Anti-infective agents

Abstract

The present invention relates to compounds or pharma- ceutically acceptable salts thereof, compositions containing them, including in combination with at least one additional therapeutic agent, and their use in therapy, e.g., in the treatment of infectious diseases or in the treatment of diseases caused by Cryptosporidium.

Description

[Technical field]

[0001] FIELD OF THE PRESENT APPLICATION The present invention relates to compounds or pharma- ceutically acceptable salts thereof, compositions containing them, including in combination with at least one additional therapeutic agent, and their use in therapeutic methods, e.g., in the treatment of infectious diseases or in the treatment of diseases caused by Cryptosporidium. [Background technology]

[0002] background Cryptosporidiosis is a diarrheal disease caused by the parasitic species Cryptosporidium. Currently, 27 species of Cryptosporidium have been identified, including 20 species that infect humans, with C. parvum or C. hominis causing the majority of human infections (Int. J. For Parasitology 2015, 45, 367-373). Cryptosporidiosis was first identified as a cause of human infection in 1976 (Gastroenterology, 1976, 70, 592-598). A recent study investigating the etiology of diarrhea in over 22,000 children under the age of five identified Cryptosporidium as the second most common cause of both diarrhea and morbidity after rotavirus (The Lancet, 2013, 382, ​​9888, 209-222).

[0003] Cryptosporidiosis is a major cause of diarrhea affecting children in many parts of the world, especially in sub-Saharan Africa and South Asia. A recent Global Enteric Multicenter Study (GEMS) estimated that there are 2.9 to 4.7 million cases in children under 24 months of age in sub-Saharan Africa and the Indian subcontinent, and that cryptosporidiosis is associated with over 200,000 deaths annually. Cryptosporidiosis is also associated with malnutrition and stunted growth. It is one of the leading causes of death from diarrheal diseases.

[0004] Cryptosporidiosis is an opportunistic infection, and individuals with immature immune systems, such as children under the age of 5, and immunocompromised individuals co-infected with HIV, are at higher risk of infection and mortality. Malnutrition in early childhood is also associated with persistent diarrhea and Cryptosporidium infection (Lancet Infect. Dis., 2015, 15, 85-94).

[0005] Nitazoxanide is the only FDA-approved drug for the treatment of cryptosporidiosis. It has been determined that the efficacy of nitazoxanide is suboptimal and is not an effective treatment for all patients infected with Cryptosporidium alone (J. Infect. Dis., 2001, 184, 103-06 and Clin. Gastroenterol. Hepatol., 2006, 4, 320-24). Nitazoxanide has also been shown in clinical trials to be ineffective in patients with Cryptosporidium-HIV coinfection who were not co-treated with HIV antiretroviral therapy (Trans. R. Soc. Trop. Med. Hyg. 1998, 92, 663-66 and BMC Infect. Dis. 2009, 9, 195).

[0006] Thus, there is a need for new anti-infective agents that are effective against Cryptosporidium, particularly agents that are suitable for use in the treatment or prevention of subjects infected with Cryptosporidium alone; immunocompromised subjects infected with Cryptosporidium, such as Cryptosporidium-HIV co-infected subjects, malnourished children under the age of one year suffering from diarrhea caused by Cryptosporidium infection, etc.

[0007] Lysyl-tRNA synthetase (LysRS or KRS) Protein synthesis is a complex, multi-step process involving many enzymes. Aminoacyl-tRNA synthetases (aaRS) catalyze the conjugation of amino acids with their associated transfer RNAs, which play a key role in protein translation. Inhibition of aaRS has been successfully exploited against bacterial infections, with one aaRS inhibitor, mupirocin, currently in clinical use for the topical treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. Mupirocin is an inhibitor of isoleucyl-tRNA synthetase (IleRS).

[0008] Recently, cladosporins have been identified as inhibitors of lysyl-tRNA synthetase (LysRS). Cladosporins are fungal secondary metabolites, as reported by Scott et. al., J. Antibiot. 1971 24, 747-755. Cladosporins have been shown to inhibit PfLysRS with over 100-fold selectivity over HsLysRS (Hoepfner et al, Cell Host Microbe, 2012, 11(6):654-63). We have reported that cladosporins and chromone analogs are inhibitors of C. parvum LysRS (Baragana et al., PNAS, 2019, 116 (4), 7015-7020).

[0009] Chromone analogs showed reduced oocyst shedding in two different mouse models of C. parvum infection (WO2017221002 A1 and Baragana et al., PNAS, 2019, 116 (4), 7015-7020). These results validate C. parvum LysRS as a promising mechanism of action for the treatment of Cryptosporidium infections. Summary of the Invention

[0010] overview The present invention relates to a compound of formula I: [ka] {In the formula, R 1 is selected from -CH3, -CF2H, or -CF3; and R 2 below: [ka] or a veterinarily or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0011] In the compounds of formula I, R 1 is preferably selected from -CH3 or -CF3. Most preferably, R 1 may be -CF3.

[0012] In the compounds of formula I, R 2 is preferably one of the following: [ka] may be also possible.

[0013] The compound of formula I has the following formula IA: [ka] {where, R 1is selected from -CH3, -CF2H or -CF3; or It may be a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0014] In the compound of formula IA, R 1 is preferably selected from -CH3 or -CF3. Most preferably, R 1 may be -CF3.

[0015] The compound of formula I has the following formula IB: [ka] {where, R 1 is selected from -CH3, -CF2H, or -CF3}, or a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug, or polymorph thereof.

[0016] In the compound of formula IB, R 1 is preferably selected from -CH3 or -CF3. Most preferably, R 1 may be -CF3.

[0017] The compound of formula I has the following formula IC: [ka] {where, R 1 is selected from -CH3, -CF2H, or -CF3}, or a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug, or polymorph thereof.

[0018] In the compound of formula IC, R 1 is preferably selected from -CH3 or -CF3. Most preferably, R 1 may be -CF3.

[0019] The compound of formula I has the following formula ID: [ka] {where, R 1 is selected from -CH3, -CF2H, or -CF3}, or a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug, or polymorph thereof.

[0020] In the compound of formula ID, R 1 is preferably selected from -CH3 or -CF3. Most preferably, R 1 may be -CF3.

[0021] The compounds of formula I have the following formula IE: [ka] {where, R 1 is selected from -CH3, -CF2H, or -CF3}, or a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug, or polymorph thereof.

[0022] In the compound of formula IE, R 1 is preferably selected from -CH3 or -CF3. Most preferably, R 1 may be -CF3.

[0023] The compound of formula I has the following formula IF: [ka] {where, R 1 is selected from -CH3, -CF2H, or -CF3}, or a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug, or polymorph thereof.

[0024] In the compound of formula IF, R 1 is preferably selected from -CH3 or -CF3. Most preferably, R 1may be -CF3.

[0025] The compound of formula I has the following formula IG: [ka] {where, R 1 is selected from -CH3, -CF2H, or -CF3}, or a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug, or polymorph thereof.

[0026] In some embodiments, the compound of formula IG may be a racemic mixture. In some embodiments, the compound of formula IG may be optically active.

[0027] In the compound of formula IG, R 1 is preferably selected from -CH3 or -CF3. Most preferably, R 1 may be -CF3.

[0028] Preferred compounds of formula I are as follows: [ka] or a veterinarily or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0029] Preferred compounds of formula I are as follows: 2-Amino-4-(trifluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one; 2-Amino-4-methyl-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one; 2-Amino-4-(difluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one; 2-Amino-6-(spiro[2.5]octan-5-ylmethyl)-4-(trifluoromethyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one; 2-Amino-4-(trifluoromethyl)-6-(((1R,3R)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one; 2-Amino-4-(trifluoromethyl)-6-(((1S,3R)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one; 2-amino-4-(trifluoromethyl)-6-(((1S,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one; or a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof; may be selected from:

[0030] The compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG have a pEC50 against Cryptosporidium parvum of 6 or more, preferably 6.5 or more, more preferably 7 or more, in particular 7.3 or 7.7 or more. 50 may have:

[0031] The compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG have a pIC value for Cryptosporidium parvum lysyl tRNA synthetase (Cp KRS) of 5 or more, preferably 5.5 or more, and more preferably 6 or more. 50 may have:

[0032] In a second aspect, there is provided a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG as hereinbefore described for use in medicine.

[0033] In a third aspect, there is provided a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG as hereinbefore described for veterinary use.

[0034] In a fourth aspect, there is provided a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG as hereinbefore described for use as a medicament.

[0035] In a further aspect, there is provided a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG as hereinbefore described for use in the treatment of an infectious disease.

[0036] In a further aspect, there is provided a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG as hereinbefore described for use in the treatment of cryptosporidiosis.

[0037] In a further aspect, there is provided a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG as hereinbefore described for use in the treatment of disease caused by Cryptosporidium. The diseases include: Cryptosporidium andersoni, Cryptosporidium baileyi, Cryptosporidium bovis, Cryptosporidium canis, Cryptosporidium chipmunk, Cryptosporidium cuniculus, Cryptosporidium ducismarci, Cryptosporidium felis, Cryptosporidium fayeri, Cryptosporidium galli, Cryptosporidium meleagridis, Cryptosporidium meleagridis, Cryptosporidium muris, Cryptosporidium monari, Cryptosporidium suis, Cryptosporidium scrofarum, Cryptosporidium tyzzeri, Cryptosporidium ubiquitum, Cryptosporidium viatorum, Cryptosporidium nasorum, Cryptosporidium parvum, Cryptosporidium hominisThe infection may be caused by a Cryptosporidium species selected from Cryptosporidium hominis, Cryptosporidium saurophilum, Cryptosporidium serpentis, and Cryptosporidium wrairi.

[0038] Another aspect relates to a method of treating a Cryptosporidium infection in a human or animal in need thereof, the method comprising administering to the human or animal a therapeutically effective amount of a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG, as hereinbefore described, or a pharma- ceutically acceptable salt thereof.

[0039] Another aspect of the present invention is a method of treating a disease caused by infection with a Cryptosporidium parasite in a human or animal in need thereof, comprising administering to the human a therapeutically effective amount of a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG, as hereinbefore described, or a pharma- ceutically acceptable salt thereof.

[0040] Another aspect of the present invention relates to the use of a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG, or a pharma- ceutically acceptable salt thereof, as hereinbefore described, in the manufacture of a medicament for use in the treatment of cryptosporidiosis or a disease caused by infection with a Cryptosporidium parasite.

[0041] Another aspect of the present invention relates to the combination of: (a) a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG, as hereinbefore described, or a pharma- ceutically acceptable salt thereof; and (b) at least one other antiparasitic agent.

[0042] Another aspect of the present invention relates to a kit comprising a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG, as hereinbefore described, or a pharma- ceutically acceptable salt thereof, and instructions for administration to a human or animal in need thereof. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Detailed Description of the Invention Terms and Definitions It will be appreciated that phrases such as "a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG, or a pharma- ceutically acceptable salt thereof" are intended to encompass a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG, a pharma- ceutically acceptable salt or solvate of a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG, or any pharma- ceutically acceptable combination thereof. Thus, as a non-limiting example used herein for illustrative purposes, "a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG, or a pharma- ceutically acceptable salt thereof" includes compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG present as solvates or hydrates, and pharma- ceutically acceptable salts thereof, and the phrase also includes mixtures of compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG with pharma- ceutically acceptable salts of compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG.

[0044] It should further be understood that references herein to compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG, or a pharma- ceutically acceptable salt thereof, include compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG as the free base or as a pharma- ceutically acceptable salt thereof.

[0045] The term "pharmaceutical acceptable" refers to compounds (including salts), materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, or other problem or complication, and are commensurate with a reasonable benefit / risk ratio.

[0046] As used herein, the term "pharmaceutical acceptable salts" refers to salts that retain the desired biological activity of the human compound and exhibit minimal undesired toxicological effects. These pharmaceutical acceptable salts may be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free acid or free base form with a suitable base or acid, respectively.

