OA20266A

OA20266A -

Info

Publication number: OA20266A
Application number: OA1202100252
Authority: OA
Inventors: Eric Brown; Vidyasagar Reddy Gantla; Nadezda Sokolova; Michael Bruno Plewe; Gregory Henkel; Kenneth Mccormack
Original assignee: Arisan Therapeutics, Inc
Priority date: 2018-12-06
Filing date: 2019-12-03
Publication date: 2022-04-14

Abstract

Compounds as exemplified by compound A are useful in the treatment of arenavirus infections and viral infections mediated by arenavirus glycoproteins. Compounds as exemplified by compound A are useful in the treatment of arenavirus infections and viral infections mediated by arenavirus glycoproteins.

Description

COMPOUNDS FOR THE TREATMENT OF ARENAVIRUS INFECTION

CROSS REFERENCES TO RELATED APPLICATIONS

This patent application is a continuation in part of and claims the benefit of priority to United States Provisional Patent Application serial number 62/776,390, filed December 6, 2018 herein incorporated by reference in its entirety for ail purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under R44 AI112097 awarded by U.S. National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO A “SEQUENCE LISTING, A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK

NOT APPLICABLE

FIELD OF THE INVENTION

The present invention relates to the use of heterocyclic compounds for inhibiting arenavirus infection in humans, other mammals, or in cell culture, to methods of treating arenavirus infection such as Lassa, Bolivian, Argentine, Venezuelan, Brazilian, Chapare and Lujo hémorrhagie fevers, to methods of inhibiting the réplication of arenaviruses, to methods of reducing the amount of arenaviruses, and to compositions that can be employed for such methods.

BACKGROUND OF THE INVENTION

Arenaviridae comprise a diverse family of 29 (and growing) négative stranded enveloped RNA viruses. Arenaviruses are divided into two groups, Old and New World, based on serological, genetic and geographical data. Old World viruses are found primarily throughout South and West Africa and include the prototypic lymphocytic choriomeningitis virus (LCMV), along with Lassa (LASV), Lujo (LUJV), Mopeia (MOPV), Ippy and Mobala (MOBV) viruses. Both LASV and LUJV can cause léthal hémorrhagie fever (HF), while LCMV infection is associated with aseptie meningitis. Lassa (LASV) alone is estimated to cause over 300,000 disease cases each year in West Africa, of which 15-20% of hospitalized patients die and survivors often suffer sequelae, including permanent bilateral hearing damage. The larger New World complex primarily located in the South American continent, is divided into 3 clades, A, B, and C, with clade B being important as many ofthe viruses in this group can cause léthal HF. Clade B HF viruses include, Junin (JUNV), Machupo (MACV), Guanarito (GTOV), Sabia (SABV) and Chapare, along with non-HF viruses such as Tacaribe (TCRV) and Amapari (AMPV). Human infection occurs through contact with the excrétions of an infected rodent or by inhalation of tiny particles soiled with rodent urine or saliva (aérosol transmission). There is also evidence of human-to-human spread primarily in nosocomial settings (e.g. hospitals). The incubation period of virus is 1 2 weeks followed by fever, general malaise, weakness, sore throat, headache, cough, diarrhea, and vomiting. These general symptoms make it difficult to differentially diagnose arenavirus infection. Poor prognosis is indicated as symptoms worsen to include pleural effusions, facial edema, neurological complications and bleeding from mucosal surfaces. Current arenavirus treatment is limited to the use of ribavirin, which is only partially effective if given early and associated with significant side effects. Although a vaccine has been developed for Junin virus its usage is primarily restricted to the most at risk populations of farm workers in Argentina and there are no approved vaccines for any other arenaviruses. Although highly désirable, prophylactic vaccines may not always be effective countermeasures against rapidly emerging, antigenically distinct new virus strains and the existing vaccine development and production strategies cannot adequately respond to the diverse family of current or emergent arenaviruses. Novel broad-spectrum antiviral drugs could therefore, provide a first line therapy and/or prophylactic not only for endemic régions of arenavirus infection but also as a safeguard against potential biological warfare agents.

Arenaviruses consist of a nucleocapsid (NP) surrounded by an envelope membrane, and the NP contains two ambisense RNA genome segments L and S that direct the synthesis of two polypeptides. The L segment encodes the RNA-dependent-RNA polymerase (RdRp) and a small Ring Finger protein Z. The S segment encodes for nucleoprotein and a glycoprotein precursor GPC that is cleaved by host proteases and undergoes post-translational modification into a mature complex composed of glycoproteins GP1 (binds host protein at the cell surface), GP2 (directs pH dépendent membrane fusion and release of genomic material in the cytoplasm) and a stable signal peptide (SSP1). The mature glycoprotein complex (GP, or referred to as glycoprotein) is formed in the viral envelope and is responsible for mediating viral entry. The Old World arenaviruses bind to host α-dystroglycan while New World arenaviruses bind to transferrin receptor 1 for entry/endocytosis into cells. Upon binding to cell surface receptors, the virus is endocytosed and directed to acidic late endosomes whereby, GP2 médiates pH dépendent membrane fusion and release of genomic material into the cytoplasm for viral réplication and transcription. Therefore, viral entry inhibitors (e.g. small molécules) that target virus GP complex or host factors are a potential therapeutic/prophylactic approach in treating patients infected with arenavirus infection. Because the HF arenavirus species are classified as BSL-4, alternative approaches are needed to identify viral entry inhibitors. To facilitate the identification of arenavirus entry inhibitors one may express arenavirus GP complex in nonpathogenic BSL-2 envelope viruses to produce single round infectious pseudoviruses whose viral entry functions are determined by the heterogeneous glycoprotein of interest. One viral expression system that may be utilized is the vesicular stomatitis virus (VSV) system, whereby the envelope protein of VSV is substituted with an envelope glycoprotein from another virus, e.g., LASV, to médiate entry of the pseudotype virion. The cell entry and infectivity properties of GP pseudotype VSV viruses hâve been shown for multiple viruses including HIV, Hepatitis B and C, Ebola, Lassa, Hanta and others lOgino, M., et al. Use of vesicular stomatitis virus pseudotypes bearing hantaan or seoul virus envelope proteins in a rapid and safe neutralization test. Clin. Diagn. Lab. Immunol.(2003) 10(1):154-60; Saha, M.N., et al. Formation of vesicular stomatitis virus pseudotypes bearing surface proteins of hepatitis B virus. J. Viral. (2005) 79(19):12566-74; Takada, A., et al. A system for functional analysis of Ebola virus glycoprotein, Proc. Natl. Acad. Sci. (1997) 94:14764-69; Garbutt, M., et al. Properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses. J. Viral. (2004) 78(10):5458-65]. The above papers are herein incorporated by reference in their entirety for ail purposes. To monitor pseudovirus infection, a reporter gene such as green fluorescent protein (GFP) or luciferase can be engineered into the pseudovirus genome, and virus infectivity in mammalian cell lines (e.g. Veto or Hek293) can be monitored using optical détection methods (e.g. plate reader) [Cote, M.; Misasi, J.; Ren, T.; Bruchez, A., Lee, K., Filone, C. M.; Hensley, L.; Li, Q.; Ory, D.; Chandran, K.; Cunningham, J., Small molécule inhibitors reveal Niemann-Pick Cl is essential for Ebola virus infection, Nature (2011) 477: 344-348, Elshabrawy, H. A., et al. Identification of a broad-spectrum antiviral amall molécule against severe scute respiratory syndrome Coronavirus and Ebola, Hendra, and Nipah Viruses by using a novel high-throughput screening assay. J. Viral. (2014) 88: 4353-4365]. The above papers are herein incorporated by reference in their entirety for ail purposes. The “pseudoviruses” may therefore be used to screen Chemical compound libraries to identify inhibitors of arenavirus cell entry while avoiding the complications of working with highly pathogenic BSL-4 agents.

The introduction of deuterium (D) into drug molécules is an attractive strategy that might help improving drug’s metabolism, pharmacokinetic and toxicity profiles. Deuterium is a stable, nontoxic, nonradioactive isotope of hydrogen. Due to the greater atomic mass, deuterium forms a stronger bond with carbon than hydrogen, making the carbon-deuterium bond much harder to break. In cases where the breaking of a carbon-hydrogen bond is partially or wholly rate-limiting step in the cytochrome P450-mediated drug metabolism, replacing hydrogen atom(s) with deuterium may slow the rate of metabolism, resulting in improved half-life, greater tolerability, improved efficacy and dosing regimen, lower side effects, and decreased toxicity [Foster, A. B. Deuterium isotope effects in studies of drug metabolism. Trends in Pharmacological Sciences (1984) 5:524-527; Anderson, K.E.; Stamler, D.; Davis, M.D., et al. Deutetrabenazine for treatment of involuntary movements in patients with tardive dyskinesia (AIM-TD): a double-blind, randomised, placebo-controlled, phase 3 trial. Lancet Psychiatry (2017) 4:595-604; Harbeson, S.; Morgan, A.; Liu, J., et al. Altering metabolic profiles of drugs by précision deutération 2: discovery of a deuterated analog of ivacaftor with differentiated pharmacokinetics for clinical development. J. Pharmacol. Exp. Ther. (2017) 362:359-367; Malmlôf, T.; Feltmann, K.; KonradssonGeuken, A., et al. Deuterium-substituted l-DOPA displays increased behavioral potency and dopamine output in an animal model of Parkinson’s disease: comparison with the effects produced by l-DOPA and an MAO-B inhibitor. J. Neural. Transm. (Vienna) (2015) 122:259-272; Mutlib, A. E.; Gerson, R. J.; Meunier, P. C., et al. The Species-Dependent Metabolism of Efavirenz Produces a Nephrotoxic Glutathione Conjugate in Rats. Toxicol. Appl. Pharmacol. (2000) 169:102-113]. The above papers are herein incorporated by reference in their entirety for ail purposes. However, in some cases, hydrogen-deuterium exchange may lead to redirecting sites of metabolism (“metabolic switching”) [Horning, M. G., et al. Metabolic switching of drug pathways as a conséquence of drug substitution. Proceedings of the Second International Conférence on Stable Isotopes (Klein. E. R. and Klein. P. D. eds) (1976) 41-54; Miwa, G. T.; Lu, A. Y. H. Kinetic isotope effects and ‘metabolic switching’ in cytochrome P450-catalyzed reactions. Bioessays (1987) 7:215219]. The above papers are herein incorporated by reference in their entirety for ail purposes. At the same time, deuterium and hydrogen are essentially the same size, and in most cases, deutération of a drug would not be expected to affect the biochemical potency or selectivity of the deuterated drug for a biological target as compared to its nondeuterated analog. The effects of deuterium modification on a drug’s metabolic and pharmacokinetic properties are not predictable even when deuterium atoms are incorporated at known sites of metabolism. One can détermine the effect of deuterium incorporation on the absorption, distribution, metabolism, excrétion and/or toxicity (ADMET) properties only by preparing and testing the actual deuterated compound.

In the present invention, entry inhibitors described were identified using an arenavirus GP pseudovirus screen and selected compounds were tested against native non-HF virus TCRV to confirm activity against réplicative arenavirus. Selected top compounds were then tested against native LASV to confirm activity against the native highly pathogenic human (HF) arenaviruses, and initial drug-like properties were assessed.

BRIEF SUMMARY OF THE INVENTION

The présent invention relates to the use of heterocyclic compounds for inhibiting arenavirus infection in humans, other mammals, or in cell culture, to methods of treating arenavirus infection such as Lassa, Bolivian, Argentine, Venezuelan, Brazilian, Chapare and Lujo hémorrhagie fevers, to methods of inhibiting the réplication of arenaviruses, to methods of reducing the amount of arenaviruses, and to compositions that can be employed for such methods.

In one embodiment, the method comprises administering to humans, other mammals, cell culture, or biological sample an effective amount of a compound represented by Structural Formula I or a pharmaceutically acceptable sait and a pharmaceutically acceptable carrier, dilutant or vehicle thereof wherein

A is independently selected from C and N;

G is independently selected from CH, CD, and N;

E is independently selected from CH, CD, and N;

J is independently selected from

N^x^ Axa

R² is independently selected from H, D, -OR³, -R⁴, -NHR¹⁰, -CONHR¹⁰;

R³ is independently selected from H, D, C-ι to C6 alkyl, C2 to C6 alkenyl, (C3 to Cw) cycloalkyl, (C2to C9) cycloheteroalkyl, -NHC(O)R⁴, -C(O)NHR¹⁰, and -C(O)R¹⁰, wherein each Ct to C6 alkyl is optionally substituted with D, halogen, -OH, -OR⁴, -NHR¹⁰;

R⁴ is independently selected from C-, to C₆ alkyl and (C₂to C₉) cycloheteroalkyl optionally substituted with D, halogen, -OH, -OR¹⁰, and NHR¹⁰;

R⁵ is independently selected from H, D, Ci to C6 alkyl, C2 to C6 alkenyl, C2 to C6 alkynyl, halogen, -OR³, -CO2R¹⁰, -NHC(O)R⁴, -C(O)NHR¹⁰, -NHR¹⁰, -CHNHR¹⁰, -CN, -CR⁴, and -C(O)R¹⁰, wherein each Ci to Ce alkyl is optionally substituted with D;

R⁶ is independently selected from H, D, halogen, -OR³, and R⁴;

R⁹ is independently selected from H, D, halogen, Ci to C₆ alkyl, and -OR¹⁰;

R¹⁰is independently selected from H, D, -OH, Ci to C₆ alkyl and C₂ to C₆ alkenyl; and when E is N, CH or CD then A is C, G is CH or CD, and J is

and when A is N then J is

R² with the proviso that the following compounds are excluded:

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the method comprises administering to humans, other mammals, cell culture, or biological sample an effective amount of a compound represented by Structural Formula I

or a pharmaceutically acceptable sait and a pharmaceutically acceptable carrier, dilutant or vehicle there of wherein

A is independently selected from C and N;

G is independently selected from CH, CD, and N;

E is independently selected from CH, CD, and N;

J is independently selected from

R² is independently selected from H, D, -OR³, -R⁴, -NHR¹⁰, -CONHR¹⁰;

R³ is independently selected from H, D, Ci to C₆ alkyl, C₂ to C₆ alkenyl, (C₃ to C₁₀) cycloalkyl, (C₂to Cg) cycloheteroalkyl, -NHC(O)R⁴, -C(O)NHR¹⁰, and -C(O)R¹⁰, wherein each Ci to Ce alkyl is optionally substituted with D, halogen, -OH, -OR⁴, -NHR¹⁰;

R⁴ is independently selected from Ci to Οθ alkyl and (C₂to C9) cycloheteroalkyl optionally substituted with D, halogen, -OH, -OR¹⁰, and NHR¹⁰;

R⁵ is independently selected from H, D, Ci to Ce alkyl, C2 to Ce alkenyl, C2 to Ce alkynyl, halogen, -OR³, -CO2R¹⁰, -NHC(O)R⁴, -C(O)NHR¹⁰, -NHR¹⁰, -CHNHR¹⁰, -CN, -CR⁴, and -C(O)R¹⁰, wherein each Ci to C₆ alkyl is optionally substituted with D;

R⁶ is independently selected from H, D, halogen, -OR³, and R⁴;

R⁹ is independently selected from H, D, halogen, -OR¹⁰, and Ci to Ce alkyl;

R¹⁰ is independently selected from H, D, -OH, Ci to Ce alkyl, and C₂ to Ce alkenyl; and when E is N, CH or CD then A is C, G is CH or CD, and J is

with the proviso that the following compounds are excluded:

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AZ °-^	A¹ Z G-^	Yl °'\^z	y.....A ⁷ OH
^x ‘1 ⁰~V	0^0⁴¾ °^	N'% O-A^	.0« H 0~γ

In another embodiment, the method comprises administering to humans, other mammals, cell culture, or biological sample an effective amount of a compound represented by Structural Formula I, or a pharmaceutically acceptable sait, and a pharmaceutically acceptable carrier, diluent, or vehicle thereof, wherein A, G, E, R², R³, R⁴, R⁵, R⁶, R⁹, and R¹⁰ are defined as above and wherein

In another embodiment, the method comprises administering to humans, other mammals, cell culture, or biological sample an effective amount of a compound represented by Structural Formula I, or a pharmaceutically acceptable sait, and a pharmaceutically acceptable carrier, diluent, or vehicle thereof, wherein A, G, J, R², R³, R⁴, R⁵, R⁶, R⁹, and R¹⁰ are defined as above and wherein

E is CH or CD.

