TW201124141A - HCV nucleoside inhibitor - Google Patents

Abstract

4-Amino-1-((2R, 3S, 4S, 5R)-5-azido-4-hydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidin-2-one (22) and prodrugs thereof are hepatitis C (HCV) polymerase inhibitors. Also disclosed are compositions and methods for inhibiting HCV and treating HCV-mediated diseases, processes for making the compounds and synthetic intermediates employed in the process.

Description

201124141 VI. Description of the Invention: [Technical Field of the Invention] The present invention provides nucleoside compounds and certain derivatives thereof, which are inhibitors of RNA-dependent RNA viral polymerase. These compounds are inhibitors of RNA-dependent RNA viral replication and are useful for the treatment of RNA-dependent RNA viral infections. It is especially useful as an inhibitor of hepatitis C virus (HCV) NS5B polymerase, as an inhibitor of HCV replication, and for the treatment of hepatitis C infection. The present invention relates to nucleoside inhibitors of HCV replicon RNA replication. In particular, the present invention relates to the use of pyrimidine nucleoside compounds as inhibitors of subgenomic HCV RNA replication and pharmaceutical compositions containing such compounds. [Prior Art] Hepatitis C virus is the leading cause of chronic liver disease worldwide. (Boyer, N. et al. J. 2000 32: 98-1 12). Patients infected with HCV have a risk of developing cirrhosis and subsequent hepatocellular carcinoma, and therefore HCV is a major indication for liver transplantation. HCV has been classified as a member of the family Flaviviridae (F/aWWr/i/ae), a family of flaviviruses, prions and hepatitis C viruses including hepatitis C (Rice, C M·, F/aWWrzWae·· JTie viruses and their replication, in: Fields Virology, Editors: Fields, BN, Knipe, DM, and Howley, PM, Lippincott-Raven Publishers, Philadelphia, Pa., Chapter 30, 93 1 -959, 1996). HCV is an envelope disease containing approximately 9.4 kb of a single-stranded RNA genome 154377.doc 201124141. The viral genome consists of a [untranslated region (UTR), an open reading frame encoding a polymeric protein precursor of about 3011 amino acid, and a short 3, UTR. The 5' UTR is the most conserved part of the HCV genome and is important for initiating and controlling the translation of polymeric proteins. Genetic analysis of HCV has identified six major genotypes with more than 30°/〇 difference in the 〇ΝΑ sequence. More than 30 subtypes have been identified. In the United States, approximately 70% of infected individuals have la-type and lb-type infections. The ib type is the most popular subtype in Asia. (X. Forns and J. Bukh, C/im.cs h.ver 1999 3:693-716 'J. Bukh et al., Sewz·”· Ζίν. Db. 1995 15:41-63). Unfortunately, 1 Type infection is more resistant to treatment than type 2 or type 3. (NN Zein, C/i«. MzcroWo/. i?ev., 2000 13:223-235). Viral structural proteins include nuclear sheath core protein (C) And two envelope glycoproteins E1 and E2. HCV also encodes two proteases: a zinc-dependent metalloproteinase encoded by the NS2-NS3 region and a serine protease encoded in the NS3 region. These proteins 8# are precursor polymerizations. The specific region of the protein is cleaved into a mature peptide. The basal part of the non-structural protein 5 (NS5B) contains rnA-dependent RNA polymerase. The remaining non-structural proteins NS4A and NS4B function and NS5A (non-structural protein 5 amine) The function of the basal end portion is still unknown. Most of the non-structural proteins encoded by the HCV RNA genome are involved in RNA replication. There are currently a limited number of approved therapies available for the treatment of HCV infection. New and existing treatments for HCV Therapeutic methods and inhibition of HCV NS5B polymerase have been reviewed in the following literature: RG Gish, heart m. hv Er Db., 1999 19:5 ; Di Besceglie, A. M. and Bacon, B. R., s 154377.doc 201124141

Scientific American, October: 1999 80-85; G. Lake-Bakaar, Current and Future Therapy for Chronic Hepatitis C Virus Liver Disease, Curr. Drug Targ. Infect Dis. 2003 3(3): 247-253; P. Hoffmann et al. People, on patent therapy for hepatitis C virus infection (1999-2002), Exp. Opin. Ther. Patents 2003 13(1 1):1707-1723 ; Μ. P. Walker et al. Promising Candidates for the treatment of chronic Inflammatory C, Exp. Opin. investing. Drugs 2003 12(8):1269-1280 ; S.-L. Tan et al., Hepatitis C Therapeutics: Current

Status and Emerging Strategies, Nature Rev. Drug Discov. 2002 1:867-881 °

La: R = C(=0)NH2 lb:R = C(=NH+)NH2 Ribavirin (la; 1-((211,311,48,5 ft)-3,4-di 5--5-hydroxymethyl-tetrahydro-furan-2-yl)-1Η-[1,2,4]triazole-3-carboxylic acid decylamine; Viremia is a synthetic non-interferon-induced A broad spectrum antiviral nucleoside analog. Ribavirin has in vitro activity against several DNA and RNA viruses including the Flaviviridae (Gary L. Davis, Gajiroaiero/o anti-y 2000 118:S104-S114). In monotherapy, ribavirin reduced serum aminotransferase levels to normal in 40% of patients, but it did not reduce serum levels of HC V-RNA. Ribavirin also exhibits significant toxicity and is known to induce poor 154377.doc 201124141 blood. Verapamil lb is a drug that is converted to la in hepatocytes. Interferon (IFN) has been used to treat chronic hepatitis in the last decade. IFN is a glycoprotein produced by infection with an immune cell reactive virus. Two different types of interferon have been identified: Type 1 includes several interferon alpha and one interferon beta, and type 2 includes interferon y. Type 1 interferons are produced primarily by infected cells and protect adjacent cells from reinfection. IFN inhibits viral replication of many viruses including HCV, and when used as a separate treatment for hepatitis C infection, IFN inhibits serum HCV-RNA to a level that is not detectable. Furthermore, IFN normalizes serum aminotransferase content. Unfortunately, the effects of IFN are short-lived. Stop treatment resulted in a 70% relapse rate and only 10-15% showed a sustained viral response to normal serum alanine transferase levels (L.-B. Davis, supra). One limitation of early IFN therapy is the rapid clearance of proteins in the blood. The chemical derivatization of IFN with polyethylene glycol (PEG) results in a pharmacokinetic profile of the protein that is substantially improved. PEGASYS® is a combination of interferon ot-2a and a 40 kD branched chain monodecyloxy PEG and PEG-INTRON® is a combination of interferon a-2b and 12 kD monodecyloxy PEG. (BA Luxon et al, Therap. 2002 24(9): 13631383; A. Kozlowski and JM Harris,·./. 2001 72:217-224) o Combination therapy with ribavirin and interferon-a for HCV Indicates the best therapy. Combination ribavirin with PEG-IFN (below) resulted in a sustained viral response in 54-56% of patients. For Type 2 and Type 3 HCVs, the SVR is 80%. (Walker, above). Unfortunately, this combination also produces side effects' resulting in clinical challenges. Depression, flu-like symptoms, and skin reactions are associated with subcutaneous IFN-α and hemolytic anemia is associated with continued treatment with ribavirin 154377.doc 201124141. A number of potential sub-targets that have been identified for drug development such as anti-Hcv treatment include, but are not limited to, NS2_NS3 auto-protease 'Ν3 proteinase::3 helicase and NS5B polymerase. RNA-dependent RRa polymerase: is required for replication of single-stranded, sense, and RNA genomes. This enzyme stimulates the interest of physicists in e-NS5B and non-nuclear* inhibitors. A nucleoside inhibitor can be used as a chain terminator or as a competitive inhibitor that interferes with the binding of a nucleotide to a polymerase. For use as a chain terminator, the nuclear analog must be taken up by the cell and converted in vivo to a triphosphate to compete for the binding site of the polymerase nucleotide. This conversion to the triphosphate is typically mediated by cellular kinases that confer additional structural requirements on the potential nuclear polymerase inhibitor. Furthermore, this direct restriction of nucleosides as inhibitors of HCV replication is limited to cell-based assays capable of in situ phosphorylation. In WO 01 90121 published on Nov. 29, 2001, j_p s〇mmad〇ssi and P_Lacolla disclose and exemplify the anti-HCV polymerization of 1, 2, and 2, -alkyl- and 2,-alkyl groups Enzyme activity. In WO 01/92282, published on Dec. 6, 2001, J.-P. Sommadossi and P. Lacolla disclose and exemplify the treatment of flaviviruses with the wide alkyl- and 2'-alkyl nucleosides of formulas 2 and 3 and Prion. G. Gosselin et al. disclose 41-alkyl nucleoside 4 and the use of 4'-alkyl nucleosides for the treatment of flaviviruses and prions, in WO 03/026675 and WO 03/026589, both of which are incorporated by reference. The method. In WO 2004003000 and WO 2004002999, both published on January 8, 2004, J.-P. Sommadossi et al. disclose I1-, 2'-, 3·- and 4·-substituted β-D and β-L. Pre-nucleoside drug. J.-P. Sommadossi et al. disclose 3'-C-methyl-ribofuranosylcytosine 3'-0- in WO 04/002422, published Jan. 8, 2004. L-proline and its use for the treatment of HCV

Idenix has been reported as a clinical trial of the related compound NM283, which is a proline 5 of cytidine analog 2 (B = cytosine). In addition, Idenix Pharmaceuticals, Ltd. is also disclosed in WO 04/04633 1 by biologically active 2·-branched β-D or β-L nucleosides or pharmaceutically acceptable salts or prodrugs thereof. Flavivirus family mutation.

B=Adenine, thymus, β-nuclear nucleus, urinary D-dose, cytidine e-nucleus, U-tele and jaundice published in WO 02/057425, published on July 25, 2002, SS Carroll et al. disclose a nucleoside inhibitor of a chemically modified RNA-dependent RNA polymerase of a carbohydrate subunit. In WO 02/05787, published July 25, 2002, SS Carroll et al. disclose related 2α-mercapto and 2β-methylribose derivatives in which the base is optionally substituted 7Η-pyrrolo[2 , 3-d] pyrimidinyl group 6. This same application discloses an example of a 3?-methyl nucleoside. S.S. Carroll et al. (·/. CTiem. 2003 278(14): 11979-11984) revealed the inhibition of HCV polymerase by 2'-0-methylcytosine nucleoside (6a). In U.S. Patent Publication No. 2004/0259934, issued Dec. 23, 2004, D. B. Olsen et al. disclose the use of nucleoside compounds to inhibit replication of the coronavirus virus and to treat coronavirus infections. 154377.doc 201124141 Method.

6 6a In WO 02/100415 (US 2003/0236216 A1), published on Dec. 19, 2002, R. R. Devos et al. disclose a 4'-substituted nucleoside compound exhibiting HCV activity. The four compounds are specifically identified to include 4'-azido compound 7a, ethynyl compound 7b, 4'-ethoxy compound 7c and V-acetyl group compound 7d. Modifications to the ribose moiety exemplified include 2'-deoxy derivatives 8a, 3'-deoxy derivatives 8b, 3'-decyloxy derivatives 8e, 3'-fluoro derivatives 8c and 2', 2· - a difluoro derivative 8d. In WO 2004/046159 (US 2004121980), published June 3, 2004, J. A. Martin et al. disclose a pre- 7a drug suitable for the treatment of HCV-mediated diseases. Both U.S. applications are hereby incorporated by reference in their entirety.

7a: R = N3 7b: R = ethynyl 7c: R = OEt 7d: R = C(=0)Me

8a: R1 = OH, R2 = R3 = R4 = H 8b: R3 = OH, R1 = R2 = R4 = H 8c: R3 = OH, R2 = F, R1 = R4 = H 8d: R1 = R2 = H, R3 = R4 = F 8e: R' = OMe, R3 = OH, R2 = R4 = H US application filed on June 11, 2002 entitled "4'-Substituted Nucleoside Derivatives as Inhibitors of HCV RNA Replication" The compounds related to the present invention are disclosed in U.S. Patent Application Serial No. 10/7,176, the entire disclosure of which is incorporated herein by reference. Both of these applications are hereby incorporated by reference in their entirety. Y.-H. Y\xn^ J^{Arch. Pharm. Res. 1985 18(5):364-35)^ shows 4·-azido-2'-deoxy-2·-fluoro-arabinofuran Synthesis and antiviral activity of glycosyl nucleosides (9: R = H, Me and Cl).

