WO2007038425A2 - Anti-viral compouinds - Google Patents

Anti-viral compouinds Download PDF

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Publication number
WO2007038425A2
WO2007038425A2 PCT/US2006/037241 US2006037241W WO2007038425A2 WO 2007038425 A2 WO2007038425 A2 WO 2007038425A2 US 2006037241 W US2006037241 W US 2006037241W WO 2007038425 A2 WO2007038425 A2 WO 2007038425A2
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compound
alkyl
alkanoyloxy
alkoxy
halo
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PCT/US2006/037241
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French (fr)
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WO2007038425A3 (en
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David M. Ferguson
John Goodell
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Regents Of The University Of Minnesota
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

  • Flaviviridae contains three genera, the flaviviruses, the pestiviruses, and the hepatitis C viruses. Many members within the Flayivirus genus are arthropod-borne pathogens that cause significant human morbidity and mortality.
  • the global emerging flaviviruses include four serotypes of dengue (DEN), yellow fever (YF), West Nile (WN), Japanese encephalitis (JE) 5 and tick-borne encephalitis (TBE) viruses ( Burke, D. S., and T. P. Monath, 2001. Flaviviruses. Lippincott William & Wilkins).
  • the World Health Organization estimated annual human cases of more than 50 million, 200,000, and 50,000 for DEN, YF, and JE viral infections, respectively.
  • the recent epidemics of WN virus in the United States have caused thousands of human cases, representing the largest meningoencephalitis outbreak in the Western Hemisphere and the largest WN virus outbreak ever reported (CDC. 2002. Provisional surveillance summary of the West Nile virus epidemic— United States, January-November 2002. MMWR Morb. Mortal. WkIy. Rep. 51:1129-1133).
  • Human vaccines are currently available only for YF, JE, and TBE viruses, and no effective antiviral therapy has been developed for treatment of flavivirus infections. Therefore, there is currently a need for anti-viral compounds that are useful for treating flavivirus infections.
  • the invention provides anti-viral compounds that have a broad spectrum of anti- flavivirus activity. Accordingly, the invention provides a compound of the invention which is a compound of formula I:
  • R 1 is (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkanoyl, (C 1 -C 6 )alkanoyloxy, (C 1 -C 6 )alkoxycarbonyl, (C 1 -C 6 )alkylaminocarbonyl, di(C 1 -C 6 )alkylamino- carbonyl, halo(C 1 -C 6 )alkyl, or R a -X-;
  • X is a direct bond, (Ci-C 6 )alkyl 5 -SO 2 -, or R a is aryl, or heteroaryl;
  • R 2 is H 5 (C ! -C 6 )alkyl, (d-C ⁇ alkoxy, (C 1 -C 6 )alkanoyl, (C 1 - C 6 )alkanoyloxy, (d-C ⁇ alkoxycarbonyl, halo(C 1 -C 6 )alkyl, (C 1 - C 6 )alkylaminocarbonylamino, di(C 1 -C 6 )alkylaminocarbonyl, (C 1 - C6)alkylsulfonylamino, or R b -Y-;
  • R b is aryl, or heteroaryl;
  • R 3 is H, (d-C ⁇ alkyl, (C 1 -C 6 )alkoxy, (d-C ⁇ alkanoyl, (Ci- C 6 )alkanoyloxy, (CrC ⁇ alkoxycarbonyl, halo(C 1 -C 6 )alkyl, or R 0 -Z-;
  • Z is a direct bond, (CrC ⁇ alkyl, or (Ci-C ⁇ alkanoyl;
  • R 0 is aryl, or heteroaryl; each bond represented by — is independently a single or a double bond; and
  • A is CH or N; wherein any aryl or heteroaryl OfR 1 -R 3 is optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (CrC ⁇ alkyl, (C 1 -C 6 )alkoxy, (C 1 - C 6 )alkanoyloxy, (CrC ⁇ alkoxycarbonyl, cyano, carboxy, nitro, trifluomethyl, trifluoromethoxy, or R d R e N; wherein R d and R e are each independently H, (C 1 - C 6 )alkanoyl, or (Ci-C 6 )alkyl; or a pharmaceutically acceptable salt thereof.
  • the invention also provides a pharmaceutical composition comprising a compound of formula I 5 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent or carrier.
  • the invention also provides a therapeutic method for treating a viral infection in an animal comprising administering to the animal, an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof.
  • the invention also provides a therapeutic method for suppressing viral RNA synthesis in an animal comprising administering to the animal, an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • the invention also provides a compound of formula (I), or a pharmaceutically acceptable salt thereof for use in medical therapy.
  • the invention also provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof to prepare a medicament useful for the treatment of a viral infection in a animal.
  • the invention also provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof to prepare a medicament useful for suppressing viral RNA synthesis in an animal.
  • the invention also provides processes and intermediates disclosed herein that are useful for preparing compounds of formula (I) or salts thereof. Certain compounds of formula (I) are useful as intermediates for preparing other compounds of formula (I).
  • FIG. 1 Identification of triaryl pyrazolone as an inhibitor of a full-length luciferase-expressing WN virus.
  • a full-length WN virus containing a luciferase reporter FL Rluc-WN ( Deas, T. S., et al., J. Virol., 2005, 4599- 4609) was used to infect Vero cells at an MOI of 0.1. The infected cells were incubated with a compound library at indicated concentrations, and assayed for Rluc activity at 24 h p.t. Values above each bar indicate the percentage of the luciferase activity derived from the compound-treated infection versus the luciferase signal derived from the mock-treated infection.
  • FL Rluc-WN luciferase reporter
  • Mycophenolic acid (MPA), a known WN virus inhibitor, was included as positive controls. Since the compound was screened at 1% DMSO, the mock-treated infection also contained this concentration of DMSO.
  • B Structure of triaryl pyraz ⁇ line, an inhibitor identified from the screening.
  • FIG. 2 Inhibition of an epidemic strain of WN virus and cytotoxicity of triaryl pyrazoline.
  • Vero cells were infected with an epidemic strain of WN virus (0.1 MOI), immediately treated with compound at indicated concentrations, and assayed for viral titers at 42 h p.i. Values above each data point indicate the percentage of viral titer from the compound-treated infection as compared with that from the mock-treated infection.
  • FIG. 3 Antiviral activities of triaryl pyrazoline against various RNA viruses. Viral titer reduction assays were performed to determine the antiviral activities of the compound. Vero cells were infected withDEN-2, YF (17D), SLE, WEE and VSV viruses (0.1 MOI), treated immediately with the compound at indicated concentrations, and assayed for virus yield in culture medium at 42 h p.i. (see details in Materials and Methods).
  • FIG. 4 Mechanism of triaryl pyrazoline-mediated inhibition of WN virus.
  • WN VLPs containing a luciferase-expressing replicon (Rluc-VLP) was used to infect Vero cells in the presence of indicated concentrations of compound. At 48 h p.i., cells were quantified for Rluc activity. The Rluc- VLP/Vero infection allows test if inhibitors block viral entry and replication.
  • Rluc-Neo-Rep A BHK-21 cell line containing WN replicon (Rluc-Neo-Rep) ( Lo, L., et al., J. Virol, 2003, 77, 12901-12906) was incubated with compound for 48 h and measured for Rluc activity.
  • the replicon-bearing cell line examines if a compound inhibits viral replication, but not viral entry.
  • C A WN reporting replicon containing an Rluc reporter (fused in-frame with the ORF; Rluc-Rep) was used to measure the effects of compound on viral translation and RNA synthesis.
  • BHK-21 cells transfected with Rluc-Rep exhibited two distinctive Rluc peaks at 2-10 h and after 24 h p.t, representing viral translation and RNA replication, respectively ( Lo, L., et al., J. Virol., 2003, 77, 10004-10014).
  • FIG. 5 Time-of-addition analyses of triaryl pyrazoline in WN virus infection. Vero cells were synchronously infected with WN virus, treated with compounds (100 ⁇ M) at indicated time points after infection, and quantified for viral yields in culture medium at 24 h p.i. Because all compound treatments contained 1% DMSO, this concentration of DMSO was added to infected cell at 0 and 20 h p.i.' to estimate its effect on viral yield production.
  • FIG. 6 Inhibition of a reporting replicon cell line of DEN-I virus.
  • a replicon of DEN-I virus (Rluc-Neo-Rep) was constructed by replacing the viral structural genes with an Rluc-Ubi-Neo-EMCV IRES fragment. The resulting DEN-I Rluc-Neo-Rep retained the N-terminal 37 amino acids of the capsid and the C-terminal 31 amino acids of the envelope protein.
  • G418 selection of the Rluc-Neo-Rep-transfected Vero cells allowed establishment of cell lines containing persistently replicating replicons.
  • FIG. 7 Inhibition of viral translation and RNA synthesis of DEN-I by triaryl pyrazoline.
  • A Two reporting replicons were constructed for DEN-I virus. One replicon contains a Rluc in-frame fused with the ORF at a position where the structural genes were deleted (DEN-I Rluc-Rep). The other replicon is identical to Rluc-Rep except that a FMDV 2A sequence was fused to the C- terminus of Rluc (DEN-I Rluc-2A-Rep).
  • BHK-21 cells were electroporated with an identical amount of DEN-I Rluc-Rep and Rluc-2A-Rep (10 ⁇ g), and assayed for Rluc activities at various time points p.t.
  • FIG. 8. Illustrates assay data for compounds of the invention.
  • halo is fluoro, chloro, bromo, or iodo.
  • Alkyl, alkoxy, etc. denote both straight and branched groups; but reference to an individual radical such as propyl embraces only the straight chain radical, a branched chain isomer such as isopropyl being specifically referred to.
  • Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic.
  • Heteroaryl encompasses a radical attached via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non- peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H, O, (C 1 -C 4 )alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived there from, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.
  • 'animal' as used herein includes mammals and birds. In one embodiment of the invention, the term animal refers to a human. It will be appreciated by those skilled in the art that compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism.
  • the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine opioid receptor binding and modulatory activity using the standard tests described herein, or using other similar tests which are well known in the art.
  • (XVC ⁇ alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl;
  • (C 3 -C 6 )cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; (C !
  • -C 6 )alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy;
  • (Ci-C 6 )alkanoyl can be acetyl, propanoyl or butanoyl;
  • (C 1 -C 6 )alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl;
  • C 2 -C 6 )alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy;
  • aryl can be phenyl, indenyl, or naphthyl;
  • R 1 A specific value for R 1 is benzoyl, 4-chlorobenzoyl, 4-nitrobenzoyl, 4- methoxybenzoyl, phenylacetyl, 4-chlorophenylacetyl, acetyl, 2,2- dimethylpropanoyl, or 4-methylphenylsulfonyl.
  • R 2 A specific value for R 2 is 4-carboxyphenyl, 4-bromophenyl, 4- methoxyphenyl, 4-chlorophenyl, 4-nitrophenyl, 4-(N ) JV-dimethylamino)phenyl, 2-naphthyl, 1-naphthyl, 2-pyrrolyl, 4-fluorophenyl, or phenyl.
  • R 3 is 2-thienyl, 2-pyrrolyl, 2-furyl, phenyl, 3- thienyl, 4-bromophenyl, 5-bromo2-thienyl, or tert-bntyl.
  • R 1 is (Ci-C 6 )alkyl, (C r C 6 )alkoxy, (d-C ⁇ alkanoyl, (d-C ⁇ alkanoyloxy, (d-C 6 )alkoxycarbonyl, haloCd-C ⁇ alkyl, or R a -X-;
  • X is a direct bond, (C 1 -C 6 )alkyl, -SO 2 -, or (d-C ⁇ alkanoyl;
  • R a is aryl, or heteroaryl
  • R b is aryl, or heteroaryl
  • R 3 is (C r C 6 )alkyl, (Ci-C 6 )alkoxy, (C 1 -C 6 )alkanoyl, (C ! -C 6 )alkanoyloxy, (C 1 -C 6 )alkoxycarbonyl, halo(C 1 -C 6 )alkyl, or R 0 -Z-; Z is a direct bond, (C 1 -C 6 )alkyl, or (C 1 -C 6 )alkanoyl;
  • R 0 is aryl, or heteroaryl; each bond represented by — is independently a single or a double bond; and
  • A is CH or N; wherein any aryl or heteroaryl OfR 1 -R 3 is optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (CrC 6 )alkyl, (C 1 -C 6 )alkoxy, (C 1 - C 6 )alkanoyloxy, (C 1 -C 6 )alkoxycarbonyl, cyano, carboxy, nitro, trifluomethyl, trifluoromethoxy, or R d R e N; wherein Rd and Re are each independently H or (C 1 - C 6 )alkyl; and pharmaceutically acceptable salts thereof.
  • a specific group of compounds are compounds of formula I that are compounds of formula (II)
  • a specific group of compounds are compounds of formula I that are compounds of formula (III)
  • a specific group of compounds are compounds of formula I that are compounds of formula (IV)
  • a specific group of compounds are compounds of formula I that are compounds of formula (V)
  • a specific value for X is (C 1 -C 6 )alkanoyl.
