AP91A - Dideoxydidehydrocarbocyclic nucleosides. - Google Patents

Dideoxydidehydrocarbocyclic nucleosides. Download PDF

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Publication number
AP91A
AP91A APAP/P/1989/000113A AP8900113A AP91A AP 91 A AP91 A AP 91A AP 8900113 A AP8900113 A AP 8900113A AP 91 A AP91 A AP 91A
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ARIPO
Prior art keywords
compound
formula
9h
yl
purin
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APAP/P/1989/000113A
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AP8800113A0 (en
Inventor
Robert Vince
Mei Huai
Peter Leslie Myers
Richard Storer
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Univ Minnesota
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Priority to US07/146,252 priority Critical patent/US4916224A/en
Application filed by Univ Minnesota filed Critical Univ Minnesota
Publication of AP8800113A0 publication Critical patent/AP8800113A0/en
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D239/00Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46Two or more oxygen, sulfur or nitrogen atoms
    • C07D239/48Two nitrogen atoms
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D239/00Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46Two or more oxygen, sulfur or nitrogen atoms
    • C07D239/50Three nitrogen atoms
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D473/00Heterocyclic compounds containing purine ring systems
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D487/00Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by C07D451/00 - C07D477/00
    • C07D487/02Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04Ortho-condensed systems

Abstract

Disclosed are compounds of formula and pharmaceutically acceptable derivatives thereof. Also disclosed are use of the compounds as antiviral and antitumour agents, pharmaceutical formulations, methods for the preparation of the compounds and intermediates

Description

- 2 -

DIDEOXYDIDEHYCROCAREOCYCtIC NUCLEOSIDES

The present invention relates to dideoxycarbocyclic nucleosideanalogues. More specifically it is concerned with carbocyclic2',3'-dideoxy-2' ,3'-didehydro purine nucleoside analogues and theiruse in therapy, in particular as antiviral agents.

In view of the similarity between viral eno host cellularfunctions it is difficult to selectively attack a virus while leavingthe host cell intact. Thus, there are relatively few agentseffective against viruses per se and it is difficult to find antiviralagents having an acceptable therapeutic index, i.e. agents which havea meaningful antiviral effect at a dose level at which the agent hasan acceptable toxicity, or side effect, profile.

One group of viruses which have recently assumed majorsignificance are the retroviruses responsible for the human acquiredimmunodeficiency syndrome (A1D5). Such viruses have previously beenreferred to by various terminologies but are new generally referred toas human immunodeficiency viruses (HIV's); two such viruses, HIV—I andHIV—II, have been reprcducibly isolated from patients suffering fromAIDS end related conditions such as AIDS related complex (ARC) andpersistent generalised lym.phadenopathy.

Although a number of nucleosides have been taught es useful inthe treatment of conditions associated with HIV infections, onlyzidovudine (AZT, Retrovir) has received regulatory approval for thetreatment of such conditions. However, it is known that zidovudinehas severe side effects, causing suppression of the tone marrowleading to a drop in the white blood cell count with consequentpronounced anaemia, end there is a need for effective agents which areless cytotoxic.

We have new found a novel class of nucleoside analogues havingantiviral activity. There is accordingly provided in a first aspect acompound of formula (I) ΔΡΟΟΟ0 9 1 bad original il» 3 //\ /

N I il

CI) HO-CH-

wherein X is hydrogen, NRR1, SR, OR or halogen; Z is hydrogen, OR2 or NRR1; R, R1 and R2 may be the same or different and are selectedfrom hydrogen, C^^alkyl and aryl;and pharmaceutically acceptable derivatives thereof.

It will be appreciated by those skilled in the art that thecompounds of formula (I) are cis compounds and further that thecyclcpentene ring of the compounds of formula (I) contain two chiralcentres (shown in formula (I) by ♦) and may thus exist in the form oftwo optical isomers (i.e. enantiomers) and mixtures thereof includingracemic mixtures. All such isomers and mixtures thereof includingracemic mixtures are included within the scope of the invention. Thusin the compounds of formula (I) either the chiral centre to which thebase is attached is in the R configuration and the chiral centre towhich the CH20H moiety is attached is in the S configuration(hereinafter the D isomer) or the chiral centre to which the base inattached is in the S configuration and that to which the CH J3H moietyis attached is in the R configuration (hereinafter the L isomer).Conveniently the compounds will be in the form of either a racemicmixture or substantially as the pure 0 isomer. The D isomers may berepresented by the formula (la) ad η η ηfl 9 1

BAD ORIGINAL /Λ/ Ν · I » Λ\ Λ / Ζ Ν Ν Η01>·\! \_/ •-· (la) where X 2nd Ζ are as defined for formula (I). Reference hereinafterto compounds of formula (I) includes compounds cf formula (la).

It will also be appreciated by those skilled in the art thatcertain of the compounds of formula (I) may exist as a number oftautomeric forms and all such tautomers are included within the scopeof the invention.

As used herein the term halogen refers to fluorine,-«chlorine,bromine and iodine; when X is halogen it is preferably chlorine.

As used herein Cx_4alkyl refers to a straight or branched chainalkyl group for example methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl and t-butyl. Conveniently C^.^alkyl is methyl.

As used herein aryl refers to any mono- or polycyclic aromaticmoiety end includes unsubstituted end substituted aryl (such asphenyl, tolyl, xylyl, anisyl) end unsubstituted and substitutedaralkyl including ar (C^_4) alkyl such as phen(C 1|)alkyl for examplebenzyl or phenethyl.

In the compounds of formula (I) Z is preferably amino.

In one preferred class of compounds of formula (I) X is OR, inparticular OH.

In a further preferred class of compounds of formula (I) X isNRR1 in particular NH2, or hydrogen.

Particularly preferred compounds of formula (I) ere those whereinZ is ι1ίΉ2 and X is H, \'H2 or, especially, OH. . Such compounds inparticular have especially desirable therapeutic indices as antiviralagents.

By "a pharmaceutically acceptable derivative” is meant anypharmaceutically acceptable salt, ester, or salt of such ester, of a

I BAD ORIGINAL

I

I 5 compound of formula (I) or any other compound which, upon administration to the recipient, is capable of providing (directly orindirectly) a compound of formula (I) or an antivirally activemetabolite or residue thereof.

Preferred esters of the compounds of formula (I) includecarboxylic acid esters in which the non-carbonyl moiety of the estergrouping is selected from hydrogen, straight or branched chain alkyl(e.g. methyl, ethyl, n-propyl, t-butyl, n-butyl), alkoxyalkyl (e.g.methoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl), aryl (e.g. phenyl optionally substituted by halogen,alkyl or Cj_4 alkoxy); sulphonate esters such as alkyl- or aralkylsulphonyl (e.g. methanesulphonyl); amino acid esters (e.g.L-valyl or L-isoleucyl) and mono-, di- or tri-phosphate esters.

With regard to the above described esters, unless otherwisespecified, any alkyl moiety present advantageously contains 1 to 18carbon atoms, particularly 1 to 4 carbon atoms. Any aryl moietypresent in such esters advantageously comprises a phenyl group.

Pharmaceutically acceptable salts of the compounds of formula (I)include those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acids includehydrochloric, hydrobromic, sulphuric, nitric, perchloric, fumaric,maleic, phosphoric, glycollic, lactic, salicylic, succinic,toluene-p-sulphonic, tartaric, acetic, citric, methanesulphonic,formic, benzoic, malonic, naphthalene-2-sulphonic and benzenesulphonicacids. Other acids such as oxalic, while not in themselvespharmaceutically acceptable, may be useful in the preparation of saltsuseful as intermediates in obtaining the compounds of the inventionand their pharmaceutically acceptable acid addition salts.

Salts derived from appropriate bases include alkali metal (e.g.sodium), alkaline earth metal (e.g. magnesium), ammonium and NR(where R is Cx_4alkyl) salts.

References hereinafter to a compound according to the inventionincludes both compounds of formula (I) and their pharmaceuticallyacceptable derivatives.

Specific compounds of formula (I) include : - (la,4t )-4-(6-Chloro-SH-purin-9-yl)-2-cyclopentenyl-carbinol; AP0 0 0 0 9 1 BAD ORIGINAL Ά 6 (la,4a)-4-(6-Hydroxy-9H-purin-9-yl)-2-cyclopentenyl-cerbinol; (Ια,4α)-4-(6-Amino-9H-purin-9-yl)-2-cyclopentenyl-carbinol; (la,4a)-4-(6-Mercapto-9H-purin-9-yl)-2-cyclopentenyl-carbinol; (la ,4a)-4-(2-Aminc-6-chloro-9H-purin-9-yl)-2-cyclopentenyl-carbin.ol; (1α,4α)-4-(2-Amino-6-hydroxy-9H-purin-9-y1)-2-cyclopentenyl-carbinol: (la ,4 a)-4-(2,6-Diamino-9H-purin-9-y1)-2-cyclopenteny1-carbinol; in the form of a racemic mixture or a sirflgle enantiomer.

The compounds of the invention either themselves possessantiviral activity and/or are metabolizable to such compounds. Inparticular these compounds are effective in inhibiting the replicationof retroviruses, including human retroviruses such as humanimmunodeficiency viruses (HIV's), the causative agents of AIDS.

Certain compounds of the invention in particular those wherein Zis H also possSess anticancer ectivity.

There is thus provided as a further aspect of the invention acompound of formula (I) or a pharmaceutically acceptable derivativethereof for use as an ective therapeutic agent in particular as anantiviral egent, for example in the treatment of retroviralinfections, or an anticancer agent.

In a further or alternative aspect there is provided a method forthe treatment of a viral infection, in particular an infection causedby a retrovirus such as HIV, in a mammal including man comprisingadministration of an effective amount of an antiviral compound offormula (I) or a pharmaceutically acceptable derivative thereof.

There is also provided in a further or alternative aspect use ofa compound of formula (I) or a pharmaceutically acceptable derivativethereof for the manufacture of a medicament for the treatment of aviral infection or use as an anticancer egent.

