WO2014164667A1 - Dengue and west nile virus protease inhibitors - Google Patents

Abstract

Compounds and methods of treating or preventing a Flavivirus infection in a subject are provided. The methods comprise administering to the subject a therapeutically effective amount of a compound as described herein. The methods are useful in treating and/or preventing Flavivirus infections such as, for example, West Nile Virus, Dengue Virus, and Japanese Encephalitis Virus. Methods of inhibiting a Flavivirus protease in a cell are also provided.

Description

Dengue and West Nile Virus Protease Inhibitors

CROSS-REFERENCE TO PRIORITY APPLICATION

This application claims priority to U.S. Provisional Application No. 61/776,047, filed March 11, 2013, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under grant number U01AI082068 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Flaviviruses, such as West Nile virus (WNV), Japanese Encephalitis virus, and Dengue virus (e.g., the four known serotypes of Dengue virus (DEN-1-4)) are significant human pathogens that cause millions of infections each year. DEN viruses cause a self-limiting disease in humans called dengue fever (DF), which is often resolved in 7-10 days. However, more severe forms of the disease, known as Dengue hemorrhagic fever (DHF) and Dengue shock syndrome (DSS), are common in areas endemic to DEN 1-4 and lead to considerable morbidity and mortality. According to World Health Organization estimates, 50-100 million cases of DEN infections in tropical and subtropical countries occur each year. WNV was introduced into the Western Hemisphere during an outbreak in the United States in 1999. Since this outbreak, WNV has spread throughout much of North America and has become a public health concern. Most WNV infections are asymptomatic; however, about 20% of cases are associated with mild flu-like symptoms. Some of these cases progress to more severe clinical manifestations, including encephalitis and/or flaccid paralysis. Currently, there are no approved vaccines or antiviral therapeutics available for either DEN- or WNV-infected humans.

SUMMARY

Provided herein are compounds for use as Flavivirus inhibitors, e.g., Flavivirus protease inhibitors. Also provided herein are methods for their use in treating a Flavivirus infection in a subject. A class of compounds described herein includes compounds of the following formula:

and pharmaceutically acceptable salts or prodrugs thereof. In these compounds, R¹, R², R³, R⁴, and R⁵ are each independently selected from hydrogen, halogen, hydroxyl, cyano, nitro, trifluoromethyl, substituted or unsubstituted Ci-C₆ alkyl, substituted or unsubstituted

heteroalkyl, substituted or unsubstituted amino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkoxyl, substituted or unsubstituted aryloxyl, substituted or unsubstituted carbonyl, or substituted or unsubstituted carboxyl; n is 0 or 1; and X is substituted carbonyl or a substituted amino. Optionally, R² and R³ can combine to form a substituted or unsubstituted heterocycle or a substituted or unsubstituted heteroaryl. Optionally, X is an ester or an amide. The compound can optionally be selected from the group consisting of:

-3-

A class of compounds described herein includes compounds of the following formula:

and pharmaceutically acceptable salts or prodrugs thereof. In these compounds, X is substituted or unsubstituted Ci-C₆ alkyl or substituted or unsubstituted amino; and Y is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. Optionally, the compound can be represented by the following structure:

where Y is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

The compound can optionally be selected from the group consisting of:

A class of compounds described herein includes compounds of the following formula:

and pharmaceutically acceptable salts or prodrugs thereof. In these compounds, n is 0 or 1; and R is hydrogen, halogen, hydroxyl, cyano, nitro, trifluoromethyl, substituted or unsubstituted Ci- C₆ alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted amino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkoxyl, substituted or unsubstituted aryloxyl, substituted or unsubstituted carbonyl, or substituted or unsubstituted carboxyl. Optionally, the compound can be represented by the following structure:

Also described herein are methods of treating or preventing a Flavivirus infection in a subject. The methods of treating or preventing a Flavivirus infection in a subject comprise administering to the subject a therapeutically effective amount of a compound as described herein.

Optionally, the Flavivirus is the West Nile Virus, Dengue Virus serotype DENV-1, Dengue Virus serotype DENV-2, Dengue Virus serotype DENV-3, Dengue Virus serotype DENV-4, or Japanese Encephalitis Virus.

The method can further comprise administering a second compound or composition, wherein the second compound or composition includes an antiviral compound. Optionally, the second compound or composition is a protease inhibitor.

Also described herein are methods of inhibiting a Flavivirus protease in a cell (e.g., a host cell). The methods of inhibiting a Flavivirus protease in a cell include contacting a cell with an effective amount of a compound as described herein. Optionally, the Flavivirus protease is DENV-2 protease or WNV protease. Optionally, the contacting is performed in vivo or in vitro.

The details of one or more embodiments are set forth in the description and drawings below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims. DESCRIPTION OF DRAWINGS

Figure 1 is a graph showing the percent inhibition of Dengue Virus 2 (DENV2) protease activity by Compounds B6, B7, and B9 at 10 μΜ and 25 μΜ as compared to a no inhibitor control ("No I") and Aprotinin ("BPTI").

Figure 2 is a graph showing the percent inhibition of DENV2 protease activity by

Compounds A15, A16, A17, A18, BIO, Bl 1, B12, and B13 at 10 μΜ and 25 μΜ as compared to a no inhibitor control ("No I") and Aprotinin ("BPTI").

Figure 3 is a graph showing the percent inhibition of DENV2 protease and West Nile Virus (WNV) protease activities by Compounds CI (labeled as CI -272), C2 (labeled as C2- 276), C3 (labeled as C3-302), C4 (labeled as C4-304), C5 (labeled as C5-310), C6 (labeled as C6-319), Dl (labeled as Dl-179), D2 (labeled as D2-180), D3 (labeled as D3-181), D4 (labeled as D4-182), D5 (labeled as D5-183), and D6 (labeled as D6-184) at 25 μΜ as compared to Aprotinin ("BPTI").

Figure 4 is a plot demonstrating the IC₅₀ determinations of Compounds B6, B7, and B9 against DENV2 protease activity.

Figure 5 is a plot demonstrating the IC₅₀ determinations of Compounds A15, A16, A17, and A18 against DENV2 protease activity.

Figure 6 is a plot demonstrating the IC₅₀ determinations of Compounds BIO, Bl 1, B12, and B13 against DENV2 protease activity.

Figure 7 is a plot demonstrating the IC₅₀ determinations of Compounds Dl, D2, D3, D4,

D5, and D6 against DENV2 protease activity.

Figure 8 is a plot demonstrating the IC₅₀ determinations of Compounds El, E2, E3, and E4 against DENV2 protease activity.

