WO2007039144A1

WO2007039144A1 - Pyrrolidine derivatives for treating viral infections

Info

Publication number: WO2007039144A1
Application number: PCT/EP2006/009236
Authority: WO
Inventors: David Haigh; Charles David Hartley; Peter David Howes; Linos Lazarides; Fabrizio Nerozzi; Stephen Allan Smith
Original assignee: Glaxo Group Limited
Priority date: 2005-09-23
Filing date: 2006-09-21
Publication date: 2007-04-12
Also published as: GB0519485D0

Abstract

Anti-viral agents of Formula (Ia): wherein A represents hydroxy; D represents 3-bromo-4-tert-butylphenyl or 5-bromo-4-tert-butyl-2-fluorophenyl; E represents 1,3-thiazol-2-yl or 5-methylisoxazol-3-yl; G represents methoxymethyl; J represents 1,3-thiazol-4-ylmethyl or 1H-pyrazol-1-ylmethyl; and salts, solvates and esters thereof; provided that when A is esterified to form -OR where R is selected from branched chain alkyl, then R is other than tert-butyl, processes for their preparation and their use in HCV treatment are provided.

Description

PYRROLIDINE DERIVATIVES FOR TREATING VIRAL INFECTIONS

FIELD OF THE INVENTION

The present invention relates to novel C(4)-methoxymethyl acyl pyrrolidine derivatives useful as anti-viral agents. Specifically, the present invention involves novel Hepatitis C Virus (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 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 5¹ noncoding region¹ RNA- A Publication of the RNA Society. 1 (5): 526-537, 1995 JuI.). 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 "3¹ 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 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.

Although the predominant HCV genotype worldwide is genotype 1 , this itself has two main subtypes, denoted 1a and 1 b. As seen from entries into the Los Alamos HCV database (www.hcv.lanl.gov) (Table 1 ) there are regional differences in the distribution of these subtypes: while genotype 1a is most abundant in the United States, the majority of sequences in Europe and Japan are from genotype 1 b. Table 1

The prevalance of genotype 1a in some regions makes it highly desirable to identify an anti-viral agent that is able to inhibit both genotype 1a and genotype 1 b. This means a wider patient pool would be able to benefit from treatment with the same agent.

Based on the foregoing, there exists a significant need to identify synthetic or biological compounds for their ability to inhibit replication of both genotype 1a and genotype 1b of HCV.

PCT publication number WO2004/037818 generically discloses certain compounds, including certain acyl pyrrolidine compounds, having HCV inhibitory activity. The assay is directed to the 1 b genotype. The compounds disclosed have the formula (I)

wherein:

A represents hydroxy;

D represents aryl or heteroaryl;

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

G represents hydrogen or C_1-6alkyl optionally substituted by one or more substituents selected from halo, OR¹, SR¹, C(O)NR²R³, CO₂H, C(O)R⁴, CO₂R⁴, NR²R³, NHC(O)R⁴,

NHCO₂R⁴, NHC(O)NR⁵R⁶, SO₂NR⁵R⁶, SO₂R⁴, nitro, cyano, aryl, heteroaryl and heterocyclyl;

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

R² and R³ are independently selected from hydrogen, C^alkyl, 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, Ci_-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/f-butyl.

Surprisingly, it has now been found that compounds according to the present invention, generically disclosed in WO2004/037818, and having a specific substitution pattern, exhibit improved properties over those compounds specifically disclosed in WO2004/037818.

SUMMARY OF THE INVENTION

The present invention involves C(2)-heteroarylmethyl-C(4)-methoxymethyl 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 provides at least one chemical entity chosen from compounds of Formula (Ia) :

wherein:

A represents hydroxy;

D represents 3-bromo-4-terf-butylphenyl or 5-bromo-4-ferf-butyl-2-fluorophenyl;

E represents 1 ,3-thiazol-2-yl or 5-methylisoxazol-3-yl;

G represents methoxymethyl;

J represents 1 ,3-thiazol-4-ylmethyl or 1 H-pyrazol-1-ylmethyl;

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

In one aspect, the relative stereochemistry of racemic compounds of Formula (Ia), is represented by Formulae (Ip) or (Iq): r ⁽e^IPla⁾tive stereochemistry ™ relative stereochemistry

wherein A, D, E, G and J are as defined above for Formula (Ia).

In a further aspect, the absolute stereochemistry of chiral compounds of Formula (Ia) is represented by Formulae (Ipp) or (Iqq):

ute stereochemistry

wherein A, D, E, G and J are as defined above for Formula (Ia).

The following substituent groups are preferred, where applicable, in respect of each of Formulae Ia, Ip, Ipp Iq and Iqq:

In one aspect, A represents hydroxy (that is, not esterified).

In one aspect J represents 1 ,3-thiazol-4-ylmethyl. In another aspect J represents 1 H- pyrazol-1-ylmethyl.

In one aspect D represents 3-bromo-4-terf-butylphenyl. In another aspect D represents or 5-bromo-4-terf-butyl-2-fluorophenyl.

In one aspect E represents 1 ,3-thiazol-2-yl. In another aspect E represents 5- methylisoxazol-3-yl.

In one aspect, the compounds of Formula (Ia) are represented by compounds of Formula

(Ip)-

(IPP).

It is to be understood that the present invention covers all combinations of aspects, suitable, convenient and preferred groups described herein. The chemical entities of the present invention exhibit an improved genotype-1a/1 b profile against HCV polymerase, and therefore have the potential to achieve efficacy in man over a broad patient population.

The term 'genotype-1a/1 b profile' means potency as an inhibitor of HCV polymerase enzyme in wildtype HCV of the 1a genotype and of the 1 b genotype. High potency in both genotypes is considered to be advantageous.

There is provided as a further aspect of the present invention at least one chemical entity chosen from compounds of Formula (Ia) and pharmaceutically acceptable salts, solvates or esters 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 at least one chemical entity chosen from compounds of Formula (Ia) and pharmaceutically acceptable salts, solvates or esters 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 at least one chemical entity chosen from compounds of Formula (Ia) and pharmaceutically acceptable salts, solvates or esters 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.

In one aspect, chemical entities useful in the present invention may be chosen from compounds of Formula (Ia) selected from the group consisting of: re/-(2R,4S,5R)-1-(3-Bromo-4-terf-butylbenzoyl)-4-(methoxymethyl)-5-(5-methyl-isoxazol-3- yl)-2-(1 ,3-thiazol-4-ylmethyl)pyrrolidine-2-carboxylic acid; re/-(2R,4S,5R)-1 -(3-Bromo-4-tert-butylbenzoyl)-4-(methoxymethyl)-2-(1 H-Pyrazol-1 - ylmethyl)-5-(1 ,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid; re/-(2R,4S,5R)-1-(5-Bromo-4-fe/t-butyl-2-fluorobenzoyl)-4-(methoxy-methyl)-2-(1H- pyrazol-1-ylmethyl)-5-(1 ,3-thiazol-2-yl)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 pharmaceutically acceptable salts of the compounds of formula (Ia). Suitable pharmaceutically acceptable salts of the compounds of formula (Ia) 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 (Ia), for example hydrates.

The present invention also relates to pharmaceutically acceptable esters of the compounds of Formula (Ia), 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), alkoxycarbonylalkyl (e.g. methoxycarbonylmethyl), acyloxyalkyl (e.g. pivaloyloxymethyl), 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.

In one aspect, the compound of Formula (Ia) is in the form of parent compound, a salt or a solvate.

As used herein, the term "pharmaceutically acceptable" used in relation to an ingredient (active ingredient such as an active ingredient, a salt thereof or an excipient) which may be included in a pharmaceutical formulation for administration to a patient, refers to that ingredient being acceptable in the sense of being compatible with any other ingredients present in the pharmaceutical formulation and not being deleterious to the recipient thereof.

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

in which A' is a protected hydroxy group, for example an alkoxy, benzyloxy or silyloxy, for example tri-(C_1-4alkyl)-silyloxy group, and D, E, G and J are as defined above for Formula (Ia), by deprotection. 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.

For example when A' is te/f-butoxy, and D, E, G and J are as defined above for Formula (Ia), by treatment with an appropriate acid, for example trifluoroacetic acid. Optionally, the reaction is carried out in a solvent, for example dichloromethane. In one aspect, the temperature is in the range 0 to 5O⁰C, suitably 20 to 30⁰C.

For example when A' is benzyloxy, and D, E, G and J are as defined above for Formula (Ia), 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. Suitably, the temperature is in the range 0 to 50⁰C. For example when A' is allyloxy, and D, E, G and J are as defined above for Formula (Ia), 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 tri(methyl)silyloxy, and D, E, G and J are as defined above for Formula (Ia), 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 (Ia) or (II) may be prepared by reaction of a compound of Formula

in which A" is hydroxy or an alkoxy, benzyloxy or a silyloxy, for example a tri-(d^alkyl)- silyloxy, group, and E, G, and J are as defined above for Formula (Ia); 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 (Ia). Preferably the reaction is carried out in a suitable solvent, for example dichloromethane, in the presence of a suitable base, for example triethylamine. Preferably, the temperature is in the range 0 to 50⁰C, more preferably 20 to 30⁰C. Optionally, the reaction may be carried out at the reflux temperature of the solvent. Compounds of Formula (Ia) or (II) may also be prepared by methylation of a compound of formula (IV)

in which A" is as defined above for Formula (III), and G' represents hydroxymethyl using a suitable base for example sodium hydride or sodium te/f-butoxide and a suitable methylating agent such as methyl iodide. Preferably the reaction is carried out in a suitable solvent, for example dimethylformamide or acetonitrile. Preferably the reaction is carried out at a temperature in the range 0 to 50⁰C, suitably 20 to 30⁰C.

Compounds of Formula (IV) may be prepared by appropriate manipulation of a compound of Formula (V)

in which A" is as defined above for Formula (III), and D, E and J are as defined above for Formula (Ia), and L represents CHO or CO₂Y wherein Y represents hydrogen or alkyl. For example, by reduction of a compound of Formula (V) in which L represents CHO or CO₂Y wherein Y represents hydrogen or alkyl, using a suitable reducing agent, for example lithium borohydride, lithium triethylborohydride, sodium borohydride, sodium triacetoxyborohydride, borane/dimethyl sulfide complex or lithium aluminium hydride, or suitable combinations thereof, in a suitable solvent or mixture thereof for example tetrahydrofuran or methanol. Preferably the reaction is carried out at a temperature in the range -78 to -20⁰C.

A compound of Formula (V) in which L represents CO₂Y wherein Y represents hydrogen may be prepared from a compound of Formula (V) in which L represents CO₂Y wherein Y represents alkyl. For example, a compound of Formula (V) in which L represents CO₂Me may be converted into a compound of Formula (V) in which L represents CO₂H by hydrolysis, for example base catalysed hydrolysis using a suitable base such as sodium methoxide in a suitable solvent such as methanol.

A compound of Formula (V) in which L represents CHO or CO₂Y wherein Y represents hydrogen or alkyl may be prepared from a compound of Formula (Vl)

in which L represents CHO or CO₂Y wherein Y represents hydrogen or alkyl, and A", E, and J are as defined above for Formula (III); 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 (Ia). Preferably the reaction is carried out in a suitable solvent, for example dichloromethane, in the presence of a suitable base, for example triethylamine. Preferably the reaction is carried out at a temperature in the range 0 to 5O⁰C, suitably 20 to 30⁰C. Optionally the reaction may be carried out under reflux.

