# Patent

## Title
Sanglifehrin derivatives and methods for their production

## Abstract

There are provided inter alia compounds of formula (I) and (II)




and their use in therapy, particularly for the treatment of viral infection.

## Background
This application is §371 application of PCT/GB2011/052524, filed Dec. 20, 2011, which in turn claims priority to GB Application 1021522.6, filed Dec. 20, 2010, and GB Application 1113626.4, filed Aug. 8, 2011. The entire disclosure of each of the foregoing applications is incorporated by reference herein.
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/or as immunosuppressants e.g. for use in prophylaxis of transplant rejection and as anti-inflammatory agents, e.g. for use in inflammatory disorders. The present invention also provides methods for their use in medicine, in particular for the treatment of HCV infection and for use as an immunosuppressant or anti-inflammatory agent, or as intermediates in the generation of further medicinally useful compounds.
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-α (plFNα) 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), also known as direct acting antiviral (DAA) 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 PPlase 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; Gaither et al., 2010). 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 represents a novel and attractive host target for treating HCV infection, and a new potential addition to current SoC or STAT-C/DAA drugs, with the aim of increasing SVR, preventing emergence of resistance and lowering treatment side effects.







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), NIM811 (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 (PPlase) 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/DAA 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-811 (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, Wring et al., 2010). CsA class-related drug-drug interactions (DDIs) via inhibition of other drug transporters such as OAT1B1 and OAT1B3 (Konig et al., 2010) may also be a concern, potentially limiting certain combinations and use in some patients undergoing treatment for co-infections such as HIV (Seden et al., 2010).
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. A92-308110 (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).




Biosynthesis of Sanglifehrins

Sanglifehrins are biosynthesised by a mixed polyketide synthase (PKS)/Non-ribosomal peptide synthetase (NRPS) (see WO2010/034243, Qu et al., 2011). The 22-membered macrolide backbone consists of a polyketide carbon chain and a tripeptide chain. The peptide chain consists of one natural amino acid, valine, and two non-natural amino acids: (S)-meta-tyrosine and (S)-piperazic acid, linked by an amide bond. Hydroxylation of phenylalanine (either in situ on the NRPS or prior to biosynthesis) to generate (S)-meta-tyrosine is thought to occur via the gene product of sfaA.
Immunosuppressive action of Sanglifehrins
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 previously 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.
Converse to this, there is also an opportunity to develop immunosuppressive agents with low toxicity for use in such areas as prophylaxis of transplant rejection, autoimmune, inflammatory and respiratory disorders, including, but not limited to, Crohn's disease, Behcet syndrome, uveitis, psoriasis, atopic dermatitis, rheumatoid arthritis, nephritic syndrome, aplastic anaemia, biliary cirrhosis, asthma, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD) and celiac disease. Sanglifehrins have been shown to have a novel mechanism of immunosuppressive activity (Zenke et al., 2001), potentially acting through dendritic cell chemokines (Immecke et al., 2011) and there is therefore an opportunity to develop agents with a mechanism of action different to current clinical agents, such as cyclosporine A, rapamycin and FK506.
Other Therapeutic Uses of Cyclophilin Inhibitors
Human Immunodeficiency Virus (HIV)
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). However, when tested clinically, DEBIO-025 only reduced HIV-1 RNA levels ≧0.5 and >1 log 10 copies/mL in nine and two patients respectively, whilst 27 of the treated patients showed no reduction in HIV-1 RNA levels (Steyn et al., 2006). Following this, DEBIO-025 was trialed in HCV/HIV coinfected patients, and showed better efficacy against HCV, and the HIV clinical trials were discontinued (see Watashi et al., 2010).
Treatment of HIV
More than 30 million people are infected by HIV-1 worldwide, with 3 million new cases each year. Treatment options have improved dramatically with the introduction of highly active antiretroviral therapy (HAART) (Schopman et al., 2010), By 2008, nearly 25 antiretroviral drugs had been licensed for treatment of HIV-1, including nine nucleoside reverse transcriptase inhibitors (NRTI), four non-nucleoside reverse transcriptase inhibitors (NNRTI), nine protease inhibitors (PI), one fusion inhibitor, one CCR5 inhibitor and one integrase inhibitor (Shafer and Schapiro, 2008). However, none of these current regimens leads to complete viral clearance, they can lead to severe side effects and antiviral resistance is still a major concern. Therefore, there still remains a need for new antiviral therapies, especially in mechanism of action classes where there are no approved drugs, such as is the case for cyclophilin inhibitors.
Hepatitis B Virus
Hepatitis B is a DNA virus of the family hepadnaviridae, and is the causative agent of Hepatitis B. As opposed to the cases with HCV and HIV, there have been very few published accounts of activity of cyclophilin inhibitors against Hepatitis B virus. Ptak et al. 2008 have described weak activity of Debio-025 against HBV (IC50 of 4.1 μM), whilst Xie et al., 2007 described some activity of CsA against HBV (IC50 >1.3 μg/mL). This is in contrast to HIV and HCV, where there are numerous reports of nanomolar antiviral activity of cyclophilin inhibitors.
Treatment of HBV
HBV infects up to 400 million people worldwide and is a major cause of chronic viral hepatitis and hepatocellular carcinoma. As of 2008, there were six drugs licensed for the treatment of HBV; interferon alpha and pegylated interferon alpha, three nucleoside analogues (lamivudine, entecavir and telbivudine) and one nucleotide analogue (adefovir dipivoxil). However, due to high rates of resistance, poor tolerability and possible side effects, new therapeutic options are needed (Ferir et al., 2008).
Inhibition of the Mitochondrial Permeability Transition Pore (mPTP)
Opening of the high conductance permeability transition pores in mitochondria initiates onset of the mitochondrial permeability transition (MPT). This is a causative event, leading to necrosis and apoptosis in hepatocytes after oxidative stress, Ca2+ toxicity, and ischaemia/reperfusion. Inhibition of Cyclophilin D (also known as Cyclophilin F) by cyclophilin inhibitors has been shown to block opening of permeability transition pores and protects cell death after these stresses. Cyclophilin D inhibitors may therefore be useful in indications where the mPTP opening has been implicated, such as muscular dystrophy, in particular Ullrich congenital muscular dystrophy and Bethlem myopathy (Millay et al., 2008, WO2008/084368, Palma et al., 2009), multiple sclerosis (Forte et al., 2009), diabetes (Fujimoto et al., 2010), amyotrophic lateral sclerosis (Martin 2009), bipolar disorder (Kubota et al., 2010), Alzheimer's disease (Du and Yan, 2010), Huntington's disease (Perry et al., 2010), recovery after myocardial infarction (Gomez et al., 2007) and chronic alchohol consumption (King et al., 2010).
Further Therapeutic Uses
Cyclophilin inhibitors have potential activity against and therefore in the treatment of infections of other viruses, such as Varicella-zoster virus (Ptak et al., 2008), Influenza A virus (Liu et al., 2009), Severe acute respiratory syndrome coronavirus and other human and feline coronaviruses (Chen et al., 2005, Ptak et al., 2008), Dengue virus (Kaul et al., 2009), Yellow fever virus (Qing et al., 2009), West Nile virus (Qing et al., 2009), Western equine encephalitis virus (Qing et al., 2009), Cytomegalovirus (Kawasaki et al., 2007) and Vaccinia virus (Castro et al., 2003).
There are also reports of utility of cyclophilin inhibitors and cyclophilin inhibition in other therapeutic areas, such as in cancer (Han et al., 2009).
General Comments on Sanglifehrins
One of the issues in drug development of compounds such as sanglifehrins is rapid metabolism and glucuronidation, leading to low oral bioavailability. This can lead to an increased chance of food effect, more frequent incomplete release from the dosage form and higher interpatient variability.
Therefore there remains a need to identify novel cyclophilin inhibitors and anti-inflammatory agents, which may have utility, particularly in the treatment of HCV infection and anti-inflammatory conditions, 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 or where immunosuppression is useful. Preferably, such cyclophilin inhibitors have improved properties over the currently available cyclophilin inhibitors, including one or more of the following properties: longer half-life or increased oral bioavailability, possibly via reduced P450 metabolism and/or reduced glucuronidation, improved water solubility, improved potency against HCV, reduced toxicity (including hepatotoxicity), 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 (and/or selectivity) 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 mutasynthetic sanglifehrin analogues which, in at least some embodiments, have reduced metabolism via P450 or glucuronidation, for example as shown by increased microsome half-life and/or improved potency against HCV, for example as shown by a low replicon EC50 and/or increased selectivity index.
There is also a need to develop novel immunosuppressive agents, which may have utility in the prophylaxis of transplant rejection, or in the treatment of autoimmune, inflammatory and respiratory disorders. Preferably, such immunosuppressants have improved properties over the known natural sanglifehrins, including one or more of the following properties: longer half-life or increased oral bioavailability, possibly via reduced P450 metabolism and/or reduced glucuronidation, improved water solubility, improved potency in immunosuppressive activity, such as might be seen in t-cell proliferation assays, reduced toxicity (including hepatotoxicity), improved pharmacological profile, such as high exposure to target organ 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. The present invention discloses novel sanglifehrin analogues which may have one or more of the above properties. In particular, the present invention discloses novel sanglifehrin analogues which, in at least some embodiments, have reduced metabolism via P450 or glucuronidation, for example as shown by increased microsome half-life and may have improved immunosuppressive potency, for example as shown by a low t-cell proliferation IC50.
SUMMARY OF THE INVENTION
The present invention provides novel sanglifehrin analogues, which have been generated by mutasynthesis. These analogues may be generated by feeding analogues of meta-tyrosine to a sanglifehrin producing organism, such as Streptomyces sp. A92-308110 (also known as DSM 9954), or more preferentially, by feeding meta-tyrosine analogues to a genetically engineered derivative of a sanglifehrin producing organism, where sfaA, or a homologue of sfaA is inactivated or deleted. As a result, the present invention provides mutasynthetic sanglifehrin analogues, 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 mutasynthetic sanglifehrin analogues and derivatives thereof according to formula (I) or formula (II) below, or a pharmaceutically acceptable salt thereof:




wherein:

R1, R2, R3, R4 and R5 independently represent H, F, Cl, Br, C2-6alkenyl or C1-10alkyl wherein one or more carbon atoms of said alkyl group are optionally replaced by a heteroatom selected from O, N and S(O)p in which p represents 0, 1 or 2 and wherein one or more carbon atoms of said alkyl group are optionally replaced by carbonyl and which alkyl group may optionally be substituted by one or more halogen atoms;

