EA044390B1 - POLYMORPHS AND SOLID FORMS OF PYRIMIDINYLAMINO-PYRAZOLE COMPOUNDS AND METHODS FOR THEIR PREPARATION - Google Patents

Description

Cross reference to related application

This application claims priority to US Provisional Application No. 62/589,276, filed November 21, 2017, which is incorporated herein by reference.

Field of technology

This invention relates to polymorphic forms of 2-methyl-2-(3-methyl-4-((4-(methylamino)-5(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)propanenitrile for applications in the treatment of peripheral and neurodegenerative diseases, including Parkinson's disease. This description also relates to methods for producing polymorphic forms.

State of the art

Combined genetic and biochemical evidence suggests a distinct function for kinases in the pathogenesis of neurodegenerative disorders (Christensen, K.V. (2017) Progress in medicinal chemistry 56:37-80; Fuji, R.N. et al (2015) Science Translational Medicine 7(273):273ral5; Taymans , J. M. et al (2016) Current Neuropharmacology 14(3):214-225). Kinase inhibitors are being investigated for the treatment of Alzheimer's disease, Parkinson's disease, ALS and other diseases (Estrada, A.A. et al. (2015) J. Med. Chem. 58(17): 6733-6746; Estrada, A.A. et al. (2013) J Med. Chem. 57:921-936; Chen, H. et al. (2012) J. Med. Chem. 55:5536-5545; Estrada, A. A. et al. (2015) J. Med. Chem. 58: 6733 -6746; Chan, B.K. et al. (2013) ACS Med. Chem. Lett. 4:85-90; US 8354420; US 8569281; US8791130; US 8796296; US 8802674; US 8809331; US 8815882; US 9145402; US 9 212173 ; US 9212186; and WO 2012/062783.

Different crystalline forms of a drug substance with different properties in the solid state may exhibit differences in bioavailability, shelf life, physicochemical properties including melting points, crystal morphology, intrinsic dissolution rates, solubility and stability, and behavior during processing. Powder X-ray diffraction (XRD) is a powerful tool in identifying different crystalline phases by their unique diffraction patterns. Other techniques such as solid state nuclear magnetic resonance (NMR) spectrometry, Raman spectrometry (RAMAN), DSC (differential scanning calorimetry) are also suitable.

The pharmaceutical industry is often faced with the phenomenon of multiple polymorphs of the same crystalline chemical compound. Polymorphism is often characterized as the ability of a drug substance, i.e. active pharmaceutical ingredient (API) exist in the form of two or more crystalline phases having different mutual arrangements and/or conformations of molecules in the crystal lattice, giving crystals with different physicochemical properties. Having the ability to reproducibly manufacture a chosen polymorphic form is a key factor in determining the success of a drug product.

Regulatory authorities around the world require reasonable efforts to identify polymorphs of a drug substance and to test the interconversion of polymorphs. Because of the often unpredictable behavior of polymorphs and their corresponding differences in physicochemical properties, reproducibility in production between batches of the same product must be demonstrated. A proper understanding of the polymorphic landscape and the nature of drug polymorphs will facilitate manufacturing reproducibility.

Atomic-level crystal structure determinations and intermolecular interactions provide important information for absolute configuration determination (enantiomers), phase identification, quality control, and process control and optimization. X-ray diffraction analysis has become widely accepted as a reliable tool for analyzing the crystal structure of pharmaceutical solids and identifying the crystal form.

The presence of a single crystal of the drug substance is preferable due to the speed and accuracy of structure determination. However, it is not always possible to obtain a crystal of the appropriate size for data acquisition. A suitable method is synchrotron X-ray powder diffraction. In such situations, the crystal structure can be solved based on X-ray powder diffraction data obtained by measurements at ambient conditions and/or at varying temperature or humidity.

There is a need to develop new polymorphic forms of medicinal substances and methods for their preparation.

Description of the invention

This description relates to crystalline polymorphic or amorphous forms of the kinase inhibitor pyrimidinylamino-pyrazole, referred to herein as a compound of formula I, and having the structure:

- 1 044390 h ₃ c-./V

N-N

H,C CH, j or pharmaceutically acceptable salts or co-crystals thereof. One aspect of the present invention is a pharmaceutical composition of a polymorphic form of a compound of formula I.

Another aspect of the present invention is the crystalline compound, 2-methyl-2-(3methyl-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-1H-pyrazol-1-yl)propanenitrile, selected of: Polymorph Form B, which exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at approximately 7.0, 7.3, 16.1, 16.3, 24.1, 25.1 and 26 ,6;

polymorph Form C, which exhibits an x-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at approximately 6.4, 15.1, 21.2, 25.7 and 27.8; and polymorph Form D, which exhibits an x-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at approximately 9.2, 14.0, 14.8, 19.7, and 20.0.

In one aspect, a form A polymorph is provided that exhibits an x-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at approximately 7.7, 9.9, 12.7, 13.6, 14.1, 15.4 , 15.9, 19.2, 20.5, 21.6, 22.4, 2z.2 and 24.7.

In one aspect, a Form B polymorph is provided that exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at approximately 7.0, 7.3, 16.1, 16.3, 24.1, 25.1 and 26.6. In some aspects, polymorph Form B has an X-ray powder diffraction pattern substantially free of peaks at 12.9 and 14.8 ± 0.05 degrees 2-theta. In some aspects, polymorph Form B is a cyclohexanol solvate. In some aspects, DSC differential scanning calorimetry of the Form B polymorph shows two melting endotherms starting at about 87.9 and 103.2°C.

In one aspect, a form C polymorph is provided that exhibits an x-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at approximately 6.4, 8.1, 8.6, 8.8, 9.9, 10.2 , 12.9, 13.8, 15.1, 15.4, 16.5, 19.8, 21.2, 22.1, 23.7, 25.7 and 27.8.

In one aspect, a form C polymorph is provided that exhibits an x-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at approximately 6.4, 15.1, 21.2, 25.7 and 27.8. In some aspects, the Form C polymorph further contains peaks at 22.1. In some aspects, the Form C polymorph further contains peaks at 16.5 and 22.1±0.05 degrees 2-theta. In some aspects, polymorph Form C has an X-ray powder diffraction pattern substantially free of peaks at 13.6 and 14.8 ± 0.05 degrees 2-theta. In some aspects, polymorph Form C is an anhydrate. In some aspects, polymorph Form C has a differential scanning calorimetry (DSC) melting endotherm starting at about 127.8°C.

In one aspect, a form D polymorph is provided that exhibits an x-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at approximately 8.0, 8.7, 9.2, 9.8, 10.4, 12.9 , 13.4, 14.0, 14.8, 16.4, 18.5, 19.7, 20.0, 20.8, 23.1, 23.3, 23.9, 25.5 and 25 ,7.

In one aspect, a form D polymorph is provided that exhibits an x-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at approximately 9.2, 14.0, 14.8, 19.7 and 20.0. In some aspects, the Form D polymorph has an X-ray powder diffraction pattern substantially free of peaks at 13.6 ± 0.05 degrees 2-theta. In some aspects, polymorph Form D is an anhydrate. In some aspects, polymorph Form D has a differential scanning calorimetry (DSC) melting endotherm starting at about 129.1 °C.

In one aspect, the crystalline compounds provided herein exhibit a mass increase of less than about 1% when subjected to an increase in relative humidity from about 0% to about 95% relative humidity for about 180 minutes.

In one aspect, the crystalline compounds provided herein are stable when exposed to temperatures up to about 40° C. and about 75% relative humidity for at least 6 months.

Another aspect of the present invention is the crystalline compound, 2-methyl-2-(3-2044390 methyl-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-1H-pyrazol-1-yl )propanenitrile, which exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in ±0.3 degrees 2-theta at 6.4, 15.1, 21.2, 25.7 and 27.8.

In another aspect, the present invention provides a crystalline compound, 2-methyl-2-(3methyl-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-1H-pyrazol-1-yl)propanenitrile, which shows an X-ray powder diffraction pattern having characteristic peaks expressed in ±0.05 degrees 2-theta at 6.4, 15.1, 21.2, 25.7 and 27.8.

One aspect of the present invention is a pharmaceutical composition containing a therapeutically effective amount of a crystalline polymorphic or amorphous form of a compound of formula I or solvates, tautomers, or pharmaceutically acceptable salts or co-crystals thereof, and a pharmaceutically acceptable carrier, glidant, diluent or excipient.

