IE84454B1 - Antiviral compounds - Google Patents

Description

PATENT SPECIFICATION (11) 34454 (21) Application No. 1988/1595 (22) Date of Filing of Application: 27/05/1988 (30) Priority Data: (31) 8712745 (32) 30/05/1987 (33) United Kingdom (GB) (45) Specification Published: 29 December 2006 (51) Int. C1. (2006) C07H 19/00 A61K 31/7042 A6lP 31/00 (54) Title: Antiviral compounds (72) Inventor: THOMAS ANTHONY KRENITSKY GEORGE WALTER KOSZALKA LYNDA ADDINGTON JONES DEVRON RANDOLPH AVERETT ALLAN RAY MOORMAN (73) Patent Granted to: THE WELLCOME FOUNDATION LIMITED, a British Company, 183- 193, Euston Road, London NW1 2BP, United Kingdom © Copyright 2006, Government of Ireland '* 84454 I R E L A N D PATENTS ACT, 1964 COMPLETE SPECIFICATION "Antivira1 Compounds" Patent Application by THE WELLCOME FOUNDATION LIMITED, a British Company of 183-193 Euston Road, London NW1 ZBP, Eng1and. " 84454 The present invention relates to certain substituted purine arabinosides, and physiologically’ acceptable derivatives thereof is: particular esters, and their use in the treatment of certain DNA virus diseases.

Varicella-herpes zoster virus (VZV) which causes chicken-pox and shingles, is a. DNA ‘virus of the herpes family. Varicella (chicken-pox) is the primary disease produced by VZV infa host without imunity; it is usually a mild illness of young children which is manifested as fever and an itching rash. Herpes zoster (shingles) is the recurrent form of the disease occurring in adults who were previously infected with the varicella-zoster virus. The clinical manifestations of this infection are characterised by neuralgia and a vesicular skin rash that is unilateral and dermatomal in distribution. Spread of inflammation may lead to paralysis or convulsions, and coma if the meninges become affected.

In immunodeficient patients the virus may disseminate causing serious often fatal illnesses. The cause of immunodeficiency may be drugs used on transplant patients or in the treatment of malignant neoplasms, or diseases, such as AIDS, which destroy the imune system, leaving the victim vulnerable to infections which would not otherwise be dangerous.

Cytomegalovirus (CMV) is another virus of the herpes family. Infection may be acquired in infancy or early adulthood and, in the foetus, intra-uterine infection is probably the commonest form of infection, but up to 90% of congenital infections are asymptomatic at birth. Primary infection of the mother during pregnancy is generally considered to pose the greatest risk to the unborn child, whereas in reactivation the foetal infections are usually clinically silent. Clinical effects range from death and gross disease (microcephaly, hepatosplenomegaly, jaundice, mental retardation) through failure to thrive, susceptability to chest and ear infections to a lack of any obvious ill effect. In young adults infection may well go unnoticed. or manifest as a glandular fever like disease resulting from close physical contact. _1b_ Equally serious infections may also occur due to reactivation of the described for VZV Such infections result in increased morbidity and fatality from retinitis, pneumonitis and gastro-intestinal disorders. dormant virus in imuno-compromised patients as infections.

It has now been found that certain purine-arabino nucleosides described below characterised by the presence of groups substituted at the 2- and 6- positions of the purine ring have potent activity against human virus infections particularly those caused by varicella zoster virus (VZV) or cytomegalovirus (CMV).

Certain substituted purine-arabino nucleosides, in particular 9-,6-D~ arab inofuranosylmethoxypurine , 9 -fi-D- arabinofuranosyl - 6-pyrrolidino - 95- purine . 9 D - arabinofuranosyl tnethylanino - 911- purine and arabinofuranosyldimethylaminopurine described hereinafter for their use in the treatment of VZV and CKV infections, have previously been described in J .Org.Chem. Vol 27 3274-9 (1962); Cancer Treatment Rep. 60(10), 1567-84, (1976); Tetrahedron 40(4). 709-13. (1984); Canada J.

Biochen. 43(1), 1-15, (1965); J.M'ed.Chen.i 12, 498-504, (1969); J. Biol.

Chem. 251(l3), 4055-61, (1976); Ann. N.Y. Acad. Sci. 284, 81-90, (1977); EP002l92; US3666856; USli»3716l3; US3758684.

