WO2009120783A1 - High affinity inhibitors of hepatitis c virus ns3/4a protease - Google Patents

Abstract

Compositions and uses thereof that inhibit HCV life cycle by inhibiting action of the HCV NS3/4A protease. An embodiment of the invention comprises a compound designated RS-2-19.

Description

HIGH AFFINITY INHIBITORS OF HEPATITIS C VIRUS

NS3/4A PROTEASE

CROSS-REFERENCE TO RELATED APPLICATION

[OOOlj This application claims priority to U.S. Provisional Application No. 61/070,684 filed March 25, 2008, the entire contents of which are hereby incorporated by reference. This invention was made using U. S. Government support under NlH grant GM57144. The government has certain rights in this invention,

BACKGROUND

1. FIELD OF THE INVENTION

[0002] The present invention relates to the hepatitis C virus and more particularly to methods and compositions that inhibit the hepatitis C virus (HCV) life cycle such as low molecular weight compounds that inhibit the HCV NS3/4A protease.

2. BACKGROUND OF THE INVENTION

[0003] Hepatitis C virus (HCV) infection represents an increasingly important global health-problem. There are approximately 3-4 million new cases of HCV infection each year, and current estimates suggest that a minimum of 3% of the world's population (approximately 170 million people) are chronically infected, and are at risk of developing liver cirrhosis and/or hepatocellular carcinoma. In the developed world, HCV infection is responsible for ~65% of all cases^' of liver cancer and accounts for ~70% of all liver transplants.

[0004] Today, in developed countries, most cases of HCV infection are acquired through the sharing of contaminated needles while injecting drugs or, to a much lesser extent, via sexual and perinatal transmission. However, in a significant number of patients the route of infection remains unknown. Before the routine screening of blood for HCV, many patients were infected by blood transfusions or treatment with infected blood products. At present, most new cases of HCV infection occur in the developing world, and it is believed that immigration will impact on HCV prevalence and subsequent disease burden in the developed world. [0005] Projections in the United States suggest that if half of all patients infected with HCV are identified, even with the most aggressive treatment at optimal doses and durations, the best possible outcome is a 24% reduction in the incidence of decompensated cirrhosis after 20 years. By 2020, the proportion of all US HCV cases with liver cirrhosis is estimated to increase from 16% to 32%, and decompensation will increase by 106% over current levels, resulting in an increased need for liver transplantation.

[0006) There are six different genotypes of HCV, with numerous subtypes. Genotype 1 is the most prevalent and most difficult to treat viral strain in Europe and North America, and represents the greatest unmet treatment need. Genotypes 2 and 3 appear to be more prevalent in the Far East. Of the other genotypes, genotype 4 is common in Africa and the Middle East, whereas genotypes 5 and 6 are predominant in South Africa and South-East Asia, respectively.

[0007] Current HCV therapies involve combinations of interferon with rivabarin which is only effective in 50% of patients, has toxic effects and is ineffective with several viral genotypes. In those cases, long term sudies indicated clearance of the virus and cure of the disease. However, the response to therapy is dependent on several factors, including viral genotype and patient characteristics.

[0008] Currently, there are no approved treatment options available for patients who have failed to respond to previous treatments.

[0009] Several new, specific anti-HCV drugs, are predicated on an understanding of the HCV life cycle, in particular the genomic organization and polyprotein processing. Thus, several agents are in development that target specific stages of the life cycle, the so-called specifically targeted antiviral therapy for HCV (STAT-C) drugs. Potential processes for viral inhibition include virus entry into the host cell, proteolytic processing, RNA replication, and the assembly and release of the new virions. Among the most promising new agents in development are the protease and polymerase inhibitors.

[0010] As set forth in Fig. 1, HCV NS3/4A protease consists of the protease domain of the viral NS3 protein which gets activated when bound to the NS4A polypeptide. The structure of the protease is shown in panel B of Fig. 1. The active NS3/4A is a serine protease that plays an essential role in the processing and maturation of the non- structural proteins in the virus, and is an important target for drug therapy. In panel A, NS indicates non-structural proteins, S represents structural proteins, and items 1, 2, 3, and 4 represent places of cleavage promoted by the NS3 protease. M. P. Manns, G. R. Foster, J. K. Rockstroh, S. Zeuzem, F. Zoulim and M. Houghton, Nature Reviews, 2007, 6, 991-1000, N. Goudreau and L. Brunet, Expert Opin. Investig. Drugs, 2005, 14(9), 1129-1 144, S. Di Marco, M, Rizzi, C. Volpari, The Journal of Biological Chemistry, 2000, 275(10), 7152-7157 10011] The HCV NS3/4A protease is one of the most important targets for drug development against HCV. Pharmaceutical companies are spending significant efforts in developing potent HCV NS3/4A protease inhibitors which can be administered alone, in combination with NS5B polymerase inhibitors, other antivirals, or other pharmaceutical or medical modality. The non-structural protein NS3 possesses a protease domain that is responsible for polyprotein processing and is a potential target for antiviral intervention. Despite the catalytic site being a shallow and largely hydrophobic groove, making it difficult to target, several compound inhibitors of the NS3 protease have been designed, for example, Telaprevir™ (VX-950), Boceprevir™ (SCH 503034), BIl 2202, MK-7009, TMC435350 and 1TMN-191.

[0012] Telaprevir™ (VX-950) from Vertex, Boceprevir™ (SCH 503034) from Schering- Plough and ITMN-191™ from Intermune/Roche are undergoing clinical trials. The structures of these three HCV NS3/4A protease inhibitors are shown in Fig. 2. |0013) The proof-of principle for this class of HCV NS3/4A protease inhibitor compounds was provided by BILN 2061. BILN 2061 provides at least a 2-3 Iogio decrease in HCV load within 48 hours. The structure of BILN 2061 is set forth in Fig. 2. However, the clinical development of BILN 2061 was stopped owing to significant side effects. . [0014] Currently, there continues to be a significant unmet need for therapies that are specific and effective against HCV disease, therapies that also have low side effect and toxicity profiles. In general, non-peptidic compounds are preferred over peptidic compounds usually due to better bioavailability and resistance to degradation. Telepravir™, Boceprevir™ as well as BILN 2061 can be considered small peptide-based compounds.