One aspect of the present invention is a method for preparing a crystalline polymorphic or amorphous form of a compound of Formula I or solvates, tautomers, or pharmaceutically acceptable salts or cocrystals thereof.

In one aspect, a method is provided for preparing a crystalline polymorph comprising heating at 50° C. or higher 2-methyl-2-(3-methyl-4-(4-(methylamino)-5(trifluoromethyl)pyrimidin-2-ylamino)-1H -pyrazol-1-yl)propanenitrile in methyl tert-butyl ether, cyclopentyl methyl ether, ethyl acetate, isopropyl acetate, or a combination thereof, and a non-polar solvent such as heptane, and then cooling the mixture, resulting in the formation of crystalline polymorphic form C, which exhibits an x-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at approximately 6.4, 15.1, 21.2, 25.7 and 27.8 degrees 2-theta. In some aspects, a seed is added to the mixture. In one aspect, a method for producing a crystalline polymorph is provided, comprising heating at 50° C. or higher an anhydrous solution of 2-methyl-2-(3-methyl-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino )-1Hpyrazol-1-yl)propanenitrile in methyl tert-butyl ether, cyclopentyl methyl ether, ethyl acetate, isopropyl acetate, or a combination thereof, and a non-polar solvent such as heptane, and then cooling the mixture, resulting in the formation of crystalline polymorph Form D, which shows an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at approximately 9.2, 14.0, 14.8, 19.7 and 20.0 degrees 2-theta. In some aspects, a seed is added to the mixture.

In one aspect, the crystalline polymorph provided herein is crushed. In another aspect, the crystalline anhydrous polymorph provided herein is ground.

In one aspect, an amorphous compound is provided, 2-methyl-2-(3-methyl-4-(4-(methylamino)-5(trifluoromethyl)pyrimidin-2-ylamino)-1H-pyrazol-1-yl)propanenitrile.

In one aspect, a method is provided for preparing an amorphous compound, 2-methyl-2-(3-methyl-4-(4(methylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-1H-pyrazol-1-yl)propanenitrile, involving heating the crystalline form of a given compound until it dissolves, followed by cooling to form an amorphous compound. In one aspect, the cooling is rapid cooling, such as in a dry ice or liquid nitrogen bath.

Brief description of graphic materials

In fig. 1 shows the patterns of mutual transformations between the polymorphic forms of the compound of formula I, forms A, B, C and D in a schematic diagram.

In fig. Figure 2 shows an overlay of X-ray diffraction patterns of the polymorphic forms of the compound of formula I, forms A, B, C, and D.

In fig. Figure 3 shows the X-ray structure of a single crystal of polymorphic form A.

In fig. Figure 4 shows the X-ray structure of a single crystal of polymorphic form C.

In fig. Figure 5 shows the X-ray structure of a single crystal of polymorphic form D.

In fig. Figure 6 shows an X-ray diffraction pattern of the PRD of polymorphic form A (anhydrous).

In fig. Figure 7 shows TGA and DSC data for polymorph Form A (anhydrous).

In fig. Figure 8 shows an X-ray diffraction pattern of the PRD of polymorphic form B (cyclohexanol solvate).

In fig. Figure 9 shows TGA and DSC data for polymorph Form B (cyclohexanol solvate).

In fig. Figure 10 shows an X-ray diffraction pattern of the PRD of polymorphic form C (anhydrous).

In fig. Figure 11 shows TGA and DSC data for polymorph Form C (anhydrous).

In fig. Figure 12 shows an X-ray diffraction pattern of the PRD of polymorphic form D (anhydrous).

In fig. Figure 13 shows TGA and DSC data for polymorph Form D (anhydrous).

In fig. Figure 14 shows a polarized light microscopy (PLM) image of shape C single crystals.

In fig. Figure 15 shows an image of the MPS of form D single crystals.

In fig. Figure 16 shows polarized light microscopy images of amorphous form E.

- 3 044390

In fig. Figure 17 shows the X-ray diffraction pattern of amorphous form E.

Definitions

Unless otherwise specified, technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention pertains and are not inconsistent.

The words include, including, contain, containing and contains, as used in this specification and claims, are intended to identify the presence of the claimed features, integers, components or steps, but they do not exclude the presence or addition of one or more other features, integers, components, stages or groups thereof.

As used herein, the term about or approximately, when used in relation to powder X-ray diffraction peak positions, refers to the inherent variability of the peaks depending on, for example, the calibration of the equipment used, the polymorph preparation method used, the age of the crystallized material, and the like. , depending on the equipment used. In this case, the measure of instrument variability was about plus/minus ±0.3 degrees 2-theta(θ). One skilled in the art having the benefit of this description will recognize the use of the term about or approximately in this context unless otherwise indicated (eg, ±0.05 degrees 2-theta). The term about or approximately with respect to other specified parameters, such as water content, _Cmax , _Tmax , AUC, intrinsic dissolution rates, temperature and time, indicates inherent variability, such as the measurement of a parameter or the achievement of a parameter. One skilled in the art, taking advantage of this description, will appreciate the variability implied by the use of the word about or approximately.

Polymorph, or polymorph, as used herein, refers to the occurrence of different crystalline forms of a compound, differing in packing or conformation/configuration, but with the same chemical composition. Crystalline forms have different arrangements and/or conformations of molecules in the crystal lattice. Solvates are crystalline forms containing either stoichiometric or non-stoichiometric amounts of solvent. If the included solvent is water, the solvate is generally known as a hydrate. Hydrates/solvates can exist as polymorphs of compounds with the same solvent content, but different crystal lattice packing or conformation. Thus, a single compound can give rise to different polymorphic forms, each form having different and distinct physical properties such as solubility profiles, melting points, hygroscopicity, particle shape, morphology, density, flowability, compactibility and/or X-ray diffraction peaks. The solubility of each polymorph may vary, so identifying the existence of pharmaceutical polymorphs is important to provide pharmaceuticals with predictable solubility profiles. It is advisable to perform characterization and testing of all solid state forms of the drug, including all polymorphic forms, as well as determination of the stability, dissolution and flow properties of each polymorphic form. Polymorphic forms of a compound can be differentiated in the laboratory using X-ray diffractometry, as well as other methods such as infrared or Raman spectrometry, or solid state NMR spectrometry. For a general overview of polymorphs and pharmaceutical applications of polymorphs, see G. M. Wall, Pharm Manuf. 3:33 (1986); J. K. Haleblian and W. McCrone, J. Pharm. Sci., 58:911 (1969); Polymorphism in Pharmaceutical Solids, Second Edition (Drugs and the Pharmaceutical Sciences), Harry G. Brittain, Ed. (2011) CRC Press (2009); and J. K. Haleblian, J. Pharm. Sci., 64, 1269 (1975), all of which are incorporated herein by reference.

The acronym PXRD stands for Powder X-ray Diffraction, an analytical technique that measures the diffraction of X-rays in the presence of a solid component to produce an X-ray diffraction pattern. The X-ray diffraction pattern can be obtained using CuKa1 radiation. Substances that are crystalline and have regular repeating arrays of atoms generate a characteristic powder diffraction pattern. Substances with similar unit cells will give X-ray diffraction patterns similar in peak positions measured at °2θ (theta). Solvates that have this property are called isostructural or isomorphic solvates.

The intensity of the reflections varies depending on the electron density causing diffraction, as well as the sample, sample preparation, and instrument parameters. Analysis of XRD data is based on the overall appearance of the measured powder diffraction pattern(s) in relation to the known response of the X-ray diffraction system used to collect the data. For diffraction peaks that may be present in a powder diffraction pattern, their positions, shape, width, and relative intensity distributions can be used to determine the type of solid state order characteristic in the powder sample. The position, shape, and intensity of any broad diffuse scattering (halos) above the instrumental background can be used to characterize the level and type of disorder in the solid state. The combination of interpretation of the solid state order and disorder present in a powder sample provides a qualitative measure of the sample's macrostructure.