The compounds of the invention having entiviral activity are alsouseful in the treatment of AIDS related conditions such asAIDS-related complex (ARC), progressive generalised lymphadenopathy(PGL), AIDS-releted neurological conditions (such as dementia ortropical paraparesis), anti-HIV antibody positive end HIV- positiveconditions, Kaposi's sarcoma and thrombocytopenia purpura.

BAD ORIGINAL

I 7

The antiviral compounds of the invention are also useful in theprevention of progression to clinical illness of individuals who areanti-HIV antibody or HIV-antigen positive and in prophylaxis followingexposure to HIV.

The antiviral compounds of formula (I) or the pharmaceuticallyacceptable derivatives thereof, may also be used for the prevention ofviral contamination of physiological fluids such as blood or semen invitro.

Certain of the compounds of formula (I) are also useful asintermediates in the preparation of other compounds of the invention.

It will be appreciated by those skilled in the art that referenceherein to treatment extends to prophylaxis as well as the treatment ofestablished infections or symptoms.

It will be further appreciated that the amount of a compound ofthe invention required for use in treatment will vary not only withthe particular compound selected but also with the route ofadministration, the nature of the condition being treated and the ageand condition of the patient and will be ultimately at the discretionof the attendant physician or veterinarian. In general however asuitable dose will be in the range of from about 1 to about 750ma/kge.g. from about 10 to about 75Cmg/kg of bodyweight per day, such as 3to about 120mg per kilogram body weight of the recipient per day,preferably in the range of 6 to 90 mg/kg/day, most preferably in therange of 15 to 60mg/kg/day.

The desired dose may conveniently be presented in a single doseor as divided doses administered at appropriate intervals, for exampleas two, three, four or more sub-doses per day.

The compound is conveniently administered in unit dosage form;for example containing 10 to 1500mg, conveniently 20 to ICOCmg, mostconveniently 50 to 70Cmg of active ingredient per unit dosage form.

Ideally the active ingredient should be administered to achievepeak plasma concentrations of the active compound of from about 1 toabout 75 μΜ, preferably about 2 to 50 μί-i, most preferably about 3 toabout 30μΜ. This may be achieved, for example, by the intravenousinjection of a 0.1 to 52 solution of the active ingredient, optionally

I 6 0 0 0 0 dV

BAD ORIGINAL £ -4 in saline, or orally administered as a bolus containing -about 1 toabout lOCmg of the active ingredient. Desirable blood levels may bemaintained by a continuous infusion to provide about 0.01 to about 5.0mg/kg/hou? or by intermittent infusions containing about 0.4 to about15 mg/kg of the active ingredient.

While it is possible that, for use in therapy, a compound of theinvention may be administered as the raw cnemical it is preferable topresent the active ingredient as a pharmaceutical formulation.

The invention thus further provides a pharmaceutical formulationcomprising a compound of formula (1) or a pharmaceutically acceptablederivative thereof together with one or more pharmaceuticallyacceptable carriers thereof and, optionally, other therapeutic and/orprophylactic ingredients. The carrier(s) must be 'acceptable' in thesense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

Pharmaceutical formulations include those suitable for oral,'rectal, nasal, topical (including buccal and sub-lingual), vaginal orX' carentersl (including intramuscular, sub-cutaneous and intravenous) administration or in a form suitable for administration by inhalationor insufflation. The formulations may, where appropriate, beconveniently presented in discrete dosage units and may be prepared byeny of the methods well known in the art of pharmacy. All methodsinclude the step of bringing into association the active compound withliquid carriers or finely divided solid carriers or both and then, ifnecessary, shaping the product into the desired formulation.

Pharmaceutical formulations suitable for oral administration mayconveniently be presented as discrete units such as capsules, cachetsor tablets each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution, a suspension or asan emulsion. The active ingredient may also be presented as a bolus,electuary or paste. Tablets and capsules for oral administration maycontain conventional excipients such as binding agents, fillers,lubricants, disintegrants, or wetting agents. The tablets may becosted according tc methods well known in the ert. Oral liquidpreparations may be in the form: of, for example, aquecus or oilysuspensions, solutions, emulsions, syrups or elixirs, or may be , , BAD ORIGINAL & η presented as a dry product for constitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, emulsifying agents,non-aqueous vehicles (which may include edible oils), or preservatives.

The compounds according to the invention may also be formulatedfor parenteral administration (e.g. by injection, for example bolusinjection or continuous infusion) and may be presented in unit doseform in ampoules, pre-filled syringes, small volume infusion or inmulti-dose containers with an added preservative. The compositionsmay take such forms as suspensions, solutions, or emulsions in oily oraqueous vehicles, and may contain formulatory agents such assuspending, stabilising and/or dispersing agents. Alternatively, theactive ingredient may be in powder form, obtained by aseptic isolationof sterile solid or by lyophilisation from solution, for constitutionwith a suitable vehicle, e.g. sterile, pyrogen-free water, beforeuse.

For topical administration to the epidermis the compoundsaccording to the invention may be formulated as ointments, creams orlotions, or as a transdermal patch. Ointments and creams may, forexample, be formulated with an aqueous or oily base with the additionof suitable thickening and/or gelling agents. Lotions may beformulated with an aqueous or oily base and will in general alsocontain one or more emulsifying agents, stabilising agents, dispersingagents, suspending agents, thickening agents, or colouring agents.

Formulations suitable for topical administration in the mouthinclude lozenges comprising active ingredient in a flavoured base,usually sucrose and acacia or tragacanth; pastilles comprising theactive ingredient in an inert base such as gelatin and glycerin orsucrose and acacia; and mouthwashes comprising the active ingredientin a suitable liquid carrier.

Pharmaceutical formulations suitable for rectal administrationwherein the carrier is a solid are most preferably presented as unitdose suppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art, and the suppositories may beconveniently formed by admixture of the active compound with the

I

BAD ORIGINAL ΔΡ 0 0 0 0 9 1 ιο Ο Ο i? softened or melted carrier(s) followed by chilling end shaping inmoulds.

Formulations suitable for vaginal administration may be presentedas pessaries, tampons, creams, gels, pastes, foams or sprayscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

For intra-nasdl administration the compounds of the invention maybe used as a liquid spray or in the form of drops.

Drops may be formulated with an aqueous or non-aqueous base alsocomprising one more more dispersing agents, solubilising agents orsuspending agents. Liquid sprays ara conveniently delivered frompressurised packs.

For administration by inhalation the compounds according to theinvention are conveniently delivered from an insufflator, nebuliser ora pressurised pack or other convenient means of delivering an aerosolspray. Pressurised packs may comprise a suitable propellant such asdichlorodifluorcmethane, trichlorofluoromethene,· dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Inthe case of a pressurised aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation,the compounds according to the invention may take the form of a drypowder composition, for example a powder mix of the compound and asuitable powder base such as lactose or starch. The powdercomposition may be presented in unit dosage form in, for example,capsules or cartridges or e.g. gelatin or blister packs from whichthe powder may be administered with the aid of an inhalator orinsufflator.

When desired the above described formulations adapted to givesustained release of the ective ingredient may be employed.

The pharmaceutical compositions according to the invention mayalso contain other active ingredients such es antimicrobial agents, orpreservatives.

The compounds of the invention may also be used in combinationwith other therapeutic agents for example other antiinfective agents. BAD ORIGINAL ffi

In particular the compounds of the invention may be employed togetherwith known antiviral agents.

The invention thus provides, in a further aspect, a combinationcomprising a compound of formula (I) or a physiologically acceptablederivative thereof together with another therapeutically active agent,in particular an antiviral agent.

The combinations referred to above may conveniently be presentedfor use in the form of a pharmaceutical formulation and thuspharmaceutical formulations comprising a combination as defined abovetogether with a pharmaceutically acceptable carrier thereof comprise afurther aspect of the invention.

Suitable therapeutic agents for use in such combinations includeacyclic nucleosides such as aciclovir, interferons such asα-interferon, renal excretion inhibitors such as probenicid,nucleoside transport inhibitors such as dipyridamole, 2',3'-dideoxynucleosides such as 2',3'-dideoxycytidine, 2' ,3'-dideoxyadenosine, 2',3'-dideoxyinosine, 2',3'-dideoxythymidineand 2',3'-dideoxy-2',3'-didehydrothymidine and immunomodulators suchas interleukin II (IL2) and granulocyte macrophage colony stimulatingfactor (GM-CSF), erythropoetin and ampligen.

The individual components of such combinations may beadministered either sequentially or simultaneously in separate orcombined pharmaceutical formulations.

When the compound of formula (I) or a pharmaceutically acceptablederivative thereof is used in combination with a second therapeuticagent active against the same virus the dose of each compound maydiffer from that when the compound is used alone. Appropriate doseswill be readily appreciated by those skilled in the art.

The comoounds of formula (I) and their pharmaceuticallyacceptable derivatives may be prepared by any method known in the artfor the preparation of compounds of analogous structure.

Suitable methods for preparing compounds of formula (I) and theirpharmaceutically acceptable derivatives are described below; thegroups X and Z are as defined above except where otherwise indicated.It will be appreciated that the following reactions may require theuse of, or conveniently may be applied to, starting materials having

I 6 0 0 0 0 dV BAD ORIGINAL $ 12 protected functional groups, and deprotection might thus be requiredas an intermediate or final step to yield the desired compound.Protection and deprotection of functional groups may be effected usingconventional means. Thus, for example, emino groups may be protectedby a group selected from aralkyl (e.g. benzyl), acyl or aryl (e.g.2,4-dinitrophenyl); subsequent removal of the protecting group beingeffected when desired by hydrolysis or hydrogenolysis as appropriateusing standard conditions. Hydroxyl groups may be protected using anyconventional hydroxyl protecting group, for example, as described in'Protective Groups in Organic Chemistry', Ed. 0. F. W. McOmie (PlenumPress, 1973) or 'Protective Groups in Organic Synthesis' by TheodoraW. Greene (John Wiley and Sons, 1981). Examples of suitable hydroxylprotecting groups include groups selected from alkyl (e.g. methyl,t-butyl or methoxymethyl), aralkyl (e.g. benzyl, diphenylmethyl ortriphenylmethyl), heterocyclic groups such as tetrahydropyranyl, acyl(e.g. acetyl or benzoyl) and silyl groups such as trialkylsilyl (e.g. Ό t-butyldimethylsilyl). The hydroxyl protecting groups may be removed by conventional techniques. Thus, for example, alkyl, silyl, acyl andheterocyclic groups may be removed by solvolysis, e.g. by hydrolysis O under acidic or besic conditions. Aralkyl groups such as X) triphenylmethyl mav similarly be removed by solvolysis, e.g. by hydrolysis under ecidic conditions. Aralkyl groups such as benzyl maybe cleaved by hydrogenolysis in the presence of a noble metal catalystsuch as palladium-cn-charcoal. Silyl groups may also conveniently beremoved using a source of fluoride ions such as tetra-n-butylammoniumfluoride.

