Monohydrate and crystalline forms of 6-[(3S,4S)-4-methyl-l-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[l,5-a]pyrazin-8-one

Abstract

The present disclosure relates to crystalline polymorphs of 6-[(3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[1,5-a]pyrazin-8-one. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] <Related Applications> This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 676,381, filed May 25, 2018, entitled "Monohydrate and Crystalline Forms of 6-[(3S,4S)-4-Methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[1,5-A]pyrazin-8-one," and U.S. Provisional Application No. 62 / 788,323, filed January 4, 2019, entitled "Monohydrate and Crystalline Forms of 6-[(3S,4S)-4-Methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[1,5-A]pyrazin-8-one," the contents of each of which are incorporated herein by reference in their entireties.

[0002] FIELD OF THE INVENTION The present disclosure relates to polymorphic forms of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 9 inhibitors (hereinafter referred to as PDE9 inhibitors). [Background technology]

[0003] Solids exist in either amorphous or crystalline form. Polymorphism refers to the various crystalline forms of a chemical substance. These crystalline forms have different structural and physical properties, such as XRPD spectra, IR spectra, and melting points. Certain polymorphic forms may have advantages over other forms and be more suitable for the manufacture and use of drug substances.

[0004] 6-[(3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[1,5-a]pyrazin-8-one (Compound 1) is a PDE9 inhibitor disclosed in WO 2017 / 005786 for treating various diseases, such as sickle cell disease. Improved forms of Compound 1 are sought, particularly with respect to enhanced solubility, oral bioavailability, and / or physical stability. Summary of the Invention

[0005] The present disclosure provides polymorphic forms of the PDE9 inhibitor 6-[(3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[1,5-a]pyrazin-8-one (Compound 1), herein referred to as Form MH1 and Form MH2. The present disclosure also provides methods for making the polymorphic forms, characterizing the polymorphic forms, pharmaceutical compositions containing the polymorphic forms, and methods for using the polymorphic forms and compositions.

[0006] One aspect of the disclosure provided herein includes a monohydrate crystalline form of 6-[(3S,4S)-4-methyl-l-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[l,5-a]pyrazin-8-one.

[0007] [ka] (Compound 1) In some embodiments, the monohydrate crystalline form is MHI and has an XRPD pattern comprising peaks at 2θ angles of about 9.1, 11.5, 16.2, 16.7, 18.2, 18.9, 19.8, 22.6, and 26.4° 2θ, each ±0.2° 2θ. In some embodiments, the monohydrate crystalline form is MHI and has an XRPD pattern substantially as shown in FIG. 2A. In some embodiments, the monohydrate crystalline form is MHI and has a differential scanning calorimetry (DSC) thermogram with a dehydration endotherm peak at about 40-100° C. and a melting endotherm peak at about 184.4° C. In some embodiments, the monohydrate crystalline form is MHI and has a DSC thermogram substantially in accordance with FIG. 5. In some embodiments, the monohydrate crystalline form is MHI and exhibits dehydration between ambient temperature and about 90° C. with a weight loss of about 4.4% in thermogravimetric analysis (TGA). In some embodiments, the monohydrate crystalline form is MH1 and has a TGA substantially in accordance with Figure 5. In some embodiments, the monohydrate crystalline form is MH1 and has a TGA of about 782 cm -1 , 1123cm -1 , 1562cm -1 , and 1655 cm -1in its infrared (IR) spectrum. In some embodiments, the monohydrate crystalline form is MHI and has an infrared spectrum substantially in accordance with Figure 3. In some embodiments, the monohydrate crystalline form is MH2 and has an XRPD pattern comprising peaks at 2θ angles of about 9.0, 11.6, 15.0, 16.0, 18.6, 19.1, 20.4, or 20.6° 2θ, each ±0.2° 2θ. In some embodiments, the monohydrate crystalline form is MH2 and has an XRPD pattern substantially as shown in Figure 7. In some embodiments, the monohydrate crystalline form is MH2 and has endothermic peaks in its differential scanning calorimetry (DSC) thermogram at about 59.1°C (±5°C) and about 184.7°C (±5°C). In some embodiments, the monohydrate crystalline form is MH2 and has a DSC thermogram substantially in accordance with Figure 9. In some embodiments, the monohydrate crystalline form is MH2 and exhibits dehydration at about 25° C. to about 100° C. with a weight loss of about 4.4% in a thermogravimetric analysis (TGA). In some embodiments, the monohydrate crystalline form is MH2 and has a TGA substantially in accordance with Figure 9. In some embodiments, the monohydrate crystalline form is at least 95, 96, 97, 98, or 99% purified.

[0008] Another aspect described herein includes a pharmaceutical composition comprising a therapeutically effective amount of the monohydrate crystalline form of any one of the embodiments described herein, and a pharmaceutically acceptable excipient. In some embodiments, the monohydrate crystalline form is present in an amount of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by weight. In some embodiments, the monohydrate crystalline form is present in an amount of at least about 91% by weight.

[0009] Another aspect described herein includes a pharmaceutical composition consisting essentially of the monohydrate crystalline form of any one of the embodiments described herein.

[0010] Another aspect described herein includes a pharmaceutical composition consisting essentially of monohydrate crystalline form MH1 of any one of the embodiments described herein.

[0011] Another aspect described herein includes a pharmaceutical composition consisting essentially of monohydrate crystalline form MH2 of any one of the embodiments described herein. In some embodiments, the composition is in the form of a tablet or capsule.

[0012] Another aspect described herein includes a process for preparing the monohydrate crystalline form of Compound 1, comprising precipitating the monohydrate crystalline form from a solution comprising Compound 1 and a solvent selected from the group consisting of n-propyl acetate, isopropyl acetate, anisole, methyl isobutyl ketone, cumene, isopropanol, 2-methyltetrahydrofuran, and combinations thereof. In some embodiments, the solvent is n-propyl acetate. In some embodiments, the process further comprises cooling the solution.

[0013] Another aspect described herein includes the monohydrate crystalline form of Compound 1 prepared by the process of any one of the embodiments described herein.

[0014] Another aspect described herein includes a method of inhibiting PDE9 activity in a patient, comprising administering to the patient the monohydrate crystalline form of any one of the embodiments described herein.

[0015] Another aspect described herein includes a method of treating sickle cell disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the monohydrate crystalline form of any one of the embodiments described herein.

[0016] Another aspect described herein is a process for preparing monohydrate Form 1 (MH1) of Compound 1, comprising: (i) dissolving Compound 1 in a first solvent to obtain a solution, (ii) adding a second solvent to obtain a mixture, and (iii) filtering the mixture to obtain a solid, wherein the first and second solvents are each independently selected from isopropyl acetate, ethanol, tetrahydrofuran, water, dichloromethane, acetonitrile, anisole, methyl isobutyl ketone, nitromethane, 1,2-dimethoxyethane, methyl ethyl ketone, n-heptane, 1,4-dioxane, n-propyl acetate, 2-propanol, acetone, cumene, N,N-dimethylformamide, dimethyl sulfoxide, and combinations thereof. In some embodiments, the method further comprises heating the solution to a temperature above room temperature at about 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80° C.

[0017] In some embodiments, the first solvent comprises 2-propanol. In some embodiments, the second solvent comprises n-heptane. In some embodiments, the first solvent in step (i) comprises water and 2-propanol. In some embodiments, the water content of the solution obtained in step (i) is about 0.5%, 1%, or 1.5%. In some embodiments, the water content of the solution obtained in step (i) is about 1%. In some embodiments, the solid obtained in step (ii) is optionally washed one or more times with n-heptane. In some embodiments, the process further comprises drying the solid after step (iii). In some embodiments, the solid is dried in a humid state.

[0018] Another embodiment described herein is a process for preparing monohydrate Form 2 (MH2) of Compound 1, comprising: a) treating Compound 1 or MH1 with a first solvent system to obtain a suspension; b) filtering the suspension to obtain a solid; c) washing the solid with heptane; and d) removing the solvent by drying to obtain MH2, wherein the solvent is a mixture of water and ethyl acetate (EtOAc) or methyl acetate (MeOAc), and is selected from 2% (v / v) EtOAc / water, 2.7% (v / v) EtOAc / water, and 7.5% (v / v) MeOAc / water. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 is a ball-and-stick diagram of the MH1 molecular structure. [Figure 2A] FIG. 2A is an experimental XRPD pattern of MH1 crystals at room temperature. [Figure 2B] Figure 2B is an overlay of the experimental XRPD pattern of MH1 at room temperature (top line) and the calculated XRPD patterns of MH1 at 293 K (middle line) and 100 K (bottom line). [Figure 3] FIG. 3 is the FTIR spectrum of MH1. [Figure 4] Figure 4 shows the Raman spectrum of MH1. [Figure 5] FIG. 5 is a TGA and DSC analysis of MH1. [Figure 6] Figure 6 is a ball-and-stick diagram of the MH2 molecular structure. [Figure 7] Figure 7 is an overlay of the experimental XRPD pattern of MH2 at room temperature and the calculated XRPD pattern of MH2 at 100K. [Figure 8] Figure 8 is an XRPD overlay of the scale-up of MH2. [Figure 9] FIG. 9 is a TGA and DSC analysis of MH2. DETAILED DESCRIPTION OF THE INVENTION

[0020] I. Polymorphs of Compound 1 The racemic form of Compound 1 and the anhydrous form of Compound 1 are described in WO2013 / 053690 and WO2017 / 005786. The anhydrous form of Compound 1 has the following structure:

[0021] [ka]

[0022] Two distinct monohydrate polymorphic forms of Compound 1 have been discovered: Monohydrate Form 1 (MH1) and Monohydrate Form 2 (MH2). The two monohydrate forms and the anhydrous form (AH) differ in their crystalline structure as determined by single-crystal X-ray powder diffraction (XRPD). The major peaks of MH1 and MH2 are identified below 30°2θ, and their relative intensities are listed in Table 1. As will be apparent to those skilled in the art, the relative intensities of the peaks in Table 1 may vary due to various factors, such as the purity of the material being analyzed, the orientation effect of the crystal in the X-ray beam, and the crystallinity of the sample. While peak positions may shift due to variations in sample height, the peak positions will essentially remain as defined in Table 1. Those skilled in the art will also understand that measurements using different wavelengths will result in different shifts according to the Bragg equation (nλ=2d sinθ). Such additional XRPD patterns generated using alternative wavelengths are alternative representations of the XRPD pattern of a crystalline material.

