallization, a seed of S-enantiomer of Compound A can be added. The obtained solid can then be filtered and isolated, yielding Compound A having enantiomeric purity of 99% or higher (undesired enantiomer <0.1%).

[0041] In any aspect or embodiment of the disclosure, the solution comprising S-enantiomer and R-enantiomer of Compound A and which is enriched in S-enantiomer, is at a temperature of: about 25°C to about 85°C, about 30°C to about 75°C, about 35°C to about 70°C, about 35°C to about 65°C, about 40°C to about 60°C, or about 40°C to about 50°C. Alternatively, the solution may be at a temperature of about 40°C to about 60°C, or about 40°C to about 50°C.

[0042] According to any aspect or embodiment of the disclosure, crystallization of racemic form of Compound A may be effected by cooling the solution, optionally to about room temperature. Alternatively, according to any aspect or embodiment, crystallization of racemic form of Compound A may be effected by cooling the solution to a temperature of: about 0°C to about 30°C, about 5°C to about 30°C, about 10°C to about 30°C, about 20°C to about 30°C, or about 0°C to about 20°C, about 0°C to about 15°C, about 0°C to about 10°C, or about 0°C to about 5°C. The cooling may alternatively be to a temperature of; about 20°C to about 30°C, or to 0 to 10°C.

[0043] The cooling may be active or passive. For example, the solution may be allowed to cool, for example, to room temperature, or actively cooled. Cooling may be effective over a period of: about 0.25 hours to about 10 hours, about 0.5 hours to about 8 hours, about 0.5 hours to about 6 hours, about 1 hour to about 5 hours, or about 2 hours to about 3 hours.

[0044] The resulting solid racemic mixture of S-Compound A and R-Compound A may be isolated and removed by filtration, decantation or by centrifuge, preferably by filtration. The solution obtained after removal of racemic Compound A (i.e. the mother liquor), typically contains S-Compound A having an enantiomeric purity of 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher or 99.5% or higher.Attorney Docket: API092-W001 (2222-238 PCT)

[0045] According to any aspect or embodiment of the disclosure, S -Compound A may be isolated from the mother liquor by crystallization. Crystallization may be effected by the use of a mixture of acetonitrile and antisolvent.

[0046] According to any aspect or embodiment of the disclosure, S -Compound A may be isolated directly from the mother liquor by combining the mother liquor with an antisolvent. Alternatively, S -enantiomer of Compound A may be isolated by partial evaporation of the mother liquor to form a concentrated mother liquor and combining the concentrated mother liquor with an antisolvent. Alternatively, the S -enantiomer of Compound A may be isolated by evaporation of the solvent from the mother liquor and crystallization from a mixture of acetonitrile and an antisolvent. Conveniently, S -enantiomer of Compound A may be isolated from the mother liquor by combining the mother liquor with an antisolvent. The mother liquor preferably contains acetonitrile in an amount of: about 2V to about 9V, about 3V to about 8V, about 4V to about 7V, about 5 V to about 7V, or about 6V of acetonitrile (relative to Compound . The mother liquor obtained after removal of racemic Compound A may be concentrate, for example by distillation, in order to achieve the preferred concentration range of acetonitrile.

[0047] According to any aspect or embodiment of the disclosure, the S-enantiomer of Compound A may be isolated directly from the mother liquor by combining the mother liquor with an antisolvent, preferably selected from: MTBE, methanol, ethyl acetate, isopropyl alcohol and heptane, or a mixture thereof, and more preferably wherein the antisolvent is methanol.

[0048] According to any aspect or embodiment of the disclosure, the mother liquor (or concentrated mother liquor) is cooled prior to combining with an antisolvent. Preferably, the mother liquor is cooled to a temperature of: about -10°C to about 15°C, about -5°C to about 10°C or about 0°C to about 10°C, prior to combining with the antisolvent.

[0049] According to any aspect or embodiment of the disclosure, the vol / vol ratio of acetonitrile and antisolvent (preferably wherein the antisolvent is methanol) for crystallizing the S-enantiomer of Compound A is in a ratio (v / v) of: about 1:1 to about 1:5, about 1:1 to about 1:4, about 1:1.5 to about 1:3, about 1:1.8 to about 1:2.5, about 1:1.8 to about 1:2.2, or about 1:2. Preferably the antisolvent is methanol, and the ratio of acetonitrile to methanol (vol / vol) is about 1:2.

[0050] According to any aspect or embodiment of the disclosure, a seed of S-enantiomer of Compound A may be added to the mixture during the addition of the antisolvent, in order to facilitate crystallization.Attorney Docket: API092-W001 (2222-238 PCT)

[0051] According to any aspect or embodiment of the disclosure, S-enantiomer of Compound A may be isolated by filtration, centrifuge or decantation, preferably by filtration.

[0052] According to any aspect or embodiment of the present disclosure, there is provided a process according for purifying benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (S-enantiomer of Compound A) from a solution comprising S-enantiomer and R-enantiomer of Compound A and which is enriched in S-enantiomer, in acetonitrile; wherein the process comprises crystallizing racemic form of Compound A from the solution by cooling; removing the racemic form of Compound A from the solution by filtration to obtain a mother-liquor which is further enriched with the S-enantiomer of Compound A, optionally concentrating the mother-liquor, combining the mother liquor with methanol to crystallize the S-enantiomer of Compound A; and isolating the S-enantiomer of Compound A from by filtration.

