RU2773843C1 - Plinabulin composites - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a method for producing plinabulin monohydrate, including combination of plinabulin and the first system of solvents to form the first mixture, where the specified first system of solvents contains ethanol, heating of the first mixture to a temperature from 50°C to 90°C until the complete dissolution of plinabulin, addition of water to the first mixture to obtain the second mixture, and cooling the second mixture to form a precipitate.

EFFECT: new production method is developed, which allows for producing plinabulin monohydrate.

23 cl, 34 dwg, 13 tbl, 8 ex

Description

[0001] This application claims priority from U.S. Provisional Application No. 62/191990 entitled "Plinabulin Compositions" filed July 13, 2015, the contents of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Technical field

[0002] The present invention relates to the fields of chemistry and medicine. More specifically, the present invention relates to forms and compositions of plinabulin and their preparation. Description of the Prior Art

[0003] Plinabulin is a synthetic analogue of diketopiperazine phenylakhistin (halimide) found in marine and terrestrial members of the genus Aspergillus. Plinabulin is structurally distinct from colchicine and its combretastatin-like analogs (eg, fosbretabulin) and binds at or near the colchicine binding site on tubulin monomers. Previous studies have shown that plinabulin induced tubulin depolymerization in vascular endothelial cells and monolayer permeability at low concentrations compared to colchicine, and that it induced apoptosis in Jurkat leukemia cells. Studies of plinabulin in monotherapy in patients with advanced malignant tumors (lung, prostate and colon cancer) have shown favorable pharmacokinetic, pharmacodynamic and safety profiles.

BRIEF DESCRIPTION OF THE INVENTION

[0004] Some embodiments relate to plinabulin monohydrate.

[0005] Other embodiments relate to plinabulin monohydrate in crystalline form.

[0006] Some embodiments relate to a plinabulin composition containing more than 90 wt. % plinabulin relative to the total weight of the composition.

[0007] Other embodiments relate to a plinabulin composition containing more than 99 wt. % plinabulin relative to the total weight of molecules in the composition, other than molecules of water, dimethylformamide, ethanol, ethyl acetate, methanol, toluene and acetic acid.

[0008] Some embodiments relate to a plinabulin composition containing plinabulin and not more than about 1.9 wt. % impurities, relative to the total weight of the composition, with the exception of water.

[0009] Other embodiments relate to a plinabulin composition containing plinabulin and not more than about 1 wt. % of impurities other than solvent molecules relative to the total weight of molecules in the composition other than solvent molecules.

[0010] Some embodiments relate to a plinabulin composition containing plinabulin and not more than about 1 wt. % impurities determined by HPLC analysis.

[0011] Some embodiments relate to a method for producing plinabulin monohydrate or a plinabulin composition.

BRIEF DESCRIPTION OF GRAPHICS

[0012] FIG. 1 is an X-ray powder diffraction (XRPD) spectrum of the crystalline form of plinabulin monohydrate.

[0013] FIG. 2 shows a snapshot of a sample containing the crystalline form of plinabulin monohydrate obtained using polarizing microscopy (PLM).

[0014] FIG. 3A shows thermogravimetric analysis (TGA) of the crystalline form of plinabulin monohydrate (Form 1); FIG. 3B demonstrates

results of the analysis of the crystalline form of plinabulin monohydrate (form 1) by digital scanning calorimetry (DSC); and FIG. 3C shows a dynamic vapor sorption (DVS) isotherm plot.

[0015] FIG. 4 is the XRPD spectrum of crystalline form 2.

[0016] FIG. 5 shows a snapshot of a sample containing crystalline form 2 obtained by PLM.

[0017] FIG. 6A shows the TGA results of Crystalline Form 2, and FIG. 6B shows the results of DSC analysis of crystalline Form 2.

[0018] FIG. 7 is the XRPD spectrum of crystalline Form 3.

[0019] FIG. 8 shows a snapshot of a sample containing Crystal Form 3 taken by PLM.

[0020] FIG. 9A shows TGA results of Crystalline Form 3; and FIG. 9B shows the results of DSC analysis of Crystal Form 3.

[0021] FIG. 10 is the XRPD spectrum of crystalline Form 4.

[0022] FIG. 11 shows a snapshot of a sample containing Crystal Form 4 taken by PLM.

[0023] FIG. 12A shows TGA results of Crystalline Form 4; and FIG. 12B shows the results of analysis of crystalline form 4 by digital scanning calorimetry (DSC).

[0024] FIG. 13 is the XRPD spectrum of crystalline Form 5.

[0025] FIG. 14 shows TGA results of crystalline form 5

[0026] FIG. 15 shows the results of DSC analysis of crystalline Form 5.

[0027] FIG. 16 is the XRPD spectrum of crystalline Form 6.

[0028] FIG. 17 shows TGA results for crystalline form 6

[0029] FIG. 18 shows the results of analysis of crystalline Form 6 by DSC.

[0030] FIG. 19 is the XRPD spectrum of crystalline Form 7.

[0031] FIG. 20 shows TGA results of crystalline form 7

[0032] FIG. 21 shows the results of analysis of crystalline Form 7 by DSC.

[0033] FIG. 22 is the XRPD spectrum of crystalline Form 8.

[0034] FIG. 23 shows the TGA results of crystalline form 8.

[0035] FIG. 24 shows the results of analysis of crystalline form 8 by DSC.

[0036] FIG. 25 is the XRPD spectrum of crystalline Form 9.

[0037] FIG. 26 shows TGA results for crystalline form 9

[0038] FIG. 27 shows the results of analysis of crystalline form 9 by DSC.

[0039] FIG. 28 shows the interconversion of polymorphic forms of plinabulin.

[0040] FIG. 29 is a flowchart for preparing a plinabulin monohydrate composition.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0041] Plinabulin, (3Z,6Z)-3-benzylidene-6-{[5-(2-methyl-2-propanyl)-1H-imidazol-4-yl]methylene}-2,5-piperazinedione, is a synthetic analogue of the natural compound phenylakhistine. Plinabulin can be readily prepared by the methods and procedures disclosed in US Pat. Nos. 7,064,201 and 7,919,497, incorporated herein by reference in their entirety. Some embodiments relate to plinabulin polymorphs and solvates (eg, hydrates) and pharmaceutical compositions containing them. Some embodiments include methods of preparation and methods of treatment. In particular, some embodiments relate to plinabulin monohydrate.

Plinabulin monohydrate

[0042] Plinabulin monohydrate (form 1) is a stable crystalline form of plinabulin. The X-ray powder diffraction (PXRD) spectrum of plinabulin monohydrate (Form 1) is essentially the same as shown in FIG. 1, the corresponding peak data of which are presented in tabular form in Table 1.

[0043] Table 1. X-Ray Powder Diffraction (PXRD) Peak Data of Plinabulin Monohydrate (Form 1)

[0044] In some embodiments, pinabulin monohydrate (Form 1) described herein includes a crystalline form characterized by an X-ray powder diffraction pattern containing at least three characteristic peaks selected from the group consisting of peaks at approximately 8.1°, 13.1°, 16.3°, 23.9°, 24.2°, 24.5° and 26.6° 2θ. In some embodiments, pinabulin monohydrate (Form 1) described herein includes a crystalline form characterized by an X-ray powder diffraction pattern containing at least peaks at about 8.1°, 13.1°, 16.3°, 23.9 °, 24.2°, 24.5° and 26.6° 2θ. In some embodiments, pinabulin monohydrate (Form 1) described herein includes a crystalline form characterized by an X-ray powder diffraction pattern containing at least peaks at about 8.1°, 13.1°, 16.1°, 16.3 °, 19.8°, 22.9°, 23.9°, 24.2°, 24.5°, 26.6° and 29.3° 2θ.