[0023] [Table 1]

[0024] i.Crystal form MH1 Form MH1 may be characterized by any of its peaks in Table 1. For example, MH1 may be characterized by any of the following peaks, specifically 9.1, 11.5, 16.2, 16.7, 18.2, 18.9, and 19.8 degrees 2-theta, each ±0.2 degrees 2-theta.

[0025] In some embodiments, the monohydrate crystalline form of Compound 1 is MH1 and has an X-ray powder diffraction (XRPD) pattern including any one or more peaks at 9.1, 11.5, 16.2, 16.7, 18.2, 18.9, and 19.8 degrees 2θ, each ±0.2 degrees 2θ.

[0026] In some embodiments, the monohydrate crystalline form of Compound 1 is MH1 and has an X-ray powder diffraction (XRPD) pattern including peaks at 9.1, 11.5, 16.2, 16.7, 18.2, 18.9, and 19.8 degrees 2θ, each ±0.2 degrees 2θ.

[0027] In some embodiments, MHCl analyzed by infrared (IR) spectroscopy has a peak at about 782 cm, as shown in FIG. -1 , 1123cm -1 , 1562cm -1 , and 1655 cm -1 (±0.5cm -1 ) characteristic absorption.

[0028] In some embodiments, MH1 analyzed by differential scanning calorimetry (DSC) thermogram exhibits a dehydration endothermic peak at about 40-100°C (±10°C) and a melting endothermic peak at about 184.4°C (±5°C), as shown in Figure 5.

[0029] In some embodiments, MH1 analyzed by thermogravimetric analysis (TGA) exhibits dehydration from ambient to about 90° C. with a weight loss of about 3.8%, as shown in FIG.

[0030] ii. Crystalline form MH2 Form MH2 may be characterized by any of its peaks in Table 1. For example, MH1 may be characterized by any of the following peaks, specifically 9.0, 11.6, 15.0, 16.0, 18.6, 19.1, 20.4, and 20.6 degrees 2-theta, each ±0.2 degrees 2-theta.

[0031] In some embodiments, the monohydrate crystalline form of Compound 1 is MH2 and has an X-ray powder diffraction (XRPD) pattern including any one or more peaks at 9.0, 11.6, 15.0, 16.0, 18.6, 19.1, 20.4, and 20.6 degrees 2θ, each ±0.2 degrees 2θ.

[0032] In some embodiments, the monohydrate crystalline form of Compound 1 is MH2 and has an X-ray powder diffraction (XRPD) pattern including peaks at 9.0, 11.6, 15.0, 16.0, 18.6, 19.1, 20.4, and 20.6 degrees 2θ, each ±0.2 degrees 2θ.

[0033] In some embodiments, MH2 analyzed by differential scanning calorimetry (DSC) thermogram exhibits endothermic peaks at about 59.1° C. (±5° C.) and about 184.7° C. (±5° C.), as shown in FIG.

[0034] In some embodiments, MH2 analyzed by thermogravimetric analysis (TGA) exhibits dehydration at about 25° C. to about 100° C. with a weight loss of about 4.4%, as shown in FIG.

[0035] In some embodiments, a crystalline form of Compound 1 (e.g., MH1 or MH2) is substantially pure. In some embodiments, crystalline form MH1 or MH2 is at least 80%, 85%, 90%, or 95% pure. In some embodiments, crystalline form MH1 or MH2 is at least 95%, 96%, 97%, 98%, or 99% pure. In some embodiments, crystalline form MH1 or MH2 contains less than 10%, 5%, 3%, or 1% impurities.

[0036] II. Pharmaceutical Compositions The present disclosure further provides pharmaceutical compositions comprising a therapeutically effective amount of any of the polymorphic forms of Compound 1 (e.g., monohydrate crystalline form MH1 or MH2, etc.) and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is for oral administration. In some embodiments, the pharmaceutical composition is in the form of a tablet or capsule.

[0037] Polymorphic forms of Compound 1 (such as, for example, monohydrate crystalline forms MH1 or MH2) can be administered alone or in combination with pharmaceutically acceptable carriers, diluents, or excipients, either in single or multiple doses. Pharmaceutical compositions according to the present disclosure can be formulated with pharmaceutically acceptable carriers or diluents, as well as other known adjuvants and excipients, according to conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy, 22nd Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 2013.

[0038] Pharmaceutical compositions for oral administration include capsules, tablets, dragees, pills, lozenges, powders, and granules.If necessary, pharmaceutical compositions may be prepared with coatings, such as enteric coatings, or may be formulated to provide controlled release of active ingredients, such as sustained or prolonged release, for example, according to methods well known in the art.Liquid dosage forms for oral administration include solutions, emulsions, suspensions, syrups, and elixirs.

[0039] Pharmaceutical compositions for parenteral administration include sterile aqueous and non-aqueous injection solutions, dispersions, suspensions, or emulsions, as well as sterile powders to be reconstituted into sterile injection solutions or dispersions before use. Other suitable administration forms include, but are not limited to, suppositories, sprays, ointments, creams, gels, inhalants, skin patches, and implants.

[0040] Typical oral dosages range from about 0.001 to about 100 mg / kg of body weight per day. Typical oral dosages range from about 0.01 to about 50 mg / kg of body weight per day. Typical oral dosages range from about 0.05 to about 10 mg / kg of body weight per day. Oral dosages are usually administered in one or more doses, typically 1-3 doses per day. The exact dosage will depend on the frequency and method of administration, the sex, weight, and relative health of the subject being treated, the nature and severity of the disease being treated, and any co-morbidities being treated, as well as other factors that will be apparent to those skilled in the art.

[0041] The formulations may be presented in unit dosage form by methods known to those skilled in the art. By way of illustration, a typical unit dosage form for oral administration may contain from about 0.01 to about 1000 mg, from about 0.05 to about 500 mg, or from about 0.5 mg to about 200 mg.

[0042] For parenteral routes such as intravenous, intrathecal, intramuscular, and the like, typical dosages are about half the dosage used for oral administration.

[0043] The present disclosure also provides a process for making a pharmaceutical composition, comprising mixing a therapeutically effective amount of any of the polymorphic forms of Compound 1 (e.g., monohydrate crystalline form MH1 or MH2, etc.) and at least one pharmaceutically acceptable carrier or diluent.

[0044] Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions, and various organic solvents. Examples of solid carriers include lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, and lower alkyl ethers of cellulose. Examples of liquid carriers include, but are not limited to, syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene, and water. Similarly, the carrier or diluent may include any sustained-release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax. The pharmaceutical composition formed by mixing the compound of the present disclosure with a pharmaceutically acceptable carrier can then be easily administered in a variety of dosage forms suitable for the disclosed administration route. The formulations can be conveniently provided in unit dosage form by methods known in the pharmaceutical arts.

[0045] Formulations of the present disclosure suitable for oral administration may be provided as discrete units, such as capsules or tablets, each containing a predetermined amount of the active ingredient and, optionally, suitable excipients. Further, orally administrable formulations may also be in the form of a powder or granules, a solution or suspension in an aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil liquid emulsion.

[0046] When a solid carrier is used for oral administration, the preparation may be in the form of tablets or pellets placed in a hard gelatin capsule in powder form, or the preparation may be in the form of a troche or lozenge. The amount of solid carrier may vary widely, but will range from about 25 mg to about 1 g per dosage unit. When a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule, or sterile injectable liquid such as an aqueous or non-aqueous suspension or solution.

[0047] The pharmaceutical compositions of the present disclosure can be prepared by conventional methods in the art. For example, tablets can be prepared by mixing the active ingredient with a conventional adjuvant and / or diluent, and then compressing the mixture in a conventional tablet machine to prepare tablets. Examples of adjuvants or diluents include corn starch, potato starch, talc, magnesium stearate, gelatin, lactose, gums, etc. Any other adjuvants or additives commonly used for purposes such as coloring agents, flavoring agents, preservatives, etc. can also be used, provided that they are compatible with the active ingredient.

[0048] In some embodiments, the pharmaceutical composition comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight of a polymorphic form of Compound 1 (such as, for example, monohydrate crystalline form MH1 or MH2). In some embodiments, the pharmaceutical composition comprises at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by weight of a polymorphic form of Compound 1 (such as, for example, monohydrate crystalline form MH1 or MH2).

[0049] In some embodiments, pharmaceutical compositions include compounds of the present disclosure in combination with one or more additional active agents, such as hydroxyurea ("HU").

[0050] III. Methods for Making Polymorphic Forms of Compound 1 Crystals can be precipitated from a solution containing a compound and a solvent. For example, crystals can be obtained from a clear solution at low temperatures. Because the solubility of most materials decreases with temperature, cooling can be used to induce supersaturation. Crystals can also be obtained by rapid evaporation.

[0051] In some embodiments, the MH1 crystals are produced in a crystallization process. In some embodiments, the MH2 crystals are produced in a crystallization process.

[0052] Crystals of Compound 1 can be prepared by dissolving Compound 1 in a solvent to obtain a saturated solution of Compound 1, and then cooling the saturated solution to precipitate the crystals.

[0053] Crystals of monohydrate form MH1 or MH2 can be prepared by precipitating crystals from a solution containing compound 1 and a solvent. The solvent can be selected from the group consisting of ethanol, tetrahydrofuran, water, dichloromethane, acetonitrile, nitromethane, 1,2-dimethoxyethane, methyl ethyl ketone, 1,4-dioxane, 2-propanol, acetone, cumene, N,N-dimethylformamide, dimethyl sulfoxide, n-propyl acetate, isopropyl acetate, anisole, methyl isobutyl ketone, 2-methyltetrahydrofuran, and combinations thereof. For example, compound 1 can be suspended in a solvent, such as n-propyl acetate, to obtain a suspension, which can be filtered to obtain a solution (e.g., mother liquor), and then the solution (e.g., mother liquor) can be cooled to precipitate crystals.