Figure 9 is a graph showing the percent inhibition of DENV2 protease and WNV protease activities by Compounds El, E2, E3, E4, F5, and F6 at 10 μΜ as compared to

Aprotinin ("BPTI").

Figure 10 is a graph showing the percent inhibition of DENV2 protease and WNV protease activities by Compounds El, E2, E3, E4, F5, and F6 at 25 μΜ as compared to

Aprotinin ("BPTI").

Figure 11 is a plot demonstrating the IC₅₀ determinations of Compounds CI, C2, C3,

C4, C5, and C6 against WNV protease activity.

Figure 12 is a plot demonstrating the IC₅₀ determinations of Compounds Dl, D2, D3, D4, D5, and D6 against WNV protease activity. Figure 13 is a graph showing the percent inhibition of DENV2 protease activity by Compounds ASH-276 (Compound C2), ASH-441, and ASH-440 at 10 μΜ.

Figure 14 is a graph showing the percent inhibition of DENV2 protease activity by Compounds ASH-276, ASH-441, and ASH-440 at 25 μΜ.

DETAILED DESCRIPTION

Described herein are compounds for use as Flavivirus inhibitors, e.g., Flavivirus protease inhibitors. Also provided herein are methods for their use in treating a Flavivirus infection in a subject comprising administering to the subject a therapeutically effective amount of the Flavivirus inhibitors described herein. The Flavivirus inhibitors are administered in an effective amount to prevent or treat Flavivirus infections such as, for example, West Nile Virus, Dengue Virus, and Japanese Encephalitis Virus.

I. Compounds

A class of protease inhibitors useful in the methods described herein includes compounds represented by Formula I:

or a pharmaceutically acceptable salt or prodrug thereof. Optionally, the compound represented by Formula I can be in the form of an ammonium salt.

In Formula I, R¹, R², R³, R⁴, and R⁵ are each independently selected from hydrogen, halogen, hydroxyl, cyano, nitro, trifluoromethyl, substituted or unsubstituted Ci-C₆ alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted amino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkoxyl, substituted or unsubstituted aryloxyl, substituted or unsubstituted carbonyl, or substituted or unsubstituted carboxyl. Exemplary substituents for substituted groups include halogen, hydroxyl, nitro, amino, trifluoromethyl, Ci-C₆ alkyl, alkoxyl, aryloxyl, aryl, and heteroaryl.

Optionally, in Formula I, R² and R³ can combine to form a substituted or unsubstituted heterocycle or a substituted or unsubstituted heteroaryl. Optionally, R² and R³ combine to form a substituted or unsubstituted oxazolyl.

Additionally, in Formula I, n is 0 or 1. Also, in Formula I, X is substituted carbonyl (e.g., an ester or an amide) or a substituted . Optionally, when X is substituted carbonyl, Formula I can be represented by Structure

or a pharmaceutically acceptable salt or prodrug thereof.

In Structure I-A, R¹, R², R³, R⁴, R⁵, and n are as defined for Formula I.

Also in Structure I-A, Y is substituted or unsubstituted amino, alkoxyl, or aryloxyl. In examples of Formula I where n is 0, the compound according to Formula I can be represented by Structure I-B:

or a pharmaceutically acceptable salt or prodrug thereof.

In Structure I-B, R¹, R², R³, R⁴, and R⁵ are as defined for Formula I.

Also in Structure I-B, Y is substituted or unsubstituted amino, alkoxyl, or aryloxyl.

In examples of Formula I where n is 1 , the compound according to Formula I can be represented by Structure I-C

or a pharmaceutically acceptable salt or prodrug thereof.

In Structure I-C, R¹, R², R³, R⁴, and R⁵ are as defined for Formula I.

Also in Structure I-C, Y is substituted or unsubstituted amino, alkoxyl, or aryloxyl.

Optionally, when X is substituted amino, Formula I can be represented by Structure I-

D:

harmaceutically acceptable salt or prodrug thereof. In Structure I-D, R¹, R², R³, R⁴, and R⁵ are as defined for Formula I.

Also in Structure I-D, R⁶ and R⁷ are each independently selected from hydrogen, substituted or unsubstituted Ci-C₆ alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

Examples of Formula I include the following compounds:

Compound B5 Compound B6

Compound B9 Compound BIO

Compound Bll Compound B12

Compound B13 Compound CI

Compound C2 (ASH-276)

Compound C5

Compound C6 Compound ASH-440

Compound ASH-441

A class of protease inhibitors useful in the methods described herein includes compounds represented by Formula II:

II

or a pharmaceutically acceptable salt or prodrug thereof.

In Formula II, X is substituted or unsubstituted Ci-C₆ alkyl or substituted or

unsubstituted amino.

Also, in Formula II, Y is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. Exemplary substituents for substituted aryl and substituted heteroaryl include halogen, hydroxyl, nitro, amino, trifluoromethyl, Ci-C₆ alkyl, alkoxyl, aryloxyl, aryl, and heteroaryl.

Optionally, Formula II can be represented by Structure II-A:

or a pharmaceutically acceptable salt

In Structure II-A, Y is as described above for Formula II.

Optionally, Formula II can be represented by Structure II-B:

or a pharmaceutically acceptable salt or prodrug thereof.

In Structure II-B, R¹, R², R³, R⁴, and R⁵ are each independently selected from hydrogen and halogen (e.g., CI, Br, I, or F). In some embodiments, R¹, R³, R⁴, and R⁵ are hydrogen and R² is chloro.

Also, in Structure II-B, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each independently selected from hydrogen, halogen, hydroxyl, cyano, nitro, trifluoromethyl, substituted or unsubstituted Ci-C₆ alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted amino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkoxyl, substituted or unsubstituted aryloxyl, substituted or unsubstituted carbonyl, and substituted or unsubstituted carboxyl. Exemplary substituents for substituted groups include halogen, hydroxyl, nitro, amino, trifluoromethyl, Ci-C₆ alkyl, alkoxyl, aryloxyl, aryl, and heteroaryl.

Optionally, Formula II can be represented by Structure II-C:

or a pharmaceutically acceptable salt or prodrug thereof.

In Structure II-C, R¹, R², R³, R⁴, and R⁵ are each independently selected from hydrogen and halogen (e.g., CI, Br, I, or F). In some embodiments, R¹, R³, R⁴, and R⁵ are hydrogen and R² is chloro.

Also, in Structure II-C, R¹¹, R¹², and R¹³ are each independently selected from hydrogen, halogen, hydroxyl, cyano, nitro, trifluoromethyl, substituted or unsubstituted Ci-C₆ alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted amino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkoxyl, substituted or unsubstituted aryloxyl, substituted or unsubstituted carbonyl, and substituted or unsubstituted carboxyl. Exemplary substituents for substituted groups include halogen, hydroxyl, nitro, amino, trifluoromethyl, Ci-C₆ alkyl, alkoxyl, aryloxyl, aryl, and heteroaryl.