B = adenine, uracil, thymine GS Jeon and V. Nair (7Wra/zei/ro« 1996 52(39): 12643-50) reveals the synthesis of 4·-azidomethyl-2',3'- Deoxyribonucleoside 10 (B = adenine, thymine, and uracil) acts as an HIV reverse transcriptase inhibitor. Several computational studies have been reported for 4'-azido-based nuclear bitterness: D, Galisteo et al. » J. Mol. Struct. 1996 384(1):25-33; J. Pepe#A > Eur. J. Med. Chem. 1996 32(10): 775-786; E. Estrada et al., / « silico studies toward the discovery of New Anti HIV Nucleoside, J. Chem. Info. Comp. Sci. 2002 42(5): 1194-1203 . I. Sugimoto et al. revealed the synthesis of 4'-ethynyl-2'-deoxycytidine nucleoside (11) and other dicarbon substituents at the f-position and HIV and herpes simplex (from sz'wp/ex Bioassay (Nucleosides and Nucleotides. 183. Synthesis of 4' β-Branched Thymidines as a New Type of Antiviral Agent, Bioorg. Med. Chem. Lett. 1999 9:385-154377.doc -10- 201124141 88). T. Wada et al. ίip% 15(1-3): 287-3 04) reveals the synthesis and anti-HIV activity of 4'-C-methyl nucleosides. In WO 01/32153, published May 10, 2001, R. Storer discloses a method of treating or preventing Flaviviridae infection by administering a oxetane dioxolane analog. In WO 02/18404, published March 7, 2002, R. Devos et al. disclose novel and known purine and pyrimidine nucleoside derivatives and their use as inhibitors of subgenomic HCV replication and contain such cores A pharmaceutical composition of a glycoside derivative. The disclosed compounds are composed of nucleosides having substituted oxime and π determinatory groups. ΕΡΑ Publication No. 352 248 discloses a variety of spectrums for the treatment of HIV, cancer therapy and liver k. L- ° 夫 南 ribosyl nucleoside nucleosides. Similar instructions are found in

Applicable in WO 88/09001 by Aktiebolaget Astra. K. Kitano et al. 1997 53(39): 13315-13322) revealed the synthesis of 4'-fluoromethyl 2-deoxy-D-un-, ritual- and therapeutic rapa pentanofuranosylcytosine and antibiotics赘 Biological activity. Non-nucleoside ectopic inhibitors of HI V reverse transcriptase have been shown to be effective for use alone or in combination with nucleoside inhibitors and with protease inhibitors. Some of the non-nucleoside HCV NS5B inhibitors that have been described and are currently in various stages of development include: benzox. Sitting class (H. Hashimoto et al. WO 01/47833 H. Hashimoto et al. WO 03/000254, PL Beauliei^ <w〇 03/020240 A2; Ρ·L_ Beaulieu et al. US 6,448,281 Bl; P. L Beaulieu et al. WO 03/007945 Al); 吲哚 (L. Beauliei^

Human WO 03/0010141 A2); benzodiazepines, such as i, (D 154377.doc 201124141

WO 01/85172 A1, filed on May 10, 2001, by Dhanak et al., WO 2002098424, filed on Jun. 7, 2002, D. Dhanak et al., October 28, 2002, filed WO 03/037262 A2; KJ Duffy et al., WO 03/099801, filed on May 23, 2003, PCT Application No. WO 2003059356, filed on Oct. 28, 2002, filed on Jun. 004052312, WO 2004052313, filed December 13, 2003, to D. C., et al., PCT Application No. WO 2004058150, filed Dec. 12, 2003, PCT Application No. WO 200401958, filed on Jan. 19, 2004, PCT Application No. WO 2005019191; JK Pratt WO 2004/041818 Al, filed on October 31, 2003.

Thiophenes, for example 2, (CK Chan et al. WO 02/100851 A2); benzothiophenes (DC Young and TR Bailey WO 00/18231); β-ketopyruvate (S. Attamura et al US 6,492,423 Bl , A. Attamura et al. WO 00/06529); pyrimidines (C. Gardelli et al. WO 02/06246 Al); pyrimidinediones (TR Bailey and DC Young WO 00/13708); triazines (K.- H. Chung et al. WO 02/079187 Al); rhodamine derivatives (T. R. Bailey and DC Young WO 00/10573, JC Jean et al. WO 01/77091 A2); 2,4-di- oxy oxyperazine Ran (R. A. Love et al. EP 256628 A2); phenylalanine derivatives (M. Wang et al. / s 154377. doc -12- 201124141 C/zew. 2003 278: 2489-2495). The thiazide which inhibits HCV NS5B has been disclosed in U.S. Patent No. 20060040927, filed on Jan. 22, 2005. Inhibitors of HCV protease required for viral replication have also been revealed (F. McPhee #^ A » Drugs of the Future 2003 28(5): 465-488; Y. S. Tsantrizos et al., drtgew. C/zem. / «i. 2003 42(12): 1356-1360). The nucleoside compounds of the invention can be used in combination with these and other polymerases and protease inhibitors. The results of these efforts have been reviewed (J. Z. Chen and Z. Hong, Targeting NS5B RNA-Dependent RNA Polymerase for Anti-HCV Chemotherapy, Curr. Drug Targ. Inf. Dis. 2003 3(3): 207-219). These non-nucleoside inhibitors are not relevant to the present invention. SUMMARY OF THE INVENTION It is an object of the present invention to provide novel nucleoside compounds for use in methods and compositions for treating a host infected with hepatitis C virus. Current currently approved therapies for the absence of prophylactic treatment of hepatitis C virus (HCV) and only for HC V are limited. It is necessary to design and develop novel pharmaceutical compounds. Surprisingly ' 2'-deoxy-2'-β-methyl-4'-azido- cytidine nucleoside or its ester is suitable for the treatment of HCV and exhibits lower toxicity when administered to a host. The invention also provides a pharmaceutical composition of the compound with at least one pharmaceutically acceptable carrier, excipient or diluent. Combination therapy has been shown to be useful in the treatment of viral diseases and novel compounds potentiate other approved and studied HCV therapeutics and the present invention provides nucleosides, or pharmaceutically acceptable, using the above disclosure 154377.doc-13·201124141: An acceptable salt, in combination with one or more pots/virions or immunomodulators, or used interchangeably (including at least one pharmaceutically acceptable agent, as appropriate) to treat HCV. Further carrier, excipient or dilution

The invention provides a nucleoside of formula I or a pharmaceutically acceptable salt thereof, and the use of such a compound for treating a host infected with HCV, wherein: R, R2 and R3 are independently selected from the group consisting of: hydrogen , COR4, C(=0)0R4 and c(=〇)CHR5NHR6; R4 is independently selected from the group consisting of: (4) Ci i8 non-branched or branched bond, (8), (4) c38 cycloalkyl, (4) Cii noisy a base group and (e) a phenyl group which is optionally substituted by one or three groups independently selected from the group consisting of c..3 alkyl, Gw alkoxy, dentate, cyano or nitro; R is hydrogen , C ^ o alkyl, phenyl or hydrazine · · 3-phenyl thiol, the phenyl optionally substituted by one or three groups selected from the group consisting of: halogen, hydroxyl 'C! 3 alkoxy, C _3 alkyl, cyano and nitro; R 6 is hydrogen or C alkoxy; or an acid addition salt thereof. The invention also provides Formula I or pharmaceutically acceptable, if used in combination with other effective antiviral agents for the treatment of HCV infection, and optionally at least one pharmaceutically acceptable carrier, excipient or diluent. Salt is used in the manufacture of 154377.doc •14· 201124141 for the treatment or prevention of HCV in a host. Without wishing to be bound by theory, it is believed that the compounds of the invention are continuously phosphorylated into 5,_〇_monophosphate, 5,_〇-diphosphate in human cells by kinase and eventually become antiviral active metabolites 5,- 〇_Triphosphate. Accordingly, another aspect of the invention provides such 5'-tantal acidified materials, i.e., compounds of formula j, wherein R1 and R3 are hydrazine and R2 is a monophosphate, diphosphate or triphosphate. The antiviral activity of a nuclear bitter inhibitor is generally the ability of the nucleoside to be absorbed in the host cell, the intracellular stability of the nucleoside to the active triphosphate 'triphosphate, and the ability of the diphosphate to interfere with the RNA synthesis activity of the viral polymerase. Combine the results. As presented by the biological examples below, the compounds of the invention are readily phosphorylated in vivo to active triphosphates and have a long intracellular triphosphate half-life, thus allowing for sustained and high concentrations of antiviral actives. [Embodiment] In one embodiment of the invention, a compound of formula I or a pharmaceutically acceptable salt thereof is provided, wherein R1, R2, R3, R4, RS&amp;R6 are as defined above. The phrase "as defined above" refers to the broadest definition of each group as provided in the definition of Formula I given above. The substituents, which are not explicitly defined in the examples, are retained in the other embodiments provided below, and the broadest definitions provided in [Inventive Content] are retained. In another embodiment of the present invention, there is provided a compound of formula I or a pharmaceutically acceptable salt thereof, wherein R2, R3 are hydrogen. In another embodiment, there is provided a compound of formula I, wherein R1 and R3 are Η and R is a single-plate acid ester, a di-acid acid ester or a tri-squaternate ester. In still another embodiment of the present invention, there is provided a compound of formula j or a pharmaceutically acceptable salt thereof, wherein R1, r2, r3 are each independently hydrogen, COR4 or C(=〇)〇 R4XR4 is as described above. In a further embodiment of the invention, 'providing a compound of formula J or a pharmaceutically acceptable salt, wherein r1, r2, r3 are each independently hydrogen, COR4 or C(=0)0r4j_r4 is a non-branched chain Or a branched chain Cii oxime group, such as a lower alkyl group, especially a methyl, ethyl, isopropyl or tert-butyl group. - In a further embodiment of the invention, there is provided a compound of formula 1 or a pharmaceutically acceptable salt thereof, wherein R1 is hydrogen; and Rule 2 and 3 are c〇R4. In still another embodiment of the present invention, there is provided a compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein Ri is hydrogen; r2&amp;r3 is c〇R4, and Μ is a non-branched or branched C, _1G An alkyl group, such as a lower alkyl group, especially methyl ethyl, isopropyl or a second butyl group. When a compound contains two R4 moieties, it is generally the same for convenience of synthesis. In still another embodiment of the present invention, there is provided a compound of formula j or a pharmaceutically acceptable salt thereof, wherein R1 and R3 are hydrogen; R2 is COR4, C(=0)0R4 or C0CH(Rs)NHR6; R4, rs&amp;r6 are as defined above. In a further embodiment of the invention, 'providing a compound of formula I or a pharmaceutically acceptable salt thereof, wherein R1 and R2 are hydrogen; R3 is COR4, c(=o)〇R4 or C0CH(RS)NHR6 And R4, ^5 and only 6 as defined above. In another embodiment of the invention 'provides a compound of formula I, wherein R2 is COCH(Rs)NHR6, and R5 is isopropyl, isobutyl or Dibutyl and R6 is hydrogen. In a preferred configuration of this embodiment, the spatial configuration of the RS group 154377.doc •16- 201124141 is (S) ', i.e., R2 is an L-aliphatic amino acid residue. R1 is usually Η' in this embodiment and R3 is Η or C〇CH(R5)NHR6, RS is isopropyl isobutyl or second butyl and R6 is hydrogen. When the compound has two such COCH(Rs)NHR6 moieties, it is usually the same amino acid for ease of synthesis. In another embodiment of the present invention, there is provided a compound of formula J, wherein R3 is COCH(Rs)NHR6, R5 is isopropyl, isobutyl or second butyl and R6 is hydrogen. In a preferred configuration of this embodiment, the space of the Rs group is configured as (S), i.e., R3 is an L-aliphatic amino acid residue. ;^ This embodiment is usually Η. In still another embodiment of the present invention, there is provided a hydrazine compound or a pharmaceutically acceptable salt thereof, wherein R1 and R3 are hydrogen; R2 is C〇R4; and r4 is as defined above. In another embodiment of the present invention, there is provided a hydrazine compound or a pharmaceutically acceptable salt thereof, wherein R1 and R3 are hydrogen; R2 is C〇R4; and r4 is C^Q non-branched chain or branched chain An alkyl group such as a lower alkyl group, especially a decyl group, an ethyl group, an isopropyl group or a tert-butyl group. In another embodiment of the present invention, a method of treating a disease mediated by a hepatitis C virus (HCV) virus, comprising administering to a mammal in need thereof a therapeutically effective amount of a compound of the formula or a medicament thereof A salt of acceptable learning 'where R 丨, ..., ..., ^, ^ and ... are as defined above. In yet another embodiment of the present invention, a method of treating a disease mediated by a hepatitis C virus (HCV) virus, comprising administering to a mammal in need thereof a therapeutically effective amount of a compound of formula I or Medically available as 154377.doc, 7 201124141