[0054] In some embodiments, preparing an MH1 or MH2 crystal comprises: (i) suspending Compound 1 in a first solvent at room temperature (RT) to form a suspension; (ii) heating the suspension obtained in step (i) to a temperature above room temperature (e.g., about 40°C to about 60°C); (iii) adding a second solvent to the suspension obtained in step (ii) and heating the mixture to a temperature above room temperature (e.g., about 40°C to about 60°C) to form a mixture; (iv) optionally filtering the mixture obtained in step (iii) to obtain a solution (e.g., a mother liquor); and (v) cooling the mixture obtained in step (iii) or the solution obtained in step (iv) to room temperature or below (e.g., about 4°C) to precipitate MH1 or MH2 crystals; wherein the first and second solvents are each independently selected from the group consisting of n-heptane, ethanol, tetrahydrofuran, water, dichloromethane, acetonitrile, nitromethane, 1,2-dimethoxyethane, methyl ethyl ketone, 1,4-dioxane, 2-propanol, acetone, cumene, N,N-dimethylformamide, dimethyl sulfoxide, n-propyl acetate, isopropyl acetate, anisole, methyl isobutyl ketone, 2-methyltetrahydrofuran, and combinations thereof.

[0055] In some embodiments, the first and second solvents are each independently selected from methyl ethyl ketone, 1,4-dioxane, 2-propanol, acetone, n-propyl acetate, isopropyl acetate, anisole, methyl isobutyl ketone, cumene, n-heptane, 2-methyltetrahydrofuran, and combinations thereof. In some embodiments, the first and second solvents are each independently selected from n-propyl acetate or n-heptane.

[0056] In some embodiments, the first and second solvents are the same. In some embodiments, the first and second solvents are different.

[0057] In some embodiments of MH1, the first solvent is selected from n-propyl acetate and the second solvent is selected from n-heptane.

[0058] In some embodiments, the volume of the first solvent used in step (i) is less than the volume of the second solvent used in step (iii). For example, the ratio of the volume of the first solvent used in step (i) to the volume of the second solvent used in step (iii) is approximately 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In one example, the ratio of the volume of the first solvent used in step (i) to the volume of the second solvent used in step (iii) is approximately 1:2.

[0059] In some embodiments, the temperature above room temperature in steps (ii) and (iii) is about 30° C. to about 100° C. In some embodiments, the temperature above room temperature is about 40° C. to about 60° C. In some embodiments, the temperature above room temperature is about 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80° C.

[0060] In some embodiments, the MH1 crystal is: (i) dissolving Compound 1 in a first solvent to obtain a solution; (ii) adding a second solvent to obtain a mixture; and (iii) filtering the mixture to obtain a crystalline solid MH1; It can be prepared by a process comprising:

[0061] In some embodiments, the first solvent is selected from methyl ethyl ketone, 2-propanol, cumene, n-propyl acetate, isopropyl acetate, anisole, methyl isobutyl ketone, n-heptane, 2-methyltetrahydrofuran, and combinations thereof. In some embodiments, the second solvent is selected from methyl ethyl ketone, 2-propanol, n-propyl acetate, isopropyl acetate, anisole, methyl isobutyl ketone, cumene, n-heptane, 2-methyltetrahydrofuran, and combinations thereof. In some embodiments, the first solvent is 2-propanol. In some embodiments, the second solvent is n-heptane.

[0062] In some embodiments, the first solvent in step (i) further comprises water. In some embodiments, the first solvent in step (i) comprises 2-propanol and water. In some embodiments, the water content of the solution obtained in step (i) is about 1%, 2%, or 3%. In some embodiments, the water content of the solution obtained in step (i) is about 1%. In some embodiments, the ratio (weight / weight) of water to 2-propanol is about 1:70.

[0063] In some embodiments, the process of step (i) is protected by nitrogen gas.

[0064] In some embodiments, step (i) is carried out at a temperature of about 25° C. to about 40° C. In some embodiments, step (i) is carried out at a temperature of about 27° C. to about 35° C.

[0065] In some embodiments, seed crystals are added after step (i) to induce crystallization of MH1. In some embodiments, the temperature of the mixture is adjusted to about 20°C to about 30°C before the seed crystals are added. In some embodiments, the temperature of the mixture is adjusted to about 22°C to about 28°C before the seed crystals are added. The weight of the seed crystals is about 0.05% to about 2% of the weight of Compound 1 added in step (i). In some embodiments, the weight of the seed crystals is about 0.05, 1%, or 2% of the weight of Compound 1 in step (i).

[0066] In some embodiments, steps (ii) and (iii) are each independently carried out at a temperature of about 20° C. to about 30° C., for example, about 22° C. to about 28° C. In some embodiments, steps (ii) and (iii) are each independently carried out at a temperature of about 22° C. to about 28° C.

[0067] In some embodiments, the solid obtained in step (iii) is optionally washed one or more times with a solvent selected from methyl ethyl ketone, 1,4-dioxane, 2-propanol, acetone, cumene, n-propyl acetate, isopropyl acetate, anisole, methyl isobutyl ketone, n-heptane, and 2-methyltetrahydrofuran, and combinations thereof. In some embodiments, the solid obtained in step (iii) is optionally washed with n-heptane. In some embodiments, the solid obtained in step (iii) or after washing with n-heptane is pressed until dry. In some embodiments, the resulting dried solid is optionally then dried under a stream of nitrogen gas. In some embodiments, the solid is further dried in a humidified environment, optionally under a stream of nitrogen gas. Various salt solutions, such as saturated solutions of sodium chloride, can provide humidified environments with various relative humidity (RH). Commercially available gas supply equipment with adjustable RH and temperature can also be used.

[0068] In some embodiments, the anhydrous (AH) form of Compound 1 can be converted to MH1 when exposed to normal laboratory air (which contains some moisture). In some embodiments, the anhydrous form of Compound 1 is left under ambient conditions (e.g., 25°C) for at least about 12 hours, about 24 hours, about 36 hours, or about 48 hours.

[0069] In some embodiments, the anhydrous (AH) or MH2 is made by a crystallization process. Drying and humid drying of the anhydrous (AH) or MH2 then produces MH1 crystals.

[0070] 1. A process in which monohydrate crystalline form MH2 is prepared, comprising: (i) treating dried Compound 1 or MH1 with a first solvent system to obtain a suspension; (ii) filtering the suspension to obtain a solid; (iii) washing the solid one or more times with a second solvent; and (iv) air-drying the solid to obtain crystalline MH2; wherein the first solvent system is selected from water, ethyl acetate (EtOAc), and methyl acetate (MeOAc), and combinations thereof, and the second solvent is selected from acetone, n-heptane, and 2-methyltetrahydrofuran.

[0071] In some embodiments, the first solvent system is a mixture of water and ethyl acetate (EtOAc) or methyl acetate (MeOAc). In some embodiments, the first solvent system is selected from 2% (v / v) EtOAc / water, 2.7% (v / v) EtOAc / water, or 7.5% (v / v) MeOAc / water. In some embodiments, the suspension is maintained at about 5° C. or about 25° C. for about 4 days.

[0072] In some embodiments, the second solvent is heptane.

[0073] In some embodiments, compound 1 or MH1 in step (i) is dried in a vacuum oven. In some embodiments, compound 1 or MH1 in step (i) is air-dried.

[0074] In one embodiment, crystalline MH2 is prepared by treating compound 1 or MH1 with a first solvent system containing 7.5% (v / v) MeOAc / water at about 5°C, filtering the solid, and then air-drying the solid.

[0075] In one embodiment, the crystalline MH2 is (i) treating Compound 1 or MH1 with a first solvent system to obtain a suspension; (ii) filtering the suspension to obtain a solid; (iii) washing the solid with heptane; and (iv) by air drying to remove the solvent to obtain MH2; Here, the solvent is a mixture of water and ethyl acetate (EtOAc) or methyl acetate (MeOAc), selected from 2% (v / v) EtOAc / water, 2.7% (v / v) EtOAc / water, and 7.5% (v / v) MeOAc / water.

[0076] IV. Methods of Using Polymorphic Forms of Compound 1 PDE9 is specifically expressed in human hematopoietic cells, including neutrophils, reticulocytes, erythroblasts, and erythroleukemia cells. Furthermore, sickle cell disease (SCD) patients exhibit a marked and significant increase in PDE9 expression in reticulocytes and neutrophils compared with healthy individuals (Almeida et al., Br J Haematol. 2008 Sep;l42(5):836-44). Evidence further supports a link between PDE9 and cell adhesion, as pharmacological PDE9 inhibition increases the adhesive properties of SCD neutrophils (Miguel et al., Inflamm Res. 2011 Jul;60(7):633-42). The mechanism for the reduction in cell adhesion by PDE9 inhibition has been shown to be mediated by increased cGMP and decreased expression of endothelial adhesion molecules. Importantly, in animal models of SCD, PDE9 inhibitor-mediated reduction in cell adhesion has the functional effect of increasing cell viability. In addition to demonstrating reduced cell adhesion comparable to that of hydroxyurea (HU), PDE9 inhibition leads to increased production of fetal non-sickle hemoglobin (HbF), which reduces the cellular concentration of abnormal hemoglobin (HbS) within red blood cells (RBCs), resulting in less polymerization of abnormal hemoglobin and its associated sequelae. The importance of increasing HbF in the treatment of SCD is evidenced by the results of large-scale studies such as the Sickle Cell Disease Collaborative Study, as well as studies in various patient cohorts outside the United States showing that HbF is one of the most important modifiers of this disease (Alsultan et al., Am J Hematol., 88(6):531-2 (2013)), and data showing that modifiers of HbF improve other hematological parameters (Akinsheye, Blood, 118(1):19-27 (2011)). Finally, Almeida et al. demonstrated that treatment with HU in combination with PDE9 inhibition in a mouse model of SCD leads to an additional beneficial amplification of HU's cGMP-elevating effect (Almeida et al., Blood. 2012 Oct 4;l20(l4):2879-88). In conclusion, PDE9 inhibition can not only regulate the expression of fetal hemoglobin production but also reduce cell adhesion, both mechanisms being key to the treatment of SCD.

[0077] One aspect of the disclosure provides methods of using any of the polymorphic forms of Compound 1 (such as the monohydrate crystalline form MH1 or MH2) and pharmaceutical compositions comprising any of the polymorphic forms of Compound 1 (such as the monohydrate crystalline form MH1 or MH2).