Optionally, Formula II can be represented by Structure II-D:

or a pharmaceutically acceptable salt or prodrug thereof.

In Structure II-D, Y is as described above for Formula II.

Optionally, Formula II can be represented by Structure II-E:

or a pharmaceutically acceptable salt or prodrug thereof.

In Structure II-E, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are as described above for Structure II-B. Optionally, in Structure II-E, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each independently selected from hydrogen, hydroxyl, nitro, and methoxyl.

Optionally, Formula II can be represented by Structure II-F:

or a pharmaceutically acceptable salt or prodrug thereof.

In Structure II-F, R¹¹, R¹², and R¹³ are as described above for Structure II-C.

Optionally, in Structure II-F, R¹¹, R¹², and R¹³ are each independently selected from halogen (e.g., Br) and Ci-C₆ alkyl (e.g., methyl).

Examples of Formula II include the following compounds:

Compound Al Compound A2 Compound A3

Compound A4 Compound Α5 Compound A6

Compound AlO Compound All Compound A12

Compound A13 Compound A14 Compound A15

Compound A16 Compound A17 Compound A18

Compound Dl Compound D2

Compound D3 Compound D4

Compound D5 Compound D6

A class of protease inhibitors useful in the methods described herein includes compounds represented by Formula I

or a pharmaceutically acce

In Formula III, n is 0 or 1. Also, in Formula III, R is hydrogen, halogen, hydroxyl, cyano, nitro, trifluoromethyl, substituted or unsubstituted Ci-C₆ alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted amino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkoxyl, substituted or unsubstituted aryloxyl, substituted or unsubstituted carbonyl, or substituted or unsubstituted carboxyl. Optionally, R is hydrogen or methoxyl.

Optionally, in examples of Formula III where n is 0, the compound according to

Formula III can be represented by Structure III-A:

or a pharmaceutically acceptable salt or prodrug thereof.

In Structure III-A, R is as described above for Formula III.

Optionally, in examples of Formula III where n is 1 , the compound according to

Formula III can be represented by Structure III-B:

or a pharmaceutically acceptable salt or prodrug thereof.

In Structure III-B, R is as described above for Formula III.

Examples of Formula III include the following compounds:

Compound El Compound E2 Compound E3

Compound E4

Further protease inhibitors useful in the methods described herein include compounds as shown below:

Compound F5 Compound F6

As used herein, the terms alkyl, alkenyl, and alkynyl include straight- and branched- chain monovalent substituents. Examples include methyl, ethyl, isobutyl, 3-butynyl, and the like. Ranges of these groups useful with the compounds and methods described herein include C1-C20 alkyl, C2-C20 alkenyl, and C2-C20 alkynyl. Additional ranges of these groups useful with the compounds and methods described herein include C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₄ alkyl, C₂-C₄ alkenyl, and C₂-C₄ alkynyl.

Heteroalkyl, heteroalkenyl, and heteroalkynyl are defined similarly as alkyl, alkenyl, and alkynyl, but can contain O, S, or N heteroatoms or combinations thereof within the backbone. Ranges of these groups useful with the compounds and methods described herein include C₁-C₂₀ heteroalkyl, C₂-C₂o heteroalkenyl, and C₂-C₂o heteroalkynyl. Additional ranges of these groups useful with the compounds and methods described herein include C₁-C₁₂ heteroalkyl, C₂-C₁₂ heteroalkenyl, C₂-C₁₂ heteroalkynyl, Ci-C₆ heteroalkyl, C₂-C₆ heteroalkenyl, C₂-C₆

heteroalkynyl, C₁-C₄ heteroalkyl, C₂-C₄ heteroalkenyl, and C₂-C₄ heteroalkynyl.

The terms cycloalkyl, cycloalkenyl, and cycloalkynyl include cyclic alkyl groups having a single cyclic ring or multiple condensed rings. Examples include cyclohexyl,

cyclopentylethyl, and adamantanyl. Ranges of these groups useful with the compounds and methods described herein include C3-C20 cycloalkyl, C3-C20 cycloalkenyl, and C3-C20 cycloalkynyl. Additional ranges of these groups useful with the compounds and methods described herein include C₅-C₁₂ cycloalkyl, C₅-C₁₂ cycloalkenyl, C₅-C₁₂ cycloalkynyl, C₅-C₆ cycloalkyl, C₅-C₆ cycloalkenyl, and C₅-C₆ cycloalkynyl.

The terms heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl are defined similarly as cycloalkyl, cycloalkenyl, and cycloalkynyl, but can contain O, S, or N heteroatoms or combinations thereof within the cyclic backbone. Ranges of these groups useful with the compounds and methods described herein include C3-C20 heterocycloalkyl, C3-C20

heterocycloalkenyl, and C₃-C₂₀ heterocycloalkynyl. Additional ranges of these groups useful with the compounds and methods described herein include C₅-C₁₂ heterocycloalkyl, C₅-C₁₂ heterocycloalkenyl, C₅-C₁₂ heterocycloalkynyl, C₅-C₆ heterocycloalkyl, C₅-C₆

heterocycloalkenyl, and C₅-C₆ heterocycloalkynyl.

Aryl molecules include, for example, cyclic hydrocarbons that incorporate one or more planar sets of, typically, six carbon atoms that are connected by delocalized electrons numbering the same as if they consisted of alternating single and double covalent bonds. An example of an aryl molecule is benzene. Heteroaryl molecules include substitutions along their main cyclic chain of atoms such as O, N, or S. When heteroatoms are introduced, a set of five atoms, e.g., four carbon and a heteroatom, can create an aromatic system. Examples of heteroaryl molecules include furan, pyrrole, thiophene, imadazole, oxazole, pyridine, and pyrazine. Aryl and heteroaryl molecules can also include additional fused rings, for example, benzofuran, indole, benzothiophene, naphthalene, anthracene, and quinoline. The aryl and heteroaryl molecules can be attached at any position on the ring, unless otherwise noted.

The alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocycloalkyl, heterocycloalkenyl, or

heterocycloalkynyl molecules used herein can be substituted or unsubstituted. As used herein, the term substituted includes the addition of an alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl,

heterocycloalkyl, heterocycloalkenyl, or heterocycloalkynyl group to a position attached to the main chain of the alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocycloalkyl, heterocycloalkenyl, or heterocycloalkynyl, e.g., the replacement of a hydrogen by one of these molecules. Examples of substitution groups include, but are not limited to, hydroxyl, halogen (e.g., F, Br, CI, or I), and carboxyl groups. Conversely, as used herein, the term unsubstituted indicates the alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocycloalkyl, heterocycloalkenyl, or heterocycloalkynyl has a full complement of hydrogens, i.e., commensurate with its saturation level, with no substitutions, e.g., linear decane (-(CH₂)₉-CH₃).

