HK1124092A

HK1124092A - Modified 4'-nucleosides as antivral agents

Info

Publication number: HK1124092A
Application number: HK09103738.2A
Authority: HK
Inventors: 杜锦发; 菲利普‧弗曼; 迈克尔‧索菲亚
Original assignee: 法莫赛特股份有限公司
Priority date: 2005-09-26
Filing date: 2006-09-26
Publication date: 2009-07-03

Description

Modified 4' -nucleosides as antiviral agents

This application is filed as PCT international patent application on 26.9.2006, in the name of american national company PHARMASSET for all countries except the united states, and designated applicants only for the united states are dupuss, philips fmann, and michael soffit, all united states citizens. This application claims priority from U.S. provisional application No. 60/720,388 filed on 26/9/2005.

Technical Field

The present invention is in the field of medicinal chemistry, and in particular, compounds, methods and compositions for treating a host infected with human immunodeficiency virus (hereinafter "HIV"), hepatitis b virus (hereinafter "HBV"), or both HIV and HBV, comprising administering an effective amount of the described β -D-and β -L-4 '-C-substituted-3' -fluoro-and 3 '-azido-3' -deoxynucleosides, or pharmaceutically acceptable salts or prodrugs thereof.

Background

Acquired Immune Deficiency Syndrome (AIDS) was identified in 1981 as a disease that severely damages the human immune system, leading almost without exception to death. In 1983, the etiological cause of AIDS was determined to be HIV.

In 1985, it was reported that the synthetic nucleoside 3 '-azido-3' -deoxythymidine (AZT) inhibits HIV replication. Since then, many other synthetic nucleosides including 2 ', 3' -dideoxyinosine (DDI), 2 ', 3' -dideoxycytidine (DDC), and 2 ', 3' -dideoxy-2 ', 3' -didehydrothymidine (D4T) have been shown to be effective against HFV. After cellular phosphorylation to 5 '-triphosphate by cellular kinases, these synthetic nucleosides were incorporated into the growing viral DNA strand, causing chain termination due to the absence of the 3' -hydroxyl group. They may also inhibit the viral enzyme reverse transcriptase.

The success of different synthetic nucleosides to inhibit HIV replication in vivo or in vitro has led many researchers to design and test nucleosides in which a heteroatom is substituted at the 3' position of the nucleoside for a carbon atom (Norbeck et al, 1989, Tetrahedron Letters, 30(46)6246, european patent application publication No. 0337713, and U.S. patent No. 5,041,449).

U.S. Pat. No. 5,047,407 and European patent application publication No. 0382526, disclose a number of racemic 2 ' -substituted-5 ' -substituted-1, 3-oxathiolane (oxathiolane) nucleosides having antiviral activity, and specifically report that the racemic mixture of the C1 ' - β isomer of 2-hydroxymethyl-5- (cytosin-1-yl) -1, 3-oxathiolane (. + -.) -BCH-189) has approximately the same activity against HIV as AZT, and is not cytotoxic at the experimental level. It has also been found that (+ -) -BCH-189 inhibits replication of AZT-resistant HIV isolates in vitro from patients that have been treated with AZT for longer than 36 weeks. The (-) -enantiomer of the isomer of BCH-189, referred to as 3TC, is highly potent against HIV and shows little toxicity. (-) cis-2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1, 3-oxathiolane ("FTC") also has potent HIV activity (Schinazi et al, 1992 antibiotic. agent and Chemotherap (antimicrobials and chemotherapy), 2423. C2431).

Recently, it has been reported that 4' -C-substituted nucleosides show potent anti-HIV activity (Siddiqui, M.A. et al J.Med.Chem. (J.Med. chem.) 2004, 47, 5041-5048; Nomura, M. et al J.Med.chem. (J.Med. chem.) 1999, 42, 2901-2908).

Another virus that causes serious human health problems is HBV. HBV is second only to tobacco as a cause of human cancer. The mechanism by which HBV induces cancer is unknown, although it is postulated that it may directly trigger tumor development, or indirectly trigger tumor development through chronic inflammation, cirrhosis, and cellular regeneration associated with infection.

After a 2 to 6 month incubation period when the host is unaware of the infection, HBV infection can result in acute hepatitis and liver damage causing abdominal pain, jaundice, and elevated blood levels of certain enzymes. HBV can cause fulminant hepatitis, a rapidly progressive, often fatal form of disease in which most of the liver is destroyed.

In western industrialized countries, high risk groups for HBV infection include those in contact with HBV carriers or their blood samples. The epidemiology of HBV is very similar to that of acquired immunodeficiency syndrome, explaining why HBV infection is common in patients with AIDS or AIDS-related syndrome (complete). However, HBV is more infectious than HIV. FTC and 3TC both show activity against HBV (Furman et al 1992 Antimicrobial Agents and chemotherapy), 2686-2692).

Human serum derived vaccines have been developed for immunizing patients against HBV. Although it has been found to be effective, the production of vaccines is also cumbersome, because the supply of human serum from chronic carriers is limited, and the purification steps are long and expensive. In addition, each batch of vaccine prepared from a different serum must be tested in chimpanzees to ensure safety. Vaccines have also been produced by genetic engineering. Daily treatment with the genetically engineered protein interferon-alpha has also shown promise.

In light of the fact that acquired immunodeficiency syndrome, AIDS-related syndrome, and hepatitis b virus have reached epidemic levels worldwide and have catastrophic effects on infected patients, there is a strong need to provide new effective agents for treating these diseases and with low toxicity to the host.

Summary of The Invention

Disclosed are compounds, their syntheses, methods and compositions for treating a host infected with HIV, HBV, or both HIV and HBV, comprising administering an effective amount of the described beta-D-and beta-L-4 '-C-substituted-3' -fluoro-and 3 '-azido-3' -deoxynucleosides, or pharmaceutically acceptable salts or prodrugs thereof.

Brief Description of Drawings

FIG. 1 shows the chemical structure of modified 4' -nucleosides as antiviral agents.

FIG. 2 is 4 '-C-ethynyl-3' -fluorothymidine (Ia, R)¹＝F，R²＝OH，R³Ethynyl) or 4 '-C-ethynyl-3' -azidothymidine (Ia, R)¹＝N₃，R²＝OH，R³Ethynyl) is used.

FIG. 3 is 4 '-C-ethynyl-3' -fluoro-2 ', 3' -dideoxynucleosides (29, R)¹F) and 3 ' -azido-2 ', 3 ' -dideoxynucleosides (29, R)¹＝N₃) A non-limiting illustrative example of a synthesis of (a).

Detailed Description

The present invention relates to methods and compositions for treating HIV, HBV, or HIV and HBV infections in a host comprising administering an effective amount of the described β -D-and β -L-4 '-C-substituted 3' -fluoro-and 3 '-azido-3' -dideoxynucleosides or their pharmaceutically acceptable salts and prodrugs thereof.

More particularly, the first aspect of the invention relates to compounds, methods and compositions for treating infection with HIV, HBV, or both HIV and HBV, comprising administering an effective amount of the described β -D-and β -L-nucleosides of formulas I and II, or pharmaceutically acceptable salts or prodrugs thereof.

