EP4646415A1 - Polymorphs of a jak1/tyk2 inhibitor and uses thereof - Google Patents

Abstract

Provided herein are solid forms, for example polymorphs, of 2 -(3 - ((7 R, 8a S) -7 -fl uo ro h exa hyd ro pyrro Io [ 1, 2-a ] pyra zi n - 2 (1 H) - yl) - 1- (5- methyl - 2 - ((I - methy l-lH-pyrazol-4-yl)amino)pyrimidin-4- yl)azetidin-3-yl)acetonitrile, which are useful for treating kinase- related diseases or disorders. The solid forms, and compositions thereof, are useful for treating diseases, including, without limitation, eye disease, such as glaucoma, ocular hypertension, ocular wound repair, neurodegenerative ocular diseases, retinal detachment, and non-ocular diseases such as neuronal damage, or skin wound repair, or inflammatory diseases, among others, in a subject. (Compound 1)

Description

POLYMORPHS OF A JAK1/TYK2 INHIBITOR AND USES THEREOF

RELATED APPLICATIONS

[0001]This application claims priority of U.S. Provisional Patent Application No. 63/465,091, filed May 9, 2023, and U.S. Provisional Patent Application No. 63/437,084, filed January 4, 2023, the entire content of each of which is incorporated herein by reference.

TECHNICAL FIELD

[0002] The present disclosure relates to solid forms, for example polymorphs, of 2-(3-((7R,8aS)-7-fluorohexahydropyrrolo[l,2-a]pyrazin-2(lH)-yl)-l-(5-methyl-2-((l-methy l-lH-pyrazol-4-yl)amino)pyrimidin-4-yl)azetidin-3-yl)acetonitrile, which are useful for treating kinase-related diseases or disorders (such as a JAK1- or TYK2-related disease or disorder). These include diseases or disorders of the eye (such as dry eye, corneal disease, retinal disease, and ocular hypertension), diseases of the skin, diseases or conditions of the respiratory system, diseases of the cardiovascular system, and diseases characterized by abnormal growth (such as cancers).

BACKGROUND

[0003] Dry eye disease (DED) is a multifactorial disorder in which the eyes respond to minor stimuli with abnormal sensations, such as dryness, blurring, foreign body sensation, discomfort, irritation, and pain. The active pharmaceutical ingredient

2-(3-((7R,8aS)-7-fluorohexahydropyrrolo[l,2-a]pyrazin-2(lH)-yl)-l-(5-methyl-2-((l -methyl-lH-pyrazol-4-yl)amino)pyrimidin-4-yl)azetidin-3-yl)acetonitrile (Compound 1) may be used to treat DED.

Compound 1 [0004] Stable solid forms of Compound 1 and compositions thereof having high purity of Compound 1 have been discovered. Stability of an active agent is useful to survive the diverse stresses of manufacturing so that the resultant administrable article of manufacture comprises a pharmaceutically acceptable purity of the active agent. High purity of an active agent is useful in, for example, avoiding administration, to subjects, of side-products of active agents resulting from manufacturing processes.

[0005] Thus, provided herein are polymorphic forms of Compound 1, methods useful in their preparation including at high purity, compositions comprising such forms, and therapeutic uses thereof.

SUMMARY

[0006] In some embodiments, provided herein are forms of Compound 1, including solid forms such as polymorphic forms.

[0007] Also disclosed are pharmaceutical compositions comprising the polymorphic forms, and methods of using the polymorphic forms for treatment of kinase related diseases or disorders.

BRIEF DESCRIPTION OF THE FIGURES

[0008] Fig. 1 shows an X-Ray Powder Diffraction (XRPD) pattern of Form 1 of Compound 1.

[0009] Fig. 2 shows XRPD patterns of polymorphs of Compound 1, prepared as in the Examples.

[0010] Fig. 3 shows XRPD patterns of polymorphs of Compound 1, prepared as in the Examples.

[0011] Fig. 4 shows an XRPD pattern of polymorph Form 1 of Compound 1 described in Table 13.

[0012] Fig. 5 shows an XRPD pattern of polymorph Form 2 of Compound 1 described in Table 14.

[0013] Fig. 6 shows an XRPD pattern of polymorph Form 3 of Compound 1 described in Table 15.

[0014] Fig. 7 shows an XRPD pattern of polymorph Form 4 of Compound 1 described in Table 16.

[0015] Fig. 8 shows an XRPD pattern of polymorph Form 5 of Compound 1 described in

Table 17. DETAILED DESCRIPTION

[0016] Polymophic forms of Compound 1 are provided herein. Compound 1 may be prepared according to the syntheses described in, for example, WO2023279105A1, the entire content of which is incorporated herein by reference, including, without limitation, Scheme 7.

[0017] Solid or polymorphic forms of Compound 1 can be used to treat or prevent kinase-related diseases or disorders. In some embodiments, these include diseases or disorders of the eye, such as dry eye, corneal damage, retinal inflammation, and ocular hypertension, diseases of the respiratory system, diseases of the cardiovascular system, and diseases characterized by abnormal growth, such as cancers, which may be referred to herein as JAK-associated diseases. In some embodiments, the solid forms or polymorphs of the compound described herein are useful: for the treatment of eye diseases including, without limitation, non-infectious uveitis, non-infectious chorioretinitis, iritis, sterile conjunctivitis, keratitis, episcleritis, dry eye diseases, meibomian gland dysfunction, allergic conjunctivitis, glaucoma, or retinal diseases; as anti-inflammatory agents; for the treatment of skin diseases; for the treatment of cardiovascular diseases; for the treatment of autoimmune disorders including, without limitation, rheumatoid arthritis, Crohn's disease, ulcerative colitis; or for the treatment of diseases characterized by abnormal growth, including, without limitation cancers, including, without limitation, prostate cancer.

Definitions

[0018] Certain terms, whether used alone or as part of a phrase or another term, are defined below.

[0019]The articles "a" and "an" refer to one or to more than one of the grammatical object of the article.

[0020] Numerical values relating to measurements are subject to measurement errors that place limits on their accuracy. For this reason, all numerical values provided herein, unless otherwise indicated, are to be understood as being modified by the term "about." Accordingly, the last decimal place of a numerical value provided herein indicates its degree of accuracy. Where no other error margins are given, the maximum margin is ascertained by applying the rounding-off convention to the last decimal place or last significant digit when a decimal is not present in the given numerical value.

[0021]The term "amelioration" means a lessening of severity of at least one indicator of a condition or disease, such as a delay or slowing in the progression of one or more indicators of a condition or disease. The severity of indicators may be determined by subjective or objective measures which are known to those skilled in the art.

[0022] The term "composition" refers to a mixture of at least two or more components.

[0023] The terms "effective amount" and "therapeutically effective amount" refer to an amount of therapeutic compound, combination of compounds, or composition, either as a single dose or as part of a series of doses, which is effective to produce a desired therapeutic effect. In general, the therapeutically effective amount can be estimated initially either in cell culture assays or in mammalian animal models, for example, in non-human primates, mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in non-human subjects and human subjects.

[0024] The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid filler, solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent, or encapsulating material, involved in carrying or transporting at least one compound described herein within or to the patient such that the compound may perform its intended function. A given carrier must be "acceptable" in the sense of being compatible with the other ingredients of a particular formulation, including the compounds described herein, and not injurious to the patient. Other ingredients that may be included in the pharmaceutical compositions described herein are known in the art and described, for example, in "Remington's Pharmaceutical Sciences" (Genaro (Ed.), Mack Publishing Co., 1985), the entire content of which is incorporated herein by reference.

