CN101043893A

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

Info

Publication number: CN101043893A
Application number: CN 200580036083
Authority: CN
Inventors: M·麦科斯; D·B·奥尔森; J·莱奥内; P·L·杜雷特
Original assignee: Merck and Co Inc
Current assignee: Merck and Co Inc
Priority date: 2004-10-21
Filing date: 2005-10-17
Publication date: 2007-09-26

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

Treatment RNA-RNA-dependent viral infection fluoridize pyrrolo-[2,3-d] pyrimidine nucleoside

Invention field

The present invention relates to fluoridize pyrrolo-[2,3-d] pyrimidine nucleoside compound and some derivant thereof, they synthetic and their purposes as RNA-RNA-dependent viral polymerase inhibitors.Chemical compound of the present invention is the inhibitor of RNA-RNA-dependent virus replication and can be used for treating the infection of RNA-RNA-dependent virus-virus.Especially, they can be used as the inhibitor of hepatitis C virus (HCV) NS5B polymerase, the inhibitor that duplicates as HCV and be used for the treatment of infection with hepatitis C virus.

Background of the present invention

Prior art level hepatitis C virus (HCV) in a lot of infected individualities that summary treatment HCV infects is to cause chronic hepatopathy, and for example the main health problem of liver cirrhosis and hepatocarcinoma estimates that infected individuality is the 2-15% of world population.Estimate sheet according to the Center for Disease Control just has 3900000 people to be infected in the U.S., approximately is five times of infection Human Immunodeficiency Viruses (HIV).According to the estimation of World Health Organization (WHO), the whole world has the people above 1700,000,000 to be infected, and has every year 300 ten thousand to 4 million peoples to be infected.In case infected, about 20% people can remove virus, and after this others will carry HCV all the life.10% to 20% chronic infection individuality finally can develop into destructive liver cirrhosis of liver or cancer.This virosis can be passed through contaminated blood and blood products, and contaminated pin infects through parenteral route, or through the property infection with by infected mother or be the offspring that mother's vertical infection of carrier is given them.Current HCV treatment of infection only limits to use separately recombinant interferon-α or with the immunization therapy of itself and the associating of nucleoside analogues 'Libaweilin ', its clinical benefit is limited.And, also there is not the HCV vaccine of determining.Therefore, urgent need can effectively be resisted the therapeutic agent of the improvement of chronic HCV infection.The state of the art that treatment HCV is infected is summarized, can be with reference to following publication: B.Dymock, etc., " Novel approaches to the treatment ofhepatitis C virus infection, " Antiviral Chemistry ﹠amp; Chemotherapy.11:79-96 (2000); H.Rosen, etc., " Hepatitis C virus:current understandingand prospects for future therapies, " Molecular Medicine Today.5:393-399 (1999); D.Moradpour, etc., " Current and evolving therapies forhepatitis C, " European J.Gastroenterol. Hepatol., 11:1189-1202 (1999); R.Bartenschlager, " Candidate Targets for Hepatitis C Virus-SpecificAntiviral Therapy, " Intervirology.40:378-393 (1997); G.M.Lauer and B.D.Walker, " Hepatitis C Virus Infection, " N.Engl.J.Med..345:41-52 (2001); B.W.Dymock, " Emerging therapies for hepatitis C Virusinfection, " Emerging Drugs.6:13-42 (2001); And C.Crabb, " Hard-WonAdvances Spark Excitement about Hepatitis C, " Science: 506-507 (2001); Be incorporated herein by reference its full content is complete.

Adopted different approach treatment HCV, comprised the serine protease (NS3 protease) that suppresses virus, unwindase and RNA-RNA-dependent polymerase (NS5B) and exploitation vaccine.

The HCV virion is tunicary positive chain RNA virus, and the single oligoribonucleotide genome sequence that is had is shown about 9600 bases, can encode to have about 3,010 amino acid whose polyproteins.The protein product of HCV gene is by structural protein C, E1 and E2 and non-structural protein NS2, and NS3, NS4, NS4B, NS5a and NS5B form.Think that non-structure (NS) protein provides catalysis means (catalytic machinery) for virus replication.NS3 protease discharges NS5B by the polyprotein chain, and it is a RNA-RNA-dependent polymerase.By in the HCV replication cycle, needing HCV NS5B polymerase as the synthetic double-stranded RNA of the strand viral RNA of template.Therefore think that in the HCV replication complex NS5B polymerase is necessary component.[referring to 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 andKinetic Analyses of NS5B RNA-Dependent RNA Polymerase of theHepatitis C Virus, " Viroloey.249:108-118 (1998)]. suppress HCV NS5B polymerase and can prevent to form double-stranded HCV RNA, therefore, constitute the approach that has much captivation of exploitation HCV specificity antivirus treatment.

The development of the HCV NS5B inhibitor that the potential HCV of treatment is infected is summarized, referring to M.P.Walker etc. and " Promising candidates for the treatment ofchronic hepatitis C, " Expert Opin.Invest.Drugs.12:1269-1280 (2003); P.Hoffmann etc., " Recent patents on experimental therapy for hepatitisC virus infection (1999-2002), " Expert Opin.Ther.Patents, " 13:1707-1723 (2003); And V.Brass, etc., " Recent developments in targetidentification against HCV, " Expert Opin.Ther.Targets," 8:295-307 (2004) .A.E.Eldrup etc. exist " Structure-Activity Relationship of PurineRibonucleosides for Inhibition of HCV RNA-Dependent RNAPolymerase " has reported among the J.Med.Chem..47:2283-2295 (2004) that suppressing HCV by the purine ribonucleotide duplicates.Continue the treatment approach of need be as the structure of HCV AG14361 different nucleoside derivates as the HCV treatment

United States Patent (USP) the 6th, 777,396 (publication on August 17th, 2004) disclose pyrrolo-[2, the 3-d] Pyrmidine nucleoside derivatives that is used for the treatment of a series of new constructions of HCV infection as the HCVNS5B AG14361.D.B.Olsen etc. exist " A 7-Deaza-Adenosine Analog is aPotent and Selective Inhibitor of HCV Replication with ExcellentPharmacokinetic Properties, " Antimicrob.Agents Chemother..48:3944-3953 in (2004) and A.E.Eldrup, Deng at " Structure-ActivityRelationship of Heterobase-Modified 2 '-C-Methyl Ribonucleosides asInhibitors of Hepatitis C Virus RNA Replication, " J.Med.Chem., 47:5284-5297 (2004). the biological property to this compounds is described.The nucleoside derivates that fluorine is introduced in the position of the C-5 that finds now at pyrrolo-[2,3-d] pyrimidine nuclear to be provided is to have good pharmacokinetics character, for example better is distributed in the more efficiently HCV rna replicon inhibitor of liver.

Therefore, an object of the present invention is to provide and fluoridize pyrrolo-[2,3-d] pyrimidine nucleoside compound and its some derivant as RNA-RNA-dependent viral polymerase inhibitors, particularly HCV NS5B AG14361.

Another object of the present invention provides fluoridizes pyrrolo-[2,3-d] pyrimidine nucleoside compound and its some derivant as RNA-RNA-dependent viral polymerase inhibitors, particularly hepatitis c viral replication inhibitor.

Another object of the present invention provides and is used for the treatment of RNA-RNA-dependent viral infection, and particularly HCV infects fluoridizes pyrrolo-[2,3-d] pyrimidine nucleoside compound and its some derivant.

Another object of the present invention provides the pharmaceutical composition of fluoridizing pyrrolo-[2,3-d] pyrimidine nucleoside compound that comprises with pharmaceutically acceptable carrier associating.

Another object of the present invention provides the pharmaceutical composition of fluoridizing pyrrolo-[2,3-d] pyrimidine nucleoside compound and its some derivant of the present invention that comprises as RNA-RNA-dependent viral polymerase inhibitors, particularly HCV NS5B AG14361.

Another object of the present invention provides the pharmaceutical composition of fluoridizing pyrrolo-[2,3-d] pyrimidine nucleoside compound and its some derivant of the present invention that comprises as the inhibitor, particularly HCV replication inhibitors of RNA-RNA-dependent virus replication.

Another object of the present invention provides and comprises and be used for the treatment of RNA-RNA-dependent viral infection, particularly the pharmaceutical composition of fluoridizing pyrrolo-[2,3-d] pyrimidine nucleoside compound and its some derivant of the present invention that infects of HCV.

Another object of the present invention is to comprise the pharmaceutical composition of fluoridizing pyrrolo-[2,3-d] pyrimidine nucleoside compound and its some derivant of the present invention of uniting with the activating agent of other anti-RNA-RNA-dependent virus, particularly anti-HCV.

Another object of the present invention provides and suppresses RNA-RNA-dependent varial polymerases, the particularly method of HCV NS5B polymerase.

Another object of the present invention provides and suppresses RNA-RNA-dependent virus replication, particularly suppresses the method that HCV duplicates.

Another object of the present invention provides treatment RNA-RNA-dependent viral infection, particularly treats the method that HCV infects.

Another object of the present invention provides and the viral combined treatment of other anti-RNA-RNA-dependent RNA-RNA-dependent virus, infects, particularly the method that infects with the activating agent therapeutic alliance HCV of other anti-HCV.

Another object of the present invention is as suppressing RNA-RNA-dependent virus replication and/or treatment RNA-RNA-dependent viral infection, that suppresses particularly that HCV duplicates and/or treat medicine that HCV infects fluoridizes pyrrolo-[2,3-d] pyrimidine nucleoside compound and its some derivant and pharmaceutical composition thereof.

Another object of the present invention provides the pyrrolo-[2 of fluoridizing of the present invention, 3-d] pyrimidine nucleoside compound and its some derivant and preparation of pharmaceutical compositions inhibition RNA-RNA-dependent virus replication and/or treatment RNA-RNA-dependent viral infection, particularly suppress the purposes that HCV duplicated and/or treated the medicine of HCV infection.

