Salt forms of organic compound

Abstract

A salt compound of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid is disclosed. Also disclosed are methods for making the salt compound and formulations of the salt compound into dosage forms for clinical use.

Description

FIELD

The disclosure relates to various formulations and compositions comprising a salt compound useful as an inhibitor of FAF-1. Also disclosed are methods for preparing the salt compound.

BACKGROUND

Ischemia means a reduction in blood flow to organs, tissues or a region thereof, caused by contraction or occlusion of one or more blood vessels. Once ischemia occurs, even if reperfusion is prompt, it is followed by various sequelae that develop due to damage of nerve cells. Such ischemia frequently occurs in coronary artery diseases, cardiovascular diseases, angina pectoris, headache or other symptoms related to blood vessel occlusion or contraction, and eventually leads to irreversible damage, i.e., necrosis of cells or tissues.

Since ischemic diseases such as myocardial infarction, arrhythmia or heart failure caused by cell damage and dysfunction during ischemia-reperfusion have a high morbidity rate, a high mortality rate, and a low complete cure rate, basic research and clinical studies have been ongoing in this field for fifty years [Wang, Q. D. et al., Cardiovasc. Res. 55:25-37, 2002]. Especially, since ischemia-reperfusion injury involves various physiological mechanisms including change of metabolism, immune response and ion homeostasis, generation of oxygen free radicals and the like, studies are ongoing in various fields related to immune modulators, cell death suppressors, ion channel modulators, etc. [Hearse, D. J. et al., Mol. Cell. Biochem. 186:177-184, 1998]. Based on such mechanistic research, there have so far been developed a number of therapeutics and surgical operations focused on novel acting sites, but no technique for protecting cardiomyocytes from ischemia-reperfusion injury has yet been commercialized. Therefore, there is a need for an agent for preventing and treating ischemic heart diseases or a heart protecting agent, which can delay the progress of ischemic damage of cardiomyocytes and reduce reperfusion-induced injuries.

In addition, it has become plain that if ischemia is relieved by recovery of blood flow, the generation of reactive oxygen species (ROS) is accelerated, which causes a remarkable decrease of glutathione and brings about more serious diseases. Similar diseases are observed when blood flow stops or recovers during various techniques of transplant surgery of various kinds of organs such as heart, liver, lung, pancreas or blood vessels, and will be a problem in incising and removing an organ as well. Reactive oxygen and reactive free radicals assumed to cause diseases are detected in the cytoplasm and organelles of cells of tissues, especially in mitochondria producing ATP as a main energy source of a cell. In mitochondria, it is observed that the above reactive molecules are mainly released through a respiratory chain, and their concentration is significantly increased during ischemia-reperfusion.

In this regard, since ischemia leads to cell death or necrosis of cells, and especially cell death occurring after reperfusion is a main cause of tissue damage, ischemic cell death is a cause for various ischemic diseases, for example brain ischemia, heart ischemia, diabetic cardiovascular disease, heart failure, myocardial hypertrophy, retinal ischemia, ischemic colitis and ischemic acute renal failure.

In brain ischemia, the depletion of an energy source due to the reduction of blood supply induces ischemic cell death. Then, the ischemic cell death excessively activates a cell membrane receptor, which is followed by various biochemical alterations including accumulation of glutamic acid and calcium, respectively outside and inside of cells, and damage of lipids, proteins and nucleic acids, and finally leads to brain tissue injury (Liu, P. K., J. Biomed. Sci. 10:4-13, 2003; Lipton, P., Physiol. Rev. 79:1431-1568, 1999; and Renolleau, S. et al., Stroke 29:1454-1460, 1998).

In cases of myocardial infarction, heart failure and arrhythmia as ischemic heart diseases, it has been reported that ischemic cell death occurs by activation of lipid enzymes triggering damage to cell membranes, and subsequent changes of pH and calcium transport [Ferrari, R. Rev. Port. Cardiol. 5:7-20, 2000; Webster, K. A. et al., J. Clin. Invest. 104:239-252, 1999; Katz, A. M. et al., J. Mol Cell. Cardiol. 2:11-20, 1985; and Vandeplassche, G. et al., Basic Res. Cardiol. 85:384-391, 1990]. In retinal ischemia, it has been known that cell death of retinal cells mediated by glutamate is mediated by ischemic cell death [Napper, G. A. et al., Vis. Neurosci. 16:149-158, 1999]. Insufficient blood supply to colon causes ischemic cell death, and then, occlusive injury of arteries due to cell necrosis and hemodynamic disorders lead to ischemic colitis as an ischemic disease [Saegesser, F. et al., Pathobiol. Annu. 9:303-337, 1979].

Meanwhile, Minocycline, which is one of the tetracycline antibiotics inhibiting ischemic cell death, has been known to be effective in ischemic diseases such as cerebral infarction [Yrjanheikki, J. et al., Proc. Natl. Acad. Sci. USA 96:13496-13500, 1999], myocardial infarction [Scarabelli, T. M. et al., J. Am. Coll. Cardiol. 43:865-874, 2004] and an ischemic acute renal failure [Wang, J. et al., J. Biol. Chem. 279:19948-19954, 2004], suggesting that ischemic cell death is a cause of the above diseases.

Further, it has been known that damage or cell death of nerve cells induced by ischemia is a main cause of various nervous system diseases such as Alzheimer's disease, Parkinson's disease, glaucoma and diabetic neuropathy, and of pathologies resulting from stroke, head trauma, neonatal hypoxia, [G. J. Zoppo et al., Drugs 54, 9 (1997); I. Sziraki et al., Neurosci. 85, 1101 (1998)].

SUMMARY

A salt compound having the formula (2) (below) is disclosed.

In a salt compound of the Formula 2:

n is 1, 2 or 3;

m is from 0 to 3 and can be a non-integer, m is typically 0, 0.5, 1, 2, or 3;

“Sol” is a solvent molecule and can be, for example, water or C2-C4 alcohol; and

X+ is a cation, and can be, for example, a potassium ion, a sodium ion, a calcium ion, magnesium ion, ammonium ion or a substituted ammonium ion.

A salt compound of the Formula 2 can be prepared by treating the free base or zwitterion of Compound 1 (described below) with, for example, potassium hydroxide, sodium hydroxide, L-arginine, calcium hydroxide, N,N,N-trimethylglycine, ammonium hydroxide, magnesium hydroxide, choline, diethylamine, L-lysine, N,N′-dibenzylethylenediamine, M-ethylglucamine, calcium acetate, 1-(2-hydroxyethyl)pyrrolidine, N-(phenylmethyl)benzeneethaneamine, ammonia, magnesium acetate, N-methylglucamine, tromethamine, 4-(2-hydroxyethyl)morpholine, 2-(diethylamino)ethanol, or 2-dimethylamino-ethanol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows XRPD diffraction of samples prepared with potassium hydroxide in different solvents. 1—from 4-methyl-pentan-2-one, 2—from diisopropyl ether, 3—from ethanol, 4—free acid.

FIG. 2 shows XRPD diffraction of samples prepared with sodium hydroxide in different solvents. 1—from ethanol, 2—from diisopropyl ether, 3—from 4-methyl-pentan-2-one, 4—free acid.

FIG. 3 shows XRPD diffraction of samples prepared with L-arginine in different solvents. 2—from diisopropyl ether, 3—from 4-methyl-pentan-2-one, 4—free acid.

FIG. 4 shows XRPD diffraction of samples prepared with calcium hydroxide in different solvents. 1—from 4-methyl-pentan-2-one, 2—from diisopropyl ether, 3—ethanol, 4—free acid.

FIG. 5 shows XRPD diffraction of samples prepared with N,N,N-trimethylglycine indifferent solvents. 1—from 4-methyl-pentan-2-one, 2—from diisopropyl ether, 3—from ethanol, 4—free acid.

FIG. 6 shows XRPD diffraction of samples prepared with ammonium hydroxide in different solvents. 1—from 4-methyl-pentan-2-one, 2—from diisopropyl ether, 3—from ethanol, 4—free acid.

FIG. 7 shows XRPD diffraction of samples prepared with magnesium hydroxide in different solvents. 1—from 4-methyl-pentan-2-one, 2—from diisopropyl ether, 3—from ethanol, 4—free acid.

FIG. 8 shows XRPD diffraction of samples prepared with choline in different solvents. 1—from 4-methyl-pentan-2-one, 2—from diisopropyl ether, 3—from ethanol, 4—free acid.

FIG. 9 shows XRPD diffraction of samples prepared with diethylamine in different solvents. 1—from 4-methyl-pentan-2-one, 2—from diisopropyl ether, 3—from ethanol, 4—free acid.

