Polymorphism of 5-aza-4'-thio-2'-deoxycytidine

Abstract

The present disclosure provides crystalline polymorphs of 5-aza-4'-thio-2'-deoxycytidine. The crystalline polymorphs may be formulated into pharmaceutical compositions, optionally in combination with additional chemotherapeutic agents. The crystalline polymorphs are useful for treating various diseases, including myelodysplastic syndromes and blood cancers such as leukemia. Processes for preparing the crystalline polymorphs of 5-aza-4'-thio-2'-deoxycytidine are also disclosed. This Abstract is intended as a scanning tool for searching in a particular technical field and is not intended to limit the invention.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Patent Application No. 63 / 055,754, filed on July 23, 2020, the content of which is incorporated herein by reference in its entirety.

Background Art

[0002] Decitabine (also known as Dacogen® or 5 - aza - 2'-deoxycytidine) is a pyrimidine nucleoside analog of cytidine that induces DNA hypomethylation by inhibiting DNA methyltransferase. Specifically, decitabine functions by incorporating into the DNA strand during replication. Then, when a DNA methyltransferase (DNMT), such as DNMT1, binds to the DNA and engages to replicate methylation to the daughter strand, the DNMT binds irreversibly to decitabine and cannot be released. Thus, the decitabine effect is division - dependent, and cells must divide for the drug to act. Therefore, cells that divide much faster than most other cells in the body (e.g., cancer cells) are more profoundly affected by decitabine. It is used in the treatment of cancers such as leukemia, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), where high methylation of DNA is important in their pathogenesis.

[0003] 5 - Aza - 4'-thio - 2'-deoxycytidine (“aza - T - dCyd”) is a thio - substituted derivative of decitabine that has been subjected to initial clinical evaluation by the National Cancer Institute (NCI). This DNMT1 inhibitor has recently attracted attention due to its high DNMT removal and inhibitory activity in cells, reduced degradation rate by cytidine deaminase, and relatively low production of toxic by - products compared to conventional compounds having a 5 - azacytidine backbone. Similar to decitabine, aza - T - dCyd can be prepared in various forms and crystal structures.

[0004] U.S. Patent No. 5,591,722 relates to 2'-deoxy-4'-thioribonucleosides and intermediates useful for treating viral diseases and describes a general formula including 5-azacitidine compounds. U.S. Patent Publication No. 2006 / 0014949 reports polymorphs of decitabine. Thottassery et al. (Cancer Chemother Pharmacol, 2014) reported aza-T-dCyd. In clinical trial NCT04167917, a Phase I trial of aza-T-dCyd in MDS and AML has been reported and is scheduled to be completed in 2025. Despite these advancements, the polymorphs of aza-T-dCyd have remained elusive thus far.

Summary of the Invention

[0005] In accordance with the objectives of the present invention, as embodied and broadly described herein, the present invention, in one embodiment, relates to crystalline polymorphs of aza-T-dCyd that may be useful for the treatment of cancers, such as, for example, MDS and leukemia.

[0006] Accordingly, there is disclosed a crystalline polymorph of 5-aza-4'-thio-2'-deoxycytidine, wherein the crystalline polymorph has a powder X-ray diffraction pattern having peaks at about 8°, about 13°, about 15°, about 17°, about 19°, about 22°, about 23°, about 26°, about 28°, about 29°, about 31°, about 33°, and about 37° 2θ.

[0007] Also disclosed is a crystalline polymorph of 5-aza-4'-thio-2'-deoxycytidine, wherein the crystalline polymorph has a powder X-ray diffraction pattern having peaks at about 6°, about 12°, about 13°, about 14°, about 16°, about 18°, about 20°, about 21°, about 22°, about 26°, about 27°, about 29°, about 30°, about 33°, about 35°, about 36°, about 39°, and about 41° 2θ.

[0008] Also disclosed is an aza-T-dCyd compound comprising a crystalline polymorph having a powder X-ray diffraction pattern including peaks at about 8°, about 13°, about 15°, about 17°, about 19°, about 22°, about 23°, about 26°, about 28°, about 29°, about 31°, about 33°, and about 37° 2θ.

[0009] Also disclosed is an aza-T-dCyd compound comprising a crystalline polymorph having a powder X-ray diffraction pattern including peaks at about 6°, about 12°, about 13°, about 14°, about 16°, about 18°, about 20°, about 21°, about 22°, about 26°, about 27°, about 29°, about 30°, about 33°, about 35°, about 36°, about 39°, and about 41° 2θ.

[0010] Also disclosed is a crystalline polymorph of aza-T-dCyd, wherein the crystalline polymorph is Form A or Form F.

[0011] Also disclosed is a pharmaceutical composition comprising a therapeutically effective amount of the disclosed crystalline polymorph and a pharmaceutically acceptable carrier.

[0012] Also disclosed is a method of treating a subject in need of treating cancer, the method comprising administering to the subject an effective amount of the disclosed crystalline polymorph, thereby treating cancer in the subject. Examples of cancer include, but are not limited to, myelodysplastic syndromes and leukemia.

[0013] Also disclosed is a method of making the disclosed crystalline polymorph or the disclosed composition.

[0014] Also disclosed is a method for making the disclosed crystalline polymorph, the method comprising subjecting aza-T-dCyd to one or more of solvent equilibration, evaporation crystallization, anti-solvent addition, thermal cycle crystallization, sonication, and vapor diffusion into a solution.

[0015] Also disclosed is a kit comprising the disclosed crystalline polymorph and one or more of (a) at least one chemotherapeutic agent, (b) instructions for administering the composition in connection with the treatment of cancer, and (c) instructions for treating cancer.

[0016] Embodiments of the present invention can be described and claimed in specific statutory classes such as the statutory classes of systems, but this is merely for convenience, and those skilled in the art will understand that each embodiment of the present invention can be described and claimed in any statutory class. Unless otherwise specified, it is never intended that any method or embodiment described herein be construed as requiring that its steps be performed in a particular order. Thus, when the claims of a method are not specifically recited in the claims or the specification as being limited to steps in a particular order, in no way is it intended that an order be inferred. This also applies to any possible non-expressive basis for interpretation, including the arrangement of steps or operation flows, the plain meaning derived from grammatical construction or punctuation, or the reasoning matters regarding the number or type of embodiments described herein.

Brief Description of the Drawings

[0017] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure.

[0018]

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[0019] Further advantages of the present invention will be described in part in the following description, will be apparent in part from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It should be understood that both the foregoing general description and the following detailed description of the invention are exemplary and explanatory only and are not restrictive of the invention as claimed.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention can be more easily understood by reference to the following detailed description of the invention and the examples included therein. The following description is merely exemplary in nature and is not intended to limit the disclosure, application, or use.

[0021] Embodiments of the present invention can be described and claimed in terms of specific statutory classes such as the statutory classes of systems, but this is for convenience only, and those skilled in the art will understand that each embodiment of the present invention can be described and claimed in terms of any statutory class. Unless otherwise specified, it is never intended that any method or embodiment described herein be construed as requiring that its steps be performed in a particular order. Thus, where the claims of a method do not specifically recite that the steps are to be limited to a particular order in the claims or the specification, no inference is intended in any respect that the order is required. This applies to any possible non-expressive basis for interpretation, including the arrangement of steps or the flow of operations, the plain meaning derived from the grammatical construction or punctuation, or the logical matters regarding the number or type of embodiments described herein.

[0022] Throughout this application, various publications are referenced. The disclosures of these publications are hereby incorporated by reference in their entirety into this application to more fully describe the state of the art relevant to this application. The disclosed references are also discussed in the context of the references and are hereby incorporated by reference herein individually and specifically for the materials contained therein. Nothing in this specification should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Further, the publication dates provided herein may be different from the actual publication dates, which may require independent verification.

[0023] A. Definitions As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, references to "functional group", "alkyl", or "residue" include mixtures of two or more such functional groups, alkyls, or residues.

[0024] As used in this specification and the claims, the term "comprising" may include embodiments "consisting of" and "consisting essentially of".

[0025] In this specification, ranges may be expressed as from a particular value of "about" and / or to another particular value of "about". When such a range is expressed, another embodiment includes from that particular value and / or to the other particular value. Similarly, when a value is expressed as an approximation, it will be understood that by use of the antecedent "about", the particular value forms another embodiment. Further, it will be understood that each endpoint of a range is significant both in relation to the other endpoint and independently of the other endpoint. In this specification several values are disclosed, and it is understood that each value, in addition to the value itself, is disclosed herein as "about" that particular value. For example, if the value "10" is disclosed, "about 10" is also disclosed. It should also be understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0026] As used herein, the terms "about" and "or about" mean that the quantity or value in question can be a value that indicates approximately or nearly some other value. As used herein, generally, unless otherwise indicated or inferred, it is understood to be a nominal value indicating a variation of ±10%. This term is intended to convey that similar values promote equivalent results or effects recited in the claims. That is, it should be understood that quantities, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and / or larger or smaller, reflecting, for example, tolerances, conversion factors, rounding, measurement errors, and other factors known to those of ordinary skill in the art, as desired. Generally, a quantity, size, formulation, parameter, or other quantity or characteristic is "about" or "approximate", whether or not expressly stated to be so. When "about" is used before a quantitative value, it should be understood that the parameter includes the particular quantitative value itself as well, unless otherwise specifically stated.

[0027] References to parts by weight of specific elements or components in a composition in this specification and the claims of the conclusion indicate the weight relationship between the element or component and any other element or component in the composition or article in which the parts by weight are expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y are present in a weight ratio of 2:5 and are present in such a ratio regardless of whether additional components are contained in the compound.

[0028] The weight percentage (wt%) of a component is based on the total weight of the formulation or composition in which the component is included, unless otherwise specifically stated.

[0029] As used herein, "EC 50 " is intended to refer to the effective concentration of a substance (e.g., a compound or drug) that is required to inhibit 50% of a biological process or a component of a process, including proteins, subunits, organelles, ribonucleoproteins, etc. In one embodiment, EC 50 may refer to the concentration of a substance required for 50% inhibition in vivo, as further defined elsewhere in this specification.

[0030] As used herein, the terms "optional" or "optionally" mean that the event or situation described thereafter may or may not occur, and the description includes both the case where the event or situation occurs and the case where it does not occur.

[0031] 5-Aza-4'-thio-2'-deoxycytidine (also known as NTX-301) refers to a modified cytidine nucleoside in which the ring oxygen on the sugar moiety of the nucleoside is replaced by sulfur. Aza-T-dCyd has the following structure.

Chemical formula

[0032] Unless otherwise noted, the term "aza-T-dCyd" includes the compound itself and its pharmaceutically acceptable salts.

[0033] The crystalline polymorphs of aza-T-dCyd refer to various crystal structures of the nucleoside. In some embodiments, the crystalline polymorphs of aza-T-dCyd refer to Form A, Form B, Form C1, Form C2, Form D1, Form D2, Form E, Form F, Form G1, Form G2, Form H, Form 1, or Form J further described herein, including the examples. In certain embodiments, the crystalline polymorph is Form A or Form F.

[0034] As used herein, the term "substantially similar to" refers to a powder X-ray diffraction pattern that is not identical to that shown herein but shares most of the major peaks within the limits of experimental error. For example, in various aspects, a substantially similar powder X-ray diffraction pattern may share at least 3 peaks, at least 4 peaks, at least 5 peaks, at least 6 peaks, at least 7, at least 8 peaks, at least 9 peaks, at least 10 peaks, or more than 10 peaks with the powder X-ray diffraction pattern disclosed herein.

[0035] The term "polymorphic form A" or "form A" refers to a crystalline form of aza-T-dCyd that exhibits an X-ray powder diffraction pattern substantially the same as that shown in Figure 11. In one embodiment, form A has an XRPD pattern having peaks at about 8°, about 13°, about 15°, about 17°, about 19°, about 22°, about 23°, about 26°, about 28°, about 29°, about 31°, about 33°, and about 37° 2θ. In another embodiment, form A has an XRPD pattern having peaks at 7.7° ± 0.3°, 13.02° ± 0.3°, 15.34° ± 0.3°, 16.78° ± 0.3°, 18.62° ± 0.3°, 19.42° ± 0.3°, 21.94° ± 0.3°, 22.90° ± 0.3°, 25.70° ± 0.3°, 26.64° ± 0.3°, 27.86° ± 0.3°, 28.63° ± 0.3°, 29.45° ± 0.3°, 31.42° ± 0.3°, 32.70° ± 0.3°, 34.72° ± 0.3°, 35.97° ± 0.3°, and 37.46° ± 0.3° 2θ. In a particular embodiment, form A has an XRPD pattern having peaks at 7.7°, 13.02°, 15.34°, 16.78°, 18.62°, 19.42°, 21.94°, 22.90°, 25.70°, 27.86°, 28.70°, 31.42°, 32.70°, and 37.46° 2θ.

[0036] The term "polymorphic form B" or "form B" refers to a crystalline form of aza-T-dCyd that exhibits an X-ray powder diffraction pattern substantially the same as that shown in Figure 12.

[0037] The term "polymorphic form C1" or "form C1" refers to a crystalline form of aza-T-dCyd that appears in a mixture with form A and exhibits an X-ray powder diffraction pattern substantially the same as that shown in Figure 12.

[0038] The term "polymorphic form C2" or "form C2" refers to a crystalline form of aza-T-dCyd that appears in a mixture with form A and exhibits an X-ray powder diffraction pattern substantially the same as that shown in Figure 12.

