5 The present invention relates to a novel compound, 3-(benzo[d]thiazol-5-yl)-5- (methylamino)-l-(2,2,2-trifluoroethyl)-lH-pyrazole-4-carboxa mide, salts thereof, processes for its preparation, compositions comprising it, and to its use in therapy, in particular in the treatment or prevention of Chagas disease.

10 Background of the Invention

Chagas disease is an anthropozoonosis due to the flagellated protozoan parasite Trypanosoma cruzi. It is transmitted to humans and other mammals by infected faeces of a blood-sucking triatominae bug through the insect sting, another skin break or 15 through mucous membranes, including conjunctiva or oral/digestive mucosa, occasionally

causing outbreaks with contaminated food. Transmission through blood transfusion, pregnancy and delivery are also possible, and less frequently, through organ transplantation or laboratory accident.

20 Chagas disease is endemic throughout much of Mexico, Central America, and South

America where an estimated 7-8 million people are infected. The triatomine bug thrives under poor housing conditions (for example, mud walls, thatched roofs), so in endemic countries, people living in rural areas are at greatest risk for acquiring infection. The recent migration of populations from countries endemic for the disease has increased the

25 geographic distribution of Chagas disease, so that it is now becoming an important

health issue in the USA and Canada and in many parts of Europe and the western Pacific.

The most common destination for migrants from Latin America is the USA, where more than three hundred thousand individuals are infected with T.cruzi. Spain has the second highest number of infected immigrants, an estimated sixty-seven thousand patients.

30 Approximately thirteen thousand die each year from the complications of Chagas-induced

heart disease - a result of the chronic infection.

Chagas disease presents itself in 2 phases. The initial, acute phase lasts for about 2 months after infection. During the acute phase, a high number of parasites circulate in 35 the blood. In most cases, symptoms are absent or mild, but can include fever, headache,

enlarged lymph glands, pallor, muscle pain, difficulty in breathing, swelling and abdominal or chest pain. Manifestations of the acute disease resolve spontaneously in about 90% of infected individuals even if the infection is not treated with trypanocidal drugs. About 60-70% of these patients will never develop clinically apparent disease. These patients have the indeterminate form of chronic Chagas disease, which is characterised by positivity for antibodies against T.cruzi in serum, a normal 12-lead electrocardiogram (ECG), and normal radiological examination of the chest, oesophagus, and colon. The remaining 30-40% of patients will subsequently develop a determinate form of chronic disease.

During the chronic phase, the parasites are hidden mainly in the heart and digestive muscle. Up to 30% of patients suffer from cardiac disorders and up to 10% suffer from digestive (typically enlargement of the oesophagus or colon), neurological or mixed alterations. In later years the infection can lead to sudden death or heart failure caused by progressive destruction of the heart muscle. There is no vaccine yet for Chagas disease. Chemotherapy options are limited: benznidazole and nifurtimox are the only trypanocidal drugs available with proven efficacy against Chagas disease. Both medicines are almost 100% effective in curing the disease if given soon after infection at the onset of the acute phase. However, the efficacy of both diminishes the longer a person has been infected. Furthermore, benznidazole and nifurtimox are not consistently used in part because of their substantial side effects (peripheral neurotoxicity, digestive system irritation and serious dermatological conditions).

Newer, safer and more efficacious treatments are in desperate need.

WO2011/045415 discloses certain beta-amyloid binding compounds for use in detecting amyloid deposits in body organs and in Alzheimer's disease.

Summary of the Invention

The present invention relates to the pyrazole derivative 3-(benzo[d]thiazol-5-yl)-5- (methylamino)-l-(2,2,2-trifluoroethyl)-lH-pyrazole-4-carboxa mide, a compound having the Formula (I):

or a salt thereof.

The present invention also relates to pharmaceutical compositions comprising a) the compound of Formula (I) or a pharmaceutically acceptable salt thereof, and b) one or more pharmaceutically acceptable carriers.

Furthermore, the present invention also relates to methods of treating Chagas disease comprising administration of a therapeutically effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof to a subject in need thereof.

According to another aspect, the invention relates to the compound of Formula (I) or a pharmaceutically acceptable salt thereof for use in therapy. In another aspect, the invention relates to the compound of Formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of Chagas disease.

In another aspect, the invention relates to the use of the compound of Formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of Chagas disease.

Detailed Description of the Invention In a first aspect, the present invention is directed to 3-(benzo[d]thiazol-5-yl)-5- (methylamino)-l-(2,2,2-trifluoroethyl)-lH-pyrazole-4-carboxa mide, the compound of formula (I):

or a salt thereof.

The name 3-(benzo[d]thiazol-5-yl)-5-(methylamino)-l-(2,2,2-trifluoroe thyl)-lH-pyrazole- 4-carboxamide was derived using ChemBioDraw Ultra software. It will be appreciated that the compound of formula (I) can also be named as 3-(l,3-benzothiazol-5-yn-5- (methylaminoVl-(2,2,2-trifluoroethyn-lH-pyrazole-4-carboxami de

It is to be understood that references herein to compounds of the invention mean the compound of formula (I) as the free base, or as a salt, for example a pharmaceutically acceptable salt.

In one aspect of the invention, the compound of formula (I) is in the form of a free base. In a further aspect of the invention, the compound of formula (I) is in the form of a pharmaceutically acceptable salt.

Salts of the compound of formula (I) include pharmaceutically acceptable salts and salts which may not be pharmaceutically acceptable but may be useful in the preparation of the compound of formula (I) and pharmaceutically acceptable salts thereof. In one aspect of the invention, the compound of formula (I) is in the form of a pharmaceutically acceptable salt. Salts may be derived from certain inorganic or organic acids.

Examples of salts are pharmaceutically acceptable salts. Pharmaceutically acceptable salts include acid addition salts. For a review on suitable salts see Berge eta/., J. Pharm. Sci., 66:1-19 (1977).

Examples of pharmaceutically acceptable acid addition salts of the compound of formula (I) include inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, orthophosphoric acid, nitric acid, phosphoric acid, or sulphuric acid, or with organic acids such as, for example, methanesu I phonic acid, ethanesulphonic acid, p-toluenesu I phonic acid, acetic acid, propionic acid, lactic acid, citric acid, fumaric acid, malic acid, succinic acid, salicylic acid, maleic acid, glycerophosphoric acid, tartaric, benzoic, glutamic, aspartic, benzenesulphonic, naphthalenesulphonic such as 2-naphthalenesuphonic, hexanoic acid or acetylsalicylic acid.

