CRYSTALLINE FORMS OF A TYK2 INHIBITOR

Abstract

The invention provides crystalline forms of a compound having the formula (1):

##STR00001##

along with methods of making the crystalline forms and pharmaceutical formulations comprising the crystalline forms.

Claims

1-7. (canceled)

8. A substantially crystalline form (Form B) of a compound of formula (1): ##STR00012## having an X-ray powder diffraction pattern characterised by the presence of major peaks at the diffraction angles (2θ) 23.2° and/or 16.7° and/or 22.6° and/or 26.6° and/or 12.0°.

9. A substantially crystalline form (Form B) of the compound of formula (1) according to claim 8 having an X-ray powder diffraction pattern characterised by the presence of major peaks at two or more, e.g. three or more, or four or more, and in particular five diffraction angles (2θ) selected from 23.2°, 16.7°, 22.6°, 26.6° and 12.0° (±0.2°).

10. A substantially crystalline form (Form B) of the compound of formula (1) according to claim 8 having an X-ray powder diffraction pattern characterised by the presence of major peaks at the diffraction angles (2θ) 23.2° and/or 16.7° and/or 22.6° and/or 26.6° and/or 12.0° (e.g. at least four and more particularly at least five of the diffraction angles), and one or more further peaks at diffraction angles (2θ) selected from 23.4°, 25.3°, 7.1°, 19.9° and 27.8° (±0.2°).

11. A method for the preparation of a substantially crystalline form (Form B) of compound (1) as defined in claim 8, which method comprises: (i) dispersing an amorphous form of compound (1) in a solvent selected from hydrocarbon solvents, halogenated hydrocarbon solvents (other than dichloromethane), methanol, isopropyl alcohol, aliphatic ketones (e.g. C.sub.1-8 ketones), non-aromatic ethers (e.g. C.sub.3-6 dialkyl and alkylcycloalkyl ethers and THF), and isopropylacetate to form a mixture; (ii) heating the mixture to a moderately elevated temperature in the range from 45-65° C. and holding the mixture at the moderately elevated temperature for a period of at least 10 hours (for example from 10 to 25 hours, e.g. about 17 hours); (iii) cooling or allowing the cooling of the mixture from the moderately elevated temperature to a lower temperature in the range from 15-30° C. (e.g. 20-30° C. such as approximately 25° C.) and holding the mixture at the lower temperature for a period of at least 2 hours (e.g. 2 to 8 hours such as approximately 4.5 hours); and (iv) optionally subjecting the mixture to a further heating and cooling cycle comprising heating the mixture to a moderately elevated temperature in the range from 45-65° C. and holding the mixture at the moderately elevated temperature for a period of at least 10 hours (for example from 10 to 25 hours, e.g. about 16 hours); cooling the mixture to a lower temperature in the range from 15-30° C. (e.g. 20-30° C. such as approximately 25° C.) and optionally holding the mixture at the lower temperature for a period of at least half an hour (e.g. up to approximately 1 hour); and (v) isolating (e.g. by filtration) the crystalline Form B of compound (1) thus formed.

12. A method for the preparation of a substantially crystalline form (Form B) of compound (1) as defined in claim 8, which method comprises: (i) dispersing a solid form of compound (1) in methylethylketone (MEK) to form a mixture; (ii) subjecting the mixture to an equilibration procedure comprising periods of heating and cooling the mixture until a suspension of crystalline Form B is formed; and optionally further equilibrating the mixture until a desired level of polymorphic purity is achieved.

13-16. (canceled)

17. A pharmaceutical composition comprising a crystalline form of a compound having the formula (1) as defined in claim 8 and a pharmaceutically acceptable excipient.

18. (canceled)

19. A pharmaceutical composition comprising a crystalline form of a compound having the formula (1) as defined in claim 9 and a pharmaceutically acceptable excipient.

20. A pharmaceutical composition comprising a crystalline form of a compound having the formula (1) as defined in claim 10 and a pharmaceutically acceptable excipient.

21. A substantially crystalline form (Form B) of the compound of formula (1) according to claim 8 having an X-ray powder diffraction pattern characterized by the presence of major peaks at the diffraction angles (2θ) 23.2°, 16.7°, 22.6°, 26.6° and 12.0°, and one or more further peaks at diffraction angles (2θ) selected from 23.4°, 25.3°, 7.1°, 19.9° and 27.8° (±0.2°), and one or more further peaks at diffraction angles (2θ) selected from 14.2°, 27.0°, 24.1°, 28.9° and 14.5° (±0.2°).

22. A substantially crystalline form (Form B) of the compound of formula (1) according to claim 8 having a DSC thermogram characterized by an endotherm with an onset and maxima at about 230° C. and about 233° C.

23. A substantially crystalline form (Form B) of the compound of formula (1) according to claim 8 having a crystalline purity of at least 90%.

24. A substantially crystalline form (Form B) of the compound of formula (1) according to claim 9 having a crystalline purity of at least 90%.

25. A substantially crystalline form (Form B) of the compound of formula (1) according to claim 10 having a crystalline purity of at least 90%.

26. A substantially crystalline form (Form B) of the compound of formula (1) according to claim 8 having a crystalline purity of at least 95%.

27. A substantially crystalline form (Form B) of the compound of formula (1) according to claim 9 having a crystalline purity of at least 95%.

28. A substantially crystalline form (Form B) of the compound of formula (1) according to claim 10 having a crystalline purity of at least 95%.

29. A method for the preparation of a substantially crystalline form (Form B) of compound (1) as defined in claim 9, which method comprises: (i) dispersing an amorphous form of compound (1) in a solvent selected from hydrocarbon solvents, halogenated hydrocarbon solvents (other than dichloromethane), methanol, isopropyl alcohol, aliphatic ketones (e.g. C.sub.1-8 ketones), non-aromatic ethers (e.g. C.sub.3-6 dialkyl and alkylcycloalkyl ethers and THF), and isopropylacetate to form a mixture; (ii) heating the mixture to a moderately elevated temperature in the range from 45-65° C. and holding the mixture at the moderately elevated temperature for a period of at least 10 hours (for example from 10 to 25 hours, e.g. about 17 hours); (iii) cooling or allowing the cooling of the mixture from the moderately elevated temperature to a lower temperature in the range from 15-30° C. (e.g. 20-30° C. such as approximately 25° C.) and holding the mixture at the lower temperature for a period of at least 2 hours (e.g. 2 to 8 hours such as approximately 4.5 hours); and (iv) optionally subjecting the mixture to a further heating and cooling cycle comprising heating the mixture to a moderately elevated temperature in the range from 45-65° C. and holding the mixture at the moderately elevated temperature for a period of at least 10 hours (for example from 10 to 25 hours, e.g. about 16 hours); cooling the mixture to a lower temperature in the range from 15-30° C. (e.g. 20-30° C. such as approximately 25° C.) and optionally holding the mixture at the lower temperature for a period of at least half an hour (e.g. up to approximately 1 hour); and (v) isolating (e.g. by filtration) the crystalline Form B of compound (1) thus formed.

30. A method for the preparation of a substantially crystalline form (Form B) of compound (1) as defined in claim 10, which method comprises: (i) dispersing an amorphous form of compound (1) in a solvent selected from hydrocarbon solvents, halogenated hydrocarbon solvents (other than dichloromethane), methanol, isopropyl alcohol, aliphatic ketones (e.g. C.sub.1-8 ketones), non-aromatic ethers (e.g. C.sub.3-6 dialkyl and alkylcycloalkyl ethers and THF), and isopropylacetate to form a mixture; (ii) heating the mixture to a moderately elevated temperature in the range from 45-65° C. and holding the mixture at the moderately elevated temperature for a period of at least 10 hours (for example from 10 to 25 hours, e.g. about 17 hours); (iii) cooling or allowing the cooling of the mixture from the moderately elevated temperature to a lower temperature in the range from 15-30° C. (e.g. 20-30° C. such as approximately 25° C.) and holding the mixture at the lower temperature for a period of at least 2 hours (e.g. 2 to 8 hours such as approximately 4.5 hours); and (iv) optionally subjecting the mixture to a further heating and cooling cycle comprising heating the mixture to a moderately elevated temperature in the range from 45-65° C. and holding the mixture at the moderately elevated temperature for a period of at least 10 hours (for example from 10 to 25 hours, e.g. about 16 hours); cooling the mixture to a lower temperature in the range from 15-30° C. (e.g. 20-30° C. such as approximately 25° C.) and optionally holding the mixture at the lower temperature for a period of at least half an hour (e.g. up to approximately 1 hour); and (v) isolating (e.g. by filtration) the crystalline Form B of compound (1) thus formed.

31. A method for the preparation of a substantially crystalline form (Form B) of compound (1) as defined in claim 9, which method comprises: (i) dispersing a solid form of compound (1) in methylethylketone (MEK) to form a mixture; (ii) subjecting the mixture to an equilibration procedure comprising periods of heating and cooling the mixture until a suspension of crystalline Form B is formed; and optionally further equilibrating the mixture until a desired level of polymorphic purity is achieved.

32. A method for the preparation of a substantially crystalline form (Form B) of compound (1) as defined in claim 10, which method comprises: (i) dispersing a solid form of compound (1) in methylethylketone (MEK) to form a mixture; (ii) subjecting the mixture to an equilibration procedure comprising periods of heating and cooling the mixture until a suspension of crystalline Form B is formed; and optionally further equilibrating the mixture until a desired level of polymorphic purity is achieved.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0285] FIG. 1 shows XRPD patterns for amorphous (2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)-anilino]oxazole-4-carboxamide. The upper trace is that of amorphous compound formed after drying in vacuo for an hour 45° C. and the lower trace is that of amorphous compound subjected to drying in vacuo at 45° C. for about another 19% hours.

[0286] FIG. 2 is a DSC and TGA thermograph overlay for the amorphous compound after initial drying in vacuo for an hour at 45° C. followed by further drying in vacuo at 45° C. for about another 19¼ hours. The upper line is the TGA thermograph and the lower line containing the peak is the DSC thermograph.

[0287] FIG. 3 shows an XRPD pattern for crystalline Form A.

[0288] FIG. 4 is an overlay of the DSC and TGA thermographs for crystalline Form A. The upper line is the TGA thermograph and the lower line containing the peak is the DSC thermograph.

[0289] FIG. 5 is a DVS isotherm profile for crystalline Form A.

[0290] FIG. 6 shows an XRPD pattern for crystalline Form B.

[0291] FIG. 7 shows DSC thermographs of three samples of crystalline Form B, denoted by lines B-1, B-2 and B-3.

[0292] FIG. 8 is a DVS isotherm profile for crystalline Form B.

[0293] FIG. 9 shows an XRPD pattern for crystalline Form C.

[0294] FIG. 10 shows a DSC thermograph comparison between crystalline Form A (line A), crystalline Form B (line B) and crystalline Form C (line C).

[0295] FIG. 11 shows an XRPD pattern for crystalline Form D.

[0296] FIG. 12 shows a DSC thermograph for crystalline form D.