SUMMARY

[0015] This invention relates to inhibitors of the HCV NS3/4A protease. In some embodiments, these inhibitors are used in therapies that are specific and effective against HCV disease; these therapies may have low side effect and toxicity profiles. [0016] Embodiments include compounds of Formula I, shown below, that inhibit the HCV NS3/4A protease (NS3/4A inhibitors).

FORMULA I wherein Z is -CH₂-X, -CH=CR₁₅-X, or

¹ are each independently selected from the group consisting of hydrogen, (Ci-C₆)alkyl, (Ci-C₆)alkoxy, (C|-C₆)haloalkyl, (Ci-C6)hydroxyalkyl, haloge

n is independently 1, 2, 3, 4, or 5

; X is Ar o CH Ar, wherein Ar is aryl or heteroaryl, either of which may be further substituted by one or more

wherein

is independently selected from the group consisting of

( alkyl,

)alkoxy,

haloalkyl,

)hydroxyalkyl, halogen,

and -

are each independently hydrogen or (Ci-C₆)alkyl; and R₁5 is -CN or -

[0017] Other embodiments include compositions comprising these compounds and one or more pharmaceutical excipients.

[0018] Other embodiments include methods for altering the physiology of a subject infected with HCV comprising administering a composition comprising a compound of

Formula I to a subject that is infected with HCV. In certain embodiments, the HCV-infectcd subject thereby achieves a therapeutic benefit. In other embodiments, the compositions or agents may be administered orally.

[0019] Other features of the invention will be apparent from the detailed description, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : HCV NS3/4A protease consists of the protease domain of the viral NS3 protein which gets activated when bound to the NS4A polypeptide.

[0021] Figure 2: Structures of HCV NS3/4A protease inhibitors BILN-2061 ; telaprevir; boceprevir; and ITMN-191.

[0022] Figure 3: Exemplary compounds according to Formula VH.

[0023] Figure 4: Exemplary compounds according to Formulas X and XI.

[0024] Figure 5: Diagramatic scheme for chemical synthesis of compounds according to

Formulas II, III, IV, and V.

[0025] Figure 6: Diagramatic scheme for chemical synthesis of exemplary compound RS-

2-5 and (E)-2-(3-chlorophenyl)ethenesulfonyl chloride..

[0026] Figure 7: Diagramatic scheme for chemical synthesis of compounds according to

Formula X.

|0027] Figure 8: Diagramatic scheme for chemical synthesis of compounds according to

Formula XI.

[0028] Figure 9: Rate of substrate hydrolysis with and without inhibitor RS-2- 19.

[0029] Figure 10: Chemical structure of exemplary compound RS-2-19.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions

[003Oj As used herein, "agent" is a non-peptide, small molecule compound according to the invention.

[0031] By "analog" is meant an agent having structural or functional homology to a reference agent.

[0032] By "cell substrate" is meant the cellular or acellular material (e.g., extracellular matrix, polypeptides, peptides, or other molecular components) that is in contact with the cell.

[0033] By "control" is meant a standard or reference condition.

[0034] By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, organ or subject. [0035] By "effective amount" is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated subject. The effective amount of an active therapeutic agent for the treatment of a disease or injury varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending clinician will decide the appropriate amount and dosage regimen. [0036] By "modifies" is meant alters. An agent that modifies a cell, substrate, or cellular environment produces a biochemical alteration in a component (e.g., polypeptide, nucleotide, or molecular component) of the cell, substrate, or cellular environment. [0037] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

[0038] As used herein, a "prodrug" is a pharmacologically inactive compound that is converted into a pharmacologically active agent by a metabolic transformation. [0039] By "subject" is meant a mammal, including, but not limited to, a human or non- human mammal, such as a bovine, equine, canine, ovine, or feline.

[0040] By "therapeutic delivery device" is meant any device that provides for the release of a therapeutic agent. Exemplary therapeutic delivery devices include osmotic pumps, indwelling catheters, delayed-release and sustained-release biomaterials. [0041] As used herein, the terms "treat," treating," "treatment," "therapeutic" and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. [0042] By "variant" is meant an agent having structural homology to a reference agent/compound but varying from the reference in its biological activity. For example, variants can comprise optimized amino acid and nucleic acid sequences that are selected using the methods described herein as having one or more desirable characteristics.

Compounds

[0043] One embodiment includes compounds having the Formula I shown below

FORMULA I wherein

or

( ₂)₂-, or -CH=CH-; R¹, R" and R"¹ are each independently selected from the group consisting of hydrogen,

ydroxyalkyl, halogen,

independently 1, 2, 3, 4, or 5; X is -Ar Or -CH₂-Ar, wherein Ar is aryl or heteroaryl, either of which may be further substituted by one or more Ri ₆, wherein Ri₆ is independently selected from the group consisting o

C₆)hydroxyalkyl, halogen and -

are each independently hydrogen or -

. Examples of substituents R', R", and R"' include

H C(CH ) Cl B CH -

-

. Where n is greater than 1, the substituents may be the same or different, and may be located at any position on the ring. [0044] The terms "alkyl", "alkoxy", "hydroxyalkyl", "alkoxyalkyl", and "alkoxycarbonyl", used alone or as part of a larger moiety include both straight and branched chains containing one to four carbon atoms, as well as cyclic structures such as cyclopropyl and cyclobutyl. Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (Pr) (including n-propyl (ⁿPr or n-Pr), isopropyl (¹Pr or i-Pr) and cyclopropyl (^cPr or C-Pr)), butyl (Bu) (including n-butyl ("Bu or n-Bu), isobutyl (¹Bu or i-Bu), tert-butyl (¹Bu or t-Bu) and cyclobutyl (^cBu or C-Bu)) and so forth. The term "alkoxy" refers to an -O-alkyl radical. The term "haloalkyl" means alkyl, substituted with one or more halogen atoms, such as trifiuoromethyl. The term "halogen" or "halo" means F, Cl, Br, or I. [0045] The term "aryl" used alone or as part of a larger moiety, refers to mono-, bi-, or tricyclic aromatic hydrocarbon ring systems having five to fourteen members, such as phenyl, 1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl. The term "aryl" also refers to rings that are optionally substituted by R)₆, defined previously. The term "aryl" may be used interchangeably with the term "aryl ring". "Aryl" also includes fused polycyclic aromatic ring systems in which an aromatic ring is fused to one or more rings. Examples include 1- naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl. Also included within the scope of the term "aryl", as it is used herein, is a group in which an aromatic ring is fused to one or more non- aromatic rings, such as in an indanyl, phenanthridinyl or tetrahydronaphthyl, where the radical or point of attachment is on the aromatic ring.