In one aspect of the invention, the compound of Formula (I) is in the form of a hydrochloride or trifluoroacetate salt. In one aspect of the invention, 3-(benzo[d]thiazol- 5-yl)-5-(methylamino)-l-(2,2,2-trifluoroethyl)-lH-pyrazole-4 -carboxamide trifluoroacetate is provided. The invention includes within its scope all possible stoichiometric and non-stoichiometric forms of the salts of the compound of formula (I). Salts may be formed using techniques well-known in the art, for example by precipitation from solution followed by filtration, or by evaporation of the solvent.

Typically, a pharmaceutically acceptable acid addition salt can be formed by reaction of a compound of formula (I) with a suitable acid (such as hydrobromic, hydrochloric, sulphuric, maleic, />toluenesulphonic, methanesu I phonic, naphthalenesulphonic or succinic acids), optionally in a suitable solvent such as an organic solvent, to give the salt which is usually isolated for example by crystallisation and filtration.

It will be appreciated that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallised. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". Solvents with high boiling points and/or solvents with a high propensity to form hydrogen bonds such as water, ethanol, /so-propyl alcohol, and /V-methyl pyrrolidinone may be used to form solvates. Methods for the identification of solvates include, but are not limited to, NMR and microanalysis. Solvates of the compound of formula (I) are within the scope of the invention. As used herein, the term solvate encompasses solvates of both a free base compound as well as any salt thereof.

Certain of the compounds of the invention may contain chiral atoms and hence may exist in one or more stereoisomeric forms. The present invention encompasses all of the stereoisomers of the compounds of the invention, including optical isomers, whether as individual stereoisomers or as mixtures thereof including racemic modifications. Any stereoisomer may contain less than 10% by weight, for example less than 5% by weight, or less than 0.5% by weight, of any other stereoisomer. For example, any optical isomer may contain less than 10% by weight, for example less than 5% by weight, or less than 0.5% by weight, of its antipode.

Certain of the compounds of the invention may exist in tautomeric forms. It will be understood that the present invention encompasses all of the tautomers of the compounds of the invention whether as individual tautomers or as mixtures thereof. The compounds of the invention may be in crystalline or amorphous form. Furthermore, some of the crystalline forms of the compounds of the invention may exist as polymorphs, all of which are included within the scope of the present invention. The most thermodynamically stable polymorphic form or forms of the compounds of the invention are of particular interest.

Polymorphic forms of compounds of the invention may be characterised and differentiated using a number of conventional analytical techniques, including, but not limited to, X-ray powder diffraction (XRPD), infrared spectroscopy (IR), Raman spectroscopy, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and solid-state nuclear magnetic resonance (ssNMR).

The present invention also includes all suitable isotopic variations of a compound of formula (I) or a pharmaceutically acceptable salt thereof. An isotopic variation of a compound of formula (I), or a pharmaceutically acceptable salt thereof, is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ 0, ¹⁸ 0, ¹⁸ F and ³⁶ CI, respectively. Certain isotopic variations of a compound of formula (I) or a salt or solvate thereof, for example, those in which a radioactive isotope such as ³ H or ¹⁴ C is incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., ³ H, and carbon-14, i.e., ¹⁴ C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., ² H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of a compound of formula (I), or a pharmaceutically salt thereof, can generally be prepared by conventional procedures such as by the illustrative methods or by the preparations described in the Examples hereafter using appropriate isotopic variations of suitable reagents.

It will be appreciated from the foregoing that included within the scope of the invention are solvates, hydrates, isomers and polymorphic forms of the compound of formula (I) and salts and solvates thereof.

Compound Preparation The compound of formula (I) and salts thereof may be prepared by the methodology described hereinafter, constituting further aspects of this invention. Accordingly, there is provided a process for the preparation of the compound of formula (I), or a salt thereof.

The compound of Formula (I) may be prepared by reaction of a compound of Formula

with an acid, for example sulphuric acid or hydrochloric acid, optionally in the presence of a suitable solvent, for example a mixture of toluene and dioxane.

The compound of Formula (II) may be prepared by reaction of a compound of Formula (III)

with a suitable amine methylating agent, for example triethylorthoformate followed by reducing agent for example sodium borohydride, or iodomethane in the presence of a suitable base, for example sodium hydride, in a suitable solvent, for example ethanol or dimethylformamide, at a convenient temperature, for example room temperature or 0°C.

The compound of Formula (III) may be prepared by reaction of a compound of Formula (IV)

with a suitable ring-closing agent, for example (2,2,2-trifluoroethyl)hydrazine, in the presence of a suitable base, for example triethylamine or DIPEA, in a suitable solvent, for example ethanol or MeOH, at a convenient temperature, for example room temperature.

The compound of Formula (IV) may be prepared by reaction of a compound of Formula (V)

with a suitable methylating agent, for example trimethylsilyldiazomethane or iodomethane, in a suitable solvent, for example a mixture of acetonitrile and methanol or DMF, in the presence of a suitable base, for example DIPEA or lithium carbonate, at a convenient temperature, for example room temperature.

The compound of Formula (V) may be prepared by reaction of a compound of Formula (VI)

with malononitrile, in a suitable solvent, for example a mixture of tetrahydrofuran and toluene, at a convenient temperature, for example room temperature.

The compound of Formula (VI) may be prepared by reaction of a compound of Formula (VII)

with a suitable halogenating agent, for example thionyl chloride or oxalyl chloride, in a suitable solvent, for example toluene or DMF, at a suitable temperature, preferably under reflux conditions. The compound of Formula (VII) is commercially available.

Examples of protecting groups that may be employed in the synthetic routes described herein and the means for their removal can be found in T. W. Greene 'Protective Groups in Organic Synthesis,' 4th Edition, J. Wiley and Sons, 2006, incorporated herein by reference as it relates to such procedures.

For any of the hereinbefore described reactions or processes, conventional methods of heating and cooling may be employed, for example temperature-regulated oil-baths or temperature-regulated hot-blocks, and ice/salt baths or dry ice/acetone baths respectively. Conventional methods of isolation, for example extraction from or into aqueous or non-aqueous solvents may be used. Conventional methods of drying organic solvents, solutions, or extracts, such as shaking with anhydrous magnesium sulphate, or anhydrous sodium sulphate, or passing through a hydrophobic frit, may be employed. Conventional methods of purification, for example crystallisation and chromatography, for example silica chromatography or reverse-phase chromatography, may be used as required. Crystallisation may be performed using conventional solvents such as ethyl acetate, methanol, ethanol, or butanol, or aqueous mixtures thereof. It will be appreciated that specific reaction times and temperatures may typically be determined by reaction-monitoring techniques, for example thin-layer chromatography and LC-MS.

Where appropriate individual isomeric forms of the compounds of the invention may be prepared as individual isomers using conventional procedures such as the fractional crystallisation of diastereoisomeric derivatives or chiral high performance liquid chromatography (chiral HPLC).

The absolute stereochemistry of compounds may be determined using conventional methods, such as X-ray crystallography.

