WO2011144924A1 - Amine or amid analogues of sanglifehrin - Google Patents

Abstract

The present invention relates to macrocyclic sanglifehrin analogues, that are useful as cyclophilin inhibitors, e.g. in the treatment of viral infection especially infection by RNA viruses, such as Hepatitis C virus (HCV) and HIV, and muscular dystrophy. The present invention also provides methods for their use in medicine, in particular for the treatment of HCV infection.

Description

AMINE OR AMID ANALOGUES OF SANGLIFEHRIN

Introduction

The present invention relates to sanglifehrin analogues, that are useful as cyclophilin inhibitors, e.g. in the treatment of viral infection especially infection by RNA viruses such as Hepatitis C virus (HCV) and HIV and muscular dystrophy. The present invention also provides methods for their use in medicine, in particular for the treatment of HCV infection.

Background of the invention

Hepatitis C

Hepatitis C virus (HCV) is a positive strand RNA virus, and infection is a leading cause of post-transfusional hepatitis. HCV is the most common chronic blood borne infection, and the leading cause of death from liver disease in United States. The World Health Organization estimates that there are more than 170 million chronic carriers of HCV infection, which is about 3% of the world population. Among the un-treated HCV-infected patients, about 70%-85% develop chronic HCV infection, and are therefore at high risk to develop liver cirrhosis and hepatocellular carcinoma. In developed countries, 50-76% of all cases of liver cancer and two- thirds of all liver transplants are due to chronic HCV infection (Manns et al, 2007).

In addition to liver diseases, chronically infected patients may also develop other chronic HCV-related diseases, and serve as a source of transmission to others. HCV infection causes non-liver complications such as arthralgias (joint pain), skin rash, and internal organ damage predominantly to the kidney. HCV infection represents an important global health-care burden, and currently there is no vaccine available for hepatitis C (Strader et al., 2004;

Jacobson et al. 2007; Manns et al., 2007 Pawlotsky, 2005; Zeuzem & Hermann, 2002).

Treatment of HCV

The current standard of care (SoC) is subcutaneous injections of pegylated interferon-a (pIFNa) and oral dosing of the antiviral drug ribavirin for a period of 24-48 weeks. Success in treatment is defined by sustained virologic response (SVR), which is defined by absence of HCV RNA in serum at the end of treatment period and 6 months later. Overall response rates to SoC depend mainly on genotype and pretreatment HCV RNA levels. Patients with genotype 2 and 3 are more likely to respond to SoC than patients infected with genotype 1 (Melnikova, 2008; Jacobson et al., 2007).

A significant number of HCV patients do not respond adequately to the SoC treatment, or cannot tolerate the therapy due to side effects, leading to frequent issues with completion of the full course. The overall clinical SVR rate of SoC is only around 50% (Melnikova, 2008).

Development of resistance is another underlying factor for failure of treatment (Jacobson et al. et al. 2007). SoC is also contraindicated in some patients who are not considered candidates for treatment, such as patients with past significant episodes of depression or cardiac disease. Side effects of the SoC, which frequently lead to discontinuation of treatment include a flu-like illness, fever, fatigue, haematological disease, anaemia, leucopaenia, thrombocytopaenia, alopecia and depression (Manns et al., 2007).

Considering the side effects associated with the lengthy treatments using SoC, development of resistance, and suboptimum overall rate of success, more efficacious and safer new treatments are urgently needed for treatment of HCV infection. The objectives of new treatments include improved potency, improved toxicity profile, improved resistance profile, improved quality of life and the resulting improvement in patient compliance. HCV has a short life cycle and therefore development of drug resistance during drug therapy is common.

Novel, specifically targeted antiviral therapy for hepatitis C (STAT-C) drugs are being developed that target viral proteins such as viral RNA polymerase NS5B or viral protease NS3 (Jacobson et al, 2007; Parfieniuk et al., 2007). In addition, novel compounds also are being developed that target human proteins (e.g. cyclophilins) rather than viral targets, which might be expected to lead to a reduction in incidence of resistance during drug therapy (Manns et al., 2007; Pockros, 2008; Pawlotsky J-M, 2005).

Cyclophilin inhibitors

Cyclophilins (CyP) are a family of cellular proteins that display peptidyl-prolyl cis-trans isomerase activity facilitating protein conformation changes and folding. CyPs are involved in cellular processes such as transcriptional regulation, immune response, protein secretion, and mitochondrial function. HCV virus recruits CyPs for its life cycle during human infection.

Originally, it was thought that CyPs stimulate the RNA binding activity of the HCV non-structural protein NS5B RNA polymerase that promotes RNA replication, although several alternative hypotheses have been proposed including a requirement for CyP PPIase activity. Various isoforms of CyPs, including A and B, are believed to be involved in the HCV life cycle (Yang et al., 2008; Appel et al., 2006; Chatterji et al., 2009) The ability to generate knockouts in mice (Colgan et al., 2000) and human T cells (Braaten and Luban, 2001 ) indicates that CyPA is optional for cell growth and survival.. Similar results have been observed with disruption of CyPA homologues in bacteria (Herrler et al., 1994), Neurospora (Tropschug et al., 1989) and Saccharomyces cerevisiae (Dolinski et al. 1997). Therefore, inhibiting CyPs represent a novel and attractive host target for treating HCV infection, and a new potential addition to current SoC or STAT-C drugs, with the aim of increasing SVR, preventing emergence of resistance and lowering treatment side effects.

Cyclosporine A, 1 DEBIO-025, 2

IIM-811 , 3 SCY-635, 4

Cyclosporine A (Inoue et al. 2003) ("CsA") and its closely structurally related non- immunosuppressive clinical analogues DEBIO-025 (Paeshuyse et al. 2006; Flisiak et al. 2008), NIM81 1 (Mathy et al. 2008) and SCY-635 (Hopkins et al., 2009) are known to bind to cyclophilins, and as cyclophilin inhibitors have shown in vitro and clinical efficacy in the treatment of HCV infection (Crabbe et al., 2009; Flisiak et al. 2008; Mathy et al. 2008; Inoue et al., 2007; Ishii et al., 2006; Paeshuyse et al., 2006). Although earlier resistance studies on CsA showed mutations in HCV NS5B RNA polymerase and suggested that only cyclophilin B would be involved in the HCV replication process (Robida et al., 2007), recent studies have suggested an essential role for cyclophilin A in HCV replication (Chatterji et al. 2009; Yang et al., 2008). Considering that mutations in NS5A viral protein are also associated with CsA resistance and that NS5A interacts with both CyPA and CypB for their specific peptidyl-prolyl cis/trans isomerase (PPIase) activity, a role for both cyclophilins in viral life cycle is further suggested (Hanoulle et al., 2009).

The anti-HCV effect of cyclosporine analogues is independent of the

immunosuppressive property, which is dependent on calcineurin. This indicated that the essential requirement for HCV activity is CyP binding and calcineurin binding is not needed. DEBIO-025, the most clinically advanced cyclophilin inhibitor for the treatment of HCV, has shown in vitro and in vivo potency against the four most prevalent HCV genotypes (genotypes 1 , 2, 3, and 4). Resistance studies showed that mutations conferring resistance to DEBIO-025 were different from those reported for polymerase and protease inhibitors, and that there was no cross resistance with STAT-C resistant viral replicons. More importantly, DEBIO-025 also prevented the development of escape mutations that confer resistance to both protease and polymerase inhibitors (Crabbe et al., 2009).

However, the CsA-based cyclophilin inhibitors in clinical development have a number of issues, which are thought to be related to their shared structural class, including: certain adverse events that can lead to a withdrawal of therapy and have limited the clinical dose levels; variable pharmacokinetics that can lead to variable efficacy; and an increased risk of drug-drug interactions that can lead to dosing issues.