A group consisting of a non-branched chain or a branched chain lower alkyl group. In another embodiment of the present invention, in one embodiment, a method of treating a disease caused by a hepatitis C disease comprising administering a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable thereof, to a need thereof Accepting that H2 and R3 are each C〇R4; R4 is a method selected from diseases mediated by the q1 scorpion venom (HCV) virus, providing a treatment by c-hepatitis disease, which comprises administering to a mammal in need thereof A therapeutically effective amount of a compound of the formula or a pharmaceutically acceptable salt thereof, wherein R1 and R3 are hydrogen; R2 is c〇R, c〇ch(r5)nhr6; and R6 is as defined above. In another embodiment of the present invention, a

R4 is selected from the group consisting of a non-branched chain or a branched chain lower alkyl group; RS is a method for treating a disease mediated by a hepatitis C virus (HCV) virus, which comprises administering to a mammal in need thereof The dose is between 1 and 1 mg per kg of patient body weight of a compound of formula I or a pharmaceutically acceptable salt thereof, wherein ri, r2, r3, R4, Rs and R6 are as defined above. In another embodiment of the invention, a method of treating a disease mediated by a hepatitis C virus (HCV) virus comprising administering a therapeutically effective amount of a compound of formula I to a mammal having the need thereof Or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, R4, RS&amp;R6 are as defined above; and at least one immune system ampoules and/or at least one antiviral agent that inhibits HCV replication. In another embodiment of the present invention, a method of treating a disease mediated by a hepatitis C disease 154377.doc 201124141 toxic (HCV) virus comprising administering a therapeutically effective amount to a mammal in need thereof a compound of formula I or a pharmaceutically acceptable salt thereof, wherein R丨, R2, R' r4, and ... are as defined above; and at least one is selected from the group consisting of interferon, interleukin I, tumor necrosis factor or community Immune system modulator of stimulating factors. Those skilled in the art will appreciate that such immune system molecules may be in their naturally occurring form or they may be chemically derivatized to impart beneficial pharmacokinetic properties. In another embodiment of the present invention, a method of treating a disease mediated by a hepatitis C virus (HCV) virus, comprising administering to a mammal in need thereof a therapeutically effective amount of a compound of formula j or A pharmaceutically acceptable salt, wherein R1, R2, R3, R4, Rs and R6 are as defined above; and interferon or chemically derived interferon. In another embodiment of the present invention, a method of treating a disease mediated by a hepatitis virus (HCV) virus comprising administering to a mammal in need thereof a therapeutically effective amount of a compound of formula j or a pharmaceutical thereof An acceptable salt, wherein Ri, R2, R3, r4, r5 are as defined above, and at least one other antiviral agent. In another embodiment of the invention, a method of treating a disease mediated by a hepatitis C virus (HCV) virus, comprising administering to a mammal in need thereof a therapeutically effective amount of a compound of formula j or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, R4 are as defined above; and at least one other HCV protease inhibitor, another nucleoside HCV polymerase inhibitor, a non-nucleoside HCV polymerase inhibitor, HCV helicase inhibitor, HCV-priming enzyme inhibitor or HC V fusion inhibitor. 154377.doc 201124141 In an embodiment of the invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I in admixture with at least one pharmaceutically acceptable carrier, diluent or excipient or A pharmaceutically acceptable salt thereof, wherein R, R2, R3, R4, "and R6 are as defined above. In another embodiment of the invention, there is provided a pharmaceutical composition comprising 500-1500 mg comprising 35- 75 wt% of a compressed tablet of a compound of formula I or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, R4, R5 &amp; R6 are as defined above; and the balance comprises at least one pharmaceutically acceptable carrier A diluent, or an excipient. In another embodiment of the invention, there is provided a pharmaceutical composition comprising 500-1500 mg of a 40-40% by weight of a compound of formula I or a pharmaceutically acceptable salt thereof. A lozenge wherein Ri, R2, R3, r4, rS&r6 are as defined above; and the balance comprises at least one pharmaceutically acceptable carrier, diluent or excipient. As used herein, A &quot;real system refers to one or more of that entity; for example, A compound refers to one or more compounds or at least one compound. Likewise, the terms &quot;one&quot;, &quot;one or more &quot; &&quot;at least one &quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot; is the first definition of each group provided in the definition of 式! As used herein, the term &quot;optional&quot; or &quot;as appropriate&quot; means that the described event or situation can occur Or may not occur, and the description includes the occurrence or non-occurrence of the event or situation. For example, 'substituting a substituted phenyl group' means that the phenyl group may be substituted or unsubstituted, and the description includes A substituted phenyl group and a substituted phenyl group thereof by 154377.doc •20·201124141. The compounds of the invention may have a symmetry center on the side chain of the carboxylate, guanamine or carbonate moiety which, when attached to the nucleoside, produces a diastereomer. All stereoisomers of the side chains of the compounds of the invention, whether in the form of a mixture or in pure or substantially pure form, are contemplated. The definition of a compound of the invention includes all optically isomer enantiomers of the knife and mixtures thereof, including racemic forms. Pure optical isomers may be prepared by stereospecific synthesis from a_D_ribose or by racemic forms by physical methods (eg, fractional crystallization, separation or crystallization of diastereomeric derivatives or by palms) Separate by column chromatography to separate. Individual optical isomers can be obtained from the racemate by conventional methods (e.g., formation of a salt with an optically active acid followed by crystallization). The term &quot;alkyl, as used herein, denotes a non-branched or branched chain hydrocarbon radical containing from 丨 to 18 carbon atoms. The term |, lower alkyl" means an unbranched or branched chain hydrocarbon group containing from 1 to 6 carbon atoms. Representative lower alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, iso Butyl, tert-butyl or pentyl. When the term &quot;burning base&quot; is used as a suffix after another term, as in &quot;phenyl phenyl or &quot;carbyl&quot; It is intended to indicate an alkyl group as defined above substituted with one to two substituents selected from other specifically named groups. Thus, for example, &quot;phenylalkyl&quot; means having one to two phenyl substituents. Alkyl' and thus includes benzyl, phenylethyl and biphenyl. &quot;Alkylaminoalkyl&quot; is an alkyl group having one to two leumino substituents. The term &quot as used herein And a dentate group, wherein the non-branched chain or the branched chain is as defined above, wherein 1, 2, 3 or more hydrogen atoms are replaced by a halogen. An example is 1-fluoroindolyl 1 - gas enthalpy Base, 丨 bromomethyl ' 丨 曱 曱 154377.doc -21- 201124141 tri-gas methyl, tribromomethyl, tri-g-ethyl, 1-Ethyl, 2- 2-iodoethyl, 2,2-dichloroethyl, trifluoromethyl, yl, 1-oxyethyl, yl, 2-bromoethyl, 2,2,2-trifluoroethyl. Methyl, 1-fluoroethyl 2-fluoroethyl, 2-ethane, benzyl, 3-bromopropyl or the term "cycloalkyl" as used herein denotes a saturated carbocyclic ring containing from 3 to 8 carbon atoms. That means cyclopropyl, cyclobutyl, cyclopentyl, benzyl or cyclooctyl. Cyclohexyl, cycloheptane As used herein, the term "cycloalkylalkyl" refers to the group rir"-, wherein R| is cycloalkyl as defined herein, and R&quot; is as defined herein. The base 'supplement that the point of attachment of the cycloalkyl group will be on the pendant group. Examples of the benzyl group include, but are not limited to, cyclopropylmethyl, cyclohexylmethyl, cyclopentylethyl. Cm naphthenic The radical —Ci·3 alkyl refers to the group r,r&quot;, wherein &amp; is C3·7% of the substituents and R is the Cl.3 extension, as defined herein. Unless otherwise stated, The term &quot;alkylene&quot; as used herein denotes a divalent saturated linear hydrocarbon group having 1 to 8 carbon atoms or a branched chain saturated divalent hydrocarbon group having 3 to 8 carbon atoms. Examples of alkylene groups include (but Not limited to) methylene, ethyl, propyl, 2, methyl, propyl, butyl, 2-ethylbutyl butyl. The term "alkenyl" as used herein means having 2 to An unsubstituted [or substituted] hydrocarbon chain having 18 carbon atoms, preferably 2 to 4 carbon atoms, having one or two olefinic double bonds, preferably an olefinic double bondExamples are vinyl, 1-propenyl, 2-propenyl (allyl) or 2-butenyl (crotyl). As used herein, the term "alkynyl" means having 2 to 18 carbon atoms [preferably 2 to 4 carbon atoms] and having one or, if possible, two ginseng bonds [preferably 154377.doc - 22- £ 201124141 A non-substituted hydrocarbon chain with a key. Examples are ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl or 3-butynyl. The term "alkoxy" as used herein denotes an unsubstituted non-branched or branched alkyloxy group, wherein the "alkyl" moiety is as defined above, such as methoxy, ethoxy, n-propyl. Oxyl, isopropyloxy, n-butyloxy, isobutyloxy, tert-butoxy, pentyloxy, hexyloxy, heptyloxy, including isomers thereof. "Lower alkoxy" as used herein denotes an alkoxy group having a &quot;lower alkyl&quot; as defined above. The term "alkylthio" or "sulfanyl" as used herein. &quot; represents a non-branched or branched (alkyl) S- group, wherein the 'alkyl' moiety is as defined above. Examples are methylthio, ethylthio, n-propylthio, isopropylsulfide Base, n-butylthio, isobutylthio or tert-butylthio. The term alkylsulfinyl&quot; and &quot;arylsulfinyl&quot; as used herein, &quot; =0) a group of R wherein R is alkyl or aryl, respectively, and alkyl and aryl are as defined herein. The terms "alkylsulfonyl" and "arylsulfonyl" as used herein mean a radical of the formula -S(=0)2R wherein R is alkyl or aryl and alkyl and aryl, respectively. As defined herein, the term "aryl" as used herein denotes optionally substituted monocyclic or polycyclic aromatic groups containing carbon and hydrogen atoms. Examples of suitable aryl groups include, but are not limited to, phenyl And a naphthyl group (e.g., 1-naphthyl or 2-naphthyl). Suitable substituents for the aryl group are selected from the group consisting of alkyl, alkenyl, alkynyl, aryloxy, cycloalkyl, anthracene Alkyl, mercaptoamine, alkoxy, amino, alkylamino, dialkylamino, halogen, haloalkyl, thiol, acetyl and chloro. 154377.doc -23- 201124141

