GB2636969A - Novel crystalline salt forms - Google Patents

Abstract

Disclosed are crystalline salt forms of the compound 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl)benzonitrile, also known as CF3CN, wherein the desired salt has a sodium, calcium, or potassium base. The salt is most preferably a crystalline sodium salt and is characterised by an XRPD pattern of Figure 6. Further disclosed are pharmaceutical preparations comprising the compound and its medical use.

Description

NOVEL CRYSTALLINE SALT FORMS

FIELD OF THE INVENTION

[0001] The present invention relates to novel crystalline salt forms of the compound 4-(6-oxo-2- (trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl)benzonitrile, also known as CF3CN, and defined herein as the compound of Formula I. The compound of Formula I has the chemical formula C18H8F3N302 and has a molecular weight of 355.28g/mol.

[0002] The invention further relates to a method of preparation of a novel crystalline salt of the compound of Formula I and to the use of the salt as a medicament. In one embodiment the novel crystalline salt form is 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d] imidazol-8-yObenzonitrile sodium salt.

BACKGROUND TO THE INVENTION

[0003] The compound tropoflavin, also known as 7,8-dihydroxyflavone (7,8-DHF), is a naturally occurring flavone found in Godmania aesculifolio, Tridax procumbent and primula tree leaves. It is known to act as a potent and selective agonist of tropomyosin receptor kinase B (TrkB), which is the main signaling receptor of neurotrophin brain-derived neurotrophic factor (BDNF). [0004] Tropoflavin has been shown to have therapeutic efficacy in several animal models including depression, Alzheimer's disease, cognitive deficits in schizophrenia, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, traumatic brain injury, cerebral ischemia, fragile X syndrome and Rett syndrome.

[0005] A derivative of tropoflavin, 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile, referred to herein as the compound of Formula I, has been shown to be useful in the treatment of several diseases and conditions.

[0006] The patent application US 2021/0292335 describes a method of preparation of the compound of Formula I and data on the use of this compound in an Alzheimer's disease mouse model.

[0007] The compound of Formula I is sparingly soluble in aqueous solutions meaning that it's use in the preparation of a pharmaceutical is limited as such formation of a saline form of the compound is desirable.

[0008] The compound of Formula I has additionally found to be poorly crystalline which causes issues with the use of the compound in a pharmaceutical formulation.

[0009] The patent application US 2021/0292335 describes that a salt form of 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile may be used, however, there is no motivation or suggestion to make or test salts of the compound of Formula I or indeed any reasonable expectation of success.

[0010] An object of the present invention is the preparation of a salt form of the compound of Formula I. Such salt forms may be useful in the preparation of medicaments where the compound of Formula I is the active pharmaceutical ingredient as the salt form of the compound may have improved physico-chemical properties compared to the free molecule.

[0011] The data presented herein demonstrates that the sodium salt of the compound of Formula I exhibits advantageous properties which render it particularly suitable for use as active principle in a medicament.

[0012] Specifically, the applicant has demonstrated, that the sodium form of 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile unexpectedly has superior properties in comparison to other salt forms. In particular the 4-(6-oxo-2- (trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl)benzonitrile sodium salt of the invention has improved solubility and stability, which are further improved with respect to the other salt forms of this same compound.

[0013] The advantages related to the sodium salt form of 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile in comparison to the free molecule form or to other saline forms, are described with reference to physicochemical analysis and characterization.

BRIEF SUMMARY OF THE DISCLOSURE

[0014] In accordance with a first aspect of the present invention there is provided a 4-(6-oxo- 2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl)benzonitrile salt wherein the salt is taken from the group consisting of sodium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile; potassium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile and calcium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile.

[0015] Preferably the salt is sodium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile. [0016] More preferably the 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d] imidazol-8-yObenzonitrile salt is in a solid form.

[0017] More preferably the 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d] imidazol-8-ypbenzonitrile salt is in a crystalline form.

[0018] In one embodiment of the present invention the 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile salt is characterized by an XRPD pattern of Figure 6.

[0019] In accordance with a second aspect of the present invention there is provided a pharmaceutical preparation comprising a 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile salt, wherein the salt is taken from the group consisting of sodium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile; potassium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile and calcium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile.

[0020] Preferably the salt is sodium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile.

[0021] In a further embodiment the pharmaceutical preparation produces an elevated blood level of 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile of between 80% and 125% compared to those obtained with a pharmaceutical preparation not comprising a salt form of 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile.

[0022] In accordance with a third aspect of the present invention there is provided a 4-(6-oxo2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile salt for use in the treatment of a disease, wherein the salt is taken from the group consisting of sodium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile; potassium 4-(6-oxo-2- (trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl)benzonitrile and calcium 4-(6-oxo-2- (trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl)benzonitrile.

[0023] Preferably the salt is sodium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile.

[0024] The compositions provided herein contain therapeutically effective amounts of one or more of the compounds provided herein that are useful in the prevention, treatment, or amelioration of one or more of the symptoms of diseases or disorders described herein and a vehicle. Vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.

[0025] In addition, the compounds may be formulated as the sole active ingredient in the composition or may be combined with other active ingredients.

[0026] The compositions contain one or more compounds provided herein. The compounds are, in some embodiments, formulated into suitable preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as topical administration, transdermal administration, nasal inhalation, and oral inhalation via nebulizers, pressurized metered dose inhalers and dry powder inhalers. In some embodiments, the compounds described above are formulated into compositions using techniques and procedures well known in the art (see, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, Seventh Edition (1999)).

[0027] In the compositions, effective concentrations of one or more compounds or derivatives thereof is (are) mixed with a suitable vehicle. The compounds may be derivatized as the corresponding salts, esters, enol ethers or esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, ion-pairs, hydrates or prodrugs prior to formulation, as described above. The concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration that treats, leads to prevention, or amelioration of one or more of the symptoms of diseases or disorders described herein. In some embodiments, the compositions are formulated for single dosage administration. To formulate a composition, the weight fraction of a compound is dissolved; suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration such that the treated condition is relieved, prevented, or one or more symptoms are ameliorated.

[0028] The active compound is included in the vehicle in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The therapeutically effective concentration may be predicted empirically by testing the compounds in in vitro and in vivo systems well known to those of skill in the art and then extrapolated therefrom for dosages for humans. Human doses are then typically fine-tuned in clinical trials and titrated to response.

[0029] The concentration of active compound in the composition will depend on absorption, inactivation and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. For example, the amount that is delivered is sufficient to ameliorate one or more of the symptoms of diseases or disorders as described herein.

[0030] In some embodiments, a therapeutically effective dosage should produce a serum concentration of active ingredient of from about 0.001 ng/ml to about 1.0 ng/ml, 2-10 ng/ml, 11 to 50 ng/ml, 51-200 ng/ml, or about 200 to 1000 ng/ml. The compositions, in other embodiments, should provide a dosage of from about 0.0001 mg to about 70 mg of compound per kilogram of body weight per day. Dosage unit forms are prepared to provide from about 0.01 mg, 0.1 mg or 1 mg to about 500 mg, or about 1000 mg, and in some embodiments from about 10 mg to about 500 mg of the active ingredient or a combination of essential ingredients per dosage unit form.

[0031] The active ingredient may be administered at once or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data or subsequent clinical testing. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

[0032] In instances in which the compounds exhibit insufficient solubility, methods for solubilizing compounds may be used such as use of liposomes, prodrugs, complexation/chelation, nanoparticles, or emulsions or tertiary templating. Such methods are known to those of skill in this art, and include, but are not limited to, using co-solvents, such as dimethylsulfoxide (DMSO), using surfactants or surface modifiers, such as TWEEN®, complexing agents such as cyclodextrin or dissolution by enhanced ionization (i.e., dissolving in aqueous sodium bicarbonate). Derivatives of the compounds, such as prodrugs of the compounds may also be used in formulating effective compositions.

[0033] Upon mixing or addition of the compound(s), the resulting mixture may be a solution, suspension, emulsion or the like. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected vehicle. The effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined.

[0034] The compositions are provided for administration to humans and animals in indication appropriate dosage forms, such as dry powder inhalers (DPIs), pressurized metered dose inhalers (pMDls), nebulizers, tablets, capsules, pills, sublingual tapes/bioerodible strips, tablets or capsules, powders, granules, lozenges, lotions, salves, suppositories, fast melts, transdermal patches or other transdermal application devices/preparations, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the compounds or derivatives thereof. The therapeutically active compounds and derivatives thereof are, in some embodiments, formulated and administered in unit-dosage forms or multiple-dosage forms. Unit-dose forms as used herein refer to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the required vehicle. Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms may be administered in fractions or multiples thereof. A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.

[0035] Liquid compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional adjuvants in a vehicle, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension, colloidal dispersion, emulsion or liposomal formulation. If desired, the composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.

[0036] Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975 or later editions thereof.

[0037] Dosage forms or compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from vehicle or carrier may be prepared. Methods for preparation of these compositions are known to those skilled in the art. The contemplated compositions may contain 0.001%-100% active ingredient, in one embodiment 0.1-95%, in another embodiment 0.4-10%.

[0038] In certain embodiments, the compositions are lactose-free compositions containing excipients that are well known in the art and are listed, for example, in the U.S. Pharmacopeia (USP) 25-NF20 (2002). In general, lactose-free compositions contain active ingredients, a binder/filler, and a lubricant in compatible amounts. Particular lactose-free dosage forms contain active ingredients, microcrystalline cellulose, pre-gelatinized starch, and magnesium stearate.

[0039] Further provided are anhydrous compositions and dosage forms including active ingredients, since water can facilitate the degradation of some compounds. For example, the addition of water (e.g., 5%) is widely accepted as a means of simulating long-term storage in order to determine characteristics such as shelf-life or the stability of formulations over time. See, e.g., Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed., Marcel Dekker, NY, N.Y., 1995, pp. 379-80. In effect, water and heat accelerate the decomposition of some compounds. Thus, the effect of water on a formulation can be of great significance since moisture and/or humidity are commonly encountered during manufacture, handling, packaging, storage, shipment, and use of formulations.

[0040] Anhydrous compositions and dosage forms provided herein can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions.

[0041] An anhydrous composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions are generally packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0042] Oral dosage forms are either solid, gel or liquid. The solid dosage forms are tablets, capsules, granules, and bulk powders. Types of oral tablets include compressed, chewable lozenges and tablets which may be enteric-coated, sugar-coated or film-coated. Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form with the combination of other ingredients known to those skilled in the art.

[0043] In certain embodiments, the formulations are solid dosage forms such as for example, capsules or tablets. The tablets, pills, capsules, troches and the like can contain one or more of the following ingredients, or compounds of a similar nature: a binder; a lubricant; a diluent; a glidant; a disintegrating agent; a coloring agent; a sweetening agent; a flavoring agent; a wetting agent; an enteric coating; a film coating agent and modified release agent. Examples of binders include microcrystalline cellulose, methyl paraben, polyalkyleneoxides, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, molasses, polyvinylpyrrolidine, povidone, crospovidones, sucrose and starch and starch derivatives. Lubricants include talc, starch, magnesium/calcium stearate, lycopodium and stearic acid. Diluents include, for example, lactose, sucrose, trehalose, lysine, leucine, lecithin, starch, kaolin, salt, mannitol and dicalcium phosphate. Glidants include, but are not limited to, colloidal silicon dioxide. Disintegrating agents include crosscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose. Coloring agents include, for example, any of the approved certified water-soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended on alumina hydrate and advanced coloring or anti-forgery color/opalescent additives known to those skilled in the art. Sweetening agents include sucrose, lactose, mannitol and artificial sweetening agents such as saccharin, and any number of spray dried flavors. Flavoring agents include natural flavors extracted from plants such as fruits and synthetic blends of compounds which produce a pleasant sensation or mask unpleasant taste, such as, but not limited to peppermint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Enteric coatings include fatty acids, fats, waxes, shellac, ammoniated shellac and cellulose acetate phthalates. Film coatings include hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate. Modified release agents include polymers such as the Eudragit° series and cellulose esters.

[0044] The compound, or derivative thereof, can be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient.

[0045] When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

[0046] The active materials can also be mixed with other active materials which do not impair the desired action, or with materials that supplement the desired action. The active ingredient is a compound or derivative thereof as described herein. Higher concentrations, up to about 98% by weight of the active ingredient may be included.

[0047] In all embodiments, tablets and capsules formulations may be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient. Thus, for example, they may be coated with a conventional enterically digestible coating, such as phenylsalicylate, waxes and cellulose acetate phthalate.

[0048] Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

[0049] Elixirs are clear, sweetened, hydroalcoholic preparations. Vehicles used in elixirs include solvents. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may contain a preservative. An emulsion is a two-phase system in which one liquid is dispersed in the form of small globules throughout another liquid. Carriers used in emulsions are non-aqueous liquids, emulsifying agents and preservatives. Suspensions use suspending agents and preservatives. Acceptable substances used in non-effervescent granules, to be reconstituted into a liquid oral dosage form, include diluents, sweeteners and wetting agents. Acceptable substances used in effervescent granules, to be reconstituted into a liquid oral dosage form, include organic acids and a source of carbon dioxide. Coloring and flavoring agents are used in all of the above dosage forms.

[0050] Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examples of preservatives include glycerin, methyl and propylparaben, benzoic acid, sodium benzoate and alcohol. Examples of non-aqueous liquids utilized in emulsions include mineral oil and cottonseed oil.

Examples of emulsifying agents include gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, Veegum and acacia. Sweetening agents include sucrose, syrups, glycerin and artificial sweetening agents such as saccharin. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Organic acids include citric and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Coloring agents include any of the approved certified water-soluble FD and C dyes, and mixtures thereof. Flavoring agents include natural flavors extracted from plants such fruits, and synthetic blends of compounds, which produce a pleasant taste sensation.

[0051] For a solid dosage form, the solution or suspension, in for example, propylene carbonate, vegetable oils or triglycerides, is in some embodiments encapsulated in a gelatin capsule. Such solutions, and the preparation and encapsulation thereof, are disclosed in U.S. Pat. Nos. 4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form, the solution, e.g., for example, in a polyethylene glycol, may be diluted with a sufficient quantity of a liquid vehicle, e.g., water, to be easily measured for administration.

[0052] Alternatively, liquid or semi-solid oral formulations may be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those set forth in U.S. Pat. Nos. RE 28,819 and 4,358,603. Briefly, such formulations include, but are not limited to, those containing a compound provided herein, a dialkylated mono-or polyalkylene glycol, including, but not limited to, 1,2-dimethoxyethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether wherein 350, 550 and 750 refer to the approximate average molecular weight of the polyethylene glycol, and one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates.

[0053] Other formulations include, but are not limited to, aqueous alcoholic solutions including an acetal. Alcohols used in these formulations are any water-miscible solvents having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol. Acetals include, but are not limited to, di(lower alkyl) acetals of lower alkyl aldehydes such as acetaldehyde diethyl acetal.

[0054] Parenteral administration, in some embodiments characterized by injection, either subcutaneously, intramuscularly or intravenously is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. The injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

[0055] Implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained (see, e.g., U.S. Pat. No. 3,710,795) is also contemplated herein.