To become apparent according to the following detailed description these and other objects.

Summary of the invention

The present invention relates to indicate the nucleoside compound of the structural formula I of stereochemical configuration

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.

The chemical compound of formula I can be used as RNA-RNA-dependent varial polymerases, the particularly inhibitor of HCVNS5B polymerase.They also are the inhibitor that duplicate of RNA-RNA-dependent virus replication, particularly HCV and can be used for treating RNA-RNA-dependent viral infection, particularly treat HCV and infect.

The present invention also comprise the pharmaceutical composition that contains this chemical compound separately or with other anti-RNA-RNA-dependent virus, the particularly pharmaceutical composition of this chemical compound of other activating agent associating of anti-HCV, and the method that suppresses RNA-RNA-dependent virus replication and treatment RNA-RNA-dependent viral infection.

Detailed Description Of The Invention

And pharmaceutically acceptable salt; Wherein

R ¹Be hydrogen or fluorine;

R ²Be fluorine or hydroxyl;

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 amino-carbonyl, phenyl C _0-2Alkyl amino-carbonyl, C _1-4Alkyl sulphonyl or phenyl C _0-2Alkyl sulphonyl;

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 by one or two wherein, hydroxyl with, cyano group, C _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 _1-4The group of alkoxy carbonyl replaces;

Formula I chemical compound can be used as the inhibitor of RNA RNA-dependent varial polymerases.They also are the inhibitor of RNA RNA-dependent virus replication, can be used for treating RNA RNA-dependent viral infection.

In an embodiment of The compounds of this invention, R ¹Be hydrogen; R ²It is hydroxyl; R ³And R ⁴Be hydrogen.

In second embodiment of The compounds of this invention, R ¹Be hydrogen; R ²It is fluorine; R ³And R ⁴Be hydrogen; Condition is to work as R ¹, R ³, R ⁴, and R ⁶Be hydrogen and R ²When being hydroxyl, R ⁵Not amino.

In the 3rd embodiment of The compounds of this invention, Ar is unsubstituted phenyl.

In the 4th embodiment of The compounds of this invention, R ⁹Be selected from hydrogen, methyl, ethyl, n-pro-pyl, isopropyl, isobutyl group, 2-methyl isophthalic acid-propyl group, methylol, mercapto methyl, carboxymethyl, carbamoyl methyl, 1-ethoxy, the 2-carboxyethyl, the 2-carbamoyl methyl, the 2-methylthio ethyl, 4-amino-the 1-butyl, 3-amino-1-propyl group, 3-guanidine radicals-1-propyl group, 1H-imidazol-4 yl methyl, phenyl, 4-hydroxy benzenes methyl, 1H-indol-3-yl methyl.In this embodiment of a class, R ⁹Be methyl or benzyl.

In the 5th embodiment of The compounds of this invention, R ¹⁰Be C _1-6Alkyl, cyclohexyl, phenyl or benzyl.In this embodiment of a class, R ¹⁰It is methyl.

In the 6th embodiment of The compounds of this invention, Ar is unsubstituted phenyl, R ⁹Be methyl or benzyl, R ¹⁰It is methyl.

Exemplary and nonrestrictive example as the The compounds of this invention of the structural formula I of the inhibitor of RNA RNA-dependent varial polymerases is 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] 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.

In one embodiment of the invention, the pyrrolo-[2 of fluoridizing of the present invention, 3-d] pyrimidine nucleoside compound can be used as the inhibitor of sense single stranded rna RNA-dependent varial polymerases, the inhibitor of sense single stranded rna RNA-dependent virus replication, and/or be used for the treatment of the dependent picornavirus infection of sense single stranded rna.In this embodiment of a class, sense single stranded rna RNA-dependent virus is flaviviridae (Flaviviridae) virus or Picornaviridae (Picornaviridae) virus.In such subclass, Picornaviridae virus is rhinovirus, poliovirus or hepatitis A virus.In such second subclass, flaviviridae is selected from hepatitis C virus, yellow fever virus, dengue virus, west nile virus, Japanese encephalitis virus, banzi virus and bovine viral diarrhea virus (BVDV).In the subclass of this subclass, flaviviridae is a hepatitis C virus.

Another aspect of the present invention relates to the method that suppresses RNA RNA-dependent varial polymerases, the method that suppresses RNA RNA-dependent virus replication, and/or the method for the mammiferous RNA RNA-dependent viral infection of this treatment of treatment needs, this method comprises the compound in structural formula I to administration treatment effective dose.

In an embodiment of this respect of the present invention, RNA RNA-dependent varial polymerases is a sense single stranded rna RNA-dependent varial polymerases.In this embodiment of a class, sense single stranded rna RNA-dependent varial polymerases is flaviviridae polymerase or Picornaviridae varial polymerases.In such subclass, the Picornaviridae varial polymerases is rhinovirus polymerase, poliovirus polymerase or hepatitis A virus polymerase.In such second subclass, the flaviviridae polymerase is selected from 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 the subclass of this subclass, the flaviviridae polymerase is the hepatitis C virus polymerase.

In second embodiment of this respect of the present invention, RNA RNA-dependent virus replication is a sense single stranded rna RNA-dependent virus replication.In this embodiment of a class, sense single stranded rna RNA-dependent virus replication is that flaviviridae duplicates or the Picornaviridae virus replication.In such subclass, the Picornaviridae virus replication is that rhinovirus replication, poliovirus are duplicated or hepatitis A virus duplicates.In such second subclass, flaviviridae duplicates and is selected from that hepatitis c viral replication, yellow fever virus duplicate, dengue virus duplicates, west nile virus is duplicated, Japanese encephalitis virus is duplicated, banzi virus duplicates and bovine viral replicability diarrhea virus duplicates.In the subclass of this subclass, flaviviridae copies as hepatitis c viral replication.

In the 3rd embodiment of this respect of the present invention, RNA RNA-dependent viral infection is a sense single stranded rna RNA-dependent viral infection.In this embodiment of a class, sense single stranded rna RNA-dependent viral infection is flaviviridae infections or Picornaviridae viral infection.In such subclass, the Picornaviridae viral infection is that rhinovirus infection, poliovirus infect or hepatitis A virus infects.In such second subclass, flaviviridae infections is selected from infection with hepatitis C virus, yellow fever virus infection, dengue virus infection, west nile virus infection, Japanese encephalitis virus infection, banzi virus infects and bovine viral diarrhea virus infects.In the subclass of this subclass, flaviviridae infections is an infection with hepatitis C virus.

Run through the application all the time, following term has the connotation of pointing out:

" alkyl ", and other groups with prefix " alk ", for example alkoxyl or alkylthio group refer to the carbochain of straight or branched, or its combination, unless in addition carbochain is defined.The example of alkyl has methyl, ethyl, propyl group, isopropyl, butyl, sec-butyl, the tert-butyl group, amyl group, isopentyl, hexyl, heptyl, octyl group, nonyl etc.Stipulating under the carbon number purpose situation, for example C _3-10, term " alkyl " also comprises cycloalkyl, and with the combination of the bonded straight or branched alkyl chain of cycloalkyl structure.

Term " cycloalkyl " refers to one group of alkyl and refers to have the alkyl saturated carbon ring of defined amount carbon atom.The example of cycloalkyl comprises cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl, suberyl or ring octyl group etc.Except as otherwise noted, cycloalkyl is generally monocycle.Except as otherwise noted, cycloalkyl is saturated.

Term " alkenyl " refers to that total carbon atom number is the straight or branched alkene (for example vinyl, acrylic, cyclobutenyl, pentenyl etc.) of any numeral in 2-6 or this scope.

Term " alkynyl " refers to that total carbon atom number is the straight or branched alkynes (for example acetenyl, propinyl, butynyl, pentynyl etc.) of any numeral in 2-6 or this scope.

Term " alkoxyl " refers to that carbon number is defined amount (C for example _1-4Alkoxyl) the straight or branched alkyl oxygen [being methoxyl group (MeO-), ethyoxyl, isopropoxy etc.] of any number or in this scope.

Term " alkylthio group " refers to that carbon number is defined amount (C for example _1-4Alkylthio group) the straight or branched alkyl sulfide [being methyl mercapto (MeS-), ethylmercapto group, iprotiazem base etc.] of any number or in this scope.

Term " alkyl amino " refers to that carbon number is defined amount (C for example _1-4Alkyl amino) the straight or branched alkylamine [being methylamino, ethylamino, isopropyl amino, tert-butyl group amino etc.] of any number or in this scope.

Term " alkyl sulphonyl " refers to that carbon number is defined amount (C for example _1-6Alkyl sulphonyl) the straight or branched alkyl sulfone of any number [is methyl sulphonyl (MeSO or in this scope ₂-), ethylsulfonyl, isopropyl sulfonyl etc.].

Term " alkoxy carbonyl " refers to that carbon number is defined amount (C for example _1-4Alkoxy carbonyl) the straight or branched ester [being methoxycarbonyl (MeOCO-), ethoxy carbonyl or butoxy carbonyl] of carboxylic acid derivates of the present invention of any number or in this scope.

Term " alkyl-carbonyl " refers to that carbon number is defined amount (C for example _1-4Alkyl-carbonyl) the straight or branched alkyl acyl [being methyl carbonyl (MeCO-), ethyl carbonyl or butyl carbonyl] of any number or in this scope.

Term " halogen " is intended to comprise halogen atom fluorine, chlorine, bromine and iodine.

Term " phosphoryl " refers to-P (O) is (OH) ₂-

The base that term " two phosphoryls " refers to have following structure:

The base that term " three phosphoryls " refers to have following structure:

Term " replacement " should be believed to comprise the multiple replacement of appointed substituent group.When multiple replacement part has been disclosed or has been required to protect, substituted compound can be independently by one or more disclosed or be required that the single or many places of the substituent group part of protecting replace.