[0078] Polymorphic forms of Compound 1 (such as the monohydrate crystalline forms MH1 or MH2) may be used to treat sickle cell disease or any disease and / or condition associated with sickle cell disease, such as anemia, sickle hemoglobin C disease (SC), beta thalassemia (beta plus thalassemia and beta zero thalassemia), vaso-occlusive disease, pain crises (sickle cell disease), splenic hemocytosis, acute chest syndrome, aplastic crisis, hemolytic crisis, prolonged pain, bacterial infection, and stroke.

[0079] In one embodiment, a polymorphic form of Compound 1 (such as the monohydrate crystalline form MH1 or MH2) is used to treat beta-thalassemia in a subject and / or to increase hemoglobin levels in a subject.

[0080] In another embodiment, a polymorphic form of Compound 1 (such as the monohydrate crystal form MH1 or MH2) is used to increase cGMP levels in cells or plasma of a subject with sickle cell disease. The cells can be, but are not limited to, red blood cells and / or white blood cells. The cGMP levels can be increased by at least 50%, 100%, or 150%. In some embodiments, the cGMP levels are increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, or 25-fold.

[0081] In another embodiment, a polymorphic form of Compound 1 (such as the monohydrate crystal form MH1 or MH2) is used to increase the number of fetal hemoglobin (HbF)-positive red blood cells in a subject with sickle cell disease. The number of HbF-positive red blood cells is increased by at least 50%, 100%, or 150%. In some embodiments, the number of HbF-positive red blood cells is increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, or 25-fold.

[0082] In another embodiment, a polymorphic form of Compound 1 (such as the monohydrate crystalline form MH1 or MH2) is used to reduce the percentage of sickle red blood cells (% sickle RBCs), the rate of congestion (% congestion), total bilirubin, or total leucocyte count in a subject with sickle cell disease. The % sickle RBCs, % congestion, total bilirubin, total leucocyte count, or spleen weight is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, or 70%.

[0083] cGMP levels can be measured using any suitable method in the art, such as an enzyme immunoassay.

[0084] As used herein, HbF-positive cells refer to red blood cells carrying HbF. HbF-positive cells can be measured from a blood sample using any suitable method in the art, such as electrophoresis and / or colorimetry.

[0085] As used herein, sickle cells, or sickle-shaped red blood cells, refer to crescent-shaped or sickle-shaped red blood cells. % sickle cells can be measured from a blood sample using any suitable method in the art.

[0086] As used herein, congestion or microvascular congestion is a severe slowing or complete cessation of blood or lymph flow through blood vessels. % congestion is the number of static (no flow) venules divided by 100 times the number of flowing venules. % congestion can be measured using any suitable method in the art.

[0087] As used herein, total bilirubin refers to both unconjugated and conjugated bilirubin. Total bilirubin levels can be measured from a blood sample using any suitable method in the art.

[0088] As used herein, a total leucocyte count or white blood cell count is a blood test that measures the number of white blood cells in the body. It can be measured from a blood sample using any suitable method in the art.

[0089] Another aspect of the present disclosure provides a method of using a polymorphic form of Compound 1 (such as the monohydrate crystalline form MH1 or MH2) in combination with at least one other active agent. They can be administered simultaneously or sequentially. They can be present as a mixture for simultaneous administration, or in separate containers for sequential administration.

[0090] As used herein, the term "co-administration" is not particularly limited and means that a polymorphic form of Compound 1 (such as the monohydrate crystalline form MH1 or MH2) and at least one other active agent are administered substantially simultaneously, for example, as a mixture or immediately following one another.

[0091] As used herein, the term "sequential administration" is not particularly limited and means that a polymorphic form of Compound 1 (such as the monohydrate crystalline form MH1 or MH2) and at least one other active agent are not administered simultaneously, but rather one after the other or in groups, with a specific time interval between administrations. The time interval may be the same or different between each administration of a polymorphic form of Compound 1 (such as the monohydrate crystalline form MH1 or MH2) and at least one other active agent, and may be selected from the ranges of, for example, 2 minutes to 96 hours, 1 to 7 days, or 1, 2, or 3 weeks. Generally, the time interval between administrations may range from minutes to several hours, such as 2 minutes to 72 hours, 30 minutes to 24 hours, or 1 to 12 hours. Further examples include time intervals in the ranges of 24 to 96 hours, 12 to 36 hours, 8 to 24 hours, and 6 to 12 hours.

[0092] The molar ratio of the polymorphic form of Compound 1 (such as monohydrate crystal form MH1 or MH2) and at least one other active agent is not particularly limited.For example, when a polymorphic form of Compound 1 (such as monohydrate crystal form MH1 or MH2) and one other active agent are combined in a composition, their molar ratio can be in the range of 1:500 to 500:1, or 1:100-100:1, or 1:50-50:1, or 1:20-20:1, or 1:5-5:1, or 1:1.When a polymorphic form of Compound 1 (such as monohydrate crystal form MH1 or MH2) and two or more other active agents are combined in a composition, the same molar ratio applies. A polymorphic form of Compound 1 (such as the monohydrate crystalline form MH1 or MH2) may comprise a given molar weight percentage of the composition, such as about 1%-10%, or about 10%-about 20%, or about 20%-about 30%, or about 30%-40%, or about 40%-50%, or about 50%-60%, or about 60%-70%, or about 70%-80%, or about 80%-90%, or about 90%-99%.

[0093] The other active agent may be a different PDE9 inhibitor of the present disclosure or HU. The other active agent may also be an antibiotic such as penicillin, a nonsteroidal anti-inflammatory drug (NSAIDS) such as diclofenac or naproxen, an analgesic such as an opioid, or folic acid.

[0094] Yet another aspect of the present disclosure provides methods of using a polymorphic form of Compound 1 (such as monohydrate crystalline form MH1 or MH2) in combination with at least one other treatment, such as, but not limited to, blood transfusion, bone marrow transplant, or gene therapy.

[0095] V. Kits and Devices The present disclosure provides various kits and devices for conveniently and / or effectively practicing the methods of the present disclosure. Typically, the kits will contain a sufficient quantity and / or number of components to allow a user to perform multiple treatments of subjects and / or conduct multiple experiments.

[0096] In one embodiment, the disclosure provides a kit for treating sickle cell disease comprising a polymorphic form of Compound 1 (such as the monohydrate crystalline form MH1 or MH2) or a combination of polymorphic forms of Compound 1 (such as the monohydrate crystalline forms MH1 and MH2), optionally in combination with other active agents such as HU, antibiotics such as penicillin, nonsteroidal anti-inflammatory drugs (NSAIDS) such as diclofenac or naproxen, analgesics such as opioids, or folic acid.

[0097] The kit may further comprise packaging and instructions and / or delivery agent for forming a pharmaceutical composition.The delivery agent may comprise saline, buffer solution, or any of the delivery agents disclosed herein.The amount of each component may be varied to allow for consistent and reproducible formulation of high-concentration saline or simple buffer solution.Components may also be varied to increase the stability of the PDE9 inhibitor compound in buffer solution over a period of time and / or under various conditions.

[0098] The present disclosure provides devices that can incorporate polymorphic forms of Compound 1, such as the monohydrate crystalline forms MH1 or MH2. These devices encompass stable formulations that can be readily delivered to subjects in need of the formulation, such as human patients with sickle cell disease or beta-thalassemia.

[0099] Non-limiting examples of the device include pumps, catheters, needles, transdermal patches, pressurized olfactory delivery devices, iontophoresis devices, and multi-layer microfluidic devices.The device can be used to deliver polymorphic forms of Compound 1 (such as monohydrate crystalline forms MH1 or MH2) according to single, multiple, or divided dosage regimens.The device can be used to deliver polymorphic forms of Compound 1 (such as monohydrate crystalline forms MH1 or MH2) throughout biological tissue, intradermally, subcutaneously, or intramuscularly. More examples of devices suitable for delivering polymorphic forms of compounds include medical devices for intravesical drug delivery as disclosed in International Publication WO2014036555, glass bottles made of type I glass as disclosed in U.S. Publication No. 20080108697, drug-eluting devices comprising a film made of a degradable polymer and an active agent as disclosed in U.S. Publication No. 20140308336, infusion devices with injection micropumps or containers containing pharmaceutically stable preparations of active agents as disclosed in U.S. Patent No. 5,716,988, reservoirs and These include, but are not limited to, implantable devices comprising a channeling member in fluid communication with a reservoir, hollow fiber-based biocompatible drug delivery devices with one or more layers, such as those disclosed in U.S. Publication No. 20090220612, implantable devices for drug delivery comprising an elongated, flexible device having a housing defining a reservoir containing a drug in solid or semi-solid form, such as those disclosed in International Publication WO2013170069, and bioabsorbable implantable devices as disclosed in U.S. Patent No. 7,326,421, the contents of each of which are incorporated herein by reference in their entirety.

[0100] VI.Definition As used herein, the articles "a" and "an" should be understood to mean "at least one," unless clearly indicated to the contrary.

[0101] The phrase "and / or," as used herein, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements, whether related or unrelated to the elements specifically identified, may optionally be present, unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and / or B," when used in combination with open-ended language such as "comprising," can, in one embodiment, refer to A without B (optionally including elements other than B); in another embodiment, refer to B without A (optionally including elements other than A); and in yet another embodiment, refer to both A and B (optionally including other elements).

[0102] As used herein, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as being inclusive, i.e., including at least one of the number or list of elements, but more than one of the number or list of elements, and optionally, also including additional, unlisted items. Only when used in terms clearly indicated to the contrary, such as "only one of" or "exactly one," or in the claims, "consisting of" refers to the inclusion of exactly one element of the number or list of elements.

[0103] As used herein generally, the term "or" should be construed to indicate exclusive alternatives (i.e., "one or the other" but not both) only when preceded by terms of exclusivity such as "either," "one," "only one," or "exactly one." When used in the claims, "consisting essentially of" should have its ordinary meaning as used in the field of patent law.

[0104] As used herein, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the specifically identified elements, may optionally be present.

[0105] Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one, optionally including more than one A, and no B (and optionally including elements other than B); in another embodiment to at least one, optionally including more than one B, and no A (and optionally including elements other than A); in yet another embodiment to at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); etc.

[0106] As used herein, all transitional phrases such as "including," "comprising," "hold," "having," "containing," "including," "having," and the like, should be understood to be open-ended, i.e., to mean including but not limited to.

