THERAPEUTIC NUCLEOSIDES

Abstract: The present invention relates to certain novel 2'-deoxy-4'-thio-purine nucleosides, physiologically functional derivatives thereof, processes for their preparation, pharmaceutical formulations containing them and to their use in therapy, particularly in the treatment or prophylaxis of viral infections.

Description: rπvτ ~ A VTJΠC NUCLEOSIDES The present invention relates to certain novel 2'-deoxy-4 , -thio-purine nucleosides, physiologically functional derivatives thereof processes for their preparation, pharmaceutical formulations containing them and to their use in therapy, particularly in the treatment or prophylaxis of viral infections. Of the DNA viruses, those of the herpes group are the sources of the most common viral illnesses in man. The group includes herpes simplex virus (HSV), varicella zoster virus (VZV), cytomegalovirus (CMV); Epstein-Barr virus (EBV) and human herpes virus 6 (HHV6). HSV 1 and HSV 2 are some of the most common infectious agents of man. Most of these viruses are able to persist in the host's neural cells; once infected, individuals are at risk of recurrent clinical manifestations of infection which can be both physically and psychologically distressing. HSV infection is often characterised by extensive and debilitating lesions of the skin, mouth and/or genitals. Primary infections may be subclinical although they tend to be more severe than infections in individuals previously exposed to the virus. Ocular infection by HSV can lead to keratitis or cataracts thereby endangering the host's sight. Infection in the newborn, in immunocompromised patients including AIDS patients or penetration of the infection into the central nervous system can prove fatal. Transmission of the virus is by direct physical contact between a host and a recipient; the spread of HSV infection is therefore considered a very significant social problem, particularly as no effective vaccine is yet available. Varicella zoster virus (VZV) is a herpes virus which causes chickenpox and shingles. Chickenpox is the primary disease produced in a host without immunity and in young children is usually a mild illness characterised by a vesicular rash and fever. Shingles or zoster is the recurrent form of the disease which occurs in adults who were previously infected with varicella-zoster virus. The clinical manifestions of shingles are characterised by neuralgia and a vescicular skin rash that is unilateral and dermatomal in distribution. Spread of inflammation may lead to paralysis or convulsions. Coma can occur if the meninges becomes affected. In immunodeficient patients VZV may disseminate causing serious or even fatal illness. VZV is of serious concern in patients receiving immunosuppressive drugs for transplant purposes or for treatment of malignant neoplasia and is a serious complication of patients with Acquired Immune Deficiency Syndrome (AIDS) due to their impaired immune system. In common with other herpes viruses, infection with CMV leads to a lifelong association of virus and host and, following a primary infection, virus may be shed for a number of years. Congenital infection following infection of the mother during pregnancy may give rise to clinical effects such as death or gross disease (microcephaly, hepatosplenomegaly, jaundice, mental retardation), retinitis leading to blindness or, in less severe forms, failure to thrive, and susceptibility to chest and ear infections. CMV infection in patients who are immunocompromised for example as a result of malignancy, treatment with immunosuppressive drugs following transplantation or infection with Human Immunodeficiency Virus (HTV) may give rise to retinitis, pneumonitis, gastrointestinal disorders and neurological diseases. CMV infection in AIDS patients is a predominant cause of morbidity as it is present in a latent form in 50-80% of the adult population and can be re-activated in immunocompromised patients. Epstein-Barr virus (EBV) causes infectious mononucleosis and hairy leukoplakis, and is also suggested as the causative agent of human cancer, such as nasopharyngeal cancer, immunoblastic lymphoma, Buriritt's lymphoma. HHV6 has been shown to be a causative agent of kidney rejection and interstitial pneumonia in kidney and bone marrow transplant patients respectively. There is also evidence of repression of stem cell counts in bone marrow transplant patients. Another group of viral pathogens of world-wide major importance are the hepatitis viruses, in particular hepatitis B virus (HBV). HBV is aetiologjcally associated with primary hepatocellular carcinoma and is thought to cause 80% of the world's liver cancer. In the United States more than ten thousand people are hospitalised for HBV illness each year, and average of 250 die with fulminant disease. The United States currently contains an estimated pool of 500,000-one million infectious carriers. Chronic active hepatitis generally develops in over 25% of carriers, and often progresses to cirrhosis. Clinical effects of infection with HBV range from headache, fever, malaise, nausea, vomiting, anorexia and abdominal pains. Replication of the virus is usually controlled by the immune response, with a course of recovery lasting weeks or months in humans, but infection may be more severe leading to persistent chronic liver disease outlined above. Of the RNA viruses, retroviruses have assumed a particular importance in recent years. Retroviruses form a sub-group of RNA viruses which, in order to replicate, must first 'reverse transcribe' the RNA of their genome into DNA ('transcription' conventionally describes the synthesis of RNA from DNA). Once in the form of DNA, the viral genome may be incorporated into the host cell genome, allowing it to take advantage of the host cell's transcription/translation machinery for the purposes of replication. Once incorporated, the viral DNA is virtually indistinguishable from the host's DNA and, in this state, the virus may persist for the life of the cell. A species of retrovirus, Human Immunodeficiency Virus (HTV), has been reproducibly isolated from humans with AIDS or with the symptoms that frequently precede AIDS. AIDS is an immunosuppressive or immunodestructive disease that predisposes subjects to fatal opportunistic infections. Characteristically, AIDS is associated with a progressive depletion of T-cells, especially the helper-inducer subset bearing the OKT surface marker. HTV is cytopathic and appears to preferentially infect and destroy T- cells bearing the OKT marker and it is now generally recognised that HTV is the etiological agent of AIDS. Another RNA virus which has been recognised as the causative agent of an increasingly serious international health problem is the non-A, non-B hepatitis virus. At least 80% of cases of chronic post-transfusional non-A, non-B hepatitis have been shown to be due to the virus now identified as hepatitis C and this virus probably accounts for virtually all cases of post-transfusional hepatitis in clinical settings where blood products are screened for hepatitis B. Whereas approximately half of the cases of acute hepatitis C infection resolve spontaneously over a period of months, the remainder become chronic and in many if not all such cases chronic active hepatitis ensues with the potential for cirrhosis and hepatocellular carcinoma. The structure of the hepatitis C virus genome has recently been elucidated and the virus has been characterised as a single stranded RNA virus with similarities to flaviviruses. Coxsackie viruses belong to the enterovirus genus. They have a single stranded RNA genome contained in an icosahedral nucleocapsid. Coxsackie virus infection is increasingly recognised as a cause of primary myocardial disease in adults and children. Coxsackie infection is also associated with meningitis, pleurodynia, herpangia, hand- feet and mouth disease, respiratory disease, eye disease, diabetes and post-viral fatigue syndrome. In the latter case viral RNA has been detected in the muscle and in menocytes. European Patent Specifications No. EP-A-421777 and No. EP-A-409575 disclose certain 4'-thio-pyrimidine nucleosides and their use as antiviral agents. Compounds of formula (I) below fall within the broad scope of the compounds disclosed in International Patent Specification No. W091/04033. However, there is no specific disclosure of the compounds of formula (I) or of their use in the treatment of viral infections. We have now surprisingly and unexpectedly found that the compounds of formula (I) below and physiologically functional derivatives thereof and solvates of any thereof are suitable for use as antiviral agents. According to the present invention, therefore, there is provided a compound of formula (I): wherein: R 1 represents: halogen -NR2R3 wherein R^ and R^, which may be the same or different, each represent hydrogen, C^alkyl, C3_6cycloalkyl, C2-6aH∞nyl, phenyl or phenylC^alkyl (where the phenyl moiety may be optionally substituted by one or more substituents selected from halogen, C^galkoxy, nitro, cyano, amino and C^galkyl), or R 2 R 3 together with the N atom to which they are attached form a 3-, 4-, 5-, 6- or 7-membered heterocyclic ring optionally containing, in addition to said nitrogen atom, one or more other hetero atoms independently selected from O and N; -S(=O) n R 4 where n is 0, 1 or 2 and R 4 represents C galkyl, C3_6cycloalkyl, C3_6cycloalkylCι_3al_kyl, C^alkoxy, phenyl or phenylC^alkyl (where the phenyl moiety may be optionally substituted by one or more substituents selected from halogen, C^alkoxy, nitro, cyano, amino and C^alkyl), or, where n is 0, R represents hydrogen; -S(=O)mOR 4a where m is 0, 1 or 2 and R 4a represents C^alkyl, C3_6cycloalkyl, C3_6cycloalkylCι_3alkyl, C^alkoxy, phenyl or phenyl Cι_3alkyl (where the phenyl moiety may be optionally substituted by one or more substituents selected from halogen, Cι_6 lkoxy, nitro, cyano, amino and Ci.galkyl); -0R5 where B represents C^galkyl, C3_6cycloalkyl, C3_6cycloalkylCι_.3alkyl, phenyl or phenyl C^alk l (where the phenyl moiety may be optionally substituted by one or more substituents selected from halogen, Cι_6alkoxy, nitro, cyano, amino and Cι. 6 alkyl); -C^alkyl, C2-6al enyl or C2_6al nyl; with the proviso that when R represents -NR 2 R 3 ,R 2 and R 3 are not both hydrogen; or a salt, ester or other physiologically functional derivative thereof or a solvate of any thereof. In a further aspect, the present invention provides compounds of formula (I) above wherein R 1 represents: -NR R 3 wherein R 2 and R 3 , which may be the same or different, each represent hydrogen, Chalky!, C3_6cycloalkyl, phenyl or phenyl Cι_3alkyl (where the phenyl moiety may be optionally substituted by one or more substituents selected from halogen, C^galkoxy, nitro, cyano, amino and C^galkyl), or R 2 R 3 together with the N - 6 e - atom to which they are attached form a 3, 4, 5, 6 or 7-membered heterocyclic ring optionally containing, in addition to said nitrogen, one or more other hetero atoms independendently selected from O and N; -S(=O) n R 4 where n is 0, 1 or 2 and R 4 represents C^alkyl, C3_6cycloalkyl, C3. 