In a first process (A), compounds of formula (I) endpharmaceutically acceptable derivatives thereof may be prepared byreacting a compound cf formula (II)

BAD ORIGINAL - 13 • NH,// \ / 2 / \\ Λ

Z N NH (II) (wherein X and Z are substituents having the meaning in formula (I) orare protected forms thereof and Y is OH or a protected form thereof) ora pharmaceutically acceptable derivative thereof with a reagentselected from formic acid and reactive derivatives thereof, followed,where necessary, by removal of unwanted groups introduced by saidreagent and/or by removal of any protecting groups present.

Examples of suitable derivatives of formic acid which may be usedin process (A) above include orthoformates (e.g. triethylorthoformate), dialkoxymethyl acetates (e.g. diethoxymethyl acetate),dithioformic acid, formamide, s-triazine or formamidine acetate.

Unwanted groups introduced by formic acid or a reactivederivative thereof may conveniently be removed by mild hydrolysis, forexample using an inorganic acid such as aqueous hydrochloric acid.

When a trialkyl orthoformate such as triethyl orthoformate isused this is conveniently also the solvent for the reaction. Othersolvents which may be used include amides (e.g. dimethylformamide ordimethylacetamide), chlorinated hydrocarbons (e.g. dichloromethane),ethers (e.g. tetrahydrofuran) or nitriles (e.g. acetonitrile).

In some cases (e.g. when a trialkyl orthoformate such as triethylorthoformate is used) the reaction may preferably be carried out inthe presence of a catalyst such as a strong acid (e.g. concentratedhydrochloric, nitric or sulphuric acid). The reaction may be effectedat a temperature in the range of “25° to +150°C, e.g. 0° to lCO^C,and conveniently at ambient temperature.

I 6 0 0 o 0 dV BAD ORIGINAL & 14

In another process (3), compounds of formula (I) end theirpharmaceutically acceptable derivatives or a protected form thereofare subjected to an interconversion reaction whereby the substituent Xinitially present is replaced by a different substituent X and,zor thegroup Z initially present is replaced by a different group Z followed,where necessary by removal of any protecting groups present.

In one embodiment of process (B), compounds of formula (I) inwhich X represents a group RR 1 (where R and R1 are as definedpreviously) may be prepared by amination of a corresponding compoundof formula (I) in which X represents a halogen atom (e.g. chlorine).The emination may be effected by reaction with a reagent HNRR1 (whereR and R1 are as defined previously) conveniently in a solvent such asan alcohol (e.g. methanol). The reaction may be carried out at anysuitable temperature and conveniently at an elevated temperature suchas under reflux or, when liquid ammonia is used, in a sealed tube atabout 50 to 80°C. Suitable conditions for the conversion of halidesto secondary and tertiary amines have also been described by I. T.Harrison et. al., Compendium of Organic Synthetic Methods,Wiley-Interscience, New York (1971) at pages 250-252.

In another embodiment of process (B), compounds of formula (I) inwhich X represents a group OR (where R is as defined previously) maybe prepared by displacement of the halogen (e.g. chlorine) atom withen appropriate anion R0“. When R represents a hydrogen etom thedisplacement reaction may be carried out by hydrolysis which may beeffected in water or in a mixture of water and a water-misciblesolvent such es an elcohol (e.g. methanol or ethanol), en ether (e.g.dioxa.n or tetrahydr ofuran), a ketone (e.g. acetone), an emide (e.g.dimethylformamide) or a sulphoxide (eg. dimethylsulphoxide),conveniently in the presence of an acid or base. Suitable acidsinclude organic acids such as p-toluenesulphcnic acid and inorganicacids such es hydrochloric, nitric or sulphuric acid. Suitable basesinclude inorganic bases such as alkali metal hydroxides or carbonates(e.g. sodium or potassium hydroxide or carbonate). Aqueous ecid orbase may also be used es the reaction solvent- The hydrolysis mayconveniently be effected at a temperature in the range “10° to +150 °C, BAD ORIGINAL $ 15 e.g. at reflux. When R represents a Cx_Halkyl or aryl group the anionRO" is conveniently formed from a corresponding alcohol ROH using aninorganic base such as an alkali metal (e.g. sodium metal) or analkali metal hydride (e.g. sodium hydride). The reaction with the insitu formed anion may conveniently be effected at ambient temperature.

In a further embodiment of process (B), compounds of formula (I)in which X represents a group SH may be prepared by reacting the halocompound of formula (I) with thiourea in a suitable solvent such as analcohol (e.g. n-propanol) at an elevated temperature (e.g. reflux)followed by alkaline hydrolysis. Suitable bases which may be usedinclude alkali metal hydroxides (e.g. sodium hydroxide). The reactionmay conveniently be carried out according to the method of G. G.Urquart et. al. Org. Syn. Coll. Vol. 2, 363(1953) eg by refluxing theintermediate product with aqueous NAOH for about 0.25 to about 5hours.

In another embodiment of process (B), compounds of formula (I) inwhich X represents a hydrogen atom may be prepared by reducing thehalo compound of formula (I) using a reducing system which will notaffect the rest of the molecule. Suitable reducing agents which maybe used to effect the desired dehalogenation reaction include zincmetal/water using the method described by 3. R. Marshall et. al., 3.Chem. Soc., 1004 (1951). Alternatively, the reaction may be effectedby photolysis in a suitable solvent such as tetrahydrofuran containingIOS triethylamine and conveniently in a Rayonet photochemical reactor(2537A) according to the method of V. Nair et. al., 3. Org. Chem., 52,1344 (1987).

In a yet further embodiment of process (B), compounds of formula(I) in which X represents a halogen atom may be prepared from adifferent halo compound of formula (I) by conventional methods ofhalide-halide exchange. Alternatively, when X is chlorine thissubstituent may be replaced by other halogen atoms by using variousp-(halo)benzene diazonium chlorides according to well-knownprocedures.

Compounds of formula (I) in which X represents a group SR where Ris a Ci-4alkyl or aryl group may be prepared from the corresponding

Aon η o 0 9 1

BA© ORIGINAL thiols using standard methods of alkylation or arylation for exampleas described in US Patent NO. 4,383,114.

Compounds of formula (I) in which Z represents a hydroxyl groupmay conveniently be prepared from a correspondig compound of formula(I) in which Z represents NH2 by reaction with nitrous acid, forexample employing the procedure used by 3. Davoll in 3. Amer. Chem.Soc., 73, 3174 (1951).

Many of the reactions described hereinabove have been extensivelyreported in the context of purine nucleoside synthesis, for example inNucleoside Analogs - Chemistry, Biology, end Medical Applications, R. T. Walker et. el., eds, Plenum Pless, New York (1979) at pages193-223, the disclosure of which is incorporated by reference herein.

Pharmaceutically acceptable salts of the compounds of theinvention may be prepared es described in US Patent No. 4,333,114, thedisclosure of which is incorporated by reference herein. Thus, forexample, when it is desired to prepare an acid addition salt of acompound of formula (I) the product of any of the above procedures maybe converted into a salt by treatment of the resulting free base witha suitable acid using conventional methods. Pharmaceuticallyacceptable acid addition salts may be prepared by reacting the freebase with an appropriate acid optionally in the presence of a suitablesolvent such as an ester (e.g. ethyl acetate) or an alcohol (e.g.methanol, ethanol or isopropanol). Inorganic basic salts may beprepared by reacting the free base with a suitable base such as analkoxide (e.g. sodium methoxide) optionally in the presence of asolvent such as an alcohol (e.g. methanol). Pharmaceuticallyacceptable salts may also be prepared from other salts, includingother pharmaceutically acceptable salts, of the compounds of formula(I) using conventional methods. A compound of formula (I) may be converted into apharmaceutically acceptable phosphate cr other ester by reaction witha phosphorylsting agent, such as POC13, or a suitable esterifyingagent, such as an acid halide or anhydride, as appropriate. An esteror salt of a compound of formula (I) may te converted to the parentcompound for example by hydrolysis.

BAD ORIGINAL 17

The compounds of formula (II) and salts thereof are novelcompounds and form a further feature of the present invention.

The compounds of formula (II) in which Z represents hydrogen orhydroxyl may be prepared directly from the compound 2a

2a by reaction with an excess of a pyrimidine of formula (III)

X

N · I 2 (III) A\ Λ

N (wherein Y is a halogen atom, e.g. chlorine and Z is hydrogen orhydroxyl) in the presence of an amine base such as triethylamine andin an alcoholic solvent (e.g. n-butanol), conveniently at reflux.

Compounds of formula (II) in which Z represents NH2 may beprepared using the compound of formula 2a by reaction with an excessof a pyrimidine of formula (IV)

X

I /Λ N · ! ;ι (iv) Λ Λ h2n n y (wherein Y is as defined in formula (III) above) under similarconditions to those described just above for the preparation ofcompounds of formula (II) in which Z represents hydrogen or hydroxylto give a compound of formula (V)

I 6 0 0 0 0 dV

BAD ORIGINAL - 18 // \ N · I 3 Λ\ Λ h2n n nhho-ch2\ which may be diazotized using a diazoniun salt ArN2+E~ (wherein Arrepresents an aromatic group, e.g. p-chlorophenyl, and E~ representsan anion, e.g. a halide such as chloride) in a solvent such as water,an organic acid such as acetic acid or a mixture thereof, convenientlyat about ambient temperature to give a compound of formula (VI) • N=N-Ar//\ / .«e*» I. Λ\ Λ

Η-,Ν N NH HO-CK, ·2\ / \ (VI). (wherein Ar is as defined just above) which may be converted to thedesired compound of formula (II) by reduction using for example areducing metal such as zinc in the presence of an acid, e.g. aceticacid. It will be appreciated that the choice of reducing agent willdepend on the nature of the group X.