[0107] As set forth in the United States Patent Office Manual of Patent Examining Procedure, only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

[0108] As used herein, "subject" or "patient" refers to any mammal (e.g., a human), such as a mammal that may be susceptible to a disease or disorder, e.g., tumor formation or cancer. Examples include humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents such as mice, rats, hamsters, or guinea pigs. In various embodiments, a subject refers to a subject that has been or is the subject of treatment, observation, or experimentation. For example, a subject may be a subject that has been diagnosed with or is otherwise known to have cancer, or a subject that is selected for treatment, observation, or experimentation based on the subject's known cancer.

[0109] As used herein, "process" and "method" can be used interchangeably.

[0110] As used herein, "treatment" or "treating" refers to the amelioration of a disease or disorder, or at least one sign or symptom thereof. "Treatment" or "treating" can refer to, for example, a reduction in the progression of a disease or disorder as determined by stabilization of at least one sign or symptom, or a reduction in the rate of progression as determined by a reduction in the rate of progression of at least one sign or symptom. In another embodiment, "treatment" or "treating" refers to delaying the onset of a disease or disorder.

[0111] As used herein, "prevention" or "preventing" refers to a reduction in the risk of acquiring or having signs or symptoms of a given disease or disorder, i.e., prophylactic treatment.

[0112] As used herein, the phrase "therapeutically effective amount" refers to an amount of a compound, material, or composition containing a compound of the present teachings that is effective to produce a desired therapeutic effect. Thus, a therapeutically effective amount treats or prevents a disease or disorder, for example, improves at least one sign or symptom of the disorder. In various embodiments, the disease or disorder is cancer.

[0113] A dash (“-”) that is not between two letters or symbols is used to indicate the point of attachment of a substituent. For example, -CONH2 is attached through a carbon atom (C).

[0114] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not. For example, "optionally substituted aryl" encompasses both "aryl" and "substituted aryl" as defined herein. With respect to any group that contains one or more substituents, those of skill in the art understand that such group is not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically impractical, and / or inherently unstable.

[0115] As used herein, the term "substantially" refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object that is "substantially the same" or "substantially identical" to a second object means that the object is either completely identical or nearly completely identical to the second object. The precise degree of acceptable deviation from absolute perfection may depend on the specific circumstances. However, generally speaking, near perfection results in having the same overall result as if absolute and complete perfection were achieved.

[0116] The use of "substantially" is equally applicable when used in a negative sense to refer to the complete or nearly complete absence of an action, characteristic, property, state, structure, item, or result. For example, a composition that is "substantially free" of an impurity is completely devoid of the impurity or nearly completely devoid of the impurity, so that the effect is the same as if it were completely devoid of the impurity. In other words, a composition that is "substantially free" of a component or element may still actually contain such item as long as there is no measurable effect of it.

[0117] Unless otherwise indicated, all numerical values ​​expressing quantities of ingredients, reaction conditions, and other properties or parameters used in the specification and claims should be understood to be modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the following specification and appended claims should be understood to be approximations. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, numerical parameters should be read in light of the number of reported significant digits and by applying ordinary rounding approaches. For example, the term "about" can include numerical values ​​of ±10%, ±5%, ±2%, ±1%, ±0.5%, or ±0.1% of the number it modifies. In various embodiments, the term "about" encompasses a ±5%, ±2%, ±1%, or ±0.5% variation of the numerical value of the number. In some embodiments, the term "about" encompasses a ±5%, ±2%, or ±1% variation of the numerical value of the number. In certain embodiments, the term "about" encompasses a ±5% variation of the numerical value of the number. In certain embodiments, the term "about" encompasses a ±2% variation of the numerical value of a number. In certain embodiments, the term "about" encompasses a ±1% variation of the numerical value of a number.

[0118] All numerical ranges herein include all numerical values ​​and ranges within the stated numerical range. As a non-limiting example, (C-C) alkyl also includes any one of C, C, C, C, C, C, C, (C-C), (C-C), (C-C), (C-C), (C-C), (C-C), (C-C), (C-C), (C-C), (C-C), (C-C), (C-C), (C-C), and (C-C) alkyl.

[0119] Further, while the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations as discussed above, the numerical values ​​set forth in the Examples section are reported as precisely as possible, with the understanding that such numerical values ​​inherently contain certain errors resulting from the measurement equipment and / or measurement techniques. [Example]

[0120] It should be understood that the following examples are intended to illustrate the present disclosure, but are not intended to limit the present disclosure.Various other examples and modifications of the foregoing description and examples will be apparent to those skilled in the art after reading this disclosure, without departing from the spirit and scope of the present disclosure, and all such examples or modifications are intended to be included within the scope of the appended claims.All publications and patents referenced herein are incorporated herein by reference in their entirety. List of abbreviations: 1 H-NMR Proton Nuclear Magnetic Resonance ATR Attenuated Total Reflection ca. Approximately DMSO dimethyl sulfoxide DSC Differential Scanning Calorimetry DVS Dynamic Water Vapor Sorption EtOAc ethyl acetate EtOH ethanol FBRM Focused Beam Reflectance Measurement GVS Gravimetric Vapor Sorption HPLC High Performance Liquid Chromatography HSM Hot Stage Microscopy ID Identification IPA Propan-2-ol IPrOAc Isopropyl acetate KF Karl Fischer MeOH Methanol MeOAc Methyl acetate N / A Not applicable PLM Polarized Light Microscopy RH Relative Humidity RT room temperature SCXRD Single Crystal X-ray Diffraction TFA trifluoroacetic acid TGA thermogravimetric analysis THF tetrahydrofuran Vol Volume VT-XRPD Variable Temperature Powder X-ray Diffraction XRPD X-ray powder diffraction

[0121] Equipment and methodology details X-ray powder diffraction (XRPD) XRPD diffractograms were collected on a Bruker D8 diffractometer using Cu Kα radiation (40 kV, 40 mA) and a theta-2theta goniometer equipped with a Ge monochromator. The incident beam passed through a 2.0 mm divergence slit, followed by a 0.2 mm anti-scatter slit and a knife edge. The diffracted beam passed through an 8.0 mm receiving slit with a 2.5° Soller slit, followed by a Lynxeye detector. The software used for data collection and analysis was Diffrac Plus XRD Commander and Diffrac Plus EVA, respectively.

[0122] Samples were run under ambient conditions using the as-received powder as a flat specimen. The samples were prepared on polished, zero-background (510) silicon wafers by gently pressing the flat surface or packing it into a cut cavity. The sample was rotated within its own plane.

[0123] Standard Pharmormix data acquisition method details are: angle range: 2-42° 2θ, step size: 0.05° 2θ, acquisition time: 0.5 seconds / step (total acquisition time: 6.40 minutes).

[0124] PANalytical Empyrean XRPD diffractograms were collected on a PANalytical Empyrean diffractometer in transmission mode using Cu Kα radiation (45 kV, 40 mA). A 0.5° slit, a 4 mm mask, and a 0.04 rad Soller slit with a focusing mirror were used on the incident beam. A PIXcel30 detector positioned on the diffracted beam was fitted with a receiving slit and a 0.04 rad Soller slit. The software used for data collection was X'Pert Data Collector with the X'Pert Operator Interface. Data were analyzed and presented using Diffrac Plus EVA or HighScore Plus.

[0125] Samples were prepared and analyzed in transmission mode in either metal or Millipore 96-well plates. X-ray transparent film was used between metal sheets on the metal well plates, and powders (approximately 1-2 mg) were used as received. Millipore plates were used to separate and analyze solids from suspensions by adding a small amount of suspension directly to the plate before filtering under light vacuum.

[0126] The scan mode for metal plates used gonioscan axis, while 2θ scan was used for Millipore plates.

[0127] Standard screening data collection details are: angular range: 2.5-32.0° 2θ, step size: 0.0130° 2θ, collection time: 12.75 seconds / step (total collection time 2.07 minutes).

[0128] Non-ambient conditions XRPD diffractograms were collected on a PANalytical Empyrean diffractometer in reflection mode using Cu Kα radiation (45 kV, 40 mA). The instrument was fitted with an Anton Paar CHC plus diffractometer fitted with graphite / Kapton windows. + The stage was mounted and equipped with air cooling and a rough vacuum pumping system using an Edwards RV3 pump. A programmable divergence slit (in automatic mode) with a fixed 10 mm incident beam mask, a nickel filter, and a 0.04 rad Soller slit were used on the incident beam. A PIXcel placed on the diffracted beam 3D The detector was fitted with a programmable (in automatic mode) anti-scatter slit and a 0.04 rad Soller slit.

[0129] The software used for data collection was X'Pert Data Collector, and data were analyzed and presented using Diffrac Plus EVA or Highscore Plus.

[0130] For variable temperature (VT) experiments, samples were prepared and analyzed in an Anton Paar chrome-plated sample holder. A heating / cooling rate of 10 °C / min was used with a 2-minute isothermal hold before starting the measurement. Measurement parameters were according to the standard screening data collection method (detailed above). Measurements were performed at temperatures of 25, 50, 75, 100, 160, and 25 °C. Samples were then reanalyzed by XRPD after 1 hour to confirm complete rehydration.

[0131] For the vacuum experiments, samples were prepared and analyzed in an Anton Paar chrome-plated sample holder. Measurement parameters were 25°C without vacuum (I2) and standard screening data collection methods (detailed above). A vacuum of approximately 50 mbar was then applied, and the sample was measured every 5 minutes for three consecutive measurements until an anhydrous pattern was obtained (up to sample I8, to confirm complete dehydration). The vacuum was then released, and the sample was analyzed for six measurements every 5 minutes (up to sample I22). The sample was then reanalyzed by XRPD after 1 hour to confirm complete rehydration.

[0132] Proton nuclear magnetic resonance ( 1 H-NMR) 1 H NMR spectra were collected on a Bruker 400 MHz instrument equipped with an autosampler and controlled by a DRX400 console. Unless otherwise stated, samples were prepared in DMSO-d6 solvent. The automated experiments were performed using a standard Bruker load experiment ( 1 H) were obtained using the ICON-NMR configuration in Topspin software. Offline analysis was performed using an ACD Spectrus Processor.

[0133] Differential scanning calorimetry (DSC) DSC data were collected on a TA Instruments Discovery DSC equipped with a 50-position autosampler. Typically, 0.5–3 mg of each sample in a pinhole-equipped aluminum pan was heated from 25°C to 300°C at 10°C / min. A 50 ml / min purge of dry nitrogen was maintained over the sample.

