Crystalline forms of 5-(3, 4-difluorobenzyl)-8-((1r, 4r)-4-methylcyclohexyl)-6, 9-dioxo-2, 5, 8-triazaspiro [3.5] nonane-2-carboxaldehyde

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-formaldehyde, compositions thereof, methods of making the same, and methods of using the same. # imgabs0 #

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the priority benefit of PCT application No. CN2022 / 116765 filed on September 2, 2022, and the disclosure of the PCT application is incorporated herein by reference in its entirety. Technical Field

[0003] 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 of making the same, and methods of using the same. Background Art

[0004] The cardiac sarcomere is composed of a network of contractile and structural proteins that regulate myocardial function. Components of the cardiac sarcomere are targets for the treatment of various cardiac diseases and disorders, for example by increasing contractile force or promoting complete relaxation to modulate systolic and diastolic function, respectively. The force and speed of cardiac contraction are major determinants of organ function and are regulated by the periodic interaction of actin and myosin. Regulation of actin and myosin binding is regulated by a network of myofilament regulatory proteins and intracellular Ca 2+ The troponin complex and tropomyosin are thin filament proteins that control actin binding sites and the availability of essential and regulatory light chains, while myosin-binding protein C regulates the position and mechanical properties of myosin.

[0005] Abnormalities in cardiac sarcomeres have been identified as drivers of various cardiac diseases and disorders, such as hypertrophic cardiomyopathy (HCM) and heart failure with preserved ejection fraction (HFpEF). Mutations in sarcomeric proteins cause disease by causing the heart muscle to contract 'too high' or 'too low'. Modulators of cardiac sarcomeres could be used to rebalance contractility and halt or reverse disease progression.

[0006] Current agents directed to cardiac sarcomeres, such as inotropes (drugs that increase the heart's contractile capacity), have poor selectivity for cardiac tissue, which results in well-recognized adverse effects that limit their use. These adverse effects include cellular damage due to increased rates of energy expenditure, exacerbation of diastolic abnormalities, and potential proarrhythmic side effects that may result from increased cytosolic Ca inotropically stimulated myocardium. 2+ Given the limitations of current agents, new approaches are needed to improve cardiac function in HCM and HFpEF.

[0007] There remains a great need for agents that utilize new mechanisms of action and that may have improved efficacy in terms of symptom relief, safety, and patient mortality, both in the short and long term. New agents with improved therapeutic indexes compared to existing agents would provide a means of achieving these clinical outcomes. Selectivity of agents for cardiac sarcomeres (e.g., by targeting cardiac myosin) has been identified as an important means of achieving 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 effect on smooth muscle myosin. The benefits of this compound include a broader therapeutic index, less effect on cardiac relaxation, better pharmacokinetics, and better safety, and therefore it offers a potential treatment for cardiac diseases and disorders.

[0008] In order to convert a drug candidate such as Compound 1 into a viable drug product, it is important to understand whether the drug candidate has polymorphic forms, and the relative stability and interconversion of these forms under conditions that may be encountered during large-scale production, transportation, storage, and preparation before use. The ability to control and produce stable polymorphs through a robust manufacturing process is critical for regulatory approval and marketing. The large-scale production process for preparing high-purity Compound 1 can be improved by using a specific crystalline form. Therefore, new crystalline forms of Compound 1 with desired chemical and physical stability and their formulations and uses are needed. Summary of the invention

[0009] In one aspect, 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 (Compound 1).

[0010] In another aspect, provided herein is a method for preparing a polymorph of Compound 1.

[0011] In another aspect, provided herein is a composition containing a polymorph of Compound 1 as described herein.

[0012] In another aspect, provided herein are methods of using a polymorph of Compound 1 to treat cardiac disease in a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1A Shown is an 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.

[0014] Figure 1B Differential scanning calorimetry (DSC) and thermal imaging 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 are shown.

[0015] Figure 1C Shown is a gravimetric vapor sorption (GVS) graph 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.

[0016] Figure 1D Shown is 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 and after storage at 40°C / 75% RH and 25°C / 97% RH for 7 days (from top to bottom: Form I after storage at 40°C / 75% RH for 7 days, Form I after storage at 25°C / 97% RH for 7 days, Form I before storage at 40°C / 75% RH and 25°C / 97% RH for 7 days).

[0017] Figure 1E Shown are the 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).

[0018] Figure 2A Shown is 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.

[0019] Figure 2B Shown are the 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.

[0020] Figure 2CShown is a GVS 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.

[0021] Figure 2D Shown is an overlay of the 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 storage at 40°C / 75% RH and 25°C / 97% RH for 8 days (from top to bottom: Form II after storage at 25°C / 97% RH for 8 days, Form II after storage at 40°C / 75% RH for 8 days, Form II before storage at 40°C / 75% RH and 25°C / 97% RH for 8 days).

[0022] Figure 2E Shown is an overlay of the 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).

[0023] Figure 3A Shown is 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.

[0024] Figure 3B Shown are 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.

[0025] Figure 3C Shown are the overlays of the 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 and after storage at 40°C / 75% RH for 7 days, and the XRPD pattern of crystalline Form II (from top to bottom: Form III after storage at 40°C / 75% RH for 7 days, Form III before storage at 40°C / 75% RH for 7 days, Form II). DETAILED DESCRIPTION

[0026] definition

[0027] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0028] As used herein, and unless otherwise indicated, the terms "about" and "approximately" when used in conjunction with a dose, amount, or weight percentage of an ingredient of a composition or dosage form, mean a dose, amount, or weight percentage that one of ordinary skill in the art considers to provide a pharmacological effect comparable to that obtained from a specified dose, amount, or weight percentage. Specifically, where applicable, the terms "about" and "approximately" when used in context, encompass doses, amounts, or weight percentages within 15% of a specified dose, amount, or weight percentage.

[0029] As used herein, the terms "polymorph", "polycrystal", "polymorphic form" or "crystalline form" refer to the crystalline form of a compound. Different polymorphs may have different physical properties, such as melting temperature, heat of fusion, solubility, dissolution rate and / or vibrational spectrum, caused by the arrangement or conformation of molecules or ions in the crystal lattice. Differences in physical properties exhibited by polymorphs may affect pharmaceutical parameters such as storage stability, compressibility, density (important in formulation and product manufacturing), and dissolution rate (an important factor in bioavailability). Differences in stability may be caused by changes in chemical reactivity (e.g., differential oxidation, causing a dosage form to change color faster when composed of one polymorph than when composed of another polymorph), mechanical changes (e.g., tablets break up into a kinetically favorable polymorph during storage, converting to a thermodynamically more stable polymorph), or both (e.g., tablets of one polymorph decompose more easily under high humidity). Due to differences in solubility / dissolution, some polymorphic transformations may result in a lack of efficacy in extreme cases, or toxicity in another extreme case. Additionally, the physical properties of the crystalline form may be important during processing; for example, one polymorph may form solvates more easily or may be difficult to filter and wash to remove impurities (e.g., ion shape and size distribution may differ between polymorphs).

[0030] As used herein, "therapeutically effective amount" indicates an amount that produces the desired pharmacological and / or physiological effect on a disorder. The effect may be therapeutic in terms of partial or complete cure of the disorder and / or side effects attributable to the disorder.

[0031] As used herein, the term "pharmaceutically acceptable carrier" and its cognates 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. As will be appreciated by those skilled in the art, a pharmaceutically acceptable carrier as described herein and any additional components should be suitable for the intended route of administration (e.g., oral, parenteral) of a particular dosage form.

[0032] The terms "treat," "treating," and "treatment" are intended to include alleviating or eliminating a condition, disease, or disorder, or one or more symptoms associated with a condition, disease, or disorder; or slowing the progression, spread, or worsening of a disease, condition, or disorder, or one or more symptoms thereof. Typically, the beneficial effects a subject obtains from a therapeutic agent do not completely cure the disease, condition, or disorder.

[0033] The term "subject" refers to an animal, including but not limited to a primate (e.g., 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, e.g., to refer to a mammalian subject, e.g., a human.

[0034] As used herein, the term "substantially as shown in..." 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 depicted herein, but that fall within the limits of experimental error or deviation when considered by a person of ordinary skill in the art.

[0035] As used herein, the term "substantially free" means that the composition comprising the 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 indicated substance.

[0036] Polymorph

[0037] In one aspect, provided herein is a polymorph of 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,

[0038]

[0039] Polymorphs may have properties such as bioavailability and stability that are suitable for medical or pharmaceutical use under certain conditions.

[0040] Polymorphs of compound 1 can provide advantages in bioavailability and stability and can be suitable for use as active agents in pharmaceutical compositions. Changes in the crystal structure of drug substances may affect the dissolution rate of the drug (which may affect bioavailability, etc.), manufacturability (e.g., ease of handling, ease of purification, ability to consistently prepare known strength doses, etc.), and stability (e.g., thermal stability, shelf life (including anti-degradation), etc.). These changes may affect the preparation or formulation methods of pharmaceutical compositions of different doses 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 can provide desired or suitable hygroscopicity or lack of hygroscopicity, provide particle size control, dissolution rate, solubility, purity, physical and chemical stability, manufacturability, yield, reproducibility, and / or process control. Therefore, polymorphs of compound 1 can provide the following advantages: improving the stability or storage of the manufacturing process of the active agent or the active agent drug form, or having bioavailability and / or stability suitable as an active agent.

[0041] It has been found that using certain conditions, such as using different solvents and / or temperatures, can produce different polymorphs of Compound 1 or solvates thereof, including crystalline forms I-III described herein, which can exhibit one or more of the advantageous properties described herein. The preparation methods of the polymorphs described herein and the characterization of these polymorphs are described in more detail below.

[0042] Form I

[0043] 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.

[0044] In some embodiments, Form I has substantially Figure 1A The XRPD pattern shown in .

[0045] The 2-theta angles and relative peak intensities observed for Form I using XRPD are shown in Table 1.

[0046] Table 1

[0047]

[0048]

[0049] In some embodiments, the crystalline Form I has a Figure 1AIn some embodiments, the peak assignments listed herein (including crystalline Form I) may vary by about ± 0.6, ± 0.4, ± 0.2, or ± 0.1 degrees 2-θ.

[0050] In some embodiments, crystalline Form I has an XRPD pattern comprising peaks at 2-θ angles of 6.0 ± 0.2, 10.2 ± 0.2, 21.6 ± 0.2, and 22.1 ± 0.2 degrees. In some embodiments, crystalline Form I has an XRPD pattern comprising additional peaks at 2-θ angles of 17.9 ± 0.2 and 24.1 ± 0.2 degrees. In some embodiments, crystalline Form I has an XRPD pattern further comprising additional peaks at 2-θ angles of 16.0 ± 0.2, 16.6 ± 0.2, 17.3 ± 0.2, 17.6 ± 0.2, and 20.5 ± 0.2 degrees. In some embodiments, crystalline Form I has a range of about 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. 2, 29.6 ± 0.2, and 30.1 ± 0.2 degrees. It is understood that peaks in the XRPD pattern other than peaks at 2-θ angles of 9 ± 0.2, 21.6 ± 0.2, 22.1 ± 0.2, 22.8 ± 0.2, 23.1 ± 0.2, 24.1 ± 0.2, 24.5 ± 0.2, 25.4 ± 0.2, 25.7 ± 0.2, 27.8 ± 0.2, 28.6 ± 0.2, 29.0 ± 0.2, 29.6 ± 0.2, and 30.1 ± 0.2 degrees may be observed, for example, due to the presence of impurities, solvents, or other polymorphs or amorphous forms in the test sample. Figure 1A Additional peaks beyond those shown in or as provided in Table 1.

[0051] In some embodiments, Form I has substantially Figure 1BThe differential scanning calorimetry (DSC) graph shown in . In some embodiments, Form I is characterized as having an endothermic onset at about 125.6° C. as determined by DSC. In some embodiments, Form I is characterized by having an endothermic onset as determined by DSC 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). In some embodiments, Form I is characterized as having an endothermic peak at about 130.2 °C as determined by DSC. In some embodiments, Form I is characterized by having 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 determined by DSC.

[0052] In some embodiments, Form I has substantially Figure 1B 04%, 0.35% ± 0.03%, 0.35% ± 0.02%, or 0.35% ± 0.01%) weight loss between 105°C and 145°C as determined by TGA.

[0053] In some embodiments, Form I has substantially Figure 1C The gravimetric vapor sorption (GVS) diagram shown in .

[0054] In some embodiments, Form I exhibits substantially no change or no change when stored for 7 days at two different temperature / RH conditions (40°C / 75% RH and 25°C / 97% RH) as determined by XRPD. In some embodiments, Form I exhibits substantially no change or no change when stored for a period of 7 days at 40°C / 75% RH as determined by XRPD. In some embodiments, Form I exhibits substantially no change or no change when stored for a period of 7 days at 25°C / 97% RH as determined by XRPD.

[0055] In some embodiments, Form I exhibits substantially no change or no change before and after GVS measurement as determined by XRPD, such as Figure 1E as shown in .

[0056] In some embodiments, Form I is 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde having 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% relative to other forms of the compound, including salts, solvates, or amorphous forms, and the purity remains substantially unchanged or unchanged as determined by HPLC when stored at 40°C / 75% RH and / or at 25°C / 97% RH for a period of 7 days.

[0057] In some embodiments of 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) apply:

[0058] (a) Form I has an XRPD pattern comprising peaks at 2-θ angles of 6.0±0.2, 10.2±0.2, 21.6±0.2, 22.1±0.2 degrees; an XRPD pattern comprising additional peaks at 2-θ angles of 17.9±0.2 and 24.1±0.2 degrees; an XRPD pattern further comprising additional peaks at 2-θ angles of 16.0±0.2, 16.6±0.2, 17.3±0.2, 17.6±0.2, and 20.5±0.2 degrees; or an XRPD pattern comprising peaks at 2-θ angles of 6.0±0.2, 10.2±0.2, 11.9±0.2, 13.7±0.2, 14.4±0.2,

[0059] 15.5±0.2, 16.0±0.2, 16.6±0.2, 17.3±0.2, 17.6±0.2, 17.9±0.2,

[0060] 19.8±0.2, 20.5±0.2, 20.9±0.2, 21.6±0.2, 22.1±0.2, 22.8±0.2,

[0061] an XRPD pattern of peaks at 2-theta angles of 23.1±0.2, 24.1±0.2, 24.5±0.2, 25.4±0.2, 25.7±0.2, 27.8±0.2, 28.6±0.2, 29.0±0.2, 29.6±0.2, and 30.1±0.2 degrees;

[0062] (b) Form I has substantially Figure 1A The XRPD pattern shown in;

[0063] (c) Form I has substantially Figure 1B The DSC diagram shown in;

[0064] (d) Form I is characterized by having an endothermic onset at 125.6 ± 2 °C as determined by DSC;

[0065] (e) Form I is characterized by having an endothermic peak at 130.2±2° C. as determined by DSC;

[0066] (f) Form I has substantially Figure 1B The TGA diagram shown in;

[0067] (g) Form I has a weight loss of about 0.35% or 0.35% ± 0.05% between 105°C and 145°C as determined by TGA; and

[0068] (h) Form I has substantially Figure 1C The GVS diagram shown in .

[0069] Form II

[0070] 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. Samples and single crystal data for Form II are provided in Table 2-D.

[0071] Table 2-D

[0072]

[0073]

[0074] 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%.

[0075] In some embodiments, Form II has substantially Figure 2A The XRPD pattern shown in .

[0076] The 2-theta angles and relative peak intensities of Form II observed using XRPD are shown in Table 2C.

[0077] Table 2C

[0078]

[0079]

[0080] In some embodiments, the crystalline Form II has a Figure 2A In some embodiments, the peak assignments listed herein (including crystalline form II) may vary by about ± 0.6 degrees, ± 0.4 degrees, ± 0.2 degrees, or ± 0.1 degrees 2-θ.

[0081] In some embodiments, crystalline Form II has an XRPD pattern comprising peaks at 2-θ angles of 5.9 ± 0.2, 11.5 ± 0.2, 11.7 ± 0.2, 17.9 ± 0.2, and 19.1 ± 0.2 degrees. In some embodiments, crystalline Form II has an XRPD pattern comprising additional peaks at 2-θ angles of 7.8 ± 0.2 and 16.2 ± 0.2 degrees. In some embodiments, crystalline Form II has an XRPD pattern further comprising additional peaks at 2-θ angles of 13.1 ± 0.2 and 19.8 ± 0.2 degrees. In some embodiments, crystalline Form II has a molecular weight comprised between 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, 20.3±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 In some embodiments, the XRPD pattern may also be calculated from single crystal data acquired for Form II. It is understood that, for example, due to the presence of impurities, solvents, or other polymorphs or amorphous forms in the test sample, peaks at 2-θ angles of 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 degrees may be observed in the XRPD pattern. Figure 2A Additional peaks beyond those shown in or as provided in Table 2C.

