EGFR inhibitor polymorphic forms

Abstract

Disclosed herein are polymorphic forms of (S)-N-(3-bromo-2-fluorophenyl)-7-((4-methylpiperazin-1-yl) methyl)-7, 8-dihydro-[1, 4] dioxin [2, 3-g] quinazolin-4-amine, as well as methods for preparing the same. # imgabs0 #

Description

Background Art

[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 hallmark genetic abnormalities encountered 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 series of potential therapies targeting EGFR or its mutant constitutively active form AEGFR (including tyrosine kinase inhibitors (TKI), monoclonal antibodies, vaccines, and RNA-based agents) are currently under development or in clinical trials for the treatment of GBM. However, so far, their efficacy in the clinic has been limited by previous and acquired drug resistance (Taylor et al. (2012) Curr. Cancer Drug Targets. 12: 197-209). A major limitation is that current therapies such as erlotinib, lapatinib, gefitinib, and afatinib are poor brain penetrants (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. WO 2020 / 190765 discloses (S)-N-(3-bromo-2-fluorophenyl)-7-((4-methylpiperazin-1-yl)methyl)-7,8-dihydro-[1,4]dioxaquino[2,3-g]quinazolin-4-amine ("Compound (I)"):

[0003]

[0004]

[0005] It exhibits both good EGFR inhibitory activity and brain penetrability. However, no polymorphic form has been identified in the present disclosure.

[0006] During drug development of an active pharmaceutical ingredient (API), the physical form of the API or its salt can affect the physical properties of the candidate drug in the formulation. Many active pharmaceutical ingredients may exist in more than one polymorphic form. In addition, the preferred form may also exist as a free base, free acid, or a pharmaceutically acceptable salt. Therefore, identifying a pharmaceutically acceptable salt form as well as a free base or free acid form for manufacture represents an important step in developing a candidate drug.

[0007] In determining or identifying preferred solid forms, preferred solid forms are generally solid forms with unpredictable physical properties. Specific solid forms (crystallization, semisolid) may be preferred because they are easily prepared, stable, etc. On the other hand, different crystalline solids may be preferred due to larger solubility and / or better pharmacokinetics. Therefore, although the pharmaceutically acceptable salt of active ingredient can provide increased solubility or increased solubility, developing specific drug forms (such as crystalline solids) may be necessary to obtain clinical drug formulations. Summary of the invention

[0008] The present application relates to polymorphs of (S)-N-(3-bromo-2-fluorophenyl)-7-((4-methylpiperazin-1-yl)methyl)-7,8-dihydro-[1,4]dioxaquino[2,3-g]quinazolin-4-amine.

[0009] In one aspect, the embodiments herein relate to polymorphic forms of Compound (I):

[0010]

[0011]

[0012] In one embodiment, the polymorphic form of Compound (I) is Form A and has an X-ray powder diffraction pattern having characteristic peaks represented 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.

[0013] In one embodiment, the polymorphic form of Compound (I) is Form B and has an X-ray powder diffraction pattern having characteristic peaks represented 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.

[0014] In one embodiment, the polymorphic form of Compound (I) is Form C and has an X-ray powder diffraction pattern having characteristic peaks represented 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.

[0015] In one embodiment, the polymorphic form of Compound (I) is Form D and has an X-ray powder diffraction pattern having characteristic peaks represented 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.

[0016] In one aspect, the embodiments herein relate to a method of purifying compound (I):

[0017]

[0018] The method comprises preparing crystals of polymorph Form A and isolating the crystals of polymorph Form A.

[0019] In one aspect, the embodiments herein relate to a method of purifying compound (I):

[0020]

[0021] The method comprises preparing crystals of polymorph Form B and isolating the polymorph Form B crystals.

[0022] In one aspect, the embodiments herein relate to a method of purifying compound (I):

[0023]

[0024] The method comprises preparing crystals of polymorph Form C and isolating the polymorph Form C crystals.

[0025] In one aspect, the embodiments herein relate to a method of purifying compound (I):

[0026]

[0027]

[0028] The method comprises preparing crystals of polymorph Form D and isolating the polymorph Form D crystals.

[0029] In one aspect, the embodiments herein relate to a pharmaceutical composition comprising a polymorphic form of Compound (I) and a pharmaceutically acceptable excipient.

[0030] In one aspect, the embodiments herein relate to a method of treating a subject having cancer, the method comprising administering to the subject one or more polymorphic Forms A, B, C, or D of Compound (I).

[0031] In one aspect, the embodiments herein relate to the use of one or more polymorphic Forms A, B, C or D of Compound (I) in the manufacture of a medicament for treating a subject suffering from cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Shown is a powder X-ray diffraction (PXRD) pattern of crystalline Form A of Compound (I).

[0033] Figure 2 shows the powder X-ray diffraction (PXRD) pattern of crystalline form B of compound (I).

[0034] Figure 3 Shown is a powder X-ray diffraction (PXRD) pattern of crystalline Form C of Compound (I).

[0035] Figure 4a shows the powder X-ray diffraction (PXRD) pattern of crystalline form D of compound (I).

[0036] Figure 4b shows the Fourier transform-Raman spectrum of crystalline form D of Compound (I). DETAILED DESCRIPTION

[0037] The present application relates to polymorphs of (S)-N-(3-bromo-2-fluorophenyl)-7-((4-methylpiperazin-1-yl)methyl)-7,8-dihydro-[1,4]dioxaquino[2,3-g]quinazolin-4-amine.

[0038] By referring to the following specific embodiments in conjunction with the drawings and examples forming a part of the present disclosure, the present disclosure can be more easily understood. It should be understood that the disclosed embodiments are not limited to the specific equipment, methods, applications, conditions or parameters described and / or shown herein, and the terms used herein are only used for the purpose of describing specific embodiments by way of example, and are not intended to limit the disclosed embodiments. In addition, unless the context clearly indicates otherwise, as used in the specification (including the appended claims), the singular forms "one", "a kind of" and "said" include plural numbers, and references to specific numerical values ​​include at least the specific value.

[0039] As used herein, the terms "comprising," "including," "having," "has," "may," "containing," and variations thereof are intended to be open transitional phrases, terms, or words that require the presence of specified ingredients / steps and allow for the presence of other ingredients / steps. However, such descriptions should also be construed as describing compositions or methods as "consisting of the recited compounds" and "consisting essentially of the recited compounds," which allow for the presence of only the recited compounds and any pharmaceutical carriers, and exclude other compounds.

[0040] definition

[0041] As used herein and unless indicated to the contrary, the following terms and phrases have the meanings set forth below.

[0042] As used herein, the term "polymorph" refers to a crystalline form of a compound. The terms "polymorph" and "crystalline form" or "form" followed by a letter identifier are used interchangeably. Such crystalline forms can be identified, inter alia, by X-ray diffraction patterns.

[0043] As used herein, "equilibrium" when used in reference to the conditions under which polymorphs of a compound are formed, refers to the physical processes that lead to thermodynamic equilibrium.

[0044] As used herein, "suspension equilibrium" when used in reference to the conditions under which polymorphs of a compound are formed, refers to the physical process that results in thermodynamic equilibrium in a suspension, typically solid particles / material dispersed in a liquid.

[0045] As used herein, "slurrying" when used to refer to conditions for forming polymorphs of a compound refers to the physical process of causing thermodynamic equilibrium in a suspension, typically solid particles / material dispersed in a liquid, by agitating the suspension with a suitable stirring device.

[0046] As used herein, "seeding" refers to the process of using a small amount of an existing crystalline form to assist in the crystallization of more of that form from a solution or suspension of a compound of interest.

[0047] Unless otherwise indicated, all chiral (enantiomers and diastereomers) and racemic forms are within the scope of the disclosed embodiments. Many geometric isomers of C=C double bonds, C=N double bonds, ring systems, etc. may also exist in the compounds, and all such stable isomers are encompassed in the present disclosure. Cis and trans (or E- and Z-) geometric isomers of the disclosed compounds are described and may be separated as a mixture of isomers or as individual isomeric forms.

[0048] The term "stereoisomer" refers to isomers of the same 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 stereoisomers that are not mirror images.

