Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
  • C07K9/001—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence having less than 12 amino acids and not being part of a ring structure
  • C07K9/003—Peptides being substituted by heterocyclic radicals, e.g. bleomycin, phleomycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
  • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
  • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
  • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
  • C07H19/067—Pyrimidine radicals with ribosyl as the saccharide radical

Definitions

• This invention is in the area of pharmaceutical chemistry and, in particular, is 2'-C-methyl-ribofuranosyl cytidine-3'-O-L-valine ester and pharmaceutically acceptable salts, derivatives and prodrugs thereof, their syntheses and their uses as an -Flaviviridae agents in the treatment of hosts, notably humans, infected with a Flaviviridae, and in particular, hepatitis C, virus.
• the Flaviviridae family of viruses comprises at least three distinct genera: pestiviruses, which cause disease in cattle and pigs; flaviviruses, which are the primary cause of diseases such as dengue fever and yellow fever; and hepaciviruses, whose sole member is HCV.
• the flavivirus genus includes more than 68 members separated into groups on the basis of serological relatedness (Calisher et al., J. Gen. Virol, 1993, 70, 37-43). Clinical symptoms vary and include fever, encephalitis and hemorrhagic fever (Fields Virology, Editors: Fields, B. N., Knipe, D. M., and Howley, P.
• Flaviviruses of global concern that are associated with human disease include the dengue hemorrhagic fever viruses (DHF), yellow fever virus, shock syndrome and Japanese encephalitis virus (Halstead, S. B., Rev. Infect. Dis., 1984, 6, 251-264; Halstead, S. B., Science, 239:476-481, 1988; Monath, T. P., New Eng. J. Med, 1988, 319, 641-643).
• DHF dengue hemorrhagic fever viruses
• Yellow fever virus yellow fever virus
• shock syndrome and Japanese encephalitis virus
• the pestivirus genus includes bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV, also called hog cholera virus) and border disease virus (BDV) of sheep (Moennig, V. et al. Adv. Vir. Res. 1992, 41, 53-98). Pestivirus infections of domesticated livestock (cattle, pigs and sheep) cause significant economic losses worldwide. BVDV causes mucosal disease in cattle and is of significant economic importance to the livestock industry (Meyers, G. and Thiel, H.- J., Advances in Virus Research, 1996, 47, 53-118; Moennig V., et al, Adv. Vir. Res. 1992, 41, 53-98). Human pestiviruses have not been as extensively characterized as the animal pestiviruses. However, serological surveys indicate considerable pestivirus exposure in humans.
• BVDV bovine viral diarrhea virus
• CSFV classical swine fever virus
• BDV border disease virus
• Pestiviruses and hepaciviruses are closely related virus groups within the Flaviviridae family.
• Other closely related viruses in this family include the GB virus A, GB virus A-like agents, GB virus-B and GB virus-C (also called hepatitis G virus, HGV).
• the hepacivirus group (hepatitis C virus; HCV) consists of a number of closely related but genotypically distinguishable viruses that infect humans. There are approximately 6 HCV genotypes and more than 50 subtypes.
• bovine viral diarrhea virus Due to the similarities between pestiviruses and hepaciviruses, combined with the poor ability of hepaciviruses to grow efficiently in cell culture, bovine viral diarrhea virus (BVDV) is often used as a surrogate to study the HCV virus.
• BVDV bovine viral diarrhea virus
• RNA viruses possess a single large open reading frame (ORF) encoding all the viral proteins necessary for virus replication. These proteins are expressed as a polyprotein that is co- and post-translationally processed by both cellular and virus-encoded proteinases to yield the mature viral proteins.
• the viral proteins responsible for the replication ofthe viral genome RNA are located within approximately the carboxy-terminal. Two-thirds ofthe ORF are termed nonstructural (NS) proteins.
• NS nonstructural
• the mature nonstructural (NS) proteins in sequential order from the amino-terminus ofthe nonstructural protein coding region to the carboxy-terminus ofthe ORF, consist of p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B.
• the NS proteins of pestiviruses and hepaciviruses share sequence domains that are characteristic of specific protein functions.
• the NS3 proteins of viruses in both groups possess amino acid sequence motifs characteristic of serine proteinases and of helicases (Gorbalenya et al. (1988) Nature 333:22; Bazan and Fletterick (1989) Virology 171 :637-639; Gorbalenya et al. (1989) Nucleic Acid Res. 17.3889-3897).
• the NS5B proteins of pestiviruses and hepaciviruses have the motifs characteristic of RNA-directed RNA polymerases (Koonin, E.V. and Dolja, V.V. (1993) Crit. Rev. Biochem. Molec. Biol. 28:375-430).
• NS3 serine proteinase is responsible for all proteolytic processing of polyprotein precursors downstream of its position in the ORF (Wiskerchen and Collett (1991) Virology 184:341-350; Bartenschlager et al. (1993) J. Virol. 67:3835-3844; Eckart et al. (1993) Biochem. Biophys. Res. Comm. 192:399-406; Grakoui et al. (1993) J. Virol. 67:2832-2843; Grakoui et al. (1993) Proc.
• NS4A protein acts as a cofactor with the NS3 serine protease (Bartenschlager et al. (1994) J. Virol. 68:5045-5055; Failla et al.
• NS5B proteins of pestiviruses and hepaciviruses have the predicted RNA-directed RNA polymerases activity (Behrens et al.(1996) EMBO J. 15:12-22; Lchmannet al.(1997) J. Virol. 71:8416-8428; Yuan et al.(1997) Biochem. Biophys. Res. Comm. 232:231-235; Hagedorn, PCT WO 97/12033; Zhong et al.(1998) J. Virol. 72.9365-9369).
• HCV hepatitis C virus
• HCV Hepatitis B Virus
• HCV is an enveloped virus containing a positive-sense single-stranded RNA genome of approximately 9.4kb.
• the viral genome consists of a 5' untranslated region (UTR), a long open reading frame encoding a polyprotein precursor of approximately 3011 amino acids, and a short 3' UTR.
• the 5' UTR is the most highly conserved part ofthe HCV genome and is important for the initiation and control of polyprotein translation.
• Translation ofthe HCV genome is initiated by a cap- independent mechanism known as internal ribosome entry. This mechanism involves the binding of ribosomes to an RNA sequence known as the internal ribosome entry site (IRES).
• IRES internal ribosome entry site
• Viral structural proteins include a nucleocapsid core protein (C) and two envelope glycoproteins, El and E2.
• HCV also encodes two proteinases, a zinc-dependent metalloproteinase encoded by the NS2- NS3 region and a serine proteinase encoded in the NS3 region. These proteinases are required for cleavage of specific regions ofthe precursor polyprotein into mature peptides.
• the carboxyl half of nonstructural protein 5, NS5B contains the RNA- dependent RNA polymerase.
• the function ofthe remaining nonstructural proteins, NS4A and NS4B, and that of NS5A remain unknown.
• a significant focus of current antiviral research is directed to the development of improved methods of treatment of chronic HCV infections in humans (Di Besceglie, A. M. and Bacon, B. R., Scientific American, Oct.: 80-85, (1999)).
• IFNs Interferons
• Flaviviridae including HCV, treatments, using interferon-based therapies.
• U.S. Patent No. 5,980,884 to Blatt et al. discloses methods for retreatment of patients afflicted with HCV using consensus interferon.
• U.S. Patent No. 5,942,223 to Bazer et al. discloses an anti-HCV therapy using ovine or bovine interferon-tau.
• U.S. Patent No. 5,928,636 to Alber et al. discloses the combination therapy of interleukin- 12 and interferon alpha for the treatment of infectious diseases including HCV.
• U.S. Patent No. 5,830,455 to Valtuena et al. discloses a combination HCV therapy employing interferon and a free radical scavenger.
• U.S. Patent No. 5,738,845 to Imakawa discloses the use of human interferon tau proteins for treating HCV.
• Other interferon-based treatments for HCV are disclosed in U.S. Patent No. 5,676,942 to Testa et al, U.S. Patent No. 5,372,808 to Blatt et al, and U.S. Patent No. 5,849,696.
• a number of patents also disclose pegylated forms of interferon, such as, U.S. Patent Nos. 5,747,646, 5,792,834 and 5,834,594 to Hoffmann-La Roche Inc; PCT Publication No. WO 99/32139 and WO 99/32140 to Enzon; WO 95/13090 and US Patent Nos. 5,738,846 and 5,711,944 to Schering; and U.S. Patent No. 5,908,621 to Glue et al.
• Interferon alpha-2a and interferon alpha-2b are currently approved as monotherapy for the treatment of HCV.
• ROFERON®-A (Roche) is the recombinant form of interferon alpha-2a.
• PEGASYS® (Roche) is the pegylated (i.e. polyethylene glycol modified) form of interferon alpha -2a.
• INTRON®A (Schering Corporation) is the recombinant form of Interferon alpha-2b
• PEG-INTRON® (Schering Corporation) is the pegylated form of interferon alpha-2b.
• interferon alpha as well as interferon beta, gamma, tau and omega are currently in clinical development for the treatment of HCV.
• INFERGEN interferon alphacon-1 by InterMune
• OMNIFERON natural interferon
• ALBUFERON Human Genome Sciences
• REBIF interferon beta- la
• Ares-Serono Omega Interferon by BioMedicine
• Oral Interferon Alpha by Amarillo Biosciences
• interferon gamma, interferon tau, and interferon gamma- lb by InterMune
• Ribavirin (l- ⁇ -D-ribofuranosyl-l-l,2,4-triazole-3-carboxamide) is a synthetic, non-interferon-inducing, broad spectrum antiviral nucleoside analog sold under the trade name, Virazole (The Merck Index, 11th edition, Editor: Budavari, S., Merck & Co., Inc., Rahway, NJ, pi 304, 1989). United States Patent No. 3,798,209 and
• Ribavirin is structurally similar to guanosine, and has in vitro activity against several DNA and RNA viruses including Flaviviridae (Gary L. Davis. Gastroenterology 118:S104-S114, 2000).
• Ribavirin reduces serum amino transferase levels to normal in 40% of patients, but it does not lower serum levels of HCV-RNA (Gary L. Davis. Gastroenterology 118:S104-S114, 2000). Thus, ribavirin alone is not effective in reducing viral RNA levels. Additionally, ribavirin has significant toxicity and is known to induce anemia. Ribavirin is not approved fro monotherapy against HCV. It has been approved in combination with interferon alpha-2a or interferon alpha-2b for the treatment of HCV. Combination of Interferon and Ribavirin
• the current standard of care for chronic hepatitis C is combination therapy with an alpha interferon and ribavirin.
• the combination of interferon and ribavirin for the treatment of HCV infection has been reported to be effective in the treatment of interferon naive patients (Battaglia, A.M. et al, Ann. Pharmacother. 34:487-494, 2000), as well as for treatment of patients when histological disease is present (Berenguer, M. et al. Antivir. Ther. 3(Suppl. 3):125-136, 1998).
• HCV-derived enzymes such as protease, helicase, and polymerase inhibitors are being developed.
• Drugs that inhibit other steps in HCV replication are also in development, for example, drugs that block production of HCV antigens from the RNA (IRES inhibitors), drugs that prevent the normal processing of HCV proteins (inhibitors of glycosylation), drugs that block entry of HCV into cells (by blocking its receptor) and nonspecific cytoprotective agents that block cell injury caused by the virus infection.
• ribozymes which are enzymes that break down specific viral RNA molecules
• antisense oligonucleotides which are small complementary segments of DNA that bind to viral RNA and inhibit viral replication
• Examples of classes of drugs that are being developed to treat Flaviviridae infections include:
• Substrate-based NS3 protease inhibitors (Attwood et al, Antiviral peptide derivatives, PCT WO 98/22496, 1998; Attwood et al, Antiviral Chemistry and Chemotherapy 1999, 10, 259-273; Attwood et al, Preparation and use of amino acid derivatives as anti-viral agents, German Patent Pub. DE 19914474; Tung et al.
• Inhibitors of serine proteases particularly hepatitis C virus NS3 protease, PCT WO 98/17679), including alphaketoamides and hydrazinoureas, and inhibitors that terminate in an electrophile such as a boronic acid or phosphonate (Llinas-Brunet et al, Hepatitis C inhibitor peptide analogues, PCT WO 99/07734) are being investigated.
• Non-substrate-based NS3 protease inhibitors such as 2,4,6-trihydroxy-3-nitro- benzamide derivatives (Sudo K. et al, Biochemical and Biophysical Research Communications, 1997, 238, 643-647; Sudo K. et al. Antiviral Chemistry and Chemotherapy, 1998, 9, 186), including RD3-4082 and RD3-4078, the former substituted on the amide with a 14 carbon chain and the latter processing apara- phenoxyphenyl group are also being investigated.
• Sch 68631 a phenanthrenequinone, is an HCV protease inhibitor (Chu M. et al., Tetrahedron Letters 37:7229-7232, 1996).
• U.S. patents disclose protease inhibitors for the treatment of HCV.
• U.S. Patent No. 6,004,933 to Spruce et al. discloses a class of cysteine protease inhibitors for inhibiting HCV endopeptidase 2.