The term cocrystal refers to a crystalline molecular complex consisting of two or more different molecular compounds, usually in a stoichiometric ratio, that are neither solvates nor simple salts. The cocrystal consists of a complex hydrogen bonded to a pharmaceutically acceptable coformer (Aitipamula, S. et al. (2012) Cryst. Growth Des. 12(5):2147-2152). Coformers include, but are not limited to, acetylsalicylic acid, trans-aconitic acid, adipic acid, L-ascorbic acid, benzoic acid, citric acid, fructose, fumaric acid, gallic acid, glucose, glutaric acid, hippuric acid, 4-hydroxybenzoic acid , maleic acid, malonic acid, mannitol, nicotinamide, nicotinic acid, phenylalanine, riboflavin, salicylic acid, succinic acid and vanillic acid.

The phrase pharmaceutically acceptable salt, as used herein, refers to pharmaceutically acceptable organic or inorganic salts of a compound of this invention. Typical examples of salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, phosphate acid, isonicotinate, lactate, salicylate, citrate acid, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, sucrose, formate, benzoate, glutamate, methanesulfonate mesylate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e. 1,1'-methylene-bis- (2-hydroxy-3-naphthoate)) salt. Other salts include acid salts such as the coformers described above. The pharmaceutically acceptable salt may contain the inclusion of another molecule, such as an acetate ion, succinate ion, or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge of the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. In cases where multiple charged atoms are part of a pharmaceutically acceptable salt, multiple counterions may be present. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.

The desired pharmaceutically acceptable salt can be prepared by any suitable method available in the art. For example, by treating the free base with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or an organic acid such as acetic acid, maleic acid, succinic acid, mandelic acid, methanesulfonic acid, fumaric acid acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidylic acid such as glucuronic acid or galacturonic acid, alpha hydroxy acid such as citric acid or tartaric acid, amino acid such as aspartic acid or glutamic acid, aromatic acid , such as benzoic acid or cinnamic acid, sulfonic acid such as p-toluenesulfonic acid or ethanesulfonic acid, or the like. Acids which are generally considered useful for the formation of pharmaceutically useful or acceptable salts from basic pharmaceutical compounds are discussed, for example, in Stahl PH, Wermuth CG, editors. Handbook of Pharmaceutical Salts; Properties, Selection and Use, ^2nd Revision (International Union of Pure and Applied Chemistry). 2012, New York: Wiley-VCH; S. Berge et al, Journal of Pharmaceutical Sciences (1977) 66(1) 1 19; P. Gould, International J. of Pharmaceutics (1986) 33 201 217; Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; Remington's Pharmaceutical Sciences, ^18th ed., (1995) Mack Publishing Co., Easton PA; and in The Orange Book (Food & Drug Administration, Washington, DC on its website). Descriptions thereof are incorporated herein by reference.

The phrase pharmaceutically acceptable means that the substance or composition must be compatible chemically and/or toxicologically with the other ingredients contained in the composition and/or with the mammal being treated.

Solvate refers to the association or complex of one or more solvent molecules and a compound of this invention. Examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, cyclohexanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. Other solvents that can form solvates include groups 2 and 3 from Q3C - Tables and List Guidance for Industry: (June 2017) US Dept. HHS, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), and Center for Biologies Evaluation and Research (CBER). Class 2 group of solvents that can form solvates are: acetonitrile, chlorobenzene, chloroform, cyclohexane, cumene, 1,2-dichloroethylene, dichloromethane, 1,2dimethoxyethane, N,N-dimethylacetamide, N,N-dimethylformamide, 1,4 -dioxane, 2-ethoxyethanol, ethylene glycol, formamide, hexane, methanol, 2-methoxyethanol, methylbutylketone, methylcyclohexane, methylisobutylketone, N-methylpyrrolidone, nitromethane, pyridine, sulfolane, tetrahydrofuran (THF), tetralin, toluene, trichlorethylene and xylene Class 3 group of solvents that can also form solvates are: acetic acid, heptane, acetone, isobutyl acetate, anisole, isopropyl acetate, 1-butanol, methyl acetate, 2-butanol, 3-methyl-1-butanol, butyl acetate, methyl ethyl ketone, t-butyl methyl ether, 2-methyl-1-propanol, dimethyl sulfoxide, pentane, ethanol, 1-pentanol, ethyl acetate, 1-propanol, ethyl ether, 2-propanol, ethyl formate, propyl acetate, formic acid and triethylamine.

The term hydrate refers to a complex in which the solvent molecule is water.

The term chiral refers to molecules that have the property of not matching when mirrored, while the term achiral refers to molecules that match when mirrored.

The term stereoisomers refers to compounds that have the same chemical structure but differ in the arrangement of atoms or groups in space.

A diastereomer refers to a stereoisomer with two or more centers of chirality, the molecules of which are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties and reactivity. Mixtures of diastereomers can be separated under high-resolution analytical techniques such as electrophoresis and chromatography.

Enantiomers refer to two stereoisomers of a compound that are non-matching mirror images of each other when superimposed.

Stereochemical definitions and conventions used herein generally follow those of S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., New York, 1994. The compounds of this invention may contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of this invention, including, but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof, such as racemic mixtures, are intended to form part of the present invention. Many organic compounds exist in optically active forms, i.e. they are capable of rotating the plane of plane-polarized light. When describing an optically active compound, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule relative to its chiral center(s).

The prefixes d and 1 or (+) and (-) are used to indicate the direction of rotation of plane-polarized light by the compound, with (-) or 1 indicating that the compound is left-handed. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. A particular stereoisomer may also be called an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur in cases where there was no stereoselectivity or stereospecificity in a chemical reaction or process. The terms racemic mixture and racemate refer to an equimolar mixture of two enantiomeric species lacking optical activity.

The term tautomer or tautomeric form refers to structural isomers with different energies that are interconvertible through a low-energy barrier. For example, protic tautomers (also known as prototropic tautomers) involve interconversions via proton migration, such as the isomerization of keto-enol and imine-enamine. Valence tautomers involve interconversions by reorganizing some of the bonding electrons.

Compound of formula I

This invention includes polymorphs and amorphous forms of the compound of formula I (CAS registration number 1374828-69-9) having the structure:

ΗΝ^ί'^

Nose. -Pch

Ν-Ν \-CN H ₄ C CH, j and called as: 2-methyl-2-(3-methyl-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2ylamino)-1H-pyrazol- 1-yl)propanenitrile (WO 2012/062783; US 8815882; US 2012/0157427, each of which is incorporated herein by reference). As used herein, a compound of formula I includes tautomers and their pharmaceutically acceptable salts or co-crystals. The compound of formula I is an API (active pharmaceutical ingredient) in compositions for use in the treatment of neurodegenerative and other disorders, with pKa upon protonation calculated at 6.7 and 2.1.

Crystallization and screening of a compound of formula I

Primary polymorph screening experiments were carried out using various crystallization or solid state phase transition methods including: addition of antisolvent, back addition to antisolvent, slow evaporation, slow cooling, suspension at room temperature (RT), suspension at 50°C, diffusion into solid-gas system, diffusion in the vapor-liquid system and polymer-induced crystallization. All these methods identified the crystal type of Form A. Polarized light microscopy (PLM) images of Form A obtained by various polymorph screening methods were collected (Example 5). The particles produced by adding antisolvent were small in size, ranging from about 20 to 50 microns (µm) in diameter, while slow evaporation, slow cooling (except THF/isooctane), vapor-liquid diffusion and polymer-induced crystallization resulted in large particles. The addition of isooctane to a solution of a compound of formula I in dichloromethane (DCM) resulted in particles with the most uniform size. The crude compound of formula I was crystallized from THF/n-heptane and then finely ground. The crystallization procedure was developed to control particle size.

A total of four crystalline forms (Forms A, B, C and D) and an amorphous Form E of the compound of formula I were obtained, including 3 anhydrates (Form A, C and D) and one solvate (Form B). Suspension competition experiments showed that form D was thermodynamically more stable at water activity aw <0.2 at RT, while form C was more stable at water activity aw >0.5 at RT. Solubility assessment at 24 hours showed that the solubility of forms A, C and D in H ₂ O at RT was 0.18, 0.14 and 0.11 mg/ml, respectively. The EVA (Dynamic Vapor Sorption) results showed that Forms A and D were non-hygroscopic, as determined by less than 0.1% reversible water uptake by EAF, and Form C was slightly hygroscopic. Some data on the characteristics and observations of crystalline forms are given in Table. 1.