6cycloalkylCι_3alkyl, C^alkoxy, phenyl or phenylC^alkyl (where the phenyl moiety may be optionally substituted by one or more substituents selected from halogen, C^alkoxy, nitro, cyano, amino and C^galkyl); or where n is O, R 4 represents hydrogen; -S(O)mOR 4 where m is 0, 1 or 2 and R 4 represents C^galkyl, C3_6cycloalkyl, C3. 6cycloalkylCι_3alkyl, C^alkoxy, phenyl or phenyl C^alkyl (where the phenyl moiety may be optionally substituted by one or more substituents selected from halogen, C^galkoxy, nitro, cyano, amino and C^galkyl); -OR 5 where R 5 represents C^galkyl, C^cycloalkyl, C3_6cycloalkylCι_3alkyl, phenyl or phenyl Cι_3alkyl (where the pyenyl moiety may be optionally substituted by one or more substituents selected from halogen, C^alkoxy, nitro, cyano, amino and Cμ eaikyi); with the proviso that when R* represents -NR R 3 , R 2 and R 3 are not both hydrogen; or a salt, ester or physiologically fimctional derivative thereof or a solvate of any thereof. In yet a further aspect, the present invention provides compounds of formula (I) wherein _\S represents halogen, -OR 5 where R 5 represents C^alkyl, C3_6cycloalkyl, C3_6cycloalkylCι_3alkyl, -NR 2 R 3 wherein R 2 and R > which may be the same or different, each represent hydrogen, C3_6cycloalkyl, Chalky!, or C2-6a enyl, or R 2 ,R 3 together with the N atom to which they are attached form a 4-, 5- or 6- membered heterocyclic ring optionally containing, in addition to said nitrogen atom, one or more other hetero atoms independently selected from O and N; -S(=O) n R 4 where n is 0 and R 4 represents hydrogen, Cμgalkyl, C3_6cycloalkyL, C3. gcycloalkylC 1.3 alkyl; with the proviso that when R 1 represents -NR 2 R 3 , R 2 and R 3 are not both hydrogen; or a salt, ester or other physiologically functional derivative thereof or a solvate of any thereof. Preferred compounds of the invention include those wherein R represents halogen, -OR 5 where R 5 represents C^alkyl, C3_6cycloalkyl, C3_6cycloalkylCμ3alkyl; -NR 2 R 3 where R 2 is hydrogen or C^alkyl and R 3 is C3_6cycloalkyl, C^galkyi, C2-6 a lk en yl; or R 2 R 3 together with the N atom to which they are attached form a 4- ,5- or 6-membered heterocyclic ring optionally containing, in addition to said nitrogen atom, one or more other hetero atoms independently selected from O and N; - S(=O)nR 4 where n is 0 and R 4 represents hydrogen or C^alkyl; or a salt, ester or other physiologically functional derivative thereof or a solvate of any thereof. Of such compounds, those compounds wherein R* represents -NR 2 R 3 where R 2 is H and R 3 is C3_6cycloalkyl are preferred. Particularly preferred compounds of formula (I) include those wherein R represents -OR 5 where R 5 represents methyl, ethyl, propyl, cyclopropyl, cyclopropylmethyl; -NR 2 R 3 where R 2 is hydrogen or methyl and R 3 is cyclopropyl, ethyl, n-propyl, isopropyl, allyl, piperidinyl, pyrrolidinyl, thio or methylthio; or salt, ester or other physiologically functional derivative thereof or a solvate of any thereof. Preferred compounds of formula (I) include:- 2-ammo-6-cyclopropylmethoxy-9-(2-deoxy-4-tMo-β-D-erythro-pentofuranosyl)-9H- purine; 2-ammo-9-(2-deoxy-4-tMo-β-D-erythro-pentofu_ranosyl)-6-methoxy-9H pι___rine; 2-ammo-9-(2-deoxy-4-tMo-β-D-erythro-pentofuranosyl)-6-metJιyltMo-9H-purme; 2-am o-9-(2-deoxy-4-tWo-β-D-erythro-pento___uranosyl)-6-(<^clopropylmethylamm 9H-purine; -amino-9-(2-deoxy-4-tMo-β-D-erythro-pentofurano_^l)-6-(ethylmethylamino)- H-purine; -am o-9-(2-deoxy-4-t o-β-D-erythro-pentofurano_^l)-6-iso-propylamino-9H-purine; 2-am o-9-(2-deoxy-4-tMo-β-D-erythro-pentofuranosyl)-6-N-piperidino-9H-purine; 2-ammo-9-(2-deoxy-4-thio-β-D-eιτthro-pentofuranosyl)-6-n-propylamino-9H-purine; 2-ammo-9-(2-deoxy-4-thio-β-D-erythro-pentofuranosyl)-6-N-pyrrolidino-9H-purine; 2-ammo-9-(2-deoxy-4-thio-β-D-erythro-pentofuranosyl)-6-aUylamino-9H-purine; and salts, esters and other physiologically functional derivatives thereof and solvates of any thereof. Especially preferred compounds of formula (I) include 2-amino-6-(cyclopropylamino)- 9-(2-deoxy-4-tMo-β-D-erythro-pentofuranosyl)-9H-purine and salts, esters and other physiologically functional derivatives thereof and solvates of any thereof. These compounds are of particular use against HBV and CMV infections of animals, which term is intended to include humans, woodchucks and ducks. As used herein the term "alkyl" as a group or part of a group means a straight or branched chain alkyl group. By the term halogen is meant chloro, bromo, fluoro or iodo, preferably chloro. The compounds of formula (I) and their physiologically functional derivatives and the solvates of any thereof are hereinafter collectively referred to as the compounds according to the invention. As used herein, the term "physiologically functional derivative" means any physiologically acceptable salt, ester, or salt of such ester, of a compoimd of formula (I) or any other compoimd which upon administration to the recipient is capable of providing (directly or indirectly) such a compound or an antivirally active metabolite or residue thereof. For example, it is within the scope of the invention to replace the H of the OH group at the 5'-position by a potentially hydrolysable group such as acyl or alkyl. Preferred esters of the compounds of formula (I) included within the scope of the invention as physiologically functional derivatives include carboxylic acid esters in which the non-carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl (for example, methyl, n-propyl, t-butyl, or n-butyl), cycloalkyl, alkoxyalkyl (for example, methoxymethyl), aralkyl (for example benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl, optionally substituted by, for example, halogen, C alkyl, or C alkoxy or amino); 1-4 1-4 sulphonate esters, such as alkyl- or aralkylsulphonyl (for example, methanesulphonyl); amino acid esters (for example, L-valyl or L-isoleucyl); and mono-, di-, or tri- phosphate esters. In such esters, unless otherwise specified, any alkyl moiety present advantageously contains from 1 to 18 carbon atoms, particularly from 1 to 6 carbon atoms, more particularly from 1 to 4 carbon atoms. Any cycloalkyl moiety present in such esters advantageously contains from 3 to 6 carbon atoms. Any aryl moiety present in such esters advantageously comprises an optionally substituted phenyl group. Any reference to any of the above compounds also includes a reference to a physiologically acceptable salt thereof. For therapeutic use, esters of compounds of formula (I) will be physiologically acceptable. However, esters which are not physiologically acceptable may also find use as intermediates. All esters, including non-physiologically acceptable esters are within the scope of the present invention. Examples of physiologically acceptable salts of the compounds of formula (I) and physiologically acceptable derivatives thereof include salts derived from an appropriate base, such as an alkali metal (for example, sodium or potassium), an alkaline earth metal (for example, magnesium or calcium), ammonium and NX4 + (wherein X is Cχ_ 4alkyl). Physiologically acceptable salts of a hydrogen atom or an amino group include salts of organic carboxylic acids such as acetic, lactic, fumaric, tartaric, malic, isethionic, lactobionic and succinic acids; organic sulphonic acids such as methanesulphonic, ethanesulphonic, benzenesulphonic and p-toluenesulphonic acids and inorganic acids such as hydrochloric, sulphuric, phosphoric and sulphamic acids. Physiologically acceptable salts of a compound having an hydroxy group consist of the + + anion of said compound in combination with a suitable cation such as Na , NH , and NX (wherein X is a C alkyl group). 4 1-4 For therapeutic use, salts of compounds of formula (I) will be physiologically acceptable, i.e. they will be salts derived from a physiologically acceptable acid or base. However, salts of acids or bases which are not physiologically acceptable may also find use, for example, as intermediates in the preparation or in the purification of a physiologically acceptable compound. All salts, whether or not derived from a physiologically acceptable acid or base, are within the scope of the present invention. The present invention also provides a compound according to the invention for use in therapy, more particularly for use as an antiviral agent, for example, for use in the treatment or prophylaxis of a hepatitis virus infection, such as an HBV infection or a herpes virus infection such as those mentioned above and more particularly CMV, HSVl, HSV2 or VZV. According to another aspect, the present invention provides compounds according to the invention for use in the treatment or prophylaxis of a retroviral infection, in particular an HIV infection. Examples of retroviral infections which may be treated or prevented in accordance with the invention include human retroviral infections such as Human Immunodeficiency Virus (HTV), for example, HTV-l or HIV-2 and Human T-cell Lymphotropic Virus (HTLV), for example HTLV-1 or HTLV-IL, infections. The compounds according to the invention are especially useful for the treatment of ADDS and related clinical conditions such as AIDS related complex (ARC), progressive generalized lymphadenopathy (PGL), Kaposi's sarcoma, thrombocytopenic purpura, AIDS-related neurological conditions, such as multiple sclerosis or tropical paraperesis, and also anti- HIV antibody-positive and HIV-positive conditions, including such conditions in asymptomatic patients. Examples of other climcal conditions which may be treated in accordance with this invention include those conditions caused by HTV, HSV 1 and 2, VZV, CMV, EBV, HHV6, HBV, coxsackie virus and hepatitis C virus infections as described above. The present invention also provides to compounds according to the present invention for use in the treatment or prophylaxis of a coxsackie virus or hepatitis C virus infection. According to another aspect, the present invention provides: (a) A method for the treatment or prevention of the symptoms or effects of a viral infection in an infected animal, for example, a mammal including a human, which comprises treating said animal with a therapeutically effective amount of a compound according to the invention. According to a particular embodiment of this aspect of the invention, the viral infection is a hepatitis infection, in particular an HBV infection or a herpes virus infection, such as CMV, HSV1, HSV2, VZV, EBV or HHV6. (b) A method for the treatment or prevention of the symptoms or effects of an HTV infection in an HTV infected animal, for example, a mammal including a human, which comprises treating said animal with a therapeutically effective amount of a compoimd according to the invention. (c) A method for the treatment or prevention of the symptoms or effect of a coxsackie virus or hepatitis C virus infection in an infected animal, for example, a mammal