WO2013029006A1

WO2013029006A1 - Dengue virus and yellow fever virus therapies

Info

Publication number: WO2013029006A1
Application number: PCT/US2012/052344
Authority: WO
Inventors: Thomas E. Shenk; Emre Koyuncu; Lillian CHIANG
Original assignee: The Trustees Of Princeton University
Priority date: 2011-08-25
Filing date: 2012-08-24
Publication date: 2013-02-28

Abstract

The present invention provides methods and compositions for treating dengue virus or yellow fever virus infections using modulators of host cell enzymes. The invention also provides methods and compositions combining such modulators of host cell enzymes with direct-acting antiviral agents.

Description

DENGUE VIRUS AND YELLOW FEVER VIRUS THERAPIES

FIELD OF THE INVENTION

[0001] This application relates to antiviral therapies for treatment of dengue virus and yellow fever virus infection.

BACKGROUND OF THE INVENTION

[0002] There is a great unmet medical need for agents that safely, effectively, and reliably treat mosquito-bourne flavivirus infections, particularly hemorrhagic diseases.

Dengue virus infections affect more than 50 million individuals per year. Dengue fever symptoms include fever, headache, muscle and joint pains, and a skin rash. In a small proportion of cases the disease develops into demgue hemorrhagic fever, resulting in bleeding, low platelet levels, and blood plasma leakage, or into dengue shock syndrome. Further, the virus has four different types. Infection with one type results in immunity to that type, but not necessarily to any other type. Currently, there is no dengue virus vaccine. Thus prevention has depended on control of the mosquito that transmits it. Treatment of acute dengue fever is supportive, using either oral or intravenous rehydration for mild cases and intravenous fluids and blood transfusion for severe cases.

[0003] Yellow fever virus is also spread by mosquitos. Once bitten, symptoms usually develop 3 to 6 days later. Yellow fever symptoms, in most cases, include fever, nausea, and pain that subsides after several days. Most people recover at this stage, but in some patients (15%), this is followed by a second "toxic phase." This stage is usually accompanied by multi-organ dysfunction, including heart, liver, and/or kidney damage. There may be bleeding disorders, hemorrhage, and brain dysfunction. This phase is fatal in 20% of cases. An effective vaccine exists, but yellow fever virus causes 200,000 illnesses and 30,000 deaths in unvaccinated populations.

[0004] In lab treatments of animal models, early administration of ribavarin improved survival rates and reduced tissue damage. However, treatment in primates using ribavarin or other antiviral drugs, including interferons, generally does not have a positive effect.

Notably, pretreatment of human cells in vitro with intereferons protects against dengue virus infection, but treatment after infection does not. The absense of an interferon response, in vitro and in vivo has been attributed to blockage of the IFN-induced signal-transduction cascade by one or more viral components (NS2A in dengue virus infection). SUMMARY OF THE INVENTION

[0005] The invention provides novel methods and compositions for treatment or prevention of dengue virus infection or yellow fever virus infection by administration of agents that target host components. The invention also provides methods and compositions combining such modulators of host cell enzymes with other antiviral agents, such as direct- acting antiviral agents.

[0006] The present invention provides a method of treating or preventing dengue virus infection or yellow fever virus infection in a mammal, comprising administering to a mammalian subject in need thereof a therapeutically effective amount of a compound, or pharmaceutically acceptable salt of said compound, wherein the compound is a compound of Formula I:

wherein R¹ and R² each independently represents H, a C₃-C6 cycloalkyl group, an aryl group, a heteroaryl group or a C1-C6 alkyl group optionally substituted by a substituent selected from a halogen, a haloalkyl, a Ci-C₆ alkyl group, a Ci-C₆ alkoxy group, a Ci-C₆ hydroxy group, a C₃-C6 cycloalkyl group, an aryl group and a heteroaryl group;

wherein R³ and R⁴ each independently represents a C1-C6 alkyl group, a C3-C6 cycloalkyl group, an aryl group or an aralkyl group, or R³ and R⁴ may form a C₃-C6 cycloalkylidene group together with the carbon atom R³ and R⁴ are attached to;

wherein R⁶ represents a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C₃-C6 cycloalkyl group or a C1-C6 alkoxy-Ci-C6 alkyl group; wherein the above-described C₃-C6 cycloalkyl group, C3-C6 cycloalkylidene group, aryl group, alkyl aryl group and heteroaryl group may be each independently substituted by a substituent selected from a halogen atom, a C1-C6 alkyl group, a C1-C6 haloalkyl group, C1-C6 hydroxy group and a C1-C6 alkoxy group;

R⁵ represents a hydrogen atom, a lower cycloalkyl group, an aryl group, a heteroaryl group or a C1-C6 alkyl group optionally substituted by a substituent selected from a lower cycloalkyl group, an aryl group and a heteroaryl group, wherein the lower cycloalkyl group, aryl group and heteroaryl group in R₅ each independently represents an unsubstituted group or a lower cycloalkyl group, an aryl group or a heteroaryl group substituted by one or two substituents selected from a halogen atom, a nitro group, a nitrile group, a Ci-C₆ alkyl group, a Ci-C₆ haloalkyl group, a Ci-C₆ alkoxy group, Ci-C₆ hydroxy group, a C₁-C6 haloalkoxy group, a C₁-C6 carboxylic acid group, a C₃-C8 cycloalkyl group, an aryl group, a heteroaryl group, an aryl ether group, an aralkyl group, -CON(R⁷)R⁸, -N(R⁷)R⁸, -N(R⁷)COR⁸, -N(R⁷)S0₂R⁸, -OCOR⁷, -OCON(R⁷)R⁸, -SR⁷, -SO₂R⁷, -S0₂N(R⁷)R⁸ and and a group with the formula:

wherein R⁷ and R⁸ each independently represents a hydrogen atom or a Ci-C₆ alkyl group; and

X represents -N(R⁷) - or -0-.

[0007] In an embodiment of the invention the compound of Formula I is Formula la:

wherein, R¹ is selected from H, unsubstituted benzene, substituted benzene having

substitutents such as F, CH₃, CH₂CH₃, CI, OCH₃, OCF₃, and CF₃; Ci_6 alkanes (such as methyl, ethyl, /-propyl, and n-propyl); and C₃_6 cycloalkanes (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl); wherein R₃ and R₄ are independently selected from H; Ci_3 alkane; and benzene group; or R³ and R⁴ taken together form a cycloalkyl of formula -(CH₂)_n- where n = 2, 3, 4 and 5; wherein R⁵ is selected from methyl; CF₃;

cyclopropyl; unsubstituted benzene; mono- and disubsituted benzene having substitutents such as F, CH₃, CH₂CH₃, CN, CH₃CHCH₃, CI, OCH₃, OC₆H₅, OCF₃, and CF₃;

unsubstituted heteroaryl groups (such as 2-, 3-, or 4-pyridine, isoxazole, pyrazole, triazole); and imidazole. [0008] In certain embodiments of the invention, in a compound of Formula I_a, R¹ is phenyl, R³ and R⁴ are methyl, and R⁵ is

[0009] In certain embodiment of the invention, the compound of Formula I is

[0010] In another embodiment, the invention provides a method of treating or preventing dengue virus infection or yellow fever virus infection in a mammal, comprising administering to a mammalian subject in need thereof a therapeutically effective amount of a compound, or pharmaceutically acceptable salt of said compound, wherein the compound is a compound of Formula (II).

wherein R¹ represents optionally halogenated C_1-6 alkyl or optionally halogenated C3-:

cycloalkyl

R² represents a substituent selected from:

wherein W represents Ci_6 alkylene, C2-6 alkenylene, C2-6 alkynylene or C3-6 cycloalkylene, wherein the alkylene, alkenylene, alkynylene or cycloalkylene may be substituted by optionally halogenated C1-3 alkyl, optionally halogenated Ci_ alkoxy, alcohol or halogen; and

R represents Ci_6 alkyl, C₃_s cycloalkyl, aryl or heteroaryl, wherein the Ci_6 alkyl, C3_s

cycloalkyl, aryl or heteroaryl may be substituted by halogen, optionally halogenated Ci_6 alkyl, optionally halogenated Ci_6 alkoxy, Ci_6 alkylsulfonyl, nitrile, benzene, Ci_6 sulfide, Ci_6 thiol, Ci_6 alcohol, amino, Ci_6 alkylamino, di-Ci_e alkylamino, alcohol-Ci_6 alkyl, amino-Ci_6 alkyl, Ci_6 alkylamino- Ci_e alkyl, di-Ci-6 alkylaminoalkyl, Ci_6 alkoxy-Ci_6 alkyl, Ci_6 alkylcarbonyl, Ci_6 alkylamide, Ci_6 alkylcarbamoyl, C3_8 cycloalkylcarbamoyl, Ci_6 alkylsulfonylamino or Ci_6 alkylaminosulfonyl;

wherein R³ represents a hydrogen atom, Ci_6 alkyl, aryl or heteroaryl;

X represents -0-, -C(R^4a)(R^4b) - or -NR⁵-;

R^4a, R^4b and R⁵ each independently represent hydrogen, Ci_6 alkyl, C₃-8 cycloalkyl, aryl or heteroaryl, wherein the Ci_6 alkyl, C₃-8 cycloalkyl, aryl or heteroaryl may be substituted by halogen, optionally halogenated Ci_6 alkyl, optionally halogenated Ci_6 alkoxy, Ci_6 alkylsulfonyl, nitrile, aryl or heteroaryl;

Yi represents -CR⁶- or -N-;

Y2 represents -CR⁷- or -N-;

Y3 represents -CR⁸- or -N-;

Y4 represents -CR⁹- or -N-; and

R⁶, R⁷, R⁸ and R⁹ each independently represent a hydrogen atom, halogen, nitrile, Ci e alkyl, C3_8 cycloalkyl, Ci_6 alkoxy, Ci_6 hydroxy, alcohol, alcohol-Ci_6 alkyl, amino, Ci_6 alkylamino, di-Ci_6 alkylamino, amino-Ci_6 alkyl, Ci_6 alkylamino-Ci_6 alkyl, di-Ci_6 alkylamino-(Ci_6)alkyl, Ci e alkoxy-Ci-6 alkyl, heterocycle, aryl, heteroaryl, Ci_6 alkylsulfonyl, Ci_<5 alkylsulfmyl, Ci_6 alkylthio, arylsulfonyl, heteroaryl sulfonyl, aryl sulflnyl, heteroaryl sulfinyl, aryl thiol, heteroaryl thiol, C_1-6 alkylcarbonyl, aryl carbonyl, heteroaryl carbonyl, Ci_6 alkylcarbonylamino, aryl amide, heteroaryl amide, Ci_6 alkylcarbamoyl, C₃_g cycloalkylcarbamoyl, heterocycle carbamoyl, aryl carbamoyl, heteroarylcarbamoyl, Ci_6 alkylsulfonylamino, arylsulfonylamino,

heteroarylsulfonylamino, Ci_6 alkylsulfamoyl, arylsulfamoyl or heteroarylsulfamoyl, wherein the alkyl, cycloalkyl, heterocycle, aryl or heteroaryl may be substituted by a group selected from the group consisting of halogen, optionally halogenated Ci_₆ alkyl, optionally halogenated Ci_6 alkoxy, Ci_6 alkylsulfonyl, carbonyl and nitrile.

[0011] In an embodiment of the invention, the compound of Formula II has the formula II_a

wherein L is selected from carbamate, urea, or amide including, for example

wherein R is selected from halo; CF₃; cyclopropyl; optionally substituted Ci_s alkyl, wherein the Ci_5 alkyl may be substituted with halo, oxo, -OH, -CN, -NH₂, CO₂H, and Ci_3 alkoxy; wherein Ri is selected from substituted phenyl where the substiuents are selected from F, CF₃, CH₃, O CH₃, or isopropyl;

wherein R₂ is CI, Ph, l-(2-pyridone), 4-isoxazole, 3-pyrazole, 4-pyrazole, 1-pyrazole, 5- (1,2,4-triazole), l-(l,2,4-triazole), 2-imidazolo, l-(2-pyrrolidone), 3-(l ,3-oxazolidin-2- one).

[0012] The chiral center at C4 can be racemic, (S), (R), or any ratio of enantiomers. In one embodiment, L is an amide. In certain embodiments, R is selected from CI, CF₃, methyl, ethyl, isopropyl and, cyclopropyl. In certain embodiments R¹ is para-substitued wherein the substituent is selected from F, CF₃, CH₃, O CH₃, or isopropyl,

[0013] In one embodiment, the compound of formula li_b is

wherein R is selected from

[0014] In another embodiment, the elongase inhibitor is a compound of formula II_C

wherein R¹ is substituted at position 2, 3, or 4 with F, or Me, or R¹ is substituted at position 4 with MeO, or CF₃, R² is CI, H, Ph, 4-isoxazole, 4-pyrazole, 3-pyrazole, 1-pyrazole, 5-(l,2,4- triazole), l-(l,2,4-triazole), 2-imidazol, l-(2-pyrrolidone), or 3-(l,3-oxazolidin-2-one).

[0015] In one embodiment the compound of formula II is

[0016] The invention further provides a method of treating or preventing dengue virus infection or yellow fever virus infection in a mammal, comprising administering to a mammalian subject in need thereof a therapeutically effective amount of a compound, or pharmaceutically acceptable salt of said compound, wherein the compound is a compound of Formula (III),

wherein, Z is

where nl , n2, and n3 are 0, 1 , or 2, and wherein

R¹ represents Ci_₆ alkyl, C₃_₈ cycloalkyl, aryl, or heteroaryl, wherein the alkyl, cycloalkyl, aryl, or heteroaryl may be substituted with a substituent selected from the group consisting of: alcohol, nitrile, carboxylic acid, carbonyl, ester, thiol, halogen, Ci_6 alkyl, halo Ci_6 alkyl, C3_g cycloalkyl, Ci_6 alkoxy, Ci_6 hydroxy, Ci_6 alcohol, halo Ci_6 alkoxy, amino (the amino being optionally substituted with 1 to 2 Ci_6 alkyls, aryls, or heteroaryls), carbamoyl (the carbamoyl being optionally substituted with 1 to 2 Ci_6 alkyls, aryls, or heteroaryls), sulfonyl (the sulfonyl being optionally substituted with one Ci_6 alkyl, aryl, or heteroaryl), Ci_6 alkylsulfinyl, arylsulfmyl, heteroarylsulfmyl, Ci_e alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, sulfamoyl (the sulfamoyl being optionally substituted with 1 to 2 Ci_6 alkyls, aryls, or heteroaryls), Ci_6 alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, Ci_6 alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, Ci_6 ester, aryl ester, heteroaryl ester, carbamoylamino (the carbamoylamino being optionally substituted with 1 to 2 Ci_₆ alkyls, aryls, or

heteroaryls), Ci_6 alkyl carbamate, aryl carbamate, heteroaryl carbamate, Ci_6 alkyl amide, aryl amide, heteroaryl amide, aryl, heteroaryl, aralkyl, heteroaralkyl, arylalkoxy, and heteroarylalkoxy;

R² represents benzene or heteroaryl, wherein the benzene or heteroaryl may be substituted with a substituent selected from the group consisting of: alcohol, nitrile, carboxylic acid, carbonyl, ester, thiol, halogen, Ci_6 alkyl, halo Ci_<5 alkyl, C -8 cycloalkyl, Ci_<5 hydroxy, Ci_6 alkoxy, Ci_6 alcohol, halo Ci_6 alkoxy, amino (the amino being optionally substituted with 1 to 2 Ci_6 alkyls, aryls, or heteroaryls), carbamoyl (the carbamoyl being optionally substituted with 1 to 2 Ci_₆ alkyls, aryls, or heteroaryls), sulfonyl (the sulfonyl being optionally substituted with one Ci_6 alkyl, aryl, or heteroaryl), Ci_6 alkylsulfmyl, arylsulfmyl, heteroarylsulfinyl, Ci_6 alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, sulfamoyl (the sulfamoyl being optionally substituted with 1 to 2 Ci_6 alkyls, aryls, or heteroaryls), Ci_6 alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, Ci_6 alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, Ci_6 ester, aryl ester, heteroaryl ester, carbamoylamino (the carbamoylamino being optionally substituted with 1 to 2 Ci_6 alkyls, aryls, or heteroaryls), Ci_e alkyl carbamate, aryl carbamate, heteroaryl carbamate, Ci_e alkylamide, arylamide, heteroarylamide, aryl (the aryl being optionally substituted with Ci_6 alkoxy and Ci_6 hydroxy), heteroaryl, aryl ether, heteroaryl ether, aralkyl, heteroaralkyl, aralkyl ether, and heteroaralkyl ether;

R³ represents a hydrogen atom, Ci_6 alkyl, C3_8 cycloalkyl, aralkyl, heteroaralkyl, aryl, or heteroaryl, wherein the alkyl, cycloalkyl, aralkyl, heteroaralkyl, aryl, or heteroaryl may be substituted with a substituent selected from the group consisting of halogen, Ci_6 alkyl, halo Ci_6 alkyl, Ci_6 alkoxy, Ci_6 hydroxy and halo Ci_6 alkoxy; and

Mi, M₂, M₃, and M₄ represent each independently a hydrogen atom or Ci_6 alkyl which may be substituted with halogen; or Mi, together with M₂, M3, and M4, forms -CH₂- or - -CH2-CH2-, or M₄, together with M₂, forms -CH₂- or -CH₂-CH₂-, provided that any two of Mi, M₂, M3, and M₄ together form -CH₂- or -CH₂-CH₂- when R³ is a hydrogen atom.

[0017] In another embodiment, the invention provides a pharmaceutical composition for treatment or prevention of dengue virus infection comprising a therapeutically effective amount of a compound or prodrug thereof, or pharmaceutically acceptable salt of said compound or prodrug; and a pharmaceutically acceptable carrier, wherein the compound is a compound of Formula I, II or III.