The term &quot;alkyl&quot; (,, alkylcarbonyl&quot;) as used herein denotes a group of the formula C(=〇)R, wherein R is hydrogen, a non-branched chain of 1 to 7 carbon atoms or Branched chain alkyl or phenyl. The term &quot;alkoxycarbonyl,&quot; and &quot;aryloxycarbonyl&quot; as used herein denotes a radical of the formula -C(=0)0R, wherein r is alkyl or aryl, respectively, and alkyl and aryl As defined herein, the term halogen means fluoro, chloro, bromo or iodo, preferably fluoro, chloro, dimethyl. The term &quot;deuterating agent&quot; as used herein means carboxylic acid anhydride, hydrazine halide or other An activated derivative. The term "anhydride" as used herein refers to a compound of the formula RC(0)-0-C(0)R, wherein r is as defined in the above paragraph. The term &quot as used herein "醯"" refers to the group RC(〇)x, where X is bromo or chloro. The term "activated derivative" as used herein refers to the initial activation of a compound in a desired chemical reaction. a transition-reactive form of a compound wherein the initial compound is only moderately reactive or non-reactive. Activation is achieved by the formation of a chemical group having a higher free energy content than the initial compound in the derivative or molecule, which enables activation The form is easier to react with another reagent. In the case of the present invention The activation of a carboxyl group is particularly important. The term oximeing agent as used herein additionally includes an agent which produces a carbonate 〇C(=0)〇R4 where R4 is as defined above. The term &quot;protecting group&quot; as used herein The protective reactive group does not participate in the undesired chemical reaction; and (b) the chemical group that is easily removed after the protection of the reactive group is no longer needed. For example, a trialkylsulfonyl group is a protecting group for the first sulfhydryl group and the acetonide is a protecting group for the diol. In the illustration of the compound given throughout this application, the substituent above the ring plane of 154377.doc -24· 201124141 is bolded (also represented by the oblique wedge bond below the ring plane to which the β asymmetric carbon belongs). The asymmetric carbon belongs to the representation) and the wedge-shaped bond of the dotted line represents the substituent (also denoted by a). The term "," or "combination therapy" as used herein refers to a plurality of drugs that are administered simultaneously or sequentially at the same time or at different times. As used herein, the term "chemically derived" An interferon molecule that is a polymer that alters the physical and/or pharmacokinetic properties of the interferon. A non-limiting list of such polymers includes a polyalkylene oxide homopolymer such as polyethylene glycol (PEG) Or polypropylene glycol (ppg), polyoxyethylated polyol (P〇ly0xyethylenated p〇ly〇1), copolymers thereof and block copolymers thereof, with the proviso that the water solubility of the block copolymer is maintained. The skilled artisan will appreciate a number of methods for linking polymers to interferons (see, for example, A. Kozlowski and JM Hards X Co„ir〇/.心/_ 2001 72(1-3): 217-24). A non-limiting list of chemically-derived IFNa encompassed by this patent includes peginterferon-&amp;2a (PEGASYS®) and polyethylene glycol interferon _a_2b (pEGINTR〇N 8). The compounds of formula I exhibit tautomerism. The tautomeric compound may exist in the form of two or more substances which are mutually converted. The proton mobile tautomer system is produced by the migration of a covalently bonded hydrogen atom between two atoms. Tautomers are generally present in equilibrium and attempts to separate individual tautomers typically result in a mixture of chemical and physical properties consistent with the mixture of compounds. The position of the equilibrium depends on the chemical characteristics of the molecule. For example, in many aliphatic aldehydes and ketones (such as acetaldehyde), the ketone type is dominant, while in phenol, the enol form is dominant. Common proton mobile tautomers include ketone/enol (-C( = 〇)_CH_〇_ 154377.doc -25- 201124141 C(-OH)=CH-), indoleamine/indenine (-C) (=0)-NH-E]-C(-0H)=N-) and hydrazine (-C(=NR)-NH-H1-C(-NHR)=N-) tautomer. The latter two are especially common in heteroaryl and heterocycles, and the invention includes all tautomeric forms of the compounds. Commonly used abbreviations include: acetonitrile (Ac), azo-bis-isobutyl decyl nitrile (AIBN), atmospheric (Atm), tert-butoxycarbonyl (Boc), di-tert-butyl pyrocarbonate or third Butoxycarbonyl anhydride (B0C20), benzyl (Bn), butyl (Bu), benzyloxycarbonyl (CBZ or Z), carbonyl diimidazole (CDI), 1,4-diazabicyclo[2.2.2 Octane (〇8-8::〇), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,8-diazabicyclo[5.4.0] eleven- 7-ene (DBU), N, N'-dicyclohexylcarbodiimide (DCC), 1,2-dioxaethane (DCE), dioxane (DCM), diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), diisobutylaluminum hydride (DIB AL or DIB AL-H), diisopropylethylamine (DIPEA), N,N-didecyl Indoleamine (DMA), 4-N,N-dimethylaminopyridine (DMAP), N,N-dimethyldecylamine (DMF), disulfoxide (DMSO), 1-(3-di Methylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), ethyl (Et), ethyl acetate (EtOAc), ethanol (EtOH), 2-ethoxy-2// - Quinoline-1-carboxylate (EEDQ), diethyl ether (Et20), six 0-(7-azabenzotriazol-1-yl)-N,N,N'N'-tetradecylhydrazine fluorophosphate (HATU), acetic acid (HOAc), 1-N-hydroxybenzotriazole (HOBt), high pressure liquid chromatography (HPLC), lithium hexamethyldiazepine (LiHMDS), decyl alcohol (MeOH), melting point (mp), MeS02-(sulfonyl or Ms), fluorenyl (Me), acetonitrile (MeCN), meta-benzoic acid (MCPBA), mass spectrometry (ms), mercapto-tert-butyl ether (MTBE), N-bromosuccinimide (NBS), N-carboxy anhydride ( NCA), N-154377.doc -26- 201124141 Gas amber imine (NCS), N-methylmorpholine (NMM), N-decylpyrrolidone (NMP), pyrchromic pyridinium (PCC) , dichromate pyrene (PDC), phenyl (Ph), propyl (Pr), ginger propyl (i-Pr), broken / square leaf (psi), ° ratio (pyr), room temperature (rt or RT), tert-butyldimethylmethylalkyl or ί-BuMe2Si (TBDMS), triethylamine (TEA or Et3N), 2,2,6,6-tetradecylpiperidine 1-oxyl (TEMPO), Trifluoromethanesulfonic acid or CF3S02-(Tf), trifluoroacetic acid (TFA), thin layer chromatography (TLC), tetrahydrofuran (THF), trimethylsulfonyl or Me3Si (TMS), p-toluenesulfonic acid Hydrate (TsOH or pTsOH), 4- Me-C6H4S02- or toluenesulfonyl (Ts), N-aminoformate-N-carboxy anhydride (UNCA). When used in the alkyl moiety, including the syllabary (Ό, 姜 (z·-), greedy, Nongsan (kN-), and the new well-known nomenclature have their conventional meanings (J. Rigaudy and DP Klesney) , TVowewc/aiwre //7 IUPAC 1979 Pergamon Press, Oxford.) The compounds of the present invention can be prepared by a variety of methods as described in the illustrative synthetic schemes shown and described below. Starting materials for the preparation of such compounds And reagents are generally available from suppliers such as Aldrich Chemical Co., or by methods known to those skilled in the art, according to the procedures described in the following reference, such as: Fiese/- /or Orgam'c iSyni/zesz'·?; Wiley &amp; Sons: New York, 1-21 ^ l KC LaRock, Comprehensive Organic Transformations * 2nd Edition Wiley-VCH, New York 1999 ! Comprehensive Organic B. Trost and I. Fleming (Editor Volumes 1-9 Pergamon, Oxford, 1991 ; Comprehensive Heterocyclic Chemistry, AR Katritzky and CW Rees (ed.) Pergamon, .Oxford 154377.doc -27- 201124141 1984 'Volume i-9' Comprehensive ffeterocyciic Chemis Try II, A. R. Katritzky and C. W. Rees (ed.) Pergamon, Oxford 1996, vol. 1-11, and wiiey &amp; Sons: New

York, 1991, vol. 40. The following synthetic reaction schemes are merely illustrative of some of the methods by which the compounds of the present invention can be synthesized, and various modifications can be made to these synthetic reaction schemes and such modifications can be suggested to those familiar with the disclosure contained in the present application. Technician. If desired, the starting materials and intermediates of the synthetic reaction scheme can be separated and purified using conventional techniques including, but not limited to, filtration, distillation, crystallization, chromatography, and the like. Such materials can be characterized using conventional methods including physical constants and spectral data. Unless stated to the contrary, the reactions described herein are preferably in the range of from about -78 C to about 150 ° C, more preferably from about 〇 ° c to about 125 ° C in an inert atmosphere at atmospheric pressure, and are most preferred. And suitably at a temperature of about room temperature (or ambient), for example about 20 °C. The chemical modification of the 2, and 3, positions of nucleosides has been studied, and the modification of the 4,_ position has been less popular, most likely due to the additional synthetic challenges associated with its synthesis.

Maag #A (Anti-HIVACtivity of 4, _Azid〇 and 4i· Methoxynucleosides, 乂Mec/ (: Xinshang· 1992 35:1440-1451) reveals 4'-azido-2-deoxyribonucleosides and 4_azido groups Synthesis of nucleosides. c O'Yang ' et al. ι992 33(1):37·4〇 and 33(1):41-44) reveals the synthesis of 4,-cyano, 4,-hydroxymethyl- and 4,_A a nucleoside substituted with a thiol nucleoside compound. These compounds have been evaluated as anti-hiv compounds. Maag et al. (supra) teach that 4,-azido-based nucleus 16c can be added to 5-methylidene-tetrahydro-furan-2- nucleoside 15 by azide 154377.doc •28·£ 201124141 iodine Wherein B is prepared as thymine, uracil, adenine or guanosine. An improved method for the preparation of 4,-azido nucleosides is disclosed in U.S. Patent Publication No. 2,5,382, issued to Feb. 17, 2005. Η

HO (15) -

RO j—16a: X = I; R = H I— 16b: X = 〇C(0)-w-C1-C6H4C1; R = c〇Ph * V — mT· r&gt; _ tt

16c: X = OH; R = H In WO 02/100415, R. Devos et al. disclose a novel 4'-substituted nucleoside derivative that inhibits the Hcv NS5B viral DNA polymerase. The addition of azide iodine is most effective on uridine 15 (B = uracil), and uridine 15 can be used by A_D. Borthwick et al. (/· you 乂199〇33(1):179 See also KJ Divakar and CB ReeSe J. Chem ~c·, / 1982 lm_1176) The method described is transformed into the corresponding mouth bite.

Process A

X HO HO r 18a: X = OH i9 18b: X = I Step i N f &gt;

PhCO rO— Me Step 4匚S:1 m-ac6H4C02- -CI-C^COj

Step 6 ^ ΛΛ.