The most frequently occurring adverse events (AEs) in patients who received DEBIO- 025 included jaundice, abdominal pain, vomiting, fatigue, and pyrexia. The most clinically important AEs were hyperbilirubinemia and reduction in platelet count (thrombocytopaenia). Peg-IFN can cause profound thrombocytopaenia and combination with DEBIO-025 could represent a significant clinical problem. Both an increase in bilirubin and decrease in platelets have also been described in early clinical studies with NIM-81 1 (Ke et al., 2009). Although the hyperbilirubinemia observed during DEBIO-025 clinical studies was reversed after treatment cessation, it was the cause for discontinuation of treatment in 4 out of 16 patients, and a reduction in dose levels for future trials. As the anti-viral effect of cyclophilin inhibitors in HCV is dose related, a reduction in dose has led to a reduction in anti-viral effect, and a number of later trials with CsA-based cyclophilin inhibitors have shown no or poor reductions in HCV viral load when dosed as a monotherapy (Lawitz et al., 2009; Hopkins et al., 2009; Nelson et al., 2009). DEBIO-025 and cyclosporine A are known to be inhibitors of biliary transporters such as bile salt export pumps and other hepatic transporters (especially MRP2/cMOAT/ABCC2) (Crabbe et al., 2009). It has been suggested that the interaction with biliary transporters, in particular MRP2, may be the cause of the hyperbilirubinaemia seen at high dose levels of DEBIO-025 (Nelson et al., 2009).

Moreover, DEBIO-025 and cyclosporine A are substrates for metabolism by cytochrome P450 (especially CYP3A4), and are known to be substrates and inhibitors of human P- glycoprotein (MDR1 ) (Crabbe et al., 2009). Cyclosporine A has also been shown to be an inhibitor of CYP3A4 in vitro (Niwa et al., 2007). This indicates that there could be an increased risk of drug-drug interactions with other drugs that are CYP3A4 substrates, inducers or inhibitors such as for example ketoconazole, cimetidine and rifampicin. In addition, interactions are also expected with drugs that are subject to transport by P-glycoprotein (e.g. digoxin), which could cause severe drug-drug interactions in HCV patients receiving medical treatments for other concomitant diseases (Crabbe et al. 2009). CsA is also known to have highly variable pharmacokinetics, with early formulations showing oral bioavailability from 1 -89% (Kapurtzak et al., 2004). Without expensive monitoring of patient blood levels, this can lead to increased prevalence of side effects due to increased plasma levels, or reduced clinical response due to lowered plasma levels.

Considering that inhibition of cyclophilins represent a promising new approach for treatment of HCV, there is a need for discovery and development of more potent and safer CyP inhibitors for use in combination therapy against HCV infection.

Sanglifehrins

Sanglifehrin A (SfA) and its natural congeners belong to a class of mixed non-ribosomal peptide/polyketides, produced by Streptomyces sp. A92-3081 10 (also known as DSM 9954) (see WO 97/02285), which were originally discovered on the basis of their high affinity to cyclophilin A (CyPA). SfA is the most abundant component in fermentation broths and exhibits approximately 20-fold higher affinity for CyPA compared to CsA. This has led to the suggestion that sanglifehrins could be useful for the treatment of HCV (WO2006/138507). Sanglifehrins have also been shown to exhibit a lower immunosuppressive activity than CsA when tested in vitro (Sanglier et al., 1999; Fehr et al., 1999). SfA binds with high affinity to the CsA binding site of CyPA (Kallen et al., 2005;).

The immunosuppressive mechanism of action of SfA is different to that of other known immunophilin-binding immunosuppressive drugs such as CsA, FK506 and rapamycin. SfA does not inhibit the phosphatase activity of calcineurin, the target of CsA (Zenke et al. 2001 ), instead its immunosuppressive activity has been attributed to the inhibition of interleukin-6 (Hartel et al., 2005), interleukin-12 (Steinschulte et al., 2003) and inhibition of interleukin-2- dependent T cell proliferation (Zhang & Liu, 2001 ). However, the molecular target and mechanism through which SfA exerts its immunosuppressive effect is hitherto unknown.

The molecular structure of SfA is complex and its interaction with CyPA is thought to be mediated largely by the macrocyclic portion of the molecule. In fact, a macrocyclic compound (hydroxymacrocycle) derived from oxidative cleavage of SfA has shown strong affinity for CyPA (Sedrani et al., 2003). X-ray crystal structure data has shown that the hydroxymacrocycle binds to the same active site of CyPA as CsA. Analogues based on the macrocycle moiety of SfA have also been shown to be devoid of immunosuppressive properties (Sedrani et al., 2003), providing opportunity for design of non-immunosuppressive CyP inhibitors for potential use in HCV therapy. These molecules will have lower molecular weight than SfA and are expected to possess improved physico-chemcial properties suitable for drug development.

One of the issues in drug development of compounds such as sanglifehrins are the low solubilities if these highly lipophilic molecules. This can lead to issues with poor bioavailability, an increased chance of food effect, more frequent incomplete release from the dosage form and higher interpatient variability. Poorly soluble molecules also present many formulation issues, such as severely limited choices of delivery technologies and increasingly complex dissolution testing, with limited or poor correlation to in vivo absorption. These issues are often sufficiently formidable to halt development of many compounds.

Other therapeutic uses of cyclophilin inhibitors

Cyclophilin inhibitors, such as CsA and DEBIO-025 have also shown potential utility in inhibition of HIV replication. The cyclophilin inhibitors are thought to interfere with function of CyPA during progression/completion of HIV reverse transcription (Ptak et al., 2008).

Cyclophilin inhibitors, such as DEBIO-025 have shown utility in animal models of muscular dystrophy, in particular Ullrich congenital muscular dystrophy and Bethlem myopathy (WO2008/084368). Specifically, they were seen to reduce mitochondrial swelling and necrotic disease manifestation in mdx mice, a model of Duchenne Muscular Dystrophy (Millay et al., 2008). They are thought to exert their effect through inhibition of cyclophilin D.

There are also reports of utility of cyclophilin inhibitors in other therapeutic areas, such as recovery after myocardial infarction (Gomez et al., 2007), and treatment of Cytomegalovirus infection (Kawasaki et al., 2007).

Therefore there remains a need to identify novel cyclophilin inhibitors, which may have utility, particularly in the treatment of HCV infection, but also in the treatment of other disease areas where inhibition of cyclophilins may be useful, such as HIV infection, Muscular Dystrophy or aiding recovery after myocardial infarction. Preferably, such cyclophilin inhibitors have improved properties over the currently available cyclophilin inhibitors, including one or more of the following properties: improved water solubility, improved pharmacological profile, such as high exposure to target organ (e.g. liver in the case of HCV) and/or long half life (enabling less frequent dosing), reduced drug-drug interactions, such as via reduced levels of CYP3A4 metabolism and inhibition and reduced (Pgp) inhibition (enabling easier multi-drug

combinations) and improved side-effect profile, such as low binding to MRP2, leading to a reduced chance of hyperbilirubinaemia, lower immunosuppressive effect, improved activity against resistant virus species, in particular CsA and CsA analogue (e.g DEBIO-025) resistant virus species and higher therapeutic index. The present invention discloses novel sanglifehrin analogues which may have one or more of the above properties. In particular, the present invention discloses novel amine derivatives, which are anticipated to have improved solubility, and therefore improved formulation.

Summary of the invention

The present invention provides novel macrocyclic sanglifehrin analogues, which have been generated by semisynthetic modification of native sanglifehrins. These analogues may be generated by dihydroxylation of a sanglifehrin, such as SfA, followed by cleavage to generate the aldehydic macrocycle, followed by further chemistry, such as reductive amination or oxidation followed by amidation, to generate molecules with a variety of substituents to replace the aldehyde. As a result, the present invention provides macrocylic amine and amide analogues of SfA, methods for the preparation of these compounds, and methods for the use of these compounds in medicine or as intermediates in the production of further compounds.