Wherein:

x is hydrogen, halogen (F, Cl, Br, I), NH₂、NHR⁴、NR⁴R⁵、NHOH、NHOR⁴、NHNH₂、NR⁴NH₂、NHNHR⁴、SH、SR⁴、S(O)R⁴、S(O)₂R⁴、OH、OR⁴、N₃CN, or CF₃；

Y is hydrogen, halogen (F, Cl, Br, I), NH₂、NHR⁴、NR⁴R⁵、NHOH、NHOR⁴、NHNH₂、NR⁴NH₂、NHNHR⁴、SH、SR⁴、S(O)R⁴、S(O)₂R⁴、OH、OR⁴、N₃、CN、CF₃Hydroxymethyl, methyl, optionally substituted or unsubstituted ethyl, optionally substituted or unsubstituted vinyl, optionally substituted or unsubstituted 2-bromovinyl, optionally substituted or unsubstituted ethynyl;

R¹is F or N₃；

R²Is OH, OR⁴、OC(O)R⁴、OP_vO_3vM_xR⁴ _yR⁵ _z、P_vO_3vM_xR⁴ _yR⁵ _z、OCH₂P_vO_3vM_xR⁴ _yR⁵ _z、OP(O)(OQ)_a(NHR⁴)_b、SH、SR⁴、S(O)R⁴、S(O)₂R⁴、SC(O)R⁴、NH₂、NHC(O)R⁴、NHR⁴、NR⁴R⁵、NHOH、NHOR⁴、NHNH₂、NR⁴NH₂Or NHNHR⁴；

R³Is F, cyano, azido, ethynyl, chloroethenyl, fluorovinyl, alkyl (C)_1-6)1 to 3 halogen-substituted alkyl (C)_1-6) Alkenyl (C)_1-6) Or alkynyl (C)_1-6) Provided that when R is¹Is N₃When R is³Is not hydroxymethyl;

z is O, S, CH₂Or C ═ CH₂；

A is N, CH, or CF; and

R⁴and R⁵Are the same or different and are lower alkyl, lower alkenyl, acyl of 1 to 17 carbons, aryl, or aralkyl, such as unsubstituted or substituted phenyl or benzyl;

m is selected from the group consisting of H⁺、Na⁺And K⁺At least one member of the group;

v has a value of 1, 2, or 3;

x, y, and z are independent of each other and have a value of 0, 1, 2, 3, or 4; and a has a value of 0 or 1, b has a value of 1 or 2, and Q is M or R⁴。

A second aspect of the invention relates to an intermediate having the structure:

wherein

X is hydrogen, F, Cl, Br, I, NH₂、NHR⁴、NR⁴R⁵、NHOH、NHOR⁴、NHNH₂、NR⁴NH₂、NHNHR⁴、SH、SR⁴、S(O)R⁴、S(O)₂R⁴、OH、OR⁴、N₃CN, or CF₃；

Y is hydrogen, F, Cl, Br, I, NH₂、NHR⁴、NR⁴R⁵、NHOH、NHOR⁴、NHNH₂、NR⁴NH₂、NHNHR⁴、SH、SR⁴、S(O)R⁴、S(O)₂R⁴、OH、OR⁴、N₃、CN、CF₃Hydroxymethyl, methyl, optionally substituted or unsubstituted ethyl, optionally substituted or unsubstituted vinyl, optionally substituted or unsubstituted 2-bromovinyl, optionally substituted or unsubstituted ethynyl;

pg is a hydroxyl protecting group including, but not limited to, trityl, dimethoxytrityl, and tert-butyl-silyl;

l is a leaving group including, but not limited to, sulfonyl, trifluorosulfonyl, unsubstituted sulfonate, substituted sulfonate, unsubstituted carbonate, substituted carbonate; and

R⁴and R⁵Are the same or different and are lower alkyl, lower alkenyl, acyl of 1 to 17 carbons, aryl, or aralkyl.

A third aspect of the present invention relates to a process for the preparation of an intermediate disclosed in the second aspect of the present invention, said process comprising:

(a) the method comprises the following steps Selectively protecting the 5 '-OH with a protecting group Rg to form a 5' -OPg group;

(b) the method comprises the following steps Activating the 3 '-OH with a leaving group L to form a 3' -OL group;

(c) the method comprises the following steps Reacting the 3 '-C with a hydroxide base to convert the 3' -C position from a ribose configuration to a xylose configuration;

(d) the method comprises the following steps Activating the 3 '-OH having xylose configuration (xylo-configuration) with a leaving group L to form a 3' -OL group;

wherein the hydroxide base includes, but is not limited to NaOH, KOH, and R⁴ ₄NOH and mixtures thereof.

A fourth aspect of the invention relates to an intermediate represented by the formula:

wherein

pg is a hydroxyl protecting group including, but not limited to, trityl, dimethoxytrityl, and tert-butyl-silyl; and

A fifth aspect of the present invention relates to a process for the preparation of an intermediate disclosed in the fourth aspect of the present invention, said process comprising:

(a) the method comprises the following steps Activating the 3 '-OH of the 5' -O-protected nucleoside with a leaving group L to form a 3 '-OL-5' -O-protected nucleoside group; followed by

(b) The method comprises the following steps Treating the 3 '-OL-5' -O-protected nucleoside with DBU (1, 8-diazabicyclo [5.4.0] undec-7-ene) or DBN (1, 5-diazabicyclo [4.3.0] non-5-ene); so as to obtain said intermediate;

wherein L includes, but is not limited to, sulfonyl, trifluorosulfonyl, substituted sulfonate, and unsubstituted sulfonate, unsubstituted carbonate, and substituted carbonate.

Various embodiments of the present invention will now be described in detail. As used in the specification herein and throughout the claims that follow, the meaning of "a", "an", and "the" includes plural references unless the context clearly dictates otherwise. Also, as used in the specification herein and throughout the claims that follow, the meaning of "in.

The terms used in this specification generally have their ordinary meaning in the art, within the context of the present invention, and in the specific context in which each term is used. Certain terms used in describing the present invention are discussed below or otherwise in the specification to provide additional guidance to the practitioner and how to make and use them in describing the compositions and methods of the present invention. For convenience, certain terms may be emphasized, such as with italics and/or reference numerals. The use of emphasis has no effect on the scope and meaning of the term; the scope and meaning of a term is the same in the same context, regardless of whether emphasis is placed thereon. It should be understood that the same thing can be expressed in more than one way. Thus, alternative terms and synonyms may be used for any one or more of the terms discussed herein, nor is any special meaning set forth, whether or not a term is set forth or discussed herein. Synonyms for certain terms are provided. Recitation of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only and in no way limits the scope and meaning of the invention or any of the described terms. Likewise, the invention is not limited to the different embodiments given in this description.

As used herein, "about" or "approximately" shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the terms "about" or "approximately" can be inferred, unless expressly stated otherwise.

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

The compounds and their pharmaceutically acceptable derivatives or pharmaceutical dosage forms containing the compounds or their derivatives are also useful in the prevention and treatment of HBV infection and other related disorders such as anti-HBV antibody-positive and HBV-positive disorders, chronic hepatitis caused by HBV, liver cirrhosis, acute hepatitis, fulminant hepatitis, chronic stable hepatitis, and fatigue. These compounds or dosage forms may also be used prophylactically to prevent or delay the progression of clinical disease in an individual who is anti-HBV antibody or HBV-antigen positive or has been exposed to HBV.

The compounds may be converted into pharmaceutically acceptable esters by reaction with a suitable esterifying agent, for example an acid halide or anhydride. The compounds or their pharmaceutically acceptable derivatives may be converted into their pharmaceutically acceptable salts in a conventional manner, for example by treatment with a suitable base. The esters or salts of the compounds may be converted to the parent compound, for example, by hydrolysis.

The term "independently" is used herein to indicate that independently applicable variables vary independently of the respective application. Thus, in compounds such as R^aXYR^aIn which R is^aIs "independent carbon or nitrogen" two R^aMay be carbon, two R^aMay be nitrogen, or one R^aMay be carbon or another R^aIs nitrogen.

