Pyrazine compounds used in treatment of parasitic protozoal infection

Abstract

FIELD: pharmaceutical composition.SUBSTANCE: compound selected from the group of compounds of the structural formulas indicated below, or a pharmaceutically acceptable salt thereof. A pharmaceutical composition containing a compound of the present invention and the use of said compound or pharmaceutical composition are also provided.EFFECT: compound of the present invention is effective against malaria and may be useful in the treatment of parasitic protozoan infections caused by the malarial parasite Plasmodiumfalciparum.17 cl, 2 dwg, 6 tbl, 11 ex

Description

[0001] FIELD OF THE INVENTION

[0002] The present application relates to compounds and their pharmaceutically acceptable salts, their compositions and their use for the treatment and prevention of systemic infections, for example for the treatment and prevention of parasitic protozoan infection such as malaria, in particular infection caused by Plasmodium falciparum:

[0003] BACKGROUND OF THE INVENTION

[0004] Parasitic infections cause a wide range of diseases of medical and veterinary significance, such as malaria in humans and coccidiosis in birds, fish, and mammals. Many of these diseases are life-threatening to the host and cause significant economic losses to livestock production.

[0005] Malaria is a disease caused by protozoan parasites of the genus Plasmodium that infect and destroy red blood cells, leading to fever, severe anemia, cerebral malaria, and, if untreated, death. There are five species of Plasmodium parasites: falciparum, vivax, ovale, Malariae, and knowlesi. Plasmodium falciparum is the most virulent. In 2019, an estimated 229 million people were infected with malaria in 87 malaria-endemic countries, and malaria caused 409,000 deaths (The global malaria report 2020: 20 years of global progress and challenges. Geneva: World Health Organization; 2020, Switzerland).

[0006] Effective oral drugs for the prevention and treatment of malaria are known in the art. For example, known treatment / prophylaxis methods include artemisinin and artemisinin-based combination therapies (ACTs). ACTs are essentially the standard treatment for Plasmodium falciparum in uncomplicated cases (WHO Malaria Manual. Geneva: World Health Organization; 2021. License: CC BY-NC-SA 3.0 IGO). However, artemisinins are associated with delayed parasite clearance, which is associated with less favorable clinical outcomes. Emerging resistance to ACTs is a particular concern.

[0007] One approach to solving this problem is the development of new therapeutic agents with novel mechanisms of action. International patent application PCT / EP2016 / 074875 (published as WO 2017 / 067881) discloses pyrazine compounds and their use in the treatment of parasitic protozoan infections. However, there is a need to develop new therapeutic agents with improved physicochemical properties, whole-cell activity, in vivo efficacy, and predictable in vitro toxicity.

[0008] SUMMARY OF THE INVENTION

[0009] In a first aspect, the present invention provides a compound of formula (I):

[0010]

[0011] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0012] each R 1 , R 2 , R 3 and R 4independently selected from the group consisting of H, halogen, C1-C6 alkyl, and a 3-7 membered cycloalkyl ring optionally containing a heteroatom selected from the group consisting of O, S, SO, SO2, NH, and N-C1-C6 alkyl; or

[0013] R 1 and R 2 together with the atom to which they are simultaneously attached, form a 3-, 4-, 5-, 6-, or 7-membered cycloalkyl ring, optionally containing a heteroatom selected from the group consisting of O, S, SO, SO2, NH, and N-C1-C6 alkyl, where each R 3 and R 4 independently selected from the group consisting of H, C1-C6 alkyl, and a 3-7 membered cycloalkyl ring optionally containing a heteroatom selected from the group consisting of O, S, SO, SO2, NH, and N-C1-C6 alkyl; or

[0014] R 1 and R 3together with the atom to which they are simultaneously attached, form a 3-, 4-, 5-, 6-, or 7-membered cycloalkyl ring, optionally containing a heteroatom selected from the group consisting of O, S, SO, SO2, NH, and N-C1-C6 alkyl, where each R 3 and R 4 is independently selected from the group consisting of H, C1-C6 alkyl, and a 3-7 membered cycloalkyl ring optionally containing a heteroatom selected from the group consisting of O, S, SO, SO2, NH, and N-C1-C6 alkyl; and where

[0015] R 5 selected from the group consisting of halogen and C1-C6 alkyl.

[0016] The second aspect of the present invention provides a compound selected from the group consisting of N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, N-(2-amino-2-methylpropyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, N-(2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide and N-((1-aminocyclopropyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide.

[0017] In a third aspect, the present invention provides a pharmaceutical composition comprising (a) a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof; and (b) a pharmaceutically acceptable excipient.

[0018] In a fourth aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof for use in the treatment of a parasitic protozoan infection.

[0019] In a fifth aspect of the present invention, there is provided the use of a compound of formula (I) or a pharmaceutical composition comprising a compound of formula (I) in the manufacture of a medicament for the treatment of a parasitic protozoan infection.

[0020] In a sixth aspect, the present invention provides a method for treating a parasitic protozoan infection in a human, comprising administering to the human a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition.

[0021] In a seventh aspect of the present invention, there is provided a combination comprising (a) a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof and (b) at least one other antimalarial agent.

[0022] In an eighth aspect of the present invention, there is provided a method for treating a parasitic protozoan infection in a human, comprising administering to the human a therapeutically effective amount of the combination.

[0023] BRIEF DESCRIPTION OF FIGURES / GRAPHIC MATERIALS

[0024] The present invention will now be described with reference to the accompanying figures / graphic materials, which are not limiting.

[0025] Fig. 1 shows the X-ray powder diffraction pattern of N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide (Example 4).

[0026] Fig. 2 shows the X-ray powder diffraction pattern of N-((1S,2R)-2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide (Example 10).

[0027] DETAILED DESCRIPTION OF IMPLEMENTATION OPTIONS

[0028] STATEMENT OF THE ESSENCE OF THE INVENTION

[0029] As described above, in one aspect of the present invention, there is provided a compound of formula (I):

[0030]

[0031] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0032] each R 1 , R 2 , R 3 and R 4 independently selected from the group consisting of H, halogen, C1-C6 alkyl, and a 3-7 membered cycloalkyl ring optionally containing a heteroatom selected from the group consisting of O, S, SO, SO2, NH, and N-C1-C6 alkyl; or

[0033] R 1 and R 2 together with the atom to which they are simultaneously attached, form a 3-, 4-, 5-, 6-, or 7-membered cycloalkyl ring, optionally containing a heteroatom selected from the group consisting of O, S, SO, SO2, NH, and N-C1-C6 alkyl, where each R 3 and R 4 independently selected from the group consisting of H, C1-C6 alkyl and a 3-7 membered cycloalkyl ring optionally containing a heteroatom selected from the group consisting of O, S, SO, SO2, NH and N-C1-C6 alkyl; or

[0034] R 1 and R 3 together with the atoms to which they are simultaneously attached, form a 3-, 4-, 5-, 6-, or 7-membered cycloalkyl ring, optionally containing a heteroatom selected from the group consisting of O, S, SO, SO2, NH, and N-C1-C6 alkyl, where each R 2 and R 4independently selected from the group consisting of H, C1-C5 alkyl, and a 3-7 membered cycloalkyl ring optionally containing a heteroatom selected from the group consisting of O, S, SO, SO2, NH, and N-C1-C6 alkyl; and where

[0035] R 5 selected from the group consisting of halogen and C1-C6 alkyl.

[0036] In one embodiment, R 5 is a halogen. In one embodiment, R 5 represents fluorine (F).

[0037] In one embodiment, R 5 is C1-C6 alkyl. In one embodiment, R 5 is methyl.

[0038] In one embodiment, R 3 represents H. In one embodiment, R 3 represents H and R 5 is a halogen, in particular fluorine. In one embodiment, R 3 represents H and R 5 is C1-C6 alkyl, in particular methyl.

[0039] In one embodiment, R 3 and R 4 represent H. In one embodiment, R 3 and R 4 represent H and R 5 is a halogen, in particular fluorine. In one embodiment, R 3 and R 4 represents H and R 5 is C1-C6 alkyl, in particular methyl.

[0040] In one embodiment, R 1 and R 2 , together with the atom to which they are simultaneously attached, form a 3, 4, 5, 6, or 7-membered cycloalkyl ring. In one embodiment, R 1 and R 2 , together with the atom to which they are simultaneously attached, form a 3, 4, 5, 6 or 7-membered cycloalkyl ring, in particular a 4-membered cycloalkyl ring and R 5 is a halogen, and in particular, fluorine. R 1 and R 2together with the atom to which they are simultaneously attached, form a 3-, 4-, 5-, 6-, or 7-membered cycloalkyl ring and R 5 is C1-C6 alkyl, in particular methyl. In one embodiment, R 1 and R 2 together with the atom to which they are simultaneously attached, form a 3, 4, 5, 6, or 7-membered cycloalkyl ring, and R 3 represents hydrogen. In one embodiment, R 1 and R 2 , together with the atom to which they are simultaneously attached, form a 3, 4, 5, 6, or 7-membered cycloalkyl ring, and R 3 and R 4 represent hydrogen. In one embodiment, R 1 and R 2 , together with the atom to which they are simultaneously attached, form a 3, 4, 5, 6 or 7-membered cycloalkyl ring, in particular a 4-membered cycloalkyl ring, R 3 and R 4 represent hydrogen, and R 5is a halogen, in particular fluorine. In one embodiment, R 1 and R 2 , together with the atom to which they are simultaneously attached, form a 3, 4, 5, 6, or 7-membered cycloalkyl ring, and R 3 and R 4 represent hydrogen. In one embodiment, R 1 and R 2 , together with the atom to which they are simultaneously attached, form a 3, 4, 5, 6, or 7-membered cycloalkyl ring, R 3 and R 4 represent hydrogen, and R 5 is C1-C6 alkyl, in particular methyl.

