HK1149017B

HK1149017B - 2',4'-substituted nucleosides as antiviral agents

Info

Publication number: HK1149017B
Application number: HK11103048.3A
Authority: HK
Inventors: 迈克尔‧J‧索菲亚; 杜锦发
Original assignee: Gilead Pharmasset Llc
Priority date: 2007-11-20
Filing date: 2008-11-17
Publication date: 2016-02-19

Abstract

Embodiments of the invention are to compounds of formulae (A), (A'), methods, and compositions for use in the treatment of viral infections. More specifically embodiments of the invention aur 2 ', 4 ' -substituted nucleoside compounds useful for the treatment of viral infections, such as HIV, HCV, and HBV infections.

Description

2 ', 4' -substituted nucleosides as antiviral agents

Technical Field

Embodiments of the present invention relate to compounds, methods and compositions for treating viral infections. More specifically, embodiments of the present invention are 2 ', 4' -substituted nucleoside compounds useful for the treatment of viral infections, such as HIV, HCV and HBV infections.

Background

Hepatitis C Virus (HCV) infection is a major health problem leading to chronic liver disease, such as cirrhosis and hepatocellular carcinoma, in a large number of infected individuals, estimated to be 2-15% of the world's population. According to the united states Disease Control center (u.s.center for Disease Control), it is estimated that 450 million infected persons exist in the united states alone. According to the world health organization, there are more than 2 billion infected individuals worldwide, with at least 3 to 4 million people infected per year. Once infected, about 20% of people are able to clear the virus, but others carry HCV for the remainder of their lives. From 10% to 20% of chronically infected individuals will eventually develop life-threatening cirrhosis or cancer. Viral diseases are transmitted parenterally by contaminated blood and blood products, contaminated needles, or by intercourse and vertical transmission from infected mothers or mother carriers to their offspring. Current treatments for HCV infection are limited to immunotherapy with recombinant interferon- α alone or in combination with the nucleoside analog ribavirin, which has limited clinical benefit because resistance develops more rapidly. Moreover, there is no established vaccine for HCV. Thus, there is an urgent need for improved therapeutic agents effective against chronic HCV infection.

The HCV virion is an enveloped positive-strand RNA virus having a single oligoribonucleotide genomic sequence of about 9600 bases encoding a polyprotein of about 3,010 amino acids. The protein products of the HCV gene consist of the structural proteins C, E1 and E2, and the non-structural proteins NS2, NS3, NS4A and NS4B, and NS5A and NS 5B. The Nonstructural (NS) protein is thought to provide a catalytic mechanism for viral replication. The NS3 protease releases NS5B, an RNA-dependent RNA polymerase, from the polyprotein chain. HCV NS5B polymerase is required to synthesize double-stranded RNA from single-stranded viral RNA as a template in the HCV replication cycle. Thus, NS5B polymerase is believed to be an essential component of the HCV replication complex (K. Ishi, et al, hepatology (Heptology), 1999, 29: 1227-1235; V. Lohmann, et al, Virology (Virology), 1998, 249: 108-118). Inhibition of HCV NS5B polymerase prevents the formation of double-stranded HCV RNA, and therefore constitutes an attractive approach to the development of HCV-specific antiviral therapies.

HCV belongs to a much larger family of viruses that share many common characteristics.

Flaviviridae (Flaviviridae) viruses

The flaviviridae family of viruses includes at least three independent genera: pestiviruses (pestiviruses), which cause diseases in cattle and pigs; yellow fever viruses (flaviviruses), which are the major cause of diseases such as dengue and yellow fever; and hepaciviruses (hepaciviruses), the only member of which is HCV. The yellow fever virus genus comprises more than 68 members, which are grouped into sets based on serological relationships (Calisher et al, J.Gen.Virol, 1993, 70, 37-43). Clinical symptoms vary and include fever, encephalitis and hemorrhagic fever (Fields Virology, eds.: Fields, b.n., Knipe, d.m., and Howley, p.m., Lippincott-Raven press (Lippincott-Raven Publishers), philadelphia, PA, 1996, chapter 31, 931-. The globally interesting genus of yellow Fever viruses associated with human diseases includes Dengue Hemorrhagic Fever virus (DHF), yellow Fever virus, shock syndrome and Japanese encephalitis virus (Halstead, S.B., Rev.Infect.Dis., 1984, 6, 251-264; Halstead, S.B., Science, 239: 476-481, 1988; Monath, T.P., New Eng.J.Med. (New England journal of medicine) 1988, 319, 641-643).

Pestiviruses include Bovine Viral Diarrhea Virus (BVDV), classical swine fever virus (CSFV, also known as hog cholera virus) and ovine Border Disease Virus (BDV) (Moennig, V., et al. adv. Vir. Res.1992, 41, 53-98). Pestivirus infection of domestic livestock (cattle, pigs and sheep) causes significant economic losses worldwide. BVDV causes mucosal disease in cattle and is of great economic importance to the livestock industry (Meyers, G. and Thiel, H.J., Advances in Virus Research, 1996, 47, 53-118; Moennig V., et al, Adv.Vir.Res.1992, 41, 53-98). Human pestiviruses have not been characterized as broadly as animal pestiviruses. However, serological investigations have shown that there is a large exposure to pestiviruses in humans.

Pestiviruses and hepaciviruses are closely related groups of viruses in the flaviviridae family. Other closely related viruses in this family include GB virus a, GB virus a-like pathogens, GB virus-B and GB virus-C (also known as hepatitis G virus, HGV). The genus hepatitis C virus (hepatitis C virus; HCV) consists of a number of closely related, but genotypically different, viruses that infect humans. There are at least 6 HCV genotypes and more than 50 subtypes. Due to the similarity between pestiviruses and hepaciviruses, and the poor ability of hepaciviruses to grow efficiently in cell culture, Bovine Viral Diarrhea Virus (BVDV) is often used as a replacement for the study of HCV virus.

The genetic organization of pestiviruses and hepaciviruses is very similar. These positive-stranded RNA viruses have a single large Open Reading Frame (ORF) that encodes all of the viral proteins required for viral replication. These proteins are expressed as polyproteins that are co-translated and post-translationally processed by both cellular and virally encoded proteolytic enzymes to produce mature viral proteins. The viral proteins responsible for viral genomic RNA replication are located approximately within the carboxy terminus. Two thirds of the ORF are called non- (NS) proteins. The genetic organization and polyprotein processing of the nonstructural protein portion of the ORFs for pestiviruses and hepaciviruses are very similar. For both pestiviruses and hepaciviruses, the mature nonstructural sequence is arranged in order from the amino terminus of the nonstructural protein coding region to the carboxy terminus of the ORF (the NS protein consists of p7, NS2, NS3, NS4A, NS4B, NS5A, and NS 5B.

The NS proteins of pestiviruses and hepaciviruses share sequence domains that are functional features of specific proteins. For example, the NS3 proteins of the viruses in both groups have the amino Acid sequence motif characteristic of serine proteolytic and helicases (Gorbalenya, et al, Nature (Nature), 1988, 333, 22; Bazan and Fletterick, Virology, 1989, 171, 637-639; Gorbalenyl, et al, Nucleic Acid Res. (Nucleic acids research), 1989, 17, 3889-3897). Similarly, the NS5B protein of pestiviruses and hepaciviruses has the motif characteristic of RNA-directed RNA polymerase (Koonin, E.V. and Dolja, V.V., Crit. Rev. biochem. molec. biol., 1993, 28, 375-.

The actual role and function of the NS proteins of pestiviruses and hepaciviruses in the life cycle of the virus are directly analogous. In both cases, NS3 serine proteolytic enzyme is responsible for all proteolytic processing of the polyprotein precursor downstream of its ORF position. (Wiskerchen and Collett, Virology, 1991, 184, 341-350; Bartenschlager et al, J.Virol. 1993, 67, 3835-3844; Eckart et al, biochem. Biophys. Res. Comm. Biochemical and biophysical research communications, 1993, 192, 399-406; Grakoui et al, J.Virol. 1993, 67, 2832-2843; Grakoui et al, Proc. Natl.Acad.Aci.USA, 1993, 909, 10583-87; Hijikoata et al, J.Virol. 1993, 67, 65-464675; Tokoui et al, J.Virol. 40267, 4016). The NS4A protein, in both cases, functions as a cofactor for the NS3 serine protease (Bartenschlager et al, J.Virol (J.Virol.) 1994, 68, 5045-. The NS3 proteins of both viruses also function as helicases (Kim et al, Biochem Biophys. Res. Comm., 1995, 215, `160-166 `; Jin and Peterson, Arch. Biochem. Biophys. (Biochemical biophysical Advances), 1995, 323, 47-53 `; Warrener and Collett, J. Virol. (J. Virol., 1995, 69, 1720-1726). Finally, the NS5B protein of pestiviruses and hepaciviruses has the expected RNA-directed RNA polymerase activity (Behrens et al, EMBO, 1996, 15, 12-22; Lechmann et al, J.Virol. (J.Virol., 1997, 71, 8416-.

Currently, there are very limited treatment options for individuals infected with hepatitis c virus. The currently approved treatment options are immunotherapy with recombinant interferon-alpha alone or in combination with the nucleoside analog ribavirin. This therapy is very limited in its clinical effectiveness, and only 50% of treated patients respond to treatment. Thus, there is a significant need for more effective and new therapies to address the unmet medical need resulting from HCV infection.

Currently, a number of potential molecular targets for drug development of direct-acting antiviral drugs for anti-HCV therapeutics have been identified, including, but not limited to, NS2-NS3 autoprotease, N3 protease, N3 helicase, and NS5B polymerase. RNA-dependent RNA polymerases are single-stranded, sense-stranded, absolutely essential for RNA genome replication, and this enzyme has attracted great interest to medicinal chemists.

HCV NS5B inhibitors as potential therapies for HCV infection have been reviewed in the following references: tan, s.l., et al, Nature rev.drug discov. (review for natural drug development), 2002, 1, 867-; walker, m.p., et al, exp.opin.investigational Drugs, 2003, 12, 1269-; ni, Z-J, et al, Current Opinion in Drug Discovery and Development, 2004, 7, 446-; beaulieu, P. L., et al, Current Opinion in Investigational Drugs, 2004, 5, 838-; wu, J., et al, Current Drug Targets-Infectious Disorders (Current Drug Targets-Infectious diseases), 2003, 3, 207-219; griffith, r.c., et al, Annual Reports in Medicinal Chemistry, 2004, 39, 223-; carrol, S., et al, Infectious Disorders-Drug Targets, 20-06, 6, 17-29. The possibility of emergence of resistant HCV strains and the need to identify pathogens with extensive genotypic coverage support the need for continued efforts to identify new and more effective nucleosides as inhibitors of HCV NS 5B. Nucleoside inhibitors of NS5b polymerase may act as unnatural substrates leading to chain termination effects, or as competitive inhibitors that compete with nucleotides binding to the polymerase. In order to function as a chain terminator, nucleoside analogs must be taken up by cells and converted in vivo to triphosphates to compete for the polymerase nucleotide binding site. This conversion to triphosphates is generally mediated by cellular kinases, which impart additional structural requirements to potential nucleoside polymerase inhibitors.

Despite the existence of effective vaccines, Hepatitis B Virus (HBV) infection remains a major public health problem worldwide, with 4 billion chronic carriers. These infected patients are exposed to the risk of developing cirrhosis and hepatocellular carcinoma (Lee, w.m.1997, n.eng.j.med., 337, 1733-. At present, it is considered that about 125 million chronic hepatitis b carriers exist in the united states alone, of which 20 million people are newly infected by contact with blood or body fluid every year.

Hepatitis b virus is the second leading cause of human cancer after tobacco. The mechanism by which HBV induces cancer is not yet understood, although it is hypothesized that it may directly trigger tumor development, or indirectly trigger tumor development through chronic inflammation, cirrhosis, and cell regeneration associated with infection.

Hepatitis b virus has reached epidemic levels worldwide. In the 2-6 month incubation period, where the host is unaware of the infection, HBV infection may lead to acute hepatitis and liver damage, which results in abdominal pain, jaundice, and elevated blood levels of certain enzymes. HBV can cause fulminant hepatitis, a rapidly developing, often fatal form of disease, in which most of the liver is damaged. Patients typically recover from acute viral hepatitis. However, in some patients, high levels of viral antigen persist in the blood for extended periods, or indeterminate periods, resulting in chronic infections. Chronic infection can lead to chronic persistent hepatitis. Patients infected with chronic persistent HBV are most common in developing countries. By the mid-1991 year, there were only about 2 hundred 2 million to 5 million chronic carriers of HBV in Asia, and almost 3 million carriers worldwide. Chronic persistent hepatitis can lead to fatigue, cirrhosis of the liver, and hepatocellular carcinoma, primary liver cancer.

In western industrialized countries, the high risk group of HBV infections include those in contact with HBV carriers or their blood samples. The epidemiology of HBV is in fact very similar to that of acquired immunodeficiency syndrome, which explains why HBV infection is common in patients with AIDS or HIV-related infections. However, HBV is more infectious than HIV.

Acquired Immune Deficiency Syndrome (AIDS) is a well-known disease that severely damages the human immune system and causes death. The cause of AIDS has been determined to be the Human Immunodeficiency Virus (HIV). To alleviate the suffering of the infected host and prolong life, new compounds and methods for treating AIDS and attacking the HIV virus are continually sought.

The foregoing references and all other references cited in this specification are hereby incorporated by reference.