Work as R ³And R ⁴Aminoacyl residue embodiment in R ⁷When in following formula, being not hydrogen,

The aminoacyl residue contains asymmetric center and is intended to comprise independent R-and S-stereoisomer and RS-diastereo-isomerism mixture.

Term " 5 '-triguaiacyl phosphate " refers to have the triguaiacyl phosphate derivant that the present invention of following universal architecture formula II fluoridizes 5 '-hydroxyl of pyrrolo-[2,3-d] pyrimidine nucleoside compound:

R wherein ¹-R ³, R ⁵And R ⁶As above definition.The compounds of this invention also is intended to the pharmaceutically acceptable salt of 5 '-Monophosphate and 5 '-bisphosphate derivant of comprising the pharmaceutically acceptable salt of triguaiacyl phosphate and having structural formula II I and IV respectively.

Term in " pharmaceutical composition " " compositions " is intended to comprise the product that comprises active component and form the inert fraction of carrier, and by any two or more compositions mixing, compound or gathering, perhaps by the disassociation of one or more compositions, the perhaps spawn that directly or indirectly obtains by other type reaction of one or more compositions or interaction.Correspondingly, pharmaceutical composition of the present invention comprises by mixing any compositions that The compounds of this invention and pharmaceutically acceptable carrier are made.

Term " is granted " and " using " chemical compound to be interpreted as be to point to the prodrug have the individuality that needs that The compounds of this invention or The compounds of this invention are provided.

Another aspect of the present invention relates to and suppresses HCV NS5B polymerase, suppresses the method that HCV duplicates, perhaps the method that infects with The compounds of this invention and one or more reagent combined treatment HCV that is used for the treatment of the HCV infection.The reagent that can effectively resist HCV like this includes but not limited to the associating of associating, Polyethylene Glycol interferon-' alpha ' and Levovirin of associating, interferon-ALPHA and Levovirin of associating, Polyethylene Glycol interferon-' alpha ' and the ribavirin of ribavirin, Levovirin (levovirin), viramidine, thymosin alpha 1, interferon beta, interferon-ALPHA, Pegylation (pegylated) interferon-ALPHA (Polyethylene Glycol interferon-' alpha '), interferon-' alpha ' and ribavirin.Interferon-ALPHA includes but not limited to recombinantinterferon 2a (as Hoffmann-LaRoche, Nutley, the Roferon interferon that NJ obtains), glycol interferon alpha 2a (Pegasys ^TM), interferon alpha 2 b (as Schering Corp., the Intron-Ainterferon that Kenilworth, NJ obtain), glycol interferon alpha 2b (PegIntron ^TM), the interferon-ALPHA product of the total interferon (as interferon alphacon-1) of recombinant and purification.The total interferon of Amgen ' s recombinant has trade name Infergen .Levovirin is the L-enantiomer of ribavirin, and it demonstrates the immunoregulatory activity that is similar to ribavirin.Viramidine represents disclosed ribavirin analog among the WO01/60379 (transferring ICN Pharmaceuticals).The method according to this invention can be used the different time of each composition in during treating of associating respectively, perhaps uses simultaneously with combining form that separate or single.Therefore, the present invention should be interpreted as comprise all these simultaneously or the therapy of alternating treatment, term administering " also make respective explanations.Be to be understood that scope that The compounds of this invention and other are used for the treatment of the reagent associating that HCV infects comprises any associating of the pharmaceutical composition that infects with any HCV of being used for the treatment of in principle.When with The compounds of this invention or its pharmaceutically acceptable salt with can effectively resist second kind of treatment reagent associating of HCV the time, the dosage the when dosage of each chemical compound can be with independent use chemical compound is identical or different.

HCV infects for treatment, also can the reagent of The compounds of this invention and HCV NS3 serpin is co-administered.HCV NS3 serine protease is essential viral enzyme, and to be described to be to suppress the good target that HCV duplicates.The substrate of HCV NS3 protease inhibitor and non-substrate base inhibitor all 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 United States Patent (USP) the 6th, in 323, No. 180.B.W.Dymock, " Emergingtherapies for hepatitis C virus infection, " Emerging Drugs has discussed the HCV NS3 protease as the target of exploitation HCV replication inhibitors and treatment HCV infection among the 6:13-42 (2001).

Ribavirin, Levovirin and viramidine can be by bringing into play its anti-hcv activity via suppressing the interior pond (pool) of endocellular enzyme inosine monophosphate dehydrogenase (IMPDH) adjusting guanylic acid cell.IMPDH is the rate-limiting enzyme of biosynthesis pathway in the guanylic acid biosynthesis from the beginning.Ribavirin is easily by the endocellular phosphorus acidylate, and a phosphoric acid derivatives is the inhibitor of IMPDH.Therefore, another effective target of HCV replication inhibitors is found in the inhibition of IMPDH representative.Thereby The compounds of this invention also can be co-administered with following substances: the IMPDH inhibitor, as disclosed VX-497 among WO 97/41211 and the WO 01/00622 (transferring Vertex); Another IMPDH inhibitor is as disclosed material among the WO 00/25780 (transferring Bristol-Myers Squibb); Perhaps mycophenolate mofetil [referring to A.C.Allison and E.M Eugui, Agents Action, 44 (Suppl.): 165 (1993)].

Be that treatment HCV infects, can The compounds of this invention and antiviral agent amantadine (1-aminoadamantan) is co-administered [summary of this medicament referring to J.Kirschbaum, Anal.Profiles-DrugSubs.12:1-36 (1983)].

Chemical compound of the present invention can also be with 2 '-C-side chain ribonucleotide unites and is used for the treatment of HCV and infects, 2 '-C-side chain ribonucleotide is disclosed in R.E.Harry-O ' kuru etc., J.Org.Chem..62:1754-1759 (1997); M.S.Wolfe etc., Tetrahedron Lett..36:7611-7614 (1995); United States Patent (USP) the 3rd, 480, No. 613 (on November 25th, 1969); International publishing WO01/90121 (calendar year 2001 November 29); International publishing WO 01/92282 (calendar year 2001 December 6 days); International publishing WO 02/32920 (on April 25th, 2002); International publishing WO04/002999 (on January 8th, 2004); International publishing WO 04/003000 (on January 8th, 2004); International publishing WO 04/002422 (on January 8th, 2004); Its content intact is incorporated herein by reference.Like this 2 '-C-side chain ribonucleotide includes, but are not limited to 2 '-the C-methylcytidine; 2 '-the C-methyluridine; 2 '-the C-methyladenosine; 2 '-the C-methylguanosine, 9-(2-C-methyl-β-D-ribofuranosyl)-2,6-diaminopurine; ribose C-2 '; the corresponding amino-acid ester of C-3 and C-5 ' hydroxyl (for example, 3 '-O-(L-is valyl)-2 '-C-methylcytidine) and corresponding optional replace 5 '-ring 1 of phosphoric acid fat derivant, the ammediol ester.

Chemical compound of the present invention also can be united with other other nucleoside with anti-HCV character and is used for the treatment of the HCV infection, and other nucleoside is disclosed in WO02/51425 (on July 4th, 2002), transfers Mitsubishi Pharma Corp.; WO01/79246, WO02/32920, WO02/48165 (on June 20th, 200) transfers Pharmasset, Ltd.; WO01/686632001 JIUYUE 20 days), transfer ICN Pharmaceuticals; WO99/43691 (on JIUYUE 2nd, 1999); WO02/18404 (on March 7th, 2002) transfers Hoffmann-LaRoche; U.S2002/0019363 (on February 14th, 2002); WO02/100415 (on December 19th, 2002); WO03/026589 (on April 3rd, 2003); WO03/026675 (on April 3rd, 2003); WO03/093290 (on November 13rd, 2003): US2003/0236216 (on December 25th, 2003); US2004/0006007 (on January 8th, 2004); WO04/011478 (on February 5th, 2004); WO04/013300 (on February 12nd, 2004); US 2004/0063658 (on April 1st, 2004); WO04/028481 (on April 8th, 2004).

Chemical compound of the present invention also can be united with the HCV AG14361 and is used for the treatment of the HCV infection, and the HCV AG14361 is disclosed in WO01/77091 (October 18 calendar year 2001), transfers Tularik, Inc.; WO01/47883 (July 5 calendar year 2001) transfers JapanTobacco, Inc.; WO02/04425 (on January 17th, 2002) transfers BoehringerIngelheim; WO02/06246 (on January 24th, 2002) transfers Istituto di Ricerchedi Biologia Moleculare P.Angeletti S.P.A.; WO02/20497 (on March 3rd, 2002).

" pharmaceutically acceptable " means carrier, diluent or excipient must be compatible with other preparation composition, and harmless to its receiver.

Be also included within the scope of the invention is to comprise the pharmaceutical composition that the present invention fluoridizes pyrrolo-[2,3-d] pyrimidine nucleoside compound and derivant and pharmaceutically acceptable carrier.Another example of the present invention is by making up the pharmaceutical composition that any above-claimed cpd and pharmaceutically acceptable carrier are made.The method of another example pharmaceutical compositions of the present invention, this method comprise any above-claimed cpd of combination and pharmaceutically acceptable carrier.