  • R 1 is (CrC 6 )alkyl, (d-C 6 )alkoxy, (C 1 -C 6 )alkanoyl, (Ci-C 6 )alkanoyloxy, (C ! -C 6 )alkoxycarbonyl, 1IaIo(C 1 -C6)alkyl, or R a -X-;
  • X is a direct bond, (Ci-C 6 )alkyl, -SO 2 -, or (C 1 -C 6 )alkanoyl;
  • R a is aryl, or heteroaryl; wherein any aryl or heteroaryl OfR 1 is optionally substituted with one or more halo, hydroxy, (Ci-C 6 )alkyl, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkanoyloxy, (C 1 - C 6 )alkoxycarbonyl, cyano, carboxy, nitro, tri
  • R 3 is phenyl, optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (C 1 -C 6 )alkyl, (C r C 6 )alkoxy, (C 1 - C 6 )alkanoyloxy, (C 1 -C 6 )alkoxycarbonyl, cyano, nitro, trifluomethyl, trifluoromethoxy, or R d R e N; wherein R d and R e are each independently H or (C 1 - C 6 )alkyl.
  • a specific value for R a is phenyl or 4-chlorophenyl.
  • a specific value for Y is a direct bond.
  • a specific value for R 2 is (C 1 -C 6 )alkyl, (d-C ⁇ alkoxy, (CrC 6 )alkanoyl,
  • R b is phenyl, optionally substituted with one or more (e.g. 1 , 2, 3, or 4) halo, hydroxy, (Q-C ⁇ alkyl, (Ci-C 6 )alkoxy, (C 1 - C 6 )alkanoyloxy, (C 1 -C 6 )alkoxycarbonyl, cyano, nitro, trifluomethyl, trifluoromethoxy, or R d R e N; wherein R d and R e are each independently H or (C 1 - C 6 )alkyl.
  • R b is phenyl, optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, (C]-C 6 )alkoxy, or nitro.
  • R b is 4-nitrophenyl, 4-chlorophenyl, 4-bromophenyl, or 4-methoxyphemyl.
  • a specific value for Z is a direct bond.
  • R 3 is (C 1 -C 6 )alkyl, (Ci-C 6 )alkoxy, (C 1 -C 6 )alkanoyl, (CrC ⁇ alkanoyloxy, (C 1 -C 6 )alkoxycarbonyl, ImIo(C 1 -C 6 )alkyl, or R 0 -Z-; Z is a .
  • R c is aryl, or heteroaryl; wherein any aryl or heteroaryl of R 3 is optionally substituted with one or more halo, hydroxy, (Q-C ⁇ alkyl, (Ci-C 6 )alkoxy, (C 1 -C 6 )alkanoyloxy, (C 1 - C 6 )alkoxycarbonyl, cyano, carboxy, nitro, trifluomethyl, trifluoromethoxy, or R d R e N; wherein R d and R e are each independently H or (C 1 -C 6 )alkyl.
  • R c is heteroaryl, optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (CrC 6 )alkyl, (C 1 - C 6 )alkanoyloxy, (C 1 -C 6 )aUcoxycarbonyl, cyano, nitro, trifluomethyl, trifluoromethoxy, or R d R 6 N; wherein R d and R e are each independently H or (C 1 - C 6 )alkyl.
  • a specific value for R 0 is phenyl, furyl, pyridyl, thienyl, pyrrolyl, imadazole, thiazole, or oxazole; optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, (d-C ⁇ alkanoyloxy, (C 1 - C 6 )alkoxycarbonyl, cyano, nitro, trifluomethyl, trifluoromethoxy, or R d R e N; wherein R d and R e are each independently H or (C 1 -C 6 )alkyl.
  • R 0 is 2-thienyl, optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (CrC ⁇ alkyl, (CrC 6 )alkoxy, (C 1 - C 6 )alkanoyloxy, (CrC ⁇ alkoxycarbonyl, cyano, nitro, trifluomethyl, trifluoromethoxy, or R d R e N; wherein R d and R e are each independently H or (C 1 - C 6 )alkyl.
  • R 0 is 2-thienyl
  • R 1 A specific value for R 1 is benzoyl, 4-chlorobenzoyl, 2,4-difluorobenzoyl, ⁇ 3,4-difluorobenzoyl, 3,5-difluorobenzoyl, 3-chlorobenzoyl, 2-chlorobenzoyl, 4- fluorobenzoyl, 3-fluorobenzoyl, 2-fluorobenzoyl, 4-nitrobenzoyl, A- methoxybenzoyl, 4-methylbenzoyl, 4-trifluoromethylbenzoyl, 3- trifluoromethylbenzoyl, phenylacetyl, 4-chlorophenylacetyl, acetyl, trimethylacetyl, p-toulenesulfonyl, diethylcarbamyl, 2-thiophenecarbonyl, 3- pyridinecarbonyl, 2-naphthylcarbonyl 5 or 3-naphthylcarbonyl.
  • R 2 A specific value for R 2 is 2-thienyl, 2-furyl, 2-pyrrolyl, 5-bromo-2- thienyl, 4-bromo-2-thienyl, 5-chloro-2-thienyl, 3-indolyl, 5-indolyl, 1-naphthyl, 2-naphthyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 3-chlorophenyl, 2- chlorophenyl, 3,5-dichlorophenyl, 4-methoxyphenyl, 4-carboxyphenyl, 4- nitrophenyl, 4-dimethylaminophenyl, 4-trifluoromethylplienyl, 3- trifluoromethylphenyl, 4-methylphenyl, 3-methylphenyl, or H.
  • R 3 A specific value for R 3 is 2-thienyl, 2-furyl, 2-pyrrolyl, 3-thienyl, 2- thiazolyl, 2-oxazolyl, 2-imidazolyl, 5-bromo-2-thienyl, 5-cliloro-2-thienyl, 3- indolyl, 1-naphthyl, 2-naphthyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, phenyl, 4- bromophenyl, 4-chlorophenyl, 4-nitrophenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-methylphenyl, 3-methylphenyl, 4-trifluoromethylphenyl, 3- trifluoromethylphenyl, fez't-butyl, methyl, or H.
  • a specific group of compounds are compounds of formula (I) wherein R 1 is Ra-X-; R2 is Rb-Y-; R3 is R c -Z-; R 3 is aryl, or heteroaryl; Rb is aryl, or heteroaryl; and R c is aryl, or heteroarylwherein any aryl or heteroaryl OfR 1 -R 3 is optionally substituted with one or more halo, hydroxy, (C 1 -C 6 )alkyl, (C 1 - Ce)alkoxy, (C 1 -C 6 )alkanoyloxy, (C 1 -C 6 )alkoxycarbonyl, cyano, carboxy, nitro, trifluomethyl, trifluoromethoxy, or R d R e N; wherein R d and R e are each independently H or (CrC ⁇ alkyl.
  • a specific group of compounds are compounds of formula (I) wherein any aryl or heteroaryl OfR 1 -R 3 is optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (C 1 -C 6 )alkyl, (C r C 6 )alkoxy, (C 1 -C 6 )alkanoyloxy, (C 1 - C 6 )alkoxycarbonyl, cyano, carboxy, nitro, trifluomethyl, trifluoromethoxy, or R d R e N; wherein R d and R» are each independently H or (Ci-C 6 )alkyl;
  • salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, ⁇ -ketoglutarate, and ⁇ -glycerophosphate.
  • Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
  • salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
  • a sufficiently basic compound such as an amine
  • a suitable acid affording a physiologically acceptable anion.
  • Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
  • the compounds of formula I can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
  • the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet.
  • a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.
  • the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.1% of active compound.
  • compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form.
  • the amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
  • the tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added.
  • binders such as gum tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as
  • the unit dosage form When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like.
  • a syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.
  • any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and devices.
  • the active compound may also be administered intravenously or intraperitoneally by infusion or injection.
  • Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms .
  • the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes, m all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like.
  • Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
  • Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.
  • the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
  • Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
  • Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.
  • the amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
  • the compound is conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.
  • the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
  • the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.
  • pyrazoline compounds of the invention can be prepared as illustrated in Schemes 1 and 2. Claisen-Schmidt condensation between a methyl aryl ketone (1) and an aromatic aldehyde (2) provides the stable chalcone intermediate (3). This reaction can conveniently be carried out in the presence of a strong base, such as NaOH, in a minimal volume bf methanol or ethanol. The Chalcone (3) is reacted with hydrazine in ethanol to provide the 3,5-diaryl pyrazoline (4). Subsequent reaction with an acid chloride yields the 1,3,5- trisubstituted pyrazoline (5).
  • a strong base such as NaOH
  • a Reagents and conditions (a) NaOH, methanol or ethanol, room temp; (b) N 2 H 4 H 2 O, ethanol, room temp; (c) acid chloride, slight heat.
  • Representative compounds of the invention can also be prepared by reduction with a suitable reducing agent (e.g. lithium aluminum hydride) as illustrated below.
  • a suitable reducing agent e.g. lithium aluminum hydride
  • Representative compounds of the invention can also be prepared by oxidation with a suitable oxidizing agent (e.g. potassium permanganate) as illustrated below.
  • a suitable oxidizing agent e.g. potassium permanganate
  • test compound The antiviral activity of a test compound can be evaluated using assay procedures that are known, or it can be evaluated using the procedures described hereinbelow.
  • Vero and BHK-21 cells were maintained in DMEM with 10% FBS in 5% CO 2 at 37 0 C.
  • a BHK-21 cell line containing a luciferase- expressing WN replicon was maintained with 1 mg/ml G418 in the culture medium ( Lo, L., et al, J. Virol, 2003, 77, 12901-12906).
  • WN virus was derived from a full-length infectious cDNA clone of an epidemic strain ( Shi, P. Y., et al., New York City. J. Virol, 2002, 76, 5847-56).
  • a full-length WN virus containing a luciferase reporter was previously constructed from an infectious cDNA clone by inserting an encephalomyocarditis virus internal ribosomal entry site (EMCV IRES)-luciferase fragment into the 3'-UTR of the WN virus genome ( Deas, T. S., et al., J. Virol., 2005, 4599-4609).
  • EMCV IRES encephalomyocarditis virus internal ribosomal entry site
  • SLE Saint Louis encephalitis
  • WEE Western Equine encephalitis
  • DEN-I reporting replicons Three types of reporting replicon were prepared for DEN-I virus Western Pacific 74 strain (GenBank accession U88535) ( Pur, B., et al., Virus Genes, 2000, 20, 57-63). Type one replicon contained a Renilla luciferase (Rluc) in-frame fused with the ORF of the genome at a position where the structural genes from nucleotide (nt) 281 to 2326 were deleted.
  • Rluc Renilla luciferase
  • Type two replicon was constructed by insertion of a foot-and-mouth disease virus (FMDV) 2A sequence ( Ryan, M. D., and J. Drew., EMBO J., 1994, 13, 928-933) immediately downstream of the Rluc reporter in the DEN-I Rluc-Rep.
  • FMDV 2A was designed to cleave the polyprotein at the N- terminus of the residual E fragment in the replicon.
  • Complementary oligoes representing the FMDV 2A sequence Ryan, M. D., and J.
  • Type three replicon was prepared to generate a stable cell line containing persistently replicating DEN-I replicon.
  • a reporting cassette containing the following sequence was directly RCR amplified from a replicon pLN-BR of bovine viral diarrhea virus (BVDV) ( Horscroft, N., et al., J. Virol., 2005, 79, 2788-96): Rluc-Ubi (ubiquitin)-Neo (Neomycin phosphotransferase)-EMCV IRES.
  • BVDV replicon was generously provided by Weidong Zhong from Valeant Pharmaceutical International Inc., Costa Mesa, CA.
  • the reporting cassette was then cloned into DEN-I Rluc-Rep at unique Not I and BwiW I sites, resulting in plasmid DEN-I Rluc-Neo-Rep.
  • DNA sequencing was performed to ensure that no mutations had occurred during PCR amplification. Standard procedures were performed for PCR and DNA cloning with modifications as previously described ( Shi, P. Y., et al., New York City. J. Virol, 2002, 76, 5847-56).
  • the IFA was performed using DEN-I immune mouse ascites fluid (ATCC, Manassas, VA) and goat anti-mouse IgG conjugated with Texas red as primary and secondary antibodies, respectively ( Shi, P. Y., et al., Virology, 2002, 296, 219-233).
  • DEN-I immune mouse ascites fluid ATCC, Manassas, VA
  • goat anti-mouse IgG conjugated with Texas red as primary and secondary antibodies
  • HTS antiviral assays For WN virus, three HTS assays (a luciferase- expressing replicon cell line, a virus-like particle (VLP) infection assay, and a reporting full-length viral infection assay) were previously established and validated for antiviral screening in a 96-well format. Since the VLP infection assay and the reporting WN virus infection assay involved infectious particles, these two assays were performed in biosafety level-3 containment. The replicon cell line-based assays (for both WN and DEN-I viruses) were performed in a biosafety level-2 laboratory because no infectious particles were involved.
  • Repreentative compounds of the invention were screened to identify WN virus inhibitors.
  • the compounds were dissolved in dimethyl sulfoxide (DMSO) and assayed at a final concentration of 1% DMSO.
  • DMSO dimethyl sulfoxide
  • a full-length Rluc-expressing WN virus was used to screen the compound library. Briefly, Vero cells were seeded at 8 * 10 4 per well of 96-well plate. At 6 h post cell seeding, cells were infected with the Rluc-expressing virus (1 MOI) and treated immediately with 30 ⁇ M of compounds. The screening concentration at 30 ⁇ M was empirically selected. The plates were assayed at 24 h p.i.
  • MTT cell proliferation assay An MTT cell proliferation assay (ATCC 5 Manassas, VA) was used to estimate potential cytotoxicity of compound. Approximately 2 x 10 4 BHK-21 or 8 * 10 4 Vero cells in 100 ⁇ l medium were seeded per well in a 96-well plate. After 6 h of incubation, 1 ⁇ l of compound dissolved in DMSO was added to cells at indicated concentrations. After 48 h of incubation, 10 ⁇ l of MTT reagent was added and cells were incubated for another 3.5 h, after which 100 ⁇ l of detergent reagent was added. The plates were swirled gently and left in dark at room temperature for 4 h.
