CN101120947B

CN101120947B - Beta-l-2'-deoxy-nucleosides for the treatment of hepatitis b

Info

Publication number: CN101120947B
Application number: CN2006101007520A
Authority: CN
Inventors: 吉尔斯·格索林; 让-路易斯·伊姆巴克; 马丁·L·布兰特
Original assignee: Montpellier Ii, University of; Centre National de la Recherche Scientifique CNRS; Idenix Pharmaceuticals LLC
Current assignee: Montpellier Ii, University of; Novartis AG; Centre National de la Recherche Scientifique CNRS
Priority date: 1998-08-10
Filing date: 1999-08-10
Publication date: 2010-11-24
Anticipated expiration: 2019-08-10
Also published as: CN1911237A; CN101120947A; RU2300381C2; CN1911237B

Abstract

This invention is directed to a method for treating a host infected with hepatitis B comprising administering an effective amount of an anti-HBV biologically active 2'-deoxy- beta -L-erythro-pentofuranonucleoside or a pharmaceutically acceptable salt or prodrug thereof, wherein the 2'-deoxy- beta -L-erythro-pentofuranonucleoside has formula (I) wherein R is selected from the group consisting of H, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative; and BASE is a purine or pyrimidine base which may be optionally substituted. The 2'-deoxy- beta -L-erythro-pentofuranonucleoside or a pharmaceutically acceptablesalt or prodrug thereof may be administered either alone or in combination with another 2'-deoxy- beta -L-erythro-pentofuranonucleoside or in combination with another anti-hepatitis B agent.

Description

β-L-2 '-deoxidation-the nucleoside that is used for the treatment of hepatitis B

The application is on August 10th, 1999 for the applying date, and application number is 200410002863.9, and denomination of invention is divided an application for the patent application of " β-L-2 '-deoxidation-nucleoside that is used for the treatment of hepatitis B ".

Related description

The application divides an application for CN99809553.2's.

The background technology of invention

The present invention relates to hepatitis B virus (being also referred to as " HBV ") Therapeutic Method field, this method comprises the reactive compound disclosed herein with one or more effective doses, or wherein a kind of pharmaceutically acceptable prodrug or its salt of chemical compound are used separately to the host who needs this treatment separately or in combination.

Except Nicotiana tabacum L., HBV is deputy aspect people's the cancer causing.Can directly cause the development of cancer though suppose it, or by chronic inflammatory disease, liver cirrhosis with cause the development of cancer indirectly with infecting relevant cell regeneration, it is unknown that HBV induces the mechanism of cancer to be still.

Hepatitis B has become in global epidemic diseases.Do not knowing infected 2-6 through the host after individual month incubation period, HBV infects may cause acute hepatitis and hepatic injury, and this may cause that some enzyme index raises in stomachache, jaundice and the blood.HBV may cause acute severe hepatitis, and can be fast-developing, and this is the disease that usually causes death, and described disease can make the major part of liver be damaged.

Typical patient can recover from the acute hepatitis phase.But high-caliber virus antigen continues development in some patients' blood, and is perhaps unstable, or periodically causes chronic infection.Chronic infection can cause chronic persistent hepatitis.The patient of chronic persistent hepatitis is very general in developing country.To mid-term in 1991,200,012,500 chronic HBV carriers are only just arranged, nearly 300,000,000 virus carriers in Africa in the whole world.Chronic persistent hepatitis can cause fatigue, hepatitis interstitialis chronica and hepatocarcinoma, primary hepatic carcinoma.

At western developed country, the group of people at high risk that HBV infects comprises the people who contacts with HBV carrier or their blood sample.(AIDS) is closely similar for the Epidemiological study of HBV and acquired immune deficiency syndrome (AIDS), and why this illustrated that HBV usually can occur in AIDS patient or the syndrome patient relevant with AIDS infects.But HBV is stronger than the infectiousness of HIV.

But, produced vaccine by genetic engineering recently, and be widely used.Unfortunately, vaccine does not have help to the people who has infected HBV.Alpha-interferon is that allow with its treatment every day by the protein of genetic engineering preparation, but the success rate of treatment has only about 1/3rd.In addition, interferon can not oral administration.

Now identified and had the more active synthetic nucleosides of anti-HBV.BCH-189, (-)-enantiomer, be known as 3TC, be U.S.P5, the claimed product of 539,116 people such as () Liotta has been used for the treatment of hepatitis by U.S.Food and Drug Administration approval.In addition, also can be referring to EP-A-0494119A1, the application people is Bio Chem Phanna, Inc..

Cis-2-methylol-5-(5-flurocytosine-1-yl)-1,3-oxygen thia pentane (＂ FTC ＂) has anti-HBV activity, referring to WO92/15308; People such as Furman, ＂ The Anti-Hepatitis BVirus Activities, Cytotoxicities, and Anabolic Profiles of the (-) and (+) Enantiomers ofcis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-oxathiolane-5-yl]-Cytosine ＂ AntimicrobialAgents AndChemotherapy, in December, 1992, p 2686-2692; With people such as Cheng, Journal of Biological Chemistry, Vol.267 (20), 13938-13942 (1992).

People such as von Janta-Lipinski are disclosed 3 '-the fluoro-modification-β-2 '-dezyribonucleoside 5 '-triguaiacyl phosphate can be used for suppressing hepatitis B polymerase (J.Med.Chem., 1998,41,2040-2046).Specifically, disclose 5 of following nucleoside '-phosphate ester is effective HBV DNA polymerase inhibitor: 3 '-deoxidation-3 '-fluoro-beta-L-thymidine (β-L-FTTP), 2 ', 3 '-dideoxy-3 '-fluoro-beta-L-5-cytidine (β-L-FdCTP), and2 ', 3 '-dideoxy-3 '-fluoro-beta-L-5-methylcystein nucleoside (β-L-FMethCTP).

(Inc.) disclosed some L-ribofuranose yl nucleosides can be used for treating cancer and virus to WO 96/13512 for Genencor International, Inc. and Lipitek.The application of this compounds for treating cancer and HIV is particularly disclosed.

U.S.P5,565,438,5,567,688 and 5,587,362 (people such as Chu) disclose 2 '-fluoro-5-methyl-β-L-1-nucleoside (L-FMAU) is used for the treatment of the application of hepatitis B and Epstein Barr virus.

Yale University and University of Georgia Research Foundation, Inc. in WO92/18517, disclose the L-FddC that is used for the treatment of hepatitis B virus (β-L-5-fluoro-2 ', 3 '-zalcitabine).

Ucleosides β-L-2 '-deoxycytidine (β-L-2 '-dC), β-L-2 '-deoxyribosylthymine (β-L-dT) and β-L-2 '-deoxyadenosine (β-L-2 '-dA) to synthesize in this area be known.1972, Antonin Holy discloses β-L-dC and β-L-dT first, " Nucleic AcidComponents and Their Analogs.CL III.Preparation of2 '-deoxy-L-Ribonucleosides of the Pyrimidine Series ＂; Collect.Czech.Chem.Commun.; (1972); 37 (12), 4072-87.People such as Morris S.Zedeck are in Mol.Phys. (1967), and 3 (4), disclose first among the 386-95 and be used for suppressing the synthetic β-L-dA of Pseudomonastestosteroni inducible enzyme.

Some 2 '-deoxidation-β-L-erythro form-furan pentose nucleoside is known, they have antitumor and antiviral activity optionally.People such as Verri disclose 2 '-deoxidation-β-L-erythro form-furan pentose nucleoside as antitumor agent and anti-herpesvirus agent (Mol.Pharmacol. (1997), 51 (1), 132-138 and Biochem.J (1997), 328 (1), 317-20).People such as Saneyoshi point out 2 '-deoxidation-L-nucleoside can be used as reverse transcription (1) inhibitor, is used to control retrovirus retrovirus and treatment AIDS, referring to Jpn.Kokal Tokkyo Koho, and JP06293645 (1994).

People such as Giovanni attempt with 2 '-deoxidation-β-L-erythro form-furan pentose nucleoside treatment pseudorabies virus (PRV), see Biochem.J., (1993), 294 (2), 381-5.

People such as Tyrsted (Biochim.Biophys.Acta (1968), 155 (2), 619-22) and people such as Bloch (J.Med.Chem., (1967), 10 (5), 908-12) studied be used for 2 of chemotherapy '-deoxidation-β-L-erythro form-furan pentose nucleoside.

(β-L-dT) can be used for suppressing herpes complex virus 1 type (HSV-1) thymidine kinase (TK) to β-L-2 ' known in the art-deoxyribosylthymine.People such as lotti point out that in WO92/08727 β-L-dT is by HSV-1TK, rather than suppress the phosphorylation of D-thymidine by people's TK selectivity.People such as Spaldari have reported the L-thymidine by herpes complex virus 1 type thymidine kinase phosphorylation, and suppress viral growth, J.Med.Chem., (1992), 35 (22), 4214-20.

In view of hepatitis B has become worldwide epidemic diseases, it has serious to the patient who infects, and usually is the result of tragedy formula, therefore to medicine new, effective low toxicity is provided to the host, with the viral infection for the treatment of the people intensive demand is arranged.

Therefore, the purpose of this invention is to provide the new method of the hepatitis B infection for the treatment of people or other host and new compositions.

Summary of the invention

The invention discloses the method for treatment people or other host's hepatitis B infection, comprise with one or more effective doses, have bioactive 2 '-deoxidation-β-L-erythro form-furan pentose nucleoside (this paper is also referred to as β-L-d-nucleoside or β-L-2 '-d-nucleoside) or its pharmaceutically acceptable salt or its prodrug, giving separately or in combination needs the patient of this treatment to use, and wherein optionally contains pharmaceutically acceptable carrier.In this manual the term of Shi Yonging " 2 '-deoxidation " be meant 2 of nucleoside '-do not have substituent group on the position.

Disclosed by the invention 2 '-deoxidation-β-L-erythro form-furan pentose nucleoside, or its pharmaceutically acceptable prodrug, or its salt, or the pharmaceutical formulation that contains these chemical compounds is used to prevent the symptom relevant with other with treating hepatitis B infection, the chronic hepatitis, liver cirrhosis, acute hepatitis, acute severe hepatitis, chronic persistent hepatitis and the fatigue that cause as anti-HBV antibody positive and the HBV-positive symptom, by HBV.These chemical compounds or preparation also can be used for prophylactically preventing or postpone to suffer from HBV antibody positive or HBV-antigen positive or the development of the clinical symptoms of the individuality that contacts with HBV.

In one embodiment of the invention, 2 '-deoxidation-β-L-erythro form-furan pentose nucleoside derivates is a following formula: compound:

Wherein R is selected from H, straight chain, side chain or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, the CO-aryloxy alkyl, the aryl that CO-replaces, alkyl sulphonyl, aryl sulfonyl, the aralkyl sulfonyl, amino acid residue, one, two or triguaiacyl phosphate, or phosphate derivative; And BASE is purine or pyrimidine bases, and they selectively are substituted.

In another embodiment of the present invention, 2 '-deoxidation-β-L-erythro form-furan pentose nucleoside derivates is the β-L-2 '-deoxyadenosine of following formula, or its pharmaceutically acceptable salt or its prodrug:

Wherein R is selected from H, and one, two or triguaiacyl phosphate, acyl group or alkyl, or stable phosphate derivative (forming stable nucleotide prodrug).

In another embodiment of the present invention, 2 '-deoxidation-β-L-erythro form-furan pentose nucleoside derivates is the β-L-2 '-deoxycytidine of following formula, or its pharmaceutically acceptable salt or its prodrug:

In another embodiment of the present invention, 2 '-deoxidation-β-L-erythro form-furan pentose nucleoside derivates is the β-L-2 '-BrdU of following formula, or its pharmaceutically acceptable salt or its prodrug:

In another embodiment of the present invention, 2 '-deoxidation-β-L-erythro form-furan pentose nucleoside derivates is the β-L-2 '-deoxyguanosine of following formula, or its pharmaceutically acceptable salt or its prodrug:

In another embodiment of the present invention, 2 '-deoxidation-β-L-erythro form-furan pentose nucleoside derivates is the β-L-2 '-deoxyinosine of following formula, or its pharmaceutically acceptable salt or its prodrug:

In another embodiment of the present invention, 2 '-deoxidation-β-L-erythro form-furan pentose nucleoside derivates is the β-L-2 '-deoxyribosylthymine of following formula, or its pharmaceutically acceptable salt or its prodrug:

In another embodiment of the present invention, 2 '-deoxidation-β-L-erythro form-furan pentose nucleoside and one or more other 2 '-deoxidation-β-L-erythro form-furan pentose nucleoside, or with one or more hepatitis B virus is demonstrated active chemical compound and replace administration or combine administration.In general, when replacing drug treatment, with the effective dose successive administration of each medicament; In conjunction with drug treatment the time, with the effective dose administration simultaneously of two or more medicaments.Used dosage depends on absorption, inactivation and the discharge rate of medicine, and the other factors known to the those skilled in the art.The numerical value that it should be noted that dosage also can change along with the order of severity of treatment symptom.Further, for a specific receiver, specific dosage and medication schedule should be according to the needs of individuality, and write a prescription or the doctor's of supervision group compound administration prescription is adjusted in time.

In another embodiment, present invention includes the method that treatment people's HBV infects, this method comprise use 2 of disclosed by the invention, HBV treatment effective dose '-prodrug of deoxidation-β-L-erythro form-furan pentose nucleoside derivates.Prodrug described here is meant the chemical compound that can change into nucleoside of the present invention after the medication in vivo.Infinite example comprises pharmaceutically acceptable salt (or being called the physiologically acceptable salt ＂ of ＂), 5 of reactive compound ' and N ⁴(cytidine) or N ⁶(adenosine) acidylate or alkylating derivant, or reactive compound 5 '-phospholipid or 5 '-ether fat.

Description of drawings

Fig. 1 illustrated with L-ribose or L-xylose and made raw material, prepares β-L-erythro form-furan pentose nucleoside (conventional method of β-L-dN).

