Novel crystalline form of pol1 inhibitor

Abstract

To provide a process for producing a single crystalline form of a compound having a specific biological activity, a single crystal, a pharmaceutical composition, and its use in a method for treating cancer.SOLUTION: A process for producing a single crystalline form of N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-a][1,8]naphthyridine-6-carboxamide (compound (I)), being form 1, wherein the process comprises preparing a mixture of polymorphic compound (I) in acetone, and subsequently isolating the resulting crystalline form 1 from the acetone, wherein the form 1 has the same powder X-ray diffraction pattern (PXRD) as that shown in the figure.SELECTED DRAWING: Figure 81

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 62 / 477,746, filed March 28, 2017, and U.S. Provisional Patent Application No. 62 / 491,635, filed April 28, 2017, each of which is incorporated by reference in its entirety herein.

[0002] The present invention relates, in part, to novel crystalline forms of N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-a][1,8]naphthyridine-6-carbaximide having specific biological activity, including, but not limited to, inhibiting cell proliferation and / or inducing cell apoptosis. The present invention also relates, in part, to methods of using such forms, methods for their preparation, and compositions thereof. Summary of the Invention

[0003] In one embodiment, compound (I):

[0004] [ka] A solid form of the compound is provided.

[0005] In one variation, the solid form is a crystalline form. In one variation, the crystalline form is specifically described herein (e.g., Forms 1-5).

[0006] Certain crystalline forms described herein may be advantageous over known solid forms of Compound (I) due to improved handling properties, reduced tendency to aggregate, improved suitability for formation of cohesive solid forms (tablets), improved long-term storage stability, better solubility, and / or improved solubility profile.

[0007] (Crystalline form) (Form 1) In one variation, the crystalline form of Compound (I) is a crystalline form of Form 1. In certain embodiments, crystalline Form 1 is advantageous because it can be prepared consistently, while other crystalline forms can be converted to crystalline Form 1 upon storage or handling. Indeed, some other forms can be readily converted to crystalline Form 1. The conditions for such interconversion are described herein. Consistent physical properties greatly facilitate the handling and preparation of solid dosage forms at a production scale. Therefore, it is advantageous to use a crystalline form that is stable under normal handling conditions. Thus, in one embodiment, crystalline Form 1 is utilized.

[0008] In some embodiments, crystalline Form 1 is characterized by having an X-ray powder diffraction pattern containing peaks at about 10.0° and 20.7° 2-theta.

[0009] In some embodiments, crystalline Form 1 is characterized by having any one or combination of X-ray powder diffraction pattern peaks at about 6.7°, 8.6°, 10.0°, 11.2°, 16.2°, 20.7°, 22.7°, 25.6°, and 27.6° 2-theta (±0.2° 2θ). In some embodiments, crystalline Form 1 is characterized by having an X-ray powder diffraction pattern substantially as shown in FIG.

[0010] In some embodiments, crystalline Form 1 is characterized by having an endothermic peak at about 212-216° C. as shown by differential scanning calorimetry. In some embodiments, crystalline Form 1 is characterized by having a differential scanning calorimetry pattern substantially as shown in FIG.

[0011] In some embodiments, crystalline Form 1 is characterized by having a thermogravimetric analysis substantially as shown in FIG.

[0012] (Form 2) In one variation, the crystalline form of Compound (I) is a crystalline form of Form 2. In some embodiments, crystalline Form 2 is characterized by having an X-ray powder diffraction pattern comprising peaks at about 20.3° and 26.4° 2-theta.

[0013] In some embodiments, crystalline Form 2 is characterized by having an X-ray powder diffraction pattern comprising peaks at about 6.7°, 11.2°, 17.5°, 19.5°, 20.3°, 21.7°, and 26.4° (±0.2) 2θ. In some embodiments, crystalline Form 2 is characterized by having an X-ray powder diffraction pattern substantially as shown in FIG.

[0014] In some embodiments, crystalline Form 2 is characterized by having a differential scanning calorimetry pattern substantially as shown in FIG.

[0015] In some embodiments, crystalline Form 2 is characterized by having a thermogravimetric analysis substantially as shown in FIG.

[0016] (Form 3) In one variation, the crystalline form of Compound (I) is crystalline form Form 3. In some embodiments, crystalline Form 3 is characterized by having an X-ray powder diffraction pattern comprising peaks at about 6.4° and 13.5° 2-theta.

[0017] In some embodiments, crystalline Form 3 is characterized by having an X-ray powder diffraction pattern comprising peaks at about 6.4°, 9.3°, 9.7°, 13.5°, 19.1°, 23.7°, and 28.0° (±0.2 2θ) 2-theta. In some embodiments, crystalline Form 3 is characterized by having an X-ray powder diffraction pattern substantially as shown in FIG.

[0018] In some embodiments, crystalline Form 3 is characterized by having a differential scanning calorimetry pattern substantially as shown in FIG.

[0019] In some embodiments, crystalline Form 3 is characterized by having a thermogravimetric analysis substantially as shown in FIG.

[0020] (Form 4) In one variation, the crystalline form of Compound (I) is crystalline form Form 4. In some embodiments, crystalline Form 4 is characterized by having an X-ray powder diffraction pattern comprising peaks at about 7.0° and 11.4° 2θ.

[0021] In some embodiments, crystalline Form 4 is characterized by having an X-ray powder diffraction pattern comprising peaks at about 6.3°, 7.0°, 9.3°, 9.7°, 11.4°, 19.1°, 25.6°, and 27.9° 2θ (±0.2° 2θ). In some embodiments, crystalline Form 4 is characterized by having an X-ray powder diffraction pattern substantially as shown in FIG.

[0022] In some embodiments, crystalline Form 4 is characterized by having a differential scanning calorimetry pattern substantially as shown in FIG.

[0023] In some embodiments, crystalline Form 4 is characterized by having a thermogravimetric analysis substantially as shown in FIG.

[0024] (Form 5) In one variation, the crystalline form of Compound (I) is crystalline form 5. In some embodiments, crystalline form 5 is characterized by having an X-ray powder diffraction pattern comprising peaks at about 9.2° and 10.3° 2-theta.

[0025] In some embodiments, crystalline Form 5 is characterized by having an X-ray powder diffraction pattern including peaks at about 9.2°, 10.3°, 11.2°, 18.1°, 19.7°, 21.2°, 23.6°, and 25.8° 2-theta (±0.2° 2θ). In some embodiments, crystalline Form 5 is characterized by having an X-ray powder diffraction pattern substantially as shown in FIG.

[0026] In some embodiments, crystalline Form 5 is characterized by an endothermic peak at about 212-213.5° C. as shown by differential scanning calorimetry. In some embodiments, crystalline Form 5 is characterized by having a differential scanning calorimetry pattern substantially as shown in FIG.

[0027] In some embodiments, crystalline Form 5 is characterized by having a thermogravimetric analysis substantially as shown in FIG.

[0028] (Manufacturing method) In one embodiment, a process for producing a crystalline form of compound (I) is provided. In one variation, the crystalline form of compound (I) is any crystalline form described herein (e.g., crystalline form 1, 2, 3, 4, and 5). In certain embodiments, the crystalline form of compound (I) is crystalline form 1 or crystalline form 5.

[0029] (Form 1) In one variation, a process for producing crystalline Form 1 is provided that includes preparing a mixture (e.g., a slurry) of multiphasic Compound (I) in acetone. In some embodiments, the mixture is temperature cycled (e.g., from about 5° C. to about 25° C. for a period of about 72 hours).

[0030] (Form 2) In one variation, a process for making crystalline Form 2 is provided that includes preparing a mixture (e.g., a slurry) of Compound (I) in 1,4-dioxane. In some embodiments, the mixture is temperature cycled (e.g., from about 5° C. to about 25° C. for a period of about 72 hours).

[0031] (Form 3) In one variation, a manufacturing process is provided for producing crystalline Form 3, comprising preparing a mixture (e.g., a slurry) of Compound (I) in dichloromethane. In some embodiments, the mixture is temperature cycled (e.g., from about 5° C. to about 25° C. for a period of about 72 hours).

[0032] (Form 4) In one variation, a process for preparing crystalline Form 4 is provided, comprising dissolving Compound (I) in dichloromethane. In some embodiments, the resulting suspension is filtered and allowed to evaporatively crystallize under ambient conditions in an uncapped vial.

[0033] (Form 5) In one variation, there is provided a process for preparing crystalline Form 5, comprising dissolving Compound (I) in dichloromethane. In some embodiments, heptane is added to the resulting suspension to precipitate the material from the solution.

[0034] (composition) In one aspect, a composition is provided that comprises the crystalline form of compound (I) described herein.In one variation, a crystalline form (e.g., crystalline form 1, 2, 3, 4, and 5) described herein and a pharmaceutically acceptable carrier are provided.In some embodiments, the composition comprises an effective amount of the crystalline form and a pharmaceutically acceptable carrier.

[0035] (Method of processing) In one aspect, a method of inhibiting cell proliferation is provided, comprising contacting a cell with an effective amount of a form of Compound (I), which is a crystalline form of Compound (I) (e.g., crystalline forms 1, 2, 3, 4, and 5). In some embodiments, the cell is a cancer cell line. In some embodiments, the cancer cell line is a breast cancer, prostate cancer, pancreatic cancer, lung cancer, hematopoietic tissue cancer, colorectal cancer, skin cancer, ovarian cancer cell line. In some embodiments, the cell is in a tumor of a subject. In some embodiments, contacting the cell induces cell apoptosis.

[0036] In one aspect, a method of treating a condition associated with abnormal cell proliferation is provided, the method comprising administering to an individual in need thereof an effective amount of a crystalline form (e.g., crystalline forms 1, 2, 3, 4, and 5) described herein. In some embodiments, the cell proliferation condition is a tumor-associated cancer. In some embodiments, the cancer is breast, prostate, pancreatic, lung, colorectal, skin, or ovarian cancer. In some embodiments, the cell proliferation condition is a non-tumor cancer. In some embodiments, the non-tumor cancer is a hematopoietic tissue cancer.

[0037] In one embodiment, a method is provided for inhibiting angiogenesis in an individual in need thereof, comprising administering to the individual an effective amount of a crystalline form described herein (e.g., crystalline forms 1, 2, 3, 4, and 5).

[0038] In one aspect, a method is provided for treating cancer or an inflammatory disorder in an individual in need thereof, comprising administering to the individual an effective amount of a crystalline form described herein (e.g., crystalline forms 1, 2, 3, 4 and 5) and administering an additional pharmaceutical agent, an additional therapeutic modality, or a combination thereof.

[0039] In some embodiments, the crystalline form and the additional pharmaceutical agent are combined in one pharmaceutical composition. In some embodiments, the administration of the additional pharmaceutical agent, the additional therapeutic modality, or a combination thereof is carried out simultaneously with the administration of the crystalline form. In some embodiments, the administration of the additional pharmaceutical agent, the additional therapeutic modality, or a combination thereof is carried out after the administration of the crystalline form. In some embodiments, the administration of the additional pharmaceutical agent, the additional therapeutic modality, or a combination thereof is carried out before the administration of the crystalline form.

[0040] (kit) In one aspect, a kit for treating or preventing an individual having cancer is provided that includes a crystalline form of Compound (I) described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) and packaging.

[0041] In one variation, a kit for the treatment or prevention in an individual with cancer is provided comprising a composition of the crystalline form described herein and packaging. [Brief description of the drawings]