HO 21 22 Use the procedure of Maag et al. (see above RCO's text) (Scheme A) 1-(4-Hydroxy-154377.doc •29- 23 201124141 5-Hydroxymercapto-3-indolyl-tetrahydro-furan 4-Amino-1 -pyrimidine-2,4-dione (18a) Preparation of 4-Amino-1-(5-azido-4-alkyl-5-ylaminomethyl-3-indole Base-tetrahydrofurfuryl-2-yl)-1Η-pyrimidin-2-one (22) (D. 0. Cicero et al., &quot;Stereoselective synthesis of novel analogs of 2'-deoxy- and 2*,3 '-dideoxynucleosides with potential antiviral activity&quot;, Bioorg. Med. Chem. Lett. 1994 4(7):861-6 i A. Iribarren, published on June 16, 1993 entitled &quot;Preparation of new (2'R )- and (2'S)-2'-deoxy-2'-C-hydrocarbyl antisense oligonucleotides useful in scientific research, therapeutics and diagnostics&quot; (EP547008 Al). In the cell-based replicon assay for HCV polymerase activity, 4-amino-1-(5-azido-4-alkyl-5-ylamino-3-methyl-tetrahydro-cod Indole-2-yl)-1 -pyrimidine-2-one (22) exhibited good activity (Table I). In addition, the compound exhibited a low degree of cytotoxicity in the assay. Table I - Maximum inhibition of HCV polymerase by compound / 〇 (100 _ maximum cytotoxicity % (100 _ 22 98.58 9.48 nucleoside derivatives are often effective antiviral agents (eg HIV, HCV, herpes simplex, CMV) and anticancer chemistry Therapeutic agents. Unfortunately, their practical utility is often limited by two factors. First, poor pharmacokinetic properties often limit the absorption of the digestive tract and the intracellular concentration of nucleoside derivatives, second, suboptimal physics Characteristic limitations can be used to enhance the choice of formulation for delivery of active ingredients. Albert introduces the term prodrug to describe a compound that lacks intrinsic biological activity but is capable of converting to an active drug by 154377.doc -30- 201124141 (A. Albert, Se/ecHve Tox /c/i, Chapman and Hall, London, 1951). Recent prodrugs have been reviewed (P. Ettmayer et al., / Med C/zew. 2004 47(10): 2393-2404; Κ· Beaumont et al. , Curr. Drug Me tab. 2003 4:461-485 ; H. Bundgaard, Design of Prodrugs: Bioreversible derivatives for various functional groups and chemical entities in Design of Prodrugs, H. Bundgaard (ed) Elsevier Science Publishers, Amerst Erdam 1985; G. M. Pauletti et al. Jdv. Drug Deliv. Rev. 1997 27:235-256; RJ Jones and N. Bischofberger, 1995 27, 1-15 and CR Wagner et al., Med· Λα. 2000 20: 417-45) Metabolic transformation can be catalyzed by specific enzymes (often hydrolases), and active compounds can also be regenerated by non-specific chemical methods. Pharmaceutically acceptable prodrugs refer to metabolism in the host (eg hydrolysis or oxidation) A compound which forms a compound of the present invention. Biotransformation should avoid formation of fragments having a toxicological tendency. Typical examples of prodrugs include compounds having a biolabile protecting group attached to a functional moiety of the active compound. Alkylation, thiolation or other lipophilic modification of the hydroxyl group on the sugar moiety is used. These normal nucleotides can be hydrolyzed or dealkylated in vivo to form the active compound. The factors limiting oral bioavailability are often Gastrointestinal absorption and excretion of the digestive tract and liver for the first time. The intercellular absorption through the gastrointestinal tract preferably requires a D greater than 0 (7.4). However, the optimization of the partition coefficient does not guarantee success. Prodrugs may have to avoid active efflux transporters in intestinal epithelial cells. Intestinal 154377.doc -31 - 201124141 Intracellular metabolism in the skin, which can lead to the passive transport or metabolite transport of metabolites through the runoff; Prodrugs must also resist unwanted biotransformation in the blood prior to the t cell or receptor. Although it is sometimes possible to rationally design a chemical modification based on the chemical functional groups present in the molecule, the chemical modification of the active compound can produce a complete physical, chemical and biological property that does not exist in the parentified mouth. Novel knife body. The multiple pathways in the right produce multiple metabolites, which can cause challenges. Because of &amp;, the identification of prodrugs remains an inaccurate and challenging practice. In addition, s flat evaluation of the pharmacokinetic properties of possible drugs may be challenging and expensive. The pharmacokinetics of the automated model can be difficult to extrapolate to humans. In U.S. Patent No. 6,846,81, issued Jan. 25, 2005, J A. Martin et al. have shown that deuterated 4,-azido nucleosides have been found to be effective prodrugs. The 22 bisindenyl derivative 23 can be prepared by the deuteration reaction of the parent nucleoside 22. The compound of the present invention is suitably prepared in one step in an aqueous organic solvent by a hydrazine reaction of 22. The solvent can be a homogeneous aqueous solution or a two phase solution. The pH of the aqueous organic solvent is maintained at 7.5 or more by neutralizing the acid generated by the deuteration reaction by adding a base. The base can be an alkali metal hydroxide or a third amine. The reaction is carried out in the presence of DMAP known in the art as a catalyst for the deuteration reaction. One of the advantages of this method is that the desired product can be obtained in the absence of a deuteration reaction of the heterocyclic base. Alternatively, the deuteration reaction is suitably between -20 and 200 ° C, but preferably at a temperature of _ 10 and 160 ° C, such as DCM, chloroform, carbon tetrachloride, B.

-32- 154377.doc S 201124141 、—% alkane, benzene, toluene, MeCN, DMF, sodium hydroxide solution or ring-turned stone wind L Φ I heart in the case of 'in the case of inorganic or organic base Corresponding brewing or liver-+p y_ Figure 4 needles into the sputum. The deuteration reaction can also be carried out in a two-phase organic-aqueous medium in the presence of a phase transfer catalyst and DMAp under Schotten Baumann conditions. Selective deuteration of the hydroxyl group can be achieved. Alternatively, the N-branched group of N, 〇, 〇_triterpene nucleosides can be selectively decomposed by desert zinc to form protected dimercapto compounds (R. Kierzek et al. TWra/ze office on leif. 1981 22 (38): 3762- 64). The selective deuteration of a particular hydroxyl group on the carbohydrate group can be conveniently achieved by an enzymatically catalyzed deuteration or desulfurization reaction. Enzyme catalysis provides mild selection conditions for organic conversion. S. M. Roberts has reviewed preparative biotransformation (J. C/zem. 5W. 7, 2001, 1475; 2000 611; 1999, 1 and 1998 157). M. Mahmoudian et al., Woiec/mo, but p/. 1999 29:229-233) reported the use of Novozyme 435, an immobilized preparation of Candida antarctica (IV) aricifca), to selectively brew 2-amino- 9-0-D-Arabid bite 5'-position of the south glycosyl-6-methoxy-9H-β ticket. Other enzymes reported to selectively degenerate 5,-hydroxyl groups include: Bacillus licheniformis (Saci/Zwi Hc/zem/orw/??) protease, immobilized lipase IM (Lipozyme IM, immobilized M.) Mwcor mie/iei) lipase), CLEC-BL (Bacillus licheniformis), Savinase (Savinase, Bacillus (5α(7!7/Μ·ί protease), Novozyme-243 (Bacillus licheniformis) Protease), Z/ca/z'gewes sp. Lipase and Lipolipase (lipoase, Novo). 154377.doc -33- 201124141 Discovered Lipolase® Enzyme Preparation (from Thermophilic Fungus/awwgiwosw ·?) lipase, Sigma Catalog No. L 0777) selectively hydrolyzes the 5'-mercapto group of the tridecyl derivative to give a 2',3'-didecyl compound. In WO 2004043894, GG Heraldsson et al. The use of thermophilic fungal lipase for the esterification of marine oils. N. Weber et al. (five w. «/. 〇/iScz·. rec/mo/. 2003 105(10): 624-626) reveals hobby The transesterification of methyl oleate catalyzed by thermal fungi. V. Bodai et al. (04dv. A. Cai. 2003 345 (6 and 7): 811-818) describes selectivity Novel hydrolase from thermophilic filamentous fungi. Other reports of position-selective enzymatic ester hydrolysis include: R. Hanson et al. A 5 Bioorg. and Med. Chem. 2000, 2681-2687 (synthesis of a lobucavir prodrug Via regioselective acylation and hydrolysis); R. Pfau et al., Xe&quot; 1999, 1817-1819 (selective hydrolysis of carbohydrate ester); A. Bianco et al., /.〇/ Mo/. C&lt;3i. 5 . 1997 209-212 (regioselective ester hydrolysis and hydrolysis for synthesis of sialic acid derivatives); Y. Ota et al, Bioscience, Biotechnology, Biochemistry (1997), 166-167 (regioselective ester hydrolysis of l, 2,3-tHhexanolylglycerol); UT Bornscheuer et al., Enzyme Microbial Technol. 1995, 578-86 (lipase catalyzed syntheses of monoacylglycerol; review); CT Goodhue et al. WO 9403625 (regioselective process for resolution of carbohydrate monoesters); NW Boaz, WO 9115470 (Separation of alcohol-ester Mixture by selective enzymatic hydrolysis);

S 154377.doc -34- 201124141 YS Sanghvi et al US2002142307 (regioselective hydrolysis of 3', 5'-di-0-levulinylnucleosides); J. Garcia et al. Org. Chem. 2002, 4513-4519 (regioselective hydrolysis of 3' , 5'-di-O-levulinylnucleosides); O. Kirk et al.

Biotransformation (1995) 91-7 (lipase catalyzed regioselective acylation and deacylation of glucose derivatives). Those skilled in the art will recognize that selective acetification can also be achieved by standard chemical methods. The selective protection of the 5,-hydroxyl group (which allows direct esterification of the 2,-hydroxyl group) or the incorporation of a second protecting group (which allows deprotection and selective brewing of the first alcohol) has been described. The compounds of the present invention can be formulated into a variety of oral dosage forms and carriers in the form of lozenges, coated lozenges, hard and soft gelatin capsules, solutions, emulsions, sugars or beta liquids. In the administration route, the compound of the present invention is effective when administered by a suppository administration form. The most convenient way to administer medication is usually oral administration using a convenient daily dosing regimen that can be adjusted depending on the severity of the disease and the patient's response to the antiviral drug. The compounds of the present invention, as well as the pharmaceutically acceptable salts thereof, are placed in the form of a pharmaceutical composition together with one or more excipients, carriers or diluents. The pharmaceutical compositions and unit dosage forms are in the form of conventional ingredients in a known ratio, and the active ingredient in unit dosage form. These medicines, suitable for effective solids, semi-solids, etc. 戍 ° Liquids such as tablets or filled emulsions, continuous release formulations or fillers such as suspensions, limbs - sputum for oral use壹·, sac, or for rectal or vagina 154377.doc •35· 201124141 Two pieces of I form use. A typical preparation will contain from about to about % by weight of activated u (w/w). The term &quot;preparation&quot; or &quot;dosage form&quot; is intended to include the active compound(s) and liquid formulations and those skilled in the art will be aware that the active ingredient may be present in the agent (4) depending on the desired dosage and the kinetic parameters of the music. The term "excipient" as used herein refers to a compound that is used to prepare a pharmaceutical composition and is generally safe and non-toxic and neither biologically nor otherwise undesirable, and includes both Beast H materials and human (4) (iv) acceptable routes. Excipients. The compounds of the present invention can be administered alone, but are usually administered in admixture with the choice of the desired standard pharmaceutical practice - or a plurality of suitable pharmaceutical excipient diluents or carriers. The &quot;pharmaceutically acceptable salt&quot; form of the active ingredient may also initially impart the desired pharmacokinetic properties of the active f-inhibited deficiency in the non-salt form, and may even positively affect the active ingredient for its in vivo treatment. The pharmacodynamics of activity. As used herein, a pharmaceutically acceptable salt, &quot;pharmaceutically acceptable salt&quot; means a salt that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. These include: (1) acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or with such as glycolic acid, pyruvic acid, lactic acid, malonic acid , fruit acid, cis-succinic acid, anti-succinic acid, tartaric acid, citric acid, 3-(4, benzyl benzoyl) benzoic acid, cinnamic acid, mandelic acid, f sulfonic acid, ethanesulfonic acid, 1&gt ; 2 · ethane · disulfonic acid, 2 - hydroxyethyl acid, benzene acid, 4. benzene acid, 2 · naphthoic acid, 4 - toluene acid, camphor acid, lauryl sulfate, gluconic acid An acid addition salt formed by an acid of a face acid, a salicylic acid, a sticky acid or the like. It is to be understood that all pharmaceutically acceptable salts include the same acid addition salts as defined herein. 154377.doc • 36 - s 201124141 Additive form (solvate) or crystalline form (polymorph). Solid form preparations include powders, troches, pills, capsules, suppositories, and dispersible granules. The solid carrier can be one or more substances which may also act as a diluent, a flavoring agent, a solubilizer, a lubricant, a suspending agent, a binder, a preservative, a tablet disintegrating agent or an encapsulating material. In the form of a powder, the carrier is usually a finely divided solid which forms a mixture with the finely divided active component. In the tablet form, the active ingredient will usually be mixed in a suitable proportion of the carrier with the necessary binding capacity and compressed into the desired shape and size. Suitable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar 'lactose, pectin, dextrin, powder, gelatin, xanthine, methylcellulose, m-methylcellulose sodium , low melting wax, cocoa butter and the like. The solid form preparation may contain, in addition to the active ingredient, a coloring agent, a flavoring agent, a stabilizer, a buffering agent, an artificial and natural sweetener, a dispersing agent, a thickening agent, a solubilizing agent, and the like. Liquid formulations which are also suitable for oral administration include liquid formulations comprising emulsions, syrups, ugly and aqueous suspensions. This includes the solid form of the liquid form preparation to be converted into a liquid form preparation immediately before use. The emulsion can be prepared, for example, in a solution in an aqueous propylene solution, such as lecithin, sorbitol oleate or gum arabic. 'Difficult suspending liquid can be finely powdered by using a viscous substance such as sac or synthetic rubber, 嵩tm Λ 曰 曰, methyl cellulose, sodium carboxymethyl sulphate and other well-known suspension heat squats. The active ingredient is prepared by dispersing in water. The compound of the present invention can be administered by adjusting the fatty acid glyceride or cocoa butter by stirring the active ingredient in an assay form. The low melting wax such as a mixture is first melted and, for example, smeared. The melted homogeneous mixture 154377.doc -37- 201124141 is then poured into a convenient size mold which is allowed to cool and solidify. The compounds of the invention may be formulated for vaginal administration. Carriers other than the active ingredient, such as pessaries, hemostatic cotton balls, creams, gels, pastes, foams or sprays, are also suitable in the art. Suitable formulations, together with pharmaceutical carriers, diluents and excipients, are described in the literature: 77ze iVaciz.ce 1995, Ε. W. Martin Editor, Mack Publishing