Fig. 2 has illustrated the rule according to accumulation and decay, L-dA, L-dC and the L-dT metabolic map in the Hep of human body G2 cell.Described cell is hatched with 10 μ M chemical compounds.

Fig. 3 illustrates β-L-dA, β-L-dT and the antiviral effect of β-L-dC in marmot chronic hepatitis model.

The detailed description of invention

Term used herein " being the individual isomer form substantially " or " with the form of separating " refer to 2 '-deoxidation-β-L-erythro form-furan pentose nucleosides is to exist with similar 95% appointment spatial configuration at least, in a preferred embodiment, reactive compound is to give the patient's medication that needs treatment with this purity at least.

The term hepatitis B symptom relevant with it refers to hepatitis B and example relevant symptom described as follows as used herein: anti-HBV antibody positive and HBV-positive symptom, the chronic hepatitis, cirrhosis, oxyhepatitis, fulminant hepatitis, chronic persistant hepatitis and the fatigue that are caused by HBV. Method of the present invention comprise use 2 '-deoxidation-β-L-erythro form-furan pentose nucleoside derivates prophylactically prevents or postpones to suffer from HBV antibody positive or HBV-antigen positive or the development of the clinical symptoms of the individuality that contacts with HBV.

Except as otherwise noted, term alkyl used herein refer to saturated straight chain, straight chain or ring-type primary, the second month in a season or tertiary hydrocarbon, be typically C₁-C ₁₈, preferred C₁-C ₆, particularly include but not limited to methyl, ethyl, propyl group, butyl, amyl group, hexyl, isopropyl, isobutyl group, sec-butyl, the tert-butyl group, isopentyl, neopentyl (amyl), tertiary pentyl, cyclopenta and cyclohexyl.

The terminology used here acyl group refers to formula-C (O) R ' part, and R ' wherein is alkyl; Aryl, alkaryl, aralkyl, heteroaryl comprises the alkoxyalkyl of methoxy; The aralkyl that comprises benzyl; Aryloxy alkyl such as phenoxymethyl; Comprise with halogen, C₁-C ₄Alkyl or C₁-C ₄The aryl of the phenyl that alkoxyl replaces, or amino acid residue. Particularly the term acyl group includes but not limited to acetyl group, propiono, bytyry, valeryl, 3-methylbutyryl base, butanedioic acid hydrogen ester, 3-chlorobenzoic acid ester, benzoyl, acetyl group, valeryl, methanesulfonates, propiono, valeryl, positive caproyl, caprylyl, capryl, dodecane acyl group, tetradecane acyl group, hexadecane acyl group, stearyl and oleoyl.

Here used term purine or pyrimidine bases include but not limited to 6-alkyl purines and N⁶-alkyl purines, N⁶The fast cry of certain animals class of-acyl group, N⁶-benzyl purine, N⁶-halo purine, N⁶-vinyl purine, N⁶-acetenyl purine, N⁶-acyl group purine, N⁶-hydroxyalkyl purine, N⁶-alkylthio purine, N²-alkyl purines, N⁴-alkyl miazines, N⁴-acyl group miazines, 4-benzyl pyrimidines, N⁴-halogen pyrimidines, N⁴-acetenyl miazines, 4-acyl group and N⁴-acyl group miazines, 4-hydroxyalkyl miazines, 4-alkylthio miazines, thymidine, cytimidine, the 6-aza-pyrimidine comprises 6-azepine cytimidine, 2-and/or 4-mercaptopyrimidine, uracil, C⁵-alkyl miazines, C⁵-benzyl pyrimidines class, C⁵-halogen pyrimidines, C⁵-vinyl pyrimidine, C⁵-acetenyl pyrimidine, C⁵-acyl group pyrimidine, C⁵-hydroxyalkyl purine, C⁵-amide groups pyrimidine, C⁵-cyanopyrimidine, C⁵-nitro-pyrimidine, C⁵-aminopyrimidine, N²-alkyl purines, N²-alkyl-6-thio-purine class, 5-azacitidine base, 5-azauracil base, Triazolopyridine base, phonetic azoles and pyridine radicals, pyrrolopyridinyl and pyrazolopyrimidine base. Necessary and when needing, can functional oxygen and the nitrogen groups on the alkali be protected. Suitable blocking group is known in the art, comprises trimethyl silyl, dimethyl hexyl silicyl; t-butyldimethylsilyl and t-butyldiphenylsilyl, trimethylphenyl, alkyl; acyl group such as acetyl group and propiono, mesyl and p-toluenesulfonyl.

Terminology used here biologically active nucleosides refers to when carrying out the test of 2.2.15 cell transfecting with hepatitis viruse, the EC that nucleosides shows₅₀It is 15 moles or still less.

Preferred alkali comprises cytimidine, 5-flurocytosine, 5-bromine cytimidine, 5-iodocytosine, uracil, 5 FU 5 fluorouracil, 5-bromouracil, 5-iodouracil, methyl uracil, thymidine, adenine, guanine, inosine, xanthine, 2,6-diaminopurine, adenine, 6-chloropurine and 2,6-dichloropurine, 6-bromine purine, 2,6-dibromo purine, 6-iodopurine, 2,6-, two iodine purines, 5-bromo vinyl cytimidine, 5-bromo vinyl uracil, 5-bromo vinyl cytimidine, 5-bromo vinyl uracil, 5-trifluoromethyl cytimidine, 5-trifluoromethyl uracil.

As disclosed in following document, 2 '-deoxidation-β-L-erythro form-furan pentose nucleosides can 5 '-phosphatide or 5 '-form of phosphorus ether provides: Kucera, L.S., N.Lyer, E.Leake, A.Raben, Modest E.J., D.L.W. and C.Piantadosi.1990.Novelmembrane-interactive ether lipid analogs that inhibit infectious HIV-1production and induce defective virus formation.AIDS Res HumRetroviruses.6:491-501; Piantadosi, C., J.Marasco C.J., S.L.morris-Natschke, K.L.Meyer, F.Gumus, J.R.Surles, K.S.Ishaq, L.S.Kucera, N.lyer, CA.Wallen, S.Piantadosi and E.J.Modest.1991-Synthesis and evaluation of novel ether lipid nucleoside conjugatesfor anti-HIV activity.J Med Chem.34:1408-1414; Hosteder, K.Y., D.D.Richman, DA.Carson, L.M.Stuhmiller, G.M.T.van Wijic, with H.vanden Bosch.1992.Greafly enhanced inhibition of human immunodeficiencyvirus type 1 replication in CEM and HT4-6C cells by31-deoxythymidinediphosphate dimyristoylglycerol, a lipid prodrug of 31-deoxythymidine.Antimicrob Agents Chemother.36:2025-2029; Hostetler, K.Y., L.M.StuInniller, H.B.Lenting, H.van den Bosch and D.D.Richman.1990.Synthesis and antiretroviral activity of phospholipid analogs ofazidothymidine and other antiviral nucleosides.J.Biol Chem.265:6112-7.

By reacting with suitable esterifying agent such as carboxylic acid halides or acid anhydrides, can be with 2 '-deoxidation-β-L-erythro form-furan pentose nucleosides changes into pharmaceutically acceptable ester. Method according to routine can change into pharmaceutically acceptable salt with nucleosides or its pharmaceutically acceptable prodrug, for example by processing with suitable acid or alkali. Also can change into described ester or salt the nucleosides of its parent, for example realize by hydrolysis.

Term pharmaceutically acceptable salt used herein or compound refer to 2 '-salt or the compound of deoxidation-β-L-erythro form-furan pentose nucleosides, they have kept the needed biologically active of parent compound, and make unwanted toxic action minimum when toxic. The limiting examples of these salt is acid-addition salts that (a) and inorganic acid (such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid etc.) form, with the salt that organic acid forms, described organic acid for example is acetic acid, oxalic acid, tartaric acid, butanedioic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid (palmoicacid), alginic acid, many glutamic acid, naphthalene sulfonic acids, naphthalenedisulfonic acid and galactosan aldehydic acid; (b) with cation (such as sodium, potassium, zinc, calcium, bismuth, barium, magnesium, aluminium, copper, cobalt, nickel, cadmium, sodium, potassium etc.) or and N, the base addition salts that the organic cation that N-dibenzyl-ethylenediamin, ammonium or ethylenediamine generate forms; Or (c) (a) and combination (b), such as tannic acid zinc salt etc.

Term prodrug used herein refers to can change in vivo after the administration compound of nucleosides. Nonrestrictive example is pharmaceutically acceptable salt (also can be called in addition " physiologically acceptable salt "), 5 of reactive compound ' and N⁴Or N⁶Acidylate or alkylating derivative, and reactive compound 5 '-phosphatide and 5 '-ether fat derivative.

The modification of reactive compound is particularly at its N⁴，N ⁶The modification of and5 '-O position can affect bioavailability and the metabolic rate of active material, therefore can control the release of active material.

The preferred embodiment of the invention is the method that the HBV for the treatment of people or other host animal infects, that the method comprises that one or more that use effective dose are selected from is following 2 '-deoxidation-β-L-erythro form-furan pentose nucleoside derivates: β-L-2 '-desoxyadenossine, β-L-2 '-deoxycytidine, β-L-2 '-BrdU, β-L-2 '-deoxyguanosine, β-L-2 '-deoxyinosine, β-L-2 '-AZT or their physiologically acceptable prodrugs, comprise phosphate, 5 ' and/or N⁶The derivative of alkylation or acyl group, or its physiologically acceptable salt, and pharmaceutically acceptable carrier is optionally arranged. The compounds of this invention or have anti-HBV activity perhaps can be metabolized to one or more compounds with anti-HBV activity. In preferred embodiments, 2 '-deoxidation-β-L-erythro form-furan pentose nucleosides is the form administration with individual isomer basically, i.e. the spatial configuration of its appointment is at least about 95%.

Nucleoside prodrugs

Arbitrary nucleosides as herein described all can be stable the nucleoside prodrugs administration, with other performance of the activity, bioavailability, stability or the nucleosides that improve nucleosides. The part of some nucleosides is known. Generally, the lipophilic modification of the alkylation of nucleosides, acidylate or other, two or triphosphoric acid esterification can improve the stability of nucleosides. Can replace the substituent example of one or more hydrogen atoms at phosphonate moiety is alkyl, aryl, and steroids, carbohydrate, comprising sucrose, 1,2-diglyceride and alcohols. Wherein have many at R.Jones and N.Bischofberger, Antiviral Research, the existing description among 27 (1995) 1-17. Above-mentioned any one can be used in combination to get a desired effect with nucleosides disclosed by the invention.

In one embodiment, can provide 2 '-deoxidation-β-L-erythro form-furan pentose nucleosides be its 5 '-prodrug of hydroxyl lipophilization. United States Patent (USP) discloses the substituent limiting examples of suitable lipophilization, and they introduce nucleosides with covalent bond, preferably 5 of nucleosides '-the O position introduces, or the preparation of lipophilization, these patents comprise U.S.P5,149,794 (on September 22nd, 1992, the people such as Yatvin); 5,194,654 (on March 16th, 1993, the people such as Hostetler); 5,223,263 (on June 29th, 1993, the people such as Hostetler); 5,256,641 (on October 26th, 1993, the people such as Yatvin); 5,411,947 (May 2 nineteen ninety-five, the people such as ostetler); 5,463,092 (October 31 nineteen ninety-five, the people such as Hostetler); 5,543,389 (on August 6th, 1996, the people such as Yatvin); 5,543,390 (on August 6th, 1996, the people such as Yatvin); 5,543,391 (on August 6th, 1996, the people such as Yatvin); With 5,554,728 (on September 10th, 1996; The people such as Basava).

That the disclosed lipophilization substituting group of aforementioned patent applications can be connected in is of the present invention 2 '-deoxidation-β-L erythro form-furan pentose nucleoside derivates, and the preparation of lipophilization disclosed, these patents comprise WO 89/02733, and WO 90/00555, and WO 91/16920, WO 91/18914, WO93/00910, WO 94/26273, and WO 96/15132, EP 0 350 287, EP 93917054.4 and WO 9I/19721.

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To nucleosides ring 3 ', 5 '-the phosphate ring of phosplate, chair form distortion equilibrium problem, thymidine phenyl ring-type 3 ', 5 '-the phosplate diastereoisomer¹The research of H NMR and X-ray crystallogram sees J.Am.Chem.Soc.109,40584064; Nerbonne, J.M., Richard, S., N﹠geot, J. and Lester, HA. (1984) New photoactivatablecyclic nucleotides produce intracellular jumps in cyclic AMP and cyclicGMP concentrations.Nature 301,74-76; Neumann, J.M., Herve ', M., Debouzy, J.C., Guerra, F.I., Gouyette, C., Dupraz, B. and Huynh-Dinh, T. (1989) Synthesis and transmembrane transport studies by NMR of aglucosyl phospholipid of thymidine.J.Am.Chem.Soc.111,4270-4277; Ohno, R., Tatsumi, N., Hirano, M., Imal, K.Mizoguchi, H., Nakamura, T., Kosaka, M., Takatuski, K., Yamaya, T., Toyama, K., Yoshida, T., Masaoka, T., Hashimoto, S., Ohshima, T., Kimura, I., Yamada, K. and Kimura, J. (1991) Treatment of myelodysplastic syndromes with orallyadministered 1-β-D-rabinoftnanosylcytosine-5 '-stearylphosphate.Oncoogy 48,451-455.