Briefly, a compound provided herein is dispersed in a solid inner matrix, e.g., polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethyleneterephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinylalcohol and cross-linked partially hydrolyzed polyvinyl acetate, that is surrounded by an outer polymeric membrane, e.g., polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetate copolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride, vinylchloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble in body fluids. The compound diffuses through the outer polymeric membrane in a release rate controlling step. The percentage of active compound contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject.

[0056] Parenteral administration of the compositions includes intravenous, subcutaneous and intramuscular administrations. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions. The solutions may be either aqueous or nonaqueous.

[0057] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.

[0058] Vehicles used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other substances.

[0059] Examples of aqueous vehicles include Sodium Chloride Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations must be added to parenteral preparations packaged in multiple-dose containers which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (Tween® 80). A sequestering or chelating agent of metal ions includes EDTA. Carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[0060] The concentration of compound is adjusted so that an injection provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight, body surface area and condition of the patient or animal as is known in the art.

[0061] The unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration must be sterile, as is known and practiced in the art.

[0062] Illustratively, intravenous or intraarterial infusion of a sterile aqueous solution containing an active compound is an effective mode of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing an active material injected as necessary to produce the desired pharmacological effect.

[0063] Injectables are designed for local and systemic administration. In some embodiments, a therapeutically effective dosage is formulated to contain a concentration of at least about 0.01% w/w up to about 90% w/w or more, in certain embodiments more than 0.1% w/w of the active compound to the treated tissue(s).

[0064] The compound may be suspended in micronized or other suitable form or may be derivatized to produce a more soluble active product or to produce a prodrug. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the condition and may be empirically determined.

[0065] Active ingredients provided herein can be administered by controlled release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, be used to provide slow or controlled release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the active ingredients provided herein.

[0066] All controlled-release products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time.

Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and can thus affect the occurrence of side (e.g., adverse) effects.

[0067] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, temperature, enzymes, water, or other physiological conditions or compounds.

[0068] In certain embodiments, the agent may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In some embodiments, a pump may be used (see, Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N Engl. J. Med. 321:574 (1989)).

but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480; 5,733,566; 5,739,108; 5,891,474; 5,922,356; 5,972,891; 5,980,945; 5,993,855; 6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363; 6,264,970; 6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358; 6,699,500 and 6,740,634. Such dosage forms can In other embodiments, polymeric materials can be used. In other embodiments, a controlled release system can be placed in proximity of the therapeutic target, i.e., thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)). In some embodiments, a controlled release device is introduced into a subject in proximity of the site of inappropriate immune activation or a tumor. Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)). The active ingredient can be dispersed in a solid inner matrix, e.g., polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethyleneterephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinylalcohol and cross-linked partially hydrolyzed polyvinyl acetate, that is surrounded by an outer polymeric membrane, e.g., polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetate copolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride, vinylchloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble in body fluids. The active ingredient then diffuses through the outer polymeric membrane in a release rate controlling step. The percentage of active ingredient contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the needs of the subject.

[0069] Of interest herein are also lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. They may also be reconstituted and formulated as solids or gels.

[0070] The sterile, lyophilized powder is prepared by dissolving a compound provided herein, or a derivative thereof, in a suitable solvent. The solvent may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, an antioxidant, a buffer and a bulking agent. In some embodiments, the excipient is selected from dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose and other suitable agent. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, at about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. In some embodiments, the resulting solution will be apportioned into vials for lyophilization. Each vial will contain a single dosage or multiple dosages of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature.

[0071] Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The precise amount depends upon the selected compound. Such amount can be empirically determined.

[0072] Topical mixtures are prepared as described for the local and systemic administration. The resulting mixture may be a solution, suspension, emulsions or the like and are formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, dermal patches or any other formulations suitable for topical administration.

[0073] The compounds or derivatives thereof may be formulated as aerosols for topical application, such as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment of inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the formulation will, in some embodiments, have mass median geometric diameters of less than 5 microns, in other embodiments less than 10 microns.

[0074] Oral inhalation formulations of the compounds or derivatives suitable for inhalation include metered dose inhalers, dry powder inhalers and liquid preparations for administration from a nebulizer or metered dose liquid dispensing system. For both metered dose inhalers and dry powder inhalers, a crystalline form of the compounds or derivatives is the preferred physical form of the drug to confer longer product stability.

[0075] In addition to particle size reduction methods known to those skilled in the art, crystalline particles of the compounds or derivatives can be generated using supercritical fluid processing which offers significant advantages in the production of such particles for inhalation delivery by producing respirable particles of the desired size in a single step. (e.g., International Publication No. W02005/025506). A controlled particle size for the microcrystals can be selected to ensure that a significant fraction of the compounds or derivatives is deposited in the lung. In some embodiments, these particles have a mass median aerodynamic diameter of about 0.1 to about 10 microns, in other embodiments, about 1 to about 5 microns and still other embodiments, about 1.2 to about 3microns.

[0076] Inert and non-flammable HFA propellants are selected from HFA 134a (1,1,1,2-tetrafluoroethane) and HFA 227e (1,1,1,2,3,3,3-heptafluoropropane) and provided either alone or as a ratio to match the density of crystal particles of the compounds or derivatives. A ratio is also selected to ensure that the product suspension avoids detrimental sedimentation or cream (which can precipitate irreversible agglomeration) and instead promote a loosely flocculated system, which is easily dispersed when shaken. Loosely fluctuated systems are well regarded to provide optimal stability for pMDI canisters. As a result of the formulation's properties, the formulation contained no ethanol and no surfactants/stabilizing agents.

[0077] The compounds may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active compound alone or in combination with other excipients can also be administered.

[0078] For nasal administration, the preparation may contain an esterified phosphonate compound dissolved or suspended in a liquid carrier, in particular, an aqueous carrier, for aerosol application. The carrier may contain solubilizing or suspending agents such as propylene glycol, surfactants, absorption enhancers such as lecithin or cyclodextrin, or preservatives.

[0079] Solutions, particularly those intended for ophthalmic use, may be formulated as 0.01% -10% isotonic solutions, pH about 5-7.4, with appropriate salts.

[0080] Other routes of administration, such as transdermal patches, including iontophoretic and electrophoretic devices, and rectal administration, are also contemplated herein.

[0081] Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those of skill in the art. For example, such patches are disclosed in U.S. Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433 and 5,860,957.

[0082] For example, dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect. Rectal suppositories are used herein mean solid bodies for insertion into the rectum which melt or soften at body temperature releasing one or more pharmacologically or therapeutically active ingredients. Substances utilized in rectal suppositories are bases or vehicles and agents to raise the melting point. Examples of bases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) and appropriate mixtures of mono-, di-and triglycerides of fatty acids. Combinations of the various bases may be used. Agents to raise the melting point of suppositories include spermaceti and wax. Rectal suppositories may be prepared either by the compressed method or by molding.

The weight of a rectal suppository, in one embodiment, is about 2 to 3 gm. Tablets and capsules for rectal administration are manufactured using the same substance and by the same methods as for formulations for oral administration.

[0083] The compounds provided herein, or derivatives thereof, may also be formulated to be targeted to a particular tissue, receptor, or other area of the body of the subject to be treated.

Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions. For non-limiting examples of targeting methods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874.

[0084] In some embodiments, liposomal suspensions, including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as carriers. These may be prepared according to methods known to those skilled in the art. For example, liposome formulations may be prepared as described in U.S. Pat. No. 4,522,811. Briefly, liposomes such as multilamellar vesicles (MLV's) may be formed by drying down phosphatidyl choline and phosphatidyl serine (7:3 molar ratio) on the inside of a flask. A solution of a compound provided herein in phosphate buffered saline lacking divalent cations (PBS) is added and the flask shaken until the lipid film is dispersed. The resulting vesicles are washed to remove unencapsulated compound, pelleted by centrifugation, and then resuspended in PBS.

[0085] The compounds or derivatives may be packaged as articles of manufacture containing packaging material, a compound or derivative thereof provided herein, which is effective for treatment, prevention or amelioration of one or more symptoms of the diseases or disorders, supra, within the packaging material, and a label that indicates that the compound or composition or derivative thereof, is used for the treatment, prevention or amelioration of one or more symptoms of the diseases or disorders, supra.

[0086] The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging products are well known to those of skill in the art. See, e.g., U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,252. Examples of packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. A wide array of formulations of the compounds and compositions provided herein are contemplated as are a variety of treatments for any disease or disorder described herein.

[0087] In human therapeutics, the physician will determine the dosage regimen that is most appropriate according to a preventive or curative treatment and according to the age, weight, stage of the disease and other factors specific to the subject to be treated. The compositions, in other embodiments, should provide a dosage of from about 0.0001 mg to about 70 mg of compound per kilogram of body weight per day. Dosage unit forms are prepared to provide from about 0.01 mg, 0.1 mg or 1 mg to about 500 mg, or about 1000 mg, and in some embodiments from about 10 mg to about 500 mg of the active ingredient or a combination of essential ingredients per dosage unit form.

[0088] The amount of active ingredient in the formulations provided herein, which will be effective in the prevention or treatment of a disorder or one or more symptoms thereof, will vary with the nature and severity of the disease or condition, and the route by which the active ingredient is administered. The frequency and dosage will also vary according to factors specific for each subject depending on the specific therapy (e.g., therapeutic or prophylactic agents) administered, the severity of the disorder, disease, or condition, the route of administration, as well as age, body, weight, response, and the past medical history of the subject.

[0089] Exemplary doses of a formulation include milligram or microgram amounts of the active compound per kilogram of subject (e.g., from about 1 microgram per kilogram to about 50 milligrams per kilogram, from about 10 micrograms per kilogram to about 30 milligrams per kilogram, from about 100 micrograms per kilogram to about 10 milligrams per kilogram, or from about 100 microgram per kilogram to about 5 milligrams per kilogram).

[0090] It may be necessary to use dosages of the active ingredient outside the ranges disclosed herein in some cases, as will be apparent to those of ordinary skill in the art. Furthermore, it is noted that the clinician or treating physician will know how and when to interrupt, adjust, or terminate therapy in conjunction with subject response.

[0091] Different therapeutically effective amounts may be applicable for different diseases and conditions, as will be readily known by those of ordinary skill in the art. Similarly, amounts sufficient to prevent, manage, treat or ameliorate such disorders, but insufficient to cause, or sufficient to reduce, adverse effects associated with the composition provided herein are also encompassed by the above-described dosage amounts and dose frequency schedules. Further, when a subject is administered multiple dosages of a composition provided herein, not all of the dosages need be the same. For example, the dosage administered to the subject may be increased to improve the prophylactic or therapeutic effect of the composition or it may be decreased to reduce one or more side effects that a particular subject is experiencing.

[0092] In certain embodiments, administration of the same formulation provided herein may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.

BRIEF SUMMARY OF THE DRAWINGS

[0093] The present invention is described with reference to the figure listed below: [0094] Figure 1 details the XRPD of Form 1 (black) and Form 2 (red); [0095] Figure 2 details the DSC of sample Form 1 (top) and Form 2 (bottom); [0096] Figure 3 details the TGA/DSC of sample Form 1 (top) and Form 2 (bottom); [0097] Figure 4 details the DVS sorption and de-sorption plot of Form 1 (top) and DVS isotherm plot of Form 1 (bottom); [0098] Figure 5 details the DVS sorption and de-sorption plot of Form 2 (top) and DVS isotherm plot of Form 2 (bottom); [0099] Figure 6 details the XRPD overlay of Form 1 (black), Form (blue), and Form 3 (red) (top) and TGA/DSC of Form 3; [00100] Figure 7 details the XRPD overlay of Form 2 (black), Ca-1 (red) K-1 (blue), K-2 (green), and K-3 (purple); [00101] Figure 8 details the XRPD overlay of Form 2 (black), Na-1 (blue), and Na-2 (red); [00102] Figure 9 details the XRPD overlay of patterns Form 2 (black), L-Arg-1 (red), Cho-1 (blue) and NH3-1 (green); [00103] Figure 10 details the XRPD overlay of patterns Form 2 (black), Tris-1 (red), Tris-2 (blue) and Tris-3 (green); [00104] Figure 11 details the XRPD overlay of Form 2 (black), Nap-1 (red), Nap-2 (blue) and Edi-1 (green); [00105] Figure 12 details the XRPD overlay of Form 2 (black), Mes-1 (green), Tos-1 (red), Tos- 2 (blue) and Mes-2 (purple); [00106] Figure 13 details the XRPD overlay of Form 2 (black), Mg-1 (red) and Mg-2 (blue) (top) and Raman overlay of Form 2 (red) and Mg-1 (purple) (bottom); [00107] Figure 14 details the XRPD overlay of Na-1 (black) and scale-up Na-1 (red) (top) and XRPD overlay of Ca-1 (black) and scale-up of Ca-1 (red) (bottom); [00108] Figure 15 details the XRPD pattern of scale-up at 3-gram Na-1 (black), Na-1 (red), and scale-up at 150 mg Na-1 (blue); [00109] Figure 16 details the XRPD overlay of pattern Na-1 (black), Na-2 (blue), Na-3 lot#DNS2181-39-6 (red), Na-5 (green), Na-(pink), Na-6 (brown), Na-7 (orange), Na-10 (teal), Na-8 (purple), and Na-11 (yellow); [00110] Figure 17 details the XRPD overlay of pattern Na-1 (black), Na-2 (blue), Na-3 (red), Na-5 (green), Na-9 (pink), Na-6 (brown), Na-7 (orange), Na-10 (teal), Na-8 (purple), and Na-11 (yellow); [00111] Figure 18 details the XRPD overlay of patterns Form 1 (blue), Form 2 (green), Form 3 (black), Form 4 (pink), Form (red), Form 8 (orange), mixture Form 1+3+11 (brown) (top) and XRPD overlay of patterns Form 1 (black), Form 2 (red), Form 3 (blue), Form 4 (green), Form 7 (pink), mixture of Form 3+9 (brown), Form 10 (orange), and a mixture of Form 1, 3 and 11 (teal) (bottom); [00112] Figure 19 details the XRPD pattern of Na-1 (black), Exp# 3 (red), and Exp# 4 (blue); and [00113] Figure 20 details the XRPD overlay of Form 1 (green), Form 2 (red), Form 2-crystalline (blue), and Form 13 (black).

DEFINITIONS

[00114] Various definitions are made throughout this document. Most words have the meaning that would be attributed to those words by one skilled in the art. Words specifically defined either below or elsewhere in this document have the meaning provided in the context of the present invention as a whole and as typically understood by those skilled in the art.

[00115] The term "substantially crystalline" means at least about 50% crystalline and ranging up to about 100% crystalline. The present invention provides a salt that is at least about 50% crystalline, at least about 60% crystalline, at least about 70% crystalline, at least about 80% crystalline, at least about 90% crystalline, at least about 95% crystalline, at least about 98% crystalline, or at least about 100% crystalline in form.

[00116] The degree or percentage of crystallinity may be determined by the skilled person using X-ray powder diffraction (XRPD). Other techniques, such as solid-state nuclear magnetic resonance (NMR), FT-IR, Raman spectroscopy, differential scanning calorimetry (DSC) and microcalorimetry, may also be used.