[0018] In another embodiment, the invention provides a pharmaceutical composition for treatment or prevention of yellow fever virus infection comprising a therapeutically effective amount of a compound or prodrug thereof, or pharmaceutically acceptable salt of said compound or prodrug; and a pharmaceutically acceptable carrier, wherein the compound is a compound of Formula I, II, or III. [0019] The invention further provide therapeutic methods that combine

administration of a compound of Formular I, II or III with a second agent that reduces or inhibits viral replication or infection. Such second agents include those that act directly on the virus or a component of the virus, as well as antiviral agents that act on a host factor.

DETAILED DESCRIPTION

[0020] The present invention is directed to methods and compositions for treatment or amelioration of dengue virus infection or yellow fever virus infection. In particular, according to the present invention, compounds are identified that inhibit replication of dengue virus and yellow fever virus. In one embodiment, the invention provides

pharmceutical compositions comprising a therapeutically effective amount of a compound or pharmaceutically acceptable salt of said compound, wherein the compound is a compound of Formula I:

wherein R¹ and R² each independently represents H, a C₃-C6 cycloalkyl group, an aryl group, a heteroaryl group or a C1-C6 alkyl group optionally substituted by a substituent selected from a halogen, a haloalkyl, a C1-C6 alkyl group, a C1-C6 alkoxy group, a Ci-C₆ hydroxy group, a C₃-C6 cycloalkyl group, an aryl group and a heteroaryl group;

wherein R³ and R⁴ each independently represents a Ci-C₆ alkyl group, a C₃-C₆ cycloalkyl group, an aryl group or an aralkyl group, or R³ and R⁴ may form a C3-C6 cycloalkylidene group together with the carbon atom R³ and R⁴ are attached to; wherein R⁶ represents a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C3-C6 cycloalkyl group or a C1-C6 alkoxy- Ci-C₆ alkyl group; wherein the above-described C₃-C6 cycloalkyl group, C₃-C6 cycloalkylidene group, aryl group, alkyl aryl group and heteroaryl group may be each independently substituted by a substituent selected from a halogen atom, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 hydroxy and a C1-C6 alkoxy group;

R⁵ represents a hydrogen atom, a lower cycloalkyl group, an aryl group, a heteroaryl group or a Ci-Ce alkyl group optionally substituted by a substituent selected from a lower cycloalkyl group, an aryl group and a heteroaryl group, wherein the lower cycloalkyl group, aryl group and heteroaryl group in R₅ each independently represents an unsubstituted group or a lower cycloalkyl group, an aryl group or a heteroaryl group substituted by one or two substituents selected from a halogen atom, a nitro group, a nitrile group, a Ci-Ce alkyl group, a Ci-Cg haloalkyl group, a Ci-C alkoxy group, Ci-C hydroxy group, a C1-C6 haloalkoxy group, a C1-C6 carboxylic acid group, a C3-C8 cycloalkyl group, an aryl group, a heteroaryl group, an aryl ether group, an aralkyl group, -CON(R⁷)R⁸, -N(R⁷)R⁸, -N(R⁷)COR⁸, -N(R⁷)S0₂R⁸, -OCOR⁷, -OCON(R⁷)R⁸, -SR⁷, - S0₂R⁷, -S0₂N(R⁷)R⁸ and and a group with the formula:

wherein R⁷ and R⁸ each independently represents a hydrogen atom or a lower alkyl group; and

X represents -N(R⁷)- or -0-.

[0021] In an embodiment of the invention the compound of Formula I is Formula I_a:

wherein Ri is selected from H, unsubstituted benzene, and substituted benzene with

substitutents selected from F, CH₃, CH₂CH₃, CI, OCH₃, OCF₃, and CF₃; Ci e alkanes (including but not limited to methyl, ethyl, -propyl, and n-propyl); and C₃_₆ cycloalkanes (cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl);

wherein R³ and R⁴ are independently selected from H; Ci_3 alkane; and benzene; or R³ and R⁴ taken together form a cycloalkyl of formula -(CH₂)_n- where n = 2, 3, 4 and 5; wherein R⁵ is selected from methyl; CF₃; cyclopropyl; unsubstituted benzene; mono- and disubsituted benzene where substitutents are selected from F, CH₃, CH₂CH₃, CN, CH₃CHCH₃, CI, OCH₃, OPh, OCF₃, and CF₃; unsubstituted heteroaryl groups (such as 2-, 3-, or 4-pyridine, isoxazole, pyrazole, triazole); and imidazole. [0022] In certain embodiments of the invention, R⁵ is one of the following substituted benzene rings:

[0023] In certain embodiments of the invention, R¹ is benzene, R³ and R⁴ are methyl, and R⁵ is one of the aforementioned substituted benzene rings.

[0024] In an embodiment of the invention, the compound of Formula I is selected from the group consisting of

[0025] Compounds of Formula I are disclosed in U.S. Patent Publication

2010/0056597 (Takahashi et al.) and indicated to be useful in treating various diseases, including circulatory diseases, neurological diseases, metabolic diseases, and infections caused by bacteria, fungi, and parasites. The compounds are also indicated to be long chain fatty acid elongase (LCE) inhibitors. According to Takahashi et al., 2009, J. Med. Chem. 52, 3142, compounds of Formula I are inhibitors of long chain fatty acid elongase 6 (ELOVL6). Both publications are incorporated by reference herein.

[0026] In another embodiment, the invention provides a compound represented by the formula (II) or a pharmaceutically acceptable salt thereof.

wherein R represents optionally halogenated Cue alkyl or optionally halogenated C₃_s

cycloalkyl

R² represents a substituent selected from:

wherein W represents Ci_6 alkylene, C2-6 alkenylene, C2-6 alkynylene or C₃-6 cycloalkylene, wherein the alkylene, alkenylene, alkynylene or cycloalkylene may be substituted by optionally halogenated C1-3 alkyl, optionally halogenated Ci_3 alkoxy, alcohol or halogen; and

R represents Ci_6 alkyl, C3-8 cycloalkyl, aryl or heteroaryl, wherein the Ci_6 alkyl, C3-8

cycloalkyl, aryl or heteroaryl may be substituted by halogen, optionally halogenated Ci_6 alkyl, optionally halogenated Ci_6 alkoxy, Ci_6 hydroxy, Ci_6 alkylsulfonyl, nitrile, benzene, Ci_6 sulfide, Cue thiol, Ci_6 alcohol, amino, Cue alkylamino, di-Ci_6 alkylamino, alcohol-Ci_6 alkyl, amino-Ci e alkyl, C e alkylamino- Cue alkyl, di-Ci_6 alkylaminoalkyl, Cue alkoxy-Ci_₆ alkyl, Cue alkylcarbonyl, Ci_₆ alkylamide, Ci_₆ alkylcarbamoyl, C₃_₈ cycloalkylcarbamoyl, Ci_6 alkylsulfonylamino or Ci_6 alkylaminosulfonyl;

wherein R³ represents a hydrogen atom, Cue alkyl, aryl or heteroaryl;

X represents -0-, -C(R^4a)(R^4b)- or -NR⁵-;

R^4a, R^4b and R⁵ each independently represent hydrogen, Ci_₆ alkyl, C₃_₈ cycloalkyl, aryl or heteroaryl, wherein the Ci_6 alkyl, C3-8 cycloalkyl, aryl or heteroaryl may be substituted by halogen, optionally halogenated Ci_6 alkyl, optionally halogenated Ci_6 alkoxy, Ci_6 alkylsulfonyl, nitrile, aryl or heteroaryl;

Y₁ represents -CR⁶- or -N-; Y2 represents -CR - or -N-; Y3 represents -CR⁸- or -N-;

Y4 represents -CR⁹- or -N-; and

R⁶, R⁷, R⁸ and R⁹ each independently represent a hydrogen atom, halogen, nitrile, Ci_6 alkyl, C₃_8 cycloalkyl, Ci_6 alkoxy, Ci_6 hydroxy, alcohol-Ci_6 alkyl, amino, Ci_6 alkylamino, di- Ci_6 alkylamino, amino-Ci_6 alkyl, Ci_6 alkylamino-Ci-e alkyl, di-Ci_6 alkylamino-(Ci_ 6)alkyl, Ci_6 alkoxy-Ci_6 alkyl, Ci_6 alcohol, heterocycle, aryl, heteroaryl, Ci_6

alkylsulfonyl, Ci_₆ alkylsulfinyl, Ci_₆ alkylthiol, arylsulfonyl, heteroaryl sulfonyl, aryl sulfmyl, heteroaryl sulfinyl, aryl thiol, heteroaryl thiol, Ci_6 alkylcarbonyl, aryl carbonyl, heteroaryl carbonyl, Ci e alkylamide, aryl amide, heteroaryl amide, Ci e alkylcarbamoyl, C₃_8 cycloalkylcarbamoyl, heterocycle carbamoyl, aryl carbamoyl, heteroarylcarbamoyl, Ci_6 alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, Ci_6

alkylsulfamoyl, arylsulfamoyl or heteroarylsulfamoyl, wherein the alkyl, cycloalkyl, heterocycle, aryl or heteroaryl may be substituted by a group selected from the group consisting of halogen, optionally halogenated Ci_6 alkyl, optionally halogenated Ci_6 alkoxy, Ci_₆ alkylsulfonyl, carbonyl and nitrile.

[0027] In an embodiment of the invention, the compound of Formula II has the formula II_a

wherein L is selected from carbamate, urea, or amide including, for example

wherein R is selected from halo; CF₃; cyclopropyl; optionally substituted Ci_₅ alkyl, wherein the Ci-5 alkyl may be substituted with halo, oxo, -OH, -CN, -NH₂, CO2H, and C1 ₃ alkoxy; wherein Ri is selected from substituted phenyl where the substiuents are selected from F, CF₃, CH₃, O CH₃, or isopropyl; wherein R₂ is CI, Ph, l-(2-pyridone), 4-isoxazole, 3-pyrazole, 4-pyrazole, 1-pyrazole, 5- (1,2,4-triazole), l-(l,2,4-triazole), 2-imidazole, 1 -(2-pyrrolidone), 3-(l ,3-oxazolidin-2- one).

[0028] The chiral center at C-4 can be racemic, (S), (R), or any ratio of enantiomers. In one embodiment, L is an amide. In certain embodiments, R is selected from CI, CF3, methyl, ethyl, isopropyl and, cyclopropyl. In certain embodiments R¹ is para-substitued wherein the substituent is selected from F, CF₃, CH₃, OCH₃, or isopropyl,

[0029] In one embodiment, the compound of formula II is

[0030] In another embodiment, the elongase inhibitor is a compound of formula II_C

(He) wherein R¹ is substituted at position 2, 3, or 4 with F, or Me, or R¹ is substituted at position 4 with MeO, or CF₃, R² is CI, H, Ph, 4-isoxazole, 4-pyrazole, 3-pyrazole, 1-pyrazole, 5-(l,2,4- triazol), l -(l ,2,4-triazole), 2-imidazole, l-(2-pyrrolidone), or 3-(l ,3-oxazolidin-2-one).

[0031] In one embodiment the compound of formula II is

[0032] Compounds of Formula II are disclosed in U.S. Patent Publication

2010/0210636 (Ishikawa et al.) and indicated to be useful in treating various diseases, including meatbolic syndrome, obesity, diabetes, malignant neoplasm, and infectious diseases caused by bacteria, fungi or parasites. The compounds are also indicated to be long chain fatty acid elongase (LCE) inhibitors. According to Mizutani et al., 2009, J. Med. Chem. 52, 7289, compounds of Formula II are inhibitors of long chain fatty acid elongase 6 (ELOVL6). Both publications are incorporated by reference herein.

[0033] The invention further provides a compound represented by the following Formula (III) or a pharmaceutically acceptable salt thereof;

wherein, Z is

where nl, n2, and n3 are 0, 1 , or 2, and wherein

R¹ represents Ci_6 alkyl, C₃_g cycloalkyl, aryl, or heteroaryl, wherein the alkyl, cycloalkyl, aryl, or heteroaryl may be substituted with a substituent selected from the group consisting of: alcohol, nitrile, carboxylic acid, carbonyl, ester, thiol, halogen, Ci_₆ alkyl, halo Ci_6 alkyl, C3_8 cycloalkyl, Ci_6 alkoxy, Ci_e hydroxy, Ci_6 alcohol, halo Ci_6 alkoxy, amino (the amino being optionally substituted with 1 to 2 Ci_6 alkyls, aryls, or heteroaryls), carbamoyl (the carbamoyl being optionally substituted with 1 to 2 Ci_6 alkyls, aryls, or heteroaryls), sulfonyl (the sulfonyl being optionally substituted with one Ci_6 alkyl, aryl, or heteroaryl), Ci_6 alkylsulfinyl, arylsulfmyl, heteroarylsulfinyl, Ci_6 alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, sulfamoyl (the sulfamoyl being optionally substituted with 1 to 2 Ci_6 alkyls, aryls, or heteroaryls), Ci_6 alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, Ci_6 alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, Ci_6 ester, aryl ester, heteroaryl ester, carbamoylamino (the carbamoylamino being optionally substituted with 1 to 2 Ci_₆ alkyls, aryls, or

R² represents benzene or heteroaryl, wherein the benzene or heteroaryl may be substituted with a substituent selected from the group consisting of: alcohol, nitrile, carboxylic acid, carbonyl, ester, thiol, halogen, Ci_6 alkyl, halo Ci_6 alkyl, C3-8 cycloalkyl, Ci_6 hydroxy, Ci_6 alkoxy, Ci_6 alcohol, halo Ci_6 alkoxy, amino (the amino being optionally substituted with 1 to 2 Ci_6 alkyls, aryls, or heteroaryls), carbamoyl (the carbamoyl being optionally substituted with 1 to 2 Ci_6 alkyls, aryls, or heteroaryls), sulfonyl (the sulfonyl being optionally substituted with one Ci_6 alkyl, aryl, or heteroaryl), Ci_6 alkylsulfinyl, arylsulfmyl, heteroarylsulfinyl, Ci_6 alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, sulfamoyl (the sulfamoyl being optionally substituted with 1 to 2 Ci_6 alkyls, aryls, or heteroaryls), Ci_6 alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, Ci_6 alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, Ci_e ester, aryl ester, heteroaryl ester, carbamoylamino (the carbamoylamino being optionally substituted with 1 to 2 Ci_6 alkyls, aryls, or heteroaryls), Ci_6 alkyl carbamate, aryl carbamate, heteroaryl carbamate, Ci_6 alkylamide, arylamide, heteroarylamide, aryl (the aryl being optionally substituted with Ci_6 alkoxy and Ci_6 hydroxy), heteroaryl, aryl ether, heteroaryl ether, aralkyl, heteroaralkyl, aralkyl ether, and heteroaralkyl ether;

R³ represents a hydrogen atom, Ci_6 alkyl, C3_g cycloalkyl, aralkyl, heteroaralkyl, aryl, or heteroaryl, wherein the alkyl, cycloalkyl, aralkyl, heteroaralkyl, aryl, or heteroaryl may be substituted with a substituent selected from the group consisting of halogen, Ci_6 alkyl, halo Ci_6 alkyl, Ci_6 alkoxy, Ci_6 hydroxy and halo Ci_6 alkoxy; and

Mi, M₂, M3, and M4 represent each independently a hydrogen atom or Ci_6 alkyl which may be substituted with halogen; or M_u together with M₂, M₃, and M₄, forms -CH₂- or -CH2-CH2-, or M₄, together with M₂, forms -CH₂- or -CH₂- CH₂-, provided that any two of Mi, M₂, M3, and M₄ together form -Cl¾- or -CH2-CH2- when R³ is a hydrogen atom.

[0034] In a particular embodiment, the compound of formula III is

wherein Ri is selected from Ci_6 alkyl (such as methyl, ethyl, isopropyl), Ci_6 alkoxy (such as OCH₃, O-isopropyl, OCF₃, OC₆H5), halogen, (such as fluoro, chloro, bromo), aryl and alkyl aryl (such as CeH₅, CH₂Ph); halogenated Ci_e alkyl (such as CF₃) and

wherein R2 is selected from C_1- alkyl (such as n-butyl, t-butyl, /?-propyl, i-butyl,and i- propyl); benzene; substituted benzene where substituents are selected from alcohol, nitrile, carboxylic acid, carbonyl, ester, thiol, Ci_6 alkyl, Ci_6 alkoxy (such as OCH₃), halogen Ci_6 alkyl (such as CF₃), Ci_6 alcohol, halo Ci_6 alkoxy (such as OCF₃), amino (the amino being optionally substituted with 1 to 2 Ci_6 alkyls, aryls, or heteroaryls), aryl, heteroaryl, aralkyl, heteroaralkyl, arylalkoxy, and heteroarylalkoxy (such as 2-pyridine; 3-pyridine; and N-methyl imidazole).

[0035] In one embodiment, Ri is selected from O-isopropyl and OCF₃. In one embodiment R2 is selected from 77-butyl, unsubstituted benzene, fluorobenzene and benzene sulfide.

[0036] In one embodiment the inhibitor of formula Ilia is

wherein R² is selected from butyl, propyl, phenyl, pyridyl, and imidazole.

0037] In one inbodiment the inhibitor of formula III is selected from

and

[0038] Compounds of Formula III are disclosed in U.S. Patent Publication

2010/0331360 (Nagase et al.). The compounds are also indicated to be long chain fatty acid elongase (LCE) inhibitors. According to Sasake et al., 2009, Bioorg. Med. Chem. 17, 5639, compounds of Formula II are inhibitors of long chain fatty acid elongase 6 (ELOVL6). Both publications are incorporated by reference herein.

[0039] The invention also provides a method of treating or ameliorating a dengue virus infection or a yellow fever infection comprising administering a therapeutically effective amount of a compound or prodrug thereof, or pharmaceutically acceptable salt or ester of said compound or prodrug, wherein the compound is a compound of Formula I, I_a, II, II_a, II_b, II_C, III, III_a, or III_b.

