Antimalarial drugs

Abstract

Another object of the present invention is to provide a novel antimalarial drug.SOLUTION: The present invention relates to novel derivatives in the manufacture of a medicament for preventing or treating malaria. In particular, the invention relates to dihydroisoquinoline derivatives useful for the preparation of pharmaceutical formulations for the inhibition of malaria parasite growth.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to novel antimalarial drugs. In particular, the present invention relates to agents useful in the preparation of pharmaceutical formulations for preventing or treating malaria, as well as methods for their use and manufacture. [Background technology]

[0002] Background of the Invention Malaria is caused by protozoan parasites of the genus Plasmodium that infect and destroy red blood cells, causing fever, severe anemia, cerebral malaria, and, if untreated, death. Plasmodium falciparum is the predominant species in sub-Saharan Africa, responsible for nearly 500,000 deaths annually. The disease burden is heaviest among African children under the age of five and pregnant women. Plasmodium vivax causes 25–40% of the global malaria burden, particularly in South and Southeast Asia and Central and South America. The other three major species known to infect humans are Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi.

[0003] Malaria is an endemic disease in many developing countries: approximately 40% of the world's population live in countries where malaria is endemic, and approximately 200 million people contract the disease each year.

[0004] Over the past 20 years, various chemical drugs have been developed for the treatment and prevention of malaria (Malaria medicines: a glass half full? Wells TN et al., 2015, Nature Reviews Drug Discovery 14:424-442). However, many of these medicines are expensive, and some exhibit significant toxicity and undesirable side effects in humans. Drugs used to treat malaria include artemisinin and its derivatives (e.g., artemether, artesunate, or dihydroartemisinin), chloroquine, hydroxychloroquine, quinine, quinidine, mefloquine, amodiaquine, atovaquone / proguanil, clindamycin, doxycycline, lumefantrine, piperaquine, pyronaridine, halofantrine, pyrimethamine-sulfadoxine, primaquine, quinacrine, and fulvic acid. Eroquine, tafenoquine, alterolane, spiro[3H-indole-3,1'-[1H]pyrido[3,4-b]indol]-2(1H)-one, 5,7'-dichloro-6'-fluoro-2',3',4',9'-tetrahydro-3'-methyl-(1'R,3'S)-] (cypargamine, KAE609, CAS Registry Number: 1193314-23-6), 2-(1,1-difluoroethyl)-5-methyl-N-[4-(pentafluoro-λ 6-sulfanyl)phenyl]-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (DSM265, CAS Registry Number: 1282041-94-4), morpholine, 4-[2-(4-cis-dispiro[cyclohexane-1,3'-[1,2,4]trioxolane-5',2"-tricyclo[3.3.1.13,7]decane]-4-ylphenoxy)ethyl]-](artefenomel, OZ439, CA S Registry Number: 1029939-86-3), 4-quinolinecarboxamide, 6-fluoro-2-[4-(4-morpholinylmethyl)phenyl]-N-[2-(1-pyrrolidinyl)ethyl]-(DDD107498, CAS Registry Number: 1469439-69-7), ethanone, 2-amino-1-[2-(4-fluorophenyl)-3-[(4-fluorophenyl)amino]-5,6-dihydro-8,8-dimethylimidazoline Zo[1,2-a]pyrazin-7(8H)-yl]-(ganaplacid, KAF-156, CAS Registry Number 1261113-96-5), 5-[4-(methylsulfonyl)phenyl]-6'-(trifluoromethyl)[3,3'-bipyridine]-2-amine (MMV390048, CAS Registry Number: 1314883-11-8), 4(1H)-quinolinone, 6-chloro-7-methoxy-2-methyl-3-[4-[4-(trifluoromethyl)phenyl]-

[0033] Examples of suitable fluoroisoquinoline include N-(3-cyano-4-fluorophenyl)-1,2,3,4-tetrahydro-1-oxo-3-(3-pyridinyl)-2-(2,2,2-trifluoroethyl)-, (3S,4S)-(SJ-733, CAS Registry Number 1424799-20-1).

[0005] Malaria remains a major health problem despite increasing international efforts to control and ultimately eradicate it. Currently, artemisinin-based combination therapy is the standard of care for uncomplicated malaria, demonstrating high efficacy in sub-Saharan Africa. However, its three-day administration is associated with significant issues with treatment adherence, resulting in significantly reduced efficacy in real-world settings. Furthermore, resistance to artemisinin and partner drugs is emerging. Therefore, given the widespread emergence of drug resistance in malaria parasites in endemic countries, new chemotherapy approaches continue to be needed.

[0006] For reasons of treatment compliance and ease of use, it is preferable to develop new drugs for the treatment of malaria that have a curative effect after a single dose, and this is also recommended by the Malaria Policy Advisory Committee (MPAC) of the World Health Organization (WHO) (Single dose treatment of malaria - current status and perspectives. Mischlinger J. et al., 2016, Expert Rev. Anti. Infect. Ther., 14:669-678). The Medicines for Malaria Venture (MMV) furthermore aims to develop a drug that provides a cure (i.e., a dose of approximately 10% parasitemia). 12 The authors recommend that the daily dose (a 1 / 2 reduction) should ideally be less than 100 mg for a 70 kg person (New developments in anti-malarial target candidate and product profiles. Burrows, JN et al., 2017, Malaria J, 16:26).

[0007] Phenotypic screening against Plasmodium falciparum identified a dihydroisoquinoline series of compounds that showed significant potency against the intraerythrocytic stage of malaria via inhibition of the parasite's sodium ion transporter, ATP-4'ase. Optimization of this series ultimately yielded (+)-SJ000557733 (hereafter referred to as (+)SJ-733), which advanced into clinical development for malaria. (+)SJ-733 and 58 other members of the same dihydroisoquinoline series have been claimed as antimalarial drugs in WO 2013 / 027196 (A New In Vivo Screening Paradigm to Accelerate Antimalarial Drug Discovery. Jimenez-Diaz MB et al., 2013, PLoS ONE 8(6):e66967).

[0008] In a Phase 1a clinical trial, human pharmacokinetic studies of SJ-733 showed significant formation of inactive N-oxide metabolites. Subsequent investigations in a Phase 1b malaria infection study in human volunteers showed that parasitological cure was not achieved after a single 600 milligram dose, requiring multiple dosing regimens (Gaur et al., 2020, Lancet Infect Dis., 20:30611-5). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2013 / 027196 [Non-patent literature]

[0010] [Non-Patent Document 1] Malaria medicines: a glass half full? Wells TN et al., 2015, Nature Reviews Drug Discovery 14:424-442 [Non-patent document 2] Single dose treatment of malaria -current status and perspectives. Mischlinger J. et al., 2016, Expert Rev. Anti. Infect. Idler., 14:669-678 [Non-patent document 3] New developments in anti-malarial target candidate and product profiles. Burrows, JN et al., 2017, Malaria J, 16:26 [Non-patent document 4] A New In Vivo Screening Paradigm to Accelerate Antimalarial Drug Discovery. Jimenez-Diaz MB et al., 2013, PLoS ONE 8(6):e66967 [Non-Patent Document 5] Gaur et al., 2020, Lancet Infect Dis., 20:30611-5 Summary of the Invention

[0011] SUMMARY OF THE INVENTION The present invention is directed to novel tetrahydroisoquinoline derivatives, pharmaceutical formulations, uses, and manufacture thereof that are useful in the treatment and / or prevention of malaria. These compounds have been unexpectedly found to offer several advantages over the current clinical candidate, (+)SJ-733. In particular, the novel compounds of the present invention, with similar or improved in vitro potency, have been found to exhibit significantly improved metabolic stability compared to (+)SJ-733, providing confidence that the predicted effective single dose in humans will be significantly lower (i.e., <500 mg for a 70 kg human), thus providing the added benefit of improved compliance due to a reduced pill burden.

[0012] A first aspect of the present invention provides a compound according to the present invention or a pharmaceutically acceptable salt thereof or a pharmaceutically active derivative thereof.

[0013] Another aspect of the invention relates to a compound according to the invention or a pharmaceutically acceptable salt thereof or a pharmaceutically active derivative thereof, for use as a medicine.

[0014] Another aspect of the invention relates to a compound according to the invention or a pharmaceutically acceptable salt thereof or a pharmaceutically active derivative thereof for use in the prevention and / or treatment of malaria.

[0015] Another aspect of the invention relates to the use of a compound according to the invention or a pharmaceutically acceptable salt thereof or a pharmaceutically active derivative thereof for the preparation of a pharmaceutical composition for the prevention and / or treatment of malaria.

[0016] Another aspect of the present invention resides in pharmaceutical formulations comprising at least one compound according to the invention or a pharmaceutically acceptable salt thereof or a pharmaceutically active derivative thereof, and a pharmaceutically acceptable carrier, diluent or excipient thereof.

[0017] Another aspect of the present invention resides in a method of preventing and / or treating malaria in a subject, the method comprising administering to a subject in need thereof a compound according to the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutically active derivative thereof.

[0018] Another aspect of the present invention provides methods for preparing compounds according to the present invention and intermediates thereof, or pharmaceutically acceptable salts thereof or pharmaceutically active derivatives thereof according to the present invention.

[0019] Another aspect of the present invention provides a method for preparing a compound of formula (I).

[0020] Another aspect of the present invention provides an intermediate of formula (III) or formula (IV-2) or formula (V-2) according to the present invention.

[0021] Another aspect of the present invention provides a process for preparing an intermediate of formula (III) according to the present invention.

[0022] Other features and advantages of the present invention will become apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS [Brief explanation of the drawings]

[0023] [Figure 1] Figure 1 shows the efficacy of compounds of the invention in a mouse SCID model of malaria compared to the reference compound (+)SJ-733, as described in Example 3. All compounds were administered orally at a dose of 1 mg / kg twice daily for 4 days (arrows). [Figure 2] FIG. 1 shows the pharmacokinetic profiles of compounds of the invention evaluated in a mouse SCID model of malaria compared to the reference compound (+)SJ-733, as described in Example 3. [Figure 3] FIG. 1 shows the oral pharmacokinetic profile of compound la in mouse, rat and dog species compared to the reference compound (+)SJ-733. [Figure 4] FIG. 1 shows the effect of compounds of the invention (1a) and (9a) on cytosolic [Na+] of Plasmodium falciparum strain 3D7 following treatment with the compounds compared to a positive control (cypargamine, KAE609) and a negative control (DMSO), as described in Example 7. [Figure 5] Figure 1 shows the effect of compound 1a on the transmission of Plasmodium falciparum from infected blood to A. stefensi mosquitoes, as determined by the number of oocysts in the mosquito midgut (IC50 = 323 nM), as described in Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description of the Invention The following paragraphs provide definitions of the various chemical moieties that make up the compounds according to the invention and are intended to apply uniformly throughout the specification and claims, unless a definition expressly set forth otherwise provides a broader definition.

[0025] The term "C1-C6 alkyl," when used alone or in combination with other terms, refers to a monovalent alkyl group having from 1 to 6 carbon atoms, including straight-chain or branched C1-C6 alkyl. This term is exemplified by groups such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, n-pentyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, and the like.

[0026] The term "C2-C6 alkenyl," when used alone or in combination with other terms, includes straight-chain or branched C2-C6 alkenyl. Specifically, it refers to a group having 2 to 6 carbon atoms and at least one or two sites of alkenyl unsaturation. It may have any number of double bonds at any available position, and the double bond configuration may be either (E) or (Z). This term includes groups such as vinyl, allyl, isopropenyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-ethyl-1-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, and the like. Among others, vinyl or ethenyl (-CH=CH2), n-2-propenyl (allyl, -CH2CH=CH2), isopropenyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and 3-methyl-2-butenyl are included.

[0027] The term "C2-C6 alkynyl," when used alone or in combination with other terms, includes straight-chain or branched C2-C6 alkynyl, which may have any available number of triple bonds at any available position. This term is exemplified by alkynyl groups having 2 to 6 carbon atoms and optionally having double bonds, such as ethynyl (-C≡CH), 1-propynyl, 2-propynyl (propargyl: -CH2C≡CH), 2-butynyl, and 2-penten-4-ynyl.

[0028] The term "heteroalkyl" refers to a C1-C 12 It refers to alkyl, preferably C1-C6 alkyl, where at least one carbon is replaced by a heteroatom selected from O, N or S, including 2-methoxyethyl and the like.

[0029] The term "monocyclic aryl" refers to an unsaturated aromatic carbocyclic group of 6 to 14 carbon atoms having a single ring (eg, phenyl).

[0030] The term "C1-C6 alkylaryl" refers to an aryl group having a C1-C6 alkyl substituent, including methylphenyl, ethylphenyl, and the like.

[0031] The term "aryl C1-C6 alkyl" refers to C1-C6 alkyl groups having an aryl substituent, including 3-phenylpropanyl, benzyl and the like.

[0032] The term "heteroaryl" refers to a monocyclic heteroaromatic, or a bicyclic or tricyclic fused-ring heteroaromatic group. Particular examples of heteroaromatic groups include optionally substituted pyridyl, pyrrolyl, pyrimidinyl, furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl, [2,3-dihydro]benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, and isobenzothienyl. Examples of suitable pyrido[3,4-b]pyridyl include pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl, tetrazolyl, 5,6,7,8-tetrahydroquinolyl, 5,6,7,8-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl, xanthenyl, and benzoquinolyl.

[0033] The term "5-membered heterocycle" refers to a 5-membered heteroaryl or a 5-membered heterocycloalkyl, examples of which include triazole, pyrazole, triazole, imidazole, and isoxazole.

[0034] The term "C1-C6 alkyl heteroaryl" refers to heteroaryl groups having a C1-C6 alkyl substituent, including methylfuryl and the like.

[0035] The term "heteroaryl C1-C6 alkyl" refers to C1-C6 alkyl groups having a heteroaryl substituent, including furylmethyl and the like.

[0036] The term "C2-C6 alkenyl aryl" refers to an aryl group having a C2-C6 alkenyl substituent, including vinyl phenyl and the like.

[0037] The term "aryl C2-C6 alkenyl" refers to C2-C6 alkenyl groups having an aryl substituent, including phenyl vinyl and the like.

[0038] The term "C2-C6 alkenyl heteroaryl" refers to heteroaryl groups having a C2-C6 alkenyl substituent, including vinylpyridinyl and the like.

[0039] The term "heteroaryl C2-C6 alkenyl" refers to C2-C6 alkenyl groups having a heteroaryl substituent, including pyridinyl vinyl and the like.

[0040] The term "C3-C8 cycloalkyl" refers to a saturated carbocyclic group of 3 to 8 carbon atoms having a single ring (e.g., cyclohexyl) or multiple condensed rings (e.g., norbornyl). C3-C8 cycloalkyl includes cyclopentyl, cyclohexyl, norbornyl, and the like.

[0041] The term "heterocycloalkyl" refers to a C3-C8 cycloalkyl group, as defined above, in which up to three carbon atoms are replaced by heteroatoms selected from the group consisting of O, S, NR, and R, which are defined as hydrogen or methyl. Heterocycloalkyl includes pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, tetrahydrofuranyl, and the like.

[0042] The term "C1-C6 alkyl C3-C8 cycloalkyl" refers to a C3-C8 cycloalkyl group having a C1-C6 alkyl substituent, including methylcyclopentyl and the like.

[0043] The term "C1-C8 cycloalkyl C1-C6 alkyl" refers to a C1-C6 alkyl group having a C3-C8 cycloalkyl substituent, including 3-cyclopentylpropyl and the like.

[0044] The term "C1-C6 alkyl heterocycloalkyl" refers to heterocycloalkyl groups having a C1-C6 alkyl substituent, including 4-methylpiperidinyl and the like.

[0045] The term "heteroaryl C1-C6 alkyl" refers to C1-C6 alkyl groups having a heterocycloalkyl substituent, including (1-methylpiperidin-4-yl)methyl and the like.

[0046] The term "carboxy" refers to the group -C(O)OH.

[0047] The term "carboxy C1-C6 alkyl" refers to C1-C6 alkyl groups having a carboxy substituent, including 2-carboxyethyl and the like.

[0048] The term "acyl" refers to the group -C(O)R, where R is H, "C1-C6 alkyl", "aryl", "heteroaryl", "C3-C8 cycloalkyl", "heterocycloalkyl", "aryl C1-C6 alkyl", "heteroaryl C1-C6 alkyl", "C3-C8 cycloalkyl C1-C6 alkyl", or "heterocycloalkyl C1-C6 alkyl", including acetyl and the like.