FIG. 10 shows XRPD diffraction of samples prepared with L-lysine in different solvents. 1—from 4-methyl-pentan-2-one, 2—from diisopropyl ether, 3—from ethanol, 4—free acid.

FIG. 11 shows XRPD diffraction of samples prepared with N,N′-dibenzylethylenediamine in different solvents. 1—from 4-methyl-pentan-2-one, 2—from diisopropyl ether, 3—from ethanol, 4—free acid.

FIG. 12 shows XRPD diffraction of samples prepared with N-ethylglucamine in different solvents. 1—from 4-methyl-pentan-2-one, 2—from diisopropyl ether, 3—from ethanol, 4—free acid.

FIG. 13 shows XRPD diffraction of samples prepared with calcium acetate in different solvents. 1—from 4-methyl-pentan-2-one, 2—from diisopropyl ether, 3—from ethanol, 4—free acid.

FIG. 14 shows XRPD diffraction of samples prepared with N-(phenylmethyl) benzeneethaneamine in different solvents. 1—from 4-methyl-pentan-2-one, 2—from diisopropyl ether, 3—from ethanol, 4—free acid.

FIG. 15 shows XRPD diffraction of samples prepared with ammonia in different solvents. 1—from 4-methyl-pentan-2-one, 2—from diisopropyl ether, 3—from ethanol, 4—free acid.

FIG. 16 shows XRPD diffraction of samples prepared with magnesium acetate in different solvents. 1—from 4-methyl-pentan-2-one, 2—from diisopropyl ether, 3—from ethanol, 4—free acid.

FIG. 17 shows XRPD diffraction of samples prepared with N-methylglucamine in different solvents. 1—from 4-methyl-pentan-2-one, 2—from diisopropyl ether, 3—from ethanol, 4—free acid.

FIG. 18 shows XRPD diffraction of samples prepared with tromethamine in different solvents. 1—from 4-methyl-pentan-2-one, 2—from diisopropyl ether, 3—from ethanol, 4—free acid.

FIG. 19 shows XRPD diffraction of samples prepared with 4-(2-hydroxyethyl)morpholine in different solvents. 1—from 4-methyl-pentan-2-one, 2—from diisopropyl ether, 3—from ethanol, 4—free acid.

FIG. 20 shows PLM analysis of potassium salt Formula 2 from a second preparation of KM-819.

FIG. 21 shows TGA analysis of Potassium salt Formula 2 from a second preparation of KM-819.

FIG. 22 shows Differential Scanning calorimetery (DSC) analysis of potassium salt Formula 2 from a second preparation of KM-819.

FIGS. 23A-23E shows HSM analysis of potassium salt Formula 2 from a second preparation of KM-819. 23A: potassium salt initial condition, 23B: 127° C.—loss of birefringence, 23C: 154° C.—initial melt, 23D: 212° C.—secondary melt, 23E: re-crystallisation.

FIG. 24 shows Fourier Transform Infra-Red (FT-IR) analysis of potassium salt Formula 2 from a second preparation of KM-819.

FIG. 25 shows Particle Size Distribution (PSD) of potassium salt Formula 2 from a second preparation of KM-819.

FIG. 26 shows a DVS change in mass plot of potassium salt Formula 2 from a second preparation of KM-819.

FIG. 27 shows a DVS isotherm plot of potassium salt Formula 2 from a second preparation of KM-819.

FIG. 28 shows PLM analysis of sodium salt from a second preparation of KM-819.

FIGS. 29A-29C show HSM analysis of sodium salt from a second preparation of KM-819. 29A—salt as prepared, 29B—at 136° C.—melt, 29C—re-crystallization.

FIG. 30 shows TGA analysis of sodium salt from a second preparation of KM-819.

FIG. 31 shows DSC analysis of sodium salt from a second preparation of KM-819.

FIG. 32 shows FT-IR analysis of sodium salt from a second preparation of KM-819.

FIG. 33 shows Sympatec PSD analysis of sodium salt from a second preparation of KM-819.

FIG. 34 shows a DVS change in mass plot of a sodium salt from a second preparation of KM-819.

FIG. 35 shows a DVS isotherm plot of sodium salt from a second preparation of KM-819.

FIG. 36 shows PLM analysis of a diethylamine salt Formula 2 from a second preparation of KM-819.

FIG. 37 shows TGA analysis of a diethylamine salt Formula 2 from a second preparation of KM-819.

FIG. 38 shows DSC analysis of a diethylamine salt Formula 2 from a second preparation of KM-819.

FIGS. 39A-E show HSM analysis of a diethylamine salt Formula 2 from a second preparation of KM-819. 39A—diethylamine salt as prepared, 39B—at 154° C.—initial melt, 39C—at 200° C.—particle movement, 39D—at 209° C.—secondary melt, 39E—re-crystallization.

FIG. 40 shows FT-IR analysis of a diethylamine salt Formula 2 from a second preparation of KM-819.

FIG. 41 shows Sympatec PSD of a diethylamine salt Formula 2 from a second preparation of KM-819.

FIG. 42 shows a DVS change in mass plot of a diethylamine salt Formula 2 from a second preparation of KM-819.

FIG. 43 shows a DVS isotherm plot of a diethylamine salt Formula 2 from a second preparation of KM-819.

FIG. 44 shows an alignment of the five XRPD diffraction patterns of salts obtained during the screen. Spectra are aligned by the 2-theta scale.

FIG. 45 shows an alignment of five distinct 1H NMR analyses observed in samples from the salt screen. The spectra are aligned via the DMSO standard peak. Pattern numbering corresponds to the numbering of the XRPD patterns, thus salts exhibiting the XRPD pattern 1 will often exhibit NMR pattern 1 as well.

DETAILED DESCRIPTION

Aminopyrazole derivatives are disclosed that inhibit ischemic cell death, and thus can be used as agents for preventing and treating ischemic diseases such as brain ischemia, heart ischemia, diabetic cardiovascular disease, heart failure, myocardial hypertrophy, retinal ischemia, ischemic colitis, ischemic acute renal failure, stroke, head trauma, Alzheimer's disease, Parkinson's disease, neonatal hypoxia, glaucoma and diabetic neuropathy, which are mediated by ischemic cell death, and as an agents for protecting organs during transplant procedures.

Compound 1 (KM-819) is a novel aminopyrazole derivative useful for the treatment of Parkinson's disease. The compound 1 (KM-819) disclosed can be synthesized as described by WO2008/051047 (hereby incorporated by reference in its entirety and for all purposes), providing a white, crystalline powder. Initial analysis of the compound 1 (KM-819), as the free acid or zwitterion, was performed to both understand more about the material and provide baseline data so that comparisons can be made between the compound 1 (KM-819) and any salts that are prepared.

One of ordinary skill in the art understands that the Compound 1 includes both a carboxylate group that can form an anion, and nitrogen centers that can form cationic quaternary amines. Thus, “Compound 1” can refer to either the free acid, or to zwitterionic forms of the compound, depending upon the pH of a solution of Compound 1.

Solubility assessment of the Compound 1 (KM-00819) was performed using water and a diverse range of organic solvents. A list of solvents suitable for use during a salt screen was determined from this assessment. At the completion of the solvent screen 1.1 eq of NaOH(aq) was added to each of the samples in order to test the ability of the samples to generate a salt form. Upon addition of the hydroxide, cloudy precipitates were formed from some samples indicating that salt formation may have occurred, and on XRPD analysis of solids isolated from salt formation, 5 distinct diffraction patterns were observed. (See, e.g., Example 8 and FIGS. 10-19 and 44.) 1H NMR analysis of samples showing diffraction patterns was used to determine which solvents would be the most suitable for use in preparing salt forms of KM-819 as explained below. FIG. 45 shows an alignment of representative 1H NMR analyses of samples from the salt screen.

Salt Screen

The salt screen was performed using approximately 25 mg of the compound 1 (KM-819) per experiment with 3 solvents and 22 bases, which were added in a ratio of 1:1.1 (free acid:base). Upon preparation the samples were matured for 5 days prior to filtration and analysis by XRPD. In instances when the salt remained completely soluble the solvent was slowly evaporated from the sample.

Aqueous solubility assessment of the salts exhibiting novel XRPD patterns was performed and any that showed complete or partial dissolution after being shaken overnight at 50° C. (at a concentration of 1.25 mg/ml) were further analyzed by 1H NMR. Some salts exhibited polymorphism as shown by differences in XRPD pattern and 1H NMR chemical shifts.

Disclosed herein are new pharmaceutically acceptable solid forms of KM-819 and salts thereof and processes of their preparation. These forms can be used to prepare salts or base and prepare formulations thereof for clinical use.