In another embodiment, the method comprises administering to humans, other mammals, cell culture, or biological sample an effective amount of a compound represented by Structural Formula I, or a pharmaceutically acceptable sait, and a pharmaceutically acceptable carrier, diluent, or vehicle thereof, wherein E, G, J, R², R³, R⁴, R⁵, R⁶, R⁹, and R¹⁰ are defined as above and wherein

A is C.

A is N.

In another embodiment, the method comprises administering to humans, other mammals, cell culture, or biological sample an effective amount of a compound represented by Structural Formula I, or a pharmaceutically acceptable sait, and a pharmaceutically acceptable carrier, diluent, or vehicle thereof, wherein A, G, E, R², R³, R⁴, R⁵, R⁹, and R¹⁰ are defined as above and wherein

In another embodiment, the method comprises administering to humans, other mammals, cell culture, or biological sample an effective amount of a compound represented by Structural Formula I, or a pharmaceutically acceptable sait, and a pharmaceutically acceptable carrier, diluent, or vehicle thereof, wherein A, G, E, R², R³, R⁴, R⁵, R⁹, and R¹⁰ are defined as above and wherein / ^CD3

K Y R² _R ^-M“^oh

J is and R⁶ is \ or ^CD3 .

In another embodiment, the method comprises of administering to humans, other mammals, cell culture, or biological sample a pharmaceutically effective amount of a pharmaceutical composition comprising a compound selected from the group of compounds described as Examples A1 to A3, B4 to B9, C10 to C26, D27 to D29, and E30 with a pharmaceutically acceptable carrier, dilutant, or vehicle.

In another embodiment the method comprises of administering a pharmaceutically effective amount of a pharmaceutical composition comprising a compound selected of Structural Formula I or a compound as shown above with a pharmaceutically acceptable carrier, dilutant, or vehicle, with an additional therapeutically effective amount of a therapeutic agent selected from the group consisting of Ribavirin, polymerase inhibitors, Favipiravir, Triazavirin, small interfering RNAs (siRNAs), vaccines, monoclonal antibodies, immunomodulators, and other arenavirus inhibitors.

In another embodiment, the invention relates to compounds of Structural Formula I

A is independently selected from C and N;

G is independently selected from CH, CD, and N;

E is independently selected from CH, CD, and N;

J is independently selected from

R² is independently selected from H, D, -OR³, -R⁴, -NHR¹⁰, -CONHR¹⁰;

R³ is independently selected from H, D, Ci to C6 alkyl, C2 to C6 alkenyl, (C3 to C10) cycloalkyl, (C2to Cg) cycloheteroalkyl, -NHC(0)R⁴, -C(O)NHR¹⁰, and -C(O)R¹⁰, wherein each C-ι to C6 alkyl is optionally substituted with D, halogen, -OH, -OR⁴, -NHR¹⁰;

R⁴ is independently selected from Ci to C₆ alkyl and (C₂to C₉) cycloheteroalkyl optionally substituted with D, halogen, -OH, -OR¹⁰, and NHR¹⁰;

R⁵ is independently selected from H, D, Ci to C6 alkyl, C2 to C6 alkenyl, C2 to C6 alkynyl, halogen, -OR³, -CO2R¹⁰, -NHC(O)R⁴, -C(O)NHR¹⁰, -NHR¹⁰, -CHNHR¹⁰, -CN, -CR⁴, and -C(O)R¹⁰, wherein each Ci to C₆ alkyl is optionally substituted with D;

R⁶ is independently selected from H, D, halogen, -OR³, and R⁴;

R⁹ is independently selected from H, D, halogen, -OR¹⁰, and Ci to C₆ alkyl;

R¹⁰ is independently selected from H, D, -OH, Ci to C₆ alkyl and C₂ to C₆ alkenyl; and when E is N, CH or CD then A is C, G is CH or CD, and J is

and when A is N then J is

with the proviso that the following compounds are excluded:

In another embodiment, the invention relates to compounds of Structural Formula I or a pharmaceutically acceptable sait, and a pharmaceutically acceptable carrier, diluent, or vehicle thereof, wherein A, G, E, R², R³, R⁴, R⁵, R⁶, R⁹, and R¹⁰ are defined as above and wherein

In another embodiment, the invention relates to compounds of Structural Formula I or a pharmaceutically acceptable sait, and a pharmaceutically acceptable carrier, diluent, or vehicle thereof, wherein A, G, E, R², R³, R⁴, R⁵, R⁶, R⁹, and R¹⁰ are defined as above and wherein έ 9“^r2

J is W

In another embodiment, the invention relates to compounds of Structural Formula I or a pharmaceutically acceptable sait, and a pharmaceutically acceptable carrier, diluent, or vehicle thereof, wherein A, G, J, R², R³, R⁴, R⁵, R⁶, R⁹, and R¹⁰ are defined as above and wherein

E is CH or CD.

In another embodiment, the invention relates to compounds of Structural Formula I or a pharmaceutically acceptable sait, and a pharmaceutically acceptable carrier, diluent, or vehicle thereof, wherein E, G, J, R², R³, R⁴, R⁵, R⁶, R⁹, and R¹⁰ are defined as above and wherein

A is C.

A is N.

In another embodiment, the invention relates to compounds of Structural Formula I or a pharmaceutically acceptable sait, and a pharmaceutically acceptable carrier, diluent, or vehicle thereof, wherein A, G, E, R², R³, R⁴, R⁵, R⁹, and R¹⁰ are defined as above and wherein

Νχ\ / ^CD3

Q J-^r2 _r AOH H^oh

J is v and R⁶ is \ or ^CD3 .

In another embodiment, the invention relates to compounds, or a pharmaceutically acceptable sait, and a pharmaceutically acceptable carrier, diluent, or vehicle thereof, selected from the group consisting of the compounds described as examples A1 to A3, B4 to B9, C10 to C26, D27 to D29, and E30.

In another embodiment, the invention relates to compounds, or a pharmaceutically acceptable sait, and a pharmaceutically acceptable carrier, diluent, or vehicle thereof, selected from the group consisting of:

DEFINITIONS

As used herein, the terms “comprising” and “including” are used in their open, nonlimiting sense.

The terms halo and/or “halogen” refer to fluorine, chlorine, bromine or iodine.

The term “(Ci to C₆)” alkyl refers to a saturated aliphatic hydrocarbon radical including straight chain and branched chain groups of 1 to 6 carbon atoms. Examples of (Ci to C₆) alkyl groups include methyl, ethyl, propyl, 2-propyl, n-butyl, /so-butyl, tert-butyl, pentyl, and the like. The terms “Me” and “methyl,” as used herein, mean a -CH₃ group. The terms “Et” and “ethyl,” as used herein, mean a -C₂H₅ group.

The term (C2 to Cg) alkenyl, as used herein, means an alkyl moiety comprising 2 to 8 carbons having at least one carbon-carbon double bond. The carbon-carbon double bond in such a group may be anywhere along the 2 to 8 carbon chain that will resuit in a stable compound. Such groups include both the E and Z isomers of said alkenyl moiety. Examples of such groups include, but are not limited to, ethenyl, propenyl, butenyl, allyl, and pentenyl. The term “allyl,” as used herein, means a -CH₂CH=CH2 group. The term, “C(R)=C(R),” as used herein, represents a carbon-carbon double bond in which each carbon is substituted by an R group, and includes both the E and Z isomers.

As used herein, the term “(C2to Cs) alkynyl” means an alkyl moiety comprising from 2 to 8 carbon atoms and having at least one carbon-carbon triple bond. The carbon-carbon triple bond in such a group may be anywhere along the 2 to 8 carbon chain that will resuit in a stable compound. Examples of such groups include, but are not limited to, ethyne, propyne, 1-butyne, 2-butyne, 1-pentyne, 2-pentyne, 1-hexyne, 2-hexyne, and 3-hexyne.

The term (Ci to Cs) alkoxy, as used herein, means an O-alkyl group wherein said alkyl group contains from 1 to 8 carbon atoms and is straight, branched, or cyclic. Examples of such groups include, but are not limited to, methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butoxy, iso-butoxy, tert-butoxy, cyclopentyloxy, and cyclohexyloxy.

The term “(C₆to Cw) aryl”, as used herein, means a group derived from an aromatic hydrocarbon containing from 6 to 10 carbon atoms. Examples of such groups include, but are not limited to, phenyl or naphthyl. The terms “Ph” and “phenyl,” as used herein, mean a -C₆H₅ group. The term “benzyl,” as used herein, means a -CH₂C₆H₅ group.

“(C₂to C₉) heteroaryl”, as used herein, means an aromatic heterocyclic group having a total of from 5 to 10 atoms in its ring, and containing from 2 to 9 carbon atoms and from one to four heteroatoms each independently selected from O, S and N, and with the proviso that the ring of said group does not contain two adjacent O atoms or two adjacent S atoms. The heterocyclic groups include benzo-fused ring Systems. Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. The (C₂ to C₉) heteroaryl groups may be Cattached or N-attached where such is possible. For instance, a group derived from pyrrole may be pyrrol-1 -yl (N-attached) or pyrrol-3-yl (C-attached). Further, a group derived from imidazole may be imidazol-1-yl (N-attached) or imidazol-3-yl (C-attached).

“(C₂to C₉) cycloheteroalkyl”, as used herein, means a non-aromatic, monocyclic, bicyclic, tricyclic, spirocyclic, or tetracyclic group having a total of from 4 to 13 atoms in its ring system, and containing from 5 to 9 carbon atoms and from 1 to 4 heteroatoms each independently selected from O, S and N, and with the proviso that the ring of said group does not contain two adjacent O atoms or two adjacent S atoms. Furthermore, such C₂ to C₉ cycloheteroalkyl groups may contain an oxo substituent at any available atom that will resuit in a stable compound. For example, such a group may contain an oxo atom at an available carbon or nitrogen atom. Such a group may contain more than one oxo substituent if chemically feasible. In addition, it is to be understood that when such a C2 to C9 cycloheteroalkyl group contains a sulfur atom, said sulfur atom may be oxidized with one or two oxygen atoms to afford either a sulfoxide or sulfone. An example of a 4 membered cycloheteroalkyl group is azetidinyl (derived from azetidine). An example of a 5 membered cycloheteroalkyl group is pyrrolidinyl. An example of a 6 membered cycloheteroalkyl group is piperidinyl. An example of a 9 membered cycloheteroalkyl group is indolinyl. An example of a 10 membered cycloheteroalkyl group is 4/7-quinolizinyl.

Further examples of such C2 to C9 cycloheteroalkyl groups include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4/7 pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3azabicyclo[3.1.0]hexanyl, 3-azabicyclo [4.1,0]heptanyl, 3/7-indolyl quinolizinyl, 3oxopiperazinyl, 4-methylpiperazinyl, 4-ethylpiperazinyl, and 1-oxo-2,8,diazaspiro[4.5]dec-8yi·

The term (C₃ to Cio) cycloalkyl group means a saturated, monocyclic, fused, spirocyclic, or polycyclic ring structure having a total of from 3 to 10 carbon ring atoms. Examples of such groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptyl, and adamantyl.

The term spirocyclic as used herein has its conventional meaning, that is, any compound containing two or more rings wherein two ofthe rings hâve one ring carbon in common. The rings of a spirocyclic compound, as herein defined, independently hâve 3 to 20 ring atoms. Preferably, they hâve 3 to 10 ring atoms. Non-limiting examples of a spirocyclic compound include spiro[3.3]heptane, spiro[3.4]octane, and spiro[4.5]decane.

The term “(C₅to C₈) cycloalkenyl” means an unsaturated, monocyclic, fused, spirocyclic ring strucures having a total of from 5 to 8 carbon ring atoms. Examples of such groups include, but are not limited to, cyclopentenyl, cyclohexenyl.

An aldéhyde group refers to a carbonyl group, -C(O)R, where R is hydrogen.

An alkoxy group refers to both an -O-alkyl and an -O-cycloalkyl group, as defined herein.

An alkoxycarbonyl refers to a -C(O)OR.

An alkylaminoalkyl group refers to an -alkyl-NR-alkyl group.

An alkylsulfonyl group refer to a -SO2 alkyl.

An amino group refers to an -NH₂ or an -NRR' group.

An aminoalkyl group refers to an -alkyl-NRR' group.

An aminocarbonyl refers to a -C(O)NRR'.

An arylalkyl group refers to -alkylaryl, where alkyl and aryl are defined herein.

An aryloxy group refers to both an -O-aryl and an -O-heteroaryl group, as defined herein.

An aryloxycarbonyl refers to -C(O)O aryl.

An arylsulfonyl group refers to a -SO₂ aryl.

A C-amido group refers to a -C(0)NRR' group.

A carbonyl group refers to a -C(O)R.

A C-carboxyl group refers to a -C(O)OR groups.

A carboxylic acid group refers to a C-carboxyl group in which R is hydrogen.

A cyano group refers to a -CN group.

A dialkylaminoalkyl group refers to an -(alkyl)N(alkyl)₂ group.

A halo or halogen group refers to fluorine, chlorine, bromine or iodine.

A haloalkyl group refers to an alkyl group substituted with one or more halogen atoms.

A heteroaryloxyl group refers to a heteroaryl-O group with heteroaryl as defined herein.

A hydroxy group refers to an -OH group.

An N-amido group refers to a -R'C(0)NR group.

An N-carbamyl group refers to a -ROC(O)NR- group.

A nitro group refers to a -NO₂ group.

An N-Sulfonamido group refers to a -NR-S(O)₂R group.

An N-thiocarbamyl group refers to a ROC(S)NR' group.

An O-carbamyl group refers to a -OC(O)NRR' group.

An O-carboxyl group refers to a RC(O)O group.

An O-thiocarbamyl group refers to a -OC(S)NRR' group.

An “oxo” group refers to a carbonyl moiety such that alkyl substituted by oxo refers to a ketone group.

A perfluoroalkyl group refers to an alkyl group where ail ofthe hydrogen atoms hâve been replaced with fluorine atoms.

A phosphonyl group refers to a -P(O)(OR)₂ group.

A silyl group refers to a -SiR₃ group.

An S-sulfonamido group refers to a -S(O)₂NR- group.

A sulfinyl group refers to a -S(O)R group.

A sulfonyl group refers to a -S(O)₂R group.

A thiocarbonyl group refers to a -C(=S)-R group.

A trihalomethanecarbonyl group refers to a Z₃CC(O) group, where Z is halogen.

A trihalomethanesulfonamido group refers to a Z₃CS(O)₂NR- group, where Z is halogen.

A trihalomethanesulfonyl group refers to a Z₃CS(O)₂ group, where Z is halogen.

A trihalomethyl group refers to a -CZ₃ group, where Z is halogen.

A C-carboxyl group refers to a -C(O)OR groups.

The term “substituted,” means that the specified group or moiety bears one or more substituents.

The term “unsubstituted,” means that the specified group bears no substituents. The term “optionally substituted” means that the specified group is unsubstituted or substituted by one or more substituents. It is to be understood that in the compounds of the present invention when a group is said to be “unsubstituted,” or is “substituted” with fewer groups than would fill the valencies of ail the atoms in the compound, the remaining valencies on such a group are filled by hydrogen. For example, if a C₆ aryl group, also called “phenyl” herein, is substituted with one additional substituent, one of ordinary skill in the art would understand that such a group has 4 open positions left on carbon atoms of the C₆ aryl ring (6 initial positions, minus one to which the remainder ofthe compound ofthe present invention is bonded, minus an additional substituent, to leave 4). In such cases, the remaining 4 carbon atoms are each bound to one hydrogen atom to fill their valencies. Similarly, if a C₆ aryl group in the présent compounds is said to be “disubstituted,” one of ordinary skill in the art would understand it to mean that the C₆ aryl has 3 carbon atoms remaining that are unsubstituted. Those three unsubstituted carbon atoms are each bound to one hydrogen atom to fill their valencies.

The term solvaté, is used to describe a molecular complex between compounds of the présent invention and solvent molécules. Examples of solvatés include, but are not limited to, compounds of the invention in combination water, isopropanol, éthanol, methanol, dimethylsulfoxide (DMSO), ethyl acetate, acetic acid, ethanolamine, or mixtures thereof. The term “hydrate” can be used when said solvent is water. It is specifically contemplated that in the présent invention one solvent molécule can be associated with one molécule ofthe compounds ofthe présent invention, such as a hydrate. Furthermore, it is specifically contemplated that in the présent invention, more than one solvent molécule may be associated with one molécule of the compounds of the présent invention, such as a dihydrate. Additionally, it is specifically contemplated that in the présent invention less than one solvent molécule may be associated with one molécule of the compounds of the présent invention, such as a hemihydrate. Furthermore, solvatés ofthe présent invention are contemplated as solvatés of compounds of the présent invention that retain the biological effectiveness of the non-hydrate form of the compounds.