X1, X2, X3, X4 and X5 independently represent C or N, and in the case of any of these groups representing N the attached substituent is absent;

with the proviso that where R1, R3, R4 and R5 all represent H and X1, X2, X3, X4 and X5 all represent C, then R2 cannot represent OH;

including any tautomer thereof; or an isomer thereof in which the C═C bond at the C26, 27 position C═C (by reference to the structure of sanglifehrin A) shown as trans is cis; 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 group 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 “inflammatory disorders” refers to the list of disorders caused by inflammation, including, but not limited to Acne vulgaris, Atherosclerosis, Asthma, Automimmune diseases (such as Acute Disseminated Encephalomyelitis (ADEM), Addison's Disease, Alopecia Areata, Ankylosing Spondylitis, Antiphospholipid Antibody Syndrome (APS), Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Autoimmune Inner Ear Disease, Bullous Pemphigoid, Coeliac Disease, Chagas Disease, Chronic Obstructive Pulmonary Disease (COPD), Crohn's Disease, Dermatomyositis, Diabetes Mellitus Type 1, Endometriosis, Goodpasture's Syndrome, Graves' Disease, Guillain-Barré Syndrome, Hashimoto's Disease, Hidradenitis Suppurativa, Kawasaki Disease, IgA Nephropathy, Idiopathic Thrombocytopenic Purpura, Interstitial Cystitis, Lupus Erythematosus, Mixed Connective Tissue Disease, Morphea, Multiple sclerosis (MS), Myasthenia Gravis, Narcolepsy, Neuromyotonia, Pemphigus Vulgaris, Pernicious Anaemia, Psoriasis, Psoriatic Arthritis, Polymyositis, Primary Biliary Cirrhosis, Rheumatoid Arthritis, Schizophrenia, Scleroderma, Sjögren's Syndrome, Stiff Person Syndrome, Temporal Arteritis, Ulcerative Colitis, Vasculitis, Vitiligo, Wegener's Granulomatosis), Inflammatory Bowel disease, Pelvic inflammatory disease, Rheumatoid arthritis and transplant rejection.
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. A92-308110. Examples include the sanglifehrin-like compounds discussed in WO97/02285 and WO98/07743, such as sanglifehrin B.
As used herein the term “mutasynthetic sanglifehrin(s)” or “mutasynthetic sanglifehrin analogue(s)” refers to chemical compounds that are structurally similar to sanglifehrin A, B, C or D but which differ slightly in composition (as in the replacement of one or more atom by another or in the presence or absence of a particular functional group), in particular, those generated by fermentation of Streptomyces sp. A92-308110 or a mutant thereof, where the culture is fed with a meta-tyrosine analogue.
As used herein the term “meta-tyrosine analogue(s)” refers to chemical compounds that are structurally similar to meta-tyrosine but which differ slightly in composition (as in the replacement of one or more atom by another or in the presence or absence of a particular functional group), in particular, those described in formula (III).
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”.
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, N,N′-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. Exemplary alkyl is C1-6 alkyl eg C1-4alkyl.
“Alkenyl” refers to an alkyl group containing two or more carbons which is unsaturated with one or more double bonds.
Examples of alkyl groups include C1-4 alkyl groups such as methyl, ethyl, n-propyl, i-propyl, and n-butyl. Examples of alkenyl groups include C2-4alkenyl groups such as —CH═CH2 and —CH2CH═CH2.
The term “treatment” includes prophylactic as well as therapeutic treatment.



FIGURE LEGEND
FIG. 1: compound structures and sanglifehrin A numbering system
FIG. 2: 1H NMR of compound 14
FIG. 3: 1H NMR of compound 15
FIG. 4: 1H NMR of compound 16
FIG. 5: 1H NMR of compound 17
FIG. 6: 1H NMR of compound 18
FIG. 7: 1H NMR of compound 19
FIG. 8: 1H NMR of compound 20



DESCRIPTION OF THE INVENTION
The present invention provides mutasynthetic sanglifehrin 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.
In certain embodiments a carbon atom of the C1-10alkyl group (e.g. C1-6alkyl group) that one or more of R1, R2, R3, R4 and R5 may represent is replaced by a heteroatom.
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 R1, R2, R3, R4 and R5 may be primary, secondary or tertiary nitrogen atoms.
If —CH3 is replaced by 0, the group formed is —OH.
When a carbon atom of the C1-10alkyl group (e.g. C1-6alkyl group) that one or more of R1, R2, R3, R4 and R5 may represent is replaced by a heteroatom, it is suitably replaced by O, S or N, especially N or O particularly O.
When any one of R1, R2, R3, R4 and R5 contains a group S(O)p, 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 a C1-10alkyl group (e.g. C1-6alkyl group) that one or more of R1, R2, R3, R4 and R5 may represent contains more than one heteroatom, these should typically be separated by two or more carbon atoms.
Suitably, a carbon atom of a C1-10alkyl group (e.g. C1-6alkyl group) that one or more of R1, R2, R3, R4 and R5 may represent is not replaced by any heteroatom or else represents OH or NH2.
When a carbon atom of the C1-10alkyl group (e.g. C1-6alkyl group) that one or more of R1, R2, R3, R4 and R5 may represent 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 sulfur or oxygen atoms.
For example one or more of R1, R2, R3, R4 and R5 may represent —COC1-3alkyl e.g. —COMe.
Suitably a carbon atom of the C1-10alkyl (e.g. C1-6alkyl) group that one or more of R1, R2, R3, R4 and R5 may represent is not replaced by a carbonyl.
The C1-10alkyl group (e.g. C1-6alkyl group) that one or more of R1, R2, R3, R4 and R5 may represent may be substituted by one or more halogen atoms. For example one or more of R1, R2, R3, R4 and R5 may represent —CF3. Alternatively one or more of R1, R2, R3, R4 and R5 may represent C1-10alkyl (e.g. C1-6alkyl) substituted by one or more (eg one) Cl or F atom (eg —CH2CH2Cl).
Suitably a C1-10alkyl group (e.g. C1-6alkyl group) of R1, R2, R3, R4 or R5 is not substituted by halogen.
When one or more of R1, R2, R3, R4 and R5 group(s) represent a C1-10alkyl group (e.g. C1-6alkyl group) suitably the group(s) represent C1-4 alkyl (e.g. C1-2 alkyl such as methyl).
In an embodiment, one or more of R1, R2, R3, R4 and R5 represent C1-6alkyl (such as C1-2 alkyl) or C2-3alkenyl e.g. one or more of R1, R2, R3, R4 and R5 represent methyl.
Suitably R1 represents H, F, Cl, CF3, OH or C1-6alkyl (e.g. methyl). Most suitably, R1 represents H or F, especially H.
Suitably R2 represents H, F, Cl, CF3, OH, NH2 or C1-6alkyl (e.g. methyl). More suitably, R2 represents H, F, OH or NH2, especially OH.
Suitably R3 represents H, F, Cl, CF3, OH or C1-6alkyl (e.g. methyl). More suitably, R3 represents H, Me or F. R3 may also represent ethyl.
Suitably R4 represents H, F, Cl, CF3, OH or C1-6alkyl (e.g. methyl). More suitably, R4 represents H or F.
Suitably R5 represents H, F, Cl, CF3, OH or C1-6alkyl (e.g. methyl). More suitably, R5 represents H or F
Suitably one or more, more suitably two or more (for example three or more) of R1, R2, R3, R4 or R5 do not represent H.
Suitably one or more, for example two or more of R1, R2, R3, R4 or R5 represent F. Suitably R3 or R4 or R3 and R4 represent F. In another embodiment R1 and R3 represent F.
In one embodiment X1 represents N (therefore R1 is absent). In another more preferable embodiment X1 represents C.
Suitably X2 represents C.
Suitably X3 represents C.
Suitably X4 represents C.
Suitably X5 represents C.
In one embodiment the compound is a compound of formula (I). In another embodiment the compound is a compound of formula (II).
In a suitable embodiment of the invention, R1 represents H, R2 represents OH, R3 represents H, R4 represents F, R5 represents H and X1, X2, X3, X4 and X5 represent C as represented by the following structure:




In a suitable embodiment of the invention, R1 represents H, R2 represents OH, R3 represents F, R4 represents F, R5 represents H and X1, X2, X3, X4 and X5 represent C as represented by the following structure:




In a suitable embodiment of the invention, R1 represents H, R2 represents OH, R3 represents F, R4 represents H, R5 represents H and X1, X2, X3, X4 and X5 represent C as represented by the following structure:




In a suitable embodiment of the invention, R1 represents H, R2 represents OH, R3 represents Me, R4 represents H, R5 represents H and X1, X2, X3, X4 and X5 represent C as represented by the following structure:




In a suitable embodiment of the invention, R1 represents H, R2 represents H, R3 represents H, R4 represents H, R5 represents H and X1, X2, X3, X4 and X5 represent C as represented by the following structure:




In a suitable embodiment of the invention, R1 represents H, R2 represents NH2, R3 represents H, R4 represents H, R5 represents H and X1, X2, X3, X4 and X5 represent C as represented by the following structure:




In a suitable embodiment of the invention, R1 represents H, R2 represents F, R3 represents H, R4 represents H, R5 represents H and X1, X2, X3, X4 and X5 represent C as represented by the following structure:




In a suitable embodiment of the invention, R1 represents H, R2 represents OH, R3 represents H, R4 represents F, R5 represents H, X1 represents N and X2, X3, X4 and X5 represent C as represented by the following structure:




which can also be represented as:





In a suitable embodiment of the invention, R1 represents H, R2 represents OH, R3 represents H, R4 represents F, R5 represents H and X1, X2, X3, X4 and X5 represent C as represented by the following structure:




In a suitable embodiment of the invention, R1 represents H, R2 represents OH, R3 represents F, R4 represents F, R5 represents H and X1, X2, X3, X4 and X5 represent C as represented by the following structure:




In a suitable embodiment of the invention, R1 represents H, R2 represents OH, R3 represents F, R4 represents H, R5 represents H and X1, X2, X3, X4 and X5 represent C as represented by the following structure:




In a suitable embodiment of the invention, R1 represents H, R2 represents OH, R3 represents Me, R4 represents H, R5 represents H and X1, X2, X3, X4 and X5 represent C as represented by the following structure:




In a suitable embodiment of the invention, R1 represents H, R2 represents H, R3 represents H, R4 represents H, R5 represents H and X1, X2, X3, X4 and X5 represent C as represented by the following structure:




In a suitable embodiment of the invention, R1 represents H, R2 represents NH2, R3 represents H, R4 represents H, R5 represents H and X1, X2, X3, X4 and X5 represent C as represented by the following structure:




In a suitable embodiment of the invention, R1 represents H, R2 represents F, R3 represents H, R4 represents H, R5 represents H and X1, X2, X3, X4 and X5 represent C as represented by the following structure:




In a suitable embodiment of the invention, R1 represents H, R2 represents OH, R3 represents Et, R4 represents H, R5 represents H and X1, X2, X3, X4 and X5 represent C as represented by the following structure:




In a suitable embodiment of the invention, R1 represents F, R2 represents OH, R3 represents F, R4 represents H, R5 represents H and X1, X2, X3, X4 and X5 represent C as represented by the following structure:




In a suitable embodiment of the invention, R1 represents H, R2 represents OH, R3 represents H, R4 represents F, R5 represents H, X1 represents N and X2, X3, X4 and X5 represent C as represented by the following structure:




which can also be represented as:





In some embodiments the double bond at the C26,27 position (by reference to the structure of sanglifehrin A) may be in the cis form instead of the trans form.
In general, the compounds of the invention are prepared by mutasynthesis.
In general, a process for preparing certain compounds of formula (I) or (II) or a pharmaceutically acceptable salt thereof comprises:

    
    
        Inoculating a fermentation broth with a culture of a sanglifehrin producer (such as Streptomyces sp. A92-308110 (also known as DSM 9954) or more preferably, a sanglifehrin producer with the sfaA gene or sfaA gene homologue inactivated or deleted;
        Feeding the fermentation broth with an meta-tyrosine analogue (as shown in formula (III))
        Allowing fermentation to continue until sanglifehrin analogues are produced
        Extracting and isolating the sanglifehrin analogue
    
    






where R7 represents H or an ester forming group such as an alkyl group, e.g.C1-6alkyl such as Me.

