Solid polymorphs of flna-binding compound and hydrochloride salts thereof

Abstract

To provide crystalline polymorphs and solvates of 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one (Compound C0105) free base as well as the mono-and dihydrochloride salts and solvates thereof, and to provide an amorphous polymorph of the dihydrochloride.SOLUTION: A solid state form is selected from (a) crystalline compound C0105 dihydrochloride monohydrate, Crystalline Form 1; (b) crystalline compound C0105 dihydrochloride monohydrate, Crystalline Form 2; (c) crystalline compound C0105 dihydrochloride dimethylacetamide solvate, Crystalline Form 3; (d) amorphous compound C0105 dihydrochloride, Crystalline Form 4; and (e) crystalline compound C0105 dihydrochloride monohydrate, Crystalline Form 5.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 62 / 808,609, filed February 21, 2019, the disclosure of which is incorporated herein by reference. Technical Field The present invention contemplates solid state polymorphs of 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one, a compound that tightly binds to the protein Filamin-A (FLNA), or polymorphs of the monohydrochloride or dihydrochloride salts of this compound. [Background technology]

[0002] 1-benzyl-8-methyl-1,4,8-triazaspiro[4.5]-decan-2-one has the structural formula shown below: [ka] Also known in the art as compounds C0105 and C0105M, they specifically bind to the protein filamin-A (FLNA). Compound C0105 inhibits the production of tritium-labeled naloxone {[ in membranes prepared from A7 cells or SK-N-MC cells according to the procedures discussed in Wang et al., PlosOne vol. 3(2):e1554 (2008). 3 The competitive (displacement) curve for inhibition of H]NLX binding was approximately 10 for the higher affinity binding site of two such sites identified. -12 ~10 -13 EC in the M range 50 Instead of this membrane preparation, the use of an N-biotinylated pentapeptide (Bn-VAKGL; SEQ ID NO: 1) present in the sequence of FLNA also yielded EC values ​​of similar magnitude to those obtained with the membrane preparation. 50 values ​​were obtained, indicating that there is a single affinity site in this pentamer.

[0003] Compound C0105 is disclosed, synthesized, and claimed in U.S. Patent No. 8,653,068. The preparation of pharma- ceutically acceptable salts of compound C0105 is also disclosed, but such salts are not specifically prepared, and the compound is eluted by HPLC and 1 It was only isolated and used as an orange / yellow gummy material that was found by H-NMR to be about 86 to about 97% pure depending on the preparation used. Contacting suitable cells with compound C0105 can, among other things, reduce inflammatory responses (U.S. Pat. No. 8,653,068), inhibit phosphorylation of tau protein and formation of tau-containing plaques (U.S. Pat. No. 10,017,736), inhibit the growth of certain cancer cells that contain enhanced amounts of one or more of phosphorylated mTOR, Akt1, ERK2, and serine 2152 phosphorylated filamin A compared to non-cancerous cells, as disclosed in U.S. Pat. No. 9,433,604, and treat Alzheimer's disease, as discussed in U.S. Pat. No. 10,222,368 and Wang et al., J Prev Alz Dis, 2020, published online February 7, 2020. Compound C0105 can be used in assays for diagnosing Alzheimer's disease (AD) in living patients, determining the efficacy of AD therapies, and treating AD, as taught in U.S. Pat. Nos. 9,354,223, 9,500,640, and 10,222,368.

[0004] When formulating a pharmaceutical composition of an active pharmaceutical ingredient (API, compound, or drug), it can be important that the drug substance is in a form that can be easily handled and processed. This can be important not only from the standpoint of having a commercially viable manufacturing process, but also from the standpoint of the subsequent production of a pharmaceutical formulation that includes the active compound. Gums are not in such a convenient form for handling and processing. The chemical stability, solid state stability, and shelf life of the active ingredient are also important factors: the drug substance and compositions containing it should be capable of being effectively stored for a significant period of time without exhibiting significant changes in the physicochemical characteristics of the active ingredient (e.g., its chemical composition, density, hygroscopicity, solubility). Furthermore, it may be important to be able to provide a drug in as pure a form as possible. In this regard, amorphous materials may present significant problems compared to crystalline materials. For example, such materials are typically more difficult to handle and formulate than crystalline materials, provide less reliable solubility, and are often found to be unstable and chemically impure. Those skilled in the art will appreciate that if the active pharmaceutical ingredient (API) could be readily obtained in a stable crystalline form, the above problems would be significantly alleviated, if not solved. Therefore, when producing a commercially viable and pharma- ceutical acceptable drug composition, it is desirable to provide the API in a substantially crystalline and stable form whenever possible. However, it should be noted that this goal is not always achievable. In fact, it is typically not possible to predict the crystallization and post-crystallization behavior of a compound from its molecular structure alone, and such behavior can often only be determined empirically. Summary of the Invention

[0005] The present invention contemplates solid state polymorphs of 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one, solvates thereof, and polymorphs of the monohydrochloride and dihydrochloride salts of this compound and solvates thereof. In one embodiment, the compound of formula III: [ka] and pharma- ceutically acceptable solvates thereof, the solid forms of which have an X-ray powder diffraction pattern of a) an X-ray powder diffraction pattern having at least one peak selected from 8.0, 13.0, 13.8, 19.1, and 20.2 2θ±0.2 degrees 2θ; or X-ray powder diffraction pattern substantially similar to that in FIG. A crystalline monohydrate form 1 characterized by: b) an X-ray powder diffraction pattern having at least one peak selected from 9.9, 11.9, 13.2, 14.2, 15.8, 20.0 and 20.4 2θ±0.2° 2θ; or X-ray powder diffraction pattern substantially similar to that in FIG. A crystalline monohydrate form 2 characterized by: c) 5.5 An X-ray powder diffraction pattern with peaks at 2θ ± 0.2 degrees 2θ, or X-ray powder diffraction pattern substantially similar to that in FIG. A crystalline dimethylacetamide solvate form 3 characterized by: (d) X-ray powder diffraction pattern substantially similar to that in FIG. an amorphous form 4 characterized by: e) An X-ray powder diffraction pattern having peaks at 22.4 2θ ± 0.2 degrees 2θ, or X-ray powder diffraction pattern substantially similar to that in FIG. A crystalline monohydrate form 5 characterized by A solid form exhibiting characteristic peaks defining said

[0006] Formula II: [ka] A crystalline form of a compound of (a) an X-ray powder diffraction pattern having at least one peak selected from 12.4, 20.5, 21.7, and 25.5 2θ±0.2 degrees 2θ; or X-ray powder diffraction pattern substantially similar to that in FIG. crystalline form 1 characterized by: (b) an X-ray powder diffraction pattern having at least one peak selected from 10.5, 13.8, and 22.7 2θ±0.2 degrees 2θ; or X-ray powder diffraction pattern substantially similar to that in FIG. crystalline form 2 characterized by: (c) an X-ray powder diffraction pattern having at least one peak selected from 13.6, 15.8, 20.8, 22.0, and 27.2 2θ±0.2 degrees 2θ; or X-ray powder diffraction pattern substantially similar to that in FIG. Crystalline form 3 characterized by: (d) an X-ray powder diffraction pattern having at least one peak selected from 11.2, 18.0, and 20.0 2θ±0.2 degrees 2θ; or X-ray powder diffraction pattern substantially similar to that in FIG. Crystalline form 4 characterized by A crystalline form selected from:

[0007] Formula I: [ka] A crystalline form of a compound of a) an X-ray powder diffraction pattern having at least one peak selected from 24.1, 26.3, and 27.3 2θ±0.2 degrees 2θ; or X-ray powder diffraction pattern substantially similar to that of FIG. crystalline form 1 characterized by: b) an X-ray powder diffraction pattern having at least one peak selected from 13.1 and 16.8 2θ±0.2 degrees 2θ; or X-ray powder diffraction pattern substantially similar to that in FIG. Crystalline form 2 characterized by: c) an X-ray powder diffraction pattern having at least one peak selected from 10.1, 14.1, and 19.3 2θ±0.2 degrees 2θ; or X-ray powder diffraction pattern substantially similar to that of FIG. Crystalline form 3 characterized by A crystalline form selected from:

[0008] In another aspect of the present invention, the present invention relates to a method for reducing or inhibiting tau protein phosphorylation, inhibiting the interaction of FLNA with α7nAChR and TLR4, and inhibiting Aβ 42 Contemplated are pharmaceutical compositions comprising the above-described crystalline or amorphous forms of one or more compounds of Formula I, II, or III, dissolved or dispersed in a physiologically tolerable carrier or diluent, in an amount effective for one or more of the following: inhibiting the interaction of α7 nAChR with α7 nAChR, inhibiting the growth of cancer cells as discussed above, reducing one or both of pain and inflammation, or treating and / or assaying for Alzheimer's disease in a living patient. To inhibit the interaction of FLNA with α7nAChR and TLR4, to reduce or inhibit tau protein phosphorylation, Aβ 42 and α7 nAChR, inhibiting the growth of cancer cells, reducing one or both of pain and inflammation, or treating and / or assaying Alzheimer's disease in a living patient, comprising administering to a host mammal a pharmaceutical composition as described above. Illustrated in the drawings forming part of this disclosure are the following figures: [Brief description of the drawings]

[0009] [Figure 1] FIG. 2 is an X-ray powder diffraction pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one dihydrochloride monohydrate, crystalline Form 1. [Diagram 2] FIG. 2 is an X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one dihydrochloride monohydrate, crystalline Form 2. [Diagram 3] FIG. 2 is an X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one dihydrochloride dimethylacetamide solvate, crystalline Form 3. [Figure 4] FIG. 2 is the X-ray powder diffraction (XRPD) pattern of amorphous 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one dihydrochloride, Form 4. [Diagram 5] FIG. 2 is an X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one dihydrochloride monohydrate, crystalline Form 5. [Figure 6] FIG. 2 is an X-ray powder diffraction pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one monohydrochloride, crystalline Form 1. [Figure 7] FIG. 2 is an X-ray powder diffraction pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one monohydrochloride, crystalline Form 2. [Figure 8] FIG. 2 is an X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one monohydrochloride, crystalline Form 3. [Figure 9] FIG. 2 is an X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one monohydrochloride, crystalline Form 4. [Figure 10] FIG. 2 is an X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one free base, crystalline Form 1. [Figure 11] FIG. 2 is an X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one free base, crystalline Form 2. [Figure 12] FIG. 2 is the X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one free base, crystalline Form 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention has several benefits and advantages. One advantage is that it provides multiple crystalline stable forms of the API. An advantage of the present invention is that one such crystalline stable form of the dihydrochloride salt of 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one is particularly useful for the preparation of solid oral compositions. Another advantage of the present invention is that it provides a stable hydrated dihydrochloride salt, among others. Another advantage of the present invention is that it provides an amorphous polymorph of the dihydrochloride salt. Further benefits and advantages will be apparent to those skilled in the art from the following description and claims.