[0049] The symbols "R" and "S" represent the configuration of substituents around a chiral carbon atom. The isomer descriptors "R" and "S" are used as described herein to indicate the atomic configuration relative to the core molecule and are intended to be used as defined in the literature (IUPAC Recommendations 1996, Pure and Applied Chemistry, 68: 2193-2222 (1996)).

[0050] The compounds of the present disclosure, their free forms and salts may exist in a variety of tautomeric forms, in which hydrogen atoms are replaced with other parts of the molecule, and the chemical bonds between the atoms of the molecule are therefore rearranged. It should be understood that all tautomeric forms, as long as they can exist, are included in the disclosed embodiments.

[0051] All ranges disclosed herein include the stated endpoints and are independently combinable (e.g., a range of "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 the exact ranges or values; they are imprecise enough to include values ​​approximating these ranges and / or values.

[0052] 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]dioxin-[2,3-g]quinazolin-4-amine.

[0053] As used herein, the term "physically stable" means that a specific 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 specified conditions (e.g., room temperature ambient humidity or 40°C / 75% relative humidity) for a specified 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 longer). In some embodiments, when subjected to specified conditions, less than 25% of the form of the compound changes into one or more different physical forms. In some embodiments, when subjected to specified conditions, 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 specific compound changes into one or more different physical forms of the specific compound. In some embodiments, no detectable amount of a specific form of a compound changes into one or more different physical forms of the compound.

[0054] As used herein, the term "chemically stable" means that the chemical structure of a particular compound does not change into another compound (e.g., decompose) when subjected to specified conditions (e.g., room temperature ambient humidity or 40°C / 75% relative humidity) for a specified 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 longer). In some embodiments, when subjected to specified conditions, less than 25% of the form of a particular compound becomes one or more other compounds. In some embodiments, when subjected to specified conditions, 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 becomes one or more other compounds. In some embodiments, there is no detectable amount of a particular compound that becomes one or more different physical forms of the particular compound.

[0055] When a value is expressed as an approximation by using the antecedent "about," it is 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 stated value, including the end values. For example, the phrase "about 8" refers to values ​​of 7.2 to 8.8, including the end values; as another example, the phrase "about 8%" refers to values ​​of 7.2% to 8.8%, including the end values. When present, all ranges are inclusive and combinable.

[0056] "Solid form" refers to a polymorphic form of a chemical compound. A solid form can exist as a crystalline solid or an amorphous solid.

[0057] "Hydrochloride" refers to a structure in which a proton ion is bonded to the most basic site on the compound (eg, a nitrogen atom) and a chloride ion is the counter anion.

[0058] If there is a discrepancy between a depicted structure and the name given to that structure, the depicted structure shall prevail. In addition, if the stereochemistry of a structure or portion of a structure is not indicated, for example, by bold or dashed lines, the structure or portion of a structure should be interpreted as encompassing all stereoisomers thereof. However, in some cases, where there is more than one chiral center, the structure and name may be represented as a single enantiomer to aid in describing the relative stereochemistry. One skilled in the art of organic synthesis will know whether a compound is prepared as a single enantiomer by the process used to prepare them.

[0059] In this specification, the term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can be interconverted through a low energy barrier. For example, proton tautomers (also called prototropic tautomers) include interconversions through proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions through the reorganization of some bonding electrons.

[0060] 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 caused by the administration of one or more prophylactic or therapeutic agents to a patient suffering from the disease. In the context of the present disclosure, the terms "treat," "treating," and "treatment" also refer to:

[0061] (i) preventing the occurrence of said disease or disorder in a mammal, particularly where such mammal is susceptible to said disorder but has not yet been diagnosed as having said disorder;

[0062] (ii) inhibiting the disease or disorder, i.e. arresting its development;

[0063] (iii) alleviate the disease or condition, i.e. cause regression of the disease or condition; or

[0064] (iv) Alleviate the symptoms caused by the disease or disorder, i.e., relieve pain without addressing the underlying disease or disorder. As used herein, the terms "disease" and "disorder" may be used interchangeably or may be different, as a particular disease or disorder may not have a known pathogen (so that an etiology has not yet been derived) and therefore has not been identified as a disease, but only as an undesirable disorder or syndrome in which a clinician has identified a more or less specific set of symptoms.

[0065] The term "effective amount" refers to an amount of a compound or other active ingredient sufficient to provide a therapeutic or preventive benefit in the treatment or prevention of a disease or sufficient to delay or minimize symptoms associated with a disease. In addition, a therapeutically effective amount with respect to a compound means an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment or prevention of a disease. Used in conjunction with a compound, the term may encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease, or enhances therapeutic efficacy or synergy with another therapeutic agent.

[0066] "Patient" or "subject" includes animals such as humans, cows, horses, sheep, lambs, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits, or guinea pigs. 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, teenager, or adult.

[0067] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent or encapsulating material, which is involved in carrying or transporting a salt of a compound useful in the disclosed embodiments or carrying or transporting it to a subject so that the compound can perform its intended function. Each carrier must be "acceptable", meaning compatible with the other ingredients of the formulation (including salts useful in the disclosed embodiments) and not harmful to the subject. Some examples of materials that can be used as pharmaceutically 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; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; 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 glycerol, 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; ethanol; phosphate buffered saline; and other nontoxic compatible substances employed in pharmaceutical formulations.

[0068] Appropriate formulations depend on the chosen route of administration. Additional details about suitable excipients for use in the pharmaceutical compositions described herein can be found in, for example, Remington: The Science and Practice of Pharmacy, Volumes I and II, Twenty-Second Edition, Loyd V. Allen, Jr., ed. (Philadelphia, PA: Pharmaceutical Press, 2012); Excipient Development for Pharmaceutical, Biotechnology, and Drug Delivery Systems, Ashok Katdare and Mahesh V. Chaubal, eds. (Boca Raton, FL: CRC Press, 2006); and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Edition (Lippincott Williams & Wilkins 1999), which are incorporated herein by reference for such disclosure.

[0069] As used herein, a pharmaceutical composition refers to a mixture of a crystalline solid polymorphic form 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.

[0070] In an embodiment, a polymorph of the free base of Compound (I) having the following structure is provided:

[0071]

[0072] Wherein the polymorph is:

[0073] (1) Form A, which has an X-ray diffraction pattern having 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; in embodiments, Form A is characterized by 2, 3, or 4 of the foregoing peaks;

[0074] (2) Form B, which has an X-ray diffraction pattern characterized by characteristic peaks with °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; in embodiments, Form B is characterized by 2, 3, 4, 5 to all of the foregoing peaks;

[0075] (3) Form C, which has an X-ray diffraction pattern characterized by characteristic peaks with °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; in embodiments, Form C is characterized by 2, 3, 4, or 5 of the foregoing peaks; or

[0076] (4) Form D, which has an X-ray diffraction pattern having 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; in embodiments, Form B is characterized by 2, 3, 4, 5 to all of the foregoing peaks.

[0077] In embodiments, the polymorph is Form A, and the X-ray diffraction pattern of Form A has characteristic peaks characterized by °2θ 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.

[0078] In embodiments, the polymorph is Form C, and the Form C X-ray diffraction pattern has characteristic peaks characterized by °2θ values ​​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, 24.3±0.2, about 24.6±0.2, and about 26.1±0.2.

[0079] In embodiments, the polymorph is Form D, and the Form D has an X-ray diffraction pattern having characteristic peaks characterized by °2θ values ​​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.

[0080] In embodiments, the polymorph is Form D, and the Form D has an X-ray diffraction pattern having characteristic peaks characterized by °2θ 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.

[0081] In embodiments, the polymorph is Form D having a melting point of about 153°C.

[0082] In an embodiment, the polymorph is Form A, such as Figure 1 and having the peak list provided in Table 4. In an embodiment, the polymorph is Form B, as shown in Figure 2a and having the peak list provided in Table 5. In an embodiment, the polymorph is Form C, as shown in Figure 3 and having the peak list provided in Table 6. In an embodiment, the polymorph is Form D, as shown in Figure 4a and having the peak list provided in Table 8.

[0083] In an embodiment, a method for forming a polymorph of compound (I) is provided, the method comprising converting an amorphous compound (I) into a polymorphic form by equilibrium, suspension equilibrium, slurrying, seeding, evaporation or a combination thereof. In an embodiment, Form A, Form B or Form C is formed from an amorphous compound (I) in the presence of water. In an embodiment, Form D is formed from an amorphous compound (I) in a non-aqueous solvent system.