Table 1

Brief description of the characteristics for the crystalline forms of the compound of formula I

Cryst allic form oter and masses in TGA (%) Endotherm at dec (°C, beginning) Form identity Solubility over 24 hours in _H2O at RT (mg/ml) Hygrosk opicity E nalpi I Δ N (J/g) a A Form .6 1 22.8 Naya Bezvod 0.18 Non-hygroscopic 1.2 9 aB Form 3.9 8 7.9, 103.2 Solv at cyclohexano la nd nd 5.3 6 аС Form ,8 1 27.8 Naya Bezvod 0.14 I'm slightly hygroscopic 4.37 9 aD Form ,5 1 29.1 Naya Bezvod 0.11 Non-hygroscopic 90.28

Differential scanning calorimetry (DSC) analysis of Forms A and C showed that Form C had a higher melting point and a higher heat of fusion (Table 1), indicating that the two forms are monotropic and Form C is more stable. Competition experiments with 1:1 suspensions of Forms A and C in various solvents always resulted in Form C, confirming that Form C was more stable than Form A. Consistent with this, Form C was obtained even when Seed form A was added to the crystallization charge.

Polymorphs of the compound of formula I

Physical characteristics as well as interconversion patterns were assessed to assess the identity of a suitable crystalline form of the compound of Formula I for further development. To date, a total of four crystalline forms A, B, C and D have been obtained. An amorphous form E has also been obtained. All crystalline forms have been characterized by powder x-ray diffraction (XRD) using the procedures of Example 6, and thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) using the procedures of Example 7. Form identification studies confirmed that Forms A, C and D were anhydrates and Form B was a cyclohexanol solvate. A brief description of the characteristics for all crystalline forms is presented in Table. 1. The thermodynamic stability relationship between the three anhydrates (form A, C and D) was investigated using suspension competition experiments. A detailed pattern of mutual transformations is depicted in a schematic diagram (shown in Fig. 1). The pattern of mutual transformations between forms C and D was associated with the effect of the solvent H2O. Form D was thermodynamically more stable at aw <0.2 at room temperature (RT, 25 ± 2°C), while form C was more stable at water activity aw >0.5 at RT. For forms A, C and D, solubility over 24 hours in H ₂ O and hygroscopicity were additionally assessed. The 24 hour solubilities of Forms A, C and D in H ₂ O at RT were measured to be 0.18, 0.14 and 0.11 mg/ml, respectively. No changes in forms were observed after 24 hour solubility assessment. Dynamic vapor sorption (DVA) data showed that Forms A and D were non-hygroscopic and Form C was slightly hygroscopic. Based on the characterization and evaluation results, both forms C and D showed excellent physicochemical properties, including high crystallinity, low TGA mass loss, and one steep DSC endotherm. However, taking into account the solvent effect of H ₂ O associated with the pattern of mutual transformations between forms C and D, it may be useful to control the water content of process solvents and the relative humidity of the environment during production and storage (aw > 0.5 for form C , aw <0.2 for form D).

A total of four crystalline forms A, B, C and D of the compound of formula I and an amorphous form E are obtained. FIG. Figure 2 shows an overlay of X-ray diffraction patterns of crystalline forms of the compound of formula I.

Interconversion of crystalline forms

Suspension competition experiments were carried out to study the interconversion patterns between the three anhydrates (form A, C and D). Since the compound of formula I showed low solubility (<2 mg/ml) in H ₂ O and non-polar solvents (eg, n-heptane, cyclohexane) and good solubility (>40 mg/ml) in other solvents, for most competition experiments mixed solvent systems were used in the suspension. Competition in suspension was first carried out between form A and C at RT and 50°C. Form C was obtained after suspension for 40 hours at RT or 2 hours at 50°C. Further suspension for an additional three weeks resulted in Form D as a new anhydrate, except when exposure to H2O occurred. Thus, competition in suspension was additionally carried out between forms C and D in different solvent systems. An H ₂ O effect was discovered, associated with the pattern of mutual transformations between forms C and D, and form D is formed under anhydrous conditions.

To understand the thermodynamic stability relationship between Form A and Form C, suspension competition experiments were performed at RT and 50°C in various solvent systems, including acetone/n-heptane, methyl tert-butyl ether (MTBE)/heptane, and water. Form A was used to saturate the appropriate solvent before filtering to obtain a nearly saturated solution. Equal amounts of samples of Forms A and C were weighed and then mixed with the prepared nearly saturated solution to form a new suspension, which was stirred with a magnetic stirrer (~1000 rpm) at RT and 50°C. The PRD pattern of the remaining solids after suspension was measured. Form C was obtained after suspension at RT for 40 hours or suspension at 50°C for 2 hours, indicating that Form C is thermodynamically more stable than Form A in the range from RT to 50°C. Further suspension for 3 weeks resulted in the formation of Form D, except when suspended in H ₂ O at 50°C. Form D was prepared by suspending Form A in MTBE/n-heptane (1:4, v/v) at RT for 1 day with the addition of a sample of Form D as seeds.

To further understand the thermodynamic stability relationship between Form C and Form D, suspension competition experiments were performed at RT and 70 °C in various solvent systems, including methyl tert -butyl ether/heptane, water, cyclohexane, and ethanol/isooctane. Equal amounts of Form C and D samples were weighed and then mixed with a nearly saturated solution of Form A in an appropriate solvent to form a suspension, which was stirred magnetically (~1000 rpm) at various temperatures. The PRD patterns of the remaining solids after suspension were obtained. Form C was obtained after suspension in H ₂ O at RT/70°C and in EtOH/isooctane (1:19) at RT, while Form D was obtained in other solvent systems. Based on these results, a pattern of mutual transformations between forms C and D was postulated, associated with the effect of the solvent H2O or EtOH. In order to study the effect of H2O in technological solvents on the pattern of mutual transformations between forms C and D, competition was carried out in a suspension of form C and form D in EtOAc/n-heptane (1:4, v/v) in the presence and absence of H saturation ₂ O. Form C was prepared in EtOAc/n-heptane (1:4, v/v) saturated with H ₂ O, while Form D was prepared in a solvent system without pretreatment. The water content in process solvents was monitored and controlled during the preparation of samples of form C or D. Competition in a suspension of form C and form D was carried out in the acetone/H ₂ O system with different water activities (aw ~ 0.2, 0.5, 0.8 ) with CT. Form D was obtained at aw ~0.2 at RT, while

- 8 044390 form C was obtained at aw ~0.5, 0.8 at RT. Water activity (or relative humidity) was monitored and controlled during the preparation and storage of Form C or D samples.

The 24-hour solubilities of Forms A, C, and D were measured in water at RT. Samples of Form A, Form C and Form D were suspended in H ₂ O at a dose concentration of 10 mg/ml. After sedimentation of the suspensions at RT for 24 h (1000 rpm), the supernatants were separated for solubility measurements by HPLC, and the residual solids were characterized by PRD. The solubility of Forms A, C, and D in H ₂ O was measured to be 0.18, 0.14 and 0.11 mg/ml, respectively. No shape changes were observed for Forms A, C, or D after 24-hour solubility assessment at CT.

Determination of single crystal structure

The crystal structures of forms A, C and D were determined by X-ray diffraction of single crystals (X-ray diffraction analysis, X-ray diffraction) according to the methods of example 4. A single crystal of form A of appropriate quality for X-ray diffraction was obtained by diffusion in a vapor-liquid system from a solvent system n-butyl acetate/cyclohexane (n -butyl acetate was the solvent, while cyclohexane was the antisolvent) at RT. Crystallographic data and information on structure refinement are listed in example 4. Determination of the characteristics of a single crystal by X-ray diffraction showed that the crystal accepted the space group P21/n with a = 5.325(2) A, b = 13.005(5) A, c = 24.778(9) A; α=90°, β=94.408 (11)°, γ=90°.

An asymmetric single crystal unit of polymorph A is shown in FIG. 3. The asymmetric unit consists of only one molecule of formula I, indicating that Form A is an anhydrate.

The hydrogen bonds in the single crystal structure of Form A show that the three-dimensional (3-D) packing of the molecules of formula I is supported by intermolecular H bonds (N3-H3-N7, N-4-N1), as well as additionally by van der Waals forces. The calculated PRD pattern is consistent with the experimental PRD pattern of form A (Fig. 6).

