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WO2006060774A2 - Inhibiteurs de la replication de flavivirus et leurs utilisations - Google Patents

Inhibiteurs de la replication de flavivirus et leurs utilisations Download PDF

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Publication number
WO2006060774A2
WO2006060774A2 PCT/US2005/043938 US2005043938W WO2006060774A2 WO 2006060774 A2 WO2006060774 A2 WO 2006060774A2 US 2005043938 W US2005043938 W US 2005043938W WO 2006060774 A2 WO2006060774 A2 WO 2006060774A2
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WIPO (PCT)
Prior art keywords
derivative
virus
flavivirus
urea
dengue
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PCT/US2005/043938
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English (en)
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WO2006060774A3 (fr
Inventor
Scott R. Gilbertson
Pedro J. Lory
Robert D. Malmstrom
Yuan-Ping Pang
Andrew T. Russo
Stanley J. Watowich
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Board Of Regents, The University Of Texas System
Mayo Foundation For Medical Education And Research
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Publication of WO2006060774A2 publication Critical patent/WO2006060774A2/fr
Publication of WO2006060774A3 publication Critical patent/WO2006060774A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/28—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C275/30—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by halogen atoms, or by nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/135—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D335/00—Heterocyclic compounds containing six-membered rings having one sulfur atom as the only ring hetero atom
    • C07D335/04—Heterocyclic compounds containing six-membered rings having one sulfur atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D335/10—Dibenzothiopyrans; Hydrogenated dibenzothiopyrans
    • C07D335/12—Thioxanthenes
    • C07D335/14—Thioxanthenes with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 9
    • C07D335/16—Oxygen atoms, e.g. thioxanthones
    • 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
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Arthropod-borne flaviviruses such as dengue virus, West Nile virus, yellow fever virus, Japanese encephalitis virus and tick borne encephalitis virus cause significant public health problems worldwide and pose threats as agents of biowarfare and bioterrorism. These viruses are endemic in many areas of the world and are emerging in other areas. The recent establishment of West Nile virus in North America demonstrates the long-term, damaging consequences that can arise from the introduction of a flavivirus disease agent into a previously non-endemic area.
  • dengue virus causes the greatest number of cases of human disease of any flavivirus, Japanese encephalitis and yellow fever are also important diseases, affecting hundreds of thousands of people each year. In addition, approximately 10,000 confirmed cases of West Nile virus infection occurred in the United States in 2003.
  • tick borne encephalitis virus and yellow fever virus two flaviviruses that can display case-fatality rates of up to 50%, have been developed for use as bioweapons.
  • Current prevention is largely focused on control of the mosquito vector (Gibbons and Vaughn, BMJ 324(7353): 1563-6, 2002).
  • the present invention includes a method of treating a flavivirus infection including administering l,3-Bis(4-nitrophenyl)urea, a derivative of 1 ,3-Bis(4-nitrophenyl)urea, 3-(lH-tetrazol-5-yl)-9H-thio-xanthen- 9-one-10,10-dioxide monohydrate, or a derivative of 3-(lH-tetrazol-5-yl)-9H- thio-xanthen-9-one-10,10-dioxide monohydrate.
  • the present invention includes a method of treating a flavivirus infection including administering l,3-Bis(4-nitrophenyl)urea, or a derivative thereof.
  • the present invention includes a method of treating a flavivirus infection including administering 3-(lH-tetrazol-5-yl)-9H-fhio- xanthen-9-one-10,10-di oxide monohydrate, or a derivative thereof.
  • the present invention includes a method of treating a flavivirus infection in a subject, the method including administering 1,3-Bis(4- nitrophenyl)urea, or a derivative thereof, to a subject infected with a flavivirus in an amount effective to inhibit replication of the flavivirus.
  • the present invention includes a method of treating a flavivirus infection in a subject, the method including administering 3-(1H- tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10-dioxide monohydrate, or a derivative thereof, to a subject infected with a flavivirus in an amount effective to inhibit replication of the flavivirus.
  • the present invention includes a method of preventing infection of a subject with a flavivirus, the method including administering 1,3- Bis(4-nitrophenyl)urea, or a derivative thereof, to the subject prior to exposure to a flavivirus.
  • the present invention includes a method of preventing infection of a subject with a flavivirus, the method including administering 3- (lH-tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10-dioxide monohydrate, or a derivative thereof, to the subject prior to exposure to a flavivirus.
  • the present invention includes a method of reducing the severity of symptoms associated with a flavivirus infection, the method including administering 1 ,3-Bis(4-nitrophenyl)urea, or a derivative thereof, to the subject prior to infection with a flavivirus.
  • the flavivirus infection is an infection with a dengue virus and wherein development of dengue hemorrhagic fever and/or dengue shock syndrome is prevented.
  • the present invention includes a method of reducing the severity of symptoms associated with a flavivirus infection, the method including administering 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one-10, 10- dioxide monohydrate, or a derivative thereof, to the subject prior to infection with a flavivirus.
  • the method of claim 8 or 9 wherein the flavivirus infection is an infection with a dengue virus and wherein development of dengue hemorrhagic fever and/or dengue shock syndrome is prevented.
  • the present invention includes a method of reducing the severity of the symptoms associated with a flavivirus infection, the method including administering 1 ,3-Bis(4-nitrophenyl)urea, or a derivative thereof, to the subject after infection with a flavivirus.
  • the flavivirus infection is an infection with a dengue virus and wherein development of dengue hemorrhagic fever and/or dengue shock syndrome is prevented.
  • the present invention includes a method of reducing the severity of the symptoms associated with a flavivirus infection, the method including administering 3-( lH-tetrazol-5-yl)-9H-thio-xanthen-9-one- 10,10- dioxide monohydrate, or a derivative thereof, to the subject after infection with a flavivirus.
  • the flavivirus infection is an infection with a dengue virus and wherein development of dengue hemorrhagic fever and/or dengue shock syndrome is prevented.
  • the present invention includes a method of inhibiting the replication of a flavivirus in a cell, the method including contacting cells with l,3-Bis(4-nitrophenyl)urea, or a derivative thereof.
  • the present invention includes a method of inhibiting the replication of a flavivirus in a cell, the method including contacting cells with 3-( 1 H-tetrazol-5-yl)-9H-thio-xanthen-9-one- 10, 10-di oxide monohydrate, or a derivative thereof.
  • the present invention includes a method of identifying an agent suitable for the treatment or prevention of a flavivirus infection, the method including contacting cells with a flavivirus and an agent that is a derivative of l,3-Bis(4-nitrophenyl)urea, wherein the production of a decreased flavivirus titer indicates the agent is suitable for the treatment or prevention of a flavivirus infection.
  • the present invention includes a method of identifying an agent suitable for the treatment or prevention of a flavivirus infection, the method including contacting cells with a flavivirus and an agent that is a derivative of 3-(l H-tetrazol-5-yl)-9H-thio-xanthen-9-one- 10,10-di oxide monohydrate, wherein the production of a decreased flavivirus titer indicates the agent is suitable for the treatment or prevention of a flavivirus infection.
  • the present invention includes a method of identifying an agent suitable for the treatment or prevention of a flavivirus infection, the method including contacting a flavivirus protease with an agent that is a derivative of l,3-Bis(4-nitrophenyl)urea, wherein an inhibition of the protease activity of the flavivirus protease indicates the agent is suitable for the treatment or prevention of a flavivirus infection.
  • the present invention includes a method of identifying an agent suitable for the treatment or prevention of a flavivirus infection, the method including contacting a flavivirus protease with an agent that is a derivative of 3-( 1 H-tetrazol-5-yl)-9H-thio-xanthen-9-one- 10, 10-di oxide monohydrate, wherein an inhibition of the protease activity of the flavivirus protease indicates the agent is suitable for the treatment or prevention of a flavivirus infection.
  • the present invention includes a method of identifying an agent suitable for the treatment or prevention of a dengue virus infection, the method including contacting the NS3 serine protease of a dengue virus with an agent that is a derivative of 1 ,3-Bis(4-nitrophenyl)urea, wherein an inhibition of the serine protease activity of the NS3 serine protease of a dengue virus indicates the agent is suitable for the treatment or prevention of a dengue virus infection.
  • the present invention includes a method of identifying an agent suitable for the treatment or prevention of a dengue virus infection, the method including contacting the NS3 serine protease of a dengue virus with an agent that is a derivative of 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10- dioxide monohydrate, wherein an inhibition of the serine protease activity of the NS3 serine protease of a dengue virus indicates the agent is suitable for the treatment or prevention of a dengue virus infection.
  • the flavivirus is selected from the group consisting of dengue 1 virus, dengue 2 virus, dengue 3 virus, dengue 4 virus, West Nile virus, Yellow Fever virus, Japanese encephalitis virus, Murray valley fever virus, Yakose virus, acea virus, Rio Bravo virus, Modoc virus, Deer tick virus, Langat virus, Powassan virus, Tick-borne encephalitis virus, and Hepatitis C virus.
  • the dengue virus is selected from the group consisting of dengue 1 virus, dengue 2 virus, dengue 3 virus, and dengue 4 virus.
  • the present invention includes a method of treating a flavivirus infection including administering l,3-Bis(4-nitrophenyl)urea, or a derivative thereof, and 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10- dioxide monohydrate, or a derivative thereof.
  • the present invention includes a method of preventing infection with a flavivirus, the method including administering 1 ,3-Bis(4- nitrophenyl)urea, or a derivative thereof, and 3-(lH-tetrazol-5-yl)-9H-thio- xanthen-9-one-10,10-di oxide monohydrate, or a derivative thereof.
  • the present invention includes a method of reducing the severity of the symptoms associated a flavivirus infection, the method including administering l,3-Bis(4-nitrophenyl)urea, or a derivative thereof, and 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10-dioxide monohydrate, or a derivative thereof.
  • the present invention includes a method of inhibiting the replication of a flavivirus in a cell, the method including contacting cells with l,3-Bis(4-nitrophenyl)urea, or a derivative thereof, and 3-(lH-tetrazol-5- yl)-9H-thio-xanthen-9-one-10,10-dioxide monohydrate, or a derivative thereof.
  • the method further includes administering one or more additional antiviral agents.
  • the method further includes contacting the cells, flavivirus protease, or NS3 serine protease with or more additional antiviral agents.
  • the present invention includes a derivative of 1,3- Bis(4-nitrophenyl)urea, wherein the derivative inhibits the replication of a flavivirus in cell culture.
  • the derivative of 1,3-Bis(4- nitrophenyl)urea may be an symmetrical urea derivative.
  • the derivative of l,3-Bis(4-nitrophenyl)urea may be an symmetrical urea derivative.
  • the derivative of 1,3-Bis(4- nitrophenyl)urea may have the structure:
  • L is a divalent linking group
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 are independently selected from H, F, an alkyne, an alkene, a ketone, an aldehyde, an ester or an amine; and
  • R 1 and R 8 are not both a moiety.
  • the present invention includes a compound having the formula:
  • the present invention includes a compound having the formula:
  • the present invention includes a derivative of 1,3- Bis(4-nitrophenyl)urea, wherein the derivative inhibits the replication of a dengue virus in cell culture.
  • the present invention includes a derivative of 1 ,3- Bis(4-nitrophenyl)urea, wherein the derivative inhibits the in vivo replication of a flavivirus.
  • the present invention includes a derivative of 1,3- Bis(4-nitrophenyl)urea, wherein the derivative inhibits the in vivo replication of a dengue virus.
  • the present invention includes a derivative of 1 ,3- Bis(4-nitrophenyl)urea, wherein the derivative inhibits a flavivirus protease.
