HK1065707B - 4--substituted nucleosides - Google Patents

Description

4' -substituted nucleosides

The present invention relates to nucleoside derivatives as inhibitors of HCV replicon RNA replication. In particular, the present invention relates to the use of purine and pyrimidine nucleoside derivatives as inhibitors of subgenomic Hepatitis C Virus (HCV) RNA replication and pharmaceutical compositions containing such compounds.

Hepatitis c virus is the leading cause of chronic liver disease worldwide. Patients infected with HCV are at risk of developing cirrhosis and subsequent hepatocellular carcinoma, and therefore HCV is the primary indication for liver transplantation. Currently there are only two approved therapies available for the treatment of HCV infection (r.g. gish, sen.liver, 1999, 19, 35). They are interferon- α monotherapy and the recent combination therapy of the nucleoside analog ribavirin (Virazole) with interferon- α.

Many of the approved drugs for the treatment of viral infections are nucleosides or nucleoside analogs, most of which inhibit viral replication by inhibiting viral polymerase after conversion to the corresponding triphosphate. This conversion to triphosphate is often mediated by cellular kinases, and therefore direct evaluation of nucleosides as inhibitors of HCV replication can be conveniently performed using only cell-based assays. But for HCV there is a lack of a true cell-based viral replication assay or animal infection model.

Hepatitis c virus belongs to the flaviviridae family. It is an RNA virus, and the RNA genome encoding the large polyprotein is processed to create the necessary replication machinery to ensure synthesis of progeny RNA. It is believed that most of the non-structural proteins encoded by the HCV RNA genome are involved in RNA replication. Lohmann et al (V.Lohmann et al, Science, 1999, 285, 110-. It is believed that the mechanism of RNA replication in these cell lines is equivalent to replication of the full-length HCV RNA genome in infected hepatocytes. The subgenomic HCV cDNA clones used to isolate these cell lines have been the basis for the development of cell-based assays for identifying nucleoside analog HCV replication inhibitors.

The compounds of formula I have been shown to be inhibitors of subgenomic hepatitis c virus replication in hepatoma cell lines. These compounds have the potential to be effective antiviral drugs for the treatment of HCV infection in humans.

The present invention relates to the use of compounds of formula I and pharmaceutically acceptable salts thereof for the treatment of diseases mediated by the Hepatitis C Virus (HCV) or for the manufacture of medicaments for such treatment,

wherein the content of the first and second substances,

r is hydrogen or- [ P (O) (OH) -O]_nH, and n is 1, 2 or 3;

R¹is alkyl, alkenyl, alkynyl, haloalkyl, alkylcarbonyl, alkoxycarbonyl, hydroxyalkyl, alkoxyalkyl, alkoxy, cyano, azido, hydroxyiminomethyl, alkoxyiminomethyl, halogen, alkylcarbonylamino, alkylaminocarbonyl, azidoalkyl, aminomethyl, alkylaminomethyl, dialkylaminomethyl or heterocyclyl;

R²is hydrogen, hydroxy, amino, alkyl, hydroxyalkyl, alkoxy, halogen, cyano or azido;

R³and R⁴Is hydrogen, hydroxy, alkoxy, halogen or hydroxyalkyl, with the proviso that R³And R⁴At least one is hydrogen; or

R³And R⁴Together represent ═ CH₂Or ═ N-OH, or

R³And R⁴Both represent fluorine;

x is O, S or CH₂；

B represents a 9-purinyl group of formula B1,

wherein the content of the first and second substances,

R⁵is hydrogen, hydroxy, alkyl, alkoxy, alkylthio, NHR⁸Halogen or SH;

R⁶is hydroxy, NHR⁸、NHOR⁹、NHNR⁸、-NHC(O)OR⁹' or SH;

R⁷is hydrogen, hydroxy, alkyl, alkoxy, alkylthio, NHR⁸Halogen, SH or cyano;

R⁸is hydrogen, alkyl, hydroxyalkyl, arylcarbonyl or alkylcarbonyl;

R⁹is hydrogen or alkyl;

R⁹' is an alkyl group; or

B represents a 1-pyrimidinyl radical of formula B2,

wherein the content of the first and second substances,

z is O or S;

R¹⁰is hydroxy, NHR⁸、NHOR⁹、NHNR⁸、-NHC(O)OR⁹' or SH;

R¹¹is hydrogen, alkyl, hydroxy, hydroxyalkyl, alkoxyalkyl, haloalkyl or halogen;

R⁸、R⁹and R⁹' is as defined above.

In which R is a phosphate group- [ P (O) (OH) -O]_nIn the compound of H, n is preferably 1. The phosphate group may be in the form of a stabilized monophosphate prodrug or other pharmaceutically acceptable leaving group capable of providing a compound wherein R is monophosphate upon in vivo administration. These "mononucleotides" can improve a property of the parent nucleotide such as activity, bioavailability or stability.

Examples of substituents which can replace one or more hydrogens in the phosphate moiety are described in c.r.wagner et al, Medicinal Research Reviews, 2000, 20(6), 417 or r.jones and n.bischofberger, Antiviral Research 1995, 27, 1. Such pro-nucleotides include alkyl and aryl phosphodiesters, steroid phosphodiesters, alkyl and aryl phosphotriesters, cyclic alkyl phosphotriesters, cyclosaligenyl (cyclosal) phosphotriesters, S-acyl-2-thioethyl (SATE) derivatives, Dithioethyl (DTE) derivatives, pivaloyloxymethyl phosphate, p-acyloxybenzyl (PAOB) phosphate, glycerolipid (diglyceride) phosphodiester, glycolipid (triglyceride) phosphotriester, dinucleoside phosphodiester, dinucleoside phosphotriester, phosphorodiamidate, cyclic phosphoramidate, mono-and phosphoramidate.

The invention also includes prodrugs or bioprecursors of the parent nucleoside which are converted in vivo into compounds wherein R is hydrogen or R²、R³And R⁴Compounds of formula I wherein at least one is hydroxy. Preferred prodrug derivatives include carboxylic acid esters in which the non-carbonyl moiety of the ester group is selected from the group consisting of straight or branched chain alkyl (e.g., methyl, n-propyl, n-butyl or tert-butyl), alkoxyalkyl (e.g., methoxymethyl), aralkyl (e.g., benzyl), aryloxyalkyl (e.g., phenoxymethyl), aryl (e.g., optionally substituted with halogen, C, etc.)_1-4Alkyl or C_1-4Alkoxy or amino substituted phenyl); sulfonates such as alkylsulfonyl or arylsulfonyl (e.g., methylsulfonyl); an amino acid ester (e.g., L-valyl or L-isoleucyl), or a pharmaceutically acceptable salt thereof. The preparation is according to the fieldKnown methods are carried out, for example, from textbooks of organic chemistry (e.g.March (1992), "advanced organic chemistry: reactions, mechanisms and structures", fourth edition John Wiley&Sons).

The term "alkyl" as used herein denotes a straight or branched chain hydrocarbon group containing from 1 to 12 carbon atoms. Preferably, the term "alkyl" denotes a straight or branched chain hydrocarbon group containing 1 to 7 carbon atoms. Most preferred are methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl or pentyl. Alkyl groups may be unsubstituted or substituted. The substituents are selected from one or more of cycloalkyl, nitro, amino, alkylamino, dialkylamino, alkylcarbonyl and cycloalkylcarbonyl.

The term "cycloalkyl" as used herein denotes an optionally substituted cycloalkyl group containing 3 to 7 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.

The term "alkoxy" as used herein denotes an optionally substituted straight or branched alkyl-oxy group, wherein the "alkyl" moiety is as defined above, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy, heptyloxy, including isomers thereof.

The term "alkoxyalkyl" as used herein denotes an alkoxy group, as defined above, bonded to an alkyl group, as defined above. Examples are methoxymethyl, methoxyethyl, methoxypropyl, ethoxymethyl, ethoxyethyl, ethoxypropyl, propoxypropyl, methoxybutyl, ethoxybutyl, propoxybutyl, butoxybutyl, tert-butoxybutyl, methoxypentyl, ethoxypentyl, propoxypentyl, including their isomers.

