WO2004060889A1 - 5-thiazole substituted 2-pyrrolidine-carboxylic acids - Google Patents

Abstract

Anti-viral agents of Formula (1a) wherein: RA represents hydroxy; RB represents 4-tert-butylbenzoyl further substituted in the meta-position by halo or C1-3alkoxy; RC represents 2-thiazolyl, 5-methylthiazol-2-yl, or 4-thiazolyl; RD represents methyl; X represents isobutyl; and salts, solvates and esters thereof; provided that when RA is esterified to form -OR where R is selected from straight or branched chain alkyl, arakyl, aryloxyalkyl, or aryl, then R is other than tert-butyl; processes for their preparation and their use in HCV treatment are provided.

Description

5-THIAZ0LE SUBSTITUTED 2-PYRRO IDINE-CARBOXYLIC ACIDS

FIELD OF THE INVENTION

The present invention relates to novel C(4)-ketone acyl pyrrolidine derivatives useful as anti-viral agents. Specifically, the present invention involves novel HCV inhibitors.

BACKGROUND 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): 71 S-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 cessa^'tion 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 1b isolates) and inter-typically (-85% aa identity between genotype 1a and 1 b 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.

PCT publication number WO2001/085720 generically discloses certain compounds, including certain acyl pyrrolidine compounds, having HCV inhibitory activity. The compounds disclosed have the formula (I)

in which A represents OR¹, NR¹R², or R¹ wherein R¹ and R² is selected from the group consisting of hydrogen, Cι-₆alkyl, optionally substituted aryl, alkylaryl, arylalkyl;

B represents hydrogen, C(O)R¹ wherein R¹ is selected from the group consisting of hydrogen, C^alkyl, aryl, alkylaryl, arylalkyl;

C represents hydrogen, d-₆alkyl, or optionally substituted aryl;

D represents OR¹, NR¹R², or R¹ wherein R¹ and R² are selected from the group consisting of hydrogen, C^alkyl, aryl, alkylaryl, arylalkyl;

E represents hydrogen, Cι-₆alkyl, optionally substituted aryl, alkylaryl, arylalkyl;

X represents C^alkyl, optionally substituted aryl, alkylaryl, arylalkyl; and n is 1 or 2 or 3.

Although disclosed generically, there are no compounds exemplified in which D is R and

R¹ is C^alkyl.

Surprisingly, it has now been found that compounds according to the present invention, generically disclosed in WO2001/085720, and having a specific substitution pattern, exhibit improved properties over those compounds specifically disclosed in WO2001/085720.

SUMMARY OF THE INVENTION

The present invention involves C(4)-ketone 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 INVENTION

The present invention (la) :

wherein:

R^A represents hydroxy; R^B represents 4-tert-butylbenzoyl further substituted in the tnete-position by halo or C,. ₃alkoxy;

R^c represents 2-thiazolyl, 5-methylthiazol-2-yl or 4-thiazolyl;

R^D represents methyl;

X represents isobutyl;

and salts, solvates and esters thereof; provided that when R^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 terf-butyl.

Preferably, R^A is hydroxy (that is, not esterified).

Preferably, R^B represents 4-tert-butylbenzoyl further substituted in the mete-position by bromo, chloro or methoxy (3-bromo-4-terf-butylbenzoyl, 4-tert-butyl-3-chlorobenzoyl, 4- terf-butyl-3-methoxybenzoyl); more preferably R^B represents 4-tert-butylbenzoyl further meta-substituted by methoxy (4-tert-butyl-3-methoxybenzoyl).

Preferably, R^c represents 2-thiazolyl or 5-methylthiazol-2-yl; more preferably, R^c represents 2-thiazolyl.

In a preferred aspect, the present invention provides compounds of Formula (la) in which R^A represents hydroxy; R^B represents 4-terf-butyl-3-methoxy-benzoyl; R^c represents 2- thiazolyl; R^D represents methyl; and X represents isobutyl; and salts, solvates and esters thereof; provided that when R^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.

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

The compounds of the present invention exhibit improved inhibitory activity against HCV polymerase in addition to good bioavailability, and therefore have the potential to achieve greater efficacy in man.

There is provided as a further aspect of the present invention a compound of Formula (la) 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 (la) 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 (la) 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, diastereoisomeric, and optically active forms.

Preferred compounds of Formula (la) useful in the present invention are selected from the group consisting of: re/-(2S,4R,5R)-1-(4-tert-Butyl-3-methoxybenzoyl)-2-isobutyl-4-acetyl-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-1-(4-tert-Butyl-3-bromobenzoyl)-2-isobutyl-4-acetyl-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-1-(4-tert-Butyl-3-chlorobenzoyl)-2-isobutyl-4-acetyl-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-1-(4-tert-Butyl-3-methoxybenzoyl)-2-isobutyl-4-acetyl-5-(5-methyl-1 ,3- thiazol-2-yl)pyrrolidine-2-carboxylic acid; and re/-(2S,4R,5R)-1-(4-tert-Butyl-3-methoxybenzoyl)-2-isobutyl-4-acetyl-5-(1 ,3-thiazol-4- yl)pyrrolidine-2-carboxylic acid and salts, solvates and esters, and individual enantiomers thereof.

More preferred compounds of Formula (la) useful in the present invention are selected from the group consisting of:

(2S,4R,5R)-1-(4-tert-Butyl-3-methoxybenzoyl)-2-isobutyl-4-acetyl-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid;

(2S,4R,5R)-1-(4-tert-Butyl-3-bromobenzoyl)-2-isobutyl-4-acetyl-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; and

(2S,4R,5R)-1-(4-tert-Butyl-3-chlorobenzoyl)-2-isobutyl-4-acetyl-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid and salts, solvates and esters 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 (la). Suitable physiologically acceptable salts of the compounds of formula (la) 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 (la), for example hydrates.

The present invention also relates to pharmaceutically acceptable esters of the compounds of Formula (la), 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, C₁-₄alkyl or C^alkoxy 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.

Preferably, the compound of Formula (la) is in the form of a free base, a salt or a solvate.

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 (la) may be prepared from a compound of Formula

in which R^A is an alkoxy, benzyloxy or silyloxy group and R^B, R^c, R^D and X are as defined above for Formula (la). For example when R^A is tert-butoxy, and R^B, R^c, R^D and X are as defined above for Formula (la), 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 20 to 30°C. For example when R^A is benzyloxy, and R^B, R^c, R^D and X are as defined above for Formula (la), 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 R^A is allyloxy, and R^B, R^c, R^D and X are as defined above for Formula (la), 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 R^A is silyloxy, and R^B, R^c, R^D and X are as defined above for Formula (la), by treatment with a suitable fluoride source for example tetrabutylammonium fluoride. The reaction is carried out in a suitable solvent, for example tetrahydrofuran.