II. Pharmaceutical Formulations

The compounds described herein or derivatives thereof can be provided in a

pharmaceutical composition. Depending on the intended mode of administration, the

pharmaceutical composition can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include a therapeutically effective amount of the compound described herein or derivatives thereof in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, or diluents. By

pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, which can be administered to an individual along with the selected compound without causing unacceptable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained.

As used herein, the term carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia,

Lippincott, Williams & Wilkins, Philadelphia Pa., 2005. Examples of physiologically acceptable carriers include buffers, such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins;

hydrophilic polymers, such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN^® (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ). Compositions containing the compound described herein or derivatives thereof suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants, such as preserving, wetting,

emulsifying, and dispensing agents. Prevention of the action of microorganisms can be promoted by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, for example, sugars, sodium chloride, and the like may also be included. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration of the compounds described herein or derivatives thereof include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein or derivatives thereof is admixed with at least one inert customary excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example, paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art. They may contain opacifying agents and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration of the compounds described herein or derivatives thereof include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include additional agents, such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.

Suspensions, in addition to the active compounds, may contain additional agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.

Compositions of the compounds described herein or derivatives thereof for rectal administrations are optionally suppositories, which can be prepared by mixing the compounds with suitable non-irritating excipients or carriers, such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and, therefore, melt in the rectum or vaginal cavity and release the active component.

Dosage forms for topical administration of the compounds described herein or derivatives thereof include ointments, powders, sprays, and inhalants. The compounds described herein or derivatives thereof are admixed under sterile conditions with a

physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required. Ophthalmic formulations, ointments, powders, and solutions are also contemplated as being within the scope of the compositions. The compositions can include one or more of the compounds described herein and a pharmaceutically acceptable carrier. As used herein, the term pharmaceutically acceptable salt refers to those salts of the compound described herein or derivatives thereof that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds described herein. The term salts refers to the relatively non-toxic, inorganic and organic acid addition salts of the compounds described herein. These salts can be prepared in situ during the isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate, methane sulphonate, and laurylsulphonate salts, and the like. These may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium,

tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. {See S.M. Barge et al., J. Pharm. Sci. (1977) 66, 1, which is incorporated herein by reference in its entirety, at least, for compositions taught therein.)

Administration of the compounds and compositions described herein or pharmaceutically acceptable salts thereof can be carried out using therapeutically effective amounts of the compounds and compositions described herein or pharmaceutically acceptable salts thereof as described herein for periods of time effective to treat a disorder. The effective amount of the compounds and compositions described herein or pharmaceutically acceptable salts thereof as described herein may be determined by one of ordinary skill in the art and includes exemplary dosage amounts for a mammal of from about 0.5 to about 200mg/kg of body weight of active compound per day, which may be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day. Alternatively, the dosage amount can be from about 0.5 to about 150mg/kg of body weight of active compound per day, about 0.5 to lOOmg/kg of body weight of active compound per day, about 0.5 to about 75mg/kg of body weight of active compound per day, about 0.5 to about 50mg/kg of body weight of active compound per day, about 0.5 to about 25mg/kg of body weight of active compound per day, about 1 to about 20mg/kg of body weight of active compound per day, about 1 to about lOmg/kg of body weight of active compound per day, about 20mg/kg of body weight of active compound per day, about lOmg/kg of body weight of active compound per day, or about 5mg/kg of body weight of active compound per day. Those of skill in the art will understand that the specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition.

III. Methods of Making the Compounds

The compounds described herein can be prepared in a variety of ways known to one skilled in the art of organic synthesis. The compounds can be synthesized using synthetic methods known in the art of synthetic organic chemistry or variations thereon as appreciated by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

Variations on Formula I, Formula II, Formula III, and Compounds F5 and F6 include the addition, subtraction, or movement of the various constituents as described for each compound. Similarly, when one or more chiral centers is present in a molecule the chirality of the molecule can be changed. Additionally, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4^th Ed., Wiley & Sons, 2006, which is incorporated herein by reference in its entirety. The synthesis and subsequent testing of various compounds as described by Formula I, Formula II, Formula III, and Compounds F5 and F6 to determine efficacy is contemplated.

Reactions to produce the compounds described herein can be carried out in solvents, which can be readily selected by one of skill in the art of organic synthesis. Solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products under the conditions at which the reactions are carried out, i.e., temperature and pressure. Reactions can be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1H or ¹³C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

The compounds according to Formula I, Formula II, and Formula III, and

Compounds F5 and F6 can be prepared according to the exemplary synthetic methods provided in Example 1.

IV. Methods of Use

The methods described herein are useful for treating viral infections, such as Flavivirus infections, in humans, e.g., including pediatric and geriatric populations, and animals, e.g., veterinary applications. The methods described herein comprise administering to a subject a therapeutically effective amount of one or more compounds as described herein or a

pharmaceutically acceptable salt or prodrug thereof. Flavivirus infections include, for example, West Nile Virus, Dengue Virus, and Japanese Encephalitis Virus. Several serotypes of Dengue Virus have been identified such as, for example, serotype DENV-1, serotype DENV-2, serotype DENV-3, and serotype DENV-4.

The methods described herein are useful for both prophylactic and therapeutic treatment of Flavivirus infections. For prophylactic use, a therapeutically effective amount of one or more compounds as described herein is administered to a subject prior to exposure (e.g., before or when traveling to a location where Flavivirus infections are possible), during a period of potential exposure to Flavivirus infections, or after a period of potential exposure to Flavivirus infections. Prophylactic administration can occur for several days to weeks prior to potential exposure, during a period of potential exposure, and for a period of time, e.g., several days to weeks, after potential exposure. Therapeutic treatment involves administering to a subject a therapeutically effective amount of one or more compounds as described herein after a

Flavivirus infection is diagnosed.

In the methods described herein, a Flavivirus infection, for example, can be further treated with one or more additional agents. Optionally, the additional agent includes an antiviral agent. Optionally, the additional agent includes a protease inhibitor. The one or more additional agents and the compounds described herein or a pharmaceutically acceptable salt or prodrug thereof can be administered in any order, including simultaneous administration, as well as temporally spaced order of up to several days apart. The methods may also include more than a single administration of the one or more additional agents and/or the compounds described herein or a pharmaceutically acceptable salt or prodrug thereof. The administration of the one or more additional agent and the compounds described herein or a pharmaceutically acceptable salt or prodrug thereof may be by the same or different routes and concurrently or sequentially.