Methods of Use

It will be appreciated by those skilled in the art that references herein to treatment refer to the treatment of established conditions. However, the compound of formula (I) and pharmaceutically acceptable salts thereof may, depending on the condition, also be useful in the prevention of certain diseases. Thus, in one embodiment, there is provided the treatment or prevention of a disease. In another embodiment, there is provided the treatment of a disease. In a further embodiment, there is provided the prevention of a disease.

There is thus provided as a further aspect of the invention the compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy.

It will be appreciated that, when the compound of formula (I) or a pharmaceutically acceptable salt thereof is used in therapy, it is used as an active therapeutic agent. There is also therefore provided the compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of Chagas disease.

There is further provided the use of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prevention of Chagas disease.

There is further provided a method of treatment or prevention of Chagas disease, which method comprises administering to a human subject in need thereof, a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof.

Compositions

While it is possible that, for use in the methods of the invention, the compound of Formula (I) or a pharmaceutically acceptable salt thereof may be administered as the bulk substance, it is preferable to present the active ingredient in a pharmaceutical formulation, for example, wherein the agent is in admixture with at least one pharmaceutically acceptable carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

Accordingly, the present invention provides a pharmaceutical composition comprising a) the compound of Formula (I) or a pharmaceutically acceptable salt thereof and b) one or more pharmaceutically acceptable carriers. The term "carrier" refers to a diluent, excipient, and/or vehicle with which an active compound is administered. The pharmaceutical compositions of the invention may contain combinations of more than one carrier. Such pharmaceutical carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, 18th Edition. The choice of pharmaceutical carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise, in addition to the carrier, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), and/or solubilizing agent(s).

The phrase "pharmaceutically acceptable", as used herein, refers to salts, molecular entities and other ingredients of compositions that are generally physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., human). Suitably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in mammals, and more particularly in humans.

A "pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes an excipient that is acceptable for veterinary use as well as human pharmaceutical use. A "pharmaceutically acceptable excipient" as used in the present application includes both one and more than one such excipient.

The present invention is further related to a pharmaceutical composition for the treatment or prevention of Chagas disease comprising the compound of Formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers. The present invention is even further related to a pharmaceutical composition comprising a) 10 to 2000 mg of the compound of Formula (I) or a pharmaceutically acceptable salt thereof, and b) 0.1 to 2 g of one or more pharmaceutically acceptable excipients. In a further aspect, the present invention relates to a pharmaceutical composition comprising a) 1 to 2000 mg of the compound of Formula (I) or a pharmaceutically acceptable salt thereof, and b) 0.1 to 2 g of one or more pharmaceutically acceptable excipients.

It will be appreciated that pharmaceutical compositions for use in accordance with the present invention may be in the form of oral, parenteral, transdermal, inhalation, sublingual, topical, implant, nasal, or enterally administered (or other mucosally administered) suspensions, capsules or tablets, which may be formulated in conventional manner using one or more pharmaceutically acceptable carriers or excipients. In one aspect, the pharmaceutical composition is formulated for oral administration.

The compounds of the invention can be administered for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.

In one aspect, oral compositions are slow, delayed or positioned release (e.g., enteric especially colonic release) tablets or capsules. This release profile can be achieved, for example, by use of a coating resistant to conditions within the stomach but releasing the contents in the colon or other portion of the GI tract wherein a lesion or inflammation site has been identified. Or a delayed release can be achieved by a coating that is simply slow to disintegrate. Or the two (delayed and positioned release) profiles can be combined in a single formulation by choice of one or more appropriate coatings and other excipients. Such formulations constitute a further feature of the present invention.

Suitable compositions for delayed or positioned release and/or enteric coated oral formulations include tablet formulations film coated with materials that are water resistant, pH sensitive, digested or emulsified by intestinal juices or sloughed off at a slow but regular rate when moistened. Suitable coating materials include, but are not limited to, hydroxypropyl methylcellulose, ethyl cellulose, cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, polymers of metacrylic acid and its esters, and combinations thereof. Plasticizers such as, but not limited to polyethylene glycol, dibutylphthalate, triacetin and castor oil may be used. A pigment may also be used to color the film. Suppositories are be prepared by using carriers like cocoa butter, suppository bases such as Suppocire C, and Suppocire NA50 (supplied by Gattefosse Deutschland GmbH, D-Weil am Rhein, Germany) and other Suppocire type excipients obtained by interesterification of hydrogenated palm oil and palm kernel oil (C ₈ -Ci ₈ triglycerides), esterification of glycerol and specific fatty acids, or polyglycosylated glycerides, and whitepsol (hydrogenated plant oils derivatives with additives). Enemas are formulated by using the appropriate active compound according to the present invention and solvents or excipients for suspensions. Suspensions are produced by using micronized compounds, and appropriate vehicle containing suspension stabilizing agents, thickeners and emulsifiers like carboxymethylcellulose and salts thereof, polyacrylic acid and salts thereof, carboxyvinyl polymers and salts thereof, alginic acid and salts thereof, propylene glycol alginate, chitosan, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylcellulose, methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, N-vinylacetamide polymer, polyvinyl methacrylate, polyethylene glycol, pluronic, gelatin, methyl vinyl ether-maleic anhydride copolymer, soluble starch, pullulan and a copolymer of methyl acrylate and 2-ethylhexyl acrylate lecithin, lecithin derivatives, propylene glycol fatty acid esters, glycerin fatty acid esters, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyethylene glycol fatty acid esters, polyoxyethylene hydrated caster oil, polyoxyethylene alkyl ethers, and pluronic and appropriate buffer system in pH range of 6.5 to 8. The use of preservatives, masking agents is suitable. The average diameter of micronized particles can be between 1 and 20 micrometers, or can be less than 1 micrometer. Compounds can also be incorporated in the formulation by using their water-soluble salt forms.

Alternatively, materials may be incorporated into the matrix of the tablet e.g. hydroxypropyl methylcellulose, ethyl cellulose or polymers of acrylic and metacrylic acid esters. These latter materials may also be applied to tablets by compression coating.

Pharmaceutical compositions can be prepared by mixing a therapeutically effective amount of the active substance with a pharmaceutically acceptable carrier that can have different forms, depending on the way of administration. Pharmaceutical compositions can be prepared by using conventional pharmaceutical excipients and methods of preparation. The forms for oral administration can be capsules, powders or tablets where usual solid vehicles including lactose, starch, glucose, methylcellulose, magnesium stearate, di-calcium phosphate, mannitol may be added, as well as usual liquid oral excipients including, but not limited to, ethanol, glycerol, and water. All excipients may be mixed with disintegrating agents, solvents, granulating agents, moisturizers and binders. When a solid carrier is used for preparation of oral compositions (e.g., starch, sugar, kaolin, binders disintegrating agents) preparation can be in the form of powder, capsules containing granules or coated particles, tablets, hard gelatin capsules, or granules without limitation, and the amount of the solid carrier can vary (between 1 mg to lg). Tablets and capsules are the preferred oral composition forms.