[0011] The present invention relates to a polymorphic form of the compound 1-benzyl-8-methyl-1,4,8-triazaspiro[4.5]-decan-2-one, the structural formula I of which is shown below: [ka] and polymorphs of its monohydrochloride and dihydrochloride salts and its pharma- ceutically acceptable solvates (pharma-ceutically acceptable solvatomorphs) are contemplated. This compound of structural formula I is also known in the art as compound C0105 (also C0105M). Such polymorphs of the free base, monohydrochloride or dihydrochloride salts, or solvates are distinguished from peaks in their X-ray powder diffraction (XRPD) spectra.

[0012] Polymorphs of solid forms of a molecule are usually defined by one or more characteristic peaks expressed as scattering angle position values ​​in 2 theta (°2θ) ±0.2°. Further characteristic data for each polymorphic form include the d-spacing, which is a measure of the distance between the planes of the crystalline form of the molecule associated with a particular °2θ peak, and the relative intensity of each of the distinguishing peaks as a percentage of the most intense peak. A characteristic scattering pattern (diffractogram) that identifies a particular crystalline polymorph may contain only a single peak unique to that particular polymorph. It is considered better practice to use 3-5 peaks, and more preferably, more than 5 peaks are used to identify a particular polymorph. Thus, if only one or two marker peaks are unique to a particular polymorph, and one or more other peaks are shared (overlapped) with peaks produced by another polymorph, the unique peak data, together with data from 1-5 overlapping peaks, can be used to provide a definitive diffractogram of the particular polymorph.

[0013] Preferably, scattering peaks having a relative intensity of at least 5% of the intensity of the most intense peak are reported as members of the characteristic scattering pattern, however, fewer and / or lower relative intensity peaks may be observed and reported for the characteristic scattering pattern. The characteristic 2 theta positions, d-spacings, and intensity percentages of the 12 proposed polymorphs of compound C0105 as the free base and its di(bis)hydrochloride and monohydrochloride salts and solvates are provided in the table below. Each °2θ measurement in the table below is ±0.2°, which is not shown in the table to improve the clarity of the table. Data is typically reported up to an approximate scattering angle of about 20 to about 30°2θ.

[0014] At least one XRPD marker peak can be used to identify a particular crystalline form of a compound known to be its free base, monohydrochloride or dihydrochloride salt, or solvate. Preferably, at least three XRPD marker peaks are used, and more preferably, five or more additional XRPD marker peaks are used if present in the diffractogram. As can be seen from the figures herein, the number of resolvable peaks can vary for different crystalline forms of the free base or hydrochloride salt. A "marker peak" is used to identify an XRPD peak that is unique to a particular polymorph and can be used to identify that polymorph. The phrase "full X-ray powder diffraction pattern" is used to describe peaks identified in a diffractogram, whether or not they are marker peaks. The phrase "peaks that overlap slightly with peaks of other forms" are peaks that can be used to identify a particular polymorph using one or more marker peaks. The phrase "pharmaceutically acceptable solvate" is used to refer to the solvent molecules that form part of the crystalline matrix of the contemplated polymorph. The solvate is named by adding the word "solvate" after the name of the solvent, except when the word "hydrate" is used when water is the solvate. A "pharmaceutically acceptable solvate" includes solvents that may be present in a pharmaceutical product in accordance with local or national drug laws and regulations, such as those by the FDA in the United States. Thus, exemplary solvates include hydrates, methanol solvates, ethanol solvates, isopropanol solvates, and dimethylacetamide solvates, etc.

[0015] Pharmaceutical compositions can be prepared by dissolving or dispersing an effective amount of a contemplated polymorph or solvate thereof in a physiologically tolerable carrier or diluent. A "physiologically tolerable carrier or diluent" is a diluent or carrier that may be present in a pharmaceutical product in accordance with local or national drug laws and regulations, such as those by the FDA in the United States. Polymorphs are referred to by the word "form" followed by an Arabic numeral. The use of numerals is merely for convenience in distinguishing one polymorph from another, and no other meaning is intended.

[0016] Characterization of crystalline morphology FIG. 1 is an X-ray powder diffraction pattern (XRPD) of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one dihydrochloride monohydrate, crystalline Form 1, obtained using a Philips Pananalytical X'Pert powder materials research diffractometer (MRD) machine over a scan range of 3° to 35° 2θ with 0.013° 2θ increments. X-rays were generated by a copper anode operated at 40 kV and 40 mA. The wavelength of the X-rays was 1.5406 Å. Table 1 lists the positions, °2θ ± 0.2° 2θ, d-spacings, and relative intensities of the peaks identified in the experimental XRPD pattern of FIG. 1. The entire list of peaks, or a unique subset thereof, and an XRPD pattern substantially similar to (i.e., discernible within experimental variability by one of ordinary skill in the art using a characterization method such as this one) Figure 1, can be used to fully characterize a crystalline form. Table 2 lists the peaks identified in the experimental XRPD pattern of Figure 1, which do not overlap with peaks in the XRPD diffraction patterns of other polymorphs and are preferred peaks for discerning a crystalline form. [Table 1] Peaks presented were selected with an intensity percentage less than 2 theta=25 and greater than 5%. [Table 2] Peaks presented were selected that were less than 2 theta=25° and had an intensity percentage greater than 5%.

[0017] C0105 Bis-HCl salt monohydrate: Form 2 Chemical formula:C 15 H 23 Cl2N3O.H2O FIG. 2 is an X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one dihydrochloride monohydrate, crystalline Form 2, obtained as described for FIG. Table 3 lists the positions, °2θ±0.2°2θ, d-spacings, and relative intensities of the peaks identified in the experimental XRPD pattern of Figure 2. The entire list of peaks, or a unique subset thereof, and an XRPD pattern substantially similar to Figure 2 (i.e., discernible within experimental variability by one of ordinary skill in the art using characterization methods such as this one), can be used to fully characterize a crystalline form.

[0018] Table 4 lists peaks idents that do not overlap with peaks in the XRPD diffraction patterns of other polymorphs and are preferred peaks for identifying crystalline forms. [Table 3] Peaks presented were selected with an intensity percentage less than 2theta=30 and greater than 5%. [Table 4] Peaks presented were selected based on their intensity percentages below 2 theta = 25 and greater than 5%, provided their positions were unique. * Peak offered as an exception.

[0019] C0105 Bis-HCl salt DMA solvate: Form 3 Chemical formula:C 15 H 23 Cl2N3O.C4H9NO FIG. 3 is an X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one dihydrochloride dimethylacetamide solvate, crystalline Form 3, obtained as described for FIG. 1, except that the peaks shown are all peaks that were detectable.

[0020] Table 5 lists the positions, °2θ±0.2 °2θ, d-spacings, and relative intensities of the peaks identified in the experimental XRPD pattern of Figure 3. The entire list of peaks, or a unique subset thereof, and an XRPD pattern substantially similar to Figure 3 (i.e., discernible within experimental variability by one of ordinary skill in the art using characterization methods such as this one), can be used to sufficiently characterize a crystalline form. Table 6 lists the peaks identified in the experimental XRPD pattern of FIG. 3 that do not overlap with peaks in the XRPD diffraction patterns of other polymorphs and are preferred peaks for identifying crystalline forms. [Table 5] The peaks presented are all that could be detected as the sample exhibited only a small number of peaks. [Table 6]

[0021] C0105 Bis-HCl salt: Form 4 - amorphous Chemical formula:C 15 H 23 Cl2N3O 4 is an X-ray powder diffraction (XRPD) pattern of amorphous 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one dihydrochloride, Form 4, obtained using a Siemens D5000 diffractometer machine over a scan range of 3° to 30.0° 2θ at 0.02° 2θ increments. X-rays were generated by a copper anode operated at 40 kV and 40 mA. No peaks indicative of an amorphous solid state form were observed.

[0022] C0105 Bis-HCl salt monohydrate: Form 5 Chemical formula:C 15 H 23 Cl2N3O.H2O FIG. 5 is an X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one dihydrochloride monohydrate, crystalline Form 5, obtained as described for FIG. 4 except that peaks presented were selected having an intensity of at least 5% of the most intense peak and less than 2θ=30°. Table 7 lists the positions, °2θ±0.2°2θ, d-spacings, and relative intensities of the peaks identified in the experimental XRPD pattern of Figure 5. The entire list of peaks, or a unique subset thereof, and an XRPD pattern substantially similar to Figure 5 (i.e., discernible within experimental variability by one of ordinary skill in the art using characterization methods such as this one), can be used to sufficiently characterize a crystalline form. Table 8 lists peaks identified in the experimental XRPD pattern of Figure 5 that do not overlap with peaks in the XRPD diffraction patterns of other polymorphs and are preferred peaks for identifying crystalline forms. Table 9 lists characteristic peaks identified in the experimental XRPD pattern of Figure 5 that may slightly overlap with peaks in the XRPD diffraction patterns of other polymorphs. [Table 7] Peaks presented were selected with an intensity percentage less than 2theta=30 and greater than 5%. [Table 8] Peaks presented were selected with an intensity percentage less than 2theta=30 and greater than 5%. [Table 9] Peaks presented were selected with an intensity percentage less than 2theta=30 and greater than 5%.

[0023] C0105 MonoHCl salt: Form 1 Chemical formula:C15 H 22 ClNO FIG. 6 is an X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one monohydrochloride, crystalline Form 1, obtained as described for FIG. 4 except that peaks presented were selected having an intensity of at least 5% of the most intense peak and less than 2θ=30°. Table 10 lists the positions, °2θ±0.2°2θ, d-spacings, and relative intensities of the peaks identified in the experimental XRPD pattern of Figure 6. The entire list of peaks, or a unique subset thereof, and an XRPD pattern substantially similar to Figure 6 (i.e., discernible within experimental variability by one of ordinary skill in the art using characterization methods such as this one), can be used to sufficiently characterize a crystalline form. Table 11 lists peaks identified in the experimental XRPD pattern of Figure 6 that do not overlap with peaks in the XRPD diffraction patterns of other polymorphs and are preferred peaks for identifying crystalline forms. Table 12 lists characteristic peaks identified in the experimental XRPD pattern of Figure 6 that may slightly overlap with peaks in the XRPD diffraction patterns of other polymorphs. [Table 10] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%. [Table 11] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%. [Table 12] Peaks presented were selected with an intensity percentage less than 2theta=30 and greater than 5%.