Summary of The Invention

Embodiments of the present invention relate to novel 2 ', 4' -substituted nucleoside derivatives for treating viral infections in mammals. Thus, in one aspect, an antiviral effective nucleoside is a 2 ', 4' -disubstituted 2 '-deoxynucleoside (β -D or β -L), its 5' -mono-phosphate ester, its 5 ', 3' -cyclic phosphate ester, its 5 '-diphosphate ester and its 5' -triphosphate ester or pharmaceutically acceptable salts (acid and base addition salts), hydrates, solvates, crystalline forms or prodrugs thereof of the general formula:

wherein

(a)R²Independently is CH₃，CH₂F，CHF₂，CF₃，F，CN，C_2-4Alkenyl radical, C_2-4Alkynyl, or C_1-4Alkyl optionally substituted with amino, hydroxyl, or 1-3 fluorine atoms;

(b) r is H, phosphate, including 5 ' -monophosphate, 5 ', 3 ' -cyclic phosphate, diphosphate, triphosphate, or a stabilized phosphate prodrug, H-phosphonate, including stabilized H-phosphonate, acyl, including optionally substituted phenyl and lower acyl, alkyl, including lower alkyl, O-substituted carboxyalkylamino or peptide derivatives thereof, sulfonate, including alkyl or arylalkylsulfonyl, including methanesulfonyl and benzyl, wherein the phenyl is optionally substituted, a lipid, including a phospholipid, an L or D-amino acid, a carbohydrate, a peptide, cholesterol, or other pharmaceutically acceptable leaving group when administered in vivo;

(c)R³independently of one another OH, H, C_1-4Alkyl radical, C_2-4Alkenyl radical, C_2-4Alkynyl, vinyl, N₃，CN，Cl，Br，F，I，NO₂，C(O)O(C_1-4Alkyl group, C (O) O (C)_1-4Alkyl group, C (O) O (C)_2-4Alkynyl group, C (O) O (C)_2-4Alkenyl), O (C)_1-10Acyl group), O (C)_1-4Alkyl), O (C)_2-4Alkenyl), SH, S (C)_1-4Acyl radical), S (C)_1-4Alkyl radical), S (C)_2-4Alkynyl), S (C)_2-4Alkenyl), SO (C)_1-4Acyl group), SO (C)_1-4Alkyl), SO (C)_2-4Alkynyl), SO (C)_2-4Alkenyl), SO₂(C_1-4Acyl), SO₂(C_1-4Alkyl), SO₂(C_2-4Alkynyl), SO₂(C_2-4Alkenyl), OS (O)₂(C_1-4Acyl group), OS (O)₂(C_1-4Alkyl), OS (O)₂(C_2-4Alkenyl) NH₂，NH(C_1-4Alkyl), NH (C)_2-4Alkenyl), NH (C)_2-4Alkynyl), NH (C)_1-4Acyl radical, N (C)_1-4Alkyl radical)₂，N(C_1-18Acyl radical)₂Wherein alkyl, alkynyl, alkenyl and vinyl are optionally substituted with: n is a radical of₃CN, 1-3 halogens (Cl, Br, F, I), NO₂，C(O)O(C_1-4Alkyl group, C (O) O (C)_1-4Alkyl group, C (O) O (C)_2-4Alkynyl group, C (O) O (C)_2-4Alkenyl), O (C)_1-4Acyl group), O (C)_1-4Alkyl), O (C)_2-4Alkenyl), SH, S (C)_1-4Acyl radical), S (C)_1-4Alkyl radical), S (C)_2-4Alkynyl), S (C)_2-4Alkenyl), SO (C)_1-4Acyl group), SO (C)_1-4Alkyl), SO (C)_2-4Alkynyl), SO (C)_2-4Alkenyl), SO₂(C_1-4Acyl), SO₂(C_1-4Alkyl), SO₂(C_2-4Alkynyl), SO₂(C_2-4Alkenyl), OS (O)₂(C_1-4Acyl group), OS (O)₂(C_1-4Alkyl), OS (O)₂(C_2-4Alkenyl) NH₂，NH(C_1-4Alkyl), NH (C)_2-4Alkenyl), NH (C)_2-4Alkynyl), NH (C)_1-4Acyl radical, N (C)_1-4Alkyl radical)₂，N(C_1-4Acyl radical)₂；

(d)R⁴Independently is H, lower alkyl, CN, vinyl, O- (lower alkyl), hydroxy lower alkyl, i.e., - (CH)₂)_pOH, wherein p is 1-6, including hydroxymethyl (CH)₂OH)，CH₂F，N₃，CH₂CN，CH₂NH₂，CH₂NHCH₃，CH₂N(CH₃)₂Ethynyl alkyne (optionally substituted), or halogen, including F, Cl, Br, or I, alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkenyl, NO₂Amino, lower alkylamino, or di (lower alkyl) amino;

(e) r and R³Together may form a 5 ', 3' -cyclic phosphate ester, including stable prodrugs thereof;

(f) base (B) is a naturally occurring or modified purine or pyrimidine base represented by the following structure:

wherein for a and b

Z is N or CR⁸；

R⁵，R⁶And R⁷Independently is H, F, Cl, Br, I, OH, OR ', SH, SR', NH₂，NHR′，NR′₂(both R' may form a saturated or unsaturated ring, or a saturated or unsaturated heterocyclic ring), C₁-C₆Lower alkyl of (A), halo (F, Cl, Br, I) C₁-C₆Lower alkyl of (C)₂-C₆Lower alkenyl of (a), halo (F, Cl, Br, I) C₂-C₆Lower alkenyl of, C₂-C₆Lower alkynyl of (C.ident.CH), halo (F, Cl, Br, I) C₂-C₆Lower alkynyl of (2), C₁-C₆Lower alkoxy of (C), halo (F, Cl, Br, I)₁-C₆Lower alkoxy of, CO₂H，CO₂R′，CONH₂，CONHR′，CONR′₂，CH＝CHCO₂H, or CH ═ CHCO₂R ', wherein R' is optionally substituted alkyl, including, but not limited to, optionally substituted C_1-20Alkyl, optionally substituted C_1-10Alkyl, optionally substituted lower alkyl; optionally substituted cycloalkyl; optionally substituted C₂-C₆Alkynyl, optionally substituted C₂-C₆Lower alkenyl, or optionally substituted acyl including, but not limited to, C (O) alkyl, C (O) (C)_1-20Alkyl radical, C (O) (C)_1-10Alkyl), or C (O) (lower alkyl), optionally substituted aryl, optionally substituted C₁-C₄Alkyl-aryloxy, heteroaryl, optionally substituted C₁-C₄Alkyl-heteroaryl, optionally substituted alkoxy C_1-20Alkyl, optionally substituted amino C_1-20Alkyl, optionally substituted fluoro C_1-20An alkyl group;

R⁸independently H, halogen (including F, Cl, Br, I), OH, OR ', SH, SR', NH₂，NHR′，NR′₂(both R' may form a saturated or unsaturated ring, or a saturated or unsaturated heterocyclic ring), NO₂，C₁-C₆Lower alkyl of (A), halo (F, Cl, Br, I) C₁-C₆Lower alkyl of (C)₂-C₆Lower alkenyl of (a), halo (F, Cl, Br, I) C₂-C₆Lower alkenyl of, C₂-C₆Lower alkynyl of (C), halo (F, Cl, Br, I) C₂-C₆Lower alkynyl, C₁-C₆Lower alkoxy of (C), halo (F, Cl, Br, I)₁-C₆Lower alkoxy of, CO₂H，CO₂R′，CONH₂，CONHR′，CONR′₂，CH＝CHCO₂H, or CH ═ CHCO₂R ', wherein R' is optionally substituted alkyl, including, but not limited to, optionally substituted C_1-20Alkyl, optionally substituted C_1-10Alkyl, optionally substituted lower alkyl; optionally substituted cycloalkyl; optionally substituted C₂-C₆Alkynyl, optionally substituted C₂-C₆Lower alkenyl, or optionally substituted acyl including, but not limited to, C (O) alkyl, C (O) (C)_1-20Alkyl radical, C (O) (C)_1-10Alkyl), or C (O) (lower alkyl), optionally substituted aryl, optionally substituted C₁-C₄Alkyl-aryloxy, heteroaryl, optionally substituted C₁-C₄Alkyl-heteroaryl, optionally substituted alkoxy C_1-20Alkyl, optionally substituted amino C_1-20Alkyl, optionally substituted fluoro C_1-20An alkyl group; or

The base (B) may be selected from groups of formula c

Wherein in relation to the structure c,

z is independently selected from N or C-G if Z is a participant of a pi bond (double bond); or, if Z is not a participant of a pi bond (double bond), Z is independently selected from O, S, Se, NR, NOR, NNR₂，CO，CS，CNR，SO，S(O)₂，SeO，Se(O)₂Or C (G)₂；

Each G is independently selected from the group consisting of: h, halogen, OR, SR, NR₂，NROR，N₃，COOR，CN，CONR₂，C(S)NR₂，C(＝NR)NR₂And R; and wherein any two adjacent Z are not simultaneously selected from O, S, and Se, or from CO, CS, CNNR, SO, S (O)₂SeO and Se (O)₂；

Wherein if X is a participant of a pi bond (double bond), then X is C; or X is CR or N if X is not a participant of a pi bond (double bond);

wherein, if R 'is a participant of a pi bond (double bond), R' is O, S, Se, NR, NOR or NNR₂(ii) a OR R 'is OR, SR, F, Cl, R, OR SeR if R' is not a participant of a pi bond (double bond); and the dotted line (- - - -) represents a possible pi or double bond;

each R is independently selected from the group consisting of: h, CF₃Optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted acyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heteroaryl, optionally substituted heterocyclyl, and optionally substituted arylalkyl; or

The base (B) may be a structure selected from the group consisting of:

structure d-n

Wherein Z, X, and R' are as defined in structure c;

the base may be a structure selected from the group consisting of: structure o-ff

Wherein G and R are as defined in structure c;

the base (B) may be of the structure gg

Wherein each Z 'is independently N (if a participant of a pi bond) or NR (if not a participant of a pi bond) and R', R, and Z are as defined in structure c;

the base (B) may be of the structure hh

Wherein each Z 'is independently N (if a pi-bonded participant) or NR (if not a pi-bonded participant), and each Z is independently CG (if a pi-bonded participant) or > C (G)2 (if not a pi-bonded participant), wherein R' and G are as defined in structure c;

the base (B) may be of structure ii

Wherein R and G are as defined in structure c;

the base may be of the structure jj

Wherein R and G are as defined in structure c; or

The base may be of the structure kk

Wherein with respect to structure kk:

r is selected from the group consisting of: hydrogen and C₁-C₃An alkyl group;

x is selected from the group consisting of: hydrogen, halogen, and OW²；

Y is selected from the group consisting of: a bond, O, and CH₂；

Q is absent orSelected from the group consisting of: o, S, and NH, with the proviso that when Q is absent, V and NH are both attached to CH₂A group;

v is selected from the group consisting of: n and C-G;

z is selected from the group consisting of: n and C-G';

g and G' are independently selected from the group consisting of: hydrogen, amino, aminocarbonyl, methylamino, dimethylamino, acylamino, alkoxyamino, -SO₃H，-SO₂NH₂Aminocarbonylamino, oxycarbonylamino, HR ' NCHR "C (O) NH-, azido, cyano, halogen, hydroxyamino, and hydrazino groups, wherein R ' is hydrogen and R" is the side chain of an amino acid or wherein R ' and R "together with the nitrogen and carbon to which the respective groups are bound respectively form a pyrrolidinyl group;

provided that V and Z are not the same;

provided that when V is C-H, Z is N;

T¹and T²Independently selected from the group consisting of: hydrogen, hydroxy, C₁-C₄-alkoxy radical, C₁-C₄-thioalkoxy, amino, substituted amino, and halogen; and

W，W¹and W and²each of which is independently selected from the group consisting of: hydrogen, C₁-C₄Alkyl, and prodrug groups; or

The base may be of structure ll

Wherein with respect to structure ll:

r is C₁-C₃An alkyl group;

x is selected from the group consisting of: hydrogen, halogen, andOW²；

q' is selected from the group consisting of: NH, O, and S;

g' is selected from the group consisting of: amino, aminocarbonyl, methylamino, dimethylamino, acylamino, -SO₃H，-SO₂NH₂Alkoxyamino, aminocarbonylamino, oxycarbonylamino, HR ' NCHR "C (O) NH-, azido, cyano, halogen, hydroxyamino, and hydrazino groups, wherein R ' is hydrogen and R" is the side chain of an amino acid or wherein R ' and R "together with the nitrogen and carbon to which the respective groups are bound respectively form a pyrrolidinyl group; y is selected from the group consisting of: a bond, O, and CH₂(ii) a And W, W¹And W and²each of which is independently selected from the group consisting of: hydrogen, C₁-C₄Alkyl, and prodrug groups; or

The base may be of structure mm

Wherein with respect to structure mm

Q is as defined for structure kk

A and B are independently selected from the group consisting of: c ═ Q, NH, and methylene optionally substituted with 1 to 2 halo groups, provided that a and B are not both NH;

d is NH, or-D-A-B-together form-N ═ CH-NH-, - (C ═ Q) -CH₂- (C ═ Q) -, - (C ═ Q) -NH- (C ═ Q) -, - (CX ') - (C ═ Q) -, or-CH ═ CH-NH-group, where X' is halogen;

each Q is independently selected from the group consisting of: o, S, and NH; r is selected from the group consisting of: hydrogen and C₁-C₃An alkyl group;

x is selected from the group consisting of: hydrogen, halogen, and OW²；

T¹And T²Independently selected from the group consisting of: hydrogen, hydroxy, C₁-C₄-alkoxy radical, C₁-C₄-thioalkoxy, amino, substituted amino, and halogen;

y is selected from the group consisting of: a bond, O, and CH₂(ii) a And W, W¹And W and²each of which is independently selected from the group consisting of: hydrogen, C₁-C₄Alkyl, and prodrug groups;

and pharmaceutically acceptable salts, tautomers, pharmaceutically acceptable salts of tautomers, salts (acid or base addition salts), hydrates, solvates, crystalline forms thereof;

optionally in combination with one or more antiviral, antibacterial or antiproliferative agents.

It is an object of embodiments of the present invention to provide compounds, methods, and compositions for treating or preventing HIV, HBV, or HCV infection in a host. It is another object of embodiments of the present invention to provide compounds, methods and compositions for treating or preventing HIV, HBV, or HCV when the host is a human, or when the host is an animal.

Detailed Description

Embodiments of the present invention relate to novel 2 ', 4' -substituted nucleoside derivatives for treating viral infections in mammals, including one or more compounds of the formula:

wherein

(a)R²Independently is CH₃，CH₂F，CHF₂，CF₃，F，CN，C_2-4Alkenyl radical, C_2-4Alkynyl, or C_1-4Alkyl optionally substituted by amino, hydroxy, or 1-3Fluorine atom substitution;

(b) r is H, phosphate, including 5 ' -monophosphate, 5 ', 3 ' -cyclic phosphate, diphosphate, triphosphate, or a stabilized phosphate prodrug, H-phosphonate, including stabilized H-phosphonate, acyl, including optionally substituted phenyl and lower acyl, alkyl, including lower alkyl, O-substituted carboxyalkylamino or peptide derivatives thereof, sulfonate, including alkyl or arylalkylsulfonyl, including methanesulfonyl and benzyl, wherein the phenyl group is optionally substituted, a lipid, including a phospholipid, an L or D-amino acid, a carbohydrate, a peptide, cholesterol, or other pharmaceutically acceptable leaving group when administered in vivo;

(d)R⁴Independently is H, lower alkyl, CN, vinyl, O- (lower alkyl), hydroxy lower alkyl, i.e., - (CH)₂)_pOH, wherein p is 1-6, including hydroxymethyl (CH)₂OH)，CH₂F，N₃， CH₂CN，CH₂NH₂，CH₂NHCH₃，CH₂N(CH₃)₂Ethynyl alkyne (optionally substituted), or halogen, including F, Cl, Br, or I, alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkenyl, NO₂Amino, lower alkylamino, or di (lower alkyl) amino;

