A crystalline form of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purin-6-yl)amino)-pyrrolidine-1-sulfonamide

Abstract

Crystalline forms of N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purin-6-yl)amino)-pyrrolidine-1-sulfonamide; corresponding pharmaceutical compositions; uses for treating or preventing cyclin-dependent kinase 2 (CDK2) mediated disorders; kits; and methods of manufacture.(I)

Description

Technical Field

[0001] This disclosure relates in its entirety to the solid form of N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide, particularly the crystalline form of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide. This disclosure also relates to pharmaceutical compositions comprising (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide in crystalline form; and pharmaceutical compositions comprising (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide in crystalline form for the treatment or prevention of cyclin-dependent kinases. 2. Use for (CDK2)-mediated diseases; kits comprising pharmaceutical compositions containing (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide in crystalline form; and methods for preparing (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide in crystalline form. Background Technology

[0002] Cyclin-dependent kinases (CDKs), including CDK2, are serine / threonine protein kinases involved in cell cycle regulation. CDK2 drives cell progression into the S and M phases of the cell cycle. Overexpression of CDK2 is associated with aberrant regulation of the cell cycle and tumor growth in various cancer types. The monomeric form of CDK2 is inactive, but it is activated when it forms a heterodimeric complex with one of its two regulatory partners, cyclin A or cyclin E. Cycin E binding to CDK2 in late G1 phase of the cell cycle is essential for the transition from G1 to S phase. Cycin A then needs to bind to CDK2 to pass through the S phase of the cell cycle. The activated CDK2-cyclin A / E complex controls the phosphorylation of various transcription factors that regulate multiple oncogenic signaling pathways that influence cell cycle progression. CDK2 activation also leads to the hyperphosphorylation and inactivation of retinoblastoma protein (pRB), a tumor suppressor protein that helps maintain cells in a quiescent state (i.e., the G0 phase of the cell cycle).

[0003] Overexpression of the CCNE1 gene, which produces cyclin E, occurs in many tumor cells, leading to these cells becoming dependent on CDK2 and cyclin E. For example, aberrant cyclin E activity has been observed in solid tumors such as breast cancer, ovarian cancer, lung cancer, colorectal cancer, gastric cancer, endometrial cancer, and bone cancer, as well as in hematologic malignancies such as leukemia and lymphoma. Furthermore, amplification and / or overexpression of cyclin E has been reported as a potential mechanism of resistance to CDK4 / 6 therapy in ER-positive HER2-negative breast cancer. Similarly, aberrant expression of cyclin A is associated with chromosomal instability and tumor proliferation, while inhibition of cyclin A leads to a slowing of tumor growth.

[0004] Inhibition of CDK2 activity remains an undeveloped therapeutic approach for cancer and other diseases associated with CDK2 activity. Despite significant efforts, no approved pharmacological agents can universally or specifically inhibit CDK2 activity. Identifying such inhibitors has been challenging, partly due to two major obstacles. First, the mechanisms of CDK2 degradation are poorly understood. Second, CDK2 inhibitors developed to date often lack the necessary specificity or otherwise exhibit off-target CDK-driven toxicity. The high sequence and structural similarity among various CDKs, and the similarity between CDK binding sites, contribute to the poor specificity and / or high toxicity of most CDK2 inhibitors. Therefore, there is a need for CDK2 inhibitors, particularly those with pharmacologically suitable properties and physical characteristics conducive to the manufacture of pharmaceutical substances and the formulation of corresponding drug products.

[0005] This disclosure addresses this significant unmet need by providing a crystalline form of the CDK2 inhibitor (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide in pharmaceutical compositions and methods suitable for treating CDK2-mediated conditions such as breast cancer and ovarian cancer. Summary of the Invention

[0006] In one aspect, this disclosure provides the crystalline form of N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide.

[0007] In another aspect, this disclosure provides the crystalline form of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)-pyrrolidine-1-sulfonamide.

[0008] In another aspect, this disclosure provides crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)-pyrrolidine-1-sulfonamide.

[0009] In another aspect, this disclosure provides a pharmaceutical composition comprising crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide, and one or more pharmaceutically acceptable excipients.

[0010] In another aspect, this disclosure provides a method for treating or preventing cyclin-dependent kinase 2 (CDK2)-mediated conditions by administering to a subject in need a pharmaceutical composition comprising a therapeutically effective amount of crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide.

[0011] In another aspect, this disclosure provides the use of a pharmaceutical composition comprising (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide in crystalline form A for the treatment or prevention of cyclin-dependent kinase 2 (CDK2) mediated conditions.

[0012] In another aspect, this disclosure provides the use of crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)-pyrrolidine-1-sulfonamide for the manufacture of medicaments for the treatment or prevention of cyclin-dependent kinase 2 (CDK2) mediated conditions.

[0013] In another aspect, this disclosure provides a kit comprising a pharmaceutical composition in crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide.

[0014] In another aspect, this disclosure provides a method for preparing crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide. Attached Figure Description

[0015] Figure 1The representative powder X-ray diffraction (PXRD) pattern of amorphous (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)-pyrrolidine-1-sulfonamide.

[0016] Figure 2 The image shows a representative differential scanning calorimetry (DSC) curve for amorphous (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)-pyrrolidine-1-sulfonamide.

[0017] Figure 3 This is a representative thermogravimetric (TGA) chromatogram of amorphous (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide.

[0018] Figure 4 The figure shows a representative gravimetric vapor adsorption (GVS) curve for amorphous (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide.

[0019] Figure 5-A Thermoellipsoid plot of the crystal structure of crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide based on single-crystal analysis and plotted at a 50% probability level.

[0020] Figure 5-B The representative powder X-ray diffraction (PXRD) pattern of crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide is shown.

[0021] Figure 6 The representative differential scanning calorimetry (DSC) curves for crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide are shown.

[0022] Figure 7The representative thermogravimetric analysis (TGA) chromatogram of crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide is shown.

[0023] Figure 8 The representative gravimetric vapor adsorption (GVS) curves for crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)-pyrrolidine-1-sulfonamide are shown.

[0024] Figure 9 The representative crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)-pyrrolidine-1-sulfonamide 13 C CPMAS spectrum. Detailed Implementation

[0025] Numerous embodiments are described in detail throughout the specification, and these embodiments will be apparent to those skilled in the art. Such embodiments are provided by way of example only and are not intended to otherwise limit the scope of the invention. Various alternatives to the described embodiments may be employed in the practice of this invention.

[0026] I. Definition

[0027] Regarding the embodiments disclosed in this specification, the following terms have the meanings described below:

[0028] The reference to “a” or “an” means “one or more” or “one or more types of”. Throughout the text, the plural and singular forms should be considered interchangeable, except for indications of quantity.

[0029] When a range is used herein to describe, for example, physical or chemical properties, it is intended to include all combinations and sub-combinations of the range and specific embodiments thereof. When referring to a number or numerical range, the use of the terms “about” or “approximately” means that the number or numerical range mentioned is an approximation within experimental variability (or within statistical experimental error), and therefore the number or numerical range will vary between 1% and 15% of the stated number or numerical range.

[0030] Unless the context otherwise requires, the use of the words “comprising” or “including” is based on and clearly understood to be interpreted as inclusive rather than exclusive, and the applicant intends to interpret each of these words in this way when interpreting the patent (including the claims below).

[0031] The term "amorphous form" refers to a compound form that lacks long-range crystalline order.

[0032] As used herein, the terms “co-administered,” “administered in combination with,” and “administered in conjunction with” cover the administration of two or more agents to a subject so that the two agents and / or their metabolites are present in the subject simultaneously. Co-administration includes simultaneous administration of a single composition, administration of a single composition at different times, or administration of a composition in which two or more agents are present.

[0033] The term “crystalline form” is intended to include all crystalline forms of a compound, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, nonsolventized polymorphs (including anhydrous polymorphs) and conformational polymorphs, as well as mixtures thereof, unless a specific crystalline form is mentioned.

[0034] The term "therapeuticly effective amount" for a pharmacological agent is the amount sufficient to achieve a beneficial or desired outcome (including clinical outcomes), and therefore will depend on how it is administered. For example, in the case of a pharmacological agent being administered to treat cancer, the therapeutically effective amount of the agent is the amount, alone or in combination with an adjunct therapy, sufficient to provide an anticancer effect in a subject compared to the response obtained without the administration of the agent.

[0035] The term “pharmaceutical acceptable” is used as an adjective in this specification, meaning that the noun it modifies is suitable for use as a pharmaceutical product or as part of a pharmaceutical product. For example, the terms “pharmaceutical acceptable carrier” or “pharmaceutical acceptable excipient” are intended to include any and all carriers or excipients suitable for mammals, particularly humans.

[0036] When used in conjunction with powder X-ray diffraction, the term "reflection" or "reflection mode" refers to the reflection (also known as the Bragg-Brentano) sampling mode.

[0037] The term “prevention” is easily understood by a general practitioner and, in relation to the treatment of a particular condition, can include the intention to have its normal meaning, and includes primary prevention to prevent the development of the condition as well as secondary prevention where the condition has already developed and which temporarily or permanently protects the patient against the aggravation or worsening of the condition or the development of new symptoms associated with the condition.

[0038] The term "solvent" refers to the crystalline phase of a compound that is physically associated with one or more molecules of a solvent. The crystalline phase of a compound that is physically associated with one or more molecules of water is called a "hydrate".

[0039] When used in conjunction with powder X-ray diffraction, the term "transmission" or "transmission mode" refers to the transmission (also known as the Debye-Scherrer) sampling mode.

[0040] The term "treatment" is easily understood by a generally skilled physician and, in relation to a specific condition, can include (1) reducing the severity or cause of the treated condition, and / or (2) alleviating or improving one or more symptoms associated with the condition. Treatment of cancer, for example, may include stabilizing (i.e., not worsening), delaying or slowing the spread or progression of cancer; prolonging survival compared to expected survival without treatment; and / or otherwise wholly or partially improving or reducing the severity of cancer.

[0041] As used herein, “enantiomer purity” refers to the relative amount (expressed as a percentage) of a particular enantiomer present relative to another enantiomer. For example, if a compound can potentially exist as a racemic mixture having (R)- or (S)- isomer configurations, the enantiomer purity relative to the (R)- or (S)- isomer is about 50%. If the compound has one isomer form preferred to the other, for example, 80% (S)- isomer and 20% (R)- isomer, the enantiomer purity of the compound relative to the (S)- isomer form is 80%. The enantiomer purity of a compound can be determined in a variety of ways, including but not limited to chromatography using chiral supports, polarization measurements using polarized light rotation, nuclear magnetic resonance spectroscopy using chiral shift reagents (including but not limited to chiral complexes containing lanthanides or Pirkle reagents), or derivatization of a compound using chiral compounds (such as Mosher acids) followed by chromatography or nuclear magnetic resonance spectroscopy.

[0042] In some embodiments, the enantiomer-enriched composition has a higher potency relative to the therapeutic effect per unit mass than a racemic mixture of the composition. Enantiomers can be separated from the mixture by methods known to those skilled in the art, including chiral high-performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred enantiomers can be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions, Wiley Interscience, New York, 1981; Eliel, Stereochemistry of Carbon Compounds, McGraw-Hill, NY, 1962; and Eliel and Wilen, Stereochemistry of Organic Compounds, Wiley-Interscience, New York, 1994.

[0043] As used herein, the terms "enantiomer-enriched" and "non-racemic" refer to compositions in which one enantiomer has a weight percentage greater than that enantiomer in a control mixture of racemic compositions (e.g., greater than 1:1 by weight). For example, an enantiomer-enriched preparation of the (R)-enantiomer means a preparation of a compound having more than 50% by weight (e.g., at least 75% by weight, or at least 80% by weight) of the (R)-enantiomer relative to the (S)-enantiomer. In some embodiments, enrichment may be significantly greater than 80% by weight, thereby providing a "substantially enantiomer-enriched" or "substantially non-racemic" preparation, which refers to a preparation of a composition in which one enantiomer has at least 85% by weight (e.g., at least 90% by weight, or at least 95% by weight) of one enantiomer relative to another. The terms "enantiomer-pure" or "substantially enantiomer-pure" refer to compositions containing at least 98% of a single enantiomer and less than 2% of a relative enantiomer.

[0044] II. Crystallization Form A

[0045] Compounds that are active pharmaceutical ingredients in pharmaceutical products may exist in different solid forms exhibiting different physical properties. These differences in physical properties can affect the manufacture and formulation of pharmaceutical products. These physical properties may include, but are not limited to: (1) filling properties, such as molar volume, density, and hygroscopicity; (2) thermodynamic properties, such as melting temperature, vapor pressure, and solubility; (3) kinetic properties, such as dissolution rate and stability (including stability under environmental conditions, especially to moisture and under storage conditions); (4) surface properties, such as surface area, wettability, interfacial tension, and shape; (5) mechanical properties, such as hardness, tensile strength, compressibility, compressibility, handling, flowability, and blendability; and (6) filtration properties. Therefore, solid forms of compounds (especially crystalline forms of compounds) that provide improvements in one or more of these physical properties relative to other solid forms of the compound are desirable. Thus, the discovery of new solid forms of pharmaceutically available compounds provides potential opportunities to improve the performance characteristics of corresponding pharmaceutical products and related manufacturing processes.

[0046] This disclosure provides a crystalline form of N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purin-6-yl)amino)pyrrolidine-1-sulfonamide. In one aspect, the crystalline form has one or more of the advantageous properties described above, relative to one or more other solid forms of the compound. In another aspect, the crystalline form is anhydrous. In yet another aspect, the crystalline form is substantially pure. As used in this specification, the term "substantially pure" means that, relative to any other solid form of the compound, the crystalline form of the compound contains at least about 90% by weight of the desired crystalline form. In one aspect, the crystalline form of the compound contains at least about 95% by weight of the desired crystalline form, relative to any other solid form of the compound. In another aspect, the crystalline form of the compound contains at least about 96% by weight of the desired crystalline form, relative to any other solid form of the compound. In yet another aspect, the crystalline form of the compound contains at least about 97% by weight of the desired crystalline form, relative to any other solid form of the compound. In another aspect, the crystalline form of the compound comprises at least about 98% by weight of the desired crystalline form, relative to any other solid form of the compound. In another aspect, the crystalline form of the compound comprises at least about 99% by weight of the desired crystalline form, relative to any other solid form of the compound.

