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  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
  • C07F9/65515—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a five-membered ring
  • C07F9/65517—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a five-membered ring condensed with carbocyclic rings or carbocyclic ring systems
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  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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  • C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
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  • C07F9/2454—Esteramides the amide moiety containing a substituent or a structure which is considered as characteristic
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  • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
  • C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
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  • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
  • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
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  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
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  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
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  • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
  • C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
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  • C07H19/24—Heterocyclic radicals containing oxygen or sulfur as ring hetero atom

Definitions

• nucleoside phosphoramidates and their use as agents for treating viral diseases. These compounds are inhibitors of RNA-dependent RNA viral replication and are useful as inhibitors of HCV NS5B polymerase, as inhibitors of HCV replication and for treatment of hepatitis C infection in mammals.
• Hepatitis C virus (HCV) infection is a major health problem that leads to chronic liver disease, such as cirrhosis and hepatocellular carcinoma, in a substantial number of infected individuals, estimated to be 2-15% of the world's population.
• chronic liver disease such as cirrhosis and hepatocellular carcinoma
• According to the World Health Organization there are more than 200 million infected individuals worldwide, with at least 3 to 4 million people being infected each year. Once infected, about 20% of people clear the virus, but the rest can harbor HCV the rest of their lives.
• Ten to twenty percent of chronically infected individuals eventually develop liver- destroying cirrhosis or cancer.
• the viral disease is transmitted parenterally by contaminated blood and blood products, contaminated needles, or sexually and vertically from infected mothers or carrier mothers to their offspring.
• Current treatments for HCV infection which are restricted to immunotherapy with recombinant interferon- ⁇ alone or in combination with the nucleoside analog ribavirin, are of limited clinical benefit.
• the HCV virion is an enveloped positive-strand RNA virus with a single oligoribonucleotide genomic sequence of about 9600 bases which encodes a polyprotein of about 3,010 amino acids.
• the protein products of the HCV gene consist of the structural proteins C, El, and E2, and the non-structural proteins NS2, NS3, NS4A and NS4B, and NS5A and NS5B.
• the nonstructural (NS) proteins are believed to provide the catalytic machinery for viral replication.
• the NS3 protease releases NS5B, the RNA-dependent RNA polymerase from the polyprotein chain.
• HCV NS5B polymerase is required for the synthesis of a double-stranded RNA from a single-stranded viral RNA that serves as a template in the replication cycle of HCV. Therefore, NS5B polymerase is considered to be an essential component in the HCV replication complex (K.
• HCV NS5B polymerase Inhibition of HCV NS5B polymerase prevents formation of the double-stranded HCV RNA and therefore constitutes an attractive approach to the development of HCV-specific antiviral therapies.
• HCV belongs to a much larger family of viruses that share many common features.
• the Flaviviridae family of viruses comprises at least three distinct genera: pestiviruses, which cause disease in cattle and pigs; flavivruses, 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, 64 1-643).
• 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
• 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 at least 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 of the viral genome RNA are located within approximately the carboxy-terminal. Two-thirds of the ORF are termed
• nonstructural (NS) proteins The genetic organization and polyprotein processing of the nonstructural protein portion of the ORF for pestiviruses and hepaciviruses is very similar.
• the mature nonstructural (NS) proteins in sequential order from the amino-terminus of the nonstructural protein coding region to the carboxy-terminus of the 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., Nature, 1988, 333, 22; Bazan and Fletterick Virology, 1989, 171, 637-639; Gorbalenya et al., Nucleic Acid Res., 1989, 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., Crir. Rev. Biochem. Molec. Biol. 1993, 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,
• the NS4A protein acts as a cofactor with the NS3 serine protease (Bartenschlager et al., J Virol. 1994, 68, 5045-5055; Failla et al., J Virol. 1994, 68, 3753-3760; Xu et al, J Virol., 1997, 71:53 12-5322).
• the NS3 protein of both viruses also functions as a helicase (Kim et al., Biochem.
• NS5B proteins of pestiviruses and hepaciviruses have the predicted RNA-directed RNA polymerases activity (Behrens et al., EMBO, 1996, 15, 12-22; Lechmann et al., J. Virol., 1997, 71, 8416-8428; Yuan et al, Biochem. Biophys. Res. Comm. 1997, 232, 231-235; Hagedorn, PCT WO 97/12033; Zhong et al, J Virol., 1998, 72, 9365-9369).
• RNA-dependent RNA polymerase is absolutely essential for replication of the single-stranded, positive sense, RNA genome and this enzyme has elicited significant interest among medicinal chemists.
• Inhibitors of HCV NS5B as potential therapies for HCV infection have been reviewed: Tan, S.-L., et al., Nature Rev. Drug Discov., 2002, 1, 867-881 ; Walker, M.P. et al., Exp. Opin. Investigational Drugs, 2003, 12, 1269-1280; Ni, Z-J., et al., Current Opinion in Drug Discovery and Development, 2004, 7, 446-459; Beaulieu, P.
• Nucleoside inhibitors of NS5B polymerase can act either as a non-natural substrate that results in chain termination or as a competitive inhibitor which competes with nucleotide binding to the polymerase.
• the nucleoside analog must be taken up by the cell and converted in vivo to a triphosphate to compete for the polymerase nucleotide binding site. This conversion to the triphosphate is commonly mediated by cellular kinases which imparts additional structural requirements on a potential nucleoside polymerase inhibitor. Unfortunately, this limits the direct evaluation of nucleosides as inhibitors of HCV replication to cell-based assays capable of in situ phosphorylation.