EXAMPLES

[0297] The invention will now be illustrated, but not limited, by reference to the specific embodiments described in the following examples.

Abbreviations

[0298] In the Examples below, the following abbreviations are used:

[0299] ACN acetonitrile

[0300] DCM dichloromethane

[0301] DMF dimethylformamide

[0302] DPPF 1,1′-bis(diphenylphosphino)ferrocene

[0303] EDCl N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide

[0304] Et.sub.3N triethylamine

[0305] EtOAc ethyl acetate

[0306] HOBt hydroxybenzotriazole

[0307] HPLC high performance liquid chromatography

[0308] LCMS liquid chromatography—mass spectrometry

[0309] MeCN acetonitrile

[0310] MeOH methanol

[0311] mL millilitres

[0312] mmol millimoles

[0313] NMP N-methylpyrrolidone

[0314] Pd(dba).sub.2 bis(dibenzylideneacetone)palladium(0)

[0315] SiO.sub.2 silica

[0316] tert-BuONO tertiary butyl nitrite

[0317] TFA trifluoroacetic acid

[0318] TLC thin layer chromatography

[0319] Analytical Conditions

[0320] NMR spectra were recorded on a Bruker 400 MHz instrument or on a JEOL ECX 400 MHz spectrometer equipped with an auto-sampler. In Example 1, the NMR spectra were recorded on the Bruker instrument unless indicated otherwise.

[0321] HPLC separations were carried out using Phenomenex LUNA-C18(2) 5p particle size, 2×50 mm columns.

[0322] X-Ray Powder Diffraction (XRPD)

[0323] X-Ray Powder Diffraction patterns were collected on a PANalytical diffractometer using Cu Kα radiation (45 kV, 40 mA), θ-θ goniometer, focusing mirror, divergence slit (½”), soller slits at both incident and divergent beam (4 mm) and a PIXcel detector. The software used for data collection was X'Pert Data Collector, version 2.2f and the data were presented using X'Pert Data Viewer, version 1.2d. XRPD patterns were acquired under ambient conditions via a transmission foil sample stage (polyimide—Kapton, 12.7 μm thickness film) under ambient conditions using a PANalytical X'Pert PRO. The data collection range was 2.994-35°2θ with a continuous scan speed of 0.202004° s-1.

[0324] Differential Scanning Calorimetry (DSC)

[0325] DSC data were collected on a PerkinElmer Pyris 6000 DSC equipped with a 45-position sample holder. The instrument was verified for energy and temperature calibration using certified indium. A predefined amount of the sample, 0.5-3.0 mg, was placed in a pin holed aluminium pan and heated at 20° C.min.sup.−1 from 30 to 350° C., or varied as experimentation dictated. A purge of dry nitrogen at 20 ml min.sup.−1 was maintained over the sample. The instrument control, data acquisition and analysis were performed with Pyris Software v11.1.1 revision H.

[0326] Thermo-Gravimetric Analysis (TGA)

[0327] TGA data were collected on a PerkinElmer Pyris 1 TGA equipped with a 20-position auto-sampler. The instrument was calibrated using a certified weight and certified Alumel and Perkalloy for temperature. A predefined amount of the sample, 1-5 mg, was loaded onto a pre-tared aluminium crucible and was heated at 20° C.min.sup.−1 from ambient temperature to 400° C. A nitrogen purge at 20 ml.Math.min.sup.−1 was maintained over the sample. Instrument control, data acquisition and analysis were performed with Pyris Software v11.1.1 revision H.

[0328] Optical Microscopy

[0329] Optical microscopy examination was undertaken using a Leica DME polarised light microscope and an Infinity 1 digital video camera for image capture. A small amount of each sample was placed onto a glass slide and dispersed as best as possible. The samples were viewed with appropriate magnification and various images recorded. The image scale bar was calibrated against an external graticule, 0.1 mm/0.002 mm DIV.

[0330] Hot Stage Microscopy (HSM)

[0331] Hot Stage Microscopy was undertaken using a Leica DME polarised light microscope combined with a Mettler-Toledo MTFP82HT hot-stage and a digital video camera for image capture. A small amount of each sample was placed onto a glass slide with individual particles separated as best as possible. The sample was viewed with appropriate magnification and partially polarised light, whilst being heated from ambient temperature typically at 20° C.min.sup.−1 unless an alternate heating rate is stated.

[0332] Dynamic Vapour Sorption Sorption isotherms were obtained using a Hiden Isochema moisture sorption analyser (model IGAsorp), controlled by IGAsorp Systems Software V6.50.48. The sample was maintained at a constant temperature (25° C.) by the instrument controls. The humidity was controlled by mixing streams of dry and wet nitrogen, with a total flow of 250 ml.Math.min.sup.−1. The instrument was verified for relative humidity content by measuring three calibrated Rotronic salt solutions (10-50-88%). The weight change of the sample was monitored as a function of humidity by a microbalance (accuracy +/−0.005 mg). A defined amount of sample was placed in a tared mesh stainless steel basket under ambient conditions. A full experimental cycle typically consisted of three scans (sorption, desorption and sorption) at a constant temperature (25° C.) and 10% RH intervals over a 0-90% range (60 minutes for each humidity level). This type of experiment should demonstrate the ability of samples studied to absorb moisture (or not) over a set of well-determined humidity ranges.

[0333] Karl Fischer Titration

[0334] Water content in a sample was determined using a Mettler Toledo Volumetric Karl Fischer Titrator. The titrant was HYDRANAL composite 5 and the solvent was HYDRANAL Methanol dry. A sample mass of ca. 0.2 g was charged and mixed for 600 seconds.

[0335] Mya4 Reaction Station

[0336] Equilibrations or crystallisations that require temperature control and/or defined heating/cooling profiles are performed in the Radley's Mya4 Reaction Station; a 4-zone reaction station with magnetic and overhead stirring capabilities and a temperature range of −30 to 180° C. on 2 to 400 ml scale mixtures. The reaction conditions required are programmed via the Mya 4 Control Pad. The temperature control is verified semi-annually in-house.

Example 1

2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]-oxazole-4-carboxamide

[0337] ##STR00004##

1A. Preparation of 5-amino-2-(2,6-dichlorophenyl)-oxazole-4-carbonitrile

[0338] ##STR00005##

[0339] 2,6-Dichlorobenzoyl chloride (10 g, 47.74 mmol) was added slowly to a solution of aminomalononitrile p-toluenesulfonate (13.3 g, 52.51 mmol) in NMP (50 mL). The reaction mixture was heated at 110° C. for 14 hours before quenching with water (100 mL) and the resulting solid was collected by filtration. The crude product was dissolved in ethyl acetate (100 mL) and washed with water (40 mL×2), and the organic layer was dried over Na.sub.2SO.sub.4. The solvent was removed to give the title compound (19 g, crude) as a white solid.

[0340] .sup.1H NMR (400 MHz, CDCl.sub.3): δ: 7.37-7.35 (m, 2H), 7.29-7.26 (m, 1H), 6.19 (s, 2H).

1B. Preparation of 5-bromo-2-(2,6-dichlorophenyl)-oxazole-4-carbonitrile

[0341] ##STR00006##

[0342] To a solution of 5-amino-4-cyano-2-(2,6-dichlorophenyl)-oxazole (9.0 g, 35.42 mmol) in CH.sub.2Br.sub.2 (50 mL) was added bromo(trimethyl)silane (13.56 g, 88.55 mmol). tert-BuONO (36.53 g, 354.20 mmol) was then added very slowly at 0° C. under a protective N.sub.2 atmosphere and the mixture was stirred at 0° C. for 2.5 hour. The reaction mixture was then concentrated under reduced pressure to remove CH.sub.2Br.sub.2, water (H.sub.2O 100 mL) was added and the resulting mixture was extracted with DCM (100 mL×3). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give a residue which was purified by column chromatography (SiO.sub.2, petroleum ether/ethyl acetate=50/1 to 10:1). The title compound (8 g, 71.03% yield) was obtained as a white solid.

1B(a). Alternative Preparation of 5-bromo-2-(2,6-dichlorophenyl)-oxazole-4-carbonitrile

[0343] To CuBr.sub.2 (880 g, 3.94 mol) in a flask at room temperature under N.sub.2 was added MeCN (7.5 L) and the resulting mixture was cooled to 0-4° C. tBuONO (90% active, 475 mL, 5.68 mol) was added followed by a solution of 5-amino-4-cyano-2-(2,6-dichlorophenyl)-oxazole (500 g, 1.97 mol) in MeCN (2.5 L) at 0-4° C. The reaction mixture was stirred at 5° C. for 30 minutes and then allowed to warm to 10° C. over 1 hour, after which time LC showed the reaction to be complete. The reaction mixture was split into two equal portions (˜5.5 L) for work-up. The first portion was quenched with 1 M HCl (aq) (7.5 L) [exotherm 20-27° C.] and was extracted with EtOAc (2×6.0 L). The extraction procedure was repeated on the second portion and the organic phases were combined (˜24 L) and dried over MgSO.sub.4, reduced in vacuo and then azeotroped with IPA (4.8 L) to give 711 g of crude material. The crude material was slurried in IPA (0.63 L) at room temperature for 2 hours under N.sub.2, filtered, washed (cold IPA, 2×0.1 L) and the resulting pale yellow solid was dried at 40° C. in vacuo for 18 hours to give 390 g (62% yield) at 87% by LC.

1C. Preparation of 4-(4-nitrobenzoyl)-1,1-dioxo-1,4-thiazinane

[0344] ##STR00007##

[0345] To a mixture of 4-nitrobenzoic acid (5 g, 29.92 mmol) and 1,4-thiazinane 1,1-dioxide hydrochloride (5.1 g, 29.92 mmol) in DMF (50 mL) was added HOBt (6.1 g, 44.88 mmol), EDCl (8.6 g, 44.88 mmol), Et.sub.3N (6.1 g, 59.84 mmol) in one portion at 15° C. under N.sub.2. The mixture was stirred at 15° C. for 14 hours. The reaction mixture was diluted with saturated Na.sub.2CO.sub.3 (300 mL) and extracted with EtOAc (150 mL×3). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give the title compound (6.5 g, crude) as a white solid.

[0346] .sup.1H NMR (400 MHz, CDCl.sub.3): δ: 8.27 (d, J=8.8 Hz, 2H), 7.55 (d, J=8.8 Hz, 2H), 4.33-3.75 (m, 4H), 3.22-2.75 (m, 4H).

1D. Preparation of 4-(4-aminobenzoyl)-1,1-dioxo-1,4-thiazinane

[0347] ##STR00008##

[0348] To a solution of 4-(4-nitrobenzoyl)-1,1-dioxo-1,4-thiazinane (5.5 g, 19.35 mmol) in MeOH (100 mL) was added Pd/C (1.0 g, 19.35 mmol) under N.sub.2. The suspension was degassed under vacuum and purged with H.sub.2 several times, and then stirred under H.sub.2 (15 psi) at 15° C. for 14 hours. The reaction mixture was filtered and the filtrate was concentrated to give the title compound (4.5 g, 91.45% yield) as a white solid.

