Anti-infective agents

Abstract

The present invention relates to a compound or a pharmaceutically acceptable salt thereof, a composition containing the compound or the pharmaceutically acceptable salt thereof, a combination comprising the compound or the pharmaceutically acceptable salt thereof and at least one additional therapeutic agent, and the use of the compound or the pharmaceutically acceptable salt thereof, the composition, the combination in therapy, and the use of the compound or the pharmaceutically acceptable salt thereof. For example, the compound is used for treating infectious diseases or treating diseases caused by cryptosporidium.

Description

Technical Field

[0001] The present invention relates to a compound or a pharmaceutically acceptable salt thereof, a composition containing the compound or a pharmaceutically acceptable salt thereof, a combination comprising the compound or a pharmaceutically acceptable salt thereof and at least one additional therapeutic agent, and the use of the compound or a pharmaceutically acceptable salt thereof, the composition or the combination in treatment, for example, in the treatment of infectious diseases or diseases caused by Cryptosporidium. Background Art

[0002] Cryptosporidiosis is a diarrheal disease caused by the parasite Cryptosporidium. Currently, there are 27 recognized Cryptosporidium species, 20 of which can infect humans, and most human infection cases are caused by Cryptosporidium parvum (C parvum) or Cryptosporidium hominis (C.hominis) (Int. J. For Parasitology 2015, 45, 367-373). In 1976, cryptosporidiosis was first identified as the cause of human infection (Gastroenterology, 1976, 70, 592-598). Recent studies have investigated the causes and effects of diarrhea in more than 22,000 children under the age of 5, and found that Cryptosporidium is the second largest cause of diarrhea and morbidity after rotavirus (The Lancet, 2013, 382, ​​9888, 209-222).

[0003] Cryptosporidiosis is a major cause of diarrhea in many parts of the world, particularly affecting children in sub-Saharan Africa and South Asia. The 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, resulting in more than 200,000 deaths each year. Cryptosporidiosis is also associated with malnutrition and stunting. It is one of the leading causes of death from diarrheal diseases.

[0004] Cryptosporidium is an opportunistic infection, and people with underdeveloped immune systems (such as children under 5 years old) and immunocompromised people co-infected with HIV are at higher risk of infection and death. Early childhood malnutrition has also been associated with persistent diarrhea and Cryptosporidium infection (Lancet Infect. Dis., 2015, 15, 85-94).

[0005] Nitazoxanide is the only drug approved by the U.S. Food and Drug Administration for the treatment of cryptosporidiosis. It has been confirmed that the efficacy of nitazoxanide is not ideal, and can not effectively treat all patients who are only infected with Cryptosporidium (J.Infect.Dis., 2001,184,103-06 and Clin.Gastroenterol.Hepatol., 2006,4,320-24). In clinical trials, nitazoxanide has also been shown to be invalid for patients with Cryptosporidium-HIV co-infection, and these patients do not receive HIV antiretroviral therapy simultaneously (Trans.R.Soc.Trop.Med.Hyg.1998,92,663-66 and BMC Infect.Dis.2009.9,195).

[0006] Therefore, there is a need for new anti-infective agents that are effective against Cryptosporidium, particularly anti-infective agents that are suitable for treating or preventing the following diseases: subjects infected with Cryptosporidium alone; immunocompromised subjects infected with Cryptosporidium, such as subjects co-infected with Cryptosporidium-HIV; and malnourished children under one year old who have diarrhea caused by Cryptosporidium infection.

[0007] Lysyl t-RNA synthetase (LysRS or KRS)

[0008] Protein synthesis is a complex multistep process involving many enzymes. Aminoacyl-tRNA synthetases (aaRS) catalyze the attachment of amino acids to their cognate transfer RNAs (tRNAs) and play a key role in protein translation. Inhibition of aaRS has been successfully used to treat bacterial infections, and one aaRS inhibitor, mupirocin, is currently in clinical use for topical treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. Mupirocin is an inhibitor of isoleucyl-tRNA synthetase (IleRS).

[0009] Recently, cladosporin was identified as an inhibitor of lysyl-tRNA synthetase (LysRS). As reported by Scott et al., J. Antibiot. 197124, 747-755, cladosporin is a secondary metabolite of fungi. Studies have shown that cladosporin has a 100-fold higher selectivity for inhibiting PfLysRS than HsLysRS (Hoepfner et al., Cell Host Microbe, 2012, 11(6): 654-63). We report that cladosporin and chromone analogs are inhibitors of Cryptosporidium parvum LysRS (Baragana et al., PNAS, 2019, 116(4), 7015-7020).

[0010] Chromone analogs showed reduced oocyst shedding in two different mouse models of Cryptosporidium parvum infection (WO2017221002A1 and Baragana et al., PNAS, 2019, 116(4), 7015-7020). These results validate that LysRS of Cryptosporidium parvum is a promising mode of action for the treatment of Cryptosporidium parvum infection. Summary of the invention

[0011] The present invention relates to a compound of formula I:

[0012]

[0013] Among them, R 1 is selected from: -CH3, -CF2H or -CF3; and

[0014] Where R 2 Selected from:

[0015] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0016] In the compounds of formula I, preferably, R 1 Can be selected from: -CH3 or -CF3. Most preferably, R 1 It can be -CF3.

[0017] In the compounds of formula I, preferably, R 2 Can

[0018] The compound of formula I may be a compound of formula IA:

[0019]

[0020] Among them, R 1 Selected from: -CH3, -CF2H or -CF3;

[0021] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0022] In the compounds of formula IA, preferably, R 1 Can be selected from: -CH3 or -CF3. Most preferably, R 1 It can be -CF3.

[0023] The compound of formula I may be a compound of formula IB:

[0024]

[0025] Among them, R 1Selected from: -CH3, -CF2H or -CF3;

[0026] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0027] In the compounds of formula IB, preferably, R 1 Can be selected from: -CH3 or -CF3. Most preferably, R 1 It can be -CF3.

[0028] The compound of formula I may be a compound of formula IC:

[0029]

[0030] Among them, R 1 Selected from: -CH3, -CF2H or -CF3;

[0031] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0032] In the compounds of formula IC, preferably, R 1 Can be selected from: -CH3 or -CF3. Most preferably, R 1 It can be -CF3.

[0033] The compound of formula I may be a compound of formula ID:

[0034]

[0035] Among them, R 1 Selected from: -CH3, -CF2H or -CF3;

[0036] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0037] In the compounds of formula ID, preferably, R 1 Can be selected from: -CH3 or -CF3. Most preferably, R 1 It can be -CF3.

[0038] The compound of formula I may be a compound of formula IE:

[0039] as well as

[0040] Among them, R 1 Selected from: -CH3, -CF2H or -CF3;

[0041] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0042] In the compounds of formula IE, preferably, R 1 Can be selected from: -CH3 or -CF3. Most preferably, R 1 It can be -CF3.

[0043] The compound of formula I may be a compound of formula IF:

[0044] as well as

[0045] Among them, R 1 Selected from: -CH3, -CF2H or -CF3;

[0046] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0047] In the compounds of formula IF, preferably, R 1 Can be selected from: -CH3 or -CF3. Most preferably, R 1 It can be -CF3.

[0048] The compound of formula I may be a compound of formula IG:

[0049]

[0050] Among them, R 1 Selected from: -CH3, -CF2H or -CF3;

[0051] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0052] In some embodiments, the compound of formula IG may be a racemic mixture. In some embodiments, optionally, the compound of formula IG may be optically active.

[0053] In the compounds of formula IG, preferably, R 1 Can be selected from: -CH3 or -CF3. Most preferably, R 1 It can be -CF3.

[0054] Preferred compounds of formula I may be selected from:

[0055]

[0056] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0057] Preferred compounds of formula I may be selected from:

[0058] 2-amino-4-(trifluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one;

[0059] 2-amino-4-methyl-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one;

[0060] 2-amino-4-(difluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one;

[0061] 2-amino-6-(spiro[2.5]octan-5-ylmethyl)-4-(trifluoromethyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one;

[0062] 2-amino-4-(trifluoromethyl)-6-(((1R,3R)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one;

[0063] 2-amino-4-(trifluoromethyl)-6-(((1S,3R)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one;

[0064] 2-amino-4-(trifluoromethyl)-6-(((1S,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one;

[0065] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0066] The pEC of the compounds of Formula I, IA, IB, IC, ID, IE, IF and / or IG against Cryptosporidium parvum 50 It may be 6 or more, preferably 6.5 or more, more preferably 7 or more, particularly 7.3 or 7.7 or more.

[0067] The pIC of the compounds of formula I, IA, IB, IC, ID, IE, IF and / or IG against Cryptosporidium parvum lysyl t-RNA synthetase (Cp KRS) 50 It may be 5 or more, preferably 5.5 or more, more preferably 6 or more.

[0068] In a second aspect, there is provided a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG as described above for use in medicine.

[0069] In a third aspect, there is provided a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG as described above for use in veterinary medicine.

[0070] In a fourth aspect, there is provided a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG as described above for use as a medicament.

[0071] In another aspect, there is provided a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG as described above for use in treating an infectious disease.

[0072] In another aspect, there is provided a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG as described above for use in treating cryptosporidiosis.

[0073] In another aspect, there is provided a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG as described above for use in treating a disease caused by Cryptosporidium. The disease may be caused by a species of Cryptosporidium selected from the group consisting of Cryptosporidium andersoni, Cryptosporidium baileyi, Cryptosporidium bovis, Cryptosporidium canis, Cryptosporidium chipmunk, Cryptosporidium cuniculus, Cryptosporidium ducismarci, Cryptosporidium felis, Cryptosporidium fayeri, Cryptosporidium galli, Cryptosporidium meleagridis, Cryptosporidium muris, Cryptosporidium spp. monari), Cryptosporidium suis, Cryptosporidium scrofarum, Cryptosporidium tyzzeri, Cryptosporidium ubiquitum, Cryptosporidium viatorum, Cryptosporidium nasorum, Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium saurophilum, Cryptosporidium serpentis, and Cryptosporidium wrairi.

[0074] Another aspect of the present invention relates to a method for treating Cryptosporidium infection in a human or animal in need thereof. The method comprises 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 described above, or a pharmaceutically acceptable salt thereof.

[0075] Another aspect of the present invention relates to a method for treating a disease caused by infection with Cryptosporidium parasites 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 described above, or a pharmaceutically acceptable salt thereof.

[0076] 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 as described above or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating Cryptosporidium infection or a disease caused by infection with Cryptosporidium parasites.

[0077] Another aspect of the invention relates to a combination of: (a) a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG as described above, or a pharmaceutically acceptable salt thereof; and (b) at least one other antiparasitic agent.