[0042] Further objects and advantages will become apparent from a consideration of the description, drawings, and examples. [Figure 1] FIG. 1 shows the powder X-ray diffraction pattern (PXRD) of the polyphasic form of N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-a][1,8]naphthyridine-6-carbaximide (Compound (I)). [Diagram 2] FIG. 2 shows polarized light microscopy (PLM) results for multiphasic N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-a][1,8]naphthyridine-6-carbaximide (Compound (I)). [Diagram 3] FIG. 3 shows the thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) thermograms of the multiphasic form of N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-a][1,8]naphthyridine-6-carbaxamide (Compound (I)). [Figure 4] FIG. 4 shows a differential scanning calorimetry (DSC) thermogram (first heating cycle) of multiphasic N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-a][1,8]naphthyridine-6-carbaxamide (Compound (I)). [Diagram 5] FIG. 5 shows a differential scanning calorimetry (DSC) thermogram (second heating cycle) of multiphasic N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-a][1,8]naphthyridine-6-carbaxamide (Compound (I)). [Figure 6]FIG. 6 shows gravimetric vapor sorption (GVS) data for multiphasic N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-a][1,8]naphthyridine-6-carbaximide (Compound (I)). [Figure 7] FIG. 7 shows the powder X-ray diffraction pattern (PXRD) of the undissolved material from the solubility screen described in Example 4. [Figure 8] FIG. 8 shows a powder X-ray diffraction pattern (PXRD) of a solvent slurry of the multiphasic form of N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-a][1,8]naphthyridine-6-carbaxamide (Compound (I)) that produced Form 1. [Figure 9] FIG. 9 shows the powder X-ray diffraction patterns (PXRD) of N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-a][1,8]naphthyridine-6-carbaxamide (Compound (I)) in multiple phases (starting material); Form 2 produced in 1,4-dioxane slurry; and Form 3 produced in dichloromethane slurry. [Figure 10] FIG. 10 shows the powder X-ray diffraction pattern (PXRD) of N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-a][1,8]naphthyridine-6-carbaxamide (Compound (I)) in a multiphase form (starting material); and Form 4 produced by a dichloromethane evaporation crystallization process. [Figure 11] FIG. 11 shows the powder X-ray diffraction patterns (PXRD) of N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-a][1,8]naphthyridine-6-carbaxamide (Compound (I)) in multiphasic form (starting material); and form 5 produced by the dichloromethane / heptane anti-solvent combination process. [Figure 12]FIG. 12 shows the powder X-ray diffraction patterns (PXRD) of the polyphasic forms (starting material) of N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-a][1,8]naphthyridine-6-carbaxamide (Compound (I)); and Form 3 from evaporated dichloromethane cooled crystallization. [Figure 13] FIG. 13 shows the powder X-ray diffraction pattern (PXRD) of the multiphase form of N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-a][1,8]naphthyridine-6-carbaximide (Compound (I)) after repeated dichloromethane evaporation crystallization experiments. [Figure 14] FIG. 14 shows the thermogravimetric analysis (TGA) and differential thermal analysis (DTA) of Form 3 from the primary screen. [Figure 15] FIG. 15 shows a differential scanning calorimetry (DSC) thermogram (first heating cycle) of Form 3 from the primary crystal morphology screen. [Figure 16] FIG. 16 shows a differential scanning calorimetry (DSC) thermogram (cooling cycle) of Form 3 from the primary crystal morphology screen. [Figure 17] FIG. 17 shows a differential scanning calorimetry (DSC) thermogram (second heating cycle) of Form 3 from the primary crystal morphology screen. [Figure 18] FIG. 18 shows the powder X-ray diffraction pattern (PXRD) of Form 1 from an acetone slurry in the primary crystal morphology screen (top); and the powder X-ray diffraction pattern (PXRD) of Form 1 from an acetone slurry in the secondary crystal morphology screen (bottom). [Figure 19] FIG. 19 shows polarized light microscopy (PLM) results for Form 1 before drying. [Figure 20] FIG. 20 shows polarized light microscopy (PLM) results of Form 1 after drying. [Figure 21] FIG. 21 shows the thermogravimetric analysis (TGA) and differential thermal analysis (DTA) of Form 1. [Figure 22] FIG. 22 shows a differential scanning calorimetry (DSC) thermogram of Form 1 (first heating cycle). [Figure 23] FIG. 23 shows a differential scanning calorimetry (DSC) thermogram of Form 1 (second heating cycle). [Figure 24] FIG. 24 shows the 1H nuclear magnetic resonance (NMR) analysis of Form 1 (CDCl3, 500 MHz). [Diagram 25] FIG. 25 shows the powder X-ray diffraction pattern (PXRD) of Form 2 from the primary crystal morphology screen (top); the powder X-ray diffraction pattern (PXRD) of Form 2 after 72 hours of slurrying (middle); and the powder X-ray diffraction pattern (PXRD) of Form 2 after 144 hours of slurrying (bottom). [Figure 26] FIG. 26 shows polarized light microscopy (PLM) results for Form 2 before drying. [Figure 27] FIG. 27 shows polarized light microscopy (PLM) results of Form 2 after drying. [Figure 28] FIG. 28 shows the thermogravimetric analysis (TGA) and differential thermal analysis (DTA) of Form 2. [Figure 29] FIG. 29 shows a differential scanning calorimetry (DSC) thermogram of Form 2 (first heating cycle). [Diagram 30] FIG. 30 shows a differential scanning calorimetry (DSC) thermogram of Form 2 (second heating cycle). [Diagram 31] FIG. 31 shows the 1H NMR analysis of Form 2 (CDCl3, 500 MHz). [Diagram 32] FIG. 32 shows the powder X-ray diffraction pattern (PXRD) of Form 3 generated from a DCM slurry of the primary screen (top); and the powder X-ray diffraction pattern (PXRD) of Form 3 generated from a DCM slurry of the secondary screen (bottom). [Diagram 33] FIG. 33 shows polarized light microscopy (PLM) results for Form 3 before drying. [Diagram 34] FIG. 34 shows polarized light microscopy (PLM) results of Form 3 after drying. [Diagram 35] FIG. 35 shows polarized light microscopy (PLM) results of Form 3 after drying. [Diagram 36] FIG. 36 is a TG / DTA thermogram of Form 3. [Figure 37] FIG. 37 shows a differential scanning calorimetry (DSC) thermogram of Form 3 (first heating cycle). [Figure 38] FIG. 38 shows a differential scanning calorimetry (DSC) thermogram of Form 3 (second heating cycle). [Figure 39] FIG. 39 shows the 1H NMR analysis of Form 3 (CDCl3, 500 MHz). [Diagram 40] FIG. 40 shows the powder X-ray diffraction pattern (PXRD) of Form 4 generated from a primary screen and repeated DCM evaporations (top 3); and the powder X-ray diffraction pattern (PXRD) of Form 4 generated from a DCM slurry of a secondary screen (bottom). [Diagram 41] FIG. 41 shows polarized light microscopy (PLM) results for Form 4. [Diagram 42] FIG. 42 shows the thermogravimetric analysis (TGA) and differential thermal analysis (DTA) of Form 4. [Diagram 43] FIG. 43 shows a differential scanning calorimetry (DSC) thermogram of Form 4 (first heating cycle). [Diagram 44] FIG. 44 shows a differential scanning calorimetry (DSC) thermogram of Form 4 (second heating cycle). [Diagram 45] FIG. 45 shows the 1H NMR analysis (CDCl3, 500 MHz) of Form 4. [Diagram 46] Figure 46 shows the powder X-ray diffraction pattern (PXRD) of Form 5 generated from a DCM anti-solvent primary screen and repeat (top); the powder X-ray diffraction pattern (PXRD) of Form 5 generated from a DCM anti-solvent secondary screen (middle); and the powder X-ray diffraction pattern (PXRD) of Form 5 generated from a DCM anti-solvent secondary screen the next day (bottom). [Figure 47] FIG. 47 shows polarized light microscopy (PLM) results of Form 5 after drying. [Figure 48] FIG. 48 shows the thermogravimetric analysis (TGA) and differential thermal analysis (DTA) of Form 5. [Figure 49] FIG. 49 shows a differential scanning calorimetry (DSC) thermogram of Form 5 (first heating cycle). [Figure 50] FIG. 50 shows a differential scanning calorimetry (DSC) thermogram of Form 5 (second heating cycle). [Figure 51] FIG. 51 shows the 1H NMR analysis (CDCl3, 500 MHz) of Form 5. [Figure 52] FIG. 52 shows the powder X-ray diffraction patterns (PXRD) of Forms 1-5 before and after stability testing. [Figure 53] FIG. 53 shows powder X-ray diffraction patterns (PXRD) from competing slurries of Form 1 and Form 5. [Figure 54] FIG. 54 shows the differential scanning calorimetry (DSC) thermograms when the competing slurry material is heptane at room temperature. [Figure 55] FIG. 55 shows a differential scanning calorimetry (DSC) thermogram when the competing slurry material is heptane at 60° C. [Figure 56] FIG. 56 shows differential scanning calorimetry (DSC) thermograms where the competing slurry material is methanol at room temperature. [Figure 57] FIG. 57 shows the differential scanning calorimetry (DSC) thermogram when the competing slurry material is methanol at 60° C. [Figure 58] FIG. 58 shows differential scanning calorimetry (DSC) thermograms when the competing slurry material is dichloromethane (DCM) at room temperature. [Figure 59] FIG. 59 shows a differential scanning calorimetry (DSC) thermogram when the competing slurry material is dichloromethane (DCM) at 35° C. [Figure 60] FIG. 60 shows the powder X-ray diffraction patterns (PXRD) of the dichloromethane competitive slurry at room temperature before drying (top); the dichloromethane competitive slurry at room temperature after drying (third from the top); and crystalline forms 1, 3 and 5 as references (second from the bottom, second from the top, and bottom, respectively). [Figure 61] Figure 61 shows the powder X-ray diffraction pattern (PXRD) of Form 1 from the secondary screen (top) and the large scale up (bottom). [Figure 62] FIG. 62 shows polarized light microscopy (PLM) results of Form 1 from large scale up after drying. [Figure 63] FIG. 63 shows the thermogravimetric analysis (TGA) and differential thermal analysis (DTA) of Form 1 from large scale scale up. [Figure 64] FIG. 64 shows a differential scanning calorimetry (DSC) thermogram of Form 1 from large scale scale-up (first heating cycle). [Figure 65] FIG. 65 shows the differential scanning calorimetry (DSC) thermogram (cooling cycle) of Form 1 from large scale scale-up. [Figure 66] FIG. 66 shows a differential scanning calorimetry (DSC) thermogram of Form 1 from large scale scale-up (second heating cycle). [Figure 67] FIG. 67 shows the 1H NMR analysis (CDCl3, 500 MHz) of Form 1 from large scale up. [Figure 68] Figure 68 shows powder X-ray diffraction patterns (PXRD) of Form 1 at week 0 (top); after 2 weeks at 40°C and 75% relative humidity (second from the top); after 4 weeks at 40°C and 75% relative humidity (third from the top); after 2 weeks at 50°C (fourth and fifth from the top); after 2 weeks at room temperature (sixth from the top); and after 4 weeks at room temperature (seventh from the top, or bottom). [Figure 69] FIG. 69 shows the 1H NMR analysis (CDCl3, 500 MHz) of Form 1 after 2 weeks of storage at 40° C. and 75% relative humidity. [Figure 70] FIG. 70 shows the 1H NMR analysis (CDCl3, 500 MHz) of Form 1 after 4 weeks of storage at 40° C. and 75% relative humidity. [Figure 71] FIG. 71 shows the 1H NMR analysis (CDCl3, 500 MHz) of Form 1 after storage at 50° C. for 2 weeks. [Figure 72] FIG. 72 shows the 1H NMR analysis (CDCl3, 500 MHz) of Form 1 after storage at 50° C. for 4 weeks. [Figure 73]FIG. 73 shows the 1H NMR analysis (CDCl3, 500 MHz) of Form 1 after two weeks of storage at room temperature. [Figure 74] FIG. 74 shows the 1H NMR analysis (CDCl3, 500 MHz) of Form 1 after 4 weeks of storage at room temperature. [Figure 75] FIG. 75 shows a high performance liquid chromatography (HPLC) purity analysis of Form 1 after two weeks at room temperature. [Figure 76] FIG. 76 shows a high performance liquid chromatography (HPLC) purity analysis of Form 1 after 4 weeks at room temperature. [Figure 77] FIG. 77 shows high performance liquid chromatography (HPLC) purity analysis of Form 1 after 2 weeks at 40° C. and 75% relative humidity. [Figure 78] FIG. 78 shows high performance liquid chromatography (HPLC) purity analysis of Form 1 after 4 weeks at 40° C. and 75% relative humidity. [Figure 79] FIG. 79 shows high performance liquid chromatography (HPLC) purity analysis of Form 1 after 2 weeks at 50° C. [Figure 80] FIG. 80 shows high performance liquid chromatography (HPLC) purity analysis of Form 1 after 4 weeks at 50° C. [Figure 81] FIG. 81 shows the powder X-ray diffraction pattern (PXRD) of Form 1. [Figure 82] FIG. 82 shows the powder X-ray diffraction pattern (PXRD) of Form 2. [Figure 83] FIG. 83 shows the powder X-ray diffraction pattern (PXRD) of Form 3. [Figure 84] FIG. 84 shows the powder X-ray diffraction pattern (PXRD) of Form 4. [Figure 85] FIG. 85 shows the powder X-ray diffraction pattern (PXRD) of Form 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Detailed Description of the Invention Other objects, features and advantages of the compounds, methods and compositions described herein will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments, are given for purposes of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0044] Some embodiments of the present invention are discussed in detail below. In describing the embodiments, specific terms are used for clarity. However, the present invention is not intended to be limited to the specific terms so selected. Those skilled in the art will recognize that other equivalent components can be used and other methods can be developed without departing from the broad concept of the present invention. All references cited anywhere in this specification are incorporated by reference as if each was incorporated individually.

[0045] As used herein, Compound (I):

[0046] JPEG2025060702000003.jpg53133 crystal forms have been described:

[0047] Compound (I) has specific biological activities, including but not limited to inhibiting cell proliferation and / or inducing cell apoptosis. Compound (I) and its pharmaceutically acceptable salts, prodrugs, active metabolites, cocrystals and pharmaceutically acceptable solvates can inhibit ribosome biogenesis by inhibiting POL1 transcription.

[0048] Methods for preparing the novel crystalline forms of Compound (I) and methods of using the same are also provided. Also provided are compositions comprising the above forms of Compound (I) in combination with other agents (e.g., one or more additional pharmaceutical agents) and methods of using the same in combination with other agents.

[0049] As is well known to those of skill in the art, variations in the salt form and / or crystal structure of a drug substance often impact the dissolution rate (which can affect bioavailability, etc.), manufacturability (e.g., ease of handling, ability to consistently prepare doses of known strengths), and stability (e.g., thermal stability, shelf life, etc.) of the pharmaceutical product, especially when formulated in a solid oral dosage form.

[0050] Through experimentation with numerous crystallization conditions (e.g., various solvents, solvent mixtures, various cooling rates, etc.), the production of the specific crystalline forms described herein is unpredictable, and therefore specific processes have been developed to consistently produce these crystalline forms. These methods have enabled the preparation and characterization of the novel crystalline forms disclosed herein. The processes for the preparation and characterization of these forms are described in more detail below. These crystalline forms of Compound (I) may have particularly desirable properties in the solid form, such as dissolution rate, absorption and stability.

[0051] For example, certain crystalline forms exhibit higher intrinsic solubility and / or faster dissolution rates than others, which may be advantageous for handling and formulation purposes.

[0052] In another aspect, crystalline forms of Compound (I) are provided, namely, crystalline forms 1, 2, 3, 4, and 5. Each of these is a crystalline form of Compound (I). These crystalline forms of Compound (I) are useful for preparing solid dosage forms of pharmaceutical compositions that exhibit the biological activity of Compound (I), including the efficacy of treating proliferative disorders as discussed herein. The novel crystalline forms may be more stable than polyphasic or amorphous forms of Compound (I).

[0053] In another aspect, the present invention provides methods of making particular crystalline forms of a salt of Compound (I), as further described herein.

[0054] In another aspect, methods are provided for treating conditions responsive to Compound (I) using the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5). Such conditions include pain, inflammation, angiogenesis, and cell proliferation (e.g., cancer).