• nucleoside phosphoramidate prodrugs have been shown to be precursors of the active nucleoside triphosphate and to inhibit viral replication when administered to viral infected whole cells (McGuigan, C, et al., J. Med. Chem., 1996, 39, 1748-1753; Valette, G., et al., J. Med. Chem., 1996, 39,
• nucleosides are limiting the utility of nucleosides as viable therapeutic agents. Also limiting the utility of nucleosides as viable therapeutic agents is their sometimes poor physicochemical and pharmacokinetic properties. These poor properties can limit the intestinal absorption of an agent and limit uptake into the target tissue or cell. To improve on their properties prodrugs of nucleosides have been employed. It has been demonstrated that preparation of nucleoside
• phosphoramidates improves the systemic absorption of a nucleoside
• the phosphoramidate moiety of these "pronucleotides” is masked with neutral lipophilic groups to obtain a suitable partition coefficient to optimize uptake and transport into the cell dramatically enhancing the intracellular concentration of the nucleoside monophosphate analog relative to administering the parent nucleoside alone.
• Enzyme-mediated hydrolysis of the phosphate ester moiety produces a nucleoside monophosphate wherein the rate limiting initial phosphorylation is unnecessary.
• U.S. Patent Application 12/053,015 which corresponds to WO 2008/121634 and US 2010/0016251, discloses a number of phosphoramidate nucleoside prodrugs, many of which show activity in an HCV assay.
• Several compounds disclosed in US 2010/0016251 were tested as a potential clinical candidate for approval by the FDA.
• a or “an” entity refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound.
• a compound refers to one or more compounds or at least one compound.
• the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.
• P* (where used) means that the phosphorus atom is chiral and that it has a corresponding Cahn-Ingold-Prelog designation of "R” or "S” which have their accepted plain meanings. Due to the chirality at phosphorus, Compound 1, as used herein, is sometime referred to the ⁇ Sp-isomer. Its diastereomeric analog is some times referred to as the Rp-isomer. Mixtures of the S P -isomer and i? P -isomer are sometimes referred to as a mixture containing 1 and the i?p-isomer.
• purified refers to the purity of a given compound.
• a compound is “purified” when the given compound is a major component of the composition, i.e., at least 50% w/w pure.
• purified embraces at least 50% w/w purity, at least 60% w/w purity, at least 70% purity, at least 80% purity, at least 85% purity, at least 90% purity, at least 92% purity, at least 94% purity, at least 96% purity, at least 97% purity, at least 98% purity, at least 99% purity, at least 99.5% purity, and at least 99.9% purity, wherein "substantially pure” embraces at least 97% purity, at least 98% purity, at least 99% purity, at least 99.5% purity, and at least 99.9% purity, wherein "substantially pure” embraces at least 97% purity, at least 98% purity, at least 99% purity, at least 99.5% purity, and at least 99.9% purity
• a specified XRPD pattern means that the peak positions shown in the XRPD pattern are substantially the same, within visual inspection or resort to selected peak listings ( ⁇ 0.2 °20).
• the intensities can vary depending on the sample.
• substantially anhydrous means that a substance contains at most 10% by weight of water, preferably at most 1% by weight of water, more preferably at most 0.5% by weight of water, and most preferably at most 0.1% by weight of water.
• a solvent or anti-solvent (as used in reactions, crystallization, etc. or lattice and/or adsorbed solvents) includes at least one of a C ⁇ to C 8 alcohol, a C 2 to C 8 ether, a C 3 to C 7 ketone, a C 3 to C 7 ester, a C ⁇ to C 2 chlorocarbon, a C 2 to C 7 nitrile, a miscellaneous solvent, a C 5 to C 12 saturated hydrocarbon, and a C 6 to C 12 aromatic hydrocarbon.
• the C ⁇ to C 8 alcohol refers to a straight/branched and/or cyclic/acyclic alcohol having such number of carbons.
• the C ⁇ to C 8 alcohol includes, but is not limited to, methanol, ethanol, n-propanol, isopropanol, isobutanol, hexanol, and cyclohexanol.
• the C 2 to C 8 ether refers to a straight/branched and/or cyclic/acyclic ether having such number of carbons.
• the C 2 to C 8 ether includes, but is not limited to, dimethyl ether, diethyl ether, di-isopropyl ether, di-n-butyl ether, methyl-t-butyl ether (MTBE), tetrahydrofuran, and dioxane
• the C 3 to C 7 ketone refers to a straight/branched and/or cyclic/acyclic ketone having such number of carbons.
• the C 3 to C 7 ketone includes, but is not limited to, acetone, methyl ethyl ketone, propanone, butanone, methyl isobutyl ketone, methyl butyl ketone, and cyclohexanone.
• the C 3 to C 7 ester refers to a straight/branched and/or cyclic/acyclic ester having such number of carbons.
• the C 3 to C 7 ester includes, but is not limited to, ethyl acetate, propyl acetate, i-propyl acetate, n-butyl acetate, etc.
• the C ⁇ to C 2 chlorocarbon refers to a chlorocarbon having such number of carbons.
• the d to C 2 chlorocarbon includes, but is not limited to, chloroform, methylene chloride (DCM), carbon tetrachloride, 1,2-dichloroethane, and tetrachloroethane.
• a C 2 to C 7 nitrile refers to a nitrile have such number of carbons.
• the C 2 to C 7 nitrile refers to a nitrile have such number of carbons.
• C 7 nitrile includes, but is not limited to, acetonitrile, propionitrile, etc.
• a miscellaneous solvent refers to a solvent commonly employed in organic chemistry, which includes, but is not limited to, diethylene glycol, diglyme
• C 5 to C 12 saturated hydrocarbon refers to a straight/branched and/or cyclic/acyclic hydrocarbon.
• the C 5 to C 12 saturated hydrocarbon includes, but is not limited to, n-pentane, petroleum ether (ligroine), n-hexane, n-heptane, cyclohexane, and cycloheptane.
• C 6 to C 12 aromatic refers to substituted and unsubstituted hydrocarbons having a phenyl group as their backbone.
• Preferred hydrocarbons include benzene, xylene, toluene, chlorobenzene, o-xylene, m-xylene, p-xylene, xylenes, and anisole.
• co-crystallates include co-crystallates of 1 in combination with salts, which embraces pharmaceutically acceptable salts.