The compound 2a may be prepared from the versatile precursor,la-acetyl=m.ino-‘3a-acetoxy-(iiethylcyclopent-2-ene tie) by hydrolysis inthe presence of a mild base, such as an alkaline earth metalhydroxide. A particularly convenient synthesis of compounds of formula (I)via 6-chloro compounds of formula (II) is outlined below. BAD ORIGINAL $ 19

δρπ η ο o 9 1 BAD ORIGINAL & 20

The compound 2a and compounds of formulae (V) and (VI) are novelintermediates and form further features of the present invention.

The compound la is a known compound described in US Patent No.4,138,562.

Where the compound of formula (I) is desired as a single isomerit may be obtained either by resolution of the final product or bystereospecific synthesis from isomerically pure starting material orany convenient intermediate.

Resolution of the final product, or an intermediate or startingmaterial therefor may be effected by any suitable method known in theart : see for example 'Stereochemistry of Carbon Compounds' by E. L.Eliel (McGraw Hill, 1962) and 'Tables of Resolving Agents' by S. H.Wilen.

One convenient method for obtaining chirally pure compounds offormula (I) is by enzymatic conversion of a racemic mixture of thecompound or a precursor thereof. By such a method both (+) and (-)compounds of formula (I) may be obtained in optically pure form.Suitable enzymes include deaminases such es adenosine deaminase.

The invention will be further described by reference to thefollowing detailed examples wherein elemental analyses were performedby M-H-W Laboratories, Phoenix, AZ. Melting points were determined ona Mel-Temp apparatus end are corrected. Nuclear magnetic resonancespectra were obtained on 3eol FX 90QFT or Nicollet NT3Q0 spectrometersend were recorded in DMSO-D6. Chemical shifts are expressed in ppmdown-field from Me^Si. IR spectra were determined as KBr pellets witha Nicollet 50XC FT-IR spectrometer, and UV spectra were determined ona Beckmann DU-8 spectrophotometer. Mass spectra were obtained with anAEI Scientific Apparatus Limited MS-30 mass spectrometer. Thin layerchromatography (TLC) was performed on 0.25mm layers of Merck silicagel (230-4C0 mesh). All chemicals and solvents are reagent gradeunless otherwise specified. The term "active ingredient" as used inthe Examples means e compound of formula (I) cr a pharmaceuticallyacceptable derivative thereof.

Example 1 (±)-(lg,4t )-4-((5-Amino-6-chloro-4-pyrimidinyl)-amino]-2- cyclopentenylcarbinol (3a) A mixture of la-acetylamino-3a-acetoxymethyl cyclopent-2-ene (la)(3.0g, 15 mmol) and aqueous barium hydroxide (0.5N, 300ml) wasrefluxed overnight. After cooling, it was neutralized with dry ice.The precipitate was filtered out, and the aqueous solution wasconcentrated to dryness. The residue was extracted with absoluteethanol and concentrated again to yield 2a as a colourless syrup 1.6g(14mmol).

To this syrup, 5-amino-4,6-dichloropyrimidine (4.59g 28 mmol),triethylamine (4.2g, 42 mmol), and n-butanol (50ml) were added and themixture was refluxed for 24 hr. The volatile solvents were removed,the residue was absorbed on silica gel (7g), packed in a flash column(4.0 x 12cm) and eluted with CHCl3-MeOH>(20:1) to yield 2.69g (742) ofcompound 3a; m.p. 130-132°C. An analytical sample was obtained byrecrystallisation from ethyl acetate (EtOAc), m.p. 134-135 °C, MS (30ev, 200°C); m/e 240 and 242 (M+ and M*+2), 209 (M+ “31), 144 (B+); IR:3600-2600 (OH), 1620, 1580 (C=C, C=N); Anal. (C 1(JH i3ClN ι,Ο) 0, Η, N.

Example 2 (±)-(la,4c )-4-((2-Aminc-6-chloro-4-pyrimidinyl)-amino]-2- cyclopentenylcarbinol (4a)

To 14 mmol of crude 2a (Example 1) 2-amino-4,6-dichloro-pyrimidine (3.74g, 22.8 mmol), triethylamine (15ml) and n-butanol(75ml) were added and the mixture was refluxed for 48 hr. Thevolatile solvents were removed, residue was treated with methanol toseparate the undissoived by- product (the double pyrimidinenucleoside). The methanol solution was absorbed on silica gel (3g)packed into a column (4.0 x 14cm) and eluted with CHC13-MeCH (40:1) toyield 1.52g (422) of crude 4a. The product was recrystalliscd fromethyl acetate to yield 4a; m.p. 132-134°C, MS (30 ev, 200 °C); m/e 240and 242 (M+ and I4++2), 209 (M+-31), 144 (3+); IR: 3600-3000 (NH2, OH), 1620,1580 (C=C, C=N); Anal. (Ci0Hi3ClNj C,H, N. ΔΡ0 0 0 0 9 1 BAD ORIGINAL ft 22

Example 3 C ±(lg y )—4— f [ (2-Amino-6-cnIorc-5-(4-chlorcphenyl)-azo]-4- pyrlmidinyl-amino)-2-cyclooentenylcarbinol (5e) A cold diazcnium salt solution was prepared from p-chlcroaniline(1.47g, 11.5 mmol) in 3N HOI (25ml) and sodium nitrite (870mg, 12.5mmol) in water (10ml). This solution was added to a mixture of 4a(2.40g, 10 mmol), acetic acid (50ml), water (5Cml) and sodium acetatetrihydrate (20g). ‘The reaction mixture was stirred overnight at roof» M. temperature. The yellow precipitate was filtered and washed with coldwater until neutral, then it was air-dried in the fcmehood to yield3.60g (942), of 5a., m-P· 229°C (dec). The analytical sample wasobtained frcm acetone-methanol (1:2), m.p. 241-243¾ (dec). MS (30ev,260¾): m/e 378 end 380 (M+ and M+ + 2), 282 (B+); IR: 3600-3000 (NH2,OH), 1520, 1580 (C=C, C=N); Anal. (CχfaHχbCl2N60) C, Η, N.

Example 4 (±)-(la,4t )-4-[(2,5-Diamino-6-chloro-4-pyrimidinyl)-amino]-2- cvclcoentenylcarbinol (oa) A mixture of 5a (379mg, 1 mmol), zinc dust (0.65g, 10 mmol),acetic acid (0.32 ml), water (15ml) and ethanol (15ml) was refluxedunder nitrogen for 3 hr. The zinc was removed and the solvents wereevaporated. The residue was absorbed on silica gel (2g), packed intoa column (2.0 x 18cm), and eluted with CHCl3-MeOH (15:1). A pinksyrup was obtained. Further purification from methanol-ether yielded6a. as pink crystals, 170mg (662), m.p. 168-170°C, MS (30 ev, 220 ¾)}m/e 255 end 257 (^ and hT + 2), 224 (^ ‘31), 159 (B+); IR: 3600-3000(\'H2, CH) 1620,1530 (0=0, C=N); Anal. (C,2H^Cl\b) C, Η, N.

Example 5 (r)-(l2,z'o)-4-(6-Chloro-9H-purin-9-yl)-2-cyclcpentenvl-carbinol (7a) A mixture cf 3a (1.30g, 5.4 mmol), triethyl orthoformate (3Cmi)and hydrochloric acid (12N, 0.50ml) was stirred overnight at roomtemperature. The solvent was evaporated at 351 C in vacuo. To theresidue was added aqueous hydrochloric acid (0.5 N, 30ml) and themixture was stirred for Ihr., the mixture was neutralised to pH 7-3with IN sodium hydroxide and adsorbed onto silica gel (3g), packed in

BAD ORIGINAL .23 . - a column (4.0 x 8cm), and eluted with CHC13-MeCH (20:1) to yield whitecrystals of 7a, 1.12g (82%). The crude product was recrystallisedfrom ethyl acetate to yield 7a, m.p 108-110°C, MS (30 ev, 200 °C); m/e250 and 252 (M+ and M+ + 2), 219 (M4-31), 154 (B+); IR; 3600-2800(OH), 1600 (C=C, C=N); Anal. (C.XHUC1N40) C, Η, N.

Example 6 (±)-(la,4g)-4-(6-Hydroxy-9H-purin-9-yl)-2-cyclopentenyl-carbinol (8a) A mixture of 7a (251mg, 1 mmol) and aqueous sodiun hydroxide(0.2N, 10ml) was refluxed for 3hr. After cooling, the reactionmixture was adjusted to pH 5-6 with acetic acid. The reaction mixturewas absorbed on silica gel (2g) packed in a column (2.0 x 11cm) andeluted with CHCl3-MeOH (10:1) to yield 105mg (45%) of 8a. The crudewhite product was recrystallised from water-methanol (3:1) to yield8a, m.p. 248-250°C (dec), Ms (30 ev, 300°C); m/e 232 (M+), 214(M+-18), 136 (B+); IRj 3600-2600 (OH), 1680,1600 (0=0, C=C, C=N);

Anal. (CuHi2N4O2) C, Η, N.

Example 7 (i)-(lg,4g)-4-(6-Amino-9H-purin-9-yl)-2-cyclopenteny1-carbinol \9a)

Liquid ammonia was passed into a bomb containing a solution of 7 a(250mg, 1 mmol) in methanol (5ml) at “80^ C. The bomb was sealed and heated at 60uC for 24hr. Ammonia and methanol were evaporated and the residue was recrystallised from water to yield off-white crystals of9a, 187mg (81%), m.p. 198-200uC. MS (30 ev, 210°C): m/e 231 (tt*), 213 (M+ -18), 135 (B+); IR: 3600-2600 (NH2, OH), 1700,1600 (C=C, C=N);

Anal. (CuH,5N30) C, Η, N.