What also be included in the scope of the present invention is to can be used for suppressing RNA RNA-dependent varial polymerases, the particularly pharmaceutical composition of HCV NS5B polymerase, and said composition comprises the The compounds of this invention and the pharmaceutically acceptable carrier of effective dose.Can be used for treating RNA RNA-dependent viral infection, particularly the pharmaceutical composition of HCV infection is also included within the scope of the invention, suppress RNA RNA-dependent varial polymerases in addition, the method of HCVNS5B polymerase particularly, and the method that treatment RNA RNA-dependent virus replication, particularly HCV duplicate also all is included in the scope of the present invention.In addition, the present invention relates to a kind of pharmaceutical composition, what said composition comprised the The compounds of this invention for the treatment of effective dose and treatment effective dose can effectively resist RNA RNA-dependent virus, particularly resists another medicament of HCV.Effectively the medicament of antagonism HCV includes but not limited to the associating of associating, Polyethylene Glycol interferon-' alpha ' and Levovirin of associating, interferon-ALPHA and Levovirin of associating, Polyethylene Glycol interferon-' alpha ' and the ribavirin of ribavirin, Levovirin, viramidine, thymosin alpha 1, HCV NS3 serpin, interferon-ALPHA, glycol interferon alpha (Polyethylene Glycol interferon-' alpha '), interferon-' alpha ' and ribavirin.Interferon-ALPHA includes but not limited to recombinantinterferon 2a (as Hoffmann-LaRoche, Nutley, the Roferon interferon that NJ obtains), glycol interferon alpha 2a (Pegasys ^TM), interferon alpha 2 b (as Schering Corp., the Intron-Ainterferon that Kenilworth, NJ obtain), glycol interferon alpha 2b (PegIntron ^TM), the interferon-ALPHA product of the total interferon (as interferon alphacon-1) of recombinant and purification.To ribavirin and the antagonism HCV active discussion referring to J.O.Saunders and S.A.Raybuck, " Inosine Monophosphate Dehydrogenase:Cohsideration of Stmcture; Kinetics; and Therapeutic Potential; " Ann.Rep.Med.Chem, 35:201-210 (2000).

Another aspect of the present invention provides fluoridizes pyrrolo-[2,3-d] pyrimidine nucleoside compound and derivant and the purposes of their pharmaceutical composition in making medicine, described medicine is used for RNA RNA-dependent virus replication, particularly HCV duplicates, and/or treatment RNA RNA-dependent viral infection, particularly HCV infect.Of the present invention useful as drug is provided on the one hand again fluoridize pyrrolo-[2,3-d] pyrimidine compound and derivant and their pharmaceutical composition, described medicine is used to suppress RNA RNA-dependent virus replication, particularly HCV duplicates, and/or treatment RNA RNA-dependent viral infection, particularly HCV infect.

Pharmaceutical composition of the present invention comprises as the compound in structural formula I of effective ingredient or its pharmaceutically acceptable salt, also can comprise pharmaceutically acceptable carrier and other optional therapeutic component.

Compositions comprises the compositions that is suitable for per os, rectum, part, parenteral (comprising subcutaneous, intramuscular and intravenous), eye (eye with), lung (per nasal or oral cavity suck) or nasal administration, although under any circumstance only route of administration will depend on the character of sanatory character and the order of severity and active component.They can be easily exist with the form of dosage unit, can prepare by known any method in the pharmaceutics field.

In actual applications, compound in structural formula I can be made up by the tight blending of conventional pharmacy hybrid technology as active component and pharmaceutical carrier.According to using required preparation, for example per os or parenteral are used (comprising intravenous) form of required preparation, and described carrier can adopt various forms.When preparation per os dosage form is used compositions, for example under the situation of suspension, elixir and solution, can use any common drug medium, for example water, glycol, oil, alcohol, flavoring agent, antiseptic, coloring agent etc. at oral liquid formulations; Perhaps at oral solid formulation for example under the situation of powder, hard and soft capsule and tablet; can use carrier such as starch, sugar, microcrystalline Cellulose, diluent, granulating agent, lubricant, binding agent, disintegrating agent etc., solid orally ingestible than liquid preparation more preferably.

Owing to be easy to use, thereby tablet and capsule be best oral dosage unit form, at this moment obviously is to adopt solid pharmacy carrier.If desired, can carry out coating to tablet with conventional aqueous or non-aqueous technology.Such compositions and preparation should contain at least 0.1% reactive compound.Certainly, the percentage ratio of reactive compound can change in these compositionss, suits at this unitary about 2% to about 60% weight range.The amount of reactive compound in the compositions that this class can be used for treating is the amount that can obtain effective dose.Described reactive compound also can be used through intranasal, for example as drop or spray application.

Tablet, pill, capsule etc. also can comprise binding agent such as tragakanta, arabic gum, corn starch or gelatin; Excipient such as dicalcium phosphate; Disintegrating agent such as corn starch, potato starch, alginic acid; Lubricant such as magnesium stearate; With sweeting agent such as sucrose, lactose or glucide.When unit dosage form was capsule, it can also comprise liquid-carrier such as fatty oil except the material of the above-mentioned type.

Can exist various other materials as coating materials or be used to change the physical aspect of dosage device.For example, can carry out coating to tablet together with Lac, sugar or two kinds.Syrup or elixir except active component, can also comprise sucrose as sweeting agent, comprise methyl parahydroxybenzoate and propyl p-hydroxybenzoate as antiseptic, comprise dyestuff and flavoring agent as cherry-flavored or orange flavor spice.

Compound in structural formula I also can parenteral administration.The solution of these reactive compounds or suspension can be by preparing with surfactant such as suitable mixing of hydroxypropyl cellulose in water.Dispersion also can prepare in the mixture in ethylene glycol, liquid polyethylene glycol and oil thereof.Under common storage and service condition, these preparations contain antiseptic to prevent growth of microorganism.

The pharmaceutical dosage form that is suitable for injecting purposes comprises sterile water solution or dispersion and the sterile powder for preparing sterilizing injecting solution or dispersion immediately.In all cases, described dosage form must be sterilized, and must be the liquid state that is easy on the injection degree.Under manufacturing and condition of storage must be stable, and must prevent by microorganism such as antibacterial and fungal contamination.Carrier can be solvent or contain for example disperse medium of water, ethanol, polyhydric alcohol (for example glycerol, propylene glycol and liquid polyethylene glycol), its stable mixture and vegetable oil.

Can adopt any suitable route of administration with to mammal, particularly the people provides the The compounds of this invention of effective dose.For example, can adopt per os, rectum, part, parenteral, eye, lung, nose etc.Dosage form comprises tablet, lozenge, dispersion, suspension, solution, capsule, emulsifiable paste, ointment, aerosol etc.Preferred dosage forms for oral administration compound in structural formula I.

During to people's dosage forms for oral administration, the dosage range of divided dose is the 0.01-1000mg/kg body weight.In one embodiment, the dosage range of divided dose is the 0.1-100mg/kg body weight.In another embodiment, the dosage range of divided dose is the 0.5-20mg/kg body weight.For dosage forms for oral administration, preferably with compositions to contain the 1.0-1000mg active component, particularly 1,5,10,15,20,25,50,75,100,150,200,250,300,400,500,600,750,800,900 and the tablet of 1000mg active component or capsular form use to regulate the patient's that treated symptom.

The effective dose of used active component can with the particular compound that is adopted, method of application, treat disease and the sanatory order of severity changes.Those skilled in the art can easily determine such dosage.Can regulate this dosage so that optimum therapeutic response to be provided.

The compounds of this invention comprises one or more center of asymmetries, therefore can be used as racemic modification and racemic mixture, single enantiomer, non-enantiomer mixture and independent diastereomer and exists.What the present invention includes five yuan of furanose rings with following structural formula and be β-D three-dimensional chemical configuration fluoridizes pyrrolo-[2,3-d] pyrimidine nucleoside compound, promptly, what wherein the C-1 of five yuan of furanose rings and the substituent group on the C-4 had β-three-dimensional chemical configuration fluoridizes pyrrolo-[2,3-d] pyrimidine nucleoside compound (" making progress " direction of representing with thick line).

Chemical compounds more as herein described comprise olefinic double bond, except as otherwise noted, otherwise refer to comprise E and two kinds of geometric isomers of Z.

Chemical compounds more as herein described can be used as tautomer such as the ketoenol tautomerization body exists.Various tautomers and composition thereof are included in the compound in structural formula I.The example that is included in interior keto-enol of The compounds of this invention scope and imine-enamine tautomerism body is as follows:

Can for example methanol or ethyl acetate or its mixture or via the chiral chromatogram that uses the optically active immobile phase compound in structural formula I be separated into their independent diastereomer from appropriate solvent by fractional crystallization for example.

Alternatively, can carry out any stereoisomer that stereospecific synthesis obtains compound in structural formula I by optical voidness initiation material or the reagent that uses configuration known.

The compounds of this invention can be used with the form of pharmaceutically acceptable salt.Term " pharmaceutically acceptable salt " refers to comprise inorganic or organic base and salt inorganic or that organic acid is made by pharmaceutically acceptable nontoxic alkali or acid.The salt that is included in the alkali compounds in term " pharmaceutically acceptable salt " scope refers to the nontoxic salts of The compounds of this invention, and it is the prepared in reaction by free alkali and suitable organic or inorganic acid usually.The representative salt of alkali compounds of the present invention includes but not limited to following salt: acetate, benzene sulfonate, benzoate, bicarbonate, disulfate, biatrate, borate, bromide, camsilate, carbonate, chloride, Clavulanate, citrate, dihydrochloride, edetate, ethanedisulphonate, estolate, esilate, fumarate, gluceptate, gluconate, glutamate, Glu, the glycollyl arsanilate, hexyl resorcin salt (hexylresorcinate), Hai Baming, hydrobromate, hydrochlorate, hydroxynaphthoate, iodide, different thiosulfate (isothionate), lactate, Lactobionate, laruate, malate, maleate, mandelate, mesylate, MB, methyl nitrate, Methylsulfate, mucate, naphthalene sulfonate, nitrate, N-methylglucosamine ammonium salt, oleate, oxalates, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, Polygalacturonate, Salicylate, stearate, sulfate, basic acetate, succinate, tannate, tartrate, the teoclate, toluene fulfonate, triethiodide and valerate.In addition, when The compounds of this invention had acidic moiety, its suitable pharmaceutically acceptable salt included but not limited to the salt derived from inorganic base, comprised aluminum, ammonium, calcium, copper, ferrum, ferrous, lithium, magnesium, manganese, mangamous, potassium, sodium, zinc etc.Particularly preferably be ammonium, calcium, magnesium, potassium and sodium salt.Salt derived from pharmaceutically acceptable organic nontoxic alkali comprises primary, the second month in a season and tertiary amine, the salt of cyclammonium and deacidite, as arginine, betanin, caffeine, choline, N, the N-dibenzyl-ethylenediamin, diethylamine, the 2-DEAE diethylaminoethanol, the 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glycosamine, chitosan, histidine, Hai Baming, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidines, polyamino resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropyl amine (TPA), trometamol etc.