[0053] According to any aspect or embodiment of the disclosure, the starting material for the purification of Compound A is a mixture of S-enantiomer and R-enantiomer of Compound A which is enriched in S-enantiomer which is obtained by reaction of benzyl 3-methyl-5-(4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)but-2-enoyl)thiophene-2-carboxylate, (2S)-l-(anthracen-9-ylmethyl)-2-((R)-hydroxy(6-methoxyquinolin-4-yl)methyl)-5-vinylquinuclidin-l-ium chloride and N-hydroxylamine or a salt thereof, and a base, in a solvent (preferably wherein the solvent is a mixture of dichloromethane and methyl tert-butylether, more preferably in a v / v ratio of 1:1). The process may comprise providing a mixture of benzyl 3-methyl-5-(4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)but-2-enoyl)thiophene-2-carboxylate and (2S)-1-(anthracen-9-ylmethyl)-2-((R)-hydroxy (6-methoxyquinolin-4-yl)methyl)-5-vinylquinuclidin-1-ium chloride in the solvent, cooling the resulting mixture to a temperature of: about -25 °C to about 0°C, about -20°C to about -5°C, or about -15°C to about -10°C, and combining the cooled mixture with hydroxylamine, and the base, preferably wherein the hydroxylamine is hydroxylamine hydrochloride. Preferably, the base is an aqueous solution of an alkali metal hydroxide, preferably lithium hydroxide, sodium hydroxide or potassium hydroxide, and more preferably wherein the base is an aqueous solution of sodium hydroxide. The reaction mixture may be maintained at a temperature of: about -25°C to about 0°C, about -20°C to about -5°C, about -15°C to about -10°C, and preferably at about -15°C to about -10°C, preferably for a period of about 1 to about 10 hours, about 2 to about 8 hours, about 3 to about 7 hours, or about 4 to about 6 hours. Following completion of the reaction, the reaction mixture may be quenched with an aqueous mineral acid, preferably hydrochloric acid. The product may be isolated from the reaction mixture by filtration, followed by layer separation to obtain an organic layer, andAttorney Docket: API092-W001 (2222-238 PCT)concentration of the organic layer. Typically, the product from this process contains, consists essentially of, or consists of, Compound A having a ratio of S -enantiomer of Compound A to R-enantiomer of Compound A is about 80:20 to about 90:10; or about 85:15 to about 89:11; or about 82:18 to about 91:9; or about 88:12 to about 90:9; or about 84:16 to about 90:10, or about 84 :16 to about 89:11; or about 85:15 to about 89:11.

[0054] In another embodiment, the present disclosure encompasses Compound A having an enantiomeric purity of 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher or 99.5% or higher.

[0055] The above Compound A of the above described enantiomeric purity can then be used to prepare Lotilaner. In embodiment, the present disclosure encompasses the use of Compound A in preparing Lotilaner. In specific embodiment, the present disclosure encompasses the use of Compound A in preparing enantiomerically pure Lotilaner.

[0056] The present disclosure encompasses a process for preparing Lotilaner comprising purifying benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl) thiophene-2-carboxylate and converting the high-pure compound to Lotilaner. The process can comprise purifying Compound A by the process described above, and converting it to Lotilaner. The conversion can comprise converting Compound A to Compound 5, and reacting Compound 5 and 2-amino-N-(2,2,2-trifluoroethyl)acetamide, or a salt thereof, such as hydrochloride, to obtain Lotilaner. Compound A is reacted with a base, preferably in the presence of a solvent, such as acetonitrile, methanol, 2-Butanol, acetone or THF; preferably acetonitrile. The reaction of Compound 5 and 2-amino-N-(2,2,2-trifluoroethyl)acetamide can be done in a presence of a coupling reagent, for example 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide ("EDC") or hydroxy benzotriazole ("HOBT"), a solvent such as MDC, toluene, acetonitrile, DMF or EtOAct- preferably ethyl acetate; and a base, such as triethylamine ("TEA"), Diisopropylethylamine ("DIPEA"), particularly DIPEA.

[0057] Lotilaner obtained by the above described process is achieved in high yield and purity. In particular embodiment, the present disclosure encompasses Lotilaner having an enantiomeric purity of at least 99.9% and having an HPLC purity at least 99.9% of prepared by the process of the present disclosure.

[0058] Having thus described the disclosure with reference to particular preferred embodiments and illustrative examples, those in the art can appreciate modifications to the disclosure as described and illustrated that do not depart from the spirit and scope of the disclosure as disclosed in the specification. The Examples are set forth to aid in understanding the disclosure but are not intended to, and should not be construed to limit its scope in any way.Attorney Docket: API092-W001 (2222-238 PCT)EXAMPLESPreparation of starting materials

[0059] Compound A can be prepared by the process as described in record IPCOM000274548D available in IPCOM database (wherein it has been referred to in IPCOM000274548D as compound 7).Preparation of Starting material: benzyl 3-methyl-5-(4,4,4-trifluoro-3-(3,4, 5-trichlorophenyl) but-2-enoyl) thiophene-2-carboxylate

[0060] 5-acetyl-3-methylthiophene-2-carboxylic acid (25 gm) and carbonyl diimidazole (24.20 gm) were added to MTBE (120 ml) was added under nitrogen atmosphere. The mass was heated to a temperature of about 48-54°C and maintained for about 2 to about 4 hours. After completion of the reaction, phenyl methanol (14.67 gm) was added dropwise and the mixture maintained for about 2 to about 4 hours. After completion of reaction, the mass was cooled to about 20-30°C and then quenched by addition of water, followed by layer separation and water washing. The separated organic layer was distilled to obtain benzyl 5-acetyl-3-methylthiophene-2-carboxylate having HPLC purity of >98%. The resultant oily residue was dissolved in methyl tert-butyl ether ("MTBE", 500 ml) under nitrogen atmosphere and was charged into reactor. Then, triethylamine (41.1 gm) and 2, 2, 2-trifluoro-l-(3, 4, 5-trichlorophenyl) ethan-l-one (37.65 gm) were added simultaneously at about 20-30°C and a clear solution was formed. The solution was heated to about 48-54°C and maintained for about 2 to 4 hours. After completion of reaction, the mass cooled to -15 and thionyl chloride (32.30 gm) was added dropwise. The reaction mass was heated to a temperature of about 20 to 30°C. The reaction mass maintained with stirring for about 2 to 3 hours. After confirming reaction completion the mass cooled to about 0°C to about 10°C and quenched with aq. sodium bicarbonate solution. The reaction mass was heated to about 20-30°C, the mass filtered through celite bed to remove inorganic impurities and layer separated. Obtained organic layer distilled and crude oily compound crystallized in IP A: water mixture. The solid isolated by filtration and dried under vacuum at 50-60°C to obtain benzyl 3-methyl-5-(4, 4, 4-trifluoro-3-(3, 4, 5-trichlorophenyl) but-2-enoyl) thiophene-2-carboxylate (58.0 gm) having HPLC purity >95%Example 1: Preparation of Compound A with improved enantiomeric purity

