Polymorphic forms of EGFR inhibitors

Abstract

Polymorphic forms of (S)—N-(3-bromo-2-fluorophenyl)-7-((4-methylpiperazin-1-yl)methyl)-7,8-dihydro-[1,4]dioxino[2,3-g]quinazolin-4-amine are disclosed herein. [Case 1] TIFF2024535380000034.tif60113

Description

[Background technology]

[0001] Glioblastoma (Glioblastoma multiforme, GBM) accounts for the majority of primary malignant brain tumors in adults. Amplification and mutation of the epidermal growth factor receptor (EGFR) gene are characteristic genetic abnormalities found in GBM (Sugawa, et al. (1990) Proc. Natl. Acad. Sci. 87:8602-8606; Ekstrand, et al. (1992) Proc. Natl. Acad. Sci. 89:4309-4313). A variety of potential therapies, including tyrosine kinase inhibitors (TKIs), monoclonal antibodies, vaccines, and RNA-based drugs that target EGFR or its mutant constitutively active form, AEGFR, are currently in development or clinical trials for the treatment of GBM. However, so far, their effectiveness in the clinic has been limited by both primary and acquired drug resistance (Taylor, et al. (2012) Curr. Cancer Drug Targets. 12: 197-209). A major limitation is the poor brain penetration of current therapies such as erlotinib, lapatinib, gefitinib, and afatinib (Razier, et al. (2010) Neuro-Oncology 12: 95-103; Reardon, et al. (2015) Neuro-Oncology 17: 430-439; Thiessen, et al. (2010) Cancer Chemother. Pharmacol. 65: 353-361).

[0002] International Publication No. WO2020 / 190765 discloses (S)—N-(3-bromo-2-fluorophenyl)-7-((4-methylpiperazin-1-yl)methyl)-7,8-dihydro-[1,4]dioxino[2,3-g]quinazolin-4-amine ("Compound (I)"): [ka] and that it exhibited both good EGFR inhibitory activity and brain penetrating ability. However, the polymorphic form was not specified in the disclosure.

[0003] During drug development of an active pharmaceutical ingredient (API), the physical form of the API or its salts can affect the physical properties of the drug candidate in the formulation. Many active pharmaceutical ingredients can exist in more than one polymorphic form. In addition, they can exist as a free base, free acid, or pharma- ceutically acceptable salt form as a preferred form. Thus, identifying the pharma-ceutically acceptable salt form for manufacture, as well as the free base or free acid form, represents an important step in the development of a drug candidate.

[0004] In determining or identifying a preferred solid form, the preferred solid form is often one that has unpredictable physical properties. A particular solid form (crystalline, semi-solid) may be preferred for ease of preparation, stability, etc. On the other hand, a different crystalline solid may be preferred for higher solubility and / or better pharmacokinetics. Thus, while a pharmaceutically acceptable salt of an active ingredient may provide high solubility or high solubility, the development of a particular drug form, such as a crystalline solid, may be required to achieve a clinical pharmaceutical formulation. Summary of the Invention

[0005] This application is directed to polymorphs of (S)—N-(3-bromo-2-fluorophenyl)-7-((4-methylpiperazin-1-yl)methyl)-7,8-dihydro-[1,4]dioxino[2,3-g]quinazolin-4-amine.

[0006] In one aspect, embodiments herein relate to polymorphic forms of Compound (I): [ka]

[0007] In one embodiment, the polymorphic form of Compound (I) is Form A and has an X-ray powder diffraction pattern with characteristic peaks expressed at values ​​of 2θ of about 4.9±0.2, about 13.9±0.2, about 22.1±0.2, and about 25.1±0.2 degrees.

[0008] In one embodiment, the polymorphic form of Compound (I) is Form B and has an X-ray powder diffraction pattern having characteristic peaks expressed at values ​​of 2-theta of about 4.8±0.2, about 9.8±0.2, about 13.8±0.2, about 14.7±0.2, about 17.7±0.2, about 20.2±0.2, about 24.1±0.2, about 24.6±0.2, and about 25.1±0.2 degrees.

[0009] In one embodiment, the polymorphic form of Compound (I) is Form C and has an X-ray powder diffraction pattern with characteristic peaks expressed at values ​​of 2θ of about 4.9±0.2, about 22.9±0.2, about 23.2±0.2, about 23.7±0.2, and about 24.3±0.2 degrees.

[0010] In one embodiment, the polymorphic form of Compound (I) is Form D and has an X-ray powder diffraction pattern with characteristic peaks expressed at values ​​of 2θ of about 15.6±0.2, about 16.9±0.2, about 19.1±0.2, about 19.5±0.2, about 22.5±0.2, and about 26.0±0.2 degrees.

[0011] In one aspect, embodiments herein provide a method for purifying Compound (I), comprising: [ka] The present invention relates to a method comprising preparing crystals of polymorphic form A and isolating the crystals of polymorphic form A.

[0012] In one aspect, embodiments herein provide a method for purifying Compound (I), comprising: [ka] The present invention relates to a method comprising preparing crystals of polymorphic form B and isolating the crystals of polymorphic form B.

[0013] In one aspect, embodiments herein provide a method for purifying Compound (I), comprising: [ka] The present invention relates to a method comprising preparing crystals of polymorphic form C and isolating the crystals of polymorphic form C.

[0014] In one aspect, embodiments herein provide a method for purifying Compound (I), comprising: [ka] The present invention relates to a method comprising preparing a crystal of polymorphic form D and isolating the crystal of polymorphic form D.

[0015] In one aspect, embodiments herein relate to a pharmaceutical composition comprising a polymorphic form of Compound (I) and a pharma- ceutically acceptable excipient.

[0016] In one aspect, embodiments herein relate to a method of treating a subject having cancer, comprising administering to the subject one or more of polymorphic Forms A, B, C, or D of Compound (I).

[0017] In one aspect, embodiments herein relate to the use of one or more of polymorphic Forms A, B, C, or D of Compound (I) in the manufacture of a medicament for treating a subject with cancer. [Brief description of the drawings]

[0018] [Figure 1] 1 shows the powder X-ray diffraction (PXRD) spectrum of crystalline form A of compound (I). [Figure 2A] 1 shows the powder X-ray diffraction (PXRD) spectrum of crystalline form B of compound (I). [Figure 2B] 1 shows the powder X-ray diffraction (PXRD) spectrum of crystalline form B of compound (I). [Diagram 3] 1 shows the powder X-ray diffraction (PXRD) spectrum of crystalline form C of Compound (I). [Figure 4A] 1 shows the powder X-ray diffraction (PXRD) spectrum of crystalline form D of compound (I). [Figure 4B] 1 shows the Fourier transform Raman spectrum of crystalline form D of Compound (I). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] This application is directed to polymorphs of (S)—N-(3-bromo-2-fluorophenyl)-7-((4-methylpiperazin-1-yl)methyl)-7,8-dihydro-[1,4]dioxino[2,3-g]quinazolin-4-amine.

[0020] The present disclosure may be more readily understood by reference to the following detailed description taken in conjunction with the accompanying drawings and examples that form a part of this disclosure. It is understood that the disclosed embodiments are not limited to the specific devices, methods, applications, conditions, or parameters described and / or illustrated herein, and that the terminology used herein is intended to describe specific embodiments by way of example only and is not intended to limit the disclosed embodiments. Also, as used herein, including the appended claims, the singular forms "a," "an," and "the" include the plural, and reference to a particular numerical value includes at least that particular value unless the context clearly dictates otherwise.

[0021] The terms "comprise," "include," "having," "has," "can," "contain," and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the specified ingredients / steps and allow for the presence of other ingredients / steps. However, such descriptions should also be construed as describing compositions or processes as "consisting of" and "consisting essentially of" the recited compounds, which allows for the presence of only the named compounds, along with any pharmaceutical carriers, and excludes other compounds.

[0022] definition As used herein, the following terms and phrases have the meanings indicated below, unless specifically stated to the contrary.

[0023] The term "polymorph" as used herein refers to a crystalline form of a compound. The terms "polymorph" and "crystal form" or "Form" followed by an alphabetical identifier are used interchangeably. Such crystalline forms may be distinguished, among other things, by X-ray diffraction patterns.

[0024] As used herein, "equilibration," when used in reference to conditions for forming a polymorph of a compound, refers to a physical process that results in thermodynamic equilibrium.

[0025] As used herein, "suspension equilibration," when used in reference to the conditions for forming a polymorph of a compound, typically refers to a physical process that results in thermodynamic equilibrium in a suspension of solid particles / materials dispersed in a liquid.

[0026] As used herein, "slurrying," when used in reference to conditions for forming a polymorph of a compound, refers to a physical process of bringing about thermodynamic equilibrium in a suspension of solid particles / materials dispersed in a liquid, typically by agitating the suspension with a suitable stirring device.

[0027] As used herein, "seeding" refers to the process of using a small amount of an existing crystalline form to aid in the crystallization of more of the compound of interest from a solution or suspension of that form.

[0028] Unless otherwise indicated, all chiral (enantiomers and diastereomers) and racemic forms are within the scope of the disclosed embodiments. Many geometric isomers, such as C=C double bonds, C=N double bonds and ring systems, can also exist in the compounds, and all such stable isomers are contemplated in this disclosure. Cis and trans (or E and Z-) geometric isomers of the compounds of the present disclosure are described and can be isolated as a mixture of isomers or as separated isomeric forms.