  • the present invention includes a derivative of 1,3- Bis(4-nitrophenyl)urea, wherein the derivative inhibits the NS3 serine protease of a dengue virus.
  • the present invention includes a derivative of 3-(1H- tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10-dioxide monohydrate, wherein the derivative inhibits the replication of a flavivirus in cell culture.
  • the derivative of 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one- 10,10-dioxide monohydrate may have the structure:
  • R7 can not be a
  • the derivative of 3-(lH-tetrazol-5-yl)-9H-thio-xanthen- 9-one-lCUO-dioxide monohydrate may have the structure:
  • R is H, a C1-C20 organic group, an amide, or a boronic acid; with the proviso that R is not a
  • the present invention includes a compound having the structure:
  • R is selected from:
  • the present invention includes a derivative of 3-(1H- tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10-dioxide monohydrate, wherein the derivative inhibits the replication of a dengue virus in cell culture.
  • the present invention includes a derivative of 3-(1H- tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10-dioxide monohydrate, wherein the derivative inhibits the in vivo replication of a flavivirus.
  • the present invention includes a derivative of 3-(1H- tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10-dioxide monohydrate, wherein the derivative inhibits the in vivo replication of a dengue virus.
  • the present invention includes a derivative of 3-(1H- tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10-dioxide monohydrate, wherein the derivative inhibits a flavivirus protease.
  • the present invention includes a derivative of 3-(1H- tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10-dioxide monohydrate, wherein the derivative inhibits the NS3 serine protease of a dengue virus.
  • the flavivirus may be dengue 1 virus, dengue 2 virus, dengue 3 virus, dengue 4 virus, West Nile virus, Yellow Fever virus, Japanese encephalitis virus, Murray valley fever virus, Yakose virus, acea virus, Rio Bravo virus, Modoc virus, Deer tick virus, Langat virus, Powassan virus, Tick-borne encephalitis virus, or Hepatitis C virus.
  • the dengue virus may be dengue 1 virus, dengue 2 virus, dengue 3 virus, or dengue 4 virus.
  • the present invention includes a composition including a derivative of l,3-Bis(4-nitrophenyl)urea and a pharmaceutically acceptable carrier. In some embodiments, the composition may further including one or more additional antiviral agents. In another aspect, the present invention includes a composition including of 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10-dioxide monohydrate, or a derivative thereof, and a pharmaceutically acceptable carrier. In some embodiments, the composition may further including one or more additional antiviral agents.
  • the present invention includes a composition including at least two agents and a pharmaceutically acceptable carrier, wherein one agent is selected from the group consisting of l,3-Bis(4-nitrophenyl)urea, a derivative of 1 ,3-Bis(4-nitrophenyl)urea, 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one- 10,10-dioxide monohydrate, and a derivative of 3-(lH-tetrazol-5-yl)-9H-thio- xanthen-9-one-10,10-dioxide monohydrate, and wherein a second agent wherein one agent is selected from the group consisting of 1,3-Bis(4- nitrophenyl)urea, a derivative of l,3-Bis(4-nitrophenyl)urea, 3-(lH-tetrazol-5- yl)-9H-thio-xanthen-9-one-10,10-di oxide monohydrate, and a derivative
  • the present invention includes a bivalent agent including l,3-Bis(4-nitrophenyl)urea or a derivative of 1,3-Bis(4- nitrophenyl)urea and 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10-dioxide monohydrate or a derivative of 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one- 10,10-dioxide monohydrate.
  • the present invention includes a bivalent agent including as one aspect l,3-Bis(4-nitrophenyl)urea, a derivative of 1,3-Bis(4- nitrophenyl)urea, 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10-dioxide monohydrate or a derivative of 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one- 10,10-dioxide monohydrate and as a second aspect an additional antiviral agent.
  • the present invention includes a bivalent agent including 1 ,3-Bis(4-nitrophenyl)urea or a derivative of 1 ,3-Bis(4- nitrophenyl)urea covalently attached to 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9- one-10,10-dioxide monohydrate or a derivative of 3-(lH-tetrazol-5-yl)-9H-thio- xanthen-9-one-10,10-di oxide monohydrate.
  • the 1 present invention includes a bivalent agent including 1 ,3-Bis(4-nitrophenyl)urea, a derivative of 1 ,3-Bis(4- nitrophenyl)urea, 3-( 1 H-tetrazol-5-yl)-9H-thio-xanthen-9-one-l 0, 10-dioxide monohydrate, or a derivative of 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one- 10,10-dioxide monohydrate covalently attached to an additiaonl antiviral agent.
  • the present invention includes a combinatorial library including at least one derivative of 1 ,3-Bis(4-nitrophenyl)urea.
  • the present invention includes a combinatorial library including at least one derivative of 3-(lH-tetrazol-5-yl)-9H-thio xanthen-9-one- 10,10-dioxide monohydrate.
  • Figure 1 presents a phylogenic tree based on sequence similarities of the flavivirus NS3 protein.
  • Figures 2A-2B presents the functional flavivirus genome organization.
  • Fig. 2A is a schematic diagram of the flavivirus genome showing position of mature forms of the proteins within the open reading frame (ORF), along with cleavage sites (V represents signal peptidase cleavage sites, "v” represents virally encoded NS2B/NS3 cleavage sites, "/' represents furin cleavage sites, and "?” represents cleavage sites of unknown specificity).
  • Fig. 2B is a cartoon representation of a mature viral particle.
  • Figure 3 is a surface representation of the apo structure of DEN2V NS3 protease, indicating the two spatially distinct binding sites targeted by virtual screening.
  • Figure 4 shows 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H- tetrazolium bromide ("MTT") toxicity assay results. Solubility limits prevented antiviral candidate ARDPOOI l from being examined at higher concentrations.
  • MTT tetrazolium bromide
  • Figure 5 shows slot-blot results for a subset of EUDOC-suggested compounds. Relative antibody signals are shown for days one, two, and three post-DEN2V infection.
  • FIG 6 presents results from West Nile virus (WNV) replicon assay, demonstrating activity of antiviral candidate ARDPOOl 1 against WNV replicons.
  • Figure 7 presents the chemical structures of the computer-predicted candidate dengue antivirals ARDPOOIl and ARDP0012.
  • Figure 8 shows representative dengue virus whole cell ELISA (DAWCE) and cytotoxicity (MTT) dose-response curves for antiviral candidate ARDPOOl 1.
  • DAWCE dengue virus whole cell ELISA
  • MTT cytotoxicity
  • Figure 9 shows decreased infectious dengue 2 virus (measured as foci- forming particles) was produced with increased concentration of antiviral candidate ARDP0012. Error bars are from duplicate independent challenge experiments.
  • the dose-response curve fit the data points with a goodness-of-fit R2 of approximately 0.95.
  • the EC50 value interpolated from the fitted dose- response curve is 18 ⁇ 2 nM.
  • Figure 10 presents trypsin reaction kinetics demonstrating that lead compounds ARDPOOl 1 and ARDP0012 did not interfere with trypsin proteolysis. Reaction kinetics were monitored by increased 405 nm absorbance due to p-nitroanilide product release. Reaction kinetics were for standard conditions (no inhibitor; solid circles), 115 uM benzamidine ("BZ"; solid triangles), 24 uM ARDPOOl 1 (open squares), and 670 uM ARDPOOl 2 (open diamonds).
  • Figure 11 presents an overview of synthetic scheme used for initial combinatorial chemical libraries developed around leads ARPDOOl 1 (top) and ARDPOO 12 (bottom).
  • Figure 12 presents an overview of the synthetic scheme for the synthesis of ureas and thioxanthones.
  • Figures 13A-13C present the chemical structures of Derivative 1 and Derivative 2 of l,3-Bis(4-nitrophenyl)urea.
  • Fig. 13 A presents the chemical structure of 1 ,3-Bis(4-nitrophenyl)urea.
  • Fig. 13B present the chemical structure of Derivative 1 of l,3-Bis(4-nitrophenyl)urea.
  • Fig. 13C present the chemical structure of Derivative 2 of l,3-Bis(4-nitrophenyl)urea.
  • a massive-throughput virtual library screen targeting several sites within the dengue virus NS3 protease resulted in the identification of several compounds with activity against a variety of flavi viruses.
  • the molecule l,3-Bis(4-nitrophenyl)urea also referred to herein as l,3-bis(4-nitrophenyl)urea
  • the molecule 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10-dioxide monohydrate, and derivatives thereof are effective agents for the treatment and prevention of flaviviral infections.
  • a flavivirus is a member of the family Flavivirus, including, but not limited to, dengue virus, West Nile virus, yellow fever virus, Japanese encephalitis virus, Kunjin virus, Murray Valley fever virus, Yakose virus, aba virus, Rio Bravo virus, Modoc virus, deer tick virus, Langat virus, Powassan virus, tick-borne encephalitis virus, St. Louis encephalitis virus, and Hepatitis C virus.
  • the flavivrus infection is a dengue virus infection.
  • a dengue virus includes, but is not limited to, a dengue 1 virus, a dengue 2 virus, a dengue 3 virus, or a dengue 4 virus.
  • the flavivrus infection is a West Nile virus infection.
  • the flavivrus infection is a Hepatitis C virus infection
  • Flaviviruses are divided into a number of serogroups based on cross- neutralization tests and genetic analysis (Kuno et al., J. Virol., 72:73-83, 1998). The family consists of approximately seventy viruses, organized into distinct phylogenetic clades (see Fig. 1) (Ryan et al., J. of General Virology 79:947-959, 1998; Gaunt et al., J. General Virology 82: 1867-1876).
  • Flaviviruses include, for example, dengue virus (DENV), West Nile virus (WNV), hepatitis C virus (HCV), yellow fever virus (YFV), Kunjin virus, Murray Valley fever virus, Yakose virus, acea virus, Rio Bravo virus, Modoc virus, deer tick virus, Langat virus, Powassan virus, tick-borne encephalitis virus, St. Louis encephalitis virus, and Japanese encephalitis virus (JEV). These mosquito and tick-borne viruses cause significant public health problems worldwide and pose clear and recognized threats as agents of biowarfare and/or bioterrorism.
  • NASH National Institutes of Health
  • Flaviviruses as Category A-C priority pathogens: dengue, West Nile virus, Japanese encephalitis virus, yellow fever virus, and members of the tick-borne encephalitis viruses (TBEVs) which include Russian spring summer encephalitis (RSSE), central European encephalitis (CEE), Kyasanur Forest disease (KFD), and Omsk hemorrhagic fever (OHF) viruses.
  • RSSE Russian spring summer encephalitis
  • CEE central European encephalitis
  • KFD Kyasanur Forest disease
  • OF Omsk hemorrhagic fever
  • Flaviviruses are endemic in many areas of the world and are emerging in others. Mosquitoes are responsible for spreading dengue throughout tropical and subtropical environments, yellow fever virus throughout tropical and subtropical Africa and South America, and Japanese encephalitis virus throughout Asia and Indonesia (Mackenzie, Emerg. Infect. Dis. 5:1-8, 1999). Mosquito-borne West Nile virus is common in parts of Africa, Asia and Europe, and has recently been introduced into the United States (Anderson et al., Science 286:2331-2333, 1999; Lanciotti et al., Science 286:2333-2337, 1999). Tick-borne flaviviruses are found primarily in central and Eastern Europe and the former Soviet Union and exists as three subtypes; Western subtype
  • CEE commonly referred to as CEE
  • Siberian subtype Siberian subtype
  • Far-eastern subtype the latter two are often collectively called RSSE (Heinz et al., Family Flaviviridae. In Virus Taxonomy, 7th International Committee for the Taxonomy of Viruses, pp. 859-878. Edited by M. H. V. Regenmortel, Fauquet, C. M., Bishop, D. H. L., Carstens, E., Estes, M. K., Lemon, S., Maniloff, J., Mayo, M. A. McGeogch, D., Pringle, C. R., and Wickner, R. B.