[0078] 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 described above, or a pharmaceutically acceptable salt thereof, and instructions for administering to a human or animal in need thereof. DETAILED DESCRIPTION

[0079] Terms and Definitions

[0080] It should be understood that phrases such as "a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG, or a pharmaceutically acceptable salt thereof" are intended to include: a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG; a pharmaceutically acceptable salt or solvate of a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG; or any pharmaceutically acceptable combination thereof. Thus, by way of non-limiting example for purposes of illustration herein, "a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG, or a pharmaceutically acceptable salt thereof" includes a pharmaceutically acceptable salt of a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG that is present as a solvate or hydrate, and the phrase also includes a mixture of a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG with a pharmaceutically acceptable salt of a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG.

[0081] It should be further understood that the compounds of Formula I, IA, IB, IC, ID, IE, IF and / or IG or pharmaceutically acceptable salts thereof referred to herein include the compounds of Formula I, IA, IB, IC, ID, IE, IF and / or IG as free bases or pharmaceutically acceptable salts thereof.

[0082] The term "pharmaceutically acceptable" refers to compounds (including salts), materials, compositions and dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0083] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the human compound and exhibits minimal adverse toxicological effects. These pharmaceutically acceptable salts can be prepared in situ during the final isolation and purification of the compound, or by reacting the purified free acid or free base form of the compound with a suitable base or acid, respectively.

[0084] In addition, pharmaceutically acceptable salts include salts described in Berge, J. Pharm. Sci., 1977, 66, 1-19, or salts described in Handbook of Pharmaceutical Salts; Properties, Selection and Use, 2nd edition, edited by PH Stahl and C GWermuth, Stahl / Wermuth: Wiley-VCH / VHCA, 2011 (see http: / / www.wiley.com / WileyCDA / WileyTitle / productCd-3906390519.html).

[0085] Where the functionality of the compound permits, suitable pharmaceutically acceptable salts of the compounds of Formula I, IA, IB, IC, ID and / or IE may be formed, including acid addition salts. Acid addition salts may be formed by reaction with a suitable acid, or may be reacted in a suitable solvent (e.g., an organic solvent) to form a salt, which may be separated by crystallization and filtration. Representative pharmaceutically acceptable acid addition salts include, but are not limited to, 4-acetamidobenzoate, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate (benzenesulfonate), benzoate, bisulfate, tartrate, butyrate, calcium edetate, camphorate, camphorsulfonate (camphorsulfonate), decanoate (decanoate), caproate (caproate), caprylate (caprylate), cinnamate, citrate, cyclamate, digluconate, 2,5-dihydroxybenzoate, disuccinate Salt, dodecyl sulfate (dodecyl sulfate), EDTA (ethylenediaminetetraacetate), dodecyl sulfate (lauryl sulfate), ethane-1,2-disulfonate (ethanedisulfonate), ethanesulfonate (ethanesulfonate), formate, fumarate, galactonate (acetalate), gentisate (2,5-dihydroxybenzoate), glucoheptonate (glucoheptonate), gluconate, glucuronate, glutamate, glutarate, glycerophosphate, glycolate, hexylnorsorbate salt, hippurate, hydrabamine (N,N'-di(dehydroabietyl)-ethylenediamine), hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoate, isobutyrate, lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, methanesulfonate (mesylate), sulfate formate, mucoside, naphthalene-1,5-disulfonate (naphthalene disulfonate), naphthalene-2-sulfonate (naphthalene sulfonate), nicotinate, nitrate, oleate, palmitate, p-aminobenzenesulfonate The compounds described herein include, but are not limited to, succinate, pyroglutamate, pyruvate, salicylate, sebacate, stearate, subacetate, succinate, sulfamate, sulfate, tannate, tartrate, theophylline (8-chlorotheophylline), thiocyanate, trisulfide, undecanoate, undecylenate, and valerate.

[0086] As used herein, the term "therapeutically effective amount" refers to any dose that improves the treatment, rehabilitation, prevention or improvement of a disease, disorder or side effect, or reduces the rate of development of a disease or disorder, compared to a corresponding person not taking the dose. An appropriate "therapeutically effective amount" will depend on a variety of factors, such as the age and weight of the person, the exact condition to be treated and its severity, the nature of the formulation and the route of administration, and is ultimately determined by the attending physician.

[0087] As used herein, the terms "treat" and "treatment" are used interchangeably herein and refer to methods of obtaining beneficial or desired results, including but not limited to a therapeutic effect. A therapeutic effect refers to the eradication or amelioration of the underlying disorder being treated. In addition, a therapeutic benefit may also be obtained by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, thereby observing an improvement in the patient, although the patient may still suffer from the underlying disorder. As used herein, the term "treat", in all verb forms, refers to alleviating, alleviating, preventing and / or controlling at least one symptom of a disorder in a human.

[0088] As used herein, the term "solvate" refers to a form of a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG or a pharmaceutically acceptable salt thereof that is usually combined with a solvent by a solvolysis reaction. This physical combination may include hydrogen bonds. Conventional solvents include water, methanol, ethanol, acetic acid, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), diethyl ether, and the like. For example, the compounds described herein may be prepared in crystalline form or may be soluble. Suitable solvates include pharmaceutically acceptable solvates, and also include stoichiometric solvates and non-stoichiometric solvates. In some examples, for example, when one or more solvent molecules are incorporated into the lattice of a crystalline solid, the solvate will be able to be separated. "Solvate" includes solution phase solvates and separable solvates. Representative solvates include hydrates, glycolates and methanolates. In certain embodiments, a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG or a pharmaceutically acceptable salt thereof may be separated, formed and / or administered in the form of a solvate.

[0089] As used herein, the term "hydrate" refers to a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG or a pharmaceutically acceptable salt thereof in a form of water. Typically, the number of water molecules contained in a compound hydrate is proportional to the number of compound molecules in the hydrate. Thus, for example, a hydrate of a compound can be represented by the formula R·xH2O, where R is a compound and x is a number greater than 0. A given compound may form more than one hydrate, for example, including a monohydrate (x is 1), a low hydrate (x is a number greater than 0 and less than 1, such as a hemihydrate (R·0.5H2O)) and a polyhydrate (x is a number greater than 1, such as a dihydrate (R·2H2O) and a hexahydrate (R·6H2O)). In certain embodiments, a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG or a pharmaceutically acceptable salt thereof may be isolated, formed and / or administered in the form of a hydrate.

[0090] It is also understood that compounds that have identical molecular formula 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."

[0091] 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". When a compound has an asymmetric center, for example, it is bonded to four different groups, it is possible to produce a pair of enantiomers. Enantiomers can be characterized by the absolute configuration of their asymmetric center and described by the R and S ordering rules of Cahn and Prelog, or by the way the molecule rotates the plane of polarized light and is designated as a right-handed enantiomer or a left-handed enantiomer (i.e., a (+) enantiomer or a (-) enantiomer, respectively). Chiral compounds can exist as a single enantiomer or a mixture thereof. A mixture containing equal proportions of enantiomers is called a "racemic mixture". In certain embodiments, the compound of formula (I) or its pharmaceutically acceptable salt may contain one or more asymmetric centers (also called chiral centers) and may therefore exist as a single enantiomer, a diastereomer, or other stereoisomers, or may also exist as a mixture thereof. Unless otherwise indicated, the compounds of Formula I or pharmaceutically acceptable salts thereof referred to herein refer to any single or mixed stereoisomers.

[0092] The term "polymorph" refers to the crystalline form of a compound (or its salt, hydrate or solvate). All polymorphs have the same elemental composition. Different crystalline forms usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shapes, optical and electrical properties, stability and solubility. Recrystallization solvents, crystallization rates, storage temperatures and other factors may lead to one crystal form being dominant. Under different conditions, crystallization can produce various polymorphs of a compound. Polymorphs of a compound of formula (I) or a pharmaceutically acceptable salt thereof can be characterized and distinguished using a variety of conventional analytical techniques, including but not limited to X-ray powder diffraction (XRPD), infrared spectroscopy (IR), Raman spectroscopy, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and solid-state nuclear magnetic resonance (ssNMR).

[0093] Compound

[0094] The present invention relates to compounds of formula I:

[0095]

[0096] Among them, R 1 is selected from: -CH3, -CF2H or -CF3; and

[0097] Where R 2Selected from:

[0098] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0099] In the compounds of formula I, preferably, R 1 Can be selected from: -CH3 or -CF3. Most preferably, R 1 It can be -CF3.

[0100] In the compounds of formula I, preferably, R 2 Can

[0101] The compound of formula I may be a compound of formula IA:

[0102]

[0103] Among them, R 1 Selected from: -CH3, -CF2H or -CF3;

[0104] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0105] In the compounds of formula IA, preferably, R 1 Can be selected from: -CH3 or -CF3. Most preferably, R 1 It can be -CF3.

[0106] The compound of formula I may be a compound of formula IB:

[0107] as well as

[0108] Among them, R 1 Selected from: -CH3, -CF2H or -CF3;

[0109] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0110] In the compounds of formula IB, preferably, R 1 Can be selected from: -CH3 or -CF3. Most preferably, R 1 It can be -CF3.

[0111] The compound of formula I may be a compound of formula IC:

[0112] as well as

[0113] Among them, R 1 Selected from: -CH3, -CF2H or -CF3;

[0114] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0115] In the compounds of formula IC, preferably, R 1 Can be selected from: -CH3 or -CF3. Most preferably, R 1 It can be -CF3.

[0116] The compound of formula I may be a compound of formula ID:

[0117]

[0118]

[0119] Among them, R 1 Selected from: -CH3, -CF2H or -CF3;

[0120] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0121] In the compounds of formula ID, preferably, R 1 Can be selected from: -CH3 or -CF3. Most preferably, R 1 It can be -CF3.

[0122] The compound of formula I may be a compound of formula IE:

[0123] as well as

[0124] Among them, R 1 Selected from: -CH3, -CF2H or -CF3;

[0125] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0126] In the compounds of formula IE, preferably, R 1 Can be selected from: -CH3 or -CF3. Most preferably, R 1 It can be -CF3.

[0127] The compound of formula I may be a compound of formula IF:

[0128] as well as

[0129] Among them, R 1 Selected from: -CH3, -CF2H or -CF3;

[0130] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0131] In the compounds of formula IF, preferably, R 1 Can be selected from: -CH3 or -CF3. Most preferably, R 1 It can be -CF3.

[0132] The compound of formula I may be a compound of formula IG:

[0133]

[0134] Among them, R 1 Selected from: -CH3, -CF2H or -CF3;

[0135] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0136] In the compounds of formula IG, preferably, R 1 Can be selected from: -CH3 or -CF3. Most preferably, R 1 It can be -CF3.

[0137] Table 1 describes exemplary compounds of Formula I.