[0024] C0105 MonoHCl salt: Form 2 Chemical formula:C 15 H 22 ClNO FIG. 7 is an X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one monohydrochloride, crystalline Form 2, obtained as described for FIG. 4 except that peaks presented were selected having an intensity of at least 5% of the most intense peak and less than 2θ=30°. Table 13 lists the positions, °2θ±0.2 °2θ, d-spacings, and relative intensities of the peaks identified in the experimental XRPD pattern of Figure 7. The entire list of peaks, or a unique subset thereof, and an XRPD pattern substantially similar to Figure 7 (i.e., discernible within experimental variability by one of ordinary skill in the art using characterization methods such as this one), can be used to sufficiently characterize a crystalline form. Table 14 lists the peaks identified in the experimental XRPD pattern of FIG. 7 that do not overlap with peaks in the XRPD diffraction patterns of other polymorphs and are preferred peaks for identifying crystalline forms. [Table 13] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%. [Table 14] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%.

[0025] C0105 MonoHCl salt: Form 3 Chemical formula:C 15 H 22 ClNO Figure 8 is an X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one monohydrochloride, crystalline Form 3, obtained as described for Figure 4 except that peaks presented were selected that had an intensity of at least 5% of the intensity of the most intense peak and were less than 30° 2θ. Table 15 lists the positions, °2θ±0.2°2θ, d-spacings, and relative intensities of the peaks identified in the experimental XRPD pattern of Figure 8. The entire list of peaks or a unique subset thereof, as well as an XRPD pattern substantially similar to FIG. 8 (i.e., discernible within experimental variability by one of ordinary skill in the art using characterization methods such as this one), can be sufficient to characterize a crystalline form. Table 16 lists peaks identified in the experimental XRPD pattern of Figure 8 that do not overlap with peaks in the XRPD diffraction patterns of other polymorphs and are preferred peaks for identifying crystalline forms. Table 17 lists characteristic peaks identified in the experimental XRPD pattern of Figure 8 that may slightly overlap with peaks in the XRPD diffraction patterns of other polymorphs. [Table 15] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%. [Table 16] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%. [Table 17] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%.

[0026] C0105 MonoHCl salt: Form 4 Chemical formula:C 15 H 22 ClNO Figure 9 is an X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one monohydrochloride, crystalline Form 4, obtained as described for Figure 4, except that the peaks presented were selected to have an intensity of at least 5% of the most intense peak and less than 30° 2θ. Table 18 lists the positions, °2θ±0.2°2θ, d-spacings, and relative intensities of the peaks identified in the experimental XRPD pattern of Figure 9. The entire list of peaks, or a unique subset thereof, as well as an XRPD pattern substantially similar to Figure 9 (i.e., discernible within experimental variability by one of ordinary skill in the art using a characterization method such as this one) can be sufficient to characterize a crystalline form. Table 19 lists peaks identified in the experimental XRPD pattern of Figure 9 that do not overlap with peaks in the XRPD diffraction patterns of other polymorphs and are preferred peaks for identifying crystalline forms. Table 20 lists characteristic peaks identified in the experimental XRPD pattern of Figure 9 that may slightly overlap with peaks in the XRPD diffraction patterns of other polymorphs. [Table 18] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%. [Table 19] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%. [Table 20] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%.

[0027] C0105 Free base: Form 1 Chemical formula:C 15 H 21 N3O FIG. 10 is an X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one free base, crystalline Form 1, obtained as described for FIG. 4 except that peaks presented were selected having an intensity of at least 5% of the most intense peak and less than 2θ=30°. Table 21 lists the positions, °2θ±0.2 °2θ, d-spacings, and relative intensities of the peaks identified in the experimental XRPD pattern of Figure 10. The entire list of peaks, or a unique subset thereof, and an XRPD pattern substantially similar to Figure 10 (i.e., discernible within experimental variability by one of ordinary skill in the art using characterization methods such as this one), can be used to sufficiently characterize a crystalline form. Table 22 lists peaks identified in the experimental XRPD pattern of Figure 10 that do not overlap with peaks in the XRPD diffraction patterns of other polymorphs and are preferred peaks for identifying crystalline forms. Table 23 lists characteristic peaks identified in the experimental XRPD pattern of Figure 10 that may slightly overlap with peaks in the XRPD diffraction patterns of other polymorphs. [Table 21] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%. [Table 22] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%. [Table 23] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%.

[0028] C0105 Free base: Form 2 Chemical formula:C 15 H 21 N3O Figure 11 is an X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one free base, crystalline Form 2, obtained as described for Figure 4 except that peaks presented were selected having an intensity of at least 5% of the intensity of the most intense peak and less than 30° 2θ. Table 24 lists the positions, °2θ±0.2°2θ, d-spacings, and relative intensities of the peaks identified in the experimental XRPD pattern of Figure 11. The entire list of peaks, or a unique subset thereof, and an XRPD pattern substantially similar to (i.e., discernible within experimental variability by one of ordinary skill in the art using a characterization method such as this one), as in Figure 11, can be used to fully characterize a crystalline form. Table 25 lists peaks identified in the experimental XRPD pattern of Figure 11 that do not overlap with peaks in the XRPD diffraction patterns of other polymorphs and are preferred peaks for discerning a crystalline form. Table 26 lists characteristic peaks identified in the experimental XRPD pattern of FIG. 11 that may overlap slightly with peaks in the XRPD diffraction patterns of other polymorphs. [Table 24] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%. [Table 25] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%. [Table 26] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%.

[0029] C0105 Free base: Form 3 Chemical formula:C 15 H 21 N3O Figure 12 is an X-ray powder diffraction (XRPD) pattern of crystalline 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one free base, crystalline Form 3, obtained as described for Figure 4 except that peaks presented were selected having an intensity of at least 5% of the intensity of the most intense peak and less than 30° 2θ. Table 27 lists the positions, °2θ±0.2°2θ, d-spacings, and relative intensities of the peaks identified in the experimental XRPD pattern of Figure 12. The entire list of peaks or a unique subset thereof, as well as an XRPD pattern substantially similar to FIG. 12 (i.e., discernible within experimental variability by one of ordinary skill in the art using characterization methods such as this one), can be sufficient to characterize a crystalline form. Table 28 lists peaks identified in the experimental XRPD pattern of Figure 12 that do not overlap with peaks in the XRPD diffraction patterns of other polymorphs and are preferred peaks for identifying crystalline forms. Table 29 lists characteristic peaks identified in the experimental XRPD pattern of Figure 12 that may slightly overlap with peaks in the XRPD diffraction patterns of other polymorphs. [Table 27] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%. [Table 28] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%. [Table 29] Peaks presented were selected that were less than 2 theta = 30° and had an intensity percentage greater than 5%.

[0030] Pharmaceutical Compositions A contemplated compound polymorph or solvatomorph may be provided for use as such in salt or free base form. An effective amount of a contemplated solid polymorph, whether in salt form or not, and whether or not in solvate form, is typically dissolved or dispersed in a physiologically tolerable carrier or diluent to form a pharmaceutical composition in which the polymorph is its API, typically the only API. Such pharmaceutical compositions bind the API to FLNA and, by way of example, inhibit tau protein phosphorylation, inhibit the interaction of FLNA with α7nAChR and TLR4, and inhibit Aβ 42 The compounds may be administered to the CNS and / or other cells in vitro or in vivo to inhibit the interaction of α7 nAChR with α7 nAChR or to reduce pain and / or inflammation. The contemplated polymorphs can be used to manufacture medicaments (pharmaceutical compositions) useful for inhibiting tau protein phosphorylation in at least mammalian cells and mammalian cell preparations. The contemplated solid polymorphic compounds can inhibit the interaction of FLNA with α7nAChR and TLR4 as well as Aβ phosphorylation in at least mammalian cells and mammalian cell preparations. 42 and α7 nAChR, or for the manufacture of a medicament useful for reducing pain and / or inflammation.

[0031] Contemplated pharmaceutical compositions include an effective amount of a contemplated solid API polymorph (free base polymorph, or monohydrochloride or dihydrochloride hydrate, or other solvatomorph) as previously discussed, dissolved or dispersed in a physiologically tolerable carrier or diluent. Such compositions can be administered to mammalian cells or cell preparations in vitro for cell culture or protein binding studies, or in vivo for live host mammals in need thereof. Contemplated compositions are typically administered multiple times to a living recipient over a period of days, weeks, or months. More commonly, contemplated compositions are administered once, twice, or more frequently daily, with the administration being repeated. Once administration of a contemplated polymorph has begun, it is contemplated that the polymorph will be administered chronically, such as for the duration of the study being conducted or for the life of the recipient.

[0032] Contemplated free base polymorphs can bind FLNA at 100 femtomolar concentrations in vitro and effectively inhibit cytokine release from LPS-stimulated astrocytes. Contemplated monohydrochloride or dihydrochloride polymorphs bind at approximately the same molar concentrations as the free base and at proportional mass percentages based on the free base. Contemplated polymorphs are more commonly used in picomolar to micromolar amounts. Thus, an effective amount of a contemplated polymorph present in a contemplated pharmaceutical composition is an amount that provides a concentration of about 100 femtomolar to about 10 micromolar in the bloodstream of a host animal or in vitro cell culture when a contemplated method of the invention is practiced. A more typical amount is about picomolar to about micromolar. An even more typical amount is about picomolar to about nanomolar. One of skill in the art can readily determine the appropriate dosage level of a contemplated compound to inhibit the desired amount of FLNA binding. The contemplated pharmaceutical compositions can be administered orally (peroral), parenterally, or by inhalation spray, in formulations containing conventional non-toxic pharma- ceutically acceptable carriers, adjuvants, and vehicles, as appropriate. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, or infusion techniques. Drug formulations are discussed, for example, in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 1975, and Liberman, HA, and Lachman, L. (eds.), Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980.