[0047] Pharmaceutically acceptable salts include, inter alia, those described in Berge, J. Pharm. Sci., 1977, 66, 1-19, or those listed in PH Stahl and CG Wermuth, editors, Handbook of Pharmaceutical Salts; Properties, Selection and Use, Second Edition Stahl / Wermuth: Wiley- VCH / VHCA, 2011 (see http: / / www.wiley.com / WileyCDA / WileyTitle / productCd-3906390519.html).

[0048] Where the functionality of the compound is taken into consideration, suitable pharma- ceutically acceptable salts of the compounds of formula I, IA, IB, IC, ID, and / or IE may be formed, including acid addition salts, which may be formed by reaction with an appropriate acid, optionally in a suitable solvent, such as an organic solvent, to provide a salt which may be isolated by crystallization or filtration.Representative pharma- ceutically 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, and 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 (glucoceptate), gluconate, glucuronate, glutamate, glutarate, glycerophosphate, glycolate, hexylresorcinate, hippurate, hydrabamine (N, N'-Di(dehydroabietyl)-ethylenediamine), hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoate, isobutyrate, lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, methanesulfonate (mesylate), methylsulfate, mucate, naphthalene-1,5-disulfonate (napadisylate), naphthalene-2-sulfonate (napsylate), nicotinate, nitrate, oleate, palmitate, p-aminobenzenesulfonate, p-aminosalicylate, pamoate Salts such as phenylacetate (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-chlorotheophylline), thiocyanate, triethiodide, undecanoate, undecylenate, and valerate.

[0049] As used herein, the term "therapeutically effective amount" means any amount that results in improved treatment, cure, prevention, or amelioration of a disease, disorder, or side effects, or a slowing of the rate of progression of a disease or disorder, when compared to a corresponding human not receiving such amount. An appropriate "therapeutically effective amount" will depend on many factors, including, for example, the age and weight of the human, the precise condition for which treatment is sought and its severity, the nature of the formulation, and the route of administration, and will ultimately be at the discretion of the attending physician.

[0050] As used herein, "treatment" and "treating" are used interchangeably herein and refer to an approach to obtain beneficial or desired results, including, but not limited to, a therapeutic effect. A therapeutic effect means the eradication or amelioration of the underlying disorder being treated. A therapeutic effect may also be achieved by the eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in a patient, even though the patient may still be afflicted by the underlying disorder. The term "treat" in all of its verb forms is used herein to mean to alleviate, alleviate, prevent, and / or manage at least one symptom of a disorder in a human.

[0051] The term "solvate" refers to a compound of I, IA, IB, IC, ID, IE, IF and / or IG associated with a solvent, usually by solvolysis, or a pharma- ceutically acceptable salt form thereof. This physical association can include hydrogen bonding. Common solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein can be prepared, for example, in crystalline form and can be solvated. Suitable solvates include pharma- ceutically acceptable solvates, and further include both stoichiometric and non-stoichiometric solvates. In certain cases, the solvate can be isolated, for example when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" encompasses both solution-phase and isolatable solvates. Exemplary solvates include hydrates, ethanolates, and methanolates. In some embodiments, the compounds of Formula I, IA, IB, IC, ID, IE, IF and / or IG, or pharma- ceutically acceptable salts thereof, may be isolated, formed, and / or administered as a solvate.

[0052] As used herein, the term "hydrate" refers to a compound of formula I, IA, IB, IC, ID, IE, IF, and / or IG in combination with water, or in the form of a pharma- ceutically acceptable salt thereof. The term "hydrate" refers to a compound in combination with water. Usually, the number of water molecules contained in a hydrate of a compound is a certain ratio to the number of compound molecules in the hydrate. Thus, a hydrate of a compound can be represented, for example, by the general formula R·xH2O, where R is the compound and x is a number greater than 0. A given compound can form more than one hydrate, including, for example, a monohydrate (x is 1), a lower hydrate (x is a number greater than 0 and less than 1, e.g., a hemihydrate (R·0.5H2O)), and a polyhydrate (x is a number greater than 1, e.g., a dihydrate (R·2H2O) and a hexahydrate (R·6H2O)). In some embodiments, the compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG, or a pharma- ceutically acceptable salt thereof, may be isolated, formed, and / or administered as a hydrate.

[0053] It should also be understood that compounds that have identical molecular formulae but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed "isomers." Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers."

[0054] Stereoisomers that are not mirror images of each other are called "diastereomers" and stereoisomers that are non-superimposable mirror images of each other are called "enantiomers". For example, if a compound has an asymmetric center, it is attached to four different groups and a pair of enantiomers is possible. Enantiomers can be characterized by the absolute configuration of the asymmetric center and described by the R- and S-sequencing rules of Cahn and Prelog or by the way the molecule rotates the plane of polarized light and are named dextrorotatory or levorotatory (i.e., (+) or (-)-isomers, respectively). Chiral compounds can exist as individual enantiomers or mixtures thereof. A mixture containing equal proportions of enantiomers is called a "racemic mixture".

[0055] In some embodiments, a compound of formula (I) or a pharma- ceutically acceptable salt thereof may contain one or more asymmetric centers (also called chiral centers) and may therefore exist as an individual enantiomer, diastereoisomer, or other stereoisomer or as a mixture thereof. Reference herein to a compound of formula I or a pharma- ceutically acceptable salt thereof refers to any single stereoisomer or mixture of stereoisomers, unless otherwise indicated.

[0056] The term "polymorph" refers to a crystalline form of a compound (or its salts, hydrates, or solvates). All polymorphs have the same elemental composition. Usually, different crystalline forms have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors may cause one crystalline form to predominate. Various polymorphs of a compound can be prepared by crystallization under different conditions. Polymorphic forms of the compound of formula (I) or its pharma- ceutically acceptable salts can be characterized and distinguished using many conventional analytical techniques, including, but not limited to, X-ray powder diffractometry (XRPD), infrared spectroscopy (IR), Raman spectroscopy, differential scanning calorimetry (DSC), thermogravimetry (TGA), and solid-state nuclear magnetic resonance (ssNMR).

[0057] compound The present invention relates to a compound of formula I: [ka] {In the formula, R 1 is selected from -CH3, -CF2H, or -CF3; and R 2 below: [ka] or a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0058] In the compounds of formula I, R 1 is preferably selected from -CH3 or -CF3. Most preferably, R 1 may be -CF3.

[0059] In the compounds of formula I, R 2 is preferably one of the following: [ka] may be also possible.

[0060] The compound of formula I has the following formula IA: [ka] {where, R 1 is selected from -CH3, -CF2H, or -CF3}, or a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug, or polymorph thereof.

[0061] In the compound of formula IA, R 1 is preferably selected from -CH3 or -CF3. Most preferably, R 1 may be -CF3.

[0062] The compound of formula I has the following formula IB: [ka] {where, R 1 is selected from -CH3, -CF2H, or -CF3}, or a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug, or polymorph thereof.

[0063] In the compound of formula IB, R 1 is preferably selected from -CH3 or -CF3. Most preferably, R 1 may be -CF3.

[0064] The compound of formula I has the following formula IC: [ka] {where, R 1 is selected from -CH3, -CF2H, or -CF3}, or a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug, or polymorph thereof.

[0065] In the compound of formula IC, R 1 is preferably selected from -CH3 or -CF3. Most preferably, R 1 may be -CF3.

[0066] The compound of formula I has the following formula ID: [ka] {where, R 1 is selected from -CH3, -CF2H, or -CF3}, or a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug, or polymorph thereof.

[0067] In the compound of formula ID, R 1 is preferably selected from -CH3 or -CF3. Most preferably, R 1 may be -CF3.

[0068] The compounds of formula I have the following formula IE: [ka] {where, R 1 is selected from -CH3, -CF2H, or -CF3}, or a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug, or polymorph thereof.

[0069] In the compound of formula IE, R 1is preferably selected from -CH3 or -CF3. Most preferably, R 1 may be -CF3.

[0070] The compound of formula I has the following formula IF: [ka] {where, R 1 is selected from -CH3, -CF2H, or -CF3}, or a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug, or polymorph thereof.

[0071] In the compound of formula IF, R 1 is preferably selected from -CH3 or -CF3. Most preferably, R 1 may be -CF3.

[0072] The compound of formula I has the following formula IG: [ka] {where, R 1 is selected from -CH3, -CF2H, or -CF3}, or a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug, or polymorph thereof.

[0073] In the compound of formula IG, R 1 is preferably selected from -CH3 or -CF3. Most preferably, R 1 may be -CF3.

[0074] Exemplary compounds of Formula I are shown in Table 1. [Table 1]

[0075] Preferred compounds of formula I are as follows: [ka] or a veterinarily or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0076] Preferred compounds of formula I are as follows: 2-Amino-4-(trifluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one; 2-Amino-4-methyl-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one; 2-Amino-4-(difluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one; 2-Amino-6-(spiro[2.5]octan-5-ylmethyl)-4-(trifluoromethyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one; 2-amino-4-(trifluoromethyl)-6-(((1R,3R)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one; or a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof; may be selected from:

[0077] Particularly preferred compounds of formula I are those 2-Amino-4-(trifluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one; 2-amino-4-(difluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one; and 2-amino-4-methyl-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one; or a veterinary or pharma- ceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof; may be selected from:

[0078] The compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG have a pEC50 against Cryptosporidium parvum of 6 or more, preferably 6.5 or more, more preferably 7 or more, in particular 7.3 or 7.7 or more. 50 may have:

[0079] The compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG have a pIC value for Cryptosporidium parvum lysyl tRNA synthetase (Cp KRS) of 5 or more, preferably 5.5 or more, and more preferably 6 or more. 50 may have:

[0080] It is understood that compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG may exist in various tautomeric forms, and all possible tautomers and pharma- ceutically acceptable salts thereof are contemplated as being within the scope of the present invention.

[0081] The compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG or their pharma- ceutically acceptable salts may be in crystalline or non-crystalline form. Furthermore, some of the crystalline forms may exist as polymorphs, all of which are included within the scope of the present invention. The most thermodynamically stable polymorphic form(s) of the compounds of formula I, IA, IB, IC, ID and / or IE or their pharma- ceutically acceptable salts are of particular interest. In one embodiment of the present invention, the compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG or their pharma- ceutically acceptable salts are crystalline.

[0082] How to use Reference to treatment as used herein should be understood to include prophylaxis, as well as symptomatic treatment by alleviation of defined symptoms of the condition, i.e., prevention or suppression. "Treating" or "treatment" of a condition, disorder or condition includes the following: (1) preventing or delaying the appearance of clinical symptoms of the condition, disorder or condition occurring in a human who may be afflicted with or susceptible to the condition, disorder or condition, but who has not yet experienced or exhibited clinical or subclinical symptoms of the condition, disorder or condition, (2) inhibiting the condition, disorder or condition, i.e., arresting, reducing or delaying the onset of the disease or its recurrence (in the case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) alleviating or attenuating the disease, i.e., regression of the condition, disorder or condition, or at least one of its clinical or subclinical symptoms.

[0083] Those skilled in the art will appreciate that references herein to treatment refer to the treatment of the specified condition. However, the compounds of general formula (I) and their pharma- ceutically acceptable salts may also be useful in the prevention (prophylaxis) of certain diseases, depending on the circumstances.

[0084] As used herein, unless otherwise indicated, "treat," "treating," or "treatment," in reference to a disease, means: (1) ameliorating the disease or one or more of the biological symptoms of the disease; (2) interfering with (a) one or more points in the biological cascade that leads to or causes the disease, or (b) one or more of the biological symptoms of the disease; (3) alleviating one or more of the symptoms or effects associated with the disease; (4) slowing the progression of the disease or one or more of the biological symptoms of the disease; and / or (5) reducing the likelihood or severity of the disease or one or more of the biological symptoms of the disease.