• salts refers to a compound comprising a cation and an anion, which can produced by the protonation of a proton-accepting moiety and/or deprotonation of a proton-donating moiety. It should be noted that protonation of the proton-accepting moiety results in the formation of a cationic species in which the charge is balanced by the presence of a physiological anion, whereas deprotonation of the proton-donating moiety results in the formation of an anionic species in which the charge is balanced by the presence of a physiological cation. This term is meant to embrace pharmaceutically acceptable salts.
• pharmaceutically acceptable salt means a salt that is pharmaceutically acceptable.
• pharmaceutically acceptable salts include, but are not limited to: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as glycolic acid, pyruvic acid, lactic acid, malonic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,
• preparation or “dosage form” is intended to include both solid and liquid formulations of the active compound and one skilled in the art will appreciate that an active ingredient can exist in different preparations depending on the desired dose and pharmacokinetic parameters.
• excipient refers to a compound that is used to prepare a pharmaceutical composition, and is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipients that are acceptable for veterinary use as well as human pharmaceutical use.
• crystalline refers to a situation where a solid sample of 1 has crystalline characteristics when determined by X-ray powder diffraction or a single crystal X-ray technique.
• crystal-like refers to a situation where a solid sample of 1 has crystalline characteristics when determined by one means, e.g., visually or by optical or polarizing microscopy, but does not have crystalline characteristics when determined by another means, e.g., x-ray powder diffraction.
• Methods of visually determining the crystallinity of a solid sample by visual or by optical or by polarizing microscopy are disclosed in USP ⁇ 695> and ⁇ 776>, both of which are incorporated by reference.
• a solid sample of 1 that is "crystal-like" may be crystalline under certain conditions but may become non-crystalline when subjected to other conditions.
• amorphous refers to a situation where a solid sample of 1 is neither crystalline nor crystal-like.
• a first embodiment is directed to a compound represented by a compound represented by formula 1 its hydrate or solvate thereof in crystalline or crystal-like form.
• the compound represented by formula 1 as its hydrate in crystalline or crystal-like form is designated as 1 ⁇ 3 ⁇ 40, where m varies in an integer or non- integer amount from about 0 to about 5.
• the compound represented by formula 1 as its solvate in crystalline or crystal-like form is designated as 1-nS, where n varies in an integer or non-integer amount from about 0 to about 3.
• the compound represented by formula 1 its hydrate or solvate might have a certain advantageous amount of adsorbed solvent (5) or water. In which case, the amount of S or water can vary from about 0 wt. % to about 10 wt.% based on the weight of the compound represented by formula 1 or its hydrate in crystalline or crystal-like form.
• a second embodiment is directed to crystalline or crystal-like 1.
• a third embodiment is directed to crystalline or crystal-like l-mH 2 0, where m varies in an integer or non-integer amount from about 0 to about 5.
• a second aspect of the third embodiment is directed to crystalline or crystallike 1 ⁇ 1 ⁇ 23 ⁇ 4 ⁇ -).
• a fourth embodiment is directed to crystalline 1 ⁇ 3 ⁇ 40.
• a first aspect of the fourth embodiment is directed to an orthorhombic crystalline 1 ⁇ 2 0, preferably having the following unit cell parameters a ⁇ 10.99 A, b ⁇ 13.09 A, and c ⁇ 20.36 A.
• a second aspect of the fourth embodiment is directed to a crystalline 1 ⁇ 2 0 having an XRPD 20-reflection (°) at about: 14.8.
• a third aspect of the fourth embodiment is directed to a crystalline 1 ⁇ 2 0 having XRPD 20-reflections (°) at about: 14.8 and 17.6
• a fourth aspect of the fourth embodiment is directed to a crystalline 1 ⁇ 2 0 having XRPD 20-reflections (°) at about: 14.8, 17.6, and 20.4
• a fifth aspect of the fourth embodiment is directed to a crystalline 1 ⁇ 2 0 having XRPD 20-reflections (°) at about: 8.7, 14.8, 17.6, and 20.4.
• a sixth aspect of the fourth embodiment is directed to a crystalline 1 ⁇ 2 0 having XRPD 20-reflections (°) at about: 8.7, 13.6, 14.8, 17.6, and 20.4.
• a seventh aspect of the fourth embodiment is directed to a crystalline 1-H 2 0 having XRPD 20-reflections (°) at about: 8.7, 11.1, 13.6, 14.8, 17.6, and 20.4.
• An eighth aspect of the fourth embodiment is directed to a crystalline 1 ⁇ 2 0 having an XRPD diffraction pattern substantially as that shown in Fig. 1.
• a fifth embodiment is directed to substantially pure crystalline 1 ⁇ 2 0.
• a sixth embodiment is directed to crystal-like 1 ⁇ 2 0.
• a seventh embodiment is directed to a composition for the treatment and/or prophylaxis of any of the viral agents using crystalline or crystal-like l 'mH 2 0 or l*nS.
• Possible viral agents include, but are not limited to: hepatitis C virus, hepatitis B virus, Hepatitis A virus, West Nile virus, yellow fever virus, dengue virus, rhinovirus, polio virus, bovine viral diarrhea virus, Japanese encephalitis virus, or those viruses belonging to the groups of Pestiviruses, hepaciviruses, or flavaviruses.
• An aspect of this embodiment is directed to a composition for the treatment of any of the viral agents disclosed herein said composition comprising a pharmaceutically acceptable medium selected from among an excipient, carrier, diluent, and equivalent medium and crystalline or crystal-like l-mE ⁇ O or 1-nS, that is intended to include its hydrates, solvates, and any crystalline forms of crystalline or crystal-like l'mH 2 0 or l*nS.
• a pharmaceutically acceptable medium selected from among an excipient, carrier, diluent, and equivalent medium and crystalline or crystal-like l-mE ⁇ O or 1-nS, that is intended to include its hydrates, solvates, and any crystalline forms of crystalline or crystal-like l'mH 2 0 or l*nS.