A compound of Formula (V) in which A" is hydroxy, may be converted to a compound of Formula (V) in which A" is an alkoxy, benzyloxy or silyloxy group by standard hydroxy protecting techniques. Similarly, a compound of Formula (V) in which A" is an alkoxy, benzyloxy or silyloxy group, may be converted to a compound of Formula (V) in which A" is hydroxy by standard deprotecting techniques. 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 (Vl) may be prepared by reaction of a compound of Formula (VII)

in which E and J are as defined above for Formula (Ia) and A" is as defined above for Formula (III) with a compound of Formula (VIII)

wherein L represents CHO or CO₂Y wherein Y represents hydrogen or alkyl. 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 (VII) and Formula (VIII) in a suitable solvent, for example toluene, xylene or acetonitrile in the absence of a catalyst.

A compound of Formula (Vl) may also be prepared in a one pot synthesis by reaction of a compound of Formula (X) (see below for structure) with a compound of Formula (VIII) and a compound of Formula E-CHO. 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. Preferably the reaction is carried out at a temperature in the range 0 to 50⁰C, suitably 20 to 30⁰C. Optionally a drying agent is used in the process, for example molecular sieves.

A compound of Formula (III) may be prepared by appropriate manipulation of a compound of Formula (IX)

in which G' represents hydroxymethyl, A" is as defined above for Formula (III), and E, and

J are as defined above for Formula (Ia), by first protecting the N-atom of the pyrrolidine ring with a suitable N-protecting group, for example benzyloxycarbonyl (CBZ) or t- butoxycarbonyl. In a similar manner to that described above in relation to conversion of compounds of Formula (IV) into compounds of Formula (II), an N-protected compound of Formula (IX) may be converted into a compound of Formula (III), in which G represents methoxymethyl and the N-atom is protected, by methylation. Deprotection of the N-atom by standard procedures results in the compound of Formula (III).

Compounds of Formula (IX) may be prepared by reduction of an optionally N-protected compound of Formula (Vl) in which L represents CO₂Y and Y represents alkyl, using a suitable reducing agent, for example lithium borohydride or sodium borohydride, in a suitable solvent for example tetrahydrofuran. Deprotection of the N-atom by standard procedures results in the compound of Formula (IX). For example, when the N-protecting group is CBZ, deprotection may be achieved by catalytic hydrogenolysis. For example, when the N-protecting group is f-butoxycarbonyl, deprotection may be achieved by treatment with a suitable acid, for example trifluoroacetic acid.

Compounds of Formula (VII) may be prepared by reaction of a compound of Formula (X)

H₂N^/^C0A"

\ (X) ^J in which J is as defined above for Formula (Ia) and A" is as defined above for Formula (III) with a compound of Formula E-CHO in which E is as defined above for Formula (Ia) in the presence of a suitable drying agent, for example magnesium sulphate, in a suitable solvent, for example dichloromethane. Preferably the reaction is carried out at a temperature in the range 0 to 50⁰C. Compounds of Formula (X) in which J is 1H-pyrazol-1-ylmethyl, and A" is hydroxy, may be prepared by treatment of a compound of (Xl)

in which J is I H-pyrazol-1-ylmethyl, and M is a metal cation, for example potassium, with a suitable acid, for example 10% aqueous hydrochloric acid, in the presence of Amberlyst 120 (H⁺).

Compounds of Formula (X) in which J is 1 H-pyrazol-1-ylmethyl, and A" is an alkoxy, benzyloxy or tri-(Ci.₄alkyl)-silyloxy group, may be prepared by treatment of a compound of Formula (X) in which J is 1 H-pyrazol-1-ylmethyl, and A" is hydroxy, by conventional esterification or protecting group procedures. For example, a compound of Formula (X) in which J is 1 H-pyrazol-1-ylmethyl, and A" is tert-butoxy may be prepared by treatment of a compound of Formula (X) in which J is I H-pyrazol-1-ylmethyl, and A" is hydroxy, with an appropriate te/f-butyl transfer agent, such as terf-butylacetate in the presence of a suitable acid catalyst, such as 70% perchloric acid.

Compounds of Formula (X) in which A" is hydroxy are known in the art.

Compounds of Formula (Xl) in which J is I H-pyrazol-1-ylmethyl, may be prepared by reaction of the compound of Formula (XII)

with 1 H-pyrazole, in the presence of a suitable base, for example potassium carbonate when M is potassium, and in the presence of a suitable solvent, such as aqueous acetonitrile. Preferably the reaction is carried out at a temperature in the range 50-70⁰C, suitably 60⁰C.

Compounds of Formula (X) in which J is 1 ,3-thiazol-4-ylmethyl and A" is an alkoxy, benzyloxy or tri-(C_1-4alkyl)-silyloxy group, may be prepared by treatment of a compound of

Formula (XIII)

in which J is 1 ,3-thiazol-4-ylmethyl and A" is an alkoxy, benzyloxy or tri-(C_1-4alkyl)-silyloxy group with an acid, for example 15% aqueous citric acid. Preferably, the reaction is carried out in a suitable solvent or mixture thereof, for example THF or water. Compounds of Formula (XIII) may be prepared by reaction of a compound of Formula

in which A" is an alkoxy, benzyloxy or tri-(C_1-4alkyl)-silyloxy group with a compound of Formula J-hal in which J is 1 ,3-thiazol-4-ylmethyl, and hal is a halo atom, preferably chloro or bromo. Preferably, the reaction is carried out in the presence of a suitable base such as potassium £-butoxide. Preferably, the reaction is carried out in a suitable solvent, for example THF. Preferably the reaction is carried out in the presence of a suitable catalyst, for example lithium iodide. Preferably the reaction is carried out at a temperature in the range -10⁰C to room temperature, suitably at 0⁰C.

Compounds of Formula D-C(O)-hal in which D is 3-bromo-4-tert-butylphenyl may be prepared by reaction of the co

with a suitable acid halide forming reagent, for example oxalyl chloride or thionyl chloride. In one aspect, the reaction is carried out in the presence of a suitable catalyst, for example dimethylformamide or diethylformamide. Optionally, the reaction is carried out in a suitable solvent, for example dichloromethane, at a temperature in the range 0 to 50⁰C, for example 20 to 30⁰C. In an alternative aspect, the reaction is carried out using thionyl chloride under reflux.

Compounds of Formula D-C(O)-hal in which D is 5-bromo-4-te/f-butyl-2-fluorophenyl may be prepared by reaction of the

with a suitable acid halide forming reagent, for example oxalyl chloride or thionyl chloride. In one aspect, the reaction is carried out in the presence of a suitable catalyst, for example dimethylformamide or diethylformamide. Optionally, the reaction is carried out in a suitable solvent, for example dichloromethane, at a temperature in the range 0 to 5O⁰C, for example 20 to 30⁰C. In an alternative aspect, the reaction is carried out using thionyl chloride under reflux.

The compound of Formula (XVI) may be prepared by reaction of the compound of Formula (XVII) )

with a suitable brominating agent, for example N-bromosuccinimide. In one aspect, the reaction is carried in the presence of a suitable acid, for example trifluoroacetic acid and concentrated sulphuric acid. In one aspect, the reaction is carried out at a temperature in the range 20-50⁰C.

The compound of Formula (XVII) may be prepared by treatment of the compound of

Formula (XVIII)

(XVIII)

with a suitable oxidising agent, for example potassium permanganate. In one aspect the reaction is carried out in a suitable solvent, for example pyridine and water. In one aspect, the reaction is carried out under reflux.

The compound of Formula (XVIII) may be prepared by reaction of the compound of Formula (XIX)

with a suitable hydride source, for example triethylsilane, optionally in the presence of a suitable catalyst such as palladium (II) acetate, optionally in the presence of a co-ligand such as 1 ,3-bis(diphenylphosphino)propane. In one aspect the reaction is carried out in a suitable solvent, for example dimethylformamide. In one aspect, the reaction is carried out at a temperature in the range 50-80⁰C.

The compound of Formula (XIX) may be prepared by reaction of the compound of Formula (XX)

with a suitable base, for example sodium hydride, in the presence of a suitable trifluoromethylsulfonylating agent, for example N-phenyl-bis(trifluoromethanesulfonimide). In one aspect the reaction is carried out in a suitable solvent, for example dimethylformamide.

The compound of Formula (XX) may be prepared by reaction of the compound of Formula

with an alkylating agent, for example te/f-butyl chloride, in the presence of a catalyst, for example zinc chloride. In one aspect, the reation is carried out at a temperature in the range 50-90⁰C.

Compounds of Formula (VIII), (XII), (XIV), (XV), (XXI), J-hal and E-CHO are known in the art or may be prepared by standard literature procedures.

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

It will be appreciated that compounds of Formula (Ia), (II), (III), (IV), (V), (Vl) and/or (IX) which exist as diastereoisomers may optionally be separated by techniques well known in the art, for example by column chromatography.

It will also be appreciated that the present invention provides a method for the interconversion of C(4)-epimers of a compound of formula (V) or (Vl) in which L represents CHO or CO₂Y wherein Y represents hydrogen or alkyl, A" is as defined above for formula (III), E, and J are as defined above for formula (Ia). For example the rel-(2R, 4S, 5R)-diastereoisomer of a compound of formula (V) and/or (Vl) may be converted into the rel-(2R, 4R, 5R)-diastereoisomer where appropriate. Such epimerisation of these rel- (4S, 5R)-diastereoisomers into the corresponding rel-(4R, 5R)-diastereoisomers may be accomplished by treatment of a compound of formula (V) and/or (Vl) with a suitable base, in the presence of a suitable solvent. For example the conversion of the rel-(4S, 5R)- diastereoisomer of a compound of Formula (V) when L represents CO₂Me into the rel-(4R, 5R)-diastereoisomer is accomplished by treatment of the rel-(4S, 5R)-diastereoisomer with a suitable base, such as sodium methoxide, in the presence of a suitable solvent, such as methanol.

It will be appreciated that racemic compounds of Formula (Ia), (II), (III), (IV), (V), (Vl) and/or (IX) 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 (Ia), (II), (III), (IV), (V), (Vl) and/or (IX) may be resolved by chiral preparative HPLC. Alternatively, racemic compounds of Formula (Ia), (II), (III), (IV), (V), (Vl) and/or (IX) which contain an appropriate acidic or basic group, such as a carboxylic acid group or amine group may be resolved by standard diastereoisomeric salt formation with a chiral base or acid reagent respectively as appropriate. Such techniques are well established in the art. For example, a racemic compound of Formula (Vl) where L is CO₂Me may be resolved by treatment with a chiral acid such as (R)-(-)-1 ,1'- binaphthyl-2,2'-diyl-hydrogen phosphate, in a suitable solvent, for example dichloromethane, isopropanol or acetonitrile. The enantiomer of Formula (Vl) may then be obtained by treating the salt with a suitable base, for example triethylamine, in a suitable solvent, for example methyl tert-butyl ether. Individual enantiomers of Formula (II), (III), (IV), (V), (Vl) and/or (IX) may then be progressed to an enantiomeric compound of Formula (Ia) by the chemistry described above in respect of racemic compounds.