The term "alkenyl" as used herein denotes an unsubstituted or substituted hydrocarbon chain radical having from 2 to 7 carbon atoms, preferably from 2 to 4 carbon atoms, and having one or two olefinic double bonds, preferably one olefinic double bond. Examples are vinyl, 1-propenyl, 2-propenyl (allyl) or 2-butenyl (crotyl).

The term "alkynyl" as used herein denotes an unsubstituted or substituted hydrocarbon chain radical having from 2 to 7 carbon atoms, preferably from 2 to 4 carbon atoms, and having one or possibly two triple bonds, preferably one triple bond. Examples are ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl or 3-butynyl.

The term "hydroxyalkyl" as used herein denotes a straight or branched chain alkyl group as defined above wherein 1, 2, 3 or more hydrogen atoms are substituted by hydroxyl groups. Examples are hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, hydroxyisopropyl, hydroxybutyl and the like.

The term "haloalkyl" as used herein denotes a straight or branched chain alkyl group as defined above wherein 1, 2, 3 or more hydrogen atoms are substituted by halogen. Examples are 1-fluoromethyl, 1-chloromethyl, 1-bromomethyl, 1-iodomethyl, trifluoromethyl, trichloromethyl, tribromomethyl, triiodomethyl, 1-fluoroethyl, 1-chloroethyl, 1-bromoethyl, 1-iodoethyl, 2-fluoroethyl, 2-chloroethyl, 2-bromoethyl, 2-iodoethyl, 2, 2-dichloroethyl, 3-bromopropyl or 2, 2, 2-trifluoroethyl and the like.

The term "alkylthio" as used herein denotes a straight or branched chain (alkyl) S-group, wherein the "alkyl" moiety is as defined above. Examples are methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio or tert-butylthio.

The term "aryl" as used herein denotes optionally substituted phenyl and naphthyl (e.g. 1-naphthyl, 2-naphthyl or 3-naphthyl). Suitable substituents for the aryl group may be selected from those known as alkyl groups, although substituents which may be selected in addition to halogen, hydroxy and optionally substituted alkyl, haloalkyl, alkenyl, alkynyl and aryloxy groups.

The term "heterocyclyl" as used herein denotes an optionally substituted saturated, partially unsaturated or aromatic monocyclic, bicyclic or tricyclic heterocyclic ring system containing one or more heteroatoms selected from nitrogen, oxygen and sulfur, which may also be fused to an optionally substituted saturated, partially unsaturated or aromatic monocyclic carbocyclic or heterocyclic ring.

Examples of suitable heterocycles are oxazolyl, isoxazolyl, furyl, tetrahydrofuryl, 1, 3-dioxolanyl, dihydropyranyl, 2-thienyl, 3-thienyl, pyrazinyl, isothiazolyl, dihydrooxazolyl, pyrimidinyl, tetrazolyl, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, pyrrolidinonyl (pyrrolidinonyl), (N-oxide) -pyridyl, 1-pyrrolyl, 2-pyrrolyl, triazolyl (e.g. 1, 2, 3-triazolyl or 1, 2, 4-triazolyl), 1-pyrazolyl, 2-pyrazolyl, 4-pyrazolyl, piperidinyl, morpholinyl (e.g. 4-morpholinyl), thiomorpholinyl (e.g. 4-thiomorpholinyl), thiazolyl, pyridinyl, dihydrothiazolyl, thiazolyl, Imidazolidinyl, pyrazolinyl, piperazinyl, 1-imidazolyl, 2-imidazolyl, 4-imidazolyl, thiadiazolyl (e.g., 1, 2, 3-thiadiazolyl), 4-methylpiperazinyl, 4-hydroxypiperidin-1-yl.

Suitable substituents for the heterocyclic group may be selected from those known as alkyl groups, although in addition optionally substituted alkyl, alkenyl, alkynyl, oxo (═ O) or aminosulfonyl groups may be selected.

The term "acyl" ("alkylcarbonyl") as used herein denotes a group of formula C (═ O) R, wherein R is hydrogen, an unsubstituted or substituted straight or branched chain hydrocarbyl group containing from 1 to 7 carbon atoms, or phenyl. Most preferred acyl groups are those wherein R is hydrogen, unsubstituted straight or branched chain hydrocarbyl containing 1 to 4 carbon atoms or phenyl.

The term halogen represents fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine, bromine.

The term "X" in the present invention represents O, S or CH₂Preferably O or CH₂. Most preferably "X" represents O.

The term "Z" in the present invention represents O or S, preferably O.

In the compound diagrams given in the present application, the tapered line: () Represents a substituent located above the plane of the ring to which the asymmetric carbon belongs, and the bold dotted line: () Represents a substituent located below the plane of the ring to which the asymmetric carbon belongs.

The compounds of formula I may exhibit stereoisomerism. These compounds may be any isomer of the compounds of formula I or a mixture of such isomers. The compounds and intermediates of the present invention having one or more asymmetric carbon atoms are available as racemic mixtures of their stereoisomers and can be resolved.

The compounds of formula I may exhibit tautomerism, which means that the compounds of the present invention may exist as two or more chemical compounds that are capable of flexible interconversion. In many cases, this merely means an exchange of a hydrogen atom between two other atoms, which hydrogen atom forms a covalent bond with one of these two other atoms. Tautomeric compounds exist in dynamic equilibrium with each other, so attempts to prepare individual substances often result in the formation of mixtures that exhibit all of the desired chemical and physical properties based on the structure of each component.

The most common types of tautomerism involve carbonyl or ketone compounds and unsaturated hydroxyl compounds or enols. The structural change is the transfer of a hydrogen atom between a carbon atom and an oxygen atom, with bond rearrangement. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the ketone form predominates; in phenol, the enol form is the major component.

Basic compounds of formula I can form pharmaceutically acceptable salts with inorganic acids such as hydrohalic acids (e.g., hydrochloric and hydrobromic acids), sulfuric, nitric and phosphoric acids, and the like, and organic acids such as acetic, tartaric, succinic, fumaric, maleic, malic, salicylic, citric, methanesulfonic, p-toluenesulfonic acid, and the like. The formation and isolation of such salts can be carried out according to methods known in the art.

Preference is given to the use of compounds of the formula I in which

R is hydrogen;

R¹is alkyl, alkenyl, alkynyl, haloalkyl, alkylcarbonyl, alkoxy, hydroxymethyl, cyano, azido, alkoxyiminomethyl, alkylcarbonylamino, alkylaminomethyl or dialkylaminomethyl;

R²is hydrogen, hydroxy, alkoxy or halogen;

R³and R⁴Is hydrogen, hydroxy, alkoxy, halogen or hydroxyalkyl, with the proviso that R³And R⁴At least one is hydrogen; or

R³And R⁴Represents fluorine;

x is O or CH₂；

B represents a 9-purinyl group B1 or a 1-pyrimidinyl group B2 as defined above.

Examples of preferred compounds are listed in the following table.

A particularly preferred group of compounds for the treatment of HCV are those of formula I-a:

wherein the content of the first and second substances,

R¹is alkyl, alkenyl, alkynyl, haloalkyl, alkylcarbonyl, alkoxy, hydroxymethyl, cyano, azido, alkoxyiminomethyl, alkylcarbonylamino, alkylaminomethyl or dialkylaminomethyl;

R²is hydrogen, hydroxy, alkoxy or halogen;

R³and R⁴Is hydrogen, hydroxy, alkoxy, halogen or hydroxyalkyl, with the proviso that R³And R⁴At least one is hydrogen; or

R³And R⁴Represents fluorine.

Examples of such particularly preferred compounds are listed in the following table.

The most preferred compounds for the treatment of HCV are listed in the following table:

the compounds of formula I can be prepared by various methods which are generally known in the field of organic chemistry, in particular in the field of nucleoside analogue synthesis. Synthetic starting materials are readily available from commercial sources, are known, or can be prepared per se by techniques known in the art. Reviews of nucleoside analogue preparations are included in the following publications:

a M Michelson "Chemistry of nucleosides and Nucleotides (The Chemistry of Nucleotides and Nucleotides)," Academic Press, New York, 1963.

