Crystalline form of 5-(3,4-difluorobenzyl)-8-((1R,4R)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde

Abstract

Provided herein are crystalline forms of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde, compositions thereof, methods for their preparation, and methods for their use.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of PCT application CN2022 / 116765, filed September 2, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Provided herein are polymorphs of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde, compositions thereof, methods for their preparation, and methods for their use. [Background technology]

[0003] Cardiac sarcomeres are composed of a network of contractile and structural proteins that regulate myocardial function. Components of cardiac sarcomeres are targets for the treatment of various cardiac diseases and conditions, for example, by increasing contractility or promoting complete relaxation to modulate systolic and diastolic function, respectively. The force and velocity of myocardial contraction are major determinants of organ function and are regulated by the cyclic interaction of actin and myosin. Regulation of actin-myosin binding is mediated by a network of myofilament regulatory proteins and intracellular Ca 2+ The troponin complex and tropomyosin are thin filament proteins that govern the availability of actin-binding sites, and essential and regulatory light chains, as well as myosin-binding protein C, regulate the positioning and mechanical properties of myosin.

[0004] Abnormalities in cardiac sarcomeres have been identified as a cause of a wide variety of cardiac diseases and conditions, including hypertrophic cardiomyopathy (HCM) and heart failure with preserved ejection fraction (HFpEF). Mutations in sarcomere proteins cause disease by rendering the myocardium "hyper" or "hypo" contractile. Modulators of cardiac sarcomeres can be used to rebalance contractility and halt or reverse the disease process.

[0005] Current drugs that target the myocardial sarcomere, such as inotropes (drugs that increase cardiac contractility), have poor selectivity for cardiac tissue and cause recognized adverse effects that limit their use. These adverse effects include cell damage caused by increased energy expenditure rates, exacerbated relaxation abnormalities, and increased cytosolic Ca in inotropically stimulated myocardium. 2+ These include the potential for proarrhythmic side effects that may be due to increased cyclic AMP levels and increased vasopressin. Given the limitations of current medications, new approaches are needed to improve cardiac function in HCM and HFpEF.

[0006] There remains a significant need for drugs that utilize novel mechanisms of action and may have favorable prognoses in terms of symptom relief, safety, and patient mortality, both short- and long-term. Novel drugs with improved therapeutic indices over current drugs offer a means to realize these clinical outcomes. Selectivity of drugs for cardiac sarcomeres (e.g., by targeting cardiac myosin) has been identified as a key means to achieve this improved therapeutic index. 5-(3,4-Difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Compound 1) is a selective allosteric inhibitor of cardiac myosin with little or no effect on smooth muscle myosin. Advantages of this compound include a broader therapeutic window, less impact on cardiac relaxation, better pharmacokinetics, and greater safety, thereby offering it the potential to treat cardiac diseases and conditions.

[0007] To translate a drug candidate such as Compound 1 into a viable pharmaceutical product, it is important to understand whether the drug candidate possesses polymorphic forms and the relative stability and interconversion of these forms under the conditions likely to be encountered during bulk production, transportation, storage, and preparation prior to use. The ability to control and generate stable polymorphs in a robust manufacturing process can be important for regulatory approval and marketing. The bulk manufacturing process for preparing highly pure Compound 1 can be improved by using specific crystalline forms. Novel crystalline forms of Compound 1 with desirable chemical and physical stability, as well as their formulations and uses, are needed. Summary of the Invention [Means for solving the problem]

[0008] In one aspect, provided herein is 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde.

[0009] In another aspect, provided herein are methods for preparing polymorphs of Compound 1.

[0010] In another aspect, provided herein are compositions comprising polymorphs of Compound 1.

[0011] In another aspect, provided herein are methods of using a polymorph of Compound 1 to treat a cardiac disorder in a subject in need thereof. [Brief explanation of the drawings]

[0012] [Figure 1A] Figure 1 shows the experimental X-ray powder diffraction (XRPD) pattern of crystalline form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde.

[0013] [Figure 1B] 1 shows differential scanning calorimetry (DSC) and thermographic analysis (TGA) graphs of crystalline form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde.

[0014] [Figure 1C] 1 shows a graph of gravimetric vapor sorption (GVS) of crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde.

[0015] [Figure 1D] Figure 1 shows an overlay of XRPD patterns of crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde before storage and after 7 days of storage at 40°C / 75% RH and 25°C / 97% RH (top to bottom: Form I after 7 days of storage at 40°C / 75%, Form I after 7 days of storage at 25°C / 97% RH, Form I after 7 days of storage at 40°C / 75% RH and 25°C / 97% RH).

[0016] [Figure 1E] XRPD patterns of crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde before and after GVS measurement (top: after GVS, bottom: before GVS).

[0017] [Figure 2A] Figure 1 shows the experimental XRPD pattern of crystalline Form II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde.

[0018] [Figure 2B] 1 shows DSC and TGA graphs of crystalline Form II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde.

[0019] [Figure 2C] 1 shows a graph of GVS of crystalline Form II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde.

[0020] [Figure 2D] Figure 1 shows an overlay of XRPD patterns of crystalline Form II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde before storage and after 8 days of storage at 40°C / 75% RH and 25°C / 97% RH (top to bottom: Form II after 8 days of storage at 25°C / 97%, Form II after 8 days of storage at 40°C / 75% RH, Form II after 8 days of storage at 40°C / 75% RH and 25°C / 97% RH).

[0021] [Figure 2E] Overlay of XRPD patterns of crystalline Form II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde before and after GVS measurement (top: after GVS, bottom: before GVS).

[0022] [Figure 3A] Figure 1 shows the experimental XRPD pattern of crystalline Form III of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde.

[0023] [Figure 3B] 1 shows DSC and TGA graphs of crystalline Form III of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde.

[0024] [Figure 3C] Figure 1 shows the overlaid XRPD patterns of crystalline Form III of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde before storage and after 7 days of storage at 40°C / 75% RH, and the XRPD pattern of crystalline Form II (top to bottom: Form III after 7 days of storage at 40°C / 75%, Form III after 7 days of storage at 40°C / 75% RH, Form II). DETAILED DESCRIPTION OF THE INVENTION

[0025] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise.

[0026] As used herein, and unless otherwise specified, the terms "about" and "approximately," when used in reference to a dose, amount, or weight percent of a component of a composition or dosage form, mean a dose, amount, or weight percent that would be recognized by one of ordinary skill in the art as providing an equivalent pharmacological effect to that obtained from the specified dose, amount, or weight percent. Specifically, where applicable, the terms "about" and "approximately," when used in this context, contemplate a dose, amount, or weight percent within 15% of the specified dose, amount, or weight percent.

[0027] As used herein, the terms "polymorph," "polymorphism," "polymorphic form," and "crystalline form" refer to crystalline forms of a compound. Different polymorphs may have different physical properties, such as melting temperature, heat of fusion, solubility, dissolution rate, and / or vibrational spectra, as a result of the arrangement or conformation of molecules or ions within the crystal lattice. Differences in physical properties exhibited by polymorphs can affect pharmaceutical parameters such as storage stability, compressibility, density (important in formulation and product manufacturing), and dissolution rate (a key factor in bioavailability). Differences in stability can be due to changes in chemical reactivity (e.g., differential oxidation, such that a dosage form consisting of one polymorph discolors more quickly than one consisting of another), mechanical changes (e.g., a tablet that shatters on storage as the kinetically favored polymorph converts to a thermodynamically more stable polymorph), or both (e.g., a tablet of one polymorph is more susceptible to disintegration at high humidity). As a result of differences in solubility / disintegration, some polymorphic transitions may be ineffective at one extreme or toxic at another. Furthermore, the physical properties of the crystalline form may be important in processing. For example, a polymorph may be more likely to form solvates or may be difficult to filter and wash free of impurities (e.g., particle shape and size distribution may differ between polymorphs).

[0028] As used herein, a "therapeutically effective amount" refers to an amount that produces the desired pharmacological and / or physiological effect for the condition. The effect may be therapeutic in terms of a partial or complete cure of the condition and / or adverse effects resulting from the condition.

[0029] As used herein, the term "pharmaceutically acceptable carrier" and its variants refer to adjuvants, binders, diluents, etc. known to those skilled in the art that are suitable for administration to an individual (e.g., a mammal or non-mammal). Combinations of two or more carriers are also contemplated. The pharmaceutically acceptable carrier(s) and any additional components described herein must be compatible for use with the intended route of administration (e.g., oral, parenteral) of the particular dosage form, as recognized by those skilled in the art.

[0030] The terms "treat," "treating," and "treatment" are intended to include alleviating or arresting a disorder, disease, or condition, or one or more symptoms associated with the disorder, disease, or condition, or slowing the progression, spread, or worsening of the disease, disorder, or condition, or one or more symptoms thereof. Often, the beneficial effects that a subject derives from a therapeutic agent do not result in a complete cure of the disease, disorder, or condition.

[0031] The term "subject" refers to an animal, including, but not limited to, a primate (e.g., a human), a mammal, a monkey, a cow, a pig, a sheep, a goat, a horse, a dog, a cat, a rabbit, a rat, or a mouse. The terms "subject" and "patient" are used interchangeably herein in reference to a mammalian subject, such as, for example, a human.

[0032] As used herein, the term "substantially as shown," when referring to, for example, an XRPD pattern, a DSC graph, a TGA graph, or a GVS graph, includes patterns or graphs that are not necessarily identical to those shown herein, but that fall within the limits of experimental error or variation recognized by one of ordinary skill in the art.

[0033] As used herein, the term "substantially free" means that a composition including a crystalline form contains less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% by weight of the specified substance(s).

[0034] polymorphism In one aspect, provided herein is 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (also referred to as Compound 1), having the structure shown below. [ka]

[0035] Polymorphs may have properties such as bioavailability and stability under particular conditions that make them suitable for medical or pharmaceutical use.

[0036] Polymorphs of Compound 1 may offer bioavailability and stability advantages and may be suitable for use as an active agent in pharmaceutical compositions. Differences in the crystalline structure of a pharmaceutical drug substance can affect the drug's dissolution rate (which may affect bioavailability, etc.), manufacturability (e.g., ease of handling, ease of purification, ability to prepare uniform doses of known strengths, etc.), and stability (e.g., thermal stability, shelf life (including resistance to degradation), etc.). Such differences can affect how pharmaceutical compositions are prepared or formulated in different dosage or delivery forms, such as solid oral dosage forms including tablets and capsules. Compared to other forms, such as non-crystalline or amorphous forms, polymorphs may offer desirable or suitable hygroscopicity or lack thereof, particle size control, dissolution rate, solubility, purity, physical and chemical stability, manufacturability, yield, reproducibility, and / or process control. Thus, polymorphs of Compound 1 may offer advantages such as improving the manufacturing process of the active agent or the stability or storage of a pharmaceutical form of the active agent, or having suitable bioavailability and / or stability as an active agent.

[0037] It has been found that the use of certain conditions, such as the use of different solvents and / or temperatures, can result in different polymorphs of Compound 1, or solvates thereof, including crystalline Forms I-III described herein, which may exhibit one or more of the favorable characteristics described herein. The process for preparing the polymorphs described herein and characterizing these polymorphs is described in more detail below.

[0038] Crystalline form I In some embodiments, provided herein is crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde.

[0039] In some embodiments, crystalline Form I has an XRPD pattern substantially as shown in Figure 1A.

[0040] The 2θ angles and relative peak intensities observed for Form I using XRPD are shown in Table 1. [Table 1]

[0041] In some embodiments, crystalline Form I has an XRPD pattern substantially as shown in FIG. 1A or shown in Table 1, exhibiting at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten peaks at 2θ angles with maximum intensity. It should be understood that relative intensities may vary depending on many factors, including sample preparation, mounting, and the instrumentation and analytical procedures and settings used to obtain the spectrum. Relative peak intensities and peak assignments may vary within experimental error. In some embodiments, peak designations described herein, including crystalline Form I, may vary by about ±0.6°, ±0.4°, ±0.2°, or ±0.1° in 2θ.

[0042] In some embodiments, crystalline Form I is characterized by having an XRPD pattern containing peaks at 6.0±0.2°, 10.2±0.2°, 21.6±0.2°, and 22.1±0.2° 2θ. In some embodiments, crystalline Form I has an XRPD pattern containing additional peaks at 17.9±0.2° and 24.1±0.2° 2θ. In some embodiments, crystalline Form I has an XRPD pattern further containing additional peaks at 16.0±0.2°, 16.6±0.2°, 17.3±0.2°, 17.6±0.2°, and 20.5±0.2° 2θ. In some embodiments, crystalline Form I has 2θ angles = 6.0±0.2°, 10.2±0.2°, 11.9±0.2°, 13.7±0.2°, 14.4±0.2°, 15.5±0.2°, 16.0±0.2°, 16.6±0.2°, 17.3±0.2°, 17.6±0.2°, 17.9±0.2°, 19.8±0.2°, 20.5±0.2°, 20.9±0.2°, 21.9±0.2°, 22.9±0.2°, 23.9±0.2°, 24.9±0.2°, 25.9±0.2°, 26.9±0.2°, 27.9±0.2°, 28.9±0.2°, 29.9±0.2°, 30.9±0.2°, 31.9±0.2°, 32.9±0.2°, 33.9±0.2°, 34.9±0.2°, 35.9±0.2°, 36.9±0.2°, 37.9±0.2°, 38.9±0.2°, 39.9±0.2°, 40.9±0.2°, 41.9±0.2°, 42.9±0.2°, 43.9±0.2°, 44.9±0.2°, 45.9±0.2°, 46.9±0.2°, 47.9±0.2°, 48.9±0.2°, 49.9±0.2°, 50.9±0.2°, 51.9±0.2°, 52 1A or listed in Table 1 may be observed due to, for example, the presence of impurities, solvents, or other polymorphs or amorphous forms present in the test sample.

[0043] In some embodiments, crystalline Form I has a differential scanning calorimetry (DSC) graph substantially as shown in Figure 1B. In some embodiments, crystalline Form I is characterized by having an endotherm onset at about 125.6°C as measured by DSC. In some embodiments, crystalline Form I is characterized by an endothermic onset at 125.6±2°C (e.g., 125.6±1.9°C, 125.6±1.8°C, 125.6±1.7°C, 125.6±1.6°C, 125.6±1.5°C, 125.6±1.4°C, 125.6±1.3°C, 125.6±1.2°C, 125.6±1.1°C, 125.6±1.0°C, 125.6±0.9°C, 125.6±0.8°C, 125.6±0.7°C, 125.6±0.6°C, 125.6±0.5°C, 125.6±0.4°C, 125.6±0.3°C, 125.6±0.2°C, or 125.6±0.1°C), as measured by DSC. In some embodiments, crystalline Form I is characterized by an endothermic peak at about 130.2° C. as measured by DSC. In some embodiments, crystalline Form I is characterized by an endothermic peak at 130.2±2°C (e.g., 130.2±1.9°C, 130.2±1.8°C, 130.2±1.7°C, 130.2±1.6°C, 130.2±1.5°C, 130.2±1.4°C, 130.2±1.3°C, 130.2±1.2°C, 130.2±1.1°C, 130.2±1.0°C, 130.2±0.9°C, 130.2±0.8°C, 130.2±0.7°C, 130.2±0.6°C, 130.2±0.5°C, 130.2±0.4°C, 130.2±0.3°C, 130.2±0.2°C, or 130.2±0.1°C), as measured by DSC.

[0044] In some embodiments, crystalline Form I has a thermographic analysis (TGA) graph substantially as shown in Figure 1B. In some embodiments, crystalline Form I exhibits a weight loss of about 0.35% or 0.35% ± 0.05% (e.g., 0.35% ± 0.04%, 0.35% ± 0.03%, 0.35% ± 0.02%, or 0.35% ± 0.01%) between 105°C and 145°C as measured by TGA.

[0045] In some embodiments, crystalline Form I has a gravitational vapor sorption (GVS) graph substantially as shown in FIG. 1C.

[0046] In some embodiments, when stored for 7 days under two different temperature / RH conditions (40°C / 75% RH and 25°C / 97% RH), crystalline Form I exhibits substantially or no change as measured by XRPD. In some embodiments, when stored for 7 days under 40°C / 75% RH, crystalline Form I exhibits substantially or no change as measured by XRPD. In some embodiments, when stored for 7 days under 25°C / 97% RH, crystalline Form I exhibits substantially or no change as measured by XRPD.

[0047] In some embodiments, crystalline Form I exhibits substantially no change as measured by XRPD before and after GVS measurements, as shown in Figure IE.

[0048] In some embodiments, crystalline Form I is 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde and has a purity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with respect to a salt, solvate, or other form, including amorphous form, of the compound, which purity is substantially unchanged or unchanged when stored at 40° C. / 75% RH and / or 25° C. / 97% RH for 7 days as measured by HPLC.

[0049] In some embodiments of crystalline Form I, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all of the following (a)-(h) are true: (a) Crystalline Form I is characterized by an XRPD pattern containing peaks at 2θ angles of 6.0±0.2, 10.2±0.2, 21.6±0.2, and 22.1±0.2°, an XRPD pattern containing additional peaks at 2θ angles of 17.9±0.2° and 24.1±0.2°, an XRPD pattern containing additional peaks at 2θ angles of 16.0±0.2, 16.6±0.2, 17.3±0.2, 17.6±0.2, and 20.5±0.2°, or an XRPD pattern containing additional peaks at 2θ angles of 6.0±0.2, 10.2±0.2, 11.9±0.2, 13.7±0.2, and 14. and 30.1 ± 0.2°. (b) Crystalline Form I has an XRPD pattern substantially as shown in Figure 1A. (c) Crystalline Form I has a DSC graph substantially as shown in Figure 1B. (d) Crystalline Form I is characterized by an endothermic onset at 125.6±2° C. as measured by DSC. (e) Crystalline Form I is characterized by an endothermic peak at about 130.2±2° C. as measured by DSC. (f) Crystalline Form I has a TGA graph substantially as shown in Figure 1B. (g) Crystalline Form I has a weight loss of about 0.35% or 0.35%±0.05% at 105°C to 145°C as measured by TGA; and (h) Crystalline Form I has a GVS graph substantially as shown in Figure 1C.

[0050] Crystalline Form II In some embodiments, provided herein is crystalline Form II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. Sample and single crystal data for crystalline Form II are shown in Table 2-D. [Table 2D]

[0051] The crystal structure of Form II was solved in the triclinic centrosymmetric space group P-1 and refined with a final R1[I>2σ(I)] value of 3.91%.

[0052] In some embodiments, crystalline Form II has an XRPD pattern substantially as shown in Figure 2A.

[0053] The 2θ angles and relative peak intensities observed for Form II using XRPD are shown in Table 2C. [Table 2C-1] [Table 2C-2]

[0054] In some embodiments, crystalline Form II has an XRPD pattern exhibiting at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten peaks at 2θ angles with maximum intensity for the XRPD pattern substantially as shown in FIG. 2A or as shown in Table 2C. It should be understood that relative intensities may vary depending on several factors, including sample preparation, mounting, and the instrumentation and analytical procedures and settings used to acquire the spectrum. Relative peak intensities and peak assignments may vary within experimental error. In some embodiments, peak assignments described herein, including crystalline Form II, may vary by about ±0.6°, ±0.4°, ±0.2°, or ±0.1° in 2θ.

[0055] In some embodiments, crystalline Form II has an XRPD pattern comprising peaks at 5.9±0.2°, 11.5±0.2°, 11.7±0.2°, 17.9±0.2°, and 19.1±0.2° 2θ. In some embodiments, crystalline Form II has an XRPD pattern comprising additional peaks at 7.8±0.2° and 16.2±0.2° 2θ. In some embodiments, crystalline Form II has an XRPD pattern further comprising additional peaks at 13.1±0.2° and 19.8±0.2° 2θ. In some embodiments, crystalline Form II has a 2θ angle of 5.9±0.2, 6.4±0.2, 6.7±0.2, 7.4±0.2, 7.8±0.2, 10.4±0.2, 11.5±0.2, 11.7±0.2, 12.4±0.2, 12.8±0.2, 13.1±0.2, 13.7±0.2, 14.1±0.2, 14.6±0.2, 15.1±0.2, 15.7±0.2, 16.2±0.2, 16.6±0.2, 16.8±0.2, 17.2±0.2, 17.9±0.2, 18.4±0.2, 18.8±0.2, 19.1±0.2, 19.4±0.2, 19.8±0.2, 20 0.3±0.2, 20.7±0.2, 21.0±0.2, 21.3±0.2, 21.8±0.2, 22.0±0.2, 22.4±0.2, 23.3±0.2, 23.6±0.2, 23.9±0.2, 24.2±0.2, 24.5±0.2, 24.8±0.2, 25.2±0.2, 25.4±0.2, 25.9±0.2, 26.3±0.2, 26.6±0.2, 27.3±0.2, 27.7±0.2, 28.1±0.2, 28.4±0.2, 28.7±0.2, 29.1±0.2, 29.6±0.2, and 30.8±0.2°. In some embodiments, the XRPD pattern may also be calculated from single crystal data obtained for crystalline Form II. It will be understood that additional peaks in the XRPD pattern other than those shown in Figure 2A or listed in Table 2C may be observed due to, for example, the presence of impurities, solvents, or other polymorphs or amorphous forms present in the test sample.

[0056] In some embodiments, crystalline Form II has a differential scanning calorimetry (DSC) graph substantially as shown in Figure 2B. In some embodiments, crystalline Form II is characterized by an endothermic onset at about 154.9°C as measured by DSC. In some embodiments, crystalline Form II is characterized by an endothermic onset at 154.9±2°C (e.g., 154.9±1.9°C, 154.9±1.8°C, 154.9±1.7°C, 154.9±1.6°C, 154.9±1.5°C, 154.9±1.4°C, 154.9±1.3°C, 154.9±1.2°C, 154.9±1.1°C, 154.9±1.0°C, 154.9±0.9°C, 154.9±0.8°C, 154.9±0.7°C, 154.9±0.6°C, 154.9±0.5°C, 154.9±0.4°C, 154.9±0.3°C, 154.9±0.2°C, or 154.9±0.1°C), as measured by DSC. In some embodiments, crystalline Form II is characterized by an endothermic peak at about 155.8° C. as measured by DSC. In some embodiments, crystalline Form II is characterized by an endothermic onset at 155.8±2°C (e.g., 155.8±1.9°C, 155.8±1.8°C, 155.8±1.7°C, 155.8±1.6°C, 155.8±1.5°C, 155.8±1.4°C, 155.8±1.3°C, 155.8±1.2°C, 155.8±1.1°C, 155.8±1.0°C, 155.8±0.9°C, 155.8±0.8°C, 155.8±0.7°C, 155.8±0.6°C, 155.8±0.5°C, 155.8±0.4°C, 155.8±0.3°C, 155.8±0.2°C, or 155.8±0.1°C), as measured by DSC.