  • Viral titer reduction assay Viral titer reduction assays were performed to examine the antiviral activities of representative compounds in WN, YF (17D), DEN-2, SLE, VSV, and WEE viruses. Approximately 9 x 10 5 Vero cells per well were seeded in a 12-well plate. After 12-h incubation, the cells were infected with individual virus (0.1 MOI) and treated immediately with compound at indicated concentrations.
  • culture medium were collected at 42 h post infection (p.i.), stored at -8O 0 C, and subjected to plaque assays on Vero cells.
  • culture medium was collected at 16-h p.i.
  • WN virus approximately 6 x 10 5 Vero cells per well were seeded in a 6-well plate and incubated for 3 days to reach full confluence. Cells were infected with 100 ⁇ l of 1 to 10 serial dilutions of the virus for 1 hour at 37 0 C. Afterwards, 3 ml of a first layer containing 0.6% Oxoid agar, BME medium with 1% FBS, 0.02% DEAE Dextran and 0.13% NaHCO 3 was added onto the infected cells.
  • Time of drug addition assay A time of addition experiment was performed to estimate the step of viral life cycle that was suppressed by the compounds. Approximately 9 x 10 5 Vero cells per well were seeded in a 12 well- plate, incubated for 12 h for cell attachment, and synchronously infected with WN virus. The infection was carried out at MOI of 5 for 1 h followed by three rounds of PBS washing to remove the unabsorbed viruses. At different time points post infection, triaryl pyrazoline was added to the infected cells at 100 ⁇ M. Culture medium were collected at 24 h p.L, stored at -8O 0 C, and subjected to plaque assay as described above. As negative controls, 1% of DMSO was added to infected cell at 0 and 20 h p.i. to estimate its effect on viral yield production.
  • Transient replicon assay A transient replicon assay was used to quantify compound-mediated inhibition of viral translation and suppression of RNA replication.
  • replicon RNA 10 ⁇ g was electroporated into BHK-21 cells (8 x 10 6 ) as previously described ( Lo, L., et al., J. Virol, 2003, 77, 10004-10014). The transfected cells were suspended in 25 ml of DMEM with 10% FBS. Cell suspension (5 x 10 5 in 167 ⁇ l per well) was added to 12- well plates, immediately treated with 30 or 100 ⁇ M of compounds, and assayed for luciferase activity at 2 and 4 h p.t.
  • an MTT assay was performed to exclude the possibility that the observed antiviral activity was due to compound-mediated cytotoxicity.
  • Vero cells were incubated with the compound for 48 h and cell viability was measured by cellular metabolism of MTT tetrazolium salt (Fig. 2B). No reduction of cell viability was observed up to 600 ⁇ M (the highest tested concentration). Similar results were obtained when BHK-21 cells were incubated with the compound in the MTT assay (data not shown). However, tiny crystals were observd under the microscope in culture medium when compound concentration reached 300 ⁇ M or higher. The results indicate that the CC 50 of the compound is >300 ⁇ M. Overall, the above data demonstrate that the compound inhibits WN virus without observable cytotoxicity in cell culture.
  • Viral titer reduction (fold) Viral titer without treatment / Viral titer with compound treatment.
  • the compound was analyzed in two non-flaviviruses, WEE virus (a plus-strand RNA alphavirus) and VSV (a negative-strand RNA rhabdovirus) .
  • WEE virus a plus-strand RNA alphavirus
  • VSV a negative-strand RNA rhabdovirus
  • the compound inhibited both WEE and VSV viruses.
  • the compound reduced viral titer of WEE and VSV by 10 and 22 folds, respectively.
  • triaryl pyrazoline can inhibit not only flaviviruses, but also other RNA viruses in cell culture.
  • VLPs were prepared by trans supplying WN structural proteins in a stable cell line containing Rluc-reporter replicons (WN Rluc-Neo-Rep, 4B). Infection of na ⁇ ve Vero cells with such Rluc-VLPs (1 MOI) could be used to examine if an inhibitor blocks viral entry and replication. Treatment of the VLP- infected cells with triaryl pyrazoline suppressed luciferase signals, indicating that the compound could inhibit viral entry and/or replication. The EC 50 value was estimated to be 14 ⁇ M (Fig. 4A).
  • the second assay was based on a BHK-21 cell line harboring the WN Rluc-Neo-Rep (Fig. 4B).
  • the reporting cell line allows testing inhibitors of viral replication (including viral translation and RNA synthesis), but not viral entry. Analyses using the WN Rluc-Neo-Rep cell line showed that the compound inhibited viral replication with an EC 5 O of 19 ⁇ M.
  • the third assay involves WN Rluc-Rep in which a luciferase reporter was in-frame fused with the ORF of the genome where the structural genes were deleted (Fig. 4C).
  • Transfection of BHK-21 cells with such replicon reveals two Rluc peaks at 1-10 h and >24 h p.t. which represent viral translation and RNA synthesis, respectively ( Lo, L., et al, J. Virol, 2003, 77, 10004-10014).
  • BHK-21 cells were transfected with WN Rluc-Rep RNA, immediately incubated the cells with compound, and assayed for luciferase activities at 2, 4, and 72 h p.t.
  • luciferase signals from the cells treated with compound were around 96-117% of those from the mock-treated cells.
  • luciferase activities were suppressed by over 95% upon compound treatment.
  • triaryl pyrazoline inhibits WN virus through suppression of viral RNA synthesis.
  • the DEN-I replicon-containing cell line was converted into an HTS assay. Seeding of 2 x 10 4 cells per well of 96- well plate and incubation of the cells for 48 h consistently showed an assay window of 1 x 10 6 to 2 x 10 6 (Rluc signal from replicon-bearing cells / background signal from naive BHK-21 cells). To validate the assay for HTS, the DEN-I Rluc-Neo-Rep cell lines were incubated with various concentrations of mycophenolic acid (MPA) and ribavirin, two known inhibitors of DEN virus ( Diamond, M. S., et al., Virology, 2002, 304, 211-21).
  • MPA mycophenolic acid
  • ribavirin two known inhibitors of DEN virus
  • DEN-I reporting replicon which can differentiate between viral translation and RNA synthesis was established. Initially, a replicon in which an Rluc replaced viral structural genes (DEN-I Rluc-Rep, Fig. 7A) was constructed.
  • the replicon Upon transfection, the replicon was expected to express a luciferase fusion protein containing N-terminal extra 40 amino acids (37 residues from capsid and 3 residues from the Not I site engineered for cloning purpose) and C-terminal extra 33 amino acids (2 residues from the BsiW I site engineered for cloning and 31 residues from envelope).
  • Transfection of BHK-21 cells with DEN-I Rluc- Rep yielded a single Rluc peak at 1 to 10 h p.t, but never a second Rluc peak (Fig. 7B).
  • IFA analysis showed no viral protein expression in the transfected cells (right panel, Fig. 7C). The results suggest that DEN-I Rluc-Rep RNA was successfully transfected into cells (as the initial translation of input RNA had occurred), but no replication had followed.
  • a FMDV 2A sequence was inserted into the original replicon to mediate a cleavage between Rluc and the C-terminal fragment of envelope protein (DEN-I Rluc-2A-Rep, Fig. 7A). Since the FMDV 2A cleaves at its C-terminus, the Rluc expressed from the Rluc-2A-Rep would be C-terminally fused to the FMDV 2 A.
  • Transfection of BHK-21 cells with DEN-I Rluc-2A-Rep yielded two luciferase peaks: the first peak during the initial 1O h p.t. and a second peak after 1O h p.t. (Fig. 7B).
  • the 2A-containing replicon replicated efficiently, as indicated by a robust second Rluc peak after 1O h p.t. (Fig. 7B) as well as IFA- positive cells at 60 h p.t. (Fig. 7C).
  • a similar luciferase kinetics with two peaks was observed upon transfection of the Rluc-2A-Rep RNA into Vero cells.
  • the mode of action of triaryl pyrazoline has been characterized in WN virus. Because the three reporting assays described above encompasses multiple, but discrete steps of the viral life cycle, they could be used to discriminate the inhibitory step(s) of any compounds amongst viral entry, replication, and virion assembly.
  • the comparable values of EC 50 S derived from the three reporting assays indicate that the compound inhibits WN infection at a step that is shared by all three HTS assays: viral replication. Further analyses using a transient replicon system showed that the compound significantly blocks RNA synthesis (by >95%) without suppression of viral translation (Figs. 4C).
  • the observed antiviral activity was solely caused by its effect on translation, which, in turn, resulted in suppression of viral RNA synthesis.
  • representative compounds of the invention also inhibited other flaviviruses, including YF (17D) and SLE viruses.
  • the compounds suppressed other plus-strand and minus-strand RNA viruses, as represented by WEE and VSV, respectively (Fig. 3).
  • the broad spectrum of antiviral activity indicates that the compound blocks a target commonly required for replication of the tested viruses. It is currently not known whether the compound exerts its functions through direct interaction with a host factor or a viral protein. A number of approaches are being explored to define the target of the compound.
  • Testing the compound in biochemistry assays may indicate whether the inhibitors directly interfere with the viral functions.
  • sequencing of resistant viruses or replicons may point to the targets of the compound.
  • High resolution mass spectra were collected from a TOF-ESI Agilant LC-MS and analyzed using the Analyst QS software.
  • Reversed-phase high-performance liquid chromatography (RP- HPLC) was performed on a Beckman Coulter 125 S System Gold using an Agilent Zorbax Eclipse XDB-C8 3.5 ⁇ M 3.0 X 150mm column monitoring UV at 254 ⁇ on a 166 detector.
  • Method A was a gradient method that ran for 14min at a flow rate of 0.5 mL/min. Over the first 8 minutes the percent ACN/water was increased from 60%-100%. Over the next 4 minutes the gradiant was decreased back to 60% ACN/water and then held for two minutes.
  • Method B was an isocratic method using 70% MeOH/water and a flow rate of 0.4 mL/min. The run duration was twenty minutes. Results were analyzed using the 32 Karat software package.
  • Example 5 The following illustrate representative pharmaceutical dosage forms, containing a compound of formula I ('Compound X 1 ), for therapeutic or prophylactic use in humans.

Abstract

The invention provides anti-viral compounds of formula (I) wherein R1-R3 and A have any of the values defined herein. The invention also provides pharmaceutical compositions comprising such compounds as well as methods for treating viral infections by administering such compounds to an animal.

Description

ANTI-VIRAL COMPOUNDS
Priority of Invention
This application claims priority to United States Provisional Application Number 60/721002 that was filed on 27 September 2005. The entire content of this provisional application is hereby incorporated herein by reference.
Background of the Invention The family Flaviviridae contains three genera, the flaviviruses, the pestiviruses, and the hepatitis C viruses. Many members within the Flayivirus genus are arthropod-borne pathogens that cause significant human morbidity and mortality. The global emerging flaviviruses include four serotypes of dengue (DEN), yellow fever (YF), West Nile (WN), Japanese encephalitis (JE)5 and tick-borne encephalitis (TBE) viruses ( Burke, D. S., and T. P. Monath, 2001. Flaviviruses. Lippincott William & Wilkins). The World Health Organization estimated annual human cases of more than 50 million, 200,000, and 50,000 for DEN, YF, and JE viral infections, respectively. The recent epidemics of WN virus in the United States have caused thousands of human cases, representing the largest meningoencephalitis outbreak in the Western Hemisphere and the largest WN virus outbreak ever reported (CDC. 2002. Provisional surveillance summary of the West Nile virus epidemic— United States, January-November 2002. MMWR Morb. Mortal. WkIy. Rep. 51:1129-1133). Human vaccines are currently available only for YF, JE, and TBE viruses, and no effective antiviral therapy has been developed for treatment of flavivirus infections. Therefore, there is currently a need for anti-viral compounds that are useful for treating flavivirus infections.
Summary of the Invention Compounds have been identified that suppress viral RNA synthesis and that are useful for treating flavivirus infections. Thus, in one embodiment the invention provides anti-viral compounds that have a broad spectrum of anti- flavivirus activity. Accordingly, the invention provides a compound of the invention which is a compound of formula I:
Figure imgf000004_0001
(I) wherein:
R1 is (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylaminocarbonyl, di(C1-C6)alkylamino- carbonyl, halo(C1-C6)alkyl, or Ra-X-;
X is a direct bond, (Ci-C6)alkyl5 -SO2-, or
Figure imgf000004_0002
Ra is aryl, or heteroaryl;
R2 is H5 (C!-C6)alkyl, (d-C^alkoxy, (C1-C6)alkanoyl, (C1- C6)alkanoyloxy, (d-C^alkoxycarbonyl, halo(C1-C6)alkyl, (C1- C6)alkylaminocarbonylamino, di(C1-C6)alkylaminocarbonyl, (C1- C6)alkylsulfonylamino, or Rb-Y-;
Y is a direct bond, -NH-C(=O)-NH-, -NH-SO2-, (C1-C6)alkyl, or (C1- C6)alkanoyl; Rb is aryl, or heteroaryl;
R3 is H, (d-C^alkyl, (C1-C6)alkoxy, (d-C^alkanoyl, (Ci- C6)alkanoyloxy, (CrC^alkoxycarbonyl, halo(C1-C6)alkyl, or R0-Z-; Z is a direct bond, (CrC^alkyl, or (Ci-C^alkanoyl; R0 is aryl, or heteroaryl; each bond represented by — is independently a single or a double bond; and
A is CH or N; wherein any aryl or heteroaryl OfR1-R3 is optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (CrC^alkyl, (C1-C6)alkoxy, (C1- C6)alkanoyloxy, (CrC^alkoxycarbonyl, cyano, carboxy, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and Re are each independently H, (C1- C6)alkanoyl, or (Ci-C6)alkyl; or a pharmaceutically acceptable salt thereof. The invention also provides a pharmaceutical composition comprising a compound of formula I5 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent or carrier.