  • Flaviviruses are enveloped viruses composed of a nucleocapsid surrounded by a lipid bilayer containing an envelope (E) glycoprotein and a non-glycosylated membrane (M) protein, which is found in infected cells as the glycosylated precursor termed premembrane (prM) (see Fig. 2B).
  • the nucleocapsid consists of the capsid (C) protein and the single-strand RNA genome.
  • the genome contains a long open reading frame that encodes a polyprotein containing the structural and non-structural (NS) viral proteins (Lindenbach et al., Flavivridae: The Viruses and Their Replication; Chapter 20 in Fundamental Virology, Ed. Knipe, D. M.; Howley, P. M.; Lippincott Williams & Wilkins, 2001 , pp.589-640).
  • Flaviviruses contain a single stranded, positive sense RNA genome, encoding three structural proteins (capsid (C), membrane (M), envelope (E)), and seven non-structural proteins (NSl, NS2A, NS2B, NS3, NS4A, NS4B, NS5) (Chambers et al., Annu Rev Microbiol 44:649-688, 1990).
  • C capsid
  • M membrane
  • E envelope
  • NSl non-structural proteins
  • the genome is initially translated as a single, large polyprotein precursor. Post-translational cleavages of the viral polyprotein precursor in the cytoplasm of host-infected cells are necessary to produce the separate structural and functional viral proteins that are essential for replication and assembly of new viral progeny.
  • cleavages are effected by both host enzymes (including signalases and furin) and by a viral protease encoded by the N-terminal third of NS3.
  • This NS3 viral protease which is a trypsin-like serine protease with a functional catalytic triad (His-Asp-Ser), is essential for viral replication.
  • Mature viral proteins are released from the polyprotein by co- and post- translational cleavage by viral and cellular proteases; the majority of polyprotein cleavage is accomplished by the viral protease NS3 and its associated NS2B cofactor (see Fig. 2A) (Ryan et al., J. of General Virology 79:947-959, 1998). Due to its critical role in both virion assembly (via release of mature C) and processing of NS proteins for production of the viral replication complex, the NS3 protease has been identified as a key target for antiviral drug development (Leyssen et al., Clinical Microbiology Reviews 13:67-82, 2000).
  • flaviviral NS3 protease is a potential target for development of therapeutic agents that prevent viral replication.
  • inhibitors of viral proteases being potential antiviral drugs can be found in the success of inhibitors of proteases of the human immunodeficiency viruses (HIV), currently the most effective treatments for humans with HIV/ AIDS (Gulick, Clin Microbiol Infect 9:186-193, 2003; Rutenber et al., J. Biol. Chem. 268:15343-1534610, 1993) and in the early promise being shown by inhibitors of the NS3 protease of Hepatitis C virus (Lin et al., J Biol Chem 279:17508- 17514, 2004).
  • HIV human immunodeficiency viruses
  • HIV proteases have attracted considerable interest as antiviral targets, since HIV, flaviviruses, alphaviruses, rhinovirus, and adenovirus are dependent on the activity of viral proteases for replication (Strauss, Virol. 1:307-384, 1990). Inhibition of the HIV protease PR has been an effective means of treating HIV infection; between 1995 and 2001 the Food and Drug Administration (FDA) approved six anti-PR peptide analogs as treatments for AIDS (Wlodawer, Annu. Rev. Med. 53:595-614, 2002). In silico methods were successfully employed to help develop several HIV protease inhibitors (Lam et al., Science 263:380-4, 1994). Analogous drug discovery efforts targeting the flavivirus HCV protease have produced an inhibitor reported to reduce viral load in humans (Lamarre et al., Nature 426:186-9, 2003).
  • the small molecule 1 ,3-Bis(4-nitrophenyl)urea, and derivatives thereof, and the small molecule 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10-dioxide monohydrate, and derivatives thereof, are effective agents for the treatment and prevention of flaviviral infections.
  • an “agent” or “compound” includes 1,3-Bis(4- nitrophenyl)urea, a derivative of l,3-Bis(4-nitrophenyl)urea, 3-(lH-tetrazol-5- yl)-9H-thio-xanthen-9-one-10,10-di oxide monohydrate, or a derivative of 3- ( 1 H-tetrazol-5-yl)-9H-thio-xanthen-9-one- 10, 10-di oxide monohydrate.
  • l,3-Bis(4-nitrophenyl)urea ((O 2 NC 6 H 4 NH 2 )CO), also known as 4', 4"- dinitrocarbanilide or 4,4'-dinitrocarbanilide is commercially available, for example, from Sigma-Aldrich.
  • Derivatives of l,3-Bis(4-nitrophenyl)urea include compounds having the general formula represented by Formula I, or a pharmaceutically acceptable salt, solvate, ester, or prodrug thereof.
  • Formula I is as provided below:
  • L is a divalent linking group such as, for example, urea, C(O),
  • organic group is used for the purpose of this invention to mean a hydrocarbon group that is classified as an aliphatic group, cyclic group, or combination of aliphatic and cyclic groups (e.g., alkaryl and aralkyl groups).
  • aliphatic group means a saturated or unsaturated linear or branched hydrocarbon group. This term is used to encompass alkyl, alkenyl, and alkynyl groups, for example.
  • alkyl group means a saturated linear or branched monovalent hydrocarbon group including, for example, methyl, ethyl, 7 ⁇ -propyl, isopropyl, tert-butyl, amyl, heptyl, and the like.
  • alkenyl group means an unsaturated, linear or branched monovalent hydrocarbon group with one or more olefinically unsaturated groups (i.e., carbon-carbon double bonds), such as a vinyl group.
  • alkynyl group means an unsaturated, linear or branched monovalent hydrocarbon group with one or more carbon-carbon triple bonds.
  • cyclic group means a closed ring hydrocarbon group that is classified as an alicyclic group, aromatic group, or heterocyclic group.
  • alicyclic group means a cyclic hydrocarbon group having properties resembling those of aliphatic groups.
  • aromatic group or “aryl group” means a mono- or polynuclear aromatic hydrocarbon group.
  • heterocyclic group means a closed ring hydrocarbon in which one or more of the atoms in the ring is an element other than carbon (e.g., nitrogen, oxygen, sulfur, etc.).
  • group and “moiety” are used to differentiate between chemical species that allow for substitution or that may be substituted and those that do not so allow for substitution or may not be so substituted.
  • group when the term “group” is used to describe a chemical substituent, the described chemical material includes the unsubstituted group and that group with nonperoxidic O, N, S, Si, or F atoms, for example, in the chain as well as carbonyl groups or other conventional substituents.
  • moiety is used to describe a chemical compound or substituent, only an unsubstituted chemical material is intended to be included.
  • alkyl group is intended to include not only pure open chain saturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl, tert-butyl, and the like, but also alkyl substituents bearing further substituents known in the art, such as hydroxy, alkoxy, alkylsulfonyl, halogen atoms, cyano, nitro, amino, carboxyl, etc.
  • alkyl group includes ether groups, haloalkyls, nitroalkyls, carboxyalkyls, hydroxyalkyls, sulfoalkyls, etc.
  • alkyl moiety is limited to the inclusion of only pure open chain saturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl, tert- butyl, and the like.
  • C 1 and C 2 may be either symmetrical or asymmetrical.
  • either of C 1 or C 2 may be absent.
  • L has the formula
  • Each of C and C may independently be an aromatic group, including, for example, a phenyl group.
  • each of C 1 and C 2 may be substituted, wherein one, two, or three carbon atoms in C 1 or C 2 may be optionally replaced with S, SO, O, F, N, NH, among other atoms, in a chemically stable arrangement.
  • a "chemically stable arrangement” refers to a compound structure that renders the compound sufficiently stable to allow manufacture and administration by methods known in the art. Typically, such compounds are stable at a temperature of 40 degrees Celsius or less, in the absence of moisture or other chemically reactive condition, for at least a week.
  • either of C 1 or C 2 or both C 1 and C 2 may be a five membered ring, as shown below:
  • each of R 1 , R 2 , R 3 , and R 4 are independently selected from H, F, NO 2 , an alkyne, an alkene, a ketone, an aldehyde, an ester or an amine.
  • a derivative of l,3-Bis(4-nitrophenyl)urea may have the structure as shown below:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 are independently selected from H, F, an alkyne, an alkene, a ketone, an aldehyde, an ester or an amine.
  • either of C 1 or C 2 or both C 1 and C 2 may be a six membered carbon ring, as shown below:
  • each of R 1 , R 2 , R 3 , R 4 , and R 5 are independently selected from H, F, an alkyne, an alkene, a ketone, an aldehyde, an ester, or an amine.
  • a derivative of 1 ,3-Bis(4-nitrophenyl)urea may have the structure as shown below:
  • each of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 are independently selected from H, F, an alkyne, an alkene, a ketone, an aldehyde, an ester, or an amine.
  • either of C 1 or C 2 or both C 1 and C 2 may be a phenyl group, as shown below:
  • each of R ] , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 are independently selected from H, F, an alkyne, an alkene, a ketone, an aldehyde, an ester, or an amine.
  • a derivative of l,3-Bis(4-nitrophenyl)urea may have the structure as shown below:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 are independently selected from H, F, an alkyne, an alkene, a ketone, an aldehyde, an ester or an amine; and
  • R 1 and R 8 are not both a moiety.
  • any R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , or R ]0 may be:
  • any R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , or R 10 may be:
  • any R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , or R )O may be:
  • Derivatives of l,3-Bis(4-nitrophenyl)urea include, for instance,
  • Derivatives of l,3-Bis(4-nitrophenyl)urea include 1,3-Bis(4- nitrophenyl)urea derivatives in which different types of ureas have been incorporated, including symmetrical or asymmetrical (also referred to herein as "unsymmetrical") ureas derivatives.
  • Symmetrical urea derivatives include symmetrical bisaromatic ureas, possessing groups with properties similar to nitro group (NO 2 ). These ureas may have benzene rings substituted with NO 2 in the ortho and meta positions as well as sulfate (SO 3 " ), acetate (OAc) and nitrile (CN) in the ortho, meta, and para positions. Both OAc and SO 3 " are electron- withdrawing groups like NO 2 and consequently provided aromatic rings with electron densities similar to the lead compound, l,3-Bis(4-nitrophenyl)urea.
  • l,3-Bis(4-nitrophenyl)urea derivatives may contain symmetrical heteroaromatic groups such as imidazole, pyridine, thiazole and triazoles ureas. These members will possess a shape and ri-cloud similar to simpler aromatics while presenting additional hydrogen bonding options for further derivatization.
  • Unsymmetrical urea derivatives (also referred to herein as asymmetrical urea derivatives) of l,3-Bis(4-nitrophenyl) include ureas with two different aromatic groups and ureas with one aromatic group and one aliphatic group.
  • the unsymmetrical bisaromatic ureas may contain combinations of the aromatic groups discussed above.
  • the aromatic/aliphatic ureas may use the aromatic groups with both acyclic and cyclic aliphatic groups.
  • Such ureas may be synthesized from commercially available amines, of which more than 200 amines are available from Aldrich alone.
  • Symmetrical urea derivatives of 1,3- Bis(4-nitrophenyl)urea include those obtained by the addition of two equivalents of an amine to a solution of phosgene or carbonyldiimidazole.
  • Such unsymmetrical ureas are accessible by stepwise addition of one amine to carbon yl diamidazole followed by addition of the second amine.