Disclosed herein are new pharmaceutically acceptable salts of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid (KM-819) in crystalline or amorphous form and methods of their production. These salts can be used to prepare other salt or free base forms of KM-819 and to prepare formulations thereof for clinical use.

The present invention relates to salt and/or solvate (hydrate) compounds according to Formula 2 below.

Structure of salt of KM-819 (Formula 2 or Salt Compound 2)

In Formula 2, n is an integer 1, 2 or 3;

m is from 0 to 3 and can be a non-integer, for example 0.5 or 1.5;

“Sol” is a solvent molecule and can be, for example, water or C2-C4 alcohol; and

X+ is a cation, and can be, for example, a potassium ion, a sodium ion, a calcium ion, magnesium ion, ammonium ion or a substituted ammonium ion.

A salt compound of the Formula 2 can be prepared by treating the free base or zwitterion of Compound 1 with, for example, potassium hydroxide, sodium hydroxide, L-arginine, calcium hydroxide, N,N,N-trimethylglycine, ammonium hydroxide, magnesium hydroxide, choline, diethylamine, L-lysine, N,N′-dibenzylethylenediamine, N-ethylglucamine, calcium acetate, 1-(2-hydroxyethyl)pyrrolidine, N-(phenylmethyl)benzeneethaneamine, ammonia, magnesium acetate, N-methylglucamine, tromethamine, 4-(2-hydroxyethyl)morpholine, 2-(diethylamino)ethanol, or 2-dimethylamino-ethanol.

In some instances of a salt compound of Formula 2 m can be 0.5 or 1. In some such instances, X+ can be potassium ion, sodium ion or quaternary methylamine or quaternary ethylamine.

Salt Compounds 2 can alternatively or additionally be in the form of solvates such as those including water, ethanol or diisopropyl ether, or a mixture of any two or three of them. The solvent molecule can be present in a non-integer ratio to either or both of water molecules and the compound 1 ion, for example 0.1, 0.2, or 0.5 solvent molecules per molecule of compound 1 ion. The solvent molecule can be present in an integer ratio to either or both of water molecules and the compound 1 ion, for example 1 or 2 solvent molecules per molecule of compound 1 ion.

Also disclosed is a process for preparing the salt compound of Formula 2:

wherein:

n is 1, 2 or 3;

m is 0 to 3;

X+ is a cation;

“Sol” is a solvent molecule;

• • the process comprising:
  • i) dissolving the free acid form of a compound of formula 2 in an organic solvent or in an organic solvent mixed with water;
  • ii) adding an excess over the stoichiometric amount of a base required to titrate the free acid compound of formula 2 to form a precipitate of the salt compound of formula 2; and
  • iii) collecting the precipitate to obtain the salt compound 2.

In such a process, the organic solvent can be 1,1-dimethoxyethane, acetonitrile, ethanol, 1,2-dichloroethane, benzonitrile, ethyl acetate, 1,4-dioxane, anisole, heptane, 2-butanol, cumene, hexane, 2-propanol, cyclohexane, isopropyl acetate, 4-methyl-pentan-2-one, dichloromethane, methanol, acetone, diisopropyl ether (DIPE), isobutyl acetate, tetralin, toluene, methylethyl ketone (MEK), N-methylpyrrolidone, tert-butylmethyl ether (TMBE), nitromethane, pyridine or tetrahydrofuran, or a mixture of any two or three of them.

In some implementations of the process, the organic solvent can be ethanol or diisopropyl ether (DIPE).

In some implementations of the process, the base can be sodium hydroxide, potassium hydroxide, magnesium hydroxide, magnesium acetate, ammonia, a salt of quaternary dimethylamine or a salt of quaternary diethylamine.

A combination of use of ethanol or DIPE as the organic solvent and sodium hydroxide, potassium hydroxide or a salt of quaternary dimethylamine or a salt of quaternary diethylamine as the base can also be used.

In some instances ethanol or DIPE is used as the organic solvent.

Water can be mixed with the organic solvent, and in some instances water can be mixed with ethanol or DIPE.

In any implementation in which water is mixed with an organic solvent, e.g. when water is mixed with a polar organic solvent, the ratio of water to polar organic solvent can range from 5:1 to 10:0.1.

The free base (or zwitterionic) Compound 1 can be dissolved in unbuffered water, a range of organic solvents, mixtures of organic solvents and mixtures of solvents with unbuffered water. The solvents assessed were 1,1-dimethoxyethane, acetonitrile, ethanol, 1,2-dichloroethane, benzonitrile, ethyl acetate, 1,4-dioxane, anisole, heptane, 2-butanol, cumene, hexane, 2-propanol, cyclohexane, isopropyl acetate, 4-methyl-pentan-2-one, dichloromethane, methanol, acetone, diisopropyl ether (DIPE), isobutyl acetate, tetralin, toluene, methylethyl ketone, N-methylpyrrolidone, tert-butylmethyl ether, nitromethane, pyridine, tetrahydrofuran. Results of example solubility tests are shown in Table 2 below.

Organic solvents can be used neat, or as a mixture of two or three or more organic solvents. Water alone can be used as a solvent for the free base (or zwitterionic) Compound 1, or water can be mixed together with one or more organic solvents. Preferably neat polar organic solvent or solvent mixture, or an aqueous mixture of a polar organic solvent, is used to dissolve the free base or zwitterionic Compound 1.

In the instance of a binary mixture, a ratio of water to organic solvent (preferably a polar organic solvent) in a solvent mixture can be from 1:10 to 1:0.1, or from 1:5-1:0.1, or from 1:2-1:0.1, or from 1:2-1:0.5, or about 1:1.

Salt Formation

The general methods for preparing the salt compounds of this disclosure are illustrated in the following Scheme.

Scheme 1 shows the synthesis of salt forms following a general route that utilizes well-established chemistry.

The free acid Compound 1 is weighed and added to a vessel and then solvent is added to the vessels. About 1.1 eq of the base prepared as a 1M stock solutions in solvent is then added. Clouding of the sample upon base addition indicates that salt formation is occurring. Post-maturation (standing for several hours at room temperature), the samples were filtered and dried in vacuo and then characterized by various methods.

The base used for salt formation can be potassium hydroxide, sodium hydroxide, L-arginine, calcium hydroxide, N,N,N-trimethylglycine, ammonium hydroxide, magnesium hydroxide, choline, diethylamine, L-lysine, N,N′-dibenzylethylenediamine, N-ethylglucamine, calcium acetate, 1-(2-hydroxyethyl)pyrrolidine, N-(phenylmethyl)benzeneethaneamine, ammonia, magnesium acetate, N-methylglucamine, tromethamine, 4-(2-hydroxyethyl)morpholine, 2-(diethylamino)ethanol or 2-dimethylamino-ethanol.

Many organic compounds exist in different solid forms that can be amorphous or in a crystalline state.

The ability of a compound to crystallize in different crystalline phases is called polymorphism. The term polymorph may include the amorphous phases (disordered), hydrates (water presents in the crystal lattice) and solvates (solvents other than water present in the crystal lattice).

Different crystalline modifications have different crystal structures and different free energies, therefore polymorphs exhibit different physico-chemical properties such as melting point, density, solubility, chemical stability and finally, bioavailability.

Examples of preferred salts of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid of the salt Compound 2 are:

The salt of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid and potassium hydroxide;

The salt of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid and sodium hydroxide;

The salt of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid and ammonium hydroxide;

The salt of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid and choline;

The salt of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid and diethylamine;

The salt of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid and L-lysine;

The salt of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid and N,N′-dibenzylethylenediamine;

The salt of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid and N-ethylglucamine;

The salt of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid and calcium acetate;

The salt of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid and N-(phenylmethyl)benzeneethaneamine;

The salt of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid and magnesium acetate;

The salt of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid and N-methylglucamine;

The salt of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid and tromethamine; and

The salt of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid and 4-(2-hydroxyethyl)morpholine.

Many solids isolated from the salt forming step exhibited diffraction patterns in XRPD analysis (indicating the solid is crystalline), and some exhibited different diffraction patterns from that of the free acid Compound 1. 1H NMR analysis of crystalline solids in many cases showed that the —COOH group in the Compound 1 had ionized.

Preferable solvents suitable for use in salt formation are those that completely dissolve the free acid Compound 1 and preferable bases for use in preparing salt Compound 2 from Compound 1 are those in which the base completely replaces the carboxylate hydrogen of Compound 1.