L Goodman "Basic Principles of nucleic acid Chemistry (Basic Principles in nucleic acid Chemistry)" Ed P O P Ts' O, Academic Press, New York, 1974, Vol.1, Chapter.2.

"Synthesis of Nucleic acid chemistry (Synthetic Procedures in Nucleic acid chemistry)" W W Zorbach and R S Tipson editors, Wiley, New York, 1973, volumes 1 and 2.

The synthesis of carbocyclic nucleosides has been reviewed in L Agrofoglio et al, Tetrahedron, 1994, 50, 10611.

Methods useful for synthesizing compounds of formula I include:

1. modification or interconversion of a related nucleoside; or

2. Constructing a heterocyclic base after glycosylation; or

3. Condensation of protected furanose, thioxfuranose or cyclopentane derivatives with a pyrimidine (B2) or purine (B1) base.

These methods are further discussed below:

1. modifications or interconversion of the relevant nucleosides.

Such methods include modification of the 9-purinyl or 1-pyrimidinyl group on the one hand, or the carbohydrate moiety on the other hand.

A. Modification of the purinyl or pyrimidinyl moiety:

a) deamination of aminopurine or aminopyrimidine nucleosides, as described in j.r.tittensor and r.t.walker, European Polymer j., 1968, 4, 39 and h.hayatsu, Nucleic Acid Research and Molecular Biology Progress (Progress in Nucleic Acid Research and Molecular Biology), 1976, volume 16, page 75.

b) The 4-hydroxy group of the 4-hydroxypyrimidine nucleoside is converted to a leaving group and displaced with a nucleophile.

Such leaving groups include halogens, such as those described in j.brokes and j.beranek, col.czech.chem.comm., 1974, 39, 3100, or 1, 2, 4-triazoles, such as those described in k.j.divakar and c.b.reece, j.chem.soc.perkin trans.i, 1982, 1171.

c) 5-substitution of pyrimidine nucleosides has been achieved using 5-metal derivatives such as 5-mercury or 5-palladium, for example as described in D.E. Bergstrom and J.L. Ruth, J.Amer.chem.Soc., 1976, 98, 1587. Fluorine can be introduced at the 5-position of pyrimidine nucleosides using reagents such as trifluoromethylhypofluorite, as described in m.j.robins, Ann New York acad.sci.1975, 255, 104.

d) Modified purine nucleosides can be prepared from corresponding purine nucleoside derivatives in which the 2-, 6-or 8-substituent is a suitable leaving group such as a halogen or sulfonate or 1, 3, 4-triazole. 6-substituted purine nucleosides can be prepared by: suitable 6-halopurine or 6- (1, 2, 4-triazol-4-yl) -purine nucleoside derivatives are treated with a suitable nucleophile, as described in VNair and A.J.FassBender, Tetrahedron, 1993, 49, 2169 and V Samano, R.W.Miles and M.J.Robins, J.Am.Chem.Soc., 1994, 116, 9331. Similarly, 8-substituted purine nucleosides can be prepared by: the corresponding 8-halopurine nucleosides are treated with appropriate nucleophiles, such as l.tai-Shun, c.jia-Chong, i.kimiko and a.c.sartorelli, j.med.chem., 1985, 28, 1481; nandanan et al, j.med.chem., 1999, 42, 1625; jansons, y, maurinhs and m.lidaks, Nucleotides, 1995, 14, 1709. Introduction of the 8-cyano substituent can be accomplished by displacement using a metal cyanide, as described in L-L. gundersen, acta. chem. scan. 1996, 50, 58. 2-modified purine nucleosides can be prepared in a similar manner as described in T.Steinbrecher, C.Wamelung, F.Oesch and A.Seidl, Angew.chem.int.Ed.Engl., 1993, 32, 404.

e) If the substituent at the 2-or 8-position of the purine nucleoside is linked via a carbon-carbon bond, for example an alkyl group, a metal catalysed cross-coupling process may be employed starting from the appropriate 2-or 8-halopurine nucleoside analogue, as described in a.a. van amerschott et al, j.med.chem., 1993, 36, 2938; nair and g.s.buenger, j.am.chem.soc., 1989, 111(22), 8502; tu, c.keane and b.e.eaton, Nucleotides, 1995, 14, 1631.

B. Modification of the carbohydrate moiety:

after introduction of a protecting group compatible with further chemical reactions:

4 ', 5 ' -didehydronucleosides can be treated with iodine azide to introduce azides at the 4 ' -position, as described, for example, in h.mag et al, j.med.chem., 1992, 35, 1440. Treatment of 4 ', 5 ' -didehydronucleosides with iodine followed by treatment with alcohol and lead carbonate can introduce an alcoholate at the 4 ' -position, for example as described in j.p.verheyden and j.g.moffatt, j.am.chem.soc., 1975, 97(15), 4386. Treatment of 4 ', 5 ' -didehydronucleosides with iodine followed by silver (I) fluoride can introduce fluoride at the 4 ' -position, as described in G.R.Owen et al, J.org.Chem., 1976, 41(8), 3010 or A.Maguire et al, J.Chem.Soc.Perkin Trans.1, 1993, 1(15), 1795. The 4 ' -formyl group can be introduced and subsequently converted to a variety of substituents including, but not limited to, 4 ' -haloalkyl, 4 ' -ethynyl, 4 ' -oximinomethyl, and 4 ' -cyano, such as described in m.nomura et al, j.med.chem., 1999, 42, 2901.

Modifications of the 2 '-hydroxy substituent or the 3' -hydroxy substituent in nucleoside analogues are possible.

The 3-hydroxy group can be converted into a leaving group such as a halogen by reaction with, for example, triphenylphosphine and tetrahaloalkanes, as described, for example, by L.De Napoli et al, Nucleotides, 1993, 12, 981, followed by reduction to give 3-deoxy sugar derivatives, as described, for example, by D.G. Norman and C.B. Reese, Synthesis 1983, 304.

3-hydroxy groups can be derivatized by conversion to a triflate group and then reduced with sodium borohydride as described in S.A. Surzhykov et al, Nucleotides, 1994, 13(10), 2283. The direct introduction of fluorine substituents can be accomplished using fluorinating agents such as diethylaminosulfur trifluoride, as described in p.herdewijn, a.van amerschot, and l.kerremans, Nucleotides, 1989, 8, 65.

Conversion of the hydroxyl substituent to a leaving group such as a halogen or sulfonate ester also allows displacement with nucleophiles such as tetrabutylammonium fluoride, lithium azide or metal cyanides, for example h.hrebabecky, a.holy and e.de Clercq, collection.czech.chem.comm.1990, 55, 1800; k.e.b.parkes and k.taylor, tet.lett., 1988, 29, 2995; pfundheller et al, helv. chim. acta, 2000, 83, 128.

The reaction of an-2 ' -keto nucleoside with a fluorinating agent such as diethylaminosulphur trifluoride can be used for the preparation of 2 ', 2 ' -difluoronucleosides, as described in d.bergstrom, e.romo and p.shum, nucleic acids, 1987, 6, 53.

2. The heterocyclic base is constructed after glycosylation.

a) Those utilizing, for example, furanosylamine derivatives are described in n.j.cusack, b.j.hildic, d.h.robinson, p.w.rugg and g.shaw, j.chem.soc.perkin trans, I, 1973, 1720 or g.shaw, r.n.warrener, m.h.maguire and r.k.ralph, j.chem.soc.1958, 2294.

b) Those methods which utilize, for example, furanosyl ureas for pyrimidine nucleosides, e.g.Farkas andc zech chem comm, 1966, 31, 291.

c) Methods for preparing purine nucleosides from imidazole nucleosides are described in l.b. townsend, chem.rev., 1967, 67, 533.

d) Wherein X is CH₂The preparation of the compounds of formula I can be carried out from 1-hydroxymethyl-4-aminocyclopentane derivatives, such as y.f. shealy and j.d. clayton, j.am. chem. soc., 1969, 91, 3075; r.vince and s.daluge, j.org.chem., 1980, 45, 531; r.c. cermak and r.vince, tet.lett, 1981, 2331; r.d. elliott et al, j.med.chem., 1994, 37, 739.