[0057] In some embodiments, crystalline Form II has a thermographic analysis (TGA) graph substantially as shown in Figure 2B. In some embodiments, crystalline Form II exhibits a weight loss before decomposition of less than 0.1% (e.g., less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, less than 0.01%) as measured by TGA.

[0058] In some embodiments, crystalline Form II has a gravitational vapor sorption (GVS) graph substantially as shown in Figure 2C.

[0059] In some embodiments, when stored for 8 days under two different temperature / RH conditions (40°C / 75% RH and 25°C / 97% RH), crystalline Form II exhibits substantially or no change as measured by XRPD. In some embodiments, when stored for 7 days under 40°C / 75% RH, crystalline Form II exhibits substantially or no change as measured by XRPD. In some embodiments, when stored for 7 days under 25°C / 97% RH, crystalline Form II exhibits substantially or no change as measured by XRPD.

[0060] In some embodiments, crystalline Form II exhibits substantially no change as measured by XRPD before and after GVS measurements, as shown in Figure 2E.

[0061] In some embodiments, crystalline Form II is 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde and has a purity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with respect to other forms, where the purity is substantially unchanged or unchanged when stored under two different temperature / RH conditions (40°C / 75% Rh and / or 25°C / 97% Rh) for 8 days as measured by HPLC.

[0062] Crystalline Form II exhibits high solubility and high stability, making it desirable for pharmaceutical applications. These properties may provide advantages such as improved dosing consistency, improved drug release upon administration, extended shelf life, and ease of storage and packaging. Crystalline Form II has high solubility in various common solvents. In some embodiments, crystalline Form II has a solubility of at least 100 mg / mL (e.g., at least 90 mg / mL, at least 80 mg / mL, at least 70 mg / mL, at least 60 mg / mL, or at least 50 mg / mL) in methanol, acetone, DMSO, acetonitrile, and THF. In some embodiments, crystalline Form II has a solubility of about 24 mg / mL (e.g., 24±5 mg / mL, 24±4 mg / mL, 24±3 mg / mL, 24±2 mg / mL, or 24±1 mg / mL) in ethanol.

[0063] Crystalline Form II exhibits high stability in a variety of solvents. In some embodiments, crystalline Form II shows no visible signs of decomposition as measured by HPLC after 24 hours of storage in a solvent, wherein the solvent is selected from the group consisting of water, methanol, isopropanol, 2-BuOH, acetone, MIBK, DMSO, MeCN, THF, 2-methyl-THF, and toluene. In some embodiments, crystalline Form II shows no visible signs of decomposition as measured by HPLC after 24 hours of storage in a solvent, wherein the solvent is selected from the group consisting of methanol, acetone, DMSO, acetonitrile, and THF. In some embodiments, crystalline Form II initially shows signs of visual decomposition in a solvent, but shows no further decomposition as measured by HPLC after 24 hours of storage in a solvent, wherein the solvent is selected from the group consisting of ethanol, tert-butyl methyl ether, and isopropyl acetate.

[0064] In some embodiments of crystalline Form II, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all of the following (a)-(h) are true: (a) Crystalline Form II has an XRPD pattern containing peaks at 2θ angles of 5.9±0.2, 11.5±0.2, 11.7±0.2, 17.9±0.2, and 19.1±0.2°, an XRPD pattern containing additional peaks at 2θ angles of 7.8±0.2 and 16.2±0.2°, an XRPD pattern containing further additional peaks at 2θ angles of 13.1±0.2 and 19.8±0.2°, or or 2θ angle = 5.9±0.2, 6.4±0.2, 6.7±0.2, 7.4±0.2, 7.8±0.2, 10.4±0.2, 11.5±0.2, 11.7±0.2, 12.4±0.2, 12.8±0.2, 13.1±0.2, 13.7±0.2, 14.1±0.2, 14.6±0.2, 15.1±0.2, 15.7±0.2, 16.2±0.2, 16.6±0.2, 1 6.8±0.2, 17.2±0.2, 17.9±0.2, 18.4±0.2, 18.8±0.2, 19.1±0.2, 19.4±0.2, 19.8±0.2, 20.3±0.2, 20.7±0.2, 21.0±0.2, 21.3±0.2, 21.8±0.2, 22.0±0.2, 22.4±0.2, 23.3±0.2, 23.6±0.2, 23.9±0.2, 24 0.2±0.2, 24.5±0.2, 24.8±0.2, 25.2±0.2, 25.4±0.2, 25.9±0.2, 26.3±0.2, 26.6±0.2, 27.3±0.2, 27.7±0.2, 28.1±0.2, 28.4±0.2, 28.7±0.2, 29.1±0.2, 29.6±0.2, and 30.8±0.2°. (b) Crystalline Form II has an XRPD pattern substantially as shown in Figure 2A. (c) Crystalline Form II has a DSC graph substantially as shown in Figure 2B. (d) Crystalline Form II is characterized by an endothermic onset at 154.9±2° C. as measured by DSC. (e) Crystalline Form II is characterized by an endothermic peak at about 155.8±2° C. as measured by DSC. (f) Crystalline Form II has a TGA graph substantially as shown in Figure 2B. (g) Crystalline Form II has a weight loss before decomposition of less than 0.1% as measured by TGA; and (h) Crystalline Form II has a GVS graph substantially as shown in Figure 2C.

[0065] Crystalline Form III Crystalline Form III of the 1,4-dioxane solvate of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde was isolated and subjected to baseline characterization to understand its solid-state properties. In some embodiments, crystalline Form III was identified as an unstable hemi-dioxane solvate, which converted to crystalline Form II after storage at 40°C / 75% RH for 7 days as determined by XRPD, as shown in Figure 3C. In some embodiments, 1 As determined by HNMR, crystalline Form III is 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde containing 0.5 mol equivalents of 1,4-dioxane.

[0066] In some embodiments, crystalline Form III has an XRPD pattern substantially as shown in Figure 3A.

[0067] The 2θ angles and relative peak intensities observed for Form III using XRPD are shown in Table 4-A. [Table 4A-1] [Table 4A-2]

[0068] In some embodiments, crystalline Form III has an XRPD pattern exhibiting at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten peaks at 2θ angles with maximum intensity in an XRPD pattern substantially as shown in FIG. 3A. It should be understood that relative intensities may vary depending on several factors, including sample preparation, mounting, and the instrumentation and analytical procedures and settings used to acquire the spectrum. Relative peak intensities and peak assignments may vary within experimental error. In some embodiments, the peak assignments comprising crystalline Form III may vary by about ±0.6°, ±0.4°, ±0.2°, or ±0.1° in 2θ.

[0069] In some embodiments, crystalline Form III has an XRPD pattern comprising peaks at 5.6±0.2°, 7.2±0.2°, 11.2±0.2°, 14.9±0.2°, and 16.8±0.2° 2θ angles. In some embodiments, crystalline Form III has an XRPD pattern comprising additional peaks at 16.4±0.2°, 18.7±0.2°, 21.9±0.2°, 22.5±0.2°, and 22.7±0.2° 2θ angles. In some embodiments, crystalline Form III has a 2θ angle of 5.6±0.2, 6.0±0.2, 7.2±0.2, 11.2±0.2, 13.9±0.2, 14.9±0.2, 15.5±0.2, 15.8±0.2, 16.4±0.2, 16.8±0.2, 17.0±0.2, 17.7±0.2, 18.7±0.2, 18.9±0.2, 19.5±0.2, 20.1±0.2, 20.6±0.2, 21.0±0.2, 21.4±0.2, 22.0±0.2, 22.1±0.2, 22.2±0.2, 22.3±0.2, 22.4±0.2, 22.5±0.2, 23.0±0.2, 23.1±0.2, 23.2±0.2, 23.3±0.2, 23.4±0.2, 24.0±0.2, 24.2±0.2, 24.4±0.2, 24.5±0.2, 25.0±0.2, 25.0±0.2, 25.1±0.2, 25.2±0.2, 25.2±0.2, 25.2±0.2, 26.0±0.2, 26.0±0.2, 26.0±0.2, 26.0±0.2, 26.0±0.2, 27.0±0.2, 27.0±0.2, 27.0±0.2, 27.0±0.2, 28.0±0.2 3A or 3B. The XRPD pattern includes peaks at 0.2, 21.6±0.2, 21.9±0.2, 22.5±0.2, 23.2±0.2, 23.4±0.2, 23.8±0.2, 24.3±0.2, 24.6±0.2, 25.1±0.2, 26.2±0.2, 26.6±0.2, 26.9±0.2, 27.3±0.2, 27.7±0.2, 27.9±0.2, 29.0±0.2, 29.2±0.2, and 29.6±0.2°. It is understood that additional peaks in the XRPD pattern other than those shown in FIG. 3A or listed in Table 4-A may be observed due to, for example, the presence of impurities, solvents, or other polymorphs or amorphous forms present in the test sample.

[0070] In some embodiments, crystalline Form III has a differential scanning calorimetry (DSC) graph substantially as shown in Figure 3B. In some embodiments, crystalline Form III is characterized by having an endothermic onset at about 81.7±4°C, a dual endothermic onset at about 112.8±4°C, or an endothermic onset at about 154.4±4°C, or any combination thereof, as measured by DSC. In some embodiments, crystalline Form III is characterized by having an endothermic peak at about 92.1±4°C, an endothermic peak at about 118.2±4°C, an endothermic peak at about 130.0±4°C, or an endothermic peak at about 155.8±4°C, or any combination thereof, as measured by DSC.

[0071] In some embodiments, crystalline Form III has a thermographic analysis (TGA) graph substantially as shown in Figure 3B. In some embodiments, crystalline Form III exhibits a weight loss of about 3.3% or 3.3% ± 0.5% (e.g., 3.3% ± 0.4%, 3.3% ± 0.3%, 3.3% ± 0.2%, 3.3% ± 0.1%) from 45°C to 101°C and / or a weight loss of about 5.2% or 5.2% ± 0.5% (e.g., 5.2% ± 0.4%, 5.2% ± 0.3%, 5.2% ± 0.2%, 5.2% ± 0.1%) from 101°C to 189°C, as measured by TGA.

[0072] In some embodiments of crystalline Form III, at least one, at least two, at least three, at least four, at least five, at least six, or all of the following (a)-(h) are true: (a) Crystalline Form III has an XRPD pattern containing peaks at 2θ angles of 5.6±0.2, 7.2±0.2, 11.2±0.2, 14.9±0.2, and 16.8±0.2°; an XRPD pattern containing peaks at 2θ angles of 5.6±0.2, 7.2±0.2, 11.2±0.2, 14.9±0.2, 16.4±0.2, 16.8±0.2, 18.7±0.2, 21.9±0.2, 22.5±0.2, and 22.7±0.2°; or an XRPD pattern containing peaks at 2θ angles of 5.6±0.2, 6.0±0.2, 7.2±0.2, 11.2±0.2, 13.9±0.2, 14.9±0.2, 15.5±0.2, 15.8±0.2°. 16.4±0.2, 16.8±0.2, 17.0±0.2, 17.7±0.2, 18.7±0.2, 18.9±0.2, 19.5±0.2, 20.1±0.2, 20.6±0.2, 21.0±0.2, 21.4±0.2, 21.6±0.2, 21.9±0.2, 22.5±0.2, 23.2± 0.2, 23.4±0.2, 23.8±0.2, 24.3±0.2, 24.6±0.2, 25.1±0.2, 26.2±0.2, 26.6±0.2, 26.9±0.2, 27.3±0.2, 27.7±0.2, 27.9±0.2, 29.0±0.2, 29.2±0.2, and 29.6±0.2°. (b) Crystalline Form III has an XRPD pattern substantially as shown in Figure 3A. (c) Crystalline Form III has a DSC graph substantially as shown in Figure 3B. (d) Crystalline Form III is characterized by an endotherm onset at about 81.7±4.0°C, an endotherm onset at about 112.8±4.0°C, or an endotherm onset at about 154.4±4.0°C, or any combination thereof, as measured by DSC. (e) Crystalline Form III is characterized by having an endothermic peak at about 92.1±4.0°C, an endothermic peak at about 118.2±4.0°C, an endothermic peak at about 130.0±4.0°C, or an endothermic onset at about 155.8±4.0°C, or any combination thereof, as measured by DSC. (f) Crystalline Form III has a TGA graph substantially as shown in Figure 3B; and (g) Crystalline Form III has a weight loss of about 3.3% or 3.3%±0.5% from 45°C to 101°C and / or a weight loss of about 5.2% or 5.2%±0.5% from 101°C to 189°C, as measured by DSC.

[0073] composition Also provided herein are compositions containing a polymorph described herein, such as crystalline Form I, crystalline Form II, crystalline Form III, or a mixture thereof. In some embodiments, the composition contains crystalline Form I. In some embodiments, the composition contains crystalline Form II. In some embodiments, the composition contains crystalline Form III. In some embodiments, the composition contains a mixture of crystalline Form I and crystalline Form II. In some embodiments, the composition further contains a pharmaceutically acceptable carrier.

[0074] In some embodiments, a composition is provided that contains crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. In some embodiments, the composition is substantially free of at least one or both of crystalline Forms II and III of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. In some embodiments, the composition is substantially free of amorphous or non-crystalline forms of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. In some embodiments, the composition is substantially free of salts of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde.

[0075] In some embodiments of a composition comprising crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde, at least about 0.1 wt%, at least about 0.3 wt%, at least about 0.5 wt%, at least about 0.8 wt%, at least about 1.0 wt%, at least about 5.0 wt%, at least about 10 wt%, at least about 20 wt%, at least about 30 wt%, at least about 40 wt%, at least about 50 wt%, at least about 60 wt%, at least about 70 wt%, at least about 80 wt%, at least about 85 wt%, at least about 90 wt%, at least about 95 wt%, at least about 96 wt%, at least about 97 wt%, at least about 98 wt%, at least about 99 wt%, or at least 99.9 wt% of the total composition is Form I. In some embodiments of the composition comprising crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde, the crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde is at least about 0.1% by weight, at least about 0.3% by weight, at least about 0.5% by weight, or at least about 0.6% by weight of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. 8% by weight, at least about 1.0% by weight, at least about 5.0% by weight, at least about 10% by weight, at least about 20% by weight, at least about 30% by weight, at least about 40% by weight, at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 85% by weight, at least about 90% by weight, at least about 95% by weight, at least about 96% by weight, at least about 97% by weight, at least about 98% by weight, at least about 99% by weight, or at least 99.9% by weight is present as crystalline Form I.

[0076] In some embodiments, a composition is provided that contains crystalline Form II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. In some embodiments, the composition is substantially free of at least one or both of crystalline Forms I and III of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. In some embodiments, the composition is substantially free of amorphous or non-crystalline forms of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. In some embodiments, the composition is substantially free of salts of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde.

[0077] In some embodiments of a composition comprising crystalline Form II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde, at least about 0.1 wt%, at least about 0.3 wt%, at least about 0.5 wt%, at least about 0.8 wt%, at least about 1.0 wt%, at least about 5.0 wt%, at least about 10 wt%, at least about 20 wt%, at least about 30 wt%, at least about 40 wt%, at least about 50 wt%, at least about 60 wt%, at least about 70 wt%, at least about 80 wt%, at least about 85 wt%, at least about 90 wt%, at least about 95 wt%, at least about 96 wt%, at least about 97 wt%, at least about 98 wt%, at least about 99 wt%, or at least 99.9 wt% of the total composition is crystalline Form II. In some embodiments of the composition comprising crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde, the crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde is at least about 0.1% by weight, at least about 0.3% by weight, at least about 0.5% by weight, or at least about 0.6% by weight of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. 8% by weight, at least about 1.0% by weight, at least about 5.0% by weight, at least about 10% by weight, at least about 20% by weight, at least about 30% by weight, at least about 40% by weight, at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 85% by weight, at least about 90% by weight, at least about 95% by weight, at least about 96% by weight, at least about 97% by weight, at least about 98% by weight, at least about 99% by weight, or at least 99.9% by weight is present as crystalline Form I.

[0078] In some embodiments, compositions are provided that contain crystalline Form III of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. In some embodiments, the compositions are substantially free of at least one or both of crystalline Forms I and II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. In some embodiments, the compositions are substantially free of amorphous or non-crystalline forms of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. In some embodiments, the composition is substantially free of salts of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde.

[0079] In some embodiments of the composition comprising crystalline Form III of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde, at least about 0.1%, at least about 0.3%, at least about 0.5%, at least about 0.8%, at least about 1.0%, at least about 5.0%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least 99.9% by weight of the total composition is Form III. In some embodiments of the composition comprising crystalline Form III of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde, the crystalline Form III of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde is at least about 0.1 wt%, at least about 0.3 wt%, at least about 0.5 wt%, or at least about 0.6 wt% of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. 8% by weight, at least about 1.0% by weight, at least about 5.0% by weight, at least about 10% by weight, at least about 20% by weight, at least about 30% by weight, at least about 40% by weight, at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 85% by weight, at least about 90% by weight, at least about 95% by weight, at least about 96% by weight, at least about 97% by weight, at least about 98% by weight, at least about 99% by weight, or at least 99.9% by weight is present as crystalline Form III.

[0080] In some embodiments, tablets or capsules are provided containing one or more of the crystalline forms described herein (e.g., crystalline Forms I, II, III, or mixtures thereof) and one or more pharmaceutically acceptable carriers. In some embodiments, tablets or capsules are provided containing substantially pure crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde and one or more pharmaceutically acceptable carriers. In some embodiments, tablets or capsules are provided containing substantially pure crystalline Form II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde and one or more pharmaceutically acceptable carriers. In some embodiments, provided are tablets or capsules containing substantially pure crystalline Form III of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde and one or more pharmaceutically acceptable carriers. In some embodiments, provided are tablets or capsules containing a mixture of crystalline Forms I and II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde and one or more pharmaceutically acceptable carriers.

[0081] Preparation method Crystalline form I In some embodiments, a method for preparing crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde is provided, comprising: (a) combining 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde with a solvent; and (b) stirring the mixture of step (a) to form a suspension. In some embodiments, step (b) comprises stirring the mixture of step (a) at a temperature between 0° C. and 30° C., between 0° C. and 20° C., between 0° C. and 10° C., or about 5° C. In some embodiments, the solvent is selected from the group consisting of ethanol, 1-propanol, 2-propanol, ethyl acetate, methyl isobutyl ketone (MIBK), ethylene glycol, a mixture of N,N-dimethylformamide (DMF) and water, a mixture of N-methylpyrrolidone (NMP) and water, and a mixture of 1,4-dioxane and water. In some embodiments, the method further comprises adding an additional solvent. In some embodiments, the method further comprises adding an anti-solvent to promote suspension formation. In some embodiments, the method further comprises filtering the suspension of step (b). In some embodiments, the method further comprises filtering the suspension of step (b) after 5 to 9 days, 6 to 8 days, or about 7 days.

[0082] In some embodiments, a method for preparing crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde is provided, comprising: (a) combining 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde with a solvent; and (b) subjecting the mixture produced in step (a) to heating / cooling cycles. In some embodiments, the solvent is selected from the group consisting of methanol, acetone, ethyl acetate, isopropyl acetate, propyl acetate, methyl ethyl ketone (MEK), tetrahydrofuran (THF), dichloromethane, acetonitrile, dimethyl sulfoxide (DMSO), nitromethane, n-heptane, water, cyclohexane, ethylene glycol, 2-methyltetrahydrofuran, a mixture of DMSO and water, a mixture of DMF and water, a mixture of NMP and water, a mixture of methanol and water, a mixture of 1-propanol and water, a mixture of 2-methoxyethanol and water, a mixture of ethyl acetate and cyclohexane, a mixture of toluene and n-heptane, a mixture of diethyl ether and n-heptane, and a mixture of MTBE and n-heptane. In some embodiments, the solvent is selected from the group consisting of methanol, acetone, ethyl acetate, isopropyl acetate, propyl acetate, methyl ethyl ketone (MEK), tetrahydrofuran (THF), dichloromethane, acetonitrile, dimethyl sulfoxide (DMSO), nitromethane, n-heptane, water, cyclohexane, ethylene glycol, 2-methyltetrahydrofuran, DMSO:HO (1:1 v / v), DMF and water (1:1 v / v), NMP and water (1:1 v / v), methanol and water (1:1 v / v), 1-propanol:water (1:1 v / v), 2-methoxyethanol:water (1:1 v / v), ethyl acetate and cyclohexane (1:1 v / v), toluene:n-heptane (1:1 v / v), diethyl ether and n-heptane (1:1 v / v), MTBE and n-heptane (1:1 v / v). In some embodiments, the heating / cooling cycle comprises cycling between room temperature and a temperature above room temperature.In some embodiments, the heating / cooling cycle comprises cycling between room temperature and a temperature higher than room temperature (e.g., 30°C to 70°C, 40°C to 60°C, or about 50°C). In some embodiments, the duration of each condition in the heating / cooling cycle is 1 to 6 hours, 2 to 5 hours, or about 4 hours. In some embodiments, in which a two-phase layer is formed in step (a), the method further comprises sonicating the mixture of step (a) and / or subjecting the mixture of step (a) to evaporation. In some embodiments, in which a two-phase layer is formed in step (a), the method further comprises sonicating the mixture of step (a), wherein the duration of the sonication is 0.5 to 3 hours, or about 1.5 hours. In some embodiments, in which a gum is formed in step (a), the method further comprises adding a solvent (e.g., a hydrocarbon solvent such as cyclohexane) to the mixture of step (a) and sonicating the mixture of step (a). In some embodiments where a gum is formed in step (a), the method further comprises adding a solvent (e.g., a hydrocarbon solvent such as cyclohexane) to the mixture of step (a) and sonicating the mixture of step (a), wherein the duration of sonication is between 2 hours and 6 hours, between 3 hours and 5 hours, or about 4 hours. In some embodiments, the method further comprises filtering the solid produced in step (b). In some embodiments, the method further comprises filtering the solid produced in step (b) after 5 to 9 days, 6 to 8 days, or about 7 days.

