Solid forms of 3-(5-fluorobenzofuran-3-yl)-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indol-7-yl)pyrrole-2,5-dione

Abstract

The present disclosure relates to solid forms of 3-(5-fluorobenzofuran-3-yl)-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indol-7-yl)pyrrole-2,5-dione, methods for its preparation, pharmaceutical compositions thereof, and its use in the treatment of disease. [Selected Figure] Figure 1

Description

Technical Field

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 572,603, filed Oct. 16, 2017, which is hereby incorporated by reference in its entirety.

[0002] The present disclosure relates to solid forms of 3-(5-fluorobenzofuran-3-yl)-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indol-7-yl)pyrrole-2,5-dione, processes for their preparation, pharmaceutical compositions thereof, and their use in the treatment of diseases.

Background Art

[0003] 3-(5-Fluorobenzofuran-3-yl)-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indol-7-yl)pyrrole-2,5-dione (“9-ING-41”) has the following chemical structure.

Chemical Formula 1

[0004] 9-ING-41 has been reported to be useful for the treatment of cancers including brain, lung, breast, ovarian, bladder, neuroblastoma, kidney, and pancreatic cancers, as well as for the treatment of traumatic brain injury.

[0005] The structure, properties, and / or bioactivity of 9-ING-41 are described in U.S. Patent No. 8,207,216, Gaisina et al. (From a Natural Product Lead to the Identification of Potent and Selective Benzofuran-3-yl-(indol-3-yl)maleimides as Glycogen Synthase Kinase 3β Inhibitors That Suppress Proliferation and Survival of Pancreatic Cancer Cells, J. Med. Chem. 2009, 52, 1853-1863) and Hilliard et al. (Glycogen synthase kinase 3β inhibitors induce apoptosis in ovarian cancer cells and inhibit in-vivo tumor growth, Anti-Cancer Drugs 2011, 22:978-985).

[0006] Novel solid-state forms of 9-ING-41 (including polymorphs and solvates) are needed. [Overview of the project]

[0007] This disclosure relates to a solid form of 9-ING-41, a process for preparing a solid form of 9-ING-41, a pharmaceutical composition having a solid form of 9-ING-41, and a therapeutic method comprising a step of administering a solid form of 9-ING-41.

[0008] In some aspects, the present disclosure relates to a solid form of 3-(5-fluorobenzofuran-3-yl)-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indole-7-yl)pyrrole-2,5-dione ("9-ING-41") which is crystalline form II.

[0009] This disclosure also provides a process for preparing the solid form of 9-ING-41.

[0010] This disclosure also provides a pharmaceutical composition having a solid form of 9-ING-41.

[0011] The disclosure also provides a method for treating a disease, comprising the step of administering a therapeutically effective amount of the disclosed 9-ING-41 in solid form to a patient who requires it. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows the X-ray powder diffraction (XRPD) map of morphology II of 9-ING-41. [Figure 2] Figure 2 shows the X-ray powder diffraction (XRPD) map of morphology II of 9-ING-41. [Figure 3] Figure 3 shows the differential scanning calorimetry (DSC) profile of 9-ING-41, form II. [Figure 4] Figure 4 shows the thermogravimetric analysis (TGA) profile of morphology II of 9-ING-41. [Modes for carrying out the invention]

[0013] This disclosure relates to the solid form of 9-ING-41, its preparation process, and pharmaceutical compositions having the solid form. This disclosure also relates to the conversion of the described solid form of 9-ING-41 to 9-ING-41, 9-ING-41 salts, and other solid forms thereof.

[0014] The name "9-ING-41" is an alternative name for 3-(5-fluorobenzofuran-3-yl)-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indole-7-yl)pyrrole-2,5-dione, and these names are used interchangeably herein.

[0015] The solid forms of 9-ING-41 according to this disclosure may have advantageous properties selected from at least one of the following: chemical or polymorphic purity, fluidity, solubility, dissolution rate, bioavailability, morphological or crystal habit, chemical stability with respect to polymorphic transformation, thermal and mechanical stability, stability against dehydration and / or storage stability, low hygroscopicity, low residual solvent content, and advantageous processing and handling properties such as compressibility and bulk density.

[0016] In this specification, it may be mentioned that crystalline morphologies are characterized by graphical data such as those “shown” in the figures. Such data include, for example, powder X-ray diffraction (XRPD), differential scanning calorimetry (DSC) thermograms, thermogravimetric analysis (TGA) profiles, and dynamic vapor sorption profiles (DVS). As is well known in the art, graphical data may provide additional technical information that further defines each solid morphology, which cannot necessarily be described by referring only to numerical values ​​or peak positions. Thus, the term “substantially shown” when referring to graphical data in the drawings herein is not necessarily identical to what is depicted herein, but as considered by those skilled in the art, it falls within the range of experimental error or deviation. Those skilled in the art will be able to easily compare the graphical data in the drawings herein with graphical data generated for an unknown crystalline morphology and determine whether the two sets of graphical data characterize the same crystalline morphology or two different crystalline morphologies.