Palomino, E., Kessle, D. and Horwitz, J.P. (1989) A dihydropyridinecarrier system for sustained delivery of 2 ', 3 '-ideoxynucleosides to the brain.J.Med.Chem.32,622-625; Perkins, R.M., Barney, S., Wittrock, R., Clark, P.H., Levin, R.Lambert, D.M., Petteway, S.IL, Serafinowska, H.T., Bailey, S.M., Jackson, S., Hamden, M.R.Ashton, R., Sutton, D., Harvey, J.J. and Brown, A.G. (1993) Activity of BRL47923 and its oral prodrug, 5B203657A against a rauscher～murine leukemia virus infection in mice.Antiviral Res.20 (Suppl.I) .84; Piantadosi, C., Marasco, C.J., Jr., Morris-Natschke, S.L., Meyer, K.L., Gumus, F., Suries, J.R., Ishaq, K.S., Kucera, L.S.Iyer, N., Wallen, C.A., Piantadosi, S. and Modest, E.J. (1991) Synthesis and evaluation of novel ether lipid nucleoside conjugates foranti-HIV-1 activity.J.Med.Chem.34,1408-1414; Pompon, A., Lefebvre, I., Imbach, J.L., Kahn, S. and Farquhar, D. (1994) Decomposition pathways of the mono-and bis (pivaloyloxymethyl) estersof azidothymidine-5 '-monophosphate in cell extract and in tissue culturemedium; An application of the on-line ISRP-cleaning ' HPLC technique.Antiviral Chem.Chemother.5,91-98; Postemark, T. (1974) Cyclic AMPand cyclic GMP.Annu.Rev.Pharmacol.14,23-33; Prisbe, E.3., Martin, J.C.M., McGee, D.P.C., Barker, M.F., Smee, D.F.Duke, A.E., Matthews, T.R. and Verheyden, J.P.J. (1986) Synthesis and antiherpes virus activityof phosphate and phosphonate derivatives of9-[(1,3-dihydroxy-2-propoxy) methyl] guanine.J.Med Chem.29,671-6751; Puech, F., Gosselin, G., Lefebvre, I., Pompon, A., Aubertin, A.M.Dim, A. and Imbach, J.L. (1993) Intracellular delivery of nucleosidemonophosphate through a reductase-mediated activation process.AntiviralRes.22,155-174; Puaeva, V.P., K ' ochkeva, S.I., Mashbits, F.D. and Eizengart, R.S. (1969) .Robins, R.K. (1984) The potential of nucleotideanalogs as inhibitors of retroviruses and tumors.Pharm.Res.11-18; Kosowsky, A., Kim.S.H., Ross and J.Wick, the esters of 1-β-D-arabinofiiranosylcytosine and itsN of M.M. (1982) Lipophilic5 '-(alkylphosphate)⁴-acyl and 2.2 '-anhydro-3 '-O-acyl derivatives as potential prodrugs.J.Med.Chem.25,171-178; Ross, W. (1961) Increased sensitivity of thewalker turnout towards aromatic nitrogen mustards carrying basic sidechains following glucose pretreatment.Biochem.Pharm.8,235-240; Ryu, E.K., Ross, R.J.Matsushita, T., MacCoss, M., Hong, C.I. and West, C.R. (1982) .Phospholipid-nucleoside conjugates.3.Synthesis and preliminarybiological evaluation of 1-β-D-arabinoftiranosylcytosine 5 ' diphosphate[-], 2-diacylglyceiols.J.Med.Chem.25,1322-1329; SaflhiU, R. and Hume, WJ. (1986) The degradation of 5-iododeoxyuridine and5-bromodeoxycridine by serum from different sources and itsconsequences for the use of these compounds for incorporation into DNA.Chem.Biol.Interact.57,347-355; Saneyoshi, M., Morozumi, M., Kodama, K., Machida, J., Kuninaka, A. and Yoshino, H. (1980) Syntheticnucleosides and nucleotides.XVI.Synthesis and biological evaluations ofa series of 1-β-D-arabinofuranosylcytosine 5 '-alkyl or arylphosphates.Chem.Pharm.Bull., 28,2915-2923; Sastry, J.K., Nehete, P.N., Khan, S., Nowak, B.J., Plunkett, W., Arlinghaus, R.B. and Farquhar, D. (1992) Membrane-permeable dideoxyuridine 5 '-monophosphate analogue inhibitshuman immunodeficiency virus infection.Mol.Pharmacol.41,441445; Shaw, J.P., Jones, R.J.Arimilli, M.N., LoujeM.S., Lee, W.A. and Cundy, K.C. (1994) Oral bicavailability of PMEA from PMEA prodrugs in maleSprague-Dawley rats.9th Annual AAPS Meeting; San Diego, CA (Abstract) .Shuto, S., Ueda, S., Imamura, S., Fukukawa, K.Matsuda, A. and Ueda, T. (1987) A facile one-step synthesis of5 '-phosphatidylnucleosides by an enzymatic two-phase reaction.Tetrahedron Lett.28,199-202; Shuto, S., Itob, H., Ueda, S., lmamura, S., Kukukawa, K., Tsujino, M., Matsuda, A. and Ueda, the nucleosides and theirantileukemic activities.Chem.Pharm.Bull. of T. (1988) A facileenzymatic synthesis of 5 '-(3-sn-phosphatidyl), 36,209-217. A kind of preferred phosphate prodrug group is S-acyl group-2-thio-ethyl group, is also referred to as " SATE ".

In conjunction with or alternating treatment

Have realized that with after the antivirotic long-term treatment, the resistance to the action of a drug may occur to the variant of HBV. The most typical resistance to the action of a drug is undergone mutation because of the encoding gene of used enzyme in viral life cycle and is formed, and most typical is HBV, archaeal dna polymerase. Recently, known that the effectiveness that resisting HBV virus infects can prolong, expands or recover, this can pass through The compounds of this invention and second, and may be with the third antiviral compound in conjunction with or alternately administration realize, described second or the third compound can bring out the sudden change different from essential drugs. In addition, also can change medicine dynamics, biodistribution or the other medicines parameter of described medicine by this combination or alternating treatment. In general, combined treatment usually than alternating treatment more preferably because it can make virus produce simultaneously multiple effect.

β-L-2 ' as herein described-dA, β-L-2 '-dC, β-L-2 '-dU, β-L-2 '-dG, β-L-2 '-dT, β-L-dI or other β-L-2 '-nucleosides, the activity of the anti-hepatitis B virus of the prodrug of these compounds, phosphate or salt by with wherein two or more nucleosides in conjunction with or be used alternatingly, can be improved. In addition, for example β-L-2 ' provided herein-dA, β-L-2 '-dC, β-L-2 '-dU, β-L-2 '-dG, β-L-2 '-dT, β-L-dI or other β-L-2 '-nucleosides and 3TC, FTC, L-FMAU, DAPD, famciclovir (famciclovir), penciclovir, BMS-200475, bis pomPMEA (adefovir, dipivoxil); Lobucavir, 9-1,3-dihydroxy-2-third oxygen methyl guanine (ganciclovir), or ribavarin combination or alternately administration.

In arbitrary embodiment of this paper; if β-L-2 ' of the present invention-nucleosides is combined or alternatively administered with become the second ucleosides of activity form or non-nucleoside reverse transcriptase inhibitors by the enzyme phosphorylated, β-L-2 ' of the present invention-nucleosides phosphorylated enzyme in vivo is different from selected for the phosphorylated of preferred second compound. Kinase whose example be thymidine kinase, cytimidine kinases, guanosine kinases, adenosine kinase, deoxycytidine kinase, 5 '-nucleotidase and deoxyguanosine kinases.

The preparation of reactive compound

Of the present invention 2 '-deoxidation-β-L-erythro form-furan pentose nucleoside derivates is known in this area, can be according to Holy, and Collect.Czech.Chem.Commun. (1972), 37 (12), 4072-87 and Mol.Phys. (1967), 3 (4), the preparation of 386-95 disclosed method.

(conventional method of β-L-dN) as shown in Figure 1 with L-ribose or L-xylose azoles feedstock production β-L-erythro form-furan pentose nucleoside.

One, two or triguaiacyl phosphate derivant of active nucleoside can be according to disclosed method preparation.Monophosphate can exist according to people such as Imal J.Org.Chem., 34 (6), the method preparation described in the 1547-1550 (in June, 1969).Bisphosphate can exist according to people such as Davisson J.Org.Chem., 52 (9), the method preparation described in the 1794-1801 (1987).Triguaiacyl phosphate can exist according to people such as Hoard J.Am.Chem.Soc., 87 (8), the method preparation described in the 1785-1788 (1965).

Experimental technique

Fusing point is with the instrument of Gallenkamp MFB-595-010M, measures with open capillaries, and not calibrated.The UV absorption spectrum is measured in ethanol with Uvikon931 (KONTRON) spectrophotometer. ¹The H-NMR spectrum is at room temperature, in DMSO-d ₆In, with Bruker AC250 or 400 spectrophotometer.Chemical shift provides with ppm, with DMSO-d ₅For 2.49ppm is worth as reference.In order to confirm arranging of proton, carried out the deuterium exchange, taken off idol test or 2D-COSY test.The multiformity of signal is represented with following manner: s (unimodal), d (bimodal), dd (doublet of doublet), t (triplet), q (quartet), br (broad peak), m (multiplet).All J-value units are hertz (Hz).The FAB mass spectrum is with just-(FAB〉0) or negative-(FAB＜0) ion pulse device, on JEOL DX300 mass spectrograph, carry out, substrate be 3-Nitrobenzol methanol (NBA) or glycerol and thioglycerol (GT) mixture (50:50, v/v).Specific rotation Perkin-Elmer241 spectropolarimeter (the long 1cm of passage), unit is 10 ^-1Degcm ²g ^-1. elementary analysis is with ＂ Service de Microanalyses du CNRS, and Division deVernaison ＂ (France) measures, and analysis result indicates with the symbol of element, its function theoretical value ± 0.4% within.Thin layer chromatography in precoating Silica Gel 60 F ₂₅₄(Merck carries out on aluminium sheet Art.5554), absorbs by UV, after reuse 10% ethanol sulphuric acid carbonization and the heating, finishes the colour developing of product.Column chromatography under atmospheric pressure, (Merck Art.9385) finishes on the post in Silica Gel 60.

The stereospecific synthesis of embodiment 12 '-deoxidation-β-L-adenosine

9-(3,5-two-O-benzoyl-β-L-xylose type furyl glycosyl) adenosine (3)

9-(2-O-acetyl group-3,5-two-O-benzoyl-β-L-xylose type furyl glycosyl) adenosine 2[referring to: Gosselin, G; Bergogne, M.-C.; Imbach, J.-L., ＂ Synthesis andAntiviral Evaluation of β-LXyloftnanosyl Nucleosides of the FiveNaturally Occuring Nucleic Acid Bases ＂, Journal of HeterocyclicChemistry, 1993,30 (Oct.-Nov.), 1229-1233] (8.30g, 16.05mmol) and 98% hydrazine hydrate (234mL, 48.5mmol) at pyridine/glacial acetic acid mixture (4/1, v/v, 170mL) solution in stirred under room temperature 22 hours.Adding acetone (40mL) makes the reaction stopped reaction and continues stirring 1 hour.Reactant mixture is reduced to half of its volume, and water (250mL) dilution is with chloroform (2x150mL) extraction.Organic layer NaHCO ₃(3x100mL) saturated aqueous solution and water (3x100mL) wash successively, and drying is filtered, and concentrate and with toluene and methanol coevaporation.Residue silicagel column chromatography (0-3%MeOH is in dichloromethane) purification. obtain from diisopropyl ether sedimentary 3(5.2g, 68%): ¹H NMR (DMSO-d ₆): δ 4.5-4.9 (m, 4H, H-2 ', H-4 ', H-5 ' and H-5 ＂), 5.64 (t, 1H, H-3 ', J _{2 ', 3 '}=J _{3 ', 4 '}=3.5Hz), 6.3 (br s, 1H, OH-2 '), 6.45 (d, 1H, H-1 ', J ₁' _{, 2}'=4.6Hz), 7.3 (br s, 2H, NH ₂-6), 7.4-7.9 (m, 10H, 2 benzoyls), 8.07 and 8.34 (2s, 2H, H-2 and H-8); Ms: substrate G/T, (FAB ⁺) m/z476[M+H] ⁺, 136[BH ₂] ⁺, (FAB ^-) m/z474[M-H] ^-, 134[B] ^-UV (95% ethanol): λ max257nm (ε 16400), 230nm (ε 29300), λ min246nm (ε 14800); [α] _D ²⁰=-64 (c1.07, CHCl ₃). elementary analysis value of calculation C ₂₄H ₂₁N ₅O ₄(M=475.45): C, 6043; H, 4.45; N, 14.73.Measured value: C, 60.41; H, 4.68; N, 14.27.

9-(3,5-two-O-benzoyl-2-deoxidation-β-L-Su Shi-furan pentose base) adenosine ( 4)

To chemical compound 3(1.00g, 2.11mmol) dry acetonitrile (65mL) solution add 4-(dimethylamino) pyridine (0.77g, 6.32mmol) and benzene oxygen sulfo-phosgene (0.44mL, 3.16mmol).This mixture stirred under room temperature 2 hours.After concentrating, residue is dissolved in (50mL) in the dichloromethane, water (2x30mL), 0.5N aqueous hydrochloric acid solution (30ml) and water (3x30mL) wash successively.With the organic layer drying, filter, be concentrated into dried.(0.78mL, 5.23mmol) and α, (AIBN, 0.112g 0.69mmol) reflux in dry diox (17mL) and directly handled in 2 hours α '-azo isobutyronitrile the intermediate of this thick sulfo-carbonylation with three (trimethylsilyl) silane hydrate.Vacuum is got rid of solvent, and residue obtains foamed pure with silicagel column chromatography (0-5%MeOH is in dichloromethane) purification 4(0.93g, 96%): ¹H NMR (DMSO-d ₆): δ 62.9-3.1 (m, 2H, H-2 ' and H-2 ＂), 4.6-4.7 (m, 3H, H4 ', H-5 and d H-5 ＂), 5.8 (br s, 1H, H-3 '), 6.43 (dd, 1H, H-1 ', J ₁' _{, 2}'=3.1Hz, J ₁' _{, 2}'=7.6Hz), 7.3 (br s, 2H, NH ₂-6), 7.4-7.9 (m, 10H, 2 benzoyls), 8.05 and 8.33 (2s, 2H, H-2 and H-8); Ms: substrate G/T (FAB+) m/z460[M+H]+, 325[S]+, 136 (BH ₂]+, (FAB ^-) m/z458 (M-H] ^-, 134[B] ^-UV (95% ethanol): λ max261nm (ε 14400), 231nm (ε 26300), λ min249nm (ε 12000); [α] _D ²⁰=-38 (c 1.04, DMSO).