A compound of Formula (II) may be prepared from a compound of Formula (III)

in which R^A is as defined above for Formula (II), and R^c, R^D and X are as defined above for Formula (la); with a suitable acylating agent, for example R^B-hal (such as 4-tert-butyl- 3-methoxyphenyl-C(O)-hal), wherein hal is a halo atom, preferably chloro or bromo, and R^B is as defined above for Formula (la). Preferably the reaction is carried out in a suitable solvent, for example dichloromethane, in the presence of a suitable base, for example triethylamine.

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

in which R^c and X are as defined above for Formula (la) and R^A is as defined above for Formula (II), with a compound of Formula (V)

wherein R^D is as defined above for Formula (la). 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 triethylamine,

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)

in which X is as defined above for Formula (la) and R^A is as defined above for Formula (II) with a compound of Formula R^c-CHO in the presence of a suitable base, for example triethylamine, in a suitable solvent, for example dichloromethane.

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

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

It will be appreciated that racemic compounds of Formula (la), (II), and/or (III) may be optionally resolved into their individual enantiomers. Such resolutions may conveniently be accomplished by standard methods known in the art. For example, a racemic compound of Formula (la), (II), and/or (III) may be resolved by chiral preparative HPLC. Alternatively, racemic compounds of Formula (la) in which R^A is hydroxy, or Formula (III), may be resolved by standard diastereoisomeric salt formation with a chiral acid or base reagent as appropriate. Such techniques are well established in the art. For example, a racemic compound of Formula (III) may be resolved by treatment with a chiral acid such as (R)-(-)-1 ,1'-binaphthyl-2,2'-diyl dihydrogen phosphate.

With appropriate manipulation and protection of any chemical functionality, synthesis of compounds of Formula (la) 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 Intermediate 1

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

A stirred mixture of 2-amino-4-methyl-pentanoic acid tert-butyl ester, hydrochloride (5.00g, 22.34 mmol), 1 ,3-thiazole-2-carboxaldehyde (2.53g, 22.34 mmol) and triethylamine (3.1 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. H NMR (CDCI₃): δ 8.46 (s, 1 H), 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/-(2S,4R,5R)-2-lsobutyl-4-acetyl-5-(1,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid, tert-butyl ester

Racemic;

Relative stereochemistry shown

To a stirred solution of Intermediate 1 (5.0 g, 17.7 mmol), methyl vinyl ketone (1.4 g, 20 mmol) and lithium bromide (2.39 g, 27.5 mmol) in anhydrous THF (3 mL) in an ice/methanol bath, was added 1 ,8-diazabicyclo[5,4,0]undec-7-ene (DBU) (5.41 mL, 35.5 mmol). The stirred reaction was gradually allowed to attain room temperature overnight.

Aqueous ammonium chloride (100 mL) was added and the resulting mixture was extracted with ethyl acetate (2 x 200 mL). The organic extracts were combined and washed with water and brine then dried (MgSO₄). The solvent was evaporated in vacuo to give the crude product. This was purified by chromatography on silica gel using cyclohexane-ethyl acetate (6:1 v/v) as eluent to provide the title compound as an oil.

MS calcd for (C₁₈H₂₈N₂O₃S + H)⁺: 353.

MS found (electrospray): (M+H)⁺ =353. ¹H NMR (CDCI₃): δ 7.70 (1 H, d), 7.25 (1 H, d), 4.80 (1H, d), 3.40 (1 H, m), 2.95 (1 H, br),

2.70 (1 H, dd), 2.20 (3H, s), 1.95 (1 H, dd), 1.70 (3H, m), 1.45 (9H, s), 0.90 (6H, m).

Intermediate 3

(2S_>4R,5R)-1-(4-tert-Butyl-3-methoxybenzoyl)-2-isobutyl-4-acetyl-5-(1,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid, tert-butyl ester

[Enantiomer A of re/-(2S,4R,5R)-1-(4-terf-Butyl-3-methoxybenzoyl)-2-isobutyl-4- acetyl-5-(1,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid, tert-butyl ester] Chiral;

Absolute stereochemistry shown

To a stirred solution of 4-tert-butyl-3-methoxybenzoyl chloride (0.38 g, 1.68 mmol) and Intermediate 2 (0.49 g, 1.4 mmol) in anhydrous dichloromethane (4 mL) was added triethylamine (0.29 mL, 2.1 mmol) and the mixture stirred at room temperature overnight. Water (20 mL) and dichloromethane (30 mL) were added. The organic phase was dried (MgSO₄) and evaporated in vacuo. The residue was purified by chromatography on silica gel using ethyl acetate-cyclohexane (90:10 v/v) then (85:15 v/v) as eluent to provide the racemate of the title compound as a gum.

Analytical chiral HPLC of the racemic compound (Chiralpak AD, using heptane:isopropanol (90:10 v/v) as eluent showed retention times of 4.45 and 5.38 minutes respectively for the two enantiomers. The enantiomers were separated on a preparative chiral HPLC Chiralpak AD column, eluting with heptane:isopropanol (95:5 v/v). The second eluting enantiomer (Enantiomer A) was collected to afford the title compound. Comparison of Enantiomer A with the material prepared according to Intermediate 7 (below) confirms the absolute stereochemistry of Enantiomer A as (2S.4R.5R).

MS calcd for (C₃₀H₄₂N₂O₅S + H)⁺: 543. MS found (electrospray): (M+H)⁺ = 543.

¹H NMR (CDCI₃): δ 7.10 (2H, m), 6.95 (1 H, dd), 6.60 (1 H, s), 5.60 (1 H, d), 3.70 (5H, m), 2.50 (2H, m), 2.30 (1 H, dd), 2.00 (3H, s), 1.90 (1 H, m), 1.80 (1 H, m), 1.55 (9H, s), 1.25 (9H, s), 1.10 (3H, d), 0.95 (3H, d).

Intermediate 4

Preparation of a seed sample of (2S,4R,5R)-2-lsobutyl-4-acetyl-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid, tert-butyl ester, (R)-(-)-1,1'-binaphthyl-2-2'-diyl dihydrogen phosphate salt

[Enantiomer A of re/-(2S,4R,5R)-2-lsobutyl-4-acetyl-5-(1 ,3-thiazol-2-yl)pyrrolidine-2- carboxylic acid, tert-butyl ester, (R)-(-)-1,1'-binaphthyl-2-2'-diyl dihydrogen phosphate salt]

Chiral;

Absolute stereochemistry shown

Intermediate 2 (100 mg, 0.28 mmol) in isopropanol (0.5 mL) was added to a solution of

(R)-(-)-1 ,1'-binaphthyl-2-2'-diyl dihydrogen phosphate (100mg, 0.29 mmol) in hot isopropanol (5.5 mL). The mixture was allowed to cool and placed at room temperature overnight, then at 4°C for a further 24 hours. The mixture was allowed to evaporate slowly over 3 days. The resulting solid was isolated by filtration and washed with isopropanol to afford the seed sample of the title compound.

