EA042744B1 - NEW POLYMORPHIC FORMS OF CYCLO(-GIS-PRO) - Google Patents

Description

Related Applications

This application claims priority under US provisional application no. 62/696,190, filed July 10, 2018, which is hereby incorporated by reference in its entirety to the extent permitted by law.

The field of technology to which the invention belongs

The present invention is directed to novel cyclo(-His-Pro) (CHP) polymorphs.

Prerequisites for the creation of the invention

Cyclo(-His-Pro), C11H14N ₄ O ₂ , is known as an anhydrous dipeptide having CAS accession number 53109-32-3. It is an endogenous cyclic dipeptide formed in vivo by the hydrolytic removal of the amino-terminal pyroglutamic acid residue from the hypothalamic thyrotropin-releasing hormone. Cyclo(-His-Pro) can be synthesized ex vivo by conventional chemical methods. It may be important for regulating the quality of glial cell function. (Grotelli et al., The Role of Cyclo(His-Pro) in Neurodegeneration, Int JMol Sci. 2016 Aug; 17(8): 1332). Cyclo(His-Pro) is ubiquitous in the central nervous system and is a key substrate for organic cation transporters that are closely associated with neuroprotection. This cyclic dipeptide can also cross the blood-brain barrier and, once in the brain, can influence various inflammatory and stress responses by modifying the Nrf2-NF-KB signaling pathway.

The crystalline anhydrous form of cyclo(-His-Pro) (anhydrous CHP or Structure 1) is a form previously reported in the literature that can be used therapeutically. However, some anhydrous forms may be unstable. Possible disadvantages of using structure 1 include: (1) obvious physical instability in environmental conditions and high humidity; and (2) potential chemical instability due to water activity leading to the formation of diastereomers of the required L,L dipeptides such as D,L-CHP, L,D-CHP or D,D-CHP. Thus, there is a need in the art for more stable forms of CHP.

Brief summary of the invention

The present invention provides a crystalline form of cyclo(-His-Pro) hydrate (CHP hydrate or Compound Structure 2) that can be characterized and distinguished from other solid forms of CHP using a variety of analytical techniques, including X-ray powder diffraction (XRPD), solid-state nuclear magnetic resonance (NMR, or ¹³ C SSNMR), Raman spectroscopy, differential scanning calorimetry (DSC), dynamic vapor sorption (DSP), and thermogravimetric analysis (TGA).

Applicant unexpectedly found that CHP hydrate (CAS RN: 2254826-95-2 (January 2019)) has higher stability compared to amorphous cyclo(-His-Pro) or structure 1. Based on this unexpected discovery, structure 2 can be used individually as a single drug, instead of structure 1, or mixtures of structure 1 and structure 2 can be used.

In one embodiment, the present invention relates to a method for isolating structure 2 by crystallization using solvents rather than the column chromatography currently used to isolate anhydrous CHP.

In another embodiment, structure 2 is stable under typical storage conditions at room temperature for about 6 months, or about 12 months, or about 18 months, or about 24 months, or about 36 months.

In another embodiment, the present invention is directed to a substantially pure substance of structure 2. In some embodiments, the substance of structure 2 is at least about 90% pure, or at least about 95%, 96%, 97%, 98%, 99%, or 100% pure. .

In one embodiment, the purity of the sample is measured by any analytical method. In one embodiment, purity is measured by high performance liquid chromatography (HPLC), X-ray powder diffraction (XRPD), pKa analysis, polarized light microscopy (PLM), thermogravimetric analysis/differential thermal analysis (TG/DTA), differential scanning calorimetry (DSC). ), Fourier Transform Infrared Spectroscopy (FT-IR), Dynamic Vapor Sorption (DSP), Variable Temperature Humidity X-Ray Powder Diffraction (VT-/VH-XRPD), 1H Nuclear Magnetic Resonance (NMR) and/or Heteronuclear NMR single-quantum coherence (HSQC). In another embodiment, the purity of the sample is determined using HPLC.

In one embodiment, the structure 2 material is at least about 90% pure, or at least about 95%, 96%, 97%, 98%, 99%, or 100% pure, as measured by HPLC.

In another embodiment, the material of structure 2 may be characterized by at least two of the following characteristics:

(a) an x-ray powder diffraction pattern containing at least two peaks selected from the following list: 10; 13.7; 17; 18.1; 20.2 and 27.3 degrees (±0.2° 2θ);

(b) pKa about 6.4;

- 1 042744 (c) birefringence with fragmented rod-shaped morphology when analyzed using polarized light microscopy;

(d) an initial weight loss of about 6.5% (0.9 water equivalents) followed by decomposition of the sample at a temperature of about 280°C when tested by thermogravimetric analysis;

(e) an endotherm with an onset of about 99° C. and a peak of about 102° C. in the first DSC heating cycle;

(f) onset of dehydration at relative humidity (RH) below about 10%, loss of about 5.8 wt% at 10 to 0% RH (0.8 water equivalents) and hydration from 0 to about 40% RH at 40° C in dynamic analysis of vapor sorption;

(g) onset of dehydration at a relative humidity below about 20%, a loss of about 6.1 wt% at a relative humidity of about 20 to 0% (0.8 equivalents of water), and rehydration at a relative humidity of 0 to about 40% at 50 °С in dynamic analysis of vapor sorption; and (h) initiating dehydration at a relative humidity below about 20%, losing about 7% by weight at about 20 to 0% relative humidity (1.0 equivalent of water), and rehydrating at 0 to about 40% relative humidity at 60° C in the dynamic analysis of vapor sorption.

The present invention also includes a pharmaceutical composition containing the material of structure 2 and excipients. Such a composition may contain from about 1% to 50%, from about 5% to 45%, from about 10% to 40%, from about 15% to 35%, from about 20% to 30%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7 %, about 8%, about 9% or about 10% (by weight) CHP hydrate. In one embodiment, the composition may contain from about 1 to 10 wt.% CHP hydrate. In one specific embodiment, the composition may contain about 4 wt.% CHP hydrate.

In another embodiment, the composition may be in the form of a tablet, capsule, caplet, liquigel, lozenge, sterile injectable solution, and the like.

Alternatively, the compositions described herein may be formulated as a powder for dissolution in a suitable vehicle, such as sterile, pyrogen-free water, prior to use. For example, a compound suitable for parenteral administration may include sterile isotonic saline containing 0.1 to 90% by weight by volume of the compound. By way of example, the solution may contain from about 5% to 20%, more preferably from about 5% to 17%, more preferably from about 8% to 14%, and most preferably about 10% of the compound.

Additional embodiments of the present compositions, methods, and the like. will be apparent from the following description, drawings, examples and claims. As can be understood from the preceding and following description, each feature described in this application, and any and every combination of two or more such features are included in the scope of this disclosure, provided that the features included in such combination do not contradict each other. In addition, any feature or combination of features may be specifically excluded from any embodiment or aspect. Additional aspects and embodiments are set forth in the following description and claims, especially when considered in conjunction with the accompanying examples and drawings.

Brief description of the drawings

The patent or application contains at least one color drawing. Copies of this patent or publication of the patent application with color drawings will be made available by the Office upon request and upon payment of the required fee.

The above features of the embodiments will be better understood with reference to the following detailed description with reference to the accompanying drawings.

Fig. 1 is an X-ray powder diffraction pattern of crystal structure 1.

Fig. 2 is an XRPD comparison of (a) Structure 1 after Dynamic Vapor Sorption (DSV) and (b) Structure 2 CHP. These results confirm that the prepared CHP material is structure 2 (ie CHP hydrate) and that structure 1 changed to structure 2 after EAF.

Fig. 3 is a comparison of X-ray powder diffraction patterns of structure 2 at 40% relative humidity before and after EAF.

Fig. 4 is an analysis of X-ray powder diffraction patterns of structure 2 after VT-DVS. It shows that the material was a mixture of structures 1 and 2.

Fig. 5 is a comparison of X-ray powder diffraction patterns of approximately 30 mg of Structure 2 stored under three conditions for seven days: 40°C/75% RH, 80°C, and under natural light.

Fig. 6 is an XRPD analysis of about 2.2 g of CHP hydrate (structure 2) which was weighed into a glass vial and placed in a vacuum oven set at 50° C. for 1 hour.

Fig. 7 is an XRPD analysis of CHP hydrate or structure 2 (blue line: sample from FIG.

- 2 042744

5), which was returned to the vacuum furnace for another 30 min. In other words, the purple line in FIG. 7 is an XRPD analysis of the product after storage of CHP hydrate (Structure 2 material) for 1.5 h at 50°C under vacuum, showing mainly Structure 2 with some admixture of Structure 1.

Fig. 8 is an XRPD analysis of CHP hydrate or structure 2 (blue line: sample from FIG. 5) which was returned to the furnace in which the temperature was raised to 80°C. The vessel was placed in the oven for an additional 18 hours. In other words, the pink line in FIG. 8 is an XRPD analysis of the product after storage of CHP hydrate (Structure 2 material) for 18 hours at 80°C, showing mainly Structure 1 with some admixture of Structure 2.

Fig. 9 is a comparison of changes in structure 2 at different temperatures. This is an overlay of the results of XRPD analysis of the sample product from FIG. 5-7 dried for three different time periods and the results were analyzed by XRPD. The green line represents the structure of sample 2 stored at 50°C for 1 h, the purple line represents the structure of sample 2 stored at 50°C for 90 min, and the pink line represents the structure of sample 2 stored at 80°C for 18 o'clock

In FIG. 10 shows the initial analysis of the resulting material (mainly structure 1). The pKa value is 6.4.

In FIG. 11 shows the results of the analysis of the obtained CHP using unpolarized light microscopy (mainly structure 1).

In FIG. 12 shows the results of the analysis of the obtained CHP using polarized light microscopy (mainly structure 1).

In FIG. 13 shows the results of analysis of structure 2 using unpolarized light microscopy and polarized light microscopy.

In FIG. 14 shows the results of a thermogravimetric analysis of the obtained CHP (mainly structure 1).

In FIG. 15 shows the results of thermogravimetric analysis/differential thermal analysis (TG/DTA) of structure 2.

In FIG. 16 shows the results of the DSC analysis of the obtained CHP (mainly structure 1) in the first heating cycle.

In FIG. 17 shows the results of a DSC analysis of the obtained CHP (mainly structure 1) in a refrigeration cycle.

In FIG. 18 shows the results of the DSC analysis of the obtained CHP (mainly structure 1) in the second heating cycle.

In FIG. 19 shows the results of the DSC analysis of structure 2 in the first heating cycle.

In FIG. 20 shows the results of a DSC analysis of structure 2 in a refrigeration cycle.

In FIG. 21 shows the results of the DSC analysis of structure 2 in the second heating cycle.

In FIG. 22 shows the results of FT-IR spectroscopy of CHP hydrate.

In FIG. 23 is an isotherm plot of EAF Structure 1 showing a 6.3% weight gain between 60% RH and 90% RH.

In FIG. 24 is an isotherm plot of EAF of Structure 1 after recrystallization in the first heating cycle, showing slight hygroscopicity with a 0.8% weight gain at 0-90% relative humidity.

Fig. 25 illustrates the results of the DSP kinetics of structure 1.

Fig. 26 illustrates the XRPD results of the material before and after EAF. Analysis of XRPD after DSP shows a new shape characteristic of structure 2.

In FIG. 27 shows the TG/DTA results of CHP hydrate, structure 2.

Fig. 28 is an EAF isotherm plot showing that CHP hydrate, the material of structure 2, did not change at 40 to 90% and 10 to 90% relative humidity.

Fig. 29 illustrates the DSP kinetics of CHP hydrate, structure 2.

Fig. 30 illustrates VT-DVS analysis after CHP hydrate, Structure 2 was subjected to a single cycle at 40°C, showing that the material began to dehydrate below 10%, losing 5.8% wt (equivalent to 0.8 mol water). The material was rehydrated at a relative humidity of 040%.

In FIG. 31 shows the results of the VT-DVS analysis after the CHP hydrate, structure 2, was subjected to a single cycle at 50°C, demonstrating that the material began to dehydrate below 20%, losing 6.1% of the mass (equivalent to 0.8 mol of water) . The material was rehydrated at a relative humidity of 0-40%.

In FIG. 32 shows the results of the VT-DVS analysis after CHP hydrate, structure 2 was subjected to a single cycle at 60°C, showing that the material began to dehydrate below 20%, losing 7% wt (equivalent to 1.0 mol water). The material was rehydrated at a relative humidity of 0-40%.

In FIG. 33 shows the results of VT-/VH-XRPD analysis of CHP hydrate, structure 2 and structure 1.

- 3 042744

In FIG. 34 shows XRPD patterns of CHP hydrate material of structure 2 after various VT-DVS conditions. The XRPD pattern of structure 1 is shown for comparison.

Fig. 35 illustrates the ¹ H-NMR spectrum of structure 1 in DMSO-d ₆ .

Fig. 36 illustrates ¹ H-NMR spectrum of structure 2 in DMSO-d ₆ .

In FIG. 37 shows a comparison of the ¹ H-NMR spectra of structure 1 (blue line) and structure 2 (red line).

In FIG. 38 shows the HSQC-NMR spectrum of structure 1.

In FIG. 39 shows the HSQC-NMR spectrum of a mixture of structures 1 and 2.

Fig. 40 illustrates the results of XRPD after initial lyophilization of structure 1.

Fig. 41 illustrates the results of XRPD after extended lyophilization (72 hours) of Structure 1, showing that the material recovered from lyophilization was predominantly amorphous according to XRPD.

Fig. 42 illustrates the results of XRPD after the initial lyophilization of structure 2 (72 hours of drying), showing that the material recovered after lyophilization was structure 2 after 72 hours of drying.

In FIG. 43 shows the approximate solubility of structure 1 in various solvents.

Fig. 44 illustrates the solubility results of Structure 1 as a function of the volume of solvent added.

Fig. 45 illustrates the polymorph (structure 1 or structure 2) when structure 1 was treated with various solvents.

In FIG. 46 shows solvents for primary screening of polymorphs.

In FIG. 47 shows XRPD patterns of the polymorph formed in various solvents - part 1.

In FIG. 48 shows XRPD patterns of the polymorph formed in various solvents - part 2.

In FIG. 49 shows XRPD patterns of the polymorph formed in various solvents - part 3.

Fig. 50 illustrates the temperature cycle of primary screening for polymorphs.

Fig. 51 is an overlay of the XRPD patterns of structure 1 (blue) and structure 2 (red).

Fig. 52 is a typical blank chromatogram.

Fig. 53 is a chromatogram of a resolving solution.

Fig. 54 is a comparison of total impurities in CHP anhydrate (structure 1) and CHP hydrate (structure 2).

Fig. 55 is the LC-MS spectrum for Structure 1 and Structure 2, which confirmed the expected m/z ratio of 235.1 corresponding to [C ₁₁ H ₁ 4N ₄ O ₂ ]H ⁺ . Both structures give the same LC-MS results as when dissolved in a solvent (water) because there is no solid form or polymorph in the solvent. LC-MS measures the solubilized CHP, not the solid form.

Fig. 56 is an HPLC-UV chromatogram of the resulting CHP. It shows that the resulting CHP was 99.5% pure.

Fig. 57 is an example of a solids XRPD pattern obtained from solvent solubility screening.

Fig. 58-60 are XRPD patterns for solids obtained by temperature cycling.

Fig. 61 is an XRPD pattern of structure 2.

Fig. 62 is an XRPD diffraction pattern obtained from re-lyophilization of CHP in water.

Fig. 63 is an XRPD diffraction pattern from a third attempt at lyophilization of CHP in water.

Fig. 64-66 are X-ray diffraction patterns associated with temperature cycling experiments in the primary screening of polymorphs.

Fig. 67 is a diffraction pattern associated with evaporation experiments in the primary screening of polymorphs.

Fig. 68 is a X-ray diffraction pattern associated with the addition of an antisolvent in the primary screening of polymorphs.

Fig. 69 is the XRPD results for structure scaling 2.

Fig. 70 is an XRPD pattern of a material obtained by heating the material of structure 2 to 80°C.

Fig. 71 is an XRPD diffraction pattern of recovered solids in an ethanol/DCM spinning experiment.

Fig. 72 is an XRPD pattern for structure 2 in a primary polymorph screen.

- 4 042744

Fig. 73 shows images of structure 2 in polarized and unpolarized light.

Fig. 74 is the results of high temperature microscopy for structure 2 CHP.

Fig. 75 presents polarized and unpolarized light microscopy images of the material after the heating step.

Fig. 76 shows the results of the TG/DTA-TG analysis of structure 2.

Fig. 77 represents the initial DSC heat cycle identifying an endothermic event with an onset of 99°C and a peak at 102°C.

Fig. 78 represents the DSC cooling cycle.

Fig. 79 represents the second DSC heating cycle.

Fig. 80 shows the FT-IR spectra of structure 2 CHP.

Fig. 81 is a diffraction pattern associated with VT-/VH-XRPD analysis.

Fig. 82 is a graph of the DSP isotherm for structure 2 CHP.

Fig. 83 is a DSP kinetic plot for CHP structure 2.

Fig. 84 illustrates a comparison of XRPD patterns.

Fig. 85 is an EAF isotherm plot for Structure 2 at 40°C.

Fig. 86 is a kinetic plot for structure 2 at 40°C.

Fig. 87 is an isotherm plot for structure 2 at 50°C.

Fig. 88 is a kinetic plot for structure 2 at 40°C.

Fig. 89 is an isotherm plot for structure 2 at 60°C.

Fig. 90 is a kinetic plot for structure 2 at 60°C.

Fig. 91 illustrates a comparison of XRPD patterns.

In FIG. 92 shows images of samples of structure 1 after dissolution.

In FIG. 93 shows images of samples of structure 2 after dissolution.

Fig. 94 are XRPD 20 diffraction patterns for samples tested at 40°C/75% RH, 80°C and natural light.

Fig. 95 is one of the HPLC chromatograms for samples for stability analysis. Stability analysis was performed for 1 week at 40°C/75% relative humidity.

Fig. 96 is one of the HPLC chromatograms of samples for stability analysis. Stability analysis was performed for 1 week at 80°C.

Fig. 97 is one of the HPLC chromatograms for samples for stability analysis. Stability analysis at room temperature was performed for 1 week.

Fig. 98 is an XRPD 20 diffraction pattern two weeks apart for pure structure 1.

Fig. 99 is an XRPD 20 diffraction pattern two weeks apart for pure structure 2.

Fig. 100 is an XRPD 20 diffraction pattern four weeks apart for pure structure 1.

Fig. 101 is an XRPD 20 diffraction pattern four weeks apart for pure structure 2.

Fig. 102 is an XRPD 20 diffraction pattern eight weeks apart for pure structure 1.

Fig. 103 is an XRPD 20 diffraction pattern eight weeks apart for pure structure 2.

Fig. 104 is an HPLC chromatogram of pure structure 1 - initial time point.

Fig. 105 is an HPLC chromatogram of the pure structure 1-2 weeks.

Fig. 106 is an HPLC chromatogram of the pure structure 1-4 weeks.

Fig. 107 is an HPLC chromatogram of the pure structure 1-8 weeks.

Fig. 108 is an HPLC chromatogram of pure structure 2 - initial time.

Fig. 109 is an HPLC chromatogram of the pure structure 2-2 weeks.

Fig. 110 is an HPLC chromatogram of the pure structure 2-4 weeks.

Fig. 111 is the XRPD results after storage at 40°C/75% RH.

Fig. 112 is a typical overlay of chromatograms of a blank, a resolving solution, and a sample solution (6 M) for testing for impurities.

Fig. 113 is an evaporative analysis in the primary screening of polymorphs.

Fig. 114 is a summary table of primary screening polymorphs.

Fig. 115 is a summary of 8 weeks of appearance stability results.

Detailed Description of Specific Embodiments

Various aspects and embodiments will now be fully described in this application. However, these aspects and embodiments can be implemented in many different forms.

- 5 042744 and should not be considered limiting; rather, these embodiments are provided so that the disclosure is complete and complete and fully conveys the scope of the present subject matter to those skilled in the art. All publications, patents and patent applications cited herein, above or below, are incorporated herein by reference in their entirety.

A) definitions.

Unless otherwise indicated, all terms and phrases used in this application include the meanings that these terms and phrases have in the art, unless otherwise expressly indicated or clearly not obvious from the context in which the term or phrase is used. While any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, specific methods and materials are described below.

Unless otherwise noted, individual numeric values are used as an approximation, as if these values were preceded by the word approximately or approximately. Similarly, the numerical values in the various ranges specified in this application, unless expressly stated otherwise, are given as approximate, as if the minimum and maximum values in the indicated ranges were preceded by the word approximately or approximately. Thus, variations above and below these ranges can also be used to achieve substantially the same results as in the case of values within the ranges. When used in this application, the terms approximately and approximately in connection with a numerical value should be understood as their simple and conventional meanings by specialists in the field of technology with which the disclosed subject matter is most closely associated or to which the indicated range or element in question belongs. The degree of deviation from a strict numerical limit depends on many factors. For example, some of the factors that may be considered include the criticality of the element and/or the effect of a given amount of deviation on the performance of the claimed subject matter, as well as other considerations known to those skilled in the art. In this context, the use of different numbers of significant digits for different numerical values is not meant to limit how the use of words about or approximately serves to expand a particular numerical value or range. Thus, as a rule, approximately or approximately expand the numerical value. In addition, the disclosure of ranges implies a continuous range, including every value between the minimum and maximum values, plus the expansion of the range provided by the use of the term approximately or approximately. Therefore, the listing of ranges of values in this application is simply intended to be used as a shorthand method of individually referring to each individual value falling within that range, and each individual value is included in the description as if it were separately disclosed herein.

The terms parenteral administration and parenterally administered are known in the art and refer to routes of administration other than enteral and topical administration, usually by injection, and include intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, but not limited to subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal administration.

The term active agent or drug as used herein refers to any chemical that elicits a biochemical response when administered to a human or animal. The drug may act as a substrate or product of a biochemical reaction, or the drug may interact with a cellular receptor and elicit a physiological response, or the drug may bind to the receptor and block it, eliciting a physiological response.

The term bioequivalent in the context of the present description refers to two compositions, products or methods in which 90% confidence intervals (CI) for AUC, partial AUC and/or Cmax range from 0.80 to 1.25.

The term CHP hydrate (or Structure 2) means the compound identified by the CAS registration number: 2254826-95-2 published on January 7, 2019.

The term substantially pure refers to a substance having an overall purity greater than 90%, or greater than 95%, or greater than 96%, or greater than 97%, or greater than 98%, or greater than 99%, or greater than 99.5%. For example, the expression substantially pure substance A means that substance A is at least 90% pure with respect to all impurities, or substance A is at least 95% pure with respect to all impurities, or substance A is at least 98% pure. for all impurities, or Substance A is at least 99% pure for all impurities.

In one embodiment, the purity of the sample is measured by any analytical method. In one embodiment, purity is measured by HPLC, X-ray powder diffraction (XRPD), pKa analysis, polarized light microscopy (PLM), thermogravimetric analysis/differential thermal analysis (TG/DTA), differential scanning calorimetry (DSC), infrared conversion spectroscopy Fourier transform (FT-IR), dynamic vapor sorption (DSP), variable temperature and humidity x-ray powder diffraction (VT-/VH- 6 042744

XRPD), 1H nuclear magnetic resonance (NMR) and/or NMR with heteronuclear single quantum coherence (HSQC).

In some embodiments, the term substantially as shown in when referring to an X-ray powder diffraction pattern or a differential scanning calorimetry pattern means a pattern that is not necessarily identical to that depicted herein, but is within experimental error or variance when considered by a person skilled in the art. in this field of technology.

The term therapeutically effective amount refers to an amount that is sufficient to effect treatment, as defined below, when administered to a human in need of such treatment. The therapeutically effective amount will vary depending on the human subject being treated, the body weight and age of the human subject, the severity of the condition, the route of administration, and the like, as can be easily determined by one skilled in the art.

B) Introduction.

The present invention is directed to a novel CHP hydrate compound, its use and preparation. It is illustrated below.

On January 2019, CHP hydrate was assigned the CAS registration number (CAS RN): 2254826-95-2 with the name in the CA index: Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(1H-imidazole -5-ylmethyl)-, hydrate (1:1), (3S, 8aS)-. The CHP hydrate has a molecular weight of 252.3 g/mol.

