WO2004076415A1 - 1- (hetero)aroyl-pyrrolidine-2-carboxylic acid derivatives useful as anti.viral agents - Google Patents

Abstract

Anti-viral agents of Formula (I) wherein: A represents hydroxy; D represents aryl or heteroaryl; E represents hydrogen, C1-6alkyl, aryl, heteroaryl or heterocyclyl; G represents SOnRa; n is 1 or 2; Ra represents optionally substituted C1-6alkyl or aryl; J represents C1-6alkyl, heterocyclylalkyl, arylalkyl or heteroarylalkyl; and salts, solvates and esters thereof; provided that when A is esterified to form -OR where R is selected from straight or branched chain alkyl, aralkyl, aryloxyalkyl, or aryl, then R is other than tert-butyl; processes for their preparation and methods of using them in HCV treatment are provided.

Description

l- (HETER0) AR0YL-PYRR0LIDINE-2-CARB0XYLIC ACID DERIVATIVES USEFUL AS ANTI-VIRAL AGENTS

FIELD OF THE INVENTION

The present invention relates to novel acyl pyrrolidine derivatives useful as anti-viral agents. Specifically, the present invention involves novel HCV inhibitors.

BACKG OUND OF THE INVENTION Infection with HCV is a major cause of human liver disease throughout the world. In the US, an estimated 4.5 million Americans are chronically infected with HCV. Although only 30% of acute infections are symptomatic, greater than 85% of infected individuals develop chronic, persistent infection. Treatment costs for HCV infection have been estimated at $5.46 billion for the US in 1997. Worldwide over 200 million people are estimated to be infected chronically. HCV infection is responsible for 40-60% of all chronic liver disease and 30% of all liver transplants. Chronic HCV infection accounts for 30% of all cirrhosis, end-stage liver disease, and liver cancer in the U.S. The CDC estimates that the number of deaths due to HCV will minimally increase to 38,000/year by the year 2010.

Due to the high degree of variability in the viral surface antigens, existence of multiple viral genotypes, and demonstrated specificity of immunity, the development of a successful vaccine in the near future is unlikely. Alpha-interferon (alone or in combination with ribavirin) has been widely used since its approval for treatment of chronic HCV infection. However, adverse side effects are commonly associated with this treatment: flu-like symptoms, leukopenia, thrombocytopenia, depression from interferon, as well as anemia induced by ribavirin (Lindsay, K.L. (1997) Hepatology 26 (suppl 1 ): 71S-77S). This therapy remains less effective against infections caused by HCV genotype 1 (which constitutes -75% of all HCV infections in the developed markets) compared to infections caused by the other 5 major HCV genotypes. Unfortunately, only -50-80% of the patients respond to this treatment (measured by a reduction in serum HCV RNA levels and normalization of liver enzymes) and, of those treated, 50-70% relapse within 6 months of cessation of treatment. Recently, with the introduction of pegylated interferon, both initial and sustained response rates have improved substantially, and combination treatment of Peg-IFN with ribavirin constitutes the gold standard for therapy. However, the side effects associated with combination therapy and the impaired response in patients with genotype 1 present opportunities for improvement in the management of this disease.

First identified by molecular cloning in 1989 (Choo, Q-L et al (1989) Science 244:359- 362), hepatitis C virus (HCV) is now widely accepted as the most common causative agent of post-transfusion non A, non-B hepatitis (NANBH) (Kuo, G et al (1989) Science 244:362-364). Due to its genome structure and sequence homology, this virus was assigned as a new genus in the Flaviviridae family. Like the other members of the Flaviviridae, such as flaviviruses (e.g. yellow fever virus and Dengue virus types 1-4) and pestiviruses (e.g. bovine viral diarrhea virus, border disease virus, and classic swine fever virus) (Choo, Q-L et al (1989) Science 244:359-3; Miller, R.H. and R.H. Purcell (1990) Proc. Natl. Acad. Sci. USA 87:2057-2061), HCV is an enveloped virus containing a single strand RNA molecule of positive polarity. The HCV genome is approximately 9.6 kilobases (kb) with a long, highly conserved, noncapped 5' nontranslated region (NTR) of approximately 340 bases which functions as an internal ribosome entry site (IRES) (Wang CY et al 'An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5' noncoding region' RNA- A Publication of the RNA Society. 1 (5): 526-537, 1995 Jul.). This element is followed by a region which encodes a single long open reading frame (ORF) encoding a polypeptide of -3000 amino acids comprising both the structural and nonstructural viral proteins.

Upon entry into the cytoplasm of the cell, this RNA is directly translated into a polypeptide of -3000 amino acids comprising both the structural and nonstructural viral proteins. This large polypeptide is subsequently processed into the individual structural and nonstructural proteins by a combination of host and virally-encoded proteinases (Rice, CM. (1996) in B.N. Fields, D.M.Knipe and P.M. Howley (eds) Virology 2^nd Edition, p931- 960; Raven Press, N.Y.). Following the termination codon at the end of the long ORF, there is a 3' NTR which roughly consists of three regions: an - 40 base region which is poorly conserved among various genotypes, a variable length poly(U)/polypyrimidine tract, and a highly conserved 98 base element also called the "3' X-tail" (Kolykhalov, A. et al (1996) J. Virology 70:3363-3371; Tanaka, T. et al (1995) Biochem Biophys. Res. Commun. 215:744-749; Tanaka, T. et al (1996) J. Virology 70:3307-3312; Yamada, N. et al (1996) Virology 223:255-261). The 3' NTR is predicted to form a stable secondary structure which is essential for HCV growth in chimps and is believed to function in the initiation and regulation of viral RNA replication.

The NS5B protein (591 amino acids, 65 kDa) of HCV (Behrens, S.E. et al (1996) EMBO J. 15:12-22), encodes an RNA-dependent RNA polymerase (RdRp) activity and contains canonical motifs present in other RNA viral polymerases. The NS5B protein is fairly well conserved both intra-typically (-95-98% amino acid (aa) identity across 1 b isolates) and inter-typically (-85% aa identity between genotype 1a and 1b isolates). The essentiality of the HCV NS5B RdRp activity for the generation of infectious progeny virions has been formally proven in chimpanzees (A. A. Kolykhalov et al.. (2000) Journal of Virology, 74(4), p.2046-2051). Thus, inhibition of NS5B RdRp activity (inhibition of RNA replication) is predicted to cure HCV infection.

Based on the foregoing, there exists a significant need to identify synthetic or biological compounds for their ability to inhibit HCV. SUMMARY OF THE INVENTION

The present invention involves novel acyl pyrrolidine compounds represented hereinbelow, pharmaceutical compositions comprising such compounds and use of the compounds in treating viral infection, especially HCV infection.