It will also be appreciated that individual enantiomeric compounds of Formula (III), (Vl) and/or (IX) may be prepared by general methods of asymmetric synthesis using, where appropriate, chiral auxiliaries or chiral catalytic reagents and additionally performing any suitable functional group interconversion step as hereinbefore described, including the addition or removal of any such chiral auxiliary. Such general methods of asymmetric synthesis are well known in the art and include, but are not restricted to, those described in "Asymmetric Synthesis," Academic Press, 1984 and/or "Chiral Auxiliaries and Ligands in Asymmetric Synthesis", Wiley, 1995. For example, suitable general chiral auxiliaries include chiral alcohols such as menthol or 1-phenylethanol; chiral oxazolidinones such as 4-benzyloxazolidin-2-one or 4-isopropyloxazolidin-2-one; chiral sultams such as camphor sultam; or chiral amines such as 1-phenylethylamine or 2-amino-2-phenylethanol. Suitable general chiral catalytic reagents include chiral basic amines and chiral ligands such as N-methylephedrine, 1-phenyl-2-(1-pyrrolidinyl)-1-propanol, 3-(dimethylamino)- 1 ,7,7-trimethylbicyclo[2.2.1 ]-heptan-2-ol, 3,4-bis(diphenylphosphanyl)-1 -(phenylmethyl)- pyrrolidine, chinchonine, chinchonidine, sparteine, hydroquinine or quinine, BINAP or chiral bis(oxazoline) (BOX) ligands and derivatives, optionally in the presence of a metal salt, for example M_mX_x where M is silver, cobalt, zinc, titanium, magnesium, or manganese, and X is halide (for example chloride or bromide), acetate, trifluoroacetate, p- toluenesulfonate, trifluoromethylsulfonate, hexafluorophosphate or nitrate, and _m and _x are 1 , 2, 3 or 4, and optionally in the presence of a base, for example triethylamine. All of these chiral auxiliaries or chiral catalytic reagents are well described in the art. General illustrative examples of the preparation of various chiral pyrrolidines by asymmetric synthesis using chiral auxiliaries or chiral catalytic reagents include, but are not limited to, those described in Angew. Chem. Int. Ed., (2002), 41 : 4236; Chem. Rev., (1998), 98: 863; J. Am. Chem. Soc, (2002), 124: 13400; J. Am. Chem. Soc, (2003), 125: 10175; Org. Lett., (2003), 5, 5043; Tetrahedron, (1995), 51 : 273; Tetrahedron: Asymm., (1995), 6: 2475; Tetrahedron: Asymm., (2001 ), 12: 1977; Tetrahedron: Asymm., (2002), 13: 2099 and Tet. Lett., (1991 ), 41 : 5817.

In a particular aspect, a chiral pyrrolidine compound of Formula (Via)

in which L¹ represents CO₂Y or CO₂Y¹ wherein Y represents hydrogen or alkyl, Y¹ represents a chiral auxiliary, and A", E, and J are as defined above for Formula (Vl), and ^* denotes an enantioenriched chiral centre can be prepared by reaction of a compound of Formula (VII), as hereinbefore defined, with a compound of Formula (Villa)

[| (Villa) in which L¹ represents a chiral ester group CO₂Y¹ wherein Y¹ represents a chiral auxiliary and thereafter optionally carrying out any conversion of CO₂Y¹ into CO₂Y by standard methods for removal of chiral auxiliaries. Such chiral ester CO₂Y¹ may be derived from a chiral alcohol Y¹OH, for example menthol, by standard esterification techniques. Preferably, the reaction of a compound of Formula (VII) with a compound of Formula (Villa) 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 (VII) and (Villa) in a suitable solvent, for example toluene, xylene or acetonitrile in the absence of a catalyst. The preparation of compounds analogous to those of Formula (Via) and (Villa) is described in Tetrahedron: Asymm., (1995), 6: 2475.

In a further aspect, a chiral (VIb)

in which L represents CO₂Y wherein Y represents hydrogen or alkyl, and A", E, and J are as defined above for Formula (Vl), and ^* denotes an enantioenriched chiral centre can be prepared by reaction of a compound of Formula (VII) with a compound of Formula (VIII) as herein before defined, under asymmetric reaction conditions. It will be appreciated by those skilled in the art that such asymmetric reaction conditions may be afforded by, for example, the inclusion in the reaction mixture of a chiral catalytic reagent as herein before defined. In one aspect, the reaction is carried out in the presence of a suitable chiral catalytic reagent, for example (-)-N-methylephedrine, and a suitable metal salt, for example manganese (II) bromide, in a suitable solvent, for example acetonitrile. Preferably the reaction is carried out at a temperature in the range -30⁰C to room temperature, suitably at -20⁰C.

In an alternative aspect, the reaction is carried out in the presence of a suitable chiral catalytic reagent, for example S-BINAP, and a suitable metal salt, for example silver acetate, in the presence of a suitable base, for example diisopropylethylamine, in a suitable solvent, for example acetonitrile optionally co-solvated with toluene. Preferably the reaction is carried out at a temperature in the range -15°C to room temperature, suitably at -5°C.

Optionally, the major chiral diastereoisomer of a compound of Formula (Via) or Formula (VIb) arising from such an asymmetric reaction may be further enantioenriched by conventional purification techniques well known in the art, for example by chromatography, or by fractional crystallisation. A favourable crystallisation method is the fractional crystallisation of a salt of the major chiral diastereoisomer, for example the hydrochloride salt or the (R)-(-)-1 ,1 '-binaphthyl-2,2'-diyl-hydrogen phosphate salt. The hydrochloride salt of a compound of Formula (Via) or Formula (VIb) may be prepared by treating a compound of Formula (Via) or Formula (VIb) with anhydrous hydrogen chloride in a suitable solvent, for example diethyl ether. Preferably the reaction is carried out at a temperature in the range -10 to 10°C.

The (R)-(-)-1 ,1 '-binaphthyl-2,2'-diyl-hydrogen phosphate salt of a compound of Formula (Via) or Formula (VIb) may be prepared as herein before described for the resolution of a racemic compound of Formula (Vl).

Optional removal of a chiral auxiliary from a group in which L¹ represents CO₂Y¹ to afford a group in which L¹ represents CO₂Y is readily accomplished by standard methods, for example treatment with a hydrolytic reagent such as sodium hydroxide or an alkoxide such as sodium methoxide as appropriate, in a suitable solvent such as methanol.

Optionally, a chiral compound of Formula (Via) or Formula (VIb) may be converted into a chiral compound of Formula (IX) in which G' represents hydroxyalkyl, and A", E, and J are as defined above for Formula (III) by treatment with suitable reagents for accomplishing the functional group interconversion of the group L or L¹ into group G'. For example a compound of Formula (Via) in which L¹ represents CO₂Y¹ and Y¹ is as defined above may be treated with a suitable reducing agent, for example lithium aluminium hydride, in a suitable solvent, for example tetrahydrofuran.

Optionally, a chiral compound of Formula (Via) or Formula (VIb) may be converted into a chiral compound of Formula (IV) in which G' represents hydroxyalkyl, by first acylating the pyrrolidine nitrogen atom as described above for the transformation of a compound of Formula (Vl) into a compound of Formula (V) and then subsequently by treatment with suitable reagents for accomplishing the functional group interconversion of the group L or L¹ into group G' as described above for the transformation of a compound of Formula (Via) or Formula (VIb) into a chiral compound of Formula (IX).

It will be appreciated that, with suitable additional conversion steps as described above, chiral compounds of Formula (Ia), (II), (IV) and/or (V) may be prepared from chiral compounds of Formula (III), (Vl) and (IX).

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

It will be appreciated by those skilled in the art that when solvents are used in reactions it is desirable to use anhydrous solvents. It is further desirable to conduct reactions under an inert atmosphere, for example under nitrogen or argon, where appropriate.

Intermediate 1 3-Bromo-4-ferf-butylbenzoyl chloride

Alternative Method A A solution of 3-bromo-4-terf-butylbenzoic acid (584 mg) in dichloromethane (10 mL) was treated with oxalyl chloride (0.396 mL) and diethyl formamide (1 drop). The mixture was stirred under nitrogen at 20⁰C for 4.5 hours. The mixture was concentrated in vacuo to give the title compound as an oil. Alternative Method B A solution of 3-bromo-4-terf-butylbenzoic acid (1.0 g) in thionyl chloride (12 mL) was stirred under nitrogen at reflux for 2 hours. The mixture was concentrated in vacuo, then twice re-evaporated from dichloromethane to give the title compound as an oil. ¹H NMR (CDCI₃): δ 8.32 (d, 1 H), 7.99 (dd, 1 H), 7.60 (d, 1 H) and 1.55 (s, 9H).

Intermediate 2

2-[N-(Diphenylmethylene)amino]-3-(1,3-thiazol-4-yl)propanoic acid, tert-butyl ester

Part A

To a cooled (ice-bath) solution of 2-[N-(diphenylmethylene)amino]ethanoic acid, tert-butyl ester (J. Org. Chem., 1982, 47, 2663; 42.3 g, 143 mmol) in dry THF (450 mL) under an atmosphere of nitrogen, was added a 1 M solution of potassium t-butoxide in THF (146 mL) dropwise (dropping funnel) over 25 minutes. The mixture was allowed to stir for a further 45 minutes in the ice-bath. Part B Independently during this time, 4-(chloromethyl)-1 ,3-thiazole hydrochloride (25.5 g, 150 mmol) was freshly converted to the free base as follows: The hydrochloride was mixed with dichloromethane (500 mL) and washed with a 5% w/v aqueous sodium bicarbonate solution (375 mL). The organic layer was separated, dried over sodium sulphate and carefully evaporated (rotary evaporator; 80 torr, water bath 25°C) to give the free base. Part C

The 4-(chloromethyl)-1 ,3-thiazole (formed in Part B) was dissolved in THF (100 mL) and added dropwise (dropping funnel) over 30 minutes to the reaction mixture from Part A, keeping the reaction at ice-bath temperature. Solid anhydrous lithium iodide (1 g, 7.5 mmol) was added directly to the reaction mixture 5 minutes after addition of the alkylating agent had started. The dropping funnel was rinsed with further dry THF (50 mL) which was added to the reaction. The reaction was stirred at ice-bath temperature for 45 minutes, allowed to warm to room temperature over 30 minutes and was stirred at room temperature for an additional 2.5 hours before being partitioned between a mixture of saturated brine (400 mL), water (200 mL) and ethyl acetate (800 mL). The organic layer was separated and the aqueous layer re-extracted with further ethyl acetate (2 x 300 mL). The combined organic layers were dried over sodium sulphate and evaporated to give the title compound (57.8 g, crude) which was used without further purification. ¹H NMR (CDCI₃): δ 8.65 (d, 1 H), 7.55-7.62 (m, 2H), 7.2-7.55 (m, 6H), 7.05 (d, 1 H), 6.78- 6.87 (m, 2H), 4.36-4.41 (m, 1 H), 3.47-3.54 (m, 1 H), 3.36-3.44 (m, 1 H) and 1.44 (s, 9H).