including a human, which comprises treating said animal with a therapeutically effective amount of a compound according to the invention. (d) A method for the prophylaxis of a viral infection, particularly an HBV, CMV, HSV1, HSV2, VZV, EBV, HHV6, hepatitis C virus or coxsackie virus infection in an animal, for example, a mammal including a human which comprises treating said animal with a therapeutically effective amount of a compound according to the invention. (e) Use of a compound according to the invention in the manufacture of a medicament for the treatment or prophylaxis of a viral infection, in particular an HBV infection, a herpes virus infection including CMV, HSV1, HSV2, VZV, EBV and HHV6, a retrovirus infection, such as, an HTV infection, a hepatitis C virus infection or a coxsackie virus infection. The compounds according to the invention may be employed alone or in combination with other therapeutic agents for the treatment of the above infections or conditions. Combination therapies according to the present invention comprise the administration of at least one compound of the formula (I) or a physiologically fimctional derivative thereof and at least one other physiologically acceptable agent. The active ingredient(s) and physiologically acceptable agents may be administered together or separately and, when administered separately this may occur simultaneously or sequentially in any order. The amounts of the active ingredient(s) and physiologically acceptable agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect. Preferably the combination therapy involves the administration of one compound of the formula (I) or a physiologically functional derivative thereof and one of the agents mentioned herein below. Examples of such further therapeutic agents include agents that are effective for the treatment of HIV infections or associated conditions such as 3'-azido-3'- deoxythymidine (zidovudine), other 2',3'-dideox nucleosides such as 2',3'- dideoxycytidine, 2',3'-dideoxyadenosine and 2',3'-dideoxyinosine, carbovir, acyclic nucleosides (for example, acyclovir), ^'-didehydrothymidine, protease inhibitors such as N-tert-butyl-dehydro-2-[-2(R)-hydroxy-4-ρhenyl-3(S)-[[N-(2-quinolylcarbonyl)-L- aspargmyl]butyl]-(4aS,8aS)-isoquinoline-3(S)-carboxamide (Ro 31-8959), oxathiolane nucleoside analogues such as (-Vcis- 1 -(2-hydroxymethyl)- 1.3 -oxathiolan-5-ylVcvtosine (3TC) or cis-l-(2-(hydrox methyl)-l,3-oxathiolan-5-yl)-5-fluoro-cytosine (FTC), 3'- deoxy-3'-fluorothymidine, 2 , ,3 , -dideoxy-5-ethynyl-3'-fluorouridine, 5-chloro-2'3'-did- eoxy-3'-fluorouridine, (-)-cis-4-[2-aιnino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cy- clopentene-1-methanol, ribavirin, 9-[4-hydroxy-2-(hydroxymethyl)but-l-yl]-guanine (H2G), tat inhibitors such as 7-chloro-5-(2-pyrryl)-3H-l,4-benzodiazepin-2(H)-one (Ro5-3335), or 7-chloro-l,3-dihydro-5-(lH-pyrrol-2-yl)-3H-l,4-benzodiazepin-2- amine (Ro24-7429), interferons such as α-interferon, renal excretion inhibitors such as probenecid. nucleoside transport inhibitors such as dipyridamole; pentoxifylline, N- Acetylcysteine (NAC), Procysteine, α-trichosanthin, phosphonoformic acid, as well as immunodulators such as interleukin π, granulocyte macrophage colony stimulating factors, erythropoetin, soluble CD and genetically engineered derivatives thereof. 4 Examples of such further therapeutic agents which are effective for the treatment of HBV infections include carbovir, oxathiolane nucleoside analogues such as (-)-cis-l-(2- hydroxymethyl)-l,3-oxathiolan-5-yl)-cytosine (3TC)) or ris-l-(2-(hydroxy-methyl)- l,3-oxathiolan-5-yl-5-fluoro-cytosine (FTC), 2 l ,3'-dideoxy-5-ethynyl-3 l -fluorouridine, S-chloro^'^'-dideoxy-S'-fluorouridine, l-(β-D-arabinofuranosyl)-5-propynyluracil, acyclovir and interferons, such as α-interferon. Examples of further therapeutic agents which are effective for the treatment of herpes virus infections are acyclovir, 9-[4- hydroxy-2-(hydroxymethyl)butyl]guanine (H2G), 9-[4-hydroxy-3-(hy- droxymethyl)- but-l-yl)guanine (penciclovir), famciclovir, the diacetate ester of penciclovir, BVaraU, 1 -(β-D-arabinofuranosyl)-5-propynyluracil, 2-[(2-amino- 1 , 6-dihydro-6-oxo-9H-purin- 9-yl)methoxy]-ethyl L-valinate, phosphonoformic acid and phosphonoacetic acid, ganciclovir, (S)-l-(3-hydroxy-2-phosphonyl methoxypropyl)-cytosine (HPMPC), Oxetanocin G, 2'-deoxy-5-iodouridine, E-5-2-bromovinyl-2'-deoxy-uridine (BVDU) and 9-(3-hydroxypropoxy) guanine. More preferably the combination therapy involves the administration of one of the above-mentioned agents and a compound within the preferred sub-group within formula (I) as described above. Most preferably the combination therapy involves the joint use of one of the above named agents together with one of the compounds of formula (I) specifically named herein. The present invention further provides pharmaceutical formulations of the compounds according to the invention, also referred to herein as active ingredients, which may be administered for therapy to a mammal including a human ("the recipient") by any suitable route appropriate to the clinical condition to be treated; suitable routes include oral (including buccal and sublingual), rectal, nasal, topical (including buccal, sublingual and transdermal), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal). It will be appreciated that the preferred route will vary with the condition, weight, age and sex of the recipient, the nature of the infection and the chosen active ingredient. The amount of a compound of the invention required for the treatment of the above - indicated utilities and indications including HBV, HTV, HSV1, HSV2, CMV, VZV, EBV, HHV6, hepatitis C virus or coxsackie virus infections, will depend on a number of factors including the severity of the condition to be treated and the identity of the recipient and will ultimately be at the discretion of the attendant physician. In general, however, for each of these utilities and indications, a suitable, effective dose is in the range 0.05 to 100 mg per kilogram body weight of the recipient per day, preferably in the range 0.1 to 50 mg per kilogram body weight per day and most preferably in the range 0.5 to 20 mg per kilogram body weight per day. An optimum dose is about 2 to 5 mg per kilogram body weight per day. For the treatment of HBV infections a suitable effective dose is preferably in the range of 0.05 to 20mg per kilogram body weight per day and for CMV infections the dose is preferably in the range of 0.5 to 20mg per kilogram per day. Unless otherwise indicated all weights of active ingredients are calculated as the parent compounds of formula (I). In the case of a or physiologically functional derivative of a compound of formula (I) or a solvate of any thereof the figures would be increased proportionately. The desired dose is preferably presented as two, three, four, five, six, or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, containing from 1 to 1500 mg, preferably from 5 to 1000 mg, most preferably from 10 to 700 mg of active ingredient per unit dosage form. Alternatively, if the condition of the recipient so requires, the dose may be administered as a continuous infusion. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation. The formulations of the present invention comprise at least one active ingredient, as defined above, together with one or more pharmaceutically acceptable carriers thereof and, optionally, one or more other therapeutic agents. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Formulations of the invention include those suitable for administration by any of the aforementioned routes which may conveniently be presented in unit dosage form and may be prepared by any method well know in the an of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product. Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in- oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary, or paste or may be contained within liposomes. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (for example, povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycollate, cross-linked povidone, crossed-linked sodium carboxmethyl cellulose), or a surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile or to be soluble or effervescent when added to liquid. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach Formulations suitable for oral use may also include buffering agents designed to neutralize stomach acidity. Such buffers may be chosen from a variety of organic or inorganic agents such as weak acids or bases admixed with their conjugated saits. A capsule may be made by filling a loose or compressed powder on an appropriate filling machine, optionally with one or more additives. Examples of suitable additives include binders such as povidone; gelatin, lubricants, inert diluents and disintegrants as for tablets. Capsules may also be formulated to contain pellets or discrete sub-units to provide slow or controlled release of the active ingredient. This can be achieved by extruding and sphero ising a wet mixture of the drag plus an extrusion aid (for example microcrystalline cellulose) plus a diluent such as lactose. The spheroids thus produced can be coated with a semi-permeable membrane (for example ethyl cellulose. Eudragit WE30D) to produce sustained release propeπies. An edible foam or whip formulation ideally comprises; 50-70% of an edible oil. particularly a vegetable oil, including corn oil, peanut oiL sunflower oil, olive oil and soybean oil; 2-10% of one or more surfactants particularly lecithin, polyols, poiyol polymer esters including glyceryl fatty acid esters, polyglyceryl fatty acid esters (e.g. decaglycerol tetraoleate), or sorbitan fatty acid esters (e.g. sorbitan monostearate); 1- 4% of a propellant which is suitable for ingestion, notably a compressed gas propellant especially nitrogen, nitrous oxide or carbon dioxide, or a gaseous hydrocarbon especially propane, butane or isobutane; 0.5-30% of one or more viscosity modifiers of particle size in the range 10-50 microns in diameter, particularly powdered sugars or colloidal silicon dioxide; and optionally 0.5-1% of one or more suitable, non-toxic colourings, flavourings or sweetners. The active ingredient is preferably present in such formulations in a concentration of 10-46%, advantageously 30%. An edible foam or whip formulation as described above may be prepared in a conventional manner, for example by mixing the edible oil, surfactant(s) and any other soluble ingredients, adding the viscosity modifiers) and milling the m ture to form a uniform dispersion and suspension. The active ingredient is blended into the milled mbcture until evenly dispersed. Finally, a metered quantity of propellant is incorporated to the mbcture after said mixture has been measured into a suitable dispensing container. Pharmaceutical formulations for topical administration according to the present invention may be formulated as an ointment, cream, suspension, lotion, powder, solution, paste, gel, spray, aerosol