[0025]The term "pharmaceutical composition" refers to a mixture of at least one compound described herein with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound, or combination thereof, to a patient or subject. Multiple techniques of administering a compound, combination, or composition, exist including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

[0026] The terms "treatment" or "treating" refer to the application of one or more specific procedures used for the amelioration of a disease. A "prophylactic" treatment, refers to reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset. [0027] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. Accordingly, for the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0028] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the described subject matter and does not pose a limitation on the scope of the subject matter otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to practicing the described subject matter.

[0029] Groupings of alternative elements or embodiments of this disclosure are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. Furthermore, a recited member of a group may be included in, or excluded from, another recited group for reasons of convenience or patentability. When any such inclusion or exclusion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0030] References have been made to patents and printed publications throughout this specification, each of which are individually incorporated herein by reference in their entirety.

[0031] It is to be understood that the embodiments of this disclosure are illustrative. Accordingly, the present disclosure is not limited to that precisely as shown and described.

Polymorphic Forms

[0032] Provided herein are polymorphic forms of Compound 1. Also provided herein is an amorphous form of Compound 1.

[0033]Thus, in some embodiments, provided herein are polymorphic forms of Compound 1, comprising one or more XRPD signals in terms of 29, ±0.2, selected from 2.8, 4.5, 5.8, 6.4, 7.3, 7.7, 8, 8.9, 9.3, 9.7, 9.9, 10.6, 10.9, 11.5, 11.9, 12.4, 12.8, 13.5, 14.3, 14.6, 14.8, 15.2, 15.8, 17.4, 17.6, 18.5, 18.9, 19.8, 21.8, 22.4, 22.9, 23.3, 24.1, 25, 25.7, 26.1, 26.5, 27.1, 27.9, 28.5, 29.1, or 29.7.

[0034]Thus, in some embodiments, provided herein are polymorphic forms of Compound 1, comprising one or more XRPD signals in terms of 29, ±0.2, selected from 2.8, 4.5, 5.8, 7.3, 7.7, 9.3, 9.7, 9.9, 11.5, 14.6, 17.4, 22.9, 28.5, or 29.7.

[0035] In some embodiments, the polymorphic form of Compound 1 is Form 1, Form 2, Form 3, Form 4, or Form 5.

[0036] In some embodiments, the polymorphic form of Compound 1 is a crystalline form of Compound 1.

[0037] In some embodiments, the polymorphic form of Compound 1 is Form 1 and includes one or more XRPD signals in terms of 29, ±0.2, selected from 7.3, 11.5, 28.5, or 29.7.

[0038] In some embodiments, the polymorphic form of Compound 1 is Form 2 and includes one or more XRPD signals in terms of 29, ±0.2, selected from 2.8, 5.8, 9.9, or 22.9.

[0039] In some embodiments, the polymorphic form of Compound 1 is Form 3 and includes one or more XRPD signals in terms of 29, ±0.2, selected from 7.7 or 17.4.

[0040] In some embodiments, the polymorphic form of Compound 1 is Form 4 and includes one or more XRPD signals in terms of 29, ±0.2, selected from 4.5, 9.3, or 9.7.

[0041] In some embodiments, the polymorphic form of Compound 1 is Form 5 and includes an XRPD signal of 14.6 ±0.2 29.

Compositions

[0042] Provided herein are compositions, comprising Compound 1 in one or more polymorphic form. Thus, in some embodiments, provided herein are pharmaceutical compositions comprising one or more polymorphic form of Compound 1, and at least one pharmaceutically acceptable carrier. In some embodiments, the composition is a solid composition. In some embodiments, the composition is an implantable composition. In some embodiments, the composition is an inhalable composition. In some embodiments, the composition is an orally ingestible composition. In some embodiments, the composition is an injectable composition. In some embodiments, the composition is a flowable powder composition. In some embodiments, the composition is a liquid composition, including, without limitation, a suspension or emulsion of the form of Compound 1. In some embodiments, the composition is a gel, cream, or ointment comprising the form of Compound 1. Processes

[0043] Also provided herein are processes for preparing the polymorphic forms of Compound 1 described herein.

[0044] Thus, in some embodiments, provided herein are methods of preparing a polymorphic form of Compound 1, comprising lyophilizing or crystallizing Compound 1 from a solvent or solvent system as described herein.

Methods

[0045] Provided herein are methods of using the polymorphic forms of Compound 1 described herein. In some embodiments, the methods include administering the form of Compound 1 to a subject.

[0046]Thus, in some embodiments, the polymorphic forms of Compound 1 described herein are useful in treating a kinase-related disease, are useful in inhibiting a kinase, or are useful in preparation of a medicament, including, without limitation, at a commercially relevant scale.

[0047] Also provided herein are methods of treating an ocular disease or condition in a subject in need thereof, comprising administering to the subject Compound 1, wherein the Compound 1 is in a form provided herein, including, Compound 1 in amorphous form, Form 1, Form 2, Form 3, Form 4, or Form 5 as prepared herein, or in a composition thereof, which may be a pharmaceutical composition, or any combination thereof, including, without limitation, more than one forms of Compound 1 provided herein. In some embodiments, Compound 1 may be provided in a purified form, including, without limitation, purification by 1, 2, 3, or more repetitions of recrystallization, lyophilization, or a combination thereof.

[0048] In some embodiments, kinase-related diseases or disorders may include JAK1- or TYK2-related diseases or disorders. In some embodiments, the diseases or disorders treated by administration of the forms of Compound 1 provided herein include ocular diseases or disorders (such as dry eye, corneal disease, retinal disease, and ocular hypertension), diseases of the skin, diseases or conditions of the respiratory system, diseases of the cardiovascular system, and diseases characterized by abnormal growth (such as cancers).

[0049] In some embodiments, kinase-related diseases or disorders may include, without limitation, eye disease, such as glaucoma, ocular hypertension, ocular wound repair, neurodegenerative ocular diseases, retinal detachment, and non-ocular diseases such as neuronal damage, or skin wound repair.

[0050] In some embodiments, provided herein are methods of treating ocular dryness, blurring, foreign body sensation, discomfort, irritation, or pain.

Kits

[0051] In some embodiments, provided herein are packaged forms of Compound 1, packaged compositions, or packaged pharmaceutical compositions, comprising a container holding a therapeutically effective amount of a form of Compound 1 described herein, and instructions for using the form of Compound 1 in accordance with one or more of the methods provided herein.

[0052] The present forms of Compound 1 and associated materials can be finished as a commercial product by the usual steps performed in the present field, for example by appropriate sterilization and packaging steps. For example, at doses of 25-35 kGy, both e- beams and gamma radiation may effectively sterilize pharmaceuticals. Alternatively, the material can be treated by UV/vis irradiation (200-500 nm), for example using photoinitiators with different absorption wavelengths (e.g., Irgacure 184, 2959), preferably water-soluble initiators (e.g., Irgacure 2959). Such irradiation is usually performed for an irradiation time of 1-60 min, but longer irradiation times may be applied, depending on the specific method. The material according to the present disclosure can be finally sterile- wrapped so as to retain sterility until use and packaged (e.g. by the addition of specific product information leaflets) into suitable containers (boxes, etc.).

[0053] According to further embodiments, the present particles can also be provided in kit form combined with other components necessary for administration of the material to the patient. For example, disclosed kits, such as for use in ocular treatments or the treatment of cancer, can further comprise, for example, administration materials.

[0054] The kits may be designed in various forms based on the specific deficiencies they are designed to treat.