[0085] As used herein, unless otherwise indicated, "prevent", "preventing" or "prevention" refers to the prophylactic administration of a drug that reduces the likelihood of onset of a disease or its biological manifestations or delays their onset. Those skilled in the art will appreciate that "prevention" is not an absolute term. In medicine, "prevention" is understood to refer to the prophylactic administration of a drug that substantially reduces the likelihood or severity of a disorder or its biological manifestations or delays the onset of such a disorder or its biological manifestations.

[0086] Thus, in one embodiment, treatment or prevention of a disease is provided. In another embodiment, treatment of a disease is provided. In another embodiment, prevention of a disease is provided.

[0087] Thus, as a further aspect of the present invention there is provided a compound of formula I, or a pharma- ceutically acceptable salt thereof, for use in therapy.Furthermore there is provided a compound of formula (I), or a pharma- ceutically acceptable salt thereof, for use as a medicament in human or veterinary therapy.

[0088] It will be understood that the compounds of Formula I, or pharma- ceutically acceptable salts thereof, when used in therapy, are used as active therapeutic agents.

[0089] For the avoidance of doubt, general references herein to "treatment" include references to curative, symptomatic and prophylactic treatment.

[0090] Human and Veterinary Use With respect to the use of the compounds of the invention in humans, the following: a pharmaceutical composition comprising a compound of formula I or a pharma- ceutically acceptable salt, solvate, hydrate, isomer, prodrug or polymorph thereof, together with one or more pharma- ceutically acceptable carriers, excipients or additives; a compound of formula I or a pharma- ceutically acceptable salt, solvate, hydrate, isomer, prodrug or polymorph thereof for use as a medicament, or a pharmaceutical composition containing any of the foregoing; a compound of formula I or a pharma- ceutically acceptable salt, solvate, hydrate, isomer, prodrug or polymorph thereof for use in medicine, or a pharmaceutical composition containing any of the foregoing; A compound of formula I or a pharma- ceutically acceptable salt, solvate, hydrate, isomer, prodrug or polymorph thereof, or a pharmaceutical composition containing any of the foregoing, for use in the treatment of an infectious disease; is provided. Preferably, the infection may be an infection caused by Cryptosporidium, such as Cryptosporidium andersonii, Cryptosporidium bayleyi, Cryptosporidium bovis, Cryptosporidium canis, Cryptosporidium chipmunk, Cryptosporidium cuniculus, Cryptosporidium zucismarsi, Cryptosporidium felis, Cryptosporidium feiri, Cryptosporidium galli, Cryptosporidium meleagridis, Cryptosporidium muris, Cryptosporidium monarii ... , Cryptosporidium suis, Cryptosporidium scropharm, Cryptosporidium tzizeri, Cryptosporidium ubiquitum, Cryptosporidium viatrum, Cryptosporidium nasorum, Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium saurophyllum, Cryptosporidium serpentis, and Cryptosporidium laili. A compound of formula I, or a pharma- ceutically acceptable salt, solvate, hydrate, isomer, prodrug, or polymorph thereof, or a pharmaceutical composition containing any of the foregoing, for use in the prophylactic treatment of an infection that may be caused by a Cryptosporidium species selected from Cryptosporidium suis, Cryptosporidium scropharm, Cryptosporidium tzizeri, Cryptosporidium ubiquitum, Cryptosporidium viatrum, Cryptosporidium nasorum, Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium saurophyllum, Cryptosporidium serpentis, and Cryptosporidium laili. Preferably, the infection may be an infection caused by Cryptosporidium.Infections include Cryptosporidium andersonii, Cryptosporidium bayleyi, Cryptosporidium bovis, Cryptosporidium canis, Cryptosporidium chipmunk, Cryptosporidium cuniculus, Cryptosporidium zucismarsi, Cryptosporidium felis, Cryptosporidium feiri, Cryptosporidium galli, Cryptosporidium meleagridis, Cryptosporidium muris, and Cryptosporidium monarii. The infection may be caused by a Cryptosporidium species selected from Cryptosporidium suis, Cryptosporidium scrophulari, Cryptosporidium tizzeri, Cryptosporidium ubiquitum, Cryptosporidium viatolume, Cryptosporidium nasorum, Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium saurophyllum, Cryptosporidium serpentis, and Cryptosporidium laili. With regard to the use of the compounds of the invention in animals, the following: veterinary compositions comprising a compound of formula I or an acceptable salt, solvate, hydrate, isomer, prodrug or polymorph thereof, together with one or more acceptable carriers, excipients or additives; A compound of formula I or an acceptable salt, solvate, hydrate, isomer, prodrug or polymorph thereof for use in veterinary medicine, or a veterinary composition containing any of the foregoing, is provided.

[0091] For the avoidance of doubt, when the use of a compound of formula I is referred to herein, this further means the use of a compound of any one of the formulae independently selected from: I, IA, IB, IC, ID, IE, IF and / or IG.

[0092] Treatment of cryptosporidiosis Thus, there is further provided a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG, or a pharma- ceutically acceptable salt, for use in the treatment or prevention of cryptosporidiosis.

[0093] Further provided is a method of treating or preventing cryptosporidiosis, the method comprising administering to a human subject in need thereof a therapeutically effective amount of a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG, or a veterinary or pharma- ceutically acceptable salt thereof. In another embodiment of the invention, a method of treating or preventing cryptosporidiosis is provided, the method comprising administering to a mammal in need thereof a therapeutically effective amount of a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG, or a veterinary or pharma- ceutically acceptable salt thereof.

[0094] According to a preferred aspect, the above mentioned uses and / or methods provide compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG, or pharma- ceutically acceptable salts thereof, which are effective against Cryptosporidium, in particular compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG suitable for use in the treatment or prophylaxis of subjects infected with Cryptosporidium alone; immunocompromised subjects infected with Cryptosporidium, such as subjects with Cryptosporidium-HIV co-infection.

[0095] Pharmaceutical Compositions According to a further aspect, the present invention provides a pharmaceutical composition comprising a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG, or a pharma- ceutically acceptable salt, solvate, hydrate, isomer, prodrug, or polymorph thereof.

[0096] The pharmaceutical composition may further comprise one or more pharma- ceutically acceptable carriers, diluents or excipients.The pharmaceutical composition may further comprise one or more additional active agents against Cryptosporidium.

[0097] Administration and Dose Range In order to select the most appropriate dosage form and route of administration likely to be appropriate for the treatment of the desired indication, compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG should be evaluated for their biopharmaceutical properties, such as solubility, solution stability (over a wide range of pH), potential dose levels and permeability, etc. Initial biopharmaceutical trials for their potential as antimalarial treatments have given positive results.

[0098] Compounds of the invention for pharmaceutical and / or veterinary use may be administered as crystalline or amorphous products. They may be obtained, for example, as solid plugs, powders, or films by methods such as precipitation, crystallization, freeze drying, spray drying, or evaporative drying. Microwave or radio frequency drying may be employed for this purpose.

[0099] They can be administered alone or in combination with one or more other compounds of the invention, or in combination with one or more other drugs (or any combination thereof). Generally, they are administered as a formulation with one or more pharma- ceutically acceptable excipients. The term "excipient" is used herein to refer to any ingredient other than the compound of the invention. The choice of excipient depends, in large part, on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. Pharmaceutically and / or veterinarily acceptable excipients include one or more of lubricants, binders, excipients, surface active agents, antioxidants, colorants, flavoring agents, preservatives, flavor enhancers, preservatives, saliva stimulants, cooling agents, co-solvents (including oils), softeners, bulking agents, antifoaming agents, surfactants, and taste masking agents.

[0100] Pharmaceutical and / or veterinary compositions suitable for the delivery of compounds of the invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).

[0101] Formulations suitable for oral administration include solids such as tablets, semi-solids or liquids; soft or hard capsules; boluses; powders; lozenges (including liquid fills); chewables; multi- and nanoparticles; gels; solid solutions; fast dispersing dosage forms; fast dissolving dosage forms; fast disintegrating dosage forms; films; ovules; sprays; buccal / mucoadhesive patches; and liquid formulations. Liquid formulations include suspensions, solutions, elixirs and syrups. Oral administration can include swallowing, so that the compound enters the digestive tract, and / or buccal, lingual or sublingual administration, where the compound enters the bloodstream directly from the mouth. Liquid formulations can be used as fillers in soft or hard capsules, and typically include a carrier, such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose or a suitable oil, and one or more emulsifying agents and / or suspending agents. Liquid formulations can also be prepared by the reconstitution of a solid, for example, from a sachet.

[0102] Formulations for oral administration can be formulated for immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release. Tablet formulations are described in "Pharmaceutical Dosage Forms: Tablets, Vol. 1", by H. Lieberman and L. Lachman, Marcel Dekker, NY, NY, 1980 (ISBN 0-8247-6918-X).

[0103] The present invention provides a pharmaceutical composition formulated for oral delivery comprising a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG as defined in any of the preceding claims or a pharma- ceutically acceptable salt, solvate or hydrate thereof, together with one or more pharma- ceutically acceptable excipients. The present invention further provides said pharmaceutical and / or veterinary compositions formulated for oral delivery as an immediate or modified release tablet formulation.

[0104] The compounds of the present invention can also be administered parenterally or by direct injection into the bloodstream, into muscle or into an internal organ.Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, intrasynovial and subcutaneous.Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.

[0105] The present invention provides a pharmaceutical composition formulated for parenteral delivery comprising a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG as defined in any of the preceding claims or a pharma- ceutically acceptable salt, solvate or hydrate thereof, together with one or more pharma- ceutically acceptable excipients. The present invention further provides said pharmaceutical and / or veterinary compositions formulated for parenteral delivery as an immediate or modified release tablet formulation suitable for intramuscular or intravenous administration.

[0106] The compound of the present invention can also be administered topically, intradermally or transdermally to the skin or mucosa.The typical formulations for this purpose include gel, hydrogel, lotion, solution, cream, ointment, dusting agent, dressing, foam, film, skin patch, wafer, implant, sponge, fiber, bandage and microemulsion.Liposome can also be used.

[0107] The compounds of the invention may be administered rectally or vaginally, for example, in the form of a suppository, pessary, or enema. Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate.

[0108] Pharmaceutical formulations containing the compounds of the invention can be formulated for immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted- and programmed-release.

[0109] Dosage Typically, a physician and / or veterinarian will determine the actual dosage that will be most suitable for an individual subject. The specific dose level and frequency of administration in any particular individual can vary and will depend on a variety of factors, including the condition being treated, the activity of the particular compound used, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex, diet, method and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual receiving the therapy.

[0110] In general, however, a suitable dose will be within the range of about 0.001 to about 50 mg / kg body weight / day, in another embodiment, about 0.001 to about 5 mg / kg body weight / day; in another embodiment, about 0.001 to about 0.5 mg / kg body weight / day, and in yet another embodiment, about 0.001 to about 0.1 mg / kg body weight / day. In another embodiment, the range can be about 0.001 to about 750 mg / kg body weight / day, within the range of 0.5 to 60 mg / kg / day, and within the range of 1 to 20 mg / kg / day.

[0111] The desired dose may conveniently be presented as a single dose or as divided doses administered at appropriate intervals, for example, one, two, three, four or more administrations per day. If the compound is administered transdermally or in sustained release form, the compound may be administered no more than once per day.

[0112] The compound is conveniently administered in unit dosage form, for example containing 0.1 to 50 mg, conveniently 0.1 to 10 mg, most conveniently 0.1 to 5 mg of active ingredient per unit dosage form. In yet another embodiment, the compound may conveniently be administered in unit dosage form, for example containing 10 to 1500 mg, 20 to 1000 mg or 50 to 700 mg of active ingredient per unit dosage form.

[0113] These dosages are based on an average human subject having a body weight of about 65 kg to 70 kg. A physician could readily determine dosages for subjects whose body weight falls outside this range, such as infants and the elderly.