HPLC: Chiralpak AD column, heptane/ethanol (85:15, v/v), Enantiomer A showed a retention time of 4.9 min, 97.5% ee. The second eluting enantiomer had a retention time of 7.2 min. Comparison of Enantiomer A with the material derived from Intermediate 6

(below) confirmed the absolute stereochemistry of the pyrrolidine base as (2S.4R.5R).

MS calcd for (C₁₈H₂₈N₂O₃S + H)⁺: 353.

MS found (electrospray): (M+H)⁺ =353.

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

Racemic;

Relative stereochemistry shown

Crude re/-(2S,4R,5R)-2-lsobutyl-4-acetyl-5-(1 ,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid, tert-butyl ester, prepared by analogy with the method described in Intermediate 2 (15g prior to chromatography) was dried overnight under vacuum. This oil was dissolved in diethyl ether (50 mL) and treated with HCI in dioxan (4M, 12 mL, 1.1 eq). The solvent was evaporated in vacuo and the residue triturated twice with ether to afford a solid. This was dissolved in hot isopropanol (-50 mL)and placed at 4°C overnight. The resulting solid was filtered and dried in vacuo to afford the title compound as a powder.

MS calcd for (Cι₈H₂₈N₂O₃S + H)⁺: 353. MS found (electrospray): (M+H)⁺ =353.

¹H NMR (CDCI₃): δ 13.90 (2H, br), 7.85 (1 H, d), 7.50 (1 H, d), 5.8 (1 H, d), 3.25 (1 H, m), 3.1 (1 H, m), 2.5 (1 H, dd), 2.35 (1 H, t), 2.25 (4H, m), 1.85 (1 H, m), 1.55 (9H, s), 1.15 (3H, d), 1.00 (3H, d). Intermediate 6

(2S,4R,5R)-2-lsobutyl-4-acetyl-5-(1 ,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid, tert- butyl ester, (R)-(-)-1,1'-binaphthyl-2-2'-diyl dihydrogen phosphate salt [Enantiomer A of re/-(2S,4R,5R)-2-lsobutyl-4-acetyl-5-(1 ,3-thiazol-2-yl)pyrrolidine-2- carboxylic acid, tert-butyl ester, (R)-(-)-1,1'-binaphthyl-2-2'-diyl dihydrogen phosphate salt]

Chiral;

Absolute stereochemistry shown

Intermediate 5 (7.04 g, 18.1 mmol), was partitioned between ethyl acetate and saturated aqueous sodium bicarbonate solution. The organic phase was separated, washed with saturated aqueous sodium bicarbonate solution, dried over sodium sulphate and evaporated to provide the pyrrolidine base (6.51 g) as an oil. To a solution of this in isopropanol (30 mL) was added a previously filtered, hot solution of (R)-(-)1 ,1'-binaphthyl- 2-2'-diyl dihydrogen phosphate (6.3 g, 18.1 mmol) in isopropanol (337 mL) followed by a few crystals of the seeding sample (Intermediate 4). The mixture was allowed to cool and placed at room temperature overnight. The mixture was concentrated by evaporation of the isopropanol (80 mL) and then placed at room temperature. The resulting solid was isolated by filtration and washed with isopropanol to afford the title compound. HPLC: Chiralpak AD column, heptane/ethanol (85:15, v/v), Enantiomer A showed a retention time of 4.9 min, 99.5% ee. The second eluting enantiomer had a retention time of 7.2 min. The absolute stereochemistry of salt Enantiomer A was confirmed by X-ray crystallography and showed the pyrrolidine base to have (2S,4R,5R) chirality. MS calcd for (C₁₈H₂₈N₂O₃S + H)⁺: 353. MS found (electrospray): (M+H)⁺ =353.

Intermediate 7

(2S,4R,5R)-1-(4-tert-Butyl-3-methoxybenzoyl)-2-isobutyl-4-acetyl-5-(1,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid, tert-butyl ester

[Enantiomer A of re/-(2S,4R,5R)-1-(4-tert-Butyl-3-methoxybenzoyl)-2-isobutyl-4- acetyl-5-(1,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid, tert-butyl ester] Chiral; Absolute stereochemistry shown

To a stirred solution of Intermediate 6 (0.24 g, 0.34 mmol) in anhydrous dichloromethane (6 mL) was added triethylamine (0.10 mL, 0.75 mmol) and 4-tert-butyl-3-methoxybenzoyl chloride (0.12 g, 0.51 mmol). The mixture stirred at room temperature overnight prior to the addition of dichloromethane and saturated aqueous sodium bicarbonate solution. The organic phase was pre-adsorbed onto silica gel and chromatographed over silica gel, eluting with cyclohexane/ethyl acetate (5:1 v/v) to afford the title compound. This material was spectroscopically identical to that prepared according to the method described in Intermediate 3. This material was 99.5% ee as determined by analytical chiral HPLC (Chiralpak AD, using heptane:isopropanol (90:10 v/v) as eluent). Retention times for the two enantiomers were 4.64 min (0.25% peak area) and 5.64 min (Enantiomer A, 99.75% peak area) respectively. Enantiomer A corresponds to (2S,4R,5R) absolute stereochemistry.

Intermediate 8

(2S,4R,5R)-1-(4-tert-Butyl-3-bromobenzoyl)-2-isobutyl-4-acetyl-5-(1,3-thiazol-2- yl)pyrrolidine-2-carb ester

Chiral; Absolute stereochemistry shown

The title compound was prepared from Intermediate 6 and 4-tert-butyl-3-bromobenzoyl chloride in a similar manner to that described for Intermediate 7. MS calcd for (C₂₉H₃₉BrN₂O₄S + H)⁺: 591/593 MS found (electrospray): (M+H)⁺ = 591/593.

Intermediate 9 (2S,4R,5R)-1 -(4-tert-Butyl-3-chlorobenzoyl)-2-isobutyl-4-acetyl-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid, tert-butyl ester Chiral A Abbssoolliute stereochemistry shown

The title compound was prepared from Intermediate 6 and 4-tert-butyl-3-chlorobenzoyl chloride in a similar manner to that described for Intermediate 7. MS calcd for (C₂₉H₃₉CIN₂O₄S + H)⁺: 547/549 MS found (electrospray): (M+H)⁺ = 547/549.

Intermediate 10

2-[N-(5-Methyl-1 ,3-thiazol-2-ylmethylene)amino]-4-methylpentanoic acid, tert-butyl ester

2-Amino-4-methylpentanoic acid tert-butyl ester, hydrochloride (4.1g, 18.3 mmol) was dissolved in dichloromethane (70 mL) and the resultant solution was washed with saturated aqueous sodium hydrogen carbonate solution. The organic phase was separated and dried over anhydrous sodium sulphate. The sodium sulphate was removed by filtration and then 5-methylthiazole-2-carboxaldehyde (J. Med. Chem., 2000, 43, 3168; 2.3 g, 18.1 mmol) was added to the filtrate, followed by additional anhydrous sodium sulphate (7g). The resultant reaction mixture was heated to reflux for 18h then allowed to cool. The reaction mixture was filtered and the filtrate concentrated under vacuum to give the title compound as an oil. ¹H NMR (CDCI₃): δ 0.92 (6H, dd), 1.46 (9H, s), 1.80 (2H, m), 2.51 (3H, s), 4.00 (1 H, m), 7.58 (1 H, s) and 8.34 (1 H, s).