[0061] Benzyl 3-methyl-5-(4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)but-2-enoyl)thiophene-2-carboxylate (50.0 gm) and (2S)-l-(anthracen-9-ylmethyl)-2-((R)-hydroxy(6-methoxyquinolin-4-yl)methyl)-5-vinylquinuclidin-l-ium chloride (5.0 gm) wereAttorney Docket: API092-W001 (2222-238 PCT)added to a solvent mixture of dichloromethane (MDC) and MTBE (ratio 1:1, total volume 1000 ml) at a temperature of 20-30°C. The mass was cooled to a temperature of from about -15°C to -10°C. Then, aqueous hydroxylamine hydrochloride solution (19.52 gm + 39.54 gm DM water) and aqueous sodium hydroxide solution (22.44 gm + 47.77 gm DM Water) were slowly added simultaneously. The resultant mass stirred for about 4 to 6 hours at a temperature of about from about -15 to about -10°C. After confirming reaction completion, raise temperature of mass to 20-30°C and the mass quenched by adding 2N aq. HC1 solution and filtered through hyflo bead to remove particles. Then, the filtered mixture was transferred to a reactor and the layers were separated. The organic layer thus obtained after layer separation was distilled to obtain benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (Compound A) having enantiomeric ratio of S-enantiomer to R- enantiomer of about 85:15.

[0062] The thus-obtained oily compound was crystallized in acetonitrile (500 ml) followed by filtration to obtain racemic benzyl 3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate. The mother liquor, which contained enriched Compound A having undesired enantiomer < 1.0% was distilled off to 6V and cooled to about 0 tolO°C °C and to this mass a seed of pure S -enantiomer of Compound A and methanol (12V) were added, so benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (Compound A, S-enantiomer) was crystallized. The solid was isolated by filtration and suck dried to obtain white to off white solid (29.0 gm) having undesired enantiomer <0.1% with an HPLC purity of >98%.Example 2: Preparation of Compound 5

[0063] Enantiomerically pure Compound A was used to prepare Compound 5, as follows:

[0064] Benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (Compound A, 88.0 gm) and sodium hydroxide (19.24 gm) were added to acetonitrile: water solvent mixture (ratio 1:1, total volume 528 ml) at a temperature of about 20°C-30°C. The reaction mass was heated to about 50°C-60°C and the mass maintained with stirring for a period of about 2 to 3 hours. After confirming reaction completion, reaction work up done by adding aqueous HC1 solution at a temperature of about 15°C-20°C until pH adjusted to a range of about 2-2.5. A solid precipitated and was isolated by filtration followed by 2-3 times washing with a mixture of acetonitrile: water to remove residual benzyl alcohol obtained as by product during reaction) (ratio 1:2, total volume 300 mlAttorney Docket: API092-W001 (2222-238 PCT)X 2-3 times). The solid was then suck-dried under vacuum to obtain wet (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylic acid (Compound 5). The obtained wet compound was dried under vacuum at about 50°C -60°C to obtain dry (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylic acid (Compound 5, 62.0 gm) as a white to off white solid having an HPLC purity >99.5% with an ee of 99.9%.Example 3: Preparation of Lotilaner

[0065] Compound 5 was used to prepare Lotilaner, as follows:

[0066] Ethyl acetate (825 ml), 2-amino-N-(2,2,2-trifluoroethyl) acetamide hydrochloride (27.71 gm) , DIPEA (23.25 gm) were added to (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylic acid (Compound 5, 55.0 gm). The mass maintained with stirring for about 30-40 minutes at a temperature of about 20°C -30°C to obtain clear solution. The solution was cooled to a temperature of about 10°C to about 15°C and to then hydroxy benzotriazole monohydrate ("HOBt.ILO" 4.86 gm) was added, followed by l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ("EDC.HQ", 27.59 gr). The reaction mass was stirred for about 4 to about 6 hours. After completion of reaction, the mass quenched by adding aqueous HC1 solution and the layer separated. The organic layer washed with aqueous NaHCO3 solution and DM water. The organic layer distilled under vacuum to obtain crude (S)-3-methyl-N-(2-oxo-2-((2,2,2-trifluoroethyl) amino)ethyl)-5-(5-(3,4,5- trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (Lotilaner) as an oily mass. The obtained crude material was crystallized from a mixture of 2-Butanol:n-heptane (ratioLlO, total volume 2118 ml) and the solid was isolated, filtered and washed with n-heptane (275 ml); then and under vacuum at about 45°C -55°C to get dry (S)-3-methyl-N-(2-oxo-2-((2,2,2-trifhroroethyl)amino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (Lotilaner, 58 gm) having HPLC purity >99.8% with an ee of >99.9%.Example 4: Preparation of Compound A with improved enantiomeric purity

[0067] Benzyl 3-methyl-5-(4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)but-2-enoyl) thiophene-2-carboxylate (50.0 gm) and (2S)-l-(anthracen-9-ylmethyl)-2-((R)-hydroxy(6-methoxyquinolin-4-yl)methyl)-5-vinylquinuclidin-l-ium chloride (5.0 gm) were added to a mixture of dichloromethane (MDC) and MTBE (ratio 1:1, total volume 1000 ml) at a temperature of 20-30°C. The mixture was cooled to a temperature of about -15°C to -10°C.Attorney Docket: API092-W001 (2222-238 PCT)Aqueous hydroxylamine hydrochloride solution [19.52 gm in 39.54 gm demineralized (DM) water] and aqueous sodium hydroxide solution (22.44 gm in 47.77 gm DM water) were slowly added simultaneously. The resultant mass was stirred for about 4 to 6 hours at a temperature of about -15 to about -10°C. Following reaction completion, the temperature of the reaction mixture was raised to 20-30°C and the mixture was quenched by the addition of 2N aq. HC1 solution. The resulting mixture was filtered through a hyflo bed to remove particles. The filtrate was transferred to a reactor and the layers were separated. The organic layer was distilled under vacuum at 40-50°C to leave ~1.5 to 2.5 volume of solvent (MDC:MTBE) along with benzyl (S)-3-methyl-5-(5-(3, 4, 5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (Compound A) enriched in S-enantiomer (about 85:15 of S-enantiomer to R-enantiomer).