[0082] In some embodiments, Form II has substantially Figure 2BThe differential scanning calorimetry (DSC) graph shown in . In some embodiments, Form II is characterized as having an endothermic onset at about 154.9° C. as determined by DSC. In some embodiments, Form II is characterized by having an endothermic onset as determined by DSC 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). In some embodiments, Form II is characterized as having an endothermic peak at about 155.8 °C as determined by DSC. In some embodiments, Form II is characterized by an endothermic peak 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 determined by DSC.

[0083] In some embodiments, Form II has substantially Figure 2B 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%) weight loss before degradation as determined by TGA.

[0084] In some embodiments, Form II has substantially Figure 2C The gravimetric vapor sorption (GVS) diagram shown in .

[0085] In some embodiments, when stored for 8 days at two different temperature / RH conditions (40°C / 75% RH and 25°C / 97% RH), Form II exhibits substantially no change or no change as determined by XRPD. In some embodiments, when stored for a period of 7 days at 40°C / 75% RH, Form II exhibits substantially no change or no change as determined by XRPD. In some embodiments, when stored for a period of 7 days at 25°C / 97% RH, Form II exhibits substantially no change or no change as determined by XRPD.

[0086] In some embodiments, Form II exhibits substantially no change or no change before and after GVS measurement as determined by XRPD, such as Figure 2E as shown in .

[0087] In some embodiments, Form II is 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde having 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% relative to other forms, and the purity remains substantially unchanged or unchanged when stored for 8 days at two different temperature / RH conditions (40°C / 75% RH and / or 25°C / 97% RH) as determined by HPLC.

[0088] Form II shows high solubility and high stability, which is ideal for pharmaceutical use. These characteristics can provide, for example, improved administration consistency, improved release of the drug during administration, longer shelf life and easier storage and packaging advantages. Form II has high solubility in a variety of common solvents. In some embodiments, 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, 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.

[0089] Form II exhibits high stability in a variety of solvents. In some embodiments, as determined by HPLC, Form II does not show visual signs of degradation after being stored in a solvent for 24 hours, 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, as determined by HPLC, Form II does not show visual signs of degradation after being stored in a solvent for 24 hours, wherein the solvent is selected from the group consisting of methanol, acetone, DMSO, acetonitrile and THF. In some embodiments, as determined by HPLC, Form II initially shows visual signs of degradation in a solvent, but does not show further degradation after being stored in a solvent for 24 hours, wherein the solvent is selected from the group consisting of ethanol, tert-butyl methyl ether and isopropyl acetate.

[0090] In some embodiments of 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) apply:

[0091] (a) Form II has an XRPD pattern comprising peaks at 2-θ angles of 5.9±0.2, 11.5±0.2, 11.7±0.2, 17.9±0.2, and 19.1±0.2 degrees; an XRPD pattern comprising additional peaks at 2-θ angles of 7.8±0.2 and 16.2±0.2 degrees; an XRPD pattern further comprising additional peaks at 2-θ angles of 13.1±0.2 and 19.8±0.2 degrees; or 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 degrees; 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, 20.3±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, an XRPD pattern of peaks at 2-theta angles of 4.2±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 degrees;

[0092] (b) Form II has substantially Figure 2A The XRPD pattern shown in;

[0093] (c) Form II has substantially Figure 2B The DSC diagram shown in;

[0094] (d) Form II is characterized by having an endothermic onset at 154.9 ± 2 °C as determined by DSC;

[0095] (e) Form II is characterized by having an endothermic peak at 155.8 ± 2 °C as determined by DSC;

[0096] (f) Form II has substantially Figure 2B The TGA diagram shown in;

[0097] (g) Form II has less than 0.1% weight loss before degradation as determined by TGA; and

[0098] (h) Form II has substantially Figure 2C The GVS diagram shown in

[0099] Form III

[0100] The crystalline Form III 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 baseline characterized to understand the properties of the solid. In some embodiments, the crystalline Form III is designated as an unstable hemi-dioxane solvate as determined by XRPD and Figure 3C As shown in , it converts to crystalline Form II after storage at 40°C / 75% RH for 7 days. 1 According to HNMR, the 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 and 0.5 mol equivalent of 1,4-dioxane.

[0101] In some embodiments, Form III has substantially Figure 3A The XRPD pattern shown in .

[0102] The 2-theta angles and relative peak intensities of Form III observed using XRPD are shown in Table 4-A.

[0103] Table 4-A

[0104]

[0105]

[0106] In some embodiments, crystalline Form III has a Figure 3A In some embodiments, the peak distribution (including crystalline form III) may vary from about ± 0.6 degrees, ± 0.4 degrees, ± 0.2 degrees, or ± 0.1 degrees 2-θ.

[0107] In some embodiments, crystalline Form III has an XRPD pattern comprising peaks at 2-θ angles of 5.6±0.2, 7.2±0.2, 11.2±0.2, 14.9±0.2, and 16.8±0.2 degrees. In some embodiments, crystalline Form III has an XRPD pattern comprising additional peaks at 2-θ angles of 16.4±0.2, 18.7±0.2, 21.9±0.2, 22.5±0.2, and 22.7±0.2 degrees. In some embodiments, crystalline Form III has a range of about 5.6±0.2, about 6.0±0.2, about 7.2±0.2, about 11.2±0.2, about 13.9±0.2, about 14.9±0.2, about 15.5±0.2, about 15.8±0.2, about 16.4±0.2, about 16.8±0.2, about 17.0±0.2, about 17.7±0.2, about 18.7±0.2, about 18.9±0.2, about 19.5±0.2, about 20.1±0.2, about 20.6±0.2, about 21.0±0.2, about 21. 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 degrees. It is understood that peaks in the XRPD pattern other than those in the XRPD pattern may be observed, for example, due to the presence of impurities, solvents, or other polymorphs or amorphous forms in the test sample. Figure 3A Additional peaks beyond those shown in or as provided in Table 4-A.

[0108] In some embodiments, Form III has substantially Figure 3B In some embodiments, the feature of Form III is as determined by DSC, having an endothermic starting point at about 81.7 ± 4 ° C, an endothermic starting point with two events at about 112.8 ± 4 ° C, or an endothermic starting point at about 154.4 ± 4 ° C, or any combination thereof. In some embodiments, the feature of Form III is as determined by DSC, 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.

[0109] In some embodiments, Form III has substantially Figure 3B. In some embodiments, 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%) between 45°C and 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%) between 101°C and 189°C as determined by TGA.

[0110] In some embodiments of 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)-(g) apply:

[0111] (a) Form III has an XRPD pattern comprising peaks at 2-theta angles of 5.6±0.2, 7.2±0.2, 11.2±0.2, 14.9±0.2, and 16.8±0.2 degrees; and (b) Form III has an XRPD pattern comprising peaks at 2-theta 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, and 19.8±0.2 degrees. or an XRPD pattern comprising peaks at 2-θ 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, an XRPD pattern of peaks at 2-theta angles of 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 degrees;

[0112] (b) Form III has substantially Figure 3A The XRPD pattern shown in;

[0113] (c) Form III has substantially Figure 3B The DSC diagram shown in;

[0114] (d) Form III is characterized by having an endothermic onset at about 81.7±4.0°C, an endothermic onset at about 112.8±4.0°C, or an endothermic onset at about 154.4±4.0°C, or any combination thereof, as determined by DSC;

[0115] (e) 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 determined by DSC;

[0116] (f) Form III has substantially Figure 3B The TGA graph shown in ; and

[0117] (g) Form III has a weight loss between 45°C and 101°C of about 3.3% or 3.3%±0.5% and / or a weight loss between 101°C and 189°C of about 5.2% or 5.2%±0.5% as determined by TGA.

[0118] Composition

[0119] Also provided herein are compositions containing polymorphs described herein, such as Form I, Form II, Form III, or mixtures thereof. In some embodiments, the composition contains Form I. In some embodiments, the composition contains Form II. In some embodiments, the composition contains Form III. In some embodiments, the composition contains a mixture of Form I and Form II. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0120] In some embodiments, a composition containing 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. 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 an amorphous or non-crystalline form 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.

[0121] In some embodiments of compositions containing 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%, 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 I. In some embodiments of the compositions containing 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% by weight, at least about 0.3% by weight, at least about 0.5% by weight, at least about 0.8% 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 90% by weight, at least about 100% by weight, at least about 150% by weight, at least about 160% by weight, at least about 170% by weight, at least about 180% by weight, at least about 190% by weight, at least about 200% by weight, at least about 210% by weight, at least about 230% by weight, at least about 240% by weight, at least about 250% by weight, at least about 260% by weight, at least about 270% by weight, at least about 280% by weight, at least about 290% by weight, at least about 300% by weight, at least about 310% by weight, at least about 320% by weight, at least about 340% by weight, at least about 360% by weight, at least about 380% by weight, at least about 390% by weight, at least about 400% by weight, 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 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde is in Form I.

[0122] In some embodiments, a composition containing 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. 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 an amorphous or non-crystalline form 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.

[0123] In some embodiments of compositions containing 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 Form II. In some embodiments of the compositions containing 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 90 wt %, at least about 100 wt %, at least about 120 wt %, at least about 140 wt %, at least about 150 wt %, at least about 160 wt %, at least about 170 wt %, at least about 180 wt %, at least about 190 wt %, at least about 200 wt %, at least about 210 wt %, at least about 230 wt %, at least about 240 wt %, at least about 250 wt %, at least about 260 wt %, at least about 270 wt %, at least about 280 wt %, at least about 290 wt %, at least about 300 wt %, at least about 310 wt %, at least about 310 wt %, at least about 320 wt %, at least about 330 wt %, at least about 340 wt %, at least about 350 wt %, at least about 360 wt %, at least about 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 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde is in Form II.

[0124] In some embodiments, a composition containing 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 provided. In some embodiments, the composition is 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 composition is substantially free of an amorphous or non-crystalline form 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.

[0125] In some embodiments of compositions containing 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 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 III. In some embodiments of the compositions containing 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 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 90 wt %, at least about 100 wt %, at least about 120 wt %, at least about 140 wt %, at least about 150 wt %, at least about 160 wt %, at least about 170 wt %, at least about 180 wt %, at least about 190 wt %, at least about 200 wt %, at least about 210 wt %, at least about 230 wt %, at least about 240 wt %, at least about 250 wt %, at least about 260 wt %, at least about 270 wt %, at least about 280 wt %, at least about 290 wt %, at least about 300 wt %, at least about 310 wt %, at least about 310 wt %, at least about 320 wt %, at least about 330 wt %, at least about 340 wt %, at least about 350 wt %, at least about 360 wt %, at least about 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 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde is in Form III.

[0126] In some embodiments, a tablet or capsule containing one or more of the crystalline forms described herein (e.g., Form I, II, III, or mixtures thereof) and one or more pharmaceutically acceptable carriers is provided. In some embodiments, a tablet or capsule 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 is provided. In some embodiments, a tablet or capsule 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 is provided. In some embodiments, a tablet or capsule 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 is provided. In some embodiments, a tablet or capsule containing a mixture of Form I and 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 is provided.

[0127] Preparation method

[0128] Form I

[0129] 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, the method comprising: (a) mixing 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 of 0°C to 30°C, 0°C to 20°C, 0°C to 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, 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 the formation of a suspension. 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.

[0130] 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, the method comprising: (a) mixing 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 a heat / cool cycle. 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:H 2In some embodiments, the heat / cold cycle comprises a cycle between room temperature and a temperature higher than room temperature. In some embodiments, the heat / cold cycle comprises a cycle between room temperature and a temperature higher than room temperature of 30°C to 70°C, 40°C to 60°C, or about 50°C. In some embodiments, the duration of each condition of the heat / cold cycle 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 evaporating the mixture of step (a). In some embodiments where a two-phase layer is formed in step (a), the method further comprises sonicating the mixture of step (a), wherein the sonication duration is 0.5 hours to 3 hours or about 1.5 hours. In some embodiments where a jelly 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 jelly 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 sonication duration is 2 hours to 6 hours, 3 hours to 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 solids produced in step (b) after 5 to 9 days, 6 to 8 days, or about 7 days.

[0131] 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, the method 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) grinding the mixture of step (a). In some embodiments, step (b) comprises grinding the mixture of step (a) for a period of 1 hour to 3 hours, 1.5 hours to 2.5 hours, or about 2 hours. In some embodiments, step (b) includes grinding the mixture of step (a) at a speed of 3000rpm to 7000rpm, 4000rpm to 6000rpm or about 5000rpm. In some embodiments in which a jelly is formed in step (a), the method further includes vacuum drying the jelly. In some embodiments in which a jelly is formed in step (a), the method further includes vacuum drying the jelly at a temperature of 10°C higher or lower than room temperature, 5°C higher or lower than room temperature, 3°C higher or lower than room temperature or about room temperature. In some embodiments in which a jelly is formed in step (a), the method further includes vacuum drying the jelly for 5 to 15 hours, 8 to 12 hours or about 10 hours. In some embodiments in which a jelly is formed in step (a), the method further includes vacuum drying the jelly. In some embodiments in which a jelly is formed in step (a), the method further includes vacuum drying the jelly. In some embodiments in which a jelly is formed in step (a), the method further includes vacuum drying the jelly at a temperature of 10°C higher or lower than room temperature, 5°C higher or lower than room temperature, 3°C higher or lower than room temperature or about room temperature. In some embodiments where a jelly is formed in step (a), the method further comprises vacuum drying the jelly for 1 to 5 days, 2 to 4 days, or about 3 days. In some embodiments involving brittle materials, the method further comprises vacuum drying a mixture containing brittle materials. In some embodiments involving brittle materials, the method further comprises vacuum drying a mixture containing brittle materials at a temperature of 10°C higher or lower than room temperature, 5°C higher or lower than room temperature, 5°C higher or lower than room temperature, 3°C higher or lower than room temperature, or about room temperature. In some embodiments involving brittle materials, the method further comprises vacuum drying a mixture containing brittle materials 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-methoxyethane-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). Form I can also be prepared using the procedures provided in Examples 1 and / or 5 herein.

[0132] 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, 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 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 non-polar 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 between cyclohexane and 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 higher or lower than room temperature, 5 ° C higher or lower than room temperature, 5 ° C higher or lower than room temperature, 3 ° C higher or lower than room temperature or about room temperature. In some embodiments, the method further comprises (5) concentrating the filtrate of step (4) under reduced pressure and (6) recrystallizing the mixture of step (5) with a second solvent at a third temperature to obtain a crystalline solid. In some embodiments, the second solvent comprises 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, wherein the volume ratio between petroleum ether and 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 higher or lower than room temperature, 5°C higher or lower than room temperature, 5°C higher or lower than room temperature, 3°C higher or lower than room temperature or about room temperature.

[0133] Form I can also be prepared using the procedures provided in Examples 1 and / or 5 herein.

[0134] Form II

[0135] 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: (1) mixing 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 of 0°C to 30°C, 0°C to 20°C, 0°C to 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, 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 the formation of a suspension. In some embodiments, the method further comprises filtering the suspension in step (2). In some embodiments, the method further comprises filtering the suspension in step (2) after 5 to 9 days, 6 to 8 days, or about 7 days.

[0136] 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) mixing 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 a heat / cool cycle. In some embodiments, the solvent is selected from the group consisting of 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, a mixture of 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 selected from the group consisting of 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), DMF and water mixture (1:1 v / v), NMP and water mixture (1:1 v / v), diethyl ether and n-heptane mixture (1:1 v / v), ethyl acetate and n-heptane mixture (1:1 v / v). In some embodiments, the heat / cold cycle comprises a mixture of 1:1 v / v, ...In some embodiments, the hot / cold cycle comprises a cycle between room temperature and a temperature above room temperature of 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 in which a two-phase layer is formed in step (a), the method further comprises sonicating the mixture of step (a) and / or evaporating the mixture of step (a). 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 sonication duration is 0.5 hours to 3 hours or about 1.5 hours. In some embodiments in which a jelly 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 jelly 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 sonication duration is 2 hours to 6 hours, 3 hours to 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.

[0137] 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) mixing 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 aliquots 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 in which a two-phase layer is formed in step (a), the method further comprises adding an additional solvent to the mixture of step (c) until a solution is formed, cooling the solution and stirring the solution at the cooling temperature. In some embodiments in which a two-phase layer is formed in step (b) and an additional solvent is added to the mixture of step (c) until a solution is formed, the temperature at 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 in which a two-phase layer is formed in step (b), the method further comprises adding an additional solvent to the mixture of step (c) until a solution is formed and cooling the solution at a rate of 0.08°C / min to 0.12°C / min, 0.09°C / min to 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: 85v / v), ethanol and water (1: 1 and 85: 15v / v), 1-propanol and water (1: 1 and 15: 85v / v), 2-propanol and water (1: 1 and 15: 85v / v), 2-butanol and water (20: 80v / v) and acetone and water (1: 1v / v). In some embodiments, the method also includes adding another solvent. In some embodiments, the method also includes adding an anti-solvent to promote the formation of a suspension. In some embodiments, the method also includes filtering the suspension in step (c). In some embodiments, the method further comprises filtering the suspension in step (c) after 13 to 17 days, 14 to 16 days, or about 15 days.In some embodiments, the method further comprises filtering the suspension in step (c) after 16 to 20 days, 17 to 19 days, or about 18 days.

