Crystallographic forms, related products and methods of N-{3-[(1S)-1-{[6-(3,4-dimethoxyphenyl)pyrazin-2-yl]amino}ethyl]phenyl}-5-methylpyridine-3-carboxamide

Abstract

This invention provides crystalline forms of N-{3-[(1S)-1-{[6-(3,4-dimethoxyphenyl)pyrazin-2-yl]amino}ethyl]phenyl}-5-methylpyridine-3-carboxamide. Pharmaceutical compositions and dosage forms containing these crystalline forms are also provided, including methods generally used for modulating kinases, and particularly for treating PAH.

Description

Technical Field

[0001] This invention relates to the crystalline form of N-{3-[(1S)-1-{[6-(3,4-dimethoxyphenyl)pyrazin-2-yl]amino}ethyl]phenyl}-5-methylpyridine-3-carboxamide, products comprising such crystalline forms, and related methods for their application and preparation. Background Technology

[0002] Receptor tyrosine kinases are transmembrane polypeptides that regulate cell regeneration, remodeling, development, and differentiation. Among receptor tyrosine kinases, platelet-derived growth factor receptor (PDGFR) is associated with lung disease, tissue fibrosis, and solid tumors.

[0003] Pulmonary hypertension (PH) is a rare condition of the pulmonary vascular system associated with high morbidity and mortality among lung diseases. The pathological features of this disease include plexiform lesions with disordered angiogenesis and abnormal proliferation of new intimal cells, which impede blood flow through the pulmonary arterioles. Known kinase receptor inhibitors (particularly known PDGFR inhibitors) are not orally available, are associated with off-target effects that can contribute to the development of PH, and / or are associated with dose-limiting side effects. Therefore, there is a need for agents that inhibit PDGFRα and / or PDGFRβ with improved potency and selectivity compared to other kinases known to involve dose-limiting side effects (e.g., cKit, FLT3, and VEGFR2).

[0004] N-{3-[(1S)-1-{[6-(3,4-dimethoxyphenyl)pyrazin-2-yl]amino}ethyl]phenyl}-5-methylpyridine-3-carboxamide, also known as GB002 or seralutinib (hereinafter referred to as "Compound 1"), is a potent and selective inhibitor of PDGFRα and PDGFRβ signaling. Compound 1 is being clinically developed as an inhaled treatment for pulmonary arterial hypertension (PAH). The amorphous form of Compound 1 has been described in U.S. Patent Nos. 9,815,815 and 10,231,966, and its spray-dried powder formulation has been described in U.S. Patent No. 9,925,184. Compound 1 has the following structure:

[0005]

[0006] Given the clinical prospects of Compound 1, there is a need for new, improved, and / or enhanced forms of Compound 1, particularly suitable for pharmaceutical products delivered by inhalation, as well as compositions comprising Compound 1 and methods relating to their manufacture and use. The present invention addresses these and related needs, as demonstrated in the following detailed description and accompanying drawings. Summary of the Invention

[0007] Solid pharmaceutical forms can exist in amorphous or crystalline states. In the crystalline form, molecules reside in three-dimensional lattice sites. When a compound recrystallizes from a solution or slurry, it can crystallize in different spatial lattice arrangements; this property is called "polymorphism," and different crystalline forms are called "polymorphs" or simply "polymorphs." Different polymorphs of a given substance can differ from each other in one or more physical properties, such as solubility and dissociation, true density, crystal shape, compressive behavior, flow properties, and / or solid-state stability. When a chemical substance exists in two (or more) polymorphic forms, the unstable form generally transforms into a more thermodynamically stable form after a sufficiently long period of time at a given temperature. When this transformation is not rapid, the thermodynamically unstable form is called the "metastable" form. Generally, the stable form exhibits the highest melting point, lowest solubility, and greatest chemical stability. However, the metastable form may exhibit sufficient chemical and physical stability under normal storage conditions to allow its commercial use. In this case, the metastable form, despite being less stable, may exhibit properties superior to the stable form, such as increased solubility or better oral bioavailability.

[0008] Therefore, in one embodiment, a novel solid crystalline form of compound 1 is provided. In a more specific embodiment, the novel solid crystalline form is two different polymorphs of compound 1, referred to herein as "form A" and "form B".

[0009] In one embodiment, compound 1 is provided in a crystalline form, wherein the crystalline form is form A, and in another embodiment, form A is substantially pure. Form A can be characterized by various analytical techniques disclosed herein, including, for example, by X-ray powder diffraction (XRPD) and the characteristic diffraction pattern produced therefrom.

[0010] In one embodiment, compound 1 is provided in a crystalline form, wherein the crystalline form is form B, and in another embodiment, it is essentially pure form B. Form B can be characterized by various analytical techniques disclosed herein, including, for example, by X-ray powder diffraction (XRPD) and the characteristic diffraction pattern produced therefrom.

[0011] In one embodiment, a crystalline form of compound 1 is provided, wherein the crystalline form is a mixture of form A and form B. As defined below, a mixture is provided when one crystalline form is present in a ratio of 5% to 95% by weight of another crystalline form (a ratio of form A to form B above or below this range is characteristic of substantially pure crystalline forms).

[0012] In another embodiment, a pharmaceutical composition is provided comprising a combination of the solid crystalline form of compound 1 and one or more pharmaceutically acceptable carriers. Such compositions can be formulated in a variety of different forms. For example, the composition can be formulated for application to the respiratory tract, such as in the form of an inhalable powder or as a dry powder. Such powder forms can be further characterized, such as by their size (e.g., by the volume distribution in which half of them are above and half below a certain diameter, abbreviated as "Dv50").

[0013] In one embodiment, the pharmaceutical composition may contain additional therapeutically active agents (i.e., in addition to the crystalline form of compound 1).

[0014] In one embodiment, the pharmaceutical composition may comprise leucine, and in a more specific embodiment, leucine coats the solid crystalline form of compound 1. In a related embodiment, the leucine-coated form is obtained by wet polishing.

[0015] In another embodiment, a solid unit dosage form comprising compound 1 in a solid crystalline form is provided. Such a dosage form refers to a pharmaceutical product in its commercially available form. For example, the unit dosage form may be in a form suitable for administration to the respiratory tract, such as in the form of an inhalable powder or as a dry powder, including capsules or blister packs containing said unit dosage form for use in conjunction with, for example, a dry powder inhaler.

[0016] In another embodiment, a method for treating a disease or condition regulated by kinase inhibition is provided, the method comprising administering to a subject in need an effective amount of a crystalline form of compound 1, a pharmaceutical composition comprising said crystalline form, or a solid unit dosage form comprising said crystalline form. In a more specific embodiment, the kinase is a tyrosine kinase, such as (but not limited to) platelet-derived growth factor PDGFR, and more specifically PDGFRα and / or PDGFRβ.

[0017] In one implementation, the disease or symptom is PAH, primary PAH, idiopathic PAH, hereditary PAH, refractory PAH, drug-induced PAH, toxin-induced PAH, or PAH with secondary diseases, and in a more specific implementation, it is PAH.

[0018] In yet another embodiment, a method is provided for preparing the solid crystalline form of compound 1 by crystallization from a solvent containing ethyl acetate. In one embodiment, the solvent may further comprise water and n-heptane or ethanol.

[0019] In one embodiment, the applicant has surprisingly discovered a method for preparing crystalline form B from crystalline form A, which involves slurrying form A of compound 1 in ethyl acetate and maintaining the temperature at about 10°C to about 45°C for a period of time from 1 minute to 90 hours. Attached Figure Description

[0020] This patent or application document contains at least one color drawing. A copy of this patent or application disclosure with a color drawing will be provided by the Patent Office upon request and payment of the necessary fees.

[0021] Figure 1 XRPD of amorphous compound 1 after heating at 100°C (lower trace) and 150°C (upper trace).

[0022] Figure 2 DSC of amorphous compound 1, heated to 320°C.

[0023] Figure 3 DSC of amorphous compound 1, heated to 200°C.

[0024] Figure 4 DSC of amorphous compound 1, Tg and ΔCp of the first scan up to 200 °C.

[0025] Figure 5 H of amorphous compound 1 1 NMR spectrum.

[0026] Figure 6 Amorphous compound 1 1 H- 13 C heteronuclear single quantum coherence (HSQC) NMR spectrum.

[0027] Figure 7 : Heating rate (q) versus 1 / Tg for amorphous compound 1.

[0028] Figure 8 X-ray powder diffraction (XRPD) patterns of the ethanol slurry (crystallized form B) described in Example 3B: starting material (upper trace 1), after 24 hours (2), after 24 hours (3), and after 72 hours with added water (lower trace 4).

[0029] Figure 9 XRPD of compound 1, form A.

[0030] Figure 10 XRPD of compound 1 in form B.

[0031] Figure 11 : DSC curve of compound 1, form A.

[0032] Figure 12 : DSC curve of compound 1, form B.

[0033] Figure 13 TGA thermal analysis chromatogram of compound 1, form A.

[0034] Figure 14 TGA thermal analysis chromatogram of compound 1, form B.

[0035] Figure 15 Infrared spectrum of compound 1, form A

[0036] Figure 16 Infrared spectrum of compound 1, form B

[0037] Figure 17 XRPD of the crystalline form A of compound 1 in ethanol at 10°C.

[0038] Figure 18 XRPD of the crystalline form A of compound 1 in ethanol at room temperature.

[0039] Figure 19 XRPD of the crystalline form A of compound A in ethanol at 45°C.

[0040] Figure 20 Solubility curves (mg / mL versus temperature) of crystalline forms A and B of compound 1 in ethyl acetate ± water.

[0041] Figure 21 Comparison of solubility curves of crystalline forms A and B of compound 1.

[0042] Figure 22 TGA of a sample containing a mixture of crystalline forms A and B of compound 1.

[0043] Figure 23 Diffraction pattern comparison: reference (top), TGA-treated sample (middle), and initial sample (bottom).

[0044] Figure 24 ORTEP diagram of compound 1 from crystal structure A.

[0045] Figure 25 XRPD 2θ diffraction pattern of compound 1 in its crystalline polymorph form A.

[0046] Figure 26 Visible light unpolarized (top) and polarized (bottom) micrographs of compound 1.

[0047] Figure 27 LC-MS spectrum of compound 1.

[0048] Figure 28 HPLC chromatogram of compound 1.

[0049] Figure 29 : TG / DTA thermal analysis chromatogram of compound 1, with the upper trace representing TG and the lower trace representing DT.

[0050] Figure 30 DSC thermal analysis of compound 1: (a) first heating step; (b) cooling step; and (c) second heating (20°C to 200°C).

[0051] Figure 31 GVS isotherm (double cycle) of compound 1.

[0052] Figure 32 GVS kinetics of compound 1.

[0053] Figure 33 XRPD 2θ diffraction patterns of compound 1 before (top) and after (bottom) lyophilization.

[0054] Figure 34 XRPD diffraction patterns of compound 1 after storage for one week under different temperature (ambient, 25℃, 40℃, 80℃) and relative humidity (ambient, 60%, 75%) conditions.

[0055] Figure 35 XRPD diffraction patterns of compound 1 before and after slurrying in various buffer solutions.

[0056] Figure 36 ORTEP diagram of the structure of compound 1, form B (50%).

[0057] Figure 37 PXRD comparison of the simulated pattern (lower part) from SCXRD with the reference pattern B (upper part).

[0058] Figure 38A and Figure 38B Mean concentration-time curves (±SD) of compound 1 over 4 hours and 72 hours, respectively. (Treatment A - test formulation, Treatment B - reference formulation). Detailed Implementation

[0059] According to this disclosure, novel solid crystalline forms of compound 1 are provided. In a more specific embodiment, the novel solid crystalline forms are two different polymorphs of compound 1; namely, form A and form B. Forms A and B differ from the amorphous form of compound 1 in lattice structure, and each form gives a unique X-ray powder diffraction (XRPD) pattern and differential scanning calorimetry (DSC) thermal analysis pattern.

[0060] As used herein, “amorphous” refers to the lack of well-ordered diffraction lines due to the absence of a repeating crystal lattice. As used herein, the amorphous form of N-{3-[(1S)-1-{[6-(3,4-dimethoxyphenyl)pyrazin-2-yl]amino}ethyl]phenyl}-5-methylpyridine-3-carboxamide can be prepared according to the method described in U.S. Patent No. 9,815,815 (see column 29, line 25 through column 31, line 11), the disclosure of which is incorporated herein by reference in its entirety.

[0061] Therefore, in one embodiment, this disclosure provides form A, characterized by XRPD plots having peaks at 5.5, 7.8, 11.0, 12.3 and 15.6 ± 0.2 degrees 2θ.

[0062] In another implementation, form A is provided, which is further characterized by being substantially as follows: Figure 3 The XRPD diagram shown is shown below.

[0063] In practice, a single polymorph (i.e., form A or form B) can be used in a substantially pure form or in the form of a mixture of polymorphs (i.e., a mixture of form A and form B).

[0064] In one embodiment, one crystalline form (i.e., form A or form B) is present in an amount exceeding 95% by weight compared to another crystalline form. Thus, substantially pure form A contains less than 5% by weight of form B. Conversely, substantially pure form B contains less than 5% by weight of form A. In a further embodiment, one crystalline form (i.e., form A or form B) is present in an amount exceeding 96%, 97%, 98%, or 99% by weight compared to another crystalline form (i.e., the other crystalline form is present in an amount less than 4%, 3%, 2%, or 1% by weight compared to another form). In another embodiment, one crystalline form (i.e., form A or form B) is present in an amount exceeding 99.2%, 99.4%, 99.6%, or 99.8% by weight compared to another crystalline form.

[0065] In another embodiment, the crystalline form of compound 1 comprises a mixture of form A and form B. As used herein, a mixture of form A and form B means that form A is present in a ratio ranging from 5% to 95% by weight compared to form B, or conversely, that form B is present in a ratio ranging from 5% to 95% by weight compared to form A. As defined above, form A or form B is considered substantially pure relative to another form when it is present in an amount exceeding 95% by weight compared to the other form.

[0066] In one implementation, form A comprises at least 80% form A.

[0067] In another implementation, form A comprises at least 90% form A.

[0068] In one embodiment, form B is provided, characterized by XRPD plots having peaks at 5.2, 6.1, 7.6, 11.5, and 12.3 ± 0.2 degrees 2θ.

[0069] In another implementation, form B is provided, which is further characterized by being substantially as follows: Figure 4 The XRPD diagram shown is shown below.