[0084] In an embodiment, there is provided a method of forming the Form A polymorph of Compound (I), the method comprising one of the following:

[0085] (a) crystallizing an amorphous compound (I) in isopropanol:water (1:1), and equilibrating the crystallized amorphous compound (I);

[0086] (b) suspending the equilibrated Form D in acetonitrile:water (9:1); or

[0087] (c) Suspension equilibrium of amorphous compound (I) in water-saturated TBME.

[0088] In an embodiment, there is provided a method of forming the Form B polymorph of Compound (I), the method comprising one of the following:

[0089] (a) suspending and equilibrating an amorphous compound (I) in water:ethanol (2:1); or

[0090] (b) Suspended in equilibrium form D in water;

[0091] In an embodiment, there is provided a method of forming the Form C polymorph of Compound (I), the method comprising recrystallizing Compound (I) from a solution in acetonitrile:water 5.5:2, followed by equilibration at 5°C.

[0092] In an embodiment, a method of forming a Form D polymorph of Compound (I) is provided, the method comprising crystallizing amorphous Compound (I) in a non-aqueous solvent system and converting the amorphous Compound (I) to Form D by slurrying, seeding, equilibration, suspension equilibration, or a combination thereof. In an embodiment, such a method employs one of the following:

[0093] (a) slurrying in ethyl acetate;

[0094] (b) seeding with Form D in ethyl acetate;

[0095] (c) slurrying in acetonitrile;

[0096] (d) Suspension equilibrium in isopropyl acetate:cyclohexane (1:2);

[0097] (e) Suspension equilibrium in methyl ethyl ketone (MEK): diisopropyl ether;

[0098] (f) suspending and balancing in methyl ethyl ketone (MEK): p-xylene (1:2) and partially evaporating the solvent;

[0099] (g) equilibrated in methylTHF:methylcyclohexane (1:2);

[0100] (h) equilibrated in isopropyl acetate:heptane (2:3);

[0101] (i) Equilibrated in isopropyl acetate:heptane (1:1);

[0102] (j) Suspension equilibrium in n-butyl acetate:heptane (5:2);

[0103] (k) suspended in TBME and seeded with Form D;

[0104] (l) Suspension equilibrium in heptane:MIBK (4:1);

[0105] (m) Suspension equilibrium in methanol:methylcyclohexane (1:4);

[0106] (n) Suspension equilibrium in trimethylamine; or

[0107] (o) Suspended in diisopropyl ether.

[0108] In an embodiment, there is provided a method of forming the Form D polymorph of Compound (I), the method comprising evaporating a solution of Compound (I) in one of:

[0109] (a) acetone:heptane (1:3);

[0110] (b) n-butyl acetate; or

[0111] (c) Methanol: diisopropyl ether (1:12).

[0112] 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, processing time, concentration and pH.

[0113] In an embodiment, each polymorphic form A, B, C and D and amorphous form of compound (I) can be converted into each other by changing the conditions described in the preparation of the specific desired polymorphic form. In some such embodiments, mutual conversion can be carried out by amorphous form. For example, in an embodiment, form A can be converted into form D by converting form A into amorphous form, and then converted from amorphous form into form D. Therefore, as indicated in the following examples, form A can be dried to the point of reforming the amorphous form of compound (I), and then conditioned to prepare form D. Similarly, form B can be converted into amorphous form, thereby converted into form D. Similarly, form C can be converted into amorphous form, thereby converted into form D. As further indicated below, the ability to produce highly crystalline solids using forms A, B and C can contribute to the purification of compound (I). Therefore, the purification method described below herein can utilize the clever mutual conversion of polymorphic forms and their amorphous forms. In some embodiments, each of forms A, B and C can be directly converted into form D by balancing under the conditions for preparing form D. Therefore, this interconversion does not require passage through the amorphous form.

[0114] As disclosed in the examples below, the polymorphic form of compound (I) has a high degree of crystallinity. Therefore, the polymorphic form is particularly suitable for assisting the purification of compound (I). In an embodiment, the method for purifying compound (I) comprises preparing a polymorph of compound (I) according to any method disclosed herein and separating crystals of the polymorph. In an embodiment, a method for purifying compound (I) is provided:

[0115]

[0116] The method comprises preparing crystals of polymorph Form A and isolating the crystals of polymorph Form A.

[0117] In an embodiment, a method of purifying compound (I) is provided:

[0118]

[0119] The method comprises preparing crystals of polymorph Form B and isolating the polymorph Form B crystals.

[0120] In an embodiment, a method of purifying compound (I) is provided:

[0121]

[0122] The method comprises preparing crystals of polymorph Form C and isolating the polymorph Form C crystals.

[0123] In an embodiment, a method of purifying compound (I) is provided:

[0124]

[0125]

[0126] The method comprises preparing crystals of polymorph Form D and isolating the polymorph Form D crystals.

[0127] In an embodiment, a pharmaceutical composition is provided, comprising a polymorphic form of Compound (I) as disclosed herein and a pharmaceutically acceptable excipient.

[0128] The pharmaceutical compositions and methods disclosed herein can be used to treat individuals in need. In an embodiment, the individual is a mammal such as a human or a non-human mammal. When applied to an animal such as a human, the composition or compound is preferably administered as a pharmaceutical composition comprising, for example, a 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 a preferred embodiment, when such pharmaceutical compositions are used for human administration, particularly for invasive administration routes (i.e., bypassing the transport or diffusion routes (such as injection or implantation) through the epithelial barrier), the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to achieve delayed release of the medicament or selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form, such as tablets, capsules (including spray capsules and gelatin capsules), granules, lyophiles for reconstruction, powders, solutions, syrups, suppositories, injections, etc. The composition can also be present in a transdermal delivery system, such as a skin patch. The composition may also be presented in a solution suitable for topical administration, such as a lotion, cream or ointment.

[0129] A pharmaceutically acceptable carrier may contain a physiologically acceptable agent, the function of which is, for example, to stabilize, increase solubility or increase the absorption of the compound. These 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 selection of a pharmaceutically acceptable carrier (including a physiologically acceptable agent) depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) may also be a liposome or other polymer matrix, into which, for example, a compound may be incorporated. For example, liposomes comprising phospholipids or other lipids are non-toxic, physiologically acceptable and metabolizable carriers, which are relatively simple to prepare and administer.

[0130] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0131] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically 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 injurious to the patient. Some examples of materials that can be used as pharmaceutically 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, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other nontoxic compatible substances used in pharmaceutical formulations.

[0132] The pharmaceutical composition (preparation) can be administered to a subject by any of a variety of routes of administration, including, for example, oral administration (e.g., as a drench, tablet, capsule (including spray capsules and gelatin capsules) as an aqueous or non-aqueous solution or suspension, bolus, powder, granules, paste for application to the tongue); absorption through the oral mucosa (e.g., sublingual); subcutaneous; transdermal (e.g., as a patch applied to the skin); and topical administration (e.g., as a cream, ointment or spray applied to the skin). The compound can also be formulated for inhalation. In an embodiment, the compound can be simply dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable for the routes of administration can be found, for example, in U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896 and the patents cited therein.

[0133] The preparation can be conveniently present in unit dosage form and can be prepared by any method well known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated, the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces the therapeutic effect. Typically, in 100%, this amount is in the range of about 1% to about 99%, preferably about 5% to about 70%, most preferably about 10% to about 30% of the active ingredient.

[0134] The method of preparing these formulations or compositions includes the step of bringing the active compound such as a compound into association with a 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.

[0135] Formulations of the polymorphic compounds disclosed herein suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), lyophiles, 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 such as gelatin and glycerin, or sucrose and acacia) and / or as a mouthwash, etc., each containing a predetermined amount of the compound of the present disclosure as the active ingredient. The composition or compound may also be administered as a bolus, electuary or paste.