[0138] 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) 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) grinding the mixture of step (a). In some embodiments, step (b) comprises grinding the mixture of step (a) for an amount of time of 1 hour to 3 hours, 1.5 hours to 2.5 hours, or about 2 hours. In some embodiments, step (b) includes grinding the mixture of step (a) at a speed of 3000rpm to 7000rpm, 4000rpm to 6000rpm or about 5000rpm. In some embodiments in which a jelly is formed in step (a), the method further includes vacuum drying the jelly. In some embodiments in which a jelly is formed in step (a), the method further includes vacuum drying the jelly at a temperature of 10°C higher or lower than room temperature, 5°C higher or lower than room temperature, 3°C higher or lower than room temperature, or about room temperature. In some embodiments in which a jelly is formed in step (a), the method further includes vacuum drying the jelly for 5 to 15 hours, 8 to 12 hours, or about 10 hours. In some embodiments in which a jelly is formed in step (a), the method further includes vacuum drying the jelly. In some embodiments in which a jelly is formed in step (a), the method further includes vacuum drying the jelly. In some embodiments in which a jelly is formed in step (a), the method further includes vacuum drying the jelly at a temperature of 10°C higher or lower than room temperature, 5°C higher or lower than room temperature, 3°C higher or lower than room temperature, or about room temperature. In some embodiments where a jelly is formed in step (a), the method further comprises vacuum drying the jelly for 1 to 5 days, 2 to 4 days, or about 3 days. In some embodiments involving brittle materials, the method further comprises vacuum drying a mixture containing brittle materials. In some embodiments involving brittle materials, the method further comprises vacuum drying a mixture containing brittle materials at a temperature of 10°C higher or lower than room temperature, 5°C higher or lower than room temperature, 3°C higher or lower than room temperature, or about room temperature. In some embodiments involving brittle materials, the method further comprises vacuum drying a mixture containing brittle materials 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, 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).

[0139] 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: (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) 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 a 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 50°C to 110°C, 60°C to 100°C, 70°C to 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 higher or lower than room temperature, 5°C higher or lower than room temperature, 3°C higher or lower than room temperature or about room temperature. In some embodiments where the first temperature is 85°C to 90°C, step (5) also includes adding the seeds of crystalline form II to the mixture of step (4) at an intermediate temperature between the first temperature and the second temperature. In some embodiments where the seeds of Form II 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 the seeds of Form II 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 and 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 in step (7) under air or under vacuum. In some embodiments, the method further comprises drying the solid in step (7) at less than 50°C.In some embodiments, the method further comprises drying the solid in step (7) at a temperature of 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, the method further comprises drying the solid in step (7) under air or under vacuum for 1 to 5 days, 2 to 4 days, or about 3 days.

[0140] Form II can also be prepared using the procedures provided in Examples 2 and / or 5.

[0141] Form III

[0142] In some embodiments, 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 is provided, the method comprising: (1) mixing 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 of 4°C to 6°C, 4.5°C to 5.5°C, or about 5°C. In some embodiments, the solvent is a mixture of 1,4-dioxane and water. In some embodiments, the solvent is 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 the formation of a suspension. In some embodiments, the method further comprises filtering the suspension in step (2). In some embodiments, the method further comprises filtering the suspension in step (2) after 5 to 9 days, 6 to 8 days, or about 7 days.

[0143] Form III can also be prepared using the procedures provided in Examples 4 and / or 5.

[0144] How to use

[0145] The crystalline forms and compositions provided herein are useful for treating or preventing a disease or disorder in an individual or subject.

[0146] When used in a prophylactic manner, the crystalline forms and compositions disclosed and / or described herein can prevent a disease or condition from developing or reduce the extent of a disease or condition that may develop in an individual or subject at risk of developing the disease or condition.

[0147] Without being bound by theory, the crystalline forms and compositions provided are believed to work by inhibiting myosin. The inhibition may reduce the number of independent myosin heads that interact with actin filaments, thereby reducing the amount of contraction. Reducing myocardial contraction is very important for treating heart disease caused by excessive contraction. In some embodiments, a method for treating or preventing heart disease in an individual or subject is provided, the method comprising administering a crystalline form or composition provided herein to an individual or subject in need. In some embodiments, a method for treating or preventing heart disease in a subject in need is provided, the method comprising administering a therapeutically effective amount of a crystalline form or composition provided herein to the subject. In some embodiments, a method for treating heart disease in a subject in need is provided, the method comprising administering a therapeutically effective amount of a crystalline form or composition provided herein to the subject. In some embodiments, a method for treating a determined or confirmed heart disease in a subject in need is provided, the method comprising administering a therapeutically effective amount of a crystalline form or composition provided herein to the subject. In some embodiments, a method for treating a determined or confirmed heart disease in a subject in need is provided, the method comprising administering a therapeutically effective amount of a crystalline form or composition provided herein to the subject. In some embodiments, a method for preventing heart disease in a subject in need is provided, the method comprising administering a therapeutically effective amount of a crystalline form or composition provided herein to the subject.

[0148] Also provided herein is the use of a crystalline form or composition provided herein in the manufacture of a drug for treating a subject's heart disease. In some aspects, a crystalline form as described herein is provided, which is used in a method for treating a human or animal body by therapy. In some embodiments, provided herein is a crystalline form for treating a human or animal body by therapy, such as form I, II or III or a mixture thereof, or a composition thereof. In some embodiments, provided herein is a crystalline form for treating or preventing heart disease, such as form I, II or III or a mixture thereof, or a composition thereof. In some embodiments, provided herein is a crystalline form for treating heart disease, such as form I, II or III or a mixture thereof, or a composition thereof. In some embodiments, provided herein is a crystalline form for treating a confirmed or confirmed heart disease, such as form I, II or III or a mixture thereof, or a composition thereof. In other embodiments, provided herein is a crystalline form for preventing heart disease, such as form I, II or III or a mixture thereof, or a composition thereof. In some embodiments, provided herein is a crystalline form for treating a disease or illness associated with HCM, such as form I, II or III or a mixture thereof, or a composition thereof. In some embodiments, provided herein are crystalline forms for treating diseases or disorders associated with secondary left ventricular wall thickening, such as Form I, II or III or mixtures thereof, or compositions thereof. In some embodiments, provided herein are crystalline forms for improving symptoms associated with heart disease, such as Form I, II or III or mixtures thereof, or compositions thereof. In other embodiments, provided herein are crystalline forms for reducing the risk of symptoms associated with heart disease, such as Form I, II or III or mixtures thereof, or compositions thereof. In other embodiments, provided herein are crystalline forms for treating diseases or disorders associated with small left ventricular chambers, chamber occlusions, high dynamic left ventricular contractions, obstructed left ventricular blood outflow, cardiac hypertrophy, small stroke volume, impaired left ventricular diastole, high left ventricular filling pressures, myocardial ischemia or cardiac fibrosis, such as Form I, II or III or mixtures thereof, or compositions thereof. In certain embodiments, provided herein are crystalline forms for treating diseases or disorders associated with left ventricular small chambers and chamber occlusion, hyperdynamic left ventricular contraction, myocardial ischemia or cardiac fibrosis, such as Form I, II or III, or mixtures thereof, or compositions thereof. In some embodiments, provided herein are crystalline forms for treating muscular dystrophy, such as Form I, II or III, or mixtures thereof, or compositions thereof. In some embodiments, provided herein are crystalline forms for treating glycogen storage diseases, such as Form I, II or III, or mixtures thereof, or compositions thereof.In other embodiments, provided herein are crystalline forms for modulating cardiac myometrium, such as inhibiting cardiac myometrium, such as forms I, II or III or mixtures thereof, or compositions thereof. In other embodiments, provided herein are crystalline forms for enhancing cardiac myosin, such as forms I, II or III or mixtures thereof, or compositions thereof.

[0149] In some embodiments, the subject is a mammal. In some embodiments, the subject is a mouse, rat, dog, cat, pig, sheep, horse, cattle or human. In some embodiments, the subject is human. In some embodiments, the subject suffers from a confirmed or diagnosed heart disease. In some embodiments, the subject suffers from a confirmed or diagnosed hypertrophic cardiomyopathy (HCM). In some embodiments, the subject is at risk of suffering from 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 a mutation in myosin heavy chain beta (MHC-β), cardiac troponin T (cTnT), tropomyosin alpha-1 chain (TPM1), cardiac myosin binding protein C (MYBPC3), cardiac troponin I (cTnI), myosin essential light chain (ELC), titin (TTN), myosin regulatory light chain 2 ventricular / 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 a mutation in MHC-β. In some embodiments, the subject has established or confirmed hypertrophic cardiomyopathy, but the genetic cause has not been determined.

[0150] In some embodiments, the subject has a high risk of progressive symptoms. In some embodiments, the subject has a high 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 suitable for surgical intervention or percutaneous ablation to treat heart disease.

[0151] In some embodiments, the heart disease is hypertrophic cardiomyopathy (HCM). In some embodiments, the heart disease is obstructive HCM. In some embodiments, the heart disease is non-obstructive HCM. In some embodiments, HCM is associated with sarcomeric mutations. In some embodiments, HCM is associated with non-sarcomeric mutations. In some embodiments, the heart disease is obstructive or non-obstructive HCM caused by sarcomeric and / or non-sarcomeric mutations. In some embodiments, the sarcomeric mutation is a mutation in myosin heavy chain β (MHC-β), cardiac troponin T (cTnT), tropomyosin α-1 chain (TPM1), cardiac myosin binding protein C (MYBPC3), cardiac troponin I (cTnI), myosin essential light chain (ELC), titin (TTN), myosin regulatory light chain 2 ventricular / 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).

[0152] In some embodiments, provided herein is a method for treating a disease or disorder associated with HCM, the method comprising administering to an individual or subject in need thereof a crystalline form or composition provided herein. In some embodiments, the disease or disorder is Fabry's Disease, Danon Disease, mitochondrial cardiomyopathy, or Noonan Syndrome.

[0153] Also provided herein 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.

[0154] In some embodiments, the heart disease is heart failure with preserved ejection fraction (HFpEF). In some embodiments, the heart disease is diastolic dysfunction. In some embodiments, the heart disease is cardiomyopathy. In some embodiments, the heart disease is primary or secondary restrictive cardiomyopathy. In some embodiments, the heart disease is a disorder or symptom caused by coronary artery disease. In some embodiments, the heart disease is myocardial infarction or angina pectoris. In some embodiments, the heart disease is left ventricular outflow tract obstruction. In some embodiments, the heart disease is hypertensive heart disease. In some embodiments, the heart disease is congenital heart disease. In some embodiments, the heart disease is myocardial ischemia and / or coronary heart disease. In some embodiments, the heart disease is diabetic heart disease. In other embodiments, the heart disease is congestive heart failure. In some embodiments, the heart disease is right heart failure. In other embodiments, the heart disease is cardiorenal syndrome. In some embodiments, the heart disease is infiltrative cardiomyopathy. In some embodiments, the heart disease is heart aging or diastolic dysfunction caused by aging or a disorder related thereto. In some embodiments, the heart disease is left ventricular hypertrophy and / or left ventricular concentric remodeling or a disorder related thereto.

[0155] In some embodiments, a method for treating a disease or disorder associated with secondary left ventricular wall thickening in an individual or subject is provided, the method comprising administering a crystalline form or composition provided herein to an individual or subject in need thereof. In some embodiments, the disease is hypertension, valvular heart disease (aortic stenosis, mitral regurgitation), metabolic syndrome (diabetes, obesity), end-stage renal disease, scleroderma, sleep apnea, amyloidosis, Fabry disease, Friedreich Ataxia, Danon disease, Noonan syndrome, or Pompe disease.

[0156] Also provided herein 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.

[0157] In some embodiments, a method of improving a symptom associated with heart disease in a subject is provided, the method comprising administering to an individual or subject in need thereof a crystalline form or composition provided herein, wherein the symptom is selected from one or more of poor or diminished cardiac elasticity, poor or diminished left ventricular relaxation during diastole, abnormal left atrial pressure (e.g., abnormally high left atrial pressure), paroxysmal or permanent atrial fibrillation, increased left atrial and pulmonary capillary wedge pressure, increased left ventricular diastolic pressure, syncope, diastolic ventricular relaxation, ventricular fibrosis, left ventricular hypertrophy, left ventricular mass, increased left ventricular wall thickness, left ventricular mid-cavity obstruction, increased mitral valve systolic anterior motion, left ventricular outflow tract obstruction, chest pain, exertional dyspnea, presyncope, abnormal exercise capacity, and fatigue.

[0158] In some embodiments, a method of reducing the risk of a symptom associated with heart disease in a subject is provided, the method comprising administering to an individual or subject in need thereof a crystalline form or composition provided herein, wherein the symptom is one or more selected from sudden cardiac death, poor or reduced cardiac elasticity, poor or reduced diastolic left ventricular relaxation, abnormal left atrial pressure (e.g., abnormally high left atrial pressure), paroxysmal or permanent atrial fibrillation, increased left atrial and pulmonary capillary wedge pressure, increased left ventricular diastolic pressure, syncope, diastolic ventricular relaxation, ventricular fibrosis, left ventricular hypertrophy, left ventricular mass, increased left ventricular wall thickness, left ventricular mid-cavity obstruction, increased systolic anterior motion of the mitral valve, left ventricular outflow tract obstruction, chest pain, exertional dyspnea, presyncope, abnormal exercise capacity, and fatigue.

[0159] In some embodiments, a method of treating a disease or condition associated with a small left ventricular chamber, chamber occlusion, hyperdynamic left ventricular contraction, obstructed left ventricular blood outflow, cardiac hypertrophy, small stroke volume, impaired left ventricular relaxation, high left ventricular filling pressure, myocardial ischemia, or cardiac fibrosis in an individual or subject is provided, the method comprising administering to the individual or subject in need thereof a crystalline form or composition provided herein.

[0160] In some embodiments, a method of treating a disease or condition associated with left ventricular small chambers and chamber occlusion, hyperdynamic left ventricular contraction, myocardial ischemia, or cardiac fibrosis in an individual or subject is provided, the method comprising administering to the individual or subject in need thereof a crystalline form or composition provided herein.

[0161] Also provided herein 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 small chambers and chamber occlusion, hyperdynamic left ventricular contraction, myocardial ischemia, or cardiac fibrosis.

[0162] In some embodiments, a method for treating a muscular dystrophy (e.g., Duchenne muscular dystrophy) in an individual or subject is provided, the method comprising administering to an 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 a muscular dystrophy (e.g., Duchenne muscular dystrophy).

[0163] In some embodiments, a method of treating a glycogen storage disease in an individual or subject is provided, the method comprising administering to an 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 a glycogen storage disease.

[0164] Also provided is a method for regulating cardiac myometrium in an individual or subject, the method comprising administering to an individual or subject in need thereof a therapeutically effective amount of at least one chemical entity as described herein. In some embodiments, a method for inhibiting cardiac myometrium is provided, the method comprising contacting the cardiac myometrium with at least one chemical entity as described herein, e.g., a crystalline form or composition provided herein. In addition, provided herein is the use of at least one chemical entity as described herein, e.g., a crystalline form or composition provided herein, in the manufacture of a medicament for inhibiting cardiac myometrium in an individual or subject.

[0165] Also provided is a method for enhancing cardiac myosin in an individual or subject, the method comprising administering to an individual or subject in need thereof a therapeutically effective amount of at least one chemical entity as described herein, e.g., a crystalline form or composition provided herein. In addition, provided herein is the use of at least one chemical entity as described herein, e.g., a crystalline form or composition provided herein, in the manufacture of a medicament for enhancing cardiac myosin in an individual or subject.

[0166] In some embodiments, the methods provided herein also include monitoring the effectiveness of treatment. Examples of indicators include, but are not limited to, improvements in one or more of the following: New York Heart Association (NYHA) functional classification, exercise capacity, cardiac elasticity, diastolic left ventricular relaxation, left atrial pressure, paroxysmal or permanent atrial fibrillation, left atrial and pulmonary capillary wedge pressure, left ventricular diastolic pressure, syncope, diastolic ventricular relaxation, ventricular fibrosis, left ventricular hypertrophy, left ventricular mass, left ventricular wall thickness, left ventricular mid-cavity obstruction, mitral valve systolic forward motion, left ventricular outflow tract obstruction, chest pain, exertional dyspnea, presyncope, abnormal exercise capacity and fatigue. These indicators can be monitored by techniques known in the art, including self-reporting; ECG, including dynamic ECG; echocardiography; cardiac MRI; CT; biopsy; cardiopulmonary exercise testing (CPET); and activity recording examination.