[0055]The forms of Compound 1 or compositions provided herein may be prepared and placed in a container for storage at ambient or elevated temperature. When the form of Compound 1 or composition is stored in a polyolefin plastic container as compared to, for example, a polyvinyl chloride plastic container, discoloration of the form (e.g., a compound in the particle) or composition may be reduced, whether suspended in a liquid composition (e.g., an aqueous or organic liquid solution), or as a solid. Without wishing to be bound by theory, the container may reduce exposure of the container's contents to electromagnetic radiation, whether visible light (e.g., having a wavelength of about 380-780 nm) or ultraviolet (UV) light (e.g., having a wavelength of about 190-320 nm (UV B light) or about 320-380 nm (UV A light)). Some containers also include the capacity to reduce exposure of the container's contents to infrared light, or a second component with such a capacity. Some containers further include the capacity to reduce the exposure of the container's contents to heat or humidity. The containers that may be used include those made from a polyolefin such as polyethylene, polypropylene, polyethylene terephthalate, polycarbonate, polymethylpentene, polybutene, or a combination thereof, especially polyethylene, polypropylene, or a combination thereof. In some embodiments, the container is a glass container, including without limitation an amber colored glass container. The container may further be disposed within a second container, for example, a paper container, cardboard container, paperboard container, metallic film container, or foil container, or a combination thereof, to further reduce exposure of the container's contents to UV, visible, or infrared light. Articles of manufacture benefiting from reduced discoloration, decomposition, or both during storage, include dosage forms that include a form of Compound 1 or composition described herein. The forms of Compound 1 or compositions provided herein may need storage lasting up to, or longer than, three months; in some cases up to, or longer than one year. The containers may be in any form suitable to contain the contents— for example, a bag, a bottle, or a box, or any combination thereof.

[0056] The compounds and processes of the invention will be better understood by reference to the following examples, which are intended as an illustration of and not a limitation upon the scope of the invention.

EXAMPLES

[0057] Unless otherwise stated, the following instrumentation and methodologies were used in the below examples.

Instrumentation and Methodologies

[0058] X-ray Powder Diffraction (XRPD): Bruker AXS D8 Advance. XRPD diffractograms were collected on a Bruker D8 diffractometer using Cu Ko radiation (40 kV, 40 mA) in reflection geometry and a 9-20 goniometer fitted with a Ge monochromator. The incident beam passes through a 2.0 mm divergence slit followed by a 0.2 mm anti-scatter slit and knife edge. The diffracted beam passes through an 8.0 mm receiving slit with 2.5° Soller slits followed by the Lynxeye Detector. The software used for data collection was Diffrac Plus XRD Commander and data analysis was HighScore Plus. Samples were run under ambient conditions as flat plate specimens using powder as received. The sample was prepared on a polished, zero-background (510) silicon wafer by gently pressing onto the flat surface or packed into a cut cavity. The sample was rotated in its own plane. The details of the standard data collection method are: Angular range: 2 to 42° 20; Step size: 0.05° 20; and Collection time: 0.5 s/step (total collection time: 6.40 min).

[0059] X-ray Powder Diffraction (XRPD): PANalytical Empyrean. XRPD diffractograms were collected on a PANalytical Empyrean diffractometer using Cu Ka radiation (45 kV, 40 mA) in transmission geometry. A 0.5° slit, 4 mm mask and 0.04 rad Soller slits with a focusing mirror were used on the incident beam. A PIXcel^3D detector, placed on the diffracted beam, was fitted with a receiving slit and 0.04 rad Soller slits. The software used for data collection was X'Pert Data Collector using X'Pert Operator Interface. The data were analyzed and presented using HighScore Plus. Samples were prepared and analyzed in either a metal or Millipore 96 well-plate in transmission mode. X-ray transparent film was used between the metal sheets on the metal well-plate and powders (approximately 1 - 2 mg) were used as received. The Millipore plate was used to isolate and analyze solids from suspensions by adding a small amount of suspension directly to the plate before filtration under a light vacuum. The scan mode for the metal plate used the gonio scan axis, whereas a 20 scan was utilized for the Millipore plate. The details of the standard screening data collection method are: Angular range: 2.5 to 32.0° 20; Step size: 0.0130° 20; and Collection time: 12.75 s/step (total collection time of 2.07 min).

[0060] The software used for data collection was X'Pert Data Collector and the data analyzed and presented using Highscore Plus.

[0061] Nuclear Magnetic Resonance (NMR): Solution State NMR. 'H NMR, ¹³C NMR and/or ¹⁹F NMR spectra were collected on a Bruker 400 MHz instrument equipped with an auto-sampler and controlled by a Avance NEO nanobay console. Samples were prepared in DMSO-de solvent, unless otherwise stated. Automated experiments were acquired using ICON-NMR configuration within Topspin software, using standard Bruker-loaded experiments (¹H, ¹³C {¹H}, DEPT135). Off-line analysis was performed using ACD Spectrus Processor.

[0062] Differential Scanning Calorimetry (DSC): TA Instruments Q2OOO. DSC data were collected on a TA Instruments Q2000 equipped with a 50 position auto-sampler. Typically, 0.5-3 mg of each sample, in a pin-holed aluminum pan, was heated at 10 °C/min from 25 °C to 300 °C. A purge of dry nitrogen at 50 mt/min was maintained over the sample. Modulated temperature DSC (MDSC) was carried out using an underlying heating rate of 2 °C/min and temperature modulation parameters of ±0.636 °C (amplitude) every 60 seconds (period). The instrument control software was Advantage for Q Series and Thermal Advantage and the data were analyzed using Universal Analysis or TRIOS.

[0063] Differential Scanning Calorimetry (DSC): TA Instruments Discovery DSC. DSC data were collected on a TA Instruments Discovery DSC equipped with a 50 position auto-sampler. Typically, 0.5-3 mg of each sample, in a pin-holed aluminum pan, was heated at 10 °C/min from 25 °C to 300 °C. A purge of dry nitrogen at 50 mL/min was maintained over the sample. The instrument control software was TRIOS and the data were analyzed using TRIOS or Universal Analysis.

[0064] Thermo-Gravimetric Analysis (TGA): TA Instruments Q500. TGA data were collected on a TA Instruments Q500 TGA, equipped with a 16 position auto-sampler. Typically, 5-10 mg of each sample was loaded onto a pre-tared aluminum DSC pan and heated at 10 °C/min from ambient temperature to 350 °C. A nitrogen purge at 60 mL/min was maintained over the sample. The instrument control software was Advantage for Q Series and Thermal Advantage and the data were analyzed using Universal Analysis or TRIOS.

[0065] Thermo-Gravimetric Analysis (TGA): TA Instruments Discovery TGA. TGA data were collected on a TA Instruments Discovery TGA, equipped with a 25 position autosampler. Typically, 5-10 mg of each sample was loaded onto a pre-tared aluminum DSC pan and heated at 10 °C/min from ambient temperature to 350 °C. A nitrogen purge at 25 mL/min was maintained over the sample. The instrument control software was TRIOS and the data were analyzed using TRIOS or Universal Analysis.

[0066] Polarised Light Microscopy (PLM): Leica LM/DM Polarised Light Microscope. Samples were analyzed on a Leica LM/DM polarised light microscope with a digital video camera for image capture. A small amount of each sample was placed on a glass slide, with or without immersion oil, and covered with a glass slip. The sample was viewed with appropriate magnification and partially polarised light, coupled to a A false-color filter. Images were captured using StudioCapture or Image ProPlus software.