The methods described herein are also useful in inhibiting a Flavivirus protease in a cell (e.g., a host cell). Optionally, the Flavivirus protease can be DENV-2 protease. Optionally, the Flavivirus protease can be WNV protease. The method of inhibiting a Flavivirus protease in a cell includes contacting a cell with an effective amount of one or more compounds as described herein. Optionally, the contacting is performed in vivo. Optionally, the contacting is performed in vitro.

V. Kits

Also provided herein are kits for treating Flavivirus infections in a subject. A kit can include any of the compounds or compositions described herein. For example, a kit can include any of the compounds according to Formula I, Formula II, and Formula III, and Compounds F5 and F6, or combinations thereof. A kit can further include one or more additional agents, such as a protease inhibitor. A kit can additionally include directions for use of the kit (e.g., instructions for treating a Flavivirus infection in a subject), a container, a means for

administering the compounds or compositions (e.g., syringe, etc.), and/or a carrier.

As used herein the terms treatment, treat, or treating refer to a method of reducing or delaying one or more symptoms of a Flavivirus infection. Thus in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity or progression of one or more symptoms of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms or signs of the Flavivirus infection in a subject as compared to a control. As used herein, control refers to the untreated condition. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.

As used herein, the terms prevent, preventing, and prevention of a disease or disorder refer to an action, for example, administration of a composition or therapeutic agent, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder, which inhibits or delays onset or severity of one or more symptoms of the disease or disorder. For example, the method is considered to be a prevention if there is a reduction or delay in onset, incidence, severity, or recurrence of a Flavivirus infection. The reduction or delay in onset, incidence, severity, or recurrence of a Flavivirus infection can be a 10%, 20%>, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels.

As used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater as compared to a control level. Such terms can include, but do not necessarily include, complete elimination.

As used herein, subject means both mammals and non-mammals. Mammals include, for example, humans; non-human primates, e.g., apes and monkeys; cattle; horses; sheep; rats; mice; pigs; and goats. Non-mammals include, for example, fish and birds.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application.

The examples below are intended to further illustrate certain aspects of the methods and compositions described herein, and are not intended to limit the scope of the claims.

EXAMPLES

Example 1: Synthesis of Compounds

Example I A: Synthesis of Compounds of Formula I

Compounds Bl, B2, B3, B4, CI, and C2 were prepared according to the general scheme and procedure below:

To a solution of N, N-diethylcyanoacetamide (0.1 mol) in toluene was added 0.2 g IRA 96 resin and an aromatic aldehyde (0.1 mol). The reaction mass was heated to reflux under azeotrope for 5-6 hours. After completion of the reaction, the mixture was filtered hot. The filtrate was evaporated completely under vacuum and the residue was purified in ethanol to afford the pure compounds. The melting points for the compounds are shown in Table 1. Table 1

Compounds B5 and B6 were prepared according to the general scheme and procedure below:

To a solution of substituted cyanoacetic ester (0.1 mol) in ethanol was added diethyl amine (0.1 mol). The reaction mass was stirred for 25-30 min. After completion of the reaction, the solid was filtered and dried in an air drier. The melting points for the compounds are shown in Table 2.

Table 2

Compound B7 was prepared according to the scheme and procedure below:

To a solution of entacapone (3.05 g, 0.01 mol) in ethanol was added 0.85 g of piperidine (0.01 mol). The reaction mass was stirred for 25-30 min. After completion of the reaction, the solid was filtered and dried in an air drier. The melting point of the resulting compound was 156.3 - 159.6 °C.

Compound CI was prepared according to the scheme and procedure below:

A round bottomed flask equipped with a stirrer, thermowell, and a dean-Stark condenser was charged with 1.83 g of 3,4-dihydroxy-5-nitrobenzaldehyde, 1.54 g of N,N-diethyl-2-cyano acetamide, 150 mg of IRA 120 resin, and 15 mL of toluene. The mixture was refiuxed at 108 - 112 °C and the formed water was removed azeotropically until the starting material was consumed (approximately 12-16 hours). The resin was removed by filtration and the reaction mass was concentrated by distilling the solvent completely under vacuum. A mixture of geometric isomers was formed with the ratio of E/Z isomers being approximately 70:30.

The concentrated mass was treated with a solution of 1% HBr in acetic acid and maintained under stirring at 90-95 °C for 1 hour. The reaction mixture was cooled to room temperature and stirred for 20-24 hours. The reaction mixture was filtered and the precipitate recrystallized from alcohol to afford 2.1 g of Pure (E)-entacapone. The melting point of the resulting compound was 163.3 - 164.9 °C.

Compound C2 was prepared according to the scheme and procedure below:

3e(Z)

The crude mixture (25 g) was treated with 500 mL toluene and 50 mL of acetic acid and heated to reflux for 4 hours. The reaction mixture was then cooled to 5 °C over the period of 3-5 hours and filtered to separate the insoluble solid and filtrate. The filtrate was concentrated and also co-evaporated with n-hexane to afford 7.5g Z-entacapone. The melting point of the resulting compound was 139.2 - 141.1 °C.

Compound Bl 1 was prepared according to the scheme and procedure below:

To a solution of N-benzyl-2-cyanoacetamide (1.74 g, 0.01 mol) in ethanol (20 mL) was added piperidine and 3, 4-di hydroxyl-5-nitrobenzaldehyde (1.83 g, 0.01 mol). The reaction mass was heated to 70-75 °C for 2-3 hours. After completion of the reaction, the solid that formed was filtered off. To the solid was added demineralized water and the mixture was neutralized using diluted HCl. The residue was filtered off to afford the pure compound, which had a melting point of 200.9 - 204.1 °C.

Compound B8 was prepared according to the scheme and procedure below:

To a solution of 2-cyano-N-phenylacetamide (1.6 g, 0.01 mol) in ethanol (20 mL) was added piperidine and 4-hydroxy-3-methoxy-5-nitrobenzaldehyde (1.97 g, 0.01 mol). The reaction mass was heated to 70-75 °C for 2-3 hours. After completion of the reaction, the solid that formed was filtered off. To the solid was added demineralized water and the mixture was neutralized using diluted HCl. The residue was filtered off to afford the pure compound, which had a melting point of 196.6 - 199.3 °C.

mpound B9 was prepared according to the scheme and procedure below:

To a solution of 2-cyano-N-phenylacetamide (1.6 g, 0.01 mol) in ethanol (20 mL) was added piperidine and 3, 4-dihydroxy-5-nitrobenzaldehyde (1.83 g, 0.01 mol). The reaction mass was heated to 70-75 °C for 2-3 hours. After completion of reaction, the solid that formed was filtered off. To the solid was added demineralized water and the mixture was neutralized using diluted HCl. The residue was filtered off to afford the pure compound, which had a melting point of 150.3 - 154.1 °C.