Example 8 (±)-(la,4c )-4-(6-Mercapto-9H-curin-9-yl)-2-cyclopenteny1-carbinol (10a) A mixture of 7a (125mg, 0.5 mmol), thiourea (40mg, 0.64 mmol) andn-propanol (5ml) was refluxed for 2hr. After cooling, the precipitatewas isolated by filtration, washed with n-propanol, and dissolved insodium hydroxide (IN, 5ml). The solution was adjusted to pH 5 withacetic acid. The crude 10a (90mg, 73%) was isolated again, m.p. APfl 0 0 0 9 1

BAD ORIGINAL 24 ¥ου 253-262°C (dec) end was recrystallised from Ν,Ν-dimethylformamide, toyield 10a> m.p. 263-265°C (dec). MS (30 ev, 290°C): m/e 248 (M+), 230(M+ -18), 152 (8+); IR: 3600-3200 (OH), 3100,2400 (SH), 1600 (C=C,C=N); Anal. (CliHi2N1<OS) C, Η, N.

Example 9 (±)-(lg,4: )-4-(2-Amino-6-chloro-9H-purin-9-y1)-2-cyclopentenyl- carbinol (13a) A mixture of 6a (1.41g, 5.5 mmol) triethyl orthoformate (30ml) -and hydrochloric acid (12N, 1.40ml) was stirred overnight. Thesuspension was dried in vacuo. Diluted hydrochloric acid (0.5N, 40ml)was added end the mixture was reacted at room temperature for lhr.

The mixture was neutralised to pH 8 with IN sodium hydroxide andabsorbed on silica gel (7.5g) packed in a column (4.0 x 10cm) andeluted by CHCl3-MeOH (20:1) to yield off-white crystals of 13a, 1.18g(80¾). The crude product was recrystallised from ethanol to yield13a, m.p. 145-147°C. MS (30 ev, 220°C): m/e 265 and 267 (M+ andM++2), 235 (M+ -30), 169 (9+); IR: 3600-2600 (NH2, OH), 1620-1580 O (C=C, C=N); Anal. (t^H^CCl.3/4 H20) C, N. o

Example 10 (t)-(lg,4c )-4-(2-Amino-6-hydroxy-9H-purin-9-y1)-2- cyclopentenyl carbinol (14a) A mixture of 13a (266mg, 1 mmol) and aqueous sodium hydroxide(0.33N) was refluxed for 5hr, absorbed onto silica gel (2g) packed ina column (2.0 x 7.5cm) and eluted with CHCl^-MeOH (5:1). The crudeproduct was recrystallised from methanol-water (1:4) to yield whitecrystals of 14a, 152mg (61Ϊ), m.p. 254-256°C (dec). MS (30 ev, 200'C): m/e 247 (M+), 217 (l·^ ~30), 151 (B+); IR: 3600-2600 (NH2, OH),1700,1600 (0=0, C=C, C=N); Anal. (C j ,Η χ3Ν -.3/4 H^) C, Η, N.

Example 11 (c)-(lc,4c )-4-(2,6-Diamino-9H-purirr-9-yl)-2-cyclcpentenyl carbinol (15a)

Liquid ammonia was passed into a solution of 13a (265mg, 1 mol)in methanol (10ml) at -20CC in a bomb. The bomb was sealed and heated BAD ORIGINAL ft - 2.5 at 75°C for 48hr. Ammonia and methanol were evaporated. The residuewas absorbed on silica gel (2g), packed in a column (2.0 x 10cm) andeluted with CHCl3-MeOH (15:1). The crude product was recrystallisedfrom ethanol to yield 196mg (80S) of 15a, m.p. 152-155¾. MS (30 ev,200°C): m/e 246 (M+), 229 (M+ "17), 216 (M+ "30), 150 (B+); IR:3600-3000 (NH2, OH), 1700,1650,1600 (C=0, C=C, C=N); Anal. (CnH14N6O)C, Η, N.

Example 12 (15,4R)-4-(2,6-Diamino-9H-purin-9-yl)-2-cyclopentenyl carbinol [(IS,4R)-4-(2,6-Diamino-9H-purin-9-yl)-2-cyclopentene methanol] (a) Intermediate 1 : (lR,2S,3R,5R)-3-[6-Amino-9H-purin-9-yl]-5- [((l,l-dimethylethyl)-dimethylsilyloxy)methyl]-l,2-cyclopentanediol(-) Aristeromycin1 (12.505g), tert-butyldimethylsilyl chloride (7.8g)and imidazole (12.96g) in dry dimethylformamide (85ml) was stirred atambient temperature for 2½ hours. The resulting solution was dilutedwith ethyl acetate (500ml), then washed with water (3x100ml) and brine(50ml) before a white solid crystallised out. This was collected byfiltration, washed with ethyl acetate, then dried in vacuo to give thetitle product (3.92g); n.m.r. (DKS0-d6) 8.15 (1H), 8.09 (1H), 7.19(2H), 5.00 (1H), 4.72 (1H), 4.69 (1H), 4.36 (1H), 3.85 (1H), 3.67(2H), 2.23 (1H), 2.09 (1H), 1.79 (1H), 0.89 (9H), 0.07 (6H). 1. Journal of the American Chemical Society 1933, vol. 105,4049-4055. (b) Intermediate 2 : (4R,3aS,6R,6aR)-4-[6-Amino-9H-purin-9-yl]-6-[((l,l-dimethylethyl)-dimethylsilyloxy)methyl]-3a,5,6,6a-tetΓahydΓO-4H-cyclopenta-l,3- dioxole-2-thione A stirred suspension of Intermediate 1 (3.45g) in dry dimethylformamide (56ml) was treated with 1,1'-thiocarbonyldiimidazole(3.3g), giving a yellow solution. After 15/z hours at ambienttemperature the resulting solution was combined with that from aprevious experiment (6£ scale), and solvent was removed byevaporation. The residual oil was diluted with ethyl acetate (100ml),then washed with water (2x20ml) and brine (2x20ml), dried (MgSO*t) and

L 6 0 0 0 0 dV

BAD ORIGINAL 26 evaporated to a yellow solid. This was washed with diethyl ether(25ml), then collected by filtration, further washed with ether(25ml), then dried in vacuo to give the title product as a pale creamsolid (3.61g); λ (ethanol) 240.Onm (e{* 459); n.m.r. (CMSO-dr)8.27 (1H), 8.13 (1H), 7.33 (2H), 5.81 (1H), 5.37 (1H), 5.28 (1H), 3.78(2H), 2.60 (1H), 2.28 (2H), 0.90 (9H), 0.09 (6H). (c) Intermediate 3 : (l'R,4'S)-9-[4-(((1,1-Dimethylethyl) dimethyIsilyloxy)methyl)-2-cyclopenten-l-yl]-9H-purin-6-amine A solution of Intermediate 2 (3.57g) in dry tetrahydrofuran (25ml) wastreated with a solution of 1,3-dimethyl-2-phenyl-l,3,2-diazaphospholidine (4.94g) in dry tetrahydrofuran (10ml), then stirredat ambient temperature for 8¾ hours. The solvent was removed byevaporation. The residual oil was combined with that from a previousexperiment (40% scale), then subjected to column chromatography onsilica (20Cg, Merck 7734), eluted with chloroform, thenchloroform-ethanol mixtures to give a white solid. This solid waswashed with diethyl ether (25ml), then collected by filtration. The * solid was further washed with ether (10ml), then dried in vacuo to

give the title Drcduct (1.47g); λ (ethanol) 261.4nm (Ε^ 443); 1H JS n.m.r. (C«SO-d6) 8.14 (1H), 8.00 (1H), 7.20 (2H), 6.12 (1H), 5.95 (1H), 5.60 (1H), 3.66 (2H), 2.96 (1H), 2.69 (1H), 1.65 (1H), 0.74(9H), 0.02 (6H). (d) Intermediate 4 : (l'R,4'S)-9-[4-(((1,1-Dimethylethyl) dimethylsilyloxy)methyl)-2-cyclopenten-l-yl]-9H-purin-6-amine,l-oxide A solution of Intermediate 3 (1.37g) in chloroform (30ml) was treatedwith 80-90% m-cnlorcperoxybensoic acid (i.29g), then stirred atambient temperature for 3 hours. Solvent was removed by evaporationand the residual gum was dissolved in ethyl acetate (10ml). A whitesolid crystallised cut. This solid end material recovered byevaporation of the filtrate wars dissolved in chloroform (100ml), thenwashed with saturated aqueous sodium bicarbonate solution (3x10ml) andbrine (2x10ml). The aqueous washings were back-extracted withchlcroforn (50ml). The combined organic solutions were dried (Mg504),

bad original A - 27 then evaporated to a solid. This solid was washed with diethyl ether (25ml), then collected by filtration. The white solid was further washed with ether (10ml), then dried in vacuo to give the title product (1.16q); λ_____ (ethanol) 235.4nm (E^_1324), 263.2nm (E^_ «ο,ΓΠΒΧ -LCfll -LCffl 248), 300.2nm (E^ 75); ΧΗ n.m.r. (CDClj ) 8.72 (1H), 8.02 (1H), 7.16(2H), 6.21 (1H), 5.87 (1H), 5.72 (1H), 3.68 (2H), 3.04 (1H), -2.82(1H), 1.74 (1H), 0.89 (9H), 0.06 (6H). (e) Intermediate 5 ; (1'R,4'S)-7-[4-(((1,1-Oimethylethyl) dimethylsilyloxy)methyl)-2-cyclopenten-l-yl]-2-imino-l,2-dihydro[l,2,4]oxadiazolo[3,2-i]-9H-purine hydrobromide A stirred, ice-chilled suspension of Intermediate 4 (1.08g) in methanol (20ml) was treated with a solution of cyanogen bromide (0.34g) in methanol (20ml) added over 5 minutes. After 15 minutes, the suspension was allowed to warm to ambient temperature, giving a solution. After 90 minutes, solvent was removed by evaporation. The residue was washed with diethyl ether (25ml), then collected by filtration. The solid was further washed with ether (25ml), then dried in vacuo to give the title product (1.37g); (ethanol) 223.2nm (E^ 530), 285.2nm (E*% 445); 1 H n.m.r. (CDClJ 10.20 (1H), 10.02

lcm 1cm J (1H), 3.37 (1H), 6.25 (1H), 6.01 (1H), 5.90 (1H), 3.69 (2H), 3.05(1H), 2.86 (1H), 1.73 (1H), 0.86 (9H), 0.03 (6H). (f) Intermediate 6 : (1'R,4'5)-9-[4-(((1,1-Oimethylethyl) dimethylsilyloxy)methyl)-2-cyclopenten-l-yl]-6-cyanoimino- 1,6-dihydro-1-methoxy-9H-purine A solution of Intermediate 5 (1.36g) in dimethylformamide (ICml) wasstirred at ambient temperature, then treated with triethylamine(1.2ml). After 40 minutes iodomethane (0.54ml) was added, giving ayellow solution. After 3¾ hours solvent was removed by evaporation.