[0049] The term "acyl C1-C6 alkyl" refers to C1-C6 alkyl groups having an acyl substituent, including 2-acetylethyl and the like.

[0050] The term "acylaryl" refers to aryl groups having an acyl substituent, including 2-acetylphenyl and the like.

[0051] The term "acyloxy" refers to the group -OC(O)R where R is H, "C1-C6 alkyl", "C2-C6 alkenyl", "C2-C6 alkynyl", "C3-C8 cycloalkyl", "heterocycloalkyl", "aryl", "heteroaryl", "aryl C1-C6 alkyl", "heteroaryl C1-C6 alkyl", "aryl C2-C6 alkenyl", "heteroaryl C2-C6 alkenyl", "aryl C2-C6 alkynyl", "heteroaryl C2-C6 alkynyl", "C3-C8 cycloalkyl C1-C6 alkyl" or "heterocyclo C1-C6 alkyl", including acetyloxy and the like.

[0052] The term "acyloxy C1-C6 alkyl" refers to alkyl groups having an acyloxy substituent, including 2-(ethylcarbonyloxy)ethyl and the like.

[0053] The term "alkoxy" refers to the group -OR, where R includes optionally substituted "C1-C6 alkyl", optionally substituted "aryl", optionally substituted "heteroaryl", optionally substituted "aryl C1-C6 alkyl", or optionally substituted "heteroaryl C1-C6 alkyl".

[0054] The term "alkoxy C1-C6 alkyl" refers to C1-C6 alkyl groups having an alkoxy substituent, including methoxyethyl and the like.

[0055] The term "alkoxycarbonyl" refers to the group -C(O)OR, where R includes "C1-C6 alkyl", "aryl", "heteroaryl", "aryl C1-C6 alkyl", "heteroaryl C1-C6 alkyl" or "heteroalkyl".

[0056] "Alkoxycarbonyl C1-C6 alkyl" refers to C1-C6 alkyl groups having an alkoxycarbonyl substituent, including 2-(benzyloxycarbonyl)ethyl and the like.

[0057] The term "aminocarbonyl" refers to the group -C(O)NRR', where R and R' are independently H, C1-C6 alkyl, aryl, heteroaryl, "aryl C1-C6 alkyl" or "heteroaryl C1-C6 alkyl", including N-phenylcarbonyl and the like.

[0058] The term "aminocarbonyl C1-C6 alkyl" refers to C1-C6 alkyl groups having an aminocarbonyl substituent, including 2-(dimethylaminocarbonyl)ethyl, N-ethylacetamidyl, N,N-diethyl-acetamidyl, and the like.

[0059] The term "acylamino" refers to the group -NRC(O)R', where R and R' are independently H, "C1-C6 alkyl", "C2-C6 alkenyl", "C2-C6 alkynyl", "C3-C8 cycloalkyl", "heterocycloalkyl", "aryl", "heteroaryl", "aryl C1-C6 alkyl", "heteroaryl C1-C6 alkyl", "aryl C2-C6 alkenyl", "heteroaryl C2-C6 alkenyl", "aryl C2-C6 alkynyl", "heteroaryl C2-C6 alkynyl", "C3-C8 cycloalkyl C1-C6 alkyl", or "heterocyclo C1-C6 alkyl", including acetylamino and the like.

[0060] The term "acylamino C1-C6 alkyl" refers to C1-C6 alkyl groups having an acyloxy substituent, including 2-(propionylamino)ethyl and the like.

[0061] The term "ureido" refers to the group -NRC(O)NR'R", where R, R, and R" are independently H, "C1-C6 alkyl", "C2-C6 alkenyl", "C2-C6 alkynyl", "C3-C8 cycloalkyl", "heterocycloalkyl", "aryl", "heteroaryl", "aryl C1-C6 alkyl", "heteroaryl C1-C6 alkyl", "aryl C2-C6 alkenyl", "heteroaryl C2-C6 alkenyl", "aryl C2-C6 alkynyl", "heteroaryl C2-C6 alkynyl", "C3-C8 cycloalkyl C2-C6 alkyl", or "heterocyclo C1-C6 alkyl", and R' and R" can, together with the nitrogen atom to which they are attached, optionally form a 3- to 8-membered heterocycloalkyl ring.

[0062] The term "ureido C1-C6 alkyl" refers to C1-C6 alkyl groups having a ureido substituent, including 2-(N'-methylureido)ethyl and the like.

[0063] The term "carbamate" refers to the group -NRC(O)OR', where R and R' are independently "C1-C6 alkyl," "C2-C6 alkenyl," "C2-C6 alkynyl," "C3-C8 cycloalkyl," "heterocycloalkyl," "aryl," "heteroaryl," "C1-C6 alkylaryl," "heteroaryl C1-C6 alkyl," "aryl C2-C6 alkenyl," "heteroaryl C2-C6 alkenyl," "aryl C2-C6 alkynyl," "heteroaryl C2-C6 alkynyl," "C3-C8 cycloalkyl C1-C6 alkyl," or "heterocycloalkyl C1-C6 alkyl," and optionally R is hydrogen.

[0064] The term "amino" refers to the group -NRR', where R and R' are independently H, "C1-C6 alkyl", "aryl", "heteroaryl", "C1-C6 alkylaryl", "C1-C6 alkylheteroaryl", "C3-C8 cycloalkyl", or "heterocycloalkyl", and R and R', together with the nitrogen atom to which they are attached, can optionally form a 3- to 8-membered heterocycloalkyl ring.

[0065] The term "amino C1-C6 alkyl" refers to alkyl groups having an amino substituent, including 2-(1-pyrrolidinyl)ethyl and the like.

[0066] The term "ammonium" refers to the positively charged -N + refers to the group RR'R", where R, R', and R" are independently "C1-C6 alkyl", "C1-C6 alkylaryl", "C1-C6 alkylheteroaryl", "C3-C8 cycloalkyl", or "heterocycloalkyl", where R and R', together with the nitrogen atom to which they are attached, can optionally form a 3- to 8-membered heterocycloalkyl ring.

[0067] The term "ammonium C1-C6 alkyl" refers to an alkyl group having an ammonium substituent, including 1-ethylpyrrolidinium and the like.

[0068] The term "halogen" refers to fluorine, chlorine, bromine and iodine atoms.

[0069] The term "sulfonyloxy" refers to the group -OSO2-R, wherein R is selected from "C1-C6 alkyl", "C1-C6 alkyl" substituted with halogen, e.g., -OSO2-CF3 group, "C2-C6 alkenyl", "C2-C6C2-C6 alkynyl", "C3-C8 cycloalkyl", "heterocycloalkyl", "aryl", "heteroaryl", "aryl C1-C6 alkyl", "heteroaryl C1-C6 alkyl", "aryl C2-C6 alkenyl", "heteroaryl C2-C6 alkenyl", "aryl C2-C6 alkynyl", "heteroaryl C2-C6 alkynyl", "C3-C8 cycloalkyl C1-C6 alkyl" or "heterocycloalkyl C1-C6 alkyl".

[0070] The term "sulfamate" refers to the group -OSO2-NRR', where R and R' are independently selected from H, "C1-C6 alkyl", "C2-C6 alkenyl", "C2-C6 alkynyl", "C3-C8 cycloalkyl", "heterocycloalkyl", "aryl", "heteroaryl", "aryl C1-C6 alkyl", "heteroaryl C1-C6 alkyl", "aryl C2-C6 alkenyl", "heteroaryl C2-C6 alkenyl", "aryl C2-C6 alkynyl", "heteroaryl C2-C6 alkynyl", "C3-C8 cycloalkyl C1-C6 alkyl", or "heterocycloalkyl C1-C6 alkyl", and the like.

[0071] The term "sulfonyloxy C1-C6 alkyl" refers to alkyl groups having a sulfonyloxy substituent, including 2-(methylsulfonyloxy)ethyl and the like.

[0072] The term "sulfonyl" refers to the group "-SO2-R", where R is selected from "aryl", "heteroaryl", "C1-C6 alkyl", "C1-C6 alkyl" substituted with halogen, e.g., -SO2-CF3 group, "C2-C6 alkenyl", "C2-C6 alkynyl", "C3-C8 cycloalkyl", "heterocycloalkyl", "aryl", "heteroaryl", "aryl C1-C6 alkyl", "heteroaryl C1-C6 alkyl", "aryl C2-C6 alkenyl", "heteroaryl C2-C6 alkenyl", "aryl C2-C6 alkynyl", "heteroaryl C2-C6 alkynyl", "C3-C8 cycloalkyl C1-C6 alkyl" or "heterocycloalkyl C1-C6 alkyl".

[0073] The term "sulfonyl C1-C6 alkyl" refers to alkyl groups having a sulfonyl substituent, including 2-(methylsulfonyl)ethyl and the like.

[0074] The term "sulfinyl" refers to the group "-S(O)-R", wherein R is selected from "C1-C6 alkyl", "C1-C6 alkyl" substituted with halogen, such as -SO-CF3 group, "C2-C6 alkenyl", "C2-C6 alkynyl", "C3-C8 cycloalkyl", "heterocycloalkyl", "aryl", "heteroaryl", "aryl C1-C6 alkyl", "heteroaryl C1-C6 alkyl", "aryl C2-C6 alkenyl", "heteroaryl C2-C6 alkenyl", "aryl C2-C6 alkynyl", "heteroaryl C2-C6 alkynyl", "C3-C8 cycloalkyl C1-C6 alkyl" or "heterocycloalkyl C1-C6 alkyl".

[0075] The term "sulfinyl C1-C6 alkyl" refers to alkyl groups having a sulfinyl substituent, including 2-(methylsulfinyl)ethyl and the like.

[0076] The term "sulfanyl" refers to the -SR group, where R is H, halogen, for example, the -SF group, and includes optionally substituted "C1-C6 alkyl", in particular "C1-C6 alkyl" substituted with halogen, such as the -S-CF3 group, "C2-C6 alkenyl", "C2-C6 alkynyl", "C3-C8 cycloalkyl", "heterocycloalkyl", "aryl", "heteroaryl", "aryl C1-C6 alkyl", "heteroaryl C1-C6 alkyl", "aryl C2-C6 alkenyl", "heteroaryl C2-C6 alkenyl", "aryl C2-C6 alkynyl", "alkynylheteroaryl", "C3-C8 cycloalkyl C1-C6 alkyl" or "heterocycloalkyl C1-C6 alkyl".

[0077] The term "sulfanyl C1-C6 alkyl" refers to C1-C5 alkyl groups having a sulfanyl substituent, including 2-(ethylsulfanyl)ethyl and the like.

[0078] The term "sulfonylamino" refers to the group -NRSO2-R', where R and R" are independently H, "C1-C6 alkyl", "C2-C6 alkenyl", "C2-C6 alkynyl", "C3-C8 cycloalkyl", "heterocycloalkyl", "aryl", "heteroaryl", "aryl C1-C6 alkyl", "heteroaryl C1-C6 alkyl", "aryl C2-C6 alkenyl", "heteroaryl C2-C6 alkenyl", "aryl C2-C6 alkynyl", "heteroaryl C2-C6 alkynyl", "C3-C8 cycloalkyl C1-C6 alkyl", or "heterocycloalkyl C1-C6 alkyl".

[0079] The term "sulfonylamino C1-C6 alkyl" refers to alkyl groups having a sulfonylamino substituent, including 2-(ethylsulfonylamino)ethyl and the like.

[0080] The term "aminosulfonyl" refers to the group -SO2-NRR', where R and R' are independently H, "C1-C6 alkyl," "C2-C6 alkenyl," "C2-C6 alkynyl," "C3-C8 cycloalkyl," "heterocycloalkyl," "aryl," "heteroaryl," "aryl C1-C6 alkyl," "heteroaryl C1-C6 alkyl," "aryl C2-C6 alkenyl," "heteroaryl C2-C6 alkenyl," "aryl C2-C6 alkynyl," "heteroaryl C2-C6 alkynyl," "C3-C8 cycloalkyl C1-C6 alkyl," or "heterocycloalkyl C1-C6 alkyl," and R and R', together with the nitrogen atom to which they are attached, can optionally form a 3- to 8-membered heterocycloalkyl ring. Aminosulfonyl groups include cyclohexylaminosulfonyl, piperidinylsulfonyl, and the like.

[0081] The term "aminosulfonyl C1-C6 alkyl" refers to C1-C6 alkyl groups having an aminosulfonyl substituent, including 2-(cyclohexylaminosulfonyl)ethyl and the like.

[0082] Unless constrained by the definition of the individual substituents, the term "substituted" refers to a group substituted with 1 to 5 substituents selected from the group consisting of "C1-C6 alkyl," "C2-C6 alkenyl," "C2-C6 alkynyl," "C3-C8 cycloalkyl," "heterocycloalkyl," "C1-C6 alkylaryl," "C1-C6 alkylheteroaryl," "C1-C6 alkylC3-C8 cycloalkyl," "C1-C6 alkylheterocycloalkyl," "acyl," "amino," "amido," "aminosulfonyl," "ammonium," "acylamino," "aminocarbonyl," "aryl," "heteroaryl," "sulfinyl," "sulfonyl," "sulfonamido," "alkoxy," "alkoxycarbonyl," "carbamate," "sulfanyl," "halogen," trihalomethyl, cyano, hydroxy, mercapto, nitro, and the like.

[0083] The term "pharmaceutically acceptable salt or complex" refers to a salt or complex of a compound according to the present invention. Examples of such salts include acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.), and salts formed with organic acids, such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, fumaric acid, maleic acid, ascorbic acid, benzoic acid, tannic acid, palmoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, and polygalacturonic acid.

[0084] A "pharmaceutically active derivative" refers to any compound that can directly or indirectly provide the activity disclosed herein upon administration to a recipient. The term "indirectly" also encompasses prodrugs that can be converted to the active form of a drug through endogenous enzymes or metabolism. Prodrugs are derivatives of the compounds of the present invention, exhibiting antimalarial activity and possessing a chemically or metabolically degradable group, and can be converted in vivo to the pharmaceutically active compounds of the present invention by solvolysis under physiological conditions. Prodrugs are converted to the compounds of the present invention under physiological conditions in vivo, for example, by reaction with enzymes, gastric acid, etc., through oxidation, reduction, hydrolysis, etc., each of which is enzymatic. These compounds can be prepared from the compounds of the present invention according to well-known methods.

[0085] The term "indirectly" also encompasses metabolites of the compounds according to the invention.

[0086] The term "metabolite" refers to any molecule derived from any of the compounds according to the invention in a cell or organism, preferably a mammal.

[0087] In the context of this invention, the pharmaceutically acceptable salts, hydrates, solvates or polymorphs and pharmaceutically active derivatives of the compounds of the invention are encompassed.

[0088] The term "malaria" includes diseases and conditions associated with infection by the malaria parasite.

[0089] As used herein, "treatment" and "treating" and the like generally refer to obtaining a desired pharmacological and physiological effect. The effect may be preventative, in that a disease, its symptoms, or condition is prevented or partially prevented, and / or may be therapeutic, in that a disease, condition, symptom, or adverse effect resulting from the disease is partially or completely cured. As used herein, the term "treatment" encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it, and (b) inhibiting the disease, i.e., preventing its onset, or alleviating the disease, i.e., causing regression of the disease and / or its symptoms or condition.

[0090] The term "effective amount" includes a "prophylactically effective amount" and a "therapeutically effective amount."

[0091] The term "prophylactically effective amount" refers to a concentration of a compound of the invention that, when administered pre-infection, i.e., before, during and / or shortly after the period of exposure to the malaria parasite, is effective to suppress, reduce the likelihood of, or prevent malaria infection or prevent delayed onset of malaria parasite disease.

[0092] The term "prevention" includes causal prevention, i.e., antimalarial activity involving preventing pre-erythrocytic development of the parasite; suppressive prevention, i.e., antimalarial activity involving inhibiting the development of blood-stage infection; and terminal prevention, i.e., antimalarial activity involving inhibiting the development of intrahepatic-stage infection. This term includes primary prevention (i.e., prevention of initial infection), in which the antimalarial compound is administered before, during, and / or after the period of exposure to the malaria parasite, and terminal prevention (i.e., to prevent recurrence or delayed onset of clinical symptoms of malaria), in which the antimalarial compound is administered toward the end of the period of exposure to the malaria parasite and / or shortly after, but before clinical symptoms. Typically, suppressive prevention is used against Plasmodium falciparum infections, while terminal prevention is used against Plasmodium vivax or the combination of Plasmodium falciparum and Plasmodium vivax.