Haskell i{ork7Acad.WSci. 1977, 284, 81) discloses the clinical efficacy of 9-B-D- arabinofuranosyladenine analogs against VZV AND CMV. This reference briefly discusses modification of this compound at the 6-postion to produce N-acylatezlanalogs, Schifi‘ base analogs, 6- hydroxy, 6-halo or 6-hydrogen analogs. There is, however, no reference to any substitution at the 2- positon of the ring system and hence to the 9-B-D-arabinofiiranosylamino6-methoxy-9H-purine of the present invention, nor to the esters of the present invention. This reference concludes that a minimal amount of substitution in the pyrimidine ring of vidarabine is allowable if in vitro antiviral activity is to be maintained. -fl-D- _ European Patent No. 0002192 relates to enzymatic synthesis of purine and purine derivative arabinonucleosides which may be used as anti-viral agents. While this citation refers to generic purine arabinosides and to a number of particular compounds which may be enzymatically produced, there is no specific reference to 9-B-D-arabinofiiranosylaminomethoxy—9H- purine nor to any of the esters of the present invention.

Thus in the first aspect of the present invention there is provided the use of a compound of formula (I): ll: .. I N/, N Q0 Rt; N ~ (1) HO O wherein R1 is methoxy and R; is hydrogen or amino, and physiologically acceptable derivatives thereof in the manufacture of a medicament for the treatment or prophylaxis of human viral infections caused by VZV.

In another aspect of the present invention there is provided 9-B-D-arabinofiiranosylamino methoxy-9H-purine or a physiologically acceptable derivative thereof.

In yet another aspect of the present invention there is provided a pharmaceutically acceptable ester of a compound of formula (I) selected from: -(5-O-(4-methylbenzoyl)-|3-D-arabinofi.Iranosyl)methoxy-9H-purine; 9-(5-O-(4-aminobenzoyl)-B-D-arabinofuranosyl)methoxy—9H-purine; or 6-methoxy(2-O-pentanoyl-B-D-arabinofuranosyl)-9H—purine.

In a further aspect of the present invention there is provided 9-B-D-arabinofuranosylamino~ -methoxy-9H-purine for use in medicine.

The following compounds are therefore preferred compounds of the present invention by virtue of their potent antiviral activity against VZV: ) 9-B-D-Arabinofiiranosylmethoxy-9E-purine, and 2) 9-B-D—Arabinofi.1ranof;y1aminornethoxy-9_lpurine.

I In a further aspect of t e present invention there are provided Pharmaceutically acceptable derivatives of the compounds of formula (I) include any pharmaceutically acceptable ether, salt, ester or salt of such ester, or any other compound which, upon administration to a human subject is capable of providing (directly or indirectly) a compound of formula (I) or an antivirally active metabolite or residue thereof.

The pharmaceutically acc ptable esters of the above compounds of formula (I) are particularly preferred since they are cgpable of providing high levels of the parent compound in the plasma of a subject after oral adniinistration. The present invention particularly provides as a preferred class of novel compounds the pharmaceutically acceptable esters of compound 1) above fomied by esterification of the 2' «, 3' - and! or 5' - hydroxy group of the arabino-sugar moiety.

The following esters and ethers are preferred novel compounds of the present invention: l .9-(S-O—(4—Methylbenzoyl)-B-D-arabinofurano syl)methoxy-9_I-_I-purine. 4.9-[5-O-(4-Nitrobenz0yl)pB—D-arabinofuranosyl]methoxypurine. . 9-[5-O-(4-Arninobenzoy\)-B-D-arabinofuranosyl]methoxy-9L1-purine. 6.6-Methoxy(2-O-pentanoyl-B-D-arabinofurano syl)—9Ij-purine. . 6-Methoxy[3,5-O—(l ,1 ,3 ,3-tetraisopropyl-1 ,3-disiloxan-1 ,3-diyl)-B-D-arabinofiiranosyl} _I1—purine.

The purine nucleosides of formula (I) and their derivatives will be hereinafter referred to as compounds according to the invention or as active ingredients.

In a further, preferred aspect of the present invention, there is provided the use of a compound according to the invention in the manufacture of a medicament for the treatment or prophylaxis caused by VZV or CMV. of human viral infections The present invention further provides a method for the treatment or prophylaxis of V2‘! and CMV infections in a human subject which comprises administering to the said human subject an effective amount of a compound according to the invention.

The method hereinbefore described includes inhibiting the replication of VZV or CMV viruses in host cells of a mammal which comprises applying an effective virus replication inhibiting amount of the compound of formula (I), or a pharmaceutically acceptable derivative thereof, cells. to the infected E 1 £ the clinical conditions causgd by V21 and CMV infections which xamp es 0 b 1: d in accordance with the invention include those referred to may e tree e above .