[0040] The compounds described herein may exist in several tautomeric forms.

Accordingly, the chemical structures depicted herein encompass all possible tautomeric forms of the illustrated compounds. Compounds of the invention may exist in various hydrated forms.

[0041] Definitions of the more commonly recited chemical groups are set forth below. Certain variables in classes of compounds disclosed herein recite other chemical groups. Chemical groups recited herein, but not specifically defined, have their ordinary meaning as would be known by a chemist skilled in the art.

[0042] A "Ci_x alkyl" (or "Ci-C χ alkyl") group is a saturated straight chain or branched non-cyclic hydrocarbon having from 1 to X carbon atoms. Representative -(Ci_8 alkanes) include -methyl, -ethyl, -rc-propyl, -ra-butyl, -rc-pentyl, -«-hexyl, -n-heptyl and -«-octyl; while saturated branched alkyls include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl and the like. A -(Ci_x alkyl) group can be substituted or unsubstituted.

[0043] The terms "halogen" and "halo" mean fluorine, chlorine, bromine and iodine.

[0044] An "aryl" group is an aromatic carbocyclic group from 6 to 14 carbon atoms having a single ring (e.g., benzene) or multiple condensed rings (e.g. , naphthalene or anthracene). Particular aryl group examples include benzene, dibenzene, naphthalene and the like. An aryl group can be substituted or unsubstituted.

[0045] A "heteroaryl" group is an aromatic ring system having one to four heteroatoms as ring atoms in a heteroaromatic ring system, wherein the remainder of the atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur and nitrogen. In certain embodiments, the heterocyclic ring system is monocyclic or bicyclic. Non-limiting examples include aromatic groups selected from the following:

[0046] wherein Q is CH₂, CH=CH, O, S or NH. Further representative examples of heteroaromatic groups include, but are not limited to, benzofuranyl, benzothienyl, indolyl, benzopyrazolyl, coumarinyl, furanyl, isothiazolyl, imidazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, thiophenyl, pyrimidinyl, isoquinolinyl, quinolinyl, pyridinyl, pyrrolyl, pyrazolyl, lH-indolyl, lH-indazolyl, benzo[d]thiazolyl and pyrazinyl. Heteroaryls can be bonded at any ring atom (i.e., at any carbon atom or heteroatom of the heteroaryl ring) A heteroaryl group can be substituted or unsubstituted. In one embodiment, the heteroaryl group is a C₃-1₀ heteroaryl ring.

[0047] A "cycloalkyl" group is a saturated or unsaturated non-aromatic carbocyclic ring. Representative cycloalkyl groups include, but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1 ,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl. A cycloalkyl group can be substituted or unsubstituted. In one embodiment, the cycloalkyl group is a C₃_g cycloalkyl group.

[0048] A "heterocyclic" group is a cyclic group having at least one ring carbon atom and one to four ring heteroatoms selected from O, S and N. Representative examples of a heterocycle include, but are not limited to: morpholine, pyrrole, pyrrolidine, thiophene, furan, thiazole, imidazole, pyrazole, triazole, piperizine, isothiazole, isoxazole, (l ,4)-dioxane, (l,3)-dioxolane, 4,5-dihydro-lH-imidazole and tetrazole. Heterocycles can also be bonded at any ring atom (i.e., at any carbon atom or heteroatom of the heteroaryl ring). A heterocyclic group can be substituted or unsubstituted. In one embodiment, the heterocycle is a 3-7 membered ring.

[0049] In one embodiment, when groups described herein are said to be "substituted," they may be substituted with any suitable substituent or substituents. Illustrative examples of substituents include those found in the exemplary compounds and embodiments disclosed herein, as well as halogen (chloro, iodo, bromo, or fluoro); Ci_6 alkyl; C₂_6 alkenyl; C₂_6 alkynyl; alcohol; Ci_6 alkoxy; amino; nitro; thio; thioether; imine; nitrile; amido;

phosphonate; phosphine; carboxylic acid; thiocarbonyl; sulfonyl; sulfonamide; ketone;

aldehyde; ester; oxygen (=0); haloalkyl (e.g., trifluoromethyl); carbocycle, which may be monocyclic or fused or non-fused polycyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl), or a heterocycle, which may be monocyclic or fused or non-fused polycyclic (e.g. , pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, or thiazinyl); carbocyclic or heterocyclic, monocyclic or fused or non- fused polycyclic aryl (e.g., phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridinyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothiophenyl, or benzofuranyl); amino (primary, secondary, or tertiary); o-lower alkyl; o-aryl, aryl; aryl-lower alkyl; C0₂CH₃; CONH₂; OCH₂CONH₂; NH₂; S0₂NH₂; OCHF₂; CF₃; OCF₃.

[0050] As used herein, the term "pharmaceutically acceptable salt(s)" refers to a salt prepared from a pharmaceutically acceptable non-toxic acid or base including an inorganic acid and base and an organic acid and base. Suitable pharmaceutically acceptable base addition salts of the compounds include, but are not limited to metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, NN-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Suitable non-toxic acids include, but are not limited to, inorganic and organic acids such as acetic, alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, formic, fumaric, furoic, galacturonic, gluconic, glucuronic, glutamic, glycolic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic, phosphoric, propionic, salicylic, stearic, succinic, sulfanilic, sulfuric, tartaric acid, and / toluenesulfonic acid. Specific non-toxic acids include hydrochloric, hydrobromic, phosphoric, sulfuric, and methanesulfonic acids. Examples of specific salts thus include hydrochloride and mesylate salts. Others are well-known in the art, See for example, Remington's Pharmaceutical Sciences, 18th eds., Mack Publishing, Easton PA (1990) or Remington: The Science and Practice of Pharmacy, 19th eds., Mack Publishing, Easton PA (1995).

[0051] As used herein and unless otherwise indicated, the term "hydrate" means a compound, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

[0052] As used herein and unless otherwise indicated, the term "solvate" means a compound, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces.

[0053] As used herein and unless otherwise indicated, the term "prodrug" means a compound derivative that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide compound. Examples of prodrugs include, but are not limited to, derivatives and metabolites of a compound that include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. In certain embodiments, prodrugs of compounds with carboxyl functional groups are the lower alkyl esters of the carboxylic acid. The carboxylate esters are conveniently formed by esterifying any of the carboxylic acid moieties present on the molecule. Prodrugs can typically be prepared using well-known methods, such as those described by Burger's Medicinal Chemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers Gmfh).

[0054] As used herein and unless otherwise indicated, the term "stereoisomer" or "stereomerically pure" means one stereoisomer of a compound, in the context of an organic or inorganic molecule, that is substantially free of other stereoisomers of that compound. For example, a stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereomerically pure compound comprises greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound. The compounds can have chiral centers and can occur as racemates, individual enantiomers or diastereomers, and mixtures thereof. All such isomeric forms are included within the embodiments disclosed herein, including mixtures thereof.

[0055] Various compounds contain one or more chiral centers, and can exist as racemic mixtures of enantiomers, mixtures of diastereomers or enantiomerically or optically pure compounds. The use of stereomerically pure forms of such compounds, as well as the use of mixtures of those forms are encompassed by the embodiments disclosed herein. For example, mixtures comprising equal or unequal amounts of the enantiomers of a particular compound may be used in methods and compositions disclosed herein. These isomers may be asymmetrically synthesized or resolved using standard techniques such as chiral columns or chiral resolving agents. See, e.g., Jacques, J., et al., Enantiomers, Racemates and

Resolutions (Wiley-Interscience, New York, 1981); Wilen, S. FL, et al., Tetrahedron 33:2725 (1977); Eliel, E. L., Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S. H., Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0056] It should also be noted that compounds, in the context of organic and inorganic molecules, can include E and Z isomers, or a mixture thereof, and cis and trans isomers or a mixture thereof. In certain embodiments, compounds are isolated as either the E or Z isomer. In other embodiments, compounds are a mixture of the E and Z isomers.

[0057] As used herein, "small molecule" refers to a substances that has a molecular weight up to 2000 atomic mass units (Daltons). Additional small molecule inhibitors can be found by screening of compound libraries and/or design of molecules that bind to specific pockets in the structures of these enzymes. The properties of these molecules can be optimized through derivitization, including iterative rounds of synthesis and experimental testing.

[0058] The present invention also provides for the use of the disclosed combinations in cell culture-related products in which it is desirable to have antiviral activity. In one embodiment, the combination is added to cell culture media. The compounds used in cell culture media include compounds that may otherwise be found too toxic for treatment of a subject. As used herein, the term "effective amount" in the context of a compound for use in cell culture-related products refers to an amount of a compound which is sufficient to reduce the viral titer in cell culture or prevent the replication of a virus in cell culture.

[0059] In an embodiment of the invention, a method of treating or ameliorating a dengue virus infection or a yellow fever infection further comprises administering an effective amount of a second agent that reduces or inhibits viral replication or infection. Such second agents include those that act directly on the virus or a component of the virus.

1. Direct acting antiviral compounds

[0060] Direct acting antiviral compounds include those that inhibit or prevent viral entry, integration, growth and/or production by effecting the function of viral proteins. Such second agents also include agents that act at least partly on host factors, including for example, microRNA antagonists, immunomodulators, microtubule inhibitors, and host metabolic inhibitors. In certain cases, such second agents may be or have been judged ineffective when used alone to treat an ongoing denge virus or yellow fever virus infection, but will nevertheless be effective when administered in a combination treatment with a compound of Formula I, II, or III.

[0061] Direct acting agents target flavivirus components. The genome of a flavivirus, such as dengue virus and yellow fever virus, is composed of a single positive-sense genomic RNA that codes for a polyprotein. The flavivirus genome encodes three structural proteins (capsid [C], premembrane [PrM], and envelope [E] proteins), which form the viral particle, and seven non-structural proteins (NSl, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), which function in the replication of the RNA genome, virion assembly, and evasion of innate immune response. The N-terminal domain of NS3, together with NS2B, contains a serine protease activity; while the C-terminal domain functions as an RNA helicase, an RNA triphosphatase, and an NTPase. The N-terminal domain of NS5 contains a methyltransferase activity, while the C-terminal domain serves as an RNA-dependent RNA polymerase. Other nonstructural proteins are required for RNA replication, including NS2A, NS2B, NS4A, and NS4B proteins, which form the scaffold for the viral replication complex.

[0062] Thus, in an embodiment of the invention, a compound of Formula I, II, or III is coadministered with a viral protease (NS2B/NS3) inhibitor, a viral helicase (NS3) inhibitor, a viral polymerase (NS5B) inhibitor, an inhibitor of a non-structural protein (NS4B, or NS5A), an inhibitor of a viral ion channel forming protein, a viral genome-specific RNAi, an internal ribosome entry site (IRES) inhibitor, or a viral entry inhibitor.

[0063] In an embodiment of the invention, a compound of Formula I, II, or III is coadministered with an NS2B/NS3 protease inhibitor such as BP2109, disclosed in Yang et al., 201 1 , Antimicrob. Agents Chemotherapy 55, 229-38.

[0064] In an embodiment of the invention, a compound of Formula I, II, or III is coadministered with a viral entry inhibitor, such as compound 6 disclosed by Wang et al., 2009, Antimicrob Agents Chemother 53, 1821-32.

[0065] In an embodiment of the invention, a compound of Formula I, II, or III is coadministered with a nucleoside analog that inhibits function of the viral polymerase. One such polymerase inhibitor currently under evaluation in patients for dengue virus infection is balapiravir. Other nucleoside analogs include, without limitation, ribavarin, taribavirin, mizoribine , viramidine, merimepodib, mycophenolate mofetil, mycophenolate. Not all such nucleoside analogs are effective for treatment of dengue virus and/or yellow fever virus. According to the invention, the effectiveness of such nucleoside analogs can be increased by combined administration with a compound of Formula I, II, or III.

2. Other Agents That Act at Least Partly on a Host Factor

2.1 Glucosidase antagonists

[0066] According to the invention, a compound of Formula I, II, or III is

coadministered with an agent that inhibits a host glucosidase enzyme. Glucosidase is involved in folding and glysocylation of the prM, E, and NS 1 dengue proteins. Glucosidase inhibitors such as castanospermin and deoxynojirimycin (DNJ) have been observed to inhibit dengue replication in vivo.

2.2 MicroRNA antagonists

[0067] MicroRNA- 122 (miR-122) is thought to stimulate viral replication of certain flaviviruses (i. e. , HCV) through interaction with the virus ' untranslated region, and an agent that inhibits that microRNA inhibits HCV. In one embodiment, a compound of Formula I, II, or III is coadministered with an agent that inhibits a microRNA that otherwise stimulates replication of dengue virus or yellow fever virus. 2.3 Immunomodulators

[0068] According to the invention, a compound of Formula I, II, or III is

coadministered with an immunomodulator effective to reduce or inhibit denge virus or yellow fever virus. Immunomodulators include several types of compounds. Non-limiting examples include inteferons (e.g., Pegasys, Pegintron, Albumin IFN-a, locteron, Peginterferon-λ, omega-IFN, medusa-IFN, belerofon, infradure, and Veldona, caspase/pan-caspase inhibitors (e.g., emricasan, nivocasan, IDN-6556, GS9450), Toll-like receptor agonists (e.g., Actilon, ANA773, IMO-2125, SD-101), cytokines and cytokine agonists and antagonists (e.g., Acto ine-2, Interleukin 29, Infliximab (cytokine TNFa blocker), IPH1101 (cytokine agonist), and other immunomodulators such as, without limitation, thymalfasin, Eltrombopag, IP1 101, SCV-07, Oglufanide disodium, CYT107, ME3738, TCM-700C, EMZ702, EGS21.

[0069] Cytokines are know to play a role in several viral hemorrhagic fevers, including dengue, and increased levels of several cytokines have been correlated with disease severity. Accordingly, a compound of Formula I, II, or III may be coadministered with tetracycline, doxycycline, or antibiotics that modulate cytokines and cytokine receptors.

2.4 Inhibitors of microtubules

[0070] In one embodiment a compound of Formula I, II, or III is coadministered with an inhibitor of microtubule polymerization. Non-limiting examples of microtubule polymerization inhibitors include colchicine, Prazosin, and mitoquinone. Farglitazar and GI262570 are PPAR-gamma inhibitors that reduce tubulin levels without affecting the polymerization of tubulin. These compounds target tubulin itself, rather than the equilibrium between tubulin and microtubules.

2.5 Host metabolism inhibitors

[0071] In another such embodiment, a compound of Formula I, II, or III is coadministered with a host metabolism inhibitor. Examples of host metabolism inhibitors include Hepaconda (bile acid and cholesterol secretion inhibitor), Miglustat

(glucosylceramide synthase inhibitor), Celgosivir (alpha glucosidase inhibitor), Methylene blue (Monoamine oxidase inhibitor), pioglitazone and metformin (insulin regulator),

Nitazoxanide (possibly PFOR inhibitor), NA255 and NA808 (Serine palmitoyltransferase inhibitor), NOV205 (Glutathione-S-transferase activator), and ADIPEG20 (arginine deiminase). [0072] In an embodiment of the invention, a compound of Formula I, II, or III is coadministered with chloroquine. Chloroquine has a modest antiviral effect on replication of viruses from diverse taxonomic families, and has been observed to inhibit repliation of dengue in vitro. Chloroquine is concentrated in endosomes, and the antiviral effect of chloroquine is thought to depend in part on the extent to which a virus utilizes endosomes for entry.

[0073] In another such embodiment, a compound of Formula I, II, or III is coadministered with an agent selected from laccase (herbal medicine), silibinin and silymarin (antioxidant, hepato-protective agent), PY 17 and JKB-122 (anti-inflammatory), CTS-1027 (matrix metalloproteinase inhibitor), Lenocta (protein tyrosine phosphatase inhibitor), Bavituximab and BMS936558 (programmed cell death inhibitor), HepaCide-I (nano- viricide), CF102 (Adenosine A3 receptor), GNS278 (inhibits viral-host protein interaction by attacking autophagy), RPIMN (Nicotinic receptor antagonist), PY 18 (possible viral maturation inhibitor), ursa and Hepaconda (bile acids, possible farnesoid X receptor), tamoxifen (anti-estrogen), Sorafenib (kinase inhibitor), KPE02001003 (unknown

mechanism).

[0074] The combination therapies of the present invention may have the advantage of producing a synergistic inhibition of viral infection or replication and, for example, allow the use of lower doses of each compound to achieve a desirable therapeutic effect. In some embodiments, the dose of one of the compounds is substantially less, e.g., 1.5, 2, 3, 5, 7, or 10-fold less, than required when used independently for the prevention and/or treatment of viral infection. In some embodiments, the dose of both agents is reduced by 1.5, 2, 3, 5, 7, or 10-fold or more. In addition to improved antiviral activity, the combination therapies of the present invention can reduce overall toxicity and undesirable side effects of the

coadministered drugs by allowing the administration of lower doses of one or more of the combined compounds while providing the desired therapeutic effect.

[0075] The combination therapies of the present invention may also reduce the potential for the development of drug-resistant mutants that can occur when, for example, direct acting antiviral agents alone are used to treat viral infection.

[0076] As used herein, the term "combination," in the context of the administration of two or more therapies to a subject, refers to the use of more than one therapy (e.g., more than one prophylactic agent and/or therapeutic agent). The use of the term "combination" does not restrict the order in which therapies are administered to a subject with a viral infection. A first therapy (e.g., a first prophylactic or therapeutic agent) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a subject with a viral infection.