[0017] In this specification, solid and crystalline forms may be referred to as “polymorphically pure” or “substantially free of other forms.” When used in this context, the expression “substantially free of other forms” is understood to mean that the solid form contains about 20% or less, about 10% or less, about 5% or less, about 2% or less, about 1% or less, or 0% of other forms of the compound in question, as measured, for example, by XRPD. Therefore, the solid form of 9-ING-41 described herein as substantially free of other solid forms is understood to contain about 80% (w / w) or more, about 90% (w / w) or more, about 95% (w / w) or more, about 98% (w / w) or more, about 99% (w / w) or more, or about 100% of the solid form of 9-ING-41 in question. Accordingly, in some embodiments of this disclosure, the solid form of 9-ING-41 described may include one or more other solid forms of 9-ING-41 in amounts of about 1% to about 20% (w / w), about 5% to about 20% (w / w), or about 5% to about 10% (w / w).

[0018] When used herein, unless otherwise specified, the XRPD peaks reported herein are measured using CuKα emission and have a value of λ = 1.5419 Å.

[0019] The modifying phrase "about" should be considered to disclose a range defined by the absolute values ​​of two endpoints. For example, the expression "about 2 to about 4" also discloses the range "2 to 4". When used to modify a single number, the term "about" refers to plus or minus 10% of the number shown, and includes the number shown. For example, "about 10%" indicates a range from 9% to 11%, and "about 1" means 0.9 to 1.1.

[0020] As used herein, the term "solvate" refers to a crystalline form in which a solvent is incorporated into the crystalline structure, unless otherwise indicated. When the solvent is water, the solvate is often called a "hydrate." The solvent in a solvate may exist in stoichiometric or non-stoichiometric quantities.

[0021] In some embodiments, this disclosure relates to a solid form of 9-ING-41.

[0022] In some embodiments, the solid form of 9-ING-41 is crystalline form II of 9-ING-41. In other embodiments, the solid form is crystalline form II of 9-ING-41, substantially devoid of other solid forms of 9-ING-41. Crystallical form II of 9-ING-41 exhibits an XRPD substantially as shown in Figure 1.

[0023] The XRPD of 9-ING-41 in crystal morphology II, shown in Figure 1, has a reflection angle (degrees 2-theta ± 0.2 degrees 2-theta), as shown in Table 1. [Table 1]

[0024] In some embodiments of this disclosure, crystalline morphology II of 9-ING-41 is characterized by an XRPD pattern having a peak at one of the angles listed in Table 1. In other embodiments, crystalline morphology II of 9-ING-41 is characterized by an XRPD pattern having multiple peaks at one of the angles listed in Table 1 above. In other embodiments, crystalline morphology II of 9-ING-41 is characterized by an XRPD pattern having two peaks selected from the angles listed in Table 1 above. In other embodiments, crystalline morphology II of 9-ING-41 is characterized by an XRPD pattern containing three peaks selected from the angles listed in Table 1 above. In other embodiments, crystalline morphology II of 9-ING-41 is characterized by an XRPD pattern containing four peaks selected from the angles listed in Table 1 above. In other embodiments, crystalline morphology I of 9-ING-41 is characterized by an XRPD pattern containing five peaks selected from the angles listed in Table 1 above. In another embodiment, the crystalline form II of 9-ING-41 is characterized by an XRPD pattern containing six peaks selected from the angles listed in Table 1 above. In another embodiment, the crystalline form II of 9-ING-41 is characterized by an XRPD pattern containing seven peaks selected from the angles listed in Table 1 above. In another embodiment, the crystalline form II of 9-ING-41 is characterized by an XRPD pattern containing eight peaks selected from the angles listed in Table 1 above. In another embodiment, the crystalline form II of 9-ING-41 is characterized by an XRPD pattern containing nine peaks selected from the angles listed in Table 1 above. In another embodiment, the crystalline form II of 9-ING-41 is characterized by an XRPD pattern containing ten peaks selected from the angles listed in Table 1 above. In another embodiment, the crystalline form II of 9-ING-41 is characterized by an XRPD pattern containing more than ten peaks selected from the angles listed in Table 1 above.