[00117] Crystalline forms of the salt of the invention may be in the form of a solvate, including but not limited to a hydrate (e.g., a monohydrate), or otherwise (e.g., in the form of an anhydrate).

[00118] "Subject," "individual" or "patient" is used interchangeably herein and refers to a vertebrate, preferably a mammal. Mammals include, but are not limited to, murines, rodents, simians, humans, farm animals, sport animals and pets.

[00119] "Treating" or "treatment" of any disease or disorder refers, in some embodiments, to ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof,). Treatment may also be considered to include preemptive or prophylactic administration to ameliorate, arrest or prevent the development of the disease or at least one of the clinical symptoms. Treatment can also refer to the lessening of the severity and/or the duration of one or more symptoms of a disease or disorder. In a further feature, the treatment rendered has lower potential for long term side effects over multiple years. In other embodiments "treating" or "treatment" refers to ameliorating at least one physical parameter, which may not be discernible by the patient. In yet other embodiments, "treating" or "treatment refers to inhibiting the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter) or both. In yet other embodiments, "treating" or "treatment" refers to delaying the onset of the disease or disorder.

[00120] "Therapeutically effective amount" means the amount of a compound that, when administered to a patient for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, adsorption, distribution, metabolism and excretion etc., of the patient to be treated.

[00121] "Vehicle" refers to a diluent, excipient or carrier with which a compound is administered to a subject. In some embodiments, the vehicle is pharmaceutically acceptable.

[00122] "Active ingredient" or "Active pharmaceutical ingredient" or "API" refers to the novel salt of the invention.

[00123] "4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile", also known as "CF3CN", referred to herein as the "compound of Formula I" has a SMILES code N#CC1=CC=C(C2=CC(C3=CC=C4C(N=C(C(F)(F)F)N4)=C302)=0)C=C1 and the structure defined below:

DETAILED DESCRIPTION OF THE INVENTION

Compound of Formula I

CN

[00124] The Examples below describe studies to systematically and identify new salt forms of the compound, to select the thermodynamically most stable polymorph or the kinetically accessible form with optimal properties, and to provide isolation conditions for initial scale-up.

[00125] The objective of this study was to systematically explore the experimental space and identify new forms of the compound, to select the thermodynamically most stable polymorph or the kinetically accessible form with optimal properties, and to provide isolation conditions for initial scale-up.

[00126] Two batches of the compound of Formula I were studied. One of the batches was labelled as Form 1 lot#22020204-63-C, the other batch was labelled as Form 2 lot#22020204-101-B. Form 1 had good crystallinity, an anhydrous with residual solvent present, hygroscopic and practically insoluble in water. Form 2 was poorly crystalline, hydrated 1:1.5 [FM:water], hygroscopic and practically insoluble in water. Upon heating Form 2 to 185°C a new form was observed, Form 4. Form 4 was poorly crystalline, anhydrous with residual water present, slightly hygroscopic and practically insoluble in water.

[00127] In the salt screen twenty new patterns were obtained from which one solvated form of the free molecule (Form 3) and nineteen potential salt forms. The two most promising salts were Na-1 and Ca-1. Na-1 is a trihydrate salt with a ratio of 1:1 [FM:CI], which is very hygroscopic and sparingly soluble in water. Ca-1 is a nonahydrate salt with a ratio of 1:0.5 [FM:CI]. It is hygroscopic and slightly soluble in water. Based on the physicochemical properties, Na-1 was the most promising salt for development.

[00128] A polymorph study was performed on both Na-1 and Form 2 to explore the polymorphic landscape. Six additional sodium salt candidates were obtained. However, all salt candidates were found to be solvated forms. Additionally, six new forms of the free molecule were identified. TGA/DSC analyses showed that these were also solvated forms and therefore not suitable.

[00129] The most promising forms obtained were Form 1, Form 2, Form 4, and Na-1.

Form 1 and 4 are anhydrous forms, but both are not scalable and reproducible. During Process R&D, Form 2 was obtained on a larger scale, which showed improved crystallinity. Moreover, this material was a dihydrate (unlike Form 2 as received which had a 1:1.5 [FM:water] ratio) and is stable at a relatively humidity above 10%. Upon scale-up, Na-1 could not be prepared reproducibly, nor prepared without formation of an impurity. Therefore, only Form 2 is both reproducible and scalable.

EXAMPLE 1: PHYSICOCHEMICAL PROPERTIES FOR THE COMPOUND OF FORMULA I [00130] The PhysChem properties were determined using ACD / Percepta software (build 3285) to get an understanding of compound of Formula I. Aqueous solubility (LogS), LogD, LogP and pKa were determined using the software. Two prediction methods were used, 1) Classic: based on >12,000 experimental values, the algorithm uses the principal of isolating carbons and 2) GALAS: based on a training set of >11,000 compounds, provides a value that is adjusted with data from the most similar compounds.

[00131] An overview of the PhysChem properties of the different compounds is displayed in Table 1. It should be noted that the GALAS prediction of the LogP has a reliability index (RI) of 0.73 which indicates a moderate reliability of the prediction (RI of 0 is very poor and RI of 1 is ideal).

Table 1. Physicochemical properties for the compound of Formula I LogS (pH 7.4) 0.06 mg/mL GALAS LogD (pH 7.4) 2.46 2.92 (RI = 0.73) LogP LogP consensus pKa acid pKa base Classic + 0.76*LogP GALAS) 3.17 ± 1.10 2.2 ± 1.3 (atom 20) 2.98 (= 0.24*LogP Classic -1.4 ± 0.7 (atom 10 and 18) 7.0 ± 0.4 (atom 20) 1.6 ± 0.4 (atom 20) [00132] LogD is the distribution coefficient of a molecule between an aqueous and lipophilic phase and is usually determined between aqueous buffers with ranging pH and octanol. LogD considers all neutral and charged forms of the molecule.

[00133] LogP is the partition coefficient of a molecule and indicates the equilibrium of unionized drug between the aqueous and octanol layer. Consequently, the LogP is equal to the value of the upper plateau of the LogD plot.

[00134] A LogD of one means that the concentration of the compound in the octanol layer is 10x higher compared to the concentration in the aqueous layer.

[00135] The compound of Formula I shows a decrease in the predicted aqueous solubility of 0.73 mg/ml to 0.02 mg/ml at pH 2.1 which is then increasing at pH 7.0 from 0.02 mg/ml to 1.1 mg/mL at pH 9.2.

[00136] The compound of Formula I was analysed by the Drug Profiler on solubility and other general absorbance issues as shown in Table 2. The compound scores as an optimal oral drug based on Lipinski's 'Rule of Five' and the lead-likeness rules. Lipinski's rule state that the molecular weight should be below 500, the number of H-bond donors should not exceed 5, the number of H-bond acceptors should not exceed 10, and the LogP does not exceed 5. The lead-likeness rules of the Percepta software state that the molecular weight should be below 460, the number of H-bond donors should not exceed 5, the number of H-bond acceptors should not exceed 9, and the LogP does not exceed 4.2. According to the Lipinski rule of 5, no violations were observed and with the lead-likeness rules also no violations were observed.

Table 2. Predicted results versus Lipinski's rule of 5 and the lead-likeness rules Lipinski's rule of 5 Lead-likeness rules Calculated Molecular weight 500 460 355.3 H-bond donors < 5 < 5 1 H-bond acceptors.s 10.s 9 5 LogP 5 4.2 3.0 [00137] Two batches of compound of Formula I were characterized using XRPD, DSC, TGA, Raman, UPLC, microscopy and DVS. The Raman spectrum will be used as reference only. An overview of the results is depicted in Table 3.

Table 3. Predicted results versus Lipinski's rule of 5 and the lead-likeness rules ID XRPD DSC (°C) TGA/DSC (wt%) UPLC purity Microscopy DVS Form Crystalline 20.5 endo 233.9 exo 0.5 residual solvent 98.6 area% Transparent particles 2-20pm Hygroscopic reversible and reproducible Form Poorly crystalline 122.6 endo 176.4 exo 199.8 exo 7.6 98.1 area% Rounded Hygroscopic reversible and reproducible 2 particles 2-10pm XRPD analysis of Form 1 and Form 2 [00138] XRPD analysis was performed using high throughput method Bruker AXS D8 DISCOVER HTS according USP <941> with a scan range of 2-45° 2e. The collected diffractogram of Form 1 and Form 2 are displayed in Figure 1. Both diffractograms were used as reference.

DSC analysis of Form 1 and Form 2 [00139] Additionally, DSC analysis was performed. Mettler Toledo DSC-3+ equipment was used, and a sample was heated from 20°C to 350°C in an aluminium (pierced) cup. Heating ramp rate of 10°C/min was applied.

[00140] The DSC result of Form 1 is represented in Figure 2 (top). Here an endothermic event is visible at Tpeak= 23.5°C and an exothermic event at Tpeak= 233.9°C. The sample was visually analysed by the Buchi Melting Point B-545 equipment. The endothermic and exothermic events showed no visual changes.

[00141] The DSC result of Form 2 is represented in Figure 2 (bottom). Here two endothermic events are visible at Tpeak= 119.6°C, and at Tpeak= 168.5°C, followed by three exothermic events at Tpeak= 176.5°C, at Tpeak= 199.8°C and at Tpeak= 287.9°C.

Cycle DSC on Form 2 [00142] On Form 2 three cycle DSC analysis were done to analyse the origin of events that were observed in the DSC. At first the sample was heated to 150°C (after the first endothermic event) then the sample was cooled to 20°C and the sample was measured by XRPD. This program was repeated, but after cooling to 20°C the sample was heated to 350°C.

The endothermic event at Tpeak= 114.0°C was not observed.

[00143] For the second cycle DSC, the sample was heated to 185°C (after the first exothermic event) then the sample was cooled to 20°C and the sample was measured by XRPD. This program was repeated, but after cooling to 20°C the sample was heated to 350°C. The endothermic event at Tpeak= 115.6°C and exothermic event at Tpeak= 171.7°C were no longer observed upon the second heating step.

[00144] The third cycle was the sample heated to 220°C (after the second exothermic event) and cooled down to 20°C and measured by XRPD. This program was repeated, but after cooling to 20°C the sample was heated to 350°C.

[00145] The samples that were measured by XRPD after the heating steps showed a change in form. When the sample was heated to 150°C the sample was dehydrated, and it resulted in amorphous solid. After heating Form 2 to 185°C and 220°C both samples resulted in the same crystalline form indicating that Form 2 recrystallizes upon heating. This new form was labelled Form 4.

TGA/DSC of Form 1 and Form 2 [00146] The result of the TGA/DSC analysis of Form 1, is displayed in Figure 3 (top). The measurement was performed using Mettler Toledo TGA/DSC-3+ equipment, a sample was heated from 20 °C to 350 °C in an aluminium (pierced) cup. A heating rate of 10 °C/min was applied. Form 1 shows residual solvent loss of 0.5wt% from 33.6 to 135.5°C and an exothermic event at Tpeak= 236.7°C.

[00147] The TGA/DSC analysis of Form 2 showed a desolvation of 7.6wt% at Tpeak= 122.6°C, and two exothermic events at Tpeak= 176.4°C and at Tpeak= 199.8°C (Figure 3 (bottom). The desolvation is a dehydration as Karl Fisher was measured on Form 2. This resulted in a water content of 8wt% on this batch. Form 2 is a hydrated form with a 1:1.5 [API:water] ratio based on the mass loss observed in the thermogram.

UPLC purity [00148] The purity of Form 1 was determined using UPLC. 10 mg of material was dissolved in 10 mL 1,3-dioxolane and the purity of Form 1 was 98.6 area% and Form 2 was 98.1 area%.

Microscopy [00149] The microscopy images of Form 1 and Form 2 were obtained by a Zeiss microscope. The powder was transferred in a cavity of an object glass, a droplet of corn oil was added to evenly spread the sample. Form 1 consisted of transparent needles between 2-20 pm. Form 2 exists of rounded particles between 2 and 10 pm. Form 1 and Form 2 show birefringence in polarized light, confirming crystallinity.

DVS

[00150] The hygroscopicity of Form 1 and Form 2 were determined using SMS DVS equipment. The sample was allowed to dry at 0 %RH for 4 hours. Subsequently the %RH was increased by steps of 10% until 95% RH, 1 hour per step. The %RH was decreased to 0 %RH again. This cycle was repeated twice. The hygroscopicity is classified based on USP <1241>.

[00151] The DVS sorption and desorption plot of Form 1 is displayed in Figure 4 (top) and the DVS isotherm plot is shown in Figure 4 (bottom). During both cycles, the sample showed a stepwise mass uptake of 3% (at 95 %RH) in response to increase of relative humidity and therefore it was classified as hygroscopic. Upon decreasing the %RH, the compound also showed a stepwise mass loss. Large hysteresis was observed between sorption and desorption, which could be related to a formation of hydrate.

[00152] The sample thermal behaviour was similar to the input compound according to TGA-DSC analysis. Particle morphology was also remained as it was for the pre-DVS sample.

Based on this information it is concluded that Form 1 was stable at high humidity.

[00153] The DVS sorption and desorption plot of Form 2 is displayed in Figure 5 (top) and the DVS isotherm plot in Figure 5 (bottom). Upon drying 3.4% mass loss is observed. During both cycles, the sample showed a stepwise mass uptake of 4.5% (at 95 %RH) in response to increase of relative humidity and therefore it was classified as hygroscopic. At an RH of 10% a relatively large step is observed indicating that hydrate formation occurs at a humidity a. 10 RH%. Upon decreasing the %RH, the compound also showed a stepwise mass loss.

[00154] XRPD data showed poor crystalline Form 2, this could be due to limited sample amount. The sample thermal behaviour was similar to the input compound according to TGA/DSC analysis. Particle morphology also remained the same as it was for the pre-DVS sample. Based on this information was concluded that Form 2 is stable at high humidity.

pKa, LogP, and LoqD determination [00155] To measure the LogP, LogD and pKa a reversed phase LC method with UV detection was used. A pH range of 1.2 -10.0 was selected using a stock buffer (25 mM citric acid, 30 mM boric acid, 20 mM sodium chloride, 25 mM Hydrochloric acid and 25 mM phosphoric acid). A total of 18 pH values were tested in this experiment. 500 pL of each pH buffer was pipetted in the vials. To each vial 500 pL saturated (with Milli-Q water) octanol and 10 pL sample solution (circa 20.1 mg/mL dissolved in DMSO) was added. These vials were capped and shaken for 24 hours at room temperature.

[00156] Afterwards, the samples were centrifuged for 10 minutes at 4000 rpm and the concentration of the compound was measured in both layers by LC. The LogD value at pH 1.8, 3.6 and 9.6 were considered outliers and therefore not used for the calculation. An overview of the results can be found in Table 4. Where 1.02 is the correction factor for the difference in volume between the octanol and aqueous layer ((500 pL octanol + 10 pL DMSO)/ 500 pL aqueous).