The residue was partitioned between ethyl acetate (100ml) and water(20ml). The organic solution was further washed with water (2x2Cml)ano brine (20ml), dried (MgSO^) and evaporated to a solid. This solidwas washed with diethyl ether (25ml), then collected by filtration.This white solid was further washed with ether (10ml), then dried in BAD ORIGINAL £ •28 Ιο vacuo to give the title product (0.865g); ^max (ethanol) 227.2nm (Ej^449), 287.Onm (E^m 544); 1H n.m.r. 8.23 (1H), 7.96 (1H), 6.24 (1H),5.85 (1H), 5.65 (1H), 4.21 (3H), 3.66 (2H), 3.C4 (1H), 2.77 (1H), 1.68(1H), 0.88 (9H), 0.05 (6H). (g) Intermediate 7 ; (1'R,4'S)-9-[4-( ((1,1-Oimethylethyl)dimethyIsilyloxy)methyl)-2-cyclopenten-l-yl]-6-methoxyamino-9H-purin-2-amine A solution of Intermediate 6 (802mg) and l,8-diazabicyclo[5,4,0]undec- 7-ene (0.45ml)l in ethanol (8Cml) was stirred and heated at reflux.

I

Heating was stopped after 9 hours, and the solution was left atembient temperature overnight. Solvent was removed by evaporation.

The residual oil was combined with that from a previous experiment (4¾scale), then subjected to column chromatography on silica (40g, Merck9385) eluted with chloroform, then chloroform-ethanol mixtures to givea foam. This foam was triturated with diethyl ether (ICml) and theresulting solid was collected by filtration. The solid was furtherwashed with ether (5ml), then dried in vacuo to give the title product ° (594mg); λ (ethanol) 282.2nm (E^ 409); ^-n.m.r. (CMSO-dJ 9.76 (1H), 7.32 (1H), 6.53 (2H), 6.08 (1H), 5.88 (1H), 5.26 (1H), 3.72(3H), 3.61 (2H), 2.90 (1H), 2.50 (1H), 1.52 (1H), 0.83 (9H), 0.02 » (6H). &amp; (h) Intermediate 8 : (1S,4R)-4-[2-Amino-6-methoxyamino-9H-purin-9-yl]-2-cyclopentene-methanol A solution of Intermediate 7 (356mg) in tetrahydrofuran (35ml) was stirred at ambient temperature then treated with tetrabutylammonium fluoride (l.Ofj solution in tetrahydrofuran, 1.4ml). After 90 minutes the reaction was quenched with water (1ml), then solvents were removed by evaporation. The residual oil was subjected to column chromatography on silica (20g, Merck 7734), eluted with chloroform, then chloroform-ethanol mixtures to give the title product as a solid (243mg); λ (pH 6 buffer) 230.2nm (E^’ 534); Ή n.m.r. (DMSO-d-) max 1cm 0 BAD ORIGINAL ft - 29 9.75 (IH), 7.39 (IH), 6.52 (2H), 6.10 (IH), 5.84 (1H), 5.27 (IH), 4.73(IH), 3.40 (2H), 2.83 (IH), 2.55 (IH), 1.52 (1H). (15,4R)-4-[2,6-Diamino-9H-purin-9-yl]-2-cyclopentenecarbinol A stirred, ice-chilled solution of Intermediate 8 (210mg) in water(10ml) and tetrahydrofuran (50ml) was treated with aluminium amalgam[from aluminium (237mg) and 0.52 aqueous mercuric chloride solution],added in small pieces over 15 minutes. After 40 minutes the stirred mixture was allowed to warm to ambient temperature. After 15 hours the i resulting mixture was filtered through kieselguhr to remove insolubles. These were washed with water:tetrahydrofuran (1:5, 60ml).

The combined filtrates were evaporated. The residue was subjected to column chromatography cn silica (lOg, Merck 9385), eluted with chloroform-ethanol mixtures to give the title product as a foam (159mg); [a]^ “81° (£1.04, methanol); ^max (pH 6 buffer) 255.0 nm (E^* 302), 280.8 nm (E^ 381), jH n.m.r. (DMSO-dJ 7.61 (1H), 6.66 lcm 1cm 0 (2H), 6.10 (1H), 5.87 (1H), 5.76 (2H), 5.38 (1H), 4.76 (1H), 3.45(2H), 2.87 (1H), 2.60 (1H), 1.60 (1H).

Example 13 (15,4R)-4-(2-Amino-6-hydroxy-9H-pijrin-9-yl)-2-cvclopentenyl carbinol (1 'R,4'5)-2-Amino-l,9-dihydro-9-[4-hydroxymethyl-2-cyclopenten-l-yl]-6H-purin-6-one A turbid solution of the title compound of Example 12 (144mg) in 0.ΙΜ pH 6 buffer (10ml) (from 28.4g disodium orthophosphate in 2 litres of water, adjusted with orthophosphoric acid) was treated with a solution

of adenosine deeminase (0.5ml, 773 units), in 502 glycerol - 0.01M potassium phosphate, pH 6.0, then stirred and warmed to 37°. After 18½ hours the resulting suspension was refrigerated. The collected solid was recrystallised from weter to give the title product as a white solid (36mg); Ctx]_-49° (c 0.5, dimethylsulphoxide); λ (pH 6. o, U — max buffer) 252.6nm (E, 531), XH n.m.r. (DMSO-ct ) 10.60 (IH), 7.60

lcm J (IH), 6.47 (2H), 6.10 (1H), 5.86 (1H), 5.33 (1H), 4.72 (1H), 3.45(2H), 2.59 (IH), 1.58 (IH). ΑΒΛη ο η o 1 BAD ORIGINAL $ 30

: u, QA

Example 14

Preparation of Enantiomers of (lx ,4g)-4-(2-Amino-6-hydroxy-9H- purin-9-y l)-2-cyclopentenylcarbinol (a) (15, 4R)-4-(2-Amino-6-hydroxy-9H-purin-9-y1)-2- cyclopentenyl carbinol

The diamino analog (lOOmg) (Example 11) was dissolved in 3ml of0.03M K2PO4 buffer (pH 7.4) with heat (50* C). The solution was cooledto room temperature and 40 units of adenosine deaminase (Sigma, TypeVI, calf intestinal mucosa) was added. After three days of incubationat room temperature a precipitate formed end was removed by filtration, yield, 18.2mg. The filtrate was concentrated to 1.5ml andrefrigerated for 2 days. Additional solid was obtained by filtration,yield, 26.8mg. The two solid fractions were recrystallized from waterand gave the pure title product m.p. 269-272¾, [z - 62.1 (£ 0.3MeOH). (b) (1R, 45)-4-(2-Amino-6-hydroxy-9H-purir>-9yl)-2- cyclopentenyl carbinol

The filtrates from the preparation of the IS, 4R isomer (Example14a) were combined and evaporated to dryness. The unchanged diaminostarting material was separated on a silica gel flash column using IOSmethanol/chloroform. The diamino compound was dissolved in 0.05MK2P0^ buffer, pH 7.4 (15ml) and SCO units of adenosine deaminase wereadded. The solution was incubated for 96 hours at 37¾. TLCindicated some unreacted product remained. The solution was heated inboiling water for 3 minutes and filtered to remove denatured protein.Another SCO units of edenosine deaminase were added and the processwas repeated. The deprcteinated solution was evaporated to drynessand the product was crystallized from water. The title product es a white24 solid was collected by filtration from water, m.p. 265-270 4. [ + 61.1 (£0.3 MsCH).

BAD ORIGINAL 31

Example 15 (±) (la, 4c )-4-(2-Amino-6-hydroxy-9H-purin-9-yl)-2-cyclopentenyl acetoxycarbinol

To a suspension of the product of Example 10 (130 mg, 0.50 mmol)and 4-dimethylaminopyridine (5mg, 0.04mmol) in a mixture ofacetonitrile (6ml) and triethylamine (0.09ml, 0.66mmol) was addedacetic anhydride (0.06ml, 0.6 mmole). The mixture was stirred at roomtemperature for 3 hrs. Methanol (1ml) was added to quench thereaction. The solution was concentrated and absorded on silica gel(1.5g), packed on a column (2.0 x 12cm), eluted with CHCl3-Me0H(20:1). The product fractions were collected and concentrated to getwhite solid. The solid product was washed with MeOH-AcOEt: yield,123mg (85%). Further purification from methanol gave the titleproduct as needle-like crystals, m.p. 237-239°C. Anal.(C13H1:,N;>03)C,H,N.