[0041] In one embodiment, the compound of formula (I) is N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide

[0042]

[0043] In one embodiment, the compound of formula (I) is in the form of the free base N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide.

[0044] In one embodiment, the compound of formula (I) is in the form of the free base N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, which has:

[0045] i) an X-ray powder diffraction pattern (XPD) substantially as shown in Fig. 1; and / or

[0046] ii) X-ray powder diffraction (XPD) pattern with specific peaks at 2θ values, ±0.1° 2θ experimental error, 6.7, 11.2, 12.7, 13.4, 16.2, 16.6, 17.9, 20.9, 26.7 and 28.2 degrees.

[0047] In one embodiment, the compound of formula (I) is N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide and is a pharmaceutically acceptable sulfuric acid salt.

[0048] In one embodiment, the compound of formula (I) is a pharmaceutically acceptable dihydrochloride.

[0049] In one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof is N-((1-aminocyclopropyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide

[0050]

[0051] In one embodiment, each R 1 and R 2 is C1-C6 alkyl. In one embodiment, each R 1 and R 2 represents C1-C6 alkyl and R 5 represents a halogen, in particular fluorine. In one embodiment, each R 1 , R 2 and R 5 is C1-C6 alkyl. In one embodiment, each R 1 and R 2 represents C1-C6 alkyl and R 3 represents H. In one embodiment, each R 1 and R 2represents C1-C6 alkyl and each R 3 and R 4 represents H. In one embodiment, each R 1 and R 2 is C1-C6 alkyl, R 5 is a halogen, in particular fluorine, and R 3 represents H. In one embodiment, each R 1 and R 2 represents C1-C6 alkyl, in particular each methyl, R 5 is a halogen, in particular fluorine, and each R 3 and R 4 represents H. In one embodiment, each R 1 , R 2 and R 5 is C1-C6 alkyl, and R 3 represents H. In one embodiment, each R 1 , R 2 and R 5 is C1-C6 alkyl, and each R 3 and R 4 represents N.

[0052] In one embodiment, the compound of formula (I) is N-(2-amino-2-methylpropyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide

[0053]

[0054] In one embodiment, R 1 and R 3 , together with the atoms to which they are simultaneously attached, form a 3, 4, 5, 6, or 7-membered cycloalkyl ring. In one embodiment, R 1 and R 3 , together with the atoms to which they are simultaneously attached, form a 3, 4, 5, 6 or 7-membered cycloalkyl ring, in particular a 6-membered cycloalkyl ring, and R 5 is a halogen, in particular fluorine. In one embodiment, R 1 and R 3 together with the atoms to which they are simultaneously attached, form a 3-, 4-, 5-, 6-, or 7-membered cycloalkyl ring, and R 5 is C1-C6 alkyl, in particular methyl.

[0055] In one embodiment, the compound of formula (I) is N-(2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide

[0056]

[0057] In one embodiment, the compound of formula (I) is N-(2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide in free base form.

[0058] In one embodiment, the compound of formula (I) is N-(2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide in free base form, which has:

[0059] i) an X-ray powder diffraction pattern (XPD) substantially as shown in Fig. 2; and / or

[0060] ii) X-ray powder diffraction (XPD) pattern with specific peaks at 2θ values, ±0.1° 2θ experimental error, 5.4, 10.8, 15.3, 16.6, 18.2, 20.1, 21.8, 22.4, 28.1 and 31.7 degrees.

[0061] In each embodiment, wherein the compound of formula (I) is N-(2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, the compound may be N-((1S,2R)-2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide.

[0062] In one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof is selected from the group consisting of N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, N-(2-amino-2-methylpropyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, N-(2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide and N-((1-aminocyclopropyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide.

[0063] In one embodiment, the present invention provides a pharmaceutically acceptable salt of N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, N-(2-amino-2-methylpropyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, N-(2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, or N-((1-aminocyclopropyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide.

[0064] DEFINITIONS

[0065] As used herein, the term "alkyl" means a saturated, straight-chain or branched hydrocarbon group. The term "C1-C6 alkyl" refers to an alkyl group containing from 1 to 6 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, sec-pentyl, 3-pentyl, and sec-isopentyl.

[0066] The term "halogen" represents a group of chlorine, iodine, bromine, or fluorine.

[0067] The term "a compound of the present invention" means any of the compounds of the invention defined above. In particular, the term as used herein includes, but is not limited to, a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof and is a reference to any of the formulas described herein.

[0068] It will further be understood that the compounds of the invention, such as the compound of formula (I), can exist in various tautomeric forms. Tautomers refer to isomeric forms of a compound that are in equilibrium with one another. The concentration of the isomeric forms will depend on the environment in which the compound is present.

[0069] As used in this document, the term "pharmaceutically acceptable" refers to those compounds (including salts), materials, compositions and dosage forms that are suitable for use in contact with the tissues of humans or animals without undue toxicity, irritation or other adverse effect / complication.

[0070] In one aspect, the present invention provides pharmaceutically acceptable salts of the compounds of formula (I). Pharmaceutically acceptable salts include, but are not limited to, those described in Berge, J. Pharm. Sci., 1977, 66, 1-19 or those listed in P. H. Stahl and C. G. Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Second Edition, John Wiley & Sons, March 2011.

[0071] If the functionality of the compound allows, suitable pharmaceutically acceptable salts of the compound of Formula (I) can be formed, which include acid addition salts or base salts. Acid addition salts can be formed by reaction with an appropriate acid, optionally in a suitable solvent, such as an organic solvent, to yield a salt that can be isolated by crystallization and filtration. Base addition salts can be formed by reaction with an appropriate base, optionally in a suitable solvent, such as an organic solvent, to yield a salt that can be isolated by crystallization and filtration.

[0072] Typical pharmaceutically acceptable acid addition salts include, but are not limited to, 4-acetamidobenzoate, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate (besylate), benzoate, bisulfate, bitartrate, butyrate, calcium edetate, camphorate, camphorsulfonate (camsylate), caprate (decanoate), caproate (hexanoate), caprylate (octanoate), cinnamate, citrate, cyclamate, digluconate, 2,5-dihydroxybenzoate, disuccinate, dodecyl sulfate (estolate), edetate (ethylenediaminetetraacetate), estolate (lauryl sulfate), ethane-1,2-disulfonate (edisylate), ethanesulfonate (esylate), formate, fumarate, galactarate (mucate), gentisate (2,5-dihydroxybenzoate), glucoheptonate (gluceptate), gluconate, glucuronate, glutamate, glutarate, glycerophosphorate, glycolate, hexylresorcinate, hippurate, hydrabamine (N,N'-di(dehydroabietyl)-ethylenediamine), hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoate, isobutyrate, lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, methanesulfonate (mesylate), methyl sulfate, mucate, naphthalene-1,5-Disulfonate (napadisylate), naphthalene-2-sulfonate (napsylate), nicotinate, nitrate, oleate, palmitate, p-aminobenzenesulfonate, p-aminosalicylate, pamoate (embonate), pantothenate, pectinate, persulfate, phenylacetate, phenylethylbarbiturate, phosphate, polygalacturonate, propionate, p-toluenesulfonate (tosylate), pyroglutamate, pyruvate, salicylate, sebacate, stearate, subacetate, succinate, sulfamate, sulfate, tannate, tartrate, theoclate (8-chlorotheophyllinate), thiocyanate, triethiodide, undecanoate, undecylenate, and valerate.

[0073] Representative pharmaceutically acceptable salts

[0074] Base additions include, but are not limited to, aluminum, 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS, tromethamine), arginine, benetamine (N-benzylphenethylamine), benzathine (N,N'-dibenzylethylenediamine), bis-(2-hydroxyethyl)amine, bismuth, calcium, chloroprocaine, choline, clemizole (1-p-chlorobenzyl-2-pyrrolyldin-1'-ylmethylbenzimidazole), cyclohexylamine, dibenzylethylenediamine, diethylamine, diethyltriamine, dimethylamine, dimethylethanolamine, dopamine, ethanolamine, ethylenediamine, L-histidine, iron, isoquinoline, lepidine, lithium, lysine, magnesium, meglumine (N-methylglucamine), piperazine, piperidine, potassium, procaine, quinine, quinoline, sodium, strontium, f-butylamine and zinc.

[0075] In one embodiment of the present invention, there is provided a pharmaceutically acceptable salt of a compound of formula (I) selected from N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide ditrifluoroacetic acid salt

[0076]

[0077] N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide hydrochloric acid salts in a 2:1 ratio

[0078] And

[0079] N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide sulfuric acid salts

[0080]

[0081] In one embodiment of the present invention, there is provided a pharmaceutically acceptable salt of the compound of formula (I), which is the hydrochloric acid salt of N-((1-aminocyclopropyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide in a 2:1 ratio,

[0082]

[0083] N-((1-aminocyclopropyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide ditrifluoroacetic acid salt in a 2:1 ratio

[0084]

[0085] In one embodiment of the present invention, there is provided a pharmaceutically acceptable salt of the compound of formula (I), which is N-(2-amino-2-methylpropyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide ditrifluoroacetic acid salt

[0086]

[0087] In one embodiment of the present invention, there is provided a pharmaceutically acceptable salt of the compound of formula (I) selected from:

[0088] N-(2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide hydrochloric acid salts in a 2:1 ratio

[0089]

[0090] and trifluoroacetic acid salts of N-(2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide in a ratio of 2:1

[0091]

[0092] The present invention includes within its scope all possible stoichiometric and non-stoichiometric forms of salts of the compounds of formula (I). As used herein, the term "therapeutically effective amount" means any amount that, compared to a corresponding human subject not receiving such amount, results in improved treatment, recovery, prevention, or alleviation of a disease, disorder, or side effect, or a reduction in the rate of progression of a disease or disorder.