[0045] As is well known in the art, due to variations in experimental data when measuring X-ray powder diffraction spectra on different instruments, peak positions are considered equal if the two theta (2θ) values coincide to within 0.2° (i.e., ± 0.2°). For example, the USP states that if the angles of the 10 strongest diffraction peaks match to within ±0.2° those of the reference sample, and the relative intensities of the peaks do not differ by more than 20%, then identity is confirmed. Accordingly, peak positions that differ by no more than 0.2° from the positions given herein are considered identical. Unless otherwise noted, all X-ray diffraction angles given herein are based on a K-alpha copper source.

[0046] FIG. 3B shows the results of analysis of the crystalline form of plinabulin monohydrate (form 1) by digital scanning calorimetry (DSC). As shown in FIG. 3B, the crystalline form of plinabulin monohydrate (Form 1) has a melting point of approximately 267°C; the crystalline form of plinabulin monohydrate (form 1) has a differential scanning calorimetry thermogram with endothermic peaks at approximately 141°C and approximately 267°C.

[0047] The crystalline form of plinabulin monohydrate (Form 1) is more stable than the other polymorphic forms. Plinabulin monohydrate (Form 1) can remain stable during DVS and drying tests compared to other polymorphs that can show mass change and degradation during tests. Plinabulin composition

[0048] Some embodiments relate to a plinabulin composition comprising more than about 50 wt. % plinabulin monohydrate (Form 1) described herein, relative to the total weight of the composition. In some embodiments, the plinabulin composition comprises more than about 75 wt. % plinabulin monohydrate (Form 1) described herein. In some embodiments, the plinabulin composition comprises more than about 90 wt. % plinabulin monohydrate described herein. In some embodiments, the plinabulin composition comprises more than about 95 wt. %) plinabulin monohydrate described herein. In some embodiments, the plinabulin composition comprises more than about 98 wt. % plinabulin monohydrate described herein. In some embodiments, the plinabulin composition comprises more than about 99 wt. % plinabulin monohydrate described herein. In some embodiments, the plinabulin composition comprises plinabulin monohydrate as described herein in an amount of from about 50% to about 99%, from about 60% to about 99%, from about 70% to about 99%, from about 80% to about 99%, from about 90% to about 99%, from about 95% to about 99%, or from about 97.5% to about 99%, based on the total weight of the composition. The remainder of the plinabulin composition may be other forms of plinabulin and/or other chemical compounds.

[0049] Some embodiments relate to a high purity plinabulin composition. In particular, some embodiments relate to a plinabulin composition containing more than about 90 wt. % plinabulin relative to the total weight of the composition. In some embodiments, the plinabulin composition comprises greater than about 92% plinabulin compound. In some embodiments, the plinabulin composition comprises more than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96.5 %, 96%, 98%, 99% or 99.6% plinabulin compound. In some embodiments, the plinabulin composition comprises more than about 99 wt. % plinabulin relative to the total weight of molecules in the composition other than the solvent molecules. In some embodiments, the plinabulin composition comprises more than about 96 wt. %, 97 wt. %, 98 wt. %, 99 wt. % or 99.6 wt. % plinabulin relative to the total weight of molecules in the composition other than the solvent molecules. In some embodiments, the solvent may be water, dimethylformamide, ethanol, ethyl acetate, methanol, toluene, and acetic acid. In some embodiments, the plinabulin in the high purity composition is present, at least in part, in the form of plinabulin monohydrate, as described above.

[0050] In some embodiments, the plinabulin composition comprises more than about 99 wt. % plinabulin relative to the total weight of molecules in the composition, other than molecules of water, dimethylformamide, ethanol, ethyl acetate, methanol, toluene and acetic acid. In some embodiments, the plinabulin composition comprises more than about 80 wt. %, 85 wt. %), 86 wt. %, 87 wt. %, 88 wt. %, 89 wt. %, 90 wt. %, 91, wt. %, 92 wt. %, 93 wt. %, 94 wt. %, 95 wt. %, 96 wt. %, 96.5 wt. %, 96 wt. %, 98 wt. %, 99 wt. % or 99.6 wt. % plinabulin relative to the total weight of molecules in the composition, other than molecules of water, dimethylformamide, ethanol, ethyl acetate, methanol, toluene and acetic acid. In some embodiments, the implementation of plinabulin in the composition is present, at least in part, in the form of plinabulin monohydrate, as described above.

[0051] In some embodiments, the plinabulin composition comprises more than about 99 wt. % plinabulin, as determined by HPLC analysis. In some embodiments, the plinabulin composition comprises more than about 80 wt. %, 85 wt. %, 86 wt. %, 87 wt. %, 88 wt. %, 89 wt. %, 90 wt. %, 91, wt. %, 92 wt. %, 93 wt. %, 94 wt. %, 95 wt. %), 96 wt. %, 96.5 wt. %, 96 wt. %, 98 wt. %, 99 wt. % or 99.6 wt. % plinabulin, as determined by HPLC analysis. In some embodiments, the implementation of plinabulin in the composition is present, at least in part, in the form of plinabulin monohydrate, as described above.

[0052] Some embodiments relate to a low impurity plinabulin composition. The term "impurity" as used herein refers to one or more components of the composition other than plinabulin and water. In some embodiments, the implementation of the impurity may include one or more chemical compounds introduced during the synthesis of plinabulin. In some embodiments, the impurity may include dimethylformamide, ethanol, ethyl acetate, methanol, toluene, acetic acid, and other residual solvent.

[0053] In some embodiments, the implementation of the plinabulin composition includes no more than about 1 wt. % impurities relative to the total weight of the composition. In some embodiments, the implementation of the plinabulin composition includes no more than about 15 wt. %, 14 wt. %, 13 wt. %, 12 wt. %, 11 wt. %, 10 wt. %, 9 wt. %, 8 wt. %, 7 wt. %, 6 wt. %, 5 wt. %, 4 wt. %, 3 wt. %, 2 wt. %, 1 wt. %, 0.97 wt. %, 0.9 wt. %, 0.8 wt. %, 0.6 wt. %, 0.4 wt. % or 0.2 wt. % impurities relative to the total weight of the composition. In some embodiments, the plinabulin composition includes no more than about 1 wt. % impurities relative to the total weight of molecules in the composition other than solvent molecules. In some embodiments, the implementation of the plinabulin composition includes no more than about 15 wt. %, 14 wt. %, 13 wt. %, 12 wt. %, 11 wt. %, 10 wt. %, 9 wt. %, 8 wt. %, 7 wt. %, 6 wt. %, 5 wt. %, 4 wt. %, 3 wt. %, 2 wt. %, 1 wt. %, 0.97 wt. %, 0.9 wt. %, 0.8 wt. %, 0.6 wt. %, 0.4 wt. % or 0.2 wt. % impurities relative to the total weight of molecules in the composition other than solvent molecules.

[0054] In some embodiments, the implementation of the plinabulin composition includes no more than about 1.9 wt. % impurities relative to the total weight of the composition, excluding water. In some embodiments, the implementation of the plinabulin composition includes no more than about 15 wt. %, 14 wt. %, 13 wt. %, 12 wt. %, 11 wt. %, 10 wt. %, 9 wt. %, 8 wt. %, 7 wt. %, 6 wt. %, 5 wt. %, 4 wt. %, 3 wt. %, 2 wt. %, 1 wt. %, 0.97 wt. %, 0.9 wt. %, 0.8 wt. %, 0.6 wt. %, 0.4 wt. % or 0.2 wt. % impurities relative to the total weight of the composition, excluding water. In some embodiments, the plinabulin composition includes no more than about 1 wt. % impurities relative to the total weight of molecules in the composition, other than solvent molecules, with the exception of water. In some embodiments, the implementation of the plinabulin composition includes no more than about 15 wt. %, 14 wt. %, 13 wt. %, 12 wt. %, 11 wt. %, 10 wt. %, 9 wt. %, 8 wt. %, 7 wt. %, 6 wt. %, 5 wt. %, 4 wt. %, 3 wt. %, 2 wt. %, 1 wt. %, 0.97 wt. %, 0.9 wt. %, 0.8 wt. %, 0.6 wt. %, 0.4 wt. % or 0.2 wt. % impurities, relative to the total mass of molecules in the composition, other than solvent molecules, with the exception of water.