[0077] As used herein, the term "effective amount" in the context of administering a therapy to a subject refers to the amount of a therapy which is sufficient to achieve one or more of the following effects: (i) reduce or ameliorate the severity of a viral infection or a symptom associated therewith; (ii) reduce the duration of a viral infection or a symptom associated therewith; (iii) prevent the progression of a viral infection or a symptom associated therewith; (iv) cause regression of a viral infection or a symptom associated therewith; (v) prevent the development or onset of a viral infection or a symptom associated therewith; (vi) prevent the recurrence of a viral infection or a symptom associated therewith; (vii) reduce or prevent the spread of a virus from one cell to another cell, or one tissue to another tissue; (ix) prevent or reduce the spread of a virus from one subject to another subject; (x) reduce organ failure associated with a viral infection; (xi) reduce hospitalization of a subject; (xii) reduce hospitalization length; (xiii) increase the survival of a subject with a viral infection; (xiv) eliminate a virus infection; and/or (xv) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

3. Characterization of Antiviral Activity of Compounds

3.1 Antiviral Activity

[0078] The present invention provides compounds for use in the prevention, management and/or treatment of viral infection. The antiviral activity of compounds against dengue virus and/or yellow fever virus can be tested using techniques described in Section 3.2 herein below.

[0079] The antiviral activities of compounds against any type, subtype or strain of virus can be assessed. For example, the antiviral activity of compounds against naturally occurring strains, variants or mutants, mutagenized viruses, reassortants and/or genetically engineered viruses can be assessed. [0080] In some embodiments, the virus achieves peak titer in cell culture or a subject in 4 hours or less, 6 hours or less, 8 hours or less, 12 hours or less, 16 hours or less, or 24 hours or less. In other embodiments, the virus achieves peak titers in cell culture or a subject in 48 hours or less, 72 hours or less, or 1 week or less. In other embodiments, the virus achieves peak titers after about more than 1 week. In accordance with these embodiments, the viral titer may be measured in the infected tissue or serum.

[0081] In some embodiments, the virus achieves in cell culture a viral titer of 10⁴ pfu/ml or more, 5 x 10⁴ pfu/ml or more, 10⁵ pfu/ml or more, 5 x 10⁵ pfu/ml or more, 10⁶ pfu/ml or more, 5 x 10⁶ pfu/ml or more, 10⁷ pfu/ml or more, 5 x 10⁷ pfu/ml or more, 10^s pfu/ml or more, 5 x 10⁸ pfu/ml or more, 10⁹ pfu/ml or more , 5 x 10⁹ pfu/ml or more, or 10¹⁰ pfu/ml or more. In certain embodiments, the virus achieves in cell culture a viral titer of 10⁴ pfu/ml or more, 5 x 10⁴ pfu/ml or more, 10⁵ pfu/ml or more, 5 x 10⁵ pfu/ml or more, 10⁶ pfu/ml or more, 5 x 10⁶ pfu/ml or more, 10⁷ pfu/ml or more, 5 x 10⁷ pfu/ml or more, 10⁸ pfu/ml or more, 5 x 10⁸ pfu/ml or more, 10⁹ pfu/ml or more , 5 x 10⁹ pfu/ml or more, or 10¹⁰ pfu/ml or more within 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, or 24 hours or less. In other embodiments, the virus achieves in cell culture a viral titer of 10⁴ pfu/ml or more, 5 x 10⁴ pfu/ml or more, 10⁵ pfu/ml or more, 5 x 10⁵ pfu/ml or more, 10⁶ pfu/ml or more, 5 x 10⁶ pfu/ml or more, 10⁷ pfu/ml or more, 5 x 10⁷ pfu/ml or more, 10^s pfu/ml or more, 5 x 10⁸ pfu/ml or more, 10⁹ pfu/ml or more , 5 x 10⁹ pfu/ml or more, or 10¹⁰ pfu/ml or more within 48 hours, 72 hours, or 1 week.

[0082] In some embodiments, the virus achieves a viral yield of 1 pfu/ml or more, 10 pfu/ml or more, 5 x 10¹ pfu/ml or more, 10² pfu/ml or more, 5xl0² pfu/ml or more, 10³ pfu/ml or more, 2.5xl0³ pfu/ml or more, 5xl0³ pfu/ml or more, 10⁴ pfu/ml or more, 2.5 xlO⁴ pfu/ml or more, 5 xlO⁴ pfu/ml or more, or 10⁵ pfu/ml or more in a subject. In certain embodiments, the virus achieves a viral yield of 1 pfu/ml or more, 10 pfu/ml or more, 5 x 1ο¹ pfu/ml or more, 10² pfu/ml or more, 5xl0² pfu/ml or more, 10³ pfu/ml or more, 2.5xl0³ pfu/ml or more, 5xl0³ pfu/ml or more, 10⁴ pfu/ml or more, 2.5 xlO⁴ pfu/ml or more, 5 xlO⁴ pfu/ml or more, or 10⁵ pfu/ml or more in a subject within 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 24 hours, or 48 hours. In certain embodiments, the virus achieves a viral yield of 1 pfu/ml or more, 10 pfu/ml or more, 10¹ pfu/ml or more, 5 x 1ο¹ pfu/ml or more, 10² pfu/ml or more, 5xl0² pfu/ml or more, 10³ pfu/ml or more, 2.5xl0³ pfu/ml or more, 5xl0³ pfu/ml or more, 10⁴ pfu/ml or more, 2.5 xlO⁴ pfu/ml or more, 5 xlO⁴ pfu/ml or more, or 10⁵ pfu/ml or more in a subject within 48 hours, 72 hours, or 1 week. In accordance with these embodiments, the viral yield may be measured in the infected tissue or serum. In a specific embodiment, the subject is immunocompetent. In another embodiment, the subject is immunocompromised or immunosuppressed.

[0083] In some embodiments, the virus achieves a viral yield of 1 pfu or more, 10 pfu or more, 5 x 10¹ pfu or more, 10² pfu or more, 5xl0² pfu or more, 10³ pfu or more, 2.5xl0³ pfu or more, 5xl0³ pfu or more, 10⁴ pfu or more, 2.5 xlO⁴ pfu or more, 5 xlO⁴ pfu or more, or 10⁵ pfu or more in a subject. In certain embodiments, the virus achieves a viral yield of 1 pfu or more, 10 pfu or more, 5 x 1ο¹ pfu or more, 10² pfu or more, 5xl0² pfu or more, 10³ pfu or more, 2.5xl0³ pfu or more, 5xl0³ pfu or more, 10⁴ pfu or more, 2.5 xlO⁴ pfu or more, 5 xlO⁴ pfu or more, or 10⁵ pfu or more in a subject within 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 24 hours, or 48 hours. In certain embodiments, the virus achieves a viral yield of 1 pfu or more, 10 pfu or more, 10¹ pfu or more, 5 x 10¹ pfu or more, 10² pfu or more, 5xl0² pfu or more, 10³ pfu or more, 2.5xl0³ pfu or more, 5xl0³ pfu or more, 10⁴ pfu or more, 2.5 xlO⁴ pfu or more, 5 xlO⁴ pfu or more, or 10⁵ pfu or more in a subject within 48 hours, 72 hours, or 1 week. In accordance with these embodiments, the viral yield may be measured in the infected tissue or serum. In a specific embodiment, the subject is immunocompetent. In another embodiment, the subject is immunocompromised or immunosuppressed.

[0084] In some embodiments, the virus achieves a viral yield of 1 infectious unit or more, 10 infectious units or more, 5 x 10¹ infectious units or more, 10² infectious units or more, 5xl0² infectious units or more, 10³ infectious units or more, 2.5xl0³ infectious units or more, 5xl0³ infectious units or more, 10⁴ infectious units or more, 2.5 xlO⁴ infectious units or more, 5 xlO⁴ infectious units or more, or 10⁵ infectious units or more in a subject. In certain embodiments, the virus achieves a viral yield of 1 infectious unit or more, 10 infectious units or more, 5 x 10¹ infectious units or more, 10² infectious units or more, 5xl0² infectious units or more, 10³ infectious units or more, 2.5xl0³ infectious units or more, 5xl0³ infectious units or more, 10⁴ infectious units or more, 2.5 xlO⁴ infectious units or more, 5 xlO⁴ infectious units or more, or 10^s infectious units or more in a subject within 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 24 hours, or 48 hours. In certain embodiments, the virus achieves a viral yield of 1 infectious unit or more, 10 infectious units or more, 10¹ infectious units or more, 5 x 10¹ infectious units or more, 10² infectious units or more, 5xl0² infectious units or more,

10³ infectious units or more, 2.5xl0³ infectious units or more, 5xl0³ infectious units or more,

10⁴ infectious units or more, 2.5 xlO⁴ infectious units or more, 5 xlO⁴ infectious units or more, or 10⁵ infectious units or more in a subject within 48 hours, 72 hours, or 1 week. In accordance with these embodiments, the viral yield may be measured in the infected tissue or serum. In a specific embodiment, the subject is immunocompetent. In another embodiment, the subject is immunocompromised or immunosuppressed. In a specific embodiment, the virus achieves a yield of less than 10⁴ infectious units. In other embodiments the virus achieves a yield of 10⁵ or more infectious units.

[0085] In some embodiments, the virus achieves a viral titer of 1 infectious unit per ml or more, 10 infectious units per ml or more, 5 x 10¹ infectious units per ml or more, 10² infectious units per ml or more, 5xl0² infectious units per ml or more, 10³ infectious units per ml or more, 2.5xl0³ infectious units per ml or more, 5xl0³ infectious units per ml or more, 10⁴ infectious units per ml or more, 2.5 xlO⁴ infectious units per ml or more, 5 xlO⁴ infectious units per ml or more, or 10⁵ infectious units per ml or more in a subject. In certain embodiments, the virus achieves a viral titer of 10 infectious units per ml or more, 5 x 10¹ infectious units per ml or more, 10² infectious units per ml or more, 5xl0² infectious units per ml or more, 10³ infectious units per ml or more, 2.5xl0³ infectious units per ml or more, 5xl0³ infectious units per ml or more, 10⁴ infectious units per ml or more, 2.5 xlO⁴ infectious units per ml or more, 5 xlO⁴ infectious units per ml or more, or 10⁵ infectious units per ml or more in a subject within 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 24 hours, or 48 hours. In certain embodiments, the virus achieves a viral titer of 1 infectious unit per mL or more, 10 infectious units per ml or more, 5 x 1ο¹ infectious units per ml or more, 10² infectious units per ml or more, 5xl0² infectious units per ml or more, 10³ infectious units per mL or more, 2.5xl0³ infectious units per ml or more, 5xl0³ infectious units per ml or more, 10⁴ infectious units per ml or more, 2.5 xlO⁴ infectious units per ml or more, 5 xlO⁴ infectious units per ml or more, or 10⁵ infectious units per ml or more in a subject within 48 hours, 72 hours, or 1 week. In accordance with these embodiments, the viral titer may be measured in the infected tissue or serum. In a specific embodiment, the subject is immunocompetent. In another embodiment, the subject is immunocompromised or immunosuppressed. In a specific embodiment, the virus achieves a titer of less than 10⁴ infectious units per ml. In some embodiments, the virus achieves 10⁵ or more infectious units per ml.

3.2 In vitro Assays to Detect Antiviral Activity

[0086] The antiviral activity of compounds may be assessed in various in vitro assays described herein or others known to one of skill in the art. In specific embodiments, compounds exhibit an activity profile that is consistent with their ability to inhibit viral replication while maintaining low toxicity with respect to eukaryotic cells, preferably mammalian cells. For example, the effect of a compound on the replication of a virus may be determined by infecting cells with different dilutions of a virus in the presence or absence of various dilutions of a compound, and assessing the effect of the compound on, e.g. , viral replication, viral genome replication, and/or the synthesis of viral proteins. Alternatively, the effect of a compound on the replication of a virus may be determined by contacting cells with various dilutions of a compound or a placebo, infecting the cells with different dilutions of a virus, and assessing the effect of the compound on, e.g. , viral replication, viral genome replication, and/or the synthesis of viral proteins. Altered viral replication can be assessed by, e.g., plaque formation. The production of viral proteins can be assessed by, e.g. , ELISA, Western blot, immunofluorescence, or flow cytometry analysis. The production of viral nucleic acids can be assessed by, e.g., RT-PCR, PCR, Northern blot analysis, or Southern blot.

[0087] In certain embodiments, compounds reduce the replication of a virus by approximately 10%, preferably 15%, 25%, 30%, 45%, 50%, 60%, 75%, 95% or more relative to a negative control (e.g., PBS, DMSO) in an assay described herein or others known to one of skill in the art. In some embodiments, compounds reduce the replication of a virus by about at least 1.5 fold, 2, fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 75 fold, 100 fold, 500 fold, or 1000 fold relative to a negative control (e.g., PBS, DMSO) in an assay described herein or others known to one of skill in the art. In other embodiments, compounds reduce the replication of a virus by about at least 1.5 to 3 fold, 2 to 4 fold, 3 to 5 fold, 4 to 8 fold, 6 to 9 fold, 8 to 10 fold, 2 to 10 fold, 5 to 20 fold, 10 to 40 fold, 10 to 50 fold, 25 to 50 fold, 50 to 100 fold, 75 to 100 fold, 100 to 500 fold, 500 to 1000 fold, or 10 to 1000 fold relative to a negative control (e.g., PBS, DMSO) in an assay described herein or others known to one of skill in the art. In other embodiments, compounds reduce the replication of a virus by about 1 log, 1.5 logs, 2 logs, 2.5 logs, 3 logs, 3.5 logs, 4 logs, 4.5 logs, 5 logs or more relative to a negative control (e.g., PBS, DMSO) in an assay described herein or others known to one of skill in the art. In accordance with these embodiments, such compounds may be further assessed for their safety and efficacy in assays such as those described in Section 3, infra.

[0088] In certain embodiments, compounds reduce the replication of a viral genome by approximately 10%, preferably 15%, 25%, 30%, 45%, 50%, 60%, 75%, 95% or more relative to a negative control (e.g. , PBS, DMSO) in an assay described herein or others known to one of skill in the art. In some embodiments, compounds reduce the replication of a viral genome by about at least 1.5 fold, 2, fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 75 fold, 100 fold, 500 fold, or 1000 fold relative to a negative control (e.g. , PBS, DMSO) in an assay described herein or others known to one of skill in the art. In other embodiments, compounds reduce the replication of a viral genome by about at least 1.5 to 3 fold, 2 to 4 fold, 3 to 5 fold, 4 to 8 fold, 6 to 9 fold, 8 to 10 fold, 2 to 10 fold, 5 to 20 fold, 10 to 40 fold, 10 to 50 fold, 25 to 50 fold, 50 to 100 fold, 75 to 100 fold, 100 to 500 fold, 500 to 1000 fold, or 10 to 1000 fold relative to a negative control (e.g. , PBS, DMSO) in an assay described herein or others known to one of skill in the art. In other embodiments, compounds reduce the replication of a viral genome by about 1 log, 1.5 logs, 2 logs, 2.5 logs, 3 logs, 3.5 logs, 4 logs, 4.5 logs, 5 logs or more relative to a negative control (e.g., PBS, DMSO) in an assay described herein or others known to one of skill in the art. In accordance with these embodiments, such compounds may be further assessed for their safety and efficacy in assays such as those described in Section 3, infra.

[0089] In certain embodiments, compounds reduce the synthesis of viral proteins by approximately 10%, preferably 15%, 25%, 30%, 45%, 50%, 60%, 75%, 95% or more relative to a negative control (e.g., PBS, DMSO) in an assay described herein or others known to one of skill in the art. In some embodiments, compounds reduce the synthesis of viral proteins by approximately at least 1.5 fold, 2, fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 75 fold, 100 fold, 500 fold, or 1000 fold relative to a negative control (e.g. , PBS, DMSO) in an assay described herein or others known to one of skill in the art. In other embodiments, compounds reduce the synthesis of viral proteins by approximately at least 1.5 to 3 fold, 2 to 4 fold, 3 to 5 fold, 4 to 8 fold, 6 to 9 fold, 8 to 10 fold, 2 to 10 fold, 5 to 20 fold, 10 to 40 fold, 10 to 50 fold, 25 to 50 fold, 50 to 100 fold, 75 to 100 fold, 100 to 500 fold, 500 to 1000 fold, or 10 to 1000 fold relative to a negative control (e.g. , PBS, DMSO) in an assay described herein or others known to one of skill in the art. In other embodiments, compounds reduce the synthesis of viral proteins by approximately 1 log, 1.5 logs, 2 logs, 2.5 logs, 3 logs, 3.5 logs, 4 logs, 4.5 logs, 5 logs or more relative to a negative control (e.g., PBS, DMSO) in an assay described herein or others known to one of skill in the art. In accordance with these embodiments, such compounds may be further assessed for their safety and efficacy in assays such as those described in Section 3.3, infra. [0090] In some embodiments, compounds result in about a 1.5 fold or more, 2 fold or more, 3 fold or more, 4 fold or more, 5 fold or more, 6 fold or more, 7 fold or more, 8 fold or more, 9 fold or more, 10 fold or more, 15 fold or more, 20 fold or more, 25 fold or more, 30 fold or more, 35 fold or more, 40 fold or more, 45 fold or more, 0 fold or more, 60 fold or more, 70 fold or more, 80 fold or more, 90 fold or more, or 100 fold or more

inhibition/reduction of viral yield per round of viral replication. In certain embodiments, compounds result in about a 2 fold or more reduction inhibition/reduction of viral yield per round of viral replication. In specific embodiments, compounds result in about a 10 fold or more inhibition/reduction of viral yield per round of viral replication.

[0091] The in vitro antiviral assays can be conducted using any eukaryotic cell, including primary cells and established cell lines. The cell or cell lines selected should be susceptible to infection by a virus of interest. Non-limiting examples of mammalian cell lines that can be used in standard in vitro antiviral assays {e.g., viral cytopathic effect assays, neutral red update assays, viral yield assay, plaque reduction assays) for the respective viruses are set out in Table 1.

[0092] Sections 3.2.1 to 3.2.6 below provide non-limiting examples of antiviral assays that can be used to characterize the antiviral activity of compounds against the respective virus. One of skill in the art will know how to adapt the methods described in Sections 3.2.1 to 3.2.6 to different cell systems.