or oil. Alternatively, a formulation may comprise a dressing such as a bandage or adhesive plaster impregnated with active ingredients and optionally one or more excipients or diluents. Compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Such patches suitably contain the active compound 1) in an optionally buffered, aqueous solution; 2) dissolved in an adhesive; or 3) dispersed in a polymer. A suitable concentration of the active compound is about 1% to 35%, preferably about 3% to 15%. As one particular possibility, the active compound may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6), 318 (1986). For infections of the eye or other external tissues, for example mouth and skin, the formulations are preferably applied as a topical ointment or cream containing the active ingredient in an amount oζ for example, 0.075 to 20% w/w, preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. When formulated in an ointment, the active ingredients may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in-water cream base or as a water-in-oil base. If desired, the aqueous phase of the cream base may include, for example, at least 40- 45% w/w of a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups such as propylene glycol, butane- 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulphoxide and related analogues. The oily phase of an emulsion formulation according to the invention may comprise merely an emulsifier (otherwise known as an emulgent), but desirably comprises a mbcture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and or fat ma Iff; up the so-cailed emulsifying ointment base which forms the oily phase of the cream foπmiiarions. Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyi alcohol, giycεryi mono-stearate and sodium lauryi sulphate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compoimd in most oϋs likeiy :o be used in pharmaceutical emuision formulations is very iow. The cream shouid preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic aikyi esters such as di-isoadipate, isocetyl stearate, propyiene glycol diester of coconui fatty acids, isopropyl myristate, decyi oleate, isopropyi paimitate. butyi stearate. 2- ethyihexyl paimitate or a blend of branched chain esters known as Crodamoi CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used. Formulations suitable for topical administration to the eye aiso include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous soivenL The ingredient is preferably present in such foimulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10%, particularly about 1.5% w/w. Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored material usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert material such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier. Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or higher fatty alcohol (e.g. hard wax, European Pharmacopoeia) or trigiycerides and saturated fatty acids (e.g. Witepsol). Formulations suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient. Suitable formulations for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulizers or insufflators. For pulmonary administration via the mouth, the particle size of the powder or droplets is typically in the range 0.5 - lOμm, preferably 1 - 5μm, to ensure delivery into the bronchial tree. For nasal administration, a particle size in the range 10 - 500μm is preferred to ensure retention in the nasal cavity. Metered dose inhalers are pressurised aerosol dispensers, typically containing a suspension or solution formulations of the active ingredient in liquified propellant. During use these devices discharge the formulation through a valve adapted to deliver a metered volume, typically from 10 - 150μl, to produce a fine particle spray containing the active ingredient. Suitable propellants include propane and butane, certain chlorofluorocarbon compounds, commonly referred to as "CFS's", for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or mixtures thereof. The formulation may additionally contain co-solvents, for example ethanol, surfactants such as oleic acid or sorbitan trioleate, antioxidants and/or suitable flavouring agents. Nebulizers are commercially available devices that transform solutions or suspensions of the active ingredient into an aerosol therapeutic mist either by means of acceleration of a compressed gas through a narrow venturi orifice, typically air or oxygen, or by means of ultrasonic agitation. Suitable formulations for use in nebulizers consist of the active ingredient in a liquid carrier and comprising up to 40% w/w of the formulation, preferably less than 20%w/w. The carrier is typically water or a dilute aqueous aicholoic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride. Optional additives include preservatives if the formulation is not prepared sterile, for example methylhydroxybenzoate, antioxidants, flavouring agents, volatile oils, buffering agents and surfactants. Suitable formulations for administration by insufflation include finely comminuted powders which may be delivered by means of an insufflator or taken into the nasal cavity in the manner of a snuff. In the insufflator, the powder is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in- situ and the powder either presneted to air drawn through the device upon inhalation or alternatively delivered by means of a manually operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent such as lactose, and an optional surfactant. The active ingredient typically comprises from 0.1 - 100% w/w of the formulation. Pressurised aerosol formulations for inhalation are preferably arranged so that each metered dose contains from 0.05 to 5 mg of a compound of the invention. Similarly, powder formulations for insufflations are so arranged that each unit dose contains from 0.5 to 50 mg. Solution or suspension formulations for nebylisation are arranged as to deliver doses between 1 and 1500 mg. The compounds according to the invention or formulations thereof may be administered by these devices once or several times daily, with one or several doses, for example three or four, being given on each occasion. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof of an active ingredient. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents. The compounds of formula (I) may be produced by various methods known in the art of organic chemistry in general and nucleoside synthesis in particular. Starting materials are either known or readily available from commercial sources or may themselves be produced by known and conventional techniques. The present invention further includes a process for the preparation of a compound of formula (I) or a salt, ester or physiologically functional derivative of a compound of formula (I) or a solvate of any thereof which comprises either: - (A) reacting a purine base of formula (II) 1 wherein R is as hereinbefore defined, or a functional equivalent thereof, with a compound of formula (III) 6 7 wherein R and R are the same or different and each represents hydrogen or a hydroxy protecting group and A is a phosphate group or salt thereof or a pyrimidine or purine moiety other than (II) or a leaving group, to form a compound of formula (I); or (B) reacting a compound of formula (TV) 6 7 8 wherein R and R are as hereinbefore defined and R represents a precursor for R as defined in formula (I) with a reagent or reagents and or under conditions 8 1 serving to convert R to the desired R group; and thereafter or simultaneously therewith effecting one or more of the following optional conversions:- (i) removing any remaining protecting group(s); (ϋ) when a compound of formula (I) is formed, converting it into a salt, ester or other physiologically functional derivative of a compound of formula (I); or (ϋi) when a salt, ester or other physiologically functional derivative of a compound of formula (I) or a solvate of any thereof is formed, converting the derivative into a compound of formula (I) or into a different derivative of the compound of formula (I). (iv) where necessary, separating the α and β anomers of the compound of formula (I) or a protected derivative thereof or of a physiologically acceptable derivative of a compound of formula (I) In the above-described processes according to the invention, the starting compounds of formulae (II), (IH) and (TV), as well as the above-mentioned agents and conditions, may be selected from those which are known in the art of nucleoside synthetic chemistry. Examples of such conversion procedures are described hereinafter for guidance and it will be understood that they may be modified in conventional manner depending on the desired compound of formula (I). In particular, where a conversion is described which would otherwise result in the undesired reaction of labile groups, then such groups may be protected in conventional manner with subsequent removal of the protecting group(s) after completion of the conversion. According to the conditions employed to effect process (A), the purine base of formula (II) and the compound of formula (IE) may be optionally protected using conventional protecting groups, such as acyl groups, in particular,- alkanoyl (for example, acetyl), substituted alkanoyl, such as alkoxyalkanoyl, aroyl (for example, benzoyl), ether groups, including trialkylsilyl groups (for example, t-butyldimethylsilyl) or other groups, such as aralkyl (for example, benzyl) or a phosphate group. Such groups may be removed by acid or base hydrolysis, hydrogenolysis, or enzymatically. Acyl groups are typically removed by base hydrolysis and silyl groups by acid hydrolysis or fluoride ion treatment. Aralkyl groups such as benzyl are advantageously removed by catalytic hydrogenolysis. Two methods are commonly employed to effect process (A), viz enzymatic and chemical. Process (A) may be effected enzymatically by, for example, reacting an appropriate purine base of formula (II), wherein R is as hereinbefore defined or a functional equivalent thereof for example, a salt or protected derivative thereof (see above), with a compound of formula (IH) wherein R and R are the same or different and each represents hydrogen or a hydroxy protecting group (see above) and A is a pyrimidine or purine moiety (other than (II)), a phosphate group or a salt thereof. In the case where A is a pyrimidine or purine moiety (other than (II)), the reaction may be carried out in the presence of (i) phosphorylase enzymes, such as purine nucleoside phosphorylase and thymidine phosphorylase and an inorganic phosphate or salt thereof - 24- or (ϋ) a transferase enzyme, for example, trans-N-deoxyribosylase. In order to obtain the compounds of the invention it is necessary when employing this method that the compound of formula (HI) should be in its β-form. The trans-N-deoxyribosylase may be isolated by standard biochemical techniques from E.coli strain SS70-8/15 which expresses Lactobacillus enzyme, available from the American Type Culture Collection (ATCC) Rockville, MD 20852-1776 