[0055] In some embodiments, the plinabulin composition comprises no more than about 0.9 wt. % of impurities other than dimethylformamide, ethanol, ethyl acetate, methanol, toluene, and acetic acid relative to the total weight of molecules in the composition other than water, dimethylformamide, ethanol, ethyl acetate, methanol, toluene, and acetic acid. In some embodiments, the plinabulin composition includes no more than about 5 wt. %, 4 wt. %, 3 wt. %, 2 wt. %, 1 wt. %, 0.97 wt. %, 0.9 wt. %, 0.8 wt. %, 0.6 wt. %, 0.4 wt. % or 0.2 wt. % of impurities other than dimethylformamide, ethanol, ethyl acetate, methanol, toluene, and acetic acid relative to the total weight of molecules in the composition other than water, dimethylformamide, ethanol, ethyl acetate, methanol, toluene, and acetic acid.

[0056] In some embodiments, the plinabulin composition includes no more than about 1 wt. % impurities as determined by HPLC analysis. In some embodiments, the plinabulin composition includes no more than about 5 wt. %, 4 wt. %, 3 wt. %, 2 wt. %, 1 wt. %, 0.97 wt. %, 0.9 wt. %, 0.8 wt. %, 0.6 wt. %, 0.4 wt. % or 0.2 wt. % impurities as determined by HPLC analysis.

How to obtain

[0057] Some embodiments relate to a process for preparing plinabulin monohydrate or a plinabulin composition described herein, comprising: mixing plinabulin and a first solvent system to form a first mixture, heating the first mixture to a temperature of about 50°C to 90°C, and cooling the first mixture to form the first precipitate.

[0058] In some embodiments, the implementation of the method further includes filtering before cooling the first mixture. In some embodiments, the method further comprises adding water to the first mixture prior to heating.

[0059] In some embodiments, the implementation of the method described herein further includes filtering the first precipitate. In some embodiments, the method described herein further comprises washing the first precipitate.

In some embodiments, the first solvent system may be water, alcohol, or a mixture of water and alcohol.

[0060] In some embodiments, the alcohol is selected from methanol, ethanol, isopropyl alcohol, t-butyl alcohol, and n-butyl alcohol; or mixtures thereof.

[0061] In some embodiments, the alcohol is ethanol.

In some embodiments, heating the first mixture includes refluxing the first mixture.

[0062] In some embodiments, the implementation of the first mixture is heated to a temperature of approximately from 70°C to 78°C. In some embodiments, the implementation of the first mixture is heated to a temperature of approximately from 60°C to 90°C. In some embodiments, the implementation of the first mixture is heated to a temperature of approximately from 60°C to 80°C. In some embodiments, the first mixture is heated to the boiling point of ethanol.

[0063] In some embodiments, the method described herein further comprises maintaining the first mixture at a reflux temperature for about 1 hour before cooling the first mixture.

In some embodiments, heating the first mixture comprises heating the first mixture to at least 65°C, and wherein cooling the first mixture comprises cooling the first mixture to a temperature of about 50°C to 60°C.

[0064] In some embodiments, cooling the first mixture includes adding water to the first mixture to form the first precipitate.

[0065] In some embodiments, cooling the first mixture includes stirring the first mixture for at least 4 hours.

In some embodiments, the method described herein further comprises analyzing the first precipitate to determine the plinabulin composition in the first precipitate.

[0066] In some embodiments, the implementation of the method described herein, further includes mixing the first precipitate and the second solvent with the formation of the second mixture and heating the second mixture to a temperature of from about 50°C to 90°C; cooling the second mixture to form a second precipitate; and filtering off the second precipitate and washing the second precipitate.

[0067] In some embodiments, the implementation of the second solvent is water, alcohol, or a mixture of water and alcohol.

[0068] In some embodiments, the alcohol is selected from methanol, ethanol, isopropyl alcohol, t-butyl alcohol, and n-butyl alcohol; or mixtures thereof. In some embodiments, the alcohol is ethanol.

[0069] In some embodiments, heating the second mixture includes refluxing the second mixture.

[0070] In some embodiments, the implementation of the second mixture is heated to a temperature of approximately from 70°C to 78°C.

[0071] In some embodiments, the method described herein includes maintaining the second mixture at a reflux temperature for about 1 hour before cooling the second mixture.

[0072] In some embodiments, the implementation of the cooling of the second mixture includes cooling the first mixture to a temperature of from about 15°C to 30°C.

[0073] In some embodiments, cooling the second mixture includes adding water to the second mixture to form a second precipitate.

[0074] In some embodiments, cooling the second mixture includes stirring the second mixture for at least 4 hours.

[0075] In some embodiments, the first precipitate is washed with alcohol, and the washing alcohol is collected and added to the second mixture prior to heating.

[0076] In some embodiments, the method described herein includes drying the second precipitate.

[0077] In some embodiments, the method described herein includes analyzing the second precipitate to determine the plinabulin composition in the second precipitate.

[0078] In some embodiments, the mixing, cooling, and filtering steps are repeated one or more times depending on the composition of the plinabulin in the second precipitate.

[0079] Some embodiments relate to a process for preparing plinabulin monohydrate or a plinabulin composition, wherein the method includes mixing plinabulin, ethanol, and water to form a mixture. In some embodiments, the implementation of the method includes a mixture.

[0080] In some embodiments, the volume ratio of ethanol to water is approximately 95:5. In some embodiments, the volume ratio of ethanol to water is about 85:15, 90:10, 95:5, 97.5:2.5, or 99:1. In some embodiments, the volume ratio of ethanol to water is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.

[0081] In some embodiments, the mixture of plinabulin and the solvent system is stirred for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 hours. In some embodiments, the mixture is stirred for at least 2 hours.

[0082] In some embodiments, the mixing or mixing is carried out at a temperature of from about 10°C to about 50°C; from about 20°C to about 40°C; from about 25°C to about 35°C or from about 28°C to about 32°C. In some embodiments, the mixing of plinabulin, ethanol and water to form a mixture or mixing of the mixture is carried out at about 20°C, 25°C, 30°C, 35°C or 40°C.

[0083] FIG. 29 shows a block diagram of one method for preparing a composition containing the plinabulin monohydrate form. As shown in FIG. 29, the plinabulin compound and ethanol are added to the reaction flask, and then the mixture is heated to 70-78°C and mixed at this temperature for about 1 hour. Additional ethanol may be added if necessary. The flask is then cooled to approximately 50-60° C., the mixture is filtered and ethanol is used as the wash solvent. The filtered solution and the washing solution are combined and water is added in an amount up to about 10% of the combined solution. The solution is then heated to 70-78°C and mixed at this temperature for approximately 1 hour. A sample is taken for XRPD analysis, and then the solution is cooled to 20±5°C and water is added to it. The mixture is filtered using water as a washing liquid. The filtered product is washed with water and then transferred to drying trays for drying at 40-50° C. for about 24 hours or longer until the product reaches the desired ethanol and water levels.

Crystal form 2 plinabulin

[0084] Some embodiments relate to crystalline form 2 plinabulin and a method for its preparation. Without wishing to be bound by any particular theory, Form 2 is believed to be a solvate of plinabulin with isopropyl alcohol (IPA).

[0085] In some embodiments, plinabulin crystalline form 2 has substantially the same XRPD spectrum as depicted in FIG. 4, the corresponding peak data of which are presented in tabular form in Table 2.

[0086] Crystal form 2 can be further characterized by substantially the same DSC thermogram as depicted in FIG. 6B. As shown in FIG. 6B, crystalline form 2 has a melting point of approximately 267°C; crystalline form 2 plinabulin has a differential scanning calorimetry thermogram with endothermic peaks at approximately 113°C and approximately 267°C.