[0070] In another implementation, form B comprises 80% form B.

[0071] In another implementation, form B comprises 90% of form B.

[0072] In one embodiment, the crystalline form of compound 1 comprises substantially pure form A or substantially pure form B.

[0073] Surprisingly, it was found that the crystalline form of compound 1 was particularly advantageous for its use as a pharmaceutical agent, especially in cases of administration by inhalation.

[0074] In one embodiment, this disclosure provides for micronizing crystalline forms to an inhalable fraction size.

[0075] In another embodiment, the inhalable fraction size is measured as median particle size.

[0076] In yet another implementation, the median granularity is less than 5 μM, in some cases 2 μM to 4 μM, and in still other cases 3 μM to 3.5 μM.

[0077] In one implementation, crystalline form A is micronized.

[0078] In another embodiment, crystalline form A is micronized without reverting to the amorphous form of compound 1.

[0079] In yet another implementation, wet polishing is used for micronization.

[0080] In another embodiment, the mixture of form A and form B of compound 1 is used for micronization in crystalline form.

[0081] In one embodiment, form A of compound 1 is characterized by having a high solubility in a mixture of ethanol and water ranging from about 2 mg / mL to about 350 mg / mL and not showing a decrease in solubility over 24 hours.

[0082] In another embodiment, form A of compound 1 is further characterized by having moderate solubility in phosphate buffer (pH 7.4) and not showing a decrease in solubility over 24 hours, in contrast to the amorphous form of compound 1, which shows a gradual decrease in solubility.

[0083] The applicants have found that their attempts to scale up amorphous compound 1 in the preparation of a pharmaceutical product for clinical trials were unsuccessful. The applicants were surprised to find that crystalline compound 1 could be scaled up with improved purity distribution.

[0084] In another embodiment, the crystalline form of compound 1 contains less than 2% by weight of total impurities, less than about 1% by weight of water, and / or less than about 0.5% by weight of residual organic solvents.

[0085] In one embodiment, solution-phase NMR spectroscopy can be used to verify the purity and chemical structure of crystal form, form A, and form B.

[0086] In another implementation, thermogravimetric analysis (TGA) can be used to verify that the polymorph is anhydrous. (Reference) Figures 7 to 8 The TGA indicates that the crystalline form is anhydrous. In one embodiment, form B of compound 1 has a slightly higher melting point than form A, as measured by differential scanning calorimetry.

[0087] The applicant has discovered a method for converting one particular crystalline polymorph of compound 1 into another. Therefore, in another embodiment, as described more in detail in the following examples, form B is believed to be an anhydrous / unsolvable solid, and form A is slowly converted to form B, indicating that forms A and B are enantiomeric. Thus, form A can also be referred to as the metastable form, since the conversion from form A to form B has been observed (but not the conversion from form B to form A), and form A has a lower melting point compared to form B. The crystal structures of crystalline forms A and B of compound 1 are determined to be different by, for example, X-ray powder diffraction (XRPD). XRPD diagrams of forms A and B are provided in Table 7 below.

[0088] In one embodiment, crystalline form A of compound 1 is slurried in ethyl acetate and maintained at a temperature of about 10°C to about 45°C for a period of time from 1 minute to 90 hours, sufficient to convert form A to form B. Samples are removed at intervals and their XRPD patterns are determined.

[0089] The present invention also provides a method for preparing a crystalline form of compound 1 containing crystals.

[0090] In one embodiment, crystallization includes dissolving amorphous compound 1 in 1,4-dioxane, lyophilizing the solution, and adding a test solvent to allow for thermal cycling and / or evaporation.

[0091] In one implementation, the test solvent is ethyl acetate.

[0092] In another embodiment, the test solvent is acetonitrile.

[0093] In yet another implementation, the test solvent is an equal volume of ethyl acetate / tBME.

[0094] In another implementation, crystallization is induced by adding an antisolvent.

[0095] In yet another implementation, the antisolvent is heptane.

[0096] In one embodiment, a method is provided for preparing form B directly from amorphous compound 1 using a first solvent and a second solvent or an antisolvent.

[0097] In yet another implementation, the first solvent is ethanol, and the second solvent or antisolvent is water.

[0098] As described above, only an amorphous form of compound 1 has previously been obtained. Substantially pure form A and substantially pure form B, as well as mixtures of forms A and B, can be obtained using the techniques disclosed in the following examples. Once obtained, such crystalline forms can be used to prepare pharmaceutical compositions comprising them in combination with one or more pharmaceutically acceptable carriers. The compositions of the present invention may also contain other therapeutic agents as described below and can be formulated, for example, using conventional solid or liquid carriers or diluents and pharmaceutical additives (e.g., excipients, binders, preservatives, stabilizers, flavorings, etc.) of a type suitable for the desired manner of administration, according to techniques well known in the field of pharmaceutical formulations.

[0099] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal, or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoresis, and transmucosal administration. Solutions or suspensions intended for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and agents for adjusting tension, such as sodium chloride or glucose. pH may be adjusted with an acid or base (such as hydrochloric acid or sodium hydroxide). Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. For the convenience of patients or treating physicians, the medication can be provided in a kit containing all the equipment required for the treatment procedure.

[0100] The crystalline form of Compound 1 of this disclosure may be administered by any suitable means, such as oral administration, as in tablets, capsules, granules, or powder; sublingual; buccal; parenteral administration, such as via subcutaneous, intravenous, intramuscular, (per)dermal, or cisterna magna injection or infusion techniques, for example, as a sterile injectable aqueous or non-aqueous solution or suspension; intranasal administration, such as by inhalation spray or blowing; topical administration, such as in the form of cream or ointment, or ocular administration, as a solution or suspension; vaginal administration, as a suppository, tampon, or cream; or rectal administration, such as in the form of a suppository, wherein the unit dose formulation contains a non-toxic, pharmaceutically acceptable carrier or diluent. The crystalline form of Compound 1 may be administered, for example, in a form suitable for immediate or prolonged release. Immediate or prolonged release may be achieved by using a suitable pharmaceutical composition comprising the crystalline form of Compound 1, or, for the purpose of prolonged release, by using a device such as a subcutaneous implant or an osmotic pump.

[0101] For application to the respiratory tract, such as by inhalation, including intranasal administration, the active compound can be administered by any method and formulation used in the art for application to the respiratory tract. Therefore, the active compound can be administered, for example, in the form of a solution, suspension, or dry powder, with the dry powder form being a preferred embodiment. Agents according to this aspect of the invention can also be administered directly to the airways in the form of an aerosol. When used as an aerosol, the compound of the invention in solution or suspension form can be packaged together with a suitable propellant (e.g., a hydrocarbon propellant such as propane, butane, or isobutane, and conventional adjuvants) in a pressurized aerosol container. The materials of the invention can also be administered in a non-pressurized form (such as in a nebulizer or sprayer).

[0102] In one embodiment of application to the respiratory tract, compound 1 is micronized in its crystalline form. In a more specific embodiment, micronization is accomplished by wet polishing or by jet milling.

[0103] In one embodiment, the micronized form is filled into a capsule for administration as a dosage form for a dry powder inhaler. In another embodiment, the micronized form is filled into a blister pack for administration as a dosage form for a dry powder inhaler. In yet another embodiment, the micronized form is directly filled into a dry powder inhaler for administration.

[0104] In one embodiment administered over the respiratory tract, the pharmaceutical composition may contain leucine as a force control agent. In a more specific embodiment, leucine is micronized (e.g., co-milled) together with the crystalline form of compound 1.

[0105] In another embodiment, leucine is coated in the solid crystalline form of compound 1, and in a more specific embodiment, compound 1 is coated in a micronized crystalline form.

[0106] In yet another embodiment, the leucine coating is obtained by micronization, and in a more specific embodiment, it is obtained by spray drying an aqueous suspension after micronizing the solid crystalline form of compound 1.

[0107] In another embodiment, the micronization step for obtaining the leucine-coated crystalline form of compound 1 is performed by jet milling.

[0108] In another embodiment, the micronization step for obtaining the leucine-coated crystalline form of compound 1 is performed by wet polishing.

[0109] In one embodiment, the leucine of compound 1 is coated in a micronized crystalline form and filled into a capsule for administration as a dosage form.

[0110] In another embodiment, the leucine-coated micronized crystalline form of compound 1 is filled into a blister pack for administration as a dosage form for use in a dry powder inhaler.

[0111] In another embodiment, the micronized crystalline form of leucine-coated compound 1 is directly filled into a dry powder inhaler for administration.

[0112] In another embodiment, the leucine-coated micronized crystalline formulation of compound 1 has a higher drug loading than the amorphous formulation.

[0113] The propellant-driven inhaled aerosols usable according to the present invention may also contain other components, such as cosolvents, stabilizers, surfactants, antioxidants, lubricants, and pH adjusters. The propellant-driven inhaled aerosols usable according to the present invention can be administered using inhalers known in the art (e.g., metered-dose inhalers). Alternatively, the agents of the present invention can be administered to the airways in the form of pulmonary surfactant formulations. Pulmonary surfactant formulations may include exogenous pulmonary surfactant formulations (e.g., ...). (Forest Laboratories) (Ross Products) and (DEY, California, USA) or synthetic lung surfactant formulations (e.g., (GlaxoWellcome Inc. and ALEC). These surfactant formulations are administered via airway infusion (i.e., after intubation) or intratracheal administration.

[0114] As another alternative, the crystalline form of compound 1 of the present invention can be applied to the airway as an inhalable powder. The powder formulation may include physiologically acceptable excipients such as amino acids (e.g., leucine), monosaccharides (e.g., glucose or arabinose), disaccharides (e.g., lactose, sucrose, and maltose), oligosaccharides and polysaccharides (e.g., dextran), polyols (e.g., sorbitol, mannitol, xylitol), salts (e.g., sodium chloride, calcium carbonate), or mixtures of these excipients. Preferably, monosaccharides or disaccharides are used, with lactose or glucose being particularly preferred, and not exclusively, in hydrated form.

[0115] Within the scope of the inhalable powder according to the invention, the excipients have a maximum average particle size of up to 250 μm, preferably 10 to 150 μm, and most preferably 15 to 80 μm. Adding a finer portion of excipient with an average particle size of 1 μm to 9 μm to the above-mentioned excipients sometimes appears suitable. These finer excipients are also selected from the possible excipients listed above. Finally, to prepare the inhalable powder according to the invention, a micronized formulation, preferably with an average particle size of 0.5 μm to 10 μm, is added to the excipient mixture. Methods for producing the inhalable powder according to the invention by grinding and micronization, and by finally mixing the components together, are known in the prior art.

[0116] In formulations intended for use in the respiratory tract (including intranasal formulations), the active compound is typically configured to have a small particle size, for example, about 5 micrometers or smaller, using techniques such as micronization. In some embodiments, sustained-release formulations of the active compound are used. In some embodiments, the active compound is administered orally as a free-flowing powder via an inhaler.

[0117] The pharmaceutical compositions and methods disclosed herein also include additional therapeutically active compounds (second agents) as described herein and / or known in the art, which are generally used together with compositions comprising compound 1 of this disclosure for the treatment of one or more pathological conditions. The combination of therapeutic agents works synergistically to achieve the treatment or prevention of the various diseases, conditions, and / or symptoms described herein.Such second agents include, but are not limited to, prostaglandins, endothelin antagonists, cytoplasmic kinase inhibitors, receptor kinase inhibitors, endothelin receptor antagonists (e.g., ambrisentan, bosentan, and sitaxsentan), PDE5 (PDE-V) inhibitors (e.g., sildenafil, tadalafil, and vardenafil), and calcium channel blockers (e.g., amlodipine, felodipine, and varepamil). Diltiazem and menthol, prostacyclin, treprost, iloprost, beraprost, nitric oxide, oxygen, heparin, warfarin, diuretics, digoxin), cyclosporine (e.g., cyclosporine A), CTLA4-Ig, antibodies (such as ICAM-3, anti-IL-2 receptor (anti-Tac), anti-CD45RB, anti-CD2, anti-CD3 (OKT-3), anti-CD4, anti-CD80, anti-CD86), agents that block the interaction between CD40 and gp39 (such as antibodies specific to CD40 and / or gp39, i.e., CD154), fusion proteins constructed from CD40 and gp39 (CD40 1g and CD8gp39), inhibitors (such as nuclear translocation inhibitors of NF-κB function, such as deoxyguanidin (DSG), cholesterol biosynthesis inhibitors such as HMG) CoA reductase inhibitors (lovastatin and simvastatin), nonsteroidal anti-inflammatory drugs (NSAIDs) (such as ibuprofen, aspirin, acetaminophen, leflunomide, and deoxyguanidine), cyclooxygenase inhibitors such as celecoxib, steroids such as prednisolone or dexamethasone, gold compounds, beta-agonists such as salbutamol, LABAs such as salmeterol, leukotriene antagonists such as montelukast, and antiproliferative agents such as methotrexate and FK506 (tacrolimus, Prograf). Mycophenolic acid esters, cytotoxic drugs such as azathioprine, VP-16, etoposide, fludarabine, doxorubicin, acridine, camptothecin, cytarabine, gemcitabine, fludeoxyuridine, phenylalanine mustard, and cyclophosphamide, antimetabolites such as methotrexate, topoisomerase inhibitors such as camptothecin, DNA alkylating agents such as cisplatin, kinase inhibitors such as sorafenib, microtubule toxins such as paclitaxel, TNF-α inhibitors such as tenidap, anti-TNF antibodies or soluble TNF receptors, hydroxyurea, and rapamycin (sirolimus or rapamune) or their derivatives.Therefore, in another embodiment, a method is provided for treating a subject's disease or condition by administering an effective amount of the solid crystalline form of Compound 1 or a pharmaceutical composition comprising it to a subject in need. As used herein, "administration" to a subject includes any route by which the solid crystalline form of Compound 1 is introduced or delivered to the subject to perform its intended function. Administration may be performed via any suitable route, including oral, intranasal, inhalation, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, or local administration. Administration includes self-administration and administration by another person. It should also be understood that the various methods of treatment or prevention of the medical condition are intended to represent "substantially," which includes complete but not complete treatment or prevention, and in which some biological or medically relevant outcome is achieved.