[0046] The term "heteroaryl", used alone or as part of a larger moiety, refers to heteroaromatic ring groups having five to fourteen members, preferably five to ten, in which one or more ring carbons, preferably one to four, are each replaced by a heteroatom such as N, O, or S. Examples of heteroaryl rings include 2-furanyl, 3-furanyl, N-imidazolyl, 2- imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2- oxadiazolyl, 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 1-pyrτolyl, 2-pyrrolyl, 3- pyrrolyl, 1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl, 2-ρyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 3-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 5-tetrazolyl, 2- triazolyl, S-triazolyl, 2-thienyl, 3-thienyl, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl, benzimidazolyl, isoquinolinyl, indazolyl, isoindolyl, acridinyl, or benzoisoxazolyl. Also included within the scope of the term "heteroaryl", as it is used herein, is a group in which a heteroaromatic ring is fused to one or more aromatic or nonaromatic rings where the radical or point of attachment is on the heteroaromatic ring. Examples include tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyridof3,4-d]pyrimidinyl The term "heteroaryl" also refers to rings that are optionally substituted by Ri₆, defined above. The term "heteroaryl" may be used interchangeably with the term "heteroaryl ring" or the term "heteroaromatic". The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl. [0047] Further embodiments include compounds according to Formula I, where Z is

as shown by Formula II below

FORMULA II wherein

are each independently selected from the group consisting of hydrogen,

)hydroxyalkyl, halogen and -

n is independently 1, 2, 3, 4, or 5. Further embodiments include compounds according to Formula II, where (R')n and (R'")_n are the same, as shown by Formula III below.

FORMULA III wherein Y is -(CH₂)₂-, or -CH=CH-; R' and R" are each independently selected from the group consisting of hydrogen, (Ci-C₆)alkyl, (C|-C₆)alkoxy, (C]-C6)haloalkyl, (Ci- C₆)hydroxyalkyl, halogen

and - NO₂; n is independently 1, 2, 3, 4, or 5.

[0048] Further embodiments include compounds according to Formula I, where Z is - CH₂-X₁ as shown by Formula IV below

where

, wherein Ar is aryl or heteroaryl, either of which may be further substituted by one or more

wherein

is independently selected from the group consisting of

( ] ₆)alkyl, (

)alkoxy, (

i 6)haloalkyl,

C₆)hydroxyalkyl, halogen,

and - N

and

are each independently hydrogen or

( J lkyl; and

CN or -

[0049] A further embodiment includes compounds according to Formula IV, where Y is -CH=CH-, and X is -Ar.

[0050] Further embodiments include compounds according to Formula I, where Z is

as shown in Formula V below

FORMULA V where Y is -(CH₂)₂-, or -CH=CH-; X is -Ar or -CH₂-Ar, wherein Ar is aryl or heteroaryl, either of which may be further substituted by one or more

wherein

is independently selected from the group consisting o

C₆)hydroxyalkyl, halogen,

, and -

₉ are each independently hydrogen or (Ci-C6)alkyl; and Ri₅ is -CN or -

CO₂R₁₉.

[0051] A further embodiment includes compounds according to Formula V where Y is -

CH-CH- and X is -Ar.

[0052] Other embodiments include compounds of Formula VI, shown below

FORMULA VI where Y is -(CHz)₂-, or -CH=CH-; and Rj-R₉ are each independently selected from the group consisting of hydrogen, (Cι-Cό)alkyl, (Ci-C₆)alkoxy, (C_rC6)haloalkyl, (Ci-C₆)hydroxyalkyl, halogen

Examples of sυbstituents

, ₉ nclude

and -CONH₂. Examples of compounds according to Formula VI are shown in Fig. 3. [00531 Further embodiments include compounds according to Formula VI, where Y is - CH-CH-, as exemplified in Formula VII below

FORMULA VII wherein Ri-R₉ arc each independently selected from the group consisting of hydrogen, (C_r C₆)alkyl, (C,-C₆)alkoxy, (C|-C₆)haloa!kyl, (Ci-C₆)hydroxyalkyl₍ halogen, -NR,₇Ri8, -COH, -

and -NO₂.

[0054] Further embodiments include compounds according to Formula VI, wherein Y is -(CH₂)₂- as shown in Formula VIII below

FORMULA VIH wherein R₁-R₉ are each independently selected from the group consisting of hydrogen, (Ct-

C₆)alkyl, (C₁-C₆)alkoxy, (Q-C^haloalkyl, (C₁C₆)hydroxyalkyl, halogen, -NR)₇R₁₈, -COH, -

CO₂R₁₉, -CONR17R18, -OH, -CN, -CF₃, and -NO₂.

[0055] Further embodiments include compounds of Formulas VI, VII, or VIII where R₂ and R₃ are identical, but are not H, and R₄=H.

[0056] Further embodiments include compounds of Formulas VI, VII, or VIII where R₄ is not H, and R₂=R₃=H.

[0057] Further embodiments include compounds of Formulas VI, VII, or VIII where R₆-

R9 are all hydrogen, and at least one of R₁ and R₅ is not H.

[0058J Exemplary specific embodiments include compounds according to Formula VII shown below:

[0059] One specific embodiment includes compounds RS-2-5 and RS-2-19, shown below.

[0060] One specific embodiment according to Formula VIII includes the compound RS- 4-2Pl, shown below.

(0061] Other embodiments include compounds of Formula IX, shown below:

FORMULA IX wherein Z is -CH₂-X or -CH=CRj₅-X; Y is -(CH₂)₂-, or -CH=CH-; R_I0-R_M are each independently selected from the group consisting of hydrogen, (C]-C₆)alkyl, (C|-C6)alkoxy, (C,-C₆)haloalkyl, (C|-C₆)hydroxyalkyl, halogen, -NR₁₇Ri₈, -COH, -CO₂Ri₉, -CONR]₇Ri₈, - OH, -CN, -CF₃, and -NO₂; Ri₅ is -CN or -CO₂Ri₉; and X is -Ar or -CH₂-Ar, wherein Ar is aryl or heteroaryl, either of which may be further substituted by one or more Ri6, wherein R|₆ is independently selected from the group consisting of (Ci-C₆)alkyl, (Ci-C6)alkoxy, (Ci- QOhaloalkyl, (d-C₆)hydroxyaIkyl, halogen, -NRi₇Ri₈, -COH, -CO₂Ri₉, -CONRi₇Ri₈, -OH, - CN, -CF₃, and -NO₂ Examples of substituents RI₀-RH include -H, -C(CH₃)₃, -Cl, -Br, -CH₃, -OCH₃, -NO₂, -NH₂, -OH, -CH₂OH, -CH₂Br, -CHO, -CO₂H, -CO₂CH₃, -CO₂Et, -CF₃, - CONHCH₃, -CN, and -CONH₂.