Pharmaceutical compositions containing the compounds of the present invention may be in any form suitable for the intended method of administration, including, for example, a solution, a suspension, or an emulsion. Liquid carriers are typically used in preparing solutions, suspensions, and emulsions. Liquid carriers contemplated for use in the practice of the present invention include, for example, water, saline, pharmaceutically acceptable organic solvent(s), pharmaceutically acceptable oils or fats, and the like, as well as mixtures of two or more thereof. The liquid carrier may contain other suitable pharmaceutically acceptable additives such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickening agents, viscosity regulators, stabilizers, and the like. Suitable organic solvents include, for example, monohydric alcohols, such as ethanol, and polyhydric alcohols, such as glycols. Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, and the like. For parenteral administration, the carrier can also be an oily ester such as ethyl oleate, isopropyl myristate, and the like. Compositions of the present invention may also be in the form of microparticles, microcapsules, liposomal encapsulates, and the like, as well as combinations of any two or more thereof.

Examples of pharmaceutically acceptable disintegrants for oral compositions useful in the present invention include, but are not limited to, starch, pre-gelatinized starch, sodium starch glycolate, sodium carboxymethylcellulose, croscarmellose sodium, microcrystalline cellulose, alginates, resins, surfactants, effervescent compositions, aqueous aluminum silicates and crosslinked polyvinylpyrrolidone.

Examples of pharmaceutically acceptable binders for oral compositions useful herein include, but are not limited to, acacia; cellulose derivatives, such as methylcellulose, carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxypropylcellulose or hydroxyethylcellulose; gelatin, glucose, dextrose, xylitol, polymethacrylates, polyvinylpyrrolidone, sorbitol, starch, pre-gelatinized starch, tragacanth, xanthane resin, alginates, magnesium-aluminum silicate, polyethylene glycol or bentonite.

Examples of pharmaceutically acceptable fillers for oral compositions include, but are not limited to, lactose, anhydrolactose, lactose monohydrate, sucrose, dextrose, mannitol, sorbitol, starch, cellulose (particularly microcrystalline cellulose), dihydro- or anhydro- calcium phosphate, calcium carbonate and calcium sulfate.

Examples of pharmaceutically acceptable lubricants useful in the compositions of the invention include, but are not limited to, magnesium stearate, talc, polyethylene glycol, polymers of ethylene oxide, sodium lauryl sulfate, magnesium lauryl sulfate, sodium oleate, sodium stearyl fumarate, and colloidal silicon dioxide.

Examples of suitable pharmaceutically acceptable flavourings for the oral compositions include, but are not limited to, synthetic aromas and natural aromatic oils such as extracts of oils, flowers, fruits (e.g., banana, apple, sour cherry, peach) and combinations thereof, and similar aromas. Their use depends on many factors, the most important being the organoleptic acceptability for the population that will be taking the pharmaceutical compositions.

Examples of suitable pharmaceutically acceptable dyes for the oral compositions include, but are not limited to, synthetic and natural dyes such as titanium dioxide, beta-carotene and extracts of grapefruit peel.

Suitable examples of pharmaceutically acceptable sweeteners for the oral compositions include, but are not limited to, aspartame, saccharin, saccharin sodium, sodium cyclamate, xylitol, mannitol, sorbitol, lactose and sucrose.

Suitable examples of pharmaceutically acceptable buffers include, but are not limited to, citric acid, sodium citrate, sodium bicarbonate, dibasic sodium phosphate, magnesium oxide, calcium carbonate and magnesium hydroxide. Suitable examples of pharmaceutically acceptable surfactants include, but are not limited to, sodium lauryl sulfate and polysorbates.

Suitable examples of pharmaceutically acceptable preservatives include, but are not limited to, various antibacterial and antifungal agents such as solvents, for example ethanol, propylene glycol, benzyl alcohol, chlorobutanol, quaternary ammonium salts, and parabens (such as methyl paraben, ethyl paraben, propyl paraben, etc.).

Suitable examples of pharmaceutically acceptable stabilizers and antioxidants include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), thiourea, tocopherol and butyl hydroxyanisole.

The compounds of the invention may also, for example, be formulated as suppositories e.g., containing conventional suppository bases for use in human or veterinary medicine or as pessaries e.g., containing conventional pessary bases.

The compounds according to the invention may be formulated for topical administration, for use in human and veterinary medicine, in the form of ointments, creams, gels, hydrogels, lotions, solutions, shampoos, powders (including spray or dusting powders), pessaries, tampons, sprays, dips, aerosols, drops {e.g., eye ear or nose drops) or pour- ons.

For application topically to the skin, the compounds of the present invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. Such compositions may also contain other pharmaceutically acceptable excipients, such as polymers, oils, liquid carriers, surfactants, buffers, preservatives, stabilizers, antioxidants, moisturizers, emollients, colorants, and flavourings. Examples of pharmaceutically acceptable polymers suitable for such topical compositions include, but are not limited to, acrylic polymers; cellulose derivatives, such as carboxymethylcellulose sodium, methylcellulose or hydroxypropylcellulose; natural polymers, such as alginates, tragacanth, pectin, xanthan and cytosan.

As indicated, the compounds of the present invention can be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, spray or nebulizer with the use of a suitable propellant, e.g., a hydrofluoroalkane such as 1,1,1,2- tetrafluoroethane (HFA 134AT) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA), or a mixture thereof. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container, pump, spray or nebulizer may contain a solution or suspension of the active compound, e.g., using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g., sorbitan trioleate.

Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound and a suitable powder base such as lactose or starch.

For topical administration by inhalation the compounds according to the invention may be delivered for use in human or veterinary medicine via a nebulizer.

The pharmaceutical compositions of the invention may contain from 0.01 to 99% weight per volume of the active material. For topical administration, for example, the composition will generally contain from 0.01-10%, more preferably 0.01-1% of the active compound.

A therapeutically effective amount of the compounds of the present invention can be determined by methods known in the art. The therapeutically effective quantities will depend on the age and on the general physiological condition of the subject, the route of administration and the pharmaceutical formulation used. The therapeutic doses will generally be between about 1 and 2000 mg/day, 5 and 2000 mg/day, 10 and 2000 mg/day and suitably between about 30 and 1500 mg/day. Other ranges may be used, including, for example, 50-500 mg/day, 50-300 mg/day, 50-100 mg/day, 100-200 mg/day, 5-100 mg/day, 5-50 mg/day. The daily dose as employed for acute or chronic human treatment will range from 0.01 to 250 mg/kg body weight, suitably 0.1-5 mg/kg body weight, suitably 0.1-10 mg/kg body weight, suitably 2-100 mg/kg body weight, or suitably 5-60 mg/kg body weight, which may be administered in one to four daily doses, for example, depending on the route of administration and the condition of the subject. When the composition comprises dosage units, each unit will contain 1 mg to 2 g of active ingredient, suitably 10 mg to 2 g of active ingredient, suitably 200 mg to 1 g of active ingredient, suitably 5 to 300 mg of active ingredient.