[0093] Similarly, the term "therapeutically effective amount" refers to a concentration of a compound that is effective in treating a malaria infection, e.g., that when administered after infection has occurred, results in a reduction in the number of parasites in the blood following microscopic examination.

[0094] As used herein, the term "subject" refers to a mammal. For example, mammals contemplated by the present invention include humans and the like.

[0095] compound According to one embodiment, a compound of formula (I): [ka] wherein X is selected from N and CH; when X is CH, R is selected from —CF, —CHF, —O-cyclopropyl, —O-isopropyl, —OCHF, —CN, —OCHCF, and —NH(C═O)CH; and when X is N, R is —CF. and pharmaceutically acceptable salts, hydrates, solvates, tautomers, polymorphs, racemic mixtures, optically active forms and pharmaceutically active derivatives thereof are provided.

[0096] In certain embodiments, the present invention provides compounds according to the invention wherein X is CH.

[0097] In certain embodiments, the present invention provides compounds according to the invention wherein X is N.

[0098] In another particular embodiment, the present invention provides compounds according to the invention where R is —CF 3 .

[0099] In another particular embodiment, the present invention provides compounds according to the invention where R is O-cyclopropyl.

[0100] In another particular embodiment, the present invention provides compounds according to the invention where R is —O-isopropyl.

[0101] In another particular embodiment, the present invention provides compounds according to the invention wherein R is —OCHF 2 .

[0102] In another particular embodiment, the present invention provides compounds according to the invention where R is —CN.

[0103] In another particular embodiment, the present invention provides compounds according to the invention wherein R is -OCH2CF3.

[0104] In another particular embodiment, the present invention provides compounds according to the invention where R is —NH(C═O)CH 3 .

[0105] In certain embodiments, N-(3-cyano-4-fluorophenyl)-1-oxo-2-(2,2,2-trifluoroethyl)-3-(6-(trifluoromethyl)pyridin-3-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide; N-(3-cyano-4-fluorophenyl)-3-(6-(difluoromethyl)pyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline- 4-Carboxamide;N-(3-cyano-4-fluorophenyl)-3-(6-cyclopropoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide;N-(3-cyano-4-fluorophenyl)-3-(6-isopropoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide N-(3-cyano-4-fluorophenyl)-3-(6-cyanopyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide;N-(3-cyano-4-fluorophenyl)-3-(6-cyanopyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide;N-(3-cyano-4-fluorophenyl)-3-(6-cyanopyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide N-(3-cyano-4-fluorophenyl)-1-oxo-3-(6-(2,2,2-trifluoroethoxy)pyridin-3-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide;N-(3-cyano-4-fluorophenyl)-1-oxo-3-(6-(2,2,2-trifluoroethoxy)pyridin-3-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide;Provided are compounds selected from the group consisting of 3-(6-acetamidopyridin-3-yl)-N-(3-cyano-4-fluorophenyl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide and N-(3-cyano-4-fluorophenyl)-1-oxo-2-(2,2,2-trifluoroethyl)-3-(2-(trifluoromethyl)pyrimidin-5-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide, as well as pharmaceutically acceptable salts, hydrates, solvates, tautomers, polymorphs, racemic mixtures, optically active forms and pharmaceutically active derivatives thereof;

[0106] In further particular embodiments, the compounds of the invention are (3S,4S)—N-(3-cyano-4-fluorophenyl)-1-oxo-2-(2,2,2-trifluoroethyl)-3-(6-(trifluoromethyl)pyridin-3-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide; (3S,4S)—N-(3-cyano-4-fluorophenyl)-3-(6-(difluoromethyl)pyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide; Isoquinoline-4-carboxamide;(3S,4S)-N-(3-cyano-4-fluorophenyl)-3-(6-cyclopropoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide;rac-(3S,4S)-N-(3-cyano-4-fluorophenyl)-3-(6-isopropoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline- 4-Carboxamide;(3S,4S)-N-(3-cyano-4-fluorophenyl)-3-(6-(difluoromethoxy)pyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide;(3S,4S)-N-(3-cyano-4-fluorophenyl)-3-(6-cyanopyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide;(3 S,4S)-N-(3-cyano-4-fluorophenyl)-3-(6-(difluoromethoxy)pyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide;(3S,4S)-N-(3-cyano-4-fluorophenyl)-1-oxo-3-(6-(2,2,2-trifluoroethoxy)pyridin-3-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide;Selected from the group consisting of (3S,4S)-3-(6-acetamidopyridin-3-yl)-N-(3-cyano-4-fluorophenyl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide and (3S,4S)-N-(3-cyano-4-fluorophenyl)-1-oxo-2-(2,2,2-trifluoroethyl)-3-(2-(trifluoromethyl)pyrimidin-5-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide, and pharmaceutically acceptable salts thereof;

[0107] In a further particular embodiment, diastereoisomers of compounds according to the invention are provided which are in the trans configuration.

[0108] In a further particular embodiment, there are provided enantiomers of compounds according to the invention in the 3S,4S configuration.

[0109] The compounds of the present invention are useful in the manufacture of a medicament for the prevention or treatment of malaria and are capable of killing and / or inhibiting the replication of the malaria parasite.

[0110] composition The present invention provides pharmaceutical compositions useful for the prevention or treatment of malaria. The present invention further provides methods of treating mammalian patients, most preferably human patients, suffering from malaria.

[0111] In another particular embodiment, there is provided a pharmaceutical formulation comprising at least one derivative according to the invention and a pharmaceutically acceptable carrier, diluent or excipient thereof.

[0112] In another particular embodiment, there is provided a pharmaceutical formulation comprising a compound of formula (I) and a further antimalarial agent as defined in the detailed description.

[0113] In another particular embodiment, a compound of formula (I) is used in combination with artemisinin and its derivatives, such as artemisinin and its derivatives (e.g., artemether, artesunate, or dihydroartemisinin), chloroquine, hydroxychloroquine, quinine, quinidine, mefloquine, amodiaquine, atovaquone / proguanil, clindamycin, doxycycline, lumefantrine, piperaquine, pyronaridine, halofantrine, pyrimethamine-sulfadoxine, primaquinone, thiazol-2- ... Quinacrine, quinacrine, ferroquine, tafenoquine, alterolane, spiro[3H-indole-3,1'-[1H]pyrido[3,4-b]indol]-2(1H)-one, 5,7'-dichloro-6'-fluoro-2',3',4',9'-tetrahydro-3'-methyl-,(1'R,3'S)-] (cypargamine, KAE609, CAS Registry Number: 1193314-23-6), 2-(1,1-difluoroethyl)-5-methyl-N-[4-(pentafluoro-λ 6-sulfanyl)phenyl]-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (DSM265, CAS Registry Number: 1282041-94-4), morpholine, 4-[2-(4-cis-dispiro[cyclohexane-1,3'-[1,2,4]trioxolane-5',2"-tricyclo[3.3.1.13,7]decane]-4-ylphenoxy] ethyl]-] (artefenomel, OZ439, CAS Registry Number: 1029939-86-3), 4-quinolinecarboxamide, 6-fluoro-2-[4-(4-morpholinylmethyl)phenyl]-N-[2-(1-pyrrolidinyl)ethyl]- (DDD107498, CAS Registry Number: 1469439-69-7), ethanone, 2-amino-1-[2-(4-fluromethyl)phenyl]- and at least one further antimalarial agent selected from: 5-[4-(methylsulfonyl)phenyl]-6'-(trifluoromethyl)[3,3'-bipyridine]-2-amine (MMV390048, CAS Registry Number: 1314883-11-8), 4(1H)-quinolinone, 6-chloro-7-methoxy-2-methyl-3-[4-[4-(trifluoromethoxy)phenoxy]phenyl]-(ELQ-300, CAS Registry Number: 1354745-52-0).

[0114] Pharmaceutical compositions of the present invention can contain one or more compounds of the present invention in any form described herein. Compositions of the present invention may further comprise one or more additional pharmaceutically acceptable ingredients, such as alum, stabilizers, antibacterial agents, buffers, colorants, flavoring agents, adjuvants, etc.

[0115] The compounds of the present invention, together with conventional adjuvants, carriers, diluents, or excipients, may be incorporated into the form of pharmaceutical compositions and unit dosages thereof, which may be used as solids, such as tablets or filled capsules, or liquids, such as solutions, suspensions, emulsions, elixirs, or filled capsules, all in the form of sterile injectable solutions for oral or parenteral (including subcutaneous) use. Such pharmaceutical compositions and unit dosage forms may contain the ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient consistent with the intended dosage range used. The compositions according to the present invention are preferably oral.

[0116] The compositions of the present invention may be liquid formulations, including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. Liquid forms suitable for oral administration may contain a suitable aqueous or non-aqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors, and the like. The compositions may also be formulated as a dry product for reconstitution with water or another suitable vehicle before use. Such liquid formulations may contain additives, including, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles, and preservatives. Suspending agents include, but are not limited to, sorbitol syrup, methylcellulose, glucose / sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Non-aqueous vehicles include, but are not limited to, edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol. Preservatives include, but are not limited to, methyl or propyl parahydroxybenzoate and sorbic acid. Further materials and processing techniques are described in Part 5 of Remington's "The Science and Practice of Pharmacy," 22nd Edition, 2012, University of the Sciences in Philadelphia, Lippincott Williams & Wilkins, incorporated herein by reference. The solid compositions of the present invention may be in the form of tablets or lozenges formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients, including, but not limited to, binders, fillers, lubricants, disintegrants, and wetting agents. Binders include, but are not limited to, syrup, acacia, gelatin, sorbitol, tragacanth, starch mucilage, and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, corn starch, calcium phosphate, and sorbitol.Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycolate. Wetting agents include, but are not limited to, sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.

[0117] Injectable compositions are typically based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers well known in the art.

[0118] The compositions of the present invention may also be formulated as suppositories and may contain suppository bases including, but not limited to, cocoa butter or glycerides. The compositions of the present invention may also be formulated for inhalation, which may be in a form including, but not limited to, solutions, suspensions, or emulsions that can be administered as a dry powder, or in the form of an aerosol using a propellant, such as dichlorodifluoromethane or trichlorofluoromethane. The compositions of the present invention may also be formulated into transdermal formulations containing aqueous or non-aqueous vehicles, including, but not limited to, creams, ointments, lotions, pastes, medicated plasters, patches, or membranes.

[0119] The compositions of the present invention may also be formulated for parenteral administration, including, but not limited to, by injection or continuous infusion. Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulatory agents, including, but not limited to, suspending agents, stabilizing agents, and dispersing agents. The compositions may also be provided in powder form for reconstitution with a suitable vehicle, including, but not limited to, sterile, pyrogen-free water.

[0120] The compositions of the present invention may also be formulated as a depot preparation which may be administered by implantation or by intramuscular injection. The compositions may be formulated with suitable polymeric or hydrophobic materials (such as as an emulsion in an acceptable oil), ion exchange resins, or as sparingly soluble derivatives (such as as a sparingly soluble salt).

[0121] The composition of the present invention can also be formulated as a liposome preparation.The liposome preparation can include liposomes that penetrate the target cell or stratum corneum and fuse with the cell membrane, thereby delivering the contents of the liposome to the cell.Another suitable formulation can use niosomes.Niosomes are lipid vesicles similar to liposomes, whose membranes are mainly composed of nonionic lipids, and some forms of niosomes are effective in transporting compounds across the stratum corneum.

[0122] The compounds of this invention can also be administered in sustained release forms or from sustained release drug delivery systems. A description of representative sustained release materials can also be found in the incorporated materials in Remington's Pharmaceutical Sciences.

[0123] Administration Mode The compositions of the present invention may be administered in any manner, including but not limited to, orally, parenterally, sublingually, transdermally, vaginally, rectally, transmucosally, topically, via inhalation, buccal or intranasal administration, or a combination thereof. Parenteral administration includes but is not limited to intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intrathecal, and intraarticular administration. The compositions of the present invention may also be administered in the form of an implant, which allows for sustained release of the composition and slow, controlled intravenous infusion. In a preferred embodiment, the compounds of the present invention are administered orally.

[0124] The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention in any way.

[0125] The dosage administered to an individual as a single or multiple doses will vary depending on a variety of factors, including pharmacokinetic properties, the patient's condition and characteristics (sex, age, weight, health, size), the severity of symptoms, concomitant therapy, frequency of treatment, and the desired effect.

[0126] Use in combination In accordance with the present invention, the compounds of the present invention and pharmaceutical formulations thereof can be administered alone or in combination with adjuncts useful in the treatment of malaria, including, but not limited to, agents useful in the treatment and / or prevention of malaria, artemisinin and its derivatives, such as artemether, artesunate, dihydroartemisinin, chloroquine, hydroxychloroquine, quinine, mefloquine, amodiaquine, atovaquone / proguanil, doxycycline, clindamycin, halofantrine, lumefantrine, pyronaridine, pyrimethamine-sulfadoxine, ferroquine, tafenoquine, piperaquine, and primaquine.

[0127] Additional coagents useful in combination with the compounds of the present invention include spiro[3H-indole-3,1'-[1H]pyrido[3,4-b]indol]-2(1H)-one, 5,7'-dichloro-6'-fluoro-2',3',4',9'-tetrahydro-3'-methyl-,(1'R,3'S)-] (cypargamine, CAS Registry Number: 1193314-23-6), 2-(1,1-difluoroethyl)-5-methyl-N-[4-(pentafluoro-λ 6-sulfanyl)phenyl]-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (DSM265, CAS Registry Number: 1282041-94-4), morpholine, 4-[2-(4-cis-dispiro[cyclohexane-1,3'-[1,2,4]trioxolane-5',2"-tricyclo[3.3.1.13,7]decane]-4-ylphenoxy)ethyl]-] (artefenomel, OZ439, CAS Registry Number: 1029939-86-3), 4-quinolinecarboxamide, 6-fluoro-2-[4-(4-morpholinylmethyl)phenyl]-N-[2-(1-pyrrolidinyl)ethyl]- (DDD107498, CAS Registry Number: 1469439-69-7), ethanone, 2-amino-1-[2-(4-fluorophenyl)-3-[(4-fluorophenyl) )amino]-5,6-dihydro-8,8-dimethylimidazo[1,2-a]pyrazin-7(8H)-yl]-(ganaplacid, KAF-156, CAS Registry Number 1261113-96-5), 5-[4-(methylsulfonyl)phenyl]-6'-(trifluoromethyl)[3,3'-bipyridine]-2-amine (MMV390048, CAS Registry Number: 1314883-11-8), 4(1H)-quinolinone, 6-chloro-7-methoxy-2-methyl-3- [4-[4-(trifluoromethoxy)phenoxy]phenyl]-(ELQ-300, CAS Registry Number: 1354745-52-0), 4-isoquinolinecarboxamide, N-(3-cyano-4-fluorophenyl)-1,2,3,4-tetrahydro-1-oxo-3-(3-pyridinyl)-2-(2,2,2-trifluoroethyl)-, (3S,4S)-(SJ-733, CAS Registry Number: 1424799-20-1), (R)-N2-(4-cyclopropyl and [(3R)-3-(4-fluorophenyl)-1-pyrrolidinyl][4-[(2R)-2-hydroxy-3-(2H-tetrazol-2-yl)propoxy]phenyl]-methanone (GSK701, CAS Registry Number 2366983-10-8).

[0128] The present invention encompasses the administration of a compound according to the invention or a pharmaceutical formulation thereof, wherein the compound or pharmaceutical formulation thereof is administered to an individual in an effective amount prior to, concurrently with, or sequentially with other therapeutic regimens or adjuncts (e.g., multi-drug regimens) useful in the treatment of malaria. Compounds of the invention or pharmaceutical formulations thereof administered simultaneously with such adjuncts can be administered in the same or different compositions and by the same or different routes of administration.

[0129] patient In an embodiment, a patient according to the present invention is a patient suffering from malaria.

[0130] In another embodiment, a patient according to the present invention is at high risk of infection with a malaria parasite.

[0131] In another embodiment, a patient according to the present invention is at high risk of infection with Plasmodium falciparum.

[0132] In another embodiment, a patient according to the present invention is at high risk of infection with Plasmodium vivax.

[0133] In another embodiment, a patient according to the present invention is at high risk of infection with Plasmodium ovale.

[0134] In another embodiment, a patient according to the present invention is at high risk of infection with Plasmodium vivax.

[0135] In another embodiment, a patient according to the present invention is at high risk of infection with Plasmodium vivax.