[0039] The term "polymorphic form D1" or "form D1" refers to a crystalline form of aza-T-dCyd that appears in a mixture with form A and exhibits an X-ray powder diffraction pattern substantially the same as that shown in Figure 12.

[0040] The term "polymorphic form D2" or "form D2" refers to a crystalline form of aza-T-dCyd that exhibits an X-ray powder diffraction pattern substantially the same as that shown in Figure 12.

[0041] The term "polymorphic form E" or "form E" refers to a crystalline form of aza-T-dCyd that exhibits an X-ray powder diffraction pattern substantially the same as that shown in Figure 14.

[0042] The term "polymorphic form F" or "form F" refers to a crystalline form of aza-T-dCyd that exhibits an X-ray powder diffraction pattern substantially the same as that shown in Figure 16. In one embodiment, form F has an XRPD pattern having peaks at about 6°, about 12°, about 13°, about 14°, about 16°, about 18°, about 20°, about 21°, about 22°, about 26°, about 27°, about 29°, about 30°, about 33°, about 35°, about 36°, about 39°, and about 41° 2θ. In one embodiment, form F has an XRPD pattern having peaks at 6.06° ± 0.3°, 12.10° ± 0.3°, 13.02° ± 0.3°, 14.38° ± 0.3°, 15.94° ± 0.3°, 17.50° ± 0.3°, 19.62° ± 0.3°, 21.18° ± 0.3°, 22.34° ± 0.3°, 26.18° ± 0.3°, 27.42° ± 0.3°, 28.50° ± 0.3°, 29.90° ± 0.3°, 32.66° ± 0.3°, 35.02° ± 0.3°, 36.30° ± 0.3°, 38.94° ± 0.3°, and 41.06° ± 0.3° 2θ. In certain embodiments, form F has an XRPD pattern having peaks at 6.06°, 12.10°, 13.02°, 14.38°, 15.94°, 17.50°, 19.62°, 21.18°, 22.34°, 26.18°, 27.42°, 28.50°, 29.90°, 32.66°, 35.02°, 36.30°, 38.94°, and 41.06° 2θ.

[0043] The term "polymorphic form G1" or "form G1" refers to a crystalline form of aza-T-dCyd that exhibits an X-ray powder diffraction pattern substantially the same as that shown in Figure 14.

[0044] The term "polymorphic form G2" or "form G2" refers to a crystalline form of aza-T-dCyd that exhibits an X-ray powder diffraction pattern substantially the same as that shown in Figure 14.

[0045] The term "polymorphic form H" or "form H" refers to a crystalline form of aza-T-dCyd that exhibits an X-ray powder diffraction pattern substantially the same as that shown in Figure 14.

[0046] The term "polymorphic form I" or "form I" refers to a crystalline form of aza-T-dCyd that exhibits an X-ray powder diffraction pattern substantially the same as that shown in Figure 14.

[0047] The term "polymorphic form J" or "form J" refers to a crystalline form of aza-T-dCyd that exhibits an X-ray powder diffraction pattern substantially the same as that shown in Figure 14.

[0048] The term "polymorphic form K" or "form K" refers to a crystalline form of aza-T-dCyd that exhibits an X-ray powder diffraction pattern substantially the same as that shown in Figure 14.

[0049] The term "polymorphic form L" or "form L" refers to a crystalline form of aza-T-dCyd that exhibits an X-ray powder diffraction pattern substantially the same as that shown in Figure 14.

[0050] As used herein, the term "subject" can be a vertebrate such as a mammal, fish, bird, reptile, or amphibian. Thus, the subject matter of the methods disclosed herein can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or sex. Thus, adult and neonatal subjects, as well as fetuses regardless of sex, are intended to be included. In one embodiment, the subject is a mammal. In a further embodiment, the mammal is a human. In one embodiment, the subject has a blood cancer. In one embodiment, the subject is an animal that can receive administration of an aza-T-dCyd composition.

[0051] As used herein, the term "treatment" refers to the medical management of a patient intended to cure, ameliorate, stabilize, or prevent a disease, condition, or disorder. This term includes active treatment, i.e., treatment specifically directed to the improvement of a disease, condition, or disorder, and causal treatment, i.e., treatment directed to the elimination of the cause of the related disease, condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed for the relief of symptoms rather than the cure of a disease, diseased condition, or disorder, prophylactic treatment, i.e., treatment directed to minimizing or partially or completely inhibiting the development of a related disease, diseased condition, or disorder, and adjuvant treatment, i.e., treatment used to supplement another specific therapy directed to the improvement of a related disease, diseased condition, or disorder. In various embodiments, this term encompasses any treatment of a subject, including mammals (e.g., humans), to (i) prevent the occurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed as having it, (ii) inhibit a disease, i.e., prevent its onset, or (iii) alleviate a disease, i.e., cause regression of the disease. In one embodiment, the subject is a mammal such as a primate, and in a further embodiment, the subject is a human. The term "subject" also includes companion animals (e.g., cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, flies, etc.).

[0052] As used herein, the term "prevent" or "preventing" refers to hindering, avoiding, precluding, averting, stopping, or obstructing something from happening, particularly by prior action. It should be understood that when "reduce," "inhibit," or "prevent" is used herein, the use of the other two terms is also expressly disclosed unless specifically indicated otherwise.

[0053] As used herein, the term "diagnosed" means having been found, by a person skilled in the art, such as a physician, to have undergone a physical examination and to have a condition that can be diagnosed or treated by a compound, composition, or method disclosed herein.

[0054] As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injections such as intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various embodiments, the preparation can be administered therapeutically, i.e., administered to treat an existing disease or condition. In further various embodiments, the preparation can be administered prophylactically, i.e., administered to prevent a disease or condition.

[0055] As used herein, the terms "effective amount" and "effective quantity" refer to an amount sufficient to achieve a desired result or to affect an undesirable condition. For example, the term "therapeutically effective amount" as used herein refers to an amount of the crystalline polymorph of aza-T-dCyd sufficient to achieve a therapeutic effect. The specific therapeutically effective dosage level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with or concurrently with the specific compound employed, and like factors well known in the medical arts. For example, it is well within the skill of the art to start the dosage of a compound at a level lower than that required to achieve the desired therapeutic effect and to increase the dosage gradually until the desired effect is achieved. If desired, the effective daily dosage can be divided into multiple dosages for administration. Consequently, single dose compositions can contain such an amount or an approximation thereof to constitute a daily dosage. If any contraindications arise, the dosage can be adjusted by the individual physician. The dosage can vary and can be administered in one or more dose administrations per day for one or several days. Guidance regarding appropriate dosages for a given class of pharmaceuticals can be found in the literature. In certain embodiments, the therapeutically effective amount is about 30 mg / m 2 ~ about 70 mg / m 2 In another embodiment, the therapeutically effective amount is about 35 mg / m 2 ~ about 45 mg / m 2 , about 45 mg / m 2 ~ about 55 mg / m 2 , or about 55 mg / m 2 ~ about 66 mg / m 2 In still further various embodiments, the formulation can be administered in a "prophylactically effective amount", i.e., an amount effective for the prevention of a disease or condition.

[0056] As used herein, "dosage form" means a pharmaceutically active material in a medium, carrier, vehicle, or device suitable for administration to a subject. A dosage form can include a disclosed compound of the invention, a product of a disclosed method of making, or a salt, solvate, or polymorph thereof, in combination with pharmaceutically acceptable excipients such as preservatives, buffers, saline, or phosphate buffered saline. Dosage forms can be made using conventional pharmaceutical manufacturing and formulation techniques. Dosage forms can include inorganic or organic buffers (e.g., sodium or potassium salts of phosphoric, carbonic, acetic, or citric acid), as well as pH adjusters (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citric or acetic acid, amino acids and their salts), antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene 9-10 nonylphenol, sodium desoxycholate), solution and / or cryo / melt stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic agents (e.g., salts or sugars), antibacterial agents (e.g., benzoic acid, phenolic acids, gentamicin), antifoaming agents (e.g., polydimethylsiloxane), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity modifiers (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethylene glycol, ethanol). Dosage forms formulated for injectable use can have a disclosed compound, a product of a disclosed method of making, or a salt, solvate, or polymorph thereof, suspended in sterile injectable saline together with a preservative.

[0057] As used herein, "kit" means a set of at least two components that make up the kit. Together, the components constitute a functional unit for a given purpose. The individual member components may be physically packaged together or separately. For example, a kit that includes instructions for using the kit may or may not physically include the instructions along with the other individual member components. Instead, the instructions may be provided in paper form, or on a computer-readable memory device, or in electronic form downloadable from an Internet website, or as a recorded presentation, and may be provided as a separate member component.

[0058] As used herein, "instructions" means a document that describes relevant information or methodology regarding the kit. Such information may include, among other things, background information, a list of components and information about their availability (such as purchase information), a simple or detailed protocol for using the kit, troubleshooting, references, technical support, and any combination of other relevant documents. The instructions may be provided in paper form, or on a computer-readable memory device, or in electronic form downloadable from an Internet website, or as a recorded presentation, either with the kit or as a separate member component. The instructions may include one or more documents and are intended to include future updates.

[0059] As used herein, the term "cancer" includes neoplasms and dysplasia. Cancer can be a hematologic cancer or a solid cancer. The term "hematologic cancer" includes neoplasms and dysplasia of blood cells. In some embodiments, the hematologic cancer is selected from the group consisting of non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and solitary plasmacytoma. The term "solid cancer" includes neoplasms and dysplasia of tissues or organs. In some embodiments, the cancer can be one or more of gastric cancer, kidney cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, lung cancer, colon cancer, breast cancer, melanoma, and pancreatic cancer.

[0060] As used herein, the term "therapeutic agent" includes any synthetic or naturally occurring composition of biologically active compounds or substances that, when administered to a living organism (human or non-human animal), induces a desired pharmacological, immunogenic, and / or physiological effect by local and / or systemic action. Thus, the term encompasses compounds or chemical substances that are traditionally regarded as drugs, vaccines, and biopharmaceuticals, including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs, etc. Examples of therapeutic agents are described in well-known references such as the Merck Index (14th Edition), Physicians’ Desk Reference (64th Edition), and The Pharmacological Basis of Therapeutics (12th Edition), and include, but are not limited to, agents; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure, or alleviation of a disease or disorder; substances that affect the structure or function of the body, or prodrugs that become biologically active or more active after being placed in a physiological environment.For example, the term "therapeutic agent" includes adjuvants; anti-infective agents such as antibiotics and antiviral agents; analgesics and combinations of analgesics, anorectics, anti-inflammatory agents, antiepileptic drugs, local and general anesthetics, hypnotics, sedatives, antipsychotics, neuroleptics, antidepressants, anxiolytics, antagonists, neuronal blockers, anticholinergic and anticholinergic agents, antimuscarinic and muscarinic agents, antiadrenergic drugs, antiarrhythmic drugs, antihypertensive agents, hormones, and nutrients, anti-arthritis drugs, anti-asthma drugs, anticonvulsants, antihistamines, antiemetics, antineoplastic drugs, antipruritics, antipyretics; antispasmodics, cardiovascular agents (including calcium channel blockers, beta blockers, beta agonists, and antiarrhythmic drugs), antihypertensive drugs, diuretics, vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostic agents; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressive agents; muscle relaxants; psychostimulants; sedatives; psychotropics; proteins, peptides, and fragments thereof (whether occurring naturally, chemically synthesized, or recombinantly produced); and nucleic acid molecules (ribonucleotides (RNA) or deoxyribonucleotides (DNA) in polymeric form of more than two nucleotides, including both double-stranded and single-stranded molecules, gene constructs, expression vectors, antisense molecules, etc.), small molecules (e.g., doxorubicin), and other biologically active macromolecules such as proteins and enzymes, including but not limited to compounds or compositions for use in all major therapeutic areas. The agents can be biologically active agents used in medicine including veterinary medicine, applications, and agriculture such as plants, and other areas. The term "therapeutic agent" also includes drugs; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure, or alleviation of a disease or disorder; or substances that affect the structure or function of the body; or prodrugs that become biologically active or more active after being placed in a given physiological environment, including but not limited to these.

[0061] As used herein, the term "chemotherapeutic agent" refers to compounds and compositions having anti-cancer properties. In one embodiment, the chemotherapeutic agent is combined with a crystalline polymorph of aza-T-dCyd. In one embodiment, the chemotherapeutic agent is selected from the group consisting of alkylating agents, antimetabolite agents, antitumor antibiotic agents, mitotic inhibitors, and mTOR inhibitors. In certain embodiments, the antitumor antibiotic agent is selected from the group consisting of doxorubicin, mitoxantrone, bleomycin, daunorubicin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin, or pharmaceutically acceptable salts thereof. In certain embodiments, the antimetabolite agent is selected from the group consisting of gemcitabine, 5-fluorouracil, capectiabine, hydroxyurea, mercaptopurine, pemetrexed, fludarabine, nelarabine, cladribine, clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, methotrexate, and thioguanine, or pharmaceutically acceptable salts thereof. In certain embodiments, the alkylating agent is selected from the group consisting of carboplatin, cisplatin, cyclophosphamide, chlorambucil, melphalan, carmustine, busulfan, lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, temozolomide, thiotepa, bendamustine, and streptozocin, or pharmaceutically acceptable salts thereof. In certain embodiments, the mitotic inhibitor is selected from the group consisting of irinotecan, topotecan, rubitecan, cabazitaxel docetaxel, paclitaxel, etopside, vincristine, exabepilone, vinorelbine, vinblastine, and teniposide, or pharmaceutically acceptable salts thereof. In certain embodiments, the mTOR inhibitor is selected from the group consisting of everolimus, sirolimus, and temsirolimus, or pharmaceutically acceptable salts thereof.