DETAILED DESCRIPTION OF THE IMVEMTIQN The present invention provides compounds of Formula (1) :

wherein:

A represents hydroxy;

D represents aryl or heteroaryl;

E represents hydrogen, C_1-6alkyl, aryl, heteroaryl or heterocyclyl;

G represents SO_nR^a wherein n is 1 or 2; and R^a is C_1-6alkyl optionally substituted by one or more substituents selected from halo, OR¹, SR¹, C(O)NR²R³, C(O)R⁴, CO₂R⁴, NR²R³, NHC(0)R⁴, NHCO₂R⁴, NHC(O)NR⁵R⁶; or R^a is aryl;

R¹ represents hydrogen, C_1-6alkyl, arylalkyl, or heteroarylalkyl;

R² and R³ are independently selected from hydrogen, C_1-6alkyl, aryl and heteroaryl; or R² and R³ together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group;

R⁴ is selected from the group consisting of d_-6alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl;

R⁵ and R⁶ are independently selected from the group consisting of hydrogen, Cι_₆alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; or R⁵ and R⁶ together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group; and

J represents C_1-6a!kyl, heterocyclylalkyl, arylalkyl or heteroarylalkyl; and salts, solvates and esters thereof; provided that when A is estehfied to form -OR where R is selected from straight or branched chain alky], aralkyl, aryloxyalkyl, or aryl, then R is other than te -butyl;

There is provided as a further aspect of the present invention a compound of Formula (1) or a physiologically acceptable salt, solvate or ester thereof for use in human or veterinary medical therapy, particularly in the treatment or prophylaxis of viral infection, particularly HCV infection.

It will be appreciated that reference herein to therapy and/or treatment includes, but is not limited to prevention, retardation, prophylaxis, therapy and cure of the disease. It will further be appreciated that references herein to treatment or prophylaxis of HCV infection includes treatment or prophylaxis of HCV-associated disease such as liver fibrosis, cirrhosis and hepatocellular carcinoma.

According to another aspect of the invention, there is provided the use of a compound of Formula (I) or a physiologically acceptable salt, solvate or ester thereof in the manufacture of a medicament for the treatment and/or prophylaxis of viral infection, particularly HCV infection.

In a further or alternative aspect there is provided a method for the treatment of a human or animal subject with viral infection, particularly HCV infection, which method comprises administering to said human or animal subject an effective amount of a compound of Formula (I) or a physiologically acceptable salt, solvate or ester thereof.

It will be appreciated that the compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic, diastereoisomehc, and optically active forms. All of these racemic compounds, enantiomers and diastereoisomers are contemplated to be within the scope of the present invention.

Preferably, D represents optionally substituted phenyl; more preferably te/t-butylphenyl optionally further substituted; especially preferred is para-te/τ-butylphenyl optionally further substituted, preferably mefø-substituted, by halo, C_1-3alkyl or C_1-3alkoxy, especially bromo, chloro, methyl or methoxy; most preferably D is meta-methoxy-para-ferf-butylphenyl (3- methoxy-4-te/t-butylphenyl).

Preferably, E is selected from the group consisting of C_1-6alkyl, aryl and heteroaryl; more preferably E represents methyl or heteroaryl, especially heteroaryl; especially preferred heteroaryl groups are pyridin-2-yl, pyrazin-2-yl, 1 ,3-thiazol-2-yl, 5-methyl-1 ,3-thiazol-2-yl, or 1 ,3-thiazol-4-yl; most preferred is 1 ,3-thiazol-2-yl. Preferably, G is SO_nR^a wherein n is 2 and R^a is C_1-4alkyl. Most preferably, n is 2 and R^a is methyl or ethyl.

In another preferred aspect, G is SO_πR^a wherein n is 1 and R^a is aryl, for example phenyl.

Preferably, J is C_1-6alkyl, arylalkyl or heteroarylalkyl; more preferably J is isobutyl, benzyl or pyridylmethyl; most preferably J is isobutyl.

It is to be understood that the present invention covers all combinations of suitable, convenient and preferred groups described herein.

As used herein unless otherwise specified, "alkyl" refers to an optionally substituted hydrocarbon group. The alkyl hydrocarbon group may be linear, branched or cyclic, saturated or unsaturated. Where the alkyl hydrocarbon group is cyclic, it will be understood that there will be a minimum of 3 carbon atoms in the group. Preferably, the group is saturated. Preferred alkyl moieties are C_1-4alkyl. Unless otherwise stated, optional substituents include C_1-6alkyl, halo, OR¹, SR¹, C(O)NR²R³, C(O)R⁴, CO₂H, CO₂R⁴, NR²R³, NHC(O)R⁴, NHCO₂R⁴, NHC(O)NR⁵R⁶, SO₂NR⁵R⁶, SO₂R⁴, nitro, cyano, oxo, and heterocyclyl.

As used herein, "aryl" refers to an optionally substituted aromatic group with at least one ring having a conjugated pi-electron system, containing up to two conjugated or fused ring systems. "Aryl" includes carbocyclic aryl and biaryl groups, all of which may be optionally substituted. Preferred "aryl" moieties are unsubstituted, monosubstituted, disubstituted or trisubstituted phenyl. Preferred "aryl" substituents are selected from the group consisting of C_1-6alkyl, halo, OR¹, C(O)NR²R³, C(O)R⁴, CO₂H, CO₂R⁴, NR²R³, NHC(O)R⁴, NHCO₂R⁴, NHC(O)NR⁵R⁶, SO₂NR⁵R⁶, SO₂R⁴, nitro, cyano, oxo, heterocyclyl, CF₃, and NO₂.

As used herein, "heteroaryl" refers to an optionally substituted, 5 or 6 membered, aromatic group comprising one to four heteroatoms selected from N, O and S, with at least one ring having a conjugated pi-electron system, containing up to two conjugated or fused ring systems. Preferred "heteroaryl" moieties are unsubstituted, monosubstituted, disubstituted or trisubstituted pyridyl and thiazolyl. Preferred "heteroaryl" substituents are selected from the group consisting of C_1-6alkyl, halo, OR¹, C(O)NR²R³, C(O)R⁴, CO₂H, CO₂R⁴, NR²R³, NHC(O)R⁴, NHCO₂R⁴, NHC(O)NR⁵R⁶, SO₂NR⁵R⁶, SO₂R⁴, nitro, cyano, oxo, heterocyclyl, CF₃, and NO₂.