One skilled in the art will appreciate that the structure of a compound may be named or identified using other commonly recognized systems of nomenclature and symbols. By way of example, a compound may be named or identified by common names, systematic or non-systematic names. Nomenclature systems and symbols commonly recognized in the field of chemistry, including but not limited to the Chemical Abstracts Service (CAS) and the International Union of Pure and Applied Chemistry (IUPAC). Accordingly, the above structure of the compound identified as pyrrolo[1,2-a] pyrazin-1,4-dione, hexahydro-3-(1Himidazol-5-ylmethyl)-, hydrate (1:1), (3S, 8aS) - according to CAS, may be designated by other names equivalent to this CAS designation.

C) General characteristics of CHP hydrate.

In one embodiment, the CHP hydrate has an X-ray powder diffraction (XRPD) pattern that includes characteristic peaks at about 13.7° 2θ, 17° 2θ, and about 27.3° 2θ. In some embodiments, the X-ray powder diffraction pattern further includes any one or more characteristic peaks at about 10° 2θ, about 13.7° 2θ, about 17° 2θ, about 18.1° 2θ, and 24.5° 2θ.

In some embodiments, CHP hydrate has a melting point of about 170 to 172°C. In one embodiment, the CHP hydrate has an X-ray powder diffraction pattern that includes any one or more characteristic peaks at about 10° 2θ, about 13.7° 2θ, about 17° 2θ, about 18.1° 2θ, about 20.2° 2θ and approximately 27.3° 2θ.

It should be understood that the relative XRPD intensities may vary depending on a number of factors including sample preparation, setup, and the instrument and analytical procedure and settings used to acquire the spectrum. Thus, the peak assignments listed here are intended to cover plus or minus 0.2° 2θ variations.

In other embodiments, the implementation of the CHP hydrate is characterized by the temperature of the onset of melting, determined using differential scanning calorimetry, approximately 170°C. In still other embodiments, the CHP hydrate is substantially free of solvent.

In some embodiments of CHP hydrate, at least one, at least two, at least three, at least four, or all of the following items (a)-(f) are used:

(a) CHP hydrate is substantially free of solvent;

(b) CHP hydrate is crystalline;

(c) CHP hydrate has a powder diffraction pattern substantially as shown in FIG. 2(b);

(d) CHP hydrate has a differential scanning calorimetry thermogram substantially as shown in FIG. 19-21;

(e) the CHP hydrate has an onset melting point, as determined by differential scanning calorimetry, of about 170° C.; and (f) the CHP hydrate is stable under storage conditions at room temperature.

In some embodiments, the CHP hydrate has at least one, at least two, or all of the following properties:

- 7 042744 (a) the character of the X-ray powder diffraction is essentially the same as shown in FIG. 2(b);

(b) the differential scanning calorimetry thermogram is essentially as shown in FIG. 19-21; and (c) the temperature of the onset of melting, determined using differential scanning calorimetry, is about 170°C.

In some embodiments, the CHP hydrate has an X-ray powder diffraction pattern including at least two of the largest peaks, as in the X-ray powder diffraction pattern shown in FIG. 2(b). In some embodiments, the CHP hydrate has an X-ray powder diffraction pattern including at least three of the largest peaks as in the X-ray powder diffraction pattern as shown in FIG. 2(b). In some embodiments, the CHP hydrate has an X-ray powder diffraction pattern including at least four of the largest peaks, as in the X-ray powder diffraction pattern shown in FIG. 2(b). In some embodiments, the CHP hydrate has an X-ray powder diffraction pattern including at least five of the largest peaks, as in the X-ray powder diffraction pattern shown in FIG. 2(b). In some embodiments, the CHP hydrate has an X-ray powder diffraction pattern including at least six of the largest peaks, as in the X-ray powder diffraction pattern shown in FIG. 2(b).

D) Pharmaceutical compositions.

In one embodiment, a pharmaceutical composition is provided comprising a substantially pure compound of structure 2 and a pharmaceutically acceptable carrier. For example, a pharmaceutical composition may contain a substantially pure compound of structure 2 in an amount of from about 1% to about 20% (w/w%) (i.e., about 1%, about 2%, about 3%, about 4%, about 5 %, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%) of the total pharmaceutical composition. As a further example, the pharmaceutical composition may contain a compound of structure 2 in an amount of about 1 to 100%, about 1 to 10%, about 10 to 20%, about 20 to 30%, about 30 to 40%, about 40 to 50%, about 50 to 60%, about 60 to 70%, about 70 to 80%, about 80 to 90%, about 90 to 100% (w/w%) of the total pharmaceutical composition. For example, the pharmaceutical composition may contain a compound of structure 2 in an amount from about 1 to 40% (wt.%) of the total amount of the pharmaceutical composition. In a specific example, the pharmaceutical composition may contain a compound of structure 2 in an amount of about 4% (wt.%) of the total pharmaceutical composition.

In another embodiment, a pharmaceutical composition is provided comprising a substantially pure compound of structure 2, another therapeutically active agent, and a pharmaceutically acceptable carrier. In one embodiment, the active agent is selected from a biomolecule, a bioactive agent, a small molecule, a drug, a prodrug, a drug derivative, a protein, a peptide, a vaccine, an adjuvant, an imaging agent (e.g., a fluorescent moiety), a polynucleotide, or a metal. In yet another embodiment, the active agent is zinc.

In one embodiment, the Structure 2 drug substance meets the ICH Q.2A purity guidelines for CHP impurity levels.

In another embodiment, the composition of the present invention may be administered by a variety of routes, including oral, topical, parenteral, intravenous, intradermal, intra-intestinal, rectal, intramuscular or intraperitoneal route.

The composition may be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form in ampoules or in multi-dose containers, with the optional addition of a preservative. The parenteral preparation may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or the like. The formulation may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and may contain agents such as suspending, stabilizing and/or dispersing agents.

For example, the parenteral formulation may be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent. Acceptable carriers and solvents that may be used include water, 0.9% saline, or other suitable aqueous vehicles.

In one embodiment, the concentration of the intravenous solution is from about to 200 mg/ml, from about 5 to 150 mg/ml, from about 10 to 100 mg/ml. In another embodiment, the concentration of the intravenous solution is about 1 mg/L, about 2 mg/L, about 3 mg/L, about 4 mg/L, about 5 mg/L, about 6 mg/L, about 7 mg/L, about 8 mg/l, about 9 mg/l, about 10 mg/l, about 11 mg/l, about 12 mg/l, about 13 mg/l, about 14 mg/l, about 15 mg/l l, about 20 mg/l, about 25 mg/l, about 30 mg/l, about 35 mg/l, about 40 mg/l, about 45 mg/l, about 50 mg/l, about 55 mg/l, about 60 mg/l, about 65 mg/l, about 70 mg/l, about 75 mg/l, about 80 mg/l, about 85 mg/l, about 90 mg/l, about 95 mg/l, about 100 mg/l, about 110 mg/l, about 120 mg/l, about 130 mg/l, about 140 mg/l, about 150 mg/l, about 160 mg/l, about 170 mg/l, about 180 mg/l l, about 190 mg/l or about 200 mg/l.

In another embodiment, the composition may be formulated as a diffusion (slow drip) formulation or intravenous bolus injection.

In yet another embodiment, a compound of structure 2 may be administered orally or in an oral formulation. Administration may be in the form of immediate release tablets and capsules, enteric coated tablets, and the like. In the manufacture of pharmaceutical compositions that include at least one compound described in this application, the active ingredient is usually diluted with an excipient and/or enclosed in such a carrier, which may be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it may be in the form of a solid, semi-solid or liquid material which acts as a solvent, carrier or medium for the active ingredient. Thus, the compositions may be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (solid or liquid), ointments, soft and hard gelatine capsules, sterile solutions. for injections and powders in sterile packaging.

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, hydroxypropylcellulose, polyvinylpyrrolidone, cellulose, USP water or sterile water, syrup base and methylcellulose. The compositions may further include lubricants such as talc, magnesium stearate and stearic acid; wetting agents; emulsifiers and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; sweeteners; and fragrances.

In some embodiments, the compositions are formulated as unit dosage forms. The term unit dosage form refers to physically discrete units suitable as unit doses for humans and other mammals, where each unit contains a predetermined amount of active material (therapeutically effective amount) calculated to produce the desired therapeutic effect, in combination with a suitable pharmaceutical excipient ( e.g. tablets, capsules, ampoules). The compounds are generally used in a pharmaceutically effective amount. In some embodiments, each unit dose contains about 1 to 100 mg of a compound of structure 2. In some embodiments, each unit dose contains about 2 to 60 mg, about 3 to 50 mg, about 4 to 40 mg, about 5 up to 30 mg, about 6 to 20 mg, about 8 to 15 mg, or about 8 to 10 mg of a compound of structure 2.

In other embodiments, each unit dose contains about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg of a compound of structure 2.

In one embodiment, the subject receives one or more unit doses per day. In yet another embodiment, the subject receives 15 mg of a compound of structure 2 per day.

To prepare solid compositions such as tablets, the active main ingredient is mixed with a pharmaceutical excipient to form a solid mixed composition containing a homogeneous mixture of the compound of the present invention. When these mixed compositions are referred to as homogeneous, this means that the active ingredient is evenly dispersed throughout the composition so that the composition can be easily divided into equally effective unit dosage forms such as tablets, pills and capsules.

The tablets or pills of the present invention may be powder-coated or otherwise formulated to provide a dosage form that provides the benefit of prolonged action or protection from the acidic environment of the stomach. For example, a tablet or pill may contain an internal dose component and an external dose component, the latter being in the form of a shell covering the first component. The two components can be separated by an enteric layer which serves to prevent disintegration in the stomach and allows the inner component to pass into the duodenum intact or delay release. Various materials can be used for such enteric layers or coatings, including a variety of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate. In one embodiment, the film coating is a polyvinyl alcohol based coating.

- 9 042744

The compounds used in the compositions and methods include the compounds described herein in any of their pharmaceutically acceptable forms, including isomers such as diastereomers and enantiomers, salts, solvates and polymorphs, as well as racemic mixtures and pure isomers of the compounds described herein, where applicable.

Suitable adjuvants include binders, fillers, disintegrants, lubricants, antioxidants, chelating agents, and colorants.

In table. 1 presents examples of compositions for oral dosage forms, including (based on the content in wt.% of these ingredients).

Table 1. Examples of compositions for oral dosage forms include (based on the content in wt.% established ingredients).

Ingredient 1 2 3 4 5 6 7 8 9 10 Hydrate C111 85 70 60 50 40 thirty 20 10 5 2 Coupling agent 9 10 20 thirty thirty 40 40 40 40 40 disintegrant 5 5 5 5 5 5 5 5 5 5 lubricant 1 1 1 1 1 1 1 1 1 1 Filler 0 14 14 14 24 24 34 44 49 52

E) Preparation of CHP hydrate.

In some embodiments, CHP hydrate is obtained by crystallization. The CHP anhydrate was dissolved in 2-2.5 volumes of a mixture of ethanol/water or acetone/water at 50°C, and then the system was cooled to 35°C. Then 0.5 volumes of methyl tert-butyl ether (MtBE) was added followed by 0.5% seed (CHP hydrate). After stirring for 2 h, the system was cooled to 5° C. in 2 h. Finally, 7 vol. MtBE and then stirred for 8 h.

Solubility evaluations made for CHP Sample Lot 1 showed high solubility in all solvent mixtures containing water, with the exception of ethanol:water:MtBE mixtures with the highest percentage of MtBE. Particularly high solubility was observed in water:acetone mixtures. The material is poorly soluble in acetone, acetonitrile and THF (<10 mg/ml). Two-point solubility experiments showed that of the two solvent systems evaluated (ethanol:water:MtBE and acetone:water), the ethanol:water:MtBE mixture produced a slightly better yield, but there was a risk of structure 1; while the acetone:water mixture allowed only structure 2 to be obtained, but there was a risk of lowering the yield.

Two series of small-scale crystallization tests (scale 500 mg) were carried out using CHP structure 1. The first series used the solvent system acetone:water, and the second series used the solvent system ethanol:water:MtBE. The antisolvent was added at 5°C or 50°C. Structure 2 was generated solely by XRPD regardless of the solvent system or anti-solvent addition temperature. The ethanol:water/MtBE system gave larger particles, especially when the solid was isolated at a higher temperature (50°C). Loss of solvent due to evaporation (especially in ethanol: water: MtBE) resulted in a much lower calculated yield compared to the weight of solid recovered after filtration.

In these crystallization development studies, a combination of cooling protocols and the addition of anti-solvents was studied in order to obtain good yields and particle uniformity. These studies showed that on a 20 g scale using a 2% crushed seed load and incremental addition of antisolvents, an eight hour soak period at 29°C followed by cooling to 5°C was the most promising protocol for particle uniformity (as assessed by light microscopy). and FBRM) and exit.

F) X-ray powder diffraction (XRPD).

In one embodiment, the present invention relates to essentially pure compound cyclo(-His-Pro) hydrate (structure 2), as shown below.

O which is characterized by an XRPD pattern containing peaks at about 17° and about 27.3° (±0.2° 2θ).

One embodiment of a substantially pure structure 2 is characterized by an X-ray powder diffraction pattern containing at least three peaks selected from the following list: 13.7; 17; 18.1; 20.2 and 27.3 degrees (±0.2° 2θ). Another embodiment is characterized by an XRPD pattern containing at least two peaks selected from the following list: 10; 13.7; 17;

18.1; 20.2 and 27.3 degrees (±0.2° 2θ).

G) Nuclear Magnetic Resonance (NMR) or 13CSSNMR.

In one embodiment, the 1 H NMR spectrum of structure 2 shows the following chemical shifts: ¹ H NMR (400 MHz; D2O) δ 7.58 (d; 1H; J= 3.2 Hz); 6.82 (d; 1H; J= 3.2 Hz); 4.42 (m; 1H); 4.12 (m; 1H); 3.36-3.47(m; 2H); 3.05 - 3.09 (m; 2H); 2.12 (br; 1H); 1.80 - 1.84 (m; 2H); 1.37 - 1.40 (m; 1H).

In one embodiment, the CHP hydrate of the present invention exhibits substantially the same ¹ H-NMR spectrum as anhydrous CHP in a DMSO-d ₆ solvent. This includes the following values: 1.7 ppm (m; 3H); 2.1 ppm (m; 1H); 2.5 ppm (s; 3H); 3.2 ppm (d; 1H); 3.5 ppm (m; 1H); 4.2 ppm (m; 2H); 7.0 ppm (s; 1H); 7.6 ppm(s; 1H); 8.1 ppm(s; 1H).

H) Infrared spectroscopy of compounds in the solid phase.

In one embodiment, the infrared spectrum of the solid structure 2 has signals at 3457, 3411 (m); 3292, 3211(m); 2976; 1658; 1633(m); and 1445-1424 (m) cm' ¹ .

I) Differential Scanning Calorimetry (DSC).

In another embodiment of the invention, it was found that the beginning of the endotherm of structure 2 lies at about 100±2°C and 171±2°C. In other embodiments, structure 2 (CHP hydrate) of the present invention shows the onset of an endotherm at a temperature of about 75°C to 100°C.

J) Dynamic vapor sorption (DSP).

In yet another embodiment, dynamic vapor sorption of structure 2 exhibits one or more of the following:

dehydration at a relative humidity of 10% with a weight loss of 6±0.2% and rehydration at a relative humidity of 0 to 40% at 40°C;

dehydration at a relative humidity below 20% with a weight loss of 6±0.2% and rehydration at a relative humidity of 0 to 40% at 50°C; and dehydration at a relative humidity below 20% with a weight loss of 7±0.2% and rehydration at a relative humidity of 0 to 40% at 60°C.

In other embodiments, the CHP hydrate of the present invention exhibits a weight loss of about 3% to 9%, or about 4% to 8.5%, or about 5% to 8%. In other embodiments, the CHP hydrate of the present invention exhibits a weight loss of about 5.5 to 7.3%.

In yet another embodiment, the CHP hydrate of the present invention exhibits a weight loss of from about 5.8% to about 7.0%.

K) Thermogravimetric analysis (TGA).

In another embodiment, TGA structure 2 shows a weight loss of 6±0.5% before decomposition.

In other embodiments, the CHP hydrate of the present invention exhibits at least one endothermic event at a temperature of about 75-85°C, which is a loss of water. In another embodiment, the CHP hydrate of the present invention exhibits at least one exothermic event at a temperature of about 115 to 120°C, which is a recrystallization event to structure 1.

L) Melting point.

In another embodiment, the melting point of structure 2 is 170±2°C.

M) Measuring the Metastable Zone Width (MSZW).

In another embodiment, the absence of precipitation at 0% MtBE indicates that the addition of an anti-solvent is required for crystallization to occur. At 10% MtBE, a metastable zone width of approximately 43.2°C was observed. At 20% MtBE, CHP remained in solution until the initial concentration reached >100 mg/mL. The metastable zone width for the 325 and 425 mg/mL experiments was determined to be about 35.2°C and about 30.7°C, respectively. Increasing the content of MtBE to 30 vol.% gave MSZW approximately 30±0.5°C (at 325 mg/ml). In addition, at 60% MtBE, MSZW was 51.4°C (at 100 mg/mL). At 80% MtBE, samples prepared at 100 mg/mL and 425 mg/mL remained in suspension throughout the experiment, even at higher temperatures. During the analysis of the width of the metastable zone, a trend was noted according to which a temperature of approximately 56-61°C was required to obtain a clear point.

P) Methods of treatment.

The structure 2 compound of the present invention can be used in therapeutically effective amounts for the treatment of various diseases and disorders such as diabetes and other metabolic diseases, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, Huntington's disease, acute renal failure (ARF), chronic renal failure ( CRF), kidney fibrosis; to provide cytoprotection against oxidative damage, to suppress inflammatory responses in the PC12 cell line, and as an appetite suppressant.

- 11 042744

Examples

The following examples are included to demonstrate certain embodiments of the present invention. However, those skilled in the art should, in light of the present invention, take into account that certain modifications are possible in the specific disclosed embodiments, and the same or similar result can be obtained without deviating from the essence and scope of the invention. Therefore, all of the information provided is to be interpreted as illustrative and not in a limiting sense.

Example 1 Conversion of structure 1 (anhydrous CHP) to structure 2 (CHP hydrate).

g material structure 1 (batch no. PS00726-55-D) was dissolved in 1 ml of water at 80°C. An additional 1 ml of water was added to counteract significant evaporation. At 80° C. a clear dark brown solution was obtained. The solution was then rapidly cooled to 50° C. and 9.5 volumes (19 ml) of acetone was added to the solution to give a pale yellow solution. No oiling or sedimentation was found. The solution was cooled to room temperature, resulting in an appreciable amount of solid precipitate. The solution was cooled to 6°C (to increase the yield) and the suspension was filtered. The solid was filter dried to prevent dehydration. Since dehydration to structure 1 occurs at or above 80°C under vacuum, drying at 50°C under vacuum was considered safe to preserve structure 2. XRPD analysis confirmed that the product was structure 2 in about 72% yield.

The CHP hydrate batch was analyzed by various methods, including X-ray powder diffraction (XRPD), pKa analysis, polarized light microscopy (PLM), thermogravimetric analysis/differential thermal analysis (TG/DTA), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), Dynamic Vapor Sorption (DSP), Variable Temperature Humidity X-Ray Powder Diffraction (VT-/VH-XRPD), 1H Nuclear Magnetic Resonance (NMR), and Heteronuclear Single Quantum Coherence (HSQC) NMR.

A) X-ray powder diffraction (XRPD).

XRPD analysis was performed on a PANalytical X'pert pro scanning samples from 3 to 35° 2θ. The material was gently crushed to release the agglomerates and loaded onto a multiwell plate with Kapton or Mylar film to support the sample. Then, the multiwell plate was placed in a diffractometer and analyzed using Cu K radiation (α1 λ=1.54060 Α; α2=1.54443 Α; β=1.39225 A; ratio α1: α2=0.5) in transmission mode (size step 0.0130° 2θ) using 40 kV/40 mA generator settings.

As shown in FIG. 1, the batch was crystalline according to XRPD and was designated structure 1. XRPD analysis was performed on a 5 g sample of CHP hydrate. The sample was weighed into a glass tube and 6 ml of ethanol/water 90:10 was added to obtain a suspension. The mixture was then stirred for about 24 hours at ambient temperature and then analyzed by XRPD. As shown in FIG. 2, this material was confirmed as structure 2 (FIG. 2(b)). As shown in FIG. 3, analysis of structure 2 after EAF showed no shape change at 40% relative humidity after EAF. As shown in FIG. 4, analysis of structure 2 showed that after DSP at various temperatures (VT-DVS), the material was a mixture of structures 1 and 2. As shown in FIG. 5, approximately 30 mg of structure 2 (black line, FIG. 5) was stored under three conditions for seven days: at 40°C/75% RH, at 80°C, and under natural light. An XPRD analysis was performed to assess changes in the shape of the material. After seven days, no change was observed in samples stored at 40°C/75% relative humidity (blue line, Fig. 5) and in samples stored under natural light (green line, Fig. 5). The sample stored at 80° C. is converted to structure 1 (red line, Fig. 5). As shown in FIG. 7, sample structure 2 (blue line in FIG. 7) was returned to the vacuum oven for another 30 minutes. XRPD analysis (purple line in Fig. 7) showed that the material was still predominantly structure 2 with some structure 1 peaks.

As shown in FIG. 6, CHP hydrate (structure 2) was weighed into a glass vessel and placed in a 50° C. vacuum oven for 1 hour. XRPD analysis (green line) showed that the material was still predominantly structure 2 with some structure 1 peaks.

As shown in FIG. 8, the input material from FIG. 5 (black line) was returned to the oven in which the temperature was raised to 80°C. The vial was placed in the oven for an additional 18 hours. XRPD analysis (pink line) showed that the material was a mixture of structures 1 and 2. There was an additional peak at 8° 2θ which was not previously observed in either structure. As shown in FIG. 9, structure 2 of FIG. 5 was dried for three different time periods and the results analyzed by XRPD. Drying the sample at 50°C for 1 h gave structure 2 with structure 1 peaks (green line), drying the sample at 50°C for 90 min also produced structure 2 with structure 1 peaks (purple line), and drying the sample at 80 °C for 18 h produced a mixture of structure 1 and structure 2 (pink line).

In FIG. 34 summarizes the various conditions under which structure 2 changes to structure 1. Processing a sample of structure 2 (black line, Fig. 34) at 80°C and 0% relative humidity (cycle 2,

- 12 042744 red line) causes the transformation of structure 2 to structure 1 within 20 min (cycle 3, blue line), while it remains as structure 2 at 0% relative humidity and ambient temperature even after 1 hour (red line ). Structure 2 is stable at ambient temperature and 40% relative humidity (cycle 1, brown line).

B) pKa analysis.

pKa analysis was performed on structure 1 using a potentiometric method, which is an acid-base titration to determine the pKa point.

As shown in FIG. 10, analysis of anhydrous CHP (structure 1) resulted in a pKa value of 6.4.

C) Optical microscopy (unpolarized light) and polarized light microscopy (PLM).

Optical microscopy was performed visually using a Linkam THM600 calibrated warming stage with an attached control box connected to an Olympus BX50 polarizing microscope equipped with a Motic camera and image capture software (Motic Images Plus 2.0). Approximately 0.5 mg of material was placed on a microscope cover slip and heated at 10° C./min with images taken at the usual intervals to document any thermal transitions. All images were recorded using a 10x lens unless otherwise noted.

(PLM) analysis was performed using an Olympus BX50 polarizing microscope equipped with a Motic camera and image capture software (Motic Images Plus 2.0). All images were recorded using a 20x lens unless otherwise noted. The presence of crystallinity (birefringence) was determined using PLM. A polarized light microscope is designed to observe and photograph specimens that are visible primarily due to their optically anisotropic properties.

Fig. 11 (non-polarized microscope) and 12 (polarized light microscope) show that the supplied CHP (basically structure 1) was birefringent with no distinct morphology. As shown in FIG. 13, structure 2 was birefringent with a fragmented rod-like morphology. In other embodiments, the CHP hydrate (structure 2) of the present disclosure exhibits a fragmented rod-like morphology in PLM.

D) Thermogravimetric analysis/differential thermal analysis (TG/DTA).

Approximately 5 mg of material was weighed into an open aluminum cuvette and loaded into a Seiko 6200/7200 Synchronous Thermogravimetric/Differential Thermal Analyzer (TG/DTA) and maintained at room temperature. The sample was then heated at a rate of 10° C./min from 20° C. to 300° C., during which time the sample mass change was recorded along with any differential thermal events (DTA). Nitrogen was used as purge gas at a flow rate of ³⁰⁰ cc/min.