6-N-(4-one methoxyl group trityl)-9-(3,5-two-O-benzoyl-2-deoxidation-β-L-Su Shi-furan pentose base) adenosine ( 5)

To chemical compound 4(0.88g, 1.92mmol) solution in dry pyridine (40mL) add 4-one methoxyl group trityl chloride (1.18g, 3.84mmol).This mixture stirred 24 hours in 60 ℃.After adding methanol (5mL), solution is concentrated to drying.Residue is dissolved in (50mL) in the dichloromethane, water (30mL), NaHCO ₃Saturated aqueous solution (30mL) and water (30mL) wash successively.With organic layer drying, filtration, concentrate and with the toluene coevaporation, obtain foamed pure 5(1.01g, 72%): ¹H NMR (CDCl ₃): δ 2.9-3.0 (m, 2H, H-2 ' and H-2 ＂), 3.62 (s, 3H, OCH ₃), 4.6-4.8 (m, 3H, H-4 ', H-5 ' and H-5 ＂), 5.85 (pt, IH, H-3 '), 6.44 (dd, 1H, H-1 ', J ₁' _{, 2}'=3.1Hz, J ₁' _{, 2 "}=7.3Hz), 6.9 (br s, 1H, NH-6), 6.7-6.8 (2m, 24H, 2 benzoyls and MMTr), 7.97 and 8.13 (2s, 2H, H-2 and H-8); Ms: substrate G/T (FAB+) m/z732[M+H]+, (FAB ^-) m/z730 (M-H] ^-UV (95% ethanol): λ max274nm (ε 12100), 225nm (ε 24200), λ min250nm (ε 5900); [α] _D ²⁰=-16 (c 1.12, DMSO).

6-N-(4-one methoxyl group trityl)-9-(2-deoxidation-β-L-Su Shi-furan pentose base) adenosine ( 6)

Chemical compound 5(0.95g 1.30mmol) handles with methanol ammonia (40mL) solution (saturated at-10 ℃), at room temperature spends the night.After concentrating, residue is dissolved in (60mL) in the dichloromethane, water (30mL) washing.Water layer merges organic layer with dichloromethane (10mL) extracting twice, and drying is filtered and concentrated.Residue silicagel column chromatography (0-5%MeOH is in dichloromethane) purification. obtain foamed pure 6: ¹H NMR (CDCl ₃): δ 2.6-2.9 (m, 2H, H-2 ' and H-2 ＂), 3.5 (br s, 1H, OH-5 '), 3.55 (s, 3H, OCH ₃), 3.9-4.0 (m, 3H, H4 ', H-5 ' and H-5 ＂), 4.5-4.6 (m, IH, H-3 '), 6.03 (dd, 1H, H-1 ', J ₁' _{, 2}'=4.0Hz, J ₁' _{, 2 "}=8.8Hz), 7.0 (br s, 1H, NH-6), 6.7-6.8 and 7.1-7.4 (2m, 14H, MMTr), 7.40 (d, 1H, OH-3 ', J _{H, OH}=10.6Hz) 7.80 and 7.99 (2s, 2H, H-2 and H-8); Ms: substrate G/T (FAB+) m/z524[M+H]+, 408[BH ₂] ⁺, (FAB ^-) m/z 1045 (M-H] ^-, 522[M-H] ^-406[B] ^-UV (95% ethanol): λ max275nm (ε 12300), λ min 247nm (ε 3600); [α] _D ²⁰=+28 (c 0.94, DMSO).

6-N-(4-one methoxyl group trityl)-9-(2-deoxidation-5-O-(4-one methoxyl group trityl)-β-L-Su Shi-furan pentose base) adenosine ( 7)

Chemical compound 6(0.62g, 1.24mmol) in dry pyridine (25mL) (0.46g 1.49mmol) at room temperature handles 16 hours with a methoxyl group trityl chloride.After adding methanol (5mL), mixture is concentrated to drying.Residue is dissolved in (60mL) in the dichloromethane, water (40mL), saturated NaHCO ₃Aqueous solution (40mL) and water (3x40mL) wash successively.Organic layer drying, filtration, concentrate and with toluene and methanol coevaporation.Residue obtains foamed by silicagel column chromatography purification (0-10%MeOH is in dichloromethane) 7(0.71g, 72%): ¹H NMR (DMSO-d ₆): δ 2.21 (d, 1H, H-2 ' J ₂' _{, 2 "}=14.3Hz), 2.6-2.7 (m, 1H, H-2 ＂), 3.1-3.3 (2m, 2H, H-5 ' and H-5 ＂), 3.64 and 3.65 (2s, 6H, 2xOCH ₃), 4.1-4.2 (m, 1H, H-4 '), 4.2-4.3 (m, 1H, H-3 '), 5.68 (d, IH, OH-3 ', J _{H, OH}=5.2Hz), 6.24 (d, 1H, H-1 ', J ₁' _{, 2 "}=7.0Hz), 6.7-6.8 and 7.1-7.3 (2m, 29H, 2MMTr and NH-6), 7.83 and 8.21 (2s, 2H, H-2 and H-8); Ms: substrate G/T, (FAB ⁺) m/z796[M+H] ⁺, 408[BH ₂] ⁺, (FAB ^-) m/z794[M-H] ^-, 406[B] ^-, UV (95% ethanol): λ max 275nm (ε 30900), λ min246nm (ε 12800); [α] _D ²⁰=+14 (c 1.03, DMSO).

6-N-(4-one methoxyl group trityl)-9-(3-O-benzoyl-2-deoxidation-5-O-(4-one methoxyl group trityl)-β-L-red-furan pentose base) adenosine ( 8)

(0.38mL, 2.49mmol) drips of solution in dry tetrahydrofuran (20mL) is added to nucleoside with the azoethane dicarboxylic ester 7(0.66g, 0.83mmol), triphenyl phasphine (0.66g, 2.49mmol) and benzoic acid (0.30g is 2.49mmol) in dry THF (20mL) in the cold soln (0 ℃).This mixture at room temperature stirred 18 hours, added methanol (1mL).Solvent is got rid of in decompression, and roughage obtains being subjected to the chemical compound of triphenyl phasphine light contamination by silicagel column chromatography purification (the 0-5% ethyl acetate is in dichloromethane) 8

6-N-(4-one methoxyl group trityl)-9-(2-deoxidation-5-O-(4-one methoxyl group trityl)-β-L-red-furan pentose base) adenosine ( 9)

Chemical compound 8At room temperature handled 24 hours with methanol ammonia (20mL) solution (saturated), then reactant mixture is concentrated to drying at-10 ℃.Residue is dissolved in (30Lm) in the dichloromethane, water (20mL) washing.Water layer merges organic layer by dichloromethane (2x20mL) extraction, and dry filter concentrates.Behind silicagel column chromatography (0-2%MeOH is in dichloromethane) purification, obtain the cystose pure compound 9(0.50g is calculated as 76% by 7): ¹H NMR (DMSO-d ₆): δ 2.2-2.3 (m, 1H, H-2 '), 2.8-2.9 (m, 1H, H-2 ＂), 3.1-3.2 (m, 2H, H-5 ' and H-5 ＂), 3.64 and 3.65 (2s, 6H, 2xOCH ₃), 3.97 (pq, IH, H-4 '), 4.4-4.5 (m, 1H, H-3 '), 5.36 (d, 1H, OH-3 ', J _{H, OH}=4.5Hz), 6.34 (t, IH, H-I ', J ₁' _{, 2}'=J ₁' _{, 2 "}=6.4Hz), 6.8-6.9 and 7.1-7.4 (2m, 29H, 2MMTr and NH-6), 7.81 and 8.32 (2s, 2H, H-2 and H-8); MS: substrate G/T, (FAB ⁺) m/z796[M+H] ⁺, 408[BH ₂] ⁺, (FAB ^-) m/z794[M-H] ^-406[B] ^-UV (95% ethanol): λ max276nm (ε 42600), λ min248nm (ε 23300); [α] _D ²⁰=+29 (c1.05, DMSO).

2 '-deoxidation-β-L-adenosine (β-L-dA)

Chemical compound 9(0.44g 0.56mmol) at room temperature handled 8 hours with 80% acetic acid aqueous solution (17mL).This mixture is concentrated to drying, and residue is dissolved in (20mL) in the water, with diethyl ether (2x15mL) washing.Concentrate water layer and with toluene and methanol coevaporation.Through silicagel column chromatography (0-12%MeOH, in dichloromethane) purification and Millex HV-4 device (0.45 μ, Millipore) obtain after filtering desirable 2 '-deoxidation-β-L-adenosine ((0.12g of β-L-dA), 83%): mp193-194 ℃ (by water crystallization) (Lit.184-185 ℃, the L-enantiomer [is consulted Robins, M.J.; Khwaja, T.A.; Robins, R.K.J.Org.Chem.1970,35,636～39] and 187-189 ℃, the D-enantiomer [is consulted: Ness, R.K.inSynthetic Procedures in Nucleic Acid Chemistry; Zorbach, W.W., Tipson, R.S., Eds.; J.Wiley and sons:New York, 1968; Vol1, pp183-187]; ¹HNMR (DMSO-d ₆): δ 2.2-2.3 and 2.6-2.7 (2m, 2H, H-2 ' and H-2 ＂), 3.4-3.6 (2m, 2H, H-5 ' and H-5 ＂), 3.86 (pq, 1H, H-4 '), 4.3-4.4 (m, 1H, H-3 '), 5.24 (t, 1H, OH-5 ', J _{H, OH}=5.8Hz), 5.30 (d, 1H, OH-3 ', J _{H, OH}=4.0Hz), 6.32 (dd, 1H, H-I ', J ₁' _{, 2}'=6.2Hz, J ₁' _{, 2 "}=7.8Hz), 7.3 (br s, 2H, NH ₂-6), 8.11 and 8.32 (2s, 2H, H-2 and H-8); Ms: substrate G/T, (FAB ⁺) m/z 252[M+H] ⁺, 136[BH ₂] ⁺, (FAB ^-) m/z 250[M-H] ^-134[B] ^-UV (95% ethanol): λ max 258nm (ε 14300), λ min 226nm (ε 2100); [α] _D ²⁰=+25 (c1.03, H ₂O), (Lit.[α] _D ²⁰=+23 (c1.0, H ₂O), the L-enantiomer [is consulted .:Robins, M.J.; Khwaja, T.A.; Robins, R.K.J Org.Chem.1970,35,636-639] and [α] _D ²⁰=-25 (c0.47, H ₂O) the D-enantiomer (is consulted .:Ness, R.K.in Synthetic Procedures in Nucleic Acid Chemistry; Zorbach, W.W., Tipson, R.S., Eds.; J.Wiley and sons:New York, 1968; Vol1, pp183-187]). elementary analysis value of calculation C ₁₀H ₁₃N ₅O ₃+ 1.5H ₂O (M=278.28): C, 43.16; H, 5.80; N, 25.17. measured value: C, 43.63; H, 5.45; N, 25.33.

The embodiment 22 '-deoxidation-β-L-adenosine (stereospecific synthesis of β-L-dA)

Reaction 1:

Precursor: L-ribose (Cultor Science Food, CAS[24259-59-4], batch RIB971 1013)

Reaction reagent: sulphuric acid 95-97% (Merck; Ref 1.00731.1000); Benzenecarbonyl chloride. (Fluka; Ref12930); Sodium sulfate (Prolabo; Ref281 11.365)

Solvent: methanol P.A. (Prolabo; Ref20847.295); Pyridine 99% (Acros; Ref131780025); Dichloromethane P.A (Merck; Ref 1.06050.6025); Acetic acid P.A. (carloerba; Ref20104298); Acetic anhydride (Fluka; Ref 45830); Alcohol 95 (Prolabo; Ref20823.293)

List of references: Recondo, E.F., and Rinderknecht, H., Eine neue, EmfacheSynthese des1-O-Acetyl-2,3,5-Tri-O-β-L-D-Ribofuranosides., Helv.Chim.Acta, 1171-1173 (1959).

L-ribose 140(150g, 1mol) (2L) solution is handled with sulphuric acid (12ml) in methanol, and places 12 hours at+4 ℃, then uses pyridine (180ml) neutralization.The evaporation back provides the methylfuran nucleoside with the serosity form 141α, the β mixture.Solution Benzenecarbonyl chloride. (580ml, 5mol) processing, cooling and mechanical agitation simultaneously with this anomers mixture in pyridine (1.3L).This solution was at room temperature placed 12 hours, then poured into ice (approximately 10L) and continue stirring.This mixture (oil-in-water) is filtered with bed of diatomaceous earth.Ethyl acetate (3L) dissolving is then used in oily water (3x3L) washing that obtains on bed of diatomaceous earth.Organic facies 5%NaHCO ₃Solution (2L) and water (2L) washing, dried over sodium sulfate is filtered, and evaporation obtains the 1-O-methyl-2,3 as underflow liquid, 5-three-O-benzoyl-α/β-L-furan nucleoside 142This oil is dissolved in acetic anhydride (560ml) and the acetic acid (240ml).After dripping concentrated sulphuric acid (80ml), make this solution under mechanical agitation, keep cold conditions (+4 ℃) 10 hours.Then under agitation pour ice (approximately 10L) into to this solution.This mixture (oily compound in the water) filters on bed of diatomaceous earth.Sticky solid water (3x3L) washing that obtains on bed of diatomaceous earth then is dissolved in the dichloromethane (2.5L).Organic facies 5%NaHCO ₃(1L) and water (2x2L) washing, dried over sodium sulfate is filtered, and evaporation obtains heavy-gravity solid 143, again by ethanol (95%) crystallization (output 225g, 44%).