[0025] In some embodiments, crystalline Form II of 9-ING-41 is characterized by an XRPD pattern having a peak at 7.8 degrees ± 0.2 degrees 2-theta. In other embodiments, crystalline Form II of 9-ING-41 is characterized by an XRPD pattern having peaks at 7.8, 12.9, 15.8 and 18.9 degrees ± 0.2 degrees 2-theta. In other embodiments, crystalline Form II of 9-ING-41 is characterized by an XRPD pattern having peaks at 12.9, 18.1, 28.0, and 28.5 degrees ± 0.2 degrees 2-theta.

[0026] In some embodiments of the present disclosure, crystalline Form II of 9-ING-41 is characterized by an XRPD pattern having peaks at three or more of 7.8, 12.9, 15.8, 18.1, 18.9, 19.5, 22.1, 22.7, 28.0 and 28.5 degrees ± 0.2 degrees 2-theta. In some embodiments of the present disclosure, crystalline Form II of 9-ING-41 is characterized by an XRPD pattern having peaks at four or more of 7.8, 12.9, 15.8, 18.1, 18.9, 19.5, 22.1, 22.7, 28.0 and 28.5 degrees ± 0.2 degrees 2-theta. In some embodiments of the present disclosure, crystalline Form II of 9-ING-41 is characterized by an XRPD pattern having peaks at five or more of 7.8, 12.9, 15.8, 18.1, 18.9, 19.5, 22.1, 22.7, 28.0 and 28.5 degrees ± 0.2 degrees 2-theta. In some embodiments of the present disclosure, crystalline Form II of 9-ING-41 is characterized by an XRPD pattern having peaks at six or more of 7.8, 12.9, 15., 18.1, 18.9, 19.5, 22.1, 22.7, 28.0 and 28.5 degrees ± 0.2 degrees 2-theta. Characterized by an XRPD pattern having peaks at seven or more of 7.8, 12.9, 15.8, 18.1, 18.9, 19.5, 22.1, 22.7, 28.0 and 28.5 degrees ± 0.2 degrees 2-theta.

[0027] In some embodiments, crystalline Form II of 9-ING-41 exhibits an XRPD pattern substantially as shown in Figure 2.

[0028] The crystalline form II of 9-ING-41 can be substantially characterized by a DSC thermogram as shown in Figure 3. As shown in Figure 3, the crystalline form II of 9-ING-41 produces an endothermic peak at 218.03 °C. When heated at a rate of 10 °C / min, the peak start temperature is 216.14 °C and the melting enthalpy is 53.441 J / g. In some embodiments of the present disclosure, the crystalline form II of 9-ING-41 is characterized by a DSC thermogram having an endothermic peak at about 218 °C. In other embodiments of the present disclosure, the crystalline form II of 9-ING-41 is characterized by a DSC melting enthalpy of about 53 J / g.

[0029] The crystalline form II of 9-ING-41 can be characterized by a TGA profile substantially as shown in Figure 4 when heated at a rate of 10 °C / min. As shown in Figure 4, when the crystalline form II of 9-ING-41 is heated at a rate of 10 °C / min, it loses about 0.3521% of its weight when heated between about 150 °C and about 250 °C.

[0030] In some embodiments of the present disclosure, the crystalline form II of 9-ING-41 is characterized by an XRPD pattern having peaks at 7.8, 12.9, 15.8, 18.1, 18.9, 19.5, 22.1, 22.7, 28.0 and 28.5 degrees ± 0.2 degrees 2-theta, and a DSC thermogram having an endothermic peak at about 218 °C when heated at a rate of 10 °C / min.

[0031] In some aspects, the present disclosure relates to a process for preparing the disclosed solid forms of 9-ING-41. In some aspects, the process comprises concentrating a solution of 9-ING-41 dissolved in a solvent or solvent mixture.

[0032] In some embodiments, the disclosure relates to a process for preparing crystalline form II of 9-ING-41. In some embodiments, the process comprises concentrating a solution of 9-ING-41 dissolved in a mixture of 3-methylbutanol / acetone in a volume ratio of about 1:1 (e.g., in a vacuum or by evaporation). In other embodiments, the process comprises cooling a warm (i.e., above 25°C) solution of 9-ING-41 dissolved in a mixture of isoamyl alcohol:acetonitrile in a volume ratio of about 10:1.