Intermediate 11

Enantiomer A of re/-(2S,4R,5R)-1-(4-tert-Butyl-3-methoxybenzoyl)-2-isobutyl-4- acetyl-5-(5-methyl-1, ine-2-carboxylic acid, tert-butyl ester

Chiral,

Relative stereochemistry shown

Procedure A: 2-[N-(5-Methyl-1,3-thiazol-2-ylmethylene)amino]-4-methylpentanoic acid, tert-butyl ester (1.0 g, 3.3 mmol; Intermediate 10) was dissolved in THF (4 mL). To this solution was added methyl vinyl ketone (0.31 mL) and lithium bromide (0.585 g). The resultant mixture was cooled to -10°C, then stirred for 10 minutes prior to the addition of DBU (1.0 mL). The resultant reaction mixture was allowed to warm to room temperature overnight, diluted with saturated aqueous ammonium chloride solution and extracted with ethyl acetate. The organic phase was separated, washed with water and brine, then dried over anhydrous sodium sulphate. The sodium sulphate was removed by filtration and the filtrate concentrated under vacuum to give an oil which was partially purified by chromatography on silica gel using cyclohexane-ethyl acetate (8:2 v/v) as eluent to afford impure re/-(2S,4R,5R)-2-lsobutyl-4-acetyl-5-(5-methyl-1 ,3-thiazol-2-yl)pyrrolidine-2- carboxylic acid, tert-butyl ester, an oil (0.5 g).

Procedure B: A solution of the impure pyrrolidine from Procedure A (0.5 g) and triethylamine (0.42 mL) in dichloromethane (10 mL) was treated with 3-methoxy-4-tert- butylbenzoyl chloride (0.34 g). The resultant reaction mixture was stirred at room temperature for 18 hours, then washed with saturated aqueous sodium hydrogen carbonate solution. The organic phase was separated, dried using a hydrophobic frit, and then evaporated to give an oil. This was purified by chromatography on silica gel using ethyl acetate-cyclohexane (2:8 v/v) as eluent to afford the racemic title compound. Procedure C: The racemic title compound was resolved using preparative chiral HPLC on a Chiralpak AD column, eluting with heptane-isopropanol (95:5 v/v) to afford the two enantiomers with retention times of 6.1 min (99.8% ee) and 7.3 min (99.2% ee) respectively. The second eluting enantiomer (Enantiomer A) was collected to afford the title compound. MS calcd for (C₃₁H₄₄N₂O₅S + H)⁺: 557 MS found (electrospray): (M+H)⁺ = 557.

¹H NMR (CDCI₃): δ 1.05 (6H, dd), 1.30 (9H, s), 1.57 (9H, s), 1.85 (3H, m), 1.99 (3H, s), 2.29 (3H, s), 2.40 (3H, m), 3.70 (3H, s), 5.44 (1 H, d), 6.59 (1 H, s), 6.89 (2H, m) and 7.12 (1 H, d).

Intermediate 12

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

The title compound was prepared from 1 ,3-thiazole-4-carboxaldehyde (Synthesis, 1987, 998; 1.97 g, 17.5 mmol) and 2-amino-4-methylpentanoic acid tert-butyl ester, hydrochloride (3.91 g, 17.5 mmol) in a similar manner to that described for Intermediate 1.

Intermediate 13 re/-(2S,4R,5R)-2-lsobutyl-4-acetyl-5-(1,3-thiazol-4-yl)pyrrolidine-2-carboxylic acid, tert-butyl ester

Racemic;

Relative stereochemistry shown

The title compound was prepared from 2-[N-(1 ,3-thiazol-4-ylmethylene)amino]-4- methylpentanoic acid, tert-butyl ester (Intermediate 13; 2.26 g, 8.04 mmol) and methyl vinyl ketone (0.62 g, 8.84 mmol) in a similar manner to that described for Intermediate 2. MS calcd for (C₁₈H₂₈N₂O₃S + H)⁺: 353. MS found (electrospray): (M+H)⁺ =353.

Intermediate 14

Enantiomer A of re/-(2S,4R,5R)-1-(4-tert-Butyl-3-methoxybenzoyl)-2-isobutyl-4- acetyl-5-(1,3-thiazol-4-yl)pyrrolidine-2-carboxylic acid, tert-butyl ester

Chiral;

Relative stereochemistry shown

4-tert-Butyl-3-methoxybenzoyl chloride (4.83 mmol) was added to a stirred solution of Intermediate 13 (4.11 mmol) and triethylamine (8.22 mmol) in anhydrous dichloromethane (15 mL) and the mixture stirred at room temperature overnight. The reaction mixture was washed with saturated aqueous sodium bicarbonate solution (15 mL) and the dichloromethane soution filtered through a hydrophobic frit and evaporated. The resulting material was purified by chromatography on silica gel using cyclohexane-ethyl acetate (80:20 v/v) to afford the racemic N-acylpyrrolidine. This compound was resolved by preparative chiral HPLC on a Chiralpak AD column, eluting with heptane-isopropanol (95:5 v/v) to afford the two enantiomers with retention times of 6.1 min (97% ee) and 10.0 min ( >99% ee) respectively. The second eluting enantiomer (Enantiomer A) was collected to afford the title compound. MS calcd for (C₃₀H₄₂N₂O₅S + H)⁺: 543. MS found (electrospray): (M+H)⁺ = 543.

Example 1 (2S,4R,5R)-1 -(4-tert-Butyl-3-methoxybenzoyl)-2-isobutyl-4-acetyl-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid

[Enantiomer A of re/-(2S,4R,5R)-1-(4-tert-Butyl-3-methoxybenzoyl)-2-isobutyl-4- acetyl-5-(1 ,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid] Chiral,

Absolute stereochemistry shown

A solution of Intermediate 3 (135 mg, 0.25 mmol) in trifluoroacetic acid (TFA) (3 mL) was placed at room temperature overnight. The solvent was removed in vacuo and the residue was sequentially redissolved in, and re-evaporated from both dichloromethane and then toluene. The residue was dissolved in methanol (5 mL), sodium hydroxide solution was added (0.36mL, 2N) and the solution placed at room temperature overnight. Hydrochloric acid (0.3 mL, 1 N) and dichloromethane (4 mL) were added and the organic phase was separated and evaporated to afford the title compound as a solid. This compound was analysed by chiral HPLC on a Chiralpak AD column using heptane:ethanol (containing 0.01% tnfluoroacetic acid) (80:20 v/v) as eluent and shown to correspond to the slow eluting enantiomer (Enantiomer A; retention time 6.49 minutes). The corresponding racemic compound showed retention times of 4.62 and 6.49 minutes respectively for the two enantiomers. MS calcd for (C₂₆H₃₄N₂O₅S + H)⁺: 486. MS found (electrospray): (M+H)⁺ = 486.