Table 4. LogD determination using LC pH Octanol layer Water layer Calculated LogD 1.16 1242.2 2.0 3.99 1.82 1234.4 8.1 3.37 2.04 1248.8 2.2 3.94 2.55 1217.4 2.3 3.92 3.14 1226.8 2.4 3.91 3.57 1255.4 7.6 3.41 4.14 1236.8 2.1 3.96 5.01 1236.6 2.2 3.94 5.43 1248.5 2.5 3.89 6.04 1305.0 14.4 3.15 [00157] LogP is the equilibrium of unionized drug between the aqueous and octanol layer. This means that LogP is equal to LogD if the compound is in its neutral form. In this case, the LogP was determined to be equal to 3.9, the plateau value at the highest LogD value. The predicted value(s) deviates from the measured value.

[00158] The pKa value is the value of the intersection point of the slope of the graph with the LogP. It can be concluded that pKa1 is <1.2. Furthermore, it can be concluded that pKa2 value is 6.2. This means that the GALAS prediction is incorrect whereas the Classic prediction is in line with the measured value (even though the value is slightly higher; 7).

EXAMPLE 2: SALT SCREEN FOR THE COMPOUND OF FORMULA I [00159] A salt study was performed to find a suitable salt with increased solubility. The salt study was conducted on Form 2 to find a solid form of the compound with enhanced aqueous solubility. The selection is based on the predicted and measured pKa of the compound (pKaacid = 6.2 [measured], pKabase = 1.6 [Classic prediction]) and on pharmaceutical acceptability.

[00160] The solvent selection is based on the solubility data and on a broad diversity of their PCA characteristics (polarity, polarizability, and hydrogen bonds), specific for the crystallization methods. The selected counterions and solvents are displayed in Table 5 and Table 6.

[00161] The salt study was performed using various crystallization techniques: Slurry at 50°C for 4 hours; Cooling crystallization 50°C-5°C; Evaporative crystallization under reduced pressure; and Thermocycle experiments.

[00162] The slurry, cooling crystallization, and evaporative crystallization were combined in a single automated screening protocol using 96-well plates. The thermocycle experiments were performed in vials using Technobis Crystall6 equipment.

[00163] Table 5. Selected counterions for salt study Counterion Base/aci Status pKa Ratio d class [FM:CI] KOH 1 Preferred 14.0 1:1.1 NaOH 1 Preferred 14.0 1:1.1 L-Arginine 1 Suitable 13.2 1:1.1 Ca(OH)2 1 Preferred 12.6 1:1.1 Mg(OH)2 1 Suitable 11.4 1:1.1 Choline 1 Accepted 11.0 1:1.1 L-Lysine 1 Suitable 10.8 1:1.1 Ammonia 1 9.3 1:1.1 Glycine 2 Suitable 9.6 1:1.1 Tromethamine 2 Accepted 8.0 1:1.1 HCI 1 Preferred -6.0 1:1.1 Sulfuric acid 1 Accepted -3.0 1:1.1 Naphtalene-1,5-disulfonic acid 2 -3.4 1:1.1 Ethane-1,2-disulfonic acid 2 Suitable -2.1 1:1.1 p-Toluenesulfonic acid 2 Accepted -1.3 1:1.1 Methanesulfonic acid 2 Accepted -1.2 1:1.1 Table 6. Selected solvents for salt screen Solvents ICH solvent class Methanol 2 Acetonitrile 2 Ethyl acetate 3 Toluene 2 2-Propanol 3 MtBE 3 Water Procedure salt study [00164] The slurry at 50°C, cooling and evaporative crystallization experiments were combined in a single automated screening protocol. 20 mg of Form 2 was dispensed in 96 wells of the master plate using Unchained Labs Protégé equipment. The selected counter ions and used solvents are listed in Tables 7, to 9. Counterions calcium hydroxide and magnesium hydroxide were added as a solid using Protégé equipment, as these are not soluble in water or methanol. Subsequently, 800 pL of solvent was added to each well. Finally, all remaining counterions were added as 1M solutions (in water or methanol). All counterions were added in a 1:1.1 molar ratio (FM:CI).

[00165] The master plate was heated to 50°C in 10 minutes and stirred for four hours at 350 rpm. Stirring was switched off after four hours to allow the solids to settle at the bottom of the wells. Subsequently, 200 pL of the mother liquid was transferred to the corresponding well of the cooling plate (at 50°C) and the evaporation plate. The cooling plate was cooled to 5°C and kept at this temperature for 16 hours. The liquids in the evaporation plate were evaporated to dryness under reduced pressure 200 mbar) at room temperature (RT). Remaining liquids of the master plate and cooling plate were removed by filter paper that absorbs the liquids and the solids were further dried under reduced pressure 200 mbar) at RT. All wells were analysed by XRPD.

Thermocycle [00166] The thermocycle experiments were performed to obtain a salt with improved crystallinity, and to gather initial aqueous salt solubility data. The experiments were conducted using a concentration of 25 mg/mL at 20 mg scale. 800 pL of water was added to each vial. Subsequently, all counterions were added in a 1:1.1 (FM:CI) molar ratio. Only calcium hydroxide and magnesium hydroxide were added as a solid, before the addition of water, due to its low solubility in water. A stirring bar was added to each vial. The samples were placed in Technobis Crysta116 equipment and the following protocol was run: the samples were heated to 80°C and then cooled to 5°C, the heating was repeated to 70, 60, 50, 40, 30 and finally 20°C with cooling to 5°C in between each heating step, final temperature was 5 °C. A hold time of 60 minutes at lowest and highest temperatures was applied and stirring was applied at 500 rpm. A heating and cooling rate of 0.5°C /minute was applied. After the experiment was finished, the pH (by pH indication paper) and solubility were measured using LC. The samples were centrifuged, and solvents were removed by pipet. The solids were dried under reduced pressure 200 mbar) at RT. All solids were analysed by XRPD.

Table 7. Bases used in the salt study Base Base class Base Ratio pKa status FM:CI 1 KOH 1 Preferred 1:1.1 14.0 2 NaOH 1 Preferred 1:1.1 14.0 3 L-Arginine 1 Suitable 1:1.1 13.2 4 Ca(OH)2 1 Preferred 1:1.1 12.6 Mg(OH)2 1 Suitable 1:1.1 11.4 6 Choline 1 Accepted 1:1.1 11.0 7 L-Lysine 1 Suitable 1:1.1 10.8 8 Ammonia 1 1:1.1 9.3 9 Glycine 2 Suitable 1:1.1 9.6 Tromethamine 2 Accepted 1:1.1 8.0 Table 8. Acids used in the salt study # Acid Acid Acid Ratio pKa class status FM:CI 1 HCI 1 Preferred 1:1.1 -6.0 2 Sulfuric acid 1 Accepted 1:1.1 -3.0 3 Naphtalene-1,5-disulfonic acid 2 - 1:1.1 -3.4 4 Ethane-1,2-disulfonic acid 2 Suitable 1:1.1 -2.1 p-Toluenesulfonic acid 2 Accepted 1:1.1 -1.3 6 Methanesulfonic acid 2 Accepted 1:1.1 -1.2 [00167] In total seven solvents were used (see Table 9). These solvents were selected based upon the available solubility data and a broad diversity of their PCA characteristics (polarity, polarizability, and hydrogen bonds). Water was only used in the thermocycle experiments.

Table 9. Used solvents for salt study Solvent Solvent class 1 Methanol 2 2 Acetonitrile 2 3 Ethyl acetate 3 4 Toluene 2 2-Propanol 3 6 MtBE 3 7 Water Results of salt study [00168] The salt screen resulted in twenty new patterns of which one new form of the free molecule and nineteen potential salt forms were assigned. All new patterns were analysed using a long measurement by XRPD (45 minutes instead of 8 minutes). No change in the patterns were observed with the longer measurement indicating that the samples are stable for at least 2 days at ambient conditions. The obtained new forms were analysed in the well plate by Raman if enough material was available.

[00169] An overview of all observed new patterns is depicted in Table 10, here the master plate slurry at 50°C crystallization for 2 hours, cooling plate is cooling crystallization from 50°C to 5°C and evaporation plate is (fast evaporation crystallization. Most new forms were obtained by slurrying, whereas least were obtained with thermocycling crystallization.

Table 10. Overview found forms in salt screen Slurry at 50°C crystallization Form 3', K-12, K23, K-36, mix K-2 K35 Form 31, Na-12.4,6 Form 315, L-Arg123 Form 31'255 Form 31.23, mix of Form 2 and 36 Form 31, Cho-13 NH3-11,2,3,4,5 NH3-12 Form 31'5 Form 31'2'3 Form 31, Iris-12, Tris-23'4 Form 31 Form 31 Cooling Fast Thermocycling crystallization evaporation crystallization crystallization Tris-32 Form 33 Mg-11, Mg-23 Form 31'23 Counter ion Potassium hydroxide Sodium hydroxide L-Arginine Calcium hydroxide Magnesium hydroxide Choline Ammonia Glycine Tromethamine Hydrogen chloride Sulfuric acid Naphthalene-1,5-disulfonic acid Ethane-1,2-disulfonic acid p-Toluene sulfonic acid Methane sulfonic acid Na-21 Na-17 Form 32 Ca-17 Form 32 NH3-17 Tris-31,2 Form 32 Form 32 Nap-12, Nap-21, Form 33 Form 31, mix Form Edi-11, Form 2 and 33 33 Form 31 Tos-11, Tos-22 Form 31 Mes-11 Mes-212 (1) methanol, (2) acetonitrile, (3) ethyl acetate, (4) toluene, (5) 2-popanol, (6) methyl tert-butyl ether, (7) water Form 3 [00170] One new form was observed with several counter ions and solvents and was therefore assigned as a new form of the free molecule, labelled as Form 3 (Figure 5 top). Form 3 was observed using three crystallization techniques, but mostly observed in slurry at 50° crystallization. Form 3 was mostly observed in combination with methanol and shaken at 50°C for 2 hours. Form 3 was also observed with acetonitrile, ethyl acetate and 2-propanol. Form 3 shows sharp peaks indicating good crystalline material (see Figure 29). The TGA/DSC analysis shows three desolvation steps with no clear DSC events indicating that Form 3 is a solvated form (Figure 5 bottom).

Aqueous solubility and pH of thermocycle salt formation experiments [00171] The solubility of the thermocycling experiments were analysed by LC and the pH of the solution was measured, see Table 11. The solubility of the compound of Formula I is higher at higher pH values (>0.1mg/mL). The solubility is comparable with the free molecule (< 0.1 mg/ml) at lower pH solutions (pH 2). Only three experiments resulted in a possible salt form, namely Na-1, Ca-1, and NH3-1. One experiment resulted in an oil. The other experiments did not show a change in form.

Table 11. Thermocycle results Exp # Counter ion Solubility pH XRPD (mg/m1) 1 Potassium hydroxide 21 8 Oil 2 Sodium hydroxide 19 8 Na-1 3 L-Arginine 9 8 Form 2 4 Calcium hydroxide 4 8 Ca-1 Magnesium hydroxide 1 8 Form 2 + peaks 6 Choline 13 8 Form 2 7 L-Lysine 1 8 Form 2 8 Ammonia 2 8 NH3-1 9 Glycine 0.2 9 Form 2 Tromethamine 2 7 Form 2 11 Hydrochloric acid <0.1 2 Form 2 12 Sulfuric acid <0.1 2 Form 2 13 Napthalene-1,5-disolfonic acid <0.1 2 Form 2 14 Ethane-1,2-disolfunonic acid <0.1 2 Form 2 p-Toluenesulfonic acid <0.1 2 Form 2 16 Methanesulfonic acid <0.1 2 Form 2 Potential salt forms [00172] All potential salt forms were analysed by TGA/DSC and Raman providing sufficient material is available.

Potential potassium salts [00173] Potential potassium salts were obtained with slurry at 50° crystallization and during the thermocycling crystallization experiment, see Table 12 and Figure 7. K-1 was obtained in acetonitrile (blue pattern in Figure 7). TGA/DSC analysis showed that K-1, K-2 and K-3 are solvated forms.

Table 12. Potential potassium salts Counter ion Form Crystallization DSC event (°C) TGA Conclusion (wt%) Potassium K-1 Slurry at 50°C 111.1 desolvation 13.0 Acetonitrile hydroxide 244.0 endo 0.2 solvate, possible 298.1 exo 0.9 salt Potassium K-2 Slurry at 50°C 142.1 desolvation 11.7 Ethyl acetate hydroxide K-3 Slurry at 50°C 239.4 exo 0.9 solvate, possible Potassium salt hydroxide 127.8 desolvation 11.4 Methyl-tert-butyl 204.7 exo ether solvate, possible salt Potential calcium salt [00174] The potential calcium salt pattern Ca-1 (red pattern in 7) was obtained with the thermocycle experiments. This pattern showed a dehydration in the TGA/DSC analysis (Table 13). Ca-1 is a hydrated form based on the mass loss observed in the thermogram.

Table 13. Potential calcium salt

DSC event (°C) TGA Conclusion

(wrio) Ca-1 Thermocycling 127.5 desolvation 15.7 Hydrated form 311.0 exo 6.1 Potential sodium salts [00175] With sodium hydroxide two potential salt forms were observed with various conditions. Na-1 (blue pattern in Figure 8) was observed with slurry at 50° crystallization in acetonitrile, toluene, and methyl-tert-butyl ether and in the thermocycle in water. Na-1 is a solvated form, see Table 14. The second pattern with sodium hydroxide that was obtained was labelled as Na-2 (red pattern in Figure 8). However, there was not enough material to be analysed by TGA/DSC.

Table 14. Potential sodium hydroxide salt Counter ion Form Crystallization DSC event (°C) TGA Conclusion (wt%) Sodium Na-1 Slurry at 50°C 66.4 desolvation 123.0 endo 2.8 Solvate/hydrate, possible salt hydroxide 11.6 Sodium Na-2 Evaporation Insufficient material Potential salt hydroxide Possible L-arginine salt [00176] The counter ion L-arginine gave one new potential salt using two solvents Counter ion Form Crystallization Calcium hydroxide was a solvated form (acetonitrile and ethyl acetate) with slurry crystallization at 50°C. L-Arg-1 based on the TGA/DSC (Table 15).

Table 15. Possible L-arginine salt Counter ion Form Crystallization DSC event (°C)

TGA Conclusion

(wrio) L-Arginine L-Arg-1 Slurry at 50°C 102.9 desolvation 8.7 Solvate, possible 167.4 endo 0.2 salt 232.4 endo 5.7 Possible choline salt [00177] The experiment with choline resulted in one new potential salt form in slurry crystallization at 50°C. In this experiment, ethyl acetate was used as solvent. Cho-1 is a solvated form based on TGA/DSC analysis, see Table 16.

Table 16. Possible choline salt Counter ion Form Crystallization DSC event (°C) TGA Conclusion (wt%) Choline Cho-1 Slurry at 50°C 106.1 desolvation 4.5 Solvate, possible 129.3 desolvation salt 192.3 recrystallization 258.3 endo 9.6 Possible ammonia salt [00178] With ammonia as counter ion one potential salt form was observed, NH3-1. This is the green pattern in Figure 9. The pattern was obtained with slurry at 50°C crystallization with methanol, acetonitrile, ethyl acetate, toluene, and 2-propanol; with cooling crystallization with acetonitrile; and in the thermocycle with water. NH3-1 is a solvated form based on the TGA/DSC analysis (Table 17).