[0114] The present invention provides a pharmaceutical composition comprising a compound of formula (I) or a pharma- ceutically acceptable salt, solvate or hydrate thereof, together with one or more pharma- ceutically acceptable excipients, formulated as a single-dose tablet suitable for oral delivery. The present invention further provides said pharmaceutical composition formulated for oral delivery as a single-dose immediate or modified release tablet formulation.

[0115] The present invention further provides a pharmaceutical composition comprising from about 0.1 to about 3000 mg, preferably from about 0.5 to about 1500 mg, more preferably from about 1 to about 750 mg, and particularly from about 5 to about 250 mg of a compound of formula (I) or a pharma- ceutically acceptable salt, solvate or hydrate thereof, together with one or more pharma- ceutically acceptable excipients, formulated as a single-dose tablet formulation for oral delivery.

[0116] For the anti-infective treatment of any of the infectious conditions set out hereinbefore, the present invention further provides a pharmaceutical composition comprising 0.1 to 3000 mg, preferably about 0.5 to about 1500 mg, more preferably about 1 to about 750 mg, and especially about 5 to about 250 mg of a compound of formula (I) or a pharma- ceutically acceptable salt, solvate or hydrate thereof, together with one or more pharma- ceutically acceptable excipients, formulated as a single-dose tablet for oral delivery as an immediate or modified release tablet formulation.

[0117] When a single megadose therapy is administered, for example to children, administration may be achieved by more than one tablet, such as 2 x 1500 mg or 3 x 1000 mg, rather than a single dose of 3000 mg tablet, where the tablets can be taken sequentially or together according to suitability.

[0118] Simultaneous administration Insofar as it is desirable to administer a combination of active compounds, as detailed herein above, for example for the purpose of treating a particular infectious disease, it is within the scope of the present invention that two or more pharmaceutical compositions, at least one of which contains a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG according to the invention as defined herein above, may be conveniently combined in the form of a kit suitable for simultaneous administration of the compositions.

[0119] Thus, a kit of the present invention comprises two or more separate pharmaceutical compositions, at least one of which contains a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG according to the invention, and a means for keeping said compositions separate, such as a container, divided bottle or divided foil packet. One example of such a kit is the familiar blister pack used for the packaging of tablets, capsules and the like.

[0120] How to use Those skilled in the art will appreciate that references herein to treatment refer to the treatment of the specified condition. However, the compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG and their pharma- ceutically acceptable salts may also be useful in the prevention (prophylaxis) of certain diseases, such as cryptosporidiosis, depending on the circumstances.

[0121] As used herein, unless otherwise indicated, "treat," "treating," or "treatment," with respect to a disease, means: (1) ameliorating the disease or one or more of the biological symptoms of the disease; (2) interfering with (a) one or more points in the biological cascade that leads to or causes the disease, or (b) one or more of the biological symptoms of the disease; (3) alleviating one or more of the symptoms or effects associated with the disease; (4) slowing the progression of the disease or one or more of the biological symptoms of the disease; and / or (5) reducing the likelihood or severity of the disease or one or more of the biological symptoms of the disease.

[0122] As used herein, unless otherwise indicated, "prevent", "preventing" or "prevention" refers to the prophylactic administration of a drug that reduces the likelihood of onset of a disease or its biological manifestations or delays their onset. Those skilled in the art will appreciate that "prevention" is not an absolute term. In medicine, "prevention" is understood to refer to the prophylactic administration of a drug that substantially reduces the likelihood or severity of a disorder or its biological manifestations or delays the onset of such a disorder or its biological manifestations.

[0123] Thus, in another aspect, there is provided treatment or prevention of a disease. In another aspect, there is provided treatment of a disease. In another aspect, there is provided prevention of a disease. Thus, as a further aspect of the invention there is provided a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG or a pharma- ceutically acceptable salt thereof for use in therapy.Furthermore there is provided a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG or a pharma- ceutically acceptable salt thereof for use as a medicament in human or veterinary therapy.

[0124] It will be understood that the compounds of general formula I, IA, IB, IC, ID, IE, IF and / or IG or pharma- ceutically acceptable salts thereof, when used in therapy, are used as active therapeutic agents. For the avoidance of doubt, general references herein to "treatment" include references to curative, symptomatic and prophylactic treatment. EXAMPLES

[0125] The present invention is illustrated herein by the following non-limiting examples. Although specific embodiments of the present invention are described below, those skilled in the art will appreciate that various changes and modifications may be made. References to preparations carried out in the same manner as other preparations, or by general methods of other preparations, may encompass variations in certain parameters, such as slight changes in time, temperature, test conditions, amounts of reagents, etc.

[0126] In some of the intermediates and examples below, starting materials are identified by reference to other intermediate or example numbers. This does not imply that the actual material (or "batch") obtained from any particular intermediate or example will necessarily be used in the subsequent steps illustrated herein, but is used as a shorthand means to display the associated compound name.

[0127] The invention is illustrated by the following non-limiting examples, in which the following abbreviations and definitions are used: Abbreviation δ Chemical shift d double line dd double double line ACN Acetonitrile BID 2 times a day DCM Dichloromethane DMF Dimethylformamide ES Low-resolution electrospray mass spectrometry Et3N Triethylamine EtOAc Ethyl acetate Eq(s) equivalent HPLC High Performance Liquid Chromatography HRMS High Resolution Mass Spectrum LCMS Liquid Chromatography Mass Spectrometry m multiplet min m / z Mass spectrum peak NMR nuclear magnetic resonance PO Oral q quartet rt room temperature S Single line SFC Supercritical Fluid Chromatography t triple line TBME Tert Butyl Methyl Ether THF Tetrahydrofuran TLC Thin Layer Chromatography

[0128] General chemical information: Equipment and methods Flash chromatography was performed using Combiflash Companion Rf (commercially available from Teledyne ISCO) and prepacked silica gel columns purchased from Teledyne ISCO. Mass-directed preparative HPLC separations were performed using a Waters HPLC (2545 binary gradient pump, 515 HPLC make-up pump, 2767 sample manager) interfaced with a Waters 2998 photodiode array and a Waters 3100 mass detector. Preparative HPLC separations were performed using a Gilson HPLC (321 pump, 819 injection module, 215 liquid handler / injector) interfaced with a Gilson 155 UV / visible detector. In both instruments, HPLC chromatographic separations were performed using a Waters XBridge C18 column (19×100 mm, 5 um particle size); using 0.1% ammonia in water (solvent A) and acetonitrile (solvent B) or 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) as mobile phases. SFC chiral separations were performed on a Waters SFC350. 1 H NMR spectra were obtained using a Bruker Avance DPX 500 spectrometer ( 1H is 500.1MHz, 13 C is 125MHz, 19 F is 470.5MHz), Bruker Avance DPX400( 1 H is 400MHz), or Bruker Avance DPX 300( 1 H was recorded using a 300 MHz NMR spectrometer. Chemical shifts (δ) are expressed in ppm, in all cases recorded using residual solvent as internal standard. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), broad (br) or combinations thereof. Coupling constants (J) were estimated to the nearest 0.1 Hz. Low-resolution electrospray (ES) mass spectra were recorded on an Advion Compact Mass Spectrometer (CMS; model Expression CMS) connected to a Dionex Ultimate 3000 UPLC system equipped with a diode array detector. HPLC chromatographic separations were performed using a Waters XBridge C18 column, 2.1×50 mm, 3.5 μm particle size, or a Waters XSelect 2.1×30 mm, 2.5 μm particle size. Compounds were eluted using a gradient of 5-95% acetonitrile / water + 0.1% ammonia or + 0.1% formic acid. Reactions were not optimized unless otherwise stated herein. Solvents and reagents were purchased from commercial suppliers and used without further purification. Dry solvents were purchased in tightly sealed bottles and stored using molecular sieves.

[0129] synthesis General scheme for the synthesis of 2-amino-4-(trifluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (compound 1) [ka]

[0130] Ethyl 2-amino-4-methyl-6-(trifluoromethyl)pyrimidine-5-carboxylate (Intermediate 1) [ka] A mixture of guanidine hydrochloride (121 g, 1.27 mol) and NaOH (189 g, 4.73 mol) in MeCN (1.5 L) was stirred under nitrogen at 0° C. for 5 h in a sealed tube. Ethyl 3-oxobutanoate (150 g, 1.15 mol) and 1,1,1,2,2-pentafluoro-2-iodoethane (312 g, 1.27 mol) were added and the reaction mixture was stirred at 15° C. for 15 h. The reaction mixture was quenched with water (3 L) and extracted with ethyl acetate (3×2 L). The combined organic layers were washed with brine (1.5 L), dried over Na2SO4, filtered, and concentrated under reduced pressure. The product was purified by column chromatography (SiO 2 , 0:100→50:50 EtOAc:petroleum ether) to give ethyl 2-amino-4-methyl-6-(trifluoromethyl)pyrimidine-5-carboxylate (288 g, 50% yield) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.64 (br. s, 2H), 4.28 (q, J =7.2 Hz, 2H), 2.35 (s, 3H), 1.27 (t, J =6.8 Hz, 3H). MS (ESI): m / z (%) 250 (80) [M+H] + .

[0131] Ethyl 2-amino-4-(bromomethyl)-6-(trifluoromethyl)pyrimidine-5-carboxylate (Intermediate 2) [ka] To a mixture of ethyl 2-amino-4-methyl-6-(trifluoromethyl)pyrimidine-5-carboxylate (180 g, 722 mmol) in AcOH (1.6 L) at 10° C. under nitrogen was added a solution of Br2 (115 g, 722 mmol) in AcOH (200 mL) and the reaction mixture was heated at 90° C. for 2 h. The reaction mixture was concentrated, then quenched with water (1 L) and extracted with ethyl acetate (3×800 mL). The combined organic layers were washed with brine (1 L), dried over Na2SO4, filtered and concentrated under reduced pressure. The product was purified by column chromatography (SiO2, 5:95→15:85 EtOAc:petroleum ether) to give ethyl 2-amino-4-(bromomethyl)-6-(trifluoromethyl)pyrimidine-5-carboxylate (370 g, 78% yield) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.95 (br. s, 2H), 4.50 (s, 2H), 4.30 (q, J =7.2 Hz, 2H), 1.28 (t, J = 7.2 Hz, 3H). MS (ESI): m / z (%) 330 / 331 (97) [M+H] + .

[0132] 3-(Trifluoromethyl)cyclohexane-1-carboxylic acid (Intermediate 3) [ka] To a solution of 3-(trifluoromethyl)benzoic acid (100 g, 526 mmol) in AcOH (1 L) was added PtO2 (10 g, 44 mmol). The suspension was degassed under reduced pressure, purged with H2, and stirred under H2 (50 psi) at 60 °C for 48 h. The reaction mixture was filtered, concentrated under reduced pressure, and azeotroped with toluene to give racemic cis 3-(trifluoromethyl)cyclohexane-1-carboxylic acid (405 g, 98% yield) as a colorless oil. 1H NMR (400 MHz, DMSO-d6) δ 12.20 (br. s, 1H), 2.47 - 2.26 (m, 2H), 2.16 - 1.92 (m, 2H), 1.87 - 1.74 (m, 2H), 1.51 - 1.08 (m, 4H).

[0133] 3-(Trifluoromethyl)cyclohexane-1-carboxamide (Intermediate 4) [ka] To a solution of racemic cis 3-(trifluoromethyl)cyclohexanecarboxylic acid (260 g, 1.33 mol) in DCM (2.5 L) at 0° C. was added (COCl) 2 (232 mL, 2.65 mol) followed by DMF (10.2 mL, 132 mmol) and the reaction mixture was stirred at 0° C. for 1 h. The reaction mixture was concentrated under reduced pressure, then dissolved in DCM (500 mL) and added to NH 3 .H 2 O (408 mL, 2.65 mol) at 0° C. The reaction mixture was stirred at 0° C. for 1 h, then concentrated under reduced pressure and azeotroped with toluene. The product was purified by column chromatography (SiO 2 , 30:70→50:50 EtOAc:petroleum ether) to give racemic cis 3-(trifluoromethyl)cyclohexanecarboxamide (200 g, 39% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.27 (s, 1H), 6.77 (s, 1H), 2.38 - 2.08 (m, 2H), 1.98 - 1.67 (m, 4H), 1.41 - 1.06 (m, 4H).