[0138] Table 1 - Exemplary compounds of Formula I

[0139]

[0140] Preferred compounds of formula I may be selected from:

[0141]

[0142] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0143] Preferred compounds of formula I may be selected from:

[0144] 2-amino-4-(trifluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one;

[0145] 2-amino-4-methyl-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one;

[0146] 2-amino-4-(difluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one; and

[0147] 2-amino-6-(spiro[2.5]octan-5-ylmethyl)-4-(trifluoromethyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one;

[0148] 2-amino-4-(trifluoromethyl)-6-(((1R,3R)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one;

[0149] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0150] Particularly preferred compounds of formula I may be selected from:

[0151] 2-amino-4-(trifluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one;

[0152] 2-amino-4-(difluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one; and

[0153] 2-amino-4-methyl-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one;

[0154] or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

[0155] The pEC of the compounds of Formula I, IA, IB, IC, ID, IE, IF and / or IG against Cryptosporidium parvum 50 It may be 6 or more, preferably 6.5 or more, more preferably 7 or more, particularly 7.3 or 7.7 or more.

[0156] Compounds of Formula I, IA, IB, IC, ID, IE, IF and / or IG inhibit the pIC of Cryptosporidium parvum lysyl t-RNA synthetase (CpKRS) 50 It may be 5 or more, preferably 5.5 or more, more preferably 6 or more.

[0157] It should be understood that the compounds of I, IA, IB, IC, ID, IE, IF and / or IG may exist in different tautomeric forms. All possible tautomers and pharmaceutically acceptable salts thereof are considered within the scope of the present invention.

[0158] The compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG, or a pharmaceutically acceptable salt thereof, may be in crystalline or amorphous form. In addition, some crystalline forms may exist as polymorphs, all of which are included within the scope of the present invention. Of particular interest is the most thermodynamically stable polymorph or polymorphic form of the compound of Formula I, IA, IB, IC, ID, IE, or a pharmaceutically acceptable salt thereof. In one aspect of the present invention, the compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG, or a pharmaceutically acceptable salt thereof, is crystalline.

[0159] Application Method

[0160] It is understood that treatment as referred to herein includes prevention by alleviating confirmed symptoms of the condition as well as palliative care, i.e., prevention or control. "Treatment" or "treatment" of a condition, disorder, or condition includes: (1) preventing or delaying the onset of clinical symptoms of the condition, disorder, or condition in a person who may be suffering from or susceptible to the condition, disorder, or condition but does not yet develop or show clinical or subclinical symptoms of the condition, disorder, or condition; (2) inhibiting the condition, disorder, or condition, i.e., preventing, reducing, or delaying the development or recurrence of the disease (in the case of maintenance treatment) or the onset of at least one clinical or subclinical symptom; or (3) relieving or ameliorating the disease, i.e., causing regression of the condition, disorder, or condition or at least one clinical or subclinical symptom thereof.

[0161] Those skilled in the art will appreciate that the treatment mentioned herein refers to the treatment of a confirmed disease. However, the compound of formula (I) and its pharmaceutically acceptable salts can also be used for the prevention (prevention) of certain diseases depending on the condition.

[0162] As used herein, "treat," "treat," or "treatment," when referring to a disease, unless otherwise indicated, means: (1) ameliorating the disease or one or more biological manifestations of a 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 biological manifestations of the disease; (3) alleviating one or more symptoms or effects associated with the disease; (4) slowing the progression of the disease or one or more biological manifestations of the disease, and / or (5) reducing the likelihood of severity of the disease or one or more biological manifestations of the disease.

[0163] Unless otherwise indicated, as used herein, the terms "prevent", "preventing" or "preventing" refer to the prophylactic administration of a drug to reduce the likelihood of developing a disease or its biological manifestation or to delay the onset of a disease or its biological manifestation. Those skilled in the art will appreciate that "prevention" is not an absolute term. In medicine, "prevention" is understood as the prophylactic administration of a drug to substantially reduce the likelihood or severity of a disorder or its biological manifestation or to delay the onset of such a disorder or its biological manifestation.

[0164] 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.

[0165] Therefore, another aspect of the present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof for use in therapy. The present invention also provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use as a therapeutic agent, the therapy being human therapy or veterinary therapy.

[0166] It will be appreciated that when a compound of formula I or a pharmaceutically acceptable salt thereof is used in therapy, it is used as an active therapeutic agent.

[0167] For the avoidance of doubt, references herein generally to "treatment" include curative treatment, palliative treatment and preventive treatment.

[0168] Human and veterinary applications

[0169] Provided is the use of the compounds of the present invention in humans:

[0170] A pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, prodrug or polymorph thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients;

[0171] A compound of formula I, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, prodrug or polymorph thereof, or a pharmaceutical composition containing any of the foregoing, for use as a medicament;

[0172] A compound of formula I, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, prodrug or polymorph thereof, or a pharmaceutical composition containing any of the foregoing, for use in medicine;

[0173] A compound of formula I, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, prodrug or polymorph thereof, or a pharmaceutical composition containing any of the foregoing, for treating an infectious disease. Preferably, the infectious disease may be an infectious disease caused by Cryptosporidium. The infectious disease may be caused by a species of Cryptosporidium, which is selected from the group consisting of Cryptosporidium andersonii, Cryptosporidium belli, Cryptosporidium bovis, Cryptosporidium canis, Cryptosporidium chipmunks, Cryptosporidium rabbits, Cryptosporidium tortoises, Cryptosporidium cates, Cryptosporidium fischeri, Cryptosporidium gallinarum, Cryptosporidium turkey, Cryptosporidium rats, Cryptosporidium marine fish, Cryptosporidium suis, Cryptosporidium sow, Cryptosporidium tyzeri, Cryptosporidium ubiquitum, Cryptosporidium traveler, Cryptosporidium fish, Cryptosporidium microsporidiosis, Cryptosporidium hominis, Cryptosporidium lizard and snake, Cryptosporidium serpentis, and Cryptosporidium verrii;

[0174] The compound of formula I, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, prodrug or polycrystal thereof, or a pharmaceutical composition containing any of the above, is used for the preventive treatment of infectious diseases. Preferably, the infectious disease may be an infectious disease caused by Cryptosporidium. The disease may be caused by a species of Cryptosporidium, which is selected from the group consisting of Cryptosporidium andersoni, Cryptosporidium belli, Cryptosporidium bovis, Cryptosporidium canis, Cryptosporidium chipmunks, Cryptosporidium rabbits, Cryptosporidium tortoises, Cryptosporidium cates, Cryptosporidium fischeri, Cryptosporidium chickens, Cryptosporidium turkeys, Cryptosporidium rats, Cryptosporidium marine fishes, Cryptosporidium suis, Cryptosporidium sows, Cryptosporidium tyzeri, Cryptosporidium ubiquitum, Cryptosporidium traveler, Cryptosporidium fishes, Cryptosporidium microsporidiosis, Cryptosporidium hominis, Cryptosporidium lizards and snakes, Cryptosporidium serpentis, and Cryptosporidium veruis. Regarding the use of the compound of the present invention in animals, it is provided:

[0175] A veterinary composition comprising a compound of formula I, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, prodrug or polymorph thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients;

[0176] The compound of formula I, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, prodrug or polymorph thereof, or a veterinary composition containing any of the foregoing, is used as a veterinary medicament.

[0177] For the avoidance of doubt, where reference is made herein to the use of a compound of formula I, this additionally refers to the use of a compound independently selected from any one of the following formulae: I, IA, IB, IC, ID, IE, IF and / or IG.

[0178] Treatment of cryptosporidiosis

[0179] Therefore, also provided is a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG or a pharmaceutically acceptable salt thereof for use in treating or preventing cryptosporidiosis.

[0180] The present invention also provides a method for treating or preventing cryptosporidiosis, which comprises administering a therapeutically effective amount of a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG, or a veterinary or pharmaceutically acceptable salt thereof, to a human subject in need thereof. In another embodiment of the present invention, a method for treating or preventing cryptosporidiosis is provided, which comprises administering a therapeutically effective amount of a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG, or a veterinary or pharmaceutically acceptable salt thereof, to a mammal in need thereof.

[0181] According to a preferred aspect, the above-mentioned use and / or method provides a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG, or a pharmaceutically acceptable salt thereof, which is effective against Cryptosporidium, in particular a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG, which is suitable for treating or preventing the following diseases: subjects infected only with Cryptosporidium; immunocompromised subjects infected with Cryptosporidium, such as subjects co-infected with Cryptosporidium and HIV.

[0182] Pharmaceutical composition

[0183] According to another aspect of the present invention, a pharmaceutical composition is provided, comprising: a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG or a pharmaceutically acceptable salt, solvate, hydrate, isomer, prodrug or polymorph thereof.

[0184] The pharmaceutical composition also includes one or more pharmaceutically acceptable carriers, diluents or excipients. The pharmaceutical composition also includes one or more active agents against Cryptosporidium.

[0185] Administration and dosage range

[0186] In order to select the most appropriate dosage form and route of administration deemed suitable for treating the desired indication, the compounds of Formula I, IA, IB, IC, ID, IE, IF and / or IG should be evaluated for biopharmaceutical properties such as solubility, solution stability (over the pH range), possible dosage levels and permeability. Preliminary biopharmaceutical testing of potential anti-malarial treatments has provided positive results.

[0187] The compounds of the present invention can be used as crystalline or non-crystalline products for pharmaceutical and / or veterinary applications. For example, the compound can be obtained as a solid plug, powder or film by methods such as precipitation, crystallization, freeze drying, spray drying or evaporative drying. Microwave or radio frequency drying can be used to obtain the compound.

[0188] The compound can be administered alone, or co-administered with one or more other compounds of the present invention, or co-administered with one or more other drugs (or any combination thereof). In general, they will be administered as a formulation with one or more pharmaceutically acceptable excipients. The term "excipient" as used herein refers to any ingredient other than the compound of the present invention. The choice of excipient depends to a large extent on various factors, such as the specific mode of administration, the effect of the excipient on solubility and stability, and the properties of the dosage form. Pharmaceutically and veterinarily acceptable excipients include one or more of the following: lubricants, binders, diluents, surfactants, antioxidants, colorants, flavorings, preservatives, flavor enhancers, preservatives, saliva stimulants, coolants, cosolvents (including oils), emollients, leavening agents, defoaming agents, surfactants, and taste masking agents.

[0189] Pharmaceutical and veterinary compositions suitable for delivering the compounds of the invention and methods for their preparation will be readily apparent to those skilled in the art. For example, such compositions and methods for their preparation may be found in Remington's Pharmaceutical Sciences ( Remington'sPharmaceutical Sciences ) , 19th edition (Mack Publishing Company, 1995).

[0190] Formulations suitable for oral administration include solid, semisolid or liquid, such as: tablets; soft or hard capsules; pills; powders; lozenges (including liquid-filled); chewable tablets; multi-particulates and nanoparticles; gels; solid solutions; rapidly dispersible dosage forms; rapidly dissolving dosage forms; rapidly disintegrating dosage forms; films; ovules; sprays; buccal / mucosal patches; and liquid preparations. Liquid preparations include suspensions, solutions, elixirs and syrups. Oral administration may include swallowing, so that the compound enters the gastrointestinal tract, and / or buccal, lingual or sublingual administration, so that the compound enters the blood directly from the mouth. Liquid preparations can be used as fillers for soft or hard capsules, typically including carriers such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose or suitable oils, and one or more emulsifiers and / or suspending agents. Liquid preparations can also be prepared by reconstituting solid preparations, such as from sachets.