Th d f formula (1) and pharmeceuticdly acceptable derivatives e compoun 0 thereof (hereafter collectively referred to as 1119 3031" instediefits) W3)’ dm 1 t d by any route appropriate to th condition to be treated. be a in s ere bl C including oral rectal "e331 topical (including buccal suita e rou es - ' subcutaneous , and Pa'r.m-3:31 (including gnuthecal and epidural) . and sublingual) . V485-"$1 intramuscular , intravenous , intradermal . ill be appreciated that the preferred routglay vary with, for example, w the condition of the recipient.

For each of the above-indicated utilities and indications the amount ,- required of an active ingredient (as above defined) will depend upon 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 will be in the range 0.1 to 250 mg per kilogram body weight of recipient per day. preferably in the range 0.1 to 100 mg per kilogram body weight per day and most preferably in the range 1 to 20 mg per kilogram body weight per day; an optimum dose is about mg per kilogram body weight per day (unless otherwise indicated all weights of active ingredient are calculated as the parent compound of formula (I); for salts and esters thereof the figures would be increased proportionately). The desired dose is preferably presented as two, three, four or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit‘ dosage forms. example, containing 5 to l00O mg, for preferably 20 to 500 mg and most preferably 100 to 400 mg of active ingredient per unit dosage form.

While it is possible for the active ingredients to be administered alone it is preferable to present them as pharmaceutical formulations. The formulations of the present invention comprise at least one active ingredient. as above defined. together with one or more acceptable carriers thereof and optionally other therapeutic ingredients. ' The carrier(s) must be "acceptable" in the sense of beingcompatibls with the other ingredients of the formulation and not deleterious to the recipients thereof.

The formulations include those suitable for oral, rectsl,nasal. topical (including buccal and sublingusl) , subcutaneous , vaginal or parenteral intravenous , ( including intramuscular, intrathecal and epidural) administration. intradermal, The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art 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 a suspension in an aqueous liquid or a 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 .

A tablet may be made by compression or moulding. optionally with one or more accessory ingredients. Compression tablets may be prepared by compressing in an suitable machine the active ingredient in a free-flowing form such as a powder or granules. optionally mixed with a binder (e.g. povidone . gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative.’ disintegrant (e.g. ' sodima starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. made by 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, hydroxypropylmethylcellulose in vafylng proportions to provide the desired release profile.

Forfiinfections of eye or other external tissues, e.g. ,_ mouth and skin, the formulations are preferably applied as a topical ointment or cream containing the active ingredient in an amount of, for example, 0.075 to 20% w/w, preferably 0.2 to 15% w/w and most preferably 0.5 to l0% w/w. formulated in an ointment, when 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.

If desired, the aqueous phase of the cream may include, least 30% w/w of a polyhydric alcohol, hydroxyl groups for example , at Le. an alcohol having two or more such as propylene glycol , butane- 1. , 3 - diol , mannitol , s orbitol , glycerol and polye thylene glycol and mixture 3 thereo f . The top ical formulations may des irably include a compound which enhance s absorption or penetration of the active‘ ingredient through the skin orb other affected areas. Examples of such dermal penetration enhancers include dimethylsulphoxide and related analogues.

The oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a‘ mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is an lipophilic emulsifier which acts as a stabilizer. to include both an oil and a fat. included together with It is also preferred Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, together with the oil and/or fat make up the so-called emulsifying ointment and the wax base which forms the oily dispersed phase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodium lauryl 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 compound in most oils likely to be used in pharmaceutical emulsion formulations is very low. Thus the cream should preferably be a non-greasy, non- staining and washable product with suitable consistency to Straight or branched chain, mono-or dibasic alkyl esters such as di-isoadipate, avoid leakage from tubes or other containers. isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate. butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol 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 also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient.

The active ingredient is preferably present in such formulations 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 flavoured base, sucrose usually and acacia or tragacanth; pastilles comprising the ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; active 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 a salicylate.

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 for example a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.

Formulations suitable for vaginal administration may be bk 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 sterile powders, described. from granules and tablets of the kind previously Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above recited, or and appropriate fraction thereof . of an active ingredient.

It should be understoodithat 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 flavour ing agents .