[0136] Use according to the present invention In one embodiment, the present invention provides a compound of formula (I) and its pharmaceutically acceptable salts, hydrates, solvates, or polymorphs and pharmaceutically active derivatives thereof, for the treatment or prevention of malaria.

[0137] In another embodiment, the present invention provides a method for preventing and / or treating malaria in a subject, the method comprising administering to a subject in need thereof an effective amount of a compound according to the invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutically active derivative thereof, or a pharmaceutical formulation thereof.

[0138] In another embodiment, the invention provides a use or method of a compound according to the invention, wherein the compound is administered in combination with an adjunct agent useful in the treatment of malaria.

[0139] In another embodiment, the present invention provides a pharmaceutical composition comprising a compound according to the present invention in combination with an adjunct agent useful in the treatment of malaria.

[0140] In another embodiment, the present invention provides a method for preparing a compound according to the invention, comprising converting a compound of formula (II) to a compound of formula (III) in the presence of isochroman-1,3-dione as shown in Scheme 1 below: [ka] where R and X are as defined herein.

[0141] In another embodiment, the present invention provides a method for preparing a compound according to the present invention, comprising converting a compound of formula (III) to a compound of formula (I) in the presence of an optionally substituted phenylamine, such as 3-cyano,4-fluoroaniline as exemplified above.

[0142] In another further embodiment, the present invention provides a process for preparing an intermediate aldehyde of formula (IIa) from the corresponding cyano derivative of formula (IIb), for example as illustrated in Scheme 1b below. [ka]

[0143] According to certain embodiments, the use of intermediate (IIb), such as intermediate (viii), results in improved yields, the use of cheaper reagents and easier purification of intermediate (IIa), especially intermediate (IIa-1).

[0144] In another embodiment, the present invention provides a method for preparing a compound according to the present invention, comprising separating the enantiomers of formula (III) by stereoselective salt recrystallization using 1 to 2 equivalents of a chirally pure amine, such as (1S,2S)-(+)-trans-1-amino-2-indanol. The specific enantiomer of formula (III) is then converted to the corresponding enantiomer of formula (I) in the presence of an optionally substituted phenylamine, such as 3-cyano,4-fluoroaniline. [ka]

[0145] In another further embodiment, the present invention relates to intermediates of formula (III) as defined herein, in particular the following compounds: 3-(6-cyclopropoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (III-1), 3-(6-methoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (I II-2), 1-oxo-2-(2,2,2-trifluoroethyl)-3-(6-(trifluoromethyl)pyridin-3-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (III-3), 1-oxo-3-(6-(2,2,2-trifluoroethoxy)pyridin-3-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (III-4), 3-(6-acetamidopyridine-3- 3-(6-(difluoromethoxy)pyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (III-5), 3-(6-cyanopyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (III-6), 3-(6-(difluoromethoxy)pyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (III-7), tetrahydroisoquinoline-4-carboxylic acid III-7), 3-(6-(difluoromethyl)pyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (III-8), and 1-oxo-2-(2,2,2-trifluoroethyl)-3-(2-(trifluoromethyl)pyrimidin-5-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (III-9).

[0146] In another further embodiment, the present invention provides an intermediate of formula (IV) as defined herein, in particular the following compound: N-(3-cyano-4-fluorophenyl)-3-(6-methoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (IV-2).

[0147] In another further embodiment, the present invention provides an intermediate of formula (V) as defined herein, in particular the following compound: N-(3-cyano-4-fluorophenyl)-3-(6-hydroxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (V-2).

[0148] All references cited herein are incorporated herein by reference in their entirety. The present invention should not be limited in scope by the specific embodiments described herein; each specific embodiment is intended as a single illustration of an individual aspect of the invention, and functionally equivalent methods and components are within the scope of the invention. The present invention will be illustrated below by several examples, which should not be construed as limiting the scope of the invention. [Example]

[0149] Example The following abbreviations have the following definitions:

[0150] HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), DCM (dichloromethane), DMSO (dimethyl sulfoxide), (LCMS (liquid chromatography mass spectrometry), NADPH (nicotinamide adenine dinucleotide phosphate reduced form), NEAA (non-essential amino acid), PBS (phosphate buffered saline), RPMI (Roswell Park Memorial Institute), SBFI (sodium-bound benzofuran isophthalate), TLC (thin layer chromatography).

[0151] The compounds of the present invention are named according to the IUPAC standard as used in the program ChemDraw (PerkinElmer, version 16.0.1.4).

[0152] All reagents and starting materials were obtained from Angene, Sigma-Aldrich, Enamine, Apollo Scientific or Fluorochem.

[0153] Example 1 Synthesis of compounds according to the present invention The compounds of the present invention can be prepared from readily available starting materials using methods and procedures well known to those skilled in the art. Where typical or preferred experimental conditions (i.e., reaction temperature, time, moles of reagents, solvent, etc.) are given, it should be understood that other experimental conditions can also be used unless otherwise specified. Optimal reaction conditions may vary depending on the particular reactants or solvents used, but such conditions can be determined by one skilled in the art using routine optimization procedures.

[0154] Compounds 1-9 of the present invention are synthesized as described in the general synthetic route set forth herein. In particular, compounds of formula (I) are synthesized according to Scheme 2 below. For clarity and consistency, the following nomenclature is used: compounds are represented by their compound number, and when an isolated trans single enantiomer of the (3S,4S) configuration is described, it corresponds to this number with an "a." When an isolated trans single enantiomer of the (3R,4R) configuration is described, it corresponds to this number with an "b."

[0155] Synthesis of (3S,4S)-N-(3-cyano-4-fluorophenyl)-3-(6-cyclopropoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (1a) [ka]

[0156] Methyl 6-cyclopropoxynicotinate (intermediate of formula (ii)) [ka]

[0157] To a stirred solution of cyclopropanol (1.2 mL, 19.3 mmol) in THF (10 mL) at 0 °C under nitrogen, sodium hydride (0.6 g, 25.8 mmol) was added portionwise, and the reaction mixture was stirred at the same temperature for 30 minutes. A THF solution (10 mL) of 6-fluoronicotinic acid methyl ester (1) (2 g, 12.9 mmol) (i) was added dropwise to the reaction mixture at 0 °C, and the reaction mixture was allowed to warm to room temperature under nitrogen over 2 hours. After complete consumption of the starting material (monitored by TLC and LCMS), excess NaH was quenched by adding saturated aqueous ammonium chloride solution (1 mL). The reaction mixture was then diluted with ethyl acetate (50 mL) and washed sequentially with water (10 mL) and saturated brine (10 mL). The organic layer was then dried over sodium sulfate, filtered, and the solvent evaporated under reduced pressure to give the crude compound as a viscous oil, which was then purified by column chromatography on silica gel (100-200 mesh, eluent 10-20% ethyl acetate in hexane) to give methyl 6-cyclopropoxynicotinate (ii) as a colorless oil (1.4 g, 7.25 mmol, 56%). 1 H NMR (400 MHz, CDCl3): δ 8.87 (d, J= 2.0 Hz, 1H), 8.16 (dd, J= 8.6 Hz, 2.2 Hz, 1H), 6.78 (d, J= 8.7 Hz, 1H), 4.29-4.26 (m, 1H), 3.90 (s, 3H), 0.83-0.78 (m, 4H). LCMS (NH4OAc:CH3CN): m / z: MH+ 194, R t =3.13 minutes.

[0158] (6-Cyclopropoxypyridin-3-yl)methanol (intermediate of formula (iii)) [ka]

[0159] To a stirred solution of methyl 6-cyclopropoxynicotinate (ii) (1.83 g, 9.5 mmol) in THF (50 mL) under nitrogen at 0° C., lithium aluminum hydride (1 M solution in THF) (14 mL, 14.2 mmol) was added, and the reaction mixture was stirred at 0° C. for 1 h. After complete consumption of the starting material (monitored by TLC and LCMS), saturated aqueous sodium sulfate (1 mL) was added to quench excess LiAlH. The reaction mixture was filtered over a bed of Celite, dried (sodium sulfate), filtered, and the filtrate was concentrated in vacuo to give the title compound (iii) (1.3 g, 7.9 mmol, 83%) as a colorless liquid. 1 H NMR (400 MHz, CDCl3): δ 8.19 (s, 1H), 7.63 (dd, J= 8.4 Hz, 2.0 Hz, 1H), 6.78 (d, J= 8.4 Hz, 1H), 4.63 (d, J= 5.2 Hz, 2H), 4.19-4.16 (m, 1H), 1.64 (t, J= 5.5 Hz, 1H), 0.80-0.76 (m, 4H). LCMS (Formic acid:CH3CN): m / z: MH+ 166, R t =1.33.

[0160] 6-Cyclopropoxynicotinaldehyde (intermediate of formula (IIa-1)) [ka]

[0161] To a stirred solution of (6-cyclopropoxypyridin-3-yl)methanol (III) (500 mg, 3.03 mmol) in dichloromethane (15 mL) under nitrogen at 0 °C, Dess-Martin periodinane (2.5 g, 6.06 mmol) was added, and the reaction mixture was warmed to room temperature and stirred for an additional 1 h. After complete consumption of the starting material (monitored by TLC and LCMS), the reaction mixture was filtered over a bed of Celite, the filtrate was concentrated in vacuo, and the resulting oil was purified by column chromatography on silica gel (100-200 mesh, eluent: 5% ethyl acetate in hexane) to afford the title compound (IIa-1) (400 mg, 2.45 mmol, 81%) as a colorless oil. 1 H NMR (400 MHz, CDCl3): δ 9.96 (s, 1H), 8.68 (d, J=2.08 Hz, 1H), 8.07 (dd, J= 22.4 Hz, 2.2 Hz, 1H), 6.84 (d, J= 8.6 Hz, 1H), 4.37-4.32 (m, 1H), 0.88-0.78 (m, 4H). LCMS (Formic acid:CH3CN): m / z: MH+ 164, R t =1.55 minutes.

[0162] Alternative method using MnO: A well-stirred solution of (6-cyclopropoxypyridin-3-yl)methanol (iii) (1.2 g, 7.3 mmol) in toluene (30 mL) under nitrogen at room temperature was treated in small portions with freshly activated, finely divided manganese(IV) oxide (1.89 g, 21.8 mmol, 3.0 equiv.), and the slurry was heated to 80 °C with continuous TLC monitoring. After 3 h, the mixture was allowed to cool to room temperature, filtered through Celite, and the filtrate was concentrated in vacuo to give a crude oil that was subjected to column chromatography (SiO, 10:1 hexane:ethyl acetate) to afford the title compound (IIa-1) as a white powder (1.06 g, 6.5 mmol, 89%).

[0163] An alternative method for synthesizing the intermediate compound of formula (IIa-1) is as follows:

[0164] 6-Cyclopropoxynicotinonitrile (intermediate of formula (viii)) [ka]

[0165] A solution of cyclopropanol (43.9 ml, 692 mmol) in THF (50 mL) was added slowly to a well-stirred suspension of sodium hydride (27.7 g, 692 mmol, 60% w / w in mineral oil) in THF (1.2 L) at 0° C. under nitrogen. After 10 min, 6-chloropyridine-3-carbonitrile (80.0 g, 577.4 mmol, 1.0 equiv.) was added dropwise over 5 min with continuous stirring. After consumption of the nitrile as determined by TLC and LCMS, the reaction mixture was quenched by the addition of saturated aqueous ammonium chloride (250 mL) and subsequently diluted with ethyl acetate (250 mL). The organic phase was separated, washed with brine (3 × 100 mL), dried (NaSO), filtered, and concentrated to give 6-cyclopropoxynicotinonitrile (viii) as a colorless oil (92.1 g, 575.01 mmol, 99% yield), which was used in the next step without further purification. H NMR (400 MHz, DMSO-d) δ 8.72 (d, J = 2.1 Hz, 1H), 8.18 (dd, J = 2.3, 8.8 Hz, 1H), 7.05 (d, J = 8.7 Hz, 1H), 4.35-4.26 (m, 1H), 0.85-0.78 (m, 2H), 0.78-0.70 (m, 2H). LCMS (NH4OAc:CH3CN): m / z: MH+ 161, R t =1.70 minutes.

[0166] 6-Cyclopropoxynicotinaldehyde (intermediate of formula (IIa-1)) [ka]

[0167] A solution of sodium hypophosphite monohydrate (192.1 g, 1.81 mol) in water (180 mL) was added to a stirred solution of 6-cyclopropoxynicotinonitrile (58.0 g, 362.5 mmol) (viii) in AcOH / pyridine (2:1, 540 mL) at room temperature. Raney-Ni (12.0 g) was then carefully added in small portions, and the resulting suspension was heated with stirring at 80° C. for 60 minutes. The mixture was then filtered through a bed of Celite (500 g), the Celite was washed with EtOAc (500 mL), and the combined filtrate was diluted with more EtOAc (1 L) and then washed with water (4×300 mL), saturated aqueous bicarbonate solution (100 mL), and brine (100 mL). The organic phase was dried (MgSO4) and concentrated in vacuo to give the title compound (IIa-1) (54.0 g, 330.94 mmol, 91%) as a colorless oil. 1H NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H), 8.78 (d, J = 2.0 Hz, 1H), 8.14 (dd, J = 2.0, 8.6 Hz, 1H), 7.02 (d, J = 8.6 Hz, 1H), 4.40-4.25 (m, 1H), 0.90-0.78 (m, 2H), 0.78-0.65 (m, 2H). LCMS (formic acid: CH3CN): m / z: MH+ 164, R t =1.55 minutes.

[0168] (E)-1-(6-cyclopropoxypyridin-3-yl)-N-(2,2,2-trifluoroethyl)methanimine (intermediate of formula (II-1)) [ka]

[0169] To a stirred solution of 6-cyclopropoxynicotinaldehyde (IIa-1) (1 g, 6.1 mmol) in acetonitrile (40 mL) under nitrogen at 0° C. was added 2,2,2-trifluoroethylamine (3.0 g, 24.4 mmol). The reaction was allowed to warm to room temperature and then stirred for 48 h, during which time the starting material was completely consumed ( 1(Monitored by 1 H NMR.) The reaction was concentrated in vacuo to give 1.2 g of crude oil (II-1), which was used in the next step without further purification.

[0170] cis / trans-(+,-)-3-(6-cyclopropoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (intermediate of formula (III-1)) [ka]

[0171] To a stirred solution of (E)-1-(6-cyclopropoxypyridin-3-yl)-N-(2,2,2-trifluoroethyl)methanimine (II-1) (1.2 g, 4.9 mmol) in dichloromethane (15 mL) at room temperature under nitrogen, homophthalic anhydride (0.8 g, 4.9 mmol) was added, and the reaction mixture was stirred for 16 h. The crude reaction was concentrated in vacuo to give a crude oil containing 1.85 g of 3-(6-cyclopropoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (III-1) as a (1:1) mixture of cis and trans isomers. The crude material was used in the next step without further purification. LCMS (formic acid: CHCN): m / z: MH+ 407, R t = 1.53 and 1.55 min.

[0172] (3S,4S)—N-(3-cyano-4-fluorophenyl)-3-(6-cyclopropoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (1a) (compound of formula (I) where X is CH and R is —O-cyclopropyl) [ka]

[0173] To a stirred solution of 3-(6-cyclopropoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (III-1) (1.55 g, 3.82 mmol) as a mixture of cis and trans isomers (1:1) in pyridine (30.0 mL) under nitrogen was added propylphosphonic anhydride (50% solution in ethyl acetate, 24.3 mL, 38.2 mmol). The mixture was then treated with 5-amino-2-fluorobenzonitrile (0.68 g, 4.96 mmol), and the reaction was heated at 60 °C for 16 h. The reaction was then cooled to room temperature, diluted with ethyl acetate (100 mL), washed with CuSO solution (3 × 10 mL), and the organic phase was dried (MgSO), filtered, and concentrated in vacuo. The resulting crude oil was diluted with MeOH (30 mL) and stirred with KCO (1.0 g) at room temperature for 4 h to induce complete epimerization to the trans isomer. The reaction mixture was then filtered through Celite and concentrated in vacuo, and the resulting oil was purified by column chromatography on silica gel (100-200 mesh; 3:7 ethyl acetate:hexane) to afford the title compound (1) (1.3 g, 2.48 mmol, 65%) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 10.79 (s, 1H), 8.05-7.98 (m, 3H), 7.84-7.82 (m, 1H), 7.56-7.46 (m, 4H), 7.30 (d, J = 7.0 Hz, 1H), 6.79 (d, J = 8.6 Hz, 1H), 5.39 (s, 1H), 4.66-4.58 (m, 1H), 4.24 (s, 1H), 4.11 (bs, 1H), 4.04-3.96 (m, 1H), 0.70 (s, 2H), 0.59 (s, 2H). LCMS (formic acid:CH3CN): m / z: MH+ 525, Rt = 1.68 min. The desired (3S,4S) trans enantiomer (1a) could be isolated by preparative chiral HPLC separation of the enantiomers (Chiralpak IC 20 x 250 mm column with 5 μm particle size, eluted with 85 / 15 / 0.1 hexane / EtOH / i-propylamine at ambient temperature using a flow rate of 18 mL / min). The compound was detected at a wavelength of 220 nm. [α] D 25 = +111.80° (c = 0.5, dichloromethane).