from June 17, 1992 under Accession No. ATCC 69016. In the case where A represents a phosphate group or a salt thereof, the reaction may be carried out in the presence of a single phosphorylase enzyme, such as purine nucleoside phosphorylase. In order to obtain the compounds of the invention it is necessary when employing this method that the compoimd of formula (IH) should be in its α-form. Protecting groups may be used in the enzymatic process but in practice have been found to be unnecessary and in some cases to be actually disadvantageous in terms of overall yield. Process (A) may be effected chemically by, for example, reacting a compound of 6 formula (II) as hereinbefore defined with a compound of formula (HI) wherein R and R are the same or different and each represents hydrogen or a hydroxy protecting group and A represents a suitable leaving group, such as a halogen atom, for example, chlorine or an acyloxy group, such as acetoxy in the presence of a catalyst, such as tin (TV) chloride or a Lewis Acid (for example, mercury dibromide or trimethyl- silyltrifluoromethanesulphonate) in a suitable solvent, such as toluene, acetonitrile, 1,2- dichloroethane or chloroform at reduced, ambient or elevated temperature such as -78° C to reflux, preferably 0° to 25°C. In contrast to the enzymatic method, it has been found that in the chemical process (a) the compounds of formula (II) and (El) may advantageously be protected (vide supra) and (b) the compound of formula (I) so formed is a mbcture of α- and β-anomers. The β-anomers of the present invention may be obtained by anomeric separation by methods well known to a skilled person or readily available in the chemical literature, for example, by silica gel column chromatography or HPLC. 1 Compounds of formula (II) wherein R is as hereinbefore defined or a functional equivalent of any thereof may be obtained commercially, for example, from the Aldrich Chemical Company or prepared by conventional methods well known to a skilled person or readily available from the chemical literature, for example, by methods the same as or analogous to those described in Robins et al-, J.Amer.Chem.Soc. 1957, 79, 490-494 and Montgomery and Temple, J.Amer.Chem.Soc, 1961, 83, 630-635. For example, the appropriate purine base may be prepared from a corresponding purine wherein the 6-substituent is a suitable leaving group, for example, chlorine, by nucleophilic displacement of said group. Thus purines wherein the 6-substituent is methoxy or cyclopropylmethoxy may be prepared by treatment of the corresponding 6- chloropurine with methanol or cyclobutanol respectively, in the presence of a base such as sodium hydride, and purines wherein the 6-substituent is cyclopropylamino, piperidinyl, or pyrrolidinyl may be prepared by treatment of the corresponding 6- chloropurine with the appropriate amine, cyclopropylamine, piperidine, or pyrrolidine respectively, in a suitable solvent. The 2-amino-6-chloro purine precursor may be obtained commercially (Aldrich Chemical Co.) or prepared by methods well known to a skilled person or readily available from the chemical literature. Compounds of formula (III) wherein A is a pyrimidine or purine moiety may conveniently be prepared by methods well known to a skilled person or readily available from the Chemical literature. For example, 2'-deoxy-4'-thiouridine may be prepared by the method described in Secrist J.A. HI, et al, J. Med. Chem., 34 2361- 2366 (1991) and 2'-deoxy-4'-thioadenine may be prepared by the method described in WO91/04033. 6 7 Compounds of formula (HI) wherein R and R are as hereinbefore defined and A represents a leaving group, such as chloro and acetoxy, may be prepared by conventional methods, typically by methods described in European Patent Specification No. EP-A-409575, the contents of which are incorporated herein by reference. Compounds of formula (IH) wherein A represents a phosphate group may be prepared chemically by methods analogous to those available in the chemical literature or from compounds of formula (HI) wherein A is a purine moiety by treatment with a phosphorylase enzyme, such as thymidine phosphorylase. 8 With regard to process (B), in the case where the precursor group, R , represents a protecting group, conventional protecting groups as hereinbefore described for process (A) may be employed. Esters according to the invention may be prepared by methods known in the art. For example, by treatment of the parent compound of formula (I) with an appropriate esterifying agent, for example, by treatment with an appropriate acid halide, for example, chloride or anhydride. Mono-, di- or tri- phosphate esters of compounds of formula (I) may be prepared from the parent compound of formula (I) by successive phosphorylation via the mono-, di- and tri- phosphate derivatives by conventional chemical means or by enzymatic means, for example using a nucleoside kinase or phosphotransferase in the presence of a nucleotide triphosphate, for example ATP. A compound of formula (I) may be converted into a corresponding physiologically acceptable ether of formula (I) by reaction with an appropriate alkylating agent in a conventional manner. The compounds of formula (I), including esters thereof may be converted into physiologically acceptable salts in a conventional manner, for example, by treatment with an appropriate base. An ester or salt of a compound of formula (I) may be converted into the parent compound by, for example, hydrolysis. The following Examples are intended for illustration only and are not intended to limit the scope of the invention in any way. The term 'active ingredient' as used in the Examples means a compound of formula (I) or a physiologically functional derivative thereof or a solvate of any thereof. A. Preparation of trans-N-deoxyribosylase (E.C. 2.4.2.6) from Esherichia coli E. coli strain SS70-8/15 was grown overnight (15-20 hr) in a rich medium, such as Luria broth, containing 150 g/mL a picillin. The bacteria were collected from the growth medium by centrifugation at 4°C and the cell pellet washed with cold, 100 mM sodium phosphate buffer, pH 6.0. A cell extract was prepared by resuspending the washed cell pellet with 0.6-0.8 volumes of cold, 100 mM sodium phosphate buffer followed by passage of the cell suspension through a French press at 12-14 Kpsi. Whole cells and cell debris were removed by centrifugation in a 70Ti rotor and 50 Krpm for 90 min. The supernatant obtained following centrifugation was the high speed supernatant (HSS). The A for the HSS was adjusted to equal 180 by addition of cold, 100 mM sodium phosphate buffer. The diluted HSS was adjusted to 0.2% PEI (polyethyleneimine), incubated at 4°C for 15-30 min and then centrifuged. The supernatant obtained following the PEI precipitation was adjusted to 30% saturation with respect to (NH ) SO , incubated at 4°C for 60-90 min and then centrifuged to pellet the protein. The protein precipitated with 30% (NH ) SO was slowly dissolved in 100 mM sodium phosphate buffer (pH 6.0) and then dialyzed against 2 to 6 liters of the same buffer. After dialysis, the precipitate that formed was removed by centrifugation. The supernatant containing enzyme was heated 5-10 min in a 60°C water bath followed by a 20 min incubation in a ice water slurry. The precipitate that formed during the heat treatment step was removed by centrifugation. The supernatant contained trans-N-deoxyribosylase which was used for nucleoside synthesis. The trans-N-deoxyribosylase activity of each enzyme preparation was quantitated using deoxyinosine and cytosine as substrates in the xanthine oxidase coupled assay system described by Cardinaud, R. 1978. Nucleoside Deoxyribosyltransferase from Lactobacillus helveticus. Methods Enzymol. 51:446-455. E. coli strain SS70-8/15 was deposited at the American Type Culture Collection, (ATCC) Rockville, MD 20852-1776 on June 17, 1992, under Accession No. ATCC 69016. B. Enzymatic preparation of 2'-deoxy-4'-thio-purine nucleosides Modified purine bases were prepared by displacement of the chlorine in 2-amino- 6-chloropurine (Aldrich) as described in US patent 506 8320, Koszalka, G.W. et al. 6-N-Substituted Derivatives of Adenine Arabinoside As Selective Inhibitors Of Varicella-Zoster Virus, Antimicrobial Agents and Chemotherapy, 35, 1991, 1437-1443 and Burns, CL. et al, Novel 6-Alkoxypurine 2',3'- dideoxynucleosides as Inhibitors of the Cytopathic Effect of the Human Immunodeficiency Virus, J. Med. Chem., 1993, 36(3), 378-384. An appropriately substituted purine base was added to 900 mL of pH 6.0 citrate buffer to give ImM purine base solution. The buffer was prepared by addition of 9.46 g (45 mmol) of citric acid to 900 ml of distilled deionized water and adjusting the final pH to 6.0 with sodium hydroxide. An α β mixture (1:1) of 2'- deoxy-4'-thiouridine (Secrist, J.A. m, et al- J. Med. Chem., 34, 2361-2366 (1991), incorporated herein by reference) was added to give a concentration of 5mM in β compound. Solution was achieved by heating the mbcture to 50°C with sonication. Trans-N-deoxyribosylase (2051 units/mL) was added to a final concentration of 5 units of enzyme mL of reaction. The reaction mbcture was maintained at 50°C. Every day for four days an equivalent portion of purine base was added. After five days the enzyme was removed by ultrafiltration. The water was removed by lyophilization. The resulting white powdery residue was sluπied with methanol (500 mL) and filtered. The solid was rinsed thoroughly with methanol (3 x 100 mL, or until no substantial UV activity was present in the filtrate). The combined filtrates were slurried with Dowex AG-1 (OH form) resin (200 mL) and filtered. The resin was rinsed with methanol until no UV activity was present in the filtrate. The solvent was removed with a rotary evaporator. The sticky residue was dissolved in 100 mL of methanol and silica gel (-20 mL) was added. The product was purified by flash chromatography, 5 x 30 cm column, 95:5 CH Cl :CH OH as eluant. The purified product was lyophilized from H O to give the nucleoside as a white powder. Example 1 2-Aιnino-9-(2-deoxy-4-t o-β-D-ervthro-pentofuranosylV6-methoxy-9H-pιιrine 2-Amino-6-methoxypurine, prepared from 2-amino-6-chloropurine (Aldrich Chemical Co., Milwaukee, WI 53233) and methanol, was added to pH 6.0 citrate buffer as described above (Example B). H NMR (DMSO-d ): δ 2.34-2.37 (m, 1), 2.52-2.58 (m, 1), 3.36-3.39 (m, 1), 3.54- 3.58 (m, 1), 3.68-3.74 (m, 1), 3.96 (s, 3), 4.45-4.48 (m, 1), 5.13 (t, 1, J=5.5), 5.30 (d, 1, J=3.8), 6.13 (t, 1, J=6.9), 6.50 (s, 2), 8.20 (s, 1). UV (pH=7) λmax 281 (e-11.0); λmax 252 (ε=9.7); λmin 263 (ε=6.5); λmin 232 (ε=10.3). (pH=13) λmax 281 (ε=10.4); λmax 252 (ε=9.0); λmin 263 (ε=6.0); λmin 233 (ε=6.0). MS (El) m/z 298 (M+H). Example 2 2-Ammo-6-(cvclopropylaminoV9-(2-deoxy-4-thio-β-D-ervthro-pentofuranosyl')-9H- purine 2-amino-6-cyclopropylaminopurine, prepared by nucleophilic displacement of the chlorine group on 2-amino-6-chloropurine (Aldrich Chemical Co., Milwaukee, WI 53233) by cyclopropylamine, was added to pH 6.0 citrate buffer as described above (Example B). mp: 98-126°C [α] 20-73.8 (c=0.50, DMF). H NMR (300 MHz, DMSO-d ) δ 0.54- 0.68 (m, 4), 2.27-2.35 (m, 1), 2.50-2.60 (m, 1), 3.01 (br, s, 1), 3.31 (m, 1), 3.51-3.59 (m, 1), 3.67-3.73 (m, 1), 4.46 (t, 1, J=3.3), 5.15 (t, 1, J=5.5), 5.27 (d, 1, J=3.8), 5.90 (s, 2), 6.10 (dd, 1, J=6.5, 7.9), 7.36 (d, 1, J=2.3), 8.01 (s, 1). UV (pH=7) λmax 285 (ε=15.2); λmin 246 (ε=6.4); sh 265 (ε=10.3)). (pH=13) λmax 297 (ε=14.2); λmin 274 (ε=7.2); sh 258 (ε=ll.l). MS (El) m z 323 (M+H). Anal. Calcd for C H N O S- 0.5 H O: C, 47.12; H, 5.78; N, 25.36; S, 9.68. Found: C, 46.87; H, 5.81; N, 25.18; S, 9.72. Example 3 2-An3ino-9-(2-deoxy-4-tMo-β-D-e hro-pentofuranosylV6-methylthio-9H-purine 2-Amino-6-methylthio-9H-purine, obtained from Sigma Chemical Co., was added to pH 6.0 citrate buffer as described above (Example B). mp: 197-199 0 C. [afo 20 - 94.4 (c = 0.50, DMF). 