As used herein, the term "enantiomerically pure" refers to a nucleoside composition that includes at least approximately 95%, and preferably approximately 97%, 98%, 99%, or 100% of a single enantiomer of the nucleoside.

As used herein, the term "substantially free of or" substantially free of "refers to a nucleoside composition that includes at least 85 or 90% by weight, preferably 95% to 98% by weight, and even more preferably 99% to 100% by weight of the designated enantiomer of the nucleoside. In a preferred embodiment, in the methods and compounds of the present invention, the compounds are substantially free of the unspecified enantiomer of the nucleoside.

Similarly, the term "isolated" refers to a nucleoside composition comprising at least 85 or 90% by weight, preferably from 95% to 98% by weight, and even more preferably from 99% to 100% by weight of the nucleoside, the remainder comprising other chemical species or enantiomers.

The term "alkyl" as used herein, unless otherwise specified, refers to a saturated straight, branched, or cyclic, typically C₁To C₁₀And specifically includes methyl, trifluoromethyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2-dimethylbutyl, and 2, 3-dimethylbutyl. The term includes both substituted and unsubstituted alkyl groups. Alkyl Groups which may optionally be substituted with one or more moieties selected from the group consisting of hydroxy, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, or any other feasible functional group which does not inhibit the pharmacological activity of the present compounds, are either unprotected, or protected as desired, as known to those skilled in the art, for example, as taught in Greene et al 1991, Protective Groups in organic Synthesis, John Wiley Sons, second edition, incorporated herein by reference.

As used herein, unless otherwise indicated, the term "lower alkyl" refers to C₁To C₄Saturated linear, branched, or, if appropriate, cyclic (e.g., cyclopropyl) alkyl, including substituted and unsubstituted forms. Lower alkyl is preferred when alkyl is a suitable moiety, unless specifically stated otherwise in the application. Similarly, when alkyl or lower alkyl is a suitable moiety, unsubstituted alkyl or lower alkyl is preferred.

The term "lower chain" as used hereinAlkenyl ", and unless otherwise specified, refers to C₂To C₄Unsaturated, linear or branched alkenyl groups, including substituted and unsubstituted forms. When alkenyl is a suitable moiety, lower alkenyl is preferred, unless specifically stated otherwise in the application. Similarly, when alkenyl or lower alkenyl is a suitable moiety, unsubstituted alkenyl or lower alkenyl groups are preferred.

The term "alkylamino" or "arylamino" refers to an amino group having one or two alkyl or aryl substituents, respectively.

The term "protected" as used herein and unless otherwise defined, refers to a group 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.

The term "aryl" as used herein, and unless otherwise specified, refers to phenyl, biphenyl, or naphthyl, and preferably phenyl. The term includes both substituted and unsubstituted moieties. The aryl group may be substituted with one or more moieties selected from the group consisting of hydroxy, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, as desired, either unprotected or protected, as known to those skilled in the art, for example, as taught in Greene et al 1991, Protective Groups in organic Synthesis, John Wiley Sons, 2 nd edition.

The term "alkaryl" or "alkylaryl" refers to an alkyl group with an aryl substituent. The term "aralkyl" or "arylalkyl" refers to an aryl group having an alkyl substituent.

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

The term "acyl" refers to carboxylic acid esters in which the non-carbonyl portion of the ester group is selected from the group consisting of:linear, branched, or cyclic alkyl or lower alkyl, alkoxyalkyl including methoxymethyl, aralkyl including benzyl, aryloxyalkyl such as phenoxymethyl, aryl including phenyl optionally substituted with halogen (F, Cl, Br, I), C₁To C₄Alkyl or C₁To C₄Alkoxy groups, including sulfonic acid esters of methanesulfonyl such as alkyl or aralkyl sulfonyl, mono-, di-or triphosphate, trityl or monomethoxytrityl, substituted benzyl, trialkylsilyl (e.g. dimethyl-tert-butylsilyl) or diphenylmethylsilyl. The aryl group of the ester preferably comprises a phenyl group.

The term "lower acyl" refers to acyl groups wherein the non-carbonyl moiety is lower alkyl.

The term "host" as used herein refers to a unicellular or multicellular organism in which a virus can replicate, including cell lines and animals, and is preferably a human. 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, in particular, primates and humans. In most animal applications of the invention, the host is a human patient. In certain indications, however, veterinary applications are expressly contemplated by the present invention.

The term "pharmaceutically acceptable salt or prodrug" is used throughout the specification and claims to describe any pharmaceutically acceptable form of the compound (such as an ester, phosphate ester, salt of an ester or related group) which provides the active compound when administered to a patient. Pharmaceutically acceptable salts include those obtained from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among many other acids well known in the pharmaceutical art. A pharmaceutically acceptable prodrug refers to a compound that is metabolized, e.g., hydrolyzed or oxidized, in the host to form a compound of the invention. Typical examples of prodrugs include compounds having biologically labile protecting groups on functional moieties of the active 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.

1. Non-limiting examples of the synthesis of 4 ' -c-ethynyl-3 ' -fluoro-and 3 ' -azidothymidine (see FIG. 2)

Treatment of thymidine with 2.2-2.5 moles of tert-butyldimethylsilyl chloride in dichloromethane in the presence of imidazole, followed by selective deprotection of the 5' -O-silyl group in 80% acetic acid in the presence of trifluoroacetic acid, gives Compound 2. After purification by silica gel column chromatography in excellent yield, 2 was oxidized in DMSO with DCC in the presence of pyridine trifluoroacetate to give aldehyde 3. Treatment of Compound 3 with aqueous Formaldehyde in the Presence of 2N NaOH in a mixture of 1, 4-dioxane and Water, followed by NaBH₄The resulting intermediate is reduced to provide diol 4. Diol 4 was selectively protected with dimethoxytrityl chloride in pyridine to give compound 5. Compound 5 was treated with tert-butyldiphenylsilyl in dichloromethane in the presence of imidazole, followed by detritylation in 80% acetic acid to give compound 6. Oxidation of alcohol 6 with DCC in DMSO in the presence of pyridine trifluoroacetate affords compound 7. Reaction of compound 7 with chloromethylene Wittig reagent followed by elimination by treatment with butyllithium gives 4' -C-ethynylnucleoside 8. Treatment of 8 with tetrabutylammonium fluoride in THF afforded 4' -C-ethynyl-thymidine 9. Treatment of 9 with DMTrCl in pyridine affords compound 10. Compound 10 was converted to 11 by treatment with MsCl in EtOH followed by NaOH. Compound 11 was treated with DAST in the presence of pyridine at reflux temperature in dichloromethane to afford 3' -fluoronucleoside (12, X ═ F). By using methanesulfonyl chloride in dichloromethane in the presence of triethylamine followed by NaN in DMF₃Treatment of 11 to give a 3' -azidonucleoside (12, X ═ N)₃). The final product, 4' -C-ethynyl-FLT (Ia, R) was obtained by treating 12 with 80% acetic acid¹＝F，R²＝OH，R³Ethynyl) and 4' -C-ethynyl-AZT (Ia, R)¹＝N₃，R²＝OH，R³Ethynyl).

Alternatively, reaction of 10 with MsCl in the presence of a base such as triethylamine followed by treatment of the resulting methanesulfonic acid with a base such as DBU or DBN gives intermediate 11'. With NaN₃Or tetrabutylammonium fluoride (TBAF) treatment 11' also affords the same intermediate 12, respectively, X ═ N₃Or X ═ F, as disclosed in Maillard, m. et al Tetrahedron Lett (tetrahedral communication) 1989, 30, 1955-. By way of example, the inventors do not wish to be limited to thymidine as described above, and by reference to US6,949,522; US6,403,568; and US 2005/0009737, each of which discloses examples of contemplated purines or pyrimidines.