[0055] In one aspect, described herein is a method for treating or preventing cancer in a mammal, comprising administering to a mammal in need thereof a therapeutically effective amount of a crystalline form of Compound (I) as described herein, or a pharma- ceutically acceptable salt, co-crystal, or solvate thereof. In another aspect, described herein is a method for treating or preventing inflammatory disease in a mammal, comprising administering to a mammal in need thereof a therapeutically effective amount of a crystalline form of Compound (I) as described herein, or a pharma-ceutically acceptable salt, co-crystal, or solvate thereof.

[0056] In yet another aspect, described herein is a method for treating or preventing a proliferative disorder in a mammal, comprising administering to a mammal in need thereof a therapeutically effective amount of a crystalline form of Compound (I) described herein or a pharma- ceutically acceptable salt, co-crystal, or solvate thereof. In another aspect, described herein is a method for treating or preventing a disease or disorder in a mammal, comprising administering to a mammal a therapeutically effective amount of a crystalline form of Compound (I) described herein.

[0057] In some embodiments, the methods, crystalline forms and compositions of Compound (I) described herein can be used as a mixture of two or more of these crystals; a mixture of crystalline forms is sometimes useful.In some embodiments, the methods and compositions described herein can be used as a mixture of one or more of these crystalline forms and additional pharmaceutical agents, as described herein.

[0058] Kits, compositions, combination therapies and unit dosage forms of the crystalline forms described herein are also provided.

[0059] (Abbreviations and definitions) The nomenclature of some of the compounds described herein can be specified using ChemDraw Ultra version 10.0, available from CambridgeSoft®.

[0060] As used herein, "amorphous" refers to a material with too little crystalline content to produce a discernible pattern by PXRD or other diffraction techniques. Glassy materials are considered amorphous. Amorphous materials do not have a true crystal lattice and are consequently glassy rather than a true solid, technically similar to a highly viscous non-crystalline liquid. Rather than a true solid, glass may be better described as a quasi-solid amorphous material. Thus, amorphous materials refer to quasi-solid glassy materials. Precipitation of compounds from solution is often affected by rapid evaporation of the solvent and may be prone to amorphous forms of the compound.

[0061] "Crystalline" as used herein refers to a material that contains certain compounds that may be hydrated and / or solvated or may form co-crystals and has sufficient crystalline content to show a distinguishable diffraction pattern by powder X-ray diffraction (PXRD) or other diffraction techniques. Crystalline materials are characterized by a number of additional analytical techniques, including infrared spectroscopy (Fourier transform IR (FTIR), differential scanning calorimetry (DSC), density, crystal group, solubility, etc.). Crystalline substances obtained from solvents by direct crystallization of a compound dissolved in solution or by interconversion of crystals obtained under different crystallization conditions may have crystals that contain the solvent. Specific solvent compositions and physical properties of crystallization (e.g., crystallization rate, temperature), collectively referred to as crystallization conditions, may govern one crystal morphology and result in a crystalline material with physical and chemical properties specific to the crystallization conditions.

[0062] The salt forms of the present invention can be formed from the parent compound, i.e., Compound (I), by the ionizable groups of the parent compound. These salts can be acid addition salts, including inorganic or organic acids, or in the case of the acidic form of the parent compound of the present invention, can be prepared from inorganic or organic bases. Often, salts are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well known in the art, such as hydrochloric acid, sulfuric acid, hydrobromic acid, acetic acid, lactic acid, citric acid, or tartaric acid to form acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines to form base salts. Methods for preparing suitable salts are well established in the art. In some cases, compounds may contain both acidic and basic functional groups, in which case they may have two ionizable groups but no net charge. Standard methods for the preparation of pharma- ceutically acceptable salts and their formulation are well known in the art and are disclosed in various references, including, for example, "Remington: The Science and Practice of Pharmacy", A. Gennaro, ed., 20th edition, Lippincott, Williams & Wilkins, Philadelphia, Pa; P. Heinrich Stahl, Camille G. Wermuth, Pharmaceutical Salts: Properties, Selection, and Use, Edition 2.

[0063] Additionally, the salt forms may be anhydrous or include a solvent such as water, in some embodiments, the salt forms include solvates, such as hydrates.

[0064] As used herein, "solvate" refers to a compound formed by solvation (combination of a solvent molecule with a molecule or ion of a solute) or an aggregate consisting of a solute ion or molecule, i.e., a compound of the present invention with one or more solvent molecules. When water is the solvent, the corresponding solvate is a "hydrate". Examples of hydrates include, but are not limited to, hemihydrate, monohydrate, dihydrate, trihydrate, hexahydrate, etc. Those skilled in the art should understand that pharma-ceutically acceptable salts of the present salt forms, and / or prodrugs, may also exist to include solvates. Solvates are typically formed either by hydration that is part of the preparation of the present salt forms, or through the natural absorption of water by the anhydrous salt forms of the present invention.

[0065] As used herein, the terms "administer", "administering", "administration" and the like refer to methods that can be used to enable delivery of the compound / composition to the desired site of biological action. These methods include, but are not limited to, oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular or infusion), topical and rectal administration. Those skilled in the art are familiar with the administration techniques that can be used. In some embodiments, the compounds and compositions described herein are administered orally.

[0066] As used herein, a "co-crystal" refers to a solid that is a crystalline single-phase material composed of two or more different molecular and / or ionic compounds, generally in a stoichiometric ratio, that are not solvates or simple salts. As used herein, the term co-crystal can encompass hydrates, solvates, and clathrates.

[0067] As used herein, "treatment", "treating" or "treating" is an approach to obtain beneficial or desired results, including clinical results. For purposes herein, beneficial or desired results include, but are not limited to, one or more of the following: reduction of another symptom resulting from a condition (e.g., cancer), reduction in the extent of disease, stabilization of a condition (e.g., prevention or delay of the worsening of a condition such as cancer), delay or delay in the progression of a condition, improvement of a disease state, reduction in the dosage of one or more other medicines required to treat a condition, improvement of the quality of life of an individual having or suspected of having a condition, and / or prolongation of life (including overall survival and progression-free survival). Reduction of the pathological results of cancer is also included in "treatment". The methods described herein contemplate any one or more of these aspects of treatment.

[0068] As used herein, "delay" with respect to a condition means to postpone, prevent, slow, delay, stabilize, and / or postpone the onset of one or more symptoms of the condition (e.g., cancer). This delay can be of varying lengths of time depending on the history of the disease and / or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can actually encompass prevention, in that the individual does not develop the condition (e.g., cancer). A method that "delays" the onset of cancer is one that reduces the likelihood of disease onset in a particular time frame and / or reduces the severity of the condition in a particular time frame, compared to not using the method. Such comparisons are usually based on clinical studies using a statistically significant number of subjects. The onset of cancer can be detected using standard methods, such as regular physical exams and x-rays. Onset can also refer to the progression of a disease that may not be detectable at first, including onset and onset.

[0069] As used herein, an individual "at risk" with respect to a condition is an individual who is at risk of developing the condition (e.g., cancer). An "at risk" individual may or may not have a detectable condition and may or may not exhibit symptoms associated with the detectable condition prior to the treatment methods described herein. "At risk" means that an individual has one or more so-called risk factors, which are measurable parameters that correlate with the progression of a condition. Individuals who have one or more of these risk factors are more likely to develop the condition than individuals who do not have these risk factors.

[0070] As used herein, "pharmaceutically acceptable" with respect to a material refers to a material that is not biologically or otherwise unsuitable, e.g., the material may be incorporated (e.g., during manufacture or administration) into a pharmaceutical composition administered to an individual without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. As used herein, the term "pharmaceutically acceptable carrier" refers to, for example, solvents, stabilizers, pH adjusters, tonicity adjusters, adjuvants, binders, diluents, etc., known to those skilled in the art that are suitable for administration to an individual (e.g., human). Combinations of two or more carriers are also contemplated. The pharmaceutically acceptable carriers and any additional components described herein should be adapted for use with the intended route of administration (e.g., oral) of the particular dosage form. Such suitability will be readily recognized by those skilled in the art, especially in light of the teachings provided herein. A pharmaceutically acceptable carrier or excipient preferably meets the necessary standards of toxicological and manufacturing testing and / or is included in the Inactive Ingredients Guide prepared by the U.S. Food and Drug Administration.

[0071] As used herein, in the context of treatment, an "effective amount" refers to an amount that produces the desired pharmacological and / or physiological effect for a particular condition (e.g., cancer) or one or more of its symptoms, and / or to completely or partially prevent the occurrence or recurrence of the condition or its symptoms, and / or to an amount that may be therapeutic with respect to partial or complete cure of the condition and / or side effects resulting from the condition (e.g., cancer). In the context of the conditions described herein (e.g., cancer), a pharmacologic or therapeutically effective amount may include, among others, an amount sufficient to reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to some extent and preferably stop) the invasion of cancer cells into peripheral organs, inhibit (i.e., slow to some extent and preferably stop) the metastasis of tumors, inhibit (i.e., slow to some extent and preferably stop) the growth of tumors, prevent and / or kill the growth of existing cancer cells, be cytostatic and / or cytotoxic, prevent impaired or lost vasodilation, reduce tumor burden, reduce morbidity and / or mortality, and alleviate to some extent one or more symptoms associated with cancer.

[0072] An effective amount may extend progression-free survival (e.g., as measured by changes in Response Evaluation Criteria in Solid Tumors, RECIST, or CA-125), provide an objective response (including a partial or complete response), increase overall survival, and / or improve one or more symptoms of cancer (e.g., as assessed by FOSI). In certain embodiments, a pharmacologic effective amount is sufficient to prevent a condition, such as when administered prophylactically to an individual. An effective amount includes eradication or amelioration of the underlying condition being treated and / or eradication or amelioration of one or more symptoms associated with the underlying condition, such that the individual reports an improvement in sensation or condition (e.g., reduced pain intensity and / or duration), even though the individual may still be suffering from the underlying disease. An effective amount also includes halting or slowing the progression of a disease (e.g., cancer), regardless of whether an improvement in the disease or condition is realized.

[0073] An "effective amount" may vary depending on the composition being administered, the condition being treated / prevented (e.g., type of cancer), the severity of the condition being treated or prevented, the age, size, weight, and relative health of the individual, the route and form of administration, the attending physician or veterinarian (if applicable), and other factors recognized by one of skill in the art in view of the teachings provided herein. An effective amount may be assessed, for example, by using data from one or more clinical, physiological, biochemical, histological, electrophysiological, and / or behavioral assessments.

[0074] As is understood in the art, an "effective amount" can be administered in one or more doses, i.e., a single dose or multiple doses may be required to achieve a desired therapeutic endpoint. An effective amount is considered in the context of administering one or more additional pharmaceutical agents, and the crystals are considered to be administered in an effective amount, such that when combined with the one or more additional pharmaceutical agents, one or more desired or beneficial results may be or have been achieved.

[0075] As used with respect to the therapeutic / prophylactic methods and uses of the crystalline forms and compositions thereof described herein, an "individual in need thereof" may be an individual diagnosed, previously treated, and / or suspected of having the condition being treated (e.g., a proliferative disease such as cancer). With respect to prevention, an individual in need thereof may also be an individual at risk for the condition (e.g., family history of the condition, lifestyle factors indicative of risk for the condition, etc.).

[0076] In some aspects, the individual is a mammal, including but not limited to a cow, a horse, a cat, a rabbit, a dog, a rodent, or a primate. In some embodiments, the mammal is a primate. In some embodiments, the primate is a human. In some embodiments, the individual is a human, including adults, children, infants, and premature infants. In some embodiments, the individual is a non-mammal. In some variations, the primate is a non-human primate, such as chimpanzees and other ape or monkey species. In some embodiments, the mammal is a farm animal, such as cows, horses, sheep, goats, and pigs; pets, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs; and the like. In some embodiments, the individual is a non-mammal, including, but not limited to, birds, and the like. The term "individual" does not denote a particular age or sex.

[0077] With respect to the crystalline forms described herein, "combination therapy" refers to a first therapy comprising the crystals in conjunction with a second therapy (e.g., surgery and / or additional agents) useful for treating, stabilizing, preventing, and / or delaying diseases and conditions. "Concomitant" administration with another compound includes administration in the same or different compositions, either sequentially, simultaneously, or consecutively, via the same or different routes. In some embodiments, the combination therapy optionally includes one or more pharma- ceutically acceptable carriers or excipients, non-pharmacologically active compounds, and / or inactive substances.

[0078] As used herein, the term "additional pharmaceutical agent" in reference to the crystalline forms described herein refers to an active agent (e.g., a drug and / or a different crystalline form) other than the particular crystalline form, which is administered to elicit a therapeutic effect. The pharmaceutical agent may be directed to a therapeutic effect related to the condition (e.g., cancer) that the crystal is intended to treat or prevent, or the pharmaceutical agent may be intended to treat or prevent the symptoms of an underlying condition (e.g., tumor growth, bleeding, ulcers, pain, enlarged lymph nodes, cough, jaundice, swelling, weight loss, cachexia, sweating, anemia, paraneoplastic phenomena, thrombosis, etc.), or to further reduce the appearance or severity of the side effects of the crystalline form.

[0079] Reference herein to "about" a value or parameter includes (and describes) variations directed to that value or parameter itself. For example, a statement referring to "about Y" includes a statement of "Y". When used in conjunction with a measured value, "about" includes a range that includes at least the uncertainty associated with the method of measuring the particular value and can include a range of 1-2 standard deviations around the specified value. When used to describe an estimate or a dosage of a compound, it includes a range of plus or minus 10% of the stated value, or in some embodiments, a range of plus or minus 5% around the stated value. In particular, when used to describe results from PXRD, the term "about" refers to a variation of the data of + / - 0.2° (i.e., °2θ). When used to describe results from DSC, the term about refers to a variation of the data of + / - 3° (i.e., °C).