Abbreviations

λ	Wavelength	Ltd	Limited
%	Percentage	M	Moles
Θ	Theta	mA	Milliamp
% RH	Percentage relative	MeOH	Methanol
	humidity
% RSD	Percentage relative	mg	Milligram
	standard deviation
° C.	Degree centigrade	mg/mL	Milligrams per
			milliliter
° C./min	Degree centigrade per	mJ	Millijoule
	minute
μL	Microliters	mL	Milliliter
μm	Microns	mL/min	Milliliter per minute
API	Active pharmaceutical	mM	millimolar
	ingredient
ATR	Attenuated total	mm	Millimeter
	reflection
Ca.	Approximately	mm/s	Millimeter per second
CCD	Charge-coupled device	MP	Megapixels
cm−1	Wave number	mW	Milliwatt
d-	Duterated dimethyl	nm	nanometer
DMSO	sulfoxide
DIPE	Diisopropyl ether	PC	Personal computer
DSC	Differential scanning	pH	Potential of Hydrogen
	calorimetry
DVS	Dynamic vapour	PLM	Polarised light
	sorption		microscopy
eq	Equivalents	PPM	Parts per million
FASSIF	Fasted state simulated	QNP	Quadruple nucleus
	intestinal fluid		probe
FESSIF	Fed state simulated	RT	Room temperature
	intestinal fluid
FT-IR	Fourier transform	SCXRD	Single crystal
	infrared spectroscopy		X-ray diffraction
g	Gram	Std	Standard deviation
		Dev
h	Hours	TGA	Thermogravametric
			analysis
1H NMR	Proton nuclear	VMD	Volume mean
	magnetic resonance		diameter
HPLC	High performance	Wt %	Weight Percentage
	liquid chromatography
HSM	Hot stage microscopy	X10	10% of particles
ICP	Inductively coupled	X50	50% of particles
	plasma
J/g	Joule/gram	X90	90% of particles
kV	Kilovolt	XRPD	X-ray powder
			diffraction

EXAMPLES

The following examples describe preparation and detailed characterization of representative embodiments.

4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid (Compound 1) was synthesized as described by WO2008/051047 to obtain a white, crystalline powder.

Samples for the Examples were synthesized by the disclosed method (Example 1, 2, 4, 5) and analyzed by XRPD, 1H NMR, HPLC chemical purity and solubility.

Instrumentation

Perkin Elmer PYRIS 1 DSC using 40 μL aluminium pans (vented). Data collections and analysis was performed using the Perkin Elmer control and analysis software v11.0.2.0468

Bruker 400 Avance spectrometer equipped with a 5 mm QNP probe. Instrument control and data collection was performed using Top Spin v1.3 with the analysis being performed using ACD Laboratories 1D NMR processor v. 12.01.

Jasco 420 FTIR using attenuated total reflectance (ATR) module. Analysis and data collection was performed using the Jasco Spectra Manager software v1.51.00 (Build 1).

Olympus BX53 microscope equipped for polarised light microscopy with 6 objective lenses (2.5×, 4×, 10×, 20×, 40× and 100×) and 1/10λ wave plate. Sony ICX252 progressive scan interline 3.3MP CCD camera. The microscope was also equipped with a Linkam LTS420 heating/freezing stage.

PLM: Data analysis and image capture via Qcapture-Pro v7 imaging software.

HSM: Data analysis and image capture via Linksys 32DV temperature control and digital video capture software.

Bruker-AXS D8 Advance XRPD using 9 mm cavity and flat plate sample holders. Instrument control and data collection was performed using a PC equipped with Diffrac Plus XRD Commander control software v2.6.1 and analysis of the recorded data was performed Eva v18,0,0,0.

SMS DVS Intrinsic dynamic vapour sorption instrument using DVS-Intrinsic control software v1.0.6.0. Analysis of the data was performed using the DVS analysis suite v7.0.13.1 macro program embedded in Microsoft Excel. Analysis was performed as a wt % change from 0-90% RH with isothermal plots also being examined.

Perkin Elmer PYRIS 1 TGA using aluminium pans (vented) in ceramic crucibles. Data analysis and collection was performed using the Perkin Elmer control and analysis software v11.0.2.0468.

Thermo-Fisher iCAP 6500 ICP-OES using iTEVA software.

Metrohm 852 Titranto combined Volumetric and Coulometric KF unit. All samples were analyzed using volumetric Karl Fischer module.

Waters-Alliance 2695 HPLC spectrometer equipped with a PDA 2996 probe. System control and processing was performed with Empower 3 software Build 3471.

HeidolphTitramax 1000 with heating module.

Example 1

4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid potassium salt Ca. 25 mg of the compound 1 (KM-00819) was weighed into a 2 mL HPLC vial prior to the addition of 1500 μL. To the resulting slurries was added 1.1 eq of potassium hydroxide in 60 ul in water (to 1M concentration). The sample was placed on to a maturation cycle for 5 days using an 8 hour cycle (4 hours at RT followed by 4 hours at 50° C.). Post-maturation the sample was re-examined and then filtered and dried in vacuo.

Example 2: 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic Acid Calcium Salt

Ca. 25 mg of the compound 1 (KM-00819) was weighed into a 2 mL HPLC vial prior to the addition of 1500 μL of a solvent as set forth in Table 1. To the resulting slurries was added 1.1 eq of Calcium hydroxide (5.6 μg) as solid. The sample was placed on to a maturation cycle for 5 days using an 8 hour cycle (4 hours at RT followed by 4 hours at 50° C.). Post-maturation the sample was re-examined and then filtered and dried in vacuo.

Example 3: Additional salts of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic Acid

The following salts of compound were prepared by same the method as Example 1 or Example 2:

TABLE 1
Formation of salts
	Amount of
	Base
	Added
	(1M soln)		Synthetic Method
Salt Former	or solid	Solvent	(General Method)
Potassium hydroxide	60 μL	Ethanol	EXAMPLE 1
Sodium hydroxide	60 μL	Ethanol	EXAMPLE 1
L-Arginine	11.12 μg	Ethanol	EXAMPLE 2
Calcium hydroxide	5.6 μg	Ethanol	EXAMPLE 2
N,N,N-Trimethylglycine	60 μL	Ethanol	EXAMPLE 1
Ammonium hydroxide	60 μL	Ethanol	EXAMPLE 1
Magnesium hydroxide	4.6 μg	Ethanol	EXAMPLE 2
Choline	60 μL	Ethanol	EXAMPLE 1
Diethylamine	60 μL	Ethanol	EXAMPLE 1
L-Lysine	60 μL	Ethanol	EXAMPLE 1
N,N′-Dibenzylethylenediamine	60 μL	Ethanol	EXAMPLE 1
N-Ethylglucamine	60 μL	Ethanol	EXAMPLE 1
Calcium acetate	60 μL	Ethanol	EXAMPLE 1
1-(2-Hydroxyethyhpyrrolidine	60 μL	Ethanol	EXAMPLE 1
N-(Phenylmethyl)	60 μL	Ethanol	EXAMPLE 1
benzeneethaneamine
Ammonia	60 μL	Ethanol	EXAMPLE 1
Magnesium acetate	60 μL	Ethanol	EXAMPLE 1
N-Methylglucamine	60 μL	Ethanol	EXAMPLE 1
Tromethamine	60 μL	Ethanol	EXAMPLE 1
4-(2-hydroxyethyl)morpholine	60 μL	Ethanol	EXAMPLE 1
2-(Diethylamino)ethanol*	60 μL	Ethanol	EXAMPLE 1
2-Dimethylamino-ethanol*	60 μL	Ethanol	EXAMPLE 1
Potassium hydroxide	60 μL	Diisopropyl Ether	EXAMPLE 1
Sodium hydroxide	60 μL	Diisopropyl Ether	EXAMPLE 1
L-Arginine	11.0 μg	Diisopropyl Ether	EXAMPLE 2
Calcium hydroxide	5.2 μg	Diisopropyl Ether	EXAMPLE 2
N,N,N-Trimethylglycine	60 μL	Diisopropyl Ether	EXAMPLE 1
Ammonium hydroxide	60 μL	Diisopropyl Ether	EXAMPLE 1
Magnesium hydroxide	3.8 μg	Diisopropyl Ether	EXAMPLE 2
Choline	60 μL	Diisopropyl Ether	EXAMPLE 1
Diethylamine	60 μL	Diisopropyl Ether	EXAMPLE 1
L-Lysine	60 μL	Diisopropyl Ether	EXAMPLE 1
N,N′-Dibenzylethylenediamine	60 μL	Diisopropyl Ether	EXAMPLE 1
N-Ethylglucamine	60 μL	Diisopropyl Ether	EXAMPLE 1
Calcium acetate	60 μL	Diisopropyl Ether	EXAMPLE 1
1-(2-Hydroxyethyhpyrrolidine	60 μL	Diisopropyl Ether	EXAMPLE 1
N-(Phenylmethyl)	60 μL	Diisopropyl Ether	EXAMPLE 1
benzeneethaneamine
Ammonia	60 μL	Diisopropyl Ether	EXAMPLE 1
Magnesium acetate	60 μL	Diisopropyl Ether	EXAMPLE 1
N-Methylglucamine	60 μL	Diisopropyl Ether	EXAMPLE 1
Tromethamine	60 μL	Diisopropyl Ether	EXAMPLE 1
4-(2-hydroxyethyl)morpholine	60 μL	Diisopropyl Ether	EXAMPLE 1
2-(Diethylamino)ethanol*	60 μL	Diisopropyl Ether	EXAMPLE 1
2-Dimethylamino-ethanol*	60 μL	Diisopropyl Ether	EXAMPLE 1
Potassium hydroxide	60 μL	4-Methylpentan-2-one	EXAMPLE 1
Sodium hydroxide	60 μL	4-Methylpentan-2-one	EXAMPLE 1
L-Arginine	16.8 μg	4-Methylpentan-2-one	EXAMPLE 2
Calcium hydroxide	5.3 μg	4-Methylpentan-2-one	EXAMPLE 2
N,N,N-Trimethylglycine	60 μL	4-Methylpentan-2-one	EXAMPLE 1
Ammonium hydroxide	60 μL	4-Methylpentan-2-one	EXAMPLE 1
Magnesium hydroxide	3.8 μg	4-Methylpentan-2-one	EXAMPLE 2
Choline	60 μL	4-Methylpentan-2-one	EXAMPLE 1
Diethylamine	60 μL	4-Methylpentan-2-one	EXAMPLE 1
L-Lysine	60 μL	4-Methylpentan-2-one	EXAMPLE 1
N,N′-Dibenzylethylenediamine	60 μL	4-Methylpentan-2-one	EXAMPLE 1
N-Ethylglucamine	60 μL	4-Methylpentan-2-one	EXAMPLE 1
Calcium acetate	60 μL	4-Methylpentan-2-one	EXAMPLE 1
1-(2-Hydroxyethyhpyrrolidine	60 μL	4-Methylpentan-2-one	EXAMPLE 1
N-(Phenylmethyl)	60 μL	4-Methylpentan-2-one	EXAMPLE 1
benzeneethaneamine
Ammonia	60 μL	4-Methylpentan-2-one	EXAMPLE 1
Magnesium acetate	60 μL	4-Methylpentan-2-one	EXAMPLE 1
N-Methylglucamine	60 μL	4-Methylpentan-2-one	EXAMPLE 1
Tromethamine	60 μL	4-Methylpentan-2-one	EXAMPLE 1
4-(2-hydroxyethyl)morpholine	60 μL	4-Methylpentan-2-one	EXAMPLE 1
2-(Diethylamino)ethanol*	60 μL	4-Methylpentan-2-one	EXAMPLE 1
2-Dimethylamino-ethanol*	60 μL	4-Methylpentan-2-one	EXAMPLE 1
*Note: after 3 days maturation these samples were observed to be completely soluble at RT so were slowly evaporated over 3 days in an attempt to generate crystals.