[0029] The term "stereoisomer" refers to isomers of identical constitution that differ in the arrangement of their atoms in space. Enantiomers and diastereomers are examples of stereoisomers. The term "enantiomer" refers to one of a pair of molecular species that are mirror images of each other and are not superimposable. The term "diastereomer" refers to a stereoisomer that is not a mirror image.

[0030] The symbols "R" and "S" represent the configuration of substituents around a chiral carbon atom. The isomeric descriptors "R" and "S" are used to indicate the atomic configurations relative to a core molecule as described herein and are intended to be used as defined in the literature (IUPAC Recommendations 1996, Pure and Applied Chemistry, 68:2193-2222 (1996)).

[0031] The compounds of the present disclosure, both in free form and salts thereof, may exist in multiple tautomeric forms in which hydrogen atoms are transposed to other parts of the molecule, resulting in a rearrangement of the chemical bonds between the atoms of the molecule. It is understood that all tautomeric forms, insofar as they may exist, are included within the disclosed embodiments.

[0032] All ranges disclosed herein are inclusive of the recited endpoints and are independently combinable (e.g., the range "100 mg to 200 mg" includes the endpoints 100 mg and 200 mg, and all intermediate values). The endpoints of the ranges and any values ​​disclosed herein are not limited to that range or value, but rather have sufficient breadth to include values ​​that approximate those ranges and / or values.

[0033] The term "API" refers to active pharmaceutical ingredient. As used herein, API refers to "Compound (I)" or (S)-N-(3-bromo-2-fluorophenyl)-7-((4-methylpiperazin-1-yl)methyl)-7,8-dihydro-[1,4]dioxino[2,3-g]quinazolin-4-amine.

[0034] As used herein, the term "physically stable" means that a particular free base or salt form does not change into one or more different physical forms (e.g., different solid forms as measured by XRPD, DSC, etc.) when subjected to a particular condition, e.g., ambient humidity at room temperature or 40°C / 75% relative humidity, for a particular period of time, e.g., 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 12 months, 18 months, 24 months or more. In some embodiments, less than 25% of the form of the compound changes into one or more different physical forms when subjected to the particular conditions. In some embodiments, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 3%, less than about 1%, less than about 0.5% of the form of a particular compound changes into one or more different physical forms of that particular compound when subjected to the particular conditions. In some embodiments, a detectable amount of a particular form of a compound does not change into one or more different physical forms of the compound.

[0035] As used herein, the term "chemically stable" means that the chemical structure of a particular compound does not change (e.g., decompose) into another compound when subjected to a particular condition, e.g., ambient humidity at room temperature, or 40°C / 75% relative humidity, for a particular period of time, e.g., 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 12 months, 18 months, 24 months or more. In some embodiments, less than 25% of the form of the particular compound changes into one or more other compounds when subjected to the particular conditions. In some embodiments, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 3%, less than about 1%, less than about 0.5% of the form of the particular compound changes into one or more other compounds when subjected to the particular conditions. In some embodiments, no detectable amount of the form of the particular compound changes into one or more different physical forms of the particular compound.

[0036] When values ​​are expressed as approximations by use of the antecedent "about," it will be understood that the particular value forms another embodiment. As used herein, "about X," where X is a numerical value, preferably refers to ±10% of the recited value. For example, the phrase "about 8" refers to a value between 7.2 and 8.8, and as another example, the phrase "about 8%" refers to a value between 7.2% and 8.8%. Where present, all ranges are inclusive and combinable.

[0037] "Solid form" refers to a polymorphic form of a compound. Solid forms can exist as crystalline solids or amorphous solids.

[0038] "Hydrogen salt" refers to a structure in which a proton is ionically bonded to the most basic site of the compound, e.g., a nitrogen atom, and chloride is the counteranion.

[0039] In the event of a discrepancy between a drawn structure and a name given to that structure, the drawn structure shall prevail. In addition, if the stereochemistry of a structure or part of a structure is not shown, for example, with bold or dashed lines, the structure or part of a structure shall be interpreted as embracing all stereoisomers of it. However, in some cases, when two or more chiral centers are present, the structure and name may be represented as a single enantiomer to help describe the relative stereochemistry. Those skilled in the art of organic synthesis will know whether compounds are prepared as single enantiomers from the methods used to prepare them.

[0040] In this description, the term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions via rearrangement of some of the bond electrons.

[0041] The terms "treat," "treating," and "treatment" refer to the amelioration or eradication of a disease or symptoms associated with a disease. In embodiments, such terms refer to minimizing the spread or worsening of a disease resulting from the administration of one or more prophylactic or therapeutic agents to a patient with such a disease. In the context of this disclosure, the terms "treat," "treating," and "treatment" also refer to (i) Specifically, preventing a disease or condition from occurring in a mammal when such mammal is susceptible to the condition but has not yet been diagnosed as having the disease or condition; (ii) inhibiting a disease or condition, i.e., preventing its onset; (iii) alleviating the disease or condition, i.e., causing the regression of the disease or condition; or (iv) Relieving symptoms resulting from a disease or condition, i.e., relieving pain without addressing the underlying disease or condition. As used herein, the terms "disease" and "condition" may be used interchangeably or may differ in that a particular malady or condition may not have a known causative agent (and thus the etiology has not yet been resolved) and thus is not yet recognized as a disease, but only as an undesirable state or syndrome in which a more or less specific set of symptoms has been identified by clinicians.

[0042] The term "effective amount" refers to an amount of a compound or other active ingredient sufficient to treat or prevent a disease, or to delay or minimize symptoms associated with a disease. Furthermore, a therapeutically effective amount for a compound refers to an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in treating or preventing a disease. When used in connection with a compound, this term can include an amount that improves overall treatment, reduces or avoids symptoms or causes of a disease, or enhances therapeutic efficacy or synergy with another therapeutic agent.

[0043] A "patient" or "subject" includes animals such as humans, cows, horses, sheep, lambs, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits, or guinea pigs. The animals can be mammals, such as non-primates and primates (e.g., monkeys and humans). In one embodiment, the patient is a human, such as a human infant, child, adolescent, or adult.

[0044] As used herein, the term "pharmaceutically acceptable carrier" means a pharma- ceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, involved in carrying or transporting a salt of a compound useful in the disclosed embodiments so that it may perform its intended function. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the salt of a compound useful in the disclosed embodiments, and not deleterious to the subject. Some examples of substances which can serve as pharma- ceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; excipients such as powdered tragacanth, malt, gelatin, talc, cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar, buffers such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffers, and other non-toxic compatible materials used in pharmaceutical formulations.

[0045] Appropriate formulations depend on the route of administration selected. Further details regarding suitable excipients for the pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Volume I and Volume II, Twenty-Second Edition, Loyd V. Allen, Jr., editor (Philadelphia, PA: Pharmaceutical Press, 2012); Excipient Development for Pharmaceutical, Biotechnology, and Drug Delivery Systems, Ashok Katdare and Mahesh V. Chaubal, editors (Boca Raton, FL: CRC Press, 2006), and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), which are incorporated herein by reference for such disclosure.

[0046] As used herein, a pharmaceutical composition refers to a mixture of a crystalline solid polymorph of Compound (I) as described herein with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. A pharmaceutical composition facilitates administration of a compound to an organism.

[0047] In embodiments, there is provided a polymorph of Compound (I), a free base, having the structure: [ka] Polymorphism is (1) Form A having an X-ray diffraction pattern with characteristic peaks characterized by values ​​of 2θ of about 4.9±0.2, about 13.9±0.2, about 22.1±0.2, and about 25.1±0.2 degrees, and in embodiments, Form A characterized by two, three, or four of the aforementioned peaks; (2) Form B having an X-ray diffraction pattern with characteristic peaks at values ​​of 2θ of about 4.8±0.2, about 9.8±0.2, about 13.8±0.2, about 14.7±0.2, about 17.7±0.2, about 20.2±0.2, about 24.1±0.2, about 24.6±0.2, and about 25.1±0.2 degrees, in embodiments characterized by 2, 3, 4, 5, or up to all of the above peaks; (3) Form C having an X-ray diffraction pattern with characteristic peaks at values ​​of 2θ of about 4.9±0.2, about 22.9±0.2, about 23.2±0.2, about 23.7±0.2, and about 24.3±0.2 degrees, in embodiments characterized by two, three, four, or five of the foregoing peaks; or (4) Form D having an X-ray diffraction pattern with characteristic peaks at values ​​of 2θ of about 15.6±0.2, about 16.9±0.2, about 19.1±0.2, about 19.5±0.2, about 22.5±0.2, and about 26.0±0.2 degrees, and in embodiments Form B characterized by 2, 3, 4, 5, or up to all of the above peaks.

[0048] In embodiments, the polymorph is Form A, which has an X-ray diffraction pattern with characteristic peaks characterized by values ​​of about 4.9±0.2, about 12.0±0.2, about 13.9±0.2, about 14.8±0.2, about 15.5±0.2, about 15.9±0.2, about 17.8±0.2, about 20.4±0.2, about 21.0±0.2, about 22.1±0.2, about 23.0±0.2, about 23.6±0.2, about 24.3±0.2, about 24.8±0.2, about 25.1±0.2, about 26.3±0.2, and about 27.3±0.2 degrees 2θ.