As shown in FIG. 14, TGA analysis of structure 1 revealed an initial weight loss of 0.6% (0.08 eq. water) at approximately 75-85°C. This 0.6% water was classified as free surface water. Two more weight losses were observed: 0.2 and 0.3% (0.03 and 0.04 equivalents of water, respectively) before the sample decomposed at about 260°C. The first weight loss may be due to a small amount of structure 2 material mixed with the dehydrated material to become structure 1. This amount is estimated to be 8% based on weight loss. The second loss of water at a temperature of about 100°C may be due to the evaporation of unbound surface water. DT analysis revealed one abrupt endothermic event with an onset of 170°C and a peak at 172°C.

As shown in FIG. 15, TGA analysis of structure 2 showed an initial weight loss of 6.5% (0.9 water equivalents) followed by sample decomposition at about 280°C. DT recording identified an endothermic event associated with initial weight loss. This was followed by an exothermic event (recrystallization to structure 1) at 118°C. The second endothermic effect, which is considered the melting of the sample, was observed at 170°C, and a peak at 172°C. In other embodiments, the CHP hydrate of the present invention exhibits at least one endothermic event at about 75-85° C., which is water loss. In another embodiment, the CHP hydrate of the present disclosure exhibits at least one exothermic event at a temperature of from about 115°C to about 120°C, which is a recrystallization event into structure 1.

E) Differential Scanning Calorimetry (DSC).

DSC was performed by adding approximately 5 mg of material, which was weighed into an aluminum DSC pan and sealed with a perforated aluminum cap. The sample cuvette was then loaded into a Seiko DSC6200 (equipped with a cooler), cooled and kept at 20°C. After obtaining a stable heat flux response, the sample and reference were heated to 250°C at a scan rate of 10°C/min and the resulting heat flux response was monitored. Nitrogen was used as purge gas at a flow rate of 50 ^cc /min.

As shown in FIG. 16, the first heat cycle of structure 1 showed a small broad endotherm starting at 75°C with a peak at 85°C. This is consistent with the 0.6% weight loss observed

- 13 042744 in TG/DTA as shown in FIG. 14. A second, large endothermic event was observed at 169°C with a peak at 171°C. This is consistent with the melting observed in TG/DTA. The first broad peak in Fig. 16 may be caused by dehydration of structure 2 to structure 1. The second peak in FIG. 16 may be the melting of structure 1.

As shown in FIG. 17, no thermal events were observed in the refrigeration cycle. As shown in FIG. 18, a very small broad peak, a potential glass transition point, was observed at about 80° C. in the second thermal cycle.

As shown in FIG. 19, the first heat cycle of structure 2 revealed an endotherm with an onset of 99°C and a peak at 102°C. Melting of the sample was noted starting from 170°C with a peak at 173°C. This agrees with the data obtained in TG/DTA. As shown in FIG. 20, no thermal events were observed in the cooling cycle of structure 2.

As shown in FIG. 21, a possible glass transition point was observed in the second heating cycle at 77° C. of CHP hydrate.

In one embodiment, the CHP hydrate of the present invention exhibits an onset of an endotherm at a temperature of about 75 to 100°C.

F) Fourier transform infrared spectroscopy (FT-IR).

FR-IR was performed on a Bruker ALPHA P spectrometer. Sufficient material was placed in the center of the spectrometer plate and spectra were obtained using the following parameters: resolution: 4 cm ^-1 , background scan time: 16 scans, sample scan time: 16 scans, collection data: 4000 to 400 cm ^-1 , resulting spectrum: transmission, and software: OPUS version 6.

As shown in FIG. 22, FT-IR structure 2 corresponds to the chemical structure. The continuous infrared spectrum of structure 2 displays signals at 3457, 3411 (m); 3292, 3211(m); 2976; 1658; 1633(m); and 1445-1424 (m) cm ^-1 . In other embodiments, the implementation of CHP and CHP hydrate of the present invention shows essentially the same IR spectrum, which shows at least nine bands from about 500 to 1660 cm ^-1 . Structure 2 has a stretching vibration at 3457 and 3411 cm ^-1 due to water (hydrate) in the crystal lattice, while structure 1 does not.

G) Dynamic vapor sorption (DSP).

DSP was performed by placing a sample of approximately 10-20 mg in a mesh vapor sorption balance and loading it into a DVS Intrinsic dynamic vapor sorption balance from Surface Measurement Systems. The sample was ramped from 40 to 90% relative humidity (RH) in 10% increments, holding the sample at each step until a stable mass was reached (dm/dt 0.004%, min step length 30 min, max step length 500 min) at 25° WITH. After completion of the sorption cycle, the sample was dried using the same procedure to 0% RH, and then a second sorption cycle was carried out back to 40% RH. Two cycles were completed. We plotted the change in mass during the sorption/desorption cycles, which made it possible to determine the hygroscopicity of the sample. An XRPD analysis of any remaining solid was then performed.

As shown in FIG. 23, the DSP isothermal plot of Structure 1 showed a weight increase of 6.3% at 60 to 90% relative humidity, indicating conversion to CHP hydrate, Structure 2. As shown in FIG. 24, upon conversion to structure 2, the material appeared only slightly hygroscopic with a maximum absorption of 0.8 wt% at 0 to 90% relative humidity, which is assumed to be surface water and not part of the crystal structure. This phenomenon is shown in Fig. 25, DSP kinetic plot of structure 1, which shows that structure 1 first hydrates to form structure 2, but does not give up water during the desorption phase, indicating that the slight 0.8% mass increase is not part of the solid structure.

As shown in FIG. 26, XRPD analysis after DSP of structure 1 showed a new shape, designated as structure 2. As shown in FIG. 27, TG/DTA analysis after EAF structure 2 showed an initial weight loss of 6.8% (0.95 eq. of water) followed by decomposition of the sample at about 280°C. The DT curve identified an endothermic event associated with weight loss, followed by recrystallization (to structure 1), an exothermic event at about 120°C, then a melt starting at 168°C and peaking at 172°C.

CHP Structure 2 was placed under several higher temperature conditions to test for dehydration/rehydration at 0-90% humidity: 25° C. (FIG. 28); 40°C (Fig. h0); 50°C (Fig. 31); and 60°C (Fig. 32).

As shown in FIG. 28, the EAF isothermal plot showed that the material of structure 2 did not change at 40 to 90% and 10 to 90% relative humidity. The material then lost 3.5 wt.% at a relative humidity below 10% and was rehydrated at a relative humidity of 0 to 90%. As shown in FIG. 29, the DSP kinetic plot shows that CHP hydrate loses 3.5% water (red line) during the desorption phase from 80 to 0% RH in 500 minutes. As shown in FIG. 30, 31 and 32, structure 2 undergoes a single cycle at 40, 50 and 60°C. A temperature of 40° C. indicated that the material began to dehydrate at below 10% relative humidity, losing approximately 5.8% by weight from 10 to 0% relative humidity (0.8 eq. of water). The material was hydrated at a relative humidity of 0 to 40% (FIG. 30). A temperature of 50° C. showed that the material began to dehydrate at a relative humidity below 20%, losing about 6.1 wt.% from 20 to 0% RH (0.8 eq.

water). The material was rehydrated from 0 to 40% RH (FIG. 31). Analysis at 60°C showed that the material began to dehydrate at a relative humidity below 20%, losing about 7 wt.% from 20 to 0% RH (1.0 eq. water). The material was rehydrated from 0 to 40% RH (FIG. 32).

In other embodiments, the CHP hydrate of the present invention exhibits a weight loss of about 3 to 9%, or about 4 to 8.5%, or about 5 to 8%. In other embodiments, the CHP hydrate of the present invention exhibits a weight loss of about 5.5 to 7.3%.

In yet another embodiment, the CHP hydrate of the present invention exhibits a weight loss of about 5.8 to 7.0%.

H) Variable Temperature Humidity X-Ray Powder Diffraction (VT/VH-XRPD).

VT-ZVH-XRPD analysis was performed on the structure 2 material. Structure 2 was present at the initial scan at 40% relative humidity and room temperature. Structure 2 remained for cycle 2 when the relative humidity was reduced to 0% at room temperature. The temperature was raised to 80°C and the sample was left for 20 minutes before scanning. The resulting diffraction pattern suggests that the resulting material was pure pattern 1. Clear pattern 1 remained after 80 minutes at 80°C and 0% relative humidity. Fig. 33 illustrates these results.

Fig. 34 shows XRPD of structure 2 material after various VT/DVS. In other embodiments, the CHP hydrate of the present invention is present at a relative humidity of at least about 0 to 90% at room temperature. In yet another embodiment, 100% of structure 2 is converted to structure 1 at 0% relative humidity at 80°C.

I) 1H Nuclear Magnetic Resonance 1H (NMR).

NMR was performed using a Bruker AVIIIHD spectrometer equipped with a DCH cryoprobe operating at 500.12 MHz for protons. The experiments were carried out in deuterated DMSO and each sample was prepared at a concentration of about 10 mM. The ¹ H-NMR spectrum of structure 1 CHP (DMSO-d6) shows that the results are consistent with the structure of the compound (Fig. 35). The ¹ H-NMR spectrum of structure 2 (DMSO-d6) is shown in FIG. 36. Structure 2 and structure 1 of CHP showed the same NMR spectrum, since both were completely dissolved in the solvent. As shown in FIG. 37, NMR was carried out on the dried material, structure 2 (pink line in Fig. 9). No change was observed in the dried material from the starting material of structure 2.

In one embodiment, the CHP hydrate of the present invention exhibits substantially the same ¹ H-NMR profile as anhydrous CHP in a DMSO-d ₆ solvent.

J) NMR with heteronuclear single quantum coherence (HSQC).

HSQC-NMR was performed for structure 2 using Bruker 500 MHz NMR equipment. DMSO-d ₆ was used as the NMR sample solvent. As shown in FIG. 38, the results of structure 1 showed that two imidazole C-H chemical shifts were 4.2 ppm.

The HSQC-NMR experiment was performed on the dried material, structure 2 (pink line in Fig. 9). As shown in FIG. 39, the results showed that the dried material (a mixture of structure 1 and structure 2) and the NMR of the material of structure 2 are identical.

K) Lyophilization.

For dissolution screening: 330 mg of structure 1 CHP was dissolved (with gentle heating) in water (3.3 ml) and distributed equally among 33 vials. These vials were then frozen at -50° C. before being lyophilized overnight. It is widely known that lyophilization of crystalline compounds produces an amorphous material which is generally more soluble than the crystalline forms of the compound.

As shown in FIG. 40, the material reconstituted after lyophilization of Structure 1 showed some crystalline peaks when analyzed by XRPD. Applicant believes that the initial peaks indicate a mixture of hydrates and anhydrous forms mixed with amorphous forms.

For another lyophilization run, each 10 mg sample shown in FIG. 40 was redissolved in 200 µl of water. The vials were frozen again at -50° C. before lyophilization for 72 hours. The material recovered after lyophilization was predominantly amorphous according to XRPD, as shown in FIG. 41.

For amorphization test: CHP hydrate (structure 2) was dissolved in distilled water. This solution was equally divided into 26 glass vials and the vials were frozen at -50° C. in preparation for lyophilization. After freezing, the samples were placed in desiccators attached to a freeze dryer and dried for approximately 48 hours. As shown in FIG. 42, the material recovered after lyophilization of structure 2 was structure 2 when analyzed by XRPD.

For another amorphization cycle, the samples were redissolved by adding 1 ml of distilled water to each vial. Then, the solutions were pipetted into 26 glass vials, 20 ml each, and another 15 ml of distilled water was added. The vials were frozen at -50°C prior to lyophilization for approximately 48 hours. After 48 hours, the samples were not completely lyophilized and the vials were returned to the desiccators and lyophilized for a further 72 hours. XRPD still contained structure 2 peaks.

In other embodiments, lyophilization of the CHP hydrate of the present invention shows that structure 2 is more stable than structure 1 and remains at least about 10-40% pure after amorphization. L. Dissolution screening 33x10 mg samples of CHP structure 1 (anhydrous form) were lyophilized in 2 ml vials and 100 μl of different solvent systems were added to each vial. Between each addition, the mixture was checked for dissolution and if no dissolution was observed, the mixture was heated to about 40°C and the test was repeated. After adding 300 μl of solvent, 100 μl aliquots were added. This procedure was continued until dissolution was observed or until 1 ml of solvent was added. If no dissolution occurred, the solids were separated by filtration and XRPD was performed. If dissolution occurred, the solvent was allowed to evaporate and XRPD was performed on the remaining solid.

In FIG. 43 shows the approximate solubility of structure 1 in various solvents, and FIG. 44 shows the results of solubility in solvents of structure 1 as a function of the volume of solvent added. As shown in FIG. 43 and 44, high solubility was observed in ethanol, methanol, K,K-dimethylacetamide, water, DMSO, trifluoroethanol, 1-propanol, and methanol/water mixtures. The samples were left closed for evaporation and the observed solids were analyzed by XRPD.

As shown in FIG. 45, XRPD analysis revealed 2 unique structures in addition to structure 1 (crystalline form) in kinetic solubility screening, although structure 3 only appeared as a mixture with structure 2. Acetone, dichloromethane (DCM), ethyl formate and MtBE gave structure 2 (hydrated form) . A mixture of crystalline and hydrated forms was observed in ethanol. Chlorobenzene gave a mixture of structures 2 and 3 and THF gave a mostly amorphous material with some peaks of structure 2. A colorless gel was visible in methanol and was not analyzed by XRPD. The solvent system formed crystals during evaporation and was identified as a potential THF solvate.

As shown in FIG. 45, 9 of the 30 solvent systems listed did not form enough solids for XRPD analysis. Sixteen of the solvents gave structure 2, structure 1 was observed in 1-propanol, a mixture of structures 1 and 2 was observed in ethanol, a mixture of structures 2 and 3 was obtained in chlorobenzene, and a mostly amorphous material with some structure 2 peaks was obtained in THF.

In FIG. 46 shows solvents for primary screening of polymorphs.

In other embodiments, the CHP hydrate of the present invention can be prepared by kinetic crystallization (2-6 hours) from 1-propanol, 2-propanol, methanol/water, acetone, acetonitrile, ethyl acetate, ethyl formate, heptane, isopropyl acetate, methyl ethyl ketone (MEK ), methyl isobutyl ketone, nitromethane, toluene and trifluoroethanol.

M) Primary screening for polymorphs.

Twenty-four (24) vials containing 40 mg anhydrous CHP were added with various solvents in 100 μl aliquots until a liquid suspension was formed. The vials were then subjected to temperature cycling between ambient and 40° C. in 4 hour cycles for 72 hours. Any remaining solids were isolated by centrifugation and analyzed by XRPD.

Fig. 46 shows the solvents used for kinetic (2-6 h) screening of polymorphs, and FIG. 50 shows the temperature cycle of the primary screening of structure 1 polymorphs.

The results show that when water is present in the sample, structure 2 predominates. Structure 1 is formed only if insufficient water is present in the sample, either from the sample itself, from the solvent system, or from the atmosphere. These results are shown in table. 24 and 25.

Example 2. Method for producing CHP hydrate.

This study was performed (1) to evaluate the transfer process of structure 1 (anhydrate, C16081735-D) to structure 2 (hydrate, structure 2); (2) to evaluate a demonstration run on a scale of approximately 100 grams using non-GMP CHP structure 1 (anhydrate) held by a designated manufacturer; (3) to produce approximately 400 grams of Structure 2 in accordance with current Good Manufacturing Practice (cGMP) regulations; and (4) to evaluate the drying conditions to ensure that the desired structure 2 (hydrate, structure 2) is obtained.

Specifications were set as follows: HPLC purity: >98.0%. Impurity profiles: H-His-OH <1.0%, each other individual impurity <1.0%. Chiral purity: >8.0%. Chiral impurities: each impurity containing >0.1% is listed. Optical isomers DL, LD, LL <1.0%, etc.

A reproducible method was developed to obtain structure 2 (cyclo(L-His-L-Pro) dipeptide hydrate) by dissolving structure 1 (anhydrate) in ethanol/H2O=9/1 (v/v) and precipitating structure 16 042744 ry 2 (hydrate) by adding MtBE. The method was successfully carried out on a batch of cGMP at a scale of 370 g. Ultimately, a total of 288 g of structure 2 (CHP hydrate) was obtained with a purity of 99.9% according to

HPLC and 100.0% chiral purity under cGMP conditions.

A) Introduction and synthesis scheme.

The method of converting structure 1 (anhydrous) to structure 2 (hydrate, structure 2) was evaluated. Two crystallization procedures have been proposed: one was to dissolve the solid anhydrate in water and precipitate by adding acetone (see Synthesis Scheme 1); the other was to dissolve the solid anhydrate in ethanol/water=9/1 and precipitate by adding MtBE (see synthesis scheme 2). The quality and yield of the two procedures recorded in Scheme 2 were evaluated to optimize performance.

Finally, an optimized process (see scheme 3) was successfully carried out to obtain 288 g of structure 2 (CHP hydrate). Planned periodic production was carried out according to scheme 3.

Scheme 1.

The first route for the synthesis of structure 2 (CHP hydrate).

Mm. :234.25

Anhydrite CHP

Scheme 2.

1. Dissolve D in water (1 volume) at 80°C

2. Cool down to 50°C

3. Slowly add acetone (9.5 volumes)

-------------------------------►

4. Slowly cool to 20-25°C

5. Cool down to 5-10°C

6. Separate the solid by filtration.

7. Dry at 50°C under vacuum Yield 72%

The second route for the synthesis of structure 2 (CHP hydrate).

M.m.: 234.25

Anhydrite CHP

1. Dissolve D in ethanol/water (9/1 v/v) at 50°C

2. Cool down to 35°C

3. Add MTBE (7.5 volumes) ------------------------------►

4. Stir at 35°C for 5 hours

5. Cool down to HS

6. Stir for 12 hours.

7. Filter and wash.

8. Dry at 50°C under vacuum

Mm. 234.25 based on dry matter

MW: 252.27 as monohydrate

Hydrate CHP

Mm. 234.25 based on dry matter

MW: 252.27 as monohydrate

Hydrate CHP

Scheme 3.

Optimized synthesis route for structure 2 (CHP hydrate) for cGMP production.

M.m.: 234.25

Anhydrate CHP

1. Add D to ethanol/water = 9 volumes/1 volume (2.5 volumes)

2. Heat to 47-53°C to dissolve

3. Slowly cool to 32-37°C

4. Slowly add MtBE (0.5 volumes) at 32-37°C ------------------------------►

5. Add seed and incubate for 2-3 hours

6. Cool to 3-8°C for 2-3 hours

7. Add MtBE (7 vol. for 8-9 hours at 3-8°C

8. Stir at 3-8°C for 8-10 hours

9. Filtered to obtain a wet product

10. Wash with ethanol/water/MtBE = 9/1/30 vol. (2 vol.)

11. Dry at 35-40°C under vacuum

Mm. 234.25 based on dry matter

MW: 252.27 as monohydrate

Hydrate CHP

Both procedures (schemes 1 and 2) were evaluated and gave the product as a hydrate with the desired structure. The chemical stability was studied under both conditions and structure 2 was stable at 50°C for 20 h. such a small minimum mixing volume. In addition, the water/acetone procedure gave only about 70% of the actual yield. The ethanol/H ₂ O/MtBE procedure was chosen for the optimization work, with an actual yield of about 90%. After an optimized crystallization procedure, the 370g scale cGMP series was successfully carried out in the laboratory for production in the kilogram scale.

B. Summary.

Two crystallization procedures with water/acetone and ethanol/H ₂ O/MtBE mixtures were evaluated to obtain structure 2 and both gave the product as a hydrate with the target structure 2. Chemical stability was studied under both conditions and the product was stable at 50° C for 20 hours. Considering that the water/acetone procedure gave only about 70% of the actual yield, the ethanol/H ₂ O/MtBE procedure was chosen for the optimization work. The temperature and rate of addition of MtBE were studied, and as a result, the procedure for adding MtBE at 5°C at a relatively low rate was chosen in order to avoid attaching a significant amount of solid to the walls of the vessel. According to the optimized crystallization procedure, one demonstration was carried out

- 17042744 cycle on a scale of 100 g, and as a result of crystallization was obtained 96.44 g of the product in the form of a structure with about 90% of the actual yield. Based on a drying stability study, the hydrated product was stable below 40° C. in a laboratory oven at -0.09 MPa.

C) Evaluation of the crystallization procedure under water/acetone conditions.

Mm. :234.25

Anhydrate CHP

1. Dissolve D in water (1 volume) at 80°C

2. Cool down to 50°C

3. Slowly add acetone (9.5 volumes) ------------------------------►

4. Slowly cool to 20-25°C

5. Cool down to 5-10°C

6. Separate the solid by filtration.

7. Dry at 50°C under vacuum

Yield 72%

Mm. 234.25 based on dry matter

MW: 252.27 as monohydrate

Hydrate CHP

Two tests were performed to evaluate the RFP crystallization procedure under water/acetone conditions (Table 2). In experiment PS03027-2, no unseeded crystallization was observed, and a solid precipitated after the addition of the seed.

The seed was added in experiment PS03027-4 and crystallization proceeded smoothly. XRPD analysis showed that the target structure 2 was obtained. The product loss in the water/acetone system was relatively high (about 20%), which corresponded to the yield in RFP (72%).

Table 2. Results of obtaining CHP hydrate

No. series Raw Materials Observation Hydrate CHP Anhydrate CHP Solvent Observation Weight XRPD Loss in ml PS03027-2 2g Series No.: PS00726-55-D-P Water / acetone No crystallization without seeding. With seeding, a solid precipitate is formed 1.15 g Structure 2 n/a PS03027-4 5 g Series No.: PS00726-55-D-P Water / acetone A solid precipitated after seeding 3.65 g Structure 2 20%

Note: The seed was prepared by resuspending structure 1 in 1 volume of water at room temperature.

D) Evaluation of an alternative crystallization procedure with a mixture of ethanol/water/MTBE.

M.m.: 234.25

Anhydrate CHP

1. Dissolve D in ethanol/water (9/1 v/v) at 50°C

2. Cool down to 35°C

3. Add MTBE (7.5 volumes) --------------------------------------►

4. Stir at 35°C for 5 hours

5. Cool down to HS

6. Stir for 12 hours.

7. Filter and wash.

8. Dry at 50°C under vacuum

Mm. 234.25 based on dry matter

MW: 252.27 as monohydrate

Hydrate CHP

One run on a 5 g scale was prepared to evaluate alternative crystallization conditions in the ethanol/water/MTBE system and obtained crystals nucleated during the addition of MTBE and XRPD showed target structure 2. Losses in the mother liquor were relatively lower (8%), than under water/acetone conditions (Table 3).

Table 3. Results of obtaining CHP hydrate

Series No. Raw Materials Observation Hydrate CHP (STRUCTURE 2) Anhydrate CHP Solvent Observation Weight XRPD Residual solvent A loss PS03027-5 5 g, Lot # PS00726-55-D- R Ethanol/Water/Mtbe Spontaneous nucleation upon addition of MtBE n/a Structure 2 Ethanol: 0.01%; Mtbe: 0.01% 8%

E) Study of chemical stability in water/acetone and ethanol/water/MtBE systems.

The chemical stability in the dissolved state for the compound was studied in various solvent systems at various temperatures (Table 4). The solution was kept at 50°C and 80°C, and the purity of the solution was determined. The compound was usually stable for at least 20 hours.

- 18 042744

Table 4. Chemical stability in water/acetone and Ethanol/water/MtBE systems

Experiment # Solvent Concentration Input T/°С Purity (%) Observation 0h 2h 20 h PS03027-8- H-1 H ₂ O 10 mg/ml PS00726-55- D-P Purity: 99.7% 80 99.82% 99.77% 99.82% No change PS03027-8- H-2 Ethanol: H ₂ O \u003d 9: 1 (o / o) 10 mg/ml 50 99.76% 99.86% 99.69% No change PS03027-8н-з H ₂ O: Acetone \u003d 0.5: 9.5 (0/0) 10 mg/ml 50 99.77% 99.72% 99.73% No change PS03027-8- H-4 (Ethanol:H ₂ O=9:1): MTBE=1:3 (v/v) 10 mg/ml 50 99.82% 99.77% 99.82% No change

F) Solubility study of CHP hydrate (structure 2).