[0136] To prepare solid dosage forms for oral administration (capsules (including spray capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers (such as sodium citrate or dicalcium phosphate) and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders, for example, carboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) wetting agents, such as Glycerol; (4) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retardants such as paraffin; (6) absorption promoters such as quaternary ammonium compounds; (7) wetting agents such as cetyl alcohol and glyceryl monostearate; (8) absorbents such as kaolin and bentonite; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof; (50) complexing agents such as modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including spray capsules and gelatin capsules), tablets and pills, the pharmaceutical composition may also contain a buffer. Solid compositions of a similar type may also be used as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0137] Tablets can be prepared by compression or molding optionally with one or more auxiliary ingredients. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), a surfactant or a dispersant. Molded tablets can be prepared by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine.

[0138] Tablets and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including spray capsules and gelatin capsules), pills and granules, may be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the field of pharmaceutical formulation. They may also be formulated, for example, using hydroxypropylmethylcellulose, other polymer matrices, liposomes and / or microspheres in different proportions to provide the desired release profile so as to provide a slow or controlled release of the active ingredient therein. They may be sterilized, for example, by filtering through a bacterial retention filter or by incorporating a sterilizing agent in the form of a sterile solid composition, which may be dissolved in sterile water or some other sterile injectable medium before use. These compositions may also optionally contain an opacifier, and may be compositions that release the active ingredient only or preferentially in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. If appropriate, the active ingredient may also be in a microencapsulated form with one or more of the above-mentioned excipients.

[0139] Liquid dosage forms that can be used for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents, cyclodextrins and their derivatives, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof.

[0140] 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.

[0141] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0142] 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 pharmaceutically acceptable carrier and with any preservatives, buffers, or propellants that may be required.

[0143] Ointments, pastes, creams and gels may contain, in addition to the active compounds, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0144] In addition to the active compound, powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants (such as chlorofluorohydrocarbons) and volatile unsubstituted hydrocarbons (such as butane and propane).

[0145] Transdermal patches have the added advantage of providing controlled delivery of the disclosed compound to the body. Such dosage forms can be prepared by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate of this flux can be controlled by providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

[0146] As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise a combination of one or more active compounds with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions prior to use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0147] Examples of suitable aqueous and non-aqueous carriers that can be used in pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Suitable fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0148] These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. By including various antibacterial and antifungal agents, for example parabens, chlorobutanol, phenol sorbic acid, etc., it is possible to ensure that the effects of microorganisms are prevented. It may also be necessary to include isotonic agents, such as sugar, sodium chloride, etc. in the composition. In addition, the extended absorption of injectable drug forms can be achieved by including agents (such as aluminum monostearate and gelatin) that delay absorption.

[0149] In some cases, in order to prolong the effect of the drug, it is necessary to slow down the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous material with poor water solubility. The absorption rate of the drug depends on its dissolution rate, which in turn may depend on crystal size and crystalline form. Alternatively, the delayed absorption of the drug form administered parenterally can be accomplished by dissolving or suspending the drug in an oil vehicle.

[0150] Injectable depot forms are prepared by forming a microencapsulated matrix of the subject compound in a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the properties of the specific polymer used, the drug release rate can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by embedding the drug in liposomes or microemulsions compatible with body tissues.

[0151] For use in the methods of the invention, the active compound is administered per se 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 pharmaceutically acceptable carrier.

[0152] The method of introduction can also be provided by a rechargeable or biodegradable device. In recent years, various slow-release polymer devices have been developed and tested in vivo for controlled delivery of drugs (including protein biopharmaceuticals). A variety of biocompatible polymers (including hydrogels) (including biodegradable and non-degradable polymers, which can be used to form implants) can be used to form implants at specific target sites to continuously release compounds.

[0153] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0154] The selected dosage level will depend upon a variety of factors, including the activity of the specific compound or combination of compounds employed, or the esters, salts or amides thereof; the route of administration; the time of administration; the rate of excretion of the specific compound employed; the duration of the treatment; other drugs, compounds and / or materials used in combination with the specific compound employed; the age, sex, weight, condition, general health and prior medical history of the patient being treated; and like factors well known in the medical arts.

[0155] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required.

[0156] For example, a physician or veterinarian may start a dose of a pharmaceutical composition or compound at a level lower than that necessary to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. "Therapeutically effective amount" means a concentration of a compound sufficient to cause the desired therapeutic effect. It is generally understood that the effective amount of a compound will vary depending on the subject's weight, sex, age, and medical history. Other factors affecting the effective amount may include, but are not limited to, the severity of the patient's condition; the condition being treated; the stability of the compound; and, if necessary, another type of therapeutic agent administered with the compound. Larger total doses may be delivered by multiple administrations of the agent. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbaeher et al. (1996) Harrison's Principles of Internal Medicine 13th edition, 1814-1882, incorporated herein by reference).

[0157] In general, a suitable daily dose of an active compound for use in the compositions and methods will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above.

[0158] If desired, the effective daily dose of the active compound can be administered as one, two, three, four, five, six or more sub-doses at appropriate intervals throughout the day, optionally in unit dosage form. In embodiments of the present disclosure, the active compound can be administered twice or three times a day. In preferred embodiments, the active compound will be administered once a day.

[0159] The patient receiving the treatment is any animal in need thereof, including primates, particularly humans; and other mammals, such as horses, cattle, pigs, sheep, cats, and dogs; poultry; and pets in general.

[0160] In embodiments, the compounds disclosed herein may be used alone or in combination with another type of therapeutic agent.

[0161] In an embodiment, a method of treating a subject with cancer is provided, the method comprising administering to the subject one or more polymorphic forms A, B, C or D of compound (I) as disclosed herein. In some such embodiments, the cancer is characterized by abnormal EGFR expression. In some such embodiments, the cancer is brain cancer.

[0162] In an embodiment, there is provided the use of one or more polymorphic forms A, B, C or D of compound (I) as disclosed herein in the manufacture of a medicament for treating a subject with cancer. In some such embodiments, the cancer is characterized by abnormal EGFR expression. In some such embodiments, the cancer is brain cancer.

[0163] In an embodiment, a method of treating a subject having a cancer associated with aberrant EGFR expression is provided, the method comprising administering to the subject one or more polymorphic Forms A, B, C or D of Compound (I) as disclosed herein. In an embodiment, the cancer is brain cancer.

[0164] In an embodiment, there is provided use of one or more polymorphic Forms A, B, C or D of Compound (I) in the manufacture of a medicament for treating a subject suffering from a cancer associated with aberrant EGFR expression. In an embodiment, the cancer is brain cancer.

[0165] In certain aspects, the present disclosure provides a method of inhibiting EGFR or AEGFR, the method comprising administering to a subject an amount of a polymorphic form of Compound (I), ie, Form A, B, C, D, or a combination thereof.

[0166] In certain aspects, the present disclosure provides a method for treating cancer, the method comprising administering to a subject in need of cancer treatment a certain amount of a polymorph of compound (I) as disclosed herein. In embodiments, the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, heart cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, fibrosarcoma, gastric cancer, gastrointestinal cancer, head, spine and 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 embodiments, the cancer is glioma, astrocytoma or glioblastoma. In embodiments, the cancer is glioblastoma. In embodiments, the cancer is glioblastoma multiforme. In embodiments, the method reduces cancer cell proliferation.

[0167] In certain aspects, the present disclosure provides a method for treating cancer in a subject, the method comprising administering to the subject a glucose metabolism inhibitor and an additional agent, wherein the glucose metabolism is a polymorph of a compound (I) of the present disclosure or a pharmaceutically acceptable salt thereof, and the additional agent is a cytoplasmic p53 stabilizer. In embodiments, cancer is bladder cancer, bone cancer, brain cancer, breast cancer, heart cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, fibrosarcoma, gastric cancer, gastrointestinal cancer, head, spine and 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 embodiments, cancer is glioma, astrocytoma or glioblastoma. In embodiments, cancer is glioblastoma. In embodiments, cancer is glioblastoma multiforme. In embodiments, the method reduces cancer cell proliferation. In embodiments, cancer is recurrent or refractory. In other embodiments, the cancer is treatment naive.