(f) the base is a naturally occurring or modified purine or pyrimidine base represented by the following structure:

wherein for a and b

Z is N or CR⁸；

R⁸independently H, halogen (including F, Cl, Br, I), OH, OR ', SH, SR', NH₂，NHR′，NR′₂(both R' may form a saturated or unsaturated ring, or a saturated or unsaturated heterocyclic ring), NO₂，C₁-C₆Lower alkyl of (2)Alkyl, halo (F, Cl, Br, I) C₁-C₆Lower alkyl of (C)₂-C₆Lower alkenyl of (a), halo (F, Cl, Br, I) C₂-C₆Lower alkenyl of, C₂-C₆Lower alkynyl of (C), halo (F, Cl, Br, I) C₂-C₆Lower alkynyl of (2), C₁-C₆Lower alkoxy of (C), halo (F, Cl, Br, I)₁-C₆Lower alkoxy of, CO₂H，CO₂R′，CONH₂，CONHR′，CONR′₂，CH＝CHCO₂H, or CH ═ CHCO₂R ', wherein R' is optionally substituted alkyl, including, but not limited to, optionally substituted C_1-20Alkyl, optionally substituted C_1-10Alkyl, optionally substituted lower alkyl; optionally substituted cycloalkyl; optionally substituted C₂-C₆Alkynyl of (2), optionally substituted C₂-C₆Lower alkenyl of (a), or optionally substituted acyl including, but not limited to, C (O) alkyl, C (O) (C)_1-20Alkyl radical, C (O) (C)_1-10Alkyl), or C (O) (lower alkyl), optionally substituted aryl, optionally substituted C₁-C₄Alkyl-aryloxy, heteroaryl, optionally substituted C₁-C₄Alkyl-heteroaryl, optionally substituted alkoxy C_1-20Alkyl, optionally substituted amino C_1-20Alkyl, optionally substituted fluoro C_1-20An alkyl group; or

The base may be selected from a group of formula c,

wherein in relation to the structure c,

z is independently selected from N or C-G if Z is a participant of a pi bond (a double bond); or, if Z is not a participant of a pi bond (double bond), Z is independently selected from O, S, Se, NR, NOR, NNR₂，CO，CS，CNR，SO，S(O)₂，SeO，Se(O)₂Or C (G)₂；

wherein, if R 'is a participant of a pi bond (double bond), R' is O, S, Se, NR, NOR or NNR₂；

OR R 'is OR, SR, F, Cl, R, OR SeR if R' is not a participant of a pi bond (double bond); and is

The dotted line (- - - -) represents a possible pi or double bond;

The base may be a structure selected from the group consisting of: structure d-n

Wherein Z, X, and R' are as defined in structure c;

Wherein G and R are as defined in structure c;

the base may be of structure gg

the base may be of the structure hh

the base may be of structure ii

Wherein R and G are as defined in structure c;

the base may be of the structure jj

Wherein R and G are as defined in structure c; or

The base may be of the structure kk

Wherein with respect to structure kk:

x is selected from the group consisting of: hydrogen, halogen, and OW²；

Y is selected from the group consisting of: a bond, O, and CH₂；

Q is absent or selected from the group consisting of: o, S, and NH, with the proviso that when Q is absent, V and NH are both attached to CH₂A group;

v is selected from the group consisting of: n and C-G;

z is selected from the group consisting of: n and C-G';

provided that V and Z are not the same;

provided that when V is C-H, Z is N;

W，W¹and W and²each independently selected from the group consisting of: hydrogen, C₁-C₄Alkyl, and prodrug groups; or

The base may be of structure ll

Wherein with respect to structure ll:

r is C₁-C₃An alkyl group;

x is selected from the group consisting of: hydrogen, halogen, and OW²；

Q' is selected from the group consisting of: NH, O, and S;

The base may be of structure mm

Wherein with respect to structure mm

Q is as defined for structure kk

d is NH, or-D-A-B-together form-N ═ CH-NH-, - (C ═ Q) -CH₂A- (C ═ Q) -, - (C ═ Q) -NH- (C ═ Q) -, - (CX ') - (C ═ Q) -, or-CH ═ CH-NH-group, wherein X' is halogen;

x is selected from the group consisting of: hydrogen, halogen, and OW²；

y is selected from the group consisting of: a bond, O, and CH₂(ii) a And W, W¹And W and²each independently selected from the group consisting of: hydrogen, C₁-C₄Alkyl, and prodrug groups;

and pharmaceutically acceptable salts, tautomers, pharmaceutically acceptable salts of tautomers, salts (acid or base addition salts), hydrates, solvates, crystalline forms thereof

Particularly preferred embodiments of the invention are exemplified, but not limited to, in tables I and II below.

TABLE I

More preferred embodiments thereof have the following structures, but the substituent patterns are the same as shown in table I.

TABLE II

More preferred embodiments thereof have the following structures, but the substituent patterns thereof are shown in table II.

Definition of

The phrase "a" or "an" entity, as defined herein, refers to one or more entities; for example, a compound refers to one or more compounds or at least one compound. Thus, the terms "a" or (or "an"), "one or more" and "at least one" may be used interchangeably herein.

The phrase "as defined herein above" refers to the first definition provided in the summary of the invention.

The terms "optional" or "optionally," as used herein, mean that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optional bond" means a bond that may or may not be present, and the description includes single, double, or triple bonds.

The term "independently" as used herein is used to indicate that a variable applies in any one instance regardless of the presence or absence of variables having the same or different definitions in the same compound. Thus, in a compound where R occurs twice and is defined as "independently carbon or nitrogen," both R's can be carbon, both R's can be nitrogen, or one R can be carbon and the other nitrogen.

The term "alkenyl" refers to an unsubstituted hydrocarbon chain radical having from 2 to 10 carbon atoms having 1 or 2 olefinic double bonds, preferably one olefinic double bond. The term "C_2-NAlkenyl "means an alkenyl group containing 2 to N carbon atoms, where N is an integer having the following value: 3, 4, 5, 6, 7, 8, 9, or 10. The term "C_2-10Alkenyl "means an alkenyl group containing 2 to 10 carbon atoms. Examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, (allyl) or 2-butenyl (crotyl).

The term "haloalkenyl" refers to an alkenyl group comprising at least one of F, Cl, Br, and I.

The term "alkyl" refers to a straight or branched chain, saturated, monovalent hydrocarbon residue containing 1 to 30 carbon atoms. The term "C_1-NAlkyl "refers to an alkyl group containing 1 to N carbon atoms, where N is an integer having the following value: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. The term "C_1-4"alkyl" refers to an alkyl group containing 1 to 4 carbon atoms. The term "lower alkyl" or "lower alkyl" refers to a straight, or branched, hydrocarbon residue containing 1 to 8 carbon atoms. "C_1-20Alkyl "as used herein refers to an alkyl group containing 1 to 20 carbon atoms. "C_1-10Alkyl "as used herein refers to an alkyl group containing from 1 to 10 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl. The term (ar) alkyl or (heteroaryl) alkyl refers to an alkyl group optionally substituted with an aryl or heteroaryl group, respectively.

The term "haloalkyl" (or "haloalkyl") means a compound comprising at least one of F, Cl, Br, and IA linear or branched alkyl group of one. The term "C_1-3Haloalkyl "refers to a haloalkyl containing 1 to 3 carbons and at least one of F, Cl, Br, and I. The term "halogenated lower alkyl" refers to a halogenated alkyl group containing 1 to 8 carbon atoms and at least one of F, Cl, Br, and I. Examples include, but are not limited to, fluoromethyl, chloromethyl, bromomethyl, iodomethyl, difluoromethyl, dichloromethyl, dibromomethyl, diiodomethyl, trifluoromethyl, trichloromethyl, tribromomethyl, triiodomethyl, 1-fluoroethyl, 1-chloroethyl, 1-bromoethyl, 1-iodoethyl, 2-fluoroethyl, 2-chloroethyl, 2-bromoethyl, 2-iodoethyl, 2, 2-difluoroethyl, 2, 2-dichloroethyl, 2, 2-dibromoethyl, 2, 2-diiodoethyl, 3-fluoropropyl, 3-chloropropyl, 3-bromopropyl, 3-iodopropyl, 2, 2, 2-trifluoroethyl, 1, 1, 2, 2, 2-pentafluoroethyl, 1-fluoro-1-chloroethyl, or 1-fluoro-1-chloro-1-bromoethyl.

The term "alkynyl" refers to a straight or branched hydrocarbon chain radical having from 2 to 10 carbon atoms, preferably from 2 to 5 carbon atoms, and having one triple bond. The term "C_2-NAlkynyl "refers to alkynyl groups containing 2 to N carbon atoms, where N is an integer having the following value: 2, 3, 4, 5, 6, 7, 8, 9, or 10. The term "C_2-4Alkynyl "refers to alkynyl groups containing 2-4 carbon atoms. The term "C_2-10Alkynyl "refers to alkynyl groups containing 2-10 carbon atoms. Examples include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, or 3-butynyl.

The term "haloalkynyl" refers to a straight or branched hydrocarbon chain radical having from 2 to 10 carbon atoms, preferably from 2 to 5 carbon atoms, and having a triple bond and at least one of F, Cl, Br, and I.

The term "cycloalkyl" refers to a saturated carbocyclic ring containing from 3 to 8 carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl. The term "C_3-7Cycloalkyl "as used herein, refers to cycloalkyl groups containing from 3 to 7 carbons in the carbocyclic ring.

The term "alkoxy" refers to an-O-alkyl group, wherein alkyl isAs defined above. Examples include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, t-butoxy. "lower alkoxy (lower alkoxy)" or "lower alkoxy (low alkoxy)" as used herein refers to an alkoxy group having a "lower alkyl" group as previously described. "C_1-10Alkoxy "means an-O-alkyl group wherein alkyl is C_1-10。

The term "haloalkoxy" refers to an-O-alkyl group, wherein the alkyl group includes at least one of F, Cl, Br, and I.

The term "halo-lower-alkoxy (lower alkoxy)" or "halo-lower-alkoxy (low alkoxy)" refers to an-O- (lower alkyl) group, wherein the lower alkyl group includes at least one of F, Cl, Br, and I.

The term "substituted," as used herein, means that one or more hydrogens on the designated atom is replaced with an option from the designated group, provided that the designated atom's normal valence is not exceeded, and that the substitution results in a stable compound.

The term "protected" as used herein and unless otherwise defined, refers to a group that is added to an oxygen, nitrogen, or phosphorus atom to prevent its further reaction or for other purposes. A wide variety of oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis. Non-limiting examples include: c (O) -alkyl, C (O) Ph, C (O) aryl, CH₃，CH₂-alkyl, CH₂-alkenyl, CH₂Ph，CH₂-aryl, CH₂O-alkyl, CH₂O-aryl, SO₂-alkyl, SO₂Aryl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and 1, 3- (1, 1,3, 3-tetraisopropyldisiloxanylidene) groups.

The term "halogen" as used herein includes fluorine, chlorine, bromine, and iodine.

The term "purine" or "pyrimidineBases include, but are not limited to, adenine, N⁶-alkylpurine, N⁶Acylpurines (in which the acyl group is C (O) (alkyl, aryl, alkylaryl, or arylalkyl), N⁶-benzylpurine, N⁶-halopurine, N⁶-vinyl purine, N⁶-acetylenic purine, N⁶Acylpurines, N6-hydroxyalkylpurines, N⁶-allylaminopurine, N⁶Thio-allylpurine, N²-alkylpurine, N²-alkyl-6-thiopurine, thymine, cytosine, 5-fluorocytosine, 5-methylcytosine, 6-azapyrimidine including 6-azacytosine, 2-and 4-mercaptopyrimidine, guanine, 5-halo-guanine including 5-fluoroguanine, C⁵Alkyl pyrimidines, C⁵-benzylpyrimidine, C⁵Halogen pyrimidines, C⁵-vinyl pyrimidine, C⁵-acetylenic pyrimidines, C⁵Acyl pyrimidines, C⁵-hydroxyalkylpurine, C⁵Amino-pyrimidines, C⁵-cyanopyrimidine, C⁵Iodine pyrimidine, C⁶Iodine-pyrimidine, C⁵-Br-vinyl pyrimidine C⁶-Br-vinyl pyrimidine C⁵-nitropyrimidine, C⁵-amino-pyrimidine, N²-alkylpurine, N²-alkyl-6-thiopurine, 5-azacytidine, 5-azauracil, triazolopyridine, imidazopyridine, pyrrolopyrimidine, and pyrazolopyrimidine. Purine bases include, but are not limited to, guanine, adenine, hypoxanthine, 2, 6-diaminopurine, and 6-chloropurine. Functional oxygen and nitrogen groups on the bases may be protected as desired. Suitable protecting groups are well known to those skilled in the art and include trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups, and acyl groups such as acetyl and propionyl, methanesulfonyl, and p-toluenesulfonyl.

The terms "tautomerism" and "tautomer" have their accepted meanings.

The term "pharmaceutically acceptable salt or prodrug" is used throughout the specification to describe any pharmaceutically acceptable form of the compound (such as an ester, phosphate ester, salt of an ester or related group) that provides the active compound when administered to a mammal. Pharmaceutically acceptable salts include those obtained from pharmaceutically acceptable inorganic or organic bases and acids. Pharmaceutically acceptable prodrugs refer to compounds which are metabolized, e.g., hydrolyzed or oxidized, in the host to form the compounds of the invention. Typical examples of prodrugs include compounds having biologically labile protecting groups on functional moieties of selected compounds. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrated, alkylated, dealkylated, acylated, deacylated, phosphorylated, dephosphorylated to yield the active compound. The compounds of the present invention have antiviral activity against HIV, HBV and HCV viruses, or are metabolized into compounds that exhibit such activity.

In cases where the compound is sufficiently basic or acidic to form a stable, non-toxic acidic or basic salt, administration of the compound as a pharmaceutically acceptable salt may be suitable. Examples of pharmaceutically acceptable salts are organic acid addition salts formed from acids which form physiologically acceptable anions, such as tosylate, mesylate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, alpha-ketoglutarate, and alpha-glycerophosphate. Suitable inorganic salts may also be formed, including sulfates, nitrates, bicarbonates, and carbonates.

Alternatively, pharmaceutically acceptable salts may be obtained, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid to provide a physiologically acceptable anion. Alkali metal (e.g., sodium, potassium or lithium) or alkaline earth metal (e.g., calcium and magnesium) salts of, for example, carboxylic acids may also be prepared.

The compounds described herein may be administered as prodrugs to increase the activity, bioavailability, stability, or otherwise alter the properties of a selected compound. Many prodrug ligands are known.

The term "host" as used herein refers to a unicellular or multicellular organism in which a virus can replicate, including, but not limited to, cell lines and animals, and preferably humans. Alternatively, the host may carry-a portion of the viral genome, the replication or function of which may be altered by the compounds of the invention. The term host specifically refers to infected cells, cells infected with all or part of the viral genome, and animals.

The compounds of the present invention may be formulated in a variety of oral dosage forms and carriers. Oral administration can be carried out as tablets, coated tablets, hard and soft gelatin capsules, solutions, emulsions, syrups or suspensions. The compounds of the present invention are effective when administered by suppository, or by other routes of administration. The most convenient mode of administration is usually oral using a convenient daily dosing regimen, which may be adjusted according to the severity of the disease and the patient's response to the antiviral drug.

One or more compounds of the present invention, and their pharmaceutically acceptable salts, and one or more conventional excipients, carriers or diluents may be placed in pharmaceutical compositions and unit dosage forms. The pharmaceutical compositions and unit dosage forms may comprise conventional ingredients in conventional proportions, with or without additional active compounds, and the unit dosage forms may comprise any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. The pharmaceutical compositions may be used for oral application as a solid, such as a tablet or filled capsule, a semisolid, a powder, a sustained release formulation or a liquid such as a suspension, an emulsion or a filled capsule; or in the form of suppositories for rectal or vaginal administration. Typical formulations contain from about 5% to about 95% of one or more active compounds (w/w). The term "formulation or" dosage form "is intended to include both solid and liquid formulations of the active compound, and those skilled in the art will appreciate that the active ingredient may be presented in different dosage forms depending on the desired dosage and pharmacokinetic parameters.