1H NMR (300 MHz, DMSO-d 6 ) δ 2.27-2.35 (m, 1), 2.50-2.60 (m, 1), 2.6 (s, 3), (m, 1), 3.54 (m, 1), 3.74 (m, 1), 4.47 (m, 1), 5.13 (t, 1, J = 6.0), 5.31 (d, 1, J = 2.0), 6. 13 (dd, 1), 6.58 (s, 2), 8.26 (s, 1). UV (PH = 7) X^ax 2 « 5 (ε = 1 5 -1); ^ 246 (ε = 6.4); sh 265 (ε = 10.3). (pH = 13) λ^ 297 (ε = 14.2); ^ 21 (ε = 7.2); sh 258 (ε = 11.1). MS (El) m/z 314 (M + H). Anal. Calcd for ^ ^5^0282 . 0.5 H O: C, 40.98; H, 5.00; N, 21.72. Found: C, 40.93; H, 4.72; N, 21.69. Example 4 2-Amino-9-(2-deoxy- -t o-β-D-e hro-pentofuranosylV6-(cvclopropylmethylamino -9H-purine 2-Amino-6-(cyclopropylmethylamino)-9H-purine, prepared from 2-amino-6-chloro- purine (Aldrich Chemical Co., Milwaukee, WI 53233) and cyclopropylmethylamine, was added to pH 6.0 citrate buffer as described above (Example B). mp: 72-76 0 C. [α] D 20 - 78.0 (c = 0.50, DMF). l H NMR (300 MHz, DMSO-d 6 ) δ 0.64-0.69 (m, 2), 0.78-0.84 (m, 2), 2.27-2.35 (m, 1), 2.49-2.58 (m, 1), 3.2 (m, 1), 3.32 (s, 3), 3.53-3.59 (m, 1), 3.66-3.74 (m, 1), 4.46 (m, 1), 5.13 (t, 1, J = 5.5), 5.27 (d, 1, J = 3.7), 5.89 (br s, 2), 6.13 (dd, 1, J = 7.9, 7.9), 8.04 (s, 1). MS (El) m/z 337 (M + H). Anal. Calcd for C 14 H2θN 6 O 2 S . 1 H 2 O: C, 47.74; H, 6.32; N, 23.53; S, 8.98. Found: C, 47.82; H, 6.17; N, 23.47; S. 9.04. Example 5 2-Ammo-9-(2-deoxy-4-tMo-β-D-ervthro-pentofuranosylV6-(ethvhι ethylamino -9H-purine 2-Amino-6-(ethylmethylamino)-9H-purine, prepared from 2-amino-6-chloropurine (Aldrich Chemical Co., Milwaukee, WI 53233) and ethylmethylamine, was added to pH 6.0 citrate buffer as described above (Example B). mp: 78-82°C. [αfo 20 - 84.0 (c = 0.50, DMF). 1H NMR (300 MHz, DMSO-d 6 ) δ 1.18 (t, 3, J = 7.0), 2.31-2.35 (m, 1), 2.49-2.53 (m, 1), 3.26 (br, s, 3), 3.3 (m, 1), 3.53- 3.59 (m, 1), 3.66-3.71 (m, 1), 3.94 (br s, 2), 4.46 (m, 1), 5.14 (t, 1, J = 5.5), 5.27 (d, 1, J = 3.7), 5.86 (br s, 2), 6.11 (dd, 1, J = 6.3, 7.9), 8.03 (s, 1). MS (El) m z 325 (M ^ H). Anal. Calcd. for Ci3H2θN 6 O 2 S . 0.5 H 2 O . 0.1 C 2 H6θ: C, 46.90; H, 6.44; N, 24.86. Found: C, 46.98; H, 6.38; N, 25.09. Example 6 2-Ammo-9-(2-deoxy-4-tMo-β-D-erv :hro-pentofuranosylV6-iso-propylamino-9H-purine 2-Amino-6-iso-propylamino-9H-purine, prepared from 2-amino-6-chloropurine (Aldrich Chemical Co., Milwaukee, WI 53233) and isopropylamine, was added to pH 6.0 citrate buffer as described above (Example B). mp: 81-85 0 C. [α] D 20 - 71.8 (c = 0.50, DMF). 1H NMR (300 MHz, DMSO-d 6 ) δ 1.16 (d, 6, J = 6.5), 2.26-2.34 (m, 1), 2.25-2.58 (m, 1), 3.3 (m, 2), 3.53-3.59 (m, 1), 3.66-3.73 (m, 1), 4.45 (m, 1), 5.14 (t, 1, J = 5.5), 5.26 (d, 1, J = 3.7), 5.84 (br s, 2), 6.10 (dd, 1, J = 8.0, 8.1), 6.95 (br s, 1H), 8.00 (s, 1). MS (El) m z 325 (M + H). Anal. Calcd. for C13H20N6O2S . 0.5 H2O . 0.5 02^0: C, 47.18; H, 6.79; N, 23.58; S, 9.00. Found: C, 46.85; H, 6.60; N, 23.69; S, 9.08. Example 7 2-Ammo-9-(2-deoxy-4-tMo-β-D-ervthro-pentofuranosyl -6-cvclopropylmethoxy- 9H-purine 2-Amino-6-cyclopropylmethoxy-9H-purine prepared from 2-amino-6-chloropurine (Aldrich Chemical Co., Milwaukee, WI 53233) and methanol, was added to pH 6.0 citrate buffer as described above (Example B). !HNMR (300 MHz, DMSO-d 6 ) δ 0.30 (m, 2), 0.58 (m, 2), 1.30 (m, 1), 2.32-2.38 (m, 1), 2.54-2.61 (m, 1), 3.3 (m, 1), 3.53-3.60 (m, 1), 3.68-3.74 (m, 1), 4.21 (d, 2, J = 7.3), 4.47 (m, 1), 5.13 (t, 1, J = 5.5), 5.30 (d, 1, J = 3.7), 6.14 (m, 1), 6.45 (s, 2), 8.20 (s, 1). MS (El) m z 338 (M + H). Example 8 2-Amino-9-(2-deoxy-4-tMo-β-D-ervthro-pentofuranosylV6-N-piperidino-9H-purine 2-Amino-6-N-piperidino-9H-purine, prepared from 2-amino-6-chloropurine (Aldrich Chemical Co., Milwaukee, WI 53233) and piperidine, was added to pH 6.0 citrate buffer as described above (Example B). [α] D 20 - 75.0 (c = 0.50, DMF). l R NMR (300 MHz, DMSO-d 6 ) δ 1.52 (br m, 4), 1.63 (m, 2), 2.27-2.35 (m, 1), 2.49-2.57 (m, 1), 3.3 (m, 1), 3.51-3.59 (m, 1), 3.66-3.74 (m, 1), 4.09 (br m, 4), 4.45 (m, 1), 5.14 (t, 1, J = 5.5), 5.27 (d, 1, J = 3.7), 5.89 (br s, 2), 6.11 (dd, 2, J = 4.4, 6.1), 8.03 (s, 1). MS (El) m z 351 (M + H). Anal. Calcd. for C 15 H22N 6 O 2 S . 0.1 H 2 O . 0.4 CH 2 C1 2 : C, 49.72; H, 6.23; N, 22.59; S, 8.62. Found: C, 50.03; H, 6.40; N, 22.20; S, 8.97. Example 9 2-Ammo-9-(2-deoxy-4-thio-β-D-e hro-pentofinanosylV6-n-propylamino-9H-purine 2-Amino-6-n-propylamino-9H-purine, prepared from 2-amino-6-chloropurine (Aldrich Chemical Co., Milwaukee, WI 53233) and propylamine, was added to pH 6.0 citrate buffer as described above (Example B). !H NMR (300 MHz, DMSO-d 6 ) δ 0.87 (t, 3, J = 7.2), 1.55 (h, 2, J = 32.4), 2.31-2.34 (m, 1), 2.49-2.53 (m, 1), 3.26 (br s, 3), 3.3 (m, 1), 3.53-3.59 (m, 1), 3.66-3.70 (m, 1), 4.46 (m, 1), 5.15 (t, 1, J = 5.5), 5.26 (d, 1, J = 3.7), 5.85 (br s, 2), 6.10 (dd, 1, J = 8.1, 8.0), 8.00 (s, 1). MS (El) m/z 325 (M + H). Anal. Calcd. for Ci 3 H 2 oN 6 O2S . 0.8 H O: C, 46.08; H, 6.43; N, 24.80. Found C, 46.17; H, 6.19; N, 24.62. Example 10 2-Amino-9-(2-deoxy-4-thio-β-D-erythro-pentofuranosyl)-6-N-pyιτolidino-9H-purine 2-Amino-6-N-pyrrolidino-9H-purine, prepared from 2-amino-6-chloropurine (Aldrich Chemical Co., Milwaukee, WI 53233) and pyrrolidine, was added to pH 6.0 citrate buffer as described above (Example B). l ΕL NMR (300 MHz, DMSO-d 6 ) δ 1.90 (br s, 4), 2.28-2.35 (m, 1), 2.49-2.57 (m, 1), 3.3 (m, 1), 3.48-3.59 (m, 1), 3.67-3.71 (m, 1), 3.90 (br s, 1), 4.46 (m, 1), 5.14 (t, 1, J = 5.5), 5.27 (d, 1, J = 3.7), 5.89 (br s, 2), 6.10 (dd, 1, J = 7.8, 7.9), 8.01 (s, 1). MS (El) m/z 337 (M + H). Anal. Calcd. for Ci4H 2 oN 6 O 2 S . 1.0 H 2 O: C, 47.44; H, 6.26; N, 23.71. Found: C, 47.51; H, 6.22; N, 23.67. Example 11 2-Ammo-9-(2-deoxy-4-tMo-β-D-e hro-pentofuranosylV6-aUylamino-9H-purine 2-Amino-6-allylamino-9H-purine, prepared from 2-amino-6-chloropurine (Aldrich Chemical Co., Milwaukee, WI 53233) and allylamine, was added to pH 6.0 citrate buffer as described above (Example B). *H NMR (300 MHz, DMSO-d 6 ) δ 2.28-2.33 (m, 1), 2.49-2.57 (m, 1), 3.3 (m, 1), 3.53-3.59 (m, 1), 3.67-3.72 (m, 1), 4.06 (br s, 1), 4.46 (m, 1), 5.01-5.12 (m, 2), 5.15 (t, 1, J = 5.5), 5.26 (d, 1, J = 3.7), 5.89 (br s, 2), 5.93 (m, 1), 6.10 (dd, 1, J = 8.0, 8.0), 7.39 (br s, 1), 8.02 (s, 1). Example 12 Tablet Formulations The following formulations A, B and C are prepared by wet granulation of the ingredients with a solution of povidone, followed by the addition of magnesium stearate and compression. Formulation A Formulation C mg tablet Active ingredient 100 Lactose 200 Starch 50 Povidone 5 Magnesium Stearate _4 359 The following formulations, D and E, are prepared by direct compression of the admixed ingredients. The lactose in formulation E is of the direct compression type (Dairy Crest - "Zeparox"). Formulation D mg tablet Active ingredient 250 Pregelatinised Starch NF 15 150 400 Formulation E mg tablet Active ingredient 250 Lactose 150 Avicel 100 500 Formulation F (Controlled Release Formulation) The formulation is prepared by wet granulation of the ingredients (below) with a solution of povidone followed by the addition of magnesium stearate and compression. m /tablet Drug release takes place over a period of about 6-8 hours and is complete after 12 hours. Example 13 Capsule Formulations Formulation A A capsule formulation is prepared by admixing the ingredients of Formulation D in Example 2 above and filling the mixture into a two-part hard gelatin capsule. ' Formulation C mg/capsule (a) Active ingredient 250 (b) Macrogol 4000 B.P. 350 600 Formulation D mg/capsule Active ingredient 250 Lecithin 100 Arachis Oil 100 450 Capsules of formulation D are prepared by dispersing the active ingredient in the lecithin and arachis oil and filling the dispersion into soft, elastic gelatin capsules. Formulation E (Controlled Release Capsule) The following controlled release capsule formulation is prepared by extruding ingredients (a), (b) and (c) using an extruder, followed by spheronisation of the extrudate and drying. The dried pellets are then coated with the release-controlling membrane (d) and filled into a two-piece, hard gelatin capsule. m ca sule Example 14 Iniectable Formulation Formulation A Active ingredient 0.200g Hydrochloric acid solution, O.1M, or Sodium hydroxide solution, 0.1M q.s. to pH 4.0 to 7.0 Sterile water q. s. to 10ml o o The active mgredient is dissolved in most of the water at 35 C-40 C and the pH adjusted to between 4.0 and 7.0 with the hydrochloric acid or sodium hydroxide as appropriate. The batch is then made up to volume with the water and filtered through a sterile micropore filter into a sterile 10ml amber glass vial (type 1) and sealed with sterile closures and overseals. Formulation B Active ingredient 0.125 Sterile, pyrogen-free, pH 7 phosphate buffer, q.s. to 25 ml Example 15 Intramuscular injection Active ingredient 0.20 g Benzyl Alcohol 0.10 g Glycofurol 75 1.45 g Water for Injection q.s. to 3.00 ml The active mgredient is dissolved in the glycofurol. The benzyl alcohol is then added and dissolved, and water added to 3 ml. The mbcture is filtered through a sterile micropore filter and sealed in sterile 3 ml amber glass vials (type 1). Example 16 Syrup Active ingredient 0.25 g Sorbitol Solution 1.50 g Glycerol 2.00 g Sodium Benzoate 0.005 g Flavor, Peach 17.42.3169 0.0125 ml Purified Water q.s. to 5.00 ml The active ingredient is dissolved in a mixture of the glycerol and most of the purified water. An aqueous solution of the sodium benzoate is then added to the solution, followed by addition of the sorbitol solution and finally the flavor. The volume is made up with purified water and mixed well. Formulation B Active ingredient 0.125 g Sterile, pyrogen-free, pH 7 phosphate buffer, q.s. to 25 ml Example 17 Suppository mg suppositorv Active ingredient (63 μm)* 250 Hard Fat, B.P. (Witepsol H15 - Dynamit Nobel) 1770 2020 *The active ingredient is used as a powder wherein at least 90% of the particles are of 63 μm diameter or less. 0 One-fifth of the Witepsol HI 5 is melted in a steam-jacketed pan at 45 C maximum. The active ingredient is sifted through a 200μm sieve and added to the molten base with mixing, using a Silverson fitted with a cutting head, until smooth dispersion is o