[0168] In embodiments, the subject is determined to be sensitive to a glucose metabolism inhibitor by a method comprising:

[0169] a. obtaining a first blood sample from a subject;

[0170] b. Arrange a ketogenic diet for the subjects;

[0171] c. obtaining a second blood sample from the subject after a period of time of ketogenic diet arrangement;

[0172] d. measuring glucose levels in the first blood sample and the second blood sample;

[0173] e. comparing the glucose level in the second blood sample with the glucose level in the first blood sample; and

[0174] f. Determining that the subject is sensitive if the glucose level in the second blood sample is reduced compared to the glucose level in the first blood sample.

[0175] In embodiments, the reduction in glucose levels between the second blood sample and the control blood sample is about or greater than 0.15 mM. In embodiments, the reduction in glucose levels between the second blood sample and the control blood sample is about or greater than 0.20 mM. In embodiments, the reduction in glucose levels between the second blood sample and the control blood sample is in the range of 0.15 mM-2.0 mM. In embodiments, the reduction in glucose levels between the second blood sample and the control blood sample is in the range of 0.25 mM-1.0 mM.

[0176] 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, 50 mg to 1600 mg of idasanutlin is administered to a subject. In embodiments, 100 mg of idasanutlin is administered to a subject. In embodiments, 150 mg of idasanutlin is administered to a subject. In embodiments, 300 mg of idasanutlin is administered to a subject. In embodiments, 400 mg of idasanutlin is administered to a subject.

[0177] In embodiments, 600 mg of edanulin is administered to the subject. In embodiments, 1600 g of edanulin is administered to the subject. In other embodiments, the MDM2 inhibitor is RO5045337, RO550378L, R06839921, SAR405838, DS-3032, DS-3032b, or AMG-232.

[0178] In embodiments, the cytoplasmic p53 stabilizer is a BCL-2 inhibitor. In embodiments, the BCL-2 inhibitor is antisense oligodeoxynucleotide G3139, mRNA antagonist SPC2996, venetoelax (ABT-199), GDC-0199, obatoclax, paclitaxel, navitoclax (ABT-263), ABT-737, NU-0129, S055746 or APG-1252.

[0179] In embodiments, the cytoplasmic p53 stabilizer is a Bcl-xL inhibitor. In embodiments, the Bcl-xL inhibitor is WEHI 539, ABT-263, ABT-199, ABT-737, sabutoclax, ATI 01, TW-37, APG-1252, or gambogic acid.

[0180] In embodiments, the glucose metabolism inhibitor and the cytoplasmic p53 stabilizer are administered in the same composition. In other embodiments, the glucose metabolism inhibitor and the cytoplasmic p53 stabilizer are administered in separate compositions.

[0181] In embodiments, the method further comprises administering an additional therapy.

[0182] Types and stages of gliomas

[0183] Primary malignant brain tumors are tumors that start in the brain or spine, collectively referred to as gliomas. Glioma is not a specific type of cancer, but a term used to describe tumors that originate from glial cells. Examples of primary malignant brain tumors include astrocytoma, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, diffuse astrocytoma, anaplastic astrocytoma, GBM, ganglioglioma, oligodendroglioma, ependymoma. According to the WHO classification of brain tumors, astrocytoma has been classified into four grades, determined by basic pathology. Features used to classify gliomas include mitosis, cell or nuclear atypia, and vascular proliferation and necrosis with pseudomedullary 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 worst prognosis.

[0184] GBM is the most common, complex, treatment-resistant and deadliest type of brain cancer, accounting for 45% of all brain cancers and diagnosed in nearly 11,000 men, women and children each year. GBM (also known as grade 4 astrocytoma and glioblastoma multiforme) is the most common type of malignant (cancerous) primary brain tumor.

[0185] They are extremely aggressive for many reasons. First, glioblastoma cells proliferate rapidly because they secrete substances that stimulate a rich blood supply. They also have the ability to invade long distances and infiltrate the normal brain by sending microscopic tendrils of the tumor alongside normal cells. There are two known types of glioblastoma. Primary GBMs are the most common form; they grow very quickly and usually cause early symptoms.

[0186] Secondary glioblastomas are less common, accounting for approximately 10% of all GBMs. They progress from low-grade diffuse astrocytomas or anaplastic astrocytomas and are more often found in younger patients. Secondary GBMs are preferentially located in the frontal lobe and have a better prognosis.

[0187] GBM is usually treated with a multimodal combination treatment regimen 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 of the tumor can be safely removed. After surgery, radiation and chemotherapy slow the growth of the remaining tumor cells. The oral chemotherapy drug temozolomide is most often used for six weeks and then monthly thereafter. Another drug, bevacizumab (called temozolomide), is also used during treatment. ). This drug attacks the tumor's ability to recruit a blood supply, often slowing or even stopping tumor growth.

[0188] New investigational treatments are also used, and these treatments may involve adding a treatment to a standard therapy or replacing a portion of a 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 brain area where the tumor is present, and nanotherapy involving spherical nucleic acids (SNAs) 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.

[0189] Embodiments of the methods and compositions discussed herein are also contemplated to be 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, spinal cord metastases, and the like.

[0190] Cytoplasmic p53 stabilizers The present inventors have demonstrated that pharmacological p53 stabilization (such as with CNS penetrating small molecules) is, for example, synergistically lethal with inhibition of EGFR-driven glucose uptake in a patient-derived primary GBM model. The present inventors have demonstrated for the first time that the non-transcriptional function of p53 plays a key role in stimulating intrinsic apoptosis in metabolic responders. Therefore, the treatment methods described herein include administering cytoplasmic p53 stabilizers in combination with glucose metabolism inhibitors. Cytoplasmic p53 stabilizers and glucose metabolism inhibitors can be administered simultaneously or sequentially with the same or different compositions. It is contemplated that a single p53 stabilizer is used in some embodiments, and more than one p53 stabilizer is used in other embodiments. For example, it is reported that Natlin is combined with ABT 737 (which binds BCL-2 and BCL-X L ) 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, p. 217). As contemplated herein, a cytoplasmic p53 stabilizer is any small molecule, antibody, peptide, protein, nucleic acid, or derivative thereof that can pharmacologically stabilize or activate p53 directly or indirectly. The stabilization of cytoplasmic p53 leads to the initiation of apoptosis in cells (such as cancer cells).

[0191] MDM2 antagonists

[0192] The protein level of p53 in the cell is strictly controlled and maintained at a low level by its negative regulator E3 ubiquitin protein ligase MDM2. In the embodiments of the methods or compositions disclosed herein, the cytoplasmic p53 stabilizer is an MDM2 antagonist / inhibitor. In some embodiments, the MDM2 antagonist is Natlin. In other embodiments, Natlin is Natlin 3 or Idanulin. 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.

[0193] Other compounds within the scope of currently known methods for binding MDM-2 include Ro-2443, MI-219, MI-713, MI-888, DS-3032b, benzodiazepine diones (e.g., TDP521252), sulfonamides (e.g., NSC279287), ehromenotriazolopyrimidines, morpholinones and piperidones (AM-8553), terphenyls, chalcones, pyrazoles, imidazoles, imidazole-indoles, isoindolinones, pyrrolidinones (e.g., PXN822), priaxons, piperidines, naturally derived prenylated xanthones, SAH-8 (stapled peptide), sMTide-02, sMTide-02a (stapled peptide), ATSP-7041 (stapled peptide), spirooligomers (alpha helix mimetics). Other compounds known to cause MDM2 protein folding include PRIMA-1MET (also known as APR-246), Aprea 102-105, PK083, PK5174, PK5196, PK7088, benzothiazole, stearic acid, and NSC319726.

[0194] BCL-2 inhibitors

[0195] 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.

[0196] Bcl-xL inhibitors

[0197] In yet other embodiments of the methods or compositions of the invention, the cytoplasmic p53 stabilizer is a Bcl-xL inhibitor. In some embodiments, the Bcl-xL inhibitor is, for example, WEHI 539, ABT-263, ABT-199, ABT-737, Sabtok, ATI 01, TW-37, APG-1252, gambogic acid, or any other Bcl-xL inhibitor.