Example 4: 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic Acid Sodium Salt

Ca. 1 g of the compound 1 was weighed into 3×100 mL vials prior to the addition of 60 mL of DIPE to each of the vessels. To the resulting suspension was added 1.1 eq of sodium hydroxide which had been prepared as 1M stock solution in water. On base addition, the reaction mixture was observed to become cloudier, which indicates that salt formation was occurring. Post-maturation (as in Example 1) the samples were filtered and dried in vacuo.

Example 5: 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic Acid Potassium Salt

The salt of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid and potassium hydroxide was prepared by using procedures analogous to those described in Example 4.

Example 6: 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic Acid and Diethylamine

The salt of 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic acid and diethylamine was prepared by using procedures analogous to those described in Example 4.

Example 7: 4-(2-((4-bromophenyl)thio)acetamido)-1-phenethyl-1H-pyrazole-3-carboxylic Acid and Diethylamine

Ca. 8 g of the free acid of Compound 1 was weighed into a 500 mL glass vessel prior to the addition of 480 mL of DIPE. To the resulting suspension was added 1.1 eq of diethylamine which had been prepared as 1M stock solution in DIPE. On base addition the sample was observed to settle out of the reaction mixture indicating that salt formation had occurred. Maturation of the sample was then performed for 4 days (8 hour cycles of 4 hours at 50° C. and 4 hours at RT). Post-maturation the sample was filtered and dried in vacuo at 40° C. for 2 days.

Example 8: Solubility in Various Solvents

Ca. 10 mg of the free acid of Compound 1 was weighed into 2 mL HPLC vials prior to the addition of solvent in 100-250 μL increments up to a maximum of 1750 μL. After each addition of solvent the samples were briefly shaken to encourage dissolution and visually inspected for signs of any remaining solid. Once the maximum volume of solvent had been added, 1.1 eq of 1M NaOH(aq) solution was also added, to give an indication as to which of the solvents would yield a suitable salt form. The samples were visually inspected prior to maturation for 2 days, after which all were inspected again. All the samples were then left to evaporate at RT to generate solid material.

Analysis of the recovered solid by XRPD post maturation showed there was a frequently observed diffraction pattern (Pattern 1) that is observed for the sodium salt prepared from most of the solvents tested. However, four additional patterns were also identified less commonly. Salts exhibiting these five patterns were further analysed by 1H NMR from which the following conclusions have been drawn:

pattern 1: Crystalline sodium salt (from the majority of solvents tested)

pattern 2: Solvate of the sodium salt (from 2-butanol and 2-propanol)

pattern 3: Possible new polymorph (from 4-methyl-pentan-2-one)

pattern 4: Sodium salt with unknown contamination (from acetone)

pattern 5: Hemi-ethanoate of the sodium salt (from ethanol).

The following list of peaks can be used to distinguish one of these patterns from another. Values of 2-theta are rounded to two decimal places.

Pattern 1 is characterized by peaks at 2-theta of 4.68, 6.54, 9.24, 10.30, 13.80, 14.54, 16.60, 18.48, 18.96, 20.60, 22.18, 23.04, 23.49, 24.83, 25.76, 26.15, 26.97, 27.72, 28.09, 28.91, 29.63, 30.71, 31.03, 31.41, 32.10, 32.45, 32.75, 33, 48, 33.76 and 34.74.

Pattern 2 is characterized by peaks at 2-theta of 7.00, 11.61, 15.75, 19.19, 20.30, 20.86, 23.19, 26.08, 26.72 and 29.29.

Pattern 3 is characterized by peaks at 2-theta of 6.71, 9.47, 10.59, 13.13, 14.06, 14.86, 16.30, 16, 90, 17.64, 18.81, 19.28, 20.92, 22.49, 23.39, 23.80, 24.73, 25.12, 26.05, 26.45, 27.27, 27.66, 28.35, 28.79, 29.20, 29.92, 31.00, 32.46, 34.01 and 35.09.

Pattern 4 is characterized by peaks at 2-theta of 3.76, 6.47, 7.46, 8.28, 8.63, 11.34, 14.49, 15.78, 18.96, 19.27, 19.97, 21.64, 22.16, 23.24, 25.67, 27.61, 29.77 and 33.27.

Pattern 5 is characterized by peaks at 2-theta of 5.30, 5.83, 7.09, 10.57, 10.97, 11.75, 13.02, 13.80, 17.38, 17.97, 18.23, 18.75, 21.26, 22.10, 23.13, 23.50, 25.17, 26.84, 27.39, 28.76, 29.14, 29.57, 30.05, 31.18, 32.15, 33.44, 35.06, 36.29 and 39.26.

Table 2 shows the results of the solubility screen.