Also have, (COOH), [OP (O) (OH) for phosphoric acid carboxylic acid in The compounds of this invention ₂] or the situation of alcohol radical under, can use the pharmaceutically acceptable ester of carboxylic acid derivates, as methyl, ethyl or oxy acid methyl neopentyl ester; Fluoridize 5 of pyrrolo-[2,3-d] pyrimidine nucleoside '-phosphoric acid derivatives (comprise 5 '-Monophosphate, 5 '-bisphosphate and 5 '-triguaiacyl phosphate) pharmaceutically acceptable prodrug ester; Or ribose C-2 ', C-3 ', and the prodrug acyl derivative of C-5 ' hydroxyl are as O-acetas or O-maleate or O-aminoacyl.The capable territory that is included becomes known for improving the bioavailability as continuing release or prodrug formulation, tissue distribution, those esters and the acyl group of dissolubility or hydrolysis properties.The five-membered ring carbonate ester derivant that also comprises the hydroxyl of C-2 ' and C-3 '.The derivant of expection easily changes into required chemical compound external.Therefore, in Therapeutic Method of the present invention, term " is granted " and " using " refers to comprise that to use clear and definite disclosed chemical compound or use not to have clear and definite disclosed but comprising the described viral infection of compounds for treating that is converted into appointed compound after human patients is used in vivo to mammal.The method of the prodrug derivant that conventional selection and preparation are suitable for example exists, and H.Bundgaard edits " Design of Prodrugs, ", and Elsevier has description in 1985, with its complete being incorporated herein by reference.

The preparation of The compounds of this invention:

The starting material that is used to prepare The compounds of this invention is 4-amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo-[2,3-d] pyrimidine (1-9), flow chart 1 has been described it and has been synthesized.

Flow chart 1

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

Steps A: the preparation (1-2) of 5-bromo-4 chloro-7H-pyrrolo-[2,3-d] pyrimidines

0 ℃ to 4 chloro-7H-pyrrolo-[2,3-d] pyrimidines in DMF (20 mL) ( 1-1) (1.53g drips N-bromosuccinimide (1.78g, solution 10.0mmol) in (10mL) in solution 10.0mmol).Reactant mixture is stirred 30 minutes then stirring at room 1 hour at 0 ℃.Add methanol (25mL), then reactant mixture was stirred other 1 hour.Evaporating solvent also obtains title compound into white solid by the recrystallizing methanol residue.

The preparation of step B:5-(trimethyl stannyl)-4 chloro-7H-pyrrolo-[2,3-d] pyrimidines (1-3)

-78 ℃ to the chemical compound that obtains by steps A in THF (25mL) (0.92g, drip in solution 4mmol) n-BuLi (the 2.5M hexane solution, 3.48mL).After adding, reactant mixture was stirred other 30 minutes at-78 ℃.In 10 minutes, in this solution, drip trimethyltin chloride in THF (8mL) (0.88g, 4.4mmol).Reactant mixture slowly returned to room temperature and in stirred overnight at room temperature.Add saturated ammonium chloride solution (60mL) and extract (3 * 70mL) with ethyl acetate.With the organic extract salt water washing that merges, Na ₂SO ₄Drying is evaporated to exsiccation.On silica gel, the residue purification obtained title compound into colorless solid.

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

To at CH ₃Add a [1-(chloromethyl)-4-fluoro-1 in the solution of the chemical compound that obtains by step B among the CN (60mL), 4-diazabicyclo [2.2.2] octane two (Tetrafluoroboric acid esters)] (SELECTFLUOR  fluorination reagent) (2.40g, 6.5mmol) and with reactant mixture stirring at room 7 hours.Leach white precipitate, filtrate is evaporated to exsiccation.On silica gel, use ethyl acetate/hexane (3: 7) as eluent with the residue purification.The fraction that will contain product is concentrated and vacuum evaporation obtains title compound into colorless solid.

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

The preparation of 2-C-methyl-3.5-two-O-(right-toluyl)-D-ribofuranose (1-6):

To the 3-O-benzyl-1 in the 35l acetonitrile, (preparation is referring to Carbohydr.Res..44:275-283 (1975) (5.0kg for 2-O-isopropylidene-3-C-methyl-α-D-glucofuranose (1-5), 15.4mol) and pyridine (3.7kg, 46.2mol) solution in add right-Benzenecarbonyl chloride. (5.2kg, 33.9mol), will be reflected at 50-55 ℃ of heating 12 hours.At 50-55 ℃ of HBF that is added in the 48wt% in the 9L water ₄The solution 6.0L (46.2mol) of (Tetrafluoroboric acid).After 2 hours, the 10L acetonitrile is removed in distillation, adds the 10L acetonitrile.Transforming at 97% o'clock, the 10L acetonitrile is removed in distillation, and reaction solution is cooled to 0-5 ℃.Be added in periodic acid solution in the 10L water (4.2kg, 18.5mol).Reaction aging after 30 minutes, is being added 35L isopropyl acetate and 10L water.Use 25L water, use 20L NaHCO subsequently ₃Aqueous solution, 15L 5% sodium thiosulfate in water, 15L water washing organic facies.The isopropyl acetate solution concentration to 10-15L, is added 40L methanol.With solution be cooled to 0 ℃ and add diisopropylamine (0.78kg, 7.7mol).0 ℃ two days later, (1N 7.7L), adds 30L isopropyl acetate and 40L water subsequently to add the HCl aqueous solution at 0-5 ℃.Use 1N HCl, NaHCO3 and salt water washing organic facies.By the azeotropic distillation drying organic facies and there is active carbon to handle.By remove by filter carbon and use isopropyl acetate with the gained solution dilution to 75L and hydrogenation (45psi, 50 ℃, 1.5kg 10%Pd/C) 24 hours.Filtrate is concentrated into 15L and adds the 60L heptane at 50 ℃.The separation of 20% isopropyl acetate washing and filtering in heptane with 10L obtains brilliant produce thing.Drying obtains the glycol 1-6 that 4.03kg needs.

¹H NMR (CDCl ₃, 400MHz): CDCl ₃Middle α: the ratio of beta isomer is about 5: 1. for main 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 accessory isomer: δ 7.95-7.90 (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..

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

Steps A: 1.2-dehydration-3.5-two-O-(right-toluyl)-2-C-methyl-α-D-ribofuranose (1-7)

The exsiccant dichloromethane (32L) of in the container of 72L, packing into, triethylamine (3.0L), and glycol (3.44kg, 90wt% is pure).With mixture heated to 30 ℃, in 40 minutes, add mesyl chloride (0.79L) then.After 1 hour, this batch product (batch) is distributed between the buffer (20L) of pH7 and methyl tertiary butyl ether(MTBE) (44L).Use 1M NaCl aqueous solution (38L) washing organic facies, be transformed into vacuum distilling toluene then, be concentrated into about 9L subsequently.Use among the direct step B of gained epoxide solution.

Step B:4-chloro-5-fluoro-7-(2-C-methyl-3,5-two-O-(right-toluyl)-β-D-ribofuranosyl)-7H-pyrrolo-[2,3-d] pyrimidines (1-8)

In room temperature at N, 4-chloro-5-fluoro-7H-pyrrolo-[2, the 3-d] pyrimidines (1-4) of N-acetic acid dimethylamide (300mL) (28.4g, in solution 0.165mol) portionwise add 60% sodium hydride (6.6g, 0.165mol).After adding, reactant mixture was stirred 1 hour at 60 ℃.In reactant mixture, be added in 1.2-dehydration-3.5-two-O-(right-toluyl)-2-C-methyl-α-D-ribofuranose (1-7) of THF (200mL) solution (63.4g, 0.166mol), with reactant mixture 60 ℃ the heating 18 hours.Reactant mixture is cooled to room temperature, pours in water (1L) and the ethyl acetate (2L).Organic extract water (500mL) washing, MgSO ₄Drying is evaporated to exsiccation.On silica gel, use the 10-40% ethyl acetate/hexane to carry out purification residue as eluent.The fraction merging that will contain product concentrates and obtains foam, directly uses in following step C.

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

Will be in the container of sealing at 4-chloro-5-fluoro-7-(the 2-C-methyl-3 in the anhydrous ammonia (300mL); 5-two-O-(right-toluyl)-β-D-ribofuranosyl)-7H-pyrrolo-[2; 3-d] pyrimidine solution (29.4g, 0.053mol) 85 ℃ the heating 48 hours.Reactant mixture is warming up to room temperature, with residue pulping in methanol (200mL), filter, filtrate is adsorbed onto on the silica gel (200g), use the 0-30% ethanol/methylene to carry out purification by chromatograph then as eluent, the fraction that will contain product merges, and evaporation obtains being solid title compound 1-9

¹H-NMR (500MHz, MeOH-d ₄): δ 8.07 (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); ¹⁹F-NMR (MeOH-d ₄): δ-170.4; Mass spectrum: 321 (M+Na) ⁺

At intermediate 1-9The C-2 position of 6-amino-7-azapurine (7-deazaadenine) ring on introduce amino can be by Zhao etc., at J.Org.Chem., synthetic method is carried out described in the 62:7832-7835 (1997), as illustrated in flow chart 2.2, the 6-diaminourea-7-azapurine ring is converted into the 7-deazaguanine system can be by people such as K.Alarcon at Tetrahedron Lett, and method is carried out described in the 41:7211-7215 (2000), and is shown in Figure 3 as flow process.