[0047] In some embodiments, this disclosure provides a crystalline form of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)-pyrrolidine-1-sulfonamide having the following chemical structure:

[0048] .

[0049] In some embodiments, this disclosure provides crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)-pyrrolidine-1-sulfonamide.

[0050] In some embodiments, crystalline form A is characterized by powder X-ray diffraction (PXRD) containing at least one peak selected from the group consisting of: 8.1 ± 0.2°2θ, 13.9 ± 0.2°2θ, 16.1 ± 0.2°2θ, 16.7 ± 0.2°2θ, and 22.6 ± 0.2°2θ. In one aspect, the powder X-ray diffraction pattern contains at least two, three, or four peaks selected from the group consisting of: 8.1 ± 0.2°2θ, 13.9 ± 0.2°2θ, 16.1 ± 0.2°2θ, 16.7 ± 0.2°2θ, and 22.6 ± 0.2°2θ. In another aspect, the powder X-ray diffraction pattern contains peaks at 8.1 ± 0.2°2θ, 13.9 ± 0.2°2θ, 16.1 ± 0.2°2θ, 16.7 ± 0.2°2θ, and 22.6 ± 0.2°2θ. In another aspect, the powder X-ray diffraction pattern also contains at least one, two, three, or four peaks selected from the group consisting of: 17.7 ± 0.2°2θ, 20.2 ± 0.2°2θ, 21.3 ± 0.2°2θ, 25.9 ± 0.2°2θ, and 29.2 ± 0.2°2θ. In another aspect, the powder X-ray diffraction pattern contains peaks at 17.7 ± 0.2°2θ, 20.2 ± 0.2°2θ, 21.3 ± 0.2°2θ, 25.9 ± 0.2°2θ, and 29.2 ± 0.2°2θ. In another aspect, the powder X-ray diffraction pattern contains peaks at 8.1±0.2°2θ, 13.9±0.2°2θ, 16.1±0.2°2θ, 16.7±0.2°2θ, 17.7±0.2°2θ, 20.2±0.2°2θ, 21.3±0.2°2θ, 22.6±0.2°2θ, 25.9±0.2°2θ, and 29.2±0.2°2θ. In another aspect, powder X-ray diffraction is reflection powder X-ray diffraction. In another aspect, powder X-ray diffraction is transmission powder X-ray diffraction. In another aspect, the powder X-ray diffraction pattern is related to... Figure 5-B The powder X-ray diffraction patterns are basically the same.

[0051] In some embodiments, form A is characterized by a differential scanning calorimetry (DSC) curve containing endothermic melting at an initial temperature of about 122°C ± 5°C. In another aspect, the endothermic process has a peak at about 128°C ± 5°C. In yet another aspect, the endothermic process comprises endothermic melting at an initial temperature of about 122°C ± 5°C and a peak at about 128°C ± 5°C. In yet another aspect, the endothermic process has an initial temperature of about 122°C ± 2°C. In yet another aspect, the endothermic process has a peak at about 128°C. In yet another aspect, the endothermic process comprises endothermic melting at an initial temperature of about 122°C ± 2°C and a peak at about 128°C ± 2°C. In yet another aspect, the endothermic process has an initial temperature of about 122°C. In yet another aspect, the endothermic process has a peak at about 128°C. In yet another aspect, the endothermic process comprises endothermic melting at an initial temperature of about 122°C and a peak at about 128°C. In another aspect, differential scanning calorimetry curves and Figure 6 The differential scanning calorimetry curves are basically the same.

[0052] In some embodiments, form A is characterized by thermogravimetric analysis (TGA) pyrometry, wherein the crystalline form exhibits a weight loss of less than about 1.0 wt% from about 25°C to about 100°C. In one aspect, the weight loss is less than about 0.5 wt%. In another aspect, the weight loss is less than about 0.1 wt%. In yet another aspect, the thermogravimetric analysis pyrometry pyrometry is compared with... Figure 7 The thermogravimetric analysis chromatograms are basically the same.

[0053] In some embodiments, form A is characterized by a gravimetric vapor adsorption (GVS) curve, wherein the crystalline form exhibits reversible moisture absorption of less than about 1.0 wt% from about 0% relative humidity to about 80% relative humidity at 25 °C ± 0.1 °C. In one aspect, the reversible moisture absorption is less than about 0.7 wt%. In another aspect, the reversible moisture absorption is less than about 0.4 wt%. In yet another aspect, the gravimetric vapor adsorption curve is compared with... Figure 8 The weight vapor adsorption curves are basically the same.

[0054] In some implementations, form A is composed of solid-state... 13 The solid-state was characterized by C NMR spectroscopy. 13 The C NMR spectrum contains at least one peak selected from the group consisting of: 161.2 ± 0.2 ppm, 137.6 ± 0.2 ppm, 113.1 ± 0.2 ppm, 72.3 ± 0.2 ppm, and 60.0 ± 0.2 ppm. In one aspect, the solid-state... 13The C NMR spectrum contains at least two, three, or four peaks selected from the following groups: 161.2 ± 0.2 ppm, 137.6 ± 0.2 ppm, 113.1 ± 0.2 ppm, 72.3 ± 0.2 ppm, and 60.0 ± 0.2 ppm. In another aspect, the solid-state... 13 The C NMR spectrum includes peaks at 161.2 ± 0.2 ppm, 137.6 ± 0.2 ppm, 113.1 ± 0.2 ppm, 72.3 ± 0.2 ppm, and 60.0 ± 0.2 ppm. In another aspect, the solid-state... 13 The C NMR spectrum includes peaks at 161.2±0.2 ppm, 153.9±0.2 ppm, 152.8±0.2 ppm, 137.6±0.2 ppm, 113.1±0.2 ppm, 72.3±0.2 ppm, 60.0±0.2 ppm, 54.9±0.2 ppm, 51.0±0.2 ppm, 47.2±0.2 ppm, 40.1±0.2 ppm, 39.1±0.2 ppm, 29.3±0.2 ppm, 23.1±0.2 ppm, 17.1±0.2 ppm, 16.5±0.2 ppm, 13.3±0.2 ppm, and 10.7±0.2 ppm. In another aspect, the solid-state... 13 C NMR spectrum and solid state in Figure 10 13 The C NMR spectra are basically the same.

[0055] In some embodiments, form A is characterized by at least two of the above-described physical characterization embodiments selected from the following: powder X-ray diffraction, differential scanning calorimetry, thermogravimetric analysis, and / or gravimetric vapor adsorption.

[0056] In some embodiments, form A is characterized by at least three of the above-described physical characterization embodiments selected from the following: powder X-ray diffraction, differential scanning calorimetry, thermogravimetric analysis, and / or gravimetric vapor adsorption.

[0057] In some embodiments, form A is characterized by at least four of the above-described physical characterization embodiments selected from the following: powder X-ray diffraction, differential scanning calorimetry, thermogravimetric analysis, and gravimetric vapor adsorption.

[0058] In some embodiments, form A is characterized by at least two of the above-described physical characterization embodiments selected from: powder X-ray diffraction, solid-state... 13 C NMR, differential scanning calorimetry, thermogravimetric analysis and / or gravimetric vapor adsorption.

[0059] In some embodiments, form A is characterized by at least three of the above-described physical characterization embodiments selected from: powder X-ray diffraction, solid-state... 13C NMR, differential scanning calorimetry, thermogravimetric analysis and / or gravimetric vapor adsorption.

[0060] In some embodiments, form A is characterized by at least four of the physical characterization embodiments selected from the following: powder X-ray diffraction, solid-state... 13 C NMR, differential scanning calorimetry, thermogravimetric analysis, and gravimetric vapor adsorption.

[0061] In some embodiments, form A is characterized by at least five of the physical characterization embodiments selected from the following: powder X-ray diffraction, solid-state... 13 C NMR, differential scanning calorimetry, thermogravimetric analysis, and gravimetric vapor adsorption.

[0062] In some implementations, form A is characterized by the following:

[0063] Powder X-ray diffraction pattern containing peaks at 8.1±0.2°2θ, 13.9±0.2°2θ, 16.1±0.2°2θ, 16.7±0.2°2θ, and 22.6±0.2°2θ; and

[0064] solid state 13 C NMR spectrum, the solid-state 13 The C NMR spectrum includes peaks at 161.2±0.2ppm, 137.6±0.2ppm, 113.1±0.2ppm, 72.3±0.2ppm, and 60.0±0.2ppm.

[0065] In some implementations, form A is characterized by the following:

[0066] Powder X-ray diffraction pattern containing peaks at 8.1±0.2°2θ, 13.9±0.2°2θ, 16.1±0.2°2θ, 16.7±0.2°2θ, and 22.6±0.2°2θ; and

[0067] Differential scanning calorimetry (DSC) curves, which include the endothermic melting at an initial temperature of approximately 122°C ± 5°C.

[0068] In another aspect, form A is further composed of solid state. 13 The solid-state was characterized by C NMR spectroscopy. 13 The C NMR spectrum includes peaks at 161.2±0.2ppm, 137.6±0.2ppm, 113.1±0.2ppm, 72.3±0.2ppm, and 60.0±0.2ppm.

[0069] In some implementations, form A is characterized by the following:

[0070] Powder X-ray diffraction pattern containing peaks at 8.1±0.2°2θ, 13.9±0.2°2θ, 16.1±0.2°2θ, 16.7±0.2°2θ, and 22.6±0.2°2θ; and

[0071] Thermogravimetric analysis pyrographs showed that the crystalline form exhibited a weight loss of less than about 1.0% by weight from about 25°C to about 100°C.

[0072] In another aspect, form A is further composed of solid state. 13 The solid-state was characterized by C NMR spectroscopy. 13 The C NMR spectrum includes peaks at 161.2±0.2ppm, 137.6±0.2ppm, 113.1±0.2ppm, 72.3±0.2ppm, and 60.0±0.2ppm.

[0073] In some implementations, form A is characterized by the following:

[0074] Powder X-ray diffraction pattern containing peaks at 8.1±0.2°2θ, 13.9±0.2°2θ, 16.1±0.2°2θ, 16.7±0.2°2θ, and 22.6±0.2°2θ; and

[0075] The gravimetric vapor adsorption curves show that the crystalline form exhibits reversible moisture absorption of less than about 1.0% by weight from about 0% relative humidity to about 80% relative humidity at 25℃±0.1℃.

[0076] In another aspect, form A is further composed of solid state. 13 The solid-state was characterized by C NMR spectroscopy. 13 The C NMR spectrum includes peaks at 161.2±0.2ppm, 137.6±0.2ppm, 113.1±0.2ppm, 72.3±0.2ppm, and 60.0±0.2ppm.

[0077] In some implementations, form A is characterized by the following:

[0078] Powder X-ray diffraction pattern containing peaks at 8.1±0.2°2θ, 13.9±0.2°2θ, 16.1±0.2°2θ, 16.7±0.2°2θ and 22.6±0.2°2θ;

[0079] Differential scanning calorimetry (DSC) curves, which include the endothermic reaction at an initial temperature of approximately 122°C ± 5°C; and

[0080] Thermogravimetric analysis pyrographs showed that the crystalline form exhibited a weight loss of less than about 1.0% by weight from about 25°C to about 100°C.

[0081] In another aspect, form A is further composed of solid state. 13 The solid-state was characterized by C NMR spectroscopy. 13 The C NMR spectrum includes peaks at 161.2±0.2ppm, 137.6±0.2ppm, 113.1±0.2ppm, 72.3±0.2ppm, and 60.0±0.2ppm.

[0082] In some implementations, form A is characterized by the following:

[0083] Powder X-ray diffraction pattern containing peaks at 8.1±0.2°2θ, 13.9±0.2°2θ, 16.1±0.2°2θ, 16.7±0.2°2θ and 22.6±0.2°2θ;

[0084] Differential scanning calorimetry (DSC) curves, which include the endothermic reaction at an initial temperature of approximately 122°C ± 5°C; and

[0085] The gravimetric vapor adsorption curves show that the crystalline form exhibits reversible moisture absorption of less than about 1.0% by weight from about 0% relative humidity to about 80% relative humidity at 25℃±0.1℃.

[0086] In another aspect, form A is further composed of solid state. 13 The solid-state was characterized by C NMR spectroscopy. 13 The C NMR spectrum includes peaks at 161.2±0.2ppm, 137.6±0.2ppm, 113.1±0.2ppm, 72.3±0.2ppm, and 60.0±0.2ppm.

[0087] In some implementations, form A is characterized by the following:

[0088] Powder X-ray diffraction pattern containing peaks at 8.1±0.2°2θ, 13.9±0.2°2θ, 16.1±0.2°2θ, 16.7±0.2°2θ and 22.6±0.2°2θ;

[0089] Thermogravimetric analysis (TGA) pyrographs showed that the crystalline form exhibited a weight loss of less than approximately 1.0% by weight from approximately 25°C to approximately 100°C; and

[0090] The gravimetric vapor adsorption curves show that the crystalline form exhibits reversible moisture absorption of less than about 1.0% by weight from about 0% relative humidity to about 80% relative humidity at 25℃±0.1℃.

[0091] In another aspect, form A is further composed of solid state. 13 The solid-state was characterized by C NMR spectroscopy.13 The C NMR spectrum includes peaks at 161.2±0.2ppm, 137.6±0.2ppm, 113.1±0.2ppm, 72.3±0.2ppm, and 60.0±0.2ppm.

[0092] In some implementations, form A is characterized by the following:

[0093] Powder X-ray diffraction pattern containing peaks at 8.1±0.2°2θ, 13.9±0.2°2θ, 16.1±0.2°2θ, 16.7±0.2°2θ and 22.6±0.2°2θ;

[0094] Differential scanning calorimetry curve, which includes the melting endothermic process with an initial temperature of approximately 122°C ± 5°C;

[0095] Thermogravimetric analysis (TGA) pyrographs showed that the crystalline form exhibited a weight loss of less than approximately 1.0% by weight from approximately 25°C to approximately 100°C; and

[0096] The gravimetric vapor adsorption curves show that the crystalline form exhibits reversible moisture absorption of less than about 1.0% by weight from about 0% relative humidity to about 80% relative humidity at 25℃±0.1℃.

[0097] In another aspect, form A is further composed of solid state. 13 The solid-state was characterized by C NMR spectroscopy. 13 The C NMR spectrum includes peaks at 161.2±0.2ppm, 137.6±0.2ppm, 113.1±0.2ppm, 72.3±0.2ppm, and 60.0±0.2ppm.