The solubility of CHP hydrate in the water/acetone and ethanol/water/MtBE systems was studied and found to be sensitive to solvent ratio and temperature under both conditions (Tables 5 and 6). The crystallization yield in the ethanol/water/MtBE system was 90% or higher.

Table 5. Solubility of structure 2 in the water/acetone system

experiment number Input Solvent Water/acetone (v/v) t/°s Solubility (mg/ml) PS03029-4-H-1 Hydrate CHP (Structure 2) 0/10 50 14 PS03029-4-H-2 0.5/9.5 49 PS03029-4-H-3 1/9 159 PS03029-4-H-4 2/8 >200 PS03029-4-H-5 4/6 >200 PS03029-4-H-6 6/4 >200 PS03029-4-H-7 8/2 >200 PS03029-4-H-8 9/1 >200 PS03029-4-H-14 0/10 5 17 PS03029-4-H-15 0.5/9.5 19 PS03029-4-H-16 1/9 48 PS03029-4-H-17 2/8 BY PS03029-4-H-18 4/6 179 PS03029-4-H-19 6/4 206 PS03029-4-H-20 8/2 >200 PS03029-4-H-21 9/1 215

Table 6. Solubility of structure 2 in the ethanol/water/MtBE system

No. experiment Input Solvent (ethanol / water = 9/1) / Mtbe (v/v) t / "s Solubility (mg/ml) PS03029-4-H-9 Hydrate CHP (structure 2) 3/0 50 >200 PS03029-4-H-10 3/1 >200 PS03029-4-H-11 3/3 101 PS03029-4-H-12 3/6 24 PS03029-4-H-13 3/9 17 PS03029-4-H-22 3/0 5 121 PS03029-4-H-23 3/1 59 PS03029-4-H-24 3/3 thirty PS03029-4-H-25 3/6 6 PS03029-4-H-26 3/9 6

Note: 1) PS03027-2-H series was used as structure 2 material.

G) Process optimization under ethanol/water/Mtbe conditions.

To further understand crystallization in the ethanol/water/MtBE system, three experiments were performed where MtBE was added at 50, 35 and 5°C, respectively. As shown in Table. 7, three experiments provided the required crystalline form and the content of residual solvents was low.

- 19 042744

Table 7. Process optimization under ethanol/water/MtBE conditions (scale 5 g)

Series No. Raw Materials Conditions Hydrate CHP (structure 2) Anhydrate CHP Solvent Weight XRPD Residual solvent Loss of exit PS03027-9 5 g, Lot # PS0072655-D-P Ethanol/ water/ Mtbe Addition of MtBE at 50°C 4.35 g Structure 2 Ethanol: 0.01%; MTBE: 0.02% 5% PS03027- eleven 5 g, Lot # PS0072655-D-P Ethanol/ water/ Mtbe Addition of MtBE at 35°C 4.49 g Structure 2 Ethanol: 0.01%; MTBE: 0.01% Not measured PS03027- 10 5 g, Lot # PS0072655-D-P Ethanol/ water/ Mtbe Addition of MtBE at 5°C 4.89 g Structure 2 Ethanol: 0.02%; MTBE: 0.02% 5%

To test the procedure on a larger scale, three experiments were carried out on a 15 g scale, as shown in Table 1. 8. During experiments with PS03027-13 and PS03027-14 (in which MtBE was added at 50 and 35°C), the problem of formation of solid deposits was observed. A significant amount of solid settles on the walls of the vessel, especially during the addition of MtBE. To avoid this problem, in experiment PS0027-15, MtBE was added at 5° C. and the addition was extended to 8 hours (4 hours in previous experiments). It turned out that there were much less deposits on the walls. Therefore, it was concluded that MtBE should be added relatively slowly at 5°C.

Table 8. Process optimization in the ethanol/water/MtBE system (scale 15 g)

Series No. Raw Materials Conditions Hydrate CHP (structure 2) Anhydrate CHP Solvent Weight XRPD Residual solvent Loss of exit PS0302713 15 g, Lot # PS0072655-D-P Ethanol/ water/ Mtbe Addition of MtBE at 50°C 7.42 g Structure 2 MTBE:0.02% Ethanol: 0.03% Not measured PS03027-14 15 g, Lot # PS0072655-D-P Ethanol/ water/ Mtbe Addition of MtBE at 35°C 11.93 g Structure 2 MTBE:0.01% Ethanol: 0.04% 6% PS03027-15 15 g, Lot # PS0072655-D-P Ethanol/ water/ Mtbe Slow addition of MtBE at 5°C 14.44 g Structure 2 MTBE:0.01% Ethanol: 0.04% 7%

H) Stress test of rapid addition of MtBE.

As shown in Table. 9, a stress test was performed for a procedure in which the addition of MtBE was carried out at a relatively high rate. An XRPD analysis of the solid was performed during the run and the data indicated that structure 2 was observed throughout the crystallization.

Table 9. Stress test of rapid addition of MtBE

Series No. Raw Materials Conditions Hydrate CHP (structure 2) Anhydrate CHP Solvent Weight XRPD Residual solvent Loss of exit PS0302717 5 g, Lot # PS00726- 55-D-P Ethanol/ water/ Mtbe Rapid addition of MtBE at 5°C 4.58 g Structure 2 MTBE:0.00% Ethanol: 0.02% 3%

I) Study of form stability in the ethanol/water/MtBE system.

To study the form stability in the ethanol/water/MtBE system, competitive purification experiments were carried out at 50, 35 and 5°C, respectively, adding 100 mg of structure 1 anhydrous solid and 100 mg of structure 2 solid. 10, mixed forms of structure 1 and structure 2 are quickly converted to structure 2 under various conditions.

- 20 042744

Table 10. Competitive experiments for a suspension of structure 1 and structure 2 in the Ethanol/water/MtBE system

experiment number Solvent (v/v) t/°s XRPD Analysis 0 hours 2 hours 6 days 7 days PS03027-16-H-1 (Ethanol:H ₂ O=9:1):MTBE=3:0 50 Structure 2 n/a n/a n/a PS03027-16-H-2 (Ethanol:H ₂ O=9:1):MTBE=2.5:0.5 Structure 2 Structure 2 Structure 2 n/a PS03027-16-H-3 (Ethanol:H ₂ O=9:1):MTBE= 1:2 Structure 2 Structure 2 n/a n/a PS03027-16-H-4 (Ethanol:H ₂ O=9:1):MTBE= 1:3 Structure 2 Structure 2 Structure 2 Structure 2 PS03027-16-H-5 (Ethanol:H ₂ O=9:1):MTBE=3:0 35 Structure 2 Structure 2 Structure 2 Structure 2 PS03027-16-H-6 (Ethanol:H ₂ O=9:1):MTBE=2.5:0.5 Structure 2 Structure 2 Structure 2 Structure 2 PS03027-16-H-7 (Ethanol:H ₂ O=9:1):MTBE= 1:2 Structure 2 Structure 2 n/a n/a PS03027-16-H-8 (Ethanol:H ₂ O=9:1):MTBE= 1:3 Structure 2 Structure 2 Structure 2 Structure 2 PS03027-16-H-9 (Ethanol:H ₂ O=9:1):MTBE=3:0 5 Structure 2 Structure 2 Structure 2 Structure 2 PS03027-16-H10 (Ethanol:H ₂ O=9:1):MTBE=2.5:0.5 Structure 2 Structure 2 Structure 2 Structure 2 PS03027-16-H- eleven (Ethanol:H ₂ O=9:1):MTBE= 1:2 Structure 2 Structure 2 Structure 2 Structure 2 PS03027-16-H12 (Ethanol:H ₂ O=9:1):MTBE= 1:3 Structure 2 Structure 2 Structure 2 Structure 2

Note: Introductory materials: 100 mg of an anhydrous solid (structure 1, lot no. PS00726-55-D) was added along with 100 mg of solid of structure 2 (batch no. PS030275/9/10/11-H).

J) Dry stability study at 35, 40, 50 and 65°C.

Drying stability was determined at 35, 40, 50 and 65°C, respectively. As shown in Table. 11, structure 2 is stable at temperatures up to 40°C for at least 4 days.

Table 11. Dry Stability at 35, 40, 50 and 65°C

experiment number Input/Form T/°C 1 day 2 day 3 day Day 4 XRPD XRPD XRPD XRPD PS03027-15-H-35 PS03027-15-H/ Structure 2 35°C Structure 2 ON ON Structure 2 PS03027-21-H-40 PS03027-21-H/ Structure 2 40°С Structure 2 Structure 2 Structure 2 Structure 2 PS03027-15-H-50 PS03027-15-H/ 50°C Structure 2 Structure 2 ON Structure 1 PS03027-15-H-65 Structure 2 65°C Structure 2 Structure 1 ON ON

K) Study of the procedure for obtaining a suspension in order to convert the crystallinity with the worst structure back to structure 2.

Excessive dryness (KF=5.4%, theoretical 7.4%) resulted in less crystallinity for batch cGMP structure 2 (structure 218001) even when dried at 35-40°C for 10 hours. To convert a solid with poor crystalline structure 2 to the required structure 2, two resuspension experiments were performed. 2 g of structure 218001-STEP5.7 was resuspended in 8 vol. mixtures of ethanol / water / MtBE = 9 vol. / 1 vol. / 30 vol. at room temperature and 5°C, and it was found that it turned into structure 2 in just 30 minutes. The solid was isolated and dried for 14 hours, during which time the shape did not change. The loss based on the concentration of the mother liquor was approximately 5% (whereas the total loss was assumed to be 10%, including losses at the flask wall and during operation). One advantage is that this procedure converts all of the solid to structure 2, although about 10% of the product may be lost.

- 21 042744

L) General route from structure 1 to structure 2.

1) Load ethanol (1.75-1.79X; 2.25 vol) into R1 under N ₂ .

2) Load purified water (0.245-0.255X; 0.25 vol) into R1 under N2.

3) Stir R1 for 5-10 minutes to obtain a mixed solution of ethanol/water=9:1 (v/v).

4) Transfer the solution from R1 to the drum.

5) Load Structure 1 (1.0 eq., 1.0X) into R1.

6) Load the ethanol/water mixture from step 4 (1.61-1.63X; 2.0 vol) into R1 under nitrogen.

7) Adjust the temperature R1 to 47-53°C under N2.

8) Stir R1 at 47-53°C for 10-60 min under N2 atmosphere.

9) Filter the solution from R1 to R2. The R2 jacket is preheated to 50-55°C.

10) Load the ethanol/water mixture from step 4 (0.39-0.41X; 0.5 vol) into R1 under N2.

11) Stir R1 for 5-30 min under N2.

12) Filter the solution from R1 to R2.

13) Bring the temperature of R2 to 47-53°C and stir for 10-60 min under N2 atmosphere. Complete dissolution is confirmed by the project manager.

14) Bring the temperature of R2 to 32-37°C under N2.

15) Slowly add MtBE (0.36-0.38X; 0.5 vol.) at 32-37°C in R2 under nitrogen.

16) Load seed crystals of structure 2 (0.015-0.025X) in R2 at 32-37°C.

17) Stir R2 at 32-37°C for 2-3 hours under N2.

18) Bring the temperature of R2 to 3-8°C in 2-3 hours under N2 atmosphere.

19) Take a sample and filter, send the precipitate for XRPD analysis (according to PS0302715-H). Criteria: if the results are within specification, perform step 20; otherwise, consult with the project manager.

20) Load MtBE (5.1-5.3X; 7 vol.) in R2 for 8 hours at 3-8°C under N2.

21) Stir R2 at 3-8°C for 8-10 hours under N2.

22) Take a sample and filter, send the precipitate for XRPD analysis (according to PS0302715-H); send the filtrate for analysis of residual structure 2: residual H in the supernatant (<1.5 wt.%).

Criteria: if the results are within specification, perform step 25; otherwise, step 23 is performed.

23) Stir R2 at 3-8°C for 4-10 hours under N2.

24) Take a sample and filter, send the precipitate for XRPD analysis (according to PS0302715-H); send the filtrate for analysis of residual H: Residual H in the supernatant (<1.5 wt.%). Criteria: If results are within specification, step 25 is performed.

25) Filter the suspension at 3-8° C. under N2.

26) Load ethanol (0.355X), purified water (0.05X) and MtBE (1.11X) into a clean drum.

27) Stir the material from step 26 to obtain a clear mixture solution.

28) Transfer the solution from step 27 (1.5-1.6X) into R2 to wash with R2.

29) Cool R2 to 3-8°C.

30) Wash the wet cake with washing solution R2 at a temperature of 3-8°C.

31) Check the quality of the wet product:

XRPD (corresponding to PS03027-15-H); HPLC >98.0% area; H-His-OH <1.0% area; Every other single impurity <1.0% area; note each impurity content >0.05% of the area with the help of OVU.

Note: Step 32 is performed in parallel.

Criteria: if the results are within specification, perform step 33.

32) Check the residual structure 2 in the mother liquor. Note the residual amount of structure 2 (% report).

33) Dry at a temperature of 35-40°C under vacuum for 10-12 hours. It is recommended to put the water bath in the oven to avoid overdrying.

34) Check the residual solvent:

Ethanol (<5000 ppm); MtBE (<5000 ppm); Water content (report).

The dry product polymorph is checked: XRPD (corresponds to PS03027-15-H). Criteria: if the results meet the specification, perform step 37; otherwise, step 35 is performed.

35) Dry at a temperature of 35-40°C under vacuum for 5-8 hours. It is recommended to put the water bath in the oven to avoid overdrying.

36) Check the residual solvent:

Ethanol (<5000 ppm); MtBE (<5000 ppm); Water content (report). The dry product polymorph is checked: XRPD (corresponds to PS03027-15-H). Criteria: If the results are within specification, perform step 37.

- 22 042744

37) Load the dry product into drums and sift.

38) Take samples for release analysis.

M) Full description.

Purity by HPLC, spectra ¹ H-NMR, TGA, DSC and XRPD are shown in table. 12.

Table 12. Characteristics of structure 2

ID connections Structure Characterization data Ή-NMR HPLC purity TGA XRPD DSC Structure 2 0, Corresponds 99.9% Input 170 Output 172 Structure 2 Two peaks 104.29°C 172.17°C

Example 3. Development of a method for crystallization of CHP hydrate.

This study was focused on developing the crystallization of CHP hydrate (structure 2). After the initial characterization of the CGP hydrate series, solubility studies, small-scale crystallization evaluation, measurement of the metastable zone width, and large-scale crystallization were carried out. The goal of the work program was to establish crystallization conditions that would allow the successful production of structure 2 CHP hydrate and that could be efficiently scaled up for production.

Solubility tests carried out on structure 1 CHP showed high solubility in all solvent mixtures containing water, with the exception of ethanol:water:MtBE mixtures with the highest percentage of MtBE. Particularly high solubility was observed for water (>200 mg/mL) and acetone:water mixtures (>200 in water:acetone [90:10]). The material was found to be poorly soluble in acetone, acetonitrile and THF (<10 mg/mL).

The two-point solubility indicates that of the two solvent systems evaluated, ethanol:water:MtBE gave somewhat better yields, but there was a risk of formation of structure 1. Acetone:water provided only structure 2, but at the expense of yield.

In one embodiment, the present invention provides the following process for producing CHP hydrate.

Approximately 20 g of Structure 1 CHP is added to a jacketed reactor preheated to 50°C.

Add 50 ml of a solvent mixture of ethanol:water (90:10 vol.%) to the vessel to achieve an initial concentration of 400 mg/ml.

Mix at 300 rpm using an overhead stirrer.

When complete dissolution is achieved, add 25 ml of MtBE to the vessel (drop by drop).

Upon completion of the addition of MtBE, 2 wt.% (400 mg) of the seed of the crushed crystal structure of 2 CHP are added to the reaction mixture.

Continue stirring at 50°C for 20 minutes.

Add another 5 ml of MtBE to the vessel (drop by drop).

70 ml of MtBE are added at a rate of 20 ml/h.

At the end of the addition, cool from 30 to 29° C. at a rate of 0.1° C./min.

Continue stirring at 29°C for 8 hours.

Cool from 29 to 5°C at a rate of 0.25°C/min.

190 ml of MtBE are added at a rate of 20 ml/h.

Continue stirring at 5°C for 12 hours.

The solid is isolated by filtration and the filter cake is washed with 40 ml of ethanol:water:MtBE (9 vol:1 vol:30 vol).

Dry the solid under vacuum at 40°C for 1 hour.

A lab-scale agitation performance was evaluated and then a plant-scale geometry was modeled to evaluate the agitation parameters that would allow a successful transfer of the process. Transferring this procedure to the 10 kg scale can be done using the following procedure.

Approximately 10 kg of Structure 1 CHP is added to a jacketed reactor preheated to 50°C.

Add 25 L of a solvent mixture of ethanol:water (90:10 vol.%) to the vessel to achieve an initial concentration of 400 mg/ml.

Stirred at 48 rpm.

When complete dissolution is achieved, add 12.5 L of MtBE to the vessel.

After the addition of MtBE was added to the experiment 2 wt.% (200 g) the seed of the crushed crystal structure 2 CHP.

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Continue stirring at 50°C for 20 minutes.

Add another 2.5 L of MtBE (drop by drop) into the container.

Add 35 l of MtBE at the rate of 10 l/h.

At the end of the addition, cool from 30 to 29° C. at a rate of 0.1° C./min.

Continue stirring at 29°C for 8 hours.

Cool from 29 to 5°C at a rate of 0.25°C/min.

Add 95 l of MtBE at the rate of 10 l/h.

Continue stirring at 5°C for 12 hours.

The solid is isolated by filtration and the filter cake is washed with 20 L of ethanol: water: MtBE (9 volumes: 1 volume: 30 volumes).

Dry the solid under vacuum at 40°C.

The use of 48 rpm is recommended as this allows power dissipation in a factory scale tank, which is more comparable to the power dissipation in a lab scale tank. The power dissipation in the factory is estimated at 0.067 W/kg, while in the laboratory it is estimated at 0.064 W/kg. The model used to estimate these parameters also suggests that the particles are likely to be in suspension.

As an alternative, the agitator can be operated at 55 rpm at the factory. This was estimated by the mixing model as NJS, the mixing rate barely corresponding to the suspension at which the particles would be suspended in the vessel. This adds to the confidence that the mixing will be efficient, but there is a risk that the particles will receive a much higher power dissipation of 0.101 W/kg.

However, it should be understood that one or more of the steps described above for producing CHP hydrate may be omitted, or the order of the steps may be changed.

C) Methods of analysis.

1) X-ray powder diffraction (XRPD).

XRPD analysis was performed on a PANalytical X'pert pro instrument, scanning samples in the range from 3 to 35° 2θ. The material was gently crushed to release the agglomerates and loaded onto a multiwell plate with mylar film to support the sample. Then, the multiwell plate was placed in a diffractometer and analyzed using Cu K radiation (α1 λ=1.54060 A; α2=1.54443 A; β=1.39225 A; ratio α1: α2=0.5) in transmission mode (size step 0.0130° 2θ) at 40 kV/40 mA generator settings.

2) Polarized light microscopy (PLM).

The presence of birefringence and particle size and morphology were assessed using an Olympus BX50 polarizing microscope equipped with a Motic camera and image capture software (Motic Images Plus 2.0). All images were recorded using a 20x lens unless otherwise noted.

3) Thermogravimetric analysis (TGA).

About 5 mg of material was prepared in an open aluminum cuvette and loaded into a synchronous thermogravimetric/differential thermal analyzer (TG/DTA) and kept at room temperature. The sample was then heated at a rate of 10° C./min from 20 to 350° C., during which time the sample mass change was recorded along with any differential thermal events (DTA). Nitrogen was used as purge gas at a flow rate of ³⁰⁰ cc/min.

4) Nuclear magnetic resonance (NMR).

NMR experiments were performed on a Bruker AVIIIHD spectrometer equipped with a DCH cryoprobe operating at 500.12 MHz for protons. The experiments were carried out in deuterated dimethyl sulfoxide and each sample was prepared at an approximate concentration of 10 mM.

5) Focused Beam Reflectance Measurement (FBRM) Focused beam reflectance measurements were performed using a Mettler Toledo D600 probe. For each crystallization, the probe was placed in the reaction vessel at the beginning of the crystallization and the nucleation and growth of the crystals was monitored. Chord length distribution and various statistics were monitored throughout using the following settings.

Electron recognition range: thin.

Scanning speed: 2 m/s.

Sample time: 10 s.

6) Crystal 16.

The Crystal 16 uses the percentage of light transmitted through a solution (or suspension) in a clear vial to create a profile of the solution (or suspension) turbidity versus time and temperature. The resulting profile was used to determine cloud points (nucleation temperature) and transparency (dissolution temperature).

7) High performance liquid chromatography with ultraviolet detection (HPLC-UV).

HPLC analysis was performed with the following equipment settings.

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Device: Dionex Ultimate 3000.

Column: LC/ 68 X-Bridge Phenyl Column (150 mm x 4.6 mm x 3.5 µm).

Column temperature: 30°C.

Autosampler temperature: 5°C.

UV wavelength: 220 nm.

Injection volume: 5 µl.

Flow rate: 1 ml/min.

Mobile phase A: 10 mM ammonium acetate

Mobile phase B: acetonitrile.

Thinner: 0.1% TFA in water.

______ Gradient program _______

Time (minutes) Solvent B (%) 0.01 0 10.3 thirty 15 95 15.1 0 25 0

C) experiment.

1) The initial characteristics of the structure of 1 CGP.

The structure of 1CHP was characterized by XRPD, ^1H- , HSCQ- and ^13C -NMR and HPLC according to the procedures detailed in section B.

2) Approximate solubility of CHP structure 1 (anhydrous).

The approximate solubility of CHP in 37 selected solvent systems (Table 13) was evaluated by the solvent addition method. The following procedure was used:

Added a sample of approximately 20 mg of structure 1 CHP in each of the 37 vials.

Each solvent/solvent mixture was added to the appropriate vial in 5 volume (100 μl) aliquots until dissolution was observed or a maximum of 2 ml of solvent was added.

Between additions, the sample was heated to 40° C. to test dissolution at elevated temperature.

If 2 ml of solvent was added without dissolving the material, the calculated solubility was below 10 mg/ml.

After adding the solvent, all 37 vials were placed in a refrigerator for rapid cooling to 2-8°C for about 18 hours.

Any solids present were isolated by centrifugation with filtration and analyzed by XRPD (if the amount of material allowed).

If no solids formed, the vials were opened and allowed to evaporate at ambient temperature.

Any particulate matter present was analyzed by XRPD (if the amount of material allowed).

Table 13. List of approximate solubility in solvents

Number Solvent Class ICH Number Solvent Class ICH 1 Water n/a 20 Water: acetonitrile (70:30) 2 2 Acetone 3 21 Water: acetonitrile (80:20) 2 3 Acetonitrile 2 22 Water: acetonitrile (90:10) 2 4 THF 2 23 Water: THF (10:90) 2 5 Water: acetone (10:90) 3 24 Water: THF (20:80) 2 6 Water: acetone (20:80) 3 25 Water: THF (30:70) 2 7 Water: acetone (30:70) 3 26 Water: THF (40:60) 2 8 Water: acetone (40:60) 3 27 Water: THF (50:50) 2 9 Water: acetone (50:50) 3 28 Water: THF (60:40) 2 10 Water: acetone (60:40) 3 29 Water: THF (70:30) 2 eleven Water: acetone (70:30) 3 thirty Water: THF (80:20) 2 12 Water: acetone (80:20) 3 31 Water: THF (90:10) 2 13 Water: acetone (90:10) 3 32 Ethanol: water (90:10): TBME [10:90] 3 14 Water: acetonitrile (10:90) 2 33 Ethanol: water (90:10): TBME [30:70] 3 15 Water: acetonitrile (20:80) 2 34 Ethanol: water (90:10): TBME [50:50] 3 16 Water: acetonitrile (30:70) 2 35 Ethanol: water (90:10): TBME [70:30] 3 17 Water: acetonitrile (40:60) 2 36 Ethanol: water (90:10): TBME [90:10] 3 18 Water: acetonitrile (50:50) 2 37 Ethanol: water (90:10) 3 19 Water: acetonitrile (60:40) 2

3) Two-point solubility.

Two-point dissolution tests were carried out at 5° C. and 50° C. in 11 solvent mixtures using the following procedure.

- 25,042,744 ml of the selected solvent mixture was added to a 1.5 ml screw cap glass vial containing a pre-weighed mass of Structure 1 CHP to obtain a suspension.