[0067] Scanning Electron Microscopy (SEM). Data were collected on a Phenom Pro Scanning Electron Microscope. A small quantity of sample was mounted onto an aluminum stub using conducting double- sided adhesive tape. A thin layer of gold was applied using a sputter coater (20 mA, 120 s). [0068] Gravimetric Vapour Sorption (GVS): SMS DVS Intrinsic. Sorption isotherms were obtained using a SMS DVS Intrinsic moisture sorption analyzer, controlled by DVS Intrinsic Control software. The sample temperature was maintained at 25 °C by the instrument controls. The humidity was controlled by mixing streams of dry and wet nitrogen, with a total flow rate of 200 mL/min. The relative humidity was measured by a calibrated Rotronic probe (dynamic range of 1.0-100 %RH), located near the sample. The weight change, (mass relaxation) of the sample as a function of %RH was constantly monitored by a microbalance (accuracy ±0.005 mg). Typically, 5-30 mg of sample was placed in a tared mesh stainless steel basket under ambient conditions. The sample was loaded and unloaded at 40 %RH and 25 °C (typical room conditions). A moisture sorption isotherm was performed as outlined below (2 scans per complete cycle). The standard isotherm was performed at 25 °C at 10 %RH intervals over a 0 - 90 %RH range. Typically, a double cycle (4 scans) was carried out. Data analysis was carried out within Microsoft Excel using the DVS Analysis Suite. Generally, the sample was recovered after completion of the isotherm and re-analyzed by XRPD.

Table 1. Method parameters for SMS DVS Intrinsic experiments.

Parameter Value

Adsorption - Scan 1 40 - 90

Desorption, Adsorption - Scan 2 90 - 0, 0 - 40

Intervals (%RH) 10

Number of Scans 4

Flow rate (ml/min) 200

Temperature (°C) 25

Stability (°C/min) 0.2

Sorption Time (hours) 6 hour time out

Number of cycles 2

[0069] Chemical Purity Determination by High Performance Liquid Chromatography (HPLC). Purity analysis was performed on an Agilent HPllOO/Infinity II 1260 series system equipped with a diode array detector and using OpenLAB software. The full method details are provided in Table 2.

Table 2. HPLC method for chemical purity determinations.

Parameter Value

Type of method Reverse phase with gradient elution Sample Preparation 0.2 mg/ml in acetonitrile : water 1: 1

Column Supelco Ascentis Express C18 2.7 pm

100 x 4.6 mm

Column Temperature (°C) 25

Injection (pL) 3

Detection: Wavelength, 255, 90

Bandwidth (nm)

Flow Rate (ml/min) 2 0.1 % TFA in water 0.085 % TFA in acetonitrile

Time (min) % Phase A % Phase B

0 95 5

Timetable 6 5 95

6.2 95 5

8 95 5

[0070] Liquid Chromatography Mass Spectrometry (LC-MS). LC-MS data were collected on an Agilent 1260 equipped with PDA and iQ mass spectrometer (single quadrupole with electrospray ionization (ESI)). Samples (~1 mg) were dissolved in 50: 50 acetonitrile: water (1 mL), giving a final concentration of approximately 1 mg/mL. The LC- MS method details are given in Table 3. Data was processed using the total ion count (TIC) chromatograms in positive and negative mode. Mass spectra for each peak were extracted at the peak apex and the ions noted were identified where possible.

Table 3. LC-MS method parameters.

Parameter Value

Type of method Reverse Phase with Gradient Elution

Sample Preparation 1.0 mg/ml in 50:50 Acetonitrile: Water

Column Supelco Ascentis Express C18, 2.7pm 100 x 4.6 mm

Column Temperature (°C) 25

Flow Rate (ml/min) 0.6

ES+ and ES- scan mode 100-1450 m/z, Fragmentor MS 110 V, Scan Time 475 ms,

Gas Temperature 325 °C, Gas flow 11 L/min, Nebulizer 40 psi, Capillary voltage: 3500 V, Splitter 1 :3 MS:waste.

Phase A 0.1 % Formic Acid in Water Phase B 0.1 % Formic Acid in Acetonitrile

Time (min) % Phase A % Phase B

0.00 95.0 5.0

Timetable 11.00 5.0 95.0

13.00 5.0 95.0

13.20 95.0 5.0

15.00 95.0 5.0

[0071] Water Determination by Karl Fischer Titration (KF). The water content of each sample was measured on a Metrohm 874 Oven Sample Processor at 150 °C with 851 Titrano Coulometer using Hydranal Coulomat AG oven reagent and nitrogen purge. Weighed solid samples were introduced into a sealed sample vial. Approximately 10 mg of sample was used per titration and duplicate determinations were made. An average of these results is presented unless otherwise stated. Data collection and analysis were performed using Tiamo software.

[0072] Thermodynamic Aqueous Solubility. Aqueous solubility was determined by suspending sufficient compound in relevant media to give a maximum final concentration of >10 mg/mL of the parent free-form of the compound. The suspension was equilibrated at 37 °C, on a Heidolph plate shaker set to 750 rpm for 24 hours. The pH of the saturated solution was then measured, and the suspension was centrifuged (13,400 rpm, 2 mins) before being filtered through a glass fiber C filter (particle retention 1.2 pm) and diluted appropriately. Quantitation was by HPLC with reference to a standard solution of approximately 0.15 mg/mL in DMSO. Different volumes of the standard diluted and undiluted sample solutions were injected. The solubility was calculated using the peak areas determined by integration of the peak found at the same retention time as the principal peak in the standard injection. Analysis was performed on an Agilent HP1100/ Infinity II 1260 series system equipped with a diode array detector and using OpenLAB software.

Table 4. HPLC method for solubility measurements.

Parameter Value

Type of method Reverse phase with gradient elution

Column Phenomenex Luna, C18 (2) 5 pm 50 x 4.6 mm

Standard Injections (pL) 1, 2, 3, 4, 5, 7

Sample Injections (pL) 2, 3, 5, 10, 15, 20

Detection: Wavelength, 260,90

Bandwidth (nm) Flow Rate (ml/min) 2

Phase A 0.1 % TFA in water

Phase B 0.085 % TFA in acetonitrile

Time (min) % Phase A % Phase B

0.0 95 5

Timetable 1.0 80 20

2.3 5 95

3.3 5 95

3.5 95 5

4.4 95 5

[0073] Ion Chromatography (IC). Data were collected on a Metrohm 930 Compact IC Flex with 858 Professional autosampler and 800 Dosino dosage unit monitor, using IC MagicNet software. Accurately weighed samples were prepared as stock solutions in a suitable solvent. Quantification was achieved by comparison with standard solutions of known concentration of the ion being analyzed. Analyzes were performed in duplicate, and an average of the values is given unless otherwise stated.

Table 5. IC method for cation chromatography.

Parameter Value

Type of method Cation exchange

Column Metrosep C 4-250 (4.0 x 250 mm)

Column Temperature (°C) Ambient

Detection Conductivity detector

1.7 mM nitric acid '^uen 0.7 mM dipicolinic acid in a 5 % acetone aqueous solution.

Table 6. IC method for anion chromatography.

Parameter Value

Type of method Anion exchange

Column Metrosep A Supp 5-150 (4.0 x 150 mm)

Column Temperature (°C) Ambient

Injection (pL) Various

Detection Conductivity detector

Flow Rate (ml/min) 0.7

3.2 mM sodium carbonate

Eluent , „ .. .. . . . . .

1.0 mM sodium hydrogen carbonate in a 5 _r % acetone aqueous solution.

[0074] Fourier Transform Infrared (FTIR). Data were collected on a Perkin-Elmer Spectrum 3 fitted with a universal ATR sampling accessory using a 1 Bounce Diamond/ZnSe crystal. Spectra were collected 4000-650 cm ¹ over 16 scans. The data were collected using Spectrum IR software and processed using ACD Spectrus Processor.