[0068] o the thus-obtained compound A solution dissolved in a mixture of MDC:MTBE was added acetonitrile (5 vol*2) and subjected to vacuum distillation under vacuum at 40-50°C to leave -1.5 to 2.5 volume of solvent (acetonitrile) along with benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (Compound A) having enantiomeric ratio of S-enantiomer to R-enantiomer of about 85:15. To the resulting solution was added acetonitrile (8.0 vol) at 40-50°C and the mixture was cooled to 20-30°C over 2-4 hr and stirred for 2 to 3 hr. The crystallized solid was filtered to obtain racemic benzyl 3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate. The resulting mother liquor, which contained enriched Compound A having undesired enantiomer < 1.0%, was distilled off to 6V under vacuum at 40-50°C. The resulting concentrated mass was cooled to about 0 to 10°C, and a seed of pure S-enantiomer of Compound A and methanol (12 vol) were added at 0 to 10°C to crystallize benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4, 5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (Compound A, S-enantiomer). This mass was further stirred at 0 to 10°C for 2 to 3 hr, solid filtered at 0 to 10°C. Wet cake obtained is suck dried for 2 to 3 hr to obtain a white to off-white solid (29.0 gm) having undesired enantiomer <0.1% with an HPLC purity of >98%. Compound A, S-enantiomer may be used to prepare Compound 5 as described above.Comparative example 1: Preparation of (S)-3-methyl-5-(5-(3, 4, 5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylic acid (Compound 5)

[0069] Benzyl 3-methyl-5-(4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)but-2-enoyl)thiophene-2-carboxylate (80.0 gm) and (2S)-l-(anthracen-9-ylmethyl)-2-((R)-Attorney Docket: API092-W001 (2222-238 PCT)hydroxy(6-methoxyquinolin-4-yl)methyl)-5-vinylquinuclidin-l-ium chloride (8.25 gm) were added to a solvent mixture of MDC:MTBE (Ratio 1:1, total volume 1600 ml) at a temperature of about 20°C to 30°C. The mass was cooled to a temperature of about -15 to -10°C. Aqueous hydroxylamine hydrochloride solution and aqueous sodium hydroxide solution were simultaneously added slowly. The obtained mass was stirred for a period of about 4 to 6 hours at a temperature of about -15°C to 10°C. After confirming reaction completion, the mass heated to 20-30°C and the mass quenched by adding 2N of aqueous HC1 solution and filtered through hyflo bed to remove particles, and the layers were separated. Then, the filtered mixture was transferred to a reactor and the layers were separated. The organic layer thus obtained after layer separation was distilled to obtain benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (Compound A) as an oily mass having enantiomeric ratio of S -enantiomer to R- enantiomer of about 85:15 and having an HPLC purity of >94%. To this mass, a mixture of methanol: water (ratio 1:1, total volume 800 ml) and sodium hydroxide (18.0 gm) at were added at about 20°C to about 30°C. The reaction mass was heated to about 50°C to about 60°C and maintained with stirring. After confirming reaction completion, the mass cooled to about 15 to about 20°C and quenched by adding aqueous HC1 solution. The solid precipitated and isolated by filtration followed by acetonitrile: water mixture slurry washing. Then the solid was suck-dried under vacuum to obtain wet (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylic acid (Compound 5) having enantiomeric ratio of S-enantiomer to R- enantiomer of about 85:15, with an HPLC purity of >94%. The obtained wet compound was dried under vacuum at about 50°C to about 60°C to get (57 gm) product.Comparative example 2: Preparation of (S)-l-phenylethan-l-aminium (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate using (S)-l-phenylethan-l-amine

[0070] (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylic acid (Compound 5, 1.0 gm) was added to ethyl acetate (10 ml) at about 20°C to about 30°C and a clear solution formed. (S)-l-phenylethan-l-amine (0.21 gm) was added and the mixture was maintained with stirring. A solid precipitated and the suspension stirred for about 5-6 hours, then the solid isolated by filtration followed by suck drying and vacuum drying at about 50°C to about 60°C to get dry (S)-l-phenylethan-l-aminium (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-Attorney Docket: API092-W001 (2222-238 PCT)3-yl)thiophene-2-carboxylate ((S)-l-phenylethan-l-aminium salt of Compound 5, 0.6 gm) as white solid having an HPLC purity >99% with ee of >98%.Comparative example 3: Preparation of (R)-l-phenylethan-l-aminium (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate using (R)-l-phenylethan-l-amine

[0071] (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylic acid (Compound 5, 8.0 gm) was added to a solvent mixture of acetonitrile: methanol (ratio 1:0.6, total volume 140 ml) at about 20°C to about 30°C and a clear solution was formed. (R)-l-phenylethan-l -amine (1.69 gm) was added, and the mixture maintained with stirring. A solid precipitated and the suspension stirred for about 10 to about 12 hours. The solid isolated by filtration followed by suck drying and vacuum drying at about 50°C to about 60°C to get dry (R)-l-phenylethan-l-aminium (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate ((R)-l-phenylethan-l-aminium salt of Compound 5, 4.0 gm) as white solid having an HPLC purity >99% with ee of >98%.Comparative example 4: Preparation of (S)-3-methyl-5-(5-(3, 4, 5-trichlorophenyl)-5-(trifluoromethyl)-4, 5- dihydroisoxazol-3-yl) thiophene-2-carboxylic acid (Compound 5)

[0072] The products obtained in Comparative example 2 and Comparative example 3 were converted to Compound 5 upon neutralization reaction using aqueous HC1, and afforded (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylic acid (Compound 5), according to the following procedure:

[0073] Toluene (76 ml) and IN aq HC1 solution (76 ml) were added to (S)-l-phenylethan-1-aminium (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (15.2 gm), under stirring. The reaction mixture was heated to a temperature of about 50°C to about 60°C and maintained with stirring for about 1-2 hours. The layer separated and the aqueous layer discarded. The organic layer concentrated and degassed to obtain (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylic acid (Compound 5, 11.5 gm) as white to off white solid having an HPLC purity >99% with ee of >97.9%.Attorney Docket: API092-W001 (2222-238 PCT)