• the crystalline or crystal-like l'HiH 2 0 or 1-nS may be independently formulated in a wide variety of oral administration dosage forms and carriers. Oral administration can be in the form of tablets, coated tablets, hard and soft gelatin capsules, solutions, emulsions, syrups, or suspensions.
• the crystalline or crystal- like 1 ⁇ 2 ⁇ or 1-nS is efficacious when administered by suppository
• the most convenient manner of administration is generally oral using a convenient daily dosing regimen which can be adjusted according to the severity of the disease and the patient's response to the antiviral medication.
• compositions and unit dosage forms may be comprised of conventional ingredients in
• compositions may be employed as solids, such as tablets or filled capsules, semisolids, powders, sustained release formulations, or liquids such as suspensions, emulsions, or filled capsules for oral use; or in the form of
• suppositories for rectal or vaginal administration.
• a typical preparation will contain from about 5% to about 95% active compound or compounds (w/w).
• the crystalline or crystal-like l-mH 2 0 or l*nS can be administered alone but will generally be administered in admixture with one or more suitable
• Solid form preparations include, for example, powders, tablets, pills, capsules, suppositories, and dispersible granules.
• a solid carrier may be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
• the carrier In powders, the carrier generally is a finely divided solid which is a mixture with the finely divided active component.
• the active component In tablets, the active component generally is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.
• Suitable carriers include but are not limited to magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
• Solid form preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like. Examples of solid formulations are exemplified in EP 0524579; US 2002/0142050; US 2004/0224917; US 2005/0048116; US
• Liquid formulations also are suitable for oral administration include liquid formulation including emulsions, syrups, elixirs and aqueous suspensions. These include solid form preparations which are intended to be converted to liquid form preparations shortly before use. Examples of liquid formulation are exemplified in U.S. Patent Nos. 3,994,974; 5,695,784; and 6,977,257.
• Emulsions may be prepared in solutions, for example, in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia.
• Aqueous suspensions can be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins,
• methylcellulose sodium carboxymethylcellulose, and other well known suspending agents.
• the crystalline or crystal-like 1 ⁇ 2 0 or l-nS may be independently formulated for administration as suppositories.
• a low melting wax such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active component is dispersed homogeneously, for example, by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and to solidify.
• the crystalline or crystal-like l-mHbO or l*nS may be independently formulated for vaginal administration. Pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate. Certain of these formulations may also be used in conjunction with a condom with or without a spermicidal agent.
• Suitable formulations along with pharmaceutical carriers, diluents and excipients are described in Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pennsylvania, which is hereby incorporated by reference.
• a skilled formulation scientist may modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration without rendering compositions containing the compounds contemplated herein unstable or compromising their therapeutic activity.
• the purified crystalline or crystal-like 1 ⁇ 2 0 or l*nS may be independently formulated in conjunction with liposomes or micelles.
• liposomes it is contemplated that the purified compounds can be formulated in a manner as disclosed in U.S. Patent Nos. 4,797,285; 5,013,556; 5,077,056;
• hepatitis C virus West Nile virus, yellow fever virus, degue virus, rhinovirus, polio virus, hepatitis A virus, bovine viral diarrhea virus and Japanese encephalitis virus.
• the term “medicament” means a substance used in a method of treatment and/or prophylaxis of a subject in need thereof, wherein the substance includes, but is not limited to, a composition, a formulation, a dosage form, and the like, comprising crystalline or crystal-like l'itiH 2 0 or l-nS. It is contemplated that crystalline or crystal-like 1 ⁇ 2 0 or l-nS in the manufacture of a medicament, for the treatment of any of the antiviral conditions disclosed herein, either alone or in combination with another compound disclosed herein.
• a medicament includes, but is not limited to, any one of the compositions contemplated by the fourth embodiment disclosed herein.
• An eighth embodiment is directed to a method of treatment and/or prophylaxis in a subject in need thereof said method comprises administering a therapeutically effective amount of crystalline or crystal-like l-mB ⁇ O or l*nS to the subject.
• a subject in need thereof is one that has any condition the result of an infection by any of the viral agents disclosed herein, which includes, but is not limited to, hepatitis C virus, West Nile virus, yellow fever virus, degue virus, rhinovirus, polio virus, hepatitis A virus, bovine viral diarrhea virus or Japanese encephalitis virus, flaviviridae viruses or pestiviruses or hepaciviruses or a viral agent causing symptoms equivalent or comparable to any of the above-listed viruses.
• the viral agents disclosed herein includes, but is not limited to, hepatitis C virus, West Nile virus, yellow fever virus, degue virus, rhinovirus, polio virus, hepatitis A virus, bovine viral diarrhea virus or Japanese encephalitis virus, flaviviridae viruses or pestiviruses or hepaciviruses or a viral agent causing symptoms equivalent or comparable to any of the above-listed viruses.
• subject means a mammal, which includes, but is not limited to, cattle, pigs, sheep, chicken, turkey, buffalo, llama, ostrich, dogs, cats, and humans, preferably the subject is a human. It is contemplated that in the method of treating a subject thereof of the ninth embodiment can be any of the compounds contemplated herein, either alone or in combination with another compound disclosed herein.
• terapéuticaally effective amount means an amount required to reduce symptoms of the disease in an individual.
• the dose will be adjusted to the individual requirements in each particular case. That dosage can vary within wide limits depending upon numerous factors such as the severity of the disease to be treated, the age and general health condition of the patient, other medicaments with which the patient is being treated, the route and form of administration and the preferences and experience of the medical practitioner involved.
• a daily dosage of between about 0.001 and about 10 g including all values in between, such as 0.001, 0.0025, 0.005, 0.0075, 0.01, 0.025, 0.050, 0.075, 0.1, 0.125, 0.150, 0.175, 0.2, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, and 9.5, per day should be appropriate in monotherapy and/or in combination therapy.