TABLE 2
Solubility Screen
	Solvent	Observation on
	Addition	Addition of	Observation Post
Solvent	(μL)	1.1eq of NaOH	Maturation	XRPD Results
1,1-Dimethoxyethane	1750	White PPT	Clear liquid with fluffy	Crystalline-
		formed	solid & powered	Pattern 1
			sediment
1,2-Dichloroethane	1750	No change	Clear liquid with	Crystalline-
			gelatinous solid &	Pattern 1
			suspensions
1,4-Dioxane	750	No change	Clear liquid	No solids
2-Butanol	1750	No change	Clear liquid with fluffy	Crystalline-
			solid & powered	Pattern 2
			sediment
2-Propanol	1750	No change	Clear liquid & white	Crystalline-
			solid	Pattern 2
4-methyl-pentan-2-one	1750	No change	Clear liquid & white	Crystalline-
			solid	Pattern 3
Acetone	1750	Clear liquid &	Clear liquid & needles	Crystalline-
		white PPT		Pattern 4
Acetonitrile	1750	No change	Clear liquid &	Crystalline-
			gelatinous solid	Pattern 1
Benzonitrile	1750	No change	Hazy suspension &	Partially
			some solids	Crystalline
Anisole	1750	No change	White solid sediment	Crystalline-
			and & some	Pattern 1
			gelatinous solid
Cumene	1750	No change	Clear liquid & white	Crystalline-
			solid	Pattern 1
Cyclohexane	1750	No change	Hazy	Crystalline-
			suspension/clear	Pattern 1
			liquid
Dichloromethane	1750	No change	Clear liquid &	Crystalline-
			gelatinous solid	Pattern 1
Diisopropyl ether	1750	No change	Clear liquid & white	Crystalline-
			solid	Pattern 1
Ethanol	1750	No change	Clear liquid & white	Crystalline-
			solid	Pattern 5
Ethyl acetate	1750	No change	Clear liquid & white	Crystalline-
			solid	Pattern 1
Heptane	1750	No change	Hazy suspension &	Crystalline-
			white solid	Pattern 1
Hexane	1750	No change	Hazy suspension &	Crystalline-
			white solid	Pattern 1
Isopropyl acetate	1750	No change	Clear liquid & white	Crystalline-
			solid	Pattern 1
Methanol	1750	Clear liquid &	Clear liquid	No solids
		white PPT
Isobutyl Acetate	1750	No change	Clear liquid &	Crystalline-
			white solid	Pattern 1
Methylethyl ketone	1750	No change	Clear liquid &	Partially
			gelatinous solid	Crystalline-
				Pattern 1
Nitromethane	1750	No change	Yellow liquid & white	Crystalline-
			solid	Pattern 1
N-Methylpyrrolidone	250	No change	Pale yellow liquid	No solids
Pyridine	1750	No change	Pale yellow liquid	No solids
tert-Butylmethyl ether	1750	No change	Clear liquid & white	Crystalline-
			solid	Pattern 1
Tetrahydrofuran	750	No change	Clear liquid	No solids
Tetralin	1750	No change	Hazy suspension &	Partially
			white solid	Crystalline-
				Pattern 1
Toluene	1750	No change	Clear liquid &	Crystalline-
			gelatinous solid	Pattern 1
Water	1750	No change	Clear liquid &	Partially
			gelatinous solid	Crystalline

Example 9: Powder X-Ray Diffraction (XRPD) Analysis

Samples were prepared by coating them onto sample holders fitted with a zero background silicone wafer (5 1 0). Analysis was performed using a Cu Kα X-Ray source which operated at 40 kV at 40 mA and a LynxEye™ detector; all samples were analyzed over the range 2-40° 20.

XRPD analysis indicated that the salts had generated crystalline solids with novel crystalline patterns compared to the free acid Compound 1 with a further sample giving a partially crystalline pattern (see Table 3 and FIGS. 1-19 and 44).

TABLE 3
XRPD assessment on solid samples isolated from the salt screen
	From Ethanol XRPD	From DIPE XRPD	From MIBK XRPD
Salt Former	Analysis	Analysis	Analysis
Potassium hydroxide	Crystalline: Pattern 1	Crystalline:	Crystalline: Pattern 3
		Pattern 2	(similar to Pattern 2)
Sodium hydroxide	Crystalline: Pattern 1	Crystalline:	Crystalline: Pattern 2
		Pattern 2
L-Arginine	Oil	Starting material	Starting material
Calcium hydroxide	Starting material	Starting material	Partially crystalline
N,N,N-Trimethylglycine	Starting material	Starting material	Starting material
Ammonium hydroxide	Crystalline: Pattern 1	Crystalline:	Crystalline: Pattern 1
		Pattern 1
Magnesium hydroxide	Starting material	Starting material	Starting material
Choline	Partially crystalline:	Partially	Partially crystalline:
	Pattern 1 +	crystalline: Pattern	Pattern 1
	starting material	1
Diethylamine	Crystalline: Pattern 1	Crystalline:	Crystalline: Pattern 1 +
		Pattern 2	extra peak
L-Lysine	Starting material +	Crystalline:	Partially crystalline
	extra peak	Pattern 1
N,N'-	Crystalline: Pattern 1	Crystalline:	Crystalline: Pattern 1
Dibenzylethylenediamine		Pattern 1
N-Ethylglucamine	Crystalline: Pattern 1	Crystalline:	Partially crystalline:
		Pattern 2 (similar	Pattern 2
		to Pattern 1)
Calcium acetate	Crystalline: Pattern 1	Starting material +	Partially crystalline:
		extra peak	Pattern 2
1-(2-	Oil	Oil	Oil
Hydroxyethyl)pyrrolidine
N-(Phenylmethyl)	Crystalline: Pattern 1	Crystalline:	Partially crystalline:
benzeneethaneamine		Pattern 2 (similar	Pattern 2
		to Pattern 1)
Ammonia	Starting material	Starting material	Starting material
Magnesium acetate	Crystalline: Pattern 1	Starting material +	Partially crystalline:
		extra peaks	Pattern 2
N-Methylglucamine	Crystalline: Pattern 1	Crystalline:	Crystalline: Pattern 1
		Pattern 1
Tromethamine	Crystalline: Pattern 1	Crystalline:	Crystalline: Pattern 1
		Pattern 1
4-(2-	Crystalline: Pattern 1	Crystalline:	Crystalline: Pattern 1
Hydroxyethyl)morpholine		Pattern 1
2-(Diethylamino)ethanol	Oil	Oil	Oil
2-Dimethylamino-ethanol	Oil	Oil	Oil

Example 10: Aqueous Solubility

Aqueous solubility assessment was performed using Ca. 5 mg of each of the solids which had shown a unique pattern by XRPD. These samples were added to aliquots of deionized water up to a maximum of 4000 μL with the samples being shaken between additions to encourage dissolution prior to a visual inspection. It was observed that at room temperature none of the samples dissolved, however after shaking the samples overnight at 50° C. five samples were observed to have undergone complete dissolution and a further seven had partially dissolved.

Ca. 50 mg of each of the salts were weighed into a 2 mL HPLC vial prior to the addition of 1 mL of deionized water. The samples were then shaken for 24 h at 25° C. before being filtered into pre-weighed filter cartridges and dried overnight in vacuo and re-weighed, from this the solubility was calculated. The experiment was also repeated at 50° C. (see Table 4).

TABLE 4
Solubility assessment of selected samples from the salt screen
Salt Former	From Ethanol	from DIPE	from MIBK
Potassium hydroxide	Complete	Complete	Partial
	dissolution	dissolution	dissolution at
	at 50° C.	at 50° C.	50° C.
Sodium hydroxide	Complete	Complete	N/A
	dissolution	dissolution
	at 50° C.	at 50° C.
Ammonium hydroxide	Partial	N/A	N/A
	dissolution
	at 50° C.
Choline	N/A	N/A	No dissolution
			observed
Diethylamine	Partial	Complete	N/A
	dissolution	dissolution
	at 50° C.	at 50° C.
L-Lysine	N/A	Partial	N/A
		dissolution
		at 50° C.
N,N′-	N/A	N/A	No dissolution
Dibenzylethyl-			observed
enediamine
N-Ethylglucamine	Partial	Partial	N/A
	dissolution	dissolution
	at 50° C.	at 50° C.
Calcium acetate	No dissolution	N/A	N/A
	observed
N-(Phenylmethyl)	No dissolution	No dissolution	N/A
benzeneethaneamine	observed	observed
Magnesium acetate	No dissolution	N/A	N/A
	observed
N-Methylglucamine	Partial	N/A	N/A
	dissolution
	at 50° C.
Tromethamine	No dissolution	N/A	N/A
	observed
4-(2-	N/A	N/A	No dissolution
hydroxyethyl)-			observed
morpholine

Example 11: 1H Nuclear Magnetic Resonance Spectroscopy (NMR)

Samples for NMR analysis were prepared by weighing 5-7 mg of sample into a 1.5 mL HPLC vial prior to dissolving in d-DMSO, the samples were then transferred to field matched 5 mm NMR tubes for analysis. Analysis of the samples was performed using the standard instrument settings.

1H NMR data for the completely soluble or partially soluble salts prepared during the screen showed all to have different chemical shifts for peaks associated with protons around the carboxylate group compared to that of the free acid. This is indicative of salt formation (peaks at 9.91, 8.22 4.38 and 4.03 of the free acid Compound 1 show the most significant changes in shifts, see Table 5. Several samples also showed solvent present in the NMRs which could either be the result of insufficient drying or the formation of solvates; water was also seen in all NMR data, however this may be preparation-related. In cases where the counter ion was visible by 1H NMR it has also been quantified (See Table 5).