Administration may be once a day, twice a day, or more often, and may be decreased during a maintenance phase of the disease or disorder, e.g. once every second or third day instead of every day or twice a day. The dose and the administration frequency will depend on the clinical signs with the reduction or absence of at least one or more, preferably more than one, clinical signs of the acute phase known to the person skilled in the art. In one aspect of the present invention, administration is once daily oral dosing.

In one aspect, the present invention provides a combination comprising a) the compound of Formula (I) or a pharmaceutically acceptable salt thereof and b) one or more further therapeutically active agents.

The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical composition and thus pharmaceutical compositions comprising a combination as defined above together with one or more pharmaceutically acceptable carriers thereof represent a further aspect of the invention.

The individual components of such combinations may be administered together or separately and, when administered separately, administration may occur simultaneously or sequentially, in any order. Such sequential administration may be close in time or remote in time. Appropriate doses of known therapeutic agents will be readily appreciated by those skilled in the art. There is also provided a process for preparing such a pharmaceutical composition which comprises admixing a compound of formula (I), or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable excipients. Aspects of the invention are illustrated by reference to, but are in no way limited by, the following Examples.

Examples Analytical Methodology H NMR

^ NMR spectra were recorded in either CDCI ₃ or DMSO-d6 on a Bruker DPX 400 MHz. The internal standard used was the residual protonated solvent at 7.26 ppm for CDCI3 or 2.49 ppm for DMSO-d6.

UPLC-MS

Column: 50mm x 2.1mm ID, 1.7 _μ ιη Acquity UPLC CSH C ₁₈

Flow Rate: lmL/min.

Temp: 40°C

UV detection range: 210 to 350nm

Mass spectrum: Recorded on a mass spectrometer using alternative-scan positive and negative mode electrospray ionization

Solvents: A: 0.1% v/v formic acid in water

B: 0.1% v/v formic acid acetonitrile

Gradient: Time (min.l A% B%

0 97 3

1.5 0 100

1.9 0 100

2.0 97 3

LCMS Column: 20mm x 2.1mm ID, 1.9 μηη Thermo Hypersil Gold Cis

Flow Rate: 1.6 mlVmin.

Temp: 45°C

UV detection range: 210 to 350nm

Mass spectrum: Recorded on a mass spectrometer using alternative-scan positive and negative mode electrospray ionization

Solvents: A: 0.02% v/v trifluoroacetic acid in water

B: 0.02% v/v trifluoroacetic acid in acetonitrile

Gradient: Time (min.l A% B%

0.02 98 2

1.9 5 95

1.91 96 4

Abbreviations

The following list provides definitions of certain abbreviations as used herein. It will be appreciated that the list is not exhaustive, but the meaning of those abbreviations not herein below defined will be readily apparent to those skilled in the art.

ACN Acetonitrile

Cy Cyclohexane

DCM Dichloromethane

DIPEA Diisopropylethylamine

DMEM Dulbecco's Modified Eagle ^' s Medium

DMSO Dimethylsulphoxide

THF Tetrahydrofuran

EtOAc Ethyl acetate

EtOH Ethanol

FBS Fetal Bovine Serum

HCI Hydrochloric acid

HEPES N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)

MeOH Methanol

Na ₂ C0 ₃ Sodium Carbonate

NaHC0 ₃ Sodium Hydrogen Carbonate

Na ₂ S0 ₄ Sodium Sulphate NH4CI Ammonium Chloride

PBS Phosphate Buffered Solution

TLC Thin Layer Chromatography Reaction Intermediates

Intermediate 1: ri -benzothiazol-S-ylfmethoxy^methylidenelpropanedinitrile

To l,3-benzothiazole-5-carboxylic acid (3.5 g, 19.5 mmol) purchased from COMBI- BLOCKS, (catalog number SS-6875) thionyl chloride (9.8 ml, 135 mmol) purchased from ALDRICH (catalog number 230464) was added and the resulting reaction mixture was refluxed at 80°C for 2 hours. After this time UPLC-MS monitoring showed complete conversion into the desired acyl chloride (the formation of the expected methyl ester MW: 193 was observed when crude reaction quenched in MeOH). The solvent was removed under vacuo (temperature of the bath: 50°C), and the residue was taken up with toluene twice to remove any trace of the thionyl chloride. The obtained crude (yellow solid, 3.9 g) was suspended in toluene (18 ml) and tetrahydrofuran (3.5 ml), then a solution of malononitrile (1.3 g, 19.5 mmol) purchased from ALDRICH (catalog number M140-7) in THF (3.5 ml) was added. The reaction mixture was cooled to 0°C; a solution of DIPEA (6.8 ml, 39 mmol) purchased from ALDRICH (catalog number 03440) in toluene (13 ml) was added dropwise. The mixture was stirred at room temperature for 40 minutes. Monitoring by UPLS-MS showed the formation of the desired product M/Z = 227.9 (M+l) and only traces of unreacted starting material. The solvent was removed under vacuo and the obtained crude (brown oil, 4.5 g) was suspended in acetonitrile (60 ml) and methanol (8 ml). The reaction mixture was cooled to 0°C, then a solution 2 M in hexane of trimethylsilyldiazomethane purchased from ALDRICH (catalog number 362832) was added (23.4 ml, 46.8 mmol ) followed by DIPEA (7 ml, 39 mmol). The mixture was stirred at room temperature for 5 hours. Unreacted starting material was observed. Trimethylsilyldiazomethane (10 ml, 19.5 mmol) was added. After 1 hour stirring at room temperature the UPLS-MS monitoring of the reaction showed 60% of conversion into the desired product M/Z = 241.2 (M+l). The reaction mixture was stirred at room temperature overnight and monitored again by UPLS-MS (80% conversion).

The reaction mixture was evaporated to dryness. The crude (6.5 g) was purified by flash- chromatography (150 g Si02, eluting with a gradient Cy:EtOAc 60:40 to 40:60), to obtain 1.2 g of the desired product [l,3-benzothiazol-5-yl(methoxy)methylidene]propanedinitrile with a purity of 80% and other 541 mg of contaminated product (50% purity).

LCMS and 1HNMR confirmed the identity of the product [l,3-benzothiazol-5- yl(methoxy)methylidene]propanedinitrile (1200 mg, 80% purity, 3.97 mmol, 20.1 % yield).