3.2.1 Viral Cytopathic Effect (CPE) Assay

[0093] CPE is the morphological changes that cultured cells undergo upon being infected by most viruses. These morphological changes can be observed easily in unfixed, unstained cells by microscopy. Forms of CPE, which can vary depending on the virus, include, but are not limited to, rounding of the cells, appearance of inclusion bodies in the nucleus and/or cytoplasm of infected cells, and formation of syncytia, or polykaryocytes (large cytoplasmic masses that contain many nuclei). For adenovirus infection, crystalline arrays of adenovirus capsids accumulate in the nucleus to form an inclusion body. [0094] The CPE assay can provide a measure of the antiviral effect of a compound. In a non-limiting example of such an assay, compounds are serially diluted (e.g. 1000, 500, 100, 50, 10, 1 μg/ml) and added to 3 wells containing a cell monolayer (preferably mammalian cells at 80-100% confluent) of a 96-well plate. Within 5 minutes, viruses are added and the plate sealed, incubated at 37°C for the standard time period required to induce near-maximal viral CPE (e.g. , approximately 48 to 120 hours, depending on the virus and multiplicity of infection). CPE is read microscopically after a known positive control drug is evaluated in parallel with compounds in each test. Non-limiting examples of positives controls are ribavirin for dengue, influenza, measles, respiratory syncytial, parainfluenza, Pichinde, Punta Toro and Venezuelan equine encephalitis viruses; cidofovir for adenovirus; pirodovir for rhinovirus; 6-azauridine for West Nile and yellow fever viruses; and alferon (interferon a-n3) for SA S virus. The data are expressed as 50% effective concentrations or approximated virus-inhibitory concentration, 50% endpoint (EC50) and cell-inhibitory concentration, 50% endpoint (IC₅₀). General selectivity index ("SI") is calculated as the IC5₀ divided by the EC50. These values can be calculated using any method known in the art, e.g. , the computer software program MacSynergy II by M.N. Prichard, K.R. Asaltine, and C. Shipman, Jr., University of Michigan, Ann Arbor, Michigan.

[0095] In one embodiment, a compound has an SI of greater than 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 1 1 , or 12, or 13, or 14, or 15, or 20, or 21 , or 22, or 23, or 24, or 25, or 30, or 35, or 40, or 45, or 50, or 60, or 70, or 80, or 90, or 100, or 200, or 300, or 400, or 500, 1,000, or 10,000. In some embodiments, a compound has an SI of greater than 10. In a specific embodiment, compounds with an SI of greater than 10 are further assessed in other in vitro and in vivo assays described herein or others known in the art to characterize safety and efficacy.

3.2.2 Neutral Red (NR) Dye Uptake Assay

[0096] The NR Dye Uptake assay can be used to validate the CPE inhibition assay (See Section 3.2.1). In a non-limiting example of such an assay, the same 96-well microplates used for the CPE inhibition assay can be used. Neutral red is added to the medium, and cells not damaged by virus take up a greater amount of dye. The percentage of uptake indicating viable cells is read on a microplate autoreader at dual wavelengths of 405 and 540 nm, with the difference taken to eliminate background. (See McManus et al., Appl. Environment. Microbiol. 31 :35-38, 1976). An EC5₀ is determined for samples with infected cells and contacted with compounds, and an IC5₀ is determined for samples with uninfected cells contacted with compounds.

3.2.3 Virus Yield Assay

[0097] Lysed cells and supematants from infected cultures such as those in the CPE inhibition assay (See section 3.2.1) can be used to assay for virus yield (production of viral particles after the primary infection). In a non- limiting example, these supematants are serial diluted and added onto monolayers of susceptible cells (e.g., Vero cells). Development of CPE in these cells is an indication of the presence of infectious viruses in the supernatant. The 90% effective concentration (EC90), the test compound concentration that inhibits vims yield by 1 logio, is determined from these data using known calculation methods in the art. In one embodiment, the EC₉₀ of compound is at least 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 20 fold, 30 fold, 40 fold, or 50 fold less than the EC90 of the negative control sample.

3.2.4 Plaque Reduction Assay

[0098] In a non-limiting example of such an assay, the vims is diluted into various concentrations and added to each well containing a monolayer of the target mammalian cells in triplicate. The plates are then incubated for a period of time to achieve effective infection of the control sample (e.g., 1 hour with shaking every fifteen minutes). After the incubation period, an equal amount of 1% agarose is added to an equal volume of each compound dilution prepared in 2x concentration. In certain embodiments, final compound

concentrations between 0.03 μ§/ιη1 to 100 μg/ml can be tested with a final agarose overlay concentration of 0.5%. The dmg agarose mixture is applied to each well in 2 ml volume and the plates are incubated for three days, after which the cells are stained with a 1.5% solution of neutral red. At the end of the 4-6 hour incubation period, the neutral red solution is aspirated, and plaques counted using a stereomicroscope. Alternatively, a final agarose concentration of 0.4% can be used. In other embodiments, the plates are incubated for more than three days with additional overlays being applied on day four and on day 8 when appropriate. In another embodiment, the overlay medium is liquid rather than semi-solid.

3.2.5 Vims Titer Assay

[0099] In this non-limiting example, a monolayer of the target mammalian cell line is infected with different amounts (e.g., multiplicity of 3 plaque forming units (pfu) or 5 pfu) of virus and subsequently cultured in the presence or absence of various dilutions of compounds (e.g. , 0.1 μg/ml, 1 μg/ml, 5 μg/ml, or 10 μg/ml). Infected cultures are harvested 48 hours or 72 hours post infection and titered by standard plaque assays known in the art on the appropriate target cell line (e.g. , Vera cells, MRC5 cells). In certain embodiments, culturing the infected cells in the presence of compounds reduces the yield of infectious virus by at least 1.5 fold, 2, fold, 3, fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 100 fold, 500 fold, or 1000 fold relative to culturing the infected cells in the absence of compounds. In a specific

embodiment, culturing the infected cells in the presence of compounds reduces the PFU/ml by at least 10 fold relative to culturing the infected cells in the absence of compounds.

[0100] In certain embodiments, culturing the infected cells in the presence of compounds reduces the yield of infectious virus by at least 0.5 loglO, 1 loglO, 1.5 loglO, 2 loglO, 2.5 loglO, 3 loglO, 3.5 loglO, 4 loglO, 4.5 loglO, 5 loglO, 5.5 loglO, 6 loglO, 6.5 loglO, 7 loglO, 7.5 loglO, 8 loglO, 8.5 loglO, or 9 loglO relative to culturing the infected cells in the absence of compounds. In a specific embodiment, culturing the infected cells in the presence of compounds reduces the yield of infectious virus by at least 1 log 10 or 2 log 10 relative to culturing the infected cells in the absence of compounds. In another specific embodiment, culturing the infected cells in the presence of compounds reduces the yield of infectious virus by at least 2 log 10 relative to culturing the infected cells in the absence of compounds.

3.2.6 Flow Cytometry Assay

[0101] Flow cytometry can be utilized to detect expression of virus antigens in infected target cells cultured in the presence or absence of compounds (See, e.g., McSharry et al , Clinical Microbiology Rev., 1994, 7:576-604). Non-limiting examples of viral antigens that can be detected on cell surfaces by flow cytometry include, but are not limited to gB, gC, gC, and gE of HSV; E protein of Japanese encephalitis; virus gp52 of mouse mammary tumor virus; gpl of varicella-zoster virus; gB of HCMV; gpl60/120 of HIV; HA of influenza;

gpl 10/60 of HHV-6; and H and F of measles virus. In other embodiments, intracellular viral antigens or viral nucleic acid can be detected by flow cytometry with techniques known in the art.

3.2.7 Approach To Identifying and Measuring Metabolic Fluxes Regulated By Viral Infection And Anti-Viral Compounds [0102] Viruses can alter cellular metabolic activity through a variety of routes. These include affecting transcription, translation, and/or degradation of mR As and/or proteins, relocalization of mRNAs and/or proteins, covalent modification of proteins, allosteric regulation of enzymes or other proteins, and alterations to the composition of protein- containing complexes that modify their activity. The net result of all of these changes is modulation of metabolic fluxes to meet the needs of the virus. Thus, metabolic flux changes represent the ultimate endpoint of the virus' efforts to modulate host cell metabolism. Fluxes that are increased by the virus are especially likely to be critical to viral survival and replication and to represent valuable drug targets. Such fluxes are used identify involved enzymes and/or to observe the effects of antiviral compounds on the flux / enzyme targets.

[0103] The essence of this kinetic flux profiling (KFP) approach is as follows:

[0104] (1) Cells (either uninfected or infected with virus) are rapidly switched from unlabeled to isotope-labeled nutrient (or vice versa); for the present purposes, preferred nutrients include uniformly or partially ¹³C-labeled or ¹⁵N-labeled glucose, glutamine, glutamate, or related compounds including without limitation pyruvate, lactate, glycerol, acetate, aspartate, arginine, and urea. Labels can include all known isotopes of H, C, N, O, P, or S, including both stable and radioactive labels. Results are dependent on the interplay between the host cell type and the viral pathogen, including the viral load and time post infection.

[0105] (2) Metabolism is quenched at various time points following the isotope- switch (e.g., 0.2, 0.5, 1, 2, 5, 10, 20, 30 min and 1 , 2, 4, 8, 12, 16, 24, 36, 48 h or a subset or variant thereof). One convenient means of metabolism quenching is addition of cold (e.g., dry-ice temperature) methanol, although other solvents and temperatures, including also boiling solvents, are possible.

[0106] (3) The metabolome, including its extent of isotope labeling, is quantified for each collected sample. One convenient means of such quantitation is extraction of metabolites from the cells followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the extract. Appropriate extraction protocols and LC-MS/MS methods are known in the art. See the following citations, which are herein incorporated by reference (Bajad et al, 2006, J Chromatogr. A 1 125:76-88; Boiling and Fiehn, 2005, Plant Physiol. 139: 1995-2005; Coulier et al , 2006, Anal Chem. 78:6573-6582; Kimball and Rabinowitz, 2006, Anal Biochem. 358:273-280; Lu et al , 2006, J. Am. Soc. Mass Spectrom. 17:37-50;Lu et al, 2007, J Am Soc Mass Spectrom. 18:898-909; Luo et al, 2007, J.

Chromatogr. A 1147: 153-164; Maharjan and Ferenci, 2003, Anal Biochem 313 : 145-154; Milne et al, 2006, Methods 39:92-103; Munger et al , 2006, PLoS Pathog. 2:el32; Olsson et al , 2004, Anal Chem. 76:2453-2461 ; Rabinowitz and Kimball, 2007, Anal Chem. 79:6167- 73; Schaub et al, 2006, Biotechnol. Prog. 22: 1434-1442; van Winden et al, 2005, FEMS Yeast Research 5 :559-568; Villas-Boas et al , 2005, Yeast 22: 1155-1169.; Wittmann et al, 2004, Anal Biochem. 327: 135-139; Wu et al , 2005, Anal Biochem. 336: 164-171 ; Yuan et al , 2006, Nat. Chem. Biol. 2:529-530).

[0107] (4) The resulting data is analyzed to determine the cellular metabolic fluxes. Further details of kinetic flux profiling (KFP) are disclosed in Munger et al, 2008, Nat. Biotech. 26: 1 179, which is incorporated by reference herein.

3.3 Characterization of Safety and Efficacy of Compounds

[0108] The safety and efficacy of compounds can be assessed using technologies known to one of skill in the art. Sections 3.4 and 3.5 below provide non- limiting examples of cytotoxicity assays and animal model assays, respectively, to characterize the safety and efficacy of compounds. In certain embodiments, the cytotoxicity assays described in Section 3.4 are conducted following the in vitro antiviral assays described in Section 3, supra. In other embodiments, the cytotoxicity assays described in Section 3.4 are conducted before or concurrently with the in vitro antiviral assays described in Section 3, supra.

[0109] In some embodiments, compounds differentially affect the viability of uninfected cells and cells infected with virus. The differential effect of a compound on the viability of virally infected and uninfected cells may be assessed using techniques such as those described in Section 3.4, infra, or other techniques known to one of skill in the art. In certain embodiments, compounds are more toxic to cells infected with a virus than uninfected cells. In specific embodiments, compounds preferentially affect the viability of cells infected with a virus. Without being bound by any particular concept, the differential effect of a compound on the viability of uninfected and virally infected cells may be the result of the compound targeting a particular enzyme or protein that is differentially expressed or regulated or that has differential activities in uninfected and virally infected cells. For example, viral infection and/or viral replication in an infected host cells may alter the expression, regulation, and/or activities of enzymes and/or proteins. Accordingly, in some embodiments, other compounds that target the same enzyme, protein or metabolic pathway are examined for antiviral activity. In other embodiments, congeners of compounds that differentially affect the viability of cells infected with virus are designed and examined for antiviral activity. Non-limiting examples of antiviral assays that can be used to assess the antiviral activity of compound are provided in Section 3, supra.

3.4 Cytotoxicity Studies

[0110] In a preferred embodiment, the cells are animal cells, including primary cells and cell lines. In some embodiments, the cells are human cells. In certain embodiments, cytotoxicity is assessed in one or more of the following cell lines: U937, a human monocyte cell line; primary peripheral blood mononuclear cells (PBMC); Huh7, a human

hepatoblastoma cell line; 293T, a human embryonic kidney cell line; and THP-1 , monocytic cells. Other non- limiting examples of cell lines that can be used to test the cytotoxicity of compounds are provided in Table 1.

[0111] Many assays well-known in the art can be used to assess viability of cells (infected or uninfected) or cell lines following exposure to a compound and, thus, determine the cytotoxicity of the compound. For example, cell proliferation can be assayed by measuring Bromodeoxyuridine (BrdU) incorporation (See, e.g., Hoshino et al, 1986, Int. J. Cancer 38, 369; Campana et al , 1988, J. Immunol. Meth. 107:79), (3H) thymidine incorporation (See, e.g., Chen, J., 1996, Oncogene 13 : 1395-403; Jeoung, J., 1995, J. Biol. Chem. 270: 18367 73), by direct cell count, or by detecting changes in transcription, translation or activity of known genes such as proto-oncogenes (e.g., fos, myc) or cell cycle markers (Rb, cdc2, cyclin A, D l , D2, D3, E, etc). The levels of such protein and mRNA and activity can be determined by any method well known in the art. For example, protein can be quantitated by known immunodiagnostic methods such as ELISA, Western blotting or immunoprecipitation using antibodies, including commercially available antibodies. mRNA can be quantitated using methods that are well known and routine in the art, for example, using northern analysis, RNase protection, or polymerase chain reaction in connection with reverse transcription. Cell viability can be assessed by using trypan-blue staining or other cell death or viability markers known in the art. In a specific embodiment, the level of cellular ATP is measured to determined cell viability.

[0112] In specific embodiments, cell viability is measured in three-day and seven-day periods using an assay standard in the art, such as the CellTiter-Glo Assay Kit (Promega) which measures levels of intracellular ATP. A reduction in cellular ATP is indicative of a cytotoxic effect. In another specific embodiment, cell viability can be measured in the neutral red uptake assay. In other embodiments, visual observation for morphological changes may include enlargement, granularity, cells with ragged edges, a filmy appearance, rounding, detachment from the surface of the well, or other changes. These changes are given a designation of T (100% toxic), PVH (partially toxic-very heavy-80%), PH (partially toxic- heavy-60%), P (partially toxic^ 0%), Ps (partially toxic-slight-20%), or 0 (no toxicity-0%), conforming to the degree of cytotoxicity seen. A 50% cell inhibitory (cytotoxic)

concentration (IC₅₀) is determined by regression analysis of these data.

[0113] Compounds can be tested for in vivo toxicity in animal models. For example, animal models, described herein and/or others known in the art, used to test the antiviral activities of compounds can also be used to determine the in vivo toxicity of these compounds. For example, animals are administered a range of concentrations of compounds. Subsequently, the animals are monitored over time for lethality, weight loss or failure to gain weight, and/or levels of serum markers that may be indicative of tissue damage (e.g. , creatine phosphokinase level as an indicator of general tissue damage, level of glutamic oxalic acid transaminase or pyruvic acid transaminase as indicators for possible liver damage). These in vivo assays may also be adapted to test the toxicity of various administration mode and/or regimen in addition to dosages.

[0114] The toxicity and/or efficacy of a compound in accordance with the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5₀/ED5₀. A compound identified in accordance with the invention that exhibits large therapeutic indices is preferred. While a compound identified in accordance with the invention that exhibits toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0115] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage of a compound identified in accordance with the invention for use in humans. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED5₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC5₀ {i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high-performance liquid chromatography. Additional information concerning dosage determination is provided in Section 5.3, infra.

3.5 Animal Models

[0116] Compounds and compositions are preferably assayed in vivo for the desired therapeutic or prophylactic activity prior to use in humans. For example, in vivo assays can be used to determine whether it is preferable to administer a compound and/or another therapeutic agent. To assess the use of a compound to prevent a viral infection, the compound can be administered before the animal is infected with the virus. In another embodiment, a compound can be administered to the animal at the same time that the animal is infected with the virus. To assess the use of a compound to treat or manage a viral infection, in one embodiment, the compound is administered after a viral infection in the animal. In another embodiment, a compound is administered to the animal at the same time that the animal is infected with the virus to treat and/or manage the viral infection. In a specific embodiment, the compound is administered to the animal more than one time.

[0117] Compounds can be tested for antiviral activity against virus in animal model systems including, but are not limited to, rats, mice, chicken, cows, monkeys, pigs, goats, sheep, dogs, rabbits, guinea pigs, etc. Preferably, such models are chosen or adapted to approximate pathogenesis of the virus in a human subject. In a specific embodiment of the invention, compounds are tested in a mouse model adapted to approximate human pathogenesis. Such model systems are widely used and well-known to the skilled artisan.