Table 17. Possible ammonia salt Counter ion Form Crystallization DSC event (°C) TGA Conclusion (wt%) Ammonia NH3-1 Slurry at 50°C 130.1 desolvation 7.9 Solvate, possible Cooling 184.9 exo salt Thermocycling 256.8 exo Possible tromethamine salts [00179] With counter ion tromethamine three potential salt forms were obtained, see Table 18 and Figure 10. Tris-1 was obtained with slurry at 50°C crystallization with acetonitrile, ethyl acetate and toluene (red pattern in Figure 10). The second potential salt form Tris-2 was obtained with cooling crystallization in acetonitrile (blue pattern in Figure 10). This pattern could not be analysed by TGA/DSC as the yield was too low. Tris-3 was obtained in the fast evaporation crystallization in methanol and acetonitrile (green pattern Figure 10). Tris-1 and Tris-3 are solvated forms based on the TGA/DSC analysis.

Table 18. Possible tromethamine salts Counter ion Form Crystallization DSC event (°C) TGA Conclusion (wt%) Tromethamine Tris-1 Slurry at 50°C 80.1 desolvation 3.8 Solvate, possible 115.0 desolvation 3.6 salt 134.8 endo 149.3 exo 184.3 exo 209.4 endo 11.2 234.4 endo 258.8 endo Tromethamine Tris-2 Cooling Not enough material Possible salt, low yield Tromethamine Tris-3 Fast 74.8 endo 135.5 melt 3.2 Solvate, possible salt evaporation Possible naphthalene-1,5-disulfonic acid salts [00180] With naphthalene-1,5-disulfonic acid two potential salt forms were observed with fast evaporation crystallization. The first salt candidate, Nap-1 was obtained in methanol (red pattern in Figure 11). The second potential salt is Nap-2, this pattern is the blue pattern in Figure 11. Nap-1 and Nap-2 were analysed by TGA/DSC, which showed that both are solvated forms. The events of Nap-2 are similar to Nap-1; however, these events are not similar to the counter ion. Nap-1 and Nap-2 seem to be related as a lot of peaks of the diffractograms are similar. An overview of the Nap-1 and Nap-2 is displayed in Table 19.

Table 19. Possible salts with naphthalene-1,5-disulfonic acid Counter ion Form Crystallization DSC event (°C) TGA Conclusion (wt%) Naphthalene- Nap-1 Fast 68.6 endo 6.3 Methanol solvate, 1,5-disulfonic evaporation 78.6 endo 0.7 possible salt acid 100.1 endo 3.5 125.8 endo 3.3 Naphthalene- Nap-2 Fast 79.2 endo 8.0 Acetonitrile 1,5-disulfonic evaporation 110.1 endo 4.2 solvate, possible acid 135.5 endo 3.7 salt Possible ethane-1,2-disulfonic salt [00181] With ethane-1,2-disulfonic acid one potential salt form was observed with fast evaporation crystallization in methanol. This pattern Edi-1 is the green pattern in Figure 11. This pattern was analysed by TGA/DSC, but the yield was low (<0.5 mg). The thermogram shows a desolvation event and therefore is a solvated form (Table 20).

Table 20. Possible ethane-1,2-disulfonic salt Counter ion Form Crystallization DSC event (°C) TGA Conclusion (wt%) Ethane-1,2- Edi-1 Fast 80.6 endo 3.9 Methanol solvate, disulfonic acid evaporation 92.6 endo possible salt Possible tosylate salt [00182] There were two potential salt forms observed with p-toluene sulfonic acid as counter ion (Table 21). The first potential salt is Tos-1, this pattern was obtained with fast evaporation crystallization in methanol (red pattern Figure 12). This pattern could not be analysed as there was not enough material for TGA/DSC. The second potential salt observed was Tos-2, the blue pattern in Figure 12. Tos-2 could also not be analysed by TGA/DSC as there was not enough material.

Table 21. Possible toluate salts Counter ion Form Crystallization DSC event (°C) TGA Conclusion (wt%) p-Toluene Tos-1 Cooling Not enough material Possible salt sulfonic acid p-Toluene Tos-2 Cooling Not enough material Possible salt sulfonic acid Possible mesylate salt [00183] The potential salt forms observed with methanesulfonic acid, Mes-1 (green pattern in Figure 12) and Mes-2 (purple pattern in Figure 12), were also not analysed by TGA/DSC as there was not enough material. Mes-1 was observed in plate cooling crystallization in methanol. Mes-2 was observed with fast evaporation crystallization in methanol and acetonitrile (Table 22).

Table 22. Possible mesylate salts Counter ion Form Crystallization DSC event (°C) TGA Conclusion (wt%) Methanesulfonic Mes-1 Cooling Not enough material Possible salt acid Methanesulfonic Mes-2 Cooling Not enough material Possible salt acid Possible magnesium salts [00184] During cooling crystallization (cooling crystallization) two potential salt forms were observed with magnesium hydroxide (Figure 13 (top)). Mg-1 was observed in methanol and Mg-2 was observed in ethyl acetate. There was not enough material to analyse by TGA/DSC. However, it was confirmed by Raman that Mg-1 is a different form than the starting material, Form 2, see Figure 13 (bottom). For Mg-2 not enough material was present to analyse by Raman. An overview of these possible salt forms is depicted in Table 23.

Table 23. Possible magnesium salts DSC event (°C) TGA Conclusion Possible salt Possible salt Counter ion Form Crystallization (wt%) Magnesium Mg-1 Cooling hydroxide Not enough material Magnesium Mg-2 Cooling hydroxide Not enough material Overview salt screen [00185] An overview of all obtained forms during the screening is depicted in Table 24.

The three potential salt forms that showed the most promising results were Na-1, NH3-1, and Ca-1. Na-1 is good crystalline and the thermogram shows a desolvation of 2.8wt% at relatively a low temperature (Tpeak = 66.4°C). NH3-1 was obtained with various crystallization techniques and solvents. NH3-1 is a solvated/hydrated form. Solvates forms are generally not desired, but hydrated forms could be developed. Ca-1 was obtained with thermocycling in water. A dehydration event was observed in the thermogram. All other potential salt forms are solvated forms.

Table 24. Overview salt screen Form Counter ion Crystallization DSC event (°C) TGA Conclusion (wt%) Form 1 N/A As received Residual solvent 236.7 exo 0.5 Anhydrous form Form 2 N/A As received 122.6 desolvation 176.4 exo 7.6 Hydrated form 199.8 exo (1:1.5 API: Water) Form 3 N/A Slurry at 50°C 0.2 Solvated Cooling 2.2 form free Fast evaporation 1.2 molecule K-11 Potassium Slurry at 50°C 111.1 desolvation 13.0 Solvated hydroxide 244.0 endo 0.2 form, 298.1 exo 0.9 possible salt K-2 Potassium Slurry at 50°C 142.1 desolvation 11.7 Solvated hydroxide 239.4 exo 0.9 form, possible salt K-3 Potassium Slurry at 50°C 127.8 desolvation 204.7 exo 11.4 Solvated form, hydroxide possible salt Ca-1 Calcium Thermocycling 127.5 desolvation 15.7 Hydrated hydroxide 311.0 exo 6.1 form, possible salt Na-1 Sodium Slurry at 50°C 66.4 desolvation 2.8 Solvated hydroxide Thermocycling 123.0 endo 11.6 form, possible salt Na-2 Sodium hydroxide Evaporation Not enough material Possible salt, low yield L-Arg-1 L-Arginine Slurry at 50°C 102.9 desolvation 8.7 Solvated 167.4 endo 0.2 form, 232.4 endo 5.7 possible salt Cho-1 Choline Slurry at 50°C 106.1 desolvation 129.3 desolvation 192.3 recryst. 4.5 Solvated form, possible salt 258.3 melt 9.6 NH3-1 Ammonia Slurry at 50°C Cooling 130.1 desolvation 184.9 exo 256.8 exo 7.9 solvated/ hydrated form, possible salt Thermocycling Tris-1 Tromethamine Slurry at 50°C 80.1 desolvation 3.8 Solvated 115.0 desolvation 134.8 endo 3.6 form, possible salt 149.3 exo 184.3 exo 209.4 endo 234.4 endo 258.8 endo 11.2 Tris-2 Tromethamine Cooling Not enough material Possible salt, low yield Tris-3 Tromethamine Fast evaporation 74.8 endo Solvated 135.5 melt 234.6endo 3.2 form, possible salt 27.6 Nap-1 Naphthalene-1,5disulfonic acid Fast evaporation 68.3 endo 77.5 endo 100.1 endo 14.2 Solvated form, possible salt 125.8 endo Nap-2 Naphthalene-1,5- Fast evaporation 79.2 endo 8.0 Solvated disulfonic acid 110.1 endo 4.2 form, 135.5 endo 3.7 possible salt Eth-1 Ethane-1,2-disulfonic acid Fast evaporation 80.6 endo 92.6 endo 3.9 Solvated form, possible salt Tos-1 p-Toluene sulfonic acid Cooling Not enough material Possible salt, low yield Tos-2 p-Toluene sulfonic acid Cooling Not enough material Possible salt, low yield Mes-1 Methanesulfonic acid Cooling Not enough material Possible salt, low yield Mes-2 Methanesulfonic acid Cooling Not enough material Possible salt, low yield Mg-1 Magnesium hydroxide Cooling Not enough material Possible salt, low yield Mg-2 Magnesium hydroxide Cooling Not enough material Possible salt, low yield Salt scale up and characterization [00186] The experiments that resulted in a hydrated candidate salts were reproduced at mg scale in order to check reproducibility, scalability and for full characterization of the material. The experimental details are displayed in Table 25 below.

[00187] The scale-up of potential salt Na-1 and Ca-1 were performed on a 150 mg scale with thermocycle crystallization. Circa 150 mg was weighed into a vial, to which 6 mL of water was added. The counter ions were added in a 1:1.1 [FM:CI] ratio.

[00188] The scale-up of Na-1 resulted in a clear solution, this was dried under reduced pressure, which yielded a solid. The scale-up of Ca-1 was centrifuged and the liquid layer was removed, and the solid was dried under reduced pressure. The solids were analysed by XRPD.

Scale-up of Na-1 was successful with one additional peak at 11.2 2e, see Figure 14 (top). The scale-up of Ca-1 showed the same peaks as Ca-1 with a different intensity and improved crystallinity, see Figure 14 (bottom).

Table 25. Experimental conditions of scale up of the candidate salts at 150 mg scale Exp. # Pattern to Experiment Counter ion Molar ratio FM:CI Solvent reproduce 1 Na-1 Thermocycle Sodium hydroxide 1:1.1 Water 80-0°C 2 Ca-1 Thermocycle Calcium hydroxide 1:1.1 Water 80-0°C [00189] Additional analytics as TGA/DSC, 1H-NMR and ion chromatography were performed in order to confirm salt formation (Table 26).

Table 26. Result overview of 150 mg scale up of the candidate salts Exp. # XRPD TGA DSC (°C) DVS IC (wt-%) 1 Na-1 + 11.5 123.7 endo Very hygroscopic; 5.6wV/0 peak 1.8 166.2 endo reproducible, 1:1 3.0 326.5 exo reversible [FM:CI] 2 Ca-1 19.6 81.6 endo Hygroscopic; 6.1wt% 9.8 131.5 endo reproducible, 1:0.5 4.0 309.1 exo reversible [FM:CI] Remarks Trihydrate Na-1 salt; 1:1 [FM:CI]; not physically stable at high humidity Nonahydrate Ca-1 salt; 1:0.5 [FM:CI]; water content depends on humidity Na-1 characterisation [00190] The TGA/DSC analysis of Na-1 shows dehydration of 11.5wt% at Tpeak= 122.7°C, an endothermic event at Tpeak= 162.0°C with a mass loss of 1.8wt%, and lastly a decomposition event at Tpeak= 326.1°C with a mass loss of 3.0wt%. The dehydration of 11.5wt% corresponds to 3 water molecules.

IC analysis on Na-1 [00191] The sodium content of Na-1 was determined using ion chromatography. The experiments are performed using a 930 Compact IC Flex. The system consists of an 800 Dosino and an 858 Professional Sample Processor. For data collection and analysis, Waters Empower 3 is used. A sodium content of 5.4% was measured indicating that the sample is a 1:1 [FM:CI] ratio. The theoretical value of a 1:1 [FM:CI] ratio would be 5.6wt% sodium.

DVS on Na-1 [00192] The hygroscopicity of Na-1 was determined using SMS DVS equipment. The sample was allowed to dry at 0 %RH for 4 hours. Subsequently the %RH was increased by steps of 10% until 95% RH, 1 hour per step. The %RH was decreased to 0 %RH again. This cycle was repeated twice. The hygroscopicity is classified based on USP <1241>.

[00193] Upon drying 3.2% mass loss was observed. During both cycles, the sample showed a gradual mass uptake of 20.8% (at 95 %RH) in response to increase of relative humidity and therefore it was classified as very hygroscopic. Upon decreasing the %RH, the compound also showed a gradual mass loss.

[00194] XRPD data showed less peaks than pre-DVS. The sample thermal behavior was changed compared to the input compound according to TGA/DSC analysis. The thermogram showed a change in the dehydration steps. Dehydration happens over three endothermic events and not two endothermic events. Post-DVS showed a mass loss of 13.9wt% visible, this corresponds with 4 molecules of water. Particle morphology remained the same as it was for the pre-DVS sample. Based on this information it is concluded that the water content of Na-1 varies depending on the humidity.

Ca-1 characterisation [00195] The TGA/DSC analysis of Ca-1 shows that dehydration is a two-step mechanism. The first endothermic event at Tpeak= 81.6°C showed a mass loss of 19.6M% and the second endothermic event at Tpeak= 131.5°C showed a mass loss of 9.8wt%. This corresponds with nine water molecules. Lastly, an exothermic event was observed at Tpeak= 309.1°C with a mass loss of 4.0wt%.

IC analysis of Ca-1 [00196] The calcium content of Ca-1 was determined using ion chromatography. The experiments are performed using a 930 Compact IC Flex. The system consists of an 800 Dosino and an 858 Professional Sample Processor. For data collection and analysis, Waters Empower 3 is used. A calcium content of 6.1% was measured indicating that the sample is a 1:0.5 [FM:CI] ratio. The theoretical value of a 1:0.5 [FM:CI] ratio would be 5.1wt°/0 calcium.

DVS of Ca-1 [00197] The hygroscopicity of Ca-1 was determined using SMS DVS equipment. The sample was allowed to dry at 0 %RH for 4 hours. Subsequently the %RH was increased by steps of 10% until 95% RH, 1 hour per step. The %RH decreased to 0 %RH again. This cycle was repeated twice. The hygroscopicity is classified based on USP <1241>.

[00198] Upon drying 34.6% mass loss is observed. During both cycles, the sample showed a stepwise mass uptake of 9.0% (at 95 %RH) in response to increase of relative humidity and therefore it was classified as hygroscopic. Upon decreasing the %RH, the compound also showed a stepwise mass loss.

[00199] XRPD data showed one peak that shifted at 13.0 2e to 13.3 2e. In contrast to the pre-DVS sample, only one desolvation event was observed post-DVS. In total the mass loss post-DVS was circa 15.6% while it was 29.4% pre-DVS. The post-DVS mass loss corresponds to 4 water molecules to the structure. Particle morphology remained the same as it was for the pre-DVS sample, however birefringence was observed after DVS. Based on this information it is concluded that the water content of Ca-1 varies depending on the humidity.