The invention also provides a therapeutic method for treating a viral infection in an animal comprising administering to the animal, an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof.
The invention also provides a therapeutic method for suppressing viral RNA synthesis in an animal comprising administering to the animal, an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
The invention also provides a compound of formula (I), or a pharmaceutically acceptable salt thereof for use in medical therapy.
The invention also provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof to prepare a medicament useful for the treatment of a viral infection in a animal.
The invention also provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof to prepare a medicament useful for suppressing viral RNA synthesis in an animal. The invention also provides processes and intermediates disclosed herein that are useful for preparing compounds of formula (I) or salts thereof. Certain compounds of formula (I) are useful as intermediates for preparing other compounds of formula (I).
Brief Description of the Figures
FIG. 1. Identification of triaryl pyrazolone as an inhibitor of a full-length luciferase-expressing WN virus. (A) A full-length WN virus containing a luciferase reporter (FL Rluc-WN) ( Deas, T. S., et al., J. Virol., 2005, 4599- 4609) was used to infect Vero cells at an MOI of 0.1. The infected cells were incubated with a compound library at indicated concentrations, and assayed for Rluc activity at 24 h p.t. Values above each bar indicate the percentage of the luciferase activity derived from the compound-treated infection versus the luciferase signal derived from the mock-treated infection. Mycophenolic acid (MPA), a known WN virus inhibitor, was included as positive controls. Since the compound was screened at 1% DMSO, the mock-treated infection also contained this concentration of DMSO. (B) Structure of triaryl pyrazόline, an inhibitor identified from the screening. FIG. 2. Inhibition of an epidemic strain of WN virus and cytotoxicity of triaryl pyrazoline. (A) Vero cells were infected with an epidemic strain of WN virus (0.1 MOI), immediately treated with compound at indicated concentrations, and assayed for viral titers at 42 h p.i. Values above each data point indicate the percentage of viral titer from the compound-treated infection as compared with that from the mock-treated infection. (B) Cytotoxicity was examined by incubation of Vero cells with the indicated concentration of the compound. After 48 h incubation, cell viability was measured by an MTT assay and presented as a percentage of colorimetric absorbance derived from untreated cells. Representative results from three independent experiments are shown.
FIG. 3. Antiviral activities of triaryl pyrazoline against various RNA viruses. Viral titer reduction assays were performed to determine the antiviral activities of the compound. Vero cells were infected withDEN-2, YF (17D), SLE, WEE and VSV viruses (0.1 MOI), treated immediately with the compound at indicated concentrations, and assayed for virus yield in culture medium at 42 h p.i. (see details in Materials and Methods).
FIG. 4. Mechanism of triaryl pyrazoline-mediated inhibition of WN virus. (A) WN VLPs containing a luciferase-expressing replicon (Rluc-VLP) was used to infect Vero cells in the presence of indicated concentrations of compound. At 48 h p.i., cells were quantified for Rluc activity. The Rluc- VLP/Vero infection allows test if inhibitors block viral entry and replication. (B) A BHK-21 cell line containing WN replicon (Rluc-Neo-Rep) ( Lo, L., et al., J. Virol, 2003, 77, 12901-12906) was incubated with compound for 48 h and measured for Rluc activity. The replicon-bearing cell line examines if a compound inhibits viral replication, but not viral entry. (C) A WN reporting replicon containing an Rluc reporter (fused in-frame with the ORF; Rluc-Rep) was used to measure the effects of compound on viral translation and RNA synthesis. BHK-21 cells transfected with Rluc-Rep exhibited two distinctive Rluc peaks at 2-10 h and after 24 h p.t, representing viral translation and RNA replication, respectively ( Lo, L., et al., J. Virol., 2003, 77, 10004-10014). Effects of compound on WN translation and RNA synthesis were reflected by luciferase signals at 2 and 4 h p.t., and 72 h p.t., respectively. Values above each data point indicate the percentage of viral titer from the compound-treated infection as compared with that from the mock-treated infection.
FIG. 5. Time-of-addition analyses of triaryl pyrazoline in WN virus infection. Vero cells were synchronously infected with WN virus, treated with compounds (100 μM) at indicated time points after infection, and quantified for viral yields in culture medium at 24 h p.i. Because all compound treatments contained 1% DMSO, this concentration of DMSO was added to infected cell at 0 and 20 h p.i.' to estimate its effect on viral yield production.
FIG. 6. Inhibition of a reporting replicon cell line of DEN-I virus. (A) A replicon of DEN-I virus (Rluc-Neo-Rep) was constructed by replacing the viral structural genes with an Rluc-Ubi-Neo-EMCV IRES fragment. The resulting DEN-I Rluc-Neo-Rep retained the N-terminal 37 amino acids of the capsid and the C-terminal 31 amino acids of the envelope protein. G418 selection of the Rluc-Neo-Rep-transfected Vero cells allowed establishment of cell lines containing persistently replicating replicons. (B) Rluc-Neo-Rep-containing cell lines were analyzed by IFA using DEN-I immune mouse ascites fluid and goat anti-mouse IgG conjugated with Texas red as primary and secondary antibodies, respectively. The same view of IFA stained with Texas red (left panel) and differential interference contrast (right panel) are presented. (C) The reporting Vero cells containing DEN-I Rluc-Neo-Rep was validated for its use as an antiviral assay in a 96-well format. The reporting cells were incubated with three compounds (MPA, ribavirin, and glycyrrhizin) and assayed for Rluc activity at 48 h post treatment. (D) The DEN-I replicon-containing cells were used to examine the antiviral activity of triacryl pyrazoline. Various concentrations of compound were assayed for its inhibition of DEN-I replication as described in (C).
FIG. 7. Inhibition of viral translation and RNA synthesis of DEN-I by triaryl pyrazoline. (A) Two reporting replicons were constructed for DEN-I virus. One replicon contains a Rluc in-frame fused with the ORF at a position where the structural genes were deleted (DEN-I Rluc-Rep). The other replicon is identical to Rluc-Rep except that a FMDV 2A sequence was fused to the C- terminus of Rluc (DEN-I Rluc-2A-Rep). (B) BHK-21 cells were electroporated with an identical amount of DEN-I Rluc-Rep and Rluc-2A-Rep (10 μg), and assayed for Rluc activities at various time points p.t. The double lines on the vertical axis indicate that the scales of the top and bottom portions of the diagram are different. (C) BHK-21 cells transfected with equal amount of DEN- 1 Rluc-Rep (left panel) and Rluc-2A-Rep (right panel) as described in (B) were analyzed by IFA at 60 h p.t. (D) The transient replicon system was used to quantify the inhibitory effects of triaryl pyrazoline on viral translation and RNA synthesis in DEN-I virus. Immediately after electroporation with DEN-I Rluc- 2A-Rep, BHK-21 cells were treated with the compound at indicated concentrations, and assayed for Rluc activities at 2, 4, and 48 h p.t. Compound- mediated inhibition of viral translation and RNA synthesis were quantified by Rluc signals at 2 and 4 h p.t., and 72 h p.t., respectively. Values above each bar indicate the percentage of the Rluc activity derived from the compound-treated infection versus the Rluc signal derived from the mock-treated infection.
FIG. 8. Illustrates assay data for compounds of the invention.
Detailed Description
The following definitions are used, unless otherwise described: halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, etc. denote both straight and branched groups; but reference to an individual radical such as propyl embraces only the straight chain radical, a branched chain isomer such as isopropyl being specifically referred to. Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. Heteroaryl encompasses a radical attached via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non- peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H, O, (C1-C4)alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived there from, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.
The term 'animal' as used herein includes mammals and birds. In one embodiment of the invention, the term animal refers to a human. It will be appreciated by those skilled in the art that compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine opioid receptor binding and modulatory activity using the standard tests described herein, or using other similar tests which are well known in the art.
Specific and preferred values listed below for radicals, substiruents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substiruents. Specifically, (XVC^alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C3-C6)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; (C!-C6)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; (Ci-C6)alkanoyl can be acetyl, propanoyl or butanoyl; (C1-C6)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl; (C2-C6)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy; aryl can be phenyl, indenyl, or naphthyl; and heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or its N-oxide). A specific value for R1 is benzoyl, 4-chlorobenzoyl, 4-nitrobenzoyl, 4- methoxybenzoyl, phenylacetyl, 4-chlorophenylacetyl, acetyl, 2,2- dimethylpropanoyl, or 4-methylphenylsulfonyl.
A specific value for R2 is 4-carboxyphenyl, 4-bromophenyl, 4- methoxyphenyl, 4-chlorophenyl, 4-nitrophenyl, 4-(N)JV-dimethylamino)phenyl, 2-naphthyl, 1-naphthyl, 2-pyrrolyl, 4-fluorophenyl, or phenyl.
A specific value for R3 is 2-thienyl, 2-pyrrolyl, 2-furyl, phenyl, 3- thienyl, 4-bromophenyl, 5-bromo2-thienyl, or tert-bntyl.
A specific group of compounds are compounds of formula I:
Figure imgf000010_0001
(I) wherein:
R1 is (Ci-C6)alkyl, (CrC6)alkoxy, (d-C^alkanoyl, (d-C^alkanoyloxy, (d-C6)alkoxycarbonyl, haloCd-C^alkyl, or Ra-X-; X is a direct bond, (C1-C6)alkyl, -SO2-, or (d-C^alkanoyl;
Ra is aryl, or heteroaryl;
R2 is (Ci-C6)alkyl, (C1-C6)alkoxy, (Ci-C^alkanoyl, (Ci-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, halo(C1-C6)alkyl, (C1-C6)alkylaminocarbonylamino, (Q-C^alkylsulfonylamino, or Rb-Y-; Y is a direct bond, -NH-C(=O)-NH-, -NH-SO2-, (Ci-C6)alkyl, or (C1-
C6)alkanoyl;
Rb is aryl, or heteroaryl;
R3 is (CrC6)alkyl, (Ci-C6)alkoxy, (C1-C6)alkanoyl, (C!-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, halo(C1-C6)alkyl, or R0-Z-; Z is a direct bond, (C1-C6)alkyl, or (C1-C6)alkanoyl;
R0 is aryl, or heteroaryl; each bond represented by — is independently a single or a double bond; and
A is CH or N; wherein any aryl or heteroaryl OfR1-R3 is optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (CrC6)alkyl, (C1-C6)alkoxy, (C1- C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, cyano, carboxy, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and Re are each independently H or (C1- C6)alkyl; and pharmaceutically acceptable salts thereof. A specific group of compounds are compounds of formula I that are compounds of formula (II)
Figure imgf000011_0001
(H).
A specific group of compounds are compounds of formula I that are compounds of formula (III)
(HI)-
A specific group of compounds are compounds of formula I that are compounds of formula (IV)
Figure imgf000011_0003
(IV).
A specific group of compounds are compounds of formula I that are compounds of formula (V)
Figure imgf000012_0001
(V).
A specific value for X is (C1-C6)alkanoyl. A specific value for X is -C(=O)-.
A specific value for R1 is (CrC6)alkyl, (d-C6)alkoxy, (C1-C6)alkanoyl, (Ci-C6)alkanoyloxy, (C!-C6)alkoxycarbonyl, 1IaIo(C1 -C6)alkyl, or Ra-X-; X is a direct bond, (Ci-C6)alkyl, -SO2-, or (C1-C6)alkanoyl; and Ra is aryl, or heteroaryl; wherein any aryl or heteroaryl OfR1 is optionally substituted with one or more halo, hydroxy, (Ci-C6)alkyl, (C1-C6)alkoxy, (C1-C6)alkanoyloxy, (C1- C6)alkoxycarbonyl, cyano, carboxy, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and R6 are each independently H or (Ci-C6)alkyl.
A specific value for R3 is phenyl, optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (C1-C6)alkyl, (CrC6)alkoxy, (C1- C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, cyano, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and Re are each independently H or (C1- C6)alkyl.
A specific value for Ra is phenyl or 4-chlorophenyl. A specific value for Y is a direct bond. A specific value for R2 is (C1-C6)alkyl, (d-C^alkoxy, (CrC6)alkanoyl,
(C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, halo(Ci-C6)alkyl, (C1- C6)alkylaminocarbonylamino, (Q-C^alkylsulfonylamino, or Rb-Y-; Y is a direct bond, -NH-C(=0)-NH-, -NH-SO2-, (CrC6)alkyl, or (Ci-C6)alkanoyl; and Rb is aryl, or heteroaryl; wherein any aryl or heteroaryl of R2 is optionally substituted with one or more halo, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-
C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, cyano, carboxy, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and Re are each independently H or (C1- C6)alkyl.
A specific value for Rb is phenyl, optionally substituted with one or more (e.g. 1 , 2, 3, or 4) halo, hydroxy, (Q-C^alkyl, (Ci-C6)alkoxy, (C1- C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, cyano, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and Re are each independently H or (C1- C6)alkyl.
A specific value for Rb is phenyl, optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, (C]-C6)alkoxy, or nitro.