[0083] In some embodiments, a method for preparing crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde is provided, comprising: (a) wetting 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde with a solvent; and (b) milling the mixture of step (a). In some embodiments, step (b) comprises milling the mixture of step (a) for 1 hour to 3 hours, 1.5 hours to 2.5 hours, or about 2 hours. In some embodiments, step (b) comprises milling the mixture of step (a) at a speed of 3000 rpm to 7000 rpm, 4000 rpm to 6000 rpm, or about 5000 rpm. In some embodiments where a gum is formed in step (a), the method further comprises drying the gum in a vacuum. In some embodiments where a gum is formed in step (a), the method further comprises drying the gum in a vacuum at a temperature 10° C. above or below room temperature, 5° C. above or below room temperature, 3° C. above or below room temperature, or at about room temperature. In some embodiments where a gum is formed in step (a), the method further comprises drying the gum in a vacuum for 5 to 15 hours, 8 to 12 hours, or about 10 hours. In some embodiments where a gum is formed in step (a), the method further comprises drying the gum in a vacuum. In some embodiments where a gum is formed in step (a), the method further comprises drying the gum in a vacuum at a temperature 10° C. above or below room temperature, 5° C. above or below room temperature, 3° C. above or below room temperature, or at about room temperature. In some embodiments where a gum is formed in step (a), the method further comprises drying the gum in a vacuum for 1 to 5 days, 2 to 4 days, or about 3 days. In some embodiments involving a brittle material, the method further comprises drying the mixture including the brittle material in a vacuum.In some embodiments involving a brittle material, the method further comprises drying the mixture including the brittle material in a vacuum at a temperature 10° C. above or below room temperature, 5° C. above or below room temperature, 5° C. above or below room temperature, 3° C. above or below room temperature, or at about room temperature. In some embodiments involving a brittle material, the method further comprises drying the mixture including the brittle material in a vacuum for 5 to 15 hours, 8 to 12 hours, or about 10 hours. In some embodiments, the solvent is selected from the group consisting of a mixture of methanol and water, a mixture of ethanol and water, a mixture of 1-propanol and water, a mixture of 2-propanol and water, a mixture of 2-butanol and water, a mixture of 2-methoxyethan-1-ol and water, a mixture of acetone and water, a mixture of 1,4-dioxane and water, and a mixture of DMSO and water. In some embodiments, the solvent is selected from the group consisting of methanol and water (1:1 and 15:85 v / v), ethanol and water (1:1 and 15:85 v / v), 1-propanol and water (98:2 and 15:85 v / v), 2-propanol and water (98:2, 1:1, and 15:85 v / v), 2-butanol and water (15:85 v / v), 2-methoxyethan-1-ol and water (1:1 v / v), acetone and water (85:15 v / v), 1,4-dioxane and water (1:1 v / v), and DMSO and water (85:15, 1:1, and 15:85 v / v). Crystalline Form I can also be prepared using the procedures provided in Examples 1 and / or 5 herein.

[0084] In some embodiments, a method for preparing crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde is provided, comprising: (1) forming a mixture of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde and a first solvent; (2) heating the mixture of step (1) to a first temperature; (3) cooling the mixture of step (2) to a second temperature; and (4) filtering the mixture of step (3) at the second temperature to obtain a crystalline solid. In some embodiments, the first solvent comprises a nonpolar organic solvent and EtOAc. In some embodiments, the first solvent comprises cyclohexane and EtOAc. In some embodiments, the first solvent comprises petroleum ether and EtOAc. In some embodiments, the first solvent is a mixture of cyclohexane and EtOAc. In some embodiments, the first solvent is a mixture of a non-polar organic solvent and EtOAc. In some embodiments, the first solvent is a mixture of petroleum ether and EtOAc. In some embodiments, the first solvent is a mixture of cyclohexane and EtOAc, wherein the volume ratio of cyclohexane to EtOAc is 3:1 to 7:1, 4:1 to 6:1, or about 5:1. In some embodiments, the first temperature is 50°C to 110°C, 60°C to 100°C, 70°C to 90°C, or about 80°C. In some embodiments, the second temperature is 10°C above or below room temperature, 5°C above or below room temperature, 5°C above or below room temperature, 3°C above or below room temperature, or about room temperature. In some embodiments, the method further includes (5) concentrating the filtrate from step (4) under reduced pressure; and (6) recrystallizing the mixture from step (5) with a second solvent at a third temperature to obtain a crystalline solid. In some embodiments, the second solvent includes petroleum ether and EtOAc. In some embodiments, the second solvent is a mixture of petroleum ether and EtOAc.In some embodiments, the second solvent is a mixture of petroleum ether and EtOAc, and the volume ratio of petroleum ether to EtOAc is 5:1 to 15:1, 7:1 to 13:1, 8:1 to 12:1, 9:1 to 11:1, or about 10:1. In some embodiments, the third temperature is 10°C above or below room temperature, 5°C above or below room temperature, 5°C above or below room temperature, 3°C above or below room temperature, or about room temperature.

[0085] Crystalline Form I can also be prepared using the procedures provided in Examples 1 and / or 5 herein.

[0086] Crystalline Form II In some embodiments, a method for preparing crystalline Form II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde is provided, comprising: (1) combining 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde with a solvent; and (2) stirring the mixture produced in step (1) to form a suspension. In some embodiments, step (2) comprises stirring the mixture of step (1) at a temperature between 0°C and 30°C, between 0°C and 20°C, between 0°C and 10°C, or about 5°C. In some embodiments, the solvent is selected from the group consisting of n-heptane, diethyl ether, propyl acetate, ethyl acetate, isopropyl acetate, MIBK, 2-propanol, ethanol, MTBE, 2-methyl-1-propanol, toluene, 1,2-dimethoxyethane, tetrahydrofuran, 2-methoxyethanol, methanol, a mixture of isopropanol and water, a mixture of 2-propanol and water, a mixture of chloroform and n-heptane, a mixture of dichloromethane and n-heptane, a mixture of cyclohexane and heptane, and a mixture of THF and water. In some embodiments, the solvent is selected from the group consisting of n-heptane, diethyl ether, propyl acetate, ethyl acetate, isopropyl acetate, methyl isobutyl ketone (MIBK), 2-propanol, ethanol, MTBE, 2-methyl-1-propanol, toluene, 1,2-dimethoxyethane, tetrahydrofuran, 2-methoxyethanol, methanol, iPA:water (95:5 v / v), 2-propanol and water (95:5 v / v), chloroform and n-heptane (1:1 v / v), dichloromethane and n-heptane (1:1 v / v), cyclohexane and n-heptane (1:1 v / v), and THF and water (1:1 v / v). In some embodiments, the method further comprises adding an additional solvent. In some embodiments, the method further comprises adding an anti-solvent to promote suspension formation. In some embodiments, the method further comprises filtering the suspension of step (2).In some embodiments, the method further comprises filtering the suspension of step (2) after 5 to 9 days, 6 to 8 days, or about 7 days.

[0087] In some embodiments, a method for preparing crystalline Form II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde is provided, comprising: (a) combining 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde with a solvent; and (b) subjecting the mixture produced in step (a) to heating / cooling cycles. In some embodiments, the solvent is ethanol, 1-propanol, 2-propanol, ethyl acetate, propyl acetate, isopropyl acetate, MIBK, 2-methyl-1-propanol, 1,2 dimethoxyethane, 2-methoxyethanol, DMF, diethyl ether, MTBE, n-heptane, cyclohexane, a mixture of ethanol and water, a mixture of 2-propanol and water, a mixture of 1,2-dimethoxyethane and water, a mixture of DMF and water, a mixture of NMP and water, diethyl ether and n-heptane. a mixture of ethyl acetate and n-heptane, a mixture of propyl acetate and n-heptane, a mixture of isopropyl acetate and n-heptane, a mixture of MIBK and n-heptane, a mixture of MEK and n-heptane, a mixture of MTBE and n-heptane, a mixture of 2-methyl-1-propanol and n-heptane, a mixture of THF and n-heptane, a mixture of 2-propanol and n-heptane, a mixture of acetone and n-heptane, and a mixture of chloroform and n-heptane.In some embodiments, the solvent is ethanol, 1-propanol, 2-propanol, ethyl acetate, propyl acetate, isopropyl acetate, MIBK, 2-methyl-1-propanol, 1,2 dimethoxyethane, 2-methoxyethanol, DMF, diethyl ether, MTBE, n-heptane, cyclohexane, ethanol and water (1:1 v / v), 2-propanol and water (95:5 and 1:1 v / v), 1,2-dimethoxyethane and water (1:1 v / v), a mixture of DMF and water (1:1 v / v), a mixture of NMP and water (1:1 v / v), a mixture of diethyl ether and n-heptane (1:1 v / v), a mixture of ethyl acetate and n-heptane (1:1 v / v), or a mixture of methyl ether and n-heptane (1:1 v / v). 1:1 v / v), a mixture of propyl acetate and n-heptane (1:1 v / v), a mixture of isopropyl acetate and n-heptane (1:1 v / v), a mixture of MIBK and n-heptane (1:1 v / v), a mixture of MEK and n-heptane (1:1 v / v), a mixture of MTBE and n-heptane (1:1 v / v), a mixture of 2-methyl-1-propanol and n-heptane (1:1 v / v), a mixture of THF and n-heptane (1:1 v / v), a mixture of 2-propanol and n-heptane (1:1 v / v), a mixture of acetone and n-heptane (1:1 v / v), and a mixture of chloroform and n-heptane (1:1 v / v). In some embodiments, the heating / cooling cycle comprises cycling between room temperature and a temperature above room temperature. In some embodiments, the heating / cooling cycle comprises cycling between room temperature and a temperature higher than room temperature (e.g., 30°C to 70°C, 40°C to 60°C, or about 50°C). In some embodiments, the duration of each condition is 1 to 6 hours, 2 to 5 hours, or about 4 hours. In some embodiments, where a two-phase layer is formed in step (a), the method further comprises sonicating the mixture of step (a) and / or subjecting the mixture of step (a) to evaporation. In some embodiments, where a two-phase layer is formed in step (a), the method further comprises sonicating the mixture of step (a), where the duration of sonication is 0.5 to 3 hours, or about 1.5 hours.In some embodiments where a gum is formed in step (a), the method further comprises adding cyclohexane to the mixture of step (a) and sonicating the mixture of step (a). In some embodiments where a gum is formed in step (a), the method further comprises adding cyclohexane to the mixture of step (a) and sonicating the mixture of step (a), wherein the duration of sonication is between 2 hours and 6 hours, between 3 hours and 5 hours, or about 4 hours. In some embodiments, the method further comprises filtering the solids produced in step (b). In some embodiments, the method further comprises filtering the solids produced in step (b) after 5 to 9 days, 6 to 8 days, or about 7 days.

[0088] In some embodiments, a method for preparing crystalline Form II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde is provided, the method comprising: (a) combining 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde with a solvent; (b) stirring the mixture produced in step (a); and (c) collecting an aliquot of the mixture produced in step (b). In some embodiments, step (b) comprises stirring the mixture of step (a) at a temperature of about 65° C. to 85° C., 70° C. to 80° C., or about 75° C. In some embodiments, step (b) comprises stirring the mixture of step (a) at a speed of 300 rpm to 700 rpm, 400 rpm to 600 rpm, or about 500 rpm. In some embodiments, step (b) comprises stirring the mixture of step (a) for 5 to 15 hours, 8 to 12 hours, or about 10 hours. In some embodiments where a two-phase layer is formed in step (b), the method further comprises adding additional solvent to the mixture of step (c) until a solution is formed, cooling the solution, and stirring the solution at the cooled temperature. In some embodiments where a two-phase layer is formed in step (b) and additional solvent is added to the mixture of step (c) until a solution is formed, the temperature to which the solution is cooled and stirred is 3°C to 7°C, 4°C to 6°C, or about 5°C. In some embodiments, where a two-phase layer is formed in step (b), the method further comprises adding additional solvent to the mixture of step (c) until a solution is formed, and cooling the solution at a rate of between 0.08° C. / min and 0.12° C. / min, between 0.09° C. / min and 0.10° C. / min, or about 0.1° C. / min. In some embodiments, the solvent is selected from the group consisting of n-heptane, cyclohexane, a mixture of methanol and water, a mixture of ethanol and water, a mixture of 1-propanol and water, a mixture of 2-propanol and water, a mixture of 2-butanol and water, and a mixture of acetone and water.In some embodiments, the solvent is selected from the group consisting of n-heptane, cyclohexane, methanol and water (1:1 and 15:85 v / v), ethanol and water (1:1 and 85:15 v / v), 1-propanol and water (1:1 and 15:85 v / v), 2-propanol and water (1:1 and 15:85 v / v), 2-butanol and water (20:80 v / v), and acetone and water (1:1 v / v). In some embodiments, the method further comprises adding an additional solvent. In some embodiments, the method further comprises adding an anti-solvent to promote suspension formation. In some embodiments, the method further comprises filtering the suspension of step (c). In some embodiments, the method further comprises filtering the suspension of step (c) after 13-17 days, 14-16 days, or about 15 days. In some embodiments, the method further comprises filtering the suspension of step (c) after 16 to 20 days, 17 to 19 days, or about 18 days.

[0089] In some embodiments, a method for preparing crystalline Form II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde is provided, comprising: (a) wetting 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde with a solvent; and (b) milling the mixture of step (a). In some embodiments, step (b) comprises milling the mixture of step (a) for a specific time period between 1 hour and 3 hours, between 1.5 hours and 2.5 hours, or about 2 hours. In some embodiments, step (b) comprises milling the mixture of step (a) at a speed of 3000 rpm to 7000 rpm, 4000 rpm to 6000 rpm, or about 5000 rpm. In some embodiments where a gum is formed in step (a), the method further comprises drying the gum in a vacuum. In some embodiments where a gum is formed in step (a), the method further comprises drying the gum in a vacuum at a temperature 10° C. above or below room temperature, 5° C. above or below room temperature, 3° C. above or below room temperature, or at about room temperature. In some embodiments where a gum is formed in step (a), the method further comprises drying the gum in a vacuum for 5 to 15 hours, 8 to 12 hours, or about 10 hours. In some embodiments where a gum is formed in step (a), the method further comprises drying the gum in a vacuum. In some embodiments where a gum is formed in step (a), the method further comprises drying the gum in a vacuum at a temperature 10° C. above or below room temperature, 5° C. above or below room temperature, 3° C. above or below room temperature, or at about room temperature. In some embodiments where a gum is formed in step (a), the method further comprises drying the gum in a vacuum for 1 to 5 days, 2 to 4 days, or about 3 days. In some embodiments involving a brittle material, the method further comprises drying the mixture including the brittle material in a vacuum.In some embodiments involving a brittle material, the method further comprises drying the mixture comprising the brittle material in a vacuum at a temperature 10° C. above or below room temperature, 5° C. above or below room temperature, 3° C. above or below room temperature, or at about room temperature. In some embodiments involving a brittle material, the method further comprises drying the mixture comprising the brittle material in a vacuum for 5 to 15 hours, 8 to 12 hours, or about 10 hours. In some embodiments, the solvent is selected from the group consisting of a mixture of methanol and water, a mixture of ethanol and water, a mixture of 1-propanol and water, a mixture of 2-propanol and water, a mixture of 2-butanol and water, a mixture of acetone and water, a mixture of 1,4-dioxane and water, a mixture of THF and water, a mixture of acetonitrile and water, a mixture of DMSO and water, or a mixture of dichloromethane and n-heptane. In some embodiments, the solvent is selected from the group consisting of methanol and water (97:3, 85:15, and 1:1 v / v), ethanol and water (97:3 and 85:15 v / v), 1-propanol and water (85:15 and 1:1 v / v), 2-propanol and water (98:2, 85:15, and 1:1 v / v), 2-butanol and water (98:2 v / v), acetone and water (1:1 v / v), 1,4 dioxane and water (99:1 and 1:1 v / v), THF and water (99:1 v / v), acetonitrile and water (1:1 v / v), DMSO and water (15:85 v / v), and dichloromethane and n-heptane (1:1 v / v).

[0090] In some embodiments, a method for preparing crystalline Form II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde comprises: (1) reacting 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde with a first (1) forming a mixture with a solvent; (2) heating the mixture of step (1) to a first temperature; (3) adding a second solvent to the mixture of step (2) at the first temperature; (4) stirring the mixture of step (3) at the first temperature; (5) cooling the mixture of step (4) to a second temperature; (6) stirring the mixture of step (5) at the second temperature; and (7) filtering the mixture of step (6) at the second temperature to obtain a crystalline solid. In some embodiments, the first solvent comprises ethanol and water. In some embodiments, the first solvent is ethanol. In some embodiments, the second solvent comprises water. In some embodiments, the second solvent is water. In some embodiments, the first temperature is between 50°C and 110°C, between 60°C and 100°C, between 70°C and 90°C, or about 80°C. In some embodiments, the first temperature is 78°C. In some embodiments, the first temperature is 85°C. In some embodiments, the first temperature is 60°C to 120°C, 70°C to 110°C, 80°C to 100°C, or about 90°C. In some embodiments, the second temperature is 10°C above or below room temperature, 5°C above or below room temperature, 3°C above or below room temperature, or about room temperature. In some embodiments where the first temperature is 85°C to 90°C, step (5) further comprises adding crystalline Form II seeds to the mixture of step (4) at an intermediate temperature between the first and second temperatures. In some embodiments where crystalline Form II seeds are added to the mixture of step (4) at an intermediate temperature, the intermediate temperature is 40°C to 60°C, 45°C to 55°C, or about 50°C. In some embodiments where crystalline Form II seeds are added to the mixture of step (4) at an intermediate temperature, the intermediate temperature is 60°C to 80°C, 65°C to 75°C, or about 70°C.In some embodiments, step (2) further comprises adding a second solvent during the heating process. In some embodiments where step (2) comprises adding a second solvent during the heating process, the second solvent is added at a temperature of 45°C to 55°C, or about 50°C, and / or at a temperature of 60°C to 70°C, or about 65°C. In some embodiments, the method further comprises drying the solid of step (7) under air or vacuum. In some embodiments, the method further comprises drying the solid of step (7) at less than 50°C. In some embodiments, the method further comprises drying the solid of step (7) at a temperature 10°C above or below room temperature, 5°C above or below room temperature, 3°C above or below room temperature, or at about room temperature. In some embodiments, the method further comprises drying the solid of step (7) under air or vacuum for 1 day to 5 days, 2 days to 4 days, or about 3 days.

[0091] Crystalline Form II can also be prepared using the procedures set forth in Examples 2 and / or 5 herein.

[0092] Crystalline Form III In some embodiments, provided is a method for preparing crystalline Form III of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde, comprising: (1) combining 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde with a solvent comprising 1,4-dioxane; and (2) stirring the mixture produced in step (1) to form a suspension. In some embodiments, step (2) comprises stirring the mixture of step (1) at a temperature between 4° C. and 6° C., between 4.5° C. and 5.5° C., or about 5° C. In some embodiments, the solvent is 1,4-dioxane and water. In some embodiments, the solvent comprises a mixture of 1,4-dioxane and water (v / v 1:1). In some embodiments, the method further comprises adding an additional solvent. In some embodiments, the method further comprises adding an anti-solvent to promote suspension formation. In some embodiments, the method further comprises filtering the suspension of step (2). In some embodiments, the method further comprises filtering the suspension of step (2) after 5 to 9 days, 6 to 8 days, or about 7 days.

[0093] Crystalline Form III can also be prepared using the procedures set forth in Examples 4 and / or 5 herein.

[0094] How to use The crystalline forms and compositions provided herein can be used to treat or prevent a disease or condition in an individual or subject.

[0095] When used prophylactically, the crystalline forms and compositions disclosed and / or described herein can prevent or reduce the severity of a disease or disorder that may develop in an individual or subject at risk of developing the disease or disorder.

[0096] Without being bound by theory, the provided crystalline forms and compositions are believed to act by inhibiting myosin. This inhibition potentially reduces the amount of contraction by reducing the number of independent myosin heads that interact with actin filaments. Reducing cardiac muscle contractility can be important for treating cardiac diseases in which excessive contractility is a problem. In some embodiments, methods for treating or preventing cardiac disease in an individual or subject include administering a crystalline form or composition provided herein to an individual or subject in need of cardiac treatment or prevention. In some embodiments, methods are provided for treating or preventing cardiac disease in a subject in need of cardiac treatment or prevention, comprising administering a therapeutically effective amount of at least one crystalline form or composition described herein to the subject. In some embodiments, methods are provided for treating cardiac disease in a subject in need of cardiac treatment, comprising administering a therapeutically effective amount of at least one crystalline form or composition described herein to the subject. In some embodiments, methods are provided for treating established or diagnosed cardiac disease in a subject in need of established or diagnosed cardiac treatment, comprising administering a therapeutically effective amount of at least one crystalline form or composition described herein to the subject. In some embodiments, methods are provided for preventing cardiac disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one crystalline form or composition described herein.

[0097] Also provided herein is the use of a crystalline form or composition provided herein in the manufacture of a medicament for treating a cardiac disease in a subject. In some aspects, a crystalline form described herein is provided for use in a method of treating the human or animal body with therapy. In some embodiments, a crystalline form such as crystalline Form I, II, or III, or a mixture thereof, or a composition thereof, is provided herein for use in a method of treating the human or animal body with therapy. In some embodiments, a crystalline form such as crystalline Form I, II, or III, or a mixture thereof, or a composition thereof, is provided herein for use in the treatment or prevention of cardiac disease. In some embodiments, a crystalline form such as crystalline Form I, II, or III, or a mixture thereof, or a composition thereof, is provided herein for use in the treatment of cardiac disease. In some embodiments, a crystalline form such as crystalline Form I, II, or III, or a mixture thereof, or a composition thereof, is provided herein for use in the treatment of established or diagnosed cardiac disease. In other embodiments, a crystalline form such as crystalline Form I, II, or III, or a mixture thereof, or a composition thereof, is provided herein for use in the prevention of cardiac disease. In some embodiments, provided herein are crystalline forms such as crystalline Form I, II, or III, or mixtures thereof, or compositions thereof, for use in treating a disease or condition associated with HCM. In some embodiments, provided herein are crystalline forms such as crystalline Form I, II, or III, or mixtures thereof, or compositions thereof, for use in treating a disease or condition associated with secondary left ventricular hypertrophy. In some embodiments, provided herein are crystalline forms such as crystalline Form I, II, or III, or mixtures thereof, or compositions thereof, for use in ameliorating symptoms associated with cardiac disease. In other embodiments, provided herein are crystalline forms such as crystalline Form I, II, or III, or mixtures thereof, or compositions thereof, for use in reducing the risk of symptoms associated with cardiac disease.In other embodiments, provided herein are crystalline forms such as crystalline Form I, II, or III, or mixtures thereof, or compositions thereof, for use in treating diseases or conditions associated with narrowing of the left ventricular cavity, cardiac chamber obstruction, hyperdynamic left ventricular contractions, impaired left ventricular ejection blood flow, cardiac hypertrophy, low cardiac output, impaired left ventricular relaxation, elevated left ventricular filling pressure, myocardial ischemia, or myocardial fibrosis. In certain embodiments, provided herein are crystalline forms such as crystalline Form I, II, or III, or mixtures thereof, or compositions thereof, for use in treating diseases or conditions associated with narrowing of the left ventricular cavity and cardiac chamber obstruction, hyperdynamic left ventricular contractions, myocardial ischemia, or myocardial fibrosis. In some embodiments, provided herein are crystalline forms such as crystalline Form I, II, or III, or mixtures thereof, or compositions thereof, for use in treating muscular dystrophy. In some embodiments, provided herein are crystalline forms such as crystalline Form I, II, or III, or mixtures thereof, or compositions thereof, for use in treating glycogen storage disease. In other embodiments, provided herein are crystalline forms, such as Form I, II, or III, or mixtures thereof, or compositions thereof, for use in modulating cardiac muscle sarcomeres, such as inhibiting cardiac muscle sarcomeres. In yet other embodiments, provided herein are crystalline forms, such as Form I, II, or III, or mixtures thereof, or compositions thereof, for use in enhancing cardiac myosin.