1H NMR (400 MHz, DMSO-d6): δ 9.6 (s, 1H), 8.5 (m, 2H), 7.76 (dd, 1H), 3.93 (s, 3H) M/Z = 241.2 (M+l)

Intermediate 2: 5-amino-3-(1.3-benzothiazol-5-yn-l-(2.2.2-trifluoroethyn-lH- pyrazole-4- carbonitrile

[l,3-benzothiazol-5-yl(methoxy)methylidene]propanedinitrile (Intermediate 1; 0.882 g, 3.7 mmol) was dissolved in ethanol (36.5 ml), then a solution of (2,2,2- trifluoroethyl)hydrazine 70% wt in water (1.3 ml, 10.4 mmol) purchased from Aldrich (catalog number 129046) was added, followed by Et ₃ N (1.3 ml, 9.3 mmol). The reaction mixture was stirred at room temperature for 40 minutes. UPLC-MS monitoring showed complete conversion into the desired product M/Z = 324.0 (M+l); an impurity M/Z = 241.2 (M+l) was also observed (starting material).

The solvent was removed under vacuo. The obtained crude was taken up in EtOAc and transferred in a separatory funnel, then an aqueous solution of NH ₄ CI was added. The two phases were separated and the aqueous layer was extracted twice with EtOAc. The combined organic layers were dried on Na ₂ S0 ₄ , filtered and concentrated under vacuo to give 1.06 g of the desired product 5-amino-3-(l,3-benzothiazol-5-yl)-l-(2,2,2- trifluoroethyl)-l pyrazole-4-carbonitrile as a yellow solid (3.07 mmol, 70% purity, 83%yield).

1H NMR (400 MHz, DMSO-d6): δ 9.48 (s, 1H), 8.47 (s, 1H), 8.29 (d, 1H), 7.91 (dd, 1H), 7.25 (m, 2H), 5.04 (dd, 2H)

Intermediate 3: 3-(l,3-benzothiazol-5-yn-5-(methylamino)-l-(2,2,2-trifluoroe thyn- lH-pyrazole-4-carbonitrile

5-amino-3-(l,3-benzothiazol-5-yl)-l-(2,2,2-trifluoroethyl ^

(Intermedaite 2; 3.28 mmol, 1.06 g) was suspended in triethylorthoformate (147 mmol, 24.5 ml) purchased from Aldrich (catalog number T60453), then p-toluensulfonic acid (0.45 mmol, 87 mg) was added. The reaction mixture was refluxed for 1 hour. UPLC-MS of the reaction showed no starting material left. The solvent was removed under vacuum, and the obtained crude was diluted with dichloromethane and washed with a saturated solution of sodium bicarbonate. The aqueous phase was extracted twice with dichloromethane. The combined organics were dried on sodium sulphate, filtered and evaporated to dryness to give a brown oil which was dissolved in ethanol (100 ml) and NaBH ₄ (15.5 mmol, 586 mg) was added at 0°C. The reaction mixture was stirred at room temperature for 30 minutes. UPLC-MS of the reaction showed complete conversion into the desired product M/Z = 338.9 (M+l).

Brine and EtOAc were added and the two phases were separated. The aqueous phase was extracted with EtOAc and the combined organics phases were evaporated to dryness. The crude was purified by flash chromatography on silica eluting with a gradient Cy:AcOEt 6:4 to 1: 1, giving 750 mg of a yellow solid which is the title desired product 3-(l,3-benzothiazol-5-yl)-5-(methylamino)-l-(2,2,2-trifluoro ethyl)-lH-pyrazole-4-carbonitri le.

1H NMR (400 MHz, DMSO-d6): δ 9.49 (s, 1H), 8.3 (d, 1H), 7.91 (dd, 1H), 7.25 (m, 1H), 5.0 (dd, 2H), 3.14 (d, 3H)

M/Z = 338.9 (M+l)

Intermediate 4: 3,5-dibromo-lH-pyrazole-4-carbonitrile

To lH-pyrazole-4-carbonitrile (6.76 g, 52.2 mmol) purchased from Aldrich (catalog number CDS008901) were added ethanol (150 mL) and water (225 mL). Then sodium acetate (29.1 g, 355 mmol) was added and the mixture was stirred until solids completely dissolved. Then bromine (10.75 mL, 209 mmol) was added dropwise. After 2 hours the reaction was complete. The reaction was diluted with water then extracted with DCM (3X150ml). The combined organic phases were washed with a saturated aqueous solution of sodilum thiosulfate (Na ₂ S203). Poor layer seperation was observed in separating funnel, so water was added until DCM layer was in the bottom. The organic phase was then dired over MgS0 ₄ , filtered and concentrated to form a solid. Purification: the solid was first dissolved in EtOAc. When the entire solid was dissolved, additional EtOAc was added to almost double the starting volume. Then hexane was added until solution turned slightly cloudy. After 30 min decantation, the small amount of precipitate formed in the flask was filtered, a dark brown oil left on the filter. The much lighter amber color solution that filtered though was then concentrated and contained mostly the desired product of 3,5-dibromo-lH-pyrazole-4-carbonitrile (8.9 g, 35.5 mmol, 68.0 % yield), it was used without further purification.

No proton in the structure (only NH), no NMR spectrum.

LCMS: the compound does not ionize.

Intermediate 5: 3,5-dibromo-l-(2,2,2-trifluoroethyn-lH-pyrazole-4-carbonitri le

Sodium hydride (60% dispersion in mineral oil) (0.365 g, 9.13 mmol) was added to 3,5- dibromo-lH-pyrazole-4-carbonitrile (Intermediate 4; 2 g, 5.98 mmol) in N,N- Dimethylformamide (DMF) (30 ml.) and stirred at room temperature for 15 minutes. The reaction became dark red. Trifluoromethanesulfonic acid 2,2,2-trifluoroethyl ester (2.94 g, 11.96 mmol) purchased from Aldrich (catalog number 752924) was then added and the reaction heated in a 70°C heating block. After 3 hrs the reaction was allowed to cool to room temperature and stirred overnight. The reaction was then concentrated directly on the blow-down unit (over 3 days) to afford a brown solid with some mineral oil residual. This material was dissolved in DCM and purified by Biotage (50g SNAP, 0-10% EtOAc/hex). The resulting white powder was pure product, 2.06 g (5.86 mmol, 95% purity, 98% yield).