The present invention also provides a process for the preparation of 9-B-D-3F3bifl0fiJF3fl05)’l'2- aminomethoxy-9H-purine comprising either: A. reacting a compound of formula (II) /"’-‘\ N (11) K Rm ~ 2: i wherein R1 is methoxy and R2 is amino with a compound formula (III) Ho O x "O (111) 0H wherein X represents a pyrimidine or purine base ‘(other than a compound of formula (II)); or B. reacting a compound of formula (IV) J N 1 N N no °\l wherein Z is a leaving group and R2 is amino, with a compound capable of introducing a methoxy group at the 6-position; and optionally thereafter or simultaneously therewith, where the resulting compound is a compound of formula (I), converting it into a pharmaceutically acceptable ester or ether thereof or where the resulting compound is a pharmaceutically acceptable ester or ether converting it into a different pharmaceutically acceptable ester or ether of or a compound of formula (I).

With regard to process A), X is advantageously a uracil base. The reaction may be effected, for example, by treatment of formulae II and III with an enzyme such as a phosphorylase enzyme, for example, uridine phosphorylase or purine nucleoside phosphorylase, or a mixture thereof preferably in the presence of a phosphate salt at an pH of 5.0-9.0 and a temperature of 15°-90°C advantageously 40°—60°C.

Regarding process B), this may be carried out in accordance with the procedure described by Reist E.J. et al., J. Org. Chem. 31 (1962) 3274-3279. A convenient leaving group is a halogen atom for example chlorine, the reaction being advantageously carried out in an organic solvent, e. g. absolute methanol, with an agent capable of providing the necessary group at the 6-position for example, an appropriate amine in the case where R1 represents an alkyl- or dialkylarnino group.

Physiologically acceptable esters and salts of the compounds of formula (I) may be prepared in conventional manner for example esters may be prepared by esterification of the parent compound with an appropriate acyl halide or anhydride. Alternatively the esters may be prepared by displacing an appropriate leaving group eg. halide with an appropriate carboxylic acid or by opening an appropriate anhydro nucleoside of the parent compound with an appropriate carboxylic acid or salt thereof.

The following Examples illustrate the present invention but should not be considered in any way limiting of the invention.

Example 1; 9-B-D-Arabinofilranosvlmethoxypurine uridine phosphorylase and purine The filtrate was adjusted to pH 10.5 with ammonium hydroxide and chromatographed on a 2.5 x 7cm column The eluted with 30% Fractions containing the product were combined and the solvent removed under vacuum. The residue was dissolved in 30! n-propanol/water (v/v) and chromatographed on Biokad P92 column (7.5 x 90 cm). containing Dowexformate resin. resin was n-propanol/water (v/v).

Product containing fractions were combined and after lyophilization yielded 0.922 g of 6-methoxypurinefl-D-arabinofuranoside as a dihydrate.

Anal. Calcd. for 0l1H14NAO52H20: Calcd: C, 41.51: H, 5.70; N, 17.60 Found: C, 41.46; H, 5.74; N, 18.13 NMR and mass spectrometry were consistent with the structure.

Example 2;" 9-B-D-Arabinofuranosvl-Laminomethoxvpurine -Amino~6-methoxypurine (prepared by nucleophilic displacement of the chlorine group on 2-aminochloropurine (Sigma Chemicals St. Louis M0) by methanol with sodium hydride in tetrahydrofuran) (6.4mmol, 1.0Sg) combined with 35ml of a uracil arabinoside solution (7.04mmo1, 1.7Sg) in 10 mM potassium phosphate and 7% n-propanol (v/v). 6.75. Purified purine nucleoside phosphorylase (18000 units) and uridine phosphorylase (1020 units) were added and the solution incubated at 37°C.

After 26 days the reaction was filtered and the filtrate chromatographed on The pH was adjusted to a column of Dowexformate resin (2 x 7cm) after adjusting the pH to 10.5 with concentrated ammonium hydroxide. The colum. was eluted with 7% n-propanol/water (v/v) and fractions containing product were combined and - solvent removed in vacuo.The residue was extracted~with:25m1 of water and t filtrate separated from the solids by centrifugation. The supernatant upon standing at ambient temperature formed crystals of 2-aminomethoxypurine B-D-arabinofuranosido which after drying in vecuo, yielded 0.327g of product.

Anal. Calcd. for Clllilslisosiio ‘- Calcd : C,ls4.44; fl.5.09;.N,23.S6 Found : C,44.49; 1-l.S.l3; N,23.S2 NMR and mass spectrometry were consistent with the structure.