• U.S. Patent No. 5,990,276 to Zhang et al. discloses synthetic inhibitors of hepatitis C virus NS3 protease. The inhibitor is a subsequence of a substrate ofthe NS3 protease or a substrate ofthe NS4A cofactor.
• restriction enzymes to treat HCV is disclosed in U.S. Patent No. 5,538,865 to Reyes et al.
• HCV inhibitor tripeptides are disclosed in US Patent Nos. 6,534,523, 6,410,531, and 6,420,380 to Boehringer Ingelheim and WO 02/060926 to Bristol Myers Squibb.
• Diaryl peptides as NS3 serine protease inhibitors of HCV are disclosed in WO 02/48172 to Schering Corporation.
• Imidazoleidinones as NS3 serine protease inhibitors of HCV are disclosed in WO 02/08198 to Schering Corporation and WO 02/48157 to Bristol Myers Squibb.
• WO 98/17679 to Vertex Pharmaceuticals and WO 02/48116 to Bristol Myers Squibb also disclose HCV protease inhibitors.
• Helicase inhibitors (Diana G.D. et al, Compounds, compositions and methods for treatment of hepatitis C, U.S. Pat. No. 5,633,358; Diana G.D. et al, Piperidine derivatives, pharmaceutical compositions thereof and their use in the treatment of hepatitis C, PCT WO 97/36554);
• S-ODN Antisense phosphorothioate oligodeoxynucleotides (S-ODN) complementary to sequence stretches in the 5' non-coding region (NCR) of the virus (Alt M. et al, Hepatology, 1995, 22, 707-717), or nucleotides 326-348 comprising the 3' end ofthe NCR and nucleotides 371-388 located in the core coding region of the HCV RNA (Alt M. et al, Archives of Virology, 1997, 142, 589-599; Galderisi U. et al, Journal of Cellular
• Inhibitors of IRES-dependent translation (Ikeda N et al. , Agent for the prevention and treatment of hepatitis C, Japanese Patent Pub. JP-08268890; Kai Y. et al. Prevention and treatment of viral diseases, Japanese Patent Pub. JP-10101591);
• Ribozymes such as nuclease-resistant ribozymes (Maccjak, D. J. et al, Hepatology 1999, 30, abstract 995) and those disclosed in U.S. Patent No. 6,043,077 to Barber et al, and U.S. Patent Nos. 5,869,253 and 5,610,054 to
• Nucleoside analogs have also been developed for the treatment of Flaviviridae infections. Idenix Pharmaceuticals disclosed the use of branched nucleosides in the treatment of flavi viruses (including HCV) and pestiviruses in International Publication Nos. WO 01/90121 and WO 01/92282.
• hepatitis C infection and flaviviruses and pestiviruses
• a method for the treatment of hepatitis C infection in humans and other host animals is disclosed in the Idenix publications that includes administering an effective amount of a biologically active 1 ', 2', 3' or 4'-branched ⁇ -D or ⁇ -L nucleosides or a pharmaceutically acceptable salt or derivative thereof, administered either alone or in combination with another antiviral agent, optionally in a pharmaceutically acceptable carrier.
• Fluoronucleosides discloses the use of certain 2'-fluoronucleosides to treat HCV.
• Nucleoside prodrugs have been previously described for the treatment of other forms of hepatitis.
• WO 01/96353 (filed June 15, 2001) to Idenix Pharmaceuticals, discloses 2'-deoxy- ⁇ -L-nucleosides and their 3 '-prodrugs for the treatment of HBV.
• U.S. Patent No. 4,957,924 to Beauchamp discloses various therapeutic esters of acyclovir.
• val-mCyd 3 '-L-valine ester of ⁇ -D-2'-C-methyl- ribofuranosyl cytidine
• HCV 3 '-L-valine ester of ⁇ -D-2'-C-methyl- ribofuranosyl cytidine
• compounds, compositions, methods and uses are provided for the treatment of flaviviridae, including HCV, that include the administration of an effective amount of val-mCyd or its salt, ester, prodrug or derivative, optionally in a pharmaceutically acceptable carrier.
• val-mCyd is used to treat any virus that replicates through an RNA-dependent RNA polymerase.
• a first embodiment provides a compound of Formula (I), ⁇ -D-2'-C- methyl-ribofuranosyl cytidine or a pharmaceutically acceptable salt thereof, and its uses in medical therapy and in the manufacture of a medicant to treat hosts, particularly humans, infected with a flavivirus or pestivirus, including HCV.
• the compound val-mCyd is converted to the parent MCyd through de- esterification in the gastrointestinal mucosa, blood or liver, and is actively transported from the gastrointestinal lumen after oral delivery into the bloodstream by an amino acid transporter function in the mucosa ofthe gastrointestinal tract.
• This accounts for the increase in oral bioavailability compared to the parent 2'-branched nucleoside that is transported primarily by a nucleoside transporter function.
• the mono or divaline ester continues to be absorbed using the amino acid transporter function. Therefore, the superior outcome of better absorption, bioavailability, and reduced competition with other nucleosides or nucleoside analogs for uptake into the bloodstream is achieved.
• val-mCyd reduced the mean HCV RNA level by 0.83 logio copies/ml (8.3 mg/kg/day) and 1.05 logio copies/ml (16.6 mg/kg/day) in seven days.
• the chimps exhibited no drug-related safety issues.
• the parent nucleoside (mCyd) framework can exist as a ⁇ -D or ⁇ -L nucleoside.
• the pharmaceutically acceptable compound is administered in a form that is at least 90% ofthe ⁇ -D enantiomer.
• val-mCyd is at least 95% ofthe ⁇ -D enantiomer.
• the valine ester also has enantiomeric forms.
• the valine moiety is at least 90% ofthe L-enantiomer.
• the valine moiety is at least 95% ofthe L-enantiomer.
• the compounds are used as racemic mixtures or as any combination of ⁇ -D or ⁇ -L parent nucleoside and L or D amino acid.
• the compound of Formula (I) is ⁇ -D-2'-C-methyl- ribofuranosyl cytidine-3'-O-L- valine ester-HCl salt.
• the compound of Formula (II), the ⁇ -D-2'-C-methyl-ribofuranosyl cytidine dihydrochloride salt is provided for administration to a host, particularly a human, infected with a flavivirus or pestivirus infection.
• a particularly preferred embodiment is the mono or dihydrochloride salt.
• a pharmaceutical composition comprising 2'-C- methyl-cytidine-3'-O-L-valine ester-or its pharmaceutically acceptable salt, including a mono or di HCI salt, ester, prodrug or derivative thereof together with a pharmaceutically acceptable carrier, excipient or diluent is provided.
• the 5'-hydroxyl group is replaced with a 5'-OR, wherein R is phosphate (including monophosphate, diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl (including lower acyl); alkyl (including lower alkyl); sulfonate ester including alkyl or arylalkyl sulfonyl including methanesulfonyl and benzyl, wherein the phenyl group is optionally substituted with one or more substituents as described in the definition of aryl given herein; a lipid, including a phospholipids; an amino acid; a carbohydrate; a peptide; cholesterol; or other pharmaceutically acceptable leaving group which when administered in vivo is capable of providing a compound wherein R is independently H or phosphate.
• R is phosphate (including monophosphate, diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl (including lower acyl); alkyl
• the active compounds ofthe present invention can be administered in combination or alternation with another agent that is active against a flavivirus or pestivirus, including HCV (including any described or referred to in the Background ofthe Invention), or other useful biological agent.
• another principal embodiment is a pharmaceutical composition that includes ⁇ -D-2'-C-methyl- ribofuranosyl cytidine-3'-O-L- valine ester-or a pharmaceutically acceptable salt (including a mono- or di-HCl salt), ester, prodrug or pharmaceutically acceptable derivative thereof, together with one or more other effective antiviral agents, optionally with a pharmaceutically acceptable carrier or diluent.
• a method in another embodiment, includes administering ⁇ -D-2'-C-methyl- ribofuranosyl cytidine-3'-O-L- valine ester or a salt, ester, prodrug or derivative thereof, in combination or in alternation with one or more second antiviral agents, optionally with a pharmaceutically acceptable carrier or diluent.
• Any second antiviral agent may be selected that imparts the desired biological effect.
• Nonlimiting examples include interferon, ribavirin, an interleukin, an NS3 protease inhibitor, a cysteine protease inhibitor, phenanthrenequinone, a thiazolidine derivative, thiazolidine, benzanilide, a helicase inhibitor, a polymerase inhibitor, a nucleotide analogue, gliotoxin, cerulenin, antisense phosphorothioate oligodeoxynucleotides, inhibitor of IRES-dependent translation, or ribozyme.
• the second antiviral agent is selected from natural interferon, interferon alpha (including interferon-alpha-2a and interferon-alpha-2b), interferon beta (including interferon beta- la), omega interferon, interferon gamma (including interferon gamma- lb), interferon tau, and consensus interferon. Any of these interferons can be stabilized or otherwise modified to improve the tolerance and biological stability or other biological properties. One common modification is pegylation (modification with polyethylene glycol). In one particular embodiment, the second antiviral agent is pegylated or unpegylated interferon 2-alpha.
• the active compound is a 3'-amino acid ester of ⁇ -D-2'-C-methyl-ribofuranosyl cytidine, wherein the amino acid can be natural or synthetic and can be in a D or L stereoconfiguration.
• the active compound is a 3 '-acyl ester of ⁇ -D-2'-C-methyl-ribofuranosyl cytidine.
• Figures la and lb are illustration of two processes for the preparation of ⁇ -D-2'-C- methyl-ribofuranosyl cytidine-3'-O-L- valine ester dihydrochloride, as described in Example 1.
• Figure 2 is a photocopy of a gel illustrating the site-specific chain termination of in vitro RNA synthesis by ⁇ -D-2'-C-methyl-ribofuranosyl cytidine triphosphate at specified guanine residues in RNA templates, as described in Example 9.
• Figure 3 is a graph ofthe the titer of bovine viral diarrhea virus (BVDV) over number of passages of BVDV infected MDBK cells, indicating eradication of a persistent BVDV infection by prolonged treatment with ⁇ -D-2'-C-methyl- ribofuranosyl cytidine (16uM) as described in Example 10. Arrows indicate points at which a portion of cells were withdrawn from drug treatment.
• BVDV bovine viral diarrhea virus
• FIGS. 4a and 4b are graphs ofthe concentration of bovine viral diarrhea virus (BVDV) in MDBK cells persistently infected with the virus, as described in
• BVDV bovine viral diarrhea virus
• Example 11 These graphs indicate the synergy between ⁇ -D-2'-C-methyl- ribofuranosyl cytidine and interferon alpha 2b (IntronA) in reducing the viral titer.
• Figure 4a is a graph ofthe effect of ⁇ -D-2'-C-methyl-ribofuranosyl cytidine and IntronA on BVDV (strain NY-1) titers in persistently infected MDBK cells over time.
• Figure 4b is a graph ofthe effect of ⁇ -D-2'-C-methyl- ribofuranosyl cytidine in combination with IntronA on BVDV (strain I-N- dlns) titers in persistently-infected MDBK cells.
• Figures 5a-d illustrate the results of experiments studying the development of resistance to ⁇ -D-2'-C-methyl-ribofuranosyl cytidine treated MDBK cells, infected with bovine viral diarrhea virus (BVDV), as described in Example 12.
• Figure 5a is a graph of a representative experiment showing the effect over twenty eight days of ⁇ -D-2'-C-methyl-ribofuranosyl cytidine or IntronA treatment on BVDV (strain I-N-dlns) titers in persistently infected MDBK cells.
• Figure 5b is a photocopy of a dish plated with infected MDBK cells that illustrates the size ofthe foci formed by phenotypes ofthe wild-type BVDV (strain I-N-dlns), versus the ⁇ -D-2'-C-methyl-ribofuranosyl cytidine-resistant BVDV (I-N-dlns 107R), indicating that the resistant virus formed much smaller foci than the wild-type, I-N-dlns strain.
• Figure 5c is a graph ofthe titer of BVDV strains I-N-dlns or I-N-dIns-107R over hours post-infection in infected MDBK cells.
• Figure 5d is a graph ofthe effect of Intron A on the BVDV viral titer yield in de novo-infected MDBK cells treated with IntronA.
• Figure 6 is a graph ofthe concentration of hepatitis C virus (Logio) in individual chimpanzees over days of treatment with ⁇ -D-2'-C-methyl-ribofuranosyl cytidine-3 '-O-L- valine ester as described in Example 13.
• Figure 7 is a graph ofthe concentration of hepatitis C virus in individual chimpanzees over days of treatment with ⁇ -D-2'-C-methyl-ribofuranosyl cytidine-3 '-O-L- valine ester as compared to baseline, as described in Example 13.
• Figure 8 is a graph of percent of total ⁇ -D-2'-C-methyl-ribofuranosyl cytidine-3 '-O-L- valine ester remaining in samples over time after incubation ofthe drug in human plasma at 4°C, 21°C, and 37°C, as described in Example 14.
• Figure 9a is a graph showing the relative levels ofthe di- and tri-phosphate derivatives of ⁇ -D-2'-C-methyl-ribofuranosyl cytidine and ⁇ -D-2'-C-methyl- ribofuranosyl uridine (mUrd) after incubation of HepG2 cells with 10 ⁇ M ⁇ - D-2'-C-methyl-ribofuranosyl cytidine over time, as described in Example 14.