1-O-acetyl group-2,3,5-three-O-benzoyl-β-L-ribofuranose 143Analysis:

mp129-130℃(EtOH95)(lit.(1)mp130-131℃)

¹H NMR (200MHz, CDCI ₃): δ 8.09-7.87 (m, 6H, H _Arom), 7.62-7.31 (m, 9H, H _Arom) 6.43 (s, 1H, H ₁), 5.91 (dd, 1H, H ₃, J _3,46.7Hz, J _3,24.9Hz), 5.79 (pd, 1H, H ₂, J _3,24,9Hz; J _1,2＜1), 4,78 (m, 2H, H ₄And H ₅), 4,51 (dd, 1, H ₅, J _5,513,1Hz, J ₅' _{, 4}5,5Hz), 2,00 (s, 3H, CH ₃CO); (with commodity 1-O-acetyl group-2,3,5-three-O-benzoyl-β-D-ribofuranose)

Mass spectral analysis (FAB+, GT) m/z445 (M-OAc)+

Elementary analysis C ₂₈H ₂₄O ₉Value of calculation C66.66H4.79; Measured value CH

Reaction 2:

Precursor: adenine (Pharma-Waldhof; Ref400134.001lot45276800)

Reaction reagent: the stannic chloride (Fluka of being fuming; Ref96558); NH3/ methanol (NH ₃Saturated methanol; See page 5); Sodium sulfate (Prolabo; Ref28111.365)

Solvent: acetonitrile (Riedel-de Hean; Ref33019; CaH ₂Distillation); Chloroform Pur (Acros; Ref22706463); Ethyl acetate Pur (Carlo erba; Ref528299)

List of references: Saneyoshi, M., and Satoh, E., Synthetic Nucleosides andNucleotides.XIII Stannic Chloride Catalyzed Ribosylation of Several6-Substituted Purines.Chem.Pharm.Bull., 27,2518-2521 (1979); Nakayama, C., and Saneyoshi, M., Synthetic Nucleosides and Nucleotides.XX.Synthesis of Various 1-β-Xylofuranosyl-5-Alkyluracils and RelatedNucleosides, Nucleosides, Nucleotides, 1,139-146 (1982).

(19.6g 144mmol) is suspended in acetonitrile (400ml) and 1-O-acetyl group-2,3,5-three-O-benzoyl-β-L-ribofuranose with adenine 143In.To this suspension add the stannic chloride of being fuming (22ml, 187mmol). after 12 hours, this reaction is concentrated to small size (approximately 100ml), adds sodium bicarbonate (110g) and water (120ml).Resulting white solid (pink salt) merges extract and filters on bed of diatomaceous earth with hot chloroform (5x200ml) extraction.Organic facies 5%NaHCO ₃Solution and water washing, dried over sodium sulfate is filtered, and evaporation obtains chemical compound 144(60g, colourless foam).This foam under agitation uses the saturated methanol of ammonia (220ml) to handle 4 days in room temperature in the reaction bulb of sealing.Reduction vaporization falls solvent, and the gained powder suspension is (400ml) in ethyl acetate, refluxes 1 hour.After the filtration, powder obtains the L-adenosine by water (220ml) recrystallization 145(24g, crystallization, 75%).

The analysis of β-L-adenosine:

Mp233-234 ℃ (water) (mp235-238 ℃ of lit. (4))

¹H NMR (200MHz, DSMO-D ₆): δ 8.34-8.12 (2s, 2H, H ₂, and H ₈), 7.37 (1s, 2H, NH ₂), 5.86 (d, 1H, H ₁', J ₁' _{, 2}' _,6.2Hz), 5.43 (m, 2H, OH ₂' and OH ₅'), 5.19 (d, 1H, OH ₃', J3.7Hz), 4.60 (m, H ₂'), 4.13 (m, 1H, H ₃') 3.94 (m, 1H, H ₄'), 3.69-3.49 (m, 2H, H ₅' _aAnd H ₅' _b), (with commodity D-adenosine)

Mass spectral analysis (FAB+, GT) m/z268 (M+H) ⁺, 136 (BH ₂) ⁺

Reaction 3:

Reaction reagent: 1,3-two chloro-1,1,3,3-tetra isopropyl disiloxane (Fluka; Ref36520); Sodium sulfate (Prolabo; Ref28111.365)

Solvent: pyridine 99% (Acros; Ref131780025), ethyl acetate Pur (Carlo erba; Ref528299); Acetonitrile (Riedel-de Hean; Ref33019)

List of references: Robins, M.J., Deng the people, Nucleic Acid Celated Compounds.42.A General Procedure for the Efficient Deoxygenation of SecondaryAlcohols.Regiospecific and Stereoselective Conversion of Ribonucleosidesto2 '-Deoxynucleosides.J.Am.Chem.Soc.105,4059-4065 (1983).

L-adenosine in being suspended in pyridine (320ml) 145(47,2g 177mmol) adds 1,3-two chloro-1,1,3, and (63ml 201mmol), at room temperature stirred this mixture 12 hours the 3-tetra isopropyl disiloxane.Evaporate pyridine, residue is allocated in ethyl acetate (1L) and 5%NaHCO ₃Among the solution (600ml).Organic facies is washed with solution (2x500ml) and the water (500ml) of 0.5N HCl, and dried over sodium sulfate is filtered evaporate to dryness.Resulting solid obtains chemical compound by the acetonitrile crystallization 146(81g, 90%).

3 ', 5 '-O-(1,1,3,3-tetra isopropyl-1,3-disiloxane base)-β-L-adenosine 146Analysis:

Mp97-98 ℃ (acetonitrile) (98 ℃ of lit. (5) D-enantiomer)

¹H NMR (200MHz, CDCl ₃): δ 8.28 and 7.95 (2s, 2H, H ₂And H ₈), 5.96 (d, 1H, J ₁' _{, 2}' 1,1Hz), 5.63 (s, 2H, NH ₂), 5.10 (dd, 1H, H ₃', J ₃' _{, 4}' 7.6Hz, J ₃' _{, 2}' 5.5Hz), 4.57 (dd, 1H, H ₂', J ₂' _{, 1}' 1.2Hz; J _2,3, 7.6Hz), 4.15-3.99 (m, 3H, H ₄', H ₅' _aAnd H ₅' _b), 3.31 (s1,1H, OH ₂'), 1.06 (m, 28H, isopropyl protons)

Mass spectral analysis (FAB-, GT) m/z508 (M-H) ^-, 134 (B) ^-(FAB+, GT) m/z510 (m+H) ⁺, 136 (BH ₂) ⁺

Reaction 4:

Reaction reagent:

Dimethyl aminopyridine 99% (Acros; Ref1482702050);

Chloro thioncarbonic acid phenylester 99% (Acros; Ref215490050);

Three (trimethylsilyl) silane, " TTMSS " (Fluka; Ref93411);

α, and α '-azoisobutyl cyanide ＂ AIBN ＂ (Fluka, ref11630);

Sodium sulfate (Prolabo; Ref28111.365)

Solvent: acetonitrile (Riedel-de Haen; Ref33019); Ethyl acetate Pur (Carlo Erba; Ref528299); Diox P.A. (Merck; Ref1.09671.1000); Dichloromethane (merck; Refl.06050.6025); Methanol (Carlo Erba; Ref309002);

List of references: Robins, M.J., Wilson, J.S., and Hansske, F., NucleicAcid Related Compounds.42.A General Procedure for the EfficientDeoxygenation of Secondary Alcohols.Regiospecific and StereoselectiveConversion of Ribonucleosides to2 '-Deoxynucleosides.J.Am.Chem.Soc., 105,4059-4065 (1983).

To chemical compound 146(34g, (10.2ml, 73mmol), this solution at room temperature stirred 12 hours 67mmol) to add acetonitrile (280ml), DMAP (16.5g135mmol) and chloro thioncarbonic acid phenylester.Evaporate solvent, residue distributes in the solution (400ml) of ethyl acetate (400ml) and 0.5N HCI.Organic layer washs with solution (400ml) and the water (2x400ml) of HCI0.5N, and dried over sodium sulfate is filtered and evaporate to dryness, obtains the light yellow solid intermediate.With this crude product 147Be dissolved in (ml) in the diox, and add AIBN (3.3g, 20mmol) and TTMSS (33ml, 107mmol).This solution progressively is heated to backflow, and stirred 2 hours.Reactant is condensed to yellow oil, carries out chromatographic isolation (eluant: methylene chloride 95/5), obtain chemical compound 148(23g, colourless foam, 70%).Aliquot is by ethanol/petroleum ether crystallization.

3 ', 5 '-O-(1,1,3,3-tetra isopropyl-1,3-disiloxane base)-2 '-deoxidation-β-L-adenosine 148Analysis:

Mp:110-111 ℃ (EtOH/ petroleum ether) (Lit. (5) mp113-114 ℃ (EtOH))

¹H NMR (200MHz, CDCl ₃): δ 8.33 and 8.03 (2s, 2H, H ₂And H ₈), 6.30 (dd, 1H, H ₁', J2.85Hz, J7.06Hz), 5.63 (s1,2H, NH ₂), 4.96 (m, IH, H ₃'), 4.50 (m, 2H, H _5aAnd H _5b), 2,68 (m, 2H, H ₂' _aAnd H ₂' _b), 1.08 (m, 28H, isopropyl protons)

Mass spectral analysis (FAB+, GT) m/z494 (M+H) ⁺, 136 (BH ₂) ⁺

Reaction 5:

Reaction reagent: ammonium fluoride (Fluka; Ref09742); Silica gel (Merck; Ref1.07734.2500)

Solvent: methanol P.A. (Prolabo; Ref20847.295); Dichloromethane P.A. (Merck; Refl.06050.6025); Alcohol 95 % (Prolabo; Ref 20823.293)

List of references: Zhang, W., and Robins, M.J., Removal of SilylProtecting Groups from Hydroxyl Functions with Anunonium Fluoride inMethanol., Tetrahedron Lett., 33,1177-1180 (192).

3 ', 5 '-O-(1,1,3,3-tetra isopropyl-1,3-disiloxane base)-2 '-deoxidation-β-L-adenosine 148(32g, 65mmol) and ammonium fluoride (32g, methanol solution mmol) stirred 2 hours under refluxing.Add silica gel, this mixture is evaporated carefully, obtain white powder.This powder is added to the top of silicagel column, with methylene chloride 9/1 eluting, merges suitable fraction, evaporation obtains white powder, then by ethanol (95%) crystallization (12.1g, 75%).

2 '-analysis of deoxidation-β-L-adenosine 149:

Mp189-190 ℃ (EtOH95) (with commodity 2 '-deoxidation-D-adenosine)

¹H NMR (200MHz, DMSO-D ₆): δ 8.35 and 8.14 (2s, 2H, H ₂And H ₈), 7.34 (s1,2H, NH ₂), 6.35 (dd, 1H, H ₁., J6.1Hz, J7.85Hz), 5.33 (d, 1H, 0H ₂', J4.0Hz), 5.28 (dd, 1H, H ₃', J4.95Hz; J6.6Hz), 4.42 (m, 1H, OH5 '), 3.88 (m, 1H, H ₄'), 3.63-3.52 (m, 2H, H ₅' _aAnd H ₅' _b), 2,71 (m, I H, H ₂' _a), 2.28 (m, 1H, H ₂' _b). (with commodity 2 '-deoxidation-D-adenosine)

α _D+ 26 ⁰(c0.5 water) (commodity 2 '-deoxidation-D-adenosine-25 ⁰(c0.5 water)).

UVλmax260nm(ε14100)(H ₂O).

Mass spectral analysis (FAB+, GT) m/z252 (M+H) ⁺, 136 (BH ₂) ⁺

The stereospecific synthesis of embodiment 32 '-deoxidation-β-L-cytidine

1-(3,5-two-O-benzoyl-β-L-xylose type furyl glycosyl) uracil ( 11)

(1.4mL 28.7mmol) joins 1-(2-O-acetyl group-3,5-two-O-benzoyl-β-L-xylose type furyl glycosyl) uracil with hydrazine hydrate 10[consult: Gosselin, G.; Bergogne, M.-C.; Imbach, J.L, ＂ Synthesis and Antiviral Evaluation of β-L-xylofiiranosyl Nucleosides of the Five Naturally Occuring NucleicAcid Bases ＂, Journal of Heterocyclic Chemistry, 1993,30 (Oct.-Nov.), 1229-1233] (4.79g, 9.68mmol) solution in pyridine (60mL) and acetic acid (15mL).This solution at room temperature stirs and spends the night.Add acetone (35mL), stirred the mixture 30 minutes.Reduction vaporization.The gained residue is obtained by silicagel column chromatography [eluent: the stepwise gradient methanol (0-4%) in dichloromethane] purification 11(3.0g, 68%) is by cyclohexane extraction/dichloromethane crystallization: mp=111-114 ℃; ¹H-NMR (DMSO-d ₆): δ 11.35 (br s, 1H, NH), 7.9-7.4 (m, 11H, 2C ₆H ₅CO, H-6), 6.38 (d, 1H, OH-2 ', J _OH-2=4.2Hz), 5.77 (d, 1H, H-I ', J ₁' _{, 2}'=1.9Hz), 5.55 (d, IH, H-5, J _5-6=8Hz), 5.54 (dd, 1H, H-3 ', J ₃' _{, 2}'=3.9Hz and J ₃' _{, 4}'=1.8Hz), 4.8 (m, 1H, H-4 '), 4.7 (m, 2H, H-5 ' and H-5 ＂), 4.3 (m, 1H, H-2 '); MS:FAB〉0 (substrate GT) m/z453 (M+H) ⁺, 105 (C ₆H ₅CO) ⁺FAB＜0 (substrate GT) m/z451 (M-H); 121 (C ₆H ₅CO ₂) ^-, 111 (B) ^-Elementary analysis value of calculation C ₂₃H ₂₀N ₂O ₈H ₂O:C, 58.09; H, 4.76; N, 5.96. measured value: C, 57.71; H, 4.42; N, 5.70.