  • 1,3-Bis(4- nitrophenyl)urea derivatives include symmetrical ureas derivatives synthesized by reaction of aniline derivates with carbonyldiimidazole (Zhang et al., J. Org. Chem. 62:6420-6423, 1997).
  • l,3-Bis(4-nitrophenyl)urea derivatives also include unsymmetrical ureas, synthesized, for example, on a solid support (Zheng and Combs, J. Comb Chem. 4:38-42, 2002). This includes, for example, when a support has been prepared with one half of the urea attached to the polymer as the initial step to perform the solid phase synthesis of ureas.
  • This carbonyl diamidazole adduct allows twenty different amines to be added to different batches of the support to provide different unsymmetrical ureas.
  • the parent thioxanthone moiety can be synthesized by reaction of 3-bromothiophenol with 2-fluorocyanobenzene followed by treatment with sulfuric acid to hydrolyze the nitrile and perform the Friedel-Crafts acylation in one step (Rewcastle et al., J. Med. Chem. 34, 491- 496, 1991; Kristensen et al., J. Org. Chem. 68, 4091-4092, 2003). This can be followed by oxidation.
  • Derivatives can be synthesized by conversion of the bromide to a variety of different groups using palladium catalyzed coupling reactions (Brase et al., Tetrahedron 59, 885-939, 2003).
  • 1 ,3-Bis(4-nitrophenyl)urea derivatives include symmetrical and unsymmetrical functional group derivatives, where each position, or a combination of positions, on the aromatic ring could be substituted with a new functional group or chemical moiety (for example, methyl, hydroxyl, amine, or aldehyde.
  • Derivatives include substitutions of a carbon position, or a combination of carbon positions, in the aromatic ring with different chemical entities that could include nitrogen, oxygen, and sulfur, among other atoms.
  • 3-( 1 H-tetrazol-5-yl)-9H-thio-xanthen-9-one- 10,10-dioxide monohydrate is commercially available, for example, from Sigma- Aldrich.
  • a derivative of 3-(lH-tetrazol-5-yl)-9H-thio- xanthen-9-one 10,10-dioxide monohydrate includes a compound represented by structural Formula II, or a pharmaceutically acceptable salt, solvate, ester, or prodrug thereof.
  • Formula II is as provided below:
  • C and C are independently selected organic groups; wherein Z is, for example, SO 2 , O, C, a nitrogen, such as for example, NH, C; and . wherein Y is, for example, O, H, S, or an imine.
  • Z is SO 2 .
  • Y is O.
  • a derivative of 3-(lH-tetrazol-5-yl)-9H-thio- xanthen-9-one 10,10-dioxide monohydrate includes a compound represented by structural Formula III, or a pharmaceutically acceptable salt, solvate, ester, or prodrug thereof.
  • Formula III is as provided below:
  • Formula III wherein Z is, for example, SO 2 , O, or a nitrogen, such as for example, NH; wherein Y is, for example, O, H, S, or an imine; and wherein each of Rl to R8 is independently selected from H, a C1-C20 organic group, including for example, an aromatic group or a heteroaromatic group, an amide, a boronic acid, an alkyl, a vinyl, a carboxylic acid derivative, or an alkynal; with the proviso that when Z is SO 2 and Y is O, then R7 can not be a moiety.
  • Z is, for example, SO 2 , O, or a nitrogen, such as for example, NH
  • Y is, for example, O, H, S, or an imine
  • each of Rl to R8 is independently selected from H, a C1-C20 organic group, including for example, an aromatic group or a heteroaromatic group, an amide, a boronic acid, an alky
  • a derivative of 3-(lH-tetrazol-5-yl)-9H-thio- xanthen-9-one 10,10-dioxide monohydrate has the structure represented by structural Formula IV, below:
  • R may be
  • Derivatives of 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10- dioxide monohydrate include derivatives obtained by the addition of an R group to one or more different positions around the aromatic ring of the parent 3-(1H- tetrazol-5-yl)-9H-thio-xanthen-9-one- 10, 10-dioxide monohydrate compound.
  • Derivatives include, for example, the addition of alkynes, alkenes, ketones, aldehydes, esters and/or amines to different positions around the parent aromotic ring.
  • the R group added can be provided by any of the many commercially available carboxy amides or boronic acids.
  • Derivatives may also include functional group derivatives, where each position, or combination of positions, on the aromatic rings could be substituted with a new functional group or chemical moiety (for example, methyl, hydroxyl, amine, or aldehyde).
  • each carbon position, or a combination of carbon positions, in the aromatic rings could be substituted with different chemical entities that could include nitrogen, oxygen, and sulfur, among other atoms.
  • R groups may be via the addition of an intermediate bromide or chlorine to different positions around the parent aromatic rings.
  • Bromothiophenols are commercially available.
  • a wide variety of additional derivatives of 3-( 1 H-tetrazol-5-yl)-9H-thio-xanthen-9-one- 10, 10-dioxide monohydrate may be obtained with palladium catalyzed couplings.
  • Derivatives of 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10- dioxide monohydrate include derivatives with a central ring structure that has six or more carbons.
  • Derivatives include, for example, compounds with a central ring that include six carbons, seven carbons, eight carbons, nine carbons, ten carbons, twelve carbons, or more.
  • a seven member carbon ring may be synthesized as detailed by Rokach et al. (U.S. Patent No. 4,536,507).
  • An agent of the present invention may demonstrate one or more of the following activities: inhibit or block the replication of a flavivirus in cell culture, inhibit or block the replication of a dengue virus in cell culture, inhibit or block the in vivo replication of a flavivirus, inhibit or block the in vivo replication of a dengue virus, inhibit or block a flavivirus protease activity, inhibit or block flaviviral NS3 protease activity, inhibit or block dengue virus NS3 serine protease activity, reduce the titer of infectious virus produced in cells challenged or infected with a flavivirus, and/or reduce the titer of infectious virus produced in cells challenged or infected with a dengue virus.
  • the activity of an agent may be assayed by any of many art known methods, including any of those described herein. Any of many cell-free or enzyme based protease activity assays may be used.
  • HCV methods that may be used include those described by Steinkuhler et al., J Virol. 70:6694-6700, 1996; Sudo et al., Antiviral Res. 32:9-18, 1996; Takeshita et aL, Anal Biochem 247:242-246, 1997; Liu et al., Anal Biochem 267:331-335, 1999; Taliani et al., Anal Biochem 240:60-67, 1996, Lemon et al. (WO 05/053516), and Lemon et al. (U.S. Paten No. 6,921,634).
  • a cell-based screening method may be used. See, for example, Lee et al., Assay Drug Dev Technol. 3(4):385-392, 2005 for an example of a high- throughput cell-based screening method for Hepatitis C virus NS3/4A protease inhibitors)
  • Activity may be assayed in one of the various animal models that are available as models for human disease.
  • West Nile virus is lethal to mice within 6-7 days using either intracerebral or intraperitoneal routes of inoculation with clear evidence of encephalitis.
  • the WNV New York 1999 strain has an LD50 of approximately 1 pfu by intraperitoneal injection.
  • Powassan virus type strain LB a BSL-3 model for CEE virus, is lethal to mice 6-8 days post-infection with an LD50 of approximately 12 pfu by the intraperitoneal route.
  • Activity may be assayed in an animal model for dengue virus infection, including, for example models using a laboratory-adapted DENV strains to cause encephalitis following intracranial inoculation of suckling mice (Sabin, Am. J. Trop. Med. Hyg. 1:30-50, 1952). Encephalitis and death have been used as endpoints in some studies for evaluating vaccine efficacy in mice, and antiviral drugs may also show efficacy in this model (Koff et al., Antimicrob Agents Chemother. 24:134-6, 1983). Immunodeficient mice models for evaluation of DENV virulence/pathogenesis may be used. See, for example, Lin et al., K562 cells. J Virol 72, 9729-37, 1998; An et al., Virology 263, 70-7, 1999; and Johnson and Roehrig, J Virol 73, 783-6, 1999.
  • the agents of the present invention may be effective against one or more of the four dengue virus serotypes: dengue-1, dengue-2, dengue-3, and dengue- 4. These viruses form an antigenically distinct subgroup within the flavivirus family (Calisher et al., J Gen Virol 70:37-43, 1989). Dengue viruses are the most common cause of arboviral disease in the world. They are found virtually throughout the tropics and cause an estimated 1-2 million clinical illnesses annually, including 250,000-500,000 cases of dengue haemorrhagic fever, a severe manifestation of dengue, and about 254,000 deaths.
  • ribavirin and inosine monophosphate dehydrogenase (EVIPDH) inhibitors have been studied as potential flavivirus antivirals (Leyssen et ah, Clinical Microbiology Reviews 13:67-82, 2000; Diamond et ah, Virology 304:211-21, 2002). While effective at suppressing viral replication in cell culture, sub-toxic concentrations of these compounds showed little sustained virus clearance in animals. The exact mechanisms of action of ribavirin and MPA are unknown; however neither compound appears to specifically inhibit viral proteins (Benarroch et ah, J Biol Chem. 279:35638-43, 2004).
  • dengue virus Following infection of its human host by the bite of an infected mosquito, dengue virus undergoes local replication, infecting several cell types, including dendritic cells. This initial infection is followed by systemic infection of monocytes, resulting in a high-level viremia that can last for several days. Infection of macrophages and monocytes in lymphoid organs and the circulation results in the malaise, fever, and rash that characterize the clinical syndrome "dengue fever" (McBride and Bielefeldt-Ohmann, Microbes Infect 2:1041-50, 2000).
  • a second peak of viremia is observed approximately one week after initial exposure; this peak can be associated with the more severe forms of the disease, including DHF, which has been associated with a strong T cell response to dengue virus infection (Rothman, Adv Virus Res 60:397-419, 2003). Although there are some indications that severe forms of dengue infections can result in neurological symptoms, encephalitis is not considered to be an important outcome of DENV infection.
  • dengue represents an ideal disease for post-exposure (and/or post-presentation) treatment with antiviral drugs.
  • dengue disease could be managed by chemotherapeutics comes from research indicating that infection of non-human primates results in a transient (usually less than four days) viremia that is at least two orders of magnitude lower than that observed in man, and that these animals do not display any signs of disease (Halstead et al., J Infect Dis. 128:7- 14, 1973; and Halstead et al., J Infect Dis. 128:15-22, 1973).
  • dengue causes the greatest number of cases of human disease of any flavivirus
  • Japanese encephalitis virus and yellow fever are also important diseases that affect hundreds of thousands of people each year.
  • approximately 10,000 confirmed cases of West Nile virus infection occurred in the United States in 2003.
  • yellow fever virus and the tick-borne encephalitis viruses which can display case-fatality rates of up to 50%, have been developed for use as bioweapons.
  • broad-spectrum antivirals that inhibit infection by dengue and other flaviviruses could combat these bioterrorist agents and significantly improve Homeland Defense and global public health.
  • West Nile virus in North America demonstrates the long-term, damaging consequences that can arise from introduction of a flavivirus disease agent into the US.
  • the broad vertebrate and invertebrate host specificity of West Nile virus may have predisposed North America to this incursion, there are multiple mosquito vectors of Japanese encephalitis virus, yellow fever virus, and dengue virus in the US, indicating that these diseases could also become established, and yellow fever was established in many US cities in the late 1800s.
  • the fear and public anxiety that West Nile virus outbreaks have generated, the speed of its spread across the US, and the association of its spread with organ donation and transfusion demonstrate that flaviviruses can terrorize a civilian population even without intentional malicious introduction.