Example 3; -(5-O-(4—Methvlbenzov1)-B-D-arabinofilranosvlmethoxv-9H-purine -(5-D-Atabinofuranosyl)methoxy-9fl-purine from Example 1 (O.283g, 1.0 mmole) was suspended in anhydrous acetonitrile (10.0 ml), pyridine (2.3 ml) was added to effect complete chloride (0.l70g, 1.1 mmole). for 24 hours under argon. solution, followed‘ by 4-methyl-benzoyl The mixture was stirred at room temperature quenched by adding isopropanol (Sml), evaporating to dryness, followed by coevaporation with ethanol (2 x 10 ml).

The residue was then purified by flash chromatography on silica gel (25.0g, 2.5 x 15 cm) with a stepped gradient of CHC1 to 1:1 CHCI : Those with an RF_0;41 (silica acetone . fractions containing product gel , CHC13:acetone/ 1:1) were combined to afford 132 mg of the desired compound: ix mp : 127-128°C; Analysis calculated for C19H20Nh06 ' 0.5 H2O:C.S5.?tx H,5.l7;N,13.69.

Found : C,55.48; H,5.36; N,l3.64.

NMR and mass spectrometry were consistent with the structure.

Example 4," -I5-O-(6-Nitrobenzov1)-B-D-arabinofixranosvl]methoxv-9H-purine -(fl-D-Arabinofuranosyl)methoxy-9fl-purine from Example 1 (0.283g, i.0mmo1e)was suspended in anhydrous acetonitrile (10.0ml), pyridine (2.3m1) was added to effect complete solution, followed by A-nitro-benzoyl chloride (O.205g, 1.1 mmole). The mixture was stirred at room temperature for 24 hours under argon, quenched by adding isopropanol (Sal) , and evaporating to dryness, followed by coevaporation with ethanol (2xl0m1). The residue was then purified by flash chromatography on silica gel (2S.Og, 2.5 x 15cm) with a stepped gradient of CHCI3 to C1-[C13 : acetone (1:1). Those fractions containing product with an R(:—().37 (silica gel, CHC13:acetone (1:1)) were combined to afford 105mg of the desired compound : mp : 202-2o3°c; Analysis calculated for CHHUNSO8 : C,50.l2; 11.3.97; N.l6.2h.

Found : 0.50.21; I-1.4.02; N,16.l6.

NHR and mass spectrometry were consistent with the structure.

Example 5;‘ -[5(4-AminobenzovluB-D-arabinofiaranosylImethoxv~9H-purine -(5-O-(4—Nitrobenxoyl)-[3-D—arabinofuranosyl) methoxy-9H-purine from Example 4 (0.35g, 0.8lmmole) was suspended in ethanol (l00.0ml) and 10% palladium-on-carbon (0.10g) was added. After alternately evacuating and charging the system with hydrogen, the reaction is shaken on a Parr apparatus at 50 psi for three hours. The mixture is then filtered through Celite and the filtrate evaporated to dryness. The residue afier evaporation was suspended in methanol and filtered to afford the desired compound as a white solid (O.285g, 88%) : , _A\_,___ - , .. __._ . mp: 19a.2oo°c; Analysis calculated for C18H19N5O6 . 0.3 H20 : C,53.l5; I-1.4.86; N,17.22, Found : C,52.96; t-11.4.64; N,17.07.

NMR and mass spectrometry were consistent with the structure ‘ -Methoxv—9-(2-O-oentanovl—B-D-arabinofiiranosvl)—9H-purine - Method 1 _1g,_ -(fl-D-Arabinofuranosyl)nethoxypurine from Example 1 (2B3mg. 1.0 mole) acetonitrile (20ml) and pyridine (5ml) were added to a three-necked round bottom flask equipped with thermometer, magnetic stirrer. argon inlet valve, and Valerie anhydride (O.2m1. l.0mmole) was added and the solution allowed to stir at 20°C for three hours. The reaction was quenched with water (2m1) and evaporated to a clear oil, then purified by flash chromatography on silica with a stepped gradient of CHC13 to C1-lC13:l'ie0H (9:1). the 5'-ester, This yielded 110 mg of the 2’-ester contaminated with a lower Fl, material. This material was further purified by preparative layer chromatography on silica with CHCI3: MeOH(9; 1) and co—evaporaled with acetone to aflord 70mg oi the 2'-ester as a clear glass.

Analysis calculated for CEHZZNAO6 0.5 (CH3)2CO c,53.l6; 8.6.37; N.14.l7.