[0080] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly indicates otherwise. The embodiments and variations described herein are understood to include "consisting of" and / or "consisting essentially of" embodiments and variations. The terms "a" and "an" do not denote a limitation of quantity, but rather indicate the presence of at least one of the referenced items. The terms "a" and "an" are used interchangeably with "one or more" or "at least one." The terms "or" or "and / or" are used as functional terms to indicate that two words or expressions are taken together or individually. The terms "comprise," "have," "include," and "contain" are intended to be construed as open-ended terms (i.e., meaning "including, but not limited to"). All range endpoints directed to the same component or property are inclusive and independently combinable.

[0081] The terms "salt forms of the invention," "these salt forms," ​​"such salt forms," ​​"salt form," and "the salt form" refer to the salt forms described herein, such as amorphous or crystalline salt forms. Additionally, the salt forms can modulate, i.e., inhibit or enhance, the biological activity of CK2 protein, Pim protein, or both, and are thereby also referred to herein as "modulators" or "CK2 and / or Pim modulators."

[0082] Unless otherwise defined or clearly indicated by context, all technical and scientific terms and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0083] Multiple forms of N-methyl-2-(4-methyl-1,4-diazepan-1-yl) Benzo[4,5]imidazo[1,2-A][1,8]naphthyridine-6-carbaximide (Compound I) 2-(4-Methyl-[1,4]diazepan-1-yl)-1,7,11b-triazabenzo[c]fluorene-6-carboxylic acid sodium salt (1A, 99 mg, 0.25 mmol), methylamine hydrochloride (25 mg, 0.38 mmol, 1.5 equiv), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 144 mg, 0.38 mmol, 1.5 equiv) and diisopropylethylamine (49 mg, 0.38 mmol, 1.5 equiv) in N,N-dimethylformamide (5.0 mL) were combined and stirred at room temperature for 1 h.

[0084] The reaction mixture was then poured into water (8 mL) and cooled to 4° C. for 16 h. The precipitate was collected and washed with 4:1 water:N,N-dimethylformamide (1 mL) and water (1 mL). The solid was suspended in water (200 μL) and 20% aqueous sodium carbonate (200 μL) was added. The mixture was stirred at room temperature for 1 h. The solid was then collected and washed with water (3×1 mL) to give the multiphasic form of Compound (I) (16 mg, 0.041 mmol, 16%) as a yellow crystalline solid.

[0085] LCMS: 100%, Rt: 1.678 min, ESMS mz 389.1 (M+H) + .1H NMR (300 MHz, CDCI3- ) δ 10.56 - 10.34 (m, 1H), 8.99 (d, J = 8.1 Hz, 1H), 8.64 (s, 1H), 7.96 (d, J = 9.0 Hz, 2H), 7.95 (d, J = 7.8 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.47 (t, J = 7.8 Hz, 1H), 6.71 (d, J = 9.0 Hz, 1H), 4.41 - 3.69 (m, 4H), 3.21 (d, J = 4.8 Hz, 3H), 3.00 - 2.81 (m, 2H), 2.74 -2.58 (m, 2H), 2.44 (s, 3H), 2.27 - 2.07 (m, 2H).

[0086] Multiphasic forms of N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-A][1,8]naphthyridine-6-carbaxamide (compound I) Compound (I) is highly crystalline as shown by PXRD analysis (see Figure 1).

[0087] Polarized light microscopy shows that compound (I) consists of small birefringent particles with no defined morphology (see FIG. 2).

[0088] Thermogravimetric analysis (TGA) shows no mass loss up to 150° C., while the DT trace shows a sharp endotherm beginning at 212.9° C. that peaks at 214.8° C. (see FIG. 3). No significant degradation is observed up to 200° C.

[0089] Differential scanning calorimetry (DSC) analysis shows a sharp, well-defined endothermic peak in the first heating cycle starting at 211.4°C and peaking at 214°C (see Figure 4). A small exothermic peak is also observed in the second heating cycle starting at 133.4°C and peaking at 143.5°C, possibly indicating recrystallization (see Figure 5). This is followed by an endothermic peak, starting at 211.3°C and peaking at 214.1°C, indicating a melting event.

[0090] Gravimetric vapor sorption (GVS) analysis also shows that compound (I) exhibits mass uptake from 0.8% to 90% relative humidity (RH), thereby suggesting that compound (I) is non-hygroscopic (see FIG. 6).

[0091] Crystalline form 1 of compound I) of N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-A][1,8]naphthyridine-6-carbaxamide Crystalline Form 1 of Compound (I) was made by preparing a mixture (e.g., a slurry) of Compound (I) in acetone by adding 200 mg of Compound (I) to 2.4 mL of acetone. The resulting slurry was then subjected to temperature cycling from about 5° C. to 25° C. for about 72 hours. This procedure was repeated using acetone, acetonitrile, tBME, dimethylformamide, ethanol, ethyl acetate, heptane, methanol, propan-2-ol, tetrahydrofuran, and toluene as the slurry solvent (see FIG. 8).

[0092] Form 1 was also reproduced on a larger scale from an acetone slurry. PXRD analysis indicates that clean formation of Form 1 is reproducible. The PXRD 2θ diffractogram is observed to resemble the multiphasic starting material with several peak differences. Most notable is the absence of a peak at approximately 2θ=12° (see FIG. 18).

[0093] PLM analysis shows particles exhibiting birefringence without a well-defined morphology both before and after drying (see Figures 19 and 20).

[0094] TGA (see Figure 21) shows no mass loss until an endothermic event with a peak at 214.1°C (DTA trace). A small increase in mass is observed around this endotherm.

[0095] DSC analysis (first heating cycle) shows an endotherm with an onset at 211.6° C. and a peak at 214.9° C. (see FIG. 22). This endothermic event is likely associated with the melting of crystallized Compound (I), with an associated enthalpy of 82.6 mJ / mg.

[0096] In the second heating cycle, an exothermic event was also observed with an onset of 141.3° C. and a peak of 151.1° C., which is attributed to the recrystallization of Form 1. A second endothermic event was observed with an onset of 211.1° C. and a peak of 214.2° C. (see FIG. 23), which is closely related to the endothermic phenomenon in the first heating cycle and may be related to the melting of crystallized Compound (I).

[0097] 1 H NMR analysis confirmed the structural integrity of Compound (I) when crystallized as Form 1 (see Figure 24).

[0098] Form 1 was reproduced on the 1 g scale by acetone slurry. PXRD analysis confirmed that Form 1 was reproducible on the 1 g scale (see Figure 61).

[0099] PLM analysis shows the presence of small birefringent particles with no clear morphology (see Figure 62).

[0100] The TGA shows no mass loss (DTA trace) until an endothermic event with a peak at 214.1° C. (See FIG. 63). A slight decrease in mass is observed around this endotherm.

[0101] DSC analysis of the first heating cycle shows an endotherm with an onset of 211.5° C. and a peak at 214.4° C. (see FIG. 64). This endothermic event is likely associated with the melting of compound (I).

[0102] Upon cooling cycle, an exothermic event was observed with an onset of 185.2° C. and peaked at 179.8° C. (See FIG. 65), likely due to recrystallization of Compound (I).

[0103] In the second heating cycle, an endotherm is observed with an onset of 211.5° C. and a peak at 214.3° C., likely due to the melting of recrystallized Compound (I) (see FIG. 66).

[0104] Figure 67 shows the results of Form 1 from a large-scale scale-up. 1 Results from H NMR analysis (CDCL 3500 MHz) are provided.

[0105] Form 1 is stable and maintains its form after storage for 7 days under ambient conditions (closed vial) and 40° C. / 75% RH (closed vial). The results of the post-storage PXRD analysis of Form 1 are shown in FIG.

[0106] Form 1 is stable and maintains its morphology after 2 and 4 weeks of storage under 40° C. / 75% RH, 50° C. and RT. The PXRD diffractograms show that Form 1 does not change morphology or undergo any change in crystallinity after stability testing (see FIG. 68).

[0107] NMR analysis (CDC13 500 MHz) provided in Figures 69-74 demonstrates that Form 1 maintains its structural integrity and does not degrade after further stability testing. Specifically, Figure 69 shows the NMR analysis of Form 1 after storage at 40°C and 75% relative humidity for 2 weeks. 1 Figure 70 shows the HMR analysis of Form 1 after storage for 4 weeks at 40°C and 75% relative humidity. 1 Figure 71 shows the HMR analysis of Form 1 after storage at 50°C for 2 weeks. 1 Figure 72 shows the HMR analysis of Form 1 after storage at 50°C for 4 weeks. 1 Figure 73 shows the HMR analysis of Form 1 after 2 weeks storage at room temperature. 1 Figure 74 shows the HMR analysis of Form 1 after 4 weeks of storage at room temperature. 1 HMR analysis is shown.

[0108] High performance liquid chromatography (HPLC) analyses provided in Table 5 and Figures 75-80 demonstrate that Form 1 maintains high levels of purity at 40°C and 75% relative humidity at both 2 and 4 week time points, 50°C and room temperature.

[0109] Specifically, Figure 75 shows the HPLC purity analysis of Form 1 after 2 weeks storage at room temperature. Figure 76 shows the HPLC purity analysis of Form 1 after 4 weeks storage at room temperature. Figure 77 shows the HPLC purity analysis of Form 1 after 2 weeks storage at 40°C and 75% relative humidity. Figure 78 shows the HPLC purity analysis of Form 1 after 4 weeks storage at 40°C and 75% relative humidity. Figure 79 shows the HPLC purity analysis of Form 1 after 2 weeks storage at 50°C. Figure 80 shows the HPLC purity analysis of Form 1 after 4 weeks storage at 50°C. Table 5: HPLC purity of Form 1 after stability testing

[0110] [Table 1]

[0111] Crystalline form 2 of N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-A][1,8]naphthyridine-6-carbaxamide (Compound I) Form 2 of Compound (I) was made by preparing a mixture (e.g., a slurry) of Compound (I) in 1,4-dioxane by adding 200 mg of Compound (I) to 2.4 mL of 1,4-dioxane. The slurry is then subjected to a temperature cycle from 5° C. to 25° C. over a period of 72 hours. The PXRD analysis of Form 2 is shown in FIG. 9.

[0112] Form 2 was largely reproduced from the 1,4-dioxane slurry. PXRD analysis shows that Form 2 is not initially reproducible after analysis after 72 hours of slurrying (Figure 25, secondary screen crystallization #1).

[0113] However, after an additional 72 hours, Form 2 is produced (see Figure 25, Secondary Screen Crystallization #2).

[0114] PLM analysis shows that Form 2 consists of small birefringent particles with well-defined morphology, both before and after drying (see Figures 26 and 27).

[0115] TG / DTA shows an initial mass loss of 0.1% until an endothermic event with an onset of 64.4° C., peaking at 74.3° C. (see FIG. 28). A mass loss of 11.6% was observed associated with this event, likely corresponding to solvent loss.

[0116] A second endotherm was then observed with an onset of 210.1° C. and a peak at 213.3° C., indicative of the melting of Compound (I). DSC analysis of the first heating cycle showed an initial exothermic event with an onset of 59.3° C. and a peak at 68.8° C., when compared to the TGA trace, which is expected to correspond to desolvation of Form 2 (see FIG. 29).

[0117] A second exothermic event was observed with an onset at 211.1°C and peaking at 213.6°C.

[0118] In the second heating cycle, an exothermic event was observed with an onset at 138.5° C. and a peak at 151.6° C. that is likely associated with the recrystallization of Compound (I) (see FIG. 30). This leads to an endothermic event with an onset at 211° C. and a peak at 213° C. that is attributed to the melting of Compound (I).

[0119] 1 H NMR analysis confirmed the structural integrity of Compound (I) in Form 2 (see Figure 31). The presence of 1,4-dioxane is seen at 3.64 ppm.

[0120] Form 2 is stable and maintains its form after 7 days of storage (closed vial) under ambient conditions. After 7 days of storage (closed vial) at 40° C. / 75% RH, Form 2 transitions to Form 1. The results of PXRD analysis of Form 2 after storage are shown in FIG.

[0121] Crystalline form 3 of N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-A][1,8]naphthyridine-6-carbaxamide (Compound I) Compound (I) Form 3 was made by preparing a mixture (e.g., a slurry) of Compound (I) in dichloromethane by adding 200 mg of Compound (I) to 400 microliters of dichloromethane. The resulting slurry was then subjected to a temperature cycle from about 5° C. to 25° C. for about 72 hours.

[0122] The PXRD analysis of Form 3 is shown in Figure 82. Figure 12 shows the PXRD analysis of dichloromethane cooling crystallization that was evaporated to leave Form 3.

[0123] Initially, production of Form 3 did not appear to be reproducible (see Figure 13). Subsequent iterations produced different forms as shown in Figure 13. A sufficient amount of Form 3 was prepared for the primary crystal morphology screen so that TG / DTA and DSC analyses could be performed before the secondary crystal morphology screen began.

[0124] TG / DTA shows an initial mass loss (DTA trace) of 0.1% before an endotherm with an onset of 60.1° C. and a peak at 66.6° C., which coincides with a weight loss of 14.9%, thereby indicating desolvation of the material (see FIG. 14). A shallow exothermic event occurs between 85.3° C. and 124.1° C., followed by an endothermic event with an onset of 207.7° C. and peaking at 212.9° C. This is expected to be associated with the melting of crystallized Compound (I).

[0125] DSC analysis of the first heating cycle shows a broad endothermic event with an onset of 54.4° C. and a peak at 61° C. (see FIG. 15).

[0126] A second endothermic event begins at 209.5° C. and peaks at 213.2° C. On the cooling cycle, a small exotherm is observed beginning at 149.1° C. and peaking at 153.5° C., potentially due to recrystallization of Compound (I) (see FIG. 16).

[0127] The second heating cycle showed an exothermic event with an onset of 138.6° C. and a peak at 154.3° C. This gave rise to a second endothermic event with an onset of 210° C. and a peak at 213.4° C., which is expected to correspond to the melting of Form 3. Figure 17 shows the DSC analysis of the second heating cycle of Form 3 material from the primary crystal morphology screen.