2. Non-limiting examples of the synthesis of 4 ' -C-ethynyl 3 ' -fluoro-and 3 ' -azido-2 ', 3 ' -dideoxynucleosides (see FIG. 3)

Compound 13 is treated with t-butyldimethylsilyl chloride in dichloromethane in the presence of imidazole, followed by removal of the chlorobenzoyl protecting group with methanolic ammonia to afford compound 15. After purification by silica gel column chromatography, compound 15 was oxidized with DCC in DMSO in the presence of pyridine trifluoroacetate to afford aldehyde 16. Treatment of compound 16 with aqueous formaldehyde in the presence of 2N NaOH in a mixture of 1, 4-dioxane and water, followed by NaBH₄The resulting intermediate is reduced to provide diol 17. Selective protection with DMTCl followed by oxidation with DCC in DMSO in the presence of pyridine trifluoroacetate gives the aldehyde 19. Reaction of 19 with chloromethylene Wittig reagent followed by elimination in the presence of butyllithium affords 4' -C-ethynyl-furanoside 20. The tetraacetate 21 is obtained by decomposition 20 with acetic anhydride in acetic acid in the presence of concentrated sulfuric acid. With a silylating base such as TMSOTf or SnCl in the presence of a Lewis acid₄Followed by deprotection with methanolic ammonia to afford 4' -C-ethynyl-xylofuranosyl) -nucleoside 23. Compound 23 is treated with acetone in the presence of catalytic amounts of HCl to afford compound 24. Compound 24 is subjected to a Barton deoxygenation to produce 2' -deoxynucleoside 25. 25 Deisopropenylation with 80% acetic acid followed by BzCl in pyridineSelectively protected to provide nucleoside 27. Treatment of compound 27 with DAST in dichloromethane at reflux temperature, followed by methanol ammonia deprotection affords the final 4' -C-ethynyl-nucleoside (29, R)¹F). Treatment of 27 with methanesulfonyl chloride in dichloromethane in the presence of triethylamine followed by reaction of the resulting methanesulfonic acid with NaN₃Reaction in DMF to give 4' -C-ethynyl-nucleoside (29, R)¹＝N₃)。

The synthetic schemes described above provide the following contemplated compounds, including, but not limited to: 4 '-C-substituted-3' -fluoro-2 ', 3' -dideoxynucleosides, 4 '-C-substituted-3' -azido-2 ', 3' -dideoxynucleosides, 4 '-C-ethynyl-3' -fluoro-2 ', 3' -dideoxynucleosides, 4 '-C-ethynyl-3' -azido-2 ', 3' -dideoxynucleosides, 4 '-C-ethynyl-3' -fluoro-3 '-deoxythymidine, and 4' -C-ethynyl-3 '-azido-3' -deoxythymidine.

The antiviral activity of the nucleoside can be administered as a direct or indirect provision of the parent compound or any derivative which itself exhibits activity when administered to the host recipient. Non-limiting examples include pharmaceutically acceptable salts (alternatively referred to as "physiologically acceptable salts") and prodrugs.

Modification of active Compounds, in particular in N⁴And the 5' -O position, can affect the bioavailability and metabolic rate of the active, thus providing control over the delivery of the active. In addition, such modifications may affect the antiviral activity of the compound, sometimes by increasing the activity on the parent compound. This can be readily assessed by preparing the derivative according to the methods described herein and testing it for antiviral activity, or other methods known to those skilled in the art.

The inventors of the present application also contemplate the use of an antivirally effective amount of any of the compounds disclosed herein, or a pharmaceutically acceptable salt or prodrug thereof.

Pharmaceutically acceptable salts and prodrugs

The term "pharmaceutically acceptable salt or prodrug" is used throughout the specification and claims to describe any pharmaceutically acceptable form of the compound (such as an ester, phosphate ester, salt of an ester or related group) which provides the active compound when administered to a patient.

Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Administration of a compound as a pharmaceutically acceptable salt may be appropriate if the compound is sufficiently basic or acidic to form a stable, non-toxic acid or base salt. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among many other acids well known in the pharmaceutical art. In particular, examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form physiologically acceptable anions, such as, but not limited to, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, α -ketoglutarate, and α -glycerophosphate. Suitable inorganic salts may also be formed, including sulfates, nitrates, bicarbonates, and carbonates.

The pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example by reacting a sufficient 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) salts of carboxylic acids can also be made.

A pharmaceutically acceptable prodrug refers to a compound that is metabolized in the host to form a compound of the invention. Typical examples of prodrugs include compounds having biologically labile protecting groups on functional moieties of the active compounds. Prodrugs include compounds that may be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrated, alkylated, dealkylated, acylated, deacylated, phosphorylated, and/or dephosphorylated to yield the active compound.

Any of the nucleosides described herein can be administered as nucleotide prodrugs to increase the activity, bioavailability, stability, or otherwise alter the properties of the nucleoside. Typically, alkylation, acylation, or other lipophilic modification of the mono-, di-, or triphosphate of the nucleoside will increase the stability of the nucleotide. Examples of substituent groups that may replace one or more hydrogens on the phosphate moiety are alkyl groups, aryl groups, steroids, sugars including sugars, 1, 2-diacylglycerol and alcohols. Many are described in Antiviral Research, 27(1995)1-17, by r.jones and n.bischofberger. Any of these can be combined with the disclosed nucleosides to achieve the desired effect.

In various embodiments, prodrugs of the nucleoside derivatives described herein, wherein R is¹Is F or N₃Involving carbon (R) at 5²) Substituted with the following groups: OH, OR⁴、OC(O)R⁴、OP_vO_3vM_xR⁴ _yR⁵ _z、P_vO_3vM_xR⁴ _yR⁵ _z、OCH₂P_vO_3vM_xR⁴ _yR⁵ _z、OP(O)(OQ)_a(NHR⁴)_b、SH、SR⁴、SC(O)R⁴、NH₂、NHC(O)R⁴、NHR⁴、NR⁴R⁵、NHOH、NHOR⁴、NHNH₂、NR⁴NH₂Or NHNHR⁴。R⁴And R⁵Are the same or different and are lower alkyl, lower alkenyl, acyl of 1 to 17 carbons, aryl, or aralkyl, such as unsubstituted or substituted phenyl or benzyl; m is selected from the group consisting of H⁺、Na⁺And K⁺A member of the group; v has a value of 1, 2, or 3; x, y, and z are independent of each other and have a value of 0, 1, 2, 3, or 4; and a has a value of 0 or 1, b has a value of 1 or 2, and Q is M or R⁴. The inventors understand that one of ordinary skill in the art should recognize that for the phosphates and phosphonates represented above, when v is 1, the sum of x, y and z is 2; when v is 2, the sum of x, y and z is 3; and when v is 3, the sum of x, y and z is 4.