[0118] Animals are infected with virus and concurrently or subsequently treated with a compound or placebo. Samples obtained from these animals {e.g., serum, urine, sputum, semen, saliva, plasma, or tissue sample) can be tested for viral replication via well known methods in the art, e.g., those that measure altered viral replication (as determined, e.g., by plaque formation) or the production of viral proteins (as determined, e.g., by Western blot, ELISA, or flow cytometry analysis) or viral nucleic acids (as determined, e.g. , by T-PC , northern blot analysis or southern blot). For quantitation of virus in tissue samples, tissue samples are homogenized in phosphate-buffered saline (PBS), and dilutions of clarified homogenates are adsorbed for 1 hour at 37°C onto monolayers of cells (e.g., Vera, CEF or MDCK cells). In other assays, histopathologic evaluations are performed after infection, preferably evaluations of the organ(s) the virus is known to target for infection. Virus immunohistochemistry can be performed using a viral-specific monoclonal antibody. Non- limiting exemplary animal models are described below (Sections 3.5.1 - 3.5.2).

[0119] The effect of a compound on the virulence of a virus can also be determined using in vivo assays in which the titer of the virus in an infected subject administered a compound, the length of survival of an infected subject administered a compound, the immune response in an infected subject administered a compound, the number, duration and/or severity of the symptoms in an infected subject administered a compound, and/or the time period before onset of one or more symptoms in an infected subject administered a compound is assessed. Techniques known to one of skill in the art can be used to measure such effects.

3.5.1 Dengue Virus

[0120] Dengue virus appears to possess mechanisms to overcome or circumvent innate immunity including type I (IFN-α/β) and/or type II (IFN-γ) interferon responses in human and NHP hosts. Since dengue virus does not generally infect laboratory animals, the majority of in vivo work with dengue virus has been conducted in AG129 mice that are deficient in interferon receptors. Resistance to interferon is regarded as highly important in dengue virus pathogenesis. (Yauch et al, 2008, Antiviral Res. 80, 87-93). Another model is a mouse model that displays antibody-dependent enhancement of infection (ADE)-induced disease. (Balsitis et al., 2010, PLoS Pathogens, 6(2), el000790). ADE is a characteristic of dengue virus pathogenesis that occurs when non-neutralising antiviral antibodies enhance viral entry into host cells, leading to increased infectivity in the host cells. This phenomenon is often observed when a person who has previously been infected with one serotype of dengue virus becomes infected many months or years later with a different serotype. In such cases, the clinical course of the disease is more severe, and these people have higher viremia compared with those in whom ADE has not occurred.

[0121] Such models can be used to assay antiviral activity of compounds in vivo. Using such models, inhibitors of host a-glucosidase have shown efficacy against dengue virus challenge in vivo. These include 6-O-butanoyl castanospermine (BuCast / Celgosivir) and N-nonyl-deoxynojirimycin (NN-DNJ). Further, as in rhesus macaques, in AG 129 mice, subcutaneous injection of ribavirin did not produce a positive response.

3.5.2 Yellow fever virus

[0122] Yellow fever virus also possesses mechanisms to overcome or circumvent innate immunity. As for dengue virus, work with yellow fever virus can be conducted in mice that are deficient for interferon responses. These models include A129 mice, which are deficient for type I (IFN-α/β) interferon response, and AG 129 mice, which are deficient for both type I and type II (IFN-γ) interferon responses. (Meier et ciL, 2008, PLoS Pathogens 5(10): el000614).

4. Pharmaceutical Compositions

[0123] Any compound described or incorporated by referenced herein may optionally be in the form of a composition comprising the compound.

[0124] In certain embodiments provided herein, compositions (including

pharmaceutical compositions) comprise a compound and a pharmaceutically acceptable carrier, excipient, or diluent.

[0125] In other embodiments, provided herein are pharmaceutical compositions comprising an effective amount of a compound and a pharmaceutically acceptable carrier, excipient, or diluent. The pharmaceutical compositions are suitable for veterinary and/or human administration.

[0126] The pharmaceutical compositions provided herein can be in any form that allows for the composition to be administered to a subject, said subject preferably being an animal, including, but not limited to a human, mammal, or non-human animal, such as a cow, horse, sheep, pig, fowl, cat, dog, mouse, rat, rabbit, guinea pig, etc., and is more preferably a mammal, and most preferably a human.

[0127] In a specific embodiment and in this context, the term "pharmaceutically acceptable carrier, excipient or diluent" means a carrier, excipient or diluent approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g. , Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0128] Typical compositions and dosage forms comprise one or more excipients. Suitable excipients are well-known to those skilled in the art of pharmacy, and non limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a patient and the specific active ingredients in the dosage form. The composition or single unit dosage form, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0129] Lactose free compositions can comprise excipients that are well known in the art and are listed, for example, in the U.S. Pharmacopeia (USP) SP (XXI)/NF (XVI). In general, lactose free compositions comprise an active ingredient, a binder/filler, and a lubricant in pharmaceutically compatible and pharmaceutically acceptable amounts.

Preferred lactose free dosage forms comprise a compound, microcrystalline cellulose, pre gelatinized starch, and magnesium stearate.

[0130] Further provided herein are anhydrous pharmaceutical compositions and dosage forms comprising one or more compounds, since water can facilitate the degradation of some compounds. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical arts as a means of simulating long term storage in order to determine characteristics such as shelf life or the stability of formulations over time. See, e.g., Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed., Marcel Dekker, NY, NY, 1995, pp. 379 80. In effect, water and heat accelerate the decomposition of some compounds. Thus, the effect of water on a formulation can be of great significance since moisture and/or humidity are commonly encountered during manufacture, handling, packaging, storage, shipment, and use of formulations. [0131] Anhydrous compositions and dosage forms provided herein can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Compositions and dosage forms that comprise lactose and at least one compound that comprises a primary or secondary amine are preferably anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected.

[0132] An anhydrous composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions are preferably packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0133] Further provided herein are compositions and dosage forms that comprise one or more agents that reduce the rate by which a compound will decompose. Such agents, which are referred to herein as "stabilizers," include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.

[0134] The compositions and single unit dosage forms can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such compositions and dosage forms will contain a prophylactically or therapeutically effective amount of a compound preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. In a preferred embodiment, the compositions or single unit dosage forms are sterile and in suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject.

[0135] Compositions provided herein are formulated to be compatible with the intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), intranasal, transdermal (topical), transmucosal, intra-synovial, ophthalmic, and rectal administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, ophthalmic, or topical administration to human beings. In a preferred embodiment, a composition is formulated in accordance with routine procedures for subcutaneous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g. , aqueous or non aqueous liquid suspensions, oil in water emulsions, or a water in oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.

[0136] The composition, shape, and type of dosage forms of the invention will typically vary depending on their use.

[0137] Generally, the ingredients of compositions provided herein are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0138] Pharmaceutical compositions provided herein that are suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990). [0139] Typical oral dosage forms provided herein are prepared by combining a compound in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, micro crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.

[0140] Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid excipients are employed. If desired, tablets can be coated by standard aqueous or nonaqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.

[0141] For example, a tablet can be prepared by compression or molding.

Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0142] Examples of excipients that can be used in oral dosage forms provided herein include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

[0143] Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms provided herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre gelatinized starch, and mixtures thereof. The binder or filler in pharmaceutical compositions provided herein is typically present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form.

[0144] Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL PH 101 , AVICEL PH 103 AVICEL RC 581 , AVICEL PH 105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, PA), and mixtures thereof. A specific binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC 581. Suitable anhydrous or low moisture excipients or additives include AVICEL PH 103™ and Starch 1500 LM.

[0145] Disintegrants are used in the compositions provided herein to provide tablets that disintegrate when exposed to an aqueous environment. Tablets that contain too much disintegrant may disintegrate in storage, while those that contain too little may not disintegrate at a desired rate or under the desired conditions. Thus, a sufficient amount of disintegrant that is neither too much nor too little to detrimentally alter the release of the active ingredients should be used to form solid oral dosage forms provided herein. The amount of disintegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art. Typical pharmaceutical compositions comprise from about 0.5 to about 15 weight percent of disintegrant, specifically from about 1 to about 5 weight percent of disintegrant.

[0146] Disintegrants that can be used in pharmaceutical compositions and dosage forms provided herein include, but are not limited to, agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, pre gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.

[0147] Lubricants that can be used in pharmaceutical compositions and dosage forms provided herein include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional lubricants include, for example, a syloid silica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore, MD), a coagulated aerosol of synthetic silica (marketed by Degussa Co. of Piano, TX), CAB O SIL (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, MA), and mixtures thereof. If used at all, lubricants are typically used in an amount of less than about 1 weight percent of the pharmaceutical compositions or dosage forms into which they are incorporated.

[0148] A compound can be administered by controlled release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos.: 3,845,770; 3,916,899; 3,536,809;

3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5, 120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of which is incorporated herein by reference. Such dosage forms can be used to provide slow or controlled release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions.

Suitable controlled release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the active ingredients of the invention. The invention thus encompasses single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled release.

[0149] All controlled release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non controlled counterparts. Ideally, the use of an optimally designed controlled release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and can thus affect the occurrence of side (e.g., adverse) effects.

[0150] Most controlled release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled release of an active ingredient can be stimulated by various conditions including, but not limited to, H, temperature, enzymes, water, or other physiological conditions or agents.

[0151] Parenteral dosage forms can be administered to patients by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Because their administration typically bypasses patients' natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.

[0152] Suitable vehicles that can be used to provide parenteral dosage forms provided herein are well known to those skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0153] Agents that increase the solubility of one or more of the compounds provided herein can also be incorporated into the parenteral dosage forms provided herein.

[0154] Transdermal, topical, and mucosal dosage forms provided herein include, but are not limited to, ophthalmic solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions, or other forms known to one of skill in the art. See, e.g. , Remington's Pharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton PA (1980 & 1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia (1985). Dosage forms suitable for treating mucosal tissues within the oral cavity can be formulated as mouthwashes or as oral gels. Further, transdermal dosage forms include "reservoir type" or "matrix type" patches, which can be applied to the skin and worn for a specific period of time to permit the penetration of a desired amount of active ingredients.

[0155] Suitable excipients (e.g., carriers and diluents) and other materials that can be used to provide transdermal, topical, and mucosal dosage forms provided herein are well known to those skilled in the pharmaceutical arts, and depend on the particular tissue to which a given pharmaceutical composition or dosage form will be applied. With that fact in mind, typical excipients include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3 diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof to form lotions, tinctures, creams, emulsions, gels or ointments, which are non toxic and pharmaceutically acceptable. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art. See, e.g., Remington's Pharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton PA (1980 & 1990).

[0156] Depending on the specific tissue to be treated, additional components may be used prior to, in conjunction with, or subsequent to treatment with a compound. For example, penetration enhancers can be used to assist in delivering the active ingredients to the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols such as ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; and various water soluble or insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate).

[0157] The pH of a pharmaceutical composition or dosage form, or of the tissue to which the pharmaceutical composition or dosage form is applied, may also be adjusted to improve delivery of one or more compounds. Similarly, the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery. Agents such as stearates can also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of one or more compounds so as to improve delivery. In this regard, stearates can serve as a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery enhancing or penetration enhancing agent. Different salts, hydrates or solvates of the compounds can be used to further adjust the properties of the resulting composition.

[0158] In certain specific embodiments, the compositions are in oral, injectable, or transdermal dosage forms. In one specific embodiment, the compositions are in oral dosage forms. In another specific embodiment, the compositions are in the form of injectable dosage forms. In another specific embodiment, the compositions are in the form of transdermal dosage forms. 5. Prophylactic and Therapeutic Methods

[0159] The present invention provides methods of preventing, treating and/or managing a viral infection, said methods comprising administering to a subject in need thereof one or more compounds. In a specific embodiment, the invention provides a method of preventing, treating and/or managing a viral infection, said method comprising

administering to a subject in need thereof a dose of a prophylactically or therapeutically effective amount of one or more compounds or a composition comprising a compound. A compound or a composition comprising a compound may be used as any line of therapy (e.g., a first, second, third, fourth or fifth line therapy) for a viral infection.

[0160] In another embodiment, the invention relates to a method for reversing or redirecting metabolic flux altered by viral infection in a human subject by administering to a human subject in need thereof, an effective amount of one or more compounds or a composition comprising one or more compounds. For example, viral infection can be treated using combinations of the enzyme inhibition compounds that produce beneficial results, e.g., synergistic effect; reduction of side effects; a higher therapeutic index.

[0161] In specific embodiments, a compound is the only active ingredient administered to prevent, treat, manage or ameliorate said viral infection. In a certain embodiment, a composition comprising a compound is the only active ingredient.

[0162] The choice of compounds to be used depends on a number of factors, including but not limited to the type of viral infection, health and age of the patient, and toxicity or side effects. For example, treatments that inhibit enzymes required for core ATP production, such as proton ATPase are not preferred unless given in a regimen that compensates for the toxicity; e.g., using a localized delivery system that limits systemic distribution of the drug.

[0163] The present invention encompasses methods for preventing, treating, and/or managing a viral infection for which no antiviral therapy is available. The present invention also encompasses methods for preventing, treating, and/or managing a viral infection as an alternative to other conventional therapies.

[0164] The present invention also provides methods of preventing, treating and/or managing a viral infection, said methods comprising administering to a subject in need thereof one or more of the compounds and one or more other therapies (e.g. , prophylactic or therapeutic agents). In a specific embodiment, the other therapies are currently being used, have been used or are known to be useful in the prevention, treatment and/or management of a viral infection. Non-limiting examples of such therapies are provided in Section 5, infra. In a specific embodiment, one or more compounds are administered to a subject in combination with one or more of the therapies described in Section 5, infra. In another embodiment, one or more compounds are administered to a subject in combination with a supportive therapy, a pain relief therapy, or other therapy that does not have antiviral activity.

[0165] The combination therapies of the invention can be administered sequentially or concurrently. In one embodiment, the combination therapies of the invention comprise a compound and at least one other therapy which has the same mechanism of action. In another embodiment, the combination therapies of the invention comprise a compound and at least one other therapy which has a different mechanism of action than the compound.

[0166] In a specific embodiment, the combination therapies of the present invention improve the prophylactic and/or therapeutic effect of a compound by functioning together with the compound to have an additive or synergistic effect. In another embodiment, the combination therapies of the present invention reduce the side effects associated with each therapy taken alone.

[0167] The prophylactic or therapeutic agents of the combination therapies can be administered to a subject in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapies can be administered concurrently to a subject in separate pharmaceutical compositions. The prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration.

5.1 Patient Population

[0168] According to the invention, compounds, compositions comprising a compound, or a combination therapy is administered to a subject suffering from a viral infection. In other embodiments, compounds, compositions comprising a compound, or a combination therapy is administered to a subject predisposed or susceptible to a viral infection. In some embodiments, compounds, compositions comprising a compound, or a combination therapy is administered to a subject that lives in a region where there has been or might be an outbreak with a viral infection. In some embodiments, the viral infection is a latent viral infection. In one embodiment, a compound or a combination therapy is administered to a human infant. In one embodiment, a compound or a combination therapy is administered to a premature human infant. In other embodiments, the viral infection is an active infection. In yet other embodiments, the viral infection is a chronic viral infection.

[0169] In certain embodiments, a compound, a composition comprising a compound, or a combination therapy is administered to a mammal which is 0 to 6 months old, 6 to 12 months old, 1 to 5 years old, 5 to 10 years old, 10 to 15 years old, 15 to 20 years old, 20 to 25 years old, 25 to 30 years old, 30 to 35 years old, 35 to 40 years old, 40 to 45 years old, 45 to 50 years old, 50 to 55 years old, 55 to 60 years old, 60 to 65 years old, 65 to 70 years old, 70 to 75 years old, 75 to 80 years old, 80 to 85 years old, 85 to 90 years old, 90 to 95 years old or 95 to 100 years old. In certain embodiments, a compound, a composition comprising a compound, or a combination therapy is administered to a human at risk for a virus infection. In certain embodiments, a compound, a composition comprising a compound, or a combination therapy is administered to a human with a virus infection. In certain embodiments, the patient is a human 0 to 6 months old, 6 to 12 months old, 1 to 5 years old, 5 to 10 years old, 5 to 12 years old, 10 to 15 years old, 15 to 20 years old, 13 to 19 years old, 20 to 25 years old, 25 to 30 years old, 20 to 65 years old, 30 to 35 years old, 35 to 40 years old, 40 to 45 years old, 45 to 50 years old, 50 to 55 years old, 55 to 60 years old, 60 to 65 years old, 65 to 70 years old, 70 to 75 years old, 75 to 80 years old, 80 to 85 years old, 85 to 90 years old, 90 to 95 years old or 95 to 100 years old. In some embodiments, a compound, a composition comprising a compound, or a combination therapy is administered to a human infant. In other embodiments, a compound, or a combination therapy is administered to a human child. In other embodiments, a compound, a composition comprising a compound, or a combination therapy is administered to a human adult. In yet other embodiments, a compound, a composition comprising a compound, or a combination therapy is administered to an elderly human.

[0170] In certain embodiments, a compound, a composition comprising a compound, or a combination therapy is administered to a pet, e.g. , a dog or cat. In certain embodiments, a compound, a composition comprising a compound, or a combination therapy is

administered to a farm animal or livestock, e.g., pig, cows, horses, chickens, etc. In certain embodiments, a compound, a composition comprising a compound, or a combination therapy is administered to a bird, e.g., ducks or chicken.