[0049] In embodiments, the polymorph is Form C, which has an X-ray diffraction pattern with characteristic peaks characterized by values ​​of 2θ of about 4.9±0.2, about 9.8±0.2, about 12.1±0.2, about 14.4±0.2, about 14.7±0.2, about 20.2±0.2, about 23.2±0.2, about 23.7±0.2, about 24.3±0.2, about 24.6±0.2, and about 26.1±0.2 degrees.

[0050] In embodiments, the polymorph is Form D, which has an X-ray diffraction pattern with characteristic peaks characterized by values ​​of 2θ of about 15.2±0.2, about 15.6±0.2, about 16.9±0.2, about 19.1±0.2, about 19.5±0.2, about 22.5±0.2, and about 26.0±0.2 degrees.

[0051] In embodiments, the polymorph is Form D, which has an X-ray diffraction pattern with characteristic peaks characterized by values ​​of about 10.2±0.2, about 13.5±0.2, about 15.2±0.2, about 15.6±0.2, about 15.9±0.2, about 16.9±0.2, 19.1±0.2, about 19.5±0.2, about 22.5±0.2, about 23.3±0.2, about 24.3±0.2, about 24.7±0.2, about 24.9±0.2, about 26.0±0.2, about 27.9±0.2, and about 29.6±0.2 degrees 2θ.

[0052] In an embodiment, the polymorph is Form D, which has a melting point of about 153°C.

[0053] In an embodiment, the polymorph is Form A as shown in Figure 1 and has a peak list shown in Table 4. In an embodiment, the polymorph is Form B as shown in Figure 2a and has a peak list shown in Table 5. In an embodiment, the polymorph is Form C as shown in Figure 3 and has a peak list shown in Table 6. In an embodiment, the polymorph is Form D as shown in Figure 4a and has a peak list shown in Table 8.

[0054] In an embodiment, a method of forming a polymorph of Compound (I) is provided, the method comprising converting amorphous Compound (I) to a polymorphic form by equilibration, suspension equilibration, slurrying, seeding, evaporation, or a combination thereof. In an embodiment, Form A, Form B, or Form C is formed from amorphous Compound (I) in the presence of water. In an embodiment, Form D is formed from amorphous Compound (I) in a non-aqueous solvent system.

[0055] In embodiments, there is provided a method of forming the Form A polymorph of Compound (I), comprising one of the following: (a) crystallizing amorphous compound (I) in isopropyl alcohol:water (1:1) and equilibrating the crystallized amorphous compound (I); (b) suspension equilibration of Form D in acetonitrile:water (9:1); or (c) Suspension equilibration of amorphous compound (I) in water-saturated TBME.

[0056] In embodiments, there is provided a method of forming the Form B polymorph of Compound (I), comprising one of the following: (a) equilibrating amorphous Compound (I) in water:ethanol (2:1); or (b) Suspension equilibration of Form D in water.

[0057] In an embodiment, there is provided a method for forming the Form C polymorph of Compound (I), comprising recrystallizing Compound (I) from a solution of 5.5:2 acetonitrile:water, followed by equilibration at 5°C.

[0058] In embodiments, there is provided a method of forming the Form D polymorph of Compound (I), comprising crystallizing amorphous Compound (I) in a non-aqueous solvent system and converting the amorphous Compound (I) to Form D via slurrying, seeding, equilibration, suspension equilibration, or a combination thereof. In embodiments, such methods employ one of the following: (a) slurrying in ethyl acetate; (b) Seeding Form D in ethyl acetate; (c) slurrying in acetonitrile; (d) Suspension equilibration in isopropyl acetate:cyclohexane (1:2); (e) Methyl ethyl ketone (MEK): Suspension equilibration in diisopropyl ether; (f) suspension equilibration in methyl ethyl ketone (MEK):p-xylene (1:2) and partial evaporation of the solvent; (g) equilibrating in methyl THF:methyl cyclohexane (1:2); (h) equilibrating in isopropyl acetate:heptane (2:3); (i) equilibration in isopropyl acetate:heptane (1:1); (j) suspension equilibration in n-butyl acetate:heptane (5:2); (k) seeding and suspension equilibrating Form D in TBME; (l) suspension equilibration in heptane:MIBK (4:1); (m) suspension equilibration in methanol:methylcyclohexane (1:4); (n) Suspension equilibration in trimethylamine; or (o) Suspension equilibration in diisopropyl ether.

[0059] In an embodiment, there is provided a method of forming the Form D polymorph of Compound (I), comprising evaporating a solution of Compound (I) in one of the following: (a) acetone:heptane (1:3); (b) n-butyl acetate, or (c) methanol:diisopropyl ether (1:12).

[0060] In embodiments, each of the above methods for forming polymorphs of Compound (I) may control or employ one or more conditions selected from temperature, stirring / mixing conditions, cooling rate, treatment time, concentration, and pH.

[0061] In embodiments, each of the polymorphic forms A, B, C, and D, as well as the amorphous form of compound (I), can be interconverted by changing the above conditions to those described in preparing a particular desired polymorphic form. In some such embodiments, the interconversion can proceed through the amorphous form. For example, in embodiments, form A can be converted to form D via conversion of form A to an amorphous form, and then conversion of the amorphous form to form D. That is, as shown in the examples below, drying of form A can be performed to the point of reforming to the amorphous form of compound (I), followed by conditions to prepare form D. Similarly, form B can be converted to an amorphous form and thence to form D. Similarly, form C can be converted to an amorphous form and thence to form D. As further shown below, the ability to produce highly crystalline solids using forms A, B, and C can aid in the purification of compound (I). Thus, the purification methods described herein below can take advantage of the facile interconversion of polymorphic forms and their amorphous forms. In some embodiments, each of forms A, B, and C can be converted directly to form D via equilibration under the conditions used to prepare form D. Thus, such interconversion does not require passing through an amorphous form.

[0062] As disclosed herein below in the Examples, polymorphic forms of Compound (I) have high crystallinity. Thus, polymorphic forms are particularly suitable for aiding in the purification of Compound (I). In an embodiment, a method of purifying Compound (I) comprises preparing a polymorph of Compound (I) by any of the methods disclosed herein and isolating the crystals of the polymorph. In an embodiment, a method of purifying Compound (I) comprises: [ka] A method is provided that includes preparing crystals of polymorphic Form A and isolating the crystals of polymorphic Form A.

[0063] In an embodiment, there is provided a method for purifying Compound (I), comprising the steps of: [ka] A process is provided that includes preparing crystals of polymorphic Form B and isolating the crystals of polymorphic Form B.

[0064] In an embodiment, there is provided a method for purifying Compound (I), comprising the steps of: [ka] A method is provided that includes preparing crystals of polymorph Form C and isolating the crystals of polymorph Form C.

[0065] In an embodiment, there is provided a method for purifying Compound (I), comprising the steps of: [ka] A method is provided that includes preparing crystals of polymorphic Form D and isolating the crystals of polymorphic Form D.

[0066] In embodiments, there is provided a pharmaceutical composition comprising a polymorphic form of Compound (I) disclosed herein and a pharma- ceutically acceptable excipient.

[0067] The pharmaceutical compositions and methods of the present disclosure may be utilized to treat an individual in need thereof. In embodiments, the individual is a mammal, such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or compound is preferably administered as a pharmaceutical composition, for example, comprising the compound and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions, such as water or physiologically buffered saline, or other solvents or vehicles, such as glycols, glycerol, oils, such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly invasive routes of administration (i.e., routes such as injection or implantation that avoid transport or diffusion through epithelial barriers), the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients may be selected, for example, to provide delayed release of the drug or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition may be in the form of tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilisates for reconstitution, powders, solutions, syrups, suppositories, injections, etc. The composition may also be present in a transdermal delivery system, such as a skin patch. The composition may also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0068] A pharma- ceutically acceptable carrier can contain a physiologically acceptable agent that acts, for example, to stabilize, increase the solubility, or increase the absorption of a compound. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers or excipients. The choice of a pharma- ceutical acceptable carrier that includes a physiologically acceptable agent depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) can be a liposome or other polymer matrix, which can, for example, incorporate a compound. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively easy to make and administer.

[0069] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0070] The phrase "pharmacologically acceptable carrier" as used herein means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Examples of materials that can function as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) fats and oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycerides, such as glycerides, ... ) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum oxide trihydrate; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffers; and (21) other non-toxic compatible substances used in pharmaceutical preparations.

[0071] Pharmaceutical compositions (preparations) can be administered to a subject by any of a number of routes of administration, including, for example, orally (e.g., as drenches such as aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue), absorption through the oral mucosa (e.g., sublingually), subcutaneously, transdermally (e.g., as a patch applied to the skin), and topically (e.g., as a cream, ointment, or spray applied to the skin). The compounds may also be formulated for inhalation. In embodiments, the compounds may simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable therefor can be found, for example, in U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, as well as patents cited therein.

[0072] The formulations may be conveniently provided in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form varies depending on the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form will generally be the amount of compound that produces a therapeutic effect. Generally, out of 100 percent, this amount will range from about 1 percent to about 99 percent of the active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0073] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0074] Formulations of the polymorphic compounds disclosed herein suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), sachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), lyophilisates, powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (using an inert base, e.g., gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, and the like, each containing a predetermined amount of a compound of the present disclosure as an active ingredient. The composition or compound may also be administered as a bolus, electuary, or paste.