[0075] Static Stability Experiments. Solid material was placed into open vials at elevated storage conditions, unless otherwise stated. These conditions were achieved using saturated salt solutions at specific temperatures within sealed containers. Storage containers were pre-equilibrated prior to input of samples.

Table 7. Salt solutions used to produce static storage conditions.

Condition Saturated Salt Solution Temperature (°C)

25 °C / 97 %RH Potassium sulfate _ 25 _

40 °C / 75 %RH Sodium chloride 40

[0076] Freeze Dryer: Telstar Lyoquest. Samples were freeze dried using a Telstar Lyoquest laboratory freeze dryer with a condenser temperature of -85 °C using a pressure below 10 mbar. Solutions to be freeze dried were filtered through a 0.45 pm Nylon filter before flash-freezing using dry ice/ acetone. The frozen samples were then connected to the freeze dryer and freeze-dried for about 20 hours. Direct contact of the sample vessel with the surrounding ambient environment provided heat energy required for freeze drying.

Example 1. Preparation of Form 1 of Compound 1

[0077] A solution of Compound 1 in dichloromethane was solvent exchanged into isopropanol. Isopropanol and water were added to provide 15 volumes of isopropanol/water (80:20) mixture. This mixture was then heated to 80 °C until full dissolution. The mixture was then cooled with stirring to 65 °C and 2.5 wt. % seed was charged. The mixture was then cooled to ~10 °C over 4 h and stirred for another 16 h. The solids were filtered, the filter cake washed with isopropanol (2 vol) and then dried in a vacuum oven at 50 °C. This gave Compound 1 as an off-white solid. The final drug substance is not hygroscopic and packaged at a temperature <25°C. Melting point: 212 °C. ^JH NMR (600 MHz, DMSO-de) 6 8.76 (s, 1H), 7.76 (s, 1H), 7.67 (s, 1H), 7.44 (s, 1H), 5.20 (dtd, J = 56.4, 6.5, 3.5 Hz, 1H), 4.10 (dd, J = 21.9, 8.0 Hz, 4H), 3.77 (s, 3H), 3.48 (ddd, J = 16.5, 10.4, 6.3 Hz, 1H), 3.04 (d, J = 2.1 Hz, 2H), 2.90 (dt, J = 10.1, 5.0 Hz, 2H), 2.69 (d, J = 10.7 Hz, 1H), 2.33 (dtd, J = 12.9, 9.8, 8.5, 3.9 Hz, 2H), 2.29-2.18 (m, 2H), 2.03 (s, 3H), 1.99 (t, J = 10.0 Hz, 1H), 1.91 (ddd, J = 27.1, 13.7, 5.3 Hz, 1H), 1.68-1.54 (m, 1H).

Example 2. Solubility of Form 1 of Compound 1

[0078] Form 1 of Compound 1 was determined to be insoluble or poorly insoluble in the following solvents or solvent systems: Methanol; Ethanol; 1-Propanol; 2-Propanol (IPA); Acetonitrile; Acetone; Methylethyl ketone (MEK) (2-Butanone); Methylisobutyl ketone (MIBK); 2-Methyl THF; Tetra hydrofuran (THF); Toluene; Ethyl acetate; Isopropyl acetate; Heptane; tert-Butyl methyl ether (TBME); 2-Methyl-l-Propanol (Isobutanol); 1-Butanol; Methanol/Water (5% v/v); Ethanol/Water (5% v/v); IPA/Water (5% v/v); Acetone/Water (10% v/v); ACN/Water (1 :2 v/v); THF/Water (30% v/v); and Water. Form 1 of Compound 1 was determined to be somewhat soluble in 1,4-Dioxane or Nitromethane, and more soluble in Dichloromethane (DCM) and Dimethyl sulfoxide (DMSO).

[0079] Surprisingly, on suspension in Nitromethane at 50 °C, cooling to 5 °C, followed by filtration, the purity, assessed by HPLC, of Form 1 of Compound 1 was found to be improved from 98.8 % having 4 impurities present at >0.1 %, to 99.3 % having 2 impurities >0.1 %. The solid form of Compound 1 was confirmed by XRPD analysis to maintain Form 1 before and after this purification method sequence.

[0080] Form 1 of Compound 1 may be rendered amorphous by dissolution in warm 1: 1 acetonitrile:water followed by freeze-drying.

Example 3. Characterization of Form 1 of Compound 1 from Example 1.

Table 8. Characterization data for Form 1 of Compound 1.

Characterization Form 1 of Compound 1

XRPD Crystalline, Pattern 1 (Form 1)

PLM Small aggregates of birefringent particles

SEM Small aggregates of polyhedral plate-like particles

¹H NMR Consistent with structure, small residual IPA peak at 1.04 ppm Consistent with structure; absorptions for weak

FT-IR nitrile stretch ca. 2237 cm , aromatic C=C stretches and N-H amine bending ca. 1624 cm ¹ -1519 cm⁴, C-H and N-H stretches, no carbonyl absorptions.

TGA Small weight loss of 0.3 % (ca. 0.07 eq. water) between 212.8 °C and 230.2 °C, before decomposition onset at ca. 250 °C

DSC Sharp endotherm (melt) 132 J/g onset temperature at 216.2 °C mDSC Amorphous form obtained after heating to 220 °C and quenching to -80 °C; Tg 91.5 °C

Slightly hygroscopic, slight hysteresis (particularly

GVS on first cycle). 0.21 % mass increase from 0 to 90

%RH. No evidence of hydrate formation. Post-GVS XRPD: Crystalline, Pattern 1.

HPLC and LC-MS Purity 98.8 %, 4 impurities >0.1 %

LC-MS: molecular ion consistent with structure

Anion IC: No Anions observed > 0.05 eq.

IC Cation IC: 0.7 wt % Na (not accurately quantified

- scanning IC run only)

Static Storage, 8 days 25 °C / 97 %RH: Crystalline, Pattern 1. Purity

25 °C / 97 %RH 98.9 %, 4

40 °C / 75 %RH impurities >0.1 %

40 °C / 75 %RH: Crystalline, Pattern 1. Purity

98.9 %, 4 impurities >0.1 %

Medium: citrate buffer pH 6, Average Solubility Thermodynamic 0.99 mg/mL According to USP classifications,

Solubility classified as very slightly soluble.

XRPD: Crystalline, Pattern 1 (no form change) after 24 hour Equilibration.

Example 4. Characterization of amorphous Compound 1

Table 9. Characterization data for amorphous Compound 1.

Characterization FCF-2270-14-01

XRPD Amorphous

1H NMR Consistent with structure, no significant trace of solvent

Amorphous form displays Tg 92.3 °C (91.5 °C in melt-

MDSC quench of crystalline Form 1 (Example 1))

Crystallization exotherm 84 J/g onset 139.9 °C

HPLC Purity 99.0 %, 4 impurities >0.1 %

Static Storage, 6 days 25 °C / 97 %RH: Poorly crystalline, Pattern 5. Very little

25 °C / 97 %RH material retained. Purity 99.0 %, 4 impurities >0.1 %

40 °C / 75 %RH 40 °C / 75 %RH: Poorly crystalline, Pattern 1. Very little material retained. Purity 98.8 %, 4 impurities >0.1 %

[0081]The amorphous material is not stable to static storage conditions under either condition (25 °C / 97 %RH or 40 °C / 75 %RH), converting to poorly crystalline materials, either Pattern 1 by XRPD (e.g., crystallizing or re-arranging to Form 1 of Compound 1) or Pattern 5 (Form 5) of Compound 1.