[0098] In some implementations, form A is an anhydrous crystalline compound.

[0099] In some embodiments, form A is substantially pure. In one aspect, form A comprises less than 10% by weight of any other crystalline form of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purin-6-yl)amino)pyrrolidine-1-sulfonamide. In another aspect, form A comprises less than 5% by weight of any other crystalline form of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purin-6-yl)amino)pyrrolidine-1-sulfonamide. In yet another aspect, form A comprises less than 4% by weight of any other crystalline form of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purin-6-yl)amino)pyrrolidine-1-sulfonamide. In another aspect, form A comprises less than 3% by weight of any other crystalline form of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purin-6-yl)amino)pyrrolidine-1-sulfonamide. In another aspect, form A comprises less than 2% by weight of any other crystalline form of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purin-6-yl)amino)pyrrolidine-1-sulfonamide. In another aspect, form A comprises less than 1% by weight of any other crystalline form of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purin-6-yl)amino)pyrrolidine-1-sulfonamide. In another respect, form A is essentially free of any other crystalline form of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide.

[0100] III. Instructions for Use

[0101] (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purin-6-yl)amino)pyrrolidine-1-sulfonamide is an inhibitor of cyclin-dependent kinase 2 (CDK2) activity. CDK2 overexpression is associated with aberrant regulation of cell cycle and tumor growth in various cancer types.

[0102] Therefore, in some embodiments, this disclosure provides a method for treating or preventing CDK2-mediated conditions in a subject by administering to a subject in need a pharmaceutical composition comprising a therapeutically effective amount of crystalline form A.

[0103] In some embodiments, this disclosure provides a method for inhibiting CDK2 activity in a subject by administering a pharmaceutical composition comprising a therapeutically effective amount of crystalline form A to a subject suffering from or susceptible to a CDK2-mediated condition.

[0104] In some embodiments, this disclosure provides a method for treating a subject's cancer by administering to a subject who has or is susceptible to cancer a pharmaceutical composition comprising a therapeutically effective amount of crystalline form A. In one aspect, the cancer is a solid tumor. In another aspect, the cancer is a hematologic cancer. In yet another aspect, the cancer is wholly or partially mediated by CDK2.

[0105] In some embodiments, this disclosure provides a method for treating a subject with cancer characterized by amplification or overexpression of the cyclin E (CCNE) gene by administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of crystalline form A. In one aspect, the cancer is characterized by amplification or overexpression of CCNE1. In another aspect, the cancer is characterized by amplification or overexpression of CCNE2. In another aspect, the cancer is characterized by amplification or overexpression of both CCNE1 and CCNE2. In another aspect, the cancer is a solid tumor cancer characterized by amplification or overexpression of CCNE1 and / or CCNE2. In another aspect, the solid tumor cancer is selected from the group consisting of breast cancer, ovarian cancer, endometrial cancer, and lung cancer. In another aspect, the solid tumor cancer is breast cancer or ovarian cancer. In another aspect, the cancer is a hematologic cancer characterized by amplification or overexpression of CCNE1 and / or CCNE2.

[0106] In some embodiments, this disclosure provides a method for treating or preventing solid tumor cancer in a subject by administering a pharmaceutical composition comprising a therapeutically effective amount of crystalline form A to the subject who has or is susceptible to cancer, wherein the solid tumor cancer is selected from the group consisting of: breast cancer, ovarian cancer, endometrial cancer, cervical cancer, uterine cancer, gastric cancer, prostate cancer, bladder cancer, lung cancer, esophageal cancer, head and neck cancer, kidney cancer, liver cancer, pancreatic cancer, thyroid cancer, colorectal cancer, and skin cancer. In one aspect, the solid tumor cancer is selected from the group consisting of: breast cancer, ovarian cancer, endometrial cancer, cervical cancer, lung cancer, colorectal cancer, and skin cancer. In another aspect, the solid tumor cancer is selected from the group consisting of: breast cancer, ovarian cancer, endometrial cancer, and lung cancer. In yet another aspect, the solid tumor cancer is breast cancer or ovarian cancer.

[0107] In some implementations, the cancer is breast cancer. In one aspect, breast cancer is selected from the group consisting of hormone receptor-positive (HR+) breast cancer, hormone receptor-negative (HR-) breast cancer, and triple-negative breast cancer. In another aspect, breast cancer is HR+ HER2- breast cancer. In another aspect, breast cancer is chemotherapy-resistant breast cancer. In another aspect, breast cancer is radiation-resistant breast cancer. In another aspect, breast cancer is characterized by amplification or overexpression of CCNE1 and / or CCNE2. In another aspect, breast cancer is advanced or metastatic breast cancer. In another aspect, the subject with breast cancer has previously been treated with a CDK4 / 6 inhibitor.

[0108] In some implementations, the cancer is ovarian cancer. In one aspect, the ovarian cancer is platinum-sensitive or platinum-resistant ovarian cancer. In another aspect, the ovarian cancer is characterized by amplification or overexpression of CCNE1 and / or CCNE2. In another aspect, the cancer is epithelial ovarian cancer. In another aspect, the ovarian cancer is serous ovarian cancer. In another aspect, the ovarian cancer is high-grade serous ovarian cancer (HGSOC). In another aspect, the ovarian cancer is advanced or metastatic ovarian cancer. In another aspect, the ovarian cancer is metastatic high-grade serous ovarian cancer (HGSOC). In yet another aspect, the subject with ovarian cancer has previously been treated with platinum-based chemotherapy.

[0109] In some implementations, the cancer is lung cancer. In one aspect, lung cancer is small cell lung cancer (SCLC). In another aspect, lung cancer is non-small cell lung cancer (NSCLC). In yet another aspect, non-small cell lung cancer (NSCLC) is squamous cell carcinoma. In yet another aspect, non-small cell lung cancer (NSCLC) is adenocarcinoma. In yet another aspect, non-small cell lung cancer (NSCLC) is large cell carcinoma.

[0110] In some embodiments, this disclosure provides a method for treating or preventing a hematologic cancer in a subject who has or is susceptible to cancer by administering a pharmaceutical composition comprising a therapeutically effective amount of crystalline form A, wherein the hematologic cancer is selected from the group consisting of: non-Hodgkin lymphoma, leukemia, multiple myeloma (MM), and myelodysplastic syndromes (MDS). In one aspect, the hematologic cancer is non-Hodgkin lymphoma (NHL). In another aspect, non-Hodgkin lymphoma (NHL) is selected from diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, mantle cell lymphoma (MCL), and marginal zone lymphoma. In another aspect, the hematologic cancer is leukemia. In another aspect, leukemia is selected from the group consisting of: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML). In another aspect, the hematologic cancer is multiple myeloma (MM). On the other hand, hematologic cancers are myelodysplastic syndromes (MDS).

[0111] In some implementations, the pharmaceutical composition comprising a therapeutically effective amount of crystalline form A is administered as a first-line therapy.

[0112] In some implementations, the pharmaceutical composition comprising a therapeutically effective amount of crystalline form A is administered as a second-line (or subsequent) therapy.

[0113] In some implementations, subjects who are given a pharmaceutical composition comprising a therapeutically effective amount of crystalline form A exhibit a partial response (PR) in response to such treatment.

[0114] In some implementations, subjects who are given a pharmaceutical composition containing a therapeutically effective amount of crystalline form A exhibit a complete response (CR) to such treatment.

[0115] In some implementations, subjects who were given a pharmaceutical composition comprising a therapeutically effective amount of crystalline form A exhibited improved progression-free survival (PFS) in response to such treatment.

[0116] In some implementations, subjects who were given a pharmaceutical composition comprising a therapeutically effective amount of crystalline form A showed an improved overall survival (OR) in response to such treatment.

[0117] PR, CR, PFS, and OR can be assessed, for example, according to the RECIST (Responsiveness Evaluation Criteria for Solid Tumors) guidelines (version 1.1).

[0118] The subjects treated are typically human or non-human mammals, especially humans. Suitable subjects may also include domestic or wild animals; companion animals (including dogs, cats, etc.); livestock (including horses, cattle and other ruminants, pigs, poultry, rabbits, etc.); primates (including monkeys such as rhesus monkeys, cynomolgus monkeys (also known as crab-eating macaques or long-tailed macaques), marmosets, tamarins, chimpanzees, macaques, etc.); and rodents (including rats, mice, gerbils, guinea pigs, etc.).

[0119] In some embodiments, this disclosure provides a crystalline form A, which is used as a drug for treating cancers that are wholly or partially mediated by CDK2.

[0120] In some embodiments, this disclosure provides the use of crystalline form A for the treatment of cancers that are wholly or partially mediated by CDK2.

[0121] In some embodiments, this disclosure provides the use of crystalline form A for manufacturing a medicament for treating cancers that are wholly or partially mediated by CDK2.

[0122] IV. Combination therapy and fixed-dose combination

[0123] The crystalline form A disclosed herein can be used as a single pharmacological agent or in combination with other pharmacological agents or technologies in the methods described above. Such combination therapies can be achieved by administering the individual components simultaneously, sequentially, or separately. These combination therapies (and corresponding combination products) utilize crystalline form A within the dosage range described in this specification and other pharmacological agents typically within their approved dosage ranges.

[0124] In some embodiments, this disclosure provides a combination suitable for treating cancers wholly or partially mediated by CDK2, wherein the combination comprises crystalline form A and a cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitor. In one aspect, the CDK4 / 6 inhibitor is selected from the group consisting of: palbociclib, abexicillin, ribociclib, lerociclib (G1T38), trilaciclib (G1T28), dalpiciclib (SHR-6390), and BPI-16350. In another aspect, the CDK4 / 6 inhibitor is selected from the group consisting of palbociclib, abexicillin, ribociclib, and dalpiciclib. In another aspect, the CDK4 / 6 inhibitor is palbociclib. In another aspect, the CDK4 / 6 inhibitor is abexicillin. In another aspect, the CDK4 / 6 inhibitor is ribociclib. In another aspect, the CDK4 / 6 inhibitor is dalpiciclib.

[0125] In some embodiments, this disclosure provides a combination suitable for treating cancers that are wholly or partially mediated by CDK2, wherein the combination comprises crystalline form A and endocrine therapy. In one aspect, the cancer is breast cancer.

[0126] In some embodiments, this disclosure provides a combination suitable for treating cancers wholly or partially mediated by CDK2, wherein the combination comprises crystalline form A and an aromatase inhibitor. In one aspect, the aromatase inhibitor is selected from the group consisting of anastrozole, letrozole, exemestane, vorozole, formestane, and fadrozole. In another aspect, the combination also comprises everolimus. In yet another aspect, the cancer is breast cancer.

[0127] In some embodiments, this disclosure provides a combination suitable for treating cancers wholly or partially mediated by CDK2, wherein the combination comprises crystalline form A and a selective estrogen receptor degrader (SERD). In one aspect, the SERD is selected from the group consisting of: fulvestrant, giredestrant (GDC-9545), amcenestrant (SAR439859), camizestrant (AZD9833), rintodestrant (G1T48), imlunestrant (LY3484356), elacestrant (RAD-1901), taragaestrant (D-0502), OP1250 (Olema), LSZ102 (Novartis), ZN-c5 (Zentalis), and SHR9549 (Jiangsu Hengrui Medicine). In another aspect, SERD is selected from the group consisting of: fulvestrant, girarubicin, carmicostigin, ilustrant, and elastin. In another aspect, SERD is fulvestrant. In another aspect, SERD is fulvestrant, and the administered combination also contains alpelisib. In another aspect, SERD is carmicostigin (AZD9833). In another aspect, SERD is carmicostigin, and the administered combination also contains alpelisib. In another aspect, the cancer is breast cancer.

[0128] In some embodiments, this disclosure provides a combination suitable for treating cancers that are wholly or partially mediated by CDK2, wherein the combination comprises crystalline form A, a selective estrogen receptor degrader (SERD), and a cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitor. In one aspect, SERDs were selected from the group consisting of: fulvestrant, giretraceutonic acid (GDC-9545), ansentraceutonic acid (SAR439859), carmisentraceutonic acid (AZD9833), retraceutonic acid (G1T48), ilulusentraceutonic acid (LY3484356), ellastrantraceutonic acid (RAD-1901), talasentraceutonic acid (D-0502), OP1250 (Olema), LSZ102 (Novartis), ZN-c5 (Zentalis), and SHR9549 (Jiangsu Hengrui Medicine); and CDK4 / 6 inhibitors were selected from the group consisting of: palbociclib, abecilibi, ribociclib, leroximate (G1T38), trilacinib (G1T28), dalcilibi (SHR-6390), and BPI-16350. In another aspect, SERD is selected from the group consisting of: fulvestrant, girarubicin, carmicutane, iliostant, and ellastrant; and CDK4 / 6 inhibitors are selected from the group consisting of: palbociclib, abeciclib, ribociclib, and dalcitabine. In another aspect, SERD is carmicutane; and CDK4 / 6 inhibitors are selected from the group consisting of: palbociclib, abeciclib, and ribociclib. In another aspect, SERD is carmicutane; and CDK4 / 6 inhibitors are selected from the group consisting of: palbociclib, abeciclib, and ribociclib. In another aspect, the cancer is breast cancer.

[0129] In some embodiments, this disclosure provides a combination suitable for treating cancers that are wholly or partially mediated by CDK2, wherein the combination comprises crystalline form A and a PROTAC estrogen receptor degrader (PROTAC ER degrader). In one aspect, the PROTAC ER degrader is vepdegestrant. In another aspect, the cancer is breast cancer.

[0130] In some embodiments, this disclosure provides a combination suitable for treating cancers wholly or partially mediated by CDK2, wherein the combination comprises crystalline form A and a selective estrogen receptor modulator (SERM). In one aspect, the SERM is selected from the group consisting of: anordrin, bazedoxifene, broparestrol, clomifene, cyclofenil, lasofoxifene, ormeloxifene, ospemifene, raloxifene, tamoxifen, and toremifene. In another aspect, the SERM is tamoxifen. In yet another aspect, the SERM is toremifene. In another respect, SERM is selected from the following groups: acolbifene, afimoxifen, enclomifene, endoxifen, and zuclomifene. On another front, SERM is selected from the following groups: azoxifene, brilanestrant, clomifenoxide, droloxifene, etacstil, fispemifene, idoxifene, levormeloxifene, miproxifene, nafoxidine, nitromifene, panomifene, pipendoxifene, trioxifene, zindoxifene, GW-7604 (Glaxo Wellcome), and NNC 45-0095 (NovoNordisk). On yet another front, the cancer is breast cancer.