[0098] In some embodiments, the subject is a mammal. In some embodiments, the subject is a mouse, rat, dog, cat, pig, sheep, horse, cow, or human. In some embodiments, the subject is a human. In some embodiments, the subject has established or diagnosed heart disease. In some embodiments, the subject has established or diagnosed hypertrophic cardiomyopathy (HCM). In some embodiments, the subject is at risk of developing heart disease. In some embodiments, the subject has a mutation that increases the risk of heart disease. In some embodiments, the subject has a mutation that increases the risk of hypertrophic cardiomyopathy (HCM). In some embodiments, the mutation is a sarcomere mutation. In some embodiments, the mutation is in myosin heavy chain beta (MHC-β), cardiac troponin T (cTnT), tropomyosin alpha-1 chain (TPM1), myosin-binding protein C cardiac type (MYBPC3), cardiac troponin I (cTnI), myosin essential light chain (ELC), titin (TTN), myosin regulatory light chain 2 cardiac / cardiac isoform (MLC-2), cardiac alpha actin, muscle LIM protein (MLP), or protein kinase AMP-activated non-catalytic subunit gamma 2 (PRKAG2). In some embodiments, the mutation is in MHC-β. In some embodiments, the subject has established or been diagnosed with hypertrophic cardiomyopathy without a known genetic etiology.

[0099] In some embodiments, the subject has an increased risk of progressive symptoms. In some embodiments, the subject has an increased risk of atrial fibrillation, ventricular tachyarrhythmia, stroke, and / or sudden death. In some embodiments, the subject has reduced exercise capacity. In some embodiments, the reduced exercise capacity is compared to an age-matched control population. In some embodiments, the subject is eligible for surgical intervention or percutaneous ablation to treat cardiac disease.

[0100] In some embodiments, the cardiac disease is hypertrophic cardiomyopathy (HCM). In some embodiments, the cardiac disease is obstructive HCM. In some embodiments, the cardiac disease is non-obstructive HCM. In some embodiments, the HCM is associated with a sarcomere mutation. In some embodiments, the HCM is associated with a non-sarcomere mutation. In some embodiments, the cardiac disease is obstructive or non-obstructive HCM caused by a sarcomere and / or non-sarcomere mutation. In some embodiments, the sarcomere mutation is a mutation in myosin heavy chain beta (MHC-β), cardiac troponin T (cTnT), tropomyosin alpha-1 chain (TPM1), myosin-binding protein C cardiac type (MYBPC3), cardiac troponin I (cTnI), myosin essential light chain (ELC), titin (TTN), myosin regulatory light chain 2 cardiac / cardiac isoform (MLC-2), cardiac alpha actin, or muscle LIM protein (MLP). In some embodiments, the sarcomeric mutation is a mutation in MHC-β. In some embodiments, the non-sarcomeric mutation is a mutation in protein kinase AMP-activated non-catalytic subunit gamma 2 (PRKAG2).

[0101] In some embodiments, provided herein are methods of treating a disease or condition associated with HCM, comprising administering a crystalline form or composition provided herein to an individual or subject in need of treatment for the disease or condition. In some embodiments, the disease or condition is Fabry disease, Danon disease, mitochondrial cardiomyopathy, or Noonan syndrome.

[0102] Also provided is the use of a crystalline form or composition provided herein in the manufacture of a medicament for treating a disease or condition associated with HCM.

[0103] In some embodiments, the cardiac disease is heart failure with preserved ejection fraction (HFpEF). In some embodiments, the cardiac disease is diastolic dysfunction. In some embodiments, the cardiac disease is cardiomyopathy. In some embodiments, the cardiac disease is primary or secondary restrictive cardiomyopathy. In some embodiments, the cardiac disease is a condition or symptom caused by coronary artery disease. In some embodiments, the cardiac disease is myocardial infarction or angina pectoris. In some embodiments, the cardiac disease is left ventricular outflow tract obstruction. In some embodiments, the cardiac disease is hypertensive heart disease. In some embodiments, the cardiac disease is congenital heart disease. In some embodiments, the cardiac disease is cardiac ischemia and / or coronary heart disease. In some embodiments, the cardiac disease is diabetic heart disease. In other embodiments, the cardiac disease is congestive heart failure. In some embodiments, the cardiac disease is right heart failure. In other embodiments, the cardiac disease is cardiorenal syndrome. In some embodiments, the cardiac disease is infiltrative cardiomyopathy. In some embodiments, the cardiac disease is or is associated with cardiac aging or diastolic dysfunction due to aging. In some embodiments, the cardiac disease is or is associated with age-related cardiomyocyte senescence or diastolic dysfunction.

[0104] In some embodiments, provided are methods for treating a disease or condition associated with secondary left ventricular wall thickening in an individual or subject, comprising administering a crystalline form or composition provided herein to an individual or subject in need of treatment for the disease or condition. In some embodiments, the disease is hypertension, valvular heart disease (aortic stenosis, mitral regurgitation), metabolic syndrome (diabetes, obesity), end-stage renal failure, scleroderma, sleep apnea, amyloidosis, Fabry disease, Friedreich's ataxia, Danon disease, Noonan syndrome, or Pompe disease.

[0105] Also provided is the use of a crystalline form or composition provided herein in the manufacture of a medicament for treating a disease or condition associated with secondary left ventricular wall thickening.

[0106] In some embodiments, methods are provided for ameliorating a symptom associated with cardiac disease in a subject, comprising administering a crystalline form or composition provided herein to an individual or subject in need of amelioration of a symptom associated with cardiac disease, wherein the symptom is one or more selected from impaired or decreased cardiac elasticity, impaired or reduced left ventricular diastolic relaxation, abnormal left atrial pressure (e.g., abnormally high left atrial pressure), paroxysmal or persistent atrial fibrillation, elevated left atrial and pulmonary capillary wedge pressure, elevated left ventricular diastolic pressure, syncope, ventricular relaxation in diastole, ventricular fibrosis, left ventricular hypertrophy, left ventricular myocardial mass, increased left ventricular wall thickness, mid-left ventricular obstruction, increased mitral valve systolic anterior motion, left ventricular outflow tract obstruction, chest pain, dyspnea on exertion, presyncope, impaired exercise capacity, and fatigue.

[0107] In some embodiments, methods are provided for reducing the risk of a symptom associated with cardiac disease in a subject, comprising administering a crystalline form or composition provided herein to an individual or subject in need of reduced risk of a symptom associated with cardiac disease, wherein the symptom is one or more selected from sudden cardiac death, insufficient or reduced cardiac elasticity, insufficient or reduced left ventricular diastolic relaxation, abnormal left atrial pressure (e.g., abnormally high left atrial pressure), paroxysmal or persistent atrial fibrillation, elevated left atrial and pulmonary capillary wedge pressure, elevated left ventricular diastolic pressure, syncope, ventricular relaxation in diastole, ventricular fibrosis, left ventricular hypertrophy, left ventricular myocardial mass, increased left ventricular wall thickness, mid-left ventricular obstruction, increased mitral valve systolic anterior motion, left ventricular outflow tract obstruction, chest pain, dyspnea on exertion, pre-syncope, impaired exercise capacity, and fatigue.

[0108] In some embodiments, methods are provided for treating a disease or condition in an individual or subject associated with narrowing of the left ventricular chamber, cardiac chamber obstruction, hyperdynamic left ventricular contraction, impaired blood flow ejected from the left ventricle, cardiac hypertrophy, low cardiac output, impaired left ventricular relaxation, elevated left ventricular filling pressure, myocardial ischemia, or myocardial fibrosis, comprising administering to a subject in need of treatment for the disease or condition a crystalline form or composition provided herein.

[0109] In some embodiments, provided are methods for treating a disease or condition associated with left ventricular chamber narrowing and chamber obstruction, hyperdynamic left ventricular contraction, myocardial ischemia, or myocardial fibrosis in an individual or subject, comprising administering to an individual or subject in need of treatment for the disease or condition a crystalline form or composition provided herein.

[0110] Also provided is the use of a crystalline form or composition provided herein in the manufacture of a medicament for treating a disease or condition associated with left ventricular chamber narrowing and chamber obstruction, hyperdynamic left ventricular contraction, myocardial ischemia, or myocardial fibrosis.

[0111] In some embodiments, provided herein are methods of treating muscular dystrophy (e.g., Duchenne muscular dystrophy) in an individual or subject, comprising administering to the individual or subject in need thereof a crystalline form or composition provided herein. Also provided herein is the use of a crystalline form or composition provided herein in the manufacture of a medicament for treating muscular dystrophy (e.g., Duchenne muscular dystrophy).

[0112] In some embodiments, a method of treating glycogen storage disease in an individual or subject comprises administering a crystalline form or composition provided herein to an individual or subject in need of glycogen storage disease treatment. Also provided herein is the use of a crystalline form or composition provided herein in the manufacture of a medicament for the treatment of glycogen storage disease.

[0113] Also provided are methods for modulating cardiac muscle sarcomeres in an individual or subject, comprising administering a therapeutically effective amount of at least one chemical entity described herein to an individual or subject in need of cardiac muscle sarcomere modulation. In some embodiments, methods for inhibiting cardiac muscle sarcomeres are also provided, comprising contacting cardiac muscle sarcomeres with at least one chemical entity described herein, such as a crystalline form or composition provided herein. Further provided herein is the use of at least one chemical entity described herein, such as a crystalline form or composition provided herein, in the manufacture of a medicament for inhibiting cardiac muscle sarcomeres in an individual or subject.

[0114] Also provided is a method for enhancing cardiac myosin in an individual or subject, comprising administering to an individual or subject in need of enhanced cardiac myosin a therapeutically effective amount of at least one chemical entity described herein, such as a crystalline form or composition provided herein. Further provided herein is the use of at least one chemical entity described herein, such as a crystalline form or composition provided herein, in the manufacture of a medicament for enhancing cardiac myosin in an individual or subject.

[0115] In some embodiments, the methods provided herein further comprise monitoring the effectiveness of the treatment. Exemplary indicators include, but are not limited to, improvement in one or more of the following: New York Heart Association (NYHA) functional class, exercise capacity, cardiac elasticity, diastolic left ventricular relaxation, left atrial pressure, paroxysmal or persistent atrial fibrillation, left atrial and pulmonary capillary wedge pressure, left ventricular diastolic pressure, syncope, diastolic ventricular relaxation, ventricular fibrosis, left ventricular hypertrophy, left ventricular myocardial mass, left ventricular wall thickness, left ventricular mid-obstruction, mitral valve systolic anterior motion, left ventricular outflow tract obstruction, chest pain, exertional dyspnea, presyncope, exercise capacity abnormalities, and fatigue. These indicators can be monitored by techniques well known in the art, including self-report, ECG, including ambulatory ECG, echocardiography, cardiac MRI, CT, biopsy, cardiopulmonary exercise testing (CPET), and actigraphy.

[0116] In some embodiments, the crystalline forms or compositions described herein reduce cardiomyocyte contractility. In some embodiments, the crystalline forms or compositions reduce cardiomyocyte contractility by more than 40%, e.g., by more than 45%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the crystalline forms or compositions reduce cardiomyocyte contractility by 40% to 90%, e.g., by 40% to 80%, 40% to 70%, 50% to 90%, 50% to 80%, or 50% to 70%. In some embodiments, the crystalline forms or compositions do not significantly alter calcium transients in cardiomyocytes. In some embodiments, the crystalline forms or compositions reduce ATPase activity in cardiomyocytes. Methods for measuring contractile force, ATPase activity, and calcium transients, for example, by calcium labeling, electrophysiological recording, and microscopic imaging, are well known in the art. In some embodiments, the crystalline forms or compositions do not significantly inhibit or induce cytochrome P450 (CYP) proteins.

[0117] In some embodiments, crystalline forms or compositions are provided that have an elimination half-life (t, calculated as ln(2) / k, where the elimination rate constant k is the linear regression of the logarithm of the concentration versus time for the last three data points of the concentration-time profile) in humans of 30 hours or less. In some embodiments, in humans, 10 hours < t 1 / 2 In some embodiments, t 1 / 2 In some embodiments, t is about 10 hours to about 30 hours, about 10 hours to about 25 hours, about 15 hours to about 30 hours, or about 15 hours to about 25 hours. 1 / 2 is about 12, 15, 18, 21, 24, 27, or 30 hours. In some embodiments, the elimination half-life of the compounds provided herein is such that the compounds are suitable for once-daily administration.

[0118] In some embodiments, the subject has a left ventricular wall that is thicker than normal before treatment. In some embodiments, the subject has a left ventricular wall thickness of more than 15 mm, for example, more than 18 mm, 20 mm, 22 mm, 25 mm, or 30 mm, before treatment. In some embodiments, the left ventricular wall thickness is reduced by more than 5%, for example, more than 8%, 10%, 12%, 15%, 20%, or 30% after treatment. Left ventricular wall thickness can be measured by methods well known in the art, such as echocardiography, CT scan, or cardiac MRI.

[0119] In some embodiments, the subject has abnormal myocardial fibrosis before treatment. In some embodiments, the abnormal myocardial fibrosis is reduced by more than 5%, for example, 8%, 10%, 12%, 15%, 20%, or more than 30% after treatment. Myocardial fibrosis can be measured by methods well known in the art, such as biopsy or cardiac MRI.

[0120] In some embodiments, the subject has reduced exercise capacity prior to treatment. In some embodiments, the subject's exercise capacity increases by more than 5%, e.g., more than 8%, 10%, 12%, 15%, 20%, or 30%, after treatment. In some embodiments, exercise capacity is measured by cardiopulmonary exercise testing (CPET). CPET measures changes in oxygen consumption (VO2max). CPET and methods for measuring VO2max are well known in the art (Malhotra et al., JACC:Heal Failure, 2016, 4(8):607-616; Guuzzi et al., J Amer College Cardiol, 2017, 70(13):1618-1636; Rowin et al., JACC:Cariovasc Imaging, 2017, 10(11):1374-1386). In some embodiments, VO2max increases by more than 1 mL / kg / m after treatment. 2 greater than, e.g., 1.2 mL / kg / m 2 , 1.4 mL / kg / m 2 , 1.5 mL / kg / m 2 , 1.7 mL / kg / m 2 , 2 mL / kg / m 2, 2.2 mL / kg / m 2 , 2.5 mL / kg / m 2 , 3 mL / kg / m 2 , 3.2 mL / kg / m 2 , or 3.5 mL / kg / m 2 Super improved.

[0121] In some embodiments, the subject has a New York Heart Association (NYHA) functional class II, III, or IV before treatment. In some embodiments, the subject has a New York Heart Association (NYHA) functional class III or IV before treatment. In some embodiments, the subject has a New York Heart Association (NYHA) functional class IV before treatment. In some embodiments, after treatment, the subject's NYHA functional class remains the same or is lowered.

[0122] In some embodiments, after treatment, the VO2max is less than or equal to 1 mL / kg / m 2 greater than, for example, 1.2 mL / kg / m 2 , 1.4 mL / kg / m 2 , 1.5 mL / kg / m 2 , 1.7 mL / kg / m 2 , or 2 mL / kg / m 2 In some embodiments, after treatment, the VO2max is greater than or equal to 2.5 mL / kg / m 2 , 3 mL / kg / m 2 , 3.2 mL / kg / m 2 , or 3.5 mL / kg / m 2 Ultra, improved, and the subject's NYHA functional class remains the same or becomes lower in NYHA functional class.

[0123] In some embodiments, following treatment, the subject's daily function and / or activity level is improved. Improved daily function and / or activity level can be measured, for example, by daily activity tracking or actigraphy, such as a wearable health monitor or activity tracker (e.g., a FITBIT® or FITBIT®-like monitor).

[0124] In some embodiments, the subject has one or more of reduced shortness of breath, reduced chest pain, reduced arrhythmia burden such as atrial fibrillation and ventricular arrhythmias, reduced incidence of heart failure, and reduced ventricular outflow tract obstruction after treatment.

[0125] Dosage The crystalline forms and compositions disclosed and / or described herein are administered at therapeutically effective doses, e.g., doses sufficient to provide treatment for a condition. While human dosage levels for the chemical entities described herein have not yet been optimized, they generally range from about 0.01 to 100 mg / kg body weight daily, in some embodiments from about 0.05 to 10.0 mg / kg body weight, and in some embodiments, from about 0.10 to 1.4 mg / kg body weight. Thus, for a 70 kg human, the dosage range is, in some embodiments, from about 0.7 to 7000 mg per day, in some embodiments, from about 3.5 to 700.0 mg per day, and in some embodiments, from about 7 to 100.0 mg per day. The amount of chemical entity administered will depend, for example, on the subject and condition being treated, the severity of the condition, the mode and schedule of administration, and the judgment of the prescribing physician. For example, an exemplary dosage range for oral administration is about 5 mg to about 500 mg per day, and an exemplary dosage range for intravenous administration is about 5 mg to about 500 mg per day, depending on the pharmacokinetics of each.

[0126] Daily dose is the total amount administered in one day. Daily dose can be administered daily, every other day, weekly, every two weeks, monthly, or at various intervals, but is not limited to this. In some embodiments, daily dose is administered for a period ranging from one day to the life of the subject. In some embodiments, daily dose is administered once a day. In some embodiments, daily dose is administered in multiple divided doses, such as two, three, or four divided doses. In some embodiments, daily dose is administered in two divided doses.

[0127] Administration of the crystalline forms and compositions described herein can be by any accepted method of administration of therapeutic agents, including, but not limited to, oral, sublingual, subcutaneous, parenteral, intravenous, intranasal, topical, transdermal, intraperitoneal, intramuscular, pulmonary, vaginal, rectal, or ocular administration. In some embodiments, the crystalline forms or compositions are administered orally or intravenously. In some embodiments, the crystalline forms or compositions disclosed and / or described herein are administered orally.

[0128] Pharmaceutically acceptable compositions include solid, semi-solid, liquid, and aerosol dosage forms, such as tablets, capsules, powders, solutions, suspensions, suppositories, and aerosol forms. The crystalline forms disclosed and / or described herein can also be administered in sustained or controlled release dosage forms (e.g., controlled / sustained release pills, depot injections, osmotic pumps, or transdermal (including electrotransport) patch forms) for extended timed administration and / or pulsed administration at a predetermined rate. In some embodiments, the compositions are provided in unit dosage forms suitable for single administration of precise doses.

[0129] The crystalline forms disclosed and / or described herein may be administered alone or in combination with one or more conventional pharmaceutical carriers or excipients (e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, croscarmellose sodium, glucose, gelatin, sucrose, magnesium carbonate). If desired, the pharmaceutical compositions may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents, and the like (e.g., sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate). Generally, pharmaceutical compositions contain from about 0.005% to 95% by weight, or from about 0.5% to 50% by weight, of a compound disclosed and / or described herein, depending on the intended mode of administration. Actual methods for preparing such dosage forms are known, or will become apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

[0130] In some embodiments, the compositions take the form of a pill or tablet, and thus may contain one or more of a diluent (e.g., lactose, sucrose, dicalcium phosphate), a lubricant (e.g., magnesium stearate), and / or a binder (e.g., starch, gum arabic, polyvinylpyrrolidine, gelatin, cellulose, cellulose derivatives) along with the crystalline forms disclosed and / or described herein. Other solid dosage forms include powders, marume, solutions or suspensions (e.g., in propylene carbonate, vegetable oils, or triglycerides) enclosed in gelatin capsules.

[0131] Liquid pharmaceutically administrable compositions can be prepared, for example, by dissolving, dispersing, or suspending the crystalline form disclosed and / or described herein and optional pharmaceutical excipients in a carrier (e.g., water, saline, aqueous dextrose, glycerol, glycol, ethanol, etc.) to form a solution or suspension. Injectables can be prepared in conventional forms: as liquid solutions or suspensions, as emulsions, or in solid forms suitable for dissolution or suspension in liquid prior to injection. The percentage of the crystalline form contained in such parenteral compositions depends, for example, on the physical properties of the crystalline form, the activity of the crystalline form, and the needs of the subject. However, percentages of active ingredient between 0.01% and 10% in solution are usable, and may be higher if the composition is solid and will later be diluted to another concentration. In some embodiments, the composition contains about 0.2-2% of the crystalline form disclosed and / or described herein in solution.

[0132] The pharmaceutical compositions of the compositions and crystalline forms disclosed and described herein may be administered to the respiratory tract as an aerosol or solution for a nebulizer, or as a micronized powder for insufflation, either alone or in combination with an inert carrier such as lactose. In such cases, the particles of the pharmaceutical composition may have diameters of less than 50 microns, or in some embodiments, less than 10 microns.

[0133] Additionally, pharmaceutical compositions may include the crystalline forms disclosed and / or described herein, as well as one or more additional drugs, pharmaceuticals, adjuvants, etc. Suitable drugs and pharmaceuticals include those described herein.

[0134] kit Also provided are articles of manufacture and kits containing any of the crystalline forms or compositions provided herein. The articles of manufacture may include a labeled container. Suitable containers include, for example, bottles, vials, and test tubes. The container may be formed from a variety of materials, such as glass or plastic. The container may hold a pharmaceutical composition provided herein. The label on the container may indicate that the pharmaceutical composition is used to prevent, treat, or suppress a condition described herein, and may indicate instructions for either in vivo or in vitro use.