¹H NMR (CDCI₃): δ 7.6 (d, 1H), 7.2 (d, 1H), 7.05 (d, 1 H), 6.95 (d, 1H), 6.5 (s, 1H), 5.75 (d, 1 H), 3.7 (s, 3H), 3.5 (m, 1 H), 3.25 (dd, 1H) 2.55 (m, 1 H) 2.15 (m, 4H), 1.95-1.85 (m, 2H), 1.64-1.55 (m, 1H), 1.3 (s, 9H), and 1.0 (2xd, 6H).

Example 2

(2S,4R,5R)-1-(4-tert-Butyl-3-bromobenzoyl)-2-isobutyl-4-acetyl-5-(1,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid

Chiral,

Absolute stereochemistry shown

The title compound, a solid, was prepared from Intermediate 8 in a similar manner to that described for Example 1.

MS calcd for (C₂₅H₃₁BrN₂O₄S + H)⁺: 535/537.

MS found (electrospray): (M+H)⁺= 535 and 537.

¹H NMR (CD₃OD): δ 7.45 (d, 1 H), 7.39 (d, 1 H), 7.31-7.49 (m, 3H), 5.68 (d, 1H), 3.79-3.70

(m, 1 H), 2.63 (dd, 1 H), 2.54 (t, 1 H), 2.47-2.37 (br. d, 1 H), 2.03 (s, 3H), 1.99 (br.d, 1 H) 1.84 (m, 1 H), 1.46 (s, 9H), 1.11 (d, 3H) and 0.98 (s, 3H). The carboxylic acid proton exchanges with the solvent. Example 3

(2S,4R,5R)-1-(4-tert-Butyl-3-chlorobenzoyl)-2-isobutyl-4-acetyl-5-(1,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid

Chiral;

Absolute stereochemistry shown

The title compound, a solid, was prepared from Intermediate 9 in a similar manner to that described for Example 1.

MS calcd for (C₂₅H₃₁CIN₂O₄S + H)⁺ 491/493.

MS found (electrospray): (M+H)⁺ = 491/493. ¹H NMR (CD₃OD): δ 7.45 (d, 1H), 7.38 (d, 1H), 7.28 (d, 1H), 7.21 (d, 1H), 7.03 (s, 1 H),

5.70 (d, 1 H), 3.76 (m, 1 H), 2.64 (dd, 1 H), 2.55 (t, 1 H), 2.49-2.38 (br. d, 1 H), 2.04 (s, 3H),

2.01 (br.d, 1 H), 1.86 (m, 1 H), 1.44 (s, 9H), 1.12 (d, 3H) and 0.99 (s, 3H). The carboxylic acid proton exchanges with the solvent.

Example 4

Enantiomer A of re/-(2S,4R,5R)-1-(4-tert-Butyl-3-methoxybenzoyl)-2-isobutyl-4- acetyl-5-(5-methyl-1,3 dine-2-carboxylic acid

Chiral;

Relative stereochemistry shown

Enantiomer A of re/-(2S,4R,5R)-1-(4-tert-Butyl-3-methoxybenzoyl)-2-isobutyl-4-acetyl-5- (5-methyl-1 ,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid, tert-butyl ester (53 mg; Intermediate 11) was treated with tnfluoroacetic acid (3 mL) and the resultant mixture was stirred at room temperature for 18 hours. The reaction mixture was concentrated under vacuum, redissolved in toluene and re-evaporated under vacuum. The resultant residue was triturated with diethyl ether to give the title compound, a solid. MS calcd for (C₂₇H₃₆N₂O₅S + H)⁺: 501 MS found (electrospray): (M+H)⁺ = 501

¹H NMR (CD₃OD): . 0.99 (3H, d), 1.10 (3H, d), 1.32 (9H, s), 1.85 (1 H, m), 2.00 (1 H, m), 2.06 (3H, s), 2.31 (3H, s), 2.30-2.70 (3H, m), 3.70 (1 H, m), 3.72 (3H, s), 5.59 (1 H, d), 6.55 (1H, s), 6.90 (1H, d). 7.00 (1H, s) and. 7.19 (1H, d). Carboxylic acid proton exchanges with the solvent. nOe NMR studies confirmed the relative stereochemistry to be (2S,4R,5R). Aanalytical chiral HPLC (Chiralpak AD, heptane-ethanol 85:15 v/v + 0.1% TFA): retention time 7.89 minutes (>98% ee).

Example 5

Enantiomer A of re/-(2S,4R,5R)-1-(4-tert-Butyl-3-methoxybenzoyl)-2-isobutyl-4- acetyl-5-(1 ,3-thiazol-4 rboxylic acid

Chiral;

Relative stereochemistry shown

The title compound, a solid, was prepared from Intermediate 14 in a similar manner to that described for Example 1.

MS calcd for (C₂₆H₃₄N₂O₅S + H)⁺: 486.

MS found (electrospray): (M+H)⁺ = 486. H NMR (CD₃OD): δ 8.67 (1 H, d), 7.24 (1 H, s), 7.16 (1H, d), 6.86 (1 H, dd), 6.51 (1 H, s),

5.57 (1 H, d), 3.72 (3H, s), 3.63 (1 H, m), 2.67 (1 H, dd), 2.51 (1 H, dd), 2.40 (1 H, m), 2.09 (1H, dd), 2.02 (3H, s), 1.86 (1H, m), 1.31 (9H, s), 1.11 (3H, d) and 1.00 (3H, d).

Carboxylic acid proton exchanged with solvent. nOe NMR studies confirmed the relative stereochemistry to be (2S,4R,5R).

Preparation of Internal Standard for pharmacokinetic test Intermediate a

4-tert-Butyl-3-methyl-benzoic acid

To 3-bromo-4-tert-butylbenzoic acid (500mg, 1.94 mmol) (Aust.J.Chem. (1990), 43(5), 807-14) in toluene (10 mL) was added N,N-dimethylformamide-di-tert-butyl acetal (1.86 mL, 7.76 mmol). The mixture was heated at 110°C for 24 hours. More N,N- dimethylformamide-di-tert-butyl acetal (2 mL, 8.38 mmol) was added and the reaction was heated at reflux for a further 24 hours. The solvent was evaporated in vacuo and the residue was purified by SPE (silica, eluting with a gradient of ether in cyclohexane, 1% to 3%) to give a colourless oil (89%). The oil was dissolved in anhydrous THF (4 mL) and cooled to -78°C under nitrogen. tert-Butyllithium (1.7M in pentane, 0.55 mL, 0.94 mmol) was added dropwise, maintaining the temperature at -70°C. After 15 min., methyl iodide (0.53 mL, 8.45mmol) was added dropwise, maintaining the temperature at below -70°C. The reaction mixture was allowed to attain room temperature over 3 hours. Saturated ammonium chloride solution was added and the mixture extracted with diethyl ether. The organic extract was dried (MgSO₄) and the solvent was evaporated in vacuo to give a brown oil. This was purified by reverse phase HPLC on a Cι₈ column using a two-solvent gradient elution with (A) water containing formic acid (0.1%) and (B) acetonitrile-water (95:5 v/v) containing formic acid (0.05%) as the eluents, and analysis of the fractions by electrospray mass spectroscopy, to give a colourless oil (38%). This oil (80mg, 0.32 mmol) was subsequently treated with tnfluoroacetic acid (5 mL) and the reaction was stirred at room temperature for 18 hours. The solvent was evaporated in vacuo and the residue was azeotroped with toluene to give the title compound as solid. MS calcd for (C₁₂H₁₆O₂ - HV : 191

MS found (electrospray): (M-H)^" = 191.