Aqueous solubility of selected salt forms [00200] The aqueous solubility of the Na-1 and Ca-1 were determined by slurrying 10 mg of the salt in 1 mL of water at room temperature for 24 hours. Subsequently, the samples were filtered (0.2 pm filter) and diluted for LC analyses. A calibration line was set-up to determine the concentration of the samples. The pH was determined using a pH indication paper.

Summary most promising forms

[00201] Form 1, Form 2, Na-1, and Ca-1 were fully characterized, and a summary is visualized in Table 27. All forms are hydrated, except for Form 1. Form 1 does have residual (res.) solvent present. Form 1 and 2 are stable at high humidity. The aqueous solubility improved with the salt formation, for the Na-1 salt it is better than the Ca-1 salt.

Pharmaceutically sodium salts are more generally accepted than calcium salts. Furthermore, the crystallinity, LC purity, and DVS results of Na-1 is better compared to Ca-1. Therefore, the Na-1 is the most promising salt. This salt is used for the polymorph screen. This is scaled up on a 3 g scale, see 0.

Table 27. Overview characteristics forms 1, 2, Na-1 and Ca-1 Form XRPD DSC (°C) TGA LC Aqueous DVS IC Conclusion (wt%) purity solubility Very hygroscopic; reproducible, not 95.6 area% reversible Hygroscopic; reproducible, 92.0 not area% reversible 0.5 res.

solvent Form 1 Crystalline 20.5 endo 233.9 exo Form Poor 122.6 endo 7.6 2 crystalline 176.4 exo 199.8 exo 123.7 endo Na-1 Crystalline 166.2 endo 326.5 exo Poor 81.6 endo 131.5 endo crystalline 309.1 exo <0.1 mg/mL Practically insoluble <0.1 mg/mL Practically insoluble 5.6w 19 mg/mL t% Sparingly 1:1 soluble [FM: CI] 6.1w 3.9 mg/mL t% Slightly 1:0.5 soluble [FM: CI] Crystalline form, containing solvent Poor crystalline hydrate [1:1.5] [FM:water] Trihydrate Na-1 salt; 1:1 [FM:CI] Nonahydrate Ca-1 salt; 1:0.5 [FM:CI] Hygroscopic 98.6 area% Reversible, reproducible Hygroscopic; reproducible, reversible 98.1 area% Ca-1 11.5 1.8 3.0 19.6 9.8 4.0 3-gram scale up of salt Na-1 [00202] The scale-up of Na-1 was performed on a 3-gram scale for the polymorph study.

Circa 3 grams of Form 2 was used. To this 60 mL of water was added and 1 M NaOH in a ratio of 1:1.1 FM:CI. The slurry was heated to 50°C and stirred for 2 hours at 50°C. The compound was completely dissolved after 2 hours, and the solution was dried under reduced pressure. The obtained solid was analysed by XRPD to confirm if the scale-up was successful. The scaleup was successful, although two additional peaks were observed at 10.4 20 and at 11.0 20 (Figure 15).

EXAMPLE 3: POLYMORPH SCREEN FOR THE COMPOUND OF FORMULA I [00203] The objective of the polymorph study is to explore the polymorphic landscape of the compound of Formula I. A combined polymorph study was conducted on the free molecule (Form 2) and salt Na-1. To perform the polymorph screen on salt Na-1, the salt was scaled to 3g.

[00204] The polymorph study was performed using different crystallization techniques: Slurry at 50°C for 2 hours; Cooling crystallization 50°C-10°C; Reversed anti-solvent addition; Shake slurry at RT for 24 hours; and Thermocycle experiments.

[00205] Slurry at 50°C, cooling crystallization, and reversed anti-solvent addition were combined in a single automated screening protocol. The thermocycle experiments were performed in vials using Technobis Crysta116 equipment. The solvents, co-solvents and anti-solvents were selected based on the available solubility data and their PCA properties (Table 28). For the thermocycle experiments the same solvents were used without the co-solvents.

Table 28. Selected solvents, co-solvents, and anti-solvents for the polymorph study Solvent Class PCA Co-solvent Class PCA (#) Anti-solvent (#) 2-Propanol 3 1 Ethanol 3 2 Dibutyl ether Acetonitrile 2 1 Ethanol 3 2 Dibutyl ether 1,3-Dioxolane - 6 Ethanol 3 2 Dibutyl ether Methanol 2 2 Ethanol 3 2 Dibutyl ether 2-Butanone 3 1 Cyclohexane 2 6 n-Heptane 2-Methyl THE 3 5 Cyclohexane 2 6 n-Heptane Fluorobenzene - 8 Cyclohexane 2 6 n-Heptane Isobutyl acetate 3 5 Cyclohexane 2 6 n-Heptane Nitromethane 2 4 Tetrahydrofuran 2 5 n-Heptane Acetic acid 3 2 Tetrahydrofuran 2 5 n-Heptane 2-Butanol 3 1 Tetrahydrofuran 2 5 n-Heptane Pyridine 2 3 Tetrahydrofuran 2 5 n-Heptane Ethyl acetate 3 5 Toluene 2 8 n-Heptane Cyclopentyl 6 Toluene 2 8 n-Heptane methyl ether (CPME) Methyl isobutyl ketone (MIBK) di-n-Propyl ether 2 5 Toluene Toluene 2 8 n-Heptane n-Heptane 2 8 Preparation of amorphous material [00206] Formation of amorphous material of the free molecule, Form 2 was attempted by fast evaporation, grinding and fast anti-solvent addition. The experimental conditions and corresponding results can be found in Table 29. None of these experiments were successful in obtaining amorphous solid.

Table 29. Attempt to obtain amorphous free molecule Exp. # Method Solvent Observations XRPD 1 Fast evaporation Dichloromethane Did not dissolve Form 1 2 Fast evaporation Methanol Did not dissolve Form 3 3 Dry grinding, 30 min. 30 Hz N/A Form 2 4 Liquid assisted grinding, 30 min. 30 Hz Methanol Form 2 Fast evaporation Acetone Did not dissolve Form 3 Procedure and XRPD results polymorph study [00207] The procedure of the various crystallization techniques and their results are described below.

Slurry 50°C, cooling, reversed anti-solvent and slurry RT crystallization [00208] 20 mg of Na-1 was weighed in half of the wells of the master plate, in the other half 20 mg of Form 2 was weighed. Subsequently 800 pL of the specified solvent / co-solvent combination was added. The combinations that were used were 100/0, 80/20, and 50/50 v/v% solvent/co-solvent. The plate was shaken at 50°C for four hours on a thermoshaker (350 rpm). Shaking was turned off and solids were allowed to settle at the bottom. Subsequently, 200 pL of the clear liquids of the master plate were transferred to the cooling plate (at 50°C), and the precipitation plate that was pre-dispensed with 400 pl of anti-solvent. Then, the cooling plate was cooled to 5°C and stored at this temperature overnight. The precipitation plate was left at ambient conditions overnight, to allow precipitation to occur. The remaining solvents of the master plate, cooling plate and precipitation plate were absorbed by filter paper. Subsequently, the solids were dried under reduced pressure (s 200 mbar) at RT. All wells were analysed by XRPD. For the slurry at room temperature (RT) experiments 10 mg of Na-1 was weighed in half of the wells of the shake slurry plate. In the other half 10 mg of Form 2 was weighed. Subsequently 400 pL of the specified solvent / co-solvent combinations was added. The combinations that were used were 100/0, 80/20, and 50/50 solvent/co-solvent. The plate was shaken at 20°C for 24 hours on a thermoshaker (350 rpm).

Thermocycle [00209] Thermocycle experiments were performed to obtain a solid form with improved crystallinity. The experiments were conducted in vials using a concentration of 25 mg/mL on a mg scale. 800 pL of specified solvent and a stirring bar was added to each vial. The samples were placed in the Technobis Crystall6 equipment and the following protocol was run: the samples were heated to 70°C and then cooled to 5°C, the heating was repeated to 60, 50, 40, 30, 20 and finally 10°C with cooling to 0°C in between each heating step, final temperature was 0°C. A hold time of 30 minutes at lowest and highest temperatures was applied and stirring was applied at 350 rpm. A heating rate of 10°C/min and cooling rate of 0.5°C/min was applied. The samples were centrifuged, and solvents were removed by pipet. The solids were dried under reduced pressure 200 mbar) at RT. The experiments are displayed in Table 30. All solids were analysed by XRPD.

[00210] Table 30. Thermocycle experiments Starting Exp # Solvent material Na-1 1 Methanol* Na-1 2 Ethyl acetate* Na-1 3 2-Methyl THE Na-1 4 1,3-Dioxolane Na-1 5 2-Propanol Na-1 6 Acetonitrile Na-1 7 2-Butanone Na-1 8 Fluorobenzene Na-1 9 Isobutyl acetate Na-1 10 Nitromethane Na-1 11 Acetic acid Na-1 12 2-Butanol Na-1 13 Pyridine Na-1 14 CPME N a-1 15 MIBK N a-1 16 di-n-Propyl ether Form 2 17 Methanol* Form 2 18 Ethyl acetate* Form 2 19 2-Methyl THE Form 2 20 1,3-Dioxolane Form 2 21 2-Propanol Form 2 22 Acetonitrile Form 2 23 2-Butanone Form 2 24 Fluorobenzene Form 2 25 Isobutyl acetate Form 2 26 Nitromethane Form 2 27 Acetic acid Form 2 28 2-Butanol Form 2 29 Pyridine Form 2 30 CPME Form 2 31 MIBK Form 2 32 di-n-Propyl ether *Deviating maximum thermocycling temperature (50°C) Results of polymorph screen -sodium salt [00211] The results of the polymorph screen of the Na-1 are depicted in Table 31. Four new potential salt forms were obtained. Na-5 was obtained with most solvent combinations when slurried at 50°C. Na-6 and Na-7 were also obtained in the slurry 50°C plate. Na-5 and Na6 were also obtained in shake slurry and thermocycle experiments. Na-8 was obtained in the precipitation plate, as well as Na-5. All new forms were analysed again by a longer XRPD measurement after four days. This is performed for better quality of the diffractogram, and to check stability of the forms. There were differences observed between some of the short measurements and the long measurements. Na-5 is metastable and converts to a new form, labelled as Na-9. Na-7 was also not stable and converted to Na-10. The XRPD patterns are displayed in Figure 17.

Table 31. XRPD results of the polymorph screen on the Na salt Solvent/ co-solvent Master Cooling Precipitatio Shake slurry plate Thermocycle plate plate n plate Reduced pressure 50°C, 4 hours 50°C-5°C Reversed anti-solvent 2-Propanol/Ethanol Na-51 2 3 LY3 Ly1 2 Na-53, mix Na-5+61 2 Na-111 Acetonitrile/Ethanol Na-51 23 Form 21, Am2 3 Form 11 1,3-Dioxolane/Ethanol Na-51 2 3 LY1 Mix Na-5+Form 21, Am2,3 PC1 Methanol/Ethanol Na-51 2 3 LY1 Anil 2,3 Amt 2-Butanone/ Na-51 2 3 Amt Form 22,3, mix Na-Cyclohexane 5+Form 21 2-Methyl THF/ Na-51 23 Form 21,2.3 Na-61 Cyclohexane Fluorobenzene/ Na-71 23 Form 21.23 Form 11 Cyclohexane Isobutyl acetate/ Na-51 23 Form 21,2,3 Na-51 Cyclohexane Nitromethane/THF Na-51 2 3 LY1 Acetic acid/THF Na-6123 2-Butanol/THF Na-51 2'3 Pyridine/THF No-51'3 Ethyl acetate/Toluene Na-51.2,3 CPME/ Toluene Na-72 MlBK/Toluene Na-71, Form-23 di-n-Propyl ether/ No-51'2,3 Toluene (1) = 100 % solvent, (2) = 80% solvent / 20% co-solvent, (3) = 50% solvent / 50 % co-solvent, LY = low yield, Am = amorphous, PC = poor crystalline [00212] The potential salt forms were analysed by TGA/DSC, see Table 32. All thermograms showed a desolvation. Based on this overview Na-1 is the most promising sodium form since all other forms have a low yield or are either solvated or meta stable.

Table 32. Overview thermal behaviour results potential Na salts polymorph screen Form DSC event (T peak, °C) TGA mass loss (wt%) Conclusion Na-1 123.7 endo 11.5 Trihydrate Na-1 salt; 166.2 endo 1.8 1:1 [FM:CI] 326.5 exo 3.0 Na-2 Not enough material Potential salt, low yield Na-3 126.6 endo 6.4 Solvated form -nitromethane 1.8 Na-4 Reclassified as Form Na-5 Meta stable form, converts to Na-9 Na-6 67.1 endo 1.5 Potential salt, solvated -acetic 3.2 residual solvent 238.4 endo 1.9 acid Na-7 Meta stable form, converts to Na-10 Results of polymorph screen -free molecule [00213] The screening results of the free molecule are depicted in Table 33. Four new forms were obtained. Form 6 and Form 8 were obtained in the slurry 50°C plate. Form 7 was obtained in the precipitation plate and the slurry 20°C plate. Form 10 was obtained in the thermocycle experiment using acetic acid. These forms were analysed by TGA/DSC. An overlay of the XRPD diffractograms is displayed in Figure 18.

Table 33. XRPD results of the polymorph screen on the free base Na-51, Na-82 Form 21,2, mix Na5+Form 23 N a-31 Na-61,2,3 Mix Form 8+11 Na-61 Na-52, Na-23, mix Na- Amt 5+Form 21 Ami.2.2 N a-31 Form 21,23 Form 21,23 Form 1+peak& Na-3+peaks1 Form 23, mix Na-2+Form 21,2 Form 11 Form 21.2,3 Precipitation plate Reversed anti-solvent Form 312 Form 312 Shake slurry plate Reduced pressure Form 31.2.3 Form 31.2.3 Form 312.3 Form 31.2.3 Form 31,2,3 Form 3+912,3 Thermo-cycle Form 31 Form 31 Form 11 Form 31 Form 31 Form 31 Form 71 Form 31 Form 31 Form 101 Form 31 Form 7+111 Form 31 Form 31 Form 31 Form 31 Solvent/ co- Master plate solvent 50°C, 4h 2-Propanol/ Form 3123 Ethanol Acetonitrile/ Form 312,3 Ethanol 1,3-Dioxolane. Mix Form Ethanol 1+3+71,23 Methanol/ Form 312,3 Ethanol 2-Butanone/ Mix Form Cyclohexane 2+31.2,3 2-Methyl THF/ Form 312.3 Cyclohexane Fluorobenzene/ Form 312.3 Cyclohexane Isobutyl acetate/ Form 312,3 Cyclohexane Nitromethane/ Form 312,3

THE

Acetic acid/THF Form 612,3 2-Butanol/THF Form 312,3 PyridinefTHF Form 312,3 Ethyl acetate/ Form 812,3 Toluene CPME/Toluene Form 312,3 Form 32 MIBK/Toluene Form 312.3 di-n-Propyl Form 312,3 ether/Toluene (1) = 100 % solvent, (2) = 80% yield, Am = amorphous Cooling plate 50°C-5°C Form 32 Form 32 Form 32.3 Form 712 Form 312.3 Form 32,3 Form 31j Form 32 Form 3123 Form 31.2.3 Form 21, Form 32,3 Form 11.2, Form 33 Form 71,2, Form 33 Form 3123 Form 31.2.3 Form 312.3 Form 3123 solvent / 20% co-solvent, (3) = 50% solvent / 50 % co-solvent, LY = low [00214] The thermal behaviour of these forms is depicted in Table 34. Form 6 showed similar events to Form 2 and 10 and is therefore considered a mixture of the two forms. The diffractogram also showed a lot of similarities between the two forms. Form 8 is a mixture of Form 7 and unknown form. Form 7 is most likely a pyridine salt [1:1]. Theoretically, for a 1:1 salt there would be 18.3wt% present and the thermogram showed a mass loss of 16.4wt% during the melt. Form 9 was only observed in combination with Form 3. Based on this overview Form 1 and Form 2 are the most promising forms of the free molecule since all other forms are solvated or not obtained as a pure form.