[0093] The appropriate "therapeutically effective amount" will depend on a number of factors including, for example, the age and weight of the human subject, the specific condition being treated and its severity, the nature of the formulation and the route of administration, and will ultimately be at the discretion of the attending physician.

[0094] The compounds of formula (I) may contain one or more asymmetric centers (also called chiral centers) and, therefore, may exist as single enantiomers, diastereomers, or other stereoisomeric forms, or as mixtures thereof. Chiral centers, such as chiral carbon atoms, may also be present in a substituent, such as an alkyl group. If the stereochemistry of a chiral center present in formula (I) or in any chemical structure illustrated herein is not specified, the structure is intended to encompass any stereoisomer and all mixtures thereof. Thus, compounds of formula (I) containing one or more chiral centers can be used as racemic modifications, including racemic mixtures and racemates, enantiomerically enriched mixtures, or as enantiomerically pure individual stereoisomers.

[0095] With regard to solvates of the compounds of the present invention or their salts that are in crystalline form, one skilled in the art will understand that pharmaceutically acceptable solvates can be formed by the incorporation of solvent molecules into the crystal lattice during crystallization. Solvates can include non-aqueous solvents such as ethanol, isopropanol, DMSO, acetic acid, ethanolamine, and ethyl acetate, or they can include water as a solvent incorporated into the crystal lattice. Solvates in which water is the solvent incorporated into the crystal lattice are commonly referred to as "hydrates." Hydrates include stoichiometric hydrates, as well as compositions containing a variable amount of water. The present invention includes all such solvates.

[0096] The present invention also includes various deuterated forms of the compound of formula (I), respectively, or a pharmaceutically acceptable salt or stereoisomer thereof. Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom. One skilled in the art knows how to synthesize the deuterated forms of the compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof according to the present invention. For example, deuterated materials such as alkyl groups can be prepared by conventional methods.

[0097] The present invention also includes isotopically labeled compounds identical to the compounds of formula (I), as defined herein, respectively, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature. Examples of isotopes that may be included in the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, iodine, and chlorine, such as 3 H, 11 C, 14 WITH, 18 F, 123 l or 125 l.

[0098] The compounds of the present invention or their pharmaceutically acceptable salts or stereoisomers that contain the above-mentioned isotopes and / or other isotopes of other atoms are within the scope of the present invention. Isotopically labeled compounds of the present invention, for example those in which radioactive isotopes such as 3N or 14 C, are useful in tissue distribution analyses of drugs and / or substrates. Tritium isotopes, i.e. 3 H, and carbon-14, i.e. 14 C, are particularly preferred due to their ease of production and detectability. Isotopes 14 With and 18 F are particularly useful in PET (positron emission tomography).

[0099] Since the compounds of the present invention are intended for use in pharmaceutical compositions, it is readily understood that each of them is present in a substantially pure form, for example, with a purity of at least 60%, in some aspects with a purity of at least 75%, in some aspects with a purity of at least 85%, and in other aspects with a purity of at least 90% or 95%, and, in particular, with a purity of at least 98% (% are indicated on a weight basis). Preparations of the compounds with impurities can be used to obtain purer forms used in pharmaceutical compositions.

[0100] The compounds of formula (I) or their pharmaceutically acceptable salts or stereoisomers may exist as solids or liquids, both of which are included in the present invention. In the solid state, the compounds of formula (I) or their pharmaceutically acceptable salts or stereoisomers may exist in either amorphous or crystalline form, or as a mixture of both. The compounds of formula (I) or their pharmaceutically acceptable salts or stereoisomers may exist in solvated form and may be formed by the incorporation of solvent molecules into the crystal lattice during crystallization. Solvates may include non-aqueous solvents such as ethanol, isopropanol, dimethyl sulfoxide (DMSO), acetic acid, ethanolamine, and ethyl acetate, or they may include water as a solvent incorporated into the crystal lattice. Solvates in which water is used as a solvent are generally referred to as "hydrates".Therefore, in an embodiment, the present invention provides solvates of the compound of formula (I), such as hydrates.

[0101] As used herein, the term "optionally" means that the event(s) described below may or may not occur, and includes both those that do and those that do not. For example, when used in conjunction with the term "substituted," i.e., "optionally substituted," it means that the substituents described below may or may not be present.

[0102] As used herein, the term "treatment" means: (1) ameliorating or preventing a condition being treated or one or more biological manifestations of the condition being treated, (2) interfering with (a) one or more points in the biological cascade that leads to or is responsible for the disease being treated, or (b) one or more biological manifestations of the disease being treated, or (3) alleviating one or more symptoms or effects associated with the condition being treated. One skilled in the art will understand that "prevention" is not an absolute term. In medicine, "prevention" means prophylactic administration of a drug for the purpose of substantially reducing the likelihood or severity of a condition or its biological manifestation, or to delay the onset of such a condition or its biological manifestation.

[0103] As used in this document, "effective amount" or "safe and effective amount" means an amount of a compound sufficient to produce a significant beneficial modification of the condition being treated, but low enough to avoid serious adverse effects (with a reasonable benefit / risk ratio) within the limits of reasonable medical judgment.

[0104] DISCLOSURE OF USE

[0105] In one aspect, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof for use in therapy.

[0106] The compounds of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof may be useful in the treatment of certain parasitic infections, such as parasitic protozoan infections caused by the malarial parasite Plasmodium falciparum, the following species of Eimeria, Pneumocytis carinii, Trypanosoma cruzi, Trypanosoma brucei or Leishmania donovani.

[0107] In particular, a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof may be useful in the treatment of infection caused by Plasmodium falciparum. Accordingly, the invention relates to methods for treating such infections. Alternatively, compounds of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof may be useful for the treatment of infection caused by Plasmodium species other than Plasmodium falciparum, which causes human malaria. For example, compounds of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof may be useful for the treatment of infection caused by Plasmodium Vivax, i.e., malaria caused by Plasmodium Vivax infection.

[0108] In one embodiment, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof for use in the treatment of a parasitic protozoan infection. In a specific embodiment, said protozoan infection is malaria or an infection caused by Plasmodium falciparum. In one embodiment, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof for use in the treatment of malaria resulting from infection with Plasmodium falciparum.

[0109] In another aspect, the present invention provides a method for treating a parasitic protozoan infection, comprising administering a pharmaceutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof to a human in need thereof. In one embodiment, said parasitic protozoan infection is malaria or an infection caused by Plasmodium falciparum.

[0110] In another aspect, the present invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof in the manufacture of a medicament for the treatment of a parasitic protozoan infection. In one embodiment, said parasitic protozoan infection is malaria or an infection caused by Plasmodium falciparum. Accordingly, the compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof can be used in the treatment of malaria. Thus, the present invention also relates to a method for the treatment of malaria, comprising administering a pharmaceutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof to a human in need thereof. Furthermore, the present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof in the manufacture of a medicament for the treatment of malaria.

[0111] Those skilled in the art will appreciate that references to treatment in this document refer to the treatment of established conditions, such as malaria. However, the compounds of the present invention may also be useful for the prevention of such diseases, for example, for the prevention of malaria. Thus, in one embodiment, the treatment or prevention of a disease such as malaria is provided. In another embodiment, the treatment of a disease such as malaria is provided. In yet another embodiment, the prevention of a disease such as malaria is provided.

[0112] In one embodiment, the malaria is multidrug-resistant malaria. Therefore, in one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof may be useful in the treatment of susceptible malaria and / or multidrug-resistant malaria.

[0113] In one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof is for use in the treatment of a parasitic protozoan infection resistant to artemisinin combination therapy. In one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof is for use in the treatment of malaria resistant to artemisinin combination therapy.

[0114] PHARMACEUTICAL COMPOSITIONS

[0115] The compounds of formula (I) and their pharmaceutically acceptable salts and stereoisomer will typically, but not necessarily, be formulated into pharmaceutical compositions prior to administration to a patient. Accordingly, in another aspect, there is provided a pharmaceutical composition comprising (a) a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof; and (b) a pharmaceutically acceptable excipient or carrier.

[0116] Suitable pharmaceutically acceptable excipients include the following types of excipients: binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, taste-masking agents, coloring agents, anti-caking agents, humectants, chelating agents, plasticizers, viscosity-enhancing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. One skilled in the art will understand that some pharmaceutically acceptable excipients may perform more than one function and may perform alternative functions depending on how many excipients are present in the formulation and what other ingredients are present in the formulation.The carrier excipient must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient. Skilled artisans possess knowledge and skill in the art to select suitable pharmaceutically acceptable excipients in suitable amounts for use in the present invention. Additionally, those skilled in the art have access to a number of references describing pharmaceutically acceptable excipients that may be helpful in selecting suitable pharmaceutically acceptable excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company), The Handbook of Pharmaceutical Additives (Gower Publishing Limited), and The Handbook of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press).

[0117] The pharmaceutical compositions of the present invention are prepared using technologies and methods known to those skilled in the art. Some of the methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing Company). For example, dosage forms include those adapted for (1) oral administration, such as tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets and wafers; (2) parenteral administration, such as sterile solutions, suspensions and powders for reconstitution; (3) transdermal administration, such as transdermal patches; (4) rectal administration, such as suppositories; and (5) inhalation, such as aerosols and solutions.