[0135] In one aspect, a kit is provided that includes the crystalline form or composition described herein and instructions for use.The kit can include instructions for use in treating heart disease in individuals or subjects who need it.The kit can also include any material or equipment that can be used to administer the crystalline form or composition, such as vial, syringe or IV bag.The kit can also include a sterile package. Use in combination

[0136] The crystalline forms and compositions described herein may be administered alone or in combination with other therapies and / or therapeutic agents useful in the treatment of the aforementioned disorders, diseases, or conditions.

[0137] The crystalline forms and compositions described and / or disclosed herein can be used in combination with one or more other therapies to treat cardiac diseases such as HCM or HFpEF. In some embodiments, the one or more therapies include therapies that attempt to slow the progression of heart failure and prevent cardiac remodeling by downregulating cardiac neurohormonal stimulation (e.g., ACE inhibitors, angiotensin receptor blockers (ARBs), beta-blockers, aldosterone receptor antagonists, or neuroendopeptidase inhibitors). In some embodiments, the one or more therapies include therapies that improve cardiac function by stimulating cardiac contractility (e.g., positive inotropic agents such as the beta-adrenergic agonist dobutamine or the phosphodiesterase inhibitor milrinone). In other embodiments, the one or more therapies include therapies that reduce cardiac preload (e.g., diuretics such as furosemide) or afterload (vasodilators of any class, including, but not limited to, calcium channel blockers, phosphodiesterase inhibitors, endothelin receptor antagonists, renin inhibitors, or smooth muscle myosin modulators).

[0138] The crystalline forms and compositions described and / or disclosed herein can be used in combination with one or more other therapies for treating HCM or HFpEF, hi some embodiments, the crystalline forms and / or compositions can be used in combination with a beta-blocker, verapamil, and / or disopyramide. [Example]

[0139] The following examples are presented to further assist in the understanding of the embodiments disclosed in this application and assume an understanding of conventional methods well known to those skilled in the art to which the examples pertain. The specific materials and conditions described herein are intended to illustrate certain aspects of the embodiments disclosed herein and should not be construed as limiting their reasonable scope.

[0140] The following abbreviations may be used in this specification: [Table 12-1] [Table 12-2]

[0141] The crystalline forms of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde are characterized by various analytical techniques including XRPD, DSC, TGA, GVS, and HPLC using the procedures described below.

[0142] XRPD 1. XRPD using a Bruker AXS D8 Advance XRPD diffractograms were collected on a Bruker D8 diffractometer using Cu Kα radiation (40 kV, 40 mA) and a θ-2θ goniometer equipped with a Ge monochromator. The incident beam passed through a 2.0 mm divergence slit, followed by a 0.2 mm anti-scatter slit and a knife edge. The diffracted beam passed through an 8.0 mm receiving slit with a 2.5° Soller slit, followed by a Lynxeye detector. The software used for data collection and analysis was Diffrac Plus XRD Commander and DiffracPlus EVA, respectively.

[0143] Samples were run under ambient conditions as flat samples using the as-received powder. Samples were prepared by lightly pressing them onto the flat surface of a polished, zero-background (510) silicon wafer or by packing them into a cut recess. The sample was rotated in its plane. Details of the standard Pharmorphix data acquisition method are as follows. Angle range: 2~42° 2θ Step size: 0.05° 2θ Acquisition time: 0.5 seconds / step (total acquisition time: 6.40 minutes) Where necessary, other methods of data collection were used with details as follows: Angle range: 2~31° 2θ Step size: 0.06° 2θ Acquisition time: 0.5 seconds / step 2. XRPD using PANalytical Empyrean

[0144] XRPD diffractograms were collected on a PANalytical Empyrean diffractometer using Cu Kα radiation (45 kV, 40 mA) in transmission geometry. The incident beam used a 0.04 rad Soller slit with a 0.5° slit, a 4 mm mask, and a focusing mirror. A PIXcel placed on the diffracted beam. 3D The detector was equipped with a receiving slit and a 0.04 rad Soller slit. The software used for data collection was X'Pert Data Collector using the X'Pert Operator Interface. Data were analyzed and displayed using Diffrac Plus EVA or HighScore Plus.

[0145] Samples were prepared in metal 96-well plates or Millipore 96-well plates and analyzed in transmission mode using X-ray transparent film between metal sheets on the metal well plates, and each powder (approximately 1–2 mg) was used as received.

[0146] The scanning mode of the metal plate was the gonioscan axis.

[0147] Details of standard screening data collection methods are as follows: Angle range: 2.5 to 32.0° 2θ Step size: 0.0130° 2θ Acquisition time: 12.75 seconds / step (total acquisition time: 2.07 minutes) X-ray single crystal diffraction (XRSD) and structure refinement [Table 13]

[0148] DSC DSC data were collected on a TA Instruments Q2000 equipped with a 50-position autosampler. Typically, 0.5–3 mg of each sample was heated in a pinhole-equipped aluminum pan from 25°C to typically 260°C at 10°C / min. A 50 ml / min dry nitrogen purge was maintained over the sample. Temperature-modulated DSC was performed using a base heating rate of 2°C / min and temperature modulation parameters of ±0.636°C (amplitude) every 60 seconds (duration). The overall details used for mDSC were as follows: 1) equilibration at -80°C; 2) 5.00-minute isotherm; 3) sampling interval of 1.00 seconds / point; 4) ±0.636°C modulation every 60 seconds; and 5) 2.00°C / min temperature ramp to 260°C. The instrument control software was Q Series Advantage and Thermal Advantage, and data were analyzed using Universal Analysis or TRIOS.

[0149] TGA TGA data were collected on a TA Instruments Q500 TGA equipped with a 16-position autosampler. Typically, 5-10 mg of each sample was placed in a pre-weighed aluminum DSC pan and heated from ambient temperature to 350 °C at 10 °C / min. A nitrogen purge was maintained over the sample at 60 ml / min. The instrument control software was Q Series Advantage and Thermal Advantage, and data were analyzed using Universal Analysis or TRIOS.

[0150] GVS 1. SMS DVS specific GVS Sorption isotherms were obtained using an SMS DVS Intrinsic moisture sorption analyzer controlled by DVS Intrinsic Control software. The instrument control maintained the sample temperature at 25 °C. Humidity was controlled by mixing dry and humid nitrogen streams with a total flow rate of 200 ml / min. Relative humidity was measured with a calibrated Rotronic probe (dynamic range 1.0–100% RH) placed near the sample. The weight change (mass relaxation) of the sample as a function of % RH was continuously monitored by a microbalance (accuracy ±0.005 mg).

[0151] Typically, 5-30 mg of sample was placed in a weighed mesh stainless steel basket under ambient conditions. Samples were loaded and unloaded at 40% RH and 25°C (typical room conditions). Moisture sorption isotherms were performed as outlined below (two scans complete one cycle). Standard isotherms were performed at 25°C over a RH range of 0-90% at 10% RH intervals. Typically, two cycles (four scans) were performed. Data analysis was performed in Microsoft Excel using the DVS Analysis Suite. The SMS DVS-specific experimental methods are described below. [Table 7]

[0152] 2. Hiden IGASorp Sorption isotherms were obtained using a moisture sorption analyzer on a Hiden IGASorp moisture sorption analyzer controlled by DVS Intrinsic Control software. Sample temperature was maintained at 25°C by a Grant LT ecocool 150 recirculating water bath. A total flow rate of 250 ml min was achieved by mixing dry and humid nitrogen streams. -1 Humidity was controlled by . Relative humidity was measured by a calibrated Vaisala RH probe (dynamic range 0-95% RH) placed near the sample. The weight change (mass relaxation) of the sample as a function of RH% was constantly monitored by a microbalance (accuracy ±0.001 mg).

[0153] Typically, 20-30 mg of sample was placed in a tared mesh stainless steel basket under ambient conditions. Samples were loaded and unloaded at 40% RH and 25°C (typical room conditions). Moisture sorption isotherms were run as outlined below (two scans give one complete cycle). Standard isotherms were run at 25°C in 10% RH intervals over the range of 0-90% RH. Typically, two cycles (four scans) were performed. Data analysis was performed within Isochema HISorp 2019 software and exported to Microsoft Excel for presentation as needed. [Table 8]

[0154] HPLC Purity analysis was performed using OpenLAb software on an Agilent HP1100 / Infinity II 1260 series system equipped with a diode array detector. Full method details are provided below. [Table 9]

[0155] The provided method was successfully transferred to an Infinity II 1260 Agilent system. A minor modification was made to the method, adding an additional minute of equilibration time at the end of the method. This additional equilibration time allows for observation of the entire gradient. [Table 10]

[0156] NMR 1 H NMR spectra were collected on a Bruker 400 MHz instrument controlled by an Avance NEO Nanobay console equipped with an autosampler. Unless otherwise noted, samples were prepared in DMSO-d6 solvent. Standard Bruker loading experiments (1 Automated experiments were acquired using a hologram (H). Offline analysis was performed using an ACD Spectrus Processor.

[0157] Static Stability Experiment For short-term (8 days or less) stability studies, solid materials were placed in open vials at high storage conditions unless otherwise noted. These conditions were achieved using saturated salt solutions in closed containers at specified temperatures. Storage containers were pre-equilibrated before sample addition. [Table 11]

[0158] For long-term (greater than 1 month) stability studies, solid materials were placed in LDPE bags and sealed in HDPE containers at elevated storage conditions unless otherwise noted. Example 1 Synthesis of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Compound 1) [ka] Step 1: Synthesis of tert-butyl 3-cyano-3-{[(3,4-difluorophenyl)methyl]amino}azetidine-1-carboxylate (1-a): [ka] To a solution of tert-butyl 3-oxoazetidine-1-carboxylate (3.0 kg, 17523.774 mmol, 1 equiv.) in i-PrOH (15.00 L) at room temperature, acetic acid (0.74 kg, 12.27 mol, 0.7 equiv.) and 1-(3,4-difluorophenyl)methanamine (3.01 kg, 21.03 mol, 1.2 equiv.) were added. After stirring at room temperature for 1 hour, trimethylsilyl cyanide (1.74 kg, 17.52 mol, 1.0 equiv.) was added to the resulting mixture. The resulting mixture was stirred overnight at room temperature. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure to remove three-quarters of the solvent, then filtered, washed with n-hexane (2 × 3 L), and dried to give 3.6 kg (first batch) of 1-a. The filtrate was concentrated, dissolved in i-PrOH (500 mL), filtered, washed with n-hexane (2 × 500 mL), and dried to give 0.4 kg (second batch) of 1-a. The two batches were combined to give 4 kg of tert-butyl 3-cyano-3-{[(3,4-difluorophenyl)methyl]amino}azetidine-1-carboxylate (yield: 70.59%) as a white solid. LRMS (ES) m / z 268 [M+H-56]. 1 H NMR (300 MHz, chloroform-d) δ 7.30-7.17 (m, 1H), 7.17-7.04 (m, 2H), 4.24 (d, J = 8.9 Hz, 2H), 3.92-3.83 (m, 2H), 3.80 (s, 2H), 2.20 (d, J = 55.9 Hz, 1H), 1.44 (s, 9H).

[0159] Step 2: Synthesis of tert-butyl 3-{2-chloro-N-[(3,4-difluorophenyl)methyl]acetamido}-3-cyanoazetidine-1-carboxylate (1-b): [ka] To a solution of tert-butyl 3-cyano-3-{[(3,4-difluorophenyl)methyl]amino}azetidine-1-carboxylate (3.0 kg, 9.28 mol, 1 eq) in DCM (15 L) was added TEA (2.82 kg, 27.83 mol, 3.0 eq) at 0 °C. Then, chloroacetyl chloride (2.62 kg, 23.20 mol, 2.5 eq) in DCM (15 L) was added over 2 h. The resulting mixture was stirred overnight at room temperature. The reaction was monitored by LCMS. The resulting mixture was washed with saturated NaHCO3 (2 × 9 L) and brine (9 L), dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography eluting with petroleum ether and EtOAc (2:1) to give 3.0 kg of tert-butyl 3-{2-chloro-N-[(3,4-difluorophenyl)methyl]acetamido}-3-cyanoazetidine-1-carboxylate (yield: 81%) as a yellow solid. LRMS (ES) m / z 344 [M+H-56]. 1 H NMR (300 MHz, chloroform-d) δ 7.26 (dt, J = 9.6, 8.3 Hz, 1H), 7.13 (ddd, J = 10.0, 7.1, 2.3 Hz, 1H), 7.08 - 6.99 (m, 1H), 4.67 (s, 2H), 4.33 (d, J = 9.8 Hz, 2H), 4.16 - 4.05 (m, 4H), 1.44 (s, 9H).

[0160] Step 3: Synthesis of tert-butyl 3-cyano-3-{N-[(3,4-difluorophenyl)methyl]-2-{[(1r,4r)-4-methylcyclohexyl]amino}acetamido}azetidine-1-carboxylate (1-c): [ka] To a solution of tert-butyl 3-{2-chloro-N-[(3,4-difluorophenyl)methyl]acetamido}-3-cyanoazetidine-1-carboxylate (3.0 kg, 7.50 mol, 1 equiv.) in MeCN (30 L), (1r,4r)-4-methylcyclohexan-1-amine (0.93 kg, 8.25 mol, 1.1 equiv.) and TEA (1.90 kg, 18.76 mol, 2.5 equiv.) were added at room temperature. The resulting mixture was stirred at 65 °C overnight. The desired product could be monitored by LCMS. The resulting mixture was cooled to room temperature and concentrated under reduced pressure. The resulting mixture was diluted with water (10 L) and extracted with EtOAc (3 × 15 L). The combined organic layers were washed with brine (2 × 10 L), dried over anhydrous NaSO, and concentrated under reduced pressure to give 3.0 kg of tert-butyl 3-cyano-3-{N-[(3,4-difluorophenyl)methyl]-2-{[(1r,4r)-4-methylcyclohexyl]amino}acetamido}azetidine-1-carboxylate as a brown solid, which was used directly in the next step without further purification. LRMS (ES) m / z 421 [M+H-56]. 1 H NMR (300 Mhz, Chloroform-d) δ 7.20 -7.06 (m, 1H), 7.01 (ddd, J = 11.0, 7.4, 2.3 Hz, 1H), 6.96 -6.85 (m, 1H), 4.89 (s, 2H), 4.48 (dd, J = 13.6, 9.4 Hz, 2H), 4.21 -3.80 (m, 3H), 3.80 (s, 2H), 1.92 -1.69 (m, 4H), 1.58 -1.27 (m, 13H), 1.14 (qd, J = 12.7, 12.2, 3.2 Hz, 2H), 0.93 (d, J = 6.4 Hz, 3H). Step 4: Synthesis of tert-butyl 5-[(3,4-difluorophenyl)methyl]-6,9-dioxo-8-[(1r,4r)-4-methylcyclohexyl]-2,5,8-triazaspiro[3.5]nonane-2-carboxylic acid (1-d): [ka] To a solution of tert-butyl 3-cyano-3-{N-[(3,4-difluorophenyl)methyl]-2-{[(1r,4r)-4-methylcyclohexyl]amino}acetamido}azetidine-1-carboxylate (3 kg, 6.30 mol, 1 equiv.) in EtOH (30 L) was added AcOH (7.56 kg, 125.90 mol, 20 equiv.) dropwise over 30 minutes at room temperature. The resulting mixture was stirred at 90° C. overnight. The reaction was monitored by LCMS. The resulting mixture was cooled to room temperature, concentrated under reduced pressure to remove 70% of the solvent, cooled to 0 °C, stirred for 15 min, filtered, washed with ice-cold ethanol (3 L), and dried to give 2.4 kg of tert-butyl 5-[(3,4-difluorophenyl)methyl]-6,9-dioxo-8-[(1r,4r)-4-methylcyclohexyl]-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (yield: 75.44%) as a pale yellow solid. LRMS (ES) m / z 422 [M+H-56]. 1 H NMR (300 Mhz, chloroform-d) δ 7.20 -7.02 (m, 2H), 7.02 -6.91 (m, 1H), 4.89 (s, 2H), 4.51 (d, J = 9.5 Hz, 2H), 4.40 (tt, J = 12.2, 3.8 Hz, 1H), 4.00 -3.91 (m, 4H), 1.86 -1.77 (m, 2H), 1.71 (dd, J = 12.1, 3.5 Hz, 2H), 1.52 (d, J = 12.4 Hz, 1H), 1.44 (s, 9H), 1.37 -1.21 (m, 2H), 1.13 (qd, J = 12.6, 3.4 Hz, 2H), 0.92 (d, J = 6.4 Hz, 3H). Step 5: Synthesis of 5-[(3,4-difluorophenyl)methyl]-8-[(1r,4r)-4-methylcyclohexyl]-2,5,8-triazaspiro[3.5]nonane-6,9-dione hydrochloride (1-e) [ka] To a solution of tert-butyl 5-[(3,4-difluorophenyl)methyl]-6,9-dioxo-8-[(1r,4r)-4-methylcyclohexyl]-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (3.4 kg, 7.12 mol, 1 equiv.) in MeOH (34 L) at −5° C., acetyl chloride (1.68 kg, 21.36 mol, 3.0 equiv.) was added dropwise over 1.5 h. The resulting mixture was stirred overnight at room temperature. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure and purified by trituration with petroleum ether and EtOAc (10:1, 12 L) to give 2.5 kg of 5-[(3,4-difluorophenyl)methyl]-8-[(1r,4r)-4-methylcyclohexyl]-2,5,8-triazaspiro[3.5]nonane-6,9-dione hydrochloride (85% yield) as an off-white solid. LRMS (ES) m / z 378 [M+H]. 1 H NMR (300 MHz, DMSO-d6) δ 10.26 (s, 1H), 9.13 (s, 1H), 7.51-7.29 (m, 2H), 7.27-7.12 (m, 1H), 5.04 (s, 2H), 4.27-4.06 (m, 5H), 3.98 (s, 2H), 1.86-1.45 (m, 6H), 1.37-1.25 (m, 1H), 1.03 (tq, J = 12.1, 7.8, 6.0 Hz, 2H), 0.86 (d, J = 6.4 Hz, 3H).

[0161] Step 6: Synthesis of 5-[(3,4-difluorophenyl)methyl]-6,9-dioxo-8-[(1r,4r)-4-methylcyclohexyl]-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Compound 1): [ka] To a solution of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (4.0 kg, 10.60 mol, 1 equiv.) in MeCN (20 L) at room temperature was added 2,2,2-trifluoroethyl formate (1.63 kg, 12.72 mol, 1.2 equiv.) and DIPEA (3.42 kg, 26.50 mol, 2.5 equiv.). The resulting mixture was stirred overnight at room temperature. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The mixture was diluted with EtOAc (10 L). The resulting mixture was quenched with NH4Cl (6 L, saturated solution) and water (6 L) and extracted with EtOAc (3 x 15 L). The combined organic layers were washed with NH4Cl(aq) (10 L) and brine (10 L), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give a crude brown oil, which was recrystallized from cyclohexane and EtOAc (5:1, 4 L, 80 °C to room temperature) and filtered to give 3 kg (first batch) of 5-[(3,4-difluorophenyl)methyl]-6,9-dioxo-8-[(1r,4r)-4-methylcyclohexyl]-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde as a pale yellow solid. The filtrate was concentrated under reduced pressure and recrystallized from petroleum ether and EtOAc (10:1, 3 L, room temperature) to give 800 g (second batch) of a pale yellow solid. The two batches were combined and dried to give 3.8 kg of crystalline Form I (mp 133 °C) of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde as a pale yellow solid. The overall yield for this process is 97%.

[0162] Characterization of crystalline form I Crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde was analyzed by XRPD, DSC, TGA, GVS, and HPLC. Table 1-A shows the 2θ angles and relative peak intensities observed for Crystalline Form I. Figure 1A shows the XRPD pattern of Crystalline Form I. [Table 1A]

[0163] Figure 1B shows the DSC and TGA graphs of crystalline Form I. As shown in the DSC graph, an endothermic onset at approximately 125.6°C and an endothermic peak at 130.2°C were observed. As shown in the TGA graph, a weight loss of 0.4% was observed between 105°C and 145°C. Figure 1C shows the GVS graph of Form I.

[0164] The purity and stability of crystalline Form I were analyzed by XRPD and HPLC. Table 1-B shows the XRPD results and the purity of crystalline Form I analyzed by HPLC before, after, and after storage at 25°C / 97%RH and 40°C / 75%RH for 7 days. As shown in Table 1-B, crystalline Form I is 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde and has a purity of at least 96.3%, which remained substantially unchanged when stored at 40°C / 75%RH and / or 25°C / 97%RH for 7 days. [Table 1B]

[0165] Example 2 Preparation of Crystalline Form II Method 1. Conversion of Form I to Form II 2.5 g of crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2I-carbaldehyde was suspended in EtOH (5 volumes, 12.5 ml). The sample was stirred at 50 °C (500 rpm). After 15 min, a solution was obtained. Some debris, likely plastic fragments, was observed and removed before continuing.

[0166] The solution was stirred at 50°C for an additional 5 minutes and then cooled to 5°C at 0.1°C / min. After stirring overnight at 5°C, an aliquot of the suspension was filtered through a filter cartridge equipped with a frit. A stream of compressed air was gently blown over the solid. A portion of the mother liquor was allowed to slowly evaporate.

[0167] The bulk sample was filtered through a Buchner funnel. A small amount of solid passed through the filter paper. The mother liquor was collected, refiltered, and added to the filter cake. The filter cake was dried under suction for 30 minutes. This solid was determined to be Form II.

[0168] Method 2 To 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (4.0 g, 9.9 mmol) in a 100 mL round-bottom flask equipped with a magnetic stir bar, ethanol (4.0 mL) was added, and the mixture was heated to 78 °C in an oil bath. Water was added to the mixture (2 mL at a time for a total of 6 mL, then added dropwise to a total of 7.0 mL) until the mixture became slightly cloudy. The mixture was heated at 78 °C for 30 minutes, after which the heat was removed and the solution was allowed to cool slowly to room temperature with stirring (approximately 300 rpm) and stirred overnight. The solid was collected by filtration and air-dried to give 3.8 g (95%) of crystalline Form II, 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde, as a white solid. LRMS (ES) m / z 406.2 (M+H). 1H NMR (400 MHz, sodium chloride-d2) δ 8.05 (s, 1H), 7.20 (q, J = 9.0 Hz, 1H), 7.11 (dd, J = 11.0, 8.0 Hz, 1H), 7.04-6.97 (m, 1H), 4.95 (d, J = 16.2 Hz, 1H), 4.81 (d, J = 16.1 Hz, 1H), 4.71 (d, J = 9.2 Hz, 1H), 4.50 (d, J = 10.7 Hz, 1H), 4.42 (td, J = 12.3, 6.2 Hz, 1H), 4.21 (d, J = 9.1 Hz, 1H), 4.10 (d, J = 10.7 Hz, 1H), 4.03 (s, 2H), 1.85 (d, J = 13.2 Hz, 2H), 1.74 (d, J = 9.8 Hz, 2H), 1.59 -1.48 (m, 2H), 1.43 -1.33 (m, 1H), 1.17 (qd, J = 12.7, 3.5 Hz, 2H), 0.95 (d, J = 6.3 Hz, 3H).