[0174] Alternatively, compound (1a) can be synthesized from an intermediate of formula (III-1) via the isolated enantiomer as follows:

[0175] a) Epimerization of cis / trans, racemic 3-(6-cyclopropoxypyridin-3-yl-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (the trans, racemic intermediate of formula III-1) [ka]

[0176] A stirred solution of a cis / trans racemic mixture of 3-(6-cyclopropoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (III-1) (230.0 g, 566.4 mmol) in glacial acetic acid (1.0 L) was heated at 100 °C for 10 h, during which time the mixture converted completely to the trans-racemate as determined by LCMS. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate (2.0 L), and washed with water (3 × 500 mL) followed by brine (3 × 100 mL). The organic layer was dried (MgSO4) and concentrated in vacuo to give a colorless gum, which was then dissolved in saturated aqueous sodium bicarbonate (250 mL) and washed with dichloromethane (3 × 100 mL). The stirred aqueous phase was then cooled to 0° C. and acidified to pH 1 by dropwise addition of concentrated hydrochloric acid, and the resulting white solid was collected by filtration and dried. Additional material was isolated from the aqueous filtrate, which was extracted with ethyl acetate (2×200 mL), and the organic phase was washed with brine (2×100 mL), dried (MgSO), filtered, and concentrated in vacuo to give an off-white solid, which was combined with the filtered solid to give the intermediate of formula (III-I′) as the trans-racemate (152.0 g, 374.1 mmol, 66%). 1H NMR (400 MHz, DMSO-d6) δ 13.12 (br s, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.89 (s, 1H), 7.54-7.41 (m, 2H), 7.33 (d, J= 6.7 Hz, 1H), 7.28 (d, J= 7.8 Hz, 1H), 6.73 (d, J = 8.6 Hz, 1H), 5.49 (s, 1H), 4.75-4.60 (m, 1H), 4.23 (s, 1H), 4.15-4.05 (m, 1H), 4.05-3.85 (m, 1H), 0.80-0.65 (m, 2H), 0.65-0.50 (m, 2H). LCMS (formic acid: CH3CN): m / z: MH+ = 407, R t = 4.3 minutes.

[0177] b) Dissolution of (+,-) trans-3-(6-cyclopropoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (intermediate of formula III-I) [ka]

[0178] To a solution of the trans racemate of the intermediate of Formula III-I' (30.0 g, 73.8 mmol) in toluene and THF (toluene:THF 4:1, 900 mL) at room temperature was added (1S,2S)-(+)-trans-1-amino-2-indanol (22.03 g, 147.6 mmol, 2.0 equiv.), and the slurry was purged with nitrogen for 10 minutes before being heated to 90°C to obtain a homogeneous solution. The solution was allowed to cool slowly to room temperature and then cooled to 5°C. After 48 h, the resulting crystals were collected by filtration, the residue was carefully washed with an ice-cold mixture of toluene and THF (toluene:THF 4:1, 2 × 30 mL), and the solid was dried to give a salt consisting of a 2:1 ratio of (1S,2S)-(+)-trans-1-amino-2-indanol:trans acid (III-I") (21.1 g, 81% yield, 95% ee relative to a single enantiomer of acid III-I). All of this material was recrystallized from toluene and THF (toluene:THF 4:1, 440 mL) to give acid III-I (18.8 g, 26.7 mmol, 72%, >99% ee [α] D 25= +82.8° (c = 0.5, dichloromethane)), affording a mixed salt of further optical enrichment. This material was then dissolved in DCM (200 mL) and hydrochloric acid (1 M, 300 mL) and added to the stirred solution at room temperature. The organic phase was separated, the aqueous phase was washed with DCM (5 × 50 mL), and the organic extracts were combined, dried (MgSO), and concentrated in vacuo to afford the (+)-dextrorotatory-(S,S)-acid of intermediate (III-I') as a single enantiomer (9.67 g, 23.8 mmol, >99% ee, 65% yield based on recovery of the single enantiomer) as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 13.08 (br s, 1H), 7.97 (d, J= 7.2 Hz, 1H), 7.89 (s, 1H), 7.54-7.41 (m, 2H), 7.33 (d, J= 8.8 Hz, 1H), 7.28 (d, J = 6.9 Hz, 1H), 6.73 (d, J= 8.6 Hz, 1H), 5.49 (s, 1H), 4.75-4.60 (m, 1H), 4.23 (s, 1H), 4.15-4.05 (m, 1H), 4.05-3.85 (m, 1H), 0.80-0.65 (m, 2H), 0.65-0.50 (m, 2H). LCMS (formic acid: CH3CN): m / z: MH+ = 407, Rt = 3.51 min. Chiral HPLC: Daicel Chiralpak IG, n-hexane / i-PrOH / TFA (70 / 30 / 0.1), flow rate = 1 mL / min, UV = 210 nm, Rt = 9.6 min (minor) and Rt = 11.8 min (major). [α] D 25 = +75.30° (c = 0.51, dichloromethane).

[0179] c) Synthesis of the enantiomer (3S,4S)—N-(3-cyano-4-fluorophenyl)-3-(6-cyclopropoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (1a) from the (+) enantiomer of trans-III-I′ (compound of formula (I) where X is CH and R is —O-cyclopropyl) [ka]

[0180] Phosphoryl chloride (2.36 mL, 25.3 mmol) was added dropwise to a well-stirred solution of (3S,4S)-3-(6-cyclopropoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (5.14 g, 12.65 mmol, >99% ee), 5-amino-2-fluorobenzonitrile (3.44 g, 25.3 mmol), DIPEA (4.0 mL, 38.0 mmol) and DMF (1.0 mL) in DCM (100 mL) at 0° C., and the reaction was then allowed to warm to room temperature. After 12 hours, the reaction was quenched by the addition of 1 M hydrochloric acid (25 mL), then washed with saturated aqueous NaHCO (2 x 50 mL), then brine (2 x 50 mL), and the organic phase was dried (MgSO), filtered, and concentrated in vacuo to give a white powder that was triturated from EtOAc, filtered, and dried to give compound (1a) as a white powder (5.17 g, 9.87 mmol, >99% ee, 78%). 1H NMR (400 MHz, DMSO-d6) δ 10.78 (s, 1H), 8.10-7.95 (m, 3H), 7.86-7.80 (m, 1H), 7.58-7.42 (m, 4H), 7.30 (d, J= 8.6 Hz, 1H), 6.80 (d, J= 8.6 Hz, 1H), 5.39 (s, 1H), 4.68-4.56 (m, 1H), 4.24 (s, 1H), 4.14-4.07 (m, 1H), 4.06-3.92 (m, 1H), 0.75-0.65 (m, 2H), 0.65-0.55 (m, 2H).LCMS (Formic acid: CH3CN): m / z: MH+ 525, Rt=1.68 min. [α] D 25 = +101.21°(c = 0.55, dichloromethane) .

[0181] Synthesis of (+ / -)-N-(3-cyano-4-fluorophenyl)-3-(6-isopropoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide)(2)(E)-1-(6-methoxypyridin-3-yl)-N-(2,2,2-trifluoroethyl)methanimine (intermediate of formula (II-2)) [ka]

[0182] To a stirred solution of 6-methoxynicotinaldehyde (IIa-2) (2.0 g, 14.6 mmol) in acetonitrile (15 mL) at 0°C under nitrogen, 2,2,2-trifluoroethanamine hydrochloride (6 g, 43.8 mmol) was added, followed by triethylamine (6.0 mL, 43.8 mmol), and the reaction mixture was stirred at room temperature for 48 h. After complete consumption of the starting material according to NMR, the reaction was concentrated in vacuo, and the residual oil was diluted with diethyl ether (50 mL), dried (MgSO), and filtered. The filtrate was concentrated in vacuo to give the title compound (II-2) as a crude oil, which was used in the next step without further purification.

[0183] Cis / trans-(+,-)-3-(6-methoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (intermediate of formula (III-2)) [ka]

[0184] A stirred solution of crude (E)-1-(6-methoxypyridin-3-yl)-N-(2,2,2-trifluoroethyl)methanimine (1 g, 6.17 mmol) (II-2) in trifluorotoluene (10 mL) under nitrogen was treated with homophthalic anhydride (1.3 g, 6.18 mmol), and the reaction mixture was heated with stirring at 130 °C for 16 h. The reaction mixture was then concentrated in vacuo to give a crude oil which was triturated with DCM / n-pentane to give the title compound (III-2) as a mixture of cis and trans isomers (1:1), which was used in the next step without further purification. LCMS (formic acid:CHCN): m / z: MH+ 376, R t = 1.53 and 1.44 min.

[0185] (+,-)-N-(3-cyano-4-fluorophenyl)-3-(6-methoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (intermediate of formula (IV-2)) [ka]

[0186] A stirred solution of crude (E)-1-(6-methoxypyridin-3-yl)-N-(2,2,2-trifluoroethyl)methanimine (II-2) (400 mg, 1.02 mmol) and 5-amino-2-fluorobenzonitrile (193 mg, 1.42 mmol) in acetonitrile (10 mL) under argon at 0 °C was treated with POCl (0.11 mL, 1.15 mmol), and the reaction mixture was heated to reflux for 2 h. The reaction mixture was concentrated in vacuo, diluted with ethyl acetate (25 mL), and treated with saturated aqueous NaHCO (10 mL). The organic phase was separated, dried (MgSO), filtered, and the filtrate was concentrated in vacuo to give a crude oil. The crude compound was diluted with MeOH (10 mL), KCO (700 mg, 5 mmol) was added, and the reaction mixture was stirred at room temperature for 4 h, during which time the compound completely epimerized to the trans isomer. The reaction mixture was filtered through a bed of Celite, and the filtrate was concentrated in vacuo to give an oil, which was subjected to column chromatography (SiO, 50–70% ethyl acetate in hexanes–5% MeOH in DCM) to give the racemic compound (IV-2) as an off-white solid (200 mg, 0.4 mmol, 40%). 1 H NMR (400 MHz, DMSO-d6): δ 10.78 (s, 1H), 8.05-8.03 (m, 1H),8.01 (d, J= 7.12 Hz, 1H), 7.91 (s, 1H), 7.85-7.81 (m, 1H), 7.56-7.44 (m, 4H), 7.3 (d, J= 8.08 Hz, 1H), 6.77 (d, J= 8.68 Hz, 1H), 5.38 (s, 1H), 4.64-4.58 (m, 1H), 4.22 (s, 1H), 4.03-3.99 (m, 1H), 3.76 (s, 3H). LCMS (formic acid:CH3CN): m / z: MH+ 499, R t =1.62 minutes.

[0187] (+,-)-N-(3-cyano-4-fluorophenyl)-3-(6-hydroxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide) (intermediate of formula (V-2)) [ka]

[0188] A solution of (rac)-N-(3-cyano-4-fluorophenyl)-3-(6-methoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (III-2) (180 mg, 0.36 mmol) in DMF (6 mL) was added and treated with LiCl (76.6 mg, 1.81 mmol) followed by paratoluenesulfonic acid (311 mg, 1.81 mmol), and the reaction mixture was heated with stirring in a microwave at 120 °C for 1 h. The cooled reaction mixture was diluted with ethyl acetate (20 mL), washed with water (10 mL), and the organic phase was dried (NaSO), filtered, and concentrated in vacuo to give a crude oil that was purified by column chromatography (SiO, eluting with 7:3 ethyl acetate:hexanes) to give the title compound (V-2) (100 mg, 0.2 mmol, 57%) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6): δ 11.47 (s, 1H), 10.75 (s, 1H), 8.03-7.98 (m, 2H), 7.83-7.80 (m, 1H), 7.56-7.48 (m, 3H), 7.35 (d, J= 7.24 Hz, 1H), 7.23 (dd, J= 9.24 Hz, 2 Hz, 1H), 7.00 (bs,1 H), 6.27 (d, J= 9.56 Hz, 1H), 5.13 (s, 1H), 4.62-4.56 (m, 1H), 4.16 (s, 1H), 3.98-3.95 (m, 1H).LCMS (Formic acid: CH3CN): m / z: MH+ 499, R t =1.76 minutes.

[0189] (+,-)-N-(3-cyano-4-fluorophenyl)-3-(6-isopropoxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (2) (compound of formula (I) where X is CH and R is O-isopropyl) [ka]

[0190] A solution of (rac)-N-(3-cyano-4-fluorophenyl)-3-(6-hydroxypyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (V-2) (100 mg, 0.2 mmol), 2-iodopropane (0.1 mL), and CsF (94 mg, 0.6 mmol) in DMF (2 mL) was stirred at room temperature for 16 h. The reaction mixture was then diluted with ethyl acetate (25 mL) and washed with water (10 mL) and brine (10 mL). The organic phase was then dried (NaSO), filtered, and concentrated in vacuo to give a crude oil, which was subjected to reverse-phase preparative HPLC chromatography to give the title compound (2) (25 mg, 0.16 mmol, 45%) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6): δ 10.79 (s, 1H), 8.04-8.00 (m, 2H), 7.92 (s, 1H), 7.84-7.82 (m, 1H), 7.56-7.46 (m, 3H), 7.40 (d, J= 9.92 Hz, 1H), 7.30 (d, 7= 7.16 Hz, 1H), 5.36 (s, 1H), 5.14-5.11 (m, 1H), 4.65- 4.59 (m, 1H), 4.22 (s, 1H), 4.00-3.94 (m, 1H), 1.22 (s, 6H). LCMS (formic acid:CH3CN): m / z:MH+ 527, R t =1.70 minutes.

[0191] Synthesis of (3S,4S)-N-(3-cyano-4-fluorophenyl)-1-oxo-2-(2,2,2-trifluoroethyl)-3-(6-(trifluoromethyl)pyridin-3-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (3a)(E)-N-(2,2,2-trifluoroethyl)-1-(6-(trifluoromethyl)pyridin-3-yl)methanimine (an intermediate of formula (II-3)) [ka]

[0192] To a stirred solution of 2-(trifluoromethyl)pyridine-5-carboxaldehyde (IIa-3) (3.0 g, 17.1 mmol) in acetonitrile (100 mL) under nitrogen at 0°C, 2,2,2-trifluoroethanamine hydrochloride (6.9 g, 51.4 mmol) was added, followed by triethylamine (7.2 mL, 51.4 mmol), and the reaction mixture was stirred at room temperature for 48 h. The reaction was then concentrated in vacuo to give 3.5 g of the title compound (II-3) as a crude oil, which was used in the next step without further purification.

[0193] 1-Oxo-2-(2,2,2-trifluoroethyl)-3-(6-(trifluoromethyl)pyridin)-3-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (intermediate for (II-3)) [ka]

[0194] A stirred solution of crude (E)-N-(2,2,2-trifluoroethyl)-1-(6-(trifluoromethyl)pyridin-3-yl)methanimine (II-3) (3.0 g, 11.7 mmol) and homophthalic anhydride (1.9 g, 11.7 mmol) in trifluorotoluene (15 mL) under nitrogen was heated at 130° C. for 16 h. The reaction was then concentrated in vacuo to give 5.0 g of crude title compound (III-3) as a ca. 1:1 mixture of cis and trans isomers, which was used in the next step without further purification.