Found : C.S2.B9; H,6.37; N,l3.96. mm and mass spectrometry were consistent with the structure. !11Eb.9$_2 -Hethoxy [3 , 5 (1 , 1 , 3 . 3-tetraisopropyldisiloxan-1 . 3-diy1)-fi-D- arabinofuranosyll purine from example 7 round bottom flask along. with 4e_<1jmethylaminopyridine (0.069. 9.47 mnlol). Dry acetonitrile (30111) and triethylarnihez (2.6Sm1) were added, and a constant addition funnel was (20ml) The reaction mixture was cooled in an 3°C ice charged with acetonitrile anhydride (l.l3m1, 5.7mmo1). bath under a flow of argon. and pentanoic Slow addition of the pentanoic anhydride solution was carried out over a period of 2 hours. The reaction mixture was then treated with methanol (lOml) and concentrated under reduced pressure. The residue was taken up in CHCl3 (2001111) and extracted with H (2xSOml) .

The combined aqueous layers were back extracted with CHCI3 (2Sm1) and the combined organic layers were dried (M5504), filtered, and concentrated to give 3.44 gms of crude product. (2.5g, 4.8 mmol) was added to a 250m _15_ combined and concentrated.

Analysis calculated for C [-1 N 0 A 6 . 0.2 C H 0 ‘ C,52.79; H,6.25; N,14.ls8.

' Found: 0.52.97; H,6.38; N,lh.60.

Example 7 : 6-Methoxy—9-[3 5-01 1 ,2, 3 , 3-tetraisopropyl- 1 ,3-disiloxan— 1 ,3-diyl)-B-d-arabmofi1ranosv1)-9H-purine -[3-D-afflblI10fiJf3n0Sy1meth0XY-9I;I-purine from Example 3 (1.0g, 3.54mmol) and imidazole (01965 g, 14.2mmol) were dissolved in dry dimethylformamide (101111) and the solution was chilled to 3°C. 1,3,-Dichloro-1,1,3,3-tetraisopropyldisiloxane (1.35ml, 3.90mmol) was then added and the mixture stirred under argon for three hours. The reaction mixture was then quenched with l-I20 (lml) and concentrated under reduced pressure. The residue was partitioned between ethyl acetate (1501111) and H20 (50ml), and the ethyl acetate layer dried (MgSO4), filtered and concentrated. The crude product was taken up in ethyl acetate and purified by flash chromatography on silica gel (25.0g, 2.5 x 15cm) in ethyl acetate. Those fractions with an R: of 0.70 (silica, ethyl acetate) provided 1.48g (80%) of the desired material as a white solid.

UV max : EtOH : 248.4nm.

NMR and mass spectrometry were consistent with the structure. _15_ The following formulations A and B are prepared by wet granulation of the ingredients with a solution of povidone, followed by addition of magnesium stearate and compression. mu.:ab.19.t ma/£318.23 (.3) Active ingredient 250 250 (b) Lactose 3.1’. 210 25 (c) Povidone n.1=. ’ 15 to V _ . 9 (33 isocuum stetéii cxymxiee V 20 12 (e) Magnesium Stearate m __i _; Eogguiatigg E ms./£b.Le.: Active ingredient L 100 Lactose 200 Starch A 50 Povidone B.P. 5 Magnesium stearate _¢_g Tablets are prepared from the foregoing ingredients (C) by wet granulation followed by compression. In an alternative preparation the Povidone B.P. may be replaced by Polyvinylpyrrolidone. _ 17 _ The following formulations, D and E, are prepared by direct compression of the admixed ingredients. The lactose in formulation 8 is of the direct compression type (Dairy Crest - "Zeparox").

Eogmulagiog Q Esteem]; Active Ingredient 250 Pregelatinised Starch NFIS 119 O at mzsszsaah Active Ingredient 250 Lactose 150 Avicel ;QQ u n F o u 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. mmtshlss (a) Active Ingredient 500 (b) Hydroxypropylmethylcellulose 112 (uethocel KAM Premium) (c) Lactose B.P. 53 (d) Povidone B.P. 28 (e) Magnesium Stearate __1 Drug release takes place over a period of about 6-8 hours and is complete after 12 hours.

Egample 5’ ; Qgpgglg Eggmglggions A capsule formulation is prepared by admixing the ingredients of Formulation D in Example 50 above and filling into a two-part hard gelatin capsule. Formulation B flnfirg) is prepared in a similar manner.