The term "excipient", as used herein, refers to a compound that is used in the preparation of a pharmaceutical composition and is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes excipients that are acceptable for both veterinary and human pharmaceutical use. The compounds of the invention may be administered alone, but will generally be administered in admixture with one or more suitable pharmaceutical excipients, diluents or carriers selected with regard to the intended route of administration and standard pharmaceutical practice.

The "pharmaceutically acceptable salt" form of the active ingredient may also initially impart desirable pharmacokinetic properties to the active ingredient which are not present in the non-salt form and may even positively influence the pharmacokinetics of the active ingredient with respect to its therapeutic activity in vivo. As used herein, the phrase "pharmaceutically acceptable salt" of a compound means a pharmaceutically acceptable salt that possesses the desired pharmacological activity of the parent compound. Such salts include (1) acid addition salts formed with inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like; or with an organic acid such as glycolic acid, pyruvic acid, lactic acid, malonic acid, maleic acid, fumaric acid, tartaric acid, citric acid, 3- (4-hydroxybenzoyl) benzoic acid, 1, 2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, salicylic acid, muconic acid, and the like, or (2) a base addition salt with a conjugate base of any of the inorganic acids listed above, wherein the conjugate base comprises a base selected from Na, pyruvic acid, lactic acid, malonic acid, maleic acid, fumaric acid, tartaric acid, citric acid, 3- (4-hydroxybenzoyl) benzoic acid, 1, 2-ethane⁺，K⁺，Mg⁺²，Ca⁺²Cationic component of NHgR '"4-g +, wherein R'" is C_1-3Alkyl, and g is a number selected from 0, 1, 2, 3, or 4. It will be understood that reference to a pharmaceutically acceptable salt includes the solvent addition forms (solvates), water addition forms (hydrates), or crystalline forms (polymorphs) of the same acid addition salt, as defined herein.

Solid form preparations include powders, tablets, pill capsules, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating substance. In powders, the carrier is usually a finely divided solid which is a mixture of the finely divided active ingredients. In tablets, the active ingredient is usually mixed with a carrier having the necessary binding capacity in suitable proportions and packaged in the shape and size desired. Suitable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, gum tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low melting wax, cocoa butter, and the like. Formulations in solid form may contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

Liquid formulations are also suitable for oral administration, and include liquid formulations including emulsions, syrups, elixirs and aqueous suspensions. These include solid form preparations which are intended to be converted, shortly before use, to liquid form preparations. Emulsions may be prepared in solution, for example in aqueous propylene glycol, or may contain emulsifying agents, for example lecithin, sorbitan monooleate or acacia. Aqueous suspensions may be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

The compounds of the present invention may be formulated for administration as suppositories. A low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active ingredient is dispersed homogeneously, for example by stirring. The molten homogeneous mixture is then poured into a suitably sized mold, allowed to cool and solidify.

The compounds of the present invention may be formulated for vaginal administration. Pessaries, tampons, creams, gels, pastes, foams or sprays containing such carriers in addition to the active ingredient are known in the art to be suitable.

Suitable formulations and pharmaceutical carriers, diluents and excipients are described in Remington: the Science and Practice of Pharmacy 1995, edited by E.W. Martin, Mack publishing company, 19 th edition, Isston, Pa, which is incorporated herein by reference. Formulation technicians can modify the formulations in the teachings of the specification to provide a number of formulations for a particular route of administration without destabilizing the compositions of the invention or containing their therapeutic activity.

Modification of the compounds of the invention to make them more soluble in water or other excipients, for example, can be readily accomplished by minor modifications (e.g., salt formulations), which are within the ability of those skilled in the art. It will also be within the skill of the art to alter the route of administration and dosing regimen of a particular compound so as to maintain the pharmacokinetics of the compounds of the present invention to achieve optimal beneficial effects in a patient.

The term "drug" means a substance for use in a method of treatment and/or prevention of a subject in need thereof, wherein the substance includes, but is not limited to, compositions, formulations, dosage forms, and the like, comprising a compound of formula I. It is contemplated that the use of a compound represented by formula I in the manufacture of a medicament for the treatment of any of the antiviral conditions disclosed herein can be any of the compounds contemplated by any aspect of the present invention, alone, or in combination with other compounds of the present invention.

The term "subject" means a mammal, including, but not limited to, cattle, pigs, sheep, chickens, turkeys, buffalos, llamas, ostriches, dogs, cats, and humans, preferably the subject is a human.

The term "therapeutically effective amount" as used herein means the amount required to reduce the symptoms of a disease in an individual. In each particular case, the dosage is adjusted according to the individual needs. The dosage may vary within a wide range depending on various factors such as the severity of the disease to be treated, the age and general health of the patient, the other drugs with which the patient is being treated, the route and form of administration, and the preferences and experience of the medical practitioner involved. For oral administration, all values between a daily dose of between about 0.1 and about 10g, including, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, and 9.5g per day should be suitable in monotherapy and/or in combination therapy. A preferred daily dosage is from about 0.5 to about 7.5g per day, and a more preferred dosage is from 1.5 to about 6.0g per day. Typically, treatment is initiated with a larger initial "loading dose" to rapidly reduce or eliminate the virus, followed by a dose reduction to a level sufficient to prevent recurrence of infection. Those skilled in the art of treating the diseases described herein will be able to determine, without undue experimentation and relying on personal knowledge, experience and the disclosure of this application, a therapeutically effective amount of a compound of the invention for treating a given disease and patient.

Therapeutic efficacy in HBV and HCV therapy can be determined by liver function and liver metabolic function tests including, but not limited to, protein levels such as serum proteins (e.g., albumin, coagulation factors, alkaline phosphatase, aminotransferases (e.g., alanine aminotransferases, aspartate aminotransferases), 5' -nucleosidases, C-glutamyl transpeptidase, etc.), the synthesis of bilirubin, the synthesis of cholesterol, and the synthesis of bile acids; the hepatic metabolic functions include, but are not limited to, carbohydrate metabolism, amino acid and ammonia metabolism. Alternatively, treatment efficacy can be monitored by measuring HBV or HCV-RNA, and the results of these tests allow the dose to be optimized. For HIV, the efficacy of treatment in HIV infection can be determined by measuring HIV-RNA levels from plasma samples and measuring the levels of CD4 cells.

The disclosed compounds or their pharmaceutically acceptable derivatives or salts, or pharmaceutical formulations containing these compounds, are useful in the prevention and treatment of HIV infection and other related conditions such as AIDS-related syndrome (ARC), Persistent Generalized Lymphadenopathy (PGL), AIDS-related neurological conditions, anti-HIV antibody-positive and HIV-positive conditions, kaposi's sarcoma, purpuric thrombocytopenia and opportunistic infectious diseases, and in addition, these compounds or dosage forms can be used prophylactically to prevent or delay the progression of clinical disease in individuals who are anti-HIV antibody or HFV-antigen positive or have been exposed to HIV.

Yet another aspect of the invention comprises administering a therapeutically effective amount of a compound represented by formula I and a therapeutically effective amount of another antiviral agent; wherein the administration is simultaneous, or alternating or sequential. It is to be understood that the time between alternating (or sequential) administrations can be in the range of 1-24 hours, which includes any subrange between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23 hours inclusive.

It is contemplated that another antiviral agent includes, but is not limited to, interferon- α, interferon-B, pegylated interferon- α, ribavirin (levovirin), viramidine, another nucleoside HIV, HBV, or HCV polymerase inhibitor, HIV, HBV, or HCV protease inhibitor, HIV, HBV, or HCV helicase inhibitor, or HIV, HBV, or HCV fusion inhibitor. When the active compound or a derivative or salt thereof is administered in combination with another antiviral agent, the activity may be increased relative to the parent compound. When the treatment is a combination treatment, the administration may be simultaneous or sequential with the nucleoside derivative. As used herein, "simultaneously administering" thus includes administering the agents simultaneously or at different times. Simultaneous administration of two or more agents can be achieved by a single formulation comprising two or more active ingredients or by nearly simultaneous administration of two or more dosage forms containing a single active agent.

In another embodiment for the treatment of HIV infection, the active compound or prodrug or pharmaceutically acceptable salt thereof may be administered in combination with or in alternation with another antiviral agent, such as another active anti-HIV agent, including, but not limited to, those of the formulas above, other agents listed below, or other agents known in the art. Generally, in combination therapy, effective doses of two or more agents are administered together, while in alternating therapy, effective doses of each agent are administered sequentially. The dosage will depend on absorption, inactivation and excretion rates of the drug as well as other factors known to those skilled in the art. It is noted that dosage values will also vary with the severity of the disease to be alleviated. It is to be understood that for any particular subject, the specific dosing regimen and schedule should be adjusted over time according to the individual need and the professional judgment of the individual administering or supervising the administration of the composition.

Non-limiting examples of antiviral agents that may be used in combination with the compounds disclosed herein include the following: saquinavir mesylateSaquinavir capsuleNuoweiwJiaxian patientNelfinavirAminoprenavirLopinavir-ritonavirRituxivirYipingwei teaKanbivirMatch inZalcitabine' HuitongziEC, SeritetTenofovir disoproxil fumarateCovincitm, vitaminDelavirdine mesylateEfavirenzHydroxyureaEnfuvirtide injectionAtazanavirRecombinant interleukin-2 powder injectionRecombinant human erythropoietin alpha injectionDarunavirAnd growth hormone

Results of the experiment

It is to be understood that the treatment referred to herein extends to the prevention as well as the treatment of existing diseases. Furthermore, as used herein, the term "treating" a viral infection also includes treating or preventing a disease or disorder associated with or mediated by a viral infection, or a clinical symptom thereof.

Another embodiment relates to a process for the preparation of a compound represented by A or A ', to a compound A or A ' obtained by said process and to a composition comprising A or A ' obtained by said process, said process comprising

(1) Deoxygenating the 2' -C-position of a compound represented by 1 or 1

To obtain a compound represented by 2 or 2',

and is

(2) Derivatizing the compound represented by 2 or 2 'to obtain a compound represented by A or A',

wherein with respect to structures 1, 1 ', 2, 2 ', A, and A '

(e) r and R3 may together form a 5 ', 3' -cyclic phosphate, including stable prodrugs thereof;

wherein for a and b

Z is N or CR⁸；

R⁵，R⁶And R⁷Independently is H, F, Cl, Br, I, OH, OR ', SH, SR', NH₂，NHR′，NR′₂(both R' may form a saturated or unsaturated ring, or a saturated or unsaturated heterocyclic ring), C₁-C₆Lower alkyl of (A), halo (F, Cl, Br, I) C₁-C₆Lower alkyl of (C)₂-C₆Lower alkenyl of (a), halo (F, Cl, Br, I) C₂-C₆Lower alkenyl of, C₂-C₆Lower alkynyl of (C.ident.CH), halo (F, Cl, Br, I) C₂-C₆Lower alkynyl of (2), C₁-C₆Lower alkoxy of (C), halo (F, Cl, Br, I)₁-C₆Lower alkoxy of, CO₂H，CO₂R′，CONH₂，CONHR′，CONR′₂，CH＝CHCO₂H, or CH ═ CHCO₂R ', wherein R' is optionally substituted alkyl, including, but not limited to, optionally substituted C_1-20Alkyl, optionally substituted C_1-10Alkyl, optionally substituted lower alkyl; optionally substituted cycloalkyl; optionally substituted C₂-C₆Alkynyl, optionally substituted C₂-C₆Lower alkenyl, or optionally substituted acyl including, but not limited to, C (O) alkyl, C (O) (C)_1-20Alkyl radical, C (O) (C)_1-10Alkyl), or C (O) (lower alkyl), optionally substituted aryl, optionally substituted C₁-C₄Alkylaryloxy, heteroaryl, optionally substituted C₁-C₄Alkyl-heteroaryl, optionally substituted alkoxy C_1-20Alkyl, optionally substituted amino C_1-20Alkyl, optionally substituted fluoro C_1-20An alkyl group;

R⁸independently of each other, is H, or H,halogen (including F, Cl, Br, I), OH, OR ', SH, SR', NH₂，NHR′，NR′₂(both R' may form a saturated or unsaturated ring, or a saturated or unsaturated heterocyclic ring), NO₂C₁-C₆Lower alkyl of (B), halo (F, Cl, Bt, I) C₁-C₆Lower alkyl of (C)₂-C₆Lower alkenyl of (a), halo (F, Cl, Br, I) C₂-C₆Lower alkenyl of, C₂-C₆Lower alkynyl of (C), halo (F, Cl, Br, I) C₂-C₆Lower alkynyl of (2), C₁-C₆Lower alkoxy of (C), halo (F, Cl, Br, I)₁-C₆Lower alkoxy of, CO₂H，CO₂R′，CONH₂，CONHR′，CONR′₂，CH＝CHCO₂H, or CH ═ CHCO₂R ', wherein R' is optionally substituted alkyl, including, but not limited to, optionally substituted C_1-20Alkyl, optionally substituted C_1-10Alkyl, optionally substituted lower alkyl; optionally substituted cycloalkyl; optionally substituted C₂-C₆Alkynyl, optionally substituted C₂-C₆Lower alkenyl, or optionally substituted acyl including, but not limited to, C (O) alkyl, C (O) (C)_1-20Alkyl radical, C (O) (C)_1-10Alkyl), or C (O) (lower alkyl), optionally substituted aryl, optionally substituted C₁-C₄Alkyl-aryloxy, heteroaryl, optionally substituted C₁-C₄Alkyl-heteroaryl, optionally substituted alkoxy C_1-20Alkyl, optionally substituted amino C_1-20Alkyl, optionally substituted fluoro C_1-20An alkyl group; or

The base may be selected from a group of formula c,

wherein in relation to the structure c,

if Z is a participant of a pi bond (double bond), Z is independently selected fromN or C-G; or, if Z is not a participant of a pi bond (double bond), Z is independently selected from O, S, Se, NR, NOR, NNR₂，CO，CS，CNR，SO，S(O)₂，SeO，Se(O)₂Or C (G)₂；

The dotted line (- - - -) represents a possible pi or double bond;

The base may be a structure selected from the group consisting of:

structure d-n

Wherein Z, X, and R' are as defined in structure c;

Wherein G and R are as defined in structure c;

the base may be of structure gg

the base may be of the structure hh

the base may be of structure ii

Wherein R and G are as defined in structure c;

the base may be of the structure jj

Wherein R and G are as defined in structure c; or

The base may be of the structure kk

Wherein with respect to structure kk:

x is selected from the group consisting of: hydrogen, halogen, and OW²；

Y is selected from the group consisting of: a bond, O, and CH₂；

v is selected from the group consisting of: n and C-G;

z is selected from the group consisting of: n and C-G';

provided that V and Z are not the same;

provided that when V is C-H, Z is N;

T¹and T²Independently selected from the group consisting of: hydrogen, hydroxy, C₁-C₄-alkoxy radical, C₁-C₄-thioalkoxy, amino, substituted amino, and halogen; and is

The base may be of structure ll

Wherein with respect to structure ll:

r is C₁-C₃An alkyl group;

x is selected from the group consisting of: hydrogen, halogen, and OW²；

Q' is selected from the group consisting of: NH, O, and S;

The base may be of structure mm

Wherein in respect of the structure mm,

q is as defined with respect to structure kk,

each Q is independently selected from the group consisting of: o, S, and NH; r is selected from the group consisting of:

hydrogen and C₁-C₃An alkyl group;

x is selected from the group consisting of: hydrogen, halogen, and OW²；

Without being limited by the examples, the following illustrative embodiments are intended to convey information related to methods of making and using the disclosed and claimed compounds.