1-(3,5-two-O-benzoyl-β-L-arabinose type furyl glycosyl) uracil ( 12)

To 1-(3,5-two-O-benzoyl-β-L-xylose type furyl glycosyl) uracil 11(8g, 17.7mL) anhydrous benzene-DMSO mixture (265mL, 6:4, add in solution v/v) anhydrous pyridine (1.4mL), dicyclohexylcarbodiimide (10.9g, 53mmol) and dichloroacetic acid (0.75mL).Resulting mixture at room temperature stirred 4 hours, then used ethyl acetate (400mL) dilution, and added oxalic acid (4.8g, 53mmol) solution in methanol (14mL).Stir after 1 hour filtering solution.Filtrate is with saturated NaCl solution (2x500mL), 3%NaHCO ₃Solution (2x500mL) and water (2x500mL) washing.Organic facies Na ₂SO ₄Drying, then reduction vaporization.The gained residue be dissolved in absolute EtOH-benzol mixture (140mL, 2:1, v/v) in, add N at 0 ℃ to this solution _aBH ₄(0.96g 26.5mmol), stirs after 1 hour, and (400mL) dilutes this solution with ethyl acetate, and after-filtration.Filtrate is washed with saturated NaCl solution (400mL) and water (400mL). organic facies Na ₂SO ₄Drying, then reduction vaporization.The thick product of gained is obtained by silicagel column chromatography purification [eluent: the stepwise gradient methanol (0-3%) in the dichloromethane] 12(5.3g, 66%) is by acetonitrile crystallization: mp=182-183 ℃; ¹H-NMR (DMSO-d ₆): δ 11.35 (br s, 1H, NH), 8.0-7.5 (m, 11H, 2C ₆H ₅CO, H-6), 6.23 (br s, 1H, OH-2 '), 6.15 (d, 1H, H-1 ', J ₁' _{, 2}'=4Hz), 5.54 (d, IH, H-5, J _5-6=8.1Hz), 5.37 (t, 1H, H-3 ', J ₃' _{, 2}'=J ₃' _{, 4}'=2.6Hz), 4.7-4.6 (m, 2H, H-5 ' and H-5 ＂), 4.5 (m, 1H, H-4 '); MS:FAB〉0 (substrate GT) m/z453 (M+H) ⁺, 341 (S) ⁺, 113 (BH ₂) ⁺' 105 (C ₆H ₅CO) ⁺FAB＜0 (substrate GT) m/z451 (M-H); 121 (C ₆H ₅CO ₂) ^-, 111 (B) ^-Elementary analysis value of calculation C ₂₃H ₂₀N ₂O ₈H ₂O:C, 61.06; H, 4.46; N, 6.19. measured value: C, 60.83; H, 4.34; N, 6.25.

1-(3,5-two-O-benzoyl-2-deoxidation-β-L-is red-arabinose type furyl glycosyl) uracil ( 13)

To 1-(3,5-two-O-benzoyl-β-L-arabinose type furyl glycosyl) uracil ( 12) (5.2g, 11.4mmoL) anhydrous dichloroethane solution (120mL) add phenoxy group sulfo-phosgene (4.7ml, 34.3mmoL) and the 4-dimethylamino pyridine (DMAP, 12.5g, 102.6mmoL).Resulting solution at room temperature with in the argon atmospher stirred 1 hour, then reduction vaporization.Residue is dissolved in the dichloromethane (300mL), and organic solution is washed successively with ice-cold 0.2N hydrochloric acid solution (3x200mL) and water (2x200mL), uses Na ₂SO ₄Drying, reduction vaporization.This product and is dissolved in (110mL) in this solvent with no Shui diox coevaporation several times.To resulting solution under argon atmospher, add three (three silicyls) silane hydrate (4.2mL, 13.7mmol) and α, α '-azo isobutyronitrile (AIBN, 0.6g, 3.76mmol).With this reactant mixture heating, under argon atmospher,, then be cooled to room temperature and reduction vaporization in 100 ℃ of stirrings 1 hour.Residue is obtained by silicagel column chromatography purification [eluent: methanol (0-5%) gradient elution] 13(2.78g, 56%) is by EtOH crystallization: mp=223-225 ℃; ¹H-NMR (DMSO-d ₆): δ 11.4 (brs, 1H, NH), 8.0-7.5 (m, 11H, 2C ₆H ₅CO, H-6), 6.28 (t, 1H, H-1 ', J=7Hz), 5.5 (m, 2H, H-1 ' and H-5), 4.6-4.4 (m, 3H, H-4 ', H-5 ' and H-5 ＂), 2.6 (m, 2H, H-2 ' and H-2 ＂); MS:FAB〉0 (substrate GT) m/z437 (M+H) ⁺, 3325 (S)+; FAB＜0 (substrate GT) m/z435 (M-H) ^-, 111 (B) ^-Elementary analysis value of calculation C ₂₃H _2-N ₂O ₇: C, 63.30; H, 4.62; N, 6.42. measured value: C, 62.98; H, 4.79; N, 6.40.

2-deoxidation-β-L-cytidine (β-L-dC)

(1.72g 4.26mmol) joins 1-(3,5-two-O-benzoyl-2-deoxidation-β-L-is red-arabinose type furyl glycosyl) uracil under argon atmospher with Lawesson ' s reagent 13(2.66g, 6.1mmol) anhydrous dichloroethanes (120mL) solution in, reaction mixture refluxed stirred 2 hours, decompression then steams solvent, residue obtains yellow foamed 4-sulfo-intermediate by silicagel column chromatography purification [eluent: the stepwise gradient ethyl acetate (0-8%) in the dichloromethane].With this sulfo-intermediate (1.5g, 3.31mmol) solution in methanol ammonia (in advance at-10 ℃ of saturated and jam-packs) is (50mL) at 100 ℃, heating then was cooled to 0 ℃ in 3 hours in the rustless steel pressure pan.Decompression steams solvent.Resulting thick product is by silicagel column chromatography purification [eluent: the stepwise gradient methanol (0-20%) in the dichloromethane].At last, suitable fraction is merged, by MillexHV-4 (0.45 μ m Millipore) filters, reduction vaporization, (β-L-dC) (0.6g, 80%) is by absolute ethanol crystallization: mp=198-199 ℃ to obtain desirable foamed 2-deoxidation-β-L-cytidine; ¹H-NMR (DMSO-d ₆): δ 7.77 (d, 1H, H-6, J _6-5=7.4Hz), 7.10 (br d, 2H, NH- ₂), 6.13 (t, 1H, H-I ', J=6.7Hz), 5.69 (d, IH, H-5, J _5-6=7.4Hz), 5.19 (d, 1H, OH-3 ', J _OH-3'=4.1Hz), 4.96 (t, 1H, OH-5 ', J _OH-5'=J _OH-5"=5.2Hz), 4.1 (m, IH, H-3 '), 3.75 (m, 1H, H-4 '), 3.5 (m, 2H, H-5 ' and H-5 ＂), 2.0 (m, IH, H-2 '), 1.9 (m, IH, H-2 ＂); MS:FAB〉0 (substrate GT) m/z228 (M+H) ⁺, 112 (8H ₂) ⁺FAB＜0 (substrate GT) m/z226 (M-H)-; [α] _D ²⁰=-69 (c0.52, DMSO) [[α] _D ²⁰=+76 (c0.55, DMSO) the commodity hydrochlorate of D-enantiomer].Elementary analysis value of calculation C ₉H ₁₃N ₃O ₄: C, 47.57; H, 5.77; N, 18.49. measured value: C, 47.35; H, 5.68; N, 18.29.

The embodiment 42 '-deoxidation-β-L-cytidine (stereospecific synthesis of β-L-dC)

2-amino-beta--L-arabinofuranosyl adenin also [1 ', 2 ': 4,5] oxazolines ( 1)

The L-arabinose (170g, 1.13mol), cyanamide (100g, 2.38mol), methanol (300ml) and 6M-NH ₄The mixture of OH (50ml) at room temperature stirred 3 days, then remained on-10 ℃ and spent the night.This product collected at suction is washed vacuum drying successively with methanol and ether.Obtain analytically pure chemical compound 1, 130g (66.0%): m.p170-172 ℃; ¹H NMR (DMSO-d ₆) δ ppm6.35 (br5,2H, NH ₂), 5.15 (d, 1H, H-1, J=5.6Hz), 5.45 (br s, 1H, OH-3), 4.70 (br s, 1H, OH-5), 4.55 (d, 1H, H-2, J=5.6Hz), 4.00 (br s, 1H, H-3), 3.65 (m, 1H, H-4), 3.25 (m, 2H, H-5, H-5 ').

Reaction reagent: L-arabinose: Fluka,〉99.5%, ref10839

Cyanamide: Fluka,〉98%, ref28330

O ^2,2'-dehydration-β-L-uridnine ( 2)

With chemical compound 1(98.8g, 0.57mol) and methyl propiolate (98ml) refluxed 5 hours at the solution of 50% aquiferous ethanol (740ml), and postcooling, concentrating under reduced pressure are to half of initial volume.Using acetone (600ml) post precipitation, the product collected at suction is with ethanol and ether washing, drying.Partly concentrated mother liquor with acetone (1000mL) precipitation concentrated solution, solid suction method collection, with acetone and ether washing, obtains another product.Total output 80g (62%) chemical compound 2, m.p.236-240 ℃; ¹H NMR (DMSO-d ₆) δ ppm7.87 (d, 1H, H-6, J=7.4Hz), 6.35 (d, 1H, H-I ', J=5.7Hz), 5.95 (d, 1H, H-5, J=7.4Hz), (5.90 d, 1H, OH-3 '), 5.20 (d, 1H, H-2 ', J=5.7Hz), 5.00 (m, 1H, OH-3 '), (4.44 brs, 1H, H-3 '), 4.05 (m, 1H, H-4 '), 3.25 (m, 2H, H-5, H-5 ').

Reaction reagent: acetylenecarboxylic acid methyl ester: FIuka,〉97%, ref81863

3 ', 5 '-two-O-benzoyl-O ^2,2'-dehydration-β-L-uridnine ( 3)

To chemical compound 2(71.1g, the 0.31mol) solution in anhydrous pyridine (1200ml) add Benzenecarbonyl chloride. (80.4ml) under 0 ℃ and argon atmospher.This reactant mixture is in room temperature and get rid of under the condition of atmospheric moisture and stirred 5 hours, adds the ethanol stopped reaction.Decompression steams solvent, and the gained residue is with toluene and absolute ethanol coevaporation.Then with this crude product mixture ethanol dilution, the collected at suction precipitation is washed successively with ethanol and ether, and drying obtains 129g (95.8%) chemical compound 3, m.p.254 ℃; ¹H NMR (DMSO-d ₆) δ ppm8.1-7.4 (m, 11H, C ₆H ₅CO, H-6), 6.50 (d, 1H, H-1 ', J=5.7Hz), 5.90 (d, 1H, H-5, J=7.5Hz), 5.80 (d, 1H, H-2 ', J=5.8Hz), 5.70 (d, 1H, H-3 ') 4.90 (m, 1H, H-4 '), 4.35 (m, 2H, H-5, H-5 ').

Reaction reagent: Benzenecarbonyl chloride.: Fluka, p.a., ref12930

3 ', 5 '-two-O-benzoyl-2 '-chloro-2 '-deoxidation-β, the L-uridnine ( 4)

At 0 ℃ to chemical compound 3(60.3g, 0.139mol) solution in dimethyl formamide (460ml) adds 3.2N-HCl/DMF solution (208ml is by in situ preparation in the solution that the 47.2ml chloroacetic chloride is added 27.3ml methanol and 133.5ml dimethyl formamide at 0 ℃).This reactant mixture stirred 1 hour under the condition of getting rid of atmospheric moisture in 100 ℃, and water (4000ml) is injected in the back cooling down.Chemical compound 4The precipitation collected at suction, wash ethyl alcohol recrystallization with water.Collect this crystallization, with cold ethanol and ether washing, drying under reduced pressure.Obtain chemical compound 4Product 60.6g (92.6%), m.p.164-165 ℃; ¹H NMR (DMSO-d ₆) δ ppm8.7 (br s, 1H, NH), 8.1-7.3 (m, 11H, C ₆H ₅CO, H-6), 6.15 (d, 1H, H-1 ', J=4.8Hz), 5.5 (m, 2H, H-5, H-2 '), 4.65 (m, 4H, H-3 ', H-4 ', H-5 ', H-5 ＂).

Reaction reagent: chloroacetic chloride: Fluka, p.a., ref00990

3,5 '-two-O-benzoyl-2 '-deoxidation-β, the L-uridnine ( 5)

Make chemical compound 4(60.28g, 0.128mol), tri-n-butyl tin hydrate (95ml) and the mixture of azo diisobutyl nitrile (0.568g) in dry toluene (720ml) under agitation refluxed 5 hours, and postcooling.Solid is collected with the suction method, with cold toluene and petroleum ether.Filtrate decompression concentrates, with the petroleum ether dilution, to obtain another part chemical compound 5, output 54.28g (97.2%): m.p.220-221 ℃; ¹H NMR (CDCl ₃) δ ppm8.91 (br s, 1H, NH), 8.1-7.5 (m, 11H, C ₆H ₅CO, H-6), 6.43 (q, 1H, H-1 ', J ₁' _{, 2}'=5.7Hz and J ₁' _{, 2 "}=8.3Hz), 5.7-5.6 (m, 2H, H-3 ' and H-5), 4.84.6 (m, 3H, H-5 ', H-5 ＂ and H-4 '), 2.8-2.7 (m, 1H, H-2 '), 2.4-2.3 (m, 1H, H-2 ＂).

Reaction reagent: tri-n-butyl tin hydrate: FIuka, 98%, ref90915

Azo diisobutyl nitrile:: FIuka, 98%, ref11630

3 ' 5 '-two-O-benzoyl-2 '-deoxidation-β, L-4-sulfo--uridnine ( 6)

With chemical compound 5(69g, 0.158mol) and the solution of Lawesson ' s reagent (74g) in anhydrous methylene chloride (3900ml) under argon atmospher, stir and spend the night.After steaming solvent, and thick residue silicagel column chromatography purification (eluant: the gradient methanol (0-2%) in the dichloromethane], obtain quantitative pure compound 6(73g): ¹H NMR (CDCl ₃) δ ppm9.5 (br s, 1H, NH), 8.1-7.4 (m, 10H, C ₆H ₅CO), 7.32 (d, 1H, H-6, J=7.7Hz), (6.30 dd, 1H, H-1 ', J=5.6Hz and J=8.2Hz), 6.22 (d, 1H, H-5, J=7.7Hz), 5.6 (m, 1H, H-3 '), 4.7 (m, 2H, H-5 ', H-5 ＂), (4.5 m, 1H, H-4 '), 2.8 (m, 1H, H-2 '), 2.3 (m, 1H, H-2 ＂).