Therefore, in a first aspect, the present invention provides macrocylic amines, macrocyclic amides and derivatives thereof according to formula (I) below, or a

pharmaceutically

(I)

wherein:

X represents CH₂ or CO;

Ri and R₂ independently represent hydrogen; or an alkyl or alkenyl group which may optionally be joined to form a saturated or unsaturated heterocyclic ring containing the nitrogen atom shown and wherein one or more carbon atoms of Ri and/or R₂ are optionally replaced by a heteroatom selected from O, N and S(0)_p in which p represents 0, 1 or 2 and wherein one or more carbon atoms of Ri and/or R₂ are optionally replaced by carbonyl; or one of Ri and R₂ represents -alkylaryl, -alkenylaryl, -alkylheteroaryl or -alkenylheteroaryl and the other represents H, alkyl or alkenyl;

including any tautomer thereof; and including a methanol adduct thereof in which a ketal is formed by the combination of the C-53 keto and the C-15 hydroxyl groups and methanol.

The above structure shows a representative tautomer and the invention embraces all tautomers of the compounds of formula (I) for example keto compounds where enol

compounds are illustrated and vice versa.

Specific tautomers that are included within the definition of formula (I) are those in which (i) the C-53 keto group forms a hemiketal with the C-15 hydroxyl, or (ii) the C-15 and C-17 hydroxyl can combine with the C-53 keto to form a ketal. All numberings use the system for the parent sanglifehrin A structure.

Definitions

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. at least one) of the grammatical objects of the article. By way of example "an analogue" means one analogue or more than one analogue.

As used herein the term "analogue(s)" refers to chemical compounds that are structurally similar to another but which differ slightly in composition (as in the replacement of one atom by another or in the presence or absence of a particular functional group).

As used herein the term "sanglifehrin(s)" refers to chemical compounds that are structurally similar to sanglifehrin A but which differ slightly in composition (as in the

replacement of one atom by another or in the presence or absence of a particular functional group), in particular those generated by fermentation of Streptomyces sp. A92-3081 10.

Examples include the sanglifehrin-like compounds discussed in WO97/02285 and WO98/07743, such as sanglifehrin B.

As used herein, the term "HCV" refers to Hepatitis C Virus, a single stranded, RNA, enveloped virus in the viral family Flaviviridae.

As used herein, the term "HIV" refers to Human Immunodeficiency Virus, the causative agent of Human Acquired Immune Deficiency Syndrome.

As used herein, the term "bioavailability" refers to the degree to which or rate at which a drug or other substance is absorbed or becomes available at the site of biological activity after administration. This property is dependent upon a number of factors including the solubility of the compound, rate of absorption in the gut, the extent of protein binding and metabolism etc. Various tests for bioavailability that would be familiar to a person of skill in the art are described herein (see also Egorin et al. 2002).

The term "water solubility" as used in this application refers to solubility in aqueous media, e.g. phosphate buffered saline (PBS) at pH 7.4, or in 5% glucose solution. Tests for water solubility are given below in the Examples as "water solubility assay".

As used herein, the term "macrocyclic amine" or "macrocyclic amide" refers to a macrocylic amine or amide referred to above as representing the invention in its broadest aspect, for example a compound according to formula (I) above, or a pharmaceutically acceptable salt thereof. These compounds are also referred to as "compounds of the invention" or

"derivatives of sanglifehrin" or "sanglifehrin analogues" and these terms are used interchangeably in the present application.

The pharmaceutically acceptable salts of compounds of the invention such as the compounds of formula (I) include conventional salts formed from pharmaceutically acceptable inorganic or organic acids or bases as well as quaternary ammonium acid addition salts. More specific examples of suitable acid salts include hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, perchloric, fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic, tartaric, citric, palmoic, malonic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, fumaric, toluenesulfonic, methanesulfonic, naphthalene-2-sulfonic, benzenesulfonic hydroxynaphthoic, hydroiodic, malic, steroic, tannic and the like. Hydrochloric acid salts are of particular interest. Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be useful in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable salts. More specific examples of suitable basic salts include sodium, lithium, potassium, magnesium, aluminium, calcium, zinc, Ν,Ν'- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N- methylglucamine and procaine salts. References hereinafter to a compound according to the invention include both compounds of formula (I) and their pharmaceutically acceptable salts.

As used herein, the term "alkyl" represents a straight chain or branched alkyl group, containing typically 1 -8 carbon atoms, for example a Ci_-6 alkyl group. "Alkenyl" refers to an alkyl group containing two or more carbons (for example 2-8 carbons e.g. 2-6 carbons) which is unsaturated with one or more double bonds.

Examples of alkyl groups include d^alkyl groups such as methyl, ethyl, n-propyl, i-propyl, and n-butyl. Examples of alkenyl groups include Ci_-4alkenyl groups such as -CH₂CH=CH2.

Examples of aryl groups include phenyl and naphthyl, especially phenyl which groups may optionally be substituted e.g. with one or more (e.g. 1 , 2 or 3) substituents e.g. selected from alkyl (eg

halogen, alkoxy (e.g. nitro, -S0₂Me, cyano or -CONH₂.

Examples of heteroaryl groups include monocyclic groups (e.g. 5 and 6 membered rings) and bicyclic rings (e.g. 9 and 10 membered rings) which are aromatic or (in the case of bicyclic rings contain at least one aromatic ring) and contain one or more heteroatoms (e.g. 1 , 2, 3 or 4) heteroatoms selected from N, O and S. Examples of 5 membered heteroaryl rings include pyrrole, furan, thiophene, oxazole, oxadiazole, thiazole and triazole. Examples of 6 membered heteroaryl rings include pyridine, pyrimidine and pyrazine. Examples of bicyclic rings include quinoline, quinazoline, isoquinoline, indole, indane, cinnoline and quinoxaline. Monocyclic heteroaryl groups are preferred. The aforementioned heteroaryl groups may be optionally substituted as described above for aryl groups.

The term "treatment" includes prophylactic as well as therapeutic treatment.

Figure Legend

Figure 1

A: HPLC Profile of Harvest Whole Broth Sample of SfA, 5 & sanglifehrin B, 7, (monitored at 240nm)

B: UV spectrum of sanglifehrin A Figure 2.

Graph showing activity of 10 against the genotype 1 b replicon in Huh5.2 cells (lower line^*, black circles) and effect on cell viability (upper line^*, grey circles) as a percentage compared to control values. ^* to the left of the crossover point at concentration of around 1.0E+01 μΜ. Description of the Invention

The present invention provides sanglifehrin macrocylic analogues, as set out above, methods for preparation of these compounds and methods for the use of these compounds in medicine.

In one embodiment, the compound is a methanol adduct thereof in which a ketal is formed by the combination of the C-53 keto and the C-15 hydroxyl groups and methanol. In another embodiment it is not.

Variable p suitably represents 0 or 1. In one embodiment p represents 0 in another embodiment p represents 1 . In another embodiment p represents 2.

When R-i or R₂ represents -alkylaryl, an example includes Ci_-2alkylaryl e.g. benzyl. When R-i or R₂ represents -alkenylaryl, an example includes C_2-3alkenylaryl e.g. - ethenylphenyl.

When R-i or R₂ represents -alkylheteroaryl, an example includes Ci_-2alkylheteraryl e.g. -methylpyridinyl.