[0134] 3-(Trifluoromethyl)cyclohexyl)methanamine (Intermediate 5) [ka] LiAlH4 (32 g, 843 mmol) was slowly added to THF (600 mL) at 0° C. under nitrogen. A solution of racemic cis 3-(trifluoromethyl)cyclohexanecarboxamide (82 g, 420 mmol) in THF (600 mL) at 0° C. was added dropwise over 30 min under nitrogen. The reaction mixture was heated at 70° C. for 12 h, then cooled to 0° C. and quenched by the dropwise addition of water (32 mL). 3.75 M NaOH (32 mL) and water (96 mL) were added and the mixture was filtered and concentrated under reduced pressure to give racemic cis 3-(trifluoromethyl)cyclohexyl)methanamine (150 g, 98% yield) as a yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 2.47 - 2.33 (m, 2H), 2.29 - 2.12 (m, 1H), 1.93 (br. d, J = 12.4 Hz, 1H), 1.87 - 1.67 (m, 4H), 1.34 - 1.04 (m, 4H), 0.90 - 0.71 (m, 2H).

[0135] 2-Amino-4-(trifluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (Compound 1) [ka] To a mixture of racemic cis 3-(trifluoromethyl)cyclohexyl)methanamine (80 g, 442 mmol) and Et3N (82 mL, 589 mmol) in MeCN (800 mL) at 0° C. under nitrogen, ethyl 2-amino-4-(bromomethyl)-6-(trifluoromethyl)pyrimidine-5-carboxylate (97 g, 294 mmol) in MeCN (200 mL) was added. The reaction mixture was stirred at 0° C. for 2 h and then heated to 40° C. for 4 h. The reaction mixture was concentrated under reduced pressure, then quenched with water (800 mL) and extracted with ethyl acetate (3×800 mL). The combined organic layers were washed with brine (700 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The product was purified by column chromatography (SiO2, 15:85→40:60 EtOAc:petroleum ether) and then the enantiomers were separated by chiral SFC (column: Daicel Chiralpak IG (250mm*50mm, 10μm); mobile phase: [hexane-EtOH]; B%: 15% to 15%, 3.9; 6100min) to give 2-amino-4-(trifluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (81g, 40% yield) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.11 - 7.77 (m, 2H), 4.48 - 4.32 (m, 2H), 3.41 - 3.23 (m, 2H), 2.29 - 2.16 (m, 1H), 1.86 - 1.73 (m, 4H), 1.65 (br. d, J = 12.4 Hz, 1H), 1.35 - 1.09 (m, 2H), 0.99 - 0.77 (m, 2H). 13 C NMR (125 MHz, DMSO-d6) δ 176.4, 163.8, 162.6, 150.8 (q, 2 J CF = 37.9 Hz), 128.0 (q, 1 J CF = 278.5 Hz), 119.9 (q, 1 JCF = 279.5 Hz), 117.9, 109.0, 51.2, 47.5, 34.8, 29.3, 28.6, 24.4, 23.6. 19 F NMR (376 MHz, DMSO-d6) δ -66.22, -72.48. [M + H] + C 15 H 17 HRMS (ESI) calculated for F6N4O: 383.1307, found: 383.1305.

[0136] General scheme for the synthesis of 2-amino-4-methyl-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (compound 2) [ka]

[0137] Ethyl 4-methyl-2-(methylthio)-6-vinylpyrimidine-5-carboxylate (Intermediate 6) [ka] To a solution of ethyl 4-chloro-6-methyl-2-(methylthio)pyrimidine-5-carboxylate (7.7 g, 31.2 mmol) in ethanol (70 mL) was added potassium vinyltrifluoroborate (5.02 g, 37.5 mmol), Et3N (5.64 mL, 40.6 mmol), and Pd(dppf)Cl2 (255 mg, 0.31 mmol), the reaction mixture was purged with nitrogen and heated at 85° C. under nitrogen for 16 h. The reaction mixture was concentrated under reduced pressure, then diluted with water (40 mL) and extracted with EtOAc (3×40 mL). The combined organic layers were washed with brine (40 mL), dried over MgSO4, filtered, and concentrated under reduced pressure. The product was purified by column chromatography (80 g silica, 0:100→30:70 EtOAc:heptane) to give ethyl 4-methyl-2-methylsulfanyl-6-vinyl-pyrimidine-5-carboxylate (6.15 g, 79% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3): δ 6.83 (dd, J = 16.8, 10.3 Hz, 1H), 6.70 (dd, J = 16.8, 2.1 Hz, 1H), 5.67 (dd, J = 10.3, 2.1 Hz, 1H), 4.41 (q, J = 7.1 MS (ES+): m / z (%) 239 (90) [M+H] + .

[0138] Ethyl 4-formyl-6-methyl-2-(methylthio)pyrimidine-5-carboxylate (Intermediate 7) [ka] To a solution of ethyl 4-methyl-2-methylsulfanyl-6-vinyl-pyrimidine-5-carboxylate (6.02 g, 25.3 mmol) in THF (40 mL) and water (10 mL) was added a solution of dipotassium dioxide (dioxo)osmium dihydrate (326 mg, 0.88 mmol) in water (10 mL). The reaction mixture was stirred at room temperature for 15 min, then a suspension of sodium (meta)periodate (21.6 g, 101 mmol) in water (10 mL) was added dropwise and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (20 mL) and passed through a hydrophobic frit. Residual osmium was quenched by the addition of corn oil (1 mL) and the organic layer was concentrated under reduced pressure. The product was purified by column chromatography (40 g silica, 0:100→60:40 EtOAc:heptane) to give ethyl 4-formyl-6-methyl-2-methylsulfanyl-pyrimidine-5-carboxylate (1.93 g, 32% yield) as a light brown oil. 1 H NMR (400 MHz, CDCl3): δ 9.94 (s, 1H), 4.45 (q, J = 5.7 Hz, 2H), 2.64 (s, 3H), 2.58 (s, 3H), 1.39 (t, J = 5.7 Hz, 3H). MS (ES+): m / z (%) 241 (94) [M+H] + .

[0139] N-Benzyl-3-(trifluoromethyl)cyclohexane-1-carboxamide (Intermediate 8) [ka] To a solution of racemic cis 3-(trifluoromethyl)cyclohexanecarboxylic acid (51 g, 259 mmol) in DCM (510 mL) at 10° C., (COCl)2 (34 mL, 389 mmol) was added, followed by DMF (0.2 mL, 2.6 mmol) and the reaction mixture was stirred at 10° C. for 1 h. The reaction mixture was concentrated under reduced pressure, azeotroped with toluene, and then dissolved in DCM (600 mL). Benzylamine (33.5 mL, 307 mmol) and Et3N (71 mL, 512 mmol) were added and the reaction mixture was stirred at 10° C. for 2 h. The reaction mixture was poured into water (1.5 L) and extracted with DCM (3×300 mL). The combined organic layers were washed with 0.5 M HCl (200 mL), aq. NaHCO3 (200 mL), and brine (200 mL), then dried over Na2SO4, filtered, and concentrated under reduced pressure. The product was purified by column chromatography (SiO2, 15:85→25:75 EtOAc:petroleum ether) to give racemic cis N-benzyl-3-(trifluoromethyl)cyclohexane-1-carboxamide (45 g, 58% yield) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.28 - 7.19 (m, 5H), 5.71 (br. s, 1H), 4.37 (d, J = 6.0 Hz, 2H), 2.11 - 2.04 (m, 3H), 1.90 - 1.88 (m, 3H), 1.56 - 1.48 (m, 2H), 1.24 - 1.23 (m, 2H). MS (ESI): m / z (%) 285 (95) [M+H] + .

[0140] (1R,3S)-N-Benzyl-3-(trifluoromethyl)cyclohexane-1-carboxamide (Intermediate 9) [ka] Racemic cis N-benzyl-3-(trifluoromethyl)cyclohexane-1-carboxamide was purified by SFC (Column: Daicel Chiralpak AY (250mm*50mm, 10μm); Mobile phase: [0.1% NH3H2O ​​MEOH]; B%: 55% to 55%, 2.6min; 360min) to give (1R,3S)-N-benzyl-3-(trifluoromethyl)cyclohexane-1-carboxamide (peak 2, 5.8g, 48% yield) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.35 - 7.23 (m, 5H), 5.73 (br. s, 1H), 4.42 (d, J = 5.6 Hz, 2H), 2.18 - 1.99 (m, 3H), 1.99 - 1.85 (m, 3H), 1.60 - 1.39 (m, 2H), 1.36 - 1.20 (m, 2H). MS (ESI): m / z (%) 285 (99) [M+H] + .

[0141] N-Benzyl-1-((1R,3S)-3-(trifluoromethyl)cyclohexyl)methanamine (Intermediate 10) [ka] To a mixture of (1R,3S)-N-benzyl-3-(trifluoromethyl)cyclohexane-1-carboxamide (5.8 g, 20 mmol) in THF (60 mL) at 10° C., LiAlH4 (1.5 g, 41 mmol) was added and the reaction mixture was heated at 75° C. for 15 h. The reaction mixture was quenched with water (1.5 mL). 3.75 M NaOH (1.5 mL) and water (4.6 mL) were added, followed by Na2SO4 (16 g), the mixture was filtered and concentrated under reduced pressure. The crude product was purified by HPLC (column: Phenomenex luna C18 150*40mm*15μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 17% to 47%, 10min) to give N-benzyl-1-((1R,3S)-3-(trifluoromethyl)cyclohexyl)methanamine (2.85g, 48% yield) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.30 - 7.17 (m, 5H), 3.73 (s, 2H), 2.52 - 2.40 (m, 2H), 2.05 - 1.91 (m, 2H), 1.90 - 1.72 (m, 3H), 1.55 - 1.40 (m, 1H), 1.30 - 1.08 (m, 2H), 0.96 - 0.74 (m, 2H). MS (ESI): m / z (%) 271 (93) [M+H] + .

[0142] ((1R,3S)-3-(Trifluoromethyl)cyclohexyl)methanamine (Intermediate 11) [ka] To a solution of N-benzyl-1-((1R,3S)-3-(trifluoromethyl)cyclohexyl)methanamine (2.85 g, 10.5 mmol) in MeOH (28 mL) was added Pd(OH)2 (737 mg, 5.25 mmol) under N2. The suspension was degassed under reduced pressure, purged with H2, and stirred under H2 (50 psi) at 20 °C for 15 h. The reaction mixture was filtered. HCl / MeOH (50 mL) was added and the filtrate was concentrated under reduced pressure. The product was triturated with EtOAc / petroleum ether (10:90), then filtered and dried under reduced pressure to give ((1R,3S)-3-(trifluoromethyl)cyclohexyl)methanamine (2.06 g, 90% yield) as a pale yellow solid. 1 H NMR (400 MHz, DMDO-d6) δ 7.99 (s, 3H), 2.77 - 2.61 (m, 2H), 2.32 - 2.27 (m, 1H), 1.96 (d, J = 12.4 Hz, 1H), 1.88 - 1.71 (m, 3H), 1.71 - 1.58 (m, 1H), 1.37 - 1.21 (m, 1H), 1.21 - 1.06 (m, 1H), 1.01 - 0.81 (m, 2H).