3. Condensation of protected furanose, thioxfuranose or cyclopentane derivatives with purine or pyrimidine derivatives.

The condensation of the protected furanose, thioxanthose or cyclopentane derivative with the appropriate purine or pyrimidine derivative can be carried out using standard procedures involving the use of Lewis acid catalysts such as mercuric bromide or tin chloride or trimethylsilyl triflate in solvents such as acetonitrile, 1, 2-dichloroethane, dichloromethane, chloroform or toluene at low, ambient or elevated temperatures. Protected furanoses or thioxanthes

Examples of condensation reactions with heavy metal derivatives (e.g. mercury chloride-based derivatives) of purine or pyrimidine derivatives are J Davoll and b.a.lowry, j.am.chem.soc., 1951, 73, 1650; j.fox, n.yung, j.davoll and g.b.brown, j.am.chem.soc, 1956, 78, 2117.

Examples of condensation reactions with alkoxypyrimidines are described in k.a. watanabe, d.h.hollenberg and j.j.fox., carbohydrates, nucleosides and Nucleotides (carbohydrates.nucleotides and Nucleotides), 1974, 1, 1.

Examples of condensation reactions with silyl derivatives of purines or pyrimidines are e.g. u.niedballa and h.vorbrggen, j.org.chem., 1976, 41, 2084; niedballa and h.vorberuggen, j.org.chem., 1974, 39, 3672; hubbard, a.s.jones and r.t.walker, Nucleic Acids res, 1984, 12, 6827.

In addition to this, the present invention is,

fusion of peracylated sugars with purines in the presence of p-toluenesulfonic acid under vacuum is described for example in t.simadate, y.ishudo and t.sato, chem.abs., 1962, 56, 11692 and w.pfleiderer, r.k.robins, chem.ber.1965, 98, 1511.

Condensation reactions have been described for example in k.a. watanabe, d.h.hollenberg and j.j.fox, Carbohydrates, Nucleosides and nucleotides (Carbohydrates nucleotides and nucleotides), 1974, 1, 1.

Examples of condensation reactions of protected cyclopentane derivatives with appropriate purine or pyrimidine derivatives are found in h.kapelleter, h.baumgartner and h.griengl, montttshchem, 1997, 128, 191 and p.wang et al, tet.lett, 1997, 38, 4207, or t.jenny et al, helv.chim.acta, 1992, 25, 1944.

Such methods often produce mixtures of anomeric nucleoside derivatives, which can be separated by standard techniques known in the art, such as recrystallization, column chromatography, high performance liquid chromatography, or supercritical fluid chromatography.

The purine derivatives and pyrimidine derivatives used in the above condensation reactions may be obtained from commercial sources or may be prepared by processes known in the art.

See, g.shaw "general heterocyclic Chemistry", Pergamon press, volume 5, chapter 4.09, page 499 and "general heterocyclic Chemistry II", Pergamon press, volume 7, chapter 7.11, page 397.

Pyrimidine derivatives are prepared as described in d.j.brown "Heterocyclic chemistry-Pyrimidines (The chemistry of Heterocyclic Compounds-The Pyrimidines)" 1962 and suppl 1, 1970, published by John Wiley and Sons, new york; brown, "general heterocyclic chemistry", Pergamon press, volume 5, chapter 4.09, page 499 and k.unheim and t.benneche, "general heterocyclic chemistry II", Pergamon press, volume 6, chapter 6.02, page 93.

The furanose derivatives can be prepared from commercially available carbohydrate starting materials, such as ribose, arabinose, xylose or lyxose in the form of D, after the introduction of chemically compatible protecting groups.

4-substituted furanoses having a carbon-containing substituent attached to the 4-position of the furanose can be prepared from the corresponding 4-formylfuranose, such as alkyl, alkenyl, alkynyl, haloalkyl, acyl, alkoxycarbonyl, hydroxyalkyl, alkoxyalkyl, cyano, hydroximinomethyl, alkoxyiminomethyl, alkylaminocarbonyl, and acyl. One such 4-formylfuranose is described in h.ohrui et al, j.med.chem., 2000, 43, 5416. 4-haloalkyl furanoses can be prepared from the corresponding 4-hydroxymethyl furanoses (e.g., K.Kitano et al, Tetrahedron, 1997, 53(39), 13315). 4-Methylfuranose can be prepared by the method described in T.Waga et al, biosci.Biotech.biochem.1993, 19(7), 408.

2, 2-Difluorofuranose derivatives may be prepared from D-glucose or D-mannose as described in R.Fernandez, M.I.Mateu, R.Echarri and S.Castillon, Tetrahedron, 1998, 54, 3523. Thiofuranose derivatives may be prepared according to literature procedures such as those described in L.Bellon, J.L.Barascut, J.L.Imbach, Nucleosides and Nucleotides (Nucleosides and Nucleotides), 1992, 11, 1467, and modified in a manner similar to the furanose analogues described above.

Cyclopentane derivatives can be prepared by methods known in the art of organic chemistry and by methods and references included in l.agrofolio et al, Tetrahedron, 1994, 50, 10611.

Nucleoside derivatives of interest are commercially available or can be synthesized as described above.

The methods discussed above are described in more detail below:

wherein R is¹Is N₃、R²And R³Compounds of formula I that are hydroxy and B is B2 can be prepared according to reaction scheme a:

procedure A

Wherein Ac is acetyl, Bz is benzoyl, R¹¹As defined above.

Compounds of formula II may be iodinated with a mixture of triphenylphosphine, iodine and pyridine, for example as described in h.mag et al, j.med.chem., 1992, 35, 1440. The acetonide protecting group may be removed by treatment with an acid, such as acetic acid, as described in j.p. verheyden et al, j.org.chem., 1970, 35(7), 2319, to give the nucleoside of formula III. 4 ', 5' -didehydronucleosides of formula V are obtained after protection of the 2 'and 3' hydroxyl groups with acetic anhydride and pyridine, elimination of hydrogen iodide with solutions of pyridine, e.g. silver fluoride, and removal of the acetyl protecting groups with solutions of ammonia in methanol, as described in J.P.Verheyden et al, J.org.Chem., 1974, 39(24), 3573. Addition of iodine azide to the double bond can be accomplished by treatment of V with a mixture of iodine chloride and sodium azide in N, N-dimethylformamide as described in h.mag et al, j.med.chem., 1992, 35, 1440 to give a nucleoside of formula VI. Protection of the hydroxyl group in VI can be accomplished by treatment of VI with pyridine solution of benzoyl chloride to give a nucleoside of formula VII, which can then be converted to a 5' -benzoyl nucleoside of formula VIII by treatment with dichloromethane solution of m-chloroperbenzoic acid, which can then be deprotected with a base such as sodium methoxide in methanol to give a nucleoside of formula IX, as described in h.mag et al, j.med.chem., 1992, 35, 1440. In the case where B2 of the compound of formula VIII is uracil or a 5 '-substituted uracil, after protection of the 3' -hydroxyl group with acetic anhydride and pyridine, the conversion to the corresponding cytidine of formula XII can be accomplished as described in A.D. Borthwick et al, J.Med.chem., 1990, 33(1), 179, wherein the nucleoside of formula X can be treated with 4-chlorophenyl dichlorophosphate and triazole to give a 4-triazolyl nucleoside of formula XI, which nucleoside XI is then treated with aqueous ammonia to give a 5-substituted cytidine of formula XII.