[0118] Similarly, the term "effective amount" or "drug effective amount" is an amount sufficient to achieve the desired therapeutic and / or preventive effect, for example, an amount resulting in the prevention or reduction of symptoms associated with the treated disease. The amount of compound 1 in solid crystalline form administered to the subject will depend on the type and severity of the disease and individual characteristics such as general health status, age, sex, weight, and tolerance to the drug. It also depends on the degree, severity, and type of the disease. Those skilled in the art can determine the appropriate dosage based on these and other factors. The compositions of the present invention may also be administered in combination with one or more other therapeutic compounds.

[0119] This administration of the crystalline form of compound 1 will elicit a response in the subject that is sought by the clinician and is related to, for example, cells, tissues, or fluids. Appropriate dose levels are administered for the treatment or prevention of symptoms mediated or associated with kinase inhibition (e.g., RTK inhibition). In some embodiments, the dose is administered in single or multiple doses from about 0.01 mg / kg to 500 mg / kg body weight / day. Accordingly, in some embodiments, the dose level is from about 0.1 mg / kg / day to about 250 mg / kg / day, while in other embodiments, the dose is from about 0.5 to about 100 mg / kg / day. Suitable dose levels include, for example, from about 0.01 mg / kg / day to 250 mg / kg / day, from about 0.05 mg / kg / day to 100 mg / kg / day, or from about 0.1 mg / kg / day to 50 mg / kg / day. Within this range, in some embodiments, the dosage is from about 0.05 mg / kg / day to 0.5 mg / kg / day, from 0.5 mg / kg / day to 5 mg / kg / day, or from 5 mg / kg / day to 50 mg / kg / day. For oral administration, the composition is provided in tablet form containing 1.0 mg to 1000 mg of active ingredient (including, but not limited to, 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, 750 mg, 800 mg, 900 mg, and 1000 mg of active ingredient). The dosage may be selected, for example, any dosage within any of these ranges, to achieve therapeutic efficacy and / or symptom adjustment of the dose to the treated subject. In some embodiments, such as those described in, for example, US 8257741, US8263128, WO 2010 / 132827, WO 2010 / 102066, WO 2012 / 040502, WO 2012 / 031129 and / or WO2010 / 102065, the compound of this disclosure is administered by inhalation once to 20 times, once to 15 times, once to 10 times, once to 5 times, once to 4 times, or once to 3 times daily, or once or twice daily. In some embodiments, the compound of this disclosure is administered once to 5 times daily.

[0120] In some implementations, a unit dose is sufficient to provide one or more of the following: (a) when administered to a subject, the concentration of compound C in the subject's plasma. max The concentration is approximately 1 ng / mL to 5000 ng / mL or the concentration of compound C in the subject's blood. max(a) approximately 1 ng / mL to 5000 ng / mL; and (b) approximately 1 ng / mL to 5000 ng / mL of the compound in the plasma of the subject 24 hours after administration, or approximately 1 ng / mL to 5000 ng / mL of the compound in the blood of the subject 24 hours after administration.

[0121] The crystalline form of compound 1 (particularly in the form of a pharmaceutical composition) may be used to treat any of a variety of diseases or conditions that would benefit from kinase inhibition, including those mediated or associated with the following kinases: such as cell cycle 2 kinase (Cdc2 kinase), c-Kit, c-ABL, p60src, AKT, VEGFR3, PDGFRα, PDGFRβ, PDGFR-αα, PDGFR-ββ, PDGFR-αβ, FGFR3, FLT-3, FYN oncogene kinase (Fyn) associated with SRC, FGR, and YES, and lymphocyte-specific protein tyrosine kinase. Enzymes (Lck), tyrosine kinases with Ig and EGF homologous domains (Tie-2), FMS (CSF-IR), KDR, EphA2, EphA3, EphA8, FLT1, FLT4, HCK, PTK5, RET, SYK, DDR1, DDR2, glycogen synthase kinase 3 (GSK-3), cyclin-dependent kinase 2 (Cdk2), cyclin-dependent kinase 4 (Cdk4), MEK1, NEK-2, CHK2, CKlε, Raf, checkpoint kinase 1 (CHK1), ribosomal S6 kinase 2 (Rsk2), and PAR-1. In particular, compounds, compositions, and methods for inhibiting tyrosine kinases such as, for example, cell division cycle 2 kinase (Cdc2 kinase), ERK1 / 2, STAT3, AKT, c-Kit, c-ABL, p60src, VEGFR3, PDGFRα, PDGFRβ, PDGFR-αα, PDGFR-ββ, PDGFR-αβ, FGFR3, FLT-3, FYN oncogene kinase (Fyn) associated with SRC, FGR, and YES, lymphocyte-specific protein tyrosine kinase (Lck), tyrosine kinase having Ig and EGF homologous domains (Tie-2), FMS (CSF-IR), KDR, EphA2, EphA3, EphA8, FLT1, FLT4, HCK, PTK5, RET, SYK, DDR1, and DDR2. In some implementations, the tyrosine kinase is a receptor tyrosine kinase (RTK), such as, for example, PDGFR, PDGFR-αα, PDGFR-ββ, PDGFR-αβ, or c-Kit, or combinations thereof.

[0122] Representative diseases or conditions treatable in the crystalline form of compound 1 or in pharmaceutical compositions containing it, including but not limited to PAH, primary PAH, idiopathic PAH, hereditary PAH, refractory PAH, BMPR2, ALK1, endothelial glycoproteins associated with hereditary hemorrhagic angioedema, endothelial glycoproteins not associated with hereditary hemorrhagic angioedema, drug-induced PAH and toxin-induced PAH, and PAH associated with or secondary to one or more of the following: systemic sclerosis, mixed connective tissue disease, cancer, refractory cancer, metastatic cancer, tumor formation, malformation, hyperplasia, developmental abnormalities, metaplasia, rhythmic dysplasia, connective tissue hyperplasia, and vascularization. Genetic disorders, pulmonary function disorders, cardiovascular function disorders, HIV infection, hepatitis, portal hypertension, pulmonary hypertension, congenital heart disease, hypoxia, chronic hemolytic anemia, persistent neonatal pulmonary hypertension, pulmonary venous occlusive disease (PVOD), pulmonary capillary hemangioma (PCH), pulmonary hypertension due to left ventricular disease, systolic dysfunction, diastolic dysfunction, valvular heart disease, lung disease, interstitial lung disease, pulmonary fibrosis, schistosomiasis, chronic obstructive pulmonary disease (COPD), sleep apnea, alveolar hypoventilation disorders, chronic exposure to high altitude, developmental abnormalities, chronic pulmonary thromboembolic hypertension (CTEPH), and other conditions with unclear multifactorial mechanisms. The treatment includes pulmonary hypertension, hematological diseases, myeloproliferative disorders, splenectomy, systemic diseases, sarcoidosis, pulmonary Langerhans cell histiocytosis, glycogen storage disease, vasculitis, metabolic disorders, Gaucher disease, thyroid diseases, tumor obstruction, fibrotic mediastinitis, and fibrotic mediastinitis; diseases such as pulmonary hypertension, congenital heart disease, hypoxia, chronic hemolytic anemia, neonatal persistent pulmonary hypertension, pulmonary venous obstructive disease (PVOD), pulmonary capillary hemangioma (PCH), left ventricular disease, pulmonary hypertension, systolic dysfunction, diastolic dysfunction, valvular disease, lung diseases, interstitial lung disease, pulmonary fibrosis, and schistosomiasis. Chronic obstructive pulmonary disease (COPD), sleep-disordered breathing, alveolar hypoventilation disorders, chronic exposure to high altitudes, developmental abnormalities, chronic pulmonary thromboembolic hypertension (CTEPH), pulmonary hypertension with unclear multifactorial mechanisms, hematologic disorders, myeloproliferative disorders, splenectomy, systemic diseases, sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, vasculitis, metabolic disorders, glycogen storage diseases, Gaucher disease, thyroid diseases, tumor obstruction, cavitary fibrosis, immune and inflammatory diseases, hyperproliferative diseases, kidney and renal diseases, bone remodeling diseases, metabolic diseases, vascular diseases, and chronic renal failure during dialysis.

[0123] In one aspect, the disease or symptom is pulmonary hypertension (PAH), and a therapeutically effective amount of the crystalline form of compound 1 is administered to a subject in need. In a specific implementation, the disease or symptom is PAH, primary PAH, idiopathic PAH, hereditary PAH, refractory PAH, drug-induced PAH, toxin-induced PAH, or PAH with secondary disease.

[0124] The present invention is further illustrated by the following embodiments, which should not be construed as limiting in any way.

[0125] Example

[0126] Example 1

[0127] Preparation of amorphous compound 1 (Prior art)

[0128] The synthesis of compound 1 is disclosed in U.S. Patent No. 9,815,815 (see column 29, line 25 through column 31, line 11), with reference to known synthetic procedures, including those disclosed in WO2008 / 058341 (corresponding to U.S. Patent No. 8,461,161). Therefore, for comparative purposes, compound 1 was prepared using known techniques as follows.

[0129] The synthesis of intermediate (S)-N-(3-(1-((6-chloropyrazin-2-yl)amino)ethyl)phenyl)-6-methylnicotinamide is described in Example 1 of U.S. Patent No. 8,461,161 (see column 107, line 64 to column 109, line 9). The synthesis of intermediate (S)-N-(3-(1-((6-chloropyrazin-2-yl)amino)ethyl)phenyl)-5-methylnicotinamide (i.e., the methyl group of nicotinamide at the 5-position instead of the 6-position) was carried out by the same procedure, as shown in the following reaction scheme:

[0130]

[0131] Compound 1 was then prepared by the Suzuki cross-coupling reaction of intermediate (S)-N-(3-(1-(((6-chloropyrazin-2-yl)amino)ethyl)phenyl)-5-methylnicotinamide with 3,4-dimethoxyphenylboronic acid pinacol, and purified by column chromatography as shown in the following reaction scheme:

[0132]

[0133] Specifically, under nitrogen atmosphere, intermediate (S)-N-(3-(1-(((6-chloropyrazin-2-yl)amino)ethyl)phenyl)-5-methylnicotinamide (from the crude product of the previous step; 1.10 kg), pinacol ester of 3,4-dimethoxyphenylboronic acid (0.82 kg), and sodium carbonate solution (0.48 kg in 1.76 L of water) were added to a mixture of toluene (8.8 L) and 1-propanol (4.4 L) and stirred for at least 30 minutes. Pd(PPh3)4 (0.14 kg) was added. The mixture was stirred for at least 10 minutes, and then heated under nitrogen atmosphere to 80 ± 5 °C for ≥12 hours with stirring. The sample was analyzed by HPLC to confirm the completion of the reaction (≤0.50% of starting material). Once the reaction was considered complete, it was cooled to 25 ± 5 °C and filtered. The reaction flask and filter were washed with ethyl acetate, and the combined filtrates were separated. The organic layer (upper part) was separated, washed with water (1 × 2.75 L) and brine (25% NaCl aqueous solution, 1 × 2.75 L), dried with anhydrous sodium sulfate (2 kg), and concentrated to dryness using a rotary evaporator (maximum temperature 50 °C). The resulting solid was redissolved in ethyl acetate (2.2 L), and silica-methylthiol (Pd scavenger, 0.44 kg) was added. The resulting slurry was stirred at 20 ± 5 °C for ≥12 hours. Stirring was continued until ≤20 ppm Pd was detected (add more silica-methylthiol if necessary). Once Pd removal was considered complete, the slurry was filtered, and the filtrate was concentrated to dryness using a rotary evaporator (maximum temperature 60 °C).

[0134]

[0135] The crude product was purified by column chromatography: Silica gel-packed glass columns (7 kg / column; 2 columns; total 14 kg) were used in a slurry of 5:95 (v / v) ethyl acetate, 99% hexane (total 30 L). The crude product was dissolved in DCM (2 L) and packed into each column (half for each column). Each column was eluted with 5:95 ethyl acetate, 99% hexane (10 L / column, total 20 L), followed by 25:75 ethyl acetate, 99% hexane (30 L / column, total 60 L), then 50:50 ethyl acetate, 99% hexane (30 L / column, total 60 L), then 75:25 ethyl acetate, 99% hexane (30 L / column, total 60 L), and finally eluted with ethyl acetate, 99% (370 L / column, total 740 L). Collect the eluent in a 10L fraction until the product is eluted, and in a 20L fraction until the product is completely eluted. Combine the fractions containing the product and concentrate to dryness using a rotary evaporator (maximum temperature 60°C). Dissolve the resulting solid (1.10 kg) in dilute hydrochloric acid (0.5 N, 7.98 L) while maintaining the temperature below 30°C. Slowly add the product-HCl aqueous solution to an aqueous sodium bicarbonate solution (9%, 12.1 L) while maintaining the temperature below 30°C. Stir the resulting slurry for at least 2 hours, filter the resulting solid through a GMP filter, and dry the filter cake in a vacuum oven at ≤50°C to provide compound 1 in amorphous form.

[0136] Example 2

[0137] Characterization of amorphous compound 1

[0138] Solid-state characterization of amorphous compound 1 (Example 1) was performed by X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), and brittleness and relaxation time analysis. The results showed that amorphous compound 1 formed only glass and did not exhibit a tendency to crystallize.

[0139] IX-ray powder diffraction (XRPD)

[0140] Two samples of amorphous compound 1 were examined by XRPD performed on a Bruker D8-Advance XRPD S / N: 202298 using the following parameters:

[0141] Configuration: θ / θBragg Brentano

[0142] Incident beam optics: Sola slit = 2°; diverging slit = 0.2 mm; anti-scattering screen = 21 mm

[0143] Detector beam optics: Solar slit = 2.5° Ni filter; anti-scattering slit = 3mm

[0144] Detector: PSD: Lynx Eye with 1° window

[0145] Tube: Voltage = 40kV, Current = 40mA

[0146] Scan parameters: 2°2θ-50°2θ. Step size: 0.049°2θ. Time per step: 1 second.

[0147] Total scan time: 16.5 minutes

[0148] The first sample was heated to 100°C in a DSC pan. It remained a powder, changing color from white to light yellow. The sample was then covered and placed at -20°C for approximately 24 hours, after which it was spread onto a Si zero-background plate. Since this sample did not liquefy, a second sample was prepared by spraying it onto the Si zero-background plate and placing it in a 150°C oven for approximately 1 hour until liquid was observed. The plate was then covered and transferred to a -20°C freezer for approximately 24 hours. The XRPD results for the two samples (100°C and 150°C) are provided below. Figure 1 The results show that they are all amorphous. The small peaks at approximately 31.8°2θ and 45.5°2θ are thought to originate from NaCl, which has its two strongest peaks at these positions.