[0033] For injectable preparations, for example, sterile injectable aqueous or oily solutions or suspensions can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example as solutions in 1,3-butanediol. When it is desired that the solid polymorphic API is in solution, the compound is typically provided as a solid, preferably free of liquid, or as a suspension in a non-solvent liquid to which a solvent, such as aqueous saline, is added to dissolve the solid API polymorph. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution, phosphate buffered saline. Liquid pharmaceutical compositions include, for example, solutions suitable for parenteral administration. Sterile aqueous solutions of the API or sterile solutions of the API in a solvent containing water, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration. In addition, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. Dimethyl-acetamide, surfactants including ionic and non-ionic detergents, polyethylene glycols can be used. Mixtures of solvents and wetting agents such as those discussed above are also useful. Sterile solutions can be prepared by dissolving a contemplated polymorph in a desired solvent system and then sterilizing the resulting solution by passing it through a membrane filter, or by dissolving a sterile compound under sterile conditions in a previously sterilized solvent.

[0034] Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the contemplated compound is usually combined with one or more excipients appropriate for the indicated route of administration. For oral administration, the compound may be mixed with lactose, sucrose, starch powder, cellulose esters of C1-C6-alkanoic acids, cellulose C1-C6-alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphates and sulfates, gelatin, gum acacia, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol, and then tableted or encapsulated for convenient administration. Such capsules or tablets may contain controlled release formulations, such as the active compound may be provided as a dispersion in hydroxypropylmethylcellulose. In the case of capsules, tablets, and pills, the dosage form may further contain a buffering agent, such as sodium citrate, magnesium or calcium carbonate, or magnesium or calcium bicarbonate. Additionally, tablets, capsules, and pills can be prepared with enteric coatings.

[0035] The mammal in need of treatment and to which the pharmaceutical composition comprising the contemplated solid polymorphic API or a solution comprising the dissolved polymorphic API is administered may be a primate, such as a human; an ape, such as a chimpanzee or a gorilla; a monkey, such as a cynomolgus monkey or a macaque; a laboratory animal, such as a rat, a mouse, or a rabbit; a companion animal, such as a dog, a cat, a horse; or a food animal, such as a cow or steer, a sheep, a lamb, a pig, a goat, a llama, etc. When in vitro mammalian cell contact is contemplated, CNS tissue cultures of cells derived from exemplary mammals are often used, as exemplified below. Preferably, the pharmaceutical composition is in unit dosage form.In such form, the composition is divided into unit doses containing an appropriate amount of active agent.The unit dosage form may be a packaged preparation, the package containing individual amounts of the preparation in, for example, vials or ampoules.

[0036] Analytical procedures Certain compounds have been analyzed by third-party elemental analysis (CHN; ASTM D5291), high-resolution mass spectrometry (MS), and 1 H-NMR, 13 Standard chemical analyses such as C-NMR and 2D-NMR spectroscopy, and infrared spectroscopy (IR) were used to identify the compounds. Similarly, physical property assays were performed using techniques such as X-ray particle diffraction (XRPD) for polymorphic determination as discussed in detail herein, melting point by differential scanning calorimetry (DSC), and mass loss by thermogravimetric analysis (TGA). Water content was determined by Karl Fischer analysis. High pressure liquid chromatography (HPLC) was used to assay the purity of the compounds.

[0037] polymorphic preparation Dihydrochloride polymorphic form 1 hydrate Preparation 1 In some cases, the synthesis of compound C0105 (C0105M) was carried out as described in U.S. Patent No. 8,653,068. An alternative synthesis was carried out using the reaction scheme depicted and discussed below. [ka]

[0038] Nt-Boc glycine was amidified with benzylamine in dichloromethane in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, triethylamine, and ethyl cyano (hydroxyimino group) acetate to form 2-amino-N-benzylacetamide, which was then converted to its hydrochloride salt by treatment with HCl in 2-propanol (iso-propanol). A flask was charged with 2-amino-N-benzylacetamide HCl salt and isopropanol (8 volumes). To this suspension was added 1-methylpiperidin-4-one (1.10 equiv.) followed by a methanol rinse (2 volumes). The heterogeneous mixture was stirred at reflux for 20 hours. HPLC analysis indicated 94% conversion after 20 hours. The solution was cooled to 45-55°C and 30% HCl (aqueous, 1.5 equiv.) was added dropwise to give a precipitate. The resulting slurry was cooled to ambient temperature and filtered after 3 hours. The isolated solid was washed with MeOH (2×1 volume) and the solid was air-dried to give the dihydrochloride salt hydrate of polymorphic form 1 in 80% yield. Its X-ray powder diffraction (XRPD) spectrum is shown in Figure 1. Ethanol, ethyl acetate, and tetrahydrofuran (THF) can also be used as solvents.

[0039] Conversion of monoHCl to diHCl hydrate salt The HCl monosalt was mixed with 8 mL of 2-propanol and the resulting suspension was stirred at about 50° C. for about 1 hour, at which point 1.6 mL of a solution of 37% HCl in 2-propanol (1.6 mL, 2 eq.) was added. The mixture was then cooled first to ambient temperature and then cooled to 2-3° C. after equilibration. After cooling, an additional 5 mL of 2-propanol was added to the sample. The resulting white precipitate was filtered and washed with 2-propanol (2×7 mL aliquots). The isolated solid was dried under vacuum at ambient temperature for about 24 hours. Approximately 2 g of monohydrate Form 1 solid material was recovered (yield: 66%). This polymorph can also be formed using ethanol, ethyl acetate, or tetrahydrofuran as the solvent instead of 2-propanol. Preparation 2 [ka]

[0040] Amide coupling of Boc-protected glycine and benzylamine Nt-boc-glycine (0.808 kg) and ethyl acetate (EtOAc, 3.615 kg, 5.0 vol) were charged to a nitrogen purged reactor and stirred until a clear solution was obtained. The solution was cooled to 0-5° C. and benzylamine (0.534 kg, 1.10 equiv.) was dosed into the reactor while maintaining the cooled temperature. The dosing bulb was rinsed with EtOAc (0.50 vol.) and the resulting EtOAc composition was added. Triethylamine (Et3N, 2.2 equiv., Et3N) was charged to the reactor, followed by an EtOAc (0.50 vol.) rinse. Propanephosphonic anhydride (T3P) (3.493 kg, 1.20 equiv.) in 50% EtOAc and an EtOAc rinse (0.50 vol.) were charged to the reactor while maintaining the temperature at ≦25° C. The reaction mixture was stirred for 20 h while maintaining the temperature at 20±5° C. HPLC analysis showed 91% conversion of the benylamine. Purified water (2.4 kg, 3.0 vol) was charged to form a two-phase mixture which was stirred for 9 minutes. The pH of the lower aqueous layer was measured to be pH=8 (target pH≧7). Phase separation was allowed to occur under quiescent conditions and the aqueous layer was removed and discarded. Aqueous hydrochloric acid (30%; 0.032 kg, 0.03 vol) in purified water (2.4 kg, 3.0 vol) was charged and the resulting two-phase mixture was stirred for 8 minutes to form the intermediate salt product. The pH of the lower aqueous layer was measured to be pH=1 (target pH≦2). Phase separation was allowed to occur under quiescent conditions and the aqueous layer was removed and discarded. HPLC analysis showed the isolated intermediate N-Boc-2-amino-N-benzylacetamide to be 97% pure. With stirring, the reaction mixture was distilled under reduced pressure at 40°±5° C. to remove approximately 6 relative volumes of volatiles. Isopropanol (2.5 kg, 4 volumes) was charged to the reactor. The reaction mixture was distilled at 40°±5° C. to remove approximately 4 relative volumes of volatiles. The reaction mixture was cooled to 20±5° C. and held.

[0041] Cleavage of Boc protecting group Isopropanol (IPA) was charged to the empty reactor (3.2 kg, 5 volumes). With stirring and cooling (15°-35° C.), hydrogen chloride gas was pressure fed into the reactor until approximately 5 equivalents were absorbed by the isopropanol. The excess hydrogen chloride gas pressure in the reactor was vented. The intermediate reaction mixture prepared above was dosed into the reactor with stirring and temperature controlled at 20°±5° C. The dosing valve was rinsed with isopropanol (0.329 kg, 0.50 vol) and the isopropanol composition was added to the reaction mixture. The reaction mixture temperature was controlled at 20±5° C. and stirred for 20 hours. HPLC analysis showed 99.4% conversion to 2-amino-N-benzylacetamide-HCl. The product slurry was filtered to collect the solid intermediate product (2-amino-N-benzylacetamide mono-HCl). The filter cake was washed twice with isopropanol: Wash 1 (0.633 kg, 1 vol), Wash 2 (0.328 kg, 0.5 vol), and a third time: Wash 3 (0.641 kg, 1 vol) with methyl tert-butyl ether (MTBE) and dried under vacuum at 20°±5° C. for 18 hours, thereby obtaining 0.760 kg of dried mono-HCl intermediate product. HPLC analysis showed the purity of the mono-HCl product to be 100.0%. The structure was 1 The dried intermediate product retained 0.23 wt % volatiles from the loss in the drying test, as confirmed by 1 H-NMR.

[0042] Cyclization of 2-amino-N-benzylacetamide-HCl with 1-methylpiperidin-4-one. 2-Amino-N-benzylacetamide monoHCl (0.5 kg, 1 eq) and absolute ethanol (3.26 kg, 8.0 vol, EtOH) were charged to a nitrogen purged reactor and stirred vigorously. 1-Methylpiperidin-4-one (0.310 kg, 1.1 eq) was charged to the reactor and rinsed with absolute ethanol (0.795 kg, 2 vol). The reactor temperature was raised to 65°±5° C. and held at that temperature, and the reaction was allowed to occur for 18 hours. HPLC analysis showed 91% conversion to the cyclized product 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one. The intermediate product slurry was cooled to 20°±5° C. and then filtered into a holding vessel, followed by the addition of an absolute ethanol rinse (0.207 kg, 0.50 vol) to provide a sol.

[0043] Conversion of the cyclization product to 2HCl·H2O polymorphic form 1 The 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decane-2-one sol and anhydrous ethanol rinse (0.201 kg, 0.50 vol) were charged to the reactor with stirring. The temperature was raised to and maintained at 50°±5° C. while aqueous hydrochloric acid 30% (0.457 kg, 1.5 eq) was slowly dosed into the reactor over 31 minutes, followed by anhydrous ethanol rinse (0.206 kg, 0.5 vol). The reaction mixture was slowly cooled to 20°±5° C. over a period of 2 hours and stirred for an additional 4 hours while maintaining the temperature. The temperature was slowly cooled to −15°±5° C. over a period of 2 hours and the reaction mixture was stirred for an additional 11 hours while maintaining the temperature. The resulting slurried product 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decan-2-one diHCl H2O was separated by filtration. The filter cake was washed with three rinses of absolute ethanol (about 2 volumes each) at 15°±5° C., then slurried twice in EtOAc (about 2 volumes each) and filtered. The final product was recovered after drying at 20°±5° C. for 23 hours. HPLC analysis showed the purity of the final product to be 100.0%. 1 H-NMR, 13 The monohydrate product had a water content of 5.5% by weight as determined by Karl Fischer titration. The polymorphic form 1 of the crystalline monohydrate product was confirmed by X-ray powder diffraction (XRPD) as compared to FIG. 1.