Wherein R is¹is-C ≡ CH, -CH ═ CHCl, -CH ═ N-OH, -CN, R²And R³Compounds of formula I that are hydroxy and B is B1 or B2 can be prepared according to reaction scheme B:

procedure B

The compound of formula XIII can be silylated with tert-butyldimethylsilyl chloride (TBSCl) and imidazole to give the tri-tert-butyldimethylsilyl compound of formula XIV. The 5 '-tert-butyldimethylsilyl ether can be deprotected with 80% acetic acid to give the 5-hydroxynucleoside XV, which can then be oxidized to the 5' -formyl nucleoside XVI using a mixture of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDAC) and dimethyl sulfoxide (DMSO) in a suitable solvent such as benzene. Alkylation of XVI with formaldehyde and sodium hydroxide gives the 4 '-hydroxymethyl compound XVII, which can be reduced to the 4' -dihydroxymethyl compound XVIII. Selectively protecting the hydroxymethyl group on the α -plane of the nucleoside with a pyridine solution of trityl chloride gives the 4' -trityl compound XIX, followed by protection of the hydroxymethyl group on the β -plane of the nucleoside with t-butyldimethylsilyl chloride (TBSCl) and imidazole gives the compound of formula XX. Deprotection of the trityl group with bromocatechol borane affords the 4 '-hydroxymethyl compound XXI, which can be oxidized with trifluoromethanesulfonic anhydride and dimethyl sulfoxide to give the 4' -formyl compound of formula XXII.

Aldehydes of formula XXII can be used as starting materials for a variety of 4' -substituted nucleosides, as depicted in scheme C:

procedure C

Aldehyde XXII is treated with hydroxylamine hydrochloride and pyridine to give a 4' -hydroxyimine of the formula XXIII. Removal of water from compound XXIII affords a 4' -cyano compound of formula XXIV. Treatment of a 4 '-formyl compound of formula XXII with chloromethylphosphonium chloride and butyllithium affords the 4' - (2-chloroethenyl) compound XXVI. Treatment of compound XXVI with butyllithium results in elimination of hydrogen chloride to give the 4' -ethynyl compound of formula XXVII. Removal of the silyl protecting group from compounds XXIII, XXVII and XXIV protected with tri-tert-butyldimethylsilyl chloride can be achieved by using a fluoride source such as a solution of ammonium fluoride in methanol or tetrabutylammonium fluoride on silica gel adsorbed in tetrahydrofuran to give the respective 4' -substituted nucleosides XXV, XXVIII and XXIX.

Appropriately protected 4 '-substituted uridines (e.g., XXIV and XXVII) can be converted to the corresponding 4' -substituted cytidine according to reaction scheme D:

procedure D

A uridine protected with tri-tert-butyldimethylsilyl (TBS) of formula XXX can be treated with triisopropylbenzenesulfonyl chloride, triethylamine and dimethylaminopyridine to give a 4-triazolyl nucleoside XXXI. The 4-triazolyl compound XXXI can be converted to the 4-amino compound XXXII using aqueous ammonia. Deprotection of the silyl group with a mixture of methanol and hydrochloric acid in dioxane gave the cytidine derivative XXXIII.

Wherein R is¹Is alkoxy, R²And R³Compounds of formula I which are hydroxy and B is 9-purinyl B1 or 1-pyrimidinyl B2 may be prepared according to the procedures described in U.S. Pat. No.3910885 to J.P.Verheyden et al.

Wherein R is¹Compounds of formula I that are trifluoromethyl, methyl, or ethynyl may be prepared as described in reaction scheme E:

procedure E

For example, coupling of an appropriately protected 4' -substituted ribofuranoside XXXIV with a silylated base in the presence of a Lewis acid such as trimethylsilyl triflate (TMSOTf) or tin tetrachloride in a suitable solvent such as acetonitrile or 1, 2-dichloroethane affords compounds of formula XXXV. The protecting group may be obtained by treating XXXV with a base such as sodium methoxide in a compatible solvent such as methanol to give a compound of formula XXXVI.

Methods for the monophosphorylation of organic compounds, including nucleosides, have been reviewed by L A Slotin, Synthesis, 1977, 737. More recently, phosphorylation of other nucleosides has been described by M Uchiyama et al, j. R Caputo et al, synlett, 1997, 739 and M Taktakishvili and V Nair, tet. Other monophosphorylation processes that can be used for nucleosides are described in C E McKenna and J Schmidhauser, J. chem.soc., chem.commun., 1979, 739, and J K Stowell and T S Widlanski, tet.lett., 1995, 1825. Synthesis of the di-and triphosphate derivatives is described in K HScheit, Nucleotide analogs, 1980, Wiley Interscience and K Burgess and D Cook, Chemical Reviews, 2000, 100, 2047.

The following examples illustrate the preparation of compounds of formula I:

example 1

According toSchemes 1 and 1aMethod for preparingCompound 1，

Scheme 1

1.1 Compounds (i)

Compound (i) was purchased from Lancaster (Cat. No.: 206-647-7, CAS 362-43-6).

1.2 Compounds (ii)

To a solution of compound (i) (1.14g, 4.0mmol) containing pyridine (0.65ml, 8.0mmol) in dioxane (20ml) was added triphenylphosphine (1.57g, 6.0mmol) and iodine (1.52g, 6.0 mmol). The mixture was stirred overnight and quenched with methanol (1 ml). The solvent was evaporated in vacuo. The residue was dissolved in ethyl acetate (200ml), which was washed with water (100ml), 10% aqueous sodium thiosulfate (100ml), brine (100ml) and dried over anhydrous magnesium sulfate. The magnesium sulfate was removed by filtration and the filtrate was evaporated in vacuo. The residue was purified by flash column chromatography on silica eluting with 1: 1 ethyl acetate/petrol to give compound (ii) as a colourless oilA slowly solidified colorless waxy solid (1.5 g); mass Spectrometry (CI) M/z 395[ M + H ]]⁺。

1.3 Compounds (iii)

Compound (iii) is prepared from compound (ii) as described in j.p. verheyden et al, j.org.chem., 1970, 35(7), 2319.

1.4 Compounds (iv)

Compound (iv) is prepared from compound (iii) as described in j.p. verheyden et al, j.org.chem., 1974, 39(24), 3573.

1.5 Compounds (v)

Compound (v) is prepared from compound (iv) as described in h.maag et al, j.med.chem., 1992, 35, 1440-1451.

1.6 Compounds (vi)

To a solution of compound (v) (482mg, 0.80mmol) in water-saturated methylene chloride (10ml) was added 55% m-chloroperbenzoic acid (1.0g, 4.95 mmol). The mixture was stirred for 2 hours. Additional m-chloroperbenzoic acid (0.50g) was added and the mixture was stirred for an additional 3 hours. Ethyl acetate (100ml) was added and the solution was washed with 10% sodium metabisulphite solution (50ml) and then saturated sodium bicarbonate solution (50 ml). The ethyl acetate layer was dried over anhydrous magnesium sulfate. The magnesium sulfate was removed by filtration and the filtrate was evaporated in vacuo. The residue was purified by flash chromatography eluting with 1: 1 ethyl acetate/petrol to give compound (vi) as a colourless glass (200 mg); mass Spectrometry (ESI) M/z 535[ M + H + CH ]₃CN]⁺。

1.7 Compounds (vii)

To a solution of compound (vi) (170mg, 0.35mmol) in methanol (2ml) was added a solution of sodium methoxide in methanol (0.5M, 0.5 ml). The solution was stirred at room temperature for 2 hours. With ion exchange resin (Amberlite IRC 50 (H)⁺)，Aldrich，cat.no.42，883-3) the solution was neutralized and stirred for 10 minutes. The resin was removed by filtration. The filtrate was evaporated in vacuo and the residue was purified by flash chromatography, eluting with 1: 1 ethyl acetate/acetone to give a colorless oil. Trituration with ethyl acetate afforded compound (vii) as a colourless solid (35 mg); mass Spectrometry (CI) M/z 286[ M + H ]]⁺。

The conversion of azidouridine derivatives to the corresponding azidocytidine derivatives (compound 1) and their hydrochloride salts is described in scheme 1 a.