Preparation of compounds

General preparation of 2 ' - (R) -2 ' -C-methyl-2 ' -deoxynucleosides

The preparation of 2 '-substituted-2' -deoxynucleosides is illustrated in scheme 1 below. Treatment of compound 1 with tiptscl followed by reaction of the resulting intermediate with monomethyloxalyl chloride gave compound 3. Using AIBN/n-BuSn₃Treatment with H3 followed by desilylation with TBAF provided the 2 '-substituted-2' -deoxynucleoside 6. Intermediate 4 may also be prepared by treatment of 2 with phocsccl followed by reductive deoxygenation, as used for preparation 4 from 3. B and R²' is as defined above.

Scheme 1

Conditions are as follows: a) TIPDSCI/imidazole; b) monomethyl-oxalyl chloride; c) AIBN/n-Bu₃SnH；d)PhOCSCI/DAMP；e)TBAF

Preparation of 2 ' - (R) -2 ' -C-methyl-2 ' -deoxyuridine

The general procedure for the preparation of 2 '-substituted-2' -deoxynucleosides, as exemplified above in scheme 2, was used to synthesize 2- (R) -2 '-C-methyl-2' -deoxyuridine. Treatment of 2' -C-methyl-uridine with TIPDSCl in pyridine or in the presence of imidazole, followed by reaction of the obtained intermediate 8 with monomethyloxalyl chloride gave compound 9 in high yield. Deoxidation of 9 by use of AIBN/n-Bu₃SnH treatment 9 was achieved to give 10, which after deprotection gave 2 ' - (R) -2 ' -C-methyl-2 ' -deoxyuridine (11).

Scheme 2

Conditions are as follows: a) TIPDSCI/imidazole; b) monomethyl-oxalyl chloride; c) AIBN/n-Bu₃SnH；d)TBAF

General preparation of 2 '- (R) -4' -azido-2 '-C-methyl-2' -deoxynucleosides

A general synthesis of 2 '- (R) -4' -azido-2 '-C-methyl-2' -deoxynucleosides is shown in scheme 3. Treatment of compound 12 with iodine in the presence of triphenylphosphine, followed by elimination in the presence of a base such as NaOMe or DBU, gives 4' -methylene-nucleoside 14. Treatment of 14 with TBSCl in the presence of imidazole for 3' -OH protection affords compound 15. Epoxidation of 15 followed by SnCl₄Using TMSN when present₃Treatment of the epoxide for ring opening also gave the 4' -azido-nucleoside 18 after deprotection of 17 by TBAF. Compound 18 can also be prepared by azido (azizo) -iodination of 14. Using ICl and NaN₃Treatment 14 gave high yields of 4 '-azido-5' -iodo-nucleoside 19. Protection of the 3 '-OH with BzCl followed by 5' -iodo-oxidation with m-chloroperbenzoic acid in the presence of m-chlorobenzoic acid provided the protected nucleoside 20. Deprotection of 20 also gives 4' -azido-nucleoside 18.

Scheme 3

Conditions are as follows: a) i is₂/Ph₃P; b) NaOMe or DBU; c) ICI/NaN₃(ii) a d) TBSCl; e) DMDO/acetone; f) TMSN₃/SnCl₄；g)TBAF；h)1.BzCl；2.MCPBA/MCBA；i)NH₃

Preparation of 2 '- (R) -4' -azido-2 '-C-methyl-2' -deoxycytidine (scheme 4)

Scheme 4 illustrates the preparation of 2 '- (R) -4' -azido-2 '-C-methyl-2' -deoxycytidine. Treatment with iodine in the presence of triphenylphosphineCompound 11, followed by NaOMe catalyzed elimination in MeOH gave high yields of compound 22. 22 and IN₃The reaction of (a) affords intermediate 27. Treatment with BzCl in pyridine gives 3 '-OH protection of 27 followed by oxidation of 5' -iodine with mCPBA in the presence of mCBA to give the protected uridine analogue 28. The target nucleoside 26 is prepared by treatment 28 with triisopropylbenzenesulfonyl chloride in the presence of DMAP, followed by treatment with ammonium hydroxide, and then with methanolic ammonia. Compound 26 can also be epoxidized intermediate 24 by treatment of compound 23 with DMDO/acetone followed by treatment with SnCl₄In the presence of TMSN₃Ring opening and conversion of uridine analogs to cytidine derivatives.

Scheme 4

Conditions are as follows: a) i is₂/Ph₃P；b)NaOMe；c)ICI/NaN₃(ii) a d) TBSCI; e) DMDO/acetone; f) TMSN₃/SnCl₄；g)1.TBSCI，2.TIPBSCI/DMAP，3.TBAF；h)1.BzCl，2.mCPBA/mCBA；i)1.TIPBSCI/DMAP，2.NH₃

Preparation of acetaldehyde intermediates 49 and 50 (scheme 5)

Treatment of compound 42 with DMTrCl in pyridine in the presence of DMAP followed by TBSCl/imidazole selectively gave very high yields of compound 44. Deoxygenation of Compound 44 was achieved by treatment of methyl chlorooxoacetate in the presence of DMAP, followed by AIBN/(TMS) in toluene₃SiH treatment to afford 2' -deoxynucleoside intermediate 45.

Scheme 5

Reagents and conditions: a) DMTrCl/DMAP/Pyr; b) TBSCl/imidazole; c. C)i.ClCOCO₂Me/DMAP/Et₃N，ii.AIBN/(TMS)₃SiH; d) TFA; e) Dess-Martin oxidation; f) I.CH₂O/NaOH，ii.NaBH₄(ii) a g) Dess-Martin oxidation; h) TBSCl/imidazole

Detritylation of 45 followed by Dess-Martin oxidation gave aldehyde 47. Aldol condensation of Compound 47 followed by NaBH₄Reduction is carried out to give diol 48. Selective oxidation of 48 by Dess-Martin reagent provided intermediate 49. The 5' -hydroxy group of 49 was protected with a silyl group to give intermediate 50.

Preparation of 4 ' -C-cyano-2 ' -methyl-2 ' -decyclidine (scheme 6)

Treatment of compound 49 with hydroxylamine hydrochloride in pyridine followed by heating with acetic anhydride in the presence of sodium acetate at 120 ℃ gives compound 52. Amination of compound 52 is achieved by treatment of 52 with tosyl chloride in the presence of N-methylpiperidine and triethylamine, followed by treatment with ammonium hydroxide to give cytidine intermediate 53. Desilylation of 53 with TEAF, and acetylation followed by treatment with methanolic ammonia yielded compound 55.

Scheme 6

Reagents and conditions: a) NH (NH)₂-HCl/Pyr；b)NaOAc/Ac₂O; c) TsCl/N-methylpiperidine/Et₃N，ii.NH₄OH；d)i.TEAF，ii.Ac₂O/Pyr；e)NH₃

Preparation of 4 ' -C-vinyl-2 ' -methyl-2 ' -deoxycytidine (scheme 7)

Treatment with methyltriphosphonium chloride in the presence of LiBu gave 4' -C-vinyl intermediate 56. Compound 58 was prepared in a similar manner. Further elimination of 58 to the 4' -C-ethynyl analog was achieved by treating 58 with LiBu. A similar procedure was used to prepare compound 53 from 52, and amination of compounds 56 and 59 gave compounds 57 and 60, respectively.

Scheme 7

Reagents and conditions: a) triphosphonium methyl chloride/LiBu; b) TsCl/N-Me-piperidine/Et₃N，ii.NH₄OH，ii.NH₄F; c) chloromethyl triphosphonium chloride/LiBu; d) LiBu

Preparation of 4 ' -C-hydroxymethyl-2 ' -methyl-2 ' -deoxycytidine (scheme 8)

Acetylation of compound 48 with acetic anhydride gave the fully protected intermediate 61. Similarly, amination of 61 followed by deprotection affords 4 ' -C-hydroxymethyl-2 ' -methyl-2 ' -deoxycytidine 62.

Scheme 8

Reagents and conditions: a) ac of₂O/Pyr; b) TsCl/N-Me-piperidine/Et₃N，ii.NH₄OH，ii.NH₄F

Preparation of 4 ' -C-allyl-and 4 ' -C-cyano-2 ' -methyl thymine (scheme 9)

Epoxidation of Compound 63 with DMDO followed by SnCl₄Treatment of the resulting epoxide with allyltrimethylsilane in the presence of the catalyst gave silyl protected intermediate 65. Treatment of 65 with methanolic ammonia followed by ammonium fluoride in MeOH affords 4' -C-allyl-nucleoside 69.

Similarly, compound 70 is prepared from intermediate 64 when trimethylsilyl cyanide is used as the nucleophile for epoxide ring opening.

Scheme 9

Reagents and conditions: a) DMDO; b) for 65, allyltrimethylsilane/SnCl₄As to 66, trimethylsilyl cyanide; NH (NH)₃/MeOH；NH₄F/MeOH

Preparation of 4 '-C-ethynyl-2' -methyl thymine (scheme 10)

Treatment of compound 64 with tris (allyl) alumina followed by desilylation gives products 73 and 74 after isolation.

Scheme 10

Reagents and conditions: a) (allyl)₃Al；b)NH₄F

Details of the experiment:

synthesis of Compound 28

From the starting nucleoside 2 '-C-methyl-2' -deoxyuridine by reaction with I₂/Ph₃P treatment, and elimination catalyzed by NaOMe, followed by NCl/NaN₃Azido-iodination was performed to prepare compound 27.

To a solution of ethanol (203.7mg 0.53mmol, 1.0eq.) in Dichloromethane (DCM) was added Triethylamine (TEA) (148 μ l, 1.06mmol, 2.0eq.) and dimethylaminopyridine (DMAP, catalytic amount). After 5 minutes, benzoyl chloride (BzCl) was added68 μ l, -.58mmol, 1.1eq.) and the reaction was monitored by LCMS (liquid chromatography-mass spectrometry). The reaction was complete after 10 minutes. Adding water and NaHCO₃And the mixture was extracted with DCM (2 ×), the organic layer was washed with brine, over Na₂SO₄Dried, filtered and concentrated. Purification on silica gel eluting with 3: 7 to 6: 4 EtOAC-heptane gave the protected iodide (180mg, 68%) as the product as a white solid.

To a solution of iodide (180mg, 0.362mmol, 1.0eq.) in DCM (18ml) and water (9ml) was added K in sequence₂HPO₄(126mg，0.724mmol，2.0eq.)，nBu₄NHSO₄(135mg, 0.398mmol, 1.1eq.) and mCBA (m-chlorobenzoic acid) (72mg, 0.398mmol, 1.1 eq.). The reaction mixture was cooled to 0 ℃ and 77% (243mg, 1.086mmol, 3.0eq.) of mCPBA (m-chloroperbenzoic acid) was added and the reaction allowed to stand to room temperature. After 14 h, LCMS indicated the reaction was complete. To the reaction mixture was added saturated NaHCO₃And Na₂SO₃Aqueous solution, and the mixture was extracted with EtOAc (100 mL). The organic layer was washed with brine and washed with Na₂SO₄Dried, filtered and concentrated. The residue was purified by FCC on silica gel eluting with 7: 3 to 5: 5 EtOAC-heptane to yield 145mg (76%) of product 28 as a white solid.

Synthesis of Compound 26

POCl in anhydrous MeCN (5ml)₃(94. mu.l, 1.009mmol, 4.0eq.) and triazole (331mg, 4.793mmol, 19.0eq.) were stirred at 0 ℃ for 5 minutes, followed by the slow addition of TEA (0.74 ml). The resulting mixture was left at 0 ℃ for 1 hour, followed by the addition of a solution of uridine (28, 163mg, 0.252mmol, 1.0eq.) in anhydrous MeCN (5ml). The reaction mixture was stirred at room temperature for 14 hours, then filtered through a pad of celite and the solid was washed with 3ml of MeCN. EtOAc (70mL) was added and saturated aqueous NaHCO₃The solution was washed with water, brine and then concentrated. The residue was co-evaporated with dioxane (20 ml). The residue was used in the next step without purification.

To a solution of triazole (159mg) in MeOH (5ml) was added sodium methoxide (-240 μ l, 25% by weight in MeOH, 4.0 eq.). After 30min, LCMS showed the reaction was complete. HCl (1.1ml, 1N, 4.0eq.) was added to the reaction mixture, and the mixture was concentrated. The residue was purified on silica gel eluting with 1% to 6% DCM-MeOH to yield 44mg (54%) of product as an oil.

The methoxy compound (40mg, 135mmol, 1.0eq.) was dissolved in 0.5N ammonia in dioxane (5 ml). The reaction mixture was heated to 120 ℃ for 3 hours in a microwave (250W, 150 PSI). The progress of the reaction was followed by LCMS. After completion of the reaction the solvent was removed and the residue was purified on silica gel eluting with DCM-EtOH from 95: 5 to 80: 20 to give 16.8mg of the desired product 26 as a syrup.

The products listed below were prepared using a similar procedure to that described above.