[0075] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharma- ceutically acceptable extracts, e.g., sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, e.g., starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) wetting agents, e.g., glycerin; (4) disintegrants, e.g., glycerin; Examples of suitable additives include agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) emollients, such as paraffin; (6) absorption enhancers, such as quaternary ammonium compounds; (7) humectants, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium dodecyl sulfate, and mixtures thereof; (50) complexing agents, such as modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be employed as fillers for soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols, and the like.

[0076] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface active agents or dispersants. Molded tablets can be made by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine.

[0077] Tablets, and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills, and granules, can be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings known in the art of pharmaceutical formulation. They can also be formulated to provide slow or controlled release of the active ingredient therein, for example, using various proportions of hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres to provide the desired release profile. They can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions can also optionally contain an opacifying agent, and can be of a composition that releases the active ingredient only, or preferentially, in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0078] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophilized products for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs.In addition to active ingredients, liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, cyclodextrin and its derivatives, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.

[0079] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0080] Suspensions may contain, in addition to the active compounds, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0081] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharma- ceutically acceptable carrier, and any preservatives, buffers, or propellants which may be required.

[0082] The ointments, pastes, creams and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0083] Powders and sprays can contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0084] Transdermal patches have the added advantage of providing controlled delivery of the compounds of the present disclosure to the body. Such dosage forms can be made by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

[0085] The phrases "parenteral administration" and "administered parenterally" as used herein refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intracerebral injection and infusion. Pharmaceutical compositions suitable for parenteral administration contain one or more active compounds in combination with one or more pharma- ceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into a sterile injectable solution or dispersion immediately prior to use, which may include antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0086] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate, etc. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0087] These compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial activity may 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 in the composition, such as sugars, sodium chloride, etc. In addition, prolonged absorption of the injectable pharmaceutical form may be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0088] In some cases, it is desirable to delay the absorption of drugs from subcutaneous or intramuscular injection in order to prolong the effect of drugs.This can be achieved by using liquid suspension of crystalline or amorphous substances that are poorly water-soluble.In this case, the absorption rate of the drug depends on its dissolution rate, which in turn depends on the crystal size and crystalline form.Alternatively, the delayed absorption of parenterally administered drug forms can also be achieved by dissolving or suspending the drug in oil vehicles.

[0089] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and polyanthrylides. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

[0090] For use in the methods of the invention, the active compounds may be provided as such or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient in combination with a pharma- ceutically acceptable carrier.

[0091] The introduction method may be provided by a rechargeable or biodegradable device. For the controlled delivery of drugs, including proteinaceous biopharmaceuticals, various sustained release polymeric devices have been developed and tested in vivo in recent years. Various biocompatible polymers, including both biodegradable and non-degradable polymers (including hydrogels), can be used to form implants for sustained release of compounds at specific target sites.

[0092] Actual dosage levels of the active ingredients in pharmaceutical compositions may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0093] The selected dosage level will depend upon a variety of factors including the activity of the particular compound or compounds, or esters, salts or amides thereof, employed, the route of administration, the time of administration, the particular excretion rate of the particular compound(s) being employed, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular compound(s) being employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0094] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required.

[0095] For example, a physician or veterinarian may begin administering a pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. "Therapeutically effective amount" refers to the concentration of a compound sufficient to elicit the desired therapeutic effect. In general, it is understood that the effective amount of a compound varies according to the subject's weight, sex, age, and medical history. Other factors that affect the effective amount include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and other types of therapeutic agents being administered together with the compound, if necessary. Multiple administrations of the drug can deliver a larger total dose. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbaeher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference).

[0096] In general, a suitable daily dose of an active compound used in the present compositions and methods will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0097] If necessary, the effective daily dose of the active compound may be administered as 1, 2, 3, 4, 5, 6 or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage form. In an embodiment of the present disclosure, the active compound may be administered two or three times a day. In a preferred embodiment, the active compound is administered once a day.

[0098] The patients receiving this treatment are animals in need of treatment, including primates, particularly humans, and other mammals such as horses, cows, pigs, sheep, cats, dogs, poultry, and pets in general.

[0099] In embodiments, the compounds disclosed herein may be administered alone or in combination with another type of therapeutic agent.

[0100] In embodiments, methods are provided for treating a subject having cancer, comprising administering to the subject one or more of polymorphic forms A, B, C, or D of Compound (I) disclosed herein. In some such embodiments, the cancer is characterized by aberrant EGFR expression. In some such embodiments, the cancer is a brain tumor.

[0101] In embodiments, there is provided the use of one or more of polymorphic forms A, B, C, or D of Compound (I) disclosed herein in the manufacture of a medicament for treating a subject having cancer. In some such embodiments, the cancer is characterized by aberrant EGFR expression. In some such embodiments, the cancer is a brain tumor.

[0102] In embodiments, there is provided a method of treating a subject having a cancer associated with aberrant EGFR expression, comprising administering to the subject one or more of polymorphic forms A, B, C, or D of Compound (I) as disclosed herein. In embodiments, the cancer is a brain tumor.

[0103] In an embodiment, there is provided the use of one or more of polymorphic forms A, B, C, or D of Compound (I) in the manufacture of a medicament for treating a subject having a cancer associated with aberrant EGFR expression. In an embodiment, the cancer is a brain tumor.

[0104] In certain aspects, the disclosure provides a method of inhibiting EGFR or AEGFR comprising administering to a subject an amount of a polymorphic form of Compound (I), Forms A, B, C, D, or a combination thereof.

[0105] In certain aspects, the disclosure provides a method of treating cancer, comprising administering to a subject in need of cancer treatment an amount of a polymorph of Compound (I) disclosed herein. In an embodiment, the cancer is bladder cancer, bone tumor, brain tumor, breast cancer, heart cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, fibrosarcoma, gastric cancer, gastrointestinal cancer, head and spine neck cancer, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, lymphoma, melanoma, multiple myeloma, pancreatic cancer, penile cancer, testicular germ cell cancer, thymoma cancer, thymic cancer, lung cancer, ovarian cancer, or prostate cancer. In an embodiment, the cancer is glioma, astrocytoma, or glioblastoma. In an embodiment, the cancer is glioblastoma. In an embodiment, the cancer is glioblastoma multiforme. In an embodiment, the method reduces the proliferation of cancer cells.

[0106] In certain aspects, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject a glucose metabolism inhibitor and an additional agent, the glucose metabolism inhibitor being a polymorph of Compound (I) of the present disclosure or a pharma- ceutically acceptable salt thereof, and the additional agent being a cytoplasmic p53 stabilizer. In an embodiment, the cancer is bladder cancer, bone tumor, brain tumor, breast cancer, heart cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, fibrosarcoma, gastric cancer, gastrointestinal cancer, head and spine neck cancer, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, lymphoma, melanoma, multiple myeloma, pancreatic cancer, penile cancer, testicular germ cell cancer, thymoma cancer, thymic cancer, lung cancer, ovarian cancer, or prostate cancer. In an embodiment, the cancer is glioma, astrocytoma, or glioblastoma. In an embodiment, the cancer is glioblastoma. In an embodiment, the cancer is glioblastoma multiforme. In embodiments, the method reduces proliferation of cancer cells. In embodiments, the cancer is relapsed or refractory. In other embodiments, the cancer is treatment naive.

[0107] In embodiments, the subject has been determined to be sensitive to a glucose metabolism inhibitor by a method comprising: a. obtaining a first blood sample from a subject; b. placing the subject on a ketogenic diet; and c. Obtaining a second blood sample from the subject after being placed on the ketogenic diet for a period of time; d. Measuring glucose levels in the first and second blood samples; e. comparing the glucose level in the second blood sample to the glucose level in the first blood sample; f. Determining that the subject is susceptible if the glucose level in the second blood sample is reduced compared to the glucose level in the first blood sample.

[0108] In an embodiment, the decrease in glucose levels between the second blood sample and the control blood sample is about 0.15 mM or more. In an embodiment, the decrease in glucose levels between the second blood sample and the control blood sample is about 0.20 mM or more. In an embodiment, the decrease in glucose levels between the second blood sample and the control blood sample is in the range of 0.15 mM to 2.0 mM. In an embodiment, the decrease in glucose levels between the second blood sample and the control blood sample is in the range of 0.25 mM to 1.0 mM.

[0109] In embodiments, the cytoplasmic p53 stabilizer is an MDM2 inhibitor. In embodiments, the MDM2 inhibitor is nutlin. In embodiments, the MDM2 inhibitor is nutlin-3 or idasanutlin. In embodiments, the subject is administered 50 mg to 1600 mg of idasanutlin. In embodiments, the subject is administered 100 mg of idasanutlin. In embodiments, the subject is administered 150 mg of idasanutlin. In embodiments, the subject is administered 300 mg of idasanutlin. In embodiments, the subject is administered 400 mg of idasanutlin.

[0110] In an embodiment, the subject is administered 600 mg of idasanutlin. In an embodiment, the subject is administered 1600 g of idasanutlin. In another embodiment, the MDM2 inhibitor is RO5045337, RO550378L R06839921, SAR405838, DS-3032, DS-3032b, or AMG-232.