  • West Nile virus is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection causes severe neurological disease and fatalities in both human and animal hosts. West Nile virus is transmitted via mosquitoes from avian reservoir hosts to vertebrate dead end hosts that include humans and horses. While endemic in humans in parts of Africa, Europe and the Middle East, recent outbreaks in Israel (1998), Romania (1996), and the United States (1999), have been associated with serious neurological pathology and fatal infections. During the last five years, West Nile virus has spread rapidly throughout the USA, Canada and Mexico, as well as appearing recently in the United Kingdom. This rapid global spread, through developed countries, has prompted widespread implementation of prevention strategies. No vaccines or therapeutic treatments for West Nile virus infections are yet available.
  • the West Nile viral protease (NS2B-NS3) is essential for post-translational processing in host-infected cells of a viral polypeptide precursor into structural and functional viral proteins and its inhibition presents a potential treatment for viral infections. See Nail et al., J Biol Chem. 279:48535-42, 2004.
  • HCV Hepatitis C virus
  • HCV chronic hepatitis
  • HCV infection causes clinically acute disease and even liver failure, however, most instances of acute infection are clinically undetectable.
  • the natural history of chronic HCV infection can vary dramatically between individuals. Some will have clinically insignificant or minimal liver disease and never develop complications. Others will have clinically apparent chronic hepatitis. Of these, some go on to develop cirrhosis, however, the exact percentages is not known. About 20% of individuals with hepatitis C who do develop cirrhosis will develop end-stage liver disease.
  • Cirrhosis caused by hepatitis C is presently the leading indication for orthotopic liver transplantation in the United States. Individuals with cirrhosis from hepatitis C are also at an increased risk of developing hepatocellular carcinoma (primary liver cancer).
  • a major problem in discussing prognosis in patients with chronic hepatitis C is that it is difficult to predict who will have a relatively benign course and who will go on to develop cirrhosis or cancer.
  • the present invention includes bivalent compounds that include as one component, l,3-Bis(4-nitrophenyl)urea or a 1,3-Bis(4- nitrophenyl)urea derivative, and as a second component, 3-(lH-tetrazol-5-yl)- 9H-thio-xanthen-9-one-10,10-dioxide monohydrate, or a 3-(lH-tetrazol-5-yl)- 9H-thio-xanthen-9-one-10,10-dioxide monohydrate derivative.
  • the present invention further includes bivalent compounds, wherein one component of the bivalent compound is 1 ,3-Bis(4-nitrophenyl)urea, a 1,3-Bis(4- nitrophenyl)urea derivative, 3-( 1 H-tetrazol-5-yl)-9H-thio-xanthen-9-one- 10,10- dioxide monohydrate, or a 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one- 10,10- dioxide monohydrate derivative, and a second component of the bivalent compound is an additional antiviral agent.
  • an additional antiviral agent can be any of the various known antiviral agents.
  • the present invention includes methods of treating and/or preventing flaviviral infections.
  • An agent of the present invention may be administered to a subject for the treatment of a flavivirus infection.
  • An agent of the present invention may be administered to a subject prior to and/or after exposure to a flavivirus.
  • An agent of the present invention may be administered to a subject prior to and/or after infection with a flavivirus.
  • An agent may be administered in an amount effective to inhibit replication of the flavivirus.
  • Agents of the present invention may be used as antiviral agents to reduce the replication and/or production of a flavivirus, such as dengue, in an infected individual.
  • Inhibition of the replication of a flavivirus may be determined, for example, by methods including, but not limited to, methods described in the examples herein.
  • An agent of the present invention may be administered in an amount effective to inhibit protease activity of a flavivirus protease, including, but not limited to, protease activity of the NS3 protease of dengue virus.
  • Inhibition of the protease activity of a flavivirus protease may be determined by methods including, but not limited to, those described in more detail in the examples herein, or by Leung et al., (J. Biol. Chem. 276:45762-45771, 2001).
  • An agent of the present agent may serve as a broad spectrum antiviral, effective for the treatment or prophylaxis of one or more flaviviral infections.
  • a "subject" or an “individual” is an organism, including, for example, a mammal.
  • a mammal may include, for example, a rat, mouse, a primate, a domestic pet (such as, but not limited to, a dog or a cat), livestock (such as, but not limited to, a cow, a horse, and a pig), or a human.
  • an agent as provided herein is meant a nontoxic but sufficient amount of the agent or composition to provide the desired effect. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, and the particular agent and its mode of administration, and the like.
  • an appropriate effective amount may be determined by one of ordinary skill in the art using only routine experimentation. Therapeutically effective concentrations and amounts may be determined for each application herein empirically by testing the compounds in known in vitro and in vivo systems, such as those described herein; dosages for humans or other animals may then be extrapolated therefrom.
  • An agent of the present invention may be administered to a subject to prevent the infection of a subject with a flavivirus.
  • Agents of the present invention may be taken as a prophylactic to prevent the development of a flavivirus infection. This may be particularly beneficial for travelers or visitors to locations were flavivirus infections are endemic or regions experiencing an outbreak or epidemic with a flavivirus.
  • An agent of the present invention may be administered to a subject to reduce the severity of the symptoms associated with a flavivirus infection.
  • the agent may be administered to a subject prior to and/or after exposure to or infection with a flavivirus.
  • the symptoms of a flavivirus infection that may be reduced in severity by the administration of an agent of the present invention may include one or more of the following: high fever, severe headache, nausea, vomiting, flushed fades, sore throat, cough, cutaneous hyperaesthesia, taste aberrations, headache, retro-orbital pain, myalgia, arthralgia, encephalitis, neurological manifestations, haemorrhagic manifestations, rash, including, but no limited to maculopapular rash and macular rash, severe haemorrhage, thrombocytopenia, consumptive coagulopathy, vascular leak syndrome, clinical shock, leucopenia, haemorrhagic manifestations (shown, for example, by positive tourniquet test, petechiae,
  • dengue haemorrhagic fever DHF
  • an agent of the present invention may be administered to prevent the development of and/or reducing the severity of the symptoms of dengue hemorrhagic fever and/or dengue shock syndrome.
  • the agents of the present invention may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the desired therapeutic outcome and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be treated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions and methods.
  • the agents of the present invention may be administered to the subject in combination with other therapeutic modalities.
  • the agents of the present invention can be administered before, during or after the administration of the other therapies.
  • the agents of the present invention may be formulated in a composition.
  • a composition may include one or more of l,3-Bis(4-nitrophenyl)urea, a derivative of l,3-Bis(4-nitrophenyl)urea, 3-(lH-tetrazol-5-yl)-9H-thio-xanthen- 9-one->10,10-dioxide monohydrate, and a derivative of 3-(lH-tetrazol-5-yl)-9H- thio-xanthen-9-one- 10, 10-dioxide monohydrate.
  • compositions including 1,3- Bis(4-nitrophenyl)urea or a derivative thereof do not include 2-hydroxy 4,6- dimethylpyrimidine.
  • the present invention includes compositions that do not include nicarbazin, an equimolecular complex of 4,4 - dinitrocarbanilide with 2-hydroxy-4,6-dimethylpyrimidine.
  • Nicarbazin has been used in the poultry industry, where it has been administered to birds to reduce the incidence of encephalopathy in young chickens (Bartov and Budowski, Poult Sci.
  • Agents of the present invention may be administered in a composition including a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, that is, the material may be administered to an individual along with an agent of the present invention without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • Agents of the present invention may be formulated in a composition along with a "carrier.”
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
  • compositions of the present invention may include additional antiviral agents, such as, for example, ribovarin, AZT, and any of the various antiviral compound disclosed in U.S. Patent No. 6,914,054, U.S. Patent Application 2005/0215486, U.S. Paten Application 2005/0245458, Beaulieu et al., J Org Chem. 70(15):5869-79, 2005; Venkatraman et al., J Med Chem.
  • additional antiviral agents such as, for example, ribovarin, AZT, and any of the various antiviral compound disclosed in U.S. Patent No. 6,914,054, U.S. Patent Application 2005/0215486, U.S. Paten Application 2005/0245458, Beaulieu et al., J Org Chem. 70(15):5869-79, 2005; Venkatraman et al., J Med Chem.
  • the agents of the present invention may be contained within a time -released composition.
  • the present invention includes methods for identifying agents suitable for the treatment or prevention of a flavivirus infection.
  • a method may include contacting cells with a flavivirus and an agent that is a derivative of 1,3- Bis(4-nitrophenyl)urea, wherein the production of a decreased flavivirus titer indicates the agent is suitable for the treatment or prevention of a flavivirus infection.
  • Such a method may include contacting cells with a flavivirus and an agent that is a derivative of 3-( 1 H-tetrazol-5-yl)-9H-thio-xanthen-9-one- 10,10- dioxide monohydrate, wherein the production of a decreased flavivirus titer indicates the agent is suitable for the treatment or prevention of a flavivirus infection.
  • Such a method may include contacting a flavivirus protease with an agent that is a derivative of l,3-Bis(4-nitrophenyl)urea, wherein an inhibition of the protease activity of the flavivirus protease indicates the agent is suitable for the treatment or prevention of a flavivirus infection.
  • Such a method may include contacting a flavivirus protease with an agent that is a derivative of 3- ( 1 H-tetrazol-5-yl)-9H-thio-xanthen-9-one- 10,10-dioxide monohydrate, wherein an inhibition of the protease activity of the flavivirus protease indicates the agent is suitable for the treatment or prevention of a flavivirus infection.
  • Methods for assaying for the inhibition of a flavivirus protease include, for example, the methods described in the examples herein.
  • the present invention includes methods for identifying agents suitable for the treatment or prevention of a dengue viral infection.
  • Such a method may include contacting the NS3 serine protease of a dengue virus with an agent that is a derivative of l,3-Bis(4-nitrophenyl)urea, wherein the inhibition of the serine protease activity of the NS3 serine protease of a dengue virus indicates the agent is suitable for the treatment or prevention of a dengue virus infection.
  • Such a method may include contacting the NS3 serine protease of a dengue virus with an agent that is a derivative of 3-(lH-tetrazol-5-yl)-9H-thio-xanthen- 9-one-10,10-di oxide monohydrate, wherein the inhibition of the serine protease activity of the NS3 serine protease of a dengue virus indicates the agent is suitable for the treatment or prevention of a dengue virus infection.
  • Methods for assaying for the inhibition of a dengue NS3 serine protease include, for example, the methods described in the examples herein and as described in Leung et al, J. Biol. Chem. 276:45762-45771, 2001.
  • the present invention includes combinatorial chemistry libraries (also referred to herein as "combinatorial libraries") that include at least one derivative of l,3-Bis(4-nitrophenyl)urea and/or at least one derivative of 3-(1H- tetrazol-5-yl)-9H-thio-xanthen-9-one-10,10-dioxide monohydrate.
  • Combinatorial chemistry libraries of the present invention may include a multiplicity of such derivatives.
  • Such combinatorial chemistry libraries may also include l,3-Bis(4-nitrophenyl)urea and/or 3-(lH-tetrazol-5-yl)-9H-thio- xanthen-9-one-10,10-dioxide monohydrate.
  • a combinatorial library of the present invention can be built around the lead antiviral l,3-Bis(4-nitrophenyl)urea.
  • a combinatorial library of the present invention can be built around the lead antiviral 3-(lH-tetrazol-5-yl)-9H-thio- xanthen-9-one- 10, 10-dioxide monohydrate.
  • a combinatorial chemistry library a large number compounds can be synthesized and screened for various possible physiological or other activities. Combinatorial chemistry allows scientists to generate large numbers of unique molecules with a small number of chemical reactions.
  • antivirals with broad-spectrum activity these libraries will be screened in cell culture for efficacy against the four dengue virus serotypes, West Nile virus, and yellow fever virus. Since poor drug-like properties, including, for example, bioavailability, pharmacokinetics, metabolism, or toxicity, are the primary reason drug leads fail to progress beyond preclinical trials, this example stresses simultaneously optimizing drug properties and compound potency. Promising antivirals will be evaluated in vitro for solubility and cytochrome P450 inhibition, and evaluated in vivo for toxicity, pharmacokinetic properties, and bioavailability.