The initial weight was chosen for each solvent system based on the approximate solubility results, as detailed in Section 2. The solvent mixtures are shown in Table 1. 14.

The vials were kept in a thermostatically controlled reaction block set at 5 or 50° C. and stirring was performed with a magnetic stirrer.

When complete dissolution was observed after 1 h, more solid structure 1 CHP was added to the experiments to form suspensions.

When mobile suspensions were formed in all vials, the reaction mixtures continued to stir at the required temperature for about 18 hours. 14.

After about 18 hours, stirring was stopped and the supernatant was filtered using 0.45 µm PVDF filter cartridges and syringes.

The supernatant concentration was analyzed by HPLC.

Each of the remaining suspensions in the vials was transferred to centrifuge tubes with a nylon filter with a pore size of 0.22 μm, and the solid particles were separated by centrifugation.

The recovered solid (where quantity allowed) was analyzed by XRPD and HPLC for purity.

Table 14. Final masses of CHP in a two-point dissolution test

Solvent Added weight (mg) 5°С 50°C Water: acetone (40:60) 211 655 Water: acetone (80:20) 383 725 Water: acetone (90:10) 365 681 Water: acetone (20:80) 100 340 Water: acetone (10:90) 100 210 Water: acetone (5:95) 250 250 Ethanol: water (90:10): TBME [20:80] 60 60 Ethanol: water (90:10): TBME [25:75] 60 60 Ethanol: water (90:10): TBME [30:70] 60 60 Ethanol: water (90:10): TBME [40:60] 100 126 Ethanol: water (90:10) 250 475

4) Small-scale crystallization.

Small-scale crystallization tests were carried out on the resulting CHP (structure 1). The purpose of these tests was to study the most suitable solvent systems, temperatures and concentrations for further crystallization studies. This was done using two different solvent systems: acetone: water and ethanol water MtBE.

Crystallization kit A: acetone:water.

Approximately 500 mg of the resulting CHP (structure 1) were weighed into 4 x 20 ml scintillation vials.

An appropriate volume of acetone:water (80:20 vol%) was added to each vial to dissolve the sample. See table. 15 for the volumes of solvent used.

Magnetic stirring elements were added to each vial and the reaction mixture was stirred at 50°C.

After 1 hour at 50° C., acetone was added dropwise to 2 of the four vials (vials 1 and 3).

The reaction mixture was allowed to stir at 50°C for about 1 hour.

After 1 hour, the vials were cooled to 5°C at a rate of 0.25°C/min.

After reaching 5° C., acetone was added dropwise to the remaining 2 vials (vials 2 and 4).

The reaction mixture was allowed to stir at 5°C for about 18 hours.

After 18 hours at 5° C., the resulting suspensions were filtered using a 47 mm Buchner funnel and Whatman Grade 1 filter paper (diameter=42.5 mm; pore size=11 μm).

Solids were transferred to pre-weighed vials and analyzed by XRPD, PLM and HPLC for chemical purity.

The filtered mother liquors were sent for concentration analysis by HPLC.

The experiments are described in Table. 15.

Crystallization kit B: ethanol: water: Mtbe.

Approximately 500 mg of the obtained CHP (structure 1) were added in 5 x 20 ml scintillation vials.

2 ml of ethanol:water (90:10 v/v%) was added to each vial to dissolve the sample.

Magnetic stirring elements were added to each vial and the reaction mixture was stirred at 50°C.

After 1 hour at 50°C, MtBE was added dropwise to 3 of the five vials (vials 1,3 and 5).

The reaction mixture was allowed to stir at 50°C for about 1 hour.

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After 1 hour at 50° C., the suspension present in vial 1 was filtered using a 47 mm Buechner funnel and Whatman Grade 1 filter paper (diameter=42.5 mm; pore size=11 µm).

The reaction mixture was filtered at 50° C. as previous results showed a potential risk of structure 1 formation after cooling at this percentage of MtBE (80%).

The remaining vials were cooled to 5°C at a rate of 0.25°C/min.

After reaching 5°C, MtBE was added dropwise to the remaining 2 vials (vials 2 and 4).

The reaction mixture was allowed to stir at 5°C for about 18 hours.

After 18 hours at 5° C., the resulting suspensions were then filtered using a 47 mm Buchner funnel and Whatman Grade 1 filter paper (diameter=42.5 mm; pore size=11 μm).

All solids were transferred to pre-weighed vials and analyzed by XRPD, PLM and HPLC for chemical purity.

The filtered mother liquors were sent for concentration analysis by HPLC.

The experiments are described in Table. 15.

Table 15. Small Scale Crystallization - Solvent Summary

Solvent system Temperature for adding anti-solvents Initial volume (acetone: water 80/20) Added acetone 1 - acetone: water (95:5) 50°С 2 ml 6 ml 2 - acetone: water (95:5) 5°C 2 ml 6 ml 3 - acetone: water (95:5) 50°C 1.65 ml 4.95 ml 4 - acetone: water (95:5) 5°C 1.65 ml 4.95 ml Solvent system Temperature for adding anti-solvents Initial volume (ethanol: water 90/10) Added TBME 1 - ethanol: water (90:10) TBME (20:80) 50°C 2 ml 8 ml 2 - ethanol: water (90:10) TBME (30:70) 5°C 2 ml 4.7 ml 3 - ethanol: water (90:10) TBME (30:70) 50°C 2 ml 4.7 ml 4 - ethanol: water (90:10) TBME (40:60) 5°C 2 ml 3 ml 5 - ethanol: water (90:10) TBME (40:60) 50°С 2 ml 3 ml

5) Measuring the width of the metastable zone (MSZW).

MSZW measurements were performed on the obtained CHP (structure 1) by examining two parameters - cooling and adding an anti-solvent.

cooling method.

The determination of MSZW by cooling was completed using Crystal 16. The procedure used is described below.

Added weighed with a known amount of CHP (structure 1) in vials with screw caps.

1 ml of the appropriate solvent system was added to each vial to form suspensions. Magnetic stirring elements were placed in each vial.

The solvent systems and masses of CHP used in each experiment are summarized in Table 1. 16. The vials were placed in the sample holder of the Crystal 16 and the desired temperature program was started. The temperature profile can be seen in Table. 17A.

The turbidity of the system was controlled everywhere, which made it possible to determine the points of transparency and turbidity.

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Table 16. Details of the experiment to determine the width of the metastable zone during cooling

Solvent Mass of CHP (mg) introductory material ethanol water (90:10) 2 Structure 2 ethanol water (90:10) TBME (20:80) Structure 2 ethanol water (90:10) TBME (40:60) Structure 2 ethanol water (90:10) TBME (80:20) Structure 2 ethanol water (90:10) 25 Structure 2 ethanol water (90:10) TBME (20:80) Structure 2 ethanol water (90:10) TBME (40:60) Structure 2 ethanol water (90:10) TBME (80:20) Structure 2 ethanol water (90:10) 100 Structure 2 ethanol water (90:10) TBME (20:80) Structure 2 ethanol water (90:10) TBME (40:60) Structure 2 ethanol water (90:10) TBME (80:20) Structure 2 ethanol water (90:10) 325 Structure 1 ethanol water (90:10) TBME (90:10) Structure 1 ethanol water (90:10) TBME (80:20) Structure 1 ethanol water (90:10) TBME (70:30) Structure 1 ethanol water (90:10) TBME (40:60) Structure 1 ethanol water (90:10) 425 Structure 2 ethanol water (90:10) TBME (20:80) Structure 2 ethanol water (90:10) TBME (40:60) Structure 2 ethanol water (90:10) TBME (80:20) Structure 2

Table 17A. Temperature profile for determining the width of the metastable zone during cooling

1. Stirring at: 700 rpm 2. Holding at: 25°C for 10 minutes 3. Heating up to: 70°C at a rate of 0.5°C/min 4. Holding at: 70°C for 1 hour 5. Cooling up to: 5°C at a rate of 0.25°C/min 6. Holding at: 5°C for 1 hour 7. Heating up to: 70°C at a rate of 0.25°C/min 8. Holding at: 70°C for 1 hour 9. Cooling up to: 5°C at a rate of 0.25°C/min 10. Holding at: 5°C for 1 hour I. Heating to: 70°C at a rate of 0.25°C/min 12. Holding at: 70°C for 1 hour 13. Stirring at: 700 rpm 14. Holding at: 20°C

a) Anti-solvent addition method.

The determination of MSZW with respect to the addition of an anti-solvent was completed using ethanol: water: MtBE. Used the following procedure.

Approximately 20 g of the resulting structure 1 CHP was transferred into a 250 ml jacketed vessel, which was preheated to 50°C.

50 ml of a solvent mixture of ethanol: water (90:10 v/v%) was added to the vessel until an initial concentration of 400 mg/ml was reached.

The reaction mixture was stirred at 50° C., about 300 rpm using an overhead stirrer.

An FBRM probe was inserted into the vessel to monitor and record nucleation and particle number.

When complete dissolution was achieved (approximately 40 min), 75 ml of MtBE was added via a peristaltic pump at a rate of 10 ml/h.

Upon completion of the addition of MtBE, the reaction mixture was cooled to 5°C at a rate of 0.25°C/min and kept at 5°C overnight.

After 9 hours at 5° C., the solid was isolated by vacuum filtration using an 83 mm Buchner funnel and Whatman Grade 1 filter paper (diameter=70 mm; pore size=11 μm).

A sample of the filter cake was taken and analyzed by XRPD.

The remaining precipitate was dried under vacuum at 35°C for 1 h.

The concentration of the filtered mother liquor was determined by HPLC.

6) Development of crystallization.

Crystallization scaling experiments were carried out in an ethanol:water:MtBE mixture. Various experimental conditions were evaluated, such as cooling rate, antisolvent addition rate and temperature, seed loading and temperature, and antisolvent ratio. The following protocols were considered.

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a) Crystallization 1.

For crystallization of 1, 2 wt.% seed crystals were added to the solution. Adding seed and

MtBE was carried out at 35°C. The suspension was cooled to 5°C at a rate of 0.25°C/min. Used the following procedure.

Approximately 20 g of the resulting structure 1 CHP was transferred into a jacketed vessel, which was preheated to 50°C.

ml of solvent mixture ethanol:water (90:10 v/v%) was added to the vial to achieve an initial concentration of 400 mg/ml.

The reaction mixture was stirred at 50° C., about 300 rpm using an overhead stirrer.

An FBRM probe was inserted into the vessel to track and record nucleation and particle number.

When complete dissolution was achieved (approximately 30 min), the reaction mixture was cooled to 35°C at a rate of 0.25°C/min and 10 ml of MtBE was added via a peristaltic pump at a rate of 10 ml/h.

Upon completion of the addition of MtBE, the reaction mixture was seeded with 400 mg of structure 2 CHP.

The system was monitored for about 2 hours at 35°C.

The reaction mixture was then cooled to 5° C. at a rate of 0.25° C./min and 140 ml of MtBE was added via a peristaltic pump at a rate of 10 ml/h (14 h) to give a final solvent ratio of 25:75 v/v% .

The solid was isolated by vacuum filtration using an 83 mm Buchner funnel and Whatman Grade 1 filter paper (diameter=70 mm; pore size=11 μm).

The filter cake was washed with 40 ml of ethanol:water:MtBE (9 vol:1 vol:30 vol), after which it was dried in vacuum at 40°C for 1 h.

The dried solid was analyzed by XRPD, TG/DTA, PLM and HPLC;

The concentration of the filtered mother liquor was determined by HPLC.

b) Crystallization 2.

For crystallization of 2, 2 wt.% seed crystals were added to the solution. The seeding and MtBE were added at 50°C. The suspension was cooled to 5°C at a rate of 0.25°C/min. Used the following procedure/

We prepared a sample of approximately 20 g of the resulting structure 1 CHP, and transferred to a vessel with a shirt, which was preheated to 50°C.

50 ml of a solvent mixture of ethanol:water (90:10 v/v%) was added to the vessel until an initial concentration of 400 mg/ml was reached.

The reaction mixture was stirred at 50° C., about 300 rpm using an overhead stirrer.

An FBRM probe was inserted into the vessel to track and record nucleation and particle number.

When complete dissolution was achieved (approximately 20 min), the reaction mixture was kept at 50°C and 10 ml of MtBE was added using a syringe pump at a rate of 10 ml/h.

Upon completion of the addition of MtBE, 400 mg of structure 2 CHP was added to the experiment.

The system was observed for about 2 hours at 50°C.

140 ml of MtBE was added via a peristaltic pump at a rate of 20 ml/h to give a final solvent ratio of 25:75 v/v%.

The reaction mixture was then cooled to 5°C at a rate of 0.25°C/min and kept at 5°C for about 9 hours.

After 9 hours at 5° C., the solid was isolated by vacuum filtration using an 88 mm Buchner funnel and Whatman Grade 1 filter paper (diameter=70 mm; pore size=11 μm).

The filter cake was washed with 40 ml of ethanol:water:MtBE (9 vol:1 vol:30 vol), after which it was dried in vacuum at 40°C for 1 h.

The dried solid was analyzed by XRPD, TG/DTA, PLM and HPLC for purity/

The concentration of the filtered mother liquor was determined by HPLC.

c) Crystallization 3.

For crystallization of 3, 2 wt.% seed crystals were added to the solution.

The seeding and MtBE were added at 35°C. The suspension was cooled to 5°C at a rate of 0.25°C/min. Used the following procedure.

We prepared a sample of approximately 20 g of the obtained structure 1 CHP and transferred into a vessel with a shirt, which was preheated to 50°C.

50 ml of a solvent mixture of ethanol:water (90:10 v/v%) was added to the vessel until an initial concentration of 400 mg/ml was reached.

The reaction mixture was stirred at 50° C., about 300 rpm using an overhead stirrer.

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An FBRM probe was inserted into the vessel to track and record nucleation and particle number.

When complete dissolution was achieved (approximately 20 minutes), the reaction mixture was cooled to 5°C at a rate of 0.25°C/min.

Nucleation was observed when the system reached 23°C.

The reaction mixture was heated to 50° C. to redissolve the material. Once dissolution was observed, the system was cooled to 25°C at a rate of 0.25°C/min.

Nucleation was observed when the system reached 25°C.

The reaction mixture was heated to 50° C. to redissolve the material. Once dissolution was observed, the system was cooled to 35° C. and 400 mg of structure 2 CHP was added to the experiment.

140 ml of MtBE was added via a peristaltic pump at a rate of 20 ml/h to give a final solvent ratio of 26:74 v/v%.

The reaction mixture was then cooled to 5°C at a rate of 0.25°C/min and kept at 5°C for about 9 hours.

The solid was isolated by vacuum filtration using an 88 mm Buchner funnel and Whatman Grade 1 filter paper (diameter=70 mm; pore size=11 μm).

The filter cake was washed with 40 ml of ethanol:water:MtBE (9 vol:1 vol:30 vol), after which it was dried in vacuum at 40°C for 1 h.

The dried solid was analyzed by XRPD, TG/DTA, PLM and HPLC for purity.

The concentration of the filtered mother liquor was determined by HPLC.

d) Crystallization 4.

For crystallization of 4, 2 wt.% seed crystals were added to the solution.

The seeding and MtBE were added at 50°C. The suspension was cooled to 5°C at a rate of 0.1°C/min. Used the following procedure.

Approximately 20 g of structure 2 CHP (material obtained from crystallization 1 and 3) was weighed and transferred into a jacketed vessel, which was preheated to 50°C.

50 ml of a solvent mixture of ethanol: water (90:10 v/v%) was added to the vessel until an initial concentration of 400 mg/ml was reached.

The reaction mixture was stirred at 50° C., about 300 rpm using an overhead stirrer.

An FBRM probe was inserted into the vessel to track and record nucleation and particle number.

When complete dissolution was achieved (approximately 20 min), the experiment was carried out at 50°C, and 20 ml of MtBE was added using a syringe pump at a rate of 20 ml/hour.

Upon completion of the addition of MtBE, 400 mg of structure 2 CHP was added to the reaction mixture.

The system was observed for approximately 2 hours at 50°C.

140 ml of MtBE was added via a peristaltic pump at a rate of 20 ml/h to give a final solvent ratio of 24:76 v/v%.

Then the reaction mixture was cooled to 5°C at a rate of 0.1°C/min.

The reaction mixture was kept at 5°C overnight.

After 8 hours at 5° C., the solid was isolated by vacuum filtration using an 88 mm Buchner funnel and Whatman Grade 1 filter paper (diameter=70 mm; pore size=11 μm).

The filter cake was washed with 40 ml of ethanol:water:MtBE (9 vol:1 vol:30 vol), after which it was dried in vacuum at 40°C for 1 h.

The dried solid was analyzed by XRPD, TG/DTA, PLM and HPLC for purity.

The concentration of the filtered mother liquor was determined by HPLC.

e) Crystallization 5.

For crystallization of 5, 2 wt.% crushed seed crystals were added to the solution. The seeding and MtBE were added at 50°C. The suspension was cooled to 5°C at a rate of 0.1°C/min. Used the following procedure.

About 20 g of a mixture of CHP structures 1 and 2 (structures 1 from CS/352/18 obtained and structure 2 from crystallization 4) was prepared and transferred to a jacketed vessel that had been preheated to 50°C.

50 ml of a solvent mixture of ethanol:water (90:10 v/v%) was added to the vessel until an initial concentration of 400 mg/ml was reached.

The reaction mixture was stirred at 50° C. at about 300 rpm using an overhead stirrer.

An FBRM probe was inserted into the vessel to track and record nucleation and particle number.

When complete dissolution was achieved (about 20 min), the experiment was carried out at 50°C, and 25 ml of MtBE was added dropwise using a syringe.

Upon completion of the addition of MtBE, 400 mg of structure 2 CHP, crushed with a pestle and mortar, was added to the reaction mixture.

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The system was observed for approximately 75 minutes at 50°C.

140 ml of MtBE was added via a peristaltic pump at a rate of 20 ml/h to give a final solvent ratio of 23:77 v/v%.

Then the reaction mixture was cooled to 5°C at a rate of 0.1°C/min.

The system was kept at 5°C overnight.

After 9 hours at 5° C., the solid was isolated by vacuum filtration using an 88 mm Buchner funnel and Whatman Grade 1 filter paper (diameter=70 mm; pore size=11 μm).

The filter cake was washed with 40 ml of ethanol:water:MtBE (9 vol:1 vol:30 vol), after which it was dried in vacuum at 40°C for 1 h.

The dried solid was analyzed by XRPD, TG/DTA, PLM and HPLC for purity.

The concentration of the filtered mother liquor was determined by HPLC.

f) Crystallization 6.

For crystallization of 6, 2 wt.% crushed seed crystals were added to the solution. The addition was carried out at 50° C. and a stepwise addition of MtBE was used. The suspension was cooled to 29°C at a rate of 0.1°C/min and kept at this temperature for 8 hours. Then the suspension was cooled to 5°C at a rate of 0.25°C/min. Used the following procedure.

About 20 g of a mixture of CHP structures 1 and 2 (structure 1 from CS/352/18 obtained and structure 2 from crystallization 5) was prepared and transferred to a jacketed vessel that had been preheated to 50°C.

50 ml of a solvent mixture of ethanol: water (90:10 v/v%) was added to the vessel until an initial concentration of 400 mg/ml was reached.

The reaction mixture was stirred at 50° C., about 300 rpm using an overhead stirrer.

An FBRM probe was inserted into the vessel to track and record nucleation and particle number.

When complete dissolution was achieved (about 20 min), the experiment was carried out at 50°C, and 25 ml of MtBE was added dropwise using a syringe.

Upon completion of the addition of MtBE, 400 mg of structure 2 CHP, crushed with a pestle and mortar, was added to the experiment.

The system was observed for approximately 10 minutes at 50°C.

The seed crystals were not preserved. An additional 5 ml of MtBE was added dropwise to the vial using a syringe.

ml MtBE was added via a peristaltic pump at a rate of 20 ml/h.

The reaction mixture was then cooled to 29° C. at a rate of 0.1° C./min.

The reaction mixture was kept at 29°С for 8 h.

After 8 hours at 29°C, the system was cooled to 5°C at a rate of 0.25°C/min.

After the temperature in the system reached 5° C., the second step of adding MtBE was started. 190 ml were added at a rate of 20 ml/h. The final ratio of solvents 15:85 vol.%.

The system was kept at 5°C for about 12 hours.

The solid was then isolated by vacuum filtration using an 88 mm Buchner funnel and Whatman Grade 1 filter paper (diameter=70 mm; pore size=11 μm).

The filter cake was washed with 40 ml of ethanol:water:MtBE (9 vol:1 vol:30 vol), after which it was dried in vacuum at 40°C for 1 h.

The dried solid was analyzed by XRPD, TG/DTA, PLM, and HPLC for purity, and by chiral HPLC.

The concentration of the filtered mother liquor was determined by HPLC.

g) Crystallization 7.

For crystallization of 7, 2 wt.% crushed seed crystals were added to the solution. The addition was carried out at 50° C. and a stepwise addition of MtBE was used. The suspension was cooled to 5°C at a rate of 0.1°C/min. Used the following procedure.

About 20 g of a mixture of CHP structures 1 and 2 (structure 1 from the obtained CS/352/18 and structure 2 from crystallization 6) was prepared and transferred to a jacketed vessel that had been preheated to 50°C.

50 ml of a solvent mixture of ethanol: water (90:10 v/v%) was added to the vessel until an initial concentration of 400 mg/ml was reached.

The reaction mixture was stirred at 50° C. at about 300 rpm using an overhead stirrer.

An FBRM probe was inserted into the vessel to track and record nucleation and particle number.

When complete dissolution was achieved (about 20 minutes), the experiment was carried out at 50° C. and 27.5 ml of MtBE was added manually (dropwise) using a syringe.

Upon completion of the addition of MtBE, 400 mg of structure 2 CHP, crushed with a pestle and mortar, was added to the reaction mixture.

- 31 042744

The system was observed for approximately 10 minutes at 50°C.

ml MtBE was added via a peristaltic pump at a rate of 20 ml/h.

Then the reaction mixture was cooled to 5°C at a rate of 0.1°C/min.

The system was kept at 5°C overnight.

After about 9 hours at 5° C., the second step of adding MtBE was started. 190 ml were added at a rate of 20 ml/h. The final ratio of solvents 15:85 vol.%.

The solid was isolated by vacuum filtration using an 88 mm Buchner funnel and Whatman Grade 1 filter paper (diameter=70 mm; pore size=11 μm).

The filter cake was washed with 40 ml of ethanol:water:MtBE (9 vol:1 vol:30 vol), after which it was dried in vacuum at 40°C for 1 h.

The dried solid was analyzed by XRPD, TG/DTA, PLM and HPLC for purity.

The concentration of the filtered mother liquor was determined by HPLC.

h) Crystallization 8.

For crystallization of 8, 5 wt.% crushed seed crystals were added to the solution. The addition was carried out at 50° C. and a stepwise addition of MtBE was used. The suspension was cooled to 5°C at a rate of 0.1°C/min. Used the following procedure.

About 20 g of a mixture of CHP structures 1 and 2 (structure 1 from obtained CS/352/18 and structure 2 from crystallization 7) was prepared and transferred to a jacketed vessel that had been preheated to 50°C.

50 ml of a solvent mixture of ethanol: water (90:10 v/v%) was added to the vessel until an initial concentration of 400 mg/ml was reached.

The reaction mixture was stirred at 50° C. at about 300 rpm using an overhead stirrer.

An FBRM probe was inserted into the vessel to track and record nucleation and particle number.

When complete dissolution was achieved (approximately 20 min), the experiment was carried out at 50° C. and 27.5 ml of MtBE was added manually (dropwise) using a syringe.

Upon completion of the addition of MtBE, 1 g of structure 2 CHP, ground with a pestle and mortar, was added to the experiment.

The system was observed for approximately 10 minutes at 50°C.

ml MtBE was added via a peristaltic pump at a rate of 20 ml/h.

Then the reaction mixture was cooled to 5°C at a rate of 0.1°C/min.

The system was kept at 5°C overnight.

After about 9 hours at 5° C., the second step of adding MtBE was started. 190 ml were added at a rate of 20 ml/h. The final ratio of solvents 15:85 vol.%.

The solid was isolated by vacuum filtration using an 88 mm Buchner funnel and Whatman Grade 1 filter paper (diameter=70 mm; pore size=11 μm).

The filter cake was washed with 40 ml of ethanol:water:MtBE (9 vol:1 vol:30 vol), after which it was dried in vacuum at 40°C for 1 h.

