CN101043893A - Fluorinated pyrrolo[2,3-d]pyrimidine nucleosides for the treatment of rna-dependent rna viral infection - Google Patents

Abstract

The present invention provides fluorinated pyrrolo[2,3,d]pyrimidine nucleoside compounds 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 infection. They are particularly useful as precursors to inhibitors of hepatitis C virus (HCV) NS5B polymerase, as precursors to inhibitors of HCV replication, and/or for the treatment of hepatitis C infection. The invention also describes pharmaeutical compositions containing such fluorinated pyrrolo[2,3-d]pyrimidine nucleoside alone or in combination with other agents active against RNA-dependent RNA viral infection, in particular HCV infection. Also disclosed are methods of inhibiting RNA-dependent RNA polymerase, inhibiting RNA-dependent RNA viral replication, and/or treating RNA-dependent RNA viral infection with the fluorinated pyrrolo[2,3-d]pyrimidine nucleoside of the present invention.

Description

Fluorinated pyrrolo[2,3-d]pyrimidine nucleosides for the treatment of RNA-dependent RNA virus infection

field of invention

The present invention relates to fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds and certain derivatives thereof, their synthesis and their use as inhibitors of RNA-dependent RNA viral polymerases. The compounds of the present invention are inhibitors of RNA-dependent RNA virus replication and are useful in the treatment of RNA-dependent RNA virus viral infections. In particular, they are useful as inhibitors of hepatitis C virus (HCV) NS5B polymerase, as inhibitors of HCV replication and for the treatment of hepatitis C virus infection.

Background of the invention

Review of the State of the Art in the Treatment of HCV Infection Hepatitis C virus (HCV) is a major health problem leading to chronic liver diseases such as cirrhosis and hepatocellular carcinoma in many infected individuals, estimated to be 2% of the world's population. -15%. The US Centers for Disease Control estimates that 3,900,000 people are infected in the US alone, roughly five times the number infected with the human immunodeficiency virus (HIV). According to World Health Organization estimates, more than 1700,000,000 people are infected worldwide, and 3 to 4 million people are infected every year. Once infected, about 20% of people clear the virus, while the remainder carry HCV for life thereafter. Ten to 20 percent of chronically infected individuals eventually develop cirrhosis or cancer, which destroys the liver. The viral disease can be transmitted parenterally through contaminated blood and blood products, contaminated needles, or sexually and vertically from infected or carrier mothers to their offspring. Current treatment of HCV infection, limited to immunotherapy using recombinant interferon-α alone or in combination with the nucleoside analog ribavirin, has limited clinical benefit. Furthermore, there is no definitive HCV vaccine. Therefore, there is an urgent need for improved therapeutic agents that are effective against chronic HCV infection. The state of the art in the treatment of HCV infection has been reviewed, with reference to the following publications: B. Dymock, et al., "Novel approaches to the treatment of hepatitis C virus infection," Antiviral Chemistry & Chemotherapy . 11:79-96 (2000) ; H. Rosen, et al., "Hepatitis C virus: current understanding and prospects for future therapies," Molecular Medicine Today .5: 393-399 (1999); D. Moradpour, et al., "Current and evolving therapies for hepatitis C," European J. Gastroenterol . Hepatol. , 11:1189-1202 (1999); R. Bartenschlager, "Candidate Targets for Hepatitis C Virus-Specific Antiviral Therapy," Intervirology . 40:378-393 (1997); GMLauer and BD Walker, "Hepatitis C Virus Infection, " N. Engl. J. Med. .345:41-52 (2001); BW Dymock, "Emerging therapies for hepatitis C Virus infection, " Emerging Drugs . 6:13-42 (2001); and C. Crabb , "Hard-Won Advances Spark Excitement about Hepatitis C," Science : 506-507 (2001); incorporated herein by reference in its entirety.

Different approaches have been taken to treat HCV, including inhibition of the viral serine protease (NS3 protease), helicase, and RNA-dependent RNA polymerase (NS5B), and the development of vaccines.

The HCV virion is an enveloped, positive-strand RNA virus with a single oligoribonucleotide genome sequence of approximately 9600 bases, capable of encoding a polyprotein of approximately 3,010 amino acids. The protein products of HCV genes consist of structural proteins C, E1 and E2 and nonstructural proteins NS2, NS3, NS4, NS4B, NS5a and NS5B. Nonstructural (NS) proteins are thought to provide the catalytic machinery for viral replication. The NS3 protease releases NS5B, an RNA-dependent RNA polymerase, from the polyprotein chain. HCV NS5B polymerase is required for the synthesis of double-stranded RNA from single-stranded viral RNA that serves as a template in the HCV replication cycle. The NS5B polymerase is therefore considered an essential component in the HCV replication complex. [See K.Ishi, etc., "Expression of Hepatitis C Virus NS5B Protein: Characterization of Its RNA Polymerase Activity and RNA Binding," Hepatology , 29:1227-1235 (1999) and V.Lohmann, etc., "Biochemical and Kinetic Analyzes of NS5B RNA-Dependent RNA Polymerase of the Hepatitis C Virus," Viroloey .249: 108-118 (1998)]. Inhibition of HCV NS5B polymerase prevents the formation of double-stranded HCV RNA and, therefore, constitutes a key to the development of HCV-specific antiviral therapies. attractive avenue.

The development of HCV NS5B inhibitors for the potential treatment of HCV infection has been reviewed, see MP Walker et al. "Promising candidates for the treatment of chronic hepatitis C," Expert Opin. Invest. Drugs. 12:1269-1280 (2003); P .Hoffmann et al., "Recent patents on experimental therapy for hepatitis C virus infection (1999-2002), " Expert Opin. Ther. Patents , "13:1707-1723 (2003); and V.Brass, et al., "Recent developments in target identification against HCV, " Expert Opin. Ther. Targets, "8: 295-307 (2004). Inhibition of HCV replication by purine ribonucleosides was reported in : 2283-2295 (2004). There is a continuing need for structurally distinct nucleoside derivatives that are HCV polymerase inhibitors as therapeutic approaches for HCV treatment

US Patent No. 6,777,396 (published August 17, 2004) discloses a series of new structural pyrrolo[2,3-d]pyrimidine nucleoside derivatives as HCV NS5B polymerase inhibitors for the treatment of HCV infection. DBOlsen etc. in "A 7-Deaza-Adenosine Analog is a Potent and Selective Inhibitor of HCV Replication with Excellent Pharmacokinetic Properties," Antimicrob.Agents Chemother .. 48:3944-3953 (2004) and AEEldrup, etc. in "Structure-Activity Relationship of Heterobase -Modified 2′-C-Methyl Ribonucleosides as Inhibitors of Hepatitis C Virus RNA Replication,” J. Med. Chem ., 47: 5284-5297 (2004). Biological properties of such compounds are described. It is now found that the introduction of fluorine at the C-5 position of the pyrrolo[2,3-d]pyrimidine nucleus provides nucleoside derivatives with excellent pharmacokinetic properties, such as more efficient HCV RNA replication with better distribution in the liver Inhibitors.

Accordingly, it is an object of the present invention to provide fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds and certain derivatives thereof useful as inhibitors of RNA-dependent RNA viral polymerases, particularly HCV NS5B polymerase inhibitors. biology.

Another object of the present invention is to provide fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds and certain derivatives thereof useful as RNA-dependent RNA viral polymerase inhibitors, especially hepatitis C virus replication inhibitors. biology.

Another object of the present invention is to provide fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds and certain derivatives thereof for use in the treatment of RNA-dependent RNA viral infections, particularly HCV infections.

Another object of the present invention is to provide a pharmaceutical composition comprising a fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compound in association with a pharmaceutically acceptable carrier.

Another object of the present invention is to provide the fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds of the present invention which are useful as RNA-dependent RNA viral polymerase inhibitors, especially HCV NS5B polymerase inhibitors and Pharmaceutical compositions of certain derivatives thereof.

Another object of the present invention is to provide the fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds of the present invention and certain compounds thereof which are useful as inhibitors of RNA-dependent RNA virus replication, especially HCV replication inhibitors. Pharmaceutical compositions of these derivatives.

Another object of the present invention is to provide a medicament comprising the fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds of the present invention and certain derivatives thereof for use in the treatment of RNA-dependent RNA viral infections, particularly HCV infections combination.

Another object of the present invention is to include the fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds of the present invention and certain derivatives thereof in combination with other active agents against RNA-dependent RNA viruses, especially against HCV pharmaceutical composition.

Another object of the present invention is to provide methods for inhibiting RNA-dependent RNA viral polymerase, particularly HCV NS5B polymerase.

Another object of the present invention is to provide methods for inhibiting the replication of RNA-dependent RNA viruses, especially HCV.

Another object of the present invention is to provide a method of treating RNA-dependent RNA virus infection, especially HCV infection.

Another object of the present invention is to provide a method for treating RNA-dependent RNA virus infections in combination with other anti-RNA-dependent RNA viruses, especially in combination with other anti-HCV active agents for HCV infection.

Another object of the present invention is a fluorinated pyrrolo[2, 3-d] Pyrimidine nucleoside compounds and certain derivatives thereof and pharmaceutical compositions thereof.

Another object of the present invention is to provide the fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds of the present invention and certain derivatives thereof and pharmaceutical compositions thereof for the preparation of inhibitory RNA-dependent RNA virus replication and/or therapeutic RNA - Dependent RNA virus infection, in particular the use of a drug that inhibits HCV replication and/or treats HCV infection.

These and other objects will become apparent from the detailed description below.