[0349] .sup.1H NMR (400 MHz, (CDCl3): δ: 7.36-7.26 (m, 2H), 6.80-6.61 (m, 2H), 4.26-4.08 (m, 4H), 4.06-3.88 (m, 2H), 3.21-2.95 (m, 4H)

1E. Preparation of 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]oxazole-4-carbonitrile

[0350] ##STR00009##

[0351] 1,4-Dioxane (13 mL) was added to a mixture of 5-bromo-4-cyano-2-(2,6-dichlorophenyl)-oxazole (500 mg, 1.57 mmol), 4-(4-aminobenzoyl)-1,1-dioxo-1,4-thiazinane (399.25 mg, 1.57 mmol) and Pd(dba).sub.2 (90.28 mg, 157 μmol), DPPF (130.56 mg, 235.5 μmol), K.sub.2CO.sub.3 (976.45 mg, 7.07 mmol) in a reaction tube which was sealed and subjected to microwave heating at 120° C. for 4 hours. The resulting reaction mixture was filtered and concentrated in vacuum, and water (30 mL) was added before extracting with DCM (50 mL×3). The combined organic phases were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give a residue which was purified by column chromatography (SiO.sub.2, petroleum ether/ethyl acetate=10/1 to 2/3). The title compound (110 mg, 14.26% yield) was obtained as a brown solid.

1F. Preparation of 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]oxazole-4-carboxamide

[0352] ##STR00010##

[0353] A mixture of 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]oxazole-4-carbonitrile (100 mg, 203.52 μmol) in H.sub.2SO.sub.4 (1 mL) at 0° C. was stirred at 15° C. for 2 hours under an N.sub.2 atmosphere. LCMS analysis after this time indicated that the reaction had gone to completion and so the reaction mixture was quenched with ice at 0° C., and then filtered. The filtrate was extracted with EtOAc (30 mL: 10 mL×3), and the combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give a residue which was purified by preparative HPLC (TFA conditions). The title compound, 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)-anilino]oxazole-4-carboxamide (25 mg, 24% yield, 99.61% purity), was obtained as a yellow solid.

[0354] .sup.1H NMR (400 MHz, (CDCl.sub.3): δ: 9.05 (s, 1H), 7.50-7.48 (m, 2H), 7.46-7.44 (m, 3H), 7.41-7.38 (m, 2H), 6.50 (s, 1H), 5.38 (s, 1H), 4.12 (s, 4H), 3.07 (s, 4H).

[0355] MS (ESI): mass calc'd. for C.sub.21H.sub.18Cl.sub.2N.sub.4O.sub.5S 508.0408.04, m/z found, 509.0 [M+H]+.

1G. Preparation of Amorphous 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]oxazole-4-carboxamide

[0356] HPLC purification of 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)-anilino]oxazole-4-carboxamide and subsequent rapid evaporation (e.g. by rotary evaporation) of solvent from eluent containing the compound gives rise to an amorphous form of the compound.

[0357] A larger scale preparation of amorphous compound was carried out by dissolving 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)-anilino]oxazole-4-carboxamide (1.436 g) in 20 volumes of THF and swirling the mixture at 25° C. to form a solution within 15 minutes. The solution was clarified (0.45 μm nylon filter) to afford a clear, yellow solution, leaving dark residue on the filter surface. The solution was reduced in vacuo at 40° C. to a yellow foamy solid within 2 minutes and dried in vacuo at 45° C. on a rotary evaporator for a further 1 hour.

[0358] A portion of the solid was sampled (Batch 1) and the bulk (Batch 2) was dried in vacuo at 45° C. for ca. 19¼ hours to afford 1.15 g of yellow, brittle solid out of the vessel.

[0359] XRPD examination (FIG. 1) revealed both the damp (Batch 1) and dry (Batch 2) lots to be amorphous.

[0360] Thermal examination (FIG. 2) of Batch 2 revealed evidence of minor endotherms in the DSC thermograph at ca. 92° C. and 111° C. leading into a significant exotherm with an onset and peak minimum at ca. 162° C. and 188° C. respectively. The TGA thermograph demonstrated a distinct, initial mass loss of 1.052 wt % from ca. 25-70° C. and a broader mass loss of 4.851 wt % from ca. 78-160° C. leading into a third, broad mass loss of 5.787 wt % from ca. 160-240° C. before the onset of decomposition.

[0361] Examination of Batch 2 by hot stage microscopy (HSM) revealed particle movement and solid contraction from ca. 83° C., leading into the onset of melting by ca. 102° C. and melt completion by ca. 145° C. with no evidence of crystallisation from the melt. Increased discolouration in the melt was evident by ca. 175° C.

[0362] .sup.1H NMR spectroscopy (Jeol) of Batch 1 revealed a residual THF and DMSO content of 7.19 and 0.50 wt % respectively. .sup.1H NMR spectroscopy of Batch 2 revealed a slightly reduced residual THF and DMSO content of 4.90 and 0.36 wt % respectively. A stochiometric THF solvate would require ca. 12.4 wt % THF present.

[0363] The total solvent content (and potentially residual water) is considered to correspond to the first two mass loss steps from 25-70° C. and 78-160° C. in the TGA thermograph with the remaining mass loss due to decomposition in the melt.

Example 2

Conversion of Amorphous 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]oxazole-4-carboxamide into Crystalline Forms

[0364] Amorphous compound (Batch 2), 24×ca. 40±2 mg, was weighed into 24 crystallisation tubes. A selection of 24 solvents, 5 vol., were charged to the solids at 25° C. and observations made. A significant number of mixtures demonstrated dissolution upon solvent addition before affording a suspension within 10-15 minutes which was considered to be a consequence of the residual THF content and/or potentially indicative of form change and crystallisation. Where suspensions remained, additional solvent, 5 vol., was charged (10 vol. total) and observations made. The mixtures were heated to 50° C. (38° C. for DCM) and immediate observations noted. The mixtures were equilibrated at temperature for ca. 17 hours and observations noted. The mixtures were cooled to 25° C. over ca. 2¼ hours, equilibrated for ca. 4½ hours and observations noted. The mixtures were heated to temperature, equilibrated for ca. 16 hours and observations noted. The mixtures were cooled to 25° C. over ca. 2¼ hours, equilibrated for ca. 1 hour, observations noted and the solids isolated.

[0365] Upon isolation, all solids were dried in vacuo under air for ca. 10 to 20 minutes and then dried in vacuo at 45° C. for 68 hours.

[0366] Observations during the solvent addition and thermal modulation are summarised in Table 1. The crystal forms resulting from the different conditions are are summarised in Table 2.

TABLE-US-00013 TABLE 1 25° C., 5 vol. 25° C., 1.sup.st 50° C.*.sup.1, 1.sup.st 50° C.*.sup.1, 1.sup.st 25° C. 2.sup.nd 50° C.*.sup.1, 2.sup.nd 25° C. ID Solvent Immediate 10-15 mins 10 vol T = 15 mins T = 17 Hrs post-cool T = 16 Hrs post-cool A Cyclohexane Susp. Susp. Susp. Susp. Susp. + YS Susp. + YS Susp. + YS Susp. + YS B Chlorobenzene Haze Gum Susp. Susp. Susp. + YS Susp. + YS Susp. + YS Susp. + YS C 2-Chlorobutane Susp. Susp. Susp. Susp. Susp. + YS Susp. + YS Susp. + YS Susp. + YS D Benzotrifluoride Susp. Susp. Susp. Susp. Susp. + YS Susp. + YS Susp. + YS Susp. + YS E Anisole Dissolved Susp. Susp. Susp. Susp. Susp. Susp. Susp. F Nitromethane Dissolved Susp. Susp. Susp. Susp. Susp. Susp. Susp. G CPME Susp. Susp. Susp. Susp. Susp. Susp. + YS Susp. + YS Susp. + YS H Heptane Susp. Susp. Susp. Susp. Susp. + YS Susp. + YS Susp. + YS Susp. + YS I TBME Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. J MIBK Haze Susp. Susp. Susp. Susp. Susp. Susp. Susp, K MEK Dissolved Susp. Susp. Susp. Susp. Susp. Susp. Susp. L IPrOAc Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. M EtOAc Dissolved Susp. Susp. Susp. Susp. Susp. Susp. Susp. N Toluene Haze Susp. Susp. Susp. Susp. + YS Susp. + YS Susp. + YS Susp. + YS O THF Dissolved Solution N/A Susp.*.sup.2 Susp. Susp. Susp. Susp. P DCM (*.sup.138° C.) Dissolved Susp. Susp. Susp. Susp. Susp. Susp. Susp. Q MeOH Gum Susp. Susp. Susp. Susp. Susp. Susp. Susp. R EtOH Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. S IPA Susp. Susp. Susp. Susp. Susp. Susp. + YS Susp. + YS Susp. + YS T MeCN Dissolved Susp. Susp. Susp. Susp. Susp. Susp. Susp. U Water Immiscible Susp. Susp. Susp. Susp. Susp. Susp. Susp. V 4:1 MeCN/water Dissolved Susp. Susp. Susp. Susp. Susp. Susp. Susp. W 4:1 THF/water Dissolved Solution N/A Solution Susp. Susp. Susp. Susp. X 4:1 IPA/water Gum Gum N/A Gum Susp. Susp. Susp. Susp. In Table 1: *.sup.1Charged additional solvent up to 10 vol. Susp. = Suspension Haze = Predominantly dissolved YS = Yellow solid deposit above the mixture level

TABLE-US-00014 TABLE 2 Form fate after equilibrations of amorphous compound at 25° C. Form fate from ID Solvent amorphous at 25° C. A Cyclohexane Low cryst. Form B B Chlorobenzene Low cryst. Form B C 2-Chlorobutane Low cryst. Form B D Benzotrifluoride Low cryst. Form B E Anisole Form E F Nitromethane Form C G CPME Low cryst. Form B H Heptane Low cryst. Form B I TBME Low cryst. Form B J MIBK Low cryst. Form B K MEK Low cryst. Form B L iPrOAc Low cryst. Form B M EtOAc Form D N Toluene Form B O THF Low cryst. Form B P DCM (*38° C.) Form C Q MeOH Low cryst. Form B R EtOH Form C S IPA Low cryst. Form B T MeCN Form C U Water Form A V 4:1 MeCN/water Form A W 4:1 THF/water Form A X 4:1 IPA/water Form A + evidence of B

[0367] Of the twenty four solvents/solvent mixtures assessed, the amorphous compound demonstrated complete/significant dissolution upon addition of fifteen solvents, 5 vol. However, within 10-15 minutes, twelve of these mixtures gave suspensions. Following extended equilibration at 50° C., suspensions were afforded from all mixtures.

[0368] The predominant XRPD pattern of the dried solids was Form B (14 hits).

[0369] Form A was isolated only from the aqueous solvent mixtures.

[0370] Form C was isolated from nitromethane, DCM, EtOH and MeCN.

[0371] Equilibration in anisole afforded a new form, identified arbitrarily as Form E.