[0167] In some embodiments, the crystalline form or composition described therein reduces the contractility of cardiomyocytes. In some embodiments, the crystalline form or composition reduces the contractility of cardiomyocytes by more than 40%, for example, more than 45%, 50%, 60%, 70%, 80% or 90%. In some embodiments, the crystalline form or composition reduces the contractility of cardiomyocytes by 40%-90%, for example, 40%-80%, 40-70%, 50%-90%, 50%-80% or 50%-70%. In some embodiments, the crystalline form or composition does not significantly change the calcium transient in cardiomyocytes. In some embodiments, the crystalline form or composition reduces the ATPase activity in cardiomyocytes. Methods for measuring contractility, ATPase activity and calcium transients are known in the art, for example, by calcium labeling, electrophysiological recording and microscopic imaging. In some embodiments, the crystalline form or composition does not significantly inhibit or induce cytochrome P450 (CYP) protein.

[0168] In some embodiments, provided herein are crystalline forms or compositions wherein the elimination half-life in humans (t 1 / 2 ; calculated as ln(2) / k, where the elimination rate constant k is 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 curve)≤30 hours. In some embodiments, 10 hours in humans≤t 1 / 2 ≤30 hours. In some embodiments, t 1 / 2 Between about 10 hours and about 30 hours, between about 10 hours and about 25 hours, between about 15 hours and about 30 hours, or between about 15 hours and about 25 hours. In some embodiments, t 1 / 2 About 12, 15, 18, 21, 24, 27 or 30 hours. In some embodiments, the elimination half-life of the compounds provided herein makes the compounds suitable for once-daily dosing.

[0169] In some embodiments, the subject has a thicker than normal left ventricular wall before treatment. In some embodiments, the subject has a left ventricular wall thickness greater than 15 mm before treatment, such as greater than 18 mm, 20 mm, 22 mm, 25 mm, or 30 mm. In some embodiments, the left ventricular wall thickness decreases by more than 5% after treatment, such as more than 8%, 10%, 12%, 15%, 20%, or 30%. The left ventricular wall thickness can be measured by methods known in the art, such as by echocardiography, CT scan, or cardiac MRI.

[0170] In some embodiments, the subject suffers from abnormal cardiac fibrosis before treatment. In some embodiments, abnormal cardiac fibrosis is reduced by more than 5%, such as more than 8%, 10%, 12%, 15%, 20% or 30% after treatment. Cardiac fibrosis can be measured by methods known in the art, such as by biopsy or cardiac MRI.

[0171] In some embodiments, the subject has reduced exercise capacity before treatment. In some embodiments, the subject's exercise capacity increases by more than 5%, such as greater 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 (VO 2 max). Measure CPET and VO 2 max method is well known in the art (Malhotra et al., JACC: Heart Failure, 2016, 4(8): 607-616; Guazzi 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, after treatment, VO 2 Max improvement exceeds 1 mL / kg / m 2 , for example, more than 1.2 mL / kg / m 2 , 1.4mL / kg / m 2 , 1.5mL / kg / m 2 , 1.7mL / kg / m 2 , 2mL / kg / m 2 2.2 mL / kg / m 2 , 2.5mL / kg / m 2 , 3mL / kg / m 2 3.2 mL / kg / m 2 or 3.5 mL / kg / m 2 .

[0172] In some embodiments, the subject has New York Heart Association (NYHA) functional class II, III, or IV prior to treatment. In some embodiments, the subject has New York Heart Association (NYHA) functional class III or IV prior to treatment. In some embodiments, the subject has New York Heart Association (NYHA) functional class IV prior to treatment. In some embodiments, the subject maintains the same NYHA functional class or has a decreased NYHA functional class after treatment.

[0173] In some embodiments, VO 2 Max improvement exceeds 1 mL / kg / m 2 , for example, more than 1.2 mL / kg / m 2 , 1.4mL / kg / m 2 , 1.5mL / kg / m 2 , 1.7mL / kg / m 2 or 2 mL / kg / m 2 And the subject has a reduced NYHA functional class after treatment. In some embodiments, VO 2 Max improvement of more than 2.5 mL / kg / m 2 , 3mL / kg / m 2 3.2 mL / kg / m 2 or 3.5 mL / kg / m 2 And the subjects maintained the same NYHA functional class or had a decreased NYHA functional class after treatment.

[0174] In some embodiments, the subject's daily function and / or activity level is improved after treatment. The improved daily function and / or activity level can be measured, for example, by diary or activity record examination, such as a wearable physical health monitor or activity tracker (e.g. or class monitor).

[0175] In some embodiments, after treatment the subject has one or more of: reduced shortness of breath, reduced chest pain, reduced arrhythmia burden (eg, atrial fibrillation and ventricular arrhythmias), reduced incidence of heart failure, and reduced ventricular outflow tract obstruction.

[0176] dose

[0177] The crystalline forms and compositions disclosed and / or described herein are administered in a therapeutically effective dose (e.g., a dose sufficient to provide treatment of a disease state). Although human dosage levels have not been optimized for the chemical entities described herein, in general, the daily dose is in the range of about 0.01 to 100 mg / kg body weight; in some embodiments, about 0.05 to 10.0 mg / kg body weight, and in some embodiments, about 0.10 to 1.4 mg / kg body weight. Therefore, for a person administered to 70 kg, in some embodiments, the dosage range will be about 0.7 to 7000 mg per day; in some embodiments, about 3.5 to 700.0 mg per day, and in some embodiments, about 7 to 100.0 mg per day. The amount of the chemical entity administered will depend on, for example, the subject and disease state being treated, the severity of the pain, 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 intravenous administration dose is about 5 mg to about 500 mg per day, each depending on pharmacokinetics.

[0178] The daily dose is the total amount administered in one day. The daily dose may be administered (but not limited to) every day, every other day, every week, every 2 weeks, every month, or at different intervals. In some embodiments, the administration period of the daily dose is between one day and the lifetime of the subject. In some embodiments, the daily dose is administered once a day. In some embodiments, the daily dose is administered in multiple divided doses, for example, in 2, 3, or 4 divided doses. In some embodiments, the daily dose is administered in 2 divided doses.

[0179] Administration of the crystalline forms and compositions described herein can be via any accepted mode of administration of therapeutic agents, including, but not limited to, oral, sublingual, subcutaneous, parenteral, intravenous, intranasal, topical, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, or intraocular 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.

[0180] Pharmaceutically acceptable compositions include solid, semisolid, liquid and aerosol dosage forms, such as tablets, capsules, powders, liquids, suspensions, suppositories and aerosol forms. Crystalline forms disclosed and / or described herein can also be administered in sustained or controlled release dosage forms (e.g., controlled / sustained release pills, reservoir injections, osmotic pumps or transdermal (including electrotransport) patch forms), with long-term timing and / or pulse administration at a predetermined rate. In some embodiments, the composition is provided in a unit dosage form suitable for single administration of precise doses.

[0181] The crystalline forms described herein can be administered alone or in combination with one or more conventional pharmaceutical carriers or excipients (e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, cross-linked sodium carboxymethyl cellulose, glucose, gelatin, sucrose, magnesium carbonate). If desired, the pharmaceutical composition may also contain a small amount of non-toxic auxiliary substances, such as wetting agents, emulsifiers, solubilizers, pH buffers, etc. (e.g., sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate). Typically, depending on the intended mode of administration, the pharmaceutical composition will contain about 0.005% to 95% by weight, or about 0.5% to 50% by weight of the compounds disclosed and / or described herein. The actual methods for preparing such dosage forms are known or understood by those skilled in the art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

[0182] In some embodiments, the composition will take the form of a pill or tablet, and thus the composition 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 acacia, polyvinyl pyrrolidine, gelatin, cellulose, cellulose derivatives) and a crystalline form disclosed and / or described herein. Other solid dosage forms include powders, pellets, solutions or suspensions (e.g., in propylene carbonate, vegetable oils, or triglycerides) encapsulated in a gelatin capsule.

[0183] Pharmaceutically administrable liquid compositions can be prepared, for example, by dissolving, dispersing or suspending the crystalline form disclosed and / or described herein and optional pharmaceutical additives in a carrier (e.g., water, saline, aqueous dextrose solution, glycerol, glycol, ethanol, etc.) to form a solution or suspension. Injectables can be prepared in conventional form, in the form of a liquid solution or suspension, in the form of an emulsion, or in a solid form suitable for dissolving or suspending in a liquid before injection. The percentage of crystalline form contained in such parenteral compositions depends on, for example, the physical properties of the crystalline form, the activity of the crystalline form, and the needs of the subject. However, a percentage of active ingredient of 0.01% to 10% in a solution can be used, and if the composition is a solid that will be diluted to another concentration subsequently, the percentage can be higher. In some embodiments, the composition will contain about 0.2% to 2% of the crystalline form disclosed and / or described herein in a solution.

[0184] The pharmaceutical compositions of the crystalline forms and compositions described herein can also be administered to the respiratory tract as an aerosol or solution for a nebulizer, or as a finely divided powder for insufflation, alone or in combination with an inert carrier, such as lactose. In such cases, the particles of the pharmaceutical composition can have a diameter of less than 50 microns, or in some embodiments, less than 10 microns.

[0185] In addition, pharmaceutical compositions may include a crystalline form disclosed and / or described herein and one or more additional agents, medicaments, adjuvants, and the like. Suitable drugs and agents include those described herein.

[0186] Pill Box

[0187] Also provided are articles and kits containing any crystalline form or composition provided herein. The articles may comprise a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The container may be formed of 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 inhibit a disorder described herein, and may also indicate instructions for in vivo or in vitro use.

[0188] In one aspect, provided herein are kits containing a crystalline form or composition described herein and instructions for use. The kit may contain instructions for use for treating a heart disease in an individual or subject in need thereof. The kit may additionally contain any materials or equipment that can be used to administer the crystalline form or composition, such as a vial, a syringe, or an IV bag. The kit may also contain sterile packaging.

[0189] combination

[0190] The crystalline forms and compositions described herein can be administered alone or in combination with other therapies and / or therapeutic agents useful for treating the above-mentioned conditions, diseases, or disorders.

[0191] Crystalline forms and compositions described and / or disclosed herein can be combined with one or more other therapies to treat heart disease, such as HCM or HFpEF. In some embodiments, one or more therapies include therapies that delay the progression of heart failure and attempt to prevent cardiac remodeling by downregulating neurohormonal stimulation of the heart (e.g., ACE inhibitors, angiotensin receptor blockers (ARBs), β-blockers, aldosterone receptor antagonists, or neuroendopeptidase inhibitors). In some embodiments, one or more therapies include therapies that improve cardiac function by stimulating myocardial contractility (e.g., positive inotropes, such as β-adrenergic agonists dobutamine or phosphodiesterase inhibitors milrinone). In other embodiments, 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 regulators).

[0192] The crystalline forms and compositions described and / or disclosed herein may be combined with one or more other therapies to treat HCM or HFpEF. In some embodiments, the crystalline forms and / or compositions may be combined with beta-blockers, verapamil and / or disopyramide.

[0193] Example

[0194] The following examples are provided to further aid in understanding the embodiments disclosed in this application, and presuppose an understanding of conventional methods well known to those of ordinary skill in the art to which the examples belong. The specific materials and conditions described below are intended to illustrate specific aspects of the embodiments disclosed herein, and should not be construed as limiting their reasonable scope.

[0195] The following abbreviations may be used in this article:

[0196]

[0197]

[0198]

[0199] The crystalline form of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde was characterized by various analytical techniques including XRPD, DSC, TGA, GVS, HPLC using the following procedure.

[0200] XRPD

[0201] 1. XRPD using Bruker AXSD8 Advance

[0202] XRPD diffractograms were collected on a Bruker D8 diffractometer using Cu Ka 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 and a 2.5 ° Soller slit, followed by a Lynxeye detector. The software used for data collection and analysis was Diffrac Plus XRD Commander and Diffrac Plus EVA, respectively.

[0203] Powders were used as received under ambient conditions and samples were handled as flat specimens. Samples were prepared on polished zero-background (510) silicon wafers by gently pressing onto a flat surface or fitting into a cutting cavity. Samples were rotated in their own plane.

[0204] Details of the standard Pharmorphix data collection method are as follows:

[0205] · Angular range: 2 to 42° 2θ

[0206] Step size: 0.05°2θ

[0207] Collection time: 0.5 sec / step (total collection time: 6.40 min)

[0208] When necessary, other data collection methods are used as detailed below:

[0209] · Angular range: 2 to 31° 2θ

[0210] Step size: 0.06°2θ

[0211] Collection time: 0.5 seconds / step

[0212] 2. XRPD using PANalytical Empyrean

[0213] XRPD diffractograms were collected in transmission geometry on a PANalytical Empyrean diffractometer using Cu Ka radiation (45 kV, 40 mA). The incident beam used a 0.5° slit, a 4 mm mask, and a 0.04 rad Soller slit with a focusing mirror. A PIXcel placed on the diffracted beam 3DThe detector was equipped with a receiving slit and a 0.04 Radsoller slit. The software used for data collection was X'Pert Data Collector using the X'Pert operator interface. Data were analyzed and presented using Diffrac Plus EVA or HighScorePlus.

[0214] Samples were prepared and analyzed in transmission mode in metal 96-well plates. X-ray transparent film was used between metal sheets on the metal well plates and the powder was used as received (approximately 1-2 mg).

[0215] The scanning pattern of the metal disk uses an angular scanning axis.

[0216] Details of the standard screening data collection method are:

[0217] Angular range: 2.5 to 32.0° 2θ

[0218] Step size: 0.0130°2θ

[0219] Collection time: 12.75 seconds / step (total collection time 2.07 minutes)

[0220] X-ray single crystal diffraction (XRSD) and structure refinement

[0221] XRSD data collection and structure refinement details

[0222]

[0223] DSC

[0224] DSC data were collected on a TA Instruments Q2000 equipped with a 50-position autosampler. Typically, 0.5-3 mg of each sample was placed in a pinhole aluminum pan and heated from 25°C to typically 260°C at 10°C / min. A 50 ml / min dry nitrogen purge was maintained over the sample. Modulated temperature DSC was performed using a basic heating rate of 2°C / min and a temperature modulation parameter of ±0.636°C (amplitude) every 60 seconds (cycle). The complete details for mDSC are: 1) equilibrate at -80.00°C; 2) isothermal for 5.00 minutes; 3) sampling interval 1.00 seconds / point; 4) modulate ±0.636°C every 60 seconds, and 5) heat to 260.00°C at a rate of 2.00°C / min. The instrument control software was Advantage for Q Series and Thermal Advantage, and the data were analyzed using Universal Analysis or TRIOS.

[0225] TGA

[0226] TGA data were collected on a TA Instruments Q500 TGA equipped with a 16-position autosampler. Typically, 5-10 mg of each sample was loaded into a pre-tared DSC aluminum pan and heated from ambient temperature to 350° C. at 10° C. / min. 60 ml / min of dry nitrogen was maintained over the sample. The instrument control software was Advantage for Q Series and ThermalAdvantage, and data were analyzed using Universal Analysis or TRIOS.

[0227] GVS

[0228] 1. GVS using SMSDVS Intrinsic

[0229] Adsorption isotherms were obtained using a SMS DVS Intrinsic moisture adsorption analyzer controlled by DVS Intrinsic Control software. The sample temperature was maintained at 25°C by instrument control. Humidity was controlled by mixing dry nitrogen flow and wet nitrogen flow with a total flow rate of 200 ml / min. Relative humidity was measured by a calibrated Rotronic probe (dynamic range of 1.0-100% RH) located near the sample. The weight change (mass relaxation) of the sample was continuously monitored by a microbalance (accuracy ± 0.005 mg) as a function of RH%.

[0230] Typically, 5-30 mg of sample is placed in a tared mesh stainless steel basket under ambient conditions. Samples are loaded and unloaded at 40% RH and 25°C (typical room conditions). Water adsorption isotherms are performed as outlined below (2 scans per complete cycle). Standard isotherms are performed at 25°C with 10% RH intervals in the range of 0-90% RH. Typically, double cycles (4 scans) are performed. Data analysis is performed in Microsoft Excel using the DVS analysis suite. The method for the SMSDVS Intrinsic experiment is as follows.

[0231] Method parameters for SMS DVS Intrinsic experiments

[0232] parameter value Adsorption-Scan 1 40-90 Desorption, adsorption-scan 2 90-0、0-40 Interval (RH%) 10 Scan times 4 Flow rate (ml / min) 200 Temperature(℃) 25 Stability (℃ / min) 0.2 Adsorption time (hours) 6 hours suspension Cycle times 2

[0233] 2.Hiden IGASorp

[0234] Adsorption isotherms were obtained using a Hiden IGASorp moisture sorption analyzer controlled by Isochema HISorp software. The sample temperature was maintained at 25 °C by a Grant LT ecocool 150 recirculating water bath. Humidity was controlled by mixing dry and wet nitrogen with a total flow rate of 250 ml.min -1 The relative humidity was measured by a calibrated Vaisala RH probe (dynamic range 0-95% RH) located near the sample. The weight change (mass relaxation) of the sample was continuously monitored as a function of RH% by a microbalance (accuracy ± 0.001 mg).