Company's 19th edition, Pennsylvania. Skilled blending scientists may modify the formulations in the teachings of this specification to provide a variety of formulations for the particular route of administration without destabilizing or compromising the therapeutic activity of the compositions of the present invention. Modification of the compounds of the invention renders them more soluble in water or other vehicles. The modification can be readily achieved by, for example, minor changes (e.g., salt formulations)&apos; which is well within the ordinary skill. It is also within the ordinary skill to alter the route of administration and dosing regimen of a particular compound in order to control the pharmacokinetics of the compounds of the invention to maximize benefit to the patient. The term &quot;therapeutically effective amount&quot; as used herein means the amount required to reduce the symptoms of a disease in an individual. The dosage can be adjusted according to individual needs in each specific case, such as the severity of the disease to be treated, the age and general health of the patient, other agents used in the treatment of the patient, the route and form of administration, and the involved &lt;opening The doctor's preferences and experience vary widely. For oral administration, a daily dose of between about G1 and about 1 G per day should be appropriate in monotherapy and/or combination therapy. Preferably, the daily dose is between about 0.5 and about 7-5 g per day, more preferably between 15 and about 154377.doc -38 · 201124141 per day. In general, treatment begins with a large initial &quot;shock dose, begins with a rapid reduction or elimination of the virus, and then reduces the dose to a level sufficient to prevent recurrence of the infection. For the treatment of sputum diseases and patients, the general technique for treating the diseases described herein. Those skilled in the art will be able to determine the therapeutically effective amount of the compound of the present invention by relying on personal knowledge, experience, and the disclosure of this application (4). Therapeutic efficacy can be determined from liver function tests including, but not limited to, serum proteins (eg albumin, coagulation +, alkaline phosphatase, aminotransferase (eg alanine transaminase, aspartate) Turn (4)), & ribozyme, γ-fascinamide-based transpeptidase, etc.), egg synthesis, synthesis of cholesterol and synthesis of cholic acid; liver metabolism functions including (3; 4) water compound metabolism , amino acid and ammonia metabolism. Alternatively, the therapeutic effect can be monitored by measuring HCV_RNA. The results of such tests are: In the examples of the present invention, the active compound or salt may be combined with another antiviral agent such as ribavirin, another nuclear HCV polymerase inhibitor, Hcv#, a polycapase. Inhibitors, HCV protease inhibitors, helicase inhibitors or HCV fusion inhibitors are administered in combination. When the active compound or a derivative or salt thereof is administered in combination with another antiviral agent, the activity may increase beyond the parent oxime. Things. Although the treatment is a combination therapy, the administration can be carried out simultaneously or continuously with respect to the nucleoside derivative. Thus, as used herein, simultaneous administration, including administration of such agents at the same time or at different times. Administration of two or more than two agents at the same time may be by a single formulation containing two or more active ingredients or by administering two or more dosage forms having a single active agent substantially simultaneously. Achieved. I54377.doc -39- 201124141 It should be understood that the treatments mentioned herein extend to the prevention and treatment of existing conditions. Furthermore, the term "treatment" of HCV infection as used herein also includes treating or preventing a disease or condition or clinical condition associated with or mediated by HCV infection. Example 1 Step 1 · 18a (2.17 g, 8.96 mmol, 1 eq.), sodium saliva (732 mg, 10.7 mmol, 1.2 eq.) and ph3p (2.82 g, 1 〇7 mm 〇1, i 2 eq.) in anhydrous The solution in THF (30 mL) was cooled in EtOAc EtOAc (EtOAc) The reaction mixture was stirred for an additional 1 Torr at 〇_5. The ice water bath was removed and the reaction mixture was stirred at RT for 7 〇 h. The reaction mixture was diluted with DCM (200 mL) and washed with EtOAc EtOAc. The aqueous layer was washed with DCM (4×5 () mL). The combined organic extracts (NaJO4) were dried, transitioned and concentrated. The residue was purified by EtOAc/EtOAc (EtOAc) elut elut elut elut elut elut elut eluting eluting eluting s, 1H), 7.59 (d, 1H), 6.19 (d, 1H), 5.75 (dd, 1H) ' 3·79 (m, 1H), 3.65 (m, 1H), 3.57 (dd, 1H) ' 3.47 (dd,1H) ' 2.69 (m,1H), 2.13 (d, 1H), 1.00 (d, 3H) Step 2 - 18b (1.84 g, 5.22 mmol) and 0.4 M sodium methoxide in MeOH (81 mL) The solution was stirred at 60 ° C for 5 h and cooled on an ice-water bath. The sulphuric acid form of DOWEX H+ was added portionwise (by treating DOWEX H+ with pyridine (10 mL/g resin), filtering and using MeOH before use Wash to 154377.doc •40- 201124141) until the pH of the solution is neutral (5-6 g total). Remove the ice water bath and stir the mixture for 5 minutes at rt. Remove the resin by filtration and use Me〇 H (1 〇〇 mL) was washed. The residue was slurried in 6% EtOH/DCM and applied to a column of 〇 2 and eluted with EtOH/DCM gradient (6-7% Wv EtOH) to give 0.942 g ( 80°/〇) 19, which is pure enough for use in the next step. Step 3 - Rat benzyl triethylene glycol (1.91 g, 8.4 mmol) , 2 equivalents) and sodium azide (546 mg, 8.4 mmo, 2 equivalents) were suspended in anhydrous MeCN (32 mL) and sonicated for several minutes. The resulting fine suspension was mixed for 3 h at RT and then Filtration to a solution of compound i9 (942 mg, 4.2 mmol, 1 eq.) in anhydrous THF (30 mL). After cooling on a water bath, iodine (1 &gt; 81 g, 7.14 mmol, 1.7 eq.) in THF (39 mL). Add 7V_acetamido_L_cysteine (69 mg, 〇〇35 mmol, 0.1 eq.) and stir the solution until the bubbles subsided. Add NMM (2'31 ml ' 21.0 mmM, 5 equivalents) and DMAp (513 Mg, 4 2 mmol '1 eq.), followed by dropwise addition of a clumpy chlorobenzene (1), 9 24 mmol, 2.2 eq. p. TLC and LC_MS analysis showed complete reaction. Add MeOH (5 mL) and after a few minutes the solvent was concentrated on a rotary evaporator to a half volume and then a solution of 〇1M Na2S2 〇3 in saturated aqueous NaHCCh (3 〇〇mL) was added with stirring and the mixture was warmed To the feet. The mixture was extracted with DCM (150 mL) and EtOAc (EtOAc &lt; The organic phase was extracted with citric acid and the aqueous phase was washed twice with DCM (2×50 ml). The DCM extract was dried (NazSCU), filtered and concentrated in vacuo. The residue was eluted with gradient EtOH/DCM (0, 0.5, 0.75, 1.0) , 1.5 and 2.0 ° / EtOH), eluted by Si 〇 2 chromatography to obtain l72 g (83%) 20a: W-NMR data (CDC13, 25 ° C): δ 8.22-7.46 (7 Η) ' 6.49- 6.39 (1Η), 5.84 (dd, 1Η), 5.55-5.47 (1Η), 3.85 (d,1H) ' 3.74 (d,1H), 3.18 (m,1H), 1.09 (d,3H). Step 4 - A solution of compound 20a (1.72 g, 3.47 mmol, 1 eq.) in EtOAc (EtOAc EtOAc (EtOAc) The mixture was combined in a 1.75 M K2HP 〇4 aqueous solution (55 mL). The two-phase system was vigorously stirred at RT and two portions containing 55% MCPBA, 10/methane benzoic acid were added at 1.5 h intervals. And 35% H20 (2x3.57 g A commercially available reagent mixture equivalent to 2x16.5 mmol or 2x3.28 equivalents of MCPBA to 2x3.3 mmol or 2x0.66 equivalents of benzoic acid. The mixture was stirred vigorously at RT for 18 h. LC-MS analysis showed &lt;96°/. Reaction. Add a solution of Na2S203.5H20 (35 g) in EtOAc (EtOAc (EtOAc) The aqueous layer was washed with aq. aq. EtOAc (EtOAc) (EtOAc). (1-2% v/v EtOH) was dissolved and purified by SiO 2 chromatography to give 1 g (56%) 20b: W-NMR (CDC13, 25 ° C): δ 8.19 (br s, 1 Η), 8.07- 7.88 (4Η), 7.65-7.36 (6Η), 6·57·6·45 s 154377.doc -42- 201124141 (1H) ' 5.64 (dd,1H), 5.51-5.42 (1H), 4.80 (m, 2H ), 3.24 (m, 1H), 1.09 (d, 3H). Step 5 - co-evaporate a 3a20b (100 mg, 〇. 19 mmol, 1 eq.) with 1,2,4 oxadiazole (131 mg, 1.9 mm 〇i, 10 eq.) from anhydrous pyridine and redissolve in anhydrous In pyridine (1 mL). The solution was cooled in an ice water bath, and a solution of &lt;RTI ID=0.0&gt;&gt; The reaction mixture was further stirred at 0-5 ° C for 5 minutes and then stirred at RT for 3 h. The reaction mixture was concentrated on a rotary evaporator to a half volume &lt;&lt;&gt;&gt;&gt;&lt;&apos;&gt;&gt; The residue was evaporated to dryness eluting with EtOAc EtOAc EtOAc (EtOAc) eluting 2.0 ° /. The impurity is 2'-α isomer. This reaction is repeated with 90 + 0 mg of compound 20b. After chromatography, 270 mg (54%, 97% purity) of 22:4· NMR (DMSO-d6, 25〇C) : δ 7.69 (d,1H), 7.14 (d,2H), 6·32 (br s,1H), 5.72 (d, 1H), 5.78 (br s,2H), 3.89 (br s, 1H), 3.74 (d, d, d, 2H), 2.54 (m, 1H), 0.77 (d, 3H). Example 2 Isobutyric acid (2 feet, 38, 48, 51^)-5-(4- Amino-2-sided oxy-2//~*» dimethyl-1-yl)-2-isobutyl decyloxy fluorenyl-2-allotyl-4-indenyl-tetrahydro-furan-3 -ester (25)

154377.doc -43- 201124141 22 (0.700 g, 2.48 mmol) in THF (7 mL) and brine (7 mL)

The pH of the solution in the solution was adjusted to approximately u with a dilute aqueous KOH solution. Slowly (dropwise) Isobutyl hydrazine chloride (1 · 〇 g) was added to the ice-cold stirred biphasic reaction mixture while maintaining the 1) 11 value at about 11 by adding a dilute K 〇 H aqueous solution if necessary. The extent of the reaction was monitored by HPLC. Adding an additional 1 quantity of 异 醯 醯 under Ηριχ means near complete conversion. The reaction mixture was allowed to stand at RT overnight. The solution was diluted with EtOAc (50 mL) and the pH of aqueous was adjusted to approximately 7.5 with concentrated HCl. The phases were separated and the organic phase was washed 3 times with water and evaporated to dryness to afford 25. Example 3 Valeric acid (2R, 3S, 4S, 5R)-5-(4-amino-2-yloxy-2H-branch-1-yl)_ 2-azido-4-hydroxy-2- Hydroxymercapto-tetrahydrofuran_3_ester (27)