[0062] The term "pharmaceutically acceptable" describes a substance that is not biologically or otherwise undesirable, i.e., it does not cause unacceptable levels of undesirable biological effects or interact in a harmful manner.

[0063] As used herein, the term "derivative" has a structure derived from the structure of a parent compound (e.g., a compound disclosed herein), the structure is sufficiently similar to that disclosed herein, and based on that similarity, it exhibits the same or similar activity and utility as the compounds of the claims or, as a precursor, is expected by those skilled in the art to induce the same or similar activity and utility as the compounds of the claims. Exemplary derivatives include salts, esters, and amides, salts of esters or amides, and N-oxides of the parent compound.

[0064] As used herein, the term "active ingredient" refers to a therapeutic agent and includes any substance other than food that is used in the prevention, diagnosis, alleviation, treatment, or cure of a disease or disorder. Stedman’s Medical Dictionary, 25th Edition (1990). The substance can be ingested orally, injected into muscle, skin, blood vessels, or body cavities, or applied topically. Mosby’s Medical, Nursing & Allied Health Dictionary, 5th Edition (1998). The agent can include any substance disclosed in at least one of the following: The Merck Index, 14th Edition (2006), Pei-Show Juo, Concise Dictionary of Biomedicine and Molecular Biology, (1996), U.S. Pharmacopeia Dictionary, 2000 Edition, Physician’s Desk Reference, 2010 Edition, Orange Book: Approved Drug Products with Therapeutic Equivalence Evaluations (April 2013), and Approved Animal & Veterinary Drug Products (Green Book) (January 2013). The term "active ingredient" includes, for example, prescription and over-the-counter active pharmaceutical ingredients (e.g., small molecules, macrocycles, peptides, etc.), vitamins, nutraceuticals, supplements (e.g., dietary, nutritional, and herbal), cosmetics, and biological agents.

[0065] The term "pharmaceutically acceptable carrier" refers to sterile aqueous solutions or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions immediately before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and organic esters for injection such as ethyl oleate, but are not limited thereto. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc. Prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents such as aluminum monostearate and gelatin which delay absorption. Injectable depot forms are prepared by forming a microcapsule matrix of the drug in biodegradable polymers such as polyactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending on the ratio of the drug to the polymer and the specific properties used, the rate of drug release can be controlled. Depot injectable formulations are also prepared by encapsulating the drug in liposomes or microemulsions compatible with body tissues. Injectable formulations can be sterilized, for example, by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media immediately before use, and by filtration through its bacterial retention filter. Suitable inert carriers can include sugars such as lactose. In certain embodiments, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0066] Unless otherwise specified, it is never intended that any method described in this specification be construed as requiring that its steps be performed in a particular order. Thus, unless the claims of the method enumerate actual instructions to follow those steps in the claims or the specification, or are otherwise specifically described as being limited to a particular order, no inference of order is intended in any way. This applies to any possible non-expressive basis for interpretation, including the arrangement of steps or operation flows, the plain meaning derived from grammatical construction or punctuation, and the logical matters regarding the number or type of embodiments described in this specification.

[0067] The components used to prepare the compositions of the present invention, and the compositions themselves used within the methods disclosed herein are disclosed. When these and other materials are disclosed herein, and combinations, subsets, interactions, groups, etc. of these materials are disclosed, specific references to each and every individual and collective combination and permutation of these compounds cannot be explicitly disclosed, but it should be understood that each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed, and several modifications that can be made to several molecules containing the compound are discussed, every combination and permutation of the compound, as well as possible modifications, are clearly contemplated, unless specifically indicated otherwise. Thus, if classes of molecules A, B, and C are disclosed, as well as classes of molecules D, E, and F, and as an example of a combination molecule, A-D is disclosed, then each is individually and collectively contemplated, i.e., A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered to be disclosed, even if each is not individually listed. Similarly, any subset or combination of these is also disclosed. Thus, for example, subgroups of A-E, B-F, and C-E would be considered to be disclosed. This concept applies to all embodiments of the present application, including but not limited to steps in the methods of making and using the compositions of the present invention. Thus, if there are various additional steps that can be performed, it should be understood that each of these additional steps can be performed by any particular embodiment or combination of embodiments of the method of the present invention.

[0068] It should be understood that the compositions disclosed herein have specific functions. In this specification, specific structural requirements for performing the disclosed functions are disclosed, and there are various structures that can perform the same functions associated with the disclosed structures, and it should be understood that these structures will typically achieve the same results.

[0069] B. Crystal Polymorphs In one embodiment, there is provided a crystalline polymorph of 5-aza-4'-thio-2'-deoxycytidine, wherein the crystalline polymorph has a powder X-ray diffraction pattern comprising peaks at about 8°, about 13°, about 15°, about 17°, about 19°, about 22°, about 23°, about 26°, about 28°, about 29°, about 31°, about 33°, and about 37° 2θ. In a further embodiment, the crystalline polymorph has an X-ray powder diffraction pattern that is substantially similar to or the same as the X-ray powder diffraction pattern shown in FIG. 11.

[0070] In one embodiment, there is provided a crystalline polymorph of 5-aza-4'-thio-2'-deoxycytidine, wherein the crystalline polymorph has a powder X-ray diffraction pattern comprising peaks at about 6°, about 12°, about 13°, about 14°, about 16°, about 18°, about 20°, about 21°, about 22°, about 26°, about 27°, about 29°, about 30°, about 33°, about 35°, about 36°, about 39°, and about 41° 2θ. In a further embodiment, the crystalline polymorph has an X-ray powder diffraction pattern that is substantially similar to or the same as the X-ray powder diffraction pattern shown in FIG. 16.

[0071] In one embodiment, the present disclosure provides an aza-T-dCyd compound comprising a crystalline polymorph having an X-ray powder diffraction pattern comprising peaks at about 8°, about 13°, about 15°, about 17°, about 19°, about 22°, about 23°, about 26°, about 28°, about 29°, about 31°, about 33°, and about 37° 2θ. In a further embodiment, the crystalline polymorph has an X-ray powder diffraction pattern that is substantially similar to or the same as the X-ray powder diffraction pattern shown in FIG. 11.

[0072] In one embodiment, the present disclosure provides an aza-T-dCyd compound comprising a crystalline polymorph having an X-ray powder diffraction pattern comprising peaks at about 6°, about 12°, about 13°, about 14°, about 16°, about 18°, about 20°, about 21°, about 22°, about 26°, about 27°, about 29°, about 30°, about 33°, about 35°, about 36°, about 39°, and about 41° 2θ. In a further embodiment, the crystalline polymorph has an X-ray powder diffraction pattern that is substantially similar to or the same as the X-ray powder diffraction pattern shown in FIG. 16.

[0073] In various embodiments, the crystalline polymorph is present in a pharmaceutical composition, together with a pharmaceutically acceptable carrier.

[0074] C. Methods of Making Crystalline Polymorphs In one embodiment, a method for making the disclosed crystalline polymorph is disclosed, the method comprising subjecting aza-T-dCyd to one or more of solvent equilibration, evaporation crystallization, anti-solvent addition, thermal cycle crystallization, sonication, and vapor diffusion into solution. In a further embodiment, the crystalline polymorph has a powder X-ray diffraction pattern comprising peaks at about 8°, about 13°, about 15°, about 17°, about 19°, about 22°, about 23°, about 26°, about 28°, about 29°, about 31°, about 33°, and about 37° 2θ. In a further embodiment, the crystalline polymorph has a powder X-ray diffraction pattern comprising peaks at about 6°, about 12°, about 13°, about 14°, about 16°, about 18°, about 20°, about 21°, about 22°, about 26°, about 27°, about 29°, about 30°, about 33°, about 35°, about 36°, about 39°, and about 41° 2θ.

[0075] In one embodiment, the method comprises only one of solvent equilibration, evaporation crystallization, anti-solvent addition, thermal cycle crystallization, sonication, and vapor diffusion into solution. In one embodiment, the method comprises exactly two of solvent equilibration, evaporation crystallization, anti-solvent addition, thermal cycle crystallization, sonication, and vapor diffusion into solution. In one embodiment, the method comprises more than two of solvent equilibration, evaporation crystallization, anti-solvent addition, thermal cycle crystallization, sonication, and vapor diffusion into solution.

[0076] D. Pharmaceutical Compositions The present disclosure provides a composition comprising a crystalline polymorph of aza-T-dCyd. Such compositions include pharmaceutical compositions comprising a therapeutically effective amount of the crystalline polymorph of aza-T-dCyd and a pharmaceutically acceptable carrier. Generally, all known or approved amounts of crystalline aza-T-dCyd can be used in the composition. In one embodiment, the crystalline aza-T-dCyd is Form A or Form F, and is about 30 mg / m 2 ~ about 70 mg / m 2is present in an amount. In certain embodiments, the crystalline polymorph of aza-T-dCyd is about 35 mg / m 2 ~ about 45 mg / m 2 about 45 mg / m 2 ~ about 55 mg / m 2 or about 55 mg / m 2 ~ about 66 mg / m 2 is present in an amount.

[0077] In one embodiment, a pharmaceutical composition is disclosed that comprises an effective amount of: (a) a crystalline polymorph having a powder X-ray diffraction pattern with peaks at about 8°, about 13°, about 15°, about 17°, about 19°, about 22°, about 23°, about 26°, about 28°, about 29°, about 31°, about 33°, and about 37° 2θ, or (b) a crystalline polymorph having a powder X-ray diffraction pattern with peaks at about 6°, about 12°, about 13°, about 14°, about 16°, about 18°, about 20°, about 21°, about 22°, about 26°, about 27°, about 29°, about 30°, about 33°, about 35°, about 36°, about 39°, and about 41° 2θ, and a pharmaceutically acceptable carrier. In a further embodiment, the crystalline polymorph has a powder X-ray diffraction pattern with peaks at about 8°, about 13°, about 15°, about 17°, about 19°, about 22°, about 23°, about 26°, about 28°, about 29°, about 31°, about 33°, and about 37° 2θ. In yet a further embodiment, the crystalline polymorph has a powder X-ray diffraction pattern with peaks at about 6°, about 12°, about 13°, about 14°, about 16°, about 18°, about 20°, about 21°, about 22°, about 26°, about 27°, about 29°, about 30°, about 33°, about 35°, about 36°, about 39°, and about 41° 2θ.

[0078] In one embodiment, the effective amount is about 35 mg / m 2 ~ about 70 mg / m 2 about 35 mg / m 2 ~ about 65 mg / m 2 about 35 mg / m 2 ~ about 55 mg / m 2 about 35 mg / m 2 ~ about 45 mg / m 2 about 40 mg / m 2 ~ about 70 mg / m 2 about 50 mg / m 2 ~ about 70 mg / m2 , about 60 mg / m 2 ~ about 70 mg / m 2 , about 40 mg / m 2 ~ about 65 mg / m 2 , about 45 mg / m 2 ~ about 60 mg / m 2 , or about 50 mg / m 2 ~ about 55 mg / m 2 thereof.

[0079] In one embodiment, the composition further comprises a chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolite agents, antitumor antibiotic agents, mitotic inhibitors, and mTOR inhibitors.

[0080] In one embodiment, the composition further comprises an alkylating agent. Examples of alkylating agents include, but are not limited to, carboplatin, cisplatin, cyclophosphamide, chlorambucil, melphalan, carmustine, busulfan, lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, temozolomide, thiotepa, bendamustine, and streptozocin, or pharmaceutically acceptable salts thereof.

[0081] In one embodiment, the composition further comprises an antimetabolite agent. Examples of antimetabolite agents include, but are not limited to, gemcitabine, 5-fluorouracil, capectiabine, hydroxyurea, mercaptopurine, pemetrexed, fludarabine, nelarabine, cladribine, clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, methotrexate, and thioguanine, or pharmaceutically acceptable salts thereof.

[0082] In one embodiment, the composition further comprises an antitumor antibiotic agent. Examples of antitumor antibiotic agents include, but are not limited to, doxorubicin, mitoxantrone, bleomycin, daunorubicin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin, or pharmaceutically acceptable salts thereof.

[0083] In one embodiment, the composition further comprises a mitotic inhibitor. Examples of mitotic inhibitors include, but are not limited to, irinotecan, topotecan, rubitecan, cabazitaxel, docetaxel, paclitaxel, etopside, vincristine, exabepilone, vinorelbine, vinblastine, and teniposide, or pharmaceutically acceptable salts thereof.

[0084] In one embodiment, the composition further comprises an mTOR inhibitor. Examples of mTOR inhibitors include, but are not limited to, everolimus, sirolimus, and temsirolimus, or pharmaceutically acceptable salts thereof.

[0085] In one embodiment, a composition comprising a crystalline polymorph of aza-T-dCyd is formulated for systemic or topical administration. Formulations for oral, topical, intravenous, or intramuscular administration are contemplated. In certain embodiments, the crystalline polymorph of aza-T-dCyd is formulated for oral administration.

[0086] In one embodiment, the pharmaceutical composition comprises an active ingredient consisting of a crystalline polymorph having a powder X-ray diffraction pattern comprising peaks at about 8°, about 13°, about 15°, about 17°, about 19°, about 22°, about 23°, about 26°, about 28°, about 29°, about 31°, about 33°, and about 37° 2θ.