As used herein, "heterocyclic" and "heterocyclyl" refer to an optionally substituted, 5 or 6 membered, saturated cyclic hydrocarbon group containing 1 or 2 heteroatoms selected from N, optionally substituted by hydrogen, Cι_-6alkyl, C(O)R⁴, SO₂R⁴, aryl or heteroaryl; O; and S, optionally substituted by one or two oxygen atoms. Preferred compounds of Formula (I) useful in the present invention are selected from the group consisting of: re/-(2S,4S,5R)-2-lsobutyl-1-(3-methoxy-4-te -butylbenzoyl)-5-(1 ,3-thiazol-2-yl)-4- methanesulphonyl-pyrrolidine-2-carboxylic acid; re/-(2S,4S,5R)-2-lsobutyl-1 -(3-methoxy-4-feτ-butylbenzoyl)-5-(1 ,3-thiazol-2-yl)-4- ethanesulphonyl-pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-2-lsobutyl-1-(3-methoxy-4-fe/1-butylbenzoyl)-5-(1 ,3-thiazol-2-yl)-4- ethanesulphonyl-pyrrolidine-2-carboxylic acid; re/-(2S,4S,5R)-2-lsobutyl-1-(3-methoxy-4-fe/t-butylbenzoyl)-5-(1 ,3-thiazol-2-yl)-4-(RS)- benzenesulphinyl-pyrrolidine-2-carboxylic acid; re/-(2S,4S,5S)-2-lsobutyl-1-(3-methoxy-4-fe/τ-butylbenzoyl)-5-methyl-4- methanesulphonyl-pyrrolidine-2-carboxylic acid;

and salts, solvates and esters, and individual enantiomers thereof.

Also included in the present invention are pharmaceutically acceptable salt complexes. The present invention also covers the physiologically acceptable salts of the compounds of Formula (I). Suitable physiologically acceptable salts of the compounds of Formula (I) include acid salts, for example sodium, potassium, calcium, magnesium and tetraalkylammonium and the like, or mono- or di- basic salts with the appropriate acid for example organic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids and inorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamic acids and the like.

The present invention also relates to solvates of the compounds of Formula (I), for example hydrates.

The present invention also relates to pharmaceutically acceptable esters of the compounds of Formula (I), for example carboxylic acid esters -COOR, in which R is selected from straight or branched chain alkyl, for example n-propyl, n-butyl, alkoxyalkyl (e.g. methoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl), aryl (e.g. phenyl optionally substituted by halogen, C_1-4alkyl or C_1-4alkoxy or amino). Unless otherwise specified, any alkyl moiety present in such esters preferably contains 1 to 18 carbon atoms, particularly 1 to 4 carbon atoms. Any aryl moiety present in such esters preferably comprises a phenyl group.

It will further be appreciated that certain compounds of the present invention may exist in different tautomeric forms. All tautomers are contemplated to be within the scope of the present invention.

Compounds of Formula (I) in which A is hydroxy may be prepared from a compound of Formula (II)

in which A is an alkoxy, benzyloxy or silyloxy group. For example when A is te/1-butoxy, and D, E, G and J are as defined above for Formula (I), by treatment with an appropriate acid, for example trifluoroacetic acid. Suitably, the reaction is carried out in a solvent, for example dichloromethane. Preferably, the temperature is in the range 0 to 50°C, more preferably 15 to 30°C.

For example when A is benzyloxy, and D, E, G and J are as defined above for Formula (I), by hydrogenolysis in the presence of a suitable catalyst for example palladium-on-carbon. Suitably, the reaction is carried out in a solvent, for example ethanol. Preferably, the temperature is in the range 0 to 50°C.

For example when A is allyloxy, and D, E, G and J are as defined above for Formula (I), by treatment with a suitable catalyst for example tetrakis(triphenylphosphine)palladium(0) and a suitable proton source, for example phenylsilane. The reaction is carried out in a suitable solvent, for example dichloromethane. For example when A is silyloxy, and D, E, G and J are as defined above for Formula (I), by treatment with a suitable fluoride source for example tetrabutylammonium fluoride. The reaction is carried out in a suitable solvent, for example tetrahydrofuran.

Compounds of Formula (I) in which A is hydroxy or a protected form thereof may also be prepared, and compounds of Formula (II) may be prepared by reaction of a compound of

Formula (III)

in which E, G, and J are as defined above for Formula (I), and A is as defined above for Formula (I) or (II) respectively; with a suitable acylating agent, for example D-C(O)-hal, wherein hal is a halo atom, preferably chloro or bromo, and D is as defined above for Formula (I). Preferably the reaction is carried out in a suitable solvent, for example dichloromethane, in the presence of a suitable base, for example thethylamine and thereafter removing any protecting group. Suitable protecting groups can be found, but are not restricted to, those found in T W Greene and P G M Wuts 'Protective Groups in Organic Synthesis', 3^rd Ed (1999), J Wiley and Sons

A compound of Formula (III) may be prepared by reaction of a compound of Formula (IV)

in which E and J are as defined above for Formula (I) and A is as defined above for Formula (II) with a compound of Formula (V)

^G (V) wherein G is as defined above for Formula (I). Preferably, the reaction is carried out in a suitable solvent, for example THF or acetonitrile, optionally in the presence of a Lewis acid catalyst, such as lithium bromide or silver acetate, and a base, such as t ethylamine, 1 ,8-diazabicyclo[5,4,0]undec-7-ene (DBU) or tetramethyl guanidine. Alternatively, the reaction is carried out in a suitable solvent, for example THF or acetonitrile, in the presence of an acid, such as acetic acid, or the reaction may be carried out by heating compounds of Formula (IV) and Formula (V) in a suitable solvent, for example toluene, xylene or acetonitrile in the absence of a catalyst.

Compounds of Formula (IV) may be prepared by reaction of a compound of Formula (VI)

H₂N ,COA

(VI)

in which J is as defined above for Formula (I) and A is as defined above for Formula (II) with a compound of Formula E-CHO in the presence of a suitable base, for example thethylamine, in a suitable solvent, for example dichloromethane.

Compounds of Formula (V), (VI) and E-CHO are known in the art or may be prepared by standard literature procedures.

Compounds of Formula (I) in which A is an ester may be prepared by esterification of a compound of Formula (I) in which A is hydroxy by standard literature procedures for esterification.

With appropriate manipulation and protection of any chemical functionality, synthesis of compounds of Formula (I) is accomplished by methods analogous to those above and to those described in the Experimental section. Suitable protecting groups can be found, but are not restricted to, those found in T W Greene and P G M Wuts 'Protective Groups in Organic Synthesis', 3^rd Ed (1999), J Wiley and Sons.

EXAMPLES Inierrrnscliiaie 1

2-[N-(1,3-Thiazol-2-ylmethylene)amino]-4-methylpentanoic acid, ferf-butyl ester

A stirred mixture of 2-amino-4-methyl-pentanoic acid terf-butyl ester, hydrochloride salt (5.00g, 22.34 mmol), 1 ,3-thiazole-2-carboxaldehyde (2.53g, 22.34 mmol) and thethylamine (3.10 mL, 22.3 mmol) in dichloromethane (60 mL) was heated under reflux under nitrogen for 19 hours. The reaction mixture was allowed to cool to room temperature, washed twice with water, dried over Na₂SO₄ and evaporated to give the title compound as an oil.