[0128] Form 3 has been prepared on a large scale from a dichloromethane slurry. PXRD analysis shows that clean formation of Form 3 is reproducible (see Figure 32).

[0129] PLM analysis shows morphology 3 consisting of small birefringent particles before drying and a mixture of particles with no distinct morphology with large rod-like particles after drying (see Figure 33, Figure 34, and Figure 35).

[0130] TG / DTA shows an initial mass loss of about 0.5% to the onset of an endothermic event with an onset of 49.2° C. and a peak of 68.5° C. (see FIG. 36). A further mass loss of about 4.5% is observed associated with this event, likely corresponding to solvent loss. A second endothermic event is observed with an onset of 207.7° C. and a peak at 212° C., indicating the melting of compound (I).

[0131] DSC analysis of the first heating cycle shows an initial broad endothermic event with an onset of 52.9° C. and a peak at 67.7° C., which when compared to the TGA trace is predicted to correspond to desolvation of Form 3 (see FIG. 37). A small exothermic event occurs with an onset of 149.6° C. and a peak of 153.2° C. The final endothermic event due to melting has an onset at 210.4° C. and a peak at 214.0° C.

[0132] On the second heating cycle, an exothermic event was observed with an onset of 151° C. and a peak at 162° C., which appears to be associated with the recrystallization of Form 3 (see FIG. 38). This resulted in an endothermic event with an onset of 209.2° C. and a peak at 214° C., indicating the melting of the crystallized Form 3.

[0133] 1 H NMR analysis shows the structural integrity of Compound (I) in Form 3 (see Figure 39). The presence of dichloromethane can be seen at 5.30 ppm.

[0134] Form 3 is stable and maintains its form after storage for 7 days under ambient conditions (closed vial). After storage for 7 days at 40° C. / 75% RH (closed vial), Form 3 transitions to Form 1. Figure 52 provides the results of PXRD analysis of Form 3 after storage.

[0135] Crystalline form 4 of N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-A][1,8]naphthyridine-6-carbaxamide (Compound I) Form 4 of Compound (I) was prepared by dissolving 200 mg of Compound (I) in 2.2 mL of dichloromethane. The resulting suspension was filtered and allowed to crystallize by evaporation at ambient conditions in an uncapped vial. The PXRD analysis of Form 4 is shown in FIG.

[0136] Form 4 has been produced on a large scale from evaporative crystallization from Compound (I) in dichloromethane.

[0137] PXRD analysis showed that the forms produced from the primary screen were not reproducible (see Figure 40). However, the secondary screen produced the same format as the primary screen repeat (see Figure 39, DCM evaporation repeat #1). This crystal form appears to be a mixture of material produced in two repeats of this experiment (see Figure 39, repeat #1 and repeat #2).

[0138] PLM images show that Form 4 consists of small birefringent particles with no well-defined morphology (see FIG. 41).

[0139] The TG / DTA of Form 4 shows a mass loss of 9.75%, coinciding with an endothermic event with an onset of 59.6° C. and peaking at 72° C. (See FIG. 42). These events indicate desolvation of the material.

[0140] The second endothermic event is likely due to melting of the material starting at 211.2° C. and peaking at 214.2° C. DSC analysis of the first heating cycle shows a small endothermic event at 46.4° C. and peaking at 62.9° C. (see FIG. 43). Comparison with the TGA trace predicts this corresponds to desolvation of Form 4. The second endothermic event due to melting of Compound (I) starts at 211° C. and peaks at 213.7° C.

[0141] In the second heating cycle, an exothermic event was observed with an onset of 160.1° C. and peaking at 166.1° C., likely associated with the recrystallization of Form 4 (see FIG. 44). This leads to an endothermic event with an onset of 209.3° C. and peaking at 213.8° C., corresponding to the melting of Compound (I).

[0142] 1 The H NMR spectrum shows the chemical integrity of Compound (I) in Form 4 (see Figure 45). The presence of dichloromethane can be seen at 5.3 ppm.

[0143] Form 4 is stable and maintains its form after storage for 7 days under ambient conditions (closed vial). After storage for 7 days at 40° C. / 75% RH (closed vial), Form 4 transitions to Form 1. Figure 52 provides the results of PXRD analysis of Form 4 after storage.

[0144] Crystalline form 5 of N-methyl-2-(4-methyl-1,4-diazepan-1-yl)benzo[4,5]imidazo[1,2-A][1,8]naphthyridine-6-carbaxamide (Compound I) Form 5 of Compound (I) was prepared by dissolving 200 mg of Compound (I) in 2.2 mL of dichloromethane. 20 mL of heptane was added to the resulting suspension to precipitate the material from solution. The PXRD analysis of Form 5 is shown in FIG.

[0145] Form 5 has been produced on a large scale. PXRD analysis confirms that Form 5 was not initially reproducible on a large scale (see Figure 46, DCM anti-solvent immediate).

[0146] The dichloromethane / heptane solution was left overnight and upon analysis a transition to Form 5 was observed (next day, DCM-antisolvent secondary screen).

[0147] PLM analysis shows that morphology 5 consists of large rod-like particles that exhibit birefringence after drying (see FIG. 47).

[0148] The TGA trace shows that Form 5 exhibits a mass loss of 0.2% up to 300° C., however this is not a true event but is likely due to an event occurring in the pan as Form 5 melts (see FIG. 48). An endothermic event is observed with an onset of 211.5° C., peaking at 213.5° C. indicating the melting of Compound (I).

[0149] DSC analysis of the first heating cycle is clean with one endothermic event with an onset of 211.4°C and a peak at 213.5°C indicating melting of the material (see Figure 49). The second heating cycle shows two exothermic peaks. The first exothermic event has an onset of 151.8°C and peaks at 165.8°C, which directly leads to a smaller exothermic event with an onset of 178.5°C and a peak of 182.7°C. An endothermic event is observed with an onset of 209.1°C and a peak at 212.2°C, which is likely due to the melting of compound (I).

[0150] 1 The H NMR spectrum shows the chemical structure of Form 5 (see Figure 51). The presence of dichloromethane is seen at 5.30 ppm.

[0151] Form 5 is stable and maintains its form after storage for 7 days under ambient conditions and at 40° C. / 75% RH (closed vial).

[0152] Pharmaceutical Compositions The crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) can be used in the preparation of compositions, such as pharmaceutical compositions, by combining the described crystalline forms with one or more pharma- ceutically acceptable carriers, excipients, stabilizers, and / or other agents known in the art, for use in the methods of treatment, methods of administration, and dosing regimens described herein.

[0153] The composition can be changed or adjusted according to the condition to be treated, the amount of compound to be administered, the condition of the individual, and other variables that will be readily apparent to those skilled in the art in light of the teachings provided herein.Crystalline forms can be formulated as solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions (e.g., when formulated with surface interaction inhibitors), suppositories, injectable solutions, and sprays.The preferred form depends on the intended mode of administration and therapeutic use.The following compositions, additives, and methods are merely illustrative and are in no way limiting.

[0154] Additives used in the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) include, for example, one or more excipients (e.g., one or more excipients), antioxidants (e.g., one or more antioxidants) or stabilizers (e.g., one or more stabilizers), preservatives (e.g., one or more preservatives), pH adjusting and buffering agents (e.g., one or more pH adjusting agents and / or buffering agents), tonicity adjusting agents (e.g., one or more tonicity adjusting agents), thickening agents (e.g., one or more thickening agents), suspending agents (e.g., one or more suspending agents), binding agents (e.g., one or more binding agents), viscosity increasing agents (e.g., one or more viscosity increasing agents), surface interaction inhibitors, etc., alone or together with one or more additional pharmaceutical agents, provided that the additional components are pharma- ceutically acceptable for the particular disease (e.g., cancer) being treated. In some embodiments, the compositions may include a combination of two or more additional components described herein (e.g., 2, 3, 4, 5, 6, 7, 8, or more additional components). In some embodiments, the composition may comprise crystalline morphology in combination with a surface interaction inhibitor that creates a physical barrier between adjacent particles.In some embodiments, the additive comprises processing agents and drug delivery modifiers and enhancers, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidinone, low melting waxes, ion exchange resins, and the like, and any combination of two or more thereof.Other suitable pharma- ceutically acceptable excipients are described in REMINGTON'S PHARMACEUTICAL SCIENCES, Merck Pub. Co., New Jersey 18th edition (1996), and REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, Lippincott Williams & Wilkins, Philadelphia, 20th edition (2003) and 21st edition (2005); Rowe, Raymond C; Sheskey, Paul J; Cook, Walter G; Fenton, Marian E., Handbook of Pharmaceutical Excipients, Seventh edition.

[0155] Compositions suitable for oral administration can include, for example, (a) an effective amount of the compound dissolved in a liquid solution (e.g., crystalline form combined with one or more solvents, with a surface interaction inhibitor), for example, in a diluent, such as water, saline, or orange juice; (b) capsules, sachets, or tablets, each containing a predetermined amount of the active ingredient as a solid or granules; (c) a suspension in a suitable liquid (including a microsuspension); (d) a suitable emulsion; and (e) a powder. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffers, wetting agents, preservatives, flavorings, and pharmacologically compatible excipients. Lozenge forms can contain the active ingredient in a flavoring, usually sucrose and acacia or tragacanth, and the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like, including troches containing such excipients as are known in the art in addition to the active ingredient.

[0156] The crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) may be enclosed in hard or soft capsules, compressed into tablets, or incorporated into beverages or foods, or otherwise incorporated into food. Capsules can be formulated by mixing the crystalline forms with an inert pharmaceutical diluent and inserting the mixture into a hard gelatin capsule of suitable size. If soft capsules are desired, a slurry of the crystalline forms with an acceptable vegetable oil, light mineral oil, or other inert oil can be encapsulated by machine into a gelatin capsule.

[0157] Liquid dosage forms for oral administration may include pharma- ceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs that contain inert diluents commonly used in the art, such as water.Such formulations may also contain adjuvants, such as surface interaction inhibitors, wetting agents, emulsifying and suspending agents, cyclodextrins, and sweeteners, flavorings, and perfumes.Those skilled in the art will recognize that the complete solvation of crystalline or amorphous solids is not encompassed by the present invention, and that to maintain the crystalline form used in a particular composition, the crystalline form must be insoluble in the carrier.

[0158] Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation compatible with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The compositions may be provided in unit-dose or multi-dose sealed containers, such as ampoules or vials, in the treatment methods, administration methods, and dosage regimens described herein (i.e., water) for injection immediately prior to use, and may be stored in a lyophilized (lyophilized) state requiring only the addition of a sterile liquid excipient. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

[0159] Injectable preparations (e.g., sterile injectable aqueous or oily suspensions) can be formulated according to known techniques using suitable dispersants or wetting agents and suspending agents. In some embodiments, the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5), such as the amorphous salt forms or crystalline forms described herein, are formulated as microsuspensions (e.g., for parenteral, oral, or other administration). Microsuspensions are thermodynamically stable dispersions of microcrystals, which may be stabilized by interfacial layers of surfactant molecules that function as dispersants (see Encyclopedia of Pharmaceutical Technology (New York: Marcel Dekker, 1992), volume 9, the contents of which are incorporated herein by reference).

[0160] The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in propylene glycol. Sterile injectable preparations may be sterile powders that are reconstituted with acceptable vehicles before administration. Among acceptable vehicles and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, mild fixed oils, including synthetic mono- or diglycerides, can be used. In addition, fatty acids such as oleic acid can be used in the preparation of injectables.

[0161] Compositions derived from the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) formulated in liquid form (oral, parenteral, or otherwise) include a pH range of any of about 3.5 to about 9.0, such as about 5.0 to about 8.0, about 6.5 to about 7.5, and about 6.5 to about 7.0. In some embodiments, the pH of the composition is formulated to be about 6 or greater, including, for example, any of about 6.5, 7, or 8 (e.g., about 8) or greater. The composition can also be made isotonic with blood by the addition of a suitable tonicity adjusting agent, such as glycerol.

[0162] The crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) can also be formulated for administration by inhalation. Compositions suitable for aerosol administration that include crystalline forms can include, for example, antioxidants, buffers, bacteriostats, and solutes, as well as aqueous and non-aqueous isotonic sterile solutions, including aqueous and non-aqueous sterile suspensions, suspending agents, solubilizers, thickeners, stabilizers, and preservatives, either alone or in combination with other suitable components, that can be made into aerosol compositions to be administered by inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, etc. Additionally, pharmaceuticals can be formulated for non-pressurized preparations, such as in a nebulizer or atomizer.

[0163] Various sustained release systems for drugs have also been devised and can be applied to the salt forms of the present invention, see, for example, U.S. Patent No. 4,983,831, the methods of which are incorporated herein by reference.

[0164] The crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) can also be formulated in the form of suppositories for rectal administration. These can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore melts in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycols.

[0165] The crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) can also be formulated for topical administration, particularly when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, skin, or lower intestinal tract. Suitable topical compositions are readily prepared for each of these areas or organs.

[0166] Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topical-transdermal patches can also be used.

[0167] Also provided are unit dosage forms comprising the compositions described herein (e.g., compositions comprising the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5)). These unit dosage forms can be stored in suitable packaging in single or multiple unit dosage forms, and can further be sterilized and sealed. For example, a pharmaceutical formulation (e.g., a dosage or unit dosage form of a pharmaceutical composition) can include (i) a crystalline form and (ii) a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition also includes one or more other compounds (or pharma- ceutically acceptable salts thereof) useful for treating a particular condition (e.g., cancer).