[0033] In other embodiments, the Disclosure encompasses pharmaceutical compositions comprising a solid form of 9-ING-41 of the Disclosure and at least one pharmaceutically acceptable excipient. pharmaceutically acceptable excipients will be known to those skilled in the art. The pharmaceutical compositions can be administered in any convenient dosage form. Typical dosage forms include tablets, capsules, caplets, reconstituteable powders, elixirs, liquids, colloids or other types of suspensions, emulsions, beads, beadlets, granules, microparticles, nanoparticles, and combinations thereof. The amount of composition administered depends on the subject being treated, the subject's body weight, the severity of the condition being treated, the method of administration, and the prescribing physician's judgment. In some embodiments, the pharmaceutical composition comprises a crystalline form II of 9-ING-41 and at least one pharmaceutically acceptable excipient.

[0034] This disclosure also encompasses sterile aqueous or organic solution formulations of 9-ING-41, the formulations being prepared from the solid form of 9-ING-41 of this disclosure. Therefore, in some embodiments, this disclosure includes a process for preparing a pharmaceutical composition which is a solution containing 9-ING-41. In some embodiments, a method for preparing a pharmaceutical composition containing a solution of 9-ING-41 includes the step of dissolving the solid form of 9-ING-41 of this disclosure in a solvent or a mixture of solvents. In some embodiments, this method includes the step of dissolving the crystalline form II of 9-ING-41 in an aqueous solvent, a non-aqueous solvent, or a mixture of aqueous and / or non-aqueous solvents. The aqueous solvent, non-aqueous solvent, or mixture of aqueous and / or non-aqueous solvents in the embodiments may contain other solubilizing components, such as polyethylene glycol, benzyl alcohol, polysorbate, tocopheryl polyethylene glycol succinate, as well as other surfactants, solubilizers, or other pharmaceutically acceptable excipients. In other embodiments, the process includes dissolving crystalline form II of 9-ING-41 in an aqueous solvent.

[0035] The solid form of 9-ING-41 as defined herein, as well as its pharmaceutical compositions or formulations, can be used as agents for the treatment of traumatic brain injury, and in particular for the treatment of cancers, including brain tumors, lung cancer, breast cancer, ovarian cancer, bladder cancer, neuroblastoma, kidney cancer, and pancreatic cancer.

[0036] While this disclosure has been described with reference to certain preferred embodiments, other embodiments will become apparent to those skilled in the art from a review of the specification. This disclosure is further described by reference to the following embodiments. It will be apparent to those skilled in the art that many modifications to both materials and methods can be made without departing from the scope of this disclosure.

[0037] Analysis method XRPD analysis XRPD analysis was performed using an X-ray diffractometer (Bruker D8 Advanced) equipped with a LynxEye detector. The instrument parameters are as follows: Scan: 3°(2θ) to 40°(2θ) Increment: 0.02°(2θ) Scanning speed: 0.3 seconds / process Voltage: 40KV Current: 40mA Rotation: On Sample Hold: Sample holder with zero background

[0038] The sample was scanned using a D2 phaser X-ray powder diffractometer (Bruker) in 0.02°(2θ) steps from 3° to 40°(2θ). The tube voltage and current were 30kV and 10mA, respectively.

[0039] TGA analysis TGA analysis was performed using a TA Instruments TGA Q500 or Discovery TGA 55 (TA Instruments, US). Samples were placed in a tar-coated open aluminum pan and heated from room temperature to the final temperature at a rate of 10°C / min.

[0040] DSC analysis DSC analysis was performed using a DSC Q200 or Discovery DSC 250 (TA Instruments, US). Weighted samples were placed in a DSC pinhole pan, and their weight was accurately recorded. Samples were heated to the final temperature at a rate of 10°C / min.

[0041] DVS analysis DVS analysis was performed using IGAsorp (HidenIsochema Ltd.). In the isothermal test, the chamber temperature was maintained at a constant 25.0 ± 1.0°C in a water bath.

[0042] Polarizing microscope (PLM) PLM analysis was performed using a polarizing microscope, ECLIPSE LV100POL (Nikon, JPN).

[0043] HPLC analysis Equipment: Agilent 1260 Infinity series Diluent: Acetonitrile Flow rate: 1.5mL / min Mobile phase: A: 0.05% TFA in water B: 0.05% TFA in acetonitrile Injection volume: 1μL Column: XDB-C18, 4.6*50mm, 1.8μm Column temperature: 40℃ Detection: 220nm Execution time: 8 minutes (2-minute delay before the next injection) Gradient (T / B%): 0.0 / 30, 6.0 / 100, 8.0 / 100

[0044] Examples Example 1: Preparation of 9-ING-41 Crude 9-ING-41 can be obtained by the general method described in U.S. Patent No. 8,207,216 and Gaisina et al. (From a Natural Product Lead to the Identification of Potent and Selective Benzofuran-3-yl-(indol-3-yl)maleimides as Glycogen Synthase Kinase 3β Inhibitors That Suppress Proliferation and Survival of Pancreatic Cancer Cells, J. Med. Chem. 2009, 52, 1853-1863).