[0191] Oral formulations can be formulated for immediate release and / or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release. Tablet formulations are described in Pharmaceutical Dosage Forms: Tablets, Vol. 1, H Lieberman and L Lachman, Marcel Dekker, NY, 1980 (ISBN 0-8247-6918-X).

[0192] The present invention provides a pharmaceutical or veterinary composition formulated for oral administration, the composition comprising a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG or a pharmaceutically acceptable salt, solvate or hydrate thereof according to any of the preceding claims, and one or more pharmaceutically acceptable excipients. The present invention also provides the pharmaceutical or veterinary composition formulated as an oral immediate release tablet formulation or a modified release tablet formulation.

[0193] The compounds of the present invention may also be administered parenterally, or directly injected into the blood, muscle or internal organs. Suitable parenteral administration methods include intravenous injection, intraarterial injection, intraperitoneal injection, intrathecal injection, intravenous injection, intraurethral injection, intrasternal injection, intracranial injection, intramuscular injection, intrasynovial injection and subcutaneous injection. Suitable parenteral administration devices include needle-type (including microneedle) syringes, needle-free syringes and infusion techniques.

[0194] The present invention provides a drug or veterinary composition for parenteral administration, the composition comprising a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG or a pharmaceutically acceptable salt, solvate or hydrate thereof according to any of the preceding claims, and one or more pharmaceutically acceptable excipients. The present invention also provides the drug or veterinary composition, which is formulated into a normal release tablet formulation or a modified release tablet formulation for parenteral administration, suitable for intramuscular injection or intravenous administration.

[0195] The compounds of the invention may also be administered topically, intradermally or transdermally to the skin or mucous membranes. Typical formulations for this purpose include gels, hydrogels, emulsions, solutions, creams, ointments, powders, dressings, foams, films, skin patches, wafers, implants, sponges, fabrics, bandages and microemulsions. Liposomes may also be used.

[0196] The compounds of the invention may be administered rectally or vaginally, for example, in the form of suppositories, pessaries or enemas. Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate.

[0197] Pharmaceutical preparations containing the compounds of the invention can be formulated for immediate release and / or modified release. Modified release preparations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release.

[0198] dose

[0199] Generally, a physician and / or veterinarian will determine the actual dosage most suitable for an individual subject. The specific dosage level and frequency of administration for any particular individual may vary and will depend on a variety of factors, including the disease being treated, the activity of the specific compound being used, the metabolic stability and duration of action of the compound, the species, age, body weight, general health, sex, diet, route and timing of administration, rate of excretion, drug combination, severity of the specific disease, and the individual being treated.

[0200] However, in general, a suitable dosage range is from about 0.001 to about 50 mg / kg per day, in another embodiment, from about 0.001 to about 5 mg / kg per day, in another embodiment, from about 0.001 to about 0.5 mg / kg per day, in another embodiment, from about 0.001 to about 0.1 mg / kg per day. In further embodiments, the range may be from about 0.001 to about 750 mg / kg, 0.5 to 60 mg / kg, and 1 to 20 mg / kg per day.

[0201] The desired dose may conveniently be administered in a single dose or in divided doses at appropriate intervals, for example once, twice, three times, four times or more per day. If the compound is administered transdermally or in sustained release form, it may be administered once or less frequently per day.

[0202] The compound can be conveniently administered in unit dosage form; for example, each unit dosage form contains 0.1 to 50 mg of active ingredient, conveniently 0.1 to 10 mg, and most conveniently 0.1 to 5 mg. In another embodiment, the compound can be conveniently administered in unit dosage form; for example, each unit dosage form contains 10 to 1500 mg, 20 to 1000 mg, or 50 to 700 mg of active ingredient.

[0203] These doses are calculated based on an average human subject weighing approximately 65 kg to 70 kg. For subjects weighing outside this range, such as infants and the elderly, the physician can readily determine the dose.

[0204] The present invention provides a pharmaceutical composition formulated into a single-dose tablet suitable for oral administration, the composition comprising a compound of formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof, and one or more pharmaceutically acceptable excipients. The present invention further provides the pharmaceutical composition formulated for oral administration in the form of a single-dose tablet of regular release or modified release.

[0205] The present invention further provides a pharmaceutical composition formulated as a single-dose tablet, which is formulated as a single-dose tablet for oral administration of a normal release or modified release, comprising about 0.1 to about 3000 mg, preferably about 0.5 to about 1500 mg, more preferably about 1 to about 750 mg, about 1 to about 750 mg, especially about 5 to about 250 mg of a compound of formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof, and one or more pharmaceutically acceptable excipients.

[0206] For the anti-infective treatment of any infectious condition identified above, the present invention further provides a pharmaceutical composition formulated as a single-dose tablet for oral administration as a normal-release single-dose tablet formulation or as a modified-release single-dose tablet formulation, comprising 0.1 to 3000 mg, preferably about 0.5 to about 1500 mg, more preferably about 1 to about 750 mg, especially about 5 to about 250 mg of a compound of formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof, and one or more pharmaceutically acceptable excipients.

[0207] In the case of single treatment by bolus administration, for example in children, the dose may be provided by more than one tablet, such as 2 x 1500 mg, or 3 x 1000 mg, rather than a single 3000 mg tablet, in which case the tablets may be taken one after the other or together, as appropriate.

[0208] Combination therapy

[0209] In view of the fact that it may be necessary to administer a combination of active compounds as described above, for example, to treat a particular infectious disease, within the scope of the present invention, 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 as defined herein according to the present invention) may conveniently be combined in the form of a kit suitable for the combined administration of the compositions.

[0210] Therefore, the kit of the present invention comprises two or more independent pharmaceutical compositions, at least one of which contains a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG as defined in the present invention, and a device for separately storing the compositions, such as a container, a divided bottle or a divided foil bag. An example of such a kit is the familiar blister package for packaging tablets, capsules, etc.

[0211] Application Method

[0212] Those skilled in the art will appreciate that the treatment referred to herein refers to the treatment of a confirmed condition. However, the compounds of Formula I, IA, IB, IC, ID, IE, IF and / or IG and their pharmaceutically acceptable salts may also be used to prevent certain diseases (e.g., cryptosporidiosis) depending on the condition.

[0213] As used herein, unless otherwise indicated, "treating," "treatment," or "treatment" with respect to a disease means: (1) ameliorating the disease or one or more biological manifestations of a 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 biological manifestations of the disease; (3) alleviating one or more symptoms or effects associated with the disease, (4) slowing the progression of the disease or one or more biological manifestations of the disease; and / or (5) reducing the likelihood of severity of the disease or a biological manifestation of the disease.

[0214] Unless otherwise indicated, as used herein, "prevention," "preventing," or "preventing" refers to the prophylactic administration of a drug to reduce the likelihood of developing a disease or a biological manifestation thereof or to delay the onset of a disease or a biological manifestation thereof. Those skilled in the art will appreciate that "prevention" is not an absolute term. In medicine, "prevention" is understood to be the prophylactic administration of a drug to substantially reduce the likelihood or severity of a disorder or a biological manifestation thereof, or to delay the onset of such a disorder or a biological manifestation thereof.

[0215] Thus, in another embodiment, treatment or prevention of a disease is provided. In another embodiment, a method of treating a disease is provided. In another embodiment, prevention of a disease is provided.

[0216] Therefore, another aspect of the invention provides a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG, or a pharmaceutically acceptable salt thereof, for use in therapy. The invention also provides a compound of formula I, IA, IB, IC, ID, IE, IF and / or IG, or a pharmaceutically acceptable salt thereof, for use as a therapeutic agent, the therapy being human therapy or veterinary therapy.

[0217] It will be appreciated that when a compound of Formula I, IA, IB, IC, ID, IE, IF and / or IG, or a pharmaceutically acceptable salt thereof, is used in therapy, it is used as an active therapeutic agent.

[0218] For the avoidance of doubt, references herein generally to "treatment" include curative treatment, palliative treatment and preventive treatment.

[0219] Example

[0220] The present invention will now be described by the following non-limiting examples. Although what is described below is a specific embodiment of the present invention, it will be appreciated by those skilled in the art that various changes and modifications can be made to the present invention. When referring to the preparation carried out in a manner similar to other preparation methods or a general method, changes in conventional parameters may be included, such as slight changes in time, temperature, processing conditions, amount of reagents, etc.

[0221] In some of the following intermediates and examples, the starting materials are identified with reference to the numbers of other intermediates or examples. This does not mean that the actual material (or "batch") obtained from any particular intermediate or example must be used for the subsequent steps exemplified herein, but is used as a shorthand way to indicate the name of the relevant compound.

[0222] The present invention is illustrated by the following non-limiting examples, in which the following abbreviations and definitions are used:

[0223] abbreviation

[0224] δ Chemical shift

[0225] d Twin Peaks

[0226] dd double peaks

[0227] ACN Acetonitrile

[0228] BID twice daily

[0229] DCM Methyl Chloroform

[0230] DMF Dimethylformamide

[0231] ES Low-resolution electrospray mass spectrometry

[0232] Et3N Triethylamine

[0233] EtOAc Ethyl acetate

[0234] Eq equivalent

[0235] HPLC High Performance Liquid Chromatography

[0236] HRMS High Resolution Mass Spectrometry

[0237] LCMS liquid chromatography mass spectrometry

[0238] m multiplet

[0239] min

[0240] m / z mass spectrum peak

[0241] NMR Nuclear Magnetic Resonance

[0242] PO service

[0243] q quartet

[0244] rt room temperature

[0245] S Single peak

[0246] SFC Supercritical Fluid Chromatography

[0247] t Triplet

[0248] TBME tert-butyl methyl ether

[0249] THF Tetrahydrofuran

[0250] TLC Thin layer chromatography

[0251] Chemical Information: Equipment and Methods

[0252] Combifliash Companion Rf (commercially available from Teledyne ISCO) and pre-packed silica gel columns purchased from Teledyne ISCO were used for flash chromatography. Waters HPLC (2545 binary gradient pump, 515HPLC supplementary pump, 2767 sample manager) connected to Waters 2998 photodiode array and Waters 3100 mass detector was used for mass directional preparative HPLC separation. Gilson HPLC (321 pumps, 819 injection modules, 215 liquid handlers / injectors) connected to Gilson 155 UV / vis detectors were used for preparative HPLC separation. Both instruments used Waters XBridge C18 chromatographic columns (19x100 mm, 5umn particle size) for high performance liquid chromatography separation, and the mobile phases were 0.1% ammonia (solvent A) and acetonitrile (solvent B), or 0.1% formic acid aqueous solution (solvent A) and acetonitrile (solvent B). SFC chiral separation was performed on Waters SFC350. On a Bruker Avance DPX 500 spectrometer ( 1 H, 500.1MHz; 13 C, 125MHz; 19F, 470.5MHz), BrukerAvance DPX 400 (1H, 400MHz) or BrukerAvance DPX 400 (1H, 300MHz) 1 H-NMR spectra. Chemical shifts (δ) are expressed in ppm, with residual solvent as an internal reference in all cases. Signal splitting patterns are described as singlets (s), doublets (d), triplet (t), quartet (q), multiplets (m), broad peaks (br), or combinations thereof. The coupling constants (J) quoted are 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 Ultimate3000UPLC system with a diode array detector. HPLC chromatographic separation was performed using a Waters XBridge C18 column (2.1x50mm, 3.5μm particle size) or WatersXSelect (2.1x30mm, 2.5μm particle size). The compounds were eluted with a gradient of 5% to 95% acetonitrile / water + 0.1% ammonia or + 0.1% formic acid. High-resolution mass spectrometry was performed using a Bruker MicroTof mass spectrometer or a Thermo Exploris 120 Orbitrap. Reactions were not optimized unless otherwise stated. Solvents and reagents were purchased from commercial suppliers and used without further purification. Purchased dry solvents were stored in sealed bottles over molecular sieves.