Crystal form 3 plinabulin

[0087] Some embodiments relate to crystalline form 3 plinabulin and a method for its preparation. Without wishing to be bound by any particular theory, Form 3 is believed to be the anhydrous form of plinabulin.

[0088] In some embodiments, crystalline Form 3 plinabulin has substantially the same XRPD spectrum as depicted in FIG. 7,

the corresponding peak data of which are presented in tabular form in Table 3.

[0089] Crystal form 3 can be further characterized by substantially the same DSC thermogram as depicted in FIG. 9B. As shown in FIG. 9B, crystalline form 3 has a melting point of approximately 264°C; crystalline form 3 has a differential scanning calorimetry thermogram with an endothermic peak at approximately 264°C.

Crystal form 4 plinabulin

[0090] Some embodiments relate to crystalline form 4 plinabulin and a method for its preparation. Without wishing to be bound by any particular theory, Form 4 is believed to be the methanol solvate of plinabulin.

[0091] In some embodiments, crystalline Form 4 plinabulin has substantially the same XRPD spectrum as depicted in FIG. 10, the corresponding peak data of which are presented in tabular form in Table 4.

[0092] Crystal form 4 can be further characterized by substantially the same DSC thermogram as depicted in FIG. 12 V. As shown in FIG. 12B, crystalline form 4 has a melting point of approximately 267°C; crystalline form 4 has a differential scanning calorimetry thermogram with endothermic peaks at approximately 113°C and at approximately 264°C.

Crystal form 5 plinabulin

[0093] Some embodiments relate to crystalline Form 5 plinabulin and a process for its preparation.

[0094] In some embodiments, crystalline Form 5 plinabulin has substantially the same XRPD spectrum as depicted in FIG. 13, the corresponding peak data of which are tabulated in Table 5.

[0095] Crystal Form 5 can further be characterized by substantially the same DSC thermogram as depicted in FIG. 15. As shown in FIG. 15, crystalline form 5 has a melting point of approximately 267°C; crystalline form 5 has a differential scanning calorimetry thermogram with endothermic peaks at approximately 70°C and at approximately 267°C.

Crystal form 6 plinabulin

[0096] Some embodiments relate to crystalline form 6 plinabulin and a method for its preparation.

[0097] In some embodiments, crystalline form 6 plinabulin has substantially the same XRPD spectrum as depicted in FIG. 16, the corresponding peak data of which are tabulated in Table 6.

[0098] Crystal form 6 can further be characterized by substantially the same DSC thermogram as depicted in FIG. 18. As shown in FIG. 18, crystalline form 6 has a melting point of approximately 267°C; crystalline form 6 has a differential scanning calorimetry thermogram with an endothermic peak at approximately 267°C.

Crystal form 7 plinabulin

[0099] Some embodiments relate to crystalline form 7 plinabulin and a method for its preparation.

[0100] In some embodiments, crystalline form 7 plinabulin has substantially the same XRPD spectrum as depicted in FIG. 19, the corresponding peak data of which are tabulated in Table 7.

[0101] Crystal form 7 can be further characterized by substantially the same DSC thermogram as depicted in FIG. 21. As shown in FIG. 21, crystalline form 7 has a melting point of approximately 267°C; crystalline form 7 has a differential scanning calorimetry thermogram with endothermic peaks at approximately 63°C and at approximately 267°C.

Crystal form 8 plinabulin

[0102] Some embodiments relate to crystalline form 8 plinabulin and a method for its preparation.

[0103] In some embodiments, crystalline Form 8 plinabulin has substantially the same XRPD spectrum as depicted in FIG.

22, the corresponding peak data of which are tabulated in Table 8.

[0104] Crystal Form 8 can further be characterized by substantially the same DSC thermogram as depicted in FIG. 24. As shown in FIG. 24, crystalline form 8 has a melting point of approximately 262°C; crystalline form 8 has a differential scanning calorimetry thermogram with endothermic peaks at approximately 74°C and at approximately 264°C.

Crystal form 9 plinabulin

[0105] Some embodiments relate to crystalline form 9 of plinabulin and a method for its preparation.

[0106] In some embodiments, crystalline Form 9 plinabulin has substantially the same XRPD spectrum as depicted in FIG. 25, the corresponding peak data of which are tabulated in Table 9.

[0107] Crystal form 9 can further be characterized by substantially the same DSC thermogram as depicted in FIG. 27. As shown in FIG. 27, crystalline form 9 has a melting point of approximately 267°C; crystalline form 9 has a differential scanning calorimetry thermogram with endothermic peaks at about 63°C, about 119°C, about 267°C and about 289°C.

Transformation of the polymorphic form of plinabulin

[0108] Plinabulin monohydrate (form 1) is the most stable polymorph of the nine polymorphs found. Plinabulin monohydrate (form 1) remains stable during the drying process and during humidity based stability studies (stable when dried to 50°C in vacuum during Saturday and Sunday, no change in solid form when exposed to humidity greater than 95% RH , within 13 days).

[0109] FIG. 28 shows how form 1 can be converted to the other eight polymorphic forms of plinabulin. For example, plinabulin monohydrate (Form 1) can be converted to Form 2 by suspending Form 1 in isopropyl alcohol (approximately 10 times the volume of Form 1) at 30° C. for 3 hours; form 1 can be converted to form 3 by suspending form 1 in ethanol (about 10 times the volume of form 1) at room temperature overnight; form 1 can be converted to form 4 by suspending form 1 in methanol (about 10 times the volume of form 1) at 30° C. overnight; form 1 can be converted to form 5 by suspending form 1 in acetonitrile (ACN) at 30°C and stirring for 3 days; form 1 can be converted to form 6 by obtaining a saturated solution of form 1 in isopropyl alcohol at 15°C and then evaporation in vacuum at 45°C; form 1 can be converted to form 7 by obtaining a saturated solution of form 1 in methanol at 45°C and then evaporation in vacuum at 45°C; form 1 can be converted to form 8 by obtaining a saturated solution of form 1 in ethyl acetate (EtOAC) at 45°and then evaporation in vacuum at 45°C; and Form 1 can be converted to Form 9 by first converting Form 1 to Form 4 and then exposing Form 4 to moisture.

[0110] FIG. 28 also shows how other forms can be converted to Form 1. For example, Forms 2, 3, and 4 can be converted to Form 1 by suspending these forms in a mixture of ethanol and water (95:5 v/v) (volume of mixture of water and ethanol approximately 10 times the volume of the original polymorphic forms) at 30°C for 2 hours; form 2 can be converted to form 6 by drying in a vacuum oven at 45° C. for 5 days; form 4 can be converted to form 7 by drying in a vacuum oven at 45° C. overnight; and Form 4 can be converted to Form 9 by exposure to high humidity.

Introduction and pharmaceutical compositions

[0111] Some embodiments include pharmaceutical compositions comprising the plinabulin polymorph described herein and a pharmaceutically acceptable carrier. Such a composition may be administered to a subject as part of a therapeutic treatment.

[0112] In some embodiments, the implementation of the composition may further include one or more pharmaceutically acceptable diluents. In some embodiments, the pharmaceutically acceptable diluent may include Kolliphor® (polyoxyl-15-hydroxystearate). In some embodiments, the pharmaceutically acceptable diluent may include propylene glycol. In some embodiments, pharmaceutically acceptable diluents may include colliphor and propylene glycol. In some embodiments, the implementation of pharmaceutically acceptable diluents may include collifor and propylene glycol, where collifor is approximately 40 wt. %, and propylene glycol is approximately 60 wt. % relative to the total mass of diluents. In some embodiments, the implementation of the composition may further include one or more other pharmaceutically acceptable excipients.