[0045] Example 2: Preparation of 9-ING-41 crystal morphology I Crystal morphology I of 9-ING-41 can be prepared as follows. Synthesis of Intermediate 1 [Case 2]

[0046] In a 3 L four-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, 6-nitro-2H-1,3-benzodioxol-5-carbaldehyde (200 g, 1.02 mol, 1.00 equivalent), ammonium acetate (200 g, 2.59 mol, 2.53 equivalents), acetic acid (2 L), and nitromethane (313 g, 5.13 mol, 5.00 equivalent) were added. The solution was stirred at 100°C for 12 hours. The reaction was repeated three times. The solution was combined and diluted with 20 L of water. The resulting solution was extracted with 3 × 10 L of ethyl acetate, and the organic layer was combined. The mixture was washed with 3 × 10 L of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. This yielded 450 g (crude) of 5-nitro-6-[(E)-2-nitroethenyl]-2H-1,3-benzodioxol (1) as a dark green solid. Synthesis of Intermediate 2 [C3]

[0047] Iron (120 g, 2.14 mol, 17.01 equivalents) was slowly added under nitrogen at 80°C to a suspension of silica gel (120 g) in 5-nitro-6-[(Z)-2-nitroethenyl]-2H-1,3-benzodioxole (30 g, 125.97 mmol, 1.00 equivalent), acetic acid (300 mL), toluene (200 mL), and cyclohexane (400 mL). The resulting black mixture was stirred at 80°C for 8 hours. The reaction was repeated 10 times. The reaction mixture was combined. The solid was filtered off. The filtrate was concentrated under vacuum, and the residue was passed through a silica gel column using ethyl acetate / petroleum ether (1 / 5). The collected fractions were combined and concentrated under vacuum to obtain 67.3 g (33%) of 2H,5H-[1,3]dioxolo[4,5-f]indole(2) as an off-white solid. Synthesis of Intermediate 3 [C4]

[0048] Sodium hydride (19.9 g, 497.50 mmol, 1.18 equivalents, 60%) was gradually added to a solution of 2H,3H,5H-fluoro[2,3-f]indole (67.3 g, 422.78 mmol, 1.00 equivalent) in N,N-dimethylformamide (1.3 L) under nitrogen at 0°C. The mixture was stirred at 0°C for 1 hour, and CH3I (70.9 g, 499.51 mmol, 1.18 equivalents) was added dropwise. The resulting solution was stirred at room temperature for 3 hours. The solution was quenched by adding 1 L of ice water. The resulting solution was extracted with 3 × 1 L of ethyl acetate, and the organic layer was combined. The mixture was washed with 3 × 1 L of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was traced on a silica gel column using ethyl acetate / petroleum ether (1 / 10). The collected fractions were combined and concentrated under vacuum to obtain 71 g (97%) of 5-methyl-2H,3H,5H-fluoro[2,3-f]indole (3) as a pale yellow solid. Synthesis of Intermediate 4 [C5]

[0049] 2-chloro-2-oxoethyl acetate (220 g, 1.61 mol, 3.96 equivalents) was added dropwise to a solution of 5-methyl-2H,3H,5H-fluoro[2,3-f]indole (70.4 g, 406.44 mmol, 1.00 equivalent) in ethyl ether (1.6 L) at 0°C under nitrogen. The resulting solution was warmed to room temperature and stirred for 4 hours. The reaction was slowly quenched by adding 2 L of ice water, and the pH of the resulting solution was adjusted to 9 with Na2CO3. The resulting mixture was extracted with 3 × 1.5 L of ethyl acetate. The organic layer was compounded, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain 92.8 g (84%) of ethyl 2-[5-methyl-2H,3H,5H-fluoro[2,3-f]indole-7-yl]-2-oxoacetate (4) as a pale yellow solid.