[0131] In some embodiments, this disclosure provides a combination suitable for treating cancers wholly or partially mediated by CDK2, wherein the combination comprises crystalline form A and an anti-HER2 agent. In one aspect, the anti-HER2 agent is an anti-HER2 monoclonal antibody. In another aspect, the anti-HER2 monoclonal antibody is trastuzumab or pertuzumab. In yet another aspect, the cancer is breast cancer.

[0132] In some embodiments, this disclosure provides a combination suitable for treating cancers wholly or partially mediated by CDK2, wherein the combination comprises crystalline form A and a poly-ADP ribose polymerase (PARP) inhibitor. In one aspect, the PARP inhibitor is selected from the group consisting of olaparib, rucaparib, niraparib, talazoparib, and saruparib (AZD5305; CAS No. 2589531-76-8). In another aspect, the PARP inhibitor is olaparib. In another aspect, the PARP inhibitor is saruparib (AZD5305). In another aspect, the cancer is breast cancer. In another aspect, the cancer is ovarian cancer.

[0133] In some embodiments, this disclosure provides a combination suitable for treating cancers wholly or partially mediated by CDK2, wherein the combination comprises a crystalline form A and a protein kinase B (Akt) inhibitor. In one aspect, the Akt inhibitor is selected from the group consisting of capivasertib (AZD5363) and ipatasertib (RG7440). In another aspect, the Akt inhibitor is capivasertib. In yet another aspect, the Akt inhibitor is ipatasertib. In yet another aspect, the cancer is breast cancer.

[0134] In some embodiments, this disclosure provides a combination suitable for treating cancers that are wholly or partially mediated by CDK2, wherein the combination comprises crystalline form A and radiotherapy.

[0135] In some embodiments, this disclosure provides a combination suitable for treating cancers that are wholly or partially mediated by CDK2, wherein the combination comprises crystalline form A and chemotherapy.

[0136] In some embodiments, this disclosure provides a combination suitable for treating breast cancer, wherein the combination comprises crystalline form A and chemotherapy. In one aspect, the chemotherapy comprises administration of a combination of cyclophosphamide and doxorubicin (“AC”). In another aspect, the chemotherapy comprises administration of a combination of cyclophosphamide, doxorubicin, and a taxane (such as paclitaxel or docetaxel) (“CAT”). In yet another aspect, the chemotherapy comprises administration of a combination of cyclophosphamide, methotrexate, and fluorouracil (or “CMF”).

[0137] In some embodiments, this disclosure provides a combination suitable for treating ovarian cancer, wherein the combination comprises crystalline form A and chemotherapy. In one aspect, chemotherapy comprises administering one or more chemotherapeutic agents selected from the group consisting of cisplatin, carboplatin, paclitaxel, docetaxel, topotecan, doxorubicin, epirubicin, and gemcitabine. In another aspect, chemotherapy comprises administering a combination of carboplatin and doxorubicin, gemcitabine, paclitaxel, or docetaxel.

[0138] In another aspect, chemotherapy includes the administration of a combination of carboplatin and paclitaxel or docetaxel. In another aspect, chemotherapy includes the administration of topotecan. In another aspect, chemotherapy includes the administration of a combination of bleomycin, etoposide, and cisplatin (BEP). In another aspect, chemotherapy includes the administration of vincristine, dactinomycin, and cyclophosphamide (VAC). In another aspect, chemotherapy includes the administration of a combination of paclitaxel, gemcitabine, and oxaliplatin.

[0139] V. Pharmaceutical Composition

[0140] The crystalline form A of this disclosure can be administered as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients. Therefore, in some embodiments, this disclosure provides a pharmaceutical composition comprising crystalline form A and at least one pharmaceutically acceptable excipient.

[0141] The excipients selected for inclusion in a particular composition will depend on factors such as the mode of administration and the form of the composition provided. Suitable pharmaceutically acceptable excipients are well known to those skilled in the art and are described, for example, in Handbook of Pharmaceutical Excipients, 6th Edition, Pharmaceutical Press, edited by Ray C. Rowe; Paul J. Sheskey; and Marian Quinn. Pharmaceutically acceptable excipients can function as, for example, adjuvants, diluents, carriers, stabilizers, flavoring agents, colorants, fillers, binders, disintegrants, lubricants, flow aids, thickeners, and coating agents. As those skilled in the art will understand, some pharmaceutically acceptable excipients can perform more than one function and can perform alternative functions, depending on the amount of excipient present in the composition and what other excipients are present in the composition.

[0142] The composition may be in the form suitable for oral administration (e.g., as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), suitable for topical administration (e.g., as creams, ointments, gels, or aqueous or oily solutions or suspensions), suitable for inhalation administration (e.g., as fine powders or liquid aerosols), suitable for inhalation administration (e.g., as fine powders), or suitable for parenteral administration (e.g., as sterile aqueous or oily solutions for intravenous, subcutaneous, or intramuscular administration), or suitable for rectal administration as a suppository. The composition can be obtained using conventional pharmaceutical excipients known in the art through conventional processes. Therefore, compositions intended for oral use may contain, for example, one or more colorants, sweeteners, flavoring agents, and / or preservatives.

[0143] The total daily dose will have to vary depending on the subject being treated, the route of administration, any co-administered therapies, and the severity of the disease being treated, and may include single or multiple doses. Specific doses may be adjusted, for example, based on factors such as: the condition being treated; the subject's age, weight, general health condition, sex, and diet; the route of administration; the dosing interval; the excretion rate; and any other drugs co-administered to the subject. Those skilled in the art to whom this disclosure is provided will be able to determine appropriate doses and regimens for administering the therapeutic agent to a subject using methods well known in the field of medicine, and to adjust such doses and regimens as needed during treatment. The crystalline form of this disclosure will generally be administered at a dose of 2.5 mg / m². 2 Animal body surface area up to 5000 mg / m² 2 Administered to warm-blooded animals at a unit dose ranging from approximately 0.05 mg / kg to 100 mg / kg based on the animal's body surface area, and this typically provides a therapeutically effective dose.

[0144] In some embodiments, this disclosure provides a pharmaceutical composition for use in a therapy, comprising crystalline form A and at least one pharmaceutically acceptable excipient.

[0145] In some embodiments, this disclosure provides pharmaceutical compositions for use in treating CDK2-mediated conditions, comprising crystalline form A and at least one pharmaceutically acceptable excipient. In one aspect, the CDK2-mediated condition is a solid tumor cancer. In another aspect, the solid tumor cancer is selected from the group consisting of: breast cancer, ovarian cancer, endometrial cancer, cervical cancer, lung cancer, colorectal cancer, and skin cancer. In another aspect, the solid tumor cancer is selected from the group consisting of: breast cancer, ovarian cancer, endometrial cancer, and lung cancer. In another aspect, the solid tumor cancer is breast cancer or ovarian cancer. In another aspect, the CDK2-mediated condition is breast cancer. In another aspect, the CDK2-mediated condition is ovarian cancer. In another aspect, the CDK2-mediated condition is endometrial cancer. In another aspect, the CDK2-mediated condition is lung cancer.

[0146] In some embodiments, this disclosure provides a pharmaceutical composition comprising crystalline form A and one or more pharmaceutically acceptable excipients. In one aspect, the composition comprises at least 90% by weight of form A relative to any other crystalline form of the compound. In another aspect, the composition comprises at least 95% by weight of form A relative to any other crystalline form of the compound. In another aspect, the composition comprises at least 96% by weight of form A relative to any other crystalline form of the compound. In another aspect, the composition comprises at least 97% by weight of form A relative to any other crystalline form of the compound. In another aspect, the composition comprises at least 98% by weight of form A relative to any other crystalline form of the compound. In another aspect, the composition comprises at least 99% by weight of form A relative to any other crystalline form of the compound. In yet another aspect, the composition comprises form A that is substantially free of any other crystalline form of the compound.

[0147] VI. Reagent Kit

[0148] This disclosure also provides a kit comprising a unit dosage form and a label or packaging insert, the unit dosage form comprising a crystalline form A contained within packaging material, the label or packaging insert indicating that the unit dosage form may be used to treat one or more of the previously described conditions.

[0149] In some embodiments, the kit includes a unit dosage form and a label or packaging insert, the unit dosage form comprising crystalline form A contained within packaging material, the label or packaging insert indicating that the pharmaceutical composition is available for the treatment of CDK2-mediated conditions. In one aspect, the CDK2-mediated condition is a solid tumor cancer. In another aspect, the solid tumor cancer is selected from the group consisting of: breast cancer, ovarian cancer, endometrial cancer, cervical cancer, lung cancer, colorectal cancer, and skin cancer. In another aspect, the solid tumor cancer is selected from the group consisting of: breast cancer, ovarian cancer, endometrial cancer, and lung cancer. In another aspect, the solid tumor cancer is breast cancer or ovarian cancer. In another aspect, the CDK2-mediated condition is breast cancer. In another aspect, the CDK2-mediated condition is ovarian cancer. In another aspect, the CDK2-mediated condition is endometrial cancer. In another aspect, the CDK2-mediated condition is lung cancer.

[0150] In some embodiments, the kit comprises: (a) a first unit dosage form comprising the crystalline form of the present disclosure; (b) a second unit dosage form comprising an agent selected from the group consisting of: CDK4 / 6 inhibitors, aromatase inhibitors, selective estrogen receptor degraders, PROTAC estrogen receptor degraders, selective estrogen receptor modulators, anti-HER2 agents, poly-ADP-ribose polymerase inhibitors, and protein kinase B inhibitors; (c) a container device for containing the first and second dosage forms; and (d) a label or package insert indicating that the first and second unit dosage forms can be used to treat CDK2-mediated conditions.

[0151] In some embodiments, the kit comprises: (a) a first unit dosage form comprising crystalline form A; (b) a second unit dosage form comprising a CDK4 / 6 inhibitor; (c) a container device for containing the first and second dosage forms; and (d) a label or package insert indicating that the first and second unit dosage forms can be used to treat CDK2-mediated conditions. In one aspect, the second unit dosage form comprises a CDK4 / 6 inhibitor selected from the group consisting of palbociclib, abecilib, ribociclib, and dalcilib. In another aspect, the CDK2-mediated conditions are selected from the group consisting of breast cancer, ovarian cancer, endometrial cancer, and lung cancer. In yet another aspect, the CDK2-mediated condition is breast cancer or ovarian cancer.

[0152] In some embodiments, the kit comprises: (a) a first unit dosage form comprising crystalline form A; (b) a second unit dosage form comprising a CDK4 / 6 inhibitor; (c) a third unit dosage form comprising a selective estrogen receptor degrader (SERD); (d) a container device for containing the first and second dosage forms; and (e) a label or package insert indicating that the first and second unit dosage forms can be used to treat CDK2-mediated conditions. In one aspect, the second unit dosage form comprises a CDK4 / 6 inhibitor selected from the group consisting of palbociclib, abecilib, rebociclib, and dalcilib; and the third unit dosage form comprises carmistran (AZD9833). In another aspect, the CDK2-mediated conditions are selected from the group consisting of breast cancer, ovarian cancer, endometrial cancer, and lung cancer. In yet another aspect, the CDK2-mediated condition is breast cancer or ovarian cancer.

[0153] Representative implementation plan:

[0154] Implementation Scheme 1: A crystalline form of N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide.

[0155] Implementation Scheme 2: A crystalline form of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide.

[0156] Implementation Scheme 3: The crystal form according to Implementation Scheme 2, wherein the crystal form is characterized by a powder X-ray diffraction pattern, the powder X-ray diffraction pattern containing at least one peak selected from the group consisting of: 8.1±0.2°2θ, 13.9±0.2°2θ, 16.1±0.2°2θ, 16.7±0.2°2θ and 22.6±0.2°2θ.

[0157] Implementation Scheme 4: The crystal form according to Implementation Scheme 2, wherein the crystal form is characterized by a powder X-ray diffraction pattern, the powder X-ray diffraction pattern containing at least three peaks selected from the group consisting of: 8.1±0.2°2θ, 13.9±0.2°2θ, 16.1±0.2°2θ, 16.7±0.2°2θ and 22.6±0.2°2θ.

[0158] Implementation Scheme 5: The crystal form according to Implementation Scheme 2, wherein the crystal form is characterized by a powder X-ray diffraction pattern containing peaks at 8.1±0.2°2θ, 13.9±0.2°2θ, 16.1±0.2°2θ, 16.7±0.2°2θ and 22.6±0.2°2θ.

[0159] Implementation Scheme 6: The crystalline form according to Implementation Scheme 5, wherein the crystalline form is characterized by a powder X-ray diffraction pattern, the powder X-ray diffraction pattern further comprising at least one peak selected from the group consisting of: 17.7±0.2°2θ, 20.2±0.2°2θ, 21.3±0.2°2θ, 25.9±0.2°2θ and 29.2±0.2°2θ.

[0160] Implementation Scheme 7: According to the crystalline form described in Implementation Scheme 5, wherein the powder X-ray diffraction pattern further includes peaks at 17.7±0.2°2θ, 20.2±0.2°2θ, 21.3±0.2°2θ, 25.9±0.2°2θ and 29.2±0.2°2θ.

[0161] Implementation Scheme 8: The crystalline form according to any one of Implementation Schemes 3 to 7, wherein the powder X-ray diffraction is performed using Cu radiation.

[0162] Implementation Scheme 9: The crystalline form according to any one of Implementation Schemes 3 to 8, wherein the powder X-ray diffraction is performed using a Bruker D8 Advantage diffractometer with reflective geometry, tube voltage of 45 kV and filament emission of 40 mA.

[0163] Implementation Scheme 10: The crystalline form according to Implementation Scheme 3, wherein the powder X-ray diffraction pattern is consistent with... Figure 5-B The powder X-ray diffraction patterns are basically the same.

[0164] Implementation Scheme 11: The crystalline form according to any one of Implementation Schemes 3 to 10, wherein the crystalline form is further characterized by a differential scanning calorimetry curve, the differential scanning calorimetry curve comprising melting endothermic reaction with an initial temperature of about 122°C ± 5°C.