The term pharmaceutically acceptable sait, as used herein, means a sait of a compound of the présent invention that retains the biological effectiveness of the free acids and bases of the specified dérivative and that is not biologically or otherwise undesirable.

The term “pharmaceutically acceptable formulation,” as used herein, means a combination of a compound of the invention, or a sait or solvaté thereof, and a carrier, diluent, and/or excipient(s) that are compatible with a compound ofthe présent invention, and is not deleterious to the récipient thereof. Pharmaceutical formulations can be prepared by procedures known to those of ordinary skill in the art. For example, the compounds of the présent invention can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include the following: fillers and extenders such as starch, sugars, mannitol, and silicic dérivatives; binding agents such as carboxymethyl cellulose and othercellulose dérivatives, alginates, gelatin, and polyvinyl pyrrolidone; moisturizing agents such as glycerol; disintegrating agents such as povidone, sodium starch glycolate, sodium carboxymethylcellulose, agar, calcium carbonate, and sodium bicarbonate; agents for retarding dissolution such as paraffin; résorption accelerators such as quaternary ammonium compounds; surface active agents such as cetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite; and lubricants such as talc, calcium and magnésium stéarate and solid polyethylene glycols. Final pharmaceutical forms may be pills, tablets, powders, lozenges, saches, cachets, dragees, or stérile packaged powders, and the like, depending on the type of excipient used. Additionally, it is specifically contemplated that pharmaceutically acceptable formulations of the présent invention can contain more than one active ingrédient. For example, such formulations may contain more than one compound according to the présent invention. Altematively, such formulations may contain one or more compounds of the présent invention and one or more additional agents that inhibit arenavirus.

The term “Arenavirus GP-inhibiting amount” as used herein, refers to the amount of a compound of the présent invention, or a sait or solvaté thereof, required to inhibit the cell entry of Arenaviruses in vivo, such as in a mammal, birds or in vitro. The amount of such compounds required to cause such inhibition can be determined without undue expérimentation using methods described herein and those known to those of ordinary skiil in the art.

The term therapeutically effective amount, as used herein, means an amount of a compound of the présent invention, or a sait thereof, that, when administered to a mammal in need of such treatment, is sufficient to effect treatment, as defined herein. Thus, a therapeutically effective amount of a compound of the présent invention, or a sait thereof, is a quantity sufficient to modulate or inhibit the activity ofthe Arenavirus GP protein such that cell entry and réplication of arenaviruses that is mediated by activity of the Arenavirus GP protein is reduced or alleviated.

The terms treat, treating, and treatment with reference to arenavirus infection, in mammals, particularly a human, include: (i) preventing the disease or condition from occurring in a subject which may be predisposed to the condition, such that the treatment constitutes prophylactic treatment for the pathologie condition; (ii) modulating or inhibiting the disease or condition, i.e., arresting its development; (iii) relieving the disease or condition, i.e., causing régression of the disease or condition; or (iv) relieving and/or alleviating the disease or condition or the symptoms resulting from the disease or condition.

Unless indicated otherwise, ail references herein to the inventive compounds include references to salts, solvatés, and complexes thereof, including polymorphs, stereoisomers, tautomers, and isotopically labeled versions thereof. For example, compounds ofthe présent invention can be pharmaceutically acceptable salts and/or pharmaceutically acceptable solvatés.

The term stereoisomers refers to compounds that hâve identical Chemical constitution, but differ with regard to the arrangement of their atoms or groups in space. In particular, the term enantiomers refers to two stereoisomers of a compound that are nonsuperimposable mirror images of one another. A pure enantiomer can be contaminated with up to about 10% ofthe opposite enantiomer.

The terms “racemic” or “racemic mixture,” as used herein, refer to a 1:1 mixture of enantiomers of a particular compound. The term diastereomers, on the other hand, refers to the relationship between a pair of stereoisomers that comprise two or more asymmetric centers and are not mirror images of one another. In accordance with a convention used in the art, the symbol is used in structural formulas herein to depict the bond that is the point of attachment of the moiety or substituent to the core or backbone structure. In accordance with another convention, in some structural formulas herein the carbon atoms and their bound hydrogen atoms are not explicitly depicted, e.g., represents a methyl group, represents an ethyl group, L- represents a cyclopentyl group, etc.

The compounds of the présent invention may hâve asymmetric carbon atoms. The carbon carbon bonds ofthe compounds ofthe présent invention may be depicted herein using a solid line (—), a solid wedge (— ), or a dotted wedge (.....^. ). The use of a solid line to depict bonds to asymmetric carbon atoms is meant to indicate that ail possible stereoisomers (e.g. spécifie enantiomers, racemic mixtures, etc.) at that carbon atom are included. The use of either a solid or dotted wedge to depict bonds to asymmetric carbon atoms is meant to indicate that only the stereoisomer shown is meant to be included. It is possible that compounds ofthe invention may contain more than one asymmetric carbon atom. In those compounds, the use of a solid line to depict bonds to asymmetric carbon atoms is meant to indicate that ali possible stereoisomers are meant to be included. For example, unless stated otherwise, it is intended that the compounds of the présent invention can exist as enantiomers and diastereomers or as racemates and mixtures thereof. The use of a solid line to depict bonds to one or more asymmetric carbon atoms in a compound of the invention and the use of a solid or dotted wedge to depict bonds to other asymmetric carbon atoms in the same compound is meant to indicate that a mixture of diastereomers is présent.

Unless otherwise defined, a substituent “R” may résidé on any atom of the ring system, assuming replacement of a depicted, implied, or expressly defined hydrogen from one of the ring atoms, so long as a stable structure is formed.

Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate using, for example, chiral high pressure liquid chromatography (HPLC). Altematively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound contains an acidic or basic moiety, an acid or base such as tartaric acid or 1-phenyl ethyl amine. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both ofthe diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to one skilled in the art. Chiral compounds ofthe invention (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% isopropanol, typically from 2 to 20%, and from 0 to 5% of an alkylamine, typically 0.1% diethylamine. Concentration ofthe eluate affords the enriched mixture. Stereoisomeric conglomérâtes may be separated by conventional techniques known to those skilled in the art. See, e.g. “Stereochemistry of Organic Compounds” by E L Eliel (Wiley, New York, 1994), the disclosure of which is incorporated herein by reference in its entirety.

Where a compound ofthe invention contains an alkenyl or alkenylene group, géométrie cisltrans (or Z/E) isomers are possible. Where the compound contains, for example, a keto or oxime group or an aromatic moiety, tautomeric isomerism (‘tautomerism’) can occur. Examples of tautomerism include keto and enol tautomers. A single compound may exhibit more than one type of isomerism. Included within the scope of the invention are ail stereoisomers, géométrie isomers and tautomeric forms of the inventive compounds, including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof. Cis/trans isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallization.

The compounds ofthe présent invention may be administered as prodrugs. Thus certain dérivatives of compounds of Formula I, which may hâve little or no pharmacological activity themselves can, when administered to a mammal, be converted into a compound of Formula (I) having the desired activity, for example, by hydrolytic cleavage. Such dérivatives are referred to as “prodrugs”. Prodrugs can, for example, be produced by replacing appropriate functionalities présent in the compounds of Formula I with certain moieties known to those skilled in the art. See, e.g. “Pro-drugs as Novel Delivery Systems”, Vol. 14, ACS Symposium Sériés (T Higuchi and W Stella) and “Bioreversible Carriers in Drug Design”, Pergamon Press, 1987 (ed. E B Roche, American Pharmaceutical Association), the disclosures of which are incorporated herein by reference in their entireties. Some examples of such prodrugs include: an ester moiety in the place of a carboxylic acid functional group; an ether moiety or an amide moiety in place of an alcohol functional group; and an amide moiety in place of a primary or secondary amino functional group. Further examples of replacement groups are known to those of skill in the art. See, e.g. “Design of Prodrugs by H Bundgaard (Elsevier, 1985), the disclosure of which is incorporated herein by reference in its entirety. It is also possible that certain compounds of Formula I may themselves act as prodrugs of other compounds of Formula I.

Salts of the présent invention can be prepared according to methods known to those of skill in the art. Examples of salts include, but are not limited to, acetate, acrylate, benzenesulfonate, benzoate (such as chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, and methoxybenzoate), bicarbonate, bisulfate, bisulfite, bitartrate, borate, bromide, butyne-1,4-dioate, calcium edetate, camsylate, carbonate, chloride, caproate, caprylate, clavulanate, citrate, decanoate, dihydrochloride, dihydrogenphosphate, edetate, edislyate, estolate, esylate, ethylsuccinate, formate, fumarate, gluceptate, gluconate, glutamate, glycollate, glycollylarsanilate, heptanoate, hexyne-1,6-dioate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, yhydroxybutyrate, iodide, isobutyrate, isothionate, lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, mesylate, metaphosphate, methanesulfonate, methylsulfate, monohydrogenphosphate, mucate, napsylate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, nitrate, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, phenylacetates, phenylbutyrate, phenylpropionate, phthalate, phospate/diphosphate, polygalacturonate, propanesulfonate, propionate, propiolate, pyrophosphate, pyrosulfate, salicylate, stéarate, subacetate, suberate, succinate, sulfate, sulfonate, sulfite, tannate, tartrate, teoclate, tosylate, triethiodode, and valerate salts.

The compounds of the présent invention that are basic in nature are capable of forming a wide variety of different salts with various inorganic and organic acids. Although such salts must be pharmaceutically acceptable for administration to animais, it is often désirable in practice to initially isolate the compound ofthe présent invention from the reaction mixture as a pharmaceutically unacceptable sait and then simply convert the latter back to the free base compound by treatment with an alkaline reagent and subsequently convert the latter free base to a pharmaceutically acceptable acid addition sait. The acid addition salts ofthe base compounds of this invention can be prepared by treating the base compound with a substantially équivalent amount of the selected minerai or organic acid in an aqueous solvent medium or in a suitable organic solvent, such as methanol or éthanol. Upon évaporation of the solvent, the desired solid sait is obtained. The desired acid sait can also be precipitated from a solution of the free base in an organic solvent by adding an appropriate minerai or organic acid to the solution.

Those compounds of the présent invention that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include the alkali métal or alkaline-earth métal salts and particularly, the sodium and potassium salts. These salts are ail prepared by conventional techniques. The Chemical bases which are used as reagents to préparé the pharmaceutically acceptable base salts of this invention are those which form non-toxic base salts with the acidic compounds of the present invention. Such non-toxic base salts include those derived from such pharmacologically acceptable cations as sodium, potassium calcium and magnésium, etc. These salts can be prepared by treating the corresponding acidic compounds with an aqueous solution containing the desired pharmacologically acceptable cations, and then evaporating the resulting solution to dryness, preferably under reduced pressure. Altematively, they may also be prepared by mixing lower alkanolic solutions of the acidic compounds and the desired alkali métal alkoxide together, and then evaporating the resulting solution to dryness in the same manner as before. In either case, stoichiometric quantifies of reagents are preferably employed in order to ensure completeness of reaction and maximum yields of the desired final product.

If the inventive compound is a base, the desired sait may be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha-hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as ptoluenesulfonic acid or ethanesulfonic acid, or the like.

If the inventive compound is an acid, the desired sait may be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary), an alkali métal hydroxide or alkaline earth métal hydroxide, or the like. Illustrative examples of suitable salts include organic salts derived from amino acids, such as glycine and arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines, such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnésium, manganèse, iron, copper, zinc, aluminum and lithium.

In the case of agents that are solids, it is understood by those skilled in the art that the inventive compounds, agents and salts may exist in different crystal or polymorphie forms, ail of which are intended to be within the scope of the présent invention and specified formulas.

The invention also includes isotopically-labeled compounds ofthe invention, wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as ²H and ³H, carbon, such as ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶CI, ³⁵CI, and ³⁷CI, fluorine, such as ¹⁸F, iodine, such as ¹²³l and ¹²⁵l, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, and sulfur, such as ³⁵S.

Certain isotopically-labeled compounds of the invention, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, ³H, and carbon-14, ¹⁴C, are particularly useful for this purpose in view of their ease of incorporation and ready means of détection. Substitution with heavier isotopes such as deuterium, ²H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, ³⁵S increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances. Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, can be useful in Positron Emission Tomography (PET) studies for examining substrate receptor occupancy.

Isotopically-labeled compounds of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described herein, using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.

The term “deuterated” refers to the replacement of one or more hydrogen atoms with a corresponding number of deuterium atoms. Unless otherwise stated, when a particular position in a compound of this invention is designated specifically as “D”, “deuterium”, being “deuterated”, or “having deuterium” (the element deuterium is represented by the letter “D” in Chemical structures and formulas and indicated with a lower case “d” in Chemical names), the position is understood to hâve deuterium at an abundance that is at least 3000 times greater than the natural abundance of deuterium, which is 0.015% (i.e., the term “D”, “d” or “deuterium” indicates at least 45% incorporation of deuterium).

The term “isotopic enrichment factor”, as used herein, means the ratio between the isotopic abundance and the natural abundance of a specified isotope.

In some embodiments, a compound of this invention has an isotopic enrichment factor for each deuterium présent at a site designated as a potential site of deutération on the compound of at least 3500 (52.5% deuterium incorporation), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).

The compounds of the présent invention may be formulated into pharmaceutical compositions as described below in any pharmaceutical form recognizable to the skilled artisan as being suitable.

Pharmaceutical compositions of the invention comprise a therapeutically effective amount of at least one compound of the présent invention and an inert, pharmaceutically acceptable carrier or diluent.

To treat or prevent diseases or conditions mediated in part or whole by arenavirus infection or viruses expressing the arenavirus glycoprotein, a pharmaceutical composition of the invention is administered in a suitable formulation prepared by combining a therapeutically effective amount (i.e., an arenavirus GP modulating, regulating, or inhibiting amount effective to achieve therapeutic efficacy) of at least one compound of the présent invention (as an active ingrédient) with one or more pharmaceutically suitable carriers, which may be selected, for example, from diluents, excipients and auxiliaries that facilitate processing of the active compounds into the final pharmaceutical préparations.

The pharmaceutical carriers employed may be either solid or liquid. Exemplary solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnésium stéarate, stearic acid and the like.

Exemplary liquid carriers are syrup, peanut oil, olive oil, water and the like. Similarly, the inventive compositions may include time-delay or time-release material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate or the like. Further additives or excipients may be added to achieve the desired formulation properties. For example, a bioavailability enhancer, such as Labrasol, Gelucire or the like, or formulator, such as CMC (carboxy-methylcellulose), PG (propyleneglycol), or PEG (polyethyleneglycol), may be added. Gelucire®, a semi-solid vehicle that protects active ingrédients from light, moisture and oxidation, may be added, e.g., when preparing a capsule formulation.

If a solid carrier is used, the préparation can be tableted, placed in a hard gelatin capsule in powder or pellet form, or formed into a troche or lozenge. The amount of solid carrier may vary, but generally will be from about 25 mg to about 1 g. If a liquid carrier is used, the préparation may be in the form of syrup, émulsion, soft gelatin capsule, stérile injectable solution or suspension in an ampoule or vial or non-aqueous liquid suspension. If a semi-solid carrier is used, the préparation may be in the form of hard and soft gelatin capsule formulations. The inventive compositions are prepared in unit-dosage form appropriate for the mode of administration, e.g. parentéral or oral administration.

To obtain a stable water-soluble dose form, a sait of a compound of the présent invention may be dissolved in an aqueous solution of an organic or inorganic acid, such as a 0.3 M solution of succinic acid or citric acid. If a soluble sait form is not available, the agent may be dissolved in a suitable co-solvent or combinations of co-solvents. Examples of suitable co-solvents include alcohol, propylene glycol, polyethylene glycol 300, polysorbate 80, glycerin and the like in concentrations ranging from 0 to 60% of the total volume. The composition may also be in the form of a solution of a sait form of the active ingrédient in an appropriate aqueous vehicle such as water or isotonie saline or dextrose solution.

Proper formulation is dépendent upon the route of administration selected. For injection, the agents of the compounds ofthe présent invention may be formulated into aqueous solutions, preferably in physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological saline buffer.

For transmucosal administration, pénétrants appropriate to the barrier to be permeated are used in the formulation. Such pénétrants are generally known in the art.

For oral administration, the compounds can be formulated by combining the active compounds with pharmaceutically acceptable carriers known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical préparations for oral use can be obtained using a solid excipient in admixture with the active ingrédient (agent), optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include: fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; and cellulose préparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a sait thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, polyvinyl pyrrolidone, Carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active agents.