1H NMR (400 MHz, CDCI ₃ ): δ 4.80 (dd, 2H)

LCMS: M/Z = 332.0 (M+l) Intermediate 6: 3-bromo-5-(methylamino)-l-(2,2,2-trifluoroethyn-lH-pyrazole- 4- carbonitrile

A 20 ml. microwave vial was charged with 3,5-dibromo-l-(2,2,2-trifluoroethyl)-lH- pyrazole-4-carbonitrile (Intermediate 5; 390 mg, 1.172 mmol), N-Methyl-2-pyrrolidone (NMP) (3 ml_), and methylamine in THF (1.5 ml_, 2M, 3.00 mmol) purchased from Aldrich (catalog number 395056). The reaction was placed in a 90 °C heating block. After 3.5 hours the reaction was cooled to room temperature, diluted with 50 ml. water and extracted with DCM and then EtOAc. The DCM efficiently extracts all desired product. The DCM layer was concentrated and then the residual NMP was removed on the high vac (1.5 days). The resulting orange oil was purified on Biotage (50g SNAP column, 10- 100% EtOAc/Hexanes). Single large peak elutes around 45% and contains product mass. By LCMS there appears to be a single isomer formed in the addition reaction.

Material used directly in the next step

1H NMR (400 MHz, CDCI ₃ ): δ 4.48 (dd, 2H), 3.26 (d, 3H)

LCMS: M/Z = 284.9 (M+l)

Intermediate 7: 3-(l,3-benzothiazol-5-yn-5-(methylamino)-l-(2,2,2-trifluoroe thyn-lH- pyrazole-4-carbonitrile

A 20 ml. microwave vial was charged with 3-bromo-5-(methylamino)-l-(2,2,2- trifluoroethyl)-lH-pyrazole-4-carbonitrile (Intermediate 6; 230 mg, 0.813 mmol), 5- (4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-l,3-benzothiaz ole (318 mg, 1.219 mmol) purchased from ASDI, 1,4-Dioxane (130 ml.) and an aqueous solution of sodium carbonate 2M (1.219 ml_, 2.438 mmol). The reaction was purged with argon for 5 min, then PdCI ₂ (dppf)-CH ₂ Cl ₂ adduct (133 mg, 0.163 mmol) purchased from Aldrich (catalog number 379670) was added. The vessel was sealed and heated in an 120°C heating block. After 20 hours the reaction was cooled to room temperature, dried with MgS0 ₄ , filtered and concentrated. The crude material was purified on Biotage (50g SNAP column, 10-100% EtOAc/Hexanes). Desired product elutes at 100% EtOAc to yield 225 mg of a pinkish solid (0.534 mmol, 80% purity, 65 %yield), used as such in the next step.

1H NMR (400 MHz, DMSO-d6): δ 9.08 (s, 1H), 8.70 (bs, 1H), 8.00 (m, 2H), 4.60 (dd, 2H), 3.36 (s, 3H).

LCMS: M/Z = 338.2 (M+l)

Example 1 : 3-(l,3-benzothiazol-5-yn-5-(methylaminoVl-(2,2,2-tnfluoroeth yn-lH-

In a 4 ml. vial 3-(l,3-benzothiazol-5-yl)-5-(methylamino)-l-(2,2,2-trifluoro ethyl)-lH- pyrazole-4-carbonitrile (Intermediate 3; 0.1 g, 0.3 mmol) was dissolved in H ₂ S0 ₄ (2 ml_). The reaction mixture was stirred for 2 hours at room temperature and subsequently, heated at 50 °C for 16 hours and poured into ice. To the aqueous was basified with Na ₂ CC>3, extracted three times with EtOAc. The organics were combined, dried over Na ₂ S0 ₄ , filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on NH-modified silica gel (11 g cartridge; gradient: 10% -60 % EtOAc in Cy) to give 3-(l,3-benzothiazol-5-yl)-5-(methylamino)-l-(2,2,2-trifluoro ethyl)- lH-pyrazole-4-carboxamide (0.057 g, 0.16 mmol, 54% yield) as a white solid. 1H NMR spectrum was consistent with the structure of title compound.

1H NMR (400 MHz, DMSO-d6): δ 9.42 (s, 1H), 8.29 (d, 1H), 8.18 (d, 1H), 7.75 (dd, 1H), 7.3 (m, 2H), 6.06 (dd, 1H), 4.98 (dd, 2H), 2.89 (d, 3H)

M/Z = 355.9 (M+l) Example 2: 3-(l,3-benzothiazol-5-yn-5-(methylamino)-l-(2,2,2-trifluoroe thyn-lH- pyrazole-4-carboxamide trifluoroacetate salt

To a suspension of 3-(l,3-benzothiazol-5-yl)-5-(methylamino)-l-(2,2,2-trifluoro ethyl)-lH- pyrazole-4-carbonitrile (Intermediate 7; 150 mg, 0.445 mmol) in toluene (2 ml.) and 1,4- Dioxane (1 ml.) in a 20 ml. reaction vial was added sulfuric acid (0.5 ml_, 9.38 mmol). The reaction was placed in a 90 °C heating block. After 25 hours the reaction was cooled to room temperature. The reaction was diluted with water (lOmL), poured into saturated bicarbonate solution (50ml_), and extracted with EtOAc (3x50ml_). The combined organic phases were dried with MgS0 ₄ , filtered and concentrated.

Gilson RP HPLC (2810, 5-50% ACN, 15min). Broad peak, but LCMS indicates very pure product. Fractions showing desired product were combined and concentrated to afford the TFA salt 44.6 mg (0.09 mmol, 95% purity, 20% yield) as a white powder.

1H NMR (400 MHz, DMSO): δ 9.42 (s, 1H), 8.28 (d, 1H), 8.18 (d, 1H), 7.75 (dd, 1H), 7.31 (m, 2H), 6.06 (m, 1H), 4.98 (dd, 2H), 2.88 (s, 3H).

LCMS: M/Z = 356.1 (M+l)

Biological Evaluation

Compounds of the invention were tested for in vitro biological activity in accordance with the following assay.

Parasite and mammalian cell cultures

NIH-3T3 cells (mouse endothelial fibroblasts) used as host-cells in the T. cruzi intracellular assay were made available by GSK-Biological Reagents and Assay Development Department (BRAD, Stevenage, UK). Cell line was grown at 37 °C, 5% C02 and >95% humidity, in DMEM (Life-Technologies) supplemented with 10% FBS (Biowest, USA), 100 U/mL penicillin (Sigma-Aldrich), 100 μg/mL streptomycine (Sigma-Aldrich), and 4 mM L-Glutamine (Sigma-Aldrich). The DMEM formulation for the assay lacked phenol red (Life-Technologies reference 31053) and was supplemented with 2% FBS, 100 U/mL penicillin, 100 μg/mL streptomycine, 2 mM L-Glutamine, 1 mM sodium- pyruvate (Life-Technologies), and 25 mM HEPES (Life-Technologies). NIH-3T3 cells required for the whole HTS campaign were produced in advance and stored frozen at - 150°C until needed. Freezing medium contained 90% FBS and 10% DMSO and cells were frozen in 5 mL cryo-vials. On average, vials cell density was 1.7x107 cells per mL and the quality control assessment indicated that the percentage of viability was in all cases over 95.5%.