A specific value for Rb is 4-nitrophenyl, 4-chlorophenyl, 4-bromophenyl, or 4-methoxyphemyl.
A specific value for Z is a direct bond.
A specific value for R3 is (C1-C6)alkyl, (Ci-C6)alkoxy, (C1-C6)alkanoyl, (CrC^alkanoyloxy, (C1-C6)alkoxycarbonyl, ImIo(C1 -C6)alkyl, or R0-Z-; Z is a . direct bond, (C1-C6)alkyl, or (C1-C6)alkanoyl; and Rc is aryl, or heteroaryl; wherein any aryl or heteroaryl of R3 is optionally substituted with one or more halo, hydroxy, (Q-C^alkyl, (Ci-C6)alkoxy, (C1-C6)alkanoyloxy, (C1- C6)alkoxycarbonyl, cyano, carboxy, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and Re are each independently H or (C1-C6)alkyl.
A specific value for Rc is heteroaryl, optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (CrC6)alkyl,
Figure imgf000013_0001
(C1- C6)alkanoyloxy, (C1-C6)aUcoxycarbonyl, cyano, nitro, trifluomethyl, trifluoromethoxy, or RdR6N; wherein Rd and Re are each independently H or (C1- C6)alkyl.
A specific value for R0 is phenyl, furyl, pyridyl, thienyl, pyrrolyl, imadazole, thiazole, or oxazole; optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, (d-C^alkanoyloxy, (C1- C6)alkoxycarbonyl, cyano, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and Re are each independently H or (C1-C6)alkyl.
A specific value for R0 is 2-thienyl, optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (CrC^alkyl, (CrC6)alkoxy, (C1- C6)alkanoyloxy, (CrC^alkoxycarbonyl, cyano, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and Re are each independently H or (C1- C6)alkyl.
A specific value for R0 is 2-thienyl.
A specific value for R1 is benzoyl, 4-chlorobenzoyl, 2,4-difluorobenzoyl, ■ 3,4-difluorobenzoyl, 3,5-difluorobenzoyl, 3-chlorobenzoyl, 2-chlorobenzoyl, 4- fluorobenzoyl, 3-fluorobenzoyl, 2-fluorobenzoyl, 4-nitrobenzoyl, A- methoxybenzoyl, 4-methylbenzoyl, 4-trifluoromethylbenzoyl, 3- trifluoromethylbenzoyl, phenylacetyl, 4-chlorophenylacetyl, acetyl, trimethylacetyl, p-toulenesulfonyl, diethylcarbamyl, 2-thiophenecarbonyl, 3- pyridinecarbonyl, 2-naphthylcarbonyl5 or 3-naphthylcarbonyl.
A specific value for R2 is 2-thienyl, 2-furyl, 2-pyrrolyl, 5-bromo-2- thienyl, 4-bromo-2-thienyl, 5-chloro-2-thienyl, 3-indolyl, 5-indolyl, 1-naphthyl, 2-naphthyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 3-chlorophenyl, 2- chlorophenyl, 3,5-dichlorophenyl, 4-methoxyphenyl, 4-carboxyphenyl, 4- nitrophenyl, 4-dimethylaminophenyl, 4-trifluoromethylplienyl, 3- trifluoromethylphenyl, 4-methylphenyl, 3-methylphenyl, methyl, or H.
A specific value for R3 is 2-thienyl, 2-furyl, 2-pyrrolyl, 3-thienyl, 2- thiazolyl, 2-oxazolyl, 2-imidazolyl, 5-bromo-2-thienyl, 5-cliloro-2-thienyl, 3- indolyl, 1-naphthyl, 2-naphthyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, phenyl, 4- bromophenyl, 4-chlorophenyl, 4-nitrophenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-methylphenyl, 3-methylphenyl, 4-trifluoromethylphenyl, 3- trifluoromethylphenyl, fez't-butyl, methyl, or H.
A specific group of compounds are compounds of formula (I) wherein R1 is Ra-X-; R2 is Rb-Y-; R3 is Rc-Z-; R3 is aryl, or heteroaryl; Rb is aryl, or heteroaryl; and Rc is aryl, or heteroarylwherein any aryl or heteroaryl OfR1-R3 is optionally substituted with one or more halo, hydroxy, (C1-C6)alkyl, (C1- Ce)alkoxy, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, cyano, carboxy, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and Re are each independently H or (CrC^alkyl.
A specific group of compounds are compounds of formula (I) wherein any aryl or heteroaryl OfR1-R3 is optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (C1-C6)alkyl, (CrC6)alkoxy, (C1-C6)alkanoyloxy, (C1- C6)alkoxycarbonyl, cyano, carboxy, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and R» are each independently H or (Ci-C6)alkyl;
In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
The compounds of formula I can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.
The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms . The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes, m all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user. Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.
The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. The compound is conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.
The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations. Peparation of Compounds of the Invention
Representative pyrazoline compounds of the invention can be prepared as illustrated in Schemes 1 and 2. Claisen-Schmidt condensation between a methyl aryl ketone (1) and an aromatic aldehyde (2) provides the stable chalcone intermediate (3). This reaction can conveniently be carried out in the presence of a strong base, such as NaOH, in a minimal volume bf methanol or ethanol. The Chalcone (3) is reacted with hydrazine in ethanol to provide the 3,5-diaryl pyrazoline (4). Subsequent reaction with an acid chloride yields the 1,3,5- trisubstituted pyrazoline (5).
Scheme 1 :
Figure imgf000018_0001
Figure imgf000018_0002
4 Scheme 2:
Figure imgf000019_0001
a Reagents and conditions: (a) NaOH, methanol or ethanol, room temp; (b) N2H4H2O, ethanol, room temp; (c) acid chloride, slight heat.
Representative compounds of the invention can also be prepared by reduction with a suitable reducing agent (e.g. lithium aluminum hydride) as illustrated below.
Figure imgf000019_0002
Representative compounds of the invention can also be prepared by oxidation with a suitable oxidizing agent (e.g. potassium permanganate) as illustrated below.
Figure imgf000019_0003
Data for representative compounds is shown in Table 1 (Figure 8). Based on the results, it appears likely that the compounds are disrupting viral replication. It should also be noted that the pyrazolines 5a and 5b show little toxicity, lending to a favorable therapeutic index in both BHK and Vero cell lines. In comparison to the known inhibitor ribavirin, it is observed that both of these pyrazolines show slightly greater activity. It is also possible that compounds with greater potency will be identified by separation of enantiomers. Antiviral Activity
The antiviral activity of a test compound can be evaluated using assay procedures that are known, or it can be evaluated using the procedures described hereinbelow. MATERIALS AND METHODS
Cells and viruses. Vero and BHK-21 cells were maintained in DMEM with 10% FBS in 5% CO2 at 370C. A BHK-21 cell line containing a luciferase- expressing WN replicon was maintained with 1 mg/ml G418 in the culture medium ( Lo, L., et al, J. Virol, 2003, 77, 12901-12906). WN virus was derived from a full-length infectious cDNA clone of an epidemic strain ( Shi, P. Y., et al., New York City. J. Virol, 2002, 76, 5847-56). A full-length WN virus containing a luciferase reporter was previously constructed from an infectious cDNA clone by inserting an encephalomyocarditis virus internal ribosomal entry site (EMCV IRES)-luciferase fragment into the 3'-UTR of the WN virus genome ( Deas, T. S., et al., J. Virol., 2005, 4599-4609). YF (17D)3 DEN-2, Saint Louis encephalitis (SLE), and Western Equine encephalitis (WEE) viruses were generously provided by Laura Kramer at Arbovirus laboratory at Wadsworth Center, Albany, NY. Vesicular Stomatitis virus (VSV) was kindly provided by Paul Masters at the Wadsworth Center.
Construction of DEN-I reporting replicons. Three types of reporting replicon were prepared for DEN-I virus Western Pacific 74 strain (GenBank accession U88535) ( Pur, B., et al., Virus Genes, 2000, 20, 57-63). Type one replicon contained a Renilla luciferase (Rluc) in-frame fused with the ORF of the genome at a position where the structural genes from nucleotide (nt) 281 to 2326 were deleted. An overlap extension approach was used to prepare a DNA fragment containing the following sequence: Barriϋ I/Asc I/T7 promoter/DEN-1 nt 1-208/Not I/ RIUC/AΪZW I/DEN- 1 nt 2327-2721. A full-length infectious clone of DEN-I virus ( Pur, B., et al., Virus Genes, 2000, 20, 57-63) was used as a template for amplifying DEN-I sequences. The PCR product was digested with BarnΑ I and Mo I (nt 2606-2611 of DEN-I) and cloned into a pBR322-based plasmid ( Shi, P. Y., et al., Virology, 2002, 296, 219-233). Next, the Xho I to Sac II fragment representing the rest of the DEN-I genome was cleaved off from the full-length DEN-I clone and ligated into the pBR322-based intermediate plasmid described above, resulting plasmid DEN-I Rluc-Rep.
Type two replicon was constructed by insertion of a foot-and-mouth disease virus (FMDV) 2A sequence ( Ryan, M. D., and J. Drew., EMBO J., 1994, 13, 928-933) immediately downstream of the Rluc reporter in the DEN-I Rluc-Rep. The FMDV 2A was designed to cleave the polyprotein at the N- terminus of the residual E fragment in the replicon. Complementary oligoes representing the FMDV 2A sequence ( Ryan, M. D., and J. Drew., EMBO J., 1994, 13, 928-933) containing a BsiW I site on both ends were annealed in a hybridization buffer (10 mM NaCl and 10 niM Tris, pH 7.5) by heating at 950C for 3 min followed by slow cooling down to room temperature. After treatment with T4 polynucleotide kinase (to phophorylate the 5 '-end of the annealed oligoes required for subsequent DNA ligation) (New England BioLabs, Beverly, MA) in T4 DNA ligase buffer at 370C for 1 h, the annealed oligoes were purified through a gel-purification column (Qiagen, Valencia, CA) and inserted into the unique BsiW I site of plasmid DEN-I Rluc-Rep, resulting in DEN-I Rluc-2A- Rep.
Type three replicon was prepared to generate a stable cell line containing persistently replicating DEN-I replicon. A reporting cassette containing the following sequence was directly RCR amplified from a replicon pLN-BR of bovine viral diarrhea virus (BVDV) ( Horscroft, N., et al., J. Virol., 2005, 79, 2788-96): Rluc-Ubi (ubiquitin)-Neo (Neomycin phosphotransferase)-EMCV IRES. The BVDV replicon was generously provided by Weidong Zhong from Valeant Pharmaceutical International Inc., Costa Mesa, CA. The reporting cassette was then cloned into DEN-I Rluc-Rep at unique Not I and BwiW I sites, resulting in plasmid DEN-I Rluc-Neo-Rep. For all constructs, DNA sequencing was performed to ensure that no mutations had occurred during PCR amplification. Standard procedures were performed for PCR and DNA cloning with modifications as previously described ( Shi, P. Y., et al., New York City. J. Virol, 2002, 76, 5847-56).
Characterization of Rluc-expressing replicon of DEN-I virus. Plasmid DNA for various DEN-I replicons was linearized with Sac II, and subjected to in vitro transcription using a T7 mMESSAGE mMACHINE1M kit (Ambion, Austin, TX) as previously described ( Lo, L., et al., J. Virol, 2003, 77, 10004-10014). The replication kinetics of replicon was examined by transfection of BHK-21 cells followed by an Rluc assay at indicated time points post transfection (p.t.)- RNA replication was also monitored by expression of viral proteins using an indirect immunofluorescence analysis (IFA). The IFA was performed using DEN-I immune mouse ascites fluid (ATCC, Manassas, VA) and goat anti-mouse IgG conjugated with Texas red as primary and secondary antibodies, respectively ( Shi, P. Y., et al., Virology, 2002, 296, 219-233). Establishment of stable cell lines containing luciferase-expressing replicon of DEN-I virus. Vero cells were transfected with DEN-I Rluc-Neo- Rep RNA and selected under G418 (1 mg/ml) from day 2 p.t. ( Lo, L., et al., J. Virol., 2003, 77, 12901-12906). After three weeks of selection, individual foci were detached using sterile cloning disks (Bel- Art Products, Pequannock, NJ) soaked with trypsin. The Rluc-Neo-Rep-containing Vero cells (absorbed onto the disks) were then transferred into a 24-well plate, amplified, and subjected to an Rluc assay and IFA analysis.
HTS antiviral assays. For WN virus, three HTS assays (a luciferase- expressing replicon cell line, a virus-like particle (VLP) infection assay, and a reporting full-length viral infection assay) were previously established and validated for antiviral screening in a 96-well format. Since the VLP infection assay and the reporting WN virus infection assay involved infectious particles, these two assays were performed in biosafety level-3 containment. The replicon cell line-based assays (for both WN and DEN-I viruses) were performed in a biosafety level-2 laboratory because no infectious particles were involved.
Repreentative compounds of the invention were screened to identify WN virus inhibitors. The compounds were dissolved in dimethyl sulfoxide (DMSO) and assayed at a final concentration of 1% DMSO. A full-length Rluc-expressing WN virus was used to screen the compound library. Briefly, Vero cells were seeded at 8 * 104 per well of 96-well plate. At 6 h post cell seeding, cells were infected with the Rluc-expressing virus (1 MOI) and treated immediately with 30 μM of compounds. The screening concentration at 30 μM was empirically selected. The plates were assayed at 24 h p.i. using an Rluc assay kit (Promega, Madison, WI) and a Veritas1M microplate luminometer (Turner Biosystem Inc., Sunnyvale, CA). Compounds exhibiting greater than 50% inhibition of Rluc activity were assayed for cytotoxicity as described below.