[0235] Typically, 20-30 mg of sample was placed in a tared mesh stainless steel basket under ambient conditions. The sample was loaded and unloaded at 40% RH and 25°C (typical room conditions). The moisture adsorption isotherm was performed as described below (scanned 2 times to obtain 1 complete cycle). The standard isotherm was performed at 25°C in the range of 0-90% RH at 10% RH intervals. Typically, a double cycle (4 scans) was performed. Data analysis was performed in Isochema HISorp 2019 software and entered into Microsoft Excel for corresponding presentation.

[0236] Method parameters for Hiden IGASorp experiment

[0237]

[0238]

[0239] HPLC

[0240] Purity analysis was performed on an Agilent HP1100 / Infinity II1260 Series system equipped with a diode array detector using OpenLAB software. Full method details are provided below.

[0241] HPLC Methods for Chemical Purity Determination

[0242]

[0243] The presented method has been successfully transferred to an Infinity II 1260 Agilent system. A minor modification was made to the method by adding an extra equilibration time of one minute at the end of the method. This extra equilibration time allowed observation of the complete gradient.

[0244] Details of the HPLC method provided

[0245]

[0246] NMR

[0247] The data were collected on a Bruker 400 MHz instrument equipped with an autosampler and controlled by an Avance NEO nanobay console. 1 H NMR spectra. Unless otherwise stated, samples were in DMSO-d 6 The experiment was prepared in solvent. The ICON-NMR configuration in Topspin software was used and the standard Bruker loading experiment ( 1 H) Acquisition of automated experiments. Offline analysis was performed using an ACD Spectrus processor.

[0248] Static stability test

[0249] For short-term (8 days or less) stability experiments, unless otherwise stated, solid materials were placed in open vials under elevated storage conditions. These conditions were achieved by using saturated salt solutions at specific temperatures in sealed containers. Storage containers were pre-equilibrated prior to sample input.

[0250] Saline solution for creating static storage conditions

[0251]

[0252]

[0253] For long-term (1 month or longer) stability experiments, unless otherwise stated, solid materials were placed in LDPE bags and sealed in HDPE containers under elevated storage conditions.

[0254] Example 1

[0255] 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)

[0256]

[0257] Step 1: Synthesis of tert-butyl 3-cyano-3-{[(3,4-difluorophenyl)methyl]amino}azetidine-1-carboxylate (1-a):

[0258]

[0259] Acetic acid (0.74 kg, 12.27 mol, 0.7 equivalent) and 1- (3,4- difluorophenyl) methylamine (3.01 kg, 21.03 mol, 1.2 equivalent) were added to a solution of tert-butyl 3-oxoazetidine-1-formate (3.0 kg, 17523.774 mmol, 1 equivalent) in i-PrOH (15.00 L) at room temperature. After stirring at room temperature for 1 hour, trimethylsilyl cyanide (1.74 kg, 17.52 mol, 1.0 equivalent) was added to the resulting mixture. The resulting mixture was stirred at room temperature overnight. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure to remove 3 / 4 solvent, then filtered, washed with n-hexane (2 × 3 L), and dried to give 3.6 kg (batch 1) 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 (batch 2) 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 as a white solid (yield: 70.59%). LRMS (ES) m / z 268 [M+H-56]. 1 H NMR (300MHz, chloroform-d) δ7.30-7.17(m,1H),7.17-7.04(m,2H),4.24(d,J=8.9Hz ,2H),3.92-3.83(m,2H),3.80(s,2H),2.20(d,J=55.9Hz,1H),1.44(s,9H).

[0260] Step 2: Synthesis of tert-butyl 3-{2-chloro-N-[(3,4-difluorophenyl)methyl]acetamido}-3-cyanoazetidine-1-carboxylate (1-b):

[0261]

[0262] 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. Chloroacetyl chloride (2.62 kg, 23.20 mol, 2.5 eq) in DCM (15 L) was then added over a period of 2 hours. The resulting mixture was stirred at room temperature overnight. The reaction was monitored by LCMS. The resulting mixture was washed with saturated NaHCO 3 (2 x 9 L) and brine (9 L), washed with anhydrous Na 2 SO 4The mixture was dried, 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 as a yellow solid (yield: 81%). 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).

[0263] 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):

[0264]

[0265] 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 eq.) in MeCN (30 L) was added (1r, 4r)-4-methylcyclohexan-1-amine (0.93 kg, 8.25 mol, 1.1 eq.) and TEA (1.90 kg, 18.76 mol, 2.5 eq.) at room temperature. The resulting mixture was stirred at 65 °C overnight. The desired product can 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) and purified by anhydrous Na 2 SO 4 The reaction mixture was dried 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 in the next step without further purification. LRMS (ES) m / z 421 [M+H-56]. 1H 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).

[0266] 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-carboxylate (1-d):

[0267]

[0268] 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 eq) in EtOH (30 L) was added AcOH (7.56 kg, 125.90 mol, 20 eq) dropwise at room temperature over a period of 30 min. 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 minutes, 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 as a pale yellow solid (yield: 75.44%). LRMS (ES) m / z 422 [M+H-56]. 1H NMR (300MHz, chloroform-d) δ7.20-7.02(m,2H),7.02-6.91(m,1H),4.89(s,2H),4.5 1(d,J=9.5Hz,2H),4.40(tt,J=12.2,3.8Hz,1H),4.00-3.91(m,4H),1.86-1 .77(m,2H),1.71(dd,J=12.1,3.5Hz,2H),1.52(d,J=12.4Hz,1H),1.44(s,9 H), 1.37-1.21 (m, 2H), 1.13 (qd, J = 12.6, 3.4Hz, 2H), 0.92 (d, J = 6.4Hz, 3H).

[0269] 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):

[0270]

[0271] 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 eq) in MeOH (34 L) was added acetyl chloride (1.68 kg, 21.36 mol, 3.0 eq) dropwise over a period of 1.5 hours at -5°C. The resulting mixture was stirred at room temperature overnight. 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 as an off-white solid (yield: 85%). LRMS (ES) m / z 378 [M+H]. 1 HNMR(300MHz,DMSO-d6)δ10.26(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=12.1,7.8,6.0Hz,2H),0.86(d,J=6.4Hz,3H).

[0272] 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):

[0273]

[0274] 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 eq) in MeCN (20 L) was added 2,2,2-trifluoroethyl formate (1.63 kg, 12.72 mol, 1.2 eq) and DIPEA (3.42 kg, 26.50 mol, 2.5 eq) at room temperature. The resulting mixture was stirred at room temperature overnight. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The resulting mixture was diluted with EtOAc (10 L). The resulting mixture was washed with NH 4 The mixture was quenched with Cl (6 L, saturated) and water (6 L), and extracted with EtOAc (3×15 L). The combined organic layers were washed with NH 4 The mixture was washed with Cl (aqueous solution) (10 L) and brine (10 L), and then with anhydrous Na 2 SO 4 Drying and concentration under reduced pressure gave 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 (batch 1) 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 (batch 2) of a pale yellow solid. The two batches were combined and dried to give 3.8 kg of Form I of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (melting point 133° C.) as a light yellow solid. The overall yield for this step was 97%.

[0275] Characterization of Crystalline Form I

[0276] 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 observed 2-θ angles and relative peak intensities of Form I. Figure 1A The XRPD pattern of Form I is shown.

[0277] Table 1-A

[0278]

[0279]

[0280] Figure 1B There are shown DSC and TGA graphs of Form I. As shown in the DSC graph, an endothermic onset point at about 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 A GVS diagram of Form I is shown.

[0281] The purity and stability of the crystalline form I were analyzed by XRPD and HPLC. Table 1-B shows the XRPD analysis results and the purity of the crystalline form I analyzed by HPLC before storage at 25°C / 97% RH for 7 days or at 40°C / 75% RH for 7 days, after storage at 25°C / 97% RH for 7 days, and after storage at 40°C / 75% RH for 7 days. As shown in Table 1-B, Form I is 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde, having a purity of at least 96.3% and the purity remains substantially unchanged as determined by HPLC when stored at 40°C / 75% RH and / or at 25°C / 97% RH for a period of 7 days.

[0282] Table 1-B

[0283]

[0284] Example 2

[0285] Preparation of Form II

[0286] Method 1: Conversion of Form I to Form II

[0287] 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-2-carbaldehyde was suspended in EtOH (5 vol, 12.5 ml). The sample was stirred at 50° C. (500 rpm). After 15 minutes, a solution was obtained. Upon identification, some fragments appeared to be a piece of plastic and were extracted before continuing.

[0288] The solution was stirred at 50°C for a further 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 cartridge equipped with a glass frit. A stream of compressed air was gently blown over the solid. A portion of the mother liquor was left to evaporate slowly.

[0289] A large sample was filtered through a Buchner funnel. A small amount of solid passed through the filter paper. The mother liquor was collected and refiltered and added to the filter cake. The filter cake was suction dried for 30 minutes. The solid was determined to be Form II.

[0290] Method 2

[0291] To a 100 mL round bottom flask with a magnetic stir bar, 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) was added ethanol (4.0 mL) and the mixture was heated to 78 ° C with an oil bath. Water (2 mL at a time, 6 mL in total, then added dropwise to a total of 7.0 mL) was added to the mixture until it became slightly turbid. The mixture was heated at 78 ° C for 30 minutes, then the heat was turned off and the solution was slowly cooled to room temperature under stirring (about 300 rpm, revolutions per minute) and stirred overnight. The solids were collected by filtration and air dried to afford 3.8 g (95%) of Form II 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). 1 H NMR (400 MHz, dichloromethane-d 2 )δ8.05(s,1H),7.20(q,J=9.0Hz,1H),7.11(dd,J=11.0,8.0Hz,1H),7.04-6.97(m,1H),4.95(d,J=16. 2Hz,1H),4.81(d,J=16.1Hz,1H),4.71(d,J=9.2Hz,1H),4.50(d,J=10.7Hz,1H),4.42(td,J=12.3,6.2H z,1H),4.21(d,J=9.1Hz,1H),4.10(d,J=10.7Hz,1H),4.03(s,2H),1.85(d,J=13.2Hz,2H),1.74(d,J= 9.8Hz,2H),1.59-1.48(m,2H),1.43-1.33(m,1H),1.17(qd,J=12.7,3.5Hz,2H),0.95(d,J=6.3Hz,3H).

[0292] Method 3

[0293] 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 with a magnetic stir bar was added ethanol (40.0 mL) and the solution was heated to 78° C. with an oil bath. To this mixture was added water (10 mL at a time, 70 mL in total) until it became slightly turbid. The mixture was heated at 78°C for 30 minutes, 3.8 g of crystalline Form II material of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (3.8 g, 9.4 mmol) was added, the heat was turned off and the solution was slowly cooled to room temperature with stirring (about 300 rpm) and stirred overnight. The solid was collected by filtration and air dried to obtain 44.8 g (97%) of Form II 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). 1 H NMR (400 MHz, methanol-d 4 )δ7.99(s,1H),7.25(dt,J=11.3,8.7Hz,2H),7.11(dd,J=8.5,4.2Hz,1H),4.93(s, 2H),4.66(d,J=9.9Hz,1H),4.42(t,J=9.7Hz,2H),4.32(tt,J=12.1,3.9Hz,1H),4.1 5(d,J=11.1Hz,1H),4.11(s,2H),1.85(d,J=13.3Hz,2H),1.81-1.71(m,2H),1.71-1 .56(m,2H),1.51-1.35(m,1H),1.14(qd,J=12.5,3.4Hz,2H),0.95(d,J=6.4Hz,3H).

[0294] Method 4: Conversion from Form I to Form II

[0295] To a crystalline Form I solid of 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) in a 250 mL round bottom flask with a magnetic stir bar was added ethanol (50.0 mL) and the solution was heated to 86° C. with an oil bath. Water (10 mL at a time, 50 mL total) was added to the mixture until it became slightly turbid. The mixture was heated at 86° C. for 30 minutes, the heat was turned off and the mixture was seeded with a small amount of crystalline Form II (100 mg) of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde at about 50° C. and the solution was slowly cooled to room temperature with stirring (about 300 rpm) and stirred overnight. The solid was collected by filtration and air dried to obtain 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).

[0296] The process was repeated on 170 g, 80 g and 100 g scales of Form I solid to yield 176 g, 74 g and 99 g, respectively.

[0297] A 2L three-necked flask with mechanical stirring was charged with 383g taken from the above batch. Ethanol (380mL) and water (100mL) were added to the mixture, and the mixture was slowly heated with a heating jacket with an internal temperature controller. The suspension became a uniform solution at 50°C, and additional water (200mL, 100mL at a time) was added. When the internal temperature reached 65°C, additional water (100mL) was added. The mixture became turbid until it reached 80°C. Water (50mL, 5mL at a time) was added to the solution. The mixture was then heated to 85°C, maintained for 10 minutes, and the heat was turned off to allow it to slowly cool to room temperature. When the mixture cooled to 70°C, the mixture was stirred for 10 minutes.

[0298] 20 mg of Form II crystalline solid was added at 40 ° C. More solid was observed when the internal temperature reached 40 ° C., and the internal temperature was maintained at 40-46 ° C. when more solid precipitated out of solution. The solid was collected by filtration when the internal temperature reached 28 ° C. The mixture was dried in a vacuum oven without heating for 3 days to obtain 373 g of off-white crystalline Form II. 1 H NMR (500 MHz, DMSO-d 6)δ7.97(s,1H),7.44-7.32(m,2H),7.10(dt,J=7.4,2.8Hz,1H),4.83(s,2H),4.51(d,J=9.6Hz,1H),4.29-4.14(m,3H),4.01(s,2H),3.97(d,J =10.7Hz,1H),1.77-1.70(m,2H),1.65-1.51(m,4H),1.34(dtq,J=14.2,7.0,3.4Hz,1H),1.03(qd,J=12.6,3.9Hz,2H),0.87(d,J=6.5Hz,3H).

[0299] Method 5: Transformation from Form I to Form II

[0300] To 2.05 kg of 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 with a stirring bar, 2000 mL of ethanol was added and heated to reflux (internal temperature 90° C.). After stirring under reflux for 5 minutes, water was added to the mixture (2000 mL in total: 400 mL at a time, 1600 mL in total, followed by the remaining 400 mL at a time of 40 mL; when the mixture is turbid, wait until it becomes homogeneous again at 90° C.). The mixture was heated at 90° C. for 10 minutes, then the heat was turned off and the temperature was gradually brought to 55° C. (precipitation was observed during the cooling stage). Then, 400 mg (melting point 155° C.) of seed crystals of crystalline Form II were added during the cooling stage (about 55° C.). The mixture was allowed to cool to room temperature. The mixture was stirred continuously at room temperature for 30 minutes, then filtered and oven dried (below 50°C) to give 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%) as an off-white solid (melting point 155.6°C).

[0301] Characterization of Crystalline Form II

[0302] 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 2-θ angles and relative peak intensities of Form II observed using XRPD. Figure 2A The XRPD pattern of Form II is shown.

[0303] Table 2-A

[0304]

[0305]

[0306] Figure 2B DSC and TGA graphs of Form II are shown. 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, a weight loss of less than 0.1% was observed before degradation. Figure 2C A GVS diagram of Form II is shown.

[0307] The purity and stability of crystalline form II are analyzed by XRPD and HPLC. Table 2-B shows the XRPD analysis results and the purity of crystalline form II analyzed by HPLC after being stored at 25°C / 97% RH for 8 days or at 40°C / 75% RH for 8 days, after being stored at 25°C / 97% RH for 8 days, and after being stored at 40°C / 75% RH for 8 days. As shown in Table 2-B, form II is 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxaldehyde with at least 98.0% purity and when stored at 40°C / 75% RH and / or at 25°C / 97% RH for 8 days, the purity remains substantially unchanged as measured by HPLC.

[0308] Table 2-B

[0309]

[0310]

[0311] Table 2-E shows the water content results, XRPD analysis results, assay results, and purity of crystalline Form II analyzed by HPLC before or after storage at 25°C / 60% RH or at 40°C / 75% RH for 1, 3, or 6 months. As shown in Table 2-E, Form II is 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde having a purity of at least 99%, and the purity remains substantially unchanged as determined by HPLC when stored at 25°C / 60% RH or at 40°C / 75% RH for a period of 6 months.

[0312] Table 2-E

[0313]

[0314] Single crystals of Form II were obtained by evaporating 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 single crystal X-ray diffraction analysis were isolated, with dimensions of approximately 0.25×0.12×0.10 mm. The crystal structure of crystalline Form II was solved and refined in the triclinic centrosymmetric space group P-1, with a final R1[I>2σ(I)] value of 3.91%. Single crystal data for Form II are provided in Table 2-D.