Phosphoric acidSalt or ester (OP)_vO_3vM_xR⁴ _yR⁵ _z) Mono- (v ═ 1), di- (v ═ 2), and tri-phosphates (v ═ 3) are included in acid, salt, or ester forms, including combinations thereof. In the case of v ═ 2, the nucleoside is substituted at the 5' -C position with R having the following structure²And (3) substitution: OP (optical fiber)₂O₆M_xR⁴ _yR⁵ _zWherein x, y and z have the meaning as defined above. Those of ordinary skill in the art will appreciate that the pure acid form consists of (OP)₂O₆H₃) Is represented by (A); pure salt form of (OP)₂O₆M₃，M＝Na⁺，K⁺Or Na⁺And K⁺Both) are represented by; and in the pure ester form from (OP)₂O₆R⁴ _yR⁵ _zWherein, as indicated above, R⁴And R⁵May be the same or different and if different, the sum of y and z does not exceed 3). Of course, it is also contemplated that the phosphate salts or esters may be in mixed form. It will be understood from the mixed forms that the phosphate moiety may be an acid (when M ═ H)⁺) When M ═ Na, salt (when M ═ Na)⁺Or K⁺(ii) a Or even Ca²⁺) When, or an ester (wherein R is⁴And R⁵Either or both of y and z of (a) have a non-zero value). Without being limited by way of example, the following structures represent preferred examples of contemplated phosphates: OPO₃H₂、OP₂O₆H₃、OP₃O₉H₄、OPO₃Na₂、OPO₃R⁴R⁵、OP₂O₆Na₃、OP₂O₆R⁴ ₂R⁵、OP₃O₉Na₄、OP₃O₉R⁴ ₃R⁵、PO₃H₂、P₂O₆H₃、P₃O₉H₄、PO₃Na₂。

Expected R⁴、R⁵Or R is⁴And R⁵Both may have the following formula: r⁶C(O)OR⁷Wherein R is⁶Is alkyl, such as lower alkyl, and R⁷Is lower alkylene (such as methylene, ethylene, propylene, and butylene, which may be unsubstituted or substituted (with hydroxyalkyl, alkoxyalkyl, or haloalkyl), provided that R⁷Oxygen bonded to the phospholipid. Without being limited by example, it is contemplated that the nucleoside is substituted at the 5' -C position with a moiety having the structure: OP (O) [ OCH ]₂OC(O)C(CH₃)₃]₂。

5' -C position and moiety (P)_vO_3vM_xR⁴ _yR⁵ _z) The combination of P of (a) results in a mono- (v ═ 1), di- (v ═ 2), or tri-phosphonate (v ═ 3) in acid, salt, or ester form, including combinations thereof. In the case of v ═ 1, the nucleoside is substituted at the 5' -C Position (PO)₃M_xR⁴ _yR⁵ _z) R of²And (4) substitution. Those of ordinary skill in the art will appreciate that the pure acid form is composed of (PO)₃H₂) Represents; pure salt form of (OPO)₃M₂，M＝Na⁺，K⁺Or Na⁺And K⁺Both) are represented by; and in the form of a pure ester of (OPO)₃R⁴ _yR⁵ _zWherein, as indicated above, R⁴And R⁵May be the same or different and if different, the sum of y and z does not exceed 2). Of course, it is also contemplated that the phosphonate may be in mixed form. It will be understood from the mixed forms that the phosphate moiety may be an acid (when M ═ H)⁺) When M ═ Na, salt (when M ═ Na)⁺Or K⁺(ii) a Or even Ca²⁺) When, or an ester (wherein R is⁴And R⁵Either or both of y and z of (a) have a non-zero value). Not to be limited by the examples, R²The following preferred examples of substituents yield the desired phosphonate salts or esters: PO (PO)₃H₂，P₂O₆H₃，P₃O₉H₄，PO₃Na₂，P₂O₆Na₃，P₃O₉Na₄，PO₃R⁴R⁵，P₂O₆R⁴ ₂R⁵，P₃O₉R⁴ ₃R⁵。

In addition, the inventors contemplate the use of phosphoramidates (OP (O) (OQ) at the 5' carbon_a(NHR⁴)_b) Prodrugs of substituted nucleoside derivatives, wherein a has a value of 0 or 1, b has a value of 1 or 2, and Q is M or R⁴。

Active nucleosides are also provided as 5 '-phosphoether lipids or 5' -ether lipids, as disclosed in the following references, which are incorporated herein by reference: kucera, l.s., et al 1990.AIDS Rex hum.retro viruses.6: 491-; pinagadosi, g., et al, 1991.j.med.chem. (journal of medical chemistry) 34: 1408.1414, respectively; hosteller, k.y., et al.1992, anim. ingredients chemither.36: 2025.2029, respectively; hosetler, k.y., et al, 1990, J biol.chem. (journal of biochemistry) 265: 61127.

non-limiting examples of U.S. patents disclosing suitable lipophilic substituents that may be covalently bound to a nucleoside, preferably at the 5' -OH position of the nucleoside or lipophilic agent, include U.S. patent nos. 5,149,794; 5,194,654, respectively; 5,223,263, respectively; 5,256,641, respectively; 5,411,947, respectively; 5,463,092, respectively; 5,543,389, respectively; 5,543,390, respectively; 5,543,391, respectively; and 5,554,728, which are incorporated herein by reference in their entirety. Foreign patent applications disclosing lipophilic substituents which may be incorporated into the nucleosides of the present invention include WO89/02733, WO 90100555, WO 91/16920, WO 91/18914, WO 93/00910, WO94/26273, WO 96/15132, EP 0350287, EP 93917054.4, and WO 91/19721.

Pharmaceutical composition

Pharmaceutical compositions based on nucleoside compounds of formulae (I) and (II) or pharmaceutically acceptable salts or prodrugs thereof can be prepared in therapeutically effective amounts for the treatment of HBV or HIV viral infection or abnormal cell proliferation, optionally in combination with pharmaceutically acceptable additives, carriers or excipients. The therapeutically effective amount may vary with the infection or disorder to be treated, its severity, the treatment plan to be used, the pharmacokinetics of the agent used and the patient being treated.

In one aspect according to the present invention, the compound is preferably formulated in admixture with a pharmaceutically acceptable carrier. In general, it is preferred to administer the pharmaceutical composition in an orally administrable form, but dosage forms may be administered via parenteral, intravenous, intramuscular, transdermal, buccal, subcutaneous, suppository or other routes. Intravenous and intramuscular dosage forms are preferably administered in sterile saline. One of ordinary skill in the art can modify the dosage form within the teachings of the specification to provide a number of dosage forms for a particular route of administration without destabilizing the composition of the invention or compromising its therapeutic activity. In particular, for example, modifications to render the desired compound more soluble in water or other carriers can be readily accomplished by routine modification (salt dosage forms, esterification, etc.).

In certain pharmaceutical dosage forms, prodrug forms of the compounds, especially including acylated (acetylated or otherwise) and ether derivatives, phosphate esters and various salt forms of the present compounds, are preferred. One of ordinary skill in the art will understand how to readily modify the present compounds into prodrug forms to facilitate delivery of the active compounds to the target site within the host organism or patient. Where applicable, the skilled artisan will also utilize favorable pharmacokinetic parameters of the prodrug form in delivering the desired compound to a target site within a host organism or patient to maximize the intended effect of the compound in treating HBV and HIV viral infections.

According to the present invention, the amount of compound included in the therapeutically active dosage form is an effective amount for the treatment of an infection or disorder, which in a preferred embodiment is an HBV or HIV viral infection. Generally, depending on the compound used, the condition or infection being treated, and the route of administration, a therapeutically effective amount of the present compound in a pharmaceutical dosage form will generally be in the range of from about 0.1mg/kg to about 100mg/kg or more, and all values and subranges therebetween. For the purposes of the present invention, according to the present invention, a prophylactically or prophylactically effective amount of the composition is within the same concentration range as described above for the therapeutically effective amount and is generally the same as the therapeutically effective amount.

Administration of the active compound can range from continuous (intravenous drip) to oral (e.g., q.i.d., b.i.d., etc.) several times a day and can include oral, topical, parenteral, intramuscular, intravenous, subcutaneous, transdermal (which can include penetration enhancing agents), buccal and suppository administration, as well as other routes of administration. Enteric-coated oral tablets may also be used to enhance the bioavailability and stability of the compounds from the oral route of administration. The most effective dosage form will depend on the pharmacokinetics of the particular agent chosen and the severity of the disease in the patient. Oral dosage forms are particularly preferred because of the ease of administration and the expected favorable patient compliance.