[0168] In various variations, the amount of the crystalline form in the composition is within any of the following ranges: about 5 to about 50 mg, about 20 to about 50 mg, about 50 to about 100 mg, about 100 to about 125 mg, about 125 to about 150 mg, about 150 to about 175 mg, about 175 to about 200 mg, about 200 to about 225 mg, about 225 to about 250 mg, about 250 to about 300 mg, about 300 to about 350 mg, about 350 to about 400 mg, about 400 to about 450 mg, or about 450 to about 500 mg. In some embodiments, the amount of the crystalline form in the composition (e.g., dosage or unit dosage form) is in the range of about 5 mg to about 500 mg, e.g., about 30 mg to about 300 mg or about 50 mg to about 200 mg. In some embodiments, the composition and carrier are suitable for oral administration. In some embodiments, the crystalline form is the only pharma- ceutically active agent for the treatment of a condition (eg, cancer) that is included in the composition.

[0169] (kit) Also provided are kits containing materials useful for treating diseases (e.g., cancer) that respond to the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5). The kits contain the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) and may optionally contain instructions for use (e.g., instructions for preparing and / or administering compositions containing the crystalline forms). Information detailing possible side effects of the compositions and other relevant information may also be included. The instructions may be in any suitable format, including, but not limited to, printed matter, videotape, computer readable disk, optical disk, or directions to internet-based instructions.

[0170] In one aspect, a kit is provided for treating an individual suffering from or susceptible to a disease or condition (e.g., cancer) described herein, comprising a first container containing a dosage amount of the formulation disclosed herein and instructions for use. The container can be any of those known to those skilled in the art and suitable for storing and delivering intravenous formulations. In certain embodiments, the kit further comprises a second container containing a pharma- ceutically acceptable carrier, diluent, adjuvant, etc. for preparing the formulation to be administered to an individual.

[0171] In some embodiments, the kit includes a labeled container. Suitable containers include, for example, bottles, vials, and test tubes. The container may be formed from a variety of materials, such as glass or plastic. The container may hold a crystalline form (e.g., crystalline forms 1, 2, 3, 4, and 5) or a composition thereof described herein. The label on the container may indicate that the crystalline form, composition, or pharma- ceutical active agent (Compound (I)) is used to treat or inhibit a condition responsive to Compound (I) (e.g., cancer), and may also indicate instructions for either in vivo or in vitro use, as described herein.

[0172] The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for practicing any of the methods described herein. In some embodiments, the kit includes the container described above and a second container containing a buffer.

[0173] The kit may include additional pharmaceutical agents for use in conjunction with the formulations described herein. In some variations, the additional pharmaceutical agents may be one or more anti-cancer agents. These agents may be provided in separate forms or may be mixed with the crystalline forms described herein if they do not reduce the effectiveness of any of the pharmaceutical forms or formulations described herein and are compatible with the route of administration. Similarly, the kit may include additional agents for adjunctive therapy or other agents known to those skilled in the art to be effective in treating or preventing the conditions described herein (e.g., cancer).

[0174] Kits containing sufficient doses of the compounds described herein (including compositions thereof) can also be provided to provide effective treatment to an individual for extended periods of time, e.g., 1-3 days, 1-5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or more.

[0175] The kit may include the compositions described herein packaged in either unit dosage form or multi-use form. The kit may include multiple units of unit dosage form. The kit may be used for any of the methods described herein, including, for example, treating an individual with cancer or delaying cancer. In certain embodiments, the kit may include a dosage of at least one formulation disclosed herein. The kit may also include a means for delivery of the composition.

[0176] (Treatment method) The crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5), or pharma- ceutically acceptable salts thereof, which may be described herein, are used in the preparation of a medicament for the treatment of a disease or condition in a mammal that would benefit from inhibition of POL1 transcription. A method of treating any of the diseases or conditions described herein in a mammal in need of such treatment comprises administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising at least one crystalline form described herein, or a pharma- ceutically acceptable salt, active metabolite, prodrug, or pharma- ceutically acceptable solvate thereof.

[0177] In some embodiments, the present invention provides a method of treating a condition associated with polymerase I transcription comprising administering to a patient in need thereof an effective amount of a crystalline form of the present invention. In another embodiment, the present invention provides a method of inhibiting polymerase I transcription comprising contacting the enzyme with an activated form of the present invention. In a further aspect, the present invention provides a method of inhibiting polymerase I transcription comprising administering to a subject a first crystalline form that is converted in vivo to an active form of the present invention.

[0178] "Conditions associated with Polymerase I transcription" include disorders and diseases in which inhibition of Polymerase I transcription provides a therapeutic benefit, such as cancer, allergies / asthma, immune system diseases and conditions, inflammation, central nervous system (CNS) diseases and conditions, cardiovascular diseases, viral infections, skin diseases, and diseases and conditions associated with unregulated angiogenesis. When general terms are used herein to describe conditions associated with Polymerase I transcription, it is understood that more specifically described conditions referenced in various diagnostic manuals and other sources are included within the scope of the invention.

[0179] The term "cancer" as used herein refers to an abnormal growth of cells that tend to grow in an uncontrolled manner and in some cases metastasize (spread). Types of cancer include, but are not limited to, solid tumors (bladder, intestine, brain, breast, endometrium, heart, kidney, lung, lymphatic tissue (lymphoma), ovary, pancreas, or other endocrine organs (thyroid), prostate, skin (melanoma) or blood tumors (leukemia, etc.). See Ding XZ et al., Anticancer Drugs. 2005 June; 16(5):467-73.Review; Chen X et al., Clin Cancer Res. 2004 Oct. 1; 10(19):6703-9, each of which is incorporated herein by reference in its entirety.

[0180] For example, it is understood that the treatment of cancer includes the treatment of all neoplasms, regardless of histopathological appearance. In particular, cancers that may be treated include myelofibrosis, cancers of the blood, including leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), cancers of the skin, including melanoma, basal cell carcinoma, and squamous cell carcinoma, cancers of the bone, liver, lung (including small cell lung tumors, non-small cell lung cancer, and bronchioloalveolar carcinoma), brain, breast, prostate, larynx, gallbladder, pancreas, rectum, bile duct, parathyroid gland, thyroid gland, adrenal gland, nervous tissue, bladder, spleen, jaw, mouth, head and neck, including nose, colon, stomach, testes, esophagus, uterus, cervix and vulva, colorectum, bronchi, bile duct, bladder, kidney, ovary, pancreas, multiple myeloma, lymphoma, basal cell carcinoma, squamous cell carcinoma, both ulcerative and papillary types, osteosarcoma, Ewing's sarcoma, small These include, but are not limited to, alveolar cell sarcoma, myeloma, giant cell tumor, pancreatic islet cell tumor, acute and chronic lymphocytic and granulocytic tumors, hairy cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuron, intestinal ganglioneuroma, hyperplastic corneal nerve tumor, marfanoid tumor, Wilms' tumor, seminoma, ovarian tumor, leiomyoma tumor, cervical dysplasia and intraepithelial carcinoma, neuroblastoma, retinoblastoma, myelodysplastic syndrome, mycosis fungicide, rhabdomyosarcoma, astrocytoma, non-Hodgkin's lymphoma, Kaposi's sarcoma, osteogenic and other sarcomas, malignant hypercalcemia, polycythemia vera, adenocarcinoma, glioblastoma multiforme, glioma, lymphoma, epidermoid carcinoma, and other carcinomas and sarcomas.

[0181] Benign tumors may also be treated with the crystalline forms of the present invention, including, but are not limited to, hemangiomas, hepatocellular adenomas, cavernous hemangiomas, focal nodular hyperplasia, acoustic neuromas, neurofibromas, bile duct adenomas, bile duct cysts, fibromas, lipomas, leiomyomas, mesotheliomas, teratomas, myxomas, nodular regenerative hyperplasia, trachoma, pyogenic granulomas, and the like, as well as hamartomatous conditions such as Peutz-Jeghers syndrome (PJS), Cowden disease, Bannayan-Riley-Ruvalcaba syndrome (BRRS), Proteus syndrome, Lhermitte-Duclos disease, and tuberous sclerosis complex (TSC).

[0182] The crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) can also be used to treat abnormal cell proliferation due to injury to bodily tissue during surgery. These injuries may occur as a result of various surgical procedures, such as joint surgery, intestinal surgery, and keloid scars. Diseases that produce fibrous tissue include emphysema. Repetitive motion disorders that can be treated using the present invention include carpal tunnel syndrome.

[0183] The crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) may also be useful in the prevention of restenosis, which is the control of the unwanted proliferation of normal cells in the vasculature in response to the introduction of a stent in the treatment of vascular disease.

[0184] The proliferative responses associated with organ transplantation that can be treated using the Pol I transcription inhibitors of the present invention include those that contribute to potential organ rejection or associated complications, particularly those that may occur during transplantation of the heart, lung, liver, kidney, and other body organs or organ systems.

[0185] The crystalline forms described herein (e.g., Forms 1, 2, 3, 4, and 5) may also be useful in the treatment of abnormal angiogenesis, including the following: abnormal angiogenesis associated with rheumatoid arthritis, cerebral edema and injury associated with ischemia-reperfusion, cortical ischemia, ovarian hyperplasia and hypervascularity, (polycystic ovary syndrome), endometriosis, psoriasis, diabetic retinopathy, and other neovascular diseases of the eye such as retinopathy of prematurity (retrolental fibroplasia), macular degeneration, corneal graft rejection, neuromuscular glaucoma, Osterweber syndrome, retinal / choroidal neovascularization, and the like. corneal neovascularization, Best's disease, myopia, optic fossa, Stargardt's disease, Paget's disease, venous occlusion, arterial occlusion, sickle cell anemia, sarcoid, syphilis, elastic yellow pseudocarotid occlusive disease, chronic uveitis / vitreous inflammation, mycobacterial infections, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, Eyes' disease, diabetic retinopathy, macular degeneration, Bechet's disease, infections causing retinitis or choroiditis, presumed histitis, pars planus, chronic retinal detachment, hyperviscosity syndrome, toxoplasmosis, exocrine gland infections, retinal detachment, hyperviscosity syndrome ... Wound and post-laser complications, diseases associated with rubecis (neovascularization of the corner), diseases resulting from abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreoretinopathy, atopic keratitis, superior marginal keratitis, pterygium keratitis, Sjogren's, acne rosacea, phyllectinulosis, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, Mooren's ulcer, Therrien marginal degeneration, marginal keratolysis, polyarteritis, Wegener's sarcoidosis, scleritis, radial keratotomy, neovascular glaucoma and Neurodegenerative diseases including retrolental fibrosis, syphilis, mycobacterial infection, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infection, shingles infection, protozoal infection, Kaposi's sarcoma, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), epilepsy, seizures, Huntington's disease, polyglutamine diseases, traumatic brain injury, ischemic and hemorrhagic stroke, cerebral ischemia, or apoptotic neurodegenerative diseases caused by traumatic injury, acute hypoxia, ischemia or glutamate neurotoxicity.

[0186] For example, it is understood that the treatment of inflammation includes, but is not limited to, acute pancreatitis, chronic pancreatitis, asthma, allergies, chronic obstructive pulmonary disease, adult respiratory distress syndrome and chronic inflammatory diseases associated with uncontrolled angiogenesis, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, psoriasis, sarcoidosis, rheumatoid arthritis, sarcoidosis, and multisystem granulomatous disease.

[0187] For example, it is understood that treatment of autoimmunity includes, but is not limited to, glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves' disease, autoimmune gastritis, diabetes, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, atopic dermatitis, chronic active hepatitis, myasthenia gravis, multiple sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, psoriasis, graft versus host disease, multiple sclerosis, or Sjogren's syndrome.

[0188] In certain embodiments, compositions comprising the crystalline forms described herein are administered for prophylactic and / or therapeutic treatment. In certain therapeutic applications, the compositions are administered to a patient already suffering from a disease or condition in an amount sufficient to cure or at least partially prevent at least one symptom of the disease or condition. Amounts effective for this use will depend on the severity and course of the disease or condition, previous treatments, the patient's health status, weight, response to the drug, and the judgment of the treating physician. Therapeutically effective amounts are optionally determined by methods including, but not limited to, dose escalation and / or dose ranging clinical trials.

[0189] In prophylactic applications, compositions comprising the crystalline forms described herein are administered to patients susceptible to or otherwise at risk for a particular disease, disorder, or condition. Such an amount is defined as a "prophylactically effective amount or dose." In this use, the exact amount also depends on the patient's health, weight, and the like. When used in a patient, the effective amount for this use will depend on the severity and course of the disease, disorder, or condition, previous treatments, the patient's health and response to the drug, and the judgment of the treating physician. In one aspect, prophylactic treatment involves administering a pharmaceutical composition comprising the crystalline forms described herein, or a pharma- ceutically acceptable salt thereof, to a mammal that has previously experienced at least one symptom of the disease being treated and is currently in remission, to prevent the recurrence of symptoms of the disease or condition.

[0190] In certain embodiments where the patient's condition does not improve, at the physician's discretion, administration of the crystalline form is administered chronically, i.e., for an extended period of time, including throughout the patient's life, to ameliorate or otherwise control or limit the symptoms of the patient's disease or condition.

[0191] In certain embodiments where the patient's condition improves, the dose of the administered drug is temporarily reduced or temporarily suspended for a certain period of time (i.e., a "drug holiday"). In certain embodiments, the length of the drug holiday is between 2 days and 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, or more than 28 days. The dose reduction during the drug holiday is, by way of example only, 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

[0192] Once improvement of the patient's condition occurs, a maintenance dose is administered as needed. In certain embodiments, the dosage or frequency of administration, or both, is then reduced as a function of symptoms, to a level at which the improved disease, disorder or condition is maintained. However, in certain embodiments, the patient requires intermittent treatment on a long-term basis upon recurrence of symptoms.

[0193] The amount of a given agent that corresponds to such an amount will vary depending on factors such as the particular crystalline form, the disease state and its severity, the identity (e.g., weight, sex) of the subject or host requiring treatment, but will nevertheless be determined according to the particular circumstances surrounding the case, including, for example, the particular agent being administered, the route of administration, the condition being treated, and the subject or host being treated.

[0194] However, in general, the dose used for adult treatment is typically in the range of 0.01mg to 5000mg per day.In one aspect, the dose used for adult treatment is about 1mg to about 1000mg per day.In one embodiment, the desired dose is conveniently presented as a single dose or as a divided dose, which is administered simultaneously or at appropriate intervals, for example as 2, 3, 4 or more subdoses per day.