Pharmaceutical préparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingrédients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnésium stéarate, and, optionally, stabilizers. In soft capsules, the active agents may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. AH formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration intranasally or by inhalation, the compounds for use according to the présent invention may be conveniently delivered in the form of an aérosol spray présentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aérosol the dosage unit may be determined by providing a valve to deliver a metered amount.

Capsules and cartridges of gelatin for use in an inhaler or insufflator and the like may be formulated containing a powder mix ofthe compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parentéral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit-dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or émulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parentéral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active agents may be prepared as appropriate oily injection suspensions. Suitable lipophilie solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycérides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the préparation of highly concentrated solutions.

Alternatively, the active ingrédient may be in powder form for constitution with a suitable vehicle, e.g. stérile pyrogen-free water, before use.

In addition to the formulations described above, the compounds ofthe présent invention may also be formulated as a depot préparation. Such long-acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobie materials (for example, as an émulsion in an acceptable oil) or ion-exchange resins, or as sparingly soluble dérivatives, for example, as a sparingly soluble sait. A pharmaceutical carrier for hydrophobie compounds is a co-solvent system comprising benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. The co-solvent system may be a VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the non-polar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute éthanol. The VPD co-solvent system (VPD: 5W) contains VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobie compounds well, and itself produces low toxicity upon systemic administration. The proportions of a co-solvent system may be suitably varied without destroying its solubility and toxicity characteristics. Furthermore, the identity ofthe cosolvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may be substituted for dextrose.

Alternatively, other delivery Systems for hydrophobie pharmaceutical compounds may be employed. Liposomes and émulsions are known examples of delivery vehicles or carriers for hydrophobie drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity due to the toxic nature of DMSO.

Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobie polymers containing the therapeutic agent. Various sustained-release materials hâve been established and are known by those skilled in the art. Sustained-release capsules may, depending on their Chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the Chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid- or gel-phase carriers or excipients. These carriers and excipients may provide marked improvement in the bioavailability of poorly soluble drugs. Examples of such carriers or excipients include calcium carbonate, calcium phosphate, sugars, starches, cellulose dérivatives, gelatin, and polymers such as polyethylene glycols. Furthermore, additives or excipients such as Gelucire®, Capryol®, Labrafil®, Labrasol®, Lauroglycol®, Plurol®, Peceol®, Transcutol® and the like may be used.

Further, the pharmaceutical composition may be incorporated into a skin patch for delivery of the drug directly onto the skin.

It will be appreciated that the actual dosages of the agents of this invention will vary according to the particular agent being used, the particular composition formulated, the mode of administration, and the particular site, host, and disease being treated. Those skilled in the art using conventional dosage détermination tests in view ofthe experimental data for a given compound may ascertain optimal dosages for a given set of conditions. For oral administration, an exemplary daily dose generally employed will be from about 0.001 to about 1000 mg/kg of body weight, with courses of treatment repeated at appropriate intervals.

Furthermore, the pharmaceutically acceptable formulations ofthe présent invention may contain a compound ofthe présent invention, or a sait or soivate thereof, in an amount of about 10 mg to about 2000 mg, or from about 10 mg to about 1500 mg, or from about 10 mg to about 1000 mg, or from about 10 mg to about 750 mg, or from about 10 mg to about 500 mg, or from about 25 mg to about 500 mg, or from about 50 to about 500 mg, or from about 100 mg to about 500 mg.

Additionally, the pharmaceutically acceptable formulations of the présent invention may contain a compound of the présent invention, or a sait or solvaté thereof, in an amount from about 0.5 w/w% to about 95 w/w%, or from about 1 w/w% to about 95 w/w%, or from about 1 w/w% to about 75 w/w%, orfrom about 5 w/w% to about 75 w/w%, or from about 10 w/w% to about 75 w/w%, or from about 10 w/w% to about 50 w/w%.

The compounds of the présent invention, or salts or solvatés thereof, may be administered to a mammal, such as a human, suffering from a condition or disease mediated by arenavirus or any virus expressing arenavirus glycoprotein, either alone or in combination with one or more compounds selected from Ribavirin, polymerase inhibitors, Favipiravir, Triazavirin, small interfering RNAs (siRNAs), vaccines, monoclonal antibodies, immunomodulators, and other arenavirus inhibitors as part of a pharmaceutically acceptable formulation, once a day, twice a day, three times a day, four times a day, or even more frequently.

The compounds ofthe présent invention, or salts or solvatés thereof, may be administered to a mammal, such as a human, suffering from a condition or disease mediated by arenavirus in combination with at least one other agent used for treatment of arenavirus selected from the group consisting of Ribavirin, viral RNA-dependent-RNApolymerase inhibitors as shown by Ng KK, Arnold JJ and Cameron CE, Structure-Function Relationships Among RNA-Dependent RNA Polymerases, Curr Top Microbiol Immunol, 2008; 320: 137-156, incorporated herein by reference in its entirety, Favipiravir, a broadspectrum inhibitor of viral RNA-Dependent RNA Polymerases, Triazavirin, a broadspectrum inhibitor of viral RNA-Dependent RNA Polymerases, small interfering RNAs (siRNAs) and microRNAs as shown by Carthew RW and Sontheimer EJ, Origins and Mechanisms ofmiRNAs and siRNAs, Nature, 2009; 136: 642-655, incorporated herein by reference in its entirety, vaccines as shown by Nablel GJ, Designing Tomorrow’s Vaccines, NEJM, 2013; 368: 551-560, incorporated herein by reference in its entirety, and immunomodulators as shown by Patil US, Jaydeokar AV and Bandawane DD, Immunomodulators: A Pharmacological Review, Internatl J Pharmacy and Pharmaceutical Sci, 2012; 4: 30-36, incorporated herein by reference in its entirety], alone or as part of a pharmaceutically acceptable formulation containing other arenavirus inhibitors, once a day, twice a day, three times a day, four times a day, or even more frequently.

Those of ordinary skill in the art will understand that with respect to the compounds of the présent invention, the particular pharmaceutical formulation, the dosage, and the number of doses given per day to a mammal requiring such treatment, are ail choices within the knowledge of one of ordinary skill in the art and can be determined without undue expérimentation.

The compounds of the présent invention are useful for modulating or inhibiting arenavirus glycoprotein (GP) both in vitro and in vivo.

Accordingly, these compounds are useful for the prévention and/or treatment of disease States associated with arenavirus infection or treating viruses expressing the arenavirus glycoprotein.

This invention also relates to a method for the treatment of arenavirus infection in mammals including a human comprising administering to said mammal an amount of a compound of the Formula I, as defined above, or a sait or solvaté thereof, that is effective in treating disease States associated with Arenavirus infection or viruses expressing the arenavirus glycoprotein.

In the following Préparations and Examples, “Ac” means acetyl, “Me” means methyl, “Et” means ethyl, “Ph” means phenyl, “Py” means pyridine, “BOC”, “Boc” or “boc” means N-tert-butoxycarbonyl, “Ns“ means 2-Nitrophenylsulfonyl, “DCM” (CH₂CI₂) means dichloromethane or methylene chloride, “dba” means dibenzylideneacetone, “DCE” means dichloroethane or ethylene chloride, “D” or “d” means deuterium, “DIAD” means diisopropylazadicarboxylate, “DIPEA” or “DIEA” means diisopropyl ethyl amine, “DMA” means Ν,Ν-dimethylacetamide, DMF means N-N-dimethyl formamide, “DMSO means dimethylsulfoxide, “DPPP” means 1,3-bis(diphenylphosphino)propane, “HOAc” means acetic acid, “IPA” means isopropyl alcohol, “NMP” means 1-methyl 2-pyrrolidinone, “TEA” means triethyl amine, “TFA” means trifluoroacetic acid, “DCM” means dichloromethane, “EtOAc” means ethyl acetate, “MgSO₄” means magnésium sulphate, “Na₂SO₄” means sodium sulphate, “MeOH” means methanol, “Et₂O” means diethyl ether, “EtOH” means éthanol, “H₂O” means water, “HCl” means hydrochloric acid, “POCI₃” means phosphorus oxychloride,“SOCI₂“ means thionylchloride, “K₂CO₃” means potassium carbonate, “THF” means tetrahydrofuran, “DBU” means 1,8-diazabicyclo[5.4.0]undec-7-ene, “LiHMDS” or “LHMDS” means lithium hexamethyldisilazide, “TBME” or MTBE means tert-butyl methyl ether, “LDA” means lithium diisopropylamide, “NBS” means /V-bromosuccinimide, “NIS” means /V-iodosuccinimide, “Xanthphos” means 4,5-bis(diphenylphosphino)-9,9dimethylxanthene; “P(Ph₃)” means triphenylphosphine, “N” means Normal, “M” means molar, “mL” means millilitre, “mmol” means millimoles, “pmol” means micromoles, “eq. means équivalent, “°C” means degrees Celsius, “Pa” means pascals,

Methods of Préparation.

Compounds of the présent invention may be prepared using the reaction routes and synthetic schemes described below, employing the techniques available in the art using starting materials that are readily available. The préparation of certain embodiments of the présent invention is described in detail in the following examples, but those of ordinary skill in the art will recognize that the préparations described may be readily adapted to préparé other embodiments ofthe présent invention. For example, the synthesis of non-exemplified compounds according to the invention may be performed by modifications apparent to those skilled in the art, e.g. by appropriately protecting interfering groups, by changing to other suitable reagents known in the art, or by making routine modifications of reaction conditions. Alternatively, other reactions referred to herein or known in the art will be recognized as having adaptability for preparing other compounds of the invention.

Scheme 1 shows a method useful for the synthesis of compounds of structural Formula I wherein G is CH, J is N, E is CH, and A is C. Compound 1-1 (X = Cl, Br, or I, and Y is F, Cl, Br, or I) can be reacted with amine R²NH2 in the presence of a base such as NaH or Cs2CO3 in a solvent such as THF or DMF to form compound 1-2. Réduction ofthe nitro group using a reducing agent such as Fe or SnCI2 in a solvent such as THF or methanol can provide aniline 1-3 which can be reacted with formic acid HCO2H or orthoester HC(OR)3to form 1-4. Coupling of 1-4 with a boronic acid or boronic ester R¹B(OR)2 using a catalyst such as [1 ,T-bis(diphenylphosphino)ferrocene]palladium(ll) dichloride in the presence of a base such as K₂CO₃ in a solvent such as dimethoxyethane can provide compound of structural Formula I.

Scheme 1

I

Scheme 2 depicts a method of synthesis of deuterated aniline 2-4 useful in preparing deuterated intermediates 1-2 for the synthesis of deuterated compounds of the invention as described in Scheme 1 above. Reaction of phénol 2-1 with deuterated alkyl halide 2-2 (X' = Br or I) in the presence of a base such as K₂CO₃ in a solvent such as N,Ndimethylformamide can afford compound 2-3. Réduction ofthe nitro group using a reducing agent such as hydrogen gas in the presence of a catalyst such as palladium on carbon in a solvent such as methanol can provide aniline 2-4.

Scheme 2

OH

2-1

D I

CD₃ 2-2

cd₃

2-4

Scheme 3 depicts a method of synthesis of deuterated aniline 3-4 useful in preparing deuterated intermediates 1-2 for the synthesis of deuterated compounds of the invention as described in Scheme 1 above. Arylation of alcohol 3-1 with diaryliodonium sait 3-2 in the presence of a base such as NaHMDS in a solvent such as pentane can provide compound 3-3 [Lindstedt, E.; Stridfeldt, E.; Olofsson, B. Mild synthesis of sterically congested alkyl aryl ethers. Org. Lett. (2016) 18: 4234-4237], The above paper is herein incorporated by reference in its entirety for ail purposes. Réduction of the nitro group using a reducing agent such as hydrogen gas in the presence of a catalyst such as palladium on carbon in a solvent such as methanol can provide aniline 3-4.

Scheme 3

Scheme 4 depicts a method useful for the synthesis of compounds of structural Formula I wherein A is N, E is CH, and J is C. Compound 4-1 (X = Cl, Br) can be coupled with a boronic acid or boronic ester R¹B(OR)2 using a catalyst such as [1,1’bis(diphenylphosphino)ferrocene] palladium(ll) dichloride in the presence of a base such as K2CO3 in a solvent such as dimethoxyethane to form 4-2 which can be treated with a halogénation reagent such as bromine or A/-bromosuccinimide (NBS), or iodine or Niodosuccinimide (NIS) to form compound 4-3 (Y = Br, I). Treatment of 4-3 with a boronic acid or boronic ester R²B(OR)2 using a catalyst such as tetrakis(triphenylphosphine)palladium in the presence of a base such as K2CO3 in a solvent such as dioxane can provide a compound of Structural Formula I. Altematively, compound 4-4 (X = Cl, Br) can react with boronic acid or boronic ester R²B(OR)2 using a catalyst such as tetrakis(triphenylphosphine) palladium in the presence of a base such as K2CO3 in a solvent such as dioxane to provide compound 4-5 which can react with a second boronic acid or boronic ester R¹B(OR)2 using a catalyst such as [1 ,Tbis(diphenylphosphino)ferrocene] palladium(ll) dichloride in the presence of a base such as K₂CO₃ in a solvent such as dimethoxyethane to provide a compound of structural Formula

Scheme 4

4-1

R¹B(OR)₂

Halogénation

4-4

R²B(OR)₂

4-5

R¹B(OR)₂

Scheme 5 depicts a method useful for the synthesis of compounds of structural Formula 1 wherein G is CH, A is C, J is N, and E is N. Compound 5-1 (X = Cl, Br, or I, and Y is F, Cl, Br, or I) can be reacted with amine R²NH₂ in the presence of a base such as

NaH or K2CO3 in a solvent such as THF or DMF to form compound 5-2. Réduction of the nitro groups using a reducing agent such as Fe or SnCI₂ in a solvent such as THF or methanol can provide aniline 5-3 which can be reacted with nitrous acid to form 5-4. Coupling of 5-4 with a boronic acid or boronic ester R¹B(OR)₂ using a catalyst such as [1 ,T-bis(diphenylphosphino)fenOcene]palladium(ll) dichloride in the presence of a base such as K₂COs in a solvent such as dimethoxyethane can provide compound of structural Formula I.

Scheme 5

Reaction Schemes 6-8 illustrate methods of synthesis of borane reagents 6-4, 7-4, 15 and 8-4 useful in preparing deuterated intermediates and final compounds ofthe invention as described in Schemes 1,4, and 5 above, to introduce R¹ and/or R² substituents.

Scheme 6 depicts a method useful for the synthesis of deuterated boronic acid or ester 6-4. Reaction of phénol 6-1 (X = Br or I) with deuterated alkyl halide 6-2 (X' = Br or I) in the presence of a base such as K₂CO₃ in a solvent such as Ν,Ν-dimethylformamide can afford compound 6-3. Compound 6-3 can be converted to a boronic acid or ester 6-4 using standard borylation reaction conditions well known to those skilled in the art. For example, metal-halogen exchange of compound 6-3 with organolithium reagent such as nButyllithium followed by treatment with trialkyl borate B(OR)₃ can provide boronic ester 6-4, which can be hydrolyzed to afford free boronic acid 6-4 (R = H).

Scheme 6

6-1 6-3 6-4

Scheme 7 depicts a method useful for the synthesis of deuterated boronic acid or ester 7-4. Reaction of phénol 7-1 (X = Br or I) with deuterated alkyl bromide 7-2 using catalyst such as nickel(lI) acetylacetonate in the presence of a base such as NaHCO₃ in a solvent such as toluene can afford compound 7-3 [Hodous, B. L. US patent application, publication number US2016/0031892, 4 Feb 2016]. The above patent is herein incorporated by reference in its entirety for ail purposes. Compound 7-3 can be converted to a boronic acid or ester 7-4 using standard borylation reaction conditions well known to those skilled in the art. For example, metal-halogen exchange of compound 7-3 with organolithium reagent such as n-Butyllithium followed by treatment with trialkyl borate B(OR)₃ can provide boronic ester 7-4, which can be hydrolyzed to afford free boronic acid 7-4 (R = H).

Scheme 7

X °³Μβγ x Q Ç OH O 7-1 7-:

B(OR)₂ Y borylation, i| ] I^CDj CD3 CD3 3 7-4

Scheme 8 depicts a method usefui for the synthesis of deuterated boronic acid or ester 8-4. Metal-halogen exchange of compound 8-1 (X = Br or I) with organolithium reagent such as n-Butyllithium followed by treatment with compound 8-2 in a solvent such as tetrahydrofuran can provide compound 8-3. Compound 8-3 can be converted to a boronic acid or ester 8-4 using standard borylation reaction conditions well known to those skilled in the art. For example, coupling of compound 8-3 with diboronyl reagent such as bis(pinacolato)diboron using a catalyst such as [1,Tbis(diphenylphosphino)ferrocene]palladium(ll) dichloride in the presence of a base such as potassium acetate in a solvent such as dioxane can provide boronic ester 8-4.