[0044] Dihydrochloride Polymorph Form 2 Monohydrate Approximately 9.6 g of compound C0105 free base was mixed with 30 mL of 2-propanol. The resulting suspension was stirred at 50° C. for about 1 hour, followed by dropwise addition of 37% aqueous HCl (9.3 mL, 3 eq.) in 10 mL of 2-propanol. A white suspension was obtained, which was stirred at 50° C. for about 1 hour, then at ambient room temperature for 1 hour, and finally at 2° C. for about 1 hour. The collected solid material was vacuum filtered and suspended in an acetone / water mixture (20% water, 8 mL). The resulting slurry was temperature cycled from ambient temperature to 40° C. in a 4 hour cycle. The resulting solid was filtered. XRPD analysis after drying indicated a new polymorphic form, which was designated as Form 2 dihydrochloride hydrate. Its XRPD spectrum is shown in FIG. 2. Dihydrochloride Polymorph Form 3 DMA Hydrate Approximately 25 mg of the HCl di-salt (from a scaled up batch of HCl mono- to di-salt conversion) was placed in a vial and a 0.1 mL aliquot of dimethylacetamide was added. Between each addition, the mixture was checked for dissolution, and if dissolution was not evident, the mixture was heated to approximately 50° C. and checked again. This procedure was continued until 2 mL of solvent had been added (no more dissolution occurred). The slurry was then subjected to temperature cycling from ambient temperature (approximately 22° C.) to 40° C. in 4 hour cycles over approximately 3 or 4 days (cooling / heating rate after the 4 hour period was 1° C. / min). The slurry was cooled, filtered, and the recovered solids were dried at ambient conditions prior to analysis. The XRPD spectrum is shown in FIG. 3.

[0045] Dihydrochloride Polymorph Form 4 – Amorphous Approximately 25 mg of the di-HCl salt was slurried in 300-1000 μL of either heptane or toluene. The mixture was then temperature cycled (40° C. to RT). The resulting solid was filtered and dried under vacuum. Analysis of the solid showed a unique XRPD spectral pattern that contained no peaks as seen in FIG. 4. It was labeled Form 4. Dihydrochloride Polymorph Form 5 Monohydrate Approximately 20 mL of methanol was added to approximately 450 mg of the di-HCl salt hydrate (Form 1). The resulting suspension was stirred at 50° C. for approximately 3 hours, and the resulting solution was filtered at ambient temperature. The isolated gel / solid mixture was dried under vacuum and mixed with approximately 0.1 mL of methanol. The resulting slurry was temperature cycled from ambient temperature to 40° C. in 4 hour cycles. The resulting solid was then filtered and dried under vacuum. XRPD spectroscopy analysis of the spectrum in FIG. 5 showed a new polymorphic form, which has been designated Form 5 monohydrate. MonoHCl salt form 1 Approximately 3.5 g of compound C0105 free base was dissolved in about 1 mL of 2-propanol at 40° C., and a solution of 1 equivalent of 37% HCl in 1.9 mL of 2-propanol (1.128 mL) was added dropwise to the mixture while stirring at 40° C. The resulting mixture was cooled from 40° C. to 5° C. at a rate of about 1° C. / min. A suspension was obtained and stirred at ambient room temperature for another 3 hours. The resulting white solid was filtered and dried at ambient conditions before analysis. About 2.5 g of material of this new polymorphic form was recovered and labeled as Form 1. Another 0.2 g of solid was obtained from the mother liquor of this preparation by stirring at 5° C. for about 24 hours, resulting in an overall yield of 34%. The XRPD spectrum of this solid polymorphic compound is shown in FIG. 6.

[0046] MonoHCl salt form 2 Approximately 500 mg of C0105M free base was dissolved in 6 mL of 2-propanol and mixed with 1 equivalent of HCl (37%) dissolved in 2 mL of 2-propanol. The resulting solution was temperature cycled over a period of 3 days (40 °C to RT in 4 hour cycles). The XRPD spectrum of this solid polymorph is shown in Figure 7. MonoHCl salt form 3 Prepared as above for Form 2, except the solid was dried under vacuum. The XRPD spectrum of this solid polymorph is shown in FIG. MonoHCl salt form 4 The mother liquor and solid material collected from the preparation of Form 2 above were mixed together and seeded with approximately 10 mg of HCl salt obtained from approximately 500 mg of C0105M free base dissolved in 6 mL of 2-propanol and mixed with 1 equivalent of HCl (37%) dissolved in 2 mL of 2-propanol. The resulting solution was temperature cycled over a period of 3 days (40° C. to RT in 4 hour cycles). The solids obtained from the temperature cycle were dried under vacuum. The resulting slurry was then temperature cycled from 40° C. to 20° C. over a period of 24 hours and analyzed by DVS from 0 to 90% RH and 90 to 0% RH in 10% increments. The XRPD spectrum of this solid polymorph is shown in FIG. 9.

[0047] Free Base Form 1 The free base material used to prepare free base crystalline Forms 1, 2, and 3 was received as a yellow gummy solid that was believed to be amorphous. This material was prepared as discussed above under the heading "Preparation 2" and was stopped prior to the final addition of hydrochloric acid at the final step shown. Approximately 500 mg of the free base material was slurried with 0.5 mL of ethyl acetate and the resulting mixture was temperature cycled over a period of 3 days (40° C. to RT in 4 hour cycles). Upon cooling, the crystalline polymorph was precipitated and collected, referred to herein as Free Base Form 1. The XRPD spectrum of this solid polymorph is shown in FIG. 10. Free Base Form 2 2.4 grams of C0105M free base gum was dissolved in 8.4 mL of methanol and 100 μL aliquots were transferred separately to 96 vials. The solvent was allowed to evaporate under ambient conditions leaving approximately 25 mg of C0105M in each of the 96 vials. 300 microliters of ethyl acetate was added, which produced a gum. Temperature cycling from ambient temperature (approximately 22° C.) to 40° C. in 4 hour cycles over approximately 3 or 4 days (cooling / heating rate after the 4 hour period was 1° C. / min) resulted in free base Form 2 crystals. The XRPD spectrum of this solid polymorph is shown in FIG. 11. Free Base Form 3 Physical manipulation of the surface of the as-received free base gummy material with a spatula resulted in a white sticky powder. HPLC analysis of the powder showed it to be 96.5% pure free base compound. Polarized light microscopy (PLM) analysis showed that the material was birefringent under polarized light and had block-like morphology. XRPD analysis showed that the material thus obtained was crystalline. The XRPD spectrum of this polymorph is shown in FIG. 12.

[0048] result procedure X-ray powder diffraction (XRPD) XRPD analysis was performed on a Siemens D5000, scanning samples from 3 to 30° (or 50° for input material) two-theta (2θ). For samples <100 mg, 10-20 mg of material was gently compressed against a glass disk and inserted into the XRPD sample holder. For samples <20 mg, 5-10 mg of material was gently compressed against a zero background disk and inserted into the XRPD sample holder. For samples >100 mg, approximately 100 mg of material was gently compressed into a plastic XRPD sample holder slightly above the level of the sample holder to ensure a smooth sample surface. Samples were then loaded into a Siemens D5000 diffractometer or a Panalytical X'Pert diffractometer operating in reflectance mode and analyzed using the following conditions: Raw data origin: Siemens-binary V2 (.RAW) Start position [°2θ] 3.0 End position [°2θ] 30.0 (or 50.0) Step size [°2θ] 0.020 Scan step time [sec] 1 Scan Type Continuous Offset [°2θ] 0.0 Divergence slit type: fixed Divergence slit size [mm] 2.00 Specimen length [mm] Various Receiving slit size [mm] 0.2 Measurement temperature [℃] 20.0 Anode material: Cu K-alpha 1 [Å] 1.54060 K-alpha2 [Å] 1.54443 K-beta [Å] 1.39225 K-A2 / K-A1 ratio 0.50 (nominal) Generator settings: 40mA, 40kV Diffractometer type D5000 Diffractometer Number 0 Goniometer radius [mm] 217.50 Incident beam monochromator None Diffracted Beam Monochromator (Graphite) Spinning None

[0049] Further XRPD analysis was performed on a Panalytical X'pert (PANalytical BV, Westborough, MA) powder, scanning samples from 3 to 35° 2θ. The material was gently ground and loaded into a multi-well plate with Kapton® polyimide film or Mylar® polyester film to support the samples. The multi-well plate was then loaded into a PANalytical diffractometer (Westborough, MA) operating in transmission mode and analyzed using the following experimental conditions: Raw Data Origin XRD Measurement (*.XRDML) Scanning axis Gonio Start position [°2θ] 3.0066 End position [°2θ] 34.9866 Step size [°2θ] 0.0130 Scan step time [sec] 18.8700 Scan Type Continuous PSD Mode: Scan PSD length [°2θ] 3.35 Offset [°2θ] 0.0 Divergence slit type: fixed Divergence slit size [mm] 1.0000 Measurement temperature [℃] 25.0 Anode material: Cu K-alpha2 [Å] 1.54060 K-alpha2 [Å] 1.54443 K-beta [Å] 1.39225 K-A2 / K-A1 ratio 0.50 Generator settings: 40mA, 40kV Goniometer radius [mm] 240.00 Distance between focus and divergence slits (Dist. Focus Diverg. Slit) [mm] 91.00 Incident beam monochromator None Spinning None

[0050] Further XRPD analyses were performed on a Bruker D2 Phaser with a zero background disc inserted into the XRPD sample holder using the following method: Start position [°2θ] 5.0000 End position [°2θ] 30.0000 Step size [°2θ] 0.04° °2θ Scan step time [sec] 0.25 Generator settings: 10mA, 30kV Slit size [mm] 0.6

[0051] Polarized Light Microscopy (PLM) The presence of crystallinity (birefringence) was determined using an Olympus® BX50 polarizing microscope equipped with a Motic® camera and image capture software (Motic® Images Plus 2.0). All images were recorded using a 20× objective unless otherwise stated. Thermogravimetric analysis (TGA) Approximately 5 mg of material was weighed into an open aluminum pan and loaded into a simultaneous thermogravimetric / differential thermal analyzer (TG / DTA) and held at room temperature. The sample was then heated from 25°C to 300°C at a rate of 10°C / min. During that period, the change in sample mass was recorded along with any differential thermal events (DTA). The purge gas was nitrogen at 100 cm 3A flow rate of 1 / min was used. Differential Scanning Calorimetry (DSC) Approximately 5 mg of material was weighed into an aluminum DSC pan and non-hermetically sealed with a perforated aluminum lid. The sample pan was then loaded into a Seiko DSC6200 (equipped with a chiller) which was cooled and held at 25° C. Once a stable heat flow response was obtained, the sample and reference were heated to approximately 240° C. at a scan rate of 10° C. / min and the resulting heat flow response was monitored. 1 H nuclear magnetic resonance (NMR) 1 H NMR studies were performed using a Bruker AV400 ( 1 The measurement was performed at a frequency of 400 MHz for each sample. 1 H NMR studies were performed in DMSO-d6 or CDCl3, and samples were prepared to a concentration of approximately 10 mg / mL.