H-NMR data of the product MS starting Material

To a solution of compound 42(1.1g, 4.26mmol) in anhydrous pyridine (50mL) was added DMTrCl (2.17g, 6.39mmol) at room temperature. The mixture was stirred for 5h, diluted with EtOAc (100mL), washed with water (25mlx4), and dried over sodium sulfate. After filtration and concentration, the residue was co-evaporated with toluene (30mL) and purified (in CH) by flash column chromatography (flash column chromatography)₂Cl₂MeOH in 0 to 5%) to give 43(1.9g, 80%).¹H NMR(CDCl₃) (ppm)9.94(s, 1H, NH), 8.17(d, 1H, J ═ 8.0Hz, H-6), 7.40-7.24(m, 9H, aromatic), 6.84(m, 4H, aromatic), 6.08(s, 1H, H-1 '), 5.28(dd, 1H, J ═ 2.0, 8.4Hz, H-5), 4.80(s, 1H, HO), 4.11-4.01(m, 2H, H-3 ' and 4 '), 3.79(s, 6H, (OCH)₃)x2)，3.61(dd，1H，J＝2.4，11.6Hz，H-5’)，3.55(dd，1H，J＝2.4，11.2Hz，H-5”)， 2.87(d，1H，J＝9.2Hz，HO)，1.32(s，3H，CH₃)。

Synthesis of Compound 44

To anhydrous CH at room temperature₂Cl₂To a solution of 43(1.9g, 3.39mmol) and imidazole (0.69g, 10.17mmol) in (20mL) was added TBSCl (0.77g, 5.08 mmol). The resulting mixture was stirred at room temperature for 48 h. Additional imidazole (0.69g) and TBSCl (0.77g) were added. The mixture was then stirred at room temperature for 72h with CH₂Cl₂Diluted (80mL), washed with water (40mLx2), and dried over sodium sulfate. After filtration and concentration, the residue was purified by flash silica gel column chromatography (0-20-35% EtOAc in hexanes) to give compound 44(2g, 87%) as a white solid.¹H NMR(CDCl₃) (ppm)8.34(s, 1H, NH), 8.16(d, 1H, J ═ 8.4Hz, H-6), 7.32-7.17(m, 9H, aromatic), 6.85(m, 4H, aromatic), 6.13(s, 1H, H-1 '), 5.08(dd, 1H, J ═ 2.4, 8.0Hz, H-5), 4.19(d, 1H, J ═ 8.4Hz, H-3'), 4.01(m, 1H, H-4 '), 3.87(dd, 1H, J ═ 2.0, 10.8Hz, H-5'), 3.80(s, 6H, (OCH)₃)x2)，3.32(dd，1H，J＝2.0，10.8Hz，H-5”)，1.19(s，3H，CH₃)，0.79(s，9H，C(CH₃)₃)，0.07(s，3H，CH₃Si)，-0.30(s，3H，CH₃Si)。

Synthesis of Compound 45

To a solution of 44(2.0g, 2.96mmol), DAMP (2.17g, 17.78mmol), and triethylamine (2.48mL, 17.78mmol) in acetonitrile (40mL) was added methyl chlorooxoacetate (1.64mL, 17.78mmol) dropwise under an ice water bath. The resulting mixture was stirred at rt for 1h, diluted with EtOAc (125mL), washed with brine (30mLx4), and dried over sodium sulfate. After filtration and concentration, the residue was co-evaporated with toluene (20mLx2) and dried under high vacuum for 10 min. The residue was then dissolved in dry toluene (40mL) and bubbled with nitrogen for 10min, to which was added TMS silyl hydride (5.49mL, 17.78mmol) and then AIBN (1.46g, 8.89 mmol). The resulting mixture was refluxed at 120 ℃ for 1.5h in a preheated oil bath and concentrated under reduced pressure. The residue was purified by flash column chromatography (0-20-35% EtOAc in hexanes) to give compound 45 as a white solid (1.3g, 67%).¹H NMR(CDCl₃) (ppm)8.15(d, 1H, J ═ 8.4Hz, H-6), 8.02(s, 1H, NH), 7.34-7.20(m, 9H, aromatic), 6.84(m, 4H, aromatic), 6.30(d, 1H, J ═ 7.6Hz, H-1 '), 5.11(dd, 1H, J ═ 2.4, 8.4Hz, H-5), 4.14(d, 1H, J ═ 8.0Hz, H-3'), 3.83-3.74(m, 8H, H-4 ', 5' and (CH)₃O)₂)，3.33(dd，1H，J＝2.4，10.8Hz，H-5”)，2.54(m，1H，H-2’)，0.98(d，3H，J＝6.8Hz，CH₃)，0.76(d，9H，C(CH₃)₃)，0.04(s，3H，CH₃Si)，-0.28(s，3H，CH₃Si)。

Synthesis of Compound 46

To anhydrous CH in ice-water bath₂Cl₂A solution of compound 45(1.3g, 1.97mmol) in (10mL) was added TFA (0.3mL, 3.95 mmol). The resulting mixture was stirred at room temperature for 1h with CH₂Cl₂(100mL) diluted with saturated NaHCO₃(30mLx2) and dried over sodium sulfate. After filtration and concentration, the residue was purified by silica gel column chromatography (EtOAc: hexane ═ 1: 1) to give compound 46(600mg, 85%) as a white solid.

Synthesis of Compound 47

Dropwise addition of anhydrous CH at 0 deg.C₂Cl₂CH was added to a solution of compound 46(3g, 8.46mmol) in (30mL)₂Cl₂15% Dess-Martin oxidant (periodinane) in (36mL, 12.73 mmol). The reaction mixture was stirred at 0 ℃ for 3h with CH₂Cl₂Diluted (150mL), washed with sodium bicarbonate solution (50mLx3) followed by saturated sodium thiosulfate solution (50mLx3) and the organic layer dried over sodium sulfate. After filtration and concentration, the residue was purified by silica gel column chromatography (MeOH: CH)₂Cl₂1: 20) to give compound 47.¹H NMR(CDCl₃)(ppm)9.79(s，1H，H-5’)，8.86(s，1H，NH)，8.26(d，1H，J＝7.2Hz，H-6)，6.15(d，1H，J＝6.0Hz，H-1’)，5.77(d，1H，J＝8.0Hz，H-5)，4.40(d，1H，J＝3.6Hz，H-4’)，4.26(t，1H，J＝3.6Hz，H-3’)，2.70(m，1H，H-2’)，0.92(s，9H，C(CH₃)₃)，0.76(d，3H，J＝7.6Hz，CH₃)，0.14(s，3H，CH₃Si)，0.13(s，3H，CH₃Si).

Synthesis of Compound 48

The obtained compound 47 was then dissolved in dioxane (100mL) and 37% formaldehyde (3mL, 36.96mmol) was added. To the obtained solution was added 2N sodium hydroxide (5mL, 10mmol) dropwise at room temperature. The resulting reaction mixture was stirred at room temperature for 15h and cooled. Sodium borohydride (890mg, 23.53mmol) was then added portionwise. The resulting mixture was stirred at room temperature for 6h and AcOH-pyridine solution (2.5: 7.5mL) and water (100mL) were added under ice water. Followed by CH₂Cl₂(50ml x4) the mixture was extracted and the organic layer was dried over sodium sulfate. After filtration and concentration, the residue was purified by silica gel column chromatography (MeOH: CH)₂Cl₂1: 40 to 1: 20) to give compound 48(2.1g, 64%) as a white solid.¹H NMR(CD₃OD)(ppm)8.30(d，1H，J＝8.0Hz，H-6)，6.31(d，1H，J＝8.0Hz，H-1’)，5.68(d，1H，J＝8.0Hz，H-5)，4.29(d，1H，J＝9.6Hz，H-3’)，3.86(d，1H，J＝12.0Hz，CH-4’)，3.78(d，1H，J＝12.0Hz，CH-4’)，3.54(d，1H，J＝12.0Hz，CH-4’)，3.45(d，1H，J＝12.0Hz，CH-4’)，2.83(m，1H，H-2’)，0.98-0.88(m，12H，CH₃-2' and C (CH)₃)₃)，0.15(s，3H，CH₃Si)，0.13(s，3H，CH₃Si)。

Synthesis of Compound 49

In one portion to CH at 0 ℃₂Cl₂To a solution of compound 48(1.4g, 3.62mmol) in THF (46: 10mL) was added Dess-Martin oxidant (periodinane) (1.8g, 4.24 mmol). The mixture was stirred at 0 ℃ for 2 h. After the temperature had risen to 10 ℃, the mixture was stirred for 1h and sodium thiosulfate (1.0g) was added. The mixture was then stirred for 30min and added on top of a silica gel column, followed by CH₂Cl₂And CH₂Cl₂It was eluted with EtOAc (3: 1) to give compound 49 (mixture of desired α/β ═ 2) as a white solid (900mg, 64%).

Synthesis of Compound 50

At room temperature to CH₂Cl₂A solution of compound 49(500mg, 1.30mmol) and imidazole (530mg, 7.8mmol) in (20mL) was added TBSCl (590mg, 3.90 mmol). The resulting mixture was stirred for 3h with CH₂Cl₂Diluted, washed with water and dried over sodium sulfate. After filtration and concentration, the residue was separated by flash column chromatography (EtOAc, 0 to 25% in hexanes) to give compound 50(270mg, 40%).

Synthesis of Compound 51

At 0 ℃ in one portion to anhydrous CH₂Cl₂A solution of compound 48(220mg, 0.57mmol) in THF (10 mL: 2mL) was added Dess-Martin oxidant (periodinane) (300mg, 0.71 mmol). The resulting reaction mixture was stirred for 2h and sodium thiosulfate (390mg) was added. Will be provided withThe mixture was stirred at 0 ℃ for 15min, then poured onto the top of a short silica gel column and washed with CH₂Cl₂EtOAc (1: 1) eluted well. The fractions were combined and concentrated in vacuo to a residue, which was then dissolved in pyridine (10mL) and NH was added₂OH-HCl (300 mg). The resulting mixture was stirred at room temperature for 15h and concentrated in vacuo. Chromatography on silica gel (MeOH: CH)₂Cl₂1: 40 to 1: 20) the obtained residue was purified to give compound 51(107mg, 47%) and the 4' - β -isomer (53mg, 18%) as a white solid.¹H NMR(CD₃OD)(ppm)8.20(d，1H，J＝8.0Hz，H-6)，7.4(s，1H，HC＝N)，6.31(d，1H，J＝8.0Hz，H-1’)，5.70(d，1H，J＝8.0Hz，H-5)，4.32(d，1H，J＝10.0Hz，H-3’)，3.84(s，2H，H-5’)，2.64(m，1H，H-2’)，0.94(d，3H，J＝7.2Hz，CH₃)，0.92(s，9H，C(CH₃)₃)，0.17(s，3H，CH₃Si)，0.13(s，3H，CH₃Si)；¹H NMR(CD₃OD) 7.71(d, 1H, J ═ 8.0Hz, H-6), 7.55(s, 1H, HC ═ N), 6.29(d, 1H, J ═ 7.6Hz, H-1 '), 5.70(d, 1H, J ═ 8.0Hz, H-5), 4.37(d, 1H, J ═ 8.0Hz, H-3'), 3.92(d, 1H, J ═ 12.4Hz, H-5 '), 3.73(d, 1H, J ═ 12.4Hz, H-5 "), 2.86(m, 1H, H-2'), 0.93-0.88(m, 12H, CH), CH-6, (%) for the minor isomers (ppm)₃-2' and C (CH)₃)₃)，0.12(s，3H，CH₃Si)，0.10(s，3H，CH₃Si).

Synthesis of Compound 52

A mixture of compound 51(160mg, 0.40mmol) and sodium acetate (123mg, 1.5mmol) in acetic anhydride (5mL) was heated at 120 ℃ for 3h and concentrated in vacuo. The residue obtained is chromatographed on a silica gel column with MeOH-CH₂Cl₂(1: 40) to give Compound 52 as a white solid (97mg, 57%).¹H NMR(CDCl₃)(ppm)10.03(s，1H，NH)，7.26(d，1H，J＝8.0Hz，H-6)，6.17(bs，1H，H-1’)，5.78(d，1H，J＝8.0Hz，H-5)，4.52(d，1H，J＝12.4Hz，H-5’)，4.42(d，1H，J＝12.4Hz，H-5”).4.19(bs，1H，H-3’)，2.86(m，1H，H-2’)，2.17(s，3H，CH₃CO)，1.03(d，1H，J＝ 7.2，CH₃)，0.95(s，9H，C(CH₃)₃)，0.15(s，6H，CH₃Si).

Synthesis of Compound 53

To anhydrous CH at room temperature₃TsCl (270mg, 1.42mmol) was added to a solution of compound 52(200mg, 0.47mmol) in CN (4mL), which contained triethylamine (0.20mL, 1.42mmol) and N-methylpiperidine (0.11mL, 0.94 mmol). The resulting mixture was stirred at room temperature for 1h and then 29% NH was added in a water bath₄OH (4 mL). The resulting mixture was stirred at room temperature for 2h and concentrated at a temperature of 35 ℃. The residue obtained is purified by column chromatography on silica gel (MeOH: CH)₂Cl₂1: 10 to 1: 4) to give compound 53 as a syrup (160mg, 89%).

Synthesis of Compound 54

A solution of compound 53(160mg, 0.42mmol) and TEAF (200mg, 1.34mmol) in MeOH-THF (2: 4mL) was stirred at room temperature for 15h and heated at 60 deg.C for 4 h. After concentration, silica gel column chromatography (MeOH: CH)₂Cl₂1: 10 to 1: 4) to give crude compound 55(100mg, crude, contaminated TEAF), which was then dissolved in anhydrous pyridine, followed by treatment with acetic anhydride, stirred at room temperature for 3h, and concentrated in vacuo. By passingSilica gel column chromatography (MeOH: CH)₂Cl₂1: 40) to give compound 54 as a syrup, which was used in the next reaction.¹H NMR(CD₃OD)(ppm)7.97(d，1H，J＝7.6Hz，H-6)，7.46(d，1H，J＝7.6Hz，H-5)，6.23(bs，1H，H-1’)，5.52(bs，1H，H-3’)，4.65(d，1H，J＝12.0Hz，H-5’)，4.62(d，1H，J＝12.0Hz，H-5”)，3.11(m， 1H，H-2’)，2.19(s，3H，CH₃CO)，2.19(s，3H，CH₃CO)，2.12(s，3H，CH₃CO)，0.97(d，3H，J＝7.2Hz，CH₃)；MSES(M+1)：393.

Synthesis of Compound 55

Compound 54 was dissolved in 7M ammonia in methanol (5mL), stirred in a sealed flask for 15h, and concentrated in vacuo. The residue obtained is chromatographed on a silica gel column with MeOH-CH₂Cl₂(1: 4) elution was performed to give compound 55(30mg, 27% from 53) as a white solid. UV (lambda)_max)273nm(MeOH)；¹H NMR(CD₃OD)(ppm)7.85(d，1H，J＝7.8Hz，H-6)，6.40(bs，1H，H-1’)，5.88(d，1H，J＝7.6Hz，H-5)，4.10(d，1H，J＝9.6Hz，H-3’)，4.00(d，1H，J＝12.4Hz，H-5’)，3.92(d，1H，J＝12.4Hz，H-5”)，2.75(m，1H，H-2’)，0.95(d，3H，J＝3.2Hz，CH₃)；MSES(M+1)：267.

Synthesis of Compound 56

To a suspension of methyltriphenylphosphonium bromide in THF (2mL) at-78 deg.C was added n-BuLi (2.2M in hexane, 0.24mL, 0.528mmol) in anhydrous THF (2 mL). Mixing the raw materialsThe mixture was stirred at 0 ℃ for 1h and compound 50(60mg, 0.12mmol) in dry THF (2mL) was added. The mixture was then stirred at room temperature for 1h, neutralized with saturated aqueous ammonium chloride, diluted with EtOAc, washed with brine, and dried over sodium sulfate. After filtration and concentration, the residue was purified by silica gel column chromatography (MeOH: CH)₂Cl₂1: 40) to give compound 56(45mg, 75%) as a white solid.