Com ound C5 was prepared according to the scheme and procedure below:

To a solution of 2-cyano-N-(4-fluorophenyl)acetamide (1.78 g, 0.01 mol) in ethanol (20ml) was added piperidine and 3,4-dihydroxy-5-nitrobenzaldehyde (1.83 g, 0.01 mol). The reaction mass was heated to 70-75 °C for 2-3 hours. After completion of the reaction, the solid that formed was filtered off. To the solid was added demineralized water and the mixture was neutralized using diluted HCl. The residue was filtered off to afford the pure compound, which had a melting point of 250.3 - 253.9 °C.

Substituted ethyl (2D)-2-cyano-3-phenylprop-2-enoates were prepared according to the scheme and procedure shown below:

1 (a-c), 1 (f-g) 5(a-f)

To a solution of ethylcyanoacetate (0.1 mol) in ethanol was added 2-3 drops of piperidine and an aromatic aldehyde (0.1 mol). The reaction mass was stirred at room temperature for 30 min. After completion of the reaction, the solid that formed was filtered off and recrystallized from ethanol to afford the pure compounds. The melting points for the compounds are shown in Table 3.

Table 3

Compounds B12, B13, C3, and C6 were prepared according to the general scheme and procedure below:

To a solution of substituted cyanoaceticester (0.1 mol) in ethanol (10 mL) was added a solution of triethylamine (0.2 mol) in ethanol. The mixture was heated at 75-80 °C for 11-12 hours. After completion of the reaction, ethanol was completely distilled off under vacuum. The residue was dissolved in dichloromethane and washed with water. The dichloromethane was distilled off completely under vacuum and the crude product was purified by column chromatography to afford the desired compound. The melting points for the compounds are shown in Table 4.

Table 4

Compound C4 was prepared according to the scheme and procedure below:

To a solution of N, N-diethylcyanoacetamide (1.40 g, 0.01 mol) in toluene was added 0.2 g IRA 96 resin and 2-nitrocinnmaldehydealdehyde (1.77g, 0.01 mol). The reaction mass was heated to reflux under azeotrope for 5-6 hours. After completion of the reaction, the mixture was filtered hot. The filtrate was evaporated completely under vacuum and the residue was crystallized from ethanol to afford the pure compound. The compound had a melting point of 86.0 - 88.3 °C.

Compound B10 was prepared according to the scheme and procedure below:

Ethyl (2E)-3-[3-(acetyl amino)-4-hydroxy-5-methoxyphenyl]-2-cyanoprop-2-enoate and para-toluenesulfonic acid were mixed in toluene. The reaction mixture was heated to reflux under azeotrope for 2-3 hours. After completion of the reaction, toluene was distilled off completely under vacuum. The residue was crystallized from ethanol to afford the pure compound, which had a melting point of 198.3 - 200.2 °C.

Example IB: Synthesis of Compounds of Formula II

Compounds according to Structures II-A, II-B, and II-C, where R² is chloro and R¹, R³, R⁴, and R⁵ are hydrogen, were prepared according to the general scheme shown below:

An exemplary procedure for Compound Al is provided below:

Preparation of l-(3-chlorophenyl)-3-[5-(benzylidene)-l-methyl-4-oxo-4, 5-dihydro-lH- imidazol-2-yl] urea (Compound Al):

To a suspension of 5.0 g of l-(3-chlorophenyl)-3-(l-methyl-4-oxo-4,5-dihydro-lH- imidazol-2-yl) urea (18.7 mmol) in 50 mL of ethanol was added 1.98 g of benzaldehyde (18.7 mmol) followed by piperidine (0.1 eq). The mixture was heated to 80 °C for 4 hours. After completion of the reaction as monitored by TLC, ethanol was removed under reduced pressure and 20 mL methanol was added to the obtained residue. The precipitated yellow solid was filtered off and recrystallization from methanol: chloroform (1 : 1) to afford the title compound with a melting point of 206.3-208.4 °C.

The following compounds shown in Table 5 were prepared according to the process described above for Compound Al :

Table 5

A2 o-hydroxy benzaldehyde 200.9-204.3

A3 p-methoxy benzaldehyde 235.0-236.3

A4 p-hydroxy benzaldehyde 184.5-188.6

A5 p-nitro benzaldehyde 169.1-170.3

A6 4-biphenyl benzaldehyde 242.7-246.1

A7 2,3-dichloro benzaldehyde 154.3-157.2

A8 3-phenoxy benzaldehyde 180.0-183.5

A9 4-bromo-2-hydroxy benzaldehyde 225.8-229.7

A10 4-hydroxy-3-methoxy benzaldehyde 190-200(decomposition)

Al l 3,4-dihydroxy-5-nitrobenzaldehyde 175.6-186.4(decomposition)

A12 3 -ethoxy-4-hydroxybenzaldehyde 196.2-200.8

A13 2-methyl benzaldehyde 194.8-196.1

A14 4-fluoro-3-phenoxy benzaldehyde 131-148.1 (decomposition)

A15 5 -bromofuraldehyde 239-241

A16 furaldehyde 224-226

A17 5 -methy lfuraldehy de 225-228

Compounds according to Formula II-D were prepared according to the general scheme shown below:

An exemplary procedure for Compound A18 is provided below:

Preparation of N-[5-benzylidene-l-methyl-4-oxo-4,5-dihydro-lH-imidazol-2-yl]acetamide (Compound A 18):

To a solution of 5.0 g of N-(l-methyl-4-oxo-4,5-dihydro-lH-imidazol-2-yl)acetamide (32.2 mmol) in 50 mL of ethanol was added 3.41 g of benzaldehyde (32.2 mmol) followed by piperidine (0.1 eq). The mixture was heated to 80 °C for 4 hours. After completion of the reaction as monitored by TLC, ethanol was removed under reduced pressure and 20 mL methanol was added to the obtained residue. The precipitated yellow solid was filtered off. The solid was recrystallized from a 1 : 1 mixture of methanol and chloroform to afford the titled compound having a melting point of 194-196 °C.