[0052] Infrared Spectroscopy (IR) Infrared spectroscopy was performed on a Bruker ALPHA P spectrometer. Sufficient material was placed in the center of the spectrometer plate and a spectrum was obtained using the following parameters: Resolution: 4cm -1 Background scan count: 16 scans Sample scan count: 16 scans Data collection: 4000~400cm -1 Result spectrum: Transmittance Software: OPUS version 6 Dynamic Vapor Sorption (DVS) Approximately 10 mg of sample was placed in a glass vapor sorption balance pan and loaded onto a DVS-1 Dynamic Vapor Sorption Balance from Surface Measurement Systems. The sample was subjected to a relative humidity (RH) gradient profile from 0% to 90% in 10% increments, with the sample maintained at each step until a stable mass was achieved (step complete at 99.5%). After completion of the sorption cycle, the sample was dried using the same procedure from 90% RH to 10% RH. The mass change during the sorption / desorption cycle was plotted, allowing the hygroscopic nature of the sample to be determined.

[0053] Karl Fischer Coulometric Titration (KF) Approximately 10-15 mg of solid material was accurately weighed into a vial. The solid was then manually introduced into the titration cell of a Mettler Toledo C30 compact titrator. The vial was reweighed after the solid was added and the mass of the solid added was entered into the instrument. The titration was started once the sample in the cell was completely dissolved. The water content was automatically calculated by the instrument as a percentage and the data was printed out. High-performance liquid chromatography (HPLC) Instrument: Agilent® 110 Column: Waters Sunfire(TM) C18 5Qm, 150*4.6mm Flow rate: 1.0mL / min Detection wavelength: 214nm Column temperature: 25℃ Injection volume: 5Q / mL Mobile phase A: 0.03% TFA in water Mobile phase B (MPB): 0.03% TFA in acetonitrile Standard / sample preparation: 1mg / mL in water gradient: Time MPB% 0 5 16 95 18 95

[0054] Ion Chromatography (IC) Equipment: Thermo / Dionex(TM) ED40 Electrochemical detector, P50 gradient pump, AS1000 Autosampler, ASRS Utra 11 4mm Suppressor Column: Dionex™ IonPac AG14A-5Qm, 3 x 150 mm Guard column: Dionex™ IonPac AG14A-5Qm, 3 x 30 mm Mobile phase: 8mM Na2CO3 / 1mL NaHCO3 Flow rate: 0.5mL / min Runtime: 15 minutes Detector suppression: 50μS, rehydrate if necessary Column temperature: 30℃ Injection volume: 25μL (sample volume can be adjusted as needed) Standard / sample preparation: 0.003mg / mL in water

[0055] pKa analysis Potentiometric parameters for pKa studies were developed by Absorption Systems® and were performed as follows: Three titrations were performed in water with ionic strength adjusted to 0.15 with KCl (ISA water). pH values ​​ranged from 3.0 to 11.5. procedure Compound titration was carried out in ISA water at 22° C. A weighed amount of TC (2.40 mg) was placed in a titration vial. ISA water (15 mL) was automatically delivered to the vial. The pH value of the solution was adjusted to 3 by automatically adding 0.5 M HCl. Titration with 0.5 M KOH was carried out automatically until a pH value of 11.5 was reached. An additional 1 mL ISA water per titration was added to the titration vial to carry out the second and third titrations, respectively. The data sets of the three titrations were combined in the Refinement Pro program to generate a multiset for pKa calculation. First, pKa measurements and calculations were performed for compound C0105 assuming the presence of two basic ionizable groups. The lower value was found to be pH 1.5. Then, pKa calculations were performed for this compound assuming the presence of one basic ionizable group. The pKa values ​​for each amine nitrogen were found to be 8.0 and 1.5, as shown below. [ka]

[0056] Evaluation of the prepared HCl monosalt A. Stability Testing Each salt formed was exposed to the following environments for one week to assess its chemical and physical stability: 40° C. / 75% RH (open vials), elevated temperature (80° C., open vials), and ambient light (approximately 22° C. in closed vials). The resulting solids were analyzed by X-ray powder diffraction (XRPD) to establish whether any changes had occurred and by HPLC to determine purity. B. Salt Disproportionation Research Each salt was slurried in water at room temperature (approximately 22° C.) and the excess solids were removed and analyzed by XRPD at 1, 24, and 48 hours. The pH of the supernatant was also monitored. C. Hydration research Slurries of each salt were made in IPA:water mixtures (95%:5%, 90%:10%, and 60%:40% water) and stirred for about 48 hours at ambient temperature (about 22° C.) The resulting solids were then analyzed by XRPD to determine if any changes in crystal morphology occurred upon slurrying. D. Thermodynamic solubility studies An attempt was made to create slurries (~20 mg) of each salt in different aqueous buffer media (PBS, FaSSIF, FeSSIF, and SGF; Biorelevant.com) for ~48 hours at ambient temperature (~22°C). The resulting samples were found to be too soluble (>200 mg / mL) in the buffer media to perform the study. See below for buffer compositions. PBS In 100 mL of deionized water, 0.014 g KH2PO4, 0.9 g NaCl, and 0.079 g Na2HPO4. Fasted-State Simulated Intestinal Fluid (FaSSIF) 0.021 g NaOH, 0.198 g NaH2PO4 (monohydrate), 0.309 g NaCl, and 0.112 g SIF powder (Biorelevant.com Ltd, London, UK) in 0.050 mL deionized water. Fed-State Simulated Intestinal Fluid (FeSSIF) 0.202 g NaOH, 0.433 g glacial acetic acid, 0.594 g NaCl, and 0.560 g SIF powder (Biorelevant.com Ltd, London, UK) in 0.050 mL deionized water. Mimic gastric fluid (SFG) 0.200 g NaCl and 0.006 g SIF powder (Biorelevant.com Ltd, London, UK) in 100 mL deionized water

[0057] Primary polymorph screening of HCl dihydrate A. Temperature Cycling To efficiently use the material, samples recovered from the solvent solubility screen were also used in the primary polymorph screen. If a suspension was obtained during the solvent solubility screen, the mixture was used directly in the polymorph screen, whereas if a solution was obtained, more solid material was added to obtain a slurry. The suspension was then subjected to temperature cycling from ambient temperature (approximately 22° C.) to 40° C. in 4-hour cycles over a period of approximately 3 or 4 days (cooling / heating rate after the 4-hour period was 1° C. / min). The mixture was filtered and the recovered solids were dried at ambient conditions before analysis, whereas the mother liquor was retained for further studies. B. Crash Cooling at 2°C and -18°C Crash cooling studies were performed by placing a saturated solution of the HCl di-salt in each of the 24 selected solvent systems at approximately 2° C. If no solid material was recovered after 72 hours, the non-aqueous solution was placed in a −18° C. environment for a minimum of 72 hours. Any solid material was then collected and analyzed by XRPD and PLM. C. Slow evaporation A slow evaporation study was performed by evaporating the saturated solution HCl di-salt at ambient temperature (about 22° C.) and pressure. Any solid material was then collected and analyzed by XRPD and PLM. D. Anti-solvent addition at ambient and 2°C Antisolvent addition studies were performed by adding the selected antisolvent (acetone or ethanol) to a saturated solution of the HCl disalt in each of the 24 selected solvent systems at ambient temperature (approximately 22° C.). Antisolvent addition was continued until there was no further precipitation or until no more antisolvent could be added. Antisolvent addition studies were also performed at reduced temperature (2° C.) on saturated solutions recovered from crash cooling experiments that did not produce solids. All E. Large-Scale HCl Dihydrate Form 2 Approximately 0.6 mL of an acetone / water (20% water) mixture was added to approximately 450 mg of the HCl di-salt (Form 1). The resulting slurry was temperature cycled from ambient temperature (approximately 22° C.) to 40° C. in 4 hour cycles over a period of approximately 4 days. The solids were filtered and XRPD analysis was performed before and after drying. F. Large-Scale Production of HCl Dihydrate Form 5 Approximately 20 mL of methanol was added to approximately 450 mg of the HCl di-salt (Form 1). The resulting suspension was stirred at 50° C. for approximately 3 hours and the resulting solution was filtered and evaporated at ambient temperature (approximately 22° C.). The resulting gel / solid mixture was dried under vacuum and mixed with approximately 0.1 mL of methanol. The slurry obtained upon evaporation was temperature cycled from ambient temperature (approximately 22° C.) to 40° C. in 4 hour cycles over approximately 1 day. The recovered solid was filtered and analyzed by XRPD analysis before and after drying.