[0050] 1H NMR (300 MHz, DMSO-d6): δ 8.28(s,4H),7.56(s,4H),7.27(s,4H),6.17(s,1H),6.08(s,8H),4.35(q,J=7.1 Hz,7H),3.85(s,11H),3.35(s,2H),1.35(t,J=7.1 Hz,11H),1.25(s,2H). Synthesis of Intermediate 5 [C6]

[0051] In a 10 L four-necked round-bottom flask, 2-bromo-4-fluorophenol (500 g, 2.62 mol, 1.00 equivalent), N,N-dimethylformamide (5 L), potassium carbonate (1253 g, 9.07 mol, 3.46 equivalents), and (2E)-4-bromobuto-2-enoate ethyl (1010 g, 5.23 mol, 2.00 equivalents) were added. The resulting solution was stirred at room temperature for 12 hours. The solid was collected by filtration. Next, 15 L of water was added to quench the reaction, and the mixture was extracted with 3 × 10 L of ethyl acetate. The organic layer was combined and washed with 4 × 20 L of brine. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was traced on a silica gel column using ethyl acetate / petroleum ether (1 / 20). The collected fractions were combined and concentrated under vacuum to obtain 500 g (63%) of ethyl(2E)-4-(2-bromo-4-fluorophenoxy)but-2-enoate (5) as a white solid. Synthesis of Intermediate 6 [C7]

[0052] In a 2 L three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, ethyl(2E)-4-(2-bromo-4-fluorophenoxy)but-2-enoate (125 g, 412.37 mmol, 1.00 equivalent), benzyltriethylazanium chloride (99 g, 434.64 mmol, 1.05 equivalent), sodium formate dihydrate (45.1 g), Pd(OAc)2 (2.9 g, 12.92 mmol, 0.03 equivalent), sodium carbonate (92 g, 868.01 mmol, 2.10 equivalent), and N,N-dimethylformamide (1.25 L) were added. The resulting solution was stirred at 80°C for 12 hours. The reaction was repeated four times. The reaction mixtures were combined, and the solid was filtered off. The filtrate was diluted with 10 L of brine and extracted with 3 × 5 L of ethyl acetate. The organic layer was combined and washed with 4 × 6 L of brine. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was passed through a silica gel column using ethyl acetate / petroleum ether (1 / 20). The collected fraction was combined and concentrated under vacuum. This yielded 258 g (crude) of ethyl 2-(5-fluoro-1-benzofuran-3-yl)acetate (6) as a pale yellow oil. Synthesis of Intermediate 7 [C8]

[0053] In a 5 L round-bottom flask, 147 g (661.53 mmol, 1.00 equivalent) of ethyl 2-(5-fluoro-1-benzofuran-3-yl), 1 L of methanol, 1 L of tetrahydrofuran, 1 L of water, and 47.7 g (1.99 mol, 3.01 equivalent) of LiOH were added. The resulting solution was stirred at room temperature for 3 hours. The reaction was repeated twice. The mixture was concentrated under vacuum and then extracted with 1 L of dichloromethane. The aqueous layer was collected, and the pH of the layer was adjusted to 1-3 with hydrogen chloride (1 mol / L). The resulting solution was extracted with 3 × 1 L of ethyl acetate, and the combined organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. This yielded 160 g (62%) of 2-(5-fluoro-1-benzofuran-3-yl)acetic acid (7) as a white solid. Synthesis of Intermediate 8 [C9]

[0054] In a 10 L round-bottom flask, 2-(5-fluoro-1-benzofuran-3-yl)acetic acid (160 g, 824.1 mmol, 1.00 equivalent), NH4Cl (436 g, 8.16 mol, 9.89 equivalents), N,N-dimethylformamide (6 L), DIEA (1064 g, 8.24 mol, 9.99 equivalents), and HATU (376 g, 988.88 mmol, 1.20 equivalents) were added. The resulting solution was stirred at room temperature for 12 hours. The resulting solution was diluted with 10 L of water. The solid was collected by filtration to obtain 126 g (78%) of 2-(5-fluoro-1-benzofuran-3-yl)acetamide (8) as a white solid. Synthesis of 9-ING-41 in crystalline form I [C10]

[0055] t-BuOK (1200 mL, 1 mol / L in THF) was added dropwise to a solution of ethyl 2-[5-methyl-2H,3H,5H-fluoro[2,3-f]indole-7-yl]-2-oxoacetate (100 g, 365.9 mmol, 1.00 equivalent), 2-(5-fluoro-1-benzofuran-3-yl)acetamide (72 g, 372.7 mmol, 1.02 equivalent), and tetrahydrofuran (3 L) at 0°C under nitrogen. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was cooled to 0°C, poured into 2 L of saturated NH4Cl (in water), and extracted with 4 × 2 L of dichloromethane. The organic layer was compounded, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was traced on a silica gel column using ethyl acetate / dichloromethane / petroleum ether (1 / 1 / 5). The collected fractions were combined and concentrated under vacuum to obtain 107.9 g (74%) of 3-(5-fluoro-1-benzofuran-3-yl)-4-[5-methyl-2H,5H-[1,3]dioxolo[4,5-f]indole-7-yl]-2,5-dihydro-1H-pyrrole-2,5-dione as a red solid. This red solid is 9-ING-41 crystalline form I. MS-ESI:[M+H]+=405.