To prepare the pharmaceutical compositions according to the invention, a therapeutically effective amount of one or more compounds according to the invention is preferably admixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compound technology to produce a dosage. Depending on the form of the preparation for administration, e.g. oral or parenteral, the carrier may take a wide variety of forms. In preparing pharmaceutical compositions in oral dosage form, any of the usual pharmaceutical media may be employed. Thus, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives including water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like may be used. For solid oral preparations such as powders, tablets, capsules, and for solid preparations such as suppositories, suitable carriers and additives including starch, sugar carriers such as dextrose, mannitol, lactose and related carriers, diluents, granulating agents, lubricants, binders, disintegrating agents and the like may be used. The tablets or capsules may be enteric coated for sustained release, if desired, by standard techniques. The use of these dosage forms can significantly affect the bioavailability of the compound in the patient.

For parenteral dosage forms, the carrier will typically comprise sterile water or aqueous sodium chloride solution, and may still include other ingredients, including those to aid dispersion. In the case of sterile water and maintained sterile, the compositions and carriers must also be sterile. Injectable suspensions may also be prepared, and in the case of suitable liquid carriers, suspending agents and the like may be employed.

Liposomal suspensions (including liposomes targeted to viral antigens) can also be prepared by conventional methods to produce pharmaceutically acceptable carriers. This may be suitable for delivering free nucleoside, acyl nucleoside or phosphate ester prodrug forms of the nucleoside compounds according to the invention.

In addition, the compounds according to the present invention may be administered in combination or alternation with one or more antiviral, anti-HBV, anti-HIV or interferon, antibacterial agents including other compounds of the present invention. Certain compounds according to the invention may be effective for increasing the biological activity of certain agents according to the invention by reducing the metabolism, catabolism or inactivation of other compounds, and thus be administered in combination for this intended effect.

Combination or alternation therapy

In another embodiment, the active compound or its derivative or salt may be administered in combination or alternation with another antiviral agent for the purpose of treating, inhibiting, preventing and/or preventing a viral infection. Typically, in combination therapy, an effective dose of two or more agents is administered together, whereas during alternating therapy, an effective dose of each agent is administered sequentially. The dosage will depend on the absorption, inactivation, and excretion rates of the drug, as well as other factors known to those skilled in the art. It should be noted that dosage values will also vary with the severity of the condition to be alleviated. It is also understood that for any particular subject, the specific dosing regimen and schedule should be adjusted over time according to the individual needs and the professional judgment of the person administering or monitoring the administration of the composition.

Non-limiting examples of antiviral agents that may be used in combination with the compounds disclosed herein include, but are not limited to, Acyclovir (ACV), ganciclovir (GCV or DHPG) and its prodrugs (e.g., valyl-ganciclovir), E-5- (2-bromovinyl) -2' -deoxyuridine (BVDU), (E) -5-vinyl-1- β -D-arabinosyluracil (arabonosyluracil) (VaraU), (E) -5- (2-bromovinyl) -1- β -D-arabinosyluracil (arabinosyluracil) (BV-araU), 1- (2-deoxy-2-fluoro- β -D-arabinosyluracil (arabinosyl)) -5-iodocytosine (D-FIAC) ("D-FIAC)), 1- (2-deoxy-2-fluoro- β -L-arabinosyl) -5-methyluracil (L-FMAU), (S) -9- (3-hydroxy-2-phosphonomethoxypropyl) adenine [ (S) -HPMPA ], (S) -9- (3-hydroxy-2-phosphonomethoxypropyl) -2, 6-diaminopurine [ OS) -HPMPDAP ], (S) -1- (3-hydroxy-2-phosphonomethoxypropyl) cytosine [ (S) -HPMPDAP, or cidofovir (cidofivir) ], and (2S, 4S) -1- [2- (hydroxymethyl) -1, 3-dioxolan-4-yl ] -5-iodouracil (L-5-IoddU), FTC, entecavir, interferon- α -, pegylated interferon- α, lamivudine (3TC), LdT (or its prodrug), LdC (or its prodrug), and adefovir, protease inhibitors (agenase, indinavir sulfate, saquinavir soft gel, saquinavir mesylate (Invirase), Kaletra, Lexiva, norvir, Reyataz, Aptivus, and nelfinavir), and non-nucleoside reverse transcriptase inhibitors (delavirdine mesylate, efavirenz (Sustiva), and nevirapine).

Non-limiting examples of additional antiviral agents that may be used in combination with the compounds disclosed herein include, but are not limited to, the (-) -enantiomer of 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1, 3-oxathiohydroxyalkyl [ (-) -FTC); the (-) -enantiomer of 2-hydroxymethyl-5- (cytosin-1-yl) -1, 3-oxathioxohydroxyalkane (3 TC); carbomer, acyclovir, interferon, famciclovir, penciclovir, AZT, DDI, DDC, L- (-) -FMAU, and D4T.

Without wishing to limit the scope of the invention, exemplary methods according to embodiments of the invention and their associated results are given below. Note that for the convenience of the reader, titles or subtitles may be used in the embodiments, which in no way should limit the scope of the invention. Moreover, certain theories are proposed and disclosed herein; however, they should in no way limit the scope of the invention, whether they are correct or incorrect, as long as the data is processed, sampled, transformed, etc. according to the invention, without any particular theory or scheme of action being considered.

Examples

Example 1.preparation of 4' -C-ethynylthymidine.

4' -C-ethynylthymidine was prepared according to literature procedures. (Nomura, M et al J. Med. chem. (J. Med. chem.) 1999, 42, 2901-.

Example 2.preparation of 4 '-C-ethynyl-5' -O- (dimethoxytrityl) thymidine (10, FIG. 2).

To a solution (1mmol) of 4' -C-ethynylthymidine in pyridine (10ml) was added dimethoxytrityl chloride (1.2mmol) at 0 ℃ and the resulting solution was stirred at room temperature for 3 h. EtOAc (100mL) is added and the solution is washed with water and dried (Na)₂SO₄). The solvent was evaporated to dryness under reduced pressure. The residue was coevaporated with toluene (2 × 20mL) and purified by silica gel column chromatography (5% MeOH in dichloromethane) to give 4 '-C-ethynyl-5' -O- (dimethoxytrityl) thymidine (10).

Example 3.preparation of 4 '-C-ethynyl-5' -O- (dimethoxytrityl) -2, 3 '-anhydrothymidine (11', FIG. 2).

To a solution of 10 (1mmol) in dichloromethane (20mL) was added triethylamine (1mL) and methanesulfonyl chloride (1.2mmol) and the solution was stirred at room temperature for 16 h. EtOAc (50mL) is added and the mixture is washed with water and dried (Na)₂SO₄). The solvent was removed and the residue was dissolved in anhydrous tetrahydrofuran (THF, 20 mL). DBU (3mmol) was added to the solution and the resulting solution was refluxed for 16 h. The solution was diluted with EtOAc (50mL) and washed with brine. The organic solution was dried (Na)₂SO₄) Removing the solvent and column-coloring the residue with silica gelPurification by chromatography (2% MeOH in dichloromethane) afforded compound 11'.

Example 4.4 '-C-ethynyl-5' -O- (dimethoxytrityl) -3 '-azido-3' -deoxythymidine (12, X ═ N)₃FIG. 2).