[0062] Further embodiments include compounds of Formula IX where Z is -CH₂-X, as shown by Formula X.

FORMULA X

Y is ~(CH₂)₂-, or -CH=CH-; R_IO-R_M are each independently selected from the group consisting of hydrogen, (Ci-C₆)alkyl, (C|-C₆)alkoxy, (Cι-C₆)haloalkyl, (d-C₆)hydroxyalkyl, halogen, -NR₁₇Ri₈, -COH, -CO₂Ri₉, -CONR₁₇Ri₈, -OH, -CN, -CF₃, and -NO₂; and X is -Ar or -CH₂-Ar, wherein Ar is aryl or heteroaryl, either of which may be further substituted by one or more

R^ is independently selected from the group consisting of (Ci-Ce)alkyl, (C,-C₆)alkoxy, (C,-C₆)haloalkyl, (C_rC₆)hydroxyalkyl, halogen, -NR₁₇R₁₈, -COH, -CO₂Ri₉, -

CONRi₇Ri₈, -OH, -CN, -CF₃, and -NO_2. Examples of compounds according to Formula X are shown in Fig. 4.

[0063] One embodiment includes compounds of Formula X wherein Y is -CH=CH- and

X is -Ar.

[0064] Further embodiments include compounds of Formula IX, where Z is -CH=CR 15-

X, as shown by Formula XI.

FORMULA XI

Y is -(CH₂)₂-, or -CH=CH-; Rιo-Ri4 are each independently selected from the group consisting of hydrogen, (Ci-C₆)alkyl, (d-C₆)alkoxy, (Ci-C₆)haloalkyl, (d-C₆)hydroxyalkyl, halogen, -NR₁₇Ri₈, -COH, -CO₂Ri₉, -CONR₁₇Ri₈, -OH, -CN, -CF₃, and -NO₂; R₁₅ is -CN or -CO₂R₁₉; and X is -Ar or -CH₂-Ar, wherein Ar is aryl or heteroaryl, cither of which may be further substituted by one or more Ri₆, wherein R|₆ is independently selected from the group consisting of (Ci-C₆)alkyl, (Ci-C₆)alkoxy, (CrC₆)haloalkyl, (C|-C₆)hydroxyalkyl, halogen, - NR17R18, -COH, -CO₂Ri₉, -CONR17R18, -OH, -CN, -CF₃, and -NO₂. Examples of compounds according to Formula XI are shown in Fig. 4. [0065] One embodiment includes compounds of Formula XI wherein Y is -CH=CH-, and X is -Ar.

[0066] Further embodiments include compounds of Formulas IX, X and XI, where at least one of R₁₃ and Rμ is not hydrogen.

[0067] Further embodiments include compounds of Formulas IX, X and XI, where at least one of Rio. Rn, and Ri₂ is not hydrogen.

[0068] Further embodiments include compounds of Formulas IX, X and XI₁ where Ar is phenyl, pyridyl, thiophenyl, thiazolyl, indolyl, pyrrolyl, furanyl, or bezothiazolyl. [0069] Further embodiments include compounds of Formulas IX, X and XI, where Ar is one of the structures shown below:

wherein R^, is independently selected from the group consisting of (Cι-C6)alkyl, (Ci- C₆)alkoxy, (Ci-C₆)haloalkyl, (C|-C₆)hydroxyalkyl, halogen, -NR₁₇R₁₈, -COH, -CO₂R₁₉, - CONR₁₇R₁₈, -OH, -CN, -CF₃, and -NO₂

[0070] Exemplary specific embodiments include compounds according to Formula IX shown below:

[0071] In certain embodiments the compounds of the present invention may have an IC₅₀ against Heptatitis C Virus NS3/4A Protease of equal to or less than 50 μM. In certain embodiments the compounds may have an ICsoof 25 μM or less, 10 μM or less, or 5 μM or less.

[0072] The compounds may be synthesized, for example, according to the synthetic schemes shown in Fig. 5 and further exemplified in Fig. 6, 7, and 8. [0073] Further embodiments include derivatives of the compounds, such as pharmaceutically acceptable salts, ether derivatives, ester derivatives, acid derivatives, and aqueous solubility altering derivatives of the active compound. Derivatives include all individual enantiomers, diastereomers, racemates, and other isomers of the compounds. Derivatives also include all polymorphs and solvates, such as hydrates and those formed with organic solvents, of these compounds. Such isomers, polymorphs, and solvates may be prepared by methods known in the art, such as by regiospecific and/or enantioselective synthesis and resolution.

[0074] Suitable salts of the compound include, but are not limited to, acid addition salts, such as those made with hydrochloric, hydrobromic, hydroiodic, perchloric, sulfuric, nitric, phosphoric, acetic, propionic, glycolic, lactic pyruvic, malonic, succinic, maleic, fumaric, malic, tartaric, citric, benzoic, carbonic cinnamic, mandelic, methanesulfonic, ethanesulfonic, hydroxyethanesulfonic, benezenesulfonic, p-toluene sulfonic, cyclohexanesulfamic, salicyclic, p-aminosalicylic, 2-phenoxybenzoic, and 2-acetoxybenzoic acid; salts made with saccharin; alkali metal salts, such as sodium and potassium salts; alkaline earth metal salts, such as calcium and magnesium salts; and salts formed with organic or inorganic ligands, such as quaternary ammonium salts.

[0075] Additional suitable salts include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium cdetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, oleate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide and valerate salts of the compound of the present invention.

Pharmaceutical compositions

[0076] A further embodiment includes pharmaceutical compositions comprising the HCV

NS3/4A protease inhibitors of Formula 1 discussed above.

[0077] In certain embodiments the compositions may include one or more than one inhibitor, and may further contain other suitable substances and excipients, including but not limited to physiologically acceptable buffering agents, stabilizers (e.g. antioxidants), flavoring agents, agents to effect the solubilization of the compound, and the like.