H9c2 (rat cardiomyocytes) were purchased at the European Cell Cultures Collection (ECACC, Salisbury, UK) and were grown at 37°C, 5% C02 and >95% humidity in DMEM (Life-Technologies) supplemented with 10% FBS (Biowest, USA), 100 U/mL penicillin (Sigma-Aldrich), 100 μg/mL streptomycine (Sigma-Aldrich), and 2 mM L-Glutamine (Sigma-Aldrich).

T. cruzi parasites from the Tulahuen strain expressing β-galactosidase were kindly provided by Dr. Buckner (University of Washington, Seattle, USA; Buckner et al., 1996). Parasites were maintained in culture by weekly infection of LLC-MK2 cells (green monkey kidney epithelial cells, purchased from the European Cell Cultures Collection (ECACC reference 85062804)) in the same DMEM formulation used for cell growth, but supplemented with 2% FBS instead. Trypomastigote forms were obtained from the supernatants of LLC-MK2 infected cultures harvested between days 5 and 9 of infection as described in Bettiol et al (2009) PLoS Negl Trop Dis 3:e384. A mass production of trypomastigotes was made in T225 cm ² flasks (Corning Inc., NY, USA) to ensure an appropriate quantity of parasites for the whole HTS campaign. The number of trypomastigotes per ml. was determined in a Neubauer cell chamber and their density brought to 1x108 trypomastigotes per ml. for freezing purposes, which was accomplished in a 90% FBS, 10% DMSO freezing medium, following standard procedures.

T. cruzi intracellular reporter assay (Primary Assay ^" )

The assay developed was adapted from the one previously described by Bettiol et al. (2009) PLoS Negl Trop Dis 3:e384, which had been used in the anti-T. cruzi HTS performed at the Broad Institute (Harvard, USA). Active compounds in this assay format will target intracellular T. cruzi amastigotes growing in NIH-3T3 murine fibroblasts, though to a shorter extent they may also target free swimming trypomastigotes and/or the host-parasite interactions required for the parasite invasion. The assay was set up in 1536-wells tissue culture surface treated plates (Greiner Cat. # 782092). The biological reagents consisted of NIH-3T3 host cells and trypomastigotes parasitic cells, thawed and straight forwardly mixed at a 1: 1 (v:v) ratio. Upon thawing, both cell types were set to a 3.3x105 cells/mL dilution in assay DMEM by means of a CASY cell counter device (Roche-Applied Science) using a 60 μιτι capillary. 6 μΙ_ of the mixture were dispensed per well (about 1x103 host-cells and 1x103 parasites) with a Multidrop Combi liquid handling apparatus (Thermo Scientific). Beforehand, a 1.67x105 trypomastigotes per ml. solution in assay DMEM had been prepared and 6 μΙ_ per well dispensed in the allotted control columns that defined a 0% parasitic growth (or 100% of inhibition). As opposed to it, the control of 0% inhibition of parasitic growth was obtained leaving the assay mixture untreated in its corresponding plate columns. In all wells the percentage of DMSO never exceeded 0.5%. Plates were lidded and incubated for four days under a saturated humidity at 37°C and 5% C0 ₂ . The substrate used for the assay fluorescence intensity (FLINT) readout was resorufin- -D-galactopiranoside (Sigma-Aldrich) at 5 μΜ per well. It was diluted in PBS supplemented with the soft detergent Igepal (Fluka) as described by Buckner FS et al (1996) Antimicrobial Agents Chemotherapy 40:2592-2597. Upon addition to the plates, the substrate solution was incubated for 4 hours at room temperature and the signal read with the EnVision microtiter plate reader (Perkin-Elmer) using the corresponding set of filters (Excitation/Emission at 531/595 nm).

This assay was run to primary screen the compound at single shot at 5 μΜ compound concentration per well. T. cruzi intracellular imaging assay (Secondary hit confirmation assay)

H9c2 cells (rat cardiomyocytes) were seeded in T-225 flasks (225 cm2 culture surface; Corning Inc., NY, USA) in DMEM-10% FBS for 4 hours to allow attachment. Cells were then washed once with PBS before infection. T. cruzi trypomastigotes, collected at days 5 to 8 after infection, from LLC-MK2 parasite infected cultures, were allowed to swim out for 4 hours at 37°C from a centrifuged pellet (2,500 rpm/10 min/room temperature). Trypomastigotes were then collected and counted in a CASY Cell Counter (Roche- Applied-Science). Trypomastigotes, in supplemented DMEM, were added to H9c2 cultures in a multiplicity of infection (MOI) of 1 and incubated for 18 hours. Cells were washed once with PBS before incubation of the infected H9c2 monolayer with trypsin (Life- Technologies) to detach cells from the flask. Cells were counted in a CASY Cell Counter (Roche-Applied-Science) using a 150 μιτι capillary and their density set at 5x104 cells per ml. in supplemented assay DMEM. Infected H9c2 cells were dispensed into the 384 wells ^Oear bottom, Poly-Lysine coated plate (Greiner, Cat. # 781946) at 50 μΙ_ per well using a Multidrop Combi liquid handling device (Thermo Scientific). After seeding them, the plates were incubated at 37°C in a humidified 5% C0 ₂ atmosphere for 72 hours. Cultures were then fixed and stained by addition of 50 μΙ_ of a solution containing 8% formaldehyde and 4 μΜ DRAQ5 DNA dye (BioStatus, UK) per well. Plates were kept light-protected and imaged one hour later.

Plates were imaged in a Perkin-Elmer Opera microscope using a 20X air objective (NA 0.4) and the following acquisition set: a 635 nm laser excitation line and a 690/50 emission detection filter for DRAQ5 detection. Five images were collected per well for reliable statistical analysis.

Automated image analysis was performed with a script developed on Acapella® High Content Imaging and Analysis Software (PerkinElmer). Three outputs were provided for each sample well: (1) number of host cells nuclei ("Cells") to determine drug-related cytotoxicity; (2) number of amastigotes per cell (" ^Am /Cell") as infection level measurement; and (3) percentage of infected cells per well ("%Infected") as a second infection marker. Results

The compounds of Example 1 and 2 had the following activity:

IC50 Modifier average IC50 μΜ n . cruzi intracellular reporter assay (Primary Assay)

Example 1 T. cruzi = 0.10 4

Example 2 T. cruzi = 0.20 10

T. cruzi intracellular imag ing assay (Secondary Assay)

IC50 Modifier average IC50 μΜ n

Example 1 AM/cell = 0.06 6

Example 2 AM/cell = 0.16 23

Example 1 Cells > 50.12 6

Example 2 Cells > 50.12 23

Example 1 Inf = 0.13 6 cells

Example 2 Inf = 0.25 23 cells