[0074] Further aspects and embodiments of the present invention are set out in the numbered clauses below:1. A process for purifying benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5- (trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (S-enantiomer of Compound A) comprising crystallizing a racemic form of Compound A while obtaining a mother-liquor enriched with the S-enantiomer of Compound A, and isolating the S- enantiomer of Compound A from the mother liquor.2. A process according to Clause 1, for purifying benzyl (S)-3-methyl-5-(5-(3,4,5- trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (S-enantiomer of Compound A) from a solution comprising S-enantiomer and R- enantiomer of Compound A and which is enriched in S-enantiomer, preferably in acetonitrile; comprising crystallizing racemic form of Compound A from the solution, to obtain a mother-liquor which is further enriched with the S-enantiomer of Compound A, and isolating the S-enantiomer of Compound A from the mother liquor.3. A process according to any of Clause 1 and Clause 2, wherein the mother-liquor enriched with the S-enantiomer of Compound A contains the S-enantiomer of Compound A in amount of at least from about 99.0% to about 99.3%.4. A process according to according to any of Clauses 1, 2, and 3, which comprises:a. obtaining a solution of crude Compound A at a certain enantiomeric ratio in a solvent selected from acetonitrile, MTBE, methanol, ethyl acetate, isopropyl alcohol and heptane or mixture thereof, and preferably wherein the solvent is acetonitrile;b. crystallizing racemic form of Compound A;c. isolating the crystallized racemic form of Compound A while the S-enantiomer of Compound A is maintained in the mother-liquor;d. crystallizing the S-enantiomer of Compound A enantiomer from the mother-liquor;ande. isolating the S-enantiomer of Compound A.5. A process according to any of Clauses 1, 2, 3, and 4, which comprises:Attorney Docket: API092-W001 (2222-238 PCT)a. obtaining a solution containing a mixture of S -enantiomer and R-enantiomer of Compound A which is enriched in S-enantiomer, preferably at an enantiomeric ratio of S:R Compound A of: about 55:45 to about 98:2, in acetonitrile;b. crystallizing racemic form of Compound A from the solution;c. isolating the crystallized racemic form of Compound A while the S-enantiomer of Compound A is maintained in the mother-liquor;d. crystallizing the S-enantiomer of Compound A enantiomer from the mother-liquor;ande. isolating the S-enantiomer of Compound A.A process according to any of Clauses 2, 3, 4, and 5, wherein solution contains, consists essentially of, or consists of, a mixture of S- and R-enantiomers of Compound A which is enriched in S-enantiomer, in a solvent which is preferably acetonitrile, preferably wherein the S- and R-enantiomers are in an enantiomeric ratio of: 60:40 to about 95:5, or about 65:35 to about 90:10; or about 70:30 to about 92:8, or about 75:25 to about 91:9, or about 80:20 to about 90:10; or about 85:15 to about 89:11.A process according to any of Clauses 2, 3, 4, 5, and 6, wherein the solution contains, consists essentially of, or consists of, a mixture of S- and R-enantiomers of Compound A in a solvent which is preferably acetonitrile, wherein the S- and R-enantiomers are in an enantiomeric ratio of: about 80:20 to about 90:10; or about 82:18 to about 91:9; or about 88:12 to about 90:9; or about 84:16 to about 90:10, or about 84 :16 to about 89:11; or about 85:15 to about 89:11.A process according to any of Clauses 2, 3, 4, 5, 6, and 7, wherein the solution contains, consists essentially of, or consists of, Compound A in a solvent which is preferably acetonitrile, wherein the enantiomeric ratio of S-enantiomer of Compound A to R-enantiomer of Compound A is about 80 to about 20, for example 85:15.A process according to any of Clauses 2, 3, 4, 5, 6, 7, and 8, wherein the solution is at a temperature of: about 25°C to about 85°C, about 30°C to about 75°C, about 35°C to about 70°C, about 35 °C to about 65 °C, about 40°C to about 60°C, or about 40°C to about 50°C.Attorney Docket: API092-W001 (2222-238 PCT)A process according to Clause 9, wherein the solution is at a temperature of from about 40°C to about 60°C, or about 40°C to about 50°C.A process according to any of Clauses 2, 3, 4, 5, 6, 7, 8, 9, and 10, wherein the crystallization of racemic form of Compound A is carried out by cooling solution, preferably to about room temperature.A process according to any of Clauses 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11, wherein the crystallization of racemic form of Compound A is carried out by cooling the solution, preferably to a temperature of: about 0°C to about 30°C, about 5°C to about 30°C, about 10°C to about 30°C, about 20°C to about 30°C, or about 0°C to about 20°C, about 0°C to about 15°C, about 0°C to about 10°C, or about 0°C to about 5°C.A process according to any of Clauses 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, wherein the crystallization of racemic form of Compound A is carried out by cooling the solution, preferably to a temperature of from about 20°C to about 30°C to 0 to 10°C.A process according to any of Clauses 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13, wherein the crystallization of racemic form of Compound A is carried out by allowing the solution to cool, or by actively cooling the solution.A process according to any of Clauses 11, 12, 13, and 14, wherein the cooling is over a period of: about 0.25 hours to about 10 hours, about 0.5 hours to about 8 hours, about 0.5 hours to about 6 hours, about 1 hour to about 5 hours, or about 2 hours to about 3 hours.A process according to any of Clauses 11, 12, 13, 14, and 15, wherein the cooled mixture is stirred at the cooled temperature for a period of: about 0.5 to about 8 hours, about 0.5 to about 6 hours, about 1 to about 4 hours, or about 2 to about 3 hours.A process according to any of Clauses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16, wherein the crystallized racemic form of Compound A is isolated, preferably by filtration, decantation or centrifuge, preferably by filtration.Attorney Docket: API092-W001 (2222-238 PCT)A process according to any of Clauses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17, wherein the S -enantiomer of Compound A is isolated from the mother liquor by crystallization.A process according to any of Clauses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, wherein the S -enantiomer of Compound A is isolated from the mother liquor by crystallization from a mixture of acetonitrile and an antisolvent.A process according to any of Clauses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19, wherein the S-enantiomer of Compound A is isolated directly from the mother liquor by combining the mother liquor with an antisolvent; or wherein the S-enantiomer of Compound A is isolated by partial evaporation of the mother liquor to form a concentrated mother liquor and combining the concentrated mother liquor with an antisolvent; or wherein the S-enantiomer of Compound A is isolated by evaporation of the solvent from the mother liquor and crystallization from a mixture of acetonitrile and an antisolvent.A process according to any of Clauses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, wherein the mother liquor is obtained from crystallization from acetonitrile of racemic form of Compound A and filtration, and optionally partial evaporation to form a concentrated mother liquor.A process according to Clause 21, wherein the mother liquor is concentrated to form a solution comprising: about 2V to about 9V, about 3V to about 8V, about 4V to about 7V, about 5V to about 7V, or about 6V of acetonitrile.A process according to any of Clauses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, wherein the S-enantiomer of Compound A is isolated from the mother liquor by combining the mother liquor or concentrated mother liquor with an antisolvent selected from: MTBE, methanol, ethyl acetate, isopropyl