[0078] In certain embodiments, the composition may be in any suitable form such as a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. The composition may include suitable parenterally acceptable carriers and/or excipients. [0079] In certain embodiments, the compositions may comprise an effective amount of an inhibitor in a physiologically-acceptable carrier. The carrier may take a wide variety of forms depending on the form of preparation desired for a particular route of administration. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin.

[0080] In certain embodiments, the inhibitor may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) or oral administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

[0081] In certain embodiments, the compositions may be in a form suitable for administration by sterile injection. To prepare such a composition, the compositions(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). For parenteral formulations, the carrier will usually comprise sterile water, though other ingredients, for example, ingredients that aid solubility or for preservation, may be included. Injectable solutions may also be prepared in which case appropriate stabilizing agents may be employed.

[0082] Formulations suitable for parenteral administration usually comprise a sterile aqueous preparation of the inhibitor, which preferably is isotonic with the blood of the recipient (e.g., physiological saline solution). Such formulations may include suspending agents and thickening agents and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. The formulations may be presented in unit-dose or multi-dose form.

[0083] Parenteral administration may comprise any suitable form of systemic delivery or localized delivery. Administration may for example be intravenous, intra-arterial, intrathecal, intramuscular, subcutaneous, intramuscular, intra-abdominal (e.g., intraperitoneal), etc., and may be effected by infusion pumps (external or implantable) or any other suitable means appropriate to the desired administration modality.

[0084] In certain embodiments, the compositions may be in a form suitable for oral administration. In compositions in oral dosage form, any of the usual pharmaceutical media may be employed. Thus, for liquid oral preparations, such as, for example, suspensions, elixirs and solutions, suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like. For solid oral preparations such as, for example, powders, capsules and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. If desired, tablets may be sugar coated or enteric coated by standard techniques. [0085] Compositions suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount of the active ingredient as a powder or granules. Optionally, a suspension in an aqueous liquor or a non-aqueous liquid may be employed, such as a syrup, an elixir, an emulsion, or a draught. Formulations for oral use include tablets containing active ingredient(s) in a mixture with pharmaceutically acceptable excipients. Such formulations are known to the skilled artisan. Excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc).. Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like. [0086] A syrup may be made by adding the inhibitor to a concentrated aqueous solution of a sugar, for example sucrose, to which may also be added any accessory ingredient(s). Such accessory ingredient(s) may include flavorings, suitable preservative, agents to retard crystallization of the sugar, and agents to increase the solubility of any other ingredient, such as a polyhydroxy alcohol, for example glycerol or sorbitol. [0087] In some embodiments, the composition may be in a form of nasal or other mucosal spray formulations (e.g. inhalable forms). These formulations comprise purified aqueous solutions of the active compounds with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal or other mucous membranes. Alternatively, they can be in the form of finely divided solid powders suspended in a gas carrier. Such formulations may be delivered by any suitable means or method, e.g., by nebulizer, atomizer, metered dose inhaler, or the like. (0088] In some embodiments, the composition may be in a form suitable for rectal administration. These formulations may be presented as a suppository with a suitable carrier such as cocoa butter, hydrogenated fats, or hydrogenated fatty carboxylic acids. [0089] In some embodiments, the composition may be in a form suitable for transdermal administration. These formulations may be prepared by incorporating the active compound in a thixotropic or gelatinous carrier such as a cellulosic medium, e.g., methyl cellulose or hydroxyethyl cellulose, with the resulting formulation then being packed in a transdermal device adapted to be secured in dermal contact with the skin of a wearer. [0090] In addition to the aforementioned ingredients, compositions of the invention may further include one or more accessory ingredient(s) selected from encapsulants, diluents, buffers, flavoring agents, binders, disintegrants, surface active agents, thickeners, lubricants, preservatives (including antioxidants), and the like.

[0091] In some embodiments, compositions may be formulated for immediate release, sustained release, delayed-onset release or any other release profile known to one skilled in the art.

[0092] In some embodiments, the pharmaceutical composition may be formulated to release the active compound substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in the central nervous system or cerebrospinal fluid; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target the site of a pathology. For some applications, controlled release formulations obviate the need for frequent dosing to sustain activity at a medically advantageous level.

[0093] Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the compound in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the inhibitor is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the inhibitor in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

[0094] In some embodiments, the composition may comprise a "vectorized" form, such as by encapsulation of the inhibitor in a liposome or other encapsulate medium, or by fixation of the inhibitor, e.g., by covalent bonding, chelation, or associative coordination, on a suitable biomolecule, such as those selected from proteins, lipoproteins, glycoproteins, and polysaccharides.

[0095] In some embodiments, the composition can be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents. Alternatively, the inhibitor may be incorporated in biocompatible carriers, implants, or infusion devices.

[0096] Materials for use in the preparation of microspheres and/or microcapsules are, e.g., biodegradable/bioerodible polymers such as polygalactia poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutamine) and, poly(lactic acid). Biocompatible carriers that may be used when formulating a controlled release parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or antibodies. Materials for use in implants can be non-biodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters) or combinations thereof).

[0097] Unless the context clearly indicates otherwise, compositions of all embodiments can comprise various pharmaceutically acceptable salts, ether derivatives, ester derivatives, acid derivatives, and aqueous solubility altering derivatives of the inhibitor. Certain embodiments can comprise all individual enantiomers, diastereomers, racemates, and other isomer of compounds of the invention. The invention also includes all polymorphs and solvates, such as hydrates and those formed with organic solvents, of this compound. Such isomers, polymorphs, and solvates may be prepared by methods known in the art, such as by regiospecific and/or enantioselective synthesis and resolution, based on the disclosure provided herein.

[0098] Suitable salts of the compound include, but are not limited to, acid addition salts, such as those made with hydrochloric, hydrobromic, hydroiodic, perchloric, sulfuric, nitric, phosphoric, acetic, propionic, glycolic, lactic pyruvic, malonic, succinic, maleic, fumaric, malic, tartaric, citric, benzoic, carbonic cinnamic, mandelic, methanesulfonic, ethanesulfonic, hydroxyethanesulfonic, benezenesulfonic, p-toluene sulfonic, cyclohexanesulfamic, salicyclic, p-aminosalicylic, 2-phenoxybenzoic, and 2-acetoxybenzoic acid; salts made with saccharin; alkali metal salts, such as sodium and potassium salts; alkaline earth metal salts, such as calcium and magnesium salts; and salts formed with organic or inorganic ligands, such as quaternary ammonium salts.