[0253] synthesis

[0254] General 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)

[0255]

[0256] 2-Amino-4-methyl-6-(trifluoromethyl)pyrimidine-5-carboxylic acid ethyl ester (Intermediate 1)

[0257]

[0258] A mixture of guanidine hydrochloride (121 g, 1.27 mol) and NaOH (189 g, 4.73 mol) dissolved in MeCN (1.5 L) was placed in a sealed tube and stirred at 0°C under nitrogen for 5 hours. Ethyl 3-oxobutyrate (150 g, 1.15 mol) and 1,1,1,2,2-pentafluoro-2-iodo-ethane (312 g, 1.27 mol) were added and the reaction mixture was stirred at 15°C for 15 hours. The reaction mixture was quenched with water (3 L) and then extracted with ethyl acetate (3×2 L). The combined organics were washed with brine (1.5 L), dried over Na2SO4, filtered, and concentrated in vacuo. The product was purified by column chromatography (SiO2, 0:100→50:50 ethyl acetate: 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 (400MHz, DMSO-d6) 67.64 (br.s, 2H), 4.28 (q, J=7.2Hz, 2H), 2.35 (s, 3H), 1.27 (t, J=6.8Hz, 3H). MS (ESI): m / z (%) 250 (80) [M+H]+.

[0259] 2-Amino-4-(bromomethyl)-6-(trifluoromethyl)pyrimidine-5-carboxylic acid ethyl ester (Intermediate 2)

[0260]

[0261] To a mixture of ethyl 2-amino-4-methyl-6-(trifluoromethyl)pyrimidine-5-carboxylate (180 g, 722 mmol) dissolved in AcOH (1.6 L) was added a solution of Br2 (115 g, 722 mmol) dissolved in AcOH (200 mL) at 10°C under nitrogen, and the reaction mixture was heated at 90°C for 2 hours. The reaction mixture was concentrated, then quenched with water (1 L) and extracted with ethyl acetate (3×800 mL). The combined organics were washed with brine (1 L), dried over Na2SO4, filtered, and concentrated in vacuo. The product was purified by column chromatography (SiO2, 5:95→15:85 ethyl acetate: 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 (400MHz, DMSO-d6) δ7.95 (br.s, 2H), 4.50 (s, 2H), 4.30 (q, J=7.2Hz, 2H), 1.28 (t, J=7.2Hz, 3H). MS (ESI): m / z (%) 330 / 331 (97) [M+H] + .

[0262] 3-(Trifluoromethyl)cyclohexane-1-carboxylic acid (Intermediate 3)

[0263]

[0264] 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 vacuum, purged with H2, and stirred at 60°C with H2 (50 psi) for 48 hours. The reaction mixture was filtered, concentrated in vacuo, and azeotroped with toluene to give racemic cis-3-(trifluoromethyl)cyclohexane-1-carboxylic acid (405 g, 98% yield) as a colorless oil. 1 H NMR (400MHz, 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).

[0265] 3-(Trifluoromethyl)cyclohexane-1-carboxamide (Intermediate 4)

[0266]

[0267] To a solution of racemic cis-3-(trifluoromethyl)cyclohexanecarboxylic acid (260 g, 1.33 mol) in DCM (2.5 L) was added (COCl)2 (232 mL, 2.65 mol) at 0°C, followed by DMF (10.2 mL, 132 mmol), and the reaction mixture was stirred at 0°C for 1 hour. The reaction mixture was concentrated under vacuum, then dissolved in DCM (500 mL), and NH3·H2O (408 mL, 2.65 mol) was added at 0°C. The reaction mixture was stirred at 0°C for 1 hour, then concentrated under vacuum and azeotroped with toluene. The product was purified by column chromatography (SiO2, 30:70→50:50 ethyl acetate: petroleum ether) to give racemic cis-3-(trifluoromethyl)cyclohexanecarboxamide (200 g, 39% yield) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ7.27 (s, 1H), 6.77 (s, 1H), 2.38-2.08 (m, 2H), 1.98-1.67 (m, 4H), 1.41-1.06 (m, 4H).

[0268] 3-(Trifluoromethyl)cyclohexyl)methylamine (Intermediate 5)

[0269]

[0270] 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) dissolved in THF (600 mL) at 0 ° C was added dropwise over 30 minutes under nitrogen. The reaction mixture was heated at 70 ° C for 12 hours, then cooled to 0 ° C and quenched by adding water (32 mL) dropwise. 3.75 M NaOH (32 mL) and water (96 mL) were added, the mixture was filtered and concentrated in vacuo to give racemic cis-3-(trifluoromethyl)cyclohexyl)methylamine (150 g, 98% yield) as a yellow oil. 1 H NMR (400MHz, DMSO-d6) δ2.47-2.33 (m, 2H), 2.29-2.12 (m, 1H), 1.93 (br.d, J=12.4Hz, 1H), 1.87-1.67 (m, 4H), 1.34-1.04 (m, 4H), 0.90-0.71 (m, 2H).

[0271] 2-Amino-4-(trifluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (Compound 1)

[0272]

[0273] To a mixture of racemic cis-3-(trifluoromethyl)cyclohexyl)methylamine (80 g, 442 mmol) and Et3N (82 mL, 589 mmol) in MeCN (200 ml) was added ethyl 2-amino-4-(bromomethyl)-6-(trifluoromethyl)pyrimidine-5-carboxylate (97 g, 294 mmol) in MeCN (200 ml) at 0°C under nitrogen. The reaction mixture was stirred at 0°C for 2 hours and then heated to 40°C for 4 hours. The reaction mixture was concentrated under vacuum, then quenched with water (800 mL) and extracted with ethyl acetate (3×800 mL). The combined organics were washed with brine (700 mL), dried over Na2SO4, filtered and concentrated under vacuum. The product was purified by column chromatography (SiO2, 15:85→40:60 ethyl acetate:petroleum ether), and then the enantiomers were separated by chiral SFC (chromatographic column: Daicel Chiralpak IG (250 mm*50 mm, 10 um); mobile phase: [hexane-ethanol] B%: 15%-15%, 3.9; 6100 minutes) to give 2-amino-4-(trifluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (81 g, yield 40%) as a yellow solid.1 H NMR (400MHz, DMSO-d6) δ8.11-7.77(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=12.4Hz, 1H), 1.35-1.09 (m, 2H), 0.99-0.77 (m, 2H). 13 C NMR (125MHz, DMSO-d6) δ176.4, 163.8, 162.6, 150.8 (q, 2 J CF =37.9Hz), 128.0(q, 1 J CF =278.5Hz), 119.9(q, 1 J CF =279.5Hz), 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. HRMS (ESI) calcd. [M+H] + C 15 H 17 F6N4O, 383.1307, found 383.1305.

[0274] General 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)

[0275]

[0276] 4-Methyl-2-(methylthio)-6-vinylpyrimidine-5-carboxylic acid ethyl ester (Intermediate 6)

[0277]

[0278] 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 vinyl trifluoroborate (5.02 g, 37.5 mmol), Et3N (5.64 mL, 40.6 mmol) and Pd(dppf)Cl2 (255 mg, 0.31 mmol) and the reaction mixture was purged with nitrogen and heated at 85°C under nitrogen for 16 hours. The reaction mixture was concentrated under vacuum and then diluted with water (40 mL) and extracted with EtOAc (3×40 mL). The combined organics were washed with brine (40 mL), dried over MgSO4, filtered and concentrated under vacuum. The product was purified by column chromatography (80 g silica gel, 0:100→30:70 ethyl acetate:heptane) to give 4-methyl-2-methylmercapto-6-vinyl-pyrimidine-5-carboxylic acid ethyl ester (6.15 g, 79% yield) as a colorless oil. 1 H NMR (400MHz, CDCl3): δ6.83 (dd, J=16.8, 10.3Hz, 1H), 6.70 (dd, J=16.8, 2.1Hz, 1H), 5.67 (dd , J=10.3, 2.1Hz, 1H), 4.41 (q, J=7.1Hz, 2H), 2.59 (s, 3H), 2.49 (s, 3H), 1.39 (t, J=7.1Hz, 3H). MS(ES+): m / z(%)239(90)[M+H] + .

[0279] 4-Formyl-6-methyl-2-(methylthio)pyrimidine-5-carboxylic acid ethyl ester (Intermediate 7)

[0280]

[0281] To 4-methyl-2-methylthio-6-vinyl-5-pyrimidinecarboxylic acid ethyl ester (6.02g, 25.3mmol) solution dissolved in THF (40mL) and water (10mL), dipotassium osmate dihydrate (326mg, 0.88mmol) solution dissolved in water (10mL) is added.Reaction mixture was stirred at room temperature for 15 minutes, then dropwise added in (meta) sodium periodate (21.6g, 101mmol) suspension dissolved in water (10mL), reaction mixture was stirred at room temperature for 2 hours.Reaction mixture was diluted with water (20mL), extracted with EtOAc (3x20mL).The organic matter merged was washed with salt water (20mL), and passed through a hydrophobic sieve plate.Semen corneum (1mL) was added to quench the residual osmium, then organic matter was concentrated under vacuum. The product was purified by column chromatography (40 g silica gel, 0:100→60:40 ethyl acetate:heptane) to give 4-formyl-6-methyl-2-methylsulfanyl-pyrimidine-5-carboxylic acid ethyl ester (1.93 g, 32% yield) as a light brown oil. 1 H NMR (400MHz, CDCl3): δ9.94 (s, 1H), 4.45 (q, J=5.7Hz, 2H), 2.64 (s, 3H), 2.58 (s, 3H), 1.39 (t, J=5.7Hz, 3H). MS(ES+): m / z(%)241(94)[M+H] + .