  • Computer-Based Antiviral Discovery Computer screening of virtual chemical libraries binding to DEN2V NS3 protease identified commercially available small molecules (1,3-bis (4-nitrophenyl) urea and 3-(lH-tetrazol-5- yl)-9H-thio- xanthen-9-one 10,10-dioxide monohydrate) as potential NS3 inhibitors. These compounds have confirmed antiviral activity in cell against DEN2V and WN replicons. Molecular modeling studies indicate that these antivirals interact with independent NS3 protease sites.
  • antivirals will serve as templates for synthetic combinatorial chemical libraries from which new therapeutics against Category A-C flaviviruses (for example, DENV, WNV, and YFV) and widespread pathogenic flaviviruses (for example, HCV) can be developed.
  • Category A-C flaviviruses for example, DENV, WNV, and YFV
  • widespread pathogenic flaviviruses for example, HCV
  • EUDOC is a computer program for the identification of drug interaction sites in macromolecules and drug leads from chemical databases.
  • EUDOC uses a lock and key method of screening by incrementally moving a possible ligand in all three dimensions and rotations through a user defined area using an Amber force field for "energy” scoring. This approach allows for the rapid and effective searching of large compound databases.
  • the two DEN2V NS3 protease structures were used as target molecules for virtual screening (Murthy et al., J MoI Biol. 301:759-67, 2000; Murthy et al., J Biol Chem. 274:5573-80, 1999). Although neither structure contained the NS2B co-factor required for protease activity, these structures were models of apo- and inhibitor- bound conformations. The spatially distinct catalytic site and the Pl pocket of both protease structures were targeted for the virtual screening (Fig. 3). There were few conformational differences between the backbone atoms of apo protease and the protease complexed to the Bowman-Birk inhibitor.
  • a virtual library of approximately two million small molecule structures was screened. To increase the likelihood of selecting small molecules active in cell culture, the virtual library was filtered to remove compounds containing formal charges. To obviate the need for a labor-intensive synthetic chemistry program, the library was additionally filtered to retain only compounds available from reputable chemical suppliers. Virtual docking computations were performed using supercomputer resources. Potential inhibitor-protease complexes were selected for the apo and inhibitor-bound protease, with each complex having predicted interaction energies of approximately -58 kilocalories/mole (kcal/mol) and -34 kcal/mol for the catalytic and Pl binding sites, respectively. The chemical structures of the lower energy complexes obtained at each site were examined, and a small set of compounds that showed no obvious detrimental reactivity, toxicity or solubility properties were purchased for cell culture toxicity and dengue antiviral activity assays.
  • LLC-MK2 rhesus monkey kidney epithelial cells were incubated 24 hours with each compound and cells examined visually for cytotoxic effects (cpe). This crude measure of cpe dosage was used to establish concentration ranges for MTT cell proliferation assays necessary to accurately and quantitatively measure cpe with high sensitivity. Briefly, 96-well plates were seeded with LLC-MK2 cells and treated with dilution series of compounds. Cells were incubated with compounds for fixed time (typically 24-72 hours), media removed, and cells treated with MTT for approximately three hours.
  • MTT substrate was then removed, the blue aqueous-insoluble product suspended in isopropyl alcohol, and samples read at 562 nanometers (nm) using an ELISA plate reader. Cytotoxic effect (cpe) was normalized against control wells treated with media and 1% DMSO. Assay conditions were optimized to establish a linear relationship between cell number and signal produced. Maximum tolerated dose (maximum concentration with no apparent cpe) and, where possible, cytotoxic concentration (CC50) were determined for each tested compound. MTT assays (Fig. 4) included ribavirin and myophenoic acid (MPA), and were highly reproducible (standard deviations approximately 7%). 5 Maximum tolerated doses measured by MTT assay were consistent with those estimated from visual inspection of cpe.
  • MPA ribavirin and myophenoic acid
  • DEN2V strain 16681
  • MOI multiplicity of infection
  • Immuno-foci staining plaque assay The quantity of infectious particles produced is one of the most important variables for predicting pathogenesis.
  • the titer of infectious DENV in cell media was quantitated as plaque forming units (pfu) per milliliter (ml) of cell culture.
  • An immunohistochemical (immuno-foci) staining method measured titers, since standard plaque methods are notoriously difficult, unreliable, and irreproducible for DENV due to its very low cpe in cell culture.
  • This immuno- foci assay followed standard plaque methods of infecting confluent monolayers of cells with serial dilutions of sample, then covering the cells with tragacanth gum approximately one hour after infected media was applied to the monolayer.
  • Antivirals referenced by EUDOC database identifiers ARDPOOI l and ARDP0012 are 1 ,3-Bis(4-nitrophenyl)urea and 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one 10,10-di oxide monohydrate, respectively (see Fig. 7). These low molecular weight compounds were readily obtained from Aldrich Chemical Co. Interestingly, nitrophenyl urea is one of two equimolar components of nicarbazin, which has been used for approximately fifty years as a starter feed supplement to prevent intestinal coccidiosis in broiler chickens.
  • NS3 protease binding sites are highly conserved among flaviviruses.
  • the antivirals ARDPOOl 1 and ARDPOO 12 were initially selected from an extensive computer-docking calculation that predicted these compounds would bind tightly to the DEN2V NS3 protease, with ARDPOOl 1 binding to the Pl site and ARDPOOl 2 binding to the protease catalytic site. Residues forming the predicted ARDPOOl 1 and ARDPOOl 2 binding sites are highly conserved among the major clades of the flavivirus family, indicating that these lead antivirals and derivatives may display antiviral activity for a broad spectrum of flaviviruses. Lead activity in West Nile virus replicon assay.
  • ARDPOOl 1 has antiviral activity against two flavivirus clades.
  • ARDPOOl 2 will be tested in a similar fashion.
  • This assay has demonstrated utility in identifying compounds that interfere directly with viral genome replication (such as polymerase or protease inhibitors), and should be particularly useful for this project.
  • a similar replicon for Japanese encephalitis virus has been developed and a DENV replicon will be produced.
  • Cell-based dengue ELISA assay A cell-based ELISA assay to detect viral proteins in infected cells is well-suited for high-throughput screening and can be performed more rapidly than virus yield assays. This assay also minimizes exposure to infectious agent, an important consideration when screening large numbers of compounds. Briefly, cell monolayers will be cultured in 96-well plates, and incubated with low-serum media containing antiviral candidates at concentrations necessary to establish dose-response curves. DENV will be added to the wells, and the plates allowed to incubate for desired time periods, after which the monolayers will be fixed with acetone: methanol.
  • Cell monolayer-associated viral antigens will be detected using antibodies available for arboviruses, and quantitated by standard ELISA protocols. Duplicate plates incubated with antiviral compounds alone will be used for MTT assays to simultaneously quantitate cell viability and cytotoxic concentration (CC50). EC50 and selectivity indices (CC50/EC50) will be calculated for each antiviral. Studies have shown that this ELISA assay can readily detect anti-DENV activity. Promising antivirals will be tested in several cell lines to ensure that cell-specific effects are not observed,, as has been reported for ribavirin. Cell-based West Nile and YF flavivirus ELISA Assays.
  • a West Nile virus replicon based assay has been developed that is well-suited high-throughput screening of antiviral compounds.
  • the assay requires only forty-eight hours of contact time with the test compounds, permitting the identification of lead compounds that could cause cytotoxic effects during longer-term incubations required for visual evaluation of cpe or other types of in vitro assays used for screening libraries of antiviral compounds.
  • the assay also eliminates essentially all biosafety concerns since no infectious virus is needed. In the context of this project, this assay provides an additional test for specificity of action of the antiviral agents. Studies shown that the anti-DENV NS3 therapeutics that are targeted for development in this example have anti-replicon activity and this assay will provide useful information as new antiviral candidate compounds are synthesized.
  • ns2B/ns3 sequences will be amplified by PCR from DEN2V (strain 16681) cDNA . Cloning, expression, and purification will follow routine procedures. Protease activity will be evaluated spectrophotometrically using peptide substrates appended to p- nitroanilide chromophores; substrates will be produced by routine solid phase synthesis. Inhibition assays will be performed in 96-well plate format, and Ki values calculated using Graphpad Prism software (Hays and Watowich, J. Biol. Chem. 278:27456-27463, 2003).
  • Cell-based NS3 protease activity assay Inhibition of DEN2V NS3 protease in cell culture will be monitored by examining release of secreted alkaline phosphatase protein (SEAP) from cells stably expressing the DEN2V NS2B/NS3/NS4A/SEAP polyprotein. Analogous cell-based assays have been constructed to successfully monitor HCV protease activity (Cho et al., J Virol Methods 72:109-15, 1998; Kakiuchi et al., Comb Chem High Throughput Screen. 6:155-60, 2003).
  • SEAP secreted alkaline phosphatase protein
  • ns2b/ns3/ns4a sequences will be amplified by PCR from DEN2V (strain 16681) cDNA, and ligated in-frame to the 5' end of seap gene.
  • the ns2b/ns3/ns4a/seap gene will be subcloned into the retrovirus shuttle vector pFB.
  • the retroviral shuttle vector, and plasmids pGag-Pol and pVSV-env will be cotransfected into 293 cells, and recombinant retrovirus recovered after approximately 48 hours.
  • Retroviruses encoding the protease reporter gene ns2b/ns3/ns4a/seap will efficiently (100%) infect mammalian cells, allowing the dengue protease reporter gene to stably integrate into the cell genome and be expressed by a strong CMV promoter.
  • Dengue NS3 protease will cleave after the NS4A cleavage site, and protease activity will be monitored by SEAP activity released into culture media.
  • Antivirals will be incubated with cells expressing the protease reporter, and reduction in SEAP levels will indicate protease inhibition.
  • cells expressing SEAP under the control of the CMV promoter will be tested to ensure that antivirals are not inhibiting SEAP transcription, translation, or activity.
  • PK pharmacokinetics
  • bioavailability assays Small animal toxicity, pharmacokinetics (PK) and bioavailability assays.
  • the development of dengue antivirals will follow a tier approach with a Go/No Go decision tree to ensure effective use of funds.
  • the tier approach allows a project team (composed of scientists from pharmacology, toxicology, pharmacokinetics and medicinal chemistry) to critically review the data collected, and increase the chances of developing successful antivirals.
  • This approach can avoid future development challenges as compounds are ranked in the early stages of the project based on efficacy, pharmacokinetic profile, bioavailability, toxicology and chemistry.
  • Tier 1 will be the small scale-up and characterization of approximately 10-20 compounds. The feasibility of the future manufacture of these compounds will be assessed by chemistry manufacturing and controls experts. Scale up will allow for PK studies to be conducted. Approximately one gram of base of each compound will be made. Evaluation of synthetic chemistry pathways indicate that it will not be problematic to produce this amount of pure (greater than 98%) compound.
  • Tier 2 will be the screening of approximately 10-20 antivirals that have been selected based on cell culture efficacy and toxicity models. These compounds will be evaluated in pharmacokinetic studies in the Sprague-Dawley rat to determine bioavailability and PK parameters. Compounds will be ranked based on PK parameters and bioavailability.