X-ray single crystal structures of form C (Fig. 4) and form D (Fig. 5) indicate interactions in crystals of these forms, and density (Table 2).

table 2

Density of crystals of forms A, C, D

Crystal form Density (calculated from X-ray diffraction data of a single crystal, g/cm ³ ) Form A 1.318 Form C 1,367 Form D 1,390

Dynamic Vapor Sorption (DVS)

In order to examine the stability of the solid form as a function of humidity, the dynamic vapor sorption isotherms (DVS) of Form A, Form C and Form D were measured at 25°C from 0 to 95% relative humidity (RH) in accordance with the procedures of the example. 8. Based on the EAF results, Form A (0.04% water absorption at 80% RH at 25°C) and Form D (0.05% water absorption at 80% RH at 25°C) were not hygroscopic, but C (0.6% water absorption at 80% RH at 25°C) was slightly hygroscopic. No shape changes were observed after the chipboard evaluation.

Powder X-ray diffraction analysis

Powder X-ray diffraction (XRD) patterns were analyzed with commercially available analytical software following the procedures of Example 6. XRD is useful for identifying different crystalline phases, polymorphs, hydrates or solvates by their unique diffraction patterns. The abscissa (horizontal axis) shows the values of 2-theta(θ) - a series of angles between the incident and diffracted beams. The y-axis (vertical axis) records the intensity of the scattered X-ray radiation recorded by the detector. This set of peaks acts as a unique signature of a crystallographic unit cell in a crystalline substance. A crystallographic unit cell is the smallest atomic-scale 3D unit that repeats periodically in three dimensions throughout the crystal. All crystalline substances are distinguished by their crystallographic unit cells (and therefore by their peak positions). By comparing the measured peak positions with those in the database, the crystalline substance can be uniquely identified. For pure substances, the positions of all peaks, as a rule, depend on three parameters: a, b, c and three angles: alpha, beta, gamma (α, β, γ), which define the elementary parallelepiped enclosing the crystallographic unit cell.

Solid forms of the compound of formula I

Form A was characterized by PRD, TGA and DSC. The X-ray image of the PRD is shown in Fig. 6 and indicates that Form A is crystalline. A report on the search for PDP peaks for Form A of Formula I is given in Table. 3. TGA and DSC data are shown in Fig. 7. A mass loss of 0.6% was observed up to 120°C in the TGA curve. The DSC result showed a steep endotherm at 122.8°C (onset temperature). On

- 9 044390 based on the low mass loss of TGA and the only steep endotherm of DSC, it is postulated that the form

A is an anhydrate.

Table 3

PDP Peak Search Report for Form A of Formula I

Pos. [°2 theta] Height [imp.] Interatomic distance [A] Rel. INT. [%] 7.7 2149 11.5 61.1 9.9 2255 9.0 64.1 12.7 1545 7.0 43.9 13.6 3517 6.5 100.0 14.1 3373 6.3 95.9 15.4 1442 5.8 41.0 15.9 2332 5.6 66.3 19.2 658 4.6 18.7 20.5 726 4.3 20.6 21.6 1076 4.1 30.6 22.4 1515 4.0 43.1 23.2 855 3.8 24.3 24.7 808 3.6 23.0

Form B was characterized by PRD, TGA and DSC and prepared by suspending Form A in isobutyl acetate/cyclohexanol (1:9, v/v) at RT for 1 week. The X-ray image of the PRD is shown in Fig. 8. A report on the search for PDP peaks for form B of formula I is given in Table. 4. TGA and DSC data are shown in Fig. 9. We observed a mass loss of 13.9% up to 120°C on the TGA curve and two endotherms at 87.9°C and 103.2°C (onset temperature) on the DSC curve. The NMR spectrum (1H) showed that the molar ratio of cyclohexanol/API was 0.5:1 (12.9 wt.%). In combination with heating and 1H NMR results, Form B is postulated to be a cyclohexanol solvate.

Table 4

PDP Peak Search Report for Form B of Formula I

Pos. [°2 theta] Height [imp.] Interatomic distance [A] Rel. int. [%] 7.0 532 12.7 25.8 7.3 444 12.2 21.5 16.1 948 5.5 46.0 16.3 551 5.4 26.7 24.1 1469 3.7 71.2 25.1 2063 3.6 100.0 26.6 489 3.4 23.7

Form C was characterized by PRD, TGA and DSC. The XRD pattern is shown in Fig. 10. A report on the search for PDP peaks for form C of formula I is given in Table. 5. TGA and DSC data are shown in Fig. 11. We observed a mass loss of 0.8% up to 120°C on the TGA curve and a steep endotherm at 127.8°C (onset temperature) on the DSC curve. Based on the low mass loss of TGA and the single steep endotherm of DSC, form C is postulated to be an anhydrate.

Table 5

PDP Peak Search Report for Form C of Formula I

Pos. [°2 theta] Height [imp.] Interatomic distance [A] Rel. int. [%] 6.4 107 13.8 2.9 8.1 173 10.9 4.7 8.6 2175 10.2 59.2 8.8 738 10.1 20.1 9.9 401 9.0 10.9 10.2 333 8.7 9.1 12.9 2135 6.9 58.1 13.8 581 6.4 15.8 15.1 1496 5.9 40.7 16.5 1952 5.4 53.1 19.8 3675 4.5 100.0 21.2 793 4.2 21.6 22.1 1958 4.0 53.3 23.7 790 3.6 21.5 25.7 1065 3.5 29.0 27.8 570 3.2 15.5

Form D seeds were prepared by suspending Form A in MTBE/n-heptane (1:4, v/v) at RT for 1 day with the addition of a sample of Form D obtained from a suspension competition experiment. The XRD pattern is shown in Fig. 12. Report on the search for PDP peaks for form D of the formula

- 10 044390

I is given in Table. 6. TGA and DSC data are shown in Fig. 13. A weight loss of 0.2% was observed before

120°C on the TGA curve and a steep endotherm at 129.1°C (onset temperature) on the DSC curve. Based on the low mass loss of TGA and the single steep endotherm of DSC, Form D is postulated to be an anhydrate.

Table 6

PDP Peak Search Report for Form D of Formula I

Pos. [°2 theta] Height [imp.] Interatomic distance [A] Rel. INT. [%] 8.0 389 11.0 3.3 8.7 3614 10.2 30.3 9.2 603 9.6 5.1 9.8 1814 9.0 15.2 10.4 981 8.5 8.2 12.9 1805 6.9 15.1 13.4 2561 6.6 21.5 14.0 2422 6.3 20.3 14.8 11925 6.0 100.0 15.6 1044 5.7 8.8 16.4 1240 5.4 10.4 18.5 1595 4.8 13.4 19.7 3884 4.5 32.6 20.0 3839 4.4 32.2 20.8 2235 4.3 18.7 21.0 1286 4.2 10.8 23.1 1285 3.9 10.8 23.3 964 3.8 8.1 23.9 1354 3.7 11.4 25.5 1040 3.5 8.7 25.7 1104 3.5 9.3

Pharmaceutical compositions and formulations

The polymorphic form of Formula I can be formulated in accordance with standard pharmaceutical practice and in accordance with the procedures of Example 9 for use in therapeutic treatment (including prophylactic treatment) in mammals, including humans. This invention relates to a pharmaceutical composition containing a compound of formula I in combination with one or more pharmaceutically acceptable carrier, glidant, diluent or excipient.

Suitable carriers, diluents, glidants and auxiliaries are well known to those skilled in the art and include substances such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic substances, gelatin, oils, solvents, water and the like.

These compositions can be prepared using conventional dissolution and mixing techniques. The compound of this invention is typically formulated into pharmaceutical dosage forms to provide an easily controlled dosage of the drug and to enable patient compliance.

The pharmaceutical composition (or composition) for use may be packaged in various forms depending on the method used to administer the drug. Typically, an article for sale includes a container having a pharmaceutical preparation in an appropriate form contained therein. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders and the like. The container may also include a tamper-evident device to prevent inadvertent access to the contents of the package. In addition, the container has a label applied to it describing the contents of the container. This label may also contain appropriate warnings.