[0169] Method 3 To 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (42.0 g, 103.6 mmol) in a 250 mL round-bottom flask equipped with a magnetic stir bar, ethanol (40.0 mL) was added, and the solution was heated to 78 °C in an oil bath. To this mixture was added water (10 mL at a time, totaling 70 mL) until the mixture became slightly cloudy. The mixture was heated at 78°C for 30 minutes, and 3.8 g of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde Form II material (3.8 g, 9.4 mmol) was added. The heat was removed, and the solution was allowed to slowly cool to room temperature with stirring (approximately 300 rpm) and stirred overnight. The solid was collected by filtration and air-dried to give 44.8 g (97%) of Form II 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde as a white solid. LRMS (ES) m / z 406.2 (M+H). 1 H NMR (400 Mhz, methanol-d4) δ 7.99 (s, 1H), 7.25 (dt, J = 11.3, 8.7 Hz, 2H), 7.11 (dd, J = 8.5, 4.2 Hz, 1H), 4.93 (s, 2H), 4.66 (d, J = 9.9 Hz, 1H), 4.42 (t, J = 9.7 Hz, 2H), 4.32 (tt, J = 12.1, 3.9 Hz, 1H), 4.15 (d, J = 11.1 Hz, 1H), 4.11 (s, 2H), 1.85 (d, J = 13.3 Hz, 2H), 1.81 -1.71 (m, 2H), 1.71 -1.56 (m, 2H), 1.51 -1.35 (m, 1H), 1.14 (qd, J = 12.5, 3.4 Hz, 2H), 0.95 (d, J = 6.4 Hz, 3H).

[0170] Method 4. Conversion of Form I to Form II To a 250 mL round-bottom flask containing 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (50.0 g, 123.3 mmol) crystalline Form I was added ethanol (50.0 mL), and the solution was heated to 86°C in an oil bath. Water was added (10 mL at a time, totaling 50 mL) to the mixture until it became slightly cloudy. The mixture was heated at 86°C for 30 minutes, removed from the heat, and seeded with a small amount of crystalline Form II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (100 mg) at approximately 50°C. The solution was slowly cooled to room temperature under stirring (approximately 300 rpm) and stirred overnight. The solid was collected by filtration and air-dried to give 44.0 g of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde as a white solid. LRMS (ES) m / z 406.2 (M+H).

[0171] This process was repeated on scales of 170 g, 80 g, and 100 g of Form I solid to yield 176 g, 74 g, and 99 g, respectively.

[0172] A 2 L three-neck flask equipped with mechanical stirring was charged with 383 g of the product from the above lot. To this mixture, ethanol (380 mL) and water (100 mL) were added, and the mixture was slowly heated using a heating mantle equipped with an internal temperature controller. When the suspension became a homogeneous solution at 50 °C, additional water (200 mL, 100 mL at a time) was added. When the internal temperature reached 65 °C, additional water (100 mL) was added. The mixture became cloudy until it reached 80 °C. To this solution, water (50 mL, 5 mL at a time) was added. The mixture was then heated to 85 °C for 10 minutes, removed from the heat, and allowed to slowly cool to room temperature. When the mixture cooled to 70 °C, 20 mg of crystalline Form II solid was added. When the internal temperature reached 40 °C, more solid was observed, and the internal temperature was maintained at 40–46 °C until more solid precipitated from solution. When the internal temperature reached 28 °C, the solid was collected by filtration. The mixture was dried in a vacuum oven without heat for 3 days to give 373 g of off-white crystalline Form II. 1 H NMR (500 MHz, DMSO-d6) δ 7.97 (s, 1H), 7.44 -7.32 (m, 2H), 7.10 (dt, J = 7.4, 2.8 Hz, 1H), 4.83 (s, 2H), 4.51 (d, J = 9.6 Hz, 1H), 4.29 -4.14 (m, 3H), 4.01 (s, 2H), 3.97 (d, J = 10.7 Hz, 1H), 1.77 -1.70 (m, 2H), 1.65 -1.51 (m, 4H), 1.34 (dtq, J = 14.2, 7.0, 3.4 Hz, 1H), 1.03 (qd, J = 12.6, 3.9 Hz, 2H), 0.87 (d, J = 6.5 Hz, 3H).

[0173] Method 5. Conversion of Form I to Form II To 2.05 kg of crystalline Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde obtained in Step 6 of Example 1 in a 10 L round-bottom flask equipped with a stir bar was added 2000 mL of ethanol and heated to reflux (internal temperature 90°C). After stirring at reflux for 5 minutes, the mixture was added with water (2000 mL total: 400 mL in 400 mL increments up to 1600 mL, then the remaining 400 mL in 40 mL increments; if the mixture became cloudy, wait until it became homogeneous again at 90°C). The mixture was heated at 90°C for 10 minutes, after which the heat was removed and the temperature was allowed to gradually reach 55°C (precipitation was observed during the cooling phase). Then, 400 mg of crystalline Form II seed crystals (melting point 155° C.) were added during the cooling stage (approximately 55° C.). The mixture was cooled to room temperature. The mixture was continuously stirred at room temperature for 30 minutes. It was then filtered and dried in an oven (below 50° C.) to obtain crystalline Form II of 5-[(3,4-difluorophenyl)methyl]-6,9-dioxo-8-[(1r,4r)-4-methylcyclohexyl]-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (1979.4 g, 96.5%) (melting point 155.6° C.) as an off-white solid.

[0174] Characterization of Crystalline Form II Crystalline Form II of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde was analyzed by XRPD, DSC, TGA, GVS, and HPLC. Table 2-A shows the 2θ angles and relative peak intensities observed for Crystalline Form II using XRPD. Figure 2A shows the XRPD pattern of Crystalline Form II. [Table 2A-1] [Table 2A-2]

[0175] Figure 2B shows the DSC and TGA graphs of crystalline Form II. As shown in the DSC graph, an endothermic onset at about 154.9°C and an endothermic peak at about 155.8°C were observed. As shown in the TGA graph, less than 0.1% weight loss was observed before decomposition. Figure 2C shows the GVS graph of crystalline Form II.

[0176] The purity and stability of crystalline Form II were analyzed by XRPD and HPLC. Table 2-B shows the XRPD results and the purity of crystalline Form II analyzed by HPLC before, after, and after storage at 25°C / 97%RH and 40°C / 75%RH for 8 days. As shown in Table 2-B, crystalline Form II is 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde and has a purity of at least 98.0%, which remained substantially unchanged when stored at 40°C / 75%RH and / or 25°C / 97%RH for 8 days. [Table 2B]

[0177] Table 2-E shows the water content results, XRPD results, assay results, and purity of crystalline Form II analyzed by HPLC before storage or after storage at 25°C / 60% RH for 8 days or at 40°C / 75% RH for 1, 3, or 6 months. As shown in Table 2-E, crystalline Form II is 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde and has a purity of at least 99%, which remained substantially unchanged when stored at 25°C / 60% RH and / or 40°C / 75% RH for 6 months, as measured by HPLC. [Table 2E]

[0178] Single crystals of Form II were obtained by evaporation of a solution of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde in 2-methoxyethanol. Crystals of suitable size and quality for analysis by single crystal X-ray diffraction, measuring approximately 0.25 x 0.12 x 0.10 mm, were isolated. The crystal structure of Form II was solved in the triclinic centrosymmetric space group P-1 and refined with a final R1[I>2σ(I)] value of 3.91%. The single crystal data for Form II are shown in Table 2-D. [Table 2D]

[0179] Example 3 Dynamic solubility and stability of crystalline form II in solvents. The solubility of Form II was analyzed in 15 common solvents. A sufficient amount of Form II was suspended in 0.5 mL of solvent for free Form II at the maximum expected concentration of Ca. = 10-100 mg / mL. The resulting suspension was then shaken on a platform shaker at 25 °C / 750 rpm for 4 hours. After equilibration, the appearance was recorded, and the pH of the saturated solution was measured for the aqueous samples only. Each sample was then centrifuged at 13.4 krpm for 5 minutes and then diluted with MeCN. Except for the sample initially diluted with n-heptane, these samples were diluted with 2-propanol instead of MeCN.

[0180] All diluted samples were then analyzed by HPLC. Each sample was further diluted based on appearance and maximum expected concentration (Error! Reference source not found). All diluted samples were injected at 10, 15, and 20 μL to obtain peak areas within the calibration curve at a diluted concentration of approximately 0.1 mg / mL. Details of the dilution ratios used for the different solvents are shown in Table 3-A. [Table 3A]

[0181] Quantitation was determined by HPLC on a standard solution of approximately 0.15 mg / ml in MeCN. Standard, diluted, and undiluted samples were injected in different volumes. Solubility was calculated using the peak area determined by integrating the peak appearing at the same retention time as the major peak in the standard injection. Although the standard prepared in isopropanol was found to be nonlinear, the peak response of the standard dissolved in isopropanol was comparable to that of the standard dissolved in MeCN, so the MeCN standard was used for quantitation of all samples.

[0182] The kinetic solubility results of Form II in 15 different solvents after 4 hours are shown in Table 3-B. [Table 3B]

[0183] 2 Values ​​quoted as approximations due to agreement between replicates only; 3 The sample at T = 4 hours was a clear solution and was therefore reported as a value greater than that based on the initial weight of the sample; 4 The T=4 hour sample was a clear solution and was therefore reported as a value greater than that based on the initial weight of the sample.

[0184] After the samples were injected to measure solubility, each diluted sample was stored at ambient conditions (25°C) for 24 hours and then re-injected with the same injection volume in the same order.

[0185] The chromatograms were then overlaid, revealing visual signs of decomposition, i.e., a decrease in the peak area of ​​Compound 1 or an increase in other peaks in the chromatography. The HPLC instrumentation details used to determine the stability of crystalline Form II are summarized in Table 3-C. The stability observations of crystalline Form II in various solvents after T=24 hours are summarized in Table 3-D. [Table 3C] [Table 3D]

[0186] The above analysis confirms the high solubility (>100 mg / mL) of crystalline Form II in methanol, acetone, DMSO, acetonitrile, and THF. Furthermore, when these samples were re-injected at T=24 hours, no visual signs of further decomposition were observed chromatographically. The sample in ethanol showed a solubility of approximately 24 mg / mL. Although replicate experiments were less reproducible than many of the other preparations and showed a later-eluting peak at both time points, this sample did not appear to decompose further over the course of 24 hours.

[0187] Unlike the samples prepared in ethanol and tert-butyl methyl ether, only the sample prepared in isopropyl acetate showed signs of decomposition, but as with the other two examples, this was seen upon initial injection and did not appear to decompose further over the course of 24 hours. The solubility in isopropyl acetate was calculated to be 22 mg / mL.

[0188] Example 4 Preparation and Analysis of Crystalline Form III 30 mg of amorphous 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde was treated with three volumes (90 μL) of 1,4-dioxane:HO (v / v 1:1), and the mixture was stirred (300 rpm) at 5° C. After a total of 7 days, the mixture was removed from stirring. An aliquot of the resulting suspension was removed with a spatula and blotted dry on filter paper before XRPD analysis to yield crystalline Form III.

[0189] Table 4-A shows the angles 2θ and relative peak intensities observed for crystalline Form III using XRPD. Figure 3A shows the XRPD pattern observed for crystalline Form III. [Table 4A-1] [Table 4A-2]

[0190] Crystalline Form III was isolated and subjected to fundamental characterization to understand its solid-state properties. Crystalline Form III was determined to be an unstable hemidioxane solvate, and as determined by XRPD, it converted to Crystalline Form II after storage at 40°C / 75% RH for 7 days, as shown in Figure 3C. Crystalline Form III was isolated and subjected to fundamental characterization to understand its solid-state properties. 1 HNMR analysis was performed. 1 As determined by H NMR, crystalline Form III was 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde containing 0.5 mol equivalents of 1,4-dioxane.

[0191] 3B shows the DSC and TGA graphs of crystalline Form III. As shown in the DSC graph, an endothermic onset at about 81.7°C, two endothermic onsets at about 112.8°C, and an endothermic onset at about 154.4°C were observed. Additionally, an endothermic peak at about 92.1°C, an endothermic peak at about 118.2°C, an endothermic peak at about 130.0°C, and an endothermic peak at about 155.8°C were observed in the DSC graph. As shown in the TGA graph, a weight loss of about 3.3% was observed.

[0192] Example 5 Polymorphism Screening 5-1 Polymorphism screening by isothermal maturation at 5°C Thirty mg of amorphous 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde was treated with three volumes (90 μL) of the selected solvent and stirred (300 rpm) at 5°C. If the resulting suspension was too thick after approximately 1.5 hours, additional solvent (20 μL) was added to the suspension. After stirring overnight at 5°C, aliquots of the suspension were removed with a spatula and blotted dry on filter paper before XRPD analysis. If the resulting suspension was too thin or close to a solution, antisolvent (three volumes, 90 μL) was added to these solutions, and the samples were then returned to stirring at 5°C. After stirring overnight at 5°C, samples that formed suspensions after antisolvent addition were analyzed by XRPD. If a solution persisted, antisolvent (6 volumes, 180 μL) was added. The sample was returned to stirring at 5° C. After a total of 7 days, the sample was removed from stirring. An aliquot of the suspension was removed with a spatula and blotted dry on filter paper before XRPD analysis. The resulting sample was analyzed by XRPD. The results are shown in Tables 5-A and 5-B below. [Table 5A-1] [Table 5A-2] [Table 5B]

[0193] 5-2 Screening for polymorphism using temperature cycle maturation (RT / 50℃) screening 30 mg of amorphous 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde was treated with three volumes (90 μL) of the selected solvent and subjected to temperature cycling maturation (RT / 50 °C, 4 h per cycle) using a platform shaker incubator. After overnight maturation, an aliquot of the suspension was removed with a spatula and blotted dry on filter paper before XRPD analysis.

[0194] If a two-phase layer was observed, the sample was sonicated for 1.5 hours. If the sample remained unchanged, it was evaporated. After evaporation, the solid was analyzed by XRPD. The gum was treated with cyclohexane (10 volumes, 300 μL) and sonicated for 4 hours. An aliquot of the suspension was removed with a spatula and blotted dry on filter paper before XRPD analysis.

[0195] The resulting suspension was analyzed by XRPD. An aliquot of the suspension was removed with a spatula and blotted dry on filter paper before analysis.

[0196] When a solution formed, antisolvent (45 μL) was added to the solution, which was then returned to temperature cycle maturation (RT / 50° C., 4 hours per cycle). Samples that were suspensions were also returned to temperature cycle maturation. After a total of 7 days, the samples were removed from maturation. Aliquots of the suspension were removed with a spatula and blotted dry on filter paper before XRPD analysis.

[0197] The results of the temperature cycle maturation (RT / 50°C) screen are shown in Table 5-C. A summary of the results from further processing of the solutions is shown in Table 5-D. A summary of additional treatments to the two-concentration system is shown in Table 5-E. [Table 5C-1] [Table 5C-2] [Table 5C-3] [Table 5D] [Table 5E]

[0198] 5-3 Polymorph screening by high temperature slurry (75℃) screening 30 mg of amorphous 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde was treated with three volumes (90 μL) of the selected solvent mixture and subjected to stirring (500 rpm) at 75° C. Observations were recorded after approximately 5 hours. Additional solvent was added to all samples.

[0199] After stirring overnight at 75°C, an aliquot of the suspension was removed with a spatula and blotted dry on filter paper before XRPD analysis. If a biphasic solution remained, additional solvent was added in 150 μL aliquots with stirring at 75°C until a solution was obtained (a total of 300 μL was added). The solution was cooled to 5°C at 0.1°C / min. The solution was stirred at 5°C. After a total of 15 or 18 days, the resulting suspension was analyzed by XRPD. The solution and biphasic solution were stirred at room temperature.

[0200] The observations and XRPD analysis are summarized in Table 5-F. [Table 5F-1] [Table 5F-2]

[0201] 5-4 Polymorph Screening by Liquid-Assisted Grinding (LAG) 30 mg of amorphous 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde was wetted with solvent (5 μL). Two grinding beads (3 mm diameter) were added and the sample was milled for 2 hours at 500 rpm using a planetary Fritsch mill (Pulverisette 6). After milling, all solids were analyzed by XRPD.

[0202] The gum was stored at ambient conditions. The gum and sample containing the brittle material were vacuum dried in a vacuum oven (approximately 200 mbar) overnight at room temperature. Observations were recorded and the sample was vacuum dried in a vacuum oven (approximately 5 mbar) overnight at room temperature. If rubber still remained, the sample was further vacuum dried in a vacuum oven (approximately 5 mbar) for 3 days at room temperature. Observations and XRPD analysis are shown in Table 5-G. Details of further processing of the rubber and brittle material are summarized in Table 5-H. [Table 5G-1] [Table 5G-2] [Table 5G-3] [Table 5H]

[0203] Example 6 Competitive slurry experiments between Form I and Form II A mechanical mixture of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde with Form I and Form II was prepared and then used in competition slurry experiments between Forms I and II to determine the most stable form over a given temperature range (5-50°C). From the competition slurries, Form II was determined to be the most stable form under the conditions examined and was observed in all experiments in which solids were isolated. These results indicate a monotonic relationship between Form I and Form II, with Form II being the thermodynamically more stable form at all temperatures. The results of the competition slurry experiments are shown in Table 6 below. [Table 6]

[0204] Example 7 Synthesis of 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Comparative Substance A) [ka] Step 1: Synthesis of 1-(tert-butyl) 3-ethyl 3-((4-chlorobenzyl)amino)azetidine-1,3-dicarboxylate [ka] To a solution of 1-tert-butyl 3-ethyl 3-aminoazetidine-1,3-dicarboxylate (10.6 g, 43.3 mmol, 1.0 equiv.) and 4-chlorobenzaldehyde (6.1 g, 43.3 mmol, 1.0 equiv.) in DCE (120.0 mL) was added AcOH (5.0 mL) at 0 °C. The mixture was allowed to warm to room temperature, and upon stirring at room temperature, two additional portions of AcOH (5.0 mL) were added (20.0 mL total) at 4 h, 8.5 h, and 9 h. To this mixture was added STAB (11.0 g, 52.0 mmol, 1.2 equiv.). The mixture was stirred overnight at room temperature, diluted with aqueous sodium bicarbonate, and extracted three times with DCM. The combined organic layers were dried over sodium sulfate and concentrated. The resulting oil was analyzed by HPLC using a 60% HPLC column. ℃ The mixture was dissolved in warm hexane and EA while stirring at RT (added dropwise until the mixture became a homogeneous solution). ℃ The mixture was cooled to rt, and the precipitate was collected by filtration and washed with cold hexane. The filtrate was concentrated, and the process was repeated again. The combined solids were dried under vacuum to give 13.8 g (86%) of 1-(tert-butyl)3-ethyl 3-((4-chlorobenzyl)amino)azetidine-1,3-dicarboxylate as a white solid. LRMS (ES) m / z 313.1 (M+H-56).

[0205] Step 2: Synthesis of tert-butyl 3-((4-chlorobenzyl)amino)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate [ka] To a stirred solution of 1-(tert-butyl) 3-ethyl 3-((4-chlorobenzyl)amino)azetidine-1,3-dicarboxylate (10.0 g, 27.1 mmol, 1.0 equiv) and 2,4-difluoroaniline (3.0 mL, 29.8 mmol, 1.1 equiv) in THF (200.0 mL) was added a solution of LHMDS (54.2 mL, 1 M THF solution, 54.2 mmol, 2.0 equiv) dropwise over 20 min at 0° C. under N2. The mixture was stirred at 0° C. for 20 min, quenched with water (50 mL), acidified to pH 5 using aqueous HCl (0.5 N), and extracted twice with EA (100 mL). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated to give 11.3 g of tert-butyl 3-((4-chlorobenzyl)amino)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate, which was used in the next step without purification. LRMS (ES) m / z 396.1 (M+H-56).

[0206] Step 3: Synthesis of tert-butyl 3-(2-chloro-N-(4-chlorobenzyl)acetamido)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate [ka] To a solution of tert-butyl 3-((4-chlorobenzyl)amino)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate (11.3 g, 23.2 mmol, 1.0 equiv.) in THF (20 L) was added TEA (4.8 mL, 34.7 mmol, 1.5 equiv.) and 2-chloroacetyl chloride (1.5 mL, 27.8 mmol, 1.2 equiv.) at 0° C. After stirring for 30 minutes at 0° C., additional TEA (4.8 mL, 34.7 mmol, 1.5 equiv.) and 2-chloroacetyl chloride (1.8 mL, 23.2 mmol, 1.0 equiv.) were added to the mixture, and the mixture was stirred at 0° C. for 30 minutes. To this mixture was added additional 2-chloroacetyl chloride (0.6 mL, 6.9 mmol, 0.3 equiv.). The mixture was gradually warmed to room temperature, stirred for 45 min, cooled to 0° C., and quenched with aqueous sodium bicarbonate. The mixture was extracted three times with EA. The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated to give 13.2 g of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate, which was used in the next step without further purification. LRMS (ES) m / z 528.1 (M+H).

[0207] Step 4: Synthesis of tert-butyl 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate [ka] To a stirred solution of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate (13.2 g, 20.1 mmol, 1.0 equiv) in DMF (40.0 mL) under N was added KCO (4.2 g, 30.1 mmol, 1.5 equiv). The resulting mixture was stirred at room temperature for 3.5 h, diluted with water, and extracted twice with EA. The combined organic layers were washed twice with water and once with brine, dried over sodium sulfate, and concentrated to give 11.7 g of tert-butyl 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate. LRMS (ES) m / z 436.1 (M+H-56).

[0208] Step 5: Synthesis of 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate [ka] To a stirred solution of tert-butyl 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (14.7 g, 29.8 mmol, 1.0 equiv.) in DCM (50.0 mL) was added TFA (25.0 mL). The resulting mixture was stirred at room temperature for 3 hours and concentrated to dryness to give 15.1 g of 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate, which was used in the next step without further purification. LRMS (ES) m / z 392.1 (M+H).