Summary of the invention

The present invention relates to the nucleoside compound of structural formula I indicating the configuration of stereochemistry

and their pharmaceutically acceptable salts; where

R ¹ is hydrogen or fluorine;

R ² is fluorine or hydroxyl;

R ³ is hydrogen, C _1-16 alkylcarbonyl, C _2-18 alkenylcarbonyl, C _1-10 alkoxycarbonyl, C _3-6 cycloalkylcarbonyl, C _3-6 cycloalkoxycarbonyl, or An aminoacyl residue of the following structural formula;

R ⁴ is hydrogen, C _1-10 alkylcarbonyl, phosphoryl or its cyclic prodrug ester, diphosphoryl, triphosphoryl, C _2-18 alkenylcarbonyl, C _1-10 alkoxycarbonyl, C _{3 -6} cycloalkylcarbonyl, C _3-6 cycloalkoxycarbonyl, CH ₂ O(C=O)C _1-4 alkyl, CH(C _1-4 alkyl)O(C=O)C _{1- 4} alkyl or aminoacyl residues of the following structural formula;

Residues of the formula

R ⁵ is amino or hydroxyl;

R ⁶ is hydrogen, amino or fluoro;

R ⁷ is hydrogen, C _1-5 alkyl or phenyl C _0-2 alkyl; and

R ⁸ is hydrogen, C _1-4 alkyl, C _1-4 acyl, benzoyl. C _1-4 alkylaminocarbonyl, phenyl C _0-2 alkylaminocarbonyl, C _1-4 alkylsulfonyl or phenyl C _0-2 alkylsulfonyl;

R ⁹ is hydrogen, C _1-5 alkyl, phenyl or benzyl, wherein the alkyl is unsubstituted or replaced by one selected from hydroxyl, methoxy, amino, carboxyl, carbamoyl, guanidino, mercapto, methylthio substituted by substituents of 1H-imidazolinyl and 1H-indol-3-yl and wherein phenyl and benzyl are unsubstituted or substituted by one or two substituents independently selected from halogen, hydroxy and methoxy ;

R ¹⁰ is hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, phenyl or benzyl, wherein alkyl and cycloalkyl are unsubstituted or are independently selected from halogen, hydroxy, carboxyl by one to three , a substituent of C _1-4 alkoxy and wherein phenyl and benzyl are unsubstituted or are independently selected from one to three halogen, hydroxyl, cyano, C _1-4 alkoxy and trifluoromethyl Substituents; Ar is unsubstituted phenyl or by one to three independently selected from halogen, C _1-4 alkyl, C _1-4 alkoxy, C _1-4 alkylthio, cyano, Nitro, amino, carboxyl, trifluoromethyl, C _1-4 alkylamino, di(C _1-4 alkyl)amino, C _1-4 alkylcarbonyl, C _1-4 alkylcarbonyloxy and C _1-4 alkoxycarbonyl;

with the proviso that when ^R1 , ^R3 , ^R4 , and ^R6 are hydrogen and ^R2 is hydroxy, ^R5 is not amino.

The compounds of formula I are useful as inhibitors of RNA-dependent RNA viral polymerases, in particular HCV NS5B polymerase. They are also inhibitors of RNA-dependent RNA virus replication, especially HCV replication and are useful in the treatment of RNA-dependent RNA virus infections, especially HCV infection.

The present invention also includes the pharmaceutical composition containing this compound alone or the pharmaceutical composition of this compound combined with other anti-RNA-dependent RNA viruses, particularly other active agents against HCV, and inhibiting RNA-dependent RNA virus replication and Methods of treating RNA-dependent RNA viral infections.

Detailed description of the invention

The present invention relates to the nucleoside compound of structural formula I indicating the configuration of stereochemistry

and their pharmaceutically acceptable salts; where

R ¹ is hydrogen or fluorine;

R ² is fluorine or hydroxyl;

R ³ is hydrogen, C _1-16 alkylcarbonyl, C _2-18 alkenylcarbonyl, C _1-10 alkoxycarbonyl, C _3-6 cycloalkylcarbonyl, C _3-6 cycloalkoxycarbonyl, or An aminoacyl residue of the following structural formula;

R ⁴ is hydrogen, C _1-10 alkylcarbonyl, phosphoryl or its cyclic prodrug ester, diphosphoryl, triphosphoryl, C _2-18 alkenylcarbonyl, C _1-10 alkoxycarbonyl, C _{3 -6} cycloalkylcarbonyl, C _3-6 cycloalkoxycarbonyl, CH ₂ O(C=O)C _1-4 alkyl, CH(C _1-4 alkyl)O(C=O)C _{1- 4} alkyl or aminoacyl residues of the following structural formula;

Residues of the formula

R ⁵ is amino or hydroxyl;

R ⁶ is hydrogen, amino or fluoro;

R ⁷ is hydrogen, C _1-5 alkyl or phenyl C _0-2 alkyl; and

R ⁸ is hydrogen, C _1-4 alkylaminocarbonyl, phenyl C _0-2 alkylaminocarbonyl, C _1-4 alkylsulfonyl or phenyl C _0-2 alkylsulfonyl;

R ⁹ is hydrogen, C _1-5 alkyl, phenyl or benzyl, wherein the alkyl is unsubstituted or replaced by one selected from hydroxyl, methoxy, amino, carboxyl, carbamoyl, guanidino, mercapto, methylthio substituted by substituents of 1H-imidazolinyl and 1H-indol-3-yl and wherein phenyl and benzyl are unsubstituted or substituted by one or two substituents independently selected from halogen, hydroxy and methoxy ;

R ¹⁰ is hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, phenyl or benzyl, wherein alkyl and cycloalkyl are unsubstituted or are independently selected from halogen, hydroxy, carboxyl by one to three , a substituent of C _1-4 alkoxy and wherein phenyl and benzyl are unsubstituted or are independently selected from one or two of halogen, hydroxy and, cyano, C _1-4 alkoxy and trifluoromethane Substituents of substituents; Ar is unsubstituted phenyl or by one to three independently selected from halogen, C _1-4 alkyl, C _1-4 alkoxy, C _1-4 alkylthio, cyano , nitro, amino, carboxyl, trifluoromethyl, C _1-4 alkylamino, di(C _1-4 alkyl)amino, C _1-4 alkylcarbonyl, C _1-4 alkylcarbonyloxy and C _1-4 alkoxycarbonyl group substitution;

with the proviso that when ^R1 , ^R3 , ^R4 , and ^R6 are hydrogen and ^R2 is hydroxy, ^R5 is not amino.

Compounds of formula I are useful as inhibitors of RNA-dependent RNA viral polymerases. They are also inhibitors of RNA-dependent RNA virus replication and are useful in the treatment of RNA-dependent RNA virus infections.

In one embodiment of the compounds of the invention, ^R1 is hydrogen; ^R2 is hydroxyl; ^R3 and ^R4 are hydrogen.

In a second embodiment of the compounds of this invention, R ¹ is hydrogen; R ² is fluoro; R ³ and R ⁴ are hydrogen; provided that when R ¹ , R ³ , R ⁴ , and R ⁶ are hydrogen and R ² When is a hydroxyl group, R ⁵ is not an amino group.

In a third embodiment of the compounds of this invention Ar is unsubstituted phenyl.

In a fourth embodiment of the compounds of the invention, ^R is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, isobutyl, 2-methyl-1-propyl, hydroxymethyl, Mercaptomethyl, carboxymethyl, carbamoylmethyl, 1-hydroxyethyl, 2-carboxyethyl, 2-carbamoylmethyl, 2-methylthioethyl, 4-amino-1-butyl , 3-amino-1-propyl, 3-guanidino-1-propyl, 1H-imidazol-4-ylmethyl, phenyl, 4-hydroxybenzyl, 1H-indol-3-ylmethyl . In a class of such embodiments, ^R9 is methyl or benzyl.

In a fifth embodiment of the compounds of this invention, R ¹⁰ is C _1-6 alkyl, cyclohexyl, phenyl or benzyl. In a class of such embodiments, R ¹⁰ is methyl.

In a sixth embodiment of the compounds of this invention, Ar is unsubstituted phenyl, ^R9 is methyl or benzyl, ^R10 is methyl.

Illustrative, non-limiting examples of compounds of the invention of structural formula I useful as inhibitors of RNA-dependent RNA viral polymerases are as follows:

2,4-Amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine;

2-Amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin--4(3H)-one;

2,4-Diamino-5-fluoro-7-(2-fluoro-2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine;

4-Amino-5-fluoro-7-(2-fluoro-2-C-methyl-β-D-ribofuranosyl)-7H-;

2-Amino-5-fluoro-7-(2-fluoro-2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine--4(3H)- ketone;

and pharmaceutically acceptable salts thereof.

In one embodiment of the invention, the fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds of the invention are useful as inhibitors of positive-sense single-stranded RNA-dependent RNA viral polymerases, positive-sense single-stranded RNA-dependent Inhibitors of RNA virus replication, and/or for the treatment of positive-sense single-stranded RNA-dependent RNA virus infections. In one class of such embodiments, the positive-sense single-stranded RNA-dependent RNA virus is a Flaviviridae virus or a Picornaviridae virus. In a subclass of this class, the picornaviridae viruses are rhinoviruses, polioviruses or hepatitis A viruses. In the second subgroup of this class, the Flaviviridae viruses are selected from the group consisting of hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, Japanese encephalitis virus, Banzi virus and bovine viral diarrhea virus (BVDV ). In a subset of this subclass, the Flaviviridae virus is the hepatitis C virus.

Another aspect of the invention relates to methods of inhibiting RNA-dependent RNA virus polymerase, methods of inhibiting replication of RNA-dependent RNA viruses, and/or methods of treating RNA-dependent RNA virus infections in mammals in need of such treatment, the The method comprises administering to a mammal a therapeutically effective amount of a compound of formula I.

In one embodiment of this aspect of the invention, the RNA-dependent RNA viral polymerase is a positive-sense single-stranded RNA-dependent RNA viral polymerase. In one class of such embodiments, the positive-sense single-stranded RNA-dependent RNA viral polymerase is a Flaviviridae viral polymerase or a Picornaviridae viral polymerase. In a subclass of this class, the picornaviridae viral polymerase is a rhinovirus polymerase, a poliovirus polymerase, or a hepatitis A virus polymerase. In the second subclass of this class, the Flaviviridae viral polymerase is selected from the group consisting of hepatitis C virus polymerase, yellow fever virus polymerase, dengue virus polymerase, West Nile virus polymerase, Japanese encephalitis virus polymerase , Banzi virus polymerase and bovine viral diarrhea virus (BVDV) polymerase. In a subset of this subclass, the Flaviviridae viral polymerase is the hepatitis C virus polymerase.

In a second embodiment of this aspect of the invention, the RNA-dependent RNA viral replication is positive-sense single-stranded RNA-dependent RNA viral replication. In one class of such embodiments, the positive-sense single-stranded RNA-dependent RNA viral replication is Flaviviridae viral replication or Picornaviridae viral replication. In a subclass of this class, picornaviridae viral replication is rhinovirus replication, poliovirus replication, or hepatitis A virus replication. In the second subgroup of this class, the Flaviviridae virus replication is selected from the group consisting of hepatitis C virus replication, yellow fever virus replication, dengue virus replication, West Nile virus replication, Japanese encephalitis virus replication, Banzi virus replication and bovine virus replication. Viral replicative diarrhea virus replication. In a subset of this subclass, Flaviviridae viruses replicate as Hepatitis C viruses.