[0282] N-Benzyl-3-(trifluoromethyl)cyclohexane-1-carboxamide (Intermediate 8)

[0283]

[0284] To a solution of racemic cis-3-(trifluoromethyl)cyclohexanecarboxylic acid (51 g, 259 mmol) in DCM (510 mL) at 10°C was added (COCl) (34 mL, 389 mmol) followed by DMF (0.2 mL, 2.6 mmol) and the reaction mixture was stirred at 10°C for 1 hour. The reaction mixture was concentrated in vacuo, 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 hours. The reaction mixture was poured into water (1.5 L) and extracted with DCM (3×300 mL). The combined organics were washed with 0.5 M HCl (200 mL), aqueous NaHCO3 (200 mL) and brine (200 mL) then dried over Na2SO4, filtered and concentrated in vacuo. The product was purified by column chromatography (SiO2, 15:85→25:75 ethyl acetate:petroleum ether) to give racemic cis-N-benzyl-3-(trifluoromethyl)cyclohexane-1-carboxamide (45 g, 58% yield) as a white solid. 1 H NMR (400MHz, CDCl3) δ7.28-7.19 (m, 5H), 5.71 (br.s, 1H), 4.37 (d, J=6.0Hz, 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] + .

[0285] (1R, 3S)-N-Benzyl-3-(trifluoromethyl)cyclohexane-1-carboxamide (Intermediate 9)

[0286]

[0287] Racemic cis-N-benzyl-3-(trifluoromethyl)cyclohexane-1-carboxamide was purified by SFC (chromatographic column: Daicel Chiralpak AY (250 mm*50 mm, 10 um); mobile phase: [0.1% NH3H2OMEOH]; B%: 55%-55%, 2.6 minutes; 360 minutes) to obtain (1R, 3S)-N-benzyl-3-(trifluoromethyl)cyclohexane-1-carboxamide (peak 2, 5.8 g, yield 48%) as a white solid. 1H NMR (400MHz, CDCl3) δ7.35-7.23 (m, 5H), 5.73 (br.s, 1H), 4.42 (d, J=5.6Hz, 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] + .

[0288] N-Benzyl-1-((1R,3S)-3-(trifluoromethyl)cyclohexyl)methanamine (Intermediate 10)

[0289]

[0290] At 10 ° C, a mixture of (1R, 3S)-N-benzyl-3-(trifluoromethyl)cyclohexane-1-carboxamide (5.8 g, 20 mmol) dissolved in THF (60 mL) was added LiAlH4 (1.5 g, 41 mmol), and the reaction mixture was heated at 75 ° C for 15 hours. 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 in vacuo. The crude product was purified by HPLC (chromatographic column: Phenomenex luna C18150*40mm*15umn; mobile phase: [water (0.1% TFA)-ACN]; B%: 17%-47%, 10 minutes) to give N-benzyl-1-((1R,3S)-3-(trifluoromethyl)cyclohexyl)methanamine (2.85 g, yield 48%) as a yellow oil. 1 H NMR (400MHz, CDCl3) δ7.30-7.17 (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] + .

[0291] ((1R,3S)-3-(Trifluoromethyl)cyclohexyl)methylamine (Intermediate 11)

[0292]

[0293] 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 vacuum, purged with H2, and stirred at 20 °C under H2 (50 psi) for 15 hours. The reaction mixture was filtered. HCl / MeOH (50 mL) was added and the filtrate was concentrated under vacuum. The product was slurried with ethyl acetate / petroleum ether (10:90), then filtered and dried in vacuo to give ((1R,3S)-3-(trifluoromethyl)cyclohexyl)methanamine (2.06 g, 90% yield) as a pale yellow solid. 1 H NMR (400MHz, DMDO-d6) δ7.99 (s, 3H), 2.77-2.61 (m, 2H), 2.32-2.27 (m, 1H), 1.96 (d, J=12.4Hz, 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).

[0294] 4-Methyl-2-(methylthio)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (Intermediate 12)

[0295]

[0296] To a solution of 4-formyl-6-methyl-2-methylmercapto-pyrimidine-5-carboxylic acid ethyl ester (1.93 g, 8.03 mmol) in DCM (40 mL) was added (1R, 3S)-(3-(trifluoromethyl)cyclohexyl]methylamine (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 minutes. Sodium triacetoxyborohydride (4.26 g, 20.1 mmol) was added portionwise and the reaction mixture was stirred for 10 minutes. The mixture was heated at 40 °C for 1 h. The reaction mixture was diluted with DCM (20 mL), washed with water (3 x 20 mL), passed through a hydrophobic frit, and concentrated in vacuo. The product was purified by column chromatography (80 g silica gel, 0:100 → 60:40 ethyl acetate:heptane) to give 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 light golden oil. 1HNMR (400MHz, CDCl3): δ4.32 (s, 2H), 3.50 (dd, J=13.9, 7.3Hz, 1H), 3.43 (dd, J=13.9, 6.9Hz, 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=6.9Hz, 2H). MS(ES+): m / z(%)360(98)[M+H] + .

[0297] 2-Amino-4-methyl-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (Compound 2)

[0298]

[0299] To a solution of 4-methyl-2-methylthio-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 potassium persulfate (5.29 g, 8.61 mmol) in water (10 mL), and the reaction mixture was stirred at room temperature for 20 hours. The reaction mixture was diluted with water (10 mL), extracted with DCM (3×10 mL), passed through a hydrophobic screen, and concentrated in vacuo. 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 hours and then concentrated in vacuo. 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) was recrystallized from ethanol as an off-white solid. 1 HNMR (400MHz, DMSO-d6): δ7.17(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=12.7Hz, 1H), 1.30-1.11(m, 2H), 0.96-0.86(m, 2H). 13C NMR (100MHz, DMSO-d6): 6173.3, 166.3, 165.3, 164.1, 127.9 (q, 1 J CF =250.4Hz), 110.8, 50.6, 47.1, 34.9, 29.3, 28.6, 24.4, 23.6, 19.6. 19 FNMR (470 MHz, DMSO-d6) δ-72.34. HRMS (ES+) calculated value C 15 H 20 ON4F3[M+H] + 329.1589, found 329.1584.

[0300] General 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)

[0301]

[0302] Ethyl 4-(difluoromethyl)-6-methyl-2-(methylthio)pyrimidine-5-carboxylate (Intermediate 13)

[0303]

[0304] A solution of 4-formyl-6-methyl-2-methylmercapto-pyrimidine-5-carboxylic acid ethyl ester (1.08 g, 4.50 mmol) in DCM (12 mL) was cooled to -15°C under nitrogen stirring. (Diethylamino)sulfur trifluoride (1.99 g, 12.4 mmol) was added slowly over 10 minutes, the reaction mixture was allowed to warm to room temperature over 2 hours, and then stirred at room temperature for 18 hours. Ice water (20 mL) and DCM (25 mL) were added, shaken, and the two-phase system was separated. The aqueous layer was extracted with DCM (3×25 mL), and the combined organics were dried (MgSO4), filtered, and concentrated in vacuo. The product was purified by column chromatography (12 g silica gel, 0:100→30:70 ethyl acetate:heptane) to give 4-(difluoromethyl)-6-methyl-2-methylmercapto-pyrimidine-5-carboxylic acid ethyl ester (870 mg, 72% yield) as a light golden viscous oil. 1 H NMR (500MHz, CDCl3) δ6.68 (t, J=54.3Hz, 1H), 4.42 (q, J=7.1Hz, 2H), 2.60 (s, 3H), 2.59 (s, 3H), 1.39 (t, J=7.2Hz, 3H). MS(ES+): m / z(%)263.1(99)[M+H] + .

[0305] (E)-4-(difluoromethyl)-6-(2-(dimethylamino)vinyl)-2-(methylthio)pyrimidine-5-carboxylic acid ethyl ester (Intermediate 14)

[0306]

[0307] To a solution of 4-(difluoromethyl)-6-methyl-2-methylmercapto-pyrimidine-5-carboxylic acid ethyl ester (770 mg, 2.94 mmol) in DMF (10 mL) was added (dimethylamino)acetaldehyde dimethyl acetal (1.95 mL, 14.7 mmol) under nitrogen followed by 1-methylimidazole (0.65 mL, 8.22 mmol) and the reaction mixture was heated at 120°C for 2 hours. The reaction mixture was cooled to room temperature and then diluted with water (10 mL) and extracted with EtOAc-TBME (5:1) (3×15 mL). The combined organics were washed with brine (15 mL), dried (MgSO4), filtered and concentrated in vacuo. The product was purified by column chromatography (12 g silica gel, 0:100→50:50 ethyl acetate:heptane) to give 4-(difluoromethyl)-6-[(E)-2-(dimethylamino)vinyl]-2-methylmercapto-pyrimidine-5-carboxylic acid ethyl ester (595 mg, yield 61%) as a pale yellow powder. 1 H NMR (500MHz, CDCl3) δ8.08 (d, J=12.3Hz, 1H), 6.56 (t, J=54.6Hz, 1H), 5.30 (d, J=12.3Hz, 1H), 4.38 (q, J=7.1Hz, 2H), 3.00 (br s, 6H), 2.55 (s, 3H), 1.38 (t, J=7.1Hz, 3H). MS(ES+): m / z(%)318.1(79)[M+H] + +m / z(%)291.0(12)[M-(N(CH3)2)+OH+H] + .

[0308] 4-(Difluoromethyl)-2-(methylthio)-6-(((1R,3S)-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (Intermediate 15)

[0309]

[0310] To a solution of sodium (meta)periodate (5.66 g, 26.5 mmol) dissolved in water (20 mL) was added dropwise a solution of 4-(difluoromethyl)-6-[(E)-2-(dimethylamino)vinyl]-2-methylmercapto-pyrimidine-5-carboxylic acid ethyl ester (2.1 g, 6.62 mmol) dissolved in THF (35 mL) at 0-5°C over 10 minutes (the exothermic temperature was kept below 10°C). The reaction mixture was stirred at room temperature for 2 hours and then diluted with DCM (30 mL). The aqueous layer was separated and extracted with DCM (2×15 mL). The combined organics were washed with brine (20 mL), dried (MgSO ), filtered, and concentrated in vacuo to 30 mL. To the solution, (1R, 3S)-3-(trifluoromethyl)cyclohexyl)methylamine (1.44 g, 7.94 mmol), acetic acid (0.05 mL) and MgSO4 (1.19 g, 9.93 mmol) were added at room temperature under nitrogen. The reaction mixture was stirred at room temperature for 10 minutes, and then sodium (triacetoxy)borohydride (4.21 g, 19.9 mmol) was added in batches. The reaction mixture was heated at 40°C for 18 hours and then cooled to room temperature. (1R, 3S)-3-(trifluoromethyl)cyclohexyl)methylamine (720 mg, 3.97 mmol) and MgSO4 (597 mg, 4.96 mmol) were further added, and the reaction mixture was stirred at room temperature for 20 minutes. Sodium (triacetoxy)borohydride (2.1 g, 9.92 mmol) was added, and the reaction mixture was heated at 40°C for 2 hours and then cooled to room temperature. The reaction mixture was diluted with DCM (20 mL), washed with water (10 mL), passed through a hydrophobic screen, dried (MgSO4), filtered, and concentrated in vacuo. The product was purified by column chromatography (40 g silica gel, 0:100→50:50 ethyl acetate: 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 minutes, and the supernatant was removed with a pipette. The sonication process 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 gel, 0:100→50:50 ethyl acetate: heptane). The purified fractions were combined with the previously purified solid and concentrated in vacuo. The resulting solid was sonicated in TBME (1.3 mL), the supernatant removed, and dried to give 4-(difluoromethyl)-2-(methylthio)-6-(((1R,3S)-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (1.19 g, 43% yield) as a yellow powder. 1H NMR (400MHz, CDCl3) δ7.19 (t, J=26.8Hz, 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), 1.100.88 (m, 2H). MS(ES+): m / z(%)396.0(86)[M+H] + .