Scheme 1a

1.8 Compounds (viii)

To a solution of compound (vi) (460mg, 0.93mmol) in pyridine (3ml) was added acetic anhydride (1ml) and the mixture was stirred for 4 hours. Ethyl acetate (100ml) was added and the mixture was washed with 2N HCl (50ml) and then saturated sodium bicarbonate solution (50 ml). The solution was dried over anhydrous magnesium sulfate. The magnesium sulfate was removed by filtration and the filtrate was evaporated in vacuo. The residue was purified by flash chromatography, eluting with 1: 1 ethyl acetate/petrol to give compound (viii) as a colourless gum (350 mg); mass Spectrometry (ESI) M/z 536[ M + H ]]⁺。

1.9 Compounds 1

To a solution of compound (viii) (1.5g, 2.8mmol) in pyridine (20ml) was added 1, 2, 4-triazole (0.97g, 14 mmol). 4-chlorophenyl dichlorophosphate (1.36ml, 8.4mmol) was then added dropwise with stirring. The mixture was stirred for 16 hours. Ethyl acetate (300ml) was added and the mixture was washed with saturated sodium bicarbonate solution (200 ml). The solution was dried over anhydrous magnesium sulfate. The magnesium sulfate was removed by filtration and the filtrate was evaporated in vacuo. The residue was purified by flash chromatography, eluting with 2: 1 ethyl acetate/petrol to give a yellow foam (850 mg). The foam was treated with dioxane (8ml),Then treated with aqueous ammonia (16ml) and stirred for 16 hours. The filtrate was evaporated in vacuo and the residue was purified by flash chromatography, eluting with 90: 18: 3: 2 dichloromethane/methanol/acetic acid/water to give compound 1 as a light brown foam (350 mg); mass Spectrometry (FAB) M/z 285[ M + H ]]⁺。

1.10 hydrochloride salt of Compound 1

Compound 1(0.40g) was dissolved in methanol and treated with a solution of hydrogen chloride in ethyl acetate. The product was isolated as a microcrystalline solid, collected by filtration and dried in vacuo to give the hydrochloride salt of compound 1 (0.22 g); mass Spectrometry (ESI) M/z 285[ M + H ]]⁺。

Example 2

The preparation of compound 2 was carried out according to the procedure of scheme 2.

Scheme 2

2.1 Compounds (ix)

Compound (ix) is prepared from compound (xiv) as described in example 3, m.nomura et al, j.med.chem., 1999, 42, 2901-.

2.2 Compounds (x)

A mixture of (ix) (600mg, 0.98mmol) and hydroxylamine hydrochloride (140mg, 1.95mmol) in pyridine was stirred at room temperature for 2 hours. The reaction mixture was evaporated in vacuo and the residue partitioned between ethyl acetate (30ml) and water (30 ml). The ethyl acetate layer was separated and dried over anhydrous magnesium sulfate. The magnesium sulfate was removed by filtration and the filtrate was evaporated in vacuo to give compound (x) as a white foam (615 mg); mass Spectrometry (ESI) M/z 630[ M + H ]]⁺。

2.3 Compounds (xi)

A mixture of compound (x) (550mg, 0.87mmol) and sodium acetate (720mg, 5.25mmol) was suspended in acetic anhydride, followed by heating at 130 ℃ for 3 hours. The reaction mixture was evaporated in vacuo and the residue partitioned between ethyl acetate (30ml) and saturated sodium bicarbonate (30 ml). The ethyl acetate layer was separated and dried over anhydrous magnesium sulfate. The magnesium sulfate was removed by filtration and the filtrate was evaporated in vacuo. The residue was purified by flash column chromatography on silica gel eluting with 1: 2 diethyl ether/hexane. The product-containing fractions were combined and evaporated in vacuo to give compound (xi) as a colorless solid (285 mg); mass Spectrometry (ESI) M/z 612[ M + H ]]⁺。

2.4 Compounds (xii)

To a solution of compound (xi) (200mg, 0.33mmol) and 1, 2, 4-triazole (115mg, 1.63mmol) in anhydrous pyridine (5ml) was added dropwise 4-chlorophenyl dichlorophosphate (160. mu.l, 0.98mmol), followed by stirring at room temperature for 16 hours. The reaction mixture was evaporated in vacuo and the residue partitioned between ethyl acetate (30ml) and 2M hydrochloric acid (30 ml). The ethyl acetate layer was separated, which was washed with saturated sodium hydrogencarbonate (30ml) and dried over anhydrous magnesium sulfate. The magnesium sulfate was filtered off and evaporated in vacuo. The residue was purified by flash column chromatography on silica eluting with 1: 1 diethyl ether/hexane followed by 2: 1 diethyl ether/hexane. The product-containing fractions were combined and evaporated in vacuo to give (xii) as a cream solid (65 mg); mass spectrum (ESI) M/z 663[ M + H ]]⁺。

2.5 Compounds (xiii)

Compound (xii) (60mg, 0.09mmol) was stirred with a solution of ammonia (2ml) in acetonitrile at room temperature for 16 hours. The reaction mixture was evaporated in vacuo and the residue partitioned between ethyl acetate (10ml) and 2M hydrochloric acid (10 ml). The ethyl acetate layer was separated and dried over magnesium sulfate. The magnesium sulfate was removed by filtration and evaporated in vacuo to give compound (xiii) as a pale yellow solid (45 mg); mass Spectrometry (ESI) M/z 611[ M + H ]]⁺。

2.6 Compound 2

To a stirred solution of compound (xiii) (40mg, 0.06mmol) in dry tetrahydrofuran (10ml) was added tetrabutylammonium fluoride (1M THF solution, 0.3ml) and stirred at room temperature for 2 hours. The solvent was evaporated in vacuo. The residue was treated with pyridine (1ml), then acetic anhydride (0.3ml) and stirred at room temperature for 4 hours. The solvent was evaporated in vacuo. The residue was treated with ethyl acetate (50ml), which was washed with dilute hydrochloric acid (30ml) and then with 5% aqueous sodium bicarbonate. The ethyl acetate layer was dried over anhydrous magnesium sulfate. The magnesium sulfate was removed by filtration and the filtrate was evaporated in vacuo. The residue was purified by flash column chromatography eluting with ethyl acetate to give an oil. The oil was dissolved in methanol (1ml) and treated with sodium methoxide (0.5M methanol solution, 0.05ml) and left at room temperature for 3 hours. The mixture was purified with ion exchange resin (Amberlite IRC 50 (H)⁺) ) neutralization. The resin was removed by filtration and the filtrate was evaporated in vacuo. The residue was dissolved in water and lyophilized to give compound 2 as an amorphous solid (7 mg).

2.7 by deprotection of compound (xi), the corresponding 4' -cyanouridine can be prepared.

Deprotection can be carried out as follows:

compound (xi) (50mg, 82 μmol) was dissolved in tetrahydrofuran, treated with silica gel with tetrabutylammonium fluoride, and then stirred at room temperature for 16 hours. The reaction mixture was filtered through Hyflo SuperCel (Fluka, cat No.56678), evaporated in vacuo and then purified by silica gel flash column chromatography eluting with dichloromethane/methanol/acetic acid/water (240: 24: 3: 2) and then dichloromethane/methanol/acetic acid/water (90: 18: 3: 2). The product-containing fractions were combined and evaporated. The residue was dissolved in methanol/water (5: 1) and applied to Duolite C225 ion exchange resin (H)⁺Form BDH, cat.no.56678) and stirred for 15 min. The resin was removed by filtration and the filtrate was evaporated in vacuo to a small volume. The product was collected by filtration and dried in vacuo to give 4' -cyano uridine as a white knotCrystalline solid (15 mg); mass Spectrometry (ESI) M/z 270[ M + H ]]⁺。

Example 3

Preparation of compound 3 was carried out according to the procedure of scheme 3.

Scheme 3

3.1 Compounds (xiv)

This compound was prepared according to the procedure described in m.nomura et al, j.med.chem., 1999, 42, 2901-.