[0372] .sup.1H NMR spectroscopy (Jeol) of Form E demonstrated residual anisole, THF and DMSO contents of 1.14, 0.86 and 0.52 wt % respectively (17.5 wt % Anisole required for 1:1 solvate).

[0373] Thermal examination of Form E revealed two endotherms peaking at 187 and 226° C., not too dissimilar to main endotherms for Form A and Form B respectively.

[0374] Thermal manipulation by heating Form E to 200° C., past the first endotherm, and then cooling, demonstrated removal of the first endotherm upon reheating. Insufficient solid remaining for XRPD examination of the thermally manipulated material.

[0375] HSM of Form E revealed particle excitement at 170° C. leading into contraction from 179° C. into a melt by 192° C. Gradual crystallisation was observed in the melt from 192° C. until the crystals melted from 224 to 230° C.

[0376] Equilibration of the amorphous compound in EtOAc afforded a new form, labelled as Form D.

[0377] .sup.1H NMR spectroscopy of Form D demonstrated a residual EtOAc and DMSO content of 4.30 and 0.15 wt % respectively (14.7 wt % EtOAc required for 1:1 solvate). There was no detectable THF observed.

[0378] Thermal examination of Form D (see FIG. 12) revealed a complex DSC thermograph, with a broad endotherm-exotherm peaking at 171° C. and 179° C. respectively (the endotherm of which was similar to Form C) before two sharper endotherms peaking at 209° C. and 229° C. (the latter of which is similar to Form B).

[0379] Thermal manipulation by heating the solid to 180° C., past the endo-exo, and then cooling, demonstrated removal of the endo-exo event upon reheating. The solid was heated to 215° C., past the second endotherm, and then cooled before reheating, demonstrating a minor endotherm at ca. 145° C. but no other events until the Form B endotherm. Insufficient solid remained for XRPD examination of the thermally manipulated material.

[0380] HSM of Form D revealed particle excitement at 162° C. leading into contraction from 168° C. into a melt by 182° C. Solids persisted and potentially crystallised within the melt until the crystals melted from 226° C. to 231° C., similar to Form E.

Example 3

Alternative Hydrolysis Conditions for Converting 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]oxazole-4-carbonitrile to 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]oxazole-4-carboxamide

[0381] In step 1F of Example 1, the partial hydrolysis of the nitrile to the carboxamide is carried out using sulphuric acid. In an alternative method, the partial hydrolysis step can be carried out using a mixture of trifluoroacetic acid (TFA) and sulphuric acid.

[0382] Accordingly, TFA (1166 mL) was added to 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]oxazole-4-carbonitrile (350 g, 7.12 mmol) and the mixture was stirred at room temperature for 30 minutes and then cooled to 0-10° C. Concentrated H.sub.2SO.sub.4 (584 mL) was added to the mixture over 30 minutes [an exothermic process] during which time the temperature was maintained in the range 0-20° C. After addition of the H.sub.2SO.sub.4 was complete, the reaction mixture was warmed to 15-25° C. and stirred for 5 hours at room temperature, after which liquid chromatography showed 3% remaining starting material. The reaction was quenched [exothermic] by addition to a mixture of c.NH.sub.3 (3.0 L) and ice (4.0 Kg) over 30 minutes, keeping the temperature under 25° C. [pH shown to be >10 after addition]. The resulting slurry was stirred for 15 minutes and then filtered, washed (H.sub.2O, 1.0 L) and pulled dry. The wet filtercake was reslurried in water (3.5 L) for 3 hours and re-filtered, washed (H.sub.2O, 1.8 L) and dried in vacuo at 50° C. overnight to give (2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]-oxazole-4-carboxamide (402 g) at a purity of 93.2% with residual starting material at a level of 0.97% (by LC). The partially purified material was slurried in MeCN (1.5 L) for 1 hour at room temperature, filtered, washed (MeCN 0.5 L) and dried in vacuo at 50° C. overnight to afford 195 g of product. The product was further purified by dissolving in DMSO (500 mL) at room temperature, polish filtering and washing (DMSO, 80 mL), and then adding to H.sub.2O (3.8 L) over 2 hours keeping the temperature under 25° C. The resulting slurry was stirred for 30 minutes and then filtered, washed (H.sub.2O, 2×900 mL) and dried in vacuo at 50° C. to give 170.8 g (44% yield) of (2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]oxazole-4-carboxamide at an activity of 95% by .sup.1H NMR assay and a purity of 97.1% by LC, 2.3% H.sub.2O by KF. Additionally, the MeCN liquors (80.9% by LC) were reworked using column chromatography to give 61.1 g of product at 93.9% by L and at 79% activity by C NMR assay.

[0383] The aqueous work-up used in this method of preparing 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]oxazole-4-carboxamide gives the compound in crystalline Form A.

[0384] Characterising Data for crystalline forms A, B, C and D

[0385] Form A

[0386] The XRPD pattern for crystalline Form A is shown in FIG. 3.

[0387] The dataset for the XRPD pattern for Form A is set out in Table 3 below.

TABLE-US-00015 TABLE 3 Diffraction list for crystalline Form A Pos. Height FWHM d-spacing Rel. Int. [°2 Th.] [cts] [°2 Th.] [Å] [%] 5.7688 68.62 0.8187 15.32049 3.88 6.8092 183.13 0.0768 12.98161 10.35 7.9153 104.54 0.1791 11.16996 5.91 10.6555 764.11 0.1023 8.30278 43.20 11.8866 174.60 0.1023 7.44547 9.87 12.3287 1580.11 0.1279 7.17948 89.33 13.7120 908.80 0.1791 6.45812 51.38 15.8998 455.66 0.1279 5.57408 25.76 16.3772 430.47 0.1023 5.41267 24.34 16.6586 1416.08 0.1279 5.32185 80.06 17.5788 439.57 0.1535 5.04531 24.85 17.9168 448.44 0.1535 4.95088 25.35 19.1711 84.18 0.1535 4.62969 4.76 19.6584 164.13 0.1791 4.51602 9.28 20.6540 1018.69 0.1535 4.30053 57.59 20.8459 798.79 0.0768 4.26137 45.16 21.1486 649.42 0.1279 4.20105 36.72 21.4463 708.71 0.1279 4.14340 40.07 21.8734 858.65 0.1791 4.06346 48.54 23.0784 1768.78 0.1791 3.85394 100.00 23.5779 740.25 0.1279 3.77341 41.85 24.1669 323.97 0.1791 3.68277 18.32 24.7073 512.14 0.1791 3.60344 28.95 25.9969 523.29 0.1791 3.42754 29.58 27.6769 413.00 0.1791 3.22318 23.35 28.4649 305.91 0.1535 3.13573 17.29 29.4695 152.94 0.1535 3.03107 8.65 30.1433 127.27 0.1791 2.96483 7.20 31.3029 117.38 0.2558 2.85760 6.64 32.5765 88.11 0.2558 2.74873 4.98 33.1996 261.01 0.1279 2.69856 14.76 34.5246 128.01 0.2558 2.59796 7.24

[0388] Thermal examination (FIG. 4) revealed a distinct endotherm in the DSC thermograph with an onset and maxima at 181.72 and 193.65° C. respectively. Prior to the main endotherm, there was a broad endotherm peaking at ca. 100° C. This broad endotherm corresponded to a mass loss of 2.582 wt % from 30 to 150° C. in the TGA thermograph and was therefore most likely water loss. A minor weight reduction of 0.148 wt % was coincident with the main endotherm. The onset of decomposition was observed from ca. 258° C.

[0389] Thermal manipulation by holding at 150° C. for 1 minute, past the broad endotherm and mass loss, revealed a DSC thermograph that was similar to the input with little evidence of the broad endotherm shown in FIG. 4. The thermal manipulation was considered to have annealed the material. The thermal manipulation had no clear impact on the XPRD diffraction pattern of the solid.

[0390] DVS examination of crystalline Form A (FIG. 5) revealed a weight reduction of ca. 2.75 wt % upon the first desorption from 50 to 0% RH and a total weight gain of ca. 3.25 wt % upon sorption from 0 to 90% RH. Repeat desorption and sorption was similar to the first steps with little evidence of hysteresis. Weight change was relatively steady between 20 and 90% RH but increased significantly between 0 and 20% RH.

[0391] Form B

[0392] The XRPD pattern for crystalline Form B is shown in FIG. 6.

[0393] The dataset for the XRPD pattern for Form B is set out in Table 4 below.

TABLE-US-00016 TABLE 4 Diffraction list for crystalline Form B Pos. Height FWHM d-spacing Rel. Int. [°2 Th.] [cts] [°2 Th.] [Å] [%] 5.5536 51.16 0.4093 15.91347 1.16 7.0873 1325.52 0.1023 12.47286 29.97 10.4640 43.68 0.3070 8.45431 0.99 11.2039 822.74 0.1023 7.89756 18.60 11.7715 761.48 0.0512 7.51803 17.21 11.9570 2374.37 0.1023 7.40181 53.68 12.5675 866.40 0.0768 7.04357 19.59 13.6281 81.45 0.2558 6.49769 1.84 14.2410 1135.66 0.1023 6.21941 25.67 14.4789 1048.35 0.1023 6.11774 23.70 14.8804 94.58 0.0768 5.95358 2.14 16.1468 782.83 0.0768 5.48940 17.70 16.7188 4080.27 0.1535 5.30285 92.24 17.3746 353.00 0.0768 5.10415 7.98 17.9708 250.49 0.1279 4.93611 5.66 18.6086 949.61 0.1279 4.76835 21.47 19.5972 161.12 0.1023 4.52999 3.64 19.9280 1231.16 0.1279 4.45552 27.83 20.1989 148.01 0.0768 4.39638 3.35 20.5954 146.21 0.1535 4.31262 3.31 21.1125 186.10 0.0768 4.20816 4.21 21.4420 200.12 0.1279 4.14422 4.52 22.0703 750.73 0.1279 4.02765 16.97 22.5561 3876.39 0.1279 3.94200 87.63 23.1885 4423.53 0.1279 3.83589 100.00 23.4376 1897.41 0.1023 3.79569 42.89 23.8914 771.49 0.0512 3.72461 17.44 24.0891 1116.32 0.1023 3.69449 25.24 24.6251 94.22 0.2047 3.61527 2.13 25.2600 1882.59 0.1535 3.52583 42.56 26.0016 388.63 0.1535 3.42692 8.79 26.6169 2851.42 0.1279 3.34909 64.46 27.0229 1125.13 0.1535 3.29968 25.44 27.8202 1212.70 0.1279 3.20691 27.41 28.6270 210.92 0.0768 3.11833 4.77 28.9129 1077.05 0.1791 3.08814 24.35 29.9652 206.97 0.1535 2.98206 4.68 30.8688 89.74 0.1279 2.89679 2.03 31.2086 146.41 0.1279 2.86602 3.31 31.8422 627.56 0.1279 2.81043 14.19 32.4630 121.11 0.2047 2.75809 2.74 32.9792 523.67 0.1535 2.71609 11.84 33.8821 381.43 0.1279 2.64575 8.62 34.4717 110.96 0.2047 2.60183 2.51

[0394] FIG. 7 shows the DSC profiles of three samples of Form B prepared by different methods.