[0087] In one embodiment, the pharmaceutical composition comprises an active ingredient consisting of a crystalline polymorph having a powder X-ray diffraction pattern containing peaks at about 6°, about 12°, about 13°, about 14°, about 16°, about 18°, about 20°, about 21°, about 22°, about 26°, about 27°, about 29°, about 30°, about 33°, about 35°, about 36°, about 39°, and about 41° 2θ.

[0088] In one embodiment, the pharmaceutical composition comprises a crystalline polymorph having a powder X-ray diffraction pattern containing peaks at about 8°, about 13°, about 15°, about 17°, about 19°, about 22°, about 23°, about 26°, about 28°, about 29°, about 31°, about 33°, and about 37° 2θ, and does not contain other crystalline polymorphs of aza-T-dCyd.

[0089] In one embodiment, the pharmaceutical composition comprises a crystalline polymorph having a powder X-ray diffraction pattern containing peaks at about 6°, about 12°, about 13°, about 14°, about 16°, about 18°, about 20°, about 21°, about 22°, about 26°, about 27°, about 29°, about 30°, about 33°, about 35°, about 36°, about 39°, and about 41° 2θ, and does not contain other crystalline polymorphs of aza-T-dCyd.

[0090] In certain embodiments, the composition comprises any convenient pharmaceutical vehicle. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, etc. can be used to form oral liquid preparations such as suspensions, elixirs, and solutions, while carriers such as starch, sugar, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants, etc. can be used to form oral solid preparations such as powders, capsules, and tablets. Tablets and capsules are preferred oral dosage units because they are easy to administer, and thus solid pharmaceutical carriers are used. Optionally, the tablets can be coated by standard aqueous or non-aqueous techniques.

[0091] E. Methods of Using the Crystalline Polymorph and Compositions Containing the Same The crystalline polymorphs and pharmaceutical compositions of the present invention are useful for the treatment or control of cancers such as blood cancers (e.g., non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and solitary myeloma) and solid tumors (e.g., gastric cancer, kidney cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, lung cancer, colon cancer, breast cancer, melanoma, and pancreatic cancer).

[0092] To treat or control cancer, the crystalline polymorphs and pharmaceutical compositions containing the crystalline polymorphs are administered to a subject in need thereof, such as a vertebrate, for example, a mammal, fish, bird, reptile, or amphibian. The subject can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not indicate a specific age or gender. Thus, it is intended to include adult and neonatal subjects, as well as fetuses regardless of gender. The subject is preferably a mammal such as a human. Before administering the crystalline polymorph or composition, the subject can be diagnosed as in need of treatment for cancer, such as a blood cancer or a solid tumor.

[0093] The crystalline polymorph or composition can be administered to the subject according to any method. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration including injections such as intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. The administration can be continuous or intermittent. The preparation can be therapeutically administered, i.e., administered to treat an existing disease or condition. The formulation can also be prophylactically administered, i.e., administered for the prevention of cancer such as a blood cancer or a solid tumor.

[0094] The therapeutically effective amount or dosage of the crystalline polymorph can vary within wide limits. Such dosage is adjusted for each particular case, including the specific compound being administered, the route of administration, the condition being treated, and the patient being treated. Generally, for oral or parenteral administration to an adult human weighing approximately 70 Kg or more, a daily dosage of about 10 mg to about 10,000 mg, preferably about 200 mg to about 1,000 mg, may be appropriate, although it may exceed the upper limit. The daily dosage can be administered as a single dose, or in divided doses, or as a continuous infusion for parenteral administration. Consequently, a single-dose composition can contain such an amount or a multiple thereof of the compound or composition to constitute a daily dose. If any contraindications arise, the dosage can be adjusted by the individual physician. The dosage can vary and can be administered in one or more doses per day, daily, for one or several days.

[0095] 1. Method of treatment The crystalline polymorphs disclosed herein are useful for the treatment or control of cancers such as blood cancers (e.g., non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and solitary plasmacytoma) and solid tumors (e.g., gastric cancer, kidney cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, lung cancer, colon cancer, breast cancer, melanoma, and pancreatic cancer). Accordingly, a method is provided that includes administering to a subject a therapeutically effective amount of the disclosed crystalline polymorph or a composition comprising the disclosed crystalline polymorph. In a further aspect, the method can be a method for treating cancer.

[0096] a. Treatment of cancer The present disclosure provides various methods of using an azathymidine-dCyd composition for the treatment of diseases such as cancer. In one embodiment, a crystalline polymorph of azathymidine-dCyd is administered to a subject for treating a blood cancer, and the subject is in need of such treatment. Various blood cancers can be treated by the composition, and in some embodiments, the blood cancer is selected from the group consisting of non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and solitary plasmacytoma. In certain embodiments, an effective amount of the crystalline polymorph of azathymidine-dCyd is administered together with additional chemotherapeutic agents such as alkylating agents, antimetabolite agents, antitumor antibiotic agents, mitotic inhibitors, or mTOR inhibitors.

[0097] Accordingly, in one embodiment, a method of treating it in a subject in need of cancer treatment, comprising administering to the subject an effective amount of a crystalline polymorph having a powder X-ray diffraction pattern comprising peaks at about 8°, about 13°, about 15°, about 17°, about 19°, about 22°, about 23°, about 26°, about 28°, about 29°, about 31°, about 33°, and about 37° 2θ, is disclosed.

[0098] In one embodiment, a method of treating it in a subject in need of cancer treatment, comprising administering to the subject an effective amount of a crystalline polymorph having a powder X-ray diffraction pattern comprising peaks at about 6°, about 12°, about 13°, about 14°, about 16°, about 18°, about 20°, about 21°, about 22°, about 26°, about 27°, about 29°, about 30°, about 33°, about 35°, about 36°, about 39°, and about 41° 2θ, is disclosed.

[0099] In one embodiment, a method of treating it in a subject in need of cancer treatment, comprising administering to the subject an effective amount of an azathymidine-dCyd compound consisting of a crystalline polymorph having a powder X-ray diffraction pattern comprising peaks at about 8°, about 13°, about 15°, about 17°, about 19°, about 22°, about 23°, about 26°, about 28°, about 29°, about 31°, about 33°, and about 37° 2θ, is disclosed.

[0100] In one embodiment, a method for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of an aza-T-dCyd compound consisting of a crystalline polymorph having a powder X-ray diffraction pattern including peaks at about 6°, about 12°, about 13°, about 14°, about 16°, about 18°, about 20°, about 21°, about 22°, about 26°, about 27°, about 29°, about 30°, about 33°, about 35°, about 36°, about 39°, and about 41° 2θ, is disclosed.

[0101] In one embodiment, the cancer is a blood cancer. Examples of blood cancers include, but are not limited to, non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and solitary plasmacytoma.

[0102] In one embodiment, the cancer is a solid tumor. Examples of solid tumors include, but are not limited to, gastric cancer, kidney cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, lung cancer, colon cancer, breast cancer, melanoma, and pancreatic cancer.

[0103] In one embodiment, the effective amount is a therapeutically effective amount. In a further embodiment, the effective amount is about 35 mg / m 2 ~ about 70 mg / m 2 、 about 35 mg / m 2 ~ about 65 mg / m 2 、 about 35 mg / m 2 ~ about 55 mg / m 2 、 about 35 mg / m 2 ~ about 45 mg / m 2 、 about 40 mg / m 2 ~ about 70 mg / m 2 、 about 50 mg / m 2 ~ about 70 mg / m 2 、 about 60 mg / m 2 ~ about 70 mg / m 2 、 about 40 mg / m 2 ~ about 65 mg / m 2 、 about 45 mg / m 2 ~ about 60 mg / m 2 、 or about 50 mg / m2 ~about 55 mg / m 2 thereof.

[0104] In one embodiment, the crystalline polymorph is present in the pharmaceutical composition.

[0105] In one embodiment, the method further comprises administering a chemotherapeutic agent to a subject.

[0106] In one embodiment, the effective amount is administered as a single dose. In a further embodiment, the effective amount is administered via multiple doses.

[0107] In one embodiment, the method further comprises identifying a subject in need of treatment for a blood cancer. In a further embodiment, the subject has been diagnosed as being in need of cancer treatment prior to the administration step.

[0108] In one embodiment, the administration is repeated administration. In a further embodiment, the administration is for a period of about 4 days to about 6 days, about 4 days to about 5 days, or about 5 days to about 6 days. In still a further embodiment, the administration is for a period of about 5 days.

[0109] In one embodiment, the administration is carried out via a treatment cycle. In a further embodiment, each treatment cycle comprises administering an effective amount of the compound for a period of about 4 days to about 6 days.

[0110] In one embodiment, the administration is carried out via a treatment process comprising a plurality of treatment cycles and a plurality of rest periods. In a further embodiment, each treatment cycle comprises administering an effective amount of the compound for a period of about 4 days to about 6 days. In still a further embodiment, each treatment cycle comprises administering an effective amount of the compound for a period of about 5 days. In yet another embodiment, each rest period comprises refraining from administering the compound for a period of about 1 day to about 10 days.

[0111] In one embodiment, administration is performed through a treatment process that includes a first treatment cycle of administering an effective amount of the crystalline polymorph for a period of about 4 days to about 6 days, a first rest period of withholding administration of the crystalline polymorph for a period of about 1 day to about 3 days, a second treatment cycle of administering an effective amount of the crystalline polymorph for a period of about 4 days to about 6 days, and a second rest period of withholding administration of the crystalline polymorph for at least about 8 days. In a further embodiment, the effective amount is administered as a single dose. In yet a further embodiment, the effective amount is administered via multiple doses. In yet another embodiment, the effective amount is administered via single doses on some days and via multiple doses on other days.

[0112] In one embodiment, administration is performed through a treatment process that includes a first treatment cycle of administering an effective amount of the crystalline polymorph for a period of about 5 days, a first rest period of withholding administration of the crystalline polymorph for a period of about 2 days, a second treatment cycle of administering an effective amount of the crystalline polymorph for a period of about 5 days, and a second rest period of withholding administration of the crystalline polymorph for at least about 9 days.

[0113] In one embodiment, the subject is diagnosed with a blood cancer, and the diagnosis can be made prior to administration of the crystalline polymorph of aza-T-dCyd. In one embodiment, the crystalline polymorph of aza-T-dCyd is administered in a single dose or over a plurality of doses. In certain embodiments, the crystalline polymorph of aza-T-dCyd is administered over repeated administrations such as a treatment cycle. In certain embodiments, aza-T-dCyd is administered over a period of about 4 to about 6 days. In certain embodiments, the crystalline polymorph of aza-T-dCyd is administered via a treatment course that includes a first treatment cycle of administering a therapeutically effective amount of the crystalline polymorph over a period of about 4 to about 6 days, a first rest period of about 1 to about 3 days during which no crystalline polymorph is administered, a second treatment cycle of administering a therapeutically effective amount of the crystalline polymorph over a period of about 4 to about 6 days, and a second rest period of at least about 8 days during which no crystalline polymorph is administered. In yet further embodiments, the crystalline polymorph of aza-T-dCyd is administered via a treatment course that includes a first treatment cycle of administering a therapeutically effective amount of the crystalline polymorph over a period of about 5 days, a first rest period of about 2 days during which no crystalline polymorph is administered, a second treatment cycle of administering a therapeutically effective amount of the crystalline polymorph over a period of about 5 days, and a second rest period of at least about 9 days during which no crystalline polymorph is administered.

[0114] 2. Use of Compounds and Compositions In one embodiment, the present invention relates to the use of the disclosed compositions. In further embodiments, the use relates to the manufacture of a medicament for the treatment of blood cancer in a subject.

[0115] In one embodiment, the use relates to a process for preparing the disclosed pharmaceutical composition for use as a medicament.

[0116] In one embodiment, the use relates to a process for preparing the disclosed pharmaceutical composition, wherein a pharmaceutically acceptable carrier is intimately mixed with a therapeutically effective amount of the compound.

[0117] In various embodiments, the use relates to the treatment of blood cancer in a subject. In one embodiment, the use is characterized in that the subject is a human. In one embodiment, the blood cancer is characterized in that it is non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, or solitary myeloma.

[0118] In a further embodiment, the use relates to the manufacture of a medicament for the treatment of blood cancer in a subject. In one embodiment, the blood cancer is characterized in that it is non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, or solitary myeloma.

[0119] 3. Manufacture of medicaments In one embodiment, the present invention relates to a method for the manufacture of a medicament for treating blood cancer in a human subject having blood cancer, the method comprising combining a therapeutically effective amount of the disclosed compound with a pharmaceutically acceptable carrier or diluent.

[0120] With respect to these uses, the method includes administration of a therapeutically effective amount of the composition to a human. The dose administered to a human needs to be sufficient to affect the therapeutic response in a human over a reasonable time frame in the context of the present invention. One of ordinary skill in the art will recognize that the dosage will depend on various factors including the condition of the human and the weight of the human.

[0121] The total amount of the compositions of the present disclosure administered in a typical treatment is, per daily dose, preferably about 10 mg / kg to about 1000 mg / kg body weight in the case of mice, and about 100 mg / kg to about 500 mg / kg body weight in the case of humans, more preferably about 200 mg / kg to about 400 mg / kg. This total amount is typically, but not necessarily, administered as a series of small doses over a period of about 1 to about 3 times per day for about 24 months, preferably over a period of about 12 months, 2 times per day.