[0171] In certain embodiments, a compound, a composition comprising a compound, or a combination therapy is administered to a primate, preferably a human, or another mammal, such as a pig, cow, horse, sheep, goat, dog, cat and rodent, in an immunocompromised state or immunosuppressed state or at risk for becoming immunocompromised or immunosuppressed. In certain embodiments, a compound, a composition comprising a compound, or a combination therapy is administered to a subject receiving or recovering from immunosuppressive therapy. In certain embodiments, a compound, a composition comprising a compound, or a combination therapy is administered to a subject that has or is at risk of getting cancer, AIDS, another viral infection, or a bacterial infection. In certain embodiments, a subject that is, will or has undergone surgery, chemotherapy and/or radiation therapy. In certain embodiments, a compound, a composition comprising a compound, or a combination therapy is administered to a subject that has cystic fibrosis, pulmonary fibrosis, or another disease which makes the subject susceptible to a viral infection. In certain embodiments, a compound, a composition comprising a compound, or a combination therapy is administered to a subject that has, will have or had a tissue transplant. In some embodiments, a compound, a composition comprising a compound, or a combination therapy is administered to a subject that lives in a nursing home, a group home (i.e., a home for 10 or more subjects), or a prison. In some embodiments, a compound, a composition comprising a compound, or a combination therapy is administered to a subject that attends school (e.g., elementary school, middle school, junior high school, high school or university) or daycare. In some embodiments, a compound, a composition comprising a compound, or a combination therapy is administered to a subject that works in the healthcare area, such as a doctor or a nurse, or in a hospital. In certain embodiments, a compound, a composition comprising a compound, or a combination therapy is administered to a subject that is pregnant or will become pregnant.

[0172] In some embodiments, a patient is administered a compound or a composition comprising a compound, or a combination therapy before any adverse effects or intolerance to therapies other than compounds develops. In some embodiments, compounds or compositions comprising one or more compounds, or combination therapies are administered to refractory patients. In a certain embodiment, refractory patient is a patient refractory to a standard antiviral therapy. In certain embodiments, a patient with a viral infection, is refractory to a therapy when the infection has not significantly been eradicated and/or the symptoms have not been significantly alleviated. The determination of whether a patient is refractory can be made either in vivo or in vitro by any method known in the art for assaying the effectiveness of a treatment of infections, using art-accepted meanings of "refractory" in such a context. In various embodiments, a patient with a viral infection is refractory when viral replication has not decreased or has increased.

[0173] In some embodiments, compounds or compositions comprising one or more compounds, or combination therapies are administered to a patient to prevent the onset or reoccurrence of viral infections in a patient at risk of developing such infections. In some embodiments, compounds or compositions comprising one or more compounds, or combination therapies are administered to a patient who are susceptible to adverse reactions to conventional therapies.

[0174] In some embodiments, one or more compounds or compositions comprising one or more compounds, or combination therapies are administered to a patient who has proven refractory to therapies other than compounds, but are no longer on these therapies. In certain embodiments, the patients being managed or treated in accordance with the methods of this invention are patients already being treated with antibiotics, anti-virals, anti-fungals, or other biological therapy/immunotherapy. Among these patients are refractory patients, patients who are too young for conventional therapies, and patients with reoccurring viral infections despite management or treatment with existing therapies.

[0175] In some embodiments, the subject being administered one or more compounds or compositions comprising one or more compounds, or combination therapies has not received a therapy prior to the administration of the compounds or compositions or combination therapies. In other embodiments, one or more compounds or compositions comprising one or more compounds, or combination therapies are administered to a subject who has received a therapy prior to administration of one or more compounds or

compositions comprising one or more compounds, or combination therapies. In some embodiments, the subject administered a compound or a composition comprising a compound was refractory to a prior therapy or experienced adverse side effects to the prior therapy or the prior therapy was discontinued due to unacceptable levels of toxicity to the subject.

5.2 Mode of Administration

[0176] When administered to a patient, a compound is preferably administered as a component of a composition that optionally comprises a pharmaceutically acceptable vehicle. The composition can be administered orally, or by any other convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g. , oral mucosa, rectal, and intestinal mucosa) and may be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, and can be used to administer the compound and pharmaceutically acceptable salts thereof.

[0177] Methods of administration include but are not limited to parenteral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin. The mode of administration is left to the discretion of the practitioner. In most instances, administration will result in the release of a compound into the bloodstream.

[0178] In specific embodiments, it may be desirable to administer a compound locally. This may be achieved, for example, and not by way of limitation, by local infusion, topical application, e.g. , in conjunction with a wound dressing, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In such instances, administration may selectively target a local tissue without substantial release of a compound into the bloodstream.

[0179] In certain embodiments, it may be desirable to introduce a compound into the central nervous system by any suitable route, including intraventricular, intrathecal and epidural injection. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.

[0180] Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant. In certain embodiments, a compound is formulated as a suppository, with traditional binders and vehicles such as triglycerides.

[0181] For viral infections with cutaneous manifestations, the compound can be administered topically. Similarly, for viral infections with ocular manifestation, the compounds can be administered ocularly.

[0182] In another embodiment, a compound is delivered in a vesicle, in particular a liposome (See Langer, 1990, Science 249: 1527 1533; Treat et al, in Liposomes in the Therapy of Infectious Disease and Bacterial infection, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353 365 (1989); Lopez Berestein, ibid. , pp. 317 327; See generally ibid.).

[0183] In another embodiment, a compound is delivered in a controlled release system (See, e.g. , Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115 138 (1984)). Examples of controlled-release systems are discussed in the review by Langer, 1990, Science 249: 1527 1533 may be used. In one embodiment, a pump may be used (See Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201 ; Buchwald et al , 1980, Surgery 88:507; Saudek et al, 1989, N. Engl. J. Med. 321 :574). In another embodiment, polymeric materials can be used (See Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23 :61 ; See also Levy et al, 1985, Science 228: 190; During et al , 1989, Ann. Neurol. 25 :351 ; Howard et al , 1989, J. Neurosurg. 71 : 105). In a specific embodiment, a controlled-release system comprising a compound is placed in close proximity to the tissue infected with a virus to be prevented, treated and/or managed. In accordance with this embodiment, the close proximity of the controlled-release system to the infection may result in only a fraction of the dose of the compound required if it is systemically administered.

[0184] In certain embodiments, it may be preferable to administer a compound via the natural route of infection of the virus against which a compound has antiviral activity. For example, it may be desirable to administer a compound of the invention into the lungs by any suitable route to treat or prevent an infection of the respiratory tract by viruses (e.g. , influenza virus). Pulmonary administration can also be employed, e.g. , by use of an inhaler or nebulizer, and formulation with an aerosolizing agent for use as a spray.

5.3 Dosages & Frequency of Administration

[0185] The amount of a compound, or the amount of a composition comprising a compound, that will be effective in the prevention, treatment and/or management of a viral infection can be determined by standard clinical techniques. In vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend, e.g. , on the route of administration, the type of invention, and the seriousness of the infection, and should be decided according to the judgment of the practitioner and each patient's or subject's circumstances. [0186] In some embodiments, the dosage of a compound is determined by extrapolating from the no observed adverse effective level (NOAEL), as determined in animal studies. This extrapolated dosage is useful in determining the maximum

recommended starting dose for human clinical trials. For instance, the NOAELs can be extrapolated to determine human equivalent dosages (HED). Typically, HED is extrapolated from a non-human animal dosage based on the doses that are normalized to body surface area (i.e., mg/m²). In specific embodiments, the NOAELs are determined in mice, hamsters, rats, ferrets, guinea pigs, rabbits, dogs, primates, primates (monkeys, marmosets, squirrel monkeys, baboons), micropigs or minipigs. For a discussion on the use of NOAELs and their extrapolation to determine human equivalent doses, See Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER), Pharmacology and Toxicology, July 2005. In one embodiment, a compound or composition thereof is administered at a dose that is lower than the human equivalent dosage (HED) of the NOAEL over a period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, three months, four months, six months, nine months, 1 year, 2 years, 3 years, 4 years or more.

[0187] In certain embodiments, a dosage regime for a human subject can be extrapolated from animal model studies using the dose at which 10% of the animals die (LD10). In general the starting dose of a Phase I clinical trial is based on preclinical testing. A standard measure of toxicity of a drug in preclinical testing is the percentage of animals that die because of treatment. It is well within the skill of the art to correlate the LD 10 in an animal study with the maximal-tolerated dose (MTD) in humans, adjusted for body surface basis to extrapolate a starting human dose. In some embodiments, the

interrelationship of dosages for one animal model can be converted for use in another animal, including humans, using conversion factors (based on milligrams per meter squared of body surface) as described, e.g., in Freireich et al , Cancer Chemother. Rep., 1966, 50:219-244. Body surface area may be approximately determined from height and weight of the patient. See, e.g. , Scientific Tables, Geigy Pharmaceuticals, Ardley, N. Y., 1970, 537. In certain embodiments, the adjustment for body surface area includes host factors such as, for example, surface area, weight, metabolism, tissue distribution, absorption rate, and excretion rate. In addition, the route of administration, excipient usage, and the specific disease or virus to target are also factors to consider. In one embodiment, the standard conservative starting dose is about 1/10 the murine LD10, although it may be even lower if other species {i.e., dogs) were more sensitive to the compound. In other embodiments, the standard

conservative starting dose is about 1/100, 1/95, 1/90, 1/85, 1/80, 1/75, 1/70, 1/65, 1/60, 1/55, 1/50, 1/45, 1/40, 1/35, 1/30, 1/25, 1/20, 1/15, 2/10, 3/10, 4/10, or 5/10 of the murine LD10. In other embodiments, an starting dose amount of a compound in a human is lower than the dose extrapolated from animal model studies. In another embodiment, an starting dose amount of a compound in a human is higher than the dose extrapolated from animal model studies. It is well within the skill of the art to start doses of the active composition at relatively low levels, and increase or decrease the dosage as necessary to achieve the desired effect with minimal toxicity.

[0188] Exemplary doses of compounds or compositions include milligram or microgram amounts per kilogram of subject or sample weight {e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 5 micrograms per kilogram to about 100 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram). In specific embodiments, a daily dose is at least 50 mg, 75 mg, 100 mg, 150 mg, 250 mg, 500 mg, 750 mg, or at least 1 g.

[0189] In one embodiment, the dosage is a concentration of 0.01 to 5000 mM, 1 to 300 mM, 10 to 100 mM and 10 mM to 1 M. In another embodiment, the dosage is a concentration of at least 5 μΜ, at least 10 μΜ, at least 50 μΜ, at least 100 μΜ, at least 500 μΜ, at least 1 mM, at least 5 mM, at least 10 mM, at least 50 mM, at least 100 mM, or at least 500 mM.

[0190] In one embodiment, the dosage is a concentration of 0.01 to 5000 mM, 1 to 300 mM, 10 to 100 mM and 10 mM to 1 M. In another embodiment, the dosage is a concentration of at least 5 μΜ, at least 10 μΜ, at least 50 μΜ, at least 100 μΜ, at least 500 uM, at least 1 mM, at least 5 mM, at least 10 mM, at least 50 mM, at least 100 mM, or at least 500 mM. In a specific embodiment, the dosage is 0.25 μg/kg or more, preferably 0.5 μg/kg or more, 1 μg/kg or more, 2 μg/kg or more, 3 μg/kg or more, 4 μg/kg or more, 5 μg/kg or more, 6 μg/kg or more, 7 μg/kg or more, 8 μg/kg or more, 9 μg/kg or more, or 10 μg/kg or more, 25 μg/kg or more, preferably 50 μg/kg or more, 100 μg/kg or more, 250 μg/kg or more, 500 μg/kg or more, 1 mg/kg or more, 5 mg/kg or more, 6 mg/kg or more, 7 mg/kg or more, 8 mg/kg or more, 9 mg/kg or more, or 10 mg/kg or more of a patient's body weight. [0191] In another embodiment, the dosage is a unit dose of 5 mg, preferably 10 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg or more. In another embodiment, the dosage is a unit dose that ranges from about 5 mg to about 100 mg, about 100 mg to about 200 μg, about 1 0 mg to about 300 mg, about 150 mg to about 400 mg, 250 μg to about 500 mg, about 500 mg to about 800 mg, about 500 mg to about 1000 mg, or about 5 mg to about 1000 mg.

[0192] In certain embodiments, suitable dosage ranges for oral administration are about 0.001 milligram to about 500 milligrams of a compound, per kilogram body weight per day. In specific embodiments of the invention, the oral dose is about 0.01 milligram to about 100 milligrams per kilogram body weight per day, about 0.1 milligram to about 75 milligrams per kilogram body weight per day or about 0.5 milligram to 5 milligrams per kilogram body weight per day. The dosage amounts described herein refer to total amounts administered; that is, if more than one compound is administered, then, in some

embodiments, the dosages correspond to the total amount administered. In a specific embodiment, oral compositions contain about 10% to about 95% a compound of the invention by weight.

[0193] Suitable dosage ranges for intravenous (i.v.) administration are about 0.01 milligram to about 100 milligrams per kilogram body weight per day, about 0.1 milligram to about 35 milligrams per kilogram body weight per day, and about 1 milligram to about 10 milligrams per kilogram body weight per day. In some embodiments, suitable dosage ranges for intranasal administration are about 0.01 pg/kg body weight per day to about 1 mg/kg body weight per day. Suppositories generally contain about 0.01 milligram to about 50 milligrams of a compound of the invention per kilogram body weight per day and comprise active ingredient in the range of about 0.5% to about 10% by weight.

[0194] Recommended dosages for intradermal, intramuscular, intraperitoneal, subcutaneous, epidural, sublingual, intracerebral, intravaginal, transdermal administration or administration by inhalation are in the range of about 0.001 milligram to about 500 milligrams per kilogram of body weight per day. Suitable doses for topical administration include doses that are in the range of about 0.001 milligram to about 50 milligrams, depending on the area of administration. Effective doses may be extrapolated from dose- response curves derived from in vitro or animal model test systems. Such animal models and systems are well known in the art. [0195] In another embodiment, a subject is administered one or more doses of a prophylactically or therapeutically effective amount of a compound or a composition, wherein the prophylactically or therapeutically effective amount is not the same for each dose. In another embodiment, a subject is administered one or more doses of a

prophylactically or therapeutically effective amount of a compound or a composition, wherein the dose of a prophylactically or therapeutically effective amount administered to said subject is increased by, e.g., 0.01 μg/kg, 0.02 μg/kg, 0.04 0.05 μΒ/kg, 0.06 μΒ/kg,

0.08 μg/kg, 0.1 μg/kg, 0.2 μg/kg, 0.25 μg/kg, 0.5 μg/kg, 0.75 μg kg, 1 μg/kg, 1.5 μg kg, 2 μg/kg, 4 μg/kg, 5 μg/kg, 10 μg/kg, 15 μg kg, 20 μg/kg, 25 μg/kg, 30 μg/kg, 35 μg/kg, 40 μg/kg, 45 μg kg, or 50 μg kg, as treatment progresses. In another embodiment, a subject is administered one or more doses of a prophylactically or therapeutically effective amount of a compound or composition, wherein the dose is decreased by, e.g., 0.01 μg/kg, 0.02 μg/kg, 0.04 μ_§¾, 0.05 μ_§¾, 0.06 μ_§¾, 0.08 μ_§¾, 0.1 μ_§¼, 0.2 μ_§^_§, 0.25 μ_§¼, 0.5 μ_§¾, 0.75 μg kg, 1 μg/kg, 1.5 μg/kg, 2 μg/kg, 4 μg/kg, 5 μg kg, 10 μg/kg, 15 μg/kg, 20 μg kg, 25 μg kg, 30 μg kg, 35 μg/kg, 40 μg/kg, 45 μg/kg, or 50 μg/kg, as treatment progresses.

[0196] In certain embodiments, a subject is administered a compound or a composition in an amount effective to inhibit or reduce viral genome replication by at least 20% to 25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85%o relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In other embodiments, a subject is administered a compound or a composition in an amount effective to inhibit or reduce viral genome replication by at least 20% to 25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85%o relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In certain embodiments, a subject is administered a compound or a composition in an amount effective to inhibit or reduce viral genome replication by at least 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 8 fold, 10 fold, 15 fold, 20 fold, or 2 to 5 fold, 2 to 10 fold, 5 to 10 fold, or 5 to 20 fold relative to a negative control as determined using an assay described herein or other known to one of skill in the art. [0197] In certain embodiments, a subject is administered a compound or a composition in an amount effective to inhibit or reduce viral protein synthesis by at least 20% to 25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85% relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In other embodiments, a subject is administered a compound or a composition in an amount effective to inhibit or reduce viral protein synthesis by at least 20% to 25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85% relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In certain embodiments, a subject is administered a compound or a composition in an amount effective to inhibit or reduce viral protein synthesis by at least 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 8 fold, 10 fold, 15 fold, 20 fold, or 2 to 5 fold, 2 to 10 fold, 5 to 10 fold, or 5 to 20 fold relative to a negative control as determined using an assay described herein or others known to one of skill in the art.

[0198] In certain embodiments, a subject is administered a compound or a composition in an amount effective to inhibit or reduce viral infection by at least 20% to 25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85% relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In some embodiments, a subject is administered a compound or a composition in an amount effective to inhibit or reduce viral infection by at least 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 8 fold, 10 fold, 15 fold, 20 fold, or 2 to 5 fold, 2 to 10 fold, 5 to 10 fold, or 5 to 20 fold relative to a negative control as determined using an assay described herein or others known to one of skill in the art.

[0199] In certain embodiments, a subject is administered a compound or a composition in an amount effective to inhibit or reduce viral replication by at least 20%> to 25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85% relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In some embodiments, a subject is administered a compound or a composition in an amount effective to inhibit or reduce viral replication by at least 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 8 fold, 10 fold, 15 fold, 20 fold, or 2 to 5 fold, 2 to 10 fold, 5 to 10 fold, or 5 to 20 fold relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In other embodiments, a subject is administered a compound or a composition in an amount effective to inhibit or reduce viral replication by 1 log, 1.5 logs, 2 logs, 2.5 logs, 3 logs, 3.5 logs, 4 logs, 5 logs or more relative to a negative control as determined using an assay described herein or others known to one of skill in the art.