In a third embodiment of this aspect of the invention, the RNA-dependent RNA viral infection is a positive-sense single-stranded RNA-dependent RNA viral infection. In one class of such embodiments, the positive-sense single-stranded RNA-dependent RNA viral infection is a Flaviviridae viral infection or a Picornaviridae viral infection. In a subclass of this class, the picornaviridae virus infection is a rhinovirus infection, a poliovirus infection, or a hepatitis A virus infection. In the second subclass of this class, the Flaviviridae viral infection is selected from the group consisting of hepatitis C virus infection, yellow fever virus infection, dengue virus infection, West Nile virus infection, Japanese encephalitis virus infection, Banzi virus infection and bovine Viral diarrhea virus infection. In a subset of this subclass, the Flaviviridae virus infection is the hepatitis C virus infection.

Throughout this application, the following terms have the indicated meanings:

"Alkyl", as well as other groups having the prefix "alk", such as alkoxy or alkylthio, refers to straight or branched carbon chains, or combinations thereof, unless the carbon chain is defined otherwise. Examples of alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl and the like. Where the number of carbon atoms is specified, eg _C3-10 , the term "alkyl" also includes cycloalkyl, and combinations of straight or branched alkyl chains combined with a cycloalkyl structure.

The term "cycloalkyl" refers to a group of alkyl groups and refers to an alkyl saturated carbocyclic ring having the specified number of carbon atoms. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl and the like. Unless otherwise specified, cycloalkyl groups are generally monocyclic. Cycloalkyl groups are saturated unless otherwise indicated.

The term "alkenyl" refers to a straight or branched chain alkene (eg vinyl, propenyl, butenyl, pentenyl, etc.) having a total carbon number of 2-6 or any number within the range.

The term "alkynyl" refers to a straight or branched chain alkyne (eg, ethynyl, propynyl, butynyl, pentynyl, etc.) with a total carbon number of 2-6 or any number within the range.

The term "alkoxy" refers to a specified number of carbon atoms (such as C _1-4 alkoxy) or any number of linear or branched alkyl oxygen within the range [ie methoxy (MeO-), ethoxy base, isopropoxy, etc.].

The term "alkylthio" refers to a specified number of carbon atoms (such as C _1-4 alkylthio) or any number of straight-chain or branched alkylthio within the range [ie methylthio (MeS-), ethylthio group, isopropylthio group, etc.].

The term "alkylamino" refers to a specified number of carbon atoms (such as C _1-4 alkylamino) or any number of linear or branched alkylamines within this range [i.e. methylamino, ethylamino, isopropylamino, baseamino, tert-butylamino, etc.].

The term "alkylsulfonyl" refers to a specified number of carbon atoms (such as C _1-6 alkylsulfonyl) or any number of straight-chain or branched alkylsulfones within this range [ie methylsulfonyl (MeSO ₂ - ), ethylsulfonyl, isopropylsulfonyl, etc.].

The term "alkoxycarbonyl" refers to a straight-chain or branched ester of a carboxylic acid derivative of the present invention having a specified number of carbon atoms (e.g. _C1-4 alkoxycarbonyl) or any number within this range [i.e. methoxy carbonyl (MeOCO-), ethoxycarbonyl or butoxycarbonyl].

The term "alkylcarbonyl" refers to a straight _- chain or branched chain alkylacyl group [i.e. methylcarbonyl (MeCO-), ethyl, or methylcarbonyl (MeCO-), ethyl carbonyl or butylcarbonyl].

The term "halogen" is intended to include the halogen atoms fluorine, chlorine, bromine and iodine.

The term "phosphoryl" refers to -P(O)(OH) ₂ -

The term "diphosphoryl" refers to a radical having the following structure:

The term "triphosphoryl" refers to a group having the following structure:

The term "substituted" shall be considered to include multiple substitutions by the indicated substituents. When multiple substituted moieties are disclosed or claimed, the substituted compounds may independently be single or multiple substituted with one or more disclosed or claimed substituent moieties.

When R in the aminoacyl residue embodiment ^{of R} and ^R is not hydrogen in the following formulae,

Aminoacyl residues contain asymmetric centers and are intended to include individual R- and S-stereoisomers as well as RS-diastereomeric mixtures.

The term "5'-triphosphate" refers to a triphosphate derivative of the 5'-hydroxyl group of the fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds of the present invention having the following general structural formula II:

wherein R ¹ -R ³ , R ⁵ and R ⁶ are as defined above. The compounds of the present invention are also intended to include pharmaceutically acceptable salts of triphosphates and pharmaceutically acceptable salts of 5'-monophosphate and 5'-bisphosphate derivatives having structural formulas III and IV, respectively.

The term "composition" in "pharmaceutical composition" is intended to include products comprising the active ingredient and an inert ingredient forming a carrier, as well as mixtures, complexes, or aggregates of any two or more ingredients, or separation of one or more ingredients. , or any product resulting directly or indirectly from any other type of reaction or interaction of one or more components. Accordingly, the pharmaceutical compositions of the present invention include any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier.

The terms "administering" and "administering" a compound are understood to mean providing a compound of the invention or a prodrug of a compound of the invention to a subject in need thereof.

Another aspect of the invention pertains to methods of inhibiting HCV NS5B polymerase, inhibiting HCV replication, or treating HCV infection using a compound of the invention in combination with one or more agents useful in the treatment of HCV infection. Such agents effective against HCV include, but are not limited to, ribavirin, levovirin, viramidine, thymosin alpha-1, interferon beta, interferon alpha, pegylated interferon alpha (pegylated interferon-α), combination of interferon-α and ribavirin, combination of pegylated interferon-α and ribavirin, combination of interferon-α and levovirin, poly The combination of ethylene glycol interferon-alpha and levovirin. Interferon alpha includes, but is not limited to, recombinant interferon alpha 2a (such as Roferon interferon available from Hoffmann-LaRoche, Nutley, NJ), pegylated interferon alpha 2a (Pegasys ^™ ), interferon alpha 2b (such as Schering Corp., Kenilworth, NJ Intron-Ainterferon), pegylated interferon alpha 2b (PegIntron ^™ ), recombinant consensus interferon (such as interferon alphacon-1) and purified interferon alpha products. Amgen's recombinant consensus interferon has the trade name Infergen(R). Levovirin is the L-enantiomer of ribavirin, which exhibits immunomodulatory activity similar to that of ribavirin. Viramidine means the ribavirin analog disclosed in WO 01/60379 (assigned to ICN Pharmaceuticals). According to the methods of the invention, the components of the combination may be administered separately at different times during the treatment period, or administered simultaneously in separate or single combinations. Accordingly, the invention is to be understood as including all such therapies of simultaneous or alternating treatment, and the term "administration" is to be construed accordingly. It is to be understood that the scope of combinations of the compounds of the present invention with other agents useful in the treatment of HCV infection includes in principle any combination with any pharmaceutical composition useful in the treatment of HCV infection. When a compound of the present invention, or a pharmaceutically acceptable salt thereof, is combined with a second therapeutic agent effective against HCV, the dosage of each compound may be the same or different than when the compound is used alone.

For the treatment of HCV infection, the compounds of the invention may also be administered in combination with agents that are HCV NS3 serine protease inhibitors. The HCV NS3 serine protease is an essential viral enzyme and has been described as an excellent target for inhibition of HCV replication. Both substrate and non-substrate-based inhibitors of HCV NS3 protease inhibitors are disclosed in WO98/22496, WO98/46630, WO99/07733, WO99/07734, WO99/38888, WO99/50230, WO99/64442, WO00/09543, WO00/59929, GB2337262, WO02/48116, WO02/48172 and US Patent No. 6,323,180. The HCV NS3 protease as a target for developing HCV replication inhibitors and treating HCV infection is discussed in B.W. Dymock, "Emerging therapies for hepatitis C virus infection," Emerging Drugs, 6:13-42 (2001).

Ribavirin, levovirin and viramidine can exert their anti-HCV effects by regulating the intracellular pool of guanine nucleotides by inhibiting the intracellular enzyme inosine monophosphate dehydrogenase (IMPDH). IMPDH is the rate-limiting enzyme of the biosynthetic pathway in de novo guanine nucleotide biosynthesis. Ribavirin is readily phosphorylated intracellularly, and its monophosphate derivatives are inhibitors of IMPDH. Therefore, inhibition of IMPDH represents another effective target for the discovery of inhibitors of HCV replication. Thus, the compounds of the present invention may also be administered in combination with an IMPDH inhibitor such as VX-497 disclosed in WO 97/41211 and WO 01/00622 (assigned to Vertex); another IMPDH inhibitor such as WO 00/00622 25780 (assigned to Bristol-Myers Squibb); or mycophenolate mofetil [see A.C. Allison and E.M Eugui, Agents Action, 44 (Suppl.): 165 (1993)].

For the treatment of HCV infection, the compounds of the present invention may be administered in combination with the antiviral agent amantadine (1-aminoadamantane) [for a review of this agent, see J. Kirschbaum, Anal. Profiles-Drug Subs. 12: 1-36 (1983) ].

The compounds of the present invention can also be used in the treatment of HCV infection in combination with 2'-C-branched ribonucleosides disclosed in R.E.Harry-O'kuru et al., J.Org.Chem .. 62: 1754-1759 (1997); M.S. Wolfe et al., Tetrahedron Lett.. 36: 7611-7614 (1995); U.S. Patent No. 3,480,613 (November 25, 1969); International Publication No. WO 01/90121 ( November 29, 2001); International Publication No. WO 01/92282 (December 6, 2001); International Publication No. WO 02/32920 (April 25, 2002); International Publication No. WO 04/002999 (January 2004 8); International Publication No. WO 04/003000 (January 8, 2004); International Publication No. WO 04/002422 (January 8, 2004); the contents of which are incorporated by reference in their entirety. Such 2'-C-branched ribonucleosides include, but are not limited to, 2'-C-methylcytidine, 2'-C-methyluridine, 2'-C-methyladenosine, 2'-C- -Methylguanosine, 9-(2-C-methyl-β-D-ribofuranosyl)-2,6-diaminopurine, ribose C-2′, C-3 and C-5′ hydroxyl corresponding Cyclic 1,3-propanediol esters of amino acid esters (e.g., 3′-O-(L-valyl)-2′-C-methylcytidine) and the corresponding optionally substituted 5′-phospholipid derivatives .