alcohol and heptane, or a mixture thereof, and more preferably wherein the antisolvent is methanol.A process according to any of Clauses 20, 21, 22, and 23, wherein the mother liquor or concentrated mother liquor is cooled to a temperature of: about -10°C to about 15°C,Attorney Docket: API092-W001 (2222-238 PCT)about -5°C to about 10°C or about 0°C to about 10°C, prior to combining with the antisolvent.A process according to any of Clauses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24, wherein the acetonitrile and antisolvent are in a ratio (v / v) of: about 1:1 to about 1:5, about 1:1 to about 1:4, about 1:1.5 to about 1:3, about 1:1.8 to about 1:2.5, about 1:1.8 to about 1:2.2, or about 1:2; and preferably wherein the antisolvent is methanol.A process according to any of Clauses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25, wherein the antisolvent is methanol and the ratio of acetonitrile and methanol (v / v) is about 1:2.A process according to any of Clauses 20, 21, 22, 23, 24, 25, and 26, further comprising adding a seed of S -enantiomer of Compound A to the mixture comprising mother liquor or concentrated mother liquor and antisolvent.A process according to any of Clauses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27, wherein the S-enantiomer of Compound A is isolated by filtration, centrifuge or decantation, preferably by filtration.A process according to any of Clauses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28, for purifying benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (S-enantiomer of Compound A) from a solution comprising S-enantiomer and R-enantiomer of Compound A and which is enriched in S-enantiomer, in acetonitrile; wherein the process comprises crystallizing racemic form of Compound A from the solution by cooling; removing the racemic form of Compound A from the solution, preferably by filtration, to obtain a mother-liquor which is further enriched with the S-enantiomer of Compound A, optionally concentrating the mother-liquor, combining the mother liquor with an antisolvent, preferably methanol; and isolating the S-enantiomer of Compound A from the mother liquor, preferably by filtration.Attorney Docket: API092-W001 (2222-238 PCT)A process according to any preceding clause, wherein the S -isomer of Compound A has an enantiomeric purity of 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher or 99.5% or higher.A process according to any preceding clause wherein the S-enantiomer of Compound A is comprised in a mixture of S-enantiomer and R-enantiomer of Compound A which is enriched in S-enantiomer, and which is obtained by reaction of benzyl 3-methyl-5-(4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)but-2-enoyl)thiophene-2-carboxylate, (2S)-1- (anthracen-9-ylmethyl)-2-((R)-hydroxy(6-methoxyquinolin-4-yl)methyl)-5-vinylquinuclidin-l-ium chloride and N-hydroxylamine or a salt thereof, and a base, in a solvent (preferably wherein the solvent is a mixture of dichloromethane and methyl tertbutylether, more preferably in a v / v ratio of 1:1).A process according to Clause 31, comprising providing a mixture of benzyl 3-methyl-5-(4,4,4-trifluoro-3-(3,4,5-trichlorophenyl)but-2-enoyl)thiophene-2-carboxylate and (2S)-l-(anthracen-9-ylmethyl)-2-((R)-hydroxy(6-methoxyquinolin-4-yl)methyl)-5-vinylquinuclidin-l-ium chloride in the solvent, cooling the resulting mixture to a temperature of: about -25°C to about 0°C, about -20°C to about -5°C, or about -15°C to about -10°C, and combining the cooled mixture with hydroxylamine, and a base, preferably wherein the hydroxylamine is hydroxylamine hydrochloride.A process according to any of Clauses 31 and 32, wherein the base is an aqueous solution of an alkali metal hydroxide, preferably lithium hydroxide, sodium hydroxide or potassium hydroxide, and more preferably wherein the base is an aqueous solution of sodium hydroxide.A process according to any of Clauses 31, 32 and 33, wherein reaction mixture is maintained at a temperature of: about -25°C to about 0°C, about -20°C to about -5°C, about -15°C to about -10°C, and preferably at about -15°C to about -10°C, preferably for a period of about 1 to about 10 hours, about 2 to about 8 hours, about 3 to about 7 hours, or about 4 to about 6 hours.A process according to any of Clauses 31, 32, 33, and 34, wherein the reaction mixture is quenched with an aqueous mineral acid, preferably hydrochloric acid.Attorney Docket: API092-W001 (2222-238 PCT)A process according to any of Clauses 31, 32, 33, 34, and 35, wherein the reaction mixture is filtered to form a biphasic mixture, separating the organic layer and concentrating the organic layer to form a product comprising S -enantiomer and R-enantiomer of Compound A, which is enriched in S -enantiomer.A process according to any preceding clause further comprising converting the S-enantiomer of Compound A to Lotilaner.A process according to Clause 37, further comprising combining the Lotilaner with at least one pharmaceutically acceptable excipient to form a pharmaceutical composition or pharmaceutical formulation.Use of the S-enantiomer of Compound A obtained according to a process of any preceding clause, in the preparation of Lotilaner, preferably for preparing enantiomerically pure Lotilaner.A process for preparing Lotilaner comprising purifying benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl) thiophene-2-carboxylate, and converting the purified compound to Lotilaner.A process according to Clause 40, comprising purifying benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydro-isoxazol-3-yl) thiophene-2-carboxylate by a process according to any of Clauses 1-36, and converting it to Lotilaner.A process according to any of Clauses 40 and 41, comprising converting benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydro-isoxazol-3-yl) thiophene-2-carboxylate to Compound 5:Attorney Docket: API092-W001 (2222-238 PCT)and reacting Compound 5 and 2-amino-N-(2,2,2-trifluoroethyl)acetamide, or a salt thereof, preferably a hydrochloride salt, to obtain Lotilaner.A process according to Clause 42, wherein the benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydro-isoxazol-3-yl) thiophene-2-carboxylate is converted to Compound 5 by reaction with a base, preferably in the presence of a solvent.A process according to Clause 43, wherein the base is an alkali metal hydroxide, preferably lithium hydroxide, sodium hydroxide or potassium hydroxide, and more preferably sodium hydroxide.A process according to any of Clauses 43 and 44, wherein the solvent is acetonitrile, methanol, 2-butanol, acetone, tetrahydrofuran (THF) and water, or mixtures thereof; and preferably wherein the solvent is acetonitrile or a mixture of acetonitrile and water, and more preferably a mixture of acetonitrile and water.A process according to any of Clauses 42, 43, 44, and 45, wherein the reaction of Compound 5 and 2-amino-N-(2,2,2-trifluoroethyl)acetamide is carried out in the presence of a coupling reagent, a solvent, and a base.A process according to Clause 46, wherein the coupling agent is l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or hydroxy benzotriazole (HOBT), and preferably EDC.A process according to any of Clauses 46 and 47, wherein the solvent is dichloromethane (MDC), toluene, acetonitrile, dimethylformamide (DMF) or ethyl acetate (EtOAc), and preferably ethyl acetate.A process according to any of Clauses 46, 47 and 48, wherein the base is triethylamine (TEA) or diisopropylethylamine (DIPEA), and preferably DIPEA.A process according to any of Clauses 46, 47, 48, and 49, wherein the base is triethylamine ("TEA") or diisopropylethylamine ("DIPEA"), and particularly DIPEA]Attorney Docket: API092-W001 (2222-238 PCT)A process according to any of Clauses 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50, wherein the Lotilaner has an enantiomeric purity of at least 99.9% and having an HPLC purity at least 99.9%.Lotilaner having an enantiomeric purity of at least 99.9% and having an HPLC purity at least 99.9%.