Reaction reagent:

Lawesson ' s reagent: FIuka,〉98%, ref61750

2 '-deoxidation-β-L-cytosine

With chemical compound 6(7.3g 0.016mol) in the solution in the saturated methanol of ammonia (73ml), heated 3 hours at 100 ℃ in stainless steel cylinder.After careful cooling, decompression steams solvent.The aqueous solution of residue washs with ethyl acetate, and evaporate to dryness.Method is prepared chemical compound like this 6Other 9 duplicate samples (every part of 7.3g) (amount to 6=73g).Merge 10 parts of residues, with the absolute ethanol dilution, and cooling, obtain as crystalline 7By leaching method (in ethyl acetate reflux 1 hour) from 6Remove residual Benzoylamide in the crystallization.Chemical compound 6Output, 28.75g (78.6%): m.p.141-145 ℃; ¹H NMR (DMSO) δ ppm8.22 and 8.00 (2brs, 2H, NH ₂), 7.98 (d, 1H, H-6, J=7.59Hz), 6.12 (t, 1H, H-1 ', J=6.5Hz and J=7.6Hz), 5.89 (d, 1H, H-5, J=7.59Hz), 5.3 (br s, 1H, OH-3 '), 5.1 (br5,1H, OH-5 '), 4.2 (m, 1H, H-3 '), 3.80 (q, 1H, H-4 ', J=3.6Hz and J=6.9Hz), 3.6-3.5 (m, 2H, H-5 ', H-5 ＂), 2.2-2.0 (m, 2H, H-2 ', H-2 ＂); FAB＜0, (GT) m/e226 (M-H) ^-110 (B) ^-FAB〉0 (GT) 228 (M+H) ⁺, 112 (B+2H) ⁺[α] _D ²⁰=-56.48 (c=1.08 is in DMSO); UV (pH7) λ max270nm (ε 10000).

Reaction reagent:

Methanol ammonia: in advance-5 ℃ saturated, envelope is tight, is kept in the refrigerator.

The embodiment 52 '-deoxidation-β-L-thymidine (stereospecific synthesis of β-L-dT)

3,5 '-two-O-benzoyl-2 '-deoxidation-5-iodo-β, the L-uridnine ( 7)

With chemical compound 5(105.8g, 0.242mol), the mixture of iodine (76.8g), CAN (66.4g) and acetonitrile (2550ml) stirred 3 hours at 80 ℃, then this reactant mixture cooled off in room temperature, formed chemical compound 7Crystallization (6.6g, 63.5%); M.p.192-194 ℃; ¹H NMR (DMSO) δ ppm.8.34 (s, 1H, NH), 8.2-7.2 (m, 11H, 2C ₆H ₅CO, H-6), 6.31 (q, 1H, H-1 ', J=5.5Hz and J=8.7Hz), 5.5 (m, 1H, H-3 '), 4.7 (m, 2H, H-5 ', H-5 ＂), 4.5 (m, 1H, H-4 '), 2.7 (m, 1H, H-2 '), 2.3 (m, 1H, H-2 ＂); FAB＜0, (GT) m/e 561 (M-H) ^-237 (B) ^-FAB〉0 (GT) 563 (M+H) ⁺[α] _D ²⁰+ 39.05 (c=1.05 is in DMSO); UV (EtOH95%) λ max=281nm (ε=9000), λ min=254nm (ε=4000), λ max=229nm (ε=31000); Elementary analysis value of calculation C ₂₃H ₁₉IN ₂O ₇: C, 49.13H, 3.41N, 4.98I, 22.57. measured value: C, 49.31H, 3.53N, 5.05I, 22.36.

Reaction reagent: iodine: Fluka, 99.8%, ref57650

Ammonium ceric nitrate (CAN): Aldrich,〉98.5%, ref21,547-3

3 ', 5 '-two-O-benzoyl-2 '-deoxidation-3-N-toluyl-β, the L-thymidine ( 9)

To chemical compound 7(86.6g, 0.154mol) in the anhydrous pyridine that comprises N-ethyl diisopropylamine (53.6ml) (1530ml) solution, adding P-Toluoyl chloride (40.6ml) at 0 ℃ in batches. reactant mixture then adds water retention and ends reaction, reactant mixture dichloromethane extraction stirring at room 2 hours.Organic facies washes with water, dried over sodium sulfate, and evaporate to dryness, obtain thick 3 ', 5 '-two-O-benzoyl-2 '-deoxidation-3-N-toluyl-5-iodo-β, the L-uridnine ( 8), it can be used for next step reaction and need not be further purified.

Crude mixture 8, acid chloride (3.44g), triphenyl phasphine (8.0g) be in the solution of N-Methyl pyrrolidone (1375ml) and triethylamine (4.3ml), stirring at room 45 minutes.Then under argon atmospher, drip tetramethyl tin (42.4ml) at 0 ℃.After 100-110 ℃ of stirring spent the night, this reactant mixture is injected water, use extracted with diethyl ether.Organic solution dried over sodium sulfate, and concentrating under reduced pressure.Residue obtains foamed chemical compound by silicagel column chromatography purification [eluant: in toluene with ethyl acetate (0-10%) gradient elution] 9(42.3g, 48.3%, the second step). ¹H?NMR(DMSO)δ?ppm.8.3-7.2(m，15H，2C ₆H ₅CO，1CH ₃C ₆H ₄CO，H-6)，6.29(t，1H，H-1′，J＝7.0Hz)，5.7(m，1H，H-3′)，4.7-4.5(m，3H，H-5′，H-5＂，H-4′)，2.7-2.6(m，2H，H-2′，H-2＂)；FAB<0，(GT)m/e567(M-H) ^-，449(M-CH ₃C ₆H ₄CO) ^-，243(B) ^-，121(C ₆H ₅COO) ^-，FAB>0(GT)1137(2M+H) ⁺，569(M+H) ⁺，325(M-B) ^-，245(B+2H) ⁺，119(CH ₃C ₆H ₅CO) ⁺。

Reaction reagent: P-Toluoyl chloride: Aldrich, 98%, ref10,663-1

Diisopropylethylamine: Aldrich,〉99.5%, ref38,7～9

N-Methyl pyrrolidone: Aldrich,〉99%, ref44,377-8

Acid chloride: Aldrich,〉99.98%, ref37,987-5

Triphenyl phasphine: Fluka,〉97%, ref93092

Tetramethyl tin: Aldrich,〉99%, ref14,647-1

2 '-deoxidation-β-L-thymidine

Chemical compound 9(42.3g, methanol solution 0.074mol) (1850ml) (this solution is saturated with ammonia) at room temperature stirred 2 days.Behind evaporating solvent, residue diluted with water and for several times with ethyl acetate washing.Separate water layer, reduction vaporization, residue with silica gel chromatography [eluant: in dichloromethane with methanol (0-10%) gradient elution] obtain 2 '-deoxidation-β-L-thymidine, crystallization in ethanol (11.62g, 64.8%).M.p.185-188 ℃; ¹H NMR (DMSO) δ ppm11.3 (s, 1H, NB), 7.70 (s, 1H, H-6), (6.2 pt, 1H, H-1 '), 5.24 (d, 1H, OH-3 ', J=4.2Hz), 5.08 (t, 1H, OH-5 ', J=5.1Hz), 4.2 (m, 1H, H-3 '), 3.7 (m, 1H, H-4 '), 3.5-3.6 (m, 2H, H-5 ', H-5 ＂), 2.1-2.0 (m, 2H, H-2 ', H-2 ＂); FAB＜0, (GT) m/e483 (2M-H); 349 (M+T-H); 241 (M-H); 125 (B) ^-FAB〉0 (GT) 243 (M+H)+, 127 (B+2H) ⁺) ⁺[α] _D ²⁰-13.0 (c=1.0 is in DMSO); UV (pH1) λ _Max=267nm (∈=9700), λ _Min=234nm (∈=2000).

Reagent:

The embodiment 62 '-deoxidation-β-L-inosine (stereospecific synthesis of β-L-dI)

β-L-dI by 2 '-(deamination of β-L-dA) is synthetic for deoxidation-β-L-adenosine, adjoining the described method of glucopyranoside series according to aforementioned 9-D-carries out (consulting: I.Iwal, T.Nishimura and B.Shimizu, Synthetic Procedures in Nucleic Acid Chemistry, W.W.Aorbach and R.S.Tipson, eds., John Wiley﹠amp; Sons, Inc.New York, vol.1, pp.135-138 (1968)).

The solution same sodium nitrite (495mg) of β-L-dA (200mg) in acetic acid (0.61ml) and water (19ml) mixture heated together, and with this mixture in stirred overnight at room temperature.Evaporated under reduced pressure then.The aqueous solution of its residue IR-120 (H+) treatment on ion exchange columns, this post water eluting is collected suitable fraction and evaporate to dryness, obtains pure β-L-dI (106mg, productive rate 53%, non-optimum yields), uses methanol crystallization.: m.p.=209 ⁰-211 ℃; UV (H ₂O), λ _Max=247nm; ¹H-NMR (DMSO-d ₆) δ 8.32 and 8.07 (2s, each 1H, H-2 and H-8), 6.32 (Pt, 1H, H-1; J=6.7Hz), 4.4 (m, 1H, H-3 '), 3.9 (m, 1H, H-4 '), 3.7-3.4 (m, by the 2H that HOD partly covers, H-5 ', 5 ＂), 2.6 and 2.3 (2m, each 1H, H-2 ' and H-2 ＂); Mass spectrum (substrate, glycerol-thioglycerol, 1:1, v/v), and FAB〉0:253 (m+H) ⁺, 137 (alkali+2H) ⁺FAB＜0:251 (m-H) ^-, 135 (alkali) ^-[α] _D ²⁰=+19.3 (c0.88, H ₂O).

Anti--HBV the activity of reactive compound

According to following detailed description, can go up the ability that reactive compound suppresses viral growth of estimating at 2.2.15 cell cultivation instrument (the HepG2 cell is changed with hepatitis virus).

To the summary of test of the antiviral effect that in this system, carries out and HBV DNA analysis and explanation at Korba and Milman, 1991, Antiviral Res., open among the 15:217.The evaluation of antiviral effect is carried out on two cell passages that separate.All caves on all plates are at one time with same density inoculation.

Because HBV DNA cell is interior and the intrinsic variation of extracellular levels, for the average level of these HBV DNA various ways in the untreated cell, suppression ratio is only just thought quite satisfied (p＜0.05) greater than 3.5 times (to HBV virion DNA) or 3.0 times (to HBV dna replication dna intermediate).Comprehensive HBV dna level can be used to calculate level in the cell of each form of HBVDNA in each cell DNA sample (making the basic value of each cell keep a constant in these trials), can confirm that thus the cell DNA of equal quantities in the different samples has comparability.

In untreated cell, the exemplary value scope of extracellular HBV virion DNA is 50-150pg/ml culture medium (meansigma methods approximately is 76pg/ml).In untreated cell, the exemplary value scope of HBV dna replication dna intermediate is 50-100 μ g/pg cell DNA (meansigma methods approximately is 74 μ g/pg cell DNAs) in the cell.In general, owing to handle with antiviral compound, compare with the decline level of HBV virion DNA, the decline level of HBV DNA is not obvious in the cell, and occur slower (Korba and Milman, 1991, AntiviralRes., 15:217).

This method is that the result with these tests carries out the hydridization analysis, analysis be with about 1.0pg cell in HBV DNA have to each cell the 2-3 genomic templates and with 1.0pg/ml extracellular HBV DNA to 3x10 ⁵Carry out under the suitable situation of virion/ml.

Embodiment 7

Present embodiment is tested the ability of the inhibition hepatitis B of the triguaiacyl phosphate derivant of β-L-dA, β-L-dC, β-L-dU, β-L-2 '-dG, β-L-dI and β-L-dT.Table 1 has provided β-L-dT (β-L-dT-TP), β-L-dC (β-L-dC-TP), ((β-L-dA-TP) triguaiacyl phosphate is to woodchuck hepatitis virus (WHV) archaeal dna polymerase, human DNA Polymerase, and the inhibition specific activity of β and γ for β-L-dU-TP) and β-L-dA for β-L-dU.

Table 1

Inhibitor	WHV?DNAIC ₅₀	DNAαK _i ^b(μM)	DNAβK _i ^b(μM)	DNAγK _i ^b(μM)
Inhibitor	WHV?DNAIC ₅₀	DNAαK _i ^b(μM)	DNAβK _i ^b(μM)	DNAγK _i ^b(μM)	β-L-dT-TP	0.34	>100	>100	>100
β-L-dA-TP	2.3	>100	>100	>100	β-L-dT-TP	0.34	>100	>100	>100
β-L-dA-TP	2.3	>100	>100	>100	β-L-dC-TP	2.0	>100	>100	>100
β-L-dU-TP	8	>100	>100	>100	β-L-dC-TP	2.0	>100	>100	>100

^aIC ₅₀: 50% inhibition concentration

^bK _iValue is measured as substrate as templa-primer with dATP with the activatory DNA of calf thymus.Inhibitor is analyzed with the Dixon chart.Under these conditions dATP is calculated the average Km value of human DNA Polymerase, be approximately 2.6 μ M.For dATP, the Km value of human DNA polymerase beta is in steady statue, is 3.33 μ M.The Km value of human DNA polymerase γ is in steady statue, is 5.2 μ M.

Embodiment 8

Present embodiment is tested through transfection Hep G-2 (2.2.15) cell the resistance of hepatitis B activity of β-L-dA, β-L-dC, β-L-dU, β-L-2 '-dG and β-L-dT.Table 2 has illustrated β-L-dA, β-L-dC, β-L-dU and the β-L-dT resistance of hepatitis B replication effect to transfection Hep G-2 (2.2.15) cell.