When R-i or R₂ represents -alkenylheteroaryl, an example includes C_{2- 3}alkenylheteroaryl e.g. -ethenylpyridinyl. Suitably Ri and R₂ independently represent hydrogen; or an alkyl or alkenyl group which may optionally be joined to form a saturated or unsaturated heterocyclic ring containing the nitrogen atom shown and wherein one or more carbon atoms of Ri and/or R₂ are optionally replaced by a heteroatom selected from O, N and S(0)_p in which p represents 0, 1 or 2 and wherein one or more carbon atoms of Ri and/or R₂ are optionally replaced by carbonyl.

According to the invention, R-i and R₂ may independently represent hydrogen; or an alkyl or alkenyl group which may optionally be joined to form a saturated or unsaturated heterocyclic ring containing the nitrogen atom shown and wherein one or more (e.g. one or two, such as one) carbon atoms of Ri and/or R₂ are optionally replaced by a heteroatom selected from O, N and S(0)_p in which p represents 0, 1 or 2 and wherein one or more (e.g. one or two, such as one) carbon atoms of Ri and/or R₂ are optionally replaced by carbonyl or one of Ri and R₂ represents -alkylaryl, -alkenylaryl, -alkylheteroaryl or -alkenylheteroaryl and the other represents H, alkyl or alkenyl.

When R-i and R₂ independently represent hydrogen; or an alkyl or alkenyl group wherein one or more (e.g. one or two such as one) carbon atoms of Ri and/or R₂ are optionally replaced by a heteroatom selected from O, N and S(0)_p and wherein one or more (e.g. one or two such as one) carbon atoms of Ri and/or R₂ are optionally replaced by carbonyl; examples include:

(a) those embodiments in which Ri represents hydrogen and R₂ represents an alkyl group;

(b) those embodiments in which Ri represents hydrogen and R₂ represents an alkenyl group;

(c) those embodiments in which R-i represents hydrogen and R₂ represents an alkyl group and wherein one or more (e.g. one or two, such as one) carbon atoms of Ri and/or R₂ are replaced by carbonyl;

(d) those embodiments in which R-i represents hydrogen and R₂ represents an alkyl group and wherein one or more (e.g. one or two, such as one) carbon atoms of Ri and/or R₂ are replaced by a heteroatom selected from O, N and S(0)_p; and

(e) those embodiments in which R-i represents hydrogen and R₂ represents an alkyl group and wherein one or more (e.g. one or two, such as one) carbon atoms of Ri and/or R₂ are replaced by a heteroatom selected from O, N and S(0)_p; and wherein one or more (e.g. one or two, such as one) carbon atoms of Ri and/or R₂ are replaced by a heteroatom selected from O, N and S(0)_p. When one of Ri and R₂ represents -alkylaryl, -alkenylaryl, -alkylheteroaryl or - alkenylheteroaryl and the other represents H, alkyl or alkenyl; examples include:

(f) those embodiments in which R-i represents hydrogen and R₂ represents an - alkylaryl group; and (g) those embodiments in which R-i represents hydrogen and R₂ represents an - alkylheteroaryl group.

When Ri and R₂ independently represent an alkyl or alkenyl group which is joined to form a saturated or unsaturated heterocyclic ring containing the nitrogen atom shown and wherein one or more (e.g. one or two, such as one) carbon atoms of Ri and/or R₂ are optionally replaced by a heteroatom selected from O, N and S(0)_p in which p represents 0, 1 or 2 and wherein one or more (eg one or two, such as one) carbon atoms of Ri and/or R₂ are optionally replaced by carbonyl; examples include:

(h) those embodiments in which R-i and R₂ independently represent an alkyl group which are joined to form a saturated heterocyclic ring containing the nitrogen atom shown;

(i) those embodiments in which R-i and R₂ independently represent an alkyl or alkenyl group which are joined to form an unsaturated heterocyclic ring containing the nitrogen atom shown;

(j) those embodiments in which Ri and R₂ independently represent an alkyl or alkenyl group which are joined to form a saturated or unsaturated heterocyclic ring containing the nitrogen atom shown and wherein one or more (e.g. one or two, such as one) carbon atoms of Ri and/or R₂ are replaced by a heteroatom selected from O, N and S(0)_p;

(k) those embodiments in which Ri and R₂ independently represent an alkyl or alkenyl group which are joined to form a saturated or unsaturated heterocyclic ring containing the nitrogen atom shown and wherein one or more (e.g. one or two, such as one) carbon atoms of Ri and/or R₂ are replaced by carbonyl; and

(I) those embodiments in which R-i and R₂ independently represent an alkyl or alkenyl group which are joined to form a saturated or unsaturated heterocyclic ring containing the nitrogen atom shown and wherein one or more (e.g. one or two, such as one) carbon atoms of Ri and/or R₂ are replaced by a heteroatom selected from O, N and S(0)_p and wherein one or more (e.g. one or two, such as one) carbon atoms of Ri and/or R₂ are replaced by carbonyl.

When a carbon atom of Ri or R₂ is replaced by a heteroatom, it is suitably replaced by O or N, especially N.

When a carbon atom of Ri or R₂ is replaced by a carbonyl, the carbonyl is suitably located adjacent to another carbon atom or a nitrogen atom. Suitably carbonyl groups are not located adjacent to sulphur or oxygen atoms. Heterocyclic rings formed when R-i and R₂ are joined typically contain 4-8 ring atoms, e.g. 5-7 ring atoms, particularly 5 or 6 ring atoms.

Heterocyclic rings formed when R-i and R₂ are joined typically contain only the nitrogen atom shown or one or two (e.g. one) additional heteroatom, especially a nitrogen or oxygen atom.

When R-i and/or R₂ contain more than one heteroatom, these should typically be separated by two or more carbon atoms.

If -CH₃ is replaced by N, the group formed is -NH₂-. If -CH₂- is replaced by N, the group formed is -NH-. If -CHR- is replaced by N the group formed is -NR-. Hence nitrogen atoms within R-i and R₂ may be primary, secondary or tertiary nitrogen atoms.

In one embodiment R-i represents hydrogen. In another embodiment, neither R-i nor R₂ represents hydrogen. In another embodiment R-i and R₂ are not both hydrogen.

Suitably R-i and R₂ are selected from hydrogen, C i_-4 alkyl and C i_-4 alkenyl.

Suitably R-i and R₂ are selected from hydrogen, C i_-4 alkyl and C i_-4 alkenyl

provided that R-i and R₂ do not both represent hydrogen.

Alternatively, suitably R-i and R₂ together with the nitrogen to which they are attached represent a 5-7 membered heterocyclic ring, such as a pyrrolidine, piperidine, morpholine or piperazine ring in which the 4-nitrogen of piperazine is optionally substituted by

In another embodiment, suitably R-i and R₂ together with the nitrogen to which they are attached represent a 5-7 membered heterocyclic ring, such as a pyrrolidine, piperidine, morpholine or piperazine ring in which the 4-nitrogen of piperazine is optionally substituted by C1 - 4alkyl, and in which a carbon atom adjacent to a nitrogen atom within the ring is replaced with carbonyl. Thus, for example, R-i and R₂ together with the nitrogen to which they are attached represent piperidinone.

In one embodiment X represents CH₂. In another embodiment X represents CO.

In one suitable embodiment of the invention, R-i and R₂ represent CH₃ e.g. as represented by the following structure:

In another suitable embodiment of the invention, R-i represents H and R₂ represents 2CH=CH₂ e.g. as represented by the following structure:

In general, the compounds of the invention are prepared by semi-synthetic derivatisation of a sanglifehrin. Sanglifehrins may be prepared using methods described in WO97/02285 and WO98/07743, which documents are incorporated in their entirety, or additional methods described herein. Semisynthetic methods for generating the sanglifehrin macrocylic aldehyde are described in US6,124,453, Metternich et al., 1999, Banteli et al., 2001 and Sedrani et al., 2003.