[0143] 4-Methyl-2-(methylthio)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (Intermediate 12) [ka] To a solution of ethyl 4-formyl-6-methyl-2-methylsulfanyl-pyrimidine-5-carboxylate (1.93 g, 8.03 mmol) in DCM (40 mL) was added (1R,3S)-(3-(trifluoromethyl)cyclohexyl]methanamine (2.33 g, 12.9 mmol), acetic acid (20 μL), and MgSO4 (1.45 g, 12.0 mmol). The reaction mixture was stirred at room temperature under nitrogen for 10 min. Sodium triacetoxyborohydride (4.26 g, 20.1 mmol) was added in portions and the reaction mixture was heated at 40° C. for 1 h. The reaction mixture was diluted with DCM (20 mL), washed with water (3×20 mL), passed through a hydrophobic frit and concentrated under reduced pressure. The product was purified by column chromatography (80 g silica, 0:100→60:40 Purification with EtOAc:heptane) gave 4-methyl-2-(methylthio)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (2.38 g, 83% yield) as a pale gold oil. 1 H NMR (400 MHz, CDCl3): δ 4.32 (s, 2H), 3.50 (dd, J = 13.9, 7.3 Hz, 1H), 3.43 (dd, J = 13.9, 6.9 Hz, 1H), 2.79 (s, 3H), 2.62 (s, 3H), 2.08 - 2.00 (m, 1H), 1.97 - 1.90 (m, 2H), 1.84 - 1.75 (m, 2H), 1.33 - 1.23 (m, 1H), 1.13 - 1.00 (m, 2H), 0.89 (t, J = 6.9 Hz, 2H). MS (ES+): m / z (%) 360 (98) [M+H] + .

[0144] 2-Amino-4-methyl-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (compound 2) [ka] To a solution of 4-methyl-2-methylsulfanyl-6-[[3-(-1R,3S)-(trifluoromethyl)cyclohexyl]methyl]-7H-pyrrolo[3,4-d]pyrimidin-5-one (2.38 g, 6.62 mmol) in MeCN (20 mL) was added a solution of oxone (5.29 g, 8.61 mmol) in water (10 mL) and the reaction mixture was stirred at room temperature for 20 h. The reaction mixture was diluted with water (10 mL), extracted with DCM (3 x 10 mL), passed through a hydrophobic frit and concentrated under reduced pressure. The residue was dissolved in 1,4-dioxane (10 mL) and a solution of ammonia in dioxane (26.5 mL, 13.2 mmol, 0.5 M) was slowly added. The reaction mixture was stirred at room temperature for 7 h and then concentrated under reduced pressure. The product was recrystallized from EtOH to give 2-amino-4-methyl-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (231 mg, 10% yield) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6): δ 7.17 (br. s, 2H), 4.29 - 4.18 (m, 2H), 3.36 - 3.31 (m, 1H), 3.27 - 3.23 (m, 1H), 2.47 (s, 3H), 2.31 - 2.20 (m, 1H), 1.84 - 1.74 (m, 4H), 1.63 (br. d, J = 12.7 Hz, 1H), 1.30 - 1.11 (m, 2H), 0.96 - 0.86 (m, 2H). 13 C NMR (100 MHz, DMSO-d6): δ 173.3, 166.3, 165.3, 164.1, 127.9 (q, 1 J CF = 250.4 Hz), 110.8, 50.6, 47.1, 34.9, 29.3, 28.6, 24.4, 23.6, 19.6. 19 F NMR (470 MHz, DMSO-d6) δ -72.34. C 15 H 20 ON4F3[M+H] +HRMS (ES+) calculated for 329.1589, found 329.1584.

[0145] General scheme for the synthesis of 2-amino-4-(difluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (compound 3) [ka]

[0146] Ethyl 4-(difluoromethyl)-6-methyl-2-(methylthio)pyrimidine-5-carboxylate (Intermediate 13) [ka] A solution of ethyl 4-formyl-6-methyl-2-methylsulfanyl-pyrimidine-5-carboxylate (1.08 g, 4.50 mmol) in DCM (12 mL) was cooled to −15° C. under nitrogen and stirring. (Diethylamino)sulfur trifluoride (1.99 g, 12.4 mmol) was added slowly over 10 min and the reaction mixture was allowed to warm to room temperature over 2 h, then stirred at room temperature for 18 h. Ice water (20 mL) and DCM (25 mL) were added and the biphasic system was shaken and separated. The aqueous layer was extracted with DCM (3×25 mL) and the combined organic layers were dried (MgSO4), filtered and concentrated under reduced pressure. The product was purified by column chromatography (12 g silica, 0:100→30:70 EtOAc:heptane) to give ethyl 4-(difluoromethyl)-6-methyl-2-methylsulfanyl-pyrimidine-5-carboxylate (870 mg, 72% yield) as a thick pearl gold oil. 1H NMR (500 MHz, CDCl3) δ 6.68 (t, J = 54.3 Hz, 1H), 4.42 (q, J = 7.1 Hz, 2H), 2.60 (s, 3H), 2.59 (s, 3H), 1.39 (t, J = 7.2 Hz, 3H). MS (ES+): m / z (%) 263.1 (99) [M+H] + .

[0147] Ethyl (E)-4-(difluoromethyl)-6-(2-(dimethylamino)vinyl)-2-(methylthio)pyrimidine-5-carboxylate (Intermediate 14) [ka] To a solution of ethyl 4-(difluoromethyl)-6-methyl-2-methylsulfanyl-pyrimidine-5-carboxylate (770 mg, 2.94 mmol) in DMF (10 mL) under nitrogen was added (dimethylamino)acetaldehyde dimethyl acetal (1.95 mL, 14.7 mmol) followed by 1-methylimidazole (0.65 mL, 8.22 mmol) and the reaction mixture was heated to 120 °C for 2 h. The reaction mixture was cooled to room temperature, then diluted with water (10 mL) and extracted with EtOAc-TBME (5:1) (3 × 15 mL). The combined organic layers were washed with brine (15 mL), dried (MgSO4), filtered and concentrated under reduced pressure. The product was purified by column chromatography (12 g silica, 0:100→50:50 EtOAc:heptane) to give ethyl 4-(difluoromethyl)-6-[(E)-2-(dimethylamino)vinyl]-2-methylsulfanyl-pyrimidine-5-carboxylate (595 mg, 61% yield) as a pale yellow powder. 1H NMR (500 MHz, CDCl3) δ 8.08 (d, J = 12.3 Hz, 1H), 6.56 (t, J = 54.6 Hz, 1H), 5.30 (d, J = 12.3 Hz, 1H), 4.38 (q, J = 7.1 Hz, 2H), 3.00 (br s, 6H), 2.55 (s, 3H), 1.38 (t, J = 7.1 Hz, 3H). MS (ES+): m / z (%) 318.1 (79) [M+H] + + m / z (%) 291.0 (12) [M-(N(CH3)2)+OH+H] + .

[0148] 4-(Difluoromethyl)-2-(methylthio)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (Intermediate 15) [ka] To a solution of sodium (meta)periodate (5.66 g, 26.5 mmol) in water (20 mL) at 0-5° C., a solution of ethyl 4-(difluoromethyl)-6-[(E)-2-(dimethylamino)vinyl]-2-methylsulfanyl-pyrimidine-5-carboxylate (2.1 g, 6.62 mmol) in THF (35 mL) was added dropwise over 10 min (keep exotherm below 10° C.). The reaction mixture was stirred at room temperature for 2 h and then diluted with DCM (30 mL). The aqueous layer was separated and extracted with DCM (2×15 mL). The combined organic layers were washed with brine (20 mL), dried (MgSO4), filtered and concentrated under reduced pressure to a volume of 30 mL. To this solution at room temperature under nitrogen was added (1R,3S)-3-(trifluoromethyl)cyclohexyl)methanamine (1.44 g, 7.94 mmol), acetic acid (0.05 mL), and MgSO4 (1.19 g, 9.93 mmol). The reaction mixture was stirred at room temperature for 10 min, then sodium (triacetoxy)borohydride (4.21 g, 19.9 mmol) was added in portions. The reaction mixture was heated at 40° C. for 18 h, then cooled to room temperature. An additional portion of (1R,3S)-3-(trifluoromethyl)cyclohexyl)methanamine (720 mg, 3.97 mmol) and MgSO4 (597 mg, 4.96 mmol) was added, and the reaction mixture was stirred at room temperature for 20 min. Sodium (triacetoxy)borohydride (2.1 g, 9.92 mmol) was added and the reaction mixture was heated at 40° C. for 2 h, then cooled to room temperature. The reaction mixture was diluted with DCM (20 mL), washed with water (10 mL), passed through a hydrophobic frit, dried (MgSO4), filtered and concentrated under reduced pressure. The product was purified by column chromatography (40 g silica, 0:100→50:50 EtOAc:heptane) to give an orange-brown oily solid. The solid was sonicated in TBME (1.7 mL), allowed to stand at room temperature for 30 min, then the supernatant was removed by pipette. This sonication procedure was repeated and the resulting solid was dried to give a yellow powder. The supernatant was concentrated and then purified by column chromatography (12 g silica, 0:100→50:50 EtOAc:heptane).The purified fractions were combined with the previously purified solid and concentrated under reduced pressure. The resulting solid was sonicated in TBME (1.3 mL), the supernatant was removed, and the solid was dried to give 4-(difluoromethyl)-2-(methylthio)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (1.19 g, 43% yield) as a yellow powder. 1 H NMR (400 MHz, CDCl3) δ 7.19 (t, J = 26.8 Hz, 1H), 4.37 (s, 2H), 3.50 - 3.35 (m, 2H), 2.60 (s, 3H), 2.06 - 1.64 (m, 6H), 1.33 - 1.10 (m, 2H), 1.10 - 0.88 (m, 2H). MS (ES+): m / z (%) 396.0 (86) [M+H] + .

[0149] 2-Amino-4-(difluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (compound 3) [ka] To a solution of 4-(difluoromethyl)-2-(methylthio)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (1.19 g, 3.01 mmol) in MeCN (20 mL) was added a solution of oxone (2.41 g, 3.91 mmol) in water (10 mL) and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with water (20 mL), extracted with DCM (3×20 mL) and passed through a hydrophobic frit. The combined organic layers were washed with brine (20 mL), dried and concentrated under reduced pressure. The residue was dissolved in 1,4-dioxane (15 mL) and cooled to 0-5° C. A chilled 0.5 M solution of ammonia in 1,4-dioxane (7.83 mL, 3.91 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the product was purified by column chromatography (80 g silica, 0:100→80:20 EtOAc:heptane). The resulting solid was sonicated in EtOH (5 mL), allowed to stand for 30 min, and then filtered. This was repeated twice and the supernatants were combined and concentrated under reduced pressure to give a solid which was purified by column chromatography (4 g silica, 0:100→80:20 EtOAc:heptane). The resulting solid was purified by preparative HPLC (20:80→95:5 MeCN:water, 0.1% ammonia modifier), then sonicated in MeCN (0.8 mL) with 2 drops of MeOH, allowed to stand at room temperature for 30 min, filtered, and dried to give 2-amino-4-(difluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (96 mg, 9% yield) as a pale pink powder. 1H NMR (500 MHz, DMSO-d6) δ 8.00 - 7.61 (m, 2H), 7.27 (t, J = 53.8 Hz, 1H), 4.46 - 4.33, (m, 2H), 3.33 - 3.25 (m, 2H), 2.32 - 2.16 (m, 1H), 1.87 - 1.72 (m, 4H), 1.70 - 1.59 (m, 1H), 1.34 - 1.21 (m, 1H), 1.21 - 1.09 (m, 1H), 0.99 - 0.84 (m, 2H). 13 C NMR (400 MHz, DMSO-d6) δ 174.9, 164.6, 164.3, 156.1 (t, 1 J CF = 22.8 Hz), 127.9 (q, 1 J CF = 278.5 Hz), 108.7, 106.2, 51.4, 47.3, 40.2, 34.8, 29.3, 28.6, 24.3, 23.6. 19 F NMR (500 MHz, DMSO-d6) -72.3, -122.0. C 15 H 18 F5N4O [M+H] + HRMS (ES+) calculated for 365.1323, found 365.1402.