The dried solid was analyzed by XRPD, TG/DTA, PLM and HPLC for purity.

The concentration of the filtered mother liquor was determined by HPLC.

i) Crystallization 9.

For crystallization of 9, 5 wt.% crushed seed crystals were added to the solution. The addition was carried out at 50° C. and a stepwise addition of MtBE was used. The suspension was cooled to 30°C at a rate of 0.1°C/min and kept at this temperature for 8 hours. Then the suspension was cooled to 5°C at a rate of 0.1°C/min. Used the following procedure.

About 20 g of a mixture of CHP patterns 1 and 2 (pattern 1 from CS/352/18 obtained and pattern 2 from crystallization 8) was prepared and transferred to a jacketed vessel that had been preheated to 50°C.

50 ml of a solvent mixture of ethanol:water (90:10 v/v%) was added to the vessel until an initial concentration of 400 mg/ml was reached.

The reaction mixture was stirred at 50° C. at about 300 rpm using an overhead stirrer.

An FBRM probe was inserted into the vessel to track and record nucleation and particle number.

When complete dissolution was achieved (about 20 min), the experiment was carried out at 50° C. and 27.5 ml of MtBE was added manually (dropwise) using a syringe.

Upon completion of the addition of MtBE, 1 g of structure 2 CHP, crushed with a pestle and mortar, was added to the reaction mixture.

The system was observed for approximately 10 minutes at 50°C.

ml MtBE was added via a peristaltic pump at a rate of 15 ml/h.

Then the reaction mixture was cooled to 30°C at a rate of 0.1°C/min.

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The system was kept at 30°С for 8 h.

After 8 hours at 30° C., cooling to 5° C. was resumed in the system at a rate of 0.1° C./min.

Started the second stage of adding MtBE. 190 ml were added at a rate of 20 ml/h. The final ratio of solvents 15:85 vol.%.

The system was kept at 5°C overnight.

After about 10 hours at 5° C., the solid was isolated by vacuum filtration using a Buchner funnel and Whatman Grade 1 filter paper (diameter=70 mm; pore size=11 μm).

The filter cake was washed with 40 ml of ethanol:water:MtBE (9 vol:1 vol:30 vol), after which it was dried in vacuum at 40°C for 1 h.

The dried solid was analyzed by XRPD, TG/DTA, PLM and HPLC for purity.

The concentration of the filtered mother liquor was determined by HPLC.

j) Crystallization 10.

For crystallization 10, the same procedure was used as for crystallization 6. Due to the size of the container, the parameters were reduced to 70% of the original experiment. A new batch of prepared CHP (structure 1) was also used in this experiment (CS/802/18).

2 wt.% ground seed crystals were added to the solution. The addition was carried out at 50° C. and a stepwise addition of MtBE was used. The suspension was cooled to 29°C at a rate of 0.1°C/min and kept at this temperature for 8 hours. Then the suspension was cooled to 5°C at a rate of 0.25°C/min. Used the following procedure.

Approximately 14 g of the resulting CHP structure 1 sample was prepared and transferred to a jacketed vessel that had been preheated to 50°C.

35 ml of a solvent mixture of ethanol:water (90:10 v/v%) was added to the vessel until an initial concentration of 400 mg/ml was reached.

The reaction mixture was stirred at 50° C. at about 300 rpm using an overhead stirrer.

An FBRM probe was inserted into the vessel to monitor and record nucleation and particle number.

When complete dissolution was achieved (approximately 20 min), the experiment was carried out at 50° C. and 17.5 ml of MtBE was added dropwise with a syringe.

Upon completion of the addition of MtBE, 280 mg of structure 2 CHP, crushed with a pestle and mortar, was added to the reaction mixture.

The system was observed for approximately 10 minutes at 50°C.

The seed crystals were not preserved. An additional 3.5 ml of MtBE was added dropwise to the vial using a syringe.

ml MtBE was added via a peristaltic pump at a rate of 20 ml/h.

The reaction mixture was then cooled to 29° C. at a rate of 0.1° C./min.

The system was kept at 29°С for 8 h.

After 8 hours at 29°C, the reaction mixture was cooled to 5°C at a rate of 0.25°C/min.

After the temperature in the system reached 5° C., the second step of adding MtBE was started. 134 ml were added at a rate of 20 ml/h. The final solvent ratio was 15:85 vol.%.

The system was kept at 5°C for about 12 hours.

The solid was then isolated by vacuum filtration using an 88 mm Buchner funnel and Whatman Grade 1 filter paper (diameter=70 mm; pore size=11 μm).

The filter cake was washed with 28 ml of ethanol:water:MtBE (9 vol:1 vol:30 vol), after which it was dried in vacuum at 40°C for 1 h.

The dried solid was analyzed by XRPD, TG/DTA, PLM, chiral HPLC and HPLC for purity.

The concentration of the filtered mother liquor was determined by HPLC.

Example 4 Determination of purity.

The purity of CHP and CHP hydrate was measured by HPLC. The chromatographic parameters of the HPLC method for measuring purity for CHP and CHP hydrate are summarized in Table 1. 17V.

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Table 17B. Chromatographic parameters of the HPLC method for measuring the purity of CHP and CHP hydrate

Column XBridge Phenyl column (150 mm * 4.6 mm * 3.5 µm, PN: 186003335) Wavelength 220 nm Column oven temperature zo°s Flow rate 1.0 ml/min Injection volume 5 µl mobile phases A: 10 mM MHDOAc in water (w/v) B: Purified acetonitrile gradient program Time (min) A% V% 0.01 100 0 10.3 70 thirty 15.0 5 95 15.1 100 0 25.0 100 0 Autosampler temperature 5°C Time chromatography 25 min Solvent for washing the needle Purified acetonitrile Diluent 0.1% TFA in water (v/v)

Preparation of assay standard solution (0.8 mg/mL anhydrous CHP or 0.85 mg/mL CHP hydrate): Approximately 40 mg of reference standard C16081735D was weighed and transferred into a 50 ml volumetric flask. Dilute to the mark with diluent and mix thoroughly (if necessary, sonicated to dissolve). Designated the solution as STD-1. A second Standard Solution was prepared for use as a Check Standard. Designated the solution as STD-2.

Preparation of Quantitative Sample Solution (0.85 mg/mL CHP Hydrate): Add a weighed sample of approximately 42.5 mg CHP Hydrate sample to a 50 ml volumetric flask. Dilute to the mark with diluent and mix thoroughly.

A typical blank chromatogram and a typical resolving solution chromatogram are shown in FIG. 52 and 53, respectively.

Example 5 Comparison of CHP Hydrate Stability.

A) Comparison of chemical stability between CHP anhydrate and CHP hydrate of structure 2.

Compounds CHP anhydrate (structure 1) and CHP hydrate (structure 2) were prepared under GMP conditions. Anhydrous CHP stability study was conducted under accelerated conditions (40±5°C/75±5% RH) for six months and at room temperature (25±5°C/60±5% RH) for 12 months . A further study of the stability of the CHP hydrate compound was carried out under accelerated conditions (40±5°C/75±5% relative humidity) for 6 months (Table 18).

Table 18. Study of the stability of the anhydrate and hydrate of CHP structure 2

Start 1 month 3 months 6 month 12 months 25°С 25°C 40°C 25°C 40°С 25°С 40°C 25°С 40°C Compound CHP anhydrate T ¹ NS ² T ¹ T ¹ t ¹ T ¹ t ¹ t ¹ NS ² Compound CHP hydrate, structure 2 t ¹ NS ² t ¹ t ¹ t ^{1 S3 S3 S3} NS ²

T ¹ = analyzed, NS ² = not planned, S ³ = planned.

Long-term storage conditions for an anhydrous CHP compound require careful handling because CHP is hygroscopic. CHP anhydrate absorbed moisture during the stability study, an increase in new impurities and a decrease in CHP anhydrate due to CHP degradation were observed (Table 19). Data on the stability of the structure of 2 CHP hydrate are shown in table. 20.

Table 19. Impurities increasing in anhydrous CHP (structure 1) under accelerated stability test conditions

Anhydrate C1 P (structure 1) at 40°C/75% relative humidity Increase in general impurities OM 1M 3m 6M 05 0% 0.27% 0.53%

Table 20. Impurities increasing in CHP hydrate (structure 2) under accelerated stability test conditions

CHP hydrate (structure 2) at 40°C/75% RH Increase in general impurities OM 1M 3m 6M 05 0% 0.00% 0.05%

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The CHP anhydrate absorbed moisture until the water content of the CHP reached 7%. Thus, CHP anhydrate tends to absorb moisture. The decomposition of CHP anhydrate was also observed.

In contrast, there were no significant qualitative changes in the stable hydrated form of structure 2 during the stability study under accelerated test conditions for 6 months.

A comparison of total impurities between anhydrous CHP and CHP hydrate (structure 2) is shown in FIG. 54.

Example 6 Screening for CHP Hydrate Polymorphs.

A polymorphism study was performed on CHP to identify any new polymorphs with improved solid state properties. As part of this study, the structure of 2CHP was identified as a stable form for development, which is a stoichiometric monohydrate. The study entailed initial analysis of the resulting CHP anhydrate (structure 1, lot: 1058707), solubility determination in 30 solvent systems, and primary polymorph screening using 24 solvent systems and four appropriate process conditions (cycling, cooling, addition and evaporation of anti-solvent). After that, a secondary screen was carried out to scale the structure of 2 CHP, including a stability assessment for 1 week and an assessment of solubility depending on pH.

The resulting material was found to be crystalline according to the XRPD results and consisted of birefringent particles with no defined morphology. From a thermal point of view, structure 1 was found to exhibit three small weight losses at the start of heating before the sample melted at 170°C. EAF analysis indicated that a shape change had occurred and suggested that the resulting material after EAF was a hydrated form of the starting material. The TG/DTA analysis of the solid post-DSP confirmed this, with the TG/DTA thermogram consistent with the thermal data of the Structure 2 material analyzed during the secondary polymorph screening.

High solubility was observed in ethanol and methanol with an approximate solubility of 100>x>50 mg/mL. Using the information collected from the dissolution screening, a primary screening of polymorphs was performed using a predominantly amorphous starting material prepared from the resulting CHP (structure 1). One new polymorph, pure pattern 1, in addition to patterns 1 and 2, was detected at primary screening.

Based on the results of the primary polymorph screening, structure 2 was scaled for further analysis. The structure 2 material was found to thermally lose 0.9 equivalents of water from the start of the TG/DTA experiment until recrystallization occurs at about 120°C. This was followed by melting at 170°C. It was found that the melt temperature was the same as for structure 1, indicating that the sample dehydrated and recrystallized to structure 1 upon heating. Although the recrystallization event was not visible on the DSC curve, evidence of recrystallization of structure 2 to structure 1 was detected using high temperature PLM microscopy. Short-term one-week stability studies of structure 2 CHP showed good chemical stability under the conditions evaluated, but XRPD analysis showed that the starting material of structure 2 was converted to structure 1 after 7 days of storage at 80°C in closed vials. The sample stored at ambient temperature and 40°C/75% RH remained as structure 2.

Longer 8-week studies of the stability of the structure of 2 CHP showed good physical stability at 40°C/75% relative humidity. XRPD analysis confirmed that pattern 2 was dominant throughout the 8 week trial.

Clear structure 1 was initially observed in a VT/VH-XRPD test of structure 2. The X-ray diffraction pattern obtained at 80°C/0% RH showed a peak profile that was similar to the material of structure 1, with some missing peaks, primarily at 17° 2Θ. Further comparison with the diffractogram of the original structure 2 showed that missing peaks were present in the diffraction pattern of structure 2. This suggests that the resulting material (designated structure 1) is a mixture of pure structure 1 and structure 2. Scale-up of pure structure 1 was not successful when heated to 50 or 80°C, but was achieved by flash evaporation in ethanol/dichloromethane , as the ethanol/DCM mixture dissolves the material in addition to azeotropic distillation of water, helping to form the anhydrous structure 1.

Stability studies over eight (8) weeks on pure CHP 1 structure showed poor physical stability at 40°C/75% RH. XRPD analysis confirmed that pure structure 1 converted to structure 2 after 14 days.

Both structure 1 and structure 2 material remained chirally pure throughout the stability evaluation. Although the study was continued on the original samples, the material undergoing the stability test from the 2 week time point was pattern 2 and not pattern 1 due to the poor stability of pure pattern 1 at this temperature and humidity.

Short term one day stability tests of pure CHP Structure 1 showed that the material converted to Structure 2 after 2 hours at 40° C./75% RH.

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Based on the observations described in this application, highlighting structure 2 as the most stable form, a work program for the development of crystallization is recommended in order to provide a reliable, scalable procedure for preparing the material of structure 2.

2) Materials.

The following materials were analyzed.

Lot/Sample ID: cyclo(-GIS-Pro) Lot: 1058707.

SFS ID: CS/298/18/1 and CS/298/18/2.

Amount received: 10 g.

Appearance: Almost white solid.

C) Methods of analysis.

1) X-ray powder diffraction (XRPD).

XRPD analysis was performed on a PANalytical X'pert pro instrument, scanning samples in the range from 3 to 35° 2θ. The material was carefully crushed to release any agglomerates and loaded onto a multiwell plate with Kapton or Mylar film to support the sample. Then, the multiwell plate was placed in a diffractometer and analyzed using Cu K radiation (α1 λ=1.54060 Α; α2=1.54443 Α; β=1.39225 A; Ratio α1: α2=0.5) in transmission mode (size step 0.0130° 2θ) using 40 kV/40 mA generator settings.

2) 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 20x lens unless otherwise noted.

3) High-temperature light microscopy.

Temperature events were monitored visually using a calibrated Linkam THM600 warming stage with an attached controller box connected to an Olympus BX50 polarizing microscope equipped with a Motic camera and image capture software (Motic Images Plus 2.0). Approximately 0.5 mg of the material was placed on a microscope coverslip and heated at 10° C./min with images taken at the usual intervals to document any thermal transitions. All images were recorded using a 10x lens unless otherwise noted.

4) Thermogravimetric analysis (TGA).

A sample of approximately 5 mg of material was prepared in an open aluminum cuvette, loaded into a thermogravimetric/differential thermal analyzer (TG/DTA) and kept at room temperature. The sample was then heated at a rate of 10° C./min from 20 to 300° C., during which time the sample mass change was recorded along with any differential thermal events (DTA). Nitrogen was used as purge gas at a flow rate of ³⁰⁰ cc/min.

5) Differential scanning calorimetry (DSC).

A weighed portion of about 5 mg of the material was prepared in an aluminum DSC cuvette and sealed with an aluminum cap with holes. The sample cuvette was then loaded into a Seiko DSC6200 (with cooler), cooled and held at 20°C. After obtaining a stable heat flux response, the sample and reference were heated to 250°C at a scanning rate of 10°C/min and the resulting heat flux response was monitored. Nitrogen was used as purge gas at a flow rate of 50 ^cc /min.

6) Infrared spectroscopy (IR).

Infrared spectroscopy was performed on a Bruker ALPHA P spectrometer. A sufficient amount of material was placed in the center of the spectrometer plate, and the spectra were obtained using the following

blowing parameters. Resolution: 4 cm ¹ Background scan time: 16 scans Sample scan time: 16 scans Data acquisition: 4000 to 400 cm ¹ Final spectrum: transmission

Software: OPUS version 6

7) Nuclear magnetic resonance (NMR).

NMR experiments were performed on a Bruker AVIIIHD spectrometer equipped with a DCH cryoprobe operating at 500.12 MHz for protons. Experiments were performed in deuterated DMSO and each sample was prepared at an approximate concentration of 10 mM.

8) Dynamic vapor sorption (DSP).

Approximately 10-20 mg of the sample was placed in a sieve cell of a vapor sorption balance and loaded into a DVS Intrinsic dynamic vapor sorption balance from Surface Measurement Systems. Was the sample subjected to

- 36 042744 linear profile from 40 to 90% relative humidity (RH) in 10% steps, holding the sample at each step until a stable mass is reached (dm/dt 0.004%, minimum step length 30 min, maximum step length 500 min) at 25°C. After completion of the sorption cycle, the sample was dried using the same procedure to 0% RH, and then a second sorption cycle was carried out back to 40% RH. Two cycles were completed. We plotted the change in mass during the sorption/desorption cycles, which made it possible to determine the hygroscopicity of the sample. An XRPD analysis of any remaining solid was then performed.

9) Dynamic vapor sorption with variable temperature (VT-DVS).

Approximately 10-20 mg of the sample was placed in a sieve vapor sorption balance and loaded onto a Dynamic Vapor Sorption Balance EAF Advantage from Surface Measurement Systems. The sample was subjected to a variable profile from 40 to 90% relative humidity (RH) in 10% increments, holding the sample at each step until a stable mass was reached (dm/dt 0.004%, min step length 30 min, max step length 500 min) at 40° WITH. After completion of the sorption cycle, the sample was dried using the same procedure to 0% RH, and then a second sorption cycle was carried out back to 40% RH. The mass change was recorded during the sorption/desorption cycles, which made it possible to determine the hygroscopicity of the sample. The experiment was repeated at 50°C and finally at 60°C. An XRPD analysis of any remaining solid was then performed.

10) X-ray powder diffraction at variable temperature and humidity (VT-/VHXRPD).

VT-/VH-XRPD analysis was performed on a Philips X'Pert Pro multifunctional diffractometer equipped with a temperature and humidity chamber. The samples were scanned in the range from 4 to 35.99° 2Θ using Cu K radiation (α1 λ=1.54060 Α; α2=1.54443 Α; β=1.39225 A; ratio α1: α2=0.5) in Bragg-Brentano geometry (step size 0.008° 2θ) using 40 kV/40 mA generator settings. The measurements were carried out at each stage of the moisture profile below /

Program.

40% RH/room temperature - initial scan.

0% RH/room temperature - initial scan, 1 hour hold, scan.

0% RH/ 80°C - initial scan, 20 minutes hold, scan.

0% RH/ 80°C - initial scan, 80 minutes exposure, scan.

11) High performance liquid chromatography - detection in the ultraviolet range (HPLC-UV).

Dionex Ultimate 3000

Tool:

Column: LC/168 X-Bridge Phenyl Column (150 mm x 4.6 mm x 3.5 µm)

Column temperature: 30°C

Autosampler Temperature: 5°C

UV wavelength:

Input volume:

220 nm µl ml/min mM ammonium acetate

Flow rate:

Mobile phase A:

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12) Liquid chromatography - mass spectroscopy (LC-MS).

Tool: Dionex Ultimate 3000 Column: ACE Excel 3 Super C18, 75 x 4.6 mm Column temperature: 30°C Input volume: 10 µl

Flow rate: 1.0 ml/min Mobile phase A: Mobile phase B: 0.1% formic acid in deionized water 0.1% formic acid in acetonitrile Diluent: acetonitrile Washing the needle: FDM range: acetonitrile, vial position #100 190-400 nm

gradient program

Time (minutes) Solvent B (%) 0.00 5 12.00 95 15.00 95 15.10 5 20.00 5

13) Chiral HPLC.

Column: Daicel IC (5.0um, 250*4.6mm)

Column temperature: 30°C

Injection volume: 5 µl

Flow rate: 1.0 ml/min Mobile phase A: 0.01% DEA in n-hexane Mobile phase B: Ethanol: methanol = 2:8 (v/v) Diluent: acetonitrile

FDM range: 220 nm

gradient program

Time (minutes) A% IN% 0.01 70 thirty 28.0 70 thirty

C) experiments.

1) Initial characteristic.

Upon receipt of the supplied cyclo(His-Pro), referred to here as CHP, initial characterization was performed using XRPD, PLM, TG/DTA, DSC, DSP (with post-XRPD analysis), 1H and HSQC NMR, HPLC (for purity and UV -spectrum), pKa and LC-MS using the techniques and methods described in section B.

2) Sample preparation for solvent solubility screening.

Lyophilization in water.

The material was prepared by lyophilization to determine the solubility in solvents as follows.

To the obtained CHP (structure 1) (330 mg) was added 3.3 ml of distilled water, obtaining a clear colorless solution.

This solution was equally divided between 33 2 ml glass vials (containing approximately 100 μl each and 10 mg of solid).

The vials were frozen at -50°C in preparation for lyophilization.

After freezing, the samples were placed in a desiccator attached to a freeze dryer and dried for approximately 18 hours. After this time, samples were taken and analyzed by XRPD to confirm the amorphous nature of the series.

Re-lyophilization in water.

Re-lyophilization was carried out as follows.

Each 10 mg sample (prepared according to section 5.2.1) was redissolved in 200 µl of distilled water.

The vials were frozen at -50°C in preparation for lyophilization.

After freezing, the samples were placed in a desiccator attached to a freeze dryer and dried for approximately 72 hours. After this time, samples were taken and analyzed by XRPD to confirm the amorphous nature of the series.

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3) Solubility screening in solvents.

To a known mass of CHP lyophilisate (10 mg, from section 5.2.2) was added 100 µl of the appropriate solvent and, if a solid remained, the vial was gently heated to about 40° C. to promote dissolution. The addition of the solvent was continued until complete dissolution of the material or until the addition of 2 ml (<5 mg/ml). The samples were opened and allowed to evaporate at room temperature. The solvents used to determine the solubility can be found in table. 21.

4) Sample preparation for primary screening of polymorphs.

a) Lyophilization in water.

The material was prepared by lyophilization for the primary screening of polymorphs as follows.

To the resulting CHP (1.04) was added 13 ml of distilled water, obtaining a clear colorless solution.

This solution was equally divided among 26 2 ml glass vials (approximately 500 μl each and 40 mg of solid).

The vials were frozen at -50°C in preparation for lyophilization.

After freezing, the samples were placed in a desiccator attached to a freeze dryer and dried for approximately 48 hours. After this time, samples were taken and analyzed by XRPD to confirm the amorphous nature of the series.

b) Re-lyophilization in water.

The second lyophilization attempt was carried out as follows.

To each 40 mg sample (prepared according to section a), 1.5 ml of distilled water was added to redissolve the material.

The vials were frozen at -50°C in preparation for lyophilization.

After freezing, the samples were placed in a desiccator attached to a freeze dryer and dried for approximately 72 hours. After this time, a sample was taken and analyzed by XRPD to confirm the amorphous nature of the series.

Table 21. List of solvents used in dissolution screening

Number Solvent ICH class 1 1-butanol h 2 2-butanol 3 3 1-propanol 3 4 2-propanol h 5 40% methanol: 60% water (vol.%) (calculated average 0.8) 2 6 95% methanol: 5% water (vol.%) (calculated average 0.2) 2 7 Acetone h 8 Acetonitrile 2 9 dichloromethane 2 10 ethanol 3 AND ethyl acetate 3 12 Ethyl formate 3 13 Heptane 3 14 Isopropyl acetate 3 15 methanol 2 16 Methyl ethyl ketone 3 17 Methyl isobutyl ketone 3 18 Ν,Ν'-dimethylacetamide 2 19 Nitromethane 2 20 tert-butyl methyl ether 3 21 THF 2 22 Toluene 2 23 Water N/A 24 Trifluoroethanol not classified 25 benzyl alcohol 2 26 Chloroform 2 27 Chlorobenzene 2 28 1,4-dioxane 2 29 2-methoxyethanol 2 thirty Dimethyl sulfoxide 3

c) Re-lyophilization in water (2).

The third lyophilization attempt was carried out as follows.

To each 40 mg sample (prepared according to section b), 1 ml of distilled water was added to redissolve the material.

Then, the solutions were pipetted into 26 glass vials, 20 ml each, and an additional 15 ml of distilled water was added.

The vials were frozen at -50°C in preparation for lyophilization.

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After freezing, the samples were placed in a desiccator attached to a freeze dryer and dried for about 120 hours. After this time, a sample was taken and analyzed by XRPD to confirm the amorphous nature of the series.

5) Primary screening of polymorphs.

Primary screening for CGP polymorphs was performed as follows.

In this experiment, 24 vials containing approximately 40 mg CHP from section b were used.

The material was suspended in an appropriate solvent/solvent mixture and the temperature changed from room temperature to 40° C. over 4 hours over 72 hours. The solvents used can be found in Table 1. 22.

The resulting solids were isolated by centrifugation filtration and analyzed by XRPD. Any new forms were analyzed by TG/DTA.

The filtered saturated solutions were then divided into three vials and used for subsequent polymorph screening experiments as shown below.

a) Evaporation.