[0149] II. Differential Scanning Calorimetry (DSC) and Glass Transition Temperature (Tg)

[0150] A sample of amorphous compound 1 was prepared in an Al Tzero disk with a standard rolled edge seal. Initial DSC assessment to determine Tg and possible crystallization and melting events was performed as follows:

[0151] (1) Heat to 100℃ at a rate of 10℃ / min;

[0152] (2) Maintain isothermal temperature for 5 minutes;

[0153] (3) Cool to -20℃ at a rate of 10℃ / min;

[0154] (4) Maintain isothermal temperature for 5 minutes;

[0155] (5) Heat to 320℃ at a rate of 10℃ / min;

[0156] (6) Cool to -20°C at a rate of 10°C / min; and

[0157] (7) Heat to 320℃ at 10℃ / min.

[0158] Initial DSC scan shown Figure 2The material decomposed at approximately 270°C with no melting peak. Post-experimental DSC examination revealed charred / darkened material. Given Tg = 89°C (midpoint), Tm was estimated at 210°C (applied as a 4 / 3 factor on the Kelvin scale). The initial DSC test was repeated with a new sample (the upper limit temperature was lowered to 200°C in steps (5) and (7) to avoid decomposition), and is shown below. Figure 3 In the middle. Heating to 320°C and 200°C under cooling and reheating showed no signs of crystallization. The first scan at 200°C is shown in Figure 4 From Figure 4 Tg and ΔCp (heat capacity) are obtained: Tg (midpoint) = 87.05℃. Based on ΔCp = 0.5066 J / gK, the configuration heat capacity is estimated to be ΔCp, config = ΔCp / 0.9 = 0.5629 J / gK.

[0159] III 1 H and 1 H- 13 12C nuclear magnetic resonance (NMR)

[0160] NMR experiments were performed on a Bruker AVIIIHD spectrometer equipped with a DCH cryopreservation probe, operating at 500.12 MHz for protons. Experiments were conducted in deuterated DMSO, with each sample prepared to a concentration of approximately 10 mM. Compound 1... 1 HNMR spectrum shown Figure 5 The sample contained residual (<0.01 equivalent) ethyl acetate peaks (4.04 ppm, 1.99 ppm and 1.17 ppm). 1 H- 13 The C heteronuclear single quantum coherence (HSQC) NMR spectrum is shown in Figure 6 The structure is consistent with the chemical structure. Two amine peaks (10.36 ppm and 7.66 ppm, not coupled with C) were observed in the aromatic region. All other peaks correspond to the CH / CH3 group.

[0161] IV Solubility

[0162] Amorphous compound 1 (180 mg) was dissolved in 1,4-dioxane (18 mL) and aliquoted into 18 vials. A test solvent / solvent system (50 μL) was added to each vial, and the solubility of the mixture was evaluated. If no significant dissolution was observed, the mixture was heated to approximately 40 °C and re-evaluated; if dissolution was still incomplete, the cycle was repeated with the addition of another 50 μL of solvent. After adding 300 μL of solvent, 100 μL of the aliquot was added. This procedure was continued until complete dissolution was observed or until 1 mL of solvent was added. Solvent solubility is shown in Table 1; complete dissolution was observed for 13 of the 17 solvent systems.

[0163] Table 1: Solubility of amorphous compound 1

[0164] solvent Solubility (mg / mL) ICH class 2-Propanol ≥200 3 acetone ≥200 3 Acetonitrile ≥200 2 Chloroform ≥200 2 ethanol ≥200 3 Ethanol / water (95:5% v / v) ≥200 n / a Ethyl acetate ≥200 3 methanol ≥200 2 Methyl ethyl ketone ≥200 3 Tetrahydrofuran ≥200 2 EtOAc / tBME (75: 25% v / v) ≥200 n / a EtOAc / tBME (50:50% v / v) Approximately 100* n / a EtOAc / tBME (25: 75% v / v) Approximately 12.5* n / a heptane ＜10 3 tert-butyl methyl ether ＜10 3 Toluene ＜10 2 water ＜10 n / a

[0165] *Samples heated to 40℃

[0166] V. Fragility and Relaxation Time

[0167] Amorphous compound 1 samples were prepared in an Al Tzero disk with a standard rolled edge seal. Brittleness and relaxation time parameters were determined by measuring Tg as a function of heating rate. Four heating rates were used: 1 °C / min, 5 °C / min, 10 °C / min, and 20 °C / min. DSC measurements were performed as follows:

[0168] (1) Heat to 100℃ at a rate of 10℃ / min;

[0169] (2) Maintain isothermal temperature for 5 minutes;

[0170] (3) Cool to -20℃ at a rate of 10℃ / min;

[0171] (4) Hold isothermally for 5 minutes; and

[0172] (5) Heat to 130℃ at 10℃ / min.

[0173] The results for Tg and scan rate are provided in Table 2.

[0174] Table 2: Brittleness and relaxation time of amorphous compounds 1-

[0175]

[0176] The graph of heating rate (q) versus 1 / Tg of amorphous compound 1 is shown in Figure 7 The slope is used to calculate the activation enthalpy (ΔH*), from which the brittle parameters m, D, and T0 are calculated using the following formulas:

[0177] ΔH*=-8.314x slope

[0178] m=ΔH* / (2.303x 8.314x T g )

[0179] D = 2.303xm 2 min / (mm min )

[0180] T0 = ​​T g x(1-m min / m)

[0181] m min =16

[0182] The calculated brittleness parameters are:

[0183] Tg = 362K or 89℃ (10℃ / min);

[0184] ΔH*=508118J / mol;

[0185] m = 73.3;

[0186] T0 = ​​283K or 10℃; and

[0187] D = 10.3 (in the commonly observed range of 7-15 for plexiglass).

[0188] Using brittleness parameters, the initial structural relaxation time is calculated according to the following formula (using γ = 0.9 and τ0 = 10). - 14 s), and measured at 25℃ for 3 months.

[0189]

[0190] Example 3

[0191] Identification of the crystalline form of compound 1

[0192] Experiments were conducted to identify and isolate the crystalline forms of compound 1, including polymorph screening as described below.

[0193] Example 3A: Polymorph Screening #1

[0194] Amorphous compound 1 (450 mg) was dissolved in 1,4-dioxane (72 mL) and aliquoted into 9 vials, which were then frozen at -50 °C and lyophilized overnight. One or more test solvents (see Table 3 for amounts) were added to the lyophilized material in the vials to attempt to form a slurry. The slurry / solution was then thermally cyclic (with stirring) for approximately 72 h at ambient temperature for 4 h followed by 40 °C for 4 h, without a specified heating / cooling rate. Any solids remaining after temperature cycling were separated by centrifugation and filtration, and the separated substances were analyzed by XRPD. The remaining mother liquor, after filtration or without solids, was divided into 3 equal portions and processed as follows:

[0195] (a) Evaporation - Remove the cap from the vial and allow the solvent to evaporate under ambient conditions;

[0196] (b) Add antisolvent – ​​Add 1 mL of antisolvent (heptane for all samples except acetonitrile and water, water and THF for acetonitrile and water, respectively) and let the samples stand overnight; and

[0197] (c) By quenching the vial to 5°C to 18°C ​​in a freezer.

[0198] XRPD analysis of any separated solids, the results of which are shown in Table 4, reveals that most solids separated after thermal cycling or drying are essentially crystalline and in a unique crystalline form A, while any solids recovered from evaporation are amorphous. Cooling or evaporation of the mother liquor yields very little solid matter, and no solids are separated by quenching at 5°C or -18°C.

[0199] Table 3: Solvents used in polymorph screening

[0200] solvent Added volume (μL) Antisolvent ICH class 1 acetone 50 3 2 Acetonitrile 50 2 3 Chloroform 50 2 4 ethanol 50 3 5 Ethyl acetate 200 3 6 EtOAc / tBME (50:50% v / v) 450 n / a 7 Methyl ethyl ketone 50 3 8 tert-butyl methyl ether 3000 3 9 water 3000 n / a 10 1-Butanol 200 heptane 3 11 Isopropanol 200 heptane 2 12 Isopropyl acetate 200 heptane 2 13 MeOH 200 THF 3 14 MIBK 200 heptane 3 15 MeOH / water (40:60% v / v) 450 THF n / a 16 MeOH / water (80:20% v / v) 200 THF 3 17 MeOH / water (95:5% v / v) 3000 THF 3 18 EtOH / tBME (25: 75% v / v) 3000 THF n / a

[0201] Table 4: XRPD analysis of the separated solids

[0202]

[0203] A = form A

[0204] Am = amorphous solid

[0205] NM = Not measured

[0206] -= No solids

[0207] * = After evaporation, dry in a vacuum oven (40℃, 3 hours).

[0208] evaporation :

[0209] Evaporation of ethyl acetate, methanol, and EtOAc / tBME (50:50) yielded crystalline form A. An amorphous solid was separated from tBME, water, and EtOAc / tBME (75:25). A weak peak for form A was observed in the diffraction pattern of the solid separated from EtOAc / tBME (25:75).

[0210] After thermal cycling :

[0211] Crystalline form A was prepared from acetonitrile, ethyl acetate, ethyl acetate / tBME (50:50% v / v), tBME, 1-butanol, 2-propanol, isopropyl acetate, and MIBK. Amorphous solids were isolated from water and MeOH / water (40:60% v / v). All other solvent systems (acetone, chloroform, ethanol, and MEK) provided only solutions (no solids) after thermal cycling. Therefore, new samples prepared using lower solvent volumes, which provided solids from acetone, ethanol, and MEK after thermal cycling, confirmed crystalline form A.

[0212] After drying :

[0213] All crystalline solids remained unchanged after drying. The amorphous solids separated from the water appeared to produce some weak peaks indicating crystalline form A upon drying, suggesting partial recrystallization.

[0214] evaporation :

[0215] Amorphous solids were separated by evaporation of ethyl acetate, EtOAc / tBME (50:50% v / v), 1-butanol, IPA, MeOH, MIBK, MeOH / water (80:20% v / v), and MeOH / water (95:5% v / v). Evaporation of all other samples yielded only viscous oils, which were dried in a vacuum oven (40°C, 3 hours). Amorphous solids were separated from ethanol and MEK.

[0216] Antisolvent addition :

[0217] When an antisolvent was added to the mother liquor, precipitation was observed in all solvent systems except water. After standing for 24 hours under ambient conditions, solids were isolated from acetone, chloroform, ethanol, MEK, tBME, 1-BuOH, isopropyl acetate, and MIBK. When heptane was added as an antisolvent, crystalline form A was prepared from ethanol, MEK, butanol, and MIBK. When heptane was added as an antisolvent, an amorphous solid was prepared from acetone, chloroform, tBME, and isopropyl acetate.

[0218] Collision cooling :

[0219] When the mother liquor was cooled to 5°C for 72 hours after thermal cycling, no solid material was produced in the sample. Further cooling to -18°C for 72 hours also did not produce any solid material.

[0220] Example 3B: Polymorph Screening #2

[0221] Dissolution in ethanol

[0222] Amorphous compound 1 was dissolved in ethanol (6 mL / g) and stirred at room temperature. Aliquots were taken after 24 and 48 hours and analyzed by XRPD, showing they were identical to the starting material. Water (0.02 mL) was then added to the suspension. After 72 hours, the obtained solid was pure crystalline form B. The XRPD traces are shown below. Figure 2 middle.

[0223] Example 3C: Polymorph Screening #3

[0224] Solvent screening

[0225] Compound 1 (80 mg) was suspended in various solvent / water mixtures, as shown in Table 5. Dissolution was observed in methanol, acetone, and THF. Water was added to the sample, which was then stirred at room temperature for 24 hours, filtered under vacuum, and the separated solids were analyzed by XRPD. The results are summarized in Table 5, showing that pure crystalline form B was isolated from ethanol, while all other crystalline forms showed no change in the form of the starting material.

[0226] Table 5: Conditions and XRPD Results for Solvent Screening

[0227] solvent Volume (mL / g) Observation results solid form Starting materials - - A + a small amount of B ethanol 7.5 suspension B Toluene 10 suspension A + a small amount of B Isopropanol 10 suspension A + a small amount of B Acetonitrile 7.5 suspension A + a small amount of B Ethyl acetate 10 suspension A + a small amount of B THF 4 solution N / A acetone 7.5 solution N / A methanol 2.5 solution N / A Methanol / Water (1 / 1) 5 Oil N / A Acetone / water (1 / 2) 22.5 Oil N / A THF / Water (1 / 1) 8 suspension A + a small amount of B Ethanol / Water (1 / 1) 7.5 suspension A + a small amount of B Isopropanol / Water (1 / 1) 7.5 suspension A + a small amount of B Acetonitrile / water (1 / 2) 15 suspension A + a small amount of B

[0228] A = form A

[0229] B = Form B

[0230] Example 3D: Polymorph Screening #4

[0231] Solid compound 1 (200 mg) was suspended in the following substances:

[0232] A mixture of ethyl acetate / n-heptane 1 / 1 (5 mL / g) + 1% water, at room temperature;

[0233] A mixture of ethyl acetate / n-heptane 1 / 1 (5 mL / g) + 1% water, at 60 °C;

[0234] Ethyl acetate (2.5 L / kg) + 2% water, 60°C; and

[0235] A mixture of ethanol and water in a 3 / 7 ratio (5 mL / g), at 60 °C.

[0236] The suspension was stirred for 24 hours and then filtered under vacuum. The obtained solids were analyzed by XRPD. Table 6 summarizes the conditions and results. The mixture of ethyl acetate and n-heptane produced a heterogeneous suspension. No change was observed after 24 hours, and the obtained solids were identical to the original mixture of polymorphs. Only ethyl acetate (+2% water) caused the solids to dissolve. More solids were added until a suspension was obtained. After 24 hours at 60°C, a thick suspension was obtained. The obtained solids were form B with trace amounts of form A. Partial solution was observed using ethanol. The addition of water (0.5 kg / L) caused the formation of a precipitate, which was separated and found to be pure form B.