Example 5. Preparation of polymorphs of Compound 1

[0082] Polymorphism screens, starting with amorphous Compound 1, produced materials with four new XRPD patterns (Pattern 2, Pattern 3, Pattern 4, and Pattern 5). An overview of the results from the polymorphism screens can be found in Table 10.

[0083] Isothermal Slurry at 5 °C (Cold Slurry). To each HPLC vial, a stirrer bar was added and an aliquot of one of the solvents or solvent systems was added as follows. About 31-32 mg of amorphous Compound 1 was combined with 5 vol (160 pL) or 10 vol (320 pL) of solvent, then stirred at 5 °C, 500 rpm, for about 24 hours. Samples in 1,4-dioxane and DMSO were stirred at 25 °C due to the high melting points of the solvents. After the initial treatment period all samples were suspensions. An aliquot of each suspension was pipetted onto an XRPD plate, the solvent allowed to evaporate, and the solid material analyzed by XRPD. For XPRD analysis of samples containing THF or 2-Me THF, an aliquot was pipetted onto a glass slide and the solvent allowed to evaporate before the solid was transferred to the XRPD plate.

[0084] Maturation. To each HPLC vial, a stirrer bar was added and an aliquot of one of the solvents or solvent systems was added as follows. About 31-32 mg of amorphous Compound 1 was combined with 5 vol (160 pL) or 10 vol (320 pL) of solvent, then shaken in a maturation chamber cycling between room temperature and 50 °C every four hours, for about 24 hours. After the initial treatment period all samples were suspensions, except the sample with DMSO solvent, which was a clear solution after 1 day. This sample was left in maturation for a further 4 days, by which time the sample had formed a suspension. An aliquot of each suspension was pipetted onto an XRPD plate, the solvent allowed to evaporate, and the solid material analyzed by XRPD. For XPRD analysis of samples containing THF or 2-Me THF, an aliquot was pipetted onto a glass slide and the solvent allowed to evaporate before the solid was transferred to the XRPD plate.

[0085] Isothermal Slurry at 50 °C (Hot Slurry). To each HPLC vial, a stirrer bar was added and an aliquot of one of the solvents or solvent systems was added as follows. About 31-32 mg of amorphous Compound 1 was combined with 5 vol (160 pL) or 10 vol (320 pL) of solvent, then stirred at 50 °C, 500 rpm, for about 21 hours. After the initial treatment period all samples were suspensions, except the sample with DMSO solvent, which was a clear solution after 1 day. This sample was left stirring at 50 °C for a further 5 days, by which time the sample had formed a suspension. An aliquot of each suspension was pipetted onto an XRPD plate, the solvent allowed to evaporate, and the solid material analyzed by XRPD. For XPRD analysis of samples containing THF or 2-Me THF, an aliquot was pipetted onto a glass slide and the solvent allowed to evaporate before the solid was transferred to the XRPD plate.

Table 10. Polymorphism screen of amorphous Compound 1.

Solvent Cold Slurry Maturation Hot Slurry Methanol Pattern 1 Pattern 1 Pattern 1

Ethanol Pattern 1 Pattern 1 Pattern 1

1-Propanol Pattern 1 Pattern 1 Pattern 1

Isopropanol Pattern 1 Pattern 1 Pattern 1

Acetonitrile Pattern 1 Pattern 1 Pattern 1

Acetone Pattern 1 Pattern 1 Pattern 1

Methylethyl ketone Pattern 1 Pattern 1 Pattern 1

Methyl isobutyl ketone Pattern 1 Pattern 1 Pattern 1

1,4-Dioxane Pattern 1 Pattern 1 Pattern 1

2-Methyl tetra hydrofuran Pattern 1 Pattern 1 Pattern 1

Tetrahydrofuran Pattern 1 Pattern 1 Pattern 1

Toluene Pattern 2 Pattern 1 Pattern 1

Dichloromethane Pattern 1 Pattern 1 Pattern 1

Dimethyl sulfoxide Pattern 1 Pattern 1 Pattern 1

(after 6 days) (after 5 days)

Nitromethane Pattern 1 Pattern 1 Pattern 1

Ethyl acetate Pattern 1 Pattern 1 Pattern 1

Isopropyl acetate Pattern 1 Pattern 1 Pattern 1

Heptane Pattern 1 Pattern 4 Pattern 4 tert-Butyl methyl ether Pattern 1 Pattern 1 Pattern 1

2-Methyl-l-Propanol Pattern 1 Pattern 1 Pattern 1

1-Butanol Pattern 1 Pattern 1 Pattern 1

Methanol/Water (5% v/v) Pattern 3 Pattern 5 Pattern 5

(poorly crystalline)

Ethanol/Water (5% v/v) Pattern 3 Pattern 2 Pattern 5

(poorly crystalline)

IPA/Water (5% v/v) Pattern 3 Pattern 2 Pattern 2

(poorly crystalline)

Acetone/Water (10% v/v) Pattern 3 Pattern 2 Pattern 1

(poorly crystalline)

ACN/Water (1 :2 v/v) Pattern s Pattern 1 Pattern 1

THF/Water (30% v/v) Pattern 3 Pattern 1 Pattern 1

(poorly crystalline)

Water Pattern 3 Pattern 2 Pattern 1 + (poorly crystalline) Pattern 5

[0086] Materials denoted as Pattern 2 were produced from cold slurry, hot slurry, and maturation techniques. Surprisingly, materials that were found to be Pattern 3 by XRPD were produced from the cold slurry technique only. Samples containing material with Pattern 4 were produced from heptane only, in both the hot slurry and in the maturation screen. Finally, materials that were denoted as Pattern 5 based on the XRPDs were produced from the maturation screen in methanol/water (5% v/v), and from the hot slurry in methanol/water (5% v/v) or ethanol/water (5% v/v). There was also a sample which was a mixture of Pattern 5 and Pattern 1, obtained from water. Pattern 5 was also observed after an amorphous sample of Compound 1 was held in static storage for six days at 25 °C I 97 %RH.

[0087] Purity of Pattern 1 samples under various conditions may be improved, relative to that of the sample from Example 1, as shown in Table 11.

Table 11. Purity comparison of Pattern 1 samples from different preparative conditions.

Conditions Purity

Example 1 Purity 98.8 %, 4 impurities >0.1 %

5 °C, Ethanol Purity 98.9 %, 4 impurities >0.1%

5 °C, ACN Purity 99.2 %, 4 impurities >0.1%

5 °C, 2-methyl-l-propanol Purity 98.9 %, 4 impurities >0.1%

Maturation, 1-propanol Purity 99.1 %, 4 impurities >0.1%

Maturation, 1-butanol Purity 99.0 %, 4 impurities >0.1%

Maturation, THF: water Purity 99.1 %, 4 impurities >0.1%

50 °C, Methanol Purity 99.0 %, 5 impurities >0.1%

50 °C, DCM Purity 99.0 %, 4 impurities >0.1%

50 °C, ACN: water Purity 99.2 %, 4 impurities >0.1%

[0088] This purity analysis suggests that the process of making amorphous material, followed by treatment in solvent to produce material with Pattern 1 by XRPD, improves the purity of the material. In particular, the presence of ACN in the solvent appears to produce the best improvement in purity. [0089] A sample with each new pattern by XRPD was selected for isolation and characterization. Isolation was performed by air-drying the sample on filter paper, or by filtration of the sample through a small frit under positive pressure, followed by drying under vacuum for about 1 hour.

[0090] Upon isolation, the sample originally identified as having Pattern 2 was found to have converted to material with Pattern 1. A second sample was isolated, but again it was found to convert to Pattern 1. Pattern 2 was therefore determined to be unstable and was not further characterized. The XRPD patterns after isolation can be found in Fig. 3.