[0134] The instrument control software was TRIOS, and data were analyzed using TRIOS or Universal Analysis.

[0135] Thermogravimetric analysis (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 placed in a pre-tared aluminum DSC pan and heated from ambient temperature to 350 °C at 10 °C / min. A 25 ml / min nitrogen purge was maintained over the sample.

[0136] The instrument control software was TRIOS, and data were analyzed using TRIOS or Universal Analysis.

[0137] Polarized Light Microscopy (PLM) The samples were studied with a Nikon SMZ1500 polarizing microscope equipped with a digital video camera connected to a DS-L2 camera control unit for image capture. Samples were observed at appropriate magnification and in partially polarized light in combination with a λ false color filter.

[0138] Hot Stage Microscopy (HSM) Hot-stage microscopy was performed using a Leica LM / DM polarizing microscope in combination with a Mettler-Toledo FP82HT hot stage and a digital video camera for image capture. A small amount of each sample was placed on a glass slide, with individual particles separated as far as possible. Samples were heated at ambient temperature, typically at 10-20°C / min, while being viewed under appropriate magnification and partially polarized light in combination with a λ false color filter. Data were collected using StudioCapture.

[0139] Gravimetric Vapor Sorption (GVS) Sorption isotherms were obtained using an SMS DVS Intrinsic moisture sorption analyzer controlled by the DVS Intrinsic control software. The sample temperature was maintained at 25 °C by the instrument control. Humidity was controlled by mixing dry and humid nitrogen streams at a total flow rate of 200 ml / min. Relative humidity was measured by a calibrated Rotronic probe (dynamic range 1.0-100% RH) positioned 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).

[0140] Typically, 5-30 mg of sample was placed in a tared mesh stainless steel cage under ambient conditions. Samples were loaded and unloaded at 40% RH and 25°C (typical room conditions). Moisture sorption isotherms were performed as outlined below (two scans per full cycle). Standard isotherms were performed at 25°C over the range of 0-90% RH in 10% RH intervals. Typically, double cycles (four scans) were performed. Data analysis was performed in Microsoft Excel using the DVS Analysis Suite.

[0141] [Table 2]

[0142] After completion of the isotherm, samples were collected and reanalyzed by XRPD.

[0143] Determination of chemical purity by HPLC Purity analysis was performed on an Agilent HP1100 series system equipped with a diode array detector and using ChemStation software. Full method details are provided in Table 3 below.

[0144] [Table 3]

[0145] Determination of water content by Karl Fischer titration (KF) The water content of each sample was measured at 1.50°C in a Metrohm 874 oven sample processor using an 851 Titrano coulometer with Hydranal Coulomat AG oven reagent and a nitrogen purge. A weighed solid sample was introduced into a sealed sample vial. Approximately 10 mg of sample was used per titration, and duplicate measurements were made. Unless otherwise stated, the average of these results is shown. Data collection and analysis were performed using Tiamo software.

[0146] thermodynamic water solubility Aqueous solubility was determined by suspending sufficient compound in the relevant medium to give a maximum final concentration of the parent free form of the compound of ≥ 200 mg / ml. The suspension was equilibrated at 25°C for 24 hours on a Heidolph plate shaker set at 750 rpm. The pH of the saturated solution was then measured, and the suspension was filtered through a glass fiber C filter (particle retention capacity 1.2 μm) and diluted appropriately. Quantitation was by HPLC based on a standard solution of approximately 0.15 mg / ml in DMSO. Different volumes of standard, diluted, and undiluted sample solutions were injected.

[0147] Solubility was calculated using peak areas determined by integration of the peak found at the same retention time as the major peak in the standard injection.

[0148] [Table 4]

[0149] Analyses were performed on an Agilent HP1100 series system equipped with a diode array detector and using ChemStation software.

[0150] Raman spectroscopy Data were collected on a Renishaw inVia Quantor. The instrument control, data analysis, and presentation software was WiRE.

[0151] Method: Excitation source, λ = 633 nm or 785 nm laser, appropriately attenuated to avoid sample degradation. Raman shift range: 100–5000 cm -1 Exposure time: 0.02-10 seconds. Integration: 1-3. Raman shift range: 180-1700 cm -1 Exposure time: 30 seconds. Accumulation: 3.

[0152] Crystal 16 A Crytl 16 crystallization system (Technobis, NL) was used to determine the solubility and metastable zone of the materials as a function of temperature.

[0153] Slurries of API at different overall concentrations were prepared by adding a known amount of solid to a known amount of cooled solvent (between 0.5-1.5 ml) and stirring at 400 rpm using a magnetic bar. The saturation temperature was measured through heating and cooling cycles from -8 to 70 °C at 0.5 °C / min.

[0154] As the temperature increased, the solids dissolved completely, and the suspension became a clear solution, with the light transmittance reaching a maximum value. This temperature was assigned as the clearing point, assumed to correspond to the saturation temperature. Next, by cooling the solution at a rate of 0.5 °C / min, the temperature at which particles first formed was detected by the decrease in light transmittance. This was assigned as the cloud point. This point was approximated by the van't Hoff equation, and the difference between the cloud point and the clearing point defined the metastable zone width (MSZW) of the system. The instrument control software was Crystallization Systems, and data were analyzed using Crystal Clear and Microsoft Excel.

[0155] Focused Beam Reflectance Measurement (FBRM) Particle size distributions were collected using an FBRM Probe G400 by collecting data every 10 seconds. Data were processed with iC FBRM SP1 software.

[0156] Single Crystal X-ray Diffraction (SCXRD) Data were collected on an Atlas CCD diffractometer equipped with a Rigaku Oxford Diffraction Supernova Dual Source, Cu at Zero, and an Oxford Cryosystems Cobra refrigerator. Data were collected using Cu Kα or Mo Kα radiation, as presented in the experimental table. Structures were solved and refined using Bruker AXS SHELXTL suite or OLEX2 crystallography software. Full details can be found in the CIF. Unless otherwise stated, hydrogen atoms attached to carbons were geometrically positioned and allowed to refine with riding isotropic displacement parameters. Hydrogen atoms attached to heteroatoms were positioned in a difference Fourier synthesis and allowed to refine freely with isotropic displacement parameters. Reference diffractograms for the crystal structures were generated using Mercury.

[0157] Example 1. Synthesis of Compound 1 Compound 1 is an optical isomer of "6-[4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[1,5-a]pyrazin-8-one" disclosed in WO 2013 / 053690. Compound 1 can be prepared by chiral selective purification from "6-[4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[1,5-a]pyrazin-8-one" prepared according to the method disclosed in WO 2013 / 053690, the contents of which are incorporated herein by reference in their entirety. Compound 1 can also be prepared by the method disclosed in WO 2017 / 005786, the contents of which are incorporated herein by reference in their entirety.

[0158] [ka]

[0159] Example 2. Crystal structure determination and characterization of MH1 Single crystal growth experiment Crystallization can be achieved by lowering the temperature of a clear solution. Because the solubility of most substances decreases with temperature, cooling can be used to induce supersaturation. The solvents used in this study were isopropyl acetate, ethanol, tetrahydrofuran, water, dichloromethane, acetonitrile, anisole, methyl isobutyl ketone, nitromethane, 1,2-dimethoxyethane, methyl ethyl ketone, 1,4-dioxane, n-propyl acetate, 2-propanol, acetone, cumene, N,N-dimethylformamide, dimethyl sulfoxide, methanol, 2-methyltetrahydrofuran, MeCN / 5% water, IPA / 5% water, EtOH / water 1:1, and THF / 10% water.

[0160] 5.0 mg of MH1 (off-white powder) was weighed into 24 HPLC vials and treated with various solvents (50 μl) at room temperature (RT). The samples were then placed at 50 °C for 5 minutes. All solutions obtained at RT or 50 °C were placed in a refrigerator at 4 °C. An additional aliquot of solvent (100 μl) was added to any suspensions, which were then placed at 50 °C for 1 hour. All remaining suspensions were then syringe filtered, and the mother liquor was placed in a refrigerator at 4 °C. All solutions obtained after adding the solvent after 1 hour were also placed in a refrigerator.

[0161] Crystals suitable for analysis were initially obtained only upon cooling from isopropyl acetate, anisole, methyl isobutyl ketone, n-propyl acetate, cumene, and 2-methyl-2-thiazolinone (2-methyl-2-thiazolinone). Crystals of sufficient size and quality for analysis by single-crystal X-ray diffraction were isolated from a saturated solution of the compound in n-propyl acetate by cooling the mother liquor, with crystals measuring approximately 0.40 × 0.15 × 0.08 mm.

[0162] The crystal structure of MH1 was determined at 293 and 100 K. The crystals were orthorhombic and in the space group P212121 with final R1 = [I>2σ(I)] = 4.25 and 3.46%, respectively, and Flack parameters = 0.02 (8) and -0.05 (7) at 293 and 100 K, respectively. The absolute stereochemistry of the compound was determined as (S,S). The compound was identified as depicted in Figure 1. The asymmetric unit contains one molecule of compound 1 and one molecule of water, both of which are fully ordered. The XRPD pattern was calculated from the crystal structure and compared with the experimental diffractogram of the material as received at room temperature. An overlay of the experimental diffractogram at room temperature (Figure 2B) and the simulated XRPD patterns of MH1 at 293 and 100 K shows that they are consistent. Any slight differences are due to lattice changes with temperature and preferred orientation.

[0163] A summary of the characterization data for MH1 is provided in Table 5 .

[0164] [Table 5]

[0165] [Table 6]

[0166] [Table 7]

[0167] Example 3. Crystal structure determination of MH2 of compound 1 Single crystal growth experiment As-supplied MH1 (1.6 g) was placed in a vacuum oven at 50 °C for 3 h. The sample was then treated with 7.5% water / MeOAc (10 vol, 16 ml) at 5 °C. After 12 h at 5 °C, the suspension was filtered, washed with heptane, and air-dried.

[0168] After washing the vial with n-heptane during the filtration procedure, crystals suitable for analysis were obtained. These crystals were used to determine the single crystal structure at 100 K.