Suitable X1, X2, X3, X4, X5, R1, R2, R3, R4 and R5 groups in Formula (III) are as defined for compounds of formula (I) and (II).
The feed may be racemic or the L-form of a compound of formula (III).
Compounds of formula (III) are either commercially available or prepared by standard organic synthetic chemistry techniques. One generic route to compounds of formula (III) is as shown in the following scheme 1:




Aldehydes of formula (IV) may be commercially available or readily synthesised by one skilled in the art. Protection and deprotection chemistry may need to be employed in generating compounds of formula (III) from compounds of formula (IV). These techniques are known to one skilled in the art and suitable protecting groups are described in Greene's Protective Groups in Organic Synthesis (Wuts and Greene, 4th Edition, 2007)
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).
A mutasynthetic sanglifehrin analogue according to the invention may be administered alone or in combination with other therapeutic agents. Co-administration of two (or more) agents may allow 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 mutasynthetic sanglifehrin analogue 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. pIFNα and/or ribavirin).
In an alternative embodiment, a mutasynthetic sanglifehrin analogue is co-administered with one or more other anti-viral agents, such as a STAT-C/DAA (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, BI 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 non-toxic 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, hydroxypropylmethyl 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, for use as an anti-inflammatory or for prophylaxis of organ transplant rejection;
        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, for use as an anti-inflammatory or for prophylaxis of organ transplant rejection;
        A method of treatment of viral infections (especially RNA virus infections) such as HCV or HIV infection, for use as an anti-inflammatory or for prophylaxis of organ transplant rejection 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, for use as an anti-inflammatory or for prophylaxis of organ transplant rejection.
        A process for producing a mutasynthetic sanglifehrin (such as a compound of formula (I) or (II)) which comprises feeding a sanglifehrin producing bacterium, such as a Streptomyces sp (eg A92-308110), a compound of formula (III) or a salt thereof, and culturing the bacterium such that a mutasynthetic sanglifehrin is produced.
        A process according to the previous paragraph wherein the sanglifehrin producing bacterium is a Streptomyces sp in which the sfaA gene or sfaA gene homologue is inactivated or deleted.
        A process according to the previous two paragraphs further comprising the step of isolating the mutasynthetic sanglifehrin.
    
    


Novel compounds of formula (III) (such as those listed in Table 1 and the acids and esters of any of the compounds of formula (III) listed in Table 1) and (IV) including their salts and esters also form an aspect of the invention.
General Methods
Materials and Methods
Bacterial Strains and Growth Conditions
The sanglifehrin producer Streptomyces sp. A92-308110 (DSM no 9954, purchased from DSMZ, Braunschweig, Germany) also termed BIOT-4253 and BIOT-4370 or its derivatives, such as BIOT-4585 are maintained on medium oatmeal agar, MAM, ISP4 or ISP2 (see below) at 28° C.
pKC1139, a standard Streptomyces-Ecoli shuttle plasmid, was obtained from the John Innes Centre, UK, and is described in Bierman et al., 1992 and Kieser et al., 2000.
BIOT-4585 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 water 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.
Meta-Tyrosine Analogues
Methyl (2S)-2-amino-3-(6-hydroxy(2-pyridyl))propanoate, L-3-aminophenylalanine methyl ester, L-4-methyl-meta-tyrosine methyl ester, L-4-fluoro-meta-tyrosine methyl ester and L-4,5-difluoro-meta-tyrosine methyl ester were purchased from Netchem (USA).
DL-3-fluorophenylalanine and L-phenylalanine were purchased from Sigma (UK).
DL-meta-tyrosine was purchased from Fluorochem (UK).
L-meta-tyrosine was purchased from Alfa Aesar (UK).
DL-4-fluorometa-tyrosine (8), DL-5-fluorometa-tyrosine (9), methyl 2-amino-3-(3-fluoro-5-hydroxyphenyl)propanoate (10), methyl 2-amino-3-(2-fluoro-5-hydroxyphenyl)propanoate (11), methyl 2-amino-3-(2-fluoro-3-hydroxyphenyl)propanoate (12) and methyl 2-amino-3-(2,6-difluoro-3-hydroxyphenyl)propanoate (13) were synthesised as follows:
DL-4-Fluoro Meta-Tyrosine (8)




To a solution of 8-1 (3 g, 19.5 mmol) in dry DCM (150 mL) was added dropwise BBr3 (4 M in DCM, 14.6 ml, 58.5 mmol) at −70° C. After the addition, the reaction mixture was stirred at −20° C. for 3 h, ice-water was added carefully, and extracted with DCM. The organic layers were washed with water and brine, dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatographed on silica to give the desired compound 8-2.
To a solution of 8-2 (0.9 g, 6.4 mmol) in acetone (40 mL) was added K2CO3 (2.2 g, 16 mmol) at room temperature. The reaction mixture was stirred at room temperature overnight. Water was added and acetone was removed under vacuum, and then extracted with EtOAc, the organic layers were washed with water and brine, dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatographed on silica to give the desired compound 8-3.
A mixture of 8-3 (1 g, 4.34 mmol), hippuric acid (860 mg, 4.80 mmol), NaOAc (400 mg) and Ac2O (2.2 mL) was stirred at 80° C. for 2 h. The yellow reaction mixture was cooled and cold EtOH (10 mL) was added, the mixture was cooled in an ice bath for 15 min and then was poured into 30 mL of ice water, chilled and the product was collected by filtration. The solid was dried in vacuo to yield 8-4.
A solution of 8-4 (300 mg, 0.8 mmol) and NaOAc (71 mg, 0.87 mmol) in MeOH (50 mL) was stirred at room temperature overnight. The solvent was removed by rotary evaporation and the reside was dissolved in 50 mL of EtOAc, the EtOAc solution was washed two times with water and concentrated to give 8-5.
A solution of 8-5 (360 mg, 0.89 mmol) in MeOH (50 mL) was hydrogenated over 10% Pd/C (77 mg) at normal pressure for 20 h. After removal of the catalyst by filtration, the solvent was evaporated to give the product 8-6.
A solution of 8-6 (210 mg) in 3 N HCl (10 mL) was refluxed for 24 h. the solution was concentrated to dryness and the residue was purified by reverse-combiflash to give the target product 8.
DL-5-fluoro meta-tyrosine (9) and methyl 2-amine-3-(3-fluoro-5-hydroxyphenyl)propanoate (10)




To a solution of 9-1 (20 g, 97.55 mmol) in tetrahydrofuran (100 mL) was added dropwise n-butyl lithium (43 mL, 2.5 M, 107.3 mmol) at −78° C. It was stirred for 30 minutes and N,N-dimethylformamide (15.1 mL, 195.1 mmol) was added at this temperature. It was stirred for another 30 minutes and the cold bath was removed. After 1 hour, the reaction was quenched with saturated aqueous ammonium chloride. The organic layer was washed with water and saturated aqueous sodium chloride, dried (sodium sulfate), filtered and concentrated. The residue was purified by chromatography on silica to give 9-2.
To a solution of 9-2 (6 g, 38.9 mmol) in dry DCM (200 mL) was added dropwise BBr3 (4 M in DCM, 30 ml, 116.8 mmol) at −70° C. After the addition, the reaction mixture was stirred at −20° C. for 3 hours, ice-water was added carefully, and extracted with DCM. The organic layers were washed with water and brine, dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatographed on silica to give the desired compound 9-3.
To a solution of methyl 2-(benzyloxycarbonylamino)-2-(dimethoxyphosphoryl)acetate (4.64 g, 14 mmol) in DCM (150 mL) was added DBU (4.26 g, 28 mmol) at room temperature. After 10 min, 9-3 (1.95 g, 14 mmol) was added and the resulting mixture was stirred at room temperature overnight. The solution was diluted with EtOAc (150 mL), separated and the organic layer was washed with 1 N HCl, dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on silica to give 9-4.
A solution of 9-4 (1 g) in MeOH (20 mL) was hydrogenated over 200 mg of 10% Pd/C at normal pressure overnight. After removal of the catalyst by filtration, the solvent was evaporated to give 10.
To a solution of 10 (300 mg, 1.4 mmol) in EtOH (30 mL) was added aq. NaOH (2 N, 4 mL), the reaction was stirred at room temperature for 30 minutes. The solvent was removed and the residue was neutralized to pH=6 with 2 N HCl and the white crystals that formed were collected by filtration to give the target compound 9.
methyl 2-amino-3-(2-fluoro-5-hydroxyphenyl)propanoate (11)




To a solution of the compound 11-1 (1.4 g, 9 mmol) in 50 mL DCM was added dropwise BBr3 (4M in DCM, 3.6 mL, 13.5 mmol) at −78° C. After the addition, the reaction was stirred at −20° C. for 4 hours. Then slow addition of ice/water, the layers was separated, the organic layers was washed with water and brine, dried over Na2SO4 and evaporated to dryness. The residue was used to next step without further purification.
To a solution of methyl 2-(benzyloxycarbonylamino)-2-(dimethoxyphosphoryl)acetate (3 g, 9 mmol) in 100 mL DCM was added DBU (2.8 g, 18 mmol) at room temperature, after 10 mins, the compound 11-2 (crude compound from last step) was added, stirred at room temperature for 2 hours. The solution was then diluted with DCM (50 mL), washed with 1N HCl (20 mL), dried over Na2SO4 and evaporated to dryness. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=5/1) to give 11-3.
A mixture of the compound 11-3 (500 mg, 1.5 mmol) in MeOH (20 mL) was hydrogenated over 50 mg of 10% Pd/C at normal pressure overnight. After removal of the catalyst by filtration, the solvent was evaporated to get the crude product, which was purified by reverse-combiflash to get 11 as a white solid.
methyl 2-amino-3-(2-fluoro-3-hydroxyphenyl)propanoate (12)




To a solution of the compound 12-1 (1.4 g, 9 mmol) in 50 mL DCM was added dropwise BBr3 (4M in DCM, 3.6 mL, 13.5 mmol) at −78° C. After the addition, the reaction was stirred at −20° C. for 4 hours. After slow addition of ice/water, the layers were separated, the organic layer was washed with water and brine, dried over Na2SO4 and evaporated to dryness. The residue was used to next step without further purification.
To a solution of methyl 2-(benzyloxycarbonylamino)-2-(dimethoxyphosphoryl)acetate (3 g, 9 mmol) in 100 mL DCM was added DBU (2.7 mL, 18 mmol) at room temperature, after 10 mins, the compound 12-2 (crude compound from last step) was added, stirred at room temperature for 2 hours. The solution was then diluted with DCM (100 mL), washed with 1N HCl (30 mL), dried over Na2SO4 and evaporated to dryness. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=5/1) to give 12-3.
A mixture of the compound 12-3 (500 mg, 1.44 mmol) in MeOH (10 mL) was hydrogenated over 100 mg of 10% Pd/C at normal pressure overnight. After removal of the catalyst by filtration, the solvent was evaporated to get the crude product, which was purified by reverse-combiflash to get the desired compound 12 as a white solid.
methyl 2-amino-3-(2,6-difluoro-3-hydroxyphenyl)propanoate (13)