Intermediate 3

2-Amino-3-(1,3-thiazol-4-yl)propanoic acid, tert-butyl ester

To a solution of 2-[N-(diphenylmethylene)amino]-3-(1 ,3-thiazol-4-yl)propanoic acid, tert- butyl ester (prepared in a similar manner to that described in Intermediate 2; 20 g) in THF

(150 mL) under argon was added a solution of citric acid in water (15% w/v, 150 mL). The mixture was stirred at room temperature for 6 hours, left overnight and then the majority of the THF was removed under reduced pressure (rotary evaporator; water bath at 25⁰C) and 1 M aqueous hydrochloric acid (60 mL) added. The mixture was extracted with diethyl ether (2 x 200 mL) and the combined ether extracts back extracted with water (50 mL). The combined aqueous layers were extracted with further diethyl ether (100 mL). All of the ether layers were discarded. The aqueous layer was then carefully adjusted to pH 9.5 with potassium carbonate, brine (100 mL) was added and the mixture extracted with diethyl ether (4 x 200 mL). These combined ether layers were dried over sodium sulphate. Removal of the solvent under reduced pressure gave the title compound, an oil. ¹H NMR (CDCI₃): δ 8.77 (d, 1 H), 7.08 (d, 1 H), 3.77-3.85 (m, 1 H), 3.22-3.32 (m, 1 H), 3.02- 3.13 (m, 1 H) and 1.42 (s, 9H). Amine protons not observed.

Intermediate 4

2-[N-(5-Methylisoxazol-3-ylmethylene)amino]-3-(1 ,3-thiazol-4-yl)propanoic acid, tert- butyl ester

To a solution of 2-amino-3-(1 ,3-thiazol-4-yl)propanoic acid, te/f-butyl ester (prepared in a similar manner to that described in Intermediate 3; 3.718 g) in dichloromethane (20 mL) was added a solution of 5-methylisoxazole-3-carboxaldehyde (2.716 g) in dichloro- methane (35 mL). The mixture was heated at 40°C for 2 hours, cooled to 20⁰C and concentrated in vacuo to give the title compound as an oil.

¹H NMR (CDCI₃): δ 8.75 (d, 1 H), 8.11 (s, 1 H), 7.01 (d, 1 H), 6.45 (s, 1 H), 4.44 (dd, 1 H), 3.57 (dd, 1 H), 3.30 (dd, 1 H), 2.44 (s, 3H) and 1.45 (s, 9H).

Intermediate 5 re/-(2R,4S,5R)-5-(5-methylisoxazol-3-yl)-2-(1,3-thiazol-4-ylmethyl)pyrrolidine-2,4- dicarboxylic acid, 2-tert-butyl ester, 4-methyl ester

shown

To a solution of 2-[N-(5-methylisoxazol-3-ylmethylene)amino]-3-(1 ,3-thiazol-4-yl)propanoic acid, te/f-butyl ester (Intermediate 4; 4.183 g) in dry THF (40 mL) was added lithium bromide (1.911 g) followed by methyl acrylate (1.486 mL). The mixture was stirred for 5 minutes at room temperature and then triethylamine (2.30 mL) was added. The mixture was stirred at room temperature for a further 18 hours. Saturated ammonium chloride solution (60 mL) was added and the mixture was extracted with ethyl acetate. The extracts were washed with saturated brine, dried (MgSO₄) and concentrated in vacuo. The residue was partially purified by chromatography on silica gel using dichloromethane- methanol (98:2 v/v) as eluent and then further purified by additional chromatography on silica gel using cyclohexane-ethyl acetate as eluent (gradient elution from 40:60 v/v to 0:100 v/v) to afford the title compound, an oil. MS calcd for (C₁₉H₂₅N₃O₅S + H)⁺: 408 MS found (electrospray): (M+H)⁺ = 408

Intermediate 6 re/-(2R,4S,5R)-1 -(3-Bromo-4-fe/t-butylbenzoyl)-5-(5-methylisoxazol-3-yl)-2-(1 ,3- thiazol-4-ylmeth ylic acid, 2-terf-butyl ester, 4-methyl ester

Racemic; Relative stereochemistry shown

To a solution of re/-(2R,4S,5R)-5-(5-methylisoxazol-3-yl)-2-(1 ,3-thiazol-4-ylmethyl)- pyrrolidine-2,4-dicarboxylic acid, 2-terf-butyl ester, 4-methyl ester (Intermediate 5; 1.235g) in dichloromethane (25 mL) was added 3-bromo-4-tert-butylbenzoyl chloride (Intermediate 1 Alternative Method B; 1.003g) followed by triethylamine (0.507 mL). The mixture was stirred at 20⁰C under nitrogen for 18 hours. Water (25 mL) was added and the organic phase was passed through a hydrophobic frit and then concentrated in vacuo. The residue was purified by chromatography on silica gel using cyclohexane-ethyl acetate as eluent (gradient elution from 90:10 v/v to 50:50 v/v) to give the title compound as a foam. MS calcd for (C₃₀H₃₆BrN₃O₆S + H)⁺: 646/648 MS found (electrospray): (M+H)⁺ = 646/648

Intermediate 7 re/-(2R,4S,5R)-1-(3-Bromo-4-tert-butylbenzoyl)-4-(hydroxymethyl)-5-(5-methyl- terf-butyl ester

A stirred solution of re/-(2R,4S,5R)-1-(3-bromo-4-terf-butylbenzoyl)-5-(5-methylisoxazol-3- yl)-2-(1 ,3-thiazol-4-ylmethyl)pyrrolidine-2,4-dicarboxylic acid, 2-tert-butyl ester, 4-methyl ester (Intermediate 6; 1.48g) in dry THF (60 mL) was cooled to -78°C under nitrogen and a 1.0 M solution of lithium aluminium hydride in ether (2.31 mL) was added dropwise. The solution was stirred at -78°C for 30 minutes and then stirred at -43°C for 3.5 hours. The reaction was quenched at -43°C by cautious addition of 1.0 M aqueous potassium carbonate solution and then allowed to warm to 20°C. The mixture was extracted with ethyl acetate; the combined extracts were washed with saturated brine, dried (MgSO₄) and concentrated in vacuo. The residue was purified by chromatography on silica gel using cyclohexane-ethyl acetate as eluent (gradient elution from 67:33 v/v to 0:100 v/v) to give the title compound as a foam. MS calcd for (C₂₉H₃₆BrN₃O₅S + H)⁺: 618/620 MS found (electrospray): (M+H)⁺ = 618/620

Intermediate 8 re/-(2R,4S,5R)-1-(3-Bromo-4-ferf-butylbenzoyl)-4-(methoxymethyl)-5-(5-methyl- isoxazol-3-yl)-2- rolidine-2-carboxylic acid, fe/t-butyl ester

Racemic; Relative stereochemistry shown

To a stirred solution of re/-(2R,4S,5R)-1-(3-bromo-4-terf-butylbenzoyl)-4-(hydroxymethyl)- 5-(5-methyl-isoxazol-3-yl)-2-(1 ,3-thiazol-4-ylmethyl)pyrrolidine-2-carboxylic acid, terf-butyl ester (Intermediate 7; 906 mg) in dry N,N-dimethylformamide (50 mL) at -10⁰C was added sodium hydride (60% dispersion in mineral oil, 97.8 mg). The resulting mixture was stirred at -10⁰C under nitrogen for 30 minutes, lodomethane (0.454 mL) was added. The mixture was warmed to 20°C and stirred for a further 18 hours. Methanol (30 mL) was added and the mixture was concentrated in vacuo. The residue was partitioned between water and ethyl acetate. The organic phase was washed with saturated brine, dried (MgSO₄) and concentrated in vacuo. The residue was purified by chromatography on silica gel using cyclohexane-ethyl acetate as eluent (gradient elution from 100:0 v/v to 0:100 v/v) to give the title compound as a foam. MS calcd for (C₃₀H₃₈BrN₃O₅S + H)⁺: 632/634 MS found (electrospray): (M+H)⁺ = 632/634

Intermediate 9

2-Amino-3-(1H-pyrazol-1-yl)propanoic acid, fe/t-butyl ester

To a stirred suspension of 2-amino-3-(1 /-/-pyrazol-1-yl)propanoic acid (10.2 g, 65.9 mmol) in terf-butyl acetate (400 mL) was added a solution of 70% aqueous perchloric acid (15.7 ml_). The mixture was allowed to stir at room temperature for 0.5 hours and was then allowed to stand for 20 hours. The reaction mixture was diluted with ethyl acetate and then neutralised using a combination of saturated aqueous sodium bicarbonate and solid sodium bicarbonate. The aqueous phase was separated off and extracted with ethyl acetate. The organic phases were combined, dried over MgSO₄ and evaporated to give the title compound, an oil. MS calcd for (C₁₀H₁₇N₃O₂ + H)⁺: 212 MS found (electrospray): (M+H)⁺ = 212

Intermediate 10

2-[N-(1 ,3-Thiazol-2-ylmethylene)amino]-3-(1 H-pyrazol-1 -yl)propanoic acid, f erf-butyl ester

A mixture of 2-amino-3-(1 H-pyrazol-1 -yl)propanoic acid, terf-butyl ester (prepared in a similar manner to that described in Intermediate 9; 8.925 g) and thiazole-2- carboxaldehyde (4.78 g) in dichloromethane (100 ml_) was treated with anhydrous MgSO₄

(18 g) and stirred at 21⁰C for 3 hours. Solids were removed by filtration and the filtrate was concentrated in vacuo to give the title compound as an oil.

¹H NMR (CDCI₃): δ 8.08 (s, 1 H), 7.91 (d, 1 H), 7.51 (d, 1 H), 7.44 (dd, 1 H), 7.34 (d, 1 H), 6.12 (t, 1 H), 4.78 (dd, 1 H), 4.50 (m, 2H), and 1.47 (s, 9H).

Intermediate 11 re/-(2R,4S,5R)-2-(1 H-Pyrazol-1 -ylmethyl)-5-(1 ,3-thiazol-2-yl)pyrrolidine-2,4- dicarboxylic acid, 2-fert-butyl ester, 4-methyl ester

Racemic; Relative stereochemistry shown

An ice cooled solution of 2-[N-(1 ,3-thiazol-2-ylmethylene)amino]-3-(1 H-pyrazol-1 -yl)- propanoic acid, tert-butyl ester (Intermediate 10; 13.13 g) in THF (200 mL) under nitrogen was treated sequentially with methyl acrylate (4.24 mL), lithium bromide (7.43 g) and finally triethylamine (8.95 mL). The resulting mixture was allowed to warm to room temperature overnight with stirring. Saturated aqueous ammonium chloride solution was added and the mixture extracted with ethyl acetate. The combined organic solutions were washed with water and brine, dried (Na₂SO₄) and evaporated in vacuo. The residue was purified by chromatography on silica gel using cyclohexane-ethyl acetate as eluent (gradient elution from 100:0 v/v to 20:80 v/v) to afford the title compound, an oil. MS calcd for (C₁₈H₂₄N₄O₄S + H)⁺: 393 MS found (electrospray): (M+H)⁺ = 393

Intermediate 12

Chiral Diastereoisomeric Salt A of re/-(2R,4S,5R)-2-(1W-pyrazol-1-ylmethyl)-5-(1,3- thiazol-2-yl)pyrrolidine-2,4-dicarboxylic acid, 2-terf-butyl ester, 4-methyl ester with (R)-(-)-1,1 '-binaphthyl-2,2'-diyl-hydrogen phosphate

Chiral; Chiral; f?e/-(2R,4S,5R) stereochemistry shown (R)-Absolute stereochemistry shown

Alternative Method A

To a solution of re/-(2R,4S,5R)-2-(1H-pyrazol-1-ylmethyl)-5-(1 ,3-thiazol-2-yl)pyrrolidine- 2,4-dicarboxylic acid, 2-tert-butyl ester, 4-methyl ester (Intermediate 11 ; 11.62 g, 29.61 mmol) in dichloromethane (50 ml.) was added portionwise with stirring (R)-I ₁I ¹- binapththyl-2,2'-diylhydrogen phosphate (10.31 g, 29.61 mmol). After 20 minutes, a clear solution was obtained, the solvent was then evaporated and the residue treated with boiling isopropyl acetate (60 ml_). The mixture was brought to reflux with stirring and a clear solution was obtained. On allowing to cool to ambient temperature with stirring, a solid precipitated and after a further 20 minutes with ice cooling, this was filtered, washed with ice-cold isopropyl acetate (2 x 30 mL) and dried at 4O⁰C under vacuum, to yield the crude diastereoisomeric salt. This salt (9.3 g) was added portionwise with stirring to boiling isopropyl acetate (45 mL) and the suspension was refluxed for 15 minutes, allowed to cool to ambient temperature and then ice cooled for a further 20 minutes with stirring. The resulting solid was filtered, washed with ice-cold isopropyl acetate (45 mL) and dried under vacuum at 40⁰C to give the title compound.