To a solution of 11' (1mmol) in anhydrous DMF (10mL) was added NaN₃(3mmol) and the mixture was stirred at 100 ℃ for 16 h. The solvent was evaporated to dryness under reduced pressure. The residue was coevaporated with toluene (2 × 20mL) and purified by silica gel column chromatography (20-50% EtOAc in hexanes) to give 4 '-C-ethynyl-5' -O- (dimethoxytrityl) -3 '-azido-3' -deoxythymidine (12, X ═ N)₃)。

Example 5.4 ' -C-ethynyl-3 ' -azido-3 ' -deoxythymidine (Ia, X ═ N)₃FIG. 2).

4 '-C-ethynyl-5' -O- (dimethoxytrityl) -3 '-azido-3' -deoxythymidine (12, X ═ N) in a 1% trifluoroacetic acid solution in dichloromethane (20mL)₃) (1mmol) of the solution was stirred at room temperature for 3h and neutralized with ammonium hydroxide. The solvent was evaporated to dryness under reduced pressure and the residue was purified by silica gel column chromatography (2-5% MeOH in dichloromethane) to give 4' -C-ethynyl-AZT (Ia, X ═ N)₃)。

Example 6.preparation of 4 '-C-ethynyl-5' -O- (dimethoxytrityl) -3 '-fluoro-3' -deoxythymidine (12, X ═ F, fig. 2).

To a solution of 11' (1mmol) in anhydrous DMF (10mL) was added tetrabutylammonium fluoride (TBAF, 3mmol) and the mixture was stirred at 100 ℃ for 16 h. The solvent was evaporated to dryness under reduced pressure. The residue was co-evaporated with toluene (2 × 20mL) and purified by silica gel column chromatography (20-50% EtOAc in hexanes) to give 4 '-C-ethynyl-5' -O- (dimethoxytrityl) -3 '-fluoro-3' -deoxythymidine (12, X ═ F).

Example 7.preparation of 4 ' -C-ethynyl-3 ' -fluoro-3 ' -deoxythymidine (Ia, X ═ F, fig. 2).

A solution of 4 '-C-ethynyl-5' -O- (dimethoxytrityl) -3 '-fluoro-3' -deoxythymidine (12, X ═ F) (1mmol) in 1% trifluoroacetic acid in dichloromethane (20mL) was stirred at room temperature for 3h and neutralized with ammonium hydroxide. The solvent was evaporated to dryness under reduced pressure and the residue was purified by silica gel column chromatography (2-5% MeOH in dichloromethane) to give 4' -C-ethynyl-FLT (Ia, X ═ F).

anti-HIV activity

Example 8 MTT method Using MT-4 cells

Test reagents (100. mu.L) were diluted in 96-well microplates. Treating MT-4 cells with HIV-1 (III)_bStrain; 100 TCID₅₀) Infected and non-infected MT-4 cells were added to the microplate so that the number of cells in each well became 10,000. The cells were cultured at 37 ℃ for 5 days. MTT (20. mu.L, 7.5mg/ml) was added to each well and the cells were further cultured for 2-3 hours. Media (120 μ L) was sampled and MTT stop solution (isopropanol containing 4% Triton X-100 and 0.04N HCl) was added to the sample. The mixture is stirred to form a nail(formazane), which is dissolved. The absorbance of the solution at 540nm was measured. Since absorbance is directly proportional to the number of living cells, the concentration of the test agent measured at a value of half the absorbance in the assay using infected MT-4 cells represents EC₅₀Whereas the test agent concentration at a value of half the absorbance measured in the assay using uninfected MT-4 cells represents CC₅₀。

Example 9 MAGI assay Using HeLa CD4/LTR- β -Gal cells

HeLa CD4/LTR- β -Gal cells were added to 96 wells so that the number of cells in each well was 10,000. After 12-24 hours, the medium was removed and diluted test agent (100 μ L) was added. Multiple HIV strains (wild strain: WT, drug-resistant strain: MDR, M184V, NL4-3, 104pre, and C; each equal to 50 TCID) were added₅₀) And introducing said cell intoCulturing for 48 hours. Cells were fixed for 5 minutes using PBS containing 1% formaldehyde and 0.2% glutaraldehyde. After the fixed cells were washed three times with PBS, the cells were stained with 0.4mg/ml X-Gal for 1 hour, and the number of blue stained cells per well was counted under a transmission stereo microscope. The test agent concentrations at which blue-stained cells were reduced in number to 50% and 90%, respectively, represent EC₅₀And EC₉₀. Cytotoxicity was measured by using HeLa CD 4/LTR-cell-Gal cells in a similar manner to that employed in the MTT method.

anti-HBV activity

Example 10 anti-HBV AD38 test

HepG2-AD38 cell line was established in a medium containing DMEM-F/12, 10% fetal bovine serum, 100 IU/mL/100. mu.g/mL penicillin/streptomycin, 50. mu.g/mL kanamycin, 0.3. mu.g/mL tetracycline, and 200. mu.g mL G418. The test medium for the HepG2-AD38 cell line contained RPMI-1640, 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 used for this test were as follows: phosphate Buffered Saline (PBS), double coated 96 well plates, DNeasy 96 tissue kit (Qiagen), QIAvac 96 vacuum manifold, Micro amp optical 96 well reaction plates (Applied Biosystems), Micro amp optical caps (Applied Biosystems), Tagman Universal PCR Master Mix (Applied Biosystems), 7700 Sequence detector (Applied Biosystems), primers and probes for HBV DNA: 1125nM forward primer (primer 1), GGA CCC CTGCTC GTG TTA CA; 1125nM reverse primer (primer 2), GAG AGA AGT CCACCA CGA GTC TAG A; and 250nM probe, FAM-TGT TGA CAA GAA TCCTCA CAA TAC CAC.

Methodology of

And (4) cell determination. The wells of a 96-well bio-coated plate are seeded with an appropriate amount of cells, such as 5X 10⁴Cells/well and 5% CO at 37 ℃₂And (5) cultivating. After 2 days, the supernatant was carefully removed, the cell layer was washed with PBS, and subsequently refreshed with assay media, which was incubatedThe cells are re-grown for 5 days with or without an appropriate amount of test compound (such as 10 μ M or a 1: 3 ratio dose response starting at 10 μ M. on day 7, the amount of supernatant such as 180 μ L is collected and stored in an appropriate container (such as in a blue rack included in the DNeasy 96 tissue kit, or at-80 ℃ or at room temperature, depending on whether the extraction step is performed immediately or at some later time.

Viral HBV DNA was extracted from cell supernatants. Supernatant samples collected on day 7 were either thawed or used as received. The proteinase K/buffer ATL working solution, which contained 2mL of proteinase K and 18mL of buffer ATL, was transferred on top of the supernatant sample. The tube was then sealed and mixed with repeated inversion. The tube was then centrifuged up to 3000rpm to collect any solution from the cap, which was then used and referred to as cap solution. The tubes were incubated at 55 ℃ for 15 minutes and then centrifuged again until 3000 rpm. To each sample was added 410. mu.L of buffer AL/E. The tube was resealed, placed in a rack, and shaken vigorously for an appropriate amount of time (such as 15 seconds), and then the tube was centrifuged until 3000 rpm. At this point, the DNeasy 96 plate was placed on top of the QIAvac 96 vacuum manifold. The cap solution was then transferred to the DNeasy 96 plate and vacuum applied for the appropriate time. An amount of buffer AW1 (such as 500 μ Ι _) was added to each well, and then the vacuum was again applied for an appropriate amount of time (such as about 1 minute). An amount of buffer AW2 (such as 500 μ L) is added to the well and the vacuum is again applied for an amount of time (such as 1 minute). The contents of the solution in the well are then agitated and the vacuum is then applied again for a certain amount of time (such as 10 minutes). The DNA was diluted by adding pre-warmed buffer AE to each well and then vacuum was added.