To a solution of 2,4-difluorophenol (2 g, 15.4 mmol) in 50 mL DMF was added K2CO3(3.2 g, 23.1 mmol) and BnBr (2.2 mL, 18.5 mmol) at 0° C. The reaction was stirred at room temperature for 2 hours. Water (100 mL) and EA (200 mL) was added, the organic layers was washed with water (50 mL) and brine (50 mL), dried over Na2SO4 and evaporated to dryness. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=10/1) to give the crude 13-1.
To a solution of the compound 13-1 (2 g, 9 mmol) in 10 mL THF was added dropwise n-BuLi (4 mL, 2.5 M) at −78° C. and stirred for 30 mins. DMF (1.3 g, 0.018 mmol) was added and stirred for 30 mins again. The cold bath was then removed and the reaction mixture was stirred at room temperature for 1 hour before being quenched with water. It was extracted with ethyl acetate (20 mL×3), dried over Na2SO4 and evaporated to dryness. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=10/1) to give 13-2 as a yellow solid.
To a solution of methyl 2-(benzyloxycarbonylamino)-2-(dimethoxyphosphoryl)acetate (728 mg, 2.2 mmol) in 20 mL DCM was added DBU (319 mg, 2.1 mmol) at room temperature. After 10 mins, the compound 13-2 (500 mg, 2 mmol) was added and stirred at room temperature for 2 hours. The solution was then diluted with DCM (50 mL), washed with 1N HCl (20 mL), dried over Na2SO4 and evaporated to dryness. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=5/1) to give 13-3 as a yellow oil.
The compound 13-3 (600 mg, 1.32 mmol) in MeOH (20 mL) was hydrogenated over 60 mg of 10% Pd/C at normal pressure overnight. After removal of the catalyst by filtration, the solvent was evaporated to get the crude product, which was purified by reverse-combiflash to get the desired compound 13 as a white solid.
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



CaCO3 (Calcitec, V/40S)
0.05
g



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



KH2PO4 (Sigma, P3786)
0.25
g



K2HPO4 (Sigma, P5379)
0.50
g



MgSO4•7H2O (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 H2O to final vol. of
** 1.00
L







pre-sterilisation pH was adjusted to pH 7.0 with 10M NaOH/10M H2SO4



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




















FeSO4•7H2O (Sigma, F8633)
5.00
g



ZnSO4•7H2O (Sigma, Z0251)
4.00
g



MnCl2•4H2O (Sigma, M8530)
2.00
g



CuSO4•5H2O (Aldrich, 20, 919-8)
0.20
g



(NH4)6Mo7O24 (Fisher scientific, A/5720/48)
0.20
g



CoCl2•6H2O (Sigma, C2644)
0.10
g



H3BO3 (Sigma, B6768)
0.10
g



KI (Alfa Aesar, A12704)
0.05
g



H2SO4 (95%) (Fluka, 84720)
1.00
mL



RO H2O 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 H2O 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






SM25-3 Medium














Ingredient




















Glycerol (Fisher scientific, G/0650/25)
40
g



Soy Peptone A3 SC (Organotechnie)
10
g



Malt extract (Difco)
21
g



to final vol. of
1
L







pre-sterilisation pH not adjusted (i.e. pH 7.0)






ISP4 Medium














Ingredient




















Soluble Starch (Difco)
10
g



K2HPO4
1
g



MgSO4•7H2O
1
g



NaCl
1
g



(NH4)2SO4
2
g



CaCO3
2
g



ISP Trace Salts Solution
1
mL



Agar
20
g



to final vol. of
1
L










Make a paste with the starch in a small volume of cold water and bring to volume of 500 m1

    
    
        Add other ingredients to solution II in 500 mls water pH should be between pH 7.0 and pH 7.4 (pH 7.3) Mix two solutions together and add agar
    
    


ISP Trace Salts














Ingredient




















FeSO4•7H2O
1
g



MnCl2•4H2O
1
g



ZnSO4•7H2O
1
g



to final vol. of
1
L







Store at 4 degrees C.






General Fermentation Method

Cryopreserved spore stocks of BIOT-4585 were thawed at room temperature. Vegetative cultures (seed cultures) were prepared by transferring 4.0 mL of spore stock into 400 mL medium SM25 in 2 L Erlenmeyer flasks with foam plug. Cultivation was carried out for 48 hours at 27° C. and 250 rpm (5.0 cm throw). From the seed culture 25 mL was transferred into 250 mL production medium SGP2+5% HP20 in 2 L Erlenmeyer flasks with foam plug. After 24 hours cultivation at 24° C. and 250 rpm (2.5 cm throw), 2 mL of a 250 mM racemic or 125 mM enantiomerically pure solution of the desired precursor in 1M hydrochloric acid and 2 mL of a 250 mM methanolic solution of DL-piperazic acid was added to each production flask to give a final 1 mM concentration of the individual enantiomers of the precursors. Cultivation was continued for further four days at 24° C. and 250 rpm (2.5 cm throw).
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 3 u, 4.6 mm×150 mm
Fitted with a Phenomenex Analytical C18 Security Guard Cartridge (KJ0-4282)
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 HP1100 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.
QC LC-MS Method
HPLC Conditions:
C18 Hyperclone BDS C18 Column 3 u, 4.6 mm×150 mm
Fitted with a Phenomenex Analytical C18 Security Guard Cartridge (KJ0-4282)
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 Analysis LC-MS Method (e.g. For Microsome Stability Assessment)
Using an API-2000, API-4000 or UPLC instrument
HPLC Conditions:
For 15: ACQUITY UPLC BEH C18 (2.1×50 mm, 1.7 μm)
For cpds 5, 14, 16, 17, 18, 19 Ultimate XB-C18 (2.1×50 mm, 5 μm) Column temp at 50° C.
Flow rate 0.6 mL/min
Solvent gradient A1 (e.g. for cpd 15):
0.2 min: 20% B
0.6 min: 95% B
1.1 min: 95% B
1.15 min: 20% B
1.5 min: stop
Solvent A is H2O-0.025% FA-1 mM NH4OAC
Solvent B is ACN-0.025% FA-1 mM NH4OAC
Solvent gradient A2 (e.g. for cpds 5, 14, 16, 17, 18, 19):
0.3 min: 10% B
0.8 min: 95% B
2.3 min: 95% B
2.31 min: 10% B
3.5 min: stop
Solvent A is H2O-0.1% FA
Solvent B is MeOH-0.1% FA
negative scan mode:
MRM Setup:













transitions [Da]



















21
1089.7 → 504.2










positive scan mode:

MRM Setup:














transitions [Da]



















5
1090.6 → 1054.6



14
1108.9 → 1072.5



15
1126.7 → 1090.0



16
1108.8 → 1072.3



17
1104.5 → 1068.5



18
1074.6 → 1038.8










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 1b I389Iuc-ubi-neo/NS3-3′/5.1 replicon (Vrolijk et al., 2003) and subcultured in cell growth medium [DMEM (Cat No. 41965039) supplemented with 10% FCS, 1% non-essential amino acids (11140035), 1% penicillin/streptomycin (15140148) and 2% Geneticin (10131027); Invitrogen] at a ratio of 1.3-1.4 and grown for 3-4 days in 75 mL 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 6500 cells/well (100 uL/well) 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% CO2, 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% CO2, 95-99% relative humidity).
For the evaluation of anti-metabolic effects, the assay medium is aspirated, replaced with 75 uL of a 5% MTS (Promega) solution in phenol red-free medium and incubated for 1.5 hours (37° C., 5% CO2, 95-99% relative humidity). Absorbance is measured at a wavelength of 498 nm (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, 25 uL of Glo Lysis Buffer (Cat. No. E2661, Promega) is added after which lysis is allowed to proceed for 5 min at room temperature. Subsequently, 50 uL of Luciferase Assay System (Cat. No. E1501, Promega) is added and the luciferase luminescence signal is quantified immediately (1000 ms 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.
For results see Example 12.
In Vitro Replicon Assay for Assessment of HCV Antiviral Activity in Genotypes 1a and 2a
The replicon cells (subgenomic replicons of genotype 1a (H77) and 2a (JFH-1)) are grown in Dulbecco's modified essential media (DMEM), 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (pen-strep), 1% glutamine, 1% non-essential amino acids, 250 μg/ml G418 in a 5% CO2 incubator at 37° C. All cell culture reagents may be purchased from Mediatech (Herndon, Va.).
The replicon cells are trypsinized and seeded at 5×103 cells per well in 96-well plates with the above media without G418. On the following day, the culture medium is replaced with DMEM containing compounds serially diluted in the presence of 5% FBS. The HCV replicon antiviral assay examines the effects of compounds in a serial of compound dilutions. Briefly, the cells containing the HCV replicon are seeded into 96-well plates. Test article is serially diluted with DMEM plus 5% FBS. The diluted compound is applied to appropriate wells in the plate. After 72 hr incubation at 37° C., the cells are processed. The intracellular RNA from each well is extracted with an RNeasy 96 kit (Qiagen). The level of HCV RNA is determined by a reverse transcriptase-real time PCR assay using TaqMan® One-Step RT-PCR Master Mix Reagents (Applied Biosystems, Foster City, Calif.) and an ABI Prism 7900 sequence detection system (Applied Biosystems) a as described previously (Vrolijk et al., 2003). The cytotoxic effects are measured with TaqMan® Ribosomal RNA Control Reagents (Applied Biosystems) as an indication of cell numbers. The amount of the HCV RNA and ribosomal RNA is then used to derive applicable IC50 values (concentration inhibiting on replicon replication by 50%).
Assessment of Microsome Metabolism (Microsome Stability Assay)
Rate of metabolism in microsomes may be tested as follows:
Mouse or human liver microsomes were diluted with buffer C (0.1 M Potassium Phosphate buffer, 1.0 mM EDTA, pH 7.4) to a concentration of 2.5 mg/mL. Microsomal stability samples were then prepared by adding 50 uL of 5 uM compound spiking solution (0.5 uL 10 mM DMSO stock solution in 9.5 uL ACN, added to 990 uL Buffer C) to 50 μL of microsomal solution (2.5 mg/mL), 110 uL Buffer C and mixed well. All samples were pre-incubated for approximately 15 minutes at 37° C. Following this, the reaction was initiated by adding 40 uL of the NADPH solution (12.5 mM) with gentle mixing. Aliquots (40 uL) were removed at 0, 15, 30, 45 and 60 minutes and quenched with ACN containing internal standard (120 uL). Protein was removed by centrifugation (4000 rpm, 15 min) and the sample plate analysed for compound concentration by LC-MS/MS. Half-lives were then calculated by standard methods, comparing the concentration of analyte wth the amount originally present.
For results see Example 13.
Assessment of Hepatocyte Stability
Cryopreserved hepatocytes, previously stored in liquid nitrogen are placed in a 37±1° C. shaking water bath for 2 min±15 sec. The hepatocytes are then added to 10× volume of pre-warmed Krebs-Henseleit bicarbonate (KHB) buffer (2000 mg/L glucose, no calcium carbonate and sodium bicarbonate, Sigma), mixed gently and centrifuged at 500 rpm for 3 minutes. After centrifugation, the supernatant is carefully removed and a 10× volume of pre-warmed KHB buffer added to resuspend the cell pellet. This is mixed gently and centrifuged at 500 rpm for 3 minutes. The supernatant is then removed and discarded. The cell viability and yield are then determined by cell counts, and these values used to generate human hepatocyte suspensions to the appropriate seeding density (viable cell density=2×106 cells/mL). A 2× dosing solution is prepared in pre-warmed KHB (1% DMSO) (200 uM spiking solution: 20 uL of substrate stock solution (10 mM) in 980 uL of DMSO, 2× dosing solution: 10 uL of 200 uM spiking solution in 990 μL of KHB (2 uM after dilution).
50 uL of pre-warmed 2× dosing solution is added to the wells and 50 uL of pre-warmed hepatocyte solution (2×106 cells/mL) added and timing started. The plate is then incubated at 37° C. 100 uL of acetonitrile containing internal standard is added to each the wells after completion of incubation time (0, 15, 30, 60 and 120 minutes) mixed gently, and 50 uL of pre-warmed hepatocyte solution added (2×106 cells/mL). At the end of the incubation, cell viability is determined. Samples are centrifuged at 4000 rpm for 15 minutes at 4° C., supernatants diluted 2-fold with ultrapure water and compound levels analysed by LC-MS/MS.
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.
Alternatively, solubility in PBS at pH7.4 may be tested as follows: A calibration curve is generated by diluting the test compounds and control compounds to 40 uM, 16 uM, 4 uM, 1.6 uM, 0.4 uM, 0.16 uM, 0.04 uM and 0.002 uM, with 50% MeOH in H2O. The standard points are then further diluted 1:20 in MeOH:PBS 1:1. The final concentrations after 1:20 dilution are 2000 nM, 800 nM, 200 nM, 80 nM, 20 nM, 8 nM, 2 nM and 1 nM. Standards are then mixed with the same volume (1:1) of ACN containing internal standard (hydroxymacrocycle, 6). The samples are centrifuged (5 min, 12000 rpm), then analysed by LC/MS.





