achieved. Maintaining the mixture at 45 C, the remaining Witepsol HI 5 is added to the suspension and stirred to ensure a homogenous mix. The entire suspension is passed through a 250μm stainless steel screen and, with continuous stirring, allowed to o o o cool to 40 C. At a temperature of 38 C to 40 C, 2.0g of the mbcture is filled into suitable 2 ml plastic moulds. The suppositories are allowed to cool to room temperature. Example 18 Pessaries mg pessarv Active ingredient (631m) 250 Anhydrate Dextrose 380 Potato Starch 363 Magnesium Stearate 7 1000 The above ingredients are mixed directly and pessaries prepared by direct compression of the resulting mbcture. Example 19 Antiviral Activity (a) HBV Assay (Method 1) Anti-HBV activity of compounds of formula (I) was determined with a high- capacity assay for assessing efficacy. Supernatants from growing HBV- producing cells (HepG2 2.2.15, P5A cell line) in 96-well plates are applied to microtiter plate wells which have been coated with a specific monoclonal antibody to HBV surface antigen (HBsAg). Virus particles present in the supernatants bind to the antibody and remain immobilized while other debris is removed by washing. These virus particles are then denatured to release HBV DNA strands which are subsequently amplified by the polymerase chain reaction and detected with a colorimetric hybrid-capture assay. Quantitation is achieved through fitting of a standard curve to dilutions of a cell supernatant with known HBV DNA content. By comparing HBV DNA levels of untreated control cell supernatants with supernatants containing a compound of formula (I), a measure of anti-HB V effectiveness is obtained. Immunoaffinity Capture of HBV: HBV producer cells, 2500 cells well, were seeded in 96-well culture dishes in RPMI/10% fetal bovine serum/2mM glutamine (RPMI/10/2:). Media were replenished on days 1, 3, 5, and 7 with dilutions of a compound of formula (I) in RPMI/10/2 to a final volume of 150 μL. Fifty uL of mouse monoclonal anti- HBsAG antibody (lOμg mL in PBS) were added to each well of a round-bottom microtiter plate. After incubation overnight at 4°C, the solutions were aspirated and replaced with 100 μL of 0.1% BSA in PBS. Samples were incubated for 2 hours at 37°C and washed three times with PBS/0.01% Tween-20 (PBS/T) using a Nunc Washer. Ten uL of 0.035% Tween 20 in PBS were then added to all wells by Pro Pette. Cell supernatants (25 μL) containing extracellular virion DNA were transferred into wells by Pro/Pette; the final Tween concentration is 0.01%. Twenty-five μL HBV standard media dilutions in RPMI/10/2 were added to 2 rows of wells to serve as an internal standard curve for quantitation, and the plates were sealed and incubated at 4°C overnight. Samples were washed 5 times with PBS/T and 2 times with PBS, aspirating the last wash. Next, 25 μL of 0.09N NaOH/0.01% NP40 were added to each well by Pro/Pette, and the sample wells were sealed and incubated at 37°C for 60 minutes. Samples were then neutralized with 25 μL of 0.09N HCl 100 mM Polymerase Chain Reaction (PCR): Polymerase chain reaction (Saiki, R.K. et al., Science, 239 (4839) 487-91 (1988)) was carried out on 5μL samples, using a Perkin Elmer PCR kit. PCR is performed in "MicroAmp tubes" in a final volume of 25 μL. Primers were chosen from conserved regions in the HBV genome, as determined by alignment of several sequences. One primer is biotinylated at the 5-prime end to facilitate hybrid-capture detection of the PCR products. All primers were purchased from Synthecell Corp., Rockville, MD 20850. Hybrid-Capture Detection of PCR Products: PCR products were detected with horse radish peroxidase-labeled oligonucleotide probes (Synthecell Corp., Rockville, MD 20850), which hybridize to biotinylated strands of denatured PCR products directly in streptavidin-coated microtiter plate wells, using essentially the method of Holodiniy. M. et al-, BioTechniques. 12 (1) 37-39 (1992). Modifications included the use of 25k PCR reaction volumes and sodium hydroxide denaturation instead of heat. Simultaneous binding of the biotin moiety to the plate-bound streptavidin during the hybridization serves to "capture" the hybrids. Unbound labeled probes were washed away before colorimetric determination of the bound (hybridized) horse radish peroxidase. Quantities of HBV DNA present in the original samples were calculated by comparison with standards. These values were then compared to those from untreated cell cultures to determine the extent of anti-HBV activity. IC (the median inhibitory concentration) is the amount of compound which produces a 50 percent decrease in HBV DNA. (b) HeLa-CD4 + cell assay for evaluating susceptibility of HTV to antiviral compounds Susceptibility of HIV to inhibitors was determined by infection of HT4-6C cell monolayers as described by Larder, B.A., Chesebro, B. & Richman, D.D. Antimicrob. Agents Chemother. 1990 34, 436-441. Briefly cells were seeded in 24-well multiwells at 5xl0 4 cells per well and incubated overnight at 37°C in growth medium (DMEMIO). Monolayers were infected with 100-200pfu of cell-free virus in 0.2ml of DMEM containing 5% fetal bovine serum plus antibiotics (DMEM5) and incubated for 1 hour at 37°C to allow virus adsorption. Following this time 0.3m, of DMEM5 (with or without inhibitor) was added to each well and cultures were incubated at 37°C for 2-3 days. Monolayers were fixed with 10% formaldehyde solution in PBS and stained with 0.25% crystal violet in order to visualize virus plaques. Individual foci of multinucleated gian cells (plaques) were apparent using this staining procedure. ID50 values were derived from plots of percent plaque reduction versus inhibitor concentration. (c) HSV Assay Herpes Simplex Virus types 1 (HSV 1) and 2 (HSV 2) were assayed in monolayers of Vero cells in multiwell trays. The virus strains used were SCI 6 and 186 for HSV-1 and HSV-2 respectively. Activity of compounds was determined in the plaque reduction assay, in which a cell monolayer was infected with a suspension of the appropriate HSV, and then overlaid with nutrient carboxymethyl cellulose in the form of a gel to ensure that there was no spread of virus throughout the culture. A range of concentrations of compound of known molarity was incorporated in the nutrient carboxymethyl cellulose overlay. Plaque numbers at each concentration is expressed as percentages of the control and a dose-response curve was drawn. (d) CMV Assay Human cytomegalovirus (HCMV) was assayed in monolayers of MRC5 cells (human embryonic lung) in multiwell trays. The standard CMV strain AD 169 was used. Activity of compounds is determined in the plaque reduction assay, in which a cell monolayer is infected with a suspension of HCMV, and then overlaid with nutrient carboxymethyl cellulose in the form of a gel to ensure that there is no spread of virus throughout the culture. A range of concentrations of compound of known molarity was incorporated in the nutrient overlay. Plaque numbers at each concentration of drug are expressed as percentage of the control and a dose-response curve is drawn. (e) MCMV Assay Murine cytomegalovirus (MCMV) was assayed in monolayers of the mouse fibroblast cell line 3T3 clone A31, cultured in multiwell trays. MCMV strain Osborn was used. Compounds activity is determined in the plaque reduction assay in which a cell monolayer is infected with a suspension of MCMV and then overlaid with nutrient carboxymethylcelluose to ensure there is no spread of virus throughout the culture. A range of compound concentrations of known molarity was incorporated in the nutrient overlay. Plaque numbers at each concentration of drug are expressed as a percentage of a control without drug and a dose- response curve is drawn. (f) VZV Assay Clinical isolates of varicella zoster virus (VZV) were assayed in monolayers of MRC-5 cells. MRC-5 cells are derived from human embryonic lung tissue. A plaque reduction assay was used in which a suspension of the virus stock was used to infect monolayers of the cells in multiwell trays. A range of concentrations of the compound under test of known molarity was added to the wells. Plaque numbers at each concentration were expressed as percentages of the control and a dose response curve was contracted. From these curves the 50% inhibitory concentration of each drug was determined. (g) HBV Assay (Method 2) Materials Virus/Cells The cell line used was derived from a hepatoblastoma cell line, Hep G2, which had been transfected with a plasmid containing four 5'-3' tandem copies of the hepatitis B virus genome, subtype ayw, to produce the cell line designated 2:2:15. (Sells et al PNAS 84 1005-1009, 1987). These cells carry the Hep B DNA both as chromasomally integrated sequences and episomally. The cells constitutively produce small amounts of virus particles. A higher virus producing clone P5A, was obtained from the 2.2.15 cells for use in the assay. Media Cells were grown in RPM1 1640 containing 0.5% penicillin and streptomycin, 2mM L-glutamine and 10% foetal calf serum. Methods Assays were performed in 24 well plates there were seeded with, approximately 2.5x10 4 cells/well and grown for 5 days at 37°C in 5% CO2, the monolayers were then incubated with RPM1 1640, 0.5% penicillin and streptomycin, 2mM L-glutamine and 2% FCS containing the test compounds at the required concentrations. Medium was replaced every 48 hrs with fresh medium containing the test compound. The plates were incubated for 10 days, the medium was removed and the cells scraped from the wells in 0.5ml of PBS, the cells were pelleted at 5000 rpm for 5 mins the supernatant discarded and the cells frozen at -20°C. The cells were thawed and resuspended in 500μl of lysis buffer (150 mM Nad, 20mM Tris HCl pH7.4, lOmM EDTA and 0.6% SDS) and 50μl of proteinase K (20mg ml) added and the samples incubated at 37°C for 2hrs. DNA was extracted on an Autogen 540 DNA extractor and dissolved in a final volume of 50μl of water. DNA was digested with the restriction enzyme Hind m at 37°C for 16hrs and the DNA fragments separated on 1% agarose gel. The separated DNA was transferred by capillary blotting to hybond N " nylon membrane (Amersham International) and, after prehybridisation, hybridised with a 32p labelled positive strand RNA transcript of the core region of the hepatitis B genome, subtype ayw, at 42°C overnight in the presence of 50% foπnamide. After extensive washing the blot was exposed to X-ray film and the intensity of the hybridisation to the replicative intermediate DNA analysed by a Milli Pore 610 imager. Results were compared to a control sample containing no test compound and a known positive compound. (h) Cell Toxicitv Cell toxicity is assessed in cell growth inhibition assay. Subconflueπt cultures of Vero cells grown on 96-well microtiter dishes are exposed to different dilutions of drag, and cell viability determined daily on replicate cultures using uptake of a tetrazolium day (MTT). The concentration required for 50% inhibition of cell viability at 96 hours is termed CCID50.