[0395] The DSC profile of a sample of crystalline Form B prepared by equilibration of Form A in nitromethane is represented by line B-1 in FIG. 7. This Form, although predominantly Form B was shown by XRPD (diffractogram not shown) to contain some Form A. DSC examination of this crystalline form shows a single endotherm with an onset and maxima at 228° C. and 232° C. respectively.

[0396] The DSC profile of a sample of crystalline Form B prepared by equilibration in MEK is represented by line B-2 in FIG. 7. The DSC thermograph shows an endotherm with an onset and maxima at 230° C. and 233° C. respectively with a preceding shoulder at 229° C. Apart from the shoulder, the profile is similar to B-1.

[0397] The DSC profile of a third sample of Form B is shown by line B-3 and features an endotherm with an onset and maxima at similar temperatures as B-1 but with a bimodal peak at 236° C.

[0398] DVS examination of Form B (see FIG. 8) demonstrated a weight reduction of ca. 0.38 wt % upon the first desorption from 50 to 0% RH and a total weight gain of ca. 0.57 wt % upon sorption from 0 to 90% RH. Repeat desorption and sorption was similar to the first steps with hysteresis of ca. 0.11 wt %, most evident between 10 and 60% RH. Weight change was relatively steady between 0 and 90% RH. XRPD examination (diffractogram not shown) of the solid isolated post-DVS at 0 and 90% RH demonstrated no clear evidence of form modification from the input version.

[0399] Form C

[0400] The XRPD pattern for crystalline Form C is shown in FIG. 9.

[0401] The dataset for the XRPD pattern for Form C is set out in Table 5 below.

TABLE-US-00017 TABLE 5 Pos. Height FWHM d-spacing Rel. Int. [°2 Th.] [cts] [°2 Th.] [Å] [%] 4.2224 394.34 0.0768 20.92706 10.84 5.5915 165.56 0.3070 15.80584 4.55 8.5015 1191.88 0.1023 10.40094 32.75 11.5069 503.74 0.1279 7.69028 13.84 12.7898 3639.38 0.1535 6.92167 100.00 13.0197 1142.87 0.0768 6.79993 31.40 14.4454 46.19 0.3070 6.13187 1.27 16.4998 1027.50 0.1279 5.37272 28.23 17.0824 215.07 0.2047 5.19077 5.91 17.8408 1579.57 0.1535 4.97180 43.40 18.0631 238.70 0.0768 4.91110 6.56 18.8181 546.23 0.1535 4.71573 15.01 20.1113 1078.39 0.1279 4.41533 29.63 20.4314 313.82 0.1023 4.34687 8.62 20.7041 233.04 0.1535 4.29023 6.40 21.3136 206.18 0.1535 4.16889 5.67 21.5831 176.93 0.0768 4.11744 4.86 22.4078 648.46 0.1279 3.96774 17.82 22.8623 1508.30 0.1279 3.88989 41.44 23.1421 622.67 0.1023 3.84348 17.11 23.6446 497.31 0.1023 3.76293 13.66 24.3253 1242.33 0.1279 3.65914 34.14 24.6942 611.51 0.0768 3.60531 16.80 24.9864 541.61 0.1023 3.56381 14.88 25.3479 531.15 0.1023 3.51380 14.59 25.7650 491.05 0.2047 3.45785 13.49 26.1378 943.67 0.1535 3.40938 25.93 27.1360 338.98 0.1279 3.28619 9.31 27.2833 314.48 0.0768 3.26878 8.64 27.7947 81.32 0.2047 3.20979 2.23 28.8544 220.37 0.3582 3.09427 6.06 29.7177 63.98 0.2047 3.00632 1.76 30.2648 230.51 0.1791 2.95321 6.33 31.1192 108.33 0.1535 2.87405 2.98 32.3467 66.62 0.1535 2.76773 1.83 33.4199 123.26 0.2558 2.68127 3.39 34.4181 156.93 0.1791 2.60576 4.31

[0402] FIG. 10 shows a comparison of the DSC profiles of Forms A, B and C, represented respectively by lines A, B and C.

[0403] The Form C used in this DSC study was prepared by equilibration in THF at 25° C. using Form A as the starting material.

[0404] The profile of Form C features a broad endotherm from 140 to 180° C. with a peak maximum at 173° C. before a Form B-similar endotherm with an onset at 224° C. and maximum at 232° C.

[0405] HSM of Form C revealed particle excitement from ca. 147° C., leading into a partial melt by 187° C. Minor crystallisation was observed in the melt until ca. 210° C. Contraction was observed from ca. 220° C. into melt completion by 237° C.

[0406] The Form C sample was thermally manipulated by holding at 180° C. on the TGA. This revealed a mass loss of 0.42 and 2.08 wt % at 25-68° C. and 86-185° C. 1S. assessment of the manipulated material revealed a thermograph that was characteristic of Form B. XRPD examination (diffractogram not shown) revealed form conversion from Form C to Form B.

[0407] Form 0

[0408] The XRPD pattern for crystalline Form D is shown in FIG. 11.

[0409] The dataset for the XRPD pattern for Form D is set out in Table 6 below.

TABLE-US-00018 TABLE 6 Pos. Height FWHM d-spacing Rel. Int. [°2 Th.] [cts] [°2 Th.] [Å] [%] 5.6206 64.42 0.6140 15.72399 2.10 6.9197 343.25 0.0768 12.77463 11.20 7.8376 303.12 0.1279 11.28042 9.89 10.4454 174.63 0.3070 8.46928 5.70 11.7494 175.66 0.6140 7.53214 5.73 12.9620 416.52 0.1023 6.83009 13.59 13.9079 3064.71 0.1023 6.36760 100.00 16.0891 670.11 0.2303 5.50894 21.87 16.6344 1104.35 0.1535 5.32955 36.03 17.9803 555.02 0.2303 4.93353 18.11 19.8856 413.47 0.1279 4.46494 13.49 21.1635 1689.30 0.1535 4.19813 55.12 21.3741 1708.82 0.1535 4.15724 55.76 22.4874 486.56 0.4093 3.95388 15.88 23.4275 1525.54 0.1535 3.79730 49.78 24.0956 598.82 0.3070 3.69351 19.54 25.1684 323.67 0.1535 3.53846 10.56 26.3897 310.41 0.2558 3.37740 10.13 27.6672 329.24 0.2558 3.22429 10.74 28.8165 131.44 0.1535 3.09825 4.29 29.7643 157.37 0.2558 3.00172 5.13 30.7387 148.30 0.3070 2.90875 4.84 31.9093 239.42 0.2558 2.80467 7.81 33.8982 184.26 0.3070 2.64453 6.01

[0410] FIG. 12 shows a DSC thermograph of Form D. The Form D was prepared by equilibration of amorphous compound in ethyl acetate at 25° C.

[0411] .sup.1H NMR spectroscopy (JEOL—spectrum not shown) of Form D demonstrated a residual EtOAc and DMSO content of 4.30 and 0.15 wt % respectively (14.7 wt % EtOAc required for 1:1 solvate).

[0412] Thermal examination (FIG. 12) revealed a complex DSC thermograph, with a broad endotherm-exotherm peaking at 171° C. and 179° C. respectively (the endotherm of which was similar to Form C) before two sharper endotherms peaking at 209° C. and 229° C. (the latter of which is similar to Form B).

[0413] Thermal manipulation (DSC trace not shown) by heating the solid to 180° C., past the endo-exo, and then cooling demonstrated removal of the endo-exo event upon reheating. The solid was heated to 215° C., past the second endotherm, and then cooled before reheating, demonstrating a minor endotherm at ca. 145° C. but no other events occurred until the Form B endotherm. Insufficient solid remained for XRPD examination of the thermally manipulated material.

[0414] HSM revealed particle excitement at 162° C. leading into contraction from 168° C. into a melt by 182° C. Solids persisted and potentially crystallised within the melt until the crystals melted from 226° C. to 231° C.

Example 4

Conversion of 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]oxazole-4-carboxamide Form a into Other Crystalline Forms

[0415] Equilibration of Form A in various solvents gives very different outcomes from equilibration of amorphous compound in the same range of solvents.

[0416] Samples of crystalline Form A, 24×ca. 50±2 mg, were weighed into 24 crystallisation tubes. A selection of 24 solvents, 0.25 ml, 5 vol., were charged to the solids at 25° C. and observations made. Where suspensions remained, additional solvent, 0.25 ml, 5 vol., was charged up to 1 ml (20 vol.) total with ca. 30 minutes to equilibrate between each solvent charge. The mixtures were heated to 50° C. (38° C. for DCM) and immediate observations noted. The mixtures were equilibrated at temperature for ca. 21 hours and observations noted. The mixtures were cooled to 25° C. over ca. 2½ hours, equilibrated for ca. 4 hours and observations noted. The mixtures were heated to temperature and equilibrated for ca. 16½ hours. Observations were noted and, where suspensions held, samples were taken. The mixtures were cooled to 25° C. over ca. 1½ hours, equilibrated for ca. 1 hour, observations noted and the solids isolated.

[0417] Upon isolation, all solids were dried in vacuo under air for ca. 10 to 20 minutes and then dried in vacuo at 45° C. for ca. 17 hours. Upon isolation at 25° C., the filtrates were retained for compound solubility which was determined by HPLC.

[0418] Observations during the solvent addition and thermal modulation are summarised in Table 7. The form fate by XRPD at temperature and 25° C., solubility and overall CP (at 25° C.) of the solids are summarised in Table 8.

[0419] Of the 24 solvents/solvent mixtures assessed, the compound remained as a suspension upon addition of 21 solvents. Of the 3 solvents where complete/partial dissolution occurred, CPME and THF gave suspensions following extended equilibration at 50° C., leaving only THF/water, 4:1, as a solution that failed to return solid from 15 vol.

[0420] Moderate compound solubility (1 to 10 mg ml.sup.−1) was observed in half of the solvents with poor compound solubility (<1 mg ml.sup.−1) observed in cyclohexane, 2-chlorobutane, heptane, TBME and water. Significant compound solubility (>10 mg ml.sup.−1) was observed in anisole, nitromethane, THF, DCM and THF/water, 4:1.

[0421] The predominant XRPD pattern of the dried solids was Form A, the input version.

[0422] Equilibration in MEK gave Form B.

[0423] Nitromethane returned predominantly Form B with very minor evidence of Form A.

[0424] Equilibration in EtOAc and MeCN returned Form A/B mixed versions.

[0425] The crystalline forms produced by equilibration in THF and DCM were found to be dependent on the temperatures. The use THF as an equilibration solvent at 50° C. afforded Form B but, upon cooling to 25° C., afforded Form C. Conversely, DCM at 38° C. afforded Form C but, upon cooling to 25° C., afforded Form B.