[0111] In an embodiment, the cytoplasmic p53 stabilizer is a BCL-2 inhibitor.In an embodiment, the BCL-2 inhibitor is antisense oligodeoxynucleotide G3139, mRNA antagonist SPC2996, venetoelast (ABT-199), GDC-0199, obatoclax, paelitaxel, navitoclax (ABT-263), ABT-737, NU-0129, S055746, or APG-1252.

[0112] In embodiments, the cytoplasmic p53 stabilizer is a Bcl-xL inhibitor. In embodiments, the Bcl-xL inhibitor is WEHI539, ABT-263, ABT-199, ABT-737, sabutoclax, ATI01, TW-37, APG-1252, or gambogic acid.

[0113] In embodiments, the glucose metabolic inhibitor and the cytoplasmic p53 stabilizer are administered in the same composition. In other embodiments, the glucose metabolic inhibitor and the cytoplasmic p53 stabilizer are administered in separate compositions.

[0114] In embodiments, the method further comprises administration of an additional therapy.

[0115] Glioma type and stage Primary malignant brain tumors are tumors that begin in the brain or spine and are collectively known as gliomas. Glioma is not a specific type of cancer, but a term used to describe tumors derived from glial cells. Examples of primary malignant brain tumors include astrocytoma, pilocytoma, multiform xanthoastrocytoma, diffuse astrocytoma, anaplastic astrocytoma, GBM, glioma, oligodendroglioma, and ependymoma. According to the WHO classification of brain tumors, astrocytomas are classified into four grades determined by the underlying pathology. Features used to classify gliomas include mitoses, cellular or nuclear atypia, and vascular proliferation and necrosis with pseudopalisard features. Malignant (or high-grade) gliomas include anaplastic glioma (WHO grade III) and glioblastoma multiforme (GBM, WHO grade IV). These are the most aggressive brain tumors with the poorest prognosis.

[0116] GBM is the most common, complex, treatment-resistant and deadliest type of brain tumor, accounting for 45% of all brain tumors, with approximately 11,000 men, women and children diagnosed each year. GBM (also known as grade 4 astrocytoma and glioblastoma multiforme) is the most common type of malignant (cancerous) primary brain tumor.

[0117] They are highly aggressive for several reasons. First, glioblastoma cells grow rapidly because they secrete substances that stimulate a rich blood supply. They also have the ability to invade and invade long distances into normal brain by sending out microscopic tentacles of the tumor in parallel with normal cells. Two types of glioblastoma are known. Primary GBM is the most common form, it grows rapidly and often causes symptoms early on.

[0118] Secondary glioblastomas are less common, accounting for approximately 10 percent of all GBMs. They progress from low-grade diffuse astrocytomas or anaplastic astrocytomas and are more likely to be seen in younger patients. Secondary GBMs are preferentially located in the frontal lobe and confer a better prognosis.

[0119] GBM is usually treated with a complex multimodal treatment plan that includes surgical removal of the tumor, radiation, and chemotherapy. First, as much of the tumor as possible is removed during surgery. The location of the tumor in the brain often determines how much can be safely removed. After surgery, radiation therapy and chemotherapy slow the growth of remaining tumor cells. Temozolomide, an oral chemotherapy drug, is most often used for six weeks, then monthly. Another drug, bevacizumab (known as Avastin®), is also used during treatment. This drug attacks the tumor's ability to recruit a blood supply, often slowing or stopping the tumor's growth.

[0120] New investigational therapies are also used, which may include adding a treatment to a standard therapy or replacing part of the standard therapy with a different treatment that may work better. Some of these treatments include immunotherapy, such as vaccine immunotherapy, or low-dose electrical pulses to the area of ​​the brain where the tumor is located, and nanotherapy, including spherical nucleic acid (SNA), such as NU-0129. In some embodiments, the methods of the present disclosure are used in combination with one or more of the aforementioned therapies.

[0121] It is contemplated that embodiments of the methods and compositions discussed herein are also applicable to other types of cancer, including, but not limited to, lung cancer, non-CNS cancers, CNS cancers, and CNS metastases, such as brain metastases, leptomeningeal metastases, choroidal metastases, and spinal metastases.

[0122] Cytoplasmic p53 stabilizers The inventors have demonstrated that pharmacological p53 stabilization, such as, for example, CNS-permeable small molecules, is synergistically lethal with inhibition of EGFR-driven glucose uptake in patient-derived primary GBM models. The inventors have demonstrated for the first time that non-transcriptional functions of p53 can have an important role in stimulating intrinsic apoptosis in metabolic response factors. Thus, the therapeutic methods described herein include administering a cytoplasmic p53 stabilizer(s) in combination with a glucose metabolic inhibitor. The cytoplasmic p53 stabilizer and the glucose metabolic inhibitor can be co-administered or sequentially administered in the same or different compositions. It is contemplated that in some embodiments, a single p53 stabilizer is used, and in other embodiments, more than one p53 stabilizer is used. For example, (BCL-2 and BCL-X L It has been reported that the combination of nutrin with ABT737 (which binds to p53) synergistically targets the balance of pro-apoptotic and anti-apoptotic proteins at the mitochondrial level, thereby promoting cell death. (Hoe et al. 2014. Nature Reviews. Vol. 13. pp. 217). As intended herein, a cytoplasmic p53 stabilizer is any small molecule, antibody, peptide, protein, nucleic acid or derivative thereof that can directly or indirectly pharmacologically stabilize or activate p53. Stabilization of cytoplasmic p53 results in priming cells, such as cancer cells, for apoptosis.

[0123] MDM2 antagonists The protein level of p53 in cells is tightly controlled and kept low by its negative regulator, E3 ubiquitin protein ligase MDM2. In an embodiment of the method or composition of the present disclosure, the cytoplasmic p53 stabilizer is an MDM2 antagonist / inhibitor. In some embodiments, the MDM2 antagonist is nutlin. In further embodiments, the nutlin is nutlin-3 or idasanutlin. In other embodiments, the MDM2 antagonist is RO5045337 (also known as RG7112), RO5503781, R06839921, SAR405838 (also known as MI-773), DS-3032, DS-3032b, or AMG-232, or any other MDM2 inhibitor.

[0124] Other compounds within the scope of the methods of the invention that are known to bind to MDM-2 include Ro-2443, MI-219, MI-713, MI-888, DS-3032b, benzodiazepinediones (e.g., TDP521252), sulfonamides (e.g., NSC279287), eflomenotriazolopyrimidines, morpholinones and piperidinones (AM-8553), terphenyls, chalcones, pyrazoles, imidazole-indoles, isoindolinones, pyrrolidinones (e.g., PXN822), priaxones, piperidines, naturally derived prenylated xanthones, SAH-8 (stapled peptides), sMTide-02, sMTide-02a (stapled peptides), ATSP-7041 (stapled peptides), spiroligomers (a-heliximics). Other compounds known to induce protein folding of MDM2 include PRIMA-1MET (also known as APR-246), Aprea102-105, PK083, PK5I74, PK5196, PK7088, benzothiazole, stictic acid, and NSC319726.

[0125] BCL-2 inhibitors In another embodiment of the method or composition of the present invention, the cytoplasmic p53 stabilizer is a BCL-2 inhibitor.In some embodiments, the BCL-2 inhibitor is, for example, antisense oligodeoxynucleotide G3139, mRNA antagonist SPC2996, venetoclax (ABT-199), GDC-0199, obatoclax, paclitaxel, navitoclax (ABT-263), ABT-737, NIT-0129, S055746, APG-1252 or any other BCL-2 inhibitor.

[0126] Bcl-xL inhibitors In further embodiments of the method or composition of the present invention, the cytoplasmic p53 stabilizer is a Bcl-xL inhibitor. In some embodiments, the Bcl-xL inhibitor is, for example, WEHI539, ABT-263, ABT-199, ABT-737, sabutoclax, ATI01, TW-37, APG-1252, gambogic acid or any other Bcl-xL inhibitor.

[0127] Evaluation method: Glucose uptake test In embodiments of the methods and compositions of the present disclosure, subjects with GBM or cancer are classified as either "metabolic responders" or "metabolic non-responders" (i.e., sensitive to glucose metabolic inhibitors). In embodiments, the classification of the subject is prior to administering to the subject a treatment comprising a glucose metabolic inhibitor and a cytoplasmic p53 stabilizer. Thus, the present disclosure provides methods for assessing cancer, classifying subjects, and determining the subject's sensitivity to treatment, which involve analysis of glucose metabolism, glycolysis, or glucose uptake. Methods for classifying subjects as metabolic responders are described in detail in Example 1. Techniques for monitoring glycolysis and glucose uptake are provided in T. TeSlaa and MATeitell. 2014. Methods in Enzymology, Volume 542, pp. 92-114, which are incorporated herein by reference.

[0128] Glycolysis is the intracellular biochemical conversion of one molecule of glucose into two molecules of pyruvate with the concomitant generation of two molecules of ATP. Pyruvate is a metabolic intermediate with several potential fates, including entry into the tricarboxylic acid (TCA) cycle in mitochondria to produce NADH and FADH2. Alternatively, pyruvate can be converted to NAD from NADH. ÷ It can be converted to lactate in the cytoplasm by lactate dehydrogenase with the simultaneous regeneration of . Increased flux through glycolysis supports the proliferation of cancer cells, for example, by providing additional energy in the form of ATP, as well as glucose-derived metabolic intermediates for nucleotide, lipid, and protein biosynthesis. Warburg (Oncologia. 1956; 9(2): 75-83) was the first to observe that proliferating tumor ceilings enhance aerobic glycolysis, the conversion of glucose to lactate in the presence of oxygen, in contrast to non-malignant cells, which primarily respire when oxygen is available.