Intermediate b

1 ,3-Thiazole-4-carboxaldehyde

A stirred mixture of 4-chloromethylthiazole (1g, 5.88 mmol) and hexamine (1.64g, 11.76 mmol, 2eq) in 50% acetic acid (10 mL) was heated at reflux for 3 hours. The reaction mixture was allowed to cool for five minutes and then concentrated hydrochloric acid (2.5 mL) was added. The resulting reaction mixture was refluxed for a further five minutes prior to dilution of the reaction mixture with water (50 mL). The resulting reaction mixture was extracted with dichloromethane (6 x 50 mL). The extracts were combined and washed once with sodium bicarbonate (50 mL), dried over Na₂SO₄ and evaporated to give the title compound as a gum.

¹H NMR (CDCI₃): δ 10.14 (s, 1 H), 8.93 (s, 1 H), 8.28 (s,1 H). Intermediate c

stirred mixture of 2-amino-4-methyl-pentanoic acid tert-butyl ester, hydrochloride (2.1g, 9.38 mmol), 1 ,3-thiazole-4-carboxaldehyde (Intermediate b) (1.06 g, 9.38 mmol) and triethylamine (1.31 mL, 9.38 mmol) in dichloromethane (25 mL) was heated under reflux under nitrogen for 20 hours. The reaction mixture was allowed to cool to room temperature, washed twice with water, dried over Na₂SO and evaporated to give the title compound as an oil. H NMR (CDCI3): δ 8.84 (s, 1 H), 8.49 (d, 1 H), 8.01 (s, 1 H), 4.00 (dd, 1 H), 1.90-1.70 (m, 2H), 1.64-1.56 (m, 1H), 1.47 (s, 9H), 0.96 (d, 3H) and 0.91 (d, 3H). Intermediate d re/-(2S,4S,5R)- 2-lsobutyl-4-(pyrazin-2-yl)-5-(1,3-thiazol-4-yl)pyrrolidine-2-carboxylic acid, tert-butyl ester

Racemic, Relative stereochemistry shown

To a cooled (0°C) stirred solution of Intermediate c (1.5 g) in anhydrous THF (30 mL) under nitrogen, was added 2-vinylpyrazine (0.859 g) followed by lithium bromide (0.938 g) and triethylamine (1.13 mL). The reaction was stirred in a cooling bath for 5 min. and then at ambient temperature overnight. Aqueous ammonium chloride was added and the resulting mixture was extracted with ethyl acetate. The extracts were combined and washed with water then dried (MgSO₄). The solvent was evaporated in vacuo to give the crude product. This was purified by chromatography on silica gel using cyclohexane-ethyl acetate (1 :1 v/v) as eluent to provide the title compound. MS calcd for (C₂₀H₂₈N₄O₂S + H)⁺ : 389 MS found (electrospray): (M+H)⁺ = 389 Intermediate e re/-(2S,4S,5R)-1-(4-tert-butyl-3-methylbenzoyl)-2-isobutyl-4-pyrazin-2-yl-5-(1,3- thiazol-4-yl)pyrrolidine-2-carboxylic acid, tert butyl ester.

4-tert-Butyl-3-methyl-benzoic acid (Intermediate a) (100 mg) was dissolved in dry dichloromethane (10 mL) and treated with oxalyl chloride (91 uL). A catalytic amount of N,N-diethyformamide (2 drops) was added and the resultant mixture stirred for 20h. The volatiles were removed in vacuo to give 4-tert-Butyl-3-methyl-benzoyl chloride as a brown oil. This oil was dissolved in dichloromethane (10 mL) and added to a solution of Intermediate d (0.168 g) and triethylamine (76 uL) in dichloromethane (10 mL). The reaction mixture was stirred for 20h. The volatiles were removed in vacuo and the residue purified by silica gel chromatography initially eluting with dichloromethane and then ethyl acetate yclohexane (1 :1 ) to give the title compound. MS calcd for (C₃₂H₄₂N₄O₃S + H)⁺ : 563 MS found (electrospray): (M+H)⁺ = 563 PK Standard Reference Compound re/-(2S,4S,5R)-1 -(4-tert-Butyl-3-methylbenzoyl)-2-isobutyl-4-(pyrazin-2-yl)-5-(1 ,3- thiazol-4-yl)pyrrolidine

Racemic,

Relative stereochemistry shown

Intermediate e (109 mg) was dissolved in tnfluoroacetic acid (2 mL). The reaction was stirred at room temperature for 7 hours. The solvent was then evaporated in vacuo and the residue was purified by SPE (Cι₈) eluting with water followed by acetonitrile to afford the title compound.

MS calcd for (C₂₈H₃ N₄O₃S + H)⁺ : 507

MS found (electrospray): (M+H)⁺ = 507

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 (la) 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 (IC₅o) 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 (la) 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 (la). A topical formulation contains suitably 0.01 to 5.0% of a compound of Formula (la). 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.

Compositions of Formula (la) 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 (la) 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 toxological effects are expected when compounds of the present invention are administered in accordance with the present invention.

ASSAYS

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

Replicon ELISA Cell Based Assay

Cells

The 5-15 subline of Huh-7 cells (Lohmann, V., Korner, F., Koch, J-O., Herian, U.,

Theilmann, L. & Bartenschlager, R., 1999, Science, 285. pp110-113 ) were used for these assays. These are human hepatocellular carcinoma cells stably transfected with an HCV replicon comprising the majority of the HCV 1 b genome with the addition of a selectable marker gene, but lacking the genes encoding for all structural proteins and non-structural protein (NS) 2. The replicon RNA is self-replicating and fully functional viral proteins are translated from it. A quantifiable and specific reduction of expressed protein in the presence of a drug can be used as a measure of replicon inhibition. Compounds

Stock solutions of compound samples were formulated to 40mM in DMSO. Method Culture step: 100μL volumes of assay medium (Dulbecco's Minimal Essential Medium (DMEM) with 4500mg/L glucose and supplemented with 10% foetal bovine serum, 100iu/mL penicillin, 100μg/mL streptomycin, 2mM L-glutamine and 1% non-essential amino acids solution) were added to each well of a 96-well tissue culture plate. The 40mM stock solutions of compound were further diluted in assay medium to twice the highest final concentration required, and 100μL aliquots were transferred into two wells in the top row of the plate. Serial doubling dilutions were then made down the plate leaving the bottom two rows compound free. A 100μL volume of Huh-7 5-15 cell suspension of 2 x 10⁵ cells /mL in assay medium was added to all wells. The plates were incubated at 37°C in a 5% C0₂ atmosphere for 72 hours.