[0165] Implementation Scheme 12: The crystalline form according to any one of Implementation Schemes 3 to 10, wherein the crystalline form is further characterized by a differential scanning calorimetry curve, the differential scanning calorimetry curve comprising melting endothermic reaction with an initial temperature of about 122°C ± 2°C.

[0166] Implementation Scheme 13: The crystalline form according to any one of Implementation Schemes 3 to 10, wherein the crystalline form is further characterized by a differential scanning calorimetry curve, the differential scanning calorimetry curve comprising a melting endothermic peak at approximately 128°C ± 5°C.

[0167] Implementation Scheme 14: The crystalline form according to any one of Implementation Schemes 3 to 10, wherein the crystalline form is further characterized by a differential scanning calorimetry curve, the differential scanning calorimetry curve containing a melting endothermic peak at approximately 128°C ± 2°C.

[0168] Implementation Scheme 15: The crystalline form according to any one of Implementation Schemes 3 to 10, wherein the crystalline form is further characterized by a differential scanning calorimetry curve, the differential scanning calorimetry curve comprising a melting endothermic curve having an initial temperature of about 122°C ± 5°C and a peak at about 128°C ± 5°C.

[0169] Implementation Scheme 16: The crystalline form according to any one of Implementation Schemes 3 to 10, wherein the crystalline form is further characterized by a differential scanning calorimetry curve, the differential scanning calorimetry curve comprising a melting endothermic curve having an initial temperature of about 122°C ± 2°C and a peak at about 128°C ± 2°C.

[0170] Implementation Scheme 17: The crystal form according to any one of Implementation Schemes 11 to 16, wherein the differential scanning calorimetry is performed on a TA Instruments Q2000 differential scanning calorimeter, wherein the sample is placed in an aluminum pan and heated to a temperature of 300°C at a rate of 10°C / min under nitrogen.

[0171] Implementation Scheme 18: According to the crystallization form described in Implementation Scheme 11, wherein the differential scanning calorimetry curve and Figure 6 The differential scanning calorimetry curves are basically the same.

[0172] Implementation Scheme 19: The crystalline form according to any one of Implementation Schemes 3 to 18, further characterized by thermogravimetric analysis pyrometry, wherein the crystalline form exhibits a weight loss of less than about 1.0% by weight from about 25°C to about 100°C.

[0173] Implementation Scheme 20: The crystalline form according to Implementation Scheme 19, wherein the weight loss is less than about 0.5% by weight.

[0174] Implementation Scheme 21: The crystalline form according to Implementation Scheme 19, wherein the weight loss is less than about 0.1% by weight.

[0175] Implementation Scheme 22: According to the crystallization form described in Implementation Scheme 19, wherein the thermogravimetric analysis pyrometry spectrum is consistent with... Figure 7 The thermogravimetric analysis chromatograms are basically the same.

[0176] Implementation Scheme 23: The crystalline form according to any one of Implementation Schemes 3 to 22, wherein the crystalline form is further characterized by a gravimetric vapor adsorption curve, wherein the crystalline form exhibits reversible moisture absorption of less than about 1.0% by weight from about 0% relative humidity to about 80% relative humidity at 25°C ± 0.1°C.

[0177] Implementation Scheme 24: The crystalline form according to Implementation Scheme 23, wherein the reversible water absorption is less than about 0.7% by weight.

[0178] Implementation Scheme 25: The crystalline form according to Implementation Scheme 23, wherein the reversible water absorption is less than about 0.4% by weight.

[0179] Implementation Scheme 26: According to the crystal form described in Implementation Scheme 23, wherein the weight vapor adsorption curve is consistent with... Figure 8 The weight vapor adsorption curves are basically the same.

[0180] Implementation Scheme 27: The crystalline form according to any one of Implementation Schemes 2 to 26, wherein the crystalline form is an anhydrous crystalline substance.

[0181] Implementation Scheme 28: A crystalline form according to any one of Implementation Schemes 3 to 10, wherein the crystalline form is further characterized by: a differential scanning calorimetry curve comprising melting endothermation at an initial temperature of about 122°C ± 5°C; and a gravimetric vapor adsorption curve wherein the crystalline form exhibits reversible moisture absorption of less than about 1.0% by weight from about 0% relative humidity to about 80% relative humidity at 25°C ± 0.1°C.

[0182] Implementation Scheme 29: A crystalline form according to any one of Implementation Schemes 3 to 10, wherein the crystalline form is further characterized by: a thermogravimetric analysis pyrometry spectroscopy, wherein the crystalline form exhibits a weight loss of less than about 1.0 wt% from about 25°C to about 100°C; and a gravimetric vapor adsorption curve, wherein the crystalline form exhibits reversible moisture absorption of less than about 1.0 wt% from about 0% relative humidity to about 80% relative humidity at 25°C ± 0.1°C.

[0183] Implementation Scheme 30: A crystalline form according to any one of Implementation Schemes 3 to 10, wherein the crystalline form is further characterized by: a differential scanning calorimetry curve comprising melting endothermation at an initial temperature of about 122°C ± 5°C; a thermogravimetric analysis pyrometry spectroscopy curve wherein the crystalline form exhibits a weight loss of less than about 1.0 wt% from about 25°C to about 100°C; and a weight vapor adsorption curve wherein the crystalline form exhibits reversible moisture absorption of less than about 1.0 wt% from about 0% relative humidity to about 80% relative humidity at 25°C ± 0.1°C.

[0184] Implementation Scheme 31: The crystalline form according to any one of Implementation Schemes 3 to 30, wherein the crystalline form is further characterized by the following: solid state 13 C NMR spectrum, the solid 13The C NMR spectrum contains one, two, three or four peaks selected from the following groups: 161.2±0.2ppm, 137.6±0.2ppm, 113.1±0.2ppm, 72.3±0.2ppm and 60.0±0.2ppm.

[0185] Implementation Scheme 32: The crystalline form according to any one of Implementation Schemes 3 to 30, wherein the crystalline form is further composed of a solid state. 13 C NMR spectroscopy characterization of the solid state 13 The C NMR spectrum includes peaks at 161.2±0.2ppm, 137.6±0.2ppm, 113.1±0.2ppm, 72.3±0.2ppm, and 60.0±0.2ppm.

[0186] Implementation Scheme 33: The crystalline form according to any one of Implementation Schemes 3 to 30, wherein the crystalline form is further composed of a solid state. 13 C NMR spectroscopy characterization of the solid state 13 The C NMR spectrum includes peaks at 161.2±0.2ppm, 153.9±0.2ppm, 152.8±0.2ppm, 137.6±0.2ppm, 113.1±0.2ppm, 72.3±0.2ppm, 60.0±0.2ppm, 54.9±0.2ppm, 51.0±0.2ppm, 47.2±0.2ppm, 40.1±0.2ppm, 39.1±0.2ppm, 29.3±0.2ppm, 23.1±0.2ppm, 17.1±0.2ppm, 16.5±0.2ppm, 13.3±0.2ppm, and 10.7±0.2ppm.

[0187] Implementation Scheme 34: The crystalline form according to Implementation Scheme 31, wherein the solid state... 13 C NMR spectrum and solid state in Figure 10 13 The C NMR spectra are basically the same.

[0188] Implementation Scheme 35: The crystalline form according to any one of Implementation Schemes 28 to 34, wherein the crystalline form is anhydrous crystalline material.

[0189] Implementation Scheme 36: The crystalline form according to any one of Implementation Schemes 3 to 35, wherein the crystalline form comprises less than 5% by weight of any other crystalline form of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)-pyrrolidine-1-sulfonamide.

[0190] Implementation Scheme 37: The crystalline form according to any one of Implementation Schemes 3 to 35, wherein the crystalline form is substantially free of any other crystalline form of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)-pyrrolidine-1-sulfonamide.

[0191] Embodiment 38: A pharmaceutical composition comprising a crystalline form according to any one of Embodiments 1 to 37 and one or more pharmaceutically acceptable excipients.

[0192] Implementation Scheme 39: A method for treating or preventing CDK2-mediated disease in a subject who has or is susceptible to such disease, the method comprising administering to the subject a therapeutically effective amount of the crystalline form according to any one of Implementation Schemes 1 to 37.

[0193] Implementation Scheme 40: The method according to Implementation Scheme 39, wherein the CDK2-mediated diseases are selected from the group consisting of: breast cancer, ovarian cancer, endometrial cancer, cervical cancer, uterine cancer, gastric cancer, prostate cancer, bladder cancer, lung cancer, esophageal cancer, head and neck cancer, kidney cancer, liver cancer, pancreatic cancer, thyroid cancer, colorectal cancer, and skin cancer.

[0194] Implementation Scheme 41: The method according to Implementation Scheme 39, wherein the CDK2-mediated condition is breast cancer.

[0195] Implementation Scheme 42: The method according to Implementation Scheme 39, wherein the CDK2-mediated condition is ovarian cancer.

[0196] Implementation Scheme 43: Use of the crystalline form according to any one of Implementation Schemes 1 to 37 for the manufacture of a medicament for the treatment or prevention of CDK2-mediated conditions.

[0197] Implementation Scheme 44: A kit comprising a unit dosage form and a label or packaging insert, the unit dosage form comprising a crystalline form according to any one of Implementation Schemes 1 to 37 contained within packaging material, the label or packaging insert indicating that the unit dosage form is capable of treating CDK2-mediated conditions.

[0198] VII. Examples

[0199] The following descriptions of experiments, processes, examples, and intermediates are intended to illustrate embodiments of this disclosure. They are in no way intended to be limiting. Other embodiments of this disclosure may be prepared using the methods described in these examples alone or in combination with techniques generally known in the art.

[0200] Example 1: (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine Preparation of (-6-yl)amino)pyrrolidine-1-sulfonamide

[0201]

[0202] (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide can be prepared as described below:

[0203] Preparation of A. 6-chloro-9-ethyl-2-fluoro-9H-purine (intermediate 2)

[0204] At 23°C, iodoethane (5.15 mL, 63.75 mmol) was added to a stirred suspension of 6-chloro-2-fluoro-9H-purine (intermediate 1, 10 g, 57.96 mmol) and potassium carbonate (10.01 g, 72.44 mmol) in dimethyl sulfoxide (DMSO, 52.8 mL). The resulting suspension was stirred at 23°C for 20 h. The reaction mixture was diluted with water, adjusted to pH 7–8 with glacial acetic acid, and then extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by rapid silica gel chromatography, eluting a gradient of 0% to 100% ethyl acetate (EtOAc) / hexane. The product fraction was concentrated to give 6-chloro-9-ethyl-2-fluoro-9H-purine (intermediate 2, 7.00 g, 60.2%). 1 ¹H NMR (chloroform-d, 300MHz) δ 1.58 (3H, t), 4.31 (2H, q), 8.11 (1H, s); m / z (ES) + [M+H] + = 201; and 6-chloro-7-ethyl-2-fluoro-7H-purine, both of which are grayish-white solids.

[0205] B.(S)-3-((9-ethyl-2-fluoro-9H-purin-6-yl)amino)-pyrrolidine-1-carboxylic acid tert-butyl ester (intermediate 3) Preparation

[0206] Under nitrogen atmosphere, 6-chloro-9-ethyl-2-fluoro-9H-purine (intermediate 2, 2.30 g, 11.47 mmol) and (S)-3-aminopyrrolidine-1-carboxylic acid tert-butyl ester (2.093 g, 11.24 mmol) were dissolved in dimethylformamide (DMF, 15 mL), cooled to 0 °C, and then N,N-diisopropylethylamine (4.01 mL, 22.93 mmol) was added dropwise. The reaction mixture was heated to 80 °C and stirred for 1 hour. The solvent was removed under reduced pressure, and the white residue was purified by rapid silica gel chromatography, eluting with a gradient of 50% to 100% EtOAc / hexane, followed by 15% methanol (MeOH) / dichloromethane (DCM). The product fraction was concentrated under reduced pressure to give (S)-3-((9-ethyl-2-fluoro-9H-purine-6-yl)amino)pyrrolidine-1-carboxylic acid tert-butyl ester (intermediate 3, 2.37 g, 59.1%), which was a white solid. 1 H NMR (300MHz, DMSO-d6) δ 1.37 – 1.45 (12H, m), 2.00 (1H, s), 2.14(1H, s), 3.16 – 3.31 (2H, m), 3.38 – 3.66 (2H, m), 4.12 (2H, q), 4.58 (1H,s), 8.18 (1H,s), 8.56 (1H,s). m / z (ES + [M+H] + = 351.

[0207] C.(S)-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrene Preparation of tert-butyl 1-carboxylate (intermediate 4)

[0208] (S)-3-((9-ethyl-2-fluoro-9H-purin-6-yl)amino)pyrrolidine-1-carboxylic acid tert-butyl ester (intermediate 3, 0.903 g, 2.58 mmol), (2R,3S)-3-aminopentan-2-ol (0.857 mL, 7.73 mmol), and N,N-diisopropylethylamine (DIEA, 1.350 mL, 7.73 mmol) were dissolved in n-butanol (6.13 mL) / dimethyl sulfoxide (0.613 mL), and the reaction mixture was heated at 100 °C. The reaction mixture was stirred at 100 °C for 65 hours, and then stirred at 120 °C for another 16 hours. The reactants were concentrated and purified by rapid C18 chromatography, eluting with a gradient of 0% to 100% acetonitrile / water (containing 0.1% formic acid additive), to give (S)-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purin-6-yl)amino)pyrrolidine-1-carboxylic acid tert-butyl ester (intermediate 4, 0.700 g, 62.7%) as a white solid. m / z (ES +[M+H] + = 434.

[0209] D.(2R,3S)-3-((9-ethyl-6-(((S)-pyrrolid-3-yl)amino)-9H-purin-2-yl)amino)pentan- Preparation of 2-ol hydrochloride (intermediate 5)

[0210] Add (S)-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purin-6-yl)amino)pyrrolidine-1-carboxylic acid tert-butyl ester (intermediate 4, 0.240 g, 0.55 mmol), dichloromethane (3 mL), and hydrogen chloride (0.692 mL, 2.77 mmol) (4 M solution in 1,4-dioxane) to a scintillation vial. Seal the vial and stir at room temperature (rt) for 16 hours. Concentrate the reaction mixture under vacuum to give (2R,3S)-3-((9-ethyl-6-(((S)-pyrrolidine-3-yl)amino)-9H-purin-2-yl)amino)pentan-2-ol·HCl (intermediate 5, 0.205 g, 100%) as a white solid. m / z (ES + [M+H] + =334.