[0091] Forms 1, 3, 4, and 5 of Compound 1, which were stable to isolation, were characterized using ^XH NMR spectroscopy, thermal analysis, purity analysis and static storage at 40 °C I 75 %RH for 6 days. A comparison of the results and subsequent material assignment can be found in Table 12. The data can be found in Data Section 6 - Characterization of New Patterns.

Table 12.

[0092] Form 3-dry was prepared via dissolution of amorphous Compound 1 in ACN: water 1 :2 v/v, followed by stirring at 5 °C to obtain the crystalline material. Form 3-dry (Pattern 3) is crystalline and has a high purity of 99.5 %, with only one major impurity present. The

NMR spectrum is consistent with the initial material, with no traces of ACN solvent. DSC analysis shows a broad endotherm at 68.9 °C, likely solvent loss, followed by a large endotherm at 147.9 °C and a sharp endotherm at 215.9 °C (consistent with the melt of Form 1). The TGA shows a weight loss of 11.4 % before 105 °C, consistent with the loss of ca. 3 eq. water. The solid form remains unchanged under storage at 40 °C I 75 %RH for 6 days and retains its chemical integrity with a high purity of 99.4 %. Pattern 3 appears to be a hydrated form, which potentially converts to the anhydrous Form 1 material above 147.9 °C.

[0093] Form 4-dry was prepared via a suspension of amorphous Compound 1 in heptane, followed by maturation cycling between 5 °C and 50 °C to obtain the crystalline material. Form 4-dry (Pattern 4) is a crystalline material and has good purity (98.8 %). The NMR spectrum is consistent with the initial material, with small traces of heptane solvent. DSC analysis shows a small endotherm at 181.5 °C and an exotherm at 184.8 °C (possibly indicating a recrystallization/form conversion), and a sharp endotherm at 216.5 °C (consistent with the melt of Form 1). The TGA shows no weight loss before the onset of degradation. The sample is stable under storage and remains as Pattern 4 at 40 °C I 75 %RH for 6 days with a high purity of 98.9 %. Pattern 4 appears to be an anhydrous form, which potentially converts to anhydrous Form 1 above 184.8 °C.

[0094] Form 5-dry was prepared via a suspension of amorphous Compound 1 in methanol:water 5 % v/v, followed by maturation cycling between 5 °C and 50 °C to obtain the crystalline material. Form 5-dry (Pattern 5) was shown to be crystalline by XRPD and had a high purity of 99.1 %. The ^XH NMR spectrum is consistent with the initial material, with no traces of methanol solvent. DSC analysis shows a broad double endotherm at 61.4 °C, followed by a large exotherm, at 142.3 °C and a sharp endotherm at 215.2 °C (consistent with the melt of the Form 1 material of Example 1). The TGA shows a weight loss of 9.6 % before 103.1 °C, consistent with the loss of ca. 2.5 eq. water. The sample partially converts to Form 1 under storage at 40 °C / 75 %RH for 6 days and maintains a high purity of 99.0 % under these accelerated storage conditions. Pattern 5 appears to be a hydrated form, which loses water before an exothermic event likely results in Form 1, followed by melting.

[0095] In summary, from the polymorphism screens, materials with new patterns by XRPD were obtained.

[0096] Form 3 of Compound 1 (Pattern 3), obtained, for example, from a cold slurry in ACN:water 1 :2 v/v, is likely to be a trihydrate, with the highest purity of the new patterns, and good stability. [0097] Form 4 of Compound 1 (Pattern 4), obtained, for example, from the maturation screen in heptane, is likely to be anhydrous, and has good stability. However, some traces of solvent remained, and the material had no obvious increase in purity compared to Form 1 of Compound 1 from Example 1.

[0098] Form 5 of Compound 1 (Pattern 5), obtained, for example, from the maturation screen in methanol: water 5 % v/v, is likely to be a hydrate, with an increased purity compared to Form 1 of Compound 1 from Example 1. Pattern 5, however, is not stable to storage at 40 °C / 75 %RH, partially converting to the same form as Form 1 of Compound 1 from Example 1.

[0099] Purity analysis was performed on a selection of samples from the polymorphism screens which displayed Pattern 1 by XRPD. The lyophilization and screening conditions appear to slightly improve the purity of these materials as compared to Form 1 of Compound 1 from Example 1.

[0100] In particular, the solvent ACN appears to produce samples that have an increase in purity; this is observed both in the slight purity increase of the Pattern 1 samples where the solvent contained ACN, and in the high purity of Form 3-dry (Pattern 3), which was obtained from ACN: water 1:2 v/v.

[0101] Amorphous Compound 1 was successfully prepared from lyophilisation from ACN: water 1 : 1 solvent, and was found to have good purity (99.0 %) and no significant traces of solvent. This process was therefore selected as useful to produce bulk amorphous Compound 1, e.g., at commercially relevant scales. However, the melt-quench cool mDSC analysis of crystalline Form 1 of Compound 1 indicates that amorphous material could also be prepared by heating the sample to about 220 °C before cooling it rapidly in an ice bath.

[0102]The polymorphism screen was performed using the amorphous Compound 1 and the original 28 process-acceptable solvent systems, in three conditions: slurrying at 5 °C, maturation cycling between 5 °C and 50 °C, and slurrying at 50 °C. While many of the materials produced from the screen had the same pattern as Form 1 of Compound 1, materials with four new patterns by XRPD were obtained. Following isolation, only three of the four materials retained their original new XRPD patterns, while one converted to have Pattern 1 upon isolation.

[0103]Table 13 includes a list of XRPD diffractogram signals for Form 1 of Compound 1; a corresponding XRPD diffractogram is shown in Fig. 4. Table 14 includes a list of XRPD diffractogram signals for Form 2 of Compound 1; a corresponding XRPD diffractogram is shown in Fig. 5. Table 15 includes a list of XRPD diffractogram signals for Form 3 of Compound 1; a corresponding XRPD diffractogram is shown in Fig. 6. Table 16 includes a list of XRPD diffractogram signals for Form 4 of Compound 1; a corresponding XRPD diffractogram is shown in Fig. 7. Table 17 includes a list of XRPD diffractogram signals for Form 5 of Compound 1; a corresponding XRPD diffractogram is shown in Fig. 8.

Table 13. Form 1 of Compound 1.

Table 14. Form 2 of Compound 1.

Table 15. Form 3 of Compound 1.

Table 16. Form 4 of Compound 1.

Table 17. Form 5 of Compound 1.