Chiral HPLC analysis was conducted via regeneration of the free base: A sample of the title compound (25 mg) was dissolved in terf-butyl methyl ether (5 mL) and treated with triethylamine (0.006 mL). After stirring at room temperature for 1 hour, the mixture was filtered and the solvent evaporated. Chiral HPLC of the resulting oil (Chiralcel OD-H, heptane-ethanol 75:25 v/v, flow rate 1 mL/min, λ = 215 nm) showed two peaks at retention time 6.50 minutes and 9.73 minutes in the ratio of 0.34:99.66 respectively, 99.32% ee. Alternative Method B Part A - Asymmetric [1,3]-Dipolar Cycloaddition

2-[N-(1 ,3-Thiazol-2-ylmethylene)amino]-3-(1 H-pyrazol-1-yl)propanoic acid, tert-butyl ester (prepared in a similar manner to that described in Intermediate 10; 20.0 g, 65.4 mmol) was dissolved in anhydrous acetonitrile (327 mL) before manganese (II) bromide (28.1 g) was added portionwise with stirring over ca 5 minutes (a slight exotherm was observed, raising the reaction temperature from 22°C to 28°C). The reaction mixture was stirred for 30 minutes and was then cooled to 22°C before (1 R, 2S)-(-)-N-methylephedrine (17.6 g) was gradually added to the stirred reaction mixture. The reaction mixture was stirred for 10 minutes at 22°C and was then cooled to -20⁰C. Methyl acrylate (9.2 mL) was added and stirring continued for 2 hours at a temperature between -23°C and -20°C. The reaction mixture was quenched at -2O⁰C by the addition of saturated ammonium chloride solution, and then extracted with ethyl acetate. The organic phase was separated, combined, dried (Na₂SO₄), filtered and evaporated to afford a residue which was re- suspended in dichloromethane, filtered and evaporated to give an impure gum (25.57 g). Chiral HPLC analysis of an aliquot of this impure gum (50 mg purified by chromatography on silica gel, eluting with cyclohexane-ethyl acetate, 1 :1 v/v; then chiral HPLC on Chiralcel OD-H, heptane-ethanol 75:25 v/v, flow rate 1ml_/min, λ = 215 nm) showed 2 peaks at 6.65 minutes and 10.3 minutes in the ratio of ca 13:87 respectively, 74% ee.

The impure gum (15 g) was purified by chromatography on silica gel using cyclohexane- ethyl acetate as eluent (gradient elution from 80:20 v/v to 0:100 v/v) to afford an oil (8.01 g). Part B - Preparation of Chiral Diastereoisomeric Salt A To a solution of the oil (Part A; 8.0 g) in dichloromethane (40 mL) was added with stirring (R)-1 ,1 '-binaphthyl-2,2'-diylhydrogen phosphate (7.69 g, 22.06 mmol). After ca 20 minutes a solution was obtained, the solvent evaporated, and the residue treated with boiling isopropyl acetate (40 mL). The mixture was brought to reflux with stirring and after 5 minutes the resulting suspension allowed to cool to ambient temperature. After further cooling (ice bath, 20 minutes) the salt was filtered, washed with ice-cold isopropyl acetate (40 mL) and dried overnight at 40⁰C under vacuum to afford the title compound. Chiral HPLC analysis of the title compound was conducted via regeneration of the free base (cf Part A, above): This showed 2 peaks at 6.57 minutes and 9.99 minutes in the ratio of 0.44:99.56 respectively, 99.12% ee.

Intermediate 13

(2R,4S,5R)-2-(1 H-Pyrazol-1 -ylmethyl)-5-(1 ,3-thiazol-2-yl)-pyrrolidine-2,4-dicarboxylic acid, 2-tert-butyl ester, 4-methyl ester

[Enantiomer A of re/-(2R,4S,5R)-2-(1H-Pyrazol-1-ylmethyl)-5-(1,3-thiazol-2-yl)- pyrrolidine-2,4-dicarboxylic acid, 2-ferf-butyl ester, 4-methyl ester]

^Chiral; Enantiomer A AAbbssoolluuttee sstteerreeoocchheemmiisεt.r.y, shown

Stereochemistry determined by X-ray crystallography

Chiral Diastereoisomeric Salt A of re/-(2R,4S,5R)-2-(1/-/-pyrazol-1-ylmethyl)-5-(1 ,3-thiazol- 2-yl)pyrrolidine-2,4-dicarboxylic acid, 2-tert-butyl ester, 4-methyl ester with (R)-(-)-1 ,1 '- binaphthyl-2,2'-diyl-hydrogen phosphate (Intermediate 12 Alternative Method A; 8.06 g, 10.88 mmol) was suspended in tert-butyl methyl ether (90 mL). Triethylamine (1.67 mL, 11.97 mmol, 1.1 equiv) was added and the mixture was stirred for 1.5 hours. The mixture was filtered to remove remaining solid material, this solid was washed with additional tert- butyl methyl ether (90 mL) and the combined filtrate solutions were evaporated to about one third volume. This slightly cloudy mixture was filtered again, washing with tert-butyl methyl ether, and then evaporated to give a clear oil which was dried under high vacuum, to afford the title compound, containing traces of residual tert-butyl methyl ether. MS calcd for (C₁₈H₂₄N₄O₄S + H)⁺: 393 MS found (electrospray): (M+H)⁺ = 393 The absolute stereochemistry of this compound was determined by X-ray crystallography and shown to be (2R,4S,5R), as drawn.

Intermediate 14

(2R,4S,5R)-1 -(3-Bromo-4-ferf-butylbenzoyl)-2-(1 H-Pyrazol-1 -yl-methyl)-5-(1 ,3-thiazol- 2-yl)pyrrolidine-2,4-dicarboxylic acid, 2-ferf-butyl ester, 4-methyl ester

[Enantiomer A of re/-(2R,4S,5R)-1-(3-Bromo-4-te/t-butylbenzoyl)-2-(1 H-Pyrazol-1 -yl- methyl)-5-(1,3-thiazol-2-yl)pyrrolidine-2,4-dicarboxylic acid, 2-tert-butyl ester, 4- methyl ester]

Chiral; Enantiomer A

Absolute stereochemistry shown

Stereochemistry determined by reference to Intermediate 13

To a solution of Enantiomer A of re/-(2R,4S,5R)-2-(1 H-Pyrazol-1 -ylmethyl)-5-(1 ,3-thiazol- 2-yl)-pyrrolidine-2,4-dicarboxylic acid, 2-terf-butyl ester, 4-methyl ester (prepared in a similar manner to that described in Intermediate 13; 3.313 g) in dichloromethane (35 mL) was added a solution of 3-bromo-4-terf-butylbenzoyl chloride (prepared in a similar manner to that described in Intermediate 1 Alternative Method B; 3.95 g) in dichloromethane (30 mL), followed by triethylamine (3.28 mL). The mixture was stirred at 20⁰C for 18 hours, then diluted with dichloromethane (250 mL), washed with water (150 mL) and dried by passage through a hydrophobic frit. The solution was concentrated in vacuo and the residue was purified by chromatography on silica gel using cyclohexane- ethyl acetate (3:1 v/v) as eluent to give the title compound as a foam. MS calcd for (C₂₉H₃₅BrN₄O₅S + H)⁺: 631/633 MS found (electrospray): (M+H)⁺ = 631/633

Intermediate 15

(2R,4S,5R)-1 -(3-Bromo-4-ferf-butylbenzoyl)-4-(hydroxymethyl)-2-(1 H-Pyrazol-1 - ylmethyl)-5-(1,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid, tert-butyl ester

[Enantiomer A of re/-(2R,4S,5R)-1-(3-Bromo-4-fe/f-butylbenzoyl)-4-(hydroxymethyl)- 2-(1 H-Pyrazol-1 -ylmethyl)-5-(1 ,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid, tert-butyl ester] Chiral; Enantiomer A Absolute stereochemistry shown Stereochemistry determined by reference to Intermediate 13

A solution of Enantiomer A of re/-(2R,4S,5R)-1-(3-bromo-4-tert-butylbenzoyl)-2-(1 /-/- pyrazol-1-yl-methyl)-5-(1 ,3-thiazol-2-yl)pyrrolidine-2,4-dicarboxylic acid, 2-terf-butyl ester, 4-methyl ester (Intermediate 14; 5.33g) in dry THF (100 mL) was stirred under nitrogen at 0⁰C. A solution of 1.0 M lithium triethylborohydride in THF (16.8 mL) was added over ca. 2 minutes. The cooling bath was removed and the mixture was stirred between 0⁰C and 20⁰C for 1 hour. 0.5 M Hydrochloric acid (200 mL) was added. The resulting mixture was extracted with ethyl acetate (2 x 250 mL). The combined extracts were washed with saturated sodium bicarbonate solution (200 mL), water (200 mL) and saturated brine (20O mL) and then dried (MgSO₄). The solution was concentrated in vacuo and the residue was purified by chromatography on silica gel using a cyclohexane-ethyl acetate as eluent (gradient elution from 80:20 v/v to 0:100) to give the title compound as a foam. MS calcd for (C₂₈H₃₅BrN₄O₄S + H)⁺: 603/605 MS found (electrospray): (M+H)⁺ = 603/605

Intermediate 16

(2R,4S,5R)-1 -(3-Bromo-4-tert-butylbenzoyl)-4-(methoxymethyl)-2-(1 H-Pyrazol-1 - ylmethyl)-5-(1,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid, te/t-butyl ester [Enantiomer A of re/-(2R,4S,5R)-1-(3-Bromo-4-tert-butylbenzoyl)-4-(methoxymethyl)- 2-(1AV-Pyrazol-1-ylmethyl)-5-(1,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid, fe/f-butyl ester]

Chiral; Enantiomer A Absolute stereochemistry shown Stereochemistry determined by reference to Intermediate 13