[0129] This mitochondrial bypass, called the Warburg effect, occurs in rapidly proliferating cells, including cancer cells, activated lymphocytes, and pluripotent stem cells. 18 It has been utilized for clinical diagnostic testing that uses positron emission tomography (PET) scanning to identify increased cellular uptake of fluorinated glucose analogs such as F-deoxyglucose.

[0130] Glycolysis therefore represents a target for therapeutic and diagnostic methods. In the context of the methods of the invention, the measurement of glucose uptake and lactate excretion by malignant cells may be useful to detect changes in glucose catabolism and / or sensitivity to glucose metabolic inhibitors. Detecting such changes is important for methods of treating GBM, reducing the risk of ineffective treatment, and reducing the chances of tumor survival. For the purposes of this disclosure, 18F-deoxyglucose PET is useful, in embodiments, as a rapid non-invasive functional biomarker to predict sensitivity to p53 activation. This non-invasive analysis may be particularly beneficial for malignant brain tumors, where pharmacokinetic / pharmacodynamic assessments are very challenging and impractical. In some cases, delayed imaging protocols with MRI fusion (41) and parametric response maps (PRM) can be used to characterize tumor function. 18 It can be useful to quantify changes in F-FDG uptake ( 42 ).

[0131] In certain embodiments, the method may involve measuring glucose uptake and lactate production. For cells in culture, glycolytic flux can be quantified by measuring glucose uptake and lactate excretion. Glucose uptake into cells is via glucose transporters (Glutl-Glut4), whereas lactate excretion is via monocarboxylate transporters (MCT1-MCT4) in the cell membrane.

[0132] Extracellular glucose and lactate Methods for detecting glucose uptake and lactate excretion include, for example, extracellular glucose or lactate kits, extracellular bioanalyzers, ECAR measurements, [3HJ-2-DG or [14CJ-2-DG uptake, 18 These include FDG uptake or 2-NBDG uptake. Commercial kits and instruments are available to quantify glucose and lactate levels in cell culture media. The detection methods by the kits are usually colorimetric or fluorometric and are compatible with standard laboratory equipment such as spectrophotometers. Bioprofile analyzers (such as Nova Biomedical) or biochemical analyzers (such as YSI Life Sciences) can measure both glucose and lactate levels in cell culture media. GlucCell (Cesco BioProduets) can only measure glucose levels in cell media. Each commercial method has a different detection protocol, but the recovery of the media for analysis is the same.

[0133] Extracellular acidification rate Glycolysis can also be determined by measuring the extracellular acidification rate (ECAR) of the surrounding medium, primarily from the excretion of lactate after conversion from pyruvate per unit time. The Seahorse Extracel Fluorescence (XF) Analyzer (Seahorse Bioscience) is a tool to measure glycolysis and oxidative phosphorylation simultaneously (by oxygen consumption) in the same cell.

[0134] Glucose analogue uptake Certain embodiments of the disclosed method include the use of glucose analogs.As is well known to those skilled in the art, to determine the glucose uptake rate by cells, a labeled isotope of glucose can be added to cell culture medium and then measured in cells after a given period of time.Exemplary types of glucose analogs for these tests include, but are not limited to, 2-deoxy-D-[1,2-3H]-glucose, 2-deoxy-D-[1-14C]-glucose, or 2-deoxy-2- 18 F-fluoro-D-glucose ( 18 In some embodiments, glucose uptake is measured using radioactive glucose analogs such as 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG) or fluorescent glucose analogs such as 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG). Measurement of radioactive glucose analog uptake requires a scintillation counter, whereas 2-NBDG uptake is typically measured by flow cytometry or fluorescence microscopy. In some embodiments, glucose uptake is measured using radioactively labeled glucose 2-deoxy-2-[fluorine-18]-fluoro-D-glucose ( 18 In a further embodiment, the uptake of F-FDG is measured. 18 Detection of F-FDG is by positron emission tomography (PET). In some embodiments, a biopsy is taken from the GBM tumor. 18 A detailed description of an example of measuring F-FDG is provided in the example below.

[0135] In certain embodiments, the method can involve comparing glucose uptake of a biological sample, such as a tumor sample, to a control. The fold increase or decrease can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more, or any range derivable therein. Alternatively, the difference in expression between the sample and the reference can be expressed as a percentage decrease or increase, such as at least or up to a 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000% difference, or any range derivable therein.

[0136] Other ways to express relative expression levels are: 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 ,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2.0,2.1,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3.0,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4.0,4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 , 7.7, 7.8, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, or any range derivable therein. In some embodiments, the level is obtained relative to a control.

[0137] Algorithms such as weighted voting programs can be used to facilitate the assessment of biomarker levels. In addition, other clinical evidence can be combined with biomarker-based testing to reduce the risk of erroneous assessment. In some embodiments, other cytogenetic assessments can be considered. EXAMPLES

[0138] The examples and embodiments described herein are for illustrative purposes only, and in some embodiments, various modifications or alterations should be included within the disclosure and scope of the appended claims.

[0139] The following abbreviations are used in the examples below: [Table 1]

[0140] PXRD peak list: vs = very strong, s = strong, m = medium, w = weak, vw = very weak intensity.

[0141] DSC: Differential scanning calorimetry was performed using a TA Instruments Q2000 instrument (sample pan with pinhole in the lid, heating rate 10 K / min). The melting point is taken as the peak maximum. Since compound (I) was found to contain about 1.2% water, some of which was released above 100°C, the sample was dried in a DSC sample pan. This was done by heating the pan with a pinhole to 100°C and holding the sample at this temperature for 10 minutes. A second scan was then performed on the dried sample, and the first scan was ignored, since the endothermic signal could be due to the evaporation of water.

[0142] Dynamic vapor sorption: DVS measurements were performed with an SPS11-100n "Sorptions Prufsystem" from ProUmid (formerly "Projekt Messtechnik"), August-Nagel-Str23, 89079 Ulm (Germany). Approximately 5-20 mg of sample was placed in an aluminum sample pan. A humidity change rate of 5% per hour was used. The applied measurement program is visualized in the figure (blue trace). Correct the presentation showing the effective water content.

[0143] Before starting the predefined humidity program, the samples were placed in an aluminum or platinum holder on top of a microbalance and allowed to equilibrate at 50% RH. The measurement program is visualized in a diagram in the report.

[0144] Hygroscopicity Classification: Hygroscopicity was classified based on mass gain at 85% RH relative to the initial mass as follows: deliquescent (sufficient water adsorbed to form a liquid), very hygroscopic (mass gain ≥ 15%), hygroscopic (mass gain < 15% but ≥ 2%), slightly hygroscopic (mass gain < 2% but ≥ 0.2%), or non-hygroscopic (mass gain < 0.2%).

[0145] Microscopy: Light microscopy was performed on a Leitz Orthoplan polarizing microscope part #130880, typically applying a magnification of 10x10.

[0146] H-NMR: Bruker DPX300 spectrometer, proton frequency of 300.13 MHz, 30° excitation pulse, 1 s recycle delay, accumulation of 16 scans, deuterated DMSO as solvent, solvent peak used for reference, chemical shifts reported on the TMS scale.

[0147] Powder X-ray diffraction: Powder X-ray diffraction was performed using Cu-K α1 The measurements were carried out on a Stoe Stadi P diffractometer equipped with a Mythen1K detector operating at 1000 Hz. The measurements were carried out at a tube voltage of 40 kV and a tube power of 40 mA. The curved Ge monochromator was fitted with a Cu-K α1 The experiment was carried out in a 30-slot 10-mm-wide 30-mm-wide 3D microscope, allowing for testing with radiation. The following parameters were set: 0.02° 2θ step size, 12 s step time, 1.5-50.5° 2θ scan range, and 1° 2θ detector step (detector mode of step-scan). For a typical sample preparation, approximately 10 mg of sample was placed between two acetate foils and mounted in a Stoe transmission sample holder. The sample was rotated during the measurement. All sample preparations and measurements were performed in an ambient air atmosphere.

[0148] Raman spectroscopy: FT-Raman spectra were recorded on a Bruker MultiRAM FT-Raman system using a near-infrared Nd:YAG laser operating at 1064 nm and a liquid nitrogen-cooled germanium detector. -1 64 scans with a resolution of 3500 to -50 cm -1However, due to the filter cutoff effect, the accumulation was within 100 cm -1 Only data above this are evaluated. The nominal laser power is typically 100 or 300 mW.

[0149] Solubility: Approximate solubility was determined by incremental addition of solvent to approximately 10 mg of compound. If the material did not dissolve by adding a total of 10 ml of solvent, the solubility is indicated as less than 1 mg / ml. Due to the experimental error inherent in this method, solubility values ​​are intended to be considered rough estimates and are intended to be used only in designing crystallization experiments.

[0150] TG-FTIR: Thermogravimetric measurements were carried out using a Netzsch Thermo-Microbalance TG209 coupled to a Bruker FTIR spectrometer Vector 22 (sample pan with pinhole, N2 atmosphere, heating rate 10 K / min).