Example 6: ROCK and JAK Assays

ROCK Kinase Inhibition Assays

[0291] All compounds are initially prepared as 10 mM stocks in anhydrous dimethylsulfoxide (DMSO). A 20 pL aliquot of the 10 mM solutions is transferred to individual wells in column 1 of a 96-well polypropylene microtiter plate (Corning #3363) and diluted with DMSO to give a final compound concentration of 4 mM. Test compounds are then serially diluted 1:5 in DMSO for an 11-point concentration response and further diluted in the assay buffer bringing all compound concentrations to a final range of 100 pM to 10 pM in 2.5% DMSO. The assay is performed in white 96-well, flat-bottom, half-area, non-binding assay plate (Corning #3542) in assay buffer consisting of 20 mM HEPES (pH 7.5), 10 mM MgCl2*5H2O, 100 pM sodium orthovanadate, 0.05% CHAPS and 0.1% bovine serum albumin. A 10 pL aliquot of compound from each well of the intermediate dilution plate and 20 pL of a 2X substrate/enzyme solution containing acceptor substrate (800 nM RSK2 peptide 10-mer having a MW of 1242.5, e.g., product number SRP0687 from Sigma-Aldrich), ROCK2 enzyme (10 nM), or ROCK1 enzyme, and 1,4-Dithiothreitol (DTT, 2 pM) are added to all wells. The reaction is initiated by the addition of 10 pL of 4x stock solution ATP (2 pM). Reactions are thoroughly mixed manually, covered, and allowed to incubate at room temperature for 75 min. Protein kinase activity is quantitated using Promega's KINASE- GLOTM luminescent Kinase Assay Kit according to the manufacturer's directions. ATP concentrations remaining in Test wells following the termination of the enzymatic reaction are compared against control wells containing equivalent amounts of DMSO containing no inhibitor (CTRL). ATP concentrations in both Test wells and CTRL wells are normalized against background (BKG) ATP concentrations in wells containing concentrations of inhibitor that completely inhibited the protein kinase under investigation (i.e. a concentration that prevented any consumption of ATP over the course of the incubation). Percent of Control (POC) values are determined for each concentration of compound tested according to the equation:

POC = ((Test well value - BKG)/ (CTRL - BKG))*100

ICso values are calculated using the following 4-parameter logistic curve-fitting algorithm: f(x) = (A+((B-A)/(l+((x/C) D))))

IC50 values are converted to Ki values using the following Cheng-Prusoff Equation: Ki = ICso / (1 + ([ATP]/Km ATP])).

A form of Compound 1 described herein, e.g., a polymorph as in Example 5, inhibits the assayed kinase at commercially or therapeutically relevant levels.

JAK Kinase Assays

[0292] Compounds are prepared in the exact same manner as described in the ROCK Kinase Assay with the exception to the substrate and enzyme. The JAK 2X substrate/enzyme solution includes an acceptor substrate (800 nM Abl peptide 12-mer having a MW of 1336.5, e.g., product number BML-P216-0001 from Enzo Life Sciences), JAK1, TYK2, JAK2 or JAK3 enzyme (10 nM) and DTT (2uM). All other steps and solutions remain identical to the ROCK Kinase Assay above. A form of Compound 1 described herein, e.g., a polymorph as in Example 5, inhibits the assayed kinase at commercially or therapeutically relevant levels.

Example 7. PTM-HTM Assay

[0293] Porcine Trabecular Meshwork cells (PTM) are isolated from freshly obtained enucleated porcine eyes. Immortalized Human Trabecular Meshwork cells (TM-1) are obtained through a kind gift from Donna Peters in the Department of Ophthalmology and Visual Sciences at the University of Wisconsin. Cells are plated onto fibronectin coated glass-bottom 96-well plates and allowed to attach overnight. Media is removed and replaced with test compound in media with 1% fetal bovine serum and incubated for various times. After incubation, cells are formaldehyde fixed, Triton solubilized, and stained. PTM cells are stained with Alexa Fluor® 488 phalloidin (F-actin) and Hoechst 33342 (nuclei). TM-1 cells are stained with anti-paxillin followed by Alexa Fluor® 488 goat-anti-mouse IgG (focal adhesions) and Hoechst 33342 (nuclei). All staining reagents are obtained through Invitrogen. Images are collected on an INCell 2200 imager with a 20X objective. The actin fiber length and total area of focal adhesions are analyzed using custom algorithms developed in the INCell Developer Toolbox, vl.9.3. Data collected are converted to percent of control (untreated cells). Curves are fit to data in GraphPad Prizm using sigmoidal doseresponse and constraining top and bottom to 100% and 0%, respectively. A form of Compound 1 described herein, e.g., a polymorph as in Example 5, improves the actin fiber length or total area of focal adhesion at commercially or therapeutically relevant levels.

Example 8. Treatments

[0104]Topical instillation ocular pharmaceutical compositions for treating inflammation or dry eye disease comprising a form of Compound 1 described herein, e.g., a polymorph as in Example 5, are prepared. When such a composition is topically administered to one or both eyes of a subject once daily, the composition decreases ocular inflammation in the eye(s) of the subject suffering from meibomian gland dysfunction (MGD) or DED.

Claims

CLAIMS What is claimed is:

1. A solid form of a compound, wherein the compound is

2. The solid form of claim 1, which is a crystalline solid form of

3. The solid form of claim 1, which is a solid form precipitated or crystallized from at least one solvent.

4. The solid form of claim 1, which is a lyophilized amorphous solid form.

5. The solid form of claim 1, which is purified.

6. The solid form of claim 1, comprising one or more XRPD signals in terms of 20, ±0.2, selected from 2.8, 4.5, 5.8, 6.4, 7.3, 7.7, 8, 8.9, 9.3, 9.7, 9.9, 10.6, 10.9, 11.5, 11.9, 12.4, 12.8, 13.5, 14.3, 14.6, 14.8, 15.2, 15.8, 17.4, 17.6, 18.5, 18.9, 19.8, 21.8, 22.4, 22.9, 23.3, 24.1, 25, 25.7, 26.1, 26.5, 27.1, 27.9, 28.5, 29.1, or 29.7.

7. The solid form of claim 1, comprising one or more XRPD signals in terms of 20, ±0.2, selected from 2.8, 4.5, 5.8, 7.3, 7.7, 9.3, 9.7, 9.9, 11.5, 14.6, 17.4, 22.9, 28.5, or 29.7.

8. The solid form of claim 1, which is a polymorphic form selected from Form 1, Form 2, Form 3, Form 4, or Form 5.

9. The solid form of claim 1, which is Form 1 and comprises one or more XRPD signals in terms of 20, ±0.2, selected from 7.3, 11.5, 28.5, or 29.7.

10. The solid form of claim 1, which is Form 2 and comprises one or more XRPD signals in terms of 20, ±0.2, selected from 2.8, 5.8, 9.9, or 22.9.

11. The solid form of claim 1, which is Form 3 and comprises one or more XRPD signals in terms of 20, ±0.2, selected from 7.7 or 17.4.

12. The solid form of claim 1, which is Form 4 and comprises one or more XRPD signals in terms of 20, ±0.2, selected from 4.5, 9.3, or 9.7.

13. The solid form of claim 1, which is Form 5 and includes an XRPD signal of 14.6 ±0.2 20.

14. The solid form of claim 1, having an XRPD pattern substantially as shown in Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, or Fig. 8.

15. The solid form of claim 1, prepared by a process including one or more of lyophilizing or crystallizing from at least one solvent.

16. A composition, comprising the solid form of one of claims 1-15 and at least one carrier.

17. The composition of claim 16, comprising the solid form of claim 1 having a purity of at least 99% EE.

18. The composition of claim 16 or claim 17, which is a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier.

19. The composition of one of claims 16-18, in the form of a solid (e.g., an implant or a flowable powder, injectable, ingestible, or inhalable), a liquid (e.g., a suspension, emulsion, injectable, ingestible, or inhalable), a gel, a cream, or an ointment.

20. A method, comprising administering the solid form of one of claims 1-15 or the composition of one of claims 16-19 to a subject.

21. The method of claim 20, which is a method of treatment in a subject in need thereof.

22. A method of inhibiting a kinase, comprising contacting the kinase with the solid form of one of claims 1-15 or the composition of one of claims 16-19.

23. The method of claim 22, which is an in vivo method.

24. The method of claim 22, which is an in vitro method.

25. A method of treating a kinase-associated disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the solid form of one of claims 1-15 or the composition of one of claims 16-19.

26. A method of treating dry eye disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the solid form of one of claims 1-15 or the composition of one of claims 16-19.