• a particular daily dosage is between about 0.01 and about 1 g per day, including all incremental values of 0.01 g (i.e., 10 mg) in between, a preferred daily dosage about 0.01 and about 0.8 g per day, more preferably about 0.01 and about 0.6 g per day, and most preferably about 0.01 and about 0.25 g per day, each of which including all incremental values of 0.01 g in between.
• treatment is initiated with a large initial "loading dose" to rapidly reduce or eliminate the virus following by a decreasing the dose to a level sufficient to prevent resurgence of the infection.
• One of ordinary skill in treating diseases described herein will be able, without undue experimentation and in reliance on knowledge, experience and the disclosures of this application, to ascertain a therapeutically effective amount of the compound disclosed herein for a given disease and patient.
• Therapeutic efficacy can be ascertained from tests of liver function including, but not limited to protein levels such as serum proteins (e.g., albumin, clotting factors, alkaline phosphatase, aminotransferases (e.g., alanine transaminase, aspartate transaminase), 5 '-nucleosidase, ⁇ -glutaminyltranspeptidase, etc.), synthesis of bilirubin, synthesis of cholesterol, and synthesis of bile acids; a liver metabolic function, including, but not limited to, carbohydrate metabolism, amino acid and ammonia metabolism. Alternatively the therapeutic effectiveness may be monitored by measuring HCV-RNA. The results of these tests will allow the dose to be optimized.
• serum proteins e.g., albumin, clotting factors, alkaline phosphatase, aminotransferases (e.g., alanine transaminase, aspartate transaminase), 5 '-nucle
• a first aspect of the eighth embodiment is directed to a method of treatment and/or prophylaxis in a subject in need thereof said method comprises administering to the subject a therapeutically effective amount of a compound represented by compound l-mH ⁇ O or l-nS and a therapeutically effective amount of another antiviral agent; wherein the administration is concurrent or alternative.
• the time between alternative administration can range between 1-24 hours, which includes any sub-range in between including, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23 hours.
• another antiviral agent examples include, but are not limited to: HCV NS3 protease inhibitors (see EP 1881001, US 2003187018, US 2005267018, WO 2003006490, WO 200364456, WO 2004094452, WO 2005028502, WO
• HCV NS5B Inhibitors see US 2004229840, US 2005154056, US 2005-98125, US 20060194749, US 20060241064, US
• HCV NS4 Inhibitors see WO 2005067900 and WO 2007070556
• HCV NS5a Inhibitors see US 2006276511, WO 2006035061, WO 2006100310, WO 2006120251, and WO 2006120252
• Toll-like receptor agonists see WO 2007093901
• other inhibitors see WO 2000006529, WO 2003101993, WO 2004009020, WO 2004014313, WO 2004014852, and WO 2004035571
• compound A shown below and disclosed in U.S. Patent No.
• TMC435 as well as, interferon- ⁇ , interferon-/! ⁇ , pegylated interferon- ⁇ , ribavirin, levovirin, viramidine, another nucleoside HCV polymerase inhibitor, a HCV non-nucleoside polymerase inhibitor, a HCV protease inhibitor, a HCV helicase inhibitor or a HCV fusion inhibitor.
• Concurrent administration as used herein thus includes administration of the agents at the same time or at different times. Administration of two or more agents at the same time can be achieved by a single formulation containing two or more active ingredients or by substantially simultaneous administration of two or more dosage forms with a single active agent.
• references herein to treatment extend to prophylaxis as well as to the treatment of existing conditions.
• treatment also includes treatment or prophylaxis of a disease or a condition associated with or mediated by HCV infection, or the clinical symptoms thereof.
• a ninth embodiment is directed to process for preparing crystalline or crystal-like ⁇ 11 ⁇ 2 ⁇ or 1'iiS, which comprises crystallizing l-mH ⁇ O or 1-nS, wherein m and n are as defined above.
• a first aspect of the ninth embodiment is directed to a process for preparing crystalline or crystal-like ⁇ 11 ⁇ 2 ⁇ or l-nS, further comprises dissolving or suspending 1 in a solvent or solvent mixture.
• a second aspect of the ninth embodiment is directed to a process for preparing crystalline or crystal-like l*mH 2 0 or l « nS, which further comprises adding seed crystals of ⁇ 11 ⁇ 2 ⁇ or l*nS.
• a third aspect of the ninth embodiment is directed to a process for preparing crystalline or crystal-like ⁇ 1 ⁇ 2 ⁇ or l-nS, which further comprises adding an anti- solvent to the solvent or solvent mixture.
• Any suitable solvent may be used that affords crystalline or crystal-like l*mH 2 0 or l*nS.
• Specific solvents contemplated include, but are not limited to, anisole, ethyl acetate; xylenes; toluene; isopropanol; acetone; dichloromethane; diethyl ether; isopropyl acetate; t-Butyl methyl ether; or combinations thereof.
• Specific combinations include, but are not limited to, anisole/ethyl acetate;
• acetate/xylenes isopropanol/xylenes; acetone/xylenes; dichloromethane/xylenes; dichloromethane/hexanes; ethyl acetate/toluene; diethyl ether/xylenes; isopropyl acetate/xylenes; isopropyl acetate/heptanes; ethyl acetate/water; t-butyl methyl ether/water; t-butyl methyl ether/ethyl ether; or t-butyl methyl ether.
• solvent combinations that also include water
• a sufficient amount of water is required to form l mH 2 0 (when m ⁇ 0), which is based on an estimation of the amount of 1 used for the crystallization or the amount of 1 contained in a mixture containing 1 and its Rp-isomer.
• commercially available solvents contain a certain amount of water that might be sufficient, alone, to provide sufficient water for the formation of crystalline or crystal-like l-mH 2 0 (when m ⁇ 0).
• a tenth embodiment is directed to a method for determining the crystallinity of crystalline or crystal-like l mH 2 0, which comprises analyzing l-mH 2 0 by XRPD or single-crystal X-ray crystallography.