Compounds of the present invention can also be used in combination with other nucleosides having anti-HCV properties for the treatment of HCV infection, other nucleosides disclosed in WO02/51425 (July 4, 2002), assigned to Mitsubishi Pharma Corp.; WO01/79246 , WO02/32920, WO02/48165 (June 20, 200), assigned to Pharmasset, Ltd.; WO01/68663, September 20, 2001), assigned to ICN Pharmaceuticals; WO99/43691 (September 2, 1999) ; WO02/18404 (7 March 2002), assigned to Hoffmann-LaRoche; U.S2002/0019363 (14 February 2002); WO02/100415 (19 December 2002); WO03/026589 (2003 April 3, 2003); WO03/026675 (April 3, 2003); WO03/093290 (November 13, 2003): US2003/0236216 (December 25, 2003); US2004/0006007 (November 2004 February 8); WO04/011478 (February 5, 2004); WO04/013300 (February 12, 2004); US 2004/0063658 (April 1, 2004); WO04/028481 (April 2004 8).

Compounds of the present invention can also be used in combination with HCV polymerase inhibitors for the treatment of HCV infection. HCV polymerase inhibitors are disclosed in WO01/77091 (October 18, 2001), assigned to Tularik, Inc.; WO01/47883 (2001 Jul. 5, 2002), assigned to Japan Tobacco, Inc.; WO02/04425 (Jan. 17, 2002), assigned to Boehringer Ingelheim; WO02/06246 (Jan. 24, 2002), assigned to Istituto di Ricerchedi Biologia Moleculare P . Angeletti S.P.A.; WO02/20497 (March 3, 2002).

"Pharmaceutically acceptable" means that the carrier, diluent or excipient must be compatible with the other formulation ingredients and not deleterious to the recipient thereof.

Also included within the scope of the invention are pharmaceutical compositions comprising the fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds and derivatives thereof of the invention and a pharmaceutically acceptable carrier. Another example of the present invention is a pharmaceutical composition prepared by combining any of the above compounds and a pharmaceutically acceptable carrier. Another example of the present invention is a method for preparing a pharmaceutical composition, the method comprising combining any of the above-mentioned compounds and a pharmaceutically acceptable carrier.

Also included within the scope of the present invention are pharmaceutical compositions useful for inhibiting RNA-dependent RNA viral polymerase, particularly HCV NS5B polymerase, comprising an effective amount of a compound of the present invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions useful for treating RNA-dependent RNA viral infections, particularly HCV infections, are also included within the scope of the invention, as are methods of inhibiting RNA-dependent RNA viral polymerases, particularly HCV NS5B polymerases, and the treatment of RNA-dependent Methods for the replication of RNA viruses, particularly HCV, are also within the scope of the invention. Furthermore, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention and a therapeutically effective amount of another agent effective against RNA-dependent RNA viruses, in particular against HCV. Agents effective against HCV include but are not limited to ribavirin, levovirin, viramidine, thymosin alpha-1, HCV NS3 serine protease inhibitors, interferon alpha, pegylated interferon alpha (peginterferon interferon-α), the combination of interferon-α and ribavirin, the combination of pegylated interferon-α and ribavirin, the combination of interferon-α and levovirin, the combination of pegylated interferon-α Alpha in combination with levovirin. Interferon alpha includes, but is not limited to, recombinant interferon alpha 2a (such as Roferon interferon available from Hoffmann-LaRoche, Nutley, NJ), pegylated interferon alpha 2a (Pegasys ^™ ), interferon alpha 2b (such as Schering Corp., Kenilworth, NJ Intron-Ainterferon), pegylated interferon alpha 2b (PegIntron ^™ ), recombinant consensus interferon (such as interferon alphacon-1) and purified interferon alpha products. For a discussion of ribavirin and its activity against HCV see JOSaunders and SA Raybuck, "Inosine Monophosphate Dehydrogenase: Cohsideration of Structure, Kinetics, and Therapeutic Potential," Ann. Rep. Med. Chem, 35: 201-210 (2000) .

Another aspect of the present invention provides the use of fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds and their derivatives and their pharmaceutical compositions in the manufacture of a medicament for the replication of RNA-dependent RNA viruses , particularly HCV replication, and/or treatment of RNA-dependent RNA virus infections, particularly HCV infections. Still another aspect of the present invention provides fluorinated pyrrolo[2,3-d]pyrimidine compounds and their derivatives and their pharmaceutical compositions useful as medicaments for inhibiting the replication of RNA-dependent RNA viruses, in particular is HCV replication, and/or treatment of RNA-dependent RNA viral infections, particularly HCV infections.

The pharmaceutical composition of the present invention contains the compound of structural formula I or a pharmaceutically acceptable salt thereof as an active ingredient, and may also contain a pharmaceutically acceptable carrier and optional other therapeutic ingredients.

Compositions include those suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), ophthalmic (ophthalmic), pulmonary (nasal or buccal inhalation) or nasal administration, although in any The most suitable route of administration in a case will depend upon the nature and severity of the condition being treated and the nature of the active ingredient. They may conveniently be presented in unit dosage form, prepared by any of the methods known in the art of pharmacy.

In practical application, the compound of structural formula I can be combined as an active ingredient and a pharmaceutical carrier by intimate mixing through conventional pharmaceutical mixing techniques. The carrier can take a variety of forms depending on the form of preparation desired for administration, eg, oral or parenteral (including intravenous). In the preparation of compositions for oral dosage forms, in the case of oral liquid preparations such as suspensions, elixirs and solutions, any usual pharmaceutical media such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents can be used. or in the case of oral solid preparations such as powders, hard and soft capsules, and tablets, carriers such as starch, sugar, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, Disintegrants and the like, solid oral preparations are more preferable than liquid preparations.

Because of their ease of administration, tablets and capsules are the most preferred oral dosage unit forms when obviously solid pharmaceutical carriers are employed. Tablets may be coated, if desired, by conventional aqueous or non-aqueous techniques. Such compositions and preparations should contain at least 0.1% active compound. The percentage of active compound in these compositions may, of course, be varied and suitably ranges from about 2% to about 60% by weight of the unit. The amount of active compound in such therapeutically useful compositions is such that an effective dosage will be obtained. The active compounds can also be administered intranasally, eg, as liquid drops or spray.

Tablets, pills, capsules, etc. may also contain binders such as tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, alginic acid; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose, or saccharin. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.

Various other materials may be present as coatings or to modify the physical form of the dosage unit. For example, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring as cherry or orange flavor.

Compounds of formula I may also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycols, liquid polyethylene glycols, and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In each case, the dosage form must be sterile and must be liquid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be protected against the contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol), stable mixtures thereof, and vegetable oils.

Any suitable route of administration may be employed to provide an effective dose of a compound of the invention to a mammal, especially a human. For example, oral, rectal, topical, parenteral, ophthalmic, pulmonary, nasal, etc. can be used. Dosage forms include tablets, lozenges, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like. The compounds of formula I are preferably administered orally.

For oral administration to humans, the dose ranges in divided doses from 0.01 to 1000 mg/kg body weight. In one embodiment, the dose range in divided doses is 0.1-100 mg/kg body weight. In another embodiment, the dose in divided doses ranges from 0.5-20 mg/kg body weight. For oral administration, the composition is preferably formulated to contain 1.0-1000 mg of active ingredient, especially 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600 , 750, 800, 900 and 1000 mg of active ingredient in the form of tablets or capsules to regulate the symptoms of the treated patients.

The effective dosage of the active ingredient employed may vary with the particular compound employed, the mode of administration, the condition being treated and the severity of the condition being treated. Such dosages can be readily determined by those skilled in the art. The dosage regimen can be adjusted to provide the optimum therapeutic response.

The compounds of the present invention contain one or more asymmetric centers and can therefore exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. The present invention includes fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds having a five-membered furanose ring with a β-D stereochemical configuration, that is, wherein C-1 of the five-membered furanose ring is Fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds with substituents on C-4 having a β-stereochemical configuration ("up" direction indicated by bold lines).

Some of the compounds described herein contain olefinic double bonds and, unless otherwise indicated, are meant to include both E and Z geometric isomers.

Some of the compounds described herein may exist as tautomers such as keto-enol tautomers. Included within the compounds of formula I are the various tautomers and mixtures thereof. Examples of keto-enol and imine-enamine tautomers included within the scope of the compounds of the present invention are as follows:

The compounds of formula I can be separated into their individual diastereomers by eg fractional crystallization from a suitable solvent such as methanol or ethyl acetate or mixtures thereof or via chiral chromatography using optically active stationary phases.

Alternatively, any stereoisomer of a compound of formula I may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known configuration.

The compounds of the present invention may be administered in the form of pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids, including inorganic or organic bases and inorganic or organic acids. Salts of basic compounds encompassed within the term "pharmaceutically acceptable salt" refer to non-toxic salts of the compounds of this invention which are usually prepared by reacting the free base with a suitable organic or inorganic acid. Representative salts of basic compounds of the invention include, but are not limited to, the following salts: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide , camphorate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, ettoate, ethanesulfonate, fumaric acid salt, glucoheptonate, gluconate, glutamate, glycolyl p-aminophenylarsinate, hexylresorcinate (hexylresorcinate), hepamine, hydrobromide, hydrochloride , xinafoate, iodide, isothiosulfate (isothionate), lactate, lactobionate, laurate, malate, maleate, mandelate, methanesulfonate, formazan bromide, methyl nitrate, methyl sulfate, mucate, naphthalene sulfonate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, balm salt (bis moxamate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, sulfate, hydroxyacetate, succinate , tannin, tartrate, theanate, tosylate, triethyl iodide and valerate. Additionally, when compounds of the present invention bear an acidic moiety, suitable pharmaceutically acceptable salts thereof include, but are not limited to, salts derived from inorganic bases, including aluminium, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, Manganese, mangamous, potassium, sodium, zinc, etc. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, cyclic amines, and basic ion exchange resins such as arginine, betaine, caffeine, choline, N,N-di Benzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, Glucosamine, histidine, hepamine, isopropylamine, lysine, methyl glucosamine, morpholine, piperazine, piperidine, polyamine resin, procaine, purine, theobromine, three Ethylamine, trimethylamine, tripropylamine, tromethamine, etc.