Claims

Attorney Docket: API092-W001 (2222-238 PCT)CLAIMS1. A process for purifying benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5- (trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (S-enantiomer of Compound A) comprising crystallizing a racemic form of Compound A while obtaining a mother-liquor enriched with the S-enantiomer of Compound A, and isolating the S- enantiomer of Compound A from the mother liquor.

2. A process according to Claim 1, for purifying benzyl (S)-3-methyl-5-(5-(3,4,5- trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (S-enantiomer of Compound A) from a solution comprising S-enantiomer and R- enantiomer of Compound A and which is enriched in S-enantiomer, preferably in acetonitrile; comprising crystallizing racemic form of Compound A from the solution, to obtain a mother-liquor which is further enriched with the S-enantiomer of Compound A, and isolating the S-enantiomer of Compound A from the mother liquor.

3. A process according to any of Claim 1 and Claim 2, wherein the mother-liquor enriched with the S-enantiomer of Compound A contains the S-enantiomer of Compound A in amount of at least from about 99.0% to about 99.3%.

4. A process according to according to any of Claims 1, 2, and 3, which comprises:a. obtaining a solution of crude Compound A at a certain enantiomeric ratio in a solvent selected from acetonitrile, MTBE, methanol, ethyl acetate, isopropyl alcohol and heptane or mixture thereof, and preferably wherein the solvent is acetonitrile;b. crystallizing racemic form of Compound A;c. isolating the crystallized racemic form of Compound A while the S-enantiomer of Compound A is maintained in the mother-liquor;d. crystallizing the S-enantiomer of Compound A enantiomer from the mother-liquor;ande. isolating the S-enantiomer of Compound A.Attorney Docket: API092-W001 (2222-238 PCT)5. A process according to any of Claims 1, 2, 3, and 4, which comprises:a. obtaining a solution containing a mixture of S -enantiomer and R-enantiomer of Compound A which is enriched in S-enantiomer, preferably at an enantiomeric ratio of S:R Compound A of: about 55:45 to about 98:2, in acetonitrile; b. crystallizing racemic form of Compound A from the solution;c. isolating the crystallized racemic form of Compound A while the S-enantiomer of Compound A is maintained in the mother-liquor;d. crystallizing the S-enantiomer of Compound A enantiomer from the mother-liquor;ande. isolating the S-enantiomer of Compound A.

6. A process according to any of Claims 2, 3, 4, and 5, wherein solution contains, consists essentially of, or consists of a mixture of S- and R-enantiomers of Compound A in a solvent which is preferably acetonitrile, and preferably wherein the S- and R-enantiomers are in an enantiomeric ratio of: 60:40 to about 95:5, or about 65:35 to about 90:10; or about 70:30 to about 92:8, or about 75:25 to about 91:9, or about 80:20 to about 90:10; or about 85:15 to about 89:11.

7. A process according to any of Claims 2, 3, 4, 5, and 6, wherein the solution contains, consists essentially of, or consists of a mixture of S- and R-enantiomers of Compound A in a solvent which is preferably acetonitrile, wherein the S- and R-enantiomers are in an enantiomeric ratio of: about 80:20 to about 90:10; or about 82:18 to about 91:9; or about 88:12 to about 90:9; or about 84:16 to about 90:10, or about 84 :16 to about 89:11; or about 85:15 to about 89:11.

8. A process according to any of Claims 2, 3, 4, 5, 6, and 7 wherein the solution is at a temperature of: about 25°C to about 85°C, about 30°C to about 75°C, about 35°C to about 70°C, about 35 °C to about 65 °C, about 40°C to about 60°C, or about 40°C to about 50°C.