[0209] Step 6: Synthesis of 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde [ka] To a solution of 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate (15.1 g, 29.9 mmol, 1 equiv.) and N,N-diisopropylethylamine (15.7 mL, 89.7 mmol, 3 equiv.) in ACN (50 mL) was added 2,2,2-trifluoroethylformate (5.8 mL, 59.8 mmol, 2 equiv.). The reaction was stirred at room temperature for 1 hour, diluted with water, and extracted with DCM. The combined organic layers were dried over sodium sulfate, concentrated, and purified by silica gel chromatography using a 0-100% EtOAc gradient followed by 0-10% MeOH in DCM. The solid was suspended in EtOH / MTBE 1:1, heated to 80 °C, cooled on ice, filtered, washed with MTBE, and dried to give 8.3 g (66%) of 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. LRMS (ES) m / z 420.1 (M+H). 1 H NMR (400 MHz, DMSO-d6) δ 7.98 (s, 1H), 7.65 (td, J = 8.8, 6.0 Hz, 1H), 7.48 -7.41 (m, 1H), 7.43 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.22 (td, J = 8.4, 2.4 Hz, 1H), 4.91 (s, 2H), 4.55 (d, J = 9.7 Hz, 1H), 4.43 (d, J = 1.6 Hz, 2H), 4.37 (d, J = 9.8 Hz, 1H), 4.28 (d, J = 10.8 Hz, 1H), 4.08 (d, J = 10.8 Hz, 1H). Example 8 Synthesis of 8-(2,4-difluorophenyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Comparative Example B) [ka]

[0210] Step 1: Synthesis of Step 1-(tert-butyl) 3-ethyl 3-((4-trifluoromethyl)benzyl)amino)azetidine-1,3-dicarboxylate [ka] To a solution of 1-tert-butyl 3-ethyl 3-aminoazetidine-1,3-dicarboxylate (15.0 g, 61.4 mmol, 1.0 equiv.) and 4-(trifluoromethyl)benzaldehyde (11.8 g, 67.5 mmol, 1.1 equiv.) in DCE (60.0 mL) was added AcOH (7.4 g, 122.8 mmol, 2.0 equiv.) and STAB (19.5 g, 92.1 mmol, 1.5 equiv.) in small portions at 0° C. The resulting mixture was stirred overnight at room temperature, adjusted to pH 8 with ammonium hydroxide, added with water (100.0 mL), and extracted twice with DCM (300.0 mL). The combined organic layers were washed twice with brine, dried over anhydrous NaSO, and concentrated under reduced pressure to give 29.6 g of 1-(tert-butyl) 3-ethyl 3-((4-(trifluoromethyl)benzyl)amino)azetidine-1,3-dicarboxylate, which was used in the next step without purification. LRMS (ES) m / z 347.1 (M+H-56).

[0211] Step 2: Synthesis of tert-butyl 3-((2,4-difluorophenyl)carbamoyl)-3-((4-(trifluoromethyl)benzyl)amino)azetidine-1-carboxylate [ka] To a stirred solution of 1-(tert-butyl) 3-ethyl 3-((4-trifluoromethyl)benzyl)amino)azetidine-1,3-dicarboxylate (29.6 g, 44.1 mmol, 1.0 equiv) and 2,4-difluoroaniline (4.9 mL, 48.5 mmol, 1.1 equiv) in THF (200.0 mL) was added a solution of LHMDS (88.3 mL, 1 M THF solution, 88.3 mmol, 2.0 equiv) dropwise over 20 min under N at 0° C. The mixture was stirred at 0° C. for 20 min, quenched with water (50 mL), acidified to pH 5 using aqueous HCl (3 N), and extracted twice with EA (100 mL). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated to give 36.2 g of tert-butyl 3-((2,4-difluorophenyl)carbamoyl)-3-((4-(trifluoromethyl)benzyl)amino)azetidine-1-carboxylate, which was used in the next step without purification. LRMS (ES) m / z 430.1 (M+H-56).

[0212] Step 3: Synthesis of tert-butyl 3-(2-chloro-N-(4-trifluoromethyl)benzyl)acetamido)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate [ka] To a solution of tert-butyl 3-((2,4-difluorophenyl)carbamoyl)-3-((4-(trifluoromethyl)benzyl)amino)azetidine-1-carboxylate (36.2 g, 40.6 mmol, 1.0 equiv.) in THF (100 mL) cooled to 0° C., TEA (8.5 mL, 60.9 mmol, 1.5 equiv.) and 2-chloroacetyl chloride (3.9 mL, 48.7 mmol, 1.2 equiv.) were added. After stirring at 0° C. for 30 minutes, additional TEA (8.5 mL, 60.9 mmol, 1.5 equiv.) and 2-chloroacetyl chloride (3.3 mL, 40.6 mmol, 1.0 equiv.) were added to the mixture, and the mixture was stirred at 0° C. for 30 minutes. To this mixture was added additional 2-chloroacetyl chloride (1.0 mL, 12.2 mmol, 0.3 equiv.). The mixture was gradually warmed to room temperature, stirred for 45 min, cooled to 0° C., and quenched with aqueous sodium bicarbonate. The mixture was extracted three times with EA. The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated to give 42.0 g of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate, which was used in the next step without further purification. LRMS (ES) m / z 562.1 (M+H).

[0213] Step 4: Synthesis of tert-butyl 8-(2,4-difluorophenyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carboxylate [ka] To a stirred solution of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate (42.0 g, 33.4 mmol, 1.0 equiv) in DMF (80.0 mL) under N was added KCO (7.0 g, 50.1 mmol, 1.5 equiv). The resulting mixture was stirred at room temperature for 3.5 h, diluted with water, and extracted twice with EA. The combined organic layers were washed twice with water and once with brine, dried over sodium sulfate, concentrated, and purified by silica gel using a gradient of 0-60% EA in hexanes as eluent to give 12.0 g (37% over 4 steps) of tert-butyl 8-(2,4-difluorophenyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carboxylate. LRMS (ES) m / z 469.7 (M+H-56).

[0214] Step 5: Synthesis of 8-(2,4-difluorophenyl)-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate [ka] To a stirred solution of tert-butyl 8-(2,4-difluorophenyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (12.0 g, 22.8 mmol, 1.0 equiv.) in DCM (12.0 mL) was added TFA (40.0 mL). The resulting mixture was stirred at room temperature for 3 hours and concentrated to dryness to give 12.3 g of 8-(2,4-difluorophenyl)-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate, which was used in the next step without further purification. LRMS (ES) m / z 426.1 (M+H).

[0215] Step 6: Synthesis of 8-(2,4-difluorophenyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde [ka] To a stirred solution of 8-(2,4-difluorophenyl)-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate (12.3 g, 22.8 mmol, 1.0 equiv.) in THF (50.0 mL) was added sodium cyanate (4.4 g, 68.4 mmol, 3.0 equiv.) and a few drops of acetic acid. The mixture was stirred at room temperature for 30 min, concentrated, and purified by silica column chromatography using a gradient of 0 to 10% MeOH in DCM as eluent to give 8.8 g (73% over two steps) of 8-(2,4-difluorophenyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. LRMS (ES) m / z 469.1 (M+H); 1 H NMR (400 Mhz, methanol-d4) δ 7.70 (d, J = 8.1 Hz, 2H), 7.61 -7.53 (m, 3H), 7.20 (ddd, J = 10.4, 8.8, 2.8 Hz, 1H), 7.12 (dddd, J = 9.1, 8.0, 2.8, 1.4 Hz, 1H), 5.15 (s, 2H), 4.53 -4.49 (m, 4H), 4.20 (d, J = 9.5 Hz, 2H).

[0216] Example 9 Synthesis of 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Comparative Substance E) Step 1: Synthesis of tert-butyl 3-cyano-3-((4-(trifluoromethyl)benzyl)amino)azetidine-1-carboxylate: [ka] To a solution of tert-butyl 3-oxoazetidine-1-carboxylate (25 g, 146.0 mmol, 1.0 equiv.) in THF (90 mL) was added a solution of acetic acid (10.5 g, 175.2 mmol, 1.2 equiv.) and (4-(trifluoromethyl)phenyl)methanamine (31.7 g, 181.1 mmol, 1.2 equiv.) in water (40.0 mL). After stirring at room temperature for 5 minutes, a solution of sodium cyanide (7.2 g, 146.0 mmol, 1.0 equiv.) in water (10 mL) was added. The mixture was heated in an oil bath at 60° C. for 18 hours, cooled to room temperature, neutralized with saturated aqueous sodium bicarbonate, and extracted with ethyl acetate (150 mL × 2). The combined organic layer was washed with brine (40 mL), dried over magnesium sulfate, and concentrated under reduced pressure. Diethyl ether / hexane (200 mL, 1:2) was added to the resulting yellow solid, and the solution was sonicated for 1 minute, cooled to 0° C., and filtered. The resulting white precipitate was washed with ice-cold diethyl ether (50 mL) and dried overnight to give tert-butyl 3-cyano-3-((4-(trifluoromethyl)benzyl)amino)azetidine-1-carboxylate (43.9 g, 85% yield). LRMS (ES) m / z 329.2 (M+H-27). 1 H NMR (400 MHz, DMSO-d6) δ 7.71 (d, J = 8.0 Hz, 2H), 7.60 (d, J = 8.0 Hz, 2H), 4.16 (d, J = 8.8 Hz, 2H), 3.92 (t, J = 7.2 Hz, 1H), 3.84 (d, J = 9.2 Hz, 2H), 3.81 (dd, J = 7.3 Hz, 2H), 1.39 (s, 9H).

[0217] Step 2: Synthesis of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-cyanoazetidine-1-carboxylate: [ka] To a solution of tert-butyl 3-cyano-3-((4-(trifluoromethyl)benzyl)amino)azetidine-1-carboxylate (3.0 g, 8.4 mmol, 1.0 equiv.) and triethylamine (1.3 g, 12.7 mmol, 1.5 equiv.) in DCM (0.2 M) cooled to 0 °C was added chloroacetyl chloride (0.95 g, 8.4 mmol, 1.0 equiv.). The mixture was stirred at 0 °C for 15 min, warmed to room temperature, and stirred for 2 h. To the mixture was added additional 2-chloroacetyl chloride (0.95 g, 8.4 mmol, 1.0 equiv.) and triethylamine (1.3 g, 12.7 mmol, 1.5 equiv.). The reaction was stirred for 2 h, quenched with saturated aqueous ammonium chloride, and the layers were separated. The aqueous layer was extracted once with DCM. The combined organic layers were dried over magnesium sulfate, concentrated, and purified by silica gel column chromatography (5% to 70% EtOAc / hexane, R f Purification by elution with 20% EtOAc / hexanes gave 3.4 g (92%) of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-cyanoazetidine-1-carboxylate. LRMS (ES) m / z 432.1 (M+H). 1 H NMR (400 MHz, DMSO-d6) δ 7.77 (d, J = 8.1 Hz, 2H), 7.60 (d, J = 8.0 Hz, 2H), 4.95 (s, 2H), 4.52 (s, 2H), 4.15 (s, 4H), 1.35 (s, 9H).

[0218] Step 3: Synthesis of tert-butyl 3-cyano-3-(2-(((1r,4r)-4-(difluoromethyl)cyclohexyl)amino)-N-(4-(trifluoromethyl)benzyl)acetamido)azetidine-1-carboxylate: [ka] To a solution of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-cyanoazetidine-1-carboxylate (0.8 g, 1.9 mmol, 1 equiv.) in acetonitrile (15 mL) was added (1r,4r)-4-(difluoromethyl)cyclohexan-1-amine hydrochloride (0.51 g, 2.8 mmol, 1.5 equiv.) and DIPEA (1.2 g, 9.3 mmol, 5 equiv.). The solution was heated at 65° C. for 4 h, at which point LCMS showed the reaction was complete. The reaction was diluted with ethyl acetate and water (1:1, 80 mL), and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over magnesium sulfate, concentrated, and purified by silica gel chromatography using a gradient of 25% to 100% ethyl acetate in hexanes as eluent to afford 0.65 g (64%) of tert-butyl 3-cyano-3-(2-(((1r,4r)-4-(difluoromethyl)cyclohexyl)amino)-N-(4-(trifluoromethyl)benzyl)acetamido)azetidine-1-carboxylate as a pale yellow oil. f =0.55 (100% ethyl acetate, silica). LRMS (ES) m / z 545.0 (M+H).

[0219] Step 4: Synthesis of tert-butyl 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carboxylate: [ka] To a solution of tert-butyl 3-cyano-3-(2-(((1r,4r)-4-(difluoromethyl)cyclohexyl)amino)-N-(4-(trifluoromethyl)benzyl)acetamido)azetidine-1-carboxylate (0.27 g, 0.50 mmol, 1.0 equiv) in ethanol (2 mL) was added acetic acid (0.18 g, 3.0 mmol, 6.0 equiv). The reaction was heated at 70° C. for 15 h, cooled to room temperature, and diluted with hexane (1.0 mL). The precipitate was collected by filtration, washed with ethanol-hexane (1:2, 2 mL), and dried to give 186 mg (69%) of tert-butyl 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carboxylate as a pale yellow solid. LRMS (ES) m / z 490.2 (M+H-56). 1 H NMR (400 MHz, DMSO-d6) δ 7.71 (d, J = 8.1 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 5.89 (td, J = 56.7, 3.8 Hz, 1H), 4.93 (s, 2H), 4.29 -4.15 (m, 3H), 4.02 (s, 2H), 3.93 (d, J = 9.5 Hz, 2H), 1.91 -1.55 (m, 7H), 1.35 (s, 9H), 1.38 -1.20 (m, 2H).

[0220] Step 5: Synthesis of 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate: [ka] To a solution of tert-butyl 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (200 mg, 0.37 mmol, 1.0 equiv.) in DCM (1.5 mL) was added TFA (1.5 mL) at room temperature. The mixture was stirred at room temperature for 1 hour, concentrated under reduced pressure, and dried under high vacuum to give 190 mg (94%) of 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate, which was used without further purification. LRMS(ES) m / z 446.2(M+H).

[0221] Step 6: Synthesis of 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Comparative Substance E): [ka] To a solution of 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate (54.0 mg, 0.10 mmol) in acetonitrile (0.6 mL) was added DIPEA (37.0 mg, 0.29 mmol, 3.0 equiv) and 2,2,2-trifluoroethyl formate (124.0 mg, 0.97 mmol, 10.0 equiv). The mixture was heated in a microwave reactor at 110 °C for 20 min, concentrated, and purified by HPLC using a gradient of 10% to 100% ACN in water (both containing 0.1% HCOOH) as eluent to give 21.0 mg (46%) of 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde as a foam. LRMS (ES) m / z 473.9 (M+H). 1 H NMR (400 MHz, DMSO-d6) δ 7.96 (s, 1H), 7.71 (d, J = 8.0 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 5.89 (td, J = 56.4, 4.5 Hz, 1H), 4.94 (s, 2H), 4.51 (d, J = 9.6 Hz, 1H), 4.30 -4.15 (m, 3H), 4.04 (s, 2H), 3.96 (d, J = 10.7 Hz, 1H), 1.95 -1.49 (m, 7H), 1.41 -1.06 (m, 2H).

[0222] Biological Example B-1: Myofibril Assay To assess the effect of the compound 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde on the ATPase activity of full-length cardiac myosin in the context of native sarcomeres, a stripped myofibril assay was performed. Bovine cardiac myofibrils were obtained by homogenizing bovine left ventricular tissue in the presence of a detergent such as Triton X-100. Such treatment removes most of the membrane and soluble cytoplasmic proteins, while leaving the actomyosin apparatus of cardiac sarcomeres intact. Myofibril preparations were prepared using Ca 2+ The Ca(II)-dependent activation of the ATPase activity of such myofibrillar preparations in the presence and absence of the compound 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde to a defined fraction (i.e., 25%, 75%) of the maximum rate. 2+ The compound 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde was evaluated for its ability to inhibit the steady-state ATPase activity of bovine cardiac myofibrils using a pyruvate kinase and lactate dehydrogenase (PK / LDH) coupled enzyme system. This assay regenerates myosin-generated ADP to ATP by oxidizing NADH, resulting in a change in absorbance at 340 nm. Prior to testing this compound, the calcium responsiveness of bovine cardiac myofibrils was evaluated to determine whether the 50% (pCa 50 ) or 75%(pCa 75The calcium concentration resulting in activation of myofibrillar ATPase activity was selected as the final condition for evaluating the inhibitory activity of this compound. All enzyme activities were measured in a pH 6.8 buffer (PM12 buffer) containing 12 mM PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid) and 2 mM magnesium chloride. The final assay conditions were 1 mg / mL bovine cardiac myofibrils, 4 U / mL pyruvate kinase, 6 U / mL lactate dehydrogenase, 50 μM ATP, 0.1 mg / mL bovine serum albumin (BSA), 10 ppm antifoam, 1 mM DTT, 0.5 mM NADH, 1.5 mM PEP, 0.6 mM EGTA, and sufficient CaCl2 to result in 50% or 75% activation of myofibrillar ATPase activity. Each test compound was prepared according to the synthetic procedures described herein. The results for the test compounds are shown in Table B-1. [Table B-1]

[0223] Comparative substances C and D have the following structures: [Table 33] The preparation methods of Comparative Examples C and D are described in WO2020 / 047447A1.

[0224] Biological Example B-2: Pharmacokinetic Single Dose Study Mouse single dose study Male C57BL / 6 mice (18-25 g, 6-8 weeks old) were obtained from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. All animals receiving IV administration had free access to food and water. IV administration was performed via the tail vein. IV administration solutions of the test substances were prepared at a concentration of 0.1 mg / mL in a 10% DMA / 20% PG / 70% HPβCD solution (40% (w / v) HPβCD aqueous solution). Oral administration suspensions were prepared by suspending the test substance at a concentration of 0.2 mg / mL in a 0.5% HPMC / 0.1% Tween® 80 aqueous solution. IV and PO dose concentrations were measured at the end of the study. If measured values ​​were within 20% of the nominal values, the nominal dose values ​​were used to calculate pharmacokinetic (PK) parameters. Groups of 15 mice received IV doses at a volume of 5 mL / kg. Another group of 15 mice received the test article at 1 mg / kg by oral gavage. The oral dose volume was 5 mL / kg. Sparse blood samples were collected from groups of three mice by retroorbital bleeding into K2EDTA microtainer tubes and kept on ice until centrifugation to obtain plasma. Each designated group of mice was bled at two time points: pre-dose (PO only), 5 minutes post-dose (IV only), 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours. Blood samples were centrifuged, and the collected plasma was stored at -80°C until analysis. Plasma samples were analyzed for test article concentration using an LC / MS / MS method. Briefly, a 50 μL aliquot of each plasma sample was mixed with 100 μL of acetonitrile containing an internal standard (IS). The mixture was vortexed and centrifuged. 10 μL of the resulting solution was injected onto a reversed-phase C18 column, and the resulting peaks were detected by LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantitation (BLQ) were treated as zero in the pharmacokinetic calculations.Composite PK parameters were estimated from a maximum of two sampling points per mouse and three mice per sampling point, and the sparse data option in WInNonLin was used for non-compartmental analysis of concentration-time data (Phoenix WInNonLin software, version 64; Pharmacology, Mount Airy, CA). The elimination rate constant (k) was calculated as the absolute value of the slope of the linear regression of the logarithm of the concentration versus time for the last three data points of the concentration-time profile. The apparent elimination half-life (t 1 / 2 ) values ​​were calculated as ln(2) / k. Area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC from the time of administration to the last measurable concentration t The AUC values ​​were calculated. ∞ values ​​and the corresponding AUC t Plasma clearance (CL) was calculated as the ratio of dose / AUC ∞ The mean residence time (MRT) was estimated by moment analysis. The volume of distribution at steady state (V ss ) and MRT ∞ × CL. As observed, the maximum concentration (C max ) and C max Time to reach (t max The bioavailability was calculated as AUC ∞ , po / AUC ∞、iv × 100% (where dAUC is the AUC value normalized for dose). Data for the test compounds are shown in Table B-2. Each test compound was prepared according to the synthetic procedures described herein. [Table B-2]

[0225] Single dose rat study Male Sprague-Dawley rats were obtained from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. Animals in the IV group had free access to water and food. Animals in the PO group were fasted overnight before dosing and fed 2 hours after dosing. The IV dosing solution was prepared at a concentration of 1 mg / mL in a 10% DMA / 50% PG / 40% HPβCD solution (40% (w / v) HPβCD aqueous solution). The oral dosing suspension was prepared by suspending the test substance at a concentration of 0.2 mg / mL in a 0.5% HPMC / 0.1% Tween® 80 aqueous solution. The concentrations of the IV and PO doses were measured at the end of the study. If the measured values ​​were within 20% of the nominal values, the nominal dose values ​​were used to calculate pharmacokinetic parameters. Three rats were intravenously administered via a bolus injection via the tail vein. Three rats per dose group were administered by oral gavage. Blood samples were collected from the jugular vein cannula pre-dose, 5 minutes (IV only), 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, and 24 hours post-dose. The blood volume was replaced with an equal volume of sterile 0.9% saline. Blood samples were centrifuged, and the collected plasma was stored at -80°C for subsequent analysis. Plasma samples were analyzed for test article concentrations using an LC / MS / MS method. Briefly, a 50 μL aliquot of each plasma sample was mixed with 100 μL of acetonitrile containing an internal standard. The mixture was vortexed and centrifuged. 10 μL of the resulting solution was injected onto a reversed-phase C18 column, and the resulting peaks were detected by LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantitation (BLQ) were treated as zero for pharmacokinetic calculations. Pharmacokinetic parameters were estimated from individual animals using noncompartmental analysis of concentration-time data (Phoenix WinNonLin software, version 64; Pharsight, Mountain View, CA). The elimination rate constant (k) was calculated as the absolute value of the slope of the linear regression of the logarithm of the concentration (log) versus time for the last three data points of the concentration-time profile. The apparent elimination half-life (t 1 / 2) values ​​were calculated as ln(2) / k. Area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC from the time of administration to the last measurable concentration t The AUC values ​​were calculated. ∞ values ​​and the corresponding AUC t and the ratio of the final detectable concentration divided by k (AUC t-∞ Plasma clearance (CL) was calculated as the sum of dose / AUC ∞ The mean residence time (MRT) was estimated by moment analysis. The volume of distribution at steady state (V ss ) is the MRT ∞ The maximum concentration (C max ) and C max Time to reach (t max The bioavailability was calculated as the AUC ∞,po / AUC∞ / average dAUC ∞,iv ×100% (where dAUC is the AUC value normalized for dose). The data for the test compounds are shown in Table B-3. Each test compound was prepared according to the synthesis procedures described herein. [Table B-3]