[0113] To obtain the compositions described herein, standard drug preparation techniques can be used, such as those disclosed in the book Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (2005), incorporated herein in its entirety. document through a link. Accordingly, some embodiments include pharmaceutical compositions comprising: (a) a safe and therapeutically effective amount of a plinabulin polymorph or pharmaceutically acceptable salts thereof; and (b) a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

[0114] Other embodiments include the co-administration of a plinabulin polymorph and an additional therapeutic agent in separate compositions or in the same composition. Therefore, some embodiments include a first pharmaceutical composition comprising: (a) a safe and therapeutically effective amount of a plinabulin polymorph or pharmaceutically acceptable salts thereof, and (b) a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof; and a second pharmaceutical composition containing: (a) a safe and therapeutically effective amount of an additional therapeutic agent and (b) a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof. Some embodiments include a pharmaceutical composition comprising: (a) a safe and therapeutically effective amount of a plinabulin polymorph or pharmaceutically acceptable salts thereof; (b) a safe and therapeutically effective amount of the additional therapeutic agent; and (c) a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

[0115] Administration of the pharmaceutical compositions described herein may be via any of the commonly used routes of administration for agents having similar uses, including, but not limited to, orally, sublingually, buccally, subcutaneously, intravenously, intranasally, topically, transdermally, intradermally , intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally or intraocularly. Oral and parenteral administration are conventional for the treatment of the indications that are the subject of the preferred embodiments.

[0116] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any possible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional vehicle or agent is incompatible with the active ingredient, their use is contemplated in therapeutic compositions. In addition, various adjuvants such as those commonly used in the art may be included. Aspects of incorporating various components into pharmaceutical compositions are described, for example, in Gilman et al. (Eds.) (1990); Goodman and Gilman: The Pharmacological Basis of Therapeutics, 8 th Ed., Pergamon Press, incorporated herein by reference in their entirety.

[0117] Some examples of substances that can serve as pharmaceutically acceptable carriers or components thereof are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and methylcellulose; powder tragacanth; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as tween emulsifiers (TWEENS); wetting agents such as sodium lauryl sulfate; dyes; flavoring agents; tabletting aids, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic solution; and phosphate buffer solutions.

[0118] The compositions described herein are preferably presented in unit dosage form. As used herein, the term "unit dosage form" is a composition containing an amount of a compound or composition suitable for administration to an animal, preferably a mammalian subject, in a single dose, in accordance with good medical practice. However, a drug in the form of a single or unit dosage form does not imply that the dosage form is administered once a day or once per course of treatment. Such dosage forms are provided for administration once, twice, three times, or more times per day, and may be administered as an infusion over a period of time (eg, from about 30 minutes to about 2-6 hours), or administered in as a continuous infusion, and may be administered more than once per course of treatment, although a single administration is not specifically excluded. One of skill in the art will recognize that a formulation is not necessarily intended to be used throughout a course of treatment, and such decisions should be made by those skilled in the art of treatment, not drug formulation.

[0119] Suitable compositions as described above may be in various forms suitable for various routes of administration, for example, oral, sublingual, buccal, nasal, rectal, topical (including transdermal and intradermal), intraocular, intracerebral, intracranial, intrathecal , intra-arterial, intravenous, intramuscular or other related routes of administration. The person skilled in the art will appreciate that oral and nasal compositions include compositions administered by inhalation and are prepared using available techniques. Depending on the particular route of administration desired, various pharmaceutically acceptable carriers well known in the art may be used. Pharmaceutically acceptable carriers include, for example, solid or liquid excipients, diluents, hydrotropes, surfactants, and encapsulating agents. Optional pharmaceutically active agents may be included that do not substantially interfere with the activity of the compound or composition. The amount of carrier used in conjunction with the compound or composition is sufficient to provide a reasonable amount of substance to administer per dose of the compound. Formulation techniques and compositions useful for use in the methods described herein are described in the following references, all of which are incorporated herein by reference: Modern Pharmaceutics, 4th Ed., Chapters 9 and 10 (Banker & Rhodes , editors, 2002); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction to Pharmaceutical Dosage Forms 8th Edition (2004).

[0120] Various oral dosage forms may be used, including solid forms such as tablets, capsules (eg, liquid gel capsules and hard gel capsules), granules, and bulk powders. Tablets may be compressed, triturated, enteric-coated, sugar-coated, film-coated or multi-compressed, may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, anti-caking agents and substances that lower the freezing point. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, substances, freezing point depressants, colorants and flavoring agents.

[0121] Pharmaceutically acceptable carriers suitable for the preparation of unit dosage forms for oral administration are well known in the art. Tablets typically contain conventional pharmaceutically compatible adjuvants such as inert diluents such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose; binders such as starch, gelatin and sucrose; disintegrants such as starch, alginic acid and croscarmellose; lubricants such as magnesium stearate, stearic acid and talc. Glidants such as silica can be used to improve the flow characteristics of the powder mixture. Dyes, such as dyes for the chemical and food industries, may be added to give appearance. Suitable adjuvants for chewable tablets are sweeteners and flavoring agents such as aspartame, saccharin, menthol, peppermint, sucrose and fruit flavors. Capsules typically contain one or more of the solid diluents disclosed above. The choice of carrier compounds depends on aspects of secondary importance, such as taste, cost and storage stability, which are not essential, and can easily be made by a person skilled in the art.

[0122] Oral compositions also include liquid solutions, emulsions, suspensions, and the like. Pharmaceutically acceptable carriers suitable for the preparation of such compositions are well known in the art. Typical carrier components for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. For suspension, typical suspending agents include methylcellulose, sodium carboxymethylcellulose, AVICEL RC-591, tragacanth, and sodium alginate; typical wetting agents include lecithin and polysorbate 80; and typical preservatives include methyl paraben and sodium benzoate. Oral liquid compositions may also contain one or more of the ingredients disclosed above, such as sweeteners, flavoring agents, and coloring agents.

[0123] Such compositions may also be coated by conventional means, typically pH or time dependent coatings, so that the described composition is released in the gastrointestinal tract near the desired topical application or at different times to increase the duration of the desired effect. Such dosage forms typically include, but are not limited to, one or more of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, ethylcellulose, Eudragit-based coatings, waxes, and shellac.

[0124] The compositions described herein may optionally include other active drug substances.

[0125] Other compositions suitable for achieving systemic delivery of the described compounds include sublingual, buccal and nasal dosage forms. Such compositions typically contain one or more soluble excipients such as sucrose, sorbitol and mannitol; and binders such as gum arabic, microcrystalline cellulose, carboxymethylcellulose and hydroxypropylmethylcellulose. Glidants, lubricants, sweeteners, colors, antioxidants, and flavoring agents disclosed above may also be included.

[0126] The composition of the liquid composition intended for topical ophthalmic use is designed so that the dosage form is suitable for topical administration to the eye. Comfort should be increased as much as possible, however, sometimes formulation considerations (eg, drug stability) may result in a less than fully optimal level of comfort. In the event that maximum comfort cannot be achieved, the composition of the fluid may be designed such that the fluid is acceptable to the patient for topical ophthalmic application. In addition, the ophthalmologically acceptable liquid may be packaged either for single use or contain a preservative to prevent contamination from multiple uses.

[0127] For ophthalmic use, solutions or medicaments are often made using physiological saline as the main carrier. Ophthalmic solutions can preferably be maintained at a comfortable pH with a suitable buffer system. The formulations may also contain conventional pharmaceutically acceptable preservatives, stabilizers and surfactants.

[0128] Preservatives that may be used in the pharmaceutical compositions disclosed herein include, but are not limited to, benzalkonium chloride, PHMB (polyhexamethylene biguanide), chlorobutanol, thiomerosal, acetate, and phenylmercury nitrate. A suitable surfactant is, for example, Tween 80. Similarly, various useful carriers can be used in the pharmaceutical compositions disclosed herein. These carriers include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropylmethylcellulose, poloxamers, carboxymethylcellulose, hydroxyethylcellulose, and purified water.