TABLE 5
1H NMR analysis
Salt Former       1H NMR Analysis
Potassium         11.82(s, 1H), 7.89(s, 1H), 7.48(d, 2H, J = 8.0 Hz), 7.37~7.18(m, 7H),
hydroxide         4.23(t, J = 8.0 Hz, 2H), 3.86(s, 2H), 3.05(t, J = 8.0 Hz, 2H).
Sodium hydroxide  11.54(s, 1H), 7.93 (s, 1H), 7.48(d, 2H, J = 8.0 Hz), 7.37~7.18(m,
                  7H), 4.26(m, 2H), 3.89(s, 2H), 3.05(m, 2H).
Ammonium          11.40(s, 1H), 7.96(s, 1H), 7.42(d, 2H, J = 4.8 Hz), 7.21(m, 7H),
hydroxide         4.26(m, 2H), 3.88(s, 2H), 3.06(m, 2H).
Diethylamine      11.34(s, 1H), 7.98(s, 1H), 7.48(d, 2H, J = 8.0 Hz), 7.21(m, 7H),
                  4.28(m, 2H), 3.90(s, 2H), 3.31(bs, 4H), 3.07(m, 2H), 2.90(m, 4H),
                  1.18(t, J = 8.0 Hz, 6H).
L-Lysine          7.85(s, 1H), 7.42(m, 2H), 7.40-7.12(m, 7H), 4.17(t, J = 8.0 Hz, 2H),
                  3.81(s, 2H), 3.08(m, 3H), 2.99(m, 3H), 2.69(m, 2H),
                  1.80~1.40(m, 4H).
N-Ethylglucamine  7.85(s, 1H), 7.42(m, 2H), 7.40~7.12(m, 7H), 4.17(t, J = 8.0 Hz, 2H),
                  3.81(s, 2H), 3.08(m, 3H), 2.99(m, 3H), 2.69(m, 2H), 1.80~1.40
                  (m, 4H).
N-Methylglucamine 7.85(s, 1H), 7.42(m, 2H), 7.40~7.12(m, 7H), 4.17(t, J = 8.0 Hz, 2H),
                  3.81(s, 2H), 3.08(m, 3H), 2.99(m, 3H), 2.69(m, 2H), 1.80~1.40
                  (m, 4H).

Example 12: Differential Scanning Calorimetry (DSC)

Ca. 1-3 mg of sample was placed onto a pre-weighed aluminium DSC pan using an analytical balance. The sample was heated from RT to Ca. 5° C. higher than the degradation point at 10° C./min under a nitrogen atmosphere. Each of the data sets were examined for any thermal events.

Example 13: Fourier Transform Infrared Spectroscopy (FT-IR)

Ca. 1-2 mg of sample was placed on to the crystal of the ATR module and secured into position. All the data generated was modified by removal of the background within the analysis software.

Example 14: Polarised Light Microscopy (PLM)

Samples were prepared on glass microscope slides using 1-2 drops of immersion oil and a glass cover slip. Optical assessment of the samples was performed using an appropriate objective lens with the polarizers being in the crossed, partially crossed and uncrossed positions.

Example 15: Hot-Stage Microscopy (HSM)

Samples were prepared on glass microscope slides and heated at 10° C./min to mimic the temperature profiles used with the TGA and DSC, up to the samples' melting point after which they were cooled to room temperature without forced cooling.

Example 16: Dynamic Vapor Sorption (DVS)

Ca. 10-15 mg of sample was weighed into a stainless steel DVS basket before submitting for analysis. The samples were analysed over the range of 0-90% RH with a maximum time of 6 hours per humidity stage. Each sample was exposed to a double cycle. XRPD analysis of all samples was performed post-DVS.

Example 17: Thermo Gravimetric Analysis (TGA)

The samples were heated from RT to 400° C. at 10° C./min (unless otherwise stated) under a stream of nitrogen gas. Each of the data sets were examined to determine mass losses and the degradation temperature of the samples.

Example 18: Inductively Coupled Plasma (ICP)

Ca. 0.10 g of test sample was digested in 5 mL nitric acid and made to volume with deionized water. The test sample was then diluted further and analyzed against a set of calibration standards to determine the sodium and potassium content.

Example 19: Karl Fischer

Ca. 0.05 g of test sample was back weighed into the KF vessel and titrated with Hydranal® Composite 5 to determine the % water content of the salt.

Example 20: Particle Size

Dispersant: Air, Lens: R3 (potassium and diethylamine) & R5 (sodium), Pressure: 4 bar, Feed velocity: 40 mm/s, Optical model: Fraunhofer, Measurement time: 5 seconds, Samples were analyzed as dry powders in duplicate with an average of the values recorded being reported.

Example 21: HPLC

Flow rate: 3.03 mL/min, Method: Isocratic, Column temperature: 25° C., Wavelength range: 190-400 nm, Solvent A: 25 mM Ammonium acetate buffer—pH 5.5 (30%), Solvent B: MeOH (70%), Injection volume: 15 μL, Run time: 20 minutes.

Example 22: FaSSIF/FeSSIF/Solubility

Ca. 25 mg of each of the salts were weighed into a 2 mL HPLC vial prior to the addition of 1 mL of Fasting State Simulated Intenstinal Fluid (FaSSIS) solution. The samples were then shaken for 24 h at 37° C. before being filtered into pre-weighed filter cartridges and dried overnight in vacuo and re-weighed, from this the solubility was calculated. The experiment was also repeated using Fed State Simulated Intestinal Fluid (FeSSIF) solution.

Example 23: pH1 Stability

Ca. 25 mg of each of the salts were weighed into a 2 mL HPLC vial prior to the addition of 1 mL of pH 1 buffer. The samples were then shaken for 4 h at 37° C. before being filtered into SPE cartridges and dried overnight.

Example 24: pH

A saturated solution of each of the salts was prepared in 5 mL of deionized water at room temperature prior to analysis.

TABLE 6
Summary of characterization of potassium salt
Technique                  Result
Yield                      93.5%
Appearance                 White powder
XRPD                       Partially crystalline-new pattern. The experimental conditions
                           were selected from the salt screen to give XRPD pattern 2;
                           since pattern 2 was not observed this might indicate polymorphism.
1H NMR                     Chemical shifts and loss of proton at ionizable center indicating
                           salt formation had occurred. Water and a trace amount of DIPE
                           were also observed.
PLM                        Birefringent needles and lathes up to 100 μm in length
HSM                        Loss of birefringence observed between 116-129° C., shrinkage/
                           change in structure of particles observed between 153-161° C.,
                           melt observed between 189-210° C. and on cooling
                           re-crystallisation occurred between 198-182° C.
TGA                        1 × mass loss prior to decomposition between 25-170° C. (4.8%)
                           loss of water, decomposition occurred at >225° C.
DSC                        3 × endotherms with onsets at 111.6° C. (loss of water), 147° C.
                           (initial melt) and 186° C. (secondary melt). Since no
                           re-crystallisation was observed between melts this may indicate
                           a mix of crystalline phases.
FT-IR                      Reference trace: closely matches the pattern of the sodium salt
Particle size (dry powder) X10: Average: 1.61, StdDev: 0.04, % RSD: 2.20
                           X50: Average: 6.48, StdDev: 0.35, % RSD: 5.46
                           X90: Average: 35.09, StdDev: 1.82, % RSD: 5.18
                           VMD: Average: 13.07, StdDev: 0.62, % RSD: 4.71
DVS                        Ca. 5.7% mass increase between 0-90% RH on the first cycle
                           and Ca. 4.7% mass increase on the second cycle.
                           Between Ca. 10-50% RH hysteresis was observed. The change
                           in sample mass observed between the 1st and 2nd cycles may
                           indicate a change in form.
XRPD post DVS              Partially crystalline-pattern is different from free acid indicating
                           a novel polymorphic form.
Karl Fischer               5.78% Water, 1:1.6 (salt: water), suggesting the material is a
                           sesiqu-hydrate
ICP                        8.03% Potassium (1:0.95 API : potassium counter ion)
Chemical purity (HPLC)     >99.9% (by peak area)
Aqueous solubility @       6.6 mg/mL
25° C.
Aqueous solubility @       16.2 mg/mL
50° C.
FaSSIF solubility @        1.5 mg/mL
37° C.
FeSSIF solubility @        1.6 mg/mL
37° C.
Observation post stability No visible change
(40° C./75% RH)
XRPD post stability        Partially crystalline-no change in form
(40° C./75% RH)
Chemical purity post       >99.9% (by peak area)
stability (40° C./
75% RH)
Observations               No visual change
post stability
(pH 1 for 4 h)
XRPD post stability        Partially crystalline: change in crystalline form compared to the
(pH 1 for 4 h)             free acid
Chemical purity post       >99.9% (by peak area)
stability (pH 1 for 4 h)
1H NMR post stability      Reduction in chemical shifts, indicating that salt form is no
(pH 1 for 4h)              longer present. Water also present.
pH of rm temp sat'd sol'n  9.21