Claims: CLAIMS 1. A compound of formula (I) R 1 wherein: Rl represents: - halogen -N R3 wherein R 2 and R^, which may be the same or different, each represent hydrogen, C^alkyl, C3_6cycloaIkyl, C2^alkenyl, phenyl or phenylC 1.3 alkyl (where the phenyl moiety may be optionally substituted by one or more substituents selected from halogen, C^alkoxy, nitro, cyano, amino and Ci^alkyl), or R 2 R^ together with the N atom to which they are attached form a 3-, 4-, 5-, 6- or 7-membered heterocyclic ring optionally containing, in addition to said nitrogen atom, one or more other hetero atoms independently selected from O and N; -S(=O) n R 4 where n is 0, 1 or 2 and R 4 represents C^galkyl, C3_6cycloalkyl, C3_6cycloalkylCι_3 alkyl, C^alkoxy, phenyl or phenylC 1.3 alkyl (where the phenyl moiety may be optionally substituted by one or more substituents selected from halogen, C^alkoxy, nitro, cyano, amino and C^galkyl), or, where n is 0, R represents hydrogen; -S(=O)mOR 4a where m is 0, 1 or 2 and R 4a represents C galkyl, C3_6cycloalkyl, C3_6cycloalkylC 1.3 alkyl, Cι_4alkoxy, phenyl or phenyl C\. 3alkyl (where the phenyl moiety may be optionally substituted by one or more substituents selected from halogen, C^alkoxy, nitro, cyano, amino and Cι_ 6 alkyl); -OR 5 where R 5 represents Cμgalkyl, C3_6cycloalkyl, C3_.5cycloalkylC 1.3 alkyl, phenyl or phenyl C^alkyl (where the phenyl moiety may be optionally substituted by one or more substituents selected from halogen, Cι_6alko y, nitro, cyano, amino and C^aUcyl); -C^alkyl, C2-6 lkenyl or C 2 _6alkynyl; with the proviso that when Rl represents -NR R^,R 2 and R^ are not both hydrogen; or a salt, ester or other physiologically functional derivative thereof or a solvate of any thereof. 2. A compound according to claim 1, where R represents halogen, -OR 5 where R 5 represents C^alkyl, C3.6cycloalkyl, C3_6cycloalkylC 1.3 alkyl; -NR 2 R3 where R 2 is hydrogen or C*"3 alkyl and * is C3_6cycloalkyl, C^alkyl, C2. galkenyl or R 2 ,R^ together with the N atom to which they are attached form a 4- ,5- or 6- membered heterocyclic ring optionally containing, in addition to said nitrogen atom, one or more other hetero atoms independently selected from O and N; -S(=O) n R 4 where n is 0 and R 4 represents hydrogen, or C^alkyl; or a salt, ester or other physiologically functional derivative thereof or a solvate of any thereof. 3. A compound according to claim 1 or 2 wherein R^ represents -NR 2 R-* where R 2 is hydrogen and R^ is C3.6cycloalkyl; or a salt, ester or other physiologically functional derivative thereof or a solvate of any thereof. 4. A compound according to claim 1 selected from 2-aιnmo-6-c^rclopropylmethoxy-9-(2-deoxy-4-thio-β-D-erythro-pentofuranosyl)- 9H-purine; 2-ammo-9-(2-deoxy-4-tMo-β-D-erythro-pentofuranosyl)-6-methoxy-9H purine; 2-ammo-9-(2-deoxy-4-tWo-β-D-erythro-pentofuranosyl)-6-methylthio-9H- purine; 2-amino-9-(2-deoxy-4-thio-β-D-erythro-pentofuranosyl)-6- (cyclopropylmethylamino)-9H-purine; 2-amino-9-(2-deoxy-4-thio-β-D-erythro-pentofuranosyl)-6-(ethylmethylamino)- 9H-purine; 2-ammo-9-(2-deoxy-4-tWo-β-D-erythro-pentoflu , anosyl)-6-iso-propylamino-9H- purine; 2-ammo-9-(2-deoxy-4-thio-β-D-erythro-pentofuranosyl)-6-N-piperidino-9H- purine; 2-aιnino-9-(2-deoxy-4-tMo-β-D-erythro-pentoftιranosyl)-6-n-propylamino-9H- purine; 2-ammo-9-(2-deoxy-4-tMo-β-D-e_rythro-pentofύranosyl)-6-N-pyrrolidino-9H- purine; 2-an__ no-9-(2-deoxy-4-tMo-β-D-erythro-pentofuranosyl)-6-aUylamino-9H- purine; and salts, esters and other physiologically functional derivatives thereof and solvates of any thereof. 5. 2-Ammo-6-(cyclopropylaιnino)-9-(2-deoxy-4-t o-β-D-e_rythro-pentofuranosyl)- 9H-purine. 6 A physiologically functional derivative of 2-amino-6-(cyclopropylamino)-9-(2- deoxy-4-t o-β-D-erythro-pentofuranosyl)-9H-purine. 7. A compound as defined according to any one of claims 1 to 5 or a physiologically functional derivative thereof for use in therapy. 8. Use of a compound as defined according to any one of claims 1 to 5 or a physiologically functional deriative thereof for the manufacture of a medicament for the treatment or prophylaxis of a viral infection. 9. Use according to claim 8 wherein the viral infection is selected from herpes viras, retroviras, hepatitis viras, coxsackie viras and hepatitis C viras infections. 10. Use according to claim 9 wherein the herpes viras infection is an infection selected from herpes simplex viras 1, herpes simplex virus 2, varicella zoster viras, cytomegaloviras, Epstein-Barr viras and human herpes viras 6 infections. 11. Use according to claim 10 wherein the herpes viras infection is a cytomegaloviras infection. 12. Use according to claim 9 wherein the hepatitis viras infection is an hepatitis B viras infection. 13. A method of treatment or prevention of the symptoms or effects of a virus infection in an infected animal which comprises treating said animal with a therapeutically effective amount of a compound as defined according to any of claims 1 to 5 or a physiologically functional derivative thereof. 14. A method according to claim 13 wherein the viras infection is selected from herpes viras, retroviras, hepatitis viras, coxsackie viras and hepatitis C viras infections. 15. A method according to claim 14 wherein the herpes viras infection is an infection selected from herpes simplex viras 1, herpes simplex viras 2, varicella zoster virus, cytomegaloviras, Epstein-Barr viras and human herpes viras 6 infections. 16. A method according to claim 15 wherein the herpes viras infection is a cytomegaloviras infection. 17. A method according to claim 14 wherein the hepatitis viras infection is a hepatitis B viras infection. 18. A pharmaceutical formulation comprising at least one compound of formula (I) as defined according to any one of claims 1 to 5 or a physiologically functional derivative thereof together with at least one pharmaceutically acceptable carrier therefore. 19. A formulation according to claim 18 in unit dosage form. 20. A formulation according to claim 19 in the form of a tablet or capsule. 21. A process for the preparation of a compound according to any one of claims 1 to 6 which comprises:- (A) reacting a purine base of formula (II) wherein R* is defined in claim 1, or a functional equivalent thereoζ with a compound of formula (HI) OR7 wherein R^ and R^ are the same or different and each represents hydrogen or a hydroxy protecting group and A is a phosphate group or salt thereof or a pyrimidine or purine moiety other than (II) or a leaving group, to form a compound of formula (I); or (B) reacting a compoimd of formula (IV) wherein R> and R^ are as hereinbefore defined and R^ represents a precursor for R 1 as defined in claim 1 with a reagent or reagents and/or under conditions serving to convert R 8 to the desired R 1 group; and thereafter or simultaneously therewith effecting one or more of the following optional conversions:- (i) removing any remaining protecting group(s); (ii) when a compound of formula (I) is formed, converting it into a salt, ester or other physiologically functional derivative of a compound of formula (I); or (iii) when a salt, ester or other physiologically functional derivative of a compoimd of formula (I) or a solvate of any thereof is formed, converting the derivative into a compound of formula (I) or into a different derivative of the compound of formula (I). (iv) where necessary, separating the α and β anomers of the compound of formula (I) or a protected derivative thereof or a physiologically acceptable derivative of a compound of formula (I).

Inventors: FREEMAN GEORGE ANDREW, KOSZALKA GEORGE WALTER, SHORT STEVEN ANDERSEN, VAN DRAANEN NANINE AGNETA, FREEMAN, GEORGE, ANDREW, KOSZALKA, GEORGE, WALTER, SHORT, STEVEN, ANDERSEN, VAN DRAANEN, NANINE, AGNETA
Applicants: DRAANEN NANINE AGNETA VAN, FREEMAN GEORGE ANDREW, KOSZALKA GEORGE WALTER, SHORT STEVEN ANDERSEN, WELLCOME FOUND, THE WELLCOME FOUNDATION LIMITED
Application year: 1993
Main classification: C07H5
IPC codes: A61K31, A61P31, C07H19