[0169] [Table 8]

[0170] Crystal structure of MH2 Crystals of MH2 were obtained by rapid evaporation while washing with heptane. Crystals of sufficient size and quality for single-crystal X-ray diffraction analysis were isolated, measuring approximately 0.65 × 0.26 × 0.18 mm. The crystal structure of MH2 was determined at 100 K. The crystals are orthorhombic and belong to the space group P212121 with a final R1 = [I > 2σ(I)] = 3.07%. The compound was identified as depicted in Figure 6. The asymmetric unit contains one molecule of compound 1 and one molecule of water, both of which are fully ordered. The absolute configuration of MH2 has been determined with C7 and C9 in the (S,S) configuration, with a Flack parameter of -0.01 (8).

[0171] XRPD patterns were calculated from the crystal structure and compared to experimental diffractograms of the MH2 material at room temperature (Figure 7). The simulated diffractograms were generally consistent with the bulk material. Any slight differences are most likely due to lattice changes with temperature and preferred orientation.

[0172] Example 4. Conversion between anhydrous (AH) and hydrated forms (MH1 and MH2) MH1 dehydration investigation MH1 was first placed in a vacuum oven at 50°C overnight. The sample was then returned to the vacuum oven and stored at 50°C for 5 days, then at 90°C for 4 hours. Aliquots were then removed from storage at 90°C and held at ambient conditions for 2 hours. At each time point, analysis was performed by TGA.

[0173] Weight loss due to water was observed in all samples after drying. This is because the samples either do not completely lose water at the temperatures studied or reabsorb water at ambient conditions. Further XRPD studies were conducted to determine the effect of vacuum and heating on the crystalline morphology.

[0174] VT XRPD MH1 was analyzed in the Empyrean at 25, 50, 75, 100, 160, and 25 °C (heating rate of 10 °C / min and waiting 2 min before starting the measurement). The sample was then reanalyzed by XRPD after 10 and 20 min to confirm rehydration.

[0175] Initially, the sample is MH1. As the temperature increases, the sample converts to the anhydrous form and is completely anhydrous by XRPD up to 75°C. Upon recooling to 25°C, peaks corresponding to MH1 are present. Upon further standing at 25°C, these peaks corresponding to MH1 become more intense while the peaks corresponding to the anhydrous form become less intense. This indicates that the anhydrous form readily converts to MH1 under ambient conditions. TGA after VT XRPD results in a 3.2% mass loss, confirming water re-uptake.

[0176] XRPD under vacuum MH1 was analyzed at 25°C without vacuum. A vacuum of approximately 50 mbar was then applied and the sample was measured every 5 minutes (to ensure complete dehydration) until an anhydrous pattern was obtained for three consecutive measurements. The sample was then analyzed for six measurements every 5 minutes before the vacuum was released and the front of the sample stage was removed.

[0177] Initially, the sample is MH1. As the sample is stored under vacuum, it converts to the anhydrous form (it is completely anhydrous by XRPD after 10 minutes). The vacuum is released and initially no change is observed for 30 minutes. The front of the sample stage is then removed, allowing ambient air to penetrate the sample stage. A peak corresponding to MH1 immediately appears, and the sample is fully hydrated by XRPD after 15 minutes. This indicates that under ambient conditions, the anhydrous form readily converts to MH1. XRPD after TGA vacuum results in a mass loss of 3.4%.

[0178] The results of drying studies confirm that it is possible to obtain the anhydrous form, but that this material rapidly converts back to the monohydrate, MH1. Compression studies of MH1 did not show conversion to the anhydrous form by XRPD.

[0179] Water activity experiment for MH1 MH1 (30.0 mg) was weighed into an HPLC vial and placed in a vacuum oven to dry over the weekend. All samples were then placed in a desiccator to ensure dryness before use. Solvent (300 μl) was added and the slurry was stirred at either 25°C or 5°C. Any solutions formed had more MH1 added to return them to the slurry. Both ethyl acetate and methyl acetate were allowed to dry before use. After 4 days, samples were analyzed by XRPD with minimal exposure to ambient conditions.

[0180] Selected samples were also characterized by XRPD, 1H NMR, TGA, and DSC after being left at ambient conditions for 2 days.

[0181] The results obtained from the water activity experiment were a w This indicates that monohydrate MH1 was obtained at a water activity of ≦0.4. w At pH = 0.5 and 0.6, monohydrate MH1 was also obtained. However, at 5 °C, a new form was observed. This form was then w= 0.7-0.9 was observed at both 5°C and 25°C. Reanalysis of selected samples of this form after 2 days at ambient conditions confirmed that it remained the same form. The new form was also a monohydrate form and was identified as MH2.

[0182] [Table 9]

[0183] Scaling up MH2 MHI (1.6 g) was placed in a vacuum oven at 50° C. for 3 hours. The sample was then treated with 7.5% water / MeOAc (10 volumes, 16 ml) at 5° C. After 12 hours at 5° C., an aliquot of the sample was analyzed by XRPD. The suspension was then filtered, washed with heptane, air-dried, and analyzed by XRPD and the appropriate technique. Upon washing the vial with heptane, crystals formed on the walls of the vial, which were analyzed by SCXRD. A summary of the characterization results is shown in Table 9.

[0184] Results and Discussion: The attempt to prepare MH2 was successful. An XRPD overlay of the scale-up of MH2 is shown in Figure 8. The TGA data showed a mass loss of 4.4% between 25 and 100 °C, corresponding to 1 equivalent (eq) of water, indicating that the sample is a monohydrate. This was confirmed by KF measurements, which detected 4.5% water in the sample. The water loss was also observed in the DSC data, which showed a broad endotherm at 59.1 °C followed by an endotherm indicating melting at 184.7 °C. This endotherm corresponds to the melting of AH and is very close to the melting point obtained when starting from MH1 (185.6 °C). 1The H-NMR spectrum is consistent with the structure and reference spectrum of MH1. GVS data showed a lack of uptake during the first sorption cycle at 40-90% RH. This was followed by a 4.8% weight loss during the desorption cycle. A 4.5% uptake was observed during the second sorption cycle. This suggested that MH2 was converted to AH during the desorption cycle, followed by conversion to the monohydrate during the sorption cycle. The converted ion was confirmed by XRPD analysis after GVS, showing that it rehydrated as MH1. The Raman spectra of MH1 and MH2 contain similar features with the main differences at 1350 and 1650 cm-1. This is expected, as the differences in Raman spectra between polymorphs are often small. The stereochemistry of MH2 was determined by SCXRD as the S,S-enantiomer (same as MH1). PLM indicates that the morphology of the sample is a mixture of irregularly shaped and mixed crystalline plates. The thermodynamic solubility of MH2 in water at 25°C is 36.5 mg / ml.

[0185] [Table 10]

[0186] Further analysis of MH1 and MH2 Example 5. XRPD examination of MH2 Procedure: Using the same procedure, a second batch of MH2 material was prepared. This sample was then analyzed by VTXRPD and XRPD under vacuum.

[0187] Results and Discussion: MH2 is converted to AH (anhydride) upon heating. A mixture of MH2 and AH was formed at 50 °C, followed by complete conversion to AH at 75 °C. After cooling to 25 °C, AH was completely converted to MH1. The formation of MH1 was confirmed by XRPD after storage at ambient conditions for 1 hour.

[0188] The MH2 sample dehydrated to AH after 5 minutes of exposure to vacuum. After 15 minutes, the vacuum was released. XRPD data was collected for 55 minutes, and the final pattern collected was a mixture of AH and MH1. The sample was reanalyzed after 1 hour, and complete rehydration to MH1 was observed.

[0189] Example 6. Drying test of two hydrates (MH1 and MH2) Procedure: MH1 and MH2 were placed in a vacuum oven at 50° C. and RT for 24 hours. Samples were then analyzed using TGA and XRPD immediately after removal and after standing under ambient conditions for 4 hours.

[0190] Results and Discussion: Samples were analyzed using XRPD and TGA immediately after removal from the oven (T=0), and then re-measured 4 hours later (T=4 h). At T=0, both MH1 and MH2 (dried at RT and 50 °C) were converted to a mixture of MH1 and AH by XRPD. A small amount of water loss was recorded by TGA for both samples, indicating that both hydrates were converted to AH during vacuum oven drying. After 4 hours at ambient conditions, the XRPD data indicated that both samples were fully converted to MH1. However, the TGA data indicated that water loss was not complete, with only a loss of approximately 1.8-2.7%. Based on these results, it appears that MH2 is converted to AH during drying, followed by MH1 upon ambient storage.

[0191] [Table 11]

[0192] Example 7. Stability study using competing slurries of two hydrates (MH1 and MH2) Procedure: Saturated solutions of the supplied MH1 (J08343) were prepared in various solvents / systems (1 ml). The saturated solutions were then filtered and used in competitive slurry experiments.

[0193] MH1 and MH2 (approximately 15 mg each) were physically mixed before treatment with the filtered saturated solution (300 μL). The sample was stirred at 25 °C for 24 h, filtered, air-dried, and analyzed by XRPD.

[0194] As this was the current crystallization solvent, the same solvent was used as for the samples in the water activity experiments with additional IPA / heptane mixtures. This procedure was also carried out for IPA / heptane at 5 and 50°C (slurrying for 3 days).

[0195] Results and Discussion: MH2 was primarily obtained during competitive slurries at 25°C (Table 11). The solvents that produced MH2 had varying water activity values. A mixture of MH1 and MH2 was obtained even when the solvent system did not contain water (i.e., no conversion to AH was observed). MH2 predominates in IPA and mixtures of IPA and heptane at 5°C. However, a mixture of MH1 and MH2, or pure MH1, was produced when higher temperatures (50°C) were utilized (Table 12).

[0196] Thus, the competitive slurries show that MH2 predominates at lower isolation temperatures and higher water activities, whereas MH1 predominates at higher isolation temperatures and lower water activities.

[0197] Further experiments were designed to increase the diversity of the solvents / systems studied while examining the effect of water activity on the resulting morphologies. This involved defining a solvent list for Phase 3 (solvent selection). Using the supplied MH1, further saturated solutions were prepared as described in the procedure section.

[0198] Some samples dissolved between competing slurries, which may be because complete saturation did not occur. MH2 again predominates at low temperatures with high water activity (Table 13). Mixtures of MH1 and MH2 were obtained from heptane at 5 °C, IPA at 50 °C, or IPA:heptane mixtures. IPrOAc:0.5% water mixtures (aw = 0.35) favored the formation of MH1 at 50 °C, but MH2 at 5 °C. Pure MH1 was also obtained when THF solvent was used at 50 °C.