A suspension of sodium tert-butoxide (0.94 g) in acetonitrile (12.5 mL) was stirred at 0°C under nitrogen. A solution of Enantiomer A of re/-(2R,4S,5R)-1-(3-bromo-4-tert-butyl- benzoyl)-4-(hydroxymethyl)-2-(1 H-pyrazol-1 -ylmethyl)-5-(1 ,3-thiazol-2-yl)pyrrolidine-2- carboxylic acid, te/f-butyl ester (Intermediate 15; 3.69 g) in a mixture of acetonitrile (6.5 mL) and dichloromethane (8 mL) was added dropwise with stirring. The resulting mixture was stirred for 5 minutes prior to the addition of iodomethane (0.762 mL). Stirring was continued, keeping the mixture between 0⁰C and 10°C for 1 hour. The mixture was poured into a mixture of 0.1 M hydrochloric acid (70 mL) and ethyl acetate (70 mL). The organic phase was collected and the aqueous phase extracted with ethyl acetate (100 ml_). The organic phases were combined, washed with water and saturated brine and dried (MgSO₄). The solution was concentrated in vacuo and the residue was purified by chromatography on silica gel using cyclohexane-ethyl acetate as eluent (gradient elution from 100:0 v/v to 0:100) to give the title compound as a foam. MS calcd for (C₂₉H₃₇BrN₄O₄S + H)⁺: 617/619 MS found (electrospray): (M+H)⁺ = 617/619

Intermediate 17 2-te/t-Butyl-4-fluoro-5-methylphenol

To 2-fluoro-5-hydroxytoluene (25 g) in te/f-butyl chloride (43 ml_) was added zinc chloride (5.4 g). The mixture was heated at 80^cC for 2 hours. The mixture was cooled to 20°C, filtered and the filtrate concentrated in vacuo. The residue was purified by chromatography on silica gel using cyclohexane-ethyl acetate as eluent (gradient elution from 100:0 v/v to 70:30 v/v) to give the title compound as a solid. MS calcd for (C₁₁H₁₅FO - H)^": 181 MS found (electrospray): (M-H)^" = 181

Intermediate 18

2-ferf-Butyl-4-fluoro-5-methylphe sulphonate

To a solution of 2-te/f-butyl-4-fluoro-5-methylphenol (Intermediate 17; 12 g) in dry dimethylformamide (200 ml.) under nitrogen at -15⁰C was added sodium hydride (60% dispersion in mineral oil, 2.9 g) in 3 portions over 5 minutes. The mixture was warmed to 0⁰C over 1 hour. N-Phenyl-bis(trifluoromethanesulfonimide) (24.8 g) was added and the mixture was stirred at 20⁰C for 1 hour. Water was added and the mixture was extracted with diethyl ether. The extract was washed with saturated brine, dried (MgSO₄) and concentrated in vacuo to give the title compound. ¹H NMR (CDCI₃): δ 7.17 (d, 1 H), 7.11 (d, 1 H), 2.27 (s, 3H) and 1.41 (s, 9H).

Intermediate 19 4-terf-Butyl-2-fluoro-1 -methyl benzene

2-te/ϊ-Butyl-4-fluoro-5-methylphenyl trifluoromethylsulphonate (Intermediate 18; 20.7 g) in dry dimethylformamide (100 ml_) was stirred under nitrogen at room temperature. Palladium acetate (300 mg) and 1 ,3-bis(diphenylphosphino)propane (540 mg) were added and the mixture was heated to 60°C. Triethylsilane (26.3 ml_) was added and the mixture was stirred at 70⁰C for 12 hours. The mixture was diluted with water and extracted with ether. The combined extracts were washed with saturated brine, dried (MgSO₄) and concentrated in vacuo. The residue was purified by chromatography on silica gel using cyclohexane as eluent to give the title compound, admixed with ca 3 mol equivalents of residual triethylsilane. This material was used in the subsequent stage without further purification.

¹H NMR (CDCI₃): δ 7.01-7.11 (m, 3H), 2.24 (s, 3H) and 1.30 (s, 9H); together with triethylsilane peaks.

Intermediate 20

4-te/t-Butyl-2-fluorobenzoic acid

4-terf-Butyl-2-fluoro-1-methylbenzene (Intermediate 19; contaminated with triethylsilane, sample estimated to contain ca 8.3g of desired compound) in pyridine (200 mL) and water (50 mL) was treated with potassium permanganate (26.7 g), and heated under reflux for 3 hours. A further portion of potassium permanganate (26.7 g) was added and the mixture was heated under reflux for 18 hours. The cooled mixture was filtered through Celite, and the filter cake was washed with 2 M sodium hydroxide solution. The combined filtrates were acidified to pH 6 with 2 M hydrochloric acid and extracted with ethyl acetate. Combined extracts were dried (MgSO₄) and concentrated in vacuo. The residue was purified by chromatography on silica gel using cyclohexane-ethyl acetate as eluent (gradient elution from 90:10 v/v to 0:100 v/v) to give the title compound as a solid. MS calcd for (C₁₁H₁₃FO₂ + H)⁺: 197 MS found (electrospray): (M+H)⁺ = 197

Intermediate 21 5-Bromo-4-terf-butyl-2-fluorobenzoic acid

A mixture of 4-te/t-butyl-2-fluorobenzoic acid (Intermediate 20; 3.5 g), trifluoroacetic acid (11 mL), N-bromosuccinimide (3.8 g) and concentrated sulphuric acid (2 mL) was heated under nitrogen at 40⁰C for 16 hours. The mixture was diluted with dichloromethane and washed with water. The organic phase was passed through a hydrophobic frit and concentrated in vacuo. The residue was purified by chromatography on silica gel using cyclohexane-ethyl acetate as eluent (gradient elution from 70:30 v/v to 0:100 v/v) to give the title compound as a solid.

MS calcd for (C₁₁H₁₂BrFO₂ - H)-: 273/275 MS found (electrospray): (M-H)^" = 273/275

Intermediate 22 5-Bromo-4-terf-butyl-2-fluoroben

To a solution of 5-bromo-4-tert-butyl-2-fluorobenzoic acid (prepared in a similar manner to that described in Intermediate 21 ; 540 mg) in dichloromethane (8 mL) was added oxalyl chloride (0.342 mL) and diethylformamide (1 drop). The mixture was stirred at 20°C for 4 hours and then concentrated in vacuo to give an oil, which was used directly without further purification.

Intermediate 23 (2R,4S,5R)-1 -(5-Bromo-4-fert-butyl-2-f luorobenzoyl)-2-(1 H-pyrazol-1 -ylmethyl)-5-(1 ,3- thiazol-2-yl)pyrrolidine-2,4-dicarboxylic acid, 2-tert-butyl ester, 4-methyl ester [Enantiomer A of re/-(2R,4S,5R)-1-(5-Bromo-4-tert-butyl-2-fluorobenzoyl)-2-(1H- pyrazol-1 -ylmethyl)-5-(1 ,3-thiazol-2-yl)pyrrolidine-2,4-dicarboxylic acid, 2-ferf-butyl ester, 4-methyl ester]

Chiral; Enantiomer A

Absolute stereochemistry shown

Stereochemistry determined by reference to Intermediate 13

Enantiomer A of re/-(2R,4S,5R)-2-(1/-/-pyrazol-1-ylmethyl)-5-(1 ,3-thiazol-2-yl)pyrrolidine- 2,4-dicarboxylic acid, 2-te/ϊ-butyl ester, 4-methyl ester (prepared in a similar manner to that described in Intermediate 13; 0.95 g) was dissolved in dichloromethane (5 mL) and triethylamine (0.44 mL) and 5-bromo-4-te/f-butyl-2-fluorobenzoyl chloride (prepared in a similar manner to that described in Intermediate 22; 0.85 g) were added. The mixture was stirred at 20⁰C for 1 hour and then at heated at 40⁰C for a further 3 hours. The mixture was concentrated in vacuo and the residue was purified by chromatography on silica gel using initially dichloromethane as eluent, followed by further elution with cyclohexane- ethyl acetate (1 :1 v/v) to give the title compound as a foam. MS calcd for (C₂₉H₃₄BrFN₄O₅S + H)⁺: 649/651 MS found (electrospray): (M+H)⁺ = 649/651

Intermediate 24

(2R,4S,5R)-1-(5-Bromo-4-tert-butyl-2-fluorobenzoyl)-4-(hydroxymethyl)-2-(1H- pyrazol-1-ylmethyl)-5-(1,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid, te/t-butyl ester

[Enantiomer A of re/-(2R,4S,5R)-1-(5-Bromo-4-terf-butyl-2-fluorobenzoyl)-4- (hydroxymethyl)-2-(1 W-pyrazol-1 -ylmethyl)-5-(1 ,3-thiazol-2-yl)pyrrolidine-2- carboxylic acid, terf-butyl ester] Chiral; Enantiomer A

Absolute stereochemistry shown

Stereochemistry determined by reference to Intermediate 13

A solution of Enantiomer A of re/-(2R,4S,5R)-1-(5-bromo-4-ferf-butyl-2-fluorobenzoyl)-2- (1 H-pyrazol-1 -ylmethyl)-5-(1 ,3-thiazol-2-yl)pyrrolidine-2,4-dicarboxylic acid, 2-terf-butyl ester, 4-methyl ester (Intermediate 23; 1.08 g) in dry THF (20 mL) was stirred under nitrogen and cooled to -78°C. A I.O M solution of lithium aluminium hydride in THF (1.7 mL) was added. The resulting mixture was stirred at -78°C for 10 minutes and then allowed to warm to -40⁰C. The reaction mixture was monitored by thin layer chromatography (silica gel plate, ethyl acetate as eluent). When no Intermediate 23 remained, the mixture was cooled to -78°C and quenched with a 1.0 M aqueous potassium carbonate solution. The mixture was warmed to 20⁰C and extracted with ethyl acetate. The combined extracts were dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by chromatography on silica gel using cyclohexane-ethyl acetate (1 :1 v/v) as eluent to give the title compound as a gum. MS calcd for (C₂₈H₃₄BrFN₄O₄S + H)⁺: 621/623 MS found (electrospray): (M+H)⁺ = 621/623

Intermediate 25

(2R,4S_>5R)-1-(5-Bromo-4-te/t-butyl-2-fluorobenzoyl)-4-(methoxymethyl)-2-(1H- pyrazol-1-ylmethyl)-5-(1,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid, tert-butyl ester [Enantiomer A of re/-(2R,4S,5R)-1-(5-Bromo-4-teAt-butyl-2-fluorobenzoyl)-4- (methoxymethyl)-2-(1 H-pyrazol-1 -ylmethyl)-5-(1, 3-thiazol-2-yl)pyrrolidine-2- carboxylic acid, tert-butyl ester]

Chiral; Enantiomer A

Absolute stereochemistry shown

Stereochemistry determined by reference to Intermediate 13

A solution of Enantiomer A of re/-(2R,4S,5R)-1-(5-bromo-4-fert-butyl-2-fluorobenzoyl)-4- (hydroxymethyl)-2-(1 H-pyrazol-1 -ylmethyl)-5-(1 ,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid, tert-butyl ester (Intermediate 24; 0.61 g) in dry acetonitrile (3 mL) was stirred at -1O⁰C.