Also, where carboxylic acid (-COOH), phosphate [-OP(O)(OH) ₂ ] or alcohol groups are present in the compounds of the present invention, pharmaceutically acceptable esters of carboxylic acid derivatives such as Methyl, ethyl or pivaloyloxymethyl esters; 5′-phosphate derivatives of fluorinated pyrrolo[2,3-d]pyrimidine nucleosides (including 5′-monophosphate, 5′-diphosphate esters and 5'-triphosphate) to pharmaceutically acceptable prodrug esters; or prodrug acyl derivatives of ribose C-2', C-3', and C-5' hydroxyl groups, such as O-acetate Or O-maleate or O-aminoacyl. Included are those esters and acyl groups known in the art to modify bioavailability, tissue distribution, solubility or hydrolysis properties for use as sustained release or prodrug formulations. Also included are five-membered ring carbonate derivatives of the C-2' and C-3' hydroxyl groups. The expected derivatives are readily converted to the desired compounds in vitro. Therefore, in the method of treatment of the present invention, the terms "administering" and "administering" are meant to include the use of a specifically disclosed compound or the use of a compound that is not specifically disclosed but is converted in vivo to the specified compound after administration to a mammal, including a human patient. Treat viral infections as described. Routine methods for selecting and preparing suitable prodrug derivatives are described, for example, in "Design of Prodrugs," edited by H. Bundgaard, Elsevier, 1985, which is incorporated herein by reference in its entirety.

Preparation of compounds of the present invention:

The starting material for the preparation of the compounds of the present invention is 4-amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (1-9) , the synthesis of which is described in Scheme 1.

Flowchart 1

Preparation of 5-fluoro-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (1-4):

Step A: Preparation of 5-bromo-4chloro-7H-pyrrolo[2,3-d]pyrimidine (1-2)

To a solution of 4chloro-7H-pyrrolo[2,3-d]pyrimidine ( 1-1 ) (1.53 g, 10.0 mmol) in DMF (20 mL) was added dropwise in (10 mL) at 0 °C Solution of N-bromosuccinimide (1.78 g, 10.0 mmol). The reaction mixture was stirred at 0 °C for 30 minutes then at room temperature for 1 hour. Methanol (25 mL) was added and the reaction mixture was stirred for an additional 1 hour. Evaporation of the solvent and recrystallization of the residue from methanol gave the title compound as a white solid.

Step B: Preparation of 5-(trimethylstannyl)-4chloro-7H-pyrrolo[2,3-d]pyrimidine (1-3)

To a solution of the compound obtained from step A (0.92 g, 4 mmol) in THF (25 mL) was added n-BuLi (2.5M in hexane, 3.48 mL) dropwise at -78°C. After the addition was complete, the reaction mixture was stirred at -78°C for an additional 30 minutes. To this solution was added trimethyltin chloride (0.88 g, 4.4 mmol) in THF (8 mL) dropwise over 10 minutes. The reaction mixture was slowly brought to room temperature and stirred overnight at room temperature. Saturated ammonium chloride solution (60 mL) was added and extracted with ethyl acetate (3 x 70 mL). _The combined organic extracts were washed with brine, dried over _Na2SO4 and evaporated to dryness. The residue was purified on silica gel to give the title compound as a colorless solid.

Step C: 5-fluoro-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (1-4)

To a solution of the compound obtained from step B in _CH3CN (60 mL) was added one portion of [1-(chloromethyl)-4-fluoro-1,4-diazabicyclo[2.2.2]octane Bis(tetrafluoroborate)] (SELECTFLUOR(R) fluorinating reagent) (2.40 g, 6.5 mmol) and the reaction mixture was stirred at room temperature for 7 hours. The white precipitate was filtered off and the filtrate was evaporated to dryness. The residue was purified on silica gel using ethyl acetate/hexane (3:7) as eluent. Fractions containing product were pooled and evaporated in vacuo to give the title compound as a colorless solid.

¹ H-NMR (500MHz, MeOH-d ₄ ): δ8.53 (s, 1H), 7.37 (d, J=2.8Hz); ¹⁹ F-NMR (DMSO-d ₆ ): δ-171.5.

Preparation of 2-C-methyl-3.5-di-O-(p-toluoyl)-D-ribofuranose (1-6):

To 3-O-benzyl-1,2-O-isopropylidene-3-C-methyl-α-D-glucofuranose (1-5) in 35 l of acetonitrile (for preparation see Carbohydr. Res. .44:275-283 (1975) (5.0kg, 15.4mol) and pyridine (3.7kg, 46.2mol) were added to a solution of p-benzoyl chloride (5.2kg, 33.9mol), and the reaction was heated at 50-55°C 12 hours. Add 48wt% HBF ₄ (tetrafluoroboric acid) solution 6.0L (46.2mol) in 9L water at 50-55 ° C. After 2 hours, distill off 10L acetonitrile, add 10L acetonitrile. When converting 97% , 10L of acetonitrile was distilled off, and the reaction solution was cooled to 0-5°C. Periodic acid solution (4.2kg, 18.5mol) in 10L of water was added. After aging the reaction for 30 minutes, 35L of isopropyl acetate and 10L of Water. Wash the organic phase with 25 L of water, followed by 20 L of NaHCO _aqueous solution, 15 L of 5% sodium thiosulfate in water, 15 L of water. Concentrate the isopropyl acetate solution to 10-15 L and add 40 L of methanol. Cool the solution to 0° C. and diisopropylamine (0.78 kg, 7.7 mol) was added. After two days at 0° C., aqueous HCl (1 N, 7.7 L) was added at 0-5° C., followed by 30 L isopropyl acetate and 40 L water. 1 N HCl was used , NaHCO3 and brine. The organic phase was dried by azeotropic distillation and treated with activated carbon. The carbon was removed by filtration and the resulting solution was diluted to 75 L using isopropyl acetate and hydrogenated (45 psi, 50 °C, 1.5 kg 10% Pd /c) 24 hours. The filtrate was concentrated to 15 L and 60 L of heptane was added at 50° C. The crystalline product was isolated by washing with 10 L of 20% isopropyl acetate in heptane by filtration. Drying gave 4.03 kg of the required diol 1 -6.

¹ H NMR (CDCl ₃ , 400 MHz): The ratio of α:β isomer in CDCl ₃ is about 5:1. For the major isomer: δ 7.95-7.90 (m, 4H), 7.26 (d, J =8.0Hz, 2H), 7.17(d, J=8.0Hz, 2H), 5.53(d, J=7.2Hz, 1H), 5.22(d, J=2.8Hz, 1H), 4.65-4.49(m, 3H ), 3.08 (d, J = 3.2Hz, 1H), 2.44 (s, 3H), 2.38 (s, 3H), 2.26 (s, 1H), 1.44 (s, 3H) ppm; for minor isomers : δ7.95-7.90(m, 4H), 7.27(d, J=8.0Hz, 2H), 7.22(d, J=8.0Hz, 2H), 5.16(d, J=5.6Hz, 1H), 5.12( d, J=5.6Hz, 1H), 4.66-4.49(m, 3H), 3.54(d, J=5.6Hz, 1H), 2.91(s, 1H), 2.43(s, 3H), 2.40(s, 3H ), 1.44 (s, 3H) ppm.

Preparation of 4-amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (1-9)

Step A: 1.2-Anhydro-3.5-di-O-(p-toluoyl)-2-C-methyl-α-D-ribofuranose (1-7)

A 72 L vessel was charged with dry dichloromethane (32 L), triethylamine (3.0 L), and diol (3.44 kg, 90 wt% pure). The mixture was heated to 30°C, then methanesulfonyl chloride (0.79 L) was added over 40 minutes. After 1 hour, the batch was partitioned between pH 7 buffer (20 L) and methyl tert-butyl ether (44 L). The organic phase was washed with 1M aqueous NaCl (38 L), then switched to vacuum distillation of toluene, followed by concentration to approximately 9 L. The resulting epoxide solution was used directly in Step B.

Step B: 4-Chloro-5-fluoro-7-(2-C-methyl-3,5-di-O-(p-toluoyl)-β-D-ribofuranosyl)-7H-pyrrolo[ 2,3-d] pyrimidine (1-8)

To a solution of 4-chloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine (1-4) (28.4 g, 0.165 mol) in N,N-dimethylacetamide (300 mL) at room temperature Portionwise 60% sodium hydride (6.6 g, 0.165 mol) was added. After the addition was complete, the reaction mixture was stirred at 60°C for 1 hour. To the reaction mixture was added a solution (63.4 g, 0.166 mol), the reaction mixture was heated at 60°C for 18 hours. The reaction mixture was cooled to room temperature and poured into water (1 L) and ethyl acetate (2 L). The organic extracts were washed with water (500 mL), dried over MgSO ₄ and evaporated to dryness. The residue was purified on silica gel using 10-40% ethyl acetate/hexanes as eluent. Fractions containing product were pooled and concentrated to give a foam which was used directly in Step C below.

Step C: 4-Amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (1-9)

4-Chloro-5-fluoro-7-(2-C-methyl-3,5-di-O-(p-toluoyl)-β- A solution of D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (29.4 g, 0.053 mol) was heated at 85°C for 48 hours. The reaction mixture was warmed to room temperature, the residue was slurried in methanol (200 mL), filtered, the filtrate was absorbed onto silica gel (200 g) and purified by chromatography using 0-30% methanol/dichloromethane as eluent , fractions containing product were combined and evaporated to give the title compound 1-9 as a solid.

¹ H-NMR (500MHz, MeOH-d ₄ ): δ8.07(s, 1H), 7.41(d, J=2.2Hz, 1H), 6.25(d, J=1.8Hz), 4.09-3.95(m, 3H), 3.82 (dd, J=2.7, 12.5Hz, 1H); ¹⁹ F-NMR (MeOH-d ₄ ): δ-170.4; Mass Spectrum: 321 (M+Na) ⁺ .

The introduction of the amino group at the C-2 position of the 6-amino-7-azapurine (7-deazaadenine) ring of the intermediate 1-9 can be carried out according to Zhao et al., in J.Org.Chem., 62:7832-7835 (1997 ) The synthetic method described in ) was carried out, as illustrated in Scheme 2. The conversion of 2,6-diamino-7-azapurine ring into 7-deazaguanine system can be carried out according to the method described in K.Alarcon et al. in Tetrahedron Lett, 41:7211-7215 (2000), as shown in flow chart 3 .

The following examples illustrate the conditions used in the preparation of compounds of the invention. The examples are not intended to limit the scope of the invention in any way, nor should they be construed as such. Those skilled in the art of nucleoside and nucleotide synthesis will readily recognize that well-known modifications to the conditions and methods of the preparative procedures described below can be made to prepare these and other compounds of the invention. All temperatures are in degrees Celsius unless otherwise stated.