ELISA step: Growth medium was removed from the plate and the cell monolayers were washed gently once with phosphate buffered saline (PBS) prior to fixing with a 1 :1 mix of acetone:methanol for 5 minutes. The plate was washed again with PBS, blotted dry and 100μL of ELISA diluent (PBS + 0.05% Tween 20 + 2% skimmed milk powder) was added to each well. The plate was incubated at 37°C for 30 minutes and the diluent removed. Each well, except one row of the compound free wells, then received 50μL of murine monoclonal antibody, diluted to 1μg/mL, raised to a non-structural protein, more specifically NS4a. The control row received 50μl_/well of diluent alone. The plate was incubated for 2 hours, the primary antibody was removed and the cell sheets washed thoroughly with PBS + 0.05% Tween 20. Rabbit anti-mouse, polyclonal antibody conjugated to horseradish peroxidase was diluted 1/1000 and 50μL was added to all wells. Following incubation for one further hour, the secondary antibody was removed and the plate was washed thoroughly in PBS/Tween. The plate was blotted dry and 50μL of orthophenylene diamine / peroxide substrate in urea buffer was added to all wells and colour development was allowed to proceed at room temperature. The reaction was stopped by the addition of 25μL per well of 2M sulphuric acid and the plates were read spectrophotometrically at 490nm.

The ELISA solutions were removed from the plates, and the cell sheets were washed with water, blotted dry and stained with 5% carbol fuchsin. After 30 minutes the stain was removed and the plates were washed with water and allowed to air dry. Data analysis The absorbance values from all compound-free wells that had received both primary and secondary antibodies were averaged to obtain a positive control value. The mean absorbance value from the compound-free wells that had not received the primary antibody was used to provide the negative (background) control value. The readings from the duplicate wells at each compound concentration were averaged and, after the subtraction of the mean background from all values, were expressed as a percentage of the positive control signal. Grafit software was used to plot the curve of percentage inhibition against compound concentration and derive the 50% inhibitory concentration (IC₅o) for the compound.

The bioavailability of the compounds was determined according to the following in vivo tests:

Pharmacokinetics in the Male CD Sprague Rat Test compound was administered both intravenously via a caudal vein at 1 mg/kg and orally via gavage at 2mg/kg to male CD Sprague rats. The dose volume was 1 mLJkg and 2mL/kg and the dose concentration was 1mg/mL in 10% DMSO; 90% water (adjusted to pHδ with IM NaOH).

Serial blood samples (c300μL) were collected from the tail vein and the animals exsanguinated under terminal anaesthesia at the last time point.

Rats were bled at the following time-points:

Two animals were bled at 5, 30, 60, 120, 240 mins post intravenous dose Two animals were bled at 15, 45, 90, 180, 360 mins post intravenous dose Two animals were bled at 5, 30, 60, 120, 240 mins post oral dose Two animals were bled at 15, 45, 90, 180, 360 mins post oral dose

The blood was mixed in heparinised containers and centrifuged to yield plasma, which was stored frozen as soon as practicable after collection. Livers were taken from the rats sacrificed at six hours and snap frozen in liquid nitrogen as soon as practicable after collection.

Analysis of plasma samples and standards

Plasma calibration standards (0, 0.5, 1 ,2,5,10,20,50,100,200,500,1000,2000 and 5000ng/mL) were prepared by the Tecan Genesis Workstation 200 via serial dilution from a 5μg/mL plasma standard using control CD Sprague rat plasma. Standard and samples were extracted whereby ice-cold acetonitrile (300μL) was mixed with plasma (100μL) and extracted on a Whatman Unifilter 96-well protein precipitation plate. The filtrate was evaporated to dryness using heated nitrogen at 40°C. Standards and samples were reconstituted in 100μL of water: acetonitrile (95:5) containing internal standard (re/- (2S,4S,5R)-1-(4-tert-Butyl-3-methylbenzoyl)-2-isobutyl-4-(pyrazin-2-yl)-5-(1 ,3-thiazol-4- yl)pyrrolidine-2-carboxylic acid)¹ (0.25μg/mL) and analysed via LCMSMS. An injection volume of 20μl was used for this work.

LCMSMS for plasma extracts was performed on an Applied Biosystems/MDS Sciex API365 (Analyst Software Version 1.2) equipped with a Turboionspray source, an Agilent LC binary pump system and a CTCPal Autosampler. Fast gradient elution was carried out on a Phenomenex Luna C18 (2) analytical 5μm column (50mm length and 2.0mm i.d.). Elution was carried out using the following gradient: time Omin %B 5, 1min %B 100, 2.5min %B 100, 2.6min %B 5, 3.5min %B 5 where mobile phase A was 0.01% formic acid and 10mM ammonium acetate in water and mobile phase B was 0.01% formic acid in acetonitrile. Separation was at room temperature, with a mobile flow rate of 400μLJmin. The flow from the analytical column was directed into the Turboionspray source of the mass spectrometer. The first 0.75min of the gradient was directed to waste. The MS settings were as follows; negative mode ionspray voltage -3800V, source temperature 400°C, declustering potential -25V, collision energy -35V. Multiple reaction monitoring (MRM) data was acquired with the transition for test compound monitoring for the parent ion m/z 485.4 transitioning to the product ion 206.1 and the transition for the internal standard monitoring for the parent ion m/z 505.2 transitioning to the product ion 271.0. The dwell time was 200ms per ion pair. Quantitation of parent test compound was based on an internal standard method with linear regression and 1/x² weighting. The limit of detection for rat plasma was 1 ng/mL.

¹ The internal standard compound may be prepared as per the experimental section headed 'Preparation of Internal Standard for Pharmacokinetic Test' hereinbefore described.

Determination of Pharmacokinetic Parameters

Non-compartmental pharmacokinetic parameters were calculated from the parent plasma concentration versus time profile by the Excel pharmacokinetic package PK Tools_2.xlt.

Results

Compound A corresponds to the racemic compound disclosed as Example 116 in

WO2001/085720, 1-[1-(4-tert-butylphenyl)-methanoyl]-4-carbamoyl-2-isobutyl-5-thiazol-2- yl-pyrrolidine-2-carboxylic acid.