Scheme 8

1. n-BuLi

8-1 8-3 8-4

Examples

Préparation of intermediates for Examples A1 to A3.

5-Bromo-/V¹-(4-isopropoxyphenyl)-4-methylbenzene-1,2-diamine

/-PrOH, 120 °C

Fe, NH₄CI, AcOH, 80 °C

To a solution of 1-bromo-5-fluoro-2-methyl-4-nitrobenzene (200 mg, 0.85 mmol) in ipropanol (2 mL), was added 4-isopropoxyaniline (129 mg, 0.85 mmol). The resulting mixture was stirred at 120 °C for 30 min under microwave irradiation. After cooling to room température, the reaction was concentrated under reduced pressure and the residue was dissolved in éthanol (0.6 mL), dioxane (0.6 mL), and water (0.3 mL). To the solution was added iron (476 mg, 8.5 mmol) and NH₄CI (457 mg, 8.5 mmol). The reaction was stirred at 80 °C for 2 hr. After cooling to room température, the reaction was filtered through a celite pad. The filtrate was concentrated under reduced pressure and the residue was poured into water and extracted with ethyl acetate. The organic phase was dried over Na₂SO4, filtered, and concentrated in vacuo. The residue was purified by SiO₂ column chromatography (hexane/EtOAc = 3:1) to give 198 mg (69.2 %) ofthe product as a white solid. LC/MS m/z: 335.13 (⁷⁹Br, M+H)⁺, 337.19 (⁸¹Br, M+H)⁺, 376.28 (⁷⁹Br, M+H+CH3CN)⁺, 378.25 (⁸¹Br, M+H+CH3CN)⁺.

5-bromo-N1-(4-(tert-butoxy)phenyl)-4-methylbenzene-1,2-diamine

The title compound was prepared from 1-bromo-5-fluoro-2-methyl-4-nitrobenzene and 4(tert-butoxy)aniline in the same manner as described for 5-Bromo-A/¹-(4-isopropoxyphenyl)4-methylbenzene-1,2-diamine. LC/MS m/z: 351.24 (⁷⁹Br, M+H+CH₃CN)⁺

6-Bromo-5-methyl-1 -[4-(propan-2-yloxy)phenyl]-1 HA ,3-benzodiazole

MeO^^OMe OMe

To a solution of 5-bromo-/V¹-(4-isopropoxyphenyl)-4-methylbenzene-1,2-diamine (50.1 mg, 0.15 mmol) in THF (1 mL), was added trimethoxymethane (18.9 mg, 0.18 mmol) followed by formic acid (100 uL). The resulting mixture was stirred at 80 °C for 2 hr. After cooling to 20 room température, the reaction was poured into water and extracted with ethyl acetate.

The organic phase was dried over Na₂SO4, filtered, and concentrated in vacuo. The residue was purified by SiO₂ column chromatography (hexane/EtOAc = 1:1) to give 42.1 mg (81.7 %) ofthe product as a white solid. LC/MS m/z\ 345.15 (⁷⁹Br, M+H)⁺, 347.21 (⁸¹Br, M+H)⁺, 386.28 (⁷⁹Br, M+H+CH3CN)⁺, 388.20 (⁸¹Br, M+H+CH3CN)⁺.

6-bromo-1-(4-(tert-butoxy)phenyl)-5-methyl-1 H-benzo[d]imidazole

Y Y>

Y o~\/

The title compound was prepared from 5-bromo-N1-(4-(tert-butoxy)phenyl)-4methylbenzene-1,2-diamine in the same manner as described for 6-Bromo-5-methyl-1-[4(propan-2-yloxy)phenyl]-1H-1,3-benzodiazole. ¹H NMR (500 MHz, DMSO-d6) δ 8.51 (s, 1 H), 7.77 (s, 1 H), 7.74 (s, 1 H), 7.57 (d, 2H), 7.21 (d, 2H), 2.47 (s, 3H), 1.37 (s, 9H). LC/MS m/z: 359.16 (⁷⁹Br, M+H+CH3CN)⁺, 361.17 (⁸¹Br, M+H+CH3CN)⁺.

Example A1 : 2-(4-(1 -(4-(tert-butoxy)phenyl)-5-methyl-1 H-benzo[d]imidazol-6yl)phenyl)propan-2-ol

To a solution of 6-bromo-1-(4-(tert-butoxy)phenyl)-5-methyl-1 H-benzo[d]imidazole (1 g, 2.79 mmol) in 1,4-dioxane (15 mL) were added (4-(2-hydroxypropan-2-yl)phenyl)boronic acid (0.502 g, 2.79 mmol), [1,1’-bis(diphenylphosphino)ferrocene]palladium(ll) dichloride (230 mg, 0.279 mmol), potassium carbonate (1.15 g, 8.4 mmol) and water (5 mL). The resulting reaction mixture was degassed with nitrogen for 10 min, then heated to 100°C overnight. Then the reaction mixture was diluted with ethyl acetate and washed with water. The organic phase was dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by SiO₂ column chromatography (hexanes I EtOAc from 7:3 to 1:4) to give 826 mg ofthe product as a colorless oil. ¹H NMR (500 MHz, DMSO-d6) δ 8.48 (s, 1H),

7.67 (s, 1H), 7.58 (d, 2H), 7.52 (d, 2H), 7.31 (s, 1H), 7.29 (d, 2H), 7.17 (d, 2H), 5.01 (s,

H), 2.32 (s, 3H), 1.46 (s, 6H), 1.34 (d, 9H). LC/MS m/z: 415.32 (M+H)⁺

Examples A2 to A3 were prepared in the same manner as described above for 2-(4-(1-(4(tert-butoxy)phenyl)-5-methyl-1 H-benzo[d]imidazol-6-yl)phenyl)propan-2-ol (Example A1 ), 5 using the appropriate aryl halide described above and the appropriate commercially available boronic acid.

Ex.	Starting materials	Product/Name	Analytical Data
A2	6-bromo-1 -(4-(tertbutoxy)phenyl)-5methyl-1Hbenzo[d]imidazole 4- isopropoxyphenylb oronic acid	οΛ^ 1 -(4-(tertbutoxy)phenyl)-6-(4isopropoxyphenyl)-5methyl-1 Hbenzo[d]imidazole	¹H NMR (500 MHz, DMSOc/6) δ 8.56 (s, 1H), 7.67 (s, 1H), 7.60 (d,2H), 7.30 (s, 1H), 7.27 (d, 2H), 7.19 (d, 2H), 6.95 (d, 2H), 4.61-4.66 (m, 1H), 2.32 (s, 3H), 1.34 (s, 9H), 1.28 (d, 6H). LC/MS m/z\ 415.40 (M+H)⁺
A3	6-Bromo-5-methyl1-[4-(propan-2yloxy)phenyl]-1/71,3-benzodiazole (4-(2- hydroxypropan-2yl)phenyl)boronic acid	2-(4-(1-(4- isopropoxyphenyl)-5- methyl-1 H- benzo[d]imidazol-6- yl)phenyl)propan-2-ol	¹H NMR (500 MHz, DMSOc/6) δ 8.57 (s, 1H), 7.69 (s, 1H), 7.56 (d, 2H), 7.50 (d, 2H), 7.31 (s, 1H), 7.29 (d, 2H), 7.10 (d, 2H), 4.65-4.70 (m, 1H), 2.34 (s, 3H), 1.46 (s, 6H), 1.28 (d, 6H). LC/MS m/z: 401.31 (M+H)⁺

Préparation of intermediates for Examples B4 to B9

6-bromoimidazo[1,2-a]pyridine-7-carbonitrile

i-PrOH 160°C uwave

To a solution of 2-amino-5-bromoisonicotinonitrile (150 mg, 0.76 mmol) in i-PrOH (2mL) is added 0.6 mL (1.5 eq) of 2-chloro-1,1-dimethoxyethane. The solution is capped tightly and heated in a microwave reactor to 160°C for 30 minutes. The mixture is cooled and evaporated in vacuo, the residue dissolved in ethyl acetate, washed with saturated aq. NaHCO₃, and evaporated in vacuo to give 0.47 g ofthe title compound, pure enough for further use. LC/MS m/z: 221.10 (M+H)⁺

6-bromo-3-iodo-7-methylimidazo[1,2-a]pyridine

To a solution of 6-bromo-7-methylimidazo[1,2-a]pyridine (100 mg, 0.47 mmol) in CH₂CI₂ (1 mL), was added 1-lodopyrrolidine-2,5-dione (84 mg, 0.47 mmol) and MeOH (0.1 mL). The resulting mixture was stirred at room température for 2 h. The reaction was poured into water and extracted with ethyl acetate. The organic phase was dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by SiO₂ column chromatography (hexane/EtOAc = 1:1 ) to give 122 mg (77 %) of the product as a white solid. LC/MS m/z: 337.00 (M+H)⁺.

6-bromo-3-iodoimidazo[1,2-a]pyridine-7-carbonitrile _D Y

Br \

I

The title compound was prepared from 6-bromoimidazo[1,2-a]pyridine-7-carbonitrile and NIS in the same manner as described for 6-bromo-3-iodo-7-methylimidazo[1,2-a]pyridine. LC/MS m/z: 348.01 (M+H)⁺.

6-bromo-3-(4-isopropoxyphenyl)-7-methylimidazo[1,2-a]pyridine

The title compound was prepared from 6-bromo-3-iodo-7-methylimidazo[1,2-a]pyridine and

4-isopropoxyboronic acid in the same manner as described for 2-(4-(1 -(4-(tertbutoxy)phenyl)-5-methyl-1 H-benzo[d]imidazol-6-yl)phenyl)propan-2-ol (Example A1 ).

LC/MS m/z: 345.20 (M+H)⁺

6-bromo-3-(4-(tert-butoxy)phenyl)-7-methylimidazo[1,2-a]pyridine

The title compound was prepared from 6-bromo-3-iodo-7-methylimidazo[1,2-a]pyridine and (4-(tert-butoxy)phenyl)boronic acid in the same manner as described for 2-(4-(1 -(4-(tert10 butoxy)phenyl)-5-methyl-1 H-benzo[d]imidazol-6-yl)phenyl)propan-2-ol (Example A1 ).

LC/MS m/z: 359.22 (M+H)⁺

2-(4-(6-bromo-7-methylimidazo[1,2-a]pyridin-3-yl)phenyl)propan-2-ol

The title compound was prepared from 6-bromo-3-iodo-7-methylimidazo[1,2-a]pyridine and 15 (4-(2-hydroxypropan-2-yl)phenyl)boronic acid in the same manner as described for 2-(4(1-(4-(tert-butoxy)phenyl)-5-methyl-1H-benzo[d]imidazol-6-yl)phenyl)propan-2-ol (Example A1). LC/MS m/z: 345.10 (M+H)⁺

Examples B4 to B9 were prepared in the same manner as described above for 2-(4-(1-(4(tert-butoxy)phenyl)-5-methyl-1 H-benzo[d]imidazol-6-yl)phenyl)propan-2-ol (Example A1 ) using the appropriate aryl halide and commercially available boronic acids. In the case of compounds which hâve identical substitutions ofthe aryl halide, 2 équivalents ofthe boronic acid are used.

Ex.	Starting materials	Product/Name	Analytical Data
B4	6-bromo-3iodoimidazo[1,2a]pyridine-7carbonitrile	IA A+yN ,0 \ 3,6- bis(4- isopropoxyphenyl)imidaz o[1,2-a]pyridine-7- carbonitrile	LC/MS m/z: 412.29 (M+H)⁺
4- isopropoxyphenylb oronic acid (2 Equivalents)
B5	6-bromo-3-(4isopropoxyphenyl)- 7- methylimidazo[1,2a]pyridine		¹H NMR (500 MHz, DMSOd6) δ 8.11 (s, 1H), 7.62 (s, 1H), 7.58 (s, 1H), 7.56 (d, 2H), 7.55 (d, 2H), 7.42 (d, 2H), 7.05 (d, 2H), 4.63-4.68 (m, 1H), 2.26 (s, 3H), 1.46 (s, 6H), 1.27 (d, 6H). LC/MS m/z: 401.31 (M+H)⁺
H0_x	AJ A
^,0 2-(4-(3-(4isopropoxyphenyl)-7methylimidazo[1,2a]pyridin-6- yl)phenyl)propan-2-ol
(4-(2- hydroxypropan-2yl)phenyl)boronic acid
B6	6-bromo-3-(4-(tertbutoxy)phenyl)-7methylimidazo[1,2a]pyridine	H0_x	O O , A A Λ	¹H NMR (500 MHz, DMSOd6) δ 8.17 (s, 1H), 7.67 (s, 1H), 7.58 (d,2H), 7.56 (s, 1H), 7.54 (d, 2H), 7.37 (d,
	(4-(2-			2H), 7.10 (d, 2H), 2.26 (s,

	hydroxypropan-2yl)phenyl)boronic acid	2-(4-(3-(4-(tert- butoxy)phenyl)-7methylimidazo[1,2a]pyridin-6- yl)phenyl)propan-2-ol	3H), 1.46 (s, 6H), 1.33 (s, 9H). LC/MS m/z: 415.34 (M+H)⁺
B7	6-bromo-3-(4-(tertbutoxy)phenyl)-7methylimidazo[1,2a]pyridine 4- isopropoxyphenylb oronic acid	AAAA AJy Ç 3-(4-(tert- butoxy)phenyl )-6-(4isopropoxyphenyl)-7methylimidazo[1,2a]pyridine	¹H NMR (500 MHz, DMSOc/6) δ 8.13 (s, 1H), 7.65 (s, IH), 7.57 (d, 2H), 7.54 (s, 1H), 7.33 (d, 2H), 7.11 (d, 2H), 6.97 (d, 2H), 4.62-4.67 (m, 1H), 2.24 (s, 3H), 1.33 (s, 9H), 1.28 (d, 6H). LC/MS m/z: 415.36 (M+H)⁺
B8	2-(4-(6-bromo-7methylimidazo[1,2a]pyridin-3yl)phenyl)propan-2ol 4- isopropoxyphenylb oronic acid	AÿSA-sN /-OH 2-(4-(6-(4isopropoxyphenyl)-7methylimidazo[1,2a]pyridin-3- yl)phenyl)propan-2-ol	¹H NMR (500 MHz, DMSOc/6) δ 8.29 (s, 1H), 8.15 (s, 1H), 7.88 (s, 1H), 7.38 (d, 2H), 7.34 (d,2H), 7.13 (d, 2H), 7.01 (d, 2H), 4.64-4.69 (m, 1H), 2.38 (s, 3H), 1.46 (s, 6H), 1.28 (d, 6H). LC/MS m/z: 401.36 (M+H)⁺
B9	2-(4-(6-bromo-7methylimidazo[1,2a]pyridin-3yl)phenyl)propan-2ol (4-(2-	^hA/AA Y /Ah 2,2-((7- methylimidazo[1,2-	LC/MS m/z: 401.37 (M+H)⁺

hydroxypropan-2yl)phenyl)boronic acid

a]pyridine-3,6- diyl)bis(4,1phenylene))bis(propan- 2-ol)

Préparation of intermediates for Examples C10 to C26 methyl 2-bromo-4-((4-isopropoxyphenyl)amino)-5-nitrobenzoate o

^οΛγυ^ΝΗ2 βΑΑ_Νη φ

_Ύο

The title compound was prepared from methyl 2-bromo-4-fluoro-5-nitrobenzoate and 45 isopropoxyaniline in the same manner as described for 5-Bromo-A/¹-(4-isopropoxyphenyl)4-methylbenzene-1,2-diamine. LC/MS m/z\ 381.01 (M+H)⁺, 421.97 (M+H+CH₃CN)⁺

5-bromo-/V¹-(4-isopropoxyphenyl)benzene-1,2-diamine

O ° j

The title compound was prepared from 4-bromo-2-fluoro-1-nitrobenzene and 410 isopropoxyaniline in the same manner as described for 5-bromo-/V¹-(4-isopropoxyphenyl)4-methylbenzene-1,2-diamine. LCMS m/z: 321.20 (⁷⁹Br, M+H)⁺, 323.19 (⁸¹Br, M+H)⁺, 362.20 (⁷⁹Br, M+H+CH3CN)⁺, 364.24(⁸¹Br, M+H+CH3CN)⁺.