Thermophilic fungal lipase phosphate buffer

To divaleric acid vinegar 26 (R&quot;=«-C4H9, 1.9 g, 3.46 mmol) in MTBE (13 mL) and phosphate buffer (15 mL, adjusted to approximately 6 5 of 5 mM sodium phosphate and 0.1 M (about 2 mL) Lipolase® (lipase from thermophilic fungi, Sigma catalog number L 0777) was added to the suspension in NaCl). The reaction mixture was warmed to 35. (: and stir for 2 h. By adding

NaHC 3 maintained the pH of the reaction mixture at 6.5. After 2 h, the reaction was carried out until completion of 8%. Add an additional 2 mL of Lipolase® and continue to mix for 6 h, • 44 - 154377.doc After 201124141 add an additional 2 mL of the aliquot of the enzyme and stir the reaction for an additional 24 h. To the solution were added acetone (10 mL), MTBE (20 mL) and brine (10 mL) and the reaction warmed to 50 °C. The phases were separated and the organic phase was extracted twice with warm MTBE. The combined organic phases were washed twice with hot brine and dried (Na2EtOAc) Example 4 Tetradecanic acid (211,3 8,48,511)-5-(4-amino-2-oxo-211-pyrimidin-1-yl)-2-azido-3-butanyloxy -4-mercapto-tetrazine-β-furan-2-yl group (28)

JiH2 c13h27cooch=ch2 HO Me Candida antarctica lipase

28: R&quot; = C13H27 29 29 (1.0 g, 3.52 mmol), tetradecanoic acid from the purpose (1.2 g, 4.57 mmol), Candida antarctica lipase immobilized on polyacrylate resin (0.30 g The suspension from Novosome, Sigma catalog number L4777) and THF (20 mL) was warmed to 60 ° C overnight. HPLC analysis indicated that the reaction was about 33% complete and an additional 2.4 mL of vinyltetradecanoate and 0.3 g of lipase were added. After an additional 48 h, the reaction was complete 50% and an additional 0.3 g of enzyme and 3 mL of vinyltetradecylate were added. After about 80 h (total reaction time), the conversion to the monoester was complete. The crude reaction mixture was filtered through CELITE® and the filter pad was washed with THF. The combined organic phases were evaporated. The residue was dissolved in MeOH (50 mL)EtOAc. The decyl alcohol solution was evaporated and the residue was dissolved in 154377.doc •45- 201124141

The mixture was washed with EtOAc (EtOAc EtOAc (EtOAc)EtOAc. Purification by chromatography on Si02. Example 5 3',5'-〇-bis(L-proline)-4'-azidodecyl-2·-deoxycytidine nucleoside (34)

#4-[(Dimethylamino) fluorenylene]-4·-azido-HC-mercapto-2,-deoxycytidine nucleoside (32) » dimethylformamidine at room temperature A solution of 22 (1.81 g, 6.42 mmol) in DMF (30 mL). The solution was evaporated under reduced pressure and co-evaporated with ethanol. The title compound 3 2 was added from the ethanol/accuracy crystal. 3',5Ά-bis[ΛΓ·(Tertibutoxycarbonyl)_L_valeric acid group]_#4_[(dimethylamino)methylene]-4,-azido-2,- , C-methyl-2,-deoxycytidine nucleoside (33). Boe VabOHQ 62 g, 7 48 154377.doc -46· 201124141 mm〇1) was continuously added to a solution of 32 (1.26 g, 3.74 mmol) in a mixture of anhydrous acetonitrile (3 〇mi) and DMF (15 ml). EDC (1.43 g, 7 48 _〇1), TEA (1 〇 4 eg, 7 μmmol) and DMAP (0.1 g). The resulting mixture was stirred at room temperature. The progress of the reaction was followed by HPLC and the reaction mixture was further fed with 〇H (0.63 g) ^ EDC (〇.72 g) &gt; TEA (0.52 ml) ^ DMAp (〇 5 g). After the starting material was completely consumed, the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate and washed with water and brine. Purification by hydrazine column chromatography (gradient 5-40% EtOAc in hexanes) afforded the title compound 33 〇3,5'-0-bis(L-proline) 4'- Azido-2'-y9-C-methyl-2,-deoxycytidine nucleoside (trihydrochloride, 34). A solution of 13 ml of 1 M HCl in EtOH was slowly added to a concentrated solution of 33 (1_6 g, 2.17 mmol) in EtOH. The reaction mixture was stirred at room temperature for 4 h and diluted with diethyl ether. The precipitate was filtered and washed with ether to afford title compound 34 as a tris. Example 6 3·, 5'-&lt;9-bis(is〇butiryl)-4·-azido-2,yC-methyl_ 2'-deoxycytidine Preparation. (isobutyric acid (2R,3S,4S,5R)-2-(4-amino-2-oxo-oxy-2//-pyrimidinyl)-2-isobutylthiooxycarbonyl-2-azide Base 4-mercapto-tetrahydrofuran_3_ester) (25).

154377.doc •47- 201124141 3·,5·-0-bis(isobutylmethyl)_4,·azido-2,_々_c•mercapto-2,-deoxycytidine nucleoside (25). 22 (1.26 g, 3.74 mmol) in anhydrous at 0C. Isobutyrate liver (1.77 g, 11.2 mmol) was added to the solution in the bite (25 ml). The reaction was followed by HPLC and the reaction was quenched with water when complete to discard excess isobutyric anhydride and removed #4. The solvent was evaporated under reduced pressure and co-evaporated with ethanol. The residue was dissolved in ethyl acetate and washed with NaHC03 brine. Purification of the title compound 25 by hydrazine column chromatography (gradient 10-40% EtOAc in hexanes). Example 7 4'-azido-3'-indole-(L-prolinyl)-2,-,C-mercapto-2,-deoxycytosine nucleoside (37)

N-BooVal-OH, EDC, TEA, DMAP, MeCN/DMF

iV4'5'-0-bis(monomethoxytriphenylmethyl)_4, ·azido-2, broad c_mercapto-2'-deoxycytidine nucleoside (35). s 154377.doc -48- 201124141 At 80° (: 22 (2.82 lv, 10 111111〇1) and ]^河1&gt;(:1(9.15§, 30 mmol) in the ratio. (50 ml) The mixture was stirred overnight. MeOH (5 mL) was evaporated and evaporated and evaporated. Chromatography (0-5 ° / MeOH in DCM) afforded the title compound 35 ° #4,5·-0-bis(monodecyloxytriphenyl)-4'-azido-3 '-[7V-(Tertibutoxycarbonyl)-L-proline)]-2'-,C-mercapto-2'-deoxycytidine nucleoside (36). To 35 (3.09 g, 3.74 mmol) Boc-Val-OH (0.8 1 g, 3.74 mmol), EDC (0.72 g, 3.74 mmol), hydrazine (0) was added continuously to a solution of a mixture of anhydrous acetonitrile (30 ml) and DMF (15 ml) • 52 ml, 3.74 mmol) and DMAP (0.07 g). The resulting mixture was spoiled at room temperature. The progress of the reaction was followed by HPLC and re-fed into the reaction mixture: 8 〇〇¥31-OH (0.4 g ), EDC (0_36 g), TEA (0.26 ml), and DMAP (0.04 g). When the starting material was completely consumed, the solvent was removed under reduced pressure. The residue was taken up in EtOAc EtOAc (EtOAc m. '-0-(L-Proline)-2'-yS-C-indolyl-2·-deoxycytidine (dihydrochloride, 37). 36 (2.4 g, 2.34 mmol) A solution of 13 ml of 1M HCl in EtOH was added slowly EtOAc EtOAc (EtOAc) 37). Example 8 154377.doc -49- 201124141 4 - Azido-3'-indole-isobutyl hydrazine 曱 · 2, _ deoxycytidine nucleoside (38) 〇

4'-azido3,-〇-isobutanyl 2,_,c_methyl_2,_deoxycytidine (38) 〇C under 35 (3.09 g, 3.74) Methyl) Isobutyric anhydride (〇89g, 561 mmol) was added to a solution of anhydrous pyridine (25 ml). The reaction was traced by hplC and when complete, the reaction was quenched with water to discard excess isobutyric anhydride. The solvent was evaporated under reduced pressure and co-evaporated with ethanol. The residue was dissolved in ethyl acetate, washed with NaHC EtOAc, brine and evaporated. The crude MMTr protected 3'-isobutyl decyl derivative was dissolved in 80% AcOH at 5 〇〇c and stirred until the protection of the MMTr group was removed. The solvent was evaporated and the residue was purifiedjjjjjjlililililililili Example 9 5Ά-α-lysine group)_4,_azido group_2,_,c_methyl_2,·deoxycytidine nucleoside (43) 154377.doc •50· 201124141

Vr? HO Me

twenty two

5'-0-Tertiary butyl fluorenyl-4 --azido-2'-y5-c, methyl-2I-deoxycytidine nucleoside (39). To a solution of 22 (2.82 g' 10 mmol) in DMF (5 ml) was added MeOH (1. 〇2 g, 15 mmol) and TBSC1 (1.95 g, 13 mm 〇1). When the starting material was consumed, the reaction mixture was quenched with MeOH (1 ml) and partitioned between ethyl acetate and water. The organic phase was washed with water, brine, and evaporated to dryness crystals 5 Ο-Second butyl fluorenyl sulphide _4'_azido bis(monodecyloxytrityl)-2,-,C-methyl-2·-deoxycytidine nucleoside (40). A mixture of 39 (3.7 g </ RTI> 9.34 mmol) of iMMTrCl (8.63 g, 28 mmol) in anhydrous pyridine (5 mL) was stirred overnight. Add 154377.doc -51 - 201124141

The MeOH (5 ml) was stirred and the mixture was stirred. The solvent was evaporated and the residue was partitioned between ethyl acetate and water. Purification of the title compound 40 by hydrazine column chromatography (0-5% EtOAc) 4'·azido-anthracene, 3,_〇_bis(monomethoxytrityl)_2_ lost c_methyl_2· deoxygenated cellulite (41) » at room temperature A solution of 40 (5.3 g, 5.64 mmol) in THF (20 mL). The reaction mixture was diluted with ethyl acetate brine, brine and evaporated. Chromatography (0-5% ethyl acetate in CHCH) gave the title compound 41 <RTI ID=0.0> (3 </RTI> <RTIgt; </RTI> <RTIgt; </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> Oxygen-based thiol) _4 · azidothiol-2·-deoxycytidine 核 核 (42) 〇 41 (3.09 g' 3.74 mmol) in anhydrous acetonitrile (30 ml) with DMF (15 Boc_Val-〇H (0.81 g, 3.74 mmol), EDC (0.72 g, 3.74 mmol), hydrazine (0.52 ml, 3.74 mmol) and DMAP (0.07 g) were continuously added to the solution in the mixture of ml. The resulting mixture was stirred at room temperature. The progress of the reaction was followed by HPLC and B 〇c_Val_ 〇H (0.4 g), EDC (0.36 g), TEA (0.26 ml) and DMAP (〇.〇4 g) were further fed into the reaction mixture. When the starting material is completely consumed, the solvent is removed under reduced pressure. The residue was dissolved in ethyl acetate and washed with water and brine. Purification by column chromatography (5-40% EtOAc EtOAc) 5'-0-(L-proline)-4·-azido-2'-y9-C-methyl-2'-deoxycytosine 154377.doc -52- 201124141 Nucleoside (II Hydrochloride, 43). A solution of 13 ml of 1 M HCl in EtOH was slowly added to a concentrated solution of 42 (2.4 g, 2.34 mmol) in EtOH. The reaction mixture was stirred at room temperature for 4 h and diluted with diethyl ether. The precipitate was filtered and washed with diethyl ether to give the title compound 43. Example 10 5·-0-Isobutylguanidino-4'-azido-2I-P-C-mercapto-2'-deoxycytidine nucleoside (44)