[0118] In one aspect, the present invention relates to a solid or liquid oral dosage form, such as a liquid, tablet, lozenge, or capsule, containing a safe and effective amount of a compound of the present invention and a carrier. The carrier can be in the form of a diluent or filler. Suitable diluents and fillers typically include lactose, sucrose, dextrose, mannitol, sorbitol, starch (e.g., corn starch, potato starch, and pregelatinized starch), cellulose and its derivatives (e.g., microcrystalline cellulose), calcium sulfate, and dibasic calcium phosphate. A liquid dosage form typically consists of a suspension or solution of the compound or a pharmaceutically acceptable salt or stereoisomer thereof in a liquid carrier, such as ethanol, olive oil, glycerol, glucose (syrup), or water (e.g., with the addition of a flavoring, suspending, or coloring agent).If the composition is in the form of a tablet or lozenge, any pharmaceutical carrier commonly used for the preparation of solid formulations can be used. Examples of such carriers include magnesium stearate, terra alba, talc, gelatin, gum arabic, stearic acid, starch, lactose, and sucrose. If the composition is in the form of a capsule, any conventional encapsulation can be used, such as the aforementioned carriers or semi-solids, such as capric acid mono- and diglycerides, Gelucire, and Labrasol, or a hard capsule shell, such as gelatin. If the composition is in the form of a soft-shell capsule, such as gelatin, any pharmaceutical carrier commonly used for the preparation of dispersions or suspensions can be used, such as aqueous gums or oils, which can be placed within the soft capsule shell.

[0119] The pharmaceutical compositions can be administered by any suitable route, such as orally (including buccal or sublingual), by inhalation, intranasally, topically (including buccal, sublingual or transdermal), parenterally (including subcutaneously, intramuscularly, intravenously or intradermally). In particular, the pharmaceutical compositions are administered orally. The pharmaceutical compositions can be presented as unit dosage forms containing a predetermined amount of the active ingredient per unit dose. In one aspect, the unit dosage compositions are compositions containing a daily dose or sub-dose or a fraction thereof of the active ingredient. Therefore, such unit doses can be administered more than once a day. In one aspect, the unit dosage compositions are compositions containing a daily dose or sub-dose (for administration more than once a day), as defined above, or a fraction thereof of the active ingredient.

[0120] The solid oral dosage form may further contain an excipient in the form of a binder. Suitable binders include starch (e.g., corn starch, potato starch, and pregelatinized starch), gelatin, gum arabic, sodium alginate, alginic acid, tragacanth, guar gum, povidone, cellulose and its derivatives (e.g., microcrystalline cellulose). The solid oral dosage form may further contain an excipient in the form of a disintegrant. Suitable disintegrants include crospovidone, sodium starch glycolate, croscarmellose, alginic acid, and sodium carboxymethylcellulose. The solid oral dosage form may further contain an excipient in the form of a lubricant. Suitable lubricants include stearic acid, magnesium stearate, calcium stearate, and talc.

[0121] In one embodiment, a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof is prepared for administration by injection either intramuscularly or subcutaneously. In one aspect, the present invention relates to an injectable composition comprising a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof. Standard formulations and manufacturing techniques can be used to prepare a suitable stable sterile injection vehicle containing a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof according to the present invention. In some aspects, the injectable pharmaceutical composition is a long-acting injectable composition and provides controlled release of a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof.

[0122] In one embodiment, a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof is prepared for administration by injection, wherein the composition comprises the compound and a pharmaceutically acceptable excipient or carrier, such as Tween 20, PEG400 and / or mannitol. In one embodiment, a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof is formulated with Tween 20, PEG400 and mannitol and is suitable as a long-acting injectable composition.

[0123] COMBINATIONS

[0124] When the compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof is used in the treatment of malaria or Plasmodium falciparum, it may be used alone or in combination with at least one other therapeutic agent, such as at least one other antiparasitic agent, for example, an antimalarial agent.

[0125] In some embodiments, the present invention relates to a combination of (a) a compound of formula (I), or a pharmaceutically acceptable salt or stereoisomer thereof; and (b) at least one other antimalarial agent. In one embodiment, the combination comprises one, two, or three additional antimalarial agents. For the avoidance of doubt, the at least one other antimalarial agent is not a compound of formula (I).

[0126] At least one other antimalarial agent is an agent under development, approved, or recommended for the treatment of malaria.

[0127] The at least one other antimalarial agent may be selected from chloroquine, mefloquine, primaquine, pyrimethamine, quinine, artemisinin, halofantrine, doxycycline, amodiaquine, atovaquone, tafenoquine, dapsone, proguanil, sulfadoxine, cycloguanil, fansidar, piperaquine, lumefantrine, artesunate, dihydroartemisinin, artemether, fosmidomycin and azithromycin.

[0128] In one embodiment, the at least one other antimalarial agent is an artemisinin agent.

[0129] At least another antimalarial drug may be tafenoquine.

[0130] In one embodiment, the additional antimalarial agents are atovaquone and proguanil. The at least one other antimalarial agent may also be selected from ferroquine, KAF156, cipargamine, DSM265, artemisone, artemisinin, artefenomel, MMV048, SJ733, P218, MMV253, PA92, DDD498, AN13762, DSM421, UCT947, ACT 451840, 6-chloro-7-methoxy-2-methyl-3-{4-[4-(trifluoromethoxy)phenoxy]phenyl}quinolin-4(1H)-one, 6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4(1H)-one, a pharmaceutical salt thereof, and a combination thereof. In one embodiment, the additional antimalarial agent is 6-chloro-7-methoxy-2-methyl-3-{4-[4-(trifluoromethoxy)phenoxy]phenyl}quinolin-4(1H)-one, 6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4(1H)-one, a pharmaceutical salt thereof, or a combination thereof.

[0131] At least one other antimalarial agent may also be selected from OZ609, OZ277 and SAR97276.

[0132] For the treatment of Plasmodium falciparum infections, at least one, two or three additional antimalarial agents are selected from the following, wherein at least one of the antimalarial agents is an artemisinin-based agent:

[0133] artemether+lumefantrine

[0134] artesunate+amodiaquine

[0135] artesunate + mefloquine

[0136] dihydroartemisinin+piperaquine

[0137] artesunate+sulfadoxine-pyrimethamine (SP)

[0138] The above-mentioned combination treatments are known as artemisinin-based combination therapies (ACTs). The choice of ACT is typically based on the results of studies of therapeutic efficacy against local strains of Plasmodium falciparum.

[0139] Treatment of Plasmodium vivax infections with ACT as described above can be used. Alternatively, chloroquine may be used as at least one other antimalarial agent, particularly in areas where chloroquine-resistant Plasmodium vivax is not present. In areas where resistant Plasmodium vivax has been identified, infections can be treated with the ACTs described above.

[0140] The combinations may conveniently be presented for use in the form of a pharmaceutical composition or formulation. Thus, also contemplated herein is a pharmaceutical composition comprising (a) a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, as described herein, together with (b) at least one other antimalarial agent and (c) one or more pharmaceutically acceptable excipients as described herein.

[0141] The compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof and at least one other therapeutic agent may be administered together or separately, in the case of separate administration, this may be carried out separately or sequentially in any order (by the same or different routes of administration).

[0142] DOSAGES

[0143] The amount of the compound of the present invention or its pharmaceutically acceptable salt or stereoisomer and the additional therapeutically active agent and the relative timing of administration will be selected to achieve the desired combined therapeutic effect and can be determined by a practicing physician.

[0144] Typical amounts administered are from about 0.1 mg to 1000 mg, in some aspects from about 0.1 mg to about 500 mg. In one embodiment, the compound of formula (1) or a pharmaceutically acceptable salt or stereoisomer thereof is administered in an amount of 200 mg.

[0145] The compounds of the present invention can be administered orally in a dosage range of, for example, 0.1 to 5000 mg, or 1 to 1500 mg, 2 to 800 mg, or 5 to 500 mg, such as 2 to 200 mg or 10 to 1000 mg, specific examples of dosages include 10, 20, 50, 80 mg and 350 mg.

[0146] In one embodiment, the therapeutically effective single dose of the compound of formula (1) is from about 10 to about 150 mg once a day for three consecutive days. In one embodiment, the compound of formula (1) or a pharmaceutically acceptable salt or stereoisomer thereof is administered in an amount of from about 50 mg to about 85 mg once a day for three consecutive days. In one embodiment, the compound of formula (1) or a pharmaceutically acceptable salt or stereoisomer thereof is administered in an amount of from about 10 mg to about 50 mg once a day for three consecutive days.

[0147] In one embodiment, a compound or a pharmaceutically acceptable salt or stereoisomer thereof selected from the group consisting of N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, N-(2-amino-2-methylpropyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, N-(2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide and N-((1-aminocyclopropyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide is administered in an amount of about 0.1 mg-1000 mg. In one embodiment, a compound or a pharmaceutically acceptable salt or stereoisomer thereof selected from the group consisting of N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, N-(2-amino-2-methylpropyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, N-(2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, and N-((1-aminocyclopropyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide is administered in an amount of about 200 mg.

[0148] However, ultimately the amount of compound administered and the type of composition used will be appropriate to the nature of the disease or physiological condition being treated and will be determined at the discretion of the physician.

[0149] Determining effective doses in this context is typically based on studies in animal models followed by human clinical trials and focuses on administration protocols that significantly reduce the manifestation or severity of symptoms or conditions of the target disease in humans. Suitable models in this regard include, for example, mice, rats, birds, pigs, cats, non-human primates, and other established animal models known in the art. Alternatively, effective doses can be determined using in vitro models (e.g., whole-cell assays to monitor the effects of various drugs on parasite growth rate).When using such models, only routine calculations and adjustments are required to determine the appropriate concentration and dose for administering a therapeutically effective amount of a compound (e.g., in amounts effective to elicit a desired immune response or alleviate one or more symptoms of the target disease).