Synthesis of Compound 57

To anhydrous CH at room temperature₃TsCl (120mg, 0.6mmol) was added to a solution of compound 56(100mg, 0.20mmol) in CN (2mL), which contained triethylamine (0.08mL, 0.6mmol) and N-methylpiperidine (0.05mL, 0.40 mmol). The resulting mixture was stirred at room temperature for 1h, and then 29% NH was added under a water bath₄OH (2 mL). The resulting mixture was stirred at room temperature for 2h and concentrated in vacuo at 35 ℃. Chromatography on silica gel (MeOH: CH)₂Cl₂1: 10-1: 4) to give the protected cytidine intermediate as a syrup (70mg, 70%). A mixture of intermediate (70mg, 0.14mmol) and ammonium fluoride (100mg, 2.82mmol) in methanol (10mL) was refluxed for 6h and refluxed for 15h after addition of additional ammonium fluoride (100mg, 2.82 mmol). After concentration in vacuo, silica gel column chromatography (MeOH: CH)₂Cl₂1: 10 to 1: 4) to give compound 57(28.6mg, 77%). UV (lambda)_max)273nm(MeOH)；¹H NMR(CD₃OD)(ppm)8.28(d，1H，J＝7.2Hz，H-6)，6.28(d，1H，J＝7.6Hz，H-1’)，5.99(dd，1H，J＝11.2，17.2Hz，H-C＝C)，5.91(d，1H，J＝7.6Hz，H-5)，5.49(dd，1H，J＝2.0，17.2Hz，H-C＝C)，5.32(dd，1H，J＝2.0，10.8Hz，H-C＝C)，4.06(d，1H，J＝10.8Hz，H-3’)，3.72(d，1H，J＝12.0Hz，H-5’)，3.55(d，1H，J＝12.0Hz，H-5”)，2.45(m，1H，H-2’)，0.87(d，3H，J＝6.8Hz，CH₃)；MSES∶268(M+1)，535(2M+1).

Synthesis of Compound 58

To a suspension of chloromethyl triphenyl phosphonium chloride (390mg, 1.12mmol) in THF (3mL) at-78 deg.C was added n-BuLi (2.2M in hexane, 0.51mL, 1.12 mmol). The mixture was stirred at 0 ℃ for 1h and compound 50(140mg, 0.28mmol) in anhydrous THF (3mL) was added. The resulting mixture was stirred at rt for 3h, neutralized with saturated ammonium chloride, diluted with EtOAc, washed with brine, and dried over sodium sulfate. After filtration and concentration, silica gel column chromatography (MeOH: CH)₂Cl₂1: 40) to give compound 58(140mg, 94%) as a white solid.

Synthesis of Compound 59

To a solution of compound 58(200mg, 0.38mmol) in anhydrous THF (10mL) at-78 deg.C was added n-BuLi (2.8mL of 1.6M in hexanes, 4.52mmol) dropwise. The mixture was then stirred at-78 ℃ for 2h, neutralized with saturated ammonium chloride solution (10mL), diluted with EtOAc (50mL), washed with brine (15mLx3), and dried over sodium sulfate. After filtration and concentration, the residue was purified by silica gel column chromatography (hexane: EtOAc ═ 4: 1) to give compound 59(180mg, 97%) as a white solid.¹H NMR(CDCl₃)(ppm)9.21(bs，1H，NH)，8.05(d，1H，J＝8.4Hz，H-6)，6.24(d，1H，J＝7.6Hz，H-1’)，5.94(dd，1H，J＝11.2，17.6Hz，H-C＝C)，5.70(dd，1H，J＝2.0，8.0Hz，H-C＝C)，5.52(dd，1H，J＝1.2，17.2Hz，H-C＝C)，5.32(d，1H，J＝8.4Hz，H-5)，4.16(d，1H，J＝10.4Hz，H-3’)，3.64(d，1H，J＝11.2Hz，H-5’)，3.57(d，1H，J＝11.6Hz，H-5”)，2.48(m，1H，H-2’)，0.95(s，9H，C(CH₃)₃) 0.91 and 0.90(m, 12H, C (CH)₃)₃And CH₃-2’)，0.12-0.10(4s，12H，CH₃Si).

Synthesis of Compound 60

To anhydrous CH at room temperature₃TsCl (120mg, 0.61mmol) was added to a solution of compound 59(100mg, 0.20mmol) in CN (3mL), which contained triethylamine (0.08mL, 0.6mmol) and N-methylpiperidine (0.05mL, 0.40 mmol). The resulting mixture was stirred at room temperature for 1h and then 29% NH was added under a water bath₄OH (2 mL). The resulting mixture was stirred at room temperature for 2h and concentrated in vacuo at 35 ℃. Chromatography on silica gel (MeOH: CH)₂Cl₂1: 20-1: 10) to give cytidine intermediate as syrup (70mg, 70%). A mixture of intermediate (70mg, 0.14mmol) and ammonium fluoride (260mg, 7.09mmol) was heated in a sealed flask at 90 deg.C for 15h and concentrated in vacuo. Chromatography on silica gel (MeOH: CH)₂Cl₂1: 4) to give compound 60(31.2mg, 85%) as a white solid. UV (lambda)_max)273nm(MeOH)；¹H NMR(CD₃OD)(ppm)8.05(d，1H，J＝7.6Hz，H-6)，6.31(d，1H，J＝8.0Hz，H-1’)，5.89(d，1H，J＝7.6Hz，H-5)，3.95(d，1H，J＝10.8Hz，H-3’)，3.88(d，1H，J＝12.4Hz，H-5’)，3.79(d，1H，J＝12.4Hz，H-5”)，3.06(s，1H，H-C≡C)，2.75(m，1H，H-2’)，0.915(d，3H，J＝6.8Hz，CH₃).

Synthesis of Compound 61

To a solution of compound 48(220mg, 0.57mmol) in anhydrous pyridine (4mL) was added acetic anhydride (0.27mL, 2.85mmol) at room temperature. The resulting mixture was stirred at room temperature for 5h and concentrated in vacuo. Chromatography on silica gel (MeOH: CH)₂Cl₂1: 40) to give compound 61 as a white solid (220mg, 82%).¹H NMR(CDCl₃)(ppm)9.80(bs，1H，NH)，7.62(d，1H，J＝8.0Hz，H-6)，6.26(d，1H，J＝7.2Hz， H-1’)，5.74(d，1H，J＝8.4Hz，H-5)，4.53(d，1H，J＝12.4Hz，HC-4’)，4.35(d，1H，J＝12.0Hz，H-5’)，4.24(d，1H，J＝12.0Hz，H-5”)，4.05(d，1H，J＝7.2Hz，H-3’)，3.99(d，1H，J＝12.0Hz，HC-4’)，2.82(m，1H，H-2”)，2.15(s，3H，CH₃CO)，2.12(s，3H，CH₃CO)，0.96(d，3H，J＝7.2Hz，CH₃)，0.90(s，9H，C(CH₃)₃)，0.11(s，3H，CH₃Si)，0.08(s，3H，CH₃Si)；MSES(M+1)：266

Synthesis of Compound 62

To a solution of compound 61(220mg, 0.47mmol) in dry acetonitrile (5mL) at room temperature was added TsCl (270mg, 1.40mmol) comprising triethylamine (0.19mL, 1.40mmol) and N-methylpiperidine (0.11mL, 0.93 mmol). The resulting mixture was stirred at room temperature for 1h and 29% NH was added₄An aqueous OH solution. The mixture was then stirred at room temperature for 15h and concentrated in vacuo. The residue was purified by silica gel column chromatography (MeOH: CH)₂Cl₂1: 40) to give the cytidine intermediate as a white solid (120mg, 67%). A mixture of intermediate (60mg, 0.156mmol) and ammonium fluoride (100mg) in methanol (5mL) was heated at 90 ℃ for 15h and concentrated in vacuo. Chromatography on silica gel (MeOH: CH)₂Cl₂1: 10-1: 4) purifying the residue obtained in order to obtainCompound 62(10mg, 24%) was given as a white solid. UV (lambda)_max)273nm(MeOH)；¹HNMR(D₂O)(ppm)8.08(d，1H，J＝7.6Hz，H-6)，6.32(d，1H，J＝8.4Hz，H-1’)，6.09(d，1H，J＝7.6Hz，H-5)，4.09(d，1H，J＝10.4Hz，H-3’)，3.83(d，1H，J＝12.8Hz，CH-4’)，3.77(d，1H，J＝12.4Hz，CH-4’)，3.72(d，1H，J＝12.4Hz，CH-4’)，3.60(d，1H，J＝12.4Hz，CH-4’)，2.87(m，1H，H-2’)，0.87(d，3H，J＝6.8Hz，CH₃).

Synthesis of Compound 64

To CH at-30 deg.C₂Cl₂A solution of substrate 63(150mg, 0.43mmol) in (9mL) was added to a cold solution of DMDO (11.33mL, 1.8eq, 0.79mmol, 0.07M from acetone) and stirred under argon for 1 h. The solvent was evaporated and the residue was dried under vacuum at 0 ℃ with vigorous stirring and dried for a further 5 min. A viscous solid 64 was formed and used in the next step without purification (-90%).

Synthesis of Compound 67

At-30 ℃ to CH₂Cl₂TMS-CH was added to a cold solution of epoxide 64(150mg, 0.41mmol) in (10mL)₂CH＝CH₂(90mg, 146uL, 1.22mmol) followed immediately by SnCl under argon₄(1.22mL of a 1M solution in CH₂Cl₂Middle, 1.22 mmol). The reaction mixture was stirred at-30 ℃ for 6h and at room temperature for 2 h. With saturated Na₂HCO₃The solution quenched the reaction and the reaction mixture was filtered through a pad of celite. The filtrate is distributed in CH₂Cl₂/H₂And O is between. Separating the organic layerSeparating, drying (Na)₂SO₄) And concentrated to dryness. With NH at room temperature₃The crude product was treated for 15h with MeOH (5mL, 7N). The solvent was removed under vacuum. The residue was purified by silica gel chromatography, eluting with 10-40% ethyl acetate/hexanes to give compound 67 as a colorless foam (85mg, 76%). Calculated mass: 410.22, measured mass: 411.10 (M)⁺+H)；¹HNMR(CDCl₃)8.52(s，NH)，7.76(s，H)，6.19(s，H)，5.89(m，H)，5.15(d，H，J＝4Hz)，5.11(s，H)，4.25(d，H，J＝9.2Hz)，3.86(d，H，J＝11.2Hz)，3.53(dd，H，J＝11.6Hz，3.2Hz)，2.28(m，H)，2.39(dd，H，J＝15.2Hz，6.8Hz)，2.12(dd，H，J＝14.4Hz，8.4Hz)，1.90(s，3H)，0.91(s，9H)0.12(s，3H)，0.11(s，3H).

Synthesis of Compound 69

To a well-dried mixture of substrate 67(30mg, 0.073mmol), ammonium fluoride (32mg, 0.73mmol) was added methanol (3mL) and refluxed at 85 ℃ for 12 h. Excess ammonium fluoride (16mg, 0.36mmol) and methanol (2mL) were added and reflux continued for another 24 h. The reaction mixture was filtered through a pad of celite and the solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography with 1-8% MeOH/CH₂Cl₂Elute to give product 69(19mg, 88%) as a colorless solid. Calculated mass: 296.32, measured mass: 297.20 (M)⁺+H)；¹H NMR(CDCl₃)8.22(s，H)，6.18(d，H，J＝8.4Hz)，5.98(m，H)，5.09(m，2H)，4.13(d，H，J＝10.8Hz)，3.76(d，H，J＝12.4Hz)，3.55(d，H，J＝12Hz)，2.66(m，H)，2.39(dd，H，J＝14.8Hz，6.4Hz)，2.17(dd，H，J＝14.6Hz，6.4Hz)，1.85(s，3H)，0.93(d，3H，J＝6.8Hz).

Synthesis of Compound 68

At-30 ℃ to CH₂Cl₂To a cold solution of epoxide 64(120mg, 0.33mmol) in (6mL) was added TMS-CN (60mg, 0.65mmol) followed immediately by SnCl under argon₄(0.65mL, 0.65mmol from CH)₂Cl₂1M solution of (1). The reaction mixture was stirred at-30 ℃ for 1h and at room temperature for 15 h. With saturated Na₂HCO₃The solution quenches the reaction mixture. The reaction mixture was filtered and the filtrate was partitioned in CH₂Cl₂/H₂And O is between. The organic layer was separated and dried (Na)₂SO₄) And concentrated to dryness. With NH at room temperature₃The crude product was treated with MeOH (7N, 5mL) for 15 h. The solvent was removed and the crude product was purified by silica gel chromatography, eluting with 4-30% ethyl acetate/hexanes to give product 68(72mg, 56%) as a colorless solid. Calculated mass: 395.20, measured mass: 396.30 (M)⁺+H)；¹H NMR(CDCl₃)8.63(s，NH)，7.28(s，H)，6.14(d，H，J＝6.4Hz)，4.36(s，H)，4.14(d，H，J＝12Hz)，3.92(d，H，J＝12.4Hz)，3.02(br s，OH)，2.83(m，H)，1.90(d，3H，J＝1.2Hz)，1.02(d，3H，J＝6.8Hz)，0.94(s，9H)，0.17(s，3H)，0.14(s，3H).

Synthesis of Compound 70

To a well dried mixture of substrate 68(35mg, 0.09mmol) and ammonium fluoride (32mg, 0.73mmol) was added MeOH (3mL) and refluxed at 85 ℃. The reaction was complete in 12 h. The solvent was removed and the crude product was dissolved in 20% MeOH/CH₂Cl₂In solution. The reaction mixture was filtered through a pad of celite. The solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography with 1-8% MeOH/CH₂Cl₂Eluted to give as colorless solidProduct 70 of (20mg, 80%). Calculated mass: 281.27. measured mass: 282.20 (M)⁺+H)；¹H NMR(CDCl₃)7.73(s，H)，6.34(d，H，J＝6.4Hz)，4.19(d，H，J＝10.4Hz)，4.02(d，H，J＝12.4Hz)，3.92(d，H，J＝12Hz)，2.73(m，H)，1.85(s，3H)，0.98(d，3H，J＝6.8Hz).

Preparation of Trialkynylaluminum

To CH at 0 ℃ under argon₂Cl₂AlCl in (7.5mL)₃(1g, 7.5mmol) to the solution was added ethynylmagnesium chloride (37.5mL from 0.6M in THF, 22.5 mmol). The reaction mixture was warmed to room temperature and stirred overnight. The resulting dark brown solution (0.14M) was used for the next step.

Synthesis of Compounds 71 and 72

Immediately at-30 ℃ CH under argon₂Cl₂To a cold solution of epoxide 64(80mg, 0.22mmol) in (6mL) was added triethylaluminum (5.2mL, from CH)₂Cl₂0.14M stock solution in THF, 0.56mmol) and stirred at-30 ℃ for 6 h. With saturated NH₄The Cl solution quenched the reaction and was filtered through a pad of celite. The filtrate was distributed over CH₂Cl₂/H₂And O is between. The organic layer was separated and dried (Na)₂SO₄). The solvent was removed and the crude product was purified by silica gel chromatography, eluting with 5-30% ethyl acetate/hexanes to give products 71 and 72 as semi-solids (57mg, 67% total). Calculated mass: 394.20. measured mass: 395.40 (M)⁺+H).