[0315] Table 2-D

[0316]

[0317]

[0318] Example 3

[0319] Kinetic solubility and stability of crystalline form II in solvents

[0320] The solubility of crystalline form II was analyzed with 15 common solvents. A sufficient amount of form II was suspended in 0.5 mL of solvent to obtain the maximum expected concentration of free form II of approximately = 10-100 mg / ml. The resulting suspension was then shaken in a platform shaker at 25 ° C / 750 rpm for 4 hours. After equilibrium, the appearance was recorded, and only the pH value of the saturated solution of the aqueous sample was measured. The 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, which was then diluted with 2-propanol instead of MeCN.

[0321] All diluted samples were then analyzed by HPLC. Samples were further diluted according to appearance and maximum expected concentration (Table 3-A). All diluted samples were injected with 10, 15 and 20 μL to obtain the peak area within the calibration curve when attempting to reach a dilution concentration of about 0.1 mg / ml. The dilution details used for different solvents are shown in Table 3-A.

[0322] Table 3-A. Form II Dilution Table

[0323]

[0324]

[0325] Quantitatively carry out by HPLC with reference to the MeCN standard solution of about 0.15mg / ml.Inject standard dilution and undiluted sample solution of different volumes.Use the peak area determined by integrating the peak found at the retention time identical with the main peak in the standard injection to calculate solubility.Find that the standard substance prepared in isopropanol is nonlinear, but the peak response between the standard substance dissolved in isopropanol and the standard substance dissolved in MeCN is comparable, so MeCN standard substance is used for all samples to be quantitatively carried out.

[0326] The kinetic solubility results of Form II in 15 different solvents after 4 hours are shown in Table 3-B.

[0327] Table 3-B

[0328]

[0329]

[0330] 2 Approximate values ​​are cited only because of agreement between replicates; 3 The sample at T = 4 hours was a clear solution and therefore reported as greater than the value based on the initial weight of the sample; 4 The sample at T = 4 hours was a clear solution and therefore reported as greater than the value based on the initial weight of the sample.

[0331] After injection of samples to determine solubility, the diluted samples were stored at ambient conditions (25°C) for 24 hours and re-injected in the same order with the same injection volumes.

[0332] The chromatograms were then overlaid and visual signs of degradation noted, i.e., loss of Compound 1 peak area or increase in other peaks in the chromatogram. The HPLC instrument details used to determine the stability of Form II are summarized in Table 3-C. The stability observations of Form II in various solvents after T = 24 hours are summarized in Table 3-D.

[0333] Table 3-C

[0334]

[0335] Table 3-D

[0336]

[0337]

[0338] The above analysis confirmed that crystalline Form II has high solubility (>100 mg / ml) in methanol, acetone, DMSO, acetonitrile and THF. In addition, when these samples were re-injected at T=24 hours, no visual signs of further degradation were observed in the chromatogram. The sample in ethanol showed a solubility of about 24 mg / mL. The reproducibility of the repeated samples was poor compared to most other formulations, but did show a later elution peak at two time points, although the sample did not appear to be further degraded within 24 hours.

[0339] Apart from the samples prepared in ethanol and tert-butyl methyl ether, only the sample prepared in isopropyl acetate showed any signs of degradation, and as with the other two examples, this was visible in the initial injection and there appeared to be no further degradation over the course of 24 hours. The solubility in isopropyl acetate was calculated to be 22 mg / mL.

[0340] Example 4

[0341] Preparation and analysis of Form III

[0342] 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 mixed with 3 vol (90 μL) of 1,4-dioxane:H 2 O (v / v 1:1) and the mixture was stirred at 5°C (300 rpm). After a total of 7 days, the stirring of the mixture was stopped. An aliquot of the resulting suspension was taken out with a spatula and blotted dry with filter paper, and then subjected to XRPD analysis to obtain Form III.

[0343] Table 4-A shows the 2-theta angles and relative peak intensities of Form III observed using XRPD. Figure 3A The observed XRPD pattern of Form III is shown.

[0344] Table 4-A

[0345] Angle / 2θ strength / % 5.6 60.3 6.0 3.3 7.2 51.4 11.2 46.8 13.9 2.7 14.9 54.8 15.5 3.4 15.8 3.3 16.4 45.8 16.8 100 17.0 3.6 17.7 14.6 18.7 20.4 18.9 4.4 19.5 4.9 20.1 10.7 20.6 12.7 21.0 11.2 21.4 3.8 21.6 14.4 21.9 27.3 22.5 30 23.2 3.4 23.4 12.5 23.8 8.2 24.3 2.2 24.6 8.7 25.1 10.4 26.2 1.5 26.6 4.1 26.9 9.9 27.3 1.8 27.7 21.9 27.9 3.5 29.0 6.6 29.2 8.9 29.6 6.1

[0346] Crystalline Form III was isolated and baseline characterized to understand the nature of the solid. Crystalline Form III was assigned to be an unstable hemi-dioxane solvate as determined by XRPD and Figure 3C As shown in , it converts to crystalline Form II after storage at 40°C / 75% RH for 7 days. Crystalline Form III is 1 HNMR analysis. 1According to HNMR, the 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 and 0.5 mol equivalent of 1,4-dioxane.

[0347] Figure 3B DSC and TGA graphs of Form III are shown. As shown in the DSC graph, an endothermic starting point at about 81.7°C, an endothermic starting point with two events at about 112.8°C, and an endothermic starting point at about 154.4°C were observed. In addition, 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.

[0348] Example 5

[0349] Polymorph screening

[0350] 5-1 Polymorph screening using isothermal maturation at 5°C

[0351] 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 3 vol (90 μL) of the selected solvent and stirred at 5°C (300 rpm). After about 1.5 hours, if the resulting suspension was too thick, 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 with filter paper before XRPD analysis. If the resulting suspension was too dilute or close to a solution, an antisolvent (3 vol, 90 μL) was added to the solutions, and the samples were then returned to 5°C for stirring. After stirring overnight at 5°C, samples of the suspension formed after the addition of the antisolvent were analyzed by XRPD. In the presence of a solution, an antisolvent (6 vol, 180 μL) was added. The samples were returned to 5°C for stirring. After a total of 7 days, stirring of the sample was stopped. An aliquot of the suspension was taken out with a spatula and blotted dry with filter paper before XRPD analysis. The resulting samples were analyzed by XRPD. The results are provided in Table 5-A and Table 5-B below.

[0352] Table 5-A

[0353]

[0354]

[0355] Table 5-B

[0356]

[0357] 5-2 Polymorph screening using temperature cycle maturation (RT / 50°C) screening

[0358] 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 3 vol (90 μL) of the selected solvent and placed for temperature cycle maturation (RT / 50°C, 4 hours per cycle) using a platform shaker incubator. After maturation overnight, an aliquot of the suspension was removed with a spatula and blotted dry with filter paper prior to XRPD analysis.

[0359] When a biphasic layer was observed, the sample was sonicated for 1.5 hours. If the sample remained unchanged, the sample was placed for evaporation. After evaporation, the solid was analyzed by XRPD. The jelly was treated with cyclohexane (10 vol, 300 μL) and sonicated for 4 hours. An aliquot of the suspension was taken out with a spatula and blotted with filter paper, and then subjected to XRPD analysis.

[0360] The obtained suspension was analyzed by XRPD. An aliquot of the suspension was taken out with a spatula and blotted dry with filter paper before analysis.

[0361] When a solution is formed, the antisolvent (45 μL) is added to the solution, followed by a return to temperature cycle maturation (RT / 50°C, 4 hours per cycle). The suspension sample is also returned to temperature cycle maturation. After a total of 7 days, the sample is stopped from maturation. An aliquot of the suspension is removed with a spatula and blotted with filter paper before XRPD analysis.

[0362] The results of the temperature cycle ripening (RT / 50°C) screening are provided in Table 5-C. A summary of the results of further processing of the solutions is provided in Table 5-D. A summary of additional processing of the two-phase system is provided in Table 5-E.

[0363] Table 5-C

[0364]

[0365]

[0366] *These samples were subsequently confirmed to be Form II using high resolution reflection geometry XRPD and DSC.

[0367] Table 5-D

[0368]

[0369] Table 5-E

[0370]

[0371] *No more jelly when stored in sealed vial.

[0372] 5-3 Polymorph screening using high temperature slurry (75°C) screening

[0373] 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 3 vol (90 μL) of the selected solvent mixture and placed under stirring at 75°C (500 rpm). After approximately 5 hours, observations were recorded. Additional solvent was added to all samples.

[0374] After stirring overnight at 75°C, an aliquot of the suspension was removed with a spatula and blotted dry with filter paper before XRPD analysis. In the case where a biphasic solution remained, additional solvent was added in 150 μL aliquots with stirring at 75°C until a solution was obtained (300 μL added in total). 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 obtained suspension was analyzed by XRPD. The solution and the biphasic solution were stirred at room temperature.

[0375] The observations and XRPD analysis are summarized in Table 5-F.

[0376] Table 5-F

[0377]

[0378]

[0379] Legend: * Gel obtained after adding another 30 μL of solvent; ** Gel obtained before adding another 30 μL of solvent; *** Suspension obtained after 15 days, **** Suspension obtained after 18 days

[0380] 5-4 Polymorph Screening Using Liquid Assisted Grinding (LAG)

[0381] 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 moistened with solvent (5 μL). Two grinding beads (3 mm in diameter) were added and the sample was ground at 500 rpm for 2 hours using a planetary Fritsch mill (Pulverisette 6). After grinding, all solids were analyzed by XRPD.

[0382] The jelly is stored under ambient conditions. The jelly and the sample containing the brittle material are vacuum dried overnight in a vacuum oven at room temperature (about 200 mbar). The observations are recorded and the sample is further vacuum dried overnight in a vacuum oven at room temperature (about 5 mbar). When the jelly is still present, the sample is further vacuum dried for 3 days in a vacuum oven (about 5 mbar) at room temperature. Observations and XRPD analysis are provided in Table 5-G. Table 5-H summarizes the further processing details of the jelly and brittle material.

[0383] Table 5-G

[0384]

[0385]

[0386]

[0387] Table 5-H

[0388]

[0389]

[0390] Example 6

[0391] Competition slurry experiment between crystalline form I and crystalline form II

[0392] A mechanical mixture of crystalline Form I and 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 prepared and then used in a competitive slurry experiment between Form I and Form II to determine the most stable form within a certain temperature range (5-50°C). Form II was determined from the competitive slurry to be the most stable form under the conditions studied and was observed in all experiments to separate solids. These results indicate that there is a monotropic relationship between Form I and Form II, and that Form II is the more thermodynamically stable form at all temperatures. Table 6 below provides the results of the competitive slurry experiments.

[0393] Table 6

[0394]

[0395]

[0396] Example 7

[0397] Synthesis of 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Comparative A)

[0398]

[0399] Step 1: Synthesis of 1-(tert-butyl) 3-ethyl 3-((4-chlorobenzyl)amino)azetidine-1,3-dicarboxylate

[0400]

[0401] AcOH (5.0 mL) was added to a solution of 3-aminoazetidine-1,3-dicarboxylic acid 1-tert-butyl 3-ethyl ester (10.6 g, 43.3 mmol, 1.0 equivalent) and 4-chlorobenzaldehyde (6.1 g, 43.3 mmol, 1.0 equivalent) in DCE (120.0 mL) at 0 ° C. After warming to room temperature and stirring at room temperature, additional AcOH (5.0 mL) was added to the mixture twice (a total of 20.0 mL) at 4 hours, 8.5 hours and 9 hours. STAB (11.0 g, 52.0 mmol, 1.2 equivalents) was added to the mixture. The mixture was stirred at room temperature overnight, diluted with aqueous sodium bicarbonate solution, and extracted three times with DCM. The combined organic layers were dried over sodium sulfate and concentrated. The obtained oil was dissolved in warm hexane and EA under stirring at 60 ° C (dropwise until the mixture becomes a uniform solution). The mixture was then cooled to 0 ° C and the precipitate was collected by filtration and washed with cold hexane. The filtrate was concentrated and the process was repeated once more. 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

[0402] 313.1(M+H-56).

[0403] Step 2: Synthesis of tert-butyl 3-((4-chlorobenzyl)amino)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate

[0404]

[0405] At 0°C in N 2To 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 LHMDS solution (54.2 mL, 1 M in THF, 54.2 mmol, 2.0 equiv) dropwise over a period of 20 min at 4 °C. 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).

[0406] Step 3: Synthesis of tert-butyl 3-(2-chloro-N-(4-chlorobenzyl)acetamido)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate

[0407]

[0408] 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 eq) in THF (20 mL) cooled to 0°C was added TEA (4.8 mL, 34.7 mmol, 1.5 eq) and 2-chloroacetyl chloride (1.5 mL, 27.8 mmol, 1.2 eq). After stirring at 0°C for 30 min, additional TEA (4.8 mL, 34.7 mmol, 1.5 eq) and 2-chloroacetyl chloride (1.8 mL, 23.2 mmol, 1.0 eq) were added to the mixture and the mixture was stirred at 0°C for 30 min. To this mixture was added additional 2-chloroacetyl chloride (0.6 mL, 6.9 mmol, 0.3 eq). The mixture was gradually warmed to room temperature, stirred for 45 minutes, 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).

[0409] 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

[0410]

[0411] In N 2 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) was added K 2 CO 3 (4.2 g, 30.1 mmol, 1.5 equiv.). The resulting mixture was stirred at room temperature for 3.5 hours, 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).

[0412] 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

[0413]

[0414] 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).

[0415] Step 6: Synthesis of 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde

[0416]

[0417] 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 eq) and N,N-diisopropylethylamine (15.7 mL, 89.7 mmol, 3 eq) in ACN (50 mL) was added 2,2,2-trifluoroethyl formate (5.8 mL, 59.8 mmol, 2 eq). 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 to 100% EtOAc gradient followed by a 0-10% MeOH in DCM gradient. 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-d 6 )δ7.98(s,1H),7.65(td,J=8.8,6.0Hz,1H),7.48-7.41(m,1H),7.43(d,J=8.5Hz,2H),7.34(d,J=8.4Hz,2H),7.22(td,J=8.4,2.4Hz, 1H), 4.91 (s, 2H), 4.55 (d, J = 9.7Hz, 1H), 4.43 (d, J = 1.6Hz, 2H), 4.37 (d, J = 9.8Hz, 1H), 4.28 (d, J = 10.8Hz, 1H), 4.08 (d, J = 10.8Hz, 1H).

[0418] Example 8

[0419] Synthesis of 8-(2,4-difluorophenyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Comparative B)

[0420]

[0421] Step 1. Synthesis of 1-(tert-butyl) 3-ethyl 3-((4-(trifluoromethyl)benzyl)amino)azetidine-1,3-dicarboxylate

[0422]

[0423] 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) at 0°C was added AcOH (7.4 g, 122.8 mmol, 2.0 equiv) and STAB (19.5 g, 92.1 mmol, 1.5 equiv) portionwise. The resulting mixture was stirred at room temperature overnight, the pH was adjusted to 8 with ammonium hydroxide, water (100.0 mL) was added and extracted twice with DCM (300.0 mL). The combined organic layers were washed twice with brine, dried over anhydrous Na 2 SO 4 Drying and concentration under reduced pressure gave 29.6 g of 3-((4-(trifluoromethyl)benzyl)amino)azetidine-1,3-dicarboxylic acid 1-(tert-butyl) 3-ethyl ester, which was used in the next step without purification. LRMS (ES) m / z 347.1 (M+H-56)

[0424] Step 2. Synthesis of tert-butyl 3-((2,4-difluorophenyl)carbamoyl)-3-((4-(trifluoromethyl)benzyl)amino)azetidine-1-carboxylate

[0425]

[0426] At 0°C in N 2 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 LHMDS solution (88.3 mL, 1 M in THF, 88.3 mmol, 2.0 equiv) dropwise over a period of 20 min at 4 °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).

[0427] Step 3. Synthesis of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate

[0428]

[0429] 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 eq) in THF (100 mL) cooled to 0°C was added TEA (8.5 mL, 60.9 mmol, 1.5 eq) and 2-chloroacetyl chloride (3.9 mL, 48.7 mmol, 1.2 eq). After stirring at 0°C for 30 min, additional TEA (8.5 mL, 60.9 mmol, 1.5 eq) and 2-chloroacetyl chloride (3.3 mL, 40.6 mmol, 1.0 eq) were added to the mixture and the mixture was stirred at 0°C for 30 min. To this mixture was added additional 2-chloroacetyl chloride (1.0 mL, 12.2 mmol, 0.3 eq). The mixture was gradually warmed to room temperature, stirred for 45 minutes, 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).

[0430] 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

[0431]

[0432] In N 2 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) was added K 2 CO 3(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 on 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).

[0433] 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

[0434]

[0435] To a stirred solution of tert-butyl 8-(2,4-difluorophenyl)-6,9-dioxol-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).

[0436] Step 6. Synthesis of 8-(2,4-difluorophenyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde

[0437]

[0438] 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 0-10% MeOH in DCM as eluent to afford 8.8 g (73% for two steps) of 8-(2,4-difluorophenyl)-6,9-dioxol-5-(4-(trifluoromethyl)benzyl)-

[0439] 2,5,8-Triazaspiro[3.5]nonane-2-carbaldehyde. LRMS(ES)m / z 469.1(M+H); 1 HNMR (400 MHz, methanol-d4) δ7.70 (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).