MTT cell proliferation assay. An MTT cell proliferation assay (ATCC5 Manassas, VA) was used to estimate potential cytotoxicity of compound. Approximately 2 x 104 BHK-21 or 8 * 104 Vero cells in 100 μl medium were seeded per well in a 96-well plate. After 6 h of incubation, 1 μl of compound dissolved in DMSO was added to cells at indicated concentrations. After 48 h of incubation, 10 μl of MTT reagent was added and cells were incubated for another 3.5 h, after which 100 μl of detergent reagent was added. The plates were swirled gently and left in dark at room temperature for 4 h. A microtiter plate reader (Molecular Devices Corporation, Sunnyvale, CA) with a 550 run filter was used to record the absorbance, from which a CC50 (compound concentration required to reduce 50% cell viability) value was estimated. Viral titer reduction assay. Viral titer reduction assays were performed to examine the antiviral activities of representative compounds in WN, YF (17D), DEN-2, SLE, VSV, and WEE viruses. Approximately 9 x 105 Vero cells per well were seeded in a 12-well plate. After 12-h incubation, the cells were infected with individual virus (0.1 MOI) and treated immediately with compound at indicated concentrations. For WN, YF (17D), DEN-2, SLE, and WEE viruses, culture medium were collected at 42 h post infection (p.i.), stored at -8O0C, and subjected to plaque assays on Vero cells. For VSV, culture medium was collected at 16-h p.i.
A double-overlayer protocol was followed for plaque assay. For WN virus, approximately 6 x 105 Vero cells per well were seeded in a 6-well plate and incubated for 3 days to reach full confluence. Cells were infected with 100 μl of 1 to 10 serial dilutions of the virus for 1 hour at 370C. Afterwards, 3 ml of a first layer containing 0.6% Oxoid agar, BME medium with 1% FBS, 0.02% DEAE Dextran and 0.13% NaHCO3 was added onto the infected cells. Two days later, 3 ml of a second layer containing 1% Noble agar, BME medium with 1% FBS, 0.02% DEAE Dextran, 0.13% NaHCO3, and 0.004% neutral red was added over the first layer. The plates were further incubated for 12 h before counting plaques. The protocol for WEE virus was identical to that for WN virus. For other viruses, the time interval between addition of the first and the second layer of agar was 1 day for VSV5 4 days for YF, and 5 days for DEN and SLE virus.
Time of drug addition assay. A time of addition experiment was performed to estimate the step of viral life cycle that was suppressed by the compounds. Approximately 9 x 105 Vero cells per well were seeded in a 12 well- plate, incubated for 12 h for cell attachment, and synchronously infected with WN virus. The infection was carried out at MOI of 5 for 1 h followed by three rounds of PBS washing to remove the unabsorbed viruses. At different time points post infection, triaryl pyrazoline was added to the infected cells at 100 μM. Culture medium were collected at 24 h p.L, stored at -8O0C, and subjected to plaque assay as described above. As negative controls, 1% of DMSO was added to infected cell at 0 and 20 h p.i. to estimate its effect on viral yield production.
Transient replicon assay. A transient replicon assay was used to quantify compound-mediated inhibition of viral translation and suppression of RNA replication. For WN virus, replicon RNA (10 μg) was electroporated into BHK-21 cells (8 x 106) as previously described ( Lo, L., et al., J. Virol, 2003, 77, 10004-10014). The transfected cells were suspended in 25 ml of DMEM with 10% FBS. Cell suspension (5 x 105 in 167 μl per well) was added to 12- well plates, immediately treated with 30 or 100 μM of compounds, and assayed for luciferase activity at 2 and 4 h p.t. (representing viral translation) and 72 h p.t. (representing RNA replication). A similar protocol was performed for DEN- 1 luciferase replicon, except that a time point at 48-h p.t. was used to quantify viral RNA replication (see details in Results). Inhibition of viral translation and suppression of RNA replication were each expressed as the percentage of luciferase signal derived from the compound-treated cells as compared with the signal derived from the untreated cells at an equivalent time point.
The screening results showed that triaryl pyrazoline {[5-(4-Chloro- phenyl)-3-thiophen-2-yl-4,5-dihydro-pyrazol-l-yl]-phenyl-methanone} inhibited luciferase activity by 57%, as compared with the mock-treated infection (with 1% DMSO) (Fig. IA). Lower concentration of the compound at 10 μM suppressed luciferase activity to a less extent by 12%. As positive controls, the Rluc signals were suppressed by 87% and 95% in the presence of 1 μM and 10 μM MPA (a known WN virus inhibitor), respectively. The results indicated that triaryl pyrazoline is an inhibitor of WN virus.
Inhibition of an epidemic strain of WN virus without cytotoxicity. To verify the primary screening results, {[5-(4-Chloro-phenyl)-3-thiophen~2-yl-4,5- dihydro-pyrazol-l-yl]-phenyl-methanone} was assayed in an authentic viral titer reduction assay. Vero cells were infected with an epidemic strain of WN virus (0.1 MOI), immediately treated with the compound, and assayed for viral yields in culture medium at 42 h p.i. The compound inhibited viral titer in a dose responsive manner (Fig. 2A). At 100 μM or higher, the compound reduced viral tire by >90%. The EC50 value was estimated to be 28 μM. Next, an MTT assay was performed to exclude the possibility that the observed antiviral activity was due to compound-mediated cytotoxicity. Vero cells were incubated with the compound for 48 h and cell viability was measured by cellular metabolism of MTT tetrazolium salt (Fig. 2B). No reduction of cell viability was observed up to 600 μM (the highest tested concentration). Similar results were obtained when BHK-21 cells were incubated with the compound in the MTT assay (data not shown). However, tiny crystals were observd under the microscope in culture medium when compound concentration reached 300 μM or higher. The results indicate that the CC50 of the compound is >300 μM. Overall, the above data demonstrate that the compound inhibits WN virus without observable cytotoxicity in cell culture.
A broad spectrum of antiviral activity against flaviviruses and other RNA viruses. To examine if {[5-(4-Chloro-phenyl)-3-thiophen-2-yl-4,5- dihydro-pyrazol-l-yl]-phenyl-methanone} inhibits other flaviviruses, viral titer reduction assays with DEN-2, YF (17D), and SLE viruses were performed. Vero cells were infected with individual virus (0.1 MOI), incubated with the compound, and quantified for viral yields in culture medium at 42 h p.i. The compound inhibited all three tested viruses (Fig. 3). For each virus, viral titer was significantly reduced when treated with 33 μM of compound; increasing concentration of the compound to 100 and 300 μM did not further suppress viral titers to a greater extent. Comparison of viral titer reduction at 100 μM concentration (Table 2) showed different sensitivities to the compound treatment among flaviviruses: with 184-, 34-, 25-, and 12-fold reduction for YF3 DEN-2,
SLE, and WN viruses, respectively.
TABLE 2. Antiviral activities of triaryl pyrazoline against various RNA virusesa
WN DEN-2 YF SLE WEE VSV
Viral titer untreated 6.5xlOy 5.5xlO5 5.7x10" 4.3xlO6 8.5xlθ7 6.0x10* (PFU/ml)
Viral titer treated (PFU/ml) 5.3xlO8 1.6xlO4 3.IxIO4 1.7xlO5 8.4xlO6 2.7xlO8
Viral titer reduction (fold) b 12 34 184 25 10 22
aVero cells were infected with indicated virus (0.1 MOI), treated with 100 μM of compound, and assayed for viral titer as described in Materials and Methods. bViral titer reduction (fold) = Viral titer without treatment / Viral titer with compound treatment.
To further test the antiviral specificity the compound was analyzed in two non-flaviviruses, WEE virus (a plus-strand RNA alphavirus) and VSV (a negative-strand RNA rhabdovirus) . Interestingly, the compound inhibited both WEE and VSV viruses. At 100 μM concentration, the compound reduced viral titer of WEE and VSV by 10 and 22 folds, respectively. The results demonstrated that triaryl pyrazoline can inhibit not only flaviviruses, but also other RNA viruses in cell culture.
Inhibition of WN virus infection through suppression of RNA synthesis. To dissect the step(s) at which {[5-(4-Chloro-phenyl)-3-thiophen-2- yl-4,5-dihydro-pyrazol-l-yl]-phenyl-methanone} suppresses WN virus infection, the inhibitor was analyzed in three replicon-based assays. These assays cover different steps of the viral life cycle and have been validated for mode-of-action analysis ( Deas, T. S., et al, J. Virol., 2005, 4599-4609). The first assay used BHK-21 cells infected with VLPs containing an Rluc-expressing replicon RNA (Fig. 4A). VLPs were prepared by trans supplying WN structural proteins in a stable cell line containing Rluc-reporter replicons (WN Rluc-Neo-Rep, 4B). Infection of naϊve Vero cells with such Rluc-VLPs (1 MOI) could be used to examine if an inhibitor blocks viral entry and replication. Treatment of the VLP- infected cells with triaryl pyrazoline suppressed luciferase signals, indicating that the compound could inhibit viral entry and/or replication. The EC50 value was estimated to be 14 μM (Fig. 4A).
The second assay was based on a BHK-21 cell line harboring the WN Rluc-Neo-Rep (Fig. 4B). The reporting cell line allows testing inhibitors of viral replication (including viral translation and RNA synthesis), but not viral entry. Analyses using the WN Rluc-Neo-Rep cell line showed that the compound inhibited viral replication with an EC5O of 19 μM.
The third assay involves WN Rluc-Rep in which a luciferase reporter was in-frame fused with the ORF of the genome where the structural genes were deleted (Fig. 4C). Transfection of BHK-21 cells with such replicon reveals two Rluc peaks at 1-10 h and >24 h p.t. which represent viral translation and RNA synthesis, respectively ( Lo, L., et al, J. Virol, 2003, 77, 10004-10014). To distinguish the inhibitory effects of the compound on viral translation and RNA synthesis, BHK-21 cells were transfected with WN Rluc-Rep RNA, immediately incubated the cells with compound, and assayed for luciferase activities at 2, 4, and 72 h p.t. (Fig. 4C). At 2 and 4 h p.t., luciferase signals from the cells treated with compound (at 33 and 100 μM) were around 96-117% of those from the mock-treated cells. In contrast, at 72 h p.t., luciferase activities were suppressed by over 95% upon compound treatment. The data suggest that triaryl pyrazoline inhibits WN virus through suppression of viral RNA synthesis.
Time of addition of triaryl pyrazoline in WN viral infection. A a time-of-addition experiment was performed to further estimate the mode of action of {[5-(4-Chloro-phenyl)-3-thiophen-2-yl-4,5-dihydro-pyrazol-l-yl]- phenyl-methanone}. Vero cells were synchronously infected with an epidemic strain of WN virus. The compound (100 μM) was added to infected cells at various time points post infection. Viral titers in the culture medium were determined at 24 h p.i. For mock treatment, 1% DMSO was added at 0 and 20 h p.i. to estimate its effect on viral yield production (because triaryl pyrazoline was tested at 1% DMSO). As shown in Fig. 5, a significant and steady level of suppression in viral titer (a reduction of 5 x 108 PFU/ml) was achieved when the compound was added during the initial 1O h of infection. Inhibitory effects gradually diminished when the compound was added between 10 to 20 h p.i. The results indicate that the compound blocks WN virus at a late stage of viral infection.
An HTS assay for screening inhibitors of DEN virus replication. To test if {[5-(4-Chloro-phenyl)-3-thiophen-2-yl-4,5-dihydro-pyrazol-l-yl]-phenyl- methanone} inhibits DEN virus through a mechanism similar to WN virus, a replicon of DEN-I virus was developed in which an Rluc-Ubi-Neo-EMCV IRES fragment substituted for viral structural genes (DEN-I Rluc-Neo-Rep, Fig. 6A). The DEN-I Rluc-Neo-Rep retained the N-terminal 37 amino acids of the capsid and the C-terminal 31 amino acids of the envelope protein. Upon transfection of Vero cells with DEN- 1 Rluc-Neo-Rep RNA, the Rluc-Ubi-Neo polyprotein driven by the DEN-I 5'-UTR' was processed through Ubi-mediated cleavage, resulting in individual Rluc and Neo protein; the NS proteins were translated through the EMCV IRES . G418 selection of the transfected cells allowed establishment of cell lines containing persistently replicating replicons. Analyses of the established cell lines showed that 100% of the cells expressed viral proteins (Fig. 6B) as well a high level of Rluc activity (Fig. 6C). Cells passaged for over two months retained robust expression of viral proteins and Rluc reporter.
The DEN-I replicon-containing cell line was converted into an HTS assay. Seeding of 2 x 104 cells per well of 96- well plate and incubation of the cells for 48 h consistently showed an assay window of 1 x 106 to 2 x 106 (Rluc signal from replicon-bearing cells / background signal from naive BHK-21 cells). To validate the assay for HTS, the DEN-I Rluc-Neo-Rep cell lines were incubated with various concentrations of mycophenolic acid (MPA) and ribavirin, two known inhibitors of DEN virus ( Diamond, M. S., et al., Virology, 2002, 304, 211-21). Both MPA and ribavirin suppressed the luciferase signal in a dose-responsive manner (Fig. 6C). In contrast, no suppression of luciferase activity was detected when incubated with glycyrrhizin, even at 300 μM (Fig. 6C). The EC50 values were estimated to be 0.3, 220, and >300 μM for MPA, ribavirin, and glycyrrhizin, respectively. The results demonstrate that the reporting cell line can be used as an HTS assay for screening inhibitors of DEN- 1 virus. Next, {[5-(4-Chloro-phenyl)-3-thiophen-2-yl-4,5-dihydro-pyrazol-l-yl]- phenyl-methanone} was tested in the DEN-I Rluc-Neo-Rep-containing cells in a 96-well plate. Incubation of the cells with compound inhibited luciferase activities (Fig. 6D). The EC5O value was estimated to be 17 μM. The results suggest that the compound inhibits DEN-I virus replication.