[0150] GENERAL ROUTES OF SYNTHESIS

[0151] The compounds of the present invention can be prepared by a variety of methods, including methods commonly known in the art of chemistry. Any previously defined variable will continue to have the previously defined meaning unless otherwise indicated. Illustrative general synthetic methods are presented in the following schemes and can be readily adapted to prepare the compounds of the present invention. Specific compounds prepared according to the experimental procedure are described in the Examples section.

[0152] DIAGRAM 1

[0153]

[0154] Steps 1 and 2 are Suzuki coupling reactions which can be carried out with the appropriate boronic acid or ester in the presence of a suitable catalyst such as PdCl2(dppf) and a base such as sodium carbonate in a suitable solvent such as isopropanol / water and at a suitable temperature such as 120°C.

[0155] Step 3 is a deesterification reaction which can be carried out under appropriate conditions, such as using LiOH-H2O in a suitable solvent mixture such as THF / H2O.

[0156] Step 4 is a coupling reaction using an appropriate acid intermediate and an appropriate amine as a coupling reaction partner, in the presence of a coupling agent such as HATU and in the presence of a base such as DIPEA, in an appropriate solvent such as DMF.

[0157] Step 5: This is deprotection, which can be carried out with a suitable acid, such as 4 M HCl in 1,4-dioxane.

[0158] EXAMPLES

[0159] The present invention will now be illustrated by the following non-limiting examples. Although specific embodiments of the invention are described below, those skilled in the art will appreciate that various changes and modifications can be made. References to preparations carried out in a similar manner or according to a general method may include variations in typical parameters such as time, temperature, processing conditions, as well as minor changes in the amounts of reagents, etc.

[0160] In some of the intermediates and examples below, starting materials are designated by reference to other intermediate or example numbers. This does not mean that the actual material from any particular intermediate or example was necessarily used in the subsequent step illustrated herein, but is used as an abbreviation for the corresponding compound.

[0161] If materials were commercially available, this is indicated in parentheses after the compound name in capital letters. Commercial reagents and solvents were used as received. All solvents used in the reaction were high-purity grade or anhydrous. Proton nuclear magnetic resonance spectra were recorded ( 1H NMR), and chemical shifts are reported in parts per million (ppm) downfield from the internal standard tetramethylsilane (TMS). Abbreviations for NMR data are: o = single, d = doublet, t = triplet, q = quartet, m = multiplet, dd = double doublet, dt = double triplet, app = apparent, br = broadened. Mass spectra were obtained using electrospray ionization (ESI) methods. All temperatures are reported in degrees Celsius.

[0162] When diastereomers are present and the absolute stereochemistry is known, the stereocenter, i.e. the chiral carbon atom, is designated R or S.

[0163] Abbreviations

[0164] AcOEt ethyl acetate

[0165] ACN acetonitrile

[0166] CDCl3 deuterated chloroform

[0167] DCM dichloromethane

[0168] DIPEA N,N-diisopropylethylamine

[0169] DMF dimethylformamide

[0170] DMSO dimethyl sulfoxide

[0171] EtOH ethanol

[0172] h hour(s)

[0173] HATU azabenzotriazoletetramethyluronium hexafluorophosphate

[0174] HCl hydrochloric acid

[0175] HPLC high performance liquid chromatography

[0176] i-PrOH isopropyl alcohol

[0177] K2CO3potassium carbonate

[0178] min minutes

[0179] MeOH methanol

[0180] NaOH sodium hydroxide

[0181] Na2CO3 sodium carbonate

[0182] NH4Cl ammonium chloride

[0183] RBF round bottom flask

[0184] room temperature

[0185] SiO2 silicon dioxide

[0186] TBME or tBuOMe tert-butyl methyl ether

[0187] TFU trifluoroacetic acid.

[0188] Intermediate 1: methyl 4-(6-chloropyrazin-2-yl)benzoate

[0189]

[0190] (4-(methoxycarbonyl)phenyl)boronic acid (Apollo Scientific, 1.0 g, 5.56 mmol), 2,6-dichloropyrazine (Combi-Blocks, 1.656 g, 11.11 mmol), and K2CO3 (Chempure, 2.304 g, 16.67 mmol) were added to 100 mL of RBF at room temperature. Then, 1,2-dimethoxyethane (30.0 mL) and water (10.0 mL) were added at the same temperature. Then, the resulting reaction mixture was degassed with nitrogen for 15 min, followed by the addition of PdCl2(dppf)-CH2Cl2 adduct (Chempure, 0.159 g, 0.194 mmol) at room temperature. The resulting reaction mixture was then heated to 65°C and stirred at the same temperature for 16 hours. The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (2×25 mL). The combined organic phase was dried over Na2SO4 (5 g), filtered, and the filtrate was concentrated under reduced pressure to yield a black solid as a crude substance.

[0191] The crude product was dissolved in 15 mL of DCM, adsorbed onto 10 g of silica gel (230-400 mesh), and purified by Biotage isolera column chromatography (silica gel 230-400 mesh, 40 g of sample, flow rate 30 mL / min). The desired compound was eluted with 0-15% ethyl acetate in pet. ether, the collected fractions were combined and concentrated under reduced pressure to give the title compound (900 mg, 3.59 mmol, yield 64.5%) as a white solid.

[0192] 1 H NMR (δ, ppm, CDCl3): 9.38 (o, 1H), 8.84 (o, 1H), 8.29 (d, 2H), 8.12 (d, 2H), 3.91 (o, 3H). [IER MS] m / z: 249 (MN + ).

[0193] Intermediate 2: Methyl 4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzoate

[0194]

[0195] Methyl 4-(6-chloropyrazine)benzoate (250 g, 1005 mmol), (5-fluoropyridin-3-yl)boronic acid (COMBI-BLOCKS, 156 g, 1106 mmol), and K2CO3 (Chempure, 417 g, 3016 mmol) were added to 10000 mL of RBF at room temperature. Then, 1,4-dioxane (5000 mL) and water (1500.0 mL) were added at the same temperature. The resulting reaction mixture was degassed with nitrogen for 15 min, followed by the addition of PdCl2(dppf)-CH2Cl2 adduct (Chempure, 28.7 g, 35.2 mmol) at room temperature. The resulting reaction mixture was then heated to 55°C and stirred at the same temperature for 3 hours. The reaction mixture was warmed to room temperature and maintained at the same temperature for 4 hours, after which a solid precipitated. The solid was filtered and washed with water (2000 mL), acetone (2500 mL), and then pet.ether (1500 mL) and dried under reduced pressure to give methyl 4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzoate (280.0 g, 897 mmol, 89% yield) as a white solid.

[0196] 1 H NMR (δ, ppm, DMSO-D6): 9.44 (o, 1H), 9.42 (o, 1H), 9.37 (o, 1H), 8.77 (d, 1H), 8.65-8.55 (m, 1H), 8.48 (d, 2H), 8.15 (d, 2H), 3.92 (o, 3H). [ESI MS] m / z: 310 (MH + ).

[0197] Intermediate 3: 4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzoic acid

[0198]

[0199] To a stirred solution of methyl 4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzoate (200.0 g, 647 mmol) in tetrahydrofuran (2500 mL) at room temperature was added lithium hydroxide monohydrate (Chempure, 81 g, 1940 mmol) in water (1000 mL). The resulting reaction mixture was stirred for 12 h at room temperature. The reaction mixture was concentrated under reduced pressure to give the crude product as a white solid. The crude product was diluted with water (500 mL) and pH adjusted to 3-4 with 1.5 N HCl (550 mL), after which a white solid was precipitated. The resulting reaction mixture was stirred for 15 min, the resulting solid was filtered and washed with water (2500 mL), then acetone (1500 mL) and dried under reduced pressure to give the title compound (187 g, 632 mmol, 98% yield) as a white solid.

[0200] 1H NMR (δ, ppm, DMSO-D6): 13.21 (br o, 1H), 9.43 (o, 1H), 9.42 (o, 1H), 9.38 (t, 1H), 8.77 (d, 1H), 8.62-8.58 (m, 1H), 8.45 (d, 2H), 8.13 (d, 2H). [ESI-MS] m / z: 296 (MH + ).

[0201] Intermediate 4: tert-butyl (1-((4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamido)methyl)cyclobutyl)carbamate

[0202]

[0203] To a suspension of 4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzoic acid (20 g, 67.7 mmol) and DIPEA (ALDRICH, 59.2 mL, 339 mmol) in N,N-dimethylformamide (DMF) (300 mL) was added HATU (FLUOROCHEM, 33.5 g, 88 mmol) (after 10 min of stirring, not dissolving). Pure powdered tert-butyl (1-(aminomethyl)cyclobutyl)carbamate (ENAMINE, 16.28 g, 81 mmol) was added, and the resulting mixture was stirred at room temperature.

[0204] The reaction mixture was diluted with AcOEt (100 mL) and 1 N NH4Cl was added until the white solid (500 mL) completely precipitated. The mixture was filtered and washed with water (2×100 mL). The solid was dried in vacuo to give the title compound (31 g, 64.9 mmol, 96%) as a white solid.

[0205] 1 H NMR (δ, ppm, CDCl3): 9.42 (d, 1H), 9.38 (t, 1H), 8.77 (d, 1H), 8.63-8.57 (m, 1H), 8.43 (d, 2H), 8.05 (d, 2H), 7.01 (bd, 1H), 3.58 (d, 2H), 2.35-2.20 (m, 2H), 2.13-2.01 (m, 2H), 1.87-1.85 (m, 2H), 1.40 (o, 9H). [IER MS] m / z: 478 (MN + ).