TABLE-US-00019 TABLE 7 Solvents used during the equilibration of Form A and observations throughout 1.sup.st 1.sup.st 1.sup.st 2.sup.nd 2.sup.nd 25° C., 25° C., 50° C.*, 50° C.*, 25° C. 50° C.*, 25° C. 5 10 25° C., 25° C., T = 15 T = 17 post- T = 16½ post- ID Solvent vol. vol. 15 vol. 20 vol. mins Hrs cool Hrs cool A Cyclohexane Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. B Chlorobenzene Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. + Susp. + YS YS C 2- Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. Chlorobutane D Benzotrifluoride Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. E Anisole Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. F Nitromethane Susp. Susp. Susp. Susp. Susp. Susp. + Susp. + Susp. + Susp. + YS YS YS YS G CPME Susp. Susp. Susp. Susp. Dissolved Susp. Susp. Susp. Susp. H Heptane Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. I TBME Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. + Susp. + YS YS J MIBK Susp. Susp. Susp. Susp. Susp. Susp. + Susp. + Susp. + Susp. + YS YS YS YS K MEK Susp. Susp. Susp. Susp. Susp. Susp. + Susp. + Susp. + Susp. + YS YS YS YS L iPrOAc Susp. Susp. Susp. Susp. Susp. Susp. + Susp. + Susp. + Susp. + YS YS YS YS M EtOAc Susp. Susp. Susp. Susp. Susp. Susp. + Susp. + Susp. + Susp. + YS YS YS YS N Toluene Susp. Susp. Susp. Susp. Susp. Susp. + Susp. + Susp. + Susp. + YS YS YS YS O THF Susp. Susp. Susp. Dissolved Solution Haze + Susp. + Susp. + Susp. + YS YS YS YS P DCM Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. (*38° C.) Q MeOH Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. + Susp. + YS YS R EtOH Susp. Susp. Susp. Susp. Susp. Susp. + Susp. + Susp. + Susp. + YS YS YS YS S IPA Susp. Susp. Susp. Susp. Susp. Susp. + Susp. + Susp. + Susp. + YS YS YS YS T MeCN Susp. Susp. Susp. Susp. Susp. Susp. + Susp. + Susp. + Susp. + YS YS YS YS U Water Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. V 4:1 Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. Susp. MeCN/water W 4:1 Susp. Haze Dissolved N/A Solution Solution Solution Solution Solution THF/water X 4:1 Susp. Susp. Susp. Susp. Susp. Susp. + Susp. + Susp. + Susp. + IPA/water YS YS YS YS Susp. = Suspension Haze = Predominantly dissolved mixture YS = Yellow solid deposit observed above the mixture level

TABLE-US-00020 TABLE 7 Form fate, solubility and CP summary of Form A following equilibration of Form A in various solvents with thermal modulation CP by Form fate by Solubility HPLC XRPD at: at 25° C. at 25° C. ID Solvent 25° C. 50° C.* (mg/ml) (%) A Cyclohexane Form A Form A    0.01 97.25 B Chlorobenzene Form A Form A    1.87 97.63 C 2-Chlorobutane Form A Form A    0.29 97.26 D Benzotrifluoride Form A Form A    0.16 96.81 E Anisole Form A Form A   10.61 98.28 F Nitromethane Form B + Form B +   20.52 98.42 evidence evidence of A of A G CPME Form A Form A    1.87 97.81 H Heptane Form A Form A    0* 97.06 I TBME Form A Form A    0.70 97.52 J MIBK Form A Form A    7.40 98.50 K MEK Form B Form B    6.08 98.09 L iPrOAc Form A Form A    6.96 98.57 M EtOAc Form A/B Form A/B    6.64 97.90 Mix Mix N Toluene Form A Form A    1.12 96.34 O THF Form C Form B   33.30 98.54 P DCM (*38° C.) Form B Form C   22.83 98.26 Q MeOH Form A Form A    2.52 98.26 R EtOH Form A Form A    5.20 98.27 S IPA Form A Form A    2.45 97.98 T MeCN Form A/B Form A/B    9.16 97.55 Mix Mix U Water Form A Form A    0.06 97.11 V 4:1 MeCN/water Form A Form A   19.78 99.24 W 4:1 THF/water No solid isolated >67 N/A X 4:1 IPA/water Form A Form A    7.65 98.90 *There was no peak detected in the chromatogram for the filtrate (×100 dilution)

Example 5

Conversion of 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]oxazole-4-carboxamide Form a into Other Crystalline Forms by Dissolution/Anti-Solvent Method

[0426] The equilibration of Form A in a range of solvents with thermal modulation indicated a range of suitable solvents and anti-solvents. Consequently, Form A was dissolved in a range of four solvents at an elevated temperature, clarified and charged with anti-solvent at the elevated temperature to assess the propensity of the compound towards anti-solvent-driven crystallisation.

[0427] 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]oxazole-4-carboxamide Form A, 4×150-152 mg, was weighed into four crystallisation tubes. Four different solvents (see Table 8) were then charged to the solids and the resulting mixtures heated to 60° C. Where suspensions persisted, additional solvent was charged to achieve dissolution. Upon dissolution of the four mixtures, the solutions were clarified and aliquots containing ca. 30 mg of the compound were charged to four crystallisation tubes.

[0428] To each of the four aliquots was charged an anti-solvent (see Table 8), ca. 5 vol., at 60° C. and immediate observations were made. Observations were made after equilibration for 1 hour before additional anti-solvent, 5 vol., was charged to all mixtures. Where suspensions or immiscible mixtures had formed, no further anti-solvent was charged. To all solutions and hazes, additional anti-solvent up to 20 vol. total charge was added.

[0429] The mixtures were equilibrated at 60° C. for another hour before cooling to 25° C. over ca. 3 hours and equilibrated for ca. 14½ hours. Most mixtures were observed to have afforded suspensions. Upon isolation, all solids were dried in vacuo under air for ca. 1 hour and then dried in the oven in vacuo at 45° C. for 70 hours.

[0430] Observations made during the anti-solvent addition and subsequent cooling operation are summarised in Table 8.

TABLE-US-00021 TABLE 8 Solvents used and observations on outcome 60° C., 10 60° C., 15 60° C., 20 60° C., 5 vol, anti-solvent vol. anti- vol. anti- vol. anti- 25° C., 20 ID Solvent Anti-solvent Immediate 1 Hr solvent solvent solvent vol, 19 Hrs A1 TH Elater, 4:1 Cyclohexane Gum Susp. Susp. N/A N/A Susp. A2 10 vol. Heptane Gum Susp. Susp. N/A N/A Gummy Susp. A3 Benzotrifluoride Susp. Susp. Susp. N/A N/A Susp. A4 2-Chlorobutane Susp. Susp. Susp. N/A N/A Susp. B1 THF Cyclohexane Gum.fwdarw.Soln. Susp. Susp. N/A N/A Susp. B2 25 vol. Heptane Gum.fwdarw.Soln. Susp. Susp. N/A N/A Susp. B3 Benzotrifluoride Soln. Susp. Susp. N/A N/A Susp. B4 2-Chlcrobutane Soln. Susp. Susp. N/A N/A Susp. C1 Nitromethane Cyclohexane Immiscible Immiscible Immiscible N/A N/A Bi. Susp. C2 50 vol. Heptane Soln. Soln. Soln. Soln. Soln. Bi. Susp. C3 Benzotrifluoride Soln. Soln. Soln. Soln. Soln. Susp. C4 2-Chlorobutane Soln. Soln. Soln. Soln. Soln. Susp. D1 MeCN/water, 4:1 Cyclohexane Immiscible Immiscible Immiscible N/A N/A Immiscible D2 55 vol. Heptane Immiscible Immiscible Immiscible N/A N/A Bi. Susa D3 Benzotrifluoride Soln. Soln. Haze Immiscible Immiscible Susp. D4 2-Chlcrobutane Soln. Soln. Haze Haze Haze Bi. Susp.

[0431] The forms produced by each of the sixteen solvent-antisolvent combinations are shown in Table 9 below. The forms were identified by their XRPD diffraction patterns.

TABLE-US-00022 TABLE 9 Forms isolated after anti-solvent addition at temperature to solutions of the compound ID Solvent Anti-solvent Form fate by XRPD A1 THF/water, 4:1 Cyclohexane Form A A2 10 vol. Heptane N/A-insufficient solid afforded A3 Benzotrifluoride Form A A4 2-Chlorobutane Form A B1 THF Cyclohexane Form B B2 25 vol. Heptane Form B B3 Benzotrifluoride Form B B4 2-Chlorobutane Form B C1 Nitromethane Cyclohexane Form B + evidence of C C2 50 vol. Heptane Form B C3 Benzotrifluoride Form B C4 2-Chlorobutane Form C + evidence of B D1 MeCN/water, 4:1 Cyclohexane N/A-no solid afforded D2 55 vol. Heptane Form A D3 Benzotrifluoride Form A D4 2-Chlorobutane Form A

[0432] Form A was generated from the aqueous-based solvent mixtures whereas Form B was predominantly generated by non-aqueous mixtures with the exception of the nitromethane/cyclohexane solvent/anti-solvent combination (which produced Form B with evidence of Form C) and nitromethane/2-chlorobutane (which produced Form C with evidence of Form B).

[0433] Rapidly Cooled, Reverse Anti-Solvent Addition Crystallisation

[0434] Anti-solvent crystallisation was carried out by dissolving 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]oxazole-4-carboxamide in a solvent with heating, clarifying the solution and adding cold anti-solvent using the following method. 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]oxazole-4-carboxamide, 4×153-155 mg, was weighed into four crystallisation tubes and a different solvent (see Table 10) was charged into each. The mixtures were heated to 60° C. to achieve dissolution and then clarified.

[0435] Three anti-solvents (see Table 10), 1.5 ml, were each charged to four crystallisation tubes and cooled to −10° C.

[0436] The clarified solutions of 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]oxazole-4-carboxamide at 60° C. were charged to the cold anti-solvents in aliquots containing ca. 50 mg of the compound (into ca. 30 vol. anti-solvent) and immediate observations were noted.

[0437] The resulting mixtures were equilibrated at −10° C. for ca. 22 hours, observations were made and the solids were isolated. All isolated solids were dried in vacuo under air for ca. 20 minutes and then dried in vacuo at 45° C. for 23½ hours.

[0438] Observations made during the anti-solvent addition and subsequent cooling operation are summarised in Table 10. The crystalline forms (as determined by XRPD) arising from each solvent-anti-solvent combination are set out in in Table 11. For comparison purposes, the crystalline forms produced by addition of anti-solvent at 60° C. (see Table 10 above) are also included in Table 11.