The following examples illustrate the condition of using in the preparation The compounds of this invention.Embodiment limits the scope of the invention unintentionally by any way, also should not do to explain in this way to it.The technical staff in the synthetic field of nucleoside and nucleotide will easily recognize and can do well-known change to the condition and the method for following preparation process, to prepare these and other chemical compound of the present invention.Except as otherwise noted, otherwise all temperature all be degree centigrade.

Flow chart 2

Embodiment 1

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

Steps A: 4-amino-5-fluoro-3-N-oxygen-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo-[2,3-d] pyrimidines (2-1)

To the 4-of 50% methanol (20mL) amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo-[2,3-d] pyrimidine solution (268mg, add in 0.899mmol) the m-chloroperoxybenzoic acid (444mg, 1.80mmol).With reactant mixture stirring at room 18 hours.Obtain title compound for twice with solvent evaporation and with residue and methylbenzene azeotropic into beige solid.

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

At 0 ℃ of 4-amino-5-fluoro-3-N-oxygen-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo-[2,3-d] pyrimidines (2-1) (160mg, solution 0.509mmol) that in the Bromine cyanide. biological solution of (3mL) in water, is added in (3mL) in the water.Gained solution was stirred 1.5 hours at 0 ℃.Evaporating solvent and and methylbenzene azeotropic.Add N to ginseng in the present, N-dimethylformamide (3.5mL) and triethylamine (0.25mL, 1.79mol), with gained solution stirring at room 45 minutes.Add iodomethane (0.25mL 4.0mmol), stirs reactant mixture 1.5 hours under room temperature lucifuge situation in batches.Evaporating solvent also adds 0.25M sodium hydrate aqueous solution (10mL) in residue, it was at room temperature stirred 30 minutes.With 1M HCl neutralization reaction mixture, with ethanol (10mL) dilution, 60 ℃ of heating 8 hours.After being cooled to room temperature, add spissated ammonium hydroxide (12mL), therefore reactant mixture is heated to 90 ℃ adds and draw Buddhist nun's nickel.After 15 minutes, by thermal reaction mixture is filtered, with solvent evaporation, on silica gel, use ethanol/methylene as eluent with the residue purification.The fraction evaporation that will contain product obtains being solid title compound 2-4

¹H-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).

Flow chart 3

Embodiment 2

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

This chemical compound by use in DMF 1, two [(dimethylamino)-methylene] hydrazines of 2-are handled chemical compound 2-4So that chemical compound to be provided 3-1, it is pressed people such as K.Alarcon at TetrahedronLett, use the 1N NaOH aqueous solution hydrolysis in DMSO to be prepared described in the 41:7211-7215 (2000) under the condition.

2-fluoro-2-C-methylribonucleotide (R among the structural formula I of the present invention ²=F) can be prepared according to effective synthetic method in nucleoside and the practice of nucleotide chemistry.Chemical compound is provided 6-3Synthetic as example, described in flow chart 4-6.D-ribose ( 4-1) at first protected.In this case, ester, for example acetas and benzoate provide suitable protecting group, but also can use substituting protecting group.Choose wantonly at solvent, diethyl ether for example, dioxanes makes D-ribose and suitable acyl halide or anhydride reactant realization esterification under the situation that oxolane and dichloromethane exist.Such conversion is well-known in the Chemistry Literature document, at Greene, and T.W., Wuts, P.G.M., " ProtectiVe Groups in Organic Synthesis, ", John Wiley ﹠amp; Sons,, Inc., can find example in 1999 by the 3rd edition.

Flow chart 4

Intermediate 4-3Can use a lot of methods to make.Use Vorbruggen reaction in flow chart 4 (" Synthesis of Nucleosides ", H.Vorbruggen and C.Ruh-Pohlenz are at Organic Reactions, vol.55, pp 1-630 is in 2000) connect nucleobase5-fluoro-4-chloro-7H-pyrrolo-[2,3-d] pyrimidine ( 1-4) the protected nucleoside 4-3 of formation.By any suitable method, for example acid or alkali catalyzed hydrolysis and ester exchange (for example using the Feldalat NM in methanol) removed the ester protecting group to provide then 4-4

Next as described in flow chart 5, introduce C-2 ' methyl.In order to control (orthogonal manipulation) C-2 ' hydroxyl by quadrature, 5 ' and the hydroxyl of 3 ' position is at first protected.This can realize by a lot of methods, and flow chart 5 is described one of them.Aforementioned reference, Greene, T.W., Wuts, P.G.M., " Protective Groups in OrganicSynthesis, ", John Wiley ﹠amp; Sons,, Inc., the 3rd edition, 1999 comprise a lot of examples; Particularly suitable is tetra isopropyl disilicon malignant alkyl subunit (tetraisopropyldisiloxanylidene) cyclic ethers 5-1

Flow chart 5

By using suitable method for oxidation, for example, Swern or Moffatt oxidation or application Dess-Martin cross iodine alkane (periodinane) the residual ionization hydroxyl oxygen are changed into ketone then 5-2The example of such method can for example, be Richard C.Larock the author in relevant Chemistry Literature, finds in " Comprehensive Organic Transfor-mations " that VCH Publishers1989 publishes.Make then 5-2With suitable organometallic reagent, for example, lithium methide and methylmagnesiumhalide reaction.Such reaction preferably at low temperature in appropriate solvent, for example carry out in oxolane and the diethyl ether.In this case, introduce methyl by the face that hinders still less, this three-dimensional control mainly produces a kind of isomer.The example of such solid control can be in relevant Chemistry Literature, for example, be that Ernest L.Eliel and Samuel H.Wilen are found in " the Stereochemistry of Organic Compounds " that published in 1994 by Wiley-IntersciencePublications the author.Fluorine-based as introducing in C-2 ' position as described in the flow chart 6 then.

Flow chart 6

Optionally protect as mentioned above 3 ' and the hydroxyl that exists of 5 ' position.Use low alkyl group or simple aromatic ester, for example acetas and benzoate are favourable.So the selective response condition so that sterically hindered C-2 ' hydroxyl remain unaffected.Choose wantonly at solvent, aromatic hydrocarbons for example, oxolane, under the situation that chloroform exists at low temperature, room temperature or heat up down by using diethylaminosulfur trifluoride (DAST) or other the fluorination reagent that suits to realize introducing fluorine.The example of such conversion has been described in U.S. Patent Publication No. 2005/0009737 (publication on January 13rd, 2005).Remove the ester protecting group by hydrolysis then, the chlorine atom that exists in nucleobase with the ammonia displacement obtains the nucleoside of needs subsequently.But by in appropriate solvent, for example to carry out last two steps operation in room temperature or under heating up be favourable to the ammonia of methanol, needs, and can use high pressure in a still.

Embodiment 3

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

Steps A:

Use P-Toluoyl chloride (22mL, 165mmol) dropwise handle D-ribose in dry DMF (80mL) (5.00g, 33mmol), triethylamine (46mL, 330mmol) and DMAP (810mg, solution 6.6mmol) also continues stirring 3 hours in room temperature.Make the reaction cancellation on ice by being poured over 300g.Dichloromethane extraction raw product behind ice-out (3 * 100mL), use anhydrous magnesium sulfate drying, filter and solvent removed in vacuo.Pulverize remaining oily residue by acetone.With the solvent decant, with solid residue by isopropyl alcohol (200mL) recrystallization.In next step, use this product.

Step B:

Use and exist as H.Vorbruggen and U.Niedballa J.Ore.Chem., the described method of 39:3654-3660 (1974) is synthesized this chemical compound.Press people such as A.B.Eldrup, J.Med. Chem., 4-chloro-5-fluoro-7H-pyrrolo-[2, the 3-d] pyrimidine that the preparation of method described in the 47:5284 (2004) is used in this transforms ( 1-4).

Step C:

Product by step B is pressed people such as K.L.Smith, Bioorg.Med.Chem.Lett, method described in the 14:3517-3520 (2004) is synthesized this chemical compound.

Step D:

Product by step C is pressed people such as G.Gaubert, Tetrahedron Lett, method described in the 45:5629-5632 (2004) is synthesized this chemical compound.

Step e:

Product by step D is pressed people such as V.L.Moore, Biochemistry, method described in the 41:14066-14075 (2002) is synthesized this chemical compound.

Step F:

Product by step e is pressed people such as V.L.Moore, Biochemistry, method described in the 4:14066-14075 (2002) is synthesized this chemical compound.

Step G:

Product by step F uses by people such as M.Gallo, Tetrahedron.57:5707-5713 (2001). the method for middle announcement is synthesized this chemical compound.

Step H:

Product by step G uses by people such as M.Akira, Chem.Pharm.Bull., 35:3967-3970 (1987).Synthetic this diester of the middle method of announcing.

Step I:

Use the product of DAST treatment step H to prepare this chemical compound according to No. 2005/0009737 described method of U.S. Patent Publication.

Step J:

By pressing United States Patent (USP) the 6th, 777, No. 395 embodiment 62, the condition of step F uses the product of methanol ammonia treatment step I to synthesize embodiment 3, and its content intact is incorporated herein by reference.

Embodiment 4

Nucleoside 5 '-triguaiacyl phosphate

According to the general step described in the Chem.Rev.100:2047 (2000) prepare nucleoside 5 of the present invention '-triguaiacyl phosphate.

Embodiment 5

Nucleoside 5 '-purification and the purity analysis of triguaiacyl phosphate derivant

30 * 100mmMono Q post (Pharmacia) that triguaiacyl phosphate is crossed with the 50mM Tris buffer system with pH8 carries out the purification of anion exchange (AX) chromatograph.Gradient is 40mM NaCl to 0.8M NaCl usually in two column volumes, 6.5mL/ minute.Collect the suitable fraction that anion-exchange chromatography obtains, by using Luna C18 250 * 21mm post (Phenomenex), flow velocity is that anti-phase (RP) chromatograph of 10mL/ minute is removed freshen.Gradient is generally 1%-95% methanol, and 14 minutes, the constant density of 5mM second triethylenetetraminehexaacetic acid ammonium (TEAA).