[0198] Assessment Method: Glucose Uptake Test

[0199] In an embodiment of the methods and compositions of the present disclosure, subjects with GBM or cancer are classified as "metabolic responders" or "metabolic non-responders", i.e., are determined to be sensitive to glucose metabolism inhibitors. In an embodiment, the classification of the subject is before the subject is administered a treatment comprising a glucose metabolism inhibitor and a cytoplasmic p53 stabilizer. Therefore, the present disclosure provides methods for assessing cancer, classifying subjects, and determining the sensitivity of subjects to treatment, the methods involving analysis of glucose metabolism, glycolysis, or glucose uptake. A method for classifying a subject as a metabolic responder is described in detail in Example 1. Technology for monitoring glycolysis and glucose uptake is provided by T.TeSlaa and MATeitell.2014.Methodsin Enzymology, Vol. 542, pp. 92-114, which is incorporated herein by reference.

[0200] Glycolysis is the intracellular biochemical conversion of one glucose molecule into two pyruvate molecules, with the simultaneous production of two ATP molecules. Pyruvate is a metabolic intermediate with several potential fates, including entry into the tricarboxylic acid (TCA) cycle within the mitochondria to produce NADH and FADH2. Alternatively, pyruvate can be converted to lactate in the cytosol by lactate dehydrogenase, with the simultaneous regeneration of NAD from NADH. + Increased flux through glycolysis supports cancer cell proliferation by providing additional energy, for example 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) first observed that proliferating tumor cells increase aerobic glycolysis, converting glucose to lactate in the presence of oxygen, in contrast to non-malignant cells that primarily respire when oxygen is available.

[0201] This mitochondrial bypass, known as the Warburg effect, occurs in rapidly proliferating cells, including cancer cells, activated lymphocytes, and pluripotent stem cells. The Warburg effect has been used to identify fluorinated glucose analogs such as fluorinated glucose analogs using positron emission tomography (PET) scanning. 18Clinical diagnostic test for increased cellular uptake of F-deoxyglucose.

[0202] Therefore, glycolysis represents a target for therapeutic and diagnostic methods. In the context of the methods of the present invention, measuring glucose uptake and lactate excretion of malignant cells can be used to detect a shift in glucose catabolism and / or sensitivity to inhibitors of glucose metabolism. Detecting such a shift is important for methods of treating GBM, methods of reducing the risk of ineffective therapies, and methods of reducing the chances of tumor survival. For the purposes of this disclosure, 18 F-deoxyglucose PET is applicable. In embodiments, it is used as a rapid noninvasive functional biomarker to predict sensitivity to p53 activation. This noninvasive analysis is particularly valuable for malignant brain tumors where pharmacokinetic / pharmacodynamic assessment is extremely difficult and impractical. In some cases, delayed imaging protocols with MRI fusion (41) and parameter response maps (PRMs) can be used to quantify tumor l8 Changes in F-FDG uptake (42).

[0203] In some aspects, the method can be related to measuring glucose uptake and lactic acid production. For cells in culture, quantitative glycolysis flux can be carried out by measuring glucose uptake and lactic acid excretion. Glucose uptake into cells is through glucose transporter (Glut1-Glut4), but lactic acid excretion is through monocarboxylate transporter (MCT1-MCT4) on cell membrane.

[0204] Extracellular glucose and lactate

[0205] 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 FDG uptake or 2-NBDG uptake. Commercially available kits and instruments can be used to quantify glucose and lactate levels in cell culture media. Kit detection methods are typically colorimetric or fluorometric and are compatible with standard laboratory equipment such as spectrophotometers. BioProfile analyzers (such as NovaBiomedical) or Biochemistry analyzers (e.g., YSI Life Sciences) can measure the levels of glucose and lactate in cell culture media. GlucCell (Cesco BioProduets) can only measure glucose levels in cell culture media. Although each commercial method has a different detection protocol, the collection of culture media for analysis is the same.

[0206] Extracellular acidification rate

[0207] Glycolysis can also be determined by measuring the extracellular acidification rate (ECAR) of the surrounding medium, which is derived primarily from the excretion of lactate per unit time following its conversion from pyruvate. The Seahorse Extracellular Flux (XF) Analyzer (Seahorse Bioscience) is a tool for simultaneously measuring glycolysis and oxidative phosphorylation (via oxygen consumption) in the same cells.

[0208] Glucose analog uptake

[0209] Certain embodiments of the disclosed method include the use of glucose analogs. As those skilled in the art are familiar with, in order to determine the glucose uptake rate of cells, labeled glucose isoforms can be added to the cell culture medium and then measured in the cell after a given time period. Exemplary types of glucose analogs used for these studies include, but are not limited to, radioactive glucose analogs, such as 2-deoxy-D-[1,2-3H]-glucose, 2-deoxy-D-[1-14C]-glucose or 2-deoxy-2- 18 F-fluoro-D-glucose ( l8 FDG), or fluorescent glucose analogs, such as 2-[N-(7-nitrobenzo-2-oxa-1,3-dioxol-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 by radiolabeled glucose 2-deoxy-2-[Fluoro-18]-fluoro-D-glucose ( 18 In another embodiment, the uptake of F-FDG is measured by positron emission tomography (PET). 18 F-FDG. In some embodiments, the biopsy is taken from a GBM tumor. 18 Detailed descriptions of examples of F-FDG are provided in the Examples below.

[0210] In certain aspects, the method can involve comparing glucose uptake of a biological sample, such as a tumor sample, to a control. The increase or decrease can be, is at least, or is 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 times, or any range derivable therein. Alternatively, the difference in expression between a sample and a reference can be expressed as a percentage decrease or increase, such as at least or at most 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.

[0211] Other ways of expressing relative expression levels have normalized or relative numbers such as 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 .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 can be relative to a control.

[0212] Algorithms such as weighted voting procedures can be used to facilitate the evaluation of biomarker levels. In addition, other clinical evidence can be combined with biomarker-based tests to reduce the risk of erroneous evaluations. In some embodiments, other cytogenetic evaluations can be considered.

[0213] Example

[0214] The examples and embodiments described herein are for illustrative purposes only, and in some embodiments, various modifications or changes are intended to be included within the scope of the present disclosure and the scope of the appended claims.

[0215] The following abbreviations are used in the examples below:

[0216]

[0217]

[0218] In the PXRD peak listing: vs = very strong intensity, s = strong intensity, m = medium intensity, w = weak intensity, and vw = very weak intensity.

[0219] DSC: Differential scanning calorimetry was performed with a TA Instruments Q2000 instrument (sample pan with small hole in the lid, heating rate 10 K / min). The melting point is understood as the peak maximum. Since compound (I) was found to contain about 1.2% water and some of this was released above 100° C., the sample was dried in the DSC sample pan by heating the pan with small hole to 100° C. and keeping the sample at this temperature for 10 minutes. A second scan was then performed with the dried sample and the first scan was disregarded since the endothermic signal was probably due to the evaporation of water.

[0220] Dynamic Vapor Sorption: DVS measurements were performed using an SPS11-100n "Sorptions Prüfsystem" from ProUmid (formerly "Projekt Messtechnik"), August-Nagel-Str. 23, 89079 Ulm (Germany). Approximately 5-20 mg of sample were placed in an aluminum sample pan. A humidity change rate of 5% per hour was used. The applied measurement procedure is visualized in the figure (blue trace). Correction shows the representation of the effective water content.

[0221] The sample is placed on an aluminum or platinum stand on top of a microbalance and allowed to equilibrate at 50% RH before starting a predetermined humidity program. The measurement procedure is visualized in the reported figures.

[0222] Hygroscopicity classification: Hygroscopicity was classified based on the mass increase relative to the initial mass at 85% RH as follows: deliquescent (adsorbs enough water to form a liquid), very hygroscopic (mass increase ≥ 15%), hygroscopic (mass increase < 15% but ≥ 2%), slightly hygroscopic (mass increase < 2% but ≥ 0.2%), or non-hygroscopic (mass increase < 0.2%).

[0223] Microscopy: Light microscopy was performed on a Leitz Orthoplan polarizing microscope part #130880, typically using a magnification of 10x10.

[0224] H-NMR: Bruker DPX300 spectrometer; proton frequency, 300.13 MHz; 30° excitation pulse; recycle delay, 1 s; 16 scans accumulated; deuterated DMSO as solvent; solvent peak used for reference; chemical shifts reported in TMS scale.