[0237] Table 6: Conditions and XRPD Results for Secondary Solvent Screening

[0238] solvent Volume (mL / g) Observation results solid form - - - A + a small amount of B <![CDATA[EtOAc / n - heptane (1% H2O)]]> 5 Uneven A + a small amount of B <![CDATA[EtOAc / n - heptane (1% H2O)]]> 5 Uneven A + a small amount of B <![CDATA[EtOAc(2%H2O)]]> 2.5 Dissolution / Suspension B+ Trace A <![CDATA[EtOH / H2O]]> 5 Partial solution / suspension B

[0239] Example 4

[0240] Comparative properties of crystalline forms A and B

[0241] I XRPD

[0242] X-ray powder diffraction (XRPD) patterns were obtained using Datacollector software on PANalytical X′Pert Pro, employing a 3152 / 63 focusing X-ray microscope and a Pixcel detector. Instrument conditions are provided below:

[0243]

[0244]

[0245] The XRPD of the crystalline form A of compound 1 obtained is shown in Figure 1. Figure 3 The XRPD of the crystalline form B of compound 1 obtained is shown in the figure. Figure 10 Table 7 lists the diffraction peaks for form A (left column) and form B (right column).

[0246] Table 7. List of XRPD peaks - Form A (left column) and Form B (right column)

[0247]

[0248]

[0249] II Differential Scanning Calorimetry

[0250] Differential scanning calorimetry (DSC) was performed on a Mettler Toledo 823E instrument using STARe V15.00 software, with an aluminum (40 μL) tray and cover, a temperature range of 35 °C–250 °C (10 °C / min), and nitrogen (60 ml / min) as the purge gas. The DSC curves are shown below. Figure 11 (Crystal form A) and Figure 12 (in crystalline form B).

[0251] III TGA

[0252] TGA thermal analysis curves of crystalline forms A and B of compound 1 were obtained using a Mettler Toledo TGA / DSC 3+ (software: STARe V16.00), an aluminum (100 μL) disk, at a temperature range of 35 °C–250 °C (10 °C / min), and with nitrogen (50 ml / min) as the purge gas. The TGA thermal analysis curve of crystalline form A of compound 1 is shown in [image / image / description]. Figure 7 The TGA thermal analysis diagram of crystalline form B of compound 1 is shown in the figure. Figure 8 middle.

[0253] IV Infrared

[0254] Infrared spectra of crystalline form A of compound 1 were recorded using a Perkin Elmer Spectrum 2 with an MIR source, a LiTaO3 detector, and an OptKBr beam splitter, using a Universal ATR Diamond attachment. Infrared spectra of crystalline form B of compound 1 were recorded using a Perkin Elmer Spectrum 100 with an MIR source, a LiTaO3 detector, and an OptKBr beam splitter, using a Universal ATR Diamond / ZnSe attachment. In both cases, the spectra were recorded at a scan rate of 0.2 and a resolution of 4, and at a depth of 4000 cm⁻¹. -1 -650cm -1 The spectral range was collected in four scans. The infrared spectrum of crystalline form A of compound 1 is shown in... Figure 9 The infrared spectrum of crystalline form B of compound 1 is shown in the figure. Figure 10 Table 8 lists the main IR peaks for form A (left column) and form B (right column).

[0255] Table 8. Major IR peaks for crystal forms A and B (sorted by intensity)

[0256]

[0257]

[0258] V slurry in ethyl acetate at various temperatures

[0259] The starting material was crystalline form A of compound 1, containing a very small amount of crystalline form B. This material was slurried in ethyl acetate and stirred under the time, volume, and temperature conditions shown in Table 9 (note that experiments conducted at 60°C resulted in complete dissolution and are not listed in Table 9). The mixture showed a very slow conversion of crystalline form A to form B at lower temperatures (10°C and room temperature). Pure form B was obtained after 89 hours at a higher temperature (45°C). Figure 11 , Figure 12 and Figure 13 The XRPD of each slurry compared to the starting material and pure polymorph is shown (at 10 °C, room temperature, and 45 °C, respectively); the results are summarized in Table 9.

[0260] Table 9 :

[0261] Ethyl acetate slurry of crystalline compound 1

[0262]

[0263] VI Solubility Study

[0264] use The solubility of crystalline forms A and B of compound 1 in ethyl acetate was determined using a parallel crystallizer. Two heating rates were used: 1 °C / min and 0.5 °C / min. The suspension was heated to 78 °C at 0.2 °C / min and cooled to 10 °C, and held at 10 °C for 2 hours. Figure 14 The obtained solubility curves (concentration versus temperature, in mg / mL) are shown, demonstrating that the solubility of crystalline forms A and B is similar. Solubility was also determined in ethyl acetate + 2% water. The suspension was heated to 78°C at 1°C / min, cooled to 10°C at 0.2°C / min, and held at 10°C for 2 hours. Figure 15 The obtained solubility curves were compared with those of pure ethyl acetate. The results showed that crystalline forms A and B had similar solubility in ethyl acetate + 2% water compared to pure ethyl acetate, and were significantly more soluble in the presence of water (2%). In both systems, crystalline form B had slightly lower solubility.

[0265] VII. DSC and TGA of Forms A and B

[0266] The crystalline forms A and B of compound 1 were analyzed by DSC and TGA; the results are shown in Table 10. DSC and TGA analyses indicate that crystalline form B is an anhydrous polymorph. Crystalline form B has a slightly higher melting point and a slightly lower enthalpy of fusion, indicating that the two polymorphs are entropy-dependent.

[0267] Table 10 :

[0268] DSC and TGA analysis of crystal forms A and B

[0269]

[0270] VII. TGA of a mixture of crystalline forms A and B

[0271] Compound 1 (20 mg), containing a mixture of crystalline forms A and B, was heated to 85 °C at a rate of 5 °C / min, held at 85 °C for 10 minutes, and then cooled to room temperature. The TGA thermal analysis is illustrated in [image / image / etc.]. Figure 22 The total weight loss was 0.25%. The resulting solids were analyzed by XRPD and shown in [data missing]. Figure 23 The images show that crystalline form B still exists after the TGA experiment (reference (top); sample after TGA (middle); and initial sample (bottom)).

[0272] IX Sample Stress

[0273] Two samples, one containing pure crystalline form A of compound 1 and the other containing a mixture of crystalline forms A and B of compound 1, were placed in open vials for 6 days at 40°C and 75% relative humidity, and then analyzed by XRPD. The results are shown in Table 11; no changes were observed by XRPD.

[0274] Table 11: XRPD observation results after stress conditions

[0275]

[0276] X freeze-dried

[0277] 10 mg of crystalline form A of compound 1 dissolved in 1 mL of 1,4-dioxane was frozen at -50 °C and then lyophilized overnight. XRPD analysis of the starting material and the lyophilized product yielded diffraction patterns confirming that lyophilization converted crystalline form A to an amorphous form. More specifically, the analysis showed that form B was an anhydrous / unsolvable solid, form A was slowly converted to form B, and forms A and B could be enantiotropic, despite having similar stability.

[0278] Example 5

[0279] Single-crystal X-ray structure of crystalline form A of compound 1

[0280] Single crystals of crystalline form A of compound 1 were obtained from dichloromethane / pentane.

[0281] Colorless crystals measuring 0.15mm × 0.08mm × 0.04mm were mounted on a freezer ring containing Paratone oil. This was done in a Cu K... α radiation Single-crystal X-ray diffraction studies were performed using a Bruker Microstar APEX IICCD diffractometer. Data were collected in a nitrogen flow at 100 K, using φ and φ0. Scanning was performed. The distance from the crystal to the detector was 40 mm, and exposure times were 5, 10, 15, 25, and 40 seconds, depending on the 2θ range per frame, using a scan width of 1.00°. Data collection was 99% complete at θ of 66.569°. A total of 13,233 reflections were collected, with coverage indices -5 ≤ h ≤ 3, -18 ≤ k ≤ 17, and -19 ≤ l ≤ 18. 4,186 reflections were found to be symmetrically independent, R... int The value is 0.0405. Indexing and cell refinement indicate the original monoclinic lattice. The space group is H21. Data were integrated using the Bruker SAINT software program and scaled using the SADABS software program. A fully phase-determined model consistent with the proposed structure was obtained by solving using the direct method (SHELXT). All non-hydrogen atoms were anisotropically refined using full matrix least squares (SHELXL-2014). All carbon-bonded hydrogen atoms were placed using a riding model. The positions of hydrogen atoms were constrained relative to their parent atoms using the appropriate HFIX command in SHELXL-2014. SQUEEZE analysis revealed no solvent-accessible voids in the structure.

[0282] Figure 24 An ORTEP diagram illustrating the crystal structure of crystalline form A of compound 1 is shown. Furthermore, the following tables provide the structural features of crystalline form A of compound 1: Table 12 summarizes the crystallographic data; Table 13 shows the bond lengths. Table 14 shows the bond angles [°]; Table 15 shows the atomic coordinates (×10). 4 and equivalent isotropic displacement parameters Table 16 shows the hydrogen coordinates (×10) 4 and isotropic displacement parameters Table 17 shows the anisotropic displacement parameters.

[0283] Table 12. Crystal data and structural refinement

[0284]

[0285]

[0286] Table 13:

[0287]

[0288]

[0289] Table 14: Bond Angles [°]

[0290]

[0291]

[0292] Table 15: Atomic coordinates (×10) 4 ) and equivalent isotropic displacement parameters ( 2 ×10 3 U(eq) is defined as orthogonalization U ij (one-third of the trajectory of the tensor)

[0293]

[0294] Table 16: Hydrogen coordinates (×10) 4 Isotropic displacement parameters

[0295]

[0296] Table 17: Anisotropic Displacement Parameters

[0297] (The anisotropic displacement factor index takes the following form:)

[0298] -2π 2 [h 2 a* 2 U 11+...+2h ka*b*U 12 ])

[0299]

[0300]

[0301] Example 6

[0302] Analysis of the crystalline form A of compound 1

[0303] IX-ray powder diffraction (XRPD)

[0304] XRPD analysis was performed on a PANalytical X′pert pro with a PIXcel detector (128 channels), scanning the sample between 3°2θ and 35°2θ. The material was gently ground to remove any clumps and loaded onto a porous plate with a Kapton or Mylar polymer film for sample support. The porous plate was then placed in a diffractometer and irradiated with Cu K (α1) radiation. Analysis was performed using an α1:α2 ratio of 0.5, with a 40 kV / 40 mA generator set in transmission mode (step size 0.0130°2θ, step time 18.87 s). Data were visualized and images generated using the HighScore Plus 4.7 desktop application (PANalytical, 2017). The XRPD 2θ diffraction pattern of crystalline form A of compound 1 is shown in [image / image / description]. Figure 25 The material is shown to be highly crystalline.

[0305] II-polarized light microscopy

[0306] The presence of crystallinity (birefringence) was determined using an Olympus BX50 polarizing microscope equipped with a Motic camera and image capture software (Motic Images Plus 2.0). Unless otherwise noted, all images were recorded at 200x magnification using a 20x objective lens. Visible unpolarized (top) and polarized (top) microscopic images of crystalline form A of compound 1 are shown below. Figure 26 In the image, the aggregated particles do not have a clear shape.

[0307] III. Liquid Chromatography-Mass Spectrometry (LC-MS)

[0308] The LC-MS of crystalline form A of compound 1 was determined using the following parameters:

[0309] ACE EXCEL3super C18 column, 3.0μm, 75mm × 4.6mm

[0310] Mobile phase A: 0.1% formic acid in H2O solution

[0311] Mobile phase B: 0.1% formic acid in MeCN solution

[0312] Diluent: 50:50 MeCN / H2O (% v / v)

[0313] Flow rate 15 mL / min

[0314] Runtime: 20 minutes

[0315] Column temperature: 30℃

[0316] Injection volume: 10 μL

[0317] PDA range: 190nm-400nm

[0318]

[0319] The LC-MS spectrum of crystalline form A of compound 1 is shown in Figure 1. Figure 27 In the middle, the observed peak is:

[0320] m / z 470.1 [M+H] + It is consistent with the chemical structure;

[0321] m / z 236.0 [M+2H] 2+ ;

[0322] m / z 256.0 [M+H+Na] 2+ ;as well as

[0323] m / z 938.9 [2M+H] + .

[0324] IV. High-Performance Liquid Chromatography (HPLC)

[0325] The following describes how to run compound 1 in its crystalline form A via PLC:

[0326] Column: Accucore RP-MS 150mm×4.6mm, 2.6μm

[0327] Column temperature: 20℃

[0328] Autosampler temperature: Ambient temperature

[0329] UV wavelength: 270nm

[0330] Injection volume: 15 μL

[0331] Flow rate: 15 mL / min

[0332] Mobile phase A: 0.1% TFA in H2O:MeCN (75:25% v / v) solution

[0333] Mobile phase B: 0.1% TFA in MeCN solution

[0334]

[0335] The HPLC chromatogram of crystalline form A of compound 1 is shown in Figure 1. Figure 28 The results confirmed that the sample was 99.3% pure. The integration results are shown in Table 18.

[0336] Table 18: HPLC Peak Integrals

[0337]

[0338] V Thermogravimetric / Differential Thermal Analyzer (TG / DTA)

[0339] Approximately 5 mg of crystalline form A of compound 1 was weighed into an open aluminum dish and loaded into a simultaneous thermogravimetric / differential thermal analysis (TG / DTA) instrument, which was maintained at room temperature. The sample was then heated from 20 °C to 350 °C at a rate of 10 °C / min, during which the change in sample weight and any differential thermal events (DTA) were recorded. Nitrogen gas was used as the purge gas at a flow rate of 300 cm⁻¹. 3 / min. The TG / DTA thermal analysis of crystalline form A of compound 1 is illustrated in the figure. Figure 29 Thermogravimetric analysis (upper trace) showed no significant mass loss prior to degradation. Differential thermal analysis (lower trace) revealed an endothermic melting event (starting at approximately 134 °C). Therefore, the melting initiation temperature of compound 1 was approximately 134 °C. A small mass increase (approximately 0.3%) was observed near the melting temperature.

[0340] VI Differential Scanning Calorimetry (DSC)

[0341] Approximately 5 mg of crystalline form A of compound 1 was weighed into an aluminum DSC pan and non-hermetically sealed with a perforated aluminum cap. The sample pan was then loaded into a Seiko DSC 6200 (equipped with a cooler) that was cooled and maintained at 20°C. Once a stable heat flow response was obtained, the sample and reference were heated to melting at a scan rate of 10°C / min, and the resulting heat flow response was monitored. Nitrogen was used as the purge gas at a flow rate of 50 cm⁻¹. 3 / min. DSC thermal analysis graph is shown in Figure 30In the diagram: (a) first heating step; (b) cooling step; and (c) second heating (20°C to 200°C). The sample was heated to melt (approximately 200°C), then cooled to 20°C, and then reheated to melt again. A rapid endothermic event due to melting was observed during the first heating step, starting at approximately 133°C, consistent with the melting initiation observed by TG / DTA. No thermal event was observed during cooling, indicating that the material remained amorphous upon cooling. A weak thermal event was observed at approximately 83°C during the second heating step, likely due to the glass transition.