• Figure 9b is a graph ofthe decay ofthe tri-phosphate derivative of ⁇ -D-2'-C- methyl-ribofuranosyl cytidine after incubation of HepG2 cells with 10 ⁇ M ⁇ - D-2'-C-methyl-ribofuranosyl cytidine over time.
• Figure 9c is a graph ofthe concentration ofthe di- and tri-phosphate derivatives of ⁇ -D-2'-C-methyl- ribofuranosyl cytidine and ⁇ -D-2'-C-methyl-ribofuranosyl uridine (mUrd) after incubation of HepG2 cells with lO ⁇ M ⁇ -D-2'-C-methyl-ribofuranosyl cytidine at increasing concentrations ofthe drug ( ⁇ M).
• Figure 10 is a graph ofthe concentration (ng/ml) of ⁇ -D-2'-C-methyl-ribofuranosyl cytidine in human serum after administration of ⁇ -D-2'-C-methyl- ribofuranosyl cytidine-3'-O-L- valine ester to patients, as described in Example 17.
• Figure 11 is a graph ofthe median change ofthe titer of hepatitis C virus in human patients after administration of ⁇ -D-2'-C-methyl-ribofuranosyl cytidine-3 '-O- L- valine ester, as described in Example 17.
• the graph indicates change from baseline in Logio HCV RNA by patient visit.
• val-mCyd 3 '-L- valine ester of ⁇ -D-2'-C-methyl- ribofuranosyl cytidine
• HCV hepatitis C virus
• compounds, compositions, methods and uses are provided for the treatment of flaviviridae, including HCV, that include the administration of an effective amount of val-mCyd or its salt, ester, prodrug or derivative, optionally in a pharmaceutically acceptable carrier.
• val-mCyd is used to treat any virus that replicates through an RNA-dependent RNA polymerase.
• the disclosed compounds or their pharmaceutically acceptable derivatives or salts or pharmaceutically acceptable formulations containing these compounds are useful in the prevention and treatment of flaviviridae (including HCV) infections and other related conditions such as anti-HCV antibody positive and HCV-positive conditions, and hepatitis C related hepatic cancer (e.g., hepatocellular carcinoma) and hepatic tumors.
• these compounds or formulations can be used prophylactically to prevent or retard the progression of clinical illness in individuals who are anti-HCV (or more generally anti-flaviviridae) antibody or HCV-antigen or flaviviridae-antigen positive, or who have been exposed to HCV or another flaviviridae virus.
• the present invention includes the following features:
• compositions comprising the 3 '-L- valine ester of ⁇ -D-2'-C-methyl-ribofuranosyl cytidine, and pharmaceutically acceptable prodrugs, derivatives and salts thereof together with a pharmaceutically acceptable carrier or diluent;
• the active compound is a 3 '-amino acid ester of ⁇ -D-2'-C-methyl-ribofuranosyl cytidine, wherein the amino acid can be natural or synthetic and can be in a D or L stereoconfiguration.
• the active compound is a 3'-acyl ester of ⁇ -D or ⁇ -L 2'-C-methyl-ribofuranosyl cytidine.
• mCyd is a specific inhibitor of genomic RNA replication.
• the intracellular 5'-triphosphate moiety, mCyd-TP appears to directly inhibit the NS5B RNA-dependent-RNA polymerase.
• Analysis of RNA synthesis in the presence of mCyd-TP suggests that mCyd-TP acts as a specific chain terminator of viral RNA synthesis through as yet unidentified mechanisms.
• Antiviral nucleosides and nucleoside analogs are generally converted into the active metabolite, the 5'- triphosphate (TP) derivatives by intracellular kinases.
• the nucleoside-TPs then exert their antiviral effect by inhibiting the viral polymerase during virus replication.
• intracellular phosphorylation converts mCyd predominantly into the active species, mCyd-triphosphate (mCyd-TP), along with low levels of mCyd- diphosphate.
• mCyd-TP mCyd-triphosphate
• mUrd-TP 2'-C-methyl-uridine TP
• Flaviviruses included within the scope of this invention are discussed generally in Fields Virology, Editors: Fields, .N., Knipe, D.M. and Howley, P.M.; Lippincott-Raven Pulishers, Philadelphia, PA; Chapter 31 (1996).
• flaviviruses include, without limitation: Absettarov; Alfuy; AIN; Aroa; Bagaza; Banzi; Bououi; Bussuquara; Cacipacore; Carey Island; Dakar bat; Dengue viruses 1, 2, 3 and 4; Edge Hill; Entebbe bat; Gadgets Gully; Hanzalova; Hypr; Ilheus; Israel turkey meningoencephalitis; Japanese encephalitis; Jugra; Jutiapa; Kadam; Karshi; Kedougou; Kokoera; Koutango; Kumlinge; Kunjin; Kyasanur Forest disease; Langat; Louping ill; Meaban; Modoc; Montana myotis leukoencephalitis; Murray valley encephalitis; Naranjal; Negishi; Ntaya; Omsk hemorrhagic fever; Phnom-Penh bat; Powassan; Rio Bravo; Rocio; Royal Farm; Russian spring-summer encephalitis; Saboya; St.
• Pestiviruses included within the scope of this invention are also discussed generally in Fields Virology (Id.). Specific pestiviruses include, without limitation: bovine viral diarrhea virus (“VDV”); classical swine fever virus (“CSFV”) also known as hog cholera virus); and border disease virus (“DV”).
• VDV bovine viral diarrhea virus
• CSFV classical swine fever virus
• DV border disease virus
• alkyl refers to a saturated straight, branched, or cyclic, primary, secondary, or tertiary hydrocarbon of typically Ci to Cio, and specifically includes CF 3 , CC1 3 , CFC1 2 , CF 2 C1, CH 2 CF 3 , CF 2 CF 3 , methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, secbutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3- methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.
• the term includes both substituted and unsubstituted alkyl groups, and particularly includes halogenated alkyl groups, and even more particularly fluorinated alkyl groups.
• moieties with which the alkyl group can be substituted are selected from the group consisting of halogen (fluoro, chloro, bro o or iodo), hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et al, Protective Groups in Organic Synthesis. John Wiley and Sons, Second Edition, 1991, hereby incorporated by reference.
• lower alkyl refers to a Ci to C 4 saturated straight, branched, or a cyclic (for example, cyclopropyl) alkyl group, including both substituted and unsubstituted forms. Unless otherwise specifically stated in this application, when alkyl is a suitable moiety, lower alkyl is preferred. Similarly, when alkyl or lower alkyl is a suitable moiety, unsubstituted alkyl or lower alkyl is preferred.
• alkylamino or arylamino refers to an amino group that has one or two alkyl or aryl substituents, respectively.
• protected refers to a group that is added to an oxygen, nitrogen, or phosphorus atom to prevent its further reaction or for other purposes.
• oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis.
• aryl refers to phenyl, biphenyl, or naphthyl, and preferably phenyl.
• the term includes both substituted and unsubstituted moieties.
• the aryl group can be substituted with any desired moiety, including, but not limited to, one or more moieties selected from the group consisting of halogen (fluoro, chloro, bromo or iodo), hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et al, Protective Groups in Organic Synthesis. John Wiley and Sons, Second Edition, 1991.
• alkaryl or alkylaryl refers to an alkyl group with an aryl substituent.
• aralkyl or arylalkyl refers to an aryl group with an alkyl substituent.
• halo as used herein, includes a specific description of chloro, bromo, iodo, and fluoro individually.
• purine or pyrimidine base includes, but is not limited to, adenine, N 6 - alkylpurines, N 6 -acylpurines (wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl), N 6 -benzylpurine, N 6 -halopurine, N 6 -vinylpurine, N -acetylenic purine, N 6 -acyl purine, N 6 -hydroxyalkyl purine, N 6 -alkylaminopurine, N 6 -thioalkyl purine, N 2 - alkylpurines, N 2 -alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine, 5- methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or 4- mercaptopyrmidine, uracil, 5-halouracil, including 5-fluorouracil, C 5
• Purine bases include, but are not limited to, guanine, adenine, hypoxanthine, 2,6- diaminopurine, and 6-chloropurine. Functional oxygen and nitrogen groups on the base can be protected as necessary or desired. Suitable protecting groups are well known to those skilled in the art, and include trimethylsilyl, dimethylhexylsilyl, t- butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups, and acyl groups such as acetyl and propionyl, methanesulfonyl, and p-toluenesulfonyl.
• acyl or O-linked ester refers to a group ofthe formula C(O)R', wherein R' is an straight, branched, or cyclic alkyl (including lower alkyl), carboxylate residue of an amino acid, aryl including phenyl, heteroaryl, alkaryl, aralkyl including benzyl, alkoxyalkyl including methoxymethyl, aryloxyalkyl such as phenoxymethyl; or substituted alkyl (including lower alkyl), aryl including phenyl optionally substituted with chloro, bromo, fluoro, iodo, Ci to C alkyl or Ci to C 4 alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl including methanesulfonyl, the mono, di or triphosphate ester, trityl or monomethoxy-trityl, substituted benzyl, alkaryl, aralkyl including
• Aryl groups in the esters optimally comprise a phenyl group.
• acyl groups include acetyl, trifluoroacetyl, methylacetyl, cyclopropylacetyl, cyclopropyl- carboxy, propionyl, butyryl, isobutyryl, hexanoyl, heptanoyloctanoyl, neo-heptanoyl, phenylacetyl, 2-acetoxy-2-phenylacetyl, diphenylacetyl, ⁇ -methoxy- ⁇ -trifluoromethyl-phenylacetyl, bromoacetyl, 2-nitro- benzeneacetyl, 4-chloro-benzeneacetyl, 2-chloro-2,2-diphenylacetyl, 2-chloro-2- phenylacetyl, trimethylacetyl, chlorodifluoroacetyl, perfluoro
• amino acid includes naturally occurring and synthetic ⁇ , ⁇ , ⁇ , or ⁇ amino acids, and includes but is not limited to, amino acids found in proteins, i.e. glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine.
• the amino acid is in the L-configuration, but can also be used in the D-configuration.
• the amino acid can be a derivative of alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl, ⁇ -alanyl, ⁇ -valinyl, ⁇ -leucinyl, ⁇ -isoleuccinyl, ⁇ -prolinyl, ⁇ -phenylalaninyl, ⁇ - tryptophanyl, ⁇ -methioninyl, ⁇ -glycinyl, ⁇ -serinyl, ⁇ -threoninyl, ⁇ -cysteinyl
• nucleoside composition that includes at least 85 or 90% by weight, preferably 95%, 98, 99% or 100% by weight, ofthe designated enantiomer of that nucleoside.
• isolated refers to a nucleoside composition that includes at least 85 or 90% by weight, preferably 95%, 98%, 99% or 100% by weight, ofthe nucleoside.
• the term host refers to an unicellular or multicellular organism in which the virus can replicate, including cell lines and animals, and preferably a human. Alternatively, the host can be carrying a part ofthe Flaviviridae viral genome, whose replication or function can be altered by the compounds ofthe present invention.
• the term host specifically refers to infected cells, cells transfected with all or part ofthe Flaviviridae genome and animals, in particular, primates (including chimpanzees) and humans. In most animal applications ofthe present invention, the host is a human patient. Veterinary applications, in certain indications, however, are clearly anticipated by the present invention (such as chimpanzees).
• pharmaceutically acceptable salt or prodrug is used throughout the specification to describe any pharmaceutically acceptable form (such as an ester, phosphate ester, salt of an ester or a related group) of a nucleoside compound which, upon administration to a patient, provides the nucleoside compound.
• Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the pharmaceutical art.
• Pharmaceutically acceptable prodrugs refer to a compound that is metabolized, for example hydrolyzed or oxidized, in the host to form the compound ofthe present invention. Typical examples of prodrugs include compounds that have biologically labile protecting groups on a functional moiety ofthe active compound.
• Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, dephosphorylated to produce the active compound.
• the compounds of this invention possess antiviral activity against a Flaviviridae, or are metabolized to a compound that exhibits such activity.
• compositions for the treatment of pestivirus, flavivirus and hepatitis C virus infection include administering an effective amount of 2'-C-methyl-cytidine-3'-O-L-valine, or a pharmaceutically acceptable salt, ester or prodrug thereof to a host in need thereof.
• a hydrochloride salt ofthe compound of Formula (I) is used.
• a dihydrochloride salt ofthe compound of Formula (I) is preferred.
• the active compound can be administered as any salt or prodrug that upon administration to the recipient is capable of providing directly or indirectly the parent compound, or that exhibits activity itself.
• the pharmaceutically acceptable salts which are alternatively referred to as "physiologically acceptable salts”
• a compound that has been alkylated, acylated or otherwise modified at the 5 '-position or on the purine or pyrimidine base thereby forming a type of "pharmaceutically acceptable prodrug”.
• the modifications can affect the biological activity ofthe compound, in some cases increasing the activity over the parent compound. This can easily be assessed by preparing the salt or prodrug and testing its antiviral activity according to the methods described herein, or other methods known to those skilled in the art.