Flowchart 2

Example 1

2,4-Diamino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (2-4)

Step A: 4-Amino-5-fluoro-3-N-oxo-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (2 -1)

To 4-amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine in 50% methanol/water (20 mL) (268mg, 0.899mmol) was added m-chloroperoxybenzoic acid (444mg, 1.80mmol). The reaction mixture was stirred at room temperature for 18 hours. The solvent was evaporated and the residue was azeotroped twice with toluene to give the title compound as a beige solid.

Step B: 2,4-Diamino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (2-4)

To a biological solution of cyanogen bromide in water (3 mL) was added 4-amino-5-fluoro-3-N-oxygen-7-(2-C-methyl-β-D A solution of -ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (2-1 ) (160 mg, 0.509 mmol). The resulting solution was stirred at 0 °C for 1.5 hours. The solvent was evaporated and azeotroped with toluene. N,N-Dimethylamide (3.5 mL) and triethylamine (0.25 mL, 1.79 mol) were added to the solution, and the resulting solution was stirred at room temperature for 45 minutes. Iodomethane (0.25 mL, 4.0 mmol) was added in portions, and the reaction mixture was stirred at room temperature for 1.5 hours in the dark. The solvent was evaporated and 0.25M aqueous sodium hydroxide solution (10 mL) was added to the residue, which was stirred at room temperature for 30 minutes. The reaction mixture was neutralized with 1M HCl, diluted with ethanol (10 mL), and heated at 60° C. for 8 hours. After cooling to room temperature, concentrated ammonium hydroxide (12 mL) was added and the reaction mixture was heated to 90° C. whereby Raney nickel was added. After 15 minutes, the residue was purified on silica gel using methanol/dichloromethane as eluent by filtering the hot reaction mixture, evaporating the solvent. Fractions containing product were evaporated to give the title compound 2-4 as a solid.

¹ H-NMR (500MHz, MeOH-d ₄ ): δ 7.3(s, 1H), 6.1(s, 1H), 4.0(m, 3H), 3.8(d, 1H), 0.9(s, 3H); mass spectrum : 314(M+1).

Flowchart 3

Example 2

2-Amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one (3-2 )

This compound was treated by treatment of compound 2-4 with 1,2-bis[(dimethylamino)-methylene]hydrazine in DMF to provide compound 3-1 , which was described by K. Alarcon et al. in Tetrahedron Lett, 41 : 7211-7215 (2000) using hydrolysis of aqueous 1 N NaOH in DMSO solution under the conditions described in DMSO.

The 2-fluoro-2-C-methyl ribonucleoside (R ² =F in the formula I) of the present invention can be prepared according to the effective synthetic methods in nucleoside and nucleotide chemistry practice. The synthesis of compound 6-3 is provided as an example, as described in Schemes 4-6. D-ribose ( 4-1 ) was first protected. In this case, esters such as acetate and benzoate provide suitable protecting groups, but alternative protecting groups may also be used. Esterification is achieved by reacting D-ribose with an appropriate acid halide or anhydride, optionally in the presence of a solvent such as diethyl ether, dioxane, tetrahydrofuran and methylene chloride. Such transformations are well known in the chemical literature and examples can be found in Greene, TW, Wuts, PGM, "ProtectiVe Groups in Organic Synthesis," John Wiley & Sons, Inc., 3rd Ed., 1999.

Flowchart 4

Intermediate 4-3 can be made using a number of methods. In Scheme 4, the Vorbruggen reaction ("Synthesis of Nucleosides", H.Vorbruggen and C.Ruh-Pohlenz, in Organic Reactions, vol.55, pp 1-630, 2000) was used to link the nucleobase 5-fluoro-4-chloro- 7H-Pyrrolo[2,3-d]pyrimidine ( 1-4 ) forms protected nucleoside 4-3. The ester protecting group is then removed by any suitable method, such as acid or base catalyzed hydrolysis and transesterification (eg using sodium methoxide in methanol) to provide 4-4 .

The C-2' methyl group is then introduced as described in Scheme 5. To enable orthogonal manipulation of the C-2' hydroxyl group, the hydroxyl groups at the 5' and 3' positions were first protected. This can be accomplished in a number of ways, one of which is depicted in Flowchart 5. The aforementioned reference, Greene, TW, Wuts, PGM, "Protective Groups in Organic Synthesis," John Wiley & Sons, Inc., 3rd Ed., 1999, contains many examples; especially suitable is tetraisopropyldisiloxane Subunit (tetraisopropyldisiloxanylidene) cyclic ether 5-1 .

Flowchart 5

The remaining free hydroxyl group is then oxidized to the ketone 5-2 by using a suitable oxidation method, eg, Swern or Moffatt oxidation or using Dess-Martin periodinane. Examples of such methods can be found in the relevant chemical literature, eg, in "Comprehensive Organic Transformations" by Richard C. Larock, VCH Publishers, 1989. 5-2 is then reacted with a suitable organometallic reagent, eg methyllithium and methylmagnesium halide. Such reactions are preferably carried out at low temperatures in suitable solvents such as tetrahydrofuran and diethyl ether. In this case, the methyl group is introduced from the less hindered face, and this stereocontrol produces mainly one isomer. Examples of such stereocontrol can be found in the relevant chemical literature, eg, in "Stereochemistry of Organic Compounds" by Ernest L. Eliel and Samuel H. Wilen, Wiley-Interscience Publications, 1994. A fluorine group is then introduced at the C-2' position as described in Scheme 6.

Flowchart 6

The hydroxyl groups present at the 3' and 5' positions can be optionally protected as described above. It is advantageous to use lower alkyl or simple aromatic esters such as acetates and benzoates. The reaction conditions are chosen such that the sterically hindered C-2' hydroxyl group remains unaffected. The introduction of fluorine is accomplished by using diethylaminosulfur trifluoride (DAST) or other suitable fluorinating reagents optionally in the presence of solvents such as aromatic hydrocarbons, tetrahydrofuran, chloroform at low temperature, room temperature or elevated temperature. Examples of such transformations are described in US Patent Publication No. 2005/0009737 (published January 13, 2005). The ester protecting group is then removed by hydrolysis followed by replacement of the chlorine atom present in the nucleobase with ammonia to yield the desired nucleoside. However, it is advantageous to carry out the last two steps in one kettle by ammonia in a suitable solvent, eg methanol, at room temperature or elevated temperature, using high pressure if necessary.

Example 3

4-Amino-5-fluoro-7-(2-fluoro-2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine

Step A:

A solution of D-ribose (5.00 g, 33 mmol), triethylamine (46 mL, 330 mmol) and DMAP (810 mg, 6.6 mmol) in anhydrous DMF (80 mL) was treated dropwise with p-toluoyl chloride (22 mL, 165 mmol) and Stirring was continued for 3 hours at room temperature. The reaction was quenched by pouring onto 300 g of ice. The crude product (3 x 100 mL) was extracted with dichloromethane after the ice had melted, dried over anhydrous magnesium sulfate, filtered and the solvent was removed in vacuo. The remaining oily residue was triturated with acetone. The solvent was decanted and the solid residue was recrystallized from isopropanol (200 mL). This product was used in the next step.

Step B:

This compound was synthesized using the method described by H. Vorbruggen and U. Niedballa in J. Ore. Chem ., 39:3654-3660 (1974). The 4-chloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine used in this transformation was prepared as described by AB Eldrup et al., in J.Med.Chem . , 47:5284 (2004) ( 1-4 ).

Step C:

This compound was synthesized from the product of step B as described in KL Smith et al., Bioorg. Med. Chem. Lett , 14:3517-3520 (2004).

Step D:

The compound was synthesized from the product of step C according to the method described in G. Gaubert et al., Tetrahedron Lett , 45:5629-5632 (2004).

Step E:

This compound was synthesized from the product of step D according to the method described in VL Moore et al., Biochemistry , 41:14066-14075 (2002).

Step F:

This compound was synthesized from the product of step E as described in VL Moore et al., Biochemistry , 4: 14066-14075 (2002).

Step G:

This compound was synthesized from the product of step F using the method published by M. Gallo et al., in Tetrahedron . 57:5707-5713 (2001).

Step H:

The product from step G was used by M. Akira et al., in Chem. Pharm. Bull ., 35:3967-3970 (1987). The diester was synthesized by the method published in.

Step I:

This compound was prepared using DAST treatment of the product of Step H as described in US Patent Publication No. 2005/0009737.

Step J:

Example 3 was synthesized by treating the product of step I with methanolic ammonia according to the conditions of US Pat. No. 6,777,395, Example 62, Step F, which is incorporated by reference in its entirety.

Example 4

nucleoside 5′-triphosphate

The nucleoside 5'-triphosphates of the invention were prepared following the general procedure described in Chem. Rev. 100:2047 (2000).

Example 5

Purification and Purity Analysis of Nucleoside 5′-triphosphate Derivatives

Triphosphate was purified by anion exchange (AX) chromatography over a 30×100 mm Mono Q column (Pharmacia) with a 50 mM Tris buffer system at pH 8. The elution gradient is typically 40mM NaCl to 0.8M NaCl in two column volumes at 6.5mL/min. Appropriate fractions from anion exchange chromatography were collected and desalted by reverse phase (RP) chromatography using a Luna C18 250 x 21 mm column (Phenomenex) at a flow rate of 10 mL/min. The elution gradient was typically 1% to 95% methanol over 14 minutes with a constant concentration of 5 mM triethylammonium acetate (TEAA).

Mass spectra of the purified triphosphates were determined by on-line HPLC mass spectrometry on a Hewlett-Packard (Palo Alto, CA) MSD 1100. RP HPLC uses Phenomenex Luna (C18(2)), 150×2mm, plus 30×2mm guard column, 3-μm particle size. A 0-50% linear gradient (15 minutes) of acetonitrile in 20 mM TEAA (triethylammonium acetate) pH 7 was implemented in negative ionization mode. Electrospray was generated with nitrogen and a pneumatic nebulizer. Take the molecular weight range of 150-900 as the sample. Molecular weights were determined using the HP Chemstation analysis package.

Purity of purified triphosphates was determined by analytical RP and AX HPLC with Phenomonex Luna or Jupiter columns (250 x 4.6 mm), 5 μm particle size typically run with a 2-70% acetonitrile gradient in 100 mM TEAA, pH 7, over 15 minutes. AX HPLC was performed on a 1.6 x 5mm Mono Q column (Pharmacia). Triphosphate was eluted with a gradient of 0-0.4M NaCl at a constant concentration of 50mM Tris at pH 8. The purity of the triphosphate is generally >80%.

biological test

The assays used to determine inhibition of HCV NS5B polymerase and HCV replication are described below.