In general, a process for preparing certain compounds of formula (I) or a pharmaceutically acceptable salt thereof comprises:

(a) dihydroxylation of sanglifehrin A across the C-26,27 double bond

(b) oxidative cleavage of the 1 ,2-diol to yield an aldehyde

(c) reductive amination of the aldehyde with ammonia or a primary or secondary amine (optionally in the form of an acid salt). This is shown retrosynthetically below:

Hence, a process for preparing compounds of the invention comprises reducing the product of reacting a compound of formula R₁R₂NH with aldehydic macrocycle.

The preparation of the aldehydic macrocycle has been described previously (Metternich et al. 1999). Briefly, a sanglifehrin, such as SfA, is dihydroxylated using modified Sharpless conditions (catalytic osmium tetroxide). The use of the chiral ligands aids in promoting selectivity. The resultant 26,27-dihydroxylsanglifehrin can then be cleaved oxidatively, using for instance sodium periodate. The resultant aldehydic macrocycle can then be used as a substrate for reductive amination. Typically the aldehyde is treated with an amine before the introduction of a reducing agent, typically sodium cyanoborohydride. The imine that results from the coupling of the aldehyde and amine is then reduced to an amine. The use of the correct stoichiometeries of amine and / or reducing agent is necessary in order to prevent reaction at the less reactive C-53 ketone.

Reductive amination is typically performed by combining the aldehydic macrocycle with the primary or secondary amine R₁R₂NH to form an imine in an inert organic solvent (such as THF) and then treating with a reducing agent such as sodium cyanoborohydride or sodium

triacetoxyborohydride. The reaction may typically be performed at room temperature in one pot so that imine formation and reduction occur concurrently. The amine R₁R₂NH may typically be employed in the form of an acid salt such as the HCI salt. The reaction mixture may be worked up with acidic aqueous medium such as ammonium chloride.

In general, a process for preparing certain other compounds of formula (I) or a

pharmaceutically acceptable salt thereof comprises:

(a) dihydroxylation of sanglifehrin A across the C-26,27 double bond

(b) oxidative cleavage of the 1 ,2-diol to yield an aldehyde

(c) oxidation of the aldehyde to a carboxylic acid ("acid macrocycle")

(d) reacting the carboxylic acid with an amine (optionally in the form of an acid salt) to form an amide.

Optionally the carboxylic acid may be employed in the form of an activated derivative, such as an anhydride or acid halide.

This is shown retrosynthetically below:

Hence, a process for preparing certain compounds of the invention comprises reacting a compound of formula R-|R₂NH with acid macrocycle. The acid macrocycle can be prepared from the aldehydic macrocycle. The preparation of the aldehydic macrocycle has been described previously (Metternich et al. 1999) and is described above. The acid macrocycle can then be coupled to an amine by standard peptide bond forming reactions. Exemplary conditions include combination of the reagents in an organic solvent (e.g. methylene chloride, dimethylformamide (DMF) or tetrahydrofuran ( THF)) in the presence of acid or base catalysis followed by aqueous work-up.

If desired or necessary, protecting groups may be employed to protect functionality in the aldehydic macrocycle, acid macrocycle or the amine, as described in T. W. Green, P. G. M. Wuts, Protective Groups in Organic Synthesis, Wiley-lnterscience, New York, 1999, 49-54, 708-71 1 .

The methanol adduct may be prepared by fermentation and isolation from broth, or may be prepared from sanglifehrin A (WO97/02285).

Amine compounds of formula R₁R₂NH are either known or may be prepared by conventional methods.

In addition to the specific methods and references provided herein a person of skill in the art may also consult standard textbook references for synthetic methods, including, but not limited to Vogel's Textbook of Practical Organic Chemistry (Furniss et al., 1989) and March's Advanced Organic Chemistry (Smith and March, 2001 ).

It is obvious to someone skilled in the art that these compounds can be synthesised de novo from commercially available compounds, i.e. total synthesis. The synthesis of the tripeptide and subsequent macrocycle formation has been described (Cabrejas et al, 1999). A process such as this could be modified to generate compounds of the invention.

Other compounds of the invention may be prepared by methods known per se or by methods analogous to those described above.

A sanglifehrin macrocycle may be administered alone or in combination with other therapeutic agents. Co-administration of two (or more) agents allows for lower doses of each to be used, thereby reducing side effect, can lead to improved potency and therefore higher SVR, and a reduction in resistance.

Therefore in one embodiment, the sanglifehrin macrocycle is co-administered with one or more therapeutic agent s for the treatment of HCV infection, taken from the standard of care treatments. This could be an interferon (e.g. pIFNa and/or ribavirin.

In an alternative embodiment, a sanglifehrin macrocycle is co-administered with one or more other anti-viral agents, such as a STAT-C (specifically targeted agent for treatment of HCV), which could be one or more of the following: Non-nucleoside Polymerase inhibitors (e.g. IDX375, VCH-222, Bl 207127, ANA598, VCH-916), Nucleoside or nucleotide polymerase inhibitors (e.g. 2'- C-methylcytidine, 2'-C-methyladenosine, R1479, PSI-6130, R7128, R1626), Protease inhibitors (e.g. BILN-2061 , VX-950(Telaprevir), SCH503034(Boceprevir), TMC435350, MK-7009,

R7227/ITMN-191 , EA-058, EA-063) or viral entry inhibitors (e.g. PRO 206). The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (compound of the invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compounds of the invention will normally be administered orally in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a nontoxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of

administration, the compositions may be administered at varying doses.

For example, the compounds of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications.

Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the

compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium

carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.

Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a

predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

Advantageously, agents such as preservatives and buffering agents can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. The dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use.

The dosage to be administered of a compound of the invention will vary according to the particular compound, the disease involved, the subject, and the nature and severity of the disease and the physical condition of the subject, and the selected route of administration. The appropriate dosage can be readily determined by a person skilled in the art.

The compositions may contain from 0.1 % by weight, preferably from 5-60%, more preferably from 10-30% by weight, of a compound of invention, depending on the method of administration.

It will be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of a compound of the invention will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the age and condition of the particular subject being treated, and that a physician will ultimately determine appropriate dosages to be used. This dosage may be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be altered or reduced, in accordance with normal clinical practice.

Further aspects of the invention include:

-A compound according to the invention for use as a pharmaceutical;

-A compound according to the invention for use as a pharmaceutical for the treatment of viral infections (especially RNA virus infections) such as HCV or HIV infection or other diseases such as muscular dystrophy;

-A pharmaceutical composition comprising a compound according to the invention together with a pharmaceutically acceptable diluent or carrier; -A pharmaceutical composition comprising a compound according to the invention together with a pharmaceutically acceptable diluent or carrier further comprising a second or subsequent active ingredient, especially an active ingredient indicated for the treatment of viral infections such as HCV or HIV infection or muscular dystrophy;

-A method of treatment of viral infections (especially RNA virus infections) such as HCV or HIV infection or muscular dystrophy which comprises administering to a subject a therapeutically effective amount of a compound according to the invention;

-Use of a compound according to the invention for the manufacture of a medicament for the treatment of viral infections such as HCV or HIV infection or muscular dystrophy.

General Methods

Materials and Methods Bacterial strains and growth conditions

The sanglifehrin producer Streptomyces sp. A92-3081 10 (DSM no 9954, purchased from DSMZ, Braunschweig, Germany) also termed BIOT-4253 and BIOT-4370 is maintained on medium oatmeal agar, MAM, or ISP2 (see below) at 28 °C.

Streptomyces sp. A92-3081 10 was grown on oatmeal agar at 28 °C for 7-10 days. Spores from the surface of the agar plate were collected into 20% w/v sterile glycerol in distilled and stored in 0.5-ml aliquots at -80 °C. Frozen spore stock was used for inoculating seed media SGS or SM25- 3. The inoculated seed medium was incubated with shaking between 200 and 300 rpm at 5.0 or 2.5 cm throw at 27 °C for 24 hours. The fermentation medium SGP-2 or BT6 were inoculated with 2.5%- 10% of the seed culture and incubated with shaking between 200 and 300 rpm with a 5 or 2.5 cm throw at 24 °C for 4-5 days. The culture was then harvested for extraction.