  • High-pressure liquid chromatographic, mass spectrometry and other equipment will be used to confirm the identity of parent compounds and metabolites, and analyze sera for any of the lead compounds that will be examined in animals, including, for example, in the rat. Since the route of administration in humans is likely to be oral, the bioavailability of these compounds is important and the following studies, among others, may be conducted: a determination of oral PK with three doses in the rat; and a determination of intravenous IV PK with three doses in the rat. Compounds with the acceptable PKs will be considered for Tier 3. Tier 3 will be the screening of antivirals in key toxicology studies, allowing for the ranking of the compounds so that optimal antiviral candidates can be selected.
  • a maximal tolerated dose (MTD) oral toxicity study in the Sprague-Dawley rat in vitro binding/ inhibition of cytochrome P-450 protein (since many drug-drug interactions are correlated with the inhibition and/or induction of cytochrome P450 enzyme activity); in vitro antimicrobial and pharmacological screens; and reverse bacterial mutation assay.
  • MTD maximal tolerated dose
  • cytochrome P-450 protein since many drug-drug interactions are correlated with the inhibition and/or induction of cytochrome P450 enzyme activity
  • in vitro antimicrobial and pharmacological screens in vitro antimicrobial and pharmacological screens
  • reverse bacterial mutation assay reverse bacterial mutation assay.
  • mice Animal challenge models. Both nonhuman primates and human-cell complemented SCID mice may be used to evaluate viremia. Industry-standard procedures for cell-based assays will be used to establish antiviral efficacy and animal models to establish compound toxicity, pharmacokinetics, and bioavailability. Small animal challenge experiments may be undertaken using either a mouse viremia or SCID mouse model. In addition, West Nile virus challenge experiments in mouse models may be undertaken to test the in vivo efficacy of broad-spectrum antivirals.
  • Measurements of antiviral activity in cell culture were determined using independent whole cell ELISA (enzyme-linked immunosorbent assay) and foci-forming antiviral assays. Measurements of cytotoxicity were measured using a standard MTT [3-(4,5- Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] toxicity assay (Mosmann, Journal of Immunological Methods 65, 55-63, 1983).
  • DEN2V proteins were monitored using mouse anti-DEN2V primary antibody, visualized by horse radish peroxidase (HRP)-conjugated anti-mouse secondary antibody, and quantitated using standard reaction protocols on an ELISA 96-well plate reader. This assay demonstrated an excellent linear response, with an R2 correlation between absorbance and number of infected cells of approximately 0.94. Graphpad Prism was used for plotting, statistical analysis, and calculation of EC50 values.
  • HRP horse radish peroxidase
  • the dose-response curve for ARDPOOl 1 was representative of the data quality obtained for ribavirin, MPA, ARDPOOIl, and ARDP0012. Dose-response curves showed several notable features, among them high reproducibility, low background noise, clear plateaus at low and high compound concentrations, and excellent goodness-of-fit statistics between the data points and fit curve.
  • Table 2 summarizes cell culture potency (EC50), cytotoxicity (CC50), and selectivity index (SI) values of tested lead compounds and controls against dengue 2 virus challenge.
  • EC50 values determined using a dengue whole cell ELISA for ribavirin and mycophenoic acid controls gave results that were in excellent agreement with literature values (Koff et al., Antimicrob Agents Chemother. 24:134-6, 1983; Diamond et al., Virology 304:211-21, 2002). Differences between the values observed and literature values could be due to different virus strains and cultured cells. Moreover, EC50 and SI values calculated in the two independent virus replication assays (dengue whole cell ELISA and foci-forming infectivity assays) showed excellent agreement in the measured rank-order of compound efficacy.
  • the foci-forming assay measured production of infectious virus, and was expected to be more difficult to decrease when significant polyprotein accumulated and thus infectious dengue virus production was the rate-limiting step. Typically, though, there was agreement between dengue protein production (whole-cell ELISA) and infectious virus production (foci-forming assay).
  • ARDPOOl 1 had the same order of magnitude effectiveness as mycophenoic acid.
  • ARDPOO 12 had an EC50 an order of magnitude lower the mycophenolic acid, making it the most potent small molecule antiviral for dengue virus reported.
  • CC50 values for ARDPOOl 1 and ARDPOOl 2 were greater than 100 micromolar (uM) (see Table 2) for experiments carried out from one to four days.
  • ARDPOOl 1 and ARDPOO 12 had very large selectivity indices and are excellent candidates for further optimization, pharmacokinetic studies, and animal safety studies.
  • Reduction of infectious DEN2V monitored by foci-forming assay The quantity of infectious particles produced is one of the most important variables for predicting pathogenesis.
  • Cultured LLC-MK2 cells were challenged with DEN2V and increasing log concentrations of potential antiviral compound. Cells were washed, and twenty-four hours post-infection the titer of infectious DEN2V in the cell supernatant was measured as foci-forming units (ffu) per ml of cell culture.
  • An immunohistochemical (immuno-foci) staining method was developed to measure virus titers, since standard plaque methods are notoriously difficult, unreliable, and irreproducible for DENV due to its very low cpe in cell culture.
  • This assay was time-consuming and labor-intensive, but provided crucial independent confirmation of the potency of the lead compounds and validation of the dengue antiviral whole-cell ELISA. Briefly, the foci-forming assay followed standard plaque methods and probing the monolayer with mouse dengue 2 virus primary antibody. Foci were visualized by staining HRP-conjugated anti-mouse secondary antibody with the VIP kit (Vector Labs). Foci were easy to observe by eye, serial dilutions gave the expected foci reductions, and replicate wells gave reproducible foci counts.
  • the twenty-four hour post-infection dose-response curve for ARDPOO 12 (Fig. 9) was representative of the data quality obtained in the antiviral foci- forming assay. This dose-response curve showed high reproducibility, low background noise, clear plateaus at low and high compound concentrations, and excellent goodness-of-fit statistics to the data points.
  • Table 2 summarizes cell culture potency (EC50) and selectivity index (SI) values obtained for the two lead compounds and controls against DEN2V with this assay.
  • ARDP0012 had low nanomolar EC50 values and SI values greater than 104.
  • ARDPOOl 1, MPA, and ribavirin had higher EC50 values in the foci-forming assay relative to ELISA, indicating these antiviral compounds were less effective at reducing virus production relative to their ability to reduce viral protein production.
  • rank-orders based on EC50 and SI for these two independent virus replication assays showed excellent agreement. Lead compounds had minimal cytotoxic effects. The cytotoxicity of
  • EUDOC-suggested lead antiviral compounds ARDPOOl 1 and ARDPOO 12 was determined by MTT cell proliferation assays (Mosmann, Journal of Immunological Methods 65:55-63, 1983). This widely used assay reproducibly and quantitatively measured cytotoxic effects in cultured cells. Briefly, 96-well plates were seeded with LLC-MK2 cells and incubated with serial dilutions of ribavirin and MPA controls, ARDPOOl 1 and ARDP0012. After fixed time (typically 24-96 hours), cells were washed and treated with MTT, and absorbance changes read on an ELISA plate reader.
  • CC50 values were determined from dose-response curves fit to replicate data points using GraphPad Prism. As reported in Table 2, CC50 values in LLC-MK2 cells treated for twenty-four hours with ARDPOOl 1 and ARDPOO 12 were 164 and 213 uM, respectively. Similar CC50 values were obtained for human hepatoma cell line Huh-7 and for cells treated 24, 48, or 96 hours. Thus, both ARDPOOl 1 and ARDP0012 had very low cytotoxicity in cultured cells, and are thus excellent candidates for pharmacokinetic and animal safety trails. The high CC50 and SI values of the lead computer-predicted antivirals indicate they are specific inhibitors, which do not interact strongly (if at all) with host cell proteases necessary for cell survival.
  • Flavivirus NS3 protease binding sites are conserved.
  • the antivirals ARDPOOl 1 and ARDPOO 12 were initially selected from an extensive computer- docking calculation that predicted these compounds would bind tightly to the DEN2V NS3 protease, with ARDPOOl 1 and ARDPOOl 2 binding to the Pl and catalytic sites, respectively. These predictions will be further tested, and the mechanism of antiviral action conclusively shown to be NS3 inhibition. Residues surrounding the predicted ARDPOOl 1 and ARDPOO 12 binding sites are highly conserved among the major clades of the flavivirus family (Table 3), suggesting that these antivirals (or more potent derivatives) may display antiviral activity for a broad spectrum of flaviviruses.
  • ARDPOOl 1 was observed to have micromolar antiviral activity when tested in West Nile virus (WNV) and hepatitis C virus (HCV) subgenomic replicon cell culture assays.
  • WNV West
  • ARDP0011 98% 88% 83% 81 % 69% 70% 64% 52°/ ARDP0012 100% 91% 89% 86% 69% 66% 74% 69°/
  • Antivirals referenced by EUDOC database identifiers ARDPOOI l and ARDP0012 are l,3-Bis(4-nitrophenyl)urea and 3-(lH-tetrazol-5-yl)-9H-thio-xanthen-9-one 10,10-dioxide monohydrate, respectively ( Figure 7). These compounds were readily obtained from Aldrich Chemical Co. Nitrophenyl urea (ARDPOOl 1) is one of two equimolar components of nicarbazin, a starter feed supplement used for approximately fifty years to prevent intestinal coccidiosis in broiler chickens.
  • ARDPOOl 1 and ARDPOOl 2 conform to Lipinski's rules and have molecular weights of approximately 300 Daltons, which are consistent with the mean molecular weight of marketed oral drugs (Wenlock et al., J. Med. Chem. 46: 1250-56, 2003), indicating that these compounds have excellent physiochemical properties for development as drugs.
  • Lead antivirals are not non-specific serine protease inhibitors.
  • the low cytotoxicity of ARDPOOI l and ARDPOO 12 strongly indicated that these compounds are not broad-spectrum inhibitors of host cell proteases required for cell survival and proliferation. However, it is possible that these compounds may have some activity against serine proteases not found in mammalian cells.
  • a standard trypsin inhibition assay (Worthington Enzyme manual) was performed to determine if high concentrations of ARDPOOl 1 or ARDPOO 12 interfered with trypsin activity (Fig. 10). Briefly, purified trypsin (Pierce) was incubated with N-benzoyl-D,L-arginine-4-nitroanilide HCL (Sigma), and progress of the reaction was followed by observing the absorbance of liberated p-nitroaniline at 405 nm (Fig. 10).
  • Benzamidine used as a standard trypsin inhibitor control, clearly suppressed the rate of reaction, whereas ARDPOOl 1 (24 uM) and ARDPOO 12 (670 uM) had no effect on the reaction within the error of the experiment (Fig. 10).
  • Calculation of kinetic parameters such as k cat , K m , and Ki; Hays and Watowich, J. Biol. Chem. 278, 27456-
  • the compound l,3-Bis(4-nitrophenyl)urea (Fig. 11) will be used as one of the initial leads around which libraries will be built.
  • the initial library will contain sets of compounds that represent different types of ureas.
  • Symmetrical bisaromatic ureas possessing groups with properties similar to nitro group (NO 2 ), will be synthesized. These ureas will have benzene rings substituted with NO 2 in the ortho and meta positions as well as sulfate (SO 3 " ), acetate (OAc) and nitrile (CN) in the ortho, meta, and para positions.
  • SO 3 " sulfate
  • OAc acetate
  • CN nitrile
  • Both OAc and SO 3 " are electron-withdrawing groups like NO 2 and consequently will provided aromatic rings with electron densities similar to the lead compound.
  • CN is electron withdrawing it possess very different steric and hydrogen bonding profiles.
  • Other members of the library will contain symmetrical heteroaromatic groups such as imidazole, pyridine, thiazole and triazoles ureas. These members will possess a shape and p-cloud similar to simpler aromatics while presenting additional hydrogen bonding options.
  • two types of unsymmetrical ureas will be present in the initial library. These will be ureas with two different aromatic groups and ureas with one aromatic group and one aliphaticgroup.