[0206] Example 1: N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, ditrifluoroacetic acid salt

[0207]

[0208] Trifluoroacetic acid (1.000 mL) was added to a solution of tert-butyl (1-((4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamido)methyl)cyclobutyl)carbamate (50 mg, 0.105 mmol) in dry dichloromethane (2 mL) under N2 atmosphere, and the resulting mixture was stirred at room temperature. After 30 min, the reaction mixture was diluted with DCM and concentrated under reduced pressure. The resulting crude product was purified by preparative HPLC (sample loaded into 1.5 mL MeOH, XBridge 19×150 mm, flow rate 17 mL / min, using ACN and H2O with 0.1% TFA as mobile phase) to yield, after collection and lyophilization, the corresponding fraction, 35 mg aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, ditrifluoroacetic acid salt (35 mg, 0.058 mmol, 55.2% yield) as a white solid.

[0209] 1H NMR (δ, h / million, DMSO-d6): 9.43 (d, 1H), 9.39-9.36 (m, 1H), 8.95-8.88 (m, 1H), 8.78 (d, 1H), 8.65-8.56 (m, 1H, 8, 1H), 8,8.2 (d, 1H), 2H), 8.07-7.98 (m, 2H), 3.67 (d, 2H), 2.28-2.10 (m, 4H), 1.99-1.76 (m, 2H). [IER MS] m / z: 378 (MH + ).

[0210] Example 2: N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazine-2-yl)benzamide, dihydrochloride

[0211]

[0212] To a suspension of tert-butyl (1-((4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamido)methyl)cyclobutyl)carbamate (100 mg, 0.209 mmol) in anhydrous 1-propanol (2 mL) under N2 atmosphere was added 6N hydrochloric acid in iPrOH (FLUOROCHEM, 2 mL, 12.00 mmol), and the resulting mixture was stirred at room temperature. After 2 h, TBME was added, and the resulting suspension was concentrated to give a cream-colored solid. This solid was treated with ACN, filtered, and washed three times. The solid was dried under a stream of air to give 80 mg of the title compound (80 mg, 0.178 mmol, 85% yield) as a cream-colored solid.

[0213] 1 H NMR (δ, ppm, DMSO-d6): 9.42 (d, 2H), 9, 9.38 (o, 1H), 9.06 (t, 1H), 8.77 (d, 1H), 8.66-8.57 (m, 1H), 8.44 (d, 2H), 8.33 (br o, 2H), 8.17 (d, 2H), 3.70 (d, 2H), 2.29-2.14 (m, 4H), 1.99-1.77 (m, 2H). [ESI-MS] m / z: 378 (MH + ).

[0214] Example 3: N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, sulfuric acid salt

[0215]

[0216] To a solution of tert-butyl (1-((4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamido)methyl)cyclobutyl)carbamate (100 mg, 0.209 mmol) in dichloromethane (DCM) (0.5 mL) and methanol (0.5 mL) at 40 °C, sulfuric acid (0.1 mL, 1.876 mmol) was added dropwise, and the reaction mixture was stirred at 40 °C. The reaction mixture was allowed to reach room temperature, tBuOMe was added, and the solid was filtered and washed with tBuOMe (2 times) to give an off-white solid (125 mg, N74910-88-1) after drying under nitrogen flow.

[0217] 1 H NMR (δ, ppm, DMSO-d6): 9.44 (o, 1H), 9.43 (o, 1H), 9.39 (t, 1H), 8.92 (t, 1H), 8.79 (d, 1H), 8.66-8.58 (m, 1H), 8.49-8.45 (m, 2H), 8.13-8.08 (m, 2H), 8.06-7.94 (m, 3H), 3.67 (d, 2H), 2.27-2.11 (m, 4H), 1.97-1.77 (m, 2H). [ESI MS] m / z: 378 (MH+).

[0218] Example 4: N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide

[0219]

[0220] To a suspension of tert-butyl (1-((4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamido)methyl)cyclobutyl)carbamate (74.27 g, 156 mmol) in dichloromethane (1 L) at room temperature, TFA (150 mL, 1947 mmol) was added dropwise over 30 min (it became a solution), and the reaction mixture was stirred at room temperature. After 16 h, water (1 L) was added and stirred for 15 min, the organic phase was separated, the aqueous phase was basified with saturated Na2CO3 solution, and a yellow solid appeared. The yellow solid was extracted with DCM / 10% MeOH (3×700 mL). The organic phases were combined, washed with saturated NaCl solution and concentrated in vacuo to give a yellow solid, which was triturated with EtOH (50 mL), filtered, washed with EtOH (25 mL) and dried to give the title compound (51 g, 135 mmol, 87%) as a crystalline yellowish solid.

[0221] 1H NMR (δ, h / million, DMSO-D6): 9.41 (d, 2H), 9.37 (t, 1H), 8.77 (d, 1H), 8, 62-8.57 (m, 1H), 8.48-8.38 (m, 3H), 8.06 (d, 2H), (d, 2H), (d, 2H 2.03 (m, 2H), 1.89 (o, 2H), 1.82-1.54 (m, 4H) [IER MS] m / z: 378 (MH + ).

[0222] Example 4a: preparation of crystalline form of N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazine-2-yl)benzamide

[0223] The X-ray powder diffraction (XRD) pattern of the crystalline form shown in Example 4 is shown in Fig. 1, and the summary of the diffraction angles is given in Table 1 below. The XRD analysis was performed on a PANalytical Empyrean powder diffractometer, model 9430 060 03001, using a PIXcel3D detector. The data acquisition conditions were as follows: radiation: Cu Kα, generator voltage: 45 kV, generator current: 40 mA, start angle: 2.0° 2θ, end angle: 40.0° 2θ, step size: 0.0263° 2θ, step time: 46.665 seconds. The sample was prepared by depositing several milligrams of the sample onto a silicon wafer (zero-background wafer), resulting in a thin powder layer. The error margin is approximately ±0.1° 2θ for each peak assignment. Peak intensities may vary from sample to sample due to preferred orientation.

[0224]

[0225] Promenu exact compound 5: tert-butyl-(1-((4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamido)methyl)cyclopropyl)carbamate

[0226]

[0227] To a solution of 4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzoic acid (300 mg, 1.016 mmol), tert-butyl (1-(aminomethyl)cyclopropyl)carbamate (Combi-Blocks, 246 mg, 1.321 mmol) and DIPEA (Sigma-Aldrich, 0.532 mL, 3.05 mmol) in N,N-dimethylformamide (DMF) (10 mL) under N2 atmosphere, HATU (Fluorochem, 541 mg, 1.422 mmol) was added, and the resulting mixture was stirred at room temperature.

[0228] After 15.5 h, water was added until a pale yellow solid (25 mL) completely precipitated. The resulting solid was washed successively with 0.5 M NaOH (25 mL) and water (2×25 mL) and then dried under a stream of air. This solid (450 mg) was purified by column chromatography (solid loading gradients of 3 g SiO2, 27 g SiO2, silica, Cy:EtOAc from 80:20 to 0:100 over 30 min, flow 30 mL / min) to give, after collection and drying of the appropriate fractions, the title compound (358 mg, 0.772 mmol, yield 76%) as a white solid.

[0229] 1 H NMR (δ, ppm, DMSO-d6): 9.41 (o, 1H), 9.40 (o, 1H), 9.37 (t, 1H), 8.77 (d, 1H), 8.63-8.56 (m, 1H), 8.52 (t, 1H), 8.45-8.39 (m, 2H), 8.04 (br d, 2H), 7.26 (o, 1H), 3.43 (d, 2H), 0.83-0.76 (m, 2H), 0.67-0.61 (m, 2H). [ESI-MS] m / z: 464 (MH + ).

[0230] Example 5: N-((1-aminocyclopropyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide dihydrochloride

[0231]

[0232] To a stirred solution of tert-butyl (1-((4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamido)methyl)cyclopropyl)carbamate (100 mg, 0.216 mmol) in dichloromethane (2 mL) at 0°C was added HCl in 1,4-dioxane (SYMAX LABORATORY, 4 N, 0.162 mL, 0.647 mmol). The reaction mixture was stirred at room temperature for 1 hour.

[0233] The reaction mixture was diluted with DCM (5 mL), and the excess solvent was concentrated under reduced pressure to give the crude product as a brown solid. The crude product was triturated with diethyl ether (3×10 mL), and the supernatant was decanted. The compound was then dried under reduced pressure to give the title compound (73 mg, 0.180 mmol, 77%) as a brown solid.

[0234] 1H NMR (δ, ppm, DMSO-d6): 9.44 (o, 1H), 9.42 (o, 1H), 9.39 (m, 1H), 8.97 (t, 1H), 8.79 (d, 1H), 8.62 (m, 1H), 8.46 (d, 2H), 8.33 (br o, 3H), 8.13 (d, 2H), 3.58 (br d, 2H), 0.93 (o, 4H). [ESI MS] m / z: 364 (MH+).

[0235] Example 6: N-((1-aminocyclopropyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, ditrifluoroacetic acid salt

[0236]

[0237] Tert-butyl (1-((4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamido)methyl)cyclopropyl)carbamate (358 mg, 0.772 mmol) was dissolved in trifluoroacetic acid (5 mL) at 0 °C, and the reaction was allowed to reach room temperature. After 14 h, the reaction mixture was concentrated under reduced pressure. The residue (500 mg) was dissolved in 3.6 mL MeOH and purified by preparative HPLC (column: X-Bridge (50 mm × 150 mm); gradient: water (0.1% TFA): acetonitrile (0.1% TFA) from 20% to 100% (8 min); 1 injection; flow rate: 80 mL / min). The product-containing fractions were collected and the solvents were evaporated in vacuo to yield 300 mg fraction 1 and 155 mg fraction 2 as colorless oils:

[0238] - Fraction 1 was suspended in ACN (10 mL) and water (10 mL) and lyophilized to give the title compound (285 mg, 0.482 mmol, yield 62.4%) as a white solid.