[0200] In certain embodiments, a subject is administered a compound or a composition in an amount effective to inhibit or reduce the ability of the virus to spread to other individuals by at least 20% to 25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75%) to 80%), or up to at least 85% relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In other embodiments, a subject is administered a compound or a composition in an amount effective to inhibit or reduce the ability of the virus to spread to other cells, tissues or organs in the subject by at least 20% to 25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85% relative to a negative control as determined using an assay described herein or others known to one of skill in the art.

[0201] In certain embodiments, a subject is administered a compound or a composition in an amount effective to inhibit or reduce viral induced lipid synthesis by at least 20% to 25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85%) relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In other embodiments, a subject is administered a compound or a composition in an amount effective to inhibit or reduce viral induced lipid synthesis by at least 20% to 25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85%) relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In certain embodiments, a subject is administered a compound or a composition in an amount effective to inhibit or reduce viral induced lipid synthesis by at least 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 8 fold, 10 fold, 15 fold, 20 fold, or 2 to 5 fold, 2 to 10 fold, 5 to 10 fold, or 5 to 20 fold relative to a negative control as determined using an assay described herein or others known to one of skill in the art.

[0202] In certain embodiments, a dose of a compound or a composition is administered to a subject every day, every other day, every couple of days, every third day, once a week, twice a week, three times a week, or once every two weeks. In other embodiments, two, three or four doses of a compound or a composition is administered to a subject every day, every couple of days, every third day, once a week or once every two weeks. In some embodiments, a dose(s) of a compound or a composition is administered for 2 days, 3 days, 5 days, 7 days, 14 days, or 21 days. In certain embodiments, a dose of a compound or a composition is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or more.

[0203] The dosages of prophylactic or therapeutic agents which have been or are currently used for the prevention, treatment and/or management of a viral infection can be determined using references available to a clinician such as, e.g., the Physicians' Desk Reference (61^st ed. 2007). Preferably, dosages lower than those which have been or are currently being used to prevent, treat and/or manage the infection are utilized in combination with one or more compounds or compositions.

[0204] As used herein, the term "effective amount" in the context of administering a therapy to a subject refers to the amount of a therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of a viral infection or a symptom associated therewith; (ii) reduce the duration of a viral infection or a symptom associated therewith; (iii) prevent the progression of a viral infection or a symptom associated therewith; (iv) cause regression of a viral infection or a symptom associated therewith; (v) prevent the development or onset of a viral infection or a symptom associated therewith; (vi) prevent the recurrence of a viral infection or a symptom associated therewith; (vii) reduce or prevent the spread of a virus from one cell to another cell, or one tissue to another tissue; (ix) prevent or reduce the spread of a virus from one subject to another subject; (x) reduce organ failure associated with a viral infection; (xi) reduce hospitalization of a subject; (xii) reduce hospitalization length; (xiii) increase the survival of a subject with a viral infection; (xiv) eliminate a virus infection; and/or (xv) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

[0205] As used herein, the terms "manage," "managing," and "management," in the context of the administration of a therapy to a subject, refer to the beneficial effects that a subject derives from a therapy, which does not result in a cure of a viral infection. In certain embodiments, a subject is administered one or more therapies to "manage" a disease so as to prevent the progression or worsening of the viral infection.

[0206] As used herein, the terms "prevent," "preventing" and "prevention" in the context of the administration of a therapy(ies) to a subject to prevent a viral infection refer to one or more of the following effects resulting from the administration of a therapy or a combination of therapies: (i) the inhibition of the development or onset of a viral infection and/or a symptom associated therewith; and (ii) the inhibition of the recurrence of a viral infection and/or a symptom associated therewith.

[0207] As used herein, the terms "treat," "treatment," and "treating" refer in the context of administration of a therapy(ies) to a subject to treat a viral infection refer to one, two, three, four, five or more of the following effects resulting from the administration of a therapy or a combination of therapies: (i) the reduction or amelioration of the severity of a viral infection and/or a symptom associated therewith; (ii) the reduction in the duration of a viral infection and/or a symptom associated therewith; (iii) the regression of a viral infection and/or a symptom associated therewith; (iv) the reduction of the titer of a virus; (v) the reduction in organ failure associated with a viral infection; (vi) the reduction in

hospitalization of a subject; (vii) the reduction in hospitalization length; (viii) the increase in the survival of a subject; (ix) the elimination of a virus infection; (x) the inhibition of the progression of a viral infection and/or a symptom associated therewith; (xi) the prevention of the spread of a virus from a cell, tissue or subject to another cell, tissue or subject; and/or (xii) the enhancement or improvement the therapeutic effect of another therapy.

[0208] For compounds which have been approved for uses other than prevention, treatment or management of viral infections, safe ranges of doses can be readily determined using references available to clinicians, such as e.g., the Physician's Desk Reference (61^st ed. 2007). [0209] The above-described administration schedules are provided for illustrative purposes only and should not be considered limiting. A person of ordinary skill in the art will readily understand that all doses are within the scope of the invention.

[0210] It is to be understood and expected that variations in the principles of invention herein disclosed may be made by one skilled in the art and it is intended that such modifications are to be included within the scope of the present invention.

[0211] Throughout this application, various publications are referenced. These publications are hereby incorporated into this application by reference in their entireties to more fully describe the state of the art to which this invention pertains. The following examples further illustrate the invention, but should not be construed to limit the scope of the invention in any way.

EXAMPLES

[0212] EXAMPLE 1 : ASSAYS OF ANTIVIRAL ACTIVITY AND

CYTOTOXICITY

[0213] Screening Assay

[0214] Compounds are evaluated in a 2-concentration test (200, 20 μg/ml unless otherwise noted). Compound-containing solutions are diluted 1 :2 when virus is added, yielding final concentrations 100 and 10 μg/ml. The standard cytopathic effect (CPE) test uses an 18 h monolayer (80-100% confluent) of the appropriate cells. Medium is drained and each of the concentrations of test compound or placebo are added, followed within 15 min by virus or virus diluent. Two wells are used for each concentration of compound for both antiviral and cytotoxicity testing. The plate is sealed and incubated for the standard time period required to induce near-maximal viral CPE.

[0215] The plate is then stained with neutral red by the method described below and the percentage of uptake indicating viable cells read on a microplate autoreader at dual wavelengths of 405 and 540 nm, with the difference taken to eliminate background. An approximated virus-inhibitory concentration, 50% endpoint (EC₅₀) and cell-inhibitory concentration, 50% endpoint (IC₅₀) are determined from which a general selectivity index is calculated: SI = (IC50) / (EC50). An SI of 3 or greater may be used as an indicator that confirmatory testing is needed. [0216] Inhibition of Viral Cytopathic Effect (CPE)

[0217] Four logio dilutions of a test compound (e.g. 1000, 100, 10, 1 μg/ml) are added in triplicate to 96 well flat-bottomed microplates containing a cell monolayer. Within 5 min, the virus is then added and the plate sealed, incubated at 37°C and CPE read microscopically when untreated infected controls develop a 3 to 4+ CPE (approximately 72 to 120 hr). A known positive control drug is evaluated in parallel with test drugs in each test. For dengue, positive control drugs include ribavirin or Infergen (interferon a). For yellow fever virus, the positive control drugs include 6-azauridine or Infergen (interferon a).

[0218] When testing with compounds found active in initial screening tests, virus assays are preformed in the same manner except one -half logio dilutions of each compound are used (for example, in triplicate or quadruple wells) containing the cell monolayer. The data are expressed as 50% effective concentrations (EC₅₀).

[0219] Increase in Neutral Red (NR) Dye Uptake

[0220] This assay may be run to validate the CPE inhibition seen in the initial test, and utilizes the same 96-well micro plates after the CPE has been read. Neutral red is added to the medium; cells not damaged by virus take up a greater amount of dye, which may be detected using a computerized micro plate autoreader. The method is described by McManus (Appl. Environment. Microbiol. 31 :35-38, 1976). An EC50 is determined from this dye uptake.

[0221] Decrease in Virus Yield Assay

[0222] Compounds considered active by CPE inhibition and by NR dye uptake may be tested as above, to determine CPE inhibition. Using the same assay plates, reduction of virus yield is determined by assaying frozen and thawed eluates from each well for virus titer by serial dilution onto new monolayers of susceptible cells. Development of CPE in these monolayers is the indication of presence of infectious virus. A known active drug may be run in parallel as a positive control. The 90% effective concentration (EC₉₀), which is that test drug concentration that inhibits virus yield by 1 logio, may be determined from these data.

[0223] ASSAYS OF CYTOTOXIC ACTIVITY

[0224] Visual Observation

[0225] In the CPE inhibition assay above, two wells of uninfected cells treated with each concentration of test compound may be run in parallel with the infected, treated wells. At the time CPE is determined microscopically, the toxicity control cells may also be examined microscopically for any changes in cell appearance compared to normal control cells run in the same plate. These changes may be enlargement, granularity, cells with ragged edges, a filmy appearance, rounding, detachment from the surface of the well, or other changes. These changes may be given a designation of T (100% toxic), PVH (partially toxic- very heavy-80%), PH (partially toxic-heavy-60%), P (partially toxic-40%), Ps (partially toxic-slight-20%), or 0 (no toxicity-0%), conforming to the degree of cytotoxicity seen. A 50% cell inhibitory (cytotoxic) concentration (IC₅₀) may be determined by regression analysis of these data.

[0226] Neutral Red Uptake

[0227] In the neutral red dye uptake phase of the antiviral test described above, toxicity control wells (which contain uninfected cells treated with each concentration of test compound) also receive neutral red and the degree of color intensity is determined spectrophotometrically. A neutral red IC5₀ (NR IC5₀) is subsequently determined.

[0228] Viable Cell Count

[0229] Compounds considered to have significant antiviral activity in the initial CPE and NR tests may be tested for their effects on cell growth. 96-well tissue culture plates are seeded with cells (sufficient to be approximately 20% confluent in the well) and exposed to varying concentrations of the test drug while the cells are dividing rapidly. The plates are then incubated in a C0₂ incubator at 37°C for 72 hr. Neutral red is added and the color intensity, indicating viable cell number, is determined spectrophotometrically. An IC5₀ is determined by regression analysis.

[0230] Data analysis

[0231] The antiviral activity of each test compound is expressed as a selectivity index (SI), which is the IC₅₀ or IC₉₀ divided by the EC₅₀. Generally, an SI of 10 or greater is indicative of positive antiviral activity, although other factors, such as a low SI for the positive control, are also taken into consideration.

[0232] EXAMPLE 2 - DENGUE VIRUS compound,

, was tested for antiviral activity against dengue virus by the assays described in Example 1. The test virus was Dengue type 2 virus, strain New Guinea C. The cell line was African green monkey kidney (Vero). Three separate trials were performed, varying the range of drug concentrations, as shown in Table 2. The control drug was Infergen (consensus interferon YM643; interferon alfacon-1) (Yasuda, S. et al., 2000, Spectrum of virus inhibition by consensus interferon YM643, Antivir Chem Chemother

11(5):337-41).

[0234] EXAMPLE 3 - YELLOW FEVER VIRUS

[0235] The same compound was tested for antiviral activity against yellow fever virus by the assays described in Example 1. The yellow fever virus was strain 17D (ATCC). The cell line was African green monkey kidney (Vero). Three separate trials were performed, varying the range of drug concentrations, as shown in Table 3. The control drug was

Infergen (consensus interferon YM643; interferon alfacon-1).

[0236] EXAMPLE 4 - WEST NILE VIRUS

[0237] The same compound was tested for antiviral activity against west nile virus by the assays described in Example 1. The test virus was strain New York Isolate. The cell line was African green monkey kidney (Vero 76). One trials was performed, varying the range of drug concentrations, as shown in Table 2. The control drug was Infergen (consensus interferon YM643; interferon alfacon-1) (Yasuda, S. et al., 2000, Spectrum of virus inhibition by consensus interferon YM643, Antivir Chem Chemother 11(5):337-41).

[0238] EXAMPLE 5 - ANIMAL MODEL

[0239] 20 mice (AG129 strain) are inoculated with 1 x 10⁷ PFU of the DENV-2 D2S 10 Dengue virus (DENV), with animals randomized between two groups (n=10 each). One group receives an elongase inhibitor of the invention, and the second group receives only vehicle. Six additional mice not inoculated with DENV, are randomly divided into two groups (n=3 each), one treated with elongase inhibitor at the same concentration as above, and one treated only with vehicle. Serum is collected from 5 animals in each of the first two groups on days 1, 2, 3, 5, and 7 days post inoculation, and DENV titers determined by RT- PCR. Morbidity is assessed by weight loss, lethargy, fur ruffling, hunched posture, and mobility limitation.

[0240] The results demonstrate a significant reduction in the viral titer as determined from the serum draws. A reduction in the incidence and severity of morbidity in DENV infected animals treated with the elongase inhibitor relative to those treated with vehicle alone, is observed, as well as an increase in the meantime to DENV-induced death.

Claims

We claim:

1. A method of treating or preventing dengue virus infection in a mammal, comprising administering to a mammalian subject in need thereof a therapeutically effective amount of a compound or prodrug thereof, or pharmaceutically acceptable salt or ester of said compound or prodrug, wherein the compound is a compound of Formula I:

wherein Ri is selected from H, unsubstitued phenyl; substituted phenyl wherein substitutents are selected from F, Me, Et, CI, OMe, OCF₃, and CF₃; Ci_6 alkyl; and C₃_6 cycloalkyl; wherein R3 and R4 are independently selected from H; Ci_3 alkyl; and phenyl; or R3 and R4 taken together form a cycloalkyl of formula -(CH2)_n- where n = 2, 3, 4 and 5; wherein R₅ is selected from methyl; CF₃; cyclopropyl; unsubtitued phenyl; mono- and

disubsituted phenyl where substitutents are selected from F, Me, Et, CN, iPr, CI, OMe, OPh, OCF₃, and CF₃; unsubstitued heteroaromatic groups; and imidazolo, or a prodrug thereof, or pharmaceutically acceptable salt or ester of said compound or prodrug thereof.

2. A method of treating or preventing yellow fever virus infection in a mammal, comprising administering to a mammalian subject in need thereof a therapeutically effective amount of a compound or prodrug thereof, or pharmaceutically acceptable salt or ester of said compound or prodrug, wherein the compound is a compound of Formula I:

wherein Ri is selected from H, unsubstitued phenyl; substituted phenyl wherein substitutents are selected from F, Me, Et, CI, OMe, OCF3, and CF3; Ci_6 alkyl; and C3-6 cycloalkyl; wherein R₃ and R4 are independently selected from H; Ci_3 alkyl; and phenyl; or R₃ and R4 taken together form a cycloalkyl of formula -(CH2)_n- where n = 2, 3, 4 and 5; wherein R5 is selected from methyl; CF₃; cyclopropyl; unsubtitued phenyl; mono- and

3. The method of claim 1 or 2, wherein R¹ is phenyl, R³ and R⁴ are methyl, and

R⁵ is

The method of claim 1 or 2, wherein the compound of Formula I

5. The method of claim 1 or 2, wherein the compound of Formula I is an inhibitor of a long chain fatty acyl elongase.

6. The method of claim 5, wherein the long chain fatty acyl elongase is long chain fatty acid elongase 6 (ELOVL6).

7. The method of claim 1 or 2, which further comprises administering an effective amount of an interferon.

8. The method of claim 1 or 2, which further comprises administering an effective amount of an agent that acts on a virus component.

9. A pharmaceutical composition for treatment or prevention of dengue virus infection comprising a therapeutically effective amount of a compound or prodrug thereof, or pharmaceutically acceptable salt of said compound or prodrug; and a pharmaceutically acceptable carrier, wherein the compound is a compound of Formula I:

H

(I)

wherein ¾ is selected from H, unsubstitued phenyl; substituted phenyl wherein substitutents are selected from F, Me, Et, CI, OMe, OCF3, and CF3; Ci_6 alkyl; and C3-6 cycloalkyl; wherein R₃ and R4 are independently selected from H; Ci_3 alkyl; and phenyl; or R₃ and R4 taken together form a cycloalkyl of formula -(CH2)_n- where n = 2, 3, 4 and 5; wherein R5 is selected from methyl; CF₃; cyclopropyl; unsubtitued phenyl; mono- and

10. A pharmaceutical composition for treatment or prevention of yellow fever virus infection comprising a therapeutically effective amount of a compound or prodrug thereof, or pharmaceutically acceptable salt of said compound or prodrug; and a pharmaceutically acceptable c ound is a compound of Formula I:

H

(I)

wherein Ri is selected from H, unsubstitued phenyl; substituted phenyl wherein substitutents are selected from F, Me, Et, CI, OMe, OCF₃, and CF₃; Ci_6 alkyl; and C₃_6 cycloalkyl; wherein R₃ and R4 are independently selected from H; Ci_3 alkyl; and phenyl; or R₃ and R4 taken together form a cycloalkyl of formula -(CH2)_n- where n = 2, 3, 4 and 5; wherein R5 is selected from methyl; CF₃; cyclopropyl; unsubtitued phenyl; mono- and

11. The pharmaceutical composition of claim 9 or 10, wherein R¹ is phenyl, R³ and R⁴ are methyl, and R⁵ is

12. The pharmaceutical composition of claim 9 or 10, wherein the compound of Formula I is

13. The pharmaceutical composition of claim 9 or 10, wherein the compound of Formula I is an inhibitor of a long chain fatty acyl elongase.

14. The pharmaceutical composition of claim 14, wherein the long chain fatty acyl elongase is long chain fatty acid elongase 6 (ELOVL6).

15. A combination of a pharmaceutical composition of claim 9 or 10, with a therapeutically effective amount of an interferon.

16. A combination of a pharmaceutical composition of claim 9 or 10, with a therapeutically effective amount of an agent that acts on a virus component.