To further compare the mode of action of {[5-(4-Chloro-phenyl)-3- thiophen-2-yl-4,5-dihydro-pyrazol-l-yl]-phenyl-methanone} in DEN and WN viruses, a DEN-I reporting replicon which can differentiate between viral translation and RNA synthesis was established. Initially, a replicon in which an Rluc replaced viral structural genes (DEN-I Rluc-Rep, Fig. 7A) was constructed. Upon transfection, the replicon was expected to express a luciferase fusion protein containing N-terminal extra 40 amino acids (37 residues from capsid and 3 residues from the Not I site engineered for cloning purpose) and C-terminal extra 33 amino acids (2 residues from the BsiW I site engineered for cloning and 31 residues from envelope). Transfection of BHK-21 cells with DEN-I Rluc- Rep yielded a single Rluc peak at 1 to 10 h p.t, but never a second Rluc peak (Fig. 7B). IFA analysis showed no viral protein expression in the transfected cells (right panel, Fig. 7C). The results suggest that DEN-I Rluc-Rep RNA was successfully transfected into cells (as the initial translation of input RNA had occurred), but no replication had followed.
To rescue RNA replication, a FMDV 2A sequence was inserted into the original replicon to mediate a cleavage between Rluc and the C-terminal fragment of envelope protein (DEN-I Rluc-2A-Rep, Fig. 7A). Since the FMDV 2A cleaves at its C-terminus, the Rluc expressed from the Rluc-2A-Rep would be C-terminally fused to the FMDV 2 A. Transfection of BHK-21 cells with DEN-I Rluc-2A-Rep yielded two luciferase peaks: the first peak during the initial 1O h p.t. and a second peak after 1O h p.t. (Fig. 7B). An identical time frame for the first Rluc peak was observed for both Rluc-2A-Rep- and Rluc- Rep-transfected cells, except that the Rluc signal derived from the 2A-containing replicon was more robust (two folds greater) than that derived from the non-2A- replicon. This was expected because the Rluc protein expressed from the non- 2A-replicon had a longer C-terminal fusion partner (33 amino acids) than that expressed from the 2A-containing replicon (19 amino acids). In addition, due to the transmenbrane nature of the C-terminal fragment of the envelope protein, it was likely that the Rluc protein was anchored onto ER membrane through its fusion partner. Remarkably, the 2A-containing replicon replicated efficiently, as indicated by a robust second Rluc peak after 1O h p.t. (Fig. 7B) as well as IFA- positive cells at 60 h p.t. (Fig. 7C). A similar luciferase kinetics with two peaks was observed upon transfection of the Rluc-2A-Rep RNA into Vero cells. These results suggest that (i) the DEN-I Rluc-2A-Rep is replication-competent, and (ii) the two luciferase peaks observed during the initial 1O h and after 1O h p.t. represent viral translation and RNA synthesis, respectively.
Using the above DEN-I Rluc-2A-Reρ, the effects of {[5-(4-Chloro- phenyl)-3-thiophen-2-yl-4,5-dihydro-pyrazol-l -yl]-phenyl-methanone} on viral translation and RNA synthesis was determined. BHK-21 cells were transfected with the Rluc-2A-Rep RNA, incubated immediately with the compound (at 33 and 100 μM), and assayed for luciferase at 2 h, 4 h, and 48 h p.t. (Fig. 7D). Multiple rounds of experimental results showed that triaryl pyrazoline consistently inhibited Rluc signals by 11-31% at 2 h and 4 h p.t. At 48 h p.t, the compound inhibited Rluc activity by approximately 90%. The results indicate that the compound inhibits DEN-I infection through weak suppression of viral translation but strong inhibition of RNA synthesis.
The mode of action of triaryl pyrazoline has been characterized in WN virus. Because the three reporting assays described above encompasses multiple, but discrete steps of the viral life cycle, they could be used to discriminate the inhibitory step(s) of any compounds amongst viral entry, replication, and virion assembly. The comparable values of EC50S derived from the three reporting assays (Table 2) indicate that the compound inhibits WN infection at a step that is shared by all three HTS assays: viral replication. Further analyses using a transient replicon system showed that the compound significantly blocks RNA synthesis (by >95%) without suppression of viral translation (Figs. 4C). In agreement with the above results, time-of-addition experiments showed that a significant suppression in viral titer requires treatment of the compound during the first 1O h p.L, after which time point the inhibitory effects gradually diminished (Fig. 5). It was previously shown that, during BHK-21 cell infection with a luciferase-expressing WN virus or a luciferase-expressing VLP, initial translation occurred during the first 7.5 h p.L, while nascent genomic RNA began to accumulate after 1O h p.i. La combination, the results strongly suggest that triaryl pyrazoline inhibits WN viras through suppression of viral RNA synthesis.
The mechanism of inhibition of triaryl pyrazoline in DEN-I virus seems to be more complex than that in WN virus. Similarly to WN virus, the compound inhibited DEN-I infection through suppression of viral replication, as suggested by its antiviral activity in a DEN-I replicon-containing cell line (Fig. 6D). However, analyses using a transient replicon system of DEN-I consistently showed that, beside a significant inhibition of viral RNA synthesis (by 90%), the compound also suppressed viral translation (by 11-31%, Fig. 6D). These results have raised the question, in the case of DEN-I virus, whether the antiviral activity was due to a dual effect of the compound on both viral translation and RNA synthesis. Alternatively, the observed antiviral activity was solely caused by its effect on translation, which, in turn, resulted in suppression of viral RNA synthesis. Besides WN and DEN viruses, representative compounds of the invention also inhibited other flaviviruses, including YF (17D) and SLE viruses. Furthermore, the compounds suppressed other plus-strand and minus-strand RNA viruses, as represented by WEE and VSV, respectively (Fig. 3). The broad spectrum of antiviral activity indicates that the compound blocks a target commonly required for replication of the tested viruses. It is currently not known whether the compound exerts its functions through direct interaction with a host factor or a viral protein. A number of approaches are being explored to define the target of the compound. Testing the compound in biochemistry assays (RdRp5 protease, helicase, and NTPase) may indicate whether the inhibitors directly interfere with the viral functions. Alternatively, if resistant viruses or replicons could be selected, sequencing of resistant viruses or replicons may point to the targets of the compound.
The invention will now be illustrated by the following non-limiting examples. Examples
All reagents were purchased from commercial suppliers such as Sigma- Aldrich Chemical Co., Lancaster Chemical Co, Acros Chemical Co., etc. ACS Reagent grade or better solvents were used without further purification unless otherwise noted. Water was purified by Millipore filtration system. Column chromatography was conducted using silica gel 60 (40-63 microns) and thin- layer chromatography was conducted using EMD Chemical silica gel 60 F254 on aluminum sheets. 1H Nuclear Magnetic Resonance spectra were collected on a Varian Mercury 300MHz instrument and a Varian Mercury 600MHz instrument using CDCl3 and DMSO-J6 as solvents. High resolution mass spectra (HRMS) were collected from a TOF-ESI Agilant LC-MS and analyzed using the Analyst QS software. Reversed-phase high-performance liquid chromatography (RP- HPLC) was performed on a Beckman Coulter 125 S System Gold using an Agilent Zorbax Eclipse XDB-C8 3.5 μM 3.0 X 150mm column monitoring UV at 254λ on a 166 detector. Method A was a gradient method that ran for 14min at a flow rate of 0.5 mL/min. Over the first 8 minutes the percent ACN/water was increased from 60%-100%. Over the next 4 minutes the gradiant was decreased back to 60% ACN/water and then held for two minutes. Method B was an isocratic method using 70% MeOH/water and a flow rate of 0.4 mL/min. The run duration was twenty minutes. Results were analyzed using the 32 Karat software package.
Example 1 2-(4-Chloro-phenyl)-l-[5-(4-nitro-phenyl)-3-thiophen-2-yl-4,5- dihydro-pyrazol-l-yl]~ethanone (5a, Table 1).
5-(4-Nitrophenyl)-3-thiophen-2-yl-4,5-dihydro-pyrazole (~5mmol) was placed in a medium sized vial and an excess amount of 2-(4-chlorophenyl)ethanoic acid chloride was added. The reaction was then heated as HCl gas evolved. After approximately 5 min the reaction was cooled and methanol (~2ml) was added. The reaction was heated to dissolve any solid and cooled. The product crystallized out which was filtered and recrystallized in methanol to yield the 1,3,5- trisubstitudesd pyrazoline. Yield = 60.8%; 1H NMR (300MHz, CDCl3) δ 3.12 (dd, J= 5.1 and 17.6 Hz, IH), 3.82 (dd, J= 11.9 and 17.6 Hz, IH), 4.04 (s, 2H)5 5.60 (dd, J= 5.1 and 11.9 Hz, IH), 7.09 (dd, J= 3.7 and 5.0 Hz, IH), 7.22 (dd, J= 1.0 and 3.7 Hz5 IH), 7.29 (m. 6H), 7.49 (dd, J= 1.0 and 5.0 Hz, IH)5 8.15 (d, J= 8.8, 2H); HRMS (C21H17ClN3O3S) [M+H]+: found m/z 426.0644, calcd 426.0673; RP- HPLC method A, tR=5.12min (>99%); method B tR=5.62min (>99%)
The intermediate 5-(4-Nitrophenyl)-3-thiophen-2-yl-4,5-dihydro-pyrazole was prepared as follows. a. 3-(4-Nitro-phenyl)-l-thiophen-2-yl-propenone (Scheme 2, Compound 3, Rx = Nitro). 2-Acetylthiophene (Scheme 2, Compound 1) (5 mmol) and 4- nitro benzaldehyde (5 mmol) were dispersed in either ethanol or methanol (~3ml). Next, two NaOH pellets (~0.24g) were added and the reaction was stirred while slightly heating until starting materials were completely dissolved. Once dissolved, the reaction was removed from heat and stirred overnight. The product precipitated out and was filtered. Washing with cold methanol and water afforded the chalcone which was used without further purification. The product was a mixture of cis and trans alkenes which convolutes the NMR spectra; Yield = 94.8%; 1H NMR (300MHz, CDCl3) δ 7.22 (dd, J= 3.9 and 4.9 Hz5 IH)5 7.52 (d, IH), 7.75 (dd, J= 1.0 and 4.9 Hz5 IH), 7.79 (d, J= 8.8, 2H), 7.82 (d, IH)5 7.90 (dd5 J= 0.98 and 3.8 Hz, IH)5 8.29 (d, J= 8.8, 2H) b. 5-(4~Nitrophenyl)-3-thiophen~2-yl-4,5-dihydro-pyrazole. 3-(4-Nitro- phenyl)-l-thiophen-2-yl-propenone (5 mmol) was stirred in ethanol (~5ml) and hydrazine monohydrate (12.5 mmol) was added. Some light heating was required for the reaction to go into solution. The resulting product was passed through a fritted filter and washed with a cold 50/50 mix of methanol/water, quickly rinsed with methanol, and then briefly air dried. The product was used in the next reaction as soon as the product appeared dry without further purification. Example 2 [5-(4-Chloro-phenyl)-3-thiophen-2-yI-4,5-dihydro-pyrazol-l-yI]- phenyl-methanone (5b, Table 1).
Using a procedure similar to that described in Example I5 except replacing the 4-nitrobenzaldehyde used in sub-part a with 4-chlorobenzaldehyde and replacing the 2-(4-chlorophenyl)ethanoic acid chloride with benzoyl chloride, the title compound was prepared; yield = 47.3%; 1H NMR (300MHz, CDCl3) 6 3.16 (dd, J= 5.0 and 17.5 Hz, IH)5 3;81 (dd, J= 11.8 and 17.5 Hz5 IH)5 5.79 (dd, J= 5.0 and 11.8 Hz5 IH)5 7.07 (dd, J= 3.7 and 5.1 Hz, IH), 7.24 (dd, J= 1.1 and 3.7 Hz5 IH), 7.29 (m, 7H)5 8.01 (d, J= 7.0, 2H); HRMS (C20H16C1N2OS) [M+H]+: found m/z 367.0643, calcd 367.0666; RP-HPLC method A, tR=4.82min (>99%); method B tR=5.82min (>99%) Example 3
Using procedures similar to those described above, the following representative compounds of the invention were prepared.
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
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Figure imgf000043_0001
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Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Example 4
Using procedures similar to those described above, the following representative compounds of the invention were prepared.
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Example 5 The following illustrate representative pharmaceutical dosage forms, containing a compound of formula I ('Compound X1), for therapeutic or prophylactic use in humans.
(i) Tablet 1 mε/tablet
Compound X= 100.0
Lactose 77.5
Povidone 15.0
Croscarmellose sodium 12.0
Microcrystalline cellulose 92.5
Magnesium stearate 3.0
300.0
(ii) Tablet 2 mg/tablet
Compound X= 20.0
Microcrystalline cellulose 410.0
Starch 50.0
Sodium starch glycolate 15.0
Magnesium stearate 5.0
500.0
Ciii) Capsule mg/capsule
Compound X= 10.0
Colloidal silicon dioxide 1.5
Lactose 465.5
Pregelatinized starch 120.0
Magnesium stearate 3.0
600.0
(ϊv) Iniection 1 (1 mg/ml) mg/ml
Compound X= (free acid form) 1.0
Dibasic sodium phosphate 12.0
Monobasic sodium phosphateθ.7
Sodium chloride 4.5
1.0 N Sodium hydroxide solution
(pH adjustment to 7.0-7.5) q.s.