[0122] The magnitude of the dose will also be determined by the route of administration, the timing and frequency, and the presence, nature, and extent of any adverse side effects that may accompany the administration of the composition, as well as the desired physiological effect. It will be understood by those skilled in the art that various conditions or disease states, particularly chronic conditions or disease states, may require long-term treatment involving multiple administrations.

[0123] Thus, in one embodiment, the present invention relates to the manufacture of a medicament comprising combining a disclosed compound, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, with a pharmaceutically acceptable carrier or diluent.

[0124] 4. Kit In one embodiment, a kit is disclosed that includes an effective amount of a disclosed crystalline polymorph and one or more of (a) at least one chemotherapeutic agent, (b) instructions for administering the composition in connection with the treatment of cancer, and (c) instructions for treating cancer. In a further embodiment, the crystalline polymorph has a powder X-ray diffraction pattern having peaks at about 8°, about 13°, about 15°, about 17°, about 19°, about 22°, about 23°, about 26°, about 28°, about 29°, about 31°, about 33°, and about 37° 2θ. In yet a further embodiment, the crystalline polymorph has a powder X-ray diffraction pattern having peaks at about 6°, about 12°, about 13°, about 14°, about 16°, about 18°, about 20°, about 21°, about 22°, about 26°, about 27°, about 29°, about 30°, about 33°, about 35°, about 36°, about 39°, and about 41° 2θ.

[0125] In one embodiment, the agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolite agents, antitumor antibiotic agents, mitotic inhibitors, and mTor inhibitors.

[0126] In one embodiment, the chemotherapeutic agent is an alkylating agent. Examples of alkylating agents include, but are not limited to, carboplatin, cisplatin, cyclophosphamide, chlorambucil, melphalan, carmustine, busulfan, lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, temozolomide, thiotepa, bendamustine, and streptozocin, or pharmaceutically acceptable salts thereof.

[0127] In one embodiment, the chemotherapeutic agent is an antimetabolite agent. Examples of antimetabolite agents include, but are not limited to, gemcitabine, 5-fluorouracil, capecitabine, hydroxyurea, mercaptopurine, pemetrexed, fludarabine, nelarabine, cladribine, clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, methotrexate, and thioguanine, or pharmaceutically acceptable salts thereof.

[0128] In one embodiment, the chemotherapeutic agent is an antitumor antibiotic agent. Examples of antitumor antibiotic agents include, but are not limited to, doxorubicin, mitoxantrone, bleomycin, daunorubicin, dactinomycin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin, or pharmaceutically acceptable salts thereof.

[0129] In one embodiment, the chemotherapeutic agent is a mitotic inhibitor. Examples of mitotic inhibitors include, but are not limited to, irinotecan, topotecan, rubitecan, cabazitaxel, docetaxel, paclitaxel, etopside, vincristine, ixabepilone, vinorelbine, vinblastine, and teniposide, or pharmaceutically acceptable salts thereof.

[0130] In one embodiment, the chemotherapeutic agent is an mTor inhibitor. Examples of mTor inhibitors include, but are not limited to, everolimus, sirolimus, and temsirolimus, or pharmaceutically acceptable salts, hydrates, solvates, or polymorphs thereof.

[0131] In various embodiments, the crystalline polymorph and the drug are co-packaged. In various further embodiments, the crystalline polymorph and the drug are co-formulated.

[0132] In various further embodiments, the crystalline polymorph and the drug are administered sequentially. In various further embodiments, the crystalline polymorph and the drug are administered simultaneously.

[0133] In various embodiments, the disorder of uncontrolled cell growth is cancer. In various further embodiments, the cancer is a blood cancer.

[0134] The following examples are set forth to provide a complete disclosure and description of the methods of making and evaluating the compounds, compositions, articles, devices, and / or methods claimed herein to those skilled in the art and are intended to be purely exemplary and not limiting of the present disclosure. Although efforts have been made to ensure accuracy with respect to numerical values (e.g., amounts, temperatures, etc.), some error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in °C or ambient temperature, and pressure is at or near atmospheric pressure.

Examples

[0135] 1. Preparation method a. Anti-solvent addition Anti-solvent crystallization experiments were carried out by combining 10 different solvents with 10 anti-solvents. The anti-solvent crystallization experiments were carried out by reverse addition, and a small amount of a solution close to saturation of aza-T-dCyd in the selected solvent was added to 20 mL of the anti-solvent and this was stirred vigorously.

[0136] Samples that did not precipitate were left at 5 °C for 3 days to induce precipitation. The precipitated solid was isolated from the mother liquor and analyzed by HT-XRPD after drying overnight in a glove box (20% relative humidity (RH)) and after drying overnight under vacuum (10 mbar). All solids were exposed to accelerated degradation conditions (25 °C / 60% relative humidity for 2 days) and re-analyzed by HT-XRPD.

[0137] b. Evaporative crystallization In the evaporative crystallization experiments from solvent mixtures, a new solution was prepared from the crystalline starting material. The solution was transferred to vials (without caps) and placed under glove box conditions (20% relative humidity / room temperature) to slowly evaporate the solvent for 3 days, followed by placing under vacuum (10 mbar) at room temperature until all the solvent had evaporated. Samples in NMP (experiment IDs ECP43 and ECP44) were dried further at 50 °C under vacuum. The resulting solids were analyzed by HT-XRPD. Subsequently, the solids were placed at 25 °C / 60% relative humidity for 2 days (AAC) and re-analyzed by XRPD.

[0138] c. Solvent equilibration Solvent equilibration experiments were carried out in 29 solvents. Solvent was added little by little to about 20 mg of aza-T-dCyd until a thin suspension was obtained. The suspension was equilibrated with continuous stirring at 5 °C for 5 days and at 25 °C for 1 day.

[0139] After equilibration time (1 day at room temperature, 5 days at 5 °C), the solid was separated by centrifugation. A portion of the solid was collected, sampled onto a 96-well plate, and dried overnight in a glove box (20% relative humidity at room temperature). The remaining solid was dried overnight under vacuum (room temperature and 10 mbar) and then sampled onto a 96-well plate. All solids were analyzed by HT-XRPD. Subsequently, all solids were exposed to accelerated degradation conditions (AAC, 25 °C / 60% relative humidity) for 2 days and re-analyzed by HT-XRPD.

[0140] d. Sonication The sonication experiment was initiated with crystalline aza-T-dCyd. Approximately 20 mg of API was weighed into a 1.8 mL vial and 5 - 10 μL of solvent was added until a paste was obtained. The paste was sonicated in an ultrasonic bath (Fisher Scientific, FB15051) for 10 minutes at room temperature. The solid was sampled, analyzed by HT-XRPD, dried under vacuum (10 mbar / room temperature overnight), and then re-analyzed. Subsequently, all solids were exposed to accelerated degradation conditions (25 °C / 60% relative humidity) for 2 days and re-analyzed by HT-XRPD.

[0141] e. Thermal cycling crystallization The thermal cycling crystallization experiment was carried out in 20 organic solvents and solvent mixtures. Approximately 25 mg of aza-T-dCyd was added to a small amount of solvent (mixture) until a thin suspension was obtained at room temperature. The mixture was then placed in a Crystal16™ reactor and subjected to the temperature profile shown in Figure 16. The sample was heated to 50 °C and cooled to 5 °C at a heating and cooling rate of 10 °C / h, and after 3 cycles, degraded at room temperature for 24 hours.

[0142] After the temperature profile, the solid was separated from the solution by centrifugation. A portion was dried at room temperature in a glove box (20% relative humidity), a portion was dried under deep vacuum (10 mbar), then sampled and analyzed by HT-XRPD. The liquid phase was also evaporated and the recovered solid was analyzed by HT-XRPD. Subsequently, all solids were exposed to accelerated degradation conditions (25 °C / 60% relative humidity for 2 days) and then subjected to HT-XRPD re-analysis.

[0143] f. Vapor diffusion Vapor diffusion experiments into the solution were carried out at room temperature. A solution nearly saturated with aza-T-dCyd was prepared in the solvent of a 1.8 mL glass vial or a 40 mL vial. The open vial containing the saturated solution was placed into a closed larger vial containing 2 - 5 mL of an anti-solvent. The sample was checked for solid formation after one week. The solid was analyzed by HT-XRPD after drying in a glove box (20% relative humidity) and after drying under vacuum (10 mbar). If no precipitation occurred, the solvent was evaporated under vacuum and the resulting solid was analyzed by HT-XRPD. Subsequently, all solids were exposed to accelerated degradation conditions (25 °C / 60% relative humidity for 2 days) and re-analyzed by HT-XRPD.

[0144] 2. Analytical methods a. HT-XRPD XRPD patterns were obtained using an Ardena SSR T2 high-throughput XRPD setup. The plates were mounted on a Bruker General Area Detector Diffraction System (GADDS) equipped with a VÅNTEC-500 gas area detector corrected for intensity and geometric variations. Calibration of the measurement accuracy (peak position) was performed using a NIST SRM1976 standard (Corundum).

[0145] Data collection was performed at room temperature using monochromatic CuKα radiation in the 2 Å region of 1.5° - 41.5°, which is the most characteristic part of the XRPD pattern. The diffraction pattern of each well was collected in two 2θ ranges (1.5° ≤ 2θ ≤ 21.5° for the first frame and 19.5° ≤ 2θ ≤ 41.5° for the second frame) with an exposure time of 90 seconds for each frame. No background subtraction or curve smoothing was applied to the XRPD patterns.

[0146] The carrier material used during XRPD analysis was transparent to X-rays and contributed only slightly to the background.

[0147] b. HR-XRPD HR-XRPD data was collected at room temperature on a D8 Advance diffractometer using Cu Kα1 radiation (1.54056 Å) equipped with a germanium monochromator. Diffraction data was collected in the 2θ range of 2 - 41.5° 2θ. Detector scans with a solid-state LynxEye detector were performed using 0.016° per step at a scan rate of 5 seconds / step. The sample was measured in an 8 mm long glass capillary with an outer diameter of 0.5 mm.

[0148] c. TGMS analysis Mass loss due to the loss of solvent or water from the crystal was determined by TGA. Weight - temperature curves and heat flow thermograms were obtained by monitoring the sample weight during heating in a TGA / DSC3 + STARe system (Mettler - Toledo GmbH, Switzerland). The TGA / DSC3 + was calibrated against indium and aluminum for temperature. A sample (about 2 mg) was weighed into a 100 μL aluminum crucible and sealed. A pinhole was opened in the seal and the crucible was heated in the TGA from 25 to 300 °C at a heating rate of 10 °C / min. Dry N 2 gas was used for purging.

[0149] The gas released from the TGA sample was analyzed by an Omnistar GSD 301 T2 mass spectrometer (Pfeiffer Vacuum GmbH, Germany). This MS is a quadrupole mass spectrometer that analyzes masses in the range of 0 - 200 amu.

[0150] d. DSC analysis Thermal events (i.e., melting, recrystallization) were obtained from DSC thermograms recorded with a Thermal Flow DSC3+ STARe system (Mettler-Toledo GmbH, Switzerland). The DSC3+ was calibrated for temperature and enthalpy with small pieces of indium (m.p. = 156.6 °C, δHf = 28.45 J / g) and zinc (m.p. = 419.6 °C, δHf = 107.5 J / g). The sample (ca. 2 mg) was sealed in a standard 40 μL aluminum pan, pierced with a pinhole, and heated from 25 °C to 300 °C at a heating rate of 10 °C / min in the DSC. To purge the DSC apparatus during the measurement, dry N 2 gas was used at a flow rate of 50 mL / min.

[0151] Cyclic DSC experiments were measured in a standard 40 μL aluminum pan, pierced with a pinhole, heated in the DSC from 25 °C to a variable temperature, and then cooled to 25 °C. The heating and cooling rates were 10 °C / min. To purge the DSC apparatus during the measurement, dry N2 gas was used at a flow rate of 50 mL / min. Subsequently, the sample was recovered and analyzed by HT-XRPD.

[0152] e.LCMS LCMS experiments were performed on an Agilent 1290 series instrument equipped with a diode array UV detector and an MSD XT single quadrupole mass detector. Mobile phases A and B were 10 mM ammonium acetate in water and acetonitrile, respectively. The column was a Waters XBridge HILIC (150x4.6 mm, 3.5 μm, pn. 186004441). Detection was at 244 nm with a 4 nm bandwidth and a UV spectrum from 200 - 400 nm. Spectral measurements were performed in positive scan mode from 100 - 800 m / z with a 500 ms scan time. The flow rate was 0.8 mL / min. The run time was 10 min. The injection volume was 5 μL at 40 °C and the autosampler temperature was 8 °C.

[0153] 3. Example 1: Characterization of the starting material Approximately 4.0 g of aza-T-dCyd was prepared and analyzed by X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermogravimetric analysis / mass spectrometry (TGMS), and liquid chromatography / mass spectrometry (LCMS). The starting material (SM) represents aza-T-dCyd that has not yet been subjected to specific crystallization conditions. Figure 1 shows high-throughput XRPD (HT-XRPD) and high-resolution XRPD (HR-XRPD) in the upper and lower patterns, respectively. The starting material contains crystals suitable for single crystal structure analysis. It crystallizes in the non-centrosymmetric monoclinic P2 1 space group and is designated as Form A. Table 1 provides the relevant dimensions of Form A.