E.o_:mJ.as.i9.n._& mxéeenesle (a) Active ingredient 250 (b) Lactose B.P. 1143 (c) Sodium Starch Glycollate 25 (d) Magnesium Stearate _2_ 420 O U 8 mzzgazaule (a) Active ingredient 250 (b) Macrogol 4000 B.P. 153 600 Capsules are prepared by melting the Macrogol 4000 BP, dispersing the active ingredient in the melt and filling the -melt into a two—part hard gelatin capsule. &uu1.l.a1:.12u_Q maéeenauls Active ingredient 250 Lecithin 100 Arachis Oil LQQ Capsules are prepared by dispersing the active ingredient in the lecithin and arachis oil and filling the dispersion into soft. elastic gelatin capsules.

Fo u 'o e Ca 5 e The following controlled release formulation is capsule prepared by followed by The dried pellets are then extruding ingredients a, b and c using an extruder, spheronisation of the extrudate and drying. _]_9_ coated with release-controlling membrane (d) and filled into a two-piece, hard gelatin capsule. magma: (a) Active Ingredient 250 (b) Microcrystalline Cellulose 125 (c) Lactose B.P. 125 » (d) Ethyl Cellulose 132 emmJ Active ingredient 0.5 Sodium chloride, analytical grade 0.9 g Thiomersal 0.001 g Purified water 100 ml pl-I adjusted 7.5 km Active Ingredient 0.200 g Sterile, pyrogen free phosphate buffer (pH 9.0) to 10 ml The active ingredient is dissolved in most of the phosphate buffer (3500 60°C‘ then made up to volume and filtered through a sterile micropore filter into a sterile 10ml amber glass vial (type 1) and sealed with sterile closures and overseals.

Active Ingredient 0.20 g Benzyl Alcohol 0.10 g Glycofurol 75 L45 g Water for Injection q.s. to 3.00 ml _ 20 _ The active ingredient is dissolved in the glycofurol. The benzyl alcohol is then added and dissolved, and water added to 3 ml. The mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml amber glass vials (type 1).

Active Ingredient 0.25 g Sorbitol Solution 1.50 g Glycerol 2.00 g Dispersible Cellulose 0.075 g Sodium Benzoate 0.005 g Flavour, Peach 17.42.3169 0.0125 ml Purified Water q.s. to 5.00 ml The sodium benzoate is dissolved in a portion of the purified water and the sorbitol solution added. The active ingredient is added and dispersed. In the glycerol is dispersed the thickener (dispersible cellulose). The two dispersions are mixed and made up to the required volume with the purified water. Further thickening is achieved as required by extra shearing of the suspension.

*The active ingredient is used as a powder wherein at least 90% of the particles are of 63pm diameter or less.

One-fifth of the Witepsol H15 is melted in a steam-jacketed pan at 45°C maximum. The active ingredient is sifted through a 200nm sieve and added to the molten base with mixing, using a silverson fitted with a cutting head, until a smooth dispersion is achieved. Maintaining the mixture at .4 .....V......................._......._.._._.... ,... -. i. _. . , /TV-7171 _21_ °C, the remaining Witepsol H15 is added to the suspension and stirred to ensure a homogenous mix. The entire suspension is passed through a 250nm stainless steel screen and. with continuous stirring, is allowed to cool to 540°C. At a temperature of 38°C to 40°C, 2.02g of the mixture is filled into suitable plastic moulds. The suppositories are allowed to cool to room temperature. m e a ‘active ingredient 63pm ' 250 Anhydrate Dextrose 380 Potato Starch 363 Magnesium Stearate __Z The above ingredients are mixed directly and pessariee prepared by direct compression of the resulting mixture. e t -V - V v The inhibitory effects of compounds on the replication of VZV (Oka strain) were assessed by an ELISA procedure (Berkowitz, F.E., and Levin, M.J. (1985). Antimicrob. Agents and Chemother. 2_&: 207-210) that was modified as follows. Infections were initiated in the presence of drug, rather than before drug addition. At the end of the three day incubation of drug and virus with uninfected cells (human diploid fibroblasts, strain MRC-5), the 96-well plates were centrifuged for 5 minutes at 200 x g to sediment detached cells prior to glutaraldehyde fixation. The present ELISA used an alkaline phosphatase-conjugated anti-human IgG as the second antibody. The rate of cleavage of p-nitrophenyl phosphate by bound alkaline phosphatase was determined as described elsewhere (Tadepalli, S.M.. Quinn. R.P., and Averett, D.R. 1986. Antimicrob. 93-93).

Uninfected cells were used to obtain the blank reaction rates. which were Agents and Chemother. 2_2: subtracted from the rates obtained with the virus present. This assay was suitable to detect progeny virus in cultures that were initially infected with 15 to 3600 infectious particles per well.