[0440] Example 9

[0441] 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 E)

[0442] Step 1: Synthesis of tert-butyl 3-cyano-3-((4-(trifluoromethyl)benzyl)amino)azetidine-1-carboxylate:

[0443]

[0444] To a solution of tert-butyl 3-oxoazetidine-1-carboxylate (25 g, 146.0 mmol, 1.0 equiv) in THF (90 mL) was added water (40.0 mL) containing 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). 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 to the mixture. The mixture was heated at 60 ° C in an oil bath for 18 hours, cooled to room temperature, neutralized by adding saturated aqueous sodium bicarbonate solution, and extracted with ethyl acetate (150 mL×2). The combined organic layers were washed with brine,

[0445] Dried over magnesium sulfate and concentrated under reduced pressure. Ether / hexane (200 mL, 1:2) was added to the yellow solid and the solution was sonicated for one minute, cooled to 0 ° C, and filtered. The resulting white precipitate was washed with ice-cold 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-d 6 )δ7.71(d,J=8.0Hz,2H),7.60(d,J=8.0Hz,2H),4.16(d,J=8.8Hz,2H),3.92( t, J=7.2Hz, 1H), 3.84 (d, J=9.2Hz, 2H), 3.81 (dd, J=7.3Hz, 2H), 1.39 (s, 9H).

[0446] Step 2: Synthesis of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-cyanoazetidine-1-carboxylate:

[0447]

[0448] Chloroacetyl chloride (0.95 g, 8.4 mmol, 1.0 equiv) was added 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. The mixture was stirred at 0 ° C for 15 minutes, warmed to room temperature and stirred for 2 hours. Additional 2-chloroacetyl chloride (0.95 g, 8.4 mmol, 1.0 equiv) and triethylamine (1.3 g, 12.7 mmol, 1.5 equiv) were added to the mixture. The reaction was stirred for 2 hours, 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%-70% EtOAc / hexanes, R f = 0.24 (20% EtOAc / hexanes) to give 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-d 6 )δ7.77(d,J=8.1Hz,2H),7.60(d,J=8.0Hz,2H),4.95(s,2H),4.52(s,2H),4.15(s,4H),1.35(s,9H).

[0449] Step 3: Synthesis of tert-butyl 3-cyano-3-(2-(((1r,4r)-4-(difluoromethyl)cyclohexyl)amino)-N-(4-(trifluoromethyl)benzyl)acetamido)azetidine-1-carboxylate:

[0450]

[0451] 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 eq) in acetonitrile (15 mL) was added (1r,4r)-4-(difluoromethyl)cyclohexan-1-amine hydrochloride (0.51 g, 2.8 mmol, 1.5 eq) and DIPEA (1.2 g, 9.3 mmol, 5 eq). The solution was heated at 65 °C for 4 hours, at which point LCMS indicated 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 light yellow oil. f = 0.55 (100% ethyl acetate, silica). LRMS (ES) m / z 545.0 (M+H).

[0452] 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:

[0453]

[0454] 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 hexanes (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 light yellow solid. LRMS (ES) m / z 490.2 (M+H-56). 1 HNMR (400 MHz, DMSO-d 6)δ7.71(d,J=8.1Hz,2H),7.48(d,J=8.0Hz,2H),5.89(td,J=56.7,3.8Hz,1H),4.93(s,2H),4.29-4 .15(m,3H),4.02(s,2H),3.93(d,J=9.5Hz,2H),1.91-1.55(m,7H),1.35(s,9H),1.38-1.20(m,2H).

[0455] 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:

[0456]

[0457] To a solution of tert-butyl 8-((lr,4r)-4-(difluoromethyl)cyclohexyl)-6,9-dioxol-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) at room temperature was added TFA (1.5 mL) and the mixture was stirred at room temperature for 1 hour, concentrated under reduced pressure, and dried under high vacuum to afford 190 mg (94%) of 8-((lr,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).

[0458] 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 E):

[0459]

[0460] To a solution of 8-((lr,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 at 110 °C in a microwave reactor 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 afford 21.0 mg (46%) of 8-((lr,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-d 6 )δ7.96(s,1H),7.71(d,J=8.0Hz,2H),7.47(d,J=8.0Hz,2H),5.89(td,J=56.4,4.5Hz,1H),4.94(s,2H),4.51( d,J=9.6Hz,1H),4.30-4.15(m,3H),4.04(s,2H),3.96(d,J=10.7Hz,1H),1.95-1.49(m,7H),1.41-1.06(m,2H).

[0461] Biological Example B-1: Myofibril Analysis

[0462] To evaluate 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 a native sarcomere context, a peeled myofibril analysis 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 membranes and most soluble cytoplasmic proteins, but leaves the cardiac sarcomeric actomyosin apparatus intact. Myofibril preparations retain the Ca-based 2+The ATPase activity of myofibril preparations was determined 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 at a given fraction (i.e., 25%, 75%) of the maximum ratio. 2+ The assay was performed at 100% concentration. The ability 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 inhibit the steady-state ATPase activity of bovine myocardial fibrils was evaluated using a pyruvate kinase and lactate dehydrogenase (PK / LDH) coupled enzyme system. The assay regenerates ADP generated by myosin into ATP by oxidizing NADH, resulting in an absorbance change at 340 nm. Prior to testing the compound, the calcium responsiveness of bovine myocardial fibrils was assessed and the myofibril system was selected to achieve 50% (pCa 50 ) or 75% (pCa 75 ) activated calcium concentration as the final condition for evaluating the inhibitory activity of the compound. All enzyme activities were measured in a buffer solution containing 12 mM PIPES (piperazine-N, N'-bis(2-ethanesulfonic acid), 2 mM magnesium chloride, pH 6.8 (PM 12 buffer). The final assay conditions were 1 mg / mL bovine myofibrils, 4 U / mL pyruvate kinase, 6 U / mL lactate dehydrogenase, 50 μM ATP, 0.1 mg / mL BSA (bovine serum albumin), 10 ppm defoamer, 1 mM DTT, 0.5 mM NADH, 1.5 mM PEP, 0.6 mM EGTA, and an amount of CaCl sufficient to achieve 50% or 75% activation of myofibril ATPase activity. 2 The test compounds were prepared according to the synthetic procedures described herein. The results for the test compounds are provided in Table B-1.

[0463] Table B-1

[0464] Compound <![CDATA[CDMF IC 15 (μM)]]> 1 1.5 Comparative A 1.8 Comparison B 1.1 Comparison C 1.1 Comparison D 1.1 Comparator E 1.3

[0465] Comparative C and Comparative D have the following structures:

[0466]

[0467]

[0468] The preparation of Comparator C and Comparator D is described in WO2020 / 047447A1.

[0469] Biological Example B-2: Single-dose pharmacokinetic study

[0470] Single-dose study in mice

[0471] Male C57BL / 6 mice (18-25g, 6-8 weeks old) were obtained from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. All IV administered animals had free access to food and water. IV administration was performed through the tail vein. An IV dose solution of the test article was prepared in a 10% DMA / 20% PG / 70% HPβCD solution (40% w / v HPβCD aqueous solution) at a concentration of 0.1 mg / mL. An oral administration suspension was prepared by suspending the test article in water containing 0.5% HPMC / 0.1% Tween 80 at a concentration of 0.2 mg / mL. The concentrations of IV and PO doses were measured at the end of the study. If the measured value was within 20% of the nominal value, the pharmacokinetic (PK) parameters were calculated using the nominal dose value. One group of 15 mice received an IV dose with a volume of 5 mL / kg. Another group of 15 mice received 1 mg / kg of the test article by oral gavage. The oral dose volume was 5 mL / kg. Diluted blood samples were collected from groups of three mice by retro-orbital bleeding and placed in K 2 EDTA micro blood collection tubes and kept on ice until centrifugation to obtain plasma. Each group of designated mice was bled at two time points. The time points were pre-dose (PO only), 5 minutes after administration (IV only), 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours and 24 hours. The blood samples were centrifuged and the collected plasma was stored at -80 ° C until analysis. The test article concentration of plasma samples was analyzed using LC / MS / MS method. Briefly, 50 μL aliquots of each plasma sample were mixed with 100 μL of acetonitrile containing internal standard (IS). The mixture was vortexed and centrifuged. Ten (10) μL of the resulting solution was injected into a reverse phase C18 column and the resulting peak was detected on an LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantitation (BLQ) were considered zero in PK calculations. Composite PK parameters were estimated from up to two sampling points per mouse and up to three mice per sampling point, and noncompartmental analysis of concentration-time data was performed using the rarefied data option of WinNonlin (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 versus time for the last three data points of the concentration-time curve. The apparent elimination half-life (t 1 / 2 ) values ​​were calculated as ln(2) / k. The area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC tValues ​​are calculated from the time of administration to the last measurable concentration. ∞ The value was calculated as the corresponding AUC t The sum of the ratios of the last detectable concentration divided by k. Plasma clearance (CL) is based on dose / AUC ∞ Calculated. Mean residence time (MRT) estimated by moment analysis. Steady state distribution volume (V ss ) is based on MRT ∞ × CL. Record the maximum observed concentration (C max ) and reach C max The time (t max ). Bioavailability was calculated as dA UC ∞,po / dAUC ∞,iv × 100%, where dAUC is the dose-normalized AUC value. Data for the test compounds are provided in Table B-2. The test compounds were prepared according to the synthetic procedures described herein.

[0472] Table B-2

[0473] Compound CL(mL / min / kg) <![CDATA[t 1 / 2 (h)]]> <![CDATA[V ss (L / kg)]]> 1 28.4 1.77 4.2 Comparison B 21.45 2.96 4.51 Comparison C 4.67 2.6 1.39 Comparator E 11.6 2.8 3.28

[0474] Single-dose study in rats

[0475] 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 provided food 2 hours after dosing. The IV dose solution was prepared in 10% DMA / 50% PG / 40% HPβCD solution (40% w / v HPβCD aqueous solution) at a concentration of 1 mg / mL. The oral administration suspension was prepared by suspending the test article in water containing 0.5% HPMC / 0.1% Tween 80 at a concentration of 0.2 mg / mL. The concentrations of IV and PO doses were measured at the end of the study. If the measured value was within 20% of the nominal value, the pharmacokinetic parameters were calculated using the nominal dose value. Three rats were administered IV by tail vein push. Three rats in each dose group were administered by oral gavage. Blood samples were collected from the jugular vein cannula before dosing, 5 minutes (IV only), 15 minutes, 30 minutes, and 1 hour, 2 hours, 4 hours, 6 hours, and 24 hours after dosing. The blood volume was replaced with an equal amount of sterile 0.9% saline. The blood samples were centrifuged and the collected plasma was stored at -80°C for subsequent analysis. The test article concentration of the plasma samples was analyzed using the LC / MS / MS method. Briefly, a 50 μL aliquot of each plasma sample was mixed with 100 μL of acetonitrile containing internal standard. The mixture was vortexed and centrifuged. Ten (10) μL of the resulting solution was injected into a reverse phase C18 column and the resulting peak was detected on an LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantitation (BLQ) were considered zero in pharmacokinetic calculations. Pharmacokinetic parameters were estimated from individual animals using non-compartmental 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 (log) of the concentration versus time for the last three data points of the concentration-time curve. 1 / 2 ) values ​​were calculated as ln(2) / k. The area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC t Values ​​are calculated from the time of dosing to the last measurable concentration. ∞ The value was calculated as the corresponding AUC t The ratio of the last detectable concentration divided by k (AUC t-∞ ) and. Plasma clearance (CL) is based on dose / AUC ∞ Calculated. Mean residence time (MRT) estimated by moment analysis. Steady state distribution volume (V ss ) is based on MRT ∞× CL. Record the maximum observed concentration (C max ) and reach C max The time (t max ). Bioavailability was calculated as the dose-normalized AUC for individual rats. ∞,po / Average dAUC ∞,iv The ratio of × 100%, where dAUC is the dose-normalized AUC value. Data for the test compounds are provided in Table B-3. The test compounds were prepared according to the synthetic procedures described herein.

[0476] Table B-3

[0477]

[0478]

[0479] Single-dose study in dogs

[0480] Treated male beagledogs (8 months-3 years old, weighing 8-13 kg) were used in this study. All IV-administered animals had free access to food and water; all PO animals were fasted overnight before dosing and fed approximately 6 hours after dosing. For animals in the PO group, pentagastrin (6.0 μg / kg, im) was administered 20 minutes before administration of the PO formulation and 1.5 hours after the second pentagastrin administration. The dosing volume was 0.024 mL / kg, and the concentration in DMSO / 1N NaOH / PBS was 250 μg / mL. 10 mL of 0.001N HCl was used to wash the gavage catheter of each animal. The IV dosing solution was prepared in 10% DMA / 50% PG / 40% HPβCD solution (40% w / v HPβCD in water) at a concentration of 1.0 mg / mL. The compound was suspended in 0.5%

[0481] The oral dose suspension was prepared in distilled water with HPMC / 0.1% Tween 80. The concentration of IV and PO doses was measured at the end of the study. If the measured value was within 20% of the nominal value, the PK parameters were calculated using the nominal dose value. Blood samples were collected by venipuncture of a peripheral vein other than the dosing vein before dosing, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 24 hours 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 before analysis. Plasma samples were analyzed for compound concentration using LC / MS / MS methods. Briefly, 50 μL aliquots of each plasma sample were mixed with 100 μL of acetonitrile containing internal standard. The mixture was vortexed and centrifuged. Ten (10) μL of the resulting solution was injected into a reverse phase C18 column and the resulting peak was detected on an LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantitation (BLQ) were considered zero in PK 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 (log) of concentration versus time for the last three data points of the concentration-time curve. The apparent elimination half-life (t 1 / 2 ) values ​​were calculated as ln(2) / k. The area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC t Values ​​are calculated from the time of dosing to the last measurable concentration. ∞ The value was calculated as the corresponding AUC t The sum of the ratios of the last detectable concentration divided by k. Plasma clearance (CL) is based on dose / AUC ∞ The mean residence time (MRT) extrapolated to infinity was estimated by moment analysis. ∞ ). ss According to MRT ∞ × CL. Record the maximum observed concentration (C max ) and reach C max The time (t max ). Since this was a crossover study, bioavailability was calculated as dAUC ∞,po / dAUC ∞,iv × 100%, where dAUC is the dose-normalized AUC value from the same animals given IV and PO doses. Data for the test compounds are provided in Table B-4. Test compounds were prepared according to the synthetic procedures described herein.

[0482] Table B-4

[0483] Compound CL(mL / min / kg) <![CDATA[t 1 / 2 (h)]]> <![CDATA[V ss (L / kg)]]> 1 7.57 9.54 4.23 Comparative A 1.59 39.17 5.2 Comparison B 2.39 45.88 6.51 Comparison C 4.92 14.66 5.91 Comparison D 3.55 23.69 6.7 Comparator E 1.49 33.1 4.1

[0484] Single-dose study in monkeys

[0485] Treated male cynomolgus monkeys (2-5 years old, weighing 2-5 kg) used in this study were obtained from Topgene Biotechnology. All IV administered animals had free access to food and water; all PO animals were fasted overnight before dosing and fed approximately 6 hours after dosing. The IV dose solution was prepared in 10% DMA / 50% PG / 40% HPβCD solution (40% w / v HPβCD aqueous solution) at a concentration of 1.0 mg / mL. The oral dose suspension was prepared by suspending the compound in distilled water containing 0.5% HPMC / 0.1% Tween 80 at a concentration of 0.2 mg / mL. The concentrations of IV and PO doses were measured at the end of the study. If the measured value is within 20% of the nominal value, the PK parameters are calculated using the nominal dose value. Blood samples were collected by venous puncture of a peripheral vein other than the dosing vein before, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 24 hours 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 before analysis. The compound concentrations of plasma samples were analyzed using the LC / MS / MS method. Briefly, 50 μL aliquots of each plasma sample were mixed with 100 μL of acetonitrile containing an internal standard. The mixture was vortexed and centrifuged. Ten (10) μL of the resulting solution was injected into a reversed phase C18 column and the resulting peak was detected on an LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantification (BLQ) were considered zero in PK calculations. PK parameters were estimated from individual animals using non-compartmental 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 (log) of the concentration versus time for the last three data points of the concentration-time curve. The apparent elimination half-life (t 1 / 2 ) values ​​were calculated as ln(2) / k. The area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC t Values ​​are calculated from the time of dosing to the last measurable concentration. ∞ The value was calculated as the corresponding AUC t The sum of the ratios of the last detectable concentration divided by k. Plasma clearance (CL) is based on dose / AUC ∞ The mean residence time (MRT) extrapolated to infinity was estimated by moment analysis. ∞ ). ss According to MRT ∞× CL. Record the maximum observed concentration (C max ) and reach C max The time (t max ). Since this was a crossover study, bioavailability was calculated as dAUC ∞,po / dAUC ∞,iv × 100%, where dAUC is the dose-normalized AUC value from the same animals given IV and PO doses. Data for the test compounds are provided in Table B-5. The test compounds were prepared according to the synthetic procedures described herein.