Example 16 (15,4R)-4-[2-amino-9H-purin-9-yl]-2-cyclopentenylcarbinol A stirred, ice-chilled solution of (lS,4R)-4-[2-amino-6-methoxyamino-9H-purin-9-ylJ-2-cyclopentene-methanol (intermediate 8,Example 12) (1.202g) in tetrahydrofuran (25Cml) and water (50ml) wastreated with aluminium amalgam (from aluminium (1.761g) and 0.5%aqueous mercuric chloride solution), added in small pieces over lhr47min. After 35 min the stirred mixture was allowed to warm toambient temperature. After 16hr 50min more aluninium amalgam (from235mg aluminium) was added over 14min. After a further 4hr lCmin theresulting mixture was filtered through kieselguhr to removeinsolubles. These were washed with tetrahydrofuran:water (5:1, 300mi). The combined filtrates were evaporated to leave a yellowfoam. The foam was subjected to column chromatography on silica(33.8g, Merck 7734) prepared in chloroform and eluted with chloroform-ethanol mixtures to give several fractions (578mg, 42Cmgand 40mg). The two larger fractions were separately crystallised fromiso-propanol. The filtrates were combined with the smallest columnfraction and subjected to preparative thin layer chromatography (Merck5717) developed three times in 10:1 chloroform:methanol. The plates AD ft 0 0 0 9 1

BAD ORIGINAL 32 ο Ο Ο Ω were eluted with ethyl acetate and ethyl acetate-ethanol (1:1) to givea brown solid (45mg). The solid was subjected to columnchromatography on silica (2.7g, Merck 7734) prepared in chloroform andeluted with chloroform-methenol-triethylamine mixtures to give a gum(17mg). Following an unsuccessful crystallisation from iso-propanoland charcoal treatment in methanol, an aqueous solution of therecovered material was freeze dried to give the title compound (15mg).iHnmr (CMSO-dg) 1.62 (1H), 2.63 (1H), 2.89 (1H), 3.45 (2H), 4.73 (1H),5.48 (1H), 5.91 (1H), 6.14 (1H), 6.50 (2H), 7.93 (1H), 8.57 (1H).

Mass spec, [MHj+ 232.

Example 17

Tablet Formulations A. The following formulation is prepared by wet granulation of theingredients with a solution of povidone in .water, drying" andscreening, followed by addition of magnesium stearate andcompression. mg/tablet (a) Active ingredient 250 (b) Lactose B.P. 210 (c) Povidone B.P. 15 (d) Sodium Starch Glycollate 20 (e) Magnesium Stearate 5 500 B. The following formulation is prepared by direct compression; thelactose is of the direct compression type.

BAD ORIGINAL 3 mg/tablet Active ingredient 250 Lactose 145 Avicel 100 Magnesium Stearate 5 500 C. (Controlled Release Formulation) The Formulation is prepared bywet granulation of the ingredients (below) with a solution oF povidonein water, drying and screening Followed by the addition oF magnesiumstearate and compression. mg/tablet (a) Active ingredient 500 (b) Hydroxypropylmethylcellulose (Methocel K4M Premium) 112 (c) Lactose B.P. 53 (d) Povidone B.P. 28 (e) Magnesium 5tearate 7 ADOn 0 0 9 1 700

Example 18

Capsule Formulation A capsule Formulation is prepared by admixing the ingredientsbelow and Filling into a two-part hard gelatin capsule. mo/capsule

Active ingredient 125

Lactose 72.5

Avicel 50

Magnesium Stearate 2.5

BAD ORIGINAL Λ _ 34

Example 19

Injectable Formulation

Active ingredient 0.200g

Sodium hydroxide solution, 0.1M q.s. to a pH of about 11.

Sterile water q.s. to 10ml

The active ingredient is suspended in some of the water (whichmay be warmed) and the pH adjusted to about 11 with a solution ofsodium hydroxide. The batch is then made up to volume and filteredthrough a sterilising grade membrane filter into a sterile lCml glassvial and sealed with sterile closures and overseals.

Example 20

Suppository mq/suppository

Active ingredient (63pm) 250

Hard Fat, BP 1770 2020

One-fifth of the hard fat is melted in a steam-jacketed pan at45°C maximum. The active ingredient is sifted through a 200 μη sieveand added to the molten base with mixing, using a high shear stirrer,until a smooth dispersion is achieved. Maintaining the mixture at45^0, the remaining hard fat is added to the suspension end stirred toensure a homogenous mix. The entire suspension is passed through a250pm stainless steel screen end, with continuous stirring, is allowedto cool to 40°C. At a temperature of 383C to 40^0, 2.02c of themixture is filled into suitable, 2ml plastic moulds. The suppositories are allowed to cool to room temperature. BAD ORIGINAL ft

Example 21 - ANTIVIRAL ACTIVITY (A) Anti-HIV Assay

Compounds of formula (I) were screened for anti-HIV activity atthe National Cancer Institute, Frederick Cancer Research Facility,Frederick, Maryland (FCRF). The following are the current screeningmode operational procedures utilized at FCRF. The protocol consistsof 3 areas, (I) preparation of infected cells and distribution to thetest plates, (II) preparation of drug dilution plates and distrubitionto the test plates, and (III) XTT assay procedure. See D. A. Scudieroet al., "A New Simplified Tetrazolium Assay for Cell Growth and DrugSensitivity in Culture," Cancer Res., 48, 4827 (1988). I. Infection and Distribution of ATH8 Cells to Microtiter Trays

Cells to be infected (a normal lymphoblastoid cell line whichexpresses CD4) are placed in 50ml conical centrifuge tubes and treatedfor 1 hr with l-2pg/ml of polybrene at 37°C. The cells are thenpelleted for 8min. at 1200 RPM. HIV virus, diluted 1:10 in media(RMP1-1640, 10% human serum or 15% fetal calf serum (FCS), with IL-2and antibiotics) is added to provide an MOI of .001. Medium alone isadded to virus-free control cells. Assuming en infectious virus titerof 10“\ an MOI of .001 represents 8 infectious virus particles per10,000 cells. About 500,000 cells/tube are exposed to 400μΐ of thevirus dilution. The resultant mixture is incubated for lhr at 37 PC inAir-C02· The infected or uninfected cells are diluted to give 1 x10“H (with human serum or 2 x 10-4 (with fetal calf serum) cells/ΙΟΟμΙ.

Infected or uninfected cells (ΙΟΟμΙ) are distributed toappropriate wells of a 96 well, U-bottom microtiter plate. Eachcompound dilution is tested in duplicate with infected ceils.Uninfected cells are examined for drug sensitivity in a single wellfor each dilution of compound. Drug-free control cells, infected ancuninfected, are run in triplicate. Wells 32 through G2 served as

l 6 0 0 0 0dV

BAD ORIGINAL 36 reagent controls and received medium only. The plates are incubatedat 37°C in Air-CO2 until the drug is added. II. Drug Dilution end Addition

Dilution plates (flat bottom 96 well, microtiter plates) aretreated overnight with phosphate buffered saline (P8S) or mediacontaining at least 13 FCS or 13 human serum (depending on the mediumused in the test), beginning the day before assay. This "blocking"procedure is used to limit the adsorption of drug to the microtitertray during the dilution process. The wells are filled completelywith the blocking solution and allowed to stand at room temperature ina humidified chamber in a hood.

The dilution process is begun by first diluting the test compound1:20. Blocked, dilution plates are prepared by flicking out theblocking solution and blotting dry on sterile gauze. All wells ofeach plate are then filled with 225μ1 of the appropriate medium using ·*»«. a Cetus liquid handling system. Twenty-five microliters (25 μΐ) of/» each 1:20 diluted compound is then manually added to row A of a > blocked and filled dilution plate. Four compounds, sufficient to supply two test plates, are added per dilutien plate. The fourcompounds are then serially diluted 10 fold from row A through row Husing the Cetus liquid handling system. The starting dilution of eachcompound in row A is, at this point, 1:200. The dilution plates arekept on ice until needed.

Using a multi-channel pipettor with 6 microtips, 100 μΐ of eachdrug dilution is transferred to the test plate which already contains100μ1 of medium plus cells. The final dilution, in the test plate,starts at 1:400 (wells 34 through GA). This dilution (to .253 C,MSQ)prevents the CMSO vehicle from interfering with cell growth.

Drug-free, infected or uninfected cells (wells B3 through G3) andreagent controls (32 through G2) receive medium alone. The final 2compounds are then transferred from wells H7 through H12 to a secondtest plate suing the same procedure. Test plates are incubated at37oC in Air-C02 for 7-14 days or until virus controls ere lysed asdetermined macrosccpicelly. bad original $ 37 III. Quantitation of Viral Cytcpathogenicity and Drug Activity A. Materials 1. A solution of 2,3-bis[2-methoxy~4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide. (XTT) - lmg/mlsolution in media without FCS. Store at 4 °C. Prepare weekly. 2. Phenazine methosulfonate (PMS) stock solution - This can beprepared and maintained frozen until needed at "20°C. It should bemade in PBS to a concentration of 15.3mg/ml. 8. Microculture Tetrozolium Assay (MTA) 1. Preparation of XTT-PM5 Solution - The XTT-PMS is preparedimmediately prior to its addition to the wells of the culture dish.

The stock PMS solution is diluted 1:100 (0.153mg/ml). Diluted PMS isadded to every ml of XTT required to give a final PMS concentration of0.02mM. A 50μ1 aliquot of the XTT-PMS mixture is added to each of theappropriate wells, and the plate is incubated for four hours at 37¾.The plate lids are removed and replaced with adhesive plate sealers(Dynatech cat 001-010-3501). The sealed plate is shaken on amicroculture plate mixer and the absorbance is determined at 45Cnm. IV. Results

Figure 1 depicts a plot of the percentage of test cells overuninfected cells (%) for both infected and uninfected cells as afunction of the increasing concentration of the compound of Example10.

The data plotted on Figure 1 permits the calculation of aneffective concentration (ECS(J) with respect to infected cells of about0.15pg/ml, an inhibitory concentration dC50) with respect to normalcells of about lOO^g/ml, and a therapeutic index (TI30)of about 667.