Dt t w t oU d a s The capability of candidate compounds to inhibit the growth of D98 cells (human) and L cells (murine) was measured by determination of cell number following three days exposure of a standard number of cells to various dilutions of compound (Rideout, J. L., Krenitslcy, ’l‘.A., Koszalka, G.W., Cohn, N.l(., Chao. E.Y. Elion. G.B., Latter, V.S., and Williams, R.B. (1982) J. Med Chem. 11: 1040-1044). The cell number was then compared to the number obtained in the absence of compound. Cell enumeration was performed by either direct particle counts following trypsinization of the monolayer, or by spectrophotometric determination of the amount of vital stain taken " up by the cells. Comparable results were obtained with both methods. a:.a..AnaJ.ys.1.s The concentration of compound resulting in 50$ of control values (ICSO) was calculated either by direct interpolation from graphs of the log of the compound concentration versus the percent of control value, or from a computer program which analyses the data according to the same algorithm.

Data in the range of 20% to 80% of control were used in these calculations.

Example VZV (|C50um) Cell Toxicity (% of control at 100nm) D-98 cells L C6115 1 0.8 96 72 ACV (Acyclovir) 20 100 Bioavailabilitv Assay & Data Two Long-Evans rats were dosed with the compound by gavage with an intragastric tube at a dose of 10mg/kg of Example 1 or the molar equivalent of Example 3, 5 and 6. The animals were housed in metabolic cages and urine was collected for the 0-24 hour and 24-48 hour periods post dose. The urines were analysed by reverse-phase I-IPLC. Results are expressed as %. Dose recovered in the urine in 48 hours as compound of Example 1.

Exam le % Dose 1 49/25 2 11.7 3 7,9 4 15.9 Claims 1' The use 01 compound of formula (I) K.

Claims (1)

1. 1. CLAIMS wherein R1 is methoxy and R2 is hydrogen or amino, and any pharmaoeutically acceptable salts, ethers, esters or salts of such esters in the manufacture of a medicament for the treatment or prophylaxis of human viral infections caused by VZV. 5 2, 9-B-D—Arabinofiiranosyl-2—amino—6-methoxy-9H-purine or a phygiologially acceptable derivative thereof. 3_ 9-B-D-Arabinofuranosy1aminomethoxy-9H-purine. 10 4_ 9-B-D-Arabinofi1ranosylaminomethoxy-9H-purine for use in medicine. 5. A pharmaceutically acceptable ester of a compound of formula (1) selected from:- 9-(5(4-Methylpenzgyl)-fl-D-arabinofuranosyl)methoxy-9fl-purine. 15 9 ~ (5- 0- (l+-Amin_obenzoy1)-;9-D- arabinofuranosyl) -6 -methoxy- 911-purine, O, 6 -Methoxy' 9 - (_2.. pentanoyl -,8-D- arabinofuranosy1)- 9fl— purine . 6. A process for the preparation of9-B-D-ArabinofilranoSy1amin0meth0xy-9H-purine or a salt, ether, ester or salt of such ester comprising either: 20 a) reacting a compound ot formula (II) 25 wherein R1 is methoxy and R2 is amino with a compound of formula (III) wherein X represents a pyrimidine or purine base (other than a compound of formula (II); or b) reacting a compoutéd of formula (IV) wherein Z is a leaving group and R2 is amino, with a compound capable of introducing a methoxy group at the 6-position; and optionally thereafter or simultaneously therewith, wherein the resulting compound is 9-B-D-Arabin0fi1ran0sy1amin0meth0xy-9H- purine, converting it into a pharmaceutically acceptable salt, esteror salt of such ester converting it into a different pharmaceutically acceptable salt, ether, ester or salt of such ester of 9-B-D-Arabinofi.1ranosylaminomethoxy-9H-purine. A pharmaceutical fon'nu]atlOn comprising 9-B-D-Arabinofixranosylaminomethoxy-9H- purine or a salt, ether, ester or salt of such ester, together with a pharmaceutically acceptable carrier. A pharmaceutical formulation as claimed in claim 7 wherein the active is 9-B-D- Arabinofuranosylaminomethoxy-9H-purine. A formulation as claimed in claims 7 or 8 in unit dosage form wherein the unit dosage of active compound is 5 to 1000g. A formulation as claimed in claims 7, 8 or 9 adapted for oral administration. A formulation as claimed in claim 10 in the form of a capsule, cachet or tablet. CRUICKSHANK & C0 .