[0342] VII. Gravimetric Vapor Adsorption (GVS)

[0343] Approximately 10-20 mg of crystalline form A of compound 1 was placed in a mesh vapor adsorption equilibrium pan and placed in a HidenAnalytical IGASorp moisture adsorption analyzer balance. The sample was subjected to a ramp curve from 40% to 90% relative humidity (RH) in 10% increments, holding the sample at each step until a steady weight was reached at 25°C (98% of steps completed, minimum step size 30 min, maximum step size 60 min). After completing the adsorption cycle, the sample was dried to 0% RH using the same procedure, eventually returning to the starting point of 40% RH. Two cycles were performed. The weight change during the adsorption / desorption cycle was plotted to determine the hygroscopicity of the sample. Figure 31 The GVS isotherm (double cycle) is shown. Figure 32 GVS kinetics are shown. The approximately 0.7% mass increase at up to 90% relative humidity indicates that the material is slightly hygroscopic. The material exhibits a Langmuir type I isotherm. No signs of recrystallization or change of form are observed. (Note that the artifacts observed during the first desorption step, approximately 300 min, are considered to be due to experimental error.)

[0344] VIII Freeze-dried

[0345] The crystalline form A (180 mg) of compound 1 was dissolved in 1,4-dioxane (18 mL). 1 mL (10 mg) of the solution was transferred to a vial, frozen at -50 °C, and then lyophilized overnight. The starting material and lyophilized product were analyzed by XRPD. The obtained XRPD 2θ diffraction pattern is shown in [Figure / image ... Figure 33 Furthermore, it was demonstrated that freeze-drying transforms the crystalline form A of compound 1 into its amorphous form.

[0346] IX physical stability

[0347] 10 mg of crystalline form A of compound 1 was weighed into vials. Two vials were then stored separately for one week under various temperature and relative humidity conditions. HPLC analysis was performed to assess changes in purity, and the results are shown in Table 19, indicating that no significant decrease in purity was observed under any test conditions.

[0348] Table 19: HPLC Analysis

[0349]

[0350] XRPD analysis was performed to detect changes in crystallinity. The XRPD diffraction pattern is shown in... Figure 34 Furthermore, it was demonstrated that the crystalline form A of compound 1 was maintained under the test conditions (i.e., it did not transform into the amorphous form); that is, it was stored for one week under different temperature (ambient temperature, 25°C, 40°C, 80°C) and relative humidity (ambient, 60%, 75%) conditions.

[0351] X thermodynamic solubility

[0352] Weigh 10 mg of crystalline form A of compound 1 into a vial and add the following substance in 1 mL:

[0353] pH 3 buffer (0.2M sodium citrate and 0.2M citric acid);

[0354] pH 4.5 buffer (0.2M sodium acetate and 0.2M acetic acid);

[0355] pH 6.8 buffer (0.2M KH2PO4 and 0.2M NaOH); and

[0356] Deionized water.

[0357] The pH value was measured after adding the buffer solution. The material was kept at ambient temperature for approximately 24 hours with stirring, and the pH was measured again. The pH values ​​are shown in Table 20, and no significant changes were observed.

[0358] Table 20: Solubility of crystalline form A of compound 1 in buffer solution

[0359]

[0360] The remaining solids were separated by filtration and analyzed by XRPD. The XRPD diffraction pattern is shown in the figure. Figure 35 The results showed that form A was isolated from all buffer systems and water. HPLC analysis of the filtered mother liquor showed that compound 1 had low concentrations (<0.05 mg / mL) in all buffer systems and water.

[0361] Example 7

[0362] Single-crystal X-ray structure of compound 1, form B

[0363] Single-crystal diffraction data were collected using a Rigaku diffractometer equipped with a MicroMax-007HF Microfocus rotating anode X-ray generator, MoKα radiation, a Pilatus 200K hybrid pixel array detector, and an Oxford Cryosystems Cryostream 700plus cryogenic system (T = -173°C). Global volume data collection was completed during the scan. The programs used were: data collection and reduction, CrysAlisPro 1.171.39.12b with absorption correction, and the Scale3Abspack scaling algorithm. The crystal structure was solved using the computer program SHELXT, and observed using the program SHELXle. Missing atoms were subsequently located from the difference Fourier synthesis and added to the atom list. The intensity of all measurements was compared to the F-value using SHELXL2018 / 3. 2 Perform least-squares refinement. Refine all non-hydrogen atoms, including anisotropic shift parameters.

[0364] The obtained ORTEP plot (50%) of atom numbering is shown in Figure 36 The absolute structure shown in the figure was randomly selected and has an R1 value of 6.7%. Compound 1, in form B, crystallizes in chiral space group P 212121 with symmetry operations:

[0365] 1′x, y, z′

[0366] 2′-x+1 / 2,-y,z+1 / 2′

[0367] 3′-x, y+1 / 2, -z+1 / 2′

[0368] 4′x+1 / 2,-y+1 / 2,-z′

[0369] Figure 37 A comparison is shown between the actual form B spectrum and a simulated spectrum from single-crystal X-ray diffraction (SCXRD) data. Both correspond to the same crystalline phase. The small shift observed is due to the different measurement temperatures.

[0370] Table 21 shows the crystal data and structural refinement of crystalline form B of compound 1. Table 22 shows the bond lengths of crystalline form B of compound 1. Table 22 shows the bond angles [°] of crystalline form B of compound 1. Table 23 shows the torsion angles [°] of crystalline form B of compound 1.

[0371] Table 21: Crystal data and structural refinement

[0372]

[0373] Table 22:

[0374]

[0375]

[0376] Table 22: Bond Angles [°]

[0377]

[0378]

[0379] Table 23: Angle of Twist [°]

[0380]

[0381] Example 8

[0382] Micronization of crystalline compound 1

[0383] For the purposes of inhalation formulations, it is desirable to obtain compound 1 with a small particle size, preferably a Dv50 of 2 μm-3 μm. To this end, various particle engineering techniques were evaluated to produce stable micronized compound 1 that maintains its crystalline form and the purity of the starting materials. The evaluation methods included:

[0384] Jet milling involves feeding powder into a grinding chamber, where compressed nitrogen gas is used to create vortex motion that promotes particle-to-particle collisions, thereby reducing particle size.

[0385] Wet milling – involves microfluidizing suspensions via high-pressure homogenization (HPH); and

[0386] Wet polishing – a combination of wet grinding of a suspension and subsequent separation by spray drying, i.e., a three-step process:

[0387] (i) Prepare the feed suspension;

[0388] (ii) Microfluidizing the suspension by high-pressure homogenization (HPH); and

[0389] (iii) Spray drying of the suspension to separate micronized particles.

[0390] Compared to jet milling, wet polishing offers advantages such as precise control over particle size distribution and a smoother final surface area, potentially enabling high-dose / pure substance formulations.

[0391] I. Particle size reduction - jet milling

[0392] By using pressurized nitrogen (Venturi pressure P) through a Venturi system 排气 Higher than grinding pressure P 研磨 The vacuum generated forces the crystalline compound A, form 1, tangentially into the grinding chamber. Compressed nitrogen is also used in nozzles within the chamber walls. The feed rate is set and controlled automatically (via a gravimetric feeder) or manually. Once inside the grinding chamber, the particles are accelerated in a helical motion by a series of peripheral jets. Compressed fluid exiting from the nozzles flows from P... 研磨 The expansion and very high rotational speed within the chamber cause micronization as slower-moving particles collide with faster-moving particles in the spiral path. While centrifugal force holds larger particles at the periphery of the grinding chamber, smaller particles are discharged from the center of the chamber along with exhaust gas. At different feed rates (F... 进料，JM Five 20g tests were conducted in a 1.5″ jet mill under the specified conditions and pressure; optimal conditions were used for a sixth, larger-scale 50g test. The conditions for each run and the analysis of the resulting micronized material are shown in Table 24.

[0393] Table 24: Product characterization of compound 1 form A obtained by jet milling and micronization crystallization

[0394]

[0395]

[0396] P = Crystalline form

[0397] A = amorphous

[0398] II. Grain Size Reduction - Wet Polishing (Wet Grinding + Spray Drying)

[0399] Step 1 - Wet grinding

[0400] An aqueous suspension (5% w / w or 10% w / w) of crystalline compound 1, form A, in water was treated via microfluidization using an HPH18 (M-110EH-30 Microfluidics Pilot) wet milling apparatus equipped with an auxiliary processing module (200 μm) and an interaction chamber (Z-shaped, 100 μm). The unit was started up with only the auxiliary processing module (200 μm) at a given pressure for the first five cycles to pre-mill the suspension. The interaction chamber (100 μm) was then introduced, and the suspension was treated at a defined pressure. Temperature was controlled and recorded using the reactor jacket. To address clogging of the micronization chamber (observed after approximately 25 cycles), the pressure was increased from 25 bar to 60 bar. Analysis of the resulting micronized material is shown in Tables 25 and 26.

[0401] Table 25: Wet Grinding

[0402]

[0403] Table 26: Wet milling - Product characterization

[0404]

[0405]

[0406] Step 2 - Spray Drying

[0407] Then use SD48 The B-290 advanced spray dryer sprays wet-milled materials to dry them. The dryer is equipped with two fluid nozzles and a high-efficiency cyclone separator to collect the dried product. The unit operates in a closed loop, where a suction pump blows in nitrogen gas at 100% capacity (the flow rate of the drying nitrogen is F). 干燥 At maximum capacity, approximately 20 kg / h). Adjust the flow rate of atomized nitrogen to 40 mm in the rotor flowmeter. Stabilize the spray dryer with water and adjust the flow rate before feeding the feed suspension. Adjust the inlet temperature to achieve the target outlet temperature. Expose the sample to different process conditions to evaluate the feed mixture flow rate (F). 进料，SD The temperature T of the drying gas at the outlet of the spray drying chamber 出口 The impact.

[0408] The material separated from the first run 1A was divided into three aliquots (1BI, 1BII, and 1BIII); runs 2A and 3A were spray-dried to produce samples 2B and 3B, respectively. The resulting materials were characterized by PSD, XRPD, amorphous content, water content, and determination of related substances. The specific surface area (SSA) of runs 2B and 3B was also characterized. Conditions and product characterization are shown in Table 27 (runs 1BI, 1BII, and 1BIII) and Table 28 (runs 2B and 3B).

[0409] Table 27: Spray drying conditions and product characterization

[0410] (Run 1BI, 1BII, and 1BIII)

[0411]

[0412]

[0413] Table 28: Spray drying conditions and product characterization

[0414] (Running 2B and 3B)

[0415]

[0416] III capsule filling

[0417] Micronized material, prepared by jet milling or wet polishing as described in Section I or II above, was filled into clear hydroxypropyl methylcellulose (HPMC) No. 3 capsules. More specifically, clear HPMC No. 3 capsules were filled using a spiral-filled Quantos unit with a nominal throughput of approximately 100 caps / hour after 100% net weight check. The Quantos unit was placed in a climate-controlled housing (CTS ClimateZone unit) and set at 20°C–25°C and 40 ± 5% relative humidity (recorded precise conditions). The micronized crystalline compound 1, form A, was sieved through a 250 μm sieve and then conditioned at 20°C–25°C and 40 ± 10% relative humidity for at least 2 hours. The conditioned material was loaded into the Quantos dispenser head. Capsules were filled with 10.0 mg and 20.0 mg, with an exclusion limit of ± 5% of the fill weight. The fill weight of each capsule was automatically recorded and classified as PASS or FAIL. After each filling cycle, FAIL-filled capsules were discarded, and PASS capsules were manually sealed. Repeat the cycle until 60 capsules are filled. It is noteworthy that, based on the same amount of powder (20 mg) occupying space within the capsule, the wet-polished powder appears to have a lower density than the jet-milled powder. A total of 8 batches were prepared using the following methods:

[0418] Three types of jet-milled powders from operations 1, 4, and 6;

[0419] 2B wet polishing powder;

[0420] Three different HPMC sizes of capsule #3 from three different suppliers: Capsugel, Ravago, and Qualicaps; and

[0421] Two filler weights (10mg and 20mg).

[0422] The materials used in each experiment, manufacturing parameters, and capsule characterization are summarized in Table 29.

[0423] Table 29: Materials, Manufacturing Parameters, and Capsule Characterization

[0424]

[0425]

[0426] JM = Jet Polishing; WP = Wet Polishing

[0427] Capsules: C = Capsugel; Q = Qualicaps; R = Ravago

[0428] AC = Amorphous content

[0429] Example 9

[0430] The effect of adding force control agent (L-leucine)

[0431] Step 1: Micronization

[0432] Following step I above, crystalline compound 1, form A (100 g), suspended in water (1900 g) (5.0% w / w), was micronized by wet milling. The suspension was pre-milled for 5 cycles using only the auxiliary processing module (200 μm), followed by 40 cycles in the interaction chamber, both at 50 bar pressure. The conditions are shown in Table 30. PSD samples were removed after each series of cycles, and the results after 45 cycles are shown in Table 31.

[0433] Table 30: Micronization Conditions

[0434]

[0435]

[0436] Table 31: Particle Size Analysis

[0437]

[0438] Step 2: Spray dry with L-leucine

[0439] The micronized material was divided into three approximately equal batches and dried using a setup similar to that described in step 2-spray drying of Example 8. Three batches (2A, 2B, and 2C) with different leucine concentrations were obtained by spray drying with an SD41 spray dryer to increase the leucine content of the coating. PSD, SEM, determination, and XRPD of the materials were evaluated. Spray drying process parameters and product characterization are shown in Table 32. XRPD showed characteristic peaks for crystalline compound form A, but not for crystalline L-leucine, indicating that the L-leucine in the spray-dried material was in an amorphous form.

[0440] Table 32: Spray drying conditions and product characterization

[0441]

[0442] Step 3: Capsule filling

[0443] Following step III above, the micronized, spray-dried material was filled into capsules. Three batches of capsules were prepared from three different spray-dried products using HMPC Capsugel No. 3 capsule shells. The capsules were filled with the following material (capsules have a rejection limit of ±5% of the filling weight):

[0444] 20mg of 2A powder is filled into 3A capsules;

[0445] 15mg of 2B powder is filled into 3B capsules; or

[0446] 15mg of 2C powder is filled into 3C capsules.