MeOH/H2O

Working

MeOH/buffer

Final



Solution
(1:1)

solution
Solution
(1:1)

solution



(uL)
(uL)

(uM)
(uL)
(uL)

(nM)

























10
mM
10
240
→
400






400
uM
50
450
→
40
20
380
→
2000




20
480
→
16
20
380
→
800


40
uM
50
450
→
4
20
380
→
200


16
uM
50
450
→
1.6
20
380
→
80


4
uM
50
450
→
0.4
20
380
→
20


1.6
uM
50
450
→
0.16
20
380
→
8


0.4
uM
50
450
→
0.04
20
380
→
2


0.04
uM
50
950
→
0.002
20
380
→
1









Test compounds are prepared as stock solutions in DMSO at 10 mM concentration. The stock solutions are diluted in duplicate into PBS, pH7.4 in 1.5 mL Eppendorf tubes to a target concentration of 100 uM with a final DMSO concentration of 1% (e.g. 4 uL of 10 mM DMSO stock solution into 396 uL 100 mM phosphate buffer). Sample tubes are then gently shaken for 4 hours at room temperature. Samples are centrifuged (10 min, 15000 rpm) to precipitate undissolved particles. Supernatants are transferred into new tubes and diluted (the dilution factor for the individual test article is confirmed by the signal level of the compound on the applied analytical instrument) with PBS. Diluted samples are then mixed with the same volume (1:1) of MeOH. Samples are finally mixed with the same volume (1:1) of ACN containing internal standard (hydroxymacrocycle, 6) for LC-MS/MS analysis.
Assessment of Cell Permeability
Cell permeability may be tested as follows: The test compound is dissolved to 10 mM in DMSO and then diluted further in buffer to produce a final 10 μM 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 Vivo Assessment of Oral and Intravenous Pharmacokinetics (Specific Method)
For sanglifehrin analogues, whole blood is analysed. Compounds are formulated in 5% ethanol/5% cremophor EL/90% saline for both p.o. and i.v. administration. Groups of 3 male CD1 mice are dosed with either 1 mg/kg i.v. or 10 mg/kg p.o. Blood samples (40 uL) are taken via saphenous vein, pre-dose and at 0.25, 0.5, 2, 8, and 24 hours, and diluted with an equal amount of dH2O and put on dry ice immediately. Samples are stored at −70° C. until analysis. The concentration of the compound of the invention or parent compound in the sample is determined via LCMS as follows: 20 uL of blood:H2O (1:1, v/v)/PK sample is added with 20 uL Internal standard (hydroxyl macrocycle, 6) at 100 ng/mL, 20 uL working solution/MeOH and 150 uL of ACN, vortexed for 1 minute at 1500 rpm, and centrifuged at 12000 rpm for 5 min. The supernatant is then injected into LC-MS/MS. The time-course of blood concentrations is plotted and used to derive area under the whole blood concentration-time curve (AUC—which is directly proportional to the total amount of unchanged drug that reaches the systemic circulation). These values are used to generate the oral bioavailability (F %) and other PK parameters where possible.
In Vitro Assessment of CytotoxiCity
Huh-7 and HepG2 cells obtained from ATCC are grown in Dulbecco's modified essential media (DMEM) containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (pen-strep) and 1% glutamine; whereas CEM cells (human T-cell leukemia cells obtained from ATCC) are grown in RPMI 1640 medium with 10% FBS, 1% pen-strep and 1% glutamine. Fresh human PBMCs are isolated from whole blood obtained from at least two normal screened donors. Briefly, peripheral blood cells are pelleted/washed 2-3 times by low speed centrifugation and resuspension in PBS to remove contaminating platelets. The washed blood cells are then diluted 1:1 with Dulbecco's phosphate buffered saline (D-PBS) and layered over 14 mL of Lymphocyte Separation Medium (LSM; cellgrow by Mediatech, Inc.; density 1.078+/−0.002 g/ml; Cat.#85-072-CL) in a 50 mL centrifuge tube and centrifuged for 30 minutes at 600×g. Banded PBMCs are gently aspirated from the resulting interface and subsequently washed 2× with PBS by low speed centrifugation. After the final wash, cells are counted by trypan blue exclusion and resuspended at 1×107 cells/mL in RPMI 1640 supplemented with 15% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 4 μg/mL PHA-P. The cells are allowed to incubate for 48-72 hours at 37° C. After incubation, PBMCs are centrifuged and resuspended in RPMI 1640 with 15% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 ug/mL streptomycin, 10 ug/mL gentamycin, and 20 U/mL recombinant human IL-2.
Compound cytotoxicity is evaluated by testing half-log concentrations of each compound in triplicate against the cells described above. Cell containing medium alone served as the cell control (CC). Huh-7 and HepG2 cells are seeded in 96-well plates at a concentration of 5×103 cells per well. On the following day, the media is aspirated, and 100 uL of corresponding media containing 5% FBS is added. Test drug dilutions are prepared at a 2× concentration in microtiter tubes and 100 uL of each concentration is placed in appropriate wells in a standard format. After 72 hours, the cells are processed for cytotoxicity assessment.
PBMCs are diluted in fresh medium and plated in the interior wells of a 96 well round bottom microplate at 5×104 cells/well in a volume of 100 L. Similarly, CEM cells are plated at 1×104 cells/well. Then, 100 uL of 2× preparations of the test drugs are added in appropriate wells in a standard format. The cultures are maintained for six to seven days and then processed for cytotoxicity determination.
Cytotoxicity is determined using CytoTox-ONETM homogeneous membrane integrity assay kit (Promega). The assay measures the release of lactate dehyrodgenase (LDH) from cells with damaged membranes in a fluorometric, homogeneous format. LDH released into the culture medium is measured with a coupled enzymatic assay that results in the conversion of resazurin into a fluorescent resorufin product. The amount of fluorescence produced is proportional to the number of lysed cells. Six serially diluted concentrations of each compound are applied to the cells to derive where applicable TC50 (toxic concentration of the drug decreasing cell viability by 50%) and TC90 (toxic concentration of the drug decreasing cell viability by 90%) values.
In Vitro Assessment of Inhibition of MDR1 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 100 uM) 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
Construction of an sfaA Deletion Mutant of Streptomyces Sp. A92-308110 (DSM9954)
1.1 Construction of the sfaA Deletion Construct
The  ̃7 kb EcoRV-StuI fragment of cosmid TL3006 (SEQ ID NO. 3) encompassing sfaA (nucleotide position 14396-21362, NCBI sequence accession number FJ809786) was excised by digestion with EcoRV and StuI and the resulting isolated fragment ligated directly into pKC1139 that had previously been digested with EcoRV and treated with shrimp alkaline phosphatase (Roche). This plasmid was designated pSGK268.
An in frame deletion of the sfaA gene contained within this clone was performed using the Red/ET recombination kit supplied by Gene Bridges (catalog number K006).







(SEQ ID NO. 1) SfaA17161f 5′-


CGCTCTGTGGCGCCTGGTTTCCAAGCGGCTCGCGGACCGGCACCGGCACA


 


TGCATAATTAACCCTCACTAAAGGGCG-3′


 


(SEQ ID NO. 2) SfaA17825r 5′-


TGGATGTATCGTCGCAGGACGCCCAGAATTCACCTGCGACGTCCTCCAGA


 