Sodium terf-butoxide (0.151 g) was added slowly, maintaining the reaction temperature at

-10⁰C and the resulting mixture was stirred at -10⁰C for 10 minutes, lodomethane

(0.122 mL) was added. The mixture was slowly warmed to 20⁰C and then stirred for a further 2 hours. The mixture was quenched with methanol and was then partitioned between water and ethyl acetate. Sodium chloride was added to facilitate phase separation. The organic phase was washed with saturated brine, dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by chromatography on silica gel using cyclohexane-ethyl acetate (1 :1 v/v) as eluent and further purified by re-chromatography on silica gel using cyclohexane-ethyl acetate (3:1 v/v) as eluent to give the title compound, a gum.

MS calcd for (C₂₉H₃₆BrFN₄O₄S + H)⁺: 635/637

MS found (electrospray): (M+H)⁺ = 635/637

Example 1 re/-(2R,4S,5R)-1-(3-Bromo-4-fert-butylbenzoyl)-4-(methoxymethyl)-5-(5-methyl- isoxazol-3-yl)-2-( lidine-2-carboxylic acid

Racemic, Relative stereochemistry shown

To a solution of re/-(2R,4S,5R)-1-(3-bromo-4-teAf-butylbenzoyl)-4-(methoxymethyl)-5-(5- methyl-isoxazol-3-yl)-2-(1 ,3-thiazol-4-ylmethyl)pyrrolidine-2-carboxylic acid, terf-butyl ester (Intermediate 8; 0.664 g) in dichloromethane (10 mL) was added trifluoroacetic acid (1O mL). The solution was set aside at room temperature for 18 hours and then concentrated in vacuo. The residue was re-evaporated from dichloromethane and then partitioned between dichloromethane and saturated sodium bicarbonate solution. The organic phase was collected and concentrated in vacuo to give the title compound as a solid. MS calcd for (C₂₆H₃₀BrN₃O₅S + H)⁺: 576/578 MS found (electrospray): (M+H)⁺ = 576/578

¹H NMR (CD₃OD): δ 9.10 (s 1H), 7.52 (d, 1H), 7.47 (d, 1 H), 7.39 (d, 1H), 7.31 (dd, 1H), 6.57 (s, 1 H), 4.73 (d, 1 H), 4.13 (d, 1 H), 3.54 (d, 1 H), 2.96 (s, 3H), 2.81 (dd, 1 H), 2.71 (dd, 1 H), 2.43 (dd, 1 H), 2.39 (s, 3H), 2.15 (t, 1 H), 1.73 (m, 1 H) and 1.50 (s, 9H). Carboxylic acid proton not observed due to exchange with solvent.

Example 2

Enantiomer A of re/-(2R,4S,5R)-1-(3-Bromo-4-fert-butylbenzoyl)-4-(methoxymethyl)-

S-tδ-methylisoxazol-S-yO^-ti^-thiazoM-ylmethyOpyrrolidine^-carboxylic acid

re/-(2R,4S,5R)-1-(3-Bromo-4-terf-butylbenzoyl)-4-(methoxymethyl)-5-(5-methyl-isoxazol-3- yl^-O .S-thiazoM-ylmethyOpyrrolidine^-carboxylic acid (Example 1 ; 600 mg) was resolved by preparative chiral HPLC on a Chiralpak AD column using heptane-ethanol (85:15 v/v) containing 0.1% trifluoroacetic acid as eluent to afford the first and second eluting enantiomers. The fractions containing the second eluting enantiomer were combined and evaporated. This crude product was dissolved in dichloromethane (10 mL), washed with saturated aqueous sodium carbonate solution, dried (hydrophobic frit) and solvent removed to give the title compound as a foam. MS calcd for (C₂₆H₃₀BrN₃O₅S + H)⁺: 576/578 MS found (electrospray): (M+H)⁺ = 576/578

¹H NMR (CD₃OD): δ 9.08 (s 1H), 7.48 (s, 1 H), 7.44 (m, 2H), 7.28 (dd, 1 H), 6.89 (s, 1 H), 4.67 (d, 1 H), 4.19 (d, 1 H), 3.55 (d, 1 H), 2.95 (s, 3H), 2.78 (dd, 1 H), 2.68 (dd, 1 H), 2.43 (dd, 1 H), 2.39 (s, 3H), 2.14 (t, 1 H), 1.70 (m, 1 H) and 1.50 (s, 9H). Carboxylic acid proton not observed due to exchange with solvent.

Example 3

(2R,4S,5R)-1 -(3-Bromo-4-tert-butylbenzoyl)-4-(methoxymethyl)-2-(1 H-Pyrazol-1 - ylmethyl)-5-(1 ,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid

[Enantiomer A of re/-(2R,4S,5R)-1 -(3-Bromo-4-te/t-butylbenzoyl)-4-(methoxymethyl)- 2-(1 H-Pyrazol-1 -ylmethyl)-5-(1 ,3-thiazol-2-yl)pyrrolidine-2 -carboxylic acid]

Chiral; Enantiomer A

Absolute stereochemistry shown

Stereochemistry determined by reference to Intermediate 13

A solution of Enantiomer A of re/-(2R,4S,5R)-1-(3-bromo-4-tert-butylbenzoyl)-4-(methoxy- methyl)-2-(1 H-Pyrazol-1-ylmethyl)-5-(1 ,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid, tert- butyl ester (Intermediate 16; 2.78g) in dichloromethane (20 mL) was treated with trifluoroacetic acid (20 mL) and stored at 20⁰C for 3 hours. The mixture was concentrated in vacuo. The residue was dissolved in dichloromethane (280 mL) and this solution was washed with water (4 x 200 mL) until washings were neutral to pH paper. The organic layer was passed through a hydrophobic frit and concentrated in vacuo to give a foam. Crystallisation from diethyl ether gave the title compound as a solid. MS calcd for (C₂₅H₂₉BrN₄O₄S + H)⁺: 561/563 MS found (electrospray): (M+H)⁺ = 561/563

¹H NMR (CDCI₃): δ 7.83 (d, 1 H), 7.64 (d, 1 H), 7.53 (d, 1 H)₁ 7.36 (d, 1H), 7.33 (d, 1 H), 7.10 (d, 1 H), 7.02 (dd, 1 H), 6.39 (t, 1 H), 5.35 (d, 1 H), 5.05 (d, 1 H), 4.90 (d, 1 H), 3.13 (dd, 1H), 3.03 (s, 3H), 2.50 (dd, 1 H), 2.32 (t, 1 H)₁ 2.14 (t, 1 H), 1.79 (m, 1 H) and 1.44 (s, 9H). Carboxylic acid proton not observed.

Example 4 (2R,4S,5R)-1-(5-Bromo-4-te/t-butyl-2-fluorobenzoyl)-4-(methoxymethyl)-2-(1H- pyrazol-1 -ylmethyl)-5-(1 ,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid [Enantiomer A of re/-(2R,4S,5R)-1-(5-Bromo-4-te/t-butyl-2-fluorobenzoyl)-4- (methoxymethyl)-2-(1 H-pyrazol-1 -ylmethyl)-5-(1 ,3-thiazol-2-yl)pyrrolidine-2- carboxylic acid]

Chiral; Enantiomer A

Absolute stereochemistry shown

Stereochemistry determined by reference to Intermediate 13

A solution of Enantiomer A of re/-(2R,4S,5R)-1-(5-bromo-4-teAf-butyl-2-fluorobenzoyl)-4- (methoxymethyl)-2-(1 H-pyrazol-1 -ylmethyO-S^I .S-thiazol^-yOpyrrolidine^-carboxylic acid, te/f-butyl ester (Intermediate 25; 0.236 g) in trifluoroacetic acid (3 mL) was stirred at 2O⁰C for 3 hours. The mixture was concentrated in vacuo. The residue was purified by chromatography on silica gel, sequentially eluting with dichloromethane, cyclohexane- ethyl acetate (1 :1 v/v), ethyl acetate, dichloromethane-methanol (95:5 v/v) and finally methanol. Fractions containing product were combined and concentrated to give the title compound as a solid. MS calcd for (C₂₅H₂₈BrFN₄O₄S + H)⁺: 579/581 MS found (electrospray): (M+H)⁺ = 579/581

¹H NMR (CDCI₃): δ 14.78 (s, 1 H), 7.84 (d, 1 H), 7.61 (d, 1 H), 7.59 (d, 1 H), 7.36 (d, 1 H), 7.10 (d, 1 H), 6.86 (d, 1 H), 6.35 (t, 1 H), 5.25 (d, 1 H), 5.03 (d, 1 H), 4.98 (d, 1 H), 3.12 (dd, 1 H), 3.01 (s, 3H), 2.67 (dd, 1 H), 2.28 (t, 1H), 2.12 (t, 1 H), 1.54 (m, 1 H) and 1.43 (s, 9H).

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 (Ia) or a pharmaceutically acceptable salt, solvate or ester thereof in admixture with one or more pharmaceutically 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 pharmaceutically 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 (Ia) 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 (Ia). A topical formulation contains suitably 0.01 to 5.0% of a compound of Formula (Ia). 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 comprising a compound of Formula (Ia) and/or a pharmaceutically acceptable salt, solvate or ester thereof 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 non-CFC propellant such as 1 ,1 ,1 ,2-tetrafluoroethane or 1 ,1 ,1 ,2,3,3,3- heptafluoropropane.

A typical suppository formulation comprises a compound of Formula (Ia) 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 nonaqueous 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 chemical entities of the invention to inhibit NS5B wildtype HCV polymerase activity, genotype 1a and genotype 1 b, may be demonstrated, for example, using the following in vitro assays: 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.

a) Genotype 1a C-Terminally Truncated (delta21) Enzyme

HCV RNA Polymerase [Recombinant NS5B with C-terminal 21 amino acid deletion and C- terminal 6His-tag (Ferrari et al. J. Virol. 73(2), 1999, 1649. 'Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli.') expressed in E. coli and purified to homogeneity] was added to 25 nM final concentration. Polymerase of genotype 1a was from strain H77 (Yanagi, M., Purcell, R. H., Emerson, S. U. & Bukh, J. (1997), Proceedings of the National Academy of Sciences, USA 94, 8738- 8743) containing a sequence change from valine to isoleucine at position 180.

Reaction Conditions were 25 nM enzyme, 1.5 μg/ml oligo-rG13/poly-rC and 0.2 μCi α-³³P- GTP in 0.5 μM GTP (20 Ci/mMol) , 20 mM Tris pH 7.5, 23 mM NaCI, 3 mM DTT, 5 mM MgCI₂, 1 mM MnCI₂.

Enzyme was diluted to 500 nM concentration in 20 mM Tris-HCI, pH 7.5, 25 mM NaCI and 3 mM DTT.

4x concentrated assay buffer mix was prepared using 1 M Tris-HCI, pH7.5 (1 ml_), 5M NaCI (0.25 ml_), 1 M DTT (0.12 mL) and Water (8.63 ml_), Total 10 ml_.

2x concentrated first reagent was prepared using 4x concentrated assay buffer mix (5μl_), 40 u/μL RNasin (0.1 μL), 20 μg/mL polyrC/biotinylated-oligorG (1.6 μl_), 500 nM enzyme (1 μL ) and Water (2.3 μL), Total 10 μL/well.

2x concentrated second reagent was prepared using 1 M MgCI₂ (0.1 μL), 1 M MnCI₂ (0.02 μL), 25 μM GTP (0.4 μL), Q-[³³P]- GTP (10 μCi/μL, 0.02μL) and water (9.5 μL), Total 10 μL/well.