3.2 Compounds (xv)

To a solution of compound (xiv) (3.0g, 6.0mmol) in pyridine (20ml) was added trityl chloride (3.2g, 11.5mmol), and the mixture was stirred at room temperature for 16 hours. The solvent was evaporated in vacuo and the residue partitioned between ethyl acetate (50ml) and 2M hydrochloric acid (50 ml). The ethyl acetate layer was separated, which was washed with brine (50ml) and dried over anhydrous magnesium sulfate. The magnesium sulfate was removed by filtration and the filtrate was evaporated in vacuo. The crude product was purified by flash column chromatography on silica eluting with 2: 1 diethyl ether/hexane. The product-containing fractions were combined and evaporated in vacuo to give compound (xv) as a white solid (2.75 g); mass Spectrometry (ESI) M/z 767[ M + H ]]⁺。

3.3 Compounds (xvi)

To a stirred solution of compound (xv) (2.75g, 3.7mmol) in dimethylformamide (20ml) were added tert-butyldimethylsilyl chloride (0.67g, 4.4mmol) and imidazole (0.91g, 13.3 mmol). The reaction was heated to 45 ℃ for 16 hours. Additional tert-butyldimethylsilyl chloride (0.67g, 4.4mmol) and imidazole (0.91g, 13.3mmol) were added and the mixture was heated to 60 ℃ for 4 hours. The solvent was removed by evaporation in vacuo and the residue partitioned between ethyl acetate and brine. The ethyl acetate layer was separated, washed with more brine and dried over anhydrous magnesium sulfate. The magnesium sulfate was removed by filtration and the filtrate was evaporated in vacuo. The remaining colorless foam was purified by flash column chromatography on silica gel eluting with 1: 2 diethyl ether/hexane. The product-containing fractions were combined and evaporated in vacuo to give compound (xvi) as a white solid (3.1 g).

3.4 Compounds (xvii)

To a stirred solution of compound (xvi) (1.5g, 1.77mmol) in anhydrous dichloromethane (50ml) was added bromocatechol borane (355mg, 1.77mmol) at 0 ℃ under nitrogen. The reaction was stirred for 15 minutes, diluted with dichloromethane (50ml) and washed with saturated sodium bicarbonate (100ml) and brine (100 ml). The dichloromethane layer was dried over anhydrous magnesium sulfate. The magnesium sulfate was removed by filtration and the filtrate was evaporated in vacuo. The residue was purified by flash column chromatography on silica eluting with 1: 1 diethyl ether/hexane. The product-containing fractions were combined and evaporated in vacuo to give compound (xvii) as a white solid (930 mg).

3.5 Compound 3

Compound 3 was prepared from compound (xvii) as described in m.nomura et al, j.med.chem., 1999, 42, 2901-.

Other compounds may be prepared according to methods described in the art, for example:

additional compounds of formula I may be prepared according to procedures analogous to those described in the prior art below:

the following assays demonstrate the ability of the compounds of formula I to inhibit HCV RNA replication, thus demonstrating their potential utility in the treatment of HCV infection.

Renilla luciferase assay

The present assay is based on the idea of using a reporter gene as a simple readout of the intracellular HCV replicon RNA levels. For this purpose, the Renilla luciferase gene was introduced into the first open reading frame of the replicon construct NK5.1 (Krieger et al, J.Virol.75: 4614), immediately after the Internal Ribosome Entry Site (IRES) sequence, and fused via the foot-and-mouth disease virus self-cleaving peptide 2A with the Neomycin Phosphotransferase (NPTII) gene (Ryan & Drew, EMBO 13: 928-. Following in vitro transcription, RNA was electroporated into human hepatoma Huh7 cells, and colonies resistant to G418 were isolated and expanded. Stably selected cell line 2209-23 was shown to contain replicative HCV subgenomic RNA, and the activity of the Renilla luciferase expressed by the replicon reflects its intracellular RNA levels.

For the assay method, Renilla luciferase HCV replicon cells (2209-23) cultured in Dulbecco's MEM (GibcoBRL cat No.31966-021) containing 5% fetal calf serum (FCS, GibcoBRL cat No.10106-169) were seeded onto 96-well plates at 5000 cells per well and incubated overnight. After 24 hours, different dilutions of the compound in growth medium were added to the cells, followed by incubation at 37 ℃ for three additional days. The assays were performed on duplicate plates, one milky white and the other transparent, in order to measure the activity and cytotoxicity of the compounds in parallel to ensure that the observed activity was not caused by a decrease in cell proliferation.

At the end of the incubation period, cells in white plates were collected and luciferase activity was measured using the dual luciferase reporter assay system (Promega cat No. eb1960). All reagents described in the following paragraphs were included in the manufacturer's kit, and the reagents were prepared according to the manufacturer's instructions. Briefly, cells were washed twice with 200 μ l phosphate buffered saline (ph7.0) (PBS) per well and lysed with 25 μ l 1 × non-working lysis buffer, followed by incubation at room temperature for 20 minutes. Add 100. mu.l of LAR II reagent to each well. The plates were then inserted into an LB 96V microplate luminometer (MicroLumatplus, Berthold) and 100X Stop & Glo reagents were injected into each well using a machine and the signals were measured using a 2-second delay, 10-second measurement program. IC50, which is the concentration of drug required to reduce replicon levels by 50% relative to untreated cell control values, can be calculated from the graph of percent reduction in luciferase activity versus drug concentration. The results are given below.

For the cytotoxicity assay, WST-1 reagent (cat No.1644807) from Roche diagnostics was used. To each well, including wells containing only medium as blank, 10. mu.l of WST-1 reagent was added. The cells were then incubated at 37 ℃ for 1 to 1.5 hours and the OD at 450nm was measured using a 96-well plate reader (wavelength of reference filter 650 nm). From the WST-1 value reduction percentage-drug concentration graph, CC50, which is the drug concentration required to reduce cell proliferation by 50% relative to untreated cell control values, can still be calculated.

As shown in the above table, the compounds of formula I have the potential to be effective as antiviral agents for the treatment of HCV infections in humans, or can be metabolized into compounds exhibiting such activity.

In another embodiment of the invention, the active compound or derivative or salt thereof may be administered in combination with another antiviral agent, such as an anti-hepatitis agent, including those of formula I. When the active compound or a derivative or salt thereof is administered in combination with another antiviral agent, the activity may be increased over the parent compound. This can be readily assessed by preparing the derivatives according to the methods described herein and testing them for anti-HCV activity.

Administration of the active compound may range from continuous administration (intravenous drip) to several times daily oral administration (e.g., q.i.d.), and other routes of administration may include oral, topical, parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may include a penetration enhancer), buccal and suppository administration.

The 4' -substituted nucleoside derivatives and their pharmaceutically acceptable salts can be used as medicaments in the form of any pharmaceutical preparations. The pharmaceutical preparations can be administered enterally, i.e. orally, for example in the form of tablets, coated tablets, dragees, hard and soft gelatine capsules, solutions, emulsions, syrups or suspensions, or rectally, for example in the form of suppositories. They can also be administered parenterally (by intramuscular, intravenous, subcutaneous or intrasternal injection or infusion techniques), for example in the form of injection solutions; nasally, e.g. in the form of a nasal spray or inhalation spray; topical application, and the like.

For the preparation of pharmaceutical preparations, the 4' -substituted nucleoside derivatives and their pharmaceutically acceptable salts can be formulated together with therapeutically inert inorganic or organic excipients to give tablets, coated tablets, dragees, hard and soft gelatine capsules, solutions, emulsions or suspensions.

The compounds of formula I may be formulated in admixture with a pharmaceutically acceptable carrier. For example, the compounds of the present invention may be administered orally in the form of pharmacologically acceptable salts. Since most of the compounds of the present invention are water soluble, they can be administered intravenously in physiological saline solution (e.g., buffered to a pH of about 7.2 to 7.5). Conventional buffers such as phosphate, bicarbonate or citrate may be used for this purpose. Of course, one of ordinary skill in the art can adjust the formulation within the teachings of the specification to provide a wide variety of formulations for a particular route of administration, so long as the compositions of the present invention are not destabilized or have reduced their therapeutic activity. Rather, modifications of the compounds of the invention to increase their solubility in water or other carriers can be accomplished, for example, by minor adjustments (salt formation, esterification, etc.), which are well within the ordinary skill in the art. It is well within the ordinary skill in the art to adjust the route of administration and dosage regimen of a particular compound to control the pharmacokinetics of the compounds of the present invention to maximize the beneficial effect on the patient.