Saturated CHP solutions were transferred into vials with a capacity of 2 ml; these vials were then opened and allowed to evaporate at ambient temperature to recover the material. All recovered material was characterized by XRPD.

b) Rapid cooling.

Saturated CHP solutions were stored at 2-8°C for 96 hours. At this time, any recovered material was analyzed by XRPD, vials were moved and stored at -20°C for 72 hours. After this time, any recovered material was analyzed by XRPD. XRPD.

c) Addition of anti-solvent at room temperature.

Up to 2 ml of the antisolvent (heptane or MtBE) was added dropwise to saturated CHP solutions. The samples were left closed at ambient temperature for 72 hours. Any solid obtained was analyzed by XRPD.

Table 22. List of solvents in the primary screening of polymorphs

Number Solvent ICH class 1 1-propanol 3 2 2-propanol 3 h 95% methanol: 5% water (vol.%) 2 4 50% methanol/ 50% TBME (vol.%) 2 5 10% methanol/ 90% TBME 2 6 Acetone 3 7 Acetonitrile 2 8 dichloromethane 2 9 ethanol 3 10 50% ethanol/ 50% TBME (vol%) 3 AND 10% ethanol/ 90% TBME 3 12 ethyl acetate 3 13 Ethyl formate 3 14 Heptane 3 15 Isopropyl acetate 3 16 Methyl ethyl ketone 3 17 Methyl isobutyl ketone h 18 Nitromethane 2 19 tert-butyl methyl ether h 20 Toluene 2 21 Trifluoroethanol not classified 22 Chlorobenzene 2 23 THF 2 24 methanol 2

6) Secondary screening of polymorphs.

a) Structure scaling 2.

Structure 2 of the CGP was scaled using the procedures outlined below.

g of the obtained CHP (structure 1) was suspended in 6 ml of a mixture of ethanol/water 90:10.

The resulting suspension was placed on a stirrer and stirred for about 24 hours.

After 24 hours, the sample was removed from the shaker and the solid was analyzed by XRPD.

The solid has been characterized in accordance with P-SFS1482-00 section 2.4.

a) Pure structure scaling 1.

i) Heating structure 2 to 80°C.

Approximately 2.2 g of structure 2 material (prepared according to section 5.6.1) was placed in a vacuum oven set at 50°C.

After 1 hour, a sample was taken and analyzed by XRPD.

The vial was returned to the vacuum oven and the temperature was raised to 60°C.

After 30 minutes, a sample was taken and analyzed by XRPD.

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The vial was returned to the vacuum oven and the temperature was raised to 80°C.

After 18 hours at 80°C, a sample was taken and analyzed by XRPD.

ii) Rapid rotary evaporation in ethanol/DCM.

A weighed portion of 1 g of the obtained structure 1 CHP was added to a round bottom flask and dissolved in 40 ml of ethanol.

After the solid had dissolved, 2 ml of dichloromethane was added to the flask using a pipette.

The flask was then attached to a rotary evaporator and rapidly evaporated at ambient temperature under vacuum.

A pale beige solid was recovered and analyzed by XRPD. 7. Estimation of pH solubility.

CHP structures 1 and 2 were prepared to evaluate pH solubility as follows.

Added weighed 100 mg of structure 1 and structure 2 CHP in 4 glass bottles of 5 ml with a screw cap.

100 μl of the selected buffer, pre-warmed to 37° C., was added to each vial.

The samples were heated to 37° C. in an incubator shaker for about 10 minutes after each volume of buffer, and sample dissolution was checked.

C) results.

1) Initial characteristic.

The initial characterization of CGP (CS/298/18) showed the following.

The material was crystalline according to XRPD (FIG. 1). The XRPD structure of the supplied CHP was designated structure 1.

The PLM analysis showed that the material exhibits birefringence without a distinct morphology. Polarized and non-polarized images can be seen in Fig. 12 and 11, respectively.

TG analysis showed that from the beginning of heating, a series of small weight losses (0.6, 0.2 and 0.3%) was observed up to about 280°C, where degradation of the sample was observed. DTA analysis identified one abrupt endothermic event beginning at 170°C and peaking at 172°C. Most likely, this is the melting of the sample. The TG/DTA thermogram can be seen in Fig. 14.

In FIG. 16 shows the first thermal run of the DSC analysis. A small wide endothermic effect was noted at 75°C and a peak at 85°C. The second endothermic effect was observed at 169°C with a peak at 171°C. This was consistent with the melt observed in TG/DT. No significant thermal events were observed in the cold cycle (FIG. 17). A very small endothermic event, most likely a weak glass transition, was noted at 75-80° C. during the second thermal cycle (FIG. 18).

Plot of the EAF isotherm in Fig. 23 showed a mass change of +6.3% at 60 to 90% relative humidity, indicating a clear change in shape.

After reshaping, the material appeared to be slightly hygroscopic with a maximum absorption of 0.8 wt% from 0 to 90% relative humidity (FIG. 24). The kinetic plot of the EAF can be seen in Fig. 25. XRPD analysis confirmed that the structure 1 introductory material recrystallized to structure 2 (originally visible in the solvent solubility screening detailed in section 3) after exposure to EAF moisture conditions. XRPD patterns can be seen in Fig. 26. Comparison of the diffraction patterns of structure 1 and structure 2 shows that a number of structure 2 peaks are also present in the resulting structure 1. This suggests that the resulting structure 1 is a potential mixture. This was further investigated by running VT/VH-XRPD on structure 2 material (according to section B.1).

TG/DTA analysis was performed on the solid recovered from EAF for Structure 2. The thermogram is shown in FIG. 27. TG analysis showed an initial weight loss of 6.8% (0.95 eq. of water) followed by sample decomposition at 280°C. The DT plot identified an endothermic event associated with the initial mass loss. This was followed by an exothermic event, most likely recrystallization of the material, followed by a second endotherm (sample melting) at 168°C.

¹ H-NMR (FIG. 5) and HSQC-NMR (FIG. 38) of the obtained CHP were consistent with the supplied structure. No imidazole CH signal was observed, probably due to slow relaxation.

Analysis of the obtained material gave a pKa value of 6.38 (FIG. 10).

The LC-MS spectrum of CHP structure 1 confirmed the expected m/z value of 235.1, which corresponds to [C ₁₁ H ₁₄ N ₄ O ₂ ]H ⁺ (FIG. 55).

The resulting CHP was 99.5% pure when analyzed by HPLC-UV. The corresponding chromatogram is shown in Fig. 56.

2) Sample preparation for solvent solubility screening.

a) Lyophilization in water.

Analysis of the solid from lyophilization in water indicated that the material was still crystalline. The 2Θ diffraction pattern of the amorphous material is shown in FIG. 40.

b) Re-lyophilization in water.

Analysis of re-lyophilized solids from water showed a predominant material.

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3) Solubility screening in solvents.

As indicated above, the solubility of lyophilized CHP was evaluated in 30 selected solvents/solvent mixtures. From the results in Table. 23, the material showed low solubility in most of the solvents/solvent mixtures used in this study. High solubility was observed in ethanol and methanol with approximate solubility estimated between 100>x>50 mg/mL. The material was also found to be soluble in water, DMSO, DMA, trifluoroethanol and both methanol/water mixtures.

The results of XRPD analysis to determine the solvent solubility of the obtained solids are presented in table. 24. An example XRPD pattern for each of the resulting structures can be seen in FIG. 57. The rest of the diffractograms are included in FIG. 58-60.

Table 23. Results of Solubility Analysis in Solvents of CHP Lyophilisate

Number Solvent Approximate solubility (mg/ml) 1 1-butanol <5 2 2-butanol <5 3 1-propanol 14.3>x>12.5 4 2-propanol 5.6>x>5.3 5 40% methanol: 60% water (vol.%) (avg. 0.8) 20>x>16.7 6 95% methanol: 5% water (vol.%) (avg. 0.2) 33.3>х>25 7 Acetone <5 8 Acetonitrile <5 9 dichloromethane <5 10 ethanol 100>x>50 AND ethyl acetate <5 12 Ethyl formate <5 13 Heptane <5 14 Isopropyl acetate <5 15 methanol 100>x>50 16 Methyl ethyl ketone <5 17 Methyl isobutyl ketone <5 18 Ν,Ν'-dimethylacetamide 33.3<х<25 19 Nitromethane <5 20 tert-butyl methyl ether <5 21 THF <5

Table 24 Summary Table of XRPD Structures in Solubility Screening in Solvents

Number Solvent XRPD Results 1 1-butanol N/A - no solids 2 2-butanol N/A - no solids 3 1-propanol Structure 1 4 2-propanol Structure 2 5 40% methanol: 60% water (vol.%) (avg. 0.8) Structure 2 6 95% methanol: 5% water (vol.%) (avg. 0.2) Structure 2 7 Acetone Structure 2 8 Acetonitrile Structure 2 9 10 And 12 13 14 15 16 Dichloromethane Ethanol Ethyl acetate Ethyl formate Heptane Isopropyl acetate Methanol Methyl ethyl ketone Structure 2 Structure 1 | Structure 2 Structure 2 Structure 2 Structure 2 Structure 2 N/A - colorless gel Structure 2 17 Methyl isobutyl ketone Structure 2 18 Ν,Ν'-dimethylacetamide N/A - no solids 19 Nitromethane Structure 2 20 tert-butyl methyl ether Structure 2 21 THF Amorphous substance with peaks Structure 2

4) Preparation of samples for primary screening of polymorphs.

2) Lyophilization in water.

Analysis of lyophilized solids from water indicated that the samples were still crystalline. The diffractogram pattern was identified as pattern 2 (FIG. 61).

b) Re-lyophilization in water.

In FIG. 62 shows a diffraction pattern obtained by re-lyophilization of CHP in water. XRPD analysis confirmed that the material was less crystalline than the previous sample shown in FIG. 61, but still contained structure 2 peaks.

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c) Re-lyophilization in water (2).

In FIG. 63 shows the X-ray diffraction pattern from the third attempt to lyophilize CHP in water.

XRPD analysis confirmed that the material was less crystalline than the sample shown in FIG.

61, but the peaks of structure 2 from FIG. 62 remained.

5) Primary screening of polymorphs.

As stated above, the propensity of CHP to exhibit polymorphism was evaluated in 24 selected solvents/solvent mixtures.

a) Temperature cycling.

The results of experiments on temperature changes during the primary screening of polymorphs are presented in table. 25. The corresponding diffractograms are shown in FIG. 64-66.

Pure structure 1 CHP was isolated from 50:50 methanol/MtBE, 10:90 methanol/MtBE, ethanol/MtBE, and from ethyl acetate, although some preferred orientation was evident in this sample.

Structure 1 was prepared from ethyl formate, acetonitrile, MEK and THF.

solvent systems provided structure 2: heptane, isopropyl acetate, MIBA, MtBE, and toluene.

The preferred orientation of the DCM-derived solid meant that the structure could not be determined.

A solid was noted from chlorobenzene, but it was not produced in sufficient quantity so XRPD analysis could not be performed.

An oil was obtained in methanol.

Solutions were obtained from the remaining solvent systems, so XRPD analysis was not performed.

Table 25 Temperature cycling in polymorph primary screening

Solvent Structure 1-propanol N/A - solution 2-propanol N/A - solution 95% methanol: 5% water (vol.%) N/A - solution 50% methanol/ 50% TBME (vol.%) Net Structure 1 10% methanol/ 50% TBME Net Structure 1 Acetone N/A - solution Acetonitrile Structure 1 dichloromethane Preferred orientation cannot be estimated ethanol N/A - solution 50% ethano/ 50% TBME (vol%) Net Structure 1 10% ethanol/ 90% TBME Net Structure 1 ethyl acetate Net Structure 1 (preferred orientation) Ethyl formate Structure 1 - poor crystallinity Heptane Structure 2 Isopropyl acetate Structure 2 Methyl ethyl ketone Structure 1 Methyl isobutyl ketone Structure 2 Nitromethane N/A - solution tert-butyl methyl ether Structure 2 Toluene Structure 2 (preferred orientation) Trifluoroethanol N/A - solution Chlorobenzene N/A - not enough solids THF Structure 1 methanol N/A - oil

b) Evaporation.

The results of experiments on evaporation during the primary screening of polymorphs are presented in table. 26 and in FIG. 113. The corresponding diffractograms are shown in FIG. 67.

Structure 1 with poor crystallinity was obtained from acetonitrile.

Structure 2 was obtained twice: once from trifluoroethanol and also from nitromethane.

Amorphous material was identified from DCM and THF.

A solid was isolated from a mixture of 10:90 ethanol/MtBE, 50:50 ethanol/MtBE, MIBK, and ethanol, but it was not produced in sufficient quantity, so XRPD analysis could not be performed.

Most solvent systems form a colorless film on the walls of the vial that cannot be analyzed by XRPD.

- 43 042744

Table 26. Evaporation in Primary Screening of Polymorphs

Solvent Structure 1-propanol N/A - colorless film on the walls of the test tube 2-propanol N/A - colorless film on the walls of the test tube 95% methanol: 5% water (vol.%) N/A - colorless film on the walls of the test tube 50% methanol/ 50% TBME (vol.%) N/A - no solution after temperature cycling 10% methanol/ 50% TBME N/A - not enough solids Acetone N/A - oil residue on the walls of the test tube Acetonitrile Structure 1 (poor crystallinity) dichloromethane amorphous substance ethanol N/A - not enough solids 50% ethano/ 50% TBME (vol.%) N/A - not enough solids 10% ethanol/ 90% TBME N/A - colorless film on the walls of the test tube ethyl acetate N/A - colorless film on the walls of the test tube Ethyl formate N/A - colorless film on the walls of the test tube Heptane N/A - colorless film on the walls of the test tube Isopropyl acetate N/A - colorless film on the walls of the test tube Methyl ethyl ketone N/A - colorless film on the walls of the test tube Methyl isobutyl ketone N/A - not enough solids Nitromethane Structure 2 (poor crystallinity) tert-butyl methyl ether N/A - colorless film on the walls of the test tube Toluene N/A - colorless film on the walls of the test tube Trifluoroethanol Structure 2 Chlorobenzene N/A - colorless film on the walls of the test tube THF amorphous substance methanol N/A - no solution after temperature cycling

c) Rapid cooling.

In table. 27 shows the results obtained with rapid cooling at both 2-8°C and 20°C in the primary screening of polymorphs.

The quench experiments were not performed in a 50:50 mixture of methanol/MTBE and methanol due to insufficient mother liquor production with temperature changes.

In the remaining experiments, only solutions were obtained, so XRPD analysis could not be performed.

Table 27 Primary Screening for Polymorphs on Rapid Cooling

Solvent Structure (2-8°С) Structure (-20°C) 1-propanol Solution Solution 2-propanol Solution Solution 95% methanol: 5% water (vol.%) Solution Solution 50% methanol/ 50% TBME (vol.%) N/A - no solution after temperature cycling N/A - no solution after temperature cycling 10% methanol/ 50% TBME Solution Solution Acetone Solution Solution Acetonitrile Solution Solution dichloromethane Solution Solution ethanol Solution Solution 50% ethano/ 50% TBME (vol%) Solution Solution 10% ethanol/ 90% TBME Solution Solution ethyl acetate Solution Solution Ethyl formate Solution Solution Heptane Solution Solution Isopropyl acetate Solution Solution Methyl ethyl ketone Solution Solution Methyl isobutyl ketone Solution Solution Nitromethane Solution Solution tert-butyl methyl ether Solution Solution Toluene Solution Solution Trifluoroethanol Solution Solution Chlorobenzene Solution Solution THF Solution Solution methanol N/A - oil obtained after temperature cycling N/A - oil obtained after temperature cycling

d) Addition of anti-solvent.

Tab. 28 shows the results obtained with the addition of an antisolvent in the primary screening of polymorphs. The corresponding diffraction pattern is shown in Fig. 68.

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Insufficient solids were obtained from acetone, DCM and 50:50 ethanol/MtBE.

Structure 1 (for ethanol) was the only pattern observed in the anti-solvent addition experiments.

Antisolvent experiments were not performed with 50:50 methanol/MTBE and methanol due to insufficient mother liquor formation with temperature changes.

Table 28 Antisolvent Addition for Primary Screening of Polymorphs

Solvent Antisolvent Structure 1-propanol TBME N/A - solution 2-propanol TBME N/A - solution 95% methanol: 5% water (vol.%) TBME N/A - solution 50% methanol/ 50% TBME (vol.%) N/A N/A - no solution after temperature cycling 10% methanol/ 50% TBME TBME N/A - solution Acetone TBME N/A - not enough solids Acetonitrile TBME N/A - solution dichloromethane TBME N/A - not enough solids ethanol TBME Structure 1 50% ethano/ 50% TBME (vol.%) TBME N/A - not enough solids 10% ethanol/ 90% TBME TBME N/A - solution ethyl acetate TBME N/A - solution Ethyl formate acetone N/A - solution Heptane TBME N/A - solution Isopropyl acetate TBME N/A - solution Methyl ethyl ketone TBME N/A - solution Methyl isobutyl ketone TBME N/A - solution Nitromethane TBME N/A - solution tert-butyl methyl ether heptane N/A - solution Toluene TBME N/A - solution Trifluoroethanol TBME N/A - solution Chlorobenzene TBME N/A - solution THF TBME N/A - solution methanol N/A - oil obtained after temperature cycling

6) Summary of primary screening for polymorphs.

In table. 29 and in FIG. 114 is a summary of the screening results for the primary polymorph. Table 29. Summary table of primary screening of polymorphs

Ch. C1 Net Structure 1 C1 Structure 1 C2 Structure 2 Rust. Solution Underdos. Not enough solid wt. Oil Am. amorphous substance Bests. Colorless film/Oil remainder - The experiment was not carried out 1 Preferred Orientation * Poor crystallinity

- 45 042744

Temperature cycling Evaporation Cooling (2-8°С) Cooling (-20°C) Addition of anti-solvents No. Solvent 1 1-propanol Rust. Bests. Rust. Rust. Rust. 2 2-propanol Rust. Bests. Rust. Rust. Rust. 3 95% methanol: 5% water (vol.%) Rust. Bests. Rust. Rust. Rust. 4 50% methanol/ 50% TBME (vol.%) Ch. C1 - - - - 5 10% methanol/ 50% TBME Ch. C1 Underdos. Rust. Rust. Rust. 6 Acetone Rust. Bests. Rust. Rust. Underdos. 7 Acetonitrile C1 C1* Rust. Rust. Rust. 8 dichloromethane 1 Am. Rust. Rust. Underdos. 9 ethanol Rust. - Rust. Rust. C1 10 50% ethanol/ 50% TBME (vol.%) Ch. C1 Underdos. Rust. Rust. Underdos. AND 10% ethanol/ 90% TBME Ch. C1 Bests. Rust. Rust. Rust. 12 ethyl acetate Ch. C1 Bests. Rust. Rust. Rust. 13 Ethyl formate C1* Bests. Rust. Rust. Rust. 14 Heptane C2 Bests. Rust. Rust. Rust. 15 Isopropyl acetate C2 Bests. Rust. Rust. Rust. 16 Methyl ethyl ketone C1 Bests. Rust. Rust. Rust. 17 Methyl isobutyl ketone C2 Underdos. Rust. Rust. Rust. 18 Nitromethane Rust. C2* Rust. Rust. Rust. 19 tert-butyl methyl ether C2 Bests. Rust. Rust. Rust. 20 Toluene C2 ¹ Bests. Rust. Rust. Rust. 21 Trifluoroethanol Rust. C2 Rust. Rust. Rust. 22 Chlorobenzene Bests. Rust. Rust. Rust. 23 THF C1 Am. Rust. Rust. Rust. 24 methanol wt. - - - -

Primary polymorph screening identified 1 new form of CHP, designated as pure structure 1, obtained after temperature cycling. Structures 1 and 2 were also reproduced in multi-solvent systems with temperature changes. Rapid cooling experiments to 2-8°C and -20°C resulted in only clear solutions. Most solvent systems gave clear solutions in anti-solvent addition experiments, but structure 1 was observed in ethanol after addition of MtBE.

7) Secondary screening of polymorphs.

a) Structure scaling 2.

In FIG. 69 shows the results of XRPD at structure scaling 2.

The XRPD pattern confirmed the formation of structure 2.

b) Net structure scaling 1.

i) Heating structure 2 to 80°C.

Pure structure 1 was not successfully obtained in this experiment. A mixture of structure 1 and structure 2 was obtained both at 50°C and at 80°C. A previously invisible peak at 8° 2 Θ appeared after the sample was kept at 50°C for 90 min. The intensity of this peak increased after 18 h at 80°C. The corresponding diffractograms are shown in Fig. 70.

ii) Rapid rotary evaporation in ethanol/DCM.

Analysis of solids recovered by flashing from ethanol/DCM indicated that the material was pure XRPD structure 1. The diffraction pattern is shown in Fig. 71.

c) Characterization of structure 2.

Analysis of the structure of 2 CHP from a mixture of ethanol/water 90:10 gave the following results.

The material was crystalline according to XRPD and matched the pattern 2 diffraction patterns from the primary polymorph screen (FIG. 72).

PLM images of the 2 CHP structure were also taken and the material was found to be birefringent with a fragmented rod-like morphology. Images in polarized and unpolarized light are shown in Fig. 73.

High temperature microscopy was also performed on the 2 CHP structure. The original image identified a rod-shaped morphology (FIG. 74). This was consistent with the images from the PLM analysis in FIG. 69. It was observed that the material began to melt at 95°C and completely melted at 101°C. Recrystallization took place at 115°C. The PLM images obtained after the heating step show that the morphology of the material was no longer well defined, but the sample was still birefringent. Polarized and non-polarized images of the material after heating can be seen in FIG. 75.

- 46 042744

Analysis of TG/DTA - TG showed an initial weight loss of 6.5% followed by decomposition of the sample at 280°C (0.9 water equivalent). The DT plot identified an endothermic event associated with the initial mass loss. This was followed by an exothermic event, most likely recrystallization of the material, followed by a second endotherm (sample melting) at 170°C (FIG. 76).

The initial thermal DSC cycle revealed an endothermic event with an onset of 99°C and a peak at 102°C (FIG. 77). This is consistent with the endotherm data observed in TGA, although the recrystallization event observed in TGA is not present in DSC. No thermal phenomena were observed in the cold cycle (Fig. 78). The second heating cycle revealed a possible glass transition at 75°C (FIG. 79).

In FIG. 80 shows Fourier transform IR spectra for structure 2 CHP. The list of peaks can be found in Table. thirty.

VT-/VH-XRPD Analysis: Structure 2 parent material remained when humidity was reduced to 0% at ambient temperature. When the temperature was raised to 80°C, the material changed shape, which was designated as pure structure 1. Pure structure 1 remained when kept at 80°C for 80 minutes. Tab. 31 shows humidity, temperature and texture identity at each stage. The corresponding diffraction pattern is shown in Fig. 81.

Analysis of the DSP structure of 2 CHP showed that the material did not change its shape from 40 to 90% or from 90 to 10%, but lost 3.5 wt.% (0.5 eq. of water) at a relative humidity below 10%. It has been noted that rehydration occurs at relative humidity between 0 and 90%. This can be seen in the EAF isotherm plot in Fig. 82. In FIG. 83 shows an EAF kinetic plot. XRPD analysis after EAF showed no shape change at 40% relative humidity. A comparison of the XRPD patterns can be seen in Fig. 84. The non-stoichiometric loss of water observed in this experiment was thought to be related to the ambient temperature (approximately 25°C), with the dehydration kinetics being too slow and hence the instrument proceeding to the next step before completion. To explore this further, variable temperature EAF experiments were carried out, detailed below.

VT-DVS analysis at 40° C. showed that the material began to dehydrate at below 10% relative humidity, losing about 5.8 wt% from 10 to 0% relative humidity (0.8 eq water). The material was subsequently rehydrated from 0 to 40% relative humidity. A graph of the EAF isotherm can be seen in Fig. 85 and the kinetic plot in FIG. 86. Analysis of the EAF at 50°C showed that the material began to dehydrate at a relative humidity below 20%, losing about 6.1 wt.% (0.8 eq. of water). The material was then rehydrated from 0 to 40% relative humidity. In FIG. 87 is a plot of the isotherm at 50°C, and FIG. 88 is a kinetic graph. VT-DVS at 60°C showed that the material began to dehydrate at a relative humidity below 20%, losing about 7 wt.% or 1.0 eq. water. The material has been observed to rehydrate at 0 to 40% relative humidity. A plot of the 60°C isotherm can be seen in FIG. 89. The kinetic plot can be seen in FIG. 90. XRPD analysis of the recovered solid showed that after EAF the material was a mixture of Structure 1 and Structure 2. Comparative XRPD patterns can be seen in FIG. 91.