[0129] Tonicity regulators may be added as needed or appropriate. These include, but are not limited to, salts such as sodium chloride, potassium chloride, mannitol and glycerin, or any other ophthalmologically acceptable tonicity regulator.

[0130] Provided that the resulting preparation is ophthalmologically stable, various buffers and pH adjustment agents can be used. For many formulations, the pH is between 4 and 9. Accordingly, buffers include acetate buffers, citrate buffers, phosphate buffers, and borate buffers. If necessary, acids or bases can be used to adjust the pH of these preparations.

[0131] Ophthalmologically acceptable antioxidants include, but are not limited to, sodium metabisulphite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole, and butylated hydroxytoluene.

[0132] Other auxiliary components that can be included in ophthalmic preparations are chelating agents. Disodium edetate (EDTA) is a useful complexing agent, but other chelating agents may be used instead or in combination with it.

[0133] For topical application, creams, ointments, gels, solutions or suspensions, etc. containing the composition disclosed herein can be used. Dosage forms for topical application may generally consist of a pharmaceutically acceptable carrier, co-solvent, emulsifier, penetration enhancer, preservative system and emollient.

[0134] For intravenous administration, the compositions described herein may be dissolved or dispersed in a pharmaceutically acceptable diluent such as saline or dextrose solution. Suitable excipients may be included to achieve the desired pH, including but not limited to NaOH, sodium carbonate, sodium acetate, HCl, and citric acid. In various embodiments, the pH of the finished composition ranges from 2 to 8, or preferably from 4 to 7. Antioxidant excipients may include sodium bisulfite, acetone sodium bisulfite, sodium formaldehyde, sulfoxylate, thiourea, and EDTA. Other non-limiting examples of suitable adjuvants in intravenous formulations may include sodium or potassium phosphate, citric acid, tartaric acid, gelatin, and carbohydrates such as dextrose, mannitol, and dextran. Additional acceptable excipients are described in Powell, et al., Compendium of Excipients for Parenteral Formulations, PDA J Pharm Sci and Tech 1998, 52 238-311 and Nema et al., Excipients and Their Role in Approved Injectable Products: Current Usage and Future Directions, PDA J Pharm Sci and Tech 2011, 65 287-332, both of which are incorporated herein by reference in their entirety. Antimicrobial agents may also be included to form a bacteriostatic or fungistatic solution, including but not limited to phenylmercury nitrate, thiomerosal, benzethonium chloride, benzalkonium chloride, phenol, cresol, and chlorobutanol.

[0135] Compositions for intravenous administration may be provided to caregivers as one or more solid dosage forms that are reconstituted shortly before administration with a suitable diluent such as sterile water, saline, or aqueous dextrose. In other embodiments, the compositions are presented in solution ready for parenteral administration. In other embodiments, the compositions are presented in a solution that must be further diluted prior to administration. In embodiments involving administering a combination of a compound described herein and another agent, such combination may be provided to caregivers as a mixture, or caregivers may mix the two agents prior to administration, or the two agents may be administered separately.

[0136] In some embodiments, a single dose of a plinabulin polymorph or other therapeutic agent may be from about 5 mg/m ² to about 150 mg/m ² body surface area, from about 5 mg/m ² to about 100 mg/m ² area body surface area, from about 10 mg/m ² to about 100 mg/m ² body surface area, from about 10 mg/m ² to about 80 mg/m ² body surface area, from about 10 mg/m ² to about 50 mg /m ² body surface area, from about 10 mg/m ² to about 40 mg/m ² body surface area, from about 10 mg/m ² to about 30 mg/m ² body surface area, from about 13.5 mg/ m ² to about 100 mg/m ² body surface area, from about 13.5 mg/m ² to about 80 mg/m ² body surface area, from about 13.5 mg/m ² to about 50 mg/m ² area body surface, from approximately 13.5 mg/m ² to approximately specifically 40 mg/m ² body surface area, from about 13.5 mg/m ² to about 30 mg/m ² body surface area, from about 15 mg/m ² to about 80 mg/m ² body surface area, from about 15 mg/m ² to about 50 mg/m ² body surface area, or about 15 mg/m ² to about 30 mg/m ² body surface area. In some embodiments, a single dose of a plinabulin polymorph or other therapeutic agent may be from about 13.5 mg/m ² to about 30 mg/m ² body surface area. In some embodiments, a single dose of a plinabulin polymorph or other therapeutic agent may be about 5 mg/m ² , about 10 mg/m ² , about 12.5 mg/m ² , about 13.5 mg/m ² , about 15 mg/m 2 m ² , approximately 17.5 mg/m ² , approximately 20 mg/m ² , approximately 22.5 mg/m ² , approximately 25 mg/m ² , approximately 27.5 mg/m ² , approximately 30 mg/m ² , about 40 mg/m ² , about 50 mg/m ² , about 60 mg/m ² , about 70 mg/m ² , about 80 mg/m ² , about 90 mg/m ² or about 100 mg/m ² area body surface.

[0137] In some embodiments, a single dose of a plinabulin polymorph or other therapeutic agent may be from about 5 mg to about 300 mg, from about 5 mg to about 200 mg, from about 7.5 mg to about 200 mg, from about 10 mg to about 100 mg, about 15 mg to about 100 mg, about 20 mg to about 100 mg, about 30 mg to about 100 mg, about 40 mg to about 100 mg, about 10 mg to about 80 mg, about 15 mg to about 80 mg, about 20 mg to about 80 mg, about 30 mg to about 80 mg, about 40 mg to about 80 mg, about 10 mg to about 60 mg, about 15 mg to about 60 mg, from about 20 mg to about 60 mg, from about 30 mg to about 60 mg, or from about 40 mg to approximately 60 mg. In some embodiments, a single dose of a plinabulin polymorph or other therapeutic agent may be from about 20 mg to about 60 mg, from about 27 mg to about 60 mg, from about 20 mg to about 45 mg, or from about 27 mg to about 45 mg. In some embodiments, a single dose of a plinabulin polymorph or other therapeutic agent may be about 5 mg, about 10 mg, about 12.5 mg, about 13.5 mg, about 15 mg, about 17.5 mg, about 20 mg, about 22 .5 mg, approximately 25 mg, approximately 27 mg, approximately 30 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, approximately 80 mg, approximately 90 mg, approximately 100 mg, approximately 125 mg, approximately 150 mg or approximately 200 mg.

[0138] The period of administration may be a course of treatment lasting several weeks, provided that the tumor is under control and that the regimen is clinically tolerated. In some embodiments, a single dose of the plinabulin polymorph or other therapeutic agent may be administered weekly, and preferably once on each of days 1 and 8 of a three-week (21-day) course of treatment. In some embodiments, a single dose of the plinabulin polymorph or other therapeutic agent may be administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, or daily for a course of treatment lasting one a week, two weeks, three weeks, four weeks or five weeks. Administration may be on the same or different days of each week of treatment.

[0139] The course of treatment may be repeated provided that the regimen is clinically tolerated. In some embodiments, the treatment is repeated n times, where n is an integer ranging from 2 to 30. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, implementation of a new course of treatment can be started immediately after the completion of the previous course of treatment. In some embodiments, a new course of treatment may be initiated after a period of time has elapsed since the completion of the previous course of treatment.

[0140] In some embodiments, the compositions described herein may be used in combination with other therapeutic agents. In some embodiments, the compositions described herein may be administered or used in combination with treatments such as chemotherapy, radiation, and biological therapy.