Table 34. Overview thermal behaviour results of the polymorph screen starting with Form 2

Form DSC event (Tpeak;°C) TGA mass loss Conclusion (wt%)

Form 1 Residual solvent 0.5 Anhydrous form 236. exoxo Form 2 122.6 dehydration 7.6 Hydrated form 176.4 exo (1:1.5 API:Water) 199.8 exo Form 3 0.2 Chanel solvated form 2.2 1.2 Form 4 Residual solvent 0.8 Anhydrous form 309.7 exo 1.5 Form 5 Only observed as mixture with Form Form 6 52.9 endo 1.0 Mixture of Form 2 and Form 10 105.7 endo 6.2 176.3 exo 0.2 197.4 exo Form 7 0.3 residual solvent Potential pyridine salt [1:1] 161.5 melt 16.4 Form 8 93.1 endo 3.5 Solvated form, mixture of Form 7 with 165.3 melt 4.5 another unknown form 193.9 exo Form 9 Only observed as mixture with Form Form 10 124.5 endo 6.3 Solvated form -acetic acid Form 11 Only observed as mixture with Form 1, 3 and 7 Hydrate formation experiments [00215] Various (premixed) water/solvent ratios were used at two temperatures in hydrate formation experiments. An overview of the experimental conditions and results are displayed in Table 35 and Table 36. All experiments were slurry experiments at either RT or 50°C for 24 hours. The concentration of the free molecule experiments was 40 mg/mL. The concentration of the Na-1 experiments was 100 mg/m L. The samples were centrifuged to separate the solid and liquid, and the liquid was removed by pipette. The solids were dried under reduced pressure 200 mbar) for 2 hours at RT. All solids were analysed by XRPD and TGA/DSC, the results are summarized in Table 35 and Table 36 below.

[00216] The experiments mostly resulted in Form 3. Form 6 was observed with a 1/99 v/v% water/methanol ratio, Form 10 was observed with a 10/90 v/v% water/methanol ratio. However, in contrast to all other Form 3 samples no endothermic event with mass loss nor other DSC signals were observed.

Table 35. Results of hydrate formation experiments starting with Form 2 DSC events (Tpeak;°C) 73.3 endo 122.0 endo 173.1 endo 191.1 exo 272.7 exo TGA (wt%) 3.9 0.4 1.3

Conclusion

Solvated form Exp. # Experiment Water/methano XRPD I [ratio v/v-%] 1 Slurry RT 1/99 Form 3 2 Slurry RT 5/95 Form 3 76.2 endo 5.5 Solvated 119.4 endo - form 190.4 exo - 3 Slurry RT 10/90 Form 10 73.7 endo 3.8 Solvated 91.6 endo - form 105.9 endo 191.1 exo 4 Slurry RT 75/25 Form 3 74.7 endo 0.5 Solvated 116.5 endo 5.5 form 223.9 exo Slurry 50°C 1/99 Form 6 + peaks Residual solvent 1.3 Anhydrous form 6 Slurry 50°C 5/95 Form 3 Residual solvent 1.4 Solvent present 7 Slurry 50°C 10/90 Form 3 71.6 endo Solvated 98.8 endo 0.7 form 8 Slurry 50°C 75/25 Form 3 - 0.1 Solvated 100.5 endo 0.9 form 224.4 exo 0.1 [00217] With the hydrate formation experiments of Na-1 two new forms were obtained, Na-12 and Na-13, see Figure 19. The thermal behaviour of both new forms is comparable to Na-1. The other experiments resulted either in Form 2 or Form 3. This indicates a dissociation of the salt.

Table 36. Results of hydrate formation experiments Na-1 Exp. # Experiment Water/Solvent XRPD DSC events TGA (wt%) Conclusion [ratio v/v-%] (Tpeak; °C) 1 Slurry RT 1/99 Na-1 PC 102.1 endo 12.7 Hydrated 325.5 exo 4.4 form 2 Slurry RT 5/95 Form 2 + 105.6 endo 13.1 Hydrated peaks 325.5 exo 4.2 form 3 Slurry RT 10/90 Na-13 PC 105.4 endo 13.4 Hydrated 326.5 exo 5.0 form 4 Slurry RT 75/25 Na-12 111.0 endo 12.9 Hydrated 325.9 exo 4.8 form Slurry 50°C 1/99 Form 3 106.3 endo 12.3 Solvated 324.0 exo 6.1 form 6 Slurry 50°C 5/95 Form 3 102.2 endo 12.2 Solvated 323.9 exo 5.4 form 7 Slurry 50°C 10/90 104.9 endo 12.2 Solvated Form 3 323.9 exo 5.7 form 8 Slurry 50°C 75/25 Form 3 + 106.9 endo 13.3 Solvated peaks 325.1 exo 5.0 form Stability after exposure to humidity [00218] In total 18 samples were placed for stability at various relative humidity; six of Form 1; six of Form 2 and six of Na-1. The relative humidity (RH) was set to 11, 33 or 54% RH. The temperature for all conditions was room temperature (RT). The goal was to determine the stability of these forms at various relative humidities since DVS data showed that the hydrated forms showed a different water content after the DVS experiment.

[00219] The samples were analysed by XRPD, LC and TGA/DSC after 3 days and 7 days (Table 37). No changes were observed between t=0, t=3 and t=7 for Form 1 and Form 2. The purity and impurities are similar at t=0 (99.3area%), t=3, and t=7 for Form 1. For Form 2 the purity and impurity are also similar between the time points, with t=0 98.5area%. For Na-1 the purity decreases at 54%RH for 7 days. From t=0 88.7area% to t=7 83.8area%. Na-1 is stable at lower relative humidities.

[00220] Table 37. Results of the stability at various conditions experiments Form Exp # Relative humidity (A) Time (days) XRPD DSC events TGA (wt%) LC purity (area%) Conclusion (r peak;° c) 1 1 11 3 Form 1 0.2 99.3 No changes, 197.2 exo - stable at 237.3 exo - RT/11%RH 1 1 11 7 Form 1 - 0.4 99.3 238.1 exo - 1 2 33 3 Form 1 - 0.1 99.3 No changes, 200.5 exo - stable at 238.6 exo - RT/33%RH 1 2 33 7 Form 1 - 03 99.3 197.5 exo - 237.6 exo - 1 3 54 3 Form 1 - 0.1 99.2 No changes, stable at 199.2 exo 237.8 exo RT/33%RH 1 3 54 7 Form 1 - 02 99.3 197.5 exo - 237.8 exo - 2 4 11 3 Form 2 106.7 endo 8.0 98.5 No changes, 177.0 exo 0.1 stable at 200.5 exo - RT/11%RH 285.0 exo - 2 4 11 7 Form 2 119.1 endo 8.1 98.6 176.3 exo 0.2 200.0 exo - 290.4 exo - 2 5 33 3 Form 2 108.7 endo 7.9 98.5 No changes, 176.4 exo - stable at 200.5 exo RT/33%RH 285.7 exo - 2 5 33 7 Form 2 109.4 endo 8.1 98.5 177.1 exo 0.2 200.2 exo -283.7 exo 2 6 54 3 Form 2 107.7 endo 7.9 98.6 No changes, 176.9 exo 0.2 stable at 200.5 exo - RT/54%RH 2 6 54 7 Form 2 112.6 endo 8.0 98.6 176.3 exo 0.2 200.0 exo -286.2 exo Na-1 7 11 3 Na-1 114.1 endo 14.5 86.4 No changes, 325.4 exo 5.2 stable at Na-1 7 11 7 Na-1 125.7 endo 14.9 88.9 RT/11%RH 326.2 exo 4.9 Na-1 8 33 3 Na-1 60.8 endo 4.3 87.2 No changes, 103.6 endo 8.3 stable at 136.1 endo 1.6 RT/33%RH 324.6 exo 3.8 Na-1 8 33 7 Na-1 116.0 endo 14.6 88.4 326.1 exo 5.0 Na-1 9 54 3 Na-1 111.4 endo 14.2 88.1 Purity 325.8 exo 5.2 decreases Na-1 9 54 7 Na-1 121.3 endo 14.4 83.8 after 7 days at 326.9 exo 4.8 54%RH Slurry/fast evaporation experiments [00221] The slurry/fast evaporation experiments were performed on the free molecule of compound Form 2. The samples were shaken for 24 hours at room temperature. All experiments remained a slurry and therefore no fast evaporation crystallization was performed.

Then the samples were centrifuged, the liquid layer was removed, and the samples dried under reduced pressure. The results are shown in Table 38. All obtained forms were already observed in previous experiments.

Table 38. Slurry/fast evaporation results Exp # Solvent Slurry/fast evaporation XRPD 1 Ethyl formate Slurry Form 1 + peaks 2 Acetone Slurry Form 3 3 Di-ethyl ether Slurry Form 1 + 2 4 Methyl-tert-butyl ether Slurry Form 3 + peaks Methyl acetate Slurry Form 11 6 Pentane Slurry Form 2 7 Dichloromethane Slurry Form 1 + 2 8 Chloroform Slurry Form 2 Slurry/forward anti-solvent experiments [00222] The slurry/forward anti-solvent experiments were performed on Form 2. Only the experiment in dimethyl sulfoxide was completely dissolved after 2 hours at 80°C. This experiment also resulted in Form 4 after antisolvent addition. Up till now, this form was only observed when Form 2 was heated to 185°C. All other forms obtained were already observed in previous experiments. The experimental information and results are depicted in Table 39.

Table 39. Slurry/forward anti-solvent results Exp # Solvent Slurry/antisolvent XRPD 1 3-Methyl-1-butanol Slurry Form 3 2 Butyl acetate Slurry Form 1 + 3 3 di-n-Butyl ether Slurry Form 1 4 o-Xylene Slurry Form 3 + 4 3-Heptanone Slurry Form 1 + 3 6 1-Pentanol Slurry Form 1 + 3 7 Sulfolane Slurry Oil 8 Dimethyl sulfoxide Anti-solvent Form 4 DMSO:Water [1:2] Overview obtained forms polymorph screen [00223] The forms as found in the polymorph study were analysed by TGA/DSC. An overview of these results is given in Table 40. The most promising forms based on the polymorph screen and solubility experiments are Form 1, Form 2, and Na-1. Form 1 is an anhydrous form; Form 2 is a hydrated form and Na-1 is a hydrated sodium salt. Form 4 is also an anhydrous form that could also be interesting for development.

Table 40. Overview obtained forms polymorph screen Form Conditions Form 1 As received Form 2 As received Form 3 Multiple conditions and solvents Form 4 Heating Form 2 to 185°C Form 5 Slurry 50°C Ethyl acetate with ethane-1,2-disulfonic acid Form 6 Slurry 50°C Acetic acid/THF Form 7 Slurry 20°C Pyridine/THF Acetic acid/THF Form 8 Slurry 50°C Pyridine/THF Form 9 Slurry 20°C 2-Methyl THF/ cyclohexane Form 10 Thermocycle Acetic acid Form 11 Multiple conditions Na-1 Slurry 50°C Multiple solvents Thermocycle in water DSC event (Tpeak C) Residual solvent 236.7 exo 122.6 desolvation 176.4 exo 199.8 exo Residual solvent 309.7 exo 52.9 endo 105.7 endo 176.3 exo 197.4 exo 161.5 melt 93.1 endo 165.3 melt 193.9 exo 124.5 endo 123.7 endo 166.2 endo 326.5 exo

TGA Conclusion

mass loss (wt%) 0.5 Anhydrous form 7.6 Hydrated form (1:1.5 API:Water) 0.2 Solvated form 2.2 1.2 0.8 Anhydrous form 1.5 Only observed as mixture with Form 4 1.0 Mixture of Form 2 and 6.2 Form 10 0.2 0.3 Solvated form -pyridine residual 1:1 ratio solvent 16.4 3.5 Solvated form, mixture of 4.5 Form 7 with another unknown form Only observed as mixture with Form 3 6.3 Solvated form -acetic acid Only observed as mixture with Form 1, 3 and 7 11.5 Trihydrate Na-1 salt; 1:1 1.8 [FM:CI] 3.0 N a-2 Fast evaporation Insufficient material Potential salt Methanol N a-3 Solubility 126.6 endo 6.4 Solvated form -Nitromethane 1.8 nitromethane N a-4 Reassigned as Form 1 N a-5 Multiple conditions Meta stable form, converts to Na-10 Na-8 Precipitation 60.5 endo 5.2 Potential salt, unclear if it is Pyridine/THF 16.7 solvated or residual - 80/20v/v% residual pyridine/THF solvent N a-9 Multiple conditions 76.9 endo 5.5 Potential salt, solvated - 139.1 endo 4.4 pyridine/THF, low yield Na-10 Multiple conditions 87.7 endo 8.5 Potential salt, solvate.

4.7 residual solvent Na-11 Thermocycle 95.0 endo 18.4 Potential salt, solvated -2- 2-Propanol 101.0 endo propanol 136.5 endo 8.8 Na-12 Hydrate formation RT 111.0 endo 12.9 Hydrated form 25/75 water/methanol 325.9 exo 4.8 Na-13 Hydrate formation RT 105.4 endo 13.4 Hydrated form 10/90 water/methanol 326.5 exo 5.0 Scale up and characterization [00224] Forms 1 and 4 were scaled to 150 mg of Form 2 using Technobis Crystalline equipment. These forms are anhydrous forms therefore the most promising. The experimental conditions and results are described in Table 41. A concentration of 40 mg/mL was used.

Obtained solids were isolated by centrifuging and dried under reduced pressure 200 mbar) at RT. The dried solids were analysed by XRPD.

Table 41. Scale up Form 1 and Form 4 Exp. # To reproduce pattern Experiment Solvent Appearance XRPD (29) 1 Form 1 Slurry at 80°C Di-n-butyl ether Red powder Form 3 + peaks at 8.76, 15.13 and 21.00 2* Form 4 Heating to 185°C N/A Red powder Form 4 3 Slurry/forward anti-solvent at 80°C DMSO/water Red slurry Form 12 Form 4 * 30 mg scale in a DSC 100p1 crucible [00225] Only the scale-up of Form 4 heating Form 2 to 185°C was successful. This solid was analysed by TGA/DSC, DSC, microscopy, DVS, and LC. The results are depicted in Table 42.