• a compound ofthe Formula (I), or a pharmaceutically acceptable salt or prodrug thereof is provided:
• the active compound is a 3 '-amino acid ester of ⁇ -D-2'-C-methyl-ribofuranosyl cytidine, wherein the amino acid can be natural or synthetic and can be in a D or L stereoconfiguration.
• the active compound is a 3 '-acyl ester of ⁇ -D-2'-C-methyl-ribofuranosyl cytidine.
• the 5'-hydroxyl group is replaced with a
• R is phosphate (including monophosphate, diphosphate, triphosphate, or a stabilized phosphate prodrug); a stabilized phosphate prodrug; acyl (including lower acyl); alkyl (including lower alkyl); sulfonate ester including alkyl or arylalkyl sulfonyl including methanesulfonyl and benzyl, wherein the phenyl group is optionally substituted with one or more substituents as described in the definition of aryl given herein; a lipid, including a phosphohpids; an amino acid; a carbohydrate; a peptide; cholesterol; or other pharmaceutically acceptable leaving group which when administered in vivo is capable of providing a compound wherein R is independently H or phosphate.
• acyl including lower acyl
• alkyl including lower alkyl
• sulfonate ester including alkyl or arylalkyl sulfonyl including methanes
• the compound ofthe present invention can be synthesized by means known in the art.
• a nucleoside, nucleoside analog, or a salt, prodrug, stereoisomer or tautomer thereof, that is disubstituted at the 2' C is prepared.
• ⁇ -D-2'-C-methyl-cytidine (4-amino-l-(3,4-dihydroxy-5- hydroxymethyl-3-C-methyl-tetrahydro-furan-2-yl)-7H-pyrimidine-2-one), which has value as an active nucleoside or is used as a process intermediate is prepared.
• 3'- ⁇ 9-valinyl ester of ⁇ -D-2'-C-methyl-cytidine (2-amino-3- methyl-butyric acid 5-(4-amino-2-oxo-2H-pyrimidin-l-yl)-4-hydroxy-4-C-methyl-2- hydroxymethyl-tetrahydro-furan-3-yl ester) or a hydrochloride salt form is prepared.
• Nucleosides, nucleoside analogs, salts or ester prodrugs prepared may be used as intermediates in the preparation of a wide variety of other nucleoside analogues, or may be used directly as antiviral and/or antineoplastic agents.
• EXAMPLE 1 SYNTHESIS OF ⁇ -D-2'-C-METHYL-RIBOFURANOSYL CYTroiNE-3'-O-L- VALINE ESTER
• the synthesis comprises reacting cytosine, BSA and SnCU/acetonitrile with 1,2,3, 5-tetra-0-benzoyl-2-C- methyl- ⁇ -D-ribofuranose (1) to form 4-amino-l-(3,4-dibenzoyloxy-5- benzoyloxymethyl-3-methyl-tetrahydro-furan-2-yl)-7H-pyrimidin-2-one (2); and reacting (2) with NaOMe/MeO ⁇ to provide 4-amino-l-(3,4-dihydroxy-5- hydroxymethyl-3-methyl-tetrahydro-furan-2-yl )-/H-pyrimidin-2-one (3), also known as 2-C-methyl- ⁇ -D-ribofuranose.
• cytosine as a starting material rather than benzoyl-cytosine improves the "atom economy" ofthe process and simplifies purification at later steps.
• next steps in this process comprise reacting (3) with Me 2 NC ⁇ (OMe) 2 in DMF to form (4), 7V-[l-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan- 2-yl)-2-oxo-l, 2-dihydro-pyrimidin-4-yl]-NN-dimethyl-formamidine, which is the amino-protected form of (3); reacting (4) with TBDPSC1 and imidazole in DCM to provide the 5'-silyl-protected form of (4) as N'- ⁇ l-[5-(tert-butyl-diphenyl- silanyloxymethyl)-3,4-dihydroxy-3-methyl-tetrahydro-furan-2-yl]-2-oxo-l, 2- dihydro-pyrimidin-4-yl ⁇ -N,N-dimethyl-fo ⁇ mamidine (5), where the use of DCM provides the advantage of having greater control over disilyl by-product
• EXAMPLE 2 SYNTHESIS OF 2'-C-METHYL-CYTIDINE-3 , -O-L- VALINE ESTER (VAL-mCyd)
• De-ionized water 100 mL was stirred in a 250 mL 3-necked round bottom flask, equipped with an overhead stirrer, a stirring shaft, a digital temperature read-out device and an argon line.
• Argon was bubbled into water for thirty minutes and D- fructose (20.0 g, 0.111 mole) was added and the solution became clear in a few minutes.
• Calcium oxide (12.5 g, 0.223 mole) was added in portions over a period of five minutes and the mixture was vigorously stirred. An exotherm was observed and reaction temperature reached 39.6 °C after 10 minutes from the start of the calcium oxide addition. After about fifteen minutes, the reaction mixture developed a yellow color that deepened with time.
• the plate was then heated, using a heat gun, until the appearance of yellowish spots on the pink background).
• the desired lactone typically appears at an Rf value of 0.33 under the above conditions. More polar by-products and unreacted material are detected in the R f value range of 0.0 to 0.2.
• the slurry mixture was stirred at 45-50 °C overnight. The mixture was then evaporated under reduced pressure and at 45-50 °C to remove 75 mL of water.
• Sodium chloride (30 g) and tetrahydrofuran (100 mL) were added to the aqueous slurry (about 75 mL) and the mixture was vigorously stirred at 25 °C for 30 minutes. The layers were separated and the aqueous layer was stirred for 10 minutes with 75 mL of fresh tetrahydrofuran. This process was repeated for three times and the tetrahydrofuran solutions were combined and stirred with 10 g of anhydrous magnesium sulfate for 30 minutes.
• Step 2 Synthesis of compound 10: 2,3,5-Tri-O-benzoyl-2-C-methyl-D- ribonic- ⁇ -lactone
• Step 3 Synthesis of compound 11: 2.3.5-Tri-O-benzoyl-2-C-methyl- ⁇ -D- ribofuranose:
• Step 4 Synthesis of compound 12: 1.2,3,5-tetra-O-benzoyl-2-C-metbyl- ⁇ -D- ribofuranose:
• Benzoyl chloride (283 mg, 2.0 mmol.) was added, over a period of five minutes, to a cold solution (5 °C) of compound 11 (480 mg, 1.0 mmol.), 4- dimethylaminopyridine (12.3 mg, 0.1 mmol.) and triethylamine (506 mg, 5.0 mmol.) in anhydrous tetrahydrofuran (5 mL).
• the reaction mixture was stirred at room temperature and under argon atmosphere overnight. HPLC analysis indicated 0.25% of un-reacted starting material.
• the reaction was quenched by adding ice-cold water (10 g) and saturated aqueous solution of sodium bicarbonate.
• Tetrahydrofuran was removed under reduced pressure and the mixture was extracted with ethyl acetate (50 mL). The organic solution was washed with water (25 mL), brine (25 mL), dried (anhydrous sodium sulfate, 12 g) and solvent removed under reduced pressure to give 650 mg of thick oily product. This crude product was stirred with 5 mL of tert-butyl methyl ether for 5 minutes and heptane (5 mL) and water (0.1 mL) were added and stirring was continued for an additional period of two hours at 20 °C.
• Step 5 Synthesis of compound 13: 4-Amino-l-(3,4-dibenzoyloxy-5- benzyloxymethyl-3-methyl-tetrahydro-furan-2-yl)-7H-pyrimidine-2-one
• Cytosine (89 g, 0.80 mol) was suspended in acetonitrile (900 ml) in a 12 L round bottomed flask equipped with a reflux condenser, overhead stirrer and an argon inlet adapter. The suspension was stirred at 20 °C under argon atmosphere and N,O- bis(trimethylsilyl)acetamide (537 ml, 2.2 mol) was added in one portion. The resulting solution was heated to 80 ° C and stirred for an additional hour at the same temperature.
• 1,2,3, 5-tetra-O-benzoyl-2-C-methyl- ⁇ -D-ribofuranose (425.0 g, 0.73 mol) was suspended in acetonitrile (4000 ml) and added to the reaction mixture. The reaction mixture became clear after a few minutes and the temperature dropped to ca. 50 ° C. Tin(IV) chloride (154 ml, 1.31 mol) was added over a period of 15 minutes and stirring was continued at 80 °. After one hour, an aliquot of reaction mixture was quenched by adding aqueous sodium bicarbonate solution and extracting the aqueous layer with ethyl acetate.
• the ethyl acetate layer was examined by TLC (silica gel, 20% ethyl acetate in heptane, R f for sugar derivative: 0.40). TLC analysis indicated the complete consumption of the sugar derivative. Desired product was detected by TLC using 10% methanol in dichloromethane (Rf : 0.37). The reaction was also monitored by HPLC (Method # 2). Reaction mixture was cooled to 20 °C and quenched by adding saturated aqueous sodium bicarbonate solution (3000 ml) over a period of 30 minutes (observed an exotherm when added the first few drops of the sodium bicarbonate solution). Solid sodium bicarbonate (1350 g) was added in portions to avoid foaming.
• Step 6 Synthesis of compound mCyd: 4-Amino-l-(3.4-dihydroxy-5-hvdroxymethyl- 3-methyl-tetrahvdro-furan-2-yl)-7H-pyrimidine-2-one
• Step 7 Synthesis of compound 14: 2-7ert-butoxycarbonylamino-3 -methyl-butyric acid 5-(4-amino-2-oxo-2H-pyrimidin-l-yl)-4-hvdroxy-2-hvdroxymethyl-4-methyl- tetrahydro-furan-3-yl ester
• the mixture was next heated at 97 ° C for 20 minutes and the tetrahydrofuran solution was added slowly through an addition funnel over a period of 2 hours, maintaining the temperature no lower than 82 °C.
• the reaction mixture was cooled to room temperature and then triethylamine and tetrahydrofuran were removed under vacuum at 30 °C.
• the solution was then neutralized with acetic acid to a p ⁇ of 7.69.
• N,N-dimethylformamidine was removed under vacuum at 35 °C and chased with ethyl acetate (2 x 200 mL).
• the crude product was stirred with ethyl acetate (500 mL) and water (300 mL). The two layers were separated and the aqueous layer was extracted with ethyl acetate (500 mL). The combined organic layers were washed with an aqueous saturated brine solution (500 mL). Next the organic layer was extracted with an aqueous solution of malonic acid (4 x 400 mL, 10wt.%). The organic layer was checked by TLC (silica, 20% methanol in dichloromethane) to make sure that all the desired product was removed from the organic layer.
• Step 8 Synthesis of val-mCyd - 2-Amino-3-methyl-butyric acid 5-(4-amino-2-oxo- 2H-pyrimidine- 1 -yl)-4-hydroxy-2 hydroxy-methyl-4-methyl-tetrahvdro-furan-3-yl ester (dihydrochloride salt)
• Identification is determined at 272 nm.
• the column used is a Waters Novapak® C18, 3.9 x 150 mm ID, 4 ⁇ m particle size, 6 ⁇ A pore size or equivalent.
• the system suitability requirement for the percent relative standard deviation for the reference standard is not more than 1.0%.
• nucleosides ofthe present invention have several chiral centers and may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, diastereomeric, polymo ⁇ hic, or stereoisomeric form, or mixtures thereof, of a compound ofthe invention, which possess the useful properties described herein. It being well known in the art how to prepare optically active forms (for example, by resolution ofthe racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).
• the 1 ' and 4' carbons ofthe nucleoside are chiral, their nonhydrogen substituents (the base and the CHOR groups, respectively) can be either cis (on the same side) or trans (on opposite sides) with respect to the sugar ring system.
• the four optical isomers therefore are represented by the following configurations (when orienting the sugar moiety in a horizontal plane such that the oxygen atom is in the back): cis (with both groups “up”, which corresponds to the configuration of naturally occurring ⁇ -D nucleosides), cis (with both groups “down”, which is a nonnaturally occurring ⁇ -L configuration), trans (with the C2' substituent "up” and the C4' substituent "down”), and trans (with the C2' substituent "down” and the C4' substituent "up”).
• the "D-nucleosides” are cis nucleosides in a natural configuration and the "L-nucleosides” are cis nucleosides in the nonnaturally occurring configuration.
• optically active materials examples include at least the following.
• the resulting diastereomers are then separated by chromatography or crystallization by virtue of their now more distinct structural differences and the chiral auxiliary later removed to obtain the desired enantiomer; vii) first- and second-order asymmetric transformations - a technique whereby diastereomers from the racemate equilibrate to yield a preponderance in solution ofthe diastereomer from the desired enantiomer or where preferential crystallization of the diastereomer from the desired enantiomer perturbs the equilibrium such that eventually in principle all the material is converted to the crystalline diastereomer from the desired enantiomer.
• kinetic resolutions this technique refers to the achievement of partial or complete resolution of a racemate (or of a further resolution of a partially resolved compound) by virtue of unequal reaction rates ofthe enantiomers with a chiral, non- racemic reagent or catalyst under kinetic conditions; ix) enantiospecific synthesis from non-racemic precursors - a synthetic technique whereby the desired enantiomer is obtained from non-chiral starting materials and where the stereochemical integrity is not or is only minimally compromised over the course ofthe synthesis; x) chiral liquid chromatography - a technique whereby the enantiomers of a racemate are separated in a liquid mobile phase by virtue of their differing interactions with a stationary phase.