The following compounds shown in Table 6 were prepared according to the process described above for Compound A18:

Table 6

Example 1C: Synthesis of Compound of Formula III

Compounds according to Formula III -A were prepared according to the general scheme shown below:

A mixture of homophthalic acid (20 g, 1.09 mol) and urea (8 g, 1.31 mol) was ground to a fine powder. The powder was then heated at 175-185 °C until melted and was then resolidified. The mixture was cooled to ambient temperature and methanol (250 mL) was added. The mixture was then heated under reflux for 20 minutes, filtered, and allowed to cool to ambient temperature. The resulting solid was collected by filtration, washed with methanol, and dried under vacuum to give homophthalimide (melting point: 235.0 - 240.0 °C).

Homophthalimide was dissolved in a mixture of ethanol (6 mL) and benzene (4 mL) and then 2 to 3 drops of piperidine was added. The resulting mixture was stirred for 10 minutes. An aldehyde, as listed in Table 7, was slowly added to the mixture and the mixture was refiuxed for 1-2 hours and cooled to 20-25 °C. The solid that formed was filtered off and washed with ethanol. The product was dried under vacuum.

The following compounds shown in Table 7 were prepared according to the process described above:

Table 7

Compounds according to Formula III-B were prepared according to the general scheme shown below:

To a solution of substituted benzylideneisoquinoline-dione (0.1 mol) in ethanol (10 mL) was added a solution of triethylamine (0.2 mol) in ethanol. The mixture was heated at 75-80 °C for 7 to 8 hours. After completion of the reaction, ethanol was completely distilled off under vacuum. The residue was dissolved in dichloromethane and washed with water. The dichloromethane was distilled off completely under vacuum and the crude product was purified by column chromatography to afford the desired compound.

The following compounds shown in Table 8 were prepared according to the process described above:

Table 8

Compound E4 was also prepared according to the following procedure:

Homophthalimide (0.5 g, 0.01 mol) was dissolved in a mixture of ethanol (6 mL) and benzene (4 mL). Piperidine (2-3 drops) was added and the mixture was stirred for 10 minutes. To this mixture was added cinnamaldehyde (0.5 g, 0.01 mol) slowly and the mixture was refluxed for 1 to 2 hours. The mixture was cooled to 20-25 °C. The solid that formed was filtered off and washed with dried ethanol. The product was dried under vacuum. The resulting product had a melting point of 227.3-231.0 °C.

Example ID: Synthesis of Compounds F5 and F6

Compound F5 (i.e., 7-[(diethylamino)methyl]-5-nitroquinolin-8-ol hydrochloride) was prepared according to the following procedure:

To a solution of 5-nitroquinolin-8-ol (2.0 g; 10.5 mmol) in 25 mL of ethanol was added 0.314 g of paraformaldehyde (10.5 mmol) and 0.766 g of diethylamine (10.5 mmol) under stirring. The mixture was heated to 50-55 °C and stirred for 4 hours. The solvent was distilled off completely. The residue obtained was converted into 7-[(diethylamino)methyl]-5- nitroquinolin-8-ol hydrochloride by dissolving the residue in isopropanol and adding isopropanolic HC1 to the solution. The precipitated product was filtered and dried at 70-75 °C for 4 hours. The resulting product had a melting point of 198.0-202.0 °C.

Compound F6 (i.e., 5-(thiomorpholin-4-ylmethyl)quinolin-8-ol hydrochloride) was prepared according to the following procedure:

To a solution of 5-(chloromethyl)quinolin-8-ol hydrochloride (5g; 0.021 mol) in 50 mL of Ν,Ν-dimethylformamide (DMF) was added thiomorpholine (4.5 g; 0.43 mol) under stirring. The mixture was heated to 60 °C for 6 hours. The solvent was distilled off completely. The obtained residue was slurried in chloroform and heated to 60 °C for 30 minutes. The product, 5- (thiomorpholin-4-ylmethyl)quinolin-8-ol hydrochloride, was filtered and oven dried at 70-75 °C for 4 hours. The resulting product had a melting point of 214-216 °C. Example 2: Dengue Virus 2 Protease Assay

Dengue Virus 2 Protease Assay with Small Molecule Inhibitors

The compounds were analyzed by in vitro protease assays performed in 96-well plates. Protease activity experiments were performed using purified viral protease NS2B-NS3-pro from DENV-2. Enzyme kinetics were performed under steady-state conditions. Standard reaction mixtures (50 μΐ,) containing 200 mM Tris HCl (pH 9.5), 6 mM NaCl, 30 % glycerol, 24 nM DENV2 protease, and 25 μΜ and /or 10 μΜ inhibitors (dissolved in DMSO) in each assay were incubated 15 minutes at 25 °C. Reactions were started by adding 5.0 μΜ of tetra-peptide substrate Benzyloxycarbonyl (Z)-Nle-Gly-Arg-Arg-AMC.

The release of free AMC (fluorescent probe) was measured using a spectrofluorometer (Molecular Devices; Silicon Valley, CA) at excitation and emission wavelengths of 380 and 460 nm, respectively. Fluorescence values obtained with the "no inhibitor" control were taken as 100%, and those in the presence of inhibitors were calculated as the percentage of inhibition of the control using Microsoft Excel and plotted using SigmaPlot software. The background of AMC in the absence of protease was subtracted before the data analysis. All assays were done in triplicate. The results of the protease inhibition assay against DENV2 protease are shown in Table 9. Compound C2 was analyzed at two separate times as shown in Table 9 (see

Compound C2 entry and Compound ASH-276 entry).

Determination of IC50 of Compounds against DENV-2 Protease

The percentages of Dengue Virus Type 2 Protease activity for the compounds were compared with the positive control (Aprotinin; "BPTI") and in the absence of inhibitors ("No

I"). Compounds having a protease activity of less of 50% (i.e., a % inhibition of greater than

50%)), as compared with the controls, were selected for IC₅₀ determination against DENV-2 protease (see Figure 1 for B6, B7, and B9; Figure 2 for A15, A16, A17, A18, B10, Bl l, B12, and B 13; Figure 3 for CI, C2, C3, C4, C5, C6, Dl, D2, D3, D4, D5, and D6; Figure 9 for El,

E2, E3, E4, F5, and F6 at 10 μΜ; Figure 10 for El, E2, E3, E4, F5, and F6 at 25 μΜ; and

Figure 13 for ASH-276 (Compound C2), ASH-441, and ASH-440 at 10 μΜ; and Figure 14 for

ASH-276 (Compound C2), ASH-441, and ASH-440 at 25 μΜ). The inhibitors were incubated with DENV2 NS2B/NS3pro (25 nM) in buffer (200 mM Tris-HCl, pH 9.5, 6 mM NaCl, 30% glycerol and 0.1% CHAPS) for 15 min. Bz-Nle-Lys-Arg-Arg-AMC (5.0 μΜ) was added to the mixture in a final volume of 100 μί. The fluorescence intensity was measured at 460 nm with excitation at 380 nm and converted to the percentage of protease activity in the absence and presence of inhibitors. The results are shown in Table 9 (all), Figure 4 (B6, B7, and B9), Figure 5 (A 15, Al 6, Al 7, and A 18), Figure 6 (BIO, B 11, B 12, and B 13), Figure 7 (Dl, D2, D3, D4, D5, and D6), and Figure 8 (El, E2, E3, and E4). In Figures 4, 5, 6, 7, and 8, the solid line is the theoretical fitting curve based on the Sigmoidal Equation. The x-values are displayed as the log of compound concentration (0-50 μΜ). All assays were done in duplicate.