Media Recipes

Water used for preparing media was prepared using Millipore Elix Analytical Grade Water Purification System

SGS Seed Medium

Ingredient (and supplier) Recipe

Glucose (Sigma, G7021 ) 7.50 g

Glycerol (Fisher scientific, G/0650/25) 7.50 g

yeast extract (Becton Dickinson, 212770) 1.35 g

malt extract (Becton Dickinson, 218630) 3.75 g

potato starch (soluble) (Signma, S2004) 7.50 g NZ-amine A (Sigma, C0626) 2.50 g toasted soy flour, Nutrisoy (ADM, 063-160) 2.50 g

L-asparagine (Sigma, A0884) 1.00 g

CaC0₃ (Calcitec, V/40S) 0.05 g

NaCI (Fisher scientific, S/3160/65) 0.05 g

KH₂PO₄ (Sigma, P3786) 0.25 g

K₂HPO₄ (Sigma, P5379) 0.50 g

MgS0₄.7H₂0 (Sigma, M7774) 0.10 g trace element solution B 1.00 ml_ agar 1.00 g

SAG471 Antifoam (GE Silicones, SAG471 ) * 0.20 ml_

RO H₂0 to final vol. of ** 1.00 L pre-sterilisation pH was adjusted to pH 7.0 with 10M NaOH/10M H₂S0₄

sterilised by heating 121 °C, 20-30 min (autoclaving) Notes

^* antifoam only used in seed fermenters, NOT seed flasks ^** final volume adjusted accordingly to account for seed volume

Trace Element Solution B

Ingredient Recipe

FeS0₄.7H₂0 (Sigma, F8633) 5.00 g

ZnS0₄.7H₂0 (Sigma, Z0251 ) 4.00 g

MnCI₂.4H₂0 (Sigma, M8530) 2.00 g

CuS0₄.5H₂0 (Aldrich, 20,919-8) 0.20 g

(ΝΗ₄)₆Μθ7θ₂4 (Fisher scientific, A/5720/48) 0.20 g

CoCI₂.6H₂0 (Sigma, C2644) 0.10 g

H3BO3 (Sigma, B6768) 0.10 g

Kl (Alfa Aesar, A12704) 0.05 g

H₂S0₄ (95%) (Fluka, 84720) 1.00 ml_

RO H₂0 to final vol. of 1.00 L SGP2 Production Medium

Ingredient Recipe

toasted soy flour (Nutrisoy) (ADM, 063-160) 20.00 g

Glycerol (Fisher scientific, G/0650/25) 40.00 g

MES buffer (Acros, 172595000) 19.52 g

SAG471 Antifoam (GE Silicones, SAG471 ) *0.20 mL

RO H₂0 to final vol. of 1.00 L pre-sterilisation pH adjusted to pH 6.8 with 10M NaOH

sterilised by heating 121 °C, 20-30 min (autoclaving)

Notes

^* final volume adjusted accordingly to account for seed

volume

^** antifoam was used only in fermentors not flasks

Analysis of culture broths by LC-UV and LC-UV-MS

Culture broth (1 mL) and ethyl acetate (1 mL) is added and mixed for 15-30 min followed by centrifugation for 10 min. 0.4 mL of the organic layer is collected, evaporated to dryness and then re-dissolved in 0.20 mL of acetonitrile.

HPLC conditions:

C18 Hyperclone BDS C18 Column 3u, 4.6 mm x 150 mm

Fitted with a Phenomenex Analytical C18 Security Guard Cartridge (KJO-4282)

Column temp at 50 °C

Flow rate 1 mL/min

Monitor UV at 240 nm

Inject 20 uL aliquot Solvent gradient:

0 min: 55% B

1 .0 min: 55% B

6.5 min: 100% B

10.0 min: 100% B

10.05 min: 55% B

13.0 min: 55% B Solvent A is Water + 0.1 % Formic Acid

Solvent B is Acetonitrile + 0.1 % Formic Acid

Under these conditions SfA elutes at 5.5 min

Under these conditions SfB elutes at 6.5 min

LCMS is performed on an integrated Agilent HP1 100 HPLC system in combination with a Bruker Daltonics Esquire 3000+ electrospray mass spectrometer operating in positive ion mode using the chromatography and solvents described above.

Synthesis

All reactions are conducted under anhydrous conditions unless stated otherwise, in oven dried glassware that is cooled under vacuum, using dried solvents. Reactions are monitored by LC-UV-MS, using an appropriate method, for instance the method described above for monitoring culture broths.

QC LC-MS method

HPLC conditions:

C18 Hyperclone BDS C18 Column 3u, 4.6 mm x 150 mm

Fitted with a Phenomenex Analytical C18 Security Guard Cartridge (KJO-4282)

Column temp at 50 °C

Flow rate 1 mL/min

Monitor UV at 210, 240 and 254 nm

Solvent gradient:

0 min: 10% B

2.0 min: 10% B

15 min: 100% B

17 min: 100% B

17.05 min: 10% B

20 min: 10% B

Solvent A is Water + 0.1 % Formic Acid

Solvent B is Acetonitrile + 0.1 % Formic Acid MS conditions

MS operates in switching mode (switching between positive and negative), scanning from 150 to 1500 amu.

In vitro replicon assay for assessment of HCV antiviral activity

Antiviral efficacy against genotype 1 HCV may be tested as follows: One day before addition of the test article, Huh5.2 cells, containing the HCV genotype 1 b l389luc-ubi-neo/NS3- 375.1 replicon (Vrolijk et al., 2003) and subcultured in cell growth medium [DMEM (Cat No. 41965039) supplemented with 10% FCS, 1 % non-essential amino acids (1 1 140035), 1 % penicillin/streptomycin (15140148) and 2% Geneticin (10131027); Invitrogen] at a ratio of 1.3- 1 .4 and grown for 3-4 days in 75cm² tissue culture flasks (Techno Plastic Products), were harvested and seeded in assay medium (DMEM, 10% FCS, 1 % non-essential amino acids, 1 % penicillin/streptomycin) at a density of 6 500 cells/well (Ι ΟΟμ-Jwell) in 96-well tissue culture microtitre plates (Falcon, Beckton Dickinson for evaluation of the anti-metabolic effect and

CulturPlate, Perkin Elmer for evaluation of antiviral effect). The microtitre plates are incubated overnight (37°C, 5% C0₂, 95-99% relative humidity), yielding a non-confluent cell monolayer. Dilution series are prepared; each dilution series is performed in at least duplicate. Following assay setup, the microtitre plates are incubated for 72 hours (37°C, 5% C0₂, 95-99% relative humidity).

For the evaluation of anti-metabolic effects, the assay medium is aspirated, replaced with 75μΙ_ of a 5% MTS (Promega) solution in phenol red-free medium and incubated for 1 .5 hours (37°C, 5% C0₂, 95-99% relative humidity). Absorbance is measured at a wavelength of 498nm (Safire², Tecan) and optical densities (OD values) are converted to percentage of untreated controls.

For the evaluation of antiviral effects, assay medium is aspirated and the cell monolayers are washed with PBS. The wash buffer is aspirated, 25μΙ_ of Glo Lysis Buffer (Cat. N°. E2661 , Promega) is added after which lysis is allowed to proceed for 5min at room temperature. Subsequently, 50μί of Luciferase Assay System (Cat. N°. E1501 , Promega) is added and the luciferase luminescence signal is quantified immediately (1000ms integration time/well, Safire², Tecan). Relative luminescence units are converted to percentage of untreated controls.