[0099] Additional suitable salts include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylatc, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate,.napsylate, nitrate, N-methylglucamine ammonium salt, oleate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide and valerate salts of the compound of the present invention. [00100] The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy.

Methods

[00101] A further embodiment includes methods for altering the physiology of a subject infected with HCV. The method comprises administering a composition comprising a compound of Formula I to a subject that is infected with HCV. [00102] In one embodiment, a preparation comprising the inhibitor formulated for oral administration is orally given to a patient infected with HCV.

[00103] In one embodiment, the preparation is administered such that the free concentration of inhibitor in serum is higher than the Ki (inhibition constant) of the inhibitor.

In another embodiment the free concentration of inhibitor in serum is 10 times the Ki. In another embodiment, the concentration of inhibitor in serum is sufficient to inhibit 90% or more of the protease molecules.

[00104] For example RS-2-5 is characterized by a Ki of 0.25 μM, and as such, a concentration of 3μM inhibits 90% of the viral protease molecules.

[00105] At these concentrations the inhibition of the NS3/4A protease arrests viral replication, and results in a drop in viral count of several orders of magnitude within one week. With continued oral administration, sufficient HCV is killed that the patient achieves extremely low and in some cases undetectable viral titers.

[00106] In another embodiment, the inhibitor is administered in combination with an antiviral agent that targets a different essential viral protein. In this context, "in combination" means that they are administered concurrently or sequentially or a combination thereof.

Combination therapy targets, inhibits or alters two or more aspects relevant to HCV lifecycle.

With combination therapy the incidence of viral escape is lessened because the virus would have to have two or more escape mutations simultaneously.

[00107] In one embodiment, a composition according to the above embodiments that inhibits the NS3/4A protease, and a composition that targets NS5B polymerase are used in combination. The NS5B polymerase is another viral protein essential for the viral life cycle.

Several HCV NS5B polymerase inhibitors are disclosed in the scientific literature.

[00108] In one embodiment, the NS3/4A inhibitor, RS-2-19, is given in combination with a polymerase inhibitor. The simultaneous inhibition of essential protein(s) accelerates viral killing and results in a drop in viral count of several orders of magnitude within one week.

With continued oral administration of RS-2-19 in combination with an HCV NS5B polymerase inhibitor, sufficient HCV is killed that the patient achieves extremely low and in some cases undetectable viral titers.

[00109] In other embodiments, the NS3/4A inhibitor according to the present invention may be administered in combination with other therapeutic agents. For example, the NS3/4A inhibitor, RS-2-19 may be administered in combination with pegylated interferon (peglFN- α). [00110] The compounds or compositions which are administered may be administered in any of many forms which are well-known to those of skill in the art. For example, they may be administered in any of a variety of art-accepted forms such as tablets, capsules, various injectable formulations, liquids for oral administration and the like, as suitable for the desired means of administration. The preparation which is administered may include one or more than one inhibitory compound, and may further contain other suitable substances and excipients, including but not limited to physiological acceptable buffering agents, stabilizers (e.g. antioxidants), flavoring agents, agents to effect the solubilization of the compound, and the like. Administration of the compounds may be effected by any of a variety of routes that are well-known to those of skill in the art, including but not limited to oral, parenteral, intravenously, via inhalation, and the like. Further, the compounds may be administered in conjunction with other appropriate treatment modalities, for example, with nutritional supplements, agents to reduce symptoms and treatment with other agents. [0011 IJ In certain embodiments, the compositions may be administered orally. Administration to human patients or other animals is generally carried out using a physiologically effective amount of a compound of the invention in a physiologically- acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin.

[00112] In certain embodiments, the compositions may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Routes of administration include, for example, subcutaneous, intravenous, intraperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the drug in the patient. Administration to human patients or other animals is generally carried out using a physiologically effective amount of a compound of the invention in a physiologically- acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin.

[00113] The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy.

[00114] For example, compositions according to the invention may be provided in a form suitable for administration by sterile injection. To prepare such a composition, the compositions(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate).

[00115] The compositions may be provided in unit dosage forms (e.g., in single-dose ampules), or in vials containing several doses and in which a suitable preservative may be added. A composition of the invention may be in any suitable form such as a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. The composition may include suitable parenterally acceptable carriers and/or excipients. [00116] The amount of the compound/agent to be administered varies depending upon the manner of administration, the age and body weight of the subject/patient, and with the subject's symptoms and condition. A compound is generally administered at a dosage that best achieves medical goals with the fewest corresponding side effects. [00117] In some embodiments, the compositions including biologically active fragments, variants, or analogs thereof, can be administered by any suitable route including, but not limited to: oral, intracranial, intracerebral, intraventricular, intraperitoneal, intrathecal, intraspinal,, topical, rectal, transdermal, subcutaneous, intramuscular, intravenous, intranasal, sub-lingual, mucosal, nasal, ophthalmic, subcutaneous, intramuscular, intravenous, intraarticular, intra-arterial, sub-arachinoid, bronchial, lymphatic, and intra-uterille administration, and other dosage forms for systemic delivery of active ingredients. [00118] Those of skill in the art will recognize that the precise quantity of such a compound to be administered will vary from case to case, and is best determined by a skilled practitioner such as a physician. For example, the amount may vary based on several characteristics of the patient, e.g. age, gender, weight, overall physical condition, extent of disease, and the like. Further, the individual characteristics of the compound itself (e.g. Ki, selectivity, ICso, solubility, bioavailability, and the like) will also play a role in the amount of compound that must be administered. However, in general, the required amount will be such that the concentration of compound in the blood stream of the patient is about equal to or larger than the IC₅₀ or K, of the compound.

[00119] The composition may be administered parenterally by injection, infusion or implantation in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and/or adjuvants. In one embodiment, the compositions are added to a retained physiological fluid, such as cerebrospinal fluid, blood, or synovial fluid. The compositions of the invention can be amenable to direct injection, application or infusion at a site of disease or injury. [00120] In one approach, a composition of the invention is provided within an implant, such as an osmotic pump, or in a graft comprising appropriately transformed cells. Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested for the controlled delivery of drugs, including proteinacious biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a bioactive factor at a particular target site.

Dosage

[00121] The administration of a compound may be by any suitable means that results in a concentration of the compound that, combined with other components, is effective in preventing, diagnosing, prognosing, ameliorating, reducing, or stabilizing a deficit or disorder.