EXAMPLES

Example 1

[0141] A sample of the plinabulin compound was stirred in ethanol and heated to reflux. Ethanol was added in portions to maintain reflux until all of the sample had dissolved to give a clear yellow solution. A total of 124.7 g of ethanol was required to completely dissolve the sample at reflux. The solution was then allowed to cool and the formation of a precipitate was observed. Precipitation was observed when the temperature of the solution was 49°C. The mixture was reheated to reflux to give a clear yellow solution. The hot solution was transferred to a larger Erlenmeyer flask, rinsing with 9.45 g of ethanol (to simulate hot filtration). To this solution, refluxing, was added 6.6 g of water (approximately 5% water in ethanol). The solution was allowed to cool slowly with stirring. A precipitate was observed when the solution cooled to 70°C. At this point, additional water (128.6 g) was added slowly causing a large amount of solid to precipitate. The solution was allowed to cool to room temperature with stirring. The solids were filtered off at 17°C and washed three times with 20 g of water. A total of 134 g of water was added to the filtrate, resulting in a cloudy solution with little solid to be filtered. An additional 137 g of water was added in an attempt to cause precipitation of additional product, but no additional solid could be isolated. The solid was dried at 40-45° C. for 3 days to give a yield of 4.73 g, 97.5%. XRPD analysis showed the product to be plinabulin monohydrate (Form 1). Karl Fischer analysis showed that the water content was 4.9%.

[0142] Analysis by the Karl Fischer method after drying at 45°C in vacuum for another 70 hours showed that the water content decreased to 4.1%. The XRPD of this sample showed that it was plinabulin monohydrate (Form 1). This sample was placed in a glove box in an open container next to an open water container and the water content was monitored. The water content measured after 4 hours was 5.0%. The measured water content after 18.5 hours was 4.8%, and the result of the Karl Fischer analysis after 51 hours was 4.9%.

Example 2

). Этот образец помещали в перчаточную камеру в открытом контейнере рядом с открытым контейнером с водой и следили за содержанием воды. Измеренное через 4 часа содержание воды составило 5,0%. Измеренное через 18,5 часов содержание воды составило 4,9%, и результат анализа методом Карла Фишера через 51 час составил 5,1%.[0143] A sample of the plinabulin compound (4.92 g) was stirred in ethanol (147.6 g) and heated to reflux (completely soluble at 75°C). The solution was then allowed to cool and the formation of a precipitate was monitored. Precipitation was observed when the temperature of the solution was 48°C. The mixture was reheated to reflux to give a clear yellow solution. 295 g of water (approximately twice the weight of ethanol) was added to the hot solution, allowing the mixture to cool during the addition. A precipitate was observed after adding approximately 150 ml of water at a temperature of 48°C. This solution was allowed to cool to room temperature. The solids were filtered off and washed three times with 20 g of water. The solid was dried at 40-45° C. for 2.5 days to give a yield of 4.82 g, 98.0%. XRPD analysis showed that the product was a mixture of plinabulin monohydrate (Form 1) (major component) and anhydrous plinabulin (Form 3) (minor component). Karl Fischer analysis showed that the water content was 5.0%. Analysis by the Karl Fischer method after drying at 45°C in vacuum for another 70 hours showed that the water content was reduced to 4.4%. The XRPD of this sample showed that it was essentially unchanged and was a mixture of Forms 1 and 3 with additional peaks (at approximately 12.26°, 15.19° and 28.79°

). This sample was placed in a glove box in an open container next to an open water container and the water content was monitored. The water content measured after 4 hours was 5.0%. The measured water content after 18.5 hours was 4.9%, and the result of the Karl Fischer analysis after 51 hours was 5.1%.

Example 3

[0144] A sample of the plinabulin compound was dissolved in 1,2-propanediol with vigorous stirring or moderate heating (50°C). After 4 hours, the sample showed peaks characteristic of form 3. After stirring over Saturday and Sunday, the sample was suspended in water and completely converted to plinabulin monohydrate (form 1), which did not show any peaks characteristic of crystalline form 3 on XRPD scans. A similar result was observed in the ethanol/water experiment, where after 1 hour and 4 hours very small peaks characteristic of form 3 were observed, and after 66 hours only form 1 was observed.

Example 4

[0145] During re-treatment, the plinabulin compound (Form 3) was dissolved in ethanol at a ratio of 1:25 (by weight) at the boil under reflux. This solution was filtered at a temperature in excess of 50° C. and the filtrate was combined with an equal weight of water to give the product. It may be desirable to reheat the finely filtered ethanol solution before adding all of the water, and add approximately 5% water (relative to ethanol) and stir the solution at approximately 70° C. to ensure conversion to Form 1. Additional water may then be added, and the mixture may be cooled to isolate the product by filtration. The sample can be dried for an extended period of time to reduce the water content obtained by Karl Fischer analysis. One of the samples was dried for three days resulting in a Karl Fischer analysis result of 4.9% water. Drying for another three days reduced the Karl Fischer result to 4.1%.

[0146] Exposing the dry product to wet conditions increased the water content to about 5%, at which point it appeared to remain constant. One of the 4.1% moisture samples was exposed to an open container of water in the glove box for 4 hours, which raised its water content to 5.0%. After a further 14.5 hours under these conditions, the measured water content of the sample was 4.8%. After a total of 51 hours, the result of the Karl Fischer analysis was 4.9%.

Example 5

[0147] A weighed portion of plinabulin with a water content determined by Karl Fischer analysis of approximately 3.1% was added to a mixture of collifor (40 wt.%)) and propylene glycol (60 wt.%). Insoluble particles formed in the solution, and it was determined that the insoluble particles were anhydrous plinabulin (Form 3). The sample was re-treated according to the steps described in FIG. 29 to form plinabulin monohydrate (Form 1). Karl Fischer analysis indicated that the water content of the recycled plinabulin was approximately 5.1%, consistent with the calculated water content of the monohydrate. Plinabulin monohydrate (form 1) completely dissolved in a mixture of collifor (40 wt.%) and propylene glycol (60 wt.%), and no insoluble particles formed in the solution. Therefore, plinabulin monohydrate (form 1) showed better solubility than plinabulin compositions containing anhydrous plinabulin (form 3).

Example 6

[0148] Plinabulin monohydrate (crystalline Form 1) was characterized by XRPD (crystalline, Fig. 1), optical microscopy (Fig. 2), DSC (Fig. 3A), TGA (Fig. 3B), and Karl Fischer method.

[0149] DSC data were obtained using a Q10 differential scanning calorimeter from TA Instruments. Typically, samples (~2 mg) were placed in sealed alodin-coated aluminum sample crucibles and scanned from 30 to 300°C at a flow rate of 10°C/min while purging with nitrogen at a flow rate of 50 ml/min. TGA data were obtained using a Q500 thermogravimetric analyzer from TA Instruments. Typically, samples (~10 mg) were placed in open pre-tared aluminum sample crucibles and scanned from 30 to 300°C at 10°C/min while purging with nitrogen at a flow rate of 60 ml/min. X-ray powder diffraction patterns were obtained using a Bruker D8 Advance instrument equipped with a Cu Kα (1.54°A) radiation source, a 9-position sample holder, and a LYNXEYE ultra-fast detector. As a rule, the duration of each scan was 180 seconds, and the range 29 was from 4 to 40°. The samples were placed on silicon plate holders with zero background. The samples were analyzed using an Aquadyne DVS-2 gravimetric water vapor sorption analyzer. The relative humidity was controlled in the range of 2-95% and the weight of the sample was continuously monitored and recorded.

[0150] XRPD showed that the substance is crystalline. DSC data showed a broad endotherm (peak maximum at 141°C, most likely water loss), a small exotherm (peak maximum at 164°C) and a sharp peak endotherm (peak maximum at 268°C). The TGA indicated a 5.26% weight loss at approximately 130° C. (most likely water loss). Analysis by the Karl Fischer method also showed that the substance contains water in an amount of 5.25 wt. %.

[0151] A sample of plinabulin monohydrate (Form 1) was placed in a vacuum oven at 50° C. overnight and over Saturday and Sunday. The sample remained stable during the drying studies and no weight change occurred during drying.

[0152] The DVS study was performed on plinabulin monohydrate (Form 1). Sample gained ~0.1% mass at 95% RH lost when dried to 0% RH with no hysteresis. The DVS sample was analyzed by XRPD, which confirmed the absence of any conversion. Fig. 3C shows a plot of the DVS isotherm. The XRPD of the pre-DVS (form 1 plinabulin monohydrate) sample is superimposed on the XRPD of the DVS sample (form 1 plinabulin monohydrate).