The EC50 and EC90 (values derived from the dose-response curve) represent the concentrations at which respectively 50% and 90% inhibition of viral replication would be observed. The CC50 (value derived from the dose-response curve) represents the

concentration at which the metabolic activity of the cells would be reduced to 50 % of the metabolic activity of untreated cells. The selectivity index (SI), indicative of the therapeutic window of the compound, is calculated as CC50/EC50.

A concentration of compound is considered to elicit a genuine antiviral effect in the HCV replicon system when, at that particular concentration, the anti-replicon effect is above the 70% threshold and no more than 30% reduction in metabolic activity is observed.

Assessment of water solubility

Water solubility may be tested as follows: A 10 mM stock solution of the sanglifehrin analogue is prepared in 100% DMSO at room temperature. Triplicate 0.01 mL aliquots are made up to 0.5 mL with either 0.1 M PBS, pH 7.3 solution or 100% DMSO in amber vials. The resulting 0.2 mM solutions are shaken, at room temperature on an IKA® vibrax VXR shaker for 6 h, followed by transfer of the resulting solutions or suspensions into 2 mL Eppendorf tubes and centrifugation for 30 min at 13200 rpm. Aliquots of the supernatant fluid are then analysed by the LCMS method as described above.

Assessment of cell permeability

Cell permeability may be tested as follows: The test compound is dissolved to 10mM in DMSO and then diluted further in buffer to produce a final 10μΜ dosing concentration. The fluorescence marker lucifer yellow is also included to monitor membrane integrity. Test compound is then applied to the apical surface of Caco-2 cell monolayers and compound permeation into the basolateral compartment is measured. This is performed in the reverse direction (basolateral to apical) to investigate active transport. LC-MS/MS is used to quantify levels of both the test and standard control compounds (such as Propanolol and Acebutolol). In vivo assessment of pharmacokinetics

In vivo assays may also be used to measure the bioavailability of a compound.

Generally, a compound is administered to a test animal (e.g. mouse or rat) both intravenously (i.v.) and orally (p.o.) and blood samples are taken at regular intervals to examine how the plasma concentration of the drug varies over time. The time course of plasma concentration over time can be used to calculate the absolute bioavailability of the compound as a percentage using standard models. An example of a typical protocol is described below.

Mice are dosed with 1 , 10, or 100 mg/kg of the compound of the invention or the parent compound i.v. or p.o.. Blood samples are taken at 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 360, 420 and 2880 minutes and the concentration of the compound of the invention or parent compound in the sample is determined via HPLC. The time-course of plasma concentrations can then be used to derive key parameters such as the area under the plasma concentration- time curve (AUC - which is directly proportional to the total amount of unchanged drug that reaches the systemic circulation), the maximum (peak) plasma drug concentration, the time at which maximum plasma drug concentration occurs (peak time), additional factors which are used in the accurate determination of bioavailability include: the compound's terminal half life, total body clearance, steady-state volume of distribution and F%. These parameters are then analysed by non-compartmental or compartmental methods to give a calculated percentage bioavailability, for an example of this type of method see Egorin et al. 2002, and references therein.

In vitro assessment of inhibition of MDRI and MRP2 transporters

To assess the inhibition and activation of the MDR1 (P-glycoprotein 1 ) and MRP2 transporters, an in vitro ATPase assay from Solvo Biotechnology Inc. can be used (Glavinas et al., 2003). The compounds (at 0.1 , 1 , 10 and 100μΜ) are incubated with MDR1 or MRP2 membrane vesicles both in the absence and presence of vanadate to study the potential ATPase activation. In addition, similar incubations are conducted in the presence of verapamil/sulfasalazine in order to detect possible inhibition of the transporter ATPase activity. ATPase activity is measured by quantifying inorganic phosphate spectrophotometrically.

Activation is calculated from the vanadate sensitive increase in ATPase activity. Inhibition is determined by decrease in verapamil/sulfasalazine mediated ATPase activity.

EXAMPLES

Example 1 - Production of sanglifehrin A and its natural congers in 15-L stirred bioreactors with secondary seed

Vegetative cultures were prepared by inoculating 0.2 mL from a spore stock of

Streptomyces sp. A92-3081 10 into 400ml_ seed medium SGS in 2-L Erlenmeyer flasks with foam plugs.

The culture flasks were incubated at 27°C, 250 rpm (2.5 cm throw) for 24 h.

From the seed culture, 300 mL was transferred into 15 litres of primary seed medium SGS containing 0.02% antifoam SAG 471 , in a 15 L Braun fermentor. The fermentation was carried out for 24 hours at 27 °C, with starting agitation set at≥ 300rpm aeration rate at 0.5 VA /M and dissolved oxygen (DO) level controlled with the agitation cascade at >30% air saturation.

From the secondary seed culture prepared in the fermentor, 600 mL was taken under aseptic conditions and transferred into 15 litres of production medium SGP-2 containing 0.02% antifoam SAG 471 , in 15 L Braun fermentor. The fermentation was carried out for 5 days at 24 °C, with starting agitation set at 300 rpm, aeration rate at 0.5 VA /M and dissolved oxygen (DO) level controlled with the agitation cascade at >30% air saturation.

SfA was seen to be produced at 10-20 mg/L in fermentation broths. Example 2 - Extraction and purification of sanglifehrin A

The whole broth (30 L) was clarified by centrifugation. The resulting cell pellet was extracted twice with ethyl acetate (2 x 10 L), each by stirring for 1 hour with overhead paddle stirrer and leaving to settle before pumping off solvent. The ethyl acetate layers were then combined (-20 L) and the solvent removed under reduced pressure at 40°C to obtain an oily residue. This oily residue was then suspended in 80:20 methanokwater (total volume of 500 ml_), and twice extracted with hexane (2 x 500 ml_). The 80:20 methanokwater fraction was then dried under reduced pressure to yield a crude dry extract which contained SfA and SfB.This extract was dissolved in methanol (100 ml), mixed with 15 g Silica gel and dried to a powder. The powder was loaded into a silica gel column (5 x 20 cm) packed in 100% CHCI₃. For every one litre of elution solvent the methanol concentration was increased stepwise by 1 % and 250 ml fractions collected. After three litres of solvent elution the methanol concentration was increased stepwise by 2% up to 8%. Fractions containing SfA and / or SfB were combined and reduced in vacuo to dryness and SfA and SfB purified by preparative HPLC. Preparative HPLC was achieved over a Waters XTerra Prep MS C18 OBD 10mm (19 x 250 mm) column running with solvent A (water) and solvent B (acetonitrile) at 20 ml/min with the following timetable: t = 0 mins, 55% B

t = 4 mins, 55% B

t = 30mins, 100% B

t = 32mins, 100% B

t = 36mins, 55% B

Fractions containing SfA were combined and taken to dryness.