Compound B corresponds to the racemic compound disclosed as Example 101 in

WO2001/085720, 1-[1-(4-tert-butylphenyl)-methanoyl]-2-isobutyl-5-thiazol-2-yl-pyrrolidine-

2,4-dicarboxylic acid.

Compounds A and B may be made according to the processes described in

WO2001/085720.

The individual enantiomers, a and b, of Compounds A and B may be prepared as follows:

Enantiomers a & b of Compound A [Enantiomers a & b derived from rel-(2S,4S,5R)-1-[1-(4-tert-butylphenyl)- methanoyl]-4-carbamoyl-2-isobutyl-5-thiazol-2-yl-pyrrolidine-2-carboxylic acid].

Chiral;

Relative stereochemistry shown

Re/-(2S,4S,5R)-1-[1-(4-tert-butylphenyl)-methanoyl]-4-carbamoyl-2-isobutyl-5-thiazol-2-yl- pyrrolidine-2-carboxylic acid (WO2001/085720, 0.11g) was resolved using preparative normal phase HPLC over a chiral support (Column 2cm x 25cm Chiralpak AD), using heptane:ethanol (containing 0.1% tnfluoroacetic acid) 80:20 (v/v) as eluent at a flow rate of 20mL/min. Fractions containing the second eluting isomer (retention time ~ 8.8min) were combined and evaporated and then the residue was partitioned between ethyl acetate and water. The organic phase was separated, washed once with water and finally evaporated to provide Enantiomer a, of the title compound (0.032g) as a solid. ¹H NMR (CD₃OD): δ 7.78(br d, 1 H), 7.52(br d, 1 H), 7.32(dd, 2H), 7.01 (dd, 2H), 5.86(d, 1 H), 3.85(m, 1 H), 2.84(t, 1 H), 2.38-2.15(m, 3H), 2.11-1.95(m, 1 H), 1 ,28(m, 9H), 1.17(d, 3H), 1.135(d, 3H). Carboxylic acid and amide protons exchanged with solvent.

Fractions containing the first eluting isomer (retention time ~ 6.1 min) were similarly combined and evaporated and then the residue was partitioned between ethyl acetate and water. The organic phase was separated, washed once with water and finally evaporated to provide Enantiomer b of the title compound (0.035g) as a solid.

Enantiomers a & b of Compound B

[Enantiomers a & b derived from rel-(2S,4S,5R)-1-[1-(4-tert-butylphenyl)- methanoyl]-2-isobutyl-5-thiazol-2-yl-pyrrolidine-2,4-dicarboxylic acid].

Chiral;

Relative stereochemistry shown

f?e/-(2S,4S,5R)-1 -[1 -(4-tert-butylphenyl)-methanoyl]-2-isobutyl-5-thiazol-2-yl-pyrrolidine- 2,4-dicarboxylic acid (WO2001/085720, 0.06g) was resolved using preparative normal phase HPLC over a chiral support (Column 2cm x 25cm Chiralpak AD) using heptane:ethanol (containing 0.1% trifluoroacetic acid) (85:15 v/v) as eluent at a flow rate of 20mlJmin. Fractions containing the second eluting isomer (retention time ~ 8.8min) were combined and evaporated to afford Enantiomer a of the title compound, a solid. Similarly, fractions containing the first eluting isomer (retention time ~ 4.7 min) provided Enantiomer b of the title compound, a solid.

The enantiomeric compounds of the present invention have surprisingly superior IC₅₀ and pharmacokinetic properties, in particular oral bioavailability and clearance, compared to Compounds A and B and the enantiomers thereof. Accordingly, the compounds of the present invention are of great potential therapeutic benefit in the treatment and prophylaxis of HCV.

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-muscarinics, anti-leukotrienes, inhibitors of cell adhesion (e.g. ICAM antagonists), anti-oxidants (eg N-acetylcysteine), cytokine agonists, cytokine 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 (la) 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

1. Compounds of Formula (la) :

wherein: R^A represents hydroxy;

R^B represents 4-tert-butylbenzoyl further substituted in the mete-position by halo or Ci- ₃alkoxy;

R^c represents 2-thiazolyl, 5-methylthiazol-2-yl, or 4-thiazolyl;

R^D represents methyl;

X represents isobutyl;

and salts, solvates and esters thereof; provided that when R^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.

2. A compound as claimed in claim 1 selected from the group consisting of: re/-(2S,4R,5R)-1-(4-tert-Butyl-3-methoxybenzoyl)-2-isobutyl-4-acetyl-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-1-(4-tert-Butyl-3-bromobenzoyl)-2-isobutyl-4-acetyl-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-1 -(4-tert-Butyl-3-chlorobenzoyl)-2-isobutyl-4-acetyl-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-1-(4-tert-Butyl-3-methoxybenzoyl)-2-isobutyl-4-acetyl-5-(5-methyl-1 ,3- thiazol-2-yl)pyrrolidine-2-carboxylic acid; and re/-(2S,4R,5R)-1-(4-tert-Butyl-3-methoxybenzoyl)-2-isobutyl-4-acetyl-5-(1 ,3-thiazol-4- yl)pyrrolidine-2-carboxylic acid and salts, solvates and esters, and individual enantiomers thereof.

3. 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 (la) as claimed in claim 1.

4. A method as claimed in claim 3 wherein the viral infection is HCV.

5. A method as claimed in claim 3 in which the compound is administered in an oral dosage form.

6. A compound of

wherein:

R^A represents hydroxy;

R^B represents 4-tert-butylbenzoyl further substituted in the mete-position by halo or Ci ₃alkoxy;

R^c represents 2-thiazolyl, 5-methylthiazol-2-yl, or 4-thiazolyl;

R^D represents methyl;

X represents isobutyl;

and salts, solvates and esters thereof; provided that when R^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; for use in medical therapy.

7. A compound as claimed in claim 6 wherein the medical therapy is the treatment of viral infection.

8. A compound as claimed in claim 7 wherein the viral infection is HCV.

9. Use of a compound of

wherein:

R^A represents hydroxy; R^B represents 4-tert-butylbenzoyl further substituted in the mete-position by halo or C^ ₃alkoxy;

R^c represents 2-thiazolyl, 5-methylthiazol-2-yl, or 4-thiazolyl;

R^D represents methyl;

X represents isobutyl;

and salts, solvates and esters thereof; provided that when R^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; in the manufacture of a medicament for the treatment of viral infection.

10. Use as claimed in claim 9, wherein the viral infection is HCV.

11. A pharmaceutical formulation comprising a compound of Formula (la) as claimed in claim 1 in conjunction with a pharmaceutically acceptable diluent or carrier.

12. A process for the preparation of a compound of Formula (la) as defined in claim 1 comprising deprotection of a

in which R^A is an alkoxy, benzyloxy or silyloxy group; R^B represents 4-tert-butylbenzoyl further substituted in the mete-position by halo or d-₃alkoxy; R^c represents 2-thiazolyl or 5-methylthiazol-2-yl; R^D represents methyl; and X represents isobutyl.