5-bromo-4-chloro-N1-(4-isopropoxyphenyl)benzene-1,2-diamine

The title compound was prepared from 1-bromo-2-chloro-5-fluoro-4-nitrobenzene and 4isopropoxyaniline in the same manner as described for 5-bromo-A/¹-(4-isopropoxyphenyl)4-methylbenzene-1,2-diamine. LC/MS m/z: 355.05 (⁷⁹Br, M+H)⁺, 357.12 (⁸¹Br, M+H)⁺

5-bromo-6-fluoro-N1 -(4-isopropoxyphenyl)benzene-1,2-diamine

The title compound was prepared from 1-bromo-2,3-difluoro-4-nitrobenzene and 4isopropoxyaniline in the same manner as described for 5-bromo-/\/¹-(4-isopropoxyphenyl)4-methylbenzene-1,2-diamine. LC/MS m/z: 339.13 (⁷⁹Br, M+H)⁺, 341.27 (⁸¹Br, M+H)⁺

5-bromo-4-fluoro-N 1 -(4-isopropoxyphenyl)benzene-1,2-diamine

The title compound was prepared from 1-bromo-2,5-difluoro-4-nitrobenzene and 4isopropoxyaniline in the same manner as described for 5-bromo-/V¹-(4-isopropoxyphenyl)4-methylbenzene-1,2-diamine. LC/MS m/z: 339.13 (⁷⁹Br, M+H)⁺, 341.22 (⁸¹Br, M+H)⁺

5-bromo-N1-(4-isopropoxyphenyl)-6-methylbenzene-1,2-diamine

The title compound was prepared from 1-bromo-3-fluoro-2-methyl-4-nitrobenzene and 4isopropoxyaniline in the same manner as described for 5-bromo-/V¹-(4-isopropoxyphenyl)4-methylbenzene-1,2-diamine. LC/MS m/z: 335.19 (M+H)⁺

5-bromo-N1-(4-isopropoxyphenyl)-4-methoxybenzene-1,2-diamine

The title compound was prepared from 1-bromo-5-fluoro-2-methoxy-4-nitrobenzene and 4isopropoxyaniline in the same manner as described for 5-bromo-A/¹-(4-isopropoxyphenyl)4-methylbenzene-1,2-diamine. LC/MS m/z: 418.30 (M+H)⁺

6-bromo-1 -[4-(propan-2-yloxy)phenyl]-1 H-î ,2,3-benzotriazole

NaNO₂, PPh₃

AcOH

To a solution of 5-bromo-A/¹-(4-isopropoxyphenyl)benzene-1,2-diamine (100 mg, 0.3 mmol) in acetic acid (3 mL), was added PPh₃ (81.6 mg, 0.3 mmol) followed by addition of sodium nitrite (25.8 mg, 0.36 mmol) at 0 °C. The reaction was warmed to room température and stirred for 1 h. The reaction was poured into water and extracted with ethyl acetate. The organic phase was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by SiO₂ column chromatography (hexane/EtOAc = 2:1 ) to give 98 mg (95%) of the product as a white solid. LC/MS m/z\ 332.07 (⁷⁹Br, M+H)⁺, 334.08 (⁸¹Br, M+H)⁺, 373.15 (⁷⁹Br, M+H+CH3CN)⁺, 375.14 (⁸¹Br, M+H+CH3CN)⁺.

methyl 6-bromo-1-(4-isopropoxyphenyl)-1 H-benzo[d][1,2,3]triazole-5-carboxylate

The title compound was prepared from methyl 2-bromo-4-((4-isopropoxyphenyl)amino)-5nitrobenzoate, sodium nitrite, and triphenylphosphine in the same manner as described for 6-bromo-1-[4-(propan-2-yloxy)phenyl]-1/7-1,2,3-benzotriazole. LC/MS m/z: 390.17 (⁷⁹Br, M+H+CH3CN)⁺, 392.16 (⁸¹Br, M+H+CH3CN)⁺.

6-bromo-5-chloro-1 -(4-isopropoxyphenyl)-1 H-benzo[d][1,2,3]triazole

The title compound was prepared from 5-bromo-4-chloro-N1-(4isopropoxyphenyl)benzene-1,2-diamine in the same manner as described for 6-bromo-1[4-(propan-2-yloxy)phenyl]-1/7-1,2,3-benzotriazole. LC/MS m/z: 368.13 (M+H)⁺, 408.91 (M+H+CH₃CN)⁺

6-bromo-7-fluoro-1 -(4-isopropoxyphenyl)-1 H-benzo[d][1,2,3]triazole

The title compound was prepared from 5-bromo-6-fluoro-N1-(4-isopropoxyphenyl)benzene1,2-diamine in the same manner as described for 6-bromo-1-[4-(propan-2-yloxy)phenyl]1/7-1,2,3-benzotriazole. ¹H NMR (500 MHz, DMSO-d6) δ 7.98 (d, 1H), 7.73-7.68 (m, 3H), 5 7.16 (d, 2H), 4.78-4.73 (m, 1 H), 1.34 (d, 6H). LC/MS m/z: 351.93 (M+H)⁺

6-bromo-5-fluoro-1 -(4-isopropoxyphenyl)-1 H-benzo[d][1,2,3]triazole

The title compound was prepared from 5-bromo-4-fluoro-N1-(4-isopropoxyphenyl)benzene1,2-diamine in the same manner as described for 6-bromo-1-[4-(propan-2-yloxy)phenyl]10 1/7-1,2,3-benzotriazole. LC/MS m/z: 352.20 (M+H)⁺, 393.19 (M+H+CH₃CN)⁺

6-bromo-1 -(4-isopropoxyphenyl)-7-methyl-1 H-benzo[d][1,2,3]triazole

The title compound was prepared from 5-bromo-N1-(4-isopropoxyphenyl)-6methylbenzene-1,2-diamine in the same manner as described for 6-bromo-1-[4-(propan-215 yloxy)phenyl]-1/7-1,2,3-benzotriazole. LC/MS m/z: 346.03 (M+H)⁺

6-bromo-1 -(4-isopropoxyphenyl)-5-methoxy-1 H-benzo[d][1,2,3]triazole

The title compound was prepared from 5-bromo-N1-(4-isopropoxyphenyl)-4methoxybenzene-1,2-diamine in the same manner as described for 6-bromo-1-[4-(propan2-yloxy)phenyl]-1/7-1,2,3-benzotriazole. LC/MS m/z: 362.13 (M+H)⁺

Examples C10 to C18 were prepared in the same manner as described above for 2-(4-(1(4-(tert-butoxy)phenyl)-5-methyl-1H-benzo[d]imidazol-6-yl)phenyl)propan-2-ol (Example A1 ), using the appropriate aryl halide described above and the appropriate commercially available boronic acid.

Ex.	Starting materials	Product/Name	Analytical Data
C1 0	methyl 6-bromo-1- (4- isopropoxyphenyl)1H- benzo[d][1,2,3]triaz ole-5-carboxylate	o JL Υ-οΎΑ θ __ methyl 1,6-bis(4isopropoxyphenyl)-1 Hbenzo[d][1,2,3]triazole-5carboxylate	LC/MS m/z: 446.34 (M+H)⁺
4- isopropoxyboronic acid
C1 1	6-bromo-5-chloro- 1-(4- isopropoxyphenyl)- 1H- benzo[d][1,2,3]triaz oie	«WN jf T 'N I 5-chloro-1,6-bis(4-	¹H NMR (500 MHz, DMSOd6) δ 8.44 (s, 1H), 7.77 (d, 2H), 7.72 (s, 1H), 7.42 (d, 2H), 7.17 (d, 2H), 7.01 (d, 2H), 4.74-4.67 (m, 2H), 1.32 (d, 6H), 1.30 (d, 6H). LC/MS

	4- isopropoxyboronic acid	isopropoxyphenyl)-1 Hbenzo[d][1,2,3]triazole	m/z: 422.15 (M+H)⁺
C1 2	6-bromo-7-fluoro-1 (4- isopropoxyphenyl)- 1H- benzo[d][1,2,3]triaz oie	i JT JL JÛU X _^o 7-fluoro-1,6-bis(4isopropoxyphenyl)-1 Hbenzo[d][1,2,3]triazole	¹H NMR (500 MHz, DMSOd6) δ 8.02 (d, 1H), 7.71 (d, 2H), 7.59- 7.54 (m, 3H), 7.14 (d, 2H), 7.04 (d, 2H), 4.77-4.66 (m, 2H), 1.33 (d, 6H), 1.30 (d, 6H). LC/MS m/z: 406.35 (M+H)⁺
4isopropoxyboronic acid
Cl 3	6-bromo-5-fluoro-1 (4- isopropoxyphenyl)- 1H- benzo[d][1,2,3]triaz oie	Ύ T > i jîjx UM/ θ 5-fluoro-1,6-bis(4isopropoxyphenyl)-1 Hbenzo[d][1,2,3]triazole	¹H NMR (500 MHz, DMSOd6) δ 8.11 (d, 1H), 7.827.78 (m, 3H), 7.56 (d, 2H), 7.17 (d, 2H), 7.04 (d, 2H), 4.76-4.66 (m, 2H), 1.33 (d, 6H), 1.30 (d, 6H). LC/MS m/z: 406.26 (M+H)⁺
4isopropoxyboronic acid
C1 4	6-bromo-1-(4isopropoxyphenyl)7-methyl-1 Hbenzo[d][1,2,3]triaz oie	Ma-N X JL k jl jCji Y ^^o 1,6-bis(4isopropoxyphenyl)-7- methyl-1 H- benzo[d][1,2,3]triazole	¹H NMR (500 MHz, DMSOd6) δ 7.98 (d, 1H), 7.58 (d, 2H), 7.30 (d, 1H), 7.26 (d, 2H), 7.13 (d,2H), 6.98 (d, 2H), 4.73-4.77 (m, 1H), 4.63-4.68 (m, 1H), 2.01 (s, 3H), 1.32 (d, 6H), 1.28 (d, 6H). LC/MS m/z: 402.29
4- isopropoxyboronic acid

			(M+H)⁺
C1 5	6-bromo-1-(4isopropoxyphenyl)5-methoxy-1 Hbenzo[d][1,2,3]triaz oie	z°vV-N XX > ΛΧΧΧ^ __^o 1,6-bis(4- isopropoxyphenyl)-5- methoxy-1 H- benzo[d][1,2,3]triazole	LC/MS m/z: 418.30 (M+H)⁺, 459.33 (M+H+CH₃CN)⁺
4isopropoxyboronic acid
C1 6	6-bromo-1-(4isopropoxyphenyl)1Hbenzo[d][1,2,3]triaz oie	W-N _ XX > HoY^ Q ___ 2-(4-(1-(4- isopropoxyphenyl)-1 Hbenzo[d][1,2,3]triazol-6yl)phenyl)propan-2-ol	¹H NMR (500 MHz, DMSOd6) δ 8.22 (d, 1H), 7.93 (s, 1H), 7.82- 7.78 (m, 3H), 7.72 (d, 2H), 7.58 (d, 2H), 7.20 (d, 2H), 5.08 (s, 1H), 4.77-4.73 (m, 1H), 1.46 (s, 6H), 1.34 (d, 6H). LC/MS m/z: 388.31 (M+H)⁺
(4-(2- hydroxypropan-2yl)phenyl)boronic acid
C1 7	6-bromo-1-(4isopropoxyphenyl)1H- benzo[d][1,2,3]triaz oie	f X > γ θ __^O 2-(3-(1-(4isopropoxyphenyl)-1 Hbenzo[d][1,2,3]triazol-6yl)phenyl)propan-2-ol	LC/MS m/z: 388.28 (M+H)⁺
(3-(2- hydroxypropan-2yl)phenyl)boronic acid
C1	6-bromo-1-(4-	/VN f X >	LC/MS m/z: 388.28 (M+H)⁺

8	isopropoxyphenyl)1H- benzo[d][1,2,3]triaz oie	6-(3-isopropoxyphenyl)1 -(4-isopropoxyphenyl)1H- benzo[d][1,2,3]triazole
(3- isopropoxyphenyl)b oronic acid

Example C19: (1,6-bis(4-isopropoxyphenyl)-1 H-benzo[d][1,2,3]triazol-5-yl)methanol

To a solution of methyl 1,6-bis(4-isopropoxyphenyl)-1 H-benzo[d][1,2,3]triazole-55 carboxylate (86 mg, 1 Eq) in THF (3 mL), was slowly added 2.6 M lithium aluminum hydride solution in THF (80 uL, 1 Eq). This mixture was stirred at room température for 3 hours, quenched slowly with cold, saturated Na2SO4 solution, and filtered through a celite pad. The filtrate was concentrated in vacuo and the residue purified by S1O2 column chromatography (hexane/EtOAc = 7:3 to 6:4) to afford 52 mg ofthe title compound. LC/MS m/z: 418.31 (M+H)⁺, 835.60 (2M+H)⁺

Example C20: 1,6-bis(4-isopropoxyphenyl)-1 H-benzo[d][1,2,3]triazole-5-carboxylic acid

To a solution of methyl 1,6-bis(4-isopropoxyphenyl)-1 H-benzo[d][1,2,3]triazole-5carboxylate (200 mg, 1 Eq), in 1:1 MeOH/THF (8 mL) is added 2 M aq. NaOH (4.25 mL). The mixture is stirred overnight, quenched by addition of 1M aq. HCl, extracted with MTBE, and the organic phase evaporated in vacuo. 10 mg ofthe residue was purified by préparative HPLC to afford 2.3 mg of the title compound. LC/MS m/z: 432.35 (M+H)⁺, 473.25 (M+H+CH₃CN)⁺

Example C21: 1,6-bis(4-isopropoxyphenyl)-1 H-benzo[d][1,2,3]triazole-5-carboxamide

To a solution of 1,6-bis(4-isopropoxyphenyl)-1 H-benzo[d][1,2,3]triazole-5-carboxylic acid (9.5 mg, 1 Eq), in DMF (0.5 mL) is added diisopropylethylamine (11 uL, 3 Eq), and HATU (12 mg, 1.5 Eq). The mixture is stirred for 1 hour, at which time ammonium chloride (5 mg, 4 Eq) is added in one portion. The mixture is stirred overnight, diluted with ethyl acetate, washed twice with 1 M aq. HCl, and evaporated in vacuo. The residue is purified by préparative HPLC to afford 5 mg ofthe title compound as a white solid. LC/MS m/z: 431.29 (M+H)⁺, 861.54 (2M+H)⁺

Example C22: (1,6-bis(4-isopropoxyphenyl)-1 H-benzo[d][1,2,3]triazol-5-yl)methanamine

The title compound was prepared from 1,6-bis(4-isopropoxyphenyl)-1 Hbenzo[d][1,2,3]triazole-5-carboxamide in the same manner as described for (1,6-bis(420266 isopropoxyphenyl)-1 H-benzo[d][1,2,3]triazol-5-yl)methanol (Example C19), stirring at reflux instead of room température. LC/MS m/z: 417.41 (M+H)⁺

Example C23: 1,6-bis(4-isopropoxyphenyl)-1 H-benzo[d][1,2,3]triazol-5-amine

To a solution of 1,6-bis(4-isopropoxyphenyl)-1 H-benzo[d][1,2,3]triazole-5-carboxamide (51 mg, 1 Eq) in t-BuOH (0.5 mL), is added triethylamine (33 uL, 2 Eq) and diphenylphosphoryl azide (25 uL, 1 Eq). The mixture is heated to 85°C and stirred for 6 hours, at which time it is diluted with ethyl acetate, washed with saturated aq. NH₄CI and water, and evaporated in vacuo. The residue is taken up in DCM (1 mL) and TFA (1 mL) is added dropwise. The resulting solution is stirred overnight then diluted with DCM, washed with saturated aq. NaHCO₃, and evaporated in vacuo. The residue is purified by S1O2 column chromatography (hexane/EtOAc = 7:3 to 1:2) to afford 7 mg of the title compound as a colorless oil. LC/MS m/z: 403.30 (M+H)⁺, 444.30 (M+H+CH₃CN)⁺

Example C24: 4,4'-(5-methoxy-1 H-benzo[d][1,2,3]triazole-1,6-diyl)diphénol

To a solution of 1,6-bis(4-isopropoxyphenyl)-5-methoxy-1H-benzo[d][1,2,3]triazole (200 mg, 0.5 mmol) in DCM (4 mL) at 0°C is added a 1M solution of BBr₃ (0.5 mL, 0.5 mmol). The mixture is allowed to warm to room température while stirring overnight, at which time it is quenched by pouring over ice, extracted twice with ethyl acetate, and evaporated in vacuo. The residue is purified by préparative HPLC to afford 13 mg of the title compound.