Pharmaceutical compositions of the polymorphic form of a compound of Formula I can be prepared for various routes and modes of administration with pharmaceutically acceptable diluents, carriers, excipients, glidants or stabilizers (Remington's Pharmaceutical Sciences (1995) 18th edition, Mack Publ. Co., Easton, PA ), in the form of a lyophilized preparation, ground powder or aqueous solution. The composition can be prepared by stirring at ambient temperature, at the appropriate pH and at the desired degree of purity, with physiologically acceptable carriers, i.e. carriers that are non-toxic to recipients at the doses and concentrations used. The pH of the formulation depends primarily on the specific application and concentration of the compound, but may range from about 3 to about 8.

Preferably, the pharmaceutical composition is sterile. In particular, formulations intended for in vivo administration must be sterile. This sterilization is easily achieved by filtration through sterile filtration membranes.

The pharmaceutical composition may generally be stored as a solid composition, tablet, pill, capsule, lyophilized composition or as an aqueous solution.

The pharmaceutical compositions of this invention are dosed and administered in a manner, i.e. quantities, concentrations, regimens, courses of treatment, carrier media and route of administration are in accordance with good medical practice. Factors to be considered in this context include the specific disorder being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the route of administration, the schedule of administration and other factors known to the practitioner.

Acceptable diluents, carriers, excipients and stabilizers are non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, ethyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; pyrocatechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as lactose, sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, including Tween 80, PLURONICS™ or polyethylene glycol (PEG), including PEG400. Active pharmaceutical ingredients can also be encapsulated in microcapsules made, for example, by coacervation or interfacial polymerization techniques, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such methods are described in Remington's Pharmaceutical Sciences 18th edition, (1995) Mack Publ. Co., Easton, PA. Other examples of dosage formulations can be found in Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, Vol 3, 2 ^nd Ed., New York, NY.

Tablets may contain one or more pharmaceutically acceptable carriers, glidants, diluents or excipients selected from microcrystalline cellulose, lactose, sodium starch glycolate and magnesium stearate.

Pharmaceutically acceptable glidants may be selected from silica, powdered cellulose, microcrystalline cellulose, metal stearates, sodium aluminosilicate, sodium benzoate, calcium carbonate, calcium silicate, corn starch, magnesium carbonate, asbestos-free talc, stearowet C, starch, starch 1500, magnesium lauryl sulfate, magnesium oxide and combinations thereof.

Pharmaceutical compositions include those suitable for the routes of administration described herein. The compositions may be conveniently provided in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Techniques and preparations in general can be found in Remington's Pharmaceutical Sciences ^18th Ed. (1995) Mack Publishing Co., Easton, PA. Such methods include the step of combining the active ingredient with a carrier that consists of one or more additional ingredients. These compositions can be prepared by uniformly and thoroughly combining the active ingredient with liquid carriers or finely divided solid carriers, or both, and then, if necessary, molding the product.

The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example a sterile injectable aqueous or oily suspension. This suspension can be formulated in accordance with methods known in the art, using suitable dispersing or wetting agents and suspending agents as previously mentioned. The sterile injectable preparation may be in the form of a solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol, or it may be prepared from a lyophilized powder. Acceptable carriers and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils can be used as a solvent or suspending medium, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injection solutions.

In another aspect, the present invention provides a method of treating a disease or condition mediated, at least in part, by leucine-rich repeat kinase 2 (LRRK2). In particular, this disclosure relates to methods of preventing or treating a disorder associated with LRRK2 in a mammal, comprising the step of administering to said mammal a therapeutically effective amount of a compound provided herein. In some embodiments, the disease or condition mediated at least in part by LRRK2 is a neurodegenerative disease, such as a central nervous system (CNS) disorder such as Parkinson's disease (PD), Alzheimer's disease (AD), dementia (including diffuse Lewy bodies and vascular dementia), amyotrophic lateral sclerosis (ALS), age-related memory dysfunction, mild cognitive impairment (eg, including the transition from mild cognitive impairment to Alzheimer's disease), disease characterized by the appearance of argyrophilic granules, lysosomal disorders (eg, type C Niemann-Pick disease, Gaucher disease) corticobasal degeneration, progressive supranuclear palsy, inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), withdrawal symptoms/relapse associated with drug addiction, L-dopa-induced dyskinesia, Huntington's disease (HD) and HIV-associated dementia (HAD). In other embodiments, the disorder is an ischemic disease of organs including, but not limited to, the brain, heart, kidneys, and liver.

In some other embodiments, the disease or condition mediated at least in part by LRRK2 is cancer. In some specific embodiments, said cancer is thyroid, kidney (including papillary kidney cancer), breast, lung, blood and prostate cancer (eg, solid tumor), leukemia (including acute myeloid leukemia (AML)), or lymphomas. In some embodiments, said cancer is kidney cancer, breast cancer, prostate cancer, blood cancer, papillary cancer, lung cancer, acute myelogenous leukemia, or multiple myeloma.

In other embodiments, the compounds described herein are used in methods of treating inflammatory diseases. In some embodiments, said disorder is an inflammatory bowel disease, such as Crohn's disease or ulcerative colitis (generally both known as inflammatory bowel disease). In other embodiments, said inflammatory disease is leprosy, amyotrophic lateral sclerosis, rheumatoid arthritis, or ankylosing spondylitis. In some embodiments, said inflammatory disease is leprosy, Crohn's disease, inflammatory bowel disease, ulcerative colitis, amyotrophic lateral sclerosis, rheumatoid arthritis, or ankylosing spondylitis.

In other embodiments, the compounds described herein are used in methods of treating multiple sclerosis, systemic lupus erythematosus, autoimmune hemolytic anemia, true red cell aplasia, idiopathic thrombocytopenic purpura (ITP), Evans syndrome, vasculitis, bullous skin diseases, type 1 diabetes mellitus, syndrome Sjogren's disease, Devic's disease and inflammatory myopathies.

Examples

Example 1. Isolation and physicochemical characteristics of the compound of formula I, 2-methyl-2-(3methyl-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-1n-pyrazol-1-yl )propanenitrile, reg. CAS No. 1374828-69-9.

The compound of formula I, prepared in accordance with example 394 of US 8815882, and compound 12 of Estrada, A.A. et al (2013) J. Med. Chem. 57:921-936, each specifically incorporated by reference, were dissolved in methyl tert-butyl ether (MTBE, 10 volumes, 200 ml) to give a brown solution. This solution was filtered through a 3M Zeta Plus activated carbon disk (R55SP, 5 cm diameter) at 3 ml/min. The filter was washed with MTBE (5 volumes, 100 ml). The clear, uncolored solution (300 ml) was evaporated to 8 volumes (160 ml) and loaded into a 500 ml reactor. Nheptane (8 volumes, 160 ml) was added at 20°C. The solution initially remained transparent, but then crystallization began after 2 minutes. The temperature was gradually increased (rate 2°C/min). Complete dissolution was achieved only at 69°C. Next, heptane (4 volumes, 80 ml) was added at 70°C; a clear solution was visually observed at 70°C. The temperature was set to 65°C (1.0°C/min); At 65 °C, seed crystals of the compound of formula I (200 mg, same loading) were added to a clear solution and they did not dissolve. The temperature was then lowered to 20°C over 8 hours. The solution was stirred at 20°C overnight. The solid was filtered and washed twice with mother liquors. The substance was dried under vacuum at 40°C for 2 hours to obtain 15.91 g of crystalline compound of formula I (yield 79.6%). The mother liquors were evaporated to dryness to obtain an additional 3.47 g (17.4% recovery).

Example 2. Growing single crystals of polymorphic form C.

Lumpy single crystals of the Form C polymorph of Formula I were prepared from an n-butyl acetate/cyclohexane solvent system (n-butyl acetate was the solvent while cyclohexane was the antisolvent) via vapor-liquid diffusion at RT. The details of the experiment are as follows.

- 13 044390

About 30 mg of a sample of Form A of Formula I was weighed into a 3 ml glass vial with 65 μl of n-butyl acetate solvent added to dissolve the entire solid sample. A small amount of Form C crystalline sample was also added to the 3 mL vial as crystal seeds. This vial was then placed in a 20 ml glass vial with 4 ml of cyclohexane antisolvent in it for vapor-liquid diffusion at ambient temperature. After 11 days, block-shaped crystals were obtained, as shown in FIG. 14.