TGCATTAATACGACTCACTATAGGGCTC-3′






Two oligonucleotides, SfaA17161f and SfaA17825r were used to amplify the neomycin marker from the FRT-PGK-gb2-neo-FRT template DNA supplied in the kit using KOD DNA polymerase. The resulting  ̃1.7 kb amplified product was isolated by gel electrophoresis and purified from the gel with QiaEX resin.
Plasmid pSGK268 was transformed into E. coli DH10B using standard techniques and selected on plates containing apramycin (50 μg/ml). Introduction of the deletion construct was performed essentially following the Gene Bridges kit protocol. A single colony was grown overnight in 2TY apramycin (50 ug/ml) and transformed with the pRedET (tet) plasmid and selected on apramycin (50 ug/ml) and tetracycline (3 ug/ml) at 30° C. A single colony was used to prepare an overnight culture of this strain in 3 ml 2TY apramycin (50 ug/ml) and tetracycline (3 mg/ml) at 30 C. 0.5 ml of this culture was used to inoculate 10 ml 2TY apramycin (50 μg/ml) and tetracycline (3 ug/ml) at 30° C. and grown to an OD600 nm  ̃0.5. 1.4 ml of this culture was transferred to each of 2 eppendorf tubes and 50 ul 10% arabinose added to one tube to induce expression of the Red/ET recombination proteins. Tubes were shaken for  ̃1 hour at 37° C. Induced and non-induced cells were pelleted in a bench top centrifuge and washed twice with chilled sterile water; resuspending and centrifuging to pellet the cells each time. The resulting pellets were suspended in about 30-40 ul of water and kept on ice. The 1.7 kb disruption fragment isolated previously was added to the induced and non-induced tubes and transferred to 1 mm Biorad electrocuvettes on ice. The samples were electroporated (Biorad Micropulser at 1.8 kV, resulting time constant  ̃4 ms) and 1 ml 2TY (no antibiotics) added and mixed to remove the cells from the cuvette. Cells were incubated for  ̃3 hours at 37° C. with shaking (1100 rpm, eppendorf thermomixer compact) before plating onto 2TY plates containing apramycin (50 ug/ml and kanamycin 25 ug/ml and incubating over night at 37° C. Colonies from the induced sample plates were streaked onto 2TY plates containing kanamycin at 50 μg/ml to purify and confirm introduction of the kanamycin resistance cassette. PCR on individual bacterial colonies was used to confirm the introduction of the cassette. Plasmids were prepared from these cultures and digested to confirm the expected plasmid pSGK270. Plasmids were then digested with NsiI to remove the marker fragment, and the remainder religated to produce the sfaA in-frame deletion construct pSGK271.
1.2 Conjugation of Streptomyces sp. A92-308110 (DSM9954) and Introduction of an sfaA Deletion
Plasmid pSGK271 was transformed into E. coli ET12567 pUZ8002 using standard techniques and selected on 2TY plates containing apramycin (50 ug/ml), kanamycin (25 ug/ml) and chloroamphenicol (10 μg/ml). The resulting strain was inoculated into 3 ml liquid 2TY containing apramycin (50 ug/ml), kanamycin (25 ug/ml) and chloroamphenicol (10 ug/ml) and incubated overnight at 37° C., 250 rpm. 0.8 ml of this culture was used to inoculate 10 ml liquid 2TY containing apramycin (50 ug/ml), kanamycin (25 ug/ml) and chloroamphenicol (10 ug/ml) in a 50 ml Falcon tube and incubated at 37° C. 250 rpm until OD600 nm  ̃0.5 was reached. The resulting culture was centrifuged at 3500 rpm for 10 minutes at 4° C., washed twice with 10 ml 2TY media using centrifugation to pellet the cells after each wash. The resulting pellet was resuspended in 0.5 ml 2TY and kept on ice before use. This process was timed to coincide with the complete preparation of Streptomyces spores described below.
Spores of Streptomyces sp. A92-308110 (DSM9954) (Biot-4370) were harvested from a 1-2 week old confluent plate by resuspending in  ̃3 ml 20% glycerol. Spores were centrifuged (5000 rpm, 10 minutes room temperature) and washed twice with 50 mM TES buffer before resuspending in 1 ml 50 mM TES buffer and splitting between 2 eppendorf tubes. These tubes were heat shocked at 50° C. for 10 minutes in a water bath before adding 0.5 ml 2TY and incubating in an Eppendorf Thermomixer compact at 37° C. for 4-5 hours.
The prepared E. coli ET12567 pUZ8002 pSGK271 and Biot-4370 were mixed at ratios 1:1 (250 uL each strain) and 1:3 (100 uL E. coli) and immediately spread on R6 plates and transferred to a 37° C. incubator. After approximately 2 hours incubation these plates were overlaid with 2 ml of sterile water containing nalidixic acid to give a final in-plate concentration of 25 ug/L. Plates were returned to the 37° C. incubator overnight before overlaying with 2 ml of sterile water containing apramycin to give a final in-plate concentration of 20-25 ug/L. Ex-conjugant colonies appearing after  ̃4-7 days were patched to ISP4 media containing apramycin (25 ug/L) and nalidixic acid (25 ug/L) and incubated at 37° C. Once adequate mycelial growth was observed strains were repatched to ISP4 media containing apramycin (25 ug/L) at 37° C. and allowed to sporulate. Strains were then subcultured three times (to promote removal of the temperature sensitive plasmid) by patching to ISP4 (without antibiotic) and incubating at 37° C. for 3-4 days. Strains were finally patched to ISP4 and incubated at 28° C. to allow full sporulation (5-7 days). Spores were harvested and serially diluted onto ISP4 plates at 28° C. to allow selection of single colonies. Sporulated single colonies were doubly patched to ISP4 plates with or without apramycin (25 ug/L) to confirm loss of plasmid and allowed to grow  ̃7 days before testing for production of sanglifehrins.
1.3 Screening Strains for Production of Sanglifehrins in Falcon Tubes
A single  ̃7 mm agar plug of a well sporulated strain was used to inoculate 7 ml of sterile SM25-3 media and incubated at 27° C. 200 rpm in a 2′′ throw shaker. After 48 hours of growth 0.7 ml of this culture was transferred to a sterilised falcon tube containing 7 ml of SGP2 media with 5% HP20 resin. Cultures were grown at 24° C. 300 rpm on a 1 inch throw shaking incubator for 5 days before harvest. 0.8 ml bacterial culture was removed and aliquoted into a 2 ml eppendorf tube ensuring adequate dispersal of the resin in throughout the culture prior to aliquoting. 0.8 ml acetonitrile and 15 ul of formic acid were added and the tube mixed for about 30 minutes. The mixture was cleared by centrifugation and 170 ul of the extract removed into a HPLC vial and analysed by HPLC.
1.4 Analysis of Strains for Reversion to Wild Type or sfaA Phenotype.
Extracts of strains were analysed by HPLC. Strains that produced sanglifehrin A and B were not analysed further as these had reverted to wild type. Strains lacking sanglifehrin A and B production showed small levels ( ̃1-2 mg/L) of a peak retention time 6.5 minutes that displayed a sanglifehrin like chromophore. Analysis by LCMS indicated this peak had a m/z 1073, −16 units from the expected m/z of sanglifehrin. It was postulated this peak was due to incorporation of phenylalanine in absence of meta-hydroxytyrosine.
Eight strains showing loss of sanglifeherin production were subsequently regrown to assess whether the potential sfaA mutation could be complemented chemically allowing a mutasynthetic process to novel sanglifehrins. Strains were grown in SM25-3 seed media for 48 hours before transferring to SGP2 production media with 5% resin. After a further 24 hours growth strains were fed in triplicate with 2 mM DL meta-hydroxytyrosine (addition of 100 ul of a 0.16M solution in 1M HCL) or 2 mM L-phenylalanine with an unfed strain used as control. Strains were also fed pipecolic acid (2 mM) in methanol) to enhance product yields. Strains were harvested after a further 4 days growth and extracted and analysed by HPLC. Meta-hydroxy tyrosine was shown to completely complement the sfaA mutation and addition of L-phenylalanine increased levels of the −16 amu compound. Strain Biot-4585 was chosen for further study as the sfaA deletion mutant.
Example 2
Other Methods for Construction of the sfaA Deletion Construct
Other methods can be used to generate sfaA deletion mutants. Examples include sfaA insertional inactivation mutants (such as example 12 from WO2010/034243 (the contents of which are herewith incorporated by reference in their entirety)). This strain was generated as described in WO2010/034243, and given the strain designation BIOT-4452.
In an alternative procedure to generate the deletion of sfaA two oligonucleotides 15209F 5′-CAGAGAATTCGCGGTACGGGGCGGACGACAAGGTGTC-3′(SEQ ID NO. 4) and 17219R5′-GCGCATGCATGTGCCGGTGCCGGTCCGCGAGCCGCTTGG-3′(SEQ ID NO. 5) are used to amplify an upstream region of homology using cosmid TL3006 (SEQ ID NO. 3) as template and KOD DNA polymerase. The amplified product is treated with T4 polynucleotide kinase (NEB) and cloned into pUC18 that has been dephosphorylated by treating with shrimp alkaline phosphatase (Roche). The resulting construct is checked by restriction digestion and thoroughly sequenced to ensure the desired sequence is generated and that errors have not been introduced during polymerase amplification. This construct is digested with EcoRI and NsiI and the products analysed by gel electrophoresis. The desired sequence-containing band (i.e. upstream homology  ̃2 kb) is excised from the gel and purified using standard procedures (QiaEX resin). A second series of oligonucleotides (SEQ ID NO. 6) 17766F 5′-CCTCATGCATCTGGAGGACGTCGCAGGTGAATTCTGGGCG-3′ and 19763R 5′-GGGCAAGCTTCTCCTGGCTGAGCTTGAACATCG-3′(SEQ ID NO. 7) are used to amplify a downstream region of homology using cosmid TL3006 (SEQ ID NO. 3) as template and KOD DNA polymerase. The amplified product is treated with T4 polynucleotide kinase (NEB) and cloned into pUC18 that has been dephosphorylated by treating with shrimp alkaline phosphatase (Roche). The resulting construct is analysed by restriction digestion and thoroughly sequenced to ensure the desired sequence is generated and that errors have not been introduced during polymerase amplification. This construct is digested with HindIII and NsiI and the products analysed by gel electrophoresis. The desired sequence-containing band (i.e. downstream homology  ̃2 kb) is excised from the gel and purified using standard procedures (QiaEX resin). Vector pKC1139 is digested with EcoRI and HindIII and the large vector fragment isolated by gel electrophoresis and purified by standard methods (QiaEX resin). The isolated upstream and downstream homology fragments are then cloned into this fragment of pKC1139 in a three-way ligation to generate the desired sfaA deletion construct.
In a further alternative procedure for generation of a sfaA deletion construct commercial gene synthesis (i.e. Genscript or other vendor) is used to generate a construct containing the desired sequence (SEQ ID NO. 8). This purchased construct is digested using BamHI and XbaI to excise the sequence of interest and the products analysed by gel electrophoresis. The desired sequence-containing band ( ̃4 kb) is excised from the gel and purified using standard procedures. Vector pKC1139 is digested with BamHI and XbaI and the large fragment isolated by gel electrophoresis and purified by standard methods. The two isolated fragments are then ligated together to generate the desired sfaA deletion construct.
These alternative sfaA deletion constructs are introduced into Streptomyces sp. A92-308110 (DSM9954) by conjugation using the methods in Example section 1.2.
Example 3
Array Feed of the sfaA Deletion Mutant
Spore stocks of a mutant disrupted in sfaA (BIOT-4452 or BIOT-4585) were prepared after growth on MAM, ISP4, ISP3 or ISP2 medium, and preserved in 20% w/v glycerol in distilled water and stored at −80° C. Vegetative cultures (seed cultures) were prepared by inoculating spore stock (1% v/v) into 7 mL seed medium (SM25 medium) in 50 mL centrifuge tubes with foam plugs. The culture tubes were incubated at 27° C., 250 rpm (5 cm throw) for 48 h. From the seed culture 10% (v/v) was transferred into 7 mL production medium SGP-2 in 50 mL centrifuge tubes with foam plugs. Cultivation was carried out at 24° C. and 300 rpm (2.5 cm throw). For production of sanglifehrin mutasynthetic analogues, 0.05 mL of a 0.32 M solution (in 1N HCl) of the feed compound (mutasynthon) was added to each tube at 24 hours post inoculation to give a final concentration of 2 mM. Additionally, 0.05 ml of a 0.32 M solution of piperazic acid (in methanol) was added to each tube at 24 hours to give a final concentration of 2 mM. Cultivation was continued for an additional four days post feeding.
Samples were extracted by transferring 0.8 ml of the whole broth into a 2 ml capped eppendorf tube. 0.8 ml of acetonitrile was added, along with 0.015 ml of formic acid. The mixture was then shaken for 30 minutes on a vibrax. The tube was then centrifuged at 13000 rpm for 10 minutes and 0.15 ml of the supernatant was removed for analysis. Extracts were analysed as described in general methods.
Table 1 shows the mutasynthons that were fed in this way, along with the LCMS H+ and Na+ adducts, anticipated molecular mass and retention time of the sanglifehrin mutasynthetic products observed. The major peaks, relating to the sanglifehrin A analogues, are shown. In all cases, LCMS peaks were also seen for the sanglifehrin B analogues (Mass −18).