¹H NMR (CDC ): δ 8.46 (s, 1H), 7.94 (d, 1 H), 7.44 (dd, 1 H), 4.07 (dd, 1 H), 1.89-1.74 (m, 2H), 1.64-1.52 (m, 1 H), 1.48 (s, 9H), 0.96 (d, 3H) and 0.90 (d, 3H).

Intermediate 2 re/^S^S^RJ^-lsobutyl-S-CI.S-thiazol^-y ^-methanesulphonyl-pyrrolidine^- carboxylic acid, 2-fert-butyl ester.

Racemic; Relative stereochemistry shown

To a stirred solution of Intermediate 1 (0.200 g, 1.41 mmol) in anhydrous THF (5 mL) under nitrogen, was added lithium bromide (0.182 g, 2.1 mmol) followed by thethylamine (0.295 mL, 2.1 mmol) and methyl vinyl sulphone (0.126 mL, 1.41 mmol). The mixture was stirred at ambient temperature for 20 hours. Aqueous ammonium chloride was added with rapid stirring and the resulting mixture was extracted twice with ethyl acetate. The combined organic layers were dried over sodium sulphate and evaporated. The residue was purified by chromatography on silica gel using cyclohexane-ethyl acetate (6:4, v/v) as eluent to provide the crude product. This was recrystallised from cyclohexane to afford the title compound as a solid.

Mass spec m/z calcd for (Cι₇H₂₈N₂O₄S₂ + H)⁺: 389. Mass spec (electrospray) Found: (M+H)⁺ 389.

Intermediate 3 re/-(2S,4S,5R)-2-lsobutyl-1-(3-methoxy-4-fert-butylbenzoyl)-5-(1,3-thiazol-2-yl)-4- methanesulphonyl-pyrrolidine-2-carboxylic acid, 2-terf-butyl ester. Racemic;

Relative stereochemistry shown

To a solution of Intermediate 2 (0.107 g, 0.275 mmol) in anhydrous dichloromethane (3 mL) under nitrogen, was added thethylamine (0.057 mL, 0.41 mmol) followed by 3- methoxy-4-te butylbenzoyl chloride* (0.093 g, 0.414 mmol) and the mixture was stirred overnight at room temperature. The reaction was quenched by the addition of water and extracted with dichloromethane. The organic fraction was dried over sodium sulphate, evaporated and purified by chromatography over silica gel and eluting with cyclohexane- ethyl acetate (9:1 then 1 :1 , v/v), providing the title compound as a solid. * Prepared from 3-methoxy-4-te -butylbenzoic acid (J. Org. Chem. (1961), 26, 1732). Mass spec m/z calcd for (C₂₉H₄₂N₂0₆S₂ + H)⁺: 579. Mass spec (electrospray) Found: (M+H)⁺ 579.

Intermediate 4 re/-(2S,4R,5R)-2-lsobutyl-5-(1,3-thiazol-2-yl)-4-ethanesulphonyl-pyrrolidine-2- carboxylic acid, 2-terf-butyl ester

Racemic;

Relative stereochemistry shown

To a stirred solution of Intermediate 1 (1.500 g, 5.32 mmol) in anhydrous THF (50 mL) under nitrogen, was added lithium bromide (0.550 g, 6.4 mmol) followed by thethylamine (0.890 mL, 6.4 mmol) and ethyl vinyl sulphone (0.555 mL, 5.32 mmol). The mixture was stirred at ambient temperature for 20 hours. Aqueous ammonium chloride was added with rapid stirring and the resulting mixture was extracted twice with ethyl acetate. The combined organic layers were dried over sodium sulphate and evaporated. The residue was purified by chromatography on silica gel using cyclohexane-ethyl acetate (8:2, v/v) as eluent. The first eluted pyrrolidine was collected to provide the title compound. Mass spec m/z calcd for (Cι₈H₃₀N₂O₄S₂ + H)⁺: 403. Mass spec (electrospray) Found: (M+H)⁺ 403.

Intermediate 5 re/-(2S,4S,5R)-2-lsobutyl-5-(1,3-thiazol-2-yl)-4-ethanesulphonyl-pyrrolidine-2- carboxylic acid, 2-tert-butyl ester Racemic,

Relative stereochemistry shown

Continued elution of the chromatography column from Intermediate 4 (above) afforded a second eluting pyrrolidine, the title compound.

Mass spec m/z calcd for (C₁₈H₃₀N₂O₄S₂ + H)⁺: 403. Mass spec (electrospray) Found: (M+H)⁺ 403.

Intermediate 6 re/-(2S,4S,5R)-2-lsobutyl-1-(3-methoxy-4-tert-butylbenzoyl)-5-(1,3-thiazol-2-yl)-4- ethanesulphonyl-pyrrolidine-2-carboxylic acid, 2-te -butyl ester

Racemic, Relative stereochemistry shown

To a solution of Intermediate 5 (0.647 g, 1.61 mmol) in anhydrous dichloromethane (20 mL) under nitrogen, was added thethylamine (0.336 mL, 2.41 mmol) followed by 3- methoxy-4-fe/t butylbenzoyl chloride* (0.550 g, 2.41 mmol) and the mixture was stirred overnight at room temperature. The reaction was quenched by the addition of water and extracted with dichloromethane. The organic fraction was dried over sodium sulphate, evaporated and purified by chromatography over silica gel and eluting with cyclohexane- ethyl acetate (6:4, v/v), providing the title compound as a solid. * Prepared from 3-methoxy-4-te/τ-butylbenzoic acid (J. Org. Chem. (1961 ), 26, 1732). Mass spec m/z calcd for (C₃₀H₄₄N₂O₆S₂ + H)⁺: 593. Mass spec (electrospray) Found: (M+H)⁺ 593.

Intermediate 7 re/-(2S,4R,5R)-2-lsobutyl-1-(3-methoxy-4-terf-butylbenzoyl)-5-(1,3-thiazol-2-yl)-4- ethanesulphonyl-pyrrolidine-2-carboxylic acid, 2-terf-butyl ester

Racemic,

Relative stereochemistry shown

To a solution of Intermediate 4 (0.384 g, 0.96 mmol) in anhydrous dichloromethane (10 mL) under nitrogen, was added thethylamine (0.199 mL, 1.43 mmol) followed by 3- methoxy-4-terf butylbenzoyl chloride* (0.327 g, 1.43 mmol) and the mixture was stirred overnight at room temperature. The reaction was quenched by the addition of water and extracted with dichloromethane. The organic fraction was dried over sodium sulphate, evaporated and purified by chromatography over silica gel and eluting with cyclohexane- ethyl acetate (6:4, v/v), providing the title compound as a solid. " Prepared from 3-methoxy-4-teτ-butylbenzoic acid (J. Org. Chem. (1961), 26, 1732). Mass spec m/z calcd for (C₃oH₄₄N₂OeS₂ + H)⁺: 593. Mass spec (electrospray) Found: (M+H) 593.