Isomer 71:¹H NMR(CDCl₃)：9.02(s，NH)，7.58(s，H)，6.31(s H)，4.10(d，H，J＝12.0Hz)，3.99(d，H，J＝12.6Hz)，3.71(d，H，J＝12.3Hz)，2.81(m，H)，1.91(s，3H)，1.01(s，3H)，0.97(s，9H)，0.17(s，3H)，0.12(s.3H).

isomer 72: 8.34(s, NH), 7.43(s, H), 6.33(d, H, J ═ 7.2Hz), 4.23(d, H, J ═ 7.2Hz), 3.91(dd, H, J ═ 12.4Hz, 4.8Hz), 3.78(dd, H, J ═ 12.0Hz, 9.2Hz), 2.79(m, H), 1.95(s, 3H), 0.98(d, H, J ═ 7.6Hz), 0.93(s, 9H), 0.18(s, 3H), 0.14(s, 3H).

Synthesis of Compound 73

To a well dried mixture of substrate 71(20mg, 0.46mmol) and ammonium fluoride (17mg, 4.56mmol) was added MeOH (3mL) and refluxed at 85 ℃. The reaction was terminated in 12h, the solvent was removed by reduced pressure and the crude product was dissolved in 20% MeOH/CH₂Cl₂And (3) solution. The reaction mixture was filtered through a pad of celite.

The solvent was removed under reduced pressure and the crude product was purified by silica gel chromatography with 1-8% MeOH/CH₂Cl₂Elute to give product 73 as a colorless solid (12mg, 94%). Calculated mass: 280.30. measured mass: 281.20 (M)⁺+H)；¹H NMR(CDCl₃)7.94(s，H)，6.23(d，H，J＝8Hz)，4.03(d，H，J＝10.8Hz)，3.90(d，H，J＝12.4Hz)，3.80(d，H，J＝12.4Hz)，3.05(s，H)，2.75(m，H)，1.85(d，3H，J＝1.2Hz)，0.95(d，3H，J＝7.2Hz).

Synthesis of Compound 74

To a well dried mixture of substrate 72(10mg, 0.03mmol) and ammonium fluoride (10mg, 0.3mmol) was added MeOH (2mL) and refluxed at 85 ℃. The reaction was terminated in 12h and the solvent was removed under reduced pressure. The crude product was dissolved in 20% MeOH/CH₂Cl₂And (3) solution. The reaction mixture was filtered through a pad of celite. The solvent was removed and the crude product was purified by silica gel chromatography with 1-8% MeOH/CH₂Cl₂Elute to give product 74 as a colorless solid (8mg, 91%). Calculated mass: 280.30. measured mass: 281.2 (M)⁺+H)；¹H NMR(CDCl₃)7.58(s，H)，6.30(d，H，J＝8.4Hz)，4.16(d，H，J＝12.2Hz)，3.89(d，H，J＝12.8Hz)，3.71(d，H，J＝12.7Hz)，3.28(s，H)，2.85(s，H)，1.90(d，3H，J＝1.3Hz)，0.96(d，3H，J＝7.0)。

In a similar manner, but using the appropriate sugar and pyrimidine or purine bases, nucleosides of the formula below can be prepared.

Biological assay

HBV AD38 assay

Materials required for the assay include the following:

HepG2-AD38 cell line.

The medium used for HepG2-AD38 included DMEM-F/12, 10% fetal bovine serum, 100IU/ml/100ug/ml penicillin/streptomycin, 50. mu.g/ml kanamycin, 0.3. mu.g/ml tetracycline, and 200. mu.g/ml G418.

The assay medium for HepG2-AD38 included DMEM-F/12, 10% fetal bovine serum, 100 IU/ml/100. mu.g/ml penicillin/streptomycin, 50. mu.g/ml kanamycin, and 200. mu.g/ml G418

Other materials include the following: phosphate Buffered Saline (PBS), Biocoated (Biocoated) 96-well plates, DNeasy 96 tissue kits (Qiagen), QIAvac 96 vacuum manifold, Micro amp optical 96-well reaction plates (Applied Biosystems), Micro amp optics caps (Applied Biosystems), Tagman Universal PCR Standard mixture (Tagman Universal PCR Master Mix) (Applied Biosystems), 7700 sequence Detector (Applied Biosystems), primers and probes for HBV DNA including 1125nM forward primer (GGA CCC CTG CTC GTG TTA CA); 1125nM reverse primer (GAGAGA AGT CCA CCA CGA GTC TAG A; and 250nM probe (FAM-TGT TGACAA GAA TCC TCA CAA TAC CAC).

HBV cell assay:

will be 5X 10⁴Cells/well were seeded in 200. mu.l medium of 96-well biocoated plates and incubated with 5% CO at 37 ℃₂The plates were incubated. After 2 days, the supernatant was carefully removed, the cell layer was washed with 200 μ Ι PBS, and subsequently refreshed with 200 μ Ι assay medium with or without test compounds in a 1: 3 ratio at 10 μ Μ or starting at 10 μ Μ (all samples should be tested in duplicate). Cells were grown for an additional 5 days. On day 7, 180 μ Ι _ of supernatant/well was collected in blue racks (included in DNeasy 96 tissue kit). Stored at-80 ℃ or used directly in the next step.

Viral HBV DNA was extracted from cell supernatants.

The samples in the blue racks were thawed. Proteinase K/buffer ATL working solution (2mL proteinase K and 18mL buffer ATL) was prepared and transferred 180. mu.l on top of the supernatant liquid in each tube of the blue rack. The tube was suitably sealed using a cap provided and mixing was performed by turning the rack upside down several times. Centrifuge at up to 3000rpm to collect any solution from the cap. Incubate at 55 ℃ for 15 minutes. Again, centrifugation was carried out at up to 3000 rpm. The cap was carefully removed and 410ul of buffer AL/E was added to each sample. The tube was sealed with a new cap, the rack was shaken vigorously up and down for 15 seconds, and centrifuged at up to 3000 rpm. The DNeasy 96 plate was placed on top of the QIAvac 96 vacuum manifold. The supernatant from step 8 was transferred to DNeasy 96 plates. Vacuum was applied for several seconds. Carefully add 500. mu.l of buffer AW1 to each well. Vacuum was applied for about 1 minute. Carefully add 500. mu.l of buffer AW2 to each well. Vacuum was applied for about 1 minute, the plate was flicked in a sink, the bottom of the DNeasy 96 plate was struck on the paper towel stack and vacuum was again applied for 10 minutes. 10ml of buffer AE were heated at 70 ℃ for several minutes. DNeasy 96 plates were placed on top of the rack of eluted microtubes RS. 100 μ l of preheated buffer AE was added to each well and vacuum was applied for 1 min to elute the DNA.

HBV real-time PCR:

a200-well HBV primer + probe mix (total 1500. mu.l) was prepared, which contained 45. mu.l of primer 1 (100. mu.M), 45. mu.l of primer 2 (100. mu.M), 20. mu.l of probe (50. mu.M), and 1390. mu.l of nuclease-free water. An optical 96-well reaction plate was selected. A100-well reaction Mix containing 1000. mu.l of a Universal PCR standard Mix (Universal PCR Master Mix), 750ul of HBV primer + probe Mix, and 250. mu.l of nuclease-free water was prepared. 20 μ l of the reaction mixture was aliquoted into each well. Mu.l of HBV DNA from each sample was added to each well. The aperture is covered with an optical cap. Centrifuge for several seconds to bring all reagents to the bottom and eliminate air bubbles. The plate was placed in a 7700 sequence detector. The reporter for FAM was selected and set to a volume of 25 μ Ι. The machine was started and allowed to run for 1 hour 56 minutes. dCt was calculated for each test compound as well as the reduction in viral load.

HCV replicon assay

Huh 7 cells (clone A cells; Apath, LLC, St. Louis, Mo.) containing HCV replicon RNA were cultured in exponential growth in Dulbecco's modified Eagle's medium (high glucose) containing 10% fetal bovine serum, 4mM L-glutamine and 1mM sodium pyruvate, 1 × non-essential amino acids, and G418(1,000 μ G/ml). Antiviral assays were performed in the same medium without G418. Cells were seeded at 1,500 cells/well in 96-well plates and test compounds were added immediately after seeding. Incubation time was 4 days. At the end of the incubation step, total cellular RNA was isolated (RNeasy 96 kit; Qiagen). Replicon RNA and internal controls were amplified in a single-step, multi-stage RT-PCR protocol (TaqMan rRNA control reagents; Applied Biosystems) as recommended by the manufacturer. HCV primers and probes were designed with Primer expression software (Primer Express software) (Applied Biosystems) and cover highly conserved 5 ' -untranslated region (UTR) sequences (sense, 5 ' -AGCCATGGCGTTAGTA(T) GAGTGT-3 ', and antisense, 5'-TTCCGCAGACCACTATGG-3'; probe, 5 ' -FAM-CCTCCAGGACCCCCCCTCCC-TAMRA-3 ').

To show the antiviral effectiveness of the compounds, the threshold RT-PCR cycles (Δ CtHCV) of the test compounds were subtracted from the mean threshold RT-PCR cycles of the non-drug controls. A.3. DELTA. Ct equals a 1-log 10 replicon RNA level reduction (equals 90% effective concentration [ EC)₉₀]). The cytotoxicity of the test compounds can also be expressed by calculating the delta CtrRNA value. Next, a Δ Δ Ct-specific parameter (Δ CtHCV- Δ CtrRNA) can be introduced, where HCV RNA levels are normalized to rRNA levels and corrected for drug-free controls.

HIV Activity

HIV screening: preliminary screening for testing PSI Compounds for antiviral HIV Activity at 50 μ M

The cells used were P4CCR51uc cells; they are human HIV indicator cells from Hela cells expressing CD4, CXCR4, CCR5, luciferase, and β -gal genes under the control of the HIV-1 LTR. P4CCR5luc cells were cultured in DMEM, 10% FBS, penicillin, streptomycin, and 500. mu.g/ml G418. Mu.l of P4CCR5-luc cells were seeded at 10,000 cells/well in 96-well opaque assay plates and incubated overnight at 37 ℃. The next day, media was aspirated from the plates and replaced with 100 μ Ι of compound freshly diluted into media at 2 × 50uM in triplicate at 37 ℃ for 4 hours. Subsequently, cells were infected with 100ul NL43 virus in the presence of 2X20ug/ml DEAE-dextran at 5ng p 24/well for 40-42 hours. Uninfected, infected without drug, and AZT controls were always present in triplicate on each plate. After infection, β -gal was quantified using the Galacto-Star kit from Applied Biosystems (Applied Biosystems) using the manufacturer's recommendations and luminescence was measured using the Victor instrument from platimum (Perkin-Elmer). Results are expressed as percent inhibition compared to untreated cells. The assay was performed in 2-3 independent experiments.

HIV-titration of PSI Activity to determine EC on P4CCR-luc cells₅₀。

P4CCR5-luc cells were seeded at 10,000 cells/well (100. mu.l) in 96-well opaque assay plates and incubated overnight at 37 ℃. The next day, the medium was aspirated from the plate and replaced with 100ul of a new diluted compound in a suitable medium (DMEM, 10% FBS, G418500 μ G/ml, penicillin/streptomycin) at 2x final concentration, at 5-fold dilution, usually from 2x100 μ M to 2x0.032um, in triplicate, at 37 ℃ for 4 hours. Next, cells were infected with 100ul NL43 wild-type or mutant virus at 5ng-20ng p 24/well for 40-42 hours in the presence of 2X20ug/ml DEAE-dextran. Uninfected and infected no-drug controls were always presented in duplicate on each plate. AZT controls were tested in parallel for each experiment. Following infection, beta-gal in cell lysates was quantified using the Galacto-Star kit from Applied Biosystems (Applied Biosystems) and luminescence was measured using a Victor apparatus from platimum (Perkin-Elmer). Use ofSpreadsheet calculation of EC₅₀(effective concentration), the EC₅₀The concentration required to inhibit 50% of the infection was calculated. The assay was performed in at least 2 independent experiments.

Toxicity

Luciferase assay

P4CCR5-luc cells were seeded at 10,000 cells/well (100ul) in 96-well opaque assay plates and incubated overnight at 37 ℃. The next day, media was aspirated from the plate and replaced with 200ul of a compound freshly diluted into the media at a 5-fold dilution from 100 μ M to 0.0062 μ M. After 4 days of incubation at 37 ℃, luciferase activity was measured in cell lysates using the Bright Glow kit from Promega and luminescence was measured using a Victor instrument from platinum-Elmer (Perkin-Elmer).

MTS assay

The human cell lines Huh 7 and HepG2 (liver), BxPC3 (pancreas) and CEM (lymph) were used for MTS assay in 96-well plates. Drugs freshly diluted in culture medium at 2 × 100 μ M, 50 μ M, 25 μ M, 10 μ M, 5 μ M, 1 μ M and 50 μ l were dispersed in triplicate in the plates. The wells at the periphery of the plate contained only 100ul of medium and were blank controls. Duplicate controls without drug were always performed in each plate. 50 μ l of cells were added to the plate, with 2000 cells per well for Huh 7, HepG2 and PxPC3, and 5000 cells per well for CEM cells. No cells were added to the periphery of the plate. The media used for Huh 7, HepG2 and BxPC3 cells was DMEM with 10% FBS, and penicillin/streptomycin, and for CEM cells was RPMI with 10% FBS, and penicillin/streptomycin. After 8 days of incubation at 37 ℃, 20 μ l of MTS dye from the CellTiter 96Aqueous single Solution Cell Proliferation Assay kit (CellTiter 96Aqueous One Solution Cell Proliferation Assay kit) from Promega was added to each well and the plates were incubated for 2h at 37 ℃. Absorbance was read at 490nm using a microplate reader E1800 from Biotek. The signal was calculated by subtracting the absorbance measured in the blank. Next, CC is determined by comparing the signal obtained with the drug-free cell control to the signal obtained with the treated cells and calculating the drug concentration required to inhibit 50% of the signal in the drug-treated wells₅₀(cytotoxic concentration) value.

Biological results

Antiviral activity

This application claims priority from U.S. provisional patent application 60/989,296 filed on 20/11/2007 and U.S. non-provisional patent application 12/271,388 filed on 14/11/2008, which are incorporated herein by reference in their entirety.

Claims

1. A compound of the formula:

or the use of a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of hepatitis B virus infection,

wherein

(a)R²Is C_1-4Alkyl optionally substituted with 1-3 fluorine atoms;

(f) the base is a naturally occurring or modified pyrimidine base represented by the following structure:

R⁵and R⁶Independently is H, OH, or NH₂。

2. The use of claim 1, wherein R²Is CH₃,CH₂F,CHF₂Or CF₃。

3. The use of claim 1, wherein the compound is

Or a pharmaceutically acceptable salt thereof.

4. The use of claim 3, wherein the medicament further comprises one or more antiviral agents, antibacterial agents, antiproliferative agents, or a combination thereof.

5. The use of claim 1, wherein the medicament further comprises one or more antiviral agents, antibacterial agents, antiproliferative agents, or a combination thereof.

6. The use of claim 1, wherein the compound is administered orally.

7. Use according to claim 6, wherein between 0.1g and 10g of the compound is administered daily in monotherapy or in combination therapy.

8. Use according to claim 7, wherein between 0.5g and 7.5g of the compound is administered per day.

9. Use according to claim 8, wherein between 1.5g and 6.0g of the compound is administered per day.

10. The use of claim 6, wherein the compound is administered at an initial loading dose followed by a reduced dose.