TABLE 1







[M − H]−
[M + Na]+
molecular
retention


mutasynthon

observed
observed
mass
time


fed
mutasynthon name
(m/z)
(m/z)
(amu)
(minutes)











2-amino-3-(4-fluoro-3- hydroxyphenyl)propanoic acid
1106.4
1130.4
1107.4
5.5


 






2-amino-3-(3-fluoro-5- hydroxyphenyl)propanoic acid
1106.4
1130.4
1107.4
5.7


 






methyl 2-amino-3-(3-fluoro- 5-hydroxyphenyl) proprionate
1106.4
1130.4
1107.4
5.7


 






(S)-methyl 2-amino-3-(3- hydroxy-4- methylphenyl)propanoate
1102.5
1126.7
1103.5
6.0


 






2-amino-3-(3- fluorophenyl)propanoic acid
1090.4
1114.5
1091  
6.1


 






methyl (2S)-2-amino-3-(3- hydroxy(2- pyridyl))propanoate
1089.5
1113.7
1090.5
4.4


 






methyl 2-amino-3-(2-fluoro- 5- hydroxyphenyl)propanoate
1106.5
1130.6
1107.5
5.5


 






methyl 2-amino-3-(2-fluoro- 3- hydroxyphenyl)propanoate
1106.5
1130.6
1107.5
5.1


 






methyl 2-amino-3-(2,6- difluoro-3- hydroxyphenyl)propanoate
1124.4
1148.5
1125.5
5.1


 






methyl 2-amino-3-(4-ethyl-3- hydroxyphenyl)propanoate
1116.7
1141.0
1117.7
7.2


 






methyl 2-amino-3-(2,4- difluoro-3- hydroxyphenyl)propanoate
1124.7
1148.8
1125.7
6.0









Example 4
Isolation of 63-Fluoro Sanglifehrin a, Compound 14
Fermentation carried out as described in general methods utilising methyl 2-amino-3-(3-fluoro-5-hydroxyphenyl)propanoate and DL-piperazic acid as precursors, both were added at 26 hours.
After harvesting the culture broths were pooled and adjusted to approx. pH 3 with formic acid and centrifuged (3300 g) for 25 mins to separate the cells and resin from the clarified broth. The clarified broth was discarded after assay having confirmed less than 5% of target compound present. The cells and resin were stirred with 2 volumes of acetonitrile for 1 hr using a magnetic stirrer. The acetonitrile extract was recovered either by centrifugation or by allowing it to settle under gravity. A second acetonitrile extraction of the cells and resin was then performed under the same conditions. The combined acetonitrile extracts were concentrated to a residual aqueous volume under reduced pressure and then adjusted to pH 6. This was extracted twice with ethyl acetate and the combined organics taken to dryness under reduced pressure to give the final crude (1.3 g).
The crude extract (1.3 g) was dissolved in ethyl acetate (2 ml) and loaded onto a silica gel column (10×2 cm) conditioned with ethyl acetate (500 ml). The column was eluted with ethyl acetate and then with stepwise increases in acetone (10%, 20%, 30%, etc. in ethyl acetate). Approx. 250 mL fractions were collected and the target compound identified by analytical LC, combined and taken to dryness. This material (278 mg) was dissolved in methanol (1.8 ml) and purified by preparative HPLC. A Waters Xterra MSC18 column (10 micron, 19 cm×250 mm) was used with solvent pumped at 21 mL/min. Solvent A was water and solvent B was acetonitrile. The column was run isocratically at 50% B for 6 minutes following the injection followed by a gradient to 100% B at 30 minutes. Pure fractions were identified by HPLC-UV and combined. These fractions were taken to dryness under reduced pressure to yield the target compound as an off-white amorphous solid (20 mg).
Example 5
Isolation of 62,63-Fluoro Sanglifehrin A, Compound 15
Fermentation carried out as described in general methods utilising methyl (S)-2-amino-3-(3,4-difluoro-5-hydroxyphenyl)propanoate and DL-piperazic acid as precursors, both were added at 26 hours.
After harvesting the culture broths were pooled and adjusted to approx. pH 3 with formic acid and centrifuged (3300 g) for 25 mins to separate the cells and resin from the clarified broth. The clarified broth was discarded after assay having confirmed less than 5% of target compound present. The cells and resin were stirred with 2 volumes of acetonitrile for 1 hr using a magnetic stirrer. The acetonitrile extract was recovered either by centrifugation or by allowing it to settle under gravity. A second acetonitrile extraction of the cells and resin was then performed under the same conditions. The combined acetonitrile extracts were concentrated to a residual aqueous volume under reduced pressure and then adjusted to pH 6. This was extracted twice with ethyl acetate and the combined organics taken to dryness under reduced pressure to give the final crude (1.6 g).
The crude extract (1.6 g) was dissolved in 2 ml ethyl acetate and loaded onto a silica gel column (10×2 cm) conditioned with 500 ml ethyl acetate. The column was eluted with ethyl acetate and then with stepwise increases in acetone (10%, 20%, 30%, etc. in ethyl acetate). Approx. 250 mL fractions were collected and the target compound identified by analytical LC, combined and taken to dryness. This material (188 mg) was dissolved in 1.8 ml methanol and purified by preparative HPLC. A Waters Xterra MSC18 column (10 micron, 19 cm×250 mm) was used with solvent pumped at 21 mL/min. Solvent A was water and solvent B was acetonitrile. The column was run isocratically at 50% B for 6 minutes following the injection followed by a gradient to 100% B at 30 minutes. These fractions were taken to dryness under reduced pressure to yield the target compound as an off-white amorphous solid (15 mg).
Example 6
Isolation of 62-Fluoro Sanglifehrin A, Compound 16
Employed methyl (S)-2-amino-3-(4-fluoro-3-hydroxyphenyl)propanoate and DL-piperazic acid precursors. Carried out in accordance with general method with exception that precursors were added at 27 hours.
After harvesting the culture broths were pooled and adjusted to approx. pH 3 with formic acid and centrifuged (3300 g) for 25 mins to separate the cells and resin from the clarified broth. The clarified broth was discarded after assay having confirmed less than 5% of target compound present. The cells and resin were stirred with 2 volumes of acetonitrile for 1 hr using magnetic stirrer. The acetonitrile extract was recovered either by centrifugation or by allowing it to settle under gravity. A second acetonitrile extraction of the cells and resin was then performed under the same conditions.
The combined acetonitrile extracts were concentrated to a residual aqueous volume under reduced pressure and then adjusted to pH 6. This was extracted twice with ethyl acetate and the combined organics taken to dryness under reduced pressure to give the final oily crude (4.2 g).
The crude extract (4.2 g) was dissolved in 4 ml ethyl acetate and loaded onto a silica gel column (15×2 cm) conditioned with 500 ml ethyl acetate. The column was eluted with ethyl acetate and then with stepwise increases in acetone (10%, 20%, 30%, etc. in ethyl acetate). Approx. 250 mL fractions were collected and the target compound identified by analytical LC, combined and taken to dryness. This material (390 mg) was dissolved in 2.4 ml methanol and purified by preparative HPLC. A Waters Xterra MSC18 column (10 micron, 19 cm×250 mm) was used with solvent pumped at 21 mL/min. Solvent A was water and solvent B was acetonitrile. The column was run isocratically at 50% B for 6 minutes following the injection followed by a gradient to 100% B at 30 minutes. Pure fractions were identified by HPLC-UV and combined. These fractions were taken to dryness under reduced pressure to yield the target compound as an off-white amorphous solid (38 mg).
Example 7
Isolation of 62-Methyl Sanglifehrin A, Compound 17
Cryopreserved spore stocks of BIOT-4585 were thawed at room temperature. Vegetative cultures (seed cultures) were prepared by transferring 0.4 mL of spore stock into 400 mL medium SM25 in 2 L Erlenmeyer flasks with foam plug. Cultivation was carried out for 48 hours at 27° C. and 250 rpm (2.5 cm throw). From the seed culture 20 mL was transferred into 400 mL production medium SGP2+5% HP20 in 2 L Erlenmeyer flasks with foam plug. After 24 hours cultivation at 24° C. and 250 rpm (2.5 cm throw), 2 mL of a 200 mM solution of methyl (S)-2-amino-3-(3-hydroxy-4-methylphenyl)propanoate in 1M hydrochloric acid and 2 mL of a 400 mM methanolic solution of DL-piperazic acid was added to each production flask to give a final 1 mM concentration of the individual enantiomers of the precursors. Cultivation was continued for further four days at 24° C. and 250 rpm (2.5 cm throw).
The culture broths were pooled and adjusted to approx. pH 3 with formic acid and centrifuged (3300 g) for 25 mins to separate the cells and resin from the clarified broth. The clarified broth was discarded after assay having confirmed less than 5% of target compound present. The cells and resin were stirred with 2 volumes of acetonitrile for 1 hr using an overhead paddle stirrer. The acetonitrile extract was recovered by allowing it to settle under gravity. A second acetonitrile extraction of the cells and resin was then performed under the same conditions. The combined acetonitrile extracts were concentrated to a residual aqueous volume under reduced pressure and then adjusted to pH 6. This was extracted twice with ethyl acetate and the combined organics taken to dryness under reduced pressure to give the final crude extract (7.6 g).
The crude extract (7.6 g) was dissolved in 5 ml ethyl acetate and loaded onto a silica gel column (15×2 cm) conditioned with 500 ml ethyl acetate. The column was eluted with ethyl acetate and then with stepwise increases in acetone (10%, 20%, 30%, etc. in ethyl acetate). Approx. 250 mL fractions were collected and the target compound identified by analytical LC, combined and taken to dryness. This material (319 mg) was dissolved in 2.4 ml methanol and purified by preparative HPLC. A Waters Xterra MSC18 column (10 micron, 19 cm×250 mm) was used with solvent pumped at 21 mL/min. Solvent A was water and solvent B was acetonitrile. The column was run isocratically at 50% B for 6 minutes following the injection followed by a gradient to 100% B at 30 minutes. Pure fractions were identified by HPLC-UV and combined. These fractions were taken to dryness under reduced pressure to yield the target compound as an off-white amorphous solid (14.9 mg).
Example 8
Isolation of 61-Deshydroxy Sanglifehrin A, Compound 18
Cryopreserved spore stocks of BIOT-4585 were thawed at room temperature. Vegetative cultures (seed cultures) were prepared by transferring 0.4 mL of spore stock into 400 mL medium SM25 in 2 L Erlenmeyer flasks with foam plug. Cultivation was carried out for 48 hours at 27° C. and 250 rpm (2.5 cm throw). From the seed culture 500 mL was transferred into 4.5 L production medium SGP2+5% HP20 in a 7 L Applikon fermenter and cultivated at 24° C., 400 rpm (cascade DOT control), 2.5 L/min air flow and 30% DOT (cascade agitation control). After 24 hours cultivation, 7.5 mL of a 667 mM solution of (S)-2-amino-3-phenylpropanoic acid in 1M hydrochloric acid was added to the fermenter to give a final 1 mM concentration of the precursor. Cultivation was continued for further four days at 24° C., 400 rpm (cascade DOT control), 2.5 L/min air flow and 30% DOT (cascade agitation control).
The culture broths were pooled and adjusted to approx. pH 3 with formic acid and centrifuged (3300 g) for 25 mins to separate the cells and resin from the clarified broth. The clarified broth was discarded after assay having confirmed less than 5% of target compound present. The cells and resin were stirred with 2 volumes of acetonitrile for 1 hr using an overhead paddle stirrer. The acetonitrile extract was recovered by allowing it to settle under gravity. A second acetonitrile extraction of the cells and resin was then performed under the same conditions, but with the second extract being recovered by centrifugation. The combined acetonitrile extracts were concentrated to a residual aqueous volume under reduced pressure and then adjusted to pH 6. This was extracted twice with ethyl acetate and the combined organics taken to dryness under reduced pressure to give the final crude (55 g).
The crude extract (55 g) was suspended in 80% methanol in water and extracted with 300 ml hexane twice. The target compound was found in methanol/water part which was taken to dryness. This dried extract (48 g) was dissolved in 30 ml ethyl acetate and loaded onto a silica gel column (20×5 cm) conditioned with 1 L ethyl acetate. The column was eluted with ethyl acetate and then with stepwise increases in acetone (10%, 20%, 30%, etc. in ethyl acetate). Approx. 250 mL fractions were collected and the target compound identified by analytical LC, combined and taken to dryness. This material (813 mg) was dissolved in methanol and purified by preparative HPLC. A Waters Xterra MSC18 column (10 micron, 19 cm×250 mm) was used with solvent pumped at 21 mL/min. Solvent A was water and solvent B was acetonitrile. The column was run isocratically at 50% B for 6 minutes following the injection followed by a gradient to 100% B at 30 minutes. Pure fractions were identified by HPLC-UV and combined. These fractions were taken to dryness under reduced pressure to yield the target compound as an off-white amorphous solid (34 mg).
Example 9
Isolation 58-des(3-hydroxyphenyl)-58-(3-hydroxy(2-pyridyl)-sanglifehrin A, Compound 19
Employed methyl (2S)-2-amino-3-(3-hydroxy(2-pyridyl))propanoate and DL-piperazic acid precursors. Carried out in accordance with general method with exception that the incubator throw during vegetative (seed) cultivation was 2.5 cm.
The culture broths were pooled and adjusted to approx. pH 3 with formic acid and centrifuged (3300 g) for 25 mins to separate the cells and resin from the clarified broth. The clarified broth was discarded after assay having confirmed less than 5% of target compound present. The cells and resin were stirred with 2 volumes of acetonitrile for 1 hr using an overhead paddle stirrer. The acetonitrile extract was recovered by allowing it to settle under gravity. A second acetonitrile extraction of the cells and resin was then performed under the same conditions. The combined acetonitrile extracts were concentrated to a residual aqueous volume under reduced pressure and then adjusted to pH 6. This was extracted twice with ethyl acetate and the combined organics taken to dryness under reduced pressure to give the final crude extract (7 g).
The crude extract (7 g) was dissolved in 4 ml ethyl acetate and loaded onto a silica gel column (15×2 cm) conditioned with 500 ml ethyl acetate. The column was eluted with ethyl acetate and then with stepwise increases in acetone (10%, 20%, 30%, etc. in ethyl acetate to 100% acetone then 1% methanol to stepwise 5% methanol in acetone). Approx. 250 mL fractions were collected and the target compound identified by analytical LC, combined and taken to dryness. This material (204 mg) was dissolved in methanol and purified by preparative HPLC. A Waters Xterra MSC18 column (10 micron, 19 cm×250 mm) was used with solvent pumped at 21 mL/min. Solvent A was water and solvent B was acetonitrile. The column was run isocratically at 50% B for 6 minutes following the injection followed by a gradient to 100% B at 30 minutes. Pure fractions were identified by HPLC-UV and combined. These fractions were taken to dryness under reduced pressure to yield the target compound as an off-white amorphous solid (4 mg).
Example 10
Isolation of 61-Deshydroxy-61-Fluoro Sanglifehrin A, Compound 20
Cryopreserved spore stocks of BIOT-4585 were thawed at room temperature. Vegetative cultures (seed cultures) were prepared by transferring 0.4 mL of spore stock into 400 mL medium SM25 in 2 L Erlenmeyer flasks with foam plug. Cultivation was carried out for 48 hours at 27° C. and 250 rpm (2.5 cm throw). From the seed culture 20 mL was transferred into 400 mL production medium SGP2+5% HP20 in 2 L Erlenmeyer flasks with foam plug. After 24 hours cultivation at 24° C. and 250 rpm (2.5 cm throw), 2 mL of a 400 mM solution of 2-amino-3-(3-fluorophenyl)propanoic acid in 1M hydrochloric acid and 2 mL of a 400 mM methanolic solution of DL-piperazic acid was added to each production flask to give a final 1 mM concentration of the individual enantiomers of the precursors. Cultivation was continued for further four days at 24° C. and 250 rpm (2.5 cm throw).
The culture broths were pooled and adjusted to approx. pH 3 with formic acid and centrifuged (3300 g) for 25 mins to separate the cells and resin from the clarified broth. The clarified broth was discarded after assay having confirmed less than 5% of target compound present. The cells and resin were stirred with 2 volumes of acetonitrile for 1 hr using an overhead paddle stirrer. The acetonitrile extract was recovered either by allowing it to settle under gravity. A second acetonitrile extraction of the cells and resin was then performed under the same conditions. A third extract was obtained by centrifugation of the residual cell and resin mix. The combined acetonitrile extracts were concentrated to a residual aqueous volume under reduced pressure and then adjusted to pH 6. This was extracted twice with ethyl acetate and the combined organics taken to dryness under reduced pressure to give the final crude extract (10.5 g).
The crude extract (10.5 g) was dissolved in 7 ml ethyl acetate and loaded onto a silica gel column (15×2 cm) conditioned with 500 ml ethyl acetate. The column was eluted with ethyl acetate and then with stepwise increases in acetone (10%, 20%, 30%, etc. in ethyl acetate). Approx. 250 mL fractions were collected and the target compound identified by analytical LC, combined and taken to dryness. This material (342 mg) was dissolved in methanol and purified by preparative HPLC. A Waters Xterra MSC18 column (10 micron, 19 cm×250 mm) was used with solvent pumped at 21 mL/min. Solvent A was water and solvent B was acetonitrile. The column was run isocratically at 53% B for 30 minutes following the injection. Pure fractions were identified by HPLC-UV and combined. These fractions were taken to dryness under reduced pressure to yield the target compound as an off-white amorphous solid (6 mg).
Example 11
Isolation of 61-Deshydroxy-61-Amino Sanglifehrin A, Compound 21
Cryopreserved spore stocks of BIOT-4585 were thawed at room temperature. Vegetative cultures (seed cultures) were prepared by transferring 0.4 mL of spore stock into 400 mL medium SM25 in 2 L Erlenmeyer flasks with foam plug. Cultivation was carried out for 48 hours at 27° C. and 250 rpm (2.5 cm throw). From the seed culture 20 mL was transferred into 400 mL production medium SGP2+5% HP20 in 2 L Erlenmeyer flasks with foam plug. After 24 hours cultivation at 24° C. and 250 rpm (2.5 cm throw), 2 mL of a 200 mM solution of methyl (S)-2-amino-3-(3-aminophenyl)propanoate in 1M hydrochloric acid was added to each production flask to give a final 1 mM concentration of the precursors. Cultivation was continued for further four days at 24° C. and 250 rpm (2.5 cm throw).
The culture broths were pooled and adjusted to approx. pH 3 with formic acid and centrifuged (3300 g) for 25 mins to separate the cells and resin from the clarified broth. The clarified broth was discarded after assay having confirmed less than 5% of target compound present. The cells and resin were stirred with 2 volumes of acetonitrile for 1 hr using an overhead paddle stirrer. The acetonitrile extract was recovered either by allowing it to settle under gravity. A second acetonitrile extraction of the cells and resin was then performed under the same conditions. A third extract was obtained by centrifugation of the residual cell and resin mix.
The combined acetonitrile extracts were concentrated to a residual aqueous volume under reduced pressure and then adjusted to pH 6. This was extracted twice with ethyl acetate and the combined organics taken to dryness under reduced pressure to give the final crude.
The crude extract is dissolved in ethyl acetate and loaded onto a silica gel column (conditioned with ethyl acetate. The column is eluted with organic solvent with increasing polarity. Approx. 250 mL fractions are collected and the target compound identified by analytical LC, combined and taken to dryness. This material is dissolved in methanol and purified by preparative HPLCPure fractions are identified by HPLC-UV and combined. These fractions are taken to dryness under reduced pressure to yield the target compound as an off-white amorphous solid.
Example 12
Biological Data—In Vitro Evaluation of HCV Antiviral Activity in the Replicon System
Compounds were analysed in the replicon assay using Huh5.2 cells as described in the General Methods. Cyclosporine A, 1, sanglifehrin A, 5, and the hydroxymacrocycle, 6 were included as a comparison.
