Example 3 - Synthesis of 8 (aldehydic macrocycle)

-dihydroxysanglifehrin, 9

sanglifehrin A (SFA), 5 26, 27-dihydroxysanglifehrin, 9 To a stirred solution of sanglifehrin A, 5 (135 mg, 0.1238 mmol), (DHQ)₂PHAL (5.76 mg, 0.0074 mmol), 2.5 wt % solution of osmium tetroxide in ie f-butyl alcohol (47 uL, 0.0037 mmol), and methanesulfonamide (23.6 mg, 0.2476 mmol) in ie f-butyl alcohol (4 ml.) were added at room temperature together with a solution of potassium ferricyanide (122.3 mg, 0.3714 mmol) and potassium carbonate (51.3 mg, 0.3714 mmol) in 4 mL of water. After stirring for 1 h, a solution of saturated aqueous sodium sulfite (187.3 mg, 1.4857 mmol) was added. The resulting mixture was stirred for 30 min and then extracted with three portions of ethyl acetate. The organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by Combiflash using reverse phase column (C18 column, A = H₂0, B = acetonitrile, t = 2 min, B = 0%; t = 4 min, B = 30%, t = 9min, B = 35%; t = 12min, B = 45%; t = 16 min, B = 70%) to afford 26,27-dihydroxysanglifehrin, 9 (102 mg, 70 %) as a white solid. QC LC-MS, RT = 5.3 mins, m/z = 1 124.8 [M+H]⁺, 1 122.7 [M- H]-

2. The preparation of the aldehydic macrocycle, 8

26, 27-dihydroxysanglifehrin, 9 aldehydic macrocycle, 8

To a solution of 26,27-dihydroxysanglifehrin, 9 (60.0 mg, 0.053 mmol) in THF and water (2:1 , 5 ml_ ) was added sodium periodate (22.8 mg,0.107 mmol). The resulting mixture was stirred at room temperature for 2 h, and saturated aqueous sodium bicarbonate was added. This mixture was extracted with three portions of ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by Combiflash using reverse phase column (C18 column, A = water, B = CH₃CN, t = 3 min, B = 0%; t = 12 min, B = 40 %; t = 17 min, B = 40%, t = 21 min, B = 70%) to afford the aldehydic macrocycle, 8 (35 mg, 90 %) as a white solid. QC LC-MS, RT = 4.0 mins, m/z = 761 .4 [M+Na]⁺, 737.3 [M-H]^" Exa

aldehydic macrocycle, 8 10

To a solution of aldehydic macrocycle, 8 (20 mg, 0.0271 mmol) in THF (4 ml) was added prop-2-en-1 -amine (3.0 ul, 0.0406 mmol) and the mixture was stirred at room temperature for 1 .5 h, then cooled to 0 °C and a solution of NaCNBH₃ in THF (20 ul, 1 mg/10 ul, 0.0318 mmol) was added. After the mixture was stirred about 16 h at room temperature, further portions of NaCNBH₃ in THF (5 ul, 1 mg/10 ul, 0.0079 mmol) was added, and stirring was continued for another 2h. The reaction was quenched by the addition of water (0.5 ml) and purified directly by preparative HPLC (Shimadzu C18 column, 20 mm x 250 mm, A = 0.05%HCO₂H/H₂O, B = CH₃CN, t = 0-3 min, 15 % B; t = 6-9 min, 35% B; t = 13-18 min., 95% B; 30 ml/min) to give about 6 mg of 10 (Yield = 28%). QC LC-MS, RT = 8.1 mins, m/z = 781.1 [M+H]⁺, 778.9 [M-H]^"

aldehydic macrocycle, 8 n

To a solution of aldehydic macrocycle, 8 (18 mg, 0.024 mmol) in THF (2.5 ml) was added dimethylamine hydrochloride (8 mg 0.098 mmol). After the mixture was stirred at room temperature for 50 min, sodium cyanoborohydride (1 .6 mg, 0.0255 mmol) in THF (1 ml) was added. The mixture was stirred for another 24h, then aq.NH₄CI (5 ml) was added; the mixture was purified by prep-HPLC (YMC-C18, 20 X 250 mm, 5 urn; 30 ml/min; 15%~45% CH₃CN + 0.5 % formic acid over 15 min gradient) to give about 3 mg of 11 . Example 6 - Biological data - In vitro evaluation of HCV antiviral activity in the replicon system Compounds were analysed in the replicon assay as described in the General Methods. Cyclosporine A, 1 and sanglifehrin B, 7 were included as a comparison.

Analysis of the activity against the replicon for 10 across a concentration gradient revealed that at 1 μΜ there was >70% inhibition of the replicon, whilst the cell viability was still 100% of the control. Therefore, 10 is proven to be active in the replicon according to standard criteria (see figure 2).

References

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All references including patent and patent applications referred to in this application are incorporated herein by reference to the fullest extent possible.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer or step or group of integers but not to the exclusion of any other integer or step or group of integers or steps.

Claims

Claims

1 . A compound according to formula (I) below, or a pharmaceutically acceptable salt

thereof:

wherein:

X represents CH₂ or CO

Ri and R₂ independently represent hydrogen; or an alkyl or alkenyl group which may optionally be joined to form a saturated or unsaturated heterocyclic ring containing the nitrogen atom shown and wherein one or more carbon atoms of Ri and/or R₂ are optionally replaced by a heteroatom selected from O, N and S(0)_p in which p represents 0, 1 or 2 and wherein one or more carbon atoms of Ri and/or R₂ are optionally replaced by carbonyl; or one of Ri and R₂ represents -alkylaryl, -alkenylaryl, -alkylheteroaryl or -alkenylheteroaryl and the other represents H, alkyl or alkenyl;

including any tautomer thereof; and including a methanol adduct thereof in which a ketal is formed by the combination of the C-53 keto and the C-15 hydroxyl groups and methanol.

2. A compound according to claim 1 wherein R-i and R₂ independently represent hydrogen; or an alkyl or alkenyl group wherein one or two carbon atoms of Ri and/or R₂ are optionally replaced by a heteroatom selected from O, N and S(0)_p and wherein one or two carbon atoms of Ri and/or R₂ are optionally replaced by carbonyl.

3. A compound according to claim 2 wherein R-i represents hydrogen and R₂ represents an alkyl group.

4. A compound according to claim 2 wherein Ri represents hydrogen and R₂ represents an alkenyl group.

5. A compound according to claim 1 wherein Ri and R₂ independently represent an alkyl or alkenyl group which is joined to form a saturated or unsaturated heterocyclic ring containing the nitrogen atom shown and wherein one or two carbon atoms of Ri and/or R₂ are optionally replaced by a heteroatom selected from O, N and S(0)_p in which p represents 0, 1 or 2 and wherein one or two carbon atoms of Ri and/or R₂ are optionally replaced by carbonyl.

6. A compound according to claim 5 wherein R-i and R₂ independently represent an alkyl group which are joined to form a saturated heterocyclic ring containing the nitrogen atom shown.

7. A compound according to claim 5 wherein R-i and R₂ independently represent an alkyl or alkenyl group which are joined to form a saturated or unsaturated heterocyclic ring containing the nitrogen atom shown and wherein one or two carbon atoms of Ri and/or R₂ are replaced by a heteroatom selected from O, N and S(0)_p.

8. A compound according to any one of claims 1 to 5 wherein if a carbon atom is replaced with a heteroatom it is replaced with N or O.

9. A compound according to any one of claims 1 to 8 wherein X represents CH₂.

10. A compound according to claim 1 which

or a pharmaceutically acceptable salt thereof.

1 1 . A compound according to any one of claims 1 to 10 for use as a pharmaceutical.

12. A compound according to any one of claims 1 to 10 for use as a pharmaceutical for the treatment of viral infections such as HCV or HIV infection or muscular dystrophy.

13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 10 together with a pharmaceutically acceptable diluent or carrier.

14. A pharmaceutical composition comprising a compound according to any one of claims 1 to 10 together with a pharmaceutically acceptable diluent or carrier further comprising a second or subsequent active ingredient.

15. A method of treatment of viral infections such as HCV or HIV infection or muscular dystrophy which comprises administering to a subject a therapeutically effective amount of a compound according to any one claims 1 to 10.

16. A process for preparing a compound according to any one of claims 1 to 10 which comprises reducing the product of reacting a compound of formula R₁R₂N, wherein R-i and R₂ are defined in any one of claims 1 to 10, with aldehydic macrocycle.

17. A process for preparing a compound according to any one of claims 1 to 10 which comprises reacting a compound of formula R₁R₂NH, wherein R-i and R₂ are defined in any one of claims 1 to 10, with acid macrocycle.