[0226] Dog single dose study Non-naive male beagle dogs (8 months to 3 years old, weighing 8 to 13 kg) were used in this study. All animals administered IV had free access to food and water. All animals administered PO were fasted overnight before dosing and fed approximately 6 hours after dosing. For animals in the PO group, pentagastrin (6.0 μg / kg, intramuscular) was administered 20 minutes before administration of the PO formulation and 1.5 hours after the second pentagastrin administration. The dose was 0.024 mL / kg, and the concentration was 250 μg / mL in DMSO / 1N NaOH / PBS. 10 mL of 0.001N HCl was used to flush the gavage catheter of each animal. The IV dosing solution was prepared at a concentration of 1.0 mg / mL in a 10% DMA / 50% PG / 40% HPβCD solution (40% (w / v) HPβCD in water). Oral dosing suspensions were prepared by suspending the compound at a concentration of 0.2 mg / mL in 0.5% HPMC / 0.1% Tween® 80 in distilled water. IV and PO dose concentrations were measured at the end of the study. If measured values ​​were within 20% of the nominal values, the nominal dose values ​​were used to calculate PK parameters. Blood samples were collected by venipuncture of a peripheral vein, excluding the dosing vein, at pre-dose, 5, 15, 30, 1, 2, 4, 6, 8, 24, and 48 hours after dosing. Blood samples were centrifuged, and the resulting plasma was frozen for bioanalysis. Plasma samples were stored at -80°C prior to analysis. Plasma samples were analyzed for compound concentration using the LC / MS / MS method described below. Briefly, a 50 μL aliquot of each plasma sample was mixed with 100 μL of acetonitrile containing an internal standard. The mixture was vortexed and centrifuged. Ten microliters of the resulting solution was injected onto a reversed-phase C18 column, and the resulting peaks were detected by LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantitation (BLQ) were treated as zero in pharmacokinetic calculations. PK parameters were estimated from individual animals using noncompartmental analysis of concentration-time data (Phoenix WinNonLin software, version 64; Pharsight, Mountain View, CA).The elimination rate constant (k) was calculated as the absolute value of the slope of the linear regression of the logarithm of the concentration (log) against time for the last three data points of the concentration-time profile. The apparent elimination half-life (t 1 / 2 ) values ​​were calculated as ln(2) / k. Area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC from the time of administration to the last measurable concentration t The AUC values ​​were calculated. ∞ values ​​and the corresponding AUC t Plasma clearance (CL) was calculated as the ratio of dose / AUC ∞ The mean residence time (MRT) was calculated from the ∞ ) was estimated by moment analysis. ss , MRT ∞ × CL. As observed, the maximum concentration (C max ) and C max Time to reach (t max ) was recorded. Because this was a crossover study, bioavailability was measured using dAUC ∞,po / dAUC ∞,iv × 100% (where dAUC is the dose-normalized AUC value from the same animal administered IV and PO doses). Data for the test compounds are shown in Table B-4. Each test compound was prepared according to the synthetic procedures described herein. [Table B-4]

[0227] Single-dose monkey study Non-naive male cynomolgus monkeys (2-5 years old, weighing 2-5 kg) used in this study were obtained from Topgene Biotechnology. All animals for IV administration had free access to food and water. All animals for PO administration were fasted overnight before dosing and fed approximately 6 hours after dosing. The IV administration solution was prepared at a concentration of 1.0 mg / mL in a 10% DMA / 50% PG / 40% HPβCD solution (40% (w / v) HPβCD in water). The oral administration suspension was prepared by suspending the compound at a concentration of 0.2 mg / mL in 0.5% HPMC / 0.1% Tween® 80 in distilled water. IV and PO dose concentrations were measured at the end of the study. If the measured values ​​were within 20% of the nominal values, the nominal dose values ​​were used to calculate PK parameters. Blood samples were collected by venipuncture of a peripheral vein, excluding the dosing vein, at pre-dose, 5, 15, 30, 1, 2, 4, 6, 8, and 48 hours after dosing. The blood samples were centrifuged, and the resulting plasma was frozen for bioanalysis. Plasma samples were stored at -80°C prior to analysis. Plasma samples were analyzed for compound concentrations using the LC / MS / MS method described below. Briefly, a 50 μL aliquot of each plasma sample was mixed with 100 μL of acetonitrile containing an internal standard. The mixture was vortexed and centrifuged. 10 μL of the resulting solution was injected onto a reverse-phase C18 column, and the resulting peaks were detected by LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantitation (BLQ) were treated as zero for pharmacokinetic calculations. PK parameters were estimated from individual animals using noncompartmental analysis of concentration-time data (Phoenix WinNonLin software, version 64; Pharsight, Mountain View, CA). The elimination rate constant (k) was calculated as the absolute value of the slope of the linear regression of the logarithm of concentration (log) versus time for the last three data points of the concentration-time profile. The apparent elimination half-life (t 1 / 2 ) values ​​were calculated as ln(2) / k. Area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC from the time of administration to the last measurable concentration t The AUC values ​​were calculated. ∞values ​​and the corresponding AUC t Plasma clearance (CL) was calculated as the ratio of dose / AUC ∞ The mean residence time (MRT) was calculated from the ∞ ) was estimated by moment analysis. ss , MRT ∞ × CL. As observed, the maximum concentration (C max ) and C max Time to reach (t max ) was recorded. Because this was a crossover study, bioavailability was measured using dAUC ∞,po / dAUC ∞,iv × 100% (where dAUC is the dose-normalized AUC value from the same animal administered IV and PO doses). Data for the test compounds are shown in Table B-5. Each test compound was prepared according to the synthetic procedures described herein. [Table B-5]

[0228] Predicted single-dose CL and V in humans ss value Allometric scaling for predicting human clearance and volume of distribution was based on simple interspecies allometric scaling of intravenous pharmacokinetic parameters in mice, rats, dogs, and cynomolgus monkeys (Boxenbaum, J Pharmackinet Biopharm 10:201-27, 1982). Human CL was predicted by extrapolating plasma intravenous clearance data from preclinical species. This prediction considered the "exponent rule" (Mahmood & Balian, Life Sci. 59:579-85, 1996), which states that if the simple allometric exponent is in the range of 0.71-0.99, a correction factor based on the maximum life span (MLP) of this species can be applied; if the simple allometric exponent is greater than 1.0, a correction factor based on brain weight (BrW) or protein binding correction, if available, can be applied. Similarly, simple allometric scaling was used to predict human volume of distribution. This method has been used successfully with a variety of drugs (Ward & Smith, Drug Metab Dispos 32:612-19, 2004; McGinnity et al., Curr Drug Metab 8:463-79, 2007). Prediction data for selected compounds is shown in Table B-6. [Table B-6]

[0229] Legend for Table B-6: SA = simple allometry; ROE = exponential rule; fu correction = correction for unbound fraction in plasma; MLP correction = maximum lifetime correction; BrW correction = brain weight correction.

[0230] Cassette administration in dogs This study involved non-naive male beagle dogs (8 months to 3 years old, weighing 8 to 14 kg) provided by Jiangsu Johnsen Bioresource Co. and / or Beijing Rixinkeji Co., Ltd. and / or Beijing Marshll Biotechnology Co., Ltd. All animals receiving IV dosing had free access to food and water. The IV dosing solution was prepared at a concentration of 0.2 mg / mL in a 10% DMA / 50% PG / 40% HPβCD solution (40% (w / v) HPβCD aqueous solution). IV dose concentrations were measured at the end of the study. If measured values ​​were within 20% of the nominal values, the nominal dose values ​​were used to calculate PK parameters. Blood samples were collected by venipuncture of a peripheral vein, excluding the dosing vein, before dosing and 5, 15, 30, 2, 4, 6, 8, and 24 hours after dosing. Blood samples were centrifuged, and the resulting plasma was frozen for bioanalysis. Plasma samples were stored at -80°C prior to analysis. Plasma samples were analyzed for compound concentrations using the LC / MS / MS method described below. Briefly, a 50 μL aliquot of each plasma sample was mixed with 100 μL of acetonitrile containing an internal standard. The mixture was vortexed and centrifuged. 10 μL of the resulting solution was injected onto a reversed-phase C18 column, and the resulting peaks were detected by LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantitation (BLQ) were treated as zero in pharmacokinetic calculations. PK parameters were estimated from individual animals using noncompartmental analysis of concentration-time data (Phoenix WinNonLin software, version 64; Pharsight, Mountain View, CA). The elimination rate constant (k) was calculated as the absolute value of the slope of the linear regression of the logarithm of the concentration (log) versus time for the last three data points of the concentration-time profile. The apparent elimination half-life (t 1 / 2 ) values ​​were calculated as ln(2) / k. Area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC from the time of administration to the last measurable concentration t The AUC values ​​were calculated. ∞ values ​​and the corresponding AUC tPlasma clearance (CL) was calculated as the ratio of dose / AUC ∞ The mean residence time (MRT) was calculated from the ∞ ) was estimated by moment analysis. ss , MRT ∞ × CL. As observed, the maximum concentration (C max ) and C max Time to reach (t max ) was recorded. Because this was a crossover study, bioavailability was measured using dAUC ∞,po / dAUC ∞,iv × 100% (where dAUC is the dose-normalized AUC value from the same animal administered IV and PO doses). Data for compound 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Compound 1) are shown in Table B-7. Each test compound was prepared according to the synthetic procedures described herein. [Table B-7]

[0231] Cassette administration in monkeys Non-naive male beagle dogs (2-5 years old, weighing 2-5 kg) provided by Topgene Biotechnology were used in this study. All animals receiving IV dosing had free access to food and water. The IV dosing solution was prepared at a concentration of 0.2 mg / mL in a 10% DMA / 50% PG / 40% HPβCD solution (40% (w / v) HPβCD in water). IV dose concentrations were measured at the end of the study. If measured values ​​were within 20% of the nominal values, the nominal dose values ​​were used to calculate PK parameters. Blood samples were collected by venipuncture of a peripheral vein, excluding the dosing vein, before dosing and 5, 15, 30, 2, 4, 6, 8, and 24 hours after dosing. Blood samples were centrifuged, and the resulting plasma was frozen for bioanalysis. Plasma samples were stored at -80°C before analysis. Plasma samples were analyzed for compound concentration using the LC / MS / MS method described below. Briefly, a 50 μL aliquot of each plasma sample was mixed with 100 μL of acetonitrile containing an internal standard. The mixture was vortexed and centrifuged. 10 μL of the resulting solution was injected onto a reversed-phase C18 column, and the resulting peaks were detected by LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantitation (BLQ) were treated as zero in pharmacokinetic calculations. PK parameters were estimated from individual animals using noncompartmental analysis of concentration-time data (Phoenix WinNonLin software, version 64; Pharsight, Mountain View, CA). The elimination rate constant (k) was calculated as the absolute value of the slope of the linear regression of the logarithm of the concentration (log) versus time for the last three data points of the concentration-time profile. The apparent elimination half-life (t 1 / 2 ) values ​​were calculated as ln(2) / k. Area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC from the time of administration to the last measurable concentration t The AUC values ​​were calculated. ∞ values ​​and the corresponding AUC t Plasma clearance (CL) was calculated as the ratio of dose / AUC ∞ The mean residence time (MRT) was calculated from the ∞) was estimated by moment analysis. ss , MRT ∞ × CL. As observed, the maximum concentration (C max ) and C max Time to reach (t max ) was recorded. Because this was a crossover study, bioavailability was measured using dAUC ∞,po / dAUC ∞,iv × 100% (where dAUC is the dose-normalized AUC value from the same animal administered IV and PO doses). Data for compound 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Compound 1) are shown in Table B-8. Each test compound was prepared according to the synthetic procedures described herein. [Table B-8]

[0232] Predicted cassette administration CL and V in humans ss value For PK data obtained from cassette IV dosing, single-species allometry was used to predict human clearance and volume of distribution. In this case, values ​​were predicted from plasma intravenous clearance data in dogs and monkeys by applying protein binding corrections (Tang, Drug Metab Dispos 33:1294-96, 2005; Patel, Journal of Pharmaceutical Research International, 22(3): 1-7, 2018). The predicted data for the compound 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Compound 1) are shown in Tables B-9 and B-10. [Table B-9] [Table B-10]

[0233] Biological Example B-3 Echocardiographic assessment of acute pharmacodynamic effects on cardiac contractility in rats. In vivo cardiac function was assessed by echocardiography in male Sprague-Dawley rats under isoflurane (1–3%) anesthesia. 2D M-mode images of the left ventricle were acquired in the parasternal long axis view before, during, and after administration of the compound 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. In vivo fractional shortening was determined by M-mode image analysis using the following formula: ((end-diastolic diameter - end-systolic diameter) / end-diastolic diameter × 100). Three pre-dose baseline M-mode images were taken at 1-minute intervals before compound administration. The compound 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde was formulated in a 0.5% hydroxypropylmethylcellulose 2910 (HPMC2910):0.1% Tween® 80 suspension and administered as a single dose (5 mL / kg) by oral gavage. One and four hours after dosing, rats were lightly anesthetized for M-mode echocardiographic measurements. Blood samples were collected simultaneously with the echocardiographic measurements to determine the plasma concentration of the corresponding compound. The obtained plasma concentrations were used to calculate the IC 50 and IC 10 The values ​​(concentrations at which the shortening fraction was 50% and 10%, respectively, of the baseline shortening fraction before administration) were estimated. The data for the compound 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Compound 1) are shown in Table B-11. [Table B-11]

[0234] Biological Example B-4 In vitro measurement of time-dependent inhibition of CYP450 enzymes The time-dependent inhibitory potency of each test compound against major human cytochrome P450 isozymes was also assessed using human liver microsomes using standard methods (Grimm et al., Drug Metab. Dispos., Jul;37(7):1355-70. doi: 10.1124 / dmd.109.026716, 2009). Pooled human microsomes and selective CYP probe substrates were used to in vitro evaluate test compounds at 25 and 50 μM as time-dependent inhibitors of seven human hepatic cytochrome P450 isozymes (CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4). Metabolite formation was quantified using LC-MS / MS. The percent inhibition of each P450 enzyme in human liver microsomes was measured as the percent reduction in activity of marker metabolite formation, as measured by LC-MS / MS, compared to the uninhibited control (=100% activity) at time 0 and after 30 minutes of incubation. The occurrence of time-dependent inhibition was then expressed as the fold change in enzyme activity at time 0 relative to the activity after 30 minutes of incubation. The time-dependent inhibition of compound 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Compound 1) against CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4-M, and CYP3A4-T is shown in Table B-12. [Table B-12A] [Table B-12B] [Table B-12C] [Table B-12D]

[0235] NA: Not applicable, CYP3A4-T: CYP3A4 activity measured with a testosterone probe substrate, CYP3A4-M: CYP3A4 activity measured with a midazolam probe substrate

[0236] For 3A4, % activity was measured using midalzolam and testosterone as probes, and 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (compound 1) did not show any signs of time-dependent inhibition, as the enzyme's activity did not change by more than 1.2-fold. However, comparative substance C showed a fold change in activity of more than 1.5-fold with both probes at 25 μM and 50 μM, suggesting that there may be some change in 3A4 activity when tested in a time-dependent format with comparative substance C. Both compounds tested did not change the activity of 1A2 and 2B6 when tested in this format. For 2C19, compound 1 showed a 1.4-fold change in activity at 25 μM, but no change in activity was observed with compound 1 at 50 μM. For 2D6, compound 1 showed a 1.3-fold change in activity at a concentration of 25 μM, but no change in activity was observed at a concentration of 50 μM.

[0237] While the above description of the compounds, uses, and methods described herein will enable one of ordinary skill in the art to make and use the compounds, uses, and methods described herein, one of ordinary skill in the art will understand and appreciate that there are variations, combinations, and equivalents to the specific embodiments herein. Thus, the compounds, uses, and methods provided herein should not be limited by the above embodiments, methods, or examples, but rather encompass all embodiments and methods within the scope and spirit of the compounds, uses, and methods provided herein.

[0238] All references cited herein are incorporated in their entirety.

Claims

1. Crystalline form of the compound in formula 1: 【Chemistry 29】

2. The crystal form according to claim 1, characterized by having an XRPD pattern that includes peaks at 2θ angles = 5.9±0.2°, 11.5±0.2°, 11.7±0.2°, 17.9±0.2°, and / or 19.1±0.2°.

3. The crystal form according to claim 2, characterized by having an XRPD pattern that includes further peaks at 2θ angles = 7.8 ± 0.2° and 16.2 ± 0.2°.

4. The crystal form according to claim 2, characterized by having an XRPD pattern that includes further peaks at 2θ angles = 13.1 ± 0.2° and 19.8 ± 0.2°.

5. The crystal form according to claim 1, characterized by having an XRPD pattern substantially shown in Figure 2A.

6. The crystal form according to claim 1, characterized by having a DSC graph substantially shown in Figure 2B.

7. The crystal form according to claim 1, characterized in that it has an endothermic start at 154.9 ± 2°C when measured by DSC.

8. The crystal form according to claim 1, characterized in that it has an endothermic peak at approximately 155.8 ± 2°C when measured by DSC.

9. The crystal form according to claim 1, characterized by having a TGA graph substantially shown in Figure 2B.

10. The crystal form according to claim 1, characterized in that it has a weight loss of less than 0.1% before decomposition when measured by TGA.

11. The crystal form according to claim 1, characterized by having a GVS graph substantially shown in Figure 2C.

12. The crystal form according to claim 1, characterized by having an XRPD pattern that includes peaks at 2θ angles = 6.0 ± 0.2°, 10.2 ± 0.2°, 21.6 ± 0.2°, and 22.1 ± 0.2°.

13. The crystal form according to claim 12, characterized by having an XRPD pattern that includes further peaks at 2θ angles = 17.9 ± 0.2° and 24.1 ± 0.2°.

14. The crystal form according to claim 12, characterized in that it has an XRPD pattern that includes further peaks at 2θ angles = 16.0±0.2°, 16.6±0.2°, 17.3±0.2°, 17.6±0.2°, and 20.5±0.2°.

15. The crystal form according to claim 1, characterized by having an XRPD pattern substantially shown in Figure 1A.

16. The crystal form according to claim 1, characterized by having a DSC graph substantially shown in Figure 1B.

17. The crystal form according to claim 1, characterized in that it has an endothermic start at 125.6 ± 2°C when measured by DSC.

18. The crystal form according to claim 1, characterized in that it has an endothermic start at 130.2 ± 2°C when measured by DSC.

19. The crystal form according to claim 1, characterized by having a TGA graph substantially shown in Figure 1B.

20. The crystal form according to claim 1, characterized in that, when measured by TGA, it has a weight loss of 0.35% ± 0.05% between 105°C and 145°C.

21. The crystal form according to claim 1, characterized by having a GVS graph substantially shown in Figure 1C.

22. A method for preparing the crystal form described in claim 2, The method comprising (1) forming a mixture of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde and a solvent selected from the group consisting of alcohol, water, and mixtures thereof, and (2) cooling the mixture from step (1).

23. The method according to claim 22, wherein the solvent comprises ethanol and water.

24. The method according to claim 22, wherein step (1) includes stirring the mixture at a temperature of 40°C to 60°C.

25. The method according to claim 22, wherein step (2) includes cooling the mixture from step (1) to a temperature of 0°C to 10°C.

26. A method for preparing the crystalline form according to claim 2, comprising: (1) forming a mixture of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde and a first solvent; (2) heating the mixture from step (1) to a first temperature of 75°C to 95°C; (3) adding a second solvent to the mixture from step (2); (4) cooling the mixture from step (3) to a second temperature of 10°C to 30°C; and (5) filtering the mixture from step (4) to obtain the crystalline form.

27. The method according to claim 26, wherein step (4) further comprises cooling the mixture from step (3) to an intermediate temperature of 50°C to 75°C, adding the crystalline seed crystal, and further cooling the mixture to the second temperature.

28. The method according to claim 26, wherein the first solvent is ethanol or a mixture of ethanol and water.

29. The method according to claim 26, wherein the second solvent is water.

30. A pharmaceutical composition comprising the crystalline form described in claim 1 and a pharmaceutically acceptable excipient.

31. A composition or pharmaceutical composition for treating heart disease in a person requiring treatment for heart disease, comprising the crystalline form described in any one of claims 1 to 21 or the pharmaceutical composition described in claim 30.

32. The composition or pharmaceutical composition according to claim 31, wherein the aforementioned heart disease is hypertrophic cardiomyopathy or heart failure with preserved ejection fraction.

33. The composition or pharmaceutical composition according to claim 32, wherein the hypertrophic cardiomyopathy is obstructive or non-obstructive, or is caused by (i) a sarcomere mutation, (ii) a non-sarcomere mutation, or (iii) both a sarcomere mutation and a non-sarcomere mutation.

34. The composition or pharmaceutical composition according to claim 31, wherein the cardiac disease is selected from the group consisting of diastolic dysfunction, primary or secondary cardiomyopathy, myocardial infarction and angina pectoris, left ventricular outflow tract obstruction, hypertensive heart disease, congenital heart disease, cardiac ischemia, coronary heart disease, diabetic heart disease, congestive heart failure, right heart failure, cardiorenal syndrome, and infiltrative cardiomyopathy, or the cardiac disease is one or more conditions selected from the group consisting of cardiac aging, age-related diastolic dysfunction, left ventricular hypertrophy, and concentric left ventricular remodeling, or is related thereto.

35. A composition or pharmaceutical composition for treating a disease or condition in a subject requiring treatment of a disease or condition selected from muscular dystrophy and glycogen storage disease, such as hypertrophic cardiomyopathy, secondary left ventricular wall thickening, narrow left ventricular cavity and cardiac chamber obstruction, hyperdynamic left ventricular contraction, myocardial ischemia, or myocardial fibrosis, comprising the crystalline form described in any one of claims 1 to 21, or the pharmaceutical composition described in claim 30.

36. The composition or pharmaceutical composition according to claim 35, wherein the disease or condition is selected from the group consisting of Fabry disease, Danon disease, mitochondrial cardiomyopathy, Noonan syndrome, hypertension, valvular heart disease, aortic stenosis, mitral regurgitation, metabolic syndrome, diabetes mellitus, obesity, end-stage renal failure, scleroderma, sleep apnea, amyloidosis, ataxia Friedrich, and Pompe disease.

37. A composition comprising the crystalline form described in any one of claims 1 to 21, or a pharmaceutical composition described in claim 30, which is a composition or pharmaceutical composition for use in a method for inhibiting cardiac sarcomeres, wherein the method comprises contacting the cardiac sarcomeres with the crystalline form or the pharmaceutical composition.

Images