Intermediate 8 re/-(2S,4S,5R)-2-lsofoutyl-5-(1 ,3-thia∑ol-2-yl)-4-(RS)-benzenesulphinyl-pyrrolidine-2- carhoxylic acid, 2-

Racemic, Relative stereochemistry shown

To a stirred solution of Intermediate 1 (141 mg, 0.5 mmole) in THF (3 mL) was added a solution of phenyl vinyl sulphoxide (76 mg, 0.5 mmole) in THF (1 mL) followed by lithium bromide (87 mg, 1 mmole) and finally thethylamine (105 μL, 0,75 mmole). The solution was stirred under nitrogen for 3 days. Saturated ammonium chloride solution (10 mL) was added and the mixture was extracted with ethyl acetate (2 x 25 mL). Extracts were washed with water and brine and dried (MgSO₄). The residue was purified by preparative plate chromatography eluting with 1 :1 (v/v) cyclohexane:ethyl acetate to give the title compound as a gum.

Mass spec m/z calcd for (C₂₂H₃oN₂O₃S₂ + H)⁺: 435. Mass spec (electrospray) Found: (M+H)⁺ 435.

Intermediate 9 re/-(2S,4S,5R)-2-lsobutyl-1 -(3-methoxy-4-terf-butylbenzoyl)-5-(1 ,3-thiazol-2-yl)-4- (RS)-benzenesulphinyl-pyrrolidine-2-carboxylic acid, 2-terf-butyl ester

Racemic;

Relative stereochemistry shown

To a solution of Intermediate 8 (70 mg, 0.16 mmole) in dichloromethane (1 mL) was added a solution of 3-methoxy-4-fe/ butylbenzoyl chloride (55 mg, 0.24 mmole) in dichloromethane ( 1 mL) followed by triethylamine (60 μL). The mixture was stirred at room temperature overnight. Further quantities of 3-methoxy-4 -tert butylbenzoyl chloride (55 mg) and triethylamine (60 μL) were added and stirring was continued for a further 24 hr. The mixture was loaded onto a SPE cartridge ( 20g silica) and eluted with 4:1 then 3:1 then 1 :1 cyclohexane:ethyl acetate to give the title compound as a colourless gum. Mass spec m/z calcd for (C₃₄H₄₄N₂O₅S₂ + H)⁺: 625. Mass spec (electrospray) Found: (M+H)⁺ 625.

Intermediate 10

2-(W-Ethylideneamino)-4-methylpentanoic acid, 2-iert-buty\ ester

The title compound was prepared from 2-amino-4-methylpentanoic acid fe/ -butyl ester hydrochlohde (4.46 g, 20 mmol) and acetaldehyde (2.24 mL, 40 mmol) by a procedure analogous to that described in Tetrahedron Asymmetry, 1991 , 2, 1231.

Intermediate 11 re/-(2S,4S,5S)-2-lsobutyl-5-methyl-4-methanesulphonyl-pyrrolidine-2-carboxylic acid, 2-terf-butyl ester

Racemic; Relative stereochemistry shown

A mixture of Intermediate 10 (2.08 g, 9.75 mmol), methyl vinyl sulphone (1.67 mL, 12.19 mmol) and lithium bromide (1.27 g, 14.63 mmol) was dissolved in dry THF (10 mL) and cooled in an ice bath. DBU (1.46 mL, 9.75 mmol) was added and the mixture stirred at room temperature for 16 hours. The solution was diluted with saturated ammonium chloride and extracted with ethyl acetate. The combined organic solutions were evaporated and the resulting gum chromatographed on silica gel using ethyl acetate- cyclohexane (gradient elution from 30:70 v/v to 50:50 v/v) to afford the title compound, a solid after crystallisation from light petroleum ether.

Intermediate 12 re/-(2S,4S,5S)-2-lsobutyl-1-(3-methoxy-4-tert-butylbenzoyl)-5-methyl-4- methanesulphonyl-pyrrolidine-2-carboxylic acid, 2-tefτ-butyl ester

Racemic;

Relative stereochemistry shown

The title compound, a solid, was prepared from Intermediate 11 and 3-methoxy-4-tert butylbenzoyl chloride in a similar manner to that described for the preparation of Intermediate 3. Mass spec m/z calcd for (C₂₇H₄₃NO₆S + H)⁺: 510. Mass spec (electrospray) Found: (M+H)⁺ 510.

Example 1 re/-(2S,4S,5R)-2-lsobutyl-1-(3-methθ2.y-4-føι -butylben_2oyl)-5-(1,3-thiasol-2-yl)-4- methanesulphonyl-pyrrolidine-2-carboxylic acid

Racemic; R Reellaattiivvee s stereochemistry shown

A solution of Intermediate 3 (0.028 g, 0.048 mmol) in trifluoroacetic acid (3 mL) was stirred at room temperature for 3 hours. The mixture was evaporated and the residue was triturated with ether to give the title compound as a solid.

Mass spec calcd for (C₂₅H₃₄N₂O₆S₂ + H)⁺ : 523

Mass spec found (electrospray) : (M+H)⁺ = 523

The relative stereochemistry was determined by NMR nOe studies to be (2S, 4S, 5R). ¹H NMR (CD₃OD): δ 7.76 (d, 1 H), 7.59 (d, 1 H), 7.22 (d, 1 H), 6.76 (dd, 1H), 6.34 (s, 1 H),

6.01 (d, 1 H), 4.67 (m, 1 H), 3.64 (s, 3H), 3.38 (s, 3H), 2.86 (d, 1 H), 2.74 (dd, 1 H), 2.29 (dd,

1 H), 2.18 (dd, 1 H), 2.03 (m, 1 H), 1.31 (s, 9H),1.12 (d, 3H), 1.11 (d, 3H).

Example 2 re/-(2S,4S,5R)-2-lsobutyl-1 -(3-methoxy-4-ferf-butylbenzoyl)-5-(1 ,3-thiazol-2-yl)-4- ethanesulphonyl-pyrrolidine-2-carboxylic acid

Racemic;

Relative stereochemistry shown

A solution of Intermediate 6 (0.052 g, 0.088 mmol) in trifluoroacetic acid (3 mL) was stirred at room temperature for 3 hours. The mixture was evaporated and the residue was triturated with ether to give the title compound as a solid.