TABLE-US-00023 TABLE 10 Solvents and anti-solvents used during the rapidly cooled, reverse addition to anti-solvent at −10° C. and observations throughout −10° C., 30 vol. anti-solvent ID Solvent Anti-solvent Immediate 5 Hrs 22 Hrs A1 THF/water, Heptane Gum Suspension Suspension A2 4:1 10 vol. Benzotrifluoride Suspension Suspension Suspension A3 2-Chlorobutane Suspension Suspension Suspension B1 THF Heptane Suspension Suspension Suspension B2 25 vol. Benzotrifluoride Suspension Suspension Suspension B3 2-Chlorobutane Suspension Suspension Suspension C1 Nitromethane Heptane Immiscible Bi. Susp. Bi. Susp. C2 50 vol. Benzotrifluoride Solution Solution Suspension C3 2-Chlorobutane Solution Solution Haze D1 MeCN/water, Heptane Immiscible Bi. Susp. Bi. Susp. D2 4:1 55 vol. Benzotrifluoride Haze Suspension Suspension D3 2-Chlorobutane Haze Suspension Suspension Haze = Predominantly solution Bi. Susp. = Biphasic with a susp. and clear solution layer

TABLE-US-00024 TABLE 11 Comparison of crystalline forms of solids arising from anti-solvent addition at 60° C. (See also Table 10) and reverse addition to anti-solvent at −10 °C Crystalline Form fate by XRPD Reverse addition Addition at to anti-solvent Solvent Anti-solvent 60° C. at −10° C. THF/water, 4:1 Heptane N/A- Low cryst. Form B 10 vol. insufficient solid Benzotrifluoride Form A Low cryst. Form B 2-Chlorobutane Form A Low cryst. Form B THF Heptane Form B Low cryst. Form B + 25 vol. evidence of C Benzotrifluoride Form B Low cryst. Form B 2-Chlorobutane Form B Low cryst. Form C Nitromethane Heptane Form B Form C 50 vol. Benzotrifluoride Form B Form C 2-Chlorobutane Form C + Form B/C mix evidence of B MeCN/water, 4:1 Heptane Form A Form A 55 vol. Benzotrifluoride Form A Form B 2-Chlorobutane Form A Form B

[0439] Rapidly cooled, reverse anti-solvent addition and subsequent equilibration at −10° C. for ca. 22 hours predominantly resulted in the formation of Form B, but with some instances of Form C or Form B/C mixed versions and only one instance of Form A.

[0440] The results demonstrate that the crystalline forms of 2-(2,6-dichlorophenyl)-5-[4-(1,1-dioxo-1,4-thiazinane-4-carbonyl)anilino]oxazole-4-carboxamide arising from solvent/anti-solvent crystallisations depends not only on the natures of the solvents but also on the temperatures at which the crystallisations are carried out.

Example 5

[0441] Conversion of Form A to Form B by Equilibration in Methylethylketone (MEK)

[0442] Crude crystalline Form A, 2.992 g (2.84 g active), and a crystalline Form B seed, ca. 3 mg, ca. 0.1 wt %, were weighed into a vessel. MEK, 6 vol., was charged into the vessel and the mixture warmed to 50° C. The resulting suspension was equilibrated at this temperature for ca. 19 hours and then cooled to 25° C. over 1½ hours. The solid was isolated via filtration followed by an MEK, 1 vol., vessel and filter wash. The washed solid was dried in vacuo under air for 1 hour and in vacuo at 45° C. for 19½ hours to afford 2.639 g of pale yellow solid, (88.2% mass recovery).

[0443] XRPD examination (diffractogram not shown) of the dried compound showed revealed a Form B diffraction pattern.

[0444] Thus, equilibration of crude crystalline Form A in MEK, 6 vol., at 50° C. successfully afforded Form B with a recovery of 88.2%, an increased active content of 97% (from 95%) and an improved CP of 98.85% (from 97.14%) on a 3 g scale.

[0445] The purity of the crystalline Form B can be improved still further by first equilibrating the Form A starting material in MeCN/water, 4:1, 4 vol., at 50° C., isolating and drying the purified Form A and then equilibrating the Form A in MEK as described above. In this way, crystalline Form B with an improved CP of 98.64%, and an active content of 99% can be obtained.

Example 6

[0446] Comparison of the Compression Stabilities of Crystalline Form a and Crystalline Form B

[0447] Compression stability tests were carried out on samples from two batches of crystalline Form A and samples from two batches of Crystalline Form B. One pair of samples of Form A and Form B was subjected to compression under 10×10.sup.4N force for 23 and 23¾ hours respectively to assess the impact upon form fate and chemical stability The other pair of samples of Form A and Form B was subjected to similar compression pressures over an extended duration of 7 days to assess the impact of prolonged compression force upon form fate and chemical stability.

[0448] XRPD examination of the Form A solids isolated post-compression demonstrated a diffraction pattern matching the input Form A but of reduced diffraction resolution following compression for both ca. 1 and 7 days, possibly due to limited sample or a reduction in the order of the solid.

[0449] XRPD examination of the Form B solids isolated post-compression demonstrated a diffraction pattern matching the input Pattern B but of reduced diffraction resolution following compression for both ca. 1 and 7 days, similar to Form A, possibly due to limited sample or a reduction in the order of the solid.

[0450] HPLC assessment of Form A, post-compression for 7 days, revealed significant chemical degradation. However, compression for ca. 1 day demonstrated no discernible chemical degradation, within experimental error.

[0451] HPLC assessment of Form B, post-compression for both ca. 1 and 7 days, revealed no change to the impurity profile of Pattern B, within experimental error.

[0452] The results of the tests showed that crystalline Form A was stable to relatively brief compression force, demonstrating no discernible chemical degradation but slightly reduced crystallinity by XRPD and DSC after 23 hours. However, compression for 7 days afforded significant chemical degradation.

[0453] On the other hand, crystalline Form B was stable to brief and prolonged compression force, demonstrating no discernible chemical degradation but slightly reduced crystallinity by XRPD and DSC.

[0454] Thus, Form B demonstrates improved stability to compression over Form A.

[0455] On the basis of the test results, it is considered that crystalline Form B would be better suited for solid formulations, and particularly those (such as tablets) that are prepared by processes involving compression steps.

[0456] Form A on the other hand may be better suited to the preparation of formulations that do not involve a compression step.

Example 7

[0457] Biological Activities

[0458] (i) TYK2 and JAK Kinase Inhibition Assays

[0459] The compound of the formula (1) was assayed for its ability to inhibit TYK2 kinase and other JAK kinases. The activity of the compound was compared with the activities of Compound A (2-(2-chloro-6-fluoro-phenyl)-5-[4-(morpholine-4-carbonyl)-phenylamino]-oxazole-4-carboxylic acid amide) and Compound B (2-(2,6-dichloro-phenyl)-5-[4-(morpholine-4-carbonyl)-phenylamino]-oxazole-4-carboxylic acid amide):

##STR00011##

which are the compounds of Examples 25 and 29 respectively in each of WO 2015/032423 and WO2018/073438.

[0460] Substrates and kinases used in the assays are identified in Table 12 below.

[0461] Kinase assays were performed at Reaction Biology Corp., Malvern, Pa., USA, using the general procedure set out below. In the assays, the ATP concentration was 100 μM and the top compound concentrations were 10 μM.

[0462] Note that the TYK2 and JAK kinase data in Table 7 on page 61 of WO 2015/032423 were generated using an assay in which the ATP concentration was 10 μM whereas, as indicated above, the assay described in the protocol below used an ATP concentration of 100 μM.

[0463] Assay: [0464] 1) Prepare indicated substrate in freshly prepared Base Reaction Buffer (20 mM Hepes pH 7.5, 10 mM MgCl.sub.2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na.sub.3VO.sub.4, 2 mM DTT, 1% DMSO). [0465] 2) Deliver cofactors (1.5 mM CaCl.sub.2, 16 ug/mL Calmodulin, 2 mM MnCl.sub.2) to the substrate solution above [0466] 3) Deliver indicated kinase into the substrate solution and gently mix [0467] 4) Deliver varying concentrations of test compound in DMSO into the kinase reaction mixture [0468] 5) Deliver .sup.33P-ATP (specific activity 0.01 μCi/μL final) into the reaction mixture to initiate the reaction [0469] 6) Incubate kinase reaction for 120 min at room temperature [0470] 7) Reactions are spotted onto P81 ion exchange filter paper (Whatman #3698-915) [0471] 8) Unbound phosphate is removed by washing filters extensively in 0.75% Phosphoric acid. [0472] 9) .sup.33P signal was determined using Typhoon phosphorimagers (GE Healthcare). After subtraction of background derived from control reactions containing inactive enzyme, IC.sub.50 values were determined using the nonlinear regression function in Prism (Graphpad software).

TABLE-US-00025 TABLE 12 Protein HUGO Genbank Protein Name symbol Substrate Accession # Accession # Clone Expression Tag JAK1 JAK1 pEY NP_002218.2 P23458 aa Baculovirus N- 866-1154 in Sf21 terminal insect cells GST tag JAK2 JAK2 pEY NP_004963 O60674 aa Baculovirus N- 809- in Sf21 terminal 1132 + insect cells GST g tag JAK3 JAK3 JAK3tide NP_000206 P52333 aa Baculovirus N- 781-1124 in Sf21 terminal insect cells GST tag TYK2 TYK2 AXLtide NP_003322.2 P29597 Aa Baculovirus N- 833-1187 in Sf21 terminal insect cells GST tag Substrates: AXLtide = [KKSRGDYMTMQIG] JAK3tide = [Ac-GEEEEYFELVKKKK-NH.sub.2] pEY = poly Glu-Tyr [Glu:Tyr (4:1), M.W. = 5,000-20,000]

[0473] The results are shown in Table 3 below.

TABLE-US-00026 TABLE 13 IC.sub.50 (nM) Selectivity v TYK2 TYK2 JAK1 JAK2 JAK3 TYK2 JAK1 JAK2 JAK3 Comparative 10 49 87 290 1 4.9 8.7 29 Compound A Comparative 5.1 26 77 271 1 5.1 15.1 53.1 Compound B Compound (1)- 1.9 20 50 212 1 10.5 26.3 111.6 Example 1

[0474] Although all tested compounds were shown to possess good TYK2 inhibitory activity, the data illustrate that the compound of the formula (1) is both more potent and more selective with respect to TYK2 (particularly towards TYK2 over JAK2 and JAK3) than the prior art compounds A and B.

[0475] (ii) Cytochrome P450 Inhibition Assays

[0476] The susceptibility of Compound (1) to potential drug-drug interactions was tested by assaying their abilities to inhibit various cytochrome P450 isoforms. Prior art Compound B (see Example 7(i) above) was also tested as a comparative example.

[0477] Test compounds, prepared and serially diluted in DMSO, were incubated at six concentrations (1% DMSO final) with pooled human liver microsomes in the presence of probe substrate for each isoform, and their effects on the metabolism of probe substrates determined. Incubations (in 96-well plates) were carried out at 37° C. in 0.1 M Tris buffer, pH 7.4, with reactions initiated by the addition of cofactor, NADPH (1 mM final concentration).

[0478] At the specified times, reactions were terminated with acetonitrile containing an analytical internal standard, samples were centrifuged and the supernatant fractions were analysed for probe substrate metabolites by mass spectrometry (LC-MS/MS). The instrument responses were normalised to internal standard and compared to the appropriate solvent controls to determine the amount of metabolite formed from the probe substrates relative to these “uninhibited” controls.

[0479] The results are reported as percentage inhibition and IC.sub.50 values (concentration resulting in a 50% reduction in probe metabolite formation) were calculated using a non-linear sigmoidal dose response equation (BioBook):

% inhibition=lowest value+(highest value−lowest value)/(1+10{circumflex over ( )}((Log IC.sub.50−X)*HillSlope))

[0480] where X=Log concentration.