Table 1

[0154] The HR-XRPD pattern of the starting material was compared with the simulated HR-XRPD pattern of the single crystal Form A and is shown in Figure 2. Form A has peaks at 7.7°, 13.02°, 15.34°, 16.78°, 18.62°, 19.42°, 21.94°, 22.90°, 25.70°, 27.86°, 28.70°, 31.42°, 32.70°, and 37.46° 2θ. Based on this comparison, the starting material is calculated to contain approximately 70% Form A and approximately 30% of other crystalline forms of aza-T-dCyd.

[0155] TGMS analysis of the starting material at 25 - 300 °C (10 °C / min) showed a 11.7% mass loss at 100 - 170 °C due to the most likely organic solvent (Figure 3). Simultaneous with the mass loss, the heat flow signal showed two endothermic events with an exothermic event in between. A third endothermic event due to the onset of melting and decomposition was observed around 195 °C.

[0156] DSC analysis of the starting material at 25 - 300 °C (10 °C / min) was consistent with the heat flow signal observed during TGMS analysis and showed two endothermic events at 131 °C and 162 °C with an exothermic event at 141 °C. The third endothermic event is the T at 196.4 °C related to the melting of the non-solvated anhydrous phaseピーク Observed in (Figure 4).

[0157] From XRPD and single crystal structure analysis, it was found that the starting material consisted of a mixture of crystalline phases. To further investigate the nature of the thermal events, two cycle DSC experiments were carried out on the starting material. One sample was heated to 170 °C and cooled to room temperature. The resulting solid was analyzed by XRPD and was found to match the simulated pattern of Form A (Figure 5). In the second cycle DSC experiment, the starting material was heated to 170 °C, cooled to 25 °C, and then heated to 300 °C (Figure 6). No thermal events were observed during cooling, and only an endothermic melting event at 194 °C was observed in the second heating cycle, confirming the melting temperature of Form A.

[0158] The chemical purity of aza-T-dCyd was evaluated by LCMS analysis. The analysis confirmed a chemical purity of 99.8% (area %) (Figure 7A and Figure 7B). The MS spectrum (positive scan mode) showed an ion with an m / z of 489.3 attributable to species [2M+H] + and a low intensity ion at m / z 245.1 attributable to species [M+H] + confirming the molecular weight of the API of 244.3 g / mol.

[0159] Aza-T-dCyd eluted from the column at 4.4 minutes and had an m / z of 489.3 [2M+H] + (Figure 8A and Figure 8B). During the development of the LCMS method, when the API was dissolved in an aqueous medium, impurities appeared over time. The impurities formed were seen in the chromatogram at 3.8 minutes and had an m / z of 263.2 [M+18] + (Figure 8A and Figure 8C).

[0160] The chemical stability of aza-T-dCyd was determined. Aza-T-dCyd was prepared in 1,4-dioxane, acetonitrile (ACN), isopropanol (IPA), and methyl ethyl ketone. Each solution was divided into three vials and incubated at room temperature for 24 hours, at 50 °C for 1 hour, or at 80 °C for 1 hour. The solutions were analyzed by HPLC at the start of the incubation time and thereafter.

[0161] The results are shown in Figure 9. T 0 For, the purity of the API was approximately 99% (area %) in each solvent. The compound remained stable in acetonitrile and 1,4-dioxane at room temperature for 24 hours and at elevated temperatures for 1 hour (purity > 95%). In IPA, the compound was significantly degraded in the solution heated at 80 °C or when stored at room temperature for 24 hours. In methyl ethyl ketone, significant degradation was observed after 1 hour at 50 °C and 80 °C, and after 24 hours of incubation at room temperature.

[0162] Additional stability tests were conducted at 5 °C. Suspensions of the starting material were prepared in water, acetonitrile, ethanol (EtOH), and isopropanol. The stock suspensions and aqueous solutions were analyzed by HPLC at regular intervals over 3 days.

[0163] The results are shown in Figure 10. The purity of aza-T-dCyd is plotted against time as area %. The data points were obtained from single measurements from the same solution. Aza-T-dCyd in ethanol and IPA remained stable for 70 hours, but in acetonitrile and water, the aza-T-dCyd purity decreased to 84% and 78% respectively over time.

[0164] The solid phase from the suspension was also evaluated for purity after 72 hours. The solids recovered from the four solvents had a purity of approximately 99% (area %). See Table 2. Thus, aza-T-dCyd appeared to be chemically stable in the solid phase after incubation at 5 °C for 70 hours.

Table 2

[0165] 4. Example 2: Generation of Amorphous Material An attempt was made to produce an amorphous material from the starting material for the polymorph screen using a lyophilized solution of aza-T-dCyd. To obtain a solution of aza-T-dCyd in an organic solvent for the lyophilization experiment, aza-T-dCyd was added to water, water / 1,4-dioxane (50 / 50), water / THF (50 / 50), and water / tert-butyl alcohol (50 / 50% (v / v)). By lyophilizing the aza-T-dCyd solution, a low-crystalline material containing impurities was obtained.

[0166] 5. Example 3: Solubility Study The thermodynamic solubility of aza-T-dCyd was determined according to the shake flask method. Suspensions of crystalline aza-T-dCyd were prepared in 25 pure solvents. Small amounts of solvent were added to aza-T-dCyd until a thin suspension was obtained. Subsequently, the samples were equilibrated at room temperature under continuous stirring for 24 hours. After equilibration, a small amount of the mother liquor was filtered and analyzed by HPLC. The concentration of the solute was determined against a calibration curve of aza-T-dCyd. The solubility values of aza-T-dCyd at room temperature are shown in Table 3 according to the United States Pharmacopeia classification (USP29). Aza-T-dCyd was soluble in high-boiling solvents such as DMF and DMA. Generally, aza-T-dCyd was slightly or very slightly soluble in polar solvents and substantially insoluble in non-polar solvents.

Table 3

[0167] 6. Example 4: Polymorph Screen A polymorph screen was performed by combining six different crystallization techniques with various pure organic solvents and solvent mixtures. Considering the poor thermal stability of aza-T-dCyd in solution and the limited stability of aza-T-dCyd in water and ketones, the screening experiment conditions were selected as follows: (1) starting the experiment with the crystalline starting material, (2) keeping the compound in solution for a limited time (less than 5 days), (3) avoiding high temperatures (less than 50 °C), (4) handling solid aza-T-dCyd in a glove box under dry conditions (relative humidity of about 20%) to avoid moisture absorption as much as possible, (5) avoiding water, restricting the use of ketones, and (6) gentle stress conditions for evaluating the physical stability of the obtained solid.

[0168] The following crystallization techniques were applied.

[0169] Solvent equilibration experiment. The solvent equilibration experiment was carried out at two temperatures, namely at room temperature for 1 day and at 5 °C for 5 days. Suspensions of aza-T-dCyd were prepared with crystalline starting materials in different solvents, and when the equilibration time was completed, the solid was separated from the mother liquor.

[0170] Evaporative crystallization experiment. Using the filtered mother liquor recovered from the solvent equilibration experiment and solvent mixtures carried out at room temperature, an evaporative crystallization experiment was set up. The solvent was slowly evaporated under ambient conditions and then further dried at 50 °C under vacuum (10 mbar).

[0171] Antisolvent experiment. The antisolvent experiment was carried out using 10 combinations of solvents and antisolvents by reverse addition. A small amount of a high-concentration solution of aza-T-dCyd was added to 20 mL of the antisolvent (1 step).

[0172] Thermal cycling experiment. The thermal cycling experiment was carried out by preparing aza-T-dCyd suspensions in different solvents and solvent mixtures at room temperature. The obtained suspensions were subjected to a temperature profile of 5 - 50 °C.

[0173] Sonication experiment. The sonication experiment was carried out by sonicating the crystalline starting material in the presence of a small amount of solvent.

[0174] Vapor diffusion experiment into solution. The vapor diffusion experiment into solution was carried out as a slow method of anti-solvent crystallization. A saturated aza-T-dCyd solution was exposed to the vapor of the anti-solvent at room temperature for 1 week.

[0175] All the obtained solids were dried overnight in a glove box at room temperature and 20% relative humidity, and then dried overnight under vacuum (10 mbar) at room temperature, and then analyzed by HT-XRPD. When the mother liquor was recovered, the mother liquor (ML) was evaporated and the recovered solid was analyzed by HT-XRPD. Subsequently, all the solids were exposed to accelerated aging conditions (25 °C / 60% relative humidity) for 2 days and re-analyzed by HT-XRPD.

[0176] Form A was the most abundant crystalline phase recovered from the screening experiments. This form was found in all crystallization methods and in a wide variety of solvents and solvent mixtures. From the solvent equilibration experiments, it was observed that Form A was obtained as a pure phase from solvents in which aza-T-dCyd was slightly soluble or very slightly soluble.

[0177] In some of the solids, in addition to the XRPD pattern of Form A, the presence of peaks already observed in the received starting material was detected, as described above. The received batch of aza-T-dCyd contained 70% Form A and 30% of other crystalline phases. The presence of the 30% other phase was most clearly highlighted by the peaks appearing in the XRPD pattern at 16.0°, 17.6°, 24.8°, 26.3°, and 34.1° 2θ. The evaluation of the solids recovered from the polymorph screening experiments attempted the assignment and classification of such impurity peaks. An overview of the XRPD patterns of the starting material, Form A, Form B, Form A + C1, Form A + C2, Form A + D1, and Form A + D2 is shown in Figure 11.

[0178] The peak at 26.3° 2θ was assigned to form B. The peak observed at 16.0° 2θ represented form C1, and the peaks at 16.0 and 17.6° 2θ were assigned to form C2. The peak observed at 24.8° 2θ was assigned to form D1, and the peaks at 24.8 and 34.1° 2θ were assigned to form D2. Based on this assignment, some solids were classified as form A+D1 / D2, A+C1 / C2 or A+B+D2.

[0179] By solvent equilibration in both DMA and DMF at room temperature and 5 °C, and also from thermal cycling experiments in DMSO / 2-ethyl-1-hexanol (50 / 50), form B was obtained as a pure phase. Form B was physically unstable and converted to form A after storage at 25 °C and 60% relative humidity.

[0180] Classes C and D were never observed as pure crystalline phases and were always mixed with form A. In most cases, these mixtures were converted to form A after storage at 25 °C and 60% relative humidity.

[0181] New forms were found from solution-based crystallization methods but no seeds of form A were present. These new forms were classified as forms E, F, G1, G2, H, I, J, K. Form E was obtained from anti-solvent addition in DMA / chloroform or evaporation crystallization from DMA / TBME (80 / 20). Form E converts to form A after storage at 25 °C and 60% relative humidity.

[0182] Form F was obtained from vapor diffusion or evaporation crystallization in various solvents. Form F was physically stable. The peaks of form F are at 6.06°, 12.10°, 13.02°, 14.38°, 15.94°, 17.50°, 19.62°, 21.18°, 22.34°, 26.18°, 27.42°, 28.50°, 29.90°, 32.66°, 35.02°, 36.30°, 38.94°, and 41.06° 2θ.

[0183] Polymorphs G1 and G2 have similar XRPD patterns, with some peaks shifted between the two polymorphs. Polymorph G1 was obtained from anti-solvent addition or sonication. Polymorph G2 was obtained from evaporation crystallization in DMA / EtOH. Both Polymorph G1 and Polymorph G2 are converted to Polymorph A after storage at 25 °C and 60% relative humidity.

[0184] Polymorph H was obtained from evaporation crystallization in several solvent mixtures. This polymorph is unstable. When obtained from NMP, Polymorph H was converted to Polymorph F. When obtained from other solvents, Polymorph H was converted to Polymorph A.

[0185] Polymorph I was obtained from evaporation crystallization from DMSO / IPA. Polymorph I converts to Polymorph A after storage at 25 °C and 60% relative humidity.

[0186] Polymorph J was obtained from vapor diffusion into a solution using DMF as the solvent and THF as the anti-solvent. Polymorph J converts to Polymorph A after storage at 25 °C and 60% relative humidity.

[0187] After evaporation crystallization from DMF, Polymorph K was observed in a mixture with Polymorph F. Polymorph K converted to Polymorph F after storage at 25 °C and 60% relative humidity.

[0188] Polymorph L was observed in the solid state after storage at 25 °C and 65% relative humidity.

[0189] The XRPD patterns for each of these novel polymorphs are shown in Figure 12.

[0190] 7. Example 5: Characterization of Novel Polymorphs of Aza-T-DCYD Each unique polymorph identified by screening was further characterized by TGMS and LCMS. Polymorphs A and F appear to be anhydrous, while the other polymorphs were solvated. Table 4 summarizes the crystallization conditions for the described polymorphs of Aza-T-dCyd (AAC indicates storage at 25 °C and 60% relative humidity). Table 5 summarizes the properties of the various Aza-T-dCyd polymorphs (AAC indicates storage at 25 °C and 60% relative humidity).

Table 4

Table 5

[0191] The morphological form A obtained from the solvent equilibration experiment at RT in TFE was used for analytical characterization. The TGMS results showed the release of approximately 0.7% of the residual solvent in the temperature range of 30 - 190 °C (Figure 12A). Endothermic events due to melting and decomposition were observed at 205 °C in the DSC trace (Figure 12B). The integrity of form A was confirmed by LCMS analysis at 100% purity (area %) (Figure 12C).

[0192] The morphological form F obtained from the evaporation crystallization experiment using DMF / acetonitrile (80 / 20, v / v) was used for characterization. The results of TGMS showed a small loss of 1.1% at 30 - 140 °C, which is most likely due to the residual solvent (Figure 15A). The DSC trace showed one endothermic event near 170 °C due to melting and decomposition (Figure 15B). The integrity of the API was confirmed by LCMS analysis at 100% purity (area %) (Figure 15C).