[0195] In one embodiment, the daily dosage suitable for the crystalline form or its pharma- ceutically acceptable salt described herein is about 0.01 to about 50 mg / kg of body weight. In some embodiments, the daily dosage or amount of active substance in the dosage form is lower or higher than the ranges indicated herein, based on many variables related to individual treatment plans. In various embodiments, the daily dosage and unit dosage vary depending on many variables, including but not limited to the activity of the crystalline form used, the disease or condition being treated, the method of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the physician's judgment.

[0196] The toxicity and therapeutic efficacy of such treatment regimens are 50 and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, and is the LD 50 and ED 50In certain embodiments, the data obtained from cell culture assays and animal studies are used to formulate a therapeutically effective daily dose range and / or therapeutically effective unit dose for use in mammals, including humans. In some embodiments, the daily dosage of the crystalline forms described herein is the ED at a dose that results in minimal toxicity. 50 In certain embodiments, the daily dosage range and / or unit dosage amount vary within this range depending upon the dosage form employed and the route of administration utilized.

[0197] Any of the foregoing aspects are embodiments in which an effective amount of a crystalline form described herein (e.g., Forms 1, 2, 3, 4, and 5) or a pharma- ceutically acceptable salt thereof is (a) administered systemically to a mammal, and / or (b) administered orally to a mammal, and / or (c) administered intravenously to a mammal, and / or (d) administered by injection to a mammal, and / or (e) administered locally to a mammal, and / or (f) administered non-systemically or locally to a mammal.

[0198] Any of the foregoing aspects are further embodiments comprising a single administration of an effective amount of a crystalline form described herein (e.g., Forms 1, 2, 3, 4, and 5), including further embodiments in which (i) a crystalline form described herein (e.g., Forms 1, 2, 3, 4, and 5) is administered once daily, or (ii) a crystalline form described herein (e.g., Forms 1, 2, 3, 4, and 5) is administered to the mammal multiple times over the course of a day.

[0199] Any of the foregoing aspects include further embodiments comprising multiple administrations of an effective amount of a crystalline form described herein (e.g., crystalline forms 1, 2, 3, 4, and 5), including further embodiments in which (i) the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) are administered continuously or intermittently: as a single dose; (ii) the time between multiple doses is every 6 hours; (iii) the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) are administered to the mammal every 8 hours; (iv) the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) are administered to the mammal every 12 hours; and (v) the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) are administered to the mammal every 24 hours. In further or alternative embodiments, the method includes a drug holiday, in which administration of the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) is temporarily suspended or the administered dose is temporarily reduced, at the end of the drug holiday, dosing is resumed. In one embodiment, the length of the drug holiday varies from 2 days to 1 year.

[0200] In certain instances, it is appropriate to administer at least one of the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5), or a pharma- ceutically acceptable salt thereof, in combination with one or more other therapeutic agents. In certain embodiments, the pharmaceutical composition further comprises one or more anti-cancer agents.

[0201] In one embodiment, the therapeutic effect of one of the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) is enhanced by administration of an adjuvant (i.e., an adjuvant by itself has minimal therapeutic benefit, but in combination with another therapeutic agent, improves the overall therapeutic benefit to the patient), or in some embodiments, the benefit experienced by the patient is increased by administering one of the forms described herein with another agent (which also comprises a treatment regimen) that also has therapeutic benefit.

[0202] A wide variety of therapeutic agents may have therapeutic additive or synergistic effects with the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5). Combination therapy including one or more crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) with one or more other therapeutic agents can be used, for example, to: (1) enhance the therapeutic effect of the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5), and / or one or more other therapeutic agents; (2) reduce the side effects exhibited by the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5), and / or one or more other therapeutic agents; and / or (3) reduce the effective dose of the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5), and / or one or more other therapeutic agents. It should be noted that combination therapy is intended to cover cases where agents are administered before or after each other (sequential therapy) and cases where agents are administered simultaneously.

[0203] Examples of such therapeutic agents that can be used in combination with the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) include, but are not limited to, anti-cell proliferation agents, anti-cancer agents, alkylating agents, antibiotic agents, antimetabolites, hormonal agents, plant-derived agents, and biologic agents.

[0204] Anti-cell proliferation agents useful in combination with the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) include, but are not limited to, retinoid acid and its derivatives, 2-methoxyestradiol, ANGIOSTATIN™ protein, ENDOSTATIN™ protein, suramin, squalamine, tissue inhibitor of metalloproteinase-I, tissue inhibitor of metalloproteinase-2, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, cartilage-derived inhibitor, paclitaxel, platelet factor 4, protamine sulfate (clupeine), sulfated chitin derivatives (prepared from queen crab shells), sulfated polysaccharide peptidoglycan complex (sp-pg), staurosporine, modulators of matrix metabolism, such as proline analogs ((l-azetidine-2-carboxylic acid (LACA), cis-hydroxyproline, d,l-3,4-dehydroproline, thiaproline, beta-aminopropyl fumarate, nitrile, 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone, methotrexate, mitoxantrone, heparin, interferon, 2 macroglobulin serum, thymidine-3, chymostatin, beta-cyclodextrin tetradecasulfate, eponemycin; fumagillin, sodium gold thiomalate, d-penicillamine (CDPT), beta-1-anticollagenase serum, alpha-2-antiplasmin, bisantrene, lobenzarit diacetate Metalloproteinase inhibitors such as sodium, n-(2-carboxyphenyl-4-chloroanthronylate disodium or "CCA", thalidomide, angostat steroid, carboxyaminol imidazole, BB94, etc. Other anti-angiogenic agents that can be used include antibodies, preferably monoclonal antibodies against the angiogenic growth factors: bFGF, aFGF, FGF-5, VEGF isoforms, VEGF-C, HGF / SF and Ang-1 / Ang-2.

[0205] Inhibitors of mTOR, PI3K, MEK, MAPK, PIM or ERK kinases are useful in combination with the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4 and 5). In particular, (R)-3-(2,3-dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3d]pyrimidine-4,7(3H,8H)-dione, an inhibitor of hedgehog kinase, is useful in combination with the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4 and 5). Proteasome inhibitors, particularly bortezomib, are useful in combination with the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4 and 5).

[0206] NAE inhibitors, VPS34 inhibitors, Aurora kinase, including Aurora A inhibitors, and EGFR inhibitors (both antibodies and kinase inhibitors) are useful in combination with the crystalline forms described herein (e.g., in combination with crystalline forms 1, 2, 3, 4, and 5).

[0207] Alkylating agents useful in combination with the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) include, but are not limited to, bischloroethylamines (chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, nitrogen mustards such as uracil mustard), aziridines (such as thiotepa), alkyl alkone sulfonates (such as busulfan), nitrosoureas (carmustine, lomustine, streptozocin, non-classical alkylating agents (altretamine, dacarbazine, and procarbazine), platinum compounds (carboplatin and cisplatin). Combination therapies including polymerase I inhibitors and alkylating agents are expected to provide therapeutic synergy in the treatment of cancer and reduce the side effects associated with these chemotherapeutic agents.

[0208] Examples of antibiotics useful in combination with the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) include, but are not limited to, anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, and anthracenedione), mitomycin C, bleomycin, dactinomycin, plicatomycin. These antibiotics interfere with cell growth by targeting different cellular components.

[0209] Anti-metabolites useful in combination with the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) include, but are not limited to, fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate, leucovorin, hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP), cytarabine, pentostatin, fludarabine phosphate, cladribine (2-CDA), asparaginase, and gemcitabine. Combination therapy with an antimetabolite is expected to have therapeutic synergistic effects against cancer and reduce the side effects associated with these chemotherapeutic agents.

[0210] Hormonal agents useful in combination with the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) include, but are not limited to, synthetic estrogens (e.g., diethylstibestrol), antiestrogens (e.g., tamoxifen, toremifene, fluoxymesterol, and raloxifene), antiandrogens (bicalutamide, nilutamide, and flutamide), aromatase inhibitors (e.g., aminoglutethimide, anastrozole, tetrazole), ketoconazole, goserelin acetate, leuprolide, megestrol acetate, and mifepristone. Combination therapy comprising the crystalline forms disclosed herein and hormonal agents is expected to have therapeutic synergistic effects on cancer and reduce the side effects associated with these chemotherapeutic agents.

[0211] Plant-derived drugs useful in combination with the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) include, but are not limited to, vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinzolidine, and vinorelbine), podophyllotoxins (e.g., etoposide (VP-16) and teniposide (VM-26)), and taxanes (e.g., paclitaxel and docetaxel). These plant-derived drugs generally act as antimitotic agents that bind to tubulin and inhibit mitosis. Podophyllotoxins, such as etoposide, are believed to disrupt DNA synthesis by interacting with topoisomerase II, resulting in DNA strand breaks. Combination therapies including the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) and plant-derived drugs are expected to have therapeutic synergistic effects against cancer and reduce the side effects associated with these chemotherapeutic agents.

[0212] In either case, regardless of the disease, disorder, or condition being treated, the overall benefit experienced by the patient is simply additive of the two therapeutic agents or the patient experiences a synergistic benefit.

[0213] In certain embodiments, different therapeutically effective doses of the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) are utilized in the formulation of pharmaceutical compositions and / or treatment regimens when administered in combination with one or more additional agents, such as additional therapeutically effective drugs, adjuvants, etc. The therapeutically effective dosages of drugs and other agents for use in combination treatment regimens are optionally determined by similar means as described above for the active agents themselves. In addition, the methods of prevention / treatment described herein include the use of metronomic dosing, i.e., providing more frequent and lower doses to minimize toxic side effects. In some embodiments, combination treatment regimens encompass treatment regimens in which administration of the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5) or pharma- ceutically acceptable salts thereof is initiated before, during, or after treatment with a second agent described herein, and continues until any time during or after the end of treatment with the second agent. Also included are treatments in which the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5), or pharma- ceutically acceptable salts thereof, and a second drug used in combination are administered at the same time or at different times, and / or with decreasing or increasing time intervals during the treatment period. Combination therapy further includes periodic treatments that are started and stopped at different times to aid in the clinical management of the patient.

[0214] It is understood that the dosage regimen for treating, preventing, or ameliorating the condition for which relief is sought will be modified according to a variety of factors (e.g., the disease, disorder, or condition from which the subject suffers; the age, weight, sex, diet, medical condition of the subject). Thus, in some instances, the dosage regimen actually used will vary and, in some embodiments, will deviate from the dosage schedule set forth herein.

[0215] For the combination therapies described herein, the dosages of the co-administered compounds will vary depending on the type of co-drug used, the particular drug being used, the disease or condition being treated, and the like. In further embodiments, when co-administered with one or more other therapeutic agents, the crystalline forms provided herein are administered either simultaneously or sequentially with the one or more other therapeutic agents.

[0216] In combination therapy, the multiple therapeutic agents (one of which is a crystalline form described herein) are administered in any order, or even simultaneously. When administration is simultaneous, the multiple therapeutic agents may be provided in a single, unified form or in multiple forms (e.g., as a single pill or two separate pills), by way of example only.

[0217] The crystalline forms (e.g., crystalline forms 1, 2, 3, 4, and 5), or pharma- ceutically acceptable salts thereof, as well as combination therapies, are administered before, during, or after the onset of a disease or condition, and the timing of administering the compositions comprising the crystalline forms can vary. Thus, in one embodiment, the crystalline forms (e.g., crystalline forms 1, 2, 3, 4, and 5) described herein are used as prophylactics and are administered continuously to subjects prone to developing a symptom or disease to prevent the onset of the disease or condition. In another embodiment, the crystalline forms (e.g., crystalline forms 1, 2, 3, 4, and 5) and compositions described herein are administered to a subject during or as soon as possible after the onset of symptoms. In certain embodiments, the crystalline forms (e.g., crystalline forms 1, 2, 3, 4, and 5) described herein are administered as soon as practicable after the onset of a disease or condition is detected or suspected, for an extended period of time required to treat the condition. In some embodiments, the length of treatment required can vary, and the length of treatment is adjusted to fit the specific needs of each subject. For example, in certain embodiments, a crystalline form described herein (eg, crystalline forms 1, 2, 3, 4, and 5) or a formulation containing a crystalline form is administered for at least 2 weeks, from about 1 month to about 5 years.

[0218] In some embodiments, the crystalline forms described herein (e.g., crystalline forms 1, 2, 3, 4, and 5), or a pharma- ceutically acceptable salt thereof, are administered in combination with chemotherapy, hormone-blocking therapy, radiation therapy, a monoclonal antibody, or a combination thereof.

[0219] The present invention will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the invention. EXAMPLES

[0220] Example 1: Synthesis of Compound (I) - Multiphasic Forms

[0221] Synthesis of ethyl 1H-benzimidazol-2-yl acetate Error! Objects cannot be created from editing field codes.

[0222] A mixture of SM1 (40 g, 370 mmol) and SM2 (73 g, 373 mmol) in ethanol (200 ml) was stirred at 90° C. overnight. The reaction was cooled to room temperature and the solvent was removed in vacuo. Water (300 ml) and DCM (500 ml) were added to the residue. The organic layer was separated, dried over Na2SO4 and the solvent was removed to give ethyl acetate 1H-benzimidazol-2-yl (40 g, 53% yield) as a pale yellow solid.

[0223] Synthesis of ethyl 2-(4-methyl-[1,4]diazepan-1-yl)-1,7,11b-triazabenzo[c]fluorene-6-carboxylate Error! Objects cannot be created from editing field codes.

[0224] To a suspension of SM1 (25.9 g, 148 mmol) and SM2 (30 g, 147 mmol) in ethanol (400 mL), SM3 (33.5 g, 394 mmol) was added and the mixture was stirred at room temperature for 20 min. The reaction mixture was heated to reflux for 6 h. The mixture was then cooled to 20 °C and the precipitate was collected by filtration to give ethyl 2-(4-methyl-[1,4]diazepan-1-yl)-1,7,11b-triazabenzo[c]fluorene-6-carboxylate (40 g, 68% yield) as a pale yellow solid.