• the stationary phase can be made of chiral material or the mobile phase can contain an additional chiral material to provoke the differing interactions; xi) chiral gas chromatography - a technique whereby the racemate is volatilized and enantiomers are separated by virtue of their differing interactions in the gaseous mobile phase with a column containing a fixed non-racemic chiral adsorbent phase; xii) extraction with chiral solvents - a technique whereby the enantiomers are separated by virtue of preferential dissolution of one enantiomer into a particular chiral solvent; xiii) transport across chiral membranes - a technique whereby a racemate is placed in contact with a thin membrane barrier.
• the barrier typically separates two miscible fluids, one containing the racemate, and a driving force such as concentration or pressure differential causes preferential transport across the membrane barrier. Separation occurs as a result ofthe non-racemic chiral nature ofthe membrane which allows only one enantiomer ofthe racemate to pass through.
• Hosts including humans, infected with pestivirus, flavivirus, HCV or another organism replicating through a RNA-dependent RNA viral polymerase, or for treating any other disorder described herein, can be treated by administering to the patient an effective amount ofthe active compound or a pharmaceutically acceptable prodrug or salt thereof in the presence of a pharmaceutically acceptable carrier or dilutent.
• the active materials can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid or solid form.
• a preferred dose ofthe compound for pestivirus, flavivirus or HCV will be in the range from about 1 to 50 mg/kg, preferably 1 to 20 mg/kg, of body weight per day, more generally 0.1 to about 100 mg per kilogram body weight ofthe recipient per day. Lower doses may be preferable, for example doses of 0.5-100 mg, 0.5-50 mg, 0.5-10 mg, or 0.5-5 mg per kilogram body weight per day. Even lower doses may be useful, and thus ranges can include from 0.1-0.5 mg per kilogram body weight per day.
• the effective dosage range ofthe pharmaceutically acceptable salts and prodrugs can be calculated based on the weight ofthe parent nucleoside to e delivered. If the salt or prodrug exhibits activity in itself, the effective dosage can be estimated as above using the weight ofthe salt or prodrug, or by other means known to those skilled in the art.
• the compound is conveniently administered in unit any suitable dosage form, including but not limited to one containing 7 to 3000 mg, preferably 70 to 1400 mg of active ingredient per unit dosage form.
• An oral dosage of 50-1000 mg is usually convenient, including in one or multiple dosage forms of 50, 100, 200, 250, 300, 400, 500, 600, 700, 800, 900 or 1000 mgs.
• Lower doses may be preferable, for example from 10-100 or 1-50 mg.
• Also contemplated are doses of 0.1-50 mg, or 0.1-20 mg or 0.1-10.0 mg.
• lower doses may be utilized in the case of administration by a non-oral route, as, for example, by injection or inhalation.
• the active ingredient should be administered to achieve peak plasma concentrations ofthe active compound of from about 0.2 to 70 ⁇ M, preferably about 1.0 to 10 ⁇ M. This may be achieved, for example, by the intravenous injection of a 0.1 to 5% solution ofthe active ingredient, optionally in saline, or administered as a bolus ofthe active ingredient.
• the concentration of active compound in the drug composition will depend on abso ⁇ tion, inactivation and excretion rates ofthe drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity ofthe condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment ofthe person administering or supervising the administration ofthe compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice ofthe claimed composition.
• the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time, with or without other anti-viral agents.
• Oral compositions will generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the pu ⁇ ose of oral therapeutic administration, the active compound can be inco ⁇ orated with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can e included as part ofthe composition.
• the tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
• a binder such as microcrystalline cellulose, gum tragacanth or gelatin
• an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
• a lubricant such as magnesium stearate or Sterotes
• a glidant such as colloidal silicon dioxide
• the compound can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like.
• a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
• the compound or a pharmaceutically acceptable prodrug or salt thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics, antifungals, anti- inflammatories, or other antivirals, including other nucleoside compounds.
• Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• the parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
• preferred carriers are physiological saline or phosphate buffered saline (PBS).
• the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems, biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Co ⁇ oration. Liposomal suspensions (including liposome's targeted to infected cells with monoclonal antibodies to viral antigens) are also preferred as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No.
• liposome formulations may be prepared by dissolving appropriate lipid(s), such as sterol phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol, in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface ofthe container.
• An aqueous solution ofthe active compound or its monophosphate, diphosphate, and/or triphosphate derivatives is then introduced into the container.
• the container is then swirled by hand to free lipid material from the sides ofthe container and to disperse lipid aggregates, thereby forming the liposomal suspension.
• nucleoside as a pharmaceutically acceptable salt is within the scope ofthe invention.
• pharmaceutically acceptable salts are organic acid addition salts formed with acids, which provide a physiological acceptable anion. These include, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, ⁇ -ketoglutarate, and ⁇ - glycerophosphate, formate, fumarate, propionate, glycolate, lactate, pyruvate, oxalate, maleate, and salicyate. Suitable inorganic salts may also be formed, including, sulfate, nitrate, bicarbonate, hydrobromate, carbonate salts, and phosphoric acid. A particularly preferred embodiment is the mono or dihydrochloride salt.
• salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
• Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
• the salt is a hydrochloride salt ofthe compound.
• the pharmaceutically acceptable salt is a dihydrochloride salt ofthe compound of Formula (I).
• the compounds of this invention possess antiviral activity against flavivirus, pestivirus or HCV, or are metabolized to a compound that exhibits such activity.
• nucleosides described herein can be administered as a nucleotide prodrug to increase the activity, bioavailability, stability or otherwise alter the properties ofthe nucleoside.
• a number of nucleotide prodrug ligands are known.
• alkylation, acylation or other lipophilic modification ofthe mono-, di- or triphosphate ofthe nucleoside reduces polarity and allows passage into cells.
• substituent groups that can replace one or more hydrogens on the phosphate moiety are alkyl, aryl, steroids, carbohydrates, including sugars, and alcohols, such as 1,2- diacylglycerol. Many are described in R. Jones and N.
• the active nucleoside can also be provided as a 5'-phosphoether lipid or a 5'- ether lipid.
• Non-limiting examples are disclosed in the following references, which are inco ⁇ orated by reference herein: Kucera, L.S., N. Iyer, E. Leake, A. Raen, Modest E.K., D.L.W., and C. Piantadosi. 1990. "Novel membrane-interactive ether lipid analogs that inhibit infectious HIV-l production and induce defective virus formation.” AIDS Res. Hum. Retro Viruses.
• Nonlimiting examples of U.S. patents that disclose suitable lipophilic substituents that can be covalently incorporated into the nucleoside, preferably at the 5'-OH position ofthe nucleoside or lipophilic preparations thereof include U.S. Patent Nos. 5,149,794 (Sep. 22, 1992, Yatvin et al); 5,194,654 (Mar. 16, 1993, Hostetler et al, 5,223,263 (June 29, 1993, Hostetler et al); 5,256,641 (Oct. 26, 1993, Yatvin et al); 5,411,947 (May 2, 1995, Hostetler et al); 5,463,092 (Oct.
• lipophilic substituents that can be attached to the nucleosides ofthe present invention, or lipophilic preparations, include WO 89/02733, W0 90/00555, W0 91/16920, W0 91/18914, W0 93/00910, W0 94/26273, W0 96/15132, EP 0 350 287, EP 93917054.4, and W0 91/19721.
• the active compounds ofthe present invention can be administered in combination or alternation with another anti-flavivirus or pestivirus agent, or in particular an anti-HCV agent.
• combination therapy effective dosages of two or more agents are administered together, whereas in alternation or sequential-step therapy, an effective dosage of each agent is administered serially or sequentially.
• the dosages given will depend on abso ⁇ tion, inactivation and excretion rates ofthe drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity ofthe condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment ofthe person administering or supervising the administration ofthe compositions.
• an anti-HCV (or anti-pestivirus or anti-flavivirus) compound that exhibits an EC 50 of 10-15 ⁇ M, or preferably less than 1-5 ⁇ M, is desirable.
• Examples include substrate-based NS3 protease inhibitors (Attwood et al, Antiviral peptide derivatives, PCT WO 98/22496, 1998; Attwood et al, Antiviral Chemistry and Chemotherapy 1999, 10, 259-273; Attwood et al, Preparation and use of amino acid derivatives as anti-viral agents, German Patent Pub. DE 19914474; Tung et al.
• Inhibitors of serine proteases particularly hepatitis C virus NS3 protease, PCT WO 98/17679), including alphaketoamides and hydrazinoureas, and inhibitors that terminate in an electrophile such as a boronic acid or phosphonate (Llinas-Brunet et al, Hepatitis C inhibitor peptide analogues, PCT WO 99/07734); Non-substrate- based NS3 protease inhibitors such as 2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al, Biochemical and Biophysical Research Communications, 1997, 238, 643-647; Sudo K. et al.
• Eglin c isolated from leech, is a potent inhibitor of several serine proteases such as S. griseus proteases A and B, ⁇ -chymotrypsin, chymase and subtilisin. Qasim M.A. et al, Biochemistry 36:1598-1607, 1997.
• U.S. patents disclosing protease inhibitors for the treatment of HCV include, for example, U.S. Patent No. 6,004,933 to Spruce et al. which discloses a class of cysteine protease inhibitors for inhibiting HCV endopeptidase 2; U.S. Patent No. 5,990,276 to Zhang et al. which discloses synthetic inhibitors of hepatitis C virus NS3 protease; U.S. Patent No. 5,538,865 to Reyes et a; WO 02/008251 to Corvas International, Inc, and WO 02/08187 and WO 02/008256 to Schering Corporation.
• HCV inhibitor tripeptides are disclosed in US Patent Nos.
• Diaryl peptides as NS3 serine protease inhibitors of HCV are disclosed in WO 02/48172 to Schering Corporation.
• Imidazolidindiones as NS3 serine protease inhibitors of HCV are disclosed in WO 02/08198 to Schering Corporation and WO 02/48157 to Bristol Myers Squibb.
• WO 98/17679 to Vertex Pharmaceuticals and WO 02/48116 to Bristol Myers Squibb also disclose HCV protease inhibitors.
• S-ODN Antisense phosphorothioate oligodeoxynucleotides (S-ODN) complementary to sequence stretches in the 5' non-coding region (NCR) of the virus (Alt M. et al, Hepatology, 1995, 22, 707-717), or nucleotides 326-348 comprising the 3' end of the NCR and nucleotides 371-388 located in the core coding region of the HCV RNA (Alt M. et al, Archives of Virology, 1997, 142, 589-599; Galderisi U. et al, Journal of Cellular Physiology, 1999, 757, 251- 257);
• Inhibitors of IRES-dependent translation (Ikeda N et al, Agent for the prevention and treatment of hepatitis C, Japanese Patent Pub. JP-08268890; Kai Y. et al. Prevention and treatment of viral diseases, Japanese Patent Pub. JP- 10101591 );
• Ribozymes such as nuclease-resistant ribozymes (Maccjak, D. J. et al, Hepatology 1999, 30, abstract 995) and those disclosed in U.S. Patent No. 6,043,077 to Barber et al, and U.S. Patent Nos. 5,869,253 and 5,610,054 to Draper et al; and
• miscellaneous compounds including 1-amino- alkylcyclohexanes (U.S. Patent No. 6,034,134 to Gold et al), alkyl lipids (U.S. Pat. No. 5,922,757 to Chojkier et al), vitamin E and other antioxidants (U.S. Pat. No. 5,922,757 to Chojkier et al), squalene, amantadine, bile acids (U.S. Pat. No. 5,846,964 to Ozeki et al), N-(phosphonoacetyl)-L-aspartic acid, (U.S. Pat. No.
• any other compound currently in preclinical or clinical development for treatment of hepatitis C virus including: Interleukin- 10 by Schering-Plough, IP-501 by Interneuron, Merimebodib (VX-497) by Vertex, AMANTADINE® (Symmetrel) by Endo Labs Solvay, HEPTAZYME® by RPI, IDN-6556 by Idun Pharma., XTL-002 by XTL., HCV/MF59 by Chiron, CIVACIR (Hepatitis C Immune Globulin) by NABI, LEVOVIRTN® by ICN/Ribapharm, VIRAMIDINE® by ICN/Ribapharm, ZADAXIN® (thymosin alpha-1) by Sci Clone, thymosin plus pegylated interferon by Sci Clone, CEPLENE® (histamine dihydrochloride) by Maxim, VX 950 / LY 570310 by Vertex/Eli Li
• a useful cell-based assay to detect HCV and its inhibition assesses the levels of replicon RNA from Huh7 cells harboring the HCV replicon. These cells can be cultivated in standard media, for example DMEM medium (high glucose, no pyruvate), supplemented with 10% fetal bovine serum, l ⁇ non-essential amino acids, Pen-Strep-Glu (100 units/liter, 100 microgram/liter, and 2.92 mg/liter, respectively), and G418 500 to 1000 microgram/milliliter). Antiviral screening assays can be done in the same medium without G418. To keep the cells in the logarithmic growth phase, cells are seeded in 96-well plates at low density (for example, 1000 cells per well).
• Replicon RNA + host RNA can then be amplified in a real-time RT-PCR (Q- RT-PCR) protocol, and quantified.