5'-〇-Isobutylguanidino-4'-azidodecyl-2'-deoxycytidine nucleoside (44). Isobutyric anhydride (0.89 g, 5.61 mmol) was added to a solution of 41 (3.09 g, 3.74 mmol) in dry water (25 ml) at EtOAc. The reaction was followed by HPLC and the reaction was quenched with water when complete to discard excess isobutyric anhydride. The solvent was evaporated under reduced pressure and co-evaporated with ethanol. The residue was dissolved in ethyl acetate, washed with NaHC03, brine and evaporated. The crude MMTr protected 3'-isobutyl decyl derivative was dissolved in 80% AcOH at 50 ° C and stirred until the protection of the MMTr group was completely removed. The solvent was evaporated and the residue was purified elut elut elut elut elut elut elut elut 154377.doc -53- 201124141 Example 11 Renii luciferase assay This assay quantifies the ability of a compound of formula I to inhibit HCV RNA replication and its potential for treating HCV infection. This assay utilizes a reporter as a simple reader for the intracellular HCV replicon RNA content. The Renilla luciferase gene was introduced into the first open reading frame of the replicon construct NK5.1 (Krieger et al., Hro/. 75:4614) immediately after the internal ribosome entry site (IRES) sequence. The self-cleaving peptide 2A from foot-and-mouth disease virus is fused with the neomycin phosphotransferase (NPTII) gene (Ryan &amp; Drew, EMBO Vol 13: 928-933). After in vitro transcription, RNA was electroporated into human liver tumor Huh7 cells and the anti-G418 community was isolated and expanded. The stably selected cell line 2209-23 contains a replicative HCV subgenomic RNA, and the activity of Renilla luciferase expressed by the replicon in the cell reflects its RNA content. The assay is performed in a double plate, one being opaque white and the other being transparent to measure the antiviral activity and cytotoxicity of the compound while ensuring that the observed activity is not due to reduced cell proliferation. Renilla luciferase HCV replicon cells (2209-23) cultured in Dulbecco's MEM (GibcoBRL Cat # 31966-021) with 5% fetal bovine serum (FCS, Gibco BRL Cat. No. 10106-169) at 5000 per well Cells were plated on 96-well plates and incubated overnight. After 24 hours, different dilutions of the compounds in the growth medium were added to the cells, which were then incubated for an additional three days at 37 °C. At the end of the incubation period, collect the cells in the white plate and use the dual luciferase reporter assay system (Promega catalog number 154377.doc -54- 201124141

Luciferase activity was measured in El960). All of the reagents described in the following paragraphs are included in the manufacturer's kit and are accompanied by the manufacturer's instructions for preparing such trials. The cells were washed with 2 μl of phosphate buffered saline (pH 7.0) (PBS) in a mother well and incubated for 2 min at room temperature after lysing the cells with 25 - &amp; passive lysis buffer. Add 1 liter of microliter LAR 1]; reagent to each well. The plate was then inserted into a Lb 96V micro-quantitative plate luminometer (MicroLumatPlus 'Berthold), and 1 μμ Stop & Glo® reagent was injected into each well and the signal was measured using a 2 second delay, 1 second measurement program. . ICw is the concentration of drug required to reduce the replicon content of 50% associated with untreated cell control values, as calculated from the percent reduction in luciferase activity versus drug concentration. The WST-1 reagent from Roche Diagnostic (catalog number 1644807) was used for cytotoxicity assays. A microliter of WST_丨 reagent was added to each well, including wells containing only medium as a blank. The cells were then incubated at 37 °C for 1 to 1.5 hours' and the 0D values were measured by a 96-well plate reader at 45 〇 nm (reference filter at 650 nm). Furthermore, cc50 is a drug concentration required to reduce cell proliferation by 〇% associated with untreated cell control values, which can be calculated from a plot of the percentage reduction in WST-1 value versus drug concentration. EXAMPLE 12 MT4/XTT Assays The compounds of the invention may also be assayed for activity against concurrent infections, e.g., individuals at risk of a gold-borne infection such as HCV are sometimes infected with HIV. For example, 'XTT is used in a variety of assays in MT-4 cells to characterize the HIV activity of a compound (Weislow et al, J Nat Cancer Inst 1989, 81 154377. doc-55-201124141, Vol. 8, 577 and the like, etc. Preferably, an assay in the presence of 40-50% human serum is included to account for the effect of protein binding. Briefly, a typical XTT assay uses human T cell line MT4 cells grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (or, if appropriate, 40-50% human serum), penicillin and streptomycin, Cells were seeded in 96-well microplates (2.104 cells/well), each well infected with 10-20 TCID50 of HIV-1丨丨丨B (wild type) or with mutations such as RT lie 100, Cys 181 or Asn 103 Mutant virus. Serially diluted test compounds were added to individual wells and the cultures were incubated at 37 t: in a CO2-rich atmosphere and the viability of the cells was determined in the fifth or sixth sputum with XTT live dye. Usually the result is expressed as ED5() μΜ. The compound of Example 1 exhibited an ED50 of about 0.6 μΜ in the XTT assay. Example 13 Intracellular Triphosphate Concentration and Half-Life The putative active substance of the compound of the present invention is β-ϋ-2'-deoxyindolyl-4'-azidocytidine nucleoside triphosphate. The stability of the triphosphate was determined in fresh human primary hepatocytes (CellzDirect or In Vitro Technologies) pre-incubated with the deuterated parent compound. Hepatocytes were typically cultured at 1.5 million cells/well on 6-well collagen coated plates (BD Biosciences) using serum-containing medium (CellzDirect or In Vitro Technologies) / 37 °C / 5% C〇2. Various hepatocyte cell lines such as Hu497, MHL-091806 and Hu504 can be used, and are therefore suitable for testing these cell lines simultaneously and calculating the average value from a range of cell lines. It is usually pre-incubated with 2 μΜ 10 μ(:/ιη1 of the deuterated precursor for 24 hours. At t〇, the cell monolayer is washed with cell culture medium to remove the extracellular matrix compound 154377.doc -56- 201124141, and the cells are The culture was re-cultured with fresh cell culture medium. The concentration of intracellular triphosphate was quantified at different time points (up to 72 hours). Convenient points were 0, 0.5, 1, 2 '4, 6, 8, 24, 48 and 72. In the hour, there are duplicate cell cultures at each time point. At appropriate time, the cells are harvested by pumping the cell culture medium and the cells are washed with cold PBS. The cells are brought into the extraction medium, such as, for example, 60% of pre-cooling ( Wv) Methanol, extracted into methanol at _2 (TC) for 24 h. The extracted sample was centrifuged to remove cell debris. The supernatant was transferred to a fresh tube, evaporated and stored in liquid nitrogen for analysis. The dried cell pellet extract is dissolved in water and filtered through nanometer (eg nan〇sep centrifugation device, palI Life Sciences). Before the HPLC analysis, the parent and its monophosphate, mono- and acid esters are filled. Unlabeled reference standard for ester form Prominent (spiked typical HPLC system using ion exchange HPLC with Whatman Partisil 10SAX (4.6 x 25 0 mm) column coupled to a radiometric detector such as β-RAM, IN/US Systems Inc. Conventional mobile phase linear gradient 〇% aqueous buffer to a flow rate of 1 ml/min to 1 〇〇〇/〇 phosphate buffer (eg 0.5M KH2P〇4/〇.8M KC1) for detection of β-RAM For radiolabeled substances, a 5:1 ratio of n〇Scint IV or UltimaFloAP (Perkin Elmer) to the column lysing agent can be used. By comparing the retention time of intracellular substances in the radioactive chromatogram with the prominent in the cell extract sample The residence time of the non-radioactive reference standard is used to identify maternal and intracellular metabolites and is detected by UV absorption 'usually at 270 nm. The time course of uptake and phosphorylation is measured in a similar manner' (for example) 2 μΜ and 10 pCi/ml of the deuterated compound of the present invention cultured human primary 154377.doc •57· 201124141 Liver', the cell is added to the compound at 72,%^λ, Ιό before the cell collection , ό and double culture at 1 hour For the determination of the dose response of the phosphorylation of the compounds of the invention, human primary hepatocytes are cultured for 24 hours using, for example, gasification test compounds at 〇, 2, 5 50, 100 and 250 μΜ. By supplementing the non-radiolabeled test The compound is brought to a final concentration. Duplicate cell cultures are typically collected after 24 hours of incubation. The average triphosphate half-life of the compounds of the present invention in the &quot;Hai assay was 2 1 · 4 hours (standard deviation 4.22 hours). The steady-state triphosphate content was about 15 ρΜ/million cells at 2 μΜ at 24 hours. Using 3 μΐ as the average volume of human hepatocytes, the concentration of triphosphate corresponds to a value substantially exceeding the Ki of the parent compound. The long half-life and high concentration of the triphosphate indicate that the antiviral activity of the active substance will be present in HCV-infected cells for a prolonged period of time after administration. This in turn means that the minimum daily minimum level will remain high, even when QD is administered, thereby minimizing the chance of suboptimal exposure to the drug, leading to the development of drug escape mutations. Similar to the long half-life of the diphosphate of the present invention, similar to 2, _p_c methyl compound PSI-6130 (pD-2, -deoxy-2, -fluoro-2, -pC-methylcytosine nucleoside III The half-life of phosphate vinegar (described below) was only 4.7 hours, and the steady-state triphosphate concentration was only 1.3 pM/million cells at 24 hours.

PSI-6130 154377.doc -58· 201124141 Example 14 A pharmaceutical composition of a compound of the invention administered via several routes is prepared as described in the Examples. Composition for oral administration (A) Ingredient % wt./wt. Active ingredient 20.0% Lactose 79.5% Magnesium stearate 0.5% The ingredients are mixed and distributed in capsules each containing about 500 to 1000 mg, for oral administration. Composition (B) Ingredient % wt./wt. Active ingredient 20.0% Magnesium stearate 0.5% Croscarmellose sodium 2.0% Lactose 76.5% PVP (polyvinylpyrrolidone) 1.0% Combine and use ingredients A solvent such as sterol is used for granulation. The formulation is then dried and formed into a tablet (containing about 20 mg of active compound) using a suitable tablet machine. Composition for oral administration (C) Ingredient % wt./wt. Active compound 1.0 g Fumaric acid 0.5 g Gasified sodium 2.0 g Hydroxybenzoic acid methyl ester 0.15 g Propyl hydroxybenzoate 0.05 g 154377.doc -59- 201124141 Sugar 25.5 g Sorbitol (70% solution) 12.85 g

Veegum K (Vanderbilt Co.) 1.0 g Flavoring agent 0.035 ml Coloring agent 0.5 mg Distilled water Appropriate amount to 100 ml These ingredients are mixed to form a suspension for oral administration. Parenteral Formulation (D) Ingredients % wt./wt. Active Ingredient 0.25 g Sodium Chloride Appropriate amount to make isotonic water for injection 100 ml Dissolve the active ingredient in one part of the water for injection. Sufficient sodium chloride is then added while stirring to make the solution isotonic. The solution was prepared and weighed together with the rest of the water for injection, filtered through a 0.2 micron filter membrane and packaged under sterile conditions. Suppository Formulation (E) Ingredient % wt./wt. Active Ingredient 1.0% Polyethylene Glycol 1000 74.5% Polyethylene Glycol 4000 24.5% The ingredients are melted together and mixed on a steam bath and poured into a total weight of 2.5 g. In the mold. The above invention has been described in detail by way of illustration and example embodiments Those skilled in the art will appreciate that variations and modifications can be made in the scope of the accompanying claims. Therefore, the above description is intended to be illustrative, and not restrictive. Therefore, the scope of the present invention should not be referred to the above description to ensure that 154377.doc-60-201124141 should be used in conjunction with the scope of the patents granted in the above-mentioned patents. Determined by scope. ^ = All patent applications and publications cited in this application are incorporated by reference in their entirety as if they had been patented, respectively. 154377.doc

Claims (1)

201124141 VII. Patent application scope: 1 · A compound of the following formula 154377.doc 201124141 IV. Designated representative map: (1) The representative representative of the case is: (none) (2) The symbol of the symbol of the representative figure is simple: 5. If there is a chemical formula in this case, please reveal the best indication of the characteristics of the invention. Chemical formula: 154377.doc