• Compound 1 may be prepared by stereoselective or non-stereoselective means.
• a stereoselective process is described below, as well as U.S. Provisional Patent Application No. 61/319,548, which is incorporated by reference.
• a non- stereoselective process is also described below, as well as in U.S. Patent Application No. 12/645,765, the subject matter of which is incorporated by reference.
• Production of 1 via the non-stereoselective process, which produces a diastereomeric mixture containing 1 and its Rp-isomer further includes, as detailed below, crystallization of the diastereomeric mixture to obtain 1 or chromatographic separation of the diastereomeric mixture by way of SMB chromatography.
• Example 1-1 Synthesis of (2R,3R,4R,5R)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyI)-5-(2-((bis(4- methoxyphenyl)(phenyI)methyl)amino)-6-methoxy-9H-purin-9-yl)-4-fluoro-4- methyltetrahydrofuran-3-ol (3):
• Example 1-4 Preparation of (S)-2-[(4-nitro-phenoxy)-phenoxy- phosphorylamino] propionic acid isopropyl ester (Mixture of Diastereomers (S, Sp)-6 and (S, R P )-6)).
• Example 1-7 Synthesis of S P -(2S)-isopropyl 2-(((((2R,3R,4R,5R)-5-(2-amino-6- methoxy-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (1): To a solution of crude phosphoramidate 7 in MeOH (2.5 mL) was added 5%
• 1 can be prepared directly from 2, as illustrated below.
• Example 2 Synthesis of S P -(2S)-isopropyl 2-(((((2R,3R,4R,5R)-5-(2-amino-6- methoxy-9H-purin-9-yI)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2- yl)methoxy)(phenoxy)phosphoryl)amino)propanoate, 1
• dichlorophosphate (2.66 g, 12.61 mmol) and anhydrous dichloromethane (40 mL).
• the amino ester salt (2.60 g, 15.53 mmol) was added to the solution and the mixture was cooled to -5°C.
• N-Methyl imidazole (7.7 mL, 97 mmol) was then added quickly via a dry syringe at -5°C and the solution was stirred at -5°C for 1 h.
• the nucleoside (1, 3.04 g, 9.7 mmol) was added from a vial in one portion at -5°C and the solid was slowly dissolved in 20 minutes. The reaction temperature was allowed to rise to ambient temperature over 2 h.
• An alternate purification method is to chemically alter the minor 3' phosphoramidate by-product in order to simplify the chromatographic separation.
• the crude phosphoramidate product is dissolved in anhydrous pyridine (5 mL/g), and is treated with 0.5 molar equivalents of t-butyldimethylsilyl chloride at ambient temperature to react selectively with the free 5' primary hydroxyl of the 3' isomer impurity. Reaction progress can be monitored by LC/MS.
• the chromatographic equipment and separation method are as follows: A 5 -cm SMB system, consisting of 8 columns, each holding 100 grams of
• CHIRALPAK ® IATM was used for processing of the diastereomeric mixture.
• the mobile phase was 100% ethyl acetate.
• the solubility of this material was 75 g/1 in the mobile phase.
• the material was dissolved in the mobile phase at 40°C with stirring.
• the extract stream from the SMB was first concentrated using a 1 ⁇ 2 sq ft horizontal thin-film evaporator (Protherm) and then dried down in the rotovap at 40°C.
• the flow rate of the raffinate stream was too low to be handled by the Protherm.
• the raffinate was directly dried down using the rotovap.
• the products were then dried to constant weight in a vacuum oven at 40°C.
• the residual level of ethyl acetate however was found to exceed the desired level ( ⁇ 0.4wt%) even after constant weight had been reached. After further drying under vacuum at 50°C, the level remained at approximately lwt%.
• the products were re-dissolved in acetone and then evaporated to dryness in the rotovap. This step effectively removed the residual ethyl acetate level to below 0.1 wt%.
• the products were then further dried in the vacuum oven to reduce the residual acetone to the desired level of ⁇ 0.4wt%.
• Extract - 1 A quantity of 6,864g of amorphous 1 was recovered with a
• a 10 L rotary evaporator flask was equipped with a mechanical stirrer. 1000 g of amorphous solid (98.9% HPLC purity, 0.25 molar equiv of water present) was added followed by ethyl acetate (1.00 L) and anisole (99% grade, 4.00 L) at ambient temperature. The suspension was rapidly stirred until all the solid dissolved (15 min). Stirring was slowed to 88 rpm. Crystalline seeds (40 mg) were added, followed by water (31 mL, 1.0 eq). The solution became cloudy after about 30 minutes and showed heavy precipitate after 3-4 h with no visible water remaining. The suspension was stirred for a total of 20 h at ambient temperature.
• the crystalline solid was collected by vacuum filtration on a 25 cm Buchner funnel. The cake washed with a 50:50 mixture of heptanes and t-butyl methyl ether (3x1 L). The cake washed easily and did not require pressing. After air-drying for 15 min, the solid was transferred to a 8x14" drying pan and dried under vacuum (0.2 mm Hg, 50°C) to a constant weight (4 h) and then held under vacuum at ambient temperature for 17 h to yield 840 g (ca 82% for the difference in hydration) of fine broken crystalline laths and needles less than 150 micrometers on a side. HPLC purity 99.7%. The apparent melting point showed shrinking starting at 88°C and melting at 93-100°C. As a hydrate, this represents a combination of dehydration to an amorphous solid and then the phase transition temperature.
• Example 6 Crystallization of 1-H 2 0 from amorphous 1 (obtained from SMB chromatography) with heptanes/ethyl acetate.
• 1 ⁇ 2 0 can be obtained by crystallization of crude mixtures of 1 and the iip-isomer.
• the Sp-isomer can also be isolated without any other substance.
• Anisole- water is a suitable solvent combination to initiate the
• phenyl dichlorophosphate (2.68 g, 12.7 mmol) and anhydrous dichloromethane (40 mL).