[0151] HPLC: HPLC was performed using an Agilent 1100 series instrument with UV detection and a Waters XTerra MSC18, 100×4.6 mm, 5 um, (FK-CC01H) column using the following parameters: [Table 2] [Table 3]

[0152] Example 1 Amorphous compound (I). A sample of compound (I), obtained by synthesis and purification as described in WO 2020 / 190765, was characterized by various techniques and determined to be amorphous. Optical microscopy images showed glass-like particles and powder X-ray diffraction (PXRD) patterns showed no sharp reflections. Thermogravimetry coupled with FT-IR spectroscopy showed the sample contained about 1.4% water. Some of the water appeared to be trapped in the void spaces of the amorphous particles and was released above its boiling point. The sample decomposed at temperatures above about 200°C. Differential scanning calorimetry (DSC) thermograms showed a glass transition at about 64°C with a ΔCp of about 0.44 J / g / °C. Dynamic vapor sorption (DVS) showed that compound (I) lost about 15% water content at the high rh end during cycling, while reaching about the same amount of water as during TG-FTIR measurements, i.e., about 1.5%. Thus, the amorphous form was considered hygroscopic. The moisture content at the end of the test was approximately 6.4%, significantly higher than at the beginning. Powder X-ray diffraction of samples recovered after DVS revealed that the samples were unchanged during the DVS experiment.

[0153] Approximate solubilities were determined at room temperature for amorphous Compound (I). These values ​​were obtained by adding a small amount of solvent to a small amount of solid, and dissolution was achieved by short stirring or shaking. These values ​​are approximations and do not necessarily correspond to thermodynamic solubility values. The ratios of the solvent mixtures correspond to volume ratios.

[0154] Initially, tests were performed on the amorphous form of compound (I), and later, after the discovery of a stable crystalline form D (Example 7 below), some tests were repeated on the crystalline form. Amorphous compound (I) is highly soluble in the most common organic solvents, such as acetic acid, ketones, alcohols, esters, and dichloromethane, and mixtures thereof. The anti-solvents are water, heptane, and TBME. The solubility of crystalline form D was found to be substantially lower than that of the amorphous form in acetonitrile and ethyl acetate. Based on available data and further experiments, cyclohexane and methylcyclohexane can also be used as anti-solvents. The approximate solubility data of crystalline form D and amorphous form of compound (I) in various solvents are summarized in Table 1. [Table 4]

[0155] Example 2 Suspension equilibration experiments Suspension equilibration experiments were performed to obtain thermodynamically stable forms (polymorphs or solvates) in a given solvent system. Compound (I) was suspended in an appropriate amount of the specified solvent or solvent mixture. Both amorphous and crystalline form D were used as starting materials. In non-aqueous systems, crystalline form D was generated from the amorphous form, and no new forms were generated when form D was the starting material. In aqueous systems, form A or form B was generated. Table 2 provides a summary of the suspension equilibration experiments with the amorphous form and form D. [Table 5]

[0156] Example 3 Evaporation experiment When crystallization in some solution experiments failed, evaporation experiments were performed. In some cases, crystallization was achieved when fresh (anti)solvent was added and suspension equilibrium experiments were performed. The results of some of these evaporation experiments are summarized in Table 3. [Table 6]

[0157] Example 4 Crystal form A The first crystalline form of Compound (I) was designated as Form A and was obtained from a suspension equilibrium experiment of the amorphous form in isopropanol-water mixtures. Form A was characterized by PXRD, TG-FTIR, H-NMR and DVS. Highly crystalline samples of Form A typically contained large amounts of solvent, and it was found that drying resulted in significant loss of crystallinity. It is believed that drying the samples to residual solvent levels below the ICH Q3C limit may lead to a completely amorphous material.

[0158] The PXRD pattern of Form A is depicted in FIG. 1. This sample was tested by PXRD before drying and it should be noted that the undried sample is highly crystalline in nature. The PXRD pattern shows strong and sharp reflections. TG-FTIR of the less dried crystalline Form A sample showed a large mass loss of 36.6% due to water and isopropanol. Given the large mass loss, it was concluded that the drying was insufficient, so the sample was further dried under vacuum at ambient temperature for about 23 hours and a new TG-FTIR was recorded. The new TG-FTIR showed a mass loss of about 1.75%, which still contained some water and isopropanol. H-NMR spectroscopy also showed about 0.1 equivalents of isopropanol, which corresponds to about 1%, in good agreement with the TG-FTIR results. However, PXRD measurement of the dried sample showed an essentially amorphous material with the most intense peaks barely visible. Table 4 below shows the peak list for crystalline Form A of Compound (I). [Table 7-1] [Table 7-2]

[0159] Form A can be reliably produced by suspension equilibration in water-saturated TBME. TG-FTIR was performed after Form A was kept in a desiccator at 75% rh, and the resulting mass loss was 8.9% due to water and some TBME. This sample was further investigated by dynamic water vapor sorption, which showed that compound (I) took up a small amount of water above 50% rh during cycling, while losing about 8% water below 50% rh. A small step was observed between 0% rh and 20% rh. Water was not completely removed during the first DVS cycle at 0% rh, which may be the reason why Form A was retained after completion of the DVS test, as demonstrated by PXRD of the post-DVS sample.

[0160] The water content varies over the full range of relative humidity, i.e., from 0% to 95%, roughly corresponding to 3 water per 10% of compound (I); however, given the low stability of the hydrates, a fixed stoichiometry was not assigned in this case.

[0161] Example 5 Crystal form B A new crystalline form was obtained from a solution of compound (I) in ethanol. Powder X-ray diffraction showed that the solid form was slightly different from form A and was designated as form B. Form B was characterized by PXRD, TG-FTIR, DVS, and H-NMR. Form B was also obtained by suspension equilibrium experiments in methanol-water and pure water as solvent systems.

[0162] The PXRD pattern of Form B is shown in Figure 2a and the peak list is shown in Table 5 below. Similar to Form A, Form B was tested by PXRD and the dried material was shown to be highly crystalline in nature. The PXRD pattern shows strong and sharp reflections. Without further drying, TG-FTIR was performed on Form B and the thermogram showed a large mass loss of 36.5% due to water, but no alcohol was detected. Given the large mass loss, it was concluded that the drying was insufficient and the sample was dried under vacuum for an additional 10 minutes at ambient temperature and a new TG-FTIR was recorded. The new TG-FTIR thermogram showed a water content of approximately 4.6%. PXRD measurement of the dried sample showed a dramatic loss of crystallinity and further drying would lead to a completely amorphous material. [Table 8]

[0163] Form B was tested by dynamic vapor sorption (DVS). When a wet sample was used, there was a large initial mass loss at 50% rh, i.e., at the beginning of the DVS test. DVS showed that once the sample was at 50% rh, Form B took up a small amount of water above 50% rh. The total moisture change during the cycle was about 13%. At the end of the DVS test, the sample was removed from the sample pan and tested by TG-FTIR and PXRD. The PXRD pattern after DVS showed a loss of crystallinity. The TG-FTIR thermogram showed the water content to be about 6.4%. A Raman spectrum was also obtained and is shown in Figure 2b.

[0164] Example 6 Crystal form C Recrystallization from a solution in acetonitrile-water 5.5:2 followed by equilibration at 5° C. afforded a new form, Form C. Form C was characterized by PXRD, TG-FTIR, H-NMR, and DVS.

[0165] The PXRD pattern of Form C is shown in Figure 3, and a selection list is shown in Table 6 below. Similar to Forms A and B, Form C was tested by PXRD before drying, which showed that it was highly crystalline in nature. The PXRD pattern shows strong and sharp reflections. TG-FTIR was performed without further drying, and the thermogram showed a large mass loss of 48% due to water. In view of the large mass loss, it was concluded that drying was insufficient, but no further drying experiments were performed at this time. [Table 9-1] [Table 9-2]

[0166] The DVS results for Form C were similar to those for Forms A and B. When using wet samples, there was a large initial mass loss at 50% rh, i.e., at the beginning of the DVS test. Dynamic water vapor sorption showed that when the samples were lightly dried (i.e., dried at 50% rh), they picked up a small amount of water above 50% rh. The total moisture content change was about 14% during the cycle, with an end moisture content of about 5.9%, but completely dry material was not achieved at 0% rh (i.e., under nitrogen for 5 hours). At the end of the DVS test, samples were removed from the sample pan and examined by TG-FTIR and PXRD. PXRD after DVS showed an almost complete loss of crystallinity. The TG-FTIR thermogram showed a moisture content of about 5.9%.

[0167] Example 7 Crystal form D During polymorphism studies performed on batches, amorphous samples were frequently obtained. The discovered crystalline forms A, B, and C showed poor physical form stability. During salt scale-up experiments, a new crystalline form of the free base was discovered when starting with a new batch. Based on the approximate solubility data, amorphous compound (I) had high solubility in ethyl acetate. When attempting to obtain the mesylate salt from a solution of the free base in ethyl acetate by adding methanesulfonic acid, a suspension formed before the acid was added. The solid product isolated from the suspension was crystalline and showed a new PXRD pattern. This new crystalline form of the free base was designated as Form D and was further characterized by chemical purity by HPLC, FT-Raman, TG-FTIR, DSC, DVS, and solubility in pure water and various buffers. Form D is a nonsolvated form and is most preferably produced in anhydrous solvents and solvent mixtures. Solubility studies were also performed in possible process solvent mixtures.