[0058] Complete physical characterization of the HCl monosalt (form 1) HPLC analysis showed the purity to be 98.8%. 1 H-NMR analysis showed a peak shift upfield compared to the free base, indicating the formation of a salt. Traces of IPA were also observed in the spectrum. A 1:1 ratio of API to chloride counterion was observed by IC analysis, suggesting the formation of a monosalt. TG / DT analysis showed a slight mass loss of 0.2% below 180° C., followed by a slight mass loss of about 0.5%, which corresponds to the endothermic event observed in the DT trace occurring at 184° C. The onset of decomposition occurs above 190° C. KF analysis indicated a moisture content of approximately 0.15%. DSC analysis showed a large endothermic event occurring at 180° C., which most likely corresponds to melting. DVS analysis showed that the material was highly hygroscopic above 70% RH. The sorption cycle showed no significant mass gain up to 60% RH (water uptake was approximately 0.27%). A constant mass gain was observed from 60% to 70% RH (2.55% water uptake at 70% RH) and a sharp mass gain was observed from 70% to 90% RH (68.57% water uptake at 90% RH). The desorption cycle showed a constant mass loss of 54.25% from 90% to 50% and another mass loss of 6.10% from 10% to 0%. A hysteresis of 7.3% was observed at 0% RH. The material deliquesced at high RH. XRPD analysis after DVS showed that this material did not match any of the forms previously observed for the HCl monosalt, a finding that supports the observation that this material deliquesced during analysis and recrystallized into a new form upon drying.

[0059] Stability Study (1 week) The results and observations of the HCl salt stability study are reported in the table below. By HPLC analysis, no significant loss of purity was observed during the stability study. XRPD analysis showed that the material retained its polymorphism during the one week stability study at ambient temperature and at 80° C. The material deliquesced at one week at 40° C. / 75% RH, which is consistent with the DVS analysis. A slight loss of crystallinity was also observed during the stability study. Results and observations from a one-week stability study of the HCl monosalt. [Table 30]

[0060] Solvent solubility screening of HCl monosalts The results of the solvent solubility screen of the HCl mono-salt are reported in the table below. The screen shows that the HCl mono-salt is poorly soluble in most of the selected solvent systems. However, very high solubility was observed in methanol, 2-propanol:water (10%), acetone:water (20%), and water, and high solubility was observed in dimethylsulfoxide. Some solubility was also observed in dimethylacetamide, ethanol, and acetone:water (5%). HCl - Solubility Screening Results Solvent Solubility at 40℃ (mg / mL) Acetone <10 Acetonitrile <10 2-Butanol <10 Cyclohexane <10 1,2-Dichloroethene <10 Dimethylacetamide approx. 10 Dimethyl sulfoxide approx. 105 Ethanol about 20 Ethyl acetate <10 Heptane <10 Isopropyl acetate <10 Methanol >200 Methyl acetate <10 Methyl ethyl ketone <10 Methyl isobutyl ketone <10 2-MeTHF <10 2-Propanol <10 2-Propanol:Water(10%) >200 tert-Butyl methyl ether <10 Tetrahydrofuran <10 Toluene <10 Acetone:water (5%) approx. 20 Acetone:water (20%) >200 Water >200

[0061] Physical Characterization of HCl Di-Salt Hydrate, Form 1 An XRPD diffractogram showed this material to be consistent with the resulting disalt. PLM analysis showed that the material was birefringent between crossed poles and had block-like morphology. HPLC analysis showed the purity to be 99.1%. 1 H-NMR analysis showed a peak shift upfield compared to the input material, indicating salt formation. Small amounts of solvent (2-propanol) were also observed in the spectrum. Furthermore, the spectrum is consistent with the analysis of the received HCl disalt. TG / DT analysis showed three successive mass losses, each at <200°C: 1) 5.4%, which corresponds to an endothermic event in the DT trace occurring at 81° C. (approximately 5.2% is expected for the monohydrate). 2) 1.7%, which corresponds to an endothermic event in the DT trace occurring at 146°C. 3) 0.6%, which corresponds to an endothermic event in the DT trace peaking at 170°C. Decomposition was observed at >200°C. A moisture content of about 5.5% was observed in the KF analysis, which was considered to be consistent with the TG analysis. DVS analysis indicated that this material was considered hygroscopic above 50% RH. The sorption cycle showed a slight mass gain of about 0.56% from 0% to 50% RH, a constant mass gain from 50% to 80% (about 3.53% water uptake at 80% RH), and a rapid mass gain from 80% to 90% RH (about 8.26% water uptake at 90% RH). The desorption cycle seemed to follow the same trend as sorption from 90% to 70% RH, followed by a constant mass loss of about 2.33% from 70% to 0%. Slight hysteresis was observed across the humidity range. The largest hysteresis was observed at 50% RH, where the difference in water uptake between the sorption and desorption profiles was 0.78%. The final dry mass (at 0% RH after the sorption and desorption profiles) was observed to be 0.13% higher than the initial dry mass. According to XRPD analysis, no significant differences in the polymorphic forms after DVS were observed.

[0062] HCl di-salt hydrate, Form 1 Stability Study (1 week) The results and observations of the stability study of the HCl di-salt are reported in the table below. HPLC analysis showed purities of 99.4% (ambient), 99.0% (40° C. / 75% RH), and 99.4% (80° C.), respectively. During the stability study of the HCl di-salt form 1 by XRPD analysis, no significant change in polymorphic form was observed. HCl Dihydrate - Form 1 One Week Stability Study Results and Observations [Table 31]

[0063] Solvent solubility screening of HCl di-salt hydrate, form 1 The results of the solvent solubility screen of the HCl di-salt are reported in the table below. The screen shows that the HCl di-salt is poorly soluble in most of the selected solvent systems, although very high solubility was observed in dimethylsulfoxide and water. High solubility was also observed in acetone:water (20%). Moderate solubility was observed in 2-propanol:water (10%) and methanol. HCl Dihydrate - Form 1 Solubility Screening Results Solvent Solubility at 40℃ (mg / mL) Acetone <10 Acetonitrile <10 2-Butanol <10 Cyclohexane <10 1,2-Dichloroethene <10 Dimethylacetamide <10 Dimethyl sulfoxide >200 Ethanol <10 Ethyl acetate <10 Heptane <10 Isopropyl acetate <10 Methanol approx. 25 Methyl acetate <10 Methyl ethyl ketone <10 Methyl isobutyl ketone <10 2-MeTHF <10 2-Propanol <10 2-Propanol:Water (10%) approx. 25 tert-Butyl methyl ether <10 Tetrahydrofuran <10 Toluene <10 Acetone:water(5%) <10 Acetone:water (20%) approx. 85 Water >200

[0064] Primary polymorph screening of HCl di-salt hydrate, form 1 The results of the primary polymorph screen for the HCl di-salt are summarized below: In the primary polymorph screen, Form 1 crystals were temperature cycled in the above solvents, the solvent was evaporated leaving the HCl di-salt in, and the solvent-HCl di-salt compositions were crash-cooled to 2° C. or crash-cooled to −18° C., and the anti-solvent was added at ambient temperature and at 2° C. Five crystalline forms were identified, as well as amorphous forms resulting from crash-cooling to 2° C. and from all solvents except dimethylacetamide and those containing added water. A total of five polymorphic crystalline forms were observed. Form 1 (hydrate) - input material, observed in numerous studies. Form 2 (hydrate) Form 2 (phase pure) - temperature cycle in acetone:water (20%) Form 2 (mixtures) - temperature cycling in acetonitrile, acetone:water (10%), and 2-propanol:water (10%) Form 3 (solvate) - temperature cycling in DMA Form 4 - Slow evaporation in heptane and toluene Form 5 (hydrate) Form 5 (phase pure) - evaporation in methanol Form 5 (mixture) - DMSO, 2-propanol, and acetone: evaporation in water (5%), crash cooling in water, and addition of anti-solvent to water Forms 1, 2, and 5 were deemed to have the highest levels of crystallinity and were selected for the secondary polymorph screening.

[0065] Polymorphic Stability Study of Form 1, Form 2, and Form 5 Approximately 20 mg of each of Form 1, Form 2, and Form 5 di-HCl salts were mixed together and suspended in the selected solvent system to obtain a slurry. Each of the resulting suspensions was then stirred at different temperatures for about 3 days. A list including study details is summarized in the table below. The recovered solids were filtered and analyzed by XRPD analysis to elucidate the major polymorphic form recovered.

[0066] Polymorphic stability study table Form Solvent Solvent (μL) Temperature 1+2 Methanol 100 Ambient environment * 1+2 IPA / Water (10%) 100 Ambient * 1+2 Cyclohexane 300 Ambient * 1+5 Methanol 80 Ambient environment * 1+5 IPA / water (10%) 80 Ambient * 1+5 Cyclohexane 200 Ambient environment * 2+5 Methanol 60 Ambient environment * 2+5 IPA / Water (10%) 60 Ambient * 2+5 Cyclohexane 200 Ambient * 1+2 Methanol 60 60℃ 1+2 IPA / water (10%) 60 60℃ 1+2 Cyclohexane 200 60℃ 1+5 Methanol 60 60℃ 1+5 IPA / water (10%) 60 60℃ 1+5 Cyclohexane 200 60℃ 2+5 Methanol 60 60℃ 2+5 IPA / water (10%) 60 60℃ 2+5 Cyclohexane 200 60℃ * Ambient temperature: approx. 22°C

[0067] Polymorphic Stability Study of Form 1, Form 2, and Form 5 Form Solvent T℃ Observation 1+2 Methanol RT * Form 2 (Tr # Form 1) 1+2 IPA / Water (10%) RT Form 2 1+2 Cyclohexane RT Form 1+Form 2 1+5 Methanol RT Form 5 1+5 IPA / Water (10%) RT Form 2 1+5 Cyclohexane RT form 1 (Tr form 5) 2+5 Methanol RT Form 5 (Tr Form 2) 2+5 IPA / Water (10%) RT Form 5 (Tr Form 2) 2+5 Cyclohexane RT Form 5 1+2 Methanol 60°C Form 5 (additional peak) 1+2 IPA / Water (10%) 60℃ Form 2 1+2 Cyclohexane 60℃ Form 1 (Tr Form 2) 1+5 Methanol 60°C Form 5 (additional peak) 1+5 IPA / Water (10%) 60℃ Form 5 1+5 Cyclohexane 60℃ Form 1 (Tr Form 5) 2+5 Methanol 60°C Form 5 (additional peak) 2+5 IPA / water (10%) 60°C Form 5 (additional peak) 2+5 Cyclohexane 60°C Form 5 (partially crystalline)

[0068] Some studies were performed using water as the solvent. The compound is highly soluble in water. If a clear solution was obtained, evaporating the solvent or slow cooling resulted in the formation of Form 1. If not enough water was added so that the slurry could not be stirred, no conversion to another polymorph was observed. When a certain amount of water is added to obtain a good stirrable suspension, Form 2 is found. Cooling or evaporating this suspension results in the well-crystalline Form 2. Only 0.8 volumes of water need to be added to obtain this suspension, 0.7 volumes results in a thick paste, and 1.0 volumes results in a clear solution. Other water-miscible solvents such as isopropanol (IPA) can be mixed with water to expand the useful range of solvent to solid that results in the Form 2 polymorph.