[0193] Form A has a higher melting temperature than form F and can be considered a more thermodynamically stable form. Both form A and form F are anhydrous.

[0194] Forms B, C2, D2, E, G1, G2, H, I, J, and K are each solvated and are converted to form A when stored at 25 °C and 60% relative humidity for 2 days.

[0195] Morphology B obtained from the solvent equilibration experiment in DMA at room temperature was further characterized. The TGMS results showed a gradual mass loss upon heating with a 25.0% mass loss from 30 to 170 °C. Due to the gradual mass loss upon heating, it is unclear at which temperature the decomposition starts. Morphology B may be a non-stoichiometric solvate that can be formed with different solvents. LCMS analysis showed a solid purity of 97.3% of aza-T-dCyd and the presence of 2.7% impurities (area %).

[0196] Morphology C2 represented two additional peaks observed in the XRPD pattern in a mixture with other morphologies. The TGMS analysis showed a 0.7% mass loss in the temperature range of 30 to 160 °C. The heat flow signal showed only one endothermic event near 190 °C, which may be related to the melting and decomposition of morphology A. In a mixture with morphology A, morphology C2 was present only in trace amounts, so the investigation regarding morphology C2 was inconclusive, and thus the nature of this morphology remained unknown. However, since the chemical purity of the whole solid sample was 100% (area %), it seems to be a true (pseudo) polymorph of aza-T-dCyd.

[0197] Morphology D2 represented two additional peaks observed in the XRPD pattern in a mixture with morphology A. The TGMS analysis of morphology A+D2 indicated that morphology D2 was most likely a solvated form. A 5.1% mass loss was observed from 90 to 170 °C. The results were inconclusive regarding the released solvent. The integrity of aza-T-dCyd with 100% chemical purity (area %) was confirmed by LCMS analysis of the mixture of morphologies.

[0198] Morphology E obtained from the evaporation crystallization experiment in DMA was further analyzed by TGMS and LCMS. The TGMS results showed a 25.8% mass loss of DMA corresponding to 1 molar equivalent of the solvent. The solvent was released stepwise from 90 to 160 °C, suggesting that morphology E is a mono-DMA solvate. An endothermic event at 200 °C, which most likely corresponds to the melting of morphology A, was recorded after desolvation. The integrity of the compound was confirmed by LCMS analysis.

[0199] Class G is an isostructural class of solvates. Forms G1 and G2 were further characterized by TGMS and LCMS. The integrity of the compound was confirmed by LCMS analysis (100% area%). Form G1 obtained from the anti-solvent addition experiment using NMP and cyclohexane was used for characterization. The TGMS results showed a stepwise mass loss of 27.5% at 90 - 160 °C. The 27.5% mass loss corresponds to approximately 1 molecule of NMP per molecule of aza-T-dCyd, and thus, form G1 may be a mono-NMP solvate. The DSC signal recorded two endothermic events near 110 and 150 °C due to solvent loss, and a third endothermic event at 200 °C that may correspond to the melting of form A. Form G2 was obtained by evaporation crystallization from DMA / ethanol 80 / 20 (v / v). The 14.6% mass loss observed by TGMS at 70 - 120 °C corresponded to 0.5 molar equivalent of DMA. This suggested that form G2 may be a hemi-DMA solvate. In the DSC signal, two endothermic events were observed near 80 and 90 °C due to solvent loss, and a third endothermic event near 195 °C due to melting and decomposition was observed.

[0200] Form H obtained from evaporation crystallization from NMP / THF (80 / 20, v / v) was used for the characterization of form H. The progressive mass loss observed by TGMS analysis was 15.3% at 30 - 180 °C, corresponding to approximately 0.5 molar equivalent of NMP. At the same time, a broad endothermic event was observed near 130 °C. Since form H was observed in experiments using various solvents, it is most likely a non-stoichiometric solvate that can incorporate various solvent molecules into its crystal structure. A second broad endothermic event due to decomposition was observed at approximately 220 °C in the DSC trace. From the TGMS data, it was unclear where the solvent loss ended and the thermal decomposition began, and the events may have overlapped partially. The solid had to be dried under vacuum at 50 °C for 24 h to obtain a dry sample. The LCMS data showed that the solid had a purity of 82% (area%), which may have affected the purity.

[0201] Form I was obtained by evaporation crystallization from DMSO / IPA (80 / 20, v / v). The TGMS data showed a progressive mass loss of 14.7% from 30 to 170 °C. The 14.7% mass loss corresponds to approximately 0.5 molar equivalents of DMSO. Form I may be a hemi-DMSO solvate. The DSC trace showed two broad endothermic events at 70 °C and 110 °C due to mass loss, and a third endothermic event at around 190 °C due to the melting and decomposition processes.

[0202] Form J, precipitated by vapor diffusion into solutions using DMF and THF, was further characterized. The TGMS data showed a stepwise mass loss of 7.6% of THF from 120 to 170 °C. The mass loss corresponds to approximately 0.3 molar equivalents of THF, and thus Form J is most likely a non-stoichiometric solvate. The DSC trace recorded two endothermic events at 120 °C and 150 °C due to solvent loss, and the third endothermic event was recorded at 200 °C, which coincides with the melting / decomposition event of Form A.

[0203] Form K was once observed in a mixture with Form F and was obtained by evaporation from a DMF solution. The mixture was further characterized. The TGMS analysis showed a mass loss of 6.3% from 30 to 160 °C, probably due to the loss of DMF. The mass loss was accompanied by a small endothermic event around 110 °C. Two large endothermic events were observed at 180 °C and 195 °C. The endotherm at 195 °C may be due to the melting and decomposition of Form A. Since Form K was in a mixture with Form F (non-solvated form), Form K is most likely a solvated form.

[0204] Form L was only observed after storage at 25 °C and 60% relative humidity and was a poor crystalline solid with a very low yield. In the TGMS analysis, a 2.8% mass loss was observed between 30 and 170 °C, followed by decomposition. The absence of thermal events in the DSC trace may be due to the small amount of sample used in the analysis. It is unclear whether the mass loss is due to solvent trapped in the crystal structure or to residual solvent. Further characterization could not be performed, and thus the properties of Form L remain unknown.

[0205] Crystals obtained from attempts to grow single crystals from an aza-T-dCyd solution in acetonitrile appeared to be an acetonitrile solvate. This phase was not observed in any of the screening experiments. The solvate crystallized in the monoclinic space group P21 / c with cell unit dimensions a = 9.2948(15), b = 7.3509(9), c = 10.2312(15) Å, β = 107.661(2)°, V = 666.10(17) Å 3 , Z = 2, and a density of 1.423 g / cm 1 3. Since only single crystals were formed (very low yield), further characterization of this form was not performed and its physical stability has not been investigated. 3

[0206] 8. Example 6: Pharmacokinetic Properties of Aza-T-DCYD The pharmacokinetic properties of aza-T-dCyd (starting material, SM, aza-T-dCyd which has not yet been subjected to specific crystallization conditions) were studied as follows.

[0207] Six female NOD-SCID mice that divided the aza-T-dCyd starting material (SM) into four groups were administered. Group 1 was the vehicle control group. In the second group, 2.0 mg / kg of the aza-T-dCyd starting material (SM) was administered once a day, and in the third group, 1.0 mg / kg of the aza-T-dCyd starting material (SM) was administered twice a day. In groups 2 and 3, the above amount of the aza-T-dCyd starting material (SM) was administered for 5 days, followed by a 2-day rest period, and then re-administered for 5 days, followed by another 9-day rest period. This cycle was repeated. In the fourth group, 1.0 mg / kg of the aza-T-dCyd starting material (SM) was administered once a day for 5 days, followed by a 2-day rest period, and this cycle was repeated. The tumor size in the mice was measured using a fluorescent agent, and the results were as shown in Figure 17.

[0208] As shown in Figure 17, the tumor size increased in Group 1 (the vehicle control group). Also, it was confirmed that the increase in tumor size was most significantly suppressed in Group 2. In contrast, although the AUC of SM in Group 3 was expected to be the same as that in Group 2, it was observed that the tumor size increased rapidly 40 days after administration. From the above, it was found that aza-T-dCyd is Cmax-dependent rather than AUC-dependent.

[0209] In addition, as shown in Figure 18 showing the results on the 43rd day, the tumor size of Group 2 (2.0 mg / kg, once a day) was significantly smaller than that of Group 1 (1.0 mg / kg, twice a day).

[0210] Furthermore, after treating blood cancer cells (Mv4-11) with the aza-T-dCyd starting material (SM), the half-maximal inhibitory concentration (IC 50 ) was measured at 1 hour, 2 hours, and 4 hours. The results are shown in Figure 19. The IC 50 measured at 1 hour was approximately 160 nM, and thus, the IC 50 at 2 hours was expected to be 80 nM, and the IC 50 at 4 hours was expected to be 20 nM. However, the measured IC 50was approximately 120 nM, which was much higher than the expected value of 80 nM. In addition, the measured IC at 4 hours 50 was approximately 80 nM, which was much higher than the expected value of 20 nM. Therefore, it was confirmed that as the exposure time of the compound to cells increased, the efficiency of the aza-T-dCyd starting material (SM) decreased significantly. This suggests that exposing a larger amount of the aza-T-dCyd starting material (SM) for a shorter period provides an efficient treatment.

[0211] Therefore, the above data suggest that crystalline polymorphs with a large dissolution profile such as Form A or Form F of the present disclosure have advantages over the aza-T-dCyd starting material (SM) and other crystalline polymorphs with inferior dissolution profiles. Further, for the same reason, the above data suggest that crystalline polymorphs such as Form A or Form F of the present disclosure exhibit an improved PK profile compared to the aza-T-dCyd starting material (SM) or other crystalline polymorphs.

[0212] 9. Example 7: Dissolution rate profiles of Form A and Form B at various pH points The dissolution rates of Form A and Form B at pH 1.2, pH 6.5, and pH 5.0 were measured and are shown in Table 6 and Figures 20 - 22.

Table 6

[0213] As shown in Table 6 and Figures 20 - 22, at pH 1.2 (the pH conditions of the stomach and large intestine), similar dissolution rates were shown for Form A and Form F, while Form A showed a more consistent dissolution rate profile compared to Form F. At pH 6.5 and pH 5 (the pH conditions of the appendix and small intestine), Form F showed a higher dissolution rate than Form A.

[0214] The above suggests that Form A can be prepared in various drug forms targeting the release of the active ingredient of the drug at about pH 1.2 (e.g., stomach or large intestine). Additionally, the above suggests that Form F can be prepared in various drug forms targeting the release of the active ingredient of the drug at about pH 5.0 - 6.5 (e.g., small intestine).

[0215] 10. Example 8: Comparison of the Pharmacokinetics of Azathioprine - T - DCyd Starting Material, Form A, and Form F The pharmacokinetic properties of azathioprine - dCyd (starting material, SM, azathioprine - dCyd that has not yet been subjected to specific crystallization conditions), Form A, and Form F were studied as follows.

[0216] Each of azathioprine - dCyd starting material (SM), Form A, and Form F was prepared in the form of capsules each mixed with microcrystalline cellulose at 8:92 (w / w), and could be administered to rats at 2 mg / kg of SM, Form A, or Form F. Each of the SM capsules, Form A capsules, and Form F capsules was administered to two male SD rats (i.e., a total of six male SD rats) at a dose of 2 mg / kg. As shown in Tables 7 - 9, the plasma concentrations of each of the SM, Form A, and Form F in the tested SD rats were measured at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after the administration of the capsules.

Table 7

Table 8

Table 9

[0217] In addition, as shown in Tables 10 - 12 below, pharmacokinetic parameters were obtained.

Table 10

Table 11

Table 12

[0218] As shown above, both Form A and Form F showed a max lowering gold C value compared to SM. In particular, Form A showed a C value max about 1.3 times higher than SM, and Form F showed a C value max about 1.4 times higher than SM. In addition, both Form A and Form B showed an AUC value about 30% higher than SM.

[0219] 11. Example 9: Comparison of the half-maximal inhibitory concentration (IC 50 ) of aza-T-DCYD starting material and Form A The K562 and HL-60 cell lines were cultured and maintained in RPMI (10% FBS, 1% penicillin-streptomycin) medium at 37 °C, 95% air, and 5% CO 2 . The K562 and HL-60 cell lines were seeded into 96-well plates at a density of 3000 cells / well (90 μl) each. Form A and SM were treated at a final concentration of 10 μM in each well by treating 10 μl using 3-fold dilutions. The cells were incubated at 37 °C, 95% air, and 5% CO 2 for 3 days. The 96-well plates were placed at room temperature for 30 minutes for equilibration. Then, 100 μl of CellTiter-Glo® Luminescent Cell Viability Assay Reagent was added to the 96 wells and incubated at room temperature for 10 minutes. Luminescence was measured using a luminometer, and the IC 50 values were analyzed using GraphPrism.

[0220] As shown in FIGS. 23 and 24, Form A had an IC 50 value about 5% lower than SM.Indicates a value and thus provides a greater effect.