[0239] - Fraction 2 was suspended in ACN (10 mL) and water (10 mL) and lyophilized to give the title compound (155 mg, 0.262 mmol, 33.9% yield) with lower purity (95% purity by HPLC) as a white solid.

[0240] 1 H NMR (δ, ppm, DMSO-D6): 9.43 (o, 1H), 9.42 (o, 1H), 9.38 (t, 1H), 8.88 (t, 1H), 8.78 (d, 1H), 8.61 (m, 1H), 8.45 (m, 2H), 8.21 (br o, 3H), 8.08 (m, 2H), 3.58 (d, 2H), 0.91 (m, 4H). [ESI MS] m / z: 364 (MH+).

[0241] Intermediate 6: tert-butyl (1-(4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamido)-2-methylpropan-2-yl)carbamate

[0242]

[0243] To a solution of 4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzoic acid (187 mg, 0.633 mmol) and DIPEA (SIGMA-ALDRICH, 0.553 mL, 3.17 mmol) in N,N-dimethylformamide (6.5 mL) was added HATU (FLUOROCHEM, 313 mg, 0.823 mmol). After stirring for 10 minutes, tert-butyl (1-amino-2-methylpropan-2-yl)carbamate (COMBI-BLOCKS, 155 mg, 0.823 mmol) was added, and the resulting mixture was stirred at room temperature.

[0244] After 16.5 h, the reaction mixture was partitioned between 20 mL of 1 N NH4Cl and 50 mL of EtOAc. The layers were separated, and the organic layer was washed with 1 N NH4Cl (20 mL) and then with brine (20 mL). The resulting mixture was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (packed in 1.5 mL DCM, 12 g SiO2, silicycle, gradients of Cy:EtOAc-EtOH 3 / 1 from 100:0 to 85:15 and to 50:50) to give, after collection and concentration of the appropriate fraction, 250 mg of tert-butyl (1-(4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamido)-2-methylpropan-2-yl)carbamate (250 mg, 0.537 mmol, 85% yield) as a white solid.

[0245] 1 H NMR (δ, ppm, DMSO-d6): 9.41 (d, 2H), 9.37 (t, 1H), 8.77 (d, 1H), 8.64 (t, 1H), 8.62-8.57 (m, 1H), 8.42 (d, 2H), 8.04 (d, 2H), 1H), 6.61 (br o, 1H), 3.42 (d, 2H), 1.45-1.34 (m, 9H),

[0246] 1.26 (o, 6H). [IER MS] m / z: 466 (MN + ).

[0247] Example 7: N-(2-amino-2-methylpropyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, ditrifluoroacetic acid salt

[0248]

[0249] To a solution of tert-butyl (1-(4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamido)-2-methylpropan-2-yl)carbamate (295 mg, 0.634 mmol) in dry dichloromethane (2 mL) under N2 atmosphere was added trifluoroacetic acid (1.000 mL), and the resulting mixture was stirred overnight.

[0250] The reaction mixture was diluted with MeOH and concentrated under reduced pressure. The residue was purified by preparative HPLC (sample loaded into 1.5 mL MeOH, XBridge 30×100 mm, using ACN:H2O (with 0.1% TFA) as mobile phase, flow rate 35 mL / min), collected and lyophilized to give 140 mg of N-(2-amino-2-methylpropyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, ditrifluoroacetic acid salt (140 mg, 0.292 mmol, yield 46.1%) as a white solid.

[0251] 1H NMR (δ, h / million, DMSO-D6): 9.42 (d, 2H), 9.38 (t, 1H), 8.88 (t, 1H), 8.77 (d, 1H), 8.63-8.56 (m, 1H), 8.48-8.41 (m, 1H), 8.48-8.41 (m, 8.62H), 2H), 7.89-7.73 (m, 1H), 7.88-7.73 (m, 1H), 3.53-3.40 (m, 2H), 1.38-1.20 (m, 6H) [IER MS] m / z: 366 (MH + ).

[0252] Intermediate 7: tert-butyl-((1R,2S)-2-(4-(6-(5-fluoropyridin-3-yl)pyrazine-2-yl)benzamido)cyclohexyl)carbamate

[0253]

[0254] To a stirred solution of 4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzoic acid (350 mg, 1.185 mmol) in N,N-dimethylformamide (15 mL) were added DIPEA (Sonia, 0.621 mL, 3.56 mmol) and HATU (GLR, 676 mg, 1.77 8 mmol) at 0 °C. The reaction mixture was stirred for 10 min at 0 °C. Later, tert-butyl ((1R,2S)-2-aminocyclohexyl)carbamate (TCI, 356 mg, 1.659 mmol) was added to the reaction. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with cold water (50 mL), after which a white solid precipitated. The resulting solid was filtered, washed with cold water (50 mL), and dried under vacuum. The crude product was adsorbed onto neutral alumina (1 g) and purified by column chromatography with Isolera (neutral alumina, 25 g), using 2% methanol in dichloromethane as the eluent.The product fractions were combined and evaporated in vacuo to give the title compound (430 mg, 0.865 mmol, 72.9% yield) as a brown solid.

[0255] 1 H NMR (δ, ppm, DMSO-d6): 9.42 (o, 1H), 9.41 (o, 1H), 9.37 (t, 1H), 8.77 (d, 1H), 8.63-8-55 (m, 1H), 8.41 (d, 2H), 8.00 (bd, 3H), 6.69 (m, 1H), 4.08 (m, 1H), 3.80 (m, 1H), 1.78 (m, 2H), 1.56 (m, 4H), 1.32 (m, 11H). [IER MS] m / z: 392 [(MN + )-100].

[0256] Example 8: N-((1S,2R)-2-aminodiclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, dihydrochloride

[0257]

[0258] A solution of tert-butyl ((1R,2S)-2-(4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamido)cyclohexyl)carbamate (0.895 g, 1.821 mmol) in a 1:9 mixture of DCM:MeOH (10 mL) under N2 atmosphere was heated at 40°C, and one portion of 37% hydrochloric acid (Sigma-Aldrich, 1 mL, 12.18 mmol) was added. The resulting mixture (solution) was stirred at 40°C overnight.

[0259] After 20 h, the reaction mixture was concentrated under reduced pressure. The resulting solid was triturated with TBME, but the resulting solid was not filterable. After concentration, the mixture was triturated with iPrOH, but the resulting solid was again not filterable.

[0260] After re-concentration, the mixture was dissolved in MeOH / DCM mixture and DCM was slowly evaporated on a rotary evaporator. The resulting suspension was stored in the refrigerator for 2 days. The suspension was filtered and washed with cold MeOH. The resulting solid was dried with air vapor to afford 800 mg of N-((1S,2R)-2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide dihydrochloride (800 mg, 1.723 mmol, 95% yield) as a white solid.

[0261] 1 H NMR (δ, ppm, DMSO-d6: 9.43 (o, 1H), 9.41 (o, 1H), 9.38 (t, 1H), 8.78 (d, 1H), 8.61 (m, 1H), 8.42 (d, 2H), 8.39 (d, 1H), 8.17 (d, 2H), 8.12 (br d, 3H), 4.33 (td, 1H), 3.48 (m, 1H), 1.90 (m, 2H), 1.67 (m, 4H), 1.42 (m, 2H). [ESI MS] m / z: 392 [(MH+)].

[0262] Example 9: N-((1S,2R)-2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, ditrifluoroacetic acid salt

[0263]

[0264] To a suspension of N-((1S,2R)-2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide (850 mg, 2.171 mmol) in dichloromethane (DCM) (40 mL) was added trifluoroacetic acid (2 mL, 26.0 mmol) (turned into a solution), and stirred at room temperature for 1 hour.

[0265] The crude product was concentrated in vacuo and the resulting solid was dried in a vacuum oven at 40°C for 18 hours to give a yellowish solid of N-((1S,2R)-2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, ditrifluoroacetic acid salt (1.36 g, 2.195 mmol, yield approximately 99%).

[0266] 1 H NMR (δ, ppm, DMSO-d6): 9.44 (o, 1H), 9.42 (o, 1H), 9.38 (m, 1H), 8.78 (d, 1H), 8.60 (m, 1H), 8.43 (d, 2H), 8.23 ​​(d, 1H), 8.12 (d, 2H), 7.82 (br d, 3H), 4.37 (m, 1H), 3.45 (m, 1H), 1.75 (m, 6H), 1.42 (m, 2H). [ESI-MS] m / z: 392 (MH + ).

[0267] Example 10: N-((1S,2R)-2-aminodiclogexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide

[0268]

[0269] To a solution of tert-butyl ((1R,2S)-2-(4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamido)cyclohexyl)carbamate (1.7 g, 3.46 mmol) in a mixture of dichloromethane (DCM) (5 mL) and methanol (5.00 mL) was added 6 N hydrochloric acid in iPrOH (FLUOROCHEM, 1.32 mL, 7.92 mmol), and stirred at room temperature. After 16 h, the solid was filtered, the solid was suspended in water and saturated Na2CO3 solution was added until basic pH was reached, the solid was extracted with 10% DCM / MeOH solution (3×10 mL), the organic phases were combined, washed with saturated NaCl, dried over Na2SO4, filtered and concentrated in vacuo to give a yellow crystalline solid of N-((1S,2R)-2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide (1.02 g, 2.61 mmol, 75% yield).

[0270] 1H NMR (δ, h / million, DMSO-d6): 9.41 (o, 2H), 9.37 (m, 1H), 8.77 (d, 1H), 8.60 (m, 1H), 8.40 (d, 2H), 8.03 (d, 2H), 8.01 (d, 1H), (m, 95), 3.06 (m, 1H), 1.84-1.46 (m, 8H), 1.32 (m, 2H). [IER MS] m / z: 392 (MH + ).