[0447] The NGI (n=3) and DUSA (n=10) of the filled capsules were analyzed, and manufacturing data and capsule characterization are shown in Table 33. Notably, the powder flow during the filling process was significantly worse for capsules with added L-leucine compared to compound 1 alone, especially for formulations with higher leucine content. Consequently, the filling weight was reduced from 20 mg to 15 mg due to powder adhesion to the inner wall of the capsule shell.

[0448] Table 33: Manufacturing parameters and capsule characterization of capsules containing compound 1+leucine

[0449]

[0450] FPD: Fine Particle Dosage

[0451] ED: Exhaustion dose (from inhaler)

[0452] NGI: Next-Generation Shocker

[0453] DUSA: Dosage Unit Sampling Device

[0454] MMAD: Mass Median Aerodynamic Diameter

[0455] FPF ED，DUSA Fine particle fraction (fine particle dose exceeds DUSA excretion dose)

[0456] FPF ED，NGI Fine particle fraction (fine particle dose exceeds the excretion dose of NGI)

[0457] GSD: Geometric Standard Deviation

[0458] Example 10

[0459] Carrier-based formulations

[0460] To investigate aerodynamic performance optimization, six carrier-based formulations were examined using three blending mechanisms: two low-shear blends (Turbula), two high-shear blends (Diosna), and two jet milling methods. Blending and capsule filling parameters are shown in Table 35, and the characterization of capsules filled with the blends is shown in Table 36. L-leucine or lactose was used as the carrier. Two grades of lactose were used:

[0461] Respitose SV003-sieved lactose crystals with a smooth surface and an average particle size >50μm (Dv10=19μm-43μm / Dv50=53μm-66μm / Dv90=75μm-106μm); bulk density = 630g / L; and

[0462] Tablettose 80 - Granular lactose, particle size 0μm-630μm (<63μm NMT 20%, <180μm 40-75%, <400μm NLT 85%, <63μm NLT 97%; average about 300μm); bulk density = 620g / L.

[0463] High shear mixing

[0464] High-shear mixing was performed using a Diosna high-shear mixer with a 0.5L bin. Lactose was sieved (Respitose SV003 = 500μm sieve; Tablettose 80 = 850μm sieve) and loaded. Crystalline compound 1 form A was sieved (using the same sieve as the lactose) and loaded, and the combined materials were mixed for 5 minutes at 450 RPM in the main impeller and 500 RPM in the shredder. Batch sizes were 20-30g, containing 60% micronized compound 1 and 40% lactose.

[0465] Low-shear mixing

[0466] Low-shear mixing was performed using a Glen Mills T2F Turbula flask with a 0.5L flask. Lactose was sieved (Respitose SV003 = 500μm sieve; Tablettose 80 = 850μm sieve) and loaded. Crystalline compound 1 form A was sieved (using the same sieve as the lactose) and loaded. The combined materials were mixed at 96 RPM for 15 minutes. Batch size was 15g, containing 60% micronized compound 1 and 40% lactose.

[0467] co-grinding

[0468] Crystalline compound 1 form A and excipients (lactose or leucine) were mixed at 96 RPM for 15 minutes using a Turbula low-shear mixer. The blended material was then fed into an MZ0502 jet mill at 60 g / h and micronized (milling pressure = 4 bar - 5.5 bar, exhaust pressure = 6 bar - 7 bar). Compound 1 (60%) was mixed with lactose (7711.01) and Compound 1 (96%) was mixed with L-leucine (7603.02).

[0469] Capsule filling

[0470] Following the aforementioned procedure, the six mixtures were filled into capsules using a Quantos spiral filler. No significant differences were observed during capsule filling for different formulations. The NGI (n=3) and DUSA (n=10) of the filled capsules were analyzed. The results are summarized in Tables 34 and 35.

[0471] Table 34: Materials and parameters of carrier-based formulations

[0472] run 1 2 3 4 5 6 Compound 1, form A(g) 18 12 9 9 21 21 Compound 1, form A (%) 60 60 60 60 60 96 excipient R T R T R Leucine Excipients (g) 12 8 6 6 14 1 Total mass (g) 30 20 15 15 35 22 Yield (%) 67 63 71 71 87 83 method HS HS LS LS JM JM T(℃) 20 20 21 20 21 20 RH (%) 51 51 51 52 50 52 Capsule number 88 63 60 57 55 54 Fill weight (target) 30 30 30 30 30 20 Fill weight (mg) 29.9±0.3 29.7±0.4 29.8±0.2 29.9±0.3 29.8±0.3 20.0±0.3

[0473] R = Respitose SV003

[0474] T = Tablettose 80

[0475] HS = High shear

[0476] LS = Low Shear

[0477] JM = Jet Milling

[0478] Table 35: Capsule characterization of carrier-based formulations

[0479]

[0480]

[0481] Example 11

[0482] High-dose crystallized dry powder inhaler (DPI) formulation

[0483] I. Wet polishing - Compound 1 alone

[0484] Two 250g batches of crystalline compound 1, form A, were each suspended in water (5L) and wet polished in a HPH18 (M-110EH-30 MicrofluidicsPilot) wet mill equipped with an auxiliary treatment module (200μm) and an interaction chamber (Z-shaped, 100μm). The unit was started at 50 bar for the first five cycles using only the auxiliary treatment module (200μm) to pre-mill the suspension. The interaction chamber (100μm) was then introduced, and the suspension was treated at 50 bar (increased to 60 or 70 bar if the system became clogged). The temperature was maintained at 18°C–28°C using the reactor jacket. The reactor stirring speed was 280 RPM (batch 1) and 390 RPM (batch 2). The first batch was micronized for 25 cycles, and the particle size distribution was analyzed after 5, 15, and 20 cycles. The second batch was micronized for 35-25 cycles, and the particle size distribution was analyzed after 15, 20, 25, and 35 cycles. The particle size distribution analysis is shown in Table 36.

[0485] Table 36: PSD analysis of compound 1 alone after wet milling

[0486]

[0487]

[0488] Two batches were then spray-dried (separately) using a Büchi spray dryer with an open-loop system. The unit was equipped with dual-fluid nozzles: a cap and an orifice diameter of 1.5 mm and 0.7 mm, respectively, and operated with nitrogen. The nitrogen-blown suction pump was set to 100% capacity (40 kg / h). The suspension feed flow rate was set to 8 ml / min (high), and the dryer outlet temperature was set to 75 ± 1 °C. The particle size distribution (PSD) and water content of the final blend of the spray-dried powders were characterized by KF, XRPD, and amorphous content (DSC), details of which are shown in Table 37. The total yield (two batches combined) was 267 g (53%).

[0489] Table 37: Spray drying conditions for compound 1 alone

[0490] parameter Batch 01 Batch 02 Input quantity (g) 5000 5000 Compound 1 (g) 250 250 <![CDATA[D 干燥 (kg / h)]]> 35 35 <![CDATA[T 入口 (℃)]]> 150-155 155-166 <![CDATA[Measured T 出口 (°C)]]> 74-76 74-76 Output cyclone separator 1(g) 124 126 Output cyclone separator 2(g) 8 9

[0491] As shown in Table 38, the particle size distribution of the two batches is 0.5μm < Dv50 < 3μm, and there is almost no difference between the two batches.

[0492] Table 38: Particle Size Distribution

[0493]

[0494] The final product showed a Dv50 of 0.6 μm with the expected amorphous content, water content, and XRPD peak, as shown in Table 39.

[0495] Table 39: Final Product Analysis

[0496]

[0497] II. Wet polishing - compound coated with L-leucine 1

[0498] Two 250g batches (250g and 238g; total 488g) of crystalline compound form A coated with approximately 2% L-leucine were each suspended in water (5L) and wet polished in a wet milling HPH18 (M-110EH-30 Microfluidics Pilot) apparatus using the same method as described in the preceding examples. The first batch was micronized for 25 cycles, and the second batch for 30 cycles. Samples were taken before the milling process and after 5, 10, 15, 20, and 25 cycles of the first batch, and after 30 cycles of the second batch. Clogging of the micronization chamber was minimized by using a pressure of 50 bar (200 μm chamber) for the first 5 cycles and a pressure of 70 bar (200 μm chamber and 100 μm chamber) for the remaining cycles. The suspensions were weighed at the beginning and end of the process to determine the process yield. The yields for the first and second batches were 99% and 97%, respectively. The conditions are summarized in Table 40, and the PSD analysis is summarized in Table 41.

[0499] Table 40: Wet polishing of compound 1 coated with L-leucine.

[0500] Micronization conditions

[0501] parameter Batch 01 Batch 02 Total input (g) 5000 5000 Total output (g) 4974 4828 Yield (%) 99 97 Compound 1 input (g) 250 238 Theoretical output (g) 249 230 Suspension temperature (°C) 19-21 21-30 Output chamber temperature (°C) 16-36 19-46 Reactor stirring speed (RPM) 320 320-390

[0502] Table 41: PSD analysis of L-leucine-coated compound 1 after wet polishing

[0503]

[0504] Then, two batches were spray-dried as described in the previous examples, and the results are summarized in Table 42. The determination of the final spray-dried product and related substances, PSD, and water content by KF and XRPD are shown in Table 43. The two sub-batch were blended to obtain the final product, with a total final yield of 137 g (68%), see Table 44.

[0505] Table 42: Spray drying conditions for L-leucine-coated compound 1

[0506] parameter Batch 01 Batch 02 Input quantity (g) 4974 4828 Compound 1 (g) 249 230 Added leucine (g) 5.0 4.9 nozzle 2.2 / 1.5 2.2 / 1.5 <![CDATA[D 干燥 (kg / h)]]> 35 35 <![CDATA[T 入口 (℃)]]> 123-161 143-166 <![CDATA[Measured T 出口 (°C)]]> 64-83 73-76 Output cyclone separator 1(g) 142 171 Output cyclone separator 2(g) 8 5 SD theoretical yield (%) 60 77

[0507] Table 43: Single Batch Analysis

[0508]

[0509] The final product showed a Dv50 of 0.6 μm with the expected amorphous content, water content, and XRPD peak, see Table 44.

[0510] Table 44: Final Product Analysis

[0511]

[0512] III capsule filling

[0513] The micronized materials prepared in steps I and II (form A of crystalline compound 1 alone and added L-leucine) were used for capsule filling experiments. HPMC No. 3 capsules (Capsugel) were filled using an MG2 FlexaLab unit (500 capsules / hour - 3000 capsules / hour) and a fully automated metering nozzle filling device. The powder was filled into a rotating product container, creating a powder bed, where a dispenser was inserted and collected to obtain the desired volume of powder. In this method, the dispenser creates a powder plug by applying compaction to the powder bed. The metered powder volume and compaction were adjusted by varying the powder bed depth in the rotating container, the dosing chamber height, and the dispenser diameter. The capsule filling process includes the following steps:

[0514] The blend is loaded into a powder hopper and formed into a powder bed in a rotating container: the powder is distributed from the hopper into the rotating container by a vibration system until a uniform powder bed is formed. The rotating container is equipped with a leveler, which prevents powder holes from forming due to immersion of the dispenser during filling. The machine is run for at least 30 minutes to allow the product to settle in the rotating container.

[0515] Feeder adjustment: The machine is operated without the MultiNETT (MG2) system, and the filling weight is checked by the weight difference during emptying. The height of the feeder is continuously adjusted until the target filling weight is achieved.

[0516] After the batch begins, the filled capsules are collected and the filling weight is measured by the weight difference of approximately 100 capsules when 5 capsules are emptied.

[0517] A summary of capsule filling parameters is shown in Table 45 (Runs 1-5) and Table 46 (Runs 6-9).

[0518] Table 45: Parameters of five capsule filling tests - Compound 1 alone

[0519]

[0520] * The product screened before capsule filling (metal sieve mesh size 30).

[0521] Table 46: Parameters of four capsule filling tests - Compound 1 + L-leucine

[0522]

[0523]

[0524] The batch analysis results are summarized in Tables 47 and 48.

[0525] Table 47: Capsule characterization of compound 1 alone

[0526]

[0527]

[0528] LC = Standard quantity

[0529] DUSA = Dosage Unit Sampling Device

[0530] ED = Exhausted dose (from inhaler device)

[0531] FPD = Fine Particle Dose

[0532] FPF ED Fine particle fraction (fine particle dose exceeds the excretion dose of NGI)

[0533] MMAD = Mass Median Aerodynamic Diameter

[0534] NGI = Next Generation Shocker

[0535] RSD = Relative Standard Deviation

[0536] GSD = Geometric Standard Deviation

[0537] Table 48: Encapsulation characterization of compound 1+L-leucine

[0538]

[0539]

[0540] Example 12

[0541] Scale-up of high-dose crystallized dry powder inhaler (DPI) formulations

[0542] Ninety percent of the purified water was added to the mixing vessel. 1.86 kg of crystalline compound 1, form B, was added to the mixing vessel, followed by the remaining purified water to obtain a 5% w / w suspension. The mixture was stirred between 200 rpm and 600 rpm for at least 2 hours until a homogeneous suspension was observed. The suspension was pre-micronized using a high-pressure homogenizer equipped with a 400 μm micronization chamber at a pressure of 70 bar. The temperature of the suspension was maintained between 15 °C and 25 °C, and the particle size distribution of compound 1 was monitored by laser diffraction. The suspension was further micronized using a 100 μm micronization chamber at a pressure of 70 bar until the particle size Dv50 of compound 1 in the suspension was less than 1 μm. 0.14 kg of L-leucine was added to the micronized suspension of compound 1 while mixing. The L-leucine was allowed to dissolve for at least 30 minutes until the suspension was homogeneous.

[0543] Assemble the PSD-1 spray dryer and configure a suitable cyclone separator and collection container for the spray-dried powder of Compound 1. After starting the program, spray-dry the micronized suspension with nitrogen drying gas under the following target (setpoint) conditions:

[0544] Process gas inlet temperature: 125℃

[0545] Process gas outlet temperature: 75℃

[0546] Process gas flow rate: 100 kg / hr

[0547] Liquid feed rate: 1.2 kg / hr

[0548] Atomizing gas flow rate: 3.3 kg / hr

[0549] Process parameters are recorded approximately every 30 minutes, and the cyclone collection container is replaced approximately every 4 hours.

[0550] Manually fill the capsules and test the aerosol properties, as summarized in Table 49 below.