[0481] The CYP450 isoforms studied, and their respective probe substrates are shown in Table 14.

TABLE-US-00027 TABLE 14 CYP450 isoform Substrate CYP1A2 Phenacetin CYP2B6 Bupropion CYP2C8 Amodiaquine CYP2C9 Diclofenac CYP2C19 S-(+)-Mephenytoin CYP2D6 Dextromethorphan CYP3A4 Midazolam CYP3A4 Testosterone

[0482] The assay results are shown in Table 15.

TABLE-US-00028 TABLE 15 CYP CY|P CYP CYP CYP CYP CYP CYP 3A4 3A4 Compound 1A2 2B6 2C8 2C19 2C9 2D6 (MID) (Test) Comparative >30 >30 >8.2 >30 4.4 >30 >30 >30 Compound B Compound >30 >30 >30 >30 26 >30 >30 >30 (1)

[0483] Although all tested compounds show good CYP inhibition profiles, the data illustrate that the compound of the formula (1) has better CYP inhibition profiles (i.e. inhibit the CYP isoforms tested to a lesser extent) than the Comparative Compound B, particularly with respect to CYP2C8 and CYP2D6.

[0484] (iii) hERG Channel Inhibition Assay

[0485] The potential for compounds to inhibit the hERG potassium channel was determined using a hERG-HEK stably transfected cell line on the Sophion Qube automated electrophysiology platform. The assay was performed at room temperature and recordings of the hERG tail current from individual cells was made using single-hole QChips.

[0486] The potency (IC.sub.50) of test compounds to inhibit the hERG channel was determined from a concentration-response curve generated from 8 test compound concentrations with up to 4 replicates per concentration.

[0487] The compound concentration was added to the test well twice to ensure complete exchange of the external buffer with the test compound. In total, compound was applied to the well for >7 minutes.

[0488] Results are shown in Table 16 below.

TABLE-US-00029 TABLE 16 IC.sub.50 Max Conc. % inhibition at Compound (uM) (uM) Max. Conc. Comparative ~30 30 50.5 Compound B Compound (1) >30 30 38.6

[0489] All three tested compounds show relatively low hERG activity, but the results demonstrate that the compound of the formula (1) has an even lower hERG liability compared to prior art comparative Compound B.

[0490] (iv) Hepatocyte Stability Assay

[0491] The compound (1) and prior art comparative Compound B were tested in hepatocyte stability assays which were performed using pooled hepatocytes from mouse (male CD-1), rat (male Sprague-Dawley), dog (male Beagle) and human (mixed gender). Test and control compounds were incubated with hepatocytes at 37° C. Aliquots were removed at 6 timepoints over a one hour period. Samples were centrifuged and the supernatant fractions analysed for parent compound by mass spectrometry (LC-MS/MS).

[0492] The amount of compound remaining (expressed as %) was determined from the MS response in each sample relative to that in the T=0 samples, and used to determine the half-life and intrinsic clearance of the compound.

[0493] Results are shown in Table 17 below.

TABLE-US-00030 TABLE 17 Mouse Rat Dog Human CL.sub.int CL.sub.int CL.sub.int CL.sub.int μL/min/ μL/min/ μL/min/ μL/min/ Com- 10.sup.6 T.sub.1/2 10.sup.6 T.sub.1/2 10.sup.6 T.sub.1/2 10.sup.6 T.sub.1/2 pound cells mins. cells mins. cells mins. cells mins. Com- parative 40.5 27 103.7 14.8 29.8 53.5 9.0 124 Com- pound B Com- pound 16.8 65.3 50.0 30.6 <3.0 >460 <3.0 >460 (1)

[0494] While comparative Compound B exhibits a good half-life in humans (over 2 hours), the data in Table 7 indicate that compound (1) has a significantly reduced rate of clearance in all four hepatocyte stability assays compared to prior art comparative Compound B and that, consequently, the half life (T.sub.1/2) of the compound of the formula (1) is even longer in all four assays than the half life of the comparative Compound B.

[0495] (v) pSTAT3 Inhibition

[0496] The compound (1) and prior art comparative Compounds A and B were tested for pSTAT3 inhibition in response to IL-22 stimulation in serum starved HT29 cells.

[0497] HT29 cells were serum starved overnight before the four test compounds were diluted to generate a 9-point semi-log dose dilution with a top concentration of 10 μM, plus vehicle control. HT29 cells were incubated with the test compounds for 20 minutes at 37° C. HT29 cells were incubated for a further 15 minutes with 10 ng/ml human IL-22 before cells were fixed with 4% PFA for 10 minutes, and 90% methanol for 30 minutes before being labelled with a phospho-STAT3Y705 antibody (CST #9145). Cells were rinsed three times using 0.5% BSA/PBS solution before being incubated with Alexa-488 anti-rabbit secondary antibodies.

[0498] Mean fluorescence intensity of phospho-STAT3 in single cells was analysed by flow cytometry using an Intellicyt iQue instrument and FlowJo software. The IC.sub.50 was determined using a four-parameter analysis following removal of background signal and normalisation to the DMSO control.

[0499] Results are shown in Table 18 below.

TABLE-US-00031 TABLE 18 % pSTAT3 IC.sub.50 inhibition Test Compound (nM) at 10 μM Comparative Compound A 170 96.9 Comparative Compound B 53 97.9 Compound (1) 16 95.9

[0500] While both comparative Compound B and Compound (1) were shown to have IC.sub.50 values against pSTAT3 inhibition of less than 100 nM, the IC.sub.50 value for Compound (1) was significantly lower than for comparative Compound B.

[0501] (vi) Human Primary CD4CD45RO+ Cells Assay

[0502] Inhibition of IL-17F production and STAT3 phosphorylation by Compound (1) and Comparative Compound B were measured in Th17 cells derived from human peripheral blood CD4CD45RO+ cells.

[0503] Fresh human Peripheral blood CD4CD45RO+ cells were purchased commercially (Generon, UK); 3 separate vials from 3 different volunteers for experimental replicates. Cells were grown in T-cell medium (Thermo Fisher) containing 10 ng/ml recombinant human IL-1B (R&D Systems), IL-23 (R&D Systems), TGF-B1 (R&D Systems) and 50 ng/ml IL-6 (R&D Systems) together with anti-CD3/CD28 magnetic Dynabeads (Thermo Fisher). These were grown for 11 days to induce expansion of Th17 cells. Prior to plating for assays cells were grown overnight in T-cell medium supplemented with human serum (1%) overnight. Media was removed and replaced with unsupplemented RPMI for 4 h prior to assay.

[0504] To measure IL-17F levels, 200,000 cells were plated into a 96 well plate and preincubated with compounds for 30 minutes followed by stimulation with recombinant IL-23 at 6.25 ng/ml and recombinant human IL-1B at 0.1 ng/ml for 48 h. Supernatants were removed and IL-17F levels measured using a commercially available ELISA kit (Thermo Fisher; BMS2037-2).

[0505] To measure pSTAT3 levels, 200,000 cells were plated into a 96 well plate and preincubated with compounds for 30 minutes followed by stimulation with recombinant IL-23 at 12.5 ng/ml for 15 minutes then lysed using cell lysis buffer. pSTAT3 levels in the lysates were measured using a commercially available ELISA kit (Thermo Fisher; 85-86102-11).

[0506] ELISAs were carried out according to manufacturers' instructions and absorbance read using a microplate reader (Thermo Fisher; Varioskan). Data were normalised to the response in untreated samples using the formula:

% of control=((Stimulated sample Conc.−unstimulated sample Conc.)×100)/(Control stimulated Conc.−control unstimulated Conc.)

[0507] Graphpad Prism 8.1.0 was used to calculate IC.sub.50 values using a Nonlinear 4 parameter logistic regression model (4PL).

[0508] The results are shown in Tables 19A and 19B below:

TABLE-US-00032 TABLE 19A IL17-F Production Inhibition Average Compound Donor 1 Donor 2 Donor 3 (nM) SD Comparative 243 217 148 203 49 Compound B Compound (1) 117 134  64 105 37

TABLE-US-00033 TABLE 19B Inhibition of STAT3 phosphorylation Average Compound Donor 1 Donor 2 Donor 3 (nM) SD Comparative 111 17 54 61 47 Compound B Compound (1)  69 29 55 51 20

[0509] While all tested compounds showed inhibition of IL17-F production and STAT3 phosphorylation, in both assays Compound (1) was shown to be more active than comparative Compound B.

[0510] Comparative Data—Conclusions

[0511] The data obtained from assays (i) to (vi) above indicate that the compound of the formula (1) has significant advantages over the structurally most similar compound (Compound B) in WO2015/032423.

[0512] Thus, compound (1) is more active than Compound B in the TYK2 kinase inhibition assay and has greater selectivity for TYK2 versus JAK1, JAK2 and JAK3 kinases than Compound B.

[0513] Compound (1) has slightly advantageous properties compared to prior art comparative Compound B in the cytochrome P450 assays, notably in the CYP2C8 and CYP2C9 assays.

[0514] Compound (1) has a reduced hERG liability compared to prior art comparative Compound B.

[0515] In the hepatocyte stability assays, Compound (1) showed a reduced rate of clearance and a consequently longer half life than comparative Compound B.

[0516] In addition, Compound (1) is more potent in inhibiting phosphorylation of STAT3 in IL-22 stimulated HT29 cells and Th17 cells compared to comparative Compound B.

[0517] Finally, Compound (1) shows a greater inhibition of IL-17F production in Th17 cells compared to comparative Compound B.

[0518] Taken together, the data indicate that Compound (1) is a highly potent and selective TYK2 kinase inhibitors and has excellent pharmacokinetic properties.

Example 7

[0519] Pharmaceutical Formulations

[0520] (i) Tablet Formulation

[0521] A tablet composition containing a crystalline form of the compound of the formula (1) as defined herein is prepared by mixing 50 mg of the compound with 197 mg of lactose (BP) as diluent, and 3 mg magnesium stearate as a lubricant and compressing to form a tablet in a known manner.

[0522] (ii) Capsule Formulation

[0523] A capsule formulation is prepared by mixing 100 mg of a crystalline form of the compound of the formula (1) as defined herein with 100 mg lactose and filling the resulting mixture into standard opaque hard gelatin capsules.

[0524] (iii) Sub-Cutaneous Injection Formulation

[0525] A composition for sub-cutaneous administration is prepared by mixing a crystalline form of the compound of the formula (1) as defined herein with pharmaceutical grade corn oil to give a concentration of 5 mg/mL. The composition is sterilised and filled into a suitable container.

EQUIVALENTS

[0526] The foregoing examples are presented for the purpose of illustrating the invention and should not be construed as imposing any limitation on the scope of the invention. It will readily be apparent that numerous modifications and alterations may be made to the specific embodiments of the invention described above and illustrated in the examples without departing from the principles underlying the invention. All such modifications and alterations are intended to be embraced by this application.