For parenteral formulations, the carrier will typically comprise sterile water or aqueous sodium chloride, although other ingredients to aid dispersion may be included. Of course, when sterile water is used and maintained sterile, the compositions and carriers must also be sterile. Suspensions for injection may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed.

Suitable excipients for tablets, coated tablets, dragees and hard gelatine capsules are, for example, lactose, maize starch and derivatives thereof, talc and stearic acid or salts thereof.

If desired, the tablets or capsules may be enteric coated or sustained release formulations prepared by standard techniques.

Suitable excipients for soft gelatine capsules are, for example, vegetable oils, waxes, fats, semi-solid and liquid polyols.

Suitable excipients for injection solutions are, for example, water, saline, alcohols, polyols, glycerol or vegetable oils.

Suitable excipients for suppositories are, for example, natural oils and hardened oils, waxes, fats, semi-liquid or liquid polyols.

Suitable excipients for enteral solutions and syrups are, for example, water, polyols, sucrose, invert sugar and glucose.

The pharmaceutical formulations of the present invention may also be provided in sustained release formulations or other suitable formulations.

The pharmaceutical preparations can also contain preserving agents, solubilizers, stabilizing agents, wetting agents, emulsifying agents, sweetening agents, coloring agents, flavoring agents, salts for regulating the osmotic pressure, buffering agents, masking agents or antioxidants.

The pharmaceutical formulation may also contain other therapeutically active agents known in the art.

The dosage can vary within wide limits and can, of course, be adjusted in each particular case to the individual requirements. For oral administration, daily dosages of about 0.01 to about 100mg/kg body weight per day should be suitable for monotherapy and/or combination therapy. Preferred daily dosages are from about 0.1 to about 500mg/kg body weight, more preferably from 0.1 to about 100mg/kg body weight and most preferably from 1.0 to about 100mg/kg body weight. Typical formulations may contain from about 5% to about 95% active compound (w/w). The daily dose may be administered in a single dose or divided doses, typically 1 to 5 times per day.

Prodrug forms of the compounds are preferred in some pharmaceutical dosage forms, including, inter alia, acylated (acetylated or other) derivatives, pyridinium esters and various salt forms of the compounds of the invention. One of ordinary skill in the art will find it easy to modify the compounds of the present invention into prodrug forms to facilitate the release of the active compound to a target site within a host organism or patient. One of ordinary skill in the art may also utilize desirable pharmacokinetic parameters of prodrug forms-where applicable-to deliver the compounds of the invention to a targeted site within a host organism or patient to maximize the intended effect of the compound.

The nucleoside derivatives or the drugs thereof can be used in monotherapy or in combination therapy, i.e. the treatment can be carried out in combination with the administration of one or more other therapeutically active substances, for example immune system modulators such as interferons, interleukins, tumor necrosis factors or colony stimulating factors; an antiviral agent or an anti-inflammatory agent. When the treatment is a combination therapy, such administration may be concurrent or sequential with the 4' -substituted nucleoside derivative. Parallel administration as used herein thus includes administration of each drug at the same time or at different times.

It will be appreciated that references herein to treatment extend to prophylaxis as well as treatment of existing conditions, and that treatment of animals includes treatment of humans as well as other mammals. In addition, as used herein, treating a Hepatitis C Virus (HCV) infection also includes treating or preventing a disease or condition associated with or mediated by Hepatitis C Virus (HCV) infection, or a clinical symptom thereof.

In the present specification, "comprising" means "including or consisting of.

The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

Claims (6)

1. The use of a compound of formula I and pharmaceutically acceptable salts thereof in the manufacture of a medicament for the treatment of a disease mediated by the hepatitis C virus HCV,

wherein the content of the first and second substances,

r is hydrogen or- [ P (O) (OH) -O]_nH, and n is 1, 2 or 3;

R¹is C_1-12Alkyl radical, C_2-7Alkene(s)Base, C_2-7Alkynyl, haloalkyl, alkylcarbonyl, alkoxycarbonyl, hydroxyalkyl, alkoxyalkyl, alkoxy, cyano, azido, hydroxyiminomethyl, alkoxyiminomethyl, halogen, alkylcarbonylamino, alkylaminocarbonyl, azidoalkyl, aminomethyl, alkylaminomethyl, dialkylaminomethyl or heterocyclyl;

R²is hydrogen, hydroxy, amino, C_1-12Alkyl, hydroxyalkyl, alkoxy, halogen, cyano or azido;

R³and R⁴Is hydrogen, hydroxy, alkoxy, halogen or hydroxyalkyl, with the proviso that R³And R⁴At least one is hydrogen; or

R³And R⁴Together represent ═ CH₂Or ═ N-OH, or

R³And R⁴Both represent fluorine;

b represents a 9-purinyl group of formula B1,

wherein the content of the first and second substances,

R⁵is hydrogen, hydroxy, C_1-12Alkyl, alkoxy, alkylthio, NHR⁸Halogen or SH;

R⁶is hydroxy, NHR⁸、NHOR⁹、NHNR⁸、-NHC(O)OR⁹' or SH;

R⁷is hydrogen, hydroxy, C_1-12Alkyl, alkoxy, alkylthio, NHR⁸Halogen, SH or cyano;

R⁸is hydrogen, C_1-12Alkyl, hydroxyalkyl, arylcarbonyl or alkylcarbonyl;

R⁹is hydrogen or C_1-12An alkyl group;

R⁹is' a C_1-12An alkyl group; or

B represents a 1-pyrimidinyl radical of formula B2,

wherein the content of the first and second substances,

z is O or S;

R¹⁰is hydroxy, NHR⁸、NHOR⁹、NHNR⁸、-NHC(O)OR⁹' or SH;

R¹¹is hydrogen, C_1-12Alkyl, hydroxy, hydroxyalkyl, alkoxyalkyl, haloalkyl or halogen;

R⁸、R⁹and R⁹' is as defined above.

2. The use according to claim 1 of a compound of formula I,

wherein the content of the first and second substances,

r is hydrogen;

R¹is C_1-12Alkyl radical, C_2-7Alkenyl radical, C_2-7Alkynyl, haloalkyl, alkylcarbonyl, alkoxy, hydroxymethyl, cyano, azido, alkoxyiminomethyl, alkylcarbonylamino, alkylaminomethyl or dialkylaminomethyl;

R²is hydrogen, hydroxy, alkoxy or halogen;

R³and R⁴Is hydrogen, hydroxy, alkoxy, halogen or hydroxyalkyl, with the proviso that R³And R⁴At least one is hydrogen; or

R³And R⁴Represents fluorine; and is

B represents a 9-purinyl group B1 or a 1-pyrimidinyl group B2 as defined in claim 1.

3. The use according to claim 1 or claim 2 of a compound of formula I-a,

wherein the content of the first and second substances,

R¹is C_1-12Alkyl radical, C_2-7Alkenyl radical, C_2-7Alkynyl, haloalkyl, alkylcarbonyl, alkoxy, hydroxymethyl, cyano, azido, alkoxyiminomethyl, alkylcarbonylamino, alkylaminomethyl or dialkylaminomethyl;

R²is hydrogen, hydroxy, alkoxy or halogen;

R³and R⁴Is hydrogen, hydroxy, alkoxy, halogen or hydroxyalkyl, with the proviso that R³And R⁴At least one is hydrogen; or

R³And R⁴Represents fluorine, or

R¹¹Is hydrogen, C_1-12Alkyl, hydroxy, hydroxyalkyl, alkoxyalkyl, haloalkyl or halogen.

4. Use of a compound according to claim 3, wherein the compound is 4 ' -C-ethynylcytidine hydrochloride (1: 1), 4 ' -C-ethoxycytidine, 4 ' -C-acetylcytidine.

5. Use of a compound according to claim 3, wherein the compound is 4' -C-azidocytidine.

6. Use of a pharmaceutical composition based on a pharmaceutically effective amount of a compound of formula I or I-a as defined in any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a disease mediated by the hepatitis c virus HCV.