[0150] General scheme for the synthesis of 2-amino-6-(spiro[2.5]octan-5-ylmethyl)-4-(trifluoromethyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (compound 4) [ka]

[0151] 2-Amino-6-(spiro[2.5]octan-5-ylmethyl)-4-(trifluoromethyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (compound 4) [ka] To a solution of spiro[2.5]octan-7-ylmethanamine hydrochloride (375 mg, 2.13 mmol) and Et3N (0.89 mL, 6.4 mmol) in MeCN (2 mL) was added ethyl 2-amino-4-(bromomethyl)-6-(trifluoromethyl)pyrimidine-5-carboxylate (350 mg, 1.07 mmol) and the reaction mixture was stirred at room temperature for 30 min and then heated at 60 °C for 2 h. The reaction mixture was concentrated under reduced pressure, then dissolved in DCM (10 mL), washed with water (10 mL) and saturated NH4Cl (10 mL), dried over MgSO4, filtered and concentrated under reduced pressure. The product was purified by column chromatography (silica, 0:100→60:40 EtOAc:heptane) to give 2-amino-6-(spiro[2.5]octan-7-ylmethyl)-4-(trifluoromethyl)-7H-pyrrolo[3,4-d]pyrimidin-5-one (192 mg, 50% yield) as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 7.91 - 7.88 (m, 2H), 4.36 (d, J = 4.1 Hz, 2H), 3.30 - 3.27 (m, 2H), 1.88 - 1.80 (m, 1H), 1.68 - 1.64 (m, 2H), 1.61 - 1.56 (m, 1H), 1.39 - 1.32 (m, 2H), 0.99 - 0.83 (m, 3H), 0.26 - 0.23 (m, 2H), 0.21 - 0.14 (m, 2H). 13 C NMR (100 MHz, DMSO-d6) δ 176.3, 163.7, 162.4, 150.7 (q, 2 J CF = 37.5 Hz), 120.4, 119.9 (q, 1 J CF = 298.1), 109.1, 51.0, 47.3, 35.3, 35.0, 29.7, 24.2, 18.2, 11.8, 11.7. 19 F NMR (470 MHz, DMSO-d6) δ -66.09. [M + H] + C 16 H 20HRMS (ESI) calculated for F3N4O: 341.1589, found: 341.1583.

[0152] 2-amino-4-(trifluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (compound 1), 2-amino-4-(trifluoromethyl)-6-(((1R,3R)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (compound 5), 2-amino-4 General scheme for the synthesis of -(trifluoromethyl)-6-(((1S,3R)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (compound 6) and 2-amino-4-(trifluoromethyl)-6-(((1S,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (compound 7). [ka]

[0153] 2-Amino-4-(trifluoromethyl)-6-((3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (Intermediate 16) [ka] To a solution of 3-(trifluoromethyl)cyclohexyl)methanamine (144 mg, 0.8 mmol) and Et3N (0.2 mL, 1.6 mmol) in MeCN (1 mL) was added ethyl 2-amino-4-(bromomethyl)-6-(trifluoromethyl)pyrimidine-5-carboxylate (130 mg, 0.4 mmol). The reaction mixture was stirred at 15° C. for 1 h and then heated to 80° C. for 1 h. The reaction mixture was concentrated under reduced pressure and then dissolved in DCM (10 mL) and washed with water (3×10 mL) and saturated NH4Cl (10 mL). The product was purified by column chromatography (SiO, 5:95→45:55 EtOAc:heptane) to give 2-amino-4-(trifluoromethyl)-6-((3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (75 mg, 49% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.98 - 7.91 (m, 2H), 4.45 - 4.34 (m, 2H), 3.41 - 3.26 (m, 2H), 2.26 - 2.22 (m, 1H), 1.86 - 1.79 (m, 4H), 1.65 (br. d, J = 11.2 Hz, 1H), 1.30 - 1.10 (m, 2H), 0.97 - 0.88 (m, 2H). MS (ES+): m / z (%) 383.2 (100) [M+H] + .

[0154] 2-Amino-4-(trifluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (Compound 1) [ka] 2-Amino-4-(trifluoromethyl)-6-((3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one was purified by SFC (column LuxCel2; solvent isopropanol, CO2; isocratic mode (15% isopropanol) and then further purified by SFC (column LuxCel2; solvent methanol, CO2; isocratic mode (10% methanol)) to give 2-amino-4-(trifluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (28 mg, 37% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.11 - 7.77 (m, 2H), 4.48 - 4.32 (m, 2H), 3.41 - 3.23 (m, 2H), 2.29 - 2.16 (m, 1H), 1.86 - 1.73 (m, 4H), 1.65 (br. d, J = 12.4 Hz, 1H), 1.35 - 1.09 (m, 2H), 0.99 - 0.77 (m, 2H). 13 C NMR (125 MHz, DMSO-d6) δ 176.4, 163.8, 162.6, 150.8 (q, 2 J CF = 37.9 Hz), 128.0 (q, 1 J CF = 278.5 Hz), 119.9 (q, 1 J CF = 279.5 Hz), 117.9, 109.0, 51.2, 47.5, 34.8, 29.3, 28.6, 24.4, 23.6. 19 F NMR (376 MHz, DMSO-d6) δ -66.22, -72.48. [M + H] + C 15 H 17 HRMS (ESI) calculated for F6N4O: 383.1307, found: 383.1305.

[0155] 2-Amino-4-(trifluoromethyl)-6-(((1R,3R)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (Compound 5) [ka] 2-Amino-4-(trifluoromethyl)-6-((3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one was purified by SFC (column LuxCel2; solvent isopropanol, CO2; isocratic mode (15% isopropanol) and then further purified by SFC (column LuxCel2; solvent methanol, CO2; isocratic mode (10% methanol)) to give 2-amino-4-(trifluoromethyl)-6-(((1R,3R)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (8 mg, 11% yield) as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 7.96 - 7.86 (m, 2H), 4.38 (d, J = 7.3 Hz, 2H), 3.59 - 3.54 (m, 1H), 3.41 - 3.37 (m, 1H), 2.23 - 2.21 (m, 1H), 1.81 - 1.79 (m, 1H), 1.58 - 1.55 (m, 3H), 1.50 - 1.41 (m, 3H), 1.35 - 1.27 (m, 2H). [M + H] + C 15 H 17 HRMS (ESI) calculated for F6N4O: 383.1314, found: 383.1339.

[0156] 2-Amino-4-(trifluoromethyl)-6-(((1S,3R)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (compound 6) [ka] 2-Amino-4-(trifluoromethyl)-6-((3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one was purified by SFC (column LuxCel2; solvent isopropanol, CO2; isocratic mode (15% isopropanol) and then further purified by SFC (column LuxCel2; solvent methanol, CO2; isocratic mode (10% methanol)) to give 2-amino-4-(trifluoromethyl)-6-(((1S,3R)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (30 mg, 40% yield) as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 7.92 - 7.89 (m, 2H), 4.44 - 4.34 (m, 2H), 3.38 - 3.31 (m, 2H), 2.28 - 2.22 (m, 1H), 1.84 - 1.78 (m, 4H), 1.66 (d, J = 12.4 Hz, 1H), 1.32 - 1.25 (m, 1H), 1.20 - 1.13 (m, 1H), 0.97 - 0.88 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 176.3, 163.8, 162.5, 150.8 (q, 2 J CF = 37.0 Hz), 127.9 (q, 1 J CF = 283.1 Hz), 119.9 (q, 1 J CF = 260.9 Hz), 118.6, 109.0, 51.2, 47.5, 34.8, 29.3, 28.6, 24.3, 23.5. [M + H] + C 15 H 17 HRMS (ESI) calculated for F6N4O: 383.1267, found: 383.1322.

[0157] 2-Amino-4-(trifluoromethyl)-6-(((1S,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (compound 7) [ka] 2-Amino-4-(trifluoromethyl)-6-((3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one was purified by SFC (column LuxCel2; solvent isopropanol, CO2; isocratic mode (15% isopropanol) and then further purified by SFC (column LuxCel2; solvent methanol, CO2; isocratic mode (10% methanol)) to give 2-amino-4-(trifluoromethyl)-6-(((1S,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (8 mg, 11% yield) as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 7.95 - 7.88 (m, 2H), 4.42 - 4.34 (m, 2H), 3.59 - 3.54 (m, 1H), 3.41 - 3.37 (m, 1H), 2.23 - 2.21 (m, 1H), 1.81 - 1.79 (m, 1H), 1.58 - 1.55 (m, 3H), 1.49 - 1.42 (m, 3H), 1.35 - 1.28 (m, 2H). [M + H] + C 15 H 17 HRMS (ESI) calculated for F6N4O: 383.1291, found: 383.1324.

[0158] Biological data and experimental results To avoid misunderstanding, activity data is pIC 50 When presented as a value, this is not the same as the IC50 value. 50 and IC 50 and IC50 of less than about 10 μM, as both are terms of the art. 50Compounds having a corresponding pIC value of greater than about 5 50 It will be readily understood by those skilled in the art that the arithmetic operations have a value.

[0159] Generally speaking, an IC of 1 μM 50 pIC with a value of 6 50 value corresponds to an IC of 100 nM 50 pIC with a value of 7 50 Equivalent to the value.

[0160] Protein expression and purification method of recombinant CpKRS The protein expression and purification methods of recombinant CpKRS are described in Baragana et al., PNAS, 2019, 116 (4), 7015-7020, and are summarized below.

[0161] C. parvum KRS had 45 residues truncated at the N-terminus. All amplicons were cloned into the pAVA0421 expression vector using the ligation-independent cloning (LIC) method. Recombinant plasmids were transformed into Rosetta BL21(DE3) competent cells and plated on LB agar plates with ampicillin, carbenicillin, and chloramphenicol selection. Clones were expressed using the autoinduction method previously reported and scaled up to 2 liter cultures in shake flasks. Cell pellets were harvested and lysed with 100 mM HEPES / 150 mM NaCl / 5% Glycerol / 20 mM Imidazole / 0.5 mM TCEP pH 7.5 / DNase / Complete inhibitor tablet using a Cell Disrupter (Constant Systems) at 30 KPSI and centrifuged at 40,000 g for 20 minutes to remove cell debris. The supernatant was loaded onto a 5 ml HiTrap Ni HP column equilibrated with buffer A (100 mM HEPES / 150 mM NaCl / 5% Glycerol / 20 mM Imidazole / 0.5 mM TCEP pH 7.5) at 5 ml / min using the AKTA Pure system. Immediately after loading, the column was washed with 10 column volumes of buffer A. A 5% step of buffer B (100 mM HEPES / 150 mM NaCl / 5% Glycerol / 500 mM Imidazole / 0.5 mM TCEP pH 7.5) was then used to wash off the His-rich contaminating proteins. A linear gradient of 5-50% B was used to elute the protein. Approximately 287 mg of protein was present in the fraction containing the CpKRS protein. The sample was then concentrated to approximately 30 ml, passed through a 0.2 μm filter, and then loaded onto a XK26 / 60 Superdex 200 column using the AKTA Pure system at 2 ml / min, 10 ml at a time, at 4° C. The eluted protein was pooled and then frozen at −80° C. The protein concentration was 1.3 mg / ml.Sometimes proteins were cleaved overnight with PreScission Protease and a second Ni column was run in breakthrough mode to remove uncleaved proteins. GST beads were then added to remove PreScission Protease. Proteins were then gel filtered as described above.