[0311] 2-Amino-4-(difluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (Compound 3)

[0312]

[0313] 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 potassium persulfate (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 screen. The combined organics were washed with brine (20 mL), dried and concentrated in vacuo. The residue was dissolved in 1,4-dioxane (15 mL) and cooled to 0-5°C. A cooled 0.5 M ammonia solution (7.83 mL, 3.91 mmol) dissolved in 1,4-dioxane was added dropwise and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum and the product was purified by column chromatography (80 g silica gel, 0:100 → 80:20 ethyl acetate: heptane). The resulting solid was sonicated in EtOH (5 mL), allowed to stand for 30 minutes and then filtered. Repeat twice, combine the supernatants, and concentrate under vacuum to obtain a solid, which was purified by column chromatography (4 g silica gel, 0:100 → 80:20 ethyl acetate: heptane). The resulting solid was purified by preparative HPLC (20:80→95:5 acetonitrile:water, 0.1% ammonia modifier), then sonicated in MeCN (0.8 mL), two drops of methanol were added, and the mixture was 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 light pink powder. 1HNMR (500MHz, DMSO-d6) δ8.00-7.61 (m, 2H), 7.27 (t, J=53.8Hz, 1H), 4.46-4.33, (m, 2H), 3.33-3.25 (m, 2H), 2.3 2-2.16 (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 (400MHz, DMSO-d6) δ174.9, 164.6, 164.3, 156.1 (t, 1 J CF =22.8Hz), 127.9(q, 1 J CF =278.5Hz), 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. HRMS (ES+) calcd. C 15 H 18 F5N4O[M+H] + 365.1323, found 365.1402.

[0314] General 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)

[0315]

[0316] 2-Amino-6-(spiro[2.5]octan-5-ylmethyl)-4-(trifluoromethyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (Compound 4)

[0317]

[0318] To a solution of spiro[2.5]octan-7-ylmethylamine hydrochloride (375 mL, 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 minutes and then heated at 60° C. for 2 hours. The reaction mixture was concentrated in vacuo, then dissolved in DCM (10 mL), washed with water (10 mL) and saturated NH4Cl (10 mL), dried over MgSO4, filtered, and concentrated in vacuo. The product was purified by column chromatography (silica gel, 0:100→60:40 ethyl acetate: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 (500MHz, DMSO-d6) δ7.91-7.88 (m, 2H), 4.36 (d, J=4.1Hz, 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 (100MHz, DMSO-d6) δ176.3, 163.7, 162.4, 150.7 (q, 2 J CF =37.5Hz), 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. HRMS (ESI) calculated value [M+H] + C 16 H 20 F3N4O 341.1589, found 341.1583.

[0319] 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- General Synthesis of Amino-4-(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)

[0320]

[0321] 2-Amino-4-(trifluoromethyl)-6-((3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (Intermediate 16)

[0322]

[0323] To a solution of 3-(trifluoromethyl)cyclohexyl)methylamine (144 mg, 0.8 mmol) and Et3N (0.2 mL, 1.6 mmol) dissolved in MeCN (1 mL) was added 2-amino-4-(bromomethyl)-6-(trifluoromethyl)pyrimidine-5-carboxylic acid ethyl ester (130 mg, 0.4 mmol). The reaction mixture was stirred at 15 ° C for 1 hour and then heated to 80 ° C for 1 hour. The reaction mixture was concentrated under vacuum 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 (SiO2, 5:95→45:55 ethyl acetate: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. 1HNMR (400MHz, DMSO-d6) δ7.98-7.91 (m, 2H), 4.45-4.34 (m, 2H), 3.41-3.26 (m, 2H), 2.26-2.2 2 (m, 1H), 1.86-1.79 (m, 4H), 1.65 (br.d, J=11.2Hz, 1H), 1.30-1.10 (m, 2H), 0.97-0.88 (m, 2H). MS (ES+): m / z (%) 383.2 (100) [M+H]+.

[0324] 2-Amino-4-(trifluoromethyl)-6-(((1R,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (Compound 1)

[0325]

[0326] 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 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, yield 37%) as a white solid. 1H NMR (400MHz, DMSO-d6) δ8.117.77 (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=12.4Hz, 1H), 1.351.09 (m, 2H), 0.990.77 (m, 2H). 1 3 C NMR (125MHz, DMSO-d6) δ176.4, 163.8, 162.6, 150.8 (q, 2 J CF =37.9Hz), 128.0(q, 1 J CF =278.5Hz), 119.9(q, 1 J CF =279.5Hz), 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. HRMS (ESI) calculated value [M+H] + C 15 H 17 F6N4O, 383.1307, found 383.1305.

[0327] 2-Amino-4-(trifluoromethyl)-6-(((1R,3R)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (Compound 5)

[0328]

[0329] 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 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, yield 11%) as a white solid. 1H 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). HRMS (ESI) calculated value [M+H] + C 15 H 17 F6N4O, 383.1314, found 383.1339.

[0330] 2-Amino-4-(trifluoromethyl)-6-(((1S,3R)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (Compound 6)

[0331]

[0332] 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 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, yield 40%) as a white solid. 1 H NMR (500MHz, DMSO-d6) δ7.92-7.89(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=12.4Hz, 1H), 1.32-1.25 (m, 1H), 1.20-1.13 (m, 1H), 0.97-0.88 (m, 2H). 13 C NMR (101MHz, DMSO-d6) δ176.3, 163.8, 162.5, 150.8 (q, 2 J CF =37.0Hz), 127.9(q, 1 J CF =283.1Hz), 119.9(q, 1 JCF =260.9 Hz), 118.6, 109.0, 51.2, 47.5, 34.8, 29.3, 28.6, 24.3, 23.5. HRMS (ESI) calculated value [M+H] + C 15 H 17 F6N4O, 383.1267, found 383.1322.

[0333] 2-Amino-4-(trifluoromethyl)-6-(((1S,3S)-3-(trifluoromethyl)cyclohexyl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-5-one (Compound 7)

[0334]

[0335] 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 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, yield 11%) as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 7.957.88 (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). HRMS (ESI) calculated value [M+H] + C 15 H 17 F6N4O, 383.1291, found 383.1324.

[0336] Biological data and experimental results

[0337] For the avoidance of doubt, when activity data are expressed as pIC 50 When the value is expressed, the value is the same as IC 50 The pIC values ​​are different. 50 and IC 50 These are all professional terms that are easy to understand for those skilled in the art. 50Compounds with values ​​less than about 10 μM have corresponding pIC 50 The value will be greater than about 5.

[0338] Generally speaking, an IC of 1 μM 50 Value equivalent to pIC 50 The IC value is 6, 100nM 50 Value equivalent to pIC 50 The value is 7.

[0339] Protein expression and purification method of recombinant CpKRS

[0340] The protein expression and purification methods of recombinant CpKRS are described in Baragana et al. (PNAS, 2019, 116(4), 7015-7020) and will be summarized as follows.

[0341] Cryptosporidium parvum KRS has 45 residues removed at the N-terminus. All amplicons were cloned into the pAVA0421 expression vector using the ligation-independent cloning (LIC) method. The recombinant plasmid was transformed into Rosetta BL21 (DE3) competent cells and cultured on LB agar plates selected with ampicillin, carbenicillin and chloramphenicol. The cloned expression was expanded to 2 liters of culture in a shaking bottle using the previously reported autoinduction method. The cell pellet was harvested and cell lysis was performed with a cell disruptor (CellDisrupter, Constant Systems) at 30KPSI using 100mM HEPES / 150mM NaCl / 5% glycerol / 20mM imidazole / 0.5mM TCEP pH7.5 / DNase / Complete inhibitor tablets), then centrifuged at 40000g 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 a rate of 5 ml / min using an AKTA purification system. Once loaded, the column was washed with 10 column volumes of buffer A. 5% buffer B (100 mM HEPES / 150 mM NaCl / 5% glycerol 500 mM imidazole / 0.5 mM TCEP pH 7.5) was used to wash away contaminating proteins rich in histidine (His). The protein was eluted using a linear gradient of 5-50% buffer B. There were approximately 287 mg of protein in the fraction containing the CpKRS protein. The sample was then concentrated to approximately 30 mL, passed through a 0.2 μm filter, and loaded onto an XK26 / 60 Superdex 200 column at 4°C at a rate of 2 mL / min, 10 mL each time, using an AKTA purification system. The eluted proteins were pooled and frozen at -80°C. The protein concentration was 1.3 mg / ml. Sometimes, the protein was cleaved overnight with PreScission protease, in which case a second Ni column separation was performed in a flow-through mode to remove uncleaved protein. GST beads were then added to remove PreScission protease. The protein was then gel filtered as described above.

[0342] Protein expression and purification method of recombinant HsKRS

[0343] The gene encoding human KRS obtained from Genscript (codons optimized for expression in E. coli) was cloned into the His PP pET15b vector using the Nde1 and Xho1 restriction enzyme sites. The resulting vector was used to transform BL21 (DE3) competent cells to express the protein. An overnight culture of 120 mL was established and cultured for 16 hours at 37°C and 200 rpm, and the next day was used to inoculate 6 L of autoinduction medium + AMP medium. The culture was cultured at 37°C for 4 hours and then at 20°C for 20 hours. Cells were harvested by centrifugation at 3500g for 30 minutes and then stored at -20°C. The resulting cell pellet was 50 g. Lysis buffer (150 mL, 25 mM HEPES / 500 mM NaCl / 20 mM imidazole / 2 mM DTT / 10% glycerol pH 7.5 / protease inhibitor tablets / DNase) was added and the cell pellet was thawed at room temperature for about 10 minutes. The slurry was then passed through a cell disruptor (Cell Disrupter, Constant Systems) set at 30KPSI to lyse the cells. The sample was then centrifuged at 40000g for 20 minutes. The supernatant was then filtered to 0.45 μm using a syringe filter. The supernatant was loaded into a 5 ml HiTrap NiHP column equilibrated with buffer A (25 mM HEPES / 500 mM NaCl / 20 mM imidazole / 2 mM DTT / 10% glycerol pH 7.5) at a rate of 5 ml / min using an AKTA purification system. The contaminating proteins rich in histidine (His) were then washed away with 5% buffer B (25 mM HEPES / 500 mM NaCl / 500 mM imidazole / 2 mM DTT / 10% glycerol pH 7.5). The protein was eluted with a gradient of 5-50% buffer B. There was about 55 mg of protein in the fraction containing the HsKRS protein. The sample was then passed through a 0.2 μm filter and loaded onto an 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 purification system at 4°C at a rate of 2 ml / min. 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. Approximately 23 mg of protein was produced.