The purity of the structure of 2 CHP by HPLC was 99.5%.

Table 30. FT-IR Peak Table of Structure 2 CHP for Ethanol/Water

3457.2 81.8 1233.3 80.9 3411.6 82.2 1223.5 81.4 3292.7 78.6 1198.3 79.2 3211.7 79.1 1161.1 83.6 3148.3 82.9 1121.0 71.5 2976.7 84.3 1083.1 74.5 2949.6 85.8 1003.9 86.4 2912.7 86.4 968.7 73.7 2879.7 86.0 942.5 82.4 2116.5 91.3 922.3 81.4 1658.1 56.5 908.0 84.9 1633.1 55.6 889.5 87.3 1567.1 77.5 862.8 85.8 1445.9 61.6 798.1 58.9 1424.3 63.4 737.6 61.6 1345.0 77.9 688.9 70.4 1330.0 82.3 628.5 56.0 1314.6 82.0 612.6 49.5 1300.0 71.8 487.4 75.4 1280.0 81.8 470.8 68.9 1268.9 79.0 429.1 59.9 1252.2 82.3

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Table 31. VT/VH-XRPD results for CHP structure 2

Exp. % r.h. Temperature Nature 1 40 room Structure 2 2 0 Structure 2 3 0 80°С Structure 1 4 0 Structure 1

8) Estimation of pH solubility.

Solubility pH evaluation for both structure 1 and structure 2 showed that dissolution occurs after addition of 3 volumes of buffer at all pH values tested (pH 1; 4; 6.8 and 7.2). The solubility of structure 1 and structure 2 was estimated as 500>x>333.3 mg/ml. A summary of the results is presented in table. 32.

Images of the samples after dissolution can be seen in Fig. 92 for the material of structure 1 and FIG. 93 for structure 2.

Table 32. Summary table of pH solubility results

Structure CHP pH µl for dissolution at 37°C Approximate solubility (mg/ml) 1 1 300 500>x>333.3 mg/ml 1 4 300 500>x>333.3 mg/ml 1 6.8 300 500>x>333.3 mg/ml 1 7.2 300 500>x>333.3 mg/ml 2 1 300 500>x>333.3 mg/ml 2 4 300 500>x>333.3 mg/ml 2 6.8 300 500>x>333.3 mg/ml 2 7.2 300 500>x>333.3 mg/ml

9) Stability studies.

a) Stability evaluation within 1 week.

XRPD 20 diffraction patterns for samples tested at 40°C/75% RH, 80°C and natural light are shown in FIG. 94. No change in shape was observed for specimens stored at 40°C/75% RH or under natural light. The structure 2 material was found to convert to structure 1 after 7 days at 80° C. as expected based on previous thermal experiments, EAF and VT/VH XRPD.

The results of HPLC purity for structure 2 CHP are presented in table. 33. There was no drop in purity after 7 days of storage under all conditions. HPLC chromatograms for testing sample stability are shown in FIG. 95-97.

Table 33 Purity Analysis Results from the 1 Week Stability Test for CHP Structure 2

Raw Material Purity (%) Conditions Purity after 1 week (%) 99.5 40°C/75% r.h. 99.5 80°С 99.4 Room temperature 99.5

b) 8-week stability assessment.

i) Two-week period.

XRPD 20 diffractograms for two week time points are shown in FIG. 98 for pure structure 1 and in FIG. 99 for structure 2. The results show that pure structure 1 was converted to structure 2 after 14 days at 40° C./75% relative humidity. After 14 days, no shape changes were observed for structure 2 CHP.

ii) Four weeks.

XRPD 20 patterns for four week time points are shown in FIG. 100 for pure structure 1 and in FIG. 101 for structure 2. The results show that the pure structure 1 material, which had previously converted to structure 2 after two weeks, remained as structure 2 after 4 weeks at 40° C./75% relative humidity. No change in shape was observed for structure 2 CHP after 4 weeks.

iii) Eight weeks.

XRPD 20 patterns for eight week time points are shown in FIG. 102 for pure structure 1 and in FIG. 103 for structure 2. The results show that the pure structure 1 material, which previously converted to structure 2 after two weeks, remained as structure 2 after 8 weeks at 40° C./75% relative humidity. No change in shape was observed for structure 2 CHP after 8 weeks.

iv) Appearance check.

Tab. 34 and FIG. 115 show the results of appearance testing from the 8 week stability study. The appearance of pure structure 1 and structure 2 remained unchanged throughout the experiment.

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Table 34: Summary of 8-Week Stability Appearance Results

Introductory structure Moment of time Appearance Net Structure 1 original pale beige Net Structure 1 2 weeks pale beige Net Structure 1 4 weeks pale beige Net Structure 1 8 weeks pale beige Structure 2 original White Structure 2 2 weeks White Structure 2 4 weeks White Structure 2 8 weeks White

v) Chiral HPLC.

HPLC analysis confirmed that the starting samples of both pure structure 1 and structure 2 were of high chiral purity. There was no change in chiral purity throughout the stability evaluation period. A summary of the results can be seen in Table. 35. HPLC chromatograms are shown in FIG. 104-110.

Table 35 Summary of Chiral HPLC Results

Introductory structure Moment of time Chiral purity (%) Net structure 1 original >99.9 2 weeks 99.9 4 weeks 99.9 8 weeks 99.9 Structure 2 original 99.9 2 weeks >99.9 4 weeks 99.9

c) 1 day stability test.

The results of XRPD when stored at 40°C/75% RH are shown in FIG. 111. Pure structure 1 was found to convert to structure 2 after 2 hours at 40°C/75% RH. The original experiment included additional time points at 4, 6, and 8 hours, but because the material converted to pattern 2 after the first time point, the experiment was stopped after 2 hours.

d) Brief description of the results.

1) Initial characteristic.

The supplied material, CHP, was found to be crystalline in XRPD and birefringent with no well-defined morphology in PLM. The material had several weight losses of 0.6%, 0.2% and 0.3% due to TG/DTA and was found to decompose when heated to 280°C. Melting is also observed at 170°C. DSC led to a small endothermic event at 75°C, which was associated with the loss of solvent (water). The second endothermic effect was observed at 169°C with a peak at 171°C. This corresponded to the melt observed in TG/DT. No significant thermal events were observed in the cold cycle, but a weak glass transition was noted at 75° C. during the second thermal cycle.

EAF data showed a mass change of +6.3% at 60 to 90% relative humidity, indicating a clear recrystallization event. After recrystallization, the material appeared slightly hygroscopic with a maximum absorption of 0.8 wt.% at a relative humidity of 0 to 90%. XRPD analysis after DSP confirmed the shape change. The diffraction pattern obtained after EAF was designated structure 2 and was identified as the hydrated form of structure 1. After EAF, TG/DTA showed that the input material of structure 2 was dehydrated on heating and lost 6.8 wt% (0.95 eq. . water). This was followed by recrystallization, after which the sample was melted at 168°C. The temperature of this melt corresponded to that of structure 1. The constancy of the melting temperature between the two samples suggests that structure 2 is a monohydrate and loses about 1 eq. water when heated before recrystallization into structure 1.

The NMR data were consistent with the provided structure and showed no solvent content.

2) Solubility in solvents.

The resulting CHP (structure 1) showed low solubility in most of the solvents/solvent mixtures used in this study. High solubility was observed in methanol and ethanol with an approximate solubility of 100>x>50 mg/ml. The material was also found to be soluble in water, DMSO, DMA, trifluoroethanol and both methanol/water mixtures. Structure 2 was the only new crystal form identified during this experiment (in addition to the resulting structure 1). Structure 2 was prepared from 17 solvent systems, while structure 1 was prepared only once from 1-propanol. A mixture of structure 1 and structure 2 has been noted for ethanol. THF provided an amorphous material with some structure 2 peaks.

3) Primary screening of polymorphs.

Primary polymorph screening identified 1 new form of CHP, designated as pure

- 49 042744 structure 1, and obtained after temperature cycling. Structures 1 and 2 were also reproduced in multi-solvent systems with temperature changes. Rapid cooling experiments at 2-8°C and -20°C gave only clear solutions. Most solvent systems gave clear solutions in anti-solvent addition experiments, but pattern 1 was observed for ethanol after MtBE addition.

4) Secondary screening of polymorphs.

Secondary polymorph screening showed successful scaling of the structure of 2CHP from an ethanol/water mixture. The analysis of the material gave the following results.

The material was crystalline according to XRPD and matched the diffraction patterns of structure 2 obtained from the primary screening of polymorphs.

PLM images of the 2 CHP structure were also taken and the material was found to be birefringent with a fragmented rod-like morphology.

The initial image taken at the beginning of the high temperature microscopy experiment revealed a rod-like morphology that matched the PLM images of Structure 2. The material started to melt at 95°C and completely melted at 101°C. Recrystallization was observed at 115°C. The PLM images taken after the heating step show that the morphology of the material was no longer well defined, but the sample still had birefringence.

TG/DTA-TG analysis showed an initial weight loss of 6.5% followed by sample decomposition at 280°C. The DT plot identified an endothermic event associated with initial weight loss. This was followed by an exothermic event, most likely recrystallization of the material, after which a second endotherm (melting of the sample) was observed at 170°C.

The initial thermal DSC cycle revealed an endothermic event with an onset of 99°C and a peak at 102°C. This is consistent with the endotherm data observed in TGA, although the recrystallization event observed in TGA is not present in DSC. No thermal events were observed in the cold cycle, but a small glass transition was observed at 75°C in the second thermal cycle.

VT-/VH-XRPD Analysis: Structure 2 parent material remained when humidity was reduced to 0% at ambient temperature. When the temperature was raised to 80°C, the material changed shape, which was designated as pure structure 1. Pure structure 1 remained when kept at 80°C for 80 minutes.

Analysis of the DSP structure of 2 CHP showed that the material did not change shape from 40 to 90% or from 90 to 10%, but lost 3.5 wt.% (0.5 eq. of water) below 10%. It has been noted that rehydration occurs at relative humidity between 0 and 90%. XRPD analysis after EAF showed no shape change at 40% relative humidity.

VT-DVS: sample dehydrated below 10% at 40°C and below 20% at 50 and 60°C. The material lost a higher percentage of water with each increase in temperature, but rehydrated from 0 to 40% RH in each experiment. A mixture of structures 1 and 2 was observed by XRPD after VT-DVS.

The purity of the structure of 2 CHP by HPLC was 99.5%.

Pure structure 1 was not successfully reproduced when heated to 80°C, a mixture of structures 1 and 2 was confirmed by XRPD. XRPD analysis also identified a previously invisible peak at 8° 2Θ after the sample was held at 50° C. for 90 minutes. The intensity of this peak increased after 18 h at 80°C. This peak was not observed in any other diffractogram obtained in this study.

Successful re-preparation of pure structure 1 was achieved by flash evaporation in ethanol/DCM.

5) 1-week stability studies.

Stability studies over 1 week showed that structure 2 exhibits good chemical stability under the evaluated conditions (40° C./75% RH, 80° C. and natural light). No change in purity was observed for any sample after 7 days.

An elevated temperature of 80° C. resulted in dehydration from structure 2 to structure 1 after 1 week, but the material still remained as structure 2 when stored at 40° C./75% relative humidity and under natural light.

6) 8-week stability studies.

An 8 week stability study showed that the pure structure 1 material exhibited poor physical stability at 40° C./75% RH, with the material converting to a hydrated structure 2 after the first (two week) time point. Although the study was continued on the original samples, the material undergoing stability testing from the 2 week time point was pattern 2 and not pattern 1 due to the poor stability of pure pattern 1 at this temperature and humidity. No change in appearance was noted at any of the time points tested, despite the shape change identified by XRPD.

In contrast, structure 2 showed good physical stability with no change in shape observed during the 8 week stability evaluation. The appearance of the material of structure 2 did not change at any stage of the experiment. Both pure structure 1 and structure 2 were found to have high chiral purity at the start of the 8 week evaluation and it remained high in all samples throughout the experiment.

7) One day stability study.

A 1-day stability evaluation confirmed that the material of pure structure 1 readily converts to structure 2 under high humidity conditions. XRPD analysis confirmed that shape change occurred after only 2 hours at 40°C/75% RH.

8) Estimation of pH solubility.

Solubility pH evaluation for both structure 1 and structure 2 showed that dissolution occurs after addition of 3 volumes of buffer at all pH values tested (pH 1; 4; 6.8 and 7.2). The solubility of structure 1 and structure 2 was estimated as 500>x>333.3 mg/ml.

e) Conclusion.

Longer 8-week studies of the stability of the structure of 2 CHP showed good physical stability at 40°C/75% relative humidity. XRPD analysis confirmed that pattern 2 was dominant throughout the 8 week trials. Eight-(8)-week stability tests of pure structure 1 CHP showed poor physical stability at 40° C./75% relative humidity. Structure 2 is the most appropriate form of CGP for further development.

Example 7 Stability Test

Purpose: to store and generate drug substance stability data, GMP mode.

Packaging: The sample was packaged at each time point in double anti-static LDPE bags, each secured with a tie. Samples packaged for the same storage conditions were stored together inside a fiber drum and covered with a metal lid. All packages were prepared in the same way and labeled. Detailed information about the description of the packaging material is presented in Table. 36.

Table 36. Description of packaging material

Item Manufacturer Series No. Size LDPE bag with antistatic properties Beyers Plastics N.V. 18031413 Length * Width * Thickness, cm = 22*10*0.0125 fiber drum VWR International VWR Part No: 16465-149 Inner length * inner diameter = 19.1 * 7.9 cm

Note: An anti-static LDPE bag was provided by STA and is cut into small pieces and heat-sealed before being used in stability tests.

Time zero will be the day the samples are placed in storage. Initial test results were derived from release testing, provided samples were stored within 30 days of release testing date, otherwise the original test will be repeated. The stability test protocol is presented in table. 37.

Table 37 Stability Protocol

Storage conditions Initial* 1M gp 6M 12 M 24 M 36 m 48 M Reserve bags Total bags 25±2°C/60±5%RH XY - X X XY XY XY XY 3 9 40±2°C/75±5%RH X X XY - - - - 2 5

Subject of analysis.

X = take a sample and test for appearance, quantity and impurities by HPLC, chiral forms and water content.

Y=take samples and test by XRPD.

-=not planned.

Test requirements: 22.5 g sample total (21 g sample total if no initial test required).

1.5 g for initial analysis if needed.

The 21 g sample will be divided into 14 parts, 1.5 g for each part, the sample for each part must be weighed in double bags of antistatic low density polyethylene (LDPE), tied with strings. Stability samples for each storage condition are stored together inside a fiber drum covered with a metal lid and then stored at 25±2°C/60±5% RH (9 bags) and 40±2°C/75±5%RH (5 packages), respectively.

Sampling time.

Samples stored no more than 6 months were taken as scheduled or within 2 working days after the scheduled time. Samples stored for more than 6 months were taken +2 business days from the scheduled date.

- 51 042744

Limitation of the period of storage and testing of the sample.

Samples from stability testing stored for one month to one year and more than one year were analyzed and completed within 10 and 20 working days after the samples were removed from the stability chamber, respectively. If time zero testing was performed, analyzes were performed within 10 working days of the start of the study.

Detailed information on the stability of structure 2 is presented in table. 38 and 39.

Table 38. Stability test results after 6 months at 25°C/60% RH

Project number: NMP-20180620 Stability Protocol No.: ST-180726100-H-01, Revision 00 Stability Sample/ Lot No.: С180726100-Н Series No: С180726100- H18001 Specification: ASU-SP-C180726100-H-1, Revision 00 Stability test conditions: 25±2°C/60±5%RH; 40±2°C/75±5%RH start date stability tests: September 28, 2018 Report No: RP-C180726100-H-03, Revision 00 Subject of test Analysis method Acceptance Criteria Initial* ¹ 25±2°C/60±5%RH ZM 6M Appearance GM-036-AT White or almost white powder almost white powder almost white powder almost white powder Quantitative content of io HPLC* AM-S180726100- H-01 Not less than 95.0% 103.4% 103.3% 103.4% Impurities, HPLC* H-His-OH <1.0% 0.07% 0.08% 0.08% Individual unspecified impurities <1.0% Note each impurity > 0.05% io ROV or ID if known Each individual impurity <0.05% Each individual impurity <0.05% Each individual impurity <0.05% chiral forms, HPLC* AM-S180726100n-oz Chiral purity: not less than 98.0% Chiral impurities: DL, LD, DD Optical isomers: <1.0% Each is noted: >0.1% impurities 100.0% 100.0% 100.0% (D,L):H/O (D,L):H/O (D,L):H/O (L,D):H/O (L,D):H/O (L,D):H/O (D,D):H/O (D,D):H/O (D,D):H/O Water content (CF) GM-016-KFT (Solvent: MeOH; Titration reagent: Mixture Hydranal #5; Reporting result 7.4% 7.1% 7.2%

Sample weight: 0.1 XRPD* GM-017-XRPD Corresponds to the standard pattern of Structure 2 (Series No.: PS03027-15-H) Corresponds to the standard pattern of Structure 2 (Series No.: PS03027-15-H) Not applicable Corresponds to the standard pattern of Structure 2 (Series No.: PS03027-15-H)

*

Claims (22)

Table 39. Stability result after 6 months at 40°C/75% RH Project number: NMP-20180620 Stability Protocol No.: ST-180726100-H-01, Revision 00 Stability Sample/ Lot No.: С180726100-Н Series No: С180726100- H18001 Specification: ASU-SP-C180726100-H-1, Revision 00 Stability test conditions: 25±2°C/60±5%RH; 40±2°C/75±5%RH Stability test start date: September 28 2018 Report No: RP-C180726100-H-03, Revision 00 Test item Analysis method Acceptance criteria Initial* ¹ 40±2°C/75±5%RH 1M ZM 6M Appearance GM-036-AT White or almost white powder Almost white powder Almost white powder Almost white powder Almost white powder Quantitative content by HPLC* AMC180726100-N- 01 Not less than 95.0% 103.4% 103.2% 103.3% 102.4% Impurities, HPLC* H-His-OH <1.0% 0.07% 0.08% 0.07% 0.06% Individual unspecified impurities <1.0% Note each impurity >0.05% by RVA or ID if known Each individual impurity <0.05% Each individual impurity <0.05% Each individual impurity <0.05% C16081735-Disomer: 0.05% Chiral forms, HPLC* AMCI80726100-H- 03 Chiral purity: not less than 98.0% Chiral impurities: DL, LD, DD Optical isomers: <1.0% Note each: >0.1% impurities 100.0% 100.0% 100.0% 99.9% (D,L):H/O (D,L):H/O (D,L):H/O (D,L):H/O (L,D):H/O (L,D):H/O (L,D):H/O (L,D):0.05% (D,D):H/O (D,D):H/O (D,D):H/O (D,D):H/O Water content (CF) GM-016-KFT (Solvent: MeOH; Titration reagent: Hydranal No. 5 mixture; Sample weight: 0.1 g Reporting result 7.4% 7.2% 7.2% 7.2% XRPD* GM-017-XRPD Corresponds to the standard pattern of Structure 2 (Series No.: PS03027-15-H) Corresponds to the standard pattern of Structure 2 (Series No.: PS03027-15-H) Not applicable Not applicable Corresponds to the standard pattern of Structure 2 (Series No.: PS03027-15-H) The result for the stability samples met the specification acceptance criteria. A typical overlay chromatogram of a blank, a resolving solution, and a sample solution (6 M) for testing for impurities are shown in FIG. 112. The embodiments described in this application are intended to be exemplary. Those skilled in the art will appreciate that changes and modifications may be made without departing from the scope of the invention as encompassed by the following claims. CLAIM 1. Cyclo(-His-Pro) hydrated compound characterized by an X-ray powder diffraction (XRPD) diffraction pattern containing peaks at 13.7; 17 and 27.3 degrees (±0.2° 2θ). oofs* ⁿ *° o 2. The compound of claim 1, wherein the XRPD pattern further includes a peak at 10 degrees (±0.2° 2θ). 3. The compound of claim 1, wherein the XRPD pattern is substantially the same as that shown in FIG. 2(b). 4. The compound of claim 1 having a differential scanning calorimetry (DSC) endotherm at a temperature of about 75 to 100°C. 5. The compound of claim 1 wherein the onset of the DSC exotherm is at a temperature of about 115 to 120°C. - 53 042744 6. The compound of claim 1 having a DSC thermogram having two endothermic peaks peaking at about 100°C and about 170°C. 7. A compound according to claim 1, characterized by at least two of the following: (a) an x-ray powder diffraction pattern containing at least two peaks from the following list: 10; 13.7; 17; 18.1; 20.2 and 27.3 degrees (±0.2° 2θ); (c) birefringence with fragmented rod-like morphology as analyzed by polarized light microscopy; (d) an initial weight loss of 6.5% (0.9 water equivalents) followed by decomposition of the sample at a temperature of about 280°C when tested by thermogravimetric analysis; (e) an endotherm with an onset of 99°C and a peak at 102°C in the first thermal cycle of DSC; (f) onset of dehydration at relative humidity (RH) below 10%, loss of approximately 6% mass from 10 to 0% RH (0.8 water equivalents), and hydration from 0 to 40% RH in a dynamic vapor sorption assay at 40°C ; and (h) initiating dehydration at below 20% relative humidity, losing about 7 wt% at 20 to 0% relative humidity (1.0 equivalent water), and rehydrating from 0 to 40% relative humidity in a dynamic vapor sorption assay at 60 °C. 8. The compound of claim 1 having a purity selected from the group consisting of at least 90%, at least about 95%, about 95%, about 96%, about 97%, about 98%, about 99%, and approximately 100%. 9. A compound according to claim 1 stable at room temperature for about 6 months, about 12 months, about 18 months, about 24 months, or about 36 months. 10. Pharmaceutical composition containing cyclo(-His-Pro) hydrate as defined in claim 1. 11. Pharmaceutical composition according to claim 10, containing from about 1 to 50%, from about 5 to 45%, from about 10 to 40%, from about 15 to 35%, from about 20 to 30%, from about 1 to 20 %, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% (by weight) cyclo(-His-Pro) hydrate. 12. Pharmaceutical composition according to claim 11, containing from about 1 to 20% (wt.%) cyclo(-HisPro) hydrate. 13. The pharmaceutical composition according to claim 11, further comprising, if necessary, an additional therapeutically active agent, a pharmaceutically acceptable carrier, or a combination thereof. 14. The pharmaceutical composition according to claim 10, which is a dosage form suitable for oral, topical, parenteral, intravenous, intradermal, intra-intestinal, rectal, intramuscular or intraperitoneal administration. 15. Pharmaceutical composition according to claim 10 formulated for parenteral administration by injection or continuous infusion. 16. The pharmaceutical composition according to claim 10, wherein the concentration of cyclo(-His-Pro) hydrate is from about 1 mg/l to 200 mg/ml. 17. Pharmaceutical composition according to claim 10 formulated as an oral unit dosage form. 18. Pharmaceutical composition according to claim 17, containing a unit dose of from about 1 to 100 mg of cyclo(-His-Pro) hydrate. 19. Pharmaceutical composition containing: a) substantially pure cyclo(-His-Pro) hydrate as defined in claim 1; b) at least one additional therapeutically active agent; And c) at least one pharmaceutically acceptable carrier. 20. The pharmaceutical composition of claim 19 wherein the active agent is a biomolecule, bioactive agent, protein, peptide, vaccine, adjuvant, imaging agent, polynucleotide, or metal. 21. The pharmaceutical composition according to claim 19, wherein the active agent is zinc. 22. The method for the synthesis of cyclo(-His-Pro) hydrate, which is defined in paragraph 1, including the steps: a) adding cyclo(-His-Pro) anhydrate to an ethanol/water mixture to form a mixture; b) heating the mixture to about 50°C to dissolve and obtain a solution; c) cooling the solution obtained in step (b) to about 35°C; d) seeding and holding for 2 to 3 hours; e) cooling to about 5°C; f) adding methyl tert-butyl ether over about 8 hours at about 5°C; g) stirring for about 8 to 10 hours at a temperature of about 5°C; h) filtration to obtain a wet product; And j) washing the wet product with ethanol/water/methyl tert-butyl ether and drying at about 35° C. under vacuum to form a cyclo(-His-Pro) hydrate compound. -