And (5) carrying out real-time PCR.

Real-time pcr sufficient HBV primer and probe solutions were prepared for 200 wells (1500 μ L total) by using the following solutions containing 100 μ M primer 1, 100 μ M primer 2, 50 μ M probe in nuclease-free water. It is also necessary to prepare a sufficient amount of a reaction mixture containing Universal PCR Master Mix, HBV primer and probe solutions, nuclease-free water. To each well of an optical 96-well reaction plate, the appropriate amount of reaction mixture and HBVDNA was added from each sample. The wells were covered with an optical cap and then they were centrifuged for an appropriate amount of time. The plate is placed in a sequence detector (such as a 7700 sequence detector) and the appropriate reporter is selected for FAM and the set volume is selected to be 25 μ L. The machine was started and at a certain period (after about 2 hours) the reduction in dCt and virus amount was calculated for each test compound.

Example 11.8 day cytotoxicity assay

HepG2 (liver). BxPC3 (pancreatic) and CEM (lymphocytic) cell lines were established in appropriate media. For example, the medium of the HepG2 cell line contains DMEM, 10% fetal bovine serum, and 100 IU/mL/100. mu.g/mL penicillin/streptomycin. Assay media for BxPC3 and CEM contained RPMI-1640, 10% fetal bovine serum, and 100 IU/mL/100. mu.g/mL penicillin/streptomycin.

Methodology amount of 2X drug dilution was added to wells of 96-well plates. 50 μ L of 2X drug dilutions were added to 96-well plates. In each assay, a "no drug" (media only) control was used to determine the minimum absorbance value and a "cell + media only" control was used for the maximum absorbance value. Solvent control was also used if the drug was dissolved in DMSO. Cells were counted and resuspended in the appropriate assay medium. It should be noted that cells should be added at 2000 cells/well. New cell suspension was added to each well and the plates were incubated with 5% CO at 37 deg.C₂The cultivation was carried out for 8 days. After 8 days incubation, MTS dye was added to each well and the plates were incubated with 5% CO at 37 ℃₂Incubate for 2 hours. The plate was then read at 490nm using an ELISA plate reader. The absorbance in control wells with medium only was calculated. The 50% inhibition value (CC) was determined by comparing the absorbance of drug-free cell control wells to the absorbance in wells containing cells and test drug₅₀)。

Claims

1. A compound comprising β -D-and β -L-nucleosides, or pharmaceutically acceptable salts or prodrugs thereof, said compound having a structure defined by formula (I) or by formula (II):

wherein:

x is hydrogen, F, Cl, Br, I, NH₂、NHR⁴、NR⁴R⁵、NHOH、NHOR⁴、NHNH₂、NR⁴NH₂、NHNHR⁴、SH、SR⁴、S(O)_bR4、OH、OR⁴、N₃CN, or CF₃(ii) a Y is hydrogen, F, Cl, Br, I, NH₂、NHR⁴、NR⁴R⁵、NHOH、NHOR⁴、NHNH₂、NR⁴NH₂、NHNHR⁴、SH、SR⁴、S(O)_bR⁴、OH、OR⁴、N₃、CN、CF₃Hydroxymethyl, methyl, optionally substituted or unsubstituted ethyl, optionally substituted or unsubstituted vinyl, optionally substituted or unsubstituted 2-bromovinyl, optionally substituted or unsubstituted ethynyl; r¹Is F or N₃；

R²Is OH, OR⁴、OC(O)R⁴、OP_vO_3vM_xR⁴ _yR⁵ _z、P_vO_3vM_xR⁴ _yR⁵ _z、OCH₂P_vO_3vM_xR⁴ _yR⁵ _z、OP(O)(OQ)_a(NHR⁴)_b、SH、SR⁴、S(O)_bR⁴、SC(O)R⁴、NH₂、NHC(O)R⁴、NHR⁴、NR⁴R⁵、NHOH、NHOR⁴、NHNH₂、NR⁴NH₂、NHNHR⁴；

z is O, S, CH₂Or C ═ CH₂；

A is N, CH, or CF; and

R⁴and R⁵Are the same or different and are lower alkyl, lower alkenyl, acyl of 1 to 17 carbons, aryl, or aralkyl;

v has a value of 1, 2, or 3;

x, y, and z are independent of each other and have a value of 0, 1, 2, 3, or 4; and

a has a value of 0 or 1, b has a value of 1 or 2, and Q is M or R⁴。

2. The compound of claim 1, wherein the nucleoside is a 4 '-C-substituted-3' -fluoro-2 ', 3' -dideoxynucleoside.

3. The compound of claim 1, wherein the nucleoside is a 4 '-C-substituted-3' -azido-2 ', 3' -dideoxynucleoside.

4. The compound of claim 1, wherein the nucleoside is a 4 ' -C-ethynyl-3 ' -fluoro-2 ', 3-dideoxynucleoside.

5. The compound of claim 1, wherein the nucleoside is a 4 '-C-ethynyl-3' -azido-2 ', 3' -dideoxynucleoside.

6. The compound of claim 1, wherein said nucleoside is 4 ' -C-ethynyl-3 ' -fluoro-3 ' -deoxythymidine.

7. The compound of claim 1, wherein the nucleoside is 4 ' -C-ethynyl-3 ' -azido-3 ' -deoxythymidine.

8. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 7 and a carrier or diluent.

9. A pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of claims 1-7 and at least one other antiviral agent.

10. A method for treating or preventing a host infected with human immunodeficiency virus, comprising administering a therapeutically effective amount of a compound of any one of claims 1-7, or a pharmaceutically acceptable salt or prodrug thereof, alone or in combination with another agent.

11. A method for the treatment or prophylaxis of a host infected with hepatitis b virus, which comprises administering a therapeutically effective amount of a compound of any one of claims 1 to 7, or a pharmaceutically acceptable salt or prodrug thereof, alone or in combination with another agent.

12. Use of an antiviral effective amount of a compound of any one of claims 1-7, or a pharmaceutically acceptable salt or prodrug thereof.

13. An intermediate of the formula:

wherein

Y is hydrogen, F, Cl, Br, I, NH₂、NHR⁴、NR⁴R⁵、NHOH、NHOR⁴、NHNH₂、NR⁴NH₂、NHNHR⁴、SH、SR⁴、S(O)R⁴、S(O)₂R⁴、OH、OR⁴、N₃、CN、CF₃Hydroxymethyl, methyl, optionally substituted or unsubstitutedAn optionally substituted or unsubstituted vinyl group, an optionally substituted or unsubstituted 2-bromovinyl group, an optionally substituted or unsubstituted ethynyl group;

pg is a hydroxyl protecting group;

l is a leaving group; and

14. A process for preparing the intermediate of claim 13, the process comprising:

(a) the method comprises the following steps Selectively protecting the 5 '-OH with a protecting group Pg to form a 5' -OPg group;

(d) the method comprises the following steps Activation of the 3 '-OH with the xylose configuration with a leaving group L forms a 3' -OL group.

15. An intermediate of the formula:

wherein

pg is a hydroxyl protecting group; and

16. A process for preparing the intermediate of claim 15, the process comprising:

(a) the method comprises the following steps Activating the 3 '-OH of the' -O-protected nucleoside with a leaving group L to form a 3 '-OL-5' -O-protected nucleoside group; followed by

(b) The method comprises the following steps Treating the 3 '-OL-5' -O-protected nucleoside with DBU or DBN; so as to obtain said intermediate;

wherein L is selected from the group consisting of sulfonyl, trifluorosulfonyl, substituted sulfonate, and unsubstituted sulfonate.