Mass spec calcd for (C₂₆H₃₆N₂O₆S₂ + H)⁺ : 537

Mass spec found (electrospray) : (M+H)⁺ = 537

The relative stereochemistry was determined by NMR nOe studies to be (2S, 4S, 5R).

¹H NMR (CD₃OD): δ 7.78 (d, 1H), 7.58 (d, 1 H), 7.22 (d, 1 H), 6.76 (dd, 1H), 6.34 (s, 1H), 6.01 (d, 1 H), 4.65 (m, 1 H), 3.64 (s, 3H), 3.16 (m, 1 H), 3.03 (m, 1 H), 2.86 (t, 1 H), 2.73 (dd,

1 H), 2.29 (dd, 1 H), 2.17 (dd, 1 H), 2.02 (m, 1 H), 1.31 (s, 9H), 1.26 (t, 3H), 1.11 (d, 6H). Example 3 re/-(2S,4R,5R)-2-lsobutyl-1-(3-methoxy-4-te -butylbenzoyl)-5-(1,3-thiazol-2-yl)-4- ethanesulphonyl-pyrrolidine-2-carboxylic acid

Racemic;

Relative stereochemistry shown

A solution of Intermediate 7 (0.028 g, 0.048 mmol) in trifluoroacetic acid (3 mL) was stirred at room temperature for 3 hours. The mixture was evaporated and the residue was triturated with ether to give the title compound as a solid. Mass spec calcd for (C₂₆H₃₆N₂O₆S₂ + H)⁺ : 537 Mass spec found (electrospray) : (M+H)⁺ = 537

The relative stereochemistry was determined by NMR nOe studies to be (2S, 4R, 5R). ¹H NMR (CD₃OD): δ 7.48 (d, 1H), 7.28 (m, 1H), 7.19 (1 H), 6.89 (dd, 1H), 6.55 (m, 1H), 5.85 (d, 1 H), 4.33 (m, 1H), 3.71 (s, 3H), 2.87 (m, 4H), 2.67 (dd, 1 H), 2.03 (m, 1H), 1.87 (m, 1 H), 1.32 (s, 9H), 1.17 (t, 3H), 1.03 (d, 3H), 1.01 (d, 3H).

Example 4 re/-(2S,4S,5R)-2-lsobutyl-1-(3-methoxy-4-tert-butylbenzoyl)-5-(1,3-thiazol-2-yl)-4- (RS)-benzenesulphinyl-pyrrolidine-2-carboxylic acid

Racemic; R Reellaattiivvee ; stereochemistry shown

The title compound was prepared from Intermediate 9 by a procedure analogous to that described for Example 1.

Mass spec m/z calcd for (C₃₀H₃6N₂O₅S₂ + H)⁺: 569.

Mass spec (electrospray) Found: (M+H)⁺ 569.

Example 5 fe/-(2S,4S,5S)-2-lsobutyl-1-(3-methoxy-4-ført-butylbenzoyl)-5-methyl-4- methanesulphonyl-pyrrolidine-2-carboxylic acid Racemic;

Relative stereochemistry shown

A solution of Intermediate 12 (0.4 g, 0.78 mmol) in trifluoroacetic acid (4 mL) was stirred at room temperature for 4 hours. The mixture was evaporated and the residue was triturated with ether and light petroleum ether to give the title compound as a solid. Mass spec calcd for (C₂₃H₃₅N0₆S + H)⁺ : 454 Mass spec found (electrospray) : (M+H)⁺ = 454

¹H NMR (CDCI₃): δ 7.33 (d, 1 H), 6.93 (d, 1 H), 6.87 (dd, 1 H), 4.76 (m, 1 H), 3.89 (s, 3H), 3.77 (m, 1 H), 3.50 (t, 1 H), 2.97 (s, 3H), 2.53 (dd, 1 H), 2.44 (dd, 1 H), 1.88 (dd, 1 H), 1.81 (m, 1 H), 1.39 (s, 9H), 1.27 (d, 3H), 1.05 (d, 6H).

The compounds according to the invention may be formulated for administration in any convenient way, and the invention therefore also includes within its scope pharmaceutical compositions for use in therapy, comprising a compound of formula (I) or a physiologically acceptable salt or solvate thereof in admixture with one or more physiologically acceptable diluents or carriers.

The compounds of the present invention can be administered by different routes including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical, transdermal, or transmucosal administration. For systemic administration, oral administration is preferred. For oral administration, for example, the compounds can be formulated into conventional oral dosage forms such as capsules, tablets and liquid preparations such as syrups, elixirs and concentrated drops.

Alternatively, injection (parenteral administration) may be used, e.g., intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds of the invention are formulated in liquid solutions, preferably, in physiologically compatible buffers or solutions, such as saline solution, Hank's solution, or Ringer's solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms can also be produced.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration, for example, may be through nasal sprays, rectal suppositories, or vaginal suppositories.

For topical administration, the compounds of the invention can be formulated into ointments, salves, gels, or creams, as is generally known in the art.

The amounts of various compounds to be administered can be determined by standard procedures taking into account factors such as the compound (IC₅₀) potency, (EC₅₀) efficacy, and the biological half-life (of the compound), the age, size and weight of the patient, and the disease or disorder associated with the patient. The importance of these and other factors to be considered are known to those of ordinary skill in the art.

Amounts administered also depend on the routes of administration and the degree of oral bioavailability. For example, for compounds with low oral bioavailability, relatively higher doses will have to be administered. Oral administration is a preferred method of administration of the present compounds.

Preferably the composition is in unit dosage form. For oral application, for example, a tablet, or capsule may be administered, for nasal application, a metered aerosol dose may be administered, for transdermal application, a topical formulation or patch may be administered and for transmucosal delivery, a buccal patch may be administered. In each case, dosing is such that the patient may administer a single dose.

Each dosage unit for oral administration contains suitably from 0.01 to 500 mg/Kg, and preferably from 0.1 to 50 mg/Kg, of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, calculated as the free base. The daily dosage for parenteral, nasal, oral inhalation, transmucosal or transdermal routes contains suitably from 0.01 mg to 100 mg/Kg, of a compound of Formula(l). A topical formulation contains suitably 0.01 to 5.0% of a compound of Formula (I). The active ingredient may be administered from 1 to 6 times per day, preferably once, sufficient to exhibit the desired activity, as is readily apparent to one skilled in the art.