[0225] [ka]

[0226] A suspension of ethyl 2-(4-methyl-[1,4]diazepan-1-yl)-1,7,11b-triazabenzo[c]fluorene-6-carboxylate (40 g, 99 mmol) in CH3NH2 / EtOH (2 M, 400 ml, 800 mmol) was heated to reflux for 6 h. The mixture was then cooled to 20° C. and the precipitate was collected by filtration to give crude 2G as a pale yellow solid (35 g, 86% yield). The crude product was dissolved in DCM (500 ml), washed with water (3 times), brine, dried and concentrated to give compound I (30.2 g, yield=78%) as a pale yellow solid.

[0227] 1H NMR (400 MHz, DMSO-d6) δ 10.18 (1H, d, J = 4.8 Hz), 8.91 (1H, d, J = 6.0 Hz), 8.58 (1H, s), 8.25 (1H, d, J = 8.8 Hz), 7.91 (1H,d, J = 8.0 Hz), 7.55 (1H, t, J = 7.6 Hz), 7.47 (1H, t, J = 7.6 Hz), 6.99 (1H, d, J = 88 Hz), 4.06 ~3.95 (4H, m), 3.03 (3H, d, J = 4.8 Hz), 2.79~2.76 (2H, m), 2.50 (2H, m), 2.28 (3H, s), 2.02 (2H, br s). LC-MS: Rt=9.160 min, 389.5[M+H] + .

[0228] Example 2: Analytical Method Powder X-ray Diffraction (PXRD)

[0229] PXRD analysis was performed on a PANalytical X'pert pro, scanning samples between 3 and 35° 2θ.

[0230] The material was packed into a multiwell plate with a Kapton polymer film to support the sample, which was then loaded into a PANalytical diffractometer operating in transmission mode and analyzed using the following experimental conditions:

[0231] [Table 2]

[0232] Polarized Light Microscope (PLM) The presence of crystallinity (birefringence) was determined using an Olympus BX50 polarizing microscope equipped with a Motic camera and image capture software (Motic Images Plus 2.0). All images were recorded using a 20x objective unless otherwise stated.

[0233] Thermogravimetric analysis (TGA) Approximately 5 mg of material was weighed into an open aluminum pan and loaded into a simultaneous thermogravimetric / differential thermal analyzer (TG / DTA) and held at room temperature. The sample was then heated from 25°C to 300°C at a rate of 10°C / min and the change in sample weight was recorded along with the differential temperature events (DTA). Nitrogen was used as the purge gas at 300 cm 3 A flow rate of 1 / min was used.

[0234] Differential Scanning Calorimetry (DSC) Approximately 5 mg of material was weighed into an aluminum DSC pan and unsealed with a perforated aluminum lid. The sample pan was then loaded into a Seiko DSC6200 (equipped with a cooler) that was cooled and held at 25° C. Once a stable heat flow response was obtained, the sample and reference were heated up to 280° C. at a scan rate of 10° C. / min and the resulting heat flow response was monitored.

[0235] 1 H nuclear magnetic resonance ( 1 H NMR) 1 H-NMR experiments were performed on a Bruker PRO500 (frequency: 500 MHz). Experiments were performed in deuterated chloroform, and each sample was prepared at a concentration of approximately 10 mM.

[0236] Gravimetric Vapor Sorption (GVS) Approximately 10-20 mg of sample was placed in a mesh vapor sorption pan and loaded onto a Hiden Analytical IGAsorp Moisture Sorption Analyzer balance.

[0237] The samples were subjected to a ramp profile of 10% increments from 40% to 90% relative humidity (RH), holding the sample at each step until a stable weight was achieved (98% step completion). After completion of the adsorption cycle, the samples were dried to 0% RH using the same procedure and finally returned to the starting point of 40% RH. The weight change during the adsorption / desorption cycle was plotted, allowing the hygroscopicity of the sample to be measured.

[0238] High performance liquid chromatography-ultraviolet detection (HPLC-UV) Instrument: HPLC - Agilent 1100 with UV detector Column: Waters X-Terra MSC18 (ISO x 4.6 x 5μm Column temperature: 30℃ Autosampler temperature: Ambient temperature UV wavelength: 220 nm Injection amount: 5μm Flow rate: 1mL / min Mobile phase A: 0.1% TFA in HO Mobile phase B: 0.1% TFA in MeCN Gradient Program:

[0239] [Table 3]

[0240] Example 3: Initial characterization Compound (I), prepared by the method described in Example 1, was characterized by PXRD, PLM, TG / DTA and DSC.

[0241] Example 4: Rapid Solvent Solubility Screen Thirteen solvents were selected for the rapid solvent solubility screen (see Table 1). 10 mg of multiphasic Compound (I) prepared according to Example 1 was placed into each of the 13 vials and 5 volume aliquots of the appropriate solvent system were added to each vial.

[0242] Between each addition, the mixture was checked for dissolution, and if dissolution was not apparent, the mixture was heated to 40° C. and checked again. This procedure was continued until dissolution was observed or until 100 volumes of solvent had been added.

[0243] [Table 4]

[0244] Example 5: Primary Crystal Morphology Screen The 13 solvents investigated in the solubility screen were used in the primary crystal form screen. Saturated solutions of Compound (I), prepared according to Example 1, were made by adding a volume of each solvent to a 15 mg sample.

[0245] These solutions were then split into four different vials for (1) cooling, (2) crash cooling, (3) evaporation, and (4) anti-solvent addition experiments. The resulting solids were analyzed by PXRD in the first instance.

[0246] Temperature Cycling Based on the results from the solubility screen, an appropriate volume of solvent was added to 15 mg of multiphasic Compound (I) prepared according to Example 1. The resulting slurry was temperature cycled from 5° C. to 25° C. on a hot plate for 72 hours.

[0247] After temperature cycling, the solid sample was separated from the solution and transferred to a PXRD plate for analysis.

[0248] Cooling (4℃) Subsamples were taken from each saturated solution vial from the primary crystal morphology screen and stored in a 4° C. refrigerator to cool.

[0249] Crash cooling (-20℃) Subsamples were taken from each saturated solution vial from the primary crystal morphology screen and stored and cooled in a -20°C freezer.

[0250] evaporation Subsamples were taken from each saturated solution vial from the primary crystal morphology screen and allowed to crystallize by evaporation in uncapped vials at room temperature conditions.

[0251] Addition of antisolvent Subsamples were taken from each saturated solution vial from the primary crystal morphology screen and antisolvent (heptane or tBME) was added in 50 mL aliquots until precipitation ceased or the vial contained a maximum volume of 2.4 mL.

[0252] Example 6: Secondary Crystal Morphology Screen Positive "hits" from the primary crystal morphology screen were scaled up to test reproducibility. Full characterization of solid samples was performed on the dried solids using PXRD, PLM, TG / DTA, and DSC.

[0253] Example 7: 7-day stability study at 40°C and 75% relative humidity 10 mg of Forms 1, 2, 3, 4 and 5 were weighed into separate vials.

[0254] Each vial was placed in an oven at 40° C. and 75% relative humidity for 7 days. Once complete, PXRD analysis was performed on each of these samples.

[0255] Example 8: Competitive Slurrying of Form 1 and Form 5 Saturated solutions of compound (I) in multiphasic form were made in two solutions of dichloromethane, heptane and methanol, respectively.

[0256] 10 mg of Form 1 and Form 5 were added to each vial. Each vial was stirred at ambient conditions for 24 hours, while the other heptane and methanol vials were stirred at 60° C. for 24 hours.

[0257] The remaining dichloromethane vial was stirred for 24 hours at 35° C. The resulting sample was analyzed by PXRD and then dried overnight on a vacuum pump prior to DSC analysis of the sample.

[0258] Example 9: Large scale scale-up of Form 1 To create a slurry, acetone (3.5 mL) was added to a vial containing 1.236 g of multiphasic Compound (I) prepared according to Example 1. The resulting solution was then subjected to temperature cycling for 24 hours.

[0259] The crystallinity morphology was then confirmed using PXRD, TGA, DSC, and PLM.

[0260] Example 10: Further stability studies - Form 1 10 mg of Form 1 was weighed into each of 12 vials. Four vials were subjected to each of the following storage conditions: (1) 40° C., open cap; (2) 75% RH, open cap; (3) 50° C., closed cap; and (4) room temperature, closed cap.

[0261] Two (2) vials were extracted at each of the two and four week time points and PXRD, NMR and HPLC analyses were performed.

[0262] Example 11: Rapid Solvent Solubility Screen Compound (I) was found to be practically insoluble (<10 mg / mL) in most of the solvents evaluated. Two solvents showed a solubility of >10 mg / mL, with Compound (I) being very soluble in dichloromethane (200 mg / mL) and sparingly soluble in methanol (12.5 mg / mL).

[0263] PXRD analysis was performed on undissolved samples from the solubility screen. The PXRD of the undissolved samples from the solubility screen all showed the same morphology with clear similarities to the multiphase morphology. The solvent is clearly evident with peaks at 2θ=12 and 28.5°.

[0264] [Table 5]

[0265] FIG. 7 provides the results from PXRD analysis of undissolved material samples from the solubility screen.

[0266] Example 12: Primary Crystal Morphology Screen Five crystalline forms of Compound (I) were discovered during the primary screen (Table 3).

[0267] Form 1 was found to be the most commonly identified form and was isolated from slurries of acetone; acetonitrile; tBME; dimethylformamide; ethanol; ethyl acetate; heptane; methanol; propan-2-ol; tetrahydrofuran; and toluene (see Figure 8).

[0268] Form 2 was isolated from a slurry in 1,4-dioxane and Form 3 was isolated from a slurry in dichloromethane (see FIG. 9).

[0269] Form 4 was isolated from a slurry in evaporating dichloromethane (see FIG. 10).

[0270] Form 5 was isolated from a slurry in dichloromethane anti-solvent (see Figure 11).

[0271] [Table 6]

[0272] Due to the insolubility of compound (I) in most solvent systems, only dichloromethane produced sufficient material for analysis in evaporation, antisolvent addition and crash-cooling experiments.

[0273] Example 13: Competitive Slurrying of Form 1 and Form 5 PXRD analysis of the wet slurry residues showed that all runs promoted the transition from Form 5 to Form 1 (see Figure 53).

[0274] Table 4 shows the DSC traces of competitive slurry samples (see Figures 54-59) compared to the DSC traces of Forms 1 and 5 identified from the secondary screen (see Figures 22 and 48, respectively).

[0275] FIG. 53 shows the PXRD of competitive slurrying of Form 1 and Form 5.

[0276] FIG. 54 shows the DCS analysis of samples from the heptane ambient temperature competitive slurry.

[0277] FIG. 55 shows the DSC analysis of a sample from a competitive slurry in heptane at 60° C.

[0278] FIG. 56 shows the DSC analysis of samples from the methanol ambient temperature competitive slurry.

[0279] FIG. 57 shows the DSC analysis of samples from the competitive slurry in methanol at 60° C.

[0280] Figure 58 shows the DSC analysis of a sample from a DCM ambient temperature slurry. It can be seen that the ambient temperature DSC trace of dichloromethane shows an initial endothermic event.

[0281] Subsequent PXRD analysis of the dried sample showed a transition to a mixture of Form 1 and Form 3 during the drying process (see Figure 60).

[0282] [Table 7]

[0283] The embodiments shown and discussed herein are intended only to teach those skilled in the art how to make and use the present invention. In describing the embodiments of the present invention, specific terms are used for the sake of clarity. However, the present invention is not intended to be limited to the specific terms so selected. The above-described embodiments of the present invention can be modified or altered without departing from the present invention, as will be appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the present invention can be practiced otherwise than as specifically described.

Claims

1. Compound (I): 【Chemistry 1】 and a crystalline form of the compound having a 2-theta powder X-ray diffraction (PXRD) pattern peaks of: (a) about 10.0° and 20.7°, the crystalline form being crystalline form 1; (b) about 20.3° and 26.4°, the crystalline form being crystalline form 2; (c) about 6.4° and 13.5°, the crystalline form being crystalline form 3; (d) about 7.0° and 11.4°, and the crystalline form is crystalline form 4; or (e) about 9.2° and 10.3°, the crystalline form being crystalline form 5 A crystalline form characterized by:

2. 2. The crystalline form of claim 1, wherein crystalline Form 1 is characterized by having powder X-ray diffraction pattern peaks at about 6.7°, 8.6°, 10.0°, 11.2°, 16.2°, 20.7°, 22.7°, 25.6°, and 27.6° 2-theta (±0.2° 2θ).

3. 81. The crystalline form of claim 1, wherein crystalline form 1 has an X-ray powder diffraction pattern substantially as shown in FIG.

4. 2. The crystalline form of claim 1, wherein crystalline form 1 is further characterized as having an endothermic peak at about 212-216° C. as shown by DSC.

5. 2. The crystalline form of claim 1, wherein crystalline Form 1 is further characterized as having an endotherm onset at about 209-212° C. as shown by DSC.

6. 2. The crystalline form of claim 1, wherein crystalline form 1 maintains its shape after storage at room temperature for 7 days.

7. 10. A pharmaceutical composition comprising the crystalline form of claim 1 and a pharma- ceutically acceptable carrier.

8. 13. A pharmaceutical composition consisting essentially of the crystalline form of claim 1.

9. The pharmaceutical composition of claim 7, wherein the crystalline form is crystalline form 1.

10. 10. The crystalline form of claim 1 which is substantially purified.

11. 10. A method of treating cancer in a subject comprising therapeutically administering a therapeutically effective amount of the composition of claim 7.

12. 12. The method of claim 11, wherein the cancer is breast, lung, colorectal, liver, pancreatic, lymph node, colon, prostate, brain, head and neck, skin, liver, kidney, blood or heart cancer.

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