[0199] MH2 is low temperature and high a w Although predominant in , MH2 can be converted to MH1 (via AH) and obtained using higher temperatures and lower water activities, so MH1 was selected for further development.

[0200] [Table 12]

[0201] [Table 13]

[0202] [Table 14]

[0203] Structural comparison of MH1 and MH2 The unit cells and asymmetric units of MH1 and MH2 were compared (Table 14, measured at 100 K). The two hydrated structures have the same space group and the same size unit cell. However, the asymmetric units are significantly different.

[0204] In MH1, the water and API (compound 1) in the asymmetric unit are involved in one OH---O intermolecular hydrogen bond, and there are also intramolecular branched asymmetric hydrogen bonds between the nitrogen atoms of the imidazopyrazine ring and the nitrogen atoms of the pyrimidine and pyrrolidine rings.

[0205] In MH2, the water molecule is located between the imidazopyrazine ring and the pyrimidine ring, so that the intramolecular hydrogen bond present in MH1 is replaced by an intermolecular interaction between water and the API.

[0206] [Table 15]

[0207] Results and Discussion: The experiments performed in this study showed that MH1 is the most stable form at ambient conditions. Solvent mixtures with water (high water activity) at lower temperatures (5 °C) should be avoided because they produce MH2. MH1 was only produced at the end of the crystallization process, but care should be taken to avoid the formation of AH or MH2.

[0208] Example 8. Solubility evaluation of MH1 Procedure: Four solvents were selected based on competitive slurry experiments in which MH1 was dominant, as well as for diversity. These solvents and water (a w =0.35) was also utilized to confirm which hydrate predominated. A mixture of solvent and heptane (1:1) was used to check for crystalline forms with good antisolvent properties.

[0209] MH1 (22 x 104 mg) was suspended in various solvents (11 x 0.5 ml) and stirred at 750 rpm for 24 hours at 5 or 50°C. Solids were isolated by filtration and centrifugation, and the liquids were analyzed by HPLC to determine their solubility (compared to prepared standards). The solids were also examined by XRPD. This procedure was also carried out sequentially for five additional solvent systems with different ratios of THF:heptane and IPA:heptane at either 5 or 50°C. The isolated solids were analyzed by XRPD, HPLC, and 1 It was investigated by HNMR.

[0210] Results and Discussion: Samples that formed clear solutions were considered to have a solubility greater than 200 mg / ml (Table 15). This was true for six samples at 50°C, including THF, ethanol, IPA, and several of these solvents in combination with water or heptane. At 5°C, solubility greater than 200 mg / ml was achieved in ethanol and mixtures of ethanol and water. Significant solubility was also measured using ethanol:heptane (1:1) (170 mg / ml) at 5°C.

[0211] The sample produced variations of MH1 and MH2, as well as mixtures of MH1 and AH, at both 5 and 50° C. Because IPA and THF gave high solubility at 50° C. (>200 mg / ml) and THF:heptane (1:1) showed MH1 at both temperatures, further solubility evaluations were performed using mixtures of IPA:heptane (1:1, 1:2, 1:3) and THF:heptane (2:1, 1:2) (Table 16).

[0212] Based on the solubility observations, it was decided to proceed with the IPA / heptane (1:3) system for antisolvent crystallization, since MH2 was not observed at 5 °C due to its low solubility. This solvent / system was chosen for the solubility and MSZW experiments to explore the temperature dependence on the solubility of MH1.

[0213] [Table 16]

[0214] [Table 17]

[0215] Example 9. Solubility studies of MH1 and MH2. Procedure: pH profiling solubility experiments were performed on MH1 and MH2. Solubility was determined solely by suspending sufficient compound in the relevant vehicle (1.00 ml) to achieve the maximum final concentration, as shown in Table 17.

[0216] [Table 18]

[0217] The suspension was equilibrated at 25°C for 24 hours on a Heidolph plate shaker set at 750 rpm. Samples were pH adjusted to within 0.1 of the desired pH unit (if possible) using 0.5M / 1M HCl and 0.2M NaOH as needed. The pH of the saturated solution was measured (if applicable) and its appearance was recorded. The suspension was filtered through a glass fiber C filter (1.2 μm particle retention) and diluted appropriately. Quantitation was performed by HPLC with reference to a standard solution of approximately 0.15 mg / ml in DMSO. Different amounts of standard, diluted, and undiluted sample solutions were injected. Solubility was calculated using the peak area determined by integration of the peak detected at the same retention time as the major peak in the standard injection.

[0218] Results and Discussion: Upon addition of vehicle to MH1-A and MH2-A, a thick paste was observed indicating that the vehicle had been absorbed, so additional vehicle was added to the sample vial and a suspension was observed.

[0219] After approximately 1 hour, MH1-B, C, and D, and MH2-C and D were clear solutions, so additional material was added to the sample vials.

[0220] During the initial pH adjustment of MH1-A, little change in pH was observed when 0.5 M HCl was used, so it was decided to use 1 M HCl as the next adjustment solution. The vial reached capacity when the sample pH was 5.02. MH1-A, with a measured pH of 6.63, also reached capacity.

[0221] [Table 19]

[0222] The solubility of sample A (initially in pH 1.2 medium) was determined to be >275 mg / mL; however, it is worth noting that the pH could not be maintained at pH 1.2 because the vial's capacity limit was reached. Therefore, the solubility values ​​obtained are for the final pH. These experiments were repeated using the reverse addition approach.

[0223] Solubility Repeat Procedure: pH 1.2 buffered media (0.4 ml) was added to two separate 7 ml vials. For each compound, material was added in small portions to the pH 1.2 media. After each compound addition, the vial was vortex mixed and the pH and appearance were recorded. Samples were adjusted to pH 1.2 with 1 M HCl to within 0.05 of the desired pH unit. This was repeated until no further material was available (approximately 400 mg of each form, resulting in a maximum concentration of approximately 1000 mg / ml if no adjustment was required).

[0224] Results and Discussion: Clear solutions were observed for both samples due to the amount of adjustment solution required, however the pH of both solutions was maintained at pH 1.2 throughout.

[0225] [Table 20]

[0226] Example 10. Crystallization process for the preparation of MH1. Dry (KF ≤ 0.1%) 2-propanol (67 kg) was charged to the reactor under N2, followed by crude solid Compound 1 (20.4 kg). Purified water (1 kg) and dry 2-propanol (3 kg) were then added, and the reactor temperature was adjusted to 27-35 °C. The resulting reaction mixture was stirred under N2 protection until all solid material was dissolved. Optionally, an in-process control (IPC) sample (KF) was run to measure the water content of the reaction mixture, and sufficient dry 2-propanol was added to bring the water content to 1.0% (verified by KF).

[0227] The temperature of the reaction mixture was adjusted to 22-28°C, and seed crystals (0.24 kg) were added. The resulting reaction mixture was stirred at 22-28°C for 0.5-2.0 hours. Using a pump such as a diaphragm pump, n-heptane (246 kg) was slowly charged to the reactor at 22-28°C, and the resulting reaction mixture was stirred at 22-28°C. The resulting reaction mixture may be stirred for 8-12 hours to achieve complete supersaturation removal and maximize yield. Optionally, an IPC sample was taken at this point to determine the water content, residual Compound 1 in the supernatant, and the purity and crystallinity of the precipitated solid.

[0228] The reaction mixture was filtered at 22-28°C, and the resulting solid was washed with n-heptane (27.8 kg). The filter cake was pressed to dryness and then dried under a stream of N2 for 1-2 hours. Optionally, an IPC sample was taken at this point to confirm the purity and crystallinity of the isolated solid.

[0229] The solids were dried over a saturated solution of sodium chloride in a drying chamber at 20-27°C under a constant nitrogen flow for 10-18 hours. The solids were removed from the dryer, sieved, and packed into drums lined with LDPE bags. A release sample confirmed moisture (KF=4.2%) and crystallinity (XRPD: MHCl) along with other purity-related release methods. Yield: 17.46 kg MHCl.

Claims

1. A monohydrate crystalline form of 6-[(3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[1,5-a]pyrazin-8-one, represented by formula 1: 【Chemistry 1】 The monohydrate crystalline form of the crystal is MH1, and has an XRPD pattern including peaks at 2θ angles of 9.1, 11.5, 16.2, 16.7, 18.2, 18.9, 19.8, 22.6, and 26.4° 2θ, each ±0.2° 2θ.

2. A crystal of the monohydrate crystalline form described in claim 1 having the XRPD pattern shown in Figure 2A. 【Chemistry 2】

3. 2. The monohydrate crystalline form of the crystal according to claim 1, which has a dehydration endothermic peak at 40 to 100°C and a melting endothermic peak at 184.4°C in a differential scanning calorimetry (DSC) thermogram.

4. 2. The monohydrate crystalline form of claim 1, having a DSC thermogram as shown in FIG. 【Transformation 3】

5. 2. The monohydrate crystalline form of the crystal of claim 1, which exhibits dehydration between ambient temperature and 90°C with a weight loss of 3.8% in thermogravimetric analysis (TGA).

6. 2. The monohydrate crystalline form of claim 1, having a TGA as shown in FIG. 【Chemistry 4】

7. A crystal of the monohydrate crystalline form described in claim 1, having characteristic absorptions at 782 cm-1, 1123 cm-1, 1562 cm-1, and 1655 cm-1 in the infrared (IR) spectrum.

8. 2. The monohydrate crystalline form of claim 1, having an infrared spectrum as shown in FIG. 【Transformation 5】

9. A pharmaceutical composition comprising the monohydrate crystalline form of the crystal according to any one of claims 1 to 8 and a pharmaceutically acceptable excipient.

10. A method for producing the monohydrate crystalline form of crystals of claim 1, comprising precipitating the monohydrate crystalline form from a solution comprising the compound of formula 1 and a solvent selected from the group consisting of n-propyl acetate, isopropyl acetate, anisole, methyl isobutyl ketone, cumene, 2-methyltetrahydrofuran, and combinations thereof. 【Transformation 6】

11. A pharmaceutical composition for inhibiting PDE9 activity in a patient, comprising the monohydrate crystalline form of a crystal according to any one of claims 1 to 8.

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