[0344] Test Method for Measuring In Vitro Inhibition of Cryptosporidium Lysyl-tRNA Synthetase (KRS)

[0345] The potency of the compound was determined based on a 10-point dose-response inhibition curve. The compound was transferred to a 384-well clear bottom plate (Greiner Bio-One, 781101) using an Echo 550 noise dispenser (Labcyte). Using an automatic dispenser, 25 μL of substrate mixture was added to all wells, and then 25 μL of enzyme mixture was added to the compound wells and the non-inhibition control wells to prepare a 50 μL reaction mixture (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; 0.5 U / mL pyrophosphatase). Complete inhibition control reactions were performed without enzyme and incubated at room temperature for 8 hours. Biomol green (50 μμL: Enzo LifeSciences) was added to stop the reaction, and the amount of free phosphate was detected by measuring the absorbance (650nm) after continuing to incubate for 20 minutes (BMG Pherastar plate reader). 0.5U / mL pyrophosphatase and 3μM pyrophosphate were used as substrates, and incubated at room temperature for 8 hours in the same test buffer (100mM HEPES, pH7.4, 20mM MgCl2, 1mM DTT) to perform a counter-screening test to eliminate the direct inhibitory effect of pyrophosphatase. Biomolgreen was added and incubated for 20 minutes before the above test. The samples were repeated in duplicate, and the data were processed and analyzed using ActivityBase (IDBS).

[0346] An assay for measuring the in vitro inhibition of human lysyl tRNA synthetase (KRS)

[0347] The potency of the compound was determined according to a 10-point dose-response inhibition curve. The compound was transferred to a 384-well clear bottom plate (Greiner Bio-One, 781101) using an Echo 550 noise dispenser (Labcyte). Using an automatic dispenser, 25 μL of substrate mixture was added to all wells, and then 25 μL of enzyme mixture was added to all wells to prepare a 50 μL reaction mixture (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; 0.5 U / mL pyrophosphatase). Complete inhibition control reactions were performed under the conditions of 100 μM control compound and incubated at room temperature for 5 hours. Biomol green (50 μL: Enzo Life Sciences) was added to stop the reaction, and the absorbance (650 nm) was measured after incubation for 20 minutes to detect the amount of free phosphate (BMG Pherastar plate reader). 0.5 U / mL pyrophosphatase and 3 μM pyrophosphate were used as substrates, and incubated at room temperature for 5 hours 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) to perform a counter-screening test to eliminate the direct inhibitory effect of pyrophosphatase. Biomol Green was added and incubated for 20 minutes before the above detection. Samples were performed in duplicate, and the data were processed and analyzed using ActivityBase (IDBS).

[0348] Test Method for Measuring In Vitro Inhibition of Cryptosporidium

[0349] EC for compounds of Formula I, IA, IB, IC, ID, IE, IF and / or IG 50 (The effective concentration of the compound causing 50% parasite death against Cryptosporidium) was measured in vitro. These experiments were performed according to the method of Besssoff et al. (Antimicrob. Agents Chemother. 2013, 57: 1804-1814). The results from these experiments are provided in "Experimental Results" below.

[0350] Assay for measuring the inhibitory effect of HepG2 cells in vitro

[0351] EC for compounds of Formula I, IA, IB, IC, ID, IE, IF and / or IG 50The effective concentration of the compound causing 50% parasite death on HepG2 (human Caucasian hepatocellular carcinoma, HPACC No. 85011430) was measured in vitro. These experiments were performed according to the method of Mersch-Sundermann V et al. (Toxicology 2004, 198: 329-340).

[0352] "Experimental Results" below provides the results from these experiments.

[0353] Measuring in vivo efficacy against Cryptosporidium

[0354] According to the method described in Antimicrob.Agents Ch., 2018, 62, 4, e01505-17, the potential efficacy of the compound of formula (I) in the Cryptosporidium mouse model was measured in vivo, as summarized below.

[0355] The NOD-SCIDγ mouse model of cryptosporidiosis was established as follows: Male NOD-SCIDγ mice (NOD.Cg-Prkdcsc id Iq tm1Wjl / SzJ, Jackson Labs) were infected with 10^5 Cryptosporidium parvum Iowa strain oocysts by oral gavage approximately 2 weeks after weaning. Four mice were used in each experimental group. This will result in chronic asymptomatic infection of the small intestine, cecum and biliary system. Fecal shedding of parasites was monitored by quantitative PCR amplification of Cryptosporidium parvum DNA. One week after infection (this is the time when parasite shedding was uniformly detected), the compound was administered by oral gavage. Treatment lasted for a total of seven days. The number of oocysts per milligram in the feces of each mouse was measured on day 8 (1 day after the last dose was completed) and day 15 (7 days after the last dose was completed). The average oocyst concentration of each group of mice was calculated. The log reduction value of oocyst shedding refers to the average reduction in oocyst shedding compared to the untreated vehicle.

[0356] These experiments were performed according to the method of CD Huston et al. (Antimicrob. Agents Ch., 2018, 62, 4, e01505-17). Table 3 provides the in vivo results.

[0357] Results from these experiments are provided below in "Experimental Results".

[0358] Biological Activity

[0359] Compounds of Formula I, IA, IB, IC, ID, IE, IF and / or IG have demonstrated Cryptosporidium inhibitory activity. Table 2 provides the Cryptosporidium parvum pEC values ​​of certain exemplary compounds of the invention tested in these Cryptosporidium in vitro inhibition assays. 50 data.

[0360] Compounds of Formula I, IA, IB, IC, ID, IE, IF and / or IG have been shown to be cell selective. Table 2 provides the HepG2 pEC values ​​of certain exemplary compounds of the invention tested in these HepG2 in vitro cytotoxicity assays. 50 data.

[0361] Compounds of Formula I, IA, IB, IC, ID, IE, IF and / or IG have been shown to have Cp KRS inhibitory activity. Table 2 provides the Cp KRS pIC values ​​of certain exemplary compounds of the invention tested in these Cryptosporidium parvum lysyl t-RNA synthetase (Cp KRS) inhibition assays. 50 data.

[0362] Table 2 - Biological results

[0363]

[0364]

[0365] Table 3 - In vivo efficacy, mean reduction in oocyst shedding compared to untreated vehicle

[0366]

[0367]

Claims

1. A compound of formula I: in, R 1 is selected from: -CH3, -CF2H or -CF3; and Where R 2 Selected from: or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

2. The compound of formula I according to claim 1, characterized in that R 1 It is -CH3.

3. The compound of formula I according to claim 1, characterized in that R 1 It is -CF3.

4. A compound of formula I according to any preceding claim, characterized in that R 2 for 5. A compound of formula I according to any preceding claim, characterized in that The compound of formula I is a compound of formula IA: Among them, R 1 Selected from: -CH3, -CF2H or -CF3; or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

6. The compound of formula IA according to claim 5, characterized in that The compound of formula IA is a compound of formula IB: as well as Among them, R 1 Selected from: -CH3, -CF2H or -CF3; or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

7. The compound of formula IA according to claim 5, characterized in that The compound of formula IA is a compound of formula IC: as well as Among them, R 1 Selected from: -CH3, -CF2H or -CF3; or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

8. A compound of formula I according to any preceding claim, characterized in that The compound of formula I is a compound of formula ID: Among them, R 1 Selected from: -CH3, -CF2H or -CF3; or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

9. The compound of formula ID according to claim 8, characterized in that The compound of formula ID is a compound of formula IE: as well as Among them, R 1 Selected from: -CH3, -CF2H or -CF3; or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

10. The compound of formula ID according to claim 8, characterized in that The compound of formula ID is a compound of formula IF: as well as Among them, R 1 Selected from: -CH3, -CF2H or -CF3; or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

11. The compound of formula IA according to claim 5, the compound of formula IB according to claim 6, the compound of formula IC according to claim 7, or the compound of formula ID according to claim 8, or the compound of formula IE according to claim 9, or the compound of formula IF according to claim 10, characterized in that R 1 It is -CH3.

12. The compound of formula IA according to claim 5, the compound of formula IB according to claim 6, the compound of formula IC according to claim 7, or the compound of formula ID according to claim 8, or the compound of formula IE according to claim 9, or the compound of formula IF according to claim 10, characterized in that R 1 It is -CF3.

13. A compound of formula I according to any one of claims 1 to 3, characterized in that The compound of formula I is a compound of formula IG: Among them, R 1 Selected from: -CH3, -CF2H or -CF3; or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

14. The compound of formula IG according to claim 13, characterized in that R 1 It is -CH3.

15. The compound of formula IG according to claim 13, characterized in that R 1 It is -CF3.

16. The compound of formula I according to claim 1, characterized in that The compound is selected from: or a veterinarily or pharmaceutically acceptable salt, hydrate, solvate, isomer, prodrug or polymorph thereof.

17. A compound according to any preceding claim, characterized in that The pEC of the compound to Cryptosporidium parvum 50 It is 6 or more, preferably 6.5 or more, more preferably 7 or more, especially 7.3 or 7.7 or more.

18. A compound according to any preceding claim, characterized in that The pIC of the compound on Cryptosporidium parvum lysyl t-RNA synthetase (Cp KRS) 50 It is 5 or more, preferably 5.5 or more, more preferably 6 or more.

19. A compound as claimed in any preceding claim for use in medicine or veterinary medicine.

20. A compound as claimed in any one of claims 1 to 18 for use in the treatment of an infectious disease.

21. A compound as claimed in any one of claims 1 to 18 for use in treating a disease caused by Cryptosporidium, optionally, the disease is caused by a species of Cryptosporidium selected from the group consisting of Cryptosporidium andersoni, Cryptosporidium belli, Cryptosporidium bovis, Cryptosporidium canis, Cryptosporidium chipmunk, Cryptosporidium rabbit, Cryptosporidium turtle, Cryptosporidium cati, Cryptosporidium fischeri, Cryptosporidium gallinarum, Cryptosporidium turkey, Cryptosporidium rat, Cryptosporidium marine fish, Cryptosporidium suis, Cryptosporidium sow, Cryptosporidium tyzeri, Cryptosporidium ubiquitum, Cryptosporidium traveller, Cryptosporidium fish, Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium lizard and snake, Cryptosporidium serpentis and Cryptosporidium viridis.

22. A compound as claimed in any one of claims 1 to 18 for use in the treatment of cryptosporidiosis.

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