[0162] Protein expression and purification method for recombinant HsKRS The gene encoding human KRS, codon optimized for expression in E. coli, was obtained from Genscript and cloned into His PP pET15b vector using Nde1 and Xho1 restriction sites. The resulting vector was used to transform BL21(DE3) competent cells for protein expression. A 120 ml overnight culture was constructed, grown at 37°C, 200 rpm for 16 hours, and used to inoculate 6 liters of autoinduction + AMP medium the next day. This culture was grown at 37°C for 4 hours and then at 20°C for 20 hours. The cells were harvested by centrifugation at 3,500g for 30 minutes and then stored at -20°C. The resulting cell pellet was 50g. Lysis buffer (150ml, 25mM HEPES / 500mM NaCl / 20mM Imidazole / 2mM DTT / 10% Glycerol pH7.5 / Protease inhibitor tablet / DNAase) was added and the pellet was allowed to thaw at room temperature for approximately 10 minutes. The slurry was then passed through a Cell Disrupter (Constant Systems) set at 30KPSI to lyse the cells. The sample was then centrifuged at 40,000g for 20 minutes. The supernatant was then filtered using a syringe filter up to 0.45μm. The supernatant was loaded onto a 5ml HiTrap Ni HP column equilibrated with Buffer A (25mM HEPES / 500mM NaCl / 20mM Imidazole / 2mM DTT / 10% Glycerol pH7.5) at 5ml / min using the AKTA Pure system. Immediately after loading, the column was washed with 10 column volumes of buffer A. A 5% step of buffer B (25 mM HEPES / 500 mM NaCl / 500 mM Imidazole / 2 mM DTT / 10% Glycerol pH 7.5) was then used to wash off His-rich contaminating proteins. A gradient of 5-50% B was used to elute the protein. Approximately 55 mg of protein was present in the fractions containing the HsKRS protein.The sample was then passed through a 0.2 μm filter and then loaded onto a XK26 / 60 Superdex 200 column equilibrated with Buffer C (25 mM HEPES / 150 mM NaCl / 2 mM DTT / 10% glycerol pH 7.0) using an AKTA Pure system at 2 ml / min at 4° C. The sample was then dialyzed into 25 mM HEPES / 500 mM NaCl / 2 mM DTT / 10% glycerol / 0.25% azide pH 7.0. The protein was concentrated to 1 mg / ml and frozen at −80° C. This yielded approximately 23 mg of protein.

[0163] An assay method for measuring in vitro inhibition of Cryptosporidium lysyl-tRNA synthetase (KRS) Compound potency was measured with 10-point dose-response inhibition curves. Compounds were transferred to 384-well clear-bottom plates (Greiner Bio-One, 781101) using an Echo 550 acoustic dispenser (Labcyte). Using an automated liquid dispenser, 50 μL reaction mixtures were prepared by adding 25 μL of substrate mix into all wells, followed by 25 μL of enzyme mix into compound and no inhibition control wells (final reaction: 100 mM HEPES, pH 7.4, 20 mM MgCl2, 1 mM DTT, 500 μM ATP, 1.2 mM L-lysine, 300 nM CpKRS, and 0.5 U / mL Pyrophosphatase). Full inhibition control reactions were performed in the absence of enzyme, and plates were incubated at room temperature for 8 hours. The reaction was stopped by the addition of Biomol green (50 μL: Enzo Life Sciences) with the amount of free phosphate detected by absorbance (650 nm) measurement (BMG Pherastar plate reader) after a further incubation of 20 min. A counterscreen assay to exclude direct inhibition of pyrophosphatase was performed using 0.5 U / mL pyrophosphatase and 3 μM pyrophosphate as substrate in the same assay buffer (100 mM HEPES, pH 7.4, 20 mM MgCl2, 1 mM DTT) for 8 h at room temperature. Biomol green was added and incubated for 20 min, after which detection was performed as described above. Samples were run in duplicate and data were processed and analyzed using ActivityBase (IDBS).

[0164] An assay method for measuring in vitro inhibition of human lysyl-tRNA synthetase (KRS) Compound potency was measured with 10-point dose-response inhibition curves. Compounds were transferred to 384-well clear-bottom plates (Greiner Bio-One, 781101) using an Echo 550 acoustic dispenser (Labcyte). Using an automated liquid dispenser, 50 μL of reaction mixture was prepared by adding 25 μL of substrate mix into all wells, followed by 25 μL of enzyme mix into all wells (final reaction: 30 mM Tris HCl, pH 8.0, 40 mM MgCl2, 140 mM NaCl, 30 mM KCl, 0.01% Brij-35, 1 mM DTT, 3.5 μM ATP, 6 μM L-lysine, 200 nM CpKRS, and 0.5 U / mL Pyrophosphatase). Full inhibition control reactions were performed in the presence of 100 μM of control compound and plates were incubated for 5 h at room temperature. Reactions were stopped by addition of Biomol green (50 μL: Enzo Life Sciences) with the amount of free phosphate detected by absorbance (650 nm) measurement (BMG Pherastar plate reader) after a further incubation of 20 min. Counterscreen assays to exclude direct inhibition of pyrophosphatase were performed using 0.5 U / mL pyrophosphatase and 3 μM pyrophosphate as substrate in the same assay buffer (30 mM Tris HCl, pH 8.0, 40 mM MgCl2, 140 mM NaCl, 30 mM KCl, 0.01% Brij-35, 1 mM DTT) for 5 h at room temperature. Biomol green was added and incubated for 20 min, followed by detection as described above. Samples were run in duplicate and data was processed and analyzed using ActivityBase (IDBS).

[0165] Assay method for measuring in vitro inhibition of Cryptosporidium EC is the effective concentration of a compound that causes 50% parasite death against Cryptosporidium, as represented by the compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG. 50In vitro measurements were performed. These experiments were performed according to the method of Besssoff et.al. Antimicrob. Agents Chemother. 2013, 57:1804-1814. The results of these experiments are provided in the following experimental results.

[0166] Assay method for measuring in vitro inhibition (cytotoxicity) of HepG2 cells EC is the effective concentration of a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG that causes 50% parasite killing against Hep G2 (Human Caucasian hepatocellular carcinoma, HPACC cat. no. 85011430). 50 In vitro measurements were performed. These experiments were performed according to the method of Mersch-Sundermann V et. al. Toxicology 2004, 198:329-340. The results of these experiments are provided in the following experimental results.

[0167] Measuring anti-Cryptosporidium efficacy in vivo In vivo measurements of the potential efficacy of compounds of formula (I) in a Cryptosporidium mouse model were performed according to the methods described in Antimicrob. Agents Ch., 2018, 62, 4, e01505-17, and are summarized below.

[0168] The NOD SCID γ mouse model of cryptosporidiosis was performed as follows: Male NOD SCID γ mice (NOD.Cg-Prkdc scid Il2rg tm1WjlMice (100-1500 / SzJ, Jackson Labs) were infected with 10^5 C. parvum Iowa strain oocysts by oral gavage 2 weeks after weaning. Four mice were used per experimental group. This established a chronic, asymptomatic infection of the small intestine, cecum, and biliary system. Fecal parasite shedding was monitored by quantitative PCR to amplify C. parvum DNA. Compounds were dosed by oral gavage 1 week after infection (when shedding was uniformly detectable). Treatment continued for a total of 7 days. The number of oocysts per milligram of stool was counted for each mouse on day 8 (1 day after completion of the last dose) and day 15 (7 days after completion of the last dose). The mean oocyst concentration was calculated for each group of mice. Log reduction is the mean reduction in oocyst shedding compared to untreated vehicle.

[0169] These experiments were performed according to the method of CD Huston et al, Antimicrob. Agents Ch., 2018, 62, 4, e01505-17. The in vivo results are provided in Table 3. The results of these experiments are provided in the following experimental results.

[0170] biological activity Compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG have demonstrated Cryptosporidium inhibitory activity. The Cryptosporidium parvum pEC activity of some of the representative compounds herein from these in vitro Cryptosporidium inhibition assay tests was 50 The data are provided in Table 2. Compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG demonstrate cell selectivity. Hep G2 pEC50 values ​​from these Hep G2 in vitro cytotoxicity studies for some of the representative compounds herein are shown in Table 1. 50 The data are provided in Table 2. Compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG have demonstrated Cp KRS inhibitory activity. The ceruloplasmin KRS pIC of some of the representative compounds herein from these Cryptosporidium parvum lysyl t-RNA synthetase (Cp KRS) inhibition assays are shown in Table 1. 50 The data are provided in Table 2.

[0171] [Table 2]

[0172] [Table 3]

Claims

1. The following equation I: 【Chemistry 1】 {During the ceremony, R 1 is, -CH 3 -CF 2 H or -CF 3 Selected from; and, R 2 The following: 【Chemistry 2】 Compounds selected from { or veterinary or pharmaceutically acceptable salts, hydrates, solvates, isomers, prodrugs or polymorphs thereof.

2. The aforementioned R 1 However, -CH 3 The compound according to claim 1, or -CF3.

3. Said R 2 However, the following 【Transformation 3】 The compound according to claim 1 or 2.

4. The compound of formula I is given by the following formula IA: 【Chemistry 4】 {wherein, R 1 is selected from -CH 3 , -CF 2 H or -CF 3}, or a veterinary or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof, the compound according to claim 1.

5. The compound of formula IA is the following compound IB: 【Transformation 5】 {In the formula, R 1 is, -CH 3 -CF 2 H or -CF 3 The compound according to claim 4, which is a compound selected from { or a veterinary or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

6. The compound of formula IA is given by the following formula IC: 【Transformation 6】 {In the formula, R 1 is, -CH 3 -CF 2 H or -CF 3 The compound according to claim 4, which is a compound selected from { or a veterinary or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

7. The compound of formula I is given by the following formula ID: 【Transformation 7】 {In the formula, R 1 is, -CH 3 -CF 2 H or -CF 3 The compound according to claim 1, which is a compound selected from { or a veterinary or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

8. The compound of formula ID is given by the following formula IE: 【Transformation 8】 {In the formula, R 1 is, -CH 3 -CF 2 H or -CF 3 The compound according to claim 7, which is a compound selected from { or a veterinary or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

9. The compound of formula ID above is given by the following formula IF: 【Chemistry 9】 {In the formula, R 1 is, -CH 3 -CF 2 H or -CF 3 The compound according to claim 7, which is a compound selected from { or a veterinary or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

10. R 1 However, -CH 3 The compound according to any one of claims 4 to 9, or -CF3.

11. The compound of formula I is given by the following formula IG: 【Chemistry 10】 {In the formula, R 1 is, -CH 3 -CF 2 H or -CF 3 The compound according to claim 1, which is a compound selected from { or a veterinary or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

12. The aforementioned R 1 However, -CH 3 The compound according to claim 11, or -CF3.

13. The aforementioned compound is as follows: 【Chemistry 11】 Alternatively, the compound according to claim 1, selected from veterinary or pharmaceutically acceptable salts, hydrates, solvates, isomers, prodrugs, or polymorphs thereof.

14. pEC for Cryptosporidium parvum of 6 or higher, preferably 6.5 or higher, more preferably 7 or higher, and particularly 7.3 or 7.7 or higher. 50 The compound according to claim 1, having the following characteristics.

15. pIC for cryptosporidiolus parvamuligyl tRNA synthetase (Cp KRS) of 5 or higher, preferably 5.5 or higher, more preferably 6 or higher. 50 The compound according to claim 1, having the following characteristics.

16. A pharmaceutical composition for pharmaceutical or veterinary use comprising the compound described in claim 1.

17. A pharmaceutical composition for the treatment of an infectious disease, comprising the compound described in claim 1.

18. A pharmaceutical composition for the treatment of diseases caused by Cryptosporidium, comprising the compound described in claim 1, wherein the disease is optionally Cryptosporidium andersonii, Cryptosporidium baileyi, Cryptosporidium bovis, Cryptosporidium canis, Cryptosporidium tipmunk, Cryptosporidium cniculus, Cryptosporidium zusismarsi, Cryptosporidium ferris, Cryptosporidium faeri, Cryptosporidium galli, Cryptosporidium mereagridis, Cryptosporidium The pharmaceutical composition is caused by a Cryptosporidium species selected from Liptosporidium muris, Cryptosporidium monali, Cryptosporidium suis, Cryptosporidium scropharum, Cryptosporidium chiseri, Cryptosporidium ubiquitum, Cryptosporidium biatum, Cryptosporidium nasorum, Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium saurophyllum, Cryptosporidium serpentis, and Cryptosporidium lyri.

19. A pharmaceutical composition for the treatment of cryptosporidiosis, comprising the compound described in claim 1.

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