[0225] Powder X-ray diffraction: The powder was analyzed by using a Cu-K α1 Powder X-ray diffraction was performed on a Stoe Stadi P diffractometer operated with radiation. Transmission measurements were performed with this instrument at 40 kV tube voltage and 40 mA tube power. A bent Ge monochromator allowed the use of Cu-Kα1 The test was performed 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 in step scan). For a typical sample preparation, about 10 mg of sample was placed between two acetate foils and mounted into a Stoe transmission sample holder. The sample was rotated during the measurement. All sample preparation and measurements were performed in an ambient air atmosphere.

[0226] Raman spectroscopy: FT-Raman spectra were recorded on a Bruker MultiRAM FT-Raman system with a near-infrared Nd:YAG laser operating at 1064 nm and a liquid nitrogen-cooled germanium detector. -1 The cumulative resolution within the range is 2cm -1 64 scans of 100 cm were evaluated; however, due to filter cutoff effects, only 100 cm -1 The above data. The nominal laser power is usually 100 or 300mW.

[0227] Solubility: Approximate solubility is determined by incremental addition of solvent to approximately 10 mg of compound. If the material does not dissolve by adding a total of at least 10 ml of solvent, the solubility is indicated to be <1 mg / ml. Due to the experimental error inherent in this method, solubility values ​​are intended to be considered rough estimates and will only be used to design crystallization experiments.

[0228] TG-FTIR: Thermogravimetric measurements were performed with a Netzsch thermomicrobalance TG 209 coupled to a Bruker FTIR spectrometer Vector 22 (sample pan with small hole, N2 atmosphere, heating rate 10 K / min).

[0229] HPLC: HPLC was performed using an Agilent 1100 series instrument with UV detection and a Waters XTerra MS C18, 100x4.6 mm, 5 um, (FK-CC01H) column using the following parameters:

[0230]

[0231]

[0232] Example 1

[0233] Amorphous compound (I).

[0234] A sample of compound (I) obtained by synthesis and purification as described in WO2020 / 190765 was characterized by various techniques and determined to be amorphous. Optical microscopy images showed glassy particles, and the powder x-ray diffraction (PXRD) pattern showed no sharp reflections. Thermogravimetric analysis coupled with FT-IR spectroscopy showed that the sample contained about 1.4% water. Some water appeared to be encapsulated in the void spaces of the amorphous particles and released above its boiling point. The sample decomposed at temperatures above about 200°C. The differential scanning calorimetry (DSC) thermogram 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 approximately the same amount of water as during the TG-FTIR measurement, i.e., approximately 1.5%, while reaching a water content of about 15% at the high rh end during the cycle. Therefore, the amorphous form is considered to be hygroscopic. The moisture content at the end of the test was about 6.4%, which was significantly higher than the moisture content at the beginning. Powder X-ray diffraction of the sample recovered after DVS revealed that the sample had not changed during the DVS experiment.

[0235] The approximate solubility of amorphous compound (I) is determined at room temperature. These values ​​are obtained by adding a small amount of solvent aliquots to a small amount of solid to achieve dissolution by stirring or agitation for a short time. These values ​​are only approximate and do not necessarily correspond to thermodynamic solubility values. The ratio of the solvent mixture corresponds to the volume ratio.

[0236] Initially, the compound (I) in amorphous form was tested, and then, after finding stable crystalline form D (Example 7 below), some tests were repeated with the crystalline form. Amorphous compound (I) is highly soluble in common organic solvents such as acetic acid, ketones, alcohols, esters and dichloromethane and their mixtures. Anti-solvents are water, heptane and TBME. It was found that the solubility of crystalline form D in acetonitrile and ethyl acetate is significantly lower than that of amorphous form. Based on available data and further experiments, diisopropyl ether, 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.

[0237] Table 1.

[0238]

[0239]

[0240] Example 2

[0241] Suspension balance experiment

[0242] Suspension equilibrium experiments are run to obtain thermodynamically stable forms (polymorphs or solvates) in a given solvent system. Compound (I) is suspended in an appropriate amount of a specified solvent or solvent mixture. Amorphous and crystalline form D are both used as starting materials. In non-aqueous systems, crystalline form D is produced from amorphous form, and when form D is the starting material, no new form is produced. In aqueous systems, form A or form B is produced. Table 2 provides a summary of suspension equilibrium experiments of amorphous form and form D.

[0243] Table 2

[0244]

[0245] Example 3

[0246] Evaporation experiment

[0247] When crystallization failed in some solution experiments, evaporation experiments were performed. In some cases, crystallization was achieved when new (anti) solvents were added and suspension equilibrium experiments were performed. The results of some of these evaporation experiments are summarized in Table 3.

[0248] Table 3

[0249]

[0250]

[0251] Example 4

[0252] Crystalline Form A

[0253] The first crystalline form of compound (I) was designated as Form A and was obtained from suspension equilibrium experiments of an amorphous form in an isopropanol-water mixture. Form A was characterized by PXRD, TG-FTIR, H-NMR and DVS. Highly crystalline samples of Form A typically contain a large amount of solvent and drying results in a significant loss of crystallinity. It is speculated that drying samples to residual solvent levels below the ICH Q3C limit may result in completely amorphous material.

[0254] Figure 1The PXRD pattern of Form A is depicted in . It should be noted that the sample was examined by PXRD before drying, and the undried sample was highly crystalline in nature. The PXRD pattern shows strong and sharp reflections. The TG-FTIR of the crystalline Form A sample that was not dried extensively showed a large mass loss of 36.6% attributable to water and isopropanol. In view of 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% and it still contained some water and isopropanol. The H-NMR spectrum also showed about 0.1 equivalents of isopropanol, which would correspond to about 1% and was in good agreement with the TG-FTIR results. However, the PXRD measurement of the dried sample showed that there was almost no substantially amorphous material with the strongest peak. Table 4 below shows a peak list of the crystalline Form A of compound (I).

[0255] Table 4

[0256]

[0257]

[0258] Form A can be reliably produced by suspension equilibrium in water-saturated TBME. TG-FTIR was performed after Form A was kept in a desiccator at 75% relative humidity, and the resulting mass loss was 8.9%, which can be attributed to water and some TBME. The sample was further examined by dynamic water vapor sorption, which showed that compound (I) only absorbed a small amount of water above 50% relative humidity, and lost about 8% of water below 50% relative humidity during the cycle. A small step was observed between 0% relative humidity and 20% relative humidity. Water was not completely removed during the first DVS cycle at 0% relative humidity, and this may be the reason why Form A was retained after the DVS test was completed, as demonstrated by PXRD of the post-DVS sample.

[0259] The water content varies over the entire range of relative humidity; ie 0% to 95% corresponds approximately to three waters (10%) per compound (I); however, in view of the poor stability of hydrates, no fixed stoichiometric ratio is specified in this case.

[0260] Example 5

[0261] Crystalline Form B

[0262] 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 from methanol-water as a solvent system and by suspension equilibrium experiments in pure water.

[0263] The PXRD pattern of Form B is shown in Figure 2a, and a peak list is provided in Table 5 below. Like Form A, Form B was shown to be highly crystalline by PXRD inspection before drying. The PXRD pattern shows strong and sharp reflections. Form B was subjected to TG-FTIR without further drying, and the thermogram showed a large mass loss of 36.5% attributable to water; however, no alcohol was detected. In view of the large mass loss, it was concluded that the drying was insufficient, and the sample was subjected to further vacuum drying for ten minutes at ambient temperature, and a new TG-FTIR was recorded. The new TG-FTIR thermogram showed that the water content was about 4.6%. The PXRD measurement of the dried sample showed a significant loss of crystallinity and further drying resulted in a completely amorphous material.

[0264] Table 5

[0265]

[0266]

[0267] Form B was examined by dynamic vapor sorption (DVS). When a wet sample was used, there was a large initial mass loss at 50% relative humidity, i.e., at the beginning of the DVS test. DVS showed that once the sample was at 50% relative humidity, Form B absorbed only a small amount of water above 50% relative humidity. The total water content changed by about 13% during the cycle. At the end of the DVS test, the sample was recovered from the sample pan and examined by TG-FTIR and PXRD. The PXRD pattern after DVS showed a loss of crystallinity. The TG-FTIR thermogram showed a water content of about 6.4%. Raman spectra were also obtained and are shown in Figure 2b.

[0268] Example 6

[0269] Crystalline Form C

[0270] Recrystallization from a solution of 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.