Composition of Formula (I) and their pharmaceutically acceptable salts which are active when given orally can be formulated as syrups, tablets, capsules and lozenges. A syrup formulation will generally consist of a suspension or solution of the compound or salt in a liquid carrier for example, ethanol, peanut oil. olive oil, glycerine or water with a flavoring or coloring agent. Where the composition is in the form of a tablet, any pharmaceutical carrier routinely used for preparing solid formulations may be used. Examples of such carriers include magnesium stearate, terra alba, talc, gelatin, acacia, stearic acid, starch, lactose and sucrose. Where the composition is in the form of a capsule, any routine encapsulation is suitable, for example using the aforementioned carriers in a hard gelatin capsule shell. Where the composition is in the form of a soft gelatin shell capsule any pharmaceutical carrier routinely used for preparing dispersions or suspensions may be considered, for example aqueous gums, celluloses, silicates or oils, and are incorporated in a soft gelatin capsule shell.

Typical parenteral compositions consist of a solution or suspension of a compound or salt in a sterile aqueous or non-aqueous carrier optionally containing a parenterally acceptable oil, for example polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil or sesame oil.

Typical compositions for inhalation are in the form of a solution, suspension or emulsion that may be administered as a dry powder or in the form of an aerosol using a conventional propellant such as dichlorodifluoromethane or trichlorofluoromethane.

A typical suppository formulation comprises a compound of Formula (I) or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent, for example polymeric glycols, gelatins, cocoa- butter or other low melting vegetable waxes or fats or their synthetic analogs.

Typical dermal and transdermal formulations comprise a conventional aqueous or non- aqueous vehicle, for example a cream, ointment, lotion or paste or are in the form of a medicated plaster, patch or membrane.

No unacceptable toxicological effects are expected when compounds of the present invention are administered in accordance with the present invention.

ASSAY The potential for compounds of the invention to inhibit NS5B wildtype HCV polymerase activity may be demonstrated, for example, using the following in vitro assay:

In Vitro Detection of inhibitors of HCV RNA-dependent RNA Polymerase Activity

Incorporation of [³³P]-GMP into RNA was followed by absorption of the biotin labelled RNA polymer by streptavidin containing SPA beads. A synthetic template consisting of biotinylated 13mer-oligoG hybridised to polyrC was used as a homopolymer substrate.

Reaction Conditions were 0.5 μM [³³P]-GTP (0.2 Ci/mMol), 1 mM Dithiothreitol, 20 mM MgCI₂, 5mM MnCI₂, 20 mM Tris-HCI, pH7.5, 1.6 μg/mL polyC/0.256 μM biotinylated oligoG13, 10% glycerol, 0.01% NP-40, 0.2 u/μL RNasin and 50 mM NaCI. HCV RNA Polymerase (Recombinant full-length NS5B (Lohmann et al, J. Virol. 71 (11), 1997, 8416 'Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity') expressed in baculovirus and purified to homogeneity) was added to 10 nM final concentration.

5x concentrated assay buffer mix was prepared using 1 M MnCI₂ (0.25 mL), glycerol ( mL), 10% NP-40 (0.025 mL) and Water (7.225 mL), Total 10 mL.

2x concentrated enzyme buffer contained 1 M-Tris-HCI, pH7.5 (0.4 mL), 5M NaCI (0.2 mL), 1 M-MgCI₂ (0.4 mL), glycerol (1 mL), 10% NP-40 (10 μL), 1 M DTT (20 μL) and water (7.97 mL), Total 10 mL

Substrate Mix was prepared using 5x Concentrated assay Buffer mix (4μL), [³³P]-GTP (10 μCi/μL, 0.02μL), 25 μM GTP (0.4 μL), 0.4 u/μL RNasin (0.04 μL), 20 μg/mL polyrC/biotinylated-oligorG (1.6 μL), and Water (3.94 μL), Total 10 μL.

Enzyme Mix was prepared by adding 1mg/ml full-length NS5B polymerase (1.5 μL) to 2.811mL 2x-concentrated enzyme buffer.

The Assay was set up using compound (1 μL), Substrate Mix (10 μL), and Enzyme Mix (added last to start reaction) (10 μL), Total 21 μL.

The reaction was performed in a U-bottomed, white, 96-well plate. The reaction was mixed on a plate-shaker, after addition of the Enzyme, and incubated for 1 h at 22°C. After this time, the reaction was stopped by addition of 40 μL 1.875 mg/ml streptavidin SPA beads in 0.1 M EDTA. The beads were incubated with the reaction mixture for 1 h at 22°C after which 120 μL 0.1 M EDTA in PBS was added. The plate was sealed, mixed centrifuged and incorporated radioactivity determined by counting in a Trilux (Wallac) or Topcount (Packard) Scintillation Counter.

After subtraction of background levels without enzyme, any reduction in the amount of radioactivity incorporated in the presence of a compound, compared to that in the absence, was taken as a measure of the level of inhibition. Ten concentrations of compounds were tested in three- or fivefold dilutions. From the counts, percentage of inhibition at highest concentration tested or IC₅₀s for the compounds were calculated using Grafit3 or Grafit4 software packages.

The exemplified compounds had an IC₅₀ of <50μM in the above described assay. Accordingly, the compounds of the invention are of potential therapeutic benefit in the treatment and prophylaxis of HCV. Preferred compounds had an IC₅₀ of <10μM, more preferably <5μM. The pharmaceutical compositions according to the invention may also be used in combination with other therapeutic agents, for example immune therapies (eg. interferon), therapeutic vaccines, antifibrotic agents, anti-inflammatory agents such as corticosteroids or NSAIDs, bronchodilators such as beta-2 adrenergic agonists and xanthines (e.g. theophylline), mucolytic agents, anti-musca nics, anti-leukot enes, inhibitors of cell adhesion (e.g. ICAM antagonists), anti-oxidants (eg N-acetylcysteine), cytokine agonists, cylokine antagonists, lung surfactants and/or antimicrobial and anti-viral agents (eg ribavirin and amantidine). The compositions according to the invention may also be used in combination with gene replacement therapy.

The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a physiologically acceptable salt or solvate thereof together with another therapeutically active agent.

The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a pharmaceutically acceptable carrier thereof represent a further aspect of the invention.

The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations. Appropriate doses of known therapeutic agents will be readily appreciated by those skilled in the art.

All publications, including but not limited to patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference as though fully set forth.

Claims

Claims

Compounds of Formula

wherein:

A represents hydroxy;

D represents aryl or heteroaryl;

E represents hydrogen, C_1-6alkyl, aryl, heteroaryl or heterocyclyl;

G represents SO_nR^a wherein n is 1 or 2; and R^a is C_1-6alkyl optionally substituted by one or more substituents selected from halo, OR¹, SR¹, C(O)NR²R³, C(O)R⁴, CO₂R⁴, NR²R³, NHC(O)R⁴, NHCO₂R⁴, NHC(O)NR⁵R⁶; or R^a is aryl;

R¹ represents hydrogen, C_1-6alkyl, arylalkyl, or heteroarylalkyl;

R² and R³ are independently selected from hydrogen, C_1-6alkyl, aryl and heteroaryl; or R² and R³ together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group;

R⁴ is selected from the group consisting of C_1-6alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl;

R⁵ and R⁶ are independently selected from the group consisting of hydrogen, C_1-6alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; or R⁵ and R⁶ together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group; and

J represents C_1-6al yl, heterocyclylalkyl, arylalkyl or heteroarylalkyl;

and salts, solvates and esters thereof; provided that when A is esterified to form -OR where R is selected from straight or branched chain alkyl, aralkyl, aryloxyalkyl, or aryl, then R is other than fe/τ-butyl.