[0056] Example 3: Preparation of 9-ING-41 crystal form II Crystallographic form II of 9-ING-41 was prepared by the slow evaporation of a solution of 9-ING-41 as follows: 29.12 mg of 9-ING-41 (form I) was weighed into a glass vial and 3 mL of 3-methylbutanol was added. 80.43 mg of 9-ING-41 (form I) was weighed into another glass vial and 3 mL of acetone was added. The resulting suspension was filtered, and 100 μL of each filtrate was mixed in the wells of a 96-well plate. The mixture was evaporated under ambient conditions in a laboratory fume hood to obtain solid 9-ING-41 crystallographic form II. This material was used to produce the XRPD shown in Figure 1.

[0057] Example 4: Preparation of 9-ING-41 crystal form II Approximately 100 mg of 9-ING-41 form I was weighed into a vial. 7 ml of a 10:1 (vol / vol) mixture of isoamyl alcohol and acetonitrile was added to the vial. The mixture was heated to 80°C to obtain a solution, which was then filtered and inoculated with form II 9-ING-41. The mixture was stirred while cooling to room temperature and crystallized. XRPD showed that the resulting solid was form II.

[0058] Example 5: Preparation of 9-ING-41 crystal form II Approximately 2.0 g of Form I was weighed into a vial. 150 ml of a 10:1 (vol / vol) mixture of isoamyl alcohol and acetonitrile was added to the vial. The mixture was heated to 90°C to obtain a solution, which was then filtered and seeded with Form II 9-ING-41. The mixture was stirred while cooling to room temperature and crystallized. The crystalline solid (1.46 g) was characterized by PLM, XRPD, DSC, and TGA. PLM showed that the crystalline solid was a crystalline mass of irregular chunks. XRPD (see Figure 2) showed that the crystalline solid was Form II. DSC showed an initial endothermic reaction at a starting temperature of 216°C due to the melting of Form II, followed by exothermic reaction due to the crystallization of Form I, followed by a second endothermic reaction at a starting temperature of 228°C (see Figure 3). This was due to the melting of Form I. TGA showed a morphology that decreased by approximately 0.4% by weight before 250°C. See Figure 4.

[0059] Example 6: Mechanical and pressure effects 10.0 mg of 9-ING-41 form II was weighed into a mortar and ground for 5 minutes. The remaining solid was collected and analyzed by XRPD. The ground solid remained as 9-ING-41 form II.

[0060] The 4.50 mg Form II was compressed into tablets. The tablets were then ground into a powder and analyzed by XRPD. Compression did not affect the crystalline morphology.

[0061] Example 7: Heat treatment - Preparation of amorphous 9-ING-41 Form II of 9-ING-41 was heated by DSC at a rate of 10°C / min to a final temperature of 260°C. The molten product was immediately placed in an ice bath and held for 15 minutes. XRPD analysis of the residue revealed that the material was amorphous.

[0062] 9-ING-41 form II was heated to a final temperature of 260°C at a rate of 10°C / min, then cooled to -40°C, reheated to 260°C at the same heating rate, and then cooled to 40°C. XRPD analysis of the residue revealed that the material was amorphous.

[0063] Example 8: Solubility study - 24 hours Suspensions were prepared by adding 1 mL each of 10 different solvents in a vial to either form I or form II of 9-ING-41. The samples were shaken at 2000 rpm in a shaker at room temperature for 24 hours. The mixtures were visually examined to confirm saturation. After 24 hours, 1 mL of each suspension was filtered into separate clear glass vials using a 0.45 μm nylon microfiltration membrane. The filtrates were analyzed by HPLC, and the remaining solids were analyzed using XRPD. The solubility results are shown in Table 2 below. [Table 2]

[0064] In experiments where morphology I was the starting material, the remaining solids remained in morphology I.

[0065] In experiments where morph II was the starting material, the remaining solid underwent a change in morphology, except in the experiments with water and sesame oil. In the water and sesame oil experiments, the remaining solid was morph II. In the ethanol experiment, the remaining solid was the solvate. In the other experiments, the solid appeared to be morph I.

[0066] Example 9: Solubility study - 1 hour Step 1: A sufficient amount of Form I was added to 1 mL of solvent (EtOH, Labrasol, benzyl benzoate, benzyl alcohol, PEG400:Tween80:EtOH (75:8:17) and PEG300:BzOH:water (65:10:25)) to prepare a suspension. The sample was shaken at 2000 rpm in a shaker at room temperature for 24 hours.