[0195] (3S,4S)—N-(3-cyano-4-fluorophenyl)-1-oxo-2-(2,2,2-trifluoroethyl)-3-(6-trifluoromethyl)pyridin-3-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (3a) (compound of formula (I) where X is CH and R is CF)

[0196] To a stirred solution of (rac)-1-oxo-2-(2,2,2-trifluoroethyl)-3-(6-(trifluoromethyl)pyridin-3-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (III-3) (2.0 g, 4.78 mmol) in pyridine (30 mL) at room temperature under nitrogen was added propylphosphonic anhydride (50% solution in ethyl acetate, 28.0 mL, 47.8 mmol), followed by 5-amino-2-fluorobenzonitrile (0.7 g, 5.23 mmol), and the reaction was heated at 50° C. for 16 hours. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate (50 mL), washed with CuSO solution (3×10 mL), dried (MgSO), filtered, and the filtrate was concentrated in vacuo. [ka]

[0197] The resulting oil was diluted with MeOH (20 mL) and stirred with KCO (3.3 g, 24.0 mmol) for 4 h, during which time only the trans epimer formed according to NMR. The suspension was then filtered through a bed of Celite, concentrated in vacuo, and the resulting oil was chromatographed (SiO, 3:7 ethyl acetate:hexane) to give the title compound (3) as a racemate (1.0 g, 1.86 mmol, 39%). The (+)-enantiomer was obtained by preparative chiral HPLC (column: Chiralpak IA, 21 × 250 mm, 5 μm particle size, operated at ambient temperature and a flow rate of 21 ml / min. Wavelength: 222 nm, run time 20 min. Mobile phase: hexane / dichloromethane / EtOH: 70 / 15 / 15) followed by lyophilization to give 200 mg of the title compound (3S,4S)—N-(3-cyano-4-fluorophenyl)-1-oxo-2-(2,2,2-trifluoroethyl)-3-(6-(trifluoromethyl)pyridin-3-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (3a) in >99% ee. 1 H NMR (400 MHz, DMSO-d6): δ 10.88 (s, 1H), 8.72 (s, 1H), 8.06-8.00 (m, 2H), 7.87-7.82 (m, 2H), 7.74 (d, J = 7.12 Hz, 1H), 7.57-7.47 (m, 3H), 7.29 (d, J = 6.28 Hz, 1H), 5.64 (s, 1H), 4.66-4.58 (m, 1H), 4.35 (s, 1H), 4.20-4.09 (m, 1H). LCMS (formic acid:CH3CN): m / z: MH+ 537, Rt=1.68. [α] D 25 = +59.07°(c= 0.536, chloroform).

[0198] (3S,4S)-N-(3-cyano-4-fluorophenyl)-1-oxo-3-(6-(2,2,2-trifluoroethoxy)pyridin-3-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (4a) Synthesis of (E)-1-(6-(2,2,2-trifluoroethoxy)pyridin-3-yl)-N-(2,2,2-trifluoroethyl)methanimine (intermediate of formula (II-4)) [ka]

[0199] To a stirred solution of 2-(trifluoromethyl)pyrimidine-5-carbaldehyde (IIa-4) (2.0 g, 9.8 mmol) in acetonitrile (50 mL) under nitrogen at 0° C., 2,2,2-trifluoroethylamine (6.5 g, 39.2 mmol) was added, and the reaction mixture was stirred at room temperature for 48 h. The reaction mixture was then concentrated in vacuo to give 2.7 g of crude title compound (II-4) as a colorless oil, which was used in the next step without further purification.

[0200] 1-Oxo-3-(6-(2,2,2-trifluoroethoxy)pyridin-3-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (intermediate of formula (III-4)) [ka]

[0201] To a stirred solution of crude (E)-1-(6-(2,2,2-trifluoroethoxy)pyridin-3-yl)-N-(2,2,2-trifluoroethyl)methanimine (II-4) (1.8 g, 6.28 mmol) in dichloromethane (25 mL) under nitrogen, homophthalic anhydride (1.12 g, 6.9 mmol) was added, and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was then concentrated in vacuo to afford 2.0 g of crude title compound (III-4) as a colorless oil, as a 1:1 mixture of cis and trans isomers, which was used in the next step without further purification. LCMS (formic acid:CHCN): m / z: MH+ 449, R t =1.83 minutes.

[0202] (3S,4S)—N-(3-cyano-4-fluorophenyl)-1-oxo-3-(6-(2,2,2-trifluoroethoxy)pyridin-3-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (4a) (compound of formula (I) where X is CH and R is OCHCF) [ka]

[0203] To a stirred solution of 1-oxo-3-(6-(2,2,2-trifluoroethoxy)pyridin-3-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (III-4) (1 g, 2.23 mmol) in pyridine (10.0 mL) at room temperature was added propylphosphonic anhydride (50% solution in ethyl acetate, 13 mL, 22.3 mmol). 5-Amino-2-fluorobenzonitrile (0.42 g, 3.12 mmol) was then added to the mixture, and the reaction mixture was heated at 50° C. with continued stirring for 16 hours. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate (50 mL), washed with CuSO solution (3×10 mL), dried (MgSO), filtered, and the filtrate was concentrated in vacuo. The resulting oil was diluted with MeOH (20 mL) and stirred with KCO (1.5 g, 10.9 mmol) for 4 h, during which time only the trans-epimer formed according to NMR. The suspension was then filtered through a bed of Celite and concentrated in vacuo. The resulting oil was subjected to chromatography (SiO, 3:7 ethyl acetate:hexane) to give the title compound (4) as a racemate (700 mg, 1.33 mmol, 55%). The (+)-enantiomer was obtained by preparative chiral HPLC (column: Chiralpak IA, 21 × 250 mm, 5 μm particle size, operated at ambient temperature and a flow rate of 21 mL / min. Wavelength: 222 nm, run time: 20 min. Mobile phase: hexane / dichloromethane / EtOH: 70 / 15 / 15) followed by lyophilization to give 250 mg of the title compound (4a) in >99% ee. 1H NMR (400 MHz, DMSO-d6): δ 10.81 (s, 1H), 8.06-7.94 (m, 3H), 7.85-7.80 (m, 1H), 7.60-7.44 (m, 4H), 7.30 (d, J = 6.72 Hz, 1H),6.95 (d, J = 8.6 Hz, 1H), 5.43 (s, 1H), 4.94-4.87 (m, 2H), 4.63-4.57 (m,1H), 4.23 (s, 1H), 4.09-4.03 (m, 1H). LCMS (Formic acid:CH3CN): m / z: MH+ 567, Rt=1.75 minutes. [α]D 25 = +69.12°(c= 0.37, dichloromethane) .

[0204] Synthesis of (3S,4S)-3-(6-acetamidopyridin-3-yl)-N-(3-cyano-4-fluorophenyl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (5a)(E)-N-(5-(((2,2,2-trifluoroethyl)imino)methyl)pyridin-2-yl)acetamide (intermediate of formula (II-5)) [ka]

[0205] To a stirred solution of N-(5-formylpyridin-2-yl)acetamide (IIa-5) (50 mg, 0.3 mmol) in acetonitrile (2 mL) under nitrogen at 0°C, 2,2,2-trifluoroethanamine hydrochloride (123 mg, 0.9 mmol) was added, followed by triethylamine (127 μL, 0.9 mmol), and the reaction mixture was stirred at room temperature for 48 h. The reaction was then concentrated in vacuo, and the resulting slurry was diluted with diethyl ether (20 mL), filtered, and then concentrated in vacuo to afford the title compound (II-5) as a crude colorless oil, which was used in the next step without further purification.

[0206] 3-(6-acetamidopyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (intermediate of formula (III-5)) [ka]

[0207] To a stirred solution of crude (E)-N-(5-(((2,2,2-trifluoroethyl)imino)methyl)pyridin-2-yl)acetamide (II-5) (50 mg, 0.2 mmol) in trifluorotoluene (5 mL) under nitrogen, homophthalic anhydride (33 mg, 0.2 mmol) was added and the reaction mixture was heated to 100 °C for 16 h. The reaction mixture was then concentrated in vacuo to give a slurry, which was triturated with n-pentane / DCM and filtered to give the title compound (III-5) as a ∼1:1 mixture of cis and trans isomers, which was used in the next step without further purification.

[0208] (3S,4S)-3-(6-acetamidopyridin-3-yl)-N-(3-cyano-4-fluorophenyl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (5a) (compound of formula (I) where X is CH and R is NHC(=O)CH) [ka]

[0209] To a stirred solution of 3-(6-acetamidopyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (95 mg, 0.22 mmol) in pyridine (5 mL) under nitrogen, propylphosphonic anhydride (0.7 mL, 2.2 mmol) was added, followed by 5-amino-2-fluorobenzonitrile (48 mL, 0.35 mmol), and the reaction mixture was heated to 50 °C for 16 h. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate (50 mL), washed with CuSO solution (3 × 10 mL), dried (MgSO), filtered, and the filtrate concentrated in vacuo. The resulting oil was diluted with MeOH (5 mL) and stirred with KCO (152 mg, 1.1 mmol) for 4 h, during which only the trans epimer formed according to NMR. The suspension was then filtered through a bed of Celite, concentrated in vacuo, and the resulting oil was chromatographed (SiO, 3:7 ethyl acetate:hexane) to give the title compound (5) as a racemate (20 mg, 0.04 mmol, 12%). The (+)-enantiomer was obtained by preparative chiral HPLC (column: Chiralpak IA, 21 × 250 mm, 5 μm particle size, operated at ambient temperature and a flow rate of 21 ml / min. Wavelength: 222 nm, run time 20 min. Mobile phase: hexane / dichloromethane / EtOH: 70 / 15 / 15), followed by lyophilization to give 8 mg of the title compound (5a) as a pale yellow solid (>99% ee). 1 H NMR (400 MHz, DMSO-d6): δ 10.80 (s, 1H), 10.46 (s, 1H), 8.10 (s, 1H), 8.06-8.04 (m, 1H),8.01 (d, J = 8.28 Hz, 1H), 7.92 (d, J = 8.72 Hz, 1H), 7.85-7.81 (m, 1H), 7.56-7.44 (m, 4H), 7.28 (d, J = 6.68 Hz, 1H), 5.41 (s, 1H), 4.65-20 4.56 (m, 1H), 4.26 (s, 1H), 4.07-4.01 (m, 1H), 2.03 (s, 3H). LCMS (formic acid: CH3CN): m / z: MH+ 526, Rt=1.55 min. [α]D 25= +137.5°(c= 0.28, EtOAc).

[0210] Synthesis of (3S,4S)-N-(3-cyano-4-fluorophenyl)-3-(6-cyanopyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (6a)(E)-5-(((2,2,2-trifluoroethyl)imino)methyl)picolinonitrile (an intermediate of formula (II-6)) [ka]

[0211] To a stirred solution of 5-formyl-2-pyridinecarbonitrile (IIa-6) (200 mg, 1.5 mmol) in methanol (6 mL) under nitrogen at 0°C, 2,2,2-trifluoroethanamine hydrochloride (818 mg, 6 mmol) was added, followed by triethylamine (1.26 mL, 9 mmol), and the reaction mixture was stirred at room temperature for 48 h. The reaction was then concentrated in vacuo, and the resulting slurry was diluted with diethyl ether (20 mL), filtered, and then concentrated in vacuo to afford the title compound (II-6) as a crude colorless oil, which was used in the next step without further purification.

[0212] 3-(6-cyanopyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (intermediate of formula (III-6)) [ka]

[0213] To a stirred solution of (E)-5-(((2,2,2-trifluoroethyl)imino)methyl)picolinonitrile (II-6) (70 mg, 0.33 mmol) in dichloromethane (5 mL) under nitrogen, homophthalic anhydride (74 mg, 0.46 mmol) was added and the reaction mixture was stirred at room temperature for 16 h. The reaction was then concentrated in vacuo to give a slurry, which was triturated with n-pentane / DCM and filtered to give the title compound (III-6) as a ∼1:1 mixture of cis and trans isomers, which was used in the next step without further purification. LCMS (formic acid:CHCN): m / z: MH+ 376, R t =1.44 minutes.

[0214] (3S,4S)—N-(3-cyano-4-fluorophenyl)-3-(6-cyanopyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (6a) (compound of formula (I) where X is CH and R is CN) [ka]

[0215] To a stirred solution of crude 3-(6-cyanopyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (III-6) (140 mg, 0.37 mmol) in pyridine (2 mL) under nitrogen, propylphosphonic anhydride (50% solution in ethyl acetate, 1.1 mL, 3.77 mmol) was added, followed by 5-amino-2-fluorobenzonitrile (61 mg, 0.45 mmol), and the reaction was heated to 50° C. for 16 h. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate (50 mL), washed with CuSO solution (3×10 mL), dried (MgSO), filtered, and the filtrate was concentrated in vacuo. The resulting oil was diluted with MeOH (4 mL) and stirred with KCO (255 mg, 1.85 mmol) for 4 h, during which time only the trans-epimer formed according to NMR. The suspension was then filtered through a bed of Celite and concentrated in vacuo. The resulting oil was subjected to chromatography (SiO, 3:7 ethyl acetate:hexane) to give the title compound (6) as a racemate (82 mg, 0.16 mmol, 45%). The (+)-enantiomer was obtained by preparative chiral HPLC (column: Chiralpak IA, 21 × 250 mm, 5 μm particle size, operated at ambient temperature and a flow rate of 21 mL / min. Wavelength: 222 nm, run time: 20 min. Mobile phase: hexane / dichloromethane / EtOH: 70 / 15 / 15), followed by lyophilization to give 27 mg of the title compound (6a) as an off-white solid in >99% ee. 1 H NMR (400 MHz, DMSO-d6): δ 10.84 (s, 1H), 8.73 (s, 1H), 8.05-7.96 (m, 3H), 7.84-7.82 (m, 1H), 7.68 (d, J = 6.48 Hz, 1H), 7.57-7.45 (m, 3H), 7.29 (d, J = 6.04 Hz, 1H), 5.63 (s, 1H), 4.65-4.59 (m, 1H), 4.32 (s, 1H), 4.17-4.11 (m, 1H). LCMS (formic acid:CH3CN): m / z: MH+ 492, Rt=1.74 min. [α]D 25 = +108.5°(c= 0.25, DMSO).

[0216] Synthesis of (3S,4S)-N-(3-cyano-4-fluorophenyl)-3-(6-(difluoromethoxy)pyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (7a) 6-(difluoromethoxy)nicotinaldehyde (intermediate of formula (IIa-7)) [ka]

[0217] To a stirred solution of 6-hydroxynicotinaldehyde (iv) (2.0 g, 16.2 mmol) in acetonitrile (60 mL) at room temperature under nitrogen was added sodium chlorodifluoroacetate (3.7 g, 24.4 mmol). After heating the reaction mixture at 85 °C for 72 h, the reaction was quenched by adding distilled water (10 mL). The mixture was extracted with ethyl acetate (3 × 20 mL). The combined extracts were dried (NaSO), filtered, and concentrated in vacuo. The resulting white gum was subjected to column chromatography (SiO, 1:5 ethyl acetate:hexane) to afford the title compound (IIa-7) (1.8 g, 10.4 mmol, 64%) as an off-white solid. 1 H MR (400 MHz, DMSO-d6): δ 10.06 (s, 1H), 8.84 (s, 1H), 8.34 (dd, J= 8.52, Hz, 2.16 Hz, 1H), 8.01-7.65 (m, 1H), 7.28 (d, J = 8.56 Hz, 1H).

[0218] (E)-1-[6-(difluoromethoxy)pyridin-3-yl)-N-(2,2,2-trifluoroethyl)methanamine (intermediate of formula (II-7)) [ka]

[0219] To a stirred solution of 6-(difluoromethoxy)nicotinaldehyde (IIa-7) (500 mg, 2.89 mmol) in acetonitrile (20 mL) under nitrogen at 0°C, 2,2,2-trifluoroethanamine hydrochloride (1.5 g, 11.5 mmol) was added, followed by triethylamine (2.4 mL, 17.3 mmol), and the reaction mixture was stirred at room temperature for 48 h. The reaction was concentrated in vacuo to give the title compound (II-7) as a crude oil, which was used in the next step without further purification.