[0271] The PXRD pattern of Form C is shown in Figure 3, and a select list is provided in Table 6 below. Like Forms A and B, Form C was examined by PXRD before drying and showed to be highly crystalline in nature. The PXRD pattern showed strong and sharp reflections. TG-FTIR was performed without further drying, and the thermogram showed a large mass loss of 48% attributable to water. Given the large mass loss, it was concluded that drying was inadequate; however, in this case, no further drying experiments were performed.

[0272] Table 6

[0273]

[0274]

[0275] The DVS results of Form C are similar to those of Forms A and B. When a wet sample is used, there is a large initial mass loss at 50% relative humidity, i.e., at the beginning of the DVS test. Dynamic water vapor sorption shows that when the sample is slightly dried (i.e., dried at 50% relative humidity), it only absorbs a small amount of water above 50% relative humidity. The total moisture content changes by about 14% during the cycle, and the moisture content at the end is about 5.9%, while completely dried material is not obtained at 0% relative humidity (i.e., for five hours under nitrogen). At the end of the DVS test, the sample is recovered from the sample tray and examined by TG-FTIR and PXRD. PXRD after DVS shows that the crystallinity is almost completely lost. The TG-FTIR thermogram shows that the moisture content is about 5.9%.

[0276] Example 7

[0277] Crystalline Form D

[0278] During the polymorphic studies carried out in batches, amorphous samples are often obtained. The crystalline forms A, B and C found show poor physical form stability. During the salt amplification experiment, a new crystalline form of the free base was found when starting with a new batch. Based on approximate solubility data, amorphous compound (I) has high solubility in ethyl acetate. When attempting to obtain mesylate from a solution of the free base in ethyl acetate by adding methanesulfonic acid, a suspension is formed before adding acid. The solid product separated from the suspension is crystalline and shows a new PXRD pattern. This new crystalline form of the free base is designated as form D, and is further characterized by the chemical purity determined by HPLC, FT-Raman, TG-FTIR, DSC, DVS, and the solubility in pure water and various buffer solutions. Form D is a non-solvate form, and is preferably prepared with anhydrous solvents and solvent mixtures. Solubility tests are also carried out in possible process solvent mixtures.

[0279] Form D is generally obtained from crystallization experiments of the solutions listed in Table 7. These experiments are generally combined with phase equilibration; that is, the suspension obtained is not immediately filtered. Since the discovery of a stable form is considered important, the suspension is stirred for several days.

[0280] Table 7

[0281]

[0282]

[0283] The powder X-ray diffraction pattern of Compound (I) (free base) Form D is shown in Figure 4a and the peak list is provided in Table 8 below.

[0284] Table 8

[0285]

[0286]

[0287] The purity of the new Form D was checked by a common standard HPLC method. The purity was found to be 99.4% (98.1% relative to the amorphous starting material). The Fourier transform (FT)-Raman spectrum of Form D is shown in Figure 4b.

[0288] A TG-FTIR thermogram was recorded for Form D, which indicated that it was a solvent-free form, since it showed only about 0.3% residual ethyl acetate despite the sample being tested after brief drying in air at ambient temperature. DSC revealed a fairly sharp melting endotherm with a peak maximum at 153°C and an enthalpy signal of about 69 J / g.

[0289] Solubility testing was performed in potential process solvent mixtures with Form D. The results are shown in Table 9 below.

[0290] Table 9

[0291]

[0292] DVS was performed on Form D. At 95% relative humidity, Form D absorbed up to 0.8% of water, and when the humidity was scanned back to 0% relative humidity, the sample lost all of its water.

[0293] Aqueous solubility was tested on the amorphous form and Form D of Compound 1. The results are shown in Table 10 below.

[0294] Table 10

[0295]

[0296] pH dependent solubility tests were performed in 0.1 M HCl, in citrate buffer and in USP phosphate buffer with nominal pH values ​​of 6.8 and 7.4. The solubility test failed at pH values ​​below 5 because all compounds dissolved during the equilibrium phase. Approximate tests showed that 55 mg of compound was easily dissolved in 1.0 ml of 0.1 M HCl. The results are summarized in Table 11 below.

[0297] Table 11

[0298]

[0299]

[0300] All documents mentioned herein are incorporated by reference in their entirety.

Claims

1. A polymorph of a free base of compound (I) having the following structure: Wherein the polymorph is: (1) Form A, which has an X-ray diffraction pattern having 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; (2) Form B, which has an X-ray diffraction pattern having 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; (3) Form C, which has an X-ray diffraction pattern having 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; or (4) Form D, which has an X-ray diffraction pattern having 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.

2. The polymorph of claim 1, wherein the polymorph is Form A, and Form A has an X-ray diffraction pattern having characteristic peaks characterized by °2θ 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.

3. The polymorph of claim 1, wherein the polymorph is Form C, and the X-ray diffraction pattern of Form C has characteristic peaks characterized by °2θ values ​​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, 24.3±0.2, about 24.6±0.2, and about 26.1±0.

2.

4. The polymorph of claim 1, wherein the polymorph is Form D, and the X-ray diffraction pattern of Form D has characteristic peaks characterized by °2θ values ​​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.

5. The polymorph of claim 1, wherein the polymorph is Form D, and the Form D has an X-ray diffraction pattern having characteristic peaks characterized by °2θ 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.

6. The polymorph of claim 1, wherein the polymorph is Form D having a melting point of about 153°C.

7. A method of forming the Form A polymorph of Compound (I) according to claim 1 or 2, the method comprising one of the following: (a) crystallizing an amorphous compound (I) in isopropanol:water (1:1), and equilibrating the crystallized amorphous compound (I); (b) suspending the equilibrated Form D in acetonitrile:water (9:1); or (c) Suspension equilibrium of amorphous compound (I) in water-saturated TBME.

8. A method of forming the Form B polymorph of Compound (I) according to claim 1, the method comprising one of the following: (a) suspending and equilibrating the amorphous compound (I) in water:ethanol (2:1); or (b) Suspended in equilibrium form D in water.

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

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

11. The method of claim 10, comprising one of the following: (a) slurrying in ethyl acetate; (b) seeding with Form D in ethyl acetate; (c) slurrying in acetonitrile; (d) Suspension equilibrium in isopropyl acetate:cyclohexane (1:2); (e) Suspension equilibrium in methyl ethyl ketone (MEK): diisopropyl ether; (f) suspending and balancing in methyl ethyl ketone (MEK): p-xylene (1:2) and partially evaporating the solvent; (g) equilibrated in methylTHF:methylcyclohexane (1:2); (h) equilibrated in isopropyl acetate:heptane (2:3); (i) Equilibrated in isopropyl acetate:heptane (1:1); (j) Suspension equilibrium in n-butyl acetate:heptane (5:2); (k) suspended in TBME and seeded with Form D; (l) Suspension equilibrium in heptane:MIBK (4:1); (m) Suspension equilibrium in methanol:methylcyclohexane (1:4); (n) Suspension equilibrium in trimethylamine; or (o) Suspended in diisopropyl ether.

12. A method of forming the Form D polymorph of Compound (I) according to any one of claims 1 or 4 to 6, the method 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).

13. A method for purifying compound (I): The method comprises preparing crystals of polymorph Form A and isolating the crystals of polymorph Form A.

14. A method for purifying compound (I): The method comprises preparing crystals of polymorph Form B and isolating the polymorph Form B crystals.

15. A method for purifying compound (I): The method comprises preparing crystals of polymorph Form C and isolating the polymorph Form C crystals.

16. A method for purifying compound (I): The method comprises preparing crystals of polymorph Form D and isolating the polymorph Form D crystals.

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

18. A method of treating a subject suffering from cancer, the method comprising administering to the subject one or more polymorphic forms A, B, C or D of compound (I) according to any one of claims 1 to 6.

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

20. The method of claim 18 or 19, wherein the cancer is brain cancer.

21. Use of one or more polymorphic forms A, B, C or D of compound (I) according to any one of claims 1 to 6 in the manufacture of a medicament for treating a subject suffering from cancer.

22. The use of claim 21, wherein the cancer is characterized by aberrant EGFR expression.

23. The use according to claim 21 or 22, wherein the cancer is brain cancer.

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