[0067] Step 2: To ensure saturation, the vial was visually inspected. A 1 mL suspension was filtered through a 0.45 μm nylon microfiltration membrane over 24 hours into another clean, clear glass vial. The filtrate was analyzed by HPLC.

[0068] Step 3: Excess form II was added to each of the six residual filtrates from Step 2 to prepare suspensions. The samples were shaken in a shaker at 2000 rpm for 1 hour. After 1 hour, the suspensions were filtered through a 0.45 μm nylon microfiltration membrane into another clean glass vial. The filtrates were analyzed by HPLC, and the remaining solids were analyzed using XRPD. [Table 3]

[0069] The remaining solids in labrasol, benzyl benzoate, benzyl alcohol, and PEG400:Tween80:EtOH (75:8:17) were in morphology I, indicating that morphology II of the solid rapidly converts to morphology I. The crystalline morphology of the remaining solids did not change in EtOH and PEG300:BzOH:water (65:10:25).

[0070] Example 10: Solubility in sesame oil The solubility of 9-ING-41 in sesame oil cannot be measured by HPLC. Solubility was estimated by adding a weighed portion of Form I (or Form II) to sesame oil. The sample was stirred at room temperature for 24 hours, and dissolution was confirmed by visual inspection of the vial. Solids were added in this manner until they no longer dissolved after 24 hours. The results are shown in Table 4 below. [Table 4]

Claims

1. A crystalline form of 3-(5-fluorobenzofuran-3-yl)-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indol-7-yl)pyrrole-2,5-dione, characterized by an X-ray powder diffraction pattern with peaks at 7.8, 12.9, 15.8, 18.1, 18.9, 28.0, and 28.5 degrees ± 0.2 degrees 2-theta on a 2-theta scale of lambda = 1.54 angstroms (Cu Kα), and a differential scanning calorimetry (DSC) thermogram with an endothermic peak at about 218°C when heated at a rate of 10°C / min.

2. 2. The crystal of claim 1, characterized by an X-ray powder diffraction pattern having peaks at 7.8, 12.9, 15.8, 18.1, 18.9, 19.5, 22.1, 22.7, 28.0, and 28.5 degrees ±0.2 degrees 2-theta on a 2-theta scale of lambda = 1.54 angstroms (Cu Kα).

3. A method for preparing the crystals according to any one of claims 1 to 2, comprising: (i) adding 3-methylbutanol to 3-(5-fluorobenzofuran-3-yl)-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indol-7-yl)pyrrole-2,5-dione to form a first suspension; (ii) adding acetone to 3-(5-fluorobenzofuran-3-yl)-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indol-7-yl)pyrrole-2,5-dione to form a second suspension; (iii) filtering the first and second suspensions separately and combining the resulting filtrates to form a mixture; (iv) evaporating the mixture to obtain the crystals; A method comprising:

4. A pharmaceutical composition comprising the crystal of any one of claims 1 to 2 and a pharmaceutically acceptable excipient.

5. 10. Use of the crystals of any one of claims 1 to 2 in the manufacture of a medicament for treating cancer in a patient.

6. 6. The use of claim 5, wherein the cancer is brain cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, neuroblastoma, kidney cancer, or pancreatic cancer.

7. 10. Use of the crystals of any one of claims 1 to 2 in the manufacture of a medicament for treating traumatic brain injury in a patient.

8. A method for preparing the crystals according to any one of claims 1 to 2, comprising: (i) adding isoamyl alcohol and acetonitrile to 3-(5-fluorobenzofuran-3-yl)-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indol-7-yl)pyrrole-2,5-dione in a volume ratio of about 10:1 and heating at 80° C. or higher to form a solution; (ii) filtering the solution and then seeding the mixture with crystals, the crystals used for seeding being crystals of 3-(5-fluorobenzofuran-3-yl)-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indol-7-yl)pyrrole-2,5-dione, characterized by an X-ray powder diffraction pattern with peaks at 7.8, 12.9, 15.8, 18.1, 18.9, 28.0, and 28.5 degrees ± 0.2 degrees 2-theta on a 2-theta scale of lambda = 1.54 angstroms (Cu Kα), and a differential scanning calorimetry (DSC) thermogram with an endothermic peak at about 218°C when heated at a rate of 10°C / minute; (iii) cooling the mixture to allow crystallization; A method comprising:

9. A crystal according to any one of claims 1 to 2 for use in the treatment of cancer.

10. 10. The crystal of claim 9, wherein the cancer is brain cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, neuroblastoma, kidney cancer, or pancreatic cancer.

11. A crystal according to any one of claims 1 to 2 for use in treating traumatic brain injury.

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