[0220] 3-(6-(difluoromethoxy)pyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (intermediate of formula (III-7))

[0221] To a stirred solution of (E)-1-(6-(difluoromethoxy)pyridin-3-yl)-N-(2,2,2-trifluoroethyl)methanimine (II-7) (1.1 g, 4.3 mmol) in dichloromethane (20 mL) at room temperature under nitrogen, homophthalic anhydride (0.7 g, 4.3 mmol) was added and the reaction mixture was stirred for 16 h. The mixture was concentrated in vacuo to give 2 g of crude material (III-7), which was used in the next step without further purification. [ka]

[0222] (3S,4S)—N-(3-cyano-4-fluorophenyl)-3-(6-(difluoromethoxy)pyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (7a) (compound of formula (I) where X is CH and R is OCHF) [ka]

[0223] To a stirred solution of crude 3-(6-(difluoromethoxy)pyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (III-7) (2 g, 4.8 mmol) in pyridine (30 mL) under nitrogen at room temperature was added propylphosphonic anhydride (50% solution in ethyl acetate, 30.0 mL, 48.1 mmol), followed by 5-amino-2-fluorobenzonitrile (0.78 g, 5.8 mmol), and the reaction was heated at 50 °C for 16 h. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate (50 mL), washed with CuSO solution (3 × 10 mL), dried (MgSO), filtered, and the filtrate was concentrated in vacuo. The resulting oil was diluted with MeOH (50 mL) and stirred with KCO (1.0 g, 7.25 mmol) for 4 h, during which time only the trans-epimer formed according to NMR. The suspension was then filtered through a bed of Celite and concentrated in vacuo. The resulting oil was chromatographed (SiO, 3:7 ethyl acetate:hexane) to give the title compound (7) as a racemate (1.1 g, 2.06 mmol, 42%). The (+)-enantiomer was obtained by preparative chiral HPLC (column: Chiralpak IA, 21 × 250 mm, 5 μm particle size, operated at ambient temperature and a flow rate of 21 mL / min; wavelength: 222 nm; run time: 20 min; mobile phase: hexane / dichloromethane / EtOH: 70 / 15 / 15), followed by lyophilization to give 205 mg of the title compound (7a) as an off-white solid in >99% ee. 1 H NMR (400 MHz, DMSO-d6): δ 10.82 (s, 1H), 8.06-8.00 (m, 3H), 7.84-7.82 (m, 1H), 7.70-7.45 (m, 4H), 7.31 (d, J= 6.92 Hz, 1H),7.06 (d, J= 8.52 Hz, 1H), 5.49 (s, 1H), 4.64-4.58 (m, 1H), 4.26 (s, 1H), 4.11-4.05 (m, 1H). LCMS (Formic acid:CH3CN): m / z: MH+ 536, R t =1.68 minutes. [α] D 25= +56.38°(c= 0.418, dichloromethane).

[0224] Synthesis of (3S,4S)-N-(3-cyano-4-fluorophenyl)-3-(6-(difluoromethyl)pyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (8a) 5-bromo-2-(difluoromethyl)pyridine (intermediate of formula (vi)) [ka]

[0225] To a stirred solution of 5-bromo-2-formylpyridine (v) (10.0 g, 53.8 mmol) in dichloromethane (100 mL) at −78° C. under nitrogen was added diethylaminosulfur trifluoride (DAST) (15.6 mL, 118.3 mmol). The reaction mixture was allowed to warm slowly to room temperature and then stirred for an additional 6 h. The reaction mixture was then diluted with dichloromethane (100 mL), washed with saturated aqueous sodium bicarbonate (3×50 mL) and brine (2×50 mL), dried (MgSO), filtered, and concentrated in vacuo to give an oil which was chromatographed (SiO, 1:5 ethyl acetate:hexanes) to give the title compound (vi) (5.6 g, 27.2 mmol, 50%) as a colorless oil. 1 H NMR (400 MHz, CDCl3): δ 8.71 (s, 1H), 7.97 (d, J= 8.36 Hz, 1H), 7.53 (d, J= 8.28 Hz, 1H), 6.73-6.45 (m, 1H).

[0226] Methyl 6-(difluoromethyl)nicotinate (intermediate of formula (ii-8)) [ka]

[0227] A solution of 5-bromo-2-(difluoromethyl)pyridine (v) (5 g, 24.03 mmol) in MeOH (50 mL) under a carbon monoxide atmosphere was treated with triethylamine (10 mL, 72 mmol) followed by 1,1'-bis-(diphenylphosphino)-ferrocenedichloropalladium(II) (1.7 g, 2.4 mmol) and heated in an autoclave at 80 °C and 60 PSI for 6 h. The reaction mixture was then allowed to cool, filtered through a bed of Celite, and the filtrate concentrated in vacuo to give a slurry that was chromatographed (SiO, 1:5 ethyl acetate:hexanes) to give the title compound (ii-8) (2.9 g, 15.5 mmol, 64%) as a brown gum. 1 H NMR (400 MHz, CDCl3): δ 9.23 (s, 1H), 8.44 (d, J= 8.08 Hz, 1H), 7.72 (d, J= 8.12 Hz, 1H), 6.80-6.53 (m, 1H), 3.97 (s, 1H). LCMS (Formic acid:CH3CN): m / z: MH+ 188, R t =1.43 minutes.

[0228] (6-(difluoromethyl)pyridin)-3-yl)methanol (intermediate of formula (iii-8)) [ka]

[0229] To a stirred solution of methyl 6-(difluoromethyl)nicotinate (ii-8) (2.0 g, 10.7 mmol) in THF (25 mL) under nitrogen at 0 °C was slowly added a 1 M solution of lithium aluminum hydride (21.0 mL, 21.0 mmol) in THF. After 2 h at 0 °C, the starting material was completely consumed, and the reaction was quenched by the slow addition of 5 mL of saturated aqueous sodium sulfate. The crude reaction mixture was then filtered through a bed of Celite, the filtrate was concentrated in vacuo, and the resulting residue was chromatographed (SiO, 1:1 ethyl acetate:hexane) to afford the title compound (iii-8) (1.6 g, 10.0 mmol, 94%) as a white waxy solid. 1H NMR (400 MHz, CDCl3): δ 8.60 (s, 1H), 7.85 (d, J= 7.68 Hz, 1H), 7.62 (d, J = 7.96 Hz, 1H), 6.76-6.48 (m, 1H), 4.78 (s, 2H). LCMS (Formic acid: CH3CN): m / z: MH+ 160, R t =1.19 minutes.

[0230] 6-(Difluoromethyl)nicotinaldehyde (intermediate of formula (IIa-8)) [ka]

[0231] To a stirred solution of (6-(difluoromethyl)pyridin-3-yl)methanol (iii-8) (1.5 g, 9.5 mmol) in dichloromethane (50 mL) under nitrogen at 0 °C, Dess-Martin periodinane (8.0 g, 19.0 mmol) was added, and the reaction mixture was allowed to warm gradually to room temperature over 2 h. The reaction mixture was then filtered through a bed of Celite, the filtrate was concentrated in vacuo, and the resulting oil was chromatographed (SiO, 1:5 ethyl acetate:hexane) to afford the title compound (IIa-8) (700 mg, 4.45 mmol) as a colorless liquid. 1 H NMR (400 MHz, DMSO-d6): δ 10.17 (s, 1H), 9.17 (s, 1H), 8.45 (d, J= 8.04 Hz, 1H), 7.92 (d, J= 8.00 Hz, 1H), 7.21-6.94 (m, 1H). LCMS (Formic acid:CH3CN): m / z: MH+ 158, R t =1.17 minutes.

[0232] (E)-1-(6-(difluoromethoxy)pyridin-3-yl)-N-(2,2,2-trifluoroethyl)methanimine (intermediate of formula (II-8)) [ka]

[0233] To a stirred solution of 6-(difluoromethyl)nicotinaldehyde (IIa-8) (500 mg, 3.18 mmol) in acetonitrile (20 mL) under nitrogen at 0°C, 2,2,2-trifluoroethylamine hydrochloride (1.7 g, 12.7 mmol) in triethylamine (0.5 mL) was added, and the reaction mixture was allowed to warm to room temperature and stirred for an additional 48 h. The reaction mixture was then concentrated in vacuo to give 1.2 g of the title compound (II-8) as a crude oil, which was used in the next step without further purification.

[0234] 3-(6-(difluoromethyl)pyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (intermediate of formula (III-8)) [ka]

[0235] To a stirred solution of crude (E)-1-(6-(difluoromethyl)pyridin-3-yl)-N-(2,2,2-trifluoroethyl)methanimine (II-8) (1.2 g, 5.2 mmol) in dichloromethane (15 mL) under nitrogen at room temperature, homophthalic anhydride (0.87 g, 5.2 mmol) was added, and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was then concentrated in vacuo to afford 2.1 g of crude title compound (III-8) as a mixture of approximately 1:1 cis and trans isomers, which was used in the next step without further purification. LCMS (formic acid:CHCN): m / z: MH+ 401, R t =1.52 minutes.

[0236] (3S,4S)—N-(3-cyano-4-fluorophenyl)-3-(6-(difluoromethyl)pyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (8a) (compound of formula (I) where X is CH and R is CHF)

[0237] To a stirred solution of crude 3-(6-(difluoromethyl)pyridin-3-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (III-8) (1.0 g, 2.5 mmol) in pyridine (10 mL) under nitrogen at room temperature was added propylphosphonic anhydride (50% solution in ethyl acetate, 15.0 mL, 25.0 mmol), followed by 5-amino-2-fluorobenzonitrile (0.45 g, 3.25 mmol), and the reaction was heated at 50° C. for 16 h. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate (50 mL), washed with CuSO solution (3×10 mL), dried (MgSO), filtered, and the filtrate was concentrated in vacuo. [ka]

[0238] The resulting oil was diluted with MeOH (50 mL) and stirred with KCO (2.0 g, 14.5 mmol) for 4 h, during which time only the trans-epimer formed according to NMR. The suspension was then filtered through a bed of Celite and concentrated in vacuo. The resulting oil was subjected to chromatography (SiO, 3:7 ethyl acetate:hexane) to give the title compound (8) as a racemate (400 mg, 0.77 mmol, 31%). The (+)-enantiomer was obtained by preparative chiral HPLC (column: Chiralpak IA, 21 × 250 mm, 5 μm particle size, operated at ambient temperature and a flow rate of 21 mL / min; wavelength: 222 nm; run time: 20 min; mobile phase: hexane / dichloromethane / EtOH: 70 / 15 / 15), followed by lyophilization to give 197 mg of the title compound (8a) as an off-white solid with >99% ee. 1H NMR (400 MHz, DMSO-d6): δ 10.84 (s, 1H), 8.61 (s, 1H), 8.06-8.00 (m, 2H), 7.85-7.83 (m, 1H), 7.68-7.46 (m, 5H), 7.28 (d, J = 7.48 Hz, 1H), 7.03-6.75 (m, 1H), 5.59 (s, 1H), 4.65-4.59 (m, 1H), 4.32 (s, 1H), 4.17-4.10 (m, 1H). LCMS (ammonium acetate: CH3CN): m / z: MH+ 519, Rt = 2.66 min of a 5 min run. [α]D 25 = +49.28°(c= 0.303, dichloromethane).

[0239] Synthesis of (3S,4S)-N-(3-cyano-4-fluorophenyl)-1-oxo-2-(2,2,2-trifluoroethyl)-3-(2-(trifluoromethyl)pyrimidin-5-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide) (9a)(E)-N-(2,2,2-trifluoroethyl)-1-(2-(trifluoromethyl)pyrimidin-5-yl)methanimine (Intermediate of Formula (IIa-9)) [ka]

[0240] To a stirred solution of 2-(trifluoromethyl)pyrimidine-5-carbaldehyde (IIa-9) (1.0 g, 5.7 mmol) in acetonitrile (20 mL) at 0 °C under nitrogen, 2,2,2-trifluoroethylamine hydrochloride (2.25 g, 22.7 mmol) in triethylamine (0.5 mL) was added. The reaction mixture was allowed to warm to room temperature and then stirred for an additional 48 h. The reaction mixture was then concentrated in vacuo to give 1.46 g of crude title compound (II-9), which was used in the next step without further purification.

[0241] 1-Oxo-2-(2,2,2-trifluoroethyl)-3-(2-(trifluoromethyl)pyrimidin-5-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (intermediate of formula (III-9)) [ka]

[0242] To a stirred solution of crude (E)-N-(2,2,2-trifluoroethyl)-1-(2-(trifluoromethyl)pyrimidin-5-yl)methanimine (II-9) (1.46 g, 4.3 mmol) in dichloromethane (15 mL) under nitrogen at room temperature was added homophthalic anhydride (920 mg, 5.67 mmol). After 16 h, the reaction was concentrated in vacuo to afford 2.3 g of crude title compound (III-9) as a ∼1:1 mixture of cis and trans isomers, which was used in the next step without further purification. LCMS (formic acid:CHCN): m / z: MH+ 420, Rt = 1.68 min.

[0243] (3S,4S)—N-(3-cyano-4-fluorophenyl)-1-oxo-2-(2,2,2-trifluoroethyl)-3-(2-(trifluoromethyl)pyrimidin-5-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (9a) (compound of formula (I) where X is N and R is CF) [ka]

[0244] To a stirred solution of crude 1-oxo-2-(2,2,2-trifluoroethyl)-3-(2-(trifluoromethyl)pyridin-5-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid (III-9) (1.95 g, 4.65 mmol) in pyridine (30.0 mL) at room temperature was added propylphosphonic anhydride (50% solution in ethyl acetate, 29.6 mL, 46.5 mmol), followed by 5-amino-2-fluorobenzonitrile (0.8 g, 6.05 mmol), and the reaction was heated at 50° C. for 16 h. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate (50 mL), washed with CuSO solution (3×10 mL), dried (MgSO), filtered, and the filtrate was concentrated in vacuo. The resulting oil was diluted with MeOH (20 mL) and stirred with KCO (1.0 g, 7.3 mmol) for 4 h, during which time only the trans-epimer formed according to NMR. The suspension was then filtered through a bed of Celite and concentrated in vacuo. The resulting oil was subjected to chromatography (SiO, 3:7 ethyl acetate:hexane) to give the title compound (9) as a racemate (1.2 g, 2.23 mmol, 48%). The (+)-enantiomer was obtained by preparative chiral HPLC (column: Chiralpak IA, 21 × 250 mm, 5 μm particle size, operated at ambient temperature and a flow rate of 21 mL / min; wavelength: 222 nm; run time: 20 min; mobile phase: hexane / dichloromethane / EtOH: 70 / 15 / 15), followed by lyophilization to give 410 mg of the title compound (9a) as an off-white solid in >99% ee. 1 H NMR (400 MHz, DMSO-d6): δ 10.84 (s, 1H), 8.86 (s, 2H), 8.05-8.01 (m, 2H), 7.84-7.82 (m, 1H), 7.57-7.47 (m, 3H), 7.33 (d, J = 6.88 Hz, 1H), 5.69 (s, 1H), 4.73-4.65 (m, 1H), 4.41 (s, 1H), 4.18-4.07 (m, 1H). LCMS (Formic acid:CH3CN): m / z: MH+ 536, Rt=1.84 min. [α]D 25 = +27.12°(c= 0.47, dichloromethane) .

[0245] Example 2 In vitro efficacy against Plasmodium falciparum (strain 3D7) Cultures of the widely used chloroquine-sensitive Plasmodium falciparum (3D7) malaria reference strain were maintained in a 1.25% suspension of human erythrocytes cultured in RPMI 1640 medium (pH 7.3) supplemented with 0.5% Albumax II (Gibco Life Technologies, San Diego, CA), 12 mM sodium bicarbonate, 0.2 mM hypoxanthine, and 20 mg / L gentamicin at 37°C in an atmosphere of 1% O2, 3% CO2 with a nitrogen balance (Human Malaria Parasites in Continuous Culture; Trager W. et al., 1976, Science 193:673-675). Growth inhibition was quantified using a fluorescent assay that utilizes the binding of SYBR Green to double-stranded DNA (Novel, rapid, and inexpensive cell-based quantification of antimalarial drug efficacy. Bennett TN et al., 2004, Antimicrob. Agents Chemother., 48:1807-1810), which emits a fluorescent signal at 528 nm after excitation at 485 nm. Mefloquine was used as a drug control to monitor the quality of the assay (Z' = 0.6-0.8, where Z' is a measure of discrimination between positive and negative controls on a screen plate). Hits were confirmed by complete efficacy curves with dose-response curves determined from a minimum of three independent experiments. Compound bioactivity was measured using the EC2000-EC2000 (effective concentration of compound causing 50% malaria parasite death). 50 The data were expressed as % inhibition. All data were processed using IDBS ActivityBase. Raw data were converted to percent inhibition by linear regression by setting the high inhibition control to 100% and the no inhibition control to 0%. Quality control criteria for a passing plate were z'>0.5, S:B>3, %CV (阻害対照なし) <15. The formula used to calculate z' is:

number

[0246] Curve fitting was performed using the following four parametric equations:

number

[0247] These data demonstrate that the compounds of the present invention exhibit half-maximal concentrations (i.e., IC ) significantly lower than the half-maximal concentration of the reference compound (+)SJ-733. 50 ) and has been shown to be able to inhibit the growth of the asexual intraerythrocytic stage of Plasmodium falciparum. Furthermore, efficacy is primarily associated with the dextrorotatory, or (+), enantiomer.