• the amino ester salt (2.58 g, 15.4 mmol) was added to the solution and the mixture was cooled to -30 °C.
• N-Methyl imidazole (6.3 g, 76 mmol) was then added quickly via a dry syringe at - 30 °C and the solution was stirred at -5°C for 20 min.
• the nucleoside (7, 2.00 g, 6.38 mmol) was added from a vial in one portion at -5°C and the solid was slowly dissolved in 20 minutes.
• reaction temperature was allowed to rise to ambient temperature and stirred for 1 h. TLC indicated approximately 95% completion.
• the reaction was diluted dichloromethane (50 mL) and washed with 1 N HCl (2x50 mL), 1 : 1 mixture of sat'd bicarbonate and brine, dried over sodium sulfate (5 g), filtered, concentrated under reduced pressure and then high vacuum to 4.5 g of crude product.
• P-NMR indicated a 1.5:1 mixture of the S P /R P isomers.
• X-Ray Powder Diffraction patterns were collected on a Bruker D8 diffractometer using Cu Ka radiation (40kV, 40mA), ⁇ -2 ⁇ goniometer, and divergence of V4 and receiving slits, a Ge monochromator and a Lynxeye detector.
• the instrument is performance checked using a certified Corundum standard (NIST 1976).
• the software used for data collection was Diffrac Plus XRD Commander v2.5.0 and the data were analysed and presented using Diffrac Plus EVA v 11.0.0.2 or v 13.0.0.2.
• Fig. 1 contains an XRPD spectrum of 1 H 2 0.
• Table 2 lists cell information, data collection parameters, and refinement data. Final positional and parameters are given in Table 3.
• Figure 2 is an ORTEP ("ORTEP-II: A Fortran Thermal Ellipsoid Plot Program for Crystal Structure Illustrations.” C.K. Johnson (1976) ORNL-5138.) representation of the 1 ⁇ 2 0 with 30% probability thermal ellipsoids displayed,
• phosphoramidate reagents are used to prepare 1, its i?p-isomer, or a diastereomeric mixture of 1 and its Rp-isomer.
• Example 12-1 Synthesis of (S)-2- ⁇ (S)-[(lR,4R,5R)-5-(2-Amino-6-methoxy- purin-9-yl)-4-(R)-fluoro-3-hydroxy-4-methyl-tetrahydro-furan-2-ylmethoxy]- phenoxy-phosphorylamino ⁇ -propionic acid isopropyi ester monohydrate (1) via (S)-isopropyl 2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino) propanoate ((S, Sp)-8) and isolation by chromatography and crystallization.
• the mixture was stirred at 0°C for additional 4 h.
• the mixture was filtered through a Buchner funnel and the collected solid triethylamine hydrochloride was rinsed with DCM (3 x 40 mL).
• the filtrate was checked by 31 P-NMR (ratio ca 1.14:1 favored the Sp-diastereomer - downfield peak) and was divided into two parts of equal weight. One of them was concentrated under reduced pressure.
• the white solid residue (31 g) was triturated in a mixture of EtOAc and hexanes (150 mL, 20:80, v/v) at RT for 17 h allowing time for dynamic resolution of the less soluble Sp-isomer.
• the white slurry was filtered and solid was rinsed with 20% EtOAc in hexanes (2x 25 mL).
• the solid (22.58 g) was checked by
• the reaction was quenched by adding 20 mL of sat NH 4 C1 at 0 °C.
• the mixture was diluted with 100 mL of ethyl acetate. Two layers were separated and aqueous layer was extracted with 50 mL of ethyl acetate. Organic layer was combined and washed with water (60 mL), sat sodium bicarbonate (2 x 60 mL), water (60 mL), brine (40 mL), and dried over sodium sulfate. Solvent was removed under reduced pressure to afford an amorphous solid residue. To the crude residue 7 mL of ethyl acetate was added and followed by 26 mL of anisole. The mixture was stirred until a solution was formed.
• Example 12-2 Synthesis of (S)-2- ⁇ (S)-[(lR,4R,5R)-5-(2-Amino-6-methoxy- purin-9-yl)-4-(R)-fluoro-3-hydroxy-4-methyl-tetrahydro-furan-2-ylmethoxy]- phenoxy-phosphoiylaminoj-propionic acid isopropyl ester monohydrate (1) via (S)-isopropyl 2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino) propanoate ((S, Sp)-8) and isolation by crystallization only.
• Phenyl phosphorodichloridate (10.0 g, 47.4 mmol) was dissolved in 60 mL of dry DCM and subsequently cooled to -78 °C.
• a premixed solution of 2,4- dinitrophenol (8.72 g, 47.4 mmol) and triethylamine (7.27 mL, 52.1 mmol) in 20 mL of DCM was slowly added at -78 °C over a period of 30 min. The reaction was brought to 0 °C and stirred for 2.5 h at this temperature before (L)-alanine isopropyl ester (7.95 g, 47.4 mmol) was added as a solid in one batch.
• the reaction was quenched by adding sat NH 4 C1 after one and half hour.
• the mixture was diluted with 20 mL of ethyl acetate.
• Organic layer was separated and aqueous layer was extracted with ethyl acetate (20 mL).
• the combined organic layer was washed with water (50 mL), sat sodium bicarbonate (2 x 40 mL), sat sodium carbonate (40 mL), water (40 mL), and brine (30 mL).
• the light yellow color organic layer was dried over sodium sulfate.
• the solution was concentrated under reduced pressure and an amorphous solid residue resulted was purified via column chromatography.
• L-alanine isopropyl ester hydrochloride salt (26.2 g, 156.3 mmol) was added as solid and then followed by triethylamine (43.7 mL, 313.4 mmol) in dichloromethane (150 mL) drop wise at 0 °C for 20 min. and the reaction mass was continued stirring at the same temperature for additional one hour.