[0221] The above description of this embodiment is provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the present disclosure. The individual elements or features of a particular embodiment are generally not limited to that particular embodiment, but are interchangeable where applicable and may be used in the selected embodiment even if not specifically shown or described. The same may also be modified in various ways. Such modifications should not be regarded as a departure from the present disclosure, and it is intended that all such modifications be included within the scope of the present disclosure. Another aspect of the present invention may be as follows. 〔1〕A crystalline polymorph of 5-aza-4’-thio-2’-deoxycytidine, wherein the crystalline polymorph has a powder X-ray diffraction pattern including peaks at about 8°, about 13°, about 15°, about 17°, about 19°, about 22°, about 23°, about 26°, about 28°, about 29°, about 31°, about 33°, and about 37° 2θ. 〔2〕The crystalline polymorph according to the above 〔1〕, wherein the crystalline polymorph has an X-ray powder diffraction pattern that is substantially similar to or the same as the X-ray powder diffraction pattern shown in Figure 11. 〔3〕A crystalline polymorph of 5-aza-4’-thio-2’-deoxycytidine, wherein the crystalline polymorph has a powder X-ray diffraction pattern including peaks at about 6°, about 12°, about 13°, about 14°, about 16°, about 18°, about 20°, about 21°, about 22°, about 26°, about 27°, about 29°, about 30°, about 33°, about 35°, about 36°, about 39°, and about 41° 2θ. 〔4〕The crystalline polymorph according to the above 〔3〕, which shows an X-ray powder diffraction pattern that is substantially similar to or the same as the X-ray powder diffraction pattern shown in Figure 16. 〔5〕An effective amount of the crystalline polymorph according to the above 〔1〕, or the crystalline polymorph according to the above 〔3〕, and a pharmaceutically acceptable carrier, a pharmaceutical composition. 〔6〕The pharmaceutical composition according to the above 〔5〕, wherein the composition contains the crystalline polymorph according to the above 〔1〕. 〔7〕The pharmaceutical composition according to the above 〔5〕, wherein the composition contains an active ingredient consisting of the crystalline polymorph according to the above 〔1〕. 〔8〕The pharmaceutical composition according to the above 〔5〕, wherein the composition contains the crystalline polymorph according to the above 〔1〕 but does not contain other crystalline polymorphs of 5-aza-4’-thio-2’-deoxycytidine. 〔9〕The pharmaceutical composition according to the above 〔5〕, wherein the composition contains the crystalline polymorph according to the above 〔3〕. 〔10〕The pharmaceutical composition according to the above 〔5〕, wherein the composition contains an active ingredient consisting of the crystalline polymorph according to the above 〔3〕. 〔11〕The pharmaceutical composition according to item 〔5〕 above, wherein the composition contains the crystalline polymorph described in item 〔3〕 above, but does not contain other crystalline polymorphs of 5-aza-4'-thio-2'-deoxycytidine. 〔12〕The effective amount is about 35 mg / m 2 to about 70 mg / m 2 The pharmaceutical composition according to any one of items 〔5〕 to 〔11〕 above. 〔13〕The pharmaceutical composition according to any one of items 〔5〕 to 〔12〕 above, wherein the composition further contains a chemotherapeutic agent. 〔14〕The pharmaceutical composition according to item 〔13〕 above, wherein the chemotherapeutic agent is selected from the group consisting of alkylating agents, antimetabolite agents, antitumor antibiotics, mitotic inhibitors, and mTOR inhibitors. 〔15〕The pharmaceutical composition according to item 〔14〕 above, wherein the antitumor antibiotic is selected from the group consisting of doxorubicin, mitoxantrone, bleomycin, daunorubicin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin, or pharmaceutically acceptable salts thereof. 〔16〕The pharmaceutical composition according to item 〔14〕 above, wherein the antimetabolite agent is selected from the group consisting of gemcitabine, 5-fluorouracil, capectiabine, hydroxyurea, mercaptopurine, pemetrexed, fludarabine, nelarabine, cladribine, clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, methotrexate, and thioguanine, or pharmaceutically acceptable salts thereof. 〔17〕The pharmaceutical composition according to item 〔14〕 above, wherein the alkylating agent is selected from the group consisting of carboplatin, cisplatin, cyclophosphamide, chlorambucil, melphalan, carmustine, busulfan, lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, temozolomide, thiotepa, bendamustine, and streptozocin, or pharmaceutically acceptable salts thereof. 〔18〕The pharmaceutical composition according to item 〔14〕 above, wherein the mitotic inhibitor is selected from the group consisting of irinotecan, topotecan, rubitecan, cabazitaxel, docetaxel, paclitaxel, etopside, vincristine, exabepilone, vinorelbine, vinblastine, and teniposide, or pharmaceutically acceptable salts thereof. 〔19〕The pharmaceutical composition according to the above

[14] , wherein the mTOR inhibitor is selected from the group consisting of everolimus, sirolimus, and temsirolimus, or pharmaceutically acceptable salts thereof. 〔20〕A 5-aza-4'-thio-2'-deoxycytidine compound comprising the crystalline polymorph according to the above [1]. 〔21〕A 5-aza-4'-thio-2'-deoxycytidine compound comprising the crystalline polymorph according to the above [3]. 〔22〕A method for treating a subject in need of treating cancer, the method comprising administering to the subject an effective amount of the crystalline polymorph according to the above [1] or the crystalline polymorph according to the above [3]. 〔23〕The method according to the above

[22] , wherein the cancer is a blood cancer. 〔24〕The method according to the above

[23] , wherein the blood cancer is selected from the group consisting of non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and solitary myeloma. 〔25〕The method according to the above

[22] , wherein the cancer is a solid cancer. 〔26〕The method according to the above

[25] , wherein the solid cancer is selected from the group consisting of gastric cancer, kidney cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, lung cancer, colon cancer, breast cancer, melanoma, and pancreatic cancer. 〔27〕The method according to any one of the above

[22] to

[26] , wherein the effective amount is a therapeutically effective amount. 〔28〕The effective amount is about 35 mg / m 2 ~about 70 mg / m 2 The method according to any one of the above

[22] to

[27] . 〔29〕The method according to any one of the above

[22] to

[28] , wherein the crystalline polymorph is present in a pharmaceutical composition. 〔30〕The method according to any one of the above

[22] to

[29] , wherein the method further comprises administering a chemotherapeutic agent to the subject. 〔31〕The method according to any one of the above

[22] to

[30] , wherein the effective amount is administered as a single dose. 〔32〕The method according to any one of the above

[22] to

[30] , wherein the effective amount is administered via a plurality of doses. 〔33〕The method according to any one of the above

[22] to

[32] , further comprising identifying a subject in need of treating a blood cancer. 〔34〕The method according to any one of the above

[22] to

[33] , wherein the subject has been diagnosed as being in need of treating cancer prior to the administering step. 〔35〕The method according to 〔22〕, wherein the administration is repeated administration. 〔36〕The method according to 〔35〕, wherein the administration is for a period of about 4 days to about 6 days. 〔37〕The method according to 〔22〕, wherein the administration is carried out through treatment cycles. 〔38〕The method according to 〔37〕, wherein each treatment cycle comprises administering the effective amount of the compound for a period of about 4 days to about 6 days. 〔39〕The method according to 〔22〕, wherein the administration is carried out through a treatment process comprising a plurality of treatment cycles and a plurality of rest periods. 〔40〕The method according to 〔39〕, wherein each treatment cycle comprises administering the effective amount of the compound for a period of about 4 days to about 6 days. 〔41〕The method according to 〔39〕, wherein each treatment cycle comprises administering the effective amount of the compound for a period of about 5 days. 〔42〕The method according to 〔39〕, wherein each rest period comprises refraining from administering the compound for a period of about 1 day to about 10 days. 〔43〕The administration is a first treatment cycle comprising administering the effective amount of the crystalline polymorph for a period of about 4 days to about 6 days, a first rest period comprising refraining from administering the crystalline polymorph for a period of about 1 day to about 3 days, a second treatment cycle comprising administering the effective amount of the crystalline polymorph for a period of about 4 days to about 6 days, a second rest period comprising refraining from administering the crystalline polymorph for a period of at least about 8 days, and is carried out through a treatment process, the method according to 〔22〕. 〔44〕The method according to 〔43〕, wherein the effective amount is administered as a single dose. 〔45〕The method according to 〔43〕, wherein the effective amount is administered via a plurality of doses. 〔46〕The method according to 〔43〕, wherein the effective amount is administered via a single dose on some days and via a plurality of doses on other days. 〔47〕The administration is a first treatment cycle comprising administering the effective amount of the crystalline polymorph for a period of about 5 days, a first rest period comprising refraining from administering the crystalline polymorph for a period of about 2 days, a second treatment cycle comprising administering the effective amount of the crystalline polymorph for a period of about 5 days, a second rest period comprising refraining from administering the crystalline polymorph for a period of at least about 9 days, and is carried out through a treatment process, the method according to 〔22〕. 〔48〕A method for preparing the crystalline polymorph described in the above-mentioned 〔1〕 or 〔3〕, which comprises subjecting aza-T-dCyd to one or more of solvent equilibration, evaporation crystallization, anti-solvent addition, thermal cycle crystallization, sonication, and vapor diffusion into a solution. 〔49〕The crystalline polymorph described in the above-mentioned 〔1〕 or the crystalline polymorph described in 〔3〕, (a) at least one chemotherapeutic agent, (b) instructions for administering the composition in relation to the treatment of cancer, and (c) instructions for treating cancer, 〔50〕The kit according to the above-mentioned 〔49〕, wherein the chemotherapeutic agent is selected from alkylating agents, antimetabolite agents, antitumor antibiotic agents, mitotic inhibitors, and mTor inhibitors. 〔51〕The kit according to the above-mentioned 〔50〕, wherein the antitumor antibiotic agent is selected from doxorubicin, mitoxantrone, bleomycin, daunorubicin, dactinomycin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin, or pharmaceutically acceptable salts thereof. 〔52〕The kit according to the above-mentioned 〔50〕, wherein the antimetabolite agent is selected from gemcitabine, 5-fluorouracil, capecitabine, hydroxyurea, mercaptopurine, pemetrexed, fludarabine, nelarabine, cladribine, clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, methotrexate, and thioguanine, or pharmaceutically acceptable salts thereof. 〔53〕The kit according to the above-mentioned 〔50〕, wherein the alkylating agent is selected from carboplatin, cisplatin, cyclophosphamide, chlorambucil, melphalan, carmustine, busulfan, lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, temozolomide, thiotepa, bendamustine, and streptozocin, or pharmaceutically acceptable salts thereof. 〔54〕The kit according to the above-mentioned 〔50〕, wherein the mitotic inhibitor is selected from irinotecan, topotecan, rubitecan, cabazitaxel, docetaxel, paclitaxel, etopside, vincristine, ixabepilone, vinorelbine, vinblastine, and teniposide, or pharmaceutically acceptable salts thereof. The kit according to

[50] , wherein the mTor inhibitor is selected from everolimus, sirolimus, and temsirolimus, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof. The kit according to

[49] , wherein the crystalline polymorph is co-packaged with the chemotherapeutic agent.

Claims

1. A crystalline form A of 5-aza-4'-thio-2'-deoxycytidine as a pure crystal, comprising: 0.3°, 26.64°±0.3°, 27.86°±0.3°, 28.63°±0.3°, 29.45°±0.3°, 31.42°±0.3°, 32.70°±0.3°, 34.72°±0.3°, 35.97°±0.3°, and 37.46°±0.3° 2θ.

2. 13. The crystalline form A of claim 1, wherein the powder X-ray diffraction pattern is as shown in FIG. [Figure 11]

3. Crystal form F of 5-aza-4'-thio-2'-deoxycytidine, , 26.18°±0.3°, 27.42°±0.3°, 28.50°±0.3°, 29.90°±0.3°, 32.66°±0.3°, 35.02°±0.3°, 36.30°±0.3°, 38.94°±0.3°, and 41.06°±0.3°2θ.

4. The crystalline form F described in claim 3, having a powder X-ray diffraction pattern as shown in Figure 16. [Figure 16]

5. 13. A pharmaceutical composition comprising an effective amount of crystalline form A of claim 1 and a pharma- ceutically acceptable carrier.

6. 13. A pharmaceutical composition comprising the crystalline form A of claim 1 for use in the treatment of cancer.

7. 7. The pharmaceutical composition for use according to claim 6, wherein the cancer is a blood cancer or a solid cancer.

8. 4. A pharmaceutical composition comprising an effective amount of crystalline form F of claim 3 and a pharma- ceutically acceptable carrier.

9. 4. A pharmaceutical composition comprising crystalline form F of claim 3 for use in the treatment of cancer.

10. 10. The pharmaceutical composition for use according to claim 9, wherein the cancer is a blood cancer or a solid cancer.

11. 2. The 5-aza-4'-thio-2'-deoxycytidine compound of claim 1, which comprises crystalline form A.

12. The 5-aza-4'-thio-2'-deoxycytidine compound of claim 3, which comprises crystalline form F.

13. 13. Use of crystalline form A of claim 1 in the manufacture of a medicament for the treatment of cancer.

14. 14. The use according to claim 13, wherein the cancer is a blood cancer.

15. 15. The use of claim 14, wherein the hematological cancer is selected from the group consisting of non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and solitary myeloma.

16. The use according to claim 13, wherein the cancer is a solid cancer.

17. 4. Use of crystalline form F of claim 3 in the manufacture of a medicament for the treatment of cancer.

18. 18. The use according to claim 17, wherein the cancer is a blood cancer.

19. 19. The use of claim 18, wherein the hematological cancer is selected from the group consisting of non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and solitary myeloma.

20. The use according to claim 17, wherein the cancer is a solid cancer.

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