• ⁇ Ct Ct te stcompound - Ctnegative
• a ⁇ Ct value of 3.3 represents a 1-log reduction in replicon production.
• recombinant interferon alpha-2a for example, Roferon-A, Hoffmann-Roche, NJ, USA
• the compounds can be tested in dilution series (typically at 100, 33, 10, 3 and 1 ⁇ M).
• the ⁇ Ct values for each concentration allow the calculation of the 50% effective concentration (EC50).
• the assay described above can be adapted to the other members ofthe Flaviviridae by changing the cell system and the viral pathogen.
• Methodologies to determine the efficacy of these antiviral compounds include modifications ofthe standard techniques as described by Holbrook MR et al. Virus Res. 2000, 69, 31; Markland W et al. Antimicrob. Agents. Chemother. 2000, 44, 859; Diamond MS et al., J. Virol. 2000, 74, 7814; Jordan I et al. J. Infect. Pis. 2000, 182, 1214; Sreenivasan V et al. J. Virol. Methods 1993, 45 (1), 1; or Baginski SG et al. Proc. Natl.
• HCV replicon system in HuH7 cells (Lohmann V et al. Science. 1999, 285 (5424), 110) or modifications thereof (Blight et al. 2000) can be used.
• Nucleic acid amplification technology is now the method of choice for identification of a large and still growing number of microorganisms such as Mycobacterium tuberculosis, human immunodeficiency virus (HIV), and hepatitis C virus (HCV) in biological samples.
• Nucleic acid amplification techniques include the polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence- based amplification (NASBA), strand-displacement amplification (SDA), and transcription-mediated amplification (TMA).
• PCR polymerase chain reaction
• LCR ligase chain reaction
• NASBA nucleic acid sequence- based amplification
• SDA strand-displacement amplification
• TMA transcription-mediated amplification
• Several FDA-approved diagnostic products inco ⁇ orate these molecular diagnostic methods (see Table below).
• Nucleic acid amplification technology tests involve not only amplification, but detection methodologies as well.
• Amplified-product detection schemes are of two basic types: heterogeneous and homogeneous.
• Heterogeneous detection is characterized by a distinct step, such as washing, designed to remove unhybridized probes from hybridized probes, whereas in homogeneous detection there is no physical separation step to remove free probe from bound probe.
• Southern blotting for example, is a heterogeneous detection technique.
• electrophoresis is used to separate amplification products by size and charge.
• the size-fractionated products are transferred to a membrane or filter by diffusion, vacuuming, or electroblotting.
• Labeled detection probes are then hybridized to the membrane-bound targets in solution, the filters are washed to remove any unhybridized probe, and the hybridized probe on the membrane is detected by any of a variety of methods.
• heterogeneous detection are based on specific capture ofthe amplification products by means of enzyme-linked immunosorbent assays (ELISAs).
• ELISAs enzyme-linked immunosorbent assays
• One method used with PCR involves labeling one primer with a hapten or a ligand, such as biotin, and, after amplification, capturing it with an antibody- or streptavidin- coated microplate.
• the other primer is labeled with a reporter such as fluorescein, and detection is achieved by adding an antifluorescein antibody, horseradish peroxidase (HRP) conjugate.
• HRP horseradish peroxidase
• the LCx probe system (Abbott Laboratories; Abbott Park, IL) and the Amplicor HIV-l test (Roche Molecular Systems Inc.; Pleasanton, CA) are systems that use heterogeneous detection methods.
• hapten-labeled oligonucleotide probes thermocycle in the ligase chain reaction. Either a capture hapten or a detection hapten is covalently attached to each ofthe four primer oligonucleotides.
• each amplified product (amplicon) has one capture hapten and one detection hapten.
• the LCx system instrument transfers the reaction to a new well where antibody-coated microparticles bind the capture haptens.
• Each microparticle is then irreversibly bound to a glass-fiber matrix.
• a wash step removes from the microparticle any probe that contains only the detection hapten.
• the LCx instrument adds an alkaline phosphatase (AP)-antibody conjugate that binds to the detection hapten.
• AP alkaline phosphatase
• a fluorigenic substrate for AP is 4-methylumbelliferyl. Dephosphorylation of 4-mefhylumbelliferyl by AP converts it to 4-methylumbelliferone, which is fluorescent.
• the Amplicor HIV-l test uses an ELISA format. After amplification by PCR, the amplicon is chemically denatured. Amplicon-specific oligonucleotide probes capture the denatured strands onto a coated microplate. The operator washes away any uninco ⁇ orated primers and unhybridized material in a wash step and then adds an avidin-HRP conjugate to each well. The conjugate binds to the biotin-labeled amplicon captured on the plate. The operator then adds 3,3',5,5'-tetramethylbenzidine (TMB), a chromogenic HRP substrate. When hydrogen peroxide is present, HRP oxidizes TMB. The signal is determined colorimetrically.
• TMB 3,3',5,5'-tetramethylbenzidine
• hybridized and nonhybridized detection probes are not physically separated in homogeneous detection systems, these methods require fewer steps than heterogeneous methods and thus are less prone to contamination.
• kits that use homogeneous detection of fluorescent and chemiluminescent labels are the TaqMan system (Applied Biosystems; Foster City, CA), BDProbeTecET system (Becton Dickinson; Franklin Lakes, NJ), QPCR System 5000 (Perkin-Elmer Co ⁇ .; Norwalk, CT), and Hybridization Protection Assay (Gen-Probe Inc.; San Diego).
• the TaqMan system detects amplicon in real time.
• the detection probe which hybridizes to a region inside the amplicon, contains a donor fluorophore such as fluoroscein at its 5' end and a quencher moiety, for example, rhodamine, at its 3' end. When both quencher and fluorophore are on the same oligonucleotide, donor fluorescence is inhibited.
• the probe is bound to the target.
• Taq polymerase displaces and cleaves the detection probe as it synthesizes the replacement strand. Cleavage ofthe detection probe results in separation ofthe fluorophore from the quencher, leading to an increase in the donor fluorescence signal.
• the amount of fluorescent signal increases as the amount of amplicon increases.
• beacons use quenchers and fluorophores also. Beacons are probes that are complementary to the target amplicon, but contain short stretches
• beacons (approximately 5 nucleotides) of complementary oligonucleotides at each end.
• the 5' and 3' ends ofthe beacons are labeled with a fluorophore and a quencher, respectively.
• a hai ⁇ in structure is formed when the beacon is not hybridized to a target, bringing into contact the fluorophore and the quencher and resulting in fluorescent quenching.
• the loop region contains the region complementary to the amplicon.
• the hairpin structure opens and the quencher and fluorophore separate, allowing development of a fluorescent signal. 14
• a fluorometer measures the signal in real time.
• the BDProbeTecET system uses a real-time detection method that combines aspects of TaqMan and molecular beacons.
• the probe has a hai ⁇ in loop structure and contains fluorescein and rhodamine labels.
• the region complementary to the target molecule is not within the loop but rather in the region 3' to the rhodamine label.
• the single-stranded loop contains a restriction site for the restriction enzyme BsoBI.
• the single-stranded sequence is not a substrate for the enzyme.
• the fluorescein and rhodamine labels are near each other before amplification, which quenches the fluorescein fluorescence.
• Strand-displacement amplification converts the probe into a double-stranded molecule.
• the BsoBI restriction enzyme can then cleave the molecule, resulting in separation ofthe labels and an increase in the fluorescent signal.
• the QPCR System 5000 employs electrochemiluminescence with ruthenium labels.
• a biotinylated primer is used. After amplification, the biotin products are captured on streptavidin-coated paramagnetic beads. The beads are transferred into an electrochemical flow cell by aspiration and magnetically held to the surface ofthe electrode. Upon electrical stimulation, the ruthenium-labeled probe emits light.
• the Hybridization Protection Assay is used in Gen-Probe's nonamplified PACE assays as well as in amplified Mycobacterium tuberculosis and Chlamydia trachomatis assays.
• the detection oligonucleotide probes in HPA are labeled with chemiluminescent acridinium ester (AE) by means of a linker arm.
• AE chemiluminescent acridinium ester
• Hybridization takes place for 15 minutes at 60°C in the same tube in which the amplification occurred.
• the selection reagent a mildly basic buffered solution added after hybridization, hydrolyzes the AE on any unhybridized probe, rendering it nonchemiluminescent.
• the AE on hybridized probes folds inside the minor groove of the double helix, thereby protecting itself from hydrolysis by the selection reagent.
• the AE emits a chemiluminescent signal upon hydrolysis by hydrogen peroxide followed by sodium hydroxide.
• a luminometer records the chemiluminescent signal for 2 seconds (a period termed a light-off) and reports the photons emitted in terms of relative light units (RLU).
• Detection-probe design is critical in all methodologies that use probes to detect amplification products. Good detection probes hybridize only to specified amplification product and do not hybridize to nonspecific products. Other key issues in optimizing detection methodologies involve the labeling of probes and the maximization of sample throughput.
• Detection probes can be labeled several different ways. Enzymatic inco ⁇ oration of P or S into the probes is the most common method for isotopic labeling. Following hybridization and washing, the signal is detected on autoradiographic film.
• probes can be enzymatically labeled with a variety of molecules.
• Biotin can be inco ⁇ orated enzymatically and then detected with streptavidin-conjugated alkaline phosphatase, using AP substrates like 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT).
• AP substrates like 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT).
• Chemiluminescent substrates such as Lumi-Phos 530 or Lumi-Phos Plus (Lumigen, Southfield, MI) can also be used with AP.
• digoxigenin-11-dUTP can be incorporated enzymatically into DNA or RNA, and antidigoxigenin AP conjugates can be used with colorimetric or chemiluminescent detection.
• reporter molecules there are numerous other types of reporter molecules, including chemiluminescent moieties such as acridinium esters. Many fluorescent moieties are available as well. Electrochemiluminescent compounds such as tris (2,2'-bipyridine) ruthenium (II) can be used also. Further discussions of these and similar techniques can be found in: Schiff ER, de Medina M, Kahn RS. Semin Liver Dis. 1999;19(Suppl 1:3-15).
• HepG2 cells are obtained from the American Type Culture Collection (Rockville, MD), and are grown in 225 cm 2 tissue culture flasks in minimal essential medium supplemented with non- essential amino acids, 1% penicillin-streptomycin. The medium is renewed every three days, and the cells are subcultured once a week. After detachment of the adherent monolayer with a 10 minute exposure to 30 L of trypsin-EDTA and three consecutive washes with medium, confluent HepG2 cells are seeded at a density of 2.5 x 10° cells per well in a 6-well plate and exposed to 10 ⁇ M of [ 3 H] labeled active compound (500 dpm/pmol) for the specified time periods.
• the cells are maintained at 37°C under a 5% CO 2 atmosphere. At the selected time points, the cells are washed three times with ice-cold phosphate-buffered saline (PBS). Intracellular active compound and its respective metabolites are extracted by incubating the cell pellet overnight at -20°C with 60% methanol followed by extraction with an additional 20 ⁇ L of cold methanol for one hour in an ice bath. The extracts are then combined, dried under gentle filtered air flow and stored at -20°C until HPLC analysis.
• PBS ice-cold phosphate-buffered saline
• Antiviral nucleosides and nucleoside analogs are generally converted into the active metabolite, the 5 '-triphosphate (TP) derivatives by intracellular kinases.
• the nucleoside-TPs then exert their antiviral effect by inhibiting the viral polymerase during virus replication.
• mCyd-TP 2'-C-methyl-cytidine-5'- triphosphate
• mUrd-TP 2'-C-methyl-uridine-5'- triphosphate
• mCyd-TP concentration at 24 hours was 4 fold lower at 10.7 ⁇ 6.7 ⁇ M.
• MDBK bovine kidney cells yielded intermediate levels of mCyd-TP (30.1 ⁇ 6.9 ⁇ M at 24 hours).
• mUrd-TP Exposure of hepatocytes to mCyd led to production of a second 5'- triphosphate derivative, mUrd-TP.
• the mUrd-TP level reached 1.9 ⁇ 1.6 ⁇ M at 24 hours, compared to 8.1 ⁇ 3.4 ⁇ M in MDBK cells and 3.2 ⁇ 2.0 ⁇ M in primary human hepatocytes.
• MDBK and HepG2 cells produced comparable total amounts of phosphorylated species (approximately 43 versus 47 ⁇ M, respectively) at 24h
• mUrd-TP comprised 19% of the total product in MDBK cells versus only 4% in HepG2 cells.
• mUrd-TP concentration increased steadily over time, however reached a plateau or declined after 24 hours.
• HepG2 assay 0.5 Ci/mmol
• human and monkey hepatocyte assay 1.0 Ci/mmol.
• the concentrations of metabolites were determined as pmoles per million cells. One pmole per million cells is roughly equivalent to 1 ⁇ M.
• ND not detected.
• the apparent intracellular half-life ofthe mCyd-TP was 13.9 ⁇ 2.2 hours in HepG2 cells and 7.6 ⁇ 0.6 hours in MDBK cells: the data were not suitable for calculating the half life of mUrd-TP.
• the phosphorylation pattern detected in primary human hepatocytes was qualitatively similar to that obtained using HepG2 or MDBK cells.