[0211] E.(S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl) Preparation of amino)pyrrolidine-1-sulfonamide

[0212] Weigh (2R,3S)-3-((9-ethyl-6-(((S)-pyrrolidine-3-yl)amino)-9H-purine-2-yl)amino)pentan-2-ol·HCl (intermediate 5, 0.482 g, 1.30 mmol) into a 40 mL scintillation vial. Add dichloromethane (20 mL) and triethylamine (0.903 mL, 6.52 mmol), and cool the reaction mixture to -78 °C. Add ethylamine sulfonyl chloride (0.177 g, 1.17 mmol) to the reaction mixture and stir for 2 hours. Add another ethylamine sulfonyl chloride (0.030 g, 0.20 mmol) and stir for 1 hour. Quench the reaction mixture with an aqueous solution of NaHCO3, heat to 25 °C, stir for 15 minutes, extract with DCM, separate the organic layer, dry over MgSO4, and concentrate to obtain a white foam. The solid was purified by rapid silica gel chromatography using 0%-20% MeOH / DCM to give (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purin-6-yl)amino)pyrrolidine-1-sulfonamide (Example 1, 0.302 g, 52.6%). 1H NMR (400MHz, DMSO-d6) 0.86 (3H,t), 0.97 – 1.1 (6H, m), 1.27 – 1.49 (4H, m), 1.69 (1H, ddd), 2.05 (1H, dt),2.20 (1H, td), 2.95 (2H, qd), 3.09 (1H, dd), 3.21 (1H, dt), 3.33 – 3.42 (1H,m), 3.54 (1H, dd), 3.58 – 3.66 (1H, m), 3.71 – 3.82 (1H, m), 3.98 (2H, q), 4.64 (2H, s), 5.96 (1H, s), 6.93 – 7.62 (2H, m), 7.73 (1H, s); m / z (ES + [M+H] + = 441.

[0213] Example 2: Analysis Method

[0214] Unless otherwise stated, the following analytical methods were used to characterize the amorphous and crystalline forms described in the examples:

[0215] A. Reflected Powder X-ray Diffraction

[0216] X-ray diffraction analysis was performed according to standard methods, which can be found, for example, Kitaigorodsky, AI (1973), Molecular Crystals and Molecules, Academic Press, New York; Bunn, CW (1948), Chemical Crystallography, Clarendon Press, London; or Klug, HP and Alexander, LE (1974), X-ray Diffraction Procedures, John Wiley & Sons, New York.

[0217] Powder X-ray diffraction (PXRD) patterns were determined by mounting the sample on a zero-background holder, single-crystal silicon, and unfolding the sample into a thin layer. PXRD was recorded using a θ-θ Bruker D8 Advantage (X-ray wavelength 1.5418 Å – nickel-filtered Cu radiation, voltage 45 kV, filament emission 40 mA). Variable divergence and anti-scattering slits, as well as 0.04° incident and diffraction Soler slits, were used. The sample was rotated during the measurement. A 0.013° step size and a 115.770 s counting time were used with a PIXcel1D detector (effective length 3.35°²). Together from 5 to 40°2 Scan the sample. PXRD images were obtained using Bragg-Brentano geometry.

[0218] Those skilled in the art will recognize that, depending on the measurement conditions, such as the equipment or machine used, PXRD patterns with one or more measurement errors can be obtained (Jenkins, R & Snyder, RL'Introduction to X-Ray Powder Diffractometry' John Wiley & Sons 1996; Bunn, CW (1948), Chemical Crystallography, Clarendon Press, London; Klug, HP & Alexander, LE (1974), X-Ray Diffraction Procedures). Those skilled in the art of powder X-ray diffraction will further recognize that the relative intensity of peaks can be affected, for example, by grain sizes greater than 30 micrometers and by non-uniform aspect ratios that may influence sample analysis. Those skilled in the art will further understand that intensity can fluctuate depending on experimental conditions and sample preparation (e.g., preferred orientation). The following definitions are used for relative intensity (%): 25%–100%, vs (very strong); 10%–25%, s (strong); 3%–10%, m (moderate); 1%–3%, w (weak).

[0219] Those skilled in the art will also recognize that the position of the reflection can be affected by the precise height of the sample within the diffractometer and the zero calibration of the diffractometer. The surface flatness of the sample may also have a slight influence. Therefore, the presented diffraction pattern data are not considered absolute values. Typically, the measurement error of the diffraction angle in a powder X-ray diffraction pattern can be approximately plus or minus 0.2. ° 2 Furthermore, this level of measurement error should be taken into account when considering PXRD data.

[0220] A reflection mode PXRD pattern can be compared with a transmission mode PXRD pattern, but those skilled in the art will recognize that the diffraction pattern can be altered, especially with respect to peak intensity.

[0221] B. Differential Scanning Calorimetry (DSC)

[0222] The initial melting point temperature (T) was determined using a TA Instruments DSC Q2000 differential scanning calorimeter. m Weigh the sample (approximately 1 mg–3 mg) into an aluminum sample dish. Fill the sample dish to the bottom and cover it with a cap featuring a pinhole. Purge the instrument with nitrogen at 50 mL / min and collect data between 25 °C and 210 °C–250 °C using a heating rate of 10 °C / min.

[0223] C. Thermogravimetric analysis (TGA)

[0224] Thermogravimetric analysis was performed using a TA Instruments TGA (model Q500). The sample (approximately 10 mg) was transferred to a balanced sample holder. The instrument was purged with nitrogen, heated to an oven at 60 mL / min, and balanced at 40 mL / min. Data were collected between room temperature and 300 °C using a heating rate of 10 °C / min. During heating, the buoyancy effect will cause the observed weight increase. This effect can be reduced by using more than 15 mg of material or by baseline subtraction on the sample curve.

[0225] D. Gravimetric vapor adsorption (GVS)

[0226] Gravimetric adsorption analysis was performed using a TA Instruments TGA (model Q5000SA). Samples (approximately 5-10 mg) were transferred to a balanced sample holder. The instrument was purged with nitrogen at 25°C, chamber flow rate 200 mL / min, and balance flow rate 10 mL / min, and data were collected at the unique peak relative humidity (%RH). The equilibration standard was established by starting at 40% RH and gradually increasing to 90% RH, then gradually decreasing to 0% RH, with a final second cycle increasing to 90% RH and returning to 0% RH. The equilibrium standard for moving to the next %RH was reached when the drift standard (dm / dt) remained below 0.002 for 10 minutes.

[0227] Hygroscopicity can be assessed, for example, according to the European Pharmacopoeia (EP) classification: non-hygroscopic: <0.2%; slightly hygroscopic: ≥0.2% and <2%; hygroscopic: ≥2% and <15%; very hygroscopic: ≥15%; deliquescent: absorbs enough water to form a liquid; all values ​​are measured as weight gain at 80% RH and 25°C.

[0228] Example 3: Amorphous (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)- 9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide

[0229] A. Amorphous (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine Preparation of (-6-yl)amino)pyrrolidine-1-sulfonamide

[0230] (i) Evaporation from acetone

[0231] 50 mg of crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide was dissolved in acetone (1.0 mL). The resulting clear, colorless solution was evaporated to dryness under vacuum using a GeneVac. A white powder was obtained. The separated material was analyzed by PXRD, which showed that the material was amorphous.

[0232] (ii) Evaporation from methanol

[0233] 50 mg of crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide was dissolved in methanol (1.0 mL). The resulting clear, colorless solution was evaporated to dryness under vacuum using a GeneVac. A gel with needle-like crystals was obtained.

[0234] (iii) Evaporation from tetrahydrofuran

[0235] The material obtained from the methanol evaporation step was dissolved in tetrahydrofuran (1.0 mL). The resulting clear, colorless solution was evaporated to dryness under vacuum using a GeneVac. A gel was obtained.

[0236] B. Amorphous (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine Physical characterization of 1-yl-amino-pyrrolidine-1-sulfonamide

[0237] Amorphous (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide was obtained from PXRD ( Figure 1 Differential scanning calorimetry (DSC) Figure 2 Thermogravimetric analysis (TGA) Figure 3 ) and weight vapor adsorption (GVS) Figure 4 ) characterization. Figure 1 The PXRD pattern shows that the amorphous compound does not have regular crystalline order. Figure 4 The GVS curve shows that the amorphous compound is hygroscopic, absorbing more than 2.0% by weight of water at an 80% relative humidity level.

[0238] Example 4: Crystallization Study

[0239] Crystallization studies are conducted as described below.

[0240] A. Acetone / water solvent system

[0241] Amorphous (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide (10 mg) was dissolved in acetone (0.2 mL) at room temperature. Water (0.8 mL) was added. The resulting clear solution was evaporated in air at room temperature to give needle-like crystals. PXRD analysis showed that the separated material was crystalline form A.

[0242] B. Ethyl acetate / heptane solvent system

[0243] Amorphous (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide (10 mg) was dissolved in ethyl acetate (0.2 mL) at room temperature. Heptane (0.8 mL) was slowly added to the clear solution, and a gel formed in the solution. After approximately 30 minutes, the gel solidified, yielding a suspension of a white solid. The suspension was stirred at room temperature for two days to obtain a slurry. The solid material separated from the slurry was analyzed by PXRD, which showed that the material was in crystalline form A.

[0244] C. Methyl tert-butyl ether solvent system

[0245] Amorphous (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide (10 mg) was suspended in methyl tert-butyl ether (0.2 ml) at room temperature. A gel formed after approximately five minutes. More methyl tert-butyl ether (0.3 ml) was added to the suspension. The gel solidified, yielding a slurry. The slurry was stirred at room temperature for two days to obtain a slurry. Analysis of the solid material separated from the slurry by PXRD showed that the material was in crystalline form A.

[0246] D. Aqueous solvent system

[0247] Amorphous (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide (10 mg) was dissolved in water (0.2 mL) at room temperature. A gel formed, and more water (0.3 mL) was added. The resulting suspension was stirred at room temperature for two days to obtain a slurry. The solid material separated from the slurry was analyzed by PXRD, which showed that the material was in crystalline form A.

[0248] E. Methyl tert-butyl ether solvent system (under form A inoculation)

[0249] Amorphous (S)-N-ethyl-3-((9-ethyl-2-((((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purin-6-yl)amino)pyrrolidine-1-sulfonamide (100 mg) was suspended in methyl tert-butyl ether (2.0 mL) at room temperature. Form A seed crystals (2 mg to 3 mg) were added. After five minutes, the amorphous material turned into a gel, while the form A seed crystals remained in solid form. After 15 minutes, the gel began to solidify, and after one hour, solid clusters formed. The solid clusters were broken up with a spatula, and the resulting slurry was stirred at room temperature for one day. Methyl tert-butyl ether was evaporated from the slurry to give a white solid. PXRD analysis showed that the separated material was crystalline form A. Thermal analysis showed that the separated material was anhydrous and had a melting point of 119 °C.

[0250] F. Ethyl acetate solvent system (under the condition of inoculation in form A)

[0251] Amorphous (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide (10 mg) was suspended in ethyl acetate (0.2 mL) at room temperature. Seed crystals of form A (approximately 1 mg) were added. After a few minutes, a white solid began to precipitate (without forming a gel). The resulting slurry was stirred at room temperature for one day. Heptane (0.20 mL) was added to increase precipitation. The white solid (5-6 mg) was collected by filtration and dried in air. PXRD analysis showed that the separated material was crystalline form A. Alternatively, form A can be obtained by evaporating ethyl acetate from the slurry without first adding heptane to the slurry.

[0252] Example 5: Crystallization Study

[0253] Crystallization studies are conducted as described below:

[0254] A. Water-based slurry

[0255] Crystalline form A (10 mg) of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide was suspended in water (0.5 ml) at room temperature and kept for one day. Acetone (1.0 ml) was then added to the suspension, and the resulting solution was evaporated in air at room temperature to give a white solid. The test was repeated for the suspension kept at room temperature for 5 days, and a white solid was again obtained. The test was repeated a third time for the suspension kept at 60°C for two hours, and a white solid was again obtained. PXRD analysis of the separated white solids after evaporation indicated that they were all form A.

[0256] B. Ethyl acetate slurry

[0257] 10 mg of crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide was suspended in ethyl acetate (0.2 ml) for one day at room temperature. Then, another 1.0 ml of ethyl acetate was added to the suspension, and the resulting solution was evaporated in air at room temperature to give a white solid. This test was repeated for the suspension held at room temperature for 5 days, and a white solid was again obtained. The test was repeated a third time for the suspension held at 60°C for two hours, and a white solid was again obtained. PXRD analysis of the separated white solids after evaporation indicated that they were all form A.

[0258] C. Methyl tert-butyl ether slurry

[0259] 10 mg of crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purin-6-yl)amino)pyrrolidine-1-sulfonamide was suspended in 0.2 ml of methyl tert-butyl ether and kept at room temperature for one day. Then, another 2.0 ml of methyl tert-butyl ether was added to the suspension, and the resulting solution was evaporated in air at room temperature to give a white solid. The test was repeated for the suspension kept at room temperature for 5 days, and a white solid was again obtained. The test was repeated a third time for the suspension kept at 60°C for two hours, and a white solid was again obtained. PXRD analysis of the separated white solids after evaporation indicated that they were all form A.

[0260] D. Isopropanol slurry

[0261] 20 mg of crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide was suspended in isopropanol (0.2 ml) at room temperature and kept for one day. Then, additional isopropanol (1.0 ml) was added to the suspension, and the resulting solution was evaporated in air at room temperature to give a white solid. This test was repeated for the suspension kept at room temperature for 5 days, and a white solid was again obtained. The test was repeated a third time for the suspension kept at 60°C for two hours, and a white solid was again obtained. PXRD analysis of the separated white solids after evaporation indicated that they were all form A.

[0262] E. Evaporation from methanol

[0263] 10 mg of crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide was dissolved in methanol (0.5 mL). The resulting solution was evaporated in air at room temperature to give a white solid. PXRD analysis of the separated white solid after evaporation indicated that it was form A. Thermal analysis showed that the separated material was anhydrous and had a melting point of 122 °C.

[0264] F. Evaporation from acetone

[0265] 10 mg of crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide was dissolved in acetone (0.5 mL). The solution was evaporated in air at room temperature to give a white solid. PXRD analysis of the separated white solid after evaporation indicated that it was form A.