[0069] Large-scale version of form 2 (10g) Scaling up was successful in reproducing Form 2 at 10 g scale with a purity of 99.4% by HPLC. However, some loss of crystallinity was observed upon drying. Without changing the polymorph during hydration studies, an increase in crystallinity was observed after DVS analysis and during stability studies at 40° C. and 75% RH. This is likely due to water incorporation into the crystal lattice. This material was less hygroscopic than Form 1 from 0% to 50% RH, but at >50% RH it appeared to be more hygroscopic than Form 1 based on DVS analysis. Approximately 0.2% water uptake was observed at 50% RH and 24.9% at 90% RH based on DVS analysis (approximately 0.6% was observed at 50% RH and 8.3% was observed at 80% RH for Form 1). Form 2 was also found to be more sensitive to higher temperatures than Form 1. After a one week stability study at 80° C., a color change (from white to off-white / orange) and a purity of about 95.5% by HPLC analysis was observed.

[0070] XRPD analysis showed that Form 2 obtained from the large scale production appears to be consistent with that seen originally. However, a loss of crystallinity was observed upon drying, which may be due to partial removal of water from the crystal lattice. PLM analysis showed plate / lathe-like morphological characteristics. The TG / DT analysis showed two successive mass losses, respectively: 0.7%, which corresponds to an endothermic event in the DT trace occurring at 70.5° C.; and 4.8% (approximately 5.2% expected for the monohydrate), which corresponds to an endothermic event at 128° C. in the DT trace. KF analysis indicated a moisture content of about 6.1%. DSC analysis showed a slight endothermic event occurring at 76.9°C, likely due to unbound solvents (IPA and / or acetone), followed by a large, broad endothermic event at 144.9°C, likely due to loss of water, and then an exothermic event at 164.6°C. Based on DVS analysis, this material was considered to be hygroscopic above 50% RH. The sorption cycle did not show any significant mass gain from 0% to 50% RH (approximately 0.2% water uptake at 50% RH), but a constant mass gain was observed from 50% to 80% RH (approximately 9.9% water uptake at 80% RH) and a rapid mass gain was observed from 80% to 90% RH (approximately 24.9% water uptake at 90% RH). The desorption cycle appeared to follow the same trend as sorption from 90% to 70%, then a progressive mass loss of 6.77% was observed from 70% to 0%. During desorption, a slight hysteresis (i.e., moisture retention) of about 0.4% RH was observed at 0% RH. XRPD analysis after DVS showed an increase in crystallinity compared to the dry input material, which may be due to the reintroduction of water into the crystal lattice and subsequent crystallization of some of the amorphous content. A purity of 99.4% was observed by HPLC analysis. IC analysis showed that the API:HCl ratio was approximately 1:2, as expected for the HCl di-salt.

[0071] Form 2 1 The H-NMR spectrum showed several additional peaks compared to Form 1, specifically a multiplet at δ: 8.91, a broad singlet at δ: 8.14, and three doublets at δ: 4.35, 3.61, and 2.84. FT-IR analysis was obtained for reference.

[0072] Stability Study (1 week) HPLC analysis showed purity of 99.5% (ambient), 99.8% (40°C / 75% RH), and 99.5% (80°C). A color change (from white to off-white / orange) was also observed in the sample stored at 80°C, which is consistent with a lower purity (95.5%). Both the observations of color change and loss of purity indicate that Form 2 is more sensitive to elevated temperatures compared to Form 1, although Form 2 appears to be more stable at ambient temperatures. XRPD analysis showed no significant change in polymorphic form during the stability study of HCl di-salt hydrate Form 2, both at ambient and at 80° C. However, an increase in crystallinity was observed at 40° C. / 75% RH (likely due to water incorporation into the crystal lattice and subsequent crystallization of some of the amorphous content).

[0073] Results and observations from a one-week stability study of the HCl di-salt. [Table 32]

[0074] hydration research The results and observations of the hydration study of the HCl di-salt (Form 2) are reported in the table below. XRPD analysis showed that no morphology changes occurred upon stirring for 24 hours in various IPA / water mixtures, indicating that higher order hydrates were not formed. Enhanced crystallinity was observed compared to the dry input material, likely due to water incorporation into the crystal lattice.

[0075] Results and Observations of Hydration Studies of HCl Dihydrate (Form 2) [Table 33]

[0076] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. The above description and examples are intended to be illustrative and should not be construed as limiting. Further variations within the spirit and scope of the invention are possible and will readily occur to those skilled in the art.

Claims

1. Use of 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decane-2-one dihydrochloride monohydrate crystals in the manufacture of pharmaceuticals, wherein the dihydrochloride monohydrate crystals are characterized by a powder X-ray diffraction pattern having all of the peaks at 8.0, 13.0, 13.8, 19.1 and 20.22θ ±0.2°2θ.

2. The use of the dihydrochloride monohydrate crystal according to Claim 1, wherein the pharmaceutical agent is used to reduce or inhibit tau protein phosphorylation in a patient.

3. The use of the dihydrochloride monohydrate crystal according to claim 1 or 2, wherein the pharmaceutical is used to inhibit the interaction between FLNA and α7nAChR in a patient.

4. The use of the dihydrochloride monohydrate crystal according to any one of claims 1 to 3, wherein the pharmaceutical is used to inhibit the interaction between FLNA and TLR4 in a patient.

5. The pharmaceutical agent is used in patients with Aβ 42 Use of the dihydrochloride monohydrate crystal according to any one of claims 1 to 4 for use in inhibiting the interaction between and α7nAChR.

6. The use of the dihydrochloride monohydrate crystal according to any one of claims 1 to 5, wherein the pharmaceutical is used to inhibit the growth of cancer cells in a patient.

7. The use of the dihydrochloride monohydrate crystal according to any one of claims 1 to 6, wherein the pharmaceutical is used to reduce pain and / or inflammation in a patient.

8. The use of the dihydrochloride monohydrate crystal according to any one of claims 1 to 7, wherein the pharmaceutical is used to treat Alzheimer's disease in a patient.

9. Use of the dihydrochloride monohydrate crystal according to any one of claims 1 to 8, wherein the powder X-ray diffraction pattern of the dihydrochloride monohydrate crystal includes a peak selected from the group consisting of 9.2, 15.4, 16.1, 19.7, 21.7, 22.8, 23.2 and 24.2 2θ ±0.2°2θ.

10. Use of the dihydrochloride monohydrate crystal according to any one of claims 1 to 8, wherein the powder X-ray diffraction pattern of the dihydrochloride monohydrate crystal includes at least three peaks selected from the group consisting of 9.2, 15.4, 16.1, 19.7, 21.7, 22.8, 23.2, and 24.2 2θ ±0.2°2θ.

11. The use of the dihydrochloride monohydrate crystal according to any one of claims 1 to 8, wherein the powder X-ray diffraction pattern of the dihydrochloride monohydrate crystal includes at least five peaks selected from the group consisting of 9.2, 15.4, 16.1, 19.7, 21.7, 22.8, 23.2, and 24.2 2θ ±0.2°2θ.

12. Use of the dihydrochloride monohydrate crystal according to any one of claims 1 to 8, wherein the powder X-ray diffraction pattern of the dihydrochloride monohydrate crystal includes the peaks 9.2, 15.4, 16.1, 19.7, 21.7, 22.8, 23.2 and 24.2 2θ ±0.2°2θ.

13. The use of the dihydrochloride monohydrate crystal according to any one of claims 1 to 8, wherein the dihydrochloride monohydrate crystal is characterized by the powder X-ray diffraction pattern shown in Figure 1 below.

14. A pharmaceutical composition comprising a therapeutically effective amount of 1-benzyl-8-methyl-1,4,8-triazaspiro-[4.5]-decane-2-one dihydrochloride monohydrate crystals and a physiologically tolerable carrier, wherein the dihydrochloride monohydrate crystals are characterized by a powder X-ray diffraction pattern having all peaks at 8.0, 13.0, 13.8, 19.1 and 20.22θ ±0.2°2θ.

15. The pharmaceutical composition according to claim 14, used to reduce or inhibit tau protein phosphorylation in a patient.

16. The pharmaceutical composition according to claim 14 or 15, used to inhibit the interaction between FLNA and α7nAChR in a patient.

17. A pharmaceutical composition according to any one of claims 14 to 16, used for inhibiting the interaction between FLNA and TLR4 in a patient.

18. Aβ in patients 42 A pharmaceutical composition according to any one of claims 14 to 17, used to inhibit the interaction between and α7nAChR.

19. A pharmaceutical composition according to any one of claims 14 to 18, used for inhibiting the growth of cancer cells in a patient.

20. A pharmaceutical composition according to any one of claims 14 to 19, used to reduce pain and / or inflammation in a patient.

21. A pharmaceutical composition according to any one of claims 14 to 20, used for treating Alzheimer's disease in a patient.

22. The pharmaceutical composition according to any one of claims 14 to 21, wherein the powder X-ray diffraction pattern of the dihydrochloride monohydrate crystal includes a peak selected from the group consisting of 9.2, 15.4, 16.1, 19.7, 21.7, 22.8, 23.2, and 24.22θ ±0.2°2θ.

23. The pharmaceutical composition according to any one of claims 14 to 21, wherein the powder X-ray diffraction pattern of the dihydrochloride monohydrate crystal includes at least three peaks selected from the group consisting of 9.2, 15.4, 16.1, 19.7, 21.7, 22.8, 23.2, and 24.2 2θ ±0.2°2θ.

24. The pharmaceutical composition according to any one of claims 14 to 21, wherein the powder X-ray diffraction pattern of the dihydrochloride monohydrate crystal includes at least five peaks selected from the group consisting of 9.2, 15.4, 16.1, 19.7, 21.7, 22.8, 23.2 and 24.2 2θ ±0.2°2θ.

25. The pharmaceutical composition according to any one of claims 14 to 21, wherein the powder X-ray diffraction pattern of the dihydrochloride monohydrate crystal includes peaks at 9.2, 15.4, 16.1, 19.7, 21.7, 22.8, 23.2 and 24.22θ ±0.2°2θ.

26. The pharmaceutical composition according to any one of claims 14 to 21, wherein the dihydrochloride monohydrate crystal is characterized by the powder X-ray diffraction pattern shown in Figure 1 below.

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