[0248] Example 3 Antimalarial in vivo efficacy of compounds according to the invention in NSG SCID mice In vivo efficacy was measured against Plasmodium falciparum Pf3D70087 / N9 (A Murine Model of falciparum-Malaria by In Vivo Selection of Competent Strains in Non-Myelodepleted Mice Engrafted with Human Erythrocytes. Angulo-Barturen I. et al., 2008, Plos One., 3(5):e2252) and female NOD-scid IL-2Ry null mice (NSG) (purchased from Charles River, France) engrafted with a minimum of 40% human erythrocytes (Improved Murine Model of Malaria Using Plasmodium falciparum Competent Strains and Non-Myelodepleted NOD-scid IICRy null Mice Engrafted with Human Erythrocytes. Jimenez-Diaz MB et al., 2009, Antimicrob Agents Chemother., 53(10):4533-4536) (Basque Center of Transfusion and Human Tissues, Galdakao, Centro de Transfusiones de la Comunidad de Castilla y León, Valladolid, Spain and Bank of Blood and Tissues, Barcelona, ​​Spain), and intravenously infected parasitized human erythrocytes were measured 72 hours before the start of drug treatment. Treatment began on day 1 of the study, when mice reached a mean parasitemia of approximately 1% (P0). Compounds were administered orally at a single dose of 10 mg / kg body weight or a daily dose of 1 mg / kg body weight for 4 days in a formulation of 1% hydroxypropyl-cyclodextrin, 40% PEG-400, 10% propylene glycol, 10% ethanol, and 40% PBS, 10 mL / kg body weight.Peripheral blood parasitemia was assessed every 24 h by flow cytometry (Attune NxT Acoustic Focusing Flow Cytometer, InvitroGen) using TER-119-phycoerythrin monoclonal antibody as a marker for mouse erythrocytes and SYTO-16, a nonselective fluorescent nucleic acid dye, to detect intraerythrocytic parasites, as previously described (Quantitative measurement of Plasmodium-infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO-16 fluorescence. Jimenez-Diaz MB et al., 2009, Cytometry A., 75(3):225-35). Treatment efficacy was estimated as the percentage of growth inhibition compared to untreated mice after one cycle (% inhibition on day 3) or two cycles (% inhibition on day 5) of parasite replication. Data analysis was performed using GraphPad Prism 7.0 (GraphPad Software). Peripheral blood samples (25 μl) were continuously collected throughout the study after the first dose, mixed with 25 μl of HO MilliQ, and immediately stored at -80°C until analysis. Drug was extracted from 10 μl of lysate obtained by protein precipitation of blood samples using standard liquid-liquid extraction methods. Samples were analyzed by LC-MS / MS for quantification on a Waters Micromass UPLC-TQD (Waters, Manchester, UK). Blood concentrations versus time were analyzed by noncompartmental analysis (NCA) using Phoenix WinNonlin vers. 7.0 (Certara) or R or Excel (Microsoft), from which exposure-related values ​​(tmax, Cmax, and AUC0-t) were estimated. Treated mice that reached the limit of detection by standard flow cytometry (<0.01% of total circulating red blood cells) with chimerism of >50% of total circulating red blood cells were retained until Day 60 of the study. During the follow-up period, parasitemia in peripheral blood was assessed every 2–3 days and analyzed by flow cytometry with a limit of quantification of 0.01%.The day of relapse (DoR) was estimated by linear interpolation of the % parasitemia on the day before P0 rise and the % parasitemia on the day after P0 was reached. Mice were considered cured if they had no detectable parasites in peripheral blood by flow cytometry with a quantification limit of 0.01% by day 60 of the study. DoR was calculated only for parasites showing growth kinetics in the blood comparable to the initial infection.

[0249] Overall, these data support that the compounds of the present invention clearly have the exposure required for antimalarial drugs via the oral route and that antimalarial efficacy is significantly improved compared to the reference compound (+)SJ-733.

[0250] Example 4 Determination of in vitro toxicity against mammalian HepG2 cells Actively growing HepG2 human cells (85011430, European Collection of Authenticated Cell Cultures) were detached from the culture surface with trypsin and counted using a Cellometer (Nexcelom). They were then cultured at 1.0 × 10 cells per ml in Minimum Essential Medium (41090, Gibco Life Technologies) supplemented with 10% fetal bovine serum (10106-169, Gibco Life Technologies) and 1% NEAA solution (11140-035, Gibco Life Technologies). 5At a density of 12 ml, 12 ml of medium was prepared per plate required. Cells were seeded (25 μl per well) into 384-well plates (781098, Greiner) with pre-stamped compound and control (DMSO and doxorubicin 50 μM) volumes (125 nl per well) using a WellMate dispenser (Thermo Scientific). Cells were incubated in a humidified incubator at 37°C and 5% CO2 for 48 hours. After incubation, 5 μl of resazurin (R7017, Sigma) (final concentration 45 μM) was added per well, and the plates were kept at 37°C for 3.5-4 hours. Plates were then read on a Pherastar LS plate reader (BMG), and the percent inhibition of each test compound was calculated using the formula: % inhibition = 100 - (((test compound - blank) / (DMSO control - blank)) x 100). Toxicity IC 50 ' was computed using the ActivityBase template.

[0251] Example 5 Microsomal endogenous clearance (Cl int ) and hepatic intrinsic clearance (Cl int ) determination Compounds of the present invention (1 μM) were incubated in suspension with human (rat or mouse) liver microsomes pooled from multiple donors (>10). Samples were collected at five time points over the course of a 45-minute experiment and analyzed by LC-MS / MS. Microsomes (final protein concentration 0.5 mg / mL), 0.1 M phosphate buffer pH 7.4, and test compound (final substrate concentration 1 μM, final DMSO concentration 0.25%) were preincubated at 37°C before the addition of NADPH (final concentration 1 mM) to initiate the reaction. A minus-cofactor control incubation was included for each test compound in which 0.1 M phosphate buffer pH 7.4 was added instead of NADPH (minus NADPH). All incubations were performed in duplicate for each test compound, and the results were averaged. Each compound was incubated for 0, 5, 15, 30, and 45 minutes. The control (minus NADPH) was incubated for only 45 minutes. The reaction was stopped at the appropriate time point by transferring the incubation to acetonitrile in a 1:3 ratio. The termination plate was centrifuged at 3,000 rpm for 20 minutes at 4°C to precipitate the proteins. After protein precipitation, the sample supernatants were combined into cassettes of up to four compounds, an internal standard was added, and the samples were analyzed by LC-MS / MS. From a plot of the In peak area ratio (composite peak area / internal standard peak area) versus time, the slope of the line was determined. The half-life (t 1 / 2 ) and intrinsic clearance (CLint) are calculated using the following formula: Desorption rate constant (k) = (-slope) Half-life (t 1 / 2 )(min)=0.693 / k Intrinsic clearance (i.e., CL int )(μL / min / mg protein) = V × 0.693 / t 1 / 2 V = incubation volume (μL) / microsomal protein (mg)

[0252] Verapamil and dextromethorphan were included in the assay as controls, and if the values ​​of these compounds were not within the specified limits, the results were rejected and the experiment was repeated.

[0253] Hepatic intrinsic clearance (Cl int ) determination Compounds of the present invention (3 μM) were incubated in suspension with cryopreserved human (rat or mouse) hepatocytes pooled from multiple donors (>10). Samples were collected at six time points over the course of a 60-minute experiment and analyzed by LC-MS / MS. After preincubation at 37°C with test compounds (final substrate concentration 3 μM, final DMSO concentration 0.25%) in Williams E medium supplemented with 2 mM L-glutamine and 25 mM HEPES, the reaction was initiated by the addition of a cryopreserved hepatocyte suspension (final cell density 0.5 × 10 viable cells / mL in Williams E medium supplemented with 2 mM L-glutamine and 25 mM HEPES). The final incubation volume was 500 μL. The reaction was terminated at the appropriate time point by transferring an aliquot of the incubation to acetonitrile in a 1:2 ratio. The termination plate was centrifuged at 3,000 rpm for 20 minutes at 4°C to precipitate proteins. After protein precipitation, sample supernatants were combined into cassettes of up to four compounds, spiked with internal standards, and analyzed by LC MS / MS. From plots of the In peak area ratio (composite peak area / internal standard peak area) versus time, the slope of the line was determined. The half-life (t 1 / 2 ) and intrinsic clearance (CLint) were calculated using the following formula: Desorption rate constant (k) = (-slope) Half-life (t 1 / 2 )(min)=0.693 / k Intrinsic clearance (i.e., CL int )(μL / min / million cells)=V×0.693 / t 1 / 2 V = incubation volume (μL) / cell number

[0254] Verapamil and umbelliferone were included in the assay as controls, and if the values ​​for these compounds were not within the specified limits, the results were rejected and the experiment was repeated. The in vitro and in vivo pharmacokinetic parameters are shown in Table 2 below. [Table 2]

[0255] Taken together, these data support that the compounds of the present invention clearly have the exposure required for antimalarial drugs via the oral route. Furthermore, the in vitro metabolic stability is improved compared to the reference compound (+)SJ-733. Furthermore, the in vivo half-life in mouse and rat species is significantly improved compared to the reference compound (+)SJ-733, consistent with the improved in vitro stability in microsome and hepatocyte incubations. Furthermore, Example compound (1a) has an improved pharmacokinetic profile in dog species compared to the reference compound, i.e., (+)SJ-733 (IV dose, 3 mg / kg, Cl = 3 mL / min / Kg, t 1 / 2 = 10 h) compared to compound (1a): (IV at 1 mg / kg, Cl = 0.26 mL / min / Kg, t 1 / 2 =41 hours).

[0256] Example 6 Determination of PK parameters in mice / rats. Male Swiss Albino mice or Sprague Dawley rats (TCGLS, India) were housed under a 12 / 12-hour light / dark cycle at 22 ± 2°C and 50 ± 20% RH with free access to food and water. Mice were randomly selected based on weight and age and fasted for 4 hours before and 2 hours after dosing. Three mice were used per administration route. For IV doses (1 mg / kg, 5 ml / kg dose), the dose was administered by injection into the lateral tail vein (rats were anesthetized using a 3% v / v isoflurane:oxygen mixture). For PO doses (3 mg / kg, 10 ml / kg dose), the dose was administered by oral gavage to conscious animals. Blood samples were collected from the saphenous vein (conscious animals) at specified time points (50 μL for mice, 100 μL for rats), collected in heparinized capillary tubes, and transferred to 0.5 mL microcentrifuge tubes. Samples were processed within 30 minutes of collection by centrifugation at 1640 g for 10 minutes at +4°C and stored at -20°C until bioanalysis. All samples were mixed with ice-cold acetonitrile containing an internal standard and centrifuged at 4000 rpm for 15 minutes at 15°C. The supernatant was then diluted in half with water and analyzed by LCMS / MS analysis to determine the analyte peak area / IS peak area (ratio). The concentrations of the compounds of the present invention (plasma) were determined by reference to a calibration curve. Pharmacokinetic parameters (Cmax, Tmax, AUC, t 1 / 2 , CL, Vd, BA) were calculated using non-compartmental analysis (Phoenix, version 6.3) using WinNonLin. The pharmacokinetic parameters for the compounds shown in Table 2 suggest that the compounds of the invention have pharmacokinetic properties relevant for the treatment of malaria via the oral route, and that the compounds have substantially longer half-lives than the reference compounds.

[0257] Example 7 Determination of the effect of compounds of the invention on cytosolic [Na+] in saponin-isolated Plasmodium falciparum (strain Dd2). The ability of compounds of the invention to function as inhibitors of Na+ / H+-ATPase, which regulates parasite cytosolic sodium concentration, was tested in the following assay. Their effect on cytosolic sodium can be measured using a sodium flux assay (Biochemical characterization and chemical inhibition of PfATP4-associated Na+). + ATPase activity in Plasmodium falciparum membranes. Rosling JEO et al., 2018, J. Biol. Chem., 293:13397). Saponin-isolated parasites were grown at a density of 1.4–1.8 × 10 8 Cells / mL were suspended in bicarbonate-free RPMI 1640 supplemented with 20 mM D-glucose, 0.2 mM hypoxanthine, 25 mM HEPES, and 25 mg / L gentamicin sulfate (pH 7.10), then treated with sodium-conjugated benzofuranisophthalic acid acetoxymethyl ester (5.5 mM) and Pluronic F-127 (0.01% w / v) for 20 minutes at 37°C. The dye-loaded cells were washed twice (12,000 g, 0.5 minutes) with bicarbonate-free RPMI and then incubated for an additional 20 minutes at 37°C to completely deesterify the dye, after which the cells were resuspended in normal saline at a final cell concentration of 1.5–2.5 × 10 cells. 7 Immediately after treatment with test compounds (5 mM) or vehicle control (0.1% v / v DMSO), dye-loaded cells were excited at 340 nm and 380 nm, and fluorescence measurements were recorded at 490 nm every 15–45 seconds. [Na+] was determined by reference to a calibration curve of the 340 / 380 nm fluorescence ratio as previously described (In situ calibration and [H+] sensitivity of the fluorescent Na+ indicator SBFI; Diarra A. et al., 2001, Am. J. Physiol. Cell Physiol., 280:1623). Figure 4 shows the intracellular [Na+] of SBFI-loaded 3D7 parasites suspended in normal saline after treatment with 1 μM concentrations of compounds 1a and 9a. +The effect of compound treatment on cytosolic sodium levels is shown in comparison to 50 nM cypargamine and DMSO controls. The data show that the increase in cytosolic sodium levels after treatment with compound + These results are consistent with inhibition of the ATPase (i.e., PfATP-4).

[0258] Overall, these data support the improved potency of the compounds of the present invention against Plasmodium falciparum in mouse, rat, and human microsomes and human hepatocytes, as well as enhanced metabolic stability, which translates into longer half-lives in mice and rats and therefore increased efficacy in the mouse malaria model. The combined effect of these increased parameters is expected to result in a significant reduction in the projected single human dose compared to (+)SJ-733.

[0259] Example 8 Determination of transmission-blocking efficacy using a standard membrane feeding assay A standard membrane feeding assay (SMFA) was performed using the Plasmodium falciparum transgenic reporter strain NF54-HGL, which expresses the firefly luciferase gene driven by the hsp70 promoter (Vos et al., 2015, Scientific Reports, 5). Stage V gametocytes were preincubated with test compounds before feeding. Test compounds were dissolved in DMSO to achieve a 10 mM stock solution and serially diluted in DMSO to achieve concentrations >1000x the final test concentration. Subsequently, 10 μl of diluted compound in DMSO was added to 990 μl of prewarmed RPMI 1640 medium supplemented with 367 mM hypoxanthine, 25 mM HEPES, and 25 mM NaHCO3 ("incomplete medium"). 40 μl of this intermediate dilution was added to 360 μl of parasite culture medium and incubated in an Eppendorf tube at 37 °C for 48 h, resulting in a final DMSO concentration of 0.1%. Then, 300 μl of the gametocyte culture / compound mix was added to 180 μl of packed red blood cells and centrifuged at 10,000 × g for 20 seconds. After carefully aspirating the supernatant, 200 μl of human serotype A was added to the pellet. Finally, 300 μl of this mixture was immediately injected into individual membrane-covered mini-feeders, where 50 female A. stephensi mosquitoes were allowed to feed for 10 minutes. Within 4 hours of feeding, unfed and partially fed mosquitoes were removed from the cages, and fed mosquitoes were maintained at 26°C and 80% humidity. Mosquitoes were then processed for luminescence measurement to determine relative oocyst intensity as previously described (Stone et al., 2014, Journal of Infectious Diseases, 5, 16). The data were analyzed by fitting a four-parameter logistic regression model (Dechering et al., 2017, Scientific Reports, 7, 1) using maximum likelihood to find the best fit, as shown in Figure 5. These data indicate that compound 1a has transmission-blocking potential (i.e., the transfer of malaria parasites from humans to mosquitoes).

Claims

[Claim 1] Formula (I): 【Chemistry 1】 wherein X is selected from N and CH, and when X is CH, R is -CF 3 , -CHF 2 , -O-cyclopropyl, -O-isopropyl, -OCHF 2 , -CN, -OCH 2 CF 3 and -NH(C=O)CH 3 When X is N, R is selected from the group consisting of -CF 3 is) and pharmaceutically acceptable salts, hydrates, solvates, tautomers, polymorphs, racemic mixtures, optically active forms and pharmaceutically active derivatives thereof.

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