[0486] Table B-5

[0487] Compound CL(mL / min / kg) <![CDATA[t 1 / 2 (h)]]> <![CDATA[V ss (L / kg)]]> 1 11.6 9.94 8.89 Comparison B 8.72 16.22 9.96 Comparison C 13.78 11.56 12.03 Comparison D 6.92 18.12 12.43 Comparator E 3.4 16 4.35

[0488] Human single dose CL and V ss Estimated value

[0489] The allometric scaling used to predict human clearance and volume of distribution is based on interspecies simple allometric scaling of intravenous pharmacokinetic parameters in mice, rats, dogs, and cynomolgus monkeys (Boxenbaum, J Pharmacokinet Biopharm 10:201-27, 1982). Human CL is predicted by extrapolating plasma intravenous clearance from preclinical species. The 'exponential rule' (Mahmood and Balian, Life Sci. 59:579-85, 1996) was tested in this prediction, where it is proposed that when the exponent of the simple allometric growth is between 0.71 and 0.99, a correction factor based on the maximum life span (MLP) of the species can be applied, and when the exponent of the simple allometric growth is greater than 1.0, a correction factor based on brain weight (BrW) can be applied, or a protein binding correction can be applied when available. In a similar manner, a simple allometric scaling is used to predict human volume of distribution. This approach has been successfully used for a variety of drugs (Ward and Smith, Drug Metab Dispos 32:612-19, 2004; McGinnity et al., Curr Drug Metab 8:463-79, 2007). Prediction data for selected compounds are provided in Table B-6.

[0490] Table B-6

[0491]

[0492] Legend for Table B-6: SA = simple allometry; ROE = exponential rule; fuCorr = unbound function in plasma correction; MLPCorr = maximum lifespan correction; BrWCorr = brain weight correction

[0493] Dog box medication

[0494] This study used treated male beagle dogs (8 months to 3 years old, weighing 8-14 kg) from Jiangsu Johnsen Bioresource Company and / or Beijing Rixinkeji Co., Ltd. and / or Beijing Marshall Biotechnology Co., Ltd. All IV-administered animals had free access to food and water. The IV dose solution was prepared in 10% DMA / 50% PG / 40% HPβCD solution (40% w / v HPβCD aqueous solution) at a concentration of 0.2 mg / mL. The concentration of the IV dose was measured at the end of the study. If the measured value was within 20% of the nominal value, the PK parameters were calculated using the nominal dose value. Blood samples were collected by venous puncture of a peripheral vein other than the administration vein before administration, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours after administration. The blood samples were centrifuged and the resulting plasma was frozen for bioanalysis. Plasma samples were stored at -80°C before analysis. The compound concentrations of plasma samples were analyzed using LC / MS / MS methods. Briefly, 50 μL aliquots of each plasma sample were mixed with 100 μL of acetonitrile containing internal standard. The mixture was vortexed and centrifuged. Ten (10) μL of the resulting solution was injected into a reverse phase C18 column and the resulting peak was detected on an LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantification (BLQ) were considered zero in PK calculations. PK parameters were estimated from individual animals using non-compartmental 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 (log) of the concentration versus time for the last three data points of the concentration-time curve. The apparent elimination half-life (t 1 / 2 ) values ​​were calculated as ln(2) / k. The area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC t Values ​​are calculated from the time of dosing to the last measurable concentration. ∞ The value was calculated as the corresponding AUC t The sum of the ratios of the last detectable concentration divided by k. Plasma clearance (CL) is based on dose / AUC ∞ The mean residence time (MRT) extrapolated to infinity was estimated by moment analysis. ∞ ). ss According to MRT ∞ × CL. Record the maximum observed concentration (C max ) and reach C max The time (t max ). Since this was a crossover study, bioavailability was calculated as dAUC∞,po / dAUC ∞,iv × 100%, where dAUC is the dose-normalized AUC value from the same animal given 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 provided in Table B-7. Test compounds were prepared according to the synthetic procedures described herein.

[0495] Table B-7

[0496] Compound CL(mL / min / kg) <![CDATA[t 1 / 2 (h)]]> <![CDATA[V ss (L / kg)]]> 1 5.12 11.8 4.48

[0497] Monkey box drug delivery

[0498] This study used treated male beagle dogs (2-5 years old, weighing 2-5 kg) from Topgene Biotechnology. All IV administered animals had free access to food and water. The IV dose solution was prepared in 10% DMA / 50% PG / 40% HPβCD solution (40% w / v HPβCD aqueous solution) at a concentration of 0.2 mg / mL. The concentration of the IV dose was measured at the end of the study. If the measured value was within 20% of the nominal value, the PK parameters were calculated using the nominal dose value. Blood samples were collected by venous puncture of a peripheral vein other than the dosing vein before administration, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours after administration. The blood samples were centrifuged and the resulting plasma was frozen for bioanalysis. Plasma samples were stored at -80°C before analysis. The compound concentration of the plasma samples was analyzed using the LC / MS / MS method. Briefly, 50 μL aliquots of each plasma sample were mixed with 100 μL of acetonitrile containing an internal standard. The mixture was vortexed and centrifuged. Ten (10) μL of the resulting solution were injected into a reverse phase C18 column and the resulting peaks were detected on an LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantitation (BLQ) were considered zero in the PK calculations. PK parameters were estimated from individual animals using non-compartmental analysis of concentration-time data (Phoenix Win NonLin 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 (log) of the concentration versus time for the last three data points of the concentration-time curve. The apparent elimination half-life (t 1 / 2 ) values ​​were calculated as ln(2) / k. The area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC t Values ​​are calculated from the time of dosing to the last measurable concentration. ∞ The value was calculated as the corresponding AUCt The sum of the ratios of the last detectable concentration divided by k. Plasma clearance (CL) is based on dose / AUC ∞ The mean residence time (MRT) extrapolated to infinity was estimated by moment analysis. ∞ ). ss According to MRT ∞ × CL. Record the maximum observed concentration (C max ) and reach C max The time (t max ). Since this was a crossover study, bioavailability was calculated as dAUC ∞,po / dAUC ∞,iv × 100%, where dAUC is the dose-normalized AUC value from the same animal given 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 provided in Table B-8. Test compounds were prepared according to the synthetic procedures described herein.

[0499] Table B-8

[0500] Compound CL(mL / min / kg) <![CDATA[t 1 / 2 (h)]]> <![CDATA[V ss (L / kg)]]> 1 13.1 8.69 8.8

[0501] Human cassette dosing CL and V ss Estimated value

[0502] For PK data from cassette IV administration, single species allometry was used to predict human clearance and volume of distribution. In this case, by applying protein binding correction, the plasma intravenous clearance prediction values ​​of dog and monkey PK were used (Tang, Drug Metab Dispos 33: 1294-96, 2005; Patel, Journal of Pharmaceutical Research International, 22 (3): 1-7, 2018). The predicted data 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) are provided in Table B-9 and Table B-10.

[0503] Table B-9

[0504]

[0505] Table B-10

[0506]

[0507] Biological Example B-3

[0508] Acute pharmacodynamic effects on myocardial contractility assessed by echocardiography in rats

[0509] In vivo cardiac function assessment was performed in male Sprague Dawley rats by echocardiography under isoflurane (1-3%) anesthesia. Two-dimensional M-mode images of the left ventricle were obtained 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 calculation:

[0510] ((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. 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 (HPMC 2910):0.1% Tween 80 suspension and delivered as a single dose (5 mL / kg) by oral gavage. One hour and four hours after dosing, rats were lightly anesthetized for M-mode echocardiography measurements. Simultaneously with the echocardiography measurements, blood samples were collected to determine the plasma concentration of the corresponding compound. The resulting plasma concentrations were used to estimate the IC 50 and IC 10 The values, i.e., the fractional shortening, are the concentrations at which the contractility before administration is 50% and 10%, respectively. The 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 provided in Table B-11.

[0511] Table B-11.IC 50 、IC 10 Value and IC 50 / IC 10 ratio

[0512] Compound <![CDATA[IC 10 (μM)]]> <![CDATA[IC 50 (μM)]]> <![CDATA[IC 50 / IC 10 ]]> 1 0.284 2.763 9.73

[0513] Biological Example B-4

[0514] In vitro determination of time-dependent inhibition of CYP450 enzymes

[0515] The time-dependent inhibitory potential of the test compounds against the major human cytochrome P450 isozymes was also evaluated using human liver microsomes using standard methods (Grimm et al., Drug Metab. Dispos., July; 37(7): 1355-70. doi: 10.1124 / dmd.109.026716, 2009). The pooled human microsomes and selective CYP probe substrates were used to evaluate the test compounds of 25 μM and 50 μM as time-dependent inhibitors of seven human liver cytochrome P450 isozymes (CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4) in vitro. LC-MS / MS was used to quantify metabolite formation. The inhibition of each P450 enzyme in human liver microsomes was measured as the percentage reduction of the activity of the marker metabolite formed by LC-MS / MS measured at time zero and after 30 minutes of incubation compared to the uninhibited control (=100% activity). The occurrence of any time-dependent inhibition was then expressed as the fold change in enzyme activity at time zero 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.

[0516] Table B-12

[0517]

[0518]

[0519] NA: not available; CYP3A4-T: CYP3A4 activity measured by testosterone probe substrate; CYP3A4-M: CYP3A4 activity measured by midazolam probe substrate

[0520] For 3A4, the activity % was measured using midazolam 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 activity of the enzyme did not change by more than 1.2-fold. However, Comparator C showed a >1.5-fold change in activity for both probes at 25 μM and 50 μM concentrations, suggesting that there may be some variation in 3A4 activity when Comparator C is tested in a time-dependent format. Both compounds tested showed no change in activity for 1A2 and 2B6 when tested in this format. For 2C19, Compound 1 showed a 1.4-fold change in activity at 25 μM concentration, but no change in activity was observed for Compound 1 at 50 μM concentration. For 2D6, compound 1 showed a 1.3-fold change in activity at 25 μM concentration, but no change in activity of compound 1 was observed at 50 μM concentration.

[0521] Although the foregoing written descriptions of the compounds, uses and methods described herein enable a person of ordinary skill to prepare and use the compounds, uses and methods described herein, a person of ordinary skill will understand and appreciate that there are variations, combinations and equivalents to the specific embodiments, methods and examples herein. Therefore, the compounds, uses and methods provided herein should not be limited by the above-mentioned embodiments, methods or examples, but rather encompass all embodiments and methods within the scope and spirit of the compounds, uses and methods provided herein.

[0522] All references disclosed herein are incorporated by reference in their entirety.

Claims

1. A crystalline form of a compound of formula 1:

2. The crystalline form of claim 1, characterized in that Having an XRPD pattern comprising peaks at 2-theta angles of 5.9±0.2, 11.5±0.2, 11.7±0.2, 17.9±0.2, and 19.1±0.2 degrees.

3. The crystalline form of claim 2, characterized in that Having an XRPD pattern comprising additional peaks at 2-theta angles of 7.8±0.2 and 16.2±0.2 degrees.

4. The crystalline form according to any one of claims 2 or 3, characterized in that Having an XRPD pattern comprising additional peaks at 2-theta angles of 13.1 ± 0.2 and 19.8 ± 0.2 degrees.

5. The crystalline form according to any one of claims 1 to 4, characterized in that Having an XRPD pattern substantially as shown in Figure 2A.

6. The crystalline form according to any one of claims 1 to 5, characterized in that Having a DSC graph substantially as shown in Figure 2B.

7. The crystalline form according to any one of claims 1 to 6, characterized in that As determined by DSC, it has an endothermic onset at 154.9 ± 2°C.

8. The crystalline form according to any one of claims 1 to 7, characterized in that As measured by DSC, it has an endothermic peak at 155.8±2°C.

9. The crystalline form according to any one of claims 1 to 8, characterized in that Having a TGA graph substantially as shown in FIG2B .

10. The crystalline form according to any one of claims 1 to 9, characterized in that There was less than 0.1% weight loss before degradation as determined by TGA.

11. The crystalline form according to any one of claims 1 to 10, characterized in that Having a GVS diagram substantially as shown in FIG. 2C .

12. The crystalline form of claim 1, characterized in that Having an XRPD pattern comprising peaks at 2-theta angles of 6.0±0.2, 10.2±0.2, 21.6±0.2, and 22.1±0.2 degrees.

13. The crystalline form of claim 12, characterized in that Having an XRPD pattern comprising additional peaks at 2-theta angles of 17.9±0.2 and 24.1±0.2 degrees.

14. The crystalline form according to any one of claims 12 or 13, characterized in that Having an XRPD pattern comprising additional peaks at 2-theta angles of 16.0±0.2, 16.6±0.2, 17.3±0.2, 17.6±0.2, and 20.5±0.2 degrees.

15. The crystalline form according to any one of claims 1 and 12-14, characterized in that Having an XRPD pattern substantially as shown in Figure 1A.

16. The crystalline form of any one of claims 1 and 12-15, characterized in that Having a DSC graph substantially as shown in Figure 1B.

17. The crystalline form of any one of claims 1 and 12-16, characterized in that As determined by DSC, it has an endothermic onset at 125.6 ± 2 °C.

18. The crystalline form of any one of claims 1 and 12-17, characterized in that As measured by DSC, it has an endothermic peak at 130.2±2°C.

19. The crystalline form of any one of claims 1 and 12-18, characterized in that Having a TGA graph substantially as shown in FIG. 1B .

20. The crystalline form of any one of claims 1 and 12-19, characterized in that As determined by TGA, there was a weight loss of 0.35% ± 0.05% between 105°C and 145°C.

21. The crystalline form of any one of claims 1 and 12-20, characterized in that Having a GVS diagram substantially as shown in FIG. 1C .

22. A method for preparing a crystalline form as claimed in any one of claims 2 to 11, 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 alcohols, water and mixtures thereof, and (2) cooling the mixture of step (1).

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

24. The method of claim 22 or 23, wherein step (1) comprises stirring the mixture at a temperature of 40°C to 60°C.

25. The method of any one of claims 22-24, wherein step (2) comprises cooling the mixture of step (1) to a temperature of 0°C to 10°C.

26. A method for preparing a crystalline form as described in any one of claims 2-11, 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 first solvent; (2) heating the mixture of step (1) to a first temperature between 75°C and 95°C; (3) adding a second solvent to the mixture of step (2); (4) cooling the mixture of step (3) to a second temperature between 10°C and 30°C; and (5) filtering the mixture of step (4) to obtain the crystalline form.

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

28. The method of claim 26 or 27, wherein the first solvent is ethanol or a mixture of ethanol and water.

29. The method of any one of claims 26-28, wherein the second solvent is water.

30. A pharmaceutical composition comprising the crystalline form of any one of claims 1-21 and a pharmaceutically acceptable excipient.

31. A method of treating heart disease in a subject in need thereof, the method comprising administering to the subject the crystalline form of any one of claims 1-21 or the pharmaceutical composition of claim 30.

32. The method of claim 31, wherein the heart disease is hypertrophic cardiomyopathy or heart failure with preserved ejection fraction.

33. The method of claim 32, wherein the hypertrophic cardiomyopathy is obstructive or non-obstructive, or is caused by: (i) sarcomeric mutations, (ii) non-sarcomeric mutations, or (iii) both sarcomeric and non-sarcomeric mutations.

34. The method of claim 31, wherein the heart disease is selected from the group consisting of diastolic dysfunction, primary or secondary restrictive cardiomyopathy, myocardial infarction and angina pectoris, left ventricular outflow tract obstruction, hypertensive heart disease, congenital heart disease, myocardial ischemia, coronary heart disease, diabetic heart disease, congestive heart failure, right heart failure, cardiorenal syndrome and infiltrative cardiomyopathy; or wherein the heart disease is or is associated with one or more conditions selected from the group consisting of: heart aging, diastolic dysfunction caused by aging, left ventricular hypertrophy and left ventricular concentric remodeling.

35. A method of treating a disease or condition associated with hypertrophic cardiomyopathy, secondary left ventricular wall thickening, small left ventricular chambers and chamber occlusion, hyperdynamic left ventricular contraction, myocardial ischemia or cardiac fibrosis, or a disease or condition selected from muscular dystrophy and glycogen storage disease in a subject in need thereof, the method comprising administering to the subject a crystalline form as described in any one of claims 1 to 21 or a pharmaceutical composition as described in claim 30.

36. The method of 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, obesity, end-stage renal disease, scleroderma, sleep apnea, amyloidosis, Friedreich's ataxia, and Pompe disease.

37. A method of inhibiting a cardiac myometrium, the method comprising contacting the cardiac myometrium with the crystalline form of any one of claims 1-21 or the pharmaceutical composition of claim 30.

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