• the reaction mixture was filtered and concentrated and was finally purified by column chromatography (20 % EtOAc/hexanes) on a silica gel to yield ((S, ⁇ 3 ⁇ 4>)-10 and (S, i? P )-10) as diastereomeric mixture (14.4 g, 25%).
• Example 12-5 Synthesis of (S)-2- ⁇ [(lR,4R,5R)-5-(2-Amino-6-methoxy-purin- 9-yl)-4-(R)-fluoro-3-hydroxy-4-methyl-tetrahydro-furan-2-ylmethoxy]- phenoxy-phosphorylamino ⁇ -propionic acid isopropyl ester diastereomeric mixture (1 and its Rp-isomer) via diastereomeric mixture of (2S)-isopropyl 2- (((2,4-dichlorophenoxy)(phenoxy)phosphoryl)amino)propanoate ((S, S P )-11 and (S, RP)-11)
• Phenyl phosphorodichloridate (10.0 g, 47.4 mmol) was dissolved in 60 mL of dry DCM and subsequently cooled to -78 °C. Slow addition of a preformed mixture of 2,4-dichlorophenol (7.73 g, 47.4 mmol) and triethylamine (7.27 mL, 52.1 mmol) in 20 mL of DCM was followed by stirring at above temperature for 30 min. The reaction was brought to 0 °C and stirred for 2.5 h at this temperature before (L)- alanine isopropyl ester (7.95 g, 47.4 mmol) was added as a solid in one batch.
• Phenyl phosphorodichloridate (10.0 g, 47.3 mmol) was dissolved in 50 mL of dry DCM and subsequently cooled to 0 °C. After addition of solid (L)-alanine isopropyl ester HCl salt (7.94 g, 47.3 mmol), the reaction mixture was cooled to -70 °C and then treated with triethylamine (13.8 mL, 94.6 mmol) dissolved in 50 mL of dry DCM. The resulting mixture was stirred for 30 min at this temperature before being allowed to warm to 0 °C.
• the reaction was quenched by addition of sat NH 4 C1 (5 mL) and diluted with 20 mL of ethyl acetate and 10 mL of water. Two layers were separated and aqueous layer was extracted with 20 mL of EtO Ac. Organic layer was washed with water (20 mL), sat sodium bicarbonate (2 x 30 mL), 5% sodium carbonate (30 mL), water (20 mL), and brine (20 mL). Organic solution was dried over sodium sulfate and concentrated to a yellow color solid residue. The residue was purified via column chromatography (silica gel, 3% methanol in DCM) to afford 279 mg (0.48 mmol, 83%) of 1 and its iip-isomer.
• Example 12-7 Synthesis of (S)-2- ⁇ (R)-[(lR,4R,5R)-5-(2-Amino-6-methoxy- purin-9-yI)-4-(R)-fluoro-3-hydroxy-4-methyl-tetrahydro-furan-2-ylmethoxy]- phenoxy-phosphoryIamino ⁇ -propionic acid isopropyl ester (Rp-isomer of 1) via (2S)-isopropyl 2-(((R)-(2-chloro-4-nitrophenoxy)(phenoxy)phosphoryl) amino)propanoate ((S, Rp)-12)
• Phenyl phosphorodichloridate (6.37 g, 30.19 mmol) was dissolved in 40 mL of dry DCM and subsequently cooled to 0 °C. After addition of solid (L)-alanine isopropyl ester (5.06 g, 30.19 mmol), the reaction mixture was cooled to -78 °C and then treated with triethylamine (8.84 mL, 63.3 mmol) dissolved in 20 mL of dry DCM. The resulting mixture was stirred for 30 min at this temperature before being allowed to warm to 0 °C.
• HCV replicon assay HCV replicon assay.
• HCV replicon RNA-containing Huh7 cells (clone A cells; Apath, LLC, St. Louis, Mo.) were kept at exponential growth in Dulbecco's modified Eagle's medium (high glucose) containing 10% fetal bovine serum, 4 mM L-glutamine and 1 mM sodium pyruvate, l x nonessential amino acids, and G418 (1,000 jug/ml).
• Antiviral assays were performed in the same medium without G418. Cells were seeded in a 96- well plate at 1,500 cells per well, and test compounds were added immediately after seeding. Incubation time 4 days.
• Replicon RNA and an internal control (TaqMan rRNA control reagents; Applied Biosystems) were amplified in a single-step multiplex RT-PCR protocol as recommended by the manufacturer.
• the HCV primers and probe were designed with Primer Express software (Applied Biosystems) and covered highly conserved 5 -untranslated region (UTR) sequences (sense, 5'-AGCCATGGCGTTAGTA(T)GAGTGT-3', and antisense, 5 '-TTCCGC AGACC ACT ATGG-3 '; probe, 5VFAM- CCTCCAGGACCCCCCCTCCC-TAMRA-3').
• the threshold RT-PCR cycle of the test compound was subtracted from the average threshold RT-PCR cycle of the no-drug control (ACtncv)- A ACt of 3.3 equals a 1-log 10 reduction (equal to the 90% effective concentration [EC90]) in replicon RNA levels.
• the cytotoxicity of the test compound could also be expressed by calculating the ACtrRNA values.
• the AACt specificity parameter could then be introduced (ACtncv ⁇
• ACtrRNA in which the levels of HCV RNA are normalized for the rRNA levels and calibrated against the no-drug control.
• Compound 1 was tested for its biological properties based on the preceding assay. The results of these tests are disclosed below.
• Replicon assay results show activity when compound 1 is assayed in combination with compound C (designated as compound 19 in US 2010/0081628, and the individual diastereomers (19a and 19b) disclosed in the same application); compound D (disclosed in US 2010/0016251); compound E (disclosed in US 12/783,680 as S P -4); ITMN-191 (disclosed in US 2009/0269305 at Example 62-1); and ANA-598 (disclosed in F. Ruebasam et al. Biorg. Med. Chem. Lett. (2008) 18: 3616-3621 as compound 3i).

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