Generally, the amount of administered agent of the invention will be empirically determined in accordance with information and protocols known in the art. Often the relevant amount will be such that the concentration of compound in the blood stream of the patient is about equal to or larger than the IC50 or Kj of the compound. Typically agents are administered in the range of about 10 to 1000 μg/kg of the recipient. Other additives may be included, such as stabilizers, bactericides, and anti-fungals. These additives are present in conventional amounts.

Kits

[00122] Another emobodiment includes kits, e.g., for the treatment, diagnosis, prophylaxis or prognosis of disease or injury. In one embodiment, the kit includes a composition according to the above embodiments containing an effective amount of a compound according to the above embodiments in unit dosage form. In some embodiments, the kit comprises an outer container or package. The kit can comprise a sterile container which contains the composition according to the above embodiments, such sterile containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments. [00123] In certain kit embodiments, a composition according to the above embodiments is provided together with instructions for administering it to a subject. Instructions may include information about the use and effects of the composition. In one embodiment, the instructions will include at least one of the following: description of composition of the invention, dosage schedule and administration protocols, precautions, warnings, indications, counter- indications, overdosage information, adverse reactions, animal pharmacology, clinical studies, and/or references.

[00124] The instructions may be printed directly on a container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, insert, or folder supplied in or with the container. Thus, the instructions may be a separate item in the kit, or be imprinted, embossed, molded or otherwise affixed to another item in the kit; instructions may be printed on an outer container and also included as an insert item in the kit.

[00125] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[00126] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[00127] Terms listed in single tense also include multiple unless the context indicates otherwise.

[00128] The examples disclosed below are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. AU publications, databases, and patents cited herein are hereby incorporated by reference for all purposes.

[00129] Methods for preparing the compounds of this invention are illustrated in the following Examples. Starting materials are made according to procedures known in the art or as illustrated herein. The following examples are provided so that the invention might be more fully understood. These examples are illustrative only and should not be construed as limiting the invention in any way. EXAMPLES

Example 1. Chemical Synthesis of RS-2-5

[00130] The synthesis of RS-2-5 is depicted in Fig. 6. The synthesis of other embodied compounds according to Formula VII and VIII follow the same synthetic scheme with different substituted sulfonyl chlorides and/or different anilines. For example, different styrene sufonylchlorides can be prepared according to the first step shown in Fig. 6. [001311 Synthesis of (E)-2-(3-chlorophenyl)ethenesulfonyl chloride (1): Sulfuryl chloride (3.2 ml, 38.6 mmol, 2 eq.) was added dropwise into a 2-neck-lOO mL round bottom flask containing 2.7 mL of DMF at 0 ⁰C. After stirring the mixture at RT for 30 min, 1- chloro-3-vinylbenzene (2.5 mL, 19.3 mmol, 1 eq.) was added and the reaction mixture was heated at 55 ⁰C for 5 h. After the reaction mixture was cooled at RT, it was poured into ice water (20 mL) and the product was extracted three times from the aqueous phase with dichloromethane (DCM). The resulting organic phase was washed once with water and brine and dried under Na₂SO₄. After the product was purified from the crude by flash column chromatography (5% EtOAc/Hex), product 1 (4.55 g, 19.3 mmol) was obtained in 44% yield. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.67 (d, J-15.2Hz, C=CH₅IH), 7.56 (s, ArH, IH), 7.50 (m, ArH, IH,), 7.43 (m, ArH, 2H), 7.24 (d, J=I 3.4Hz, C=CH, IH₁). DEPT 135 (100 MHz, CDCl₃) δ (ppm): 143.5, 132.5, 131.2, 130.7, 128.8, 127.4.

[00132] Synthesis of (E)-N,2-bis(3-chlorophenyl)-N-((E)-3-chlorostyrylsulfonyl)

Ethencsulfonamide (RS-2-5): Compound 1 (140 mg, 0.6 mmol, 2 eq.) and 3- chlorobenzenamine (38.7 mg, 0.3 mmol, 1 eq.) were poured into a 50 mL round bottom flask and dissolved with 6 mL of acetonitrile. After all components were dissolved, TEA (0.1 mL, 0.66 mmol, 2.2 eq.) was added and the reaction mixture was stirred at RT for 12 h. Water was added and the solid formed was filtered and washed twice with AN. After drying the product, RS-2-5 (99.4 mg, 0.19 mmol) was obtained in 63% yield. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.54 (t, J=I .5Hz, ArH, 2H,), 7.48 (m, ArH, IH), 7.45 (m, ArH, 3H), 7.41 (m, ArH, C=CH, 5H), 7.35 (d, J=8.1 Hz₁ ArH, IH), 7.33 (t, J= 1.97, ArH, IH), 7.27 (d, J=I 5.4 Hz, C=CH, 2H), 7.20 (ddd, , J=0.8, 1.7, 7.8Hz, ArH.lH).

[001331 Exemplary compounds according to Formula VII and VIII are described in the following table. Entries where n = 1 describe compounds according to Formula VII, entries where n = 2 describe compounds according to Formula VIII.

Example 2. Chemical Synthesis of Derivatives of Formula X.

[00134] The synthesis of exemplary compounds according Io Formula X are shown in

Fig. 7. As set forth in Fig. 7, the synthesis of compounds of Formula X starts with the reaction of substituted sulfonyl chlorides (for example, prepared according to step 1 of

Example 1) and anilines. The reaction is carried out in the presence of a lM-solution of sodium carbonate at the pH range of 6.5-7.0 for about 2h. When the aniline has

Ri=R₂=COOH, an additional step with 2N HCl it is required to acidify the carboxylate intermediate formed to produce the monosulfonylated compound 2.

[00135] For all the other substituents, simple filtration of the solid formed is enough to isolate the product formed from the reaction mixture.

[00136] In the second step, as depicted in Fig. 7, an amide intermediate is initially formed by treating compound 2 with NaH and, then this reactive intermediate is allowed to react with electrophile X-CH2-Br to form the final product.

[00137] Exemplary compounds according to Formula X are described in the following table.