• HepG2 cells were cultured in 12-well plates as described above and exposed to various concentrations of drugs as taught by Pan-Zhou X-R, Cui L, Zhou X-J, Sommadossi J-P, Darley-Usmer VM. "Differential effects of antiretroviral nucleoside analogs on mitochondrial function in HepG2 cells" Antimicrob. Agents Chemother. 2000; 44:496-503. Lactic acid levels in the culture medium after 4 day drug exposure were measured using a Boehringer lactic acid assay kit. Lactic acid levels were normalized by cell number as measured by hemocytometer count.
• Cells were seeded at a rate of between 5 x 10 and 5 x 10 4 /well into 96-well plates in growth medium overnight at 37°C in a humidified CO 2 (5%) atmosphere. New growth medium containing serial dilutions of the drugs was then added. After incubation for 4 days, cultures were fixed in 50% TCA and stained with sulforhodamineB. The optical density was read at 550 nm. The cytotoxic concentration was expressed as the concentration required to reduce the cell number by 50% (CCso).
• mCyd Conventional cell proliferation assays were used to assess the cytotoxicity of mCyd and its cellular metabolites in rapidly dividing cells.
• the inhibitory effect of mCyd was determined to be cytostatic in nature since mCyd showed no toxicity in confluent cells at concentrations far in excess ofthe corresponding CC 50 for a specific cell line.
• mCyd was not cytotoxic to rapidly growing Huh7 human hepatoma cells or H9c2 rat myocardial cells at the highest concentration tested (CC 50 >250 ⁇ M).
• the mCyd CC 50 values were 132 and 161 ⁇ M in BHK-21 hamster kidney and HepG2 human hepatoma cell lines, respectively.
• the CC50 for mCyd in HepG2 cells increased to 200 ⁇ M when the cells were grown on collagen-coated plates for 4 or 10 days.
• CC 50 values of 35-36 ⁇ M were derived when ribavirin was tested in HepG2 and Huh7 cells.
• the CC 50 of mCyd was 36 ⁇ M.
• a similar CC 5 0 value (34 ⁇ M) was determined for mCyd against MT-4 human T-lymphocyte cells.
• mCyd was mostly either non-cytotoxic or weakly cytotoxic (CC 50 >50 to >200 ⁇ M) to numerous other cell lines of human and other mammalian origin, including several human carcinoma cell lines, in testing conducted by the National Institutes of Health (NIH) Antiviral Research and Antimicrobial Chemistry Program. Exceptions to this were rapidly proliferating HFF human foreskin fibroblasts and MEF mouse embryo fibroblasts, where mCyd showed greater cytotoxicity (CC 50 s 16.9 and 2.4 ⁇ M, respectively). Again, mCyd was much less toxic to stationary phase fibroblasts.
• cytotoxic effect of increasing amounts of mCyd on cellular DNA or RNA synthesis was examined in HepG2 cells exposed to [ 3 H]-thymidine or [ 3 H]-uridine.
• the CC 5 0S of mCyd required to cause 50% reductions in the inco ⁇ oration of radiolabeled thymidine and uridine into cellular DNA and RNA were 112 and 186 ⁇ M, respectively.
• Ribavirin inco ⁇ orated into both DNA and RNA at levels 10-fold higher than mCyd.
• Table 4a Cellular Nucleic Acid Synthesis and Inco ⁇ oration Studies in HepG2 Cells (10 ⁇ M Drug and Nucleoside Controls)
• Human bone marrow cells were collected from normal healthy volunteers and the mononuclear population was separated by Ficoll-Hypaque gradient centrifugation as described previously by Sommadossi J-P, Carlisle R. "Toxicity of 3'-azido-3'- deoxythymidine and 9-(l,3-dihydroxy-2-propoxymethyl)guanine for normal human hematopoietic progenitor cells in vitro" Antimicrobial Agents and Chemotherapy 1987; 31:452-454; and Sommadossi J-P, Schinazi RF, Chu CK, Xie M-Y.
• the assay was performed essentially as described by Baginski, S. G.; Pevear, D. C; Seipel, M.; Sun, S. C. C; Benetatos, C. A.; Chunduru, S. K.; Rice, C. M. and M. S. Collett "Mechanism of action of a pestivirus antiviral compound" PNAS USA 2000, 97(14), 7981-7986.
• MDBK cells ATCC were seeded onto 96-well culture plates (4,000 cells per well) 24 hours before use.
• BVDV strain NADL, ATCC
• MOI multiplicity of infection
• PFU plaque forming units
• anemia and neutropenia are the most common drug-related clinical toxicities associated with the anti-HIV drug zidovudine (ZDV) or the ribavirin (RBV) component ofthe standard of care combination therapy used for HCV treatment. These toxicities have been modeled in an in vitro assay that employed bone marrow cells obtained from healthy volunteers (Sommadossi J-P, Carlisle R. Antimicrob. Agents Chemother. 1987;31(3): 452-454).
• ZDV has been previously shown to directly inhibit human granulocyte- macrophage colony-forming (CFU-GM) and erythroid burst-forming (BFU-E) activity at clinically relevant concentrations of 1-2 ⁇ M in this model (Berman E, et al. Blood 1989;74(4): 1281-1286; Yoshida Y, Yoshida C. AIDS Res. Hum. Retroviruses 1990;6(7):929-932.; Lerza R, et al. Exp. Hematol 1997;25(3):252-255; Dornsife RE, Averett DR. Antimicrob. Agents Chemother. 1996;40(2):514-519; Kurtzberg J, Carter SG. Exp. Hematol.
• Table 5 Bone Marrow Toxicity of mCyd in Granulocyte Macrophage Progenitor and Erythrocyte Precursor Cells
• Antiviral nucleoside analogs approved for HIV therapy such as ZDV, stavudine (d4T), didanosine (ddl), and zalcitabine (ddC) have been occasionally associated with clinically limiting delayed toxicities such as peripheral neuropathy, myopathy, and pancreatitis (Browne MJ, et al. J. Infect. Dis. 1993;167(l):21-29; Fischl MA, et al. Ann. Intern. Med. 1993;18(10):762-769.; Richman DD, et al. N. Engl. J. Med. 1987;317(4): 192-197; Yarchoan R, et al. Lancet 1990;336(8714):526- 529).
• ZDV stavudine
• ddl didanosine
• ddC zalcitabine
• the cellular DNA polymerases are responsible for normal nuclear and mitochondrial DNA synthesis and repair. Nucleoside analog triphosphates are potential inhibitors of DNA polymerases and hence could disrupt critical cell functions. In particular, the inhibition of human polymerase ⁇ , the enzyme responsible for mitochondrial DNA synthesis, has been linked to defects in mitochondrial function (Lewis, W., E. S. Levine, et al. Proceedings ofthe National Academy of Sciences, USA 1996 93(8): 3592-7). Experiments were undertaken to determine if mCyd-TP inhibited human DNA polymerases. As shown in Table 7 mCyd-TP was not a substrate for human DNA polymerases ⁇ , ⁇ , or ⁇ .
• HepG2 or huh7 cells were treated with compounds for 4 days, data represent at least three independent experiments b.
• HepG2 cells were treated with compounds for 6 and 14 days, data represents at least three independent experiments c.
• HepG2 cells were treated with compounds for 14 days d. 17% cells (11 of 64) contained 1 or 2 enlarged mitochondria out of 25 in two independent experiments nd, not determined
• Compounds can exhibit anti-flavivirus or pestivirus activity by inhibiting flavivirus or pestivirus polymerase, by inhibiting other enzymes needed in the replication cycle, or by other pathways.
• the effective concentration was determined in duplicate 24-well plates by plaque reduction assays.
• Cell monolayers were infected with 100 PFU/well of virus. Then, serial dilutions of test compounds in MEM supplemented with 2% inactivated serum and 0.75% of methyl cellulose were added to the monolayers. Cultures were further incubated at 37°C for 3 days, then fixed with 50% ethanol and 0.8% Crystal Violet, washed and air-dried. Then plaques were counted to determine the concentration to obtain 90% virus suppression.
• MDBK cells were seeded onto 24-well plates (2 x 105 cells per well) 24 hours before infection with BVDV (NADL strain) at a multiplicity of infection (MOI) of 0.1 PFU per cell.
• Serial dilutions of test compounds were added to cells in a final concentration of 0.5% DMSO in growth medium. Each dilution was tested in triplicate. After three days, cell cultures (cell monolayers and supernatants) were lysed by three freeze-thaw cycles, and virus yield was quantified by plaque assay.
• MDBK cells were seeded onto 6-well plates (5 x 105 cells per well) 24 h before use.
• BVDV-based cell-protection assay This assay measures the ability of mCyd to protect growing MDBK bovine kidney cells from destruction by a cytopathic NADL strain of BVDV. The cytotoxicity ofthe test drug on uninfected cells was measured in parallel.
• ribavirin showed no clear antiviral effect: significant (50% or more) cell protection was not achieved in most assays as the cytotoxicity of ribavirin overlapped and masked the protective effect.
• ribavirin gave a CC 50 of 4.3 ⁇ 0.6 ⁇ M and an EC 50 > 4.3 ⁇ M in the CPA.
• Table 8b CCsn Test Results for ⁇ -D-2'-C-methyl-cvtidine (Compound G . 3'-O- valinyl ester of ⁇ -D-2'-C-methyl-cytidine dihydrochloride salt (Compound M). and ⁇ -
• Cell lines utilized include MT-4 for HIV; Vero 76, African green monkey kidney cells for SARS; BHK for Bovine Viral Diarrhea Virus; Sb-1 for poliovirus Sabin type-1; CVB-2, CVB-3, CVB-4, and CVA-9 for Coxsackieviruses B-2, B-3, B- 4 and A-9; and REO-1 for double-stranded RNA viruses.
• kidney cells for SARS BHK for Bovine Viral Diarrhea Virus
• Sb-1 for poliovirus Sabin type-1
• CVB-2, CVB-3, CVB-4, and CVA-9 for Coxsackieviruses B-2, B-3, B- 4 and A-9
• REO-1 for double-stranded RNA viruses.
• the overall antiviral potency of mCyd was determined against different strains of BVDV and both cytopathic (cp) and noncytopathic (ncp) biotypes in cell protection assays as well as in plaque reduction and yield reduction assays.
• the latter assays measure the output of infectious virus from cells and hence provide a stringent test of antiviral efficacy.
• the different data sets from all three assays show agreement as summarized in Table 9.
• the range of 50% and 90% effective inhibitory concentration (EC 5 o and EC 9 o) values for mCyd was 0.3 to 2.8 ⁇ M and 0.87 to 4.8 ⁇ M, respectively.
• EXAMPLE 6 IN VITRO ANTIVIRAL ACTIVITY AGAINST OTHER POSITIVE-STRAND RNA VIRUSES mCyd has been tested for efficacy against positive-strand RNA viruses other than BVDV. Data obtained are summarized in Table 9 and 10. Against flaviruses, mCyd showed modest activity. The composite EC50 ranges (in ⁇ M) determined from both sites were: West Nile virus (46-97); Yellow Fever virus (9-80); and Dengue virus (59-95). For mCyd against the alpha virus, Venezuelan equine encephalitis virus, EC50 values were 1.3 - 45 ⁇ M.
• mCyd was generally inactive against all RNA and DNA viruses tested except for the positive-strand RNA viruses.
• mCyd was also found to have no activity against HIV in MT-4 human T lymphocyte cells or HBV in HepG2.2.15 cells.
• cp, cytopathic virus; ncp noncytopathic virus l-NADL cp and I-N-dlns ncp represent recombinant BVDV viruses
• HFF human foreskin fibroblast
• Daudi Burkitt's B-cell lymphoma
• MDCK canine kidney cells
• CV-1 African green monkey kidney cells
• KB human nasopharyngeal carcinoma
• MA-104 Rhesus monkey kidney cells
• LLC-MK2 Rhesus monkey kidney cells
• A549 Human lung carcinoma cells
• MEF mouse embryo fibroblast
• Vero African green monkey kidney cells
• HeLa human cervical adenocarcinoma cells.
• EC 5 o 50% effective concentration.
• CC 50 50% cytotoxic concentration.
• the cell protection assay format was used to test the effect of increasing the amount of BVDV virus on the EC50 of mCyd.
• Increasing the multiplicity of infection (MOI) of BVDV in this assay from 0.04 to 0.16, caused the EC 50 of mCyd to increase linearly from 0.5 ⁇ M to approximately 2.2 ⁇ M.
• the effect of discontinuing treatment with mCyd was tested in MDBK cells persistently infected with a noncytopathic strain (strain I-N-dlns) of BVDV. Upon passaging in cell culture, these cells continuously produce anywhere from 10 6 to >10 7 infectious virus particles per ml of media. This virus can be measured by adding culture supernatants from treated MDBK (BVDV) cells to uninfected MDBK cells and counting the number of resultant viral foci after disclosure by immunostaining with a BVDV-specific antibody.
• strain I-N-dlns noncytopathic strain
• mCyd treatment results in a marked increase in total cellular RNA content as cells grow, protected from the cytopathic effects of BVDV. This is coupled with a marked decrease in the production of BVDV RNA due to mCyd. Conversely, in the absence of mCyd, total cellular RNA actually decreases as BVDV RNA rises due to the destruction ofthe cells by the cytopathic virus.