  • the unsymmetrical bisaromatic ureas will contain combinations of the aromatic groups discussed above.
  • the aromatic/aliphatic ureas will use the aromatic groups with both acyclic and cyclic aliphatic groups.
  • the symmetrical ureas can be obtained by the addition of two equivalents of an amine to a solution of phosgene or carbonyldiimidazole.
  • the asymmetrical ureas are accessible by stepwise addition of one amine to carbonyl diamidazole followed by addition of the second amine.
  • the synthesis can be carried out in solution or on solid support. As initial efforts will be to synthesize only a few hundred-member libraries, most of the initial work will be done in solution. Several dozen members of the initial
  • ARDPOOl 1 -based library have been synthesized and are available for testing. At least about 200 additional derivatives will be systematically synthesized and tested.
  • Derivatives will be synthesized by conversion of the bromide to a variety of different groups using palladium catalyzed coupling reactions (Brase et al., Tetrahedron 59:885-939, 2003). One may be concerned about attempting to do this chemistry on a molecule containing sulfur. However, Feringa has shown that thioxanthones of this type readily undergo the palladium catalyzed Suzuki reaction (Schoevaars et al., J. Org. Chem. 62:4943-4948, 1997).
  • the lead antivirals will serve as templates for focused combinatorial chemical libraries. Compound activity and toxicity will be evaluated in cell culture, and drug-like properties (including, for example, toxicity, bioavailability, and pharmacokinetics) evaluated in animal models. Iterative cycles of synthesis, testing, and quantitative structure-activity relationship (QSAR) analysis will rapidly improve compound potency and drug-like properties, allowing us to select optimal antivirals for subsequent IND-enabling studies.
  • QSAR quantitative structure-activity relationship
  • the lead antivirals will be used as templates for focused synthetic combinatorial chemical libraries and to aggressively evaluate compound activity and toxicity in cell culture, and drag-like properties in animal models. Since poor drug-like properties (such as, for example, bioavailability, pharmacokinetics, metabolism, and toxicity) are a primary reason drag leads fail to progress beyond preclinical trials, the Accelerated Preclinical Optimization Project outlined below emphasizes simultaneously optimizing drag properties and compound potency. These metrics will guide quantitative structure-activity relationship (QSAR) analysis to improve subsequent chemical libraries.
  • QSAR quantitative structure-activity relationship
  • the Preclinical Optimization Project is an integrated element of a comprehensive Product Development Plan to produce antiviral drags to combat DENV following bioterrorist incidents or natural epidemics.
  • This example will optimize novel dengue antivirals and generate optimized antivirals for IND-enabling studies. To reduce the chance that an antiviral compound might fail in clinical trials, this example will systematically evaluate drug-like properties as part of the lead optimization process. In addition, compound stability, non-specific binding, and mechanism of action will be evaluated.
  • Category B-C flaviviruses will be used to establish a combined ordinal ranking (for example, the Kruskal-Wallis test) of compound potency, with only compounds with acceptable selectivity indexes retained.
  • a final filtering step, designed to select optimal compounds for expensive IND-enabling studies, will involve detailed review of broad-spectrum antiviral activity, cytochrome P450 interactions, and animal toxicology, pharmacokinetic, and bioavailability data. Approximately equal numbers of compounds derived from separate classes of antiviral leads will be retained at each filter point. This will maximize compound diversity and likely enable optimized antiviral drugs to be jointly administered to synergistically target independent protease inhibitor sites. Binding of antivirals to independent protease sites will be confirmed with in vitro competition assays. As observed for HIV, inhibiting multiple viral sites can significantly delay development of resistant strains.
  • DENV selectivity indices (SI, defined as CC5O/EC5O) will be determined for each compound within the focused chemical libraries.
  • SI defined as CC5O/EC5O
  • the two lead compounds had highly encouraging SI values in cell culture against DEN2V, West Nile virus, and HCV, and are predicted to bind to distinct areas within the NS3 protease cleavage site.
  • Several hundred derivatives of each lead compound will be synthesized and their ability to inhibit DENV replication tested.
  • EC50 values will be calculated for each compound.
  • a cell- based MTT assay to determine CC50 values for each tested compound.
  • SI values will be calculated for each derivative and compared to the lead compound to discover more potent antivirals.
  • West Nile virus and Yellow fever virus selectivity indices will be determined for derivatives within each focused chemical library. Compounds from each chemical library that inhibit DEN2V replication will be subsequently tested for their ability to inhibit West Nile virus and yellow fever virus replication. Virus-specific SI values will be calculated for each derivative and compared to the lead compound to discover more potent broad-spectrum antivirals.
  • the drug-like properties of broad-spectrum antivirals will be determined within each focused chemical library. Poor drug-like properties (such as, for example, bioavailability, pharmacokinetics, metabolism, and toxicity) are the primary reason drug leads fail to progress beyond preclinical trials, thus this example stresses optimizing drug-like properties in addition to compound potency. Promising antivirals will be evaluated for solubility, cytochrome P450 inhibition, specificity, toxicity, pharmacokinetic properties, and bioavailability in animals. This information will guide development of improved antivirals for final IND-enabling studies.
  • Derivative 1 (PLORJ)OO l_044_l) has an EC 50 of approximately 0.8 uM and a SI of greater than 56.
  • Derivative 2 (PLORJ)OO l_042_l) has an EC 50 of approximately 3 uM and a SI of greater than 33. EC 50 and SI were determined as described in Examples 1 and 2. Derivative 1 and Derivative 2 are shown in Fig. 13.
  • ureas possessing groups with properties similar to nitrite group (NO 2 ) were synthesized. These ureas have benzene rings substituted with NO2 in the ortho and meta positions as well as sulfate (SO 3 " ), acetate (OAc) and nitrile (CN) in the ortho, meta and para positions.
  • Both OAc and SO 3 " are electron-withdrawing groups like NO 2 and consequently provide aromatic rings with electron densities similar to the lead compound. Additionally, their hydrogen bonding properties are similar to NO2.
  • Other members of the library contain symmetrical heteroaromatic groups such as imidazole, pyridine, thiazole and triazoles ureas. These members possess a shape and I ⁇ -cloud similar to simpler aromatics while presenting additional hydrogen bonding options. All the necessary ureas were synthesized from commercially available amines. There are more than 200 amines available from Aldrich alone. This was done using either phosgene or carbonyldiamidazole (Nieuwenhuijzen et al., Tetrahedron Lett.
  • a series of novel tricyclic thioxanth-9-one-10,10-di oxide derivatives have been prepared for subsequent evaluation as anti-viral agents.
  • a regioselective synthesis of the novel core substrate 3-chlorothioxanth-9-one- 10,10-dioxide was achieved in 85% yield over three steps without the need for chromatographic purification.
  • Subsequent microwave-assisted coupling methodology afforded the desired novel 3-substituted tricyclic compounds.
  • Reported syntheses for similar 3-halo-thioxanthenone ring systems utilize as first step the reaction of the corresponding m-halo-benzenethiol substrate 2 with 2-halo-benzoic acid 3, which, upon treatment with mineral acid, afford the desired Friedel-Crafts product 3-halo-thioxanthenone ring system 4 (Scheme 2) (Rokach et aL, EU Patent Appl. 0 000 978 Al, 1979; Hodson et ah, ' US Patent 4,103,015, 1978; Miller et aL, US Patent 5,346,917, 1994; Mahishi et aL, J.
  • Oxidation of 8 to the desired key sulfone substrate 9 required fine tuning of the existing oxidative protocols reported for similar deactivated aromatic ring systems (Table 4) (Balicki et al., Prakt. Chem., 1999, 341, 184-18; Su,
  • Microwave assisted reactions were performed on a CEM Discover Reactor in 10 mL reaction tubes.
  • Copper powder and Pd(PPh 3 ) 4 were purchase from Aldrich.
  • the title compound was synthesized employing the general reaction and work-up methodology described for the synthesis of 10 (Table 6) using compound 8 (100.0 mg; 0.359 mmol), 4-carboxyphenylboronic acid (71.4 mg; 0.43 mmol), tetrakis (triphenylphosphine) palladium (12.4 mg; 0.01 mmol; 3.0 mol %) and Cs 2 CO 3 (431.0 ⁇ l; 0.431 mmol; 1.0 M solution). After the usual aqueous work-up the crude yellow solid was triturated from DIPE to yield the pure title compound (80.0 mg; 61%) as a beige solid.

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Abstract

La présente invention a trait à des composés présentant une activité contre une variété de flavivirus, à leurs utilisations, et à des procédés pour l'identification de tels composés.
PCT/US2005/043938 2004-12-02 2005-12-02 Inhibiteurs de la replication de flavivirus et leurs utilisations WO2006060774A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2443892A (en) * 2007-01-13 2008-05-21 Shahnaz Perveen A process for the preparation of "Gaschem " (N-4- nitrophenyl-N'-4'-nitrophenylurea), which is a urease and a chymotrypsin enzyme inhibitory drug
EP1994491A4 (fr) * 2006-03-16 2010-11-17 Univ Oregon Health & Science Inhibition des flavivirus par des sultames et composes apparentes
EP2308514A2 (fr) 2007-03-23 2011-04-13 to-BBB Holding B.V. Conjugées pour le transport des médicaments à travers la barrière hémato-encéphalique
JP2016504274A (ja) * 2012-11-08 2016-02-12 中国科学院理化技術研究所 チオキサントンオキシド系誘導物、製造方法及びその応用
CN113429238A (zh) * 2021-07-23 2021-09-24 甘肃省农业科学院旱地农业研究所 一种有机肥及其制备方法

Non-Patent Citations (2)

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Title
DATABASE CAPLUS [Online] COATES ET AL.: 'New Therapeutic agents of the quinoline series. Introduction and II. Methoxy-, hydroxy-, and alkylpyridylquinolines', XP003009984 Database accession no. (1944:520) & J. CHEM. SOCIETY 1943, pages 406 - 413 *
DATABASE CAPLUS [Online] COOK A.H. ET AL.: 'New Therapeutic agents of the quinoline series, Introduction and IV. Lutidylquinolines', XP003009983 Database accession no. (1944:521) & J. CHEM. SOCIETY 1943, pages 413 - 417 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1994491A4 (fr) * 2006-03-16 2010-11-17 Univ Oregon Health & Science Inhibition des flavivirus par des sultames et composes apparentes
US8003674B2 (en) 2006-03-16 2011-08-23 Oregon Health & Science University Flavivirus inhibition by sultams and related compounds
GB2443892A (en) * 2007-01-13 2008-05-21 Shahnaz Perveen A process for the preparation of "Gaschem " (N-4- nitrophenyl-N'-4'-nitrophenylurea), which is a urease and a chymotrypsin enzyme inhibitory drug
GB2443892B (en) * 2007-01-13 2010-11-24 Shahnaz Perveen "Gaschem " a urease and alpha-chymotrypsin enzyme inhibito ry drug
EP2308514A2 (fr) 2007-03-23 2011-04-13 to-BBB Holding B.V. Conjugées pour le transport des médicaments à travers la barrière hémato-encéphalique
JP2016504274A (ja) * 2012-11-08 2016-02-12 中国科学院理化技術研究所 チオキサントンオキシド系誘導物、製造方法及びその応用
KR101807575B1 (ko) * 2012-11-08 2018-01-18 테크니컬 인스티튜트 오브 피직스 앤 케이스트리 오브 더 차이니스 아카데미 오브 사이언시스 티옥산톤 옥사이드계 유도체, 제조 방법 및 그 적용
CN113429238A (zh) * 2021-07-23 2021-09-24 甘肃省农业科学院旱地农业研究所 一种有机肥及其制备方法

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