Table 2: Exemplary Compounds According to Formula X

Example 3. Synthesis of Derivatives of Formula XI

[00138] The synthesis of exemplary compounds according to Formula XI is shown by example in Fig. 8. As set forth in Fig. 8, the synthesis of compounds of Formula XI begins with the reaction of an aniline derivative with an α-cyano aldehyde (3) (prepared for example according to the methods described by Xiao et al. (Bioorganic & Medicinal Chemistry, vol. 15, no'. 12, pp. 4212-4219, 2007) or Du et al. (Organic Letters , vol. 8, no. 26, pp. 5919-5922, 2006) to form an enamine (4). In the last step, the enamine (4) is treated with NaH and then allowed to react with a sulfonyl chloride. The sulfonyl chloride may be prepared for example according to step 1 of Example 1. The synthesis of other embodied compounds follows the same process with different aldehydes, anilines, or sulfonyl chlorides.

Example 4. Inhibition of HCV NS3/4A Protease, determination of IC^ [00139] The HCV protease inhibition assay was performed using a commercially available fluorogenic 5-FAM/QXL™520 FRET substrate pair (Ac-DE-Dap(QXL520)-EE- Abu-ψ-[COO]-AS-C(5-FAMsp)-NH2) purchased from ANASPEC, San Jose, CA. The change in fluorescence intensity was monitored in a Cary Eclipse fluorescence spectrophotometer (Varian) with 490 and 520 nm for excitation and emission wavelengths respectively. Kinetic measurements were performed at 25 ⁰C in a buffer solution prepared with component D (2X assay buffer) and component F (DTT) of the Sensolyte™490 HCV protease assay kit purchased from ANASPEC. The protease (final concentration of 8.81 nM) was incubated with inhibitor (final concentration of 5 μM) for 10 minutes at room temperature and the reaction was initiated by adding the substrate (a volume corresponding to a final concentration of 5 μM). The residual enzyme activity V/Vo was calculated as the ratio of the initial rate of substrate hydrolysis in the presence of the compound and the initial rate of hydrolysis of the control (without inhibitor). The IC₅₀ was calculated by the formula IC50 = C(V/Vo)/(l-(V/Vo)), where C is the concentration. For example, the IC50 determination of compound RS-2-19 (Fig. 10) is shown in Fig. 9. IC₅₀ values for exemplary compounds are shown in the following table.

Table 3: IC₅₀ of exemplary compounds

[00140] While the invention has been described and illustrated with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various adaptations, changes, modifications, substitutions, deletions, or additions of procedures and protocols may be made without departing from the spirit and scope of the invention. It is intended, therefore, that the invention be defined by the scope of the claims that follow and that such claims be interpreted as broadly as is reasonable.

Claims

CLAIMSWE CLAIM:

1. Λ compound having the formula

wherein Z

X, or

Y

R¹, R" and R'" are each independently selected from the group consisting of hydrogen, (Ci- C₆)alkyl,

)alkoxy,

( i ₆)haloalkyl,

( i ₆)hydroxyalkyl, halogen,

n is independently 1, 2, 3, 4, or 5;

X is -Ar or -CH₂-Ar, wherein Ar is aryl or heteroaryl, either of which may be further substituted by one or more Ri₆, wherein Ri₆ is independently selected from the group consisting of )alkyl,

( )alkoxy,

)haloalkyl,

hydroxyalkyl, halogen, -

are each independently hydrogen or (Ci-C₆)alkyl; and

2. A compound according to claim 1 , wherein Z is

3. A compound according to claim 1 , wherein Z is -CH₂-X.

4. A compound according to claim 1 , wherein Z is

5. A compound according to one of claims 1-4, wherein R'_ R" and R'" are each independently selected from the group consisting o

, ( s)₃, ,

6. A compound according to claim 2, having the formula

wherein R₁-R₉ are each independently selected from the group consisting of hydrogen, (C_f )alkyl, ( i ₆)alkoxy,

haloalkyl,

( ι ₆)hydroxyalkyl, halogen, -NR₁₇RiS,

7. A compound according to claim 6, having the formula

8. A compound according to any one of claims 6-7, wherein R₂ and R₃ are identical, but are not H, and R₄=H.

9. A compound according to any one of claims 6-7, wherein R₂ is not H, and

10. A compound according to any one of claim 6-7, wherein R₄ is not H, and

1 1. A compound according to any one of claims 6-10, wherein R&-R.9 are all hydrogen, and at least one of R| and R$ is not H.

12. A compound according to claim 7, having a formula selected from the group consisting of:

13. A compound according to claim 12, selected from the group consisting of

14. A compound according to claim 1 having the formula:

wherein Z is -CH₂-X or -CH=CR)₅-X; and

Rιo-Ri₄ are each independently selected from the gτoup consisting of hydrogen, (Ci-C₆)alkyl, (C|-C₆)alkoxy, (C|-C₆)haloalkyl, (Cι-C₆)hydroxyalkyl, halogen,

and

Ri ₅ is -CN or -CO₂H; and X is -Ar or -CH₂-Ar, wherein Ar is aryl or heteroaryl, either of which may be further substituted by one or more , wherein is independently selected from the group

consisting of alkyl, (

)alkoxy,

( ₆)haloalkyl,

( i ₆)hydroxyalkyl, halogen, -

15. A compound according to claim 14, wherein Z is -CH₂-X.

16. A compound according to claim 14, wherein Z is

17. A compound according to any one of claims 14-16, wherein at least one o

is not hydrogen.

18. A compound according to any one of claims 14-16, wherein at least one of

and Rj₂ is not hydrogen.

19. A compound according to any one of claims 14-18, wherein Ar is selected from the group consisting of phenyl, pyridyl, thiophenyl, thiazolyl, indolyl, pyrrolyl, furanyl, and bezothiazolyl.

20. A compound according to any one of claims 14-18, wherein X is selected from the group consisting of:

21. A compound according to claim 15 selected from the group consisting of:

22. A compound according to any one of claims 1 -21 having an IC₅₀ against HCV protease of equal to or less than 50μM.

23. A pharmaceutically acceptable salt of a compound according to any one of claims 1-22.

24. A pharmaceutical composition comprising a compound according to any one of claims 1- 23 and a pharmaceutically acceptable carrier or pharmaceutical excipient.

25. A method of altering the physiology of a subject infected with HCV comprising administering a composition comprising a compound according to any one of claims 1-23 to the subject.

26. A method of treating a subject infected with HCV comprising administering a composition comprising a compound according to any one of claims 1-23 to the subject.

27. A method of preventing HCV infection comprising administering a composition comprising a compound according to any one of claims 1-23 to a subject.