Table 42. Result overview of 30 mg scale up of Form 4 Exp. # DSC events TGA (wt%) LC purity DVS Microscopy Conclusion (Tpeak;°C) (area%) 1 - 0.8 98.6 Slightly Rounded Anhydrous 309.7 exo - hygroscopic; particles form reproducible, between 2 reversible and 10 pm [00226] Also, the stability of Form 1, Form 2, Form 4, and Na-1 were determined. The stability of the forms was determined by storing the forms under accelerated conditions: 40°C/75%RH for 7 days in an open vial. After 7 days, the solids were analysed by XRPD and TGA/DSC. No changes were observed after the stress conditions. An overview of the forms and their characteristics is displayed in Table 43.

Table 43. Stability experiments Exp# XRPD t=0 DSC events TGA (wt%) XRPD t=7d DSC events TGA (wt%) (Tpeak,t) (Tpeak, °C) 2 Form 1 - 0.5 Form 1 0.5 236.7 endo - 235.6 exo 0.1 3 Form 2 122.6 endo 7.6 Form 2 126.9 endo 8.5 176.4 exo - 176.7 exo 0.1 199.8 exo - 200.1 exo - 4 Form 4 - 0.8 Form 4 - 0.4 309.7 exo - 296.9 exo 1.7 Na-1 122.7 endo 11.5 Na-1 65.8 endo 3.4 162.0 endo 1.8 121.4 endo 11.3 326.1 exo 3.0 325.7 exo 4.1 [00227] The aqueous solubility was determined in water, phosphate buffer pH 7.4 and simulated fluids FaSSIF, FeSSIF, and FaSSGF (Table 44). A slurry was prepared by adding 1 mL of water, phosphate buffer pH 7.4, FaSSIF. FeSSIF, and FaSSGF to 10 mg of Form 1. For Na-1, a slurry was prepared by adding 0.5 mL of water, phosphate buffer pH 7.4, FaSSIG, FeSSIF, and FaSSGF to 5 mg of Na-1. The pH was monitored using pH indicating paper. After 1 hour and 24 hours, the sample was filtered using a 0.20 pm filter, diluted and measured on LC. Isolated solids were analysed by XRPD. Form 1 is practically insoluble in the simulated fluids and in the phosphate buffer at pH 7.4. There were no changes observed between the 1 hour and 24 hours solubility. The obtained solids were either a mixture of Form 1 with Form 2 and/or 3, or in Form 1. Na-1 is slightly soluble in FaSSIF and pH buffer 7.4 after 1 hour. The solubility increases slightly for the FaSSIF after 24 hours. The solubility increases to sparingly soluble in pH buffer 7.4 after 24 hours. Na-1 is practically insoluble in FeSSIF and FaSSGF, there is no difference observed between 1 hour and 24 hours. The obtained solids resulted either in Form 2 or poor crystalline material. Na-1 is not stable in these buffers.

Table 44. Aqueous solubility results Solvent pH 1 hour Solubility (mg/mL) XRPD pH 24 hours Solubility (mg/mL) XRPD (28) medium FM FaSSIF 6 <0.01 Form 1 + 2 6 <0.01 Form 2 Form 1 + 3 FeSSIF 7 0.02 Form 1 7 0.02 Form 1 + peaks at 8.06 and 9.11 FaSSGF 2 0.01 Form 1+ 2 2 <0.01 Form 2 Phosphate buffer pH 7 <0.01 Form 1 7 <0.01 Form 2 + peaks at 7.4 3.78 and (50 mM) 8.03 Na-1 FaSSIF 8 4.32 Oil 8 7.12 Oil FeSSIF 7 0.03 Form 2 + peaks 7 0.02 Poor crystalline FaSSGF 5 <0.01 Poor crystalline 5 <0.01 Poor crystalline Phosphate buffer pH 8 3.55 Oil 8 17.94 Oil 7.4 (50 mM) Characterization of crystalline Form 2 during from Process R&D [00228] During Process R&D the free molecule is made using pyridine. Two batches that were produced and analyzed by XRPD and TGA. The XRPD diffractograms are depicted in Figure 20. Form 2-crystalline was a more crystalline solid of Form 2. Form 13 is a pyridine solvate.

[00229] Form 2 has better crystallinity compared to the same form found earlier, it was further characterized by Karl Fisher and TGA/DSC. The Karl Fisher analysis showed a water content of 9.0wt%. The TGA/DSC analysis showed a similar thermal behaviour as Form 2.

However, dehydration in the TGA/DSC shows a mass loss of 9.3wt%, which was 7.6wt% for Form 2. This higher water content corresponds to a dihydrate form: The dehydration step in the thermogram corresponds to 1:2 ratio of free molecule to water. The formation of the dihydrate could explain the increase in crystallinity. Moreover, an additional peak at 12.54 20 was observed in XRPD, possibly related to the formation of the dihydrate. This indicates that Form 2 a reproducible form and crystallinity can be increased within the process department.

DVS on Form 2-crystalline [00230] The hygroscopicity of Form 2-crystalline was measured by Dynamic Vapour Sorption (DVS). The sample was placed at 40% relative humidity (RH) for one hour. Then, the RH was increased by steps of 10% until 95%RH was reached, one hour per step. After that, the RH was decreased to 0%RH using the same rate. This cycle was repeated resulting in one cycle from 40%RH to 95%RH and one cycle 0%RH to 95%RH and back to 40%RH. respectively. The hygroscopicity is classified based on USP <1241>.

[00231] During the first cycle, the material showed a stepwise a mass uptake of 0.2% (at %RH) in response to increase of relative humidity and is therefore classified as slightly hygroscopic according to USP <1241>. Upon decreasing the RH, the compound showed a stepwise mass loss. Below 10%RH it starts losing its hydrated form. However, this does not happen fully within one hour of drying. The second cycle is comparable with the first cycle. The cycle is reversible and reproducible.

[00232] The XRPD pattern is similar, but the intensity is less due to less material. The mass loss of the thermogram is similar to pre-DVS analysis. Based on this data it can be concluded that Form 2 is stable at high humidity.

Raman on Form 2-crystalline [00233] The Raman spectrum of Form 2-crystalline was obtained for reference purposes.

Raman measurement was performed using Thermo Scientific DXR3 SmartRaman spectrometer. The spectrometer is equipped with Laser DXR 785 nm HP laser, a DXR 785 nm filter and DXR 785 nm full range grating (900 lines/mm). A scan range of 50 -3350 cm-1 was applied, 32 scans of 2 seconds per scan, 5.5-8.3 cm-1 resolution.

Scale-up Na-1 using Form 2-crystalline [00234] The Na-1 Form 2-crystalline showed an LCMS impurity of 10%. This sodium salt was obtained from free molecule batch 22020204-101-B. The formation of Na-1 was repeated with this higher purity batch. Circa 540 mg of compound was weighed to which 1:1.1 [FM:CI] 1M NaOH was added. Considering the 9wt% water that was present. The experiment was conducted in water. The sample was heated to 50°C and stirred for 2 hours. The liquid was dried under reduced pressure 200) at 40°C. The solid was analysed by XRPD, TGA/DSC and LC. The XRPD showed fewer peaks than Na-1. The thermogram is similar to Na-1, however the water content difference is 3wt% compared to the previous Na-1 batch. LCMS data showed that there is still 5.6 area% of an impurity is present. The purity of the batch is 92.3 area%. Thus, it is concluded that Na-1 is not suitable for scale-up.

Determination of the most stable polymorphic form Overview of promising forms [00235] Form 1, 2 and 4 are the most interesting forms. Form 1 and 4 are both anhydrous. Based on the gathered data, Form 4 is the most favourable form as it is only slightly hygroscopic. Even though Form 2 is not as recommended due to the API:water ratio of 1:1.5, it is the only form that was scalable and reproducible. Na-1 is the least favourable due to instability at high RH and the increase of an impurity at 54%RH after 7 days. However, the aqueous solubility is better than that of Form 1, 2, and 4. Possibly, a scale-up starting from purer sample could result less impurities of the Na-1 salt. An overview of the characteristics is displayed in Table 45.

Table 45. Overview characteristics Form 1, 2, 4 and Na-1 Aqueous Hygroscopic solubility (mg/mL) <0.1 Hygroscopic Reversible, reproducible (3% uptake) <0.1 Hygroscopic; reproducible, reversible (4.5% when dried, from hydrated form 1.5%)

N/A Stable dihydrate

above 10% relative humidity Slightly hygroscopic; reproducible, reversible (11% uptake) Form Conditions Form 1 As received Form 2 As received Form 2- Synthesised crystalline during Process R&D Form 4 Heating Form 2 to 185°C; Slurry/forwar d anti-solvent in DMSO/water @80°C Na-1 Slurry 50°C Multiple solvents Thermocycle in water DSC TGA mass event loss (wt%) (Tpeak;°C) Residual 0.5 solvent 236.7 exo 122.6 7.6 dehydrati on 176.4 exo 199.8 exo 112.6 9.3 dehydrati on 170.9 endo 181.2 exo 199.4 exo Residual 0.8 <0.1 solvent 1.5 309.7 exo

Conclusion

Anhydrous form Hydrated form (1:1.5 API:Water) Dihydrate form (1:2 API: Water) Anhydrous form Trihydrate Na- 123.7 endo 166.2 endo 326.5 exo 11.5 19 Very hygroscopic; 1.8 reproducible, not 3.0 reversible (21% uptake) Competitive slurry [00236] Competitive slurry experiments were performed on Form 1 and Form 4 with 10 mg of each form. These experiments were conducted in DMSO/water [1:2] (500pL), acetonitrile (500pL), and DMSO (200pL) with forward anti-solvent ethanol (400pL) [1:2] at two temperatures, room temperature and at 50°C. The samples were shaken for 20 hours. Ethanol was added to the slurry in DMSO as anti-solvent in experiment 3 and 6, but after addition of ethanol the compound was completely dissolved indicating that ethanol acted as a co-solvent rather than an anti-solvent. The obtained solids were isolated by centrifuging and dried under reduced pressure 200 mbar) at 40°C. The dried solids were analysed by XRPD.

Table 46. Competitive slurry conditions and results Exp. # Solvent Temperature Observations XRPD 1 DMSO/water* [1:2] RT Slurry Form 14 2 Acetonitrile RT Slurry Form 3 3 DMSO/ethanol** [1:2] RT Clear solution after addition anti-solvent Form 12 4 DMSO/water* [1:2] 50°C Slurry Form 14 Acetonitrile 50°C Slurry Form 3 6 DMSO/ethanol** [1:2] 50°C Clear solution after addition anti-solvent Form 12 * pre-mixed; ** forward anti-solvent [00237] The competitive slurry resulted in a form transition to either Form 3 or a new form. Based on these experiments there is no data of which form is the most stable form. The two new forms were obtained are Form 12 and 14. The solids were analysed by TGA/DSC, see Table 47. Form 14 shows two endothermic events, first at Tpeak= 77.6°C with a mass loss of 0.3wt%. The second at Tpeak= 158.2°C with a mass loss of 10.Owt% indicating that it is a solvated form. This sample was analysed by 1H-NMR, and this shows that it is a DMSO solvate.

Form 12 shows two endothermic events. The first event at Tpeak= 79.2°C, with a mass loss of 0.5wt%. The second event at Tpeak= 134.1°C, with a mass loss of 14.1wt%. Form 12 is a solvated form.

Table 47. Characteristics competitive slurry experiments

Exp# XRPD DSC TGA (wt%) Conclusion

(Tpeak,°C) 1 77.6 endo 0.3 Solvated form Form 14 158.2 endo 10.0 6 79.2 endo 0.5 Solvated form Form 12 134.1 endo 14.1 Re-slurry in water [00238] To assess the form stability in water compound Form 2-crystalline, Form 13, and Form 14 were re-slurried at 50°C for 16 hours. Afterwards the samples were centrifuged and the solid was isolated. The isolated solids were dried under reduced pressure. Then measured by XRPD and TGA/DSC, see Table 48. These experiments show that Form 13 can be converted to Form 2 with a re-slurry. Therefore, it can be concluded that Form 2 is a stable and reproducible form.

Table 48. Re-slurry experiments with results DSC (°C) TGA (wt%) 9.3 Conclusion Exp # Starting XRPD 112.6 endo 170.9 endo 181.2 exo Dihydrate Form 2 material 222 Form 2 199.4 exo 245 Form 13 - 1.6 Solvated Form 92.5 endo 2.0 13 121.4 endo 10.5 4 Form 14 77.6 endo 0.3 Solvated Form 158.2 endo 10.0 14 1 Form 2- Form 2 112.6 endo 9.3 Remained crystalline 170.9 endo - Form 2 181.2 exo - 199.4 exo - 2 Form 13 Form 2 118.7 endo 9.0 Successful 169.7 endo 0.2 conversion to 181.1 exo Form 2 199.4 exo 3 Form 14 Form 15 75.8 endo 0.6 Re-slurry in 122.8 endo 3.4 water 160.57 exo 1.7 unsuccessful

Conclusion and recommendations

[00239] A salt screen on the compound of Formula I was performed to identify a salt with improved PhysChem properties, of which aqueous solubility is most important. During the salt screen 19 salt candidates were found, but after characterization most turned out to be solvated.

The two most promising salt forms were Ca-1 and Na-1. After characterization of both salts, Nat was found to have the most promising physical properties.

[00240] The most promising forms of the free molecule were Form 1, Form 2, and Form 4. Form 1 and 4 are anhydrous forms, whereas Form 2 is a dihydrate. The process to prepare Form 1 and Form 4 was not scalable. Form 4 could be reproducibly formed by heating Form 2 to 185°C. This is not a desired method to use on a larger scale (>1 g). Therefore, only Form 2 is both reproducible and scalable and therefore recommended for development.

Claims (10)

1. CLAIMS1. A 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile salt wherein the salt is taken from the group consisting of sodium 4-(6-oxo-2- (trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl)benzonitrile; potassium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile and calcium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile.
2. A 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile salt according to claim 1, wherein the salt is sodium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile.
3. A 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile salt according to claim 1 or claim 2, wherein the salt is in a solid form.
4. A 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile salt according to any of the preceding claims, wherein the salt is in a crystalline form.
5. A 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile salt according to claim 2, characterized by an XRPD pattern of Figure 6.
6. A pharmaceutical preparation comprising a 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile salt, wherein the salt is taken from the group consisting of sodium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile; potassium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile and calcium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile.
7. A pharmaceutical preparation according to claim 6, wherein the salt is sodium 4-(6-oxo2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile.
8. A pharmaceutical preparation according to either claim 6 or claim 7, wherein the preparation produces an elevated blood level of 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile of between 80% and 125% compared to those obtained with a pharmaceutical preparation not comprising a salt form of 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile. 2. 3. 4. 5. 6. 7. 8.
9. 9. A 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile salt for use in the treatment of a disease, wherein the salt is taken from the group consisting of sodium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile; potassium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8- d]imidazol-8-yl)benzonitrile and calcium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile.
10. 10. A 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile salt for use according to claim 9, wherein the salt is sodium 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl) benzonitrile.