[0168] Form D was generally obtained from crystallization experiments from the solutions listed in Table 7. These experiments were often combined with phase equilibration, i.e., the resulting suspensions were not immediately filtered. The suspensions were stirred for several days, as it was believed to be important to find a stable form. [Table 10]

[0169] The powder X-ray diffraction pattern of Compound (I) (free base) Form D is shown in FIG. 4a and the peak list is provided in Table 8 below. [Table 11]

[0170] The purity of the new Form D was tested by a generic standard HPLC method. The purity was found to be 99.4% (vs. 98.1% for the amorphous starting material). The Fourier transform (FT)-Raman spectrum of Form D is shown in Figure 4b.

[0171] A TG-FTIR thermogram was recorded for Form D, which showed only about 0.3% residual ethyl acetate even though the sample was briefly dried in air at ambient temperature before testing, indicating it was a solvent-free form. DSC revealed a fairly sharp melting endotherm with a peak maximum at 153° C. and an enthalpy signal of about 69 J / g.

[0172] Solubility testing was performed in potential process solvent mixtures of Form D. The results are shown in Table 9 below. [Table 12]

[0173] DVS was performed on Form D. At 95% relative humidity, Form D took up a maximum of 0.8% water, and when scanned at 0% rh humidity the sample lost all water.

[0174] The aqueous solubility of amorphous and Form D of Compound I was tested, and the results are shown in Table 10 below. [Table 13]

[0175] pH-dependent solubility tests were performed in 0.1 M HCl, citrate buffer, and USP phosphate buffer with nominal pH values ​​of 6.8 and 7.4. Solubility tests at pH values ​​below 5 failed because all compounds dissolved in the equilibrium phase. A rough test showed that 55 mg of compound was readily dissolved in 1.0 ml of 0.1 M HCl. The results are summarized in Table 11 below. [Table 14]

[0176] All documents mentioned herein are incorporated by reference in their entirety.

Claims

1. A crystalline polymorphic form of Compound (I) as a free base, having the following structure: 【Chemical 1】 The polymorphic form of the crystal is (1) A crystalline form A having an X-ray diffraction pattern with characteristic peaks characterized by 2θ values ​​of about 4.9±0.2, about 13.9±0.2, about 22.1±0.2, and about 25.1±0.2 degrees; (2) A crystalline form B having an X-ray diffraction pattern with characteristic peaks characterized by 2θ values ​​of about 4.8±0.2, about 9.8±0.2, about 13.8±0.2, about 14.7±0.2, about 17.7±0.2, about 20.2±0.2, about 24.1±0.2, about 24.6±0.2, and about 25.1±0.2 degrees; (3) A crystalline form C having an X-ray diffraction pattern with characteristic peaks characterized by 2θ values ​​of about 4.9±0.2, about 22.9±0.2, about 23.2±0.2, about 23.7±0.2, and about 24.3±0.2 degrees; or (4) A crystalline form of the polymorphic form, which is a crystalline form of Form D having an X-ray diffraction pattern with characteristic peaks characterized by 2θ values ​​of about 15.6±0.2, about 16.9±0.2, about 19.1±0.2, about 19.5±0.2, about 22.5±0.2, and about 26.0±0.2 degrees.

2. 2. The crystalline polymorphic form of claim 1, wherein the crystalline polymorphic form is Form A, and wherein the crystalline polymorphic form has an X-ray diffraction pattern with characteristic peaks characterized by values ​​of about 4.9±0.2, about 12.0±0.2, about 13.9±0.2, about 14.8±0.2, about 15.5±0.2, about 15.9±0.2, about 17.8±0.2, about 20.4±0.2, about 21.0±0.2, about 22.1±0.2, about 23.0±0.2, about 23.6±0.2, about 24.3±0.2, about 24.8±0.2, about 25.1±0.2, about 26.3±0.2, and about 27.3±0.2 degrees 2θ.

3. 2. The crystalline polymorphic form of claim 1, wherein the crystalline polymorphic form is Form C, and wherein Form C has an X-ray diffraction pattern with characteristic peaks characterized by values ​​of 2θ of about 4.9±0.2, about 9.8±0.2, about 12.1±0.2, about 14.4±0.2, 14.7±0.2, about 20.2±0.2, about 23.2±0.2, about 23.7±0.2, about 24.3±0.2, about 24.6±0.2, and about 26.1±0.2 degrees.

4. 2. The crystalline polymorphic form of claim 1, wherein the crystalline polymorphic form is crystalline Form D, and wherein crystalline Form D has an X-ray diffraction pattern with characteristic peaks characterized by values ​​of 2θ of about 15.2±0.2, about 15.6±0.2, about 16.9±0.2, about 19.1±0.2, about 19.5±0.2, about 22.5±0.2, and about 26.0±0.2 degrees.

5. 2. The crystalline polymorphic form of claim 1, wherein the crystalline polymorphic form is Form D crystalline, and wherein Form D crystalline has an X-ray diffraction pattern with characteristic peaks characterized by values ​​of about 10.2±0.2, about 13.5±0.2, about 15.2±0.2, about 15.6±0.2, about 15.9±0.2, about 16.9±0.2, 19.1±0.2, about 19.5±0.2, about 22.5±0.2, about 23.3±0.2, about 24.3±0.2, about 24.7±0.2, about 24.9±0.2, about 26.0±0.2, about 27.9±0.2, and about 29.6±0.2 degrees 2θ.

6. 2. The crystalline polymorphic form of claim 1, wherein the crystalline polymorphic form is crystalline form D and has a melting point of about 153°C.

7. A method for forming crystals of Form A of compound (I) according to claim 1 or 2, comprising: (a) crystallizing amorphous Compound (I) in isopropyl alcohol:water (1:1) and equilibrating the crystallized amorphous Compound (I); (b) suspension equilibrating crystals of Form D in acetonitrile:water (9:1); or (c) suspension-equilibrating amorphous Compound (I) in water-saturated TBME.

8. A method for forming crystals of Form B of compound (I) according to claim 1, comprising: (a) suspension equilibration of amorphous Compound (I) in water:ethanol (2:1); or (b) suspension-equilibrating crystals of Form D in water.

9. 10. A method for forming crystals of Form C of compound (I) according to claim 1 or 3, comprising recrystallizing compound (I) from a solution of 5.5:2 acetonitrile:water, followed by equilibration at 5°C.

10. 10. A method for forming crystals of Form D of Compound (I) according to any one of claims 1 and 4-6, comprising crystallizing amorphous Compound (I) in a non-aqueous solvent system and converting the amorphous Compound (I) to crystals of Form D via slurrying, seeding, equilibration, suspension equilibration, or a combination thereof.

11. (a) slurrying in ethyl acetate; (b) seeding crystals of Form D in ethyl acetate; (c) slurrying in acetonitrile; (d) suspension equilibration in isopropyl acetate:cyclohexane (1:2); (e) Methyl ethyl ketone (MEK): Suspension equilibration in diisopropyl ether; (f) suspension equilibration in methyl ethyl ketone (MEK):p-xylene (1:2) and partial evaporation of the solvent; (g) equilibrating in methyl THF:methyl cyclohexane (1:2); (h) equilibrating in isopropyl acetate:heptane (2:3); (i) equilibration in isopropyl acetate:heptane (1:1); (j) suspension equilibration in n-butyl acetate:heptane (5:2); (k) seeding and suspension equilibrating crystals of Form D in TBME; (l) suspension equilibration in heptane:MIBK (4:1); (m) suspension equilibration in methanol:methylcyclohexane (1:4); (n) suspension equilibration in trimethylamine; or (o) suspension equilibrating in diisopropyl ether.

12. A method for forming crystals of Form D of compound (I) according to any one of claims 1 and 4 to 6, comprising: (a) acetone:heptane (1:3); (b) n-butyl acetate, or (c) evaporating a solution of Compound (I) in one of: methanol:diisopropyl ether (1:12).

13. A method for purifying compound (I), comprising the steps of: 【Chemistry 2】 The method comprising preparing crystals of polymorphic form A; and isolating said crystals of polymorphic form A.

14. A method for purifying compound (I), comprising the steps of: 【Chemistry 3】 preparing crystals of polymorphic form B; and isolating said crystals of polymorphic form B.

15. A method for purifying compound (I), comprising the steps of: 【Chemistry 4】 preparing crystals of polymorphic Form C; and isolating said crystals of polymorphic Form C.

16. A method for purifying compound (I), comprising the steps of: 【Chemistry 5】 preparing crystals of polymorphic Form D; and isolating said crystals of polymorphic Form D.

17. A pharmaceutical composition comprising a crystalline polymorphic form of compound (I) according to any one of claims 1 to 6 and a pharmaceutically acceptable excipient.

18. 10. A pharmaceutical for treating cancer in a subject in need thereof, comprising one or more crystalline polymorphic forms A, B, C, or D of compound (I) according to any one of claims 1 to 6.

19. 19. The pharmaceutical product of claim 18, wherein the cancer is characterized by aberrant EGFR expression.

20. The pharmaceutical product of claim 18, wherein the cancer is a brain tumor.

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