Table 9

Example 3: West Nile Virus Protease Assay

West Nile Virus Protease Assay with Small Molecule Inhibitors

Protease activity experiments were performed using purified viral protease NS2B-NS3- pro from WNV according to the methods described in Example 2. The results of the protease inhibition assay against WNV protease are shown in Table 10. The percentages of WNV protease activity for the compounds were compared with Aprotinin, the positive control ("BPTI") (see Figure 3 for CI , C2, C3, C4, C5, C6, Dl , D2, D3, D4, D5, and D6; Figure 9 for El , E2, E3, E4, F5, and F6 at 10 μΜ; and Figure 10 for El , E2, E3, E4, F5, and F6 at 25 μΜ). The IC₅₀ values were determined for the compounds as described above and are shown in Table 10, Figure 11 (CI , C2, C3, C4, C5, and C6), and Figure 12 (Dl , D2, D3, D4, D5, and D6).

Table 10

The compounds and methods of the appended claims are not limited in scope by the specific compounds and methods described herein, which are intended as illustrations of a few aspects of the claims and any compounds and methods that are functionally equivalent are within the scope of this disclosure. Various modifications of the compounds and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compounds, methods, and aspects of these compounds and methods are specifically described, other compounds and methods are intended to fall within the scope of the appended claims. Thus a combination of steps, elements, components, or constituents may be explicitly mentioned herein; however, all other

combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

Claims

WHAT IS CLAIMED IS:

A compound of the following formula:

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

Y is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

3. The compound of claim 2, wherein the compound is selected from group consisting of:

5. A compound of the following formula:

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

R is hydrogen, halogen, hydroxyl, cyano, nitro, trifluoromethyl, substituted or unsubstituted Ci-C₆ alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted amino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkoxyl, substituted or unsubstituted aryloxyl, substituted or unsubstituted carbonyl, or substituted or unsubstituted carboxyl.

6. The compound of claim 5, wherein the compound is selected from group consisting of:

7. A method of treating or preventing a Flavivirus infection in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the following formula:

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

R¹, R², R³, R⁴, and R⁵ are each independently selected from hydrogen, halogen, hydroxyl, cyano, nitro, trifluoromethyl, substituted or unsubstituted Ci-C₆ alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted amino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkoxyl, substituted or unsubstituted aryloxyl, substituted or unsubstituted carbonyl, or substituted or unsubstituted carboxyl;

n is 0 or 1 ; and

X is substituted carbonyl or a substituted amino,

wherein R² and R³ can optionally combine to form a substituted or unsubstituted heterocycle or a substituted or unsubstituted heteroaryl.

8. The method of claim 7, wherein the compound has the following structure:

9. A method of treating or preventing a Flavivirus infection in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the following formula:

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

X is substituted or unsubstituted Ci-C₆ alkyl or substituted or unsubstituted amino; and Y is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

10. A method of treating or preventing a Flavivirus infection in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the following formula:

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

n is 0 or 1 ; and

R is hydrogen, Ci-C₆ alkyl, or alkoxyl.

11. A method of treating or preventing a Flavivirus infection in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the following formula:

or a pharmaceutically acceptable salt or prodrug thereof.

12. A method of treating or preventing a Flavivirus infection in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the following formula:

or a pharmaceutically acceptable salt or prodrug thereof.

13. The method of any of claims 7-12, wherein the Flavivirus is the West Nile Virus.

14. The method of any of claims 7-12, wherein the Flavivirus is Dengue Virus serotype DENV-1.

15. The method of any of claims 7-12, wherein the Flavivirus is Dengue Virus serotype DENV-2.

16. The method of any of claims 7-12, wherein the Flavivirus is Dengue Virus serotype DENV-3.

17. The method of any of claims 7-12, wherein the Flavivirus is Dengue Virus serotype DENV-4.

18. The method of any of claims 7-12, wherein the Flavivirus is Japanese Encephalitis Virus.

19. The method of any of claims 7-18, further comprising administering a second compound or composition, wherein the second compound or composition includes an antiviral compound.

20. The method of claim 19, wherein the second compound or composition is a protease inhibitor.

21. A method of inhibiting a Flavivirus protease in a cell, comprising:

contacting a cell with an effective amount of a compound of the following structure:

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

R¹, R², R³, R⁴, and R⁵ are each independently selected from hydrogen, halogen, hydroxyl, cyano, nitro, trifluoromethyl, substituted or unsubstituted Ci-C₆ alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted amino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkoxyl, substituted or unsubstituted aryloxyl, substituted or unsubstituted carbonyl, or substituted or unsubstituted carboxyl;

n is 0 or 1 ; and

X is substituted carbonyl or a substituted amino,

wherein R² and R³ can optionally combine to form a substituted or unsubstituted heterocycle or a substituted or unsubstituted heteroaryl.

22. The method of claim 21, wherein the compound has the following structure:

A method of inhibiting a Flavivirus protease in a cell, comprising:

contacting a cell with an effective amount of a compound of the following structure

pharmaceutically acceptable salt or prodrug thereof, wherein:

X is substituted or unsubstituted Ci-C₆ alkyl or substituted or unsubstituted amino; and Y is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

A method of inhibiting a Flavivirus protease in a cell, comprising:

contacting a cell with an effectiyg amount of a compound of the following structure:

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

n is 0 or 1 ; and

R is hydrogen, Ci-C₆ alkyl, or alkoxyl.

A method of inhibiting a Flavivirus protease in a cell, comprising:

contacting a cell with an effective amount of a compound of the following structure

or a pharmaceutically acceptable salt or prodrug thereof.

A method of inhibiting a Flavivirus protease in a cell, comprising:

contacting a cell with an effective amount of a compound of the following structure

or a pharmaceutically acceptable salt or prodrug thereof.

27. The method of any of claims 21-26, wherein the Flavivirus protease is DENV-2 protease.

28. The method of any of claims 21-26, wherein the Flavivirus protease is WNV protease.

29. The method of any of claims 21-28, wherein the contacting is performed in vivo.

30. The method of any of claims 21-28, wherein the contacting is performed in vitro.