Example 3. Growing single crystals of polymorphic form D.

Lumpy single crystals of polymorph Form D of Formula I were prepared from a solvent system of acetone/n-heptane (1:10, v/v) by slow evaporation at RT. The details of the experiment are as follows.

About 30 mg of a sample of Form A of Formula I was weighed into a 4 ml culture flask (44.6 mm x 14.65 mm) with 0.2 ml n-butyl acetate and 2.0 ml n-heptane added to dissolve the entire solid sample. A small amount of Form D crystalline sample was also added to the 4 mL vial as crystal seeds. The vial was then placed in a fume hood to allow slow evaporation at ambient temperature. After 15 days, block-shaped crystals were obtained, as shown in FIG. 15.

Example 4. Determination of single crystal structure.

Colorless blocky single crystals were collected from a Form C single crystal sample or a Form D single crystal sample and placed in Paratone-N (an oil-based cryoprotectant). The crystals were placed on a Mylar loop in a random orientation and immersed in a stream of nitrogen at 150 K. Preliminary study and data acquisition were performed on an Agilent SuperNova® diffractometer (Cu/Κα λ=1.54178 A) and data were analyzed using the CrysAlisPro® software package (Agilent, Version: 1.171.38.41).

The details of single crystal shape C data acquisition are as follows: The cell parameters and orientation matrix for data acquisition were extracted and refined using CrysAlisPro® software using set angles from 6568 reflections in the range 4.0790° <θ<70.0660°. Data were collected to a maximum diffraction angle (θ) of 70.266° at 150.2(2) K. The data set was 99.9% complete and had an average Ι/σ of 19.4 and D min (Cu) of 0.82 A.

The details of Shape C single crystal data reduction are as follows: Frames were integrated using CrysAlisPro® software, version: 1.171.38.41. A total of 12,836 reflections were recorded, of which 6,205 were unique. Lorentz and polarization corrections were applied to the data. Empirical absorption correction was performed using spherical harmonics, implemented in the SCALE3 ABSPACK scaling algorithm. The absorption coefficient μ of this material is 0.964 mm ^-1 at a given wavelength (λ = 1.54178 A) and the minimum and maximum transmittances are 0.80956 and 1.00000. The intensities of equivalent reflections were averaged. The agreement factor for intensity-based averaging was 2.08%.

The structure of form C was solved in space group C2/c by direct methods using the ShelXS™ structure solver (Sheldrick, GM (2008). Acta Cryst. A64:112-122) and refined using the ShelXS™ optimization package, version 2014/ 7, using the full least squares matrix on F ² contained in OLEX2 (Dolomanov, OV, et al., (2009) J. Appl. Cryst. 42:339-341). All non-hydrogen atoms were optimized anisotropically. The positions of hydrogen atoms bonded to carbon atoms were calculated geometrically and refined using the rider model, but hydrogen atoms bonded to nitrogen atoms were refined freely according to Fourier maps.

The data acquisition details of the Form D single crystal are as follows: The cell parameters and orientation matrix for data acquisition were extracted and refined using CrysAlisPro® software using fitted angles from 30349 reflections in the range 4.0180° <θ<70.5190°. Data were collected to a maximum diffraction angle (θ) of 70.562° at 150 K. The data set was 89.9% complete and had an average Ι/σ of 29.3 and D min (Cu) of 0.82 A.

The details of single crystal data reduction of shape D are as follows: Frames were integrated using CrysAlisPro® software, version: 1.171.38.41. A total of 47,670 reflections were recorded, of which 11,179 were unique. Lorentz and polarization corrections were applied to the data. Empirical absorption correction was performed using spherical harmonics, implemented in the SCALE3 ABSPACK scaling algorithm. Absorption coefficient μ. of this material is 0.980 mm ^-1 at a given wavelength (λ = 1.54178 A) and the minimum and maximum transmittances are 0.83622 and 1.00000. The intensities of equivalent reflections were averaged. The agreement factor for intensity-based averaging was 2.69%.

The structure of form D was solved in space group Pca2 ₁ by direct methods using the ShelXS™ structure solver and refined using the ShelXS™ optimization package, version 2014/7, using the full least squares matrix on F ² contained in OLEX2. All non-hydrogen atoms were optimized anisotropically. Hydrogen atom positions

- 14 044390 were calculated geometrically and refined using a rider model.

Table 7

Single Crystal X-Ray Diffraction (PCA) Instrument Parameters

Device Agilent SuperNova X-ray source SuperNova Microfocus X-ray Source (Cu/Κα: 1.54184 A) 50 kV, 0.8 mA Detector Eos CCD detector (Detector resolution: 16.0450 pixels ^mm'1 ) Goniometer Carr's Four-circle protractor Devices for maintaining low temperatures Oxford Cryosystems Software CrysAlisPro (version: 1.171.38.41)

Polymorphic forms of the compound of Formula I were solved using the ShelXT structure solver (Sheldrick, GM (2015). Acta Cryst. A71, 3-8) (internal phasing method) and optimized using the SHELXL-2015 optimization package (Sheldrick, GM (2015) Acta Cryst. A71, 38)) (complete matrix of least squares on F ² ) contained in OLEX2 (Dolomanov, O. V. et al., OLEX2: a complete structure solution, refinement and analysis program. J. Appl. Cryst 2009, 42, 339-341). The calculated PRD pattern was obtained from Mercury (Macrae, C. F., et al., Appl. Cryst. (2006) 39:453-457) and crystal structure representations were generated using Diamond. Single crystal X-ray diffraction data were collected at 296 K on a Bruker D8 VENTURE diffractometer (Mo/Κα radiation, λ=0.71073 A). In table Figure 8 shows crystallographic data and structure optimization of forms A, C and D.

- 15 044390

Table 8

Crystallographic data and optimization of the structure of single crystals of polymorphic forms A, C and D of formula I.

Options Form A Form C Form D Empirical formula C14H16F3N7 Ci ₄ H ₁₆ F ₃ N ₇ SmNshchRzMu Molecular mass 339.34 339.34 339.34 Temperature 296 K 150.2(2) K 150 K Wavelength Μο/Κα (λ=0.71073 A) Cu/Κα (λ= 1.54178 A) Cu/Κα (λ= 1.54178 A) Syngony, space group Monoclinic, R21/P Monoclinic, C2/s Orthorhombic, Pca2i Unit cell dimensions a=5.325(2) A b=13.005(5) A c=24.778(9) A a=90° ^=94.408(11)° y=90° a=13.7032(3) A b=17.5697(4) A c=27.4196(6) A a=90° β=91.982(2)° γ=99° a=17.63410(10) A b=14.03430(10) A c=26.2102(2) A a=90° β=90° ^90° Volume 1710.7(11) A ³ 6597.6(3) A ³ 6486.56(8) A ³ Z, Calculated Density 4, 1.318 g/cm ³ 16, 1.367 g/cm ³ 16, 1.390 g/cm ³ Absorption coefficient 0.108 mm ^-1 0.964 mm ^-1 0.980 mm ^-1 F(000) 704.0 2816.0 2816.0 Crystal size 0.6 x 0.5 x 0.2 mm ³ 0.4 x 0.4 x 0.3 mm ³ 0.6 x 0.5 x 0.2 mm ³ 2 Theta band for data acquisition from 4.548° to 57.89° from 6.45° to 140.532° from 6.744° to 141.124° Limit indices -6<h<6 -16<k< 16 -32 < 1 < 33 -13 <h< 16 -21 <k< 15 -31 <1<33 -21 <h<21 -16<k< 14 -23 <1<31 Collected Reflections/Independent Reflections 28803/3825 [R(int) = 0.0509] 12836/6205 [Ri _n t=0.0208, Rsigma ⁼ 0.0267] 47670/11179 [Rint=0.0269, R _s igma= 0.0214] Completeness 84.57% 98.24% 89.80% Optimization method Complete least squares matrix F ² Complete least squares matrix F ² Complete least squares matrix F ² Data/limitations/parameters 3825/0/221 6205/0/441 11179/1/881 Adequacy of the model for F ² 1,081 1.038 1.031 Final R indices [I > 2 sigma(I)] Ri=0.0993, wR ₂ =0.2464 Ri=0.0461, wR ₂ =0.1241 Ri=0.0320, wR ₂ =0.0857 Final R indexes [all data] Ri=0.0518, wR ₂ =0.1281 Ri=0.0339, wR ₂ =0.0872 The biggest difference between peak and trough 0.74/-0.78 e. A ³ 0.85/-0.37 eA ³ 0.19/-0.21 eA ³

Single crystals of Form C and Form D were prepared and analyzed by single crystal X-ray diffraction (XRD). The single crystal structures of form C and form D were determined successfully.