[0271] Example 10a: Crystal form of N-((1S,2R)-2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazine-2-yl)benzamide

[0272] The X-ray powder diffraction (XPD) pattern of the crystalline form shown in Example 11 is shown in Fig. 3, and the summary of the diffraction angles is given in Table 2 below. The XPD analysis was performed on a PANalytical Empyrean powder diffractometer, model 9430 060 03001, using a PIXcel3D detector. The data acquisition conditions were as follows: radiation: Cu Kα:, generator voltage: 45 kV, generator current: 40 mA, start angle: 2.0° 2θ, end angle: 40.0° 2θ, step size: 0.0263° 2θ, step time: 46.665 seconds. The sample was prepared by depositing several milligrams of the sample onto a silicon wafer (zero-background wafer), resulting in a thin powder layer. The error margin is approximately ±0.1° 2θ for each peak assignment. Peak intensities may vary from sample to sample due to preferred orientation.

[0273]

[0274] BIOLOGICAL AND PHYSICAL-CHEMICAL DATA

[0275] The compounds of the present invention can be tested in one of several biological and physicochemical assays to determine the concentration of the compound required to provide a desired pharmacological effect. The assays are described below.

[0276] In vitro activity

[0277] Growth Inhibition Assay of P. falciparum Sybr green Parasite growth was determined by staining the minor groove of DNA with SYBR Green I, which labels other parasites. Parasite growth was determined by staining the minor groove of DNA with SYBR Green I, which labels other parasites. The DNA-SYBR complex absorbs light, reaching a peak at λ = 498 nm, and emits light at a peak at λ = 522 nm.

[0279] Methodology

[0280] Briefly, cultured red blood cells (RBCs) parasitized with strain 3D7A (0.5% parasitemia and 2% hematocrit in RPMI-1640 supplemented with 25 mL of albumax and 150 mM hypoxanthine per 500 mL) were subjected to 3-fold serial dilutions of the compound. The plates were incubated for 48 hours at 37°C, 5% CO2, 5% O2, 90% N2. After 48 hours of incubation, SyberGreen I solution was added (2 times), and the plates were incubated for 30 minutes at 37°C, 5% CO2, 5% O2, 90% N2, and then frozen at -80°C. The plates were then read in EnVision (EnVision Multilabel Plate Reader, Perkin Elmer). Data (pIC50) were generated using ActivityBase (IDBS) and graphs were verified using Spotfire.

[0281] Results

[0282] The results are shown in Table 3 below. Inhibition of parasite growth in the whole-cell assay (infected human erythrocytes) in vitro is directly related to a reduction in parasitemia in vivo.

[0283]

[0284] In vivo efficacy

[0285] In Vivo Efficacy Analysis of P. falciparum.

[0286] Antimalarial efficacy in vivo was determined using a P. falciparum mouse model according to the procedure described in: Jimenez-Díaz, M.B., Mulet, T., Viera, S., Gómez, V., Garuti, H., Ibañez, J., Alvarez-Doval, A., Schlutz, D.L., Martinez, A., Improved Murine Model of Malaria Using Plasmodium falciparum (Competent Strains and Non-Myelodepleted NOD-scid IL2R_null Mice Engrafted with Human Erythrocytes) Antimicrob. Agents Chemother 2009, 53 (10), 4533–4536.

[0287] Results

[0288] The aim of the study was to evaluate the therapeutic efficacy of samples against Plasmodium falciparum Pf3D7 0087 / N9 , growing in the peripheral blood of NODscidlL2Ry mice 0087 / N9, which were transplanted with human red blood cells. Levels of the compounds were measured in serial peripheral blood samples obtained from each mouse in the efficacy experiment during the first 23 hours after the first dose. The area under the curve of compound levels was used to estimate exposure in the blood during the first 23 hours after the first dose (AUC 0-23 ч ).

[0289] Effect of Examples 1, 2, 5, 7, 8 and 10 on P. falciparum Pf3D7 0087 / N9 were assessed using microscopy and flow cytometry.

[0290] The results are shown in Table 4 below.

[0291]

[0292] Evaluation of solubility in a biorelevant environment

[0293] The equilibrium solubility of the compounds was measured at room temperature after 4 h equilibration in fasting simulated intestinal fluid (FaSSIF) at pH 6.5.

[0294] Solvents and buffers

[0295] HPLC-grade organic solvents were used. Ultrapure water (Milli-Q grade) was used. Buffers were prepared using ultrapure water.

[0296] Methodology

[0297] 1 mg of the solid compound was weighed into a 4 ml glass vial and 1 ml of FaSSIF was added.

[0298] 1 mg of the same solid was weighed into a 4 mL glass vial and 1 mL of dimethyl sulfoxide (DMSO) was added to prepare a standard solution of known concentration.

[0299] The FaSSIF sample was left on a shaker (900 rpm) for 4 hours at room temperature.

[0300] After 4 hours, the resulting suspension was transferred to a 96-well Multiscreen HTS-PCF solubility plate and vacuum filtration was used to separate the residual solid and filtrate.

[0301] The filtrate was analyzed by LC-UV.

[0302] A set of 3 internal standards with known solubility (atovaquone, nimesulide and warfarin 2, 20 and 140 mg / mL, respectively) were tested using the same procedure together with the compounds to assess process suitability.

[0303] LC-UV analysis for analytical quantitative analysis

[0304] Quantitative determination of the compound concentration in the filtrate was performed using LC-UV with a single-point calibration of a known compound concentration in DMSO. The dynamic range of the analysis was 1–1000 μg / mL.

[0305] Data Analysis

[0306] LC-UV data analysis was performed using an internal Excel macro. The solubility (mg / mL) of each compound was calculated from the peak areas of the sample and standard.

[0307] Results

[0308] The results are shown in Table 5 below. Aqueous solubility of a compound is an important drug characteristic affecting oral bioavailability. The FaSSIF medium simulates the fluids in the intestinal compartment, from which most drugs are absorbed. Values ​​with limited intestinal solubility can lead to incomplete and variable drug absorption. Therefore, it is of interest to estimate the amount of dissolved drug in the gastrointestinal tract.

[0309]

[0310] Evaluation of solubility in aqueous buffer

[0311] The kinetic solubility in water was measured at room temperature after 1 hour equilibration in aqueous phosphate-buffered saline (PBS) at pH 7.4 and determined by measuring the solute concentration in the solution after precipitation from the DMSO stock solution.

[0312] Solvents and buffers

[0313] HPLC-grade organic solvents were used. Ultrapure water (Milli-Q grade) was used. Buffers were prepared using ultrapure water.

[0314] Methodology

[0315] 5 mL of 10 mM DMSO stock solution was dispensed into a well of a 96-well Multiscreen HTS-PCF soluble filter plate and 95 mL of PBS was added.

[0316] The sample plate was left to equilibrate for 1 hour at room temperature.

[0317] Vacuum filtration was used to filter the solution into a 96-well collection plate.

[0318] The filtrate was analyzed using HPLC-CAD (charged aerosol detector).

[0319] Calibration standards of ketoconazole and primidone were prepared by serial dilutions in DMSO at concentrations ranging from 0.016 to 4.5 mg / mL to obtain a calibration curve.

[0320] LC-UV Analysis for Analytical Quantitative Analysis

[0321] Quantitative determination of the compound concentration in the filtrate was carried out using HPLC-DZA using the peak areas of the DZA sample and calibration coefficients.

[0322] Data Analysis

[0323] HPLC-DZA data analysis was performed using an internal Excel macro. The concentration (mM) and solubility (μg / mL) for each compound were calculated based on sample peak areas and calibration coefficients.

[0324] Results

[0325] The results are shown in Table 6 below.

[0326]

[0327]

Claims

1. A compound selected from: or a pharmaceutically acceptable salt thereof.

2. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is 3. The compound of claim 2, wherein the compound is in the form of a free base.

4. The compound according to item 3, which has i) an X-ray powder diffraction pattern (XPD) substantially as shown in Fig. 1; and / or ii) X-ray powder diffraction (XPD) pattern with specific peaks at 2θ values, ±0.1° 2θ experimental error, 6.7, 11.2, 12.7, 13.4, 16.2, 16.6, 17.9, 20.9, 26.7 and 28.2 degrees.

5. The compound according to claim 2 in the form of pharmaceutically acceptable sulfuric acid.

6. The compound according to claim 2 in the form of a pharmaceutically acceptable dihydrochloride.

7. The compound according to claim 2 in the form of a pharmaceutically acceptable salt of ditrifluoroacetic acid.

8. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is 9. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is 10. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is 11. The compound according to claim 10, which is in the form of a free base.

12. The compound according to item 11, which has i) an X-ray powder diffraction (XPD) pattern substantially as shown in Fig. 2; and / or ii) X-ray powder diffraction (XPD) pattern with specific peaks at 2θ values, ±0.1° 2θ experimental error, 5.4, 10.8, 15.3, 16.6, 18.2, 20.1, 21.8, 22.4, 28.1 and 31.7 degrees.

13. A compound or a pharmaceutically acceptable salt thereof selected from the group consisting of N-((1-aminocyclobutyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, N-(2-amino-2-methylpropyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, N-(2-aminocyclohexyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide, and N-((1-aminocyclopropyl)methyl)-4-(6-(5-fluoropyridin-3-yl)pyrazin-2-yl)benzamide.

14. A pharmaceutical composition for the treatment of malaria, comprising (a) a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13 and (b) a pharmaceutically acceptable excipient.

15. A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13 for the treatment of malaria.

16. The use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13 or a pharmaceutical composition according to claim 14 in the manufacture of a medicinal product for the treatment of a parasitic protozoan infection, wherein the parasitic protozoan infection is malaria.

17. The use according to claim 16, wherein the parasitic protozoan infection is caused by Plasmodium falciparum.

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