2. A compound as claimed in claim 1 selected from the group consisting of: re/-(2S,4S,5R)-2-lsobutyl-1-(3-methoxy-4-tert-butylbenzoyl)-5-(1 ,3-thiazol-2-yl)-4- methanesulphonyl-pyrrolidine-2-carboxylic acid; re/-(2S,4S,5R)-2-lsobutyl-1-(3-methoxy-4-te -butylbenzoyl)-5-(1 ,3-thiazol-2-yl)-4- ethanesulphonyl-pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-2-lsobutyl-1-(3-methoxy-4-te/ -butylbenzoyl)-5-(1 ,3-thiazol-2-yl)-4- ethanesulphonyl-pyrrolidine-2-carboxylic acid; re/-(2S,4S,5R)-2-lsobutyl-1-(3-methoxy-4-ferf-butylbenzoyl)-5-(1 ,3-thiazol-2-yl)-4-(RS)- benzenesulphinyl-pyrrolidine-2-carboxylic acid; re/-(2S,4S,5S)-2-lsobutyl-1-(3-methoxy-4-feι-butylbenzoyl)-5-methyl-4- methanesulphonyl-pyrrolidine-2-carboxylic acid;

and salts, solvates and esters, and individual enantiomers thereof.

3. A compound as claimed in claim 1 wherein D represents para-terf-butylphenyl optionally further mete-substituted, by halo, C_1-3alkyl or C_1-3alkoxy.

4. A method of treating or preventing viral infection which comprises administering to a subject in need thereof, an effective amount of a compound of Formula (I)

wherein:

A represents hydroxy;

D represents aryl or heteroaryl;

E represents hydrogen, C_1-6alkyl, aryl, heteroaryl or heterocyclyl;

G represents SO_nR^a wherein n is 1 or 2; and R^a is C_1-6alkyl optionally substituted by one or more substituents selected from halo, OR¹, SR¹, C(O)NR²R³, C(O)R⁴, CO₂R⁴, NR²R³, NHC(O)R⁴, NHCO₂R⁴, NHC(O)NR⁵R⁶; or R^a is aryl;

R¹ represents hydrogen, C_1-6alkyl, arylalkyl, or heteroarylalkyl;

R² and R³ are independently selected from hydrogen, C_1-6alkyl, aryl and heteroaryl; or R² and R³ together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group;

R⁴ is selected from the group consisting of C_1-6alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; R⁵ and R⁶ are independently selected from the group consisting of hydrogen, C_1-6alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; or R⁵ and R⁶ together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group; and

J represents C_1-6alkyl, heterocyclylalkyl, arylalkyl or heteroarylalkyl;

and salts, solvates and esters thereof; provided that when A is esterified to form -OR where R is selected from straight or branched chain alkyl, aralkyl, aryloxyalkyl, or aryl, then R is other than te/τ-butyl.

5. A method as claimed in claim 4 which involves inhibiting HCV.

6. A method as claimed in claim 4 in which the compound is administered in an oral dosage form.

7. A compound of Formula (I)

wherein:

A represents hydroxy;

D represents aryl or heteroaryl;

E represents hydrogen, C_1-6alkyl, aryl, heteroaryl or heterocyclyl;

G represents SO_πR^a wherein n is 1 or 2; and R^a is C_1-6alkyl optionally substituted by one or more substituents selected from halo, OR¹, SR¹, C(O)NR²R³, C(O)R⁴, CO₂R⁴, NR²R³, NHC(O)R⁴, NHCO₂R⁴, NHC(O)NR⁵R⁶; or R^a is aryl;

R¹ represents hydrogen, C_1-6alkyl, arylalkyl, or heteroarylalkyl;

R² and R³ are independently selected from hydrogen, C_1-6alkyl, aryl and heteroaryl; or R² and R³ together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group;

R⁴ is selected from the group consisting of C _-6alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; R⁵ and R⁶ are independently selected from the group consisting of hydrogen, C_1-6alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; or R⁵ and R⁶ together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group; and

J represents C_1-6alkyl, heterocyclylalkyl, arylalkyl or heteroarylalkyl;

and salts, solvates and esters thereof; provided that when A is esterified to form -OR where R is selected from straight or branched chain alkyl, aralkyl, aryloxyalkyl, or aryl, then R is other than fe τ-butyl; for use in medical therapy.

8. A compound as claimed in claim 7 wherein the medical therapy is the treatment of viral infection.

9. A compound as claimed in claim 8 wherein the viral infection is HCV.

10. Use of a compound of Formula (I)

wherein: A represents hydroxy;

D represents aryl or heteroaryl;

E represents hydrogen, C_1-6alkyl, aryl, heteroaryl or heterocyclyl;

G represents SO_nR^a wherein n is 1 or 2; and R^a is C_1-6alkyl optionally substituted by one or more substituents selected from halo, OR¹, SR¹, C(O)NR²R³, C(O)R⁴, CO₂R⁴, NR²R³, NHC(O)R⁴, NHCO₂R⁴, NHC(O)NR⁵R⁶; or R^a is aryl;

R¹ represents hydrogen, C_1-6alkyl, arylalkyl, or heteroarylalkyl;

R² and R³ are independently selected from hydrogen, C_1-6alkyl, aryl and heteroaryl; or R² and R³ together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group;

R⁴ is selected from the group consisting of C_1-6alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; R⁵ and R⁶ are independently selected from the group consisting of hydrogen, C_1-6alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; or R⁵ and R⁶ together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group; and

J represents C_1-6alkyl, heterocyclylalkyl, arylalkyl or heteroarylalkyl;

and salts, solvates and esters thereof; provided that when A is esterified to form -OR where R is selected from straight or branched chain alkyl, aralkyl, aryloxyalkyl, or aryl, then R is other than fe/f-butyl; in the manufacture of a medicament for the treatment of viral infection.

11. Use as claimed in claim 10, wherein the viral infection is HCV.

12. A pharmaceutical formulation comprising a compound of Formula (I) as defined in claim 1 in conjunction with a pharmaceutically acceptable diluent or carrier.

13. A process for the preparation of a compound of Formula (I) as defined in claim 1 , comprising treatment of a compound of Formula (II)

in which A is tert-butoxy, and D, E, G and J are as defined for Formula (I), with an acid.