An earlier assay carried out at the Southern Research Instituteyielded at TIi0 of about 200 when MT-2 cells were cultured withH9/HTLV-III3. BAD ORIGINAL gp .33

The inhibitory concentrations against HIV determined as describedebove for the compounds of Examples 7, 9, 10, 11 and 14(b; are shownin Table 1. TABLE 1

Compound Example Cell Line ED50 IDbO nb0 9a 7 MT-2 2.3 50 21.4 13a 9 MT-2 0.41 6.97 17.3 14a 10 MT-2 0.15 100 667 15a 11 MT-2 2.9 > 125 > 42.7 (-) 14a 14 (b) CEM 0.66 189 284

Π ή 3QA

The compounds of Examples 5 and 8 also showed antiviral activityin this screen. (5) Activity against Feline Leukemia Virus

Antiviral screening for activity against FeLV-FAIDS was performed in 96-well plates (Corning) using 81C indicator cells in Iscove'sModified Dulbecco's medium supplemented with 10S heat-inactivatedfetal bovine serum (F3S). Twenty hours prior to the assay, the plateswere seeded with the SIC cells at 5 x 103 cells/well. On the day ofthe assay, the cells were pretreated for 30 minutes at 37¾ with DEAE-dextran (25pg/ml) in 0.1ml Hanks balanced salt solution. This wasremoved end then O.Lml of growth medium containing 32 TCID^j ofFeLV-FAIDS, or 0.1ml cf growth medium alone, was added to each well.The virus was allowed to adsorb for 1 hour, then 0.1 ml of test orpositive control compound (2',31-dideoxycytidine; dcC), or growth

BAD ORIGINAL A - 39 - medium was added. Plates were incubated at 37¾. Cells were fedfresh growth medium containing compound on Day 4 post-infection.

Culture medium was completely changed and replaced with fresh mediumcontaining compound on Day 7 post-infectin. On Day 10 post-infectionthe cells were fixed with formalin, stained with 0.12 CoomassieBrilliant Blue R-250 and observed microscopically for CPE and drugcytotoxicity.

The compound of Example 10 had an ED50 of 1.9pg/ml.

CC) Activity against Murine AIDS

Falcon 6-well tissue culture plates were seeded with 1.75 x 10 5 cells per well in total volume of 2.5ml EMEM containing 52 heat- inactivated FBS. Twenty hours after the cells were seeded, the mediumwas decanted and 2.5ml DEAE-dextran (25pg/ml in phosphate-buff eredsaline) was added to each well. The cultures were incubated at 37¾for 1 hour, after which the DEAE-dextran solution was decanted and thecell layers rinsed once with 2.5ml PBS. Normal cell controls wererefed with 2.5ml medium alone (no virus or drug). Drug controlcultures received 2.5ml of medium containing drug but no virus.Virus-infected control cultures received 0.5ml of the appropriatedilution of stock CAS-BR-M to produce countable plaques plus 2.0mlmedium. The test samples received 0.5ml of the appropriate virusdilution plus 2.0ml medium of the drug dilution. Six concentrationsof the test compound diluted in serial half-log10 dilutions weretested. Three concentrations of the positive control drug, ddC, weretested. Triplicate wells for each concentration of test compound and6 virus and 6 cell control cultures were included in each assay. OnDay 3 post-virus inoculation toxicity of the drug for the SC-1 cellswas determined by microscopic examination of stained duplicate celland drug control cultures. The remaining test and control cultureswere irradiated with an ultraviolet lamp for 20 seconds and XC cellswere added to each culture (5 x 105 cells/well in 2.5ml EMEMcontaining 102 heat-inactivated FBS). On Day 3 post-UV irradiation,the cultures were fixed with formalin and stained with crystal violet.

The plaques were counted with the aid of a dissection microscope. BAD ORIGINAL ¢, AP0 0 0 0 9 1 40

Antiviral activity in the CAS-BR-M plaque reduction was expressedin terms of the reduction in the mean number of plaques counted in thedrug-treated, virus-infected cultures compared with the mean number ofplaques counted in the untreated, virus-infected control cultures(percent of control). The compound of Example 10 had an ED 501.lpg/ml. (D) Activity against Simian retrovirus 5AIDS (SRV-2)

Antiviral screening against the SAIDS virus (D/Washington) wasperformed by a syncytia-inhibicion assay on Raji cells. The drug wasdiluted in complete Iscove's medium and then 100μΐ of each dilutionwas added to the appropriate wells of a 96-well plate. Activelygrowing Raji cells, 5 x 103 cells in 50μΐ of complete Iscove's medium,were then added to each well. This was followed by the addition of50μ1 of clarified supernate from an SRV-2/Raji cell co-culture. DDCwas included in this assay as the positive control drug. Plates wereincubated at 37°C in a humidified atmosphere containing 52 CO 2·Syncytia were counted on Day 7 post-infection. Drug toxicity wasascertained by comparing viable cell counts of the uninfected, drug-treated sample to the viability of the uninfected, untreated control.The compound of Example 10 had an ED50 of 2.8pg/ml. (E) Activity against Visna Maedi Virus

The antiviral activity against Visna Maedi Virus (VMV) strainWLC-1, was determined by measuring reduction of virus-specificimmunohistochemical staining. Monolayers of sheep choroid plexuscells were infected with VMV end overlaid with serial dilutions oftest compounds. After incubation for five days, the monolayers werefurther incubated with virus specific antisera conjugated to horseradish peroxidase (HRP). Subsequent incubation of the monolayers witha chrcmagenic substrate of HRP, strains ereas of virus replication.These discrete foci were counted and the concentration of testcompound required to reduce the number of foci to 502 of that of druguntreated controls calculated.

The compound of Example 13 had an Ε0 = ;) 0.2;_g/ml. bad original - 4.] -

Example 22

CYTOTOXIC ACTIVITY

The compounds of Examples 5, 7 and 8 showed cytotoxic activitywhen tested against P388 mouse leukemia cell culture assay asdescribed by R. G. Alonquist and R. Vince, £. Med. Chem, 16, 1396(1973). The EDbQ's (pg/ml) obtained were :-

Example 5 12

Example 7 40

Example 8 3

Having now particularly described and ascertained our said inventionand in what manner the same is to be performed we declare that. AP000091

BAD ORIGINAL

Claims (18)

42- CLAIMS:
1. A compound of formula (I) • N I a • · / / \\ / \ /Z N N (I) ho-ch2 ·. Wherein X is hydrogen, NRR1, SR, OR or halogen; Z is hydrogen, OR2 or NRR1; R, R1 and R2 may be the same or different and are selected fromhydrogen, C^.^alkyl end aryl; and pharmaceutically acceptable derivatives thereof.
2. A compound of formula (I) according to claim 1 andpharmaceutically acceptable salts thereof. ,-r
3. A compound according to Claim 1 or Claim 2 wherein in the z""' compound of formula (I) Z is H, OH or NH2 t;
4. A compound according to eny one of Claims 1 to 3 wherein Z is «r · nh2.
5. A compound according to any one of Claims 1 to 4 wherein X ishydrogen, chloro, NH2, SH or OH.
6. A compound according to any one of Claims 1 to 5 wherein X is CH.
7. A compound according to eny cne of Claims 1 to 5 wherein X is Hor \H2. BAD ORIGINAL A - 43-
8- A compound selected from:- (la,4c ) -4-(6-Chloro-9H-purin-9-yl)-2-cyclopentenyl carbinol;(Ια,4a) -4-(6-Hydroxy-9H-purin-9-yl)-2-cyclopentenyl carbinol;(la,4a) -4-(6-Amino-9H-purin-9-yl)-2-cyclapentenyl carbinol; (la,4a) -4-(6-Mercapto-9H-purin-9-yl)-2-cyclopentenyl carbinol;(Ια,4a) -4-(2,6-Diemino-9H-purin-9-yl)-2-cyclopentenyl carbinol;(la,4a) -4-(2-Amino-6-chloro-9H-purin-9-yl)-2-cyclopentenyl carbinol; and (la,4a) -4-(2-Amino-9H-purin-9-yl)-2-cyclopentenyl carbinol.
9. (la,4a) -4-(2-Amino-6-hydroxy-9H-purin-9-yl)-2-cyclopentenyl carbinol.
10. A compound according to any one of Claims 1 to 9 in the form ofsubstantially a racemic mixture.
11. A compound according to any one of Claims 1 to 9 consistingsubstantially of an optical isomer.
12. A compound according to any one of Claims 1 to 9 consistingsubstantially of the D isomer.
13. A compound of formula (I) as defined in anyone of claims 1 to 12or a pharmaceutically acceptable derivative thereof for use as anactive therapeutic agent.
14. A compound of formula (I) as defined in any one of Claims 1 to 12or a pharmaceutically acceptable derivative thereof for use in themanufacture of a medicament for the treatment of a viral infection. AP0 0 0 0 9 1
15. A pharmaceutical formulation comprising a compound of formula (I)as defined in any one of Claims 1 to 12 or a pharmaceuticallyacceptable derivative thereof together with a pharmaceuticallyacceptable carrier therefor. BAD ORIGINAL 0 4
16. A pharmaceutical formulation comprising a compound of formula (I)as defined in any one of Claims 1 to 12 or a pharmaceuticallyacceptable salt thereof together with a pharmaceutically acceptablecarrier therefor.
17. A pharmaceutical formulation according to Claim 14 ε -nallycomprising a further therapeutic agent.
18. A compound of formula (II) // \ / 2 N · I 9 Λ\ Λ Z N NH Y-
\ _ / •-· w;» lixt wherein X is hydrogen, NRRp SR, OR, halogen or protected formsthereof; Y is CH or a protected form thereof; Z is hydrogen, OR2, NRR1 or protected forms thereof; R^1 and R2 may be the same or different and are selected fromhydrogen, Cj_4al!<yl and aryl and pharmaceutically acceptable derivatives thereof. sca\lh:;-< holdsλ : t; r. 15 r : r λ p p l ; c BAD ORIGINAL p . Ό . Re n -
APAP/P/1989/000113A 1988-01-20 1989-01-19 Dideoxydidehydrocarbocyclic nucleosides. AP91A (en)

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