[0266] G. Evaporation from acetonitrile

[0267] 10 mg of crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide was dissolved in acetonitrile (0.5 mL). The solution was evaporated in air at room temperature to give a white solid. PXRD analysis of the separated white solid after evaporation indicated that it was form A.

[0268] H. Evaporation from tetrahydrofuran

[0269] 10 mg of crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide was dissolved in tetrahydrofuran (0.5 mL). The solution was evaporated in air at room temperature to give a white solid. PXRD analysis of the separated white solid after evaporation indicated that it was form A.

[0270] I. Evaporation from dichloromethane

[0271] 10 mg of crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purin-6-yl)amino)pyrrolidine-1-sulfonamide was dissolved in 0.5 mL of dichloromethane. The solution was evaporated in air at room temperature to give a white solid. PXRD analysis of the separated white solid after evaporation indicated that it was form A.

[0272] Example 6: Physical characterization of crystalline form A

[0273] Form A was characterized using various techniques, including single-crystal analysis and PXRD (…). Figure 5-B Differential scanning calorimetry (DSC) Figure 6 Thermogravimetric analysis (TGA) Figure 7 ), weight vapor adsorption (GVS) Figure 8 )and 13 C solid-state NMR ( Figure 9 ).

[0274] A. Single crystal analysis

[0275] Single-crystal analysis was performed on form A. A single, clear, colorless needle-like crystal of form A was obtained by slow evaporation of an ethyl acetate solution of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)-pyrrolidine-1-sulfonamide. Suitable crystals were selected and mounted on Hampton rings in perfluoroether oil. X-ray diffraction data at 100 K were collected using a Cryostream 800 (Oxford Cryosystem, UK) in ω-scan mode with an XtaLab Synergy-S (Rigaku, Japan) equipped with a Cu Kα microfocusing source (50 kV, 0.01 mA) and a Hypix-Arc 100 detector. The diffraction patterns were initially indexed, and the total number of runs and images was calculated based on a strategy from the program CrysAlisPro1.171.42.46a (Rigaku, Japan). Data simplification, scaling, and absorption correction were performed using CrysAlisPro 1.171.42.46a (Rigaku, Japan). Numerical absorption correction based on Gaussian integrals over a multifaceted crystal model, implemented in the SCALE3 ABSPACK scaling algorithm, and empirical absorption correction using spherical harmonics were used to correct the integral and scaled data.

[0276] The structure is parsed using a dual-method approach via the ShelXT (Sheldrick, 2015) structure parser, and further parsed using version 2018 / 3 of ShelXL (Sheldrick, 2015) within Olex2 (Dolomanov, 2009) for F. 2 Refinement is performed using full matrix least squares minimization. All non-hydrogen atoms are refined anisotropically. All hydrogen atoms connected to oxygen and nitrogen atoms are located in the differential Fourier map and refined isotropically. All other hydrogen atoms are geometrically determined and refined isotropically.

[0277] Crystal structure analysis showed that form A crystallized in the trigonal space group P3(1) with Z'=1. The asymmetric unit contains one molecule of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)-pyrrolidine-1-sulfonamide, thus the crystal structure is anhydrous form A. The parameters found by Flack (Flack, 1999) and Hooft (Hooft, 2010) were 0.01(3) and -0.016(4), respectively. The crystallographic data are listed in Table 1-A. Figure 5-AThe image shows a thermal ellipsoidal plot of the crystal structure drawn with a 50% probability level.

[0278] Table 1-A

[0279]

[0280] References :

[0281] •Dolomanov, OV, Bourhis, J., Gildea. RJ, Howard, JAK, Puschmann, HJ Appl. Cryst. 42 (2009) 339-431.

[0282] •Hooft, RWW, Straver, LH, Spek, AL, J. Appl. Cryst. 43 (2010)665-668.

[0283] •Flack, HD, Bernardinelli, G. Acta Cryst. A55 (1999) 908-915.

[0284] •Sheldrick, GM, Acta Cryst. C71 (2015) 3-8.

[0285] B.PXRD

[0286] Figure 5-B A representative PXRD plot of form A is shown using reflection geometry measurements. Figure 5-B The PXRD pattern confirms that form A is crystalline. Table 1-B below lists the... Figure 5-B Selected peaks identified in the PXRD plot.

[0287] Table 1-B

[0288]

[0289] The following definitions are used for relative intensity (%): 25%-100%, vs (very strong); 10%-25%, s (strong); 3%-10%, m (moderate); 1%-3%, w (weak).

[0290] Form A shows characteristic peaks at 8.1±0.2°2θ, 13.9±0.2°2θ, 16.1±0.2°2θ, 16.7±0.2°2θ, and 22.6±0.2°2θ. Form A also shows additional characteristic peaks at 17.7±0.2°2θ, 20.2±0.2°2θ, 21.3±0.2°2θ, 25.9±0.2°2θ, and 29.2±0.2°2θ.

[0291] C.DSC

[0292] Figure 6 Display a representative differential scanning calorimetry (DSC) thermogram of format A. Exothermic events are plotted upwards. Figure 6 The melting endothermic process shown has an onset temperature of approximately 122 °C and an enthalpy of approximately 105 J / g. The obtained DSC values ​​can vary by up to ±5 °C, depending on the instrument used, how the sample was prepared, and batch-to-batch variability.

[0293] D.TGA

[0294] Figure 7 The representative thermogravimetric analysis (TGA) chromatogram of form A is shown. Form A exhibits a weight loss of less than about 1.0% by weight when heated from about 25°C to 100°C, which confirms that form A is anhydrous.

[0295] E.DVS

[0296] Figure 8 The diagram shows a representative gravimetric vapor adsorption (GVS) curve for form A. Form A exhibits reversible moisture absorption of approximately 0.3 wt% at 25 °C ± 0.1 °C between 0% and 80% relative humidity. The desorption curve indicates that form A loses moisture at a rate similar to that gained during adsorption, with a limited hysteresis. No change in form was observed by PXRD after the GVS experiment.

[0297] Section 5.11 of the European Pharmacopoeia 6.0 provides the following hygroscopicity classifications based on specified analytical methods:

[0298]

[0299] 1 Percentage of water absorbed at 25°C / 80% RH during the first adsorption cycle of the adsorption isotherm.

[0300] F. 13 C solid-state NMR

[0301] pass 13 C solid-state NMR analysis, form A. The obtained... 13The C20 solid-state NMR spectrum is consistent with that of sample A, which is in a highly crystalline form with Z'=1.

[0302] Approximately 100 mg of Form A was loaded into a 4 mm Magic Angle Spinning (MAS) rotor, which was then placed within a 4 mm HX probe (H12138_0092) mounted on a Bruker Avance IIIHD solid-state NMR spectrometer (Dopey). The rotor was rotated around the magic angle at a frequency of 12 kHz. A series of data were recorded as the cycle delay value increased. 1 H-direct polarization (DP) MAS spectra. When the spectral intensity no longer increases, the cyclic delay value is recorded and used in subsequent experiments. The cyclic delay measured above is used to record... 1 H- 13 C-cross polarization (CP) MAS spectrum. Using a contact pulse length of 2 ms. 1 The external reference for the H spectrum shows an isotropic peak for adamantane at 1.85 ppm, and... 13 The C-spectrum external reference peak at 176.03 ppm is attributed to the carbonyl carbon of glycine. All spectra are stored on the spectrometer.

[0303] As the loop delay value increases (1 second, 5 seconds, 10 seconds, 20 seconds, 30 seconds, 45 seconds, 60 seconds), the recording format A... 1 HDPMAS spectrum. Recorded with a 20-second circular delay, the initial spectral intensity showed no further increase. 1 H DPMAS spectrum. Figure 9 Display based on 1 The H DPMAS experimental results were recorded in the form of a 20-second loop delay. 13 C CPMAS spectrum. The peaks in the spectrum are very sharp, which is consistent with the high crystallinity of the sample. By comparing the number of analytical peaks (18) with the number of carbon atoms in the molecular formula of compound (18), the number of molecules in the asymmetric unit cell (Z') was determined to be 1.

[0304] Form A shows characteristic peaks at 161.2±0.2ppm, 137.6±0.2ppm, 113.1±0.2ppm, 72.3±0.2ppm, and 60.0±0.2ppm. Form A shows additional characteristic peaks at 161.2±0.2ppm, 153.9±0.2ppm, 152.8±0.2ppm, 137.6±0.2ppm, 113.1±0.2ppm, 72.3±0.2ppm, 60.0±0.2ppm, 54.9±0.2ppm, 51.0±0.2ppm, 47.2±0.2ppm, 40.1±0.2ppm, 39.1±0.2ppm, 29.3±0.2ppm, 23.1±0.2ppm, 17.1±0.2ppm, 16.5±0.2ppm, 13.3±0.2ppm, and 10.7±0.2ppm.

[0305] G. Unit cell parameters

[0306] High-resolution X-ray powder diffraction ("HR-XRPD") data of the crystalline form A of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide was collected at room temperature using a germanium monochromator with Cu Kα1 radiation (1.54056 Å) on a D8 Advance diffractometer. Diffraction data were collected in the 2θ range of 2.15–41.5°. Detector scans were performed on a solid-state LynxEye detector at a scan rate of 15 sec / step using 0.0157° steps. The sample was measured in an 8 mm long glass capillary with an outer diameter of 0.5 mm. The analyzed material was polymorphically pure. No crystalline impurities were detected.

[0307] Cell parameters and crystal system were obtained using the LSI-Index (Coelho, 2003; Coelho & Kern, 2005) indexing procedure. The space group was selected based on the crystal's reflection conditions and density. Cell parameters, purity, and instrument parameters were further refined using the full powder map decomposition method (Pawley, 1981). The following fitting criteria were used:

[0308] •Y o , m and Y c , m These are the observed data and calculated data at data point m, respectively.

[0309] • M is the number of data points.

[0310] • P is the number of parameters.

[0311] •wm This assigns a weight to data point m. For counting statistics, w is given. m =1 / σ(Y o , m ) 2 , where σ(Y) o , m ) is Y o , m Error in

[0312] ; ;

[0313]

[0314] The calculated cell parameters and related information are summarized in Table 1-C below.

[0315] Table 1-C

[0316]

[0317] Example 7: CDK1, CDK2 and CDK4 activities (NanoBRET assay in MCF-7 cells)

[0318] In the breast cancer cell line MCF-7, (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purin-6-yl)amino)pyrrolidine-1-sulfonamide was tested to evaluate its inhibitory effect on CDK1, CDK2, and CDK4 activities. MCF-7 cells transiently transfected with CDK1:CCNB1, CDK2:CCNE1, or CDK4:CCND1 were harvested in complete medium at a density of 1E5 cells / mL and seeded at 20 µL / well in 384-well Greiner 784080 plates using Multidrop Combi and incubated at 37°C and 5% CO2. -2 - Incubate overnight at room temperature. The next day, drain the medium from the wells using a Bluewasher centrifuge (Bluecatbio), then add 10 µL / well of phenol red-free OptiMEM using a Multidrop Combi. Then, dispense the test compounds into the wells using an Echo instrument (555 / 655, Beckman Coulter). Immediately after adding the compounds, dispense the relevant NanoBRET using a Tecan HP300 dispenser. ™ The tracer was dispensed into the CDK1 well (12.5 nl, 400 µM, NanoBRET). ™ TE tracer K-9), CDK2 well (12.5 nl, 200 µM, NanoBRET) ™TE tracer K-9) and CDK4 well (8 nmol, 100 µM, NanoBRET) ™ The plate was incubated with TE Nano-Glo (K-7) at 37°C and 5% CO₂ for 2 hours. After cooling to room temperature for 10 minutes, 5 µL / well of TE Nano-Glo was added at 2.4 µM and 1:500 (N₂162 Promega), respectively. ® Substrate / inhibitor. Incubate the plate under low light for 10 minutes, then use NanoBRET. ™ The filter modules (460±80nm / 610nm-LP) were read on a Pherastar FS plate reader (BMG Technologies). The ratios were normalized to a control, and the IC50 of the test compounds was determined using Genedata Screener software. 50 value.

[0319] IC50 of the compound in Example 1 50 The values ​​are reported in Table 2 below. The data confirm that the compound has nanomolar potency against CDK2 and is selective for CDK2 relative to CDK1 and CDK4.

[0320] Table 2

[0321]

[0322] 1 IC 50 Report after a single measurement (n=1) or as the average of multiple measurements (n>1).

[0323] Example 8: NPM phosphorylation (imaging assay in MCF-7 cells)

[0324] (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypentan-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide was tested to assess the role of CDK2 inhibition in the cellular context. Specifically, phosphorylation of nucleophosphoryl protein (NPM) was measured to determine whether the compound downregulated NPM phosphorylation in MCF-7 cells. During the cell cycle, the CDK2-cyclin E complex phosphorylates NPM at Thr199, a prerequisite for initiating centrosome replication.

[0325] MCF-7 cells at a density of 2.5E5 cells / mL in complete culture medium were seeded at 40 µL / well into 384-well Greiner 781090 plates using Multidrop Combi and incubated at 37°C and 5% CO2. -2Incubate overnight at room temperature. Then, using an Echo instrument (555 / 655, Beckman Coulter), dispense the test compounds into the wells and incubate the plate at 37°C and 5% CO₂ for 2 hours. Fix the cells by adding 40 µL / well of 8% paraformaldehyde and incubate at room temperature for 10 minutes. Wash the plate three times with 50 µL / well of PBS using a Biotek EL406 washer, then infiltrate in 0.3% Triton X100 PBS solution for 10 minutes at room temperature. After washing as before, block the plate at room temperature with 30 µL / well of 2% BSA (w / v) PBS-T solution for at least 30 minutes. After aspirating the blocking solution, seal the plate and incubate overnight at 4°C in 20 µL / well of primary antibody (CST#3541, 1 / 400, PBS-T containing 0.05% BSA). The plate was washed three times with 50 µL / well PBS-T, and then incubated for one hour at room temperature in the dark with 20 µL / well secondary antibody solution (1 / 500 Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen A11008) and 1 / 10000 Hoechst 33342 (Invitrogen H21492) in 0.05% BSA in PBS-T solution). After washing in PBS as before, each well was left in 40 µL PBS, the plate was sealed, and imaged on a Cell Insight imaging system (Thermo) with 10x objectives and 6 fields of view per well. Cells containing 4N DNA were selected using Hoechst staining. Phosphate-NPM levels were normalized to control, and the IC50 of the test compounds was determined using Genedata Screener software. 50 value.