Hewlett-Packard (Palo Alto, CA) on the MSD 1100 with the mass spectrum of online HPLC mass spectroscopy purification triguaiacyl phosphate.RP HPLC Phenomenex Luna (C18 (2)), 150 * 2mm adds 30 * 2mm guard column, 3-μ m granularity.In the negative ionization model, implement the 0-50% linear gradient (15 minutes) of 20mM TEAA (second triethylenetetraminehexaacetic acid ammonium) the pH7 solution of acetonitrile.Produce electrojet with nitrogen and pneumatic nebulizer.Molecular weight ranges with 150-900 is a sample.With HP Chemstation analysis programme bag determining molecular weight.

The purity of purification triguaiacyl phosphate is measured by analyzing with RP and AX HPLC, and (250 * 4.6mm), 5 μ m granularities are used in 100mM TEAA usually, 2-70% acetonitrile gradient running in 15 minutes among the pH7 to have Phenomonex Luna or Jupiter post.AX HPLC carries out on 1.6 * 5mm Mono Q post (Pharmacia).With 50mM Tris, the constant density of pH8,0-0.4M NaCl gradient elution triguaiacyl phosphate.The purity of triguaiacyl phosphate generally＞80%.

Biological test

Describe below to be used to measure and suppress the test that HCV NS5B polymerase and HCV duplicate.

Measure the effectiveness of The compounds of this invention in the test below as the inhibitor of HCV NS5B RNA RNA-dependent polymerase (RdRp).

A. suppress HCV NS5B polymerase assay:

This test is used to measure the ability that pyrrolo-[2,3-d] pyrimidine nucleoside triguaiacyl phosphate suppresses the enzymatic activity of RNA RNA-dependent polymerase (NS5B) on heteromerism (heteromeroc) RNA template of hepatitis C virus (HCV) of fluoridizing of the present invention.

Step:

Test buffer condition: (50 μ L-total amount/reactant)

20mM?Tris，pH7.5

50μM?EDTA

5mM?DTT

2mM?MgCl ₂

80mM KCl

0.4U/ μ L RNAsin (Promega, storing solution are 40 units/μ L)

0.75 μ gt500 (transcribe the 500-nt RNA that make with T7 runoff, have sequence) from the genomic NS2/3 of hepatitis C zone

1.6 μ g purification hepatitis C NS5B (aminoacid by the terminal truncate of 21 C-forms)

1 μ M A, C, U, GTP (ribonucleoside triphosphote ester admixture)

[α- ³²P]-GTP or [α- ³³P]-GTP

Under up to the various concentration of 100 μ M ultimate densities, test the ribonucleoside triphosphote ester.

Preparation comprises the reaction buffer of the proper volume of enzyme and template t500.Ribonucleoside triphosphote ester suction of the present invention is moved on in the hole of 96 orifice plates.Preparation comprises ribonucleoside triphosphote ester (NTP ' s) mixture of radioactive label GTP, and its suction is moved on in the hole of 96 orifice plates.Add enzyme-template reaction solution initiation reaction, and reaction was at room temperature carried out 1-2 hour.

Add 20 μ L 0.5M EDTA, pH8.0 cancellation reaction.Comprise blank assay, in the blank assay cancellation solution was joined among the NTPs before adding reaction buffer.

Reactant point after the 50 μ L cancellation is dripped on the DE8l filter disc (Whatman), and make its dry 30 minutes.Use the 0.3M ammonium formate, pH8 (150mL is used in each washing, and the cpm in the 1mL washing liquid washes 6 times usually less than 100) washing filter disc.In scintillation counter, in the 5mL scintillation solution, filter disc is counted.

Suppress percentage ratio by following formula calculating: suppress %=[1-(cpm in the blank reaction of the cpm-in the test reaction)/(cpm in the cpm-blank reaction in the control reaction)] * 100.

The representational compound exhibits of testing in HCV NS5B polymerase assay less than 50 micromolar IC ₅₀Value.

B. suppress the test of HCV rna replicon:

Also estimate the influence that The compounds of this invention duplicates HCV RNA in containing cultivation hepatocarcinoma (HuH-7) cell of sub-gene group HCV replicon.The details of test is as described below.This replicon is tested at V.Lohmann, F.Komer, J-O.Koch, U.Herian, L.Theilmann and R.Bartenschlager, " Replication of a Sub-genomicHepatitis C Virus RNAs in a Hepatoma Cell Line, " ScienceRevise on the basis of content described in 28 5:110 (1999).

Experimental program:

This test is the original position ribonuclease protecting, measures (SPA) based on the flat board of affine flicker.With 10, the 100-200 μ L that 000-40,000 cell join in the 96 hole cytostar plates (Amersham) contains in the culture medium of 0.8mg/mL G418.At 0-18 hour, with the various concentration in 1%DMSO chemical compound is joined in the cell up to 100 μ M, cultivated then 24-96 hour.With cell fixation (20 minutes, 10% formalin), saturatingization (20 minutes, 0.25%TritonX-100/PBS) and and strand ³³P rna probe hybridization (spending the night 50 ℃), described strand ³³Contained (+) chain NS5B (or other gene) complementation in PRNA and the rna virus cdna group.With cell washing, to handle with RNAse, washing is heated to 65 ℃, and counts in Top-Count.The minimizing of reading count per minute (cpm) is as the inhibition to duplicating.

The people HuH-7 hepatoma carcinoma cell that is selected to contain sub-genome duplication has cytoplasm rna, and this cytoplasm rna is made of HCV 5 ' untranslated region (NTR), neomycin selected marker, EMCV IRES (internal ribosome entry site) and HCV non-structural protein NS3-NS5B and the 3 ' NTR that follows thereafter.

The representational compound exhibits of testing in this replicated test less than 100 micromolar EC ₅₀Value.

C. endocellular metabolism test:

Also estimated The compounds of this invention enter human hepatoma cell strain and in cell, be converted into corresponding nucleoside 5 '-single-, two-and the ability of triguaiacyl phosphate

With two kinds of cell strains, HuH-7 and HBIlOA are used for the endocellular metabolism research of The compounds of this invention.HuH-7 is a human hepatoma cell strain, and HBIlOA represents the clone strain by the HuH-7 cell acquisition that has HCV bicistronic mRNA replicon.With every 60-millimeter culture dish 1.5 * 10 ⁶Cell is applied to the HuH-7 cell the complete Dulbecco ' s improvement Eagle ' s culture medium that contains 10% hyclone, the HBIlOA cell is applied in the same culture medium that contains G418 (0.8mg/mL), so that when adding chemical compound, 80% cell fusion is arranged.Tritiated compound was hatched in cell culture medium 3 or 23 hours with 2 μ M.Collecting cell is with phosphate-buffer saline washing, counting.Then cell is used 70% methanol, 20m tritiated compound EDTA, 20mm EGTA extracts and is centrifugal.With the lysate drying, use ion pair anti-phase (C-18) HPLC with Waters Millenium system that online β-RAM scintillation detector is connected on (IN/USSystems) measure radiolabeled nucleotide.HPLC mobile phase (a) has the 10mm potassium phosphate of 2mm tetrabutylammonium hydroxide and (b) comprises 50% methanol of the 10mm potassium phosphate with 2mm tetrabutylammonium hydroxide.By relatively carrying out peak identification with respect to the retention time of standard.Activity is expressed as 10 ⁶The nucleotide of detected picomole in HuH-7 or the HBIlOA cell.

Filter and also estimate cytotoxicity and the antiviral specificity that the present invention fluoridizes pyrrolo-[2,3-d] pyrimidine nucleoside compound in (counterscreens) in that the following stated is counter.

C. instead filter:

Tested the present invention in the test below and fluoridized the inhibition ability of pyrrolo-[2,3-d] pyrimidine nucleoside compound human DNA polymerase.

A. to the inhibition of human DNA polymerase α and β:

Reaction condition:

50 μ L reaction volumes

The reaction buffer components:

20mM?Tris-HCl，pH7.5

200 μ g/mL bovine serum albumins

100mM?KCl

The 2mM beta-mercaptoethanol

10mM?MgCl ₂

1.6μM?dA，dG，dC，dTTP

α- ³³P-dATP

Enzyme and template:

0.05mg/mL the sub-dna profiling of breach milt

0.01U/ μ L archaeal dna polymerase α or β

The preparation of the sub-dna profiling of breach milt:

With 5 μ L 1M MgCl ₂Join in the 500 μ L activation fish sperm DNAs (USB 70076); Be heated to 37 ℃, and add the exonuclease I II (GibcoBRL18013-011) of 30 μ L 65U/ μ L;

Hatched 5 minutes at 37 ℃;

By being heated to 65 ℃ of 10 minutes cessation reactions;

50-100 μ L aliquot is installed in Bio-spin 6 chromatographic columns (Bio-Rad 732-6002), and this chromatographic column has been used 20mM Tris-HCl, and pH7.5 carries out balance;

By with 1,000Xg carried out eluting in centrifugal 4 minutes;

Collect eluate, and measure absorbance, to determine concentration at 260nm.

Dna profiling is diluted to 20mM Tris-HCl, and the proper volume of pH7.5 is diluted to the proper volume of 20mM Tris-HCl with enzyme, contains 2mM beta-mercaptoethanol and 100mMKCl.Template and enzyme suction are moved in microcentrifugal tube or 96 orifice plates.Also the blank reactant of getting rid of enzyme and the control reaction thing of getting rid of test compounds have been prepared with enzyme dilution buffer liquid and test compounds solvent respectively.With reaction buffer initiation reaction with said components.Reactant was hatched 1 hour at 37 ℃.Add 20 μ L 0.5M EDTA cancellation reaction.Drip on the Whatman DE81 filter disc reactant point after the 50 μ L cancellation also air-dry.With the ammonium formate cyclic washing filter disc of 150mL 0.3M pH8, up to 1mL washing liquid＜100cpm.Filter disc with 150mL absolute ethanol washing twice, is washed once with the 150mL absolute ether, and drying is also counted in the 5mL scintillation solution.