LC/MS m/z: 334.27 (M+H)⁺

Example C25: 1,6-bis(4-isopropoxyphenyl)-5-vinyl-1 H-benzo[d][1,2,3]triazole

Step 1: 1,6-bis(4-isopropoxyphenyl)-1 H-benzo[d][1,2,3]triazole-5-carbaldehyde

To a solution of (1,6-bis(4-isopropoxyphenyl)-1 H-benzo[d][1,2,3]triazol-5-yl)methanol (30 mg, 0.072 mmol) is added DCM (0.5 mL) and MnO₂ (12 mg, 0.14 mmol). The mixture is stirred overnight, filtered through a celite pad, and the filtrate evaporated to afford 24 mg of the title compound, which is not purified further. LC/MS m/z: 416.27 (M+H)⁺

Step 2: 1,6-bis(4-isopropoxyphenyl)-5-vinyl-1 H-benzo[d][1,2,3]triazole

To a suspension of methyltriphenylphosphonium iodide (40 mg, 0.1 mmol) in THF (1 mL) at 0°C is added a 1.6 M solution of n-butyllithium (0.06 mL, 0.1 mmol). The mixture is stirred for 30 minutes at 0°C, at which time a solution of 1,6-bis(4-isopropoxyphenyl)-1 H15 benzo[d][1,2,3]triazole-5-carbaldehyde (24 mg, 0.057 mmol) in THF (0.5 mL) is added, and stirring is continued for 3 hours at room température. The mixture is quenched with aq. NH₄CI, extracted with ethyl acetate, and the organics evaporated in vacuo. The residue is purified by préparative HPLC to afford 12.9 mg ofthe title compound. LC/MS m/z: 414.29 (M+H)⁺

Example C26: 5-ethyl-1,6-bis(4-isopropoxyphenyl)-1 H-benzo[d][1,2,3]triazole

A solution of 1,6-bis(4-isopropoxyphenyl)-5-vinyl-1 H-benzo[d][1,2,3]triazole (11.5 mg, 0.028 mmol) in MeOH (0.5 mL) is purged of air by drawing a vacuum and backfilling with 5 nitrogen twice. 10% palladium on carbon (5 mg) is then added, and the atmosphère replaced with hydrogen by drawing a vacuum and backfilling twice with a hydrogen balloon. The mixture is stirred overnight, diluted with ethyl acetate, filtered through a celite pad, and the fïltrate evaporated in vacuo to afford 10 mg of the title compound, pure enough for use without further purification. ¹H NMR (500 MHz, DMSO-c/6) δ 8.05 (s, 1H), 7.74 (d, 2H), 7.49 10 (s, 1 H), 7.29 (d, 2H), 7.15 (d, 2H), 6.98 (d, 2H), 4.70-4.72 (m, 1 H), 4.65-4.67 (m, 1 H), 2.692.74 (m, 2H), 1.31 (d, 6H), 1.29 (d, 6H), 1.08 (t, 3H). LC/MS m/z: 416.33 (M+H)⁺

Example D27: 3,6-bis(4-isopropoxyphenyl)-7-methylimidazo[1,2-a]pyrimidine

Step 1: 6-bromo-7-methylimidazo[1,2-a]pyrimidine

XV

The title compound was prepared from 5-bromo-4-methylpyrimidin-2-amine in the same manner as described for 6-bromoimidazo[1,2-a]pyridine-7-carbonitrile. ¹H NMR (500 MHz, CDCI₃) δ 8.57 (s, 1 H), 7.85 (s, 1 H), 7.49 (s, 1 H), 2.79 (s, 3H). LC/MS m/z: 214.25 (M+H)⁺

Step 2: 6-(4-isopropoxyphenyl)-7-methylimidazo[1,2-a]pyrimidine

The title compound was prepared from 6-bromo-7-methylimidazo[1,2-a]pyrimidine and 4isopropoxyphenylboronic acid in the same manner as described for 2-(4-(1 -(4-(tertbutoxy)phenyl)-5-methyl-1 H-benzo[d]imidazol-6-yl)phenyl)propan-2-ol (Example A1 ).

LC/MS m/z: 268.25 (M+H)⁺

Step 3: 3-iodo-6-(4-isopropoxyphenyl)-7-methylimidazo[1,2-a]pyrimidine

The title compound was prepared from 6-(4-isopropoxyphenyl)-7-methylimidazo[1,2a]pyrimidine and NIS in the same manner as described for 6-bromo-3-iodo-7methylimidazo[1,2-a]pyridine. LC/MS m/z: 394.23

Step 4: 3,6-bis(4-isopropoxyphenyl)-7-methylimidazo[1,2-a]pyrimidine

The title compound was prepared from 3-iodo-6-(4-isopropoxyphenyl)-7-methylimidazo[1,2a]pyrimidine and 4-isopropoxyphenylboronic acid in the same manner as described for 2(4-(1-(4-(tert-butoxy)phenyl)-5-methyl-1H-benzo[d]imidazol-6-yl)phenyl)propan-2-ol (Example A1 ). ¹H NMR (500 MHz, CDCI₃) δ 8.31 (s, 1 H), 7.74 (s, 1 H), 7.40 (d, 2H), 7.22 (d, 2H), 6.98 (d, 2H), 6.96 (d, 2H), 4.66-4.56 (m, 2H), 2.55 (s, 3H), 1.38 (d, 6H), 1.36 (d, 6H). LC/MS m/z: 402.36 (M+H)⁺

Example D28: 3-(4-(tert-butoxy)phenyl)-6-(4-isopropoxyphenyl)-7-methylimidazo[1,2a]pyrimidine

The title compound was prepared from 3-iodo-6-(4-isopropoxyphenyl)-7-methylimidazo[1,2a]pyrimidine and 4-terf-butoxyphenylboronic acid in the same manner as described for 2(4-(1-(4-(tert-butoxy)phenyl)-5-methyl-1H-benzo[d]imidazol-6-yl)phenyl)propan-2-ol (Example A1 ). LC/MS m/z: 416.35 (M+H)⁺

Example D29: 2-(4-(6-(4-isopropoxyphenyl)-7-methylimidazo[1,2-a]pyrimidin-3yl)phenyl)propan-2-ol

The title compound was prepared from 3-iodo-6-(4-isopropoxyphenyl)-7-methylimidazo[1,210 a]pyrimidine and (4-(2-hydroxypropan-2-yl)phenyl)boronic acid in the same manner as described for 2-(4-(1-(4-(tert-butoxy)phenyl)-5-methyl-1 H-benzo[d]imidazol-6yl)phenyl)propan-2-ol (Example A1 ). LC/MS m/z: 402.39 (M+H)⁺

Example E30: 3-(4-(tert-butoxy)phenyl)-6-(4-(isopropoxy-d₇)phenyl)-7-methylimidazo[1,2a]pyridine

Step 1: 4-(3-(4-(tert-butoxy)phenyl)-7-methylimidazo[1,2-a]pyridin-6-yl)phenol

To a microwave reactor vial is added 6-bromo-3-(4-(tert-butoxy)phenyl)-7methylimidazo[1,2-a]pyridine (25 mg, 0.07 mmol), 4-hydroxyphenylboronic acid (11 mg, .083 mmol, 1.2 Eq), Pd(dppf)CI₂ (6 mg, 10 mol %), and potassium carbonate (30 mg, .210 mmol, 3 Eq). 1.5 mL 1,4-dioxane and 0.5 mL water are then added, and the mixture degassed by bubbling nitrogen for 5 minutes. The vial is then tightly capped and subjected to microwave irradiation at 80 °C for 1 hour. The resulting mixture is diluted with ethyl acetate and the aqueous layer extracted 3X with ethyl acetate, followed by drying over Na₂SO₄ and évaporation to yield the crude product. The crude materiai is purified using silica gel flash chromatography with 100% ethyl acetate as eluent to give 20 mg of the title compound as a light brown oil. LC/MS m/z: 373.35 (M+H)⁺

Step 2: 3-(4-(tert-butoxy)phenyl)-6-(4-(isopropoxy-d₇)phenyl)-7-methylimidazo[1,2a]pyridine

To a solution of 4-(3-(4-(tert-butoxy)phenyl)-7-methylimidazo[1,2-a]pyridin-6-yl)phenol (142 mg, 0.38 mmol) in acetonitrile is added potassium carbonate (0.105 g, 0.76 mmol, 2 Eq). The mixture is stirred at reflux for 30 minutes, at which time c/7-isopropyl bromide (57 uL, 1.5 Eq) is added in one portion. The mixture is stirred at reflux overnight, then evaporated to dryness. The residue is dissolved in ethyl acetate, washed twice with water, dried over Na₂SO₄ and evaporated to give the crude solid, which is purified using silica gel flash chromatography with 3:7 hexanes : ethyl acetate as eluent to give 42 mg of the title compound. LC/MS m/z: 422.33 (M+H)⁺

Following schemes 2, 3, 6-8, and procedures above using the appropriate starting materials, the following examples can be made.

Utilizing a VSV pseudotype system expressing arenavirus glycoproteins (pseudotyped viruses here to referred to as LASV-p, MACV-p, JUNV-p, GTOV-p and

TCRV-p) and the Renilla luciferase reporter gene heterocyclic compounds were screened to identify individual compounds that inhibit infectivity of the pseudotyped viruses but not the native VSV virus expressing the VSV glycoprotein. VSV viruses expressing the VSV glycoprotein or pseudotyped with LASV, MACV, JUNV, GTOV and TCRV glycoproteins (LASV-p, MACV-p, JUNV-p, GTOV-p and TCRV-p) were generated in cultured HEK-293T cells (ATCC CRL-3216), which were grown in 10 cm dishes in DMEM supplemented with 10% FBS, 1X Pen-Strep, non-essential amino acids and L-glutamine. When cells reached approximately 80% confluency, they were transfected with a mixture of 15 pg ofthe pCAGGS plasmid encoding the desired glycoprotein and 45 pl of PEI (polyethylenimine) transfection reagent (PEI MAX, Polysciences Inc., #24765). The cells were incubated with the solution for 5 hours at 37°C at 5% CO₂ then washed and the mixture replaced with supplemented DMEM and incubated at 37°C at 5% CO2 for approximately 16-18 hours. Subsequently cells were infected with approximately 50 pl of VSV reporter virus whereby the VSV glycoprotein was replaced with a luciferase reporter gene. The cells were infected for 1 hour, then washed 1X with PBS and incubated in supplemented media. 24 hours post-infection, supematant was collected, clarified by centrifugation and filtration through a 0.45um filter, aliquoted and stored at -80°C. Both VSV-Luciferase and arenavirus glycoproteins pseudotypes were titrated for luminescence activity in Vero cells as described in the Luciferase assay protocol (below). Vero cells (ATCC: CCL-81) were grown in clear 384 well plates (3000 cells/well) in supplemented DMEM media. After incubating overnight at 37°C and 5% CO2, cells were treated with compounds at desired concentrations and pseudotyped virus in assay media. Assay media consisted of 50% Opti-MEM, 50% DMEM, with 1% FBS, Pen-Strep, non-essential amino acids and Lglutamine. Each ofthe viral supematants generated was diluted (from 1:100 to 1:2000) to give similar luminescence signal / background values of > 200. Final DMSO concentration in the compound testing wells was kept < 1% and control wells were treated with assay media and 1% DMSO. Cells were incubated for 24 hours at 37°C and 5% CO₂. The compound-virus mixture was aspirated off the cells 24 hours post-infection and washed 1X with PBS. Cells were then lysed using 20 μΙ of lysis buffer from a Luciferase kit diluted according to manufacturées instructions. After incubating for approximately 20 minutes, 5 μΙ of cell lysate was transferred to an opaque white plate, and mixed with 12.5 μΙ of

Coelenterazine diluted in buffer. This mixture was incubated at room température for 10 minutes on a plate shaker and then the luminescence was read using a plate reader (Beckman Coulter DTX 880 multimode detector with an émission of 535 nm).

Luminescence signais were obtained for compound containing and control wells to détermine % activity (inhibition of luciferase signal) for each compound.

Cytotoxicity Screening

Active compounds in the pseudotype assays were also evaluated for cytotoxicity over a period of 3 days. Compounds were serially diluted and added to Vero cells (4000 cells/well) with final DMSO concentration maintained at 1% in growth media consisting of minimal essential media (MEM) with 1% FBS. The plates were incubated at 37 °C for 3 days, and then dead cells were removed by washing with Phosphate buffered saline (PBS). CPE was assessed by staining cells with neutral red dye for 1 hour and then destaining with a solution of 50% éthanol/1% acetic acid solution. Absorbances were read at 540 nm and 690 nm on a Spectramax Plus 384 spectrophotometer. Data were analyzed as (540 nm - 690 nm) and then compared to untreated Controls to obtain % cell survival.

Réplicative LASV inhibitory activity plaque assays

Confluent or near-confluent cell culture monolayers in 12-well disposable cell culture plates were prepared. Cells were maintained in MEM or DMEM supplemented with 10% FBS. For antiviral assays the same medium was used but with FBS reduced to 2% or less and supplemented with 1 % penicillin/streptomycin. The test compounds were prepared at seven half log 10 final concentrations, (01-10 μΜ) in 2X MEM or 2X DMEM. Test compounds and positive control compounds (favipiravir or ribavirin) were run in parallel in biological triplicates. The assay was initiated by first removing growth media from the 12-well plates of cells, which was challenged with compound at a given concentration and 0.01 MOI of virus or about 50 to 100 plaque forming units (pfu). Cells were incubated for 60 min: 100 pL inoculum/ well, at 37°C, 5% CO₂ with constant gentle rocking. Virus inoculum was removed, cells washed and overlaid with either 1% agarose or 1% methylcellulose diluted 1:1 with 2XMEM and supplemented with 2% FBS and 1% penicillin/streptomycin and along with the corresponding drug concentration. Cells were incubated at 37°C with 5% CO₂ for 5 days. The overlay was then removed and plates stained with 0.05% crystal violet in 10% buffered formalin for approximateiy twenty minutes at room température. The plates were washed, dried and the number of plaques counted. The number of plaques in each set of compound dilution was converted to a percentage relative to the untreated virus control. The 50% effective (EC₅₀, virus-inhibitory) concentrations were then calculated by linear régression analysis. The quotient of CC₅₀ divided by EC₅₀ gives the selectivity index (SI) value. Compounds showing SI values >10 were considered active.

Réplicative LASV Viral Yield Réduction Assay

The VYR test is a direct détermination ofthe concentration ofthe test compound that inhibits virus réplication. Compound and virus were added to Vero cells for 3-4 days at which point the supematant was removed and tested for infectious particles. The supematant was titrated Iog10 dilutions of virus using 3 or 4 microwells per dilution on fresh monolayers of Vero cells in 96-well plates. Wells were scored for presence or absence of virus after distinct CPE is observed. Plotting the inhibitor concentration versus logw of virus produced at each concentration allows calculation ofthe 90% effective concentration by linear régression. In addition, compounds were run at different concentrations in parallel on Vero cells without virus to détermine cytotoxic CC₅o value. The selectivity index (SI) was calculated as a ratio Of CC50/EC90.

Réplicative Tacaribe virus testing

Selected compounds were tested against native replicating Tacaribe (TCRV) virus (TRVL-11573, BEI Resources) using an ELISA-based assay. Vero cells (ATCC: CCL-81) were grown in a 96-well format (5000 cells/well) in supplemented DMEM media. After overnight incubation, cells were treated with TCRV and compounds at desired concentrations, in MEM media with 1% FBS and suppléments. Final DMSO concentration in the compound testing wells was kept < 1% and control wells were treated with TCRV or media and 1% DMSO. After 5 days of incubation at 37°C in 5% CO₂, cells were fixed with 2% paraformaldéhyde for 45 minutes and then washed with PBS. Subsequently the cells were permeabilized with 0.25% triton-X, then TCRV was detected using ELISA with the following protocol. Monoclonal Anti-Junin Virus antibody (BEI #NR 41860), which cross reacts with TCRV nucleoprotein, was used to stain cells. After washing, cells were treated with biotin conjugated secondary antibody and subsequently, streptavidin conjugated horseradish peroxidase. TMB substrate was added to the wells, and the reaction was stopped using 2M sulfuric acid. The absorbance was read using a plate reader (Beckman Coulter DTX 880 multimode detector with an émission of 450 nm). OD readings were obtained for compound containing and control wells to détermine % activity for each compound.