Determination of the characteristics of a single crystal by X-ray diffraction confirmed that form C crystallizes in the monoclinic system and space group C2/c with unit cell parameters {a = 13.7032(3) A, b = 17.5697(4) A, c = 27.4196 (6) A; α=90°, β=91.982 (2)°, γ=90°}. Cell volume V was calculated as 6597.6(3) A ³ . The asymmetric unit consists of two molecules, indicating that form C is an anhydrate. The calculated density of Form C is 1.367 g/cm ³ . The unit cell of a single crystal consists of sixteen molecules.

Determination of the characteristics of a single crystal by X-ray diffraction confirmed that form D crystallizes in the orthorhombic system and space group Pca21 with unit cell parameters {a = 17.63410(10) A, b = 14.03430(10) A, c = 26.2102(2 ) A; α=90°, β=90°, γ=90°}. The cell volume V was calculated to be 6486.56(8) A ³ The asymmetric unit consists of four molecules, indicating that form D is an anhydrate. The calculated density of Form D is 1.390 g/cm ³ . The unit cell of a single crystal consists of sixteen molecules.

Example 5: Polarized light microscopy (PLM).

Images of the MPS were obtained using an Axio Lab.A1 upright microscope with a ProgRes® CT3 camera at CT.

Example 6 Powder X-ray diffractometry (XRD) under ambient conditions.

- 16 044390

X-ray diffraction patterns of the PRD were read using an X-ray powder diffractometer

PANalytical Empyrean with PDP parameters according to table. 9.

Table 9

PRD tool parameters

Options Empyrean X' Pert3 X-ray wavelength Si, ka; Kαί (A): 1.540598 Ka2 (A): 1.544426 intensity ratio Ka2/Ka1: 0.50 Si, ka; Kαί (A): 1.540598 Ka2 (A): 1.544426 ratio intensity Ka2/Ka1: 0.50 X-ray tube settings 45 kV, 40 mA 45 kV, 40 mA Divergence slot Automatic Automatic Scan mode Continuous Continuous Scan range (°2TH) 3° ~ 40° 3°~40° Step size (°2TN) 0.0167 0.0263 Step scan time (s) 18 50 Test time (s) 5 min 30 s 5 min 04 s

Example 7. TGA and DSC test.

TGA data were collected using TGA model TA Q500/Q5000 from TA Instruments. DSC was performed using TA Q200/Q2000 from TA Instruments. The detailed parameters used are listed in Table. 10.

Table 10

TGA and DSC parameters

Options TGA DSK Way Linear change Linear change Sample cuvette Platinum, open Aluminum, corrugated Temperature CT - desired temperature 25 °C - desired temperature Heating rate 10 °C/min 10 °C/min Purge gas ν ₂ ν ₂

Example 8. Chipboard testing.

The chipboard was measured using SMS (Surface Measurement Systems) DVS Intrinsic. Relative humidity at 25°C was calibrated using LiCl, Mg(NO3) ₂ and KCl spreading points. Chipboard test parameters are listed in table. eleven.

Table 11

Chipboard parameters

Options Chipboard Temperature 25 C Sample weight 10-20 mg Gas and flow rate N2, 200 ml/min dm/dt 0.002%/min Min. duration stability Dm/dt 10 minutes Max, equilibrium time 180 minutes RH range from 0% RH to 95% RH OB step size 10% RH from 0-90% RH, then 5% RH from 90-95% OB

Example 9. Formulation of a dosage form of a crystalline compound of formula I.

After crystallization, the polymorphic forms of the compound of formula I can be formulated into a dosage form by dry granulation using a roller compactor and then by a tableting operation. Additional ingredients in the tablets may include microcrystalline cellulose (Avicel® PH, FMC BioPolymer), lactose (FastFlo®, Foremost Farms USA), sodium starch glycolate (EXPLOTAB®, JRS Pharma), or magnesium stearate (Hyqual®, Macron Fine Chemicals).

Example 10. Amorphous form E.

A 0.5 g sample of solid 2-methyl-2-(3-methyl-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2ylamino)-1H-pyrazol-1-yl)propanenitrile was heated until no liquid was observed, and then added to a test tube cooled to -45 °C, which led to the formation of a glassy solid.

Claims (19)

th substance. Birefringence was not observed in polarized light microscopy (PLM) (Fig. 16). PXRD analysis showed no inflection peaks but a characteristic halo indicative of an amorphous solid (Figure 17). Thermal DSC analysis showed an exothermic crystallization event with onset at 77.8°C followed by extensive endothermic melting indicating crystalline melting with onset at 122.8°C. Although the present invention has been described in some detail by illustration and example for the purpose of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the present invention. Accordingly, all suitable modifications and equivalents may be considered to fall within the scope of this invention as defined by the claims that follow. The disclosures of all patents and scientific literature cited herein are expressly incorporated herein by reference in their entirety. CLAIM 1. The crystalline compound, 2-methyl-2-(3-methyl-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-1H-pyrazol-1-yl)propanenitrile, which is polymorph form C, which exhibits an x-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at 6.4, 15.1, 21.2, 25.7 and 27.8±0.05. 2. The crystalline compound of claim 1, which is a form C polymorph that exhibits an x-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at 6.4, 8.1, 8.6, 8.8, 9.9, 10.2, 12.9, 13.8, 15.1, 15.4, 16.5, 19.8, 21.2, 22.1, 23.7, 25.7 and 27, 8±0.05. 3. A crystalline compound according to claim 1, wherein the compound is in pure form. 4. The crystalline compound according to claim 1, for which an X-ray powder diffraction pattern was obtained using CuKa1 radiation. 5. The crystalline compound of claim 1, wherein the compound is a form C polymorph having an X-ray powder diffraction pattern free of peaks at 13.6 and 14.8±0.05 degrees 2-theta. 6. The crystalline compound of claim 1, wherein the compound is a form C polymorph further containing peaks at 16.5 and 22.1 ±0.05 degrees 2-theta. 7. The crystalline compound according to claim 1, in which polymorphic form C is an anhydrate. 8. The crystalline compound according to claim 1, which is characterized by the X-ray powder diffraction pattern shown in FIG. 10. 9. The crystalline compound of claim 1, for which DSC differential scanning calorimetry shows a melting endotherm starting at about 127.8°C. 10. The crystalline compound of claim 1, exhibiting a mass increase of less than about 1% when subjected to an increase in relative humidity from about 0% to about 95% relative humidity for about 180 minutes. 11. The crystalline compound of claim 1 which is stable when exposed to temperatures up to about 40° C. and about 75% relative humidity for at least 6 months. 12. The crystalline compound, 2-methyl-2-(3-methyl-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-1H-pyrazol-1-yl)propanenitrile, which is polymorph Form D, which exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at 9.2, 14.0, 14.8, 19.7 and 20.0±0.05. 13. The crystalline compound of claim 12, which is a Form D polymorph that exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at 8.0, 8.7, 9.2, 9.8, 10.4, 12.9, 13.4, 14.0, 14.8, 16.4, 18.5, 19.7, 20.0, 20.8, 23.1, 23.3, 23, 9, 25.5 and 25.7±0.05. 14. The crystalline compound according to claim 12, wherein the compound is in pure form. 15. The crystalline compound according to claim 12, for which an X-ray powder diffraction pattern was obtained using CuKa1 radiation. 16. The crystalline compound of claim 12, exhibiting a mass increase of less than about 1% when subjected to an increase in relative humidity from about 0% to about 95% relative humidity for about 180 minutes. 17. The crystalline compound of claim 12 which is stable when exposed to temperatures up to about 40° C. and about 75% relative humidity for at least 6 months. 18. The crystalline compound of claim 12, wherein the compound is a Form D polymorph having an X-ray powder diffraction pattern free of peaks at 13.6 ± 0.05 degrees 2-theta. 19. The crystalline compound according to claim 12, in which the polymorphic form D is -