Water for injection q.s. ad 1 mL
(v) Iniection 2 (10 mg/mD mg/ml
Compound X= (free acid form) 10.0
Monobasic sodium phosphateθ.3
Dibasic sodium phosphate 1.1
Polyethylene glycol 400 200.0
01 N Sodium hydroxide solution
(pH adjustment to 7.0-7.5) q.s.
Water for injection q.s. ad 1 mL (Vi) Aerosol mg/can
Compound X= 20.0
Oleic acid 10.0
Trichloromonofluoromethane 5,000.0 Dichlorodifluoromethane 10,000.0
Dichlorotetrafluoroethane 5,000.0
The above formulations may be obtained by conventional procedures well known in the pharmaceutical art. All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

CLAIMSWhat is claimed is:
1. A compound of formula I :
Figure imgf000064_0001
(i) wherein:
R1 is (C1-C6)alkyl, (Ci-<_*)alkoxy, (C!-C6)alkanoyl, (C1-C6)alkanoyloxy, (Ci-C6)alkoxycarbonyl, (C1-C6)alkylaminocarbonyl, di(C1-C6)alkylamino- carbonyl, ImIo(C1 -C6)alkyl, or Ra-X-; X is a direct bond, (d-C6)alkyl, -SO2-, or (d-C6)alkanoyl;
Ra is aryl, or heteroaryl;
R2 is H, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)alkanoyl, (C1- C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, halo(C1-C6)alkyl, (C1- C6)alkylammocarbonylamino, di(C1-C6)alkylaminocarbonyl, (C1- C6)alkylsulfonylamino, or Rb-Y-;
Y is a direct bond, -NH-C(=O)-NH-, -NH-SO2-, (CrC6)alkyl3 or (C1- C6)alkanoyl;
Rb is aryl, or heteroaryl;
R3 is H, (Ci-C6)alkyl, (CrC6)alkoxy, (C1-C6)alkanoyl, (C1- C6)alkanoyloxy, (CrC6)alkoxycarbonyl, halo(C1-C6)alkyl, or R0-Z-;
Z is a direct bond, (C1-C6)alkyl, or (Ci-C^alkanoyl;
R0 is aryl, or heteroaryl; each bond represented by — is independently a single or a double bond; and A is CH or N; wherein any aryl or heteroaryl OfR1-R3 is optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (C1-C6)alkyl, (C1-C6)alkoxy, (C1- C6)alkanoyloxy,
Figure imgf000064_0002
cyano, carboxy, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and Re are each independently H, (C1- C6)alkanoyl, or (C1-C6)alkyl; or a pharmaceutically acceptable salt thereof.
2. The compound of claim 1 wherein the compound of formula I is a compound of formula (II)
Figure imgf000065_0001
(II).
3. The compound of claim 1 wherein the compound of formula I is a compound of formula (III)
Figure imgf000065_0002
(III).
4. The compound of claim 1 wherein the compound of formula I is a compound of formula (IV)
Figure imgf000065_0003
(IV).
5. The compound of claim 1 wherein the compound of formula I is a compound of formula (V)
Figure imgf000066_0001
(V).
6. The compound of any one of claims 1-5 wherein X is (C1-C6)alkanoyl.
7. The compound of any one of claims 1 -5 wherein X is -C(=O)-.
8. The compound of any one of claims 1-5 wherein R1Is (CpC^alkyl, (C1- C6)alkoxy5 (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, halo(Ci-C6)alkyl, or Ra-X-; X is a direct bond, (C^C^alkyl, -SO2-, or (C1-
C6)alkanoyl; and Ra is aryl, or heteroaryl; wherein any aryl or heteroaryl OfR1 is optionally substituted with one or more halo, hydroxy, (Ci-C6)alkyl, (C1- C6)alkoxy, (CrC6)alkanoyloxy, (C1-C6)alkoxycarbonyl, cyano, carboxy, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and Re are each independently H or (C i-C6)alkyl.
9. The compound of any one of claims 1 -8 wherein R3 is phenyl, optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (C1-C6)alkyl, (C1- C6)alkoxy, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, cyano, nitro, trifluomethyl, trifluoromethoxy, or RaReN; wherein Rd and Re are each independently H or (Ci-C6)alkyL
10. The compound of any one of claims 1 -8 wherein Ra is phenyl or 4- chlorophenyl.
11. The compound of any one of claims 1-10 wherein Y is a direct bond.
12. The compound of any one of claims 1-10 wherein R2 is (Q-C^alkyl, (CrC6)alkoxy, (CrC6)alkanoyl, (C1-C6)alkanoyloxy, (CrC^alkoxycarbonyl, ImIo(C1 -C6)alkyl, (C1-C6)aUcylaminocarbonylamino, (C1-C6)alkylsulfonylamino, or Rb-Y-; Y is a direct bond, -NH-CC=O)-NH-, -NH-SO2-, (d-C6)alkyl, or (Cj- C6)alkanoyl; and Rb is aryl, or heteroaryl; wherein any aryl or heteroaryl of R2 is optionally substituted with one or more halo, hydroxy, (C1-C6)alkyl, (C1- C6)alkoxy, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, cyano, carboxy, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and Re are each independently H or (C1-C6)alkyl.
13. The compound of any one of claims 1-11 wherein Rb is phenyl, optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (C1- C6)alkyl, (Ci-C6)alkoxy, (Ci-C6)alkanoyloxy, (CrC^alkoxycarbonyl, cyano, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and Re are each independently H or (C1-C6)alkyl.
14. The compound of any one of claims 1-11 wherein Rb is phenyl, optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, (C1-C6)alkoxy, or nitro.
T5. The compound of any one of claims 1-11 wherein Rb is 4-nitrophenyl, 4- chlorophenyl, 4-bromophenyl, or 4-methoxyphemyl.
16. The compound of any one of claims 1-15 wherein Z is a direct bond.
17. The compound of any one of claims 1-15 wherein R3 is (Ci-C6)alkyl, (C!-C6)alkoxy, (Ci-C^alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, halo(C1-C6)alkyl, or R0-Z-; Z is a direct bond, (CrC6)alkyl, or (C1-C6)alkanoyl; and R0 is aryl, or heteroaryl; wherein any aryl or heteroaryl of R3 is optionally substituted with one or more halo, hydroxy, (Ci-C6)alkyl, (C1-C^aIkOXy, (C1- C6)alkanoyloxy, (Ci-C6)alkoxycarbonyl, cyano, carboxy, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and Re are each independently H or (C1- C6)alkyl.
18. The compound of any one of claims 1-16 wherein R0 is heteroaryl, optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (C1- C6)alkyl,
Figure imgf000068_0001
(Ci-C6)alkanoyloxy, (CrC6)alkoxycarbonyl, cyano, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and Reare each independently H or (C1-C6^IlCyI.
19. The compound of any one of claims 1-16 wherein Rc is phenyl, furyl, pyridyl, thienyl, pyrrolyl, imadazole, thiazole, or oxazole; optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy,
Figure imgf000068_0002
(C1-C6)alkoxy, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, cyano, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and Re are each independently H or (C1- C6)alkyl.
20. The compound of any one of claims 1-16 wherein R0 is 2-thienyl, optionally substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, (C1- C6)alkyl, (C1-C6)alkoxy, (Ci-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, cyano, nitro, trifluomethyl, trifluoromethoxy, or RdReN; wherein Rd and Re are each independently H or (C1-C6)alkyl.
21. The compound of any one of claims 1-16 wherein Rc is 2-thienyl.
22. The compound of any one of claims 1 -5 wherein R1 is benzoyl, 4- chlorobenzoyl, 2,4-difluorobenzoyl, 3,4-difluorobenzoyl, 3,5-difluorobenzoyl, 3- chlorobenzoyl, 2-chlorobenzoyl, 4-fluorobenzoyl, 3-fluorobenzoyl, 2- fluorobenzoyl, 4-nitrobenzoyl, 4-methoxybenzoyl, 4-methylbenzoyl, 4- trifluoromethylbenzoyl, 3-trifluoromethylbenzoyl, phenylacetyl, 4- chlorophenylacetyl, acetyl, trimethylacetyl, />-toulenesulfonyl, diethylcarbamyl, 2-thiophenecarbonyl, 3-pyridinecarbonyl, 2-naphthylcarbonyl, or 3- naphthylcarbonyl.
23. The compound of any one of claims 1-5 and 22 wherein R2 is 2-thienyl, 2-furyl, 2-pyrrolyl, 5-bromo-2-thienyl, 4-bromo-2 -thienyl, 5-chloro-2-thienyl, 3- indolyl, 5-indolyl, 1-naphthyl, 2-naphthyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 2- pyridyl, 3 -pyridyl, 4-pyridyl, phenyl, 4-fluorophenyl, 4-chlorophenyl, 4- bromophenyl, 3-chlorophenyl, 2-chlorophenyl, 3,5-dichlorophenyl, 4- methoxyphenyl, 4-carboxyphenyl, 4-nitrophenyl, 4-dimethylarninophenyl, 4- trifluoronαethylphenyl, 3-trifluoromethylphenyl, 4-methylphenyl, 3- methylphenyl, methyl, or H.
24. The compound of any one of claims 1-5, 22, and 23 wherein R3 is 2- thienyl, 2-furyl, 2-pyrrolyl, 3-thienyl, 2-thiazolyl, 2-oxazolyl, 2-imidazolyl, 5- bromo-2-thienyl, 5-chloro-2-thienyl, 3-indolyl, 1-naphthyl, 2-naphthyl, 2- pyridyl, 3-pyridyl, 4-pyridyl, phenyl, 4-bromophenyl, 4-chlorophenyl, 4- nitrophenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-methylphenyl, 3- methylphenyl, 4-trifluoromethylphenyl, 3-trifluoromethylphenyl, tert-hutyl, methyl, or H.
25. The compound of any one of claims 1-5 wherein R1 is Ra-X-j R2 is Rb-Y-; R3 is R0-Z-; Ra is aryl, or heteroaryl; Rb is aryl, or heteroaryl; and Rc is aryl, or heteroaryl wherein any aryl or heteroaryl OfR1-R3 is optionally substituted with one or more halo, hydroxy, (CrC6)alkyl, (C1-C6)alkoxy, (C1- C6)alkanoyloxy, (C1-C6)aUcoxycarbonyl, cyano, carboxy, nitro, trifluomethyl, trifluoromethoxy, or RdR2N; wherein Rd and Re are each independently H or (C1- C6)alkyl.
26. A pharmaceutical composition comprising a compound as described in any one of claims 1-25 and a pharmaceutically acceptable diluent or carrier.
27. A method for suppressing viral RNA synthesis in an animal comprising administering to the animal, an effective amount of a compound as described in any one of claims 1-25.
28. A therapeutic method for treating a viral infection in an animal comprising administering to the animal, an effective amount of a compound as described in any one of claims 1-25.
29. The method of claim 28 wherein the viral infection is a flaviviral infection.
30. The method of claim 28 wherein the viral infection is dengue, yellow fever, West Nile, Japanese encephalitis, or tick-borne encephalitis virus.
31. The method of claim 28 wherein the viral infection is West Nile virus.
32. A compound as described in any one of claims 1-25 for use in medical therapy or diagnosis.
33. The use of a compound as described in any one of claims 1-25 to prepare a medicament for suppressing viral RNA synthesis in an animal.
34. The use of a compound as described in any one of claims 1-25 to prepare a medicament for treating a viral infection in an animal.
35. The use of claim 34 wherein the viral infection is a flaviviral infection.
36. The use of claim 34 wherein the viral infection is dengue, yellow fever, West Nile, Japanese encephalitis, or tick-borne encephalitis virus.
37. The use of claim 34 wherein the viral infection is West Nile virus.
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008137408A1 (en) 2007-04-30 2008-11-13 Genentech, Inc. Pyrazole inhibitors of wnt signaling
WO2010103488A1 (en) * 2009-03-12 2010-09-16 Institut Pasteur Dengue virus-like particle and uses thereof
US8293771B2 (en) 2008-10-02 2012-10-23 Respivert Ltd. p38 MAP kinase inhibitors
US8293748B2 (en) 2008-10-02 2012-10-23 Respivert Ltd. p38 MAP kinase inhibitors
US8299073B2 (en) 2008-12-11 2012-10-30 Respivert Ltd. P38 MAP kinase inhibitors
WO2013006308A3 (en) * 2011-07-01 2013-05-10 Merck Patent Gmbh Dihydropyrazoles, pharmaceutical compositions thereof and their use for the treatment of fertility disorders
US8642773B2 (en) 2009-04-03 2014-02-04 Respivert Ltd. P38MAP kinase inhibitor
US8809372B2 (en) 2011-09-30 2014-08-19 Asana Biosciences, Llc Pyridine derivatives
US9199975B2 (en) 2011-09-30 2015-12-01 Asana Biosciences, Llc Biaryl imidazole derivatives for regulating CYP17
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3837851A (en) * 1973-01-15 1974-09-24 Ibm Photoconductor overcoated with triarylpyrazoline charge transport layer

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3837851A (en) * 1973-01-15 1974-09-24 Ibm Photoconductor overcoated with triarylpyrazoline charge transport layer

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