Calculate the percentage ratio that suppresses by following formula: suppress %=[1-(cpm in the blank reaction of the cpm-in the test reaction)/(cpm in the blank reaction of the cpm-in the control reaction)] * 100.

B. to the inhibition of human DNA polymerase γ:

In the reaction that comprises following compositions, tested inhibition ability: 0.5ng/ μ L enzyme to human DNA polymerase γ; 10 μ m dATP, dGTP, dCTP and TTP; 2 μ Ci/ react [a- ³³P]-dATP and 0.4 μ g/ μ L activation fish sperm DNA (available from US Biochemical), containing 20mM Tris pH8,2mM beta-mercaptoethanol, 50mM KCl, 10mM MgCl ₂In the buffer of 0.1 μ g/ μ L BSA.Make to be reflected at 37 ℃ and to carry out 1 hour, then by adding the ultimate density cancellation reaction of 0.5M EDTA to 142mM.Quantitative by anion exchange filtration combination and scinticounting to formed product.At concentration determination chemical compound up to 50 μ M.

In following test, measured the present invention and fluoridized the ability that pyrrolo-[2,3-d] pyrimidine nucleoside compound suppresses HIV infection and HIV diffusion.

The c.HIV experimental infection:

Variant with the HeLa Magi cell of expressing CXCR4 and CCR5 carry out this test, and (β-gal) expression is selected according to low background beta galactosidase.Made cell infection 48 hours, (Bedford MA) carries out quantitatively the β-gal product that derives from integration (integrated) HIV-1 LTR promoter for Galactolight Plus, Tropix with chemical luminous substrate.Titration (in repeated trials) mortifier in the twice serial dilution that starts from 100 μ m; Calculating is with respect to the inhibition percentage ratio under each concentration of contrast infection.

D. the inhibition that HIV is spread:

By United States Patent (USP) the 5th, 413, No. 999 (May 9 nineteen ninety-five) and J.P.Vacca's etc. Proc.Natl.Acad.Sci., method described in the 91:4096-4100 (1994) test The compounds of this invention suppresses the ability of Human Immunodeficiency Viruses (HIV) diffusion, and the content intact of described document is incorporated herein by reference.

In the test described in the following test, screened the present invention and fluoridized the cytotoxicity of pyrrolo-[2,3-d] pyrimidine nucleoside compound cultivation hepatocarcinoma (HuH-7) cell that contains sub-gene group HCV replicon based on the MTS cell.H.Nakabayashi's etc. Cancer Res., 42:3858 has described the HuH-7 cell strain in (1982).

E. cell toxicity test:

Prepare cell culture in suitable culture medium, concentration is about 1.5 * 10 ⁵Individual cell/mL is used for 3 days suspension culture of hatching, and concentration is 5.0 * 10 ⁴Individual cell/mL is used for 3 days adhere-wall culture of hatching.99 μ L cell cultures are transferred in the hole of 96 hole tissue culture treated plates, add the test compounds of the 100 times ultimate densities of 1 μ L in DMSO.With flat board at 37 ℃ and 5%CO ₂Hatch one section special time under the condition.After nurturing period, in each hole, add 20 μ L CellTiter, 96 Aqueous One Solution Cell Proliferation test reagents (MTS) (Promega), again with flat board at 37 ℃ and 5%CO ₂Cultivation the longest is a period of time of 3 hours under the condition.Mix in dull and stereotyped so that each hole of vibration, reads absorbance under the 490nm with plate reader.With facing the standard curve that the known cell number of adding before the MTS reagent is made suspended culture cell.The metabolic activity cell is decomposed into MTS and replaces by the first moon, and the first moon is for absorbing at 490nm.The absorbance of 490nm under the chemical compound existence is compared with the absorbance of the cell that does not add any chemical compound.

Reference: Cory, A.H etc., " Use of an aqueous soluble tetrazolium/formazanassay for cell growth assays inculture, " Cancer Commun.3:207 (1991).

Following test is used to measure the activity of The compounds of this invention to other RNA RNA-dependent virus.

A. measure chemical compound and resist rhinoviral extracorporeal antivirus effect activity (CPE inhibition test):

Experimental condition is described in Sidwell and Huffman, " Use of disposablemicrotissue culture plates for antiviral and interferon inductionstudies, " Appl.Microbiol.22:797-801 in the article of (1971).

Virus:

Described in Sidwell and Huffman reference, use 2 type rhinovirus (RV-2) HGP strains and KB cell and culture medium (0.1%NaHCO ₃, antibiotic-free).Take from the cotton rod of throat of adult male with slight febris acuta upper respiratory disease by the virus of ATCC acquisition.9 type rhinovirus (RV-9) 211 strains and 14 type rhinovirus (RV-14) Tow strains are also by Rockville, and the American Type Culture Collection (ATCC) of MD obtains.RV-9 takes from people's throat washing liquid, and RV-14 takes from the cotton rod of throat of the Young Adults with upper respiratory disease.These two kinds of viruses and human cervical cancer 1 epithelium sample cancerous cell HeLa Ohio-1 cell (Dr.FredHayden, UniV.of VA) are used together.With having 5% hyclone (FBS) and 0.1%NaHCO ₃MEM (Eagle ' s minimum essential medium) as growth medium.The antiviral test media of whole three kinds of Virus Types all is to have 5%FBS, 0.1%NaHCO ₃, 50 μ g gentamycin/mL and 10mM MgCl ₂MEM.

2000 μ g/mL are the maximum concentrations that are used to test The compounds of this invention.After adding test-compound about 5 minutes, virus is joined in the bread board.Make suitable control sample simultaneously.With bread board at humid air, 5%CO ₂, cultivate under 37 ℃ of conditions.Metamorphosis by the microscopic examination control cells is monitored cytotoxicity.The regression analysis of virus CPE data and toxicity contrasting data has provided ED50 (50% effective dose) and CC50 (50% cytotoxicity concentration).Pass through formula: SI=CC50 ÷ ED50 calculates selectivity index (SI).

B. measure the extracorporeal antivirus effect activity (CPE inhibition test) of chemical compound and yellow fever neat to anti-dengue class:

The test detailed content provides in above-mentioned Sidwell and Huffman list of references.

Virus:

Obtain dengue fever 2 type virus N ew Guinea strains from Center for Disease Control (CDC).Cultivate virus (Vero) and carry out antiviral test (MA-104) with two strain African green monkey kidney cells.Obtain yellow fever virus 17D strain of producing from infected Mus brain and the banzi virus H336 strain that separates from South Africa heating boy's serum from ATCC.The Vero cell is all used in these two kinds of viruses and test.

Cell and culture medium:

Have 5%FBS and 0.1%NaHCO ₃, do not have in antibiotic 199 culture medium MA-104 of use cell (Bio Whittaker, Inc., Walkersville, MD) and Vero cell (ATCC).

The test media that is used for dengue fever, yellow fever and banzi virus is MEM, 2%FBS, 0.18%NaHCO ₃With 50 μ g gentamycin/mL.

The antiviral test of The compounds of this invention is carried out described in Sidwell and Huffman list of references, is similar to above-mentioned rhinovirus antiviral test.For various viruses, after 5-6 days, obtain suitable CPE (CPE) record.

C. measure the extracorporeal antivirus effect activity (CPE inhibition test) of chemical compound to west nile virus:

The test detailed content provides in the above-mentioned Sidwell and Huffman list of references of quoting.Obtain to take from west nile virus, the New York separator of crow brain from Center for Disease Control (CDC).Make growth of Vero cell and as above use.Test media is MEM, 1%FBS, 0.1%NaHCO ₃With 50 μ g gentamycin/mL.

The antiviral test of The compounds of this invention is carried out according to method described in the Sidwell and Huffman list of references, is similar to those methods that are used for the rhinovirus active testing.After 5-6 days, obtain suitable CPE (CPE) record.

D. measure the extracorporeal antivirus effect activity (the red picked-up test in center) of chemical compound to rhinovirus, yellow fever, dengue fever, Ban Qi and west nile virus:

After as above carrying out CPE inhibition test, re-use " Microtiter Assay forInterferon:Microspectrophotometric Quantitation of CytopathicEffect, " Appl.Environ.Microbiol.31:35-38 the cytopathy detection method described in (1976).With EL309 type tablet reader (Bio-Tek Instruments Inc.) to the test panel reading.As above calculate ED50 value and CD50 value.

The embodiment of pharmaceutical preparation

As the specific embodiments of the Orally administered composition of The compounds of this invention,, so that the total amount of 580-590mg to be provided, it is put in No. 0 hard gelatine capsule with the lactose formulated of chemical compound after abundant pulverizing of 50mg embodiment 1 or embodiment 2.

Though invention has been described and illustrate with reference to its specific embodiments, those skilled in the art will recognize can carry out various variations, improvement and replacement to it under the prerequisite that does not deviate from the spirit and scope of the present invention.For example, treat the deduction of various variations of the serious infectious people's of HCV reaction as required, the effective dose beyond the preceding preferred dose listed in the text also is suitable for.Equally, viewed pharmacological reaction also can according to and rely on selected concrete reactive compound or whether have pharmaceutical carriers and preparation type and used method of application change, can expect the variation or the result difference of these expections according to purpose of the present invention and practice.Therefore, the present invention is only limited by the scope of claims, and these claim suitably keep the scope of broad.

Claims

1, the nucleoside compound of structural formula I

And pharmaceutically acceptable salt; Wherein

R ¹Be hydrogen or fluorine;

R ²Be fluorine or hydroxyl;

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 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

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

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.