[0153] A sample of plinabulin monohydrate (Form 1) was placed in a high humidity environment for 13 days and remained stable without changing its weight. Pre-DVS and past DVS samples did not show any change on XRPD.

[0154] The Form 2 solid DVS test showed that the Form 2 sample lost approximately 7% weight at approximately 80% RH, as shown in FIG. 6C and the DVS sample was different from the pre-DVS sample based on the results of the XRPD analysis.

[0155] The Form 3 solid DVS test showed that the Form 3 sample lost approximately 0.2% weight at 90% RH, as shown in FIG. 9C and the DVS sample was different from the pre-DVS sample based on the results of the XRPD analysis.

[0156] The Form 4 solid DVS test showed that the Form 3 sample lost weight during the test, and the Form 4 DVS isotherm plot is shown in FIG. 12C. The DVS sample was different from the pre-DVS sample based on the results of the XRPD analysis.

[0157] Plinabulin monohydrate (Form 1) remained stable during DVS and drying tests. In comparison, other polymorphs were unstable and showed weight changes during DVS tests. The test results showed that form 1 is more stable than other polymorphic forms.

Example 7

[0158] The plinabulin compound was suspended in 15 different solvents/solvent mixtures as shown in Table 9 at 15°C and 45°C for 3 days for gravimetric solubility measurement. In each experiment, approximately 70 mg of a solid was added to a tube followed by the addition of 0.7 ml of the appropriate solvents. Then the suspension was centrifuged, centrifutate was added to pre-weighed test tubes and evaporated to dryness in a vacuum. The tubes with the remaining solid were weighed again to calculate the solubility. The compound is highly soluble in THF (tetrahydrofuran) and has moderate to low solubility in other tested solvents. Solubility data are presented in Table 10.

[0159] During gravimetric solubility analyses, plinabulin monohydrate (form 1) was suspended in various solvents at 15°C and at 45°C. After centrifugation, the resulting wet solids were analyzed by XRPD. Samples other than Form 1 were dried in a vacuum oven and reanalyzed by XRPD. Forms 2, 3, 4, 5, 6 and 7 were observed. These results are shown in Table 11.

[0160] Evaporative crystallization experiments were performed by evaporating (at room temperature) solutions of plinabulin monohydrate in various solvents. These solutions were obtained by gravimetric solubility analysis at 15°C and at 45°C and had different concentrations. The resulting solids were analyzed by XRPD. Form 8 was observed. These results are shown in Table 12.

[0161] Six cooling crystallization experiments and one suspension experiment were conducted. The results of these experiments are shown in Table 13.

Example 8

[0162] Investigated the conversion of various crystalline forms to other forms in the scale of 200 mg.

[0163] A scale-up experiment with Form 3 was performed on a 200 mg scale by suspending solid Form 1 in 2 ml of ethanol at room temperature overnight to form Form 3

[0164] A scale-up experiment with form 2 was performed on a 200 mg scale by suspending solid form 1 in 2 ml isopropyl alcohol at 30° C. overnight.

[0165] A scale-up experiment with form 4 was performed on a 200 mg scale by suspending solid form 1 in 2 ml of MeOH at 30° C. overnight.

[0166] Solid form 3 was suspended in a mixture of EtOH : water in a ratio of 95:5 at 30°C for 2 hours and analyzed by XRPD to demonstrate the conversion to form 1.

[0167] Solid Form 2 was suspended (2071-16-1) in EtOH:water 95:5 at 30°C for 2 hours and analyzed by XRPD to demonstrate conversion to form.

[0168] Solid form 4 was suspended in a mixture of EtOH : water in a ratio of 95:5 at 30°C for 2 hours and analyzed by XRPD to demonstrate the conversion to form 1.

[0169] Plinabulin monohydrate (Form 1) remained stable after drying at 50° C. under vacuum over Saturday and Sunday. No change in solid form was observed when Form 1 was exposed to high humidity (>95% RH) for 13 days. Form 1 has been shown to be stable during the manufacturing process, including the drying process. Form 1 was also stable under various humidity conditions. On the other hand, the crystalline forms were converted to Form I monohydrate upon exposure to moisture. Therefore Form 1 is the most stable crystalline form and the most promising form for the manufacturing process.

Claims (27)

1. A method for producing plinabulin monohydrate, including: combining plinabulin and the first solvent system to form a first mixture, wherein said first solvent system contains ethanol; heating the first mixture to a temperature of 50 °C to 90 °C until plinabulin is completely dissolved; adding water to the first mixture to form a second mixture; and cooling the second mixture to form a precipitate. 2. The method of claim 1, further comprising filtering the first mixture before adding water. 3. The method according to any one of paragraphs. 1, 2, additionally including filtering off the precipitate. 4. The method according to any one of paragraphs. 1-3, further comprising washing the precipitate. 5. The method according to any one of paragraphs. 1-4, where the first mixture is heated to a temperature of 70°C to 78°C. 6. The method according to any one of paragraphs. 1-5, further comprising maintaining the first mixture at 70°C to 78°C for 1 hour before adding water. 7. The method according to any one of paragraphs. 1-6, where heating the first mixture includes heating the first mixture to at least 65°C. 8. The method according to any one of paragraphs. 1-7, where cooling the second mixture includes stirring the first mixture for at least 4 hours. 9. The method according to any one of paragraphs. 1-7, further including analysis of the precipitate by X-ray powder diffraction analysis. 10. The method according to any one of paragraphs. 1-9, further comprising heating the second mixture to a temperature of 50°C to 90°C. 11. The method of claim 10 wherein heating the second mixture comprises refluxing the second mixture. 12. The method according to any one of paragraphs. 10, 11, where the second mixture is heated to a temperature of 70 °C to 78 °C. 13. The method according to any one of paragraphs. 10, 11, further comprising maintaining the second mixture at reflux temperature for 1 hour before cooling the second mixture. 14. The method according to any one of paragraphs. 10-13, where the cooling of the second mixture includes cooling to a temperature of from 15 °C to 30 °C. 15. The method according to any one of paragraphs. 10-14, wherein cooling the second mixture includes stirring the second mixture for at least 4 hours. 16. The method according to any one of paragraphs. 10-15 where the precipitate is washed with alcohol and the washing alcohol is collected and added to the second mixture before heating. 17. The method according to any one of paragraphs. 10-16, further comprising drying the precipitate. 18. The method according to any one of paragraphs. 10-17, further comprising an analysis of the precipitate to determine the composition of the plinabulin in the precipitate. 19. The method according to any one of paragraphs. 1-18, in which plinabulin monohydrate is characterized by an X-ray powder diffraction pattern containing at least three characteristic peaks selected from the group consisting of peaks at 8.1°, 13.1°, 16.3°, 23.9°, 24 .2°, 24.5° and 26.6° ± 0.2° 2θ. 20. The method according to any one of paragraphs. 1-18, in which plinabulin monohydrate is characterized by an X-ray powder diffraction pattern containing at least peaks at 8.1°, 13.1°, 16.3°, 23.9°, 24.2°, 24.5° and 26 .6° ± 0.2° 2θ. 21. The method according to any one of paragraphs. 1-18, in which plinabulin monohydrate is characterized by an X-ray powder diffraction pattern containing at least peaks at 8.1°, 13.1°, 16.1°, 16.3°, 19.8°, 22.9°, 23 .9°, 24.2°, 24.5°, 26.6° and 29.3° ± 0.2° 2θ. 22. The method according to any one of paragraphs. 1-18, in which plinabulin monohydrate has a melting point of 267 °C. 23. The method according to any one of paragraphs. 1-18, in which plinabulin monohydrate has a differential scanning calorimetry thermogram with endothermic peaks at 141°C and 267°C.

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