[0551] Table 49 - Aerosol Properties

[0552]

[0553]

[0554] Example 13

[0555] High-dose amorphous dry powder inhaler (DPI) formulations

[0556] Distribute 3.25 kg of crystalline compound 1, form A, and 3.25 kg of L-leucine. Distribute sufficient water and ethanol to obtain a 1.26% w / w solution. While mixing, add L-leucine to water in a stainless steel treatment tank. Allow L-leucine to dissolve in water for at least 1 hour until a visually clear solution is obtained. Purge the treatment tank with nitrogen and add dehydrated alcohol to the L-leucine solution. Add compound 1 and mix the contents for at least 1 hour until a visually clear to slightly turbid solution is obtained.

[0557] Assemble the PSD-1 spray dryer and configure a suitable cyclone separator and collection container for spray-drying the powder of Compound 1. Install two 0.2 μm filters in series in the feed line between the solution tank and the spray dryer nozzle. Measure the filter integrity (i.e., bubble point) after the process is complete. After starting the program, spray-dry the solution with nitrogen drying gas using the following target (setpoint) conditions:

[0558]

[0559] Process parameters are recorded approximately every 10 minutes, and the cyclone collection container is replaced approximately every 16 hours.

[0560] The Harro Hoflinger Modu-C capsule filling machine, capsule polisher, and metal detector were assembled and graded along with all process consumables. The spray-dried compound 1 powder was loaded into the hopper and stirred using an angled two-blade agitator. Encapsulation was performed using the Automated Quality Inspection (AMV) sorting limit of + / - 7.5% of the target fill weight. Capsule fill weight and closure length were measured throughout the encapsulation process to verify that the average capsule weight was within 0.5 mg of the AMV system and that the capsules were adequately sealed. The aerosol properties of the capsules were tested and summarized in Table 50 below.

[0561] Table 50 - Aerosol Properties

[0562]

[0563] Example 14

[0564] Solubility of Compound 1 in various forms

[0565] The solubility of crystalline compound 1 form A, amorphous compound 1, L-leucine, and a mixture of compound 1 / L-leucine spray-dried powder (SDP) was determined in phosphate buffer and ethanol / water mixture.

[0566] Sample preparation

[0567] The following test solutions were used to detect samples of crystalline compound 1 form A, amorphous compound 1, compound 1 / L-leucine SPD (70:30), compound 1 / L-leucine SPD (50:50), and L-leucine. The results are shown in Tables 51 and 52.

[0568] The phosphate buffer solution contains NaH2PO4·H2O (0.345 g), sodium hydroxide aqueous solution (0.2 M, 10.001 mL), sodium chloride (0.576 g), and water (100.0 mL).

[0569] Ethanol / Water (30 / 70) contains ethanol (30.0g) and water (70.0g);

[0570] Ethanol / Water (40 / 60) contains ethanol (40.0g) and water (60.0g);

[0571] Ethanol / Water (50 / 50) contains ethanol (50.0g) and water (50.0g); and

[0572] Ethanol / water (45 / 55) contains ethanol (4.50 g) and water (5.51 g).

[0573] Table 51: Solubility results in 25 mM phosphate buffer at pH 7.4

[0574]

[0575]

[0576] The solubility results in ethanol / water at 22°C are shown in Table 52.

[0577] Table 52: Solubility in ethanol / water

[0578]

[0579] T Top layer after centrifugation

[0580] B The bottom layer after centrifugation

[0581] *Results outside the calibration curve

[0582] Example 15

[0583] In vivo studies

[0584] Three in vivo PKPD studies were used to evaluate the pharmacology of the test formulation (i.e., capsules containing crystalline compound 1, form B, of Example 12) and the reference formulation (i.e., capsules containing amorphous compound 1, of Example 13). These head-to-head studies showed that passive inhalation delivery of the test formulation (once daily for 3 days) produced significantly higher lung exposure compared to the reference formulation. Overall, the lung exposure of the test formulation was approximately twice that of the reference formulation. The test formulation inhibited PGFFB and SCF-induced phosphorylation of PDGFR and cKIT. Immediately after administration, the test formulation exhibited stronger inhibition of PDGFR and cKIT phosphorylation. For the test formulation, this target binding persisted for 8 hours post-administration, while for the reference formulation, the inhibition was reversed, corresponding to the lung level. The results of these studies are shown in Table 53.

[0585] Table 53: Summary of PKPD for Test Formulation and Reference Formulation

[0586]

[0587] Example 16

[0588] Clinical trial results

[0589] A phase 1 study was conducted in the crossover study to evaluate the bioavailability of the test formulation (crystalline compound 1, form B) compared to the reference formulation (amorphous compound 1) identified in Example 15. The study design is described below. In short, it is a 2-part, 2-treatment, 2-period, randomized, open-label, crossover design. Participants were required to participate in parts 1 and 2 and receive a single oral inhaled dose of both formulations.

[0590] Cycle 1

[0591] On day 1, subjects were administered a single oral inhalation dose of either the test formulation of crystalline compound 1 (1 × 40 mg capsules of 93% w / w formulation) or a single oral inhalation dose of the reference formulation of amorphous form of compound 1 (3 × 15 mg capsules of 50% w / w formulation) under fasting conditions. This was followed by a 3-day washout period.

[0592] Cycle 2

[0593] Subjects underwent crossover on day 4. Subjects who received the test formulation in cycle 1 received the reference formulation in cycle 2, and vice versa. Administration was repeated under the same fasting conditions. Pharmacokinetic (PK) assessments were performed 72 hours after each administration. After administration in cycle 2, subjects were restricted to day 7 for safety and PK assessments.

[0594] result

[0595] Twenty-two subjects were enrolled, and 21 completed both cycles 1 and 2. Both formulations were well tolerated, and no significant abnormalities were observed in vital signs, ECG, or laboratory results.

[0596] Dosage

[0597] The amount of compound 1 administered to each capsule was determined by gravimetric analysis of the amount of powder dispensed from the device (weighing the device before and after administration) to confirm the actual dose of dispersion. The administered weight was multiplied by the content of compound 1 in each formulation to determine the release amount for each subject.

[0598] PK Analysis

[0599] Figure 38A and Figure 38B The average concentration-time curves (±SD) of compound 1 over 4 hours and 72 hours are shown, respectively (treatment A = test formulation, treatment B = reference formulation).

[0600] like Figure 38A and Figure 38B As shown, the concentration-time curve of the test formulation differed from that of the reference formulation. The availability and extent of compound 1 in systemic circulation changed; that is, C... max The concentration was reduced to approximately 1 / 10. When the amount of compound 1 was normalized to a fine particle dose, the AUC of the test formulation was 82% of that of the reference formulation. The test formulation was found to prolong lung exposure and produce a more favorable PK curve, with a lower Cmax and prolonged AUC when matched for systemic exposure.

[0601] Other embodiments can be provided by combining the various embodiments described above. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in and / or listed in the patent application data sheets are incorporated herein by reference in their entirety. Further embodiments may be provided by modifying various aspects of the embodiments to incorporate the concepts of the respective patents, applications, and publications, if necessary.

[0602] In light of the detailed description above, these and other changes may be made to these embodiments. Generally, the terminology used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed in this specification and claims, but rather as encompassing all possible embodiments and the full scope of equivalents conferred by such claims. Therefore, the claims are not limited by this disclosure.

[0603] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference.

[0604] This application claims priority to U.S. Provisional Application No. 63 / 185,996, filed May 7, 2021, which is incorporated herein by reference in its entirety.

Claims

1. A solid crystalline form of N-{3-[(1S)-1-{[6-(3,4-dimethoxyphenyl)pyrazin-2-yl]amino}ethyl]phenyl}-5-methylpyridine-3-carboxamide, wherein the crystalline form is form B, and form B is characterized by having XRPD plots with peaks at 5.2, 6.1, 7.6, 11.5, and 12.3 ± 0.2 degrees 2θ.

2. The solid crystalline form according to claim 1, characterized in that... Basically, it is an XRPD diagram as shown in Figure 10.

3. The solid crystalline form according to claim 1, wherein the solid crystalline form comprises at least 80% form B.

4. The solid crystalline form according to claim 3, wherein the solid crystalline form comprises at least 90% form B.

5. The solid crystalline form according to any one of claims 1-2, wherein the crystalline form is substantially pure form B.

6. A pharmaceutical composition comprising a solid crystalline form according to any one of claims 1-5 and a combination of one or more pharmaceutically acceptable carriers.

7. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition comprises an additional therapeutically active compound.

8. The pharmaceutical composition according to claim 6, wherein the composition is formulated for application to the respiratory tract.

9. The pharmaceutical composition according to claim 6, wherein the composition is in the form of an inhalable powder.

10. The pharmaceutical composition according to claim 6, wherein the composition is in the form of a dry powder.

11. The pharmaceutical composition of claim 9, wherein the inhalable powder comprises particles having a Dv50 of 2µm to 3µm.

12. The pharmaceutical composition of claim 9, wherein the inhalable powder has a median aerodynamic diameter of 0.9 µm to 4.0 µm.

13. The pharmaceutical composition of claim 9, wherein the inhalable powder is obtained by wet milling and micronization in an aqueous solution.

14. The pharmaceutical composition of claim 9, wherein the inhalable powder is obtained by jet milling micronization.

15. The pharmaceutical composition of claim 9, wherein the inhalable powder has more than 90% of the initial crystalline form.

16. The pharmaceutical composition of claim 9, wherein the inhalable powder has an initial crystalline form of more than 75%.

17. The pharmaceutical composition of claim 6, wherein one or more pharmaceutically acceptable carriers comprise lactose.

18. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition further comprises leucine.

19. The pharmaceutical composition of claim 18, wherein leucine coats the solid crystalline form.

20. The pharmaceutical composition of claim 19, wherein the leucine-coated solid crystalline form is obtained by adding leucine to a wet-milled crystalline suspension prior to spray drying.

21. A pharmaceutical dosage form comprising a pharmaceutical composition according to any one of claims 6 to 20.

22. The pharmaceutical dosage form of claim 21, wherein the dosage form is a capsule for administration using a dry powder inhaler.

23. The drug dosage form of claim 21, wherein the dosage form is a blister pack for administration with a dry powder inhaler.

24. The pharmaceutical dosage form of claim 21, wherein the dosage form is a powder for administration using a dry powder inhaler.

25. A solid unit dosage form comprising a solid crystalline form according to any one of claims 1 to 5.

26. The solid unit dosage form of claim 25, wherein the dosage form is formulated for application to the respiratory tract.

27. The solid unit dosage form according to claim 25, wherein the dosage form is in the form of an inhalable powder.

28. The solid unit dosage form according to claim 25, wherein the dosage form is in the form of a dry powder.

29. The solid unit dosage form of claim 27, wherein the inhalable powder comprises particles having a Dv50 of 2µm to 3µm.

30. The solid unit dosage form of claim 27, wherein the inhalable powder has a median aerodynamic diameter of 0.9 µm to 4.0 µm.

31. The solid unit dosage form according to claim 27, wherein the inhalable powder is obtained by wet milling and micronization in an aqueous solution.

32. The solid unit dosage form according to claim 27, wherein the inhalable powder is obtained by jet milling micronization.

33. The solid unit dosage form according to claim 27, wherein the inhalable powder has more than 90% of the initial crystalline form.

34. The solid unit dosage form according to claim 27, wherein the inhalable powder has an initial crystalline form of more than 75%.

35. The solid unit dosage form according to claim 25, wherein the solid unit dosage form further comprises leucine.

36. The solid unit dosage form according to claim 35, wherein leucine coats the solid crystalline form.

37. The solid unit dosage form according to claim 36, wherein the leucine-coated solid crystalline form is obtained by adding leucine to a wet-milled crystalline suspension prior to spray drying.

38. The solid unit dosage form of claim 25, wherein the dosage form is a capsule for administration using a dry powder inhaler.

39. The solid unit dosage form of claim 25, wherein the dosage form is a blister pack for administration with a dry powder inhaler.

40. The solid unit dosage form of claim 25, wherein the dosage form is a powder for administration using a dry powder inhaler.

41. Use of the solid crystalline form according to any one of claims 1-5, the pharmaceutical composition according to any one of claims 6 to 20, the pharmaceutical dosage form according to any one of claims 21-24, or the solid unit dosage form according to any one of claims 25 to 40 in the preparation of a medicament for treating diseases or conditions regulated by platelet-derived growth factor receptor (PDGFR) inhibition.

42. The use according to claim 41, wherein the disease or symptom is PAH, primary PAH, idiopathic PAH, hereditary PAH, refractory PAH, drug-induced PAH, toxin-induced PAH, or PAH with secondary diseases.

43. The use according to claim 42, wherein the disease or symptom is PAH.

44. A method for preparing a solid crystalline form of N-{3-[(1S)-1-{[6-(3,4-dimethoxyphenyl)pyrazin-2-yl]amino}ethyl]phenyl}-5-methylpyridine-3-carboxamide by crystallization from a solvent comprising 98% ethyl acetate and 2% water, wherein the crystalline form is form B, and form B is characterized by having XRPD plots with peaks at 5.2, 6.1, 7.6, 11.5, and 12.3 ± 0.2 degrees 2θ.

45. A method for preparing a solid crystalline form of N-{3-[(1S)-1-{[6-(3,4-dimethoxyphenyl)pyrazin-2-yl]amino}ethyl]phenyl}-5-methylpyridine-3-carboxamide by crystallization from ethanol or a solvent comprising 30% ethanol and 70% water, wherein the crystalline form is form B, and form B is characterized by having XRPD plots with peaks at 5.2, 6.1, 7.6, 11.5, and 12.3 ± 0.2 degrees 2θ.

46. ​​A method for preparing form B of N-{3-[(1S)-1-{[6-(3,4-dimethoxyphenyl)pyrazin-2-yl]amino}ethyl]phenyl}-5-methylpyridine-3-carboxamide, the method comprising slurrying form A of N-{3-[(1S)-1-{[6-(3,4-dimethoxyphenyl)pyrazin-2-yl]amino}ethyl]phenyl}-5-methylpyridine-3-carboxamide in ethyl acetate and maintaining the temperature at 45°C for a period of 87 hours, wherein form B is characterized by having an XRPD plot with peaks at 5.2, 6.1, 7.6, 11.5, and 12.3 ± 0.2 degrees 2θ, and form A is characterized by an XRPD plot substantially as shown in Figure 9.

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