Selectivity





index


Name
EC50 (μM)
CC50 (μM)
(CC50/EC50)


















Cyclosporine A, 1
0.2
4.3
21.5


Sanglifehrin A, 5
0.318
9.1
28.7


Hydroxymacrocycle, 6
8.4
83.6
9.9


14
0.135
12.8
121


15
0.195
16.6
88


16
0.89
29.7
32


17
0.083
11.6
143


18
3.4
11.7
3.5


19
24.3
48.1
3.5









As can be seen, 14, 15, 16 and 17 are all very potent in the Huh5.2 replicon assay (as shown by the low EC50), with good selectivity against the cell line (as shown by a high selectivity index). The previously described sanglifehrin A, 5, is less potent than 14, 15 and 17 at HCV inhibition, and cyclosporine A, 1 is less potent and both 1 and 5 have poorer selectivity indices.
Example 13
Microsome Stability
Stability of the compounds in human and mouse liver microsomes was analysed as described in the General Methods. Sanglifehrin A, 5 was included as a comparison.













Human liver
Mouse liver



microsome
microsome



half life
half life


Name
(mins)
(mins)

















Sanglifehrin A, 5
6.38
6.15


14
9.32
9.78


15
10.81
9.22


16
6.61
5.48


17
9.50
9.67


18
7.64
3.46


19
10.37
4.71









As can be seen, the compounds of the invention, 14, 15, 16, 17, 18 and 19 all have increased stability in human liver microsomes when compared to sanglifehrin A (5) and 14, 15 and 17 are also all more stable in mouse liver microsomes.
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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

1. A compound selected from:















which can also be represented as



















which can also be represented as:




including any tautomer thereof; or an isomer thereof in which the C26, 27 C═C bond shown as trans is cis; 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 group and methanol;
or a pharmaceutically acceptable salt thereof.

2. A compound selected from:



including any tautomer thereof; or an isomer thereof in which the C26, 27 C═C bond shown as trans is cis; 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 group and methanol;
or a pharmaceutically acceptable salt thereof.

3. A compound according to claim 1 for use as a pharmaceutical.

4. A compound according to claim 1 for use as a pharmaceutical for the treatment of viral infections such as HCV or HIV infection or muscular dystrophy.

5. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable diluent or carrier.

6. The pharmaceutical composition according to claim 5 further comprising a second or subsequent active ingredient.

7. A pharmaceutical composition comprising a compound according to claim 2 and a pharmaceutically acceptable diluent or carrier.

8. The pharmaceutical composition according to claim 7 further comprising a second or subsequent active ingredient.