The effectiveness of the compounds of the invention as inhibitors of HCV NS5B RNA-dependent RNA polymerase (RdRp) was determined in the following assay.

A. Inhibition of HCV NS5B polymerase test:

This test is used for determining that fluorinated pyrrolo [2,3-d] pyrimidine nucleoside triphosphate of the present invention inhibits the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) in different numbers (heteromeroc) RNA Capacity for enzymatic activity on the template.

step:

Test buffer conditions: (50μL-total amount/reactant)

20mM Tris, pH7.5

50 μM EDTA

5mM DTT

2mM _MgCl2

80mM KCl

0.4U/μL RNAsin (Promega, stock solution is 40 units/μL)

0.75 μgt500 (a 500-nt RNA made by T7 runoff transcription with sequences from the NS2/3 region of the hepatitis C genome)

1.6 μg purified hepatitis C NS5B (formed from 21 C-terminal truncated amino acids)

1 μM A, C, U, GTP (mixture of nucleoside triphosphates)

[α- ^32P ]-GTP or [α- ^33P ]-GTP

Nucleoside triphosphates were tested at various concentrations up to a final concentration of 100 μM.

Prepare an appropriate volume of reaction buffer including enzyme and template T500. The nucleoside triphosphates of the invention were pipetted into wells of a 96-well plate. A mixture of nucleoside triphosphates (NTP's) including radiolabeled GTP was prepared and pipetted into wells of a 96-well plate. The reaction was initiated by adding the enzyme-template reaction solution and allowed to proceed at room temperature for 1-2 hours.

The reaction was quenched by adding 20 μL of 0.5M EDTA, pH 8.0. A blank experiment was included in which the quenching solution was added to the NTPs prior to the addition of the reaction buffer.

50 [mu]L of the quenched reaction was spotted onto a DE81 filter disc (Whatman) and allowed to dry for 30 minutes. Wash the filter disc with 0.3M ammonium formate, pH 8 (150 mL for each wash, until the cpm in 1 mL of the wash solution is less than 100, usually 6 washes). Filters were counted in 5 mL of scintillation fluid in a scintillation counter.

Percent inhibition was calculated by the following formula: % inhibition = [1 - (cpm in test reaction - cpm in blank reaction)/(cpm in control reaction - cpm in blank reaction)] x 100.

Representative compounds tested in the HCV NS5B polymerase assay showed _IC50 values of less than 50 micromolar.

B. Inhibition of HCV RNA replication test:

Compounds of the invention were also evaluated for their effect on hepatitis C virus RNA replication in cultured hepatocarcinoma (HuH-7) cells containing a subgenomic HCV replicon. The details of the test are described below. This replicon test is in V.Lohmann, F.Komer, JO.Koch, U.Herian, L.Theilmann and R.Bartenschlager, "Replication of a Sub-genomic Hepatitis C Virus RNAs in a Hepatoma Cell Line," Science 28 5: 110 (1999), modified from what was stated in 110 (1999).

Experimental program:

The assay is an in situ ribonuclease protected, affinity scintillation based plate assay (SPA). 10,000-40,000 cells were added to 100-200 μL of medium containing 0.8 mg/mL G418 in 96-well cytostar plates (Amersham). Compounds were added to cells at various concentrations up to 100 [mu]M in 1% DMSO at 0-18 hours and then incubated for 24-96 hours. Cells were fixed (20 min, 10% formalin), permeabilized (20 min, 0.25% TritonX-100/PBS) and hybridized (overnight, 50°C) with a single-stranded ³³ P RNA probe, which ³³ PRNA is complementary to the (+) strand NS5B (or other genes) contained in the RNA virus genome. Cells were washed, treated with RNAse, washed, heated to 65°C, and counted in a Top-Count. A decrease in counts per minute (cpm) was read as inhibition of replication.

Human HuH-7 hepatoma cells selected to contain a subgenomic replicon harbor cytoplasmic RNA consisting of HCV 5' untranslated region (NTR), neomycin selectable marker, EMCV IRES (internal ribosome entry site ) and HCV nonstructural protein NS3-NS5B followed by 3'NTR.

Representative compounds tested in this replicate assay showed _EC50 values of less than 100 micromolar.

C. Intracellular metabolism test:

The compounds of the present invention were also evaluated for their ability to enter human liver cancer cell lines and be converted into the corresponding nucleoside 5'-mono-, di- and triphosphates in the cells

Two cell lines, HuH-7 and HBI10A, were used for intracellular metabolism studies of compounds of the present invention. HuH-7 is a human liver cancer cell line, and HBI10A represents a clone obtained from HuH-7 cells in which the HCV bicistronic replicon exists. Spread HuH-7 cells with 1.5×10 ⁶ cells per 60-mm culture dish to the complete Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, and spread HBI10A cells to the medium containing G418 (0.8mg/ mL) such that 80% of the cells were confluent at the time of compound addition. Tritiated compounds were incubated at 2 μM in cell culture medium for 3 or 23 hours. Cells were harvested, washed with phosphate-buffered saline, and counted. Cells were then extracted with 70% methanol, 20 mM tritiated compound EDTA, 20 mM EGTA and centrifuged. Lysates were dried and radiolabeled nucleotides were assayed using ion-pair reversed-phase (C-18) HPLC on a Waters Millenium system (IN/US Systems) coupled to an on-line β-RAM scintillation detector. HPLC mobile phase (a) 10 mm potassium phosphate with 2 mm tetrabutylammonium hydroxide and (b) 50% methanol containing 10 mm potassium phosphate with 2 mm tetrabutylammonium hydroxide. Peak identification was performed by comparing retention times relative to standards. Activity is expressed as picomoles of nucleotides detected in ¹⁰⁶ HuH-7 or HBI10A cells.

The cytotoxicity and antiviral specificity of the fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds of the invention were also evaluated in counterscreens as described below.

C. Anti-filtering:

The inhibitory ability of the fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds of the present invention on human DNA polymerase was tested in the following assay.

a. Inhibition of human DNA polymerase α and β:

Reaction conditions:

50μL reaction volume

Reaction Buffer Components:

20mM Tris-HCl, pH7.5

200μg/mL bovine serum albumin

100mM KCl

2mM β-mercaptoethanol

10mM _MgCl2

1.6 μM dA, dG, dC, dTTP

α- ³³ P-dATP

Enzymes and templates:

0.05mg/mL Gap Fish Sperm DNA Template

0.01U/μL DNA polymerase α or β

Preparation of gapped fish sperm DNA template:

Add 5 μL 1M MgCl ₂ to 500 μL activated fish sperm DNA (USB 70076); heat to 37°C, and add 30 μL 65U/μL exonuclease III (GibcoBRL18013-011);

Incubate at 37°C for 5 minutes;

The reaction was terminated by heating to 65 °C for 10 minutes;

Load a 50-100 μL aliquot onto a Bio-spin 6 column (Bio-Rad 732-6002) equilibrated with 20 mM Tris-HCl, pH 7.5;

Elution was performed by centrifugation at 1,000×g for 4 minutes;

The eluate was collected and the absorbance was measured at 260nm to determine the concentration.

Dilute the DNA template to an appropriate volume of 20mM Tris-HCl, pH 7.5, and dilute the enzyme to an appropriate volume of 20mM Tris-HCl containing 2mM β-mercaptoethanol and 100mM KCl. Pipette template and enzyme into microcentrifuge tubes or 96-well plates. Blank reactions excluding enzymes and control reactions excluding test compounds were also prepared in enzyme dilution buffer and test compound solvent, respectively. The reaction was initiated with a reaction buffer having the above composition. Reactions were incubated at 37°C for 1 hour. Add 20 μL of 0.5M EDTA to quench the reaction. 50 μL of the quenched reaction was spotted onto Whatman DE81 filter discs and air dried. Wash the filter piece repeatedly with 150mL of 0.3M ammonium formate pH8 until 1mL of the washing solution is <100cpm. The filter was washed twice with 150 mL of absolute ethanol and once with 150 mL of absolute ether, dried and counted in 5 mL of scintillation fluid.

Percent inhibition was calculated by the following formula: % inhibition = [1 - (cpm in test reaction - cpm in blank reaction)/(cpm in control reaction - cpm in blank reaction)] x 100.

b. Inhibition of human DNA polymerase gamma:

Inhibition of human DNA polymerase gamma was tested in reactions containing the following components: 0.5 ng/μL enzyme; 10 μM dATP, dGTP, dCTP and TTP; 2 μCi/reaction [a- ³³ P]-dATP and 0.4 μg/ μL of activated fish sperm DNA (purchased from US Biochemical) in a buffer containing 20 mM Tris pH 8, 2 mM β-mercaptoethanol, 50 mM KCl, 10 mM MgCl ₂ and 0.1 μg/μL BSA. The reaction was allowed to proceed for 1 hour at 37°C and then quenched by adding 0.5M EDTA to a final concentration of 142mM. The product formed was quantified by anion exchange filter binding and scintillation counting. Compounds were tested at concentrations up to 50 μM.

Percent inhibition was calculated by the following formula: % inhibition = [1 - (cpm in test reaction - cpm in blank reaction)/(cpm in control reaction - cpm in blank reaction)] x 100.

The ability of fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds of the invention to inhibit HIV infection and HIV spread was determined in the assay described below.

c. HIV infectivity test:

The assay was performed with a variant of HeLa Magi cells expressing CXCR4 and CCR5, selected for low background β-galactosidase (β-gal) expression. Cells were infected for 48 hours and β-gal product from the integrated HIV-1 LTR promoter was quantified using a chemiluminescent substrate (Galactolight Plus, Tropix, Bedford, MA). Inhibitors were titrated (in replicates) in two-fold serial dilutions starting at 100 [mu]M; percent inhibition at each concentration was calculated relative to control infection.

d. Inhibition of HIV spread:

Compounds of the invention were tested for inhibition of human immunodeficiency virus by the methods described in U.S. Patent No. 5,413,999 (May 9, 1995) and JPVacca et al. , Proc. (HIV) diffusion capacity, the contents of which are incorporated by reference in their entirety.