9. A process according to any of Claims 2, 3, 4, 5, 6, 7, and 8, wherein the crystallization of racemic form of Compound A is carried out by cooling solution, preferably to about room temperature.Attorney Docket: API092-W001 (2222-238 PCT)10. A process according to any of Claims 2, 3, 4, 5, 6, 7, 8, and 9, wherein the crystallization of racemic form of Compound A is carried out by cooling the solution, preferably to a temperature of: about 0°C to about 30°C, about 5°C to about 30°C, about 10°C to about 30°C, about 20°C to about 30°C, or about 0°C to about 20°C, about 0°C to about 15°C, about 0°C to about 10°C, or about 0°C to about 5°C.

11. A process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, wherein the crystallized racemic form of Compound A is isolated, preferably by filtration, decantation or centrifuge, preferably by filtration.

12. A process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11, wherein the S- enantiomer of Compound A is isolated from the mother liquor by crystallization.

13. A process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, wherein the S -enantiomer of Compound A is isolated from the mother liquor by crystallization from a mixture of acetonitrile and an antisolvent.

14. A process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13, wherein the S -enantiomer of Compound A is isolated directly from the mother liquor by combining the mother liquor with an antisolvent; or wherein the S -enantiomer of Compound A is isolated by partial evaporation of the mother liquor to form a concentrated mother liquor and combining the concentrated mother liquor with an antisolvent; or wherein the S -enantiomer of Compound A is isolated by evaporation of the solvent from the mother liquor and crystallisation from a mixture of acetonitrile and an antisolvent.

15. A process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, wherein the mother liquor is obtained from crystallisation from acetonitrile of racemic form of Compound A and filtration, and optionally partial evaporation to form a concentrated mother liquor.Attorney Docket: API092-W001 (2222-238 PCT)16. A process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15, wherein the S -enantiomer of Compound A is isolated from the mother liquor by combining the mother liquor or concentrated mother liquor with an antisolvent selected from: MTBE, methanol, ethyl acetate, isopropyl alcohol and heptane, or a mixture thereof, and more preferably wherein the antisolvent is methanol.

17. A process according to any of Claims 14, 15 and 16, wherein the mother liquor or concentrated mother liquor is cooled to a temperature of: about -10°C to about 15°C, about -5°C to about 10°C or about 0°C to about 10°C, prior to combining with the antisolvent.

18. A process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17, wherein the acetonitrile and antisolvent are in a ratio (v / v) of: about 1:1 to about 1:5, about 1:1 to about 1:4, about 1:1.5 to about 1:3, about 1:1.8 to about 1:2.5, about 1:1.8 to about 1:2.2, or about 1:2; and preferably wherein the antisolvent is methanol.

19. A process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, wherein the S-enantiomer of Compound A is isolated by filtration, centrifuge or decantation, preferably by filtration.

20. A process according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19, for purifying benzyl (S)-3-methyl-5-(5-(3,4,5-trichlorophenyl)-5- (trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate (S-enantiomer of Compound A) from a solution comprising S-enantiomer and R-enantiomer of Compound A and which is enriched in S-enantiomer, in acetonitrile; wherein the process comprises crystallizing racemic form of Compound A from the solution by cooling; removing the racemic form of Compound A from the solution, preferably by filtration, to obtain a mother-liquor which is further enriched with the S-enantiomer of Compound A, optionally concentrating the mother-liquor, combining the mother liquor with an antisolvent, preferably methanol; and isolating the S-enantiomer of Compound A from the mother liquor, preferably by filtration.Attorney Docket: API092-W001 (2222-238 PCT)21. A process according to any preceding claim, wherein the S-isomer of Compound A has an enantiomeric purity of 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher or 99.5% or higher.

22. A process according to any preceding claim further comprising converting the S- enantiomer of Compound A to Lotilaner.

23. Use of the S-enantiomer of Compound A obtained according to a process of any preceding claim, in the preparation of Lotilaner, preferably for preparing enantiomerically pure Lotilaner.

24. A process for preparing Lotilaner comprising purifying benzyl (S)-3-methyl-5-(5-(3,4,5- trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxylate, and converting the purified compound to Lotilaner.

25. A process according to Claim 24, comprising purifying benzyl (S)-3-methyl-5-(5-(3,4,5- trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydro-isoxazol-3-yl)thiophene-2-carboxylate by a process according to any of Claims 1-21, and converting it to Lotilaner.

26. A process according to any of Claims 24 and 25, comprising converting benzyl (S)-3- methyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydro-isoxazol-3- yl)thiophene-2-carboxylate to Compound 5:and reacting Compound 5 and 2-amino-N-(2,2,2-trifluoroethyl)acetamide, or a salt thereof, preferably a hydrochloride salt, to obtain Lotilaner.

27. A process according to Claim 26, wherein the benzyl (S)-3-methyl-5-(5-(3,4,5- trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydro-isoxazol-3-yl) thiophene-2-Attorney Docket: API092-W001 (2222-238 PCT)carboxylate is converted to Compound 5 by reaction with a base (preferably wherein the base is an alkali metal hydroxide, preferably lithium hydroxide, sodium hydroxide or potassium hydroxide, and more preferably sodium hydroxide.), preferably in the presence of a solvent (preferably wherein the solvent is acetonitrile, methanol, 2- butanol, acetone, tetrahydrofuran (THF), water, or mixtures thereof; preferably wherein the solvent is acetonitrile or a mixture of acetonitrile and water, and more preferably wherein the solvent is a mixture of acetonitrile and water).

28. A process according to any of Claims 26 and 27, wherein the reaction of Compound 5 and 2-amino-N-(2,2,2-trifluoroethyl)acetamide is carried out in the presence of a coupling reagent [preferably wherein the coupling reagent is: l-ethyl-3-(3- dimethylaminopropyl)carbodiimide ("EDC") or hydroxy benzotriazole ("HOBT"), and more preferably EDC], and a solvent [preferably wherein the solvent is dichloromethane (MDC), toluene, acetonitrile, dimethylformamide (DMF) or ethyl acetate (EtOAc), morc - preferably ethyl acetate]; and a base [preferably wherein the base is triethylamine ("TEA") or diisopropylethylamine ("DIPEA"), and particularly DIPEA].

29. A process according to any of Claims 22, 23, 24, 25, 26, and 27, wherein the Lotilaner has an enantiomeric purity of at least 99.9% and having an HPLC purity at least 99.9%.

30. Lotilaner having an enantiomeric purity of at least 99.9% and having an HPLC purity at least 99.9%.

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