[0326] IC50 of the compound in Example 1 50 The value is 0.061µM (the average of multiple measurements).

[0327] Example 9: POLR2A Ser2 phosphorylation (imaging assay in MCF-7 cells)

[0328] (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purin-6-yl)amino)pyrrolidine-1-sulfonamide was tested to assess its effect on CDK9 activity in the cellular background. Specifically, phosphorylation of POLR2A Ser2 was measured to determine whether the compound downregulated POLR2A Ser2 phosphorylation in MCF-7 cells. During the cell cycle, CDK9 is a component of a multiprotein complex that phosphorylates POLR2A Ser2, which leads to transcriptional elongation.

[0329] MCF-7 cells at a density of 1.25E5 cells / mL in complete culture medium were seeded at 40 µL / well into 384-well Greiner 781090 plates using Multidrop Combi and incubated at 37°C and 5% CO2. -2 - Incubate overnight at room temperature. Then, dispense the test compounds using an Echo instrument (555 / 655, Beckman Coulter) and incubate the plates at 37°C and 5% CO₂ for 2 hours. Fix T cells by adding 40 µL / well of 8% paraformaldehyde and incubate at room temperature for 10 minutes. Wash the plates three times with 50 µL / well of PBS using a Biotek EL406 washer, then infiltrate in 0.3% Triton X100 PBS solution for 10 minutes at room temperature. After washing as before, block the plates at room temperature with 30 µL / well of 2% BSA (w / v) PBS-T solution for more than 30 minutes. After aspirating the blocking solution, seal the plates and incubate overnight at 4°C in 20 µL / well of primary antibody (CST#13499, 1 / 1000, PBS-T containing 0.05% BSA). The plate was washed three times with 50 µL / well PBS-T, and then incubated for one hour at room temperature in the dark with 20 µL / well secondary antibody solution (1 / 500 Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen A11008) and 1 / 10000 Hoechst 33342 (Invitrogen H21492) in 0.05% BSA in PBS-T solution). After washing in PBS as before, each well was left in 40 µL PBS, the plate was sealed, and imaged on a Cell Insight imaging system (Thermo) with 10x objectives and two fields of view per well. POLR2A Ser2 levels were normalized to control, and the IC50 of the test compounds was determined using Genedata Screener software. 50 value.

[0330] IC50 of the compound in Example 1 50 Value >19.3µM (average of multiple measurements).

[0331] Example 10: Cell proliferation in MCF-7 and OVCAR3 cell lines (EdU assay)

[0332] The (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide was tested to evaluate its inhibitory effect on cell proliferation in the ovarian cell line OVCAR3, which was expanded from breast cancer cell lines MCF-7 and CCNE1.

[0333] Using WellMate, MCF-7 and OVCAR3 cells in RPMI supplemented with 10% fetal bovine serum were seeded into 384-well plates (Greiner, Kremsmunster, Austria; 781091), 30 µL / well. MCF7 and OVCAR3 cells were seeded at 800 and 1200 cells / well, respectively. The test compound was added to the wells using an Echo 555 liquid processor, and the plates were incubated for two days at 37°C and 5% CO2. On day 2, EdU was measured according to the manufacturer's protocol (Thermo Fisher, C10351). Cells were read on an Acumen eX3 instrument. The IC50 of the test compound was determined in Genedata by assessing the ratio of signal intensity at 488 nm to signal area at 405 nm. 50 value.

[0334] IC50 of the compound in Example 1 50 The values ​​are reported in Table 3.

[0335] Table 3

[0336]

[0337] 1 IC 50 Report after a single measurement (n=1) or as the average of multiple measurements (n>1).

[0338] Example 11: Effects on the cell cycle in the OVCAR3 cell line (24-hour compound administration)

[0339] (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide was tested in the CCNE1-amplified ovarian cell line OVCAR3 to assess its effects on the G1 and S phases of the cell cycle and its potential off-target effects on the G2 and M phases of the cell cycle.

[0340] OVCAR3 cells were seeded at 60,000 cells / well in RPMI containing 10% fetal bovine serum in 24-well plates, with four wells per sample. The test compound was diluted with dimethyl sulfoxide (DMSO) in 96-well plates and then added to the wells containing cells. The 24-well plates were incubated at 37°C and 5% CO2 for 20-24 hours. EdU was measured the following day according to the manufacturer's protocol (Thermo Fisher, C10425 EdU Alexa Fluor 488). Antibody staining was performed using PE mouse anti-cleavage PARP (BD Biosciences catalog number 552933) and 4',6-diamidindo-2-phenylindole (DAPI). Cells were then analyzed by flow cytometry.

[0341] The compound of Example 1 induced G1 / S cell cycle arrest, inhibited pRB phosphorylation, and suppressed cell proliferation. Treatment of OVCAR3 cells with the compound of Example 1 at concentrations of 0.03 µM, 0.1 µM, 0.3 µM, 1 µM, and 3 µM increased cell populations in both the G0 and G1 phases.

[0342] Example 12: pRB phosphorylation in the OVCAR3 cell line (Western blot analysis)

[0343] Western blot analysis of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide was used to assess its inhibitory effect on pRB phosphorylation in the CCNE1-amplified ovarian cell line OVCAR3. pRB is a tumor suppressor protein that inhibits cell cycle progression. However, once hyperphosphorylated, pRB is inactivated and the cell cycle can continue.

[0344] On day 1, OVCAR3 cells were seeded at 60 cells / well in 24-well plates using RPMI containing 10% fetal bovine serum. The test compound was diluted in DMSO in 96-well plates and then added to the cells. The treated cells were incubated at 37°C and 5% CO2 for 20-24 hours. On day 2, the cells were washed once with phosphate-buffered saline (PBS) and scraped onto 1% SDS lysis buffer. Pierce was used... ™The protein lysate was quantified using the BCA Protein Assay Kit (ThermoFisher 23225). Equivalent micrograms of protein were diluted in 1x Novex LDS Sample Buffer (Thermo Fisher NP0008) supplemented with reducing agent (ThermoFisher NP0009). The gel sample was loaded into NuPAGE 4% to 12% Bis-Tris buffer and electrophoresed at 200 volts. The sample was then transferred to nitrocellulose using standard wet transfer in 1x NuPAGE Transfer Buffer 10% MeOH. The membrane was blocked for one hour in blocking buffer containing 5% milk PBST (PBS containing 0.2% Tween). The blot was incubated overnight at 4°C with primary anti-Rb (S780 phosphate) antibody (Abcam ab173289) in blocking buffer. After washing three times in PBST, the membrane was incubated with the secondary antibody (CST anti-rabbit IgG, HRP-linked antibody #7074) in blocking buffer at room temperature for one hour. After washing three times in PBST, the membrane was then incubated using SuperSignal. ™ The West Dura Extended Duration Substrate (Thermo Fisher 3707) develops the signal and images it in a chemiluminescence imager.

[0345] Western blot analysis showed that the compound from Example 1 downregulated pRB phosphorylation in the OVCAR3 cell line, further confirming the cell cycle effect reported in Example 11.

[0346] The compounds of Example 1 were similarly analyzed by Western blotting to assess the regulation of pSer2 of RNAPII (a CDK9 phosphate substrate). No regulation of pSer2 of RNAPII was observed for the compounds, indicating that the compounds do not affect CDK9 activity.

[0347] Example 13: Cell proliferation in OVCAR3 and SKOV3 cell lines (CyQUANT) ™ (Measurement)

[0348] Imaging-based assays were used to investigate changes in cell number in OVCAR3 cells (CCNE1 expanded ovarian cancer cell line) and SKOV3 cells (non-expanded ovarian cancer cell line) treated with (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide.

[0349] Cells were seeded at 500 cells per well in 30 μL of RPMI-1640 / 10% FBS / 1x L-Glu in 384-well plates (Perkin-Elmer 6057300) and incubated with the test compound at 37°C and 5% CO2 for 7 days. Cells were then stained using the CyQuant Direct Cell Proliferation Assay Kit (Thermo Cat#C7026) according to the kit protocol. The stained cells were imaged using an Operetta High Content Imaging System to generate cell counts, and the IC50 was calculated relative to the DMSO control. 50 value.

[0350] IC50 of the compound in Example 1 50 The values ​​are reported in Table 4. This compound inhibited cell proliferation more effectively in CCNE-1-amplified OVCAR3 cells than in non-amplified SKOV3 cell lines.

[0351] Table 4

[0352]

[0353] 1 IC 50 Report after a single measurement (n=1) or as the average of multiple measurements (n>1).

[0354]

[0355] Although specific embodiments and examples have been described above, these embodiments and examples are merely illustrative and do not limit the scope of this disclosure. Changes and modifications may be made based on common art without departing from the broader scope of the disclosure as defined in the following claims. For example, any embodiment described herein may be combined with any other suitable embodiment described herein to provide additional embodiments.

Claims

1. A crystalline form of N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide.

2. A crystalline form of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)pyrrolidine-1-sulfonamide.

3. The crystalline form according to claim 2, wherein the crystalline form is characterized by a powder X-ray diffraction pattern comprising one, two, three, or four peaks selected from the group consisting of: 8.1±0.2°2θ, 13.9±0.2°2θ, 16.1±0.2°2θ, 16.7±0.2°2θ, and 22.6±0.2°2θ.

4. The crystalline form according to claim 3, wherein the powder X-ray diffraction is performed using Cu radiation.

5. The crystalline form according to claim 3 or claim 4, wherein the powder X-ray diffraction is performed using a Bruker D8 Advantage diffractometer operating with reflective geometry, a tube voltage of 45 kV, and a filament emission of 40 mA.

6. The crystalline form according to any one of claims 3 to 5, wherein the crystalline form is further characterized by differential scanning calorimetry (DSC) curves, the DSC curves comprising melting endothermic reaction with an initial temperature of about 122°C ± 5°C.

7. The crystalline form according to any one of claims 3 to 5, wherein the crystalline form is further characterized by differential scanning calorimetry (DSC) curves, the DSC curves comprising melting endothermic curves having a peak at approximately 128°C ± 5°C.

8. The crystalline form according to any one of claims 3 to 5, wherein the crystalline form is further characterized by differential scanning calorimetry (DSC) curves, the DSC curves comprising melting endothermic curves having an initial temperature of about 122°C ± 5°C and a peak at about 128°C ± 5°C.

9. The crystalline form according to any one of claims 6 to 8, wherein the differential scanning calorimetry is performed on a TA Instruments Q2000 differential scanning calorimeter, wherein the sample is placed in an aluminum pan and heated to a temperature of 300°C at a rate of 10°C / min under nitrogen.

10. The crystalline form according to any one of claims 3 to 9, wherein the crystalline form is further characterized by thermogravimetric analysis pyrometry, wherein the crystalline form exhibits a weight loss of less than about 1.0% by weight from about 25°C to about 100°C.

11. The crystalline form according to any one of claims 3 to 10, wherein the crystalline form is further characterized by a gravimetric vapor adsorption curve, wherein the crystalline form exhibits reversible moisture absorption of less than about 1.0% by weight from about 0% relative humidity to about 80% relative humidity at 25°C ± 0.1°C.

12. The crystalline form according to any one of claims 3 to 5, wherein the crystalline form is further characterized by the following: Differential scanning calorimetry (DSC) curves, wherein the DSC curves include endothermic melting at an initial temperature of approximately 122°C ± 5°C; and The gravimetric vapor adsorption curve shows that the crystalline form exhibits reversible moisture absorption of less than about 1.0% by weight from about 0% relative humidity to about 80% relative humidity at 25°C ± 0.1°C.

13. The crystalline form according to any one of claims 3 to 5, wherein the crystalline form is further characterized by the following: Thermogravimetric analysis pyrograph, wherein the crystalline form exhibits a weight loss of less than about 1.0 wt% from about 25 °C to about 100 °C; and The gravimetric vapor adsorption curve shows that the crystalline form exhibits reversible moisture absorption of less than about 1.0% by weight from about 0% relative humidity to about 80% relative humidity at 25°C ± 0.1°C.

14. The crystalline form according to any one of claims 3 to 5, wherein the crystalline form is further characterized by the following: Differential scanning calorimetry curve, wherein the differential scanning calorimetry curve includes melting endothermic reaction with an initial temperature of approximately 122°C ± 5°C; Thermogravimetric analysis pyrograph, wherein the crystalline form exhibits a weight loss of less than about 1.0 wt% from about 25 °C to about 100 °C; and The gravimetric vapor adsorption curve shows that the crystalline form exhibits reversible moisture absorption of less than about 1.0% by weight from about 0% relative humidity to about 80% relative humidity at 25°C ± 0.1°C.

15. The crystalline form according to any one of claims 3 to 14, wherein the crystalline form further comprises a solid state. 13 C NMR spectroscopy characterization of the solid state 13 The C NMR spectrum contains one, two, three or four peaks selected from the following groups: 161.2±0.2ppm, 137.6±0.2ppm, 113.1±0.2ppm, 72.3±0.2ppm and 60.0±0.2ppm.

16. The crystalline form according to any one of claims 3 to 15, wherein the crystalline form is an anhydrous crystalline form.

17. The crystalline form according to any one of claims 3 to 16, wherein the crystalline form comprises less than 5% by weight of any other crystalline form of (S)-N-ethyl-3-((9-ethyl-2-(((2R,3S)-2-hydroxypent-3-yl)amino)-9H-purine-6-yl)amino)-pyrrolidine-1-sulfonamide.

18. A pharmaceutical composition comprising a crystalline form according to any one of claims 1 to 17, and one or more pharmaceutically acceptable excipients.

19. A method for treating or preventing CDK2-mediated disease in a subject who has or is susceptible to such disease, the method comprising administering to the subject a therapeutically effective amount of the crystalline form according to any one of claims 1 to 17.

20. Use of the crystalline form according to any one of claims 1 to 17 for the manufacture of a medicament for the treatment or prevention of CDK2-mediated conditions.

21. A kit comprising a unit dosage form and a label or packaging insert, the unit dosage form comprising a crystalline form according to any one of claims 1 to 17 contained within packaging material, the label or packaging insert indicating that the unit dosage form is capable of treating CDK2-mediated conditions.

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