The assay was set up using compound (1 μL in 100% DMSO), first reagent (10 μL), and second reagent (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 60 μL 1.5 mg/ml streptavidin SPA beads (Amersham) in 0.1 M EDTA in PBS. The beads were incubated with the reaction mixture for 1 h at 22°C after which 100 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 per minute, percentage of inhibition at highest concentration tested or IC₅₀S for the compounds were calculated using GraFit 3, GraFit 4 or GraFit 5 (Erithacus Software Ltd.) software packages or a data evaluation macro for Excel based on XLFit Software (IDBS).

b) Genotype 1b Full-Length Enzyme Reaction Conditions were 0.5 μM [³³P]-GTP (20 Ci/mMol), 1 mM Dithiothreitol, 20 mM MgCI₂, 5mM MnCI_2, 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 4 nM final concentration.

5x concentrated assay buffer mix was prepared using 1 M MnCI₂ (0.25 mL), glycerol (2.5mL), 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 1O 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), 40 u/μL RNasin (0.1 μL), 20 μg/mL polyrC/biotinylated-oligorG (1.6 μL), and Water (3.94 μL), Total 10 μL.

Enzyme Mix was prepared by adding 1 mg/ml full-length NS5B polymerase (1.5 μL) to 2.81 mL 2x-concentrated enzyme buffer.

The Assay was set up using compound (1μL), 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 1h 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 GraFit 3, GraFit 4 or GraFit 5 software packages or a data evaluation macro for Excel based on XLFit Software (IDBS).

The potential for compounds of the invention to inhibit NS5B wildtype HCV polymerase activity, genotype 1a and genotype 1 b may be demonstrated, for example, using the following cell based assays:

Replicon ELISA cell based assay

Method

100 μl_ of medium containing 10% FCS were added to each well of clear, flat-bottomed 96 well microplates, excepting wells in the top row. Test compound was diluted in assay medium to twice the final required starting concentration from a 40 mM stock solution in DMSO. 200 μl_ of the starting dilution were introduced into two wells each in the top row and doubling dilutions made down the plate by the sequential transfer of 100 μl_ aliquots with thorough mixing in the wells; the final 100 μl_ were discarded. The two bottom rows were not used for compound dilutions. Huh-7 HCV replicon cell monolayers nearing confluency were stripped from growth flasks with versene-trypsin solution and the cells were resuspended in assay medium at either 2 x 10⁵ cells/mL (sub-line 5-15; genotype 1b; Lohmann, V., Korner, F., Koch, J-O., Herian, U., Thielmann, L. And Bartenschlager, R., 1999, Science, 285, pp 1 10-113) or at 3 x 10⁵ cells/mL (genotype 1a; Gu, B., Gates, A.T., Isken, O., Behrens, S.E.and Sarisky, R.T., J. Virol., 2003, 77, 5352-5359). 100 μl_ of cell suspension were added to all wells and the plates incubated at 37°C for 72 hours in a 5% CO₂ atmosphere.

Following incubation, the assay medium was aspirated from the plates. The cell sheets were washed by gentle immersion in phosphate buffered saline (PBS), which was then aspirated off, and fixed with acetoneimethanol (1 :1 ) for 5 minutes. Following a further wash with PBS, 100 μL of ELISA diluent (PBS + 0.05% v/v Tween 20 + 2% w/v skimmed milk powder) were added to all wells and the plates incubated at 37°C for 30 minutes on an orbital platform. The diluent was removed and each well then received 50 μL of a 1/200 dilution of anti-HCV specific, murine, monoclonal antibody (either Virostat #1872 or #1877), except for wells in one of the compound-free control rows which received diluent alone to act as negative controls. The plates were incubated at 37°C for 2 hours and washed 3 times with PBS/0.05% Tween 20, then 50 μL of horseradish peroxidase conjugated, anti-mouse, rabbit polyclonal serum (Dako #P0260), diluted 1/1000, were added to all wells. The plates were incubated for a further hour, the antibody removed and the cell sheets washed 5 times with PBS/Tween and blotted dry. The assay was developed by the addition of 50 μl_ of ortho-phenylenediamine/peroxidase substrate in urea/citrate buffer (SigmaFast, Sigma #P-9187) to each well, and colour allowed to develop for up to 15 minutes. The reaction was stopped by the addition of 25 μl_ per well of 2 M sulphuric acid and the plates were read at 490 nm on a Fluostar Optima spectrophotometer.

The substrate solution was removed and the plates were washed in tap water, blotted dry and the cells stained with 5 % carbol fuchsin in water for 30 minutes. The stain was discarded and the cell sheets washed, dried and examined microscopically to assess cytotoxicity. 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. The quantifiable and specific reduction of expressed protein detected by the ELISA in the presence of a drug can be used as a measure of replicon inhibition. GraFit software (Erithacus Software Ltd.) was used to plot the curve of percentage inhibition against compound concentration and derive the 50% inhibitory concentration (IC₅₀) for the compound.

Results

Activity ranges

GenotvDe 1a Genotype 1 b enzyme ^* <0.75 μM # <0.20 μM

_** 0.75 - 1.00 μM ## 0.20 - 0.50 μM

_*** >1.00 μM >0.50 μM replicon + <10.00 μM <0.15 μM cell-based ++ 10.00 - 100 μM 0.15 - 10.00 μM

+++ >100 μM >10.00 μM

Compound A corresponds to the racemic compound disclosed as Example 11 in

WO2004/037818, re/-(2S,4S_l5R)-2-isobutyl-1-(3-methoxy-4-tert-butylbenzoyl)-4- methoxymethyl-5-(1 ,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid.

Compound B corresponds to the enantiomeric compound disclosed as Example 15 in WO2004/037818, (2S,4S,5R)-2-isobutyl-1-(3-methoxy-4-tert-butylbenzoyl)-4- methoxymethyl-5-(1 ,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid.

Compound C corresponds to the enantiomeric compound disclosed as Example 17 in

WO2004/037818, (2S,4S,5R)-2-isobutyl-1-(3-bromo-4-tert-butylbenzoyl)-4-ethoxymethyl-

5-(1 ,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid. Compound D corresponds to the racemic compound disclosed as Example 24 in

WO2004/037818, re/-(2S,4S,5R)-2-isobutyl-1 -(3-methoxy-4-terf-butylbenzoyl)-4- methoxymethyl-5-(5-methyl-1 ,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid.

Compound E corresponds to the enantiomeric compound disclosed as Example 25 in

WO2004/037818, Enantiomer A of re/-(2S_I4S,5R)-2-isobutyl-1-(3-methoxy-4-terf- butylbenzoyl)-4-methoxymethyl-5-(5-methyl-1 ,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid.

Compound F corresponds to the racemic compound disclosed as Example 33 in

WO2004/037818, re/-(2R,4S,5R)-2-benzyl-1 -(3-methoxy-4-tert-butylbenzoyl)-4- methoxymethyl-5-( 1 , 3-th iazol-2-yl )-pyrrolid ine-2-carboxyl ic acid .

Compound G corresponds to the enantiomeric compound disclosed as Example 49 in WO2004/037818, Enantiomer A of re/-(2S,4S,5R)-2-isobutyl-1-(3-methoxy-4-tert- butylbenzoyl)-4-ethoxymethyl-5-(5-methylisoxazol-3-yl)pyrrolidine-2-carboxylic acid.

Compounds A, B, C, D, E, F and G may be made according to the processes described in WO2004/037818.

Structures of Compounds A-G are shown below for the avoidance of doubt. shown

The compounds of the present invention which have been tested demonstrate a surprisingly superior genotype- 1 a/1 b profile, as shown by the IC₅₀ values in the enzyme and cell-based assays across both of the 1a and 1 b genotypes of HCV, compared to Compounds A - G. 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 at least one chemical entity chosen from compounds of formula (Ia) and pharmaceutically acceptable salts, solvates or esters thereof, together with at least one other 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 at least one pharmaceutically acceptable diluent or 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.

The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the following claims:

Claims

1. At least one chemical entity chosen from compounds of Formula (Ia):

wherein:

A represents hydroxy;

D represents 3-bromo-4-te/f-butylphenyl or 5-bromo-4-terf-butyl-2-fluorophenyl;

E represents 1 ,3-thiazol-2-yl or 5-methylisoxazol-3-yl;

G represents methoxymethyl;

J represents 1 ,3-thiazol-4-ylmethyl or 1 H-pyrazol-1-yl methyl;

and salts, solvates and esters thereof; provided that when A is esterified to form -OR where R is selected from branched chain alkyl, then R is other than terf-butyl.

2. At least one chemical entity chosen from compounds of Formula (Ia) as defined in claim 1 selected from the group consisting of: re/-(2R,4S,5R)-1-(3-Bromo-4-terf-butylbenzoyl)-4-(methoxymethyl)-5-(5-methyl-isoxazol-3- yl)-2-(1 ,3-thiazol-4-ylmethyl)pyrrolidine-2-carboxylic acid; re/-(2R,4S,5R)-1 -(3-Bromo-4-terf-butylbenzoyl)-4-(methoxymethyl)-2-(1 /-/-Pyrazol-1 - ylmethyl)-5-(1 ,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid; re/-(2R,4S,5R)-1 -(5-Bromo-4-tert-butyl-2-fluorobenzoyl)-4-(methoxy-methyl)-2-(1 H- pyrazol-1-ylmethyl)-5-(1 ,3-thiazol-2-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 at least one chemical entity chosen from compounds of Formula (Ia) and salts, solvates and esters thereof 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 chemical entity is administered in an oral dosage form.

6. At least one chemical entity chosen from compounds of Formula (Ia)

wherein:

A represents hydroxy;

D represents 3-bromo-4-terf-butylphenyl or 5-bromo-4-terf-butyl-2-fluorophenyl;

E represents 1 ,3-thiazol-2-yl or 5-methylisoxazol-3-yl;

G represents methoxymethyl;

J represents 1 ,3-thiazol-4-ylmethyl or 1 H-pyrazol-1-ylmethyl;

and salts, solvates and esters thereof; provided that when A is esterified to form -OR where R is selected from branched chain alkyl, then R is other than te/t-butyl;

for use in medical therapy.

7. At least one chemical entity chosen from compounds of Formula (Ia) and salts, solvates and esters thereof as claimed in claim 6, wherein the medical therapy is the treatment of viral infection.

8. At least one chemical entity chosen from compounds of Formula (Ia) and salts, solvates and esters thereof as claimed in claim 7 wherein the viral infection is HCV.

9. Use of at least one chemical entity chosen from compounds of Formula (Ia)

wherein:

A represents hydroxy;

D represents 3-bromo-4-tert-butylphenyl or 5-bromo-4-terf-butyl-2-fluorophenyl; E represents 1 ,3-thiazol-2-yl or 5-methylisoxazol-3-yl;

G represents methoxymethyl;

J represents 1 ,3-thiazol-4-ylmethyl or 1 H-pyrazol-1-ylmethyl;

and salts, solvates and esters thereof; provided that when A is esterified to form -OR where R is selected from branched chain alkyl, then R is other than terf-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 at least one chemical entity chosen from compounds of Formula (Ia) and salts, solvates and esters thereof as claimed in claim 1 in conjunction with at least one pharmaceutically acceptable diluent or carrier.

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

in which A' is a protected hydroxy group, and D, E, G and J are as defined in claim 1 for Formula (Ia).