• the accumulation of intracellular BVDV RNA was monitored in MDBK cells 18-hours post infection (after approximately one cycle of virus replication) using Real Time RT-PCR.
• the inhibition constant (K,) for mCyd-TP was 0.16 ⁇ M and the Michaelis-Menten constant (K m ) for CTP was 0.03 ⁇ M.
• Inhibition of RNA synthesis by mCyd-TP required the presence of a cognate G residue in the RNA template.
• the effect of mCyd-TP on RNA synthesis in the absence of CTP was investigated in more detail using a series of short (21mer) synthetic RNA templates containing a single G residue, which was moved progressively along the template. Analysis ofthe newly synthesized transcripts generated from these templates in the presence of mCyd-TP revealed that RNA elongation continued only as far as the G residue, then stopped ( Figure 2).
• RNA synthesis stopped at the first G residue encountered by the polymerase.
• EXAMPLE 10 ERADICATION OF A PERSISTENT BVDV INFECTION
• the first study performed in MDBK cells persistently infected with the New York-1 (NY-1) strain of BVDV, compared the effect of monotherapy with either mCyd (8 ⁇ M) or interferon alpha 2b (200 IU/ml), or the two drugs in combination (Figure 4A).
• 8 ⁇ M mCyd alone reduced viral titers by approximately 3.5 logio after one passage to a level that was maintained for two more passages.
• Interferon alpha 2b alone was essentially inactive against persistent BVDV infection (approximately 0.1 logio reduction in virus titer) despite being active against de novo BVDV infection.
• the combination of mCyd plus interferon alpha 2b reduced virus to undetectable levels by the second passage and clearly showed better efficacy to either monotherapy.
• resistant virus typically has a 10 fold or more lower titer than the wild-type virus and is easily suppressed by co-therapy with IntronA (Figure 5A).
• Figure 5A The phenotype ofthe virus that re-emerged was remarkably different from the initial wild-type virus: as shown in Figure 5B, it yielded much smaller foci (typically, 3 to 10 times smaller in diameter then those ofthe wild-type virus). This phenotype did not change after prolonged passaging in culture in the presence ofthe inhibitor (at least 72 days), however, it quickly reverted to the wild-type phenotype (large foci) after the discontinuation ofthe treatment.
• RT-PCR sequencing ofthe resistant mutant was used to identify the mutation responsible for resistance. Sequencing efforts were focused on the NS5B RNA- dependent RNA polymerase region of BVDV, which was assumed to be the likely target for a nucleoside inhibitor. A specific S405T amino-acid substitution was identified at the start ofthe highly conserved B domain motif of the polymerase. The B domain is part ofthe polymerase active site and is thought to be involved in nucleoside binding (Lesburg, C. A., M. B. Cable, et al. Nature Structural Biology 1999 6(10): 937-43). Resistance to nucleosides has been mapped to this domain for other viruses such as HBV (Ono et s ⁇ , JClin Invest.
• HBV Ono et s ⁇ , JClin Invest.
• S405T mutant BVDV was refractory to mCyd up to the highest concentrations that could be tested (ECso>32 ⁇ M), but was also significantly impaired in viability compared to wild-type virus.
• the S405T mutant exhibited a 1-2 logio lower titer than wild-type BVDV and produced much smaller viral plaques.
• the mutant virus showed a marked reduction in the rate of a single cycle of replication (> 1000-fold lower virus titer at 12h), and accumulated to about 100 fold lower levels than the wild-type virus even after 36 h of replication (Figure 5C). The virus also quickly reverted to wild-type virus upon drug withdrawal.
• the mutant was also more sensitive (-40 fold) to treatment with IFN alpha 2b than wild- type as shown in Figure 5D.
• Chimpanzees chronically infected with HCV are the most widely accepted animal model of HCV infection in human patients (Lanford, R. E., C. Bigger, et al. 7/ ⁇ rJ2001 42(2): 117-26; Grakoui, A., H. L. Hanson, et al. Hepatology 2001 33(3): 489-95).
• a single in vivo study ofthe oral administration of val-mCyd in the chimpanzee model of chronic hepatitis C virus infection has been conducted.
• Table 11 Summary of Val-mCyd In Vivo Activity Study in the Chimpanzee Model of Chronic HCV Infection
• HCV RNA Serum levels of HCV RNA throughout the study were determined independently by two clinical hospital laboratories. HCV RNA was assayed using a quantitative RT-PCR nucleic acid amplification test (Roche Amplicor HCV Monitor Test, version 2.0). This assay has a lower limit of detection (LLOD) of 600 IU/mL and a linear range of 600-850,000 IU/mL.
• LLOD lower limit of detection
• HCV viral loads were (i) very similar among all five animals and all 3 dose groups, and (ii) very stable over the 3-week pretreatment period.
• the mean pretreatment logio viral load and standard deviations among the five individual animals were 5.8 ⁇ 0.1 (site 1) and 5.6 ⁇ 0.1 (site 2). These data indicated that the c.v. (coefficient of variance) ofthe assay is only around 2% at both sites.
• the largest fluctuation in HCV viral load seen in any animal during pretreatment was approximately 0.3 logio.
• the two data sets agree well, confirming the reliability ofthe assay.
• the difference in viral load between the two sites was generally 0.3 logio or less, similar to the fluctuation observed during the pretreatment period.
• the discrepancy was closer to 0.5 logio.
• the viral load drop seen in response to therapy varied from 0.436 (animal 501, site 1) to 1.514 logio (animal 497, site 2). The latter corresponds to a change in HCV viral load from 535,000 (pretreatment) to 16,500 (day 7) genomes per ml.
• Table 12 Summary of Changes in Baseline Logio HCV RNA Viral Load During Therapy
• Chimpanzees were monitored by trained veterinarians throughout the study for weight loss, temperature, appetite, and general well being, as well as for blood chemistry profile and CBCs. No adverse events due to drug were noted. The drug appeared to be well tolerated by all four treated animals. All five animals lost some weight during the study and showed some aspartate aminotransferase (AST) elevations, but these are normal occurrences related to sedation procedures used, rather than study drug. A single animal experienced an alanine aminotransferase (ALT) flare in the pretreatment period prior to the start of dosing, but the ALT levels diminished during treatment. Thus, this isolated ALT event was not attributable to drug.
• AST aspartate aminotransferase
• Val-mCyd was incubated in human plasma at 0, 21 or 37°C and samples analyzed at various time points up to 10 hours ( Figure 8). At 37°C, val- mCyd was effectively converted to mCyd, with only 2% ofthe input val-mCyd remaining after 10 hours. The in vitro half-life of val-mCyd in human plasma at 37°C was 1.81 hours.
• mCyd In studies ofthe in vitro stability of mCyd in human plasma, or upon treatment with a crude preparation enriched in human cytidine/deoxycytidine deaminase enzymes, mCyd remained essentially unchanged and no deamination to the uridine derivative of mCyd (mUrd) occurred after incubation at 37°C. Only in rhesus and cynomologus monkey plasma was limited deamination observed. Incubation of mCyd at 37°C in cynomologus monkey plasma yielded 6.1 and 13.0% of mUrd deamination product after 24 and 48 hours, respectively, under conditions where control cytidine analogs were extensively deaminated.
• mCyd-DP minor amounts of mCyd- 5'-diphosphate, mCyd-DP, roughly 10% the amount ofthe corresponding TP, were seen in all three cell types. Lesser amounts of mUrd-DP were detected only in two cell types, primary human hepatocytes and MDBK cells. No monophosphate (MP) metabolites were detected in any cell type. There was no trace of any intracellular mUrd and no evidence for the formation of liponucleotide metabolites such as the 5'- diphosphocholine species seen upon the cellular metabolism of other cytidine analogs.
• MP monophosphate
• Figure 9 shows the decay profile of mCyd-TP determined following exposure of HepG2 cells to 10 ⁇ M [ 3 H]-mCyd for 24 hours.
• the apparent intracellular half- life ofthe mCyd-TP was 13.9 ⁇ 2.2 hours in HepG2 cells and 7.6 ⁇ 0.6 hours in MDBK cells: the data were not suitable for calculating the half life of mUrd-TP.
• the long half life of mCyd-TP in human hepatoma cells supported the notion of once-a- day dosing for val-mCyd in clinical trials for HCV therapy.
• Phosphorylation of mCyd occurred in a dose-dependent manner up to 50 ⁇ M drug in all three cell types, as shown for HepG2 cells in Figure 9C.
• the phosphorylation pattern detected in primary human hepatocytes was qualitatively similar to that obtained using HepG2 or MDBK cells.
• mCyd-TP In addition to the intracellular active moiety, mCyd-TP, cells from different species have been shown to produce variable and lesser amounts of a second triphosphate, mUrd-TP, via deamination of intracellular mCyd species.
• the activity of mUrd-TP against BVDV NS5B RNA-dependent RNA polymerase has not been tested to date but is planned.
• mUrd (a) is about 10-fold less potent than mCyd against BVDV; (b) has essentially no antiviral activity against a wide spectrum of other viruses; and (c) is negative when tested at high concentrations in a variety of cytotoxicity tests (including bone marrow assays, mitochondrial function assays and inco ⁇ oration into cellular nucleic acid). Based on these results, it appeared that the contribution of mUrd to the overall antiviral activity or cytotoxicity profile of mCyd is likely to be minor. Extensive toxicology coverage for the mUrd metabolite of mCyd exists from subchronic studies conducted with val-mCyd in the monkey.
• EXAMPLE 15 CELLULAR PATHWAYS FOR METABOLIC ACTIVATION
• Cytidine is a natural substrate of cytosolic uridine-cytidine kinase (UCK), the pyrimidine salvage enzyme responsible for conversion of Cyd to Cyd-5'-monophosphate (CMP).
• UCK cytosolic uridine-cytidine kinase
• CMP Cyd-5'-monophosphate
• the intracellular phosphorylation of mCyd to mCyd-TP was reduced in the presence of cytidine or uridine in a dose-dependent fashion with EC 50 values of 19.17 ⁇ 4.67 ⁇ M for cytidine and 20.92 ⁇ 7.10 ⁇ M for uridine.
• deoxycytidine a substrate for the enzyme deoxycytidine kinase (dCK)
• dCK deoxycytidine kinase
• EXAMPLE 16 PATHWAYS FOR THE CELLULAR BIOSYNTHESIS OF mURD-TP
• mUrd-TP is a minor metabolite arising to varying extents in cells from different species. mUrd does not originate via extracellular deamination of mCyd since mUrd was not seen in the cell medium which also lacks any deamination activities.
• the cellular metabolism data are consistent with the idea that mUrd-TP arises via the biotransformation of intracellular mCyd species.
• the antiviral effect of val-mCyd was assessed by (i) the proportion of patients with a > 1.0 logio decrease from baseline in HCV RNA level at Day 15, (ii) the time to a > 1.0 logio decrease in serum HCV RNA level, (iii) the change in HCV RNA level from Day 1 to Day 15, (iv) the change in HCV RNA level from Day 1 to Day 29, (v) the proportion of patients who experience return to baseline in serum HCV RNA level by Day 29, and (vi) the relationship of val-mCyd dose to HCV RNA change from Day 1 to Day 15.
• Plasma concentrations of mCyd, mUrd and Val-mCyd were measured by a HPLC/MS/MS methodology with a lower limit of quantitation (LOQ) at 20 ng/ml.
• the pharmacokinetics of mCyd was analyzed using a non-compartmental approach. As presented in the tables below, the principal pharmacokinetic parameters were comparable on day 1 and day 15, indicative of no plasma drug accumulation after repeated dosing. The plasma exposure also appeared to be a linear function of dose. As shown in the tables below, principal pharmacokinetic parameters of drug exposure (Cmax and AUC) doubled as doses escalated from 50 to 100 mg.
• Serum HCV RNA levels were determined with the use ofthe Amplicor HCV MonitorTM assay v2.0 (Roche Molecular Systems, Branchburg, NJ, USA), which utilizes polymerase chain reaction (PCR) methods.
• the lower limit of quantification (LLOQ) with this assay was estimated to be approximately 600 IU/mL and the upper limit of quantification (ULOQ) with this assay was estimated to be approximately 500,000 IU/mL.
• Serum samples for HCV RNA were obtained at screening (Day -42 to -7) to determine eligibility for the study.
• the Screening serum HCV RNA values must be > 5 logio IU/mL by the Amplicor HBV MonitorTM assay at the central study laboratory.
• serum samples for HCV RNA were obtained at Baseline (Day 1), and at every protocol-stipulated post-Baseline study visit (Days 2, 4, 8, 11, 15, 16, 17, 22, and 29). Serum samples for HCV RNA were also collected during protocol-stipulated follow-up visits for patients prematurely discontinued from the study.
• the antiviral activity associated with the first two cohorts (50 and 100 mg per day) in the ongoing study is summarized in the following tables and graphs. Although the duration of dosing was short (15 days) and the initial dose levels low, there were already apparent effects on the levels of HCV RNA in the plasma of infected patients.
• Table 16 Summary Statistics of Change From Baseline (Day 1 " ) in LogioHCV RNA
• Figure 11 depicts the Median Change From Baseline In Logio HCV RNA By Visit

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