Fluorinated pyrrolo[2,3-d]pyrimidine nucleoside compounds of the invention were screened for their activity on cultured hepatoma (HuH-7) cells containing a subgenomic HCV replicon in the MTS cell-based assay described in the following assay. Cytotoxicity. The HuH-7 cell line is described in H. Nakabayashi et al., Cancer Res ., 42:3858 (1982).

e. Cytotoxicity test:

Prepare cell cultures in appropriate media at a concentration of approximately 1.5 x ¹⁰ cells/mL for suspension cultures in 3-day incubations and 5.0 x ¹⁰ cells/mL for stickers in 3-day incubations wall culture. 99 μL of cell culture was transferred to wells of a 96-well tissue culture treated plate and 1 μL of test compound at 100-fold final concentration in DMSO was added. Incubate the plate at 37 °C and 5% _CO2 for a specific period of time. After the incubation period, 20 μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS) (Promega) was added to each well and the plates were incubated at 37° C. and 5% CO ₂ for a period of up to 3 hours. The plate was shaken to mix the wells and the absorbance at 490 nm was read with a plate reader. A standard curve of suspension cultured cells was prepared using the known number of cells immediately before the addition of MTS reagent. Metabolically active cells break down MTS to formazan, which absorbs at 490nm. Absorbance at 490 nm in the presence of compounds was compared to the absorbance of cells without any added compound.

Reference: Cory, AH et al., "Use of an aqueous soluble tetrazolium/formazana assay for cell growth assays inculture," Cancer Commun . 3: 207 (1991).

The following assay was used to determine the activity of compounds of the invention against other RNA-dependent RNA viruses.

a. Determination of the in vitro antiviral activity of the compound against rhinovirus (cytopathic effect inhibition test):

Experimental conditions are described in the article by Sidwell and Huffman, "Use of disposable microtissue culture plates for antiviral and interferon induction studies," Appl. Microbiol . 22:797-801 (1971).

Virus:

Rhinovirus type 2 (RV-2) HGP strain was used with KB cells and medium (0.1% _NaHCO3 , no antibiotics) as described in the Sidwell and Huffman reference. Viruses obtained by ATCC were obtained from throat swabs of adult males with mild acute febrile upper respiratory illness. Rhinovirus type 9 (RV-9) 211 strain and rhinovirus type 14 (RV-14) Tow strain were also obtained from the American Type Culture Collection (ATCC) in Rockville, MD. RV-9 was obtained from human throat washes, and RV-14 was obtained from throat swabs of young adults with upper respiratory disease. These two viruses were used together with human cervical epithelioid carcinoma cells HeLa Ohio-1 cells (Dr. Fred Hayden, UniV. of VA). MEM (Eagle's minimal basal medium) with 5% fetal bovine serum (FBS) and 0.1% _NaHCO3 was used as growth medium. The antiviral test medium for all three virus types was MEM with 5% FBS, 0.1% NaHCO ₃ , 50 μg gentamicin/mL and 10 mM MgCl ₂ .

2000 μg/mL was the highest concentration used to test the compounds of the invention. Virus was added to the assay plate approximately 5 minutes after the addition of the test compound. At the same time, appropriate control samples were prepared. The test plate was cultured under the conditions of humid air, 5% CO ₂ , and 37°C. Cytotoxicity was monitored by microscopic observation of morphological changes in control cells. Regression analysis of virus CPE data and toxicity control data gave ED50 (50% effective dose) and CC50 (50% cytotoxic concentration). The selectivity index (SI) is calculated by the formula: SI=CC50÷ED50.

b. measure the in vitro antiviral activity (CPE inhibition test) of compound against dengue fever Banzi and yellow fever:

Experimental details are given in the aforementioned Sidwell and Huffman reference.

Virus:

Obtain the New Guinea strain of dengue type 2 virus from the Centers for Disease Control. The virus (Vero) was cultured with two lines of African green monkey kidney cells and tested for anti-virus (MA-104). The yellow fever virus 17D strain prepared from infected mouse brains and the Banzi virus H336 strain isolated from the sera of febrile boys in South Africa were obtained from the ATCC. Both viruses and assays use Vero cells.

Cells and media:

MA-104 cells (Bio Whittaker, Inc., Walkersville, MD) and Vero cells (ATCC) were used in 199 medium with 5% FBS and 0.1% _NaHCO3 , without antibiotics.

The test medium for dengue, yellow fever and Banzi virus was MEM, 2% FBS, 0.18% NaHCO ₃ and 50 μg gentamicin/mL.

Antiviral testing of compounds of the invention was performed as described in the Sidwell and Huffman reference, similar to the rhinovirus antiviral testing described above. Appropriate cytopathic effect (CPE) records were obtained after 5-6 days for each virus.

c. measure the in vitro antiviral activity (CPE inhibition test) of compound to West Nile virus:

Experimental details are given in the Sidwell and Huffman reference cited above. West Nile virus, New York isolates from crow brain were obtained from the Centers for Disease Control. Vero cells were grown and used as above. The test medium was MEM, 1% FBS, 0.1% NaHCO ₃ and 50 μg gentamicin/mL.

Antiviral testing of the compounds of the present invention was performed according to the methods described in the Sidwell and Huffman reference, similar to those used for rhinovirus activity testing. Appropriate cytopathic effect (CPE) records were obtained after 5-6 days.

d. Determination of in vitro antiviral activity of compounds against rhinovirus, yellow fever, dengue fever, Banzi and West Nile virus (center red uptake test):

After the CPE inhibition assay was performed as above, the cytopathic assay described in "Microtiter Assay for Interferon: Microspectrophotometric Quantitation of Cytopathic Effect," Appl. Environ. Microbiol . 31:35-38 (1976) was used. Assay plates were read with a model EL309 miniplate reader (Bio-Tek Instruments Inc.). ED50 and CD50 values were calculated as above.

Examples of Pharmaceutical Formulations

As a specific embodiment of the oral composition of the compound of the present invention, 50 mg of the compound of Example 1 or Example 2 is used to prepare the preparation together with fully pulverized lactose to provide a total amount of 580-590 mg, which is packed into a No. 0 hard in gelatin capsules.

Although the invention has been described and illustrated with reference to specific embodiments thereof, those skilled in the art will appreciate that various changes, modifications and substitutions can be made thereto without departing from the spirit and scope of the invention. . For example, effective doses other than the preferred doses listed above may be suitable, as inferred from the variable response of humans in need of treatment for severe HCV infection. Likewise, the observed pharmacological responses may also vary according to and depend upon the particular active compound chosen or the presence or absence of a pharmaceutical carrier as well as the type of formulation and mode of administration used, and such expected changes or results may be expected in light of the objectives and practice of the present invention difference. Accordingly, the invention is to be limited only by the scope of the appended claims, which claims are afforded the broadest.

Claims (7)

1, the nucleoside compound of structural formula I

And pharmaceutically acceptable salt; Wherein

R ¹Be hydrogen or fluorine;

R ²Be fluorine or hydroxyl;

R ³Be hydrogen, C _1-16Alkyl-carbonyl, C _2-18Alkenyl carbonyl, C _1-10Alkoxy carbonyl, C _3-6Naphthene base carbonyl, C _3-6Cyclo alkoxy carbonyl, or the aminoacyl residue of following structural formula;

R ⁴Be hydrogen, C _1-10Alkyl-carbonyl, phosphoryl or its ring-type prodrug ester, two phosphoryls, three phosphoryls, C _2-18Alkenyl carbonyl, C _1-10Alkoxy carbonyl, C _3-6Naphthene base carbonyl, C _3-6Cyclo alkoxy carbonyl, CH ₂O (C=O) C _1-4Alkyl, CH (C _1-4Alkyl) O (C=O) C _1-4The aminoacyl residue of alkyl or following structural formula;

The residue of following structural formula

R ⁵Be amino or hydroxyl;

R ⁶Be hydrogen, amino or fluorine;

R ⁷Be hydrogen, C _1-5Alkyl or phenyl C _0-2Alkyl; With

R ⁸Be hydrogen, C _1-4Alkyl, C _1-4Acyl group, benzoyl.C _1-4Alkyl amino-carbonyl, phenyl C _0-2Alkyl amino-carbonyl, C _1-4Alkyl sulphonyl or phenyl C _0-2Alkyl sulphonyl;

R ⁹Be hydrogen, C _1-5Alkyl, phenyl or benzyl, wherein alkyl does not replace or is selected from hydroxyl, methoxyl group by one, amino, carboxyl, carbamyl, guanidine radicals, sulfydryl, methyl mercapto, the substituent group of 1H-imidazolinyl and 1H-indol-3-yl replace and phenyl and benzyl does not replace or independently be selected from halogen by one or two, the substituent group replacement of hydroxyl and methoxyl group wherein;

R ¹⁰Be hydrogen, C _1-6Alkyl, C _3-6Cycloalkyl, phenyl or benzyl, wherein alkyl and cycloalkyl do not replace or independently are selected from halogen, hydroxyl, carboxyl, C by one to 3 _1-4The substituent group of alkoxyl replaces and phenyl and benzyl does not replace or independently be selected from halogen, hydroxyl, cyano group, C by to three wherein _1-4The substituent group of alkoxyl and trifluoromethyl replaces; Ar is unsubstituted phenyl or is independently selected from halogen, C by one to 3 _1-4Alkyl, C _1-4Alkoxyl, C _1-4Alkylthio group, cyano group, nitro, amino, carboxyl, trifluoromethyl, C _1-4Alkyl amino, two (C _1-4Alkyl) amino, C _1-4Alkyl-carbonyl, C _1-4Alkyl-carbonyl oxygen base and C _1-4Alkoxy carbonyl; Condition is to work as R ¹, R ³, R ⁴, and R ⁶Be hydrogen and R ²When being hydroxyl, R ⁵Not amino.

2, the chemical compound of claim 1, wherein R ¹Be hydrogen; R ²It is hydroxyl; R ³And R ⁴Be hydrogen.

3, the chemical compound of claim 1, wherein R ¹Be hydrogen; R ²It is fluorine; R ³And R ⁴Be hydrogen.

4, the chemical compound of claim 1 is

2,4-amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo-[2,3-d] pyrimidine;

2-amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo-[2,3-d] pyrimidine--4 (3H)-ketone;

2,4-diaminourea-5-fluoro-7-(2-fluoro-2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo-[2,3-d] pyrimidine;

4-amino-5-fluoro-7-(2-fluoro-2-C-methyl-β-D-ribofuranosyl)-7H-;

2-amino-5-fluoro-7-(2-fluoro-2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo-[2,3-d] pyrimidine--4 (3H)-ketone;

And pharmaceutically acceptable salt.

5, comprise the chemical compound of claim 1 and the pharmaceutical composition of pharmaceutically acceptable carrier.

6, the purposes of the compounds for treating mammal infection with hepatitis C virus of claim 1.

7, the compound of claim 1 is used for the treatment of the purposes of the medicine of mammal infection with hepatitis C virus.