Table 2

Chemical compound	The HBV virion ^aEC ₅₀(μM)	HBV?Ri ^bEC ₅₀(μM)	Cytotoxicity 1C ₅₀(μM)	Selectivity index 1C ₅₀/EC ₅₀
Chemical compound	The HBV virion ^aEC ₅₀(μM)	HBV?Ri ^bEC ₅₀(μM)	Cytotoxicity 1C ₅₀(μM)	Selectivity index 1C ₅₀/EC ₅₀	β-L-dT	0.05	0.05	>200	>4000
β-L-dC	0.05	0.05	>200	>4000	β-L-dT	0.05	0.05	>200	>4000
β-L-dC	0.05	0.05	>200	>4000	β-L-dA	0.05	0.05	>200	>2000
β-L-dI	1.0	1.0	>200	>200	β-L-dA	0.05	0.05	>200	>2000
β-L-dI	1.0	1.0	>200	>200	β-L-dU	5.0	5.0	>200	>40

^aExtracellular DNA

^bThe intermediate that duplicates (DNA in the cell)

Embodiment 9

With 2.2.15 raji cell assay Raji β-L-dA, β-L-dC and β-L-dT in conjunction with the time to the effect of hepatitis B virus growth.The results are shown in table 3.

Table 3

Compositions	Ratio	EC ₅₀
Compositions	Ratio	EC ₅₀	L-dC+L-dT	1:3	0.023
L-dC+L-dT	1:1	0.053	L-dC+L-dT	1:3	0.023
L-dC+L-dT	1:1	0.053	L-dC+L-dT	3:1	0.039
L-dC+L-dA	1:30	0.022	L-dC+L-dT	3:1	0.039
L-dC+L-dA	1:30	0.022	L-dC+L-dA	1:10	0.041
L-dC+L-dA	1:3	0.075	L-dC+L-dA	1:10	0.041
L-dC+L-dA	1:3	0.075	L-dT+L-dA	1:30	0.054
L-dT+L-dA	1:10	0.077	L-dT+L-dA	1:30	0.054
L-dT+L-dA	1:10	0.077	L-dT+L-dA	1:3	0.035

Anti--HBV the activity of various combinations all has synergism.In addition, the combination of L-dA+L-dC+L-dT also has synergism in this model.

Embodiment 10

Present embodiment measured β-L-dA and β-L-dC separately or when being used in combination in the 2.2.15 cell to the inhibitory action of hepatitis B replication.The results are shown in table 4.

Table 4

^aβ-L-2 '-deoxyadenosine (μ M)	^bβ-L-2 '-deoxycytidine (μ M)	Suppression ratio %	^cC.I.
^aβ-L-2 '-deoxyadenosine (μ M)	^bβ-L-2 '-deoxycytidine (μ M)	Suppression ratio %	^cC.I.	0.5		90
0.05		24		0.5		90
0.05		24		0.005		1
	0.5	95		0.005		1
	0.5	95			0.05	40
	0.005	10			0.05	40
	0.005	10		0.05	0.05	80	0.34
0.05	0.005	56	0.20	0.05	0.05	80	0.34

^aβ-L-2 '-deoxyadenosine (μ M)	^bβ-L-2 '-deoxycytidine (μ M)	Suppression ratio %	^cC.I.
^aβ-L-2 '-deoxyadenosine (μ M)	^bβ-L-2 '-deoxycytidine (μ M)	Suppression ratio %	^cC.I.	0.05	0.0005	50	0.56
0.005	0.05	72	0.35	0.05	0.0005	50	0.56
0.005	0.05	72	0.35	0.005	0.005	54	0.35
0.005	0.0005	30	0.16	0.005	0.005	54	0.35
0.005	0.0005	30	0.16	0.0005	0.05	50	0.83
0.0005	0.005	15	0.28	0.0005	0.05	50	0.83
0.0005	0.005	15	0.28	0.0005	0.0005	0	N.A.

^aβ-L-2 '-deoxyadenosine: IC ₅₀=0.09 μ M

^bβ-L-2 '-deoxycytidine: IC ₅₀=0.06 μ M

^cThe explanation of association index value has synergism (＜1), adduction (=1), and antagonism (〉 1)

Embodiment 11

This embodiment has measured L-dA, L-dT and L-dC to the chronically infected woodchuck of woodchuck hepatitis virus (WHV) (Marmota monar) Hepadnavirus infection effect.This HBV animal models infected extensively is used, and the verified model that can be used as the antiviral agent evaluation of anti-HBV.

Method:

Test group (n=3 animal/medicine group, n=4 animal/matched group)

The 1st group of vehicle Control

The 2nd group of lamivudine (1amivudine) be (10mg/kg/ days) (3TC)

3-6 organizes L-dA (0.01,0.1,1.0,10mg/kg/ days)

7-10 organizes L-dT (0.01,0.1,1.0,10mg/kg/ days)

11-14 organizes L-dC (0.01,0.1,1.0,10mg/kg/ days)

Medicine oral administration every day gavage is administered once, the 0th, 1,3,7,14,21,28 days and after treatment+1 ,+3 ,+7 ,+14 ,+28 and get blood sample+56 days the time.With dot blot quantitative assay PCR, estimate activity and toxicity with the minimizing of WHV DNA in the serum.

The result is in Fig. 3 and table 5 explanation.

Table 5

LdA in marmot chronic HBV infection model, the antiviral activity of LdT and LdC

¹LdA, LdT and LdC be oral administration 10mg/kg once a day

²Limit detection is every milliliter of serum 1ng/ml WHV-DNA

The data declaration L-dA of this body inner model, L-dT and L-dC have high activity.At first, the load of virus reduces to and can not detect (L-dT), or almost can not detect (L-dA, level L-dC).Secondly, L-dA in this model, L-dT and L-dC have better activity than 3TC (lamivudine).The 3rd, in two weeks after stopping the L-dT medication, do not detect the virus levels bounce-back.The 4th, dose response curve points out that under the dosage of L-dA and L-dC awave increase explanation antiviral activity and L-dT are similar.The 5th, all the weight of animals of accepting medicine increase, and do not have to find the toxicity relevant with medicine.

The toxicity of chemical compound

By whether investigating viewed antivirus action because the general action of pair cell survival ability carries out oxicity analysis.Used method is to measure β-L-dA, β-L-dC and the cell growth of β-L-dT in human bone marrow clorogenic test, and compares with lamivudine, the results are shown in Table 6.

Table 6

Chemical compound	CFU-GM(μM)	BFU-E(μM)
Chemical compound	CFU-GM(μM)	BFU-E(μM)	β-L-dA	>10	>10
β-L-dC	>10	>10	β-L-dA	>10	>10
β-L-dC	>10	>10	β-L-dT	>10	>10
β-L-dU	>10	>10	β-L-dT	>10	>10
β-L-dU	>10	>10	Lamivudine	>10	>10

Preparation of drug combination

Suffers from any disease described herein, the people who comprises hepatitis B can treat with following method: β-2 '-deoxidation-β-L-erythro form-furan pentose nucleoside of using effective dose to the patient, for example β-2 '-deoxyadenosine, β-2 '-deoxycytidine, β-2 '-BrdU, β-2 '-deoxyguanosine and β-2 '-deoxyribosylthymine, or their pharmaceutically acceptable prodrug or salt, and there are pharmaceutically acceptable carrier or diluent to exist.Active substance can any suitable administration, for example with form oral administration, parenteral, intravenous, intradermal, the subcutaneous or topical of liquid or solid.

The amount of the reactive compound that contains in pharmaceutically acceptable carrier or diluent is enough to give the patient treatment effective dose, suppressing virus duplicating in vivo, and causes obvious toxic and side effects can for the patient who is treated.So-called " amount of suppression " is meant that the amount of active component is enough to produce the inhibitory action that each test for example as herein described can be measured.

Under above-mentioned all conditions, preferred chemical compound dosage preferably in the 1-20mg/kg weight range, gives receiver 0.1 to about 100mg/kg body weight at about 1-50mg/kg every day more common every day.The weight of the nucleoside parent that the effective dosage ranges of pharmaceutically acceptable prodrug can discharge is that calculate on the basis.If prodrug itself also demonstrates activity, its effective dose can be determined with the weight of prodrug as stated above, or determine with other method known in the art.

Chemical compound can include, but are not limited to contain 7-3000mg in the per unit dosage form easily with suitable unit dosage form administration, preferably contains the 70-1400mg active component.Usually oral dose is that 50-1000mg is easily.

It is desirable to, can make the peak concentration of reactive compound in blood plasma reach about 0.2-70 μ M, preferably approximately 1.0-10 μ M behind the delivery of active ingredients.For example the solution of the active component by intravenous injection 0.1-5% realizes that described solution can be in saline, perhaps with the big medicine group form administration of active component.

The concentration of reactive compound depends on absorption, inactivation and the discharge rate of described medicine in pharmaceutical composition, and the known other factors of those skilled in the art.The numerical value that it should be noted that dosage also can change along with the order of severity of treatment symptom.Further, for a specific receiver, specific dosage and medication schedule should be according to the needs of individuality, and write a prescription or the doctor's of supervision group compound administration prescription is adjusted in time.Here said concentration range only illustrates, not the scope of the compositions of requirement for restriction protection.But the active component single administration, also dosage that can be less is divided into administration for several times with changeable interval.

The preferred mode of administration of reactive compound is oral.Oral compositions contains inert diluent or edible carrier usually.They can wrap in the gelatine capsule, or are pressed into tablet.In order to carry out the treatment of oral medication, can be reactive compound and excipient fusion, and use with tablet, tablet or capsular form.As the part of compositions, can also comprise pharmaceutically compatible adhesive and/or adjuvant material.

Tablet, pill, capsule, tablet etc. can contain following any composition, or the chemical compound of similarity is arranged: adhesive such as microcrystalline Cellulose, Tragacanth or gelatin; Excipient such as starch or lactose; Disintegrating agent such as alginic acid, Primogel or corn starch; Lubricant such as magnesium stearate or Sterotes; Glidant such as silicon dioxide colloid; Sweeting agent such as sucrose or glucide; Flavoring agent such as Herba Menthae, methyl salicylate or orange flavoring.When unit dosage form is capsule, except above-mentioned material, wherein can also contain liquid-carrier such as fatty oil.In addition, unit dosage form can contain other various materials that can improve the unitary physical form of dosage, for example sugar-coat, Lac or other enteric reagent.

Component administration among all right elixir of The compounds of this invention, suspending agent, syrup, thin film, the chewing gum etc.Except reactive compound, also can contain sucrose and some antiseptic, dyestuff and pigment in the syrup as sweeting agent, and flavoring agent.

The compounds of this invention or its pharmaceutically acceptable derivates or its salt also can mix with other active substance that does not damage required effect, or mix with the material that supplementary function is arranged, these materials for example are antibiotic, antibacterial, antiinflammatory, protease inhibitor, or other nucleoside or non-nucleoside antiviral agent.Parenteral, intradermal, solution or suspension subcutaneous or topical application can contain following component: sterile diluent such as water for injection, saline solution, miscella, Polyethylene Glycol, glycerol, propylene glycol or other synthetic; Antibacterial such as benzyl alcohol or methyl parahydroxybenzoate; Antioxidant such as ascorbic acid or bisulfites; Chelating agen such as ethylenediaminetetraacetic acid; Buffer agent such as acetate, citrate or phosphate and reagent such as sodium chloride or glucose that can adjustment of tonicity.The mother solution preparation can be encapsulated in the container of ampoule, disposable syringe or the multidose made with glass or plastics.

If intravenous administration, preferred carrier is normal saline or phosphate buffered solution (PBS).

In preferred embodiments, reactive compound with can protect this chemical compound, make its carrier that can in health, not disappear fast preparation together, for example the dosage form of sustained release comprises implant and microcapsule delivery system.Can use polymer biodegradable, that have biocompatibility, as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen protein, poly-adjacent ester (polyorthoesters) and polylacetic acid.The method for preparing these preparations is that this area is commonly used.Used material can have been bought at Aiza Corporation.

The suspension of liposome (comprising that described cell has the monoclonal antibody at virus antigen to the liposome of infection cell as target) also is preferred pharmaceutical carrier.These can prepare according to methods known in the art, for example, according to U.S.P4, the method preparation described in 522,811.For example; the preparation of liposome can prepare according to following method: suitable liposome (for example stearyl PHOSPHATIDYL ETHANOLAMINE, stearyl phosphatidylcholine, arachadoyl phosphatidylcholine and cholesterol) is dissolved in inorganic solvent; evaporate then, stay the thin film of dried lipid article at vessel surface.Then, in container, add reactive compound or one, two and/or the aqueous solution of triguaiacyl phosphate derivant.Afterwards, with hand moving formation vortex, make the liposome material from the chamber wall upstream from, and disperse the liposome aggregation, thereby form liposome suspension.

Invention has been described with reference to embodiment preferred.By the detailed description of description preamble as can be known, for those skilled in the art, various variants of the present invention and improvement are conspicuous.All these variants and improvement are all within protection scope of the present invention.

Claims

1. β-the L-2 ' shown in the following formula of effective dose-deoxyribosylthymine:

Or its pharmaceutically acceptable salt is used for suppressing application in the medicine that duplicates of viruses of human hepatitis B the host who infects hepatitis B in preparation.

2. the application of claim 1, wherein β-L-2 '-deoxyribosylthymine exists with at least 95% appointment spatial configuration.

3. the application of claim 1, its Chinese medicine also contain the pharmaceutically acceptable carrier that is fit to the oral administration form.

4. the application of claim 1, its Chinese medicine are unit dosage form.

5. the application of claim 4, wherein unit dosage form contains β-L-2 '-deoxyribosylthymine of 50-1000mg.

6. the application of claim 4, wherein unit dosage form is tablet, pill or capsular form.

7. the application of claim 6, wherein unit dosage form is a tablet form.

8. the application of claim 6, wherein unit dosage form is a pill.

9. the application of claim 6, wherein unit dosage form is a capsule form.

10. the application of claim 3, wherein pharmaceutically acceptable carrier contains microcrystalline Cellulose and/or magnesium stearate.

11. each application in the aforementioned claim, wherein the host is the people.