Microsomal Assays

In addition to the ability of compounds to demonstrate broad inhibitory activity against arenaviruses in vitro, compounds must also hâve certain drug-like properties for them to be used to inhibit arenaviruses and provide methods of treatment for arenavirus infection in mammals in vivo. Such compounds may exhibit drug-like properties including but not limited to Chemical stability against metabolic dégradation by liver microsomal CYP p450 enzymes, cell permeability and oral bioavailability (if the drug is to delivered orally) and lack of inhibition ofthe hERG ion channel, which is associated with cardiac safety [Kerns, E.H. Li, D. Drug-like Properties: Concepts, Structure Design and Methods from ADME to Toxicity Optimization, (2008) Academie Press, Burlington MA], The above publication is herein incorporated by reference for ail purposes. To characterize drug-like properties ofthe Chemical sériés example compounds were evaluated for metabolic stability in human, mouse, guinea pig, monkey, rat, mouse, or dog liver microsome assays (Table 4), and for inhibition ofthe hERG ion channel (Table 5). Compounds exhibiting > 60% remaining of parent indicate attractive Chemical stability. The démonstration of good microsomal stability in human and nonhuman species facilitâtes the ability to test and optimize compounds in preclinical animal studies.

A reaction premixture was set up, containing 1 uM compound of interest, 1 mg/mL liver microsomes of desired species, 2.1 mM MgCB and 0.1 M sodium phosphate buffer, pH 7.4. This premixture was incubated at 37°C for 30 minutes with gentle agitation to allow the compound to be completely dissolved in the mixture. Then freshly made NADPH solution in 0.1 M sodium phosphate buffer was added at a concentration of 2mM to start the reaction. A ‘Time 0’ sample (30 uL) was taken out immediately after addition of NADPH and added to 140uL cold acetonitrile containing 1uM of pre-decided internai standard. The rest ofthe reaction mixture was incubated at 37°C for the remaining time period. Test compounds were left in the reaction mixture for 60 minutes before ‘Time 60’ sample was added to acetonitrile with internai standard. The control compound (Verapamil for human, monkey and dog LM, Lidocaine for Guinea pig LM, and diphenhydramine for rat and mouse LM) was incubated in reaction mixture for 15 minutes before ‘Time 15’ samples were collected and added to cold acetonitrile with internai standard. The samples were then spun in a centrifuge for 10 minutes at 4000 rpm, supernatant was collected and mixed with equal parts distilled water. These were then analyzed on a Varian 500-MS.

hERG Channel Assay

Drugs belonging to different classes hâve been shown to be associated with QT prolongation and in some cases serious ventricular arrhythmias. The most common mechanism for these adverse events is the inhibition of one or more cardiac potassium channels, in particular hERG. This current is important for cardiac myocyte repolarization and is a common target for drugs that prolong the QT interval. Test articles in this study were therefore, characterized to détermine their ability to inhibit the hERG channel. Ion channel activity was measured using a stably transfected Chinese Hamster Ovary (CHO) cell line expressing the hERG mRNA. The pharmacology of this cloned channel expressed in the CHO cell line is very similar to that observed in native tissue. Cells were cultured in DMEM/F12 containing 10% FBS, 1% penicillin/streptomycin and 500 pg/ml G418. Before testing, cells were harvested using Accumax (Innovative Cell Technologies). For electrophysiological recordings, the following solutions were used: External solution: 2 mM CaCI2; 2 mM MgCI2; 4 mM KCI; 150 mM NaCI; 10 mM Glucose; 10 mM HEPES; 305-315 mOsm; pH 7.4 (adjusted with 5M NaOH); Internai solution: 140 mM KCI; 10 mM MgCI2; 6 mM EGTA; 5 mM HEPES-Na; 5 mM ATP-Mg; 295-305 mOsm; pH 7.25 (adjusted with 1M KOH). Whole cell recordings were performed using PX 7000A (Axon Instruments) with AVIVA’s SealChip™ technology. Cells were voltage clamped at a holding potential of -80 mV. The hERG current was then activated by a depolarizing step to -50 mV for 300 ms. This first step at -50 mV was used as a baseline for measuring peak amplitude ofthe tail current. Next, a voltage step to +20 mV was applied for 5 s to activate the channels. Finally, a step back to 5 50 mV for 5 s removed activation and the deactivating tail current was recorded.

External solution containing 0.1% DMSO (vehicle) was applied to the cells to establish a baseline. After allowing the current to stabilize for 3 to 10 min, test articles were applied. Test article solutions were added to cells in 4 separate additions. Cells were kept in test solution until effect ofthe test article reached steady state, to a maximum of 12 min. Next, 1 μΜ cisapride (positive control) was added. Finally, washout with External Solution was performed until the recovery current reached steady state. Data analysis was performed using DataXpress (Axon Instruments), Clampfit (Axon Instruments) and Origin (OriginLab Corporation) software.

Table 1. Pseudotyped Virus Activity. Example compounds and their observed inhibitory activities are shown as EC50 values for LASV-p, MACV-p, JUNV-p, TCRVp and GTOV-p (VSV-p EC50 values were ail > 10 uM) and CC₅₀ for cytotoxicity; nd: not determined.

Ex.	LASV-p EC50 (nM)	MACV-p EC50 (nM)	JUNV-p EC50 (nM)	TCRV-p EC50 (nM)	GTOV-p EC50 (nM)	VSV-p EC50 (nM)	CC50 (uM)
A1	0.83	0.12	0.87	0.29	nd	>10,000	37.71
A2	0.38	0.14	0.15	0.15	0.05	>10,000	7.3
A3	0.75	0.17	.40	0.24	nd	>10,000	4.5
B4	1.32	0.25	0.21	0.12	0.31	>10,000	6.71
B5	0.53	0.16	0.18	0.16	nd	>10,000	3.87
B6	0.7	0.13	0.22	0.16	0.11	>10,000	4.32

B7	0.9	0.42	0.31	0.16	0.12	>10,000	8.14
B8	2.91	0.61	0.76	0.32	0.94	>10,000	27.76
B9	>25	>25	>25	16.15	nd	>10,000	nd
C10	20.6	4.05	11.02	12.27	nd	>10,000	>100
Cl 1	2.29	0.19	0.10	0.77	nd	>10,000	>100
C12	1.31	0.55	0.15	15	nd	>10,000	>100
C13	4.0	0.66	0.27	1.53	nd	>10,000	>100
C14	2.44	0.28	0.17	4.70	nd	>10,000	>100
C15	23.42	7.09	3.06	11.37	nd	>10,000	98.44
C16	3.02	1.72	0.45	3.67	nd	>10,000	4.12
C17	15.77	>25	12.18	>25	nd	>10,000	10.46
C18	>25	>25	>25	>25	nd	>10,000	nd
C19	22.5	3.82	0.75	8.0	nd	>10,000	81.71
C20	>25	>25	>25	>25	nd	>10,000	nd
C21	>25	>25	>25	>25	nd	>10,000	nd
C22	>25	>25	>25	>25	nd	>10,000	nd
C23	6.72	0.53	0.29	0.96	nd	>10,000	42.99
C24	4.62	>25	18.97	18.40	nd	>10,000	32.21
C25	6.78	0.3	1.47	8.01	nd	>10,000	3.87
C26	3.9	0.04	0.1	5.92	nd	>10,000	3.59
D27	0.75	0.85	0.78	0.20	nd	>10,000	2.18
D28	0.30	0.31	0.40	0.15	nd	>10,000	12.32

D29	1.84	0.87	1.03	1.27	nd	>10,000	42.10
E30	0.39	0.09	0.09	nd	nd	nd	3.8

Table 2. Comparison of Pseudotyped Versus Réplicative TCRV Inhibitory

Activities. Example compounds and their observed inhibitory activities (EC50) against pseudotyped or réplicative TCRV.

Example	TCRV-p EC₅₀(nM)	TCRV EC₅₀(nM)
A1	0.24	0.89
A2	0.12	0.33
A3	0.21	0.87
B4	0.16	0.74
B5	0.10	0.88
B6	0.10	0.95
B7	0.20	0.74
B8	0.32	2.16
C11	0.61	0.84
E30	0.2	nd

A very close corrélation was surprisingly discovered between the pseudotyped and réplicative virus inhibitory activities by the compounds of the invention.

Table 3. Inhibition of Native Lassa Virus. Example compounds and their observed inhibitory activities and selectivity indices (SI) in both réplicative LASV plaque and viral yield réduction (VYR) assays.

Example	Plaque Assay EC50 (uM)	VYR Assay ECgo (uM)	SI90
B8	<.003	<0.003	>9,000
B7	<.003	<0.001	>11,000
A1	<.003	<0.001	>33,000
A2	<.003	<0.001	>13,000
E30	nd	<0.014	>1,510

AH five compounds demonstrated very potent ECsoand EC90 of less than 1-3 nM for compounds A1, A2, B7, and B8 in the plaque and VYR assay formats and EC₉₀ of 15 less than 14 nM for compound E30 in the VYR assay format. SI90 values (obtained from the VYR assay data) were >1510 clearly indicating compound efficacy was due to antiviral activity and not cytotoxic effects. The results shown in tables 1-3 confirm the activity ofthe compounds against arenaviruses including the réplicative LASV and also strongly validâtes the approach for identifying bona fide HF arenavirus inhibitors through the utilization of pseudotyped virus assays.

Table 4. Multi-species Microsomal Stability. Percent parent compound remaining at 60 minutes in liver microsomes

Ex.	Mouse	Monkey	Dog	Human	Guinea	Rat
A1	82.7	8.6	>95	68.3	>95	93.8
A2	93.9	53	>95	95	>95	>95
A3	25.4	nd	nd	89.9	67.6	nd
B4	37.5	4.9	83.3	88.1	55.45	70.6
B5	16.4	nd	nd	86.7	7.4	nd
B6	81.7	nd	nd	81.5	61.5	nd
B7	92.9	22.9	>95	>95	>95	>95
B8	77.5	33.7	77.57	87.6	67.7	93
C11	>95	nd	nd	>95	>95	nd
C12	90.3	nd	nd	>95	>95	nd
C13	nd	nd	nd	79.5	nd	nd
C14	>95	nd	nd	>95	>95	nd
C16	57.4	nd	nd	72.3	61.9	nd
E30	>95	74.4	>95	>95	>95	>95

The results of multi-species microsomal stability studies (Table 4) showed that deuterated compound E30 demonstrated improved metabolic stability in monkey liver microsome assay as compared to its non-deuterated analog B7, thus showing good microsomal stability in both human and nonhuman species.

Table 5: hERG Channel Assay

Example	% Inhibition atlS uM
A1	<10
B7	<10
B8	<10
C11	<10

These data indicate lack of hERG channel inhibition suggesting good potential for cardiac safety.

Table 6: Mouse Pharmacokinetic Parameters

Example	Clearance (mL/min/kg)	Oral halflife (hr)	Oral CMax (ug/mL)	Oral T- Max (hr)	Oral AUC_t. 0 (ug/mL*h)	Vd (L/kg)	% Oral Bioavailability
B7	13.7	13.2	5.5	2	70.4	35.7	30
A1	0.8	17	20.4	0.5	182.6	17.6	35

Compounds were dosed in mice by intravenous (3 mg/kg) and oral (30 mg/kg) routes to détermine pharmacokinetic parameters. IV time points included 0.083, 0.25, 0.5, 1, 2, 6 and 24h and oral time points included 0.5, 1,2, 4, 6, 8 and 24h. Blood was drawn from 3 mice per time point. Plasma was isolated and measured by LC/MS/MS on a Varian 500LC/MS. Both compounds demonstrated low first pass liver clearance which is in agreement with high levels of compound remaining after 1 hour in mouse liver microsome (Table 4). Both compounds demonstrate reasonable oral bioavailability and long half-lives suitable for once a day dosing. Finally, the volume of distribution (Vd) values indicate compound is being taken up into tissue that further supports good oral biodistribution for targeting arenavirus infection.

Mice were able to tolerate both compounds dosed orally daily for 3 days up to at least (the highest dose tested) 100 mg/kg once a day. There were no clinical signs of overt toxicity as determined by daily monitoring of weight, température and behavior. On day 4 (24hrs after last dose) plasma and liver samples were collected from dosed animais to measure compound levels. Livers were homogenized in a 1:1 w/v of phosphate buffered saline. Both plasma and liver extracts were measured by LC/MS/MS on a Varian 500-MS (Table n

Table 7: Compound Concentrations 24 Hours Post Final Administration

Example	24hr plasma concentration (ug/mL)	24hr liver concentration (ug/g liver)
B7	9.1	63.4
A1	8.8	240.3

Taken together the results show that the compounds ofthe invention exhibit potent, broadspectrum inhibition of HF arenaviruses and attractive drug-like properties for utilization as treatments for viral infections that are mediated by arenavirus glycoproteins.

Claims

WHAT IS CLAIMED IS:

1. A method of treating infections associated with viruses of the Arenaviridae enveloped virus family, or any virus expressing Arenavirus glycoproteins to médiate cell entry comprising administering a pharmaceutically effective dose of a compound of structural formula I:

or a pharmaceutically acceptable sait and a pharmaceutically acceptable carrier, dilutant or vehicle there of wherein

A is independently selected from C and N;

G is independently selected from CH, CD, and N;

E is independently selected from CH, CD, and N;

J is independently selected from and

R² is independently selected from -OR³, -R⁴, -NHR¹⁰, -CONHR¹⁰;

R³ is independently selected from H, D, Ci to C₆ alkyl, C₂ to C₆ alkenyl, -NHC(O)R⁴-C(O)NHR¹⁰, and -C(O)R¹°, wherein each Ci to Ce alkyl is optionally substituted with D, halogen, -OH, -OR⁴, -NHR¹⁰;

R⁴ is independently selected from Ci to Ce alkyl and (C₂to C₉) cycloheteroalkyl optionally substituted with D, halogen, -OH, -OR¹⁰, and NHR¹⁰;

WHAT IS CLAIMED IS:

1. A compound of structural formula I:

or a pharmaceutically acceptable sait thereof wherein

A is independently selected from C and N;

G is independently selected from CH, CD, and N;

E is independently selected from CH, CD, and N;

J is independently selected from

R² is independently selected from -OR³, -R⁴, -NHR¹⁰, -CONHR¹⁰;

R³ is independently selected from H, D, Ci to C₆ alkyl, C₂ to Ce alkenyl, -NHC(O)R⁴-C(O)NHR¹⁰, and -C(O)R¹⁰, wherein each Ci to Ce alkyl is optionally substituted with D, halogen, -OH, -OR⁴, -NHR¹⁰;

R⁴ is independently selected from Ci to C₆ alkyl and (C₂to C9) cycloheteroalkyl optionally substituted with D, halogen, -OH, -OR¹⁰, and NHR¹⁰;

R⁵ is independently selected from H, D, Ci to Ce alkyl, C2 to Ce alkenyl, C2 to Ce alkynyl, halogen, -OR³, -CO2R¹⁰, -NHC(O)R⁴, -C(O)NHR¹⁰, -NHR¹⁰, -CHNHR¹⁰, -CN, -CR⁴, and -C(O)R¹⁰, wherein each Ci to C₆ alkyl is optionally substituted with D;

R⁶ is independently selected from halogen, -OR³, and R⁴;

R⁹ is independently selected from H, D, halogen, -OR¹⁰, and Ci to C₆ alkyl;

R¹⁰ is independently selected from H, D, -OH, Ci to C₆ alkyl and C2 to Ce alkenyl;

and when E is N, CH or CD then A is C, G is CH or CD, and J is

5 and when A is N then J is

with the proviso that the following compounds are excluded:
4. The compound of claim 1 wherein E is CH or CD.

5
5. The compound of claim 1 wherein A is C.
6. The compound of claim 1 wherein A is N.

The compound of claim 3 wherein R⁶ is

CD₃

-1-4θΗ ^_K°^H
8. The compound of claim 2 wherein R is \ or .

CD₃

-^-q_Z_ l’O γθϋ₃
9. The compound of claims 2 and 3 wherein R² is \ or ^cp3 .
10. A compound, according to claim 1, selected from the group consisting of:

11.The compound of claim 10, wherein the compound is selected from the group
12. A pharmaceutical composition comprising the compound of anyone of claims from 1 to

20 11 or a pharmaceutically acceptable sait thereof and a pharmaceutically acceptable carrier, dilutant or vehicle.
13. A compound according to any one of claims from 1 to 11 or a pharmaceutical composition according to claim 12 for use as a médicament.
14. A compound according to any one of claims from 1 to 11 or a pharmaceutical composition according to claim 12 for use in a method of treating infections associated with viruses of the Arenaviridae enveloped virus family, or any virus expressing Arenavirus glycoproteins to médiate cell entry.
15. A compound according to any one of claims from 1 to 11 or a pharmaceutical composition according to claim 12 for use in a method according to claim 14, wherein a pharmaceutically acceptable dose ofthe compound of claim 1 is administered with a pharmaceutically acceptable dose of at least one of the compounds selected from

10 Ribavirin, polymerase inhibitors, Favipiravir, Triazavirin, small interfering RNAs (siRNAs), vaccines, monoclonal antibodies, and immunomodulators.