TABLE 7
Summary of characterization of sodium salt
Technique           Result
Yield               101.3%
Appearance          White powder
XRPD                Crystalline - matches the pattern of the sodium salt
                    prepared from DIPE
1H NMR              Chemical shifts and loss of proton at carboxylate,
                    indicating salt formation had occurred. Water was
                    also observed.
PLM                 Typically the sample is comprised of agglomerates
                    of birefringent needles up to 20 μm in length.
HSM                 The sample was observed to melt between
                    98-123° C. and on cooling re-crystallisation
                    occurred between 134-132° C.
TGA                 1× mass loss prior to decomposition between
                    25-80° C. (7.7%) - loss of water, decomposition
                    occurred at >240° C.
DSC                 2× endotherms with onsets at 157.5° C. (initial melt)
                    and 159.9° C. (secondary melt). Since no
                    re-crystallisation was observed between melts this
                    may indicate a mix of crystalline phases.
FT-IR               Reference trace: closely matches the pattern of the
                    potassium salt
Particle size       X10: Average: 6.40, StdDev: 0.33, % RSD: 5.20
(dry powder)        X50: Average: 118.75, StdDev: 5.88, % RSD: 4.95
                    X90: Average: 304.01, StdDev: 22.91, % RSD: 7.54
                    VMD: Average: 140.17, StdDev: 9.09, % RSD: 6.48
DVS                 Ca. 11.7% mass increase between 0-90% RH on
                    each cycle. Between Ca. 50-80% RH hysteresis was
                    observed. Between 0-10% there was a mass increase
                    of Ca. 6.5-6.7% (ca. 1.7 eq water) which indicates a
                    hydrate formation.
XRPD post DVS       Crystalline - no change in form
Karl Fischer        7.03% Water, 1:1.9 (salt:water), this suggest the
                    material is a di-hydrate
ICP                 6.22% Sodium (1:1.24 API:sodium counter ion)
Chemical purity     >99.9% (by peak area)
(HPLC)
Aqueous solubility  2.3 mg/mL
@ 25° C.
Aqueous solubility  2.4 mg/mL
@ 50° C.
FaSSIF solubility   4.0 mg/mL
@ 37° C.
FeSSIF solubility   3.8 mg/mL
@ 37° C.
Observation post    No visible change
stability
(40° C./75% RH)
XRPD post stability Crystalline - no change in form
(40° C./75% RH)
Chemical purity     >99.9% (by peak area)
post stability
(40° C./75% RH)
Observations post   No visual change
stability
(pH 1 for 4 h)
XRPD post stability Partially crystalline: change in crystalline
(pH 1 for 4 h)      form to that of the free acid
Chemical purity     >99.9% (by peak area)
post stability
(pH 1 for 4 h)
1H NMR post         No chemical shifts, indicating that salt form
stability           is no longer present.
(pH 1 for 4 h)      Water also present.
pH of rm temp       10.31
sat'd sol'n

TABLE 8
Summary of characterization of diethylamine salt
Technique                   Result
Yield                       92.6%
Appearance                  White powder
XRPD                        Crystalline-Pattern 1. The experimental conditions used
                            should have prepared the Pattern 2 diethylamine salt as
                            generated during the salt screen, this indicates Pattern 1 may
                            be a more stable form.
1H NMR                      Chemical shifts and loss of proton at ionizable centre
                            indicating salt formation had occurred. Diethylamine was
                            present in a ratio of 1:1.0 (API to counter ion). Water was also
                            observed.
PLM                         The sample is comprised of birefringent lathes up to 50 μm in
                            length with agglomerates.
HSM                         Particle movement observed between 72-145° C. prior to an
                            initial melt between 145-154° C., a secondary melt was
                            observed between 199-209° C., re-crystallisation occurred on
                            cooling between 117-84° C.
TGA                         2 x mass loss prior to decomposition between Ca. 110-165° C.
                            (5.5%) and 165-290° C. (17.6%) these mass losses could be
                            loss of counter ion. Decomposition occurred at >290° C.
DSC                         1 x endotherm with an onset at 214.5° C. (melt). A base line
                            shift/broad endotherm is also observed between 85-130° C.
                            which could be due to the dissociation of the counter ion-this
                            would indicate instability of the salt form above 85° C.
FT-IR                       Reference trace: pattern is dissimilar to the other salts
                            prepared indicating a different structural arrangement
Particle size (dry powder)  X10: Average: 1.62, StdDev: 0.04, % RSD: 2.62
                            X50: Average: 6.06, StdDev: 0.12, % RSD: 1.99
                            X90: Average: 15.94, StdDev: 0.62, % RSD: 3.86
                            VMD: Average: 7.86, StdDev: 0.34, % RSD: 4.32
DVS                         Ca. 0.1% mass increase between 0-90% RH on the first cycle
                            and second cycles. No significant hysteresis was observed
                            during the experiment
XRPD post DVS               Crystalline-no change in form
Karl Fischer                0.21% water, 1:0.1 (salt:water)-this shows the sample is
                            anhydrous
Chemical purity (HPLC)      >99.9% (by peak area)
Aqueous solubility @        5.1 mg/mL
25° C.
Aqueous solubility @        9.2 mg/mL
50° C.
FaSSIF solubility @ 37° C.  1.3 mg/mL
FeSSIF solubility @ 37° C.  0.1 mg/mL
Observation post stability  No visible change
(40° C./75% RH)
XRPD post stability         Crystalline-no change in form
(40° C./75% RH)
Chemical purity post        >99.9% (by peak area)
stability (40° C./75% RH)
Observations post stability No visual change
(pH 1 for 4 h)
XRPD post stability         Partially crystalline: change in crystalline form from
(pH 1 for 4 h)              that of free acid
Chemical purity post        >99.9% (by peak area)
stability
(pH 1 for 4 h)
1H NMR post stability       Reduction in chemical shifts, indicating that salt form is no
(pH 1 for 4 h)              longer present. Water also observed.
pH of rm temp sat'd sol'n   8.08

Formulations

The salts Formulae 2 disclosed are not stable under acidic conditions, and therefore formulations for clinical use should be prepared with suitable buffering and/or coating so as to survive under the conditions in the stomach (e.g. an “enteric coated” formulation), or so as to be administered by other than an oral route (e.g. by injection or patch).

Preparing the salts Formulae 2 in dosage forms for oral administration, injection, administration by trans-dermal patch and the like, including excipients such as flavorings, buffers, carriers and the like, and packaging of the dosage forms are considered to be within the skill of the ordinary artisan. See, e.g. Remington: the Science and Practice of Pharmacy, 22nd Ed., c. 2013 by Pharmaceutical Press, hereby incorporated by reference in its entirety and for all purposes. The formulations should be prepared and administered so as to provide a dose in the range from 1-1000 mg/day to a subject, or to provide a dose range from 1-100 mg/day to a subject, or a dose range from 10-100 mg/day to a subject.

Claims

1. A salt compound having the formula (2):

2. The salt compound of claim 1 in which X+ is potassium ion, sodium ion or quaternary dimethylamine or quaternary diethylamine.

3. The salt compound of claim 1, in which m is 0.5 or 1.

4. The salt compound of claim 2, in which m is 0.5 or 1.

5. The salt compound of claim 1, wherein the solvent is 4-methyl-pentan-2-one, ethanol or diisopropyl ether.

6. The salt compound of claim 2, wherein the solvent is ethanol or diisopropyl ether.

7. A process for preparing the salt compound of Formula 2:

8. The process of claim 7, in which the organic solvent is ethanol or diisopropyl ether (DIPE) or 4-methyl-pentan-2-one.

9. The process of claim 7, in which the base is sodium hydroxide, potassium hydroxide or a salt of quaternary dimethylamine or a salt of quaternary diethylamine.

10. The process of claim 8, in which the base is sodium hydroxide, potassium hydroxide or a salt of quaternary dimethylamine or a salt of quaternary diethylamine.

11. The process of claim 7, in which the solvent is water mixed with a polar organic solvent.

12. The process of claim 11, in which the ratio of water to polar organic solvent ranges from 5:1 to 10:0.1.

13. The process of claim 8, in which the solvent is water mixed with a polar organic solvent.

14. The process of claim 13, in which the ratio of water to polar organic solvent ranges from 5:1 to 10:0.1.

15. The process of claim 9, in which the solvent is water mixed with a